An ionizable lipid, a cationic lipopeptide, a lipid nanoparticle, and methods of making and using the same

Abstract

The application provides an ionizable lipid, a cationic lipopeptide, a lipid nanoparticle and a preparation method and application thereof. The application provides an ionizable lipid shown in formula (a), a cationic lipopeptide shown in formula (b), and a lipid nanoparticle containing the ionizable lipid shown in formula (a) and / or the cationic lipopeptide shown in formula (b), which has good biocompatibility, high in-vitro and in-vivo transfection efficiency, and a good clinical application prospect, and (T-L3)m-Hp (formula b).

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and relates to drug delivery technology, in particular to a lipid nanoparticle based on an ionizable lipid and a cationic lipopeptide as well as a preparation method and application thereof. BACKGROUND

[0002] In the treatment of human diseases, traditional small molecule drugs and antibody drugs usually directly act on proteins to achieve their therapeutic purposes. However, small molecule drugs and antibody drugs are difficult to regulate a large number of pathogenic proteins, and therefore have certain limitations in the treatment of certain diseases. Therefore, by regulating the expression of the upstream genes of these proteins, and then precisely regulating the expression level of the proteins as a new treatment strategy, such a strategy is generally referred to as gene therapy. With the in-depth study of molecular biology, nucleic acid drugs have become an important core in gene therapy. These drugs usually include plasmid DNA, mRNA, siRNA, ASO, miRNA, etc. Generally speaking, nucleic acid drugs used for gene therapy usually have the following functions: encoding disease-related proteins, silencing specific gene expression, regulating protein function, mediating gene transcription activation, etc. Based on its special function, nucleic acid drugs bring a glimmer of hope for the treatment of diseases that have no therapeutic drugs.

[0003] In 2018, with the approval of a therapeutic siRNA drug by the FDA, nucleic acid drugs have shown a broader prospect in clinical applications. However, the negative charge of nucleic acid drugs, their sensitivity to nucleases in the body, and the need to exert their effects in cells limit their clinical applications. Therefore, constructing a suitable delivery system to circumvent the above-mentioned adverse factors and improve the efficacy of nucleic acid drugs has become the key.

[0004] Lipid nanoparticles (LNP) are one of the most important nucleic acid delivery systems at present, which are simple to prepare and have good biocompatibility and biodegradability. LNP usually contains ionizable or cationic lipids, auxiliary lipids, cholesterol and PEG lipids. Among them, the ionizable or cationic lipid molecules are the core of the LNP delivery system, and their molecular structure plays a decisive role in the delivery efficiency, targeting, formulation stability, etc. of the whole liposome nanoparticle. Since different types of nucleic acid substances and different target-specific deliveries have different requirements for the delivery system, in order to meet the different needs of gene therapy, new lipid molecules need to be further developed. SUMMARY

[0005] The present application aims at the deficiencies of the prior art, and provides ionizable lipids, cationic lipopeptides, lipid nanoparticles, and a preparation method and application thereof. The lipid nanoparticles provided by the present application have good biocompatibility and high in vitro and in vivo transfection efficiency, and have a good clinical application prospect.

[0006] The present invention provides an ionizable lipid, which is a compound of formula (a), a pharmaceutically acceptable salt of a compound of formula (a), a stereoisomer of a compound of formula (a), a tautomer of a compound of formula (a), a solvate of a compound of formula (a), a chelate of a compound of formula (a), a non-covalent complex of a compound of formula (a), or a prodrug of a compound of formula (a).

[0007]

[0008] Among them, R2, R3, R4, and R5 are independently selected from C6-C. 24 C6-C substituted with alkyl or substituent groups 24 Alkyl, C6-C 24 C6-C substituents of alkenyl or substituent groups 24 alkenyl, C6-C 24 Alkyne group, substituted C6-C 24 alkynyl group;

[0009] Where L1 and L2 are optional divalent functional groups, and A is an optional substituted carbon atom, C1-C 24 Alkyl, C2-C 24 alkenyl, ynyl, C 3-10 cycloalkyl, C 6-12 Aryl, 3-10 membered heterocyclic groups and 5-14 membered heteroaryl groups or combinations thereof;

[0010] In a specific embodiment of the present invention, L1 and L2 are selected from non-existent, C 1-10 Alkylene, C 2-10 imidene group, C 2-10 Ethyne group, C 3-10 cycloalkyl, C 6-12 aryl, 5-14 membered heteroaryl, 3-10 membered heterocyclic, or a combination of two or more of these groups; the C 1-10 Alkylene, C 2-10 imidene group, C 2-10 Ethyne group, C 3-10 cycloalkyl, C 6-12 Aryl, 5-14 membered heteroaryl, 3-10 membered heterocyclic, or combinations of two or more of these groups may be unspecified or optionally separated by carbonyl, O, S, or N atoms; the C 1-10 Alkylene, C 2-10 imidene group, C 2-10 Ethyne group, C 3-10 cycloalkyl, C 6-12 Aryl, 5-14 membered heteroaryl, 3-10 membered heterocyclic, or combinations of two or more of these groups may be optionally C-shaped. 1-6 Alkyl, C3-6 cycloalkyl, halogen atom, haloC 1-6 alkyl group; optionally, the C 1-6 alkyl group or haloC 1-6 alkyl group can form a C 3-6 cycloalkyl, L1and L2are connected to A through any functional group or covalent bond;

[0011] Preferably, L1and L2are selected from -(CH2) q - -(CH2) q -C(=O)-, -(CH2) q -NH-C(=O)-, -(CH2) q -NCH3-C(=O)-, -(CH2) q -C(=O)-NH-, -(CH2) q -C(=O)-NCH3-, wherein q is selected from an integer from 0 to 18, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18.

[0012] In a specific embodiment of the present application, A is substituted with at least one ester group.

[0013] In a specific embodiment of the present application, the present application further provides an ionizable lipid compound represented by (formula a1):

[0014]

[0015] wherein B is a linear or branched alkyl group of C 1-6 .

[0016] In a further specific embodiment of the present application, L1and L2are independently selected from -(CH2) q - wherein q is selected from an integer from 0 to 18, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18; A is a carbon atom; B is a linear or branched alkyl group of C 1-4 .

[0017] In a specific embodiment of the present application, the present application further provides an ionizable lipid compound represented by (formula a2):

[0018]

[0019] wherein p, q are independently selected from an integer from 0 to 18, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18; B is a linear or branched alkyl group of C 1-4 .

[0020] In another embodiment of the present application, L1and L2are selected from -(CH2) q -C(=O)-NH-, q is selected from an integer from 0 to 18, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18; A is C 1-24 alkyl or C 3-6 cycloalkyl or a combination thereof.

[0021] In an embodiment of the present application, the present application further provides an ionizable lipid compound represented by formula (a3):

[0022]

[0023] wherein p, q are independently selected from an integer from 0 to 18, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18;

[0024] A is selected from

[0025]

[0026] wherein n is an integer from 0 to 18.

[0027] In the chemical structure of formula (a) in the present application, the bond is not specified in configuration, i.e. if there are stereoisomers in the chemical structure, the bond may be or or contain both configurations. and In the chemical structure of the compound described in the present application, the bond is not specified in configuration, i.e. it can be in Z configuration or E configuration, or contain both configurations.

[0028] In some embodiments, the ionizable lipid described above is prepared by reductive amination, comprising obtaining a compound represented by formula (a) according to the following reaction formula:

[0029]

[0030] Each substituent is defined as described above, and those skilled in the art know that the aldehyde in the reaction raw material, such as R2CHO, has one CH2group less than R2in the product.

[0031] In some embodiments, the ionizable lipid described above is prepared by amide condensation, comprising obtaining a compound represented by formula (a) according to the following reaction formula:

[0032]

[0033] wherein, The following is referred to as intermediate A.

[0034] In a specific embodiment of the present application, the intermediate A is prepared by reductive amination method:

[0035]

[0036] or alkylation method:

[0037]

[0038] In some embodiments, the intermediate A is prepared by adding the materials under ice bath conditions, and then reacting at room temperature. The reaction time is 12-72h, preferably 20-48h.

[0039] In some embodiments, the solvent system of the reaction includes but is not limited to tetrahydrofuran, dichloromethane, acetonitrile, ethyl acetate, N,N-dimethylformamide, methanol.

[0040] Preferably, the solvent of the reaction is dichloromethane, acetonitrile.

[0041] In some embodiments, the following compounds are included:

[0042]

[0043] The present application provides an amphiphilic cationic lipopeptide, which is a compound shown in formula (b), a pharmaceutically acceptable salt of the compound shown in formula (b), a stereoisomer of the compound shown in formula (b), a tautomer of the compound shown in formula (b), a solvate of the compound shown in formula (b), a chelate of the compound shown in formula (b), a non-covalent complex of the compound shown in formula (b), or a prodrug of the compound shown in formula (b);

[0044] (T-L3)m-H(b)

[0045] wherein, T is R6R7N- or R6R7C-;

[0046] wherein, m is an integer of 1 or 2 or 3;

[0047] wherein R6, R7 are independently selected from C6-C 24 alkyl, C6-C 24 alkyl, C6-C 24 alkenyl, C6-C 24 alkenyl, C6-C 24 alkynyl, C6-C 24 alkynyl.

[0048] wherein L3 is selected from the group consisting of absent, C 1-10 alkylene, C 2-10 alkenylene, C 2-10 alkynylene, C 3-10 cycloalkyl, C 6-12 aryl, 5-14 membered heteroaryl, 3-10 membered heterocyclyl, or a combination of two or more of these groups; said C 1-10 alkylene, C 2-10 alkenylene, C 2-10 alkynylene, C 3-10 cycloalkyl, C 6-12 aryl, 5-14 membered heteroaryl, 3-10 membered heterocyclyl, or a combination of two or more of these groups can be interrupted by carbonyl, O, S, N atoms; said C 1-10 alkylene, C 2-10 alkenylene, C 2-10 alkynylene, C 3-10 cycloalkyl, C 6-12 aryl, 5-14 membered heteroaryl, 3-10 membered heterocyclyl, or a combination of two or more of these groups can be optionally substituted with C 1-6 alkyl, C 3-6 cycloalkyl, halogen atom, halogenated C 1-6 alkyl group; optionally, said C 1-6 alkyl or halogenated C 1-6 alkyl group can form a C 3-6 cycloalkyl group, L1 and L2 are connected to H through any functional group or covalent bond;

[0049] Preferably, L3 is selected from the group consisting of -(CH2) q -, -(CH2) q -C(=O)-, -(CH2) q -NH-C(=O)-, -(CH2) q -NCH3-C(=O)-, -(CH2) q -C(=O)-NH-, -(CH2) q -C(=O)-NCH3-, wherein q is selected from an integer from 0 to 18, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18;

[0050] wherein H is a head formed by a dendrimeric polypeptide.

[0051] Preferably, the polypeptide head is a dendrimeric polypeptide consisting of arginine and / or lysine through amide bond.

[0052] Preferably, the number of amino acids of the constituent tree-shaped polypeptide is 2-5, and further preferably the number of amino acids is 3. The low-generation lipopeptide material is simple and easy to control in the synthesis process, which is conducive to industrialized production. At the same time, the low-generation tree-shaped molecular polypeptide constructed lipopeptide can also be chemically self-assembled to play a role. Compared with high-generation, it has lower toxicity and is safer to use. The low-generation tree-shaped molecular polypeptide constructed lipopeptide has a lower number of charges, which is easy to release nucleic acids.

[0053] Preferably, the compound of formula (b) is of formula (b1):

[0054]

[0055] wherein R6, R7are independently selected from C6-C 24 alkyl, C6-C 24 alkyl, C6-C 24 alkyl, C6-C 24 alkyl, C6-C 24 alkyl, C6-C 24 alkyl, C6-C

[0056] L3 is selected from -(CH2) q -, q is selected from an integer from 1 to 6, preferably 1, 2, 3, 4, 5 or 6;

[0057] wherein RH1and RH2are independently selected from lysine and arginine:

[0058] Lysine and arginine are connected to the corresponding N atom by an amide bond.

[0059] In some embodiments, formula (b1) includes the following compounds:

[0060]

[0061] The present application also provides a preparation method of the above-mentioned cationic lipopeptide, comprising obtaining a compound of formula (b) according to the following reaction formula:

[0062]

[0063] wherein: The following is referred to as intermediate B.

[0064] In some embodiments, in the process of intermediate B compound, the material is added at room temperature, and then the reaction is carried out under the condition of heating at 70°C oil bath. The reaction time is 18-96h, and preferably 48-72h.

[0065] In some embodiments, in the preparation of intermediate B, the material is added under ice bath condition, and then the reaction is carried out at room temperature. The reaction time is 12-72h, preferably 20-48h.

[0066] In some embodiments, the solvent system of the reaction includes, but is not limited to, tetrahydrofuran, dichloromethane, acetonitrile, ethyl acetate, N,N-dimethylformamide, methanol.

[0067] Preferably, the solvent of the reaction is ethyl acetate, dichloromethane, N,N-dimethylformamide.

[0068] The present application provides a nanoparticle composition, which comprises a lipid component, and optionally comprises a cargo; wherein the lipid component contains a compound of the present application.

[0069] In some embodiments, the nanoparticle composition provided by the present application comprises ionizable lipid, cationic lipopeptide, and optionally auxiliary lipid, sterol, PEG lipid, and bioactive substance.

[0070] In some embodiments, the nanoparticle composition provided by the present application comprises ionizable lipid, cationic lipopeptide, sterol, PEG lipid, and bioactive substance; further preferably, the composition does not comprise auxiliary lipid.

[0071] The cationic lipopeptide of the present application can replace auxiliary lipid in a small proportion when constructing a nanoparticle composition, achieving high encapsulation efficiency and delivery efficiency, for example, the use amount ratio of cationic lipopeptide to ionizable lipid is 1:40-1:20, preferably 1:32 (molar ratio), while the use amount ratio of traditional auxiliary lipid such as DSPC to ionizable lipid is 1:4.

[0072] Further, the ionizable lipid is one or more of the ionizable lipids provided by the present application.

[0073] Further, the cationic lipopeptide is one or more of the cationic lipopeptides provided by the present application.

[0074] Further, the auxiliary lipid is one or more of DSPC, DOPC, DPPC, DOPG, DPPG, DOPE, POPC, POPE, DPPE, DMPE, DSPE, SOPE, HSPC, EPC, DOPS, SM, DMPC, DMPG, DSPG, DEPC, POPG, DEPE, DLPE; preferably, the auxiliary lipid is DSPC, DOPE.

[0075] Further, the sterol is cholesterol;

[0076] Further, the PEG lipid is one or more of DMG-PEG, DSPE-PEG, DPPE-PEG, DMA-PEG; preferably, the PEG lipid is DMG-PEG-2000.

[0077] In some embodiments, the mole percentage of the ionizable lipid is 0.1-100%. Preferably, it is 30-60%.

[0078] In some embodiments, the mole percentage of the cationic lipopeptide is 0-99.9%. Preferably, it is 0.5-5%.

[0079] In some embodiments, the mole percentage of the helper lipid is 0-99.9%. Preferably, it is 10-20%.

[0080] In some embodiments, the mole percentage of the sterol is 0-99.9%. Preferably, it is 20-50%.

[0081] In some embodiments, the mole percentage of the PEG lipid sterol is 0-99.9%. Preferably, it is 0.5-5%.

[0082] The lipid nanoparticle provided by the present application, the bioactive substance is a nucleic acid drug.

[0083] Further, the nucleic acid drug includes but is not limited to one or more of siRNA, mRNA, microRNA, circular mRNA, snRNA, snoRNA, tRNA, rRNA, gRNA, shRNA, piRNA, rasiRNA, hnRNA, long non-coding RNA, plasmid DNA, ceDNA, mini circle DNA, antisense oligonucleotide (ASOs).

[0084] The present application provides a method for preparing a lipid nanoparticle combination, comprising the following steps:

[0085] (1) Mix 10 mg / mL of ionizable lipid, cationic lipopeptide or helper lipid, cholesterol, PEG lipid ethanol solution according to the set mole ratio to obtain a lipid mixture solution;

[0086] (2) Quickly add 1-6 times the volume of the lipid solution to a buffer solution containing a nucleic acid drug with pH=4, vortex to mix, to obtain a lipid nanoparticle solution;

[0087] (3) Use a 10 kD ultrafiltration tube to ultrafiltrate at 4°C to obtain a lipid nanoparticle.

[0088] Preferably, the buffer solution in step (2) is a sodium citrate buffer.

[0089] Preferably, the volume of the buffer solution in step (2) is 3-fold.

[0090] In another aspect, the application provides a pharmaceutical composition containing a compound of the application or a nanoparticle composition of the application, and optionally a pharmaceutically acceptable excipient, such as a carrier, adjuvant, or vehicle.

[0091] In another aspect, the application provides the use of a compound of the application, a nanoparticle composition of the application, or a pharmaceutical composition of the application, in the manufacture of a medicament for treating, diagnosing, or preventing a disease.

[0092] In another aspect, the application provides the use of a compound of the application, a nanoparticle composition of the application, or a pharmaceutical composition of the application, in the manufacture of a medicament for delivering a biologically active substance.

[0093] In another aspect, the application provides a method of treating, diagnosing, or preventing a disease in a subject, comprising administering to the subject a compound of the application, a nanoparticle composition of the application, or a pharmaceutical composition of the application.

[0094] In another aspect, the application provides a compound of the application, a nanoparticle composition of the application, or a pharmaceutical composition of the application, for use in treating, diagnosing, and / or preventing a disease.

[0095] In another aspect, the application provides a method of delivering a biologically active substance in a subject, comprising administering to the subject a compound of the application, a nanoparticle composition of the application, or a pharmaceutical composition of the application.

[0096] In another aspect, the application provides a compound of the application, a nanoparticle composition of the application, or a pharmaceutical composition of the application, for use in delivering a biologically active substance.

[0097] In particular embodiments, the biologically active substance is selected from one or more of a therapeutic agent, a prophylactic agent, or a diagnostic agent; preferably, the therapeutic agent, prophylactic agent, or diagnostic agent is a nucleic acid.

[0098] Definitions

[0099] Chemical Definitions

[0100] The definitions of specific functional groups and chemical terms are described in more detail below.

[0101] When a range of values is listed, it is intended to include each value and sub-range within the range. For example, "C 1-6 "alkyl" includes C1, C2, C3, C4, C5, C6, C 1-6 , C 1-5 , C 1-4 , C 1-3 , C 1-2, C 2-6 , C 2-5 , C 2-4 , C 2-3 , C 3-6 , C 3-5 , C 3-4 , C 4-6 , C 4-5 and C 5-6 alkyl.

[0102] In the present application, alkyl refers to a straight chain or branched saturated hydrocarbon group having the corresponding number of carbon atoms, such as "C 1-6 alkyl" refers to a straight chain or branched saturated hydrocarbon group having 1 to 6 carbon atoms, or "C 6-24 alkyl" refers to a straight chain or branched saturated hydrocarbon group having 6 to 24 carbon atoms. In some embodiments, C 1-4 alkyl and C 1-2 alkyl are preferred. C 1-6 Examples of alkyl groups include: methyl (Ci), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), t-butyl (C4), sec-butyl (C4), isobutyl (C4), n-pentyl (C5), 3-pentyl (C5), amyl (C5), neopentyl (C5), 3-methyl-2-butyl (C5), t-amyl (C5), and n-hexyl (C6). The term "C 1-6 alkyl" also includes heteroalkyl groups, wherein one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by a heteroatom (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). An alkyl group can be optionally substituted by one or more substituents, e.g., by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. Conventional alkyl abbreviations include: Me (-CH3), Et (-CH2CH3), iPr (-CH(CH3)2), nPr (-CH2CH2CH3), n-Bu (-CH2CH2CH2CH3), or i-Bu (-CH2CH(CH3)2). In other embodiments, "C 6-24 alkyl" is preferred, such as C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, and preferably straight chain alkyl.

[0103] In the present application, alkenyl refers to a straight chain or branched hydrocarbon group having the corresponding number of carbon atoms and at least one carbon-carbon double bond, such as "C 2-6 alkenyl" refers to a straight chain or branched hydrocarbon group having 2 to 6 carbon atoms and at least one carbon-carbon double bond. In some embodiments, C 2-4 alkenyl is preferred. C 2-6Examples of alkenyl groups include ethenyl (C2), 1- propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. The term "C 2-6 Alkenyl" also includes heteroalkenyl groups wherein one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by a heteroatom (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). Alkenyl groups can be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. In other embodiments "C 6-24 Alkenyl" is preferred, such as C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, and preferably straight chain alkenyl, preferably containing multiple alkenyl bonds.

[0104] "C 2-6 Alkynyl" refers to a straight or branched chain hydrocarbon group having 2 to 6 carbon atoms, at least one carbon-carbon triple bond, and optionally one or more carbon-carbon double bonds. In some embodiments, C 2-4 Alkynyl is preferred. C 2-6 Examples of alkynyl groups include, but are not limited to, ethynyl (C2), 1- propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), pentynyl (C5), hexynyl (C6), and the like. The term "C 2-6 Alkynyl" also includes heteroalkynyl groups wherein one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by a heteroatom (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). Alkynyl groups can be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. In other embodiments "C 6-24 Alkynyl" is preferred, such as C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, and preferably straight chain alkynyl.

[0105] "C 1-10 Alkylene" refers to a divalent group formed by removing two hydrogens from a C 1-10 alkyl group and can be substituted or unsubstituted. In some embodiments, C 1-4 Alkylene, C 2-4 Alkylene and C 1-3Alkylene is preferred. Unsubstituted alkylene groups include, but are not limited to, methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), butylene (-CH2CH2CH2CH2-), pentylene (-CH2CH2CH2CH2CH2-), hexylene (-CH2CH2CH2CH2CH2CH2-), and the like. Exemplary substituted alkylene groups, e.g., alkylene groups substituted with one or more alkyl (methyl) groups, include, but are not limited to, substituted methylene (-CH(CH3)-, -C(CH3)2-), substituted ethylene (-CH(CH3)CH2-, -CH2CH(CH3)-, -C(CH3)2CH2-, -CH2C(CH3)2-), substituted propylene (-CH(CH3)CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH2CH(CH3)-, -C(CH3)2CH2CH2-, -CH2C(CH3)2CH2-, -CH2CH2C(CH3)2-), and the like. 2-

[0106] "C 2-10 Alkenylene" refers to a divalent radical formed by removing two hydrogens from a C 2-10 alkene, and can be substituted or unsubstituted. In some embodiments, C 2-4 Alkenylene is particularly preferred. Exemplary unsubstituted alkenylene groups include, but are not limited to, ethenylene (-CH=CH-) and propenylene (e.g., -CH=CHCH2-, -CH2-CH=CH-). Exemplary substituted alkenylene groups, e.g., alkenylene groups substituted with one or more alkyl (methyl) groups, include, but are not limited to, substituted ethylene (-C(CH3)=CH-, -CH=C(CH3)-), substituted propenylene (-C(CH3)=CHCH2-, -CH=C(CH3)CH2-, -CH=CHCH(CH3)-, -CH=CHC(CH3)2-, -CH(CH3)-CH=CH-, -C(CH3)2-CH=CH-, -CH2-C(CH3)=CH-, -CH2-CH=C(CH3)-), and the like.

[0107] "C 2-10 Alkynylene" refers to a divalent radical formed by removing two hydrogens from a C 2-10 alkyne, and can be substituted or unsubstituted. In some embodiments, C 2-4 Alkynylene is particularly preferred. Exemplary alkynylene groups include, but are not limited to, ethynylene (-CºC-), substituted or unsubstituted propynylene (-CºCCH2-), and the like.

[0108] "C​0-6 Alkylene" refers to a chemical bond and "C 1-6 Alkylene", "C 0-4 Alkylene" refers to a chemical bond and "C 1-4 Alkylene".

[0109] "Halo" or "halogen" means fluoro (F), chloro (CI), bromo (Br), and iodo (I).

[0110] Thus, "C 1-6 Haloalkyl" refers to "C 1-6 Alkyl" groups that are substituted with one or more halogen groups. In some embodiments, C 1-4 Haloalkyl is particularly preferred, more preferably C 1-2 Haloalkyl. Exemplary haloalkyl groups include, but are not limited to: -CF3, -CH2F, -CHF2, -CHFCH2F, -CH2CHF2, -CF2CF3, -CCI3, -CH2CI, -CHCI2, 2,2,2-trifluoro-l,l-dimethyl-ethyl, and the like. The haloalkyl group can be substituted at any available point of attachment, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0111] "C 3-10 Cycloalkyl" refers to a non-aromatic cyclic hydrocarbon group having from 3 to 10 ring carbon atoms and zero heteroatoms, optionally containing 1, 2, or 3 double or triple bonds. In some embodiments, C 5-10 Cycloalkyl, C 3-7 Cycloalkyl, and C 3-6 Cycloalkyl is particularly preferred, more preferably C 5-7 Cycloalkyl, and C 5-6Cycloalkyl groups. Cycloalkyl groups also include ring systems in which the aforementioned cycloalkyl ring is fused with one or more aryl or heteroaryl groups, wherein the bonding point is on the cycloalkyl ring, and in such cases, the number of carbons continues to represent the number of carbons in the cycloalkyl system. Cycloalkyl groups also include the aforementioned cycloalkyl ring in which substituents on any non-adjacent carbon atoms are linked to form a bridged ring, together forming a polycyclic alkane sharing two or more carbon atoms. Cycloalkyl groups also include the aforementioned cycloalkyl ring in which substituents on the same carbon atom are linked to form a ring, together forming a polycyclic alkane sharing one carbon atom. Exemplary cycloalkyl groups include, but are not limited to: cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cyclohepttrienyl (C7), etc. The cycloalkyl group may be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0112] “C 3-10 "Cycloalkylene" refers to the alkylene oxide that has had C removed. 3-10 The cycloalkyl group is a divalent group formed by another hydrogen atom, and can be substituted or unsubstituted. In some embodiments, C 3-6 Cycloalkylene and C 3-4 Cycloalkylene compounds are particularly preferred, especially cyclopropylene compounds.

[0113] "3-10 membered heterocyclyl" refers to a saturated or unsaturated group of 3 to 10 members having ring carbon atoms and 1 to 5 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon, wherein optionally containing 1, 2, or 3 double or triple bonds. In heterocyclyl groups containing one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valence permits. In some embodiments, 5-10 membered heterocyclyl groups are preferred, which are 5 to 10 membered non-aromatic ring systems having ring carbon atoms and 1 to 5 ring heteroatoms; in some embodiments, 3-7 membered heterocyclyl groups are preferred, which are 3 to 7 membered non-aromatic ring systems having ring carbon atoms and 1 to 4 ring heteroatoms; preferably 5-7 membered heterocyclyl groups, which are 5 to 7 membered non-aromatic ring systems having ring carbon atoms and 1 to 3 ring heteroatoms; preferably 3-6 membered heterocyclyl groups, which are 3 to 6 membered non-aromatic ring systems having ring carbon atoms and 1 to 3 ring heteroatoms; preferably 4-6 membered heterocyclyl groups, which are 4 to 6 membered non-aromatic ring systems having ring carbon atoms and 1 to 3 ring heteroatoms; more preferably 5-6 membered heterocyclyl groups, which are 5 to 6 membered non-aromatic ring systems having ring carbon atoms and 1 to 3 ring heteroatoms. Heterocyclyl also includes ring systems in which the above heterocyclyl ring is fused with one or more cycloalkyl rings, wherein the point of attachment is on the heterocyclyl ring, or with one or more aryl or heteroaryl rings, wherein the point of attachment is on the heterocyclyl ring; and in such cases the number of ring members continues to designate the number of ring members in the heterocyclyl ring system. Heterocyclyl also includes ring systems in which the above heterocyclyl ring, wherein any non-adjacent carbon or nitrogen atoms on which substituents are attached form a bridge ring, together form a polycycloalkyl that shares two or more carbon or nitrogen atoms. Heterocyclyl also includes ring systems in which the above heterocyclyl ring, wherein substituents on the same carbon atom are connected to form a ring, together form a polycycloalkyl that shares one carbon atom. Exemplary 3-membered heterocyclyl groups containing one heteroatom include, but are not limited to: aziridinyl, oxiranyl, thiorenyl. Exemplary 4-membered heterocyclyl groups containing one heteroatom include, but are not limited to: azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclyl groups containing one heteroatom include, but are not limited to: pyrrolidinyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, pyrrolinyl, and pyrrol-2,5-dione. Exemplary 5-membered heterocyclyl groups containing two heteroatoms include, but are not limited to: pyrazolidinyl, dioxolanyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclyl groups containing three heteroatoms include, but are not limited to: triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing one heteroatom include, but are not limited to: piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl.Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, without limitation, piperazinyl, morpholinyl, dithianyl, dioxanyl. Exemplary 6-membered heterocyclyl groups containing three heteroatoms include, without limitation, hexahydrotriazinanyl. Exemplary 7-membered heterocyclyl groups containing one heteroatom include, without limitation, azepinyl, oxepinyl, and thiepinyl. Exemplary 5-membered heterocyclyl groups fused to a C6aryl ring (also referred to herein as 5,6-bicyclic heterocyclyl groups) include, without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, benzoxazolinonyl, and the like. Exemplary 6-membered heterocyclyl groups fused to a C6aryl ring (also referred to herein as 6,6-bicyclic heterocyclyl groups) include, without limitation, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like. Heterocyclyl groups also include heterocyclyl groups described above that share one or two atoms with a cycloalkyl, heterocyclyl, aryl, or heteroaryl group, forming a bridged or spirocyclic ring, and the shared atoms can be carbon or nitrogen atoms as valence permits. Heterocyclyl groups also include heterocyclyl groups described above that can be optionally substituted with one or more substituents, for example, with one to five substituents, one to three substituents, or one substituent.

[0114] "C 6-10 Aryl" means a monocyclic or polycyclic (e.g., bicyclic) 4n+2 aromatic ring system (e.g., having 6 or 10 p-electrons shared in a cyclic array) having from 6-10 ring carbon atoms and zero heteroatoms. In some embodiments, the aryl group has six ring carbon atoms ("C6 aryl"; e.g., phenyl). In some embodiments, the aryl group has ten ring carbon atoms ("C10 aryl"; e.g., naphthyl, e.g., 1-naphthyl and 2-naphthyl). Aryl also includes ring systems in which the above-described aryl ring is fused with one or more cycloalkyl or heterocyclyl rings, and the point of attachment is on the aryl ring, in which case the number of carbon atoms designates the number of carbon atoms in the aryl ring system. Aryl groups can be optionally substituted with one or more substituents, for example, with one to five substituents, one to three substituents, or one substituent. 10 Aryl" means a monocyclic or polycyclic (e.g., bicyclic) 4n+2 aromatic ring system (e.g., having 6 or 10 p-electrons shared in a cyclic array) having from 6-10 ring carbon atoms and zero heteroatoms. In some embodiments, the aryl group has six ring carbon atoms ("C6 aryl"; e.g., phenyl). In some embodiments, the aryl group has ten ring carbon atoms ("C10 aryl"; e.g., naphthyl, e.g., 1-naphthyl and 2-naphthyl). Aryl also includes ring systems in which the above-described aryl ring is fused with one or more cycloalkyl or heterocyclyl rings, and the point of attachment is on the aryl ring, in which case the number of carbon atoms designates the number of carbon atoms in the aryl ring system. Aryl groups can be optionally substituted with one or more substituents, for example, with one to five substituents, one to three substituents, or one substituent.

[0115] "5-14 membered heteroaryl" refers to a radical of a 5-14 membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a cyclic array) having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur. In heteroaryl groups containing one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valence permits. Heteroaryl bicyclic systems can include one or more heteroatoms in one or both rings. Heteroaryl also includes ring systems wherein an above-described heteroaryl ring is fused with one or more cycloalkyl or heterocyclyl rings, and the point of attachment is on the heteroaryl ring, in which case the number of carbon atoms indicates the number of carbon atoms in the heteroaryl ring system. In some embodiments, 5-10 membered heteroaryl is preferred, which is a 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms. In other embodiments, 5-6 membered heteroaryl is particularly preferred, which is a 5-6 membered monocyclic or bicyclic 4n+2 aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms. Exemplary 5-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyrrolyl, furanyl, and thiophenyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, but are not limited to, triazolyl, oxadiazolyl (e.g., 1,2,4-oxadiazolyl), and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, but are not limited to, tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyridinyl or pyridinone. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, but are not limited to, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, but are not limited to, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzoimidazolyl, benzoxazolyl, benzoisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzoisothiazolyl, benzothiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, but are not limited to, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. A heteroaryl group can be optionally substituted with one or more substituents, e.g., with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0116] "Heteroaralkyl" means an alkyl group substituted with one or more heteroaryl groups.

[0117] "Alkoxy" refers to the oxygen ether of straight chain or branched chain alkyl groups, i.e., -O-alkyl. Similarly, "methoxy" refers to -O-CH3.

[0118] "Optionally substituted" means that the group can be substituted with the indicated substituent or can be unsubstituted.

[0119] The divalent groups formed by removing two hydrogens from the above defined alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl groups are collectively referred to as "alkylene groups". The cyclic groups formed from the above defined cycloalkyl, heterocyclyl, aryl, and heteroaryl groups are collectively referred to as "cycloalkyl groups".

[0120] The above defined alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl groups are optionally substituted groups.

[0121] Exemplary substituents on a carbon atom include, but are not limited to: halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR aa , -ON(R bb )2, -N(R bb )2, -N(R bb )3 + X - , -N(OR cc )R bb , -SH, -SR aa , -SSR cc , -C(=O)R aa , -CO2H, -CHO, -C(OR cc )2, -CO2R aa , -OC(=O)R aa , -OCO2R aa , -C(=O)N(R bb )2, -OC(=O)N(R bb )2, -NR bb C(=O)R aa , -NR bb CO2R aa , -NR bb C(=O)N(R bb )2, -C(=NR bb )R aa , -C(=NR bb )OR aa , -OC(=NR bb )R aa , -OC(=NR bb )OR aa , -C(=NR bb )N(R bb )2, -OC(=NRbb )N(R bb )2, -NR bb C(=NR bb )N(R bb )2, -C(=O)NR bb SO2R aa , -NR bb SO2R aa , -SO2N(R bb )2, -SO2R aa , -SO2OR aa , -OSO2R aa , -S(=O)R aa , -OS(=O)R aa , -Si(R aa )3, -OSi(R aa )3, -C(=S)N(R bb )2, -C(=O)SR aa , -C(=S)SR aa , -SC(=S)SR aa , -SC(=O)SR aa , -OC(=O)SR aa , -SC(=O)OR aa , -SC(=O)R aa , -P(=O)2R aa , -OP(=O)2R aa , -P(=O)(R aa )2, -OP(=O)(R aa )2, -OP(=O)(OR cc )2, -P(=O)2N(R bb )2, -OP(=O)2N(R bb )2, -P(=O)(NR bb )2, -OP(=O)(NR bb )2, -NR bb P(=O)(OR cc )2, -NR bb P(=O)(NR bb )2, -P(R cc )2, -P(R cc )3, -OP(R cc )2, -OP(R cc )3, -B(R aa )2, -B(OR cc )2, -BR aa (OR cc), alkyl, haloalkyl, alkenyl, ynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl, wherein each alkyl, alkenyl, ynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. dd Group substitution;

[0122] Or the two hydrogen-bearing groups on the carbon atom: =O, =S, =NN(R) bb )2、=NNR bb C(=O)R aa =NNR bb C(=O)OR aa =NNR bb S(=O)2R aa =NR bb or = NOR cc replace;

[0123] R aa Each of them is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl, or two R aa Groups are combined to form heterocyclic or heteroaryl rings, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. dd Group substitution;

[0124] R bb Each is independently selected from: hydrogen, -OH, -OR aa -N(R) cc )2、-CN、-C(=O)R aa -C(=O)N(R) cc )2、-CO2R aa -SO2R aa -C(=NR) cc OR aa -C(=NR) cc )N(R cc )2、-SO2N(R cc )2、-SO2R cc -SO2OR cc -SOR aa -C(=S)N(R) cc )2、-C(=O)SR cc -C(=S)SR cc -P(=O)2R aa -P(=O)(R aa )2、-P(=O)2N(R cc )2、-P(=O)(NR cc2. Alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl and heteroaryl, or two R bb Groups are combined to form heterocyclic or heteroaryl rings, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. dd Group substitution;

[0125] R cc Each is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl, or two R cc Groups are combined to form heterocyclic or heteroaryl rings, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. dd Group substitution;

[0126] R dd Each is independently selected from: halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR ee -ON(R) ff )2、-N(R ff )2,、-N(R ff )3 + X - -N(OR) ee )R ff -SH, -SR ee -SSR ee -C(=O)R ee -CO2H, -CO2R ee -OC(=O)R ee -OCO2R ee -C(=O)N(R) ff )2、-OC(=O)N(R ff )2、-NR ff C(=O)R ee -NR ff CO2R ee -NR ff C(=O)N(R ff )2、-C(=NR ff OR ee -OC(=NR) ff )R ee -OC(=NR) ff OR ee -C(=NR) ff )N(R ff )2、-OC(=NR ff )N(R ff)2, -NR ff C(=NR ff )2, -C(=NR ff )2, -C(=O)R ff SO2R ee , -SO2N(R ff )2, -SO2R ee , -SO2OR ee , -OSO2R ee , -S(=O)R ee , -Si(R ee )3, -OSi(R ee )3, -C(=S)N(R ff )2, -C(=O)SR ee , -C(=S)SR ee , -SC(=S)SR ee , -P(=O)2R ee , -P(=O)(R ee )2, -OP(=O)(R ee )2, -OP(=O)(OR ee )2, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R gg groups, or two geminal R dd substituents can be combined to form =O or =S;

[0127] each R ee is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl, and heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R gg groups;

[0128] each R ff is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or two R ff groups combine to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R gg groups;

[0129] each R gg is independently halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OC 1-6 alkyl, -ON(C 1-6 alkyl)2, -N(C1-6 alkyl)2, -N(C 1-6 alkyl)3 + X - , -NH(C 1-6 alkyl)2 + X - , -NH2(C 1-6 alkyl) + X - , -NH3 + X - , -N(OC 1-6 alkyl)(C 1-6 alkyl), -N(OH)(C 1-6 alkyl), -NH(OH), -SH, -SC 1-6 alkyl, -SS(C 1-6 alkyl), -C(=O)(C 1-6 alkyl), -CO2H, -CO2(C 1-6 alkyl), -OC(=O)(C 1-6 alkyl), -OCO2(C 1-6 alkyl), -C(=O)NH2, -C(=O)N(C 1-6 alkyl)2, -OC(=O)NH(C 1-6 alkyl), -NHC(=O)(C 1-6 alkyl), -N(C 1-6 alkyl)C(=O)(C 1-6 alkyl), -NHCO2(C 1-6 alkyl), -NHC(=O)N(C 1-6 alkyl)2, -NHC(=O)NH(C 1-6 alkyl), -NHC(=O)NH2, -C(=NH)O(C 1-6 alkyl), -OC(=NH)(C 1-6 alkyl), -OC(=NH)OC 1-6 alkyl, -C(=NH)N(C 1-6 alkyl)2, -C(=NH)NH(C 1-6 alkyl), -C(=NH)NH2, -OC(=NH)N(C 1-6 alkyl)2, -OC(NH)NH(C 1-6 alkyl), -OC(NH)NH2, -NHC(NH)N(C 1-6 alkyl)2, -NHC(=NH)NH2, -NHSO2(C 1-6 alkyl), -SO2N(C 1-6 alkyl)2, -SO2NH(C 1-6 alkyl), -SO2NH2, -SO2C1-6 alkyl, -SO2OC 1-6 alkyl, -OSO2C 1-6 alkyl, -SOC 1-6 alkyl, -Si(C 1-6 alkyl)3, -OSi(C 1-6 alkyl)3, -C(=S)N(C 1-6 alkyl)2, C(=S)NH(C 1-6 alkyl), C(=S)NH2, -C(=O)S(C 1-6 alkyl), -C(=S)SC 1-6 alkyl, -SC(=S)SC 1-6 alkyl, -P(=O)2(C 1-6 alkyl), -P(=O)(C 1-6 alkyl)2, -OP(=O)(C 1-6 alkyl)2, -OP(=O)(OC 1-6 alkyl)2, C 1-6 alkyl, C 1-6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 cycloalkyl, C6-C 10 aryl, C3-C7 heterocyclyl, C5-C 10 heteroaryl; or two geminal R gg substituents can combine to form =O or =S; wherein X - is a counterion.

[0130] Exemplary substituents on a nitrogen atom include, but are not limited to: hydrogen, -OH, -OR aa , -N(R cc )2, -CN, -C(=O)R aa , -C(=O)N(R cc )2, -CO2R aa , -SO2R aa , -C(=NR bb )R aa , -C(=NR cc )OR aa , -C(=NR cc )N(R cc )2, -SO2N(R cc )2, -SO2R cc , -SO2OR cc , -SOR aa , -C(=S)N(R cc )2, -C(=O)SR cc , -C(=S)SR cc , -P(=O)2R aa , -P(=O)(Raa )2, -P(=O)2N(R cc )2, -P(=O)(NR cc )2, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or two R cc groups bound to a nitrogen atom combine to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R dd groups, and wherein R aa , R bb , R cc , and R dd are as described above.

[0131] “Nucleic acid” refers to a single- or double-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecule and hybrid molecules thereof.

[0132] In the present application, “cationic lipid” or “ionizable cationic lipid” have the same meaning and refer to a lipid molecule that is able to carry a positive charge under physiological pH conditions. In some embodiments, the cationic lipid is an amino lipid.

[0133] In some embodiments, the cationic lipid is selected from SM-102, ALC-0315, ALC- 0519, Dlin-MC3-DMA (also known as MC3), DODMA, DLin-KC2-DMA, DlinDMA, preferably SM-102; SM-102 (Heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6- (undecyloxy)hexyl)amino)octanoate) has the following structure:

[0134]

[0135] "Neutral lipid" refers to a lipid molecule that does not carry a charge under certain pH conditions, for example physiological pH conditions. Examples of neutral lipids include, but are not limited to, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE).

[0136] "Structural lipid" refers to a lipid that enhances nanoparticle stability by filling the interstitial space between lipids, often seen with steroids. Steroids are compounds having a cyclopentanoperhydrophenanthrene class of carbon skeleton. In a preferred embodiment, the steroid is selected from the group consisting of cholesterol, sitosterol, coprostanol, fecosterol, brassicasterol, ergosterol, tomatidine, ursolic acid, alpha-tocopherol, stigmasterol, avenasterol, ergocalciferol, or campesterol.

[0137] "Polymeric lipid" refers to a molecule that contains both a polymeric moiety and a lipid moiety. In some embodiments, the polymeric lipid is a polyethylene glycol (PEG) lipid.

[0138] "Nanolipid particle" refers to a particle that contains a lipid component and has a nanoscale size.

[0139] "Biodegradable group" refers to a functional group that contains a biodegradable bond, such as an ester, a disulfide bond, and an amide, among others. Biodegradation can affect the process of clearing a compound from the body.

[0140] Other definitions

[0141] The term "treatment" as used herein refers to reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. The term "treatment" as used herein refers to the act of treating as the verb, the latter being as just defined.

[0142] The term "pharmaceutically acceptable salt" as used herein denotes those carboxylic acid salts, amino acid addition salts of the compounds of the present application which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without an undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio, effective for their intended use, including, (where possible) the zwitterion forms of the compounds of the present application.

[0143] Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals, hydrides or organic amines. Examples of metals used as a cation are sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines are N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine and procaine.

[0144] Base addition salts of acidic compounds can be prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt. The free acid form can be regenerated by contacting the salt form with a sufficient amount of an acid to produce the free acid form. The free acid and base forms can be regenerated by

[0145] The salt can be a sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide salt prepared from inorganic acids such as hydrochloric acid, nitric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, and the like. Representative salts include the following: hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthoate, mesylate, gluconate, lactobionate, laurylsulcalte, and isethionate, and the like. The salt can also be prepared from organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, alkyldioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. Representative salts include acetate, propionate, caprylate, isobutyrate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, naphthoate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate, maleate, tartrate, mesylate, and the like. Pharmaceutically acceptable salts can include cations based on alkali and alkaline earth metals, for example, sodium, lithium, potassium, calcium, magnesium, and aluminum, as well as ammonium, quaternary ammonium, and amine cations such as, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. Salts of amino acids can also be encompassed (e.g., arginate, gluconate, galacturonate, and the like (see, e.g., Berge S.M. et al., "Pharmaceutical Salts," J. Pharm. Sci., 1977; 66: 1-19, incorporated herein by reference).

[0146] A "subject" for administration includes, but is not limited to, a human (i.e., a male or female of any age group, e.g., a pediatric subject (e.g., an infant, a child, an adolescent) or an adult subject (e.g., a young adult, a middle-aged adult, or an elderly adult)) and / or a non-human animal, e.g., a mammal, e.g., a primate (e.g., a cynomolgus monkey, a rhesus monkey), a bovine, a porcine, a equine, an ovine, a caprine, a rodent, a feline, and / or a canine. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. The terms "human," "patient," and "subject" are used interchangeably herein.

[0147] "Disease," "disorder," and "condition" are used interchangeably herein.

[0148] The term "treatment" as used herein includes an action that occurs while the subject is suffering from a particular disease, disorder or condition, that reduces the severity of the disease, disorder or condition, or retards or slows the progression of the disease, disorder or condition ("therapeutic treatment"), and also includes an action that occurs before the subject begins to suffer from a particular disease, disorder or condition ("prophylactic treatment").

[0149] Generally, an "effective amount" of a compound refers to an amount that is sufficient to elicit the desired biological response. As will be appreciated by those of ordinary skill in the art, the effective amount of a compound of the application can vary depending on such factors as the biological endpoint, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the age, health, and symptoms of the subject. An effective amount includes both therapeutic and prophylactic effective amounts.

[0150] The term "therapeutically effective amount" of a compound, as used herein, refers to an amount that is sufficient to provide a therapeutic benefit in the treatment of a disease, disorder or condition, or to delay or minimize one or more symptoms associated with the disease, disorder or condition. A therapeutically effective amount of a compound refers to the amount of therapeutic agent, alone or in combination with other therapies, that when administered to a subject in need, provides a therapeutic benefit in the treatment of a disease, disorder or condition. The term "therapeutically effective amount" can encompass an amount that improves overall prophylaxis or enhances the prophylactic effect of another prophylactic agent.

[0151] The term "prophylactically effective amount" of a compound, as used herein, refers to an amount that is sufficient to prevent a disease, disorder or condition, or to prevent one or more symptoms associated with a disease, disorder or condition, or to prevent the recurrence of a disease, disorder or condition. A prophylactically effective amount of a compound refers to the amount of therapeutic agent, alone or in combination with other agents, that when administered to a subject in need, provides a prophylactic benefit in the prevention of a disease, disorder or condition. The term "prophylactically effective amount" can encompass an amount that improves overall prophylaxis or enhances the prophylactic effect of another prophylactic agent.

[0152] "Combination" and related terms refer to administration of a compound of the application and another therapeutic agent simultaneously or sequentially. For example, a compound of the application can be administered simultaneously or sequentially with another therapeutic agent in separate unit dosage forms, or administered simultaneously with another therapeutic agent in a single unit dosage form.

[0153] Benefits:

[0154] 1. The present application introduces an amino acid analogue structure to obtain a new type of ionizable lipid. The amide bond in the ionizable lipid structure can be rapidly hydrolyzed by enzymes in vivo, easily metabolized and cleared, has good biocompatibility and biodegradability, and has good clinical application prospects.

[0155] 2. The ionizable lipids provided by the application are prepared by simple reductive amination and amide condensation reactions, and ionizable lipids with different branched chain lengths are synthesized. The raw material cost is low, the synthesis steps are simple, the product separation is convenient, and the product is easy to store.

[0156] 3. The cationic lipopeptides provided by the application are prepared by simple substitution and amide condensation reactions, contain dendritic structures, have high gene delivery efficiency, have amphiphilic structures, and have good biocompatibility.

[0157] 4. The lipid nanoparticles prepared from the ionizable lipids, cationic lipopeptides or auxiliary lipids, sterols and PEG lipids provided by the application can effectively deliver nucleic acid drugs such as siRNA and mRNA.

[0158] 5. The preparation method of the lipid nanoparticles provided by the application is convenient and fast, has low equipment requirements, and has reliable process, and is suitable for biological and pharmaceutical industrialization. BRIEF DESCRIPTION OF DRAWINGS

[0159] Figure 1 is a particle size characterization graph of different LNPs in Example 12.

[0160] Figure 2 is a potential characterization graph of different LNPs in Example 12.

[0161] Figure 3 is the encapsulation efficiency of different LNPs in Example 13.

[0162] Figure 4 is the average fluorescence intensity of EGFP-mRNA-LNP transfection in Example 14.

[0163] Figure 5 is the gene silencing efficiency of siLuc-LNP in Example 15. DETAILED DESCRIPTION

[0164] The technical solutions of the application will be described in detail below through specific examples, but the protection scope of the application is not limited to the examples. In the specific embodiments of the application, the technical means or methods not specifically described are conventional technical means or methods in the art. The materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained from commercial channels.

[0165] Example 1 Preparation of NL-1

[0166] Step 1: Precisely weigh H-Lys-OMe-2HCI (1.00 g, 4.28 mmol), dodecanal (4.22 g, 30.02 mmol) into a three-necked flask, vacuumize, and protect with nitrogen. Add 100 mL of dichloromethane, slowly add DIPEA (4.50 mL, 25.74 mmol) under ice bath condition, after completely dissolved, add glacial acetic acid (3.00 mL, 51.90 mmol), after reaction for 2 h, add sodium triacetoxyborohydride (8.27 g, 39.03 mmol), reaction for 48 h at room temperature, quench the reaction with 1 M NaOH, and repeatedly wash with saturated sodium bicarbonate solution, 0.1% dilute HCl solution, and saturated NaCl solution for three times, respectively. Add anhydrous sodium sulfate to dry and remove water. Vacuum rotary evaporation to obtain NL-1 crude product.

[0167] Step 2: Purify the NL-1 crude product in step 1 by column chromatography, elute with EA:MeOH = 70:1 (V / V) to obtain ionizable lipid NL-1.

[0168] 1 H NMR (300 MHz, CDCl3) δ 3.69 (d, J = 21.7 Hz, 3H), 3.44-2.23 (m, 11H), 2.19-1.00 (m, 86H), 0.86 (q, J = 5.3, 4.4 Hz, 12H).

[0169] Preparation of intermediate A in Example 2

[0170] Step 1: Precisely weigh 6-aminohexanoic acid (1.00 g, 7.62 mmol), dodecanal (4.22 g, 22.87 mmol), sodium triacetoxyborohydride (6.47 g, 30.49 mmol) into a three-necked flask, vacuumize, and protect with nitrogen. Dissolve in 75 mL of anhydrous acetonitrile, and react for 5 days at room temperature. Stop the reaction, remove the solvent by vacuum rotary evaporation, dissolve the product in dichloromethane, and wash with ultrapure water for three times. Add anhydrous sodium sulfate to dry and remove water. Vacuum rotary evaporation to obtain intermediate A crude product.

[0171] Step 2: Purify the intermediate A crude product in step 1 by column chromatography, elute with DCM:D1 = 6:1 (V / V), and D1 = DCM:MeOH:NH3-H2O = 75:22:3 to obtain intermediate pure product.

[0172] Preparation of H-6-12 in Example 3

[0173] Step 1: Precision weighing of trans-1,4-cyclohexanediamine (0.11 g, 0.97 mmol), intermediate A (1.00 g, 2.14 mmol), HOBT (0.46 g, 3.40 mmol), EDCI (0.65 g, 3.40 mmol) into a three-necked flask, vacuumizing, nitrogen protection. Adding 50 mL dichloromethane dissolving, slowly adding DIPEA (1.70 mL, 9.72 mmol) under ice bath, reacting at room temperature for 24 h. After the reaction is completed, sequentially washing with saturated sodium bicarbonate solution, 0.1% dilute hydrochloric acid solution, saturated NaCl solution repeatedly three times. Adding anhydrous sodium sulfate to dry and remove water. Vacuum rotary evaporation to obtain H-6-12 crude product.

[0174] Step 2: Purifying the H-6-12 crude product in step 1 by column chromatography, eluting with DCM: D1 = 8:1 (V / V), D1 = DCM:MeOH:NH3·H2O = 75:22:3, to obtain ionizable lipid H-6-12.

[0175] 1 H NMR (300 MHz, CDCl3) δ 5.39 (d, J = 8.1 Hz, 2H), 3.74 (s, 2H), 2.40 (t, J = 7.7 Hz, 8H), 2.12 (d, J = 7.5 Hz, 4H), 1.98 (d, J = 8.0 Hz, 4H), 1.74-1.33 (m, 20H), 1.25 (s, 80H), 0.87 (s, 12H).

[0176] Example 4: Preparation of Y-6-12

[0177] Step 1: Precision weighing of ethylenediamine hydrochloride (0.13 g, 0.97 mmol), intermediate A (1.00 g, 2.14 mmol), HOBT (0.46 g, 3.40 mmol), EDCI (0.65 g, 3.40 mmol) into a three-necked flask, vacuumizing, nitrogen protection. Adding 50 mL dichloromethane dissolving, slowly adding DIPEA (1.70 mL, 9.72 mmol) under ice bath, reacting at room temperature for 24 h. After the reaction is completed, sequentially washing with saturated sodium bicarbonate solution, 0.1% dilute hydrochloric acid solution, saturated NaCl solution repeatedly three times. Adding anhydrous sodium sulfate to dry and remove water. Vacuum rotary evaporation to obtain Y-6-12 crude product.

[0178] Step 2: Purifying the Y-6-12 crude product in step 1 by column chromatography, eluting with DCM: D1 = 8:1 (V / V), D1 = DCM:MeOH:NH3·H2O = 75:22:3, to obtain ionizable lipid Y-6-12.

[0179] 1H NMR (300 MHz, CDC13) δ 6.42 (s, 1H), 3.45 - 3.29 (m, 4H), 2.44 - 2.38 (m, 8H), 2.18 (t, J = 7.5 Hz, 4H), 2.04 (s, 4H), 1.25 (s, 92H), 0.91 - 0.81 (m, 12H).

[0180] Example 5: Preparation of Intermediate B

[0181] Step 1: Precisely weigh N-tert-butoxycarbonyl ethylenediamine (2 g, 12.48 mmol), stearyl bromide (9.16 g, 27.46 mmol), anhydrous potassium carbonate (4.31 g, 31.21 mmol), potassium iodide (0.21 g, 1.25 mmol) into a three-necked flask, dissolve with 200 mL of ethyl acetate, condense reflux, heat at 70°C oil bath for 48 h. After the reaction is completed, wash with pure water, saturated NaCl solution repeatedly three times. Add anhydrous sodium sulfate to dry and remove water. Vacuum rotary evaporation to obtain Boc-Intermediate B crude product.

[0182] Step 2: Purify the Boc-Intermediate B crude product in step 1 by column chromatography, elute with DCM:MeOH = 100:1 (V / V) to obtain Boc-Intermediate B pure product.

[0183] Step 3: Precisely weigh Boc-Intermediate B 2.0 g into a three-necked flask, vacuum, nitrogen protection. Add 4 mL of dichloromethane and 4 mL of trifluoroacetic acid under ice bath, react for 2 h. After the reaction is completed, remove the solvent by vacuum rotary evaporation, dissolve with dichloromethane, wash with saturated sodium bicarbonate solution three times, add anhydrous sodium sulfate to dry and remove water. Vacuum rotary evaporation to obtain Intermediate B pure product.

[0184] Example 6: Preparation of G2R-2-18

[0185] Step 1: Precisely weigh H-Lys-OMe-2HCl (1.00 g, 4.29 mmol), Boc-Arg(Pbf)-OH (6.78 g, 12.87 mmol), HOBT (1.74 g, 12.87 mmol), HBTU (4.88 g, 12.87 mmol) into a three-necked flask, vacuumize, and protect with nitrogen. Add 60 mL of DMF to dissolve, slowly add DIPEA (5.98 mL, 34.32 mmol) under ice bath, and react at room temperature for 24 h. After the reaction is completed, remove the solvent by vacuum rotary evaporation, dissolve in ethyl acetate, and repeatedly wash with saturated sodium bicarbonate solution, 0.1% dilute HCl solution, and saturated NaCl solution, respectively, three times. Add anhydrous sodium sulfate to dry and remove water. Vacuum rotary evaporation is performed to obtain OMe-G2R-(Boc)Pbf crude product. Purification is performed by column chromatography, elution is performed with DCM:MeOH=70:1 (V / V), and OMe-G2R-(Boc)Pbf pure product is obtained.

[0186] Step 2: Precisely weigh OMe-G2R-(Boc)Pbf 2.00 g into a round-bottom flask, add NaOH methanol solution (2 g of NaOH dissolved in 50 mL of methanol), react under ice bath conditions for 6 h, and add 1M HCl to adjust the pH to 2-3. Remove the solvent by vacuum rotary evaporation, dissolve in ethyl acetate, repeatedly wash with saturated NaCl solution three times, add anhydrous sodium sulfate to dry and remove water. Remove the solvent by vacuum rotary evaporation to obtain OH-G2R-(Boc)Pbf pure product.

[0187] Step 3: Precisely weigh OH-G2R-(Boc)Pbf (1.24 g, 1.06 mmol), intermediate B (0.50 g, 0.89 mmol), HOBT (0.36 g, 2.66 mmol), EDCI (0.51 g, 2.66 mmol) into a three-necked flask, vacuumize, and protect with nitrogen. Add 50 mL of dichloromethane to dissolve, slowly add DIPEA (1.23 mL, 7.08 mmol) under ice bath, and react at room temperature for 24 h. After the reaction is completed, repeatedly wash with saturated sodium bicarbonate solution, 0.1% dilute HCl solution, and saturated NaCl solution, respectively, three times. Add anhydrous sodium sulfate to dry and remove water. Vacuum rotary evaporation is performed to obtain G2R-(Boc)Pbf-2-18 crude product. Purification is performed by column chromatography, elution is performed with DCM:MeOH=50:1 (V / V), and G2R-(Boc)Pbf-2-18 pure product is obtained.

[0188] Step 4: Precisely weigh G2R-(Boc)Pbf-2-18 0.20 g into a three-necked flask, vacuumize, and protect with nitrogen. Add 2 mL of dichloromethane and 2 mL of trifluoroacetic acid under ice bath, and react for 12 h. After the reaction is completed, remove the solvent by vacuum rotary evaporation, wash with ethyl acetate for three times, and obtain the cationic lipopeptide G2R-2-18.

[0189] 1 H NMR (300 MHz, DMSO) δ 9.08 - 7.03 (m, 15H), 4.20 (s, 2H), 3.78 (d, J = 38.2 Hz, 5H), 3.02 (d, J = 48.0 Hz, 10H), 1.23 (s, 78H), 0.89 - 0.80 (m, 6H).

[0190] Example 7: Preparation of G2K-2-18

[0191] Step 1: Precisely weigh H-Lys-OMe·2HCl (2.00 g, 8.58 mmol), Boc-Lys(Boc)-OH (7.43 g, 21.45 mmol), HOBT (3.48 g, 25.74 mmol), and HBTU (9.76 g, 25.74 mmol) into a three-necked flask, vacuumize, and protect with nitrogen. Dissolve in 100 mL of DMF, slowly add DIPEA (11.21 mL, 64.35 mmol) under ice bath, and react for 24 h at room temperature. After the reaction is completed, remove the solvent by vacuum rotary evaporation, dissolve in ethyl acetate, and wash with saturated sodium bicarbonate solution, 0.1% diluted hydrochloric acid solution, and saturated sodium chloride solution for three times in sequence. Dry and remove water by adding anhydrous sodium sulfate. Vacuum rotary evaporation obtains the crude product of OMe-G2K-(Boc). Purify by column chromatography, elute with PE:EA = 5:1 (V / V), and obtain the pure product of OMe-G2K-(Boc).

[0192] Step 2: Precisely weigh OMe-G2K-(Boc) 2.00 g into a round-bottom flask, add a methanol solution of NaOH (2 g of NaOH dissolved in 50 mL of methanol), react for 6 h under ice bath, and adjust the pH to 2-3 by adding 1M hydrochloric acid. Remove the solvent by vacuum rotary evaporation, dissolve in ethyl acetate, wash with saturated sodium chloride solution for three times, dry and remove water by adding anhydrous sodium sulfate. Remove the solvent by vacuum rotary evaporation, and obtain the pure product of OH-G2K-(Boc)Pbf.

[0193] Step 3: Precisely weigh OH-G2K-(Boc) (0.85 g, 1.06 mmol), intermediate B (0.50 g, 0.89 mmol), HOBT (0.36 g, 2.66 mmol), EDCI (0.51 g, 2.66 mmol) into a three-necked flask, vacuumize, and protect with nitrogen. Add 50 mL of dichloromethane, slowly add DIPEA (1.23 mL, 7.08 mmol) under ice bath, and react at room temperature for 24 h. After the reaction is completed, wash with saturated sodium bicarbonate solution, 0.1% dilute hydrochloric acid solution, and saturated NaCl solution for three times, respectively. Dry with anhydrous sodium sulfate to remove water. Vacuum rotary evaporation obtains G2K-(Boc)-2-18 crude product. Purify by column chromatography, elute with DCM:MeOH = 50:1 (V / V), to obtain G2K-(Boc)-2-18 pure product.

[0194] Step 4: Precisely weigh G2K-(Boc)-2-18 0.20 g into a three-necked flask, vacuumize, and protect with nitrogen. Add 2 mL of dichloromethane and 2 mL of trifluoroacetic acid under ice bath, and react for 2 h. After the reaction is completed, remove the solvent by vacuum rotary evaporation, wash with ethyl ether for three times, to obtain cationic lipopeptide G2K-2-18.

[0195] 1 H NMR (300 MHz, DMSO) δ 8.66 (d, J = 7.3 Hz, 1H), 8.50 (d, J = 16.3 Hz, 2H), 8.20 (s, 4H), 7.89 (s, 4H), 4.36-3.42 (m, 5H), 2.91 (d, J = 98.0 Hz, 12H), 1.23 (s, 82H), 0.90-0.80 (m, 6H). Example 8: Preparation of N1-2-18

[0196] Step 1: Precisely weigh Fmoc-Lys-OMe·HCl (5.00 g, 11.94 mmol), Boc-Lys(Boc)-OH (4.55 g, 13.13 mmol), HATU (5.45 g, 14.32 mmol) into a three-necked flask, vacuumize, and protect with nitrogen. Add 80 mL of DMF, slowly add DIPEA (8.32 mL, 47.74 mmol) under ice bath, and react at room temperature for 16 h. After the reaction is completed, remove the solvent by vacuum rotary evaporation, dissolve with ethyl acetate, wash with saturated sodium bicarbonate solution, 0.1% dilute hydrochloric acid solution, and saturated NaCl solution for three times, respectively. Dry with anhydrous sodium sulfate to remove water. Vacuum rotary evaporation obtains N1-Fmoc crude product. Purify by column chromatography, elute with DCM:MeOH = 100:1 (V / V), to obtain N1-Fmoc pure product.

[0197] Step 2: Precisely weigh N1-Fmoc 2.50 g into a three-necked flask, vacuumize, and protect with nitrogen. Add 8 mL of DMF and 2 mL of piperidine under ice bath, and react for 0.5 h. After the reaction is completed, remove the solvent by vacuum rotary evaporation, and purify by column chromatography with DCM:MeOH = 60:1 (V / V) as eluent to obtain the pure product of N1.

[0198] Step 3: Precisely weigh N1 (1.72 g, 3.52 mmol), Boc-Arg(Pbf)-OH (2.22 g, 4.22 mmol), HOBT (0.71 g, 5.28 mmol), and HBTU (2.00 g, 5.28 mmol) into a three-necked flask, vacuumize, and protect with nitrogen. Add 60 mL of DMF to dissolve, slowly add DIPEA (4.90 mL, 28.14 mmol) under ice bath, and react for 24 h at room temperature. After the reaction is completed, remove the solvent by vacuum rotary evaporation, dissolve in ethyl acetate, and wash with saturated sodium bicarbonate solution, 0.1% diluted hydrochloric acid solution, and saturated sodium chloride solution in sequence for three times. Dry with anhydrous sodium sulfate to remove water. Vacuum rotary evaporation is performed to obtain the crude product of OMe-N1-Arg-(Boc)Pbf. Purify by column chromatography with DCM:MeOH = 60:1 (V / V) as eluent to obtain the pure product of OMe-N1-Arg-(Boc)Pbf.

[0199] Step 4: Precisely weigh OMe-N1-Arg-(Boc)Pbf 2.00 g into a round-bottom flask, add a methanol solution of NaOH (2 g of NaOH dissolved in 50 mL of methanol), and react for 6 h under ice bath. Add 1M hydrochloric acid to adjust the pH to 2-3. Remove the solvent by vacuum rotary evaporation, dissolve in ethyl acetate, wash with saturated sodium chloride solution for three times, and dry with anhydrous sodium sulfate to remove water. Remove the solvent by vacuum rotary evaporation to obtain the pure product of OH-N1-Arg-(Boc)Pbf.

[0200] Step 5: Precisely weigh OH-N1-Arg-(Boc)Pbf (0.63 g, 0.64 mmol), Intermediate B (0.30 g, 0.53 mmol), HOBT (0.22 g, 1.59 mmol), EDCI (0.31 g, 1.59 mmol) into a three-necked flask, vacuumize, and protect with nitrogen. Add 50 mL of dichloromethane, slowly add DIPEA (0.74 mL, 4.25 mmol) under ice-bath, and react for 24 h at room temperature. After the reaction is completed, wash with saturated sodium bicarbonate solution, 0.1% dilute hydrochloric acid solution, and saturated sodium chloride solution for three times, respectively. Dry with anhydrous sodium sulfate and remove water. Vacuum rotary evaporation to obtain N1-(Boc)Pbf-2-18 crude product. Purify by column chromatography, elute with DCM:MeOH=50:1 (V / V) to obtain N1-(Boc)Pbf-2-18 pure product.

[0201] Step 6: Precisely weigh N1-(Boc)Pbf-2-18 0.20 g into a three-necked flask, vacuumize, and protect with nitrogen. Add 2 mL of dichloromethane and 2 mL of trifluoroacetic acid under ice-bath, and react for 2 h. After the reaction is completed, remove the solvent by vacuum rotary evaporation, wash with ethyl ether for three times to obtain cationic lipopeptide N1-2-18.

[0202] 1 H NMR (300 MHz, DMSO) δ 8.69 (d, J = 7.3 Hz, 1H), 8.51 (d, J = 21.2 Hz, 2H), 8.21 (s, 5H), 7.93 (s, 3H), 7.34 (s, 2H), 4.43 - 3.44 (m, 5H), 3.08 (s, 10H), 2.74 (s, 2H), 1.23 (s, 80H), 0.90 - 0.79 (m, 6H).

[0203] Example 9: Preparation of M1-2-18

[0204] Step 1: Precisely weigh H-Lys(Fmoc)-OMe-HCl (4.00 g, 9.55 mmol), Boc-Lys(Boc)-OH (3.97 g, 11.46 mmol), HOBT (1.94 g, 14.32 mmol), EDCI (2.75 g, 14.32 mmol) into a three-necked flask, vacuumize, and protect with nitrogen. Add 150 mL of dichloromethane, slowly add DIPEA (16.63 mL, 95.49 mmol) under ice bath, and react at room temperature for 24 h. After the reaction is completed, remove the solvent by vacuum rotary evaporation, dissolve in ethyl acetate, and repeatedly wash with saturated sodium bicarbonate solution, 0.1% dilute HCl solution, and saturated NaCl solution, respectively. Dry and remove water by adding anhydrous sodium sulfate. Vacuum rotary evaporation gives M1-Fmoc crude product. Purify by column chromatography, elute with DCM:MeOH=100:1 (V / V), and obtain M1-Fmoc pure product.

[0205] Step 2: Precisely weigh M1-Fmoc 2.00 g into a three-necked flask, vacuumize, and protect with nitrogen. Add 8 mL of DMF and 2 mL of piperidine under ice bath, and react for 0.5 h. After the reaction is completed, remove the solvent by vacuum rotary evaporation, purify by column chromatography, elute with DCM:MeOH=60:1 (V / V), and obtain M1 pure product.

[0206] Step 3: Precisely weigh M1 (1.37 g, 2.82 mmol), Boc-Arg(Pbf)-OH (1.78 g, 3.38 mmol), HOBT (0.57 g, 4.22 mmol), and HBTU (1.60 g, 4.22 mmol) into a three-necked flask, vacuumize, and protect with nitrogen. Add 60 mL of DMF, slowly add DIPEA (3.93 mL, 22.52 mmol) under ice bath, and react at room temperature for 24 h. After the reaction is completed, remove the solvent by vacuum rotary evaporation, dissolve in ethyl acetate, and repeatedly wash with saturated sodium bicarbonate solution, 0.1% dilute HCl solution, and saturated NaCl solution, respectively. Dry and remove water by adding anhydrous sodium sulfate. Vacuum rotary evaporation gives N1-Fmoc crude product. Purify by column chromatography, elute with DCM:MeOH=60:1 (V / V), and obtain OMe-M1-Arg-(Boc)Pbf pure product.

[0207] Step 4: Precisely weigh OMe-M1-Arg-(Boc)Pbf 2.00 g into a round bottom flask, add NaOH in methanol solution (2 g NaOH dissolved in 50 mL of methanol), react for 6 h under ice bath condition, add 1 M HC1 to adjust pH to 2-3. Remove the solvent by vacuum rotary evaporation, dissolve with ethyl acetate, wash with saturated NaCl solution for three times, add anhydrous sodium sulfate to dry and remove water. Remove the solvent by vacuum rotary evaporation to obtain the pure product of OH-M1-Arg-(Boc)Pbf.

[0208] Step 5: Precisely weigh OH-M1-Arg-(Boc)Pbf (1.04 g, 1.06 mmol), intermediate B (0.50 g, 0.89 mmol), HOBT (0.36 g, 2.66 mmol), EDCI (0.51 g, 2.66 mmol) into a three-necked flask, vacuumize, and protect with nitrogen. Add 50 mL of dichloromethane to dissolve, slowly add DIPEA (1.23 mL, 7.08 mmol) under ice bath, and react for 24 h at room temperature. After the reaction is completed, wash with saturated sodium bicarbonate solution, 0.1% HC1 solution, and saturated NaCl solution for three times in sequence. Add anhydrous sodium sulfate to dry and remove water. Remove the solvent by vacuum rotary evaporation to obtain the crude product of M1-(Boc)Pbf-2-18. Purify by column chromatography, elute with DCM:MeOH = 50:1 (V / V) to obtain the pure product of M1-(Boc)Pbf-2-18.

[0209] Step 6: Precisely weigh M1-(Boc)Pbf-2-18 0.20 g into a three-necked flask, vacuumize, and protect with nitrogen. Add 2 mL of dichloromethane and 2 mL of trifluoroacetic acid under ice bath, and react for 2 h. After the reaction is completed, remove the solvent by vacuum rotary evaporation, wash with ethyl ether for three times to obtain the cationic lipopeptide M1-2-18.

[0210] 1 H NMR (300 MHz, DMSO) δ 8.76 - 7.79 (m, 11H), 7.34 (s, 2H), 4.19 (s, 2H), 3.76 (d, J = 26.4 Hz, 3H), 3.08 (s, 10H), 2.75 (s, 2H), 1.23 (s, 80H), 0.89 - 0.81 (m, 6H).

[0211] Example 10: Preparation of mRNA-LNP

[0212] A 10 mg / mL solution of ionizable lipids, DSPC, cholesterol, and DMG-PEG-2000 in ethanol was mixed thoroughly at a predetermined molar ratio (ionizable lipids: cofactor lipids: cholesterol: PEG lipids = 48:12:38.5:1.5) to obtain a lipid mixture. The lipid mixture was then rapidly added to three volumes of a pH 4 citrate buffer containing mRNA and vortexed (mass ratio = 15:1). Ultrafiltration was performed using a 10 kDa ultrafiltration tube at 4°C to obtain mRNA-LNP.

[0213] Table 1

[0214]

[0215] Example 11: Preparation of siRNA-LNP

[0216] A 10 mg / mL solution of NL-1, cationic lipopeptides, cholesterol, and DMG-PEG-2000 in ethanol was mixed thoroughly at a predetermined molar ratio (ionizable lipids: cationic lipopeptides: cholesterol: PEG lipids = 48:1.5:38.5:1.5) to obtain a lipid mixture solution. The lipid mixture solution was then rapidly added to three volumes of a pH 4 citrate buffer solution containing siRNA and vortexed (N:P = 10:1). Ultrafiltration was performed using a 10 kDa ultrafiltration tube at 4°C to obtain siRNA-LNP.

[0217] Table 2

[0218]

[0219] Example 12: Particle size potential determination of LNP

[0220] Take the above LNPs and place them in a centrifuge tube. Dilute with PBS buffer to 1 mL and place in a Malvern plastic sample cell and a potential sample cell. Place the tube in the slot of a laser particle size analyzer and measure the particle size and potential of each LNP at 25°C. Perform three parallel measurements.

[0221] The results are as follows Figure 1 , Figure 2 As shown. Figure 1 The LNPs shown have a particle size range of 80-200 nm and exhibit excellent nucleic acid delivery performance. Figure 2 As shown, at pH = 7.4, the zeta potential of LNP is between 0 and -4 mV, which can reduce electrostatic interactions with other substances.

[0222] Example 13: Encapsulation efficiency determination of LNP

[0223] Using StrandBrite TMGreen Fluorimetric RNA Quantitation Kit kit and multifunctional microplate reader SpectraMax M2e were used to determine the LNP encapsulation rate. LNP was treated with 2% Triton-x-100 as a demulsifier to obtain the total nucleic acid amount. The encapsulation rate was calculated by determining the amount of free nucleic acid before demulsification and the total nucleic acid amount.

[0224] The structure is shown as Figure 3 The measured encapsulation rate of LNP is between 80% and 98%, which is a good nucleic acid delivery carrier.

[0225] Example 14: Transfection efficiency determination of mRNA-LNP

[0226] Logarithmic growth phase Hela cells were inoculated in a 24-well plate, 4x10 4 cells per well, and cultured at 37°C, 5% CO2, saturated humidity incubator for 24h. When the cell density reached more than 60%, the culture medium was discarded, and the cells were washed twice with PBS. 500μL of EGFP mRNA-LNP (2μg / mL) solution prepared with double-antibody-free DMEM basic medium was added to each well, and three replicate wells were set for each group. Double-antibody-free DMEM basic medium was used as a blank control group, and commercially available DLin-MC3-DMA-LNP was used as a control. The culture medium was discarded 6 hours after administration, and 500μL of DMEM complete medium containing 10% fetal bovine serum was added to each well for continued culture for 18 hours. The EGFP fluorescence expression of cells in each group was observed using an inverted fluorescence microscope, and the average fluorescence intensity was calculated using ImageJ.

[0227] The results are shown in Figure 4 Each group had a high fluorescence intensity, significantly higher than that of commercially available DLin-MC3-DMA-LNP, indicating that mRNA-LNP had a high transfection efficiency and good transfection performance.

[0228] Example 15: Transfection efficiency determination of siRNA-LNP

[0229] Logarithmic growth phase 4T1-Luc cells were inoculated in a 96-well plate, 8x10 3The cells were incubated at 37°C in a 5% CO2 saturated humidity incubator for 24 h. When the cell density reached more than 60%, the culture medium was discarded, and the cells were washed twice with PBS. Then, 100 μL of siLuc-LNP (containing 0.15 μg siLuc per well) solution prepared with DMEM basic medium without double antibodies was added to each well. Six replicate wells were set for each group. RPMI-1640 basic medium without double antibodies was used as a blank control group, and commercially available DLin-MC3-DMA-LNP was used as a positive control. After 6 h of administration, the culture medium was discarded, and 100 μL of RPMI-1640 complete medium containing 10% fetal bovine serum was added to each well for further incubation for 18 h. The luciferase gene expression was determined using a luciferase reporter gene detection kit, and the gene silencing efficiency was calculated.

[0230] The results, as shown in Table 1, indicated that the gene silencing efficiency of each group was 50%-70%, which was significantly better than that of commercially available DLin-MC3-DMA-LNP, indicating that siRNA-LNP had high transfection efficiency and good transfection performance. Figure 5

[0231] As described above, although the present application has been shown and described with reference to specific preferred embodiments, it is to be understood that various modifications and changes can be made to it without departing from the spirit and scope of the application defined in the appended claims.​

Claims

1. A nanoparticle composition comprising a lipid component and optionally a loading agent; wherein, The lipid component contains ionizable lipids and cationic lipopeptides, with the ratio of cationic lipopeptides to ionizable lipids being 1:40 to 1:

20. The cationic lipopeptides are compounds of formula (b1) or pharmaceutically acceptable salts of compounds of formula (b1). (b1)； R6 and R7 are independently selected from C6. C 24 alkyl; L3 is selected from -(CH2) q - q is selected from integers from 1 to 6, where RH1 and RH2 are independently selected from lysine and arginine: or Lysine and arginine are linked to their corresponding nitrogen atoms via amide bonds.

2. The nanoparticle composition of claim 1, wherein the cationic lipopeptide is: (G2R-2-18) (G2K-2-18) （N1-2-18） （M1-2-18） 3. The nanoparticle composition of claim 1 or 2, wherein the nanoparticle composition comprises ionizable lipids, cationic lipopeptides, sterols, PEG lipids, and bioactive substances.

4. The nanoparticle composition of claim 3, wherein the composition does not contain auxiliary lipids.

5. The nanoparticle composition of claim 4, wherein the ionizable lipid is an ionizable lipid compound of formula (a2) or a pharmaceutically acceptable salt: （a2） in, p = 0, q = 4, B = C 1-4 Straight-chain or branched alkyl groups, R 2-5 Independently selected from C6 C 24 alkyl.

6. The nanoparticle composition of claim 5, wherein the ionizable lipid is: (NL-1)。 7. The nanoparticle composition according to any one of claims 3-6, wherein the bioactive substance is a nucleic acid drug.

8. The nanoparticle composition of claim 7, wherein the nucleic acid drug is selected from siRNA, mRNA, microRNA, circular mRNA, snRNA, snoRNA, tRNA, rRNA, gRNA, shRNA, piRNA, rasiRNA, hnRNA, longnon RNA, etc. One or more of the following: coding RNA, plasmid DNA, ceDNA, mini circle DNA, and antisense oligonucleotides.

9. A pharmaceutical composition comprising the nanoparticle composition according to any one of claims 1-8, and optionally pharmaceutically acceptable excipients.

10. Use of the nanoparticle composition of any one of claims 1-8 or the pharmaceutical composition of claim 9 in the preparation of a medicament for treating, diagnosing or preventing a disease.

11. Amphiphilic cationic lipopeptides, selected from: (G2R-2-18) （N1-2-18） （M1-2-18）

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