Novel compounds and targeted delivery uses thereof

Abstract

Disclosed herein are novel compounds and compositions comprising the compounds, lipid particles comprising the novel compounds and other lipids, such as phospholipids, structural lipids, and PEG lipids, and the use of the compounds or lipid particles in delivering therapeutic agents to a subject, target organ, or target cell.

Description

Technical Field

[0001] The present disclosure provides novel compounds and lipid particles comprising these compounds. The present disclosure also provides uses of the compounds and lipid particles for targeted delivery. The present disclosure also provides methods for preparing the compounds and lipid particles. Background Art

[0002] Therapeutic nucleic acids such as RNA have broad application prospects in the prevention and treatment of various diseases or disorders. Different types of nucleic acids (such as small interfering RNA (siRNA), microRNA (miRNA), small activating RNA (saRNA), single guide RNA (sgRNA), messenger RNA (mRNA), self-amplifying RNA, circular RNA, antisense oligonucleotide (ASO), ribonucleoprotein (RNP), ssDNA and dsDNA) can be used to regulate specific genes at the DNA level, mRNA level or protein level, so that precise expression regulation of these genes can be achieved.

[0003] However, the problems currently faced by therapeutic nucleic acids are:

[0004] 1. How to protect nucleic acids from being digested by nucleases;

[0005] 2. How to effectively deliver nucleic acids into cells;

[0006] 3. How to improve the efficiency of nucleic acids in endosomal escape;

[0007] 4. How to deliver therapeutic nucleic acids to target cells and / or tissues of interest.

[0008] Therefore, there is still a need to develop compounds, compositions and methods to facilitate the delivery of therapeutic nucleic acids. Summary of the Invention

[0009] The present disclosure discloses novel compounds and lipid particles comprising these compounds. The present disclosure also discloses uses of the compounds and lipid particles for targeted delivery. The present disclosure also discloses methods for preparing the compounds and lipid particles.

[0010] In one aspect, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0011]

[0012] Wherein

[0013] L a and L cEach independently selected from a bond, an alkyl group, an alkenyl group, an alkynyl group, and a heteroalkyl group, wherein the alkyl, alkenyl, alkynyl, and heteroalkyl groups are optionally substituted with one or more groups independently selected from halogen, hydroxy, cyano, and amino;

[0014] L b Selected from a bond, -NR a -, -O-, -S-, an alkyl group, an alkenyl group, an alkynyl group, a heteroalkyl group, a heteroalkenyl group, a heteroalkynyl group, a cycloalkyl group, a heterocyclic group, an aryl group, and a heteroaryl group, wherein the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups are optionally substituted with one or more groups independently selected from halogen, hydroxy, cyano, and amino, or

[0015] L b Is covalently linked to R 1 To form

[0016] Provided that when two of L a , L b , and L c Are bonds, then the remaining one is not a bond;

[0017] R a Selected from hydrogen, R 5 , an alkyl group, an alkenyl group, an alkynyl group, a heteroalkyl group, a heteroalkenyl group, a heteroalkynyl group, a cycloalkyl group, a heterocyclic group, an aryl group, and a heteroaryl group, wherein the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups are optionally substituted with one or more groups independently selected from halogen, hydroxy, cyano, cycloalkyl, heterocyclic, aryl, heteroaryl, and R 6 ;

[0018] R 1 Is a group having the formula (I-a):

[0019]

[0020] R 2 Is a group having the formula (I-b):

[0021]

[0022] R 3 Is a group having the formula (I-c):

[0023]

[0024] R 4 Is a group having the formula (I-d):

[0025]

[0026] Wherein

[0027] R 1a , R 2a , R 3a and R 4a each of is independently a bond or an alkyl group;

[0028] R 1b , R 2b , R 3b and R 4b each of which is independently a bond, alkyl or heteroalkyl, wherein alkyl and heteroalkyl are optionally substituted with one or more groups independently selected from hydroxy, amino, oxo, thioxo and imino; and

[0029] R 1c , R 2c , R 3c and R 4c each of which is independently selected from alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl, wherein alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl are optionally substituted with one or more groups independently selected from halogen, hydroxy, oxo, cyano, cycloalkyl, heterocyclyl, aryl, and heteroaryl, and alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl are optionally interrupted by one or more groups independently selected from cycloalkyl, heterocyclyl, aryl, and heteroaryl; and

[0030] R 5 is a group having the following formula (Ie):

[0031]

[0032] Each R 6 is independently a group having the following formula (If):

[0033]

[0034] in

[0035] R 5a and R 6a each of is independently a bond or an alkyl group;

[0036] R 5b and R 6b each of which is independently a bond, alkyl or heteroalkyl, wherein alkyl and heteroalkyl are optionally substituted with one or more groups independently selected from hydroxy, amino, oxo, thioxo and imino; and

[0037] R 5c and R 6cEach of which is independently selected from alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl and heteroalkynyl, wherein the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl and heteroalkynyl are optionally substituted with one or more groups independently selected from halogen, hydroxy, oxo and cyano, and the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl and heteroalkynyl are optionally interrupted with one or more groups independently selected from cycloalkyl, heterocyclic, aryl and heteroaryl.

[0038] In one aspect, the present disclosure provides a compound of formula (A) or a pharmaceutically acceptable salt thereof:

[0039]

[0040] Wherein

[0041] R 2 is

[0042] R 3 is

[0043] R 4 is

[0044] Each W is independently selected from O, S or NR b ;

[0045] Each Y is independently selected from O, S, NR c , N(R c )Z(W), N(R c )N(R c ) or N(R c )N(R c )Z(W);

[0046] Each Z is independently selected from C, S or S(O);

[0047] Each m is independently 0 or 1;

[0048] Each p is independently 1 or 2;

[0049] Q is 1, 2 or 3;

[0050] R b and R c Each of which is independently selected from hydrogen, alkyl, alkoxycarbonyl, alkanoyl, aroyl or sulfonyl; and

[0051] R 2c , R 3c and R 4cEach of which is independently selected from alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl and heteroalkynyl, wherein the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl and heteroalkynyl are optionally substituted by one or more groups independently selected from halogen, hydroxy, oxo and cyano, and the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl and heteroalkynyl are optionally interrupted by one or more groups independently selected from cycloalkyl, heterocyclic, aryl and heteroaryl.

[0052] In one aspect, the present disclosure provides a compound having formula (B), formula (C), formula (D) or formula (E):

[0053]

[0054]

[0055] wherein

[0056] Each W is independently selected from O, S or NR b ;

[0057] Each Y is independently selected from O, S, NR c , N(R c )Z(W), N(R c )N(R c ) or N(R c )N(R c )Z(W);

[0058] Each Z is independently selected from C, S or S(O);

[0059] Each n is independently 0, 1, 2, 3, 4 or 5;

[0060] Each m is independently 0, 1, 2 or 3;

[0061] Each p is independently 1, 2, 3 or 4; and

[0062] R b and R c Each of which is independently selected from hydrogen, alkyl, alkoxycarbonyl, acyl or sulfonyl.

[0063] In one aspect, the present disclosure provides a lipid particle comprising a compound having formula (I), formula (A), formula (B), formula (C), formula (D) or formula (E) or a pharmaceutically acceptable salt thereof. In one aspect, the present disclosure provides a loaded lipid particle comprising the lipid particle of the present disclosure and a therapeutic agent. In one aspect, the present disclosure provides a pharmaceutical composition comprising the loaded lipid particle of the present disclosure and a pharmaceutically acceptable excipient.

[0064] In one aspect, the present disclosure provides a method of delivering a therapeutic agent to a subject, which comprises the following steps:

[0065] i) encapsulating a therapeutic agent into the lipid particles of the present disclosure; and

[0066] ii) administering the lipid particles encapsulating the therapeutic agent to a subject.

[0067] In one aspect, the present disclosure provides a method of delivering a therapeutic agent to a subject, comprising administering to the subject a lipid-loaded particle of the present disclosure or a pharmaceutical composition of the present disclosure.

[0068] In one aspect, the present disclosure provides a method of treating a disease or disorder in a subject, comprising administering to the subject an effective amount of a lipid-loaded particle of the present disclosure or a pharmaceutical composition of the present disclosure.

[0069] In one aspect, the present disclosure provides the use of the lipid particles of the present disclosure, the lipid-loaded particles of the present disclosure, or the pharmaceutical compositions of the present disclosure in the manufacture of a medicament for treating a disease or disorder.

[0070] In one aspect, the present disclosure provides a method of expressing a protein in a subject, comprising:

[0071] i) encapsulating a polynucleotide encoding the protein into the lipid particles of the present disclosure; and ii) administering the lipid particles encapsulating the polynucleotide to the subject.

[0072] In one aspect, the present disclosure provides a method for preparing a compound of formula (I), formula (A), formula (B), formula (C), formula (D) or formula (E) or a pharmaceutically acceptable salt thereof, the method comprising:

[0073] A) mixing a compound of formula (II) with a compound of formula (III);

[0074]

[0075] wherein,

[0076] each R 7 is independently selected from hydrogen, alkyl, heteroalkyl, haloalkyl and hydroxyalkyl;

[0077] each L a 、L b 、L c 、R 1b and R 1c has the same definition as above;

[0078] B) refluxing the mixture until the reaction is complete, thereby obtaining a compound of formula (I), formula (A),

[0079] formula (B), formula (C), formula (D) or formula (E).

[0080] In one aspect, the present disclosure provides a method for preparing a compound of formula (I), formula (A), formula (B), formula (C), formula (D) or formula (E) or a pharmaceutically acceptable salt thereof, the method comprising:

[0081] i) mixing a compound of formula (II) and a compound of formula (IV):

[0082]

[0083] wherein R 1c has the same definition as above;

[0084] ii) mixing the product of step (i) with a compound of formula (III); and

[0085] iii) refluxing the mixture until the reaction is complete to obtain a compound of formula (I), formula (A), formula (B), formula (C), formula (D) or formula (E). BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 Shows the changes in the physical characteristics of the test lipid particles over two weeks.

[0087] Figure 2 Shows the in vitro and in vivo delivery characteristics of the test lipid particles on day 0 and day 14.

[0088] Figure 3 Shows the in vitro and in vivo delivery characteristics of formulations 1 to 4.

[0089] Figure 4 Shows the in vitro delivery characteristics and cell viability of the test formulation.

[0090] Figure 5 Shows A) in vivo fluorescence intensity imaging of FLuc mRNA delivered by the test formulation in mice; and B) the in vivo delivery characteristics of the test formulation.

[0091] Figure 6 Shows the in vitro delivery characteristics and cell viability of lipid particles containing the test compound.

[0092] Figure 7 Shows A) in vivo fluorescence intensity imaging of FLuc mRNA delivered by lipid particles containing the test compound in mice; and B) the characteristics of in vivo delivery of lipid particles containing the test compound to muscle.

[0093] Figure 8Shows A) imaging of the liver, spleen, and lungs after in vivo delivery by lipid particles containing a test compound; B) characteristics of lipid particles containing a test compound upon in vivo delivery to the liver; C) tissue distribution of FLuc mRNA delivery for testing lipid particles targeting the liver.

[0094] Figure 9 Shows A) imaging of the liver, spleen, and lungs after in vivo delivery by lipid particles containing a test compound; B) characteristics of lipid particles containing a test compound upon in vivo delivery to the spleen; C) tissue distribution of FLuc mRNA delivery for testing lipid particles targeting the spleen.

[0095] Figure 10 Shows A) imaging of the liver, spleen, and lungs after in vivo delivery by lipid particles containing a test compound; B) characteristics of lipid particles containing a test compound upon in vivo delivery to the lungs; C) tissue distribution of FLuc mRNA delivery for testing lipid particles targeting the lungs.

[0096] Figure 11 Shows the expression level of hEPO after in vivo delivery by lipid particles containing a test compound.

[0097] Figure 12 Shows the rate of change in drug concentration in plasma and lung lavage fluid between the test group and the control group after in vivo delivery of the test lipid particles.

[0098] Figure 13 Shows the antibody titers induced by a single in vivo immunization with the RAV - G mRNA vaccine delivered by the test lipid particles.

[0099] Figure 14 Shows the recognition ability of the test antibody - conjugated lipid particle preparation for different cell surface markers. Detailed Description

[0100] Reference will now be made in detail to certain embodiments of the present disclosure, examples of which are illustrated in the accompanying structures and chemical formulas. While the present disclosure will be described in conjunction with the enumerated embodiments, it should be understood that they are not intended to limit the present disclosure to those embodiments. Instead, the present disclosure is intended to cover all alternatives, modifications, and equivalents that may be included within the scope of the present disclosure as defined by the claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which can be used in the practice of the present disclosure. The present disclosure is in no way limited to the methods and materials described. If one or more of the incorporated references and similar materials are different from or conflict with the present application, including but not limited to the defined terms, term usage, the described techniques, etc., then the present disclosure shall prevail. All references, patents, patent applications cited in the present disclosure are hereby incorporated by reference in their entirety herein.

[0101] Definition

[0102] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the following terms are intended to have the following meanings:

[0103] As used in the specification and claims, unless the context clearly dictates otherwise, the singular forms "a", "an", and "the" include plural referents. Thus, for example, reference to "a compound" includes both a single compound and a plurality of different compounds.

[0104] The term "about" as used herein is intended to indicate that the recited value should not be construed as an absolute value and should also take into account measurement errors, variations between batches, and / or variations between equipment.

[0105] When used in this specification and claims, the words "comprising", "containing", and "including" are intended to specify the presence of the stated features, integers, components, or steps, but they do not preclude the presence or addition of one or more other features, integers, components, steps, or groups thereof.

[0106] It should be understood that the "compounds" of the present disclosure can exist in solvated and non-solvated forms, such as hydrated forms, solid forms, and the present disclosure is intended to cover all such solvated and non-solvated forms. It should also be understood that the "compounds" of the present disclosure can exist in the form of pharmaceutically acceptable salts. In some embodiments, the "compounds" of the present disclosure are ionizable lipids. In some embodiments, the "compounds" of the present disclosure can exist as cationic lipids at physiological pH.

[0107] The definitions of specific functional groups and chemical terms are described in more detail below. For the purposes of the present disclosure, chemical elements are identified according to the Periodic Table of the Elements inside cover of the CAS version in the 75th Edition of the Handbook of Chemistry and Physics, and the general definitions of specific functional groups are as described therein. In addition, the general principles of organic chemistry as well as specific functional groups and reactivity are described in the following references: Organic Chemistry, Thomas Sorrell, 2nd Edition, University Science Books, Sausalito, 2006; Advanced Organic Chemistry by Smith and March March, 6th Edition, John Wiley & Sons, Inc., New York, 2007; Larock, Comprehensive Organic Transformations, 3rd Edition, VCH Publishers, Inc., New York, 2018; Carruthers, Some Modern Methods of Organic Synthesis, 4th Edition, Cambridge University Press, Cambridge, 2004; the entire contents of each of these references are incorporated herein by reference.

[0108] Linking substituents are described at various places in the present disclosure. If a linking group is explicitly required by the structure, the Markush variables listed for that group are to be understood as linking groups. For example, if a linking group is required by the structure and the Markush group definition for that variable lists "alkyl", it is to be understood that the "alkyl" represents a linking alkylene.

[0109] When a bond on a substituent is shown crossing a bond connecting two atoms in a ring, the substituent may be bonded to any atom in the ring. When a substituent is listed without indicating through which atom it is bonded to the remainder of the compound of a given molecular formula, the substituent may be bonded through any atom in that molecular formula. Combinations of substituents and / or variables are permitted provided that such combinations result in a stable compound.

[0110] When any variable (e.g., R i ) appears more than once in any constituent or molecular formula of a compound, its definition at each occurrence is independent of its definition at every other occurrence. Thus, for example, if a group is shown to be substituted with 0 - 2 R i moieties, the group may optionally be substituted with up to two R i moieties, and each occurrence of R i is independently selected from Ri Definition. In addition, combinations of substituents and / or variables are permitted only if the combination results in a stable compound.

[0111] As used herein, the term "C i-j " represents a range of carbon atom numbers, where i and j are integers, and the range of carbon atom numbers includes the endpoints (i.e., i and j) and every integer point therebetween, and where j is greater than i. For example, C 1-6 represents a range of 1 to 6 carbon atoms, including 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, and 6 carbon atoms. In some embodiments, the term "C 1-24 " represents 1 to 24, particularly 2 to 24, particularly 4 to 24, particularly 6 to 24, particularly 8 to 22, particularly 10 to 20, particularly 10 to 18, or particularly 12 to 18 carbon atoms.

[0112] As used herein, the term "alkyl", whether as part of another term or used independently, refers to a saturated straight-chain or branched-chain hydrocarbon group, which may optionally be independently substituted by one or more of the substituents described below. The term "C i-j alkyl" refers to an alkyl group having i to j carbon atoms. In some embodiments, the alkyl group contains 1 to 24 carbon atoms. In some embodiments, the alkyl group contains 1 to 23 carbon atoms. In some embodiments, the alkyl group contains 1 to 22 carbon atoms. In some embodiments, the alkyl group contains 1 to 21 carbon atoms. In some embodiments, the alkyl group contains 1 to 20 carbon atoms, 1 to 19 carbon atoms, 1 to 18 carbon atoms, 1 to 17 carbon atoms, 1 to 16 carbon atoms, 1 to 15 carbon atoms, 1 to 14 carbon atoms, 1 to 13 carbon atoms, or 1 to 12 carbon atoms. In some embodiments, the alkyl group contains 12 to 18 carbon atoms. In some embodiments, the alkyl group contains 12 to 17 carbon atoms, 12 to 16 carbon atoms, 12 to 15 carbon atoms, or 12 to 14 carbon atoms. In some embodiments, the alkyl group contains 12 to 19 carbon atoms, 12 to 20 carbon atoms, 12 to 21 carbon atoms, 12 to 22 carbon atoms, 12 to 23 carbon atoms, or 12 to 24 carbon atoms. In some embodiments, the alkyl group contains 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, 6 carbon atoms, 7 carbon atoms, 8 carbon atoms, 9 carbon atoms, 10 carbon atoms, 11 carbon atoms, 12 carbon atoms, 13 carbon atoms, 14 carbon atoms, 15 carbon atoms, 16 carbon atoms, 17 carbon atoms, 18 carbon atoms, 19 carbon atoms, 20 carbon atoms, 21 carbon atoms, 22 carbon atoms, 23 carbon atoms, or 24 carbon atoms. "C 1-10Examples of "alkyl" include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl. "C 1-6 Examples of "C 1-6 alkyl" are methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, and the like.

[0113] The alkyl can be further substituted by substituents that independently replace one or more hydrogen atoms on one or more carbons of the alkyl. Examples of such substituents can include, but are not limited to, acyl, alkyl, alkenyl, alkynyl, oxo, halogen, hydroxy, alkoxy, haloalkyl, haloalkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, phosphate, phosphonate, phosphinate, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino); acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, mercapto, alkylthio, arylthio, thiocarboxylate, sulfate, alkanesulfinyl, sulfonate, sulfamoyl, sulfonamide, nitro, trifluoromethyl, cyano, nitro, azide, heterocyclic group, alkylaryl, or aromatic or heteroaromatic moiety. The alkenyl, alkynyl, cycloalkyl, heterocyclic group, aryl, and heteroaryl described below can also be similarly substituted.

[0114] As used herein, the term "alkenyl", whether as part of another term or used independently, refers to a straight-chain or branched-chain hydrocarbon group having at least one carbon-carbon double bond, which may optionally be independently substituted by one or more substituents described herein, and includes groups having "cis" and "trans" orientations or having "E" and "Z" orientations. In some embodiments, the alkenyl contains 2 to 24 carbon atoms. In some embodiments, the alkenyl contains 2 to 23 carbon atoms. In some embodiments, the alkenyl contains 2 to 22 carbon atoms, 2 to 21 carbon atoms, 2 to 20 carbon atoms, 2 to 19 carbon atoms, 2 to 18 carbon atoms, 2 to 17 carbon atoms, 2 to 16 carbon atoms, 2 to 15 carbon atoms, 2 to 14 carbon atoms, 2 to 13 carbon atoms, 2 to 12 carbon atoms, 2 to 11 carbon atoms, and in some embodiments, the alkenyl contains 2 carbon atoms. In some embodiments, the alkenyl contains 12 to 18 carbon atoms. In some embodiments, the alkenyl contains 12 to 17 carbon atoms, 12 to 16 carbon atoms, 12 to 15 carbon atoms, or 12 to 14 carbon atoms. In some embodiments, the alkenyl contains 12 to 19 carbon atoms, 12 to 20 carbon atoms, 12 to 21 carbon atoms, 12 to 22 carbon atoms, 12 to 23 carbon atoms, or 12 to 24 carbon atoms. In some embodiments, the alkenyl contains 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, 6 carbon atoms, 7 carbon atoms, 8 carbon atoms, 9 carbon atoms, 10 carbon atoms, 11 carbon atoms, 12 carbon atoms, 13 carbon atoms, 14 carbon atoms, 15 carbon atoms, 16 carbon atoms, 17 carbon atoms, 18 carbon atoms, 19 carbon atoms, 20 carbon atoms, 21 carbon atoms, 22 carbon atoms, 23 carbon atoms, or 24 carbon atoms. In some embodiments, the alkenyl contains one or more "Z" carbon-carbon double bonds. Examples of alkenyl include, but are not limited to, vinyl, propenyl, butenyl, pentenyl, 1-methyl-2-buten-1-yl, 5-hexenyl, and the like. In some embodiments, the alkenyl has at least one carbon-carbon double bond. In some embodiments, the alkenyl has at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 carbon-carbon double bonds. In some embodiments, two or more carbon-carbon double bonds in the alkenyl are conjugated. In some embodiments, two or more carbon-carbon double bonds in the alkenyl are not conjugated. In some embodiments, two or more carbon-carbon double bonds in the alkenyl are isolated, cumulative, or conjugated. The term "alkenyl", whether as part of another term or used independently, also means to include a straight-chain or branched-chain hydrocarbon group having at least one carbon-carbon double bond and at least one carbon-carbon triple bond.

[0115] As used herein, the term "alkynyl", whether as part of another term or used independently, refers to a straight-chain or branched-chain hydrocarbon group having at least one carbon-carbon triple bond, which may optionally be independently substituted by one or more substituents described herein. In some embodiments, the alkynyl group contains 2 to 24 carbon atoms. In some embodiments, the alkynyl group contains 2 to 23 carbon atoms. In some embodiments, the alkynyl group contains 2 to 22 carbon atoms, 2 to 21 carbon atoms, 2 to 20 carbon atoms, 2 to 19 carbon atoms, 2 to 18 carbon atoms, 2 to 17 carbon atoms, 2 to 16 carbon atoms, 2 to 15 carbon atoms, 2 to 14 carbon atoms, 2 to 13 carbon atoms, 2 to 12 carbon atoms, 2 to 10 carbon atoms, and in some embodiments, the alkynyl group contains 2 carbon atoms. In some embodiments, the alkynyl group contains 12 to 18 carbon atoms. In some embodiments, the alkynyl group contains 12 to 17 carbon atoms, 12 to 16 carbon atoms, 12 to 15 carbon atoms, or 12 to 14 carbon atoms. In some embodiments, the alkynyl group contains 12 to 19 carbon atoms, 12 to 20 carbon atoms, 12 to 21 carbon atoms, 12 to 22 carbon atoms, 12 to 23 carbon atoms, or 12 to 24 carbon atoms. In some embodiments, the alkynyl group contains 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, 6 carbon atoms, 7 carbon atoms, 8 carbon atoms, 9 carbon atoms, 10 carbon atoms, 11 carbon atoms, 12 carbon atoms, 13 carbon atoms, 14 carbon atoms, 15 carbon atoms, 16 carbon atoms, 17 carbon atoms, 18 carbon atoms, 19 carbon atoms, 20 carbon atoms, 21 carbon atoms, 22 carbon atoms, 23 carbon atoms, or 24 carbon atoms. Examples of alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, and the like. In some embodiments, the alkynyl group has at least one carbon-carbon triple bond. In some embodiments, the alkynyl group has at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 carbon-carbon triple bonds. In some embodiments, two or more carbon-carbon triple bonds in the alkynyl group are conjugated. In some embodiments, two or more carbon-carbon triple bonds in the alkynyl group are not conjugated. The term "alkynyl", whether as part of another term or used independently, also means a straight-chain or branched-chain hydrocarbon group having at least one carbon-carbon triple bond and at least one carbon-carbon double bond.

[0116] As used herein, the term "alkoxy", whether as part of another term or used independently, refers to an alkyl group as defined above attached to the parent molecule through an oxygen atom. The term "C" i-j"Alkoxy" means that the alkyl portion of the alkoxy group has from i to j carbon atoms. In some embodiments, the alkoxy group contains from 1 to 10 carbon atoms. In some embodiments, the alkoxy group contains from 1 to 9 carbon atoms. In some embodiments, the alkoxy group contains from 1 to 8 carbon atoms, 1 to 7 carbon atoms, 1 to 6 carbon atoms, 1 to 5 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms or 1 to 2 carbon atoms. "C 1-6 Examples of "alkoxy" include, but are not limited to, methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), tert-butoxy, pentyloxy, n-hexyloxy, and the like.

[0117] As used herein, the term "amino" means -NH2. In some embodiments, the amino group may be substituted on the nitrogen with any possible substituent.

[0118] As used herein, the term "aryl", whether as part of another term or used independently, refers to monocyclic and polycyclic systems having a total of 5 to 20 ring members, wherein at least one ring in the system is an aromatic ring, and wherein each ring in the system contains 3 to 12 ring members. Examples of "aryl" include, but are not limited to, phenyl, biphenyl, naphthyl, anthracenyl, etc., which may contain one or more substituents. As used herein, groups in which an aromatic ring is fused to one or more additional rings are also included within the scope of the term "aryl". In the case of polycyclic systems, although all rings may be aromatic rings (e.g., quinoline), it is only necessary for one of the rings to be an aromatic ring (e.g., 2,3-dihydroindole). The second ring may also be fused, bridged or spirocyclic. Examples of polycyclic aryls include, but are not limited to, benzofuranyl, indanyl, phthalimido, naphthyridinyl, phenanthridinyl or tetrahydronaphthyl, etc. The aryl group may be substituted at one or more ring positions with the above substituents.

[0119] As used herein, the term "cycloalkyl", whether as part of another term or used alone, refers to a monovalent non-aromatic, saturated or partially unsaturated monocyclic and polycyclic system in which all ring atoms are carbon and contain at least three ring-forming carbon atoms. In some embodiments, the cycloalkyl can contain 3 to 12 ring-forming carbon atoms, 3 to 11 ring-forming carbon atoms, 3 to 10 ring-forming carbon atoms, 3 to 9 ring-forming carbon atoms, 3 to 8 ring-forming carbon atoms, 3 to 7 ring-forming carbon atoms, 3 to 6 ring-forming carbon atoms, 3 to 5 ring-forming carbon atoms, 3 to 4 ring-forming carbon atoms, 4 to 12 ring-forming carbon atoms, 4 to 11 ring-forming carbon atoms, 4 to 10 ring-forming carbon atoms, 4 to 9 ring-forming carbon atoms, 4 to 8 ring-forming carbon atoms, 4 to 7 ring-forming carbon atoms, 4 to 6 ring-forming carbon atoms, 4 to 5 ring-forming carbon atoms. The cycloalkyl can be saturated or partially unsaturated. The cycloalkyl can be substituted. In some embodiments, the cycloalkyl can be a saturated cyclic alkyl. In some embodiments, the cycloalkyl can be a partially unsaturated cyclic alkyl containing at least one double bond or triple bond in its ring system.

[0120] In some embodiments, the cycloalkyl can be monocyclic or polycyclic. Examples of monocyclic cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, and cyclododecyl.

[0121] In some embodiments, the cycloalkyl can be a saturated or partially unsaturated polycyclic (e.g., bicyclic and tricyclic) carbocyclic system, which can be arranged as a fused-ring, spiro-ring, or bridged-ring system. As used herein, the term "fused ring" refers to a ring system in which two rings share two adjacent atoms, the term "spiro ring" refers to a ring system in which two rings are connected by a single shared atom, and the term "bridged ring" refers to a ring system in which two rings share three or more atoms. Examples of fused carbocyclics include, but are not limited to, naphthyl, benzopyrenyl, anthracenyl, acenaphthylenyl, fluorenyl, etc. Examples of spiro carbocyclics include, but are not limited to, spiro[5.5]undecyl, spirodienyl, spiro[3.6]decyl, etc. Examples of bridged carbocyclics include, but are not limited to, bicyclo[1,1,1]pentenyl, bicyclo[2,2,1]heptenyl, bicyclo[2,2,1]heptyl, bicyclo[2,2,2]octyl, bicyclo[3,3,1]nonyl, bicyclo[3,3,3]undecyl, etc.

[0122] As used herein, the term "cyano" refers to -CN.

[0123] As used herein, the term "oxo" refers to =O, which replaces two hydrogen atoms attached to an atom. For example, oxo-substituted ethyl is CH3C(=O)- or -C(=O)CH2-.

[0124] As used herein, the term "thioxo" refers to =S, which replaces two hydrogen atoms attached to an atom. For example, thioxo-substituted ethyl is CH3C(=S)- or -C(=S)CH2-.

[0125] As used herein, the term "imino" refers to =NR, which replaces two hydrogen atoms attached to an atom, where R refers to hydrogen or any possible substituent on the nitrogen. For example, imino-substituted ethyl is CH3C(=NR)- or -C(=NR)CH2-.

[0126] As used herein, the term "halogen" refers to an atom selected from fluorine (or fluoro), chlorine (or chloro), bromine (or bromo), and iodine (or iodo).

[0127] As used herein, the term "haloalkyl", whether as part of another term or used independently, refers to an alkyl group having one or more halogen substituents. Examples of haloalkyl include, but are not limited to, trifluoromethyl (-CF3), pentafluoroethyl (-C2F5), difluoromethyl (-CHF2), trichloromethyl (-CCl3), dichloromethyl (-CHCl2), pentachloroethyl (-C2Cl5), etc.

[0128] As used herein, the term "hydroxyalkyl", whether as part of another term or used independently, refers to an alkyl group having one or more hydroxy substituents. Examples of hydroxyalkyl include, but are not limited to, hydroxymethyl (-CH2OH), 1-hydroxyethyl (-CH(OH)CH3), 2-hydroxyethyl (-CH2CH2OH), 1,2-dihydroxyethyl (-CH(OH)CH2OH), 1-hydroxy-n-propyl (-CH(OH)CH2CH3), 2-hydroxy-n-propyl (-CH2CH(OH)CH3), 3-hydroxy-n-propyl (-CH2CH2CH2OH), 2-hydroxy-2-propyl (-C(OH)(CH3)2), 1-hydroxy-2-propyl (-C(CH3)CH2OH), 1,2-dihydroxypropyl (-CH(OH)CH(OH)CH3), 1,3-dihydroxypropyl (-CH(OH)CH2CH2OH), 2,3-dihydroxypropyl (-CH2CH(OH)CH2OH), 1,2,3-trihydroxypropyl (-CH(OH)CH(OH)CH2OH), etc.

[0129] As used herein, the term "haloalkoxy", whether as part of another term or used independently, refers to an alkoxy group having one or more halogen substituents. Thus, the term "halo-C i-j alkoxy", whether as part of another term or used independently, refers to a C i-j alkoxy group having one or more halogen substituents. Examples of haloalkoxy groups include, but are not limited to, -O-CF3, -O-C2F5, -O-CHF2, -O-CCl3, -O-CHCl2, -O-C2Cl5, etc.

[0130] As used herein, the term "heteroatom" refers to nitrogen (N), oxygen (O), sulfur (S), and includes any oxidized form of nitrogen or sulfur, as well as any quaternized form of basic nitrogen (including N-oxides).

[0131] As used herein, the terms "heteroalkyl", "heteroalkenyl", or "heteroalkynyl", whether as part of another term or used independently, refer to an alkyl, alkenyl, or alkynyl group containing one or more heteroatoms. Thus, the terms "hetero-C i-j alkyl", "hetero-C i-j alkenyl", or "hetero-C i-j alkynyl", whether as part of another term or used independently, refer to a C i-j alkyl, C i-j alkenyl, or C i-j alkynyl group containing one or more heteroatoms. For example, the term "hetero-C 1-6 alkyl", whether as part of another term or used independently, refers to a C 1-6 alkyl group containing one or more heteroatoms. In some embodiments, the heteroalkyl, heteroalkenyl, or heteroalkynyl group contains at least one heteroatom. In some embodiments, the heteroalkyl, heteroalkenyl, or heteroalkynyl group contains at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten heteroatoms. In some embodiments, two or more of the heteroatoms in the heteroalkyl, heteroalkenyl, or heteroalkynyl group are the same. In some embodiments, two or more of the heteroatoms in the heteroalkyl, heteroalkenyl, or heteroalkynyl group are different. In some embodiments, two or more of the heteroatoms in the heteroalkyl, heteroalkenyl, or heteroalkynyl group are directly bonded. In some embodiments, two or more of the heteroatoms in the heteroalkyl, heteroalkenyl, or heteroalkynyl group are not directly bonded.

[0132] As used herein, the term "heteroaryl", whether as part of another term or used independently, refers to an aryl group having one or more heteroatoms in addition to carbon atoms. The heteroaryl group can be monocyclic. Examples of monocyclic heteroaryl groups include, but are not limited to, thienyl, furyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, azolyl, iso azolyl, diazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, benzofuranyl, and pteridinyl. The heteroaryl also includes polycyclic groups, in which the heteroaryl ring is fused to one or more aryl, heteroaryl, cycloaliphatic, or heterocyclic rings, and the linking group or point of attachment is located on the heteroaryl ring or another ring. Examples of polycyclic heteroaryls include, but are not limited to, indolyl, isoindolyl, benzothienyl, benzofuranyl, benzo[1,3]dioxolyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzothiazolyl, quinolinyl, isoquinolinyl, dihydroquinolinyl, dihydroisoquinolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phen azinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like.

[0133] As used herein, the term "heterocyclic group" refers to a saturated or partially unsaturated carbocyclic group, in which one or more ring atoms are heteroatoms independently selected from oxygen, sulfur, nitrogen, phosphorus, etc., and the remaining ring atoms are carbon, and one or more of the ring atoms may optionally be independently substituted by one or more substituents. In some embodiments, the heterocyclic group is a saturated heterocyclic group. In some embodiments, the heterocyclic group is a partially unsaturated heterocyclic group having one or more double bonds in its ring system. In some embodiments, the heterocyclic group may contain any oxidized form of carbon, nitrogen, or sulfur, as well as any quaternized form of basic nitrogen. Where possible, the heterocyclic group may be carbon-linked or nitrogen-linked. In some embodiments, the heterocycle is carbon-linked. In some embodiments, the heterocycle is nitrogen-linked. For example, the group derived from pyrrole can be pyrrol-1-yl (nitrogen-linked) or pyrrol-3-yl (carbon-linked). In addition, the group derived from imidazole can be imidazol-1-yl (nitrogen-linked) or imidazol-3-yl (carbon-linked).

[0134] The heterocyclic group can be monocyclic. Examples of monocyclic heterocyclic groups include, but are not limited to, oxetanyl, 1,1-dioxothietanyl pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothienyl, azetidinyl, pyrrolyl, furanyl, thienyl, pyrazolyl, imidazolyl, triazolyl, azolyl, thiazolyl, piperidinyl, piperazinyl, morpholinyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, pyridone, pyrimidinone, pyrazinone, pyrimidinone, pyridazinone, pyrrolidinyl, triazinone, and the like.

[0135] A heterocyclic group can be polycyclic, including fused-ring, spiro-ring, and bridged-ring systems. A fused heterocyclic group includes a group in which a heterocyclic group is fused to a saturated, partially unsaturated, or fully unsaturated (i.e., aromatic) carbocyclic or heterocyclic ring. Examples of fused heterocyclic groups include, but are not limited to, a phenyl-fused ring or a pyridyl-fused ring, such as quinolinyl, isoquinolinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, chromanyl, isochromanyl, indolyl, isoindolyl, indolizinyl, indazolyl, purinyl, benzofuranyl, isobenzofuranyl, benzimidazolyl, benzothiophenyl, benzothiazolyl, carbazolyl, phenazinyl, phenothiazinyl, phenanthridinyl, imidazo[1,2-a]pyridyl, [1,2,4]triazolo[4,3-a]pyridyl, [1,2,3]triazolo[4,3-a]pyridyl, and the like. Examples of spiro heterocyclic groups include, but are not limited to, spiropyranyl, spiro azinyl, 5-aza-spiro[2.4]heptyl, 6-aza-spiro[2.5]octyl, 6-aza-spiro[3.4]octyl, 2-oxa-6-aza-spiro[3.3]heptyl, 2-oxa-6-aza-spiro[3.4]octyl, 6-aza-spiro[3.5]nonyl, 7-aza-spiro[3.5]nonyl, 1-oxa-7-aza-spiro[3.5]nonyl, and the like. Examples of bridged heterocyclic groups include, but are not limited to, 3-aza-bicyclo[3,1,0]hexyl, 8-aza-bicyclo[3,2,1]octyl, 1-aza-bicyclo[2,2,2]octyl, 2-aza-bicyclo[2,2,1]heptyl, 1,4-diazabicyclo[2,2,2]octyl, and the like.

[0136] As used herein, the term "hydroxyl" refers to -OH.

[0137] As used herein, the term "mercapto" refers to -SH.

[0138] As used herein, the term "alkoxycarbonyl" refers to alkyl-O-C(=O)-. In some embodiments, the alkoxycarbonyl can be further substituted on the alkyl with any of the above possible substituents. Examples of alkoxycarbonyl include, but are not limited to, tert-butoxycarbonyl, benzyloxycarbonyl, allyloxycarbonyl, and 9-fluorenylmethoxycarbonyl.

[0139] As used herein, the term "sulfonyl" refers to R-SO2-, where R is hydrogen or any possible substituent on sulfur. Examples of sulfonyl include, but are not limited to, p-toluenesulfonyl, p-bromobenzenesulfonyl, 2- or 4-nitrobenzenesulfonyl, trifluoromethanesulfonyl, methanesulfonyl, and 5-(dimethylamino)naphthalene-1-sulfonyl.

[0140] As used herein, the term "acyl", whether used as part of another term or independently, refers to RC(=O)-, where R is hydrogen or any possible substituent on carbon. Examples of acyl groups include, but are not limited to, formyl, acetyl, trifluoroacetyl, and benzoyl. The term "alkylacyl" refers to alkyl-C(=O)-, where the alkyl is optionally substituted with any of the possible substituents described above. The term "arylacyl" refers to aryl-C(=O)-, where the aryl is optionally substituted with any of the possible substituents described above.

[0141] As used herein, the term "partially unsaturated" refers to a group that includes at least one double bond or triple bond. The term "partially unsaturated" is intended to cover rings having multiple sites of unsaturation, but is not intended to include aromatic (i.e., fully unsaturated) moieties.

[0142] As used herein, the term "substituted", whether or not preceded by the term "optionally", means that one or more hydrogens on the designated moiety are replaced by a suitable substituent. It should be understood that "substituted", "substituted by", or "substituted with" includes the implicit condition that such substitution is in accordance with the allowed valence of the atom being substituted and that the substitution results in a stable or chemically feasible compound, e.g., one that does not spontaneously undergo transformation, such as by rearrangement, cyclization, elimination, etc. Unless otherwise specified, an "optionally substituted" group can have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure can be substituted by more than one substituent selected from a designated group, the substituents at each position can be the same or different. One of ordinary skill in the art will understand that, if appropriate, the substituent itself can be substituted. Unless specifically stated as "unsubstituted", references herein to chemical moieties should be understood to include substituted variants. For example, a reference to an "aryl" group or moiety implicitly includes both substituted and unsubstituted variants.

[0143] As used herein, the term "interrupted", whether or not preceded by the term "optionally", means that one or more covalent bonds of a designated moiety are replaced by a suitable linking group, but not at the termini. In some embodiments, the replaced bond is a carbon-carbon bond. In some embodiments, the replaced bond is a carbon-heteroatom bond. Unless otherwise specified, an "optionally interrupted" moiety may have a suitable linking group at each replaceable position of the moiety, and when more than one position in any given structure can be interrupted by more than one linking group selected from the designated moieties, the linking groups at each position may be the same or different. In some embodiments, an alkyl group "interrupted" by a cycloalkyl group means an alkyl-cycloalkyl-alkyl. In some embodiments, an alkenyl group "interrupted" by a cycloalkyl group means an alkenyl-cycloalkyl-alkyl, alkenyl-cycloalkyl-alkenyl, alkenyl-cycloalkyl-alkynyl, alkyl-cycloalkyl-alkenyl, or alkynyl-cycloalkyl-alkenyl. In some embodiments, an alkynyl group "interrupted" by a cycloalkyl group means an alkynyl-cycloalkyl-alkyl, alkynyl-cycloalkyl-alkenyl, alkynyl-cycloalkyl-alkynyl, alkyl-cycloalkyl-alkynyl, or alkenyl-cycloalkyl-alkynyl. In some embodiments, an alkyl group "interrupted" by a heterocyclic group means an alkyl-heterocyclic-alkyl. In some embodiments, an alkenyl group "interrupted" by a heterocyclic group means an alkenyl-heterocyclic-alkyl, alkenyl-heterocyclic-alkenyl, alkenyl-heterocyclic-alkynyl, alkyl-heterocyclic-alkenyl, or alkynyl-heterocyclic-alkenyl. In some embodiments, an alkynyl group "interrupted" by a heterocyclic group means an alkynyl-heterocyclic-alkyl, alkynyl-heterocyclic-alkenyl, alkynyl-heterocyclic-alkynyl, alkyl-heterocyclic-alkynyl, or alkenyl-heterocyclic-alkynyl. In some embodiments, an alkyl group "interrupted" by an aryl group means an alkyl-aryl-alkyl. In some embodiments, an alkenyl group "interrupted" by an aryl group means an alkenyl-aryl-alkyl, alkenyl-aryl-alkenyl, alkenyl-aryl-alkynyl, alkyl-aryl-alkenyl, or alkynyl-aryl-alkenyl. In some embodiments, an alkynyl group "interrupted" by an aryl group means an alkynyl-aryl-alkyl, alkynyl-aryl-alkenyl, alkynyl-aryl-alkynyl, alkyl-aryl-alkynyl, or alkenyl-aryl-alkynyl. In some embodiments, an alkyl group "interrupted" by a heteroaryl group means an alkyl-heteroaryl-alkyl. In some embodiments, an alkenyl group "interrupted" by a heteroaryl group means an alkenyl-heteroaryl-alkyl, alkenyl-heteroaryl-alkenyl, alkenyl-heteroaryl-alkynyl, alkyl-heteroaryl-alkenyl, or alkynyl-heteroaryl-alkenyl. In some embodiments, an alkynyl group "interrupted" by a heteroaryl group means an alkynyl-heteroaryl-alkyl, alkynyl-heteroaryl-alkenyl, alkynyl-heteroaryl-alkynyl, alkyl-heteroaryl-alkynyl, or alkenyl-heteroaryl-alkynyl.

[0144] As used herein, the term "pharmaceutically acceptable" refers to those compounds, lipid particles, lipid-loaded particles, materials, compositions, and / or dosage forms that, within the scope of reasonable medical judgment, are suitable for contact with the tissues of humans and other animals without excessive toxicity, irritation, allergic response, or other problems or complications, and are commensurate with a reasonable benefit / risk ratio. In some embodiments, pharmaceutically acceptable compounds, lipid particles, lipid-loaded particles, materials, compositions, and / or dosage forms refer to those that are approved by a regulatory agency (such as the US Food and Drug Administration, the National Drug Administration, or the European Medicines Agency) or listed in an accepted pharmacopoeia (such as the US Pharmacopeia, the Chinese Pharmacopeia, or the European Pharmacopeia) for use in animals, and more specifically in humans.

[0145] As used herein, "pharmaceutically acceptable salt" or "medicinal salt" refers to a derivative of a compound in which the parent compound is modified by converting an existing acidic moiety (such as a carboxyl group, etc.) or basic moiety (such as an amine, base, etc.) into its salt form. In many cases, the compounds of the present disclosure are capable of forming acid addition salts and / or base salts by virtue of the presence of an amino group, a base, or a similar group. And "pharmaceutically acceptable salt" includes acid addition salts or base salts that retain the biological effectiveness and properties of the parent compound, which are generally not biologically or otherwise undesirable. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. described pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1-19. Pharmaceutically acceptable salts of the compounds provided herein include salts derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or with organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, lactic acid, trifluoroacetic acid, benzoic acid, cinnamic acid, mandelic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, malonic acid, fumaric acid, citric acid, malic acid, maleic acid, tartaric acid, succinic acid, or methanesulfonic acid, or salts formed by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipates, alginates, ascorbates, aspartates, benzenesulfonates, benzoates, bisulfates, borates, butyrates, camphorates, camphorsulfonates, citrates, cyclopentanepropionates, digluconates, dodecyl sulfates, ethanesulfonates, formates, fumarates, glucoheptanoates, glycerophosphates, gluconates, hemisulfates, heptanoates, hexanoates, hydroiodides, 2-hydroxyethanesulfonates, lactates, lactobionates, laurates, lauryl sulfates, malates, maleates, malonates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmitates, pamoates, pectates, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propionates, stearates, succinates, sulfates, tartrates, thiocyanates, p-toluenesulfonates, undecanoates, valerates, etc. In some embodiments, the inorganic acids from which salts can be derived include, for example, hydrochlorides, sulfates, phosphates, etc. In some embodiments, the organic acids from which salts can be derived include, for example, maleates, fumarates, oxalates, p-toluenesulfonates, succinates, L-(+)-tartrates, monoadipates, hemiadipates, etc.

[0146] The term “pharmaceutical composition” refers to a mixture of one or more compounds of the present disclosure or one or more lipid particles of the present disclosure or lipid particle-loaded lipid particles with other chemical components such as pharmaceutically acceptable diluents, excipients or carriers. The purpose of the pharmaceutical composition is to facilitate the administration of the compound, lipid particle or lipid particle-loaded lipid particle to a subject.

[0147] As used herein, the term "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, used to carry or transport a compound provided herein from one location, body fluid, tissue, organ (internal or external), or part of the body to another location, body fluid, tissue, organ, or part of the body. A pharmaceutically acceptable excipient or carrier can be a vehicle, diluent, excipient, or other material that can be used to contact animal tissue without producing excessive toxicity or adverse reactions. Non-limiting examples of pharmaceutically acceptable excipients or carriers include sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; tragacanth; malt; gelatin; talc; cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as polyethylene glycol and propylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; isotonic saline; Ringer's solution; ethanol; phosphate buffer solutions; non-toxic compatible lubricants, such as sodium lauryl sulfate and magnesium stearate; coloring agents; release agents; coating agents; sweetening, flavoring, and aromatic agents; preservatives; antioxidants; ion exchange agents; alumina; aluminum stearate; lecithin; self-emulsifying drug delivery systems (SEDDS), such as d-α-tocopheryl polyethylene glycol 1000 succinate; surfactants for pharmaceutical dosage forms, such as Tween or other similar polymeric delivery matrices; serum proteins, such as human serum albumin; glycine; sorbic acid; potassium sorbate; partial glyceride mixtures of saturated vegetable fatty acids; water, salts, or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and zinc salts; colloidal silica; magnesium trisilicate; polyvinylpyrrolidone; cellulose-based materials; polyacrylates; waxes; and polyethylene-polypropylene block polymers. Cyclodextrins, such as α-, β-, and γ-cyclodextrins, or chemically modified derivatives, such as hydroxyalkyl cyclodextrins, including 2- and 3-hydroxypropyl cyclodextrins, or other solubilizing derivatives can also be used to enhance the delivery of the compounds described herein. Pharmaceutically acceptable excipients or carriers useful in the present disclosure include those commonly known in the art, such as those disclosed in Remington Pharmaceutical Sciences, Mack Pub. Co., New Jersey (1991), which reference is incorporated herein by reference.

[0148] As used herein, "administration" of the disclosed compounds, lipid particles, or lipid particle-loaded compositions encompasses delivery of the compounds, lipid particles, or lipid particle-loaded compositions, or their prodrugs or other pharmaceutically acceptable derivatives, to a subject using any suitable formulation or route of administration discussed herein.

[0149] As used herein, the term "delivery" means providing an entity to a destination. For example, delivery of a therapeutic and / or prophylactic agent to a subject may involve administering a lipid particle composition comprising the therapeutic and / or prophylactic agent to the subject (e.g., by intravenous, intramuscular, intradermal, or subcutaneous routes). Administering a lipid particle or a composition comprising a lipid particle to a mammal or mammalian cell may involve contacting one or more cells with the lipid particle or composition.

[0150] As used herein, the term "enhanced delivery" means delivery of more (e.g., at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, or at least 100-fold more) of a therapeutic and / or prophylactic agent to a target tissue of interest (e.g., liver, lung, spleen, or muscle) or a target cell of interest (e.g., hepatocyte, lung cell, spleen cell, or muscle cell) by a lipid particle compared to the level of delivery of the therapeutic and / or prophylactic agent to the target tissue of interest (e.g., liver, lung, spleen, or muscle) or the target cell of interest (e.g., hepatocyte, lung cell, spleen cell, or muscle cell) by a control lipid particle (e.g., a lipid particle comprising DLin-MC3-DMA). The level of delivery of a therapeutic and / or prophylactic agent to a particular tissue or cell can be measured by comparing the amount of the therapeutic and / or prophylactic agent in the tissue or cell to the total amount of the therapeutic and / or prophylactic agent in the tissue or cell, comparing the amount of the therapeutic and / or prophylactic agent in the tissue or cell to the weight of the tissue, comparing the amount of protein produced in the tissue or cell to the total amount of protein in the tissue or cell, comparing the amount of protein produced in the tissue to the weight of the tissue or cell, or comparing the amount of the therapeutic and / or prophylactic agent delivered to the tissue or cell to the total amount of the therapeutic and / or prophylactic agent administered. It should be understood that enhanced delivery of a therapeutic and / or prophylactic agent to a target tissue or cell need not be determined in the subject being treated; it can be determined in a surrogate such as an animal model (e.g., a mouse or rat model). In certain embodiments, regardless of the route of administration, lipid particle compositions comprising a compound of formula (I), formula (A), formula (B), formula (C), formula (D), or formula (E) have substantially the same level of delivery enhancement. For example, certain compounds disclosed herein exhibit similar delivery enhancement when used for intravenous or intramuscular delivery of a therapeutic and / or prophylactic agent.

[0151] As used herein, the term "selective delivery" refers to the delivery of more (e.g., at least 1.5-fold more, at least 2-fold more, at least 3-fold more, at least 4-fold more, at least 5-fold more, at least 6-fold more, at least 7-fold more, at least 8-fold more, at least 9-fold more, at least 10-fold more) of a therapeutic and / or prophylactic agent to a target tissue of interest (e.g., liver, lung, spleen, or muscle) or a target cell of interest (e.g., hepatocyte, lung cell, spleen cell, or muscle cell) compared to an off-target tissue (e.g., liver, lung, spleen, or muscle) or an off-target cell of interest (e.g., hepatocyte, lung cell, spleen cell, or muscle cell). The level of delivery of a therapeutic and / or prophylactic agent to a particular tissue or cell can be measured by comparing the amount of the therapeutic and / or prophylactic agent in the tissue or cell to the total amount of the therapeutic and / or prophylactic agent in the tissue or cell, comparing the amount of the therapeutic and / or prophylactic agent in the tissue or cell to the weight of the tissue or cell, comparing the amount of protein produced in the tissue or cell to the total amount of protein in the tissue or cell, or comparing the amount of protein produced in the tissue or cell to the weight of the tissue or cell. It should be understood that the ability of lipid particles to specifically deliver a therapeutic and / or prophylactic agent to a target tissue or cell need not be determined in the subject being treated; it can be determined in a surrogate such as an animal model (e.g., a mouse or rat model).

[0152] The terms "effective amount", "pharmaceutically effective amount", or "therapeutically effective amount" refer to an amount of a therapeutic and / or prophylactic agent, or a compound or pharmaceutical composition described herein, that is delivered to a tissue or cell and that is sufficient to prevent, treat, reduce, and / or ameliorate the symptoms and / or the underlying cause of any disorder or disease in a subject, or an amount of an agent sufficient to produce a desired effect on a target cell, such as, for example: a decrease in cell migration, an increase or inhibition in the expression of a target nucleic acid in a cell as compared to the normal expression level of the nucleic acid detected in the absence of the therapeutic and / or prophylactic agent or the compound or pharmaceutical composition described herein that is delivered to the tissue or cell. In one embodiment, a "pharmaceutically effective amount" or "therapeutically effective amount" is an amount sufficient to reduce or eliminate the symptoms of a disease. In another embodiment, a pharmaceutically effective amount or therapeutically effective amount is an amount sufficient to overcome the disease itself. In certain specific embodiments, a "pharmaceutically effective amount" or "therapeutically effective amount" is an amount capable of effectively achieving a detectable killing or inhibition of the growth or spread of cancer cells, a reduction in the size or number of tumors, or other measures of the level, stage, progression, or severity of cancer. The pharmaceutically effective amount or therapeutically effective amount will vary depending on the subject and disorder being treated, the weight and age of the subject, the severity of the disorder, the particular composition or excipient selected, the dosing regimen to be followed, the timing of administration, the mode of administration, etc., all of which can be readily determined by one of ordinary skill in the art. A sufficient therapeutic effect does not necessarily occur upon administration of a single dose and may occur only after administration of a series of doses. The specific dose will vary depending on, for example, the particular compound selected, the type of subject and its age / underlying health or risk of health condition, the dosing regimen to be followed, the severity of the disease, whether administered in combination with other agents, the timing of administration, the tissue to which administered, and the physical delivery system carrying the dose. Thus, the pharmaceutically effective amount or therapeutically effective amount can be administered in one or more administrations. For example, but not limited to, in the case of treating cancer, a pharmaceutically effective amount or therapeutically effective amount of an agent refers to an amount of the agent that alleviates, ameliorates, mitigates, or eliminates one or more symptoms of cancer in a patient.

[0153] As used herein, the term "treatment" refers to reversing, alleviating, delaying the onset of, or inhibiting the progression of a disease or disorder or one or more of its symptoms, as described herein. In some embodiments, treatment can be initiated after the appearance of one or more symptoms. In other embodiments, treatment can be initiated in the absence of symptoms. For example, a susceptible individual can be treated prior to the appearance of symptoms (e.g., based on a symptom history and / or based on genetic or other predisposing factors). Treatment can also continue after the symptoms have subsided, for example, to prevent or delay their recurrence.

[0154] The term "subject" as used herein includes, but is not limited to, humans (i.e., males or females of any age group, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged or elderly)), and / or other primates (e.g., cynomolgus monkeys, rhesus monkeys); mammals, including commercially relevant mammals such as cows, pigs, horses, sheep, goats, rabbits, hamsters, mice, cats, and / or dogs; and / or birds, including commercially relevant birds such as chickens, ducks, geese, quails, and / or turkeys. In some embodiments, the subject has been or may be diagnosed with a disease or disorder. In some embodiments, the subject is not diagnosed with a disease or disorder.

[0155] Lipid

[0156] In one aspect, the present disclosure provides novel compounds useful as lipids.

[0157] The lipid has at least one of the following characteristics: a hydrophilic head group with different pKas, a cationic monoamine, diamine, triamine, oligoamine / polyamine, imidazole, pyridine, guanidine, and a hydrophobic tail. In some embodiments, the lipid is an ionizable lipid. In some embodiments, the lipid is a cationic lipid.

[0158] As used herein, the term "cationic lipid" includes lipids having an amino head and one or more fatty chains, which can be protonated at physiological pH to form a cationic lipid. In some embodiments, the cationic lipid is an amino lipid. Lipids having one or more groups that can be protonated or deprotonated or lipids that act as zwitterions are also included. In some embodiments, the lipids of the present disclosure have at least one protonatable group such that the lipid is positively charged at a first pH equal to or lower than physiological pH (e.g., equal to or lower than pH 7.4) and neutral at a second pH (e.g., equal to or higher than physiological pH). In some embodiments, the lipids of the present disclosure have at least two or at least three protonatable groups. It should be understood that the addition or removal of protons according to pH is an equilibrium process, and the reference to charged (e.g., protonated) or neutral lipids refers to the nature of the predominant species (e.g., more than 50%, 60%, 70%, 80%, 90%, 95%, or 99%), and does not require all lipids to be present in a charged or neutral form.

[0159] In some embodiments, the pKa of the protonatable group of the cationic lipids of the present disclosure is in the range of about 4 to about 11. In some embodiments, when incorporated into lipid particles, the lipid has a pKa of about 4 to about 7, about 5 to about 7, or about 5.5 to about 6.8. In some embodiments, lipids having such a pKa will be cationic at lower pHs, while the particles will be mostly (although not completely) surface neutralized at physiological pH (e.g., at pH 7.4). In some embodiments, at least some of the nucleic acids associated with the outer surface of the particles comprising lipids having such a pKa will lose their electrostatic interactions at physiological pH and be removed by simple dialysis; thereby greatly reducing the susceptibility of the particles to clearance. The pKa of the lipids within the lipid particles can be measured, for example, by using the fluorescent probe 2-(p-toluidino)-6-naphthalene sulfonic acid (TNS) using the method described by Cullis et al. (1986) Chem Phys Lipids 40, 127-144.

[0160] In some embodiments, the lipids of the present disclosure are advantageously used in lipid particles. In some embodiments, the lipid particles are used for in vivo delivery of a therapeutic agent to cells. In some embodiments, the lipid particles are used for in vivo delivery of a therapeutic agent to tissues.

[0161] In another aspect, the lipids of the present disclosure are compounds of formula (I) or pharmaceutically acceptable salts thereof:

[0162] wherein

[0163] L a and L c are each independently selected from a bond, alkyl, alkenyl, alkynyl, and heteroalkyl, wherein the alkyl, alkenyl, alkynyl, and heteroalkyl are optionally substituted with one or more groups independently selected from halogen, hydroxy, cyano, and amino;

[0164] L b is selected from a bond, -NR a -, -O-, -S-, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl, wherein the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl are optionally substituted with one or more groups independently selected from halogen, hydroxy, cyano, and amino, or

[0165] L b is covalently linked to R 1 to form

[0166] provided that when L a 、L b and L cWhen two of them are keys, the remaining one is not a key;

[0167] R a is selected from hydrogen, R 5 , alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclic group, aryl and heteroaryl, wherein the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclic group, aryl and heteroaryl are optionally independently substituted by one or more groups selected from halogen, hydroxy, cyano, cycloalkyl, heterocyclic group, aryl, heteroaryl and R 6 ;

[0168] R 1 is a group having the following formula (I-a):

[0169]

[0170] R 2 is a group having the following formula (I-b):

[0171]

[0172] R 3 is a group having the following formula (I-c):

[0173]

[0174] R 4 is a group having the following formula (I-d):

[0175]

[0176] wherein

[0177] R 1a 、R 2a 、R 3a and R 4a each independently is a bond or alkyl;

[0178] R 1b 、R 2b 、R 3b and R 4b each independently is a bond, alkyl or heteroalkyl, wherein the alkyl and heteroalkyl are optionally independently substituted by one or more groups selected from hydroxy, amino, oxo, thio and imino; and

[0179] R 1c 、R 2c 、R 3c and R 4cEach is independently selected from alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl, wherein the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl are optionally substituted by one or more groups independently selected from halogen, hydroxy, oxo, cyano, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, and the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl are optionally interrupted by one or more groups independently selected from cycloalkyl, heterocycloalkyl, aryl, and heteroaryl; and

[0180] R 5 is a group having the formula (I-e):

[0181]

[0182] Each R 6 is independently a group having the formula (I-f):

[0183]

[0184] wherein

[0185] R 5a and R 6a each is independently a bond or alkyl;

[0186] R 5b and R 6b each is independently a bond, alkyl, or heteroalkyl, wherein the alkyl and heteroalkyl are optionally substituted by one or more groups independently selected from hydroxy, amino, oxo, thio, and imino; and

[0187] R 5c and R 6c each is independently selected from alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl, wherein the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl are optionally substituted by one or more groups independently selected from halogen, hydroxy, oxo, and cyano, and the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl are optionally interrupted by one or more groups independently selected from cycloalkyl, heterocycloalkyl, aryl, and heteroaryl.

[0188] In some embodiments, L a and L c are each independently alkyl. In some embodiments, L a and L c are each independently C 1-6 alkyl.

[0189] In some embodiments, L a and L c are each independently methyl, ethyl, or propyl.

[0190] In some embodiments, L b is -NR a -.

[0191] In some embodiments, R a is selected from hydrogen, R 5 , alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclic group, aryl and heteroaryl, wherein the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclic group, aryl and heteroaryl are optionally independently substituted by one or more groups selected from halogen, hydroxy, cyano, cycloalkyl, heterocyclic group, aryl, heteroaryl and R 6 .

[0192] In some embodiments, R a is C 1-6 heteroalkyl.

[0193] In some embodiments, R a is C 1-6 alkyl optionally substituted by one or more groups independently selected from hydroxy, cycloalkyl and heteroaryl.

[0194] In some embodiments, L b is selected from

[0195] In some embodiments, L b is selected from -S-, -O-, -O-alkyl-O-, cycloalkyl and heterocyclic group.

[0196] In some embodiments, L b is selected from -S-, -O-

[0197] In some embodiments, L b is

[0198] In some embodiments, -L a -L b -L c - is selected from

[0199] In some embodiments, L b is alkyl or heteroalkyl, covalently linked to R 1 to form L a is a bond, alkyl or heteroalkyl, and R 1 is a bond, alkyl or heteroalkyl.

[0200] In some embodiments, is

[0201] In some embodiments, L c is methyl.

[0202] In some embodiments, at least two of R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are the same.

[0203] In some embodiments, at least three of R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are the same.

[0204] In some embodiments, at least four of R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are the same.

[0205] In some embodiments, at least two of R 1 , R 2 , R 3 , and R 4 are different.

[0206] In some embodiments, one or more of R 1 , R 2 , R 3 , and R 4 are independently alkyl optionally substituted with one or more hydroxyl groups.

[0207] In some embodiments, one or more of R 1 , R 2 , R 3 , and R 4 are independently methyl,

[0208] In some embodiments, one or more of R 1a , R 2a , R 3a , R 4a , R 5a , and R 6a are independently a bond, methyl, ethyl, propyl, butyl, or pentyl.

[0209] In some embodiments, R 1b , R 2b , R 3b , R 4b , R 5b and R 6b are independently selected from alkyl and heteroalkyl, where the alkyl and heteroalkyl are optionally substituted with one or more groups independently selected from hydroxy, oxo, thio, and imino.

[0210] In some embodiments, one or more of R 1b , R 2b , R 3b , R 4b , R 5b and R 6b are independently where

[0211] each W is independently selected from O, S, or NR b ;

[0212] each Y is independently selected from O, S, NR c , N(R c )Z(W), N(R c )N(R c ) or N(R c )N(R c )Z(W);

[0213] each Z is independently selected from C, S, or S(O);

[0214] each m is independently 0, 1, 2, or 3;

[0215] each p is independently 1, 2, 3, or 4; and

[0216] each of R b and R c is independently selected from hydrogen, alkyl, alkoxycarbonyl, alkanoyl, aroyl, and sulfonyl.

[0217] In some embodiments, R 1 is

[0218] R 2 is

[0219] R 3 is

[0220] R 4 is

[0221] R 5 , if present, is

[0222] R 6 , if present, is

[0223] Each W is independently selected from O, S or NR b ;

[0224] Each Y is independently selected from O, S, NR c , N(R c )Z(W), N(R c )N(R c ) or N(R c )N(R c )Z(W);

[0225] Each Z is independently selected from C, S or S(O);

[0226] Each m is independently 0 or 1;

[0227] Each p is independently 1 or 2;

[0228] R b and R c each independently selected from hydrogen, alkyl, alkoxycarbonyl, alkanoyl, aroyl and sulfonyl; and

[0229] R 1c , R 2c , R 3c , R 4c , R 5c and R 6c each, if present, independently selected from alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl and heteroalkynyl, where alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl and heteroalkynyl are optionally independently substituted by one or more groups selected from halogen, hydroxy, oxo and cyano, and alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl and heteroalkynyl are optionally interrupted by one or more groups independently selected from cycloalkyl, heterocyclic, aryl and heteroaryl.

[0230] In some embodiments, L b is -NR a -;

[0231] R a is selected from hydrogen, R 5 , alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl and heteroalkynyl, where alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl and heteroalkynyl are substituted by one or more groups independently selected from halogen, hydroxy, cyano, cycloalkyl, heterocyclic, aryl, heteroaryl and R 6 ;

[0232] R 1 is

[0233] R 2 is

[0234] R 3 is

[0235] R 4 is

[0236] R 5 , if present, is

[0237] R 6 , if present, is

[0238] Each W is independently selected from O, S or NR b ;

[0239] Each Y is independently selected from O, S, NR c , N(R c ), Z(W), N(R c ), N(R c ), or N(R c ), N(R c ), Z(W);

[0240] Each Z is independently selected from C, S or S(O);

[0241] Each m is independently 0 or 1;

[0242] Each p is 1 or 2;

[0243] R b and R c each independently selected from hydrogen, alkyl, alkoxycarbonyl, alkanoyl, aroyl and sulfonyl; and

[0244] R 1c , R 2c , R 3c , R 4c , R 5c and R 6cEach of which, if present, is independently selected from alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl and heteroalkynyl, wherein the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl and heteroalkynyl are optionally substituted by one or more groups independently selected from halogen, hydroxy, oxo and cyano, and the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl and heteroalkynyl are optionally interrupted by one or more groups independently selected from cycloalkyl, heterocycloalkyl, aryl and heteroaryl.

[0245] In some embodiments, L b is -NR a -;

[0246] R a is selected from R 5 , alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl and heteroalkynyl, wherein the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl and heteroalkynyl are substituted by R 6 and are optionally substituted by one or more additional groups independently selected from halogen, hydroxy, cyano, cycloalkyl, heterocycloalkyl, aryl and heteroaryl;

[0247] R 1 is selected from alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl and heteroalkynyl, wherein the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl and heteroalkynyl are optionally substituted by one or more groups independently selected from halogen, hydroxy, cyano, cycloalkyl, heterocycloalkyl, aryl and heteroaryl;

[0248] R 2 is

[0249] R 3 is

[0250] R 4 is

[0251] R 5 , if present, is

[0252] R 6 , if present, is

[0253] Each W is independently selected from O, S or NR b ;

[0254] Each Y is independently selected from O, S, NR c , N(R c )Z(W), N(R c )N(R c ) or N(R c )N(R c )Z(W);

[0255] Each Z is independently selected from C, S or S(O);

[0256] Each m is independently 0 or 1;

[0257] Each p is either 1 or 2;

[0258] R b and R c Each of is independently selected from hydrogen, alkyl, alkoxycarbonyl, alkanoyl, aroyl or sulfonyl; and

[0259] R 2c , R 3c , R 4c , R 5c and R 6c Each of, if present, is independently selected from alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl, wherein alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl are optionally substituted with one or more groups independently selected from halogen, hydroxy, oxo, and cyano, and alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl are optionally interrupted by one or more groups independently selected from cycloalkyl, heterocyclyl, aryl, and heteroaryl.

[0260] In some embodiments, the compound has the following formula (A):

[0261]

[0262] in

[0263] R 2 for

[0264] R 3 for

[0265] R 4 for

[0266] Each W is independently selected from O, S or NR b ;

[0267] Each Y is independently selected from O, S, NR c 、N(R c )Z(W), N(R c )N(R c ) or N(R c )N(R c )Z(W);

[0268] Each Z is independently selected from C, S or S(O);

[0269] Each m is independently 0 or 1;

[0270] Each p is independently 1 or 2;

[0271] Q is 1, 2 or 3;

[0272] R b and R c each independently selected from hydrogen, alkyl, alkoxycarbonyl, alkanoyl, aroyl or sulfonyl; and

[0273] R 2c 、R 3c and R 4c each independently selected from alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl and heteroalkynyl, wherein the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl and heteroalkynyl are optionally substituted with one or more groups independently selected from halogen, hydroxy, oxo and cyano, and the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl and heteroalkynyl are optionally interrupted with one or more groups independently selected from cycloalkyl, heterocyclic, aryl and heteroaryl.

[0274] In some embodiments, the compound has the formula (B), formula (C), formula (D) or formula (E):

[0275]

[0276] wherein

[0277] each W is independently selected from O, S or NR b ;

[0278] each Y is independently selected from O, S, NR c , N(R c )Z(W), N(R c )N(R c ) or N(R c )N(R c )Z(W);

[0279] each Z is independently selected from C, S or S(O);

[0280] each n is independently 0, 1, 2, 3, 4 or 5;

[0281] each m is independently 0, 1, 2 or 3;

[0282] each p is independently 1, 2, 3 or 4; and

[0283] R b and R c each independently selected from hydrogen, alkyl, alkoxycarbonyl, acyl or sulfonyl.

[0284] In some embodiments, each is independently selected from

[0285] In some embodiments, R 1b 、R 2b 、R 3b 、R 4b 、R 5b and R 6b are independently selected from

[0286] In some embodiments, one or more of R 1b 、R 2b 、R 3b 、R 4b 、R 5b and R 6b are independently selected from

[0287] In some embodiments, one or more of R 1c 、R 2c 、R 3c 、R 4c 、R 5c and R 6c are independently selected from alkyl, alkenyl, and alkynyl, where the alkyl, alkenyl, and alkynyl are optionally substituted with one or more halogens.

[0288] In some embodiments, one or more of R 1c 、R 2c 、R 3c 、R 4c 、R 5c and R 6c are independently selected from -CH2-O-alkyl, -CH2-O-alkenyl, -CH2-O-alkynyl, -CH2-O-heteroalkyl, -CH2-O-heteroalkenyl, and -CH2-O-heteroalkynyl.

[0289] In some embodiments, each of the alkyl, alkenyl, and alkynyl independently contains 6 to 18 carbon atoms.

[0290] In some embodiments, each alkenyl independently contains one or more Z-olefins.

[0291] In some embodiments, one or more of R 1c 、R 2c 、R 3c 、R 4c 、R 5c and R 6c are independently selected from

[0292]

[0293]

[0294] In some embodiments, R 1 、R 2 、R 3 、R 4 、R 5 and R 6 one or more of which are independently selected from

[0295]

[0296]

[0297]

[0298]

[0299] In some embodiments, -R 1b -R 1c 、-R 2b -R 2c 、-R 3b -R 3c 、-R 4b -R 4c 、-R 5b -R 5c and -R 6b -R 6c each, if present, does not contain two heteroatoms directly bonded to each other.

[0300] In some embodiments, -R 1b -R 1c 、-R 2b -R 2c 、-R 3b -R 3c 、-R 4b -R 4c 、-R 5b -R 5c and -R 6b -R 6c one or more of which, if present, contain independently selected from -N(R c )-N(R c )-, -N(R c )-S(O)-, -N(R c )-S(O)2-, -N(R c)-O-, -S(O)-N(R c )-, -S(O)2-N(R c )-, -S(O)-O-, -S(O)2-O-, -O-N(R c )-, -O-S(O)-, -O-S(O)2- and -O-O- groups.

[0301] In some embodiments, -R 1b -R 1c , -R 2b -R 2c , -R 3b -R 3c , -R 4b -R 4c , -R 5b -R 5c and -R 6b -R 6c one or more of, if present, contain -N(R c )-N(R c )- or -S(O)2-N(R c ).

[0302] In some embodiments, the compounds having formula (I), formula (A), formula (B), formula (C), formula (D) or formula (E) are the compounds listed in Table 1.

[0303] For illustrative purposes, exemplary compounds of the present disclosure and their structure codes are shown in Table 1 below.

[0304] Table 1 Exemplary Compounds and Their Structure Codes

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311] The compounds provided herein are described with reference to both general formulas and specific compounds. In addition, the compounds of the present disclosure can exist in a variety of different forms or derivatives, including but not limited to, stereoisomers, racemic mixtures, regioisomers, tautomers, salts, prodrugs, soft drugs, active metabolite derivatives (active metabolites), solvated forms, different crystal forms or polymorphs, all of which are within the scope of the present disclosure.

[0312] The compounds of the present disclosure may contain one or more asymmetric centers and thus can exist in various stereoisomeric forms, such as enantiomers and / or diastereomers. Accordingly, the compounds of the present disclosure and their compositions can be in the form of a single enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers. In certain embodiments, the compounds of the present disclosure are enantiomerically pure compounds. In certain embodiments, a mixture of enantiomers or diastereomers is provided.

[0313] The term "enantiomer" refers to two stereoisomers of a compound that are mirror images of each other and are non-superimposable. The term "diastereomer" refers to a pair of optical isomers that are not mirror images of each other. Diastereomers have different physical properties, such as melting point, boiling point, spectral properties and reactivity.

[0314] In addition, certain compounds as described herein can have one or more double bonds, which can exist as Z or E isomers, unless otherwise specified. The present disclosure also encompasses compounds as individual isomers substantially free of other isomers or as mixtures of various isomers such as racemic mixtures of enantiomers. In addition to the compounds themselves described above, the present disclosure also encompasses compositions comprising one or more compounds.

[0315] As used herein, the term "isomer" includes any and all geometric and stereoisomers. For example, "isomer" includes cis and trans isomers, E- and Z-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, their racemic mixtures, and other mixtures thereof, all of which fall within the scope of the present invention. For example, in some embodiments, a stereoisomer can be provided substantially free of one or more corresponding stereoisomers and can also be referred to as "stereochemically enriched".

[0316] In the case of preferring a specific enantiomer, in some embodiments, the enantiomer can be provided as being substantially free of the opposite enantiomer and can also be referred to as "optically enriched". As used herein, "optically enriched" means that a compound consists of a significantly greater proportion of one enantiomer. In certain embodiments, the compound consists of at least about 90% by weight of the preferred enantiomer. In other embodiments, the compound consists of at least about 95%, 98% or 99% by weight of the preferred enantiomer. The preferred enantiomer can be separated from a racemic mixture by any method known to those skilled in the art (including chiral high performance liquid chromatography (HPLC) and the formation and crystallization of chiral salts), or can be prepared by asymmetric synthesis. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen, S.H. et al., Tetrahedron 33:2725 (1977); Eliel, E.L., Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); Wilen, S.H., Tables of Resolving Agents and Optical Resolutions, page 268 (edited by E.L. Eliel, University of Notre Dame Press, University of Notre Dame, 1972).

[0317] The compounds of the present disclosure can also exist in different tautomeric forms, and all such forms are included within the scope of the present disclosure. The terms "tautomer" or "tautomeric form" refer to structural isomers of different energies that can be interconverted by a low energy barrier. The existence and concentration of the isomeric forms depend on the environment in which the compound is located and may vary, for example, depending on whether the compound is a solid or in an organic or aqueous solution. For example, proton tautomers (also known as prototropic tautomers) include interconversions by proton migration, such as keto-enol, amide-imino acid, lactam-lactim, imine-enamine isomerization and cyclic forms, where the proton can occupy two or more positions in the heterocyclic system. Valence tautomers include interconversions that occur through the reorganization of some bonding electrons. Tautomers can be in equilibrium or can be stereochemically locked into one form by appropriate substitution. Unless otherwise stated, a compound identified by name or structure as a specific tautomeric form in the present disclosure is intended to include other tautomeric forms.

[0318] The present disclosure also aims to include all isotopes of atoms in the compounds. Isotopes of an atom include atoms having the same atomic number but different mass numbers. For example, unless otherwise specified, hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, bromine, or iodine in the compounds of the present disclosure also means to include their isotopes, such as but not limited to 1 H, 2 H, 3 H, 11 C, 12 C, 13 C, 14 C, 14 N, 15 N, 16 O, 17 O, 18 O, 31 P, 32 P, 32 S, 33 S, 34 S, 36 S, 17 F, 18 F, 19 F, 35 Cl, 37 Cl, 79 Br, 81 Br, 124 I, 127 I and 131 I. In some embodiments, hydrogen includes protium, deuterium, and tritium. In some embodiments, carbon includes 12 C and 13 C.

[0319] Synthesis of Compounds

[0320] The synthesis of the compounds provided herein (including their pharmaceutically acceptable salts) is illustrated in the synthetic schemes in the Examples. The compounds provided herein can be prepared using any known organic synthesis techniques and can be synthesized according to any of many possible synthetic routes. Therefore, these schemes are merely illustrative and do not mean to limit other possible methods that can be used to prepare the compounds provided herein. Additionally, the steps in the schemes are for better illustration and can be appropriately changed. The embodiments of the compounds in the Examples were synthesized for research purposes and for possible submission to regulatory agencies.

[0321] The reactions for preparing the compounds of the present disclosure can be carried out in a suitable solvent, which can be readily selected by those skilled in the art of organic synthesis. A suitable solvent can substantially not react with the starting materials (reactants), intermediates or products at the temperature at which the reaction is carried out (e.g., a temperature within the range from the freezing temperature to the boiling temperature of the solvent). A given reaction can be carried out in one solvent or a mixture of more than one solvent. Those skilled in the art can select a suitable solvent for a particular reaction step according to the specific reaction step.

[0322] The preparation of the compounds of the present disclosure may involve the protection and deprotection of various chemical groups. Those skilled in the art can readily determine whether protection and deprotection are needed and select appropriate protecting groups. For the chemistry of protecting groups, see, for example, T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 3rd Edition, Wiley & Sons, Inc., New York (1999), which reference is incorporated herein by reference in its entirety.

[0323] The reaction can be monitored according to any suitable method known in the art. For example, it can be monitored by spectroscopic means such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C), infrared spectroscopy, spectrophotometry (e.g., ultraviolet-visible light), mass spectrometry, or by chromatographic methods such as high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LCMS) or thin layer chromatography (TLC) to monitor the formation of the product. The compounds can be purified by those skilled in the art by various methods, including high performance liquid chromatography (HPLC) (“Preparative LC-MS Purification: Improved Compound Specific Method Optimization” Karl F. Blom, Brian Glass, Richard Sparks, Andrew P. Combs J. Combi. Chem. 2004, 6(6), 874 - 883, which reference is incorporated herein by reference in its entirety) and normal phase silica gel chromatography.

[0324] The structures of the compounds in the examples are characterized by nuclear magnetic resonance (NMR) or / and liquid chromatography-mass spectrometry (LC-MS). The NMR chemical shifts (δ) are in 10 -6Given in units of (ppm). Recorded on a Bruker instrument (400 MHz or 500 MHz) in CDCl3, CDOD3 or DMSO-d6 solution 1 1H-NMR spectra (reported in ppm), using tetramethylsilane (TMS) as the reference standard (0.0 ppm).

[0325] In one aspect, the present disclosure provides a method for preparing a compound of formula (I), formula (A), formula (B), formula (C), formula (D) or formula (E) or a pharmaceutically acceptable salt thereof, the method comprising:

[0326] A) Mixing a compound of formula (II) and a compound of formula (III):

[0327]

[0328] wherein,

[0329] each R 7 is independently selected from hydrogen, alkyl, heteroalkyl, haloalkyl and hydroxyalkyl;

[0330] each L a 、L b 、L c 、R 1b and R 1c have the same definitions as above; and

[0331] B) Refluxing the mixture until the reaction is complete to obtain a compound of formula (I), formula (A), formula (B), formula (C), formula (D) or formula (E).

[0332] In one aspect, the present disclosure provides a method for preparing a compound of formula (I), formula (A), formula (B), formula (C), formula (D) or formula (E) or a pharmaceutically acceptable salt thereof, the method comprising:

[0333] i) Mixing a compound of formula (II) and a compound of formula (IV):

[0334]

[0335] wherein R 1c has the same definition as above;

[0336] ii) Mixing the product of step (i) with a compound of formula (III); and

[0337] iii) Refluxing the mixture until the reaction is complete to obtain a compound of formula (I), formula (A), formula (B), formula (C), formula (D) or formula (E).

[0338] In some embodiments, the compounds having formula (IV) are selected from

[0339]

[0340] In some embodiments, the compounds having formula (II) are selected from

[0341] In some embodiments, the compounds having formula (III) are selected from

[0342]

[0343]

[0344] In some embodiments, the compounds having formula (III) are selected from:

[0345]

[0346]

[0347]

[0348] In some embodiments, the method further comprises a method for preparing a compound having formula (II) from a compound having formula (V), wherein L b is -NR a - and R a is not hydrogen:

[0349]

[0350] wherein R a is not hydrogen.

[0351] In some embodiments, the method further comprises a method for preparing a compound having formula (V) from a compound having formula (VI), wherein R a is not hydrogen:

[0352]

[0353] In some embodiments, the method further comprises a method for preparing a compound having formula (VI) from a compound having formula (VII):

[0354]

[0355] Unless otherwise specified, the reactions of the present disclosure are generally carried out under a positive pressure of nitrogen or argon or in an anhydrous solvent with a drying tube, and the reaction flask is usually equipped with a rubber septum for introducing substrates and reagents by syringe. Glassware is dried in an oven and / or by heat.

[0356] Lipid Particle

[0357] The present disclosure also provides lipid particles comprising one or more of the above lipids. The lipid particles include, but are not limited to, lipid nanoparticles (LNPs), liposomes, lipid complexes, and lipid polyplexes (LPPs). In some embodiments, the lipid particles are lipid nanoparticles. The present disclosure also provides methods for preparing lipid particles.

[0358] The lipid particles of the present disclosure may further comprise one or more additional lipids and / or other components such as sterols. Other lipids may be included in the lipid particles of the present disclosure for various purposes, such as preventing lipid oxidation or attaching ligands to the particle surface. Any lipid can be present in the lipid particles of the present disclosure, including amphiphilic, neutral, cationic, and anionic lipids, which can be used alone or in combination. Examples of additional lipid components that can be present are described below.

[0359] In some embodiments, the lipid particles comprise a lipid of the present disclosure (e.g., a compound having formula (I), formula (A), formula (B), formula (C), formula (D), or formula (E), or any compound in Table 1). In some embodiments, the lipid particles comprise two or more lipids of the present disclosure. In some embodiments, the mole fraction of the lipid of the present disclosure (e.g., a compound having formula (I), formula (A), formula (B), formula (C), formula (D), or formula (E), or any compound in Table 1) is about 5 to 75%, about 10 to 75%, about 15 to 75%, about 20 to 75%, about 25 to 75%, about 30 to 75%, about 35 to 75%, about 40 to 75%, about 10 to 70%, about 15 to 70%, about 20 to 70%, about 25 to 70%, about 30 to 70%, about 35 to 70%, about 40 to 70%, about 10 to 65%, about 15 to 65%, about 20 to 65%, about 25 to 65%, about 30 to 65%, about 35 to 65%, about 40 to 65%, about 45 to 65%, about 10 to 60%, about 15 to 60%, about 20 to 60%, about 25 to 60%, about 30 to 60%, about 35 to 60%, about 40 to 60%, about 45 to 60%, about 10 to 55%, about 15 to 55%, about 20 to 55%, about 25 to 55%, about 30 to 55%, about 35 to 55%, about 40 to 55%, about 10 to 50%, about 15 to 50%, about 20 to 50%, about 25 to 50%, about 30 to 50%, about 35 to 50%, about 40 to 50%, about 10 to 45%, about 15 to 45%, about 20 to 45%, about 25 to 45%, about 30 to 45%, about 35 to 45%, about 40 to 45%, about 10 to 40%, about 15 to 40%, about 20 to 40%, about 25 to 40%, about 30 to 40%, about 35 to 40%, about 45 to 50%, about 45 to 55%, about 45 to 60%, about 45 to 65%, about 45 to 70%, about 45 to 75%, about 50 to 55%, about 50 to 60%, about 55 to 65%, about 55 to 70%, about 55 to 75%, about 55 to 60%, about 55 to 65%, about 55 to 70%, about 55 to 75%, about 60 to 65%, about 60 to 70%, or about 60 to 75% of the total lipids present in the lipid particles. In some embodiments, the mole fraction of the lipid of the present disclosure (e.g., a compound having formula (I), formula (A), formula (B), formula (C), formula (D), or formula (E), or any compound in Table 1) is about 10% to 75%, about 40% to 75%, or about 40% to 70% of the total lipids present in the lipid particles. In some embodiments, the mole fraction of the lipid of the present disclosure (e.g., a compound having formula (I), formula (A), formula (B), formula (C), formula (D), or formula (E), or any compound in Table 1) is about 40 - 50% of the total lipids present in the lipid particles.In some embodiments, the mole fraction of the lipids of the present disclosure (e.g., a compound having Formula (I), Formula (A), Formula (B), Formula (C), Formula (D), or Formula (E), or any compound of Table 1) is about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 64%, 65%, 66%, 67%, 68%, 69%, or 70% of the total lipids present in the lipid particles.

[0360] In some embodiments, the lipid particles comprise neutral lipids. The term "neutral lipid" refers to any one of a number of lipid species that exist in an uncharged or neutral zwitterionic form at a selected pH. In some embodiments, the neutral lipid is a phospholipid. Examples of phospholipids include, but are not limited to, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphate-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, and any mixture thereof. In some embodiments, the lipid particles comprise one neutral lipid. In some embodiments, the lipid particles comprise two or more neutral lipids. In some embodiments, the neutral lipid is selected from 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and any mixture thereof. In some embodiments, the mole fraction of the neutral lipid is about 1 to 40%.In some embodiments, the mole fraction of neutral lipid is about 1 to 35%, about 5 to 30%, about 5 to 25%, about 5 to 20%, about 5 to 15%, about 4 to 15%, about 3 to 15%, about 2 to 15%, about 1 to 15%, about 5 to 10%, about 4 to 10%, about 3 to 10%, about 2 to 10% or about 1 to 10% of the total lipids present in the lipid particle. In some embodiments, the mole fraction of neutral lipid is about 1 to 35%, about 2 to 15% or about 3 to 15% of the total lipids present in the lipid particle. In some embodiments, the mole fraction of neutral lipid is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 15%, 20%, 25% or 30% of the total lipids present in the lipid particle.

[0361] In some embodiments, the lipid particles comprise a structured lipid. In some embodiments, the structured lipid is a sterol, a sterol derivative, or any mixture thereof. Examples of sterols or sterol derivatives include, but are not limited to, cholesterol, coprosterol, sitosterol, β-sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, α-tocopherol, and mixtures thereof. In some embodiments, the structured lipid is cholesterol. In some embodiments, the structured lipid comprises cholesterol and a corticosteroid (such as prednisolone, dexamethasone, prednisone, and hydrocortisone) or a combination thereof. In some embodiments, the lipid particles comprise cholesterol. In some embodiments, the mole fraction of the structured lipid is about 5 to 50% of the total lipids present in the lipid particles. In some embodiments, the mole fraction of the structured lipid is about 10 to 50%, about 15 to 50%, about 20 to 50%, about 21 to 50%, about 22 to 50%, about 23 to 50%, about 24 to 50%, about 25 to 50%, about 26 to 50%, about 27 to 50%, about 28 to 50%, about 29 to 50%, about 30 to 50%, about 31 to 50%, about 32 to 50%, about 33 to 50%, about 34 to 50%, about 35 to 50%, about 36 to 50%, about 37 to 50%, about 38 to 50%, about 39 to 50%, or about 40 to 50% of the total lipids present in the lipid particles. In some embodiments, the mole fraction of the structured lipid is about 10 to 45%, about 15 to 45%, about 25 to 45%, about 30 to 45%, or about 35 to 45% of the total lipids present in the lipid particles. In some embodiments, the mole fraction of the structured lipid is about 15% to 50%, about 20% to 50%, or about 22% to 50% of the total lipids present in the lipid particles. In some embodiments, the mole fraction of the structured lipid is about 20%, 20.1%, 20.2%, 20.3%, 20.4%, 20.5%, 20.6%, 20.7%, 20.8%, 20.9%, 21%, 21.1%, 21.2%, 21.3%, 21.4%, 21.5%, 21.6%, 21.7%, 21.8%, 21.9%, 22%, 22.1%, 22.2%, 22.3%, 22.4%, 22.5%, 22.6%, 22.7%, 22.8%, 22.9%, 23%, 23.1%, 23.2%, 23.3%, 23.4%, 23.5%, 23.6%, 23.7%, 23.8%, 23.9%, 24%, 24.1%, 24.2%, 24.3%, 24.4%, 24.5%, 24.6%, 24.7%, 24.8%, 24.9%, 25%, 25.1%, 25.2%, 25.3%, 25.4%, 25.5%, 25.6%, 25.7%, 25.8%, 25.9％、26％、26.1％、26.2％、26.3％、26.4％、26.5％、26.6％、26.7％、26.8％、26.9％、27％、27.1％、27.2％、27.3％、27.4％、27.5％、27.6％、27.7％、27.8％、27.9％、28％、28.1％、28.2％、28.3％、28.4％、28.5％、28.6％、28.7％、28.8％、28.9％、29％、29.1％、29.2％、29.3％、29.4％、29.5％、29.6％、29.7％、29.8％、29.9％、30％、30.1％、30.2％、30.3％、30.4％、30.5％、30.6％、30.7％、30.8％、30.9％、31％、31.1％、31.2％、31.3％、31.4％、31.5％、31.6％、31.7％、31.8％、31.9％、32％、32.1％、32.2％、32.3％、32.4％、32.5％、32.6％、32.7％、32.8％、32.9％、33％、33.1％、33.2％、33.3％、33.4％、33.5％、33.6％、33.7％、33.8％、33.9％、34％、34.1％、34.2％、34.3％、34.4％、34.5％、34.6％、34.7％、34.8％、34.9％、35％、35.1％、35.2％、35.3％、35.4％、35.5％、35.6％、35.7％、35.8％、35.9％、36％、36.1％、36.2％、36.3％、36.4％、36.5％、36.6％、36.7％、36.8％、36.9％、37％、37.1％、37.2％、37.3％、37.4％、37.5％、37.6％、37.7％、37.8％、37.9％、38％、38.1％、38.2％、38.3％、38.4％、38.5％、38.6％、38.7％、38.8％、38.9％、39％、39.1％、39.2％、39.3％、39.4％、39.5％、39.6％、39.7％、39.8％、39.9％、40％、40.1％、40.2％、40.3％、40.4％、40.5％、40.6％、40.7％、40.8％、40.9％、41％、41.1％、41.2％、41.3％、41.4％、41.5％、41.6％、41.7％、41.8％、41.9％、42％、42.1％、42.2％、42.3％、42.4％、42.5％、42.6％、42.7％、42.8％、42.9％、43％、43.1%, 43.2%, 43.3%, 43.4%, 43.5%, 43.6%, 43.7%, 43.8%, 43.9%, 44%, 44.1%, 44.2%, 44.3%, 44.4%, 44.5%, 44.6%, 44.7%, 44.8%, 44.9%, 45%, 45.1%, 45.2%, 45.3%, 45.4%, 45.5%, 45.6%, 45.7%, 45.8%, 45.9%, 46%, 46.1%, 46.2%, 46.3%, 46.4%, 46.5%, 46.6%, 46.7%, 46.8%, 46.9%, 47%, 47.1%, 47.2%, 47.3%, 47.4%, 47.5%, 47.6%, 47.7%, 47.8%, 47.9%, 48%, 48.1%, 48.2%, 48.3%, 48.4%, 48.5%, 48.6%, 48.7%, 48.8%, 48.9%, 49%, 49.1%, 49.2%, 49.3%, 49.4%, 49.5%, 49.6%, 49.7%, 49.8%, 49.9%, 50%, 50.1%, 50.2%, 50.3%, 50.4%, 50.5%, 50.6%, 50.7%, 50.8%, 50.9%, 51%, 51.1%, 51.2%, 51.3%, 51.4%, 51.5%, 51.6%, 51.7%, 51.8%, 51.9%, 52%, 52.1%, 52.2%, 52.3%, 52.4%, 52.5%, 52.6%, 52.7%, 52.8%, 52.9%, 53%, 53.1%, 53.2%, 53.3%, 53.4%, 53.5%, 53.6%, 53.7%, 53.8%, 53.9%, 54%, 54.1%, 54.2%, 54.3%, 54.4%, 54.5%, 54.6%, 54.7%, 54.8%, 54.9% or 55%.

[0362] In some embodiments, the lipid particles comprise lipids selected to reduce aggregation of the lipid particles during formation, which can be achieved by steric stabilization of the particles to prevent charge-induced aggregation during formation. Examples of lipids that reduce aggregation of the particles during formation include, but are not limited to, polyethylene glycol (PEG)-modified lipids, monosialoganglioside Gm1, and polyamide oligomers (PAO). Other compounds having uncharged, hydrophilic, steric moieties such as PEG or Gm1 that prevent aggregation during formulation can also be conjugated to the lipids for use in the methods and compositions of the present disclosure. In some embodiments, the lipid that reduces aggregation of the particles during formation is a surfactant. In some embodiments, the surfactant is a PEG-modified lipid. Examples of PEG-modified lipids include, but are not limited to, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. In some embodiments, the PEG-modified lipid is 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol (DMG-PEG), 1,2-distearoyl-rac-glycerol-3-methoxypolyethylene glycol (DSG-PEG), N-(methylpolyoxyethylene-carbonyl)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (PEG-DSPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)] (DOPE-PEG), or any mixture thereof. Generally, the mole fraction of the lipid component selected to reduce aggregation is about 1 to 15% of the total lipids present in the lipid particles. In some embodiments, the mole fraction of the lipid component selected to reduce aggregation is about 1 to 10%, about 1 to 7%, about 1 to 5%, or about 0.5 to 5% of the total lipids present in the lipid particles. In some embodiments, the mole fraction of the lipid component selected to reduce aggregation is about 1 to 5%, about 1 to 2.5%, or about 1.5 to 2% of the total lipids present in the lipid particles. In some embodiments, the mole fraction of the lipid component selected to reduce aggregation is about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, or 4% of the total lipids present in the lipid particles.

[0363] As used herein, the term "PEG", whether as part of another term or used alone, refers to polyethylene glycol. The term "PEGm" or "PEG-m" (where m is an integer) refers to a polyethylene glycol molecule or moiety having a molecular weight of m. For example, "PEG2000" or "PEG-2000" refers to a polyethylene glycol molecule or moiety having a molecular weight of 2000. In some embodiments, the PEG is PEG700, PEG800, PEG900, PEG1000, PEG1100, PEG1200, PEG1300, PEG1400, PEG1500, PEG1600, PEG1700, PEG1800, PEG1900, PEG2000, PEG2100, PEG2200, PEG2300, PEG2400, PEG2500, PEG2600, PEG2700, PEG2800, PEG2900 or PEG3000. In some embodiments, the PEG is PEG2000.

[0364] In some embodiments, based on the total lipids present in the lipid particle, the lipid particles of the present disclosure comprise: a compound having formula (I), formula (A), formula (B), formula (C), formula (D) or formula (E) in a mole fraction of about 10-65%; a neutral lipid of the present disclosure in a mole fraction of about 5-30%; a structural lipid of the present disclosure in a mole fraction of about 15-50%; and a lipid component of the present disclosure selected to reduce aggregation in a mole fraction of about 0.5-5%.

[0365] In some embodiments, based on the total lipids present in the lipid particle, the lipid particles of the present disclosure comprise: a compound having formula (I), formula (A), formula (B), formula (C), formula (D) or formula (E) in a mole fraction of about 20-65%; a neutral lipid of the present disclosure in a mole fraction of about 5-25%; a structural lipid of the present disclosure in a mole fraction of about 25-50%; and a lipid component of the present disclosure selected to reduce aggregation in a mole fraction of about 1-5%.

[0366] In some embodiments, based on the total lipids present in the lipid particle, the lipid particles of the present disclosure comprise: a compound having formula (I), formula (A), formula (B), formula (C), formula (D) or formula (E) in a mole fraction of about 40-65%; a neutral lipid of the present disclosure in a mole fraction of about 5-15%; a structural lipid of the present disclosure in a mole fraction of about 25-50%; and a lipid component of the present disclosure selected to reduce aggregation in a mole fraction of about 1-2.5%.

[0367] In some embodiments, based on the total lipids present in the lipid particles, the lipid particles of the present disclosure comprise: a compound having formula (I), formula (A), formula (B), formula (C), formula (D), or formula (E) at a mole fraction of about 40 - 50%; a neutral lipid of the present disclosure at a mole fraction of about 5 - 15%; a structural lipid of the present disclosure at a mole fraction of about 40 - 50%; and a lipid component of the present disclosure selected to reduce aggregation at a mole fraction of about 1.5 - 2%.

[0368] In some embodiments, based on the total lipids present in the lipid particles, the lipid particles of the present disclosure comprise: a compound having formula (I), formula (A), formula (B), formula (C), formula (D), or formula (E) at a mole fraction of about 10 - 75%; a neutral lipid of the present disclosure at a mole fraction of about 1 - 35%; a structural lipid of the present disclosure at a mole fraction of about 15 - 50%; and a lipid component of the present disclosure selected to reduce aggregation at a mole fraction of about 0.5 - 5%.

[0369] In some embodiments, based on the total lipids present in the lipid particles, the lipid particles of the present disclosure comprise: a compound having formula (I), formula (A), formula (B), formula (C), formula (D), or formula (E) at a mole fraction of about 40 - 75%; a neutral lipid of the present disclosure at a mole fraction of about 2 - 15%; a structural lipid of the present disclosure at a mole fraction of about 20 - 50%; and a lipid component of the present disclosure selected to reduce aggregation at a mole fraction of about 1 - 5%.

[0370] In some embodiments, based on the total lipids present in the lipid particles, the lipid particles of the present disclosure comprise: a compound having formula (I), formula (A), formula (B), formula (C), formula (D), or formula (E) at a mole fraction of about 40 - 70%; a neutral lipid of the present disclosure at a mole fraction of about 3 - 15%; a structural lipid of the present disclosure at a mole fraction of about 20 - 50%; and a lipid component of the present disclosure selected to reduce aggregation at a mole fraction of about 1 - 2.5%.

[0371] In some embodiments, based on the total lipids present in the lipid particles, the lipid particles of the present disclosure comprise: a compound having formula (I), formula (A), formula (B), formula (C), formula (D), or formula (E) at a mole fraction of about 40 - 70%; a neutral lipid of the present disclosure at a mole fraction of about 3 - 15%; a structural lipid of the present disclosure at a mole fraction of about 22 - 50%; and a lipid component of the present disclosure selected to reduce aggregation at a mole fraction of about 1.5 - 2.5%.

[0372] In some embodiments, based on the total lipids present in the lipid particles, the lipid particles of the present disclosure comprise:

[0373] Compounds of formula (I), formula (A), formula (B), formula (C), formula (D) or formula (E) having a molar fraction of about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or 70%;

[0374] Neutral lipids of the present disclosure having a molar fraction of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 15%, 20%, 25% or 30%;

[0375] The mole fractions are approximately 20%, 20.1%, 20.2%, 20.3%, 20.4%, 20.5%, 20.6%, 20.7%, 20.8%, 20.9%, 21%, 21.1%, 21.2%, 21.3%, 21.4%, 21.5%, 21.6%, 21.7%, 21.8%, 21.9%, 22%, 22.1%, 22.2%, 22.3%, 22.4%, 22.5%, 22.6%, 22.7%, 22.8%, 22.9%, 23%, 23.1%, 23.2%, 23.3%, 23.4%, 23.5%, 23.6%, 23.7%, 23.8%, 23.9%, 24%, 24.1%, 24.2%, 24.3%, 24.4%, 24.5%, 24.6%, 24.7%, 24.8%, 24.9%, 25%, 25.1%, 25.2%, 25.3%, 25.4%, 25.5%, 25.6%, 25.7%, 25.8%, 25.9%, 26%, 26.1%, 26.2%, 26.3%, 26.4%, 26.5%, 26.6%, 26.7%, 26.8%, 26.9%, 27%, 27.1%, 27.2%, 27.3%, 27.4%, 27.5%, 27.6%, 27.7%, 27.8%, 27.9%, 28%, 28.1%, 28.2%, 28.3%, 28.4%, 28.5%, 28.6%, 28.7%, 28.8%, 28.9%, 29%, 29.1%, 29.2%, 29.3%, 29.4%, 29.5%, 29.6%, 29.7%, 29.8%, 29.9%, 30%, 30.1%, 30.2%, 30.3%, 30.4%, 30.5%, 30.6%, 30.7%, 30.8%, 30.9%, 31%, 31.1%, 31.2%, 31.3%, 31.4%, 31.5%, 31.6%, 31.7%, 31.8%, 31.9%, 32%, 32.1%, 32.2%, 32.3%, 32.4%, 32.5%, 32.6%, 32.7%, 32.8%, 32.9%, 33%, 33.1%, 33.2%, 33.3%, 33.4%, 33.5%, 33.6%, 33.7%, 33.8%, 33.9%, 34%, 34.1%, 34.2%, 34.3%, 34.4%, 34.5%, 34.6%, 34.7%, 34.8%, 34.9%, 35%, 35.1%, 35.2%, 35.3%, 35.4%, 35.5%, 35.6%, 35.7%, 35.8%, 35.9%, 36%, 36.1%, 36.2%, 36.3%, 36.4%, 36.5%, 36.6%, 36.7%, 36.8%, 36.9%, 37%, 37.1％、37.2％、37.3％、37.4％、37.5％、37.6％、37.7％、37.8％、37.9％、38％、38.1％、38.2％、38.3％、38.4％、38.5％、38.6％、38.7％、38.8％、38.9％、39％、39.1％、39.2％、39.3％、39.4％、39.5％、39.6％、39.7％、39.8％、39.9％、40％、40.1％、40.2％、40.3％、40.4％、40.5％、40.6％、40.7％、40.8％、40.9％、41％、41.1％、41.2％、41.3％、41.4％、41.5％、41.6％、41.7％、41.8％、41.9％、42％、42.1％、42.2％、42.3％、42.4％、42.5％、42.6％、42.7％、42.8％、42.9％、43％、43.1％、43.2％、43.3％、43.4％、43.5％、43.6％、43.7％、43.8％、43.9％、44％、44.1％、44.2％、44.3％、44.4％、44.5％、44.6％、44.7％、44.8％、44.9％、45％、45.1％、45.2％、45.3％、45.4％、45.5％、45.6％、45.7％、45.8％、45.9％、46％、46.1％、46.2％、46.3％、46.4％、46.5％、46.6％、46.7％、46.8％、46.9％、47％、47.1％、47.2％、47.3％、47.4％、47.5％、47.6％、47.7％、47.8％、47.9％、48％、48.1％、48.2％、48.3％、48.4％、48.5％、48.6％、48.7％、48.8％、48.9％、49％、49.1％、49.2％、49.3％、49.4％、49.5％、49.6％、49.7％、49.8％、49.9％、50％、50.1％、50.2％、50.3％、50.4％、50.5％、50.6％、50.7％、50.8％、50.9％、51％、51.1％、51.2％、51.3％、51.4％、51.5％、51.6％、51.7％、51.8％、51.9％、52％、52.1％、52.2％、52.3％、52.4％、52.5％、52.6％、52.7％、52.8％、52.9％、53％、53.1％、53.2％、53.3％、53.4％、53.5％、53.6％、53.7％、53.8％、53.9％、54％、54.1％、54.2％、54.3%, 54.4%, 54.5%, 54.6%, 54.7%, 54.8%, 54.9% or 55% of the structured lipids of the present disclosure;.

[0376] A lipid component of the present disclosure selected to reduce aggregation in a molar fraction of about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9% or 4%.

[0377] In some embodiments, the lipid particles of the present disclosure comprise components in the molar fractions listed in the following table.

[0378]

[0379] In some embodiments, the lipid particles include a targeting moiety. In some embodiments, the targeting moiety is conjugated to the lipid particle. In some embodiments, the targeting moiety is conjugated to the lipid particle directly or via a linker. In some embodiments, the targeting moiety is an antibody or a ligand.

[0380] Lipid particles can be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy (TEM) or scanning electron microscopy (SEM)) can be used to examine the morphology and size distribution of lipid particle compositions. Dynamic light scattering (DLS) or potentiometry (e.g., potentiometric titration) can be used to measure the ζ potential. DLS can also be used to determine particle size. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can also be used to measure various characteristics of lipid particles, such as particle size, polydispersity index, and ζ potential. The DLS measurement error may depend on various factors such as the scattering angle and multiple scattering. The typical error of DLS measurement is 5%.

[0381] The average size of the lipid particles can be between tens of nanometers and hundreds of nanometers, e.g., as measured by DLS. For example, the average size of the lipid particles can be from about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the average size of the lipid particles can be from about 50 nm to about 100 nm, from about 50 nm to about 90 nm, from about 50 nm to about 80 nm, from about 50 nm to about 70 nm, from about 50 nm to about 60 nm, from about 60 nm to about 100 nm, from about 60 nm to about 90 nm, from about 60 nm to about 80 nm, from about 60 nm to about 70 nm, from about 70 nm to about 100 nm, from about 70 nm to about 90 nm, from about 70 nm to about 80 nm, from about 80 nm to about 100 nm, from about 80 nm to about 90 nm, or from about 90 nm to about 100 nm. In some embodiments, the average size of the lipid particles can be from about 70 nm to about 100 nm. In some embodiments, the average size of the lipid particles can be about 80 nm. In some embodiments, the average size of the lipid particles can be about 100 nm.

[0382] The lipid particles can be relatively uniform. The polydispersity index (PDI) can be used to indicate the uniformity of the lipid particles, e.g., the particle size distribution of the lipid particles. A smaller PDI (e.g., less than 0.3) generally indicates a narrower particle size distribution. In some embodiments, the lipid particles of the present disclosure can have a PDI of from about 0 to about 0.30, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, or 0.30. In some embodiments, the PDI of the lipid particles of the present disclosure can be from about 0.05 to about 0.20.

[0383] The ζ potential of lipid particles can be used to indicate the electromotive force. For example, the ζ potential can describe the surface charge of lipid particles. Lipid particles with relatively low charge (positive or negative) are generally desirable because substances with higher charge may have undesirable interactions with cells, tissues, and other elements in the body. In some embodiments, the ζ potential of the lipid particles of the present disclosure can be from about -10 mV to about +25 mV, from about -10 mV to about +20 mV, from about -10 mV to about +15 mV, from about -10 mV to about +10 mV, from about -10 mV to about +5 mV, from about -10 mV to about 0 mV, from about -10 mV to about -5 mV, from about -5 mV to about +25 mV, from about -5 mV to about +20 mV, from about -5 mV to about +15 mV, from about -5 mV to about +10 mV, from about -5 mV to about +5 mV, from about -5 mV to about 0 mV, from about 0 mV to about +25 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +25 mV, from about +5 mV to about +20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV.

[0384] Composition

[0385] In another aspect, the present disclosure also provides a loaded lipid particle, which refers to a composition comprising one or more of the above lipid particles and one or more therapeutic agents. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0386] In some embodiments, the therapeutic agent is encapsulated in the aqueous interior of the lipid particle. In some embodiments, the therapeutic agent is present in one or more lipid layers of the lipid particle. In some embodiments, the therapeutic agent is bound to the outer or inner lipid surface of the lipid particle.

[0387] As used herein, the term "therapeutic agent" can include any molecule or compound capable of exerting a desired effect on a cell, tissue, organ, or subject. Such an effect can be biological, physiological, or cosmetic. The therapeutic agent can be any type of molecule or compound, including but not limited to nucleic acids, peptides, and polypeptides, such as antibodies (e.g., polyclonal antibodies, monoclonal antibodies, antibody fragments, humanized antibodies, recombinant antibodies, recombinant human antibodies, and Primatized TM antibodies), cytokines, growth factors, apoptosis factors, differentiation-inducing factors, cell surface receptors and their ligands, hormones, and small molecules, including organic small molecules or compounds. In some embodiments, the therapeutic agent is a nucleic acid.

[0388] As used herein, the term "nucleic acid" is intended to include any oligonucleotide or polynucleotide. Fragments containing up to 50 nucleotides are generally referred to as oligonucleotides, and longer fragments are referred to as polynucleotides. In some embodiments, the oligonucleotide has a length of 10 - 50 nucleotides. In some embodiments, the oligonucleotide has a length of 10 - 40 nucleotides. In some embodiments, the oligonucleotide has a length of 20 - 30 nucleotides. In some embodiments, the oligonucleotide has a length of 20 - 30 nucleotides. In some embodiments, the oligonucleotide has a length of 10, 11, 12, 13, 14, 15, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides.

[0389] As used herein, the terms "polynucleotide" and "oligonucleotide" refer to polymers or oligomers of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars, and intersugar (backbone) linkages. The terms "polynucleotide" and "oligonucleotide" also include polymers or oligomers or portions thereof containing non-naturally occurring monomers having similar functionality. Such modified or substituted oligonucleotides are generally superior to the natural form because they possess properties such as enhanced cellular uptake and increased stability in the presence of nucleases. As used herein, the term "nucleotide" includes a sugar (deoxyribose (DNA) or ribose (RNA)), a base, and a phosphate group. Nucleotides are joined together by phosphate groups. "Bases" include purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, as well as synthetic derivatives of purines and pyrimidines, including but not limited to modifications that place new reactive groups (e.g., but not limited to amines, alcohols, thiols, carboxylates, and alkyl halides).

[0390] The nucleic acids present in the compositions of the present disclosure (e.g., lipid nanoparticles loaded with nucleic acids) include any known form of nucleic acid. The nucleic acids used herein include, but are not limited to, single-stranded DNA or RNA, or double-stranded DNA or RNA, and DNA-RNA hybrids. Examples of double-stranded DNA include, but are not limited to, structural genes, genes including control regions and termination regions, and self-replicating systems such as viral or plasmid DNA. Examples of double-stranded RNA include, but are not limited to, siRNA and other RNA interference reagents. Single-stranded nucleic acids include, but are not limited to, antisense oligonucleotides, ribozymes, microRNAs, and oligonucleotides that form triplexes. The nucleic acids used herein also include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, or non-naturally occurring and have binding properties similar to those of a reference nucleic acid. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2'-O-methyl ribonucleotides, and peptide nucleic acids (PNAs). Unless specifically restricted, the term encompasses nucleic acids containing known analogs of natural nucleotides that have binding properties similar to those of a reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms, and complementary sequences, as well as the explicitly recited sequence. The nucleic acids present in the compositions of the present disclosure (e.g., lipid nanoparticles containing nucleic acids) may include one or more modifications.

[0391] The nucleic acids of the present disclosure can have various lengths, typically depending on the specific form of the nucleic acid. In some embodiments, the nucleic acid has a length of 10 - 5000 nucleotides. In some embodiments, the nucleic acid has a length of about 4000 nucleotides. In some embodiments, the nucleic acid has a length of about 3000 nucleotides. In some embodiments, the nucleic acid has a length of about 2500 nucleotides. In some embodiments, the nucleic acid has a length of about 2000, 1900, 1800, 1700, 1600, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 750, 700, 650, 600, 550, 450, 400, 350, 300, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 nucleotides.

[0392] In some embodiments, the therapeutic agent (e.g., nucleic acid) is RNA. In some embodiments, the therapeutic agent (e.g., nucleic acid) is DNA. In some embodiments, the therapeutic agent (e.g., nucleic acid) is small interfering RNA (siRNA), microRNA (miRNA), small activating RNA (saRNA), single guide RNA (sgRNA), messenger RNA (mRNA), self-amplifying RNA, circular RNA, antisense oligonucleotide (ASO), ribonucleoprotein (RNP), ssDNA, dsDNA, or any mixture thereof.

[0393] In certain embodiments, the therapeutic agent is mRNA. The mRNA can encode any polypeptide of interest, including any naturally occurring or non-naturally occurring or otherwise modified polypeptide. The polypeptide encoded by the mRNA can be of any size and can have any secondary structure or activity. In some embodiments, the polypeptide encoded by the mRNA can be a "therapeutic protein", which refers to a polypeptide that has a therapeutic effect when expressed in a cell. For example, the therapeutic protein can be a protein required to enhance or replace a naturally occurring protein of interest. In some embodiments, the polypeptide encoded by the mRNA is an antibody, antigen, cytokine, clotting factor, or enzyme, or a functional fragment thereof.

[0394] In some embodiments, the mRNA is designed to have a 5' untranslated region (UTR), an open reading frame (ORF), and a 3' UTR. In some embodiments, the mRNA can be prepared in vitro by any method known in the art. For example, the mRNA used in the exemplary embodiments disclosed herein can be prepared using the TranscriptAid T7 High Yield Transcription kit (Thermo K0441). Using the TranscriptAid T7 High Yield Transcription kit, linear double-stranded DNA is used as a template to generate the mRNA of interest, and a certain proportion of pseudouridine and capping reagents are added for in vitro transcription. In vitro transcription conditions: Configure the reaction system according to the kit instructions, react at 37 °C for 0.5 - 2 hours, digest the transcript with DNase for 30 minutes, and purify the transcript using the Monarch RNA Cleanup kit (NEB T2040L).

[0395] Table 2 mRNA sequences of interest

[0396]

[0397]

[0398]

[0399]

[0400]

[0401]

[0402] Table 3.UTR

[0403]

[0404]

[0405] In some embodiments, the therapeutic agent is siRNA. The siRNA may be capable of selectively knocking down or downregulating the expression of a gene of interest. For example, siRNA can be selected to silence a gene associated with a particular disease, disorder, or condition when a lipid particle composition comprising the siRNA is administered to a subject in need thereof. The siRNA can comprise a sequence complementary to the mRNA sequence encoding the gene or protein of interest. In some embodiments, the siRNA can be an immunomodulatory siRNA.

[0406] In some embodiments, the therapeutic agent is a vaccine, a compound that elicits an immune response (e.g., a nucleic acid encoding a polypeptide or a polypeptide). Vaccines include compounds and formulations capable of providing immunity against one or more conditions associated with an infectious disease and can include mRNA encoding an antigen and / or epitope derived from an infectious disease. Vaccines also include compounds and formulations that direct an immune response against cancer cells and can include mRNA encoding an antigen, epitope, and / or neoepitope derived from a tumor cell.

[0407] The “encapsulation efficiency” of a therapeutic agent refers to the amount of the therapeutic agent that is encapsulated or otherwise bound to the lipid particles after preparation relative to the initial amount provided. Desirably, the encapsulation efficiency is high (e.g., greater than 80%, greater than 85%, greater than 90%, or greater than 95%). For example, the encapsulation efficiency can be measured by comparing the amount of the therapeutic agent in a solution containing the lipid particles before and after disrupting the lipid particles with one or more organic solvents or detergents. Fluorescence can be used to measure the amount of free therapeutic agent (e.g., RNA) in the solution. In some embodiments, the encapsulation efficiency of the therapeutic agent can be at least 50%, such as at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency can be at least 80%. In certain embodiments, the encapsulation efficiency can be at least 90%.

[0408] As used herein, "encapsulation", "encapsulated", "payload", and "association" can refer to complete, substantial, or partial encapsulation, confinement, enclosure, or wrapping. As used herein, "encapsulation" or "association" can refer to the process of confining a separate nucleic acid within a lipid particle and / or establishing a physicochemical relationship between the separate nucleic acid and the lipid particle.

[0409] As described herein, nucleic acids and polynucleotides can include one or more alternative components that confer useful properties, including increased stability and / or not substantially inducing an innate immune response in the cells into which the polynucleotide is introduced. For example, compared to the corresponding unmodified polynucleotide or nucleic acid, the alternative polynucleotide or nucleic acid exhibits reduced degradation in the cells into which the polynucleotide or nucleic acid is introduced. These alternatives can improve the efficiency of protein production, the retention of the polynucleotide within the cell, and / or the viability of the contacted cells, as well as have reduced immunogenicity.

[0410] The amount of therapeutic agent in the lipid particle can depend on the size, composition, desired target, and / or application of the lipid particle, or other characteristics, as well as the characteristics of the therapeutic agent. For example, the amount of useful RNA in the lipid particle can depend on the size, sequence, and other features of the RNA. The relative amounts of the therapeutic agent and other elements (such as lipids) in the lipid particle can also vary. In some embodiments, the mass ratio of lipid (e.g., cationic lipid, neutral lipid, structural lipid such as sterol, and lipid selected to reduce aggregation such as surfactant) to therapeutic agent (e.g., RNA) in the loaded lipid particle can be from about 5:1 to about 60:1, such as about 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, and 60:1. For example, the mass ratio of lipid to therapeutic agent can be from about 10:1 to about 50:1. In some embodiments, the mass ratio of lipid to therapeutic agent is about 40:1. In some embodiments, the mass ratio of lipid to therapeutic agent is about 20:1. In some embodiments, the mass ratio of lipid to therapeutic agent is calculated by dividing the total mass of cationic lipid, neutral lipid, structural lipid such as sterol, and surfactant (excluding solvent) by the mass of the anhydrous therapeutic agent (e.g., RNA). For example, absorption spectroscopy (e.g., ultraviolet-visible spectroscopy) can be used to measure the amount of therapeutic agent in the lipid particle composition.

[0411] In another aspect, the present disclosure also provides a pharmaceutical composition comprising the loaded lipid particle provided herein and a pharmaceutically acceptable excipient.

[0412] Pharmaceutically acceptable excipients are conventional pharmaceutical excipients in the art and can be prepared in a manner well known in the pharmaceutical field. Some examples of materials that can be used as pharmaceutically acceptable excipients or carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; (4) tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) diols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) alcohols, such as ethanol and propanol; (20) phosphate buffer solution; and (21) other non-toxic compatible substances used in pharmaceutical formulations, such as acetone.

[0413] The pharmaceutical composition may contain pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity modifiers, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc.

[0414] The form of the pharmaceutical composition depends on many criteria, including but not limited to the route of administration, the degree of the disease, or the dose to be administered.

[0415] The pharmaceutical composition can be formulated for oral, nasal, rectal, transdermal, intravenous, or intramuscular administration. Depending on the desired route of administration, the pharmaceutical composition can be formulated in the form of tablets, capsules, pills, dragees, powders, granules, sachets, cachets, lozenges, suspensions, emulsions, solutions, syrups, aerosols (as solids or in liquid media), sprays, ointments, pastes, creams, lotions, gels, patches, inhalants, or suppositories.

[0416] The pharmaceutical composition can be formulated to provide rapid, sustained or delayed release of the active ingredient after administration to a patient by procedures known in the art. In some embodiments, the pharmaceutical composition is formulated in a sustained release form. In some embodiments, the extended period of time can be about 1 hour to 24 hours, 2 hours to 12 hours, 3 hours to 8 hours, 4 hours to 6 hours, 1 to 2 days or longer. In certain embodiments, the extended period of time is at least about 4 hours, at least about 8 hours, at least about 12 hours or at least about 24 hours. The pharmaceutical composition can be formulated in the form of tablets. For example, the rate of release of the active agent can be controlled not only by the dissolution of the active agent in the gastrointestinal fluid and subsequent diffusion from the tablet or pellet (independent of pH), but also by the physical processes of tablet disintegration and erosion. In some embodiments, the polymeric materials disclosed in the following references can be used for sustained release: "Medical Applications of Controlled Release", Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); "Controlled Drug Bioavailability", "Drug Product Design and Performance", Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J Macromol. Sci. Rev. Macromol Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. neurosurg. 71:105. The above references are incorporated herein by reference in their entirety.

[0417] In certain embodiments, the pharmaceutical compositions of the present disclosure can be administered at a dose level sufficient to deliver from about 0.0001 mg / kg to about 10 mg / kg (such as from about 0.0001 mg / kg to about 10 mg / kg, from about 0.001 mg / kg to about 10 mg / kg, from about 0.005 mg / kg to about 10 mg / kg, from about 0.01 mg / kg to about 10 mg / kg, from about 0.05 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, from about 1 mg / kg to about 10 mg / kg, from about 2 mg / kg to about 10 mg / kg, from about 5 mg / kg to about 10 mg / kg, from about 0.0001 mg / kg to about 5 mg / kg, from about 0.001 mg / kg to about 5 mg / kg, from about 0.005 mg / kg to about 5 mg / kg, from about 0.01 mg / kg to about 5 mg / kg, from about 0.05 mg / kg to about 5 mg / kg, from about 0.1 mg / kg to about 5 mg / kg, from about 1 mg / kg to about 5 mg / kg, from about 2 mg / kg to about 5 mg / kg, from about 0.0001 mg / kg to about 2.5 mg / kg, from about 0.001 mg / kg to about 2.5 mg / kg, from about 0.005 mg / kg to about 2.5 mg / kg, from about 0.01 mg / kg to about 2.5 mg / kg, from about 0.05 mg / kg to about 2.5 mg / kg, from about 0.1 mg / kg to about 2.5 mg / kg, from about 1 mg / kg to about 2.5 mg / kg, from about 2 mg / kg to about 2.5 mg / kg, from about 0.0001 mg / kg to about 1 mg / kg, from about 0.001 mg / kg to about 1 mg / kg, from about 0.005 mg / kg to about 1 mg / kg, from about 0.01 mg / kg to about 1 mg / kg, from about 0.05 mg / kg to about 1 mg / kg, from about 0.1 mg / kg to about 1 mg / kg, from about 0.0001 mg / kg to about 0.25 mg / kg, from about 0.001 mg / kg to about 0.25 mg / kg, from about 0.005 mg / kg to about 0.25 mg / kg, from about 0.01 mg / kg to about 0.25 mg / kg, from about 0.05 mg / kg to about 0.25 mg / kg or from about 0.1 mg / kg to about 0.25 mg / kg) of a therapeutic agent, wherein a dose of 1 mg / kg provides 1 mg of the therapeutic agent and / or prophylactic agent per 1 kg of subject body weight.

[0418] In certain embodiments, the pharmaceutical composition can be formulated into unit dosage forms. The term "unit dosage form" refers to physically discrete units suitable as unit doses for human subjects and other mammals, each unit containing a predetermined amount of the active substance calculated to produce the desired therapeutic effect in association with a suitable pharmaceutical carrier. In some embodiments, a dose of about 0.001 mg / kg to about 10 mg / kg of the therapeutic agent can be administered. In other embodiments, a dose of about 0.005 mg / kg to about 2.5 mg / kg of the therapeutic agent can be administered. In certain embodiments, a dose of about 0.1 mg / kg to about 1 mg / kg of the therapeutic agent can be administered. In other embodiments, a dose of about 0.05 mg / kg to about 0.25 mg / kg of the therapeutic agent can be administered. The dose can be administered one or more times per day in the same or different amounts to achieve the desired level of therapeutic, diagnostic, prophylactic, or imaging effect. For example, the desired dose can be delivered three times a day, twice a day, once a day, every other day, every third day, weekly, bi-weekly, tri-weekly, or every four weeks.

[0419] In some embodiments, the lipid-loaded particles or pharmaceutical compositions thereof comprising a therapeutic agent can be used in the development of nucleic acid vaccines, protein replacement therapies, nucleic acid immunotherapies, and the like. In some embodiments, the lipid-loaded particles or pharmaceutical compositions thereof comprising a therapeutic agent can be used to treat a disease or disorder. In some embodiments, the lipid-loaded particles or pharmaceutical compositions thereof comprising a therapeutic agent can be used to manufacture a medicament for treating a disease or disorder.

[0420] Use of Lipid Particles

[0421] In another aspect, the present disclosure provides a method of delivering a therapeutic agent to a subject. In some embodiments, the method comprises the steps of:

[0422] i) encapsulating the therapeutic agent into a lipid particle of the present disclosure; and

[0423] ii) administering the lipid particle encapsulating the therapeutic agent to the subject.

[0424] In another aspect, the present disclosure provides a method of delivering a therapeutic agent to a subject. In some embodiments, the method comprises administering to the subject a lipid-loaded particle of the present disclosure or a pharmaceutical composition comprising the lipid-loaded particle.

[0425] In some embodiments, a therapeutic agent is delivered to a cell of a subject, wherein the cell is selected from a muscle cell, a lung cell, a liver cell, a spleen cell, a kidney cell, a heart cell, a skin cell, a hair cell, a nail cell, a bone cell, an artery cell, a vein cell, a diaphragm cell, a larynx cell, a stomach cell, an intestinal cell, a ureter cell, a bladder cell, a lymph node cell, a bone marrow cell, a thymus cell, a brain cell, a spinal cord cell, a nerve cell, a pituitary cell, a thyroid cell, an adrenal cell, a penis cell, a vagina cell, a prostate cell, and a uterine cell. In some embodiments, a therapeutic agent is delivered to a cell of a subject, wherein the cell is selected from a liver cell, a lung cell, a spleen cell, a muscle cell, and a brain cell.

[0426] In another aspect, the present disclosure provides a method of treating a disease or disorder in a subject, which comprises administering to the subject an effective amount of the lipid nanoparticles of the present disclosure or a pharmaceutical composition comprising the lipid nanoparticles.

[0427] In another aspect, the present disclosure provides the use of the lipid nanoparticles of the present disclosure, the lipid nanoparticles loaded with a payload of the present disclosure, or a pharmaceutical composition comprising the lipid nanoparticles loaded with a payload in the manufacture of a medicament for treating a disease or disorder.

[0428] In another aspect, the present disclosure provides a method of expressing a protein in a subject. In some embodiments, the method comprises:

[0429] i) encapsulating a polynucleotide encoding the protein into a lipid nanoparticle of the present disclosure; and ii) administering the lipid nanoparticle encapsulating the polynucleotide to the subject.

[0430] In some embodiments, the protein is an antibody, an antigen, a cytokine, a clotting factor, or an enzyme, or a functional fragment thereof.

[0431] As used herein, "expression" of a nucleic acid sequence refers to the translation of a nucleic acid (e.g., mRNA) into a polypeptide or protein and / or the post-translational modification of the polypeptide or protein.

[0432] Examples

[0433] For illustrative purposes, the following examples are included. However, it should be understood that these examples do not limit the invention and are merely intended to present a method of practicing the disclosure. Those skilled in the art will recognize that the described chemical reactions can be readily adapted to prepare many other compounds of the disclosure, and alternative methods of preparing the compounds of the disclosure are considered to fall within the scope of the disclosure. For example, the synthesis of compounds not exemplified in the disclosure can be successfully carried out by modifications that will be apparent to those skilled in the art, e.g., by appropriately protecting interfering groups, by utilizing other suitable reagents known in the art in addition to those described, and / or by making conventional modifications to the reaction conditions. Alternatively, other reactions disclosed herein or known in the art will be considered to have applicability for preparing other compounds of the disclosure.

[0434] For illustrative purposes, a general synthetic scheme for preparing the compounds of the disclosure and key intermediates is shown below. Those skilled in the art will understand that other synthetic schemes can be used to synthesize the compounds of the invention. Although specific starting materials and reagents are described in the general scheme and discussed below, other starting materials and reagents can be readily substituted to provide various derivatives and / or reaction conditions. In addition, many of the compounds prepared by the methods described below can be further modified using conventional chemistry well known to those skilled in the art.

[0435] Example 1: General Scheme 1

[0436]

[0437] Wherein

[0438] each W is independently selected from O, S, and NH;

[0439] each Y is independently selected from O, S, and NH;

[0440] each n is independently 1, 2, or 3;

[0441] each m is 0 or 1;

[0442] each p is 1 or 2.

[0443] General Procedure

[0444] A mixture of compound S1-1, S1-2, 2,6-di-tert-butyl-4-methylphenol (BHT), acetonitrile (ACN), and / or acetic acid is added to a sealed container. The mixture is heated at 60 - 80 °C for 18 - 36 hours. The mixture is then cooled to room temperature and purified by column chromatography to obtain the desired lipid.

[0445] Example 1.1: Synthesis of Compound 1

[0446]

[0447] Step 1: In an ice bath, while stirring, add Jones reagent (20 g) to a solution of compound 1-2-1 (11.6 g, 100 mmol) in acetone (500 mL). Allow the mixture to warm to room temperature and maintain at the same temperature for 3 hours. Then add BHT (220 mg, 1.0 mmol). Concentrate the mixture and extract with ether. Compound 1-2-2 is used directly without further purification.

[0448] Step 2: At room temperature, while stirring, add dicyclohexylcarbodiimide (DCC, 2.1 g, 10 mmol) and 4-dimethylaminopyridine (DMAP, 0.12 g, 1 mmol) to a solution of compound 1-2-2 (1.3 g, 10 mmol), compound 1-2-3 (1.7 g, 10 mmol) and 4-methylmorpholine (1.2 g, 10.5 mmol) in DCM (100 mL). Stir the mixture overnight and then filter. Add BHT (22 mg, 0.1 mmol) to the filtrate. Concentrate the mixture to obtain the crude product. The crude product is purified by silica gel column chromatography (eluent: hexane containing 0 - 10% ethyl acetate, v / v), and concentrated under reduced pressure to obtain compound 1-2 (1.8 g, yield 60%) as a colorless oil.

[0449] For compound 1-2 1 H NMR: (400 MHz, chloroform-d) δ 6.50 (d, J = 17.3 Hz, 1H), 6.21 (dd, J = 17.4, 10.5 Hz, 1H), 5.92 (d, J = 10.5 Hz, 1H), 4.68 (s, 2H), 4.16 (t, J = 6.7 Hz, 2H), 1.63 (dd, J = 7.4, 6.6 Hz, 3H), 1.27 (d, J = 19.2 Hz, 18H), 0.87 (t, J = 6.8 Hz, 3H).

[0450] Step 3: Add compound 1-1 (36 mg, 0.25 mmol) and acetic acid (1 drop) to a solution of compound 1-2 (280 mg, 1 mmol, containing BHT) in ACN (2 mL). Reflux the mixture for 48 hours until thin layer chromatography (TLC) indicates the completion of the reaction. Dilute the mixture with dichloromethane (DCM) (10 mL) and concentrate under reduced pressure to obtain the crude product. The crude product is purified by silica gel column chromatography (eluent: DCM containing 0 - 10% methanol, v / v), and concentrated under reduced pressure to obtain compound 1 (188 mg, yield 60%) as a colorless oil.

[0451] For compound 1 11H NMR: (400 MHz, chloroform-d) δ 4.60 (s, 8H), 4.14 (t, J = 6.8 Hz, 8H), 2.80 (t, J = 7.3 Hz, 8H), 2.55 (t, J = 7.0 Hz, 8H), 2.52–2.15 (m, 8H), 1.63 (p, J = 6.9 Hz, 11H), 1.34–1.23 (m, 68H), 0.88 (t, J = 6.7 Hz, 12H).

[0452] Example 1.2: Synthesis of Compound 2

[0453]

[0454] To a solution of Compound 2-2 (280 mg, 1 mmol, containing BHT, prepared according to the methods of Steps 1 and 2 of Example 1.1) in ACN (2 mL) was added Compound 2-1 (40 mg, 0.25 mmol) and acetic acid (1 drop). The mixture was refluxed for 48 hours until TLC indicated the completion of the reaction. The mixture was diluted with DCM (10 mL) and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (eluent: DCM containing 0-10% methanol, v / v), and concentrated under reduced pressure to obtain Compound 2 (207 mg, yield 65%) as a colorless oil.

[0455] For Compound 2 1 1H NMR: (400 MHz, chloroform-d) δ 4.60 (s, 8H), 4.14 (t, J = 6.8 Hz, 8H), 2.79 (d, J = 7.1 Hz, 8H), 2.68–2.27 (m, 16H), 1.63 (t, J = 7.0 Hz, 12H), 1.28 (d, J = 16.5 Hz, 74H), 0.88 (t, J = 6.7 Hz, 14H).

[0456] Example 1.3: Synthesis of Compound 3

[0457]

[0458] To a solution of Compound 3-2 (280 mg, 1 mmol, containing BHT, prepared according to the methods of Steps 1 and 2 of Example 1.1) in ACN (2 mL) was added Compound 3-1 (44 mg, 0.25 mmol) and acetic acid (1 drop). The mixture was refluxed for 48 hours until TLC indicated the completion of the reaction. The mixture was diluted with DCM (10 mL) and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (eluent: DCM containing 0-10% methanol, v / v), and concentrated under reduced pressure to obtain Compound 3 (190 mg, yield 60%) as a colorless oil.

[0459] Of Compound 3 1 H NMR: (400 MHz, chloroform-d) δ 4.53 (s, 8H), 4.07 (t, J = 6.8 Hz, 9H), 2.73 (t, J = 6.9 Hz, 9H), 2.49 (t, J = 6.9 Hz, 10H), 2.40 (dt, J = 19.4, 6.8 Hz, 7H), 1.57 (q, J = 7.0 Hz, 12H), 1.20 (d, J = 8.0 Hz, 79H), 0.81 (t, J = 6.7 Hz, 15H).

[0460] Example 1.4: Synthesis of Compound 4

[0461]

[0462] To a solution of Compound 4-2 (280 mg, 1 mmol, containing BHT, prepared according to the methods of Steps 1 and 2 of Example 1.1) in ACN (2 mL) was added Compound 4-1 (50 mg, 0.25 mmol) and acetic acid (1 drop). The mixture was refluxed for 48 hours until TLC indicated the completion of the reaction. The mixture was diluted with DCM (10 mL) and concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (eluent: DCM containing 0 - 10% methanol, v / v), and concentrated under reduced pressure to obtain Compound 4 (180 mg, yield 55%) as a colorless oil.

[0463] Of Compound 4 1 H NMR: (400 MHz, chloroform-d) δ 4.60 (s, 8H), 4.14 (t, J = 6.8 Hz, 8H), 2.98 (s, 2H), 2.79 (p, J = 7.0 Hz, 8H), 2.55 (t, J = 6.8 Hz, 9H), 2.38 (dt, J = 32.7, 7.2 Hz, 7H), 1.99 (s, 3H), 1.63 (p, J = 6.9 Hz, 12H), 1.28 (d, J = 16.2 Hz, 70H), 0.88 (t, J = 6.7 Hz, 13H).

[0464] Example 1.5: Synthesis of Compound 5

[0465]

[0466] To a solution of compound 5-2 (280 mg, 1 mmol, containing BHT, prepared by the methods of Steps 1 and 2 of Example 1.1) in ACN (2 mL) was added compound 5-1 (53 mg, 0.25 mmol) and acetic acid (1 drop). The mixture was refluxed for 48 h until TLC indicated the completion of the reaction. The mixture was diluted with DCM (10 mL) and concentrated under reduced pressure to give the crude product. The crude product was purified by silica gel column chromatography (eluent: DCM containing 0 - 10% methanol, v / v), and concentrated under reduced pressure to give compound 5 (166 mg, yield 50%) as a colorless oil.

[0467] of compound 5 1 1H NMR: (400 MHz, chloroform-d) δ 6.96 (s, 1H), 6.91 (s, 1H), 4.60 (d, J = 6.9 Hz, 8H), 4.38 (t, J = 6.8 Hz, 1H), 4.20 (t, J = 6.8 Hz, 1H), 4.13 (t, J = 6.8 Hz, 9H), 3.66 (d, J = 10.7 Hz, 2H), 2.95 (t, J = 6.7 Hz, 2H), 2.78 (t, J = 7.3 Hz, 6H), 2.52 (t, J = 7.2 Hz, 6H), 2.39 (q, J = 10.6, 7.8 Hz, 6H), 1.63 (p, J = 6.9 Hz, 12H), 1.35–1.24 (m, 72H), 0.88 (t, J = 6.7 Hz, 14H).

[0468] Example 1.6: Synthesis of compound 6

[0469]

[0470] Step 1: A solution of compound 6-2-1 (0.92 g, 10 mmol), compound 6-2-2 (1.72 g, 10 mmol) and sulfuric acid (100 mg, 98%) in toluene (100 mL) was heated at 150 °C under N2 atmosphere. The mixture was refluxed in a Dean-Stark apparatus for 1.5 h. The mixture was cooled to room temperature, washed twice with saturated aqueous NaHCO3 and then with brine. The aqueous layer was extracted with DCM. The combined organic layers were concentrated to give the crude compound 6-2-3, which was used directly without further purification.

[0471] Step 2: At room temperature, while stirring, add DCC (1.05 g, 5 mmol) and DMAP (0.06 g, 0.5 mmol) to a solution of compound 6-2-3 (1.23 g, 5 mmol), compound 6-2-4 (0.36 g, 5 mmol) and 4-methylmorpholine (0.6 g, 5.03 mmol) in DCM (50 mL). Stir the mixture overnight and then filter. Add BHT (11 mg, 0.05 mmol) to the filtrate. Concentrate the mixture to obtain the crude product. The crude product was purified by silica gel column chromatography (eluent: hexane containing 0 - 10% ethyl acetate, v / v), and concentrated under reduced pressure to obtain compound 6-2 (0.7 g, yield 48%) as a colorless oil.

[0472] Step 3: Add compound 6-1 (36 mg, 0.25 mmol) to a solution of compound 6-2 (300 mg, 1 mmol, containing BHT) in ACN (2 mL). Reflux the mixture for 48 hours until TLC indicates the completion of the reaction. Dilute the mixture with DCM (10 mL) and concentrate under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (eluent: DCM containing 0 - 10% methanol, v / v), and concentrated under reduced pressure to obtain compound 6 (151 mg, yield 45%) as a colorless oil.

[0473] For compound 6 1 1H NMR: (400 MHz, chloroform-d) δ 4.10 (t, J = 6.9 Hz, 8H), 3.70 (s, 6H), 3.23 (s, 1H), 2.95 (s, 2H), 2.84–2.68 (m, 12H), 2.50 (q, J = 7.5, 6.9 Hz, 4H), 1.63 (t, J = 7.0 Hz, 9H), 1.36–1.19 (m, 75H), 0.91–0.75 (m, 15H).

[0474] Example 1.7: Synthesis of compound 7

[0475]

[0476] Step 1: DMAP (698 mg, 5.7 mmol) was added to a solution of compound 7-2-1 (10 g, 57.1 mmol) and compound 7-2-2 (10.3 g, 59.9 mmol) in DCM (100 mL). Under N2 atmosphere, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 21.9 g, 114.2 mmol) was added portionwise to this mixture at <10 °C. The mixture was warmed to room temperature and stirred at the same temperature for 16 h. The reaction was quenched with saturated aqueous NaHCO3 (100 mL). The aqueous layer was extracted with ethyl acetate (200 mL). The combined organic layers were washed with 5% aqueous citric acid (100 mL) and brine (100 mL), and then with brine (100 mL). The organic layer was dried over Na2SO4 and concentrated to give 18.5 g of crude compound 7-2-3. Compound 7-2-3 was used directly without further purification.

[0477] of compound 7-2-3 1 1H NMR: (400 MHz, DMSO-d6) δ 7.15 (t, J = 6.2 Hz, 1H), 4.02 (t, J = 6.5 Hz, 2H), 3.64 (d, J = 6.2 Hz, 2H), 1.55 (t, J = 6.9 Hz, 2H), 1.38 (s, 8H), 1.34 (s, 2H), 1.24 (s, 19H), 0.85 (t, J = 6.5 Hz, 4H).

[0478] Step 2: HCl (4 M in ethyl acetate, 93 mL) was added to a solution of compound 7-2-3 (18.5 g, 56.1 mmol, crude) in ethyl acetate (93 mL) under N2 atmosphere at <10 °C. The mixture was warmed to room temperature and stirred at the same temperature for 16 h. TLC indicated consumption of the starting material. The mixture was cooled to 5 °C and stirred at the same temperature for 2 h. The mixture was filtered, and the residue was washed with ethyl acetate and dried to give 12.5 g of crude compound 7-2-4. Compound 7-2-4 was used directly without further purification.

[0479] of compound 7-2-4 1 1H NMR: (400 MHz, DMSO-d6) δ 8.63 (s, 3H), 4.11 (t, J = 6.6 Hz, 2H), 3.73 (s, 2H), 1.57 (p, J = 6.7 Hz, 2H), 1.23 (s, 17H), 0.84 (t, J = 6.6 Hz, 3H).

[0480] Step 3: Under N2 atmosphere, at <10 °C, DMAP (698 mg, 5.7 mmol) was added to a solution of compound 7-2-4 (5 g, 18.9 mmol) and compound 7-2-5 (1.9 g, 20.7 mmol) in DCM (50 mL). A solution of N,N-diisopropylethylamine (DIEA, 4.9 g, 37.8 mmol) in DCM (5 mL) was added dropwise to this mixture at the same temperature. The mixture was warmed to room temperature and stirred at the same temperature for 3 hours. TLC indicated the consumption of the starting materials. The reaction was quenched with saturated aqueous NaHCO3 (50 mL). The aqueous layer was extracted with ethyl acetate (100 mL). The combined organic layers were washed with 5% aqueous citric acid (50 mL) and brine (50 mL), and then with brine (100 mL). The organic layer was dried over Na2SO4 and concentrated to give the crude product. The crude product was purified by silica gel column chromatography (eluent: petroleum ether containing 20% ethyl acetate, v / v), and concentrated under reduced pressure to give compound 7-2 (4.4 g, 27% yield over 3 steps).

[0481] of compound 7-2 1 1H NMR: (400 MHz, DMSO-d6) δ 8.51 (t, J = 6.0 Hz, 1H), 6.29 (dd, J = 17.1, 10.2 Hz, 1H), 6.11 (dd, J = 17.1, 2.1 Hz, 1H), 5.63 (dd, J = 10.2, 2.1 Hz, 1H), 4.04 (t, J = 6.6 Hz, 2H), 3.90 (d, J = 5.9 Hz, 2H), 1.57 (s, 2H), 1.24 (s, 16H), 0.85 (t, J = 6.6 Hz, 3H).

[0482] Step 4: Under N2 atmosphere, compound 7-2 (97 mg, 0.34 mmol) was added to a 2 mL vial containing compound 7-1 (10 mg, 0.07 mmol), and then ACN (0.2 mL), BHT (2 mg) and acetic acid (3 mg) were added. The mixture was heated at 70 °C for 16 hours. TLC indicated that the reaction was incomplete. Then the mixture was heated at 85 °C for 24 hours, and TLC indicated the completion of the reaction. The mixture was purified by silica gel column chromatography (eluent: DCM containing 0 - 10% methanol and 0.5% NH4OH, v / v), and concentrated under reduced pressure to give compound 7 (18 mg, 24% yield).

[0483] of compound 7 11H NMR: (400 MHz, chloroform-d) δ 7.71 (t, J = 5.6 Hz, 4H), 4.09 (t, J = 6.8 Hz, 8H), 3.98 (d, J = 5.6 Hz, 8H), 2.75 (t, J = 6.1 Hz, 8H), 2.51 (t, J = 7.1 Hz, 5H), 2.44 (t, J = 6.0 Hz, 9H), 1.71 (s, 3H), 1.63 (q, J = 7.0 Hz, 9H), 1.28 (d, J = 15.9 Hz, 67H), 0.88 (t, J = 6.8 Hz, 12H). HR-MS: [M+H] + = 1279.03769

[0484] Example 1.8: Synthesis of Compound 8

[0485]

[0486] Step 1: Add toluene (20 mL) to a 100 mL container containing Compound 8-2-1 (4.0 g, 56.3 mmol), BHT (1.25 g, 5.6 mmol) and Compound 8-2-2 (6.0 g, 28.4 mmol). Heat the mixture at 80 °C for 3 days. TLC (eluent: hexane / ethyl acetate = 10 / 1) indicates that the reaction is substantially complete. The mixture is purified by silica gel column chromatography (eluent: hexane containing 0 - 33% ethyl acetate, v / v), and concentrated under reduced pressure to obtain Compound 8-2 (4.38 g, yield 55%).

[0487] Step 2: Add acetic acid (3 μL) to a 2 mL container containing Compound 8-1 (20 mg, 0.14 mmol), Compound 8-2 (187 mg, 0.66 mmol) and BHT (15 mg). Heat the mixture at 75 °C for 24 hours. Then add Compound 8-2 (95 mg, 0.34 mmol) and acetic acid (3 μL). Then heat the mixture at 75 °C for another 24 hours. TLC (eluent: DCM / MeOH / NH4OH = 10 / 1 / 1) indicates that the reaction is substantially complete. The mixture is purified by silica gel column chromatography (eluent: DCM containing 1 - 1.6% methanol and 0.5% NH4OH, v / v), and concentrated under reduced pressure to obtain Compound 8 (134.8 mg, yield 76%).

[0488] of Compound 8 11H NMR: (400 MHz, chloroform-d) δ 10.05 (s, 4H), 8.41 (t, J = 5.7 Hz, 4H), 3.23 (q, J = 7.4, 6.9 Hz, 8H), 2.68 (t, J = 5.6 Hz, 8H), 2.53 (t, J = 5.6 Hz, 8H), 2.42 (s, 4H), δ 1.74 (m, 7H), 1.53 (p, J = 7.2 Hz, 8H), 1.27 (d, J = 11.4 Hz, 70H), 0.88 (t, J = 6.7 Hz, 12H). HR-MS: [M+H] + = 1275.20592.

[0489] Example 1.9: Synthesis of Compound 9

[0490]

[0491] Under N2 atmosphere, to a 2 mL vial containing Compound 9-1 (10 mg, 0.06 mmol) was added Compound 9-2 (97 mg, 0.34 mmol, prepared according to the method of Steps 1 to 3 of Example 1.7), then ACN (0.2 mL), BHT (2 mg) and acetic acid (2.5 mg) were added. The mixture was heated at 73 °C for 40 h. TLC indicated the completion of the reaction. The mixture was purified by silica gel column chromatography (eluent: DCM containing 0 - 10% methanol and 0.5% NH4OH, v / v), and concentrated under reduced pressure to give Compound 9 (23 mg, yield 31%).

[0492] 1H NMR of Compound 9 1 1H NMR: (400 MHz, chloroform-d) δ 7.72–7.65 (m, 4H), 4.09 (t, J = 6.7 Hz, 8H), 3.98 (d, J = 5.5 Hz, 8H), 3.70–3.61 (m, 2H), 2.81 - 2.68 (m, 10H), 2.52 (t, J = 7.1 Hz, 4H), 2.44 (t, J = 6.1 Hz, 8H), 1.70 - 1.66 (m, 4H), 1.63 (p, J = 6.9 Hz, 8H), 1.38–1.19 (m, 64H), 0.88 (t, J = 6.7 Hz, 12H).

[0493] Example 1.10: Synthesis of Compound 10

[0494]

[0495] Under N2 atmosphere, to a 2 mL container containing Compound 10-1 (10 mg, 0.06 mmol), Compound 10-2 (93 mg, 0.35 mmol, prepared by a method similar to Steps 1 to 3 of Example 1.7) was added, then ACN (0.2 mL), BHT (2 mg), and acetic acid (2.5 mg) were added. The mixture was heated at 73 °C for 40 hours. TLC indicated the completion of the reaction. The mixture was purified by silica gel column chromatography (eluent: DCM containing 0 - 10% methanol and 0.5% NH4OH, v / v), and concentrated under reduced pressure to obtain Compound 10 (18 mg, yield 25%).

[0496] of Compound 10 1 1H NMR: (400 MHz, chloroform-d) δ 7.74 (t, J = 5.6 Hz, 4H), 4.10 (t, J = 6.8 Hz, 8H), 3.99 (d, J = 5.4 Hz, 8H), 2.76 (t, J = 6.1 Hz, 8H), 2.47 (dt, J = 25.8, 6.7 Hz, 14H), 1.63 (q, J = 7.1 Hz, 10H), 1.31 (d, J = 7.7 Hz, 20H), 1.26 (s, 45H), 1.07 - 0.98 (m, 3H) 0.88 (t, J = 6.7 Hz, 12H). HR-MS: [M + H] + = 1293.06248.

[0497] Example 1.11: Synthesis of Compound 28

[0498]

[0499] To a 5 mL container containing Compound 28-1 (200 mg, 0.95 mmol), BHT (104 mg), and Compound 28-2 (1.22 g, 4.5 mmol, prepared by a method similar to Steps 1 to 3 of Example 1.7), acetic acid (12 mg) was added. The mixture was heated at 80 °C for 48 hours. TLC (eluent: DCM / MeOH / NH4OH = 10 / 1 / 1) indicated the completion of the reaction. The mixture was purified by silica gel column chromatography (eluent: DCM containing 1.4 - 1.5% methanol and 0.5% NH4OH, v / v), and concentrated under reduced pressure to obtain Compound 28 (490 mg, yield 27%).

[0500] of Compound 28 11H NMR: (400 MHz, chloroform-d) δ 7.91 (t, J = 5.7 Hz, 3H), 7.01 (s, 2H), 4.07 (t, J = 6.8 Hz, 8H), 3.97 (d, J = 5.5 Hz, 8H), 3.86 (s, 1H), 2.75 (t, J = 6.1 Hz, 8H), 2.62 (s, 4H), 2.45 (dt, J = 16.3, 6.5 Hz, 12H), 1.77–1.66 (m, 3H), 1.60 (q, J = 7.1 Hz, 9H), 1.28 (d, J = 12.6 Hz, 61H), 0.88 (t, J = 6.7 Hz, 12H). HR-MS: [M+H] + = 1290.00711.

[0501] Example 1.12: Synthesis of Compound 29

[0502]

[0503] To a 5 mL vessel containing Compound 29-1 (600 mg, 4.13 mmol), BHT (182 mg) and Compound 29-2 (4.75 g, 19.8 mmol, prepared according to the method of Step 1 of Example 2.4) were added acetic acid (50 mg) and ACN (0.6 mL). The mixture was heated at 90 °C for 2 days. TLC (eluent: DCM / MeOH / NH4OH = 10 / 1 / 1) indicated that the reaction was substantially complete. The mixture was purified by flash column chromatography on silica gel (eluent: DCM containing 1.4 - 2.4% methanol and 0.5% NH4OH, v / v), and concentrated under reduced pressure to give Compound 29 (2.80 g, yield 61%).

[0504] For Compound 29 1 1H NMR: (400 MHz, chloroform-d) δ 7.07 (t, J = 5.6 Hz, 4H), 3.28–3.11 (m, 8H), 2.70 (t, J = 6.2 Hz, 7H), 2.42 (dt, J = 17.1, 7.0 Hz, 8H), 2.33 (t, J = 6.1 Hz, 9H), 2.24 (s, 3H), 1.61 (q, J = 7.1 Hz, 4H), 1.49 (p, J = 7.4 Hz, 8H), 1.27 (d, J = 9.1 Hz, 68H), 0.88 (t, J = 6.7 Hz, 12H). HR-MS: [M+H] + = 1103.08503.

[0505] Example 1.13: Synthesis of Compound 46

[0506]

[0507] To a 5 mL container containing compound 46-1 (200 mg, 0.95 mmol), BHT (104 mg), and compound 46-2 (1.29 g, 4.55 mmol, prepared according to the method of Steps 1 to 3 of Example 1.7) was added acetic acid (12 mg). The mixture was heated at 80 °C for 48 hours. TLC (eluent: DCM / MeOH / NH4OH = 10 / 1 / 1) indicated that the reaction was substantially complete. The mixture was purified by flash column chromatography on silica gel (eluent: DCM containing 1.3 - 1.4% methanol and 0.5% NH4OH, v / v), and concentrated under reduced pressure to obtain compound 46 (262 mg, yield 21%).

[0508] of compound 46 1 H NMR: (400 MHz, chloroform-d) δ 7.90 (t, J = 5.7 Hz, 2H), 7.02 (s, 2H), 4.07 (t, J = 6.8 Hz, 8H), 3.97 (d, J = 5.7 Hz, 9H), 2.75 (t, J = 6.0 Hz, 7H), 2.70 (s, 3H), 2.45 (dt, J = 12.4, 6.5 Hz, 14H), 1.73 (t, J = 6.7 Hz, 3H), 1.60 (q, J = 6.8 Hz, 9H), 1.28 (d, J = 11.9 Hz, 63H), 0.88 (t, J = 6.8 Hz, 12H). HR-MS: [M+H] + = 1345.06869.

[0509] Example 1.14: Synthesis of compound 47

[0510]

[0511] To a 5 mL container containing compound 47-1 (200 mg, 1.26 mmol), BHT (138 mg), and compound 47-2 (1.71 g, 6.03 mmol, prepared according to the method of Steps 1 to 3 of Example 1.7) was added acetic acid (15 mg). The mixture was heated at 80 °C for 50 hours. TLC (eluent: DCM / MeOH / NH4OH = 10 / 1 / 1) indicated that the reaction was substantially complete. The mixture was purified by flash column chromatography on silica gel (eluent: DCM containing 1.6 - 1.9% methanol and 0.5% NH4OH, v / v), and concentrated under reduced pressure to obtain compound 47 (370 mg, yield 23%).

[0512] of compound 47 11H NMR: (400 MHz, chloroform-d) δ 7.74 (t, J = 5.6 Hz, 4H), 4.10 (t, J = 6.8 Hz, 8H), 3.99 (d, J = 5.4 Hz, 8H), 2.76 (t, J = 6.1 Hz, 8H), 2.47 (dt, J = 25.8, 6.7 Hz, 14H), 1.63 (q, J = 7.1 Hz, 10H), 1.31 (d, J = 7.7 Hz, 20H), 1.26 (s, 45H), 1.07 - 0.98 (m, 3H) 0.88 (t, J = 6.7 Hz, 12H). HR-MS: [M+H] + = 1293.06248.

[0513] Example 1.15: Synthesis of Compound 50

[0514]

[0515] Step 1: Under N2 atmosphere, add Compound 50-2-1 (1.6 g, 7.3 mmol) and triethylamine (1.86 g, 18.4 mmol) to DCM (20 mL). Cool the mixture to 5 °C. Dropwise add Compound 50-2-2 (1.0 g, 6.1 mmol). Warm the mixture to room temperature and stir at the same temperature for 16 h. TLC (eluent: DCM) indicates the formation of the product. The mixture is purified by silica gel column chromatography to give Compound 50-2 (0.7 g, yield 37%).

[0516] 1H NMR of Compound 50-2 1 1H NMR: (400 MHz, chloroform-d) δ 6.51 (dd, J = 16.5, 9.9 Hz, 1H), 6.23 (d, J = 16.6 Hz, 1H), 5.93 (d, J = 9.9 Hz, 1H), 4.44 (s, 1H), 3.00 (q, J = 6.8 Hz, 2H), 1.53 (t, J = 7.2 Hz, 2H), 1.25 (s, 24H), 0.87 (t, J = 6.7 Hz, 3H).

[0517] Step 2: Add BHT (15 mg) and acetic acid (10 mg) to a solution of Compound 50-1 (20 mg, 0.14 mmol) and Compound 50-2 (209 mg, 0.69 mmol) in ACN (0.4 mL). Heat the mixture at 75 °C for 40 h. TLC (eluent: DCM / MeOH = 10 / 1) indicates the consumption of the starting materials. The mixture is purified by silica gel column chromatography and concentrated under reduced pressure to give Compound 50 (86 mg, yield 46%).

[0518] 1H NMR of Compound 50 11H NMR: (400 MHz, chloroform-d) δ 5.47 (s, 3H), 3.19 (t, J = 6.0 Hz, 8H), 3.08 (q, J = 6.6 Hz, 8H), 2.92 (t, J = 6.0 Hz, 8H), 2.80 (s, 3H), 2.56 (t, J = 6.8 Hz, 5H), 2.48 (s, 3H), 2.38–2.09 (m, 6H), 2.03 (s, 3H), 1.84 (s, 4H), 1.57 (p, J = 7.5 Hz, 8H), 1.25 (s, 90H), 0.97–0.81 (m, 12H).

[0519] Example 1.16: Synthesis of Compound 51

[0520]

[0521] Step 1: Under N2 atmosphere, Compound 51-2-1 (1 g, 4.38 mmol) and Compound 51-2-2 (2.8 g, 43.8 mmol) were added to methanol (3 mL). The mixture was stirred at room temperature for 40 h. TLC (eluent: DCM / MeOH = 10 / 1) indicated incomplete consumption of the starting materials. Compound 51-2-2 (2.8 g, 43.8 mmol) was added to the mixture. The mixture was heated at 60 °C for 16 h. TLC (eluent: PE) indicated consumption of the starting materials. The mixture was concentrated and diluted with water (10 mL). The mixture was stirred for 30 min and filtered. The residue was washed and dried to give Compound 51-2-3 (540 mg).

[0522] For Compound 51-2-3 1 1H NMR: (400 MHz, chloroform-d) δ 6.82 (s, 1H), 3.90 (s, 2H), 2.14 (t, J = 7.6 Hz, 2H), 1.62 (t, J = 7.3 Hz, 2H), 1.26 (d, J = 10.9 Hz, 18H), 0.87 (t, J = 6.7 Hz, 3H). HRMS: [M+H] + 229.22922.

[0523] Step 2: Under N2 atmosphere, add compound 51-2-3 (0.54 g, 2.4 mmol) and DIEA (0.62 g, 4.8 mmol) to DCM (20 mL). Cool the mixture to 5 °C. Add compound 51-2-4 (0.26 g, 2.9 mmol) dropwise at 5 - 10 °C. Warm the mixture to room temperature and stir for 2 h at the same temperature. TLC (eluent: DCM / MeOH = 10 / 1) indicates consumption of the starting materials. The mixture is purified by silica gel column chromatography to give the crude product. Add the crude product to water (10 mL) and stir for 30 min, then dry to obtain compound 51-2 (510 mg). HRMS: [M+H] + 283.24978.

[0524] Step 3: Add BHT (15 mg) and acetic acid (10 mg) to a solution of compound 51-1 (20 mg, 0.14 mmol) and compound 51-2 (187 mg, 0.66 mmol) in ethanol (0.4 mL). Heat the mixture at 80 °C for 16 h. TLC (eluent: DCM / MeOH / NH4OH = 10 / 1 / 0.05) indicates consumption of the starting materials. The mixture is purified by flash silica gel column chromatography and concentrated under reduced pressure to give compound 51 (86 mg, yield 49%).

[0525] For compound 51 1 H NMR: (400 MHz, chloroform-d) δ 2.89–2.56 (m, 5H), 2.45 (d, J = 6.1 Hz, 8H), 2.26 (q, J = 6.8, 5.9 Hz, 8H), 2.16–2.05 (m, 2H), 1.63 (t, J = 7.5 Hz, 7H), 1.56–1.43 (m, 2H), 1.37–1.11 (m, 59H), 0.95–0.73 (m, 12H). HRMS: [M+H] + 1275.09531.

[0526] Example 1.17: Synthesis of compound 54

[0527]

[0528] Step 1: At room temperature, add a mixture of compound 54-2-1 (7.1 g, 100 mmol) in 1,2-dichloroethane (20 mL) to a solution of compound 54-2-2 (13 g, 102 mmol) in 1,2-dichloroethane (20 mL). Stir the mixture at the same temperature for 2 h. TLC indicates that the starting materials have been substantially consumed.

[0529] Step 2: A solution of compound 54-2-4 (10.2 g, 54.7 mmol) in 1,2-dichloroethane (20 mL) was added dropwise to the mixture of Step 1. The mixture was stirred for 1 hour. The mixture was diluted with DCM (50 mL) and washed with water (30 mL). The organic layer was dried over Na2SO4 and concentrated. The residue was purified by flash column chromatography on silica gel (eluent: hexane containing 0 - 5% ethyl acetate) and concentrated under reduced pressure to give compound 54-2-5 (5.0 g, yield 31% over 2 steps).

[0530] Step 3: Triethylamine (4.75 g, 47.0 mmol) was added to a solution of compound 54-2-5 (5.0 g, 15.6 mmol) in ACN (125 mL) at room temperature. The mixture was stirred at the same temperature for 1.5 hours. TLC indicated that the starting material had been substantially consumed. The mixture was concentrated at 40 °C. Water (200 mL) was added to the mixture, and the mixture was stirred for 0.5 hour. The mixture was filtered, and the residue was washed with ACN (15 mL) to give compound 54-2 (3.5 g, yield 79%).

[0531] For compound 54-2 1 H NMR: (400 MHz, chloroform-d) δ 7.82 (s, 1H), 7.09 (dd, J = 17.0, 10.4 Hz, 1H), 6.52 (d, J = 17.0 Hz, 1H), 5.87 (d, J = 10.7 Hz, 1H), 4.16 (t, J = 6.7 Hz, 2H), 1.73–1.59 (m, 2H), 1.39–1.17 (m, 19H), 0.87 (t, J = 6.7 Hz, 3H). HR-MS: [M+H] + = 284.23291.

[0532] Step 4: A mixture of compound 54-1 (20 mg, 0.14 mmol), BHT (15 mg), and compound 54-2 (235 mg, 0.83 mmol) in THF (0.2 mL) was stirred at room temperature for 1 hour. TLC (eluent: DCM / MeOH / NH4OH = 10 / 1 / 0.05) indicated the completion of the reaction. The mixture was purified by flash column chromatography on silica gel (eluent: DCM containing 0 - 2.5% methanol and 0.5% NH4OH) and concentrated under reduced pressure to give compound 54 (127 mg, yield 76%).

[0533] For compound 54 11H NMR: (400 MHz, chloroform-d) δ 9.00 (s, 3H), 4.06 (t, J = 6.9 Hz, 8H), 2.71 (dt, J = 29.1, 6.5 Hz, 15H), 2.46 (t, J = 7.2 Hz, 4H), 2.28 (d, J = 10.9 Hz, 4H), 2.11 (s, 4H), 1.59 (q, J = 6.9 Hz, 12H), 1.20 (d, J = 6.5 Hz, 72H), 0.81 (t, J = 6.7 Hz, 12H). HR-MS: [M+H] + = 1279.04739.

[0534] Example 1.18: Synthesis of Compound 55

[0535]

[0536] Step 1: Under N2 atmosphere, to Compound 55-2-2 (0.2 g, 2.8 mmol) was added Compound 55-2-1 (0.83 g, 3.4 mmol) and BHT (31 mg). The mixture was stirred at 80 °C for 16 h. TLC indicated consumption of the starting materials. The mixture was cooled to room temperature, diluted with ACN (20 mL), stirred for 2 h, and filtered. The residue was washed with ACN to give Compound 55-2-3 (0.8 g).

[0537] Step 2: At room temperature, triethylamine (0.95 g, 9.4 mmol) was added to a solution of Compound 55-2-3 (1.0 g, 3.14 mmol) in ACN (25 mL). The mixture was stirred at the same temperature for 1.5 h. TLC indicated that the starting materials had been substantially consumed. The mixture was concentrated at 40 °C. The mixture was purified by flash column chromatography on silica gel (eluent: petroleum ether containing 0 - 6.3% ethyl acetate), and concentrated under reduced pressure to give Compound 55-2 (0.60 g, yield 68%).

[0538] For Compound 55-2 1 1H NMR: (400 MHz, chloroform-d) δ 8.53 (s, 1H), 6.61 (dd, J = 17.1, 10.2 Hz, 1H), 6.49 (dd, J = 16.9, 1.5 Hz, 1H), 5.89 (dd, J = 10.1, 1.5 Hz, 1H), 2.71 (t, J = 7.5 Hz, 2H), 1.65 (h, J = 8.5, 8.0 Hz, 2H), 1.41–1.17 (m, 21H), 0.88 (t, J = 6.7 Hz, 3H).

[0539] Step 3: A mixture of compound 55-1 (20 mg, 0.14 mmol), BHT (15 mg), and compound 55-2 (250 mg, 0.89 mmol) in THF (2 mL) and DCM (1 mL) was stirred at room temperature for 0.5 h. The mixture was purified by flash column chromatography on silica gel (eluent: DCM containing 0 - 3.3% methanol and 0.5% NH4OH), and concentrated under reduced pressure to give compound 55 (75 mg, yield 43%).

[0540] For compound 55 1 H NMR: (400 MHz, chloroform-d) δ 10.00 (s, 3H), 2.77 (t, J = 6.0 Hz, 8H), 2.67 (h, J = 6.0 Hz, 15H), 2.52 (t, J = 7.1 Hz, 4H), 2.32 (s, 4H), 2.25–2.10 (m, 4H), 1.60 (q, J = 9.9, 7.2 Hz, 12H), 1.25 (s, 76H), 0.93–0.81 (m, 12H). HR-MS: [M / 2 + H] + = 636.06442, [M + H] + = 1271.12962, [M + Na] + = 1294.11383.

[0541] Example 1.19: Synthesis of compound 56

[0542]

[0543] Step 1: Under N2 atmosphere, compound 56-2-2 (9.72 g, 100 mmol) and PEG-350 (1 g) were added to DCM (100 mL). The mixture was cooled to 0 °C. A solution of compound 56-2-1 (9.05 g, 100 mmol) in DCM (20 mL) was added dropwise to the mixture at 0 - 5 °C over 1 h. The mixture was warmed to room temperature and stirred for 2 h. TLC (quenched with compound 56-2-4, eluent: petroleum ether / ethyl acetate = 10 / 1) indicated the formation of the product. The mixture was filtered through diatomaceous earth to give crude compound 56-2-3.

[0544] Step 2: Under N2 atmosphere, compound 56-2-3 was cooled to 5 °C. A solution of compound 56-2-4 (18.5 g, 100 mmol) in DCM (50 mL) was added dropwise to the cooled compound 56-2-3 at 5 - 10 °C. The mixture was warmed to room temperature and stirred for 2 h. The mixture was purified by column chromatography on silica gel (eluent: petroleum ether containing 2 - 10% ethyl acetate), and concentrated under reduced pressure to give compound 56-2 (7.5 g, yield 25%).

[0545] of Compound 56-2 1 H NMR: (400 MHz, chloroform-d) δ 10.68 (t, J = 5.4 Hz, 1H), 9.28 (s, 1H), 6.50 (dd, J = 16.9, 1.0 Hz, 1H), 6.23 (dd, J = 16.9, 10.3 Hz, 1H), 5.92 (dd, J = 10.3, 1.1 Hz, 1H), 3.65 (td, J = 7.2, 5.3 Hz, 2H), 1.74–1.61 (m, 2H), 1.40–1.15 (m, 20H), 0.87 (t, J = 6.8 Hz, 3H).

[0546] Step 3: To a solution of Compound 56-1 (100 mg, 0.69 mmol), BHT (50 mg), and acetic acid (50 mg) in ACN (2 mL) under N2 atmosphere was added Compound 56-2 (1.12 g, 3.3 mmol). The mixture was stirred at room temperature for 16 h. TLC (eluent: DCM / methanol = 10 / 1) indicated that the starting materials had been substantially consumed. The mixture was purified by silica gel column chromatography and concentrated under reduced pressure to give Compound 56 (850 mg, yield 65%).

[0547] of Compound 56 1 H NMR: (400 MHz, chloroform-d) δ 10.70–10.29 (m, 8H), 3.57 (td, J = 7.3, 5.3 Hz, 8H), 3.00–2.83 (m, 4H), 2.73 (t, J = 5.8 Hz, 8H), 2.60 (t, J = 5.7 Hz, 8H), 2.53 (d, J = 8.6 Hz, 7H), 1.96 (q, J = 7.7 Hz, 4H), 1.63 (t, J = 7.3 Hz, 8H), 1.48–1.10 (m, 80H), 0.87 (t, J = 6.7 Hz, 13H). HRMS: [M+H] + = 1339.01158

[0548] Example 2: General Scheme 2

[0549]

[0550] wherein

[0551] each W is independently selected from O, S, and NH;

[0552] each Y is independently selected from O, S, and NH;

[0553] each n is independently 1, 2, or 3;

[0554] each m is 0 or 1;

[0555] Each p is 1 or 2.

[0556] General procedure

[0557] General procedure 1: Heat an ethanol solution containing compound S2-1 at 60 - 80 °C. Add compound S2-2 to the mixture. After the reaction is complete, cool the mixture to room temperature, then wash and dry to obtain the selectively mono-substituted intermediate S2-3.

[0558] General procedure 2: Add a mixture of intermediate S2-3, compound S2-4 (containing ACN and / or acetic acid), and BHT to a sealed container. Heat the mixture at 60 - 80 °C for 12 - 24 hours, then cool to room temperature. The mixture is purified by column chromatography to obtain compound S2-5.

[0559] Example 2.1: Synthesis of compound 11

[0560]

[0561] Step 1: Add compound 11-4-1 (280 mg, 1.0 mmol), anhydrous DCM (6 mL), and compound 11-4-2 (104 mg, 1.1 mmol) to a container in sequence at room temperature. Stir the mixture for 5 minutes, then cool it in an ice bath. Add triethylamine (211 mg, 2.1 mmol) to the mixture. Let the mixture warm up to room temperature and stir at the same temperature for 5 hours. TLC (eluent: petroleum ether / DCM = 3 / 1, stained with KMnO4) indicates that the reaction is basically complete. The mixture is purified by silica gel column chromatography (eluent: petroleum ether containing 0 - 33% DCM, v / v), and concentrated under reduced pressure to obtain compound 11-4 as a colorless oil (220 mg, yield 65%).

[0562] For compound 11-4 1 1H NMR: (400 MHz, chloroform-d) δ 6.33 (dd, J = 17.3, 1.6 Hz, 1H), 6.05 (dd, J = 17.4, 10.4 Hz, 1H), 5.74 (dd, J = 10.4, 1.5 Hz, 1H), 5.34–5.20 (m, 2H), 4.08 (t, J = 6.8 Hz, 2H), 1.94 (q, J = 6.4 Hz, 4H), 1.59 (p, J = 6.8 Hz, 2H), 1.22 (q, J = 8.0, 5.5 Hz, 24H), 0.81 (t, J = 6.7 Hz, 3H).

[0563] Step 2: To a solution of Compound 11-1 (9.46 g, 65.13 mmol) in ethanol (600 mL) heated at 65 °C (internally) was added Compound 11-2 (2.0 g, 12.80 mmol). The mixture was heated at 70 - 75 °C (externally) overnight. The mixture was cooled to room temperature and then concentrated under reduced pressure. The residue was diluted with DCM (500 mL), and while stirring, brine (400 mL) was added thereto. The mixture was heated at 35 °C (internally) and stirred for 10 minutes, then allowed to stand for separation. The aqueous layer was extracted again with DCM (500 mL), repeating 4 - 5 times. TLC (eluent: DCM / MeOH = 5:1, with 3 drops of NH4OH added) indicated the completion of extraction. The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure to obtain Compound 11-3 (3.47 g, yield 97%).

[0564] Step 3: Under a N2 atmosphere, Compound 11-4 (308 mg, 0.95 mmol) and BHT (5 mg) were added to a 4 mL container containing Compound 11-3 (80 mg, 0.27 mmol). The mixture was heated at 70 °C for 48 hours. TLC indicated the completion of the reaction. The mixture was purified by silica gel column chromatography (eluent: DCM containing 0 - 10% methanol, v / v) and concentrated under reduced pressure to obtain Compound 11 (250 mg, yield 74%).

[0565] For Compound 11 1 1H NMR: (400 MHz, chloroform-d) δ 5.44–5.26 (m, 5H), 4.05 (q, J = 6.4 Hz, 6H), 3.58 (dd, J = 6.9, 3.4 Hz, 1H), 2.97–2.83 (m, 1H), 2.72 (dt, J = 24.8, 7.0 Hz, 5H), 2.57 (dt, J = 14.3, 7.3 Hz, 1H), 2.01 (q, J = 6.4 Hz, 12H), 1.61 (t, J = 7.0 Hz, 10H), 1.28 (q, J = 8.2, 4.7 Hz, 91H), 0.87 (t, J = 6.6 Hz, 14H).

[0566] Example 2.2: Synthesis of Compound 12

[0567]

[0568] Step 1: In an ice bath, add anhydrous DCM (200 mL), NaHCO3 (7.43 g, 88.5 mmol), compound 12-4-1 (9.6 g, 35.7 mmol), and compound 12-4-2 (4 g, 44.2 mmol) to a container in sequence. Warm the mixture to room temperature and stir at the same temperature for 2 hours. TLC (stained with I2) indicates the completion of the reaction. Quench the reaction with water (200 mL) and stir for 10 minutes. Wash the organic layer twice with saturated aqueous NaHCO3 solution (200 mL), and then wash with water (200 mL). Dry and concentrate the organic layer to obtain crude compound 12-4 (11.4 g, 87%).

[0569] of compound 12-4 1 1H NMR: (400 MHz, chloroform-d) δ 6.29 (dd, J = 17.0, 1.5 Hz, 1H), 6.10 (dd, J = 17.0, 10.3 Hz, 1H), 5.68–5.48 (m, 2H), 3.39–3.31 (m, 2H), 1.56 (p, J = 7.1 Hz, 2H), 1.28 (s, 29H), 0.90 (t, J = 6.7 Hz, 3H).

[0570] Step 2: Add compound 12-2 (2.0 g, 12.97 mmol) to a solution of compound 12-1 (9.46 g, 65.13 mmol) in ethanol (600 mL) heated (internally) at 65 °C. Heat the mixture overnight at 70 - 75 °C (externally). Cool the mixture to room temperature and then concentrate it under reduced pressure. Dilute the residue with DCM (500 mL) and add brine (400 mL) thereto while stirring. Heat the mixture at 35 °C (internally) and stir for 10 minutes, then let it stand for separation. Extract the aqueous layer with DCM (500 mL) again, repeating 4 - 5 times. TLC (eluent: DCM / MeOH = 5:1, adding 3 drops of NH4OH) indicates the completion of the extraction. Dry the combined organic layers over Na2SO4 and concentrate under reduced pressure to obtain compound 12-3 (3.4 g, yield 95%).

[0571] Step 3: Add compound 12-4 (384 mg, 1.19 mmol) and BHT (5 mg) to compound 12-3 (100 mg, 0.33 mmol). Heat the mixture for 48 hours. TLC indicates the completion of the reaction. Dilute the mixture with DCM (10 mL) and concentrate under reduced pressure. Purify the residue by silica gel column chromatography (eluent: DCM containing 0 - 10% methanol, v / v) and concentrate under reduced pressure to obtain compound 12 (140 mg, yield 45%) as a colorless oil.

[0572] of compound 121 1H NMR: (400 MHz, chloroform-d) δ 5.88–5.71 (m, 1H), 5.49–5.25 (m, 6H), 5.05–4.81 (m, 2H), 4.05 (q, J = 6.4 Hz, 7H), 3.58 (td, J = 7.0, 3.6 Hz, 1H), 2.90 (dt, J = 14.5, 7.4 Hz, 1H), 2.73 (dt, J = 25.2, 6.9 Hz, 6H), 2.57 (dt, J = 14.4, 7.3 Hz, 2H), 2.43 (q, J = 7.0, 6.4 Hz, 13H), 2.36–1.91 (m, 27H), 1.61 (t, J = 7.1 Hz, 12H), 1.30 (ddd, J = 17.8, 10.8, 5.4 Hz, 96H), 0.88 (t, J = 6.7 Hz, 12H).

[0573] Example 2.3: Synthesis of Compound 13

[0574]

[0575] Step 1: To a solution of Compound 13-1 (9.46 g, 65.13 mmol) in ethanol (600 mL) heated (internally) at 65 °C was added Compound 13-2 (2.2 g, 11.94 mmol). The mixture was heated (externally) at 70 - 75 °C overnight. The mixture was cooled to room temperature and then concentrated under reduced pressure. The residue was diluted with DCM (500 mL), and while stirring, brine (400 mL) was added thereto. The mixture was heated (internally) at 35 °C and stirred for 10 minutes, and then allowed to stand for separation. The aqueous layer was extracted again with DCM (500 mL), repeating 4 - 5 times. TLC (eluent: DCM / MeOH = 5:1, adding 3 drops of NH4OH) indicated the completion of extraction. The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure to obtain Compound 13-3 (3.5 g, yield 97%).

[0576] Step 2: To Compound 13-3 (100 mg, 0.30 mmol) were added Compound 13-4 (350 mg, 1.08 mmol) and BHT (5 mg). The mixture was heated for 48 hours. TLC indicated the completion of the reaction. The mixture was diluted with DCM (10 mL) and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: DCM containing 0 - 5% methanol, v / v) and concentrated under reduced pressure to obtain Compound 13 as a colorless oil (198 mg, yield 70%).

[0577] of Compound 13 11H NMR: (400 MHz, chloroform-d) δ 4.05 (q, J = 6.6 Hz, 6H), 3.58 (s, 1H), 2.91 (dt, J = 14.3, 7.5 Hz, 1H), 2.72 (dt, J = 23.3, 6.7 Hz, 7H), 2.42 (h, J = 6.4 Hz, 10H), 2.24 (d, J = 48.6 Hz, 6H), 1.68–1.52 (m, 9H), 1.25 (s, 112H), 0.88 (t, J = 6.8 Hz, 12H).

[0578] Example 2.4: Synthesis of Compound 14

[0579]

[0580] Step 1: In an ice bath, anhydrous DCM (300 mL), NaHCO3 (11.14 g, 132.6 mmol), Compound 14-4-1 (14.15 g, 76.3 mmol), and Compound 12-4-2 (6.0 g, 66.3 mmol) were successively added to a container. The mixture was warmed to room temperature and stirred at the same temperature for 2 hours. TLC (stained with I2) indicated the completion of the reaction. The reaction was quenched with water (300 mL) and stirred for 10 minutes. The organic layer was washed twice with saturated aqueous NaHCO3 solution (300 mL), and then washed with water (300 mL). The organic layer was dried and concentrated to obtain crude Compound 14-4 (8.4 g, 47%).

[0581] For Compound 14-4 1 1H NMR: (400 MHz, chloroform-d) δ 6.27 (d, J = 16.9 Hz, 1H), 6.09 (dd, J = 17.0, 10.2 Hz, 1H), 5.64 (t, J = 11.1 Hz, 2H), 3.32 (q, J = 6.8 Hz, 2H), 1.53 (p, J = 7.1 Hz, 2H), 1.26 (s, 24H), 0.88 (t, J = 6.6 Hz, 3H).

[0582] Step 2: To a solution of Compound 14-1 (1.85 g, 13.01 mmol) in ethanol (100 mL) heated at 65 °C (internally) was added Compound 14-2 (0.40 g, 2.17 mmol). The mixture was heated at 70 - 75 °C (externally) overnight. The mixture was cooled to room temperature and then concentrated under reduced pressure. The residue was diluted with DCM (300 mL), and while stirring, tap water (300 mL) was added thereto. The mixture was heated at 35 °C (internally) and stirred for 10 minutes, then allowed to stand for separation. Extraction was repeated 3 - 4 times. TLC (eluent: DCM / MeOH = 10:1, adding 1 drop of NH4OH) indicated the completion of extraction. The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure to obtain Compound 14-3 (0.63 g, yield 68%).

[0583] Step 3: To a 2 mL vial containing Compound 14-3 (20 mg, 0.06 mmol) was added Compound 14-4 (51.6 mg, 0.22 mmol), BHT (14 mg), and acetic acid (3 μL). The mixture was heated at 80 °C for 60 hours. TLC (eluent: DCM / MeOH = 10 / 1, containing NH4OH) indicated that the reaction was substantially complete. The mixture was purified by silica gel column chromatography (eluent: DCM containing 1 - 1.6% methanol and 0.5% NH4OH, v / v) and concentrated under reduced pressure to obtain Compound 14 (17 mg, yield 27%).

[0584] For Compound 14 1 1H NMR: (400 MHz, chloroform-d) δ 6.93 (t, J = 5.6 Hz, 1H), 3.74–3.55 (m, 1H), 3.38–3.08 (m, 5H), 2.98 (dq, J = 15.1, 7.4 Hz, 1H), 2.69 (dddd, J = 35.5, 21.3, 13.6, 6.6 Hz, 5H), 2.49–2.11 (m, 11H), 2.07–1.59 (m, 4H), 1.46 (dh, J = 16.5, 8.5, 7.9 Hz, 10H), 1.26 (d, J = 8.9 Hz, 73H), 0.87 (t, J = 6.7 Hz, 13H).

[0585] Example 2.5: Synthesis of Compound 15

[0586]

[0587] Step 1: To a solution of Compound 15-1 (1.27 g, 8.93 mmol) in ethanol (100 mL) heated at 65 °C (internally) was added Compound 15-2 (0.40 g, 1.49 mmol, 85% purity). The mixture was heated at 70 - 75 °C (externally) overnight. The mixture was cooled to room temperature and then concentrated under reduced pressure. The residue was diluted with DCM (300 mL), and tap water (300 mL) was added thereto while stirring. The mixture was heated at 35 °C (internally) and stirred for 10 minutes, and then allowed to stand for separation. Extraction was repeated 3 - 4 times. TLC (eluent: DCM / MeOH = 10:1, with 1 drop of NH4OH added) indicated the completion of extraction. The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure to give Compound 15-3 (0.40 g, yield 35%).

[0588] Step 2: To a 2 mL vial containing Compound 15-3 (20 mg, 0.05 mmol) was added Compound 15-4 (58.5 mg, 0.18 mmol) and BHT (13 mg). The mixture was heated at 70 °C for 40 hours. TLC (eluent: DCM / MeOH = 40 / 1) indicated that the reaction was substantially complete. The mixture was purified by flash column chromatography on silica gel (eluent: DCM containing 0 - 5% methanol, v / v) and concentrated under reduced pressure to give Compound 15 (45.6 mg, yield 54%).

[0589] For Compound 15 1 1H NMR: (400 MHz, chloroform-d) δ 4.05 (qd, J = 6.9, 2.3 Hz, 5H), 3.59 (s, 1H), 2.94 (s, 1H), 2.75 (tt, J = 19.9, 9.3 Hz, 4H), 2.41 (t, J = 7.1 Hz, 6H), 2.34–2.10 (m, 4H), 1.94–1.56 (m, 13H), 1.26 (s, 110H), 0.88 (t, J = 6.7 Hz, 12H).

[0590] Example 2.6: Synthesis of Compound 16

[0591]

[0592] Step 1: To a solution of Compound 16-1 (1.145 g, 9.77 mmol) in ethanol (100 mL) heated at 65 °C (inside), Compound 16-2 (0.30 g, 1.63 mmol) was added. The mixture was heated at 70 - 75 °C (outside) overnight. The mixture was cooled to room temperature and then concentrated under reduced pressure. The residue was diluted with DCM (300 mL), and brine (300 mL) was added thereto with stirring. The mixture was heated at 35 °C (inside) and stirred for 10 minutes, and then allowed to stand for separation. Extraction was repeated 3 - 4 times. TLC (eluent: DCM / MeOH = 5:1, with 2 drops of NH4OH added) indicated the completion of extraction. The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure to obtain Compound 16-3 (0.47 g, yield 95%).

[0593] Step 2: To a 2 mL vial containing Compound 16-3 (20 mg, 0.07 mmol), Compound 16-4 (61 mg, 0.25 mmol, prepared according to the method of Step 1 of Example 2.4), BHT (15 mg), and acetic acid (3 μL) were added. The mixture was heated at 80 °C for 105 hours. TLC (eluent: DCM / MeOH = 10 / 1, containing NH4OH) indicated the completion of the reaction. The mixture was purified by silica gel column chromatography (eluent: DCM containing 1 - 1.3% methanol and 0.5% NH4OH, v / v), and concentrated under reduced pressure to obtain Compound 16 (45 mg, yield 66%).

[0594] For Compound 16 1 1H NMR: (400 MHz, chloroform-d) δ 7.12–6.96 (m, 1H), 6.93 (t, J = 5.5 Hz, 1H), 3.67–3.48 (m, 1H), 3.10 (dq, J = 26.9, 6.6 Hz, 6H), 2.91–2.81 (m, 1H), 2.54 (d, J = 37.5 Hz, 9H), 2.44–2.22 (m, 11H), 2.16 (dd, J = 14.0, 6.4 Hz, 2H), 2.08–1.85 (m, 2H), 1.49–1.30 (m, 9H), 1.20 (d, J = 11.0 Hz, 80H), 0.81 (t, J = 6.8 Hz, 14H).

[0595] Example 2.7: Synthesis of Compound 17

[0596]

[0597] Step 1: Compound 17-3 was prepared according to the method of Step 1 of Example 2.6.

[0598] Step 2: To a 2 mL vial containing compound 17-3 (19.6 mg, 0.07 mmol) was added compound 17-4 (78.6 mg, 0.24 mmol) and BHT (17.2 mg). The mixture was heated at 70 °C for 67 h and then at 90 °C for 23 h. TLC (eluent: DCM / MeOH = 20 / 1) indicated that the reaction was substantially complete. The mixture was purified by flash column chromatography on silica gel (eluent: DCM containing 0 - 5% methanol, v / v), and concentrated under reduced pressure to give compound 17 (40.8 mg, yield 49%).

[0599] of compound 17 1 1H NMR: (400 MHz, chloroform-d) δ 4.05 (q, J = 7.2 Hz, 5H), 3.62 (dt, J = 11.9, 6.5 Hz, 1H), 2.94 (dt, J = 14.2, 7.3 Hz, 2H), 2.84 (q, J = 6.6 Hz, 2H), 2.77 (t, J = 6.9 Hz, 5H), 2.65–2.58 (m, 1H), 2.47 (dq, J = 13.8, 7.5, 7.0 Hz, 7H), 2.40–2.17 (m, 2H), 1.71–1.53 (m, 6H), 1.25 (s, 106H), 0.88 (t, J = 6.8 Hz, 12H).

[0600] Example 2.8: Synthesis of compound 18

[0601]

[0602] Step 1: To a solution of compound 18-1 (0.61 g, 5.86 mmol) in ethanol (100 mL) heated at 65 °C (internally) was added compound 18-2 (0.18 g, 0.98 mmol). The mixture was heated at 70 - 75 °C (externally) overnight. The mixture was cooled to room temperature and then concentrated under reduced pressure. The residue was diluted with DCM (200 mL), and water (200 mL) was added thereto with stirring. The mixture was heated at 35 °C (internally) and stirred for 10 min, and then allowed to stand for separation. Extraction was repeated 3 - 4 times. TLC (eluent: DCM / MeOH = 10:1, with 1 drop of NH4OH added) indicated that the extraction was complete. The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure to give compound 18-3 (0.25 g, yield 50%).

[0603] Step 2: To a 2 mL vial containing compound 18-3 (20 mg, 0.07 mmol) was added compound 18-4 (59 mg, 0.25 mmol, prepared according to the method of Step 1 of Example 2.4) and BHT (17.2 mg). The mixture was heated at 70 °C for 67 h and then at 90 °C for 23 h. TLC (eluent: DCM / MeOH = 20 / 1) indicated that the reaction was substantially complete. The mixture was purified by flash column chromatography on silica gel (eluent: DCM containing 0 - 5% methanol, v / v), and concentrated under reduced pressure to give compound 18 (40.8 mg, yield 49%).

[0604] of compound 18 1 H NMR: (400 MHz, chloroform-d) δ 6.94 (t, J = 5.7 Hz, 2H), 6.77 (t, J = 5.8 Hz, 1H), 3.66 (d, J = 9.6 Hz, 1H), 3.51 (dhept, J = 10.1, 5.1 Hz, 4H), 3.24–3.13 (m, 6H), 3.02 (s, 2H), 2.89–2.72 (m, 6H), 2.71–2.52 (m, 4H), 2.37 (tq, J = 14.5, 9.0, 7.3 Hz, 7H), 1.54–1.38 (m, 9H), 1.26 (d, J = 8.7 Hz, 79H), 0.87 (t, J = 6.7 Hz, 15H).

[0605] Example 2.9: Synthesis of compound 19

[0606]

[0607] Step 1: Compound 19-3 was prepared according to the method of Step 1 of Example 2.8.

[0608] Step 2: To a 2 mL vial containing compound 19-3 (20 mg, 0.07 mmol) was added compound 19-4 (85.1 mg, 0.26 mmol) and BHT (14.3 mg). The mixture was heated at 70 °C for 60 h. TLC (eluent: DCM / MeOH = 20 / 1) indicated that the reaction was substantially complete. The mixture was purified by flash column chromatography on silica gel (eluent: DCM containing 0 - 5% methanol, v / v), and concentrated under reduced pressure to give compound 19 (54.1 mg, yield 62%).

[0609] of compound 19 11H NMR: (400 MHz, chloroform-d) δ 4.05 (q, J = 6.6 Hz, 5H), 3.60 (s, 1H), 3.49 (t, J = 6.2 Hz, 4H), 3.00 (s, 1H), 2.84 (t, J = 7.3 Hz, 5H), 2.67 (t, J = 6.0 Hz, 3H), 2.46 (t, J = 7.3 Hz, 7H), 2.39–2.26 (m, 2H), 1.61 (h, J = 6.1 Hz, 7H), 1.46–1.39 (m, 4H), 1.26 (s, 97H), 0.88 (t, J = 6.7 Hz, 12H).

[0610] Example 2.10: Synthesis of Compound 20

[0611]

[0612] Step 1: Compound 20-3 was prepared by the method of Step 2 in Example 2.2.

[0613] Step 2: Under a N2 atmosphere, compound 20-4 (310 mg, 0.96 mmol, prepared by the method of Step 1 in Example 2.1) and BHT (5 mg) were added to a 4 mL container containing compound 20-3 (80 mg, 0.27 mmol). The mixture was heated at 70 °C for 48 h. TLC indicated the completion of the reaction. The mixture was purified by silica gel column chromatography (eluent: DCM containing 0-10% methanol, v / v), and concentrated under reduced pressure to obtain compound 20 (230 mg, yield 68%).

[0614] For Compound 20 1 1H NMR: (400 MHz, chloroform-d) δ 5.88–5.71 (m, 1H), 5.49–5.25 (m, 6H), 5.05–4.81 (m, 2H), 4.05 (q, J = 6.4 Hz, 7H), 3.58 (td, J = 7.0, 3.6 Hz, 1H), 2.90 (dt, J = 14.5, 7.4 Hz, 1H), 2.73 (dt, J = 25.2, 6.9 Hz, 6H), 2.57 (dt, J = 14.4, 7.3 Hz, 2H), 2.43 (q, J = 7.0, 6.4 Hz, 13H), 2.36–1.91 (m, 27H), 1.61 (t, J = 7.1 Hz, 12H), 1.30 (ddd, J = 17.8, 10.8, 5.4 Hz, 96H), 0.88 (t, J = 6.7 Hz, 12H).

[0615] Example 2.11: Synthesis of Compound 21

[0616]

[0617] Step 1: To a solution of compound 21-1 (1.44 g, 9.91 mmol) in ethanol (100 mL) heated at 65 °C (inside) was added compound 21-2 (0.40 g, 1.75 mmol). The mixture was heated at 70 - 75 °C (outside) overnight. The mixture was cooled to room temperature and then concentrated under reduced pressure. The residue was diluted with DCM (200 mL), and water (200 mL) was added thereto while stirring. The mixture was heated at 35 °C (inside) and stirred for 10 minutes, and then allowed to stand for separation. Extraction was repeated 3 - 4 times. TLC (eluent: DCM / MeOH = 5:1, with 3 drops of NH4OH added) indicated the completion of extraction. The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure to obtain compound 21-3 (0.43 g, yield 66%).

[0618] Step 2: Under a N2 atmosphere, compound 21-4 (270 mg, 0.94 mmol, prepared according to the method of Step 1 of Example 2.1) and BHT (5 mg) were added to a 4 mL container containing compound 21-3 (100 mg, 0.27 mmol). The mixture was heated at 70 °C for 48 hours. TLC indicated the completion of the reaction. The mixture was purified by silica gel column chromatography (eluent: DCM containing 0 - 10% methanol, v / v) and concentrated under reduced pressure to obtain compound 21 (220 mg, yield 67%).

[0619] For compound 21 1 H NMR: (400 MHz, chloroform-d) δ 5.45–5.21 (m, 5H), 4.05 (td, J = 6.8, 4.3 Hz, 6H), 3.89–3.74 (m, 1H), 3.53–3.34 (m, 4H), 2.98–2.82 (m, 1H), 2.74 (td, J = 7.0, 4.5 Hz, 6H), 2.62–2.35 (m, 16H), 2.00 (hept, J = 5.9, 5.2 Hz, 12H), 1.58 (dt, J = 18.9, 7.0 Hz, 12H ), 1.47–1.07 (m, 90H), 0.87 (t, J = 6.7 Hz, 13H).

[0620] Example 2.12: Synthesis of compound 22

[0621]

[0622] Step 1: Compound 22-3 was prepared according to the method of Step 2 of Example 2.1.

[0623] Step 2: Under an N2 atmosphere, compound 22-4 (350 mg, 1.09 mmol) and BHT (10 mg) were added to a 4 mL container containing compound 22-3 (100 mg, 0.33 mmol). The mixture was heated at 70 °C for 48 h. TLC indicated completion of the reaction. The mixture was purified by silica gel column chromatography (eluent: DCM containing 0-10% methanol, v / v), and concentrated under reduced pressure to give compound 22 (230 mg, 55% yield).

[0624] of compound 22 1 1H NMR: (400 MHz, chloroform-d) δ 5.80 (ddt, J = 16.9, 10.2, 6.7 Hz, 1H), 5.46–5.23 (m, 13H), 5.05–4.88 (m, 2H), 4.17–3.94 (m, 6H), 3.58 (ddt, J = 10.4, 6.9, 3.4 Hz, 1H), 3.02–2.83 (m, 1H), 2.82–2.65 (m, 12H), 2.56 (q, J = 7.4, 6.8 Hz, 2H), 2.51–2.11 (m, 21H), 2.05 (q, J = 6.7 Hz, 16H), 1.61 (dd, J = 10.6, 4.3 Hz, 12H), 1.49–1.10 (m, 71H), 0.89 (t, J = 6.8 Hz, 12H).

[0625] Example 2.13: Synthesis of compound 23

[0626]

[0627] Step 1: Compound 23-3 was prepared by the method of Step 2 in Example 2.2

[0628] Step 2: Under an N2 atmosphere, compound 23-4 (360 mg, 1.12 mmol) and BHT (10 mg) were added to a 4 mL container containing compound 23-3 (100 mg, 0.33 mmol). The mixture was heated at 70 °C for 48 h. TLC indicated completion of the reaction. The mixture was purified by silica gel column chromatography (eluent: DCM containing 0-10% methanol, v / v), and concentrated under reduced pressure to give compound 23 (221 mg, 56% yield).

[0629] of compound 23 11H NMR: (400 MHz, chloroform-d) δ 5.45–5.26 (m, 12H), 4.05 (q, J = 7.1 Hz, 6H), 3.60 (s, 1H), 2.93 (dt, J = 13.9, 7.5 Hz, 3H), 2.75 (dt, J = 17.7, 6.7 Hz, 15H), 2.54–2.20 (m, 14H), 2.05 (q, J = 6.8 Hz, 14H), 1.88 (s, 5H), 1.62 (p, J = 6.7 Hz, 9H), 1.46–1.19 (m, 72H), 0.88 (td, J = 6.8, 4.5 Hz, 14H).

[0630] Example 2.14: Synthesis of Compound 24

[0631]

[0632] Step 1: Compound 24-3 was prepared by the method of Step 1 of Example 2.11.

[0633] Step 2: Under a N2 atmosphere, compound 24-4 (350 mg, 1.09 mmol) and BHT (10 mg) were added to a 4 mL container containing compound 24-3 (120 mg, 0.32 mmol). The mixture was heated at 70 °C for 48 h. TLC indicated the completion of the reaction. The mixture was purified by silica gel column chromatography (eluent: DCM containing 0-10% methanol, v / v), and concentrated under reduced pressure to give compound 24 (205 mg, yield 50%).

[0634] For Compound 24 1 1H NMR: (400 MHz, chloroform-d) δ 5.48–5.24 (m, 14H), 4.06 (q, J = 7.2 Hz, 6H), 3.79 (dq, J = 9.9, 5.1 Hz, 1H), 2.87 (dq, J = 17.0, 9.7, 8.5 Hz, 3H), 2.77 (t, J = 6.4 Hz, 7H), 2.68 (dt, J = 13.0, 6.3 Hz, 5H), 2.44 (dt, J = 13.2, 7.7 Hz, 7H), 2.29 (d, J = 6.9 Hz, 4H), 2.13–2.03 (m, 12H), 1.59 (dt, J = 17.9, 6.9 Hz, 18H), 1.43–1.20 (m, 88H), 0.88 (td, J = 6.8, 4.1 Hz, 16H).

[0635] Example 2.15: Synthesis of Compound 25

[0636]

[0637] Step 1: To a solution of compound 25-1 (1.0 g, 8.76 mmol) in ethanol (100 mL) heated internally at 65 °C was added compound 25-2 (0.27 g, 1.46 mmol). The mixture was heated externally at 70 - 75 °C overnight. The mixture was cooled to room temperature and then concentrated under reduced pressure. The residue was diluted with DCM (200 mL), and while stirring, diluted aqueous NaOH solution (200 mL) was added thereto. The mixture was heated internally at 35 °C and stirred for 10 minutes, then allowed to stand for separation. Extraction was repeated 3 - 4 times. The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure to obtain compound 25-3 (0.26 g, yield 60%).

[0638] Step 2: Under a N2 atmosphere, to a 4 mL container containing compound 25-3 (120 mg, 0.40 mmol) was added compound 25-4 (350 mg, 0.96 mmol, prepared according to the method of Step 1 of Example 2.1) and BHT (10 mg). The mixture was heated at 70 °C for 48 hours. TLC indicated completion of the reaction. The mixture was purified by silica gel column chromatography (eluent: DCM containing 0 - 10% methanol, v / v) and concentrated under reduced pressure to obtain compound 25 (215 mg, yield 56%).

[0639] For compound 25 1 1H NMR: (400 MHz, chloroform-d) δ 5.51–5.21 (m, 5H), 4.05 (t, J = 7.1 Hz, 6H), 3.57 (s, 2H), 2.90 (dt, J = 25.7, 9.3 Hz, 5H), 2.64 (dt, J = 24.3, 7.2 Hz, 5H), 2.53–2.18 (m, 14H), 1.99 (dt, J = 16.0, 8.3 Hz, 15H), 1.76 (t, J = 14.1 Hz, 5H), 1.61 (t, J = 7.1 Hz, 10H), 1.49–1.00 (m, 106H), 0.87 (t, J = 6.6 Hz, 15H).

[0640] Example 2.16: Synthesis of compound 26

[0641]

[0642] Step 1: Compound 26-3 was prepared according to the method of Step 1 of Example 2.15.

[0643] Step 2: Under N2 atmosphere, compound 26-4 (309 mg, 0.96 mmol) and BHT (10 mg) were added to a 4 mL container containing compound 26-3 (120 mg, 0.40 mmol). The mixture was heated at 70 °C for 48 h. TLC indicated the completion of the reaction. The mixture was purified by silica gel column chromatography (eluent: DCM containing 0 - 6.3% methanol, v / v), and concentrated under reduced pressure to obtain compound 26 (260 mg, yield 69%).

[0644] of compound 26 1 1H NMR: (400 MHz, chloroform-d) δ 5.35 (tt, J = 11.3, 6.3 Hz, 8H), 4.06 (q, J = 6.8 Hz, 4H), 3.66–3.48 (m, 1H), 2.92 (dt, J = 13.1, 7.6 Hz, 2H), 2.77 (t, J = 6.5 Hz, 5H), 2.61 (dd, J = 12.6, 6.4 Hz, 3H), 2.48–2.22 (m, 7H), 2.05 (q, J = 6.8 Hz, 10H), 1.86 (d, J = 13.5 Hz, 2H), 1.62 (p, J = 7.1 Hz, 9H), 1.49–1.14 (m, 59H), 0.88 (td, J = 6.8, 4.3 Hz, 10H).

[0645] Example 2.17: Synthesis of compound 27

[0646]

[0647] Step 1: Compound 27-3 was prepared by the method of Step 1 in Example 2.3

[0648] Step 2: Acetic acid (14.6 mg) was added to a 2 mL container containing compound 27-3 (400 mg, 1.2 mmol), BHT (53.4 mg) and compound 27-4 (1.24 g, 4.4 mmol, prepared by the method of Steps 1 to 3 in Example 1.7). The mixture was heated at 80 °C for 42 h. TLC (eluent: DCM / MeOH / NH4OH = 10 / 1 / 1) indicated the completion of the reaction. The mixture was purified by flash silica gel column chromatography (eluent: DCM containing 1.4 - 24% methanol and 0.5% NH4OH, v / v), and concentrated under reduced pressure to obtain compound 27 (390 mg, yield 27%).

[0649] of compound 27 11H NMR: (400 MHz, chloroform-d) δ 7.87 (t, J = 5.5 Hz, 1H), 7.68 (t, J = 5.5 Hz, 2H), 4.15 - 4.01 (m, 6H), 4.01 - 3.88 (m, 4H), 3.68 (dt, J = 17.8, 8.6 Hz, 1H), 3.03 - 2.91 (m, 1H), 2.74 (t, J = 6.0 Hz, 4H), 2.66 (dt, J = 14.1, 7.3 Hz, 2H), 2.59 - 2.39 (m, 9H), 2.40–2.19 (m, 5H), 1.81 - 1.66 (m, 4H), 1.62 (p, J = 6.9 Hz, 6H), 1.52 - 1.04 (m, 68H), 0.87 (q, J = 5.9, 5.5 Hz, 12H). HR-MS: [M+H] + = 1180.00680。

[0650] Example 2.18: Synthesis of Compound 30

[0651]

[0652] Step 1: Compound 30-3 was prepared in a similar manner to Step 2 of Example 2.1.

[0653] Step 2: Under a N2 atmosphere, BHT (5 mg) and Compound 30-4 (340 mg, 1.05 mmol, prepared according to the method of Step 1 of Example 2.1) were added to a 4 mL container containing Compound 30-3 (80 mg, 0.29 mmol). The mixture was heated at 70 °C for 48 h. TLC indicated the completion of the reaction. The mixture was purified by flash column chromatography on silica gel (eluent: DCM containing 0 - 10% methanol, v / v), and concentrated under reduced pressure to obtain Compound 30 (295 mg, yield 81%).

[0654] For Compound 30 1 1H NMR: (400 MHz, chloroform-d) δ 5.44–5.26 (m, 5H), 4.05 (q, J = 6.4 Hz, 6H), 3.58 (dd, J = 6.9, 3.4 Hz, 1H), 2.97–2.83 (m, 1H), 2.72 (dt, J = 24.8, 7.0 Hz, 5H), 2.57 (dt, J = 14.3, 7.3 Hz, 1H), 2.01 (q, J = 6.4 Hz, 12H), 1.61 (t, J = 7.0 Hz, 10H), 1.28 (q, J = 8.2, 4.7 Hz, 91H), 0.87 (t, J = 6.6 Hz, 14H). HR-MS: [M+H] + = 1240.15427。

[0655] Example 2.19: Synthesis of Compound 31

[0656]

[0657] Step 1: Compound 31-3 was prepared by the method of Step 1 in Example 2.3.

[0658] Step 2: Under N2 atmosphere, BHT (5 mg) and Compound 31-4 (308 mg, 0.95 mmol, prepared by the method of Step 1 in Example 2.1) were added to a 4 mL container containing Compound 31-3 (80 mg, 0.24 mmol). The mixture was heated at 70 °C for 48 h. TLC indicated the completion of the reaction. The mixture was purified by flash column chromatography on silica gel (eluent: DCM containing 0 - 10% methanol, v / v), and concentrated under reduced pressure to give Compound 31 (250 mg, yield 74%).

[0659] For Compound 31 1 1H NMR: (400 MHz, chloroform-d) δ 5.49–5.22 (m, 6H), 4.05 (q, J = 6.5 Hz, 6H), 3.58 (td, J = 6.8, 3.4 Hz, 1H), 2.91 (dt, J = 14.3, 7.5 Hz, 1H), 2.72 (dt, J = 23.7, 6.9 Hz, 6H), 2.43 (q, J = 7.6 Hz, 13H), 2.34–2.12 (m, 6H), 2.00 (dh, J = 11.6, 6.5 Hz, 11H), 1.63 (s, 10H), 1.30 (dt, J = 16.3, 9.4 Hz, 91H), 0.88 (t, J = 6.7 Hz, 13H). [M+H] + = 1297.21294.

[0660] Example 2.20: Synthesis of Compound 32

[0661]

[0662] Step 1: Compound 32-3 was prepared by a method similar to that of Step 1 in Example 2.3.

[0663] Step 2: Under N2 atmosphere, BHT (5 mg) and Compound 32-4 (259 mg, 0.80 mmol, prepared by the method of Step 1 in Example 2.1) were added to a 4 mL container containing Compound 32-3 (80 mg, 0.22 mmol). The mixture was heated at 70 °C for 48 h. TLC indicated the completion of the reaction. The mixture was purified by flash column chromatography on silica gel (eluent: DCM containing 0 - 10% methanol, v / v), and concentrated under reduced pressure to give Compound 32 (220 mg, yield 74%).

[0664] of Compound 32 1 H NMR: (400 MHz, chloroform-d) δ 5.45–5.23 (m, 6H), 4.05 (q, J = 6.5 Hz, 6H), 3.58 (td, J = 6.7, 3.4 Hz, 1H), 2.90 (dt, J = 14.3, 7.5 Hz, 1H), 2.80–2.36 (m, 19H), 2.33–1.87 (m, 20H), 1.60 (q, J = 7.0 Hz, 11H), 1.51–1.06 (m, 102H), 0.88 (t, J = 6.7 Hz, 14H). [M+H] + = 1325.24099

[0665] Example 2.21: Synthesis of Compound 33

[0666]

[0667] Step 1: Compound 33-3 was prepared in a similar manner to Step 1 of Example 2.3

[0668] Step 2: Under N2 atmosphere, BHT (5 mg) and Compound 33-4 (301 mg, 0.93 mmol, prepared according to the method of Step 1 of Example 2.1) were added to a 4 mL container containing Compound 33-3 (100 mg, 0.26 mmol). The mixture was heated at 70 °C for 48 h. TLC indicated the completion of the reaction. The mixture was purified by flash column chromatography on silica gel (eluent: DCM containing 0 - 10% methanol, v / v), and concentrated under reduced pressure to obtain Compound 33 (250 mg, yield 69%).

[0669] of Compound 33 1 H NMR: (400 MHz, chloroform-d) δ 5.49–5.12 (m, 5H), 4.05 (q, J = 6.6 Hz, 6H), 3.58 (td, J = 6.9, 3.5 Hz, 1H), 2.91 (dt, J = 14.3, 7.5 Hz, 1H), 2.72 (dt, J = 23.2, 6.8 Hz, 6H), 2.64–2.52 (m, 2H), 2.52–2.19 (m, 17H), 2.01 (q, J = 6.4 Hz, 14H), 1.61 (t, J = 7.0 Hz, 11H), 1.28 (dd, J = 18.7, 7.3 Hz, 99H), 0.88 (t, J = 6.7 Hz, 13H). [M+H] + = 1297.21294

[0670] Example 2.22: Synthesis of Compound 34

[0671]

[0672] Step 1: Compound 34-3 was prepared in a similar manner to Step 1 of Example 2.3.

[0673] Step 2: Under a N2 atmosphere, BHT (5 mg) and compound 34-4 (280 mg, 0.87 mmol, prepared according to the method of Step 1 of Example 2.1) were added to a 4 mL container containing compound 34-3 (100 mg, 0.24 mmol). The mixture was heated at 70 °C for 48 h. TLC indicated the completion of the reaction. The mixture was purified by flash column chromatography on silica gel (eluent: DCM containing 0-10% methanol, v / v), and concentrated under reduced pressure to obtain compound 34 (197 mg, yield 59%).

[0674] For compound 34 1 H NMR: (400 MHz, chloroform-d) δ 5.48–5.23 (m, 5H), 4.05 (q, J = 6.6 Hz, 6H), 3.58 (td, J = 7.5, 7.1, 3.7 Hz, 1H), 2.91 (dt, J = 14.5, 7.5 Hz, 1H), 2.83–2.14 (m, 26H), 2.01 (q, J = 6.4 Hz, 12H), 1.80–1.53 (m, 10H), 1.28 (dd, J = 20.0, 7.6 Hz, 109H), 0.87 (t, J = 6.7 Hz, 14H). [M+H] + = 1381.31640.

[0675] Example 2.23: Synthesis of compound 35

[0676]

[0677] Step 1: Compound 35-3 was prepared in a similar manner to Step 1 of Example 2.15.

[0678] Step 2: Under a N2 atmosphere, BHT (5 mg) and compound 35-4 (383 mg, 1.19 mmol, prepared according to the method of Step 1 of Example 2.1) were added to a 4 mL container containing compound 35-3 (120 mg, 0.50 mmol). The mixture was heated at 70 °C for 48 h. TLC indicated the completion of the reaction. The mixture was purified by flash column chromatography on silica gel (eluent: DCM containing 0-10% methanol, v / v), and concentrated under reduced pressure to obtain compound 35 (188 mg, yield 42%).

[0679] For compound 35 11H NMR: (400 MHz, chloroform-d) δ 5.51–5.21 (m, 6H), 4.05 (td, J = 6.8, 2.8 Hz, 6H), 3.57 (ddt, J = 10.4, 6.7, 3.4 Hz, 2H), 2.90 (ddt, J = 15.1, 11.7, 5.8 Hz, 5H), 2.75–2.17 (m, 17H), 2.01 (q, J = 5.9 Hz, 14H), 1.82–1.03 (m, 102H), 0.88 (t, J = 6.6 Hz, 14H). [M+H] + = 887.82994。

[0680] Example 2.24: Synthesis of Compound 36

[0681]

[0682] Step 1: Compound 36-3 was prepared in a similar manner to Step 1 of Example 2.15.

[0683] Step 2: Under a N2 atmosphere, BHT (5 mg) and Compound 36-4 (343 mg, 1.06 mmol, prepared according to the method of Step 1 of Example 2.1) were added to a 4 mL container containing Compound 36-3 (120 mg, 0.44 mmol). The mixture was heated at 70 °C for 48 h. TLC indicated the completion of the reaction. The mixture was purified by flash column chromatography on silica gel (eluent: DCM containing 0-10% methanol, v / v), and concentrated under reduced pressure to give Compound 36 (191 mg, yield 47%).

[0684] 1H NMR of Compound 36 1 1H NMR: (400 MHz, chloroform-d) δ 5.45–5.27 (m, 5H), 4.05 (td, J = 6.8, 2.7 Hz, 6H), 3.64–3.30 (m, 2H), 2.90 (ddt, J = 22.2, 11.1, 5.7 Hz, 5H), 2.72–2.18 (m, 18H), 1.99 (qd, J = 12.0, 11.2, 7.3 Hz, 15H), 1.83–1.55 (m, 15H), 1.52–1.06 (m, 105H), 0.87 (t, J = 6.7 Hz, 15H). [M+H] + = 915.84710。

[0685] Example 2.25: Synthesis of Compound 37

[0686]

[0687] Step 1: Compound 37-3 was prepared in a similar manner to Step 1 of Example 2.3.

[0688] Step 2: Add BHT (1 mg), compound 37-4 (137 mg, 0.32 mmol), and ACN (0.3 mL) to a vial containing compound 37-3 (30 mg, 0.09 mmol). Evacuate the mixture with an N2 atmosphere and decant three times. Heat the mixture at 70 °C for 2 - 3 days. TLC indicates completion of the reaction. Purify the mixture by silica gel column chromatography (eluent: DCM containing 0 - 3% methanol, v / v), and concentrate under reduced pressure to obtain compound 37 (110 mg, yield 76%).

[0689] For compound 37 1 1H NMR: (400 MHz, chloroform-d) δ 4.37 (q, J = 6.3 Hz, 6H), 3.57 (s, 1H), 2.91 (dt, J = 14.3, 7.4 Hz, 1H), 2.83–2.63 (m, 5H), 2.63–2.08 (m, 25H), 1.25 (s, 23H), 0.87 (t, J = 6.8 Hz, 3H).

[0690] Example 2.26: Synthesis of compound 38

[0691]

[0692] Step 1: Prepare compound 38-3 by a method similar to that of Step 1 in Example 2.3

[0693] Step 2: Add BHT (1 mg), compound 38-4 (170 mg, 0.33 mmol), and ACN (0.3 mL) to a vial containing compound 38-3 (30 mg, 0.09 mmol). Evacuate the mixture with an N2 atmosphere and decant three times. Heat the mixture at 70 °C for 2 - 3 days. TLC indicates completion of the reaction. Purify the mixture by silica gel column chromatography (eluent: DCM containing 0 - 3% methanol, v / v), and concentrate under reduced pressure to obtain compound 38 (100 mg, yield 58%).

[0694] For compound 38 1 1H NMR: (400 MHz, chloroform-d) δ 4.37 (q, J = 6.4 Hz, 6H), 3.57 (s, 1H), 2.91 (dt, J = 14.0, 7.4 Hz, 1H), 2.78–2.66 (m, 5H), 2.56–2.15 (m, 25H), 1.25 (s, 24H), 0.88–0.84 (m, 3H).

[0695] Example 2.27: Synthesis of compound 39

[0696]

[0697] Step 1: Prepare compound 39-3 by a method similar to that of Step 1 in Example 2.15.

[0698] Step 2: At room temperature, add BHT (10.8 mg) and compound 39-4 (380.8 mg, 1.19 mmol) to a 5 mL container containing compound 39-3 (120 mg, 0.50 mmol). Heat the mixture at 70 °C for 40 h. TLC (eluent: DCM / MeOH = 15 / 1) indicates that the reaction is basically complete. The mixture is purified by silica gel automatic column chromatography (eluent: DCM containing 0 - 3% methanol, v / v), and concentrated under reduced pressure to obtain compound 39 (190 mg, yield 43%).

[0699] For compound 39 1 H NMR: (400 MHz, chloroform-d) δ 5.45–5.25 (m, 8H), 4.06 (q, J = 6.7 Hz, 4H), 3.58 (td, J = 8.4, 7.5, 4.1 Hz, 1H), 2.92 (dt, J = 13.1, 7.6 Hz, 2H), 2.77 (t, J = 6.5 Hz, 5H), 2.60 (p, J = 5.6 Hz, 2H), 2.48–2.19 (m, 7H), 2.05 (q, J = 6.8 Hz, 9H), 1.85 (d, J = 12.7 Hz, 1H), 1.62 (p, J = 6.9 Hz, 7H), 1.54–1.42 (m, 3H), 1.40–1.21 (m, 46H), 0.89 (td, J = 6.8, 3.7 Hz, 10H). [M + H] + = 883.80336.

[0700] Example 2.28: Synthesis of compound 40

[0701]

[0702] Step 1: Prepare compound 40-3 by a method similar to that of Step 1 in Example 2.15.

[0703] Step 2: At room temperature, add BHT (10.8 mg) and compound 40-4 (341.3 mg, 1.06 mmol) to a 5 mL container containing compound 40-3 (120 mg, 0.44 mmol). Heat the mixture at 70 °C for 40 h. TLC (eluent: DCM / MeOH = 15 / 1) indicates that the reaction is basically complete. The mixture is purified by silica gel automatic column chromatography (eluent: DCM containing 0 - 3% methanol, v / v), and concentrated under reduced pressure to obtain compound 40 (250 mg, yield 62%).

[0704] Of compound 40 1 H NMR: (400 MHz, chloroform-d) δ 5.45–5.23 (m, 8H), 4.06 (q, J = 6.7 Hz, 4H), 3.58 (td, J = 6.8, 3.5 Hz, 1H), 2.92 (dt, J = 13.1, 7.6 Hz, 2H), 2.77 (t, J = 6.5 Hz, 5H), 2.60 (dt, J = 12.6, 5.1 Hz, 2H), 2.48–2.19 (m, 7H), 2.05 (q, J = 6.8 Hz, 9H), 1.92–1.80 (m, 1H), 1.62 (p, J = 6.9 Hz, 7H), 1.54–1.16 (m, 55H), 0.88 (td, J = 6.8, 4.4 Hz, 10H). [M+H] + = 911.82438

[0705] Example 2.29: Synthesis of compound 41

[0706]

[0707] Step 1: Compound 41-3 was prepared in a similar manner to that in Step 1 of Example 2.15

[0708] Step 2: At room temperature, BHT (10.8 mg) and compound 41-4 (282.6 mg, 0.88 mmol) were added to a 5 mL container containing compound 41-3 (120 mg, 0.37 mmol). The mixture was heated at 70 °C for 40 h. TLC (eluent: DCM / MeOH = 15 / 1) indicated that the reaction was substantially complete. The mixture was purified by silica gel automatic column chromatography (eluent: DCM containing 0 - 3% methanol, v / v), and concentrated under reduced pressure to obtain compound 41 (170 mg, yield 48%)

[0709] Of compound 41 1 H NMR: (400 MHz, chloroform-d) δ 5.46–5.26 (m, 9H), 4.12–4.01 (m, 4H), 3.58 (s, 1H), 3.30 (s, 1H), 2.92 (dt, J = 14.4, 7.6 Hz, 2H), 2.77 (t, J = 6.5 Hz, 5H), 2.66–2.53 (m, 2H), 2.47–2.19 (m, 8H), 2.05 (q, J = 6.9 Hz, 9H), 1.89 (s, 1H), 1.61 (dd, J = 13.4, 6.6 Hz, 13H), 1.47–1.17 (m, 63H), 0.88 (td, J = 6.8, 4.1 Hz, 10H). [M+H] + = 967.88990

[0710] Example 2.30: Synthesis of Compound 42

[0711]

[0712] Step 1: Compound 42-3 was prepared by a method similar to that in Step 1 of Example 2.15.

[0713] Step 2: At room temperature, BHT (10.8 mg) and Compound 42-4 (260.3 mg, 0.81 mmol) were added to a 5 mL container containing Compound 42-3 (120 mg, 0.34 mmol). The mixture was heated at 70 °C for 40 h. TLC (eluent: DCM / MeOH = 15 / 1) indicated that the reaction was basically complete. The mixture was purified by silica gel automatic column chromatography (eluent: DCM containing 0 - 3% methanol, v / v), and concentrated under reduced pressure to obtain Compound 42 (203.7 mg, yield 60%).

[0714] For Compound 42 1 1H NMR: (400 MHz, chloroform-d) δ 5.46–5.24 (m, 9H), 4.06 (q, J = 6.9 Hz, 4H), 3.58 (tt, J = 7.4, 3.6 Hz, 1H), 3.33 (s, 1H), 2.92 (dt, J = 13.2, 7.5 Hz, 2H), 2.77 (t, J = 6.5 Hz, 5H), 2.60 (p, J = 5.7 Hz, 3H), 2.49–2.19 (m, 8H), 2.05 (q, J = 6.8 Hz, 9H), 1.87 (d, J = 13.1 Hz, 1H), 1.62 (p, J = 7.6, 6.9 Hz, 9H), 1.50–1.16 (m, 69H), 0.88 (td, J = 6.8, 4.1 Hz, 10H). [M+H] + = 995.91986.

[0715] Example 2.31: Synthesis of Compound 43

[0716]

[0717] Step 1: Compound 43-3 was prepared by a method similar to that in Step 1 of Example 2.15.

[0718] Step 2: At room temperature, add BHT (10.8 mg) and compound 43-4 (241.2 mg, 0.75 mmol) to a 5 mL container containing compound 43-3 (120 mg, 0.31 mmol). Heat the mixture at 70 °C for 40 h. TLC (eluent: DCM / MeOH = 15 / 1) indicates that the reaction is basically complete. The mixture is purified by silica gel automatic column chromatography (eluent: DCM containing 0 - 3% methanol, v / v), and concentrated under reduced pressure to obtain compound 43 (212.1 mg, yield 66%).

[0719] of compound 43 1 H NMR: (400 MHz, chloroform-d) δ 5.46–5.24 (m, 10H), 4.06 (q, J = 6.8 Hz, 4H), 3.58 (td, J = 7.2, 3.8 Hz, 1H), 3.33 (s, 1H), 2.92 (dt, J = 13.2, 7.6 Hz, 2H), 2.77 (t, J = 6.5 Hz, 5H), 2.60 (dt, J = 12.9, 5.7 Hz, 3H), 2.46–2.20 (m, 7H), 2.05 (q, J = 6.8 Hz, 9H), 1.86 (d, J = 15.4 Hz, 2H), 1.62 (p, J = 6.9 Hz, 9H), 1.52–1.15 (m, 75H), 0.88 (td, J = 6.8, 4.2 Hz, 11H). [M+H] + = 1023.94772.

[0720] Example 2.32: Synthesis of compound 44

[0721]

[0722] Step 1: Compound 44-3 is prepared by a method similar to that of Step 1 in Example 2.15.

[0723] Step 2: At room temperature, add BHT (10.8 mg) and compound 44-4 (343.87 mg, 1.07 mmol) to a 5 mL container containing compound 44-3 (120 mg, 0.45 mmol). Heat the mixture at 70 °C for 40 h. TLC (eluent: DCM / MeOH = 15 / 1) indicates that the reaction is basically complete. The mixture is purified by silica gel automatic column chromatography (eluent: DCM containing 0 - 3% methanol, v / v), and concentrated under reduced pressure to obtain compound 44 (230 mg, yield 57%).

[0724] of compound 44 11H NMR: (400 MHz, chloroform-d) δ 5.81 (ddt, J = 16.9, 10.2, 6.7 Hz, 1H), 5.45–5.28 (m, 8H), 5.06–4.84 (m, 2H), 4.06 (q, J = 6.7 Hz, 4H), 3.58 (ddt, J = 10.4, 7.0, 3.2 Hz, 1H), 3.34 (s, 1H), 2.92 (dt, J = 13.2, 7.6 Hz, 2H), 2.77 (t, J = 6.5 Hz, 5H), 2.60 (dt, J = 12.6, 5.5 Hz, 3H), 2.47–2.21 (m, 7H), 2.05 (q, J = 6.7 Hz, 11H), 1.85 (d, J = 12.9 Hz, 1H), 1.62 (p, J = 6.8 Hz, 7H), 1.48 (q, J = 11.9, 10.8 Hz, 2H), 1.42–1.18 (m, 47H), 0.89 (t, J = 6.8 Hz, 7H). [M+H] + = 909.81634。

[0725] Example 2.33: Synthesis of Compound 45

[0726]

[0727] Step 1: Compound 45-3 was prepared by a method similar to that of Step 1 in Example 2.15.

[0728] Step 2: At room temperature, BHT (10.8 mg) and Compound 45-4 (258.86 mg, 0.81 mmol) were added to a 5 mL container containing Compound 45-3 (120 mg, 0.34 mmol). The mixture was heated at 70 °C for 40 h. TLC (eluent: DCM / MeOH = 15 / 1) indicated that the reaction was basically complete. The mixture was purified by silica gel automatic column chromatography (eluent: DCM containing 0 - 3% methanol, v / v), and concentrated under reduced pressure to obtain Compound 45 (184 mg, yield 55%).

[0729] For Compound 45 1 1H NMR: 11H NMR (400 MHz, chloroform-d) δ 5.48–5.23 (m, 8H), 4.05 (td, J = 6.8, 5.3 Hz, 4H), 3.82 (q, J = 5.7 Hz, 1H), 3.48–3.34 (m, 3H), 2.90 (dt, J = 14.0, 7.1 Hz, 1H), 2.75 (dt, J = 15.0, 6.9 Hz, 8H), 2.64–2.35 (m, 10H), 2.05 (q, J = 6.9 Hz, 9H), 1.59 (dp, J = 20.6, 6.9 Hz, 7H), 1.41–1.17 (m, 50H), 0.88 (td, J = 6.9, 4.2 Hz, 9H). [M+H] + = 997.90395。

[0730] Example 2.34: Synthesis of Compound 48

[0731]

[0732] Step 1: While stirring, Compound 48-2 (1.04 g) was added to a solution of Compound 48-1 (1.3 g) in anhydrous ethanol (6.5 mL). The mixture was heated at 70 °C for 16 h. TLC (eluent: DCM / methanol = 15 / 1, stained with I2) indicated the formation of the product.

[0733] Step 2: The mixture from Step 1 was concentrated under reduced pressure and diluted with ethyl acetate (10 mL). HCl (10 mL, 4 M, in ethyl acetate) was added dropwise to the mixture. The mixture was stirred overnight at room temperature. The mixture was filtered and the residue was dissolved in water and ethyl acetate. A saturated aqueous NaHCO3 solution was added to the mixture until pH = 10 - 12. The mixture was extracted with ethyl acetate. The combined organic layers were washed with brine and concentrated to give Compound 48-3 (1.32 g, 80% yield).

[0734] 1H NMR of Compound 48-3 1 1H NMR: (400 MHz, chloroform-d) δ 3.68 (ddt, J = 11.0, 7.8, 3.8 Hz, 1H), 3.07 (d, J = 11.4 Hz, 1H), 2.85 (d, J = 11.8 Hz, 1H), 2.59 (d, J = 5.8 Hz, 2H), 2.38–2.17 (m, 4H), 1.94 (td, J = 11.6, 2.5 Hz, 2H), 1.76–1.68 (m, 3H), 1.51–1.18 (m, 22H), 0.88 (t, J = 6.7 Hz, 4H).

[0735] Step 3: Add a drop of acetic acid to a mixture of compound 48-3 (75 mg), BHT (31 mg) and compound 48-4 (160 mg). Heat the mixture at 70 °C for 1 day in a sealed tube. Cool the mixture to room temperature and concentrate it under reduced pressure. The residue was purified by silica gel column chromatography (eluent: DCM containing 0-5% methanol) to give compound 48 (168 mg, yield 81%).

[0736] of compound 48 1 H NMR: 1 H NMR (400 MHz, chloroform-d) δ 4.03 (t, J = 6.8 Hz, 4H), 3.73 (dq, J = 13.6, 6.7, 4.6 Hz, 2H), 3.11 (d, J = 11.2 Hz, 1H), 2.92 (d, J = 11.5 Hz, 1H), 2.72 (t, J = 7.1 Hz, 4H), 2.43–2.27 (m, 7H), 2.22 (d, J = 7.0 Hz, 2H), 2.07–1.97 (m, 1H), 1.71 (dd, J = 13.4, 3.4 Hz, 2H), 1.61 (p, J = 6.8 Hz, 4H), 1.52–1.37 (m, 3H), 1.26 (d, J = 6.5 Hz, 49H), 0.87 (t, J = 6.7 Hz, 9H).

[0737] Example 2.35: Synthesis of compound 49

[0738]

[0739] Add a drop of acetic acid to a mixture of compound 49-3 (75 mg, prepared according to the method of steps 1 to 2 of Example 2.34), BHT (31 mg) and compound 49-4 (179 mg). Heat the mixture at 70 °C for 1 day in a sealed tube. Cool the mixture to room temperature and concentrate it under reduced pressure. The residue was purified by silica gel column chromatography (eluent: DCM containing 0-5% methanol) to give compound 49 (175 mg, yield 78%).

[0740] of compound 49 1 H NMR: 11H NMR (400 MHz, chloroform-d) δ 4.04 (t, J = 6.8 Hz, 4H), 3.75 (q, J = 6.0 Hz, 1H), 3.13 (d, J = 11.6 Hz, 2H), 2.94 (d, J = 11.4 Hz, 1H), 2.73 (t, J = 7.1 Hz, 4H), 2.46–2.29 (m, 7H), 2.22 (d, J = 7.0 Hz, 2H), 2.05 (t, J = 11.6 Hz, 1H), 1.77–1.67 (m, 2H), 1.61 (p, J = 6.9 Hz, 4H), 1.55–1.39 (m, 3H), 1.26 (d, J = 7.4 Hz, 58H), 0.88 (t, J = 6.6 Hz, 9H).

[0741] Example 2.36: Synthesis of Compound 52

[0742]

[0743] Step 1: Compound 52-3 was prepared by the method of Step 2 in Example 2.1.

[0744] Step 2: Under a N2 atmosphere, at room temperature, BHT (15 mg) and acetic acid (7 mg) were added to a solution of Compound 52-3 (30 mg, 0.096 mmol) and Compound 52-4 (117.3 mg, 0.386 mmol, prepared by the method of Step 1 in Example 1.15) in ACN (0.2 mL). The mixture was heated at 75 °C for 40 h. TLC (eluent: DCM / MeOH = 10 / 1) indicated that the starting materials had been substantially consumed. The mixture was purified by silica gel column chromatography and concentrated under reduced pressure to give Compound 52 (67 mg, yield 55%).

[0745] 1H NMR of Compound 52 1 1H NMR: (400 MHz, chloroform-d) δ 5.78 (t, J = 6.1 Hz, 1H), 3.65 (td, J = 8.7, 7.5, 4.0 Hz, 1H), 3.44–3.26 (m, 2H), 3.19 (dt, J = 19.3, 5.2 Hz, 7H), 3.07 (q, J = 7.1 Hz, 7H), 2.99–2.81 (m, 4H), 2.75–2.63 (m, 5H), 2.55 (dt, J = 28.9, 7.4 Hz, 3H), 2.46–2.20 (m, 2H), 2.00 (d, J = 11.9 Hz, 5H), 1.58 (dt, J = 12.5, 4.7 Hz, 6H), 1.26 (d, J = 5.9 Hz, 81H), 0.94–0.73 (m, 12H). [M+H] + = 1212.00789.

[0746] Example 2.37: Synthesis of Compound 53

[0747]

[0748] Step 1: Compound 53-3 was prepared by the method of Step 2 in Example 2.1.

[0749] Step 2: Under N2 atmosphere, at room temperature, BHT (15 mg) and acetic acid (10 mg) were added to a solution of compound 53-3 (30 mg, 0.096 mmol) and compound 53-4 (101.2 mg, 0.36 mmol, prepared by the method of Steps 1 to 2 in Example 1.16) in ACN (0.2 mL). The mixture was heated at 80 °C for 16 h. TLC (eluent: DCM / MeOH = 10 / 1 + 0.5% NH4OH) indicated that the starting materials had been substantially consumed. The mixture was purified by silica gel column chromatography and concentrated under reduced pressure to obtain compound 53 (50 mg, yield 43%).

[0750] For Compound 53 1 1H NMR: (400 MHz, chloroform-d) δ 3.70 (s, 1H), 3.07–2.87 (m, 1H), 2.68 (t, J = 5.6 Hz, 2H), 2.48–2.31 (m, 6H), 2.31–2.19 (m, 5H), 2.15 (d, J = 18.3 Hz, 2H), 1.62 (d, J = 8.0 Hz, 7H), 1.26 (d, J = 5.9 Hz, 48H), 0.94–0.76 (m, 12H). HRMS: [M+H] + 1149.01684.

[0751] Example 2.38: Synthesis of Compounds 57 and 58

[0752] Compounds 57 and 58 were synthesized by a method similar to that in Example 2.37 using different starting materials and modified conditions.

[0753]

[0754] 1-((3-((3-Aminopropyl)(methyl)amino)propyl)amino)decane-2-ol (50 mg, 1.0 equiv) and N'-acryloylpalmitohydrazide (193.7 mg, 3.6 equiv) were added to a 5 ml reaction flask, and BHT (10 mg), AcOH (6 μL) and 0.5 ml of anhydrous ethanol were added. The mixture was stirred at 80 °C for 24 h. The crude product was purified by flash column chromatography (DCM~DCM / MeOH = 20 / 1 + 0.5% aqueous ammonia) to obtain the target compound (78 mg, yield 39.5%).

[0755] Of compound 57 1 H NMR: (400 MHz, DMSO-d6) δ 9.88–9.65 (m, 5H), 3.53 (s, 1H), 3.05 (d, J = 33.9 Hz, 4H), 2.79 (s, 1H), 2.66 (d, J = 15.9 Hz, 7H), 2.37–2.18 (m, 7H), 2.10 (t, J = 7.4 Hz, 5H), 1.73 (s, 4H), 1.63–1.43 (m, 6H), 1.43–1.32 (m, 3H), 1.23 (s, 74H), 0.92–0.72 (m, 12H). HRMS: m / z calculated for C 74 H 147 N9O7 [M+H] + 1275.1423, found 1275.17927.

[0756]

[0757] To a 2 ml reaction flask, 1-(3-((3-aminopropyl)(methyl)amino)propyl)octan-2-ol (80 mg, 1.0 equiv) and N'-acryloylpalmitohydrazide (342 mg, 3.6 equiv) were added, along with BHT (10 mg), AcOH (3 μl) and 0.8 ml of anhydrous ethanol. The mixture was stirred at 80 °C for 24 h. The crude product was purified by flash column chromatography (DCM ∼ DCM / MeOH = 50 / 1 + 0.5% ammonia water) to give the target compound (95 mg, yield 26.04%, HPLC (ELSD): 96.15%).

[0758] Of compound 58 1 H NMR: (400 MHz, chloroform-d) δ 3.70 (dt, J = 7.5, 3.6 Hz, 1H), 2.69 (t, J = 5.7 Hz, 4H), 2.59 (tdd, J = 11.8, 10.7, 8.5, 5.1 Hz, 3H), 2.50–2.34 (m, 10H), 2.27 (pd, J = 11.8, 10.9, 4.6 Hz, 12H), 2.11 (s, 3H), 1.62 (tt, J = 10.9, 6.9 Hz, 10H), 1.54 (s, 2H), 1.35–1.21 (m, 70H), 0.93–0.83 (m, 12H). HRMS: m / z calculated for C 72 H 143 N9O7 [M+H] + 1247.1110, found 1247.13610.

[0759] Other compounds of the present disclosure are synthesized in a similar manner, using modified conditions and different starting materials. Exemplary starting materials are shown in Tables 4-6.

[0760] Table 4. Exemplary Amino Starting Materials

[0761]

[0762]

[0763] Each of the exemplary amino starting materials shown in Table 4 above can be used as a substitute for compound S1-1 in General Scheme 1 or compound S2-1 in General Scheme 2.

[0764] Each of the exemplary amino starting materials shown in Table 4 above is commercially available or can be synthesized by the methods described in Example 3 below.

[0765] Table 5. Exemplary Epoxide Starting Materials

[0766]

[0767]

[0768] Each of the exemplary epoxide starting materials shown in Table 5 above can be used as a substitute for compound S2-2 in General Scheme 2.

[0769] Table 6. Exemplary Acrylic Acid Starting Materials

[0770]

[0771]

[0772] Each of the exemplary acrylic acid starting materials shown in Table 6 above can be used as a substitute for compound S1-2 in General Scheme 1 or compound S2-4 in General Scheme 2.

[0773] Each of the exemplary acrylic acid starting materials shown in Table 6 above is commercially available or can be synthesized by the above methods.

[0774] Exemplary lipids synthesized by a method similar to General Scheme 1 or General Scheme 2, where the starting materials shown in Tables 4-6 are selected, and their structure codes are shown in Table 7 below.

[0775] Table 7. Structure Codes of Exemplary Lipids and Their Starting Materials

[0776]

[0777]

[0778]

[0779]

[0780] Example 3: Synthesis of Amino Starting Materials

[0781]

[0782] Example 3.1: Synthesis of Compound S3-3

[0783] Triethylamine (42.51 g, 420 mmol) was added to a solution of compound S3-1 (26.25 g, 200 mmol) in tetrahydrofuran (THF) (800 mL). The mixture was cooled and stirred in an ice bath. While stirring in the ice bath, a cold solution of compound S3-2 (98.53 g, 400 mmol) in THF (200 mL) was added dropwise to the mixture. The mixture was allowed to warm to room temperature and was kept at the same temperature overnight. The mixture was concentrated and purified by silica gel column chromatography (eluent: petroleum ether containing 0 - 50% ethyl acetate to ethyl acetate containing 0 - 5% methanol and 0.5% NH4OH, v / v), and concentrated under reduced pressure to obtain compound S3-3 (42 g, yield 63%).

[0784] For compound S3-3 1 1H NMR: (400 MHz, chloroform-d) δ 5.19 (s, 2H), 3.20 (q, J = 6.3 Hz, 4H), 2.66 (t, J = 6.5 Hz, 4H), 1.66 (p, J = 6.5 Hz, 4H), 1.43 (s, 18H).

[0785] Example 3.2: Synthesis of Compound A2

[0786]

[0787] Step 1: Ground K2CO3 (8.28 g, 59.9 mmol) was added to a solution of compound S3-3 (13.24 g, 39.9 mmol) in ACN (100 mL). The mixture was stirred for 30 minutes, and then ethyl iodide (6.23 g, 39.9 mmol) was added thereto. The mixture was stirred at room temperature overnight. The mixture was filtered, the filtrate was concentrated and purified by silica gel column chromatography (eluent: petroleum ether containing 0 - 50% ethyl acetate to ethyl acetate containing 0 - 5% methanol and 0.5% NH4OH, v / v), and concentrated under reduced pressure to obtain compound A2-1 (9 g, yield 63%).

[0788] For compound A2-1 11H NMR: (400 MHz, chloroform-d) δ 5.34 (s, 2H), 3.16 (q, J = 6.3 Hz, 4H), 2.56–2.36 (m, 6H), 1.62 (p, J = 6.6 Hz, 4H), 1.42 (s, 18H), 1.01 (t, J = 7.1 Hz, 3H).

[0789] Step 2: HCl (4 M in methanol, 50 mL) was added to compound A2-1 (9 g, 25 mmol). The mixture was stirred at room temperature for 2 h. The mixture was concentrated under reduced pressure to give crude compound A2 (4.2 g, containing water).

[0790] of compound A2 1 1H NMR: (400 MHz, deuterium oxide) δ 2.98–2.27 (m, 10H), 1.60 (dtt, J = 14.4, 12.3, 7.1 Hz, 4H), 0.95 (dt, J = 18.1, 7.2 Hz, 3H).

[0791] Example 3.3: Synthesis of compound A3

[0792]

[0793] Step 1: Ground K2CO3 (8.28 g, 59.9 mmol) was added to a solution of compound S3-3 (13.24 g, 39.9 mmol) in ACN (100 mL). The mixture was stirred for 30 min, then compound A3-1 (4.99 g, 39.9 mmol) and NaI (5.98 g, 39.9 mmol) were added thereto. The mixture was stirred at room temperature for 2 days. The mixture was filtered, the filtrate was concentrated and purified by silica gel column chromatography (eluent: petroleum ether containing 0 - 50% ethyl acetate to ethyl acetate containing 0 - 5% methanol, v / v), and concentrated under reduced pressure to give compound A3-2 (5.9 g, yield 40%).

[0794] of compound A3-2 1 1H NMR: (400 MHz, chloroform-d) δ 5.10 (t, J = 5.9 Hz, 2H), 3.65 (t, J = 5.2 Hz, 2H), 3.42 (s, 1H), 3.17 (q, J = 6.5 Hz, 4H), 2.67 (t, J = 5.2 Hz, 2H), 2.61 (t, J = 6.9 Hz, 4H), 1.70 (p, J = 6.8 Hz, 4H), 1.42 (s, 18H).

[0795] Step 2: Add HCl (4 M in methanol, 30 mL) to compound A3-2 (5.24 g, 19 mmol). Stir the mixture at room temperature for 2 hours. Concentrate the mixture under reduced pressure to obtain crude compound A3 (4.0 g, containing water).

[0796] For compound A3 1 1H NMR: (400 MHz, methanol-d4) δ 3.95 (t, J = 5.0 Hz, 2H), 3.50–3.33 (m, 6H), 3.10 (t, J = 7.5 Hz, 4H), 2.23 (p, J = 8.1 Hz, 4H).

[0797] Example 3.4: Synthesis of compound A4

[0798]

[0799] Step 1: Add compound A4-1 (3.37 g, 40 mmol) and acetic acid (0.2 mL) to a solution of compound S3-3 (13.26 g, 40 mmol) in DCM (200 mL). Stir the mixture in an ice bath for 60 minutes, then add NaBH(CH3COO)3 (12.72 g, 120 mmol) thereto. Stir the mixture at room temperature overnight. Quench the reaction with saturated NaHCO3. Extract the mixture with DCM. Concentrate the combined organic layers and purify by silica gel column chromatography (eluent: petroleum ether containing 0 - 50% ethyl acetate to ethyl acetate containing 0 - 5% methanol, v / v), and concentrate under reduced pressure to obtain compound A4-2 (14 g, yield 88%).

[0800] For compound A4-2 1 1H NMR: (400 MHz, chloroform-d) δ 5.39 (s, 2H), 3.16 (q, J = 6.3 Hz, 4H), 2.46 (dd, J = 14.8, 8.1 Hz, 7H), 2.04 (qd, J = 8.0, 4.0 Hz, 2H), 1.95–1.76 (m, 2H), 1.64 (qd, J = 8.6, 4.2 Hz, 6H), 1.43 (s, 18H).

[0801] Step 2: Add HCl (4 M in methanol, 100 mL) to compound A4-2 (14 g, 35 mmol). Stir the mixture at room temperature for 2 hours. Concentrate the mixture under reduced pressure to obtain crude compound A4 (12 g, containing water).

[0802] For compound A4 11H NMR: (400 MHz, methanol-d4) δ 3.24–3.15 (m, 6H), 3.01 (t, J = 7.6 Hz, 4H), 2.79 (hept, J = 7.6 Hz, 1H), 2.21–2.06 (m, 6H), 2.00–1.78 (m, 4H).

[0803] Example 3.5: Synthesis of Compound A5

[0804]

[0805] Step 1: To a solution of Compound S3-3 (13.26 g, 40 mmol) in DCM (200 mL) was added Compound A5-1 (3.84 g, 40 mmol) and acetic acid (0.2 mL). The mixture was stirred in an ice bath for 60 minutes, and then NaBH(CH3COO)3 (12.72 g, 120 mmol) was added thereto. The mixture was stirred at room temperature overnight. The reaction was quenched with saturated NaHCO3. The mixture was extracted with DCM. The combined organic layers were concentrated and purified by silica gel column chromatography (eluent: petroleum ether containing 0 - 50% ethyl acetate to ethyl acetate containing 0 - 5% methanol, v / v), and concentrated under reduced pressure to obtain Compound A5-2 (7.0 g, yield 42%).

[0806] For Compound A5-2 1 1H NMR: (400 MHz, methanol-d4) δ 7.03 (s, 2H), 3.69 (s, 2H), 3.06 (t, J = 6.9 Hz, 4H), 2.48 (t, J = 7.1 Hz, 4H), 1.63 (p, J = 6.9 Hz, 4H), 1.43 (s, 18H).

[0807] Step 2: To Compound A5-2 (7.0 g, 17 mmol) was added HCl (4 M in methanol, 60 mL). The mixture was stirred at room temperature for 2 hours. The mixture was concentrated under reduced pressure to obtain crude Compound A5 (7.0 g, containing water).

[0808] For Compound A5 1 1H NMR: (400 MHz, DMSO-d6) δ 8.29 (t, J = 5.8 Hz, 5H), 7.77 (s, 2H), 4.50 (s, 2H), 3.33–2.66 (m, 8H), 2.21–1.94 (m, 4H).

[0809] Example 4: Lipid Particles Containing Different Neutral Lipids

[0810] Example 4.1: Preparation of mRNA-Loaded Lipid Particles

[0811] Step 1: Dissolve FLuc mRNA in citrate buffer (pH 4) and adjust the mRNA concentration to 0.2 mg / mL to obtain an aqueous layer.

[0812] Step 2: Dissolve the test compound, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, and DMG-PEG2000 at the desired mole fractions (shown in Table 8 below) in absolute ethanol and adjust the total lipid concentration to 10 mg / mL to obtain an organic layer.

[0813] Table 8. Mole fractions of components in lipid particles

[0814] Lipid Particle Number Test Compound (Compound 1) Neutral Lipid Cholesterol DMG-PEG2000 1 62.5％ 9% (DOPE) 27％ 1.5％ 2 50％ 10% (DSPC) 38.5％ 1.5％

[0815] Step 3: Mix the aqueous layer and the organic layer at a ratio of 3:1 (v / v) through a microfluidic device ( Ignite TM ) at a total flow rate of 12 mL / min. Dilute the mixture 10-fold with PBS buffer (pH 7.4). Separate ethanol by tangential flow filtration (Repligen, TFF). Concentrate the solution to 0.1 mg / mL (mRNA concentration) and filter through a 0.22 μm millipore filter to obtain mRNA-containing lipid particles.

[0816] The average particle size and polydispersity index (PDI) of exemplary lipid particles were measured by DLS using a Malvern Zetasizer.

[0817] Using (ThermoFisher Scientific), the total RNA concentration and free RNA concentration were measured based on fluorescence measurement. The encapsulation efficiency (EE) was calculated as follows:

[0818]

[0819] The total RNA concentration was measured by appropriately diluting the test lipid particles with 1xTE buffer containing 0.2% Triton-X 100. The free RNA concentration was measured by appropriately diluting the test lipid particles with 1xTE buffer.

[0820] Figure 1Shows the changes in the physical characteristics of the test lipid particles over two weeks (size: average particle diameter; PDI: average polymer dispersity index; and encapsulation: efficiency of encapsulating RNA). The physical characteristic parameters of each of these three physical characteristic parameters of the lipid particles on day 0 are used as the base values for the corresponding normalization. Each normalization value on days 3, 5, 7, 10, and 15 is the ratio of the physical characteristic parameters of the lipid particles on that day to the physical characteristic parameters of the lipid particles on day 0. The normalization value of each of these three physical characteristic parameters on day 0 is 1.

[0821] Example 4.2: In vitro delivery test

[0822] OVCAR3 cells with good growth conditions were digested with 0.25% trypsin. The medium was resuspended and mixed 5 times with a pipette. Take the cell suspension (20 μL) on a counting plate and count with a Countstar cell counter. The cells were seeded in a 96-well plate at 10,000 cells per well and 100 μL of medium. The cells were cultured overnight and transfected with the test lipid particles containing 25 ng of FLuc mRNA. The test lipid particles and the control were diluted to a total volume of 100 μL with medium. Remove the medium from the cell culture plate and add 100 μL of the test lipid particles. Each test lipid particle was transfected into 2 - 3 wells. The blank control group had 3 wells, each with 100 μL of medium. The culture plate was placed in a cell culture incubator. 24 hours after cell transfection, the ONEGLO TM kit (Promega, catalog number E7120) was melted at room temperature for 1 hour, and 5X CellTiter-Fluor TM reagent (20 μL per well) was added to the cell culture plate. The cell culture plate was placed on a shaker and incubated at 37 °C at 300 rpm for 30 min. Fluorescence was measured with a microplate reader (EX390 nm / EM505 nm). Then, the ONEGLO TM reagent (100 μL per well) was added at room temperature for 3 minutes, and luminescence was measured with a microplate reader. The resulting relative light units (“RLU”) are shown as Figure 2 (in vitro).

[0823] Example 4.3: In vivo delivery test

[0824] Six-week-old male C57BL / 6 mice weighing approximately 20 g were selected and housed in a specific pathogen-free (SPF) facility. All animal experiments were conducted in strict accordance with the guidelines of the National Institutes of Health and animal ethics. Two or three mice were randomly selected per group (n = 2 or 3), and lipid particles containing FLuc mRNA prepared as described above were administered at a dose of 0.25 mg / kg by intramuscular injection ("IM"). Six hours later, each mouse was injected intraperitoneally with 100 μL of 30 mg / mL D-luciferin (potassium salt). Ten minutes later, the total fluorescence intensity of each mouse was observed and recorded using an in vivo imaging system. The total fluorescence intensity (p / s) was as Figure 2 (in vivo) shown.

[0825] Figure 2 showed the in vitro and in vivo delivery characteristics of the test lipid particles on day 0 and day 14 as described above.

[0826] It can be seen that lipid particles containing the disclosed compounds and different neutral lipids can effectively and safely deliver nucleic acids to cells or subjects and stably express the nucleic acids.

[0827] Example 5: Lipid particles of different formulations

[0828] Test lipid particles were prepared using the method of steps 1 to 3 of Example 4.1, and the molar fractions are shown in the following table 9. Table 10 shows the physical characteristics of the test lipid particles.

[0829] Table 9. Molar fractions of components in lipid particles

[0830]

[0831]

[0832] Table 10. Physical characteristics of test lipid particles

[0833]

[0834]

[0835] In vitro and in vivo delivery tests were conducted using the methods of Examples 4.2 and 4.3. Figure 3 、 Figure 4 and Figure 5 showed the in vitro and in vivo delivery characteristics of the test lipid particles, respectively.

[0836] Example 6: Lipid particles with different lipids (test compounds)

[0837] Use specific mole fraction test compound (46%), DSPC (8%), cholesterol (43.9%) and DMG-PEG2000 (2.1%) (preparation A) or test compound (70%), DSPC (4%), cholesterol (23.9%) and DMG-PEG2000 (2.1%) (preparation B), adopt the method of embodiment 4.1 step 1 to step 3 to prepare test lipid particles.Table 11 shows the physical characteristics of test lipid particles.

[0838] Table 11. Physical characteristics of the tested lipid particles

[0839]

[0840]

[0841] Using DLin-MC3-DMA (DLin-MC3-DMA (50%), DSPC (10%), cholesterol (38.5%) and DMG-PEG2000 (1.5%)) as positive controls, the methods of Examples 4.2 and 4.3 were used to perform in vitro and in vivo delivery experiments. In vivo delivery experiments were performed by both tail vein injection and intramuscular injection. Figure 6 The in vitro delivery properties and cell viability of the tested lipid particles were shown. Figure 7 The in vivo delivery characteristics of the tested lipid particles are shown, as indicated by muscle expression. Table 12 shows the distribution of in vivo delivery to the liver, spleen, and lung. Figure 8 , Figure 9 and Figure 10 Imaging, FLuc mRNA expression intensity and organ distribution of liver, spleen and lung of exemplary lipid particles targeting liver, spleen and lung are shown respectively. The lung efficacy (total flux, p / s) measured for LNPs with compounds 53, 57 and 58 were 1.15E+07, 1.51E+08 and 1.80E+08, respectively.

[0842] Table 12. FLuc mRNA expression intensity in liver, spleen and lung

[0843]

[0844]

[0845] Example 7: Delivery of Nucleic Acids by Exemplary Lipid Particles

[0846] Using specific molar fractions of the test compound (46%), DSPC (8%), cholesterol (43.9%) and DMG-PEG2000 (2.1%), the method of Step 1 to Step 3 of Example 4.1 was used to prepare test lipid particles containing hEPO mRNA.

[0847] Six-week-old male C57BL / 6 mice weighing approximately 20 g were selected and housed in an SPF breeding room. All animal experiments were carried out strictly in accordance with the guidelines of the National Institutes of Health and animal ethics. Three mice were randomly selected from each group (n = 3), and the lipid particles containing hEPO mRNA prepared as described above were administered by intravenous injection (tail) at a dose of 0.3 mg / kg. Six hours later, serum was isolated from the blood samples and analyzed using a commercially available ELISA kit (ab274397, Abcam).

[0848] Using the above experiment, lipid particles with exemplary test compounds (A1EP10AO18B, A1EP10O18B, A1EP10AO18A, and A1EP10O18A), a positive control (DLin-MC3-DMA), and a negative control (PBS) were tested. The hEPO expression level (mIU / mL) was as Figure 11 shown below.

[0849] Example 8: Efficient delivery of nucleic acids to the lung

[0850] A CTGF antibody analog vector was constructed by ligating the codon-optimized CTGF antibody gene in the following order: heavy chain variable region, GGGGSGGGGSGGGGS, light chain variable region, and Fc region. The 5’UTR and 3’UTR sequences are shown in SEQ ID NO:7-9 and SEQ ID NO:10-13, respectively. Using specific mole fractions of Compound 8 (46%), DSPC (8%), cholesterol (43.9%), and DMG-PEG2000 (2.1%), the method of Steps 1 to 3 of Example 4.1 was used to prepare test lipid particles containing mRNA encoding the CTGF antibody.

[0851] The test lipid particles containing CTGF antibody mRNA were diluted to 0.25 mg / mL with PBS and administered intravenously (tail) to C57 mice at a dose of 100 μL. The test lipid particles were administered 2 times a week for a total of 4 times. On the 2nd day after the last administration, EDTA-anticoagulated whole blood and lung lavage fluid were collected. The concentrations of CTGF antibody in plasma and lung lavage fluid were measured by the following steps: 50 ng of CTGF protein was coated overnight on a 96-well plate in carbonate buffer at 4 °C; the coated wells were washed 3 times with PBST; 100 μL of 0.5% BSA-PBS buffer was added to the coated wells and incubated at 37 °C for 1 hour; the coated wells were washed 3 times with PBST; 100 μL of plasma, lung lavage fluid or CTGF antibody standard was added to the coated wells and incubated at 37 °C for 2 hours; the coated wells were washed 3 times with PBST; horseradish peroxidase (HRP)-labeled anti-human antibody (Sino Biological) was added to the coated wells and incubated at 37 °C for 1 hour; the coated wells were washed 3 times with PBST; HRP chromogenic substrate solution (invitrogen) was added. Figure 12 The fold change in the concentration of CTGF antibody in plasma and lung lavage fluid between the test group mice and the control group mice ("blank": treated with PBS) was shown.

[0852] Example 9: Effective delivery of nucleic acid to induce immune response

[0853] Using the mole fractions shown in Table 13, the test lipid particles containing RAV-G mRNA were prepared by the method of steps 1 to 3 of Example 4.1.

[0854] Six-week-old male Balb / C mice weighing about 20 g were selected and housed in an SPF breeding room. All animal experiments were strictly carried out in accordance with the guidelines of the National Institutes of Health and animal ethics. 5 μg of test lipid particles were administered to each mouse at a dose of 50 μL per mouse, and the same volume of buffer 1 was administered in the negative control group. On the 7th and 14th days after immunization, blood samples were harvested by retro-orbital bleeding and allowed to stand at 4 °C for 30 minutes. Serum was separated by centrifugation at 4000 rpm at 4 °C and stored in the original solution at -80 °C for subsequent ELISA titer detection.

[0855] Coat a 96-well ELISA plate overnight at 4 °C with 100 μL / well of PBS buffer containing 2 μg / mL RAV-G. Wash the plate three times, block it with 300 μL / well of blocking buffer for 2 hours at 37 °C, and then wash it three times. Add 100 μL / well of test serum serially diluted at ratios of 1:100, 1:400, 1:1600, 1:3200, 1:6400…1:2048000, incubate at 37 °C for 60 minutes, and then wash four times. Dilute the HRP-labeled anti-mouse IgG H&L antibody 1000-fold, add 100 μL / well, incubate at 37 °C for 60 minutes, and wash four times. After the chromogenic substrate develops color and is subsequently quenched, measure the signal using a microplate reader at an absorbance of 450 nm. As Figure 13 shown, the antibody titers (“IgG titers”) from the test groups induced by single immunization with different lipid particles delivering RAV-G mRNA vaccine were higher than those of the positive control group.

[0856] Table 13. Mole fractions of components in lipid particles

[0857] Formulation Number Test Compound DSPC Cholesterol DMG-PEG2000 Formulation 38 46% (Compound 22) 8％ 43.9％ 2.1％ Formulation 39 43% (Compound 22) 8％ 47.4％ 1.6％ Formulation 40 46% (Compound 22) 10％ 42.4％ 1.6％ Formulation 41 45% (Compound 22) 8％ 45.3％ 1.7％ Formulation 42 47% (Compound 22) 7％ 44.4％ 1.6％ Formulation 43 45% (Compound 22) 6％ 47.4％ 1.6％

[0858] Example 10: Antibody-conjugated LNP targeting CD8+ T cells.

[0859] Using specific mole fractions of compound 8 (46%), DSPC (8%), cholesterol (43.9%), and DMG-PEG2000 (2.1%), prepare test lipid particles containing tdTomato mRNA by the method of steps 1 to 3 of Example 4.

[0860] Conjugate the test lipid particles containing tdTomato mRNA with mouse anti-cd8 antibody (LNP-CD8Ab) or mouse immunoglobulin G2B (LN-IgG2B). Dilute the lipid particle-antibody conjugate to 0.5 mg / ml in PBS and administer 150 μL of the diluted lipid particle-antibody conjugate to C57 mice by intravenous injection (tail). Sacrifice the mice 16 hours after injection, excise the spleen and place it in a cell strainer (200 mesh). Prepare a cell suspension by sieving the spleen through the cell strainer using a syringe plunger. Fluorescently label the cells with different cell marker antibodies as shown in Table 14. Mix 50 μL of the cell suspension (about 10 5 cells) with 0.25 μL of the staining antibody and incubate in the dark on ice for 30 minutes. Wash the cells with 1 mL of PBS, centrifuge at 400 g for 30 min, discard the supernatant, and repeat these processes once. Resuspend the cells in 200 μL of 4% paraformaldehyde and analyze by flow cytometry. Figure 14Shows the recognition ability of antibody-conjugated lipid particle preparations for different cell surface markers. That is, Figure 14 shows the percentage of cells recognized by cell marker antibodies. In the "T / ALL" section, in the test groups treated with PBS, LNP (lipid particles containing tdTomato mRNA), LNP-CD8Ab, and LN-IgG2B, among all the observed splenocytes, approximately 24%, 21%, approximately 21%, and approximately 24% of T cells were recognized by their corresponding cell surface markers, respectively. In the "CD4 + T / T" section, in the test groups treated with PBS, LNP, LNP-CD8Ab, and LN-IgG2B, among all the observed T cells, approximately 56%, approximately 63%, approximately 62%, and approximately 66% of CD4 + T cells were recognized by their corresponding cell surface markers. In the "CD8 + T / T" section, in the test groups treated with PBS, LNP, LNP-CD8Ab, and LN-IgG2B, among all the observed T cells, approximately 38%, approximately 31%, approximately 0%, and approximately 28% of CD8 + T cells were recognized by their corresponding cell surface markers. The results show that among different preparations, the lipid particles labeled with CD8 antibody can effectively bind to the corresponding CD8+ cell surface, thereby blocking the specific binding and labeling of the staining antibody to these CD8+ cells, resulting in low or undetectable CD8+ cell signals in the test.

[0861] Table 14. Fluorescently labeled antibodies

[0862] Antibody Supplier Catalog Number Ms CD3e FITC 145-2C11 100μg BD Biosciences 553061 Ms CD4 PerCP-Cy5.5 RM4-5 100μg BD Biosciences 550954 Ms CD8a PE-Cy7 53-6.7 100μg BD Biosciences 552877 Ms CD45 APC-Cy7 30-F11 100μg BD Biosciences 557659

[0863] TROP2-CAR mRNA was transcribed in vitro by the method described in the present disclosure. 25 μg of TROP2-CAR-Ab / LNP was administered to C57 mice by intravenous injection (tail), and the same volume of PBS was used as a negative control. To prepare cell suspensions, mouse spleens were isolated on the 3rd, 7th, and 14th days after injection and sieved through a cell filter (200 mesh) using a syringe plunger. 50 μL of cell suspension (about 10 5 cells) was mixed with 0.25 μL of staining antibody and incubated in the dark on ice for 30 minutes. The cells were washed with 1 mL of PBS, centrifuged at 400 g for 30 min, the supernatant was discarded, and these processes were repeated once. The cells were resuspended in 200 mL of 4% paraformaldehyde. The percentage of TROP2-CAR-positive T cells was analyzed by flow cytometry.

[0864] Example 11: TNS protocol

[0865] Prepare the test lipid nanoparticles using the method of steps 1 to 3 of Example 4.1 with the molar fractions shown in Table 15 below.

[0866] Table 15. Molar fractions of components in lipid particles

[0867] Formulation Number Test Compound DSPC Cholesterol DMG-PEG2000 21 46% (Compound 22) 8％ 43.9％ 2.1％ 22 46% (Compound 8) 8％ 43.9％ 2.1％ 23 46% (Compound 53) 8％ 43.9％ 2.1％ 24 46% (Compound 57) 8％ 43.9％ 2.1％

[0868] Dilute the obtained LNP / mRNA formulation with a series of buffers containing 10 mM HEPES, 10 mM MES (4-morpholineethanesulfonic acid), 10 mM ammonium acetate, and 130 mM NaCl, where the pH ranges from 2.5 to 11. Gently shake and mix the TNS probe (200 μM stock solution in distilled water) with the above solution for 5 min to make the final volume 100 μL, which contains lipids and the probe. Measure the fluorescence of each well using a Tecan microplate reader, where Ex = 321 nm and Em = 445 nm, and normalize the data to the value at pH 2.5. The pH at half-maximum fluorescence represents the pKa of the formulation. The pKa values observed for the test lipid particles are shown in Table 16. This indicates that the test lipid nanoparticles have significant potential in effectively delivering the desired nucleic acid into cells.

[0869] Table 16. TNS pKa of components in lipid particles

[0870] Test Compound (Structure Code) pKa (TNS) Compound 8 (A1NCN12) 6.385 Compound 22 (A1EP10O18A) 6.280 Compound 53 (A1EP10NNCC13) 6.302 Compound 57 (A1EP10NNCC16) 6.756

[0871] The foregoing description is considered to illustrate only the principles of the present disclosure. Moreover, since many modifications and variations are obvious to those skilled in the art, it is not desired to limit the invention to the exact structures and methods described above. Accordingly, all suitable modifications and equivalents may be considered to fall within the scope of the invention as defined by the claims.

Claims

1. A compound of the following formula (I) or a pharmaceutically acceptable salt thereof: wherein L a and L c each independently selected from a bond, an alkyl group, an alkenyl group, an alkynyl group, and a heteroalkyl group, wherein the alkyl group, the alkenyl group, the alkynyl group, and the heteroalkyl group are optionally substituted with one or more groups independently selected from halogen, hydroxy, cyano, and amino; L b selected from a bond, -NR a -, -O-, -S-, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclic group, aryl and heteroaryl, wherein the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclic group, aryl and heteroaryl are optionally independently substituted by one or more groups selected from halogen, hydroxy, cyano and amino, or L b is covalently linked to R 1 to form The condition is that when two of L a , L b and L c are keys, the remaining one is not a key; R a selected from hydrogen, R 5 , alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclic group, aryl and heteroaryl, wherein the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclic group, aryl and heteroaryl are optionally independently substituted with one or more groups selected from halogen, hydroxy, cyano, cycloalkyl, heterocyclic group, aryl, heteroaryl and R 6 ; R 1 is a group having the following formula (I-a): R 2 is a group having the following formula (I-b): R 3 is a group having the following formula (I-c): R 4 is a group having the following formula (I-d): wherein R 1a 、R 2a 、R 3a and R 4a each independently is a key or an alkyl group; R 1b 、R 2b 、R 3b and R 4b each independently is a key, an alkyl or a heteroalkyl, wherein the alkyl and heteroalkyl are optionally substituted by one or more groups independently selected from hydroxy, amino, oxo, thio and imino; and R 1c 、R 2c 、R 3c and R 4c each independently selected from alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl, wherein the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl are optionally substituted by one or more groups independently selected from halogen, hydroxy, oxo, cyano, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, and the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl are optionally interrupted by one or more groups independently selected from cycloalkyl, heterocycloalkyl, aryl, and heteroaryl; and R 5 is a group having the following formula (I-e): Each R 6 independently is a group having the following formula (I-f): wherein R 5a and R 6a each independently is a key or an alkyl group; R 5b and R 6b each independently is a key, an alkyl group or a heteroalkyl group, wherein the alkyl group and the heteroalkyl group are optionally substituted by one or more groups independently selected from hydroxyl, amino, oxo, thio and imino; and R 5c and R 6c each independently selected from alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl, wherein the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl are optionally substituted with one or more groups independently selected from halogen, hydroxy, oxo, and cyano, and the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl are optionally interrupted by one or more groups independently selected from cycloalkyl, heterocyclic, aryl, and heteroaryl.

2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein L a and L c are each independently alkyl, preferably C 1-6 alkyl.

3. The compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein L a and L c are each independently methyl, ethyl or propyl.

4. The compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, wherein L b is -NR a -.

5. The compound of claim 4 or a pharmaceutically acceptable salt thereof, wherein R a is selected from hydrogen, R 5 , alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl, wherein the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally independently substituted by one or more groups selected from halogen, hydroxy, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl and R 6 .

6. The compound of claim 4 or 5, or a pharmaceutically acceptable salt thereof, wherein R a is C 1-6 heteroalkyl.

7. A compound according to claim 4 or 5, or a pharmaceutically acceptable salt thereof, wherein R a is C 1-6 alkyl optionally substituted by one or more groups independently selected from hydroxy, cycloalkyl and heteroaryl.

8. The compound of claim 4 or 5 or a pharmaceutically acceptable salt thereof, wherein L b is selected from 9. The compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, wherein L b is selected from -S-, -O-, -O-alkyl-O-, cycloalkyl and heterocyclic group.

10. The compound of claim 9 or a pharmaceutically acceptable salt thereof, wherein L b is selected from -S- 11. The compound of claim 10 or a pharmaceutically acceptable salt thereof, wherein L b is 12. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein -L a -L b -L c - selected from 13. A compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, wherein L b is alkyl or heteroalkyl, which is covalently linked to R 1 to form L a is a bond, alkyl or heteroalkyl, and R 1 is a bond, alkyl or heteroalkyl.

14. The compound of claim 13 or a pharmaceutically acceptable salt thereof, wherein is 15. The compound of claim 14 or a pharmaceutically acceptable salt thereof, wherein L c is methyl.

16. A compound according to any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof, wherein at least two of R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are the same.

17. The compound of claim 16 or a pharmaceutically acceptable salt thereof, wherein at least three of R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are the same.

18. The compound of claim 17 or a pharmaceutically acceptable salt thereof, wherein at least four of R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are the same.

19. A compound according to any one of claims 1 to 15 or a pharmaceutically acceptable salt thereof, wherein at least two of R 1 , R 2 , R 3 and R 4 are different.

20. A compound according to any one of claims 1 to 19 or a pharmaceutically acceptable salt thereof, wherein R 1 , R 2 , R 3 and R 4 are independently one or more of an alkyl group optionally substituted by one or more hydroxyl groups.

21. The compound of claim 20 or a pharmaceutically acceptable salt thereof, wherein R 1 , R 2 , R 3 and R 4 are independently one or more of methyl, 22. A compound according to any one of claims 1 to 21 or a pharmaceutically acceptable salt thereof, wherein R 1a , R 2a , R 3a , R 4a , R 5a and R 6a are independently of one another a bond, methyl, ethyl, propyl, butyl or pentyl.

23. A compound according to any one of claims 1 to 22 or a pharmaceutically acceptable salt thereof, wherein R 1b , R 2b , R 3b , R 4b , R 5b and R 6b are independently selected from one or more of alkyl and heteroalkyl, wherein the alkyl and heteroalkyl are optionally substituted by one or more groups independently selected from hydroxy, oxo, thio and imino.

24. The compound of claim 23 or a pharmaceutically acceptable salt thereof, wherein R 1b , R 2b , R 3b , R 4b , R 5b and R 6b are independently one or more of wherein Each W is independently selected from O, S or NR b ; Each Y is independently selected from O, S, NR c , N(R c )Z(W), N(R c )N(R c ) or N(R c )N(R c )Z(W); each Z is independently selected from C, S or S(O); each m is independently 0, 1, 2 or 3; each p is independently 1, 2, 3 or 4; and R b and R c each independently selected from hydrogen, alkyl, alkoxycarbonyl, alkanoyl, aroyl and sulfonyl.

25. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R 1 is R 2 For R 3 For R 4 For R 5 , if present, is R 6 , if present, is Each W is independently selected from O, S or NR b ; Each Y is independently selected from O, S, NR c , N(R c )Z(W), N(R c )N(R c ) or N(R c )N(R c )Z(W); each Z is independently selected from C, S or S(O); each m is independently 0 or 1; each p is independently 1 or 2; R b and R c each independently selected from hydrogen, alkyl, alkoxycarbonyl, alkanoyl, aroyl and sulfonyl; and R 1c 、R 2c 、R 3c 、R 4c 、R 5c and R 6c each, if present, is independently selected from alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl, wherein the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl are optionally substituted with one or more groups independently selected from halogen, hydroxy, oxo, and cyano, and the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl are optionally interrupted by one or more groups independently selected from cycloalkyl, heterocyclic, aryl, and heteroaryl.

26. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein L b is -NR a -; R a selected from hydrogen, R 5 , alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl and heteroalkynyl, wherein the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl and heteroalkynyl are independently substituted with one or more groups selected from halogen, hydroxy, cyano, cycloalkyl, heterocyclic group, aryl, heteroaryl and R 6 ; R 1 For R 2 For R 3 For R 4 For R 5 , if present, is R 6 , if present, is Each W is independently selected from O, S or NR b ; Each Y is independently selected from O, S, NR c , N(R c )Z(W), N(R c )N(R c ) or N(R c )N(R c )Z(W); each Z is independently selected from C, S or S(O); each m is independently 0 or 1; each p is 1 or 2; R b and R c each independently selected from hydrogen, alkyl, alkoxycarbonyl, alkanoyl, aroyl and sulfonyl; and R 1c 、R 2c 、R 3c 、R 4c 、R 5c and R 6c each, if present, is independently selected from alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl, wherein the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl are optionally substituted with one or more groups independently selected from halogen, hydroxy, oxo, and cyano, and the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl are optionally interrupted by one or more groups independently selected from cycloalkyl, heterocyclic, aryl, and heteroaryl.

27. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein L b is -NR a -; R a Selected from R 5 , alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl and heteroalkynyl, wherein the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl and heteroalkynyl are substituted by R 6 and optionally substituted by one or more additional groups independently selected from halogen, hydroxy, cyano, cycloalkyl, heterocyclic, aryl and heteroaryl; R 1 selected from alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl and heteroalkynyl, wherein the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl and heteroalkynyl are optionally substituted by one or more groups independently selected from halogen, hydroxy, cyano, cycloalkyl, heterocyclic group, aryl and heteroaryl; R 2 For R 3 For R 4 For R 5 , if present, is R 6 , if present, is Each W is independently selected from O, S or NR b ; Each Y is independently selected from O, S, NR c , N(R c )Z(W), N(R c )N(R c ) or N(R c )N(R c )Z(W); each Z is independently selected from C, S or S(O); each m is independently 0 or 1; each p is 1 or 2; R b and R c each independently selected from hydrogen, alkyl, alkoxycarbonyl, alkanoyl, aroyl or sulfonyl; and R 2c 、R 3c 、R 4c 、R 5c and R 6c Each of which, if present, is independently selected from alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl, wherein the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl are optionally substituted with one or more groups independently selected from halogen, hydroxy, oxo, and cyano, and the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, and heteroalkynyl are optionally interrupted by one or more groups independently selected from cycloalkyl, heterocyclic, aryl, and heteroaryl.

28. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound has the following formula (A): wherein R 2 For R 3 For R 4 For Each W is independently selected from O, S or NR b ; Each Y is independently selected from O, S, NR c , N(R c )Z(W), N(R c )N(R c ) or N(R c )N(R c )Z(W); each Z is independently selected from C, S or S(O); each m is independently 0 or 1; each p is independently 1 or 2; Q is 1, 2 or 3; R b and R c each independently selected from hydrogen, alkyl, alkoxycarbonyl, alkanoyl, aroyl or sulfonyl; and R 2c 、R 3c and R 4c each independently selected from alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl and heteroalkynyl, wherein the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl and heteroalkynyl are optionally substituted with one or more groups independently selected from halogen, hydroxy, oxo and cyano, and the alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl and heteroalkynyl are optionally interrupted by one or more groups independently selected from cycloalkyl, heterocyclic, aryl and heteroaryl.

29. The compound of claim 23 or a pharmaceutically acceptable salt thereof, wherein the compound has formula (B), formula (C), formula (D) or formula (E): wherein Each W is independently selected from O, S or NR b ; Each Y is independently selected from O, S, NR c , N(R c )Z(W), N(R c )N(R c ) or N(R c )N(R c )Z(W); each Z is independently selected from C, S or S(O); each n is independently 0, 1, 2, 3, 4 or 5; each m is independently 0, 1, 2 or 3; each p is independently 1, 2, 3 or 4; and R b and R c each independently selected from hydrogen, alkyl, alkoxycarbonyl, acyl or sulfonyl.

30. A compound according to any one of claims 24 to 29 or a pharmaceutically acceptable salt thereof, wherein each is independently selected from and 31. The compound of claim 30 or a pharmaceutically acceptable salt thereof, wherein R 1b and R 2b and R 3b and R 4b and R 5b and R 6b are independently selected from 32. The compound of claim 23 or a pharmaceutically acceptable salt thereof, wherein R 1b 、R 2b 、R 3b 、R 4b 、R 5b and R 6b are independently selected from one or more of 33. A compound according to any one of claims 1 to 32 or a pharmaceutically acceptable salt thereof, wherein one or more of R 1c , R 2c , R 3c , R 4c , R 5c and R 6c are independently selected from alkyl, alkenyl and alkynyl, wherein the alkyl, alkenyl and alkynyl are optionally substituted by one or more halogens.

34. A compound according to any one of claims 1 to 32 or a pharmaceutically acceptable salt thereof, wherein R 1c , R 2c , R 3c , R 4c , R 5c and R 6c are independently selected from one or more of -CH2-O-alkyl, -CH2-O-alkenyl, -CH2-O-alkynyl, -CH2-O-heteroalkyl, -CH2-O-heteroalkenyl, and -CH2-O-heteroalkynyl.

35. The compound of claim 33 or 34 or a pharmaceutically acceptable salt thereof, wherein each of the alkyl, alkenyl and alkynyl independently contains 6 to 18 carbon atoms.

36. The compound of claim 33 or 34 or a pharmaceutically acceptable salt thereof, wherein each alkenyl independently contains one or more Z-olefins. The compound according to any one of claims 1 to 36 or a pharmaceutically acceptable salt thereof, wherein R 1c 、R 2c 、R 3c 、R 4c 、R 5c and R 6c one or more of which are independently selected from The compound of any one of claims 1 to 37 or a pharmaceutically acceptable salt thereof, wherein R 1 、R 2 、R 3 、R 4 、R 5 and R 6 one or more of which are independently selected from 39. A compound according to any one of claims 1 to 38 or a pharmaceutically acceptable salt thereof, wherein -R 1b -R 1c , -R 2b -R 2c , -R 3b -R 3c , -R 4b -R 4c , -R 5b -R 5c and R 6b -R 6c , if present, each do not contain two heteroatoms directly bonded to each other. A compound according to any one of claims 1 to 38 or a pharmaceutically acceptable salt thereof, wherein -R 1b -R 1c 、-R 2b -R 2c 、-R 3b -R 3c 、-R 4b -R 4c 、-R 5b -R 5c and R 6b -R 6c one or more of, if present, independently selected from -N(R c )-N(R c )-, -N(R c )-S(O)-, -N(R c )-S(O)2-, -N(R c )-O-, -S(O)-N(R c )-, -S(O)2-N(R c )-, -S(O)-O-, -S(O)2-O-, -O-N(R c )-, -O-S(O)-, -O-S(O)2- and -O-O- one or more groups.

41. The compound of claim 40 or a pharmaceutically acceptable salt thereof, wherein -R 1b -R 1c -R 2b -R 2c -R 3b -R 3c -R 4b -R 4c -R 5b -R 5c and -R 6b -R 6c one or more of, if present, contain -N(R c )-N(R c )- or -S(O)2-N(R c )-.

42. The compound of claim 1 or a pharmaceutically acceptable salt thereof, selected from any one listed in the following table:

43. A lipid particle comprising the compound of any one of claims 1 to 42 or a pharmaceutically acceptable salt thereof.

44. The lipid particle of claim 43, wherein the lipid particle further comprises neutral lipid.

45. The lipid particle of claim 44, wherein the neutral lipid is selected from 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-doundecanoyl-sn-glycero-phosphocholine (DUPC), 1,2-dioleoyl-sn-glycero-3-phosphate-rac-(1-glycerol) sodium salt (DOPG), 1,2-di-O-octadecenyl-5-glycero-3-phosphocholine (18:0 diether PC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16Lyso PC), 1-oleoyl-2-cholesteryl succinyl-5-glycero-3-phosphocholine (OChemsPC), and any mixture thereof.

46. The lipid particle of any one of claims 43 to 45, wherein the lipid particle further comprises a sterol, a sterol derivative, or any mixture thereof.

47. The lipid particle of claim 46, wherein the sterol is cholesterol, β-sitosterol, stigmasterol, ergosterol, brassicasterol, coprosterol, or campesterol.

48. The lipid particle of any one of claims 43 to 47, wherein the lipid particle further comprises a surfactant.

49. The lipid particle of claim 48, wherein the surfactant is selected from 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol (DMG-PEG), 1,2-distearoyl-rac-glycerol-3-methoxypolyethylene glycol (DSG-PEG), N-(methylpolyoxyethylene-carbonyl)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (PEG-DSPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)] (DOPE-PEG), and any mixture thereof.

50. The lipid particle of any one of claims 43 to 49, wherein based on the total amount of the lipid particle, the lipid particle comprises, in mole fraction: 10-75% of the compound of any one of claims 1 to 42; 1-35% of neutral lipid; 15-50% of a sterol, a sterol derivative, or any mixture thereof; and 0.5-5% of a surfactant.

51. The lipid particle of any one of claims 43 to 50, wherein based on the total amount of the lipid particle, the lipid particle comprises, in mole fraction: 40-75% of the compound of any one of claims 1 to 42; 2-15% of neutral lipid; 20 - 50% of a sterol, a sterol derivative, or any mixture thereof; and 1 - 5% of a surfactant.

52. The lipid particle according to any one of claims 43 to 51, wherein, based on the total amount of the lipid particle, the lipid particle comprises, in mole fraction: 40 - 70% of the compound according to any one of claims 1 to 42; 3 - 15% of DSPC; 20 - 50% of cholesterol; and 1 - 2.5% of DMG - PEG.

53. The lipid particle according to any one of claims 43 to 52, wherein, based on the total amount of the lipid particle, the lipid particle comprises, in mole fraction: 40 - 70% of the compound according to any one of claims 1 to 42; 3 - 15% of DSPC; 22 - 50% of cholesterol; and 1.5 - 2.5% of DMG - PEG.

54. The lipid particle according to any one of claims 43 to 53, wherein the lipid particle further comprises a targeting moiety conjugated to the lipid particle.

55. The lipid particle of claim 54, wherein the targeting moiety is conjugated to the lipid particle directly or through a linker.

56. The lipid particle of claim 54 or 55, wherein the targeting moiety is an antibody or a ligand.

57. A loaded lipid particle comprising the lipid particle according to any one of claims 43 - 56 and a therapeutic agent.

58. The loaded lipid particle of claim 57, wherein the therapeutic agent is a nucleic acid.

59. The loaded lipid particle of claim 58, wherein the nucleic acid is selected from small interfering RNA (siRNA), microRNA (miRNA), small activating RNA (saRNA), single - guide RNA (sgRNA), messenger RNA (mRNA), self - amplifying RNA, circular RNA, antisense oligonucleotide (ASO), ribonucleoprotein (RNP), ssDNA, dsDNA, and any mixture thereof.

60. The loaded lipid particle of claim 59, wherein the nucleic acid is an mRNA encoding a therapeutic protein.

61. The loaded lipid particle of claim 60, wherein the therapeutic protein is an antibody, an antigen, a cytokine, a clotting factor, or an enzyme, or a functional fragment thereof.

62. The loaded lipid particle of claim 61, wherein the therapeutic protein is an antigen or a functional fragment thereof.

63. The loaded lipid particle according to any one of claims 57 to 62, wherein the mass ratio of the lipid particle to the therapeutic agent is in the range of 10:1 to 50:

1.

64. A pharmaceutical composition comprising the loaded lipid particle according to any one of claims 57 to 63 and a pharmaceutically acceptable excipient.

65. A method of delivering a therapeutic agent to a subject, comprising the steps of: i) encapsulating the therapeutic agent into the lipid particle according to any one of claims 43 to 56; and ii) administering the lipid particle encapsulating the therapeutic agent to the subject.

66. A method of delivering a therapeutic agent to a subject, comprising administering the loaded lipid particle according to any one of claims 57 to 63 or the pharmaceutical composition of claim 64 to the subject.

67. The method of claim 65 or 66, wherein the therapeutic agent is delivered into cells of a subject, and the cells are selected from muscle cells, lung cells, liver cells, spleen cells, kidney cells, heart cells, skin cells, hair cells, nail cells, bone cells, artery cells, vein cells, diaphragm cells, larynx cells, stomach cells, intestinal cells, ureter cells, bladder cells, lymph node cells, bone marrow cells, thymus cells, brain cells, spinal cord cells, nerve cells, pituitary cells, thyroid cells, adrenal cells, penile cells, vaginal cells, prostate cells, and uterine cells.

68. The method of claim 67, wherein the cells are selected from liver cells, lung cells, spleen cells, muscle cells, and brain cells.

69. A method of treating a disease or disorder in a subject, comprising administering to the subject an effective amount of a lipid-loaded particle of any one of claims 57 to 63 or the pharmaceutical composition of claim 64.

70. Use of a lipid particle of any one of claims 43 to 56, a lipid-loaded particle of any one of claims 57 to 63, or the pharmaceutical composition of claim 64 in the manufacture of a medicament for treating a disease or disorder.

71. A method of expressing a protein in a subject, comprising: i) encapsulating a polynucleotide encoding the protein into a lipid particle of any one of claims 43 to 56; and ii) administering the lipid particle encapsulating the polynucleotide to the subject.

72. The method of claim 71, wherein the protein is an antibody, antigen, cytokine, clotting factor, or enzyme, or a functional fragment thereof.

73. The method of claim 72, wherein the protein is an antigen or a functional fragment thereof.

74. A method of preparing a compound of any one of claims 1 to 42 or a pharmaceutically acceptable salt thereof, the method comprising: A) mixing a compound having formula (II) with a compound having formula (III); wherein Each R 7 is independently selected from hydrogen, alkyl, heteroalkyl, haloalkyl, and hydroxyalkyl; Each L a 、L b 、L c 、R 1b and R 1c have the same definitions as in claim 1; and B) refluxing the mixture until the reaction is complete, thereby obtaining a compound of any one of claims 1 to 42.

75. A method of preparing a compound of any one of claims 1 to 42 or a pharmaceutically acceptable salt thereof, the method comprising: i) mixing a compound having formula (II) with a compound having formula (IV); wherein R 1c has the same definition as in claim 1; ii) mixing the product of step (i) with a compound having formula (III); and iii) refluxing the mixture until the reaction is complete, thereby obtaining a compound of any one of claims 1 to 42.

76. The method of claim 75, wherein the compound having formula (IV) is selected from 77. The method of any one of claims 74 to 76, wherein the compound having formula (II) is selected from 78. The method of any one of claims 74 to 77, wherein the compound having formula (III) is selected from 79. The method of any one of claims 74 to 78, wherein the compound having formula (III) is selected from: The method according to any one of claims 74 to 79, wherein the method further comprises a method for preparing a compound of formula (II) from a compound of formula (V), wherein L b is -NR a - and R a is not hydrogen: wherein R a is not hydrogen.

81. The method of claim 80, wherein the method further comprises a method for preparing a compound of formula (V) from a compound of formula (VI) below, wherein R a is not hydrogen:

82. The method of claim 81, wherein the method further comprises a method for preparing a compound having formula (VI) from a compound having formula (VII):

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