Ionizable cationic lipid targeting immune cells, composition comprising same and use thereof

Abstract

The invention belongs to the field of medicines, particularly relates to the technical field of targeted delivery lipids, and particularly discloses an ionizable cationic lipid targeting immune cells, a composition containing the ionizable cationic lipid and application of the ionizable cationic lipid. The immune cell targeting cationic lipid provided by the invention has a structure as shown in formula (I), can be used for RNA targeting delivery, and can significantly enhance the targeting effect of mRNA drugs on immune cells. # imgabs0 #

Description

Technical Field

[0001] The present disclosure belongs to the field of medicine, and particularly relates to ionizable cationic lipids targeting immune cells, compositions containing the same, and uses thereof. Background Art

[0002] Immune cells refer to cells that participate in or are related to immune responses. They play a crucial role in protecting the body from infections and diseases in the human body. Targeting immune cells can achieve precise drug delivery, reduce the distribution of drugs in non-target cells, and reduce the toxic and side effects of drugs. Targeted delivery of nucleic acids to immune cells provides the possibility for the development of new immunotherapies. For example, in situ CAR-T or CAR-M therapies in vivo, by directly delivering mRNA encoding chimeric antigen receptors (CARs) to T cells or macrophages, avoid the complex in vitro processes of traditional CAR cell therapies, reduce the preparation difficulty and cost; at the same time, the targeted immune cell technology can efficiently and safely engineer T cells or macrophages according to the individual differences and disease characteristics of patients, making them express CARs against cancer cells and achieving personalized treatment.

[0003] In the field of pharmacy, effective targeted delivery of small molecule drugs, polypeptides, proteins, nucleic acids, etc. is a persistent problem. For the delivery of nucleic acids, it faces great challenges due to its low cell permeability and high sensitivity to degradation by nucleases (such as RNAase).

[0004] Compositions containing cationic lipids, liposomes, and liposome complexes (lipoplexes) can effectively deliver bioactive substances such as small molecule drugs, polypeptides, proteins, and nucleic acids to cells and / or intracellular compartments as transport mediators. These compositions generally include one or more cationic and / or ionizable lipids, neutral lipids, structural lipids, and polymer-conjugated lipids. Cationic and / or ionizable lipids include, for example, amine-containing lipids that can be easily protonated. Although many such lipid-containing nanoparticle compositions have been demonstrated, safety, efficacy, and specificity still need to be improved. Notably, the increased complexity of lipid nanoparticles (LNPs) complicates their production and may increase their toxicity, which is a major concern that may limit their clinical application. For example, nucleic acid drugs represented by patisiran (trade name onpattro®) require pre-administration of steroids and antihistamines in patients to eliminate unnecessary immune responses (T. Coelho, D. Adams, A. Silva, et al., Safety and efficacy of RNAitherapy for transthyretin amyloidosis, N Engl J Med, 369 (2013) 819-829.).

[0005] Therefore, there is an urgent need to develop improved cationic lipid compounds that facilitate the delivery of therapeutic and / or prophylactic agents such as nucleic acids to immune cells, and compositions containing the same. Summary of the Invention

[0006] The present disclosure provides ionizable cationic lipids that can target immune cells, compositions containing the same, and uses thereof. The ionizable cationic lipids provided by the present disclosure can be used to deliver therapeutic or prophylactic agents such as nucleic acid molecules, small molecule compounds, polypeptides, or proteins. The preparation method is simple, the targeting to immune cells is strong, it can carry drug active ingredients to transfect cells with a high transfection efficiency, and has low cytotoxicity, which can improve the delivery efficiency and safety.

[0007] The technical solutions provided by the present disclosure may include, for example: [1] A (cationic lipid) compound, or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer, wherein the compound has the structure shown in formula (I): , Wherein: R1 is a substituted or unsubstituted N-containing heterocycle; R2 and R3 are each independently a substituted or unsubstituted C 10-16A straight-chain or branched-chain alkene or alkane, where R2 and R3 are the same or different; L1 and L2 are each independently an unsubstituted C 2-8 straight-chain alkylene; M1 is -(CH2) n -, -C(O)NH-, or -NHC(O)-, where n is 0, 1, 2, 3, or 4; M2 and M3 are each independently any one of -CH=CH-, -C(O)O-, and -C(O)N-, and M2 and M3 are the same or different.

[0008] In the compound of formula (I) provided by the present invention, the number of carbon atoms of R2 and R3 can each independently be 10, 11, 12, 13, 14, 15, 16, or can also be a range formed by any two of the above values, or any intermediate value (an integer) within that range.

[0009] In the compound of formula (I) provided by the present invention, R2 and R3 can each independently be a straight-chain alkyl, a branched-chain alkyl, a straight-chain alkenyl, or a branched-chain alkenyl.

[0010] In the compound of formula (I) provided by the present invention, the number of carbon atoms of L1 and L2 can each independently be 2, 3, 4, 5, 6, 7, 8, or can also be a range formed by any two of the above values, or any intermediate value (an integer) within that range.

[0011] [2] The compound according to [1], or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer, where R1 is , or ; and / or, R2 is , , , , or an unsubstituted C 10-16 straight-chain alkane; and / or, R3 is , , , , or an unsubstituted C 10-16 straight-chain alkane; and / or, L1 is -(CH2)2-, -(CH2)3-, -(CH2)5-, or -(CH2)8-; and / or, L2 is -(CH2)2-, -(CH2)3-, -(CH2)5-, or -(CH2)8-; and / or, M1 is -CH2- or -NHC(O)-; and / or, M2 is -CH=CH-, -C(O)O- or -C(O)N-; and / or, M3 is -CH=CH-, -C(O)O- or -C(O)N-.

[0012] [3] The compound according to [1] or [2], or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, wherein the compound has any one of the structures of YK-1501 to YK-1518 as follows: , , , , , , , , , , , , , , , , and .

[0013] Preferably, the compound has the structure of any one of YK-1503, YK-1504, YK-1505, YK-1507, YK-1510, YK-1511, YK-1513, YK-1515 and YK-1516.

[0014] [4] A carrier, wherein the carrier contains cationic lipid, and the cationic lipid includes the compound according to any one of [1]-[3], or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer.

[0015] [5] The carrier according to [4], wherein the cationic lipid accounts for 25% - 75% of the carrier in terms of molar percentage. That is, based on the total molar amount of the carrier, the content of the cationic lipid is 25 - 75 mol%.

[0016] For example, the content of the cationic lipid in the carrier can be 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, 55 mol%, 60 mol%, 65 mol%, 70 mol%, 75 mol%, or can also be a range composed of any two of the above values, or any intermediate value within this range.

[0017] [6] The carrier according to [4] or [5], wherein the carrier further comprises neutral lipid; and / or, the carrier further comprises structural lipid; and / or, the carrier further comprises polymer-conjugated lipid.

[0018] [7] The carrier according to [6], wherein the neutral lipid is selected from any one or a combination of at least two of the group consisting of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol and their derivatives; and / or, the structural lipid is selected from any one or a combination of at least two of the group consisting of cholesterol, non-sterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol and corticosteroid; and / or, the polymer-conjugated lipid is selected from any one or a combination of at least two of the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol and PEG-modified dialkylglycerol.

[0019] [8] The carrier according to [6] or [7], wherein the neutral lipid is selected from any one or a combination of at least two of the group consisting of: 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dielaidoyl-sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-di-O-octadecenoyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-cholesteryl succinyl-sn-glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3-phosphocholine, 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, dipalmitoyl phosphatidylglycerol, palmitoyl oleoyl phosphatidylethanolamine, distearoyl-phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, dimyristoyl phosphoethanolamine, 1-stearoyl-2-oleoyl-stearoylethanolamine, 1-stearoyl-2-oleoyl-phosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine and lysophosphatidylethanolamine.

[0020] [9] The carrier according to any one of [6]-[8], wherein the neutral lipid is selected from 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine and / or 1,2-distearoyl-sn-glycero-3-phosphocholine.

[0021]

[10] The carrier according to [6] or [7], wherein the structural lipid is cholesterol.

[0022]

[11] The carrier according to [6] or [7], wherein the polymer-conjugated lipid is selected from any one or a combination of at least two of the group consisting of: distearoyl phosphatidylethanolamine polyethylene glycol 2000, dimyristoyl glycerol-3-methoxy polyethylene glycol 2000, and methoxy polyethylene glycol ditetradecylacetamide.

[0023]

[12] The carrier according to any one of [6]-

[11] , wherein the molar percentage of the neutral lipid in the carrier is 5%-25%; and / or, the molar percentage of the structural lipid in the carrier is 15%-65%; and / or, the molar percentage of the polymer-conjugated lipid in the carrier is 0.5%-10%.

[0024] That is, based on the total molar amount of the carrier, the content of the neutral lipid is 5-25 mol%; and / or, the content of the structural lipid is 15-65 mol%; and / or, the content of the polymer-conjugated lipid is 0.5-10 mol%.

[0025] For example, in the carrier, the content of the neutral lipid can be 5 mol%, 10 mol%, 15 mol%, 20 mol%, 25 mol%, or can also be a range composed of any two of the above values, or any intermediate value within this range.

[0026] For example, in the carrier, the content of the structural lipid can be 15 mol%, 20 mol%, 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, 55 mol%, 60 mol%, 65 mol%, or can also be a range composed of any two of the above values, or any intermediate value within this range.

[0027] For example, in the carrier, the content of the polymer-conjugated lipid can be 0.5 mol%, 1 mol%, 2 mol%, 3 mol%, 4 mol%, 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, or can also be a range composed of any two of the above values, or any intermediate value within this range.

[0028]

[13] The carrier according to any one of [4]-

[12] , wherein in the carrier, the molar ratio of the cationic lipid to the neutral lipid is 1:1-15:1; and / or, in the carrier, the molar ratio of the cationic lipid to the structural lipid is 0.6:1-3:1; and / or, in the carrier, the molar ratio of the cationic lipid to the polymer-conjugated lipid is 4.5:1-32.5:1.

[0029]

[14] The carrier according to any one of [4]-

[13] , wherein in the carrier, 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).

[0030]

[15] The carrier according to

[14] , wherein in the carrier, the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is (35-49):(7.5-15):(35-5):(1-5).

[0031]

[16] The carrier according to

[14] or

[15] , wherein in the carrier, the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is 49:10:39.5:1.5.

[0032]

[17] The carrier according to any one of [4]-

[16] , wherein the cationic lipid further comprises one or more other cationic lipid compounds.

[0033] "Other cationic lipid compounds" refers to cationic lipid compounds other than the compound of formula (I) provided in the present disclosure, or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer. Any cationic lipid compound that can be used in the preparation of carriers (especially carriers for loading bioactive molecules such as nucleic acids, proteins, peptides, etc.) in the art can be applicable to the present invention, and there is no particular limitation on its source, and it can be obtained through commercial channels or prepared according to the prior art.

[0034]

[18] A composition, wherein the composition comprises an active ingredient and a carrier, and the carrier is the carrier according to any one of [4]-

[17] .

[0035] "Active ingredient" refers to an active molecule having a certain function or effect, such as an active molecule having a disease treatment / prevention function (such as a small molecule drug, a therapeutic / preventive nucleic acid, a therapeutic / preventive protein or peptide, etc.), an active molecule having a biomarker function (such as GFP or its encoding gene, etc.). "Carrier" refers to a molecule or composition used to load an active ingredient and play roles such as protection and transportation. Generally, the carrier itself does not have a function or activity, but the selection of the carrier has a certain influence on the activity of the active ingredient.

[0036]

[19] The composition according to

[18] , wherein the composition is a nanoparticle preparation, and the average particle size of the nanoparticle preparation is 10 nm - 300 nm; the polydispersity index (PDI) of the nanoparticle preparation is ≤ 0.5. The average particle size and polydispersity index of the nanoparticle preparation and the like can be measured by existing methods. For example, they can be detected by using instruments such as a laser particle size analyzer.

[0037]

[20] The composition according to

[18] or

[19] , wherein the average particle size of the nanoparticle preparation is 40 nm - 240 nm; the polydispersity index of the nanoparticle preparation is ≤ 0.4.

[0038] For example, the average particle size of the nanoparticle preparation can be 40 nm, 50 nm, 60 nm, 70 nm, 72 nm, 74 nm, 76 nm, 78 nm, 80 nm, 82 nm, 84 nm, 86 nm, 88 nm, 90 nm, 92 nm, 94 nm, 96 nm, 98 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 180 nm, 200 nm, 220 nm, 240 nm, or can also be a range formed by any two of the above values, or any intermediate value within this range.

[0039] For example, the polydispersity index of the nanoparticle preparation can be 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, or can also be a range formed by any two of the above values, or any intermediate value within this range.

[0040]

[21] The composition according to any one of

[18] -

[20] , wherein the active ingredient comprises a therapeutic agent or a prophylactic agent.

[0041]

[22] The composition according to any one of

[18] -

[21] , wherein the mass ratio of the carrier to the therapeutic agent or prophylactic agent is 10:1 - 30:1.

[0042] For example, the mass ratio of the carrier to the therapeutic agent or prophylactic agent can be 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, 30:1, or can also be a range formed by any two of the above ratios, or any intermediate ratio within this range.

[0043]

[23] The composition according to

[22] , wherein the mass ratio of the carrier to the therapeutic agent or prophylactic agent is 12.5:1 - 20:1.

[0044]

[24] The composition according to

[23] , wherein the mass ratio of the carrier to the therapeutic or prophylactic agent is 13:1 - 17:1.

[0045]

[25] The composition according to any one of

[21] -

[24] , wherein the therapeutic or prophylactic agent is a vaccine or a compound capable of eliciting an immune response.

[0046]

[26] The composition according to

[25] , wherein the therapeutic or prophylactic agent is selected from any one or a combination of at least two of the group consisting of nucleic acids, small molecule compounds, polypeptides, or proteins.

[0047]

[27] The composition according to

[26] , wherein the therapeutic or prophylactic agent is a nucleic acid.

[0048]

[28] The composition according to

[27] , wherein the therapeutic or prophylactic agent is ribonucleic acid (RNA).

[0049]

[29] The composition according to

[28] , wherein the ribonucleic acid is selected from any one or a combination of at least two of the group consisting of: small interfering RNA, asymmetric interfering RNA, microRNA, Dicer-substrate RNA, small hairpin RNA, messenger RNA.

[0050]

[30] The composition according to

[29] , wherein the ribonucleic acid is messenger RNA.

[0051]

[31] The composition according to any one of

[18] -

[30] , wherein the composition further comprises a pharmaceutically acceptable excipient and / or diluent.

[0052]

[32] Use of the compound according to any one of [1] - [3], or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, or the carrier according to any one of [4] -

[17] , or the composition according to any one of

[18] -

[31] in the preparation of a drug.

[0053]

[33] The use according to

[32] , wherein the active ingredient of the drug is selected from any one or a combination of at least two of the group consisting of nucleic acids, small molecule compounds, polypeptides, or proteins.

[0054] Preferably, the drug is a nucleic acid drug (i.e., a drug with a nucleic acid as the active ingredient). The nucleic acid used in this nucleic acid drug can be the nucleic acid selected from the aforementioned therapeutic or prophylactic agents, which will not be elaborated herein.

[0055]

[34] Use of the compound according to any one of [1]-[3], or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, or the carrier according to any one of [4]-

[17] in enhancing cell transfection efficiency and / or reducing cytotoxicity.

[0056]

[35] The use according to

[34] , wherein the use is for enhancing cell transfection efficiency and / or reducing cytotoxicity in vitro.

[0057]

[36] Use of the compound according to any one of [1]-[3], or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, or the carrier according to any one of [4]-

[17] in enhancing the targeting of nucleic acid to a target, and / or in enhancing the expression level of nucleic acid in the target, wherein the target is selected from any one or a combination of at least two of the group consisting of a target organ, a target tissue and a target cell.

[0058]

[37] The use according to

[36] , wherein the target organ or target tissue is selected from any one or a combination of at least two of the group consisting of spleen, liver, lymph and muscle; and / or, the target cell is selected from any one or a combination of at least two of the group consisting of B cell, NK cell, DC cell, T cell and macrophage.

[0059]

[38] The use according to

[37] , wherein the target organ or target tissue is spleen; and / or, the target cell is selected from any one or a combination of at least two of the group consisting of B cell, DC cell, T cell and macrophage.

[0060]

[39] Use of the compound according to any one of [1]-[3], or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, or the carrier according to any one of [4]-

[17] , or the composition according to any one of

[18] -

[31] in the preparation of a medicament for treating a disease or disorder in a subject in need thereof.

[0061]

[40] The use according to

[39] , wherein the disease or disorder is characterized by a malfunction or abnormality of a protein or polypeptide.

[0062]

[41] The use according to

[39] or

[40] , wherein the disease or disorder is selected from any one or a combination of at least two of the group consisting of: infectious diseases (such as diseases caused by viral infections), cancers and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.

[0063]

[42] Use according to

[39] , wherein the subject is a mammal.

[0064]

[43] Use according to

[42] , wherein the subject is a human.

[0065]

[44] Use according to any one of

[39] -

[43] , wherein the drug is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally or by inhalation.

[0066]

[45] Use according to

[44] , wherein the drug is administered subcutaneously.

[0067]

[46] Use according to any one of

[39] -

[45] , wherein the dosage of the drug is such that a therapeutic or prophylactic agent in a dose of about 0.001 mg / kg to about 10 mg / kg is administered to the subject.

[0068] It should be understood that in the above technical solutions, the uses provided by the present disclosure can include both therapeutic and diagnostic uses, and can also include non-therapeutic and non-diagnostic uses. For example, therapeutic uses can include using the compounds provided by the present disclosure, or their N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers, or using the carriers provided by the present disclosure to package the (drug) active ingredient and deliver it to the target organ / tissue / cell, or using the compositions of the present disclosure to deliver the active ingredient contained therein to the target organ / tissue / cell, so as to achieve the effects of treating diseases, improving symptoms, regulating physiological activities in the body, etc.; diagnostic uses can include packaging the active ingredient for disease diagnosis in the compounds provided by the present disclosure, or their N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers or carriers, so that the active ingredient is delivered to the target organ / tissue / cell, thereby achieving the purpose of disease diagnosis; non-therapeutic / non-diagnostic purposes can include encapsulating the active ingredient using the compounds provided by the present disclosure, or their N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers, or using the carriers provided by the present disclosure, so as to deliver it to the target organ / tissue / cell for non-therapeutic and non-diagnostic purposes such as scientific research, detection, etc. (such as conducting research on disease mechanisms, research on drug action mechanisms, developing new drugs, drug screening, etc.).

[0069] The beneficial effects of the present disclosure at least include: The cationic lipid compounds and lipid compositions of the present disclosure (which can also be referred to as "carriers" in the present disclosure) can be used for encapsulating drug active ingredients such as nucleic acids (such as mRNA, etc.).

[0070] The mRNA-LNP composition prepared from the cationic lipid of the present disclosure has at least the following advantages: it can significantly increase the protein expression level in a subject (such as a mouse), and at the same time has significant spleen targeting, and can significantly increase the protein expression level in vivo ( in vivo ) and in vitro ( in intro ); moreover, compared with the ionizable cationic lipid using the prior art, the mRNA-LNP composition using the cationic lipid compound of the present disclosure significantly increases the percentage of immune cells expressing antigens in the spleen, indicating that the spleen and immune cell targeting of the mRNA-LNP composition prepared from the cationic lipid of the present disclosure is significantly improved. Brief Description of the Drawings

[0071] In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the drawings of the present disclosure will be briefly introduced below. It should be understood that the drawings described below are only some exemplary embodiments of the present disclosure and do not limit the present disclosure.

[0072] Figure 1 Showing the total fluorescence intensity in mice after intravenous injection of an mRNA-LNP composition encapsulating Fluc-mRNA prepared based on YK-1502, YK-1503, YK-1504, YK-1505, YK-1507, YK-1509, YK-1510, YK-1511, YK-1513, YK-1515, YK-1516, YK-1517, SM-102, MC3, 9322-O17S, 76-017Se, and C16 for 6 hours.

[0073] Figure 2 Showing the fluorescence intensity in the liver of mice after intravenous injection of an mRNA-LNP composition encapsulating Fluc-mRNA prepared based on YK-1502, YK-1503, YK-1504, YK-1505, YK-1507, YK-1509, YK-1510, YK-1511, YK-1513, YK-1515, YK-1516, YK-1517, SM-102, MC3, 9322-O17S, 76-017Se, and C16 for 6 hours.

[0074] Figure 3To show the fluorescence intensity in the spleens of mice 6 hours after intravenous injection of an mRNA-LNP composition encapsulating Fluc-mRNA prepared based on YK-1502, YK-1503, YK-1504, YK-1505, YK-1507, YK-1509, YK-1510, YK-1511, YK-1513, YK-1515, YK-1516, YK-1517, SM-102, MC3, 9322-O17S, 76-017Se, and C16 into the mice.

[0075] Figure 4 Flow cytometry experimental graphs (eGFP) of DC cells and macrophages in the spleens of mice 24 hours after intravenous injection of an mRNA-LNP composition encapsulating Fluc-mRNA prepared based on MC3, YK-1505, and YK-1507 respectively, and a blank control into the mice. Detailed implementation manners

[0076] To make the purposes, technical solutions, and advantages of the embodiments of the present disclosure clearer, the technical solutions of the embodiments of the present disclosure will be described below with reference to the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present disclosure, rather than all of the embodiments. Based on the described embodiments of the present disclosure, those of ordinary skill in the art can implement in other specific forms without departing from the basic attributes and gist of the present disclosure. It should be understood that, on the premise of no conflict, any and all implementation manners of the present disclosure can be combined with the technical features in any other implementation manner or multiple other implementation manners to obtain additional implementation manners. The present disclosure includes such additional implementation manners obtained by such combinations.

[0077] All publications and patents mentioned in the present disclosure are hereby incorporated into the present disclosure in their entirety by reference. If the uses or terms used in any incorporated publication and patent conflict with the uses or terms used in the present disclosure, then the uses and terms of the present disclosure shall prevail.

[0078] The chapter titles used in the present disclosure are only for the purpose of organizing the article and should not be construed as a limitation on the subject matter described.

[0079] Unless otherwise specified, all technical terms and scientific terms used in the present disclosure have the ordinary meanings in the field to which the claimed subject matter belongs. If there are multiple definitions for a certain term, then the definition in the present disclosure shall prevail.

[0080] Except as used in working examples or otherwise indicated, all numbers expressing quantitative properties such as amounts in the specification and claims are to be understood as being modified in all instances by the term "about". It should also be understood that any numerical range recited in this disclosure is intended to include all sub-ranges within that range and any combination of the endpoints of that range or sub-ranges. When a numerical range is disclosed in this disclosure, the above range is considered continuous and includes the minimum and maximum values of that range, as well as every value therebetween. Further, when the range refers to integers, every integer between the minimum and maximum values of that range is included. Additionally, when multiple ranges are provided to describe features or characteristics, those ranges may be combined. In other words, unless otherwise specified, all ranges disclosed in this disclosure should be understood to include any and all sub-ranges subsumed therein.

[0081] As used in this disclosure, words such as "comprising", "containing" or "including" and the like mean that the elements appearing before that word encompass the elements recited after that word and their equivalents, without excluding elements not recited. The terms "containing", "including" or "comprising" used in this disclosure can be open, semi-closed and closed. In other words, the above terms also include "consisting essentially of" or "consisting of".

[0082] The term "pharmaceutically acceptable" as used in this disclosure means that a compound or composition is chemically and / or toxicologically compatible with the other components of the formulation and / or with a human or mammal to which it is administered for the prevention or treatment of a disease or disorder.

[0083] The term "subject" or "patient" as used in this disclosure includes mammals, such as common laboratory animals (e.g., mice, rats, guinea pigs, rabbits, pigs, monkeys, etc.), and also humans.

[0084] The term "treatment" as used in this disclosure means administering one or more pharmaceutical substances to a patient or subject suffering from a disease or having symptoms of said disease, for the purpose of curing, alleviating, reducing, ameliorating or affecting said disease or the symptoms of said disease. In the context of this disclosure, unless specifically stated to the contrary, the term "treatment" may also include prevention.

[0085] The term "solvate" as used in this disclosure refers to a complex formed by the compound of formula (I) or a pharmaceutically acceptable salt thereof with a solvent (such as ethanol or water). It should be understood that any solvate of the compound of formula (I) used in the treatment of a disease or disorder, although it may provide different properties (including pharmacokinetic properties), once absorbed into the subject, will yield the compound of formula (I), such that the use of the compound of formula (I) respectively encompasses the use of any solvate of the compound of formula (I).

[0086] The term "hydrate" refers to the case where the solvent in the above-mentioned term "solvate" is water.

[0087] It should be further understood that the compound of formula (I) or a pharmaceutically acceptable salt thereof can be isolated in the form of a solvate, and thus any such solvate is included within the scope of the present disclosure. For example, the compound of formula (I) or a pharmaceutically acceptable salt thereof can exist in an unsolvated form or in a solvated form formed by combination with a pharmaceutically acceptable solvent (such as water, ethanol, etc.).

[0088] The term "pharmaceutically acceptable salt" refers to relatively non-toxic, inorganic acid or organic acid addition salts of the compounds of the present disclosure. For example, see S. M. Berge et al., "Pharmaceutical Salts", J. Pharm. Sci. 1977, 66, 1-19. Among them, inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid or nitric acid, etc.; organic acids such as 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, pamoic acid, pectinic acid, 3-phenylpropionic acid, picric acid, pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, aminosulfonic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, 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, glucoheptonic acid, glycerophosphoric acid, 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 compound represented by formula (I).

[0089] The nitrogen-containing compound of formula (I) of the present disclosure can be converted into an N-oxide by treatment with an oxidizing agent (such as m-chloroperbenzoic acid, hydrogen peroxide, ozone). Therefore, under the conditions where the valence state and structure permit, the compounds claimed in the present disclosure include not only the nitrogen-containing compounds represented by the structural formula, but also their N-oxide derivatives.

[0090] Certain compounds of the present disclosure may exist in the form of one or more stereoisomers. Stereoisomers include geometric isomers, diastereoisomers, and enantiomers. Accordingly, the compounds claimed in the present 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 lower side effects than other stereoisomers. Single stereoisomers and optically active mixtures can be obtained by methods such as chiral source synthesis, chiral catalysis, and chiral resolution. Racemates can be resolved by chromatographic resolution or chemical resolution. For example, chiral acid resolving agents such as chiral tartaric acid and chiral malic acid can be added to form salts with the compounds of the present disclosure, and separation can be carried out by utilizing the physicochemical properties of the products such as different solubilities.

[0091] The present disclosure also includes all suitable isotopic variants of the compounds of the present disclosure. Isotopic variants are defined as compounds in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass commonly found or predominantly present in nature. Examples of isotopes that can be introduced into the compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, and oxygen, such as 2 H (deuterium), 3 H (tritium), 11 C, 13 C, 14 C, 15 N, 17 O, and 18 O.

[0092] The term "alkyl" in the present disclosure refers to a branched and straight-chain saturated aliphatic monovalent hydrocarbon group including a specified number of carbon atoms. The term "alkylene" in the present disclosure refers to a branched and straight-chain saturated aliphatic divalent hydrocarbon group including a specified number of carbon atoms. C n-m refers to a group including from n to m carbon atoms. For example, C 2-5 alkylene includes C2 alkylene, C3 alkylene, C4 alkylene, and C5 alkylene.

[0093] The alkyl (or alkylene) may be unsubstituted, or the alkyl (or alkylene) may be substituted, where at least one hydrogen is replaced by another chemical group. Available substituting groups may include halogen, ester group, cyano group, sulfonyl group, etc.

[0094] "Therapeutically effective amount" refers to the amount of a therapeutic agent that, when administered to a patient, can improve a disease or symptom. "Prophylactically effective amount" refers to the amount of a prophylactic agent that, when administered to a subject, can prevent a disease or symptom. The amount of the therapeutic agent constituting the "therapeutically effective amount" or the amount of the prophylactic agent constituting the "prophylactically effective amount" varies with the therapeutic agent / prophylactic agent, the disease state and its severity, the age, weight, etc. of the patient / subject to be treated / prevented. Those of ordinary skill in the art can routinely determine the therapeutically effective amount and the prophylactically effective amount based on their knowledge and the present disclosure.

[0095] In the present disclosure, when the name of a compound is inconsistent with the structural formula, the structural formula shall prevail.

[0096] It should be understood that the term "compounds of the present disclosure" used herein may include, depending on the context: compounds of formula (I), their N-oxides, their solvates, their pharmaceutically acceptable salts, their stereoisomers, and mixtures thereof.

[0097] The term "cationic lipid" used in the present disclosure refers to a lipid that is positively charged at a selected pH value or range.

[0098] Cationic lipids are prone to bind to negatively charged nucleic acids, i.e., interact with the negatively charged phosphate groups in nucleic acids through electrostatic forces to form lipid nanoparticles (LNPs).

[0099] When the inventors studied and screened a large number of lipid compounds, they found that it is very difficult to obtain cationic lipid compounds suitable for use as nucleic acid drug carriers that simultaneously meet the following conditions: (1) having a structural difference from the cationic lipids currently commonly used in the prior art for loading nucleic acid drugs; (2) having high transfection efficiency and low cytotoxicity; and (3) having high expression and sustained expression in vivo.

[0100] In long-term studies, the inventors unexpectedly found some compounds, such as YK-1503, YK-1504, YK-1505, YK-1507, YK-1510, YK-1511, YK-1513, YK-1515, and YK-1516 in the present disclosure, etc., which can deliver nucleic acids with significantly improved intracellular transfection efficiency, significantly reduced cytotoxicity, significantly increased expression levels in animals, and spleen targeting compared with the cationic lipids in the prior art.

[0101] The present disclosure is at least based on the following findings: The cationic lipid compounds of the present disclosure can be used to deliver nucleic acids, small molecule compounds, polypeptides, or proteins. Compared with the known cationic lipid compounds, the cationic lipid compounds of the present disclosure exhibit higher transfection efficiency and smaller cytotoxicity, with a significantly increased expression level in the animal spleen, thus improving the delivery efficiency.

[0102] The second aspect of the present disclosure provides a composition comprising a carrier, the carrier comprising a cationic lipid, the cationic lipid comprising the above-mentioned compound of formula (I) or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer.

[0103] In one embodiment, the composition is a nanoparticle formulation, and the average size of the nanoparticle formulation is 10 nm - 300 nm, preferably 40 nm - 240 m; the polydispersity index of the nanoparticle formulation ≤ 0.5, preferably ≤ 0.4.

[0104] Cationic lipid In one embodiment of the composition / carrier of the present disclosure, the cationic lipid is one or more selected from the above-mentioned compound of formula (I) or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer. In one embodiment, the cationic lipid is the above-mentioned compound of formula (I). For example, the cationic lipid is the compound. In one preferred embodiment, the cationic lipid is compounds YK-1501 to YK-1518, and in one preferred embodiment, the cationic lipid is compounds YK-1503, YK-1504, YK-1505, YK-1507, YK-1510, YK-1511, YK-1513, YK-1515 or YK-1516.

[0105] In another embodiment of the composition / carrier of the present disclosure, the cationic lipid comprises: (a) one or more selected from the above-mentioned compound of formula (I) or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer; (b) one or more other ionizable lipid compounds different from (a). Among them, the (b) cationic lipid compound can be a commercially available cationic lipid or a cationic lipid compound reported in the literature. For example, the (b) cationic lipid compound can be SM-102 in CN201080026228.8, can also be MC3 in CN201080026228.8, and can also be C16 in CN202380010167.3.

[0106] In one embodiment, the molar ratio of the cationic lipid in the carrier is 25% - 75%, such as 30%, 40%, 50%, 55%, 60%, 65%, 70%.

[0107] The carrier can be used for the delivery of (drug) active ingredients such as therapeutic agents and / or prophylactic agents. The active ingredient can be encapsulated in the carrier or combined with the carrier in any form.

[0108] For example, examples of the therapeutic / preventive agent may be one or more of nucleic acid molecules, small molecule compounds, polypeptides, or proteins. The nucleic acids include, but are 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, etc.

[0109] Neutral lipid The carrier may comprise a neutral lipid. A neutral lipid in the present disclosure refers to a lipid that exists in an uncharged form or in a neutral ionic form at a selected pH value or range. This neutral lipid may regulate nanoparticle fluidity to form a lipid bilayer by promoting lipid phase transitions and improve efficiency, while also potentially affecting the specificity of the target organ.

[0110] In one embodiment, the molar ratio of the cationic lipid to the neutral lipid is about 1:1 - 15:1, such as 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. Again, for example, in a preferred embodiment, the molar ratio of the cationic lipid to the neutral lipid is about 4.9:1.

[0111] For example, the neutral lipid may include one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol, and its derivatives.

[0112] The carrier component of the composition containing the cationic lipid may include one or more neutral lipid-phospholipids, such as one or more (poly)unsaturated lipids. The phospholipids can assemble into one or more lipid bilayers. Generally, the phospholipids may include a phospholipid moiety and one or more fatty acid moieties.

[0113] The neutral lipids can be selected from the non-limiting group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lyso-phosphatidylcholine, and sphingomyelin. The fatty acids can be selected from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Also covered are non-natural species including natural species with modifications and substitutions, said modifications and substitutions including branching, oxidation, cyclization, and alkynes. For example, phospholipids can be functionalized with one or more alkynes (e.g., alkenyls in which one or more double bonds are replaced by triple bonds) or crosslinked with the one or more alkynes. Under appropriate reaction conditions, the alkynyl groups may undergo copper-catalyzed cycloaddition reactions upon exposure to azides. These reactions can be used to functionalize the lipid bilayer of the composition to facilitate membrane penetration or cell recognition, or to couple the composition with useful components such as targeting or imaging moieties (e.g., dyes).

[0114] Neutral lipids that can be used in these compositions can be selected from the non-limiting group consisting of: 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-doundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesteryl succinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl oleoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof.

[0115] In some embodiments, the neutral lipid comprises DSPC. In certain embodiments, the neutral lipid comprises DOPE. In some embodiments, the neutral lipid comprises both DSPC and DOPE.

[0116] Structural lipid The carrier of the composition containing cationic lipids may also include one or more structural lipids. Structural lipids in the present disclosure refer to lipids that enhance the stability of nanoparticles by filling the gaps between lipids.

[0117] In one embodiment, the molar ratio of the cationic lipid to the structural lipid is about 1:1 - 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, 2.0:1.

[0118] Structural lipids may be selected from, but not limited to, the group consisting of: cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, 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.

[0119] Polymer-conjugated lipids The carrier of the composition containing cationic lipids may also include one or more polymer-conjugated lipids. Polymer-conjugated lipids mainly refer to polyethylene glycol (PEG)-modified lipids. The hydrophilic PEG stabilizes the LNP, regulates the nanoparticle size by restricting lipid fusion, and increases the half-life of the nanoparticles by reducing non-specific interactions with macrophages.

[0120] In one embodiment, the polymer-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, PEG-modified dialkylglycerol. The molecular weight of the PEG-modified PEG is typically 350 - 5000 Da.

[0121] For example, the polymer-conjugated lipid is selected from one or more of the following: distearoyl phosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), dimyristoyl glycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000), and methoxy polyethylene glycol ditetradecylacetamide (ALC-0159).

[0122] In one embodiment of the composition / carrier of the present disclosure, the polymer-conjugated lipid is DMG-PEG2000.

[0123] In one embodiment of the composition / carrier of the present disclosure, the carrier comprises neutral lipids, structural lipids, and polymer-conjugated lipids, and 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), such as (30 - 49):(7.5 - 15):(35 - 55):(1 - 5), and more preferably (40 - 49):(8 - 12):(39 - 45):(1 - 3). The sum of the molar percentages of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is 100.

[0124] In one embodiment of the composition / carrier of the present disclosure, the carrier comprises neutral lipids, structural lipids, and polymer-conjugated lipids, and the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is 40:10:48.5:1.5 or 49:10:39.5:1.5.

[0125] Therapeutic agent and / or prophylactic agent The composition may comprise one or more therapeutic agents and / or prophylactic agents. In one embodiment, the mass ratio of the carrier to the therapeutic agent or prophylactic agent is 10:1 - 30:1, such as 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, 25:1.

[0126] In one embodiment, the mass ratio of the carrier to the therapeutic agent or prophylactic agent is 12.5:1 - 20:1, preferably 13 - 17:1, and more preferably 15:1.

[0127] The therapeutic agent or prophylactic agent includes, but is not limited to, one or more of nucleic acid molecules, small molecule compounds, polypeptides, or proteins.

[0128] For example, the therapeutic agent or prophylactic agent is a vaccine or a compound capable of eliciting an immune response.

[0129] The carrier of the present disclosure can deliver a therapeutic agent and / or a prophylactic agent to mammalian cells or organs. Accordingly, the present disclosure also provides methods for treating diseases or disorders in mammals in need thereof, which methods include administering to a mammal a composition comprising a therapeutic agent and / or a prophylactic agent and / or contacting mammalian cells with the composition. Correspondingly, the present disclosure provides the use of a compound of the present disclosure or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, or a composition of the present disclosure in the preparation of a medicament for treating diseases or disorders in a subject in need thereof.

[0130] The present disclosure also provides uses of the compounds of the present disclosure or their N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers, or the compositions of the present disclosure in the preparation of nucleic acid drugs, vaccines, chemical drugs, polypeptide drugs or protein drugs.

[0131] Therapeutic and / or prophylactic agents include bioactive substances and are alternatively referred to as "active agents". A therapeutic and / or prophylactic agent can be a substance that causes a desired change in a cell or organ after delivery to the cell or organ or in other body tissues or systems. Such agents can be used to treat one or more diseases, disorders, or conditions. In some embodiments, the therapeutic and / or prophylactic agent is a small molecule drug that can be used to treat a specific disease, disorder, or condition.Examples of drugs useful in the compositions include, but are not limited to, antineoplastics (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, and streptozotocin), antitumor agents (e.g., actinomycin D, vincristine, vinblastine, cytosine 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), β-adrenergic blockers (e.g., propranolol, timolol, and labetalol), antihypertensives (e.g., clonidine and hydralazine), antidepressants (e.g., imipramine, amitriptyline, and doxepin), antispasmodics (e.g., phenytoin), antihistamines (e.g., diphenhydramine, chlorpheniramine, and promethazine), antibiotics / antibacterials (e.g., gentamycin, ciprofloxacin, and cefoxitin), antifungals (e.g., miconazole, terconazole, econazole, isoconazole, butaconazole, clotrimazole, itraconazole, nystatin, naftifine, and amphotericin B), antiparasitics, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma agents, vitamins, sedatives, and imaging agents.

[0132] In some embodiments, the therapeutic and / or prophylactic agent is a cytotoxin, a radioactive ion, a chemotherapeutic agent, a vaccine, a compound that elicits an immune response, and / or another therapeutic and / or prophylactic agent. A cytotoxin or cytotoxic agent includes any reagent 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, dihydroxy anthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids such as maytansinol, rachelmycin (CC-1065), and analogs or homologs thereof. 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 capable of providing immunity against one or more conditions associated with infectious diseases such as influenza, measles, human papillomavirus (HPV), rabies, meningitis, whooping cough, tetanus, plague, hepatitis, and tuberculosis, and may include, for example, mRNA encoding a pathogenic antigen and / or its epitope; vaccines may also include compounds and formulations that direct an immune response against cancer cells and may include, for example, mRNA encoding a tumor cell-derived antigen, epitope, and / or neoepitope. Compounds that elicit an immune response may include vaccines, corticosteroids (e.g., dexamethasone), and other species. In some embodiments, a vaccine and / or compound that elicits an immune response is administered intramuscularly by a composition comprising a compound according to formula (I), (IA), (IB), (II), (IIa), (IIb), (IIc), (IId), (IIe), (IIf), (IIg), or (III) (e.g., compound 3, 18, 20, 25, 26, 29, 30, 60, 108-112, or 122).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., mechlorethamine, thiotepa, chlorambucil, CC-1065, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiammineplatinum(II) (DDP), cisplatin), anthracyclines (e.g., daunorubicin (formerly known as daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly known as actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotics (e.g., vincristine, vinblastine, taxol, and maytansinoids).

[0133] In other embodiments, the therapeutic and / or prophylactic agent is a protein. Therapeutic proteins that can be used in the nanoparticles of the present 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, interferons, heparin, hepatitis B surface antigen, typhoid vaccine, and cholera vaccine.

[0134] 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 in the form of an oligonucleotide chain or can be incorporated into an oligonucleotide chain. Exemplary polynucleotides used in accordance with the present disclosure include, but are not limited to, one or more of the following: deoxyribonucleic acid (DNA); ribonucleic acid (RNA), including messenger mRNA (mRNA), its hybrids; RNAi-inducing factors; RNAi factors; siRNA; shRNA; miRNA; antisense RNA; ribozymes; catalytic DNA; RNA that induces triple helix formation; aptamers, etc. In some embodiments, the therapeutic agent and / or prophylactic agent is RNA. The RNA that can be used in the compositions and methods described in the present disclosure can be selected from the group consisting of, but not limited to, shortmer, antagomir, antisense RNA, ribozyme, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), short hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), and mixtures thereof. In certain embodiments, the RNA is mRNA.

[0135] In certain embodiments, the therapeutic agent and / or prophylactic agent is mRNA. The mRNA can encode any polypeptide of interest, including any naturally occurring or non-naturally occurring or otherwise modified polypeptide. The polypeptide encoded by the mRNA can have any size and can have any secondary structure or activity. In some embodiments, the polypeptide encoded by the mRNA can have a therapeutic effect when expressed in a cell.

[0136] In other embodiments, the therapeutic agent and / or prophylactic agent is siRNA. The siRNA is capable of selectively reducing the expression of a gene of interest or downregulating the expression of the gene. For example, the siRNA can be selected such that after administering a composition comprising the siRNA to a subject in need, the gene associated with a particular disease, disorder, or condition is silenced. The siRNA can comprise a sequence complementary to the mRNA sequence encoding the gene or protein of interest. In some embodiments, the siRNA can be an immunomodulatory siRNA.

[0137] In certain embodiments, the therapeutic agent and / or prophylactic agent is sgRNA and / or cas9 mRNA. The 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.

[0138] In some embodiments, the therapeutic and / or prophylactic agent is an shRNA or its encoding vector or plasmid. The shRNA can be produced inside the target cell after delivery of the appropriate construct into the nucleus. The constructs and mechanisms associated with shRNA are well known in the relevant art.

[0139] disease or disorder The compositions / carriers of the present disclosure can deliver a therapeutic or prophylactic agent to a subject or patient. The therapeutic or prophylactic agent includes, but is not limited to, one or more of nucleic acid molecules, small molecule compounds, polypeptides, or proteins. Accordingly, the compositions of the present disclosure can be used to prepare nucleic acid drugs, gene vaccines, small molecule drugs, polypeptide, or protein drugs. Due to the wide variety of the above-mentioned therapeutic or prophylactic agents, the compositions of the present disclosure can be used to treat or prevent a variety of diseases or disorders.

[0140] In one embodiment, the disease or disorder is characterized by a malfunctioning or abnormal protein or polypeptide activity.

[0141] For example, the disease or disorder 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.

[0142] 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.

[0143] Other components The composition can include one or more components other than those described in the foregoing section. For example, the composition can include one or more hydrophobic small molecules, such as vitamins (e.g., vitamin A or vitamin E) or sterols.

[0144] The composition can also include one or more permeability enhancing molecules, carbohydrates, polymers, surface modifying agents, or other components. The permeability enhancing molecule can be, for example, the molecule described in U.S. Patent Application Publication No. 2005 / 0222064. The carbohydrates can include simple sugars (e.g., glucose) and polysaccharides (e.g., glycogen and its derivatives and analogs).

[0145] Surface modifiers can include, but are not limited to, anionic proteins (such as bovine serum albumin), surfactants (such as cationic surfactants, such as dimethyldioctadecylammonium bromide), sugars or sugar derivatives (such as cyclodextrin), nucleic acids, polymers (such as heparin, polyethylene glycol, and poloxamer), mucolytics (such as acetylcysteine, artemisia, bromelain, papain, clerodendrum, bromhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin β4, dornase alfa, neltenexine, and erdosteine), and DNAases (such as rhDNAase). The surface modifier can be disposed within and / or on the surface of the nanoparticles of the composition (such as by coating, adsorption, covalent linkage, or other methods).

[0146] The composition can also include one or more functionalized lipids. For example, the lipid can be functionalized with an alkynyl group that may undergo a cycloaddition reaction when exposed to an azide under appropriate reaction conditions. Specifically, the lipid bilayer can be functionalized in this manner with one or more groups that can effectively promote membrane penetration, cell recognition, or imaging. The surface of the composition can also be conjugated with one or more useful antibodies. Functional groups and conjugates useful for targeted cell delivery, imaging, and membrane penetration are well known in the art.

[0147] In addition to these components, the composition may include any substances that can be used in pharmaceutical compositions. For example, the composition may include one or more pharmaceutically acceptable excipients or auxiliary components, such as but not limited to one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, granulation aids, disintegrants, fillers, glidants, liquid vehicles, binders, surfactants, isotonic agents, thickeners or emulsifiers, buffers, lubricants, oils, preservatives, flavoring agents, coloring agents, etc. Excipients such as 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, A.R. Gennaro; Lippincott, Williams&Wilkins, Baltimore, MD, 2006).

[0148] Examples of diluents may include but not limited to calcium carbonate, sodium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dried starch, corn starch, powdered sugar, and / or combinations thereof.

[0149] In some embodiments, the composition comprising one or more lipids described in the present disclosure may further include one or more adjuvants, such as glucopyranosyl lipid adjuvant (GLA), CpG oligodeoxynucleotides (e.g., class A or B), poly(I:C), aluminum hydroxide, and Pam3CSK4.

[0150] The compositions of the present disclosure can be formulated into solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, ointments, elixirs, syrups, solutions, emulsions, suspensions, injections, aerosols. The compositions of the present disclosure can be prepared by methods well known in the pharmaceutical art. For example, a sterile injectable solution can be prepared by incorporating the required amount of the therapeutic or prophylactic agent with the various other required components described above into a suitable solvent such as sterile distilled water, and then filter sterilizing. Surfactants can also be added to facilitate the formation of a uniform solution or suspension.

[0151] For example, the compositions of the present disclosure can be administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally or by inhalation. In some embodiments, the composition is administered subcutaneously.

[0152] The compositions of the present disclosure are administered in a therapeutically effective amount, which can vary not only with the particular reagent 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, a dose of about 0.001 mg / kg to about 10 mg / kg of the therapeutic or prophylactic agent can be administered to a subject (preferably a mammal, such as a human).

[0153] Examples The present disclosure will be further described below in conjunction with examples, but the present disclosure is not limited to the following examples. The implementation conditions used in the examples can be further adjusted according to different requirements of specific use, and the implementation conditions not specified are conventional conditions in this industry. In the specific examples of the present disclosure, the raw materials used can all be obtained commercially. Unless otherwise specified, all temperatures are given in degrees Celsius. The technical features involved in each implementation manner of the present disclosure can be combined with each other as long as they do not conflict with each other.

[0154] In the following examples, the meanings represented by the abbreviated letters are as follows: NaBH4: Sodium borohydride; Boc2O: Di-tert-butyl dicarbonate; DMAP: 4-Dimethylaminopyridine; EDCI: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; HATU: 2-(7-Azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate; DIEA: N,N-Diisopropylethylamine; DCM: Dichloromethane; DMF: N,N-Dimethylformamide; THF: Tetrahydrofuran; MeOH: Methanol In the following examples, unless otherwise specified, the operations are all at room temperature (25±5°C).

[0155] Example 1: Synthesis of a cationic lipid compound 1.1 Synthesis of intermediate INT-1

[0156]

[0157] Step 1: Synthesis of INT-1-PM1 (S)-1-Amino-3-chloro-2-propanol hydrochloride (10.0 g, 68.48 mmol) was dissolved in dichloromethane (150 mL), triethylamine (27.72 g, 273.95 mmol) was added, and then di-tert-butyl dicarbonate (55.30 g, 253.40 mmol) was slowly added dropwise. The temperature was raised to 40 °C and the reaction was carried out for 24 h. The reaction was monitored by TLC until the raw materials were completely reacted. Heating was stopped, and the reaction was quenched by adding saturated aqueous sodium bicarbonate solution. The layers were separated, the aqueous phase was extracted with dichloromethane twice, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography, and the product was collected and concentrated to obtain INT-I-PM1 (9.10 g, 43.40 mmol, 63.3%). C8H 16 ClNO3, MS(ES):m / z(M+H + )210.1。

[0158] Step 2: Synthesis of INT-1-PM2 INT-I-PM1 (2.81 g, 13.40 mmol) was dissolved in acetonitrile (40 mL), and then pyrrolidine (1.14 g, 16.08 mmol), potassium carbonate (5.55 g, 40.20 mmol) and potassium iodide (0.44 g, 2.68 mmol) were added in sequence. The temperature was raised to 70 °C and the reaction was carried out for 8 h. The reaction was monitored by TLC until the raw materials were completely reacted. Heating was stopped, filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography, and the product was collected and concentrated to obtain INT-I-PM2 (2.43 g, 9.94 mmol, 74.2%). C 12 H 24 N2O3, MS(ES):m / z(M+H + )245.2。

[0159] Step 3: Synthesis of INT-1 INT-I-PM2 (2.43 g, 9.94 mmol) and a 1,4-dioxane solution of hydrochloric acid (24 mL) were added, and the reaction was carried out at room temperature for 2 h. The reaction was monitored by LCMS until the raw materials were completely reacted. The reaction solution was concentrated under reduced pressure, and the obtained residue was dissolved in methanol and then concentrated under reduced pressure. This was repeated 3 times to obtain INT-1 (2.05 g, crude product). C7H 16 N2O, MS(ES):m / z(M+H + )145.2。

[0160] 1.2 Synthesis of intermediate INT-2

[0161] Step 1: Synthesis of INT-2 Dissolve 2 - hexyldecanol (24.24 g, 99.97 mmol) in cyclohexane (250 mL), then add 6 - bromohexanoic acid (23.40 g, 119.97 mmol) and p - toluenesulfonic acid hydrate (0.28 g, 1.49 mmol). Install a water separator and react at 110 °C for 7 h. Monitor the reaction by TLC until the raw materials are completely reacted. Stop heating, add saturated aqueous sodium bicarbonate solution to the reaction system to quench the reaction, separate the layers, extract the aqueous phase with n - hexane once, combine the organic phases, dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain INT - 2 (41.42 g, 98.73 mmol, 98.7%). C 22 H 43 BrO2, MS(ES): m / z(M + H + ) 419.2.

[0162] 1.3 Synthesis of Intermediate INT - 3

[0163] Step 1: Synthesis of INT - 3 Dissolve n - decanol (25.00 g, 157.94 mmol) in cyclohexane (250 mL), then add 4 - bromobutyric acid (31.65 g, 189.53 mmol) and p - toluenesulfonic acid hydrate (0.30 g, 1.57 mmol). Install a water separator and react at 110 °C for 8 h. Monitor the reaction by TLC until the raw materials are completely reacted. Stop heating, add saturated aqueous sodium bicarbonate solution to the reaction system to quench the reaction, separate the layers, extract the aqueous phase with n - hexane once, combine the organic phases, dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain INT - 3 (47.31 g, 153.96 mmol, 97.4%). C 14 H 27 BrO2, MS(ES): m / z(M + H + ) 307.1.

[0164] 1.4 Synthesis of Intermediate INT - 4

[0165] Step 1: Synthesis of INT - 4 Dissolve heptadecane-9-ol (5.00 g, 19.49 mmol) in dichloromethane (50 mL), then add 6-bromohexanoic acid (3.80 g, 19.49 mmol), EDCI (5.60 g, 29.24 mmol) and DMAP (0.47 g, 3.889 mmol), react at room temperature for 16 h, monitor the reaction by TLC until the raw materials are completely reacted. Add saturated aqueous sodium bicarbonate solution to the reaction system to quench the reaction, separate the layers, extract the aqueous phase with dichloromethane once, combine the organic phases, dry over anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and purify the obtained residue by silica gel column chromatography. Collect the product and concentrate to obtain INT-4 (8.00 g, 18.45 mmol, 94.6%). C 23 H 45 BrO2, MS(ES): m / z(M+H + ) 433.2.

[0166] 1.5 Synthesis of Intermediate INT-5

[0167] Step 1: Synthesis of INT-5 Dissolve 2-octyldecanol (25.00 g, 92.42 mmol) in cyclohexane (250 mL), then add 6-bromohexanoic acid (21.63 g, 110.90 mmol) and p-toluenesulfonic acid hydrate (0.26 g, 1.38 mmol), install a water separator, react at 110 °C for 8 h, monitor the reaction by TLC until the raw materials are completely reacted. Stop heating, add saturated aqueous sodium bicarbonate solution to the reaction system to quench the reaction, separate the layers, extract the aqueous phase with n-hexane once, combine the organic phases, dry over anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and obtain INT-5 (40.19 g, 89.80 mmol, 97.1%). C 24 H 47 BrO2, MS(ES): m / z(M+H + ) 447.2.

[0168] 1.6 Synthesis of Intermediate INT-6

[0169] Step 1: Synthesis of INT-6 Under nitrogen protection, 1-hexadecylamine (10.0 g, 41.41 mmol) and TEA (12.57 g, 124.24 mmol) were successively dissolved in DCM (100 ml), and the temperature was lowered to 0 °C. A solution of acryloyl chloride (4.49 g, 49.69 mmol) in dichloromethane (40 ml) was added dropwise. After the addition, the reaction was carried out at room temperature. The reaction was monitored by TLC. After the reaction was completed, the system was cooled to 5 °C, and the reaction was quenched by adding an aqueous solution of NaHCO3. The aqueous phase was extracted with DCM twice, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography, and the product was collected and concentrated to obtain INT-6 (9.78 g, 33.09 mmol, 79.9%). C 19 H 37 NO, MS(ES):m / z(M+H + )296.3。

[0170] 1.7 Synthesis of Intermediate INT-7

[0171]

[0172] Step 1: Synthesis of INT-7-PM1 INT-7-SM (47.00 g, 0.43 mol) was dissolved in dichloromethane (350 mL). Triethylamine (97.10 g, 0.96 mol) and Boc anhydride (279.40 g, 1.28 mol) were added at room temperature, and the reaction was stirred overnight at room temperature. After the reaction was completed, a saturated aqueous solution of sodium bicarbonate was added, and the mixture was extracted with dichloromethane. After the organic phases were combined, they were washed with pure water, and then the organic phase was dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the product was purified by silica gel chromatography (0% - 50% ethyl acetate / hexane) to obtain INT-7-PM1 (44.00 g, 0.21 mol, 48.8%). C8H 16 ClNO3, MS (ES): m / z (M + H + ) = 210.70。

[0173] Step 2: Synthesis of INT-7-PM2 2-Methyl-3 H-Imidazole (5.00 g, 60.90 mmol) and INT-7-PM1 (15.32 g, 73.08 mmol) were dissolved in DMF (50 mL). Potassium carbonate (25.25 g, 182.70 mmol) and potassium iodide (2.02 g, 12.18 mmol) were added at room temperature, and the mixture was stirred at 75 ºC. The reaction was monitored by TLC. After completion of the reaction, the mixture was diluted with pure water and extracted with ethyl acetate. The combined organic phases were washed with saturated aqueous sodium chloride solution and then dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the product INT-7-PM2 (5.05 g, 19.78 mmol, 32.5%) was purified by silica gel chromatography (0% - 20% methanol / dichloromethane). C 12 H 21 N3O3, MS (ES): m / z (M + H + ) = 256.05.

[0174] Step 3: Synthesis of INT-7 INT-7-PM2 (5.00 g, 19.58 mmol) was dissolved in a 1,4-dioxane solution of hydrochloric acid (100 mL), and the mixture was stirred overnight at room temperature. After completion of the reaction, the solution was concentrated under reduced pressure to obtain the crude product INT-7 (5.00 g) with hydrochloride salt, which was directly used in the next step.

[0175] 1.8 Synthesis of YK-1501

[0176]

[0177]

[0178] Step 1: Synthesis of YK-1501-PM1 Didecylamine (20.00 g, 67.21 mmol) was dissolved in DMF (100 mL). Boc-β-alanine (12.71 g, 67.21 mmol), HATU (38.33 g, 100.81 mmol) and DIEA (26.05 g, 201.63 mmol) were added, and the mixture was reacted at room temperature for 3 h. The reaction was monitored by LCMS until the raw materials were completely reacted. The reaction was quenched by adding water, and the mixture was extracted twice with ethyl acetate. The combined organic phases were washed three times with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography, and the product was collected and concentrated to obtain YK-1501-PM1 (23.40 g, 49.91 mmol, 74.2%). C 28 H 56 N2O3, MS(ES):m / z(M+H+ ) 469.4。

[0179] Step 2: Synthesis of YK-1501-PM2 Add YK-1501-PM1 (23.40 g, 49.91 mol) and a 1,4-dioxane solution of hydrochloric acid (120 mL), react at room temperature for 2 h, monitor the reaction by LCMS until the raw materials are completely reacted. Concentrate the reaction solution under reduced pressure, dissolve the obtained residue in methanol and then concentrate under reduced pressure for 3 times to obtain YK-1501-PM2 (18.40 g, crude product). C 23 H 48 N2O, MS(ES): m / z(M+H + ) 369.4。

[0180] Step 3: Synthesis of YK-1501-PM3 Under nitrogen protection, successively dissolve YK-1501-PM2 (7.70 g, 20.89 mmol) and TEA (6.34 g, 62.66 mmol) in DCM (80 ml), cool down to 0 °C and dropwise add a dichloromethane (20 ml) solution of acryloyl chloride (2.27 g, 25.06 mmol). After the addition, raise the temperature to room temperature and react. Monitor the reaction by TLC. After the reaction is completed, cool the system to 5 °C, add an aqueous solution of NaHCO3 to quench the reaction, extract the aqueous phase with DCM twice, combine the organic phases, dry over anhydrous sodium sulfate, filter, rotary evaporate the filtrate under reduced pressure, purify the obtained residue by silica gel column chromatography, collect the product and concentrate to obtain YK-1501-PM3 (5.88 g, 13.91 mmol, 66.6%). C 26 H 50 N2O2, MS(ES): m / z(M+H + ) 423.4。

[0181] Step 4: Synthesis of YK-1501 Dissolve INT-1 (90 mg, 0.62 mmol) in xylene (3 mL), then successively add YK-1501-PM3 (659 mg, 1.56 mmol) and potassium carbonate (258 mg, 1.87 mmol), heat up to 90 °C and react for 3 days, monitor the reaction by LCMS until the raw materials are completely reacted. Stop heating, filter, concentrate the filtrate under reduced pressure, purify the obtained residue by silica gel column chromatography, collect the product and concentrate to obtain YK-1501 (35 mg, 0.03 mmol, 5.6%).

[0182] C 59 H 116 N6O5, MS(ES): m / z(M+H + ) 989.6。 11H NMR (400 MHz, CDCl3) δ 3.58 – 3.42(m, 5H), 3.34 – 3.21 (m, 5H), 3.21 – 3.14 (m, 4H), 3.01 – 2.68 (m, 5H), 2.57– 2.47 (m, 5H), 2.33 (s, 4H), 2.13 (s, 4H), 1.50 (d, J = 7.1 Hz, 9H), 1.39 –1.23 (m, 58H), 0.90 – 0.86 (t, 12H). 1.9 Synthesis of YK-1502

[0183]

[0184] Step 1: Synthesis of YK-1502-PM1 Dissolve INT-1 (90 mg, 0.62 mmol) in xylene (3 mL), then successively add YK-1501-PM3 (237 mg, 0.56 mmol) and potassium carbonate (258 mg, 1.87 mmol). Heat the mixture to 90 °C and react for 3 h. Monitor the reaction by LCMS until the raw materials are completely reacted. Stop heating, filter, and concentrate the filtrate under reduced pressure. The obtained residue is purified by silica gel column chromatography, and the product is collected and concentrated to obtain YK-1502-PM1 (150 mg, 0.26 mmol, 42.3%). C 33 H 66 N4O3, MS(ES):m / z(M+H + )567.5。

[0185] Step 2: Synthesis of YK-1502 Dissolve YK-1502-PM1 (150 mg, 0.26 mmol) in xylene (3 mL), then successively add INT-6 (93 mg, 0.31 mmol) and potassium carbonate (109 mg, 0.79 mmol). Heat the mixture to 90 °C and react for 3 days. Monitor the reaction by LCMS until the raw materials are completely reacted. Stop heating, filter, and concentrate the filtrate under reduced pressure. The obtained residue is purified by silica gel column chromatography, and the product is collected and concentrated to obtain YK-1502 (30 mg, 0.03 mmol, 13.1%).

[0186] C 52 H 103 N5O4, MS(ES):m / z(M+H + )862.8。 11H NMR (400 MHz, CDCl3) δ 3.49 (d, J = 47.3 Hz, 2H), 3.18 (dd, J = 9.5, 4.1 Hz, 6H), 2.87 – 2.63 (m, 6H), 2.56 – 2.40 (m, 4H), 2.33 (dt, J = 9.8, 5.4 Hz, 3H), 1.51 (s, 6H), 1.31 – 1.24 (m, 55H), 0.90 – 0.86 (t, 9H). 1.10 Synthesis of YK-1503

[0187]

[0188] Step 1: Synthesis of YK-1503-PM1 Dissolve INT-1 (0.71 g, 4.92 mmol) in acetonitrile (25 mL), then successively add INT-2 (1.65 g, 3.93 mmol), potassium carbonate (2.04 g, 14.76 mmol) and potassium iodide (0.16 g, 0.98 mmol). Heat the mixture to 70 °C and react for 7 h. Monitor the reaction by LCMS until the raw materials are completely reacted. Stop heating, filter, concentrate the filtrate under reduced pressure. The obtained residue is purified by silica gel column chromatography, and the product is collected and concentrated to obtain YK-1503-PM1 (0.70 g, 1.44 mmol, 29.4%). C 29 H 58 N2O3, MS(ES): m / z(M+H + ) 483.4.

[0189] Step 2: Synthesis of YK-1503 Dissolve YK-1503-PM1 (650 mg, 1.34 mmol) in acetonitrile (10 mL), then successively add INT-3 (413 mg, 1.34 mmol), potassium carbonate (558 mg, 4.03 mmol) and potassium iodide (44 mg, 0.26 mmol). Heat the mixture to 70 °C and react for 8 h. Monitor the reaction by LCMS until the raw materials are completely reacted. Stop heating, filter, concentrate the filtrate under reduced pressure. The obtained residue is purified by silica gel column chromatography, and the product is collected and concentrated to obtain YK-1503 (390 mg, 0.54 mmol, 40.8%).

[0190] C 43 H 84 N2O3, MS(ES): m / z(M+H + ) 709.6. 11H NMR (400 MHz, CDCl3) δ 4.08 – 4.02(m, 2H), 3.96 (d, J = 5.8 Hz, 2H), 3.22 – 2.96 (m, 5H), 2.60 – 2.34 (m, 6H),2.34 – 2.26 (t, 4H), 2.08 – 1.98 (m, 4H), 1.82 – 1.70 (m, 2H), 1.67 – 1.57(m, 5H), 1.49 – 1.39 (m, 2H), 1.38 – 1.18 (m, 41H), 0.90 – 0.86 (t, 9H). 1.11 Synthesis of YK-1504

[0191] Step 1: Synthesis of YK-1504 Dissolve INT-1 (0.71 g, 4.92 mmol) in acetonitrile (25 mL), then successively add INT-2 (1.65 g, 3.93 mmol), potassium carbonate (2.04 g, 14.76 mmol) and potassium iodide (0.16 g, 0.98 mmol). Heat the mixture to 70 °C and react for 7 h. Monitor the reaction by LCMS until the raw materials are completely reacted. Stop heating, filter, concentrate the filtrate under reduced pressure. The obtained residue is purified by silica gel column chromatography, and the product is collected and concentrated to obtain YK-1504 (0.26 g, 0.31 mmol, 6.4%).

[0192] C 51 H 100 N2O5, MS(ES):m / z(M+H + )821.7。 1 1H NMR (400 MHz, CDCl3) δ 4.43 – 4.38(m, 1H), 3.96 (d, J = 5.8 Hz, 4H), 3.43 (dd, J = 17.3, 8.0 Hz, 3H), 3.34 (t,J = 13.8 Hz, 2H), 2.90 (dd, J = 12.9, 8.6 Hz, 1H), 2.70 – 2.47 (m, 4H), 2.33– 2.29 (t, 4H), 2.19 – 2.11 (m, 4H), 1.70 – 1.47 (m, 10H), 1.37 – 1.26 (m,52H), 0.90 – 0.86 (t, 12H). 1.12 Synthesis of YK-1505

[0193] Step 1: Synthesis of YK-1505 Dissolve INT-1 (80 mg, 0.55 mmol) in acetonitrile (7 mL), and then successively add INT-4 (480 mg, 1.10 mmol), potassium carbonate (229 mg, 1.66 mmol) and potassium iodide (18 mg, 0.11 mmol). Heat the mixture to 70 °C and react for 8 h. Monitor the reaction by LCMS until the raw materials are completely reacted. Stop heating, filter, concentrate the filtrate under reduced pressure, and purify the obtained residue by silica gel column chromatography. Collect the product and concentrate to obtain YK-1505 (19 mg, 0.02 mmol, 4.0%).

[0194] C 53 H 104 N2O5, MS(ES):m / z(M+H + )849.7。 1 H NMR (400 MHz, CDCl3) δ 4.88 – 4.81(m, 2H), 3.45 – 3.43 (dd, J = 27.9, 16.8 Hz, 5H), 2.94 – 2.69 (m, 6H), 2.32 –2.27 (dd, J = 14.3, 6.9 Hz, 4H), 2.16 – 2.11 (m, 4H), 1.69 – 1.46 (m, 16H),1.40 – 1.25 (m, 52H), 0.89 – 0.86 (t, 12H). 1.13 Synthesis of YK-1506

[0195]

[0196] Step 1: Synthesis of YK-1506-PM1 Dissolve INT-1 (0.96 g, 6.65 mmol) in DMF (20 mL), and then successively add INT-5 (1.48 g, 3.32 mmol) and potassium carbonate (1.84 g, 13.31 mmol). Heat the mixture to 70 °C and react for 7 h. Monitor the reaction by LCMS until the raw materials are completely reacted. Stop heating, filter, concentrate the filtrate under reduced pressure, and purify the obtained residue by silica gel column chromatography. Collect the product and concentrate to obtain YK-1506-PM1 (0.24 g, 0.46 mmol, 7.0%). C 31 H 62 N2O3, MS(ES):m / z(M+H + )511.4。

[0197] Step 2: Synthesis of YK-1506 Dissolve YK-1506-PM1 (200 mg, 0.39 mmol) in DMF (5 mL), then successively add INT-3 (180 mg, 0.58 mmol), potassium carbonate (162 mg, 1.17 mmol) and potassium iodide (64 mg, 0.39 mmol). Heat the mixture to 70 °C and react for 8 h. Monitor the reaction by LCMS until the raw materials are completely reacted. Stop heating, filter, concentrate the filtrate under reduced pressure. The obtained residue is purified by silica gel column chromatography, and the product is collected and concentrated to obtain YK-1506 (40 mg, 0.05 mmol, 13.8%).

[0198] C 45 H 88 N2O5, MS(ES):m / z(M+H + )737.6。 1 H NMR (400 MHz, CDCl3) δ 4.14 – 4.01(m, 3H), 3.96 (d, J = 5.8 Hz, 2H), 3.52 – 3.23 (m, 2H), 3.17 (dd, J = 14.5,6.6 Hz, 1H), 2.97 – 2.52 (m, 4H), 2.55 – 2.35 (m, 3H), 2.34 – 2.26 (m, 2H),2.03 – 1.91 (m, 1H), 1.87 (dd, J = 22.5, 5.4 Hz, 3H), 1.61 (ddd, J = 18.0,13.4, 5.4 Hz, 6H), 1.47 – 1.37 (m, 2H), 1.32 – 1.25 (m, 39H), 0.89 – 0.86 (s,9H). 1.14 Synthesis of YK-1507

[0199] Step 1: Synthesis of YK-1507 Dissolve INT-I (0.96 g, 6.65 mmol) in DMF (20 mL), then successively add INT-5 (1.48 g, 3.32 mmol) and potassium carbonate (1.84 g, 13.31 mmol). Heat the mixture to 70 °C and react for 7 h. Monitor the reaction by LCMS until the raw materials are completely reacted. Stop heating, filter, concentrate the filtrate under reduced pressure. The obtained residue is purified by silica gel column chromatography, and the product is collected and concentrated to obtain YK-1507 (0.12 g, 0.13 mmol, 2.0%). C 55 H 108N2O5, MS(ES): m / z(M+H + ) 877.8 1 H NMR (400 MHz, CDCl3) δ 3.96 (d, J = 5.8 Hz, 4H), 2.61 – 2.41 (m, 4H), 2.34 – 2.26 (m, 4H), 2.06 – 1.99 (m, 3H), 1.70 – 1.56 (m, 7H), 1.51 – 1.40 (m, 4H), 1.34 – 1.26 (m, 62H), 0.89 – 0.86 (t, 12H). 1.15 Synthesis of YK-1508

[0200] Step 1: Synthesis of YK-1508 Dissolve INT-I (200 mg, 1.38 mmol) in DMF (20 mL), then successively add linoleyl bromide (456 mg, 1.38 mmol), potassium carbonate (383 mg, 2.77 mmol) and potassium iodide (46 mg, 0.27 mmol). Heat to 70 °C and react for 8 h. Monitor the reaction by LCMS until the raw materials are completely reacted. Stop heating, filter, concentrate the filtrate under reduced pressure. The obtained residue is purified by silica gel column chromatography, and the product is collected and concentrated to obtain YK-1508 (80 mg, 0.12 mmol, 8.9%). C 43 H 80 N2O, MS(ES): m / z(M+H + ) 641.6 1 H NMR (400 MHz, CDCl3) δ 5.42 – 5.29 (m, 8H), 3.30 (s, 2H), 3.00 (d, J = 8.3 Hz, 1H), 2.85 – 2.75 (m, 7H), 2.10 – 2.02 (m, 11H), 1.65 – 1.61 (m, 3H), 1.42 – 1.22 (m, 34H), 0.90 – 0.85 (s, 6H). 1.16 Synthesis of YK-1509

[0201] Step 1: Synthesis of YK-1509-PM1 Dissolve INT-7 (1.00 g, crude product) and INT-2 (1.60 g, 3.90 mmol) in acetonitrile (10 mL). Add potassium carbonate (1.60 g, 11.70 mmol) and potassium iodide (130 mg, 0.80 mmol) at room temperature. Stir the reaction at 75 ºC and monitor by LCMS. After completion of the reaction, filter, and wash the filter cake with a small amount of acetonitrile. After rotary evaporation of the filtrate, purify by silica gel chromatography (0% - 50% methanol / dichloromethane containing 10% ammonia water) to obtain the product YK-1509-PM1 (200 mg, 0.41 mmol, two-step yield 10.5%), C 29 H 55 N3O3, MS (ES): m / z (M + H + ) = 494.80. Step 2: Synthesis of YK-1509 Dissolve YK-1509-PM1 (100 mg, 0.20 mmol) and INT-3 (75 mg, 0.24 mmol) in acetonitrile (2 mL). Add potassium carbonate (84 mg, 0.60 mmol) and potassium iodide (7 mg, 0.040 mmol) at room temperature. Stir the reaction at 75 ºC and monitor by LCMS. After completion of the reaction, filter, and wash the filter cake with a small amount of acetonitrile. After rotary evaporation of the filtrate, purify by silica gel chromatography (0% - 50% methanol / dichloromethane containing 10% ammonia water) to obtain the product YK-1509 (35 mg, 0.049 mmol, 24.3%), C 43 H 81 N3O5, MS (ES): m / z (M + H + ) = 721.15.

[0202] 11H NMR (400 MHz, CDCl3) δ 7.86 (s, 1H), 7.41 (s, 1H), 4.27 - 4.18 (m, 2H), 4.11 - 4.04 (m, 2H), 3.99 - 3.86 (m, 3H), 3.62 - 3.51 (m, 2H), 3.49 - 3.37 (m, 2H), 3.18 - 3.02 (m, 5H), 2.90 - 2.82 (m, 3H), 2.51 - 2.43 (m, 2H), 2.37 - 2.27 (m, 2H), 2.22 - 2.12 (m, 1H), 2.01 - 1.90 (m, 1H), 1.71 - 1.56 (m, 6H), 1.34 - 1.22 (m, 38H), 0.92 - 0.84 (m, 9H). 1.17 Synthesis of YK-1510

[0203] Step 1: Synthesis of YK-1509-PM1 Dissolve INT-7 (1.28 g, crude product) and INT-2 (4.20 g, 10.02 mmol) in acetonitrile (10 mL), add potassium carbonate (3.60 g, 26.05 mmol) and potassium iodide (172 mg, 1.04 mmol) at room temperature, stir and react at 75 ºC, monitored by LCMS. After the reaction is completed, filter, and wash the filter cake with a small amount of acetonitrile. After the filtrate is evaporated to dryness, purify by silica gel chromatography (0% - 50% methanol / dichloromethane containing 10% ammonia water) to obtain the product YK-1510 (550 mg, 0.66 mmol, overall yield of two steps 13.2%), C 51 H 97 N3O5, MS (ES): m / z (M + H + ) = 833.35.

[0204] 1 1H NMR (400 MHz, CDCl3) δ 7.56 (d, J J = 17.6 Hz, 2H), 4.28 (t, J= 7.5 Hz, 4H), 4.01 - 3.90 (m, 5H), 2.82 (s, 3H), 2.38 - 2.26 (m, 5H), 1.94 - 1.83 (m, 4H), 1.73 - 1.55 (m, 7H), 1.50 - 1.38 (m, 5H), 1.35 - 1.17 (m, 49H), 0.94 - 0.83 (m, 12H). 1.18 Synthesis of YK-1511

[0205] Step 1: Synthesis of YK-1511 Dissolve INT-7 (150 mg, crude product) and INT-4 (512 mg, 1.18 mmol) in acetonitrile (2 mL). Add potassium carbonate (361 mg, 2.61 mmol) and potassium iodide (17.3 mg, 0.10 mmol) at room temperature, and stir the reaction at 75 ºC, monitored by LCMS. After the reaction is completed, filter, and wash the filter cake with a small amount of acetonitrile. After the filtrate is concentrated by rotary evaporation, purify it by silica gel chromatography (0% - 50% methanol / dichloromethane containing 10% ammonia water) to obtain the product YK-1511 (8 mg, 0.044 mmol, overall yield of two steps 7.5%), C 53 H 101 N3O5, MS (ES): m / z (M + H + ) = 861.40.

[0206] 1 H NMR (400 MHz, CDCl3) δ 7.54 (d, J = 17.6 Hz, 2H), 4.95 - 4.75 (m, 2H), 4.19 - 4.00 (m, 3H), 3.56 - 2.93 (m, 3H), 2.86 - 2.76 (m, 3H), 2.38 - 2.20 (m, 4H), 2.07 - 1.82 (m, 3H), 1.73 - 1.58 (m, 6H), 1.56 - 1.38 (m, 12H), 1.34 - 1.17 (m, 50H), 0.95 - 0.76 (m, 12H). 1.19 Synthesis of YK-1512

[0207] Step 1: Synthesis of YK-1512-PM1 Dissolve INT-7 (1.00 g, crude product) and INT-5 (1.75 g, 3.92 mmol) in DMF (10 mL). Add potassium carbonate (1.60 g, 11.70 mmol) and potassium iodide (130 mg, 0.80 mmol) at room temperature. Stir the reaction at 75 ºC and monitor by LCMS. After the reaction is completed, dilute with pure water, extract with ethyl acetate. Combine the organic phases, wash with saturated sodium chloride aqueous solution, and then dry the organic phase with anhydrous sodium sulfate. Rotate and evaporate the solvent under reduced pressure, and purify by silica gel chromatography (0% - 50% methanol / dichloromethane containing 10% ammonia water) to obtain the product YK-1512-PM1 (300 mg, 0.57 mmol, two-step yield 14.5%), C 31 H 59 N3O3, MS (ES): m / z (M + H + ) = 523.03. Step 2: Synthesis of YK-1512 Dissolve YK-1512-PM1 (150 mg, 0.29 mmol) and INT-3 (89 mg, 0.29 mmol) in DMF (2 mL). Add potassium carbonate (120 mg, 0.87 mmol) and potassium iodide (7 mg, 0.040 mmol) at room temperature. Stir the reaction at 75 ºC and monitor by LCMS. After the reaction is completed, dilute with pure water, extract with ethyl acetate. Combine the organic phases, wash with saturated sodium chloride aqueous solution, and then dry the organic phase with anhydrous sodium sulfate. Rotate and evaporate the solvent under reduced pressure, and purify by silica gel chromatography (0% - 50% methanol / dichloromethane containing 10% ammonia water) to obtain the product YK-1512 (60 mg, 0.080 mmol, 27.6%), C 45 H 85 N3O5, MS(ES): m / z (M + H + ) = 749.46.

[0208] 1 H NMR (400 MHz, CDCl3) δ 6.99 - 6.87 (m, 2H), 4.10 - 4.03 (m, 4H), 3.99 - 3.91 (m, 4H), 2.45 (s, 3H), 2.35 - 2.26 (m, 4H), 1.67 - 1.57 (m, 7H), 1.48 - 1.38 (m, 2H), 1.37 - 1.21 (m, 49H), 0.93 - 0.84 (m, 9H). 1.20 Synthesis of YK-1513

[0209] Step 1: Synthesis of YK-1513 Dissolve INT-7 (500 mg, crude product) and INT-5 (1.75 g, 3.92 mmol) in DMF (10 mL). Add potassium carbonate (1.60 g, 11.70 mmol) and potassium iodide (130 mg, 0.80 mmol) at room temperature, and stir the reaction at 75 ºC while monitoring by LCMS. After the reaction is completed, dilute with pure water, extract with ethyl acetate. Combine the organic phases, wash with saturated sodium chloride aqueous solution, and then dry the organic phase with anhydrous sodium sulfate. Rotate the solvent off under reduced pressure, and purify by silica gel chromatography (0% - 50% methanol / dichloromethane containing 10% ammonia water) to obtain the product YK-1513 (100 mg, 0.11 mmol, two-step yield 5.6%), C 55 H 105 N3O5, MS(ES): m / z (M + H + ) = 889.41.

[0210] 1 H NMR (400 MHz, CDCl3) δ 6.96 (d, J = 15.0 Hz, 2H), 3.99 (d, J = 5.8 Hz,4H), 3.95 - 3.82 (m, 2H), 2.61 - 2.49 (m, 2H), 2.49 - 2.43 (m, 4H), 2.38 -2.27 (m, 4H), 1.70 - 1.60 (m, 6H), 1.55 - 1.39 (m, 5H), 1.37 - 1.21 (m, 63H),0.96 - 0.84 (m, 12H). 1.21 Synthesis of YK-1514

[0211] Step 1: Synthesis of YK-1514 INT-7 (200 mg, crude) and compound A (516 mg, 1.57 mmol) were dissolved in DMF (2 mL). Potassium carbonate (434 mg, 3.14 mmol) and potassium iodide (33 mg, 0.20 mmol) were added at room temperature, and the mixture was stirred at 75 ºC. The reaction was monitored by LCMS. After completion of the reaction, the mixture was diluted with pure water and extracted with ethyl acetate. The combined organic phases were washed with saturated sodium chloride aqueous solution and then dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the product YK-1514 (20 mg, 0.14 mmol, two-step yield 3.1%) was purified by silica gel chromatography (0% - 50% methanol / dichloromethane containing 10% ammonia water), C 43 H 77 N3O, MS (ES): m / z(M + H + ) = 653.03.

[0212] 1 H NMR (400 MHz, CDCl3) δ 6.94 (d, J = 15.0 Hz, 2H),5.48 - 5.32 (m, 8H),3.34 (s, 4H), 3.03 (d, J = 6.4 Hz, 2H), 2.86 (d, J = 6.8 Hz, 3H), 2.42 (s, 3H),2.17 – 2.03 (m, 11H), 1.67 (s, 3H), 1.44 - 1.24 (m, 34H), 0.92 (t, J = 6.8 Hz,6H). 1.22 Synthesis of YK-1515

[0213]

[0214]

[0215] Step 1: Synthesis of YK-1515-PM1 Using (S)-1-amino-3-chloro-2-propanol hydrochloride (10.00 g, 68.49 mmol), (Boc)2O (55.30 g, 253.40 mmol), and TEA (27.72 g, 273.90 mmol) as starting materials, dissolve them in dichloromethane (50.0 mL), react at 40 °C for 24 hours, and monitor the reaction completion by TLC. Slowly add the reaction solution to saturated aqueous sodium bicarbonate, stir for 10 minutes, separate the layers, extract twice with dichloromethane, combine the organic phases, dry over anhydrous sodium sulfate, filter, and concentrate by rotary evaporation. Load the sample by wet method, elute with hexane / ethyl acetate = 0 - 18%, and concentrate by rotary evaporation to obtain YK-1515-PM1 (9.1 g, 43.40 mmol, 63.3%).

[0216] Step 2: Synthesis of YK-1515-PM2 Using YK-1515-PM1 (3.10 g, 14.78 mmol), N-methylpiperazine (1.77 g, 17.74 mmol), K2CO3 (6.13 g, 44.35 mmol), and KI (0.49 g, 2.95 mmol) as starting materials, dissolve them in acetonitrile (40.0 mL), heat to 70 °C, react for 12 hours, and monitor the reaction completion by TLC. Stop the reaction, filter, wash with dichloromethane, and concentrate by rotary evaporation. Dissolve in a small amount of dichloromethane, load the sample by wet method, elute with dichloromethane / methanol = 0 - 15%, and concentrate by rotary evaporation to obtain YK-1515-PM2 (3.28 g, 12.00 mmol, 81.1%). C 13 H 27 N3O3, MS(ES): m / z(M+H + ) 274.2.

[0217] Step 3: Synthesis of YK-1515-PM3 Weigh YK-1515-PM2 (3.28 g, 12.00 mmol) and slowly add it to a 1,4-dioxane solution of hydrochloric acid (30 ml) at room temperature. React at room temperature for 12 hours and monitor the reaction completion by TLC. Concentrate by rotary evaporation to obtain the crude product YK-1515-PM3 (3.05 g, crude product). C8H 19 N3O, MS(ES): m / z(M+H + ) 174.2.

[0218] Step 4: Synthesis of YK-1515-PM4 and YK-1515 Using YK-1515-PM3 (0.83 g, 3.95 mmol), INT-2 (1.32 g, 3.16 mmol), K2CO3 (1.64 g, 11.87 mmol), and KI (131 mg, 0.79 mmol) as raw materials, dissolve them in acetonitrile (20.0 mL), heat to 70 °C, react for 12 hours, and check by TLC that the reaction is complete. Stop the reaction, filter, wash with dichloromethane, and evaporate to dryness. Dissolve with a small amount of dichloromethane, load the sample by wet method, with dichloromethane / methanol = 0 - 16%, and evaporate to dryness to obtain YK-1515-PM4 (582 mg, 1.14 mmol, 28.7%). C 30 H 61 N3O3, MS(ES): m / z(M+H + )512.2. With dichloromethane / methanol = 0 - 8%, evaporate to dryness to obtain YK-1515 (370 mg, 0.44 mmol, 10.9%). C 52 H 103 N3O5, MS(ES): m / z(M+H + )850.4.

[0219] 1 H NMR (400 MHz, CDCl3) δ 4.00 (d, J = 5.8 Hz, 4H), 3.13 (s, 11H), 2.74(s, 3H), 2.62 (d, J = 7.6 Hz, 2H), 2.37 (t, J = 7.3 Hz, 4H), 1.83 (s, 5H), 1.70(m, J = 24.2, 8.2 Hz, 7H), 1.44 (m, J = 14.6, 7.1 Hz, 5H), 1.30 (s, 49H), 0.92(t, J = 6.7 Hz, 12H). 1.23 Synthesis of YK-1516

[0220] Step 1: Synthesis of YK-1516 Using YK-1515-PM3 (100 mg, 0.47 mmol), INT-2 (413 mg, 0.95 mmol), K2CO3 (197 mg, 1.43 mmol), and KI (15.8 mg, 0.09 mmol) as starting materials, they were dissolved in acetonitrile (5.0 mL). The temperature was raised to 70 °C and the reaction was carried out for 12 hours. The reaction was complete as monitored by TLC. The reaction was stopped, filtered, washed with dichloromethane, and concentrated by rotary evaporation. The residue was dissolved in a small amount of dichloromethane and loaded onto a column (eluent: dichloromethane / methanol = 0 - 39%). After concentration by rotary evaporation, YK-1516 (55 mg, 0.06 mmol, 13.1%) was obtained. C 54 H 107 N3O5, MS(ES): m / z(M+H + )879.2。

[0221] 1 H NMR (400 MHz, CDCl3) δ 4.93 - 4.81 (m, 2H), 2.71 (d, J = 73.1 Hz, 11H), 2.57 - 2.43 (m, 5H), 2.33 (t, J = 7.4 Hz, 4H), 1.82 - 1.58 (m, 9H), 1.54 (d, J = 5.2 Hz, 9H), 1.47 - 1.19 (m, 54H), 0.91 (t, J = 6.6 Hz, 12H). 1.24 Synthesis of YK-1517

[0222] Step 1: Synthesis of YK-1517 Using YK-1515-PM3 (512 mg, 1.00 mmol), INT-3 (307 mg, 1.00 mmol), K2CO3 (414 mg, 3.00 mmol), and KI (33 mg, 0.20 mmol) as starting materials, they were dissolved in acetonitrile (10.0 mL). The temperature was raised to 70 °C and the reaction was carried out for 8 hours. The reaction was complete as monitored by TLC. The reaction was stopped, filtered, washed with dichloromethane, and concentrated by rotary evaporation. The residue was dissolved in a small amount of dichloromethane and loaded onto a column (eluent: dichloromethane / methanol = 0 - 16%). After concentration by rotary evaporation, YK-1517 (133 mg, 0.18 mmol, 18.0%) was obtained. C 44 H 87 N3O5, MS(ES): m / z(M+H + )738.2。

[0223] 1 1H NMR (400 MHz, CDCl3) δ 4.08 (t, J J = 6.8 Hz, 2H), 3.99 (d, J J = 5.8 Hz, 2H), 2.81 - 2.38 (m, 18H), 2.38 - 2.28 (m, 4H), 1.80 (m, J J = 12.5, 5.4 Hz, 2H), 1.72 - 1.58 (m, 5H), 1.51 (m, J J = 13.4, 6.5 Hz, 3H), 1.31 (d, J J = 14.3 Hz, 41H), 0.91 (t, J J = 6.6 Hz, 9H). 1.25 Synthesis of YK-1518

[0224] Step 1: Synthesis of YK-1518-PM1 Using 2-hexyldecanol (5.00 g, 20.62 mmol), N-Boc-6-aminohexanoic acid (5.70 g, 24.64 mmol), EDCI (5.00 g, 26.60 mmol), DMAP (756 mg, 6.22 mmol) as raw materials, dissolved in dichloromethane (50.0 mL), reacted at room temperature for 12 hours, and the reaction was complete by TLC. Column chromatography was performed with hexane / ethyl acetate = 0 - 50%, and the solvent was evaporated to obtain YK-1518-PM1 (8.6 g, 18.87 mmol, 91.5%). C 27 H 53 NO4, MS(ES): m / z(M+H + ) 456.2.

[0225] Step 2: Synthesis of YK-1518-PM2 Weighed YK-1518-PM1 (8.60 g, 18.87 mmol) and slowly added it to HCl / dioxane (50 ml) at room temperature, reacted at room temperature for 12 hours, and the reaction was complete by TLC. The solvent was evaporated to obtain the crude product YK-1518-PM2 (6.50 g, 18.28 mmol, 96.8%). C 22 H 45 NO2, MS(ES): m / z(M+H + ) 356.2.

[0226] Step 3: Synthesis of YK-1518-PM3 Using YK-1518-PM2 (2.00 g, 5.62 mmol), INT-2 (2.12 g, 5.06 mmol), and K2CO3 (2.33 g, 26.60 mmol) as raw materials, dissolve them in acetonitrile (20.0 mL), heat to 70 °C, react for 12 hours, and check by TLC that the reaction is complete. Stop the reaction, filter, wash with dichloromethane, and evaporate to dryness. Dissolve with a small amount of dichloromethane, load the sample by wet method, with dichloromethane / methanol = 0 - 10%, and evaporate to dryness to obtain YK-1518-PM3 (1.6 g, 2.31 mmol, 40.9%). C 45 H 89 NO4, MS(ES): m / z(M+H + ) 694.3

[0227] Step 4: Synthesis of YK-1518-PM4 Weigh CDI (555 mg, 3.44 mmol) as the raw material, dissolve it in dichloromethane (10.0 mL), and slowly add YK-1518-PM3 (600 mg, 0.98 mmol) at 0 °C. After addition, restore to room temperature and stir for 5 hours. Check by TLC that the reaction is complete. Stop the reaction, wash three times with water, extract with dichloromethane by liquid separation, dry, filter, and evaporate to dryness. Obtain YK-1518-PM4 (350 mg, 0.50 mmol, 51.4%). C 48 H 89 N3O5, MS(ES): m / z(M+H + ) 788.6

[0228] Step 5: Synthesis of YK-1518 Using YK-1518-PM4 (350 mg, 0.50 mmol), YK-1515-PM3 (182 mg, 1.24 mmol), and K2CO3 (207 mg, 1.50 mmol) as raw materials, dissolve them in acetonitrile (5.0 mL), heat to 70 °C, react for 12 hours, and check by TLC that the reaction is complete. Stop the reaction, filter, wash with dichloromethane, and evaporate to dryness. Dissolve with a small amount of dichloromethane, load the sample by wet method, with dichloromethane / methanol (10% ammonia water) = 0 - 10%, and evaporate to dryness to obtain YK-1518 (15 mg, 0.02 mmol, 3.8%). C 53 H 104 N4O6, MS(ES):m / z(M+H + ) 893.7

[0229] 11H NMR (400 MHz, CDCl3) δ 6.53 (s, 1H), 4.7 (d, J J = 5.8 Hz, 4H), 3.23(s, 11H), 2.75 (s, 3H), 2.71 (d, J J = 7.6 Hz, 2H), 2.43 (t, J J = 7.3 Hz, 4H), 1.83(s, 5H), 1.75 (m, J J = 24.2, 8.2 Hz, 8H), 1.42 (m, J J = 14.6, 7.1 Hz, 4H), 1.25-1.34 (m, 49H), 0.92 (t, J J = 6.7 Hz, 12H). 1.26 Synthesis of C16 Synthesized according to the synthesis procedure of C16 in CN 202380010167.3 to obtain 44 mg of C16.

[0230] 1.27 Synthesis of 9322-O17S Synthesized according to the synthetic route of 9322-O17S in Imidazole-Based Synthetic Lipidoids for In Vivo mRNA Delivery into Primary T Lymphocytes, Xuewei Zhao et al., Angew Chem Int Ed Engl., 59(45):20083-20089 to obtain 55 mg of 9322-O17S.

[0231] 1.28. Synthesis of 76-017Se Synthesized according to the synthetic method of 76-O17Se in In Vitro Engineering Chimeric Antigen Receptor Macrophages and T Cells by Lipid Nanoparticle-Mediated mRNA Delivery, Zhongfei Ye et al., ACS Biomater Sci Eng., 2022 Feb 14;8(2):722-733 to obtain 38 mg of 76-O17Se.

[0232] Example 2: Optimization of mRNA-LNP formulation The specific operations of the cell transfection experiment used in this example include: Step 1: Cell resuscitation and subculture: Resuscitate Jurkat cells and culture and subculture them in a culture dish until the required number of cells is obtained.

[0233] Step 2: Plating: Digest and count the cells in the culture dish, and plate 150,000 cells per well in a 12-well plate, and culture overnight until the cells adhere to the wall.

[0234] Step 3: Cell transfection: Add the mRNA-LNP preparations encapsulating eGFP-mRNA with different carriers into the cell culture medium in the 12-well plate (add 1.5 μg of the mRNA-LNP preparation per well) respectively. After continuing to culture for 24 hours, observe under a fluorescence microscope, and evaluate the transfection efficiency according to the fluorescence intensity.

[0235] 2.1 Optimization of the ratio of carrier (liposome) to mRNA Step 1: According to the molar ratio of cationic lipid: DSPC: cholesterol: DMG-PEG2000 of 49:10:39.5:1.5, dissolve the cationic lipids YK-1503, YK-1504, YK-1505, and YK-1507 synthesized in Example 1, DSPC (A.V.T. (Shanghai) Pharmaceutical Technology Co., Ltd.), cholesterol (A.V.T. (Shanghai) Pharmaceutical Technology Co., Ltd.), and DMG-PEG2000 in ethanol to obtain Solution A. Rapidly add Solution A to the citrate buffer (pH = 4.5 ± 0.5) by the ethanol injection method, and vortex for 30 s to obtain the ethanol-lipid solution.

[0236] Step 2: Dilute eGFP-mRNA (Shanghai Qifa Experimental Reagent Co., Ltd.) in the citrate buffer (pH = 4.5 ± 0.5) to obtain an aqueous eGFP-mRNA solution.

[0237] Step 3: According to the mass ratio of carrier: mRNA of 5:1, 10:1, 15:1, 20:1, 30:1, and 35:1 respectively, use a microfluidic device to mix the ethanol-lipid solution prepared in Step 1 with the aqueous mRNA solution prepared in Step 2 at a flow rate of 10 mL / min to obtain the corresponding liposome solutions. After diluting the liposome solutions to 10 times the volume with PBS, ultrafiltrate using a 300KDa ultrafiltration tube to remove ethanol. Then, make up the volume to a suitable volume with PBS and filter through a 0.2 μm sterile filter to obtain the mRNA-LNP preparations encapsulating eGFP-mRNA with a molar ratio of cationic lipid (YK-1503, YK-1504, YK-1505, or YK-1507) / DSPC / cholesterol / DMG-PEG2000 of 49:10:39.5:1.5.

[0238] The results of the cell transfection experiment showed that when the mass ratio of the carrier to mRNA was in the range of 10:1 - 30:1, the corresponding mRNA-LNP compositions all had good transfection effects. Among them, the best transfection effect was achieved at 15:1, while appropriate transfection effects could not be obtained when the mass ratios were 5:1 and 35:1.

[0239] 2.2 Optimization of the ratio of cationic lipid to neutral lipid mRNA-LNP compositions encapsulating eGFP-mRNA were prepared according to a method similar to that in 2.1, in which the molar ratios of the cationic lipids (YK-1503, YK-1504, YK-1505 or YK-1507) to the neutral lipid DSPC were adjusted to 1:1, 3:1, 3.5:1, 4:1, 4.9:1, 10:1, 15:1 and 20:1, respectively.

[0240] The cell transfection experiment showed that when the molar ratio of the cationic lipid to the neutral lipid was in the range of 1:1 - 15:1, the corresponding mRNA-LNP compositions could all transfect cells, and the highest transfection efficiency was achieved at 4.9:1.

[0241] 2.3 Optimization of the proportion of polymer-conjugated lipid in the carrier mRNA-LNP compositions encapsulating eGFP-mRNA were prepared according to a method similar to that in 2.1, in which the cationic lipids were YK-1503, YK-1504, YK-1505 or YK-1507, and the molar percentages of the polymer-conjugated lipid DMG-PEG2000 in the carrier were 0.5%, 1.5%, 2.5%, 3.5%, 5%, 10% and 15%, respectively.

[0242] The results of the cell transfection experiment showed that when the molar percentage of the polymer-conjugated lipid in the carrier was in the range of 0.5% - 10%, the corresponding mRNA-LNP compositions could all transfect cells. The highest transfection efficiency was achieved at 1.5%, and the lowest at 10%.

[0243] 2.4 Optimization of the proportions of each component in the carrier mRNA-LNP preparations encapsulating eGFP-mRNA were prepared according to a method similar to that in 2.1. In step 1, the molar ratios of the cationic lipids (YK-1503, YK-1504, YK-1505 or YK-1507), the neutral lipid DSPC, the structural lipid cholesterol and the polymer-conjugated lipid DMG-PEG2000 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.

[0244] From the cell transfection experiments, it is known that transfection can occur when the molar ratios of cationic lipids, neutral lipids, structural lipids, and polymer-conjugated lipids are 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 effects are achieved within the range of (35 - 49):(7.5 - 15):(35 - 55):(1 - 5). Among them, when the molar ratio is 49:10:39.5:1.5, the transfection effect is the best.

[0245] Example 3: Cell Transfection of mRNA-LNP Formulations Encapsulating eGFP-mRNA The specific operations for cell transfection used in this example include: Step 1: Cell resuscitation and passage: Resuscitate Jurkat cells and culture and passage them in a culture dish to the required cell number.

[0246] Step 2: Plating: Digest and count the cells in the culture dish, and plate 150,000 cells per well in a 12-well plate, and culture overnight until the cells adhere to the wall.

[0247] Step 3: Cell transfection: Add 1.5 μg of the mRNA-LNP formulations encapsulating eGFP-mRNA prepared in Example 2 (where the cationic lipids are YK-1503, YK-1504, YK-1505, or YK-1507 respectively) to the cell culture medium in the 12-well plate, continue to culture for 24 hours, and then observe under a fluorescence microscope. Examine the transfection efficiency based on the fluorescence intensity.

[0248] Based on the transfection efficiency results, select mRNA-LNP formulations with the following ratios for the subsequent examples: the mass ratio of vector to mRNA is 15:1; the molar ratio of cationic lipid to neutral lipid is 4.9:1; the molar proportion of polymer-conjugated lipid in the liposome is 1.5%; the molar ratio of cationic lipid, neutral lipid, structural lipid, and polymer-conjugated lipid is 49:10:39.5:1.5.

[0249] Example 4: Preparation of mRNA-LNP Formulations Table 1 Cationic Lipid Compounds 4.1 Prepare the corresponding ethanol lipid solutions of the cationic lipids in Table 1 according to the method in Step 1 of Example 2.1. 4.2 Dilute eGFP-mRNA (Shanghai Qifa Experimental Reagent Co., Ltd.) or Fluc-mRNA (Shanghai Qifa Experimental Reagent Co., Ltd.) in citrate buffer (pH = 4.5 ± 0.5) to obtain the corresponding aqueous mRNA solution. 4.3 Using a microfluidic device, mix the ethanol lipid solution obtained in 4.1 with the aqueous eGFP-mRNA solution or Fluc mRNA solution obtained in 4.2 at a flow rate of 10 mL / min in a volume ratio of 1:3 to prepare the corresponding liposome solution with a mass ratio of carrier (liposome) to mRNA of approximately 15:1. After diluting the liposome solution to 10 times its volume with PBS, ultrafiltration is performed using a 300KDa ultrafiltration tube to remove ethanol. Then, it is made up to a suitable volume with PBS and filtered through a 0.2 μm sterile filter to obtain mRNA-LNP preparations encapsulating eGFP-mRNA or Fluc-mRNA with a molar ratio of cationic lipid:DSPC:cholesterol:DMG-PEG2000 of 49:10:39.5:1.5.

[0253] Example 5: Determination of the particle size, polydispersity index (PDI), and encapsulation efficiency of mRNA-LNP Use dynamic light scattering and a Malvern laser particle size analyzer to measure the particle size and polydispersity index (PDI).

[0254] Take 10 μL of the mRNA-LNP solution prepared in Example 4, dilute it to 1 mL with RNase-free deionized water, add it to the sample cell, and measure each sample 3 times. The measurement conditions are: 90° scattering angle, 25 °C; According to the manufacturer's instructions, use the Quant-iT™ RiboGreen® RNA Quantification Assay Kit (ThermoFisher Scientific, UK) to determine the encapsulation efficiency of the LNP. The detection results are shown in Table 2: Table 2 Particle size, polydispersity index (PDI), and encapsulation efficiency of mRNA-LNP

[0255] As can be seen from Table 2, the particle size of the nano-lipid particles prepared in Example 4 is between 70 - 90 nm, and all can be used for mRNA delivery. The polydispersity index is less than 0.15, indicating good particle size uniformity. And it has a high encapsulation efficiency, with the encapsulation efficiency being greater than 90%.

[0256] Example 6: In vitro ( in vitro ) delivery performance and toxicity of LNP The methods of cell resuscitation, passage and plating refer to Steps 1 and 2 of Example 3.

[0257] In the 96-well plate containing Jurkat cells obtained in Step 2, an appropriate volume of Jurkat cell culture medium was supplemented, and the mRNA-LNP preparation containing 0.3 μg of Fluc-mRNA (prepared in Example 4) was added to the 96-well plate. After continuous culture for 24 h, the corresponding reagents were added according to the instructions of the Gaussia Luciferase Assay Kit (Thermo Fisher), and the relative fluorescence intensity of each well was detected by the IVIS fluorescence detection system; finally, 10 μL of CCK-8 solution was added to each well of the above-mentioned plate after 24-hour culture. After incubating the culture plate in the incubator for 1 hour, the absorbance at 450 nm was measured by an enzyme-linked immunosorbent assay (ELISA) reader to detect the cell viability. The results of relative fluorescence intensity and cell viability are shown in Table 3.

[0258] Table 3 Fluorescence detection results of Fluc-mRNA

[0259] There were significant differences in the relative fluorescence intensity of the above mRNA-LNP compositions (which can reflect the translation efficiency of mRNA). The mRNA-LNP compositions prepared by YK-1503, YK-1504, YK-1505, YK-1507, YK-1510, YK-1511, YK-1513, YK-1515 and YK-1516 had significantly higher relative fluorescence intensity than the mRNA-LNP compositions prepared by SM-102, MC3, 9322-O17S, 76-017Se and C16. Specifically: 1. The cell transfection efficiency of the mRNA-LNP compositions prepared by YK-1503, YK-1504, YK-1505, YK-1507, YK-1510, YK-1511, YK-1513, YK-1515 and YK-1516 was significantly improved compared with the representative cationic lipids in the prior art. For example, the cell transfection efficiency of YK-1507 could reach 2.8 times that of SM-102 and 6.0 times that of MC3.

[0260] 2. The cell transfection efficiency of the mRNA-LNP compositions prepared by YK-1503, YK-1504, YK-1505 and YK-1507 was significantly improved compared with the 76-017Se cationic lipid with a pyrrolidine head structure. For example, the cell transfection efficiency of YK-1507 could reach 4.9 times that of 76-017Se.

[0261] 3. The cell transfection efficiency of the mRNA-LNP compositions prepared from YK-1510, YK-1511, and YK-1513 is significantly improved compared to that of the 9322-O17S cationic lipid which also has a 2-methylimidazole head structure. For example, the cell transfection efficiency of YK-1513 can reach 4.6 times that of 9322-O17S.

[0262] 4. The cell transfection efficiency of the mRNA-LNP compositions prepared from YK-1515 and YK-1516 is significantly improved compared to that of the C16 cationic lipid which also has a piperazine head structure. For example, the cell transfection efficiency of YK-1516 can reach 1.8 times that of C16.

[0263] Example 7: In vivo ( in vivo ) Delivery performance The Fluc-mRNA-LNP composition prepared in Example 4 was intravenously injected into female BALB / c albino mice aged 4-6 weeks and weighing 17-19 g (at a dose of approximately 5 μg Fluc-mRNA / mouse). After 6 hours of administration, the fluorescence imaging substrate was injected into the mice by intraperitoneal injection. The mice were allowed to move freely for 5 minutes, and then the total radiation intensity of the protein expressed by the mRNA carried by the mRNA-LNP composition in the mice was detected by an IVIS Spectrum small animal in vivo imager (corresponding to the fluorescence protein expression intensity, i.e., the protein expression level). After sampling, the mice were sacrificed by cervical dislocation and dissected, and the internal organs liver and spleen of the mice were precisely separated. The total radiation intensity of the protein expressed by Fluc-mRNA in each organ of the mice was detected by an IVIS Spectrum small animal in vivo imager (corresponding to the fluorescence protein expression intensity, i.e., the protein expression level). The results of in vivo imaging of mice and protein expression detection in the liver and spleen are shown in Table 4 and Figures 1-3 .

[0264] Table 4 Experimental data of in vivo and organ imaging of mice

[0265] The mRNA-LNP composition prepared from YK-1503, YK-1504, YK-1505, YK-1507, YK-1510, YK-1511, YK-1513, YK-1515 and YK-1516 can efficiently deliver mRNA to the spleen, and the delivery effect is significantly enhanced compared with SM-102, MC3, 9322-O17S, 76-017Se and C16. Specifically: compared with the mRNA-LNP compositions prepared from the prior art ionizable cationic lipids (SM-102, MC3, 9322-O17S, 76-017Se, C16 and YK-301), the mRNA-LNP compositions prepared from YK-1503, YK-1504, YK-1505, YK-1507, YK-1510, YK-1511, YK-1513, YK-1515 and YK-1516 of the present disclosure have significantly enhanced total radiation intensity in the spleen and total in vivo radiation intensity. For example, for the mRNA-LNP composition prepared from YK-1507, the total in vivo radiation intensity is 1.8 times, 2.2 times, 2.2 times, 2.4 times and 1.9 times that of the mRNA-LNP compositions prepared from SM-102, MC3, 9322-O17S, 76-017Se and C16 respectively, and the total radiation intensity in the spleen is 4.0 times, 5.1 times, 2.6 times, 2.7 times and 2.7 times that of the mRNA-LNP compositions prepared from SM-102, MC3, 9322-O17S, 76-017Se and C16 respectively.

[0266] Example 8: Targeting of mRNA-LNP Composition to Mouse Spleen Cells 1. Inject the eGFP-mRNA compositions with different cationic lipids prepared in Example 4 into female C57BL / 6 mice aged 4 - 6 weeks and weighing 17 - 19 g via the tail vein (the dosage is about 20 μg eGFP-mRNA per mouse), and sacrifice the mice by cervical dislocation 24 hours after administration and then perform dissection immediately to precisely isolate the spleens of the mice.

[0267] 2. Prepare single cells 1) Grind the isolated spleen tissue to make the spleen tissue single-celled and pass through a cell sieve.

[0268] 2) Add 10 times the volume (about 4 mL) of red blood cell lysate to lyse and remove the red blood cells in the tissue.

[0269] 3) Count the cells and take 5×10 6 cells into a flow tube (ensure that the number of cells taken from each sample is the same).

[0270] 3. Detection of immune cells in spleen tissue 1) Add 100 μL of surface antibody MIX to each single-cell suspension (the composition of the surface antibody MIX is shown in Table 5, and incubate in the dark at room temperature for 15 minutes (one negative control).

[0271] Table 5 Reagents and sources of the surface antibody MIX used in the flow cytometry experiment of mouse spleen cells

[0272] 2) Add 2 mL of PBS, centrifuge at 500 g for 5 minutes, and discard the supernatant.

[0273] 3) Resuspend the cells with 200 μL of PBS, (after filtering through a 200-mesh nylon mesh), and detect on the flow cytometer Cytoflex S. Analyze the percentage of GFP content in each cell. The detection order of each cell line is as follows: Proportion of GFP in T cells: CD45 + →CD3 + →GFP + Proportion of GFP in B cells: CD45 + →CD3 - CD19 + →GFP + Proportion of GFP in DC cells: CD45 + →CD11c + →GFP + Proportion of GFP in macrophages: CD45 + →F4 / 80 + →GFP + Set the mice injected with an equal volume of 0.9% sodium chloride solution as the blank control group.

[0274] The percentage of eGFP-positive cells in mouse spleen cells is shown in Table 6 and Figure 4 .

[0275] Table 6 Percentage of eGFP-positive cells in mouse spleen cells (%)

[0276] From the data in Table 6 and Figure 4It can be seen that the mRNA-LNP composition prepared from the cationic lipid containing the present disclosure can significantly increase the ratio of antigen-expressing cells among immune cells (T cells, B cells, DC cells, and macrophages) in the spleen. Compared with the mRNA-LNP composition prepared from SM-102 and MC3, it can significantly increase the ratio of antigen-expressing cells among immune cells (T cells, B cells, DC cells, and macrophages) in the spleen. For example, the mRNA-LNP prepared from YK-1507 increased the ratio of eGFP-positive cells of T cells, B cells, DC cells, and macrophages in the spleen by 1.8 times, 1.5 times, 2.4 times, and 5.0 times compared with the mRNA-LNP prepared from MC3.

[0277] The preferred embodiments of the present disclosure have been described in detail above. However, the present disclosure is not limited thereto. Within the technical concept scope of the present disclosure, various simple modifications can be made to the technical solutions of the present disclosure, including combining each technical feature in any other suitable manner. These simple modifications and combinations should also be regarded as the content of the present disclosure and fall within the protection scope of the present disclosure.

Claims

1. 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): ， Wherein: R1 is a substituted or unsubstituted N-containing heterocycle; R2 and R3 are each independently a substituted or unsubstituted C 10-16 linear or branched olefin or alkane, and R2 and R3 are the same or different; L1 and L2 are each independently an unsubstituted C 2-8 linear alkylene group; M1 is -(CH2) n -, -C(O)NH-, or -NHC(O)-, where n is 0, 1, 2, 3, or 4; M2 and M3 are each independently any one of -CH=CH-, -C(O)O- and -C(O)N-, and M2 and M3 are the same or different.

2. The cationic lipid compound according to claim 1, or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, wherein, R1 is , or ; and / or, R2 is , , , , or unsubstituted C 10-16 linear alkane; and / or, R3 is , , , , or unsubstituted C 10-16 linear alkane; And / or, L1 is -(CH2)2-, -(CH2)3-, -(CH2)5- or -(CH2)8-; And / or, L2 is -(CH2)2-, -(CH2)3-, -(CH2)5- or -(CH2)8-; And / or, M1 is -CH2- or -NHC(O)-; And / or, M2 is -CH=CH-, -C(O)O- or -C(O)N-; And / or, M3 is -CH=CH-, -C(O)O- or -C(O)N-.

3. The cationic lipid compound according to claim 1 or 2, or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, wherein, The compound has any one of the following structures: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 and 。 4. A carrier, wherein, The carrier contains a cationic lipid, wherein the cationic lipid includes the cationic lipid compound according to any one of claims 1-3, or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer.

5. The carrier according to claim 4, wherein, The molar percentage of the cationic lipid in the carrier is 25% - 75%.

6. The carrier according to claim 4, wherein The carrier further contains a neutral lipid; And / or, the carrier further contains a structural lipid; And / or, the carrier further contains a polymer-conjugated lipid.

7. The carrier according to claim 6, wherein The neutral lipid is selected from any one or a combination of at least two of the group consisting of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol and their derivatives; And / or, the structural lipid is selected from any one or a combination of at least two of the group consisting of: cholesterol, non-sterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, α-tocopherol and corticosteroid; And / or, the polymer-conjugated lipid is selected from any one or a combination of at least two of the group consisting of: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol and PEG-modified dialkylglycerol.

8. The carrier according to claim 7, wherein The neutral lipid is any one or a combination of at least two selected from the group consisting of: 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dielaidoyl-sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-di-O-octadecenoyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-cholesteryl succinyl-sn-glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3-phosphocholine, 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, dipalmitoyl phosphatidylglycerol, palmitoyl oleoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, dimyristoyl phosphoethanolamine, 1-stearoyl-2-oleoyl-stearoylethanolamine, 1-stearoyl-2-oleoyl-phosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine and lysophosphatidylethanolamine.

9. The carrier according to claim 7, wherein The neutral lipid is selected from 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine and / or 1,2-distearoyl-sn-glycero-3-phosphocholine.

10. The carrier according to claim 7, wherein, The structural lipid is cholesterol.

11. The carrier according to claim 7, wherein The polymer-conjugated lipid is any one or a combination of at least two selected from the group consisting of: distearoyl phosphatidylethanolamine polyethylene glycol 2000, dimyristoyl glycerol-3-methoxy polyethylene glycol 2000 and methoxy polyethylene glycol ditetradecylacetamide.

12. The carrier according to claim 6, wherein, The molar percentage of the neutral lipid in the carrier is 5% - 25%; and / or, the molar percentage of the structural lipid in the carrier is 15% - 65%; and / or, the molar percentage of the polymer-conjugated lipid in the carrier is 0.5% - 10%.

13. The carrier according to claim 12, wherein In the carrier, the molar ratio of the cationic lipid to the neutral lipid is 1:1 - 15:1; And / or, in the carrier, the molar ratio of the cationic lipid to the structural lipid is 0.6:1 - 3:1; And / or, in the carrier, the molar ratio of the cationic lipid to the polymer-conjugated lipid is 4.5:1 - 32.5:

1.

14. The carrier according to any one of claims 4-13, wherein, In the carrier, 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).

15. The carrier according to claim 14, wherein, In the carrier, the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is (35 - 49):(7.5 - 15):(35 - 5):(1 - 5).

16. The carrier according to claim 15, wherein, In the carrier, the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is 49:10:39.5:1.

5.

17. The carrier according to claim 4, wherein, The cationic lipid further includes one or more other cationic lipid compounds.

18. A composition, wherein, The composition includes an active ingredient and a carrier, and the carrier is the carrier according to any one of claims 4 - 17.

19. The composition according to claim 18, wherein, The composition is a nanoparticle preparation, and the average particle size of the nanoparticle preparation is 10 nm - 300 nm; the polydispersity index of the nanoparticle preparation is ≤0.

5.

20. The composition according to claim 19, wherein, The average particle size of the nanoparticle preparation is 40 nm - 240 nm; the polydispersity index of the nanoparticle preparation is ≤0.

4.

21. The composition according to claim 18, wherein, The active ingredient includes a therapeutic agent or a prophylactic agent.

22. The composition according to claim 21, wherein, The mass ratio of the carrier to the therapeutic agent or the prophylactic agent is 10:1 - 30:

1.

23. The composition according to claim 22, wherein, The mass ratio of the carrier to the therapeutic agent or the prophylactic agent is 12.5:1 - 20:

1.

24. The composition according to claim 23, wherein, The mass ratio of the carrier to the therapeutic agent or the prophylactic agent is 13:1 - 17:

1.

25. The composition according to any one of claims 21 - 24, wherein the therapeutic agent or the prophylactic agent is a vaccine or a compound capable of eliciting an immune response.

26. The composition according to claim 25, wherein, The therapeutic agent or the prophylactic agent is selected from any one or a combination of at least two of the group consisting of nucleic acids, small molecule compounds, polypeptides, or proteins.

27. The composition according to claim 26, wherein, The therapeutic agent or the prophylactic agent is a nucleic acid.

28. The composition according to claim 27, wherein, The therapeutic agent or the prophylactic agent is ribonucleic acid.

29. The composition according to claim 28, wherein, The ribonucleic acid is selected from any one or a combination of at least two of the group consisting of: small interfering RNA, asymmetric interfering RNA, microRNA, Dicer-substrate RNA, small hairpin RNA, messenger RNA.

30. The composition according to claim 29, wherein, The ribonucleic acid is messenger RNA.

31. The composition according to claim 18, wherein, The composition further includes a pharmaceutically acceptable excipient and / or diluent.

32. Use of the compound according to any one of claims 1 - 3 or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer, or the carrier according to any one of claims 4 - 17, or the composition according to any one of claims 18 - 31 in the preparation of a drug.

33. The use according to claim 32, wherein The active component of the drug is selected from any one or a combination of at least two of the group consisting of nucleic acids, small molecule compounds, polypeptides, or proteins. Use of a compound according to any one of claims 1-3, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, or a carrier according to any one of claims 4-17, in enhancing cell transfection efficiency and / or reducing cytotoxicity.

35. The use according to claim 34, wherein, The use is for enhancing cell transfection efficiency and / or reducing cytotoxicity in vitro.

36. Use of the compound according to any one of claims 1-3, or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, or the carrier according to any one of claims 4-17, in enhancing the targeting of nucleic acid to a target, and / or in enhancing the expression level of nucleic acid in the target, wherein, The target is selected from any one or a combination of at least two of the group consisting of a target organ, a target tissue, and a target cell.

37. The use according to claim 36, wherein The target organ or target tissue is selected from any one or a combination of at least two of the group consisting of the spleen, liver, lymph, and muscle; and / or, the target cell is selected from any one or a combination of at least two of the group consisting of B cells, NK cells, DC cells, T cells, and macrophages.

38. The use according to claim 37, wherein The target organ or target tissue is the spleen; and / or, the target cell is selected from any one or a combination of at least two of the group consisting of B cells, DC cells, T cells, and macrophages.

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