Lipids, nanoparticles containing the same, and their use

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 of related applications] This application claims priority and interest in U.S. Provisional Patent Application No. 63 / 350,215, filed on 8 June 2022, the entirety of which is incorporated herein by reference.

[0002] 1. Field of the present invention

[0003] This disclosure relates generally 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 technology]

[0004] 2. Explanation of related technologies

[0005] It is widely recognized that among the various reagents used to transfect cells with bioactive agents such as nucleic acids, those based on delivery via lipid nanoparticles (e.g., liposomes) are the most effective. This is primarily 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 fuses with other bilayers, such as the cell membrane, allowing the contents of the liposome to be delivered into the cell.

[0006] Lipid nanoparticles are used for drug delivery due to their unique properties. Lipid nanoparticles enclose an aqueous solution region within a hydrophobic membrane, preventing the dissolved hydrophilic solute from easily passing through the lipids. Hydrophobic chemicals can dissolve in the membrane, thus allowing lipid nanoparticles to carry both hydrophobic and hydrophilic molecules. Lipid nanoparticles can be used in combination with drugs, nucleic acids, and other bioactive agents to deliver these substances for the treatment and / or prevention of diseases.

[0007] Recently, lipid nanoparticles have been used in COVID-19 mRNA vaccines. mRNA vaccines have proven effective in controlling COVID-19, and clinical trials are underway in many countries to evaluate mRNA vaccines for 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 that they can be manufactured rapidly, allowing for the targeting of variants of infectious pathogens. However, the low thermal stability of mRNA vaccines severely limits their storage and distribution. For example, the COVID-19 mRNA vaccines manufactured by Moderna and Pfizer BioNTech can only be stored for 6 months at -20°C and -80°C, respectively. Furthermore, Moderna's mRNA COVID-19 vaccine is stable for only 12 hours at room temperature. Similarly, Pfizer BioNTech's mRNA COVID-19 vaccine is stable for only 2 hours at room temperature.

[0008] Therefore, related technologies require novel lipid molecules for the production of lipid nanoparticles for delivering therapeutic agents (e.g., nucleic acids). [Overview of the project] [Problems that the invention aims to solve]

[0009] This disclosure provides novel cationic lipids for forming nanoparticles for nonviral transport 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 problem]

[0010] A first aspect of this disclosure relates to a lipid of formula (I). Here, JPEG0007909622000001.jpg2050R1 contains one or more hydroxyl groups, -CH2OH groups. JPEG0007909622000002.jpg728, or an alkyl or cycloalkyl group which may be substituted with an -NR2 group, m and n are independent integers between 0 and 12. R2 and R3 are independently H, alkenyl, and R a -(C=O)O(CH2)R a -O(C=O)R b , or -(C=O)OR b And, R a This is -CR'(COOR'')2 or -CR'(COOR'')(COOR'''), R, R b R', R'', and R''' are independently either H or alkyl.

[0011] According to embodiments of this disclosure, the lipid of formula (I) may be any of the following: JPEG0007909622000003.jpg204138JPEG0007909622000004.jpg179138JPEG0007909622000005.jpg52138

[0012] A second aspect of this disclosure relates to lipids of formula (II). JPEG0007909622000006.jpg4065 Here, R1 and R3 are alkyl groups which may be independently substituted with one or more hydroxyl groups. R2 is H or -O(C=O)R', and R' is alkyl. m and n are independent integers between 1 and 10.

[0013] According to embodiments of this disclosure, the lipid of formula (II) is It could also be any of the following: JPEG0007909622000007.jpg27124

[0014] A third aspect of this disclosure relates to the lipid of formula (III). JPEG0007909622000008.jpg3880 Here, m and n are independent integers between 1 and 10. R1, R2, R3, and R4 are independently either H or alkyl.

[0015] According to embodiments of this disclosure, the lipid of formula (III) has the following structure. JPEG0007909622000009.jpg54101

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

[0017] Furthermore, or optionally, the lipid nanoparticles of this disclosure further include a therapeutic agent disposed within the hydrophilic core or outer lipid bilayer shell of the nanoparticle. The therapeutic agent is located within the hydrophilic core if hydrophilic, or within the lipid shell if hydrophobic. The therapeutic agent may be the 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 comprise 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 comprise mRNA of the envelope (E) protein of dengue virus encapsulated within the hydrophilic core.

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

[0020] Please understand that both the general description above and the detailed description below are illustrative and are intended to provide a further explanation of the invention as requested. [Brief explanation of the drawing]

[0021] This specification will be better understood by reading the following detailed description in reference to the attached drawings.

[0022] [Figure 1] This shows protein expression after administration of the SARS-CoV-2 S protein (BA.5) mRNA-LNP complex in vitro and in vivo. (A) Cellular ELISA data indicating successful mRNA transfection, (B) Binding activity of immunoserum to the target as evaluated by ELISA, and (C) Maximum half-life inhibitory concentration (IC50) in the serum of immunized mice.

[0023] [Figure 2] This shows protein expression after administration of the SARS-CoV-2 S protein (WT) mRNA-LNP complex in vitro and in vivo. (A) Cellular ELISA data indicating successful mRNA transfection, (B) Binding activity of immunoserum to target as evaluated by ELISA.

[0024] [Figure 3] This study analyzes the function of the DENV2 E mRNA-LNP complex. (A) Protein expression level, (B) Serum collected from mice 6 weeks after initial immunization and analyzed by ELISA for dengue virus-specific antibody response, and (C) Maximum half-limit inhibitory concentration (IC50) in the serum of immunized mice. [Modes for carrying out the invention]

[0025] The detailed descriptions provided below in relation to the attached drawings are intended to be descriptive of this disclosure and are not intended to represent the only form in which this disclosure may be constructed or used.

[0026] For the purposes of this invention, lipid nanoparticles refer to particles formed by a hydrophilic nucleus covered with a lipid shell, which 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 located within the hydrophilic nucleus if hydrophilic, or within the lipid shell if hydrophobic.

[0027] For convenience, the specific terms used in the specification, examples, and appended claims are set forth herein. Unless otherwise defined, all technical and scientific terms used herein shall have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs.

[0028] Unless otherwise required by context, singular terms are understood to include plurals, and plural terms are understood to include singulars. Specifically, as used herein and in the claims, the singular “a” and “an” refer to multiple objects unless otherwise explicitly indicated by the context. Also, as used herein and in the claims, the terms “at least one” and “one or more” have the same meaning and include one, two, three, or more.

[0029] While the numerical ranges and parameters representing the broad scope of this invention are approximations, the numerical values ​​shown in specific examples are reported as accurately as possible. However, all numerical values ​​inherently contain errors that inevitably arise from the standard deviation of the measurements in each test. Furthermore, as used herein, the term “about” generally means within 10%, 5%, 1%, or 0.5% of a given value or range. Alternatively, “about” means within the acceptable mean standard error as considered by those skilled in the art. Except for operational examples / work examples, or unless expressly specified, all numerical ranges, quantities, values, and percentages such as quantities, durations, temperatures, operating conditions, and ratios of quantities of materials disclosed herein should be understood to be modified by the term “about.” Accordingly, unless otherwise indicated, the numerical parameters described in this disclosure and the appended claims are approximations that may be modified as needed. At a minimum, each numerical parameter should be interpreted using common rounding techniques, taking into account the reported number of significant figures.

[0030] The atoms, parts, or groups described herein may be unsubstituted or substituted, to the extent that their valence permits, unless otherwise specified. The term “may be substituted” means substituted or unsubstituted. Unless otherwise specified, when used to describe a chemical structure or part, the term “substituted” refers to a derivative of that structure or part, in which one or more hydrogen atoms of that structure or part are substituted with one or more non-hydrogen atoms or groups, such as halo, hydroxyl, alkyl, aryl, amino, or alkylamino. The term “substituted” includes substitution with all permissible substituents of an organic compound, any of the substituents described herein forming a stable compound. The present invention intends to use any and all such combinations 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, 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 3-10A cycloalkyl group is a ring carbon atom (C). 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 5-6 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). 3-6 Examples of cycloalkyl groups include, as mentioned earlier, C 5-6 In addition to cycloalkyl groups, examples include cyclopropyl (C3) and cyclobutyl (C4). 3-8 Examples of cycloalkyl groups include, as mentioned earlier, C 3-6 Examples include cycloalkyl groups, 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 It is a cycloalkyl group. In certain embodiments, the cycloalkyl group is a substituted C 3-10 It is a cycloalkyl compound. The carbocyclyl may be partially unsaturated.

[0033] The term "alkenyl" refers to a radical of a straight-chain or branched hydrocarbon group without triple bonds, having 2 to 20 carbon atoms and one or more carbon-carbon double bonds ("C 2-20 In some embodiments, the alkenyl group has 18 carbon atoms ("C"). 18An alkenyl group has 18 carbon atoms and 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 It is an alkenyl. In certain embodiments, the alkenyl group is substituted with C 2-20 It is Alkenil.

[0034] It is also understood that compounds having the same molecular formula but different properties, or different order of atomic bonding or arrangement of atoms in space, are called "isomers." Isomers with different arrangements 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 of each other but cannot be superimposed are called "enantiomers." If a compound has a chiral center, for example, if it is bonded to four different groups, a pair of enantiomers is possible. Enantiomers can be characterized by the absolute configuration of their chiral center and are described by Cahn and Prelog's R- and S-ordering rules, or by the way the molecule rotates in plane-polarized light and are expressed as right-handed or left-handed rotation ((+) or (-) isomers, respectively). Chiral compounds can exist as individual enantiomers or mixtures 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 they are generally understood by those skilled in the art to which this invention pertains. In case of any conflict, this specification (including definitions) shall prevail.

[0036] 1.Compound

[0037] The compounds described herein may have the structure of the following formula (I). JPEG0007909622000010.jpg2050

[0038] In formula (I), R1 is one or more hydroxyl, -CH2OH, JPEG0007909622000011.jpg720, or an alkyl or cycloalkyl which may be substituted with 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 hydroxyl group. In further embodiments, R1 is propyl substituted with two hydroxyl 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 yet another embodiment, R1 is This is ethyl substituted with JPEG0007909622000012.jpg729.

[0039] Furthermore, or alternatively, m and n are independently integers from 0 to 12, and R2 and R3 are independently H, alkenil, R a -(C=O)O(CH2)R a -O(C=O)R b , or -(C=O)OR b And here, 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 And here, R a It is -CR'(COOR'')2, where R' is methyl and R'' is -C8H 17In 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 And here, R a It is -CR'(COOR'')2, where 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), where R' is methyl and R'' is -C8H 17 In a further embodiment, m is 10, n is 6, R2 is H, and R3 is R a (-CR'(COOR')2), where 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''')), where R' is -CH3 and 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), where R' is methyl and R'' is -C8H 17 That is the case.

[0040] According to embodiments of this disclosure, the lipid of formula (I) may be any of the following: JPEG0007909622000013.jpg181139JPEG0007909622000014.jpg201139JPEG0007909622000015.jpg52138

[0041] According to one preferred embodiment of the present disclosure, the lipid of formula (I) has the following structure. JPEG0007909622000016.jpg2566

[0042] According to another preferred embodiment of the present disclosure, the lipid of formula (I) has the following structure. JPEG0007909622000017.jpg2476

[0043] The compounds described herein may have the structure of the following formula (II). JPEG0007909622000018.jpg4065

[0044] In formula (II), m and n are independently integers between 1 and 10, and R1 and R3 are independently alkyl groups which may be substituted with one or more hydroxyl groups. In some embodiments, m and n are independently 6, R1 is a propyl group substituted with one hydroxyl group, and R3 is -C6H 13 In other embodiments, m is 10, n is 6, R1 is propyl substituted with one hydroxyl group, and R3 is -C6H 13 That is the case.

[0045] Alternatively, 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 That is the case.

[0046] According to embodiments of this disclosure, the lipid of formula (II) may be any of the following: JPEG0007909622000019.jpg27126

[0047] The compounds described herein may have the structure of the following formula (III). JPEG0007909622000020.jpg3880

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

[0049] According to embodiments of this disclosure, the lipid of formula (III) has the following structure. JPEG0007909622000021.jpg3466

[0050] 2. Lipid nanoparticles

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

[0052] Furthermore, or optionally, the lipid nanoparticles may further comprise a therapeutic agent disposed within an outer lipid bilayer shell or a hydrophilic core. The therapeutic agent resides within the hydrophilic core if hydrophilic, or within the lipid shell if hydrophobic. The therapeutic agent may also be the 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] This disclosure also provides a method for intracellularly delivering a drug of interest (e.g., a therapeutic agent) to cells. According to some embodiments of this disclosure, the lipid nanoparticles of the present invention are preloaded with a nucleic acid of interest (e.g., mRNA of the SARS-CoV-2 spike protein) intended to be delivered to target cells, and one or more drugs to facilitate contact with the target cells and subsequent transfection. Preferably, the lipid nanoparticles of the present invention allow the encapsulated nucleic acid to reach the target cells and subsequently transfect the target cells. Thus, after delivery, the nucleic acid encodes one or more target proteins within the target cells. 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, osteocytes, stem cells, mesenchymal stem cells, nerve cells, cardiac cells, adipocytes, vascular smooth muscle cells, skeletal muscle cells, B cells, T cells, leukocytes, granulocytes, fibroblasts, reticulocytes, and the like. According to embodiments of this disclosure, lipid nanoparticles preloaded with the nucleic acid of interest are successfully taken up by T cells, which then transfect the T cells to express the protein of interest encoded by the delivered nucleic acid of interest (e.g., mRNA of the SARS-CoV-2 spike protein) (e.g., the SARS-CoV-2 spike protein). In certain embodiments, the protein of interest is produced at a higher level than the control group (i.e., baseline levels of cells not treated with the lipid nanoparticles of the present invention).According to embodiments of this disclosure, the target protein is at least 1 to 100,000 times greater than that of the control group, 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, 5,0 00, 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, preferably at least 5 to 50,000 times the control group, for example, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 55, 60 times the control group , 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, more preferably at least 10 to 10,000 times the control group. The target cell (e.g., T cell) expresses the protein at 0x, 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, and 10,000x of the control group. In some embodiments, the expression level of the target protein remains detectable over a sustained period, such as 1 day, 2 days, 3 days, 4 days, 5 days, or more than a week.

[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 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.

[0058] 3.2 Treatment of Diseases

[0059] This disclosure also provides a method for treating and / or preventing a disease in a subject. The method involves administering an effective amount of the lipid nanoparticles of the present invention, pre-filled with a therapeutic agent, to a target tissue in a subject in order to treat and / or prevent a disease.

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

[0061] In some embodiments, the method comprises administering lipid nanoparticles of the Disclosure, pre-filled with viral nucleic acids, to a subject so that the encapsulated viral nucleic acids of interest are delivered to a target tissue of the subject (e.g., lungs, liver, etc.), expressed in the target tissue, and act as an antigen to induce a controlled level of immune response in the subject, thereby immunizing the subject and preventing the subject from subsequently becoming infected with the virus and / or developing a disease caused by the viral infection (e.g., severe acute respiratory syndrome, SARS).

[0062] Furthermore, or optionally, the lipid nanoparticles of this disclosure may be formulated in combination with one or more additional carriers, excipients, or stabilizers. The lipid nanoparticles of this disclosure may be administered in accordance with current medical practice, taking into account the subject's clinical condition, site and method of administration, administration schedule, the subject's age, sex, weight, and other factors related to the clinical condition. An effective dose for the purposes of this disclosure may be determined by relevant factors known to those skilled in the art of clinical research, pharmacology, clinical practice, and medicine. In some embodiments, the dose is effective in stabilizing, improving, or eliminating the symptoms of the disease to at least some extent, or in preventing disease progression. For example, an appropriate dose and administration regimen will induce at least transient proteinogenesis.

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

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

[0065] In some embodiments, the lipid nanoparticles of the Disclosure are formulated to be suitable for sustained release of nucleic acids contained therein. Such sustained-release lipid nanoparticles can be administered to subjects at long intervals between doses. For example, the lipid nanoparticles of the Disclosure can be administered to subjects daily, twice daily, or every other day. In some preferred embodiments, the lipid nanoparticles are administered to subjects once a week, twice a week, every 10 days, every 2 weeks, every 3 weeks, or every 4 weeks, once a month, every 6 weeks, every 8 weeks, every 2 months, every 3 months, every 4 months, every 6 months, every 8 months, every 9 months, or annually. The Specification also considers compositions formulated for depot administration (e.g., intramuscular, subcutaneous, etc.) for long-term delivery or release of nucleic acids.

[0066] Furthermore, this specification also considers lyophilized compositions comprising one or more of the lipid nanoparticles disclosed herein. The lyophilized compositions of this disclosure may be reconstituted before administration or in vivo. For example, the lyophilized composition may be formulated into a suitable dosage form (e.g., an intradermal dosage form such as a disc, rod, or membrane) and administered in such a manner that the dosage form is rehydrated in vivo over time by the body fluids of the individual.

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

[0068] Examples

[0069] material and method

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

[0071] 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, cells were fixed with 4% paraformaldehyde / PBS for 15 minutes, followed by permeabilization 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. Subsequently, 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 (PBST0.1) containing 0.1% Tween-20, 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 using an ELISA reader (Versa Max Tunable Microplate Reader, Molecular Devices).

[0072] Mouse immunization

[0073] A group of 6-8 week old BALB / c mice were immunized at the start of the experiment (day 0) by intramuscular (im) injection of a specified mRNAP-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), followed by additional boost injections at weeks 2 and 4, respectively. Serum samples were collected 4, 6, and 8 weeks after the initial immunization (day 0) and examined for binding activity to FLS-WT or FLS-BA.5 protein and neutralization activity by pseudoviral assay.

[0074] Pseudovirus neutralization assay

[0075] Blood samples were collected from mice six weeks after the initial boost, and serum was used to measure neutralizing activity against BA.5 SARS-CoV-2 pseudovirus. The pseudovirus neutralization assay was performed using SARS-CoV-2 pseudolentivirus expressing 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 semi-maximal inhibitory concentration (IC) was measured. 50 The average IC for each experimental group was calculated using nonlinear regression with Prism software version 8.1.0 (GraphPad Software Inc.). 50 The values ​​were determined from three independent experiments.

[0076] Plaque reduction neutralizing titer (PRNT) assay

[0077] Serum from animals injected with DENV2 E mRNA-LNP was serially diluted in PBS and pre-incubated with 100 plaque-forming units (PFUs) of DENV2 at 37°C for 1 hour. The mixture was then 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 a further 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 immunoserum / number of plaques without immunoserum)]. 50% plaque reduction (PRNT) 50 The values ​​were calculated using Prism software. DENV2 strain 16881 was used in this study.

[0078] Example 1: Synthesis of the compound of the present invention

[0079] Generally, the compounds of this disclosure are synthesized according to the procedures outlined in Schemes 1 to 5, and the amines required in step 7 of Scheme 1 are listed in Table 1.

[0080] Scheme 1. General procedure for disubstituted malonic acid ester compounds JPEG0007909622000022.jpg72145

[0081] Table 1. List of amines JPEG0007909622000023.jpg22155

[0082] Scheme 2. General alkylation of aminomalonic acid ester compounds JPEG0007909622000024.jpg25128

[0083] Scheme 3. Synthesis of AS-CL-19 JPEG0007909622000025.jpg39151

[0084] Scheme 4. Asymmetric synthesis of disubstituted malonic acid ester compounds JPEG0007909622000026.jpg23154

[0085] Scheme 5. Synthesis of AS-CL-30 and AS-CL-31 JPEG0007909622000027.jpg64149

[0086] Compound 1a: Dioctyl malonate (1a) (Step 1) JPEG0007909622000028.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 (600MHz, CDCl3) δ4.12(t,J=6.6Hz,4H), 3.35(s,2H), 1.63-1.60(m,4H), 1.32-1.26(m,20H), 0.86(t,J=7.2Hz,6H). 13 C NMR (150MHz, CDCl3) δ166.7, 65.6, 41.7, 31.7, 29.1(x2), 28.4, 25.8, 22.6, 14.0.

[0088] Compound 1b: Didecylmalonate JPEG0007909622000029.jpg15128

[0089] Compound 1b was synthesized according to the general procedure of Step 1 of Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR (600MHz, CDCl3) δ4.12(t,J=6.6Hz,4H), 3.35(s,2H), 1.65-1.60(m,4H), 1.33-1.25(m,28H), 0.87(t,J=7.2Hz,6H). 13 C NMR (150MHz, CDCl3) δ166.6, 65.6, 41.6, 31.8, 29.4(x2), 29.2, 29.1, 28.4, 25.7, 22.6, 14.0.

[0090] Compound 1c: Diundecylmalonate JPEG0007909622000030.jpg14128

[0091] Compound 1c was synthesized according to the general procedure of Step 1 of Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR (600MHz, CDCl3) δ4.12(t,J=6.6Hz,4H), 3.35(s,2H), 1.65-1.59(m,4H), 1.32-1.25(m,32H), 0.87(t,J=7.2Hz,6H). 13 C NMR (150MHz, CDCl3) δ170.3, 65.6, 46.3, 32.0, 29.7, 29.6, 29.4, 29.3, 28.6, 25.9, 22.8, 14.2, 13.7.

[0092] Compound 2a: Dioctyl 2-methylmalonate (Step 2) JPEG0007909622000031.jpg1664

[0093] At 0°C, NaH (430 mg, 10.8 mmol) was added to a solution of dioctyl malonate (4.43 g, 13.5 mmol) in THF, followed by the addition of MeI (0.66 mL, 10.8 mmol). 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 (600MHz, CDCl3) δ4.15-4.08(m,4H), 3.42(q,J=7.2Hz,1H), 1.65-1.57(m,4H), 1.41(d,J=7.2Hz,3H), 1.33-1.28(m,20H), 0.88(t,J=7.2Hz,6H). 13 C NMR (150MHz, CDCl3) δ170.2, 65.5, 46.2, 31.8, 29.2 (x2), 28.5, 25.8, 22.6, 14.1, 13.6.

[0094] Compound 2b: Didecyl 2-methylmalonate JPEG0007909622000032.jpg1676

[0095] Compound 2b was synthesized according to the general procedure of step 2 of scheme 1. The title compound was obtained as a colorless oil. 1 H NMR (600MHz, CDCl3) δ4.14-4.09(m,4H), 3.42(q,J=7.2Hz,1H), 1.64-1.60(m,4H), 1.41(d,J=7.2Hz,3H), 1.32-1.25(m,28H), 0.87(t,J=7.2Hz,6H). 13 C NMR (150MHz, CDCl3) δ170.2, 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 JPEG0007909622000033.jpg1785

[0097] Compound 2c was synthesized according to the general procedure of step 2 of scheme 1. The title compound was obtained as a colorless oil. 1 H NMR (600MHz, CDCl3) δ4.12-4.07(m,4H), 3.40(q,J=7.2Hz,1H), 1.61-1.59(m,6H), 1.39(d,J=7.2Hz,3H), 1.35-1.25(m,30H), 0.85(t,J=7.2Hz,6H). 13 C NMR (150MHz, CDCl3) δ170.3, 65.6, 46.3, 32.0, 29.7, 29.6, 29.4, 29.3, 28.6, 25.9, 22.8, 14.2, 13.7.

[0098] Compound 2d: Dioctyl 2-ethyl malonate JPEG0007909622000034.jpg1765

[0099] Compound 2d was synthesized according to the general procedure of step 2 of scheme 1. The title compound was obtained as a colorless oil. 1H NMR (600MHz, CDCl3) δ4.14-4.08(m,4H), 3.24(t,J=7.2Hz,1H), 1.94-1.89(m,2H),1 .64-1.58(m,4H), 1.35-1.26(m,20H), 0.95(t,J=7.2Hz,3H), 0.87(t,J=7.2Hz,6H). 13 C NMR (150MHz, CDCl3) δ169.5, 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-propyl malonate JPEG0007909622000035.jpg1972

[0101] Compound 2e was synthesized according to the general procedure of step 2 of scheme 1. The title compound was obtained as a colorless oil. 1 H NMR(600MHz,CDCl3)δ4.15-4.08(m,4H), 3.33(t,J=7.8Hz,1H), 1.89-1.85(m,2H), 1 .64-1.60(m,4H), 1.37-1.27(m,22H), 0.93(t,J=7.2Hz,6H), 0.88(t,J=7.2Hz,6H). 13 C NMR (150MHz, CDCl3) δ169.7, 65.4, 51.9, 31.7, 30.8, 29.1, 28.5, 25.8, 22.6, 20.6 14.0, 13.7. MS(ESI):m / z [M+Na] + 393.2973(C 22 H 42 O4Na).

[0102] Compound 2f: Dioctyl 2-(6-(benzyloxy)hexyl)malonate JPEG0007909622000036.jpg1869

[0103] Compound 2f was synthesized according to the general procedure of step 2 of scheme 1. The title compound was obtained as a colorless oil. 1H NMR (600MHz, CDCl3) δ7.35-7.26(m,5H), 4.49(s,2H), 4.15-4.08(m,4H), 3.45(t,J=6.6Hz,2H), 3.3 1(t,J=6.6Hz,1H), 1.90-1.86(m,2H), 1.64-1.59(m,6H), 1.33-1.29(m,26H), 0.86(t,J=7.2Hz,6H). 13 C NMR (150MHz, CDCl3) δ169.6, 138.5, 128.3, 127.6, 127.5, 72.9, 70.3, 65.4, 52.1, 31.8, 29.6, 29.2(x2), 29.1, 28.7, 28.5, 27.3, 25.9, 25.8, 22.6, 14.1.

[0104] Compound 2g: 1-decyl-3-octyl-2-methylmalonate JPEG0007909622000037.jpg1947

[0105] Two g of the compound was synthesized according to the general procedure of step 2 in Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR (600MHz, CDCl3) δ4.15-4.08(m,4H), 3.42(q,J=7.2Hz,1H), 1.65-1.60(m,4H), 1.41(d,J=7.2Hz,3H), 1.33-1.26(m,24H), 0.89-0.87(m,6H). 13 C NMR (150MHz, CDCl3) δ170.2, 65.5, 64.2, 31.9, 31.8, 29.6, 29.3, 29.25, 29.21, 28.5, 25.8, 22.7, 22.6, 14.10, 14.08, 13.6.

[0106] Compound 2H:1-hexyl3-octyl2-methylmalonate JPEG0007909622000038.jpg2042

[0107] Compound 2H was synthesized according to the general procedure of step 2 in Scheme 1. The title compound was obtained as a colorless oil. 1H NMR (600MHz, CDCl3) δ4.15-4.08(m,4H), 3.42(q,J=7.2Hz,1H), 1.65-1.60(m,4H), 1.41(d,J=7.2Hz,3H), 1.30-1.27(m,16H), 0.89-0.87(m,6H). 13 C NMR (150MHz, CDCl3) δ170.3, 65.5, 46.2, 31.8, 31.4, 29.2 (x2), 28.5, 28.4, 25.8, 25.5, 22.6, 22.5, 14.1, 14.0, 13.6.

[0108] Compound 2i: Didecyl 2-ethyl malonate JPEG0007909622000039.jpg1878

[0109] Compound 2i was synthesized according to the general procedure of step 2 of scheme 1. The title compound was obtained as a colorless oil. 1 H NMR(600MHz,CDCl3)δ4.14-4.09(m,4H), 3.25(t,J=7.2Hz,1H), 1.95-1.90(m,2H), 1 .64-1.60(m,4H), 1.32-1.25(m,26H), 0.96(t,J=7.2Hz,3H), 0.87(t,J=7.2Hz,6H). 13 C NMR (150MHz, CDCl3) δ169.7, 65.5, 53.7, 32.0, 29.6 (x2), 29.4, 29.3, 28.6, 25.9, 22.8, 22.3, 14.2, 12.0.

[0110] Compound 3a: Dioctyl 2-((benzyloxy)methyl)-2-methylmalonate (Step 3) JPEG0007909622000040.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, followed by the addition of benzyl chloromethyl ether (0.56 mL, 4.08 mmol). 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 (600MHz, CDCl3) δ7.36-7.27(m,5H), 4.53(s,2H), 4.10(t,J=6.6Hz,4H), 3.81( s,2H), 1.60-1.57(m,4H), 1.53(s,3H), 1.30-1.25(m,20H), 0.88(t,J=7.2Hz,6H). 13 C NMR (150MHz, CDCl3) δ170.7, 138.0, 128.3, 127.5, 127.4, 73.4, 72.7, 65.5, 54.9, 31.8, 29.2 (x2), 28.5, 25.8, 22.6, 18.5, 14.1.

[0112] Compound 3b: Dioctyl 2-(4-(benzyloxy)butyl)-2-methylmalonate JPEG0007909622000041.jpg2363

[0113] Compound 3b was synthesized according to the general procedure of step 3 of scheme 1. The title compound was obtained as a colorless oil. 1 H NMR(600MHz,CDCl3)δ7.35-7.27(m,5H), 4.48(s,2H), 4.09(t,J=6.6Hz,4H), 3.46(t,J=6.6Hz,2 H)1.88-1.86(m,2H), 1.65-1.58(m,6H), 1.40(s,3H), 1.29-1.26(m,22H), 0.88(t,J=7.2Hz,6H); 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 JPEG0007909622000042.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 JPEG0007909622000043.jpg2172

[0117] [[ID=2*]] 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 JPEG0007909622000044.jpg2163

[0119] Compound 3e 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 - 7.26 (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 JPEG0007909622000045.jpg2461

[0121] Compound 3f was synthesized according to the procedure of General Step 3 of 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 JPEG0007909622000046.jpg2373

[0123] Compound 3g was synthesized according to the procedure of General Step 3 of 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, 29.6, 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 JPEG0007909622000047.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 JPEG0007909622000048.jpg1962

[0127] Compound 3i was synthesized according to the general procedure of step 3 of scheme 1. The title compound was obtained as a colorless oil. 1 H NMR(600MHz,CDCl3)δ7.35-7.27(m,5H), 4.48(s,2H), 4.09(t,J=6.6Hz,4H), 3.46(t,J=6.6Hz,2H), 1. 95-1.87(m,4H), 1.65-1.57(m,6H), 1.38-1.26(m,30H), 0.88(t,J=6.6Hz,6H), 0.80(t,J=6.6Hz,3H). 13 C NMR (150MHz, CDCl3) δ172.0, 138.6, 128.4, 127.6, 127.5, 73.0, 70.0, 65.2, 58.1, 31.9, 31.5, 30.1, 29.6(x2), 29.4, 29.3, 28.6, 25.9, 25.2, 22.7, 20.7, 14.1, 8.5. MS(ESI):m / z [M + +H] + 575.4675(C 36 H 63 O5).

[0128] Compound 4a: Dioctyl 2-(hydroxymethyl)-2-methylmalonate (Step 4) JPEG0007909622000049.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 an 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. 1H NMR (600MHz, CDCl3) δ4.13(t,J=6.6Hz,4H), 3.83(s,2H), 1.64-1.59(m,4H), 1.43(s,3H), 1.28-1.26(m,20H), 0.87(t,J=7.2Hz,6H). 13 C NMR (150MHz, CDCl3) δ171.7, 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 JPEG0007909622000050.jpg2157

[0131] Compound 4b was synthesized according to the general procedure of step 4 of scheme 1. The title compound was obtained as a colorless oil. 1 H NMR(600MHz,CDCl3)δ4.09(t,J=6.6Hz,4H), 3.64(t,J=6.6Hz,2H), 1.88-1.85(m ,2H), 1.62-1.55(m,8H), 1.40(s,3H), 1.29-1.27(m,20H), 0.87(t,J=7.2Hz,6H). 13 C NMR (150MHz, CDCl3) δ172.5, 65.4, 62.4, 53.8, 35.2, 32.8, 31.8, 29.2(x2), 28.5, 25.8, 22.6, 20.6, 19.9, 14.1.

[0132] Compound 4c: Dioctyl 2-(6-hydroxyhexyl)-2-methylmalonate JPEG0007909622000051.jpg2268

[0133] Compound 4c was synthesized according to the general procedure of step 4 of scheme 1. The title compound was obtained as a colorless oil. 1H NMR (600MHz, CDCl3) δ4.09(t,J=6.6Hz,4H), 3.63(t,J=6.6Hz,2H), 1.86-1.83(m ,2H), 1.62-1.54(m,6H), 1.39(s,3H), 1.31-1.27(m,26H), 0.88(t,J=7.2Hz,6H). 13 C NMR (150MHz, CDCl3) δ65.3, 62.9, 53.8, 35.5, 32.7, 31.8, 29.7(x2), 29.2, 28.5, 25.6, 25.5, 24.3, 22.6, 19.9, 14.1.

[0134] Compound 4d: Dioctyl 2-(8-hydroxyoctyl)-2-methylmalonate JPEG0007909622000052.jpg2379

[0135] Compound 4d was synthesized according to the general procedure of step 4 of scheme 1. The title compound was obtained as a colorless oil. 1 H NMR (600MHz, CDCl3) δ4.11-4.07(m,4H), 3.63(t,J=6.6Hz,2H), 1.85-1.82(m,2 H), 1.61-1.54(m,6H), 1.39(s,3H), 1.29-1.27(m,30H), 0.88(t,J=7.2Hz,6H). 13 C NMR (150MHz, CDCl3) δ65.3, 63.0, 53.8, 35.6, 32.8, 31.8, 29.8 (x2), 29.34, 29.3, 29.2, 28.5, 25.9, 25.7, 24.3, 22.6, 19.9, 14.1.

[0136] Compound 4e: Dioctyl 2-ethyl-2-(6-hydroxyhexyl)malonate JPEG0007909622000053.jpg2267

[0137] Compound 4e was synthesized according to the general procedure of step 4 of scheme 1. The title compound was obtained as a colorless oil. 1H NMR (600MHz, CDCl3) δ4.08-4.06(m,4H), 3.61-3.59(m,2H), 1.89(q,J=7.2Hz,2H), 1.85-1.83(m,2H) , 1.59-1.52(m,6H), 1.32-1.22(m,24H), 1.15-1.13(m,2H), 0.87-0.85(m,6H), 0.79(t,J=7.2Hz,3H). 13 C NMR (150MHz, CDCl3) δ172.1, 65.2, 63.0, 58.1, 32.7, 31.8, 31.7, 29.7, 29.3, 29.2, 28.6, 25.9, 25.7, 25.3, 24.0, 22.7, 14.1, 8.5.

[0138] Compound 4f: Dioctyl 2-(6-hydroxyhexyl)-2-propylmalonate JPEG0007909622000054.jpg2463

[0139] Compound 4f was synthesized according to the general procedure of step 4 of Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR(600MHz,CDCl3)δ4.08(t,J=6.6Hz,4H), 3.62(t,J=6.6Hz,2H), 1.87-1.82(m,4H), 1.64-1. 53(m,6H), 1.36-1.25(m,26H), 1.20-1.13(m,2H), 0.91(t,J=7.2Hz,3H), 0.88(t,J=7.2Hz,6H). 13 C NMR (150MHz, CDCl3) δ172.0, 65.1, 62.9, 57.6, 34.5, 32.6, 31.7, 29.6, 29.2, 29.1, 28.5, 25.8, 25.7, 25.4, 24.0, 22.6, 17.4, 14.4, 14.0.

[0140] Compound 4g: Dioctyl 2-(6-hydroxyhexyl)malonate JPEG0007909622000055.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 [[ID=!!]]JPEG0007909622000056.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) - JPEG0007909622000057.jpg2367 [[ID=!!]]

[0145] Note: There seems to be an inconsistent "!!" in the original text which is preserved as is in the translation. It's not clear what it represents exactly. If it's an error in the original, it should be corrected for a more accurate translation.Compound 4i was synthesized according to the general procedure of step 4 of scheme 1. The title compound was obtained as a colorless oil. 1 H NMR(600MHz,CDCl3)δ4.09(t,J=6.6Hz,4H), 3.62(t,J=6.6Hz,2H), 1.85-1.83(m,2H), 1. 62-1.59(m,6H), 1.58-1.52(m,2H), 1.39(s,3H), 1.30-1.26(m,20H), 0.89-0.86(m,6H). 13 C NMR(150MHz,CDCl3)δ172.6, 65.3(x2), 62.9, 53.8, 35.5, 32.7, 31.8, 31.4, 29 .7, 29.2(x2), 28.5, 28.4, 25.9, 25.5(x2), 24.3, 22.6, 22.5, 19.9, 14.1, 14.0.

[0146] Compound 4J: Didecyl 2-ethyl-2-(4-hydroxybutyl)malonate JPEG0007909622000058.jpg2162

[0147] Compound 4j was synthesized according to the general procedure of step 4 of scheme 1. The title compound was obtained as a colorless oil. 1 H NMR(600MHz,CDCl3)δ4.09(t,J=6.6Hz,4H), 3.63(t,J=6.6Hz,2H), 1.95-1.87(m,4H) , 1.61-1.55(m,6H), 1.34-1.24(m,30H), 0.87(t,J=6.6Hz,6H), 0.81(t,J=6.6Hz,3H). 13 C NMR (150MHz, CDCl3) δ171.9, 65.2, 62.5, 58.1, 32.8, 31.9, 31.4, 29.6 (x2), 29.3, 29.2, 28.5, 25.9, 25.4, 22.7, 20.3, 14.1, 8.5.

[0148] Compound 5a: Dioctyl 2-(((6-bromohexanoyl)oxy)methyl)-2-methylmalonate (Step 5) JPEG0007909622000059.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 target product 5a (520 mg, 0.95 mmol, 80%) as a colorless oil. 1 H NMR(600MHz,CDCl3)δ4.45(s,2H), 4.12(t,J=6.6Hz,4H), 3.39(t,J=6.6Hz,2H), 2.31(t,J=7.2Hz,2H), 1.88 -1.83(m,2H), 1.65-1.59(m,6H), 1.48(s,3H), 1.47-1.44(m,2H), 1.43-1.26(m,22H), 0.88(t,J=7.2Hz,6H). 13 C NMR (150MHz, CDCl3) δ172.8, 170.0, 66.4, 65.9, 53.9, 33.9, 33.4, 32.4, 31.9, 29.3(x2), 28.5, 27.7, 25.9, 24.1, 22.7, 18.4, 14.2.

[0150] Compound 5b: Dioctyl 2-(((8-bromooctanoyl)oxy)methyl)-2-methylmalonate JPEG0007909622000060.jpg2071

[0151] Compound 5b was synthesized according to the general procedure of step 5 of Scheme 1. The title compound was obtained as a colorless oil. 1H NMR(600MHz,CDCl3)δ4.44(s,2H), 4.12(t,J=6.6Hz,4H), 3.40(t,J=6.6Hz,2H), 2.29(t,J=7.2Hz,2H), 1.87 -1.82(m,2H), 1.63-1.59(m,6H), 1.48(s,3H), 1.44-1.40(m,2H), 1.33-1.27(m,26H), 0.88(t,J=7.2Hz,6H). 13 C NMR (150MHz, CDCl3) δ173.1, 170.0, 66.3, 65.9, 53.9, 34.1, 33.9, 32.8, 31.9, 29.3(x2), 29.0, 28.5, 28.4, 28.0, 25.9, 24.8, 22.7, 18.4, 14.2. MS(ESI):m / z [M+Na] + 599.2912, [M+Na] 2+ 601.2890(C 29 H 53 O6BrNa).

[0152] Compound 6a: Dioctyl 2-(4-iodobutyl)-2-methylmalonate (Step 6) JPEG0007909622000061.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, followed by the addition of I2 (670 mg, 2.643 mmol). 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 H NMR (600MHz, CDCl3) δ4.10(t,J=6.6Hz,4H), 3.18(t,J=6.6Hz,2H), 1.87-1.80(m ,4H), 1.62-1.58(m,4H), 1.41(s,3H), 1.34-1.22(m,22H), 0.88(t,J=7.2Hz,6H). 13C NMR (150MHz, CDCl3) δ172.3, 65.4, 53.6, 34.4, 33.5, 31.8, 29.2, 28.5, 25.8, 25.3, 22.6, 19.9, 14.1, 6.2.

[0154] Compound 6b: Dioctyl 2-(6-iodohexyl)-2-methylmalonate JPEG0007909622000062.jpg2160

[0155] Compound 6b was synthesized according to the general procedure of step 6 of Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR (600MHz, CDCl3) δ4.10-4.07(m,4H), 3.16(t,J=6.6Hz,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=7.2Hz,6H). 13 C NMR (150MHz, CDCl3) δ172.6, 65.4, 53.8, 35.5, 33.5, 31.8, 30.3, 29.3, 29.2, 28.9, 28.6, 25.9, 24.2, 22.7, 20.0, 14.1, 6.9. MS(ESI):m / z [M+Na] + 561.2406(C 25 H 47 O4NaI).

[0156] Compound 6c: Dioctyl 2-(8-iodooctyl)-2-methylmalonate JPEG0007909622000063.jpg2065

[0157] Compound 6c was synthesized according to the general procedure of step 6 of Scheme 1. The title compound was obtained as a colorless oil. 1H NMR (600MHz, CDCl3) δ4.11-4.04(m,4H), 3.16(t,J=7.2Hz,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=7.2Hz,6H). 13 C NMR (150MHz, CDCl3) δ172.6, 65.3, 53.8, 35.6, 33.5, 31.8, 30.5, 29.8, 29.23, 29.21(x2), 28.55, 28.5, 25.9, 24.3, 22.7, 19.9, 14.1, 7.1. MS(ESI):m / z [M+Na] + 603.2876(C 28 H 53 O4NaI).

[0158] Compound 6d: Dioctyl 2-ethyl-2-(6-iodohexyl)malonate JPEG0007909622000064.jpg2162

[0159] Compound 6d was synthesized according to the general procedure of step 6 of Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR (600MHz, CDCl3) δ4.09(t,J=6.6Hz,4H), 3.16(t,J=7.2Hz,2H), 1.91(q,J=7.2Hz,2H), 1.86-1.83(m,2H), 1.81-1.78(m,2H) ), 1.62-1.58(m,4H), 1.40-1.37(m,2H), 1.33-1.24(m,22H), 1.16-1.14(m,2H), 0.87(t,J=7.2Hz,6H), 0.80(t,J=7.8Hz,3H). 13 C NMR (150MHz, CDCl3) δ172.0, 65.2, 58.1, 33.5, 31.9, 31.7, 30.4, 29.3, 29.2, 28.9, 28.6, 26.0, 25.3, 23.9, 22.7, 14.2, 8.5, 6.9.

[0160] Compound 6e: Dioctyl 2-(6-iodohexyl)-2-propylmalonate JPEG0007909622000065.jpg2460

[0161] Compound 6e was synthesized according to the general procedure of step 6 of Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR (600MHz, CDCl3) δ4.09(t,J=6.6Hz,4H), 3.16(t,J=6.6Hz,2H), 1.88-1.84(m,4H), 1.62-1.57(m,6H), 1.38-1.26(m,26H), 1.18-1.12(m,2H). 13 C NMR (150MHz, CDCl3) δ172.0, 65.1, 57.6, 34.6, 33.4, 32.2, 31.8, 30.3, 29.2, 29.1, 28.8, 28.5, 25.9, 23.9, 22.6, 17.4, 14.4, 14.1, 6.9.

[0162] Compound 6f: Dioctyl 2-(6-iodohexyl)malonate JPEG0007909622000066.jpg2059

[0163] Compound 6f was synthesized according to the general procedure of step 6 of Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR(600MHz,CDCl3)δ4.13-4.06(m,4H), 3.30-3.26(m,1H), 3.16-3.12(m,2H), 1.87-1. 85(m,2H), 1.81-1.76(m,2H), 1.61-1.58(m,4H), 1.36-1.25(m,26H), 0.87-1.83(m,6H). 13 C NMR (150MHz, CDCl3) δ169.6, 65.5, 52.1, 33.4, 31.8, 30.2, 29.2 (x2), 28.7, 28.6, 28.2, 27.2, 25.9, 22.7, 14.1, 6.9.

[0164] Compound 6g: 1-decyl 3-octyl 2-(6-iodohexyl)-2-methylmalonate JPEG0007909622000067.jpg2163

[0165] Six g of the compound was synthesized according to the general procedure of step 6 of Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR (600MHz, CDCl3) δ4.09-4.03(m,4H), 3.13(t,J=6.6Hz,2H), 1.82-1.76(m,4H) ), 1.60-1.55(m,4H), 1.40-1.32(m,5H), 1.31-1.18(m,28H), 0.86-0.84(m,6H). 13 C NMR (150MHz, CDCl3) δ172.5, 65.3, 53.7, 35.5, 33.4, 31.9, 31.8, 30.3, 29.6, 29.3, 29.22, 29.21, 29.18, 28.8, 28.5, 25.9, 24.1, 22.7, 22.6, 19.9, 14.11, 14.09, 6.8.

[0166] Compound 6h: 1-Hexyl 3-Octyl 2-(6-iodohexyl)-2-methylmalonate JPEG0007909622000068.jpg2059

[0167] Compound 6h was synthesized according to the general procedure of step 6 of Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR (600MHz, CDCl3) δ4.11-4.05(m,4H), 3.15(t,J=6.6Hz,2H), 1.84-1.77(m,4H ), 1.60-1.56(m,4H), 1.40-1.35(m,5H), 1.33-1.18(m,20H), 0.88-0.85(m,6H). 13 C NMR (150MHz, CDCl3) δ172.6, 65.4, 53.8, 35.5, 33.5, 31.8, 31.4, 30.3, 29.3, 29.2, 28.9, 28.6, 28.5, 25.9, 25.6, 24.2, 22.7, 22.6, 20.0, 14.1, 14.0, 7.0.

[0168] Compound 6i: Didecyl 2-ethyl-2-(4-iodobutyl)malonate JPEG0007909622000069.jpg2163

[0169] Compound 6i was synthesized according to the general procedure of step 6 of Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR (600MHz, CDCl3) δ4.10(t,J=6.6Hz,4H), 3.17(t,J=6.6Hz,2H), 1.95-1.91(q,J=6.6Hz,2H), 1.8 7-1.81(m,4H), 1.63-1.58(m,4H), 1.32-1.25(m,30H), 0.87(t,J=6.6Hz,6H), 0.82(t,J=6.6Hz,3H). 13C NMR (150MHz, CDCl3) δ171.8, 65.4, 58.0, 33.7, 32.0, 30.6, 29.7 (x2), 29.4, 29.3, 28.6, 26.0, 25.4, 25.0, 22.8, 14.2, 8.6, 6.3.

[0170] Compound 7a: Dioctyl 2-(((6-((2-hydroxyethyl)amino)hexanoyl)oxy)methyl)-2-methylmalonate (Step 7) JPEG0007909622000070.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 the mixture was dried over MgSO4. The crude product was purified by silica gel column chromatography using 10% MeOH / 1% NH4OH in DCM to obtain the target product 7a (325 mg, 0.61 mmol, 69%) as a colorless oil. 1H NMR(600MHz,CDCl3)δ4.44(s,2H), 4.12(t,J=6.6Hz,4H), 3.64(t,J=5.4Hz,2H), 2.77(t,J=5.4Hz,2H), 2.62(t,J=6.6Hz,2H), 2.3 0(t,J=7.2Hz,2H), 2.18(br.s,2H), 1.62-1.59(m,6H), 1.52-1.49(m,2H), 1.48(s,3H), 1.36-1.26(m,22H), 0.88(t,J=7.2Hz,6H). 13 C NMR (150MHz, CDCl3) δ172.9, 169.9, 66.2, 65.8, 60.7, 53.8, 51.2, 49.2, 33.9, 31.7, 29.6, 29.1(x2), 28.4, 26.7, 25.7, 24.6, 22.6, 18.2, 14.0. MS(ESI):m / z [M+H] + 530.4044(C 29 H 56 NO7).

[0172] Compound 7b: Dioctyl 2-(((8-((2-hydroxyethyl)amino)octanoyl)oxy)methyl)-2-methylmalonate JPEG0007909622000071.jpg22128

[0173] Compound 7b was synthesized according to the general procedure of step 7 of Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR(600MHz,CDCl3)δ4.44(s,2H), 4.12(t,J=6.6Hz,4H), 3.63(t,J=5.4Hz,2H), 2.77(t,J=5.4Hz,2H), 2.60(t,J=7.2Hz, 2H), 2.28(t,J=7.2Hz,2H), 1.63-1.58(m,6H), 1.48-1.46(m,2H), 1.46(s,3H), 1.30-1.26(m,26H), 0.88(t,J=7.2Hz,6H). 13C NMR(150MHz,CDCl3)δ173.2, 170.0, 66.2, 65.9, 60.9, 53.9, 51.0, 49.4, 34.1, 31.8, 30.1, 29.2(x2), 29.1, 29.0, 28.5, 27.1 25.8, 24.8, 22.7, 18.3, 14.1. MS(ESI):m / z [M+H] + 558.4363(C 31 H 60 NO7).

[0174] Compound 7c: Heptadecan-9-yl-8-((2-hydroxyethyl)amino)octanoate JPEG0007909622000072.jpg19128

[0175] A mixed solution of 8-bromooctanoic acid (0.9 g, 4.03 mmol) and heptadecan-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 general procedure of step 7 of Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR(600MHz,CDCl3)δ4.84-4.79(m,1H), 3.60(t,J=5.4Hz,2H), 2.71(t,J=5.4Hz,2H), 2.56(t,J=7.2Hz,2H ), 2.22(t,J=7.2Hz,2H), 1.58-1.56(m,2H), 1.46-1.45(m,6H), 1.27-1.21(m,30H), 0.83(t,J=7.2Hz,6H). 13C NMR (150MHz, CDCl3) δ173.6, 74.1, 60.6, 51.3, 49.6, 34.6, 34.1, 31.8, 29.9, 29.5, 29.48, 29.2, 29.17, 29.1, 27.1, 25.3, 25.1, 22.6, 14.1. MS(ESI):m / z [M+H] + 442.4264(C 27 H 56 NO3).

[0177] Compound 7d: Dioctyl 2-(6-((2-hydroxyethyl)amino)hexyl)-2-methylmalonate JPEG0007909622000073.jpg2173

[0178] Compound 7d was synthesized according to the general procedure of step 7 of Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR(600MHz,CDCl3)δ4.08(t,J=6.6Hz,4H), 3.63(t,J=4.8Hz,2H), 2.76(t,J=4.8Hz,2H), 2.60(t,J=7.2Hz,2H) , 1.86-1.82(m,2H), 1.62-1.57(m,4H), 1.48-1.44(m,2H), 1.38(s,3H), 1.31-1.25(m,26H), 0.86-0.84(m,6H). 13 C NMR (150MHz, CDCl3) δ172.6, 65.3, 60.8, 53.7, 51.1, 49.5, 35.5, 31.8, 30.0, 29.8, 29.2 (x2), 28.5, 27.1, 25.6, 24.3, 22.6, 19.9, 14.1.

[0179] Compound 7e: Dioctyl 2-(8-((2-hydroxyethyl)amino)octyl)-2-methylmalonate JPEG0007909622000074.jpg24128

[0180] Compound 7e was synthesized according to the general procedure of step 7 of Scheme 1. The title compound was obtained as a colorless oil. 1H NMR(600MHz,CDCl3)δ4.10-4.04(m,4H), 3.64(t,J=4.8Hz,2H), 2.76(t,J=4.8Hz,2H), 2.61(t,J=7.2Hz,2H), 1. 83-1.80(m,2H), 1.61-1.56(m,4H), 1.49-1.45(m,2H), 1.37(s,3H), 1.34-1.18(m,30H), 0.86(t,J=7.2Hz,2H). 13 C NMR (150MHz, CDCl3) δ172.6, 65.2, 60.6, 53.7, 51.1, 49.5, 35.54, 31.8, 29.8(x2), 29.4, 29.3, 29.2(x2), 28.5, 27.2, 25.8, 24.3, 22.6, 19.9, 14.0. MS(ESI):m / z [M+H] + 514.4461(C 30 H 60 NO. 5).

[0181] Compound 7f: Dioctyl 2-(4-((3-hydroxypropyl)amino)butyl)-2-methylmalonate JPEG0007909622000075.jpg2274

[0182] Compound 7f was synthesized according to the general procedure of step 7 of Scheme 1. 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=5.4Hz,2H), 2.85(t,J=5.4Hz,2H), 2.59(t,J=7.2Hz,2H), 1.84-1.8 1(m,2H), 1.68-1.66(m,2H), 1.59-1.56(m,4H), 1.48-1.45(m,2H), 1.37(s,3H), 1.28-1.24(m,22H), 0.86(t,J=6.6Hz,6H). 13 C NMR (150MHz, CDCl3) δ172.5, 65.4, 64.3, 53.8, 50.0, 49.5, 35.4, 31.8, 30.6, 30.0, 29.2 (x2), 28.5, 25.9, 22.7, 22.1, 19.9, 14.1. MS(ESI):m / z [M+H] +472.3994(C 27 H 54 NO. 5).

[0183] Compound 7g: Dioctyl 2-(6-((3-hydroxypropyl)amino)hexyl)-2-methylmalonate JPEG0007909622000076.jpg2279

[0184] 7 g of the compound was synthesized according to the general procedure of step 7 of Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR(600MHz,CDCl3)δ4.05(t,J=6.6Hz,4H), 3.78-3.76(m,2H), 2.88-2.86(m,2H), 2.61-2.59(m,2H), 1.79-1.78(m ,2H), 1.71-1.69(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 (150MHz, CDCl3) δ172.6, 65.3, 63.8, 53.8, 49.6, 49.5, 35.5, 31.8, 30.2, 29.7, 29.3, 29.2, 29.1, 28.5, 27.0, 25.9, 24.3, 22.6, 19.9, 14.1. MS(ESI):m / z [M+H] + 500.4310(C 29 H 58 NO. 5).

[0185] Compound 7h: Dioctyl 2-ethyl-2-(6-((3-hydroxypropyl)amino)hexyl)malonate JPEG0007909622000077.jpg2179

[0186] Compound 7h was synthesized according to the general procedure of step 7 of Scheme 1. The title compound was obtained as a colorless oil. 1H NMR(600MHz,CDCl3)δ4.08(t,J=6.6Hz,4H), 3.80(t,J=5.4Hz,2H), 2.86(t,J=5.4Hz,2H), 2.58(t,J=7.2Hz,2H), 1.92(q,J=7.2Hz,2H), 1.89-1.83(m ,2H), 1.69-1.67(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=6.6Hz,6H), 0.80(t,J=7.2Hz,3H). 13 C NMR (150MHz, CDCl3) δ172.1, 65.2, 64.6, 58.1, 50.2, 49.8, 31.9, 31.7, 30.6, 29.9(x2), 29.3(x2), 28.6, 27.1, 26.0, 25.3, 24.0, 22.7, 14.2, 8.5. MS(ESI):m / z [M+H] + 514.4466(C 30 H 60 NO. 5).

[0187] Compound 7i: Dioctyl 2-(6-((3-hydroxypropyl)amino)hexyl)-2-propylmalonate JPEG0007909622000078.jpg2580

[0188] Compound 7i was synthesized according to the general procedure of step 7 of Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR(600MHz,CDCl3)δ4.07(t,J=6.6Hz,4H), 3.78(t,J=4.8Hz,2H), 2.88(t,J=4.8Hz,2H), 2.60(t,J=7.2Hz,2H), 1.84-1.81(m,4H), 1.72 -1.70(m,2H), 1.60-1.56(m,4H), 1.50-1.44(m,2H), 1.31-1.22(m,24H), 1.18-1.10(m,4H), 0.9(t,J=7.2Hz,3H), 0.86(t,J=6.6Hz,6H). 13C NMR (150MHz, CDCl3) δ172.1, 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 NO. 5).

[0189] Compound 7J: Dioctyl 2-(6-((4-hydroxybutyl)amino)hexyl)-2-methylmalonate JPEG0007909622000079.jpg2284

[0190] Compound 7j was synthesized according to the general procedure of step 7 of Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR(600MHz,CDCl3)δ4.06(t,J=6.6Hz,4H), 3.55(t,J=5.4Hz,2H), 2.64(t,J=5.4Hz,2H), 2.58(t,J=7.2Hz,2H), 1.82-1.79(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=7.2Hz,6H). 13 C NMR (150MHz, CDCl3) δ172.6, 65.4, 62.6, 53.8, 49.6, 49.4, 35.6, 32.6, 31. 8, 29.8, 29.5, 29.3, 29.2, 28.7, 28.6, 27.1, 25.9, 24.3, 22.7, 19.9, 14.1. MS(ESI):m / z [M+H] + 514.4468(C 30 H 60 NO. 5).

[0191] Compound 7K: Dioctyl 2-(6-((5-hydroxypentyl)amino)hexyl)-2-methylmalonate JPEG0007909622000080.jpg24128

[0192] Compound 7k was synthesized according to the general procedure of step 7 of Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR(600MHz,CDCl3)δ4.07(t,J=6.6Hz,4H), 3.60(t,J=6.6Hz,2H), 2.58(t,J=6.6Hz,2H), 2.55(t,J=7.2Hz,2H), 1.83-1.80(m,2H), 1.60-1.54(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=7.2Hz,6H). 13 C NMR (150MHz, CDCl3) δ172.6, 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 NO. 5).

[0193] Compound 7L: Dioctyl 2-(6-((6-hydroxyhexyl)amino)hexyl)-2-methylmalonate JPEG0007909622000081.jpg21128

[0194] Compound 7L was synthesized according to the general procedure of step 7 of Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR(600MHz,CDCl3)δ4.05(t,J=6.6Hz,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=7.2Hz,6H). 13C NMR (150MHz, CDCl3) δ172.6, 65.3, 62.5, 53.8, 49.9, 49.8, 35.5, 32.7, 31.8, 29 .8, 29.7(x2), 29.2(x2), 28.5, 27.2, 27.1, 25.9, 25.7, 24.3, 22.6, 19.9, 14.1.

[0195] Compound 7m: Dioctyl 2-(6-((2,3-dihydroxypropyl)amino)hexyl)-2-methylmalonate JPEG0007909622000082.jpg2074

[0196] Compound 7m was synthesized according to the general procedure of step 7 of Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR(600MHz,CDCl3)δ4.06(t,J=6.6Hz,4H), 3.75-3.72(m,1H), 3.66-3.65(m,1H), 3.56-3.53(m,1H), 2.75-2.73(m,1H), 2.65-2.61(m,1) H), 2.59-2.52(m,2H), 1.82-1.78(m,2H), 1.59-1.54(m,4H), 1.45-1.42(m,2H), 1.34(s,3H), 1.28-1.18(m,26H), 0.85(t,J=7.2Hz,6H). 13 C NMR (150MHz, CDCl3) δ1172.6, 69.7, 65.8, 65.3, 53.8, 52.4, 49.9, 35.6, 31.8, 29.9, 29.8, 29.2 (x2), 28.5, 27.0, 25.9, 24.3, 22.7, 19.9, 14.1. MS(ESI):m / z [M+H] + 516.4252(C 29 H 58 NO6).

[0197] Compound 7n: dioctyl 2-(6-(((1s,4s)-4-hydroxycyclohexyl)amino)hexyl)-2-methylmalonate JPEG0007909622000083.jpg2071

[0198] Compound 7n was synthesized according to the general procedure of step 7 of Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR(600MHz, CDCl3)δ4.07(t,J=6.6Hz,4H), 3.95-3.94(m,1H), 2.78-2.71(m,3H), 1.8 2-1.70(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 (150MHz, CDCl3) δ172.4, 65.8, 65.2, 55.4, 53.6, 46.3, 35.4, 31.7, 31.0, 29.6, 29.1 (x2), 29.0, 28.4, 27.0, 26.2, 25.7, 24.2, 22.5, 19.8, 14.0. MS(ESI):m / z [M+H] + 540.4614(C 32 H 62 NO. 5).

[0199] Compound 7o: 1-Decyl 3-octyl 2-(6-((3-hydroxypropyl)amino)hexyl)-2-methylmalonate JPEG0007909622000084.jpg2181

[0200] Compound 7o was synthesized according to the general procedure of step 7 of Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR(600MHz,CDCl3)δ4.07(t,J=6.6Hz,4H), 3.78(t,J=5.4Hz,2H), 2.86(t,J=5.4Hz,2H), 2.59(t,J=7.2Hz,2H), 1.84-1. 78(m,2H), 1.70-1.66(m,2H), 1.61-1.55(m,4H), 1.46-1.42(m,2H), 1.37(s,3H), 1.32-1.18(m,30H), 0.87-0.85(m,6H). 13C NMR (150MHz, CDCl3) δ172.6, 65.3, 64.3, 53.8, 49.9, 49.7, 35.6, 31.9, 31.8, 30.5, 29.8, 29. 7, 29.6, 29.3, 29.26, 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 NO. 5).

[0201] Compound 7p: 1-Hexyl 3-octyl 2-(6-((3-hydroxypropyl)amino)hexyl)-2-methylmalonate JPEG0007909622000085.jpg2276

[0202] Compound 7p was synthesized according to the general procedure of step 7 of Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR(600MHz,CDCl3)δ4.07(t,J=6.6Hz,4H), 3.79(t,J=5.4Hz,2H), 2.86(t,J=5.4Hz,2H), 2.58(t,J=7.2Hz,2H), 1.83-1. 80(m,2H), 1.69-1.66(m,2H), 1.60-1.57(m,4H), 1.45-1.43(m,2H), 1.37(s,3H), 1.36-1.19(m,22H), 0.87-0.85(m,6H). 13 C NMR (150MHz, CDCl3) δ172.6, 65.3, 64.4, 53.8, 50.1, 49.8, 35.6, 31.8, 31.4, 30.5, 29.8 , 29.7, 29.24, 29.22, 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 NO. 5).

[0203] Compound 7q: dioctyl 2-(6-(((1s,4s)-4-(hydroxymethyl)cyclohexyl)amino)hexyl)-2-methylmalonate JPEG0007909622000086.jpg1879

[0204] Compound 7q was synthesized according to the general procedure of step 7 of Scheme 1. The title compound was obtained as a colorless oil. 1 H NMR(600MHz,CDCl3)δ4.07(t,J=6.6Hz,4H), 3.51(d,J=6.6Hz,2H), 2.71-2.70(m,1H), 2.57(t,J=7.2Hz,2H), 1.82-1.80(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(150MHz,CDCl3)δ172.6, 65.8, 65.3, 54.4, 53.8, 46.8, 37.8, 35.5, 31.8, 2 9.8, 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 NO. 5).

[0205] Compound 7r: Dioctyl 2-(6-(((4-hydroxycyclohexyl)methyl)amino)hexyl)-2-methylmalonate JPEG0007909622000087.jpg2075

[0206] Compound 7r was synthesized according to the general procedure of step 7 of Scheme 1. The title compound was obtained as a yellow oily substance. 1H NMR(600MHz,CDCl3)δ4.04-4.02(m,4H), 3.91(br.s,0.5H), 3.49-3.45(m,0.5H), 2.55-2.50(m,2H), 2.46-2.45(m,1H), 2.39-2.38(m, 1H), 1.93-1.91(m,1H), 1.79-1.75(m,3H), 1.67-1.65(m,1H), 1.59-1.35(m,12H), 1.33(s,3H), 1.29-1.15(m,26H), 0.83-0.81(m,6H). 13 C NMR (150MHz, CDCl3) δ172.5, 70.7, 66.6, 65.2, 55.8, 55.3, 53.7, 50.0, 49.9, 36.8, 36.3, 35.5, 35.2, 32.1, 31.7, 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 NO. 5).

[0207] Compound 7s: Didecyl 2-ethyl-2-(4-((3-hydroxypropyl)amino)butyl)malonate JPEG0007909622000088.jpg21128

[0208] Compound 7s was synthesized according to the general procedure of step 7 of Scheme 1. The title compound was obtained as a yellow oily substance. 1 H NMR(600MHz,CDCl3)δ4.09-4.05(m,4H), 3.78-3.77(m,2H), 2.87-2.85(m,2H), 2.61(t,J=7.2Hz,2H), 1.90(q,J=7.8Hz,2H), 1.86-1 .83(m,2H), 1.70-1.68(m,2H), 1.59-1.55(m,4H), 1.50-1.48(m,2H), 1.27-1.15(m,30H), 0.87-0.84(m,6H), 0.79(t,J=7.8Hz,3H). 13C NMR (150MHz, CDCl3) δ171.9, 65.3, 64.0, 58.0, 49.8, 49.4, 31.9, 31.6, 30.5, 29.9, 29.6 (x2), 29.4, 29.3, 28.6.25.9, 25.4, 22.7, 21.8, 14.1, 8.5. MS(ESI):m / z [M+H] + 542.4770(C 32 H 64 NO. 5).

[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) JPEG0007909622000089.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 overnight at 30°C, the solvent was removed, and the residue was dissolved in DCM. The organic solvent was washed with water and brine, and the mixture was dried over MgSO4. The crude product was purified by silica gel column chromatography using 5% MeOH / 1% NH4OH in DCM to obtain compound AS-CL-01 (60 mg, 0.058 mmol, 39%) as a pale yellow oil. 1 H NMR(600MHz,CDCl3)δ4.43(s,2H), 4.12(t,J=6.6Hz,4H), 3.50(t,J=5.4Hz,2H), 2.55(t,J=5.4Hz,2H), 2.44-2.40(m,4 H), 2.29-2.26(m,4H), 1.62-1.58(m,12H), 1.47(s,6H), 1.44-1.38(m,4H), 1,29-1.25(m,48H), 0.87(t,J=7.2Hz,12H). 13C NMR (150MHz, CDCl3) δ173.2, 173.0, 170.0(x2), 66.3, 65.9(x2), 58.4, 55.5, 53.9(x2), 53.8, 53.7(x2), 34.1, 34.0, 31. 8(x2), 29.3, 29.2(x4), 29.1, 28.5(x2), 27.3, 27.2, 27.0, 26.9, 25.8(x2), 24.9, 24.8, 22.7(x2), 18.3(x2), 14.1(x2). MS(ESI):m / z [M+H] + 1026.7822(C 58 H 108 NO 13 ).

[0211] Compound AS-CL-02: Dioctyl 2-(((6-((8-(heptadecane-9-yloxy)-8-oxooctyl)(2-hydroxyethyl)amino)hexanoyl)oxy)methyl)-2-methylmalonate JPEG0007909622000090.jpg3172

[0212] Compound AS-CL-02 was synthesized according to the general procedure of step 8 in Scheme 2. The title compound was obtained as a colorless oil. 1 H NMR(600MHz,CDCl3)δ4.84-4.81(m,1H), 4.42(s,2H), 4.10(t,J=6.6Hz,4H), 3.48(t,J=5.4Hz,2H), 2.53(t,J=5.4Hz,2H), 2.40((t,J=7 .2Hz,2H), 2.27-2.23(m,4H), 1.61(m,8H), 1.48-1.45(m,4H), 1.45(s,3H), 1.42-1.37(m,6H), 1.28-1.22(m,54H), 0.86-0.83(m,12H). 13C NMR (150MHz, CDCl3) δ173.6, 173.1, 169.9, 66.2, 65.8(x2), 58.3, 55.5, 53.9, 53.8(x2), 34.7, 34.1, 34.0, 31.9, 31.8, 29.52, 29.5 0,29.26,29.22(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-(heptadecane-9-yloxy)-8-oxooctyl)(2-hydroxyethyl)amino)octyl)-2-methylmalonate JPEG0007909622000091.jpg3275

[0214] Compound AS-CL-03 was synthesized according to the general procedure of step 8 in Scheme 2. The title compound was obtained as a colorless oil. 1 H NMR(600MHz,CDCl3)δ4.86-4.82(m,1H), 4.10-4.04(m,4H), 3.49(t,J=5.4Hz,2H), 2.54(t,J=5.4Hz,2H), 2.41-2.39(m,4H), 2.25(t,J=7.2 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). 13C NMR (150MHz, CDCl3) δ173.6, 172.6, 74.1, 65.3, 58.4, 55.5, 53.9, 53.87, 53.8, 35.6, 34.7, 34.2, 31.9, 31.8, 29.9, 29.6, 29.5, 2 9.4, 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 JPEG0007909622000092.jpg3474

[0216] Compound AS-CL-04 was synthesized according to the general procedure of step 8 in Scheme 2. The title compound was obtained as a colorless oil. 1 H NMR (600MHz, CDCl3) δ4.06-4.05(m,4H), 3.47(t,J=4.8Hz,2H), 2.52(t,J=4.8Hz,2H), 2.40-2.37( m,4H), 1.81-1.79(m,4H), 1.58-1.54(m,6H), 1.37(s,6H), 1.35-1.24(m,62H), 0.85-0.83(m,12H). 13 C NMR (150MHz, CDCl3) δ172.57, 172.53, 65.26, 65.24, 58.4, 55.5, 53.9, 53.8, 53.77, 53.7, 35.58, 35.55, 31.8(x2), 29.91, 29.89, 29.5, 29.4, 29.21, 29.18, 28.5(x2), 27.5, 27.3, 27.2, 27.1, 25.9(x2), 24.4, 24.3, 22.6(x2), 19.9(x2), 14.1(x2). MS(ESI):m / z [M+H] +938.8026(C 56 H 108 NO9).

[0217] Compound AS-CL-05: Dioctyl 2-(6-(decyl(2-hydroxyethyl)amino)hexyl)-2-methylmalonate JPEG0007909622000093.jpg2978

[0218] Compound AS-CL-05 was synthesized according to the general procedure of step 8 in Scheme 2. The title compound was obtained as a colorless oil. 1 H NMR(600MHz,CDCl3)δ4.09-4.07(m,4H), 3.50(t,J=5.4Hz,2H), 2.55(t,J=5.4Hz,2H), 2.42-2.40(m,4H), 1. 84-1.81(m,2H), 1.61-1.57(m,4H), 1.43-1.39(m,4H), 1.38(s,3H), 1.29-1.25(m,40H), 0.88-0.85(m,9H). 13 C NMR (150MHz, CDCl3) δ172.6, 65.4, 58.4, 55.5, 53.9, 53.84, 53.82, 35.6, 32.0, 31.9, 30.0, 29.7, 29.6, 29.4, 29.3(x2), 29.2(x2), 28.6, 27.5, 27.3, 27.2, 27.1, 25.9, 24.4, 22.74, 22.7, 20.0, 14.17, 14.14. MS(ESI):m / z [M+H] + 626.5725(C 38 H 76 NO. 5).

[0219] Compound AS-CL-06: Dioctyl 2-(6-((8-(heptadecane-9-yloxy)-8-oxooctyl)(2-hydroxyethyl)amino)hexyl)-2-methylmalonate JPEG0007909622000094.jpg3575

[0220] Compound AS-CL-06 was synthesized according to the general procedure of step 8 in Scheme 2. The title compound was obtained as a colorless oil.1 H NMR(600MHz,CDCl3)δ4.85-4.83(m,1H), 4.07-4.05(m,4H), 3.48(t,J=5.4Hz,2H), 2.53(t,J=5.4Hz,2H), 2.41-2.38(m,4H), 2.25(t,J=7.2 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 C NMR (150MHz, CDCl3) δ173.6, 172.6, 74.1, 65.3, 58.4, 55.5, 53.9(x2), 53.8, 35.6, 34.7, 34.2, 31.9, 31.8, 30.0, 29.6, 29 .5, 29.3, 29.27(x2), 29.24(x2), 28.6, 27.4, 27.3, 27.2, 27.1, 25.9, 25.4, 25.2, 24.4, 22.70, 22.68, 19.9, 14.14, 14.12. MS(ESI):m / z [M+H] + 866.7800(C 53 H 104 NO7).

[0221] Compound AS-CL-07: Dioctyl 2-(((6-(Decyl(2-hydroxyethyl)amino)hexanoyl)oxy)methyl)-2-methylmalonate JPEG0007909622000095.jpg2880

[0222] Compound AS-CL-07 was synthesized according to the general procedure of step 8 in Scheme 2. The title compound was obtained as a colorless oil. 1 H NMR (600MHz, CDCl3) δ4.43(s,2H), 4.11(t,J=6.6Hz,4H), 3.50(t,J=5.4Hz,2H), 2.55, (t,J=4.8Hz,2H), 2.43-2.40(m, 4H), 2.28(t,J=7.2Hz,2H), 1.61-1.59(m,6H), 1.47(s,3H), 1.44-1.40(m,4H), 1.27-1.24(m,36H), 0.88-0.85(m,9H).13 C NMR (150MHz, CDCl3) δ173.0, 170.0, 66.3, 65.9, 58.4, 55.5, 53.9, 53.7, 34.0, 31.9, 31.8, 29.7 (x2 ), 29.6, 29.4(x2), 29.2, 28.5, 27.5, 27.2, 27.0, 26.9, 25.8, 24.8, 22.7, 22.6, 18.3, 14.15, 14.12. MS(ESI):m / z [M+H] + 670.5623(C 39 H 76 NO7).

[0223] Compound AS-CL-08: Dioctyl 2-(6-((2-hydroxyethyl)(octyl)amino)hexyl)-2-methylmalonate JPEG0007909622000096.jpg2875

[0224] Compound AS-CL-08 was synthesized according to the general procedure of step 8 in Scheme 2. The title compound was obtained as a colorless oil. 1 H NMR(600MHz,CDCl3)δ4.09-4.07(m,4H), 3.50(t,J=5.4Hz,2H), 2.55(t,J=5.4Hz,2H), 2.42-2.40(m,4H), 1. 84-1.81(m,2H), 1.60-1.57(m,4H), 1.40-1.37(m,4H), 1.37(s,3H), 1.29-1.20(m,36H), 0.88-0.85(m,9H). 13 C NMR (150MHz, CDCl3) δ172.6, 65.3, 58.4, 55.5, 53.9, 53.84, 53.82, 35.6, 31.9, 31.8, 29.9, 29. 6, 29.4, 29.3, 29.2, 28.6, 27.5, 27.3, 27.2, 27.1, 25.9, 24.4, 22.7, 22.6, 19.9, 14.15, 14.13. MS(ESI):m / z [M+H] + 598.5402(C 36 H 72 NO. 5).

[0225] Compound AS-CL-09: Dioctyl 2-(6-(decyl(3-hydroxypropyl)amino)hexyl)-2-methylmalonate JPEG0007909622000097.jpg2775

[0226] Compound AS-CL-09 was synthesized according to the general procedure of step 8 in 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 NO. 5).

[0227] Compound AS-CL-10: Dioctyl 2-(6-(decyl(2-hydroxyethyl)amino)hexyl)malonate JPEG0007909622000098.jpg2977

[0228] Compound AS-CL-10 was synthesized according to the general procedure of step 8 in Scheme 2. The title compound was obtained as a colorless oil. 1H NMR(600MHz,CDCl3)δ4.12-4.05(m,4H), 3.49(t,J=5.4Hz,2H), 3.28(t,J=7.8Hz,1H), 2.53(t,J=5.4Hz,2H), 2.40 (t,J=7.2Hz,4H), 1.87-1.84(m,2H), 1.62-1.57(m,4H), 1.42-1.36(m,4H), 1.29-1.23(m,40H), 0.86-0.84(m,9H). 13 C NMR(150MHz,CDCl3)δ169.7, 65.5, 58.4, 55.5, 53.9, 53.8, 52.1, 32.0, 31.8, 29.7, 29.6(x2), 29 .4, 29.23(x2), 29.22, 28.7, 28.6, 27.5, 27.4, 27.2(x2), 27.1, 25.9, 22.7, 22.6, 14.15, 14.12. MS(ESI):m / z [M+H] + 612.5555(C 37 H 74 NO. 5).

[0229] Compound AS-CL-11: Dioctyl 2-(6-(decyl(3-hydroxypropyl)amino)hexyl)malonate JPEG0007909622000099.jpg2878

[0230] Compound AS-CL-11 was synthesized according to the general procedure of step 8 in Scheme 2. The title compound was obtained as a colorless oil. 1 H NMR (600MHz, CDCl3) δ4.11-4.05(m,4H), 3.74(t,J=5.4Hz,2H), 3.27(t,J=7.8Hz,1H), 2.59-2.58(m,2H), 2.36 -2.34(m,4H), 1.86-1.81(m,2H), 1.63-1.57(m,6H), 1.43-1.39(m,4H), 1.31-1.20(m,40H), 0.85-0.83(m,9H). 13C NMR(150MHz,CDCl3)δ169.6, 65.4, 64.8, 55.3, 54.2, 54.1, 52.1, 31.9, 31.8, 29.62(x2), 29.60(x2 ), 29.3, 29.2(x2), 28.7, 28.5, 27.8, 27.5, 27.3, 27.2, 26.8, 26.7, 25.8, 22.7, 22.6, 14.09, 14.07. MS(ESI):m / z [M+H] + 626.5719(C 38 H 76 NO. 5).

[0231] Compound AS-CL-12: dioctyl 2-(6-((3-(dimethylamino)propyl)amino)hexyl)-2-methylmalonate JPEG0007909622000100.jpg2181

[0232] Compound AS-CL-12 was synthesized according to the general procedure of step 7 in Scheme 2. The title compound was obtained as a colorless oil. 1 H NMR(600MHz,CDCl3)δ4.06(t,J=6.6Hz,4H), 2.81(t,J=6.6Hz,2H), 2.67(t,J=7.2Hz,2H), 2.41(t,J=6.6Hz,2H ), 2.24(s,6H), 1.82-1.75(m,4H), 1.59-1.55(m,6H), 1.36(s,3H), 1.31-1.24(m,26H), 0.85(t,J=7.2Hz,6H). 13 C NMR (150MHz, CDCl3) δ172.5, 65.3, 58.8, 53.7, 49.1(x2), 45.5, 35.5, 31.8, 29.6, 29.2(x2), 28.7, 28.5, 26.9, 25.9, 25.6, 24.2, 22.7, 19.9, 14.1. MS(ESI):m / z [M+H] + 527.4779(C 31 H 63 N2O4).

[0233] Compound AS-CL-13: Dioctyl 2-(6-(decyl(3-(dimethylamino)propyl)amino)hexyl)-2-methylmalonate JPEG0007909622000101.jpg2776

[0234] Compound AS-CL-13 was synthesized according to the general procedure of 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 JPEG0007909622000102.jpg30128

[0236] Compound AS-CL-14 was synthesized according to the general procedure of step 8 in Scheme 2. The title compound was obtained as a colorless oil. 1H NMR(600MHz,CDCl3)δ4.06(t,J=6.6Hz,4H), 3.52-3.51(m,2H), 2.42-2.39(m,6H), 1.82-1.79(m ,2H), 1.62-1.54(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 C NMR (150MHz, CDCl3) δ172.6, 65.3, 62.6, 54.6, 53.8, 53.7, 53.6, 35.6, 32.7, 31.9, 31.8, 29.9, 29.7, 29. 6, 29.5, 29.3, 29.2(x2), 28.5, 27.7, 27.5, 26.2, 25.9, 25.8, 25.7, 24.4, 22.7, 22.6, 19.9, 14.13, 14.11. MS(ESI):m / z [M+H] + 654.6039(C 40 H 80 NO. 5).

[0237] Compound AS-CL-15: Diundecyl 2-(6-(decyl(2-hydroxyethyl)amino)hexyl)-2-methylmalonate JPEG0007909622000103.jpg29128

[0238] Compound AS-CL-15 was synthesized according to the general procedure of 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), 3.50(t,J=5.4Hz,2H), 2.55(t,J=5.4Hz,2H), 2.41(t,J=7.2Hz,4H) , 1.84-1.81(m,2H), 1.60-1.57(m,4H), 1.41-1.38(m,4H), 1.37(s,3H), 1.29-1.20(m,52H), 0.88-0.85(m,9H). 13C NMR (150MHz, CDCl3) δ172.6, 65.3, 58.4, 55.5, 53.9, 53.84, 53.80, 35.6, 32.0, 29.9, 29. 7, 29.65, 29.60, 29.4, 29.3, 28.6, 27.5, 27.3, 27.2, 27.1, 25.9, 24.4, 22.7, 19.9, 14.2. MS(ESI):m / z [M+H] + 710.6655(C 44 H 88 NO. 5).

[0239] Compound AS-CL-16: Dioctyl 2-(6-(decyl(3-hydroxypropyl)amino)hexyl)-2-ethylmalonate JPEG0007909622000104.jpg2980

[0240] Compound AS-CL-16 was synthesized according to the general procedure of 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), 3.77(t,J=4.8Hz,2H), 2.62(t,J=4.8Hz,2H), 2.39(t,J=7.2Hz,4H), 1.90(q,J=7.2Hz,2H), 1.85-1.82 (m,2H), 1.67-1.65(m,2H), 1.61-1.56(m,4H), 1.46-1.42(m,4H), 1.30-1 .24(m,38H), 1.15-1.11(m,2H), 0.88-0.86(m,9H), 0.79(t,J=7.2Hz,3H). 13 C NMR (150MHz, CDCl3) δ172.0, 65.2, 64.8, 58.1, 55.3, 54.2, 54.1, 32.0, 31.8, 31.7, 29.9, 29.7, 29.6, 29. 4, 29.3, 29.2(x2), 28.6, 27.8, 27.6, 27.4, 26.8, 26.7, 25.9, 25.2, 24.0, 22.73, 22.69, 14.2, 14.1, 8.5. MS(ESI):m / z [M+H] + 654.6036(C 40 H 80 NO. 5).

[0241] Compound AS-CL-17: Dioctyl 2-(6-(decyl(2,3-dihydroxypropyl)amino)hexyl)-2-methylmalonate JPEG0007909622000105.jpg31128

[0242] Compound AS-CL-17 was synthesized according to the general procedure of 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), 3.73-3.68(m,2H), 3.48-3.45(m,1H), 2.57-2.54(m,1H), 2.51-2.46(m,2H), 2.4 1-2.37(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 JPEG0007909622000106.jpg33128

[0244] Compound AS-CL-18 was synthesized according to the general procedure of step 8 in Scheme 2. The title compound was obtained as a colorless oil. 1H NMR(600MHz,CDCl3)δ4.07(t,J=6.6Hz,4H), 3.97(br.s,1H), 2.53-2.46(m,5H), 1.83-1.80(m, 4H), 1.68-1.56(m,8H), 1.51-1.41(m,6H), 1.37(s,3H), 1.31-1.17(m,40H), 0.87-0.85(m,9H). MS(ESI):m / z [M+H] + 680.6183(C 42 H 82 NO. 5).

[0245] Compound AS-CL-19: Dioctyl 2-(6-(4-(dimethylamino)butanamide)hexyl)-2-methylmalonate JPEG0007909622000107.jpg25128

[0246] Compound AS-CL-19 was synthesized according to the procedure in 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 hours. 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 H NMR(600MHz,CDCl3)δ7.84-7.82(m,2H), 7.71-7.69(m,2H), 4.08(t,4H), 3.67(t,2H), 1.84-1. 81(m,2H), 1.66-1.64(m,2H), 1.61-1.57(m,4H), 1.37(s,3H), 1.34-1.25(m,26H), 0.87(t,6H). 13 C NMR (150MHz, CDCl3) δ172.5, 168.4, 133.8, 132.1, 123.1, 65.3, 53.7, 37.9, 3 5.5, 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(600MHz,CDCl3)δ4.08(t,4H), 2.67(t,2H), 1.85-1.82(m,2H), 1.62-1. 57(m,4H), 1.43-1.41(m,2H), 1.38(s,3H), 1.31-1.25(m,26H), 0.87(t,6H). 13 C NMR (150MHz, CDCl3) δ172.6, 65.3, 53.8, 42.1, 35.5, 33.7, 31.9, 29.8, 29.6, 29.5, 29.3, 29.2, 28.5, 26.7, 25.9, 24.3, 22.7, 19.9, 14.1.

[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 overnight at 0°C. 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(600MHz,CDCl3)δ6.41(br.s,1H), 4.08(t,J=6.6Hz,4H), 3.20(q,J=6.6Hz,2H), 2.31(t,J=7.2Hz,2H), 2.24(4,J=7.2Hz,2H), 2.21(s, 6H), 1.84-1.82(m,2H), 1.81-1.76(m,2H), 1.61-1.58(m,4H), 1.47-1.45(m,2H), 1.38(s,3H), 1.31-2.20(m,26H), 0.87(t,J=7.2Hz,6H). 13C NMR (150MHz, CDCl3) δ172.7, 172.4, 65.2, 58.7, 53.6, 45.1, 39.3, 35.4, 34 .7, 31.7, 29.5, 29.1, 29.0, 28.4, 26.6, 25.7, 24.1, 23.1, 22.5, 19.8, 14.0. MS(ESI):m / z [M+H] + 555.4727(C 32 H 63 N2O5).

[0249] Compound AS-CL-20: Dioctyl 2-(6-(decyl(5-hydroxypentyl)amino)hexyl)-2-methylmalonate JPEG0007909622000108.jpg31128

[0250] Compound AS-CL-20 was synthesized according to the general procedure of step 8 in Scheme 2. The title compound was obtained as a colorless oil. 1 H NMR (600MHz, CDCl3) δ4.07(t,J=6.6Hz,4H), 3.63-3.61(m,2H), 2.52-2.46(m,6H), 1.83-1 .80(m,2H), 1.60-1.46(m,12H), 1.40-1.34(m,5H), 1.31-1.18(m,40H), 0.87-0.85(m,9H). 13 C NMR (150MHz, CDCl3) δ172.6, 65.4, 62.6, 53.9, 53.8, 53.7, 35.6, 32.4, 31.9, 31.8, 29.8, 29.7, 29.6, 29. 5, 29.4, 29.3, 29.2, 28.6, 27.6, 27.3, 26.1, 26.0(x2), 25.9, 24.3, 23.7, 22.72, 22.68, 19.9, 14.2, 14.1. MS(ESI):m / z [M+H] + 668.6194(C 41 H 82 NO. 5).

[0251] Compound AS-CL-21: Dioctyl 2-(6-((6-(decanoyloxy)hexyl)(3-hydroxypropyl)amino)hexyl)-2-methylmalonate JPEG0007909622000109.jpg38128

[0252] Compound AS-CL-21 was synthesized according to the general procedure of 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 JPEG0007909622000110.jpg32128

[0254] Compound AS-CL-22 was synthesized according to the general procedure of step 8 in Scheme 2. The title compound was obtained as a colorless oil. 1H NMR(600MHz,CDCl3)δ4.08(t,J=6.6Hz,4H), 3.78(t,J=5.4Hz,2H), 2.61(t,J=5.4Hz,2H), 2.38(t,J=7.8Hz,4H), 1.86-1.82(m,4H), 1.6 8-1.63(m,2H), 1.61-1.57(m,4H), 1.47-1.41(m,4H), 1.32-1.22(m,38H), 1.18-1.11(m,4H), 0.91(t,J=7.2Hz,3H), 0.88-0.86(m,9H). 13 C NMR(150MHz,CDCl3)δ172.1, 65.2, 64.9, 57.7, 55.4, 54.3, 54.2, 34.6, 32.4, 32.0, 31.9, 30.0, 29.70, 29.69, 2 9.67, 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 NO. 5).

[0255] Compound AS-CL-23: Dioctyl 2-(6-(dodecyl(3-hydroxypropyl)amino)hexyl)-2-methylmalonate JPEG0007909622000111.jpg2878

[0256] Compound AS-CL-23 was synthesized according to the general procedure of 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), 3.78(t,J=5.4Hz,2H), 2.61(t,J=5.4Hz,2H), 2.39-2.37(m,4H), 1.84-1.8 1(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). 13C NMR (150MHz, CDCl3) δ172.6, 65.3, 64.9, 55.4, 54.3, 54.2, 53.8, 35.6, 32.0, 31.8, 29.9, 29.7(x2), 29 .4, 29.3(x3), 29.2(x2), 28.6, 27.8, 27.6, 27.4, 26.9, 26.8, 25.9, 24.4, 22.7, 22.6, 19.9, 14.2, 14.1. MS(ESI):m / z [M+H] + 668.6181(C 41 H 82 NO. 5).

[0257] Compound 9a: Decyloctylmalonate (Step 9) JPEG0007909622000112.jpg2561

[0258] A solution of meldrumic acid (2.5 g, 17.35 mmol) and octanol (2.26 g, 17.35 mmol) in toluene was refluxed for 4 hours. 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 hours. 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 H NMR (600MHz, CDCl3) δ4.11(t,J=6.6Hz,4H), 3.34(s,2H), 1.64-1.59(m,4H), 1.32-1.24(m,24H), 0.87-0.85(m,6H). 13 C NMR (150MHz, CDCl3) δ166.7, 65.7, 41.7, 31.9, 31.8, 29.6, 29.3, 29.25, 29.20, 28.5, 25.8, 22.7, 22.6, 14.11, 14.08. MS(ESI):m / z [M+Na] + 379.2823(C 21 H 40 O4Na).

[0259] Compound 9b: Hexyloctylmalonate JPEG0007909622000113.jpg2349

[0260] Compound 9b was synthesized according to the procedure in step 9 of scheme 4. The title compound was obtained as a colorless oil. 1 H NMR (600MHz, CDCl3) δ4.10(t,J=6.6Hz,4H), 3.33(s,2H), 1.61-1.59(m,4H), 1.31-1.26(m,16H), 0.85-0.84(m,6H). 13 C NMR (150MHz, CDCl3) δ166.7, 65.6, 41.7, 31.8, 31.4, 29.2 (x2), 28.5, 28.4, 25.8, 25.5, 22.6, 22.5, 14.1, 14.0. MS(ESI):m / z [M+Na] + 323.2190(C 17 H 32 O4Na).

[0261] Compound AS-CL-24:1-Decyl 3-octyl 2-(6-(Decyl(3-hydroxypropyl)amino)hexyl)-2-methylmalonate JPEG0007909622000114.jpg2882

[0262] Compound AS-CL-24 was synthesized according to the procedure in Scheme 4 and the 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), 3.78(t,J=5.4Hz,2H), 2.62(t,J=5.4Hz,2H), 2.38(t,J=7.8Hz,4H), 1.84-1. 81(m,2H), 1.67-1.63(m,2H), 1.62-1.57(m,4H), 1.47-1.41(m,4H), 1.38(s,3H), 1.29-1.18(m,44H), 0.88-0.86(m,9H). 13C NMR(150MHz,CDCl3)δ172.6, 65.4, 64.9, 55.4, 54.3, 54.2, 53.8, 35.6, 32.0, 31.9, 30.0, 29.70, 29.69, 29.67, 2 9.63, 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 NO. 5).

[0263] Compound AS-CL-25: Dioctyl 2-(6-(decyl(6-hydroxyhexyl)amino)hexyl)-2-methylmalonate JPEG0007909622000115.jpg29128

[0264] Compound AS-CL-25 was synthesized according to the general procedure of step 8 in Scheme 2. The title compound was obtained as a colorless oil. 1 H NMR(600MHz,CDCl3)δ4.07(t,J=6.6Hz,4H), 3.61(t,J=6.6Hz,2H), 2.43-2.39(m,6H), 1.83-1.80( m,2H), 1.59-1.53(m,6H), 1.45-1.38(m,6H), 1.37(s,3H), 1.36-1.19(m,44H), 0.87-0.85(m,9H). 13 C NMR(150MHz,CDCl3)δ172.6, 65.4, 62.9, 54.12, 54.01, 53.95, 53.82, 35.6, 32.8, 32.0, 31.8, 29.9, 29.7, 29.6, 2 9.4, 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 NO. 5).

[0265] Compound AS-CL-26: Dioctyl 2-(4-(decyl(3-hydroxypropyl)amino)butyl)-2-methylmalonate JPEG0007909622000116.jpg3282

[0266] Compound AS-CL-26 was synthesized according to the general procedure of 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), 3.76(t,J=5.4Hz,2H), 2.60(t,J=5.4Hz,2H), 2.39-2.36(m,4H), 1.86-1.8 3(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 NO. 5).

[0267] Compound AS-CL-27: 1-Hexyl 3-octyl 2-(6-(decyl(3-hydroxypropyl)amino)hexyl)-2-methylmalonate JPEG0007909622000117.jpg2980

[0268] Compound AS-CL-27 was synthesized according to the procedure in Scheme 4 and the general step 8 of Scheme 2. The title compound was obtained as a colorless oil. 1H NMR(600MHz,CDCl3)δ4.08(t,J=6.6Hz,4H), 3.78(t,J=5.4Hz,2H), 2.61(t,J=5.4Hz,2H), 2.38(t,J=7.2Hz,4H), 1.84-1. 81(m,2H), 1.66-1.64(m,2H), 1.60-1.58(m,4H), 1.45-1.42(m,4H), 1.38(s,3H), 1.32-1.18(m,36H), 0.89-0.86(m,9H). 13 C NMR(150MHz,CDCl3)δ172.6, 65.4, 64.9, 55.4, 54.3, 54.2, 53.8, 35.6, 32.0, 31.9, 31.5, 30.0, 29.69, 29.68, 29.66, 2 9.4, 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 NO. 5).

[0269] Compound AS-CL-28: Didecyl 2-(6-(decyl(3-hydroxypropyl)amino)hexyl)-2-methylmalonate JPEG0007909622000118.jpg29128

[0270] Compound AS-CL-28 was synthesized according to the general procedure of step 8 in 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=5.4Hz,2H), 2.61(t,J=5.4Hz,2H), 2.37(t,J=7.2Hz,4H), 1.83-1. 81(m,2H), 1.67-1.63(m,2H), 1.61-1.56(m,4H), 1.46-1.41(m,4H), 1.37(s,3H), 1.32-1.18(m,48H), 0.87-0.85(m,9H). 13C NMR (150MHz, CDCl3) δ172.6, 65.3, 64.9, 55.4, 54.3, 54.2, 53.8, 35.6 (x2), 32.0, 30.0, 29.69, 29.67, 29.6 5, 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 NO. 5).

[0271] Compound AS-CL-29: Dioctyl 2-(6-((6-((2-hexyldecanoyl)oxy)hexyl)(3-hydroxypropyl)amino)hexyl)-2-methylmalonate JPEG0007909622000119.jpg3974

[0272] Compound AS-CL-29 was synthesized according to the general procedure of step 8 in Scheme 2. The title compound was obtained as a colorless oil. 1 H NMR(600MHz,CDCl3)δ4.07(t,J=6.6Hz,4H), 4.04(t,J=6.6Hz,2H), 3.77(t,J=5.4Hz,2H), 2.61(t,J=5.4Hz,2H), 2.39-2.36(m,4H), 2.31-2.27(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). 13C NMR (150MHz, CDCl3) δ167.8, 172.6, 65.3, 64.9, 64.1, 55.3, 54.22, 54.19, 53.8, 45.9, 35.6, 32.6 (x2), 31.9, 31.8, 31.7, 30.0, 29.6, 29.5, 29. 3, 29.28, 29.26, 29.24, 28.8, 28.6, 27.8, 27.5, 27.48, 27.43, 27.2, 26 .9, 26.8, 26.0, 25.9, 24.4, 22.72, 22.69, 22.65, 19.9, 14.2, 14.1 (x2). MS(ESI): m / z [M+H] + 838.7495(C 51 H 100 NO7).

[0273] Compound AS-CL-30: Bis(6-((6-(decanoyloxy)hexyl)(3-hydroxypropyl)amino)hexyl)2,2-dihexylmalonate JPEG0007909622000120.jpg4175

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

[0275] A mixed solution of malonic acid (2 g, 19.21 mmol), 6-bromohexane-1-ol (7.65 g, 42.26 mmol), EDCI (8.1 g, 42.26 mmol), and DMAP (0.47 g, 3.84 mmol) in DCM was stirred overnight at room temperature. After washing with 2N HCl (aqueous solution) and evaporation, 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 H NMR(600MHz,CDCl3)δ4.14(t,J=6.6Hz,4H), 3.40(t,J=6.6Hz,4H), 3.36(s,2H ), 1.88-1.84(m,4H), 1.69-1.64(m,4H), 1.50-1.44(m,4H), 1.41-1.35(m,4H). 13C NMR (150MHz, CDCl3) δ166.6, 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. After washing with saturated NH4Cl (aqueous solution) and evaporation, the residue was purified by silica gel column chromatography using EA / Hex (1 / 25) to obtain compound 11 (1 g, 67%) as a yellow oily substance. 1 H NMR(600MHz,CDCl3)δ4.10(t,J=6.6Hz,4H), 3.40(t,J=6.6Hz,2H), 1.86-1.83(m,8H), 1.65-1.60(m, 4H), 1.48-1.43(m,4H), 1.38-1.33(m,4H), 1.33-1.22(m,12H), 1.17-1.09(m,4H), 0.88-0.86(m,6H). 13 C NMR (150MHz, CDCl3) δ172.0, 64.8, 57.7, 33.5, 32.6, 32.2, 31.5, 29.5, 28.3, 27.7, 25.1, 23.9, 22.5, 14.0. MS(ESI):m / z [M+Na] + 619.1967, [M+Na] 2+ 621.1942, 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 overnight at 50°C. 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(600MHz,CDCl3)δ4.06(t,J=6.6Hz,4H), 4.03(t,J=6.6Hz,4H), 3.76(br.s,4H), 2.60(t,J=5.4Hz,4H), 2.37(t,J=7.8Hz,8H), 2.26 (t,J=7.8Hz,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 (150MHz, CDCl3) δ174.0, 172.1, 65.0, 64.8, 64.3, 57.7, 55.3, 54.2(x2), 34.4, 32.3, 31.9, 31.6, 29.6, 29 .5, 29.3(x2), 29.2(x2), 28.7, 28.6, 27.9, 27.2(x2), 26.8, 26.0, 25.9, 25.0, 24.0, 22.7, 22.6, 14.14, 14.09. MS(ESI):m / z [M+H] + 1095.9487(C 65 H 127 N2O 10 ).

[0278] Compound AS-CL-31: Bis(6-(decyl(3-hydroxypropyl)amino)hexyl)2,2-dihexylmalonate JPEG0007909622000121.jpg2970

[0279] Compound AS-CL-31 was synthesized according to the procedure for AS-CL-30. The title compound was obtained as a colorless oil. 1 H NMR(600MHz,CDCl3)δ4.09-4.06(m,4H), 3.78-3.76(m,4H), 2.62-2.38(m,4H), 2.38(br.s,8H), 1.85-1.83(m,4H), 1 .66-1.65(m,4H), 1.63-1.58(m,4H), 1.49-1.41(m,8H), 1.36-1.22(m,48H), 1.14-1.11(m,4H), 0.86-0.85(m,12H). 13 C NMR (150MHz, CDCl3) δ172.2, 65.1, 64.9, 57.8, 55.3, 54.3, 54.2, 32.3, 32.0, 31.6, 29.7 (x2) , 29.66, 29.6, 29.4, 28.6, 27.9, 27.6, 27.2, 26.9, 26.8, 26.0, 24.0, 22.8, 22.6, 14.2, 14.1. MS(ESI):m / z [M+H] + 868.0482(C 53 H 107 N2O6).

[0280] Compound AS-CL-32: Dioctyl 2-(6-(decyl((1s,4s)-4-(hydroxymethyl)cyclohexyl)amino)hexyl)-2-methylmalonate JPEG0007909622000122.jpg30128

[0281] Compound AS-CL-32 was synthesized according to the general procedure of step 8 in Scheme 2. The title compound was obtained as a colorless oil. 1 H NMR(600MHz,CDCl3)δ4.07(t,J=6.6Hz,4H), 3.59(d,J=7.2Hz,2H), 2.53-2.45(m,5H), 1.83-1.80(m,2H), 1. 78-1.71(m,2H), 1.61-1.55(m,6H), 1.45-1.38(m,6H), 1.37(s,3H), 1.32-1.18(m,42H), 0.87-0.85(m,9H). 13C NMR (150MHz, CDCl3) δ172.6, 65.3, 64.4, 59.4, 53.8, 50.6, 50.4, 36.1, 35.6, 31.9, 31.8, 29.9, 29 .7, 29.6, 29.4, 29.24, 29.22, 28.6, 27.6, 27.4, 25.9, 24.6, 24.4, 22.72, 22.67, 19.9, 14.2, 14.1. MS(ESI):m / z [M+H] + 694.6339(C 43 H 84 NO. 5).

[0282] Compound AS-CL-33: Dioctyl 2-(6-(decyl((4-hydroxycyclohexyl)methyl)amino)hexyl)-2-methylmalonate JPEG0007909622000123.jpg30128

[0283] Compound AS-CL-33 was synthesized according to the general procedure of step 8 in Scheme 2. The title compound was obtained as a colorless oil. 1 H NMR (600MHz, CDCl3) δ4.07-4.01(m,4H), 3.88(br.s,0.5H), 3.48-3.46(m,0.5H), 2.27-2.25(m,4H), 2.13-2.12(m,1H) ), 2.06-2.05(m,1H), 1.92-1.90(m,1H), 1.80-1.77(m,3H), 1.63-1.48(m,7H), 1.41-1.14(m,48H), 0.84-1.81(m,9H). 13 C NMR (150MHz, CDCl3) δ172.5, 71.2, 67.3, 65.2, 61.0, 60.2, 54.9, 54.8, 54.7, 53.7, 35.6, 35.5, 35.4, 34.7, 32.2, 31.9, 31.8, 2 9.9, 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 NO. 5).

[0284] Compound AS-CL-34: Didecyl 2-ethyl-2-(4-((6-((2-hexyldecanoyl)oxy)hexyl)(3-hydroxypropyl)amino)butyl)malonate JPEG0007909622000124.jpg3682

[0285] Compound AS-CL-34 was synthesized according to the general procedure of step 8 in Scheme 2. The title compound was obtained as a colorless oil. 1 H NMR(600MHz,CDCl3)δ4.09-4.02(m,6H), 3.76-3.75(m,2H), 2.61(br.s,2H), 2.39(br.s,4H), 2.29-2.27(m,1H), 1.90( q,J=7.8Hz,2H), 1.87-1.84(m,2H), 1.65-1.55(m,10H), 1.47-1.12(m,60H), 0.87-0.85(m,12H), 0.79(t,J=7.8Hz,3H). 13 C NMR (150MHz, CDCl3) δ176.7, 171.9, 65.2, 64.6, 64.0, 58.0, 55.1, 54.3, 54.0, 45.9, 32.6, 31.93, 31.90, 31.7, 29.6 (x2), 29.5, 29.4, 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 JPEG0007909622000125.jpg2874

[0287] Compound AS-CL-35 was synthesized according to the general procedure of step 8 in Scheme 2. The title compound was obtained as a colorless oil. 1H NMR(600MHz,CDCl3)4.09-4.06(m,4H), 3.76(t,J=4.8Hz,2H), 2.64(br.s,2H), 2.43-2.40(m,4H), 1.90(q,J=7.8Hz,2H), 1.87-1.84(m,2H) ), 1.68-1.66(m,2H), 1.60-1.56(m,4H), 1.50-1.45(m,4H), 1.28-1.24(m,42H), 1.16-1.10(m,2H), 0.87-0.85(m,9H), 0.79(J=7.8Hz,3H). 13 C NMR (150MHz, CDCl3) δ171.9, 65.3, 64.4, 58.1, 55.0, 54.2, 53.9, 32.0, 31.7, 29.7, 29 .6(x2), 29.4, 29.3, 28.6, 27.8, 27.5, 27.0, 26.7, 25.9, 25.4, 22.7, 22.1, 14.1, 8.5. MS(ESI):m / z [M+H] + 682.6341(C 42 H 84 NO. 5).

[0288] Example 2: Preparation and Characterization of Nucleic Acid-Loaded Lipid Nanoparticles

[0289] 2.1 Preparation of mRNA-lipid nanoparticle (mRNA-LNP) complexes

[0290] LNPs consisting of commercially available ionized lipids were formulated with a total lipid concentration of 50 mM MC3-, SM-102, AS-CL05, AS-CL09, AS-CL28, or AS-CL35. The LNPs were 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 the specified molar ratio.

[0291] LNP is NanoAssmblr TM(Precision NanoSystems) The mRNA was assembled using the Ignite microfluidic mixing device. Two different mRNA targets were encapsulated in LNPs: (i) mRNA of SARS-CoV-2 S protein from WT and Omicron BA.5, and (ii) mRNA encoding the DENV2 serotype envelope (E) protein (DENV2 E mRNA). For this purpose, Spark NanoAssmblr TM Before mixing with Precision NanoSystems, mRNA was dissolved in 50 mM sodium acetate buffer (pH 4.5) at a constant NP lipid:mRNA ratio of 6.5. A 16 μL aliquot of the organic phase and a 32 μL aliquot of the aqueous phase were mixed and injected into 48 μL of PBS (pH 7.4). Next, the LNP was further diluted with 96 μL of DPBS (pH 7.4) and dialyzed against PBS.

[0292] 2.2 Physiological and chemical properties of mRNA-LNP complexes

[0293] The mRNA-LNP complexes from Example 2.1 were diluted 100-fold in PBS (pH 7.4) and transferred to 384-well microplates. Size and polydispersity index (PDI) were measured by dynamic light scattering (DLS). Inclusion efficiency was evaluated by disrupting each complex with 1% Triton X-100 and releasing the mRNA cargo. The results are summarized in Table 2.

[0294] Table 2. Physiological and chemical properties of the mRNA-LNP complex from Example 2.1 JPEG0007909622000126.jpg65134

[0295] The data in Table 2 confirms that the cationic lipids of the present invention, AS-CL05, AS-CL09, AS-CL28, and AS-CL35, can each bind with other helper lipids to form nanoparticles. The physical properties of AS-CL05-LNP, AS-CL09-LNP, AS-CL28-LNP, and AS-CL35-LNP, such as size and polydispersity index (PDI), were similar to those of other LNPs made from commercially available cationic lipids. The average particle size of the mRNA-LNP complexes was 70-90 nm, and the PDI values ​​of all complexes were less than 0.3, indicating uniform distribution in 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 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 levels, followed by MC3, SM102, AS-CL05, AS-CL28, and AS-CL35 (Figures 1A and 2A).

[0298] After confirming that each mRNA-LNP complex could successfully induce protein expression in cell-based assays, immunogenicity was evaluated in BALB / c mice. Mice administered with physiological saline served as a negative control group. ELISA-based methods were used to evaluate the binding of serum antibodies to WT recombinant S protein and BA.5 recombinant S protein. Antibodies induced by WT mRNA-AS-CL09 LNP and BA.5 mRNA-AS-CL09 LNP showed the highest binding efficiency, while complexes formed by MC3, SM-102, AS-CL05, AS-CL28, and AS-CL35, respectively, 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 serum collected from vaccinated animals. In the pseudovirus neutralization experiment, serum from mice immunized with the BA.5 mRNA-AS-CL09 LNP complex showed the highest neutralizing ability. Compared to the MC3 and AS-CL05 groups, serum from mice immunized with the BA.5 mRNA-AS-CL09 LNP complex showed a 9-fold higher neutralizing antibody titer against the BA.5 pseudovirus. Furthermore, LNPs containing AS-CL09 showed a superior neutralizing antibody profile compared to SM-102 LNPs (Figure 1C and Table 3). Therefore, the type of ionized lipid in the LNP significantly influenced the final result of serum neutralizing activity, with AS-CL09 demonstrating the best performance among the lipids tested.

[0301] Table 3. Semi-maximal inhibitory concentration (IC) of mRNA-LNP complex in Example 2.1 using SARS-CoV-2 pseudovirus. 50 ) evaluation JPEG0007909622000127.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 DENV2 E protein (DB32-6). The results showed that 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 mRNA-LNP complexes by intramuscular injection according to the procedure described in the "Materials and Methods" section, and serum samples were collected at 6 weeks and the binding activity of neutralizing antibodies was evaluated by ELISA. Serum from mice inoculated with DENV2 E mRNA-AS-CL09 LNP showed higher binding activity to DENV2 serotype viruses compared to serum from mice inoculated with DENV2 E mRNA-SM-102LNP (Figure 3B). The neutralizing activity of DENV antibodies against BHK-21 cells was evaluated using a plaque reduction neutralizing titer (PRNT) assay. Neutralizing antibodies were detected at high levels in mice injected with DENV2 E mRNA-AS-CL09 LNP. In these samples, the PRNT50 value was approximately 24,725, which was approximately 1.4 times higher than the value in DENV2 E mRNA-SM-102LNP serum (Figure 3C and Table 4).

[0305] Table 4. Semi-maximal inhibitory concentration (IC) of the DENV2 E mRNA-AS-CL09 LNP complex as evaluated by the PRNT assay. 50 ) JPEG0007909622000128.jpg41128

[0306] Based on these results, we concluded that mRNA-LNPs containing AS-CL09 ionized lipids showed higher antibody production after intramuscular vaccination compared to mRNA-LNP complexes containing SM-102.

[0307] In summary, 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 equivalent to other LNPs composed of commercially available lipids (i.e., MC3-, SM-102, or ALC-0315). Therefore, 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 production of LNPs for gene and drug delivery.

[0308] The above description of embodiments is for illustrative purposes only, and it will be understood that various modifications 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. Various embodiments of the present invention are described above with some degree of specificity or by reference to one or more individual embodiments, but those skilled in the art can make numerous modifications to the disclosed embodiments without departing from the spirit or scope of this disclosure.

Claims

【Request Item 1】

2. Lipid nanoparticles having a hydrophilic core and an outer lipid bilayer shell formed of one or more lipids as described in claim 1.

3. The lipid nanoparticle according to claim 2, further comprising a therapeutic agent disposed within the hydrophilic core or the outer lipid bilayer shell of the lipid nanoparticle.

4. The lipid nanoparticle according to claim 3, wherein the therapeutic agent is the nucleic acid of a viral protein.

5. The lipid nanoparticle according to claim 4, wherein the nucleic acid is the mRNA of the SARS-CoV-2 spike protein.

6. The lipid nanoparticle according to claim 4, wherein the nucleic acid is mRNA of the envelope protein of dengue virus.

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