Lipid and lipid nanoparticle formulations

Abstract

The present invention relates generally to lipids, lipid nanoparticle formulations, and methods for their use in delivering nucleic acids, such as messenger RNA.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to PCT application filed on April 1, 2024 (application number: PCT / CN2024 / 085221), the entire contents of which are incorporated herein by reference.

[0003] background

[0004] Lipid nanoparticles (LNPs) have been applied to the development and delivery of mRNA vaccines. Their role is to act as a carrier or delivery tool to deliver mRNA (messenger RNA) into cells, enabling the mRNA to guide the cells to produce specific proteins, such as viral antigens, thereby triggering an immune response.

[0005] Lipids are the core component of lipid nanoparticles (LNPs), responsible for encapsulating substances such as nucleic acids. Classic formulations typically require the synergistic effect of multiple lipids, such as ionizable cationic lipids, cofactor lipids, cholesterol, and polyethylene glycol (PEG) lipids. Classic liposome formulations usually contain cholesterol and phospholipids, sometimes with small amounts of other cofactors. The types of lipids used and their respective molar ratios affect the particle size, nucleic acid encapsulation efficiency, zeta potential, and length of the encapsulated nucleic acids of the lipid nanoparticles. The application of novel lipids requires individual optimization. Furthermore, the structure and composition of lipids also determine the tissue type targeted by the lipid nanoparticles.

[0006] Therefore, in order to further promote the development of this important nucleic acid delivery platform, it is urgent to develop more novel lipid structures.

[0007] summary

[0008] In one respect, compounds of formula I are provided:

[0009] I

[0010] in

[0011] L 1 C 1-10 Alkylene or C 2-10 Heteroalkylene; wherein, the C 2-10 Heteroalkylene compounds are optionally R 6 replace;

[0012] L 2 C 1-10 Alkylene or C 2-10 Heteroalkylene; wherein, the C 2-10 Heteroalkylene compounds are optionally R 6 replace;

[0013] X is -CH2-, -NR3 -、-N (R 3 )2 + -, -O-, -O-CH2CH2-O- or -NR 3 -(CH2) m -NR 3 -;

[0014] m is an integer from 1 to 6;

[0015] R 1 R 2 and R 3 Each independently is hydrogen, C 1-20 Alkyl, C 2-20 alkenyl, C 2-20 alkynyl group, C 2-20 Heteroalkyl, , or ;

[0016] Wherein C 1-20 Alkyl, C 2-20 alkenyl, C 2-20 alkynyl or C 2-20 Each heteroalkyl group is independently and optionally surrounded by 1 to 5 halogen, cyano, or -OR groups. 4 -SR 4 -NR 4 2. -N(R) 4 )3 + Oxygen, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl or C 1-6 Halogenated alkyl substitution;

[0017] R a Independently selected from hydrogen, C 1-12 Alkyl or -C(O)-C 1-12 alkyl;

[0018] R b Independently selected from hydrogen, C 1-12 Alkyl or -C(O)-C 1-12 alkyl;

[0019] R 4 Independently selected from hydrogen, C 1-12 Alkyl, C 2-12 alkenyl or C 2-12 alkynyl group; wherein each C 1-12 Alkyl, C 2-12 alkenyl or C 2-12 The alkynyl group is independently and optionally surrounded by 1 to 5 halogens, cyano groups, -OH groups, or -SR groups. 5 -NR 52. -N(R) 5 )3 + Or oxygen substitution;

[0020] R 5 Independently selected from hydrogen, C 1-12 Alkyl, C 2-12 alkenyl or C 2-12 alkynyl group; wherein each C 1-12 Alkyl, C 2-12 alkenyl or C 2-12 The alkynyl group is independently and optionally surrounded by 1 to 5 halogens, cyano groups, -OH, -SH, -NH2, -NH (C 1-6 Alkyl), -N (C) 1-6 alkyl)2、-N (C) 1-6 Alkyl)3 + Or oxygen substitution;

[0021] R 6 Selected independently , , or ;

[0022] L is independently selected from C 1-10 Alkylene or C 3-10 Heteroalkylene;

[0023] R 7 Independently selected from hydrogen, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl or C 1-6 Halogenated alkyl; wherein each C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl or C 1-6 The haloalkyl group is independently and optionally surrounded by 1 to 5 halogens, cyano groups, -OH, -NH2, -NH (C 1-6 Alkyl), -N (C) 1-6 alkyl)2、-N (C) 1-6 Alkyl)3 + Oxygen, C 1-6 Alkoxy or C 1-6 Halogenated alkoxy groups;

[0024] n is an integer independently between 1 and 20;

[0025] p is an integer independently between 1 and 6;

[0026] R is independently selected from hydrogen, -ZC 1-20 Alkyl, -ZC 2-20 alkenyl, -ZC 2-20Alkyne group, -Z-heterocyclic group, -Z 1 -C 1-6 Alkylene-ZC 1-20 Alkyl, -Z 1 -C 1-6 Alkylene-ZC 2-20 alkenyl, -Z 1 -C 1-6 Alkylene-ZC 2-20 alkynyl group, -Z 1 -C 1-6 alkylene-Z-heterocyclic group, -Z 1 -C 2-6 imide-ZC 1-20 Alkyl, -Z 1 -C 2-6 imide-ZC 2-20 alkenyl, -Z 1 -C 2-6 imide-ZC 2-20 alkynyl group, -Z 1 -C 2-6 Ethyne-ZC 1-20 Alkyl, -Z 1 -C 2-6 Ethyne-ZC 2-20 alkenyl, or -Z 1 -C 2-6 Ethyne-ZC 2-20 alkynyl group;

[0027] Z is independently selected from chemical bonds, -O-, -NR. 4 -, -S-, -SS-, -C (O)-, -C(O)O-, -OC (O)-, -C (O)NR 4 -、-S (O)-、-S (O)2-、-NR 4 C(O)-、-NR 4 C (O)O-、-NR 4 C (O)NR 4 -、-NR 4 S(O)- or -S(O)2NR 4 -;as well as

[0028] Z 1 Independently selected from -O- and -NR 4 -, -S-, -SS-, -C (O)-, -C (O)O-, -C (O)NR 4 -、-S (O)-、-S(O)2-、-NR 4 C(O)-、-NR 4 C (O)O-、-NR 4 C (O)NR4 -、-NR 4 S(O)- or -S(O)2NR 4 -; and each Some contain at least 6 linear atoms.

[0029] The present invention also provides lipid nanoparticles comprising one or more compounds disclosed herein. In some embodiments, the compounds shown herein may be used alone or in combination with other lipid components, such as neutral lipids, charged lipids, steroids (including, for example, all sterols) and / or their analogues, and / or polymer-conjugated lipids, to form lipid nanoparticles for delivering therapeutic agents. In some embodiments, the lipid nanoparticles may be used to deliver nucleic acids, such as antisense RNA and / or messenger RNA. Furthermore, the present invention provides methods for treating or preventing various diseases or conditions using such lipid nanoparticles, such as conditions caused by infectious pathogens or protein deficiencies.

[0030] The present invention also provides pharmaceutical compositions comprising the lipid nanoparticle compositions and therapeutic agents described herein. In some embodiments, the pharmaceutical composition further comprises one or more components selected from neutral lipids, charged lipids, steroids, and polymer-conjugated lipids. Such compositions can be used to prepare lipid nanoparticles for the delivery of therapeutic agents.

[0031] The present invention also provides a method for delivering mRNA to mammalian cells, the method comprising administering to a subject a composition comprising lipid nanoparticles containing compounds as disclosed herein and mRNA.

[0032] The present invention also provides a method for preventing a disease or condition, comprising administering lipid nanoparticles as disclosed herein to a subject in need, wherein the lipid nanoparticles comprise one or more therapeutic or preventative agents, such as mRNA. Specific Implementation

[0034] definition

[0035] The technical embodiments are described below. It should be noted that such descriptions are not intended to limit the scope of protection of the present invention, but are specific illustrations as exemplary embodiments.

[0036] Unless otherwise stated, the following words, phrases, and symbols used in this specification generally have the following meanings.

[0037] A hyphen "-" (when not between two letters or symbols) is used to indicate the connection site of a substituent. For example, -C(O)NH2 indicates a connection via a carbon atom. Hyphens at the beginning or end of chemical groups are merely for convenience; the conventional meaning of a chemical group remains unchanged regardless of whether it includes one or more hyphens. A wavy line above a line in a structural formula indicates that the location is the connection site of a substituent. Unless otherwise required by chemical properties or structure, the order in which chemical groups are written or named does not indicate or imply any directionality.

[0038] The prefix "Cu-v" in the terminology indicates that the following group contains u to v carbon atoms. For example, "C 1-6 "Alkyl" indicates that the alkyl group contains 1 to 6 carbon atoms.

[0039] In this document, the reference to the word "about" for a numerical value or parameter includes and describes embodiments designed for that value or parameter itself. In some embodiments, the term "about" includes a specified range of ±10% of the amount; in other embodiments, the term "about" includes a specified range of ±5% of the amount; and in still other embodiments, the term "about" includes a specified range of ±1% of the amount. Furthermore, the expression "about X" also includes a description of "X" itself. Additionally, unless the context explicitly requires otherwise, the singular forms "an / a" and "the" both have plural meanings. For example, references to "the compound" include multiple such compounds; while "the detection method" encompasses one or more detection methods and their equivalents known to those skilled in the art.

[0040] The term "alkyl" refers to a straight-chain or branched saturated hydrocarbon chain. In this document, alkyl groups refer to chains with 1 to 20 carbon atoms (i.e., C10-C20). 1-20 Alkyl groups), 1 to 8 (i.e., C15 and C25) 1-8 Alkyl groups), 1 to 6 (i.e., C16) 1-6 Alkyl groups) or 1 to 4 (i.e., C14) 1-4 Alkyl groups. Common examples of alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, 2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl, and 3-methylpentyl. When an alkyl group with a specific number of carbon atoms is named by its chemical name or molecular formula, all positional isomers with that number of carbon atoms are included; for example, "butyl" includes n-butyl (i.e., -(CH2)3CH3), sec-butyl (i.e., -CH(CH3)CH2CH3), isobutyl (i.e., -CH2CH(CH3)2), and tert-butyl (i.e., -C(CH3)3); "propyl" includes n-propyl (i.e., -(CH2)2CH3) and isopropyl (i.e., -CH(CH3)2).

[0041] The term "alkenyl" refers to a group containing at least one carbon-carbon double bond and having 2 to 20 carbon atoms (i.e., C24-C24-C24).2-20 alkenyl), 2 to 8 carbon atoms (i.e., C) 2-8 alkenyl), 2 to 6 carbon atoms (i.e., C) 2-6 Alkenyl group or 2 to 4 carbon atoms (i.e., C44) 2-4 Alkyl groups (alkenyl). Examples of alkenyl groups include vinyl, propenyl, and butadienyl (including 1,2-butadienyl and 1,3-butadienyl).

[0042] The term "alkynyl" refers to a group containing at least one carbon-carbon triple bond and having 2 to 20 carbon atoms (i.e., C24-C24-C24). 2-20 alkynyl group), 2 to 8 carbon atoms (i.e., C 2-8 alkynyl group), 2 to 6 carbon atoms (i.e., C64) 2-6 (alkynyl group) or 2 to 4 carbon atoms (i.e., C46) 2-4 Alkyl groups (alkynyl groups). The term also includes groups containing both a triple bond and a double bond.

[0043] The term "alkoxy" refers to an "alkyl-O-" group. Examples of alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexyloxy, and 1,2-dimethylbutoxy.

[0044] The term "haloalkoxy" refers to a group in which one or more hydrogen atoms of the alkoxy group described above are replaced by a halogen.

[0045] The term "alkylthio" refers to an "alkyl-S-" group.

[0046] The term "acyl" refers to a group of the general formula -C(O)-R, where R is hydrogen, alkyl, cycloalkyl, heterocyclic, aryl, heteroalkyl, or heteroaryl; each of these groups may optionally be substituted as defined herein. Examples of acyl groups include formyl, acetyl, cyclohexyl, cyclohexylmethylcarbonyl, and benzoyl.

[0047] The term "amide group" refers to both "C-amide" and "N-amide" groups, where "C-amide" refers to the group -C(O)NR. y R z "N-amide" refers to the -NR group y C(O)R z , where R y and R z Each group is independently selected from hydrogen, alkyl, aryl, haloalkyl, or heteroaryl groups; each of these groups may be optionally substituted as defined herein.

[0048] The term "amino" refers to the -NR group. y R z , where R y and R zEach group is independently selected from hydrogen, alkyl, haloalkyl, aryl, or heteroaryl groups; each of these groups may be optionally substituted as defined herein.

[0049] The term "amidinium" refers to the -C(NH)(NH2) group.

[0050] The term "aryl" refers to an aromatic carbocyclic group having a monocyclic (e.g., monocyclic) or polycyclic (e.g., bicyclic or tricyclic) structure, including fused systems. The aryl groups described herein contain 6 to 20 cyclic carbon atoms (i.e., C atoms). 6-20 aryl), 6 to 12 cyclic carbon atoms (i.e., C 6-12 aryl group or 6 to 10 cyclic carbon atoms (i.e., C46) 6-10 Aryl groups. Typical examples of aryl groups include phenyl, naphthyl, fluorenyl, and anthracene. It should be noted that the aryl groups defined herein do not cover or overlap with heteroaryl groups as defined separately below. If one or more aryl groups are fused with a heteroaryl group, the resulting ring system shall be defined as a heteroaryl group; if one or more aryl groups are fused with a heterocyclic group, the resulting ring system shall be a heterocyclic group.

[0051] The term "azido" refers to the -N3 group.

[0052] The term "carbamoyl" refers to the "O-carbamoyl" group (-OC(O)NR). y R z (group), and "N-carbamoyl" group (-NR) y C(O)OR z (group), where R y and R z The groups are independently selected from hydrogen, alkyl, aryl, haloalkyl, or heteroaryl groups; each of these groups may be optionally substituted as defined herein.

[0053] The term "carboxyl group" refers to the -C(O)OH group.

[0054] The term “carboxylic acid ester” refers to a group of the general formula -OC(O)R and -C(O)OR, wherein R is hydrogen, alkyl, cycloalkyl, heterocyclic, aryl, heteroalkyl or heteroaryl; each of the above groups may optionally be substituted as defined herein.

[0055] The terms "cyano" or "nitrile" refer to the -CN group.

[0056] The term "cycloalkyl" refers to a saturated or partially unsaturated cycloalkyl group having a monocyclic or polycyclic structure (including fused, bridged, and spirocyclic systems). The "cycloalkyl" comprises a cycloalkenyl group (i.e., a cyclic group having at least one double bond). As defined herein, a cycloalkyl group has 3 to 20 cyclic carbon atoms (C60-C60). 3-20 cycloalkyl groups, 3 to 12 cyclic carbon atoms (C 3-12 cycloalkyl groups, 3 to 10 cyclic carbon atoms (C3-10 cycloalkyl groups, 3 to 8 cyclic carbon atoms (C 3-8 cycloalkyl groups or 3 to 6 cyclic carbon atoms (C 3-6 (Cycloalkyl). Typical examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0057] The term "guanidinyl" refers to -NHC(NH)(NH2).

[0058] The term "hydrazine group" refers to -NHNH2.

[0059] The term "imino" refers to a -C(NR)R group, wherein each R is an alkyl, cycloalkyl, heterocyclic, aryl, heteroalkyl, or heteroaryl group; each R may optionally be substituted as defined herein.

[0060] The terms “halogen” or “halogenated” include fluorine, chlorine, bromine, and iodine. “Halogenated alkyl” refers to a straight-chain or branched alkyl group as defined above, in which one or more hydrogen atoms are substituted with a halogen. For example, when residues are substituted with multiple halogens, a prefix corresponding to the number of halogen groups attached may be used. Dihaloalkyl and trihaloalkyl refer to alkyl groups substituted with two (“di”) or three (“tri”) halogen groups, respectively, which may be the same or different. Examples of halogenated alkyl groups include difluoromethyl (-CHF2) and trifluoromethyl (-CF3).

[0061] The term "heteroalkyl" refers to a group in which one or more carbon atoms (and attached hydrogen atoms) in an alkyl group are independently substituted by the same or different heteroatoms. The term "heteroalkyl" includes straight-chain or branched saturated chains containing carbon atoms and heteroatoms. For example, one, two, or three carbon atoms may be independently substituted by the same or different heteroatom groups, such as "C...". 2-10The "heteroalkyl" group comprises 3 to 9 carbon atoms and 1 to 3 heteroatoms, such that the number of carbon atoms and heteroatoms in the heteroalkyl chain is 2 to 10. Heteroatomic groups include, but are not limited to, -NR-, -O-, -S-, -S(O)-, -S(O)2-, etc., where R is a hydrogen atom, alkyl, aryl, cycloalkyl, heteroalkyl, heteroaryl, or heterocyclic group, and each of these groups may optionally be substituted as defined herein. The term "heteroalkylene" refers to a divalent heteroalkyl group. A "heteroalkylene" group must contain at least one carbon atom and one heteroatomic group within the chain. Examples of non-limiting heteroalkyl groups include... : -CH2OCH2-, OCH2-, -CH(CH3)OCH2-, -CH2CH2OCH2-, -CH2CH2OCH2CH2OCH2-, -CH2SCH2-, -CH(CH3)SCH2-, -CH2CH2SCH2- , -CH2CH2SCH2CH2SCH2-, -CH2S(O)2CH2-, -CH(CH3)S(O)2CH2-, -CH2CH2S(O)2CH2-, -CH2CH2S(O)2CH2CH2OCH2-, -CH2NR y CH2-, -NR y CH2CH2-、-CH(CH3)NR y CH2-、-CH2CH2NR y CH2-、-CH2CH2NR y CH2CH2NR y CH2- etc., where R y The group can be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, heteroalkyl, or heteroaryl; each of the above groups may be optionally substituted as defined herein. In this document, heteroalkyl includes 1 to 10 carbon atoms, 1 to 8 carbon atoms, or 1 to 4 carbon atoms; and 1 to 3 heteroatoms, 1 to 2 heteroatoms, or 1 heteroatom.

[0062] The term "heteroaryl" refers to an aromatic group having a monocyclic, polycyclic, or polyfused-ring structure, wherein one or more heteroatoms selected independently from nitrogen, oxygen, and sulfur are attached to the ring. The heteroaryls described herein comprise 1 to 20 cyclic carbon atoms (i.e., C atoms). 1-20 heteroaryl), 3 to 12 cyclic carbon atoms (i.e., C 3-12 (heteroaryl) or 3 to 8 cyclic carbon atoms (i.e., C) 3-8(Heteroaryl); and 1 to 5 heteroatoms, 1 to 4 heteroatoms, 1 to 3 cyclic heteroatoms, 1 to 2 cyclic heteroatoms, or 1 cyclic heteroatomum independently selected from nitrogen, oxygen, and sulfur. Examples of heteroaryl include pyrimidinyl, purine, pyridinyl, pyridazinyl, benzothiazolyl, and pyrazolyl. Examples of fused heteroaryl rings include, but are not limited to, benzo[d]thiazolyl, quinolinyl, isoquinolinyl, benzo[b]thiophene, indazole, benzo[d]imidazolyl, pyrazolo[1,5-a]pyridinyl, and imidazo[1,5-a]pyridinyl, wherein the heteroaryl group can be linked by any ring of the fused system. Any monocyclic or polycyclic aromatic ring containing at least one heteroatom, regardless of its connection to the rest of the molecule (i.e., linked by any fused ring), is considered a heteroaryl. Heteroaryl does not include and does not overlap with the term "aryl" as defined above.

[0063] The term "heterocyclic group" refers to a saturated or unsaturated cyclic alkyl group having one or more independent cyclic heteroatoms selected from nitrogen, oxygen, and sulfur. This term encompasses heterocyclic alkenyl groups (i.e., heterocyclic groups containing at least one double bond), bridged heterocyclic groups, fused heterocyclic groups, and spirocyclic groups. Heterocyclic groups can be monocyclic or multicyclic structures, wherein the multiple rings are connected by fusion, bridging, or spirocyclic linkages. Any non-aromatic ring containing at least one heteroatom is considered a heterocyclic group, regardless of whether it is linked to other groups via carbon atoms or heteroatoms. Furthermore, the term "heterocyclic group" is intended to cover any non-aromatic ring containing at least one heteroatom, which may be fused with aryl or heteroaryl rings and is not limited in its connection to other parts of the molecule. The heterocyclic groups described herein contain 2 to 20 cyclic carbon atoms (i.e., C atoms). 2-20 Heterocyclic groups), 2 to 12 ring carbon atoms (i.e., C 2-12 Heterocyclic group), 2 to 10 ring carbon atoms (i.e., C 2-10 Heterocyclic group), 2 to 8 ring carbon atoms (i.e., C 2-8 Heterocyclic group), 3 to 12 ring carbon atoms (i.e., C 3-12 Heterocyclic group), 3 to 8 ring carbon atoms (i.e., C 3-8 Heterocyclic group) or 3 to 6 ring carbon atoms (i.e., C 3-6A heterocyclic group (HCH) contains 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 heteroatom independently selected from nitrogen, sulfur, or oxygen. Typical examples of heterocyclic groups include pyrrolidinyl, piperidinyl, piperazineyl, oxoheterobutyl, dioxopentyl, azirrobutyl, and morpholinyl. The term "bridged heterocyclic group" refers to a four- to ten-membered ring structure connected by two non-adjacent atoms to one or more four- to ten-membered rings containing at least one heteroatom (each heteroatom independently selected from nitrogen, oxygen, and sulfur). Bridged heterocyclic groups include bicyclic and tricyclic systems. The term "spirocyclic group" refers to a three- to ten-membered heterocyclic group and one or more additional three- to ten-membered rings (cycloalkyl or heterocyclic groups) formed by a single shared atom. Typical examples of spiroheterocyclic groups include bicyclic / tricyclic systems, such as 2-oxa-7-azaspiro[3.5]nonyl, 2-oxa-6-azaspiro[3.4]octyl, and 6-oxa-1-azaspiro[3.3]heptyl. Fused heterocyclic groups include, but are not limited to, 1,2,3,4-tetrahydroisoquinolinyl, 4,5,6,7-tetrahydrothieno[2,3-c]pyridyl, indololinyl, and isoindololinyl, wherein the heterocyclic group can be linked by any ring in the fused ring system.

[0064] The term "hydroxyl" or "hydroxyl group" refers to the -OH group.

[0065] The term "oxo" refers to (=O) or (O).

[0066] The term "nitro" refers to the -NO2 group.

[0067] The term "sulfonyl" refers to a group with the general formula -S(O)2R, where R is an alkyl, haloalkyl, heterocyclic, cycloalkyl, heteroaryl, or aryl group. Examples of sulfonyl groups include methanesulfonyl, ethanesulfonyl, benzenesulfonyl, and toluenesulfonyl.

[0068] The term "alkylsulfonyl" refers to a group with the general formula -S(O)2R, where R is an alkyl group.

[0069] The term "alkylsulfinyl" refers to a group with the general formula -S(O)R, where R is an alkyl group.

[0070] The term "thiocyanate" refers to the -SCN group.

[0071] The term "thiol" refers to the -SH group.

[0072] The terms “thiocarbonyl” or “thion” refer to the (=S) or (S) group.

[0073] Certain commonly used alternative chemical names may be used. For example, divalent groups, such as divalent "alkyl" groups, divalent "heteroalkyl" groups, and divalent "aryl" groups, may also be referred to as "alkylene", "heteroalkylene", or "arylene", respectively. Furthermore, unless otherwise explicitly stated, when a combination of groups (such as arylalkyl) is referred to as a single group in this document, the last group mentioned includes the atoms to which that group is attached to the rest of the molecule.

[0074] The terms "optional" or "optionally" indicate that the event or situation described below may or may not occur, and the description includes both instances where the event or situation occurs and instances where it does not occur. Furthermore, the term "optional substitution" means that one or more hydrogen atoms on a specified atom or group may be substituted by other non-hydrogen groups, or may remain unchanged.

[0075] Some compounds exist as tautomers. These tautomers exist in a dynamic equilibrium. For example, an amide-containing compound may be in dynamic equilibrium with its imine tautomer. Regardless of the type of tautomer shown or its equilibrium state, those skilled in the art will recognize that these compounds contain both amide and imine tautomers. Therefore, amide-containing compounds are generally considered to contain their imine tautomers, and similarly, imine-containing compounds are generally considered to contain their amide tautomers.

[0076] Any chemical formula or structure described herein is intended to cover both unlabeled and isotopically labeled forms of the compounds. The structure of an isotopically labeled compound is consistent with the chemical formula provided herein, except that one or more atoms are replaced with atoms having a specific atomic mass or mass number. Examples of isotopes that may be incorporated into the compounds disclosed herein include, but are not limited to, isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, such as, but not limited to: 2 H (deuterium, D) 3 H (tritium) 11 C 13 C 14 C 15 N、 18 F, 31 P, 32 P, 35 S, 36 Cl and 125 I. This invention relates to various isotope-labeled compounds, such as those containing... 3 H, 13 C and 14Compounds containing radioactive isotopes such as C. These isotope-labeled compounds can be used in metabolic studies, reaction kinetic studies, detection or imaging techniques such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT), including the determination of drug or substrate tissue distribution, and can also be used in fields such as radiotherapy.

[0077] This invention also includes "deuterated analogues" of Formula I compounds, wherein one to n hydrogen atoms bonded to a carbon atom are replaced by deuterium atoms, where n is the number of hydrogen atoms in the molecule. These compounds exhibit stronger metabolic resistance and thus effectively prolong the half-life of Formula I compounds when administered to mammals, particularly humans. See Foster, "Deuterium Isotope Effects in Studies of Drug Metabolism," Trends Pharmacol. Sci. 5(12):524-527 (1984). These compounds can be synthesized using methods known in the art, for example, using starting materials with one or more hydrogen atoms replaced by deuterium.

[0078] The deuterium-labeled or deuterium-substituted therapeutic compounds described herein may exhibit superior DMPK (drug metabolism and pharmacokinetics) properties, involving distribution, metabolism, and excretion (ADME). Substitution with heavier isotopes such as deuterium may offer certain therapeutic advantages, such as prolonged in vivo half-life through enhanced metabolic stability, reduced dose requirements, and / or improved therapeutic index. 18 F-labeled compounds can be used in positron emission tomography (PET) or single-photon emission computed tomography (SPECT) studies. The isotope-labeled compounds and their prodrugs described herein can generally be prepared by replacing non-isotope-labeled reagents with readily available isotope-labeled reagents using the methods described in existing protocols or the accompanying examples. It should be understood that, herein, deuterium is considered a substituent in compounds of formula I.

[0079] The concentration of such heavier isotopes (especially deuterium) can be defined by isotope enrichment factors. In the compounds described in this invention, any atom not specifically labeled as a particular isotope should be understood as representing any stable isotope of that atom. Unless otherwise stated, when a position is explicitly labeled "H" or "hydrogen," that position should be understood as hydrogen with a naturally abundant isotopic composition. Therefore, in the compounds described in this invention, any atom explicitly labeled as deuterium (D) refers to the element deuterium.

[0080] In most cases, the compounds of the present invention are capable of forming acid salts and / or base salts by containing amino and / or carboxyl groups or similar groups.

[0081] The present invention also provides pharmaceutically acceptable salts, hydrates, solvates, tautomers, polymorphs, and prodrugs of the said compounds. "Pharmaceutically acceptable" or "physiologically acceptable" means compounds, salts, compositions, dosage forms, and other materials that can be used to prepare pharmaceutical formulations suitable for veterinary or human use.

[0082] The term "pharmaceutically acceptable salt" for a given compound refers to a salt that retains the biological activity and properties of the original substance and has no adverse effects on biology or otherwise. "Pharmaceutically acceptable salt" or "physiologically acceptable salt" includes, but is not limited to, inorganic acid salts and organic acid salts. If the compound described herein is obtained as an acid addition salt, the free base can be obtained by alkalizing the acidic salt solution. Conversely, if the product is a free base, the addition salt, particularly a pharmaceutically acceptable addition salt, can be prepared according to the conventional procedure for preparing acid addition salts from basic compounds by dissolving the free base in a suitable organic solvent and treating the solution with acid. Those skilled in the art are familiar with various synthetic methods for preparing non-toxic pharmaceutically acceptable acid addition salts. Pharmaceutically acceptable acid addition salts can be prepared from inorganic or organic acids. Inorganic acid salts include hydrochlorides, hydrobromic acids, sulfates, nitrates, phosphates, etc. Salts derived from organic acids include acetates, propionates, glycolates, pyruvates, oxalates, malates, malonates, succinates, maleates, fumarates, tartrates, citrates, benzoates, cinnamates, mandelates, methanesulfonates, ethanesulfonates, p-toluenesulfonates, and salicylates. Similarly, pharmaceutically acceptable base addition salts can be prepared from inorganic or organic bases. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, such as alkylamines (i.e., NH2(alkyl)), dialkylamines (i.e., HN(alkyl)2), trialkylamines (i.e., N(alkyl)3), substituted alkylamines (i.e., NH2(substituted alkyl)), di(substituted alkyl)amines (i.e., HN(substituted alkyl)2), tri(substituted alkyl)amines (i.e., N(substituted alkyl)3), alkenylamines (i.e., NH2(alkenyl)), dienylamines (i.e., HN(alkenyl)) Suitable amines include, but are not limited to, isopropylamine, trimethylamine (i.e., N(alkenyl)3), substituted alkenylamines (i.e., NH2(substituted alkenyl)), di(substituted alkenyl)amines (i.e., HN(substituted alkenyl)2), tri(substituted alkenyl)amines (i.e., N(substituted alkenyl)3), mono / di / tricycloalkylamines (i.e., NH2(cycloalkyl), HN(cycloalkyl)2, N(cycloalkyl)3), mono / di / triarylamines (i.e., NH2(aryl), HN(aryl)2, N(aryl)3), or mixed amines. Specific examples of suitable amines include, but are not limited to, isopropylamine, trimethylamine, diethylamine, triisopropylamine, tri-n-propylamine, ethanolamine, 2-dimethylaminoethanol, piperazine, piperidine, morpholine, N-ethylpiperidine, etc.

[0083] The term "substitution" refers to the replacement of one or more hydrogen atoms on a specified atom or group with a non-hydrogen substituent, not exceeding the atom's normal valence state. Such substituents include, but are not limited to: alkyl, alkenyl, alkynyl, alkoxy, acyl, amino, amide, amidyl, aryl, azide, carbamoyl, carboxyl, carboxyl ester, cyano, guanidinyl, halogen, haloalkyl, haloalkoxy, heteroalkyl, heteroaryl, heterocyclic, hydroxyl, hydrazine, imino, oxo, nitro, alkylsulfinyl, sulfonic acid, alkylsulfonyl, thiocyanate, mercapto, thion, or combinations thereof. Polymers derived by an infinite stacking of substituents or similar infinitely extended structures (e.g., an aryl group substituted with a substituted alkyl group, which itself is substituted with a substituted aryl group, which is further substituted with a substituted heteroalkyl group, and so on) are not included within the scope of this document. Unless otherwise stated, the maximum number of successive substitutions in the compounds described herein is three. For example, an aryl group sequentially substituted by two other substituted aryl groups is limited to a structure of ((substituted aryl)substituted aryl)substituted aryl. Similarly, the above definition is not intended to include unacceptable substitution patterns (e.g., a methyl group substituted with five fluorine atoms or a heteroaryl group having two adjacent oxygen ring atoms). Such unacceptable substitution patterns are well known to those skilled in the art. When used to modify chemical groups, the term “substituted” may refer to other chemical groups as defined herein. Unless otherwise stated, when a group is described as “optionally substituted,” all substituted elements of that group are themselves unsubstituted. For example, in some embodiments, the term “substituted alkyl” refers to an alkyl group having one or more substituents, including hydroxyl, halogen, alkoxy, cycloalkyl, heterocyclic, aryl, and heteroaryl. In other embodiments, one or more substituents may be further substituted with halogen, alkyl, haloalkyl, hydroxyl, alkoxy, cycloalkyl, heterocyclic, aryl, or heteroaryl, and each of these groups is substituted. The substituents may be further substituted with halogens, alkyl groups, haloalkyl groups, alkoxy groups, hydroxy groups, cycloalkyl groups, heterocyclic groups, aryl groups, or heteroaryl groups, and each of these groups is unsubstituted.

[0084] As used herein, the terms "pharmaceutical carrier" or "pharmaceutical excipient" include any and all solvents, dispersion media, coating agents, antimicrobial agents, antifungal agents, isotonic agents, and sustained-release agents. The use of such media and reagents in pharmaceutically active substances is common knowledge in the art. Except where conventional media or dosage forms are incompatible with the active ingredient, their use in therapeutic compositions is covered by this invention. Additional active ingredients may also be added to the composition.

[0085] The term "solvent" refers to a complex formed by the interaction of a solvent and a compound. Salt solvates of the compounds described herein are also provided. Similarly, hydrates of the compounds described herein are also included.

[0086] As used herein, the term "encapsulation efficiency" refers to the percentage of a therapeutic or prophylactic ingredient encapsulated in a nanoparticle formulation relative to the initial total amount added during the preparation of the formulation. For example, if 97 mg of a total therapeutic and / or prophylactic dose of 100 mg is encapsulated in the nanoparticle composition, the encapsulation efficiency can be expressed as 97%. It should be noted that the term "encapsulation" as used herein may refer to different meanings such as complete, substantial or partial encapsulation, closure, enclosure, or encapsulation.

[0087] As used herein, the term “expression” of a nucleic acid sequence refers to the translation of mRNA into a polypeptide or protein and / or the post-translational modification of the polypeptide or protein.

[0088] As used herein, the term “in vitro” refers to an event that occurs in an artificial environment, such as in a test tube or reaction vessel, cell culture, petri dish, etc., rather than in a living organism (e.g., an animal, plant, or microorganism).

[0089] As used in this article, the term "in vivo" refers to events that occur within an organism (such as an animal, plant, microorganism, or its cells or tissues).

[0090] As used herein, the term "extracorporeal" refers to an event that occurs outside of an organism, such as an animal, plant, or microorganism or its cells or tissues. Extracorporeal events can occur in environments that are virtually unchanged compared to the natural (e.g., in vivo) environment.

[0091] As used herein, the term "isomer" refers to any geometric isomer, tautomer, zwitterion, stereoisomer, enantiomer, or diastereomer of a compound. A compound may contain one or more chiral centers and / or double bonds, and therefore may have stereoisomers, such as double-bonded isomers (i.e., geometric E / Z isomers) or diastereomers (such as enantiomers (i.e., (+) or (-) isomers) or cis-trans isomers). This invention covers all isomers of the compound, including stereopure forms (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) and mixtures of enantiomers and stereoisomers, such as racemates. Methods for obtaining mixtures of enantiomers and stereoisomers of a compound and for resolving them into individual enantiomers or stereoisomers are well known in the art.

[0092] As used herein, the term "lipid component" refers to a component in a nanoparticle composition that contains one or more lipids. For example, a lipid component may include one or more cationic / ionizable, polyethylene glycol-modified, structured lipids, or other lipids, such as phospholipids.

[0093] As used herein, the term "linking group" refers to a chemical group that connects two molecular structures, such as the linking portion between two nucleosides in a cap-type compound. Such linking groups may contain one or more groups, including but not limited to phosphate groups (such as phosphoric acid, borophosphate, thiophosphate, selenophosphate, and phosphonic acid), alkyl groups, amide groups, or glycerol groups. For example, two nucleosides in a cap-type structure may be linked by a triphosphate group or by a chain structure containing two phosphate groups and one borophosphate group. The "route of administration" described herein includes methods of delivering the drug composition to a subject, such as intravenous injection, intramuscular injection, intradermal injection, and subcutaneous injection. The choice of route of administration can be for targeted delivery to specific regions or systems (e.g., achieving precise localization).

[0094] As used herein, the term "modification" refers to something that is not naturally occurring. For example, RNA may be a modified RNA, meaning it contains one or more non-naturally occurring nucleobases, nucleosides, nucleotides, or linkers. In the terminology of this art, a "modified" substance may also be referred to as an "altered" substance. Such substances can be modified or altered at the chemical, structural, or functional levels. For example, a modified nucleobase substance may contain one or more non-naturally occurring substituents.

[0095] As used herein, the term “nitrogen-to-phosphorus ratio” refers to the molar ratio of nitrogen atoms ionized in lipids within a physiological pH range to phosphate groups in RNA, for example, in nanoparticle compositions comprising lipid components and RNA.

[0096] The term "nanoparticle composition" as used in this article refers to a composition containing one or more lipid components. These nanoparticle compositions typically have sizes in the micrometer range or even smaller and may contain lipid bilayer structures. Specifically, nanoparticle compositions encompass various forms, including lipid nanoparticles (LNPs), liposomes (such as lipid vesicles), and lipid complexes. For example, lipid bilayer structures with a diameter not exceeding 500 nanometers belong to this category of nanoparticle compositions.

[0097] As used in this article, the term "naturally occurring" refers to something that exists in the natural environment without human intervention.

[0098] As used herein, the term “patient” means a subject who may seek or need treatment, needs to receive treatment, is receiving treatment, will receive treatment, or is being cared for by a trained professional for a particular disease or condition.

[0099] As used herein, the term "PEG lipid" or "PEGylated lipid" refers to lipids containing polyethylene glycol components.

[0100] As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that, within reasonable medical judgment, are suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems / complications, and that meet a reasonable benefit / risk ratio.

[0101] As used herein, the term "pharmaceutical excipient" refers to any other component besides the compounds described herein (e.g., a carrier capable of suspending, complexing, or dissolving the active ingredient) that is substantially non-toxic and non-inflammatory in the patient body. Excipients may include, but are not limited to: anti-adhesives, antioxidants, adhesives, coating agents, pressure aids, disintegrants, dyes (colorants), emollients, emulsifiers, fillers (diluents), film-forming agents or coating agents, flavoring agents, fragrances, flow enhancers (flow improvers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and hydrated water. Exemplary excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium hydrogen phosphate, calcium stearate, croscarmellose sodium, croscarmellose, citric acid, croscarmellose polyvinylpyrrolidone, cysteine, ethyl cellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silica, sodium carboxymethyl cellulose, sodium citrate, sodium carboxymethyl starch, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (α-tocopherol), vitamin C, xylitol, and other substances disclosed herein.

[0102] In this specification, for convenience, the structural formula of a compound may sometimes represent only one isomer, but the scope of this invention covers all isomers of the compound, such as geometric isomers, optical isomers based on asymmetric carbon, stereoisomers, tautomers, etc.; however, it should be understood that not all isomers have the same level of activity. Furthermore, the compound may exhibit polymorphism. It should be noted that any crystal form, mixture of crystal forms, or its anhydrous or hydrated form is included within the scope of this invention.

[0103] The terms "polymorphic crystal," "polymorph," or "crystal form" refer to the crystal structures formed by the ability of a compound (or its salts or solvates) to crystallize in different crystalline arrangements, all of which have the same elemental composition. Different crystal forms typically have different X-ray diffraction patterns, infrared spectra, melting points, densities, hardness, crystal shapes, optical and electrical properties, stability, and solubility.

[0104] Factors such as recrystallization solvent, crystallization rate, and storage temperature may cause a certain crystal form to dominate, and polymorphs of the compound can be prepared by crystallization under different conditions.

[0105] The compound composition may also comprise salts of one or more compounds. The salts may be pharmaceutically acceptable salts. As used herein, a "pharmaceutically acceptable salt" refers to a derivative of the disclosed compound, which is formed by converting an existing acidic or basic group in the parent compound into a salt form (e.g., by reacting a free base with a suitable organic acid). Pharmaceutically acceptable salts include, but are not limited to: inorganic or organic acid salts of basic residues (such as amines); alkali metal or organic salts of acidic residues (such as carboxylic acids), etc. Typical acid addition salts include: acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, gluconate, glyceryl phosphate, hemisulfate, heptaate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, lacturonate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, dihydroxynaphthalate, pectate, persulfate, 3-phenylpropionate, phosphate, picrate, neopentanoate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate, etc.

[0106] Typical alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc., as well as salts formed from non-toxic ammonium, quaternary ammonium, and amine cations, including but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, etc. Pharmaceutically acceptable salts of this invention include conventional non-toxic salts formed from parent compounds, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of this invention can be synthesized by conventional chemical methods from parent compounds containing a basic or acidic moiety. Typically, such salts are prepared by reacting the free acidic or basic form of these compounds with a stoichiometric amount of a suitable base or acid in water, an organic solvent, or a mixture of both; typically, a non-aqueous medium, such as diethyl ether, ethyl acetate, ethanol, isopropanol, or acetonitrile, is preferred. The list of suitable salts can be found in the following references: Remington Pharmaceutical Science (17th edition, Mack Publishing Company, Easton, Pa., 1985, p. 1418), Pharmaceutical Salts: Properties, Selection and Applications (edited by P.H. Stahl and C.G. Wermuth, Wiley-VCH, 2008), and Berge et al.’s article in the Journal of Pharmaceutical Science (Vol. 66, pp. 1–19, 1977), each of which is cited in its entirety in this article.

[0107] compound

[0108] In one respect, compounds of formula I are provided.

[0109] I-1

[0110] in

[0111] L 1 C 1-10 Alkylene or C 2-10 Heteroalkylene; wherein, the C 2-10 Heteroalkylene compounds are optionally R 6 replace;

[0112] L 2 C 1-10 Alkylene or C 2-10 Heteroalkylene; wherein, the C 2-10 Heteroalkylene compounds are optionally R 6 replace;

[0113] X is -CH2-, -NR 3 -、-N (R 3 )2 + -, -O-, -O-CH2CH2-O- or -NR 3 -(CH2) m -NR 3-;

[0114] m is an integer from 1 to 6;

[0115] R 1 R 2 and R 3 Each independently is hydrogen, C 1-20 Alkyl, C 2-20 alkenyl, C 2-20 alkynyl group, C 2-20 Heteroalkyl, , or ;

[0116] Wherein C 1-20 Alkyl, C 2-20 alkenyl, C 2-20 alkynyl or C 2-20 Each heteroalkyl group is independently and optionally surrounded by 1 to 5 halogen, cyano, or -OR groups. 4 -SR 4 -NR 4 2. -N(R) 4 )3 + Oxygen, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl or C 1-6 Halogenated alkyl substitution;

[0117] R a Independently selected from hydrogen, C 1-12 Alkyl or -C(O)-C 1-12 alkyl;

[0118] R b Independently selected from hydrogen, C 1-12 Alkyl or -C(O)-C 1-12 alkyl;

[0119] R 4 Independently selected from hydrogen, C 1-12 Alkyl, C 2-12 alkenyl or C 2-12 alkynyl group; wherein each C 1-12 Alkyl, C 2-12 alkenyl or C 2-12 The alkynyl group is independently and optionally surrounded by 1 to 5 halogens, cyano groups, -OH groups, or -SR groups. 5 -NR 5 2. -N(R) 5 )3 + Or oxygen substitution;

[0120] R 5 Independently selected from hydrogen, C 1-12 Alkyl, C 2-12alkenyl or C 2-12 alkynyl group; wherein each C 1-12 Alkyl, C 2-12 alkenyl or C 2-12 The alkynyl group is independently and optionally surrounded by 1 to 5 halogens, cyano groups, -OH, -SH, -NH2, -NH (C 1-6 Alkyl), -N (C) 1-6 alkyl)2、-N (C) 1-6 Alkyl)3 + Or oxygen substitution;

[0121] R 6 Selected independently , , or ;

[0122] L is independently selected from C 1-10 Alkylene or C 3-10 Heteroalkylene;

[0123] R 7 Independently selected from hydrogen, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl or C 1-6 Halogenated alkyl; wherein each C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl or C 1-6 The haloalkyl group is independently and optionally surrounded by 1 to 5 halogens, cyano groups, -OH, -NH2, -NH (C 1-6 Alkyl), -N (C) 1-6 alkyl)2、-N (C) 1-6 Alkyl)3 + Oxygen, C 1-6 Alkoxy or C 1-6 Halogenated alkoxy groups;

[0124] n is an integer independently between 1 and 20;

[0125] p is an integer independently between 1 and 6;

[0126] R is independently selected from hydrogen, -ZC 1-20 Alkyl, -ZC 2-20 alkenyl, -ZC 2-20 Alkyne group, -Z-heterocyclic group, -Z 1 -C 1-6 Alkylene-ZC 1-20 Alkyl, -Z 1 -C 1-6 Alkylene-ZC 2-20 alkenyl, -Z1 -C 1-6 Alkylene-ZC 2-20 alkynyl group, -Z 1 -C 1-6 alkylene-Z-heterocyclic group, -Z 1 -C 2-6 imide-ZC 1-20 Alkyl, -Z 1 -C 2-6 imide-ZC 2-20 alkenyl, -Z 1 -C 2-6 imide-ZC 2-20 alkynyl group, -Z 1 -C 2-6 Ethyne-ZC 1-20 Alkyl, -Z 1 -C 2-6 Ethyne-ZC 2-20 alkenyl, or -Z 1 -C 2-6 Ethyne-ZC 2-20 alkynyl group;

[0127] Z is independently selected from chemical bonds, -O-, -NR4-, -S-, -SS-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)NR 4 -、-S (O)-、-S (O)2-、-NR 4 C(O)-、-NR 4 C (O)O-、-NR 4 C (O)NR 4 -、-NR 4 S(O)- or -S(O)2NR 4 -;

[0128] Z 1 Independently selected from -O- and -NR 4 -, -S-, -SS-, -C (O)-, -C (O)O-, -C (O)NR 4 -、-S (O)-、-S(O)2-、-NR 4 C(O)-、-NR 4 C (O)O-、-NR 4 C (O)NR 4 -、-NR 4 S(O)- or -S(O)2NR 4 -; and each Some contain at least 6 linear atoms.

[0129] In some embodiments, each Ra Independently hydrogen or -C(O)-C 1-12 Alkyl group. In some embodiments, R a -C(O)-C 1-6 Alkyl group. In some embodiments, R a It is -C(O)CH3.

[0130] In some embodiments, R a It is hydrogen.

[0131] In some embodiments, X is -NR 3 -

[0132] In some embodiments, R 3 yes Or C 1-20 Alkyl groups are optionally surrounded by 1 to 5 -OR 4 -NR 4 2 or -N(R) 4 )3 + replace.

[0133] In some embodiments, R 3 yes .

[0134] In some embodiments, R 3 It is C 1-20 Alkyl group. In some embodiments, R 3 It is C 1-6 Alkyl group. In some embodiments, R 3 It is a methyl group.

[0135] In some embodiments, R 3 It is C 1-20 Alkyl group, optionally surrounded by 1 to 5 -OR 4 -NR 4 2 or -N (R 4 )3 + Replacement. In some embodiments, R 3 It is C 1-6 Alkyl group, wherein the alkyl group is surrounded by 1 to 3 -OH, -N (CH3)3 + Or -N (CH2CH2OH)2 substitution.

[0136] In some embodiments, R 3 Selected from: , , , , , , and .

[0137] In some embodiments, X is -N(CH3)-.

[0138] In some embodiments, X is -N (R 3 )2 + - In some embodiments, X is -N(CH3)2 + - In some embodiments, X is -N(CD3)2 + -

[0139] In some embodiments, X is -CH2-.

[0140] In some embodiments, X is -O-.

[0141] In some embodiments, X is -O-CH2CH2-O-.

[0142] In some embodiments, X is -NR 3 - (CH2) m -NR 3 - where m is an integer from 1 to 6. In some embodiments, X is -NR 3 - (CH2) m -NR 3 - where m is 2, 3 or 4.

[0143] In some embodiments, X is -N(CH3)-CH2CH2-N(CH3)-, -N(CH3)-(CH2)3-N(CH3)-, or -N(CH3)-(CH2)4-N(CH3)-.

[0144] In some embodiments, each R 4 Independently hydrogen or C 1-12 Alkyl, the C 1-12 The alkyl group is optionally substituted with 1 to 3 -OH groups.

[0145] In some embodiments, R 1 It is hydrogen, C 1-20 alkyl or .

[0146] In some embodiments, R 2 It is hydrogen, C 1-20 alkyl or .

[0147] In some embodiments, R 1 and R 2 Each independently is hydrogen, C 1-20 alkyl or .

[0148] In some embodiments, R1 and R 2 Each can be independently hydrogen, -CH3, or .

[0149] In some embodiments, R 1 and R 2 Each independently is hydrogen, C 1-20 alkyl or X is -NR 3 -;R 3 for Or C 1-20 Alkyl groups are optionally surrounded by 1 to 5 -OR 4 -NR 4 2 or -N (R 4 )3 + replace.

[0150] In some embodiments, each Y is -NH- or -N(CH3)-.

[0151] In some embodiments, each Y is -NH-.

[0152] In some embodiments, each n is 2-16.

[0153] In some embodiments, each n is 6-16.

[0154] In some embodiments, each n is 6-12.

[0155] In some embodiments, each R is independently hydrogen, -ZC 1-20 Alkyl, -ZC 2-20 alkenyl, -Z 1 -C 1-6 Alkylene-ZC 1-20 Alkyl, -Z 1 -C 1-6 Alkylene-ZC 2-20 alkenyl or -Z 1 -C 1-6 Alkyl-Z-heterocyclic group.

[0156] In some embodiments, each Z is independently a chemical bond, -O-, -NR. 4 C (O)-, -C(O)O-, or -OC (O)-.

[0157] In some embodiments, each Z is independently a chemical bond, -O-, -NR. 4 C(O)- or -OC(O)-.

[0158] In some embodiments, each Z is independently a chemical bond, -O-, -NHC(O)-, -C(O)O-, or -OC(O)-.

[0159] In some embodiments, each Z is independently a chemical bond, -O-, -NHC(O)-, or -OC(O)-.

[0160] In some embodiments, each Z is independently -NHC(O)- or -OC(O)-.

[0161] In some embodiments, each Z is independently a chemical bond, -O- or -OC(O)-.

[0162] In some embodiments, each Z 1 Independently -O- or -OC (O)-.

[0163] In some embodiments, each Z 1 Yes -O-.

[0164] In some embodiments, Z is a chemical bond and Z 1 It is -OC (O)-. In some embodiments, Z is -O- and Z 1 Yes -O-.

[0165] In some embodiments, R is hydrogen and the value of n ranges from 6 to 16. In some embodiments, R is hydrogen and n is 6, 8, 10, 12, or 14. In some embodiments, R is hydrogen and n is 8, 10, or 12. In some embodiments, R is hydrogen and n is 10 or 12.

[0166] In some embodiments, R is -ZC 2-20 Alkenyl group, wherein Z is a chemical bond. In some embodiments, R is -C. 6-14 Alkenyl. In some embodiments, R is -C6 alkenyl, -C8 alkenyl, -C... 10 alkenyl, -C 12 alkenyl or -C 14 Alkenyl. In some embodiments, n is 4, 6, 8, or 10 and R is -C. 6-14 Alkenyl. In some embodiments, n is 4, 6, 8, or 10 and R is -C6 alkenyl, -C8 alkenyl, or -C6 alkenyl. 10 alkenyl, -C 12 alkenyl or -C 14 Alkenyl. In some embodiments, R is -ZC. 1-20 Alkyl group, where Z represents -NR 4 C(O)-. In some embodiments, R is -NHC(O)-C. 1-20 Alkyl group. In some embodiments, R is -NHC(O)-C 10-20 Alkyl group. In some embodiments, R is -NHC(O)-C 12-16Alkyl group. In some embodiments, n is 2, 4, 6, 8, or 10 and R is -NHC(O)-C 1-20 Alkyl group. In some embodiments, n is 2, 4, 6, 8, or 10 and R is -NHC(O)-C. 10-20 Alkyl group. In some embodiments, n is 2, 4, 6, 8, or 10 and R is -NHC(O)-C. 12-16 alkyl.

[0167] In some embodiments, R is -ZC 1-20 Alkyl group, wherein Z is -OC(O)-. In some embodiments, R is -OC(O)-C 1-20 Alkyl group. In some embodiments, R is -OC(O)-C. 10-20 Alkyl group. In some embodiments, R is -OC(O)-C. 12-16 Alkyl group. In some embodiments, n is 2, 4, 6, 8, or 10 and R is -OC(O)-C. 1-20 Alkyl group. In some embodiments, n is 2, 4, 6, 8, or 10 and R is -OC(O)-C. 8-20 Alkyl group. In some embodiments, n is 2, 4, 6, 8, or 10 and R is -OC(O)-C. 12-16 alkyl.

[0168] In some embodiments, R is -ZC 1-20 Alkyl group, wherein Z is -OC(O)-. In some embodiments, R is -OC(O)-C 1-20 Alkyl group. In some embodiments, R is -OC(O)-C. 10-20 Alkyl group. In some embodiments, R is -OC(O)-C. 12-16 Alkyl group. In some embodiments, n is 4, 6, 8, or 10 and R is -OC(O)-C. 1-20 Alkyl group. In some embodiments, n is 4, 6, 8, or 10 and R is -OC(O)-C. 8-20 Alkyl group. In some embodiments, n is 4, 6, 8, or 10 and R is -OC(O)-C. 12-16 alkyl.

[0169] In some embodiments, R is -ZC 1-20 Alkyl group, wherein Z is -C(O)O-. In some embodiments, R is -C(O)OC. 1-20 Alkyl group. In some embodiments, R is -C(O)OC. 10-20 Alkyl group. In some embodiments, R is -C(O)OC. 12-16 Alkyl group. In some embodiments, n is 4, 6, 8, or 10 and R is -C(O)OC. 1-20Alkyl group. In some embodiments, n is 4, 6, 8, or 10 and R is -C(O)OC. 8-20 Alkyl group. In some embodiments, n is 4, 6, 8, or 10 and R is -C(O)OC. 12-16 alkyl.

[0170] In some embodiments, R is -Z 1 -C 1-6 alkylene-Z-heterocyclic group, wherein Z 1 R is -OC(O)-, and Z is a chemical bond. In some embodiments, R is -OC(O)-C. 1-6 Alkylene-heterocyclic group. In some embodiments, R is -OC(O)-C 1-6 Alkylene-1,2-dithionecyclopentane. In some embodiments, n is 4, 6, 8, or 10 and R is -OC(O)-C. 1-6 Alkylene-heterocyclic group. In some embodiments, n is 4, 6, or 8 and R is -OC(O)-C. 1-6 Alkylene-1,2-dithionecyclopentane.

[0171] In some embodiments, R is -Z 1 -C 1-6 Alkylene-ZC 1-20 Alkyl, wherein Z 1 It is -O- and Z is -O-. In some embodiments, R is -OC. 1-6 Alkylene-OC 1-20 Alkyl group. In some embodiments, R is -O-CH2-OC. 4-12 Alkyl group. In some embodiments, R is -O-CH2-O-C4 alkyl, -O-CH2-O-C6 alkyl, -O-CH2-O-C8 alkyl, or -O-CH2-OC alkyl. 10 Alkyl or -O-CH2-OC 12 Alkyl group. In some embodiments, n is 4, 6, 8, or 10 and R is -OC. 1-6 Alkylene-OC 1-20 Alkyl group. In some embodiments, n is 4, 6, 8, or 10 and R is -O-CH2-O-C4 alkyl, -O-CH2-O-C6 alkyl, -O-CH2-O-C8 alkyl, or -O-CH2-OC alkyl. 10 Alkyl or -O-CH2-OC 12 Alkyl group. In some embodiments, R is -O-CH(CH3)-OC. 1-20 Alkyl group. In some embodiments, R is -O-CH(CH3)-OC. 4-12Alkyl group. In some embodiments, R is -O-CH(CH3)-O-C4 alkyl, -O-CH(CH3)-O-C6 alkyl, -O-CH(CH3)-O-C8 alkyl, or -O-CH(CH3)-OC alkyl. 10 Alkyl or -O-CH (CH3)-OC 12 Alkyl group. In some embodiments, n is 4, 6, 8, or 10 and R is -O-CH(CH3)-O-C4 alkyl, -O-CH(CH3)-O-C6 alkyl, -O-CH(CH3)-O-C8 alkyl, or -O-CH(CH3)-OC alkyl. 10 Alkyl or -O-CH (CH3)-OC 12 alkyl.

[0172] In some embodiments, R is -Z 1 -C 1-6 Alkylene-ZC 2-20 alkenyl, of which Z 1 R is -O- and Z is -O-. In some embodiments, R is -OC. 1-6 Alkylene-OC 2-20 Alkenyl group. In some embodiments, R is -O-CH2-OC. 2-20 Alkenyl group. In some embodiments, R is -O-CH2-OC. 4-12 Alkenyl. In some embodiments, R is -O-CH2-O-C4 alkenyl, -O-CH2-O-C6 alkenyl, -O-CH2-O-C8 alkenyl, or -O-CH2-OC. 10 alkenyl or -O-CH2-OC 12 Alkenyl group. In some embodiments, n is 4, 6, 8, or 10 and R is -OC. 1-6 Alkylene-OC 2-20 Alkenyl group. In some embodiments, n is 4, 6, 8, or 10 and R is -O-CH2-OC. 2-20 Alkenyl group. In some embodiments, n is 4, 6, 8, or 10 and R is -O-CH2-OC. 4-12 Alkenyl. In some embodiments, n is 4, 6, 8, or 10 and R is -O-CH2-O-C4 alkenyl, -O-CH2-O-C6 alkenyl, -O-CH2-O-C8 alkenyl, or -O-CH2-OC. 10 alkenyl or -O-CH2-OC 12 Alkenyl group.

[0173] In some embodiments, R is hydrogen, -C 2-20 alkenyl, -OC(O)-C 1-20 Alkyl, -C(O)OC 1-20 Alkyl, -OC(O)-C1-6 alkylene-heterocyclic group, -O-CH2-OC 1-20 Alkyl, -O-CH (CH3)-OC 1-20 Alkyl or -O-CH2-OC 2-20 Alkenyl. In some embodiments, n is in the range of 6-16 and R is hydrogen, -C 2-20 alkenyl, -OC(O)-C 1-20 Alkyl, -C(O)OC 1-20 Alkyl, -OC(O)-C 1-6 alkylene-heterocyclic group, -O-CH2-OC 1-20 Alkyl, -O-CH (CH3)-OC 1-20 Alkyl or -O-CH2-OC 2-20 Alkenyl. In some embodiments, n is 4, 6, 8, or 10 and R is -C 6-14 alkenyl, -OC(O)-C 10-20 Alkyl, -C(O)OC 10-20 Alkyl, -OC(O)-C 1-6 alkylene-heterocyclic group, -O-CH2-OC 4-12 Alkyl, -O-CH (CH3)-OC 4-12 Alkyl or -O-CH2-OC 4-12 Alkenyl group.

[0174] In some embodiments, R is hydrogen, -C 2-20 alkenyl, -OC(O)-C 1-20 Alkyl, -OC(O)-C 1-6 alkylene-heterocyclic group, -O-CH2-OC 1-20 Alkyl, -O-CH (CH3)-OC 1-20 Alkyl or -O-CH2-OC 2-20 Alkenyl. In some embodiments, n is 6-16 and R is hydrogen, -C 2-20 alkenyl, -OC(O)-C 1-20 Alkyl, -OC(O)-C 1-6 alkylene-heterocyclic group, -O-CH2-OC 1-20 Alkyl, -O-CH (CH3)-OC 1-20 Alkyl or -O-CH2-OC 2-20 Alkenyl. In some embodiments, n is 4, 6, 8, or 10 and R is -C 6-14 alkenyl, -OC(O)-C 10-20 Alkyl, -OC(O)-C 1-6 alkylene heterocyclic group, -O-CH2-OC 4-12Alkyl, -O-CH (CH3)-OC 4-12 Alkyl or -O-CH2-OC 4-12 Alkenyl group.

[0175] In some embodiments, the group Selected from:

[0176] , , , , , , , , , , , , , , , , , , , , , , , , , , .

[0177] In some embodiments, compounds selected from Table 1A are provided, or pharmaceutically acceptable salts, stereoisomers, or mixtures thereof.

[0178] Table 1A. Compounds

[0179]

[0180] Lipid nanoparticles and compositions

[0181] The term "lipid nanoparticle" refers to particles that reach the nanoscale (e.g., 1-1000 nm) in at least one dimension, comprising one or more compounds of Formula I, or pharmaceutically acceptable salts, stereoisomers, or mixtures thereof (e.g., compounds in Table 1A, or pharmaceutically acceptable salts, stereoisomers, or mixtures thereof). Nanoparticle compositions include, but are not limited to, types such as lipid nanoparticles (LNPs), liposomes, lipid vesicles, and lipid complexes. In some embodiments, the nanoparticle composition is a vesicle comprising one or more lipid bilayers. In some embodiments, the nanoparticle composition consists of two or more concentric bilayer structures separated by an aqueous compartment. The lipid bilayer may be functionalized and / or cross-linked, and the lipid bilayer may contain one or more ligands, proteins, or channels.

[0182] In some embodiments, lipid nanoparticles are contained in formulations that can be used to deliver active agents or therapeutic agents (such as nucleic acids, e.g., mRNA) to target sites (such as cells, tissues, organs, tumors, etc.). In some embodiments, the lipid nanoparticles comprise nucleic acids. Such lipid nanoparticles are typically composed of a compound of Formula I or a pharmaceutically acceptable salt, stereoisomer, or mixture of stereoisomers thereof (e.g., compounds in Table 1A or pharmaceutically acceptable salts, stereoisomers, or mixtures of stereoisomers thereof) with one or more excipients, including steroids or lipids, such as neutral lipids, charged lipids (e.g., cationic lipids, steroids, or polymer-conjugated lipids).

[0183] The term "cationic lipid" refers to lipids capable of carrying a positive charge. Exemplary cationic lipids contain one or more positively charged amine groups. These lipids are ionizable, and therefore can exist in a positively charged or neutral form depending on the pH value. Under different pH conditions, the ionization of cationic lipids affects the surface charge of lipid nanoparticles. This charge state not only affects plasma protein uptake, blood clearance, and tissue distribution (Semple, SC et al., Adv. Drug Deliv Rev 32:3-17 (1998)), but also the ability to form endosomal non-bilayer structures, properties that are crucial for intracellular delivery of nucleic acids (Hafez, IM et al., Gene Ther 8:1188-1196 (2001)).

[0184] In one specific embodiment, the present invention provides a nanoparticle composition or formulation comprising the compounds of the present invention, phospholipids, structural lipids, polyethylene glycol (PEG) lipids, or combinations thereof.

[0185] As used herein, the term "phospholipid" refers to a lipid containing a phosphate group and one or more carbon chains (such as unsaturated fatty acid chains). Phospholipids may contain one or more multiple bonds (such as double or triple bonds). Non-limiting examples of phospholipids include: 1,2-dilinoleoyl-sn-glycerol-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC), 1,2-cocoacyl-sn-glycerol-3-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), and 1,2-di-O-octadecenyl-OT-glycerol-3-phosphocholine (18:0Diether). 1,2-Oleoyl-2-cholesterolyl-sn-glycerol-3-phosphate choline (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphate choline (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidonicyl-sn-glycerol-3-phosphate choline, 1,2-docosahexaenoyl-sn-glycerol-3-phosphate choline, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-diphydanyl-sn-glycerol-3-phosphate ethanolamine (ME 16.0) PE), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-docosahexaenooyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin and combinations thereof.

[0186] In some embodiments, the phospholipid is DOPE. In some embodiments, the phospholipid is DSPC.

[0187] In some embodiments, the composition comprises a structural lipid. Non-limiting examples of structural lipids include cholesterol, coccosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassosterol, tomatoamine, ursolic acid, α-tocopherol, and combinations thereof. In one embodiment, the structural lipid is cholesterol.

[0188] In some embodiments, the composition comprises PEG lipids. Non-limiting examples of PEG lipids include PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and combinations thereof.

[0189] In one specific embodiment, the nanoparticle composition of the present invention comprises a compound of formula I, 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC), cholesterol, and dimyristoylglycerol-PEG2000 (DMG-PEG2K).

[0190] The components of the nanoparticle composition (such as DMG-PEG2K, DSPC, cholesterol, and the compound of formula I) can be combined in a specific molar ratio or mass ratio. In one embodiment, the molar ratio of DMG-PEG2K, DSPC, cholesterol, and the compound of formula I is (0.2-10):(1-50):(5-75):(3-85). In another embodiment, the molar ratio is adjusted to (0.2-10):(1-50):(5-75):(5-80). In other embodiments, the molar ratio of DMG-PEG2K, DSPC, cholesterol, and the compound of formula I is (0.5-8):(4-40):(10-70):(15-60). Furthermore, in some other embodiments, the molar ratio is adjusted to (1.0-5):(4-30):(15-70):(20-60).

[0191] This invention has discovered that most of the materials in this series have special organ targeting properties. As shown in Table 2, L0865, L0866, L0878, L0879, L0884, L0885, and L0886 have lung targeting properties, while L0901, L0908, L0909, and L0928 have spleen targeting properties.

[0192] As described herein, nanoparticle compositions may optionally contain therapeutic or preventative agents. These drugs can be encapsulated either within the lipid portion of lipid nanoparticles or within the aqueous space of the lipid nanoparticles (which may be partially or completely encapsulated by lipids). This encapsulation effectively prevents enzymatic degradation or other adverse effects (such as triggering adverse immune responses) of the drug due to host organism or cellular mechanisms.

[0193] The terms "therapeutic agent" or "prophylactic agent" refer to any substance that, when administered to a subject, has a therapeutic, diagnostic, and / or preventive effect, and / or induces the expected biological and / or pharmacological effect. Non-limiting examples of therapeutic agents or prophylactic agents include, but are not limited to, small molecule drugs, proteins, cells, or nucleic acids.

[0194] In some embodiments, the therapeutic or preventative agent is a nucleic acid, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Examples of RNA include, but are not limited to: small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), circular RNA (circRNA), messenger RNA (mRNA), and combinations thereof.

[0195] In some embodiments, the therapeutic or preventative agent is mRNA, which may optionally include one or more of the following structures: stem-loop, chain-terminating nucleoside, poly(A) sequence, polyadenosine signal, and 5' cap structure.

[0196] In some embodiments, the nanoparticle composition has a desired nitrogen-to-phosphorus ratio. As used herein, the term "nitrogen-to-phosphorus ratio" is the molar ratio of ionizable nitrogen atoms (within the physiological pH range) in lipids to phosphate groups in RNA, for example, in a nanoparticle composition comprising a lipid component and RNA.

[0197] In some embodiments, the nitrogen-to-phosphorus ratio ranges from 5 to 100. In some embodiments, the nitrogen-to-phosphorus ratio ranges from 10 to 60.

[0198] mRNA can be synthesized using any of the various known methods. For example, mRNA can be synthesized via in vitro transcription (IVT). In short, IVT typically uses a linear or circular DNA template containing a promoter, a mixture of ribonucleotide triphosphates, a buffer system that may contain dithiothreitol and magnesium ions, and a suitable RNA polymerase (e.g., T3, T7, or SP6 type RNA polymerase), DNase I, pyrophosphatase, and / or RNase inhibitors. Specific experimental conditions will vary depending on the application requirements.

[0199] In some embodiments, mRNA is prepared using an in vitro transcription method using a DNA template. A suitable DNA template typically has a promoter for in vitro transcription, such as a T3, T7, or SP6 promoter, followed by the desired nucleotide sequence for mRNA and a terminator.

[0200] mRNA can be either unmodified or modified. Typically, mRNA modifications are intended to improve its stability. Modifications to mRNA can include, but are not limited to, modifications to RNA nucleotides. Therefore, modified mRNA can include, for example, backbone modifications, sugar modifications, or base modifications. In some embodiments, the mRNA encoding the protein may be synthesized from naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides), including but not limited to purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), and nucleotide-modified analogs or derivatives of these purines and pyrimidines, such as 1-methyladenine, 2-methyladenine, 2-methylthio-N6-isopentenyladenine, N6-methyladenine, N6-isopentenyladenine, 2-thiocytosine, 3-methylcytosine, 4-acetylcytosine, 5-methylcytosine, 2,6-diaminopurine, 1-methylguanine, 2-methylguanine, 2,2-dimethylguanine, 7-methylguanine, inosine, 1-methylinosine, pseudouracil (5- Uracil), dihydrouracil, 2-thiouracil, 4-thiouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thiouracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxyuracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methylpseudouracil, quinoside, 13-D-mannosylquinoside, Y base, phosphoramide ester, thiophosphate ester, peptide nucleotide, methylphosphonate, 7-deazoguanosine, 5-methylcytosine, and inosine.

[0201] In some embodiments, the mRNA may contain RNA backbone modifications. Generally, backbone modification refers to the chemical modification of the phosphate groups in the nucleotide backbone of RNA. Exemplary backbone modifications include, but are not limited to, the following types: methylphosphonates, methylphosphonamide esters, phosphoramide esters, thiophosphates (e.g., cytidine 5'-O-(1-thiophosphate)), boron phosphates, positively charged guanidine groups, etc. These modifications are achieved by replacing the original phosphodiester bonds with other anionic, cationic, or neutral groups.

[0202] In some embodiments, mRNAs may contain glycosylation modifications. Typical glycosylation modifications involve chemically modifying the sugar groups of the contained nucleotides, including but not limited to the following modifications: 2'-deoxy-2'-fluoro oligonucleotides (2'-fluoro-2'-deoxycytidine-5'-triphosphate, 2'-fluoro-2'-deoxyuridine-5'-triphosphate), 2'-deoxy-2'-deamino oligonucleotides (2'-amino-2'-deoxycytidine-5'-triphosphate, 2'-amino-2'-deoxyuridine-5'-triphosphate), 2'-O- Alkyl oligonucleotides, 2'-deoxy-2'-C-alkyl oligonucleotides (2'-O-methylcytidine-5'-triphosphate, 2'-methyluridine-5'-triphosphate), 2'-C-alkyl oligonucleotides and their isomers (2'-cytarabine-5'-triphosphate, 2'-cytarabine-5'-triphosphate) or azidotriphosphates (2'-azido-2'-deoxycytidine-5'-triphosphate, 2'-azido-2'-deoxyuridine-5'-triphosphate).

[0203] In some embodiments, mRNA may contain modifications of nucleotide bases (base modifications). Nucleotides containing modified bases are also called base-modified nucleotides. Examples of such base-modified nucleotides include, but are not limited to: 2-amino-6-chloropurine ribonucleoside-5'-triphosphate, 2-aminoadenosine-5'-triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5-aminoallylcytidine-5'-triphosphate, 5-aminoallyluridine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, 5-bromouridine-5'-triphosphate, 5-iodocytidine-5'-triphosphate, 5-iodouridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, and 5-methyluridine-5'-triphosphate. 6-azacytidine-5'-triphosphate, 6-azauridine-5'-triphosphate, 6-chloropurine ribonucleoside-5'-triphosphate, 7-deadenosine-5'-triphosphate, 7-deoxyguanosine-5'-triphosphate, 8-azaadenosine-5'-triphosphate, 8-azidoadenosine-5'-triphosphate, benzimidazole ribonucleoside-5'-triphosphate, N1-methyladenosine-5'-triphosphate, N1-methylguanosine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, O6-methylguanosine-5'-triphosphate, pseudouridine-5'-triphosphate, puromycin-5'-triphosphate, or flavin-5'-triphosphate.

[0204] Typically, mRNA synthesis involves adding a "cap" at the N-terminus (5' end) and a "tail" at the C-terminus (3' end). The cap is crucial for providing resistance against degradation by most endonucleases present in eukaryotic cells. The tail protects the mRNA from degradation by exonucleases.

[0205] Therefore, in some embodiments, the mRNA contains a 5' cap structure. The 5' cap structure is typically added in the following steps: First, an RNA terminal phosphatase removes one terminal phosphate group from the 5' nucleotide, leaving two terminal phosphates; subsequently, guanosine triphosphate (GTP) is linked to the terminal phosphates via guanylate transferase to form a 5'5'5 triphosphate bond; finally, the nitrogen atom at position 7 of guanine is methylated by a methyltransferase. Examples of cap structures include, but are not limited to: m7G(5')ppp(5')A, G(5')ppp(5)A, and G(5)ppp(5')G.

[0206] In some embodiments, the mRNA includes a 3' poly(A) tail. The poly(A) tail at the 3' end of the mRNA typically contains about 10 to 300 adenosine nucleotides (e.g., about 10 to 200, about 10 to 175, about 10 to 150, about 10 to 125, about 10 to 100, about 10 to 75, about 20 to 70, or about 20 to 60 adenosine nucleotides). In some embodiments, the mRNA encoding an antibody (such as heavy chain and light chain-encoded mRNA) includes a 3' poly(C) tail. A suitable poly(C) tail at the 3' end of the mRNA typically contains about 10 to 200 cytidine nucleotides (e.g., about 10 to 150, about 10 to 100, about 20 to 70, about 20 to 60, or about 10 to 40 cytidine nucleotides). The polycytosine tail can be attached to the end of the polyadenylated tail, or it can completely replace the polyadenylated tail.

[0207] In some embodiments, the mRNA includes a 5' and / or 3' untranslated region. In some embodiments, the 5' untranslated region includes one or more regulatory elements that affect mRNA stability or translation efficiency, such as iron-responsive elements. In some embodiments, the 5' untranslated region is approximately 50 to 500 nucleotides in length (e.g., approximately 50 to 400 nucleotides, approximately 50 to 300 nucleotides, approximately 50 to 200 nucleotides, or approximately 50 to 100 nucleotides).

[0208] In some embodiments, the 5' region of the mRNA contains a sequence encoding a signal peptide, such as those described herein. In a particular embodiment, a signal peptide derived from human growth hormone (hGH) is integrated into this 5' region. Typically, the signal peptide coding sequence is linked directly or indirectly to the heavy or light chain coding sequence via its N-terminus.

[0209] In various embodiments, the lipid nanoparticles in the composition have an average diameter of about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70-100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 nm to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or about 30 nm, 35 nm, 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, and are substantially non-toxic. In some embodiments, when nucleic acids are present in lipid nanoparticles, they are resistant to degradation by nucleases in aqueous solutions.

[0210] The properties of nanoparticle compositions can be closely related to their constituent components. For example, a nanoparticle composition containing cholesterol as a structural lipid may exhibit different properties than a composition using other structural lipids. Similarly, the properties of a composition may also depend on the absolute content or relative proportion of each component. For instance, a nanoparticle composition containing a higher molar fraction of phospholipids may have different properties than a nanoparticle composition containing a lower molar fraction of phospholipids. Furthermore, differences in preparation methods and process conditions can also lead to variations in the composition's properties.

[0211] Nanoparticle compositions can be characterized using a variety of methods. For example, microscopy techniques (such as transmission electron microscopy or scanning electron microscopy) can be used to observe the morphological characteristics and particle size distribution of nanoparticle compositions. Dynamic light scattering or potentiometric analysis (such as potentiometric titration) can be used to measure the zeta potential. Furthermore, dynamic light scattering techniques can also be used to determine particle size distribution.

[0212] The nanoparticle composition can be relatively homogeneous. The polydispersity index can be used to indicate the uniformity of the nanoparticle composition, such as the particle size distribution. A smaller polydispersity index (e.g., less than 0.3) generally indicates a narrower particle size distribution. The nanoparticle compositions disclosed in this invention may have a polydispersity index of about 0 to about 0.25, for example, 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, or 0.25. In some embodiments, the polydispersity index of the nanoparticle composition may be about 0.10 to about 0.20.

[0213] The zeta potential of a nanoparticle composition can be used to characterize its electrokinetic potential. For example, the zeta potential can be used to describe the surface charge of the nanoparticle composition. It is generally desirable for nanoparticle compositions to have a relatively low charge (whether positive or negative) because substances with higher charges may interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of the nanoparticle composition of the present invention can be about -20 mV to about +40 mV, about -20 mV to about +35 mV, about -20 mV to about +30 mV, about -20 mV to about +25 mV, about -20 mV to about +20 mV, about -20 mV to about +15 mV, about -20 mV to about +10 mV, about -20 mV to about +5 mV, about -20 mV to about 0 mV, about -20 mV to about -5 mV, about -20 mV to about -10 mV, about -20 mV to about -15 mV, about -15 mV to about +40 mV, about -15 mV to about +35 mV, about -15 mV to about +30 mV, about -15 mV to about +25 mV, about -15 mV to about +20 mV, about -15 mV to about +15 ... mV to approximately +10 mV, approximately -15 mV to approximately +5 mV, approximately -15 mV to approximately 0 mV, approximately -15 mV to approximately -5 mV, approximately -15 mV to approximately -10 mV, approximately -10 mV to approximately +40 mV, approximately -10 mV to approximately +35 mV, approximately -10 mV to approximately +30 mV, approximately -10 mV to approximately +25 mV, approximately -10 mV to approximately +20 mV, approximately -10 mV to approximately +15 mV, approximately -10 mV to approximately +10 mV, approximately -10 mV to approximately +5 mV, approximately -10 mV to approximately 0 mV, approximately -10 mV to approximately -5 mV, approximately -5 mV to approximately +40 mV, approximately -5 mV to approximately +35 mV, approximately -5 mV to approximately +30 mV, approximately -5 mV to approximately +25 mV, approximately -5 mV to approximately +20 mV, approximately -5 mV to approximately +15 mV, approximately -5 mV to approximately +10 mV, approximately -5 mV to approximately +5 mV, approximately -5 mV to approximately 0 mV, approximately 0 mV to approximately +40 mV, approximately 0 mV to approximately +35 mV, approximately 0 mV to approximately +30 mV, approximately 0 mV to approximately +25 mV, approximately 0 mV to approximately +20 mV, approximately 0 mV to approximately +15 mV, approximately 0 mV to approximately +10 mV, approximately 0 mV to approximately +5 mV, approximately 5 mV to approximately +40 mV, approximately 5 mV to approximately +35 mV, approximately 5 mV to approximately +30 mV, approximately 5 mV to approximately +25 mV, approximately 5 mV to approximately +20 mV, approximately 5 mV to approximately +15 mV, approximately 5 mV to approximately +10 mV, approximately 10mV to +40 mV, about 10 mV to +35 mV, about 10 mV to +30 mV, about 10 mV to +25 mV, about 10 mV to +20 mV, about 10 mV to +15 mV, about 15 mV to +40 mV, about 15 mV to +35 mV, about 15 mV to +30 mV, about 15 mV to +25 mV, about 15 mV to +20 mV, about 20 mV to +40 mV, about 20 mV to +35 mV, about 20 mV to +30 mV, about 20 mV to +25 mV, about 25 mV to +40 mV, about 25 mV to +35 mV, about 25 mV to +30 mV, about 30 mV to +40 mV, about 30 mV to +35 mV.

[0214] Encapsulation efficiency of therapeutic and / or preventative agents refers to the ratio of the amount of therapeutic and / or preventative agents encapsulated or otherwise bound to the nanoparticle composition after preparation to the initial feed amount. Ideally, the encapsulation efficiency should be high (e.g., close to 100%). Encapsulation efficiency can be measured by comparing the content of therapeutic and / or preventative agents in solution before and after cleavage of the nanoparticle composition by one or more organic solvents or detergents. The amount of free therapeutic and / or preventative agents (such as RNA) in solution can be quantified using fluorescence detection. The nanoparticle compositions described herein can achieve an encapsulation efficiency of at least 50% for therapeutic and / or preventative agents, such as 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 some embodiments, the encapsulation efficiency can be at least 90%.

[0215] Treatment methods and uses

[0216] "Treatment" or "therapy" refers to a method that achieves a beneficial or intended effect (including clinical efficacy) through specific means. Intended clinical efficacy may include one or more of the following: a) inhibiting the development of a disease or condition (e.g., reducing one or more symptoms caused by the disease or condition, and / or narrowing the scope of the condition); b) alleviating or delaying the development of one or more clinical symptoms associated with the disease or condition (e.g., stabilizing the condition, preventing or delaying the deterioration or progression of the condition, and / or preventing or delaying the spread of the disease or condition (e.g., metastasis or infection)); and / or c) alleviating the disease, i.e., alleviating clinical symptoms (e.g., improving the state of the disease, achieving partial or complete remission of the disease or condition, enhancing the efficacy of other medications, delaying disease progression, improving quality of life, and / or prolonging survival).

[0217] "Prevention" or "avoidance" refers to any interventions taken to prevent the occurrence of clinical symptoms of a disease or condition. In some embodiments, the relevant compound may be administered to subjects (including humans) who are at risk of the disease or condition or have a relevant family history of the disease or condition.

[0218] In some embodiments, the compounds provided herein can be used to prepare lipid nanoparticles for administration of vaccines, such as mRNA vaccines. Therefore, this document provides a method for preventing a disease or condition, comprising administering the lipid nanoparticles disclosed herein to a subject in need, wherein the lipid nanoparticles comprise one or more small molecule drugs, proteins, cells, and / or nucleic acids (such as DNA and mRNA).

[0219] "Subject" refers to an animal, such as a mammal (including humans), that has become or will become a subject of treatment, observation, or experimentation. The methods described herein can be used for human treatment and / or veterinary applications. In some embodiments, the subject is a mammal; in one embodiment, the subject is a human.

[0220] The "therapeutic effective amount" or "effective amount" of the compound (or its pharmaceutically acceptable salt, tautomer, stereoisomer, mixture of stereoisomers, prodrug, or deuterated analogue) described herein refers to an amount sufficient to achieve a therapeutic effect when administered to a subject, providing therapeutic benefits such as relief of symptoms or slowing disease progression. Therapeutic effective amounts may vary depending on the individual subject, the disease or condition being treated, the subject's weight and age, the severity of the disease or condition, and the method of administration; this dosage can be readily determined by one of ordinary skill in the art.

[0221] The methods described herein are applicable to both in vivo and in vitro cell populations. “In vivo” refers to the internal environment of a living individual, such as an animal or human. In this context, the methods can be used for therapeutic purposes in an individual. “In vitro” refers to a state outside the living environment. Examples of in vitro cell populations include in vitro cell cultures and biological samples (such as bodily fluids or tissue samples obtained from an individual), which can be obtained using methods well known in the art. Exemplary bodily fluid samples include blood, cerebrospinal fluid, urine, and saliva. In this context, the compounds and compositions described herein can be used for a variety of purposes, including therapeutic and experimental purposes. For example, the compounds and compositions described herein can be used in in vitro experiments to determine the optimal administration regimen and / or dosage of the compounds of the invention for a specific indication, cell type, individual, and other parameters. Information obtained from such applications can be used for experimental purposes or in clinical settings to develop in vivo treatment regimens. Other in vitro uses of the compounds and compositions described herein may be described below or are known to those skilled in the art. Selected compounds may be further characterized to determine their safety or tolerable doses in human or non-human subjects, and such characteristics can be detected using commonly used methods well known to those skilled in the art.

[0222] According to one embodiment of the present invention, a method for delivering a therapeutic or preventative agent to mammalian cells is provided, the method comprising contacting the cells with a nanoparticle composition of the present invention comprising the therapeutic or preventative agent.

[0223] Drug composition and administration method

[0224] The compounds described herein are typically administered in the form of pharmaceutical compositions. Therefore, the pharmaceutical compositions provided herein comprise one or more of the compounds described herein, their pharmaceutically acceptable salts, tautomers, stereoisomers, mixtures of stereoisomers, prodrugs, or deuterated analogs, and one or more pharmaceutically acceptable media selected from carriers, adjuvants, and excipients. Suitable pharmaceutically acceptable excipients may include, for example, inert solid diluents and fillers, diluents (including sterile aqueous solutions and various organic solvents), penetration enhancers, solubilizers, and adjuvants. The methods for preparing such compositions are well-known techniques in the pharmaceutical field. Specific details can be found in Remington's Pharmaceutical Sciences (17th edition, Mace Publishing Co., Philadelphia, Pa., 1985) and Modern Pharmaceutics (3rd edition, Marcel Dekker, Inc., edited by GS Banker and CT Rhodes).

[0225] In terms of application, the compounds of the present invention (typically in the form of lipid nanoparticles combined with a therapeutic agent) can be applied directly as a raw material chemical or formulated into a pharmaceutical composition for application. The pharmaceutical compositions of the present invention comprise a compound of formula I, and one or more pharmaceutically acceptable carriers, diluents, or excipients. The amount of the compound of formula I in the composition should be sufficient to effectively form lipid nanoparticles and deliver the therapeutic agent (e.g., for the treatment of a specific target disease or condition). Those skilled in the art can readily determine suitable concentrations and dosages.

[0226] In some embodiments, the lipid nanoparticles further comprise phospholipids, polyethylene glycol lipids, cholesterol, or combinations thereof. In some embodiments, the phospholipid is 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC). In some embodiments, the polyethylene glycol lipid is dimyristoylglycerol-PEG2000 (DMG-PEG2000).

[0227] The lipid nanoparticles disclosed in this invention are typically administered via a parenteral route. As used herein, the term "parenteral" includes subcutaneous injection, intravenous injection, intramuscular injection, intradermal injection, intravaginal injection, intranasal injection, intrasternal injection, or infusion techniques.

[0228] Pharmaceutical compositions intended for parenteral administration are typically specially formulated to ensure that the active ingredient contained herein is bioavailable when administered to a subject. Compositions administered to a subject or patient are typically in the form of one or more dose units.

[0229] dose

[0230] The specific dosage level of the therapeutic agent (such as mRNA or other nucleic acids) for a particular subject in this application depends on a variety of factors, including the activity of the specific compound used, the subject's age, weight, general health status, sex, diet, timing of administration, route of administration, excretion rate, and the severity of the specific disease suffered by the subject receiving treatment. For example, the dosage may be expressed as milligrams (mg / kg) of the therapeutic agent (such as mRNA or other nucleic acids) per kilogram of subject body weight. A dosage range of approximately 0.1 to 150 mg / kg is preferred. In some embodiments, a dosage range of 0.1 to 100 mg / kg may be appropriate. In other embodiments, a dosage range of 0.5 to 60 mg / kg may be appropriate. Standardized dosage based on subject weight is particularly useful when adjusting the dosage among subjects with significant differences in body size (e.g., when the drug is used simultaneously in children and adults, or when it is necessary to convert the effective dosage for non-human subjects such as dogs to a dosage suitable for human subjects).

[0231] The daily dose can also be described as the total amount of therapeutic agent (e.g., nucleic acid such as mRNA) administered per dose or daily. The daily dose can be between about 1 mg and 4000 mg, about 2000 mg and 4000 mg / day, about 1 to 2000 mg / day, about 1 to 1000 mg / day, about 10 to 500 mg / day, about 20 to 500 mg / day, about 50 to 300 mg / day, about 75 to 200 mg / day, or about 15 to 150 mg / day.

[0232] The compounds or compositions thereof of this application may be administered using any of the suitable methods described above, once, twice, three times, or four times daily. Furthermore, administration or treatment using therapeutic agents (such as nucleic acids like mRNA) may continue for several days; for example, a treatment cycle typically lasts at least 7, 14, or 28 days. Treatment cycles are well-known in cancer chemotherapy, and cycles are typically spaced between rest periods of approximately 1 to 28 days (commonly about 7 or 14 days). In other embodiments, treatment cycles may also be performed continuously.

[0233] In one specific embodiment, the method includes administering an initial daily dose of approximately 1 to 800 mg of a therapeutic agent (such as a nucleic acid like mRNA) to the subject, and gradually increasing the dose until clinical efficacy is achieved. The dose may be increased in increments of approximately 5 mg, 10 mg, 25 mg, 50 mg, or 100 mg. The dose may be increased daily, every other day, twice a week, or once a week.

[0234] Compound Synthesis

[0235] The compounds can be prepared using the methods disclosed herein and their conventional modifications. These modifications will be apparent to those skilled in the art based on the disclosure herein and methods known in the art. In addition to the methods described herein, conventional and well-known synthetic methods can also be used. The synthesis of the typical compounds described herein can be carried out as described in the following examples. If available reagents are available, they can be purchased commercially (e.g., from Sigma-Aldrich or other chemical reagent suppliers).

[0236] It should be understood that, given typical process conditions (i.e., reaction temperature, reaction time, reactant molar ratio, solvent, pressure, etc.), other process conditions may be used unless otherwise specified. Optimal reaction conditions may vary depending on the specific reactants or solvents used, but those skilled in the art can determine such conditions through conventional process optimization.

[0237] Furthermore, to prevent unintended reactions of certain functional groups, conventional protecting groups are typically required. The appropriate protecting groups for different functional groups and their corresponding protecting and deprotecting conditions are well known in the art. For example, the application methods of various protecting groups are described in detail in "Greene's Protective Groups in Organic Synthesis" (Wuts, PGM, Greene, TW, & Greene, TW (2006). Hoboken, NJ, Wiley-Interscience) and its cited references.

[0238] Furthermore, the compounds disclosed in this invention may contain one or more chiral centers. Therefore, such compounds can be prepared or isolated as pure stereoisomers, i.e., individual enantiomers, diastereomers, or mixtures rich in stereoisomers, if desired. Unless otherwise stated, all such stereoisomers (and enriched mixtures) are included within the scope of this invention. Pure stereoisomers (or enriched mixtures) can be prepared using, for example, optically active starting materials or stereoselective reagents known in the art. Alternatively, racemic mixtures of such compounds can be separated using, for example, chiral column chromatography, chiral resolving agents, etc.

[0239] The starting materials for the following reactions are typically known compounds, or can be prepared by known methods or their obvious modifications. For example, many starting materials are available from commercial suppliers such as Aldrich Chemical Co. (Milwaukee, Wisconsin, USA), Bachem (Torrance, California, USA), Emka-Chemce, or Sigma (St. Louis, Missouri, USA). Other starting materials can be prepared according to methods described in standard reference materials or their obvious modifications, such as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-15 (John Fieser's Reagents for Organic Synthesis). John Wiley, and Sons (1991), Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplements (Elsevier Science Publishers, 1989), Organic Reactions, Volumes 1-40 (John Wiley, and Sons, 1991), Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplements (Elsevier Science Publishers, 1989), John Wiley, and Sons, 1991. John Wiley, and Sons, 1991; March's Advanced Organic Chemistry (John Wiley, and Sons, 1991) John Wiley, and Sons (5th edition, 2001) and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989).

[0240] General Synthesis Method I

[0241] The following reaction routes illustrate a general method that can be used to synthesize the compounds disclosed herein.

[0242] Example I

[0243]

[0244] Suitable starting materials and reagents are commercially available or prepared by those skilled in the art using known methods. For any compound shown in Example I, it should be understood that various derivatives can be obtained by functional group conversion at any step. In some embodiments, the various substituents of Formula I are as defined herein. However, derivatization of the compound prior to any reaction step, and / or further derivatization of the resulting reaction product, can yield various compounds of Formula I or their pharmaceutically acceptable salts, stereoisomers, or mixtures of stereoisomers. Suitable starting materials and reagents are commercially available or prepared by those skilled in the art using known methods. After each reaction step, the intermediates or final compounds can be recovered using conventional techniques (such as neutralization, extraction, precipitation, chromatography, filtration, etc.) and optionally purified. Those skilled in the art can also prepare the compounds involved in this invention using other improved methods.

[0245] Furthermore, the compounds disclosed in this invention may contain one or more chiral centers. Therefore, such compounds can be prepared or isolated as pure stereoisomers (i.e., individual enantiomers or diastereomers) or as mixtures rich in stereoisomers, if desired. Unless otherwise stated, all such stereoisomers (and enriched mixtures) are included within the scope of this invention. Pure stereoisomers (or enriched mixtures) can be prepared using, for example, optically active starting materials or stereoselective reagents known in the art. Alternatively, racemic mixtures of such compounds can be separated by chiral column chromatography, chiral resolving agents, etc. It should be understood that the various isomers of Formula I can also be separated by methods known in the art.

[0246] Universal synthesis

[0247] Typical examples of the compounds described herein can be synthesized using the general reaction route described herein. As described herein, the general route can be modified by replacing the starting materials with other starting materials having similar structures, thereby yielding different products. The synthetic descriptions provided below aim to illustrate, through numerous examples, how starting materials can be varied to generate corresponding products. For target products with well-defined substituents, the required starting materials can usually be determined through structural analysis. Starting materials are generally available from commercial sources or synthesized using publicly available methods. For the synthesis of the compounds described in the embodiments of this invention, the types of substituents can be determined by analyzing the structure of the compound to be synthesized. In conjunction with the embodiments herein, the structure of the final product can usually clearly identify the type of starting material required through a simple structural analysis process. Overall, the compounds described herein are generally stable at room temperature and atmospheric pressure and can be isolated using conventional methods.

[0248] Example

[0249] The following examples illustrate specific embodiments of the present invention. Those skilled in the art should understand that the technical solutions disclosed in the following examples demonstrate good feasibility in practical applications of the present invention and can therefore be considered specific implementations of the present invention. However, based on the content of the present invention, those skilled in the art should recognize that numerous modifications can be made to the disclosed specific embodiments to obtain the same or similar results without departing from the spirit and scope of the present invention.

[0250] List of abbreviations and acronyms

[0251]

[0252] 1. Preparation of ionizable lipids

[0253]

[0254] Example L0851: N, N', N'', N'''-((2S, 2'S, 2''S, 2'''S)-((((S)-3-(2-hexyloctamido)-2-hydroxypropyl)azanediyl)bis(propane-3,1-diyl))bis(azanetriyl))tetra(2-hydroxypropane-3,1-diyl))tetra(2-hexyloctamido)

[0255]

[0256]

[0257] Step 1: tert-butyl(S)-(3-chloro-2-hydroxypropyl)carbamate

[0258] Triethylamine (3.808 mL, 27.39 mmol) was added to a methanol solution (120 mL) of (S)-1-amino-3-chloroprop-2-ol hydrochloride, followed by di-tert-butyl carbonate (6.375 g, 28.76 mmol). The resulting solution was stirred continuously at room temperature for 30 minutes. After removing methanol under reduced pressure, the residue was dissolved in ethyl acetate (200 mL), and the resulting solution was washed twice with an aqueous solution of ammonium chloride (100 mL). The organic phase was dried over sodium sulfide, filtered, and concentrated under reduced pressure. The residue was purified by rapid chromatography to give the target product as a white solid (4.90 g, 85.36% yield). HRMS (ESI, m / z): [M+Na] + calcd. For: C8H 16 ClNO3, 232.07109; Found: 232.07289.

[0259]

[0260] Step 2: Add N to a 2 mL solution of tert-butyl(S)-(3-chloro-2-hydroxypropyl)carbamate (0.731 g, 3.49 mmol) in acetonitrile. 1 -(3-aminopropyl)propane-1,3-diamine (72 mg, 0.533 mmol), potassium iodide (19 mg, 0.102 mmol), and potassium carbonate (0.525 g, 4.00 mmol). The resulting suspension was heated to 60 °C and stirred continuously for 24 hours. The reaction mixture was then filtered and washed with tetrahydrofuran (20 mL). The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography to give the target product as a white solid (377 mg, 71.0% yield). HRMS (ESI, m / z): [M+H] + calcd.For: C 46 H 93 N8O 15 , 997.67549; Found: 997.65960.

[0261]

[0262] Step 3: The compound obtained in the above steps (66 mg, 0.066 mmol) and triisopropylsilane (81 μL, 0.394 mmol) were added to a dichloromethane solution of trifluoroacetic acid (1.5 mL, v / v = 1 / 2). After stirring the reaction mixture at room temperature for 2 hours, the solvent was removed under reduced pressure, and the residue was dissolved in dimethylformamide (2 mL), followed by the addition of triethylamine (92 μL, 0.660 mmol) and 2,5-dioxopyrrolidone-1-yl-2-hexyl octanoate (0.139 g, 0.427 mmol). The resulting mixture was stirred at room temperature for 12 hours. After the reaction was complete, the mixture was diluted with dichloromethane (50 mL), washed twice with an aqueous sodium bicarbonate solution (10 mL), dried over sodium sulfide, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give a white solid product (22 mg, yield 21.6%). 1 H NMR (400 MHz, Chloroform-d) δ 6.94 - 6.38(m, 5H), 3.90 - 3.68 (m, 5H), 3.61 - 3.04 (m, 15H), 2.70 - 2.34 (m, 14H),2.15 - 2.06 (m, 4H), 1.76 - 1.50 (m, 14H), 1.44 - 1.34 (m, 10H), 1.32 - 1.16(m, 80H), 0.86 (t, J = 6.8 Hz, 30H). HRMS (ESI, m / z): [M+H] + calcd. ForC 91 H 183 N8O 10 , 1548.40517; found: 1548.41117..

[0263]

[0264] Example L0852: N, N', N'', N'''-((2S, 2'S, 2''S, 2'''S)-((((S)-3-dodecanoamide-2-hydroxypropyl)azanediyl)bis(propane-3,1-diyl))bis(nitrotriyl))tetra(2-hydroxypropane-3,1-diyl))tetra(dodecanoamide)

[0265] Compound L0852 was synthesized using a similar method to compound L0851. 1H NMR (400 MHz, Chloroform-d) δ 6.73 - 6.47 (m, 5H), 3.83 - 3.66 (m, 5H), 3.43 (ddd, J = 13.6, 7.2, 4.0Hz, 5H), 3.18 - 3.01 (m, 5H), 2.66 - 2.30 (m, 18H), 2.19 (t, J = 7.6 Hz,10H), 1.67 - 1.52 (m, 14H), 1.36 - 1.18 (m, 81H), 0.87 (t, J = 6.8 Hz, 15H).HRMS (ESI, m / z): [M+H]+ calcd. For: C 81 H 162 N8O 10 , 1408.24867; Found: 1408.24985.

[0266]

[0267] Example L0853: N, N', N'', N'''-((2S, 2'S, 2''S, 2'''S)-(((methylazanediyl)bis(propane-3,1-diyl))bis(nitrotriyl))tetra(2-hydroxypropane-3,1-diyl))tetra(decanoamide)

[0268] Compound L0853 was synthesized using a similar method to compound L0851. 1 H NMR (400 MHz, Chloroform-d) δ 6.72 - 6.35 (m, 4H), 3.83 - 3.72 (m, 4H), 3.50 - 3.37 (m, 4H), 3.16 -3.03 (m, 4H), 2.67 - 2.51 (m, 6H), 2.49 - 2.40 (m, 6H), 2.40 - 2.23 (m, 7H), 2.19 (t, J = 7.6 Hz, 8H), 1.75 - 1.51 (m, 12H), 1.35 - 1.18 (m, 48H), 0.86(t, J = 6.8 Hz, 12H). HRMS (ESI, m / z): [M+H] + calcd. For C 59 H 120 N7O8,1054.91929; found: 1054.92052.

[0269]

[0270] Example L0854: N, N', N'', N'''-((2S, 2'S, 2''S, 2'''S)-(((methylazinediyl)bis(propane-3,1-diyl))bis(nitrotriyl))tetra(2-hydroxypropane-3,1-diyl))tetra(tetradecanoamide)

[0271] Compound L0854 was synthesized using a similar method to compound L0851. 1 H NMR (400 MHz, Chloroform-d) δ 6.74 - 6.22 (m, 4H), 3.85 - 3.71 (m, 4H), 3.54 - 3.37 (m, 4H), 3.17 -3.00 (m, 4H), 2.72 - 2.52 (m, 6H), 2.51 - 2.41 (m, 6H), 2.41 - 2.25 (m, 7H), 2.19 (t, J = 7.6 Hz, 8H), 1.75 - 1.49 (m, 12H), 1.37 - 1.14 (m, 80H), 0.87(t, J = 6.8 Hz, 12H). HRMS (ESI, m / z): [M+H] + calcd. For C 75 H 152 N7O8,1279.16969; found: 1279.17239.

[0272]

[0273] Example L0855: N, N', N'', N'''-((2S, 2'S, 2''S, 2'''S)-(((methylazanediyl)bis(propane-3,1-diyl))bis(azanetriyl))tetra(2-hydroxypropane-3,1-diyl))tetra(2-hexyloctamide)

[0274] Compound L0855 was synthesized using a similar method to compound L0851. 1H NMR (400 MHz, Chloroform-d) δ 6.82 - 6.55 (m, 4H), 3.96 - 3.76 (m, 4H), 3.43 (ddd, J = 14.0, 6.0, 3.6Hz, 4H), 3.31 - 3.15 (m, 6H), 2.84 - 2.65 (m, 5H), 2.65 - 2.46 (m, 6H), 2.38 (dd, J = 13.2, 2.8 Hz, 4H), 2.16 - 2.07 (m, 4H), 1.99 - 1.90 (m, 2H), 1.82 -1.72 (m, 2H), 1.64 - 1.51 (m, 8H), 1.47 - 1.35 (m, 8H), 1.35 - 1.14 (m, 64H), 0.86 (t, J = 6.8 Hz, 24H). HRMS (ESI, m / z): [M+H] + calcd. For C 75 H 152 N7O8,1279.16969; found: 1279.17093.

[0275]

[0276] Example L0856: N, N', N'', N'''-((2S, 2'S, 2''S, 2'''S)-(((methylazanediyl)bis(propane-3,1-diyl))bis(nitrotriyl))tetra(2-hydroxypropane-3,1-diyl))tetra(2-hexyldecanoamide)

[0277] Compound L0856 was synthesized using a similar method to compound L0851. 1H NMR (400 MHz, Chloroform-d) δ 6.82 - 6.57 (m, 4H), 3.90 - 3.76 (m, 4H), 3.47 - 3.36 (m, 4H), 3.36 -3.00 (m, 8H), 2.76 - 2.64 (m, 4H), 2.55 (dd, J = 13.6, 10.0 Hz, 6H), 2.36 (dd, J = 13.2, 3.2 Hz, 4H), 2.16 - 2.05 (m, 4H), 1.97 - 1.82 (m, 2H), 1.79 -1.67 (m, 2H), 1.63 - 1.50 (m, 8H), 1.44 - 1.34 (m, 8H), 1.33 - 1.16 (m, 80H),0.86 (td, J = 6.8, 2.4 Hz, 24H). HRMS (ESI, m / z): [M+H] + calcd. For C 83 H 168 N7O8,1391.29489; found: 1391.29719.

[0278]

[0279] Example L0857: N, N', N'', N'''-((2S, 2'S, 2''S, 2'''S)-(((methylazanediyl)bis(propane-3,1-diyl))bis(nitrotriyl))tetra(2-hydroxypropane-3,1-diyl))tetra(dodecanoamide)

[0280] Compound L0857 was synthesized using a similar method to compound L0851. 1 H NMR (400 MHz, Chloroform-d) δ 6.45 (t, J = 6.0 Hz, 3H), 3.83 - 3.68 (m, 4H), 3.45 (ddd, J = 14.0, 6.4,3.2 Hz, 4H), 3.20 - 3.01 (m, 4H), 2.65 - 2.27 (m, 16H), 2.18 (t, J = 7.6 Hz,11H), 1.75 - 1.51 (m, 12H), 1.39 - 1.12 (m, 64H), 0.86 (t, J = 6.8 Hz, 12H).HRMS (ESI, m / z): [M+H] +calcd. For: C 67 H 136 N7O8, 1167.04449; Found; 1167.04615.

[0281]

[0282] Example L0858: N, N', N'', N'''-((2S, 2'S, 2''S, 2'''S)-((((S)-3-decanoamido-2-hydroxypropyl)azanediyl)bis(propane-3,1-diyl))bis(azanetriyl))tetra(2-hydroxypropane-3,1-diyl))tetra(decanoamide)

[0283] Compound L0858 was synthesized using a similar method to compound L0851. 1 H NMR (400 MHz, Chloroform-d) δ 6.72 - 6.31 (m, 5H), 3.88 - 3.72 (m, 5H), 3.58 - 3.49 (m, 1H), 3.46 -3.35 (m, 4H), 3.19 - 3.03 (m, 5H), 2.66 - 2.36 (m, 18H), 2.27 - 2.16 (m,10H), 1.69 - 1.52 (m, 14H), 1.39 - 1.10 (m, 60H), 0.87 (t, J = 6.8 Hz, 15H).HRMS (ESI, m / z): [M+H] + calcd. For C 71 H 143 N8O 10 , 1268.09217; found: 1268.09387.

[0284]

[0285] Example L0859: N, N', N'', N'''-((2S, 2'S, 2''S, 2'''S)-(((methylazanediyl)bis(ethane-2,1-diyl))bis(nitrotriyl))tetra(2-hydroxypropane-3,1-diyl))tetra(decanoamide)

[0286] Compound L0859 was synthesized using a similar method to compound L0851. 1H NMR (400 MHz, Chloroform-d) δ 6.40 - 5.99 (m, 4H), 3.87 - 3.70 (m, 4H), 3.56 - 3.40 (m, 4H), 3.26 -3.07 (m, 4H), 2.90 - 2.79 (m, 2H), 2.72 - 2.62 (m, 2H), 2.56 - 2.30 (m, 13H), 2.22 - 2.16 (m, 8H), 1.62 (p, J = 7.2, 6.8 Hz, 8H), 1.39 - 1.14 (m, 48H), 0.87 (t, J = 6.8 Hz, 12H). HRMS (ESI, m / z): [M+H] + calcd. For C 57 H 116 N7O8,1026.88799; found: 1026.88894.

[0287]

[0288] Example L0860: N, N', N'', N'''-((2S, 2'S, 2''S, 2'''S)-(((methylazanediyl)bis(ethane-2,1-diyl))bis(nitrotriyl))tetra(2-hydroxypropane-3,1-diyl))tetra(dodecanoamide)

[0289] Compound L0860 was synthesized using a similar method to compound L0851. 1 H NMR (400 MHz, Chloroform-d) δ 6.58 - 6.12 (m, 4H), 3.91 - 3.66 (m, 4H), 3.50 - 3.35 (m, 4H), 3.23 -2.97 (m, 4H), 2.87 - 2.38 (m, 13H), 2.38 - 2.09 (m, 14H), 1.70 - 1.51 (m,8H), 1.42 - 1.06 (m, 64H), 0.86 (t, J = 6.8 Hz, 12H).

[0290] Synthesis of ionizable lipids L0861-L0932. Step 1: Boc protection of the amino group of (S) or (R)-1-amino-3-chloropropyl-2-ol.

[0291]

[0292] Synthesis of tert-butyl(S)-(3-chloro-2-hydroxypropyl)carbamate. Triethylamine (3.808 mL, 27.39 mmol) was added to a methanol (120 mL) solution of (S)-1-amino-3-chloropropane-2-ol hydrochloride, followed by di-tert-butyl carbonate (6.375 g, 28.76 mmol). The resulting solution was stirred at room temperature for 30 min. Methanol was evaporated under reduced pressure, and the residue was dissolved in ethyl acetate (200 mL). The resulting solution was washed twice with an aqueous solution of ammonium chloride (100 mL). The organic phase was dried over sodium sulfide, filtered, and concentrated under reduced pressure. The residue was purified by rapid chromatography to give the target product (S)-CAA-Boc as a white solid (4.90 g, yield 85.36%). HRMS (ESI, m / z): [M+Na] + calcd. For: C8H 16 ClNO3,232.07109; Found: 232.07289.

[0293]

[0294] Synthesis of tert-butyl(R)-(3-chloro-2-hydroxypropyl)carbamate. Triethylamine (6.520 g, 64.436 mmol) was added to a methanol (200 mL) solution of (R)-1-amino-3-chloropropane-2-ol hydrochloride, followed by di-tert-butyl carbonate (15.470 g, 78.880 mmol). The resulting solution was stirred at room temperature for 30 min. Methanol was evaporated under reduced pressure, and the residue was dissolved in ethyl acetate (200 mL). The resulting solution was washed twice with an aqueous solution of ammonium chloride (100 mL). The organic phase was dried over sodium sulfide, filtered, and concentrated under reduced pressure. The residue was purified by rapid chromatography to give the target product (R)-CAA-Boc as a white solid (8.190 g, yield 60.6%). HRMS (ESI, m / z): [M+Na] + calcd. For: C8H 16 ClNO3,232.07109; Found: 232.07183.

[0295]

[0296] Step 2: SN2 reaction between the amine head and Boc-protected 1-amino-3-chloropropane-2-ol. Tert-butyl (S)-(3-chloro-2-hydroxypropyl)carbamate (0.629 g, 3.0 mmol, equivalent to 1.5 equivalents of the free protons on the amine group) was dissolved in acetonitrile (2 mL), and then N2 was added to the solution. 1 -(2-aminoethyl)-N1 1,2-Methylethane-1,2-diamine (59 mg, 0.500 mmol), potassium iodide (16 mg, 0.100 mmol), and potassium carbonate (0.414 g, 3.00 mmol) were used. The resulting suspension was heated to 60 °C and stirred continuously for 24 hours. After the reaction was complete, the reaction mixture was filtered, washed with tetrahydrofuran (20 mL), concentrated under reduced pressure to remove the solvent, and the residue was purified by silica gel column chromatography to give the target product as a white solid (258 mg, yield 63.7%). HRMS (ESI, m / z): [M+H] + calcd. For C 37 H 76 N7O 12 , 810.55465; found810.55384.

[0297]

[0298] Step 3: Synthesis of carboxylic acids containing acetal linkers.

[0299] Synthesis of 1,52OHTBDPS: TBDPSCl (30,000 mmol, 8.246 g) was added to a solution of 1,5-pentanediol (120.000 mmol, 12.498 g) in dichloromethane (100 mL), and the mixture was stirred at room temperature for 2 hours. The mixture was then diluted with 100 mL of dichloromethane and washed successively with ammonium chloride aqueous solution (10 mL × 3 times) and saturated brine (10 mL × 3 times). The organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography to give compound 1,52OHTBDPS as a colorless oil (7.978 g, yield 77.6%). HRMS (ESI, m / z): [M+H] + calcd. For C 21 H 31 O2Si, 343.20878; found343.20846;

[0300] Synthesis of 4AE5CTBDPS: Acetaldehyde (15.022 mmol, 0.622 g) was added to a mixture of TMSCl (54.626 mmol, 5.935 g) and compound 1,52OHTBDPS (13.656 mmol, 4.678 g). The mixture was stirred at room temperature for 2 hours, and excess TMSCl was removed under reduced pressure. The residue was dissolved in 50 mL of anhydrous dichloromethane and slowly added dropwise over 0.5 hours to a 200 mL dichloromethane solution containing DIPEA (34.141 mmol, 4.412 g) and n-butanol (27.313 mmol, 2.024 g). The resulting mixture was stirred at room temperature for 2 hours, diluted with 200 mL of dichloromethane, and washed successively with ammonium chloride aqueous solution (50 mL × 3 times) and saturated brine (50 mL × 3 times). The organic phase was dried over anhydrous sodium sulfate and filtered, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 4AE5CTBDPS as a colorless oil (3.863 g, yield 63.9%). HRMS (ESI, m / z): [M+Na] + calcd. For C 27 H 42 NaO3Si,465.27954; found 465.28023

[0301] Synthesis of 4AE5C-OH: 4AE5CTBDPS (8.726 mmol, 3.863 g) and TBAF3H2O (21.815 mmol, 6.883 g) were mixed and dissolved in 20 mL of tetrahydrofuran. The mixture was stirred at room temperature for 6 hours. After the reaction was complete, the tetrahydrofuran was removed, and the residue was dissolved in 200 mL of dichloromethane. The residue was washed successively with ammonium chloride aqueous solution (50 mL × 3 times) and saturated brine (50 mL × 3 times). The organic phase was dried over anhydrous sodium sulfate and filtered. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 4AE5C-OH as a colorless oil (1.032 g, yield 57.9%). HRMS (ESI, m / z): [M+Na] + calcd. For C 11 H 24 NaO3, 227.16176; found 227.16219.

[0302] Synthesis of 4AE5C: Potassium bromide (5.541 mmol, 0.659 g) was dissolved in 5.0 mL of water, and the pH of the solution was adjusted to 10–11 with saturated sodium carbonate. Then, 4AE5C-OH (5.541 mmol, 1.132 g) and TEMPO (0.554 mmol, 0.087 g) were added to the solution, followed by sodium hypochlorite (11.082 mmol, 15.608 mL) at room temperature. The resulting mixture was stirred at room temperature for 0.5 hours, then the pH was adjusted to 5–6 with 1 M hydrochloric acid aqueous solution, and extracted three times with dichloromethane (DCM) (50 mL each time). The combined organic phases were dried over anhydrous sodium sulfate and filtered, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give a colorless oily product 4AE5C (0.596 g, 49.3%). HRMS (ESI, m / z): [M+Na] + calcd. For C 11 H 22 NaO4, 241.14103; found 241.14125.

[0303]

[0304] Step 4: Synthesis of N-hydroxysuccinimide ester 4AE5C-NHS. At 0 °C, EDCI (4.095 mmol, 0.785 g) was added to anhydrous dichloromethane (20 mmol) containing 4AE5C (2.730 mmol, 0.596 g), DMAP (0.546 mmol, 0.067 g), and N-hydroxysuccinimide (5.460 mmol, 0.628 g). The resulting solution was stirred at 0 °C for 30 minutes, then transferred to room temperature and stirred overnight. The reaction mixture was then diluted with 50 mL of dichloromethane and washed successively with ammonium chloride aqueous solution (10 mL, 3 times) and saturated brine (10 mL, 3 times). The organic phase was dried over anhydrous sodium sulfate and filtered, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give a white solid 4AE5C-NHS (1.325 g, yield 80.9%). HRMS (ESI, m / z): [M+Na] + calcd. For C 15 H 25 NNaO6, 338.15741; found338.15779.

[0305]

[0306] Step 5: Synthesis of N-hydroxysuccinimide carbonate 12-OH-NHS. 12-OH (0.932 g, 5.00 mmol) and pyridine (0.396 g, 5.00 mmol) were dissolved in anhydrous dichloromethane (50 mL), followed by the addition of di(2,5-dioxopyrrolidone-1-yl) carbonate (5.5 mmol, 1.409 g). The resulting solution was stirred at room temperature for 2 hours. The reaction mixture was then washed with ammonium chloride aqueous solution (10 mL, 3 times), and the organic phase was dried over anhydrous sodium sulfate. The solvent was removed by filtration under reduced pressure. The residue was purified by silica gel column chromatography to give a white solid 12-OH-NHS (1.325 g, 80.9% yield).

[0307]

[0308] Step 6: Ring-opening reaction of the amine with the alkyl epoxide. 1,2-Epoxydodecane (0.922 g, 5.0 mmol) was added to 50 mL of isopropanol solution containing 2,2'-diamino-N-methyldiethylamine (2.930 g, 25.00 mmol). The mixture was then stirred at 70 °C for 2 days. After the reaction was complete, the mixture was diluted with dichloromethane (DCM) and then washed successively with saturated ammonium chloride and saturated sodium chloride solutions. The organic phase was dried over anhydrous sodium sulfate. The solution was filtered and the solvent was removed under reduced pressure. The residue was passed through a CombiFlash system (DCM / Ultra = 1:1, R... f Purification with approximately 0.5 g / mL yielded a colorless oily product, 22-Ep12. HRMS (ESI, m / z): [M+H] + calcd. For C 17 H 40 N3O, 302.31659; found:302.31607.

[0309]

[0310] Step 7: Synthesis of L0902 via amide bond formation. Compound (S)-22-CAA-Boc (0.060 mmol) and triisopropylsilane (74 μL, 0.36 mmol) were added to a dichloromethane solution of trifluoroacetic acid (1.5 mL, v / v = 1 / 2). The reaction mixture was stirred at room temperature for 2 hours, and the solvent was removed under reduced pressure. The residue was dissolved in dimethylformamide (2 mL), followed by the addition of triethylamine (84 μL, 0.60 mmol) and 4AF5C-NHS (0.288 mmol, 0.087 g). The resulting mixture was stirred at room temperature for 12 hours, diluted with dichloromethane (50 mL), and washed twice with an aqueous sodium bicarbonate solution (10 mL). The organic phase was dried over sodium sulfide, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give the target product (22 mg, yield 21.6%) as a white solid. L0902: 1 H NMR (400 MHz, Chloroform-d) δ 6.43 (s,4H), 4.64 (s, 8H), 3.83 - 3.68 (m, 4H), 3.57 - 3.47 (m, 16H), 3.41 (ddd, J =13.6, 6.0, 3.2 Hz, 4H), 3.17 - 3.03 (m, 4H), 2.89 - 2.69 (m, 4H), 2.56 - 2.38(m, 10H), 2.23 (t, J = 7.2 Hz, 13H), 1.77 - 1.67 (m, 8H), 1.65 - 1.51 (m,16H), 1.42 - 1.32 (m, 8H), 0.91 (t, J = 7.2 Hz, 12H). HRMS (ESI, m / z):[M+H] + calcd. For C 57 H 116 N7O 16 , 1154.84731; found 1154.84750.

[0311]

[0312] Step 8: Synthesis of L0896 via the formation of carbamate bonds. Compound (S)-22-CAA-Boc (0.060 mmol) and triisopropylsilane (74 μL, 0.36 mmol) were added to a solution of trifluoroacetic acid in dichloromethane (1.5 mL, v / v = 1 / 2). The reaction mixture was stirred at room temperature for 2 hours, and the solvent was removed under reduced pressure. The residue was dissolved in dimethylformamide (2 mL), followed by the addition of triethylamine (84 μL, 0.60 mmol) and 12-OH-NHS (0.288 mmol, 0.094 g). The resulting mixture was stirred at room temperature for 12 hours, diluted with dichloromethane (50 mL), washed twice with an aqueous sodium bicarbonate solution (10 mL), dried over sodium sulfide, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give the target product (22 mg, yield 21.6%) as a white solid. L0896: 1 H NMR (400 MHz, Chloroform-d) δ 5.53 (s,4H), 4.04 (t, J = 6.8 Hz, 8H), 3.83 - 3.55 (m, 4H), 3.44 - 3.22 (m, 4H), 3.19- 2.93 (m, 4H), 2.93 - 2.16 (m, 19H), 1.60 (p, J = 6.8 Hz, 8H), 1.35 - 1.19(m, 72H), 0.87 (t, J = 6.8 Hz, 12H). HRMS (ESI, m / z):[M+H] + calcd. ForC 69 H 140 N7O 12 , 1259.05545; found 1259.05467.

[0313]

[0314] Synthesis of L0861. (S)-33-CAA-Boc was synthesized according to method 2. HRMS (ESI, m / z): [M+H] + calcd. For C 39 H 80 N7O 12, 838.58595; found 838.58663. Synthesis of 26BDA: Succinic anhydride (6.01 g, 60 mmol) was added to 150 mL of anhydrous dichloromethane solution containing 26-OH (6.51 g, 50 mmol) and DMAP (9.17 g, 75 mmol) at 0 °C. The reaction mixture was then heated to room temperature and stirred for 12 hours, followed by dilution with 200 mL of dichloromethane and washing with 1 M hydrochloric acid aqueous solution (50 mL × 3 times). The organic phase was dried over sodium sulfide, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give a pale yellow oily substance 26BDA (10.952 g, yield 95.1%). 26BDA: HRMS (ESI, m / z): [M+Na] + Calcium phosphate (C12H23O4), 231.15909; Found: 253.14069. 26BDA-NHS was synthesized using a similar scheme to step 4, followed by an amide bond formation reaction to give the final product L0861. L0861: 1 H NMR (400 MHz, Chloroform-d) δ 6.99 - 6.66 (m, 4H), 4.82 (p, J = 6.4Hz, 4H), 3.89 - 3.67 (m, 4H), 3.44 (ddd, J = 14.0, 6.4, 3.2 Hz, 4H), 3.08(dt, J = 13.2, 6.0 Hz, 4H), 2.74 - 2.29 (m, 32H), 2.21 (s, 3H), 1.77 - 1.41(m, 20H), 1.34 - 1.15 (m, 56H), 0.85 (t, J = 6.8 Hz, 24H). HRMS (ESI, m / z):[M+H] + calcd. For C 83 H 160 N7O 16 , 1511.19161; found: 1510.98502.

[0315]

[0316] The synthesis method for L0862 is similar to that for L0861. L0862: 1H NMR (400 MHz, Chloroform-d) δ6.98 - 6.71 (m, 4H), 4.77 (p, J = 6.4 Hz, 4H), 3.86 - 3.69 (m, 4H), 3.43 (ddd, J = 14.0, 6.4, 3.2 Hz, 4H), 3.16 - 3.02 (m, 4H), 2.74 - 2.30 (m, 32H), 2.21 (s, 3H), 1.71 - 1.40 (m, 20H), 1.34 - 1.19 (m, 24H), 0.99 - 0.66 (m, 24H). HRMS (ESI, m / z): [M+H] + calcd. For C 67 H 128 N7O 16 , 1286.94121; found:1286.93641.

[0317]

[0318] The synthesis method for L0863 is similar to that for L0902. L0863: 1 H NMR (400 MHz, Chloroform-d) δ6.54 - 6.08 (m, 4H), 3.81 - 3.60 (m, 4H), 3.47 - 3.34 (m, 4H), 3.27 - 3.06(m, 4H), 3.01 - 2.10 (m, 19H), 2.09 - 1.94 (m, 4H), 1.65 - 1.48 (m, 8H), 1.47- 1.11 (m, 72H), 0.85 (t, J = 6.7 Hz, 24H). HRMS (ESI, m / z): [M+H] + calcd.For C 73 H 148 N7O8, 1251.13839; found: 1251.13923.

[0319]

[0320] The synthesis method for L0864 is similar to that for L0902. L0864: 1H NMR (400 MHz, Chloroform-d) δ7.05 - 6.35 (m, 5H), 3.96 - 3.63 (m, 5H), 3.52 - 3.37 (m, 5H), 3.28 - 3.15(m, 5H), 2.90 - 1.81 (m, 23H), 1.79 - 1.48 (m, 14H), 1.44 - 1.19 (m, 110H), 0.93 - 0.79 (m, 30H). HRMS (ESI, m / z): [M+H] + calcd. For C 101 H 203 N8O 10 ,1689.56502; found: 1689.56146.

[0321]

[0322] The synthesis method for L0865 is similar to that for L0902. L0865: 1 H NMR (400 MHz, Chloroform-d) δ6.55 (t, J = 6.0 Hz, 4H), 3.83 - 3.68 (m, 4H), 3.44 (ddd, J = 14.0, 6.4, 3.6Hz, 4H), 3.18 - 3.00 (m, 4H), 2.68 - 2.29 (m, 16H), 2.28 - 2.07 (m, 11H), 1.60 (p, J = 7.6 Hz, 12H), 1.36 - 1.16 (m, 56H), 0.86 (t, J = 6.8 Hz, 12H).HRMS (ESI, m / z): [M+H] + calcd. For C 63 H 128 N7O8, 1110.98189; found: 1110.98170.

[0323]

[0324] The synthesis method for L0866 is similar to that for L0902. L0866: 1H NMR (400 MHz, Chloroform-d) δ6.69 - 6.25 (m, 4H), 3.85 - 3.70 (m, 4H), 3.46 (ddd, J = 14.0, 6.8, 3.6 Hz,4H), 3.10 (ddd, J = 13.2, 7.2, 5.2 Hz, 4H), 2.85 - 2.05 (m, 27H), 1.77 - 1.54(m, 12H), 1.39 - 1.17 (m, 72H), 0.87 (t, J = 6.8 Hz, 12H). HRMS (ESI, m / z):[M+H] + calcd. For C 71 H 144 N7O8, 1223.10709; found: 1223.10876.

[0325]

[0326] Synthesis of L0867. Synthesis of 26BDA: Succinic anhydride (60.1 g, 60 mmol) was added to 150 mL of anhydrous dichloromethane solution containing 8-OH (6.51 g, 50 mmol) and DMAP (9.17 g, 75 mmol) at 0 °C. The reaction mixture was then heated to room temperature and stirred for 12 hours, followed by dilution with 200 mL of dichloromethane and washing with 1 M hydrochloric acid aqueous solution (50 mL × 3 times). The organic phase was dried over sodium sulfide, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give a pale yellow oily product 8BDA (11.104 g, yield 96.4%). 8BDA: HRMS (ESI, m / z): [M+H] + calcd.For: C 12 H 23 O 4, 231.15909; Found: 231.15860. 8BDA-NHS was synthesized using a similar scheme to step 4, followed by an amide bond formation reaction to obtain the final product L0867. 8BDA-NHS: HRMS (ESI, m / z): [M+H] + calcd. For: C 16 H 26 NO 6, 328.17546; Found: 328.17560. L0867: 1H NMR (400 MHz,Chloroform-d) δ 7.06 - 6.71 (m, 2H), 4.02 (td, J = 6.8, 2.0 Hz, 4H), 3.92 -3.84 (m, 2H), 3.77 - 3.66 (m, 3H), 3.59 (t, J = 6.8 Hz, 5H), 3.44 - 3.31 (m,4H), 3.10 - 2.97 (m, 2H), 2.77 - 2.68 (m, 10H), 2.63 (t, J = 6.8 Hz, 4H),2.57 - 2.24 (m, 21H), 1.69 - 1.49 (m, 12H), 1.36 - 1.16 (m, 40H), 0.85 (t, J= 6.8 Hz, 12H). HRMS (ESI, m / z): [M+H] + calcd. For C 67 H 128 N7O 16 , 1286.94121;found: 1286.94282。

[0327]

[0328] L0868: 1 H NMR (400 MHz, Chloroform-d) δ 6.25 (t, J = 6.0 Hz, 4H),3.84 - 3.64 (m, 4H), 3.43 (ddd, J = 14.0, 6.4, 3.6 Hz, 4H), 3.22 - 2.99 (m,4H), 2.89 - 2.76 (m, 2H), 2.70 - 2.60 (m, 2H), 2.55 - 2.38 (m, 9H), 2.34 -2.10 (m, 14H), 1.62 (p, J = 7.2 Hz, 8H), 1.37 - 1.17 (m, 56H), 0.87 (t, J =6.8 Hz, 12H). HRMS (ESI, m / z): s[M+H] + calcd. For C 61 H 124 N7O 8, 1082.95059; found1082.94971。

[0329]

[0330] The synthesis method for L0869 is similar to that for L0902. L0869: 1 H NMR (400 MHz, Chloroform-d) δ6.25 (t, J = 6.0 Hz, 4H), 3.84 - 3.64 (m, 4H), 3.43 (ddd, J = 14.0, 6.4, 3.6Hz, 4H), 3.22 - 2.99 (m, 4H), 2.89 - 0.87 (t, J = 6.8 Hz, 12H). HRMS (ESI, m / z): [M+H) + calcd. ForC 69 H 140 N7O 8, 1195.07579; found 1195.07242.

[0331]

[0332] The synthesis method for L0870 is similar to that for L0902. L0870: 1 H NMR (400 MHz, Chloroform-d) δ6.88 - 6.50 (m, 5H), 3.86 - 3.69 (m, 5H), 3.49 - 3.37 (m, 5H), 3.09 (dt, J =13.6, 6.4 Hz, 5H), 2.67 - 2.34 (m, 16H), 2.19 (t, J = 7.6 Hz, 10H), 1.60 (p,J = 7.2 Hz, 14H), 1.35 - 1.17 (m, 70H), 0.86 (t, J = 6.8 Hz, 15H). HRMS (ESI,m / z): [M+H] + calcd. For C 76 H 153 N8O 10, 1338.17042; found 1338.16880.

[0333]

[0334] The synthesis method for L0871 is similar to that for L0902. L0871: 1 H NMR (400 MHz, Chloroform-d) δ6.65 (t, J = 6.0 Hz, 5H), 3.91 - 3.71 (m, 5H), 3.49 - 3.36 (m, 5H), 3.18 -2.99 (m, 5H), 2.72 - 2.35 (m, 16H), 2.28 - 2.12 (m, 10H), 1.61 (p, J = 7.2Hz, 14H), 1.40 - 1.12 (m, 90H), 0.87 (t, J = 6.8 Hz, 15H). HRMS (ESI, m / z):[M+H] + calcd. For C 86 H 173 N8O 10 , 1478.32692; found: 1478.32490.

[0335]

[0336] Synthesis of L0872. The preparation of (R)-22-CAA-Boc was carried out using a similar procedure to step 2. (R)-22-CAA-Boc: HRMS (ESI, m / z): [M+H] + calcd. For C 37 H 76 N7O 12 , 810.55465; found 810.55449. L0872 is synthesized using a scheme similar to step 7. L0872: 1 H NMR (400 MHz, Chloroform-d) δ 6.31 (t,J = 6.0 Hz, 4H), 3.85 - 3.65 (m, 4H), 3.42 (ddd, J = 14.0, 6.4, 3.6 Hz, 4H), 3.21 - 3.02 (m, 4H), 2.88 - 2.79 (m, 2H), 2.68 (t, J = 10.8 Hz, 2H), 2.56 -2.12 (m, 23H), 1.61 (p, J = 7.2 Hz, 8H), 1.35 - 1.17 (m, 48H), 0.86 (t, J =6.8 Hz, 12H). HRMS (ESI, m / z): [M+H] +calcd. For C 57 H 116 N7O 8, 1026.88799; found1026.88726.

[0337]

[0338] The synthesis method for L0873 is similar to that for L0872. L0873: 1 H NMR (400 MHz, Chloroform-d) δ6.25 (t, J = 5.6 Hz, 4H), 3.83 - 3.61 (m, 4H), 3.42 (ddd, J = 14.0, 6.4, 3.6Hz, 4H), 3.21 - 3.00 (m, 4H), 2.90 - 0.86 (t, J = 6.8 Hz, 12H). HRMS (ESI, m / z): [M+H) + calcd. ForC 65 H 132 N7O 8, 1139.01319; found 1139.01255.

[0339]

[0340] The synthesis method for L0874 is similar to that for L0872. L0874: 1 H NMR (400 MHz, Chloroform-d) δ6.21 (t, J = 6.0 Hz, 4H), 3.84 - 3.63 (m, 4H), 3.56 - 3.33 (m, 4H), 3.11 (dt,J = 13.2, 6.4 Hz, 4H), 2.89 - HRMS (ESI, m / z): [M+2H]2+ calcd. For C 73 H 148 N7O8, 626.07283; found626.07114.

[0341]

[0342] Synthesis of L0875. (R)-33H-CAA-Boc was synthesized using a similar scheme as in step 2. (R)-33H-CAA-Boc:HRMS (ESI, m / z): [M+H] + calcd. For C 46 H 93 N8O 15 , 997.67549; found 997.67538.. L0875 is synthesized using a scheme similar to step 7. L0875: 1 H NMR (400 MHz, Chloroform-d) δ 6.66 (q,J = 5.6 Hz, 5H), 3.86 - 3.66 (m, 5H), 3.47 - 3.37 (m, 5H), 3.17 - 3.01 (m,5H), 2.74 - 2.31 (m, 18H), 2.28 - 2.14 (m, 10H), 1.69 - 1.50 (m, 14H), 1.35 -1.18 (m, 60H), 0.86 (t, J = 6.8 Hz, 15H). HRMS (ESI, m / z): [M+H] + calcd. ForC 71 H 143 N8O 10 , 1268.09217; found 1268.09346.

[0343]

[0344] The synthesis method for L0876 is similar to that for L0875. L0876: 1H NMR (400 MHz, Chloroform-d) δ6.63 (t, J = 5.6 Hz, 5H), 3.92 - 3.69 (m, 5H), 3.47 - 3.39 (m, 5H), 3.21 -2.99 (m, 5H), 2.73 - 2.31 (m, 17H), 2.20 (td, J = 7.6, 4.0 Hz, 10H), 1.60 (h,J = 7.2 Hz, 14H), 1.38 - 1.16 (m, 80H), 0.87 (t, J = 6.8 Hz, 15H). HRMS (ESI,m / z): [M+H] + calcd. For C 81 H 163 N8O 10 , 1408.24861; found 1408.24773.

[0345]

[0346] The synthesis method for L0877 is similar to that for L0875. L0877: 1 H NMR (400 MHz, Chloroform-d) δ6.64 (t, J = 7.6 Hz, 5H), 3.92 - 3.69 (m, 5H), 3.50 - 3.36 (m, 5H), 3.19 -3.01 (m, 5H), 2.81 - 2.29 (m, 18H), 2.20 (t, J = 7.6 Hz, 10H), 1.72 - 1.50(m, 14H), 1.43 - 1.11 (m, 100H), 0.87 (t, J = 6.8 Hz, 15H). HRMS (ESI, m / z):[M+H] + calcd. For C 91 H 183 N8O 10 , 1549.40852; found 1549.40669.

[0347]

[0348] The synthesis method of L0878 is similar to that of L0902. L0878: 1H NMR (400 MHz, Chloroform-d) δ6.43 (s, 4H), 4.63 (s, 8H), 3.82 - 3.65 (m, 4H), 3.50 (dt, J = 15.2, 6.4 Hz,16H), 3.40 (ddd, J = 14.0, 6.4, 3.6 Hz, 4H), 3.09 (dt, J = 13.2, 6.4 Hz, 4H), 2.92 - 2.63 (m, 5H), 2.54 - 2.39 (m, 9H), 2.22 (t, J = 7.3 Hz, 13H), 1.77 -1.65 (m, 8H), 1.65 - 1.47 (m, 16H), 1.36 - 1.17 (m, 56H), 0.86 (t, J = 6.8Hz, 12H). HRMS (ESI, m / z): [M+H] + calcd. For C 81 H 164 N7O 16 , 1491.22291; found1491.22192.

[0349]

[0350] The synthesis method for L0879 is similar to that for L0902. L0879: 1 H NMR (400 MHz, Chloroform-d) δ6.89 (t, J = 5.6 Hz, 4H), 4.63 (s, 8H), 3.93 - 3.76 (m, 4H), 3.50 (dt, J =13.6, 6.4 Hz, 16H), 3.41 (ddd, J = 14.0, 6.4, 3.2 Hz, 4H), 3.33 - 2.97 (m,9H), 2.81 - 2.64 (m, 5H), 2.62 - 2.45 (m, 6H), 2.38 - 2.14 (m, 11H), 2.13 -1.77 (m, 4H), 1.77 - 1.46 (m, 24H), 1.36 - 1.18 (m, 56H), 0.86 (t, J = 6.8Hz, 12H). HRMS (ESI, m / z): [M+H] + calcd. For C 83 H 168 N7O 16, 1519.25421; found1519.25345.

[0351]

[0352] The synthesis method for L0880 is similar to that for L0902. L0880: 1 H NMR (400 MHz, Chloroform-d) δ6.70 (t, J = 6.0 Hz, 5H), 4.64 (s, 10H), 3.87 - 3.65 (m, 5H), 3.61 - 3.34 (m,25H), 3.16 - 3.01 (m, 5H), 2.87 - 2.33 (m, 19H), 2.30 - 2.17 (m, 10H), 1.80 -1.46 (m, 34H), 1.37 - 1.18 (m, 70H), 0.87 (t, J = 6.8 Hz, 15H). HRMS (ESI, m / z): [M+H] + calcd. For C 101 H 203 N8O 20 , 1849.51417; found 1849.51020.

[0353]

[0354] Synthesis of L0881. Carboxylic acid 6AF5C was synthesized using a scheme similar to that in step 3. 6AF5C: HRMS (ESI, m / z): [M+Na] + calcd. For C 12 H 24 NaO4, 255.15668; found 255.15843. The ester 6AF5C-NHS was synthesized using a similar scheme to step 4. 6AF5C-NHS: HRMS (ESI, m / z): [M+NH4] + calcd. For C 16 H 31 N2O6, 347.21766; found 347.21829. L0881 was synthesized using a scheme similar to step 7. L0881((S)22-CAA-6AF5C) 1H NMR (400 MHz, Chloroform-d) δ 6.45 (d, J = 16.8 Hz, 4H), 4.63 (s,8H), 3.84 - 3.67 (m, 4H), 3.62 - 3.27 (m, 20H), 3.10 (dt, J = 13.4, 6.4 Hz,4H), 2.96 - 2.58 (m, 6H), 2.58 - 2.38 (m, 9H), 2.22 (t, J = 7.6 Hz, 12H), 1.79 - 1.44 (m, 24H), 1.36 - 1.25 (m, 24H), 0.87 (t, J = 6.8 Hz, 12H). HRMS(ESI, m / z): [M+H] + calcd. For C 65 H 132 N7O 16 , 1266.97251; found 1266.97104.

[0355]

[0356] The synthesis method of L0882 is similar to that of L0881. L0882: 1 H NMR (400 MHz, Chloroform-d) δ6.65 (t, J = 6.0 Hz, 4H), 4.63 (s, 8H), 3.83 - 3.69 (m, 4H), 3.60 - 3.34 (m,20H), 3.14 - 3.03 (m, 4H), 2.69 - 2.39 (m, 12H), 2.39 - 2.31 (m, 4H), 2.31 -2.10 (m, 11H), 1.74 - 1.23 (m, 52H), 0.87 (t, J = 6.8 Hz, 12H). HRMS (ESI, m / z): [M+H] + calcd. For C 67 H 136 N7O 16 , 1295.00381; found 1295.00313.

[0357]

[0358] The synthesis method for L0883 is similar to that for L0881. L0883: 1H NMR (400 MHz, Chloroform-d) δ7.04 - 6.51 (m, 5H), 4.64 (s, 10H), 3.91 - 3.64 (m, 5H), 3.62 - 3.28 (m,25H), 3.20 - 3.02 (m, 5H), 2.95 (s, 2H), 2.72 - 2.15 (m, 26H), 1.78 - 1.41(m, 34H), 1.40 - 1.24 (m, 34H), 0.88 (t, J = 6.8 Hz, 15H). HRMS (ESI, m / z):[M+H] + calcd. For C 81 H 163 N8O 20 , 1568.19781; found 1568.19720.

[0359]

[0360] Synthesis of L0884. (R)-33-CAA-Boc was synthesized using a scheme similar to that in step 4. (R)-33-CAA-Boc:HRMS (ESI, m / z): [M+H] + calcd. For C 39 H 80 N7O 12 , 838.58595; found 838.58727. L0884 was synthesized using a scheme similar to step 7. L0884: 1 H NMR (400 MHz, Chloroform-d) δ 6.55 (t, J= 5.6 Hz, 4H), 3.85 - 3.68 (m, 4H), 3.44 (ddd, J = 14.0, 6.4, 3.6 Hz, 4H), 3.19 - 2.99 (m, 4H), 2.69 - 2.28 (m, 16H), 2.28 - 2.06 (m, 11H), 1.74 - 1.49(m, 12H), 1.34 - 1.16 (m, 48H), 0.86 (t, J = 6.8 Hz, 12H). HRMS (ESI, m / z):[M+H] + calcd. For C 59 H 120 N7O 8,1054.91929; found 1054.91906.

[0361]

[0362] The synthesis method for L0885 is similar to that for L0884. L0885: 1 H NMR (400 MHz, Chloroform-d) δ6.53 (t, J = 6.0 Hz, 4H), 3.83 - 3.67 (m, 4H), 3.44 (ddd, J = 14.0, 6.4, 3.6Hz, 4H), 3.17 - 3.00 (m, 4H), 2.68 - 2.27 (m, 16H), 2.26 - 2.02 (m, 11H), 1.73 - 1.47 (m, 12H), 1.35 - 1.15 (m, 56H), 0.86 (t, J = 6.8 Hz, 12H). HRMS(ESI, m / z): [M+H] + calcd. For C 63 H 128 N7O 8, 1110.98189; found 1110.98366.

[0363]

[0364] The synthesis method for L0886 is similar to that for L0884. L0886: 1 H NMR (400 MHz, Chloroform-d) δ6.59 (t, J = 5.6 Hz, 4H), 3.82 - 3.64 (m, 4H), 3.43 (ddd, J = 13.6, 6.4, 3.6Hz, 4H), 3.20 - 2.99 (m, 4H), 2.67 - 2.24 (m, 16H), 2.24 - 2.00 (m, 11H), 1.59 (p, J = 7.2 Hz, 12H), 1.34 - 1.16 (m, 64H), 0.85 (t, J = 6.8 Hz, 12H).HRMS (ESI, m / z): [M+H] + calcd. For C 67 H 136 N7O8, 1167.04449; found 1167.04670.

[0365]

[0366] The synthesis method of L0887 is similar to that of L0881. L0887((R)-33-CAA-6AF5C) 1 H NMR (400 MHz, Chloroform-d) δ 6.58 (t, J = 6.0 Hz, 4H), 4.63 (s, 8H), 3.85 - 3.67 (m, 4H), 3.61 - 3.34 (m, 20H), 3.17 - 3.00 (m, 4H), 2.67 - 2.27 (m, 17H), 2.27 - 2.18(m, 10H), 1.81 - 1.45 (m, 28H), 1.37 - 1.24 (m, 24H), 0.86 (t, J = 6.8 Hz12H). HRMS (ESI, m / z): [M+H] + calcd. For C 67 H 136 N7O 16 , 1295.00381; found:1295.00593.

[0367]

[0368] The synthesis method of L0888 is similar to that of L0881. L0888((R)-22-CAA-6AF5C) 1 H NMR (400 MHz, Chloroform-d) δ 6.39 (s, 4H), 4.63 (s, 8H), 3.84 - 3.64 (m, 4H), 3.63 - 3.28 (m, 20H), 3.20 - 2.98 (m, 4H), 2.91 - 2.37 (m, 15H), 2.34 - 2.15 (m, 12H), 1.83 - 1.47 (m, 24H), 1.38 - 1.23 (m, 24H), 0.87 (t, J = 6.8 Hz, 12H). HRMS(ESI, m / z): [M+H] + calcd. For C 65 H 132 N7O 16 , 1266.97251; found 1266.97383.

[0369]

[0370] The synthesis method of L0889 is similar to that of L0881. L0889((R)-33H-CAA-6AF5C) 1 H NMR (400 MHz, Chloroform-d) δ 6.68 (t, J = 6.0 Hz, 5H), 4.63 (s, 10H), 3.74 (tt, J = 8.0,3.6 Hz, 5H), 3.65 - 3.29 (m, 25H), 3.19 - 2.99 (m, HRMS (ESI, m / z): [M+H] + calcd. For C 81 H 163 N8O 20 ,1568.19781; found 1568.19717.

[0371]

[0372] The synthesis method for L0890 is similar to that for L0896. L0890: 1 H NMR (400 MHz, Chloroform-d) δ5.75 - 5.32 (m, 4H), 4.05 (t, J = 6.8 Hz, 8H), 3.84 - 3.52 (m, 4H), 3.52 -2.94 (m, 8H), 2.95 - 2.01 (m, 19H), 1.60 (p, J = 7.2 Hz, 8H), 1.38 - 1.18 (m,40H), 0.87 (t, J = 6.8 Hz, 12H). HRMS (ESI, m / z): [M+H] + calcd. ForC 53 H 108 N7O 12 , 1034.80505; found 1034.80567.

[0373]

[0374] The synthesis method for L0891 is similar to that for L0896. L0891: 1H NMR (400 MHz, Chloroform-d) δ6.05 - 5.34 (m, 4H), 4.04 (t, J = 6.8 Hz, 8H), 3.86 - 3.59 (m, 4H), 3.45 -2.91 (m, 8H), 2.84 - 2.11 (m, 19H), 1.81 - 1.48 (m, 12H), 1.35 - 1.20 (m,40H), 0.87 (t, J = 6.8 Hz, 12H). HRMS (ESI, m / z):[M+H] + calcd. For C 55 H 112 N7O 12 ,1062.83635; found 1062.83812.

[0375]

[0376] The synthesis method for L0892 is similar to that for L0896. L0892: 1 H NMR (400 MHz, Chloroform-d) δ5.98 - 5.35 (m, 5H), 4.14 - 3.94 (m, 10H), 3.89 - 3.58 (m, 5H), 3.41 - 3.24(m, 5H), 3.08 - 2.88 (m, 5H), 2.81 - 2.23 (m, 18H), 1.77 - 1.44 (m, 14H), 1.38 - 1.19 (m, 50H), 0.87 (t, J = 6.8 Hz, 15H). HRMS (ESI, m / z):[M+H] + calcd. For C 66 H 133 N8O 15 , 1277.98849; found 1277.98860.

[0377]

[0378] The synthesis method for L0893 is similar to that for L0896. L0893: 1H NMR (400 MHz, Chloroform-d) δ5.76 - 5.38 (m, 4H), 4.04 (t, J = 6.8 Hz, 8H), 3.85 - 3.62 (m, 4H), 3.42 -3.25 (m, 4H), 3.12 - 2.96 (m, 4H), 2.94 - 2.23 (m, 19H), 1.60 (p, J = 6.8 Hz,8H), 1.36 - 1.19 (m, 56H), 0.87 (t, J = 6.8 Hz, 12H). HRMS (ESI, m / z):[M+H] + calcd. For C 61 H 124 N7O 12 , 1146.93025; found 1146.93024.

[0379]

[0380] The synthesis method for L0894 is similar to that for L0896. L0894: 1 H NMR (400 MHz, Chloroform-d) δ6.02 - 5.47 (m, 4H), 4.04 (t, J = 6.8 Hz, 8H), 3.87 - 3.62 (m, 4H), 3.46 -3.25 (m, 4H), 3.13 - 2.86 (m, 4H), 2.75 - 2.14 (m, 18H), 1.82 - 1.48 (m,12H), 1.41 - 1.14 (m, 56H), 0.87 (t, J = 6.8 Hz, 12H). HRMS (ESI, m / z):[M+H] + calcd. For C 63 H 128 N7O 12 , 1174.96155; found 1174.96035.

[0381]

[0382] The synthesis method for L0895 is similar to that for L0896. L0895: 1H NMR (400 MHz, Chloroform-d) δ5.88 - 5.29 (m, 5H), 4.19 - 3.96 (m, 10H), 3.91 - 3.57 (m, 5H), 3.42 - 3.21(m, 5H), 3.12 - 2.90 (m, 5H), 2.78 - 2.21 (m, 18H), 1.75 - 1.47 (m, 14H), 1.36 - 1.21 (m, 70H), 0.87 (t, J = 6.8 Hz, 15H). HRMS (ESI, m / z):[M+H] + calcd. For C 76 H 153 N8O 15 , 1418.14499; found 1418.14710.

[0383]

[0384] The synthesis method for L0897 is similar to that for L0896. L0897: 1 H NMR (400 MHz, Chloroform-d) δ5.80 (s, 4H), 4.04 (t, J = 7.2 Hz, 8H), 3.85 - 3.67 (m, 4H), 3.46 - 3.25 (m,4H), 3.06 - 2.87 (m, 4H), 2.64 - 2.26 (m, 16H), 2.16 (s, 3H), 1.60 (p, J =7.2 Hz, 12H), 1.39 - 1.13 (m, 72H), 0.87 (t, J = 6.8 Hz, 12H). HRMS (ESI, m / z):[M+H] + calcd. For C 71 H 144 N7O 12 , 1287.08675; found 1287.08794.

[0385]

[0386] The synthesis method for L0898 is similar to that for L0896. L0898: 1H NMR (400 MHz, Chloroform-d) δ5.97 - 5.36 (m, 5H), 4.17 - 3.89 (m, 10H), 3.88 - 3.58 (m, 5H), 3.42 - 3.23(m, 5H), 3.17 - 2.92 (m, 5H), 2.86 - 2.27 (m, 18H), 1.79 - 1.47 (m, 14H), 1.45 - 1.05 (m, 90H), 0.87 (t, J = 6.8 Hz, 15H). HRMS (ESI, m / z):[M+H] + calcd. For C 86 H 173 N8O 15 , 1558.30149; found 1558.30355.

[0387]

[0388] Synthesis of L0899. Carboxylic acid 6AE5C was synthesized using a scheme similar to that in step 3. 6AE5C: HRMS (ESI, m / z): [M+Na] + calcd. For C 13 H 26 NaO4, 269.17233; found 269.17236. Ester 6AE5C-NHS was synthesized using a similar scheme to step 4. 6AE5C-NHS: HRMS (ESI, m / z): [M+H] + calcd. For C 17 H 29 NNaO6, 366.18871; found 366.18838. L0899 was synthesized using a scheme similar to step 7. L0899: 1H NMR (400MHz, Chloroform-d) δ 6.46 (t, J = 5.9 Hz, 4H), 4.61 (q, J = 5.2 Hz, 4H), 3.82- 3.63 (m, 4H), 3.60 - 3.49 (m, 8H), 3.48 - 3.29 (m, 12H), 3.16 - 2.99 (m,4H), 2.86 - 2.59 (m, 5H), 2.52 - 2.34 (m, 9H), 2.32 - 2.11 (m, 13H), 1.75 -1.62 (m, 8H), 1.62 - 1.44 (m, 16H), 1.35 - 1.19 (m, 36H), 0.85 (t, J = 6.8Hz, 12H). HRMS (ESI, m / z):[M+H] + calcd. For C 69 H 140 N7O 16 , 1323.03511; found1323.03421.

[0389]

[0390] The synthesis method for L0900 is similar to that for L0899. L0900: 1 H NMR (400 MHz, Chloroform-d) δ6.63 (t, J = 6.0 Hz, 4H), 4.63 (q, J = 5.2 Hz, 4H), 3.83 - 3.69 (m, 4H), 3.63- 3.50 (m, 8H), 3.50 - 3.31 (m, 12H), 3.15 - 3.02 (m, 4H), 2.63 - 2.28 (m,17H), 2.23 (t, J = 7.6 Hz, 11H), 1.80 - 1.45 (m, 28H), 1.36 - 1.22 (m, 36H), 0.87 (t, J = 6.8 Hz, 12H). HRMS (ESI, m / z):[M+H + calcd. For C 71 H 144 N7O 16 ,1351.06641; found 1351.06898.

[0391]

[0392] The synthesis method for L0901 is similar to that for L0899. L0901: 1 H NMR (400 MHz, Chloroform-d) δ6.99 - 6.58 (m, 4H), 4.63 (q, J = 5.2 Hz, 5H), 3.92 - 3.66 (m, 5H), 3.64 -3.51 (m, 10H), 3.50 - 3.26 (m, 15H), 3.16 - 3.03 (m, 5H), 2.85 - 2.72 (m,4H), 2.60 - 2.34 (m, 14H), 2.24 (t, J = 7.2 Hz, 10H), 1.81 - 1.40 (m, 34H),1.33 - 1.23 (m, 45H), 0.87 (t, J = 6.8 Hz, 15H). HRMS (ESI, m / z): [M+H] + calcd. For C 86 H 173 N8O 20, 1638.27607; found 1638.27613.

[0393]

[0394] The synthesis method for L0903 is similar to that for L0902. L0903: 1 H NMR (400 MHz, Chloroform-d) δ6.65 (t, J = 6.0 Hz, 4H), 4.63 (s, 8H), 3.80 - 3.70 (m, 4H), 3.51 (dt, J =9.6, 6.4 Hz, 16H), 3.42 (ddd, J = 13.6, 6.4, 3.2 Hz, 4H), 3.08 (ddd, J =13.2, 7.2, 5.2 Hz, 4H), 2.60 - 2.32 (m, 16H), 2.27 - 2.14 (m, 11H), 1.75 -1.49 (m, 28H), 1.41 - 1.30 (m, 8H), 0.90 (t, J = 7.6 Hz, 12H). HRMS (ESI, m / z):[M+H] + calcd. For C 59 H 120 N7O 16, 1182.87861; found 1182.87842.

[0395]

[0396] The synthesis method for L0904 is similar to that for L0902. L0904: 1 H NMR (400 MHz, Chloroform-d) δ7.04 - 6.47 (m, 5H), 4.63 (s, 10H), 3.84 - 3.65 (m, 5H), 3.51 (dt, J = 9.2,6.4 Hz, 22H), 3.45 - 3.35 (m, 5H), 3.14 - 3.02 (m, 5H), 2.83 - 2.70 (m, 3H), 2.65 - 2.31 (m, 15H), 2.30 - 2.14 (m, 10H), 1.78 - 1.48 (m, 34H), 1.41 - 1.31(m, 10H), 0.90 (t, J = 7.2 Hz, 15H). HRMS (ESI, m / z): [M+H] + calcd. ForC 71 H 143 N8O 20 , 1428.04131; found 1428.04214.

[0397]

[0398] Synthesis of L0905. Carboxylic acid 4AE5C was synthesized using a scheme similar to that in step 3. 1,52OH TBDPS: HRMS (ESI, m / z): [M+H] + calcd. For C 21 H 31 O2Si , 343.20878; found 343.20846;4AE5CTBDPS: HRMS (ESI, m / z): [M+Na] + calcd. For C 27 H 42 NaO3Si, 465.27954; found465.28023; 4AE5C-OH: HRMS (ESI, m / z): [M+Na] + calcd. For C 11 H 24NaO3, 227.16176; found 227.16219; 4AE5C: HRMS (ESI, m / z): [M+Na] + calcd. For C 11 H 22 NaO4, 241.14103; found 241.14125. The ester 4AE5C-NHS was synthesized using a similar scheme to step 4. 4AE5C-NHS:4AE5C-NHS: HRMS (ESI, m / z): [M+Na] + calcd.For C 15 H 25 NNaO6, 338.15741; found 338.15779. L0905 was synthesized using a similar scheme to step 7. L0905: 1 H NMR (400 MHz, Chloroform-d) δ 6.51 (t, J = 6.0 Hz, 4H), 4.61 (q, J = 5.2 Hz, 4H), 3.80 - 3.66 (m, 4H), 3.60 - 3.49 (m, 8H), 3.45 - 3.30 (m, 12H), 3.08 (dt, J = 12.8, 6.0 Hz, 4H), 2.85 - 2.76 (m, 2H), 2.65 (t, J = 10.8 Hz, 2H), 2.52 - 2.38 (m, 9H), 2.38 -2.10 (m, 14H), 1.74 - 1.62 (m, 8H), 1.61 - 1.46 (m, 16H), 1.38 - 1.28 (m,8H), 1.25 (d, J = 5.2 Hz, 12H), 0.88 (t, J = 7.2 Hz, 12H). HRMS (ESI, m / z):[M+H] + calcd. For C 61 H 124 N7O 16 , 1210.90991; found 1210.90934.

[0399]

[0400] The synthesis method for L0906 is similar to that for L0905. L0906 1H NMR (400 MHz, Chloroform-d) δ6.69 (t, J = 6.0 Hz, 4H), 4.61 (q, J = 5.2 Hz, 4H), 3.79 - 3.69 (m, 4H), 3.61- 3.50 (m, 8H), 3.46 - 3.32 (m, 12H), 3.07 (dt, J = 13.2, 6.0 Hz, 4H), 2.65 -2.08 (m, 27H), 1.78 - 1.41 (m, 28H), 1.39 - 1.29 (m, 8H), 1.25 (d, J = 5.2Hz, 12H), 0.88 (t, J = 7.2 Hz, 12H). HRMS (ESI, m / z):[M+H) + calcd. ForC 63 H 128 N7O 16 , 1238.94121; found 1238.94205.

[0401]

[0402] The synthesis method of L0907 is similar to that of L0905. L0907((S)-33H-CAA-4AE5C) 1 H NMR (400 MHz, Chloroform-d) δ 6.80 (t, J = 6.0 Hz, 5H), 4.62 (q, J = 5.2 Hz, 5H), 3.82 -3.69 (m, 5H), 3.61 - 3.50 (m, 10H), 3.45 - 3.34 (m, 15H), 3.07 (p, J = 6.0Hz, 5H), 2.68 - 2.29 (m, 18H), 2.22 (t, J = 7.2 Hz, 10H), 1.75 - 1.44 (m,34H), 1.40 - 1.29 (m, 10H), 1.26 (d, J = 5.2 Hz, 15H), 0.89 (t, J = 7.2 Hz,15H). HRMS (ESI, m / z):[M+H] + calcd. For C 76 H 153 N8O 20 , 1498.11956; found1498.12173.

[0403]

[0404] Synthesis of L0908. Carboxylic acid 3M1BF5C was synthesized using a scheme similar to that in step 3. 1,52OHTBDPS: HRMS(ESI, m / z): [M+H] + calcd. For C 21 H 31 O2Si , 343.20878; found 343.20846;3M1BF5CTBDPS: HRMS (ESI, m / z): [M+Na] + calcd. For C 27 H 42 NaO3Si, 465.27954;found 465.27936. 3M1BF5C-OH: HRMS (ESI, m / z): [M+Na] + calcd. For C 11 H 24 NaO3,227.16176; found 227.16183. 3M1BF5C: HRMS (ESI, m / z): [M+Na] + calcd. ForC 11 H 22 NaO3, 241.14103; found 241.14046. The ester 3M1BF5C-NHS was synthesized using a similar scheme to step 4. 3M1BF5C-NHS: HRMS (ESI, m / z): [M+Na] + calcd. For C 15 H 25 NNaO6, 338.15741; found 338.15723. L0908 was synthesized using a similar scheme to step 7. L0908: 1H NMR (400 MHz, Chloroform-d) δ 6.46 (t, J = 6.0 Hz, 4H), 4.61 (s, 8H), 3.78 - 3.65 (m, 4H), 3.51 (t, J= 6.8 Hz, 16H), 3.39 (ddd, J = 14.0, 6.4, 3.2 Hz, 4H), 3.08 (dt, J = 13.2,6.4 Hz, 4H), 2.86 - 2.77 (m, 2H), 2.66 (t, J = 11.2 Hz, 2H), 2.45 (q, J =6.4, 3.6 Hz, 9H), 2.36 - 2.07 (m, 14H), 1.74 - 1.54 (m, 20H), 1.43 (q, J =6.8 Hz, 8H), 0.87 (d, J = 6.8 Hz, 24H). HRMS (ESI, m / z):[M+H] + calcd. ForC 61 H 124 N7O 16 , 1210.90991; found 1210.90941.

[0405]

[0406] The synthesis method for L0909 is similar to that for L0908. (S)-33-CAA-Boc: HRMS (ESI, m / z): [M+H] + calcd. For C 39 H 80 N7O 12 , 838.58595; found 838.58663. L0909: 1H NMR (400 MHz, Chloroform-d) δ 6.70 (t, J = 6.0 Hz, 4H), 4.60 (s, 8H), 3.76 - 3.68 (m, 4H), 3.50 (t, J = 6.8 Hz, 16H), 3.40 (ddd, J = 13.6, 6.4, 0.86 (d, J = 6.8 Hz, 24H). HRMS (ESI, m / z):[M+H) + calcd. For C 63 H 128 N7O 16, 1238.94121; found 1238.94123.

[0407]

[0408] The synthesis method for L0910 is similar to that for L0908. (S)-33H-CAA-Boc: HRMS (ESI, m / z): [M+H] + calcd. For C 46 H 93 N8O 15 , 997.67549; found 997.67538. L0910: 1 H NMR (400 MHz, Chloroform-d) δ 6.72 (t, J = 5.6 Hz, 5H), 4.61 (s, 10H), 3.80 - 3.67 (m, 5H), 3.50 (t, J = 6.8 Hz, 20H), 3.39 (ddd, J = 13.6, 6.4, 3.2 Hz, 5H), 3.12 - 2.99(m, 5H), 2.61 - 2.29 (m, 18H), 2.21 (t, J = 7.2 Hz, 10H), 1.72 - 1.49 (m,29H), 1.43 (q, J = 6.8 Hz, 10H), 0.87 (d, J = 6.8 Hz, 30H). HRMS (ESI, m / z): [M+H] +calcd. For C 76 H 153 N8O 20 , 1498.11956; found 1498.11767.

[0409]

[0410] Synthesis of L0911. 33-EP12 was synthesized using a scheme similar to step 6. 33-EP12: HRMS (ESI, m / z): [M+H] + calcd. For C 19 H 44 N3O, 330.34789; found: 330.34774. (S)-CAA-33-Ep12-Boc was synthesized using a scheme similar to that in step 2. (S)-33-Ep12-CAA-Boc: HRMS (ESI, m / z): [M+H] + calcd. For C 43 H 89 N6O 10 , 849.66347; found 849.66239. L0911 is synthesized using a scheme similar to step 7. L0911: 1 H NMR (400 MHz, Chloroform-d) δ 6.73 - 6.40 (m, 3H), 3.84 - 3.72(m, 3H), 3.66 - 3.58 (m, 1H), 3.54 - 3.42 (m, 3H), 3.14 - 2.99 (m, 3H), 2.71- 2.21 (m, 19H), 2.19 (t, J = 8.0 Hz, 6H), 1.72 - 1.54 (m, 10H), 1.33 - 1.19(m, 54H), 0.86 (t, J = 6.8 Hz, 12H). HRMS (ESI, m / z): [M+H] + calcd. ForC 58 H 119 N6O7, 1011.91348; found 1011.91430.

[0411]

[0412] The synthesis method for L0912 is similar to that for L0911. L0912: 1H NMR (400 MHz, Chloroform-d) δ6.68 - 6.45 (m, 3H), 3.83 - 3.71 (m, 3H), 3.66 - 3.58 (m, 1H), 3.54 - 3.41(m, 3H), 3.15 - 2.98 (m, 3H), 2.74 - 2.26 (m, 16H), 2.26 - 2.05 (m, 9H), 1.73- 1.52 (m, 10H), 1.33 - 1.18 (m, 58H), 0.86 (t, J = 6.8 Hz, 12H). HRMS (ESI,m / z): [M+Na] + calcd. For C 61 H 124 N6NaO7, 1075.94292; found 1075.94280.

[0413]

[0414] The synthesis method for L0913 is similar to that for L0911. L0913: 1 H NMR (400 MHz, Chloroform-d) δ6.69 - 6.39 (m, 3H), 3.84 - 3.70 (m, 3H), 3.66 - 3.58 (m, 1H), 3.55 - 3.41(m, 3H), 3.15 - 2.96 (m, 3H), 2.70 - 2.24 (m, 16H), 2.18 (t, J = 7.6 Hz, 9H), 1.71 - 1.51 (m, 10H), 1.35 - 1.17 (m, 64H), 0.86 (t, J = 6.8 Hz, 12H). HRMS(ESI, m / z): [M+H] + calcd. For C 64 H 131 N6O7, 1096.00738; found 1096.00577.

[0415]

[0416] The synthesis method for L0914 is similar to that for L0911. L0914: 1H NMR (400 MHz, Chloroform-d) δ6.63 - 6.40 (m, 3H), 3.82 - 3.69 (m, 3H), 3.66 - 3.58 (m, 1H), 3.54 - 3.41(m, 3H), 3.13 - 2.98 (m, 3H), 2.65 - 2.26 (m, 16H), 2.18 (t, J = 7.6 Hz, 9H), 1.61 (p, J = 7.6 Hz, 10H), 1.35 - 1.18 (m, 70H), 0.87 (t, J = 6.8 Hz, 12H).HRMS (ESI, m / z): [M+H] + calcd. For C 67 H 137 N6O7, 1138.05433; found 1138.05511.

[0417]

[0418] L0915 was synthesized using a similar method to L0911. (R)-CAA-33-Ep12-Boc: HRMS (ESI, m / z):[M+H] + calcd. For C 43 H 89 N6O 10 , 849.66347; found 849.66302. L0915 was synthesized using a method similar to L0911. L0915((R)-CAA-33-Ep12-C10) 1 H NMR (400 MHz, Chloroform-d) δ 6.68 -6.35 (m, 3H), 3.84 - 3.70 (m, 3H), 3.66 - 3.58 (m, 1H), 3.55 - 3.41 (m, 3H), 3.05 (dq, J = 13.2, 7.2, HRMS (ESI, m / z): [M+H] + calcd. For C 58 H 119N6O7, 1011.91348; found1011.91439.

[0419]

[0420] The synthesis method for L0916 is similar to that for L0915. L0916: 1 H NMR (400 MHz, Chloroform-d) δ6.71 - 6.43 (m, 3H), 3.85 - 3.69 (m, 3H), 3.68 - 3.59 (m, 1H), 3.55 - 3.39(m, 3H), 3.06 (dp, J = 13.2, 6.4 HRMS (ESI, m / z): [M+H] + calcd. For C 61 H 125 N6O7, 1053.96043; found1053.95968.

[0421]

[0422] The synthesis method for L0917 is similar to that for L0915. L0917: 1 H NMR (400 MHz, Chloroform-d) δ6.71 - 6.42 (m, 3H), 3.84 - 3.70 (m, 3H), 3.66 - 3.57 (m, 1H), 3.53 - 3.39(m, 3H), 3.15 - 2.96 (m, 3H), 2.78 - 2.26 (m, 16H), 2.26 - 2.03 (m, 9H), 1.61(p, J = 7.6 Hz, 10H), 1.34 - 1.17 (m, 64H), 0.86 (t, J = 6.8 Hz, 12H). HRMS(ESI, m / z): [M+H] + calcd. For C 64 H 131 N6O7, 1096.00738; found 1096.00688.

[0423]

[0424] The synthesis method for L0918 is similar to that for L0915. L0918: 1 H NMR (400 MHz, Chloroform-d) δ6.73 - 6.36 (m, 3H), 3.84 - 3.70 (m, 3H), 3.67 - 3.59 (m, 1H), 3.54 - 3.40(m, 3H), 3.05 (ddd, J = 13.2, 10.0, HRMS (ESI, m / z): [M+H] + calcd. For C 67 H 137 N6O7, 1138.05433; found1138.05384.

[0425]

[0426] Synthesis of L0919. 22-EP12 was synthesized using a scheme similar to step 6. (S)-CAA-22-Ep12-Boc was synthesized using a scheme similar to step 2. (S)-CAA-22-Ep12-Boc: HRMS (ESI, m / z): [M+H] + calcd. ForC 41 H 85 N6O 10 , 821.63217. L0919 was synthesized according to a protocol similar to procedure 7. L0919: 1H NMR (400 MHz,Chloroform-d) δ 6.51 - 6.20 (m, 3H), 3.80 - 3.68 (m, 3H), 3.64 - 3.56 (m,1H), 3.53 - 3.34 (m, 3H), 3.21 - 2.99 (m, 3H), 2.90 - 2.10 (m, 25H), 1.62 (p,J = 7.2 Hz, 6H), 1.48 - 1.17 (m, 54H), 0.86 (t, J = 6.8 Hz, 12H). HRMS (ESI,m / z): [M+H] + calcd. For C 56 H 115 N6O7, 983.88218; found 983.88414.

[0427]

[0428] The synthesis method for L0920 is similar to that for L0919. L0920: 1 H NMR (400 MHz, Chloroform-d) δ6.52 - 6.15 (m, 3H), 3.83 - 3.69 (m, 3H), 3.65 - 3.59 (m, 1H), 3.54 - 3.35(m, 3H), 3.22 - 3.01 (m, 3H), 2.93 - 2.08 (m, 25H), 1.62 (p, J = 7.2 Hz, 6H), 1.48 - 1.18 (m, 60H), 0.87 (t, J = 6.8 Hz, 12H). HRMS (ESI, m / z): [M+H] + calcd. For C 59 H 121 N6O7, 1025.92913; found 1025.92809.

[0429]

[0430] The synthesis method of L0921 is similar to that of L0919. L0921: ¹H NMR (400 MHz, Chloroform-d) δ 6.54 - 6.17 (m, 3H), 3.85 - 3.66 (m, 3H), 3.63 - 3.56 (m, 1H), 3.56 - 3.35 (m, 3H), 3.24 - 2.99 (m, 3H), 2.90 - 1.97 (m, 25H), 1.62 (p, J = 7.2 Hz, 6H), 1.49 - 1.19 (m, 66H), 0.87 (t, J = 6.8 Hz, 12H). HRMS (ESI, m / z): [M+H] + calcd. For C 62 H 127 N6O7, 1067.97608; found 1067.97976.

[0431]

[0432] The synthesis method of L0922 is similar to that of L0919. L0922: 1 H NMR (400 MHz, Chloroform-d) δ6.47 - 6.12 (m, 3H), 3.89 - 3.65 (m, 3H), 3.64 - 3.55 (m, 1H), 3.53 - 3.35(m, 3H), 3.11 (dp, J = 12.8, 6.4 Hz, 3H), 2.89 - 2.00 (m, 25H), 1.62 (p, J =7.2 Hz, 6H), 1.50 - 1.17 (m, 72H), 0.87 (t, J = 6.8 Hz, 12H). HRMS (ESI, m / z): [M+H] + calcd. For C 65 H 133 N6O7, 1110.02303; found 1110.02325.

[0433]

[0434] The synthesis method for L0923 is similar to that for L0919. (R)-22-Ep12-CAA-Boc: HRMS (ESI, m / z):[M+H] + calcd. For C 41 H 85N6O 10 , 821.63217. L0923: 1 H NMR (400 MHz, Chloroform-d) δ 6.45- 6.13 (m, 3H), 3.80 - 3.67 (m, 3H), 3.63 - 3.55 (m, 1H), 3.53 - 3.36 (m,3H), 3.21 - 2.96 (m, 3H), 2.90 - 2.06 (m, 25H), 1.62 (p, J = 7.2 Hz, 6H), 1.47 - 1.38 (m, 2H), 1.33 - 1.20 (m, 52H), 0.86 (t, J = 6.8 Hz, 12H). HRMS(ESI, m / z): [M+H] + calcd. For C 56 H 115 N6O7, 983.88218; found 983.88418.

[0435]

[0436] The synthesis method for L0924 is similar to that for L0923. L0924: 1 H NMR (400 MHz, Chloroform-d) δ6.48 - 6.13 (m, 3H), 3.83 - 3.68 (m, 3H), 3.64 - 3.56 (m, 1H), 3.54 - 3.36(m, 3H), 3.11 (dp, J = 12.4, 6.0 Hz, 3H), 2.90 - 1.98 (m, 25H), 1.62 (p, J =7.6, 7.2 Hz, 6H), 1.49 - 1.16 (m, 60H), 0.87 (t, J = 6.8 Hz, 12H). HRMS (ESI,m / z): [M+H] + calcd. For C 59 H 121 N6O7, 1025.92913; found 1025.92821.

[0437]

[0438] The synthesis method for L0925 is similar to that for L0923. L0925: 1H NMR (400 MHz, Chloroform-d) δ6.52 - 6.10 (m, 3H), 3.83 - 3.65 (m, 3H), 3.64 - 3.55 (m, 1H), 3.52 - 3.35(m, 3H), 3.11 (dp, J = 12.8, 6.4 Hz, 3H), 2.91 - 1.97 (m, 25H), 1.62 (p, J =7.2 Hz, 6H), 1.49 - 1.15 (m, 66H), 0.87 (t, J = 6.8 Hz, 12H). HRMS (ESI, m / z): [M+H] + calcd. For C 62 H 127 N6O7, 1067.97608; found 1067.97657.

[0439]

[0440] The synthesis method for L0926 is similar to that for L0923. L0926: 1 H NMR (400 MHz, Chloroform-d) δ6.54 - 6.18 (m, 3H), 3.85 - 3.65 (m, 3H), 3.63 - 3.54 (m, 1H), 3.53 - 3.35(m, 3H), 3.11 (dp, J = 12.4, 6.0 Hz, 3H), 2.90 - 2.08 (m, 25H), 1.62 (p, J =7.2 Hz, 6H), 1.50 - 1.15 (m, 72H), 0.87 (t, J = 6.8 Hz, 12H). HRMS (ESI, m / z): [M+Na] + calcd. For C 65 H 132 NaN6O7, 1132.00552; found 1132.00506.

[0441]

[0442] Synthesis of L0927. Carboxylic acid 3M1BE5C was synthesized using a scheme similar to that in step 3. 3M1BE5CTBDPS: HRMS(ESI, m / z): [M+NH4] + calcd. For C 28 H 48NO3Si, 474.33980; found 474.33935; HRMS(ESI, m / z): [M+Na] + calcd. For C 28 H 44 NaO3Si, 479.29519; found 479.29498;3M1BE5C-OH: HRMS (ESI, m / z): [M+Na] + calcd. For C 12 H 26 NaO3, 241.17741; found241.17659; 3M1BE5C: HRMS (ESI, m / z): [M+Na] + calcd. For C 12 H 24 NaO4, 255.15668; found 255.15689. 3M1BE5C-NHS was synthesized using a similar scheme to step 4. 3M1BE5C-NHS: HRMS (ESI, m / z): [M+Na] + calcd. For C 16 H 27 NNaO6, 352.17306; found 352.17320. L0927 was synthesized using a similar scheme to step 7. L0927: 1 H NMR (400 MHz, Chloroform-d) δ 6.42 (t, J = 6.0Hz, 4H), 4.62 (q, J = 5.2 Hz, 4H), 3.82 - 3.68 (m, 4H), 3.57 (dq, J = 9.2,6.8 Hz, 8H), 3.39 (qd, J = 6.8, 3.6 Hz, 12H), 3.17 - 3.02 (m, 4H), 2.86 -2.77 (m, 2H), 2.70 - 2.61 (m, 2H), 2.50 - 2.40 (m, 9H), 2.36 - 2.12 (m, 14H),1.76 - 1.62 (m, 12H), 1.62 - 1.52 (m, 8H), 1.43 (q, J = 6.8 Hz, 8H), 1.26 (d,J = 5.2 Hz, 12H), 0.88 (d, J = 6.8 Hz, 24H). HRMS (ESI, m / z): [M+H] +calcd.For C 65 H 132 N7O 16 , 1266.97251; found 1266.97192.

[0443]

[0444] The synthesis method for L0928 is similar to that for L0927. L0928: 1 H NMR (400 MHz, Chloroform-d) δ6.61 (t, J = 6.0 Hz, 4H), 4.63 (q, J = 5.2 Hz, 4H), 3.83 - 3.71 (m, 4H), 3.64- 3.53 (m, 8H), 3.51 - 3.34 (m, 12H), 3.09 (dt, J = 13.2, 6.0 Hz, 4H), 2.67 -2.50 (m, 6H), 2.49 - 2.40 (m, 6H), 2.35 (dd, J = 13.2, 3.2 Hz, 4H), 2.23 (t,J = 7.6 Hz, 11H), 1.79 - 1.51 (m, 24H), 1.44 (q, J = 6.8 Hz, 8H), 1.27 (d, J= 5.2 Hz, 12H), 0.89 (d, J = 6.8 Hz, 24H). HRMS (ESI, m / z): [M+H] + calcd. ForC 67 H 136 N7O 16 , 1295.00381; found 1295.00250.

[0445]

[0446] The synthesis method for L0929 is similar to that for L0927. L0929: 1H NMR (400 MHz, Chloroform-d) δ6.74 (s, 5H), 4.64 (q, J = 5.2 Hz, 5H), 3.90 - 3.65 (m, 5H), 3.58 (dq, J =9.2, 6.8 Hz, 10H), 3.52 - 3.34 (m, 15H), 3.17 - 3.03 (m, 5H), 2.72 - 2.16 (m,28H), 1.76 - 1.64 (m, 16H), 1.64 - 1.54 (m, 12H), 1.44 (q, J = 6.8 Hz, 10H), 1.28 (d, J = 5.2 Hz, 15H), 0.89 (d, J = 6.8 Hz, 30H). HRMS (ESI, m / z): [M+H] + calcd. For C 81 H 163 N8O 20 , 1568.19781; found 1568.19702.

[0447]

[0448] Synthesis of L0930: MeI was added to 1.5 mL of MeCN solution containing L0857. The mixture was then stirred continuously at 70 °C for 1 day. After the reaction was completed, the solvent was removed under reduced pressure. The residue was purified by a CombiFlash system (DCM / Ultra = 2:1, Rf ≈ 0.5) to give a yellow oily product L0930 (6 mg, yield 10.7%). 1 H NMR (400MHz, Chloroform-d) δ 7.48 - 6.83 (m, 4H), 4.22 - 3.62 (m, 6H), 3.62 - 3.28(m, 6H), 3.27 - 3.01 (m, 8H), 3.00 - 2.37 (m, 14H), 2.33 - 2.11 (m, 8H), 1.69- 1.50 (m, 8H), 1.45 - 1.16 (m, 64H), 0.87 (t, J = 6.8 Hz, 12H). HRMS (ESI,m / z): [MI] + calcd. For C 68 H 138 N7O8, 1181.06014; found: 1181.06378.

[0449]

[0450] Synthesis of L0931: MeI was added to 1.5 mL of MeCN solution containing L0866. The mixture was then stirred continuously at 70 °C for 1 day. After the reaction was completed, the solvent was removed under reduced pressure. The residue was purified by a CombiFlash system (DCM / Ultra = 2:1, Rf ≈ 0.5) to give a yellow oily product L0931 (10 mg, yield 17.9%). 1 H NMR (400MHz, Chloroform-d) δ 7.08 - 6.82 (m, 2H), 5.55 - 5.28 (m, 2H), 4.35 - 3.54(m, 6H), 3.55 - 3.00 (m, 11H), 2.98 - 1.72 (m, 26H), 1.70 - 1.53 (m, 8H),1.52 - 0.97 (m, 75H), 0.87 (t, J = 6.8 Hz, 12H). HRMS (ESI, m / z): [MI] + calcd. For C 72 H 146 N7O8, 1237.12274; found: 1237.12486.

[0451]

[0452] The synthesis method of L0932 is similar to that of L0902. L0932 1 H NMR (400 MHz, Chloroform-d) δ6.52 - 6.13 (m, 4H), 3.84 - 3.60 (m, 4H), 3.48 - 3.34 (m, 4H), 3.28 - 3.08(m, 4H), 2.99 - 2.11 (m, 19H), 2.11 - 1.95 (m, 4H), 1.65 - 1.49 (m, 8H), 1.49- 1.12 (m, 88H), 0.85 (t, J = 6.8 Hz, 24H). HRMS (ESI, m / z): [M+H] + calcd.For C 81 H 164 N7O8, 1363.26359; found: 1363.26372.

[0453] The compounds in Table 1A below were prepared using the corresponding starting materials according to the procedures and schemes described above. The characterization data of the relevant compounds are shown below.

[0454] Table 1B. Yield and characterization data of compounds

[0455]

[0456] 2. Preparation of nucleic acid drug lipid nanoparticles

[0457] 2.1. Lipid solubility

[0458] This lipid nanoparticle formulation contains four lipid components: cationic lipids (such as compound L0853), phospholipids (such as 1,2-distearatel-sn-glycerol-3-phosphocholine (DSPC), 1,2-palmitoyl-sn-glycerol-3-phosphocholine (DPPC), phosphatidylcholine (PC), 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), and cholesterol (Ch)). ol (Sigma-Aldrich (Shanghai) Trading Co., Ltd.), PEGylated lipids (such as DMG-PEG2000 (Beijing Jiankai Technology Co., Ltd.), ALC-0159 (Beijing Jiankai Technology Co., Ltd.), mPEG-DMPE (Aladdin Reagent (Shanghai) Co., Ltd.), mPEG-DSPE (Aladdin Reagent (Shanghai) Co., Ltd.), mPEG-STA (Aladdin Reagent (Shanghai) Co., Ltd.), mPEG-DPPE (Aladdin Reagent (Shanghai) Co., Ltd.), mPEG-PS (Aladdin Reagent (Shanghai) Co., Ltd.)).

[0459] Taking the lipid nanoparticle (LNP) encapsulation formulation of compound L0853 as an example, four lipids were dissolved in the organic solvent ethanol (Aladdin Reagent (Shanghai) Co., Ltd.), and then ultrasonically dispersed until the solution was clear and transparent, with no obvious insoluble or flocculent matter. Filtration was performed using a 0.22 μm filter if necessary. Four lipid stock solutions were prepared and obtained: 5 mg / mL mPEG5000-DSPE, 5 mg / mL DSPC, 10 mg / mL Chol, and 10 mg / mL compound L0853.

[0460] 2.2. Preparation of working solution for nucleic acid drugs

[0461] Taking the preparation method of compound L0853 as an example, the operation was carried out in a biosafety cabinet. 23.59 μL of 1 mg / mL FLuc-mRNA nucleic acid drug stock solution (Shanghai Zhaowei Technology Development Co., Ltd.) was taken out and transferred to a nuclease-free plastic centrifuge tube. 566.16 μL of 50 mM citrate-sodium citrate buffer (pH=4) was added and the solution was gently mixed by pipetting to obtain a 0.04 mg / mL FLuc-mRNA nucleic acid drug working solution. Then, all the solution was aspirated and transferred to a KDL syringe with a needle (2 mL) for later use.

[0462] 2.3. Preparation of mixed lipid working solution

[0463] Taking the preparation of compound L0853 as an example, in a clean formulation room, 23.45 μL of mPEG5000-DSPE stock solution (5 mg / mL), 29.50 μL of DSPC stock solution (5 mg / mL), 25.21 μL of Chol stock solution (10 mg / mL), and 85.62 μL of L0853 stock solution (10 mg / mL) were respectively added to a nuclease-free plastic centrifuge tube, followed by the addition of 59.48 μL of ethanol. The mixed lipid solution was then gently pipetted to obtain the mixed lipid working solution. In this working solution, the concentration of lipid L0853 is approximately 3.64 mM, the total lipid concentration is approximately 7.50 mM, and the molar ratio of the four lipids is mPEG5000-DSPE:DSPC:Chol:L0853 = 1.40:11.15:38.95:48.50. The N / P ratio of ionizable lipids to nucleic acids is 30:1. All the mixed lipid working solution was aspirated and transferred to a 1 mL KDL syringe with a needle for later use.

[0464] 2.4. Preparation of Nucleic Acid Lipid Nanoparticles

[0465] Taking the formulation of compound L0853 as an example, syringes containing nucleic acid drug working solution and mixed lipid working solution were placed in different bayonets of an aqueous phase syringe pump and an organic phase syringe pump (Baoding Lange Constant Flow Pump Co., Ltd., dLSP-520). The two ends of an enzyme-free Y-type mixer (T-connector) were connected to the syringe ports containing the nucleic acid drug working solution and the mixed lipid working solution, respectively. The perfusion volume of the aqueous phase syringe pump was set to 549.75 μL, the flow rate to 4.5 mL / min, and the syringe specification to be a 2 mL Kangdelai syringe. The perfusion volume of the organic phase syringe pump was set to 183.25 μL, the flow rate to 1.5 mL / min, and the syringe specification to be a 1 mL Kangdelai syringe. After confirming that the synthesis device is installed and the parameters are set correctly, press the start button of the injection pump to control the nucleic acid drug working solution and the mixed lipid working solution to be introduced into the Y-type mixer at a flow rate ratio of 3:1 and at a certain angle to achieve mixing, and instantly generate liposome nanoparticles encapsulating nucleic acid drugs. Use a nuclease-free plastic centrifuge tube to collect the liposome nanoparticle mixture solution at the end of the Y-type mixer, observe the appearance of the formulation and record it accurately.

[0466] 2.5. Removal of Organic Solvents

[0467] The lipid nanoparticle mixture prepared by Y-type mixer contains approximately 25% (v / v) ethanol, an organic solvent. Accurately pipette 648 μL of the lipid nanoparticle mixture obtained in step 4 and gently transfer it to a clean, UV-treated, heart-shaped ground glass flask (10 mL). Remove the ethanol by rotary evaporation using a rotary evaporator (Jinan Oulaibo Scientific Instruments Co., Ltd., RE-2000E) under vacuum at room temperature. During rotary evaporation, closely observe for phenomena such as boiling, precipitation, and changes in the appearance of the formulation, and accurately record the phenomena and the start and end times of the rotary evaporation. Note: The total flow rate of the aqueous and ethanol phases ranges from 4 mL / min to 20 mL / min.

[0468] 2.6. Static incubation of lipid nanoparticles

[0469] Taking the above formulation of compound L0853 as an example, after rotary evaporation in step 5, the lipid nanoparticle solution in the flask is gently transferred to a nuclease-free plastic centrifuge tube and allowed to stand for about 15 minutes. The lipid nanoparticle solution is then brought to a final volume of 486 μL using a pipette tip. The solvent used for volume adjustment is nuclease-free pure water (Millipore Corporation, Milli-Q Direct8 purification system). This yields a lipid nanoparticle formulation encapsulating nucleic acid drugs.

[0470] The obtained nucleic acid-lipid nanoparticle formulation (excluding losses) contained approximately 0.04 mg / mL FLuc-mRNA, approximately 1.28 mg / mL of compound L0853, and a total lipid concentration of approximately 2.06 mg / mL. 470 μL of the obtained lipid-nucleic acid nanoparticle formulation was used to verify the transfection effect in small animals; 8.5 μL of the obtained lipid-nucleic acid nanoparticle formulation was used to detect the nanoparticle size and dispersion index (PDI); and 7.5 μL of the obtained lipid-nucleic acid nanoparticle formulation was used for gel electrophoresis analysis of the degree of nucleic acid encapsulation.

[0471] 2.7. Displacement solvent for dialysis or ultrafiltration

[0472] The nucleic acid lipid nanoparticle solution prepared in step 4 or 5 can be further purified by dialysis to remove any residual ethanol and replace the solvent with another solution, such as an isotonic phosphate buffer. In a clean formulation room, the nucleic acid lipid nanoparticle solution is placed into a dialysis membrane with a molecular weight cutoff of 8 kD and dialyzed against an isotonic phosphate buffer for 3 to 4 hours at room temperature. The volume ratio of the dialysis solution to the nucleic acid lipid nanoparticle solution should be at least greater than 500:1. After dialysis, the lipid nanoparticle solution in the dialysis membrane is gently transferred to a nuclease-free plastic centrifuge tube for later use.

[0473] The nucleic acid lipid nanoparticle solution prepared in step 4 or 5 can be subjected to ultrafiltration to further remove any residual ethanol and replace the solvent with another solution, such as an isotonic phosphate buffer solution. In a clean preparation room, the nucleic acid lipid nanoparticle solution is diluted with the replacement solvent and slowly mixed, then allowed to stand for 15-30 minutes to ensure thorough mixing and equilibration. The solution is then transferred to a 50 mL ultrafiltration tube (pore size 10-100 kDa) and concentrated by centrifugation at 4000 g for 5-30 minutes at room temperature. After centrifugation, the lipid nanoparticle solution is gently transferred to nuclease-free plastic centrifuge tubes for later use.

[0474] 2.8. Add auxiliary materials and / or cryoprotectants

[0475] Excipients and / or cryoprotectants, such as sucrose, can be added to the nucleic acid lipid nanoparticle formulation obtained in step 6 or 7. Taking sucrose (Aladdin Reagent (Shanghai) Co., Ltd.) as an example, 40% sucrose solution is added to the nucleic acid lipid nanoparticle formulation at a volume ratio of 3:1. After thorough mixing, a nucleic acid lipid nanoparticle formulation containing 10% sucrose is obtained. This is then further aliquoted into nuclease-free centrifuge tubes and stored in ice boxes at 4°C, -20°C, and / or -80°C for later use. After the specified storage time, the lipid nanoparticles are thawed and brought to room temperature for physicochemical property testing and in vivo efficacy verification.

[0476] 2.9. Concentration of Nucleic Acid Lipid Nanoparticle Solution

[0477] The nucleic acid lipid nanoparticle solution prepared in steps 6, 7, and 8 can be concentrated by rotary evaporation. Specifically, in a clean formulation room, the nucleic acid lipid nanoparticle solution is gently transferred to a clean, UV-treated, heart-shaped ground glass flask. Under room temperature or heating (not exceeding 40°C), vacuum rotary evaporation is performed to remove water from the solution and concentrate the formulation. The rotary evaporation concentration time varies depending on the formulation and can range from 1 minute to 2 hours.

[0478] 3. Gel electrophoresis analysis of nucleic acid encapsulation degree

[0479] 3.1. Gel Preparation: First, prepare a 1% agarose solution. Weigh 0.8 g of agarose powder (BioFroxx) into an Erlenmeyer flask, add 80 mL of TAE buffer, shake well, and heat in a microwave oven until the agarose powder is completely dissolved. After heating, allow the hot solution to stand at room temperature, and then cool slowly.

[0480] 3.2. Secure the clean glue-making mold into the glue-making slot.

[0481] 3.3. When the agarose solution cools to 40°C to 50°C, add 8 μL of SYBR Safe dye (Thermo Fisher Scientific), shake well, and then pour the agarose solution into the molding die in one go, taking care to avoid air bubbles. Then, fix a clean molding comb onto the molding die.

[0482] 3.4. Let the gel stand at room temperature for 30 to 60 minutes, gently pull out the gel casting comb, transfer the agarose gel to the electrophoresis tank, and add TAE buffer.

[0483] 3.5. Add 5× loading buffer to the gel sample, mix thoroughly, and then add it to the gel well located at the negative electrode.

[0484] 3.6. After loading the sample, tighten the electrophoresis tank lid and connect the electrophoresis apparatus (Bio-Ray Biomedical Products (Shanghai) Co., Ltd., PowerPac). TM (Basic), set the parameters to 80 V, 30 min, and press the start button.

[0485] 3.7. The agarose gel electrophoresis results were imaged and analyzed using a gel imaging system (Shanghai Tianneng Life Science Co., Ltd., Tanon 1600).

[0486] 4. Nucleic acid encapsulation efficiency detected by fluorescence method

[0487] Using fluorescence methods (such as those using Thermo Fisher Scientific's Quant-iT) TMThe RiboGreen RNA Quantitative Reagent Kit can determine the encapsulation efficiency of the nucleic acid lipid nanoparticle formulations prepared in steps 2.6, 2.7, and 2.8. Taking the RiboGreen assay kit as an example, the specific operation is as follows: Prepare the low-range detection working solution and mRNA standard solution according to the instructions. Take 3 μL of the nucleic acid lipid nanoparticle solutions prepared in steps 2.6, 2.7, and 2.8, add 297 μL of the kit's accompanying Tris-EDTA buffer (TE buffer), mix thoroughly, and then pipette 100 μL into a black-background 96-well plate. Add 100 μL of the RiboGreen low-range detection working solution. Take 100 μL of mRNA standard solutions of different concentrations into a black-background 96-well plate, and then add 100 μL of the RiboGreen low-range detection working solution. Gently pipette to mix, incubate in the dark for 5 minutes, and immediately use a multi-functional microplate reader (BMG LABTECH, FLUOstarOmega) to detect and record the fluorescence signal intensity. By fitting a standard curve and performing calculations, the content of free mRNA not encapsulated by lipid nanoparticles was calculated, and then divided by the total amount of mRNA detected in the wells to obtain the nucleic acid encapsulation rate of the nucleic acid lipid nanoparticle formulation.

[0488] 5. Detection of particle size, potential, osmotic pressure, and electron microscopy imaging of lipid nanoparticle formulations.

[0489] 5.1. Particle size characterization of nucleic acid lipid nanoparticle formulations

[0490] Take 8.5 μL of nucleic acid lipid nanoparticle solution (containing approximately 0.04 mg / mL mRNA), add 200 μL of ultrapure water, gently mix with a pipette tip, and then use a Malvern dynamic light scattering instrument (Malvern Instruments Ltd., ZEN3700, UK) to detect the particle size of the nanoparticle formulation by dynamic light scattering (DLS).

[0491] 5.2. Potential characterization of nucleic acid lipid nanoparticle formulations

[0492] Take 20 μL of nucleic acid lipid nanoparticle solution (containing approximately 0.04 mg / mL mRNA), add 780 μL of ultrapure water, gently pipette and mix, and then use a Malvern dynamic light scattering instrument (model ZEN3700) to detect the potential of the nanoparticle formulation.

[0493] 5.3. Osmotic Pressure Characterization of Nucleic Acid Lipid Nanoparticle Formulation

[0494] Take 50 μL of nucleic acid lipid nanoparticle solution (containing approximately 0.04 mg / mL mRNA) and measure the osmotic pressure of the nanoformulation using a freezing point osmoremeter (Gonotec GmbH, OSMOMAT 3000-D).

[0495] 5.4. Simultaneous Characterization of Particle Size and Potential in Nucleic Acid Lipid Nanoparticle Formulations

[0496] Take 27.5 μL of nucleic acid lipid nanoparticle solution (containing approximately 0.04 mg / mL mRNA) and add 200 μL of ultrapure water. Gently pipette the sample and mix thoroughly. Detect the particle size of the nanoparticles using a Malvern dynamic light scattering (DLS) instrument (Malvern Instruments Ltd., ZEN3700). After detection, transfer the sample to an EP tube, add 600 μL of ultrapure water, gently mix with a pipette tip, and then measure the potential of the nanoparticles using the Malvern DLS instrument.

[0497] 5.5. Electron Microscopy Sample Preparation and Characterization

[0498] Place the carbon grid face up in the center of the nuclease-free EP tube cap. Use a pipette to add 10 μL of the prepared TEM sample (nanoparticle formulation) onto the carbon grid. Allow it to stand at room temperature until the water in the sample evaporates and the carbon grid is semi-dry (ideally with only a thin water film on the grid). Then, use a pipette to add 10 μL of nuclease-free water to the carbon grid surface. After standing for 10 seconds, remove the droplets from the carbon grid surface with filter paper. Repeat the washing process twice with nuclease-free water. Add 10 μL of negative staining solution to the carbon grid surface and stain for 30 seconds. Then, remove the negative staining solution with filter paper and wash the sample twice with nuclease-free water. Allow the sample to stand and air dry until all water on the carbon grid surface has evaporated. The sample preparation is now complete. Image the sample using a lanthanum hexaboride transmission electron microscope (FEI Corporation, FEITecnai G2 12, 120 kV).

[0499] 6. Investigation of the transfection effect and cytotoxicity of nucleic acid lipid nanoparticle formulations in cells.

[0500] 6.1. Cell Seeding Plate

[0501] A549 cells were digested and collected one day prior to cell administration, and the cell concentration was adjusted to 2 × 10⁻⁶. 5 Cells / mL were added to a 96-well plate, with 200 μL of cell culture added to each well, resulting in a cell concentration of 2 × 10⁶ cells / mL. 5 The cell culture solution was prepared at a concentration of 10 cells / mL, resulting in approximately 4 × 10⁶ cells per well in the 96-well plate. 4 Count 1. DC2.4 cells were then digested and collected, and the cell concentration was adjusted to 1 × 10⁻⁶. 5Cells / mL were added to a 96-well plate, with 200 μL of cell culture added to each well, resulting in a cell concentration of 1×10⁶ cells / mL. 5 The cell culture solution was prepared at a concentration of 10 cells / mL, resulting in approximately 2 × 10⁶ cells per well in the 96-well plate. 4 Cells were placed in a cell culture incubator (Singapore Escotech Technology Co., Ltd., CLM-170B-8-NF) and incubated at 37°C with 5% CO2 for 24 hours.

[0502] 6.2. Cell-based drug delivery

[0503] On the day of cell administration, the culture medium (Thermo Fisher Scientific) in the 96-well plate was aspirated, and 200 μL of PBS was added to each well to rinse the cells. After washing the cells with PBS, the PBS (Thermo Fisher Scientific) was aspirated. 180 μL of Opti-MEM (Thermo Fisher Scientific) was added to each well of the drug administration group.

[0504] Different volumes of the formulation were diluted with the buffer solution used for the formulation or the buffer solution used for dialysis. Four dosage concentrations were prepared for each formulation. 20 μL of the diluted nano-formulation was added to each well of a 96-well plate (three replicates for each concentration). The dosage concentrations of the formulation were approximately 50 ng / well, 100 ng / well, 200 ng / well, and 300 ng / well containing mRNA.

[0505] Place the 96-well cell culture plate in a cell culture incubator and incubate at 37°C and 5% CO2 for 4 hours. Remove the culture medium, add 200 μL of DMEM complete culture medium to each well, and continue incubation for 20 hours for detection.

[0506] 6.3. Cell transfection detection

[0507] The D-Luciferin (Shanghai Qifa Experimental Reagent Co., Ltd.) stock solution was diluted 99:1 with DMEM complete medium (9900 μL complete medium + 100 μL 25 mg / mL D-Luciferin stock solution) to make the working solution concentration 250 μg / mL (25 μg per well).

[0508] Remove the 96-well cell culture plate and aspirate the supernatant. Before imaging, add 100 μL of D-Luciferin working solution to the 96-well plate, incubate at 37°C for 5 min, and then perform imaging analysis using a microplate reader.

[0509] 6.4. Cytotoxicity Detection

[0510] The CCK-8 working solution was prepared by diluting the CCK-8 test reagent stock solution (Shanghai Beyotime Biotechnology Co., Ltd.) at a ratio of 9:1 using DMEM complete culture medium (900 μL complete culture medium + 100 μL CCK-8 test reagent).

[0511] Remove the 96-well cell culture plate and aspirate the supernatant. Add 100 μL of CCK-8 working solution to each well before assay. Incubate at 37°C with 5% CO2 for 2 hours, then analyze using a microplate reader. Calculate cell viability to reflect cytotoxicity.

[0512] 7. The silencing effect of nucleic acid lipid nanoparticles on cellular siRNA delivery

[0513] 7.1 Cell Seeding

[0514] One day prior to cell drug administration, a suspension of HeLa-EGFP cells (a clonal cell line stably expressing EGFP fluorescent protein) in logarithmic growth phase was collected at a density of 1 × 10⁻⁶ cells per well. 5 400 μL of cells were dispensed into each well of a 24-well plate. The plate was then incubated at 37°C in a 5% CO2 cell culture incubator (Cells Technology Ltd., Singapore, CLM-170B-8-NF) for 24 hours.

[0515] 7.2 Cell drug delivery

[0516] On the day of cell administration, the culture medium in the wells of the culture plate was removed first, and the cells were rinsed with 200 μL of PBS solution (Thermo Fisher Scientific) in each well, followed by aspiration of the PBS. 400 μL of opti-MEM medium (Thermo Fisher Scientific) was added to each well of the administration group, and the nucleic acid lipid nanoparticle formulation was diluted with opti-MEM to a final volume of 300 μL, with a concentration of 1.6 ng / μL. 100 μL of nucleic acid lipid nanoparticle formulation containing 160 ng of EGFP-siRNA (using EGFP-siRNA as the model siRNA) was added to each well, with three replicates per sample. Four hours after administration, the medium was replaced with complete growth medium, and incubation continued for 18–24 hours.

[0517] 7.3 Detection of Cell Silencing Effect

[0518] Cells were digested and collected. Fluorescence intensity of live cells in the FITC channel of each well was measured using a flow cytometer (Beckman Coulter, CytoFLEX FlowCytometer), and the geometric mean fluorescence intensity of EGFP-positive cells in replicates was calculated. The siRNA silencing efficiency (i.e., the percentage reduction in average cell fluorescence intensity) was calculated using the following formula:

[0519] siRNA silencing efficiency (%) = (Geometric mean fluorescence intensity of cells without nucleic acid nanoparticle complex - Geometric mean fluorescence intensity of cells with nucleic acid nanoparticle complex) / Geometric mean fluorescence intensity of cells without nucleic acid nanoparticle complex × 100%

[0520] 8. Delivery of nucleic acid lipid nanoparticle formulations in mice

[0521] 8.1 Study on the delivery effect of FLuc-mRNA loaded on lipid nanoparticles in mice

[0522] After formulation was completed, the drug was administered via intramuscular injection (rectus femoris muscle of the mouse thigh), intravenous injection (tail vein of the mouse), intraperitoneal injection, or subcutaneous injection (subcutaneous injection in the back). Each mouse received approximately 75 μL of the drug, for a total of 3 mice. In vivo imaging (PerkinElmer, IVIS Lumina III) was performed a certain time after injection. 10-15 minutes before imaging, 200 μL of 15 mg / mL D-Luciferin working solution was injected intraperitoneally, and imaging analysis was performed using an in vivo imaging system.

[0523] 8.2 Study on the delivery effect of FLuc-circ-mRNA loaded on lipid nanoparticles in mice

[0524] Lipid nanoparticles loaded with nucleic acid drugs were prepared according to step 2, replacing FLuc-mRNA with FLuc-circ-RNA (Shanghai Zhaowei Technology Development Co., Ltd.). After formulation, the drugs were administered via intramuscular injection (rectus femoris muscle of mouse thigh), intravenous injection (tail vein of mouse), intraperitoneal injection, or subcutaneous injection (subcutaneous injection of the back), with approximately 75 μL of formulation injected into each mouse, for a total of 3 mice. In vivo imaging (PerkinElmer, IVIS Lumina III) was performed a certain time after injection. 10-15 minutes before imaging, 200 μL of 15 mg / mL D-Luciferin working solution was injected intraperitoneally, and imaging analysis was performed using an in vivo imaging system.

[0525] 8.3 Study on the co-delivery effect of nucleic acid lipid nanoparticles loaded with FLuc-mRNA and Cy5-siRNA in mice.

[0526] Lipid nanoparticles loaded with nucleic acid drugs were prepared according to step 2, replacing FLuc-mRNA with a mixture of FLuc-mRNA (Shanghai Zhaowei Technology Development Co., Ltd.) and Cy5-siRNA. Each mouse received a dose of 3 μg FLuc-mRNA and 3 μg Cy5-siRNA. After a certain period of time, imaging was performed using an in vivo imaging system (PerkinElmer, IVIS Lumina III). To detect FLuc-mRNA expression, 200 μL of 15 mg / mL D-Luciferin working solution was injected intraperitoneally 10–15 minutes before imaging, followed by in vivo imaging analysis.

[0527] 9. Study on the delivery effect of lipid nanoparticle-loaded compounds in mice

[0528] IR780 powder was weighed and dissolved in the organic solvent DMSO to prepare a 2 mg / mL IR780 (DMSO) stock solution. DMG-PEG2000, DSPC, cholesterol, ionizable lipids, and the IR780 stock solution were added according to the molar ratio of each material, and ethanol was added to make the volume ratio of organic phase to aqueous phase 1:3. Lipid nanoparticles loaded with compound IR780 were prepared according to step 2, except that the nucleic acid drug working solution was replaced with a buffer solution. A free IR780 solution was used as a control group.

[0529] 10. Toxicity study of nucleic acid lipid nanoparticle formulation in mice.

[0530] Three hours after administration of the formulation, whole blood was collected from the outer canthus of mice. A portion of the whole blood was mixed with an anticoagulant for routine blood count (CBC) analysis, while another portion was centrifuged at 4°C and 5000 rcf for 5 minutes. The supernatant was transferred to a clean 1.5 mL centrifuge tube for blood biochemistry analysis. Samples were stored at room temperature and, within one hour, CBC and blood biochemistry parameters were analyzed using an automated animal hematology analyzer (Shenzhen Mindray Animal Medical Technology Co., Ltd., BC-2800 Vet) and an automated animal biochemistry analysis system (Shenzhen Mindray Animal Medical Technology Co., Ltd., BS-240 Vet).

[0531] 11. Study on the immune effects of nucleic acid lipid nanoparticle formulations in mice.

[0532] 11.1. In vivo immune experiments

[0533] Lipid nanoparticles encapsulating nucleic acid drugs were prepared according to step 2. The mRNA was replaced with Delta Spike full-length mRNA (Shanghai Zhaowei Technology Development Co., Ltd.). After formulation, the drugs were administered to mice at a dose of 5 μg mRNA per mouse. On day 14 after the first immunization, whole blood was collected from mice via the lateral canthus sampling method, and the drug was repeated for a second immunization. On day 14 after the second immunization, whole blood was again collected via the lateral canthus sampling method. The whole blood was centrifuged at 5000 rcf for 5 min, and the supernatant was collected. The supernatant was then centrifuged again at 10000 rcf for 5 min to obtain serum, which was aliquoted into tubular tubes and stored at -20℃.

[0534] 11.2. Detect the level of specific antibodies using ELISA or ELISA titer assay.

[0535] 11.2.1 Coating: Add 100 μL of 0.2 μg / 100 μL Delta-S protein (Suzhou Nearshore Protein Technology Co., Ltd.) dilution to each well and incubate overnight at 4 ℃.

[0536] 11.2.2 Washing: Add 200 μL of PBST (PBS buffer solution containing 0.05% Tween-20 at pH 7.4) to each well and wash three times.

[0537] 11.2.3 Blocking: Add 200 μL of 5% (w / v) BSA-PBS solution (BSA bovine serum albumin, Beijing Bio-Sens Biotechnology Co., Ltd.) to each well and shake at 50 rcf for 2 h at room temperature.

[0538] 11.2.4 Washing: Add 200 μL of PBST to each well and wash once.

[0539] 11.2.5 Sample Addition: Add 100 μL of serum diluted 200-fold with PBS to each well (i.e., containing 0.5 μL of stock serum). (For ELISA titers, the serum should be diluted 1×10⁻⁶ times.) 4 times, 1×10 5 times, 1×10 6 times, 1×10 7 (Times), shaken on a shaker at 50 rcf for 2 hours at room temperature.

[0540] 11.2.6 Washing: Add 200 μL of PBST to each well and wash three times.

[0541] 11.2.7 Add detection antibody: Add 100 μL of HRP-labeled goat anti-mouse IgG detection antibody diluted 1000 times (i.e., containing 0.1 μL of antibody stock solution) (Sangon Biotech (Shanghai) Co., Ltd.) to each well, and shake on a shaker at 50 rcf for 1 h at room temperature.

[0542] 11.2.8 Washing: Add 200 μL of PBST to each well and wash three times.

[0543] 11.2.9 Add TMB (Beijing Solarbio Biotechnology Co., Ltd.): Add 50 μL of TMB mixture with a volume ratio of 1:1 (A:B) to each well. React for 5 min (first blood ELISA), 7 min 58 sec (second blood ELISA), and react for about 5 min (second blood ELISA titer).

[0544] 11.2.10 Termination with phosphoric acid (Aladdin Reagent (Shanghai) Co., Ltd.): Add 50 μL of 1 mol / L phosphoric acid solution to each well to terminate the reaction.

[0545] 11.2.11 Detection: Detection was performed using a multi-functional microplate reader.

[0546] 11.3. Detection of neutralizing antibody levels using a pseudovirus neutralization experiment.

[0547] 11.3.1 Cell seeding: 293T-ACE2 cells (Fubai Ao (Suzhou) Biomedical Technology Co., Ltd.) were seeded in 300 μL DMEM medium (containing 10% heat-inactivated FBS and no antibiotics) containing 1×10⁻⁶ cells. 5 A concentration of 1 × 10⁶ cells per well was used to seed the cells in a 48-well plate. 5 After labeling each cell with information such as "cell number, cell type-generation, culture medium type, date, and name" on a 48-well plate, the cells were placed in a cell culture incubator at 37°C and 5% CO2 for 18 hours.

[0548] 11.3.2 Before performing the pseudovirus transfection operation, prepare an ice box, place the serum to be tested on ice to rehydrate, take the virus out of the -25 ℃ freezer and place it on ice to thaw, after thawing, mix the serum and pseudovirus evenly, and centrifuge with a handheld centrifuge.

[0549] 11.3.3 Examination of neutralizing antibodies in serum at different serial dilutions: Pseudovirus (Fubai Ao (Suzhou) Biomedical Technology Co., Ltd.) and mouse serum were diluted separately using complete culture medium and mixed thoroughly. The mixture was then diluted 40-fold, 100-fold, 250-fold, and 625-fold according to the final concentration required for cell incubation. 150 μl of the pseudovirus-serum mixture was added to each well. Five wells containing only the pseudovirus dilution were set up as positive controls.

[0550] 11.3.4 After removing the 48-well cell culture plate from the CO2 incubator, aspirate the culture medium with a waste pump. Mix the serum and pseudovirus for about 20 minutes. Add 150 μl of the mixture to each well and record the corresponding position of each group. Set up 5 wells with only 150 μl of culture medium as negative controls.

[0551] 11.3.5 After disinfecting with alcohol spray, place the 48-well cell culture plate in an incubator and incubate for 18-24 hours.

[0552] 11.3.6 18-24 h after pseudovirus transfection, the 48-well plate was removed from the CO2 incubator and the transfection results were observed under a fluorescence inverted microscope (Carl Zeiss, Primovert, Germany). Cells from each well were collected into 1.5 mL centrifuge tubes and centrifuged at 300 G for 5 min using a refrigerated centrifuge (Eppendorf, 5910R, Germany). The supernatant was aspirated with a waste pump, and 100-200 μL of PBS was added to each 1.5 mL centrifuge tube to resuspend the cells. The cells were placed on ice and detected using a flow cytometer (Beckman Coulter, CytoFLEX Flow Cytometer).

[0553] result

[0554] Various lipid nanoparticle formulations were prepared and tested using some of the compounds listed in Table 1A. Each formulation contained PEG lipids (such as mPEG5000-DSPE and DMG-PEG2000), phospholipids (such as DSPC and PC), structural lipids (such as cholesterol), the ionizable lipid compounds described in this invention, and mRNA. The test results are detailed in Table 2 below.

[0555]

[0556]

[0557]

[0558]

[0559]

[0560]

[0561]

[0562]

[0563]

[0564]

[0565]

[0566] As shown in Table 2, the tested formulations all exhibited small particle size and narrow particle size distribution (PDI), and these compounds demonstrated excellent in vivo delivery efficiency.

[0567] Unless otherwise stated, 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 pertains.

[0568] The exemplary content described herein is applicable even if no specific elements or limitations are disclosed. For example, terms such as "comprising," "including," and "containing" should be interpreted broadly and without limitation. Furthermore, the terms and expressions used herein are descriptive only and not restrictive; their use is not intended to exclude equivalents of the features or portions thereof shown. However, it should be noted that various modifications within the scope of the claims are possible.

[0569] Therefore, it should be understood that although the present invention has been specifically described through specific embodiments and optional features, those skilled in the art can modify, improve, and change the specific content disclosed herein, and such modifications, improvements, and changes should all be considered to fall within the protection scope of the present invention. The materials, methods, and embodiments provided herein are merely illustrative and are not intended to limit the scope of application of the present invention.

[0570] This document provides a broad and general description of the invention. Each more specific species and subgenus falling within the scope of this general disclosure constitutes a part of this invention. This provision covers situations where, in the general description of the invention, any subject matter is excluded from that class (genus) by proviso or negative limitation, regardless of whether the excluded content is explicitly listed herein.

[0571] Furthermore, when features or aspects are described in terms of Markush groups, those skilled in the art will recognize that the present invention is therefore also described with respect to any individual member or subgroup of members of that Markush group.

[0572] All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety as if each reference were cited individually. In the event of any conflict, this specification (including definitions) shall prevail.

[0573] It should be noted that although the present invention has been described in conjunction with the above embodiments, the foregoing description and embodiments are intended to illustrate rather than limit the scope of the invention. Other aspects, advantages, and modifications within the scope of the present invention will be apparent to those skilled in the art.

Claims

1. A compound of Formula I: ###0001### wherein I m is an integer from 1 to 6; L 1 is C 1-10 alkylene or C 2-10 heteroalkylene; wherein, The C 2-10 heteroalkylene is optionally substituted with R 6 substituted; L 2 C 1-10 alkylene or C 2-10 heteroalkylene; wherein said C 2-10 heteroalkylene is optionally substituted with R 6 substituted with R X is -CH2-, -NR 3 -, -O-, -O-CH2CH2-O- or -NR 3 + -, -O-, -O-CH2CH2-O- or -NR 3 m -, -O-, -O-CH2CH2-O- or -NR 3 -;​​ n is independently an integer between 1-20; R 1 , R 2 , and R 3 are each independently hydrogen, C 1-20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl, C 2-20 heteroalkyl, , or ; wherein said C 1-20 alkyl, C 2-20 alkenyl, C 2-20 alkynyl, or C 2-20 heteroalkyl is each independently optionally substituted with 1 to 5 halogen, cyano, -OR 4 , -SR 4 , -NR 4 2, -N(R 4 )3 + , oxo, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, or C 1-6 haloalkyl; R a independently selected from hydrogen, C 1-12 alkyl or -C(O)- C 1-12 alkyl; R b independently selected from hydrogen, C 1-12 alkyl or -C(O)- C 1-12 alkyl; R 4 independently selected from hydrogen, C 1-12 1-6alkyl, C 2-12 2-6alkenyl or C 2-12 2-6alkynyl; wherein each C 1-12 1-6alkyl, C 2-12 2-6alkenyl or C 2-12 2-6alkynyl is independently optionally substituted with 1 to 5 halogen, cyano, -OH, -SR 5 , -NR 5 2, -N(R 5 )3 + or oxo; R 5 independently selected from hydrogen, C 1-12 alkyl, C 2-12 alkenyl, or C 2-12 alkynyl; wherein each C 1-12 alkyl, C 2-12 alkenyl, or C 2-12 alkynyl is independently optionally substituted with 1 to 5 halogen, cyano, -OH, -SH, -NH2, -NH(C 1-6 alkyl), -N(C 1-6 alkyl)2, -N(C 1-6 alkyl)3 + or oxo; R 6 independently selected from , , or ; L is independently selected from C 1-10 alkylene or C 3-10 heteroalkylene; R 7 is independently selected from hydrogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, or C 1-6 haloalkyl; wherein each C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, or C 1-6 haloalkyl is independently optionally substituted with from 1 to 5 halogen, cyano, -OH, -NH2, -NH(C 1-6 alkyl), -N(C 1-6 alkyl)2, -N(C 1-6 alkyl)3 + , oxo, C 1-6 alkoxy, or C 1-6 haloalkoxy; p is independently an integer between 1-6; 13. The compound of claim 6, wherein X is -N(CH3)-. R is independently selected from hydrogen, -Z-C 1-20 alkyl, -Z-C 2-20 alkenyl, -Z-C 2-20 alkynyl, -Z 1 -C 1-6 alkylene-Z-C 1-20 alkyl, -Z 1 -C 1-6 alkylene-Z-C 2-20 alkenyl, -Z 1 -C 1-6 alkylene-Z-C 2-20 alkynyl, -Z 1 -C 1-6 alkylene-Z-heterocyclyl, -Z 1 -C 2-6 alkenylene-Z-C 1-20 alkyl, -Z 1 -C 2-6 alkenylene-Z-C 2-20 alkenyl, -Z 1 -C 2-6 alkenylene-Z-C 2-20 alkynyl, -Z 1 -C 2-6 alkynylene-Z-C 1-20 alkyl, -Z 1 -C 2-6 alkynylene-Z-C 2-20 alkenyl, or -Z 1 -C 2-6 alkynylene-Z-C 2-20 alkynyl; Z is independently selected from a chemical bond, -0-, -NR4-, -S-, -S-S-, -C(O)-, -C(0)0-, -OC(O)-, -C(0)NR 4 -, -S(O)-, -S(0)2-, -NR 4 C(O)-, -NR 4 C(0)0-, -NR 4 C(0)NR 4 -, -NR 4 S(O)- or -S(0)2NR 4 -; and Z 1 independently selected from -O-, -NR 4 -, -S-, -S-S-, -C(O)-, -C(O)O-, -C(O)NR 4 -, -S(O)-, -S(O)2-, -NR 4 C(O)-, -NR 4 C(O)O-, -NR 4 C(O)NR 4 -, -NR 4 S(O)- or -S(O)2NR 4 ; and each moieties comprise at least 6 linear atoms.

2. The compound of claim 1, wherein each R a is independently hydrogen or -C(O)-C 1-12 alkyl.

3. The compound of claim 1 or 2, wherein R a is -C(O)-C 1-6 alkyl.

4. The compound of claim 1 or 2, wherein R a is -C(O)CH3.

5. The compound of claim 1 or 2, wherein R a is hydrogen.

6. The compound of any one of the preceding claims, wherein X is -NR 3 - 7. The compound of claim 6, wherein R 3 is or C 1-20 alkyl optionally substituted with 1 to 5 -OR 4 , -NR 4 2 or -N(R 4 )3 + .

8. The compound of claim 7, wherein R 3 is .

9. The compound of claim 7, wherein R 3 is C 1-20 alkyl. In some embodiments, R 3 is C 1-6 alkyl. In some embodiments, R 3 is methyl.

10. The compound of claim 7, wherein R 3 is C 1-20 alkyl optionally substituted with 1 to 5 -OR 4 , -NR 4 2, or -N(R 4 )3 + .

11. The compound of claim 7, wherein R 3 is C 1-6 alkyl, which can be substituted with 1 to 3 -OH, -N(CH3)3 + or N(CH2CH2OH)2.

12. The compound of claim 7, wherein R 3 is selected from: , , , , , , , and .

17. The compound of claim 14, wherein X is -CH2-.

14. The compound according to any one of claims 1 to 5, wherein X is -N(R 3 )2 + - 15. The compound of claim 14, wherein X is -N(CH3)2 + - 16. The compound of claim 14, wherein X is -N(CD3)2 + - 18. The compound of any one of claims 1-5, wherein X is -0-.

19. The compound of any one of claims 1-5, wherein X is -0-CH2CH2-0-.

22. The compound of any one of claims 1-5, wherein X is -N(CH3)-CH2CH2-N(CH3)-, -N(CH3)-(CH2)3-N(CH3)-, or -N(CH3)-(CH2)4-N(CH3)-.

20. The compound according to any one of claims 1 to 5, wherein X is -NR 3 - (CH2) m -NR 3 -, wherein m is an integer from 1 to 6.

21. The compound according to any one of claims 1 to 5, wherein X is -NR 3 - (CH2) m -NR 3 - wherein m is 2, 3 or 4.

29. The compound of any one of the preceding claims, wherein each Y is -NH- or -N(CH3)-.

23. The compound of any one of the preceding claims, wherein each R 4 independently is hydrogen or C 1-12 alkyl optionally substituted with 1 to 3 -OH.

24. The compound according to any one of the preceding claims, wherein R 1 is hydrogen, C 1-20 alkyl or .

25. The compound according to any one of the preceding claims, wherein R 2 is hydrogen, C 1-20 alkyl or .

26. The compound of claim 24, wherein R 1 and R 2 each independently is hydrogen, C 1-20 alkyl or .

27. The compound of claim 24, wherein R 1 and R 2 are each independently hydrogen, methyl or .

28. The compound of claim 24, wherein R 1 and R 2 are each independently hydrogen, C 1-20 alkyl or ; X is -NR 3 -; R 3 is or C 1-20 alkyl optionally substituted with 1 to 5 -OR 4 , -NR 4 2 or -N(R 4 )3 + .

30. The compound of claim 29, wherein each Y is -NH-.

31. The compound of any one of the preceding claims, wherein each n is from 2 to 16, preferably from 6 to 16, or more preferably from 6 to 12.

35. The compound of claim 33, wherein each Z is independently a bond, -0-, -NHC(O)-, -C(0)0-, or -OC(O)-.

32. The compound of any one of the preceding claims, wherein each R is independently hydrogen, -Z-C 1-20 alkyl, -Z-C 2-20 alkenyl, -Z 1 -C 1-6 alkylene-Z-C 1-20 alkyl, -Z-C 1 -C 1-6 alkylene-Z-C 2-20 alkenyl, or -Z 1 -C 1-6 alkylene-Z-heterocyclyl.

33. The compound of any one of the preceding claims, wherein each Z is independently a bond, -O-, -NR 4 C (O)-, -C(O)O-, or -OC (O)-.

34. The compound of claim 33, wherein each Z is independently a bond, -O-, -NR 4 C (O)- or -OC(O)-.

36. The compound of claim 33, wherein each Z is independently a bond, -0-, -NHC(O)-, or -OC(O)-.

37. The compound of claim 33, wherein each Z is independently -NHC(O)- or -OC(O)-.

38. The compound of claim 33, wherein each Z is independently a bond, -0-, or -OC(O)-.

43. The compound of any one of the preceding claims, wherein R is hydrogen and n is from 6-16, preferably n is 6, 8, 10, 12, or 14, more preferably n is 8, 10, or 12, more preferably n is 10 or 12.

39. The compound of any one of the preceding claims, wherein each Z 1 is independently -O- or -OC(O)-.

40. The compound of claim 39, wherein each Z 1 is -O-.​ 41. The compound of claim 39, wherein Z is a bond and Z is -OC(O)-. 1 is -OC(O)-.

42. The compound of claim 39, wherein Z is -O- and Z 1 is -O-.

74. A compound selected from Table 1A.

44. The compound according to any one of claims 1 to 42, wherein R is -Z-C 2-20 alkenyl, wherein Z is a bond.

45. The compound of claim 44, wherein R is -C 6-14 alkenyl, preferably R is C6 alkenyl, C8 alkenyl, C 10 alkenyl, C 12 alkenyl, or C 14 alkenyl.

46. The compound of claim 45, wherein n is 4, 6, 8, or 10, and R is -C 6-14 alkenyl, preferably R is C6 alkenyl, C8 alkenyl, C 10 alkenyl, C 12 alkenyl, or C 14 alkenyl.

47. The compound according to any one of claims 1 to 42, wherein R is -Z-C 1-20 alkyl, and Z is -NR 4 C(O)-, preferably R is -NHC(O)-C 1-20 alkyl, -NHC(O)-C 10-20 alkyl, or -NHC(O)-C 12-16 alkyl.

48. The compound of claim 47, wherein n is 2, 4, 6, 8, or 10, and R is -NHC(O)-C 1-20 alkyl, preferably R is -NHC(O)-C 10-20 alkyl, or -NHC(O)-C 12-16 alkyl.

49. The compound according to any one of claims 1 to 42, wherein R is -Z-C 1-20 alkyl, and Z is -OC(O)-, preferably R is -OC(O)-C 1-20 alkyl, -OC(O)-C 10-20 alkyl, or -OC(O)-C 12-16 alkyl.

50. The compound of claim 49, wherein n is 2, 4, 6, 8, or 10, R is -OC(O)-C 1-20 alkyl, preferably R is -OC(O)-C 8-20 alkyl or -OC(O)-C 12-16 alkyl.

51. The compound according to any one of claims 1 to 42, wherein R is -Z-C 1-20 alkyl, and Z is -C(O)O-.

52. The compound of claim 51, wherein R is -C(O)O-C 1-20 alkyl, preferably -C(O)O-C 10-20 alkyl or -C(O)O-C 12-16 alkyl.

53. The compound of claim 51, wherein n is 4, 6, 8, or 10, and R is -C(O)O-C 1-20 alkyl, preferably R is -C(O)O-C 8-20 alkyl, or -C(O)O-C 12-16 alkyl.

54. The compound according to any one of claims 1 to 42, wherein R is -Z 1 -C 1-6 alkylene-Z-heterocyclyl, wherein Z 1 is -OC(O)-, and Z is a bond.

55. The compound of claim 54, wherein R is -OC(O)-C 1-6 alkylene-heterocyclyl, preferably -OC(O)-C 1-6 alkylene-1,2-dithiolane.

56. The compound of claim 54, wherein n is 4, 6, 8, or 10, and R is -OC(O)-C 1-6 alkylene-heterocyclyl, preferably R is -OC(O)-C 1-6 alkylene-1,2-dithiolane.

57. The compound according to any one of claims 1 to 42, wherein R is -Z 1 -C 1-6 alkylene-Z-C 1-20 alkyl, and Z 1 is -O-, Z is -O-.

58. The compound of claim 57, wherein R is -O-C 1-6 alkylene-O-C 1-20 alkyl, preferably -O-CH2-O-C 4-12 alkyl.

59. The compound of claim 57, wherein R is O-CH2-O-C4 alkyl, O-CH2-O-C6 alkyl, O-CH2-O-C8 alkyl, O-CH2-O-C 10 alkyl, or O-CH2-O-C 12 alkyl.

60. The compound of claim 57, wherein n is 4, 6, 8, or 10, and R is -O-C 1-6 alkylene-O-C 1-20 alkyl, preferably R is O-CH2-O-C4 alkyl, O-CH2-O-C6 alkyl, O-CH2-O-C8 alkyl, O-CH2-O-C 10 alkyl or O-CH2-O-C 12 alkyl.

61. The compound of claim 57, wherein R is -O-CH(CH3)-O-C 1-20 alkyl.

62. The compound of claim 61, wherein R is -O-CH(CH3)-O-C 4-12 alkyl, preferably O-CH(CH3)-O-C4alkyl, O-CH(CH3)-O-C6alkyl, O-CH(CH3)-O-C8alkyl, O-CH(CH3)-O-C 10 alkyl, or O-CH(CH3)-O-C 12 alkyl.

63. The compound of claim 61, wherein n is 4, 6, 8, or 10, and R is O-CH(CH3)-O-C4 alkyl, O-CH(CH3)-O-C6 alkyl, O-CH(CH3)-O-C8 alkyl, O-CH(CH3)-O-C10 alkyl, O-CH(CH3)-O-C12 alkyl, O-CH(CH3)-O-C14 alkyl, O-CH(CH3)-O-C16 alkyl, or O-CH(CH3)-O-C18 alkyl. 10 alkyl or O-CH(CH3)-O-C 12 alkyl.

64. The compound according to any one of claims 1 to 42, wherein R is -Z 1 -C 1-6 alkylene-Z-C 2-20 alkenyl, wherein Z 1 is -O- and Z is -O-.

65. The compound according to claim 64, wherein R is -O-C 1-6 alkylene-O-C 2-20 alkylene-O-C 2-20 alkylene-O-C 4-12 alkylene-O-C 10 alkylene-O-C 12 alkylene-O-C 66. The compound of claim 64, wherein n is 4, 6, 8, or 10, and R is -O-C 1-6 alkylene-O-C 2-20 alkylene-O-C 2-20 alkylene-O-C 4-12 alkylene-O-C 10 alkylene-O-C 12 alkylene-O-C 67. The compound according to any one of claims 1 to 32, wherein R is hydrogen, -C 2-20 alkenyl, -OC(O)-C 1-20 alkyl, -C(O)O-C 1-20 alkyl, -OC(O)-C 1-6 alkylene-heterocyclyl, -O-CH2-O-C 1-20 alkyl, -O-CH(CH3)-O-C 1-20 alkyl or -O-CH2-O-C 2-20 alkenyl.

68. The compound of claim 67, wherein n is 6 to 16 and R is hydrogen, -C 2-20 alkenyl, -OC (O)-C 1-20 alkyl, -C(O)O-C 1-20 alkyl, -OC (O)-C 1-6 alkylene-heterocyclyl, -O-CH2-O-C 1-20 alkyl, -O-CH(CH3)-O-C 1-20 alkyl or -O-CH2-O-C 2-20 alkenyl.

69. The compound of claim 67, wherein n is 4, 6, 8, or 10, and R is -C 6-14 alkenyl, -OC(O)-C 10-20 alkyl, -C(O)O-C 10-20 alkyl, -OC(O)-C 1-6 alkylene-heterocyclyl, -O-CH2-O-C 4-12 alkyl, -O-CH(CH3)-O-C 4-12 alkyl or -O-CH2-O-C 4-12 alkenyl.

70. The compound according to any one of claims 1 to 32, wherein R is hydrogen, -C 2-20 alkenyl, -OC(O)-C 1-20 alkyl, -OC(O)-C 1-6 alkylene-heterocyclyl, -O-CH2-O-C 1-20 alkyl, -O-CH(CH3)-O-C 1-20 alkyl or -O-CH2-O-C 2-20 alkenyl.

71. The compound of claim 70, wherein n is 6 to 16 and R is hydrogen, -C 2-20 alkenyl, -OC (O)-C 1-20 alkyl, -OC (O)-C 1-6 alkylene-heterocyclyl, -O-CH2-O-C 1-20 alkyl, -O-CH(CH3)-O-C 1-20 alkyl or -O-CH2-O-C 2-20 alkenyl.

72. The compound of claim 70, wherein n is 4, 6, 8, or 10, and R is -C 6-14 alkenyl, -OC(O)-C 10-20 alkyl, -OC(O)-C 1-6 alkylene-heterocyclyl, -O-CH2-O-C 4-12 alkyl, -O-CH(CH3)-O-C 4-12 alkyl or -O-CH2-O-C 4-12 alkenyl.

73. The compound according to any one of claims 1 to 32, wherein the structure is selected from: , , , , , , , , , , , , , , , , , , , , , , , , , and .

75. A composition comprising a compound of any one of claims 1-74.

76. The composition of claim 75, further comprising a phospholipid, a structural lipid, a polyethylene glycol (PEG) lipid, or a combination thereof. ​ 77. The composition of claim 75 or 76, wherein the phospholipid is selected from the group consisting of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2- heneicosanoyl-sn-glycero-3-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC), 1,2-di-O-octadecenyl-OT-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1- hexadecyl-sn-glycero-3-phosphocholine (CI 6 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3- phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-docosahexaenoyl-sn- glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2- diphatanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 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-docosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3- phospho-rac-(l-glycerol) sodium salt (DOPG), sphingomyelin, and combinations thereof.

78. The composition of any one of claims 75-77, wherein the structural lipid is selected from the group consisting of cholesterol, cholestanol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidol, ursolic acid, a-tocopherol, and combinations thereof.

79. The composition of any one of claims 75-78, wherein the PEG lipid is selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and combinations thereof.

80. The composition of any one of claims 75-79, comprising the compound of any one of claims 1-74, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and dimyristylglycerol-PEG2000 (DMG-PEG2K).

81. The composition of claim 80, wherein the molar ratio of DMG-PEG2K, DSPC, cholesterol, and the compound is (0.2 to 10): (1 to 50): (5 to 75): (3 to 85).

82. The composition of any one of claims 75-81, which is a lipid nanoparticle composition.

83. The composition of claim 82, further comprising a therapeutic or prophylactic agent.

84. The composition of claim 83, wherein the therapeutic or prophylactic agent can be a small molecule drug, a protein, a cell, or a nucleic acid.

85. The composition of claim 84, wherein the therapeutic or prophylactic agent is a nucleic acid.

86. The composition of claim 85, wherein the nucleic acid is a deoxyribonucleic acid (DNA).

87. The composition of claim 86, wherein the nucleic acid is a ribonucleic acid (RNA).

88. The composition of claim 87, wherein the RNA is selected from the group consisting of a small interfering RNA (siRNA), an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), a Dicer substrate RNA (dsRNA), a small hairpin RNA (shRNA), a circular RNA (circRNA), a messenger RNA (mRNA), and a combination thereof.

89. The composition of claim 88, wherein the mRNA comprises one or more of a stem loop, a chain terminating nucleoside, a poly(A) sequence, a polyadenylation signal, and a 5’ cap structure.

90. The composition of any one of claims 86-89, which has a molar ratio of amine groups (N) to phosphate groups (P) (N / P ratio) of 5 to 100.

91. A method of delivering a therapeutic or prophylactic agent to a mammalian cell, the method comprising contacting the cell with the composition of any one of claims 75-90 comprising the therapeutic or prophylactic agent.

92. The method of claim 91, wherein the cell is in a patient in need of treatment or prevention of a disease or disorder, and wherein the method comprises administering the composition to the patient.

93. The method of claim 92, wherein the mode of administration is selected from the group consisting of intravenous injection, intramuscular injection, intradermal injection, subcutaneous injection, intravaginal administration, and intranasal administration.

94. The method of any one of claims 91-93, wherein the therapeutic or prophylactic agent is an RNA selected from the group consisting of a small interfering RNA (siRNA), an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), a Dicer substrate RNA (dsRNA), a small hairpin RNA (shRNA), a circular RNA (circRNA), a messenger RNA (mRNA), and a combination thereof.

95. The method of any one of claims 91-94, wherein the patient has a cancer or an infectious disease.

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