Polyamino cationic lipids containing a urethane structure, compositions comprising the same and uses

Abstract

The application belongs to the field of medicine, and discloses a polyamino cationic lipid containing a urethane structure, a composition containing the same and purposes, and specifically discloses a cationic lipid shown in formula (I). The cationic lipid provided by the application can be used for nucleic acid targeted delivery, can reduce liver enrichment, and can significantly enhance the spleen targeting of nucleic acid drugs.

Description

Technical Field

[0001] This disclosure pertains to the pharmaceutical field and specifically relates to polyamino cationic lipids containing a carbamate structure, compositions containing such lipids, and their uses. Background Technology

[0002] In the pharmaceutical field, efficient targeted delivery of small molecule drugs, peptides, proteins, and nucleic acids remains a persistent challenge. The delivery of nucleic acids, in particular, faces significant challenges due to their low cell permeability and high sensitivity to degradation by nucleases such as RNases.

[0003] Compositions containing cationic lipids, liposomes, and lipoplexes serve as transport media, effectively delivering bioactive substances such as small molecule drugs, peptides, proteins, and nucleic acids into cells and / or intracellular compartments. These compositions typically contain one or more cationic and / or ionizable lipids, neutral lipids, structural lipids, and polymer-conjugated lipids. Cationic and / or ionizable lipids include, for example, readily ionizable amine-containing lipids. Although various such lipid-containing nanoparticle compositions have been demonstrated, their safety, efficacy, and specificity remain to be improved. Notably, the increased complexity of lipid nanoparticles (LNPs) complicates their production and may increase their toxicity, a major concern that could limit their clinical application. For example, nucleic acid drugs, exemplified by the small interfering RNA drug Patisiran (trade name onpattro®), require prior administration of steroids and antihistamines to patients to eliminate unwanted immune responses.

[0004] Recent studies have designed ionizable lipids for selective mRNA delivery in the spleen, thereby reducing off-target accumulation in organs such as the liver. Nevertheless, further research is needed to optimize LNP formulations, improve their efficacy and safety, and ultimately meet the stringent standards required for medical applications. Summary of the Invention

[0005] The technical problem this disclosure aims to solve is to overcome the shortcomings of existing technologies, such as poor safety or targeted delivery of ionizable lipids and the tendency for off-target accumulation in organs such as the liver. This disclosure provides polyamino cationic lipids containing carbamate structures, compositions containing them, and their uses. The cationic lipids provided in this disclosure are ionizable cationic lipids containing multiple amino and hydroxyl groups, capable of delivering therapeutic or preventative agents such as nucleic acid molecules, small molecule compounds, peptides, or proteins. They are characterized by simple preparation methods, strong targeting to the spleen, and the ability to carry active pharmaceutical ingredients to transfect cells with high transfection efficiency, while exhibiting low cytotoxicity. This improves the efficiency and safety of spleen-targeted drug delivery in vivo.

[0006] To achieve the above objectives, a first aspect of this disclosure provides a cationic lipid compound or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer, wherein the cationic lipid compound has the structure shown in formula (I):

[0007] ,

[0008] in:

[0009] R1 is a substituted or unsubstituted N-containing group, wherein the N-containing group is cyclic or non-cyclic;

[0010] R2 and R3 are each independently substituted or unsubstituted C. 5-25 Straight-chain or branched olefins, or substituted or unsubstituted C 5-25 Straight-chain or branched-chain alkanes;

[0011] L1 represents substituted or unsubstituted C. 1-5 Straight-chain alkylene;

[0012] L2 and L3 are independently substituted or unsubstituted C. 2-8 Straight-chain alkylene;

[0013] L4 is the replacement for C 1-10 Straight-chain alkylene groups, wherein the substituents are selected from -OH;

[0014] M1 and M2 are independently -OC(O)- or -C(O)O-.

[0015] In this disclosure, "each independently" means that the (multiple) positions referred to are selected from the corresponding range of group structures, which may be the same or different.

[0016] The inventors discovered through extensive research that, in the compound of formula (I) provided in this disclosure (i.e., the "cationic lipid compound provided in this disclosure"), when L4 and R1 have specific structures, the compound can be used to prepare lipid compositions (drug carriers) with good spleen targeting. In contrast, the selection of L1, L2, L3, and R2 and R3 can be more flexible.

[0017] According to a preferred embodiment of the present disclosure, in the compound of formula (I), R1 can be an N-containing group with a total number of 2-10 C atoms and N atoms, preferably with 1-3 N atoms.

[0018] For example, in compound (I), the total number of C and N atoms in R1 can be 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0019] For example, in compound (I), the number of N atoms in R1 can be 1, 2, or 3.

[0020] Preferably, R1 is connected to L4 through one of its N atoms. When R1 has more than one N atom, it can be connected to L4 through any one of its N atoms.

[0021] In some preferred embodiments, R1 is a straight-chain or branched group containing N, preferably a branched group containing N.

[0022] In some preferred embodiments, R1 is a cyclic N-containing group, preferably a ternary to ten-membered ring. When R1 has more than one N atom, at least one N atom is present in the cyclic structure, and preferably all N atoms are present in the cyclic structure.

[0023] Preferably, when R1 is a cyclic N-containing group, it can be a cyclic structure (a ring composed of a total of 3-10 N atoms and C atoms), or it can be a cyclic structure further including branches (a ring composed of a portion of N atoms and C atoms, and having at least one branch containing N atoms and / or C atoms on the ring, and the total number of N atoms and C atoms on the cyclic structure and the branch is 3-10).

[0024] According to a preferred embodiment of this disclosure, R1 is... , , , , and And any of their derived structures. A “derived structure” refers to a structure formed by further substitution or reaction with other compounds based on the above structures.

[0025] For example, in some preferred embodiments, R1 can be selected from any of the following structures:

[0026] , where R 1-1 Selected from C 1-5 Any one or a combination of straight-chain or branched alkyl groups and halogens, or the default;

[0027] , where R 1-2 Selected from C 1-5 Any one or a combination of straight-chain or branched alkyl groups and halogens, or the default;

[0028] , where R 1-3 Selected from C 1-5 Any one or a combination of straight-chain or branched alkyl groups and halogens, or the default;

[0029] , where R 1-4 Selected from C 1-5Any one or a combination of straight-chain or branched alkyl groups and halogens, or the default;

[0030] , where R 1-5 and R 1-6 Each is independently selected from C 1-5 The straight-chain or branched alkyl group and any one or a combination of at least two of the halogens, or the default.

[0031] In the compound of formula (I), the number of C atoms in L4 can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0032] In a preferred embodiment of this disclosure, the substituent of L4 may be (at least one) -OH.

[0033] In some particularly preferred embodiments, L4 is .

[0034] According to a preferred embodiment of this disclosure, R2 is substituted or unsubstituted C. 8-20 Straight-chain or branched alkanes. For example, the number of carbon atoms in R2 can be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. When R2 is a substituted C... 8-20 When the alkanes are straight-chain or branched, the substituents may include any or at least a combination of halogens.

[0035] The inventors of this disclosure have also discovered in their research that when at least one of R2 and R3 is a branched alkyl group, the lipid composition (drug carrier) using the compound of formula (I) exhibits better spleen targeting (i.e., in the preferred embodiment of this disclosure, at least one of R2 and R3 is a branched alkyl group). The following will further illustrate this by using both straight-chain and branched alkyl groups as examples, but those skilled in the art should be able to deduce from this the structure and effects of the preferred compound of formula (I) provided in this disclosure when R3 can be a straight-chain alkyl group, and understand that it also falls within the scope of this disclosure.

[0036] In some particularly preferred embodiments, R2 is unsubstituted C 10-20 Branched alkanes (most preferred) ) or unreplaced C 8-16 Straight-chain alkanes.

[0037] According to a preferred embodiment of this disclosure, R3 is substituted or unsubstituted C. 8-20 Straight-chain or branched alkanes. For example, the number of carbon atoms in R3 can be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. When R3 is a substituted C... 8-20 For straight-chain or branched alkanes, the substituents may include at least halogens.

[0038] Preferably, R3 is unsubstituted C. 8-20 Branched alkanes.

[0039] In some particularly preferred embodiments, R3 is unsubstituted C. 10-20 Branched alkanes (most preferred) or ).

[0040] In the compound of formula (I), the number of C atoms in L1 can be 1, 2, 3, 4 or 5.

[0041] In a preferred embodiment of this disclosure, when L1 is a substituted C 1-5 When the chain is alkylene, the substituent may include at least any one of the halogens or a combination of at least two of them.

[0042] Preferably, L1 can be unsubstituted C 1-5 Straight-chain alkylene (most preferably -(CH2)2-).

[0043] In the compound of formula (I), the number of C atoms in L2 and L3 can be 2, 3, 4, 5, 6, 7 or 8, respectively.

[0044] In a preferred embodiment of this disclosure, when L2 and / or L3 are substituted C 2-8 When the chain is alkylene, the substituent may include any or a combination of at least two halogens.

[0045] In some preferred embodiments, L2 is unsubstituted C 3-5 Straight-chain alkylene (most preferably -(CH2)3- or -(CH2)5-).

[0046] In some preferred embodiments, L3 is unsubstituted C 3-5 Straight-chain alkylene (most preferably -(CH2)5-).

[0047] In some preferred embodiments, M1-C(O)O-.

[0048] In some preferred embodiments, M2 is -C(O)O-.

[0049] According to some further preferred embodiments, the cationic lipid compound may have the structure shown in formula (II):

[0050] ,

[0051] In equation (II), the selection range and preferred selection range of R1, R2, R3, L1, L2 and L3 are the same as those in equation (I) above, and will not be repeated here.

[0052] According to some particularly preferred embodiments of this disclosure, the compound represented by formula (I) is any one of the following compounds (represented by YK-2001 to YK-2018):

[0053] ,

[0054] ,

[0055] ,

[0056] ,

[0057] ,

[0058] ,

[0059] ,

[0060] ,

[0061] ,

[0062] ,

[0063] ,

[0064] ,

[0065] ,

[0066] ,

[0067] ,

[0068] ,

[0069] ,

[0070] .

[0071] A second aspect of this disclosure provides a composition comprising a cationic lipid, the cationic lipid comprising the cationic lipid compound of the first aspect or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer.

[0072] The compositions provided in this disclosure can be used as delivery carriers (in this disclosure, the compositions may also be referred to as "carriers", "liposomes", etc.) to achieve targeted delivery of active ingredients (such as pharmaceutical active ingredients) in vivo, especially for targeted delivery to the spleen.

[0073] To further improve targeted delivery, other types of lipids may be added to the composition. According to a preferred embodiment of this disclosure, the composition further comprises any one or a combination of at least two of neutral lipids, structural lipids, and polymer-conjugated lipids.

[0074] According to a preferred embodiment of this disclosure, the composition satisfies any one or a combination of at least two of the following conditions (1) to (4):

[0075] (1) The molar ratio of the cationic lipid to all lipid compounds in the composition is 0.25:1-0.75:1 (e.g., 0.25:1, 0.3:1, 0.35:1, 0.4:1, 0.45:1, 0.5:1, 0.55:1, 0.6:1, 0.65:1, 0.7:1, 0.75:1, or any range of any two of the above ratios, or any intermediate ratio within that range).

[0076] (2) The molar ratio of the cationic lipid to the neutral lipid is 1:1-15:1 (e.g., 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 9.5:1, 10:1, 10.5:1, 11:1, 11.5:1, 12:1, 12.5:1, 13:1, 13.5:1, 14:1, 14.5:1, 15:1, or any range of any two of the above ratios, or any intermediate ratio within that range).

[0077] (3) The molar ratio of the structural lipid to all lipid compounds in the composition is 0.15:1-0.65:1 (e.g., 0.15:1, 0.2:1, 0.25:1, 0.3:1, 0.35:1, 0.4:1, 0.45:1, 0.5:1, 0.55:1, 0.6:1, 0.65:1, or any range of any two of the above ratios, or any intermediate ratio within that range).

[0078] (4) The molar ratio of the polymer conjugated lipid to all lipid compounds in the composition is 0.005:1-0.1:1 (e.g. 0.005:1, 0.008:1, 0.01:1, 0.015:1, 0.02:1, 0.025:1, 0.03:1, 0.035:1, 0.4:1, 0.045:1, 0.05:1, 0.055:1, 0.06:1, 0.065:1, 0.07:1, 0.075:1, 0.08:1, 0.085:1, 0.09:1, 0.095:1, 0.1:1, or any range of any two of the above ratios, or any intermediate ratio within that range).

[0079] In some preferred embodiments, the composition satisfies any one or a combination of at least two of the following conditions (1) to (4):

[0080] (1) The molar ratio of the cationic lipid to all lipid compounds in the composition is 0.35:1, 0.4:1, 0.45:1, 0.49:1 or 0.65:1;

[0081] (2) The molar ratio of the cationic lipid to the neutral lipid is 3:1, 3.5:1, 4:1, 4.9:1 or 10:1;

[0082] (3) The molar ratio of the structural lipid to all lipid compounds in the composition is 0.2:1, 0.25:1, 0.395:1, 0.435:1, 0.485:1 or 0.535:1;

[0083] (4) The molar ratio of the polymer conjugated lipid to all lipid compounds in the composition is 0.015:1, 0.025:1, 0.035:1 or 0.05:1.

[0084] According to a preferred embodiment of this disclosure, the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid in the composition is (25-75):(5-25):(15-65):(0.5-10). That is, in the composition, the cationic lipid can be 25-75 molar parts, the neutral lipid can be 5-25 molar parts, the structural lipid can be 15-65 molar parts, and the polymer-conjugated lipid can be 0.5-10 molar parts. It should be noted that the sum of the molar parts of the cationic lipid, neutral lipid, structural lipid, and polymer-conjugated lipid in the composition should be 100.

[0085] For example, in the composition, the cationic lipid can be 25 moles, 30 moles, 35 moles, 40 moles, 45 moles, 50 moles, 55 moles, 60 moles, 65 moles, 70 moles, 75 moles, or a range consisting of any two of the above values, or any intermediate value within that range.

[0086] For example, in the composition, the neutral lipids can be 5 moles, 8 moles, 10 moles, 12 moles, 15 moles, 18 moles, 20 moles, 22 moles, 25 moles, or a range consisting of any two of the above values, or any intermediate value within that range.

[0087] For example, in the composition, the structural lipid can be 15 moles, 20 moles, 25 moles, 30 moles, 35 moles, 40 moles, 45 moles, 50 moles, 55 moles, 60 moles, 65 moles, or a range consisting of any two of the above values, or any intermediate value within that range.

[0088] For example, in the composition, the polymeric conjugated lipid can be 0.5 moles, 1 mole, 2 moles, 3 moles, 4 moles, 5 moles, 6 moles, 7 moles, 8 moles, 9 moles, 10 moles, or any range of any two of the above values, or any intermediate value within that range.

[0089] In a particularly preferred embodiment, the molar ratio of the cationic lipid, neutral lipid, structural lipid, and polymer conjugated lipid in the composition is 49:10:39.5:1.5.

[0090] According to a preferred embodiment of this disclosure, the composition further satisfies any one or a combination of at least two of the following conditions (5) to (7):

[0091] (5) The neutral lipids are selected from any one or a combination of at least two of the group consisting of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide and sterol;

[0092] Preferably, the neutral lipid is selected from any or at least a combination of the following: 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline, 1,2-dimyristoyl-sn-glycerol-3-phosphate choline, 1,2-dioleoyl-sn-glycerol-3-phosphate choline, 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline, 1,2-distearateoyl-sn-glycerol-3-phosphate choline, 1,2-diundecanoyl-sn-glycerol-3-phosphate choline, 1-palmitoyl-2-oleoyl 1,2-Di-O-octadecenyl-sn-glycerol-3-phosphate choline, 1-oleoyl-2-cholesterolylhemisuccinoyl-sn-glycerol-3-phosphate choline, 1-hexadecyl-sn-glycerol-3-phosphate choline, 1,2-dilinyl-sn-glycerol-3-phosphate choline, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate choline, 1,2-bis(docosahexaenooyl)-sn-glycerol-3-phosphate choline, 1,2-dioleoyl-sn- Glycerol-3-phosphate ethanolamine, 1,2-diphydanoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-distearatel-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinolenoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docohexanoyl)-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate -rac-(1-glycerol) sodium salt, dipalmitoylphosphatidylglycerol, palmitoyloleoylphosphatidylethanolamine, distearyl-phosphatidyl-ethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphoethanolamine, 1-stearoyl-2-oleoyl-stearoylethanolamine, 1-stearoyl-2-oleoylphosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine and lysophosphatidylethanolamine;

[0093] More preferably, the neutral lipid is 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine or 1,2-distearate-sn-glycerol-3-phosphate choline;

[0094] (6) The structural lipids are selected from any one or at least two of the following groups: cholesterol, nonsterols, sitosterol, ergosterol, campesterol, stigmasterol, brassinosteroids, tomatine, ursolic acid, α-tocopherol and corticosteroids;

[0095] Preferably, the structural lipid is cholesterol;

[0096] (7) The polymer conjugated lipid is selected from any one or a combination of at least two of the following: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol and PEG-modified dialkylglycerol.

[0097] Preferably, the polymeric conjugated lipid is selected from any one or a combination of at least two of the following: distearylphosphatidylethanolamine polyethylene glycol 2000, 1,2-dimyristoyl-sn-glycerol-3-methoxy polyethylene glycol 2000 and methoxy polyethylene glycol bistetradecylacetamide;

[0098] More preferably, the polymer conjugated lipid is 1,2-dimyristoyl-sn-glycerol-3-methoxy polyethylene glycol 2000.

[0099] According to a preferred embodiment of this disclosure, the composition further includes one or more other ionizable lipid compounds.

[0100] This disclosure provides a third aspect of a pharmaceutical composition comprising a carrier and an active ingredient, wherein the carrier comprises the composition described in the second aspect.

[0101] According to a preferred embodiment of this disclosure, the active ingredient comprises a therapeutic agent and / or a preventative agent.

[0102] According to a preferred embodiment of this disclosure, the pharmaceutical composition further includes pharmaceutically usable excipients.

[0103] In some preferred embodiments, the pharmaceutically available excipients may include excipients or diluents.

[0104] According to a preferred embodiment of this disclosure, the pharmaceutical composition satisfies any one or a combination of at least two of the following conditions (1) to (3):

[0105] (1) The pharmaceutical composition is a nanoparticle formulation, wherein the average particle size of the nanoparticle formulation is 70 nm-180 nm; and the polydispersity index of the nanoparticle formulation is less than 0.10.

[0106] (2) The mass ratio of the carrier to the active component is 10:1-30:1;

[0107] (3) The active component is selected from any one of the groups consisting of nucleic acids, small molecule compounds, polypeptides or proteins, or at least a combination of two.

[0108] In this disclosure, the active ingredient is preferably a nucleic acid. The pharmaceutical composition of this disclosure may contain any nucleic acid commonly used in nucleic acid pharmaceuticals as an active ingredient, such as RNA or DNA, preferably RNA.

[0109] According to some preferred embodiments of this disclosure, the nucleic acid is selected from any or a combination of at least two of the following: small interfering RNA, asymmetric interfering RNA, microRNA, Dicer-substrate RNA, small hairpin RNA, and messenger RNA.

[0110] According to some preferred embodiments of this disclosure, the mass ratio of the carrier to the active component is 15:1 to 20:1. For example, the mass ratio of the carrier to the active component can be 15:1, 15.5:1, 16:1, 16.5:1, 17:1, 17.5:1, 18:1, 18.5:1, 19:1, 19.5:1, 20:1, or any range formed by any two of the above ratios, or any intermediate ratio within that range.

[0111] This fourth aspect of disclosure provides the use of the cationic lipid compound of the first aspect or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, or the composition of the second aspect, or the pharmaceutical composition of the third aspect, in the preparation of a medicament for treating a disease or condition.

[0112] Thanks to the properties of the cationic lipid compounds provided in this disclosure, the above uses may include their use in the preparation of medicaments having spleen-targeting properties for the treatment of diseases or conditions.

[0113] This disclosure does not impose any particular limitation on the diseases or conditions described herein; any disease or condition that can be treated by targeted administration of drugs to the spleen (especially nucleic acid drugs) falls within the scope of this disclosure. According to a preferred embodiment of this disclosure, the disease or condition is characterized by dysfunction or abnormality of a protein or polypeptide.

[0114] In some preferred embodiments, the disease or condition is selected from any one or a combination of at least two of the following: infectious diseases, cancer, proliferative diseases, genetic diseases, autoimmune diseases, neurodegenerative diseases, cardiovascular and cerebrovascular diseases, renal and vascular diseases, and metabolic diseases.

[0115] Furthermore, this disclosure also provides a method for treating a disease or condition, which may include administering to a subject in need a medicament prepared using a cationic lipid compound or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer as described in the first aspect of this disclosure, or a composition as described in the second aspect, or a pharmaceutical composition as described in the third aspect.

[0116] Furthermore, this disclosure also provides a method for improving the in vivo delivery targeting (preferably spleen targeting) of a drug, which may include loading the active ingredient of the drug onto a carrier (or at least as part of a carrier) using a cationic lipid compound or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer as described in the first aspect of this disclosure, or a composition as described in the second aspect.

[0117] Furthermore, this disclosure also provides a method for targeted delivery of an active ingredient to the spleen or spleen cells, the method comprising loading the active ingredient onto a carrier (or at least as part of a carrier) using a cationic lipid compound or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer as described in the first aspect of this disclosure, or a composition as described in the second aspect.

[0118] It should be understood that the uses / methods provided in this disclosure may include therapeutic and diagnostic uses / methods, as well as non-therapeutic and non-diagnostic uses / methods. For example, therapeutic uses / methods may include using the compounds provided in this disclosure, or their N-oxides, solvates, pharmaceutically acceptable salts, or stereoisomers, or using the compositions provided in this disclosure to package and deliver the (pharmaceutical) active ingredient to a target organ / tissue / cell, or using the pharmaceutical compositions of this disclosure to deliver the active ingredient contained therein to a target organ / tissue / cell, thereby achieving the effects of treating diseases, improving symptoms, regulating physiological activities in the body, etc.; diagnostic uses may include packaging the active ingredient for disease diagnosis in the compounds provided in this disclosure, or their N-oxides, solvates, etc. The active ingredient is delivered to target organs / tissues / cells via a compound, a pharmaceutically acceptable salt or stereoisomer, or a composition thereof, thereby achieving the purpose of disease diagnosis. Non-therapeutic / non-diagnostic purposes may include encapsulating the active ingredient with the compound provided in this disclosure, or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, or with the lipid composition provided in this disclosure, thereby delivering it to target organs / tissues / cells for scientific research, detection, and other non-therapeutic and non-diagnostic purposes (such as conducting disease mechanism research, drug action mechanism research, new drug development, drug screening, etc.).

[0119] Terminology Definition

[0120] All publications and patents mentioned in this disclosure are incorporated herein by reference in their entirety. In the event of any conflict between the use or terminology used in any publications and patents incorporated by reference and the use or terminology used in this disclosure, the use and terminology of this disclosure shall prevail.

[0121] The chapter titles used in this article are for organizational purposes only and should not be construed as limiting the subject matter.

[0122] Unless otherwise specified, all technical and scientific terms used herein have their usual meaning in the field to which the claimed subject matter pertains. Where multiple definitions exist for a term, the definition herein shall prevail.

[0123] Unless otherwise indicated in the working embodiments or elsewhere, all numerical values ​​for quantitative properties such as dosages set forth in the specification and claims should be understood to be modified by the term "about" in all cases. It should also be understood that any numerical range enumerated in this disclosure is intended to include all subranges within that range and any combination of the endpoints of that range or subranges. When a numerical range is disclosed herein, the range is considered continuous and includes both the minimum and maximum values ​​of the range, and every value between such minimum and maximum. Further, when a range refers to an integer, it includes every integer between the minimum and maximum values ​​of the range. Furthermore, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0124] Furthermore, in this disclosure, when numerical ranges are used in the general formula and / or structural formula of a compound, it means that the number of the corresponding groups within that numerical range can be any natural number within that range, such as "C". A-B "" refers to any integer number of carbon atoms within the range from the starting point to the ending point, where A and B are both integers; for example, C 1-5 The number of carbon atoms indicates 1, 2, 3, 4, or 5; that is, when combined with other groups in the general formula and / or structural formula of a compound to form various possible compounds, C A-B It can be used in conjunction with any group containing carbon atoms to specify the number of carbon atoms, such as C. 1-5 Alkyl / alkylene compounds represent various possibilities of alkyl / alkylene compounds having 1 carbon, 2 carbon, 3 carbon, 4 carbon, and / or 5 carbon.

[0125] In the structures disclosed herein, the dashed lines next to the substituent chemical groups (such as R1, R2, R3, L4, etc.) only represent the connection sites between the group and the general formula core structure, and are not chemical bonds. They are used to clarify the binding position of the substituent on the core and do not participate in the formation of chemical bonds.

[0126] As used herein, the words “comprising,” “containing,” or “including” mean that the element preceding the word encompasses the elements listed following the word and their equivalents, without excluding elements not described. The terms “containing,” “including,” or “comprising” as used herein can be open-ended, semi-closed, or closed-ended. In other words, the above terms also include “consistently composed of” or “composed of.”

[0127] The term “pharmaceutically acceptable” in this disclosure means that a compound or composition is chemically and / or toxicologically compatible with other components constituting the formulation and / or with humans or mammals for the prevention or treatment of diseases or conditions.

[0128] The terms “subject” or “patient” in this disclosure include both humans and mammals. In this document, “subject” or “patient” also appears in some instances as “object”.

[0129] As used herein, the term "treatment" refers to the administration of one or more pharmaceutical substances to a patient or subject suffering from a disease or having symptoms of said disease, in order to cure, alleviate, reduce, improve, or affect said disease or its symptoms. As used herein, the term includes prevention of the worsening of said disease, condition, or related symptoms. It should be understood that treatment, as used herein, may not be effective for all subjects to be treated. However, preferably, the term should require that a statistically significant proportion of subjects suffering from the disease or condition described herein can be successfully treated. Statistical significance can be determined by a variety of well-known statistical assessment tools, such as confidence interval determination, p-value determination, t-test, Mann-Whitney test, etc. In some embodiments, treatment includes inhibiting the proliferation of cancer cells, preferably including killing cancer cells. Preferably, treating cancer is reducing the tumor and / or cancer cell burden in the subject. As those skilled in the art will understand, the effectiveness of cancer treatment depends on a variety of factors, including, for example, cancer stage and cancer type. It is also preferred that cancer treatment further includes at least one of chemotherapy, immunotherapy, surgery, and radiation therapy.

[0130] The term "prevention" refers to maintaining health associated with the disease or condition described herein in a subject for a period of time. It should be understood that this period of time may depend on the amount of medication administered and individual factors of the subject. It should be understood that prevention may not be effective in all subjects treated. However, preferably, the term requires the effective prevention of a statistically significant proportion of a group or population of subjects suffering from the disease or condition described herein or its accompanying symptoms. Statistical significance can be determined by various well-known statistical assessment tools, such as confidence interval determination, p-value determination, t-test, Mann-Whitney test, etc. In the case of cancer treatment, prevention specifically relates to preventing cancer development, preventing metastasis formation, and / or preventing recurrence, preferably involving the prevention of metastasis formation and / or preventing recurrence.

[0131] The term "infectious disease" refers to a disease state or condition caused by a foreign organism (e.g., microorganism, parasite, etc.) that enters a subject, multiplies, and elicits a response in the subject (e.g., inflammatory response, immune response, etc.). In some implementations, infectious diseases are caused by viruses (or viral particles), bacteria, or fungi. Examples of "infectious diseases" include, but are not limited to, diseases caused by coronaviruses (e.g., SARS-CoV-2), influenza viruses, hepatitis viruses (e.g., hepatitis B virus), immunodeficiency viruses (e.g., human immunodeficiency virus), rabies virus, papillomavirus (e.g., human papillomavirus), respiratory syncytial virus (RSV), herpesviruses (e.g., herpes simplex virus type 1, herpes simplex virus type 2, varicella-zoster virus, Epstein-Barr virus, cytomegalovirus, etc.), pneumonia (e.g., viral pneumonia, bacterial pneumonia, etc.), Rift Valley fever (RVF), yellow fever, etc.

[0132] "Therapeutic effective amount" is the amount of a therapeutic agent that, when administered to a patient, improves the disease or symptoms. "Prophylactic effective amount" is the amount of a preventive agent that, when administered to a subject, prevents the disease or symptoms. The amount of a therapeutic agent constituting a "therapeutic effective amount" or a preventive agent constituting a "prophylactic effective amount" varies depending on the therapeutic / preventive agent, the disease state and its severity, the age and weight of the patient / subject to be treated / prevented, etc. Those skilled in the art can determine the therapeutic and prophylactic effective amounts conventionally based on their knowledge and this disclosure.

[0133] The term "solvent" in this disclosure refers to a complex formed by the combination of a compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof with a solvent (e.g., ethanol or water). It should be understood that any solvate of a compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof used in the treatment of a disease or condition, although it may provide different properties (including pharmacokinetic properties), will, once absorbed into a subject, yield a compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof, such that the use of a compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof respectively encompasses the use of any solvate of a compound of formula (I).

[0134] The term "hydrate" refers to the case where the solvent in the aforementioned term "solvent" is water.

[0135] It should be further understood that compounds of formula (I) or pharmaceutically acceptable salts or stereoisomers thereof can be isolated as solvates, and therefore any such solvates are included within the scope of this disclosure. For example, compounds of formula (I) or pharmaceutically acceptable salts thereof may exist in an unsolvated form or in a solvated form formed by combining with pharmaceutically acceptable solvents (such as water, ethanol, etc.).

[0136] The term "pharmaceutically acceptable salt" refers to a relatively non-toxic inorganic or organic acid addition salt of the compounds disclosed herein. Inorganic acids include, for example, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, or nitric acid; organic acids include, formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, hexanoic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)-benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, bamoic acid, pectinic acid, 3-phenylpropionic acid, picric acid, etc. Pteropenic acid, 2-hydroxyethanesulfonic acid, itaconic acid, aminosulfonic acid, trifluoromethanesulfonic acid, dodecyl sulfate, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheponic acid, glycerophosphate, aspartic acid, sulfosalicylic acid, etc. For example, HCl (or hydrochloric acid), HBr (or hydrobromic acid solution), methanesulfonic acid, sulfuric acid, tartaric acid, or fumaric acid can be used to form pharmaceutically acceptable salts with the compounds shown in formula (I).

[0137] The nitrogen-containing compounds of formula (I) of this disclosure can be converted into N-oxides by treatment with an oxidizing agent (e.g., m-chloroperoxybenzoic acid, hydrogen peroxide, ozone). Therefore, provided that the valence state and structure allow, the compounds claimed in this disclosure include not only the nitrogen-containing compounds shown in the structural formula, but also their N-oxide derivatives.

[0138] Some of the compounds disclosed herein can exist in the form of one or more stereoisomers. Stereoisomers include geometric isomers, diastereomers, and enantiomers. Therefore, the compounds claimed in this disclosure also include racemic mixtures, single stereoisomers, and optically active mixtures. Those skilled in the art should understand that one stereoisomer may have better efficacy and / or fewer side effects than other stereoisomers. Single stereoisomers and optically active mixtures can be obtained by chiral source synthesis, chiral catalysis, chiral resolution, etc. Racemates can be chirally resolved by chromatographic or chemical resolution. For example, the compounds of this disclosure can be separated by adding chiral acid resolving reagents such as chiral tartaric acid or chiral malic acid to form salts, utilizing the physicochemical properties of the products, such as differences in solubility.

[0139] This disclosure also includes all suitable isotopic variants of the compounds disclosed herein. An isotopic variant is defined as a compound in which at least one atom is replaced by an atom having the same atomic number but whose atomic mass differs from that of atoms commonly or predominantly found in nature. Examples of isotopes that can be introduced into the compounds of this disclosure include isotopes of hydrogen, carbon, nitrogen, and oxygen, respectively, for example… 2 H (deuterium) 3 H (tritium) 11 C 13 C 14 C 15 N、 17 O and 18 O.

[0140] The term "alkyl" in this disclosure refers to a branched or straight-chain saturated aliphatic monovalent hydrocarbon group having a specified number of carbon atoms. The term "alkylene" in this disclosure refers to a branched or straight-chain saturated aliphatic divalent hydrocarbon group having a specified number of carbon atoms. n-m This refers to groups that include carbon atoms from n to m. For example, C 2-5 Alkylenes include C2 alkylenes, C3 alkylenes, C4 alkylenes, and C5 alkylenes.

[0141] In this disclosure, when the name of a compound differs from its structural formula, the structural formula shall prevail.

[0142] It should be understood that the term "compound of the present disclosure" as used herein may, depending on the context, include: compounds of formula (I), their N-oxides, their solvates, their pharmaceutically acceptable salts, their stereoisomers, and mixtures thereof.

[0143] The term “cationic lipid” as used in this article refers to lipids that are positively charged at a selected pH value or range.

[0144] Cationic lipids readily bind to negatively charged nucleic acids, that is, they interact with the negatively charged phosphate groups in nucleic acids through electrostatic forces to form lipid nanoparticles (LNPs).

[0145] The inventors discovered through long-term research that screening suitable cationic lipid compounds that meet the following criteria is extremely difficult: structurally different from existing cationic lipids, possessing high transfection efficiency and low cytotoxicity, and exhibiting high and sustained expression in mice. Through extensive research, the inventors discovered several compounds, preferably YK-2001, YK-2002, YK-2003, YK-2005, YK-2006, YK-2007, YK-2008, YK-2009, YK-2011, YK-2012, YK-2014, YK-2015, YK-2017, and YK-2018, which, when used for targeted drug delivery in vivo, can improve intracellular transfection efficiency and significantly enhance spleen targeting in animals compared to existing cationic lipids.

[0146] This disclosure is based on at least the following findings:

[0147] The cationic lipid compounds disclosed herein can be used to deliver nucleic acid molecules, small molecule compounds, peptides, or proteins. Compared to known cationic lipid compounds, the cationic lipid compounds disclosed herein exhibit higher transfection efficiency and lower cytotoxicity, and significantly increased expression levels in animal spleens, thereby improving delivery efficiency.

[0148] cationic lipids

[0149] In one embodiment of the composition / carrier disclosed herein, the cationic lipid is one or more selected from the compounds of formula (I) above, or their N-oxides, solvates, pharmaceutically acceptable salts, or stereoisomers. In one embodiment, the cationic lipid is selected from the compounds of formula (I) above. For example, in a preferred embodiment, the cationic lipid is compounds YK-2001 to YK-2018, and in another preferred embodiment, the cationic lipid is compounds YK-2001, YK-2002, YK-2003, YK-2005, YK-2006, YK-2007, YK-2008, YK-2009, YK-2011, YK-2012, YK-2014, YK-2015, YK-2017, and YK-2018.

[0150] In another embodiment of the composition / carrier disclosed herein, the cationic lipid comprises: (a) one or more selected from the compounds of formula (I) above, or their N-oxides, solvates, pharmaceutically acceptable salts, or stereoisomers; and (b) one or more other ionizable lipid compounds different from (a). (b) The cationic lipid compound may be a commercially available cationic lipid or a cationic lipid compound reported in the literature. For example, (b) the cationic lipid compound may be SM-102 of CN102625696B, or MC3 of CN102625696B.

[0151] In one embodiment, the cationic lipid accounts for 25%-75% of the molar ratio of the carrier, for example, 25%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, and 75%.

[0152] This carrier can be used for the delivery of active ingredients such as therapeutic and / or preventative agents. The active ingredient can be encapsulated within the carrier or bound to the carrier in any form.

[0153] For example, examples of the therapeutic agent or the preventive agent may be one or more of nucleic acid molecules, small molecule compounds, peptides, or proteins. The nucleic acid includes, but is not limited to, single-stranded DNA, double-stranded DNA, and RNA. Suitable RNAs include, but are not limited to, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), and mixtures thereof.

[0154] neutral lipids

[0155] The carrier may contain neutral lipids. In this disclosure, neutral lipids refer to lipids present in a charge-free or neutral ionic form within a selected pH range. These neutral lipids may modulate the flowability of nanoparticles to form a lipid bilayer and improve efficiency by promoting lipid phase transitions, and may also affect the specificity of target organs.

[0156] In one embodiment, the molar ratio of the cationic lipid to the neutral lipid is about 1:1 to 15:1, for example, about 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, and 1:1. In another preferred embodiment, for example, the molar ratio of the cationic lipid to the neutral lipid is about 4.9:1.

[0157] For example, neutral lipids may include one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterols and their derivatives.

[0158] The carrier component of a composition comprising cationic lipids may include one or more neutral lipid-phospholipids, such as one or more (poly)unsaturated lipids. Phospholipids may assemble into one or more lipid bilayers. Generally, phospholipids may include a phospholipid moiety and one or more fatty acid moieties.

[0159] Neutral lipids may be selected from the non-restrictive group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin. Fatty acids may be selected from the non-restrictive group consisting of lauric acid, myristic acid, myristenoic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, erucic acid, phytic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, benzanoic acid, docosapentaenoic acid, and docosahexaenoic acid. Also encompassing are non-natural species including natural species with modifications and substitutions, such modifications and substitutions include branching, oxidation, cyclization, and alkynes. For example, phospholipids may be functionalized with or crosslinked with one or more alkynes (e.g., alkenyl groups with one or more double bonds replaced by triple bonds). Under appropriate reaction conditions, the alkyne group may undergo a copper-catalyzed cycloaddition reaction upon exposure to azides. These reactions can be used to functionalize the lipid bilayer of a composition to facilitate membrane permeation or cell recognition, or to conjugate the composition with useful components such as targeting or imaging components (e.g., dyes).

[0160] The neutral lipids that can be used in these compositions may be selected from the non-limiting group of the following: 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-diundecanoyl-sn-glycerol-3-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphocholine (18:0 Diether 1,2-Oleoyl-2-cholesterolylhemisuccinoyl-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-bis(docohexanoyl)-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-distearyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinolenoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docohexanoyl-sn-glycerol-3-phosphate ethanolamine), 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatid ...phosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylphosphatidylphosphatidylphosphatidylphosphatidylphosphatidylphosphatidylphosphatidylphosphat Phosphatidylethanolamine (POPE), distearate-phosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), 1-stearoyl-2-oleoylphosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof.

[0161] In some embodiments, neutral lipids include DSPC. In some embodiments, neutral lipids include DOPE. In some embodiments, neutral lipids include both DSPC and DOPE.

[0162] structural lipids

[0163] The carrier of the composition comprising cationic lipids may also include one or more structural lipids. In this disclosure, structural lipids refer to lipids that enhance the stability of nanoparticles by filling the gaps between lipids.

[0164] In one embodiment, the molar ratio of the cationic lipid to the structural lipid is about 1:1 to 5:1, for example, about 1.0:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, or 2.0:1.

[0165] Structural lipids may be selected from, but are not limited to, the group consisting of: cholesterol, nonsterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol, corticosteroids, and mixtures thereof. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid includes cholesterol and corticosteroids (such as prednisolone, dexamethasone, prednisone, and hydrocortisone) or combinations thereof.

[0166] Polymer conjugated lipids

[0167] The carrier of the composition containing cationic lipids may also include one or more polymer-conjugated lipids. Polymer-conjugated lipids primarily refer to polyethylene glycol (PEG)-modified lipids. Hydrophilic PEG stabilizes LNPs, modulates nanoparticle size by restricting lipid fusion, and increases the half-life of nanoparticles by reducing non-specific interactions with macrophages.

[0168] In one embodiment, the polymeric conjugated lipid is selected from one or more of the following: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. The molecular weight of the PEG-modified PEG is typically 350-5000 Da.

[0169] For example, the polymeric conjugated lipid is selected from one or more of the following: distearate phosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), 1,2-dimyristoyl-sn-glycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000), and methoxy polyethylene glycol bis(tetradecyl)acetamide (ALC-0159).

[0170] In one embodiment of the composition / carrier disclosed herein, the polymeric conjugated lipid is DMG-PEG2000.

[0171] In one embodiment of the composition / carrier disclosed herein, the carrier comprises neutral lipids, structural lipids, and polymer-conjugated lipids, wherein the molar ratio of the cationic lipids, neutral lipids, structural lipids, and polymer-conjugated lipids is (25-75):(5-25):(15-65):(0.5-10), for example (30-49):(7.5-15):(35-55):(1-5), more preferably (40-49):(8-12):(39-45):(1-3). The total molar percentage of the cationic lipids, neutral lipids, structural lipids, and polymer-conjugated lipids is 100.

[0172] In one embodiment of the composition / carrier disclosed herein, the carrier comprises neutral lipids, structural lipids, and polymer-conjugated lipids, wherein the molar ratio of the cationic lipids, the neutral lipids, the structural lipids, and the polymer-conjugated lipids is 40:10:48.5:1.5 or 49:10:39.5:1.5.

[0173] Other ionizable lipids

[0174] In this disclosure, other ionizable lipids can be ionizable lipids from any source, and can be ionizable cationic lipids or ionizable anionic lipids. This disclosure does not particularly limit the source of other ionizable lipids; for example, they can be commercially available ionizable lipids or ionizable lipids reported in the literature. In some examples, other ionizable lipid compounds can be SM-102 in CN201080026228.8, MC3 in CN201080026228.8, YK-009 in CN202210034449.4, or C16 in CN202380010167.3.

[0175] Therapeutic agents and / or preventative agents

[0176] The composition may include one or more therapeutic and / or preventive agents as active components. In one embodiment, the mass ratio of the carrier to the therapeutic or preventive agent is 10:1 to 30:1, for example, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, or 25:1.

[0177] In one embodiment, the mass ratio of the carrier to the therapeutic or preventative agent is 12.5:1-20:1, preferably 13-17:1, and more preferably 15:1.

[0178] The therapeutic or preventive agent includes, but is not limited to, one or more of nucleic acid molecules, small molecule compounds, polypeptides, or proteins.

[0179] For example, the therapeutic or preventative agent is a vaccine or compound that can elicit an immune response.

[0180] The carriers disclosed herein can deliver therapeutic and / or preventative agents to mammalian cells or organs; therefore, this disclosure also provides methods for treating diseases or conditions in mammals in need, including administering a composition comprising therapeutic and / or preventative agents to the mammal and / or contacting mammalian cells with the composition. Accordingly, this disclosure provides the use of the compounds of this disclosure or their N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers, or compositions of this disclosure in the preparation of medicaments for treating diseases or conditions in subjects in need.

[0181] This disclosure also provides the use of the compounds of this disclosure or their N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers, or compositions thereof, in the preparation of nucleic acid drugs, vaccines, chemical drugs, peptide drugs, or protein drugs.

[0182] Therapeutic agents and / or preventive agents include bioactive substances and are alternatively referred to as "active agents". Therapeutic agents and / or preventive agents can be substances that, upon delivery to a cell or organ, induce a desired change in that cell or organ or other body tissue or system. Such species can be used to treat one or more diseases, conditions, or illnesses. In some embodiments, therapeutic agents and / or preventive agents are small molecule pharmaceutical products that can be used to treat a specific disease, condition, or illness.Examples of pharmaceuticals that can be used in a composition include, but are not limited to, anti-hypertrophic agents (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, and streptozotocin), and antitumor agents (e.g., actinomycin D, vincristine, vinblastine, cytosine arabinoside). Arabinoside, anthracycline, alkylating agents, platinum compounds, antimetabolites and nucleoside analogs such as methotrexate and purine and pyrimidine analogs, anti-infectives, local anesthetics (e.g., dibucaine and chlorpromazine), beta-adrenergic blockers (e.g., propranolol, timolol, and labetalol), antihypertensives (e.g., clonidine and hydralazine), antidepressants (e.g., imipramine, amitriptyline, and doxepin), anticonvulsants (e.g., phenytoin), antihistamines (e.g., diphenhydramine, chlorpheniramine, and promethazine), antibiotics / antibacterial agents (e.g., gentamicin, ciprofloxacin, and cefoxitin), antifungal agents (e.g., miconazole, terconazole, econazole, isoconazole, butaconazole, clotrimazole, itraconazole, nystatin, naftifine, and amphotericin B), antiparasitic agents, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma medications, vitamins, sedatives, and imaging agents.

[0183] In some implementations, the therapeutic and / or prophylactic agents are cytotoxins, radioactive ions, chemotherapeutic agents, vaccines, compounds that elicit an immune response, and / or another therapeutic and / or prophylactic agent. Cytotoxins or cytotoxic agents include any agent that is harmful to cells. Examples include, but are not limited to, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, and dihydroxyanthraquinone. Anthracindione, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids such as maytansinol, rachelmycin (CC-1065), and their analogues or homologues. Radioactive ions include, but are not limited to, iodine (e.g., iodine-125 or iodine-131), strontium-89, phosphorus, palladium, cesium, iridium, phosphate, cobalt, yttrium-90, samarium-153, and praseodymium. Examples of vaccines include compounds and formulations that provide immunity against one or more conditions associated with infectious diseases such as influenza, measles, human papillomavirus (HPV), rabies, meningitis, pertussis, tetanus, plague, hepatitis, and tuberculosis. These may include, for example, mRNA encoding pathogenic antigens and / or their epitopes. Vaccines may also include compounds and formulations that direct an immune response against cancer cells, such as mRNA encoding tumor cell-derived antigens, epitopes, and / or novel epitopes. Compounds that elicit an immune response may include vaccines, corticosteroids (e.g., dexamethasone), and other species.Other therapeutic and / or prophylactic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, and 5-fluorouracil dacarbazine), alkylating agents (e.g., nitrogen mustard, thiotepa, chlorambucil, lactamazole (CC-1065), melphalan, carmustine (BSNU), lomustine (CCNU), and cyclophosphamide. Phosphoramide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine cycloplatin (II) (DDP, cisplatin), anthracyclines (e.g. daunomycin (formerly known as daunomycin) and doxorubicin), antibiotics (e.g. dactinomycin (formerly known as actinomycin), bleomycin, mithramycin and antramycin (AMC)), and antimitotic agents (e.g. vincristine, vinblastine, paclitaxel and levothyroxine).

[0184] In other embodiments, the therapeutic and / or preventative agents are proteins. Therapeutic proteins that may be used in the nanoparticles of this disclosure include, but are not limited to, gentamicin, amikacin, insulin, erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), factor VIR, luteinizing hormone-releasing hormone (LHRH) analogs, interferon, heparin, hepatitis B surface antigen, typhoid vaccine, and cholera vaccine.

[0185] In some embodiments, the therapeutic agent is a polynucleotide or nucleic acid (e.g., ribonucleic acid or deoxyribonucleic acid). The broadest meaning of the term "polynucleotide" includes any compound and / or substance that is an oligonucleotide chain or can be incorporated into an oligonucleotide chain. Exemplary polynucleotides used according to this disclosure include, but are not limited to, one or more of the following: deoxyribonucleic acid (DNA); ribonucleic acid (RNA), including messenger mRNA (mRNA) and its hybrids; RNAi inducible factors; RNAi factors; siRNA; shRNA; miRNA; antisense RNA; ribonuclease; catalytic DNA; RNA that induces triple helix formation; aptamers, etc. In some embodiments, the therapeutic and / or preventive agent is RNA. The RNA that can be used in the compositions and methods described herein can be selected from, but is not limited to, the group consisting of: shortmer, antagomir, antisense RNA, ribonuclease, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), and mixtures thereof. In some implementations, the RNA is mRNA.

[0186] In some embodiments, the therapeutic and / or preventative agent is mRNA. The mRNA may encode any polypeptide of interest, including any polypeptide that is naturally or non-naturally present or otherwise modified. The polypeptide encoded by the mRNA may have any size and may possess any secondary structure or activity. In some embodiments, the polypeptide encoded by the mRNA may have a therapeutic effect when expressed in cells.

[0187] In other embodiments, the therapeutic and / or preventative agent is siRNA. siRNA is capable of selectively reducing or downregulating the expression of a gene of interest. For example, the siRNA may be chosen such that, upon administration of a composition comprising the siRNA to a subject in need, a gene associated with a specific disease, symptom, or condition is silenced. The siRNA may contain a sequence complementary to the mRNA sequence encoding the gene or protein of interest. In some embodiments, the siRNA may be an immunomodulatory siRNA.

[0188] In some implementations, the therapeutic and / or preventative agents are sgRNA and / or cas9 mRNA. sgRNA and / or cas9 mRNA can be used as gene editing tools. For example, the sgRNA-cas9 complex can affect the mRNA translation of cellular genes.

[0189] In some implementations, the therapeutic and / or prophylactic agent is shRNA or its encoding vector or plasmid. shRNA can be generated within the target cell after delivery of an appropriate construct into the nucleus. Constructs and mechanisms associated with shRNA are well known in the relevant field.

[0190] Disease or ailment

[0191] The compositions / carriers disclosed herein can deliver therapeutic or preventative agents to subjects or patients. These therapeutic or preventative agents include, but are not limited to, one or more of nucleic acid molecules, small molecule compounds, peptides, or proteins. Therefore, the compositions disclosed herein can be used to prepare nucleic acid drugs, gene vaccines, small molecule drugs, peptide or protein drugs. Due to the wide variety of such therapeutic or preventative agents, the compositions disclosed herein can be used to treat or prevent a variety of diseases or conditions.

[0192] In one embodiment, the disease or condition is characterized by dysfunctional or abnormal protein or polypeptide activity.

[0193] For example, the disease or condition is selected from the group consisting of: infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.

[0194] In one embodiment, the infectious disease is selected from diseases caused by coronavirus, influenza virus, or HIV virus, pediatric pneumonia, Rift Valley fever, yellow fever, rabies, and various herpes diseases.

[0195] Other components

[0196] The composition may include one or more components other than those described in the foregoing sections. For example, the composition may include one or more hydrophobic small molecules, such as vitamins (e.g., vitamin A or vitamin E) or sterols.

[0197] The composition may also include one or more permeability-enhancing molecules, carbohydrates, polymers, surface modifiers, or other components. Permeability-enhancing molecules may be, for example, those described in U.S. Patent Application Publication No. 2005 / 0222064. Carbohydrates may include simple sugars (e.g., glucose) and polysaccharides (e.g., glycogen and its derivatives and analogs).

[0198] Surface modifiers may include, but are not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyl dioctadecyl ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), and mucolytics (e.g., acetylcysteine, artemisia, bromelain, papain, clerodendrum, bromhexine, carbocisteine, and eprazinone). The composition may contain mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin β4, streptococcal DNase α (dornasealfa), neltenexine, and erdosteine), and DNases (e.g., rhDNase). Surface modifiers may be placed within and / or on the surface of the nanoparticles of the composition (e.g., by coating, adsorption, covalent bonding, or other methods).

[0199] The composition may also contain one or more functionalized lipids. For example, the lipids may be functionalized with an alkynyl group, which may undergo a cycloaddition reaction when exposed to an azide under appropriate reaction conditions. Specifically, the lipid bilayer can be functionalized in this way with one or more groups that can effectively promote membrane permeation, cell recognition, or imaging. The surface of the composition may also be conjugated to one or more useful antibodies. Functional groups and conjugates that can be used for targeted cell delivery, imaging, and membrane permeation are well known in the art.

[0200] In addition to these components, the composition may include any substance that can be used in a pharmaceutical composition. For example, the composition may include one or more pharmaceutically acceptable excipients or auxiliary ingredients, but not limited to one or more solvents, dispersion media, diluents, dispersants, suspending agents, granulation agents, disintegrants, fillers, flow aids, liquid media, binders, surfactants, isotonic agents, thickeners or emulsifiers, buffers, lubricants, oils, preservatives, flavoring agents, coloring agents, etc. Excipients include, for example, starch, lactose, or dextrin. Pharmaceutically acceptable excipients are well known in the art (see, for example, Remington's *The Science and Practice of Pharmacy*, 21st edition, ARGennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006).

[0201] Examples of diluents may include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar and / or combinations thereof.

[0202] In some embodiments, compositions comprising one or more lipids described herein may further comprise one or more adjuvants, such as glucopyranosyl lipid adjuvants (GLA), CpG oligodeoxyribonucleotides (e.g., class A or class B), poly(I:C), aluminum hydroxide, and Pam3CSK4.

[0203] The compositions disclosed herein can be formulated into solid, semi-solid, liquid, or gaseous preparations, such as tablets, capsules, ointments, elixirs, syrups, solutions, emulsions, suspensions, injections, and aerosols. The compositions disclosed herein can be prepared using methods well known in the pharmaceutical industry. For example, a sterile injectable solution can be prepared by incorporating the desired amount of the therapeutic or prophylactic agent with the various other components described above into a suitable solvent, such as sterile distilled water, followed by filtration and sterilization. Surfactants may also be added to promote the formation of a homogeneous solution or suspension.

[0204] For example, the compositions of this disclosure can be administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation. In some embodiments, the compositions are administered subcutaneously.

[0205] The compositions disclosed herein are administered in therapeutically effective amounts, which can vary not only with the specific agent selected, but also with the route of administration, the nature of the disease being treated, and the age and condition of the patient, and can ultimately be determined by the attending physician or clinician. For example, the therapeutic or prophylactic agent can be administered to a subject (preferably a mammal, such as a human) at a dose of about 0.001 mg / kg to about 10 mg / kg.

[0206] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of this disclosure.

[0207] All reagents and raw materials used in this disclosure are commercially available.

[0208] The positive and progressive effects of this disclosure are as follows: the cationic lipid compounds and lipid compositions disclosed herein can be used for the encapsulation of active pharmaceutical ingredients such as nucleic acids (e.g., mRNA). The mRNA-LNP composition prepared from the cationic lipids of this disclosure can significantly increase protein expression levels in mice, and also has significant spleen targeting, which can significantly increase protein expression levels both in vivo and in vitro. Attached Figure Description

[0209] To more clearly illustrate the technical solutions of the embodiments of this disclosure, the accompanying drawings of this disclosure will be briefly introduced below. It should be understood that the drawings described below are only some exemplary embodiments of this disclosure, and not limitations on this disclosure.

[0210] Figure 1 The particle size of the mRNA-LNP compositions encapsulated with Fluc-mRNA prepared based on YK-2001 to YK-2018, SM-102, MC3, lipid 43, 10-5-C8C10, lipid 11, lipid 149 and lipid 170 is shown.

[0211] Figure 2 The relative fluorescence intensity of mRNA-LNP compositions encapsulated with Fluc-mRNA prepared based on YK-2001, YK-2002, YK-2003, YK-2005, YK-2006, YK-2007, YK-2008, YK-2009, YK-2011, YK-2012, YK-2014, YK-2015, YK-2017, YK-2018, SM-102, MC3, lipid 43, 10-5-C8C10, lipid 11, lipid 149, and lipid 170 after transfection into HEK293T cells is shown.

[0212] Figure 3This shows the mean radiation intensity in the spleen of mice 6 hours after intravenous injection of mRNA-LNP compositions encapsulating Fluc-mRNA prepared based on YK-2001, YK-2002, YK-2003, YK-2005, YK-2006, YK-2007, YK-2008, YK-2009, YK-2011, YK-2012, YK-2014, YK-2015, YK-2017, YK-2018, SM-102, MC3, lipid 43, 10-5-C8C10, lipid 11, lipid 149, and lipid 170.

[0213] Figure 4 This study demonstrates the ratio of average radiation intensity in the spleen of mice to that in the liver 6 hours after intravenous injection of mRNA-LNP compositions encapsulating Fluc-mRNA prepared based on YK-2001, YK-2002, YK-2003, YK-2005, YK-2006, YK-2007, YK-2008, YK-2009, YK-2011, YK-2012, YK-2014, YK-2015, YK-2017, and YK-2018.

[0214] Figure 5 Imaging images of mouse liver and spleen 6 h after intravenous injection of mRNA-LNP compositions encapsulating Fluc-mRNA prepared based on YK-2001, YK-2003, YK-2005, YK-2006, YK-2008, YK-2009 and YK-2011, respectively. Detailed Implementation

[0215] To make the objectives, technical solutions, and advantages of the embodiments of this disclosure clearer, the technical solutions of the embodiments of this disclosure will be described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this disclosure, and not all of them. Based on the described embodiments of this disclosure, those skilled in the art can implement them in other specific forms without departing from the basic attributes and spirit of this disclosure. It should be understood that, without conflict, any and all embodiments of this disclosure can be combined with technical features of any other embodiment or multiple other embodiments to obtain other embodiments. This disclosure includes other embodiments obtained by such combinations.

[0216] The present disclosure is further described below with reference to embodiments, but the present disclosure is not limited to the following embodiments. The implementation conditions used in the embodiments can be further adjusted according to different requirements of specific applications, and the implementation conditions not specified are conventional conditions in the industry. In the specific embodiments of the present disclosure, the raw materials used are all commercially available. Unless otherwise stated, all temperatures are given in degrees Celsius. The technical features involved in the various embodiments of the present disclosure can be combined with each other as long as they do not conflict with each other.

[0217] In the following embodiments, the abbreviations have the following meanings:

[0218] Boc2O: ditert-butyl dicarbonate; TEA: triethylamine; K2CO3: potassium carbonate; KI: potassium iodide; HCl: hydrogen chloride; DMAP: 4-dimethylaminopyridine; EDCI: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; DCM: dichloromethane; DMF: N,N-dimethylcarbamate; ACN: acetonitrile; CDI: N,N'-carbonyldiimidazole.

[0219] Example 1: Synthesis of cationic lipid compounds

[0220] 1.1 Synthesis of intermediate INT-1

[0221]

[0222]

[0223] Step 1: Synthesis of INT-1-PM1

[0224] (S)-1-amino-3-chloro-2-propanol hydrochloride (24.00 g, 164.37 mmol) was dissolved in dichloromethane (250 mL), and triethylamine (66.53 g, 657.49 mmol) was added. Then, di-tert-butyl dicarbonate (143.50 g, 657.49 mmol) was slowly added dropwise. The mixture was heated to 40 °C and reacted for 24 h. The reaction was monitored by TLC until the reactants had completely reacted. Heating was stopped, and the reaction was quenched by adding saturated sodium bicarbonate aqueous solution. The mixture was separated, and the aqueous phase was extracted twice with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (0-15% ethyl acetate / n-hexane). The product was collected and concentrated to give INT-I-PM1 (22.02 g, 105.02 mmol, 63.9%). C8H 16 ClNO3, MS(ES): m / z (M+H + ) 210.1.

[0225] Step 2: Synthesis of INT-1-PM2

[0226] INT-I-PM1 (5.00 g, 23.85 mmol) was dissolved in acetonitrile (50 mL), followed by the sequential addition of tetrahydropyrrole (2.04 g, 28.62 mmol), potassium carbonate (9.89 g, 71.54 mmol), and potassium iodide (0.79 g, 4.77 mmol). The mixture was heated to 70 °C and reacted for 8 h, monitored by TLC until the reactants had completely reacted. Heating was stopped, the mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (0-15% methanol / dichloromethane), and the product was collected and concentrated to obtain INT-I-PM2 (4.56 g, 18.66 mmol, 78.3%). 12 H 24 N₂O₃, MS(ES): m / z (M+H) + 245.2.

[0227] Step 3: Synthesis of INT-1

[0228] Add INT-I-PM2 (4.56 g, 18.66 mmol) and a 1,4-dioxane solution (20 mL) of hydrochloric acid, and react at room temperature for 2 h. Monitor the reaction with LC-MS until the reactants have completely reacted. Concentrate the reaction solution under reduced pressure, dissolve the residue in methanol, and then concentrate under reduced pressure again. Repeat this process three times to obtain INT-1 (3.95 g, crude product). C7H 16 N₂O, MS(ES): m / z (M+H) + 145.2.

[0229] 1.2 Synthesis of intermediate INT-2

[0230]

[0231] Step 1: Synthesis of INT-2-PM1

[0232] Using INT-1-PM1 (5.00 g, 23.85 mmol) as a starting material, INT-2-PM1 (4.61 g, 16.86 mmol, 70.7%) was obtained by following the synthesis method of INT-1-PM2. 13 H 27 N3O3, MS(ES): m / z (M+H + 274.2.

[0233] Step 2: Synthesis of INT-2

[0234] Using INT-2-PM1 (4.61 g, 16.86 mmol) as a starting material, INT-2 (4.40 g, crude product) was obtained by following the synthesis method of INT-1. C8H 19N3O, MS(ES): m / z (M+H + 174.1.

[0235] 1.3 Synthesis of intermediate INT-3

[0236]

[0237] Step 1: Synthesis of INT-3-PM1

[0238] Using INT-1-PM1 (2.00 g, 9.54 mmol) as a starting material, INT-3-PM1 (0.86 g, 3.00 mmol, 31.5%) was obtained by following the synthesis method of INT-1-PM2. 15 H 30 N₂O₃, MS(ES): m / z (M+H) + 287.4.

[0239] Step 2: Synthesis of INT-3

[0240] Using INT-3-PM1 (0.86 g, 3.00 mmol) as a starting material, INT-3 (0.78 g, crude product) was obtained following the synthesis method of INT-1. 10 H 22 N₂O, MS(ES): m / z (M+H) + 187.3.

[0241] 1.4 Synthesis of intermediate INT-4

[0242]

[0243] Step 1: Synthesis of INT-4-PM1

[0244] Using INT-1-PM1 (2.00 g, 9.54 mmol) as a starting material, INT-4-PM1 (1.25 g, 4.59 mmol, 48.1%) was obtained by following the synthesis method of INT-1-PM2. 14 H 28 N₂O₃, MS(ES): m / z (M+H) + 273.4.

[0245] Step 2: Synthesis of INT-4

[0246] Using INT-4-PM1 (1.25 g, 4.59 mmol) as a starting material, INT-4 (1.12 g, crude product) was obtained following the synthesis method of INT-1. C9H 20 N₂O, MS(ES): m / z (M+H)+ 173.3.

[0247] 1.5 Synthesis of intermediate INT-5

[0248]

[0249] Step 1: Synthesis of INT-5-PM1

[0250] Using INT-1-PM1 (2.00 g, 9.54 mmol) as a starting material, INT-5-PM1 (1.18 g, 5.41 mmol, 56.7%) was obtained by following the synthesis method of INT-1-PM2. 10 H 22 N₂O₃, MS(ES): m / z (M+H) + ) 219.3.

[0251] Step 2: Synthesis of INT-5

[0252] Using INT-5-PM1 (1.18 g, 5.41 mmol) as a starting material, INT-5 (1.02 g, crude product) was obtained following the synthesis method of INT-1. C5H 14 N₂O, MS(ES): m / z (M+H) + 119.2.

[0253] 1.6 Synthesis of intermediate INT-6

[0254]

[0255] Step 1: Synthesis of INT-6-PM1

[0256] Using INT-1-PM1 (2.00 g, 9.54 mmol) as a starting material, INT-6-PM1 (0.85 g, 3.45 mmol, 36.2%) was obtained by following the synthesis method of INT-1-PM2. 12 H 26 N₂O₃, MS(ES): m / z (M+H) + 247.2.

[0257] Step 2: Synthesis of INT-6

[0258] Using INT-6-PM1 (0.85 g, 3.45 mmol) as a starting material, INT-6 (0.92 g, crude product) was obtained following the synthesis method of INT-1. C7H 18 N₂O, MS(ES): m / z (M+H) + 147.2.

[0259] 1.7 Synthesis of intermediate INT-7

[0260]

[0261] 6-Bromhexanoic acid (10.00 g, 51.27 mmol) was dissolved in dichloromethane (150 mL), followed by the addition of n-decyl alcohol (8.11 g, 51.27 mmol), EDCI (14.74 g, 76.90 mmol), and DMAP (1.25 g, 10.25 mmol). The reaction was carried out at room temperature for 16 h, and the reaction was monitored by TLC until the reactants had completely reacted. The reaction was quenched by adding saturated sodium bicarbonate solution. The mixture was separated, and the aqueous phase was extracted once with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (0-4% ethyl acetate / n-hexane). The product was collected and concentrated to give INT-7 (15.01 g, 44.76 mmol, 87.3%). 16 H 31 BrO2, MS(ES): m / z (M+H + 335.3.

[0262] 1.8 Synthesis of intermediate INT-8

[0263]

[0264] Step 1: Synthesis of INT-8-PM1

[0265] 6-Bromhexanoic acid (10.00 g, 51.27 mmol) was dissolved in dichloromethane (150 mL), followed by the addition of heptadecano-9-ol (13.15 g, 51.27 mmol), EDCI (14.74 g, 76.90 mmol), and DMAP (1.25 g, 10.25 mmol). The reaction was carried out at room temperature for 16 h, and the reaction was monitored by TLC until the reactants had completely reacted. The reaction was quenched by adding saturated sodium bicarbonate solution. The mixture was separated, and the aqueous phase was extracted once with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (0-5% ethyl acetate / n-hexane). The product was collected and concentrated to give INT-8-PM1 (19.38 g, 44.70 mmol, 87.2%). 23 H 45 BrO2, MS(ES): m / z (M+H + 433.2.

[0266] Step 2: Synthesis of INT-8-PM2

[0267] Ethanolamine (0.30 g, 4.91 mmol) was dissolved in acetonitrile (20 mL), followed by the sequential addition of INT-8-PM1 (5.32 g, 12.28 mmol), potassium carbonate (2.04 g, 14.73 mmol), and potassium iodide (0.082 g, 0.49 mmol). The mixture was heated to 70 °C and reacted for 7 h, monitored by LC-MS until the reactants had completely reacted. Heating was stopped, the mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (0-9% methanol / dichloromethane), and the product was collected and concentrated to obtain INT-8-PM2 (2.85 g, 3.72 mmol, 75.7%). 48 H 95 NO5, MS(ES): m / z (M+H + 766.5.

[0268] Step 3: Synthesis of INT-8

[0269] INT-8-PM2 (2.85 g, 3.72 mmol) was dissolved in dichloromethane (25 mL), cooled in an ice bath, and CDI (1.81 g, 11.16 mmol) in dichloromethane (20 mL) was added dropwise. The reaction was carried out at room temperature for 16 h, and the reaction was monitored by LC-MS until the starting material was minimal. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (0-18% ethyl acetate / n-hexane). The product was collected and concentrated to obtain INT-8 (2.01 g, 2.34 mmol, 62.8%). 52 H 97 N3O6, MS(ES): m / z (M+H + 860.8.

[0270] 1.9 Synthesis of intermediate INT-9

[0271]

[0272] Step 1: Synthesis of INT-9-PM1

[0273] Ethanolamine (0.30 g, 4.91 mmol) was dissolved in acetonitrile (20 mL), followed by the sequential addition of INT-8-PM1 (2.13 g, 4.91 mmol), potassium carbonate (2.04 g, 14.73 mmol), and potassium iodide (0.082 g, 0.49 mmol). The mixture was heated to 70 °C and reacted for 7 h, monitored by LC-MS until the reactants had completely reacted. Heating was stopped, the mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (0-25% methanol / dichloromethane), and the product was collected and concentrated to obtain INT-9-PM1 (1.66 g, 4.01 mmol, 81.7%). 25H 51 NO5, MS(ES): m / z (M+H + 414.5.

[0274] Step 2: Synthesis of INT-9-PM2

[0275] INT-9-PM1 (1.66 g, 4.01 mmol) was dissolved in acetonitrile (16 mL), followed by the sequential addition of INT-7 (1.35 g, 4.01 mmol), potassium carbonate (1.66 g, 12.04 mmol), and potassium iodide (0.067 g, 0.40 mmol). The mixture was heated to 70 °C and reacted for 7 h, monitored by LC-MS until the reactants had completely reacted. Heating was stopped, the mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (0-11% methanol / dichloromethane), and the product was collected and concentrated to obtain INT-9-PM2 (1.93 g, 2.89 mmol, 72.0%). 41 H 81 NO5, MS(ES): m / z (M+H + 668.6.

[0276] Step 3: Synthesis of INT-9

[0277] Using INT-9-PM2 (1.93 g, 2.89 mmol) as a starting material, INT-9 (1.25 g, 1.64 mmol, 56.8%) was obtained by following the synthesis method of INT-8. C 45 H 83 N3O6, MS(ES): m / z (M+H + 762.6.

[0278] 1.10 Synthesis of intermediate INT-10

[0279]

[0280] Using YK-009 (3.00 g, 4.59 mmol) from CN114044741B as a starting material, INT-10 (2.95 g, 3.94 mmol, 86.0%) was obtained according to the synthesis method of INT-8. 44 H 81 N3O6, MS(ES): m / z (M+H + 748.6.

[0281] 1.11 Synthesis of YK-2001

[0282]

[0283] INT-8 (400 mg, 0.46 mmol) was dissolved in N,N-dimethylcarbamate (5 mL), followed by the sequential addition of INT-1 (67 mg, 0.46 mmol) and potassium carbonate (193 mg, 1.39 mmol). The mixture was heated to 70 °C and reacted for 5 h, monitored by LC-MS until the reactants had completely reacted. Heating was stopped, the mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (0-10% methanol / dichloromethane), and the product was collected and concentrated to obtain YK-2001 (279 mg, 0.30 mmol, 64.1%). 56 H 109 N3O7, MS(ES): m / z (M+H + 936.8. 1 H NMR (400 MHz, CDCl3) δ 5.28 (s, 1H), 4.92 – 4.80 (m, 2H), 4.13 – 4.05 (t, 2H), 3.87 – 3.73 (m, 1H), 3.44 – 3.32 (m, 1H), 3.23 – 3.06 (m, 1H),2.82 – 2.62 (m, 5H), 2.60 – 2.38 (m, 6H), 2.32 – 2.22 (t, 4H), 1.84 – 1.77(m, 4H), 1.64 (dd, J = 15.3, 7.6 Hz, 4H), 1.56 – 1.41 (m, 12H), 1.35 – 1.20 (m, 54H), 0.91 – 0.85 (t, 12H).

[0284] 1.12 Synthesis of YK-2002

[0285]

[0286] Using INT-9 (382 mg, 0.50 mmol) and INT-1 (80 mg, 0.55 mmol) as raw materials, YK-2002 (260 mg, 0.31 mmol, 61.9%) was obtained according to the synthesis method of YK-2001. C 49 H 95 N3O7, MS(ES): m / z (M+H + 838.7. 1H NMR(400 MHz, CDCl3) δ 5.32 – 5.21 (m, 1H), 4.90 – 4.81 (m, 1H), 4.15 – 4.00 (m,4H), 3.85 – 3.75 (m, 1H), 3.47 – 3.33 (m, 1H), 3.19 – 3.06 (m, 1H), 2.77 – 2.60 (m, 5H), 2.57 – 2.40 (m, 6H), 2.40 – 2.23 (m, 5H), 1.85 – 1.72 (m, 4H), 1.67 – 1.55 (m, 6H), 1.47 (ddd, J = 23.0, 13.7, 7.0 Hz, 8H), 1.38 – 1.17 (m, 41H), 0.97 – 0.80 (t, 9H).

[0287] 1.13 Synthesis of YK-2003

[0288]

[0289] Using INT-8 (232 mg, 0.27 mmol) and INT-2 (47 mg, 0.27 mmol) as raw materials, YK-2003 (115 mg, 0.12 mmol, 44.2%) was obtained according to the synthesis method of YK-2001. 57 H 112 N4O7, MS(ES): m / z (M+H + 965.8. 1 HNMR (400 MHz, CDCl3) δ 5.28 (s, 1H), 4.85 (dd, J = 12.5, 6.2 Hz, 1H), 4.16 –4.04 (m, 2H), 3.85 – 3.74 (m, 1H), 3.37 (d, J = 7.5 Hz, 1H), 3.18 – 3.03 (m,1H), 2.79 – 2.62 (m, 4H), 2.61 – 2.41 (m, 10H), 2.37 (d, J = 6.8 Hz, 4H), 2.29(dd, J= 13.1, 5.4 Hz, 3H), 1.68 – 1.57 (m, 4H), 1.56 – 1.40 (m, 13H), 1.37 –1.20 (m, 53H), 0.95 – 0.77 (t, 12H).

[0290] 1.14 Synthesis of YK-2004

[0291]

[0292] Using INT-9 (215 mg, 0.28 mmol) and INT-2 (49 mg, 0.28 mmol) as raw materials, YK-2004 (88 mg, 0.10 mmol, 36.0%) was obtained according to the synthesis method of YK-2001. 50 H 98 N4O7, MS(ES): m / z (M+H + 867.7. 1 H NMR(400 MHz, CDCl3) δ 5.30 – 5.18 (m, 1H), 4.92 – 4.81 (m, 1H), 4.18 – 3.97 (m,4H), 3.87 – 3.72 (m, 1H), 3.44 – 3.32 (m, 1H), 3.18 – 3.04 (m, 1H), 2.74 –2.61 (m, 4H), 2.47 (dd, J = 19.2, 11.8 Hz, 10H), 2.36 (d, J = 6.9 Hz, 2H), 2.33 –2.21 (m, 6H), 1.69 – 1.56 (m, 6H), 1.47 (ddd, J = 20.5, 12.5, 5.8 Hz, 9H), 1.38– 1.16 (m, 42H), 0.95 – 0.82 (t, 9H).

[0293] 1.15 Synthesis of YK-2005

[0294]

[0295] Using INT-8 (400 mg, 0.46 mmol) and INT-3 (87 mg, 0.46 mmol) as raw materials, YK-2005 (285 mg, 0.29 mmol, 62.6%) was obtained according to the synthesis method of YK-2001. 59 H 115N3O7, MS(ES): m / z (M+H + 978.8. 1 HNMR (400 MHz, CDCl3) δ 5.24 (s, 1H), 4.93 – 4.80 (m, 2H), 4.16 – 4.04 (m,2H), 3.83 – 3.55 (m, 2H), 3.39 (dd, J = 15.4, 6.7 Hz, 1H), 3.15 – 2.92 (m, 2), 2.76 – 2.58 (m, 4H), 2.56 – 2.39 (m, 5H), 2.35 – 2.12 (m, 6H), 1.75 – 1.56(m, 9H), 1.47 (ddd, J = 23.2, 17.1, 9.8 Hz, 15H), 1.37 – 1.16 (m, 51H), 0.97 –0.74 (m, 15H).

[0296] 1.16 Synthesis of YK-2006

[0297]

[0298] Using INT-9 (203 mg, 0.27 mmol) and INT-3 (60 mg, 0.32 mmol) as raw materials, YK-2006 (166 mg, 0.19 mmol, 70.8%) was obtained according to the synthesis method of YK-2001. 52 H 101 N3O7, MS(ES): m / z (M+H + 880.8. 1 HNMR (400 MHz, CDCl3) δ 5.25 (s, 1H), 4.90 – 4.83 (m, 1H), 4.16 – 4.01 (m,4H), 3.75 (t, J = 14.2 Hz, 1H), 3.49 (s, 1H), 3.39 (t, J= 8.1 Hz, 1H), 3.13 –2.95 (m, 1H), 2.78 – 2.58 (m, 4H), 2.56 – 2.40 (m, 5H), 2.34 – 2.16 (m, 5H),1.73 – 1.56 (m, 11H), 1.55 – 1.39 (m, 11H), 1.37 – 1.15 (m, 42H), 0.94 – 0.79 (m, 12H).

[0299] 1.17 Synthesis of YK-2007

[0300]

[0301] Using INT-8 (400 mg, 0.46 mmol) and INT-4 (80 mg, 0.46 mmol) as raw materials, YK-2007 (317 mg, 0.33 mmol, 70.7%) was obtained according to the synthesis method of YK-2001. 58 H 113 N3O7, MS(ES): m / z (M+H + 964.8. 1 HNMR (400 MHz, CDCl3) δ 5.30 – 5.19 (m, 1H), 4.94 – 4.79 (m, 2H), 4.14 – 4.06(m, 2H), 3.74 (dd, J = 10.2, 6.2 Hz, 1H), 3.49 (s, 1H), 3.42 – 3.33 (m, 1H), 3.14 – 3.03 (m, 1H), 2.84 – 2.74 (m, 2H), 2.71 – 2.58 (m, 5H), 2.48 – 2.34(m, 5H), 2.31 – 2.21 (t, 4H), 1.75 – 1.57 (m, 13H), 1.55 – 1.39 (m, 12H), 1.29 (p, J = 7.4 Hz, 51H), 0.98 – 0.77 (t, 12H).

[0302] 1.18 Synthesis of YK-2008

[0303]

[0304] Using INT-9 (203 mg, 0.27 mmol) and INT-4 (55 mg, 0.32 mmol) as raw materials, YK-2008 (171 mg, 0.20 mmol, 74.1%) was obtained according to the synthesis method of YK-2001. 51 H 99 N3O7, MS(ES): m / z (M+H + 866.7. 1 H NMR(400 MHz, CDCl3) δ 5.36 – 5.26 (m, 1H), 4.91 – 4.80 (m, 1H), 4.16 – 4.01 (m,4H), 3.80 (dd, J = 10.2, 5.5 Hz, 1H), 3.52 – 3.33 (m, 1H), 3.17 – 3.06 (m, 1H), 2.91 – 2.81 (m, 2H), 2.79 – 2.61 (m, 5H), 2.54 – 2.38 (m, 5H), 2.32 – 2.22(m, 4H), 1.79 – 1.56 (m, 14H), 1.56 – 1.39 (m, 9H), 1.37 – 1.14 (m, 42H), 0.94 – 0.81 (t, 9H).

[0305] 1.19 Synthesis of YK-2009

[0306]

[0307] Using INT-8 (400 mg, 0.46 mmol) and INT-5 (55 mg, 0.46 mmol) as raw materials, YK-2009 (278 mg, 0.31 mmol, 65.7%) was obtained according to the synthesis method of YK-2001. C 54 H 107 N3O7, MS(ES): m / z (M+H + 910.8. 1 HNMR (400 MHz, CDCl3) δ 5.29 (d, J = 6.0 Hz, 1H), 4.92 – 4.81 (m, 3H), 4.16 –4.05 (m, 2H), 3.77 (ddd, J= 13.9, 6.8, 3.7 Hz, 1H), 3.46 – 3.27 (m, 1H), 3.17– 3.03 (m, 1H), 2.73 – 2.64 (m, 2H), 2.50 – 2.43 (m, 4H), 2.37 (dd, J = 12.4,10.3 Hz, 1H), 2.33 – 2.27 (m, 9H), 2.24 (dd, J = 13.1, 4.4 Hz, 2H), 1.68 – 1.57(m, 4H), 1.55 – 1.40 (m, 12H), 1.33 – 1.20 (m, 51H), 0.93 – 0.83 (t, 12H).

[0308] Synthesis of YK-2010 1.20

[0309]

[0310] Using INT-9 (203 mg, 0.27 mmol) and INT-5 (38 mg, 0.32 mmol) as raw materials, YK-2010 (150 mg, 0.18 mmol, 69.3%) was obtained according to the synthesis method of YK-2001. 47 H 93 N3O7, MS(ES): m / z (M+H + 812.7. 1 H NMR (400 MHz, CDCl3) δ 5.32 (s, 1H), 4.89 – 4.79 (m, 1H), 4.40 (s, 3H), 4.18 – 3.99 (m, 4H), 3.85 – 3.73 (m, 1H), 3.43 – 3.32 (m, 1H), 3.17 – 3.02 (m, 1H), 2.75 – 2.65 (m, 2H), 2.51 – 2.36 (m, 7H), 2.37 – 2.31 (m, 8H), 2.31 – 2.25(m, 1H), 1.71 – 1.56 (m, 6H), 1.47 (ddd, J = 17.1, 10.5, 4.1 Hz, 9H), 1.39 –1.18 (m, 41H), 0.97 – 0.79 (t, 9H).

[0311] 1.21 Synthesis of YK-2011

[0312]

[0313] Using INT-8 (400 mg, 0.46 mmol) and INT-6 (68 mg, 0.46 mmol) as raw materials, YK-2011 (300 mg, 0.32 mmol, 68.8%) was obtained according to the synthesis method of YK-2001. C 56 H 111 N3O7, MS(ES): m / z (M+H + 938.8. 1 HNMR (400 MHz, CDCl3) δ 5.27 (d, J = 6.6 Hz, 1H), 4.94 – 4.79 (m, 2H), 4.19 –4.06 (m, 2H), 3.76 (dd, J = 10.5, 5.4 Hz, 1H), 3.39 (ddd, J = 9.5, 6.8, 3.7 Hz,1H), 3.16 – 3.04 (m, 1H), 2.78 – 2.36 (m, 12H), 2.32 – 2.23 (t, 4H), 1.70 –1.57 (m, 4H), 1.55 – 1.38 (m, 12H), 1.35 – 1.16 (m, 51H), 1.14 – 0.97 (m,7H), 0.91 – 0.79 (t, 12H).

[0314] 1.22 Synthesis of YK-2012

[0315]

[0316] Using INT-9 (203 mg, 0.27 mmol) and INT-6 (47 mg, 0.32 mmol) as raw materials, YK-2012 (153 mg, 0.18 mmol, 68.4%) was obtained according to the synthesis method of YK-2001. C 49 H 97 N3O7, MS(ES): m / z (M+H + 840.7. 1H NMR(400 MHz, CDCl3) δ 5.32 – 5.21 (m, 1H), 4.90 – 4.79 (m, 1H), 4.17 – 4.01 (m,4H), 3.81 – 3.66 (m, 1H), 3.44 – 3.32 (m, 1H), 3.14 – 3.00 (m, 1H), 2.75 –2.22 (m, 16H), 1.73 – 1.55 (m, 6H), 1.55 – 1.38 (m, 8H), 1.36 – 1.15 (m,42H), 1.10 – 0.98 (m, 7H), 0.92 – 0.79 (t, 9H).

[0317] 1.23 Synthesis of YK-2013

[0318]

[0319] Using INT-10 (683 mg, 0.91 mmol) and INT-1 (132 mg, 0.91 mmol) as raw materials, YK-2013 (342 mg, 0.41 mmol, 45.4%) was obtained according to the synthesis method of YK-2001. C 48 H 93 N3O7, MS(ES): m / z (M+H + 824.7. 1 HNMR (400 MHz, CDCl3) δ 5.38 – 5.31 (m, 1H), 4.12 – 4.01 (m, 4H), 3.96 (d, J =5.8 Hz, 2H), 3.85 – 3.67 (m, 2H), 3.44 – 3.35 (m, 1H), 3.16 – 3.07 (m, 1H), 2.77 – 2.62 (m, 5H), 2.59 – 2.26 (m, 12H), 1.84 – 1.70 (m, 6H), 1.66 – 1.56 (m, 5H), 1.49 – 1.37 (m, 3H), 1.34 – 1.29 (m, 8H), 1.29 – 1.23 (m, 36H), 0.92 – 0.84 (t, 9H).

[0320] Synthesis of YK-2014 1.24

[0321]

[0322] Using INT-10 (585 mg, 0.78 mmol) and INT-2 (135 mg, 0.78 mmol) as raw materials, YK-2014 (391 mg, 0.46 mmol, 58.6%) was obtained according to the synthesis method of YK-2001. 49 H 96 N4O7, MS(ES): m / z (M+H + 853.7. 1 HNMR (400 MHz, CDCl3) δ 5.33 (d, J = 6.2 Hz, 1H), 4.47 (s, 3H), 4.14 – 4.01 (m,4H), 3.96 (d, J = 5.7 Hz, 1H), 3.80 (ddd, J = 13.1, 6.7, 3.6 Hz, 1H), 3.44 – 3.29(m, 1H), 3.18 – 3.05 (m, 1H), 2.67 (dd, J = 12.2, 6.4 Hz, 4H), 2.48 (dt, J =14.0, 8.1 Hz, 9H), 2.40 – 2.23 (m, 8H), 1.80 – 1.68 (m, 2H), 1.67 – 1.55 (m,5H), 1.52 – 1.39 (m, 3H), 1.37 – 1.20 (m, 43H), 0.95 – 0.80 (t, 9H).

[0323] Synthesis of 1.25 YK-2015

[0324]

[0325] Using INT-10 (585 mg, 0.78 mmol) and INT-3 (146 mg, 0.78 mmol) as raw materials, YK-2015 (453 mg, 0.52 mmol, 66.9%) was obtained according to the synthesis method of YK-2001. 51 H 99 N3O7, MS(ES): m / z (M+H + 866.7. 1 HNMR (400 MHz, CDCl3) δ 5.32 (d, J = 6.8 Hz, 1H), 4.20 – 4.01 (m, 4H), 3.96 (d, J= 5.8 Hz, 1H), 3.83 – 3.65 (m, 1H), 3.45 – 3.29 (m, 1H), 3.15 – 2.93 (m, 1H), 2.57 (dddd, J = 27.6, 20.6, 14.5, 6.4 Hz, 9H), 2.38 – 2.13 (m, 6H), 1.84 – 1.54(m, 12H), 1.53 – 1.38 (m, 7H), 1.37 – 1.16 (m, 43H), 0.98 – 0.76 (m, 12H).

[0326] Synthesis of YK-2016 1.26

[0327]

[0328] Using INT-10 (585 mg, 0.78 mmol) and INT-4 (135 mg, 0.78 mmol) as raw materials, YK-2016 (367 mg, 0.43 mmol, 55.1%) was obtained according to the synthesis method of YK-2001. 50 H 97 N3O7, MS(ES): m / z (M+H + 852.7. 1 HNMR (400 MHz, CDCl3) δ 5.41 – 5.23 (m, 1H), 4.14 – 4.01 (m, 4H), 3.96 (d, J =5.7 Hz, 1H), 3.80 – 3.68 (m, 1H), 3.46 – 3.27 (m, 1H), 3.17 – 3.02 (m, 1H), 2.84 – 2.56 (m, 7H), 2.53 – 2.24 (m, 9H), 1.83 – 1.51 (m, 16H), 1.51 – 1.38 (m, 3H), 1.38 – 1.12 (m, 44H), 0.99 – 0.79 (t, 9H).

[0329] Synthesis of YK-2017 1.27

[0330]

[0331] Using INT-10 (585 mg, 0.78 mmol) and INT-5 (92 mg, 0.78 mmol) as raw materials, YK-2017 (278 mg, 0.35 mmol, 44.5%) was obtained according to the synthesis method of YK-2001. C 46 H 91 N3O7, MS(ES): m / z (M+H + 798.7. 1 HNMR (400 MHz, CDCl3) δ 5.35 (d, J = 7.1 Hz, 1H), 4.86 (s, 3H), 4.14 – 4.02 (m,3H), 3.96 (d, J = 5.8 Hz, 2H), 3.82 – 3.73 (m, 1H), 3.42 – 3.31 (m, 1H), 3.18 –3.03 (m, 1H), 2.72 – 2.62 (m, 2H), 2.52 – 2.19 (m, 15H), 1.85 – 1.69 (m, 2H), 1.68 – 1.56 (m, 5H), 1.51 – 1.38 (m, 3H), 1.38 – 1.20 (m, 43H), 0.98 – 0.77 (t, 9H).

[0332] Synthesis of YK-2018 (1.28)

[0333]

[0334] Using INT-10 (585 mg, 0.78 mmol) and INT-6 (114 mg, 0.78 mmol) as raw materials, YK-2018 (411 mg, 0.50 mmol, 63.6%) was obtained according to the synthesis method of YK-2001. C 48 H 95 N3O7, MS(ES): m / z (M+H + 826.8. 1 HNMR (400 MHz, CDCl3) δ 5.37 – 5.25 (m, 1H), 4.13 – 4.02 (m, 4H), 3.96 (d, J=5.8 Hz, 2H), 3.76 – 3.68 (m, 1H), 3.45 – 3.31 (m, 1H), 3.15 – 3.03 (m, 1H), 2.78 – 2.23 (m, 16H), 1.79 – 1.69 (m, 2H), 1.68 – 1.54 (m, 5H), 1.49 – 1.38 (m, 3H), 1.34 – 1.23 (m, 43H), 1.08 – 0.98 (t, 6H), 0.92 – 0.81 (t, 9H).

[0335] 1.29 Synthesis of Lipid 43

[0336] Following the synthesis method for lipid 43 in PCT / CN2024 / 092807, 186 mg of lipid 43 was synthesized.

[0337] Synthesis of 1.30 10⁻⁵-C₈C₁₀

[0338] Referring to the synthesis method of 14-5-C8C10 in CN202310373284.8, N,N-diethylethylenediamine in Example 13 was replaced with N,N-dimethylethylenediamine to synthesize 120 mg of 10-5-C8C10.

[0339] 1.31 Synthesis of Lipid 11

[0340] Following the synthesis method of Lipid 149 in PCT / CN2024 / 092807, the intermediates 8-oxooctyl-2-hexyldecanoate and 1-(2-aminoethyl)piperidine were replaced with 8-oxooctanoate-9-heptadecyl ester and 4-methyl-1-piperazine ethylamine, respectively, to synthesize 170 mg of Lipid 11.

[0341] 1.32 Synthesis of Lipid 149

[0342] Following the synthesis method of Lipid 149 in PCT / CN2024 / 092807, 205 mg of Lipid 149 was synthesized.

[0343] 1.33 Synthesis of Lipid 170

[0344] Following the synthesis method of Lipid 170 in PCT / CN2024 / 092807, 130 mg of Lipid 170 was synthesized.

[0345] Example 2: mRNA-LNP formulation

[0346] 2.1 Ratio of carrier (liposome) to mRNA

[0347] Step 1: Following a molar ratio of cationic lipids:DSPC:cholesterol:DMG-PEG2000 of 49:10:39.5:1.5, the cationic lipids YK-2001, YK-2005, YK-2007, and YK-2011 synthesized in Example 1 were dissolved in ethanol with DSPC (Aivert (Shanghai) Pharmaceutical Technology Co., Ltd.), cholesterol (Aivert (Shanghai) Pharmaceutical Technology Co., Ltd.), and DMG-PEG2000, respectively, to obtain solution A. Solution A was rapidly added to citrate buffer (pH approximately 4.5) using the ethanol injection method, and vortexed for 30 seconds to obtain an ethanol solution of the lipids.

[0348] Step 2: Dilute Fluc-mRNA (firefly luciferase messenger RNA, GenBank Accession M15077.1) (Shanghai Qifa Experimental Reagent Co., Ltd.) in citrate buffer (pH approximately 4.5) to obtain an aqueous solution of Fluc-mRNA.

[0349] Step 3: Using a microfluidic device, the ethanol lipid solution prepared in Step 1 and the mRNA aqueous solution prepared in Step 2 were mixed at a flow rate of 10 mL / min according to the vector:mRNA mass ratios of 10:1, 15:1, 20:1, and 30:1, respectively, to prepare the corresponding liposome solutions. The liposome solutions were diluted 10 times with PBS and then ultrafiltered using a 300 kDa ultrafiltration tube to remove ethanol. The solution was then brought to a suitable volume with PBS and filtered through a 0.2 μm sterile filter to obtain mRNA-LNP formulations encapsulated with Fluc-mRNA at a molar ratio of cationic lipids (YK-2001, YK-2005, YK-2007, or YK-2011) / DSPC / cholesterol / DMG-PEG2000 of 49:10:39.5:1.5.

[0350] Cell transfection experiments showed that all mRNA-LNP compositions had good transfection effects, with the best transfection effect observed at a ratio of 15:1.

[0351] 2.2 Ratio of cationic lipids to neutral lipids

[0352] mRNA-LNP compositions encapsulated with Fluc-mRNA were prepared according to a method similar to that in 2.1, wherein the molar ratio of cationic lipids (YK-2001, YK-2005, YK-2007 or YK-2011) to neutral lipids DSPC was adjusted to 1:1, 3:1, 3.5:1, 4:1, 4.9:1, 10:1 and 15:1, respectively.

[0353] Cell transfection experiments showed that all the corresponding mRNA-LNP compositions could transfect cells, with the highest transfection efficiency being 4.9:1.

[0354] 2.3 Proportion of polymer-conjugated lipids in the carrier

[0355] mRNA-LNP compositions encapsulated with Fluc-mRNA were prepared according to a method similar to that in 2.1, wherein the cationic lipids were YK-2001, YK-2005, YK-2007 or YK-2011, and the molar percentages of the polymer conjugated lipid DMG-PEG2000 in the carrier were 0.5%, 1.5%, 2.5%, 3.5%, 5% and 10%, respectively.

[0356] Cell transfection experiments showed that the corresponding mRNA-LNP compositions could all transfect cells, with the highest transfection efficiency at 1.5%.

[0357] 2.4 Proportion of each component in the carrier

[0358] The mRNA-LNP formulation encapsulating Fluc-mRNA was prepared according to a method similar to that in 2.1, wherein the molar ratios of cationic lipids (YK-2001, YK-2005, YK-2007 or YK-2011), neutral lipid DSPC, structural lipid cholesterol, and polymer conjugated lipid DMG-PEG2000 in step 1 were 75:5:15:5, 65:8:25:2, 49:10:39.5:1.5, 45:10:43.5:1.5, 45:25:20:10, 40:10:48.5:1.5, 35:10:53.5:1.5, and 25:5:65:5, respectively.

[0359] Cell transfection experiments showed that transfection was achieved with molar ratios of cationic lipids, neutral lipids, structural lipids, and polymer-conjugated lipids of 75:5:15:5, 65:8:25:2, 49:10:39.5:1.5, 45:10:43.5:1.5, 45:25:20:10, 40:10:48.5:1.5, 35:10:53.5:1.5, and 25:5:65:5. Good transfection results were observed within the range of (35-49):(7.5-15):(35-55):(1-5), with the best transfection effect observed at a molar ratio of 49:10:39.5:1.5.

[0360] Example 3: Cell transfection with mRNA-LNP formulation encapsulated with Fluc-mRNA

[0361] Step 1: Cell resuscitation and passage: Resuscitate HEK293T cells and passage them in culture dishes to the required number of cells.

[0362] Step 2: Plating: Digest and count the cells in the culture dish, and plate 10,000 cells per well in a 96-well plate. Incubate overnight until the cells adhere.

[0363] Step 3: Cell transfection: 1.5 μg of the mRNA-LNP preparation containing Fluc-mRNA prepared in Example 2 (the cationic lipids being YK-2001, YK-2005, YK-2007, or YK-2011, respectively) was added to the cell culture medium of a 96-well plate. After culturing for 24 hours, the transfection efficiency was assessed by observing the fluorescence intensity under a fluorescence microscope.

[0364] Based on the transfection efficiency results, the following mRNA-LNP formulations were selected for the examples described below: the mass ratio of vector to mRNA was 15:1; the molar ratio of cationic lipids to neutral lipids was 4.9:1; the molar ratio of polymer-conjugated lipids to liposomes was 1.5%; and the molar ratio of cationic lipids, neutral lipids, structural lipids, and polymer-conjugated lipids was 49:10:39.5:1.5.

[0365] Example 4: Preparation of mRNA-LNP formulation

[0366] The structures and preparation methods of YK-2001 to YK-2018 are described in Example 1; the structures and preparation methods of other cationic lipid compounds are described in Table 1 below.

[0367] Table 1 Cationic lipid compounds

[0368]

[0369] 4.1 Prepare the corresponding ethanol lipid solutions of YK-2001 to YK-2018 and the cationic lipid compounds in Table 1 according to the method in step 1 of Example 2.1.

[0370] 4.2 Dilute Fluc-mRNA in citrate buffer (pH approximately 4.5) to obtain the corresponding aqueous mRNA solution.

[0371] 4.3 Using a microfluidic device, the ethanol lipid solution obtained in 4.1 and the Fluc mRNA aqueous solution obtained in 4.2 were mixed at a volume ratio of 1:3 at a flow rate of 10 mL / min to prepare the corresponding liposome solution with a carrier (liposome) to mRNA mass ratio of approximately 15:1. The liposome solution was diluted 10 times with PBS and then ultrafiltered using a 300 kDa ultrafiltration tube to remove ethanol. The solution was then brought to a suitable volume with PBS and filtered through a 0.2 μm sterile filter to obtain an mRNA-LNP formulation encapsulated with Fluc-mRNA, with a molar ratio of cationic lipids:DSPC:cholesterol:DMG-PEG2000 of 49:10:39.5:1.5.

[0372] Example 5: Determination of mRNA-LNP particle size, polydispersity index (PDI), and encapsulation efficiency

[0373] Particle size and polydispersity index (PDI) were determined using a Malvern laser particle size analyzer based on dynamic light scattering.

[0374] Take 25 μL of the mRNA-LNP solution prepared in Example 4, dilute it to 125 μL with 0.9% physiological saline, add it to the sample well, and repeat the measurement 3 times for each sample. The measurement conditions are: 90° scattering angle, 25 °C.

[0375] According to the manufacturer's instructions, the encapsulation efficiency of LNPs was determined using the Quant-IT Ribogreen RNA Quantification Kit (ThermoFisher Scientific, UK). The results are shown in Table 2. Figure 1 :

[0376] Table 2. Particle size, polydispersity index (PDI), and encapsulation efficiency of mRNA-LNP

[0377]

[0378] As shown in Table 2, the nanolipid particles prepared in Example 4 have particle sizes between 70 and 180 nm, all of which can be used for mRNA delivery. The polydispersity index is less than 0.20, indicating good particle size uniformity. Furthermore, they exhibit high encapsulation efficiency, all exceeding 90%.

[0379] Example 6: In vitro delivery performance and toxicity of LNP

[0380] The methods for cell resuscitation, passage, and plating are the same as those in Step 1 and Step 2 of Example 3.

[0381] In step 2, an appropriate volume of cell culture medium was added to the 96-well plate containing HEK293T cells. An mRNA-LNP preparation containing 0.3 μg Fluc-mRNA (prepared in Example 4) was added to each well. After culturing for 24 h, the appropriate reagents were added according to the kit instructions (Gaussia Luciferase Assay Kit, Invitrogen, 0221001405). The relative fluorescence intensity of each well was detected using an IVIS fluorescence detection system. Finally, 10 μL of CCK-8 solution was added to each well of the 24-hour cultured plate. After incubating the plate in an incubator for 1 hour, the absorbance at 450 nm was measured using a microplate reader to determine cell viability. The cell viability results are shown in Table 3 and... Figure 2 .

[0382] Table 3 Fluorescence detection results of Fluc-mRNA

[0383]

[0384] The relative fluorescence intensity (corresponding to mRNA translation efficiency) of the above mRNA-LNP compositions showed significant differences. The mRNA-LNP compositions prepared from YK-2001, YK-2002, YK-2003, YK-2005, YK-2006, YK-2007, YK-2008, YK-2009, YK-2011, YK-2012, YK-2014, YK-2015, YK-2017, and YK-2018 exhibited higher relative fluorescence intensity than those prepared from SM-102, MC3, lipid 43, 10⁻⁵-C8C10, lipid 11, lipid 149, and lipid 170, and all showed cell viability greater than 85%. Specifically:

[0385] 1. The cell transfection efficiency of the mRNA-LNP compositions prepared by YK-2001, YK-2002, YK-2003, YK-2005, YK-2006, YK-2007, YK-2008, YK-2009, YK-2011, YK-2012, YK-2014, YK-2015, YK-2017, and YK-2018 is significantly improved compared to representative cationic lipids in the prior art. For example, the cell transfection efficiency of YK-2005 can reach 2.9 times that of SM-102 and 5.4 times that of MC3.

[0386] 2. The cell transfection efficiency of the mRNA-LNP compositions prepared by YK-2001, YK-2002, YK-2003, YK-2005, YK-2006, YK-2007, YK-2008, YK-2009, YK-2011, YK-2012, YK-2014, YK-2015, YK-2017, and YK-2018 was significantly improved compared to cationic lipids with the same carbamate structure, namely lipids 43, 10-5-C8C10, lipid 11, lipid 149, and lipid 170. For example, the cell transfection efficiency of YK-2005 was 3.2 times that of lipid 43, 4.6 times that of 10-5-C8C10, 7.0 times that of lipid 11, 6.7 times that of lipid 149, and 3.1 times that of lipid 170.

[0387] Example 7: In vivo delivery performance of LNP

[0388] The Fluc-mRNA-LNP composition prepared in Example 4 was injected via tail vein into 6-8 week old female BALB / c albino mice weighing 15-20g (approximately 5 μg Fluc-mRNA / mouse). Six hours after administration, the fluorescent imaging substrate was injected intraperitoneally. The mice were allowed free movement for 5 minutes, and then the mean radiation intensity (corresponding to fluorescent protein expression intensity, i.e., protein expression level) of the protein expressed by the mRNA carried by the mRNA-LNP composition in the mice was detected using an IVIS Spectrum small animal in vivo imaging system. After sampling, the mice were euthanized by cervical dislocation and dissected, and the liver and spleen were precisely separated. The mean radiation intensity (corresponding to fluorescent protein expression intensity, i.e., protein expression level) of the protein expressed by Fluc-mRNA in each mouse organ was detected using an IVIS Spectrum small animal in vivo imaging system. The results of protein expression detection in the mouse liver and spleen are shown in Table 4. Figure 3 , Figure 4 and Figure 5 .

[0389] Table 4. Data from mouse organ imaging experiments

[0390]

[0391] The mRNA-LNP compositions prepared from YK-2001, YK-2002, YK-2003, YK-2005, YK-2006, YK-2007, YK-2008, YK-2009, YK-2011, YK-2012, YK-2014, YK-2015, YK-2017 and YK-2018 can efficiently deliver mRNA to the spleen, and the delivery effect is significantly enhanced compared with SM-102, MC3, lipid 43, 10-5-C8C10, lipid 11, lipid 149 and lipid 170. Specifically, compared with existing ionizable cationic lipids (SM-102, MC3, lipid 43, 10⁻⁵-C₈C₁₀, lipid 11, lipid 149, and lipid 170), the mRNA-LNP compositions prepared by YK-2001, YK-2002, YK-2003, YK-2005, YK-2006, YK-2007, YK-2008, YK-2009, YK-2011, YK-2012, YK-2014, YK-2015, YK-2017, and YK-2018 of this disclosure exhibit significantly enhanced spleen mean radiation intensity and in vivo mean radiation intensity. For example... The average radiation intensity in the spleen of the mRNA-LNP composition prepared by YK-2005 was 12.9 times, 16.1 times, 5.1 times, 5.7 times, 18.9 times, 15.1 times, and 4.6 times that of the mRNA-LNP compositions prepared by SM-102, MC3, lipid 43, 10-5-C8C10, lipid 11, lipid 149, and lipid 170, respectively. The average radiation intensity in the liver was only 0.2 times, 0.3 times, 0.2 times, 0.3 times, 0.5 times, 0.4 times, and 0.2 times that of the mRNA-LNP compositions prepared by SM-102, MC3, lipid 43, 10-5-C8C10, lipid 11, lipid 149, and lipid 170, respectively.

[0392] Meanwhile, the mRNA-LNP compositions prepared from YK-2001, YK-2002, YK-2003, YK-2005, YK-2006, YK-2007, YK-2008, YK-2009, YK-2011, YK-2012, YK-2014, YK-2015, YK-2017 and YK-2018 disclosed herein exhibit spleen average radiation intensity that is 6.4 times, 3.9 times, 8.5 times, 7.9 times, 6.6 times, 4.9 times, 7.0 times, 7.1 times, 6.3 times, 4.3 times, 3.7 times, 4.5 times, 4.7 times and 3.9 times that of the liver, respectively, demonstrating a significantly enhanced spleen targeting effect.

Claims

1. A cationic lipid compound or a pharmaceutically acceptable salt or stereoisomer thereof, characterized in that, The cationic lipid compound has the structure shown in formula (I): ， in: R1 is , , , , and any one of them; R2 is or unreplaced C 8-12 Straight-chain alkanes; R3 is or ; L1 is -(CH2)2-; L2 is -(CH2)3- or -(CH2)5-; L3 is -(CH2)5-; L4 is ; M1 is -C(O)O-; M2 is -C(O)O-.

2. The cationic lipid compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein, The compound represented by formula (I) is any of the following compounds: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 。 3. A composition, characterized in that, The composition comprises cationic lipids, including the cationic lipid compound of claim 1 or 2 or a pharmaceutically acceptable salt or stereoisomer thereof.

4. The composition according to claim 3, wherein, The composition further comprises any one or a combination of at least two of neutral lipids, structural lipids, and polymeric conjugated lipids.

5. The composition according to claim 4, wherein, The composition satisfies any one or a combination of at least two of the following conditions (1) to (4): (1) The molar ratio of the cationic lipid to all lipid compounds in the composition is 0.25:1-0.75:1; (2) The molar ratio of the cationic lipid to the neutral lipid is 1:1 to 15:1; (3) The molar ratio of the structural lipid to all lipid compounds in the composition is 0.15:1 to 0.65:1; (4) The molar ratio of the polymer conjugated lipid to all lipid compounds in the composition is 0.005:1-0.1:

1.

6. The composition according to claim 4, wherein, The composition satisfies any one or a combination of at least two of the following conditions (1) to (4): (1) The molar ratio of the cationic lipid to all lipid compounds in the composition is 0.35:1, 0.4:1, 0.45:1, 0.49:1 or 0.65:1; (2) The molar ratio of the cationic lipid to the neutral lipid is 3:1, 3.5:1, 4:1, 4.9:1 or 10:1; (3) The molar ratio of the structural lipid to all lipid compounds in the composition is 0.2:1, 0.25:1, 0.395:1, 0.435:1, 0.485:1 or 0.535:1; (4) The molar ratio of the polymer conjugated lipid to all lipid compounds in the composition is 0.015:1, 0.025:1, 0.035:1 or 0.05:

1.

7. The composition according to claim 4, wherein, In the composition, the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer conjugated lipid is (25-75):(5-25):(15-65):(0.5-10).

8. The composition according to claim 4, wherein, In the composition, the molar ratio of the cationic lipid, neutral lipid, structural lipid, and polymer conjugated lipid is 49:10:39.5:1.

5.

9. The composition according to any one of claims 4-8, wherein, The composition further satisfies any one or a combination of at least two of the following conditions (5) to (7): (5) The neutral lipids are selected from any one or a combination of at least two of the group consisting of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide and sterol; (6) The structural lipids are selected from any one or at least two of the following groups: cholesterol, nonsterols, sitosterol, ergosterol, campesterol, stigmasterol, brassinosteroids, tomatine, ursolic acid, α-tocopherol and corticosteroids; (7) The polymer conjugated lipid is selected from any one or a combination of at least two of the following: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol and PEG-modified dialkylglycerol.

10. The composition according to claim 9, wherein, The composition further satisfies any one or a combination of at least two of the following conditions (5) to (7): (5) The neutral lipid is selected from any one or a combination of at least two of the following: 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline, 1,2-dimyristoyl-sn-glycerol-3-phosphate choline, 1,2-dioleoyl-sn-glycerol-3-phosphate choline, 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline, 1,2-distearate-sn-glycerol-3-phosphate choline, 1,2-diundecanoyl-sn-glycerol-3-phosphate choline, 1-palmitoyl-2-oleoyl -sn-glycerol-3-phosphate choline, 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline, 1-oleoyl-2-cholesterolylhemisuccino-sn-glycerol-3-phosphate choline, 1-hexadecyl-sn-glycerol-3-phosphate choline, 1,2-dilinanoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidanoyl-sn-glycerol-3-phosphate choline, 1,2-bis(docohexanoyl-sn-glycerol-3-phosphate choline), 1,2-dioleoyl-sn-glycerol-3-phosphate choline 1,2-Diphylanoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-distearatel-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-bis(docosahexaenooyl)-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate -rac-(1-glycerol) sodium salt, dipalmitoylphosphatidylglycerol, palmitoyloleoylphosphatidylethanolamine, distearyl-phosphatidyl-ethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphoethanolamine, 1-stearoyl-2-oleoyl-stearoylethanolamine, 1-stearoyl-2-oleoylphosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine and lysophosphatidylethanolamine; (6) The structural lipid is cholesterol; (7) The polymer conjugated lipid is selected from any one or a combination of at least two of the following: distearylphosphatidylethanolamine polyethylene glycol 2000, 1,2-dimyristic-sn-glycerol-3-methoxy polyethylene glycol 2000 and methoxy polyethylene glycol bistetradecylacetamide.

11. The composition according to claim 10, wherein, The neutral lipid is 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine or 1,2-distearate-sn-glycerol-3-phosphate choline; Alternatively, the conjugated lipid of the polymer is 1,2-dimyristoyl-sn-glycerol-3-methoxy polyethylene glycol 2000.

12. The composition according to claim 4, wherein, The composition also includes one or more other ionizable lipid compounds.

13. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a carrier and an active ingredient, wherein the carrier comprises the composition of any one of claims 3-12.

14. The pharmaceutical composition according to claim 13, wherein, The active ingredient comprises therapeutic agents and / or preventive agents; And / or, the pharmaceutical composition may further include pharmaceutically available excipients.

15. The pharmaceutical composition according to claim 13, wherein, The pharmaceutical composition satisfies any one or a combination of at least two of the following conditions (1) to (3): (1) The pharmaceutical composition is a nanoparticle formulation, wherein the average particle size of the nanoparticle formulation is 70 nm-180 nm; and the polydispersity index of the nanoparticle formulation is less than 0.

10. (2) The mass ratio of the carrier to the active component is 10:1-30:1; (3) The active component is selected from any one of the groups consisting of nucleic acids, small molecule compounds, polypeptides or proteins, or at least a combination of two.

16. The composition according to claim 15, wherein, The nucleic acid is selected from any one or a combination of at least two of the following groups: small interfering RNA, asymmetric interfering RNA, microRNA, Dicer-substrate RNA, small hairpin RNA, and messenger RNA; Alternatively, the mass ratio of the carrier to the active component is 15:1-20:

1.

17. Use of the cationic lipid compound of claim 1 or 2 or a pharmaceutically acceptable salt or stereoisomer thereof, or the composition of any one of claims 3-12, or the pharmaceutical composition of any one of claims 13-16 in the preparation of a spleen-targeting medicament for the treatment of a disease or condition.

18. The use according to claim 17, wherein, The disease or condition is characterized by the dysfunction or abnormality of proteins or polypeptides.

19. The use according to claim 18, characterized in that, The disease or condition is selected from any one or a combination of at least two of the following groups: infectious diseases, cancer, proliferative diseases, genetic diseases, autoimmune diseases, neurodegenerative diseases, cardiovascular and cerebrovascular diseases, renal and vascular diseases, and metabolic diseases.

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