Ionizable cationic lipids targeting immune cells, compositions containing same, and uses thereof

Abstract

The present disclosure belongs to the field of medicine, and specifically relates to the field of targeted lipid delivery technology, and specifically discloses ionizable cationic lipids targeting immune cells, compositions comprising the same, and uses thereof. The cationic lipids targeting immune cells provided by the present disclosure have a structure shown in formula (I), can be used for RNA targeted delivery, and can significantly enhance the targeting effect of mRNA drugs on immune cells.

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 are cells involved in or associated with immune responses. They play a key role in protecting the human body from infection and disease. Targeting immune cells enables precise drug delivery, reduces drug distribution in non-target cells, and reduces drug toxicity and side effects. Targeted immune cell delivery of nucleic acids opens up the possibility of developing novel immunotherapies, such as in situ CAR-T or CAR-M therapies. By directly delivering mRNA encoding chimeric antigen receptors (CARs) to T cells or macrophages, this approach avoids the complex in vitro processes of traditional CAR cell therapies, reducing preparation difficulty and cost. Furthermore, targeted immune cell technology can efficiently and safely engineer T cells or macrophages based on individual patient differences and disease characteristics, enabling them to express CARs targeting cancer cells, thus achieving personalized treatment.

[0003] In the pharmaceutical field, the effective targeted delivery of small molecule drugs, peptides, proteins, and nucleic acids remains a persistent challenge. The delivery of nucleic acids is particularly challenging due to their low cell permeability and high sensitivity to degradation by nucleases (e.g., RNAase).

[0004] Compositions containing cationic lipids, liposomes, and lipoplexes serve as transport vehicles that can effectively deliver bioactive substances such as small molecule drugs, peptides, proteins, and nucleic acids to cells and / or intracellular compartments. These compositions generally contain one or more cationic and / or ionizable lipids, neutral lipids, structured lipids, and polymer-conjugated lipids. Cationic and / or ionizable lipids include, for example, amine-containing lipids that can be easily protonated. Although a variety of such lipid-containing nanoparticle compositions have been demonstrated, safety, efficacy, and specificity remain to be improved. Notably, the increased complexity of lipid nanoparticles (LNPs) complicates their production and may increase their toxicity, a major concern that may limit their clinical application. For example, nucleic acid drugs such as 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 comprising the same. Summary of the Invention

[0006] The present disclosure provides ionizable cationic lipids capable of targeting immune cells, compositions comprising the same, and uses thereof. The ionizable cationic lipids provided herein 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 of immune cells is strong, the lipids can carry active pharmaceutical ingredients and transfect cells with high transfection efficiency, and the lipids have low cytotoxicity, thereby improving delivery efficiency and safety.

[0007] The technical solutions provided by the present disclosure may include, for example:

[0008] [1] A (cationic lipid) compound, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound has the structure represented by formula (I):

[0009] ,

[0010] in:

[0011] R1 is a substituted or unsubstituted N-containing heterocycle;

[0012] R2 and R3 are each independently substituted or unsubstituted C 10-16 a linear or branched olefin or alkane, R2 and R3 being the same or different;

[0013] L1 and L2 are each independently unsubstituted C 2-8 straight-chain alkylene;

[0014] M1 is -(CH2) n -, -C(O)NH- or -NHC(O)-, wherein n is 0, 1, 2, 3 or 4;

[0015] 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.

[0016] In the compound of formula (I) provided by the present invention, the number of carbon atoms of R2 and R3 can be independently 10, 11, 12, 13, 14, 15, or 16, or can be a range consisting of any two of the above values, or any intermediate value (integer) in the range.

[0017] In the compound of formula (I) provided by the present invention, R2 and R3 can each independently be a straight-chain alkyl group, a branched-chain alkyl group, a straight-chain alkenyl group or a branched-chain alkenyl group.

[0018] In the compound of formula (I) provided by the present invention, the number of carbon atoms of L1 and L2 can be independently 2, 3, 4, 5, 6, 7, or 8, or can be a range consisting of any two of the above values, or any intermediate value (integer) in the range.

[0019] [2] The compound according to [1], or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, wherein R1 is 、 or ;

[0020] and / or, R2 is 、 、 、 、 or unsubstituted C 10-16 straight-chain alkanes;

[0021] and / or, R3 is 、 、 、 、 or unsubstituted C 10-16 straight-chain alkanes;

[0022] and / or, L1 is -(CH2)2-, -(CH2)3-, -(CH2)5- or -(CH2)8-;

[0023] and / or, L2 is -(CH2)2-, -(CH2)3-, -(CH2)5- or -(CH2)8-;

[0024] and / or, M1 is -CH2- or -NHC(O)-;

[0025] and / or, M2 is -CH=CH-, -C(O)O-, or -C(O)N-;

[0026] and / or, M3 is -CH=CH-, -C(O)O-, or -C(O)N-.

[0027] [3] The compound according to [1] or [2], or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound has any one of the following structures of YK-1501 to YK-1518:

[0028] 、

[0029] 、

[0030] 、

[0031] 、

[0032] 、

[0033] 、

[0034] 、

[0035] 、

[0036] 、

[0037] 、

[0038] 、

[0039] 、

[0040] 、

[0041] 、

[0042] 、

[0043] 、

[0044] and

[0045] .

[0046] 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.

[0047] [4] A carrier, wherein the carrier comprises a cationic lipid, wherein the cationic lipid comprises the compound described in any one of [1] to [3], or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof.

[0048] [5] The carrier according to [4], wherein the molar percentage of the cationic lipid in the carrier is 25%-75%. That is, based on the total molar amount of the carrier, the content of the cationic lipid is 25-75 mol%.

[0049] For example, the content of 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 it can be a range consisting of any two of the above values, or any intermediate value in the range.

[0050] [6] The vector according to [4] or [5], wherein the vector further comprises a neutral lipid;

[0051] and / or, the carrier further comprises a structured lipid;

[0052] And / or, the carrier further comprises a polymer-conjugated lipid.

[0053] [7] The carrier according to [6], wherein the neutral lipid is any one or a combination of at least two selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol and their derivatives;

[0054] and / or, the structured lipid is selected from any one or a combination of at least two of the group consisting of: cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol and corticosteroids;

[0055] 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.

[0056] [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 following groups: 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-diondecanoyl-sn-glycero-phosphocholine, 1,2-diundecanoyl-sn-glycero-phosphocholine, -Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3-phosphocholine, 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diacarriedoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-di Oleoyl-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-bisdocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero- 3-phospho-rac-(1-glycerol) sodium salt, dipalmitoylphosphatidylglycerol, palmitoyloleoylphosphatidylethanolamine, distearoyl-phosphatidyl-ethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphoethanolamine, 1-stearoyl-2-oleoyl-stearoylethanolamine, 1-stearoyl-2-oleoyl-phosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine.

[0057] [9] The carrier according to any one of [6] to [8], wherein the neutral lipid is selected from 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine and / or 1,2-distearoyl-sn-glycero-3-phosphocholine.

[0058]

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

[0059]

[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 following groups: distearoylphosphatidylethanolamine polyethylene glycol 2000, dimyristoylglycerol-3-methoxypolyethylene glycol 2000 and methoxypolyethylene glycol ditetradecanoyl acetamide.

[0060]

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

[11] , wherein the molar percentage of the neutral lipid in the carrier is 5% to 25%;

[0061] and / or, the molar percentage of the structural lipid in the carrier is 15%-65%;

[0062] And / or, the molar percentage of the polymer-conjugated lipid in the carrier is 0.5%-10%.

[0063] 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%.

[0064] For example, the content of neutral lipids in the carrier can be 5 mol%, 10 mol%, 15 mol%, 20 mol%, 25 mol%, or a range consisting of any two of the above values, or any intermediate value in the range.

[0065] For example, the content of structural lipids in the carrier 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 it can be a range consisting of any two of the above values, or any intermediate value in the range.

[0066] 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 it can be a range consisting of any two of the above values, or any intermediate value in the range.

[0067]

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

[12] , wherein the molar ratio of the cationic lipid to the neutral lipid in the carrier is 1:1-15:1;

[0068] and / or, in the carrier, the molar ratio of the cationic lipid to the structural lipid is 0.6:1-3:1;

[0069] And / or, in the carrier, the molar ratio of the cationic lipid to the polymer-conjugated lipid is 4.5:1-32.5:1.

[0070]

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

[13] , wherein the molar ratio of the cationic lipid, the neutral lipid, the structural lipid and the polymer-conjugated lipid in the carrier is (25-75):(5-25):(15-65):(0.5-10).

[0071]

[15] The carrier according to

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

[0072]

[16] The carrier according to

[14] or

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

[0073]

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

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

[0074] "Other cationic lipid compounds" refers to cationic lipid compounds other than the compounds of formula (I) provided herein, or N-oxides, solvates, pharmaceutically acceptable salts, or stereoisomers thereof. Any cationic lipid compound known in the art that can be used to prepare carriers (especially carriers for carrying biologically active molecules such as nucleic acids, proteins, and peptides) can be used in the present invention. There is no particular limitation on the source of such cationic lipid compounds, which can be obtained commercially or prepared in accordance with existing techniques.

[0075]

[18] A composition comprising an active ingredient and a carrier, wherein the carrier is the carrier described in any one of [4] to

[17] .

[0076] An "active ingredient" refers to an active molecule with a specific function or effect, such as an active molecule with disease treatment or prevention capabilities (e.g., small molecule drugs, therapeutic or preventive nucleic acids, therapeutic or preventive proteins or peptides), or an active molecule with biomarker capabilities (e.g., GFP or its encoding gene). A "carrier" refers to a molecule or composition used to carry the active ingredient for protection, transportation, or other purposes. While a carrier itself typically lacks functionality or activity, its choice of carrier can have a significant impact on the activity of the active ingredient.

[0077]

[19] The composition according to

[18] , wherein the composition is a nanoparticle preparation, the average particle size of the nanoparticle preparation is 10 nm to 300 nm, and the polydispersity index (PDI) of the nanoparticle preparation is ≤ 0.5. The average particle size and polydispersity index of the nanoparticle preparation can be measured by existing methods, for example, using a laser particle size analyzer or other instrument.

[0078]

[20] The composition according to

[18] or

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

[0079] For example, the average particle size of the nanoparticle preparation can be 40nm, 50nm, 60nm, 70nm, 72nm, 74nm, 76nm, 78nm, 80nm, 82nm, 84nm, 86nm, 88nm, 90nm, 92nm, 94nm, 96nm, 98nm, 100nm, 110nm, 120nm, 130nm, 140nm, 150nm, 180nm, 200nm, 220nm, 240nm, or it can be a range consisting of any two of the above values, or any intermediate value in the range.

[0080] 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, or 0.4, or a range consisting of any two of the above values, or any intermediate value in the range.

[0081]

[21] The composition according to any one of

[18] to

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

[0082]

[22] The composition according to any one of

[18] to

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

[0083] For example, the mass ratio of the carrier to the therapeutic or preventive agent can be 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1, or a range consisting of any two of the above ratios, or any intermediate ratio within the range.

[0084]

[23] The composition according to

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

[0085]

[24] The composition according to

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

[0086]

[25] The composition according to any one of

[21] -

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

[0087]

[26] The composition according to

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

[0088]

[27] The composition according to

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

[0089]

[28] The composition according to

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

[0090]

[29] The composition according to

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

[0091]

[30] The composition according to

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

[0092]

[31] The composition according to any one of

[18] to

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

[0093]

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

[17] , or the composition of any one of

[18] to

[31] in the preparation of a medicament.

[0094]

[33] The use according to

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

[0095] Preferably, the drug is a nucleic acid drug (i.e., a drug whose active ingredient is a nucleic acid). The nucleic acid used in the nucleic acid drug can be the nucleic acid selected in the aforementioned therapeutic agent or preventive agent, which will not be described in detail here.

[0096]

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

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

[0097]

[35] The use according to

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

[0098]

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

[17] , in improving the targeting of a nucleic acid to a target, and / or in increasing the expression level of a nucleic acid in a 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.

[0099]

[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;

[0100] 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.

[0101]

[38] The use according to

[37] , wherein the target organ or target tissue is the spleen;

[0102] 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.

[0103]

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

[17] , or a composition as described in any one of

[18] to

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

[0104]

[40] The use according to

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

[0105]

[41] The use according to

[39] or

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

[0106]

[42] The use according to

[39] , wherein the subject is a mammal.

[0107]

[43] The use according to

[42] , wherein the subject is a human.

[0108]

[44] The use according to any one of

[39] -

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

[0109]

[45] The use according to

[44] , wherein the drug is administered subcutaneously.

[0110]

[46] The use according to any one of

[39] -

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

[0111] It should be understood that in the above technical solutions, the uses provided by the present disclosure may include both therapeutic and diagnostic uses as well as non-therapeutic and non-diagnostic uses. For example, therapeutic uses may include using the compounds provided herein, or their N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers, or using the carriers provided herein to package (drug) active ingredients and deliver them to target organs / tissues / cells, or using the compositions provided herein to deliver the active ingredients contained therein to target organs / tissues / cells, thereby achieving effects such as treating diseases, ameliorating symptoms, and regulating physiological activities in the body. Diagnostic uses may include packaging active ingredients for disease diagnosis in the compounds provided herein, or their N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers, or carriers, so that the active ingredients are delivered to target organs / tissues / cells, thereby achieving the purpose of disease diagnosis. Non-therapeutic / non-diagnostic purposes may include using the compounds provided herein, or their N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers, or using the carriers provided herein to encapsulate active ingredients and deliver them to target organs / tissues / cells for non-therapeutic and non-diagnostic purposes such as scientific research and testing (e.g., disease mechanism research, drug action mechanism research, new drug development, drug screening, etc.).

[0112] The beneficial effects of the present disclosure include at least:

[0113] The cationic lipid compounds and lipid compositions disclosed herein (also referred to as "carriers" in the present disclosure) can be used to encapsulate active pharmaceutical ingredients such as nucleic acids (such as mRNA, etc.).

[0114] The mRNA-LNP composition prepared by the cationic lipid disclosed in the present invention has at least the following advantages: it can significantly increase the protein expression in the subject (such as mouse) and has significant spleen targeting, which can significantly improve the in vivo ( in vivo ) and in vitro ( intro ) protein expression; and, compared with the ionizable cationic lipids of the prior art, the mRNA-LNP composition using the cationic lipid compound of the present invention significantly increased the percentage of immune cells expressing the antigen in the spleen, indicating that the mRNA-LNP composition prepared by the cationic lipid of the present invention has significantly improved spleen and immune cell targeting. BRIEF DESCRIPTION OF THE DRAWINGS

[0115] 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 are not limitations of the present disclosure.

[0116] Figure 1 The total fluorescence intensity in mice 6 hours after intravenous injection of mRNA-LNP compositions 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 was shown.

[0117] Figure 2 The fluorescence intensity in the mouse liver of the 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 is shown 6 hours after intravenous injection into mice.

[0118] Figure 3 The fluorescence intensity in the spleen of mice is shown 6 hours after intravenous injection of mRNA-LNP compositions 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 mice.

[0119] Figure 4 Flow cytometry analysis of DCs and macrophages in the spleen of mice 24 hours after intravenous injection of mRNA-LNP compositions encapsulating Fluc-mRNA prepared based on MC3, YK-1505, and YK-1507, as well as blank controls. DETAILED DESCRIPTION

[0120] In order to make the purpose, 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 them in other specific forms without departing from the basic attributes and purport of the present disclosure. It should be understood that, under the premise of no conflict, any and all embodiments of the present disclosure can be combined with the technical features in any other embodiment or multiple other embodiments to obtain another embodiment. The present disclosure includes other embodiments obtained by such a combination.

[0121] All publications and patents mentioned in this disclosure are hereby incorporated into the present disclosure in their entirety by reference. If the purposes or terms used in any publications and patents incorporated by reference conflict with the purposes or terms used in this disclosure, then the purposes and terms of this disclosure shall prevail.

[0122] The section headings used in this disclosure are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0123] Unless otherwise specified, all technical and scientific terms used in this disclosure have the common meanings in the field to which the claimed subject matter belongs. If there are multiple definitions for a term, the definition in this disclosure shall prevail.

[0124] Except in the working examples or otherwise noted, all numbers of quantitative properties such as dosage stated in the specification and claims should be understood to be modified by the term "about" in all cases. It should also be understood that any numerical range listed in the present disclosure is intended to include all subranges within the range and any combination of the respective endpoints of the range or subrange. When a numerical range is disclosed in the present disclosure, the above range is considered to be continuous and includes the minimum and maximum values ​​of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be merged. In other words, unless otherwise indicated, all ranges disclosed in the present disclosure should be understood to include any and all subranges included therein.

[0125] As used in this disclosure, words such as "include," "comprising," or "including" and the like mean that the elements preceding the word include the elements listed after the word and their equivalents, without excluding unlisted elements. The terms "include," "comprising," or "including" as used in this disclosure may be open, semi-closed, or closed. In other words, the above terms also encompass "consisting essentially of" or "consisting of."

[0126] The term "pharmaceutically acceptable" in this disclosure means that the compound or composition is chemically and / or toxicologically compatible with the other ingredients that make up the formulation and / or with the human or mammal for which it is used to prevent or treat a disease or condition.

[0127] The term "subject" or "patient" in the present disclosure includes mammals, such as common experimental animals (such as mice, rats, guinea pigs, rabbits, pigs, monkeys, etc.), and humans.

[0128] The term "treatment" as used in this disclosure refers to the administration of one or more pharmaceutical substances to a patient or subject suffering from a disease or symptoms of the disease in order to cure, alleviate, relieve, ameliorate or affect the disease or symptoms of the disease. In the context of this disclosure, unless specifically stated to the contrary, the term "treatment" may also include prevention.

[0129] The term "solvate" as used in this disclosure refers to a complex formed by the combination of a compound of formula (I) or a pharmaceutically acceptable salt thereof with a solvent (e.g., ethanol or water). It should be understood that any solvate of a compound of formula (I) used in the treatment of a disease or condition, although potentially providing different properties (including pharmacokinetic properties), will yield the compound of formula (I) once absorbed into a subject, such that use of a compound of formula (I) encompasses the use of any solvate of the compound of formula (I).

[0130] The term "hydrate" refers to the above-mentioned term "solvate" in which the solvent is water.

[0131] It should be further understood that the compound of formula (I) or its pharmaceutically acceptable salt can be isolated in the form of a solvate, and therefore any such solvate is included within the scope of this disclosure. For example, the compound of formula (I) or its pharmaceutically acceptable salt can exist in an unsolvated form or in a solvated form in combination with a pharmaceutically acceptable solvent (such as water, ethanol, etc.).

[0132] The term "pharmaceutically acceptable salt" refers to a relatively non-toxic, inorganic or organic acid addition salt of a compound of the present disclosure. For example, see SM Berge et al. "Pharmaceutical Salts", J. Pharm. Sci. 1977, 66, 1-19. Inorganic acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, or nitric acid; organic acids include formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, hexanoic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)-benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, 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 a pharmaceutically acceptable salt with the compound represented by formula (I).

[0133] The nitrogen-containing compounds of formula (I) disclosed herein can be converted into N-oxides by treatment with an oxidizing agent (e.g., m-chloroperbenzoic acid, hydrogen peroxide, ozone). Therefore, under conditions where valence and structure permit, the compounds claimed in the present disclosure include not only the nitrogen-containing compounds shown in the structural formula, but also their N-oxide derivatives.

[0134] Certain compounds disclosed herein may exist in the form of one or more stereoisomers. Stereoisomers include geometric isomers, diastereomers, and enantiomers. Therefore, the compounds claimed in the present disclosure also include racemic mixtures, single stereoisomers, and optically active mixtures. It should be understood by those skilled in the art 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 chiral source synthesis, chiral catalysis, chiral resolution, and the like. Racemates can be chirally resolved by chromatographic resolution or chemical resolution. For example, chiral acid resolution reagents such as chiral tartaric acid and chiral malic acid can be added to the compounds disclosed herein to form salts, and the physical and chemical properties of the products, such as different solubility, can be used for separation.

[0135] The present disclosure also includes all suitable isotopic variants of the disclosed compounds. An isotopic variant is defined as a compound in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually or predominantly found in nature. Examples of isotopes that can be introduced into the disclosed compounds 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.

[0136] The term "alkyl" in this disclosure refers to a monovalent saturated aliphatic hydrocarbon group including branched and straight chains with the specified number of carbon atoms. The term "alkylene" in this disclosure refers to a divalent saturated aliphatic hydrocarbon group including branched and straight chains with the specified number of carbon atoms. n-m It refers to a group having n to m carbon atoms. For example, C 2-5 The alkylene group includes C2 alkylene, C3 alkylene, C4 alkylene and C5 alkylene.

[0137] The alkyl (or alkylene) group may be unsubstituted, or the alkyl (or alkylene) group may be substituted, wherein at least one hydrogen is replaced by another chemical group. Useful substituent groups may include halogen, ester, cyano, sulfonyl, and the like.

[0138] A "therapeutically effective amount" is an amount of a therapeutic agent that, when administered to a patient, ameliorates a disease or symptom. A "prophylactically effective amount" is an amount of a prophylactic agent that, when administered to a subject, prevents a disease or symptom. The amount of a therapeutic agent that constitutes a "therapeutically effective amount" or the amount of a prophylactic agent that constitutes a "prophylactically effective amount" will vary depending on the therapeutic / prophylactic agent, the disease state and its severity, the age and weight of the patient / subject to be treated / prevented, etc. A person of ordinary skill in the art can routinely determine a therapeutically effective amount and a prophylactically effective amount based on their knowledge and this disclosure.

[0139] In the present disclosure, when the name of a compound is inconsistent with the structural formula, the structural formula shall prevail.

[0140] It should be understood that the term "compounds of the present disclosure" used in the present disclosure may include, depending on the context, compounds of formula (I), N-oxides thereof, solvates thereof, pharmaceutically acceptable salts thereof, stereoisomers thereof, and mixtures thereof.

[0141] As used in this disclosure, the term "cationic lipid" refers to a lipid that is positively charged at a selected pH value or range.

[0142] Cationic lipids can easily bind to negatively charged nucleic acids, that is, they interact with the negatively charged phosphate groups in nucleic acids through electrostatic forces to form lipid nanoparticles (LNPs).

[0143] The inventors discovered through research and screening of a large number of lipid compounds that it is very difficult to obtain a cationic lipid compound suitable for use as a nucleic acid drug carrier that simultaneously meets the following conditions: (1) structural differences from the cationic lipids currently used to load nucleic acid drugs in the prior art; (2) high transfection efficiency and low cytotoxicity; and (3) high and sustained expression in vivo.

[0144] During long-term research, the inventors unexpectedly discovered some compounds, such as YK-1503, YK-1504, YK-1505, YK-1507, YK-1510, YK-1511, YK-1513, YK-1515 and YK-1516 disclosed in the present invention, 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 cationic lipids in the prior art.

[0145] This disclosure is based on at least the following findings:

[0146] The cationic lipid compounds disclosed herein can be used to deliver nucleic acids, small molecules, polypeptides, or proteins. Compared to known cationic lipid compounds, the cationic lipid compounds disclosed herein exhibit higher transfection efficiency and lower cytotoxicity, with significantly increased expression in the spleen of animals, thereby improving delivery efficiency.

[0147] The second aspect of the present disclosure provides a composition comprising a carrier, wherein the carrier comprises a cationic lipid, and the cationic lipid comprises the above-mentioned compound of formula (I) or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer.

[0148] In one embodiment, the composition is a nanoparticle preparation, the average size of the nanoparticle preparation is 10 nm to 300 nm, preferably 40 nm to 240 nm; the polydispersity coefficient of the nanoparticle preparation is ≤0.5, preferably ≤0.4.

[0149] Cationic lipids

[0150] In one embodiment of the composition / carrier of the present disclosure, the cationic lipid is selected from one or more of the compounds of formula (I) or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer. In one embodiment, the cationic lipid is selected from the compounds of formula (I) above. For example, the cationic lipid is a compound. In a preferred embodiment, the cationic lipid is compound YK-1501 to YK-1518, and in a preferred embodiment, the cationic lipid is compound YK-1503, YK-1504, YK-1505, YK-1507, YK-1510, YK-1511, YK-1513, YK-1515 or YK-1516.

[0151] In another embodiment of the composition / carrier of the present disclosure, the cationic lipid comprises: (a) one or more compounds selected from the above-mentioned formula (I) compound or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer; (b) one or more other ionizable lipid compounds different from (a). Wherein, the cationic lipid compound (b) can be a commercially available cationic lipid or a cationic lipid compound reported in the literature. For example, the cationic lipid compound (b) can be SM-102 in CN201080026228.8, or MC3 in CN201080026228.8, or C16 in CN202380010167.3.

[0152] In one embodiment, the molar ratio of the cationic lipid to the carrier is 25%-75%, such as 30%, 40%, 50%, 55%, 60%, 65%, 70%.

[0153] The carrier can be used to deliver (pharmaceutical) 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.

[0154] For example, examples of the therapeutic / preventive agent can be one or more of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein. The nucleic acid includes, but is not limited to, single-stranded DNA, double-stranded DNA, and RNA. Suitable RNA includes, but is 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.

[0155] neutral lipids

[0156] The carrier may comprise a neutral lipid. A neutral lipid in the present disclosure refers to a lipid that exists in a non-charged form or in a neutral ionic form at a selected pH value or range. The neutral lipid may regulate the mobility of the nanoparticles by promoting lipid phase transition to form a lipid bilayer and improve efficiency, while also potentially affecting the specificity of the target organ.

[0157] In one embodiment, the molar ratio of the cationic lipid to the neutral lipid is about 1: 1-15: 1, for example, about 15: 1, 14: 1, 13: 1, 12: 1, 11: 1, 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3: 1, 2: 1 and 1: 1. For example, in a preferred embodiment, the molar ratio of the cationic lipid to the neutral lipid is about 4.9: 1.

[0158] For example, the neutral lipids may include one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterols, and derivatives thereof.

[0159] The carrier component of the composition comprising cationic lipids can include one or more neutral lipids-phospholipids, such as one or more (many) unsaturated lipids.Phospholipids can be assembled into one or more lipid bilayers.In general, phospholipids can include a phospholipid moiety and one or more fatty acid moieties.

[0160] Neutral lipids can be selected from the non-limiting group of free following composition: phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine and sphingomyelin. Fatty acid can be selected from the non-limiting group of free following composition: 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 contemplated are non-natural species including natural species with modification and replacement, including branching, oxidation, cyclization and alkynes. For example, phospholipids can be functionalized or cross-linked with one or more alkynes (e.g., one or more double bonds are replaced by a triple bond alkenyl) with one or more alkynes. Under appropriate reaction conditions, alkynyl may undergo copper-catalyzed cycloaddition reactions when exposed to azide. These reactions can be used to functionalize the lipid bilayer of the composition to facilitate membrane permeation or cellular recognition, or to couple the composition to useful components such as targeting or imaging moieties (eg, dyes).

[0161] Neutral lipids useful 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-Dicondecanoyl-sn-glycero-3-phosphocholine (DUPC), 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-Di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dialinolenoyl-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), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylglycerol (DPPG), Phosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE) and mixtures thereof.

[0162] 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.

[0163] Structured lipids

[0164] The carrier of the composition comprising cationic lipids may further comprise 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.

[0165] In one embodiment, the molar ratio of the cationic lipid to the structural lipid is about 1:1-5:1, e.g., 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.

[0166] The structured lipid can be selected from, but is not limited to, the group consisting of cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol, corticosteroids, and mixtures thereof. In some embodiments, the structured lipid is cholesterol. In some embodiments, the structured lipid includes cholesterol and corticosteroids (such as prednisolone, dexamethasone, prednisone, and hydrocortisone) or a combination thereof.

[0167] Polymer-conjugated lipids

[0168] The carrier of the composition comprising cationic lipids can also include one or more polymer conjugated lipids. Polymer conjugated lipids mainly refer to lipids modified by polyethylene glycol (PEG). Hydrophilic PEG stabilizes LNPs, regulates nanoparticle size by limiting lipid fusion, and increases the half-life of nanoparticles by reducing nonspecific interactions with macrophages.

[0169] 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 generally 350-5000 Da.

[0170] For example, the polymer-conjugated lipid is selected from one or more of the following: distearoylphosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), dimyristoylglycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000) and methoxy polyethylene glycol ditetradecanoyl acetamide (ALC-0159).

[0171] In one embodiment of the composition / vector of the present disclosure, the polymer-conjugated lipid is DMG-PEG2000.

[0172] In one embodiment of the composition / vector of the present disclosure, the vector comprises a neutral lipid, a structural lipid, and a polymer-conjugated lipid, wherein 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), for example (30-49):(7.5-15):(35-55):(1-5), more preferably (40-49):(8-12):(39-45):(1-3). The total molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is 100.

[0173] In one embodiment of the composition / vector of the present disclosure, the vector comprises a neutral lipid, a structural lipid and a polymer-conjugated lipid, 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.

[0174] Therapeutic and / or preventive agents

[0175] The composition can include one or more therapeutic and / or prophylactic agents. In one embodiment, the mass ratio of the carrier to the therapeutic or prophylactic agent is 10:1-30:1, for example, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1.

[0176] In one embodiment, the mass ratio of the carrier to the therapeutic agent or preventive agent is 12.5:1-20:1, preferably 13-17:1, and more preferably 15:1.

[0177] The therapeutic agent or preventive agent includes, but is not limited to, one or more of a nucleic acid molecule, a small molecule compound, a polypeptide or a protein.

[0178] For example, the therapeutic or prophylactic agent is a vaccine or a compound capable of eliciting an immune response.

[0179] The vectors disclosed herein can deliver therapeutic and / or prophylactic agents to mammalian cells or organs. Therefore, the present disclosure further provides methods for treating diseases or conditions in mammals in need thereof, comprising administering a composition comprising a therapeutic and / or prophylactic agent to the mammal and / or contacting the mammalian cells with the composition. Accordingly, the present disclosure provides the use of a compound disclosed herein, or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer, or a composition disclosed herein, in the preparation of a medicament for treating a disease or condition in a subject in need thereof.

[0180] The present disclosure also provides use of the compound of the present disclosure or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, or the composition of the present disclosure in the preparation of nucleic acid drugs, vaccines, chemical drugs, polypeptide drugs or protein drugs.

[0181] Therapeutic and / or prophylactic agents include biologically active substances and are alternatively referred to as "active agents." A therapeutic and / or prophylactic agent can be a substance that, upon delivery to a cell or organ, causes a desired change in that cell or organ, or in other body tissues or systems. Such species can be used to treat one or more diseases, disorders, or conditions. In some embodiments, a 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 that can be used in the composition include, but are not limited to, antineoplastic agents (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, and streptozotocin), antitumor agents (e.g., actinomycin D, vincristine, vinblastine, cytosine arabinoside, arabinoside), anthracyclines, alkylating agents, platinum compounds, antimetabolites, and nucleoside analogs, such as methotrexate and purine and pyrimidine analogs), anti-infectives, local anesthetics (e.g., dibucaine and chlorpromazine), beta-adrenergic blocking agents (e.g., propranolol, timolol, and labetalol), antihypertensives (e.g., clonidine and hydralazine), antidepressants (e.g., imipramine, amitriptyline, and doxepin), anticonvulsants (e.g., phenytoin), antihistamines

[0013] These agents include, but are not limited to, illicit drugs (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 medications, vitamins, sedatives, and imaging agents.

[0182] 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. Cytotoxins or cytotoxic agents include any agent that is detrimental 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 anthracenedione, daptomycin ... The invention relates to a novel radioactive ion. The radioactive ion includes, but is not limited to, iodine (e.g., iodine-125 or iodine-131), strontium-89, phosphorus, palladium, cesium, iridium, phosphate, cobalt, yttrium-90, samarium-153, and praseodymium. Examples of vaccines include compounds and formulations that can provide immunity against one or more conditions associated with infectious diseases such as influenza, measles, human papillomavirus (HPV), rabies, meningitis, pertussis, tetanus, plague, hepatitis, and tuberculosis, for example, may include mRNA encoding pathogenic antigens and / or their epitopes; vaccines may also include compounds and formulations that guide an immune response against cancer cells, for example, may include mRNA encoding tumor cell-derived antigens, epitopes, and / or new epitopes. Compounds that induce an immune response may include vaccines, corticosteroids (e.g., dexamethasone), and other species. In some embodiments, vaccines and / or compounds capable of eliciting an immune response are administered intramuscularly via a composition comprising a compound according to Formula (I), (IA), (IB), (II), (IIa), (IIb), (IIc), (IId), (IIe), (IIg), (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, razithromycin (CC-1065), melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, ...

[0014] Examples of the present invention include phosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP, cisplatin), anthracyclines such as daunorubicin (formerly daunomycin) and doxorubicin, antibiotics such as dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC), and antimitotic agents such as vincristine, vinblastine, taxol, and maytansines.

[0183] 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, interferon, heparin, hepatitis B surface antigen, typhoid vaccine, and cholera vaccine.

[0184] In some embodiments, the therapeutic agent is a polynucleotide or nucleic acid (such as ribonucleic acid or deoxyribonucleic acid). The broadest meaning of the term "polynucleotide" includes any compound and / or substance that is 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: deoxyribonucleic acid (DNA); ribonucleic acid (RNA), including messenger mRNA (mRNA), its hybrid; RNAi inducing factor; RNAi factor; siRNA; shRNA; miRNA; antisense RNA; ribozyme; catalytic DNA; RNA that induces triple helix formation; aptamer, etc. In some embodiments, the therapeutic agent and / or preventive agent is RNA. The RNA that can be used in the compositions and methods described in the present disclosure can be selected from, but is not limited to, the group consisting of: shortmer, antagomir, antisense RNA, ribozyme, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA) and mixtures thereof. In certain embodiments, the RNA is mRNA.

[0185] In certain embodiments, the therapeutic and / or preventive agent is mRNA. The mRNA can encode any polypeptide of interest, including any natural 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.

[0186] In other embodiments, therapeutic and / or preventive agent is siRNA. siRNA can selectively reduce the expression of a gene of interest or lower the expression of the gene. For example, the selection of siRNA can make the gene silencing relevant to a particular disease, disease or condition after the composition comprising the siRNA is administered to a subject in need. siRNA can comprise a sequence complementary to the mRNA sequence of a gene of interest or protein. In some embodiments, siRNA can be an immunomodulatory siRNA.

[0187] In certain embodiments, the therapeutic and / or prophylactic agent is an sgRNA and / or cas9 mRNA. The sgRNA and / or cas9 mRNA can be used as a gene editing tool. For example, the sgRNA-cas9 complex can affect the mRNA translation of a cellular gene.

[0188] In some embodiments, therapeutic and / or preventive agent is shRNA or its encoding vector or plasmid.shRNA can be produced inside target cell after appropriate construct is delivered to the nucleus.Construct and mechanism relevant to shRNA are well-known in the related art.

[0189] Disease or condition

[0190] The compositions / vectors disclosed herein can deliver therapeutic or prophylactic agents to a subject or patient. The therapeutic or prophylactic agents include, but are not limited to, one or more of nucleic acid molecules, small molecule compounds, polypeptides, or proteins. Therefore, the compositions disclosed herein can be used to prepare nucleic acid drugs, gene vaccines, small molecule drugs, polypeptide or protein drugs. Due to the wide variety of therapeutic or prophylactic agents described above, the compositions disclosed herein can be used to treat or prevent a variety of diseases or conditions.

[0191] In one embodiment, the disease or disorder is characterized by a malfunction or aberrant protein or polypeptide activity.

[0192] 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.

[0193] In one embodiment, the infectious disease is selected from the group consisting of diseases caused by coronavirus, influenza virus, or HIV virus, pediatric pneumonia, Rift Valley fever, yellow fever, rabies, and various herpes.

[0194] Other components

[0195] The composition may include one or more components in addition to those described in the preceding section.For example, the composition may include one or more hydrophobic small molecules, such as vitamins (eg, vitamin A or vitamin E) or sterols.

[0196] The composition may also include one or more permeability enhancing molecules, carbohydrates, polymers, surface modifiers, or other components. Permeability enhancing molecules may be, for example, molecules described in U.S. Patent Application Publication No. 2005 / 0222064. Carbohydrates may include simple sugars (e.g., glucose) and polysaccharides (e.g., glycogen and its derivatives and analogs).

[0197] Surface-altering agents may include, but are not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyldioctadecyl ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrins), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytics (e.g., acetylcysteine, artemisia, bromelain, papain, clerodendrum, bromhexine, carbocisteine, eprazinone), , mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin beta 4, streptococcal DNase alpha, neltenexine, and erdosteine) and DNA enzymes (e.g., rhDNA enzyme). The surface-altering agent can be disposed within and / or on the surface of the nanoparticles of the composition (e.g., by coating, adsorption, covalent attachment, or other methods).

[0198] The composition can also include one or more functionalized lipids. For example, the lipid can be functionalized with an alkynyl group, which may undergo a cycloaddition reaction when exposed to an azide under appropriate reaction conditions. Specifically, the lipid bilayer can be functionalized with one or more groups that effectively promote membrane penetration, cell recognition, or imaging in this way. The surface of the composition can also be coupled to one or more useful antibodies. Functional groups and conjugates that can be used for targeted cell delivery, imaging, and membrane penetration are well known in the art.

[0199] In addition to these components, the composition can include any substance that can be used in the pharmaceutical composition. For example, the composition can include one or more pharmaceutically acceptable excipients or auxiliary ingredients, such as but not limited to one or more solvents, dispersion media, diluents, dispersing aids, suspension aids, granulation aids, disintegrants, fillers, glidants, liquid vehicles, adhesives, surfactants, isotonic agents, thickeners or emulsifiers, buffers, lubricants, oils, preservatives, flavorings, 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, ARGennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006).

[0200] Examples of diluents can include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, dibasic calcium phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and / or combinations thereof.

[0201] In some embodiments, a composition comprising one or more lipids described herein may further comprise one or more adjuvants, such as glucopyranosyl lipid adjuvant (GLA), CpG oligodeoxyribonucleotides (e.g., class A or class B), poly(I:C), aluminum hydroxide, and Pam3CSK4.

[0202] Compositions of the present disclosure can be made into preparations in the form of solid, semisolid, liquid or gas, such as tablets, capsules, ointments, elixirs, syrups, solutions, emulsions, suspensions, injections, aerosols. Compositions of the present disclosure can be prepared by methods well known in the pharmaceutical field. For example, sterile injection solutions can be prepared by mixing the required amount of therapeutic agent or preventive agent with the required various other components mentioned above into a suitable solvent such as sterile distilled water, and then filter sterilizing. Surfactants can also be added to promote the formation of uniform solutions or suspensions.

[0203] 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.

[0204] The compositions of the present disclosure are administered in a therapeutically effective amount, which may vary not only with the specific agent selected, but also with the route of administration, the nature of the disease being treated, and the age and condition of the patient, and may ultimately be determined at the discretion of the attending physician or clinician. For example, a therapeutic or prophylactic agent may be administered to a subject (preferably a mammal, such as a human) at a dose of about 0.001 mg / kg to about 10 mg / kg.

[0205] Example

[0206] The present disclosure is further described below in conjunction with the 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 the different requirements of specific use. The implementation conditions not specified are conventional conditions in the industry. In the specific examples of the present disclosure, the raw materials used can be obtained commercially. Unless otherwise stated, all temperatures are given in degrees Celsius. The technical features involved in the various embodiments of the present disclosure can be combined with each other as long as they do not conflict with each other.

[0207] In the following embodiments, the abbreviations have the following meanings:

[0208] NaBH4: sodium borohydride; Boc2O: di-tert-butyl dicarbonate; DMAP: 4-dimethylaminopyridine; EDCI: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; HATU: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate; DIEA: N,N-diisopropylethylamine; DCM: dichloromethane; DMF: N,N-dimethylformamide; THF: tetrahydrofuran; MeOH: methanol

[0209] In the following examples, unless otherwise specified, all operations were performed at room temperature (25±5°C).

[0210] Example 1: Synthesis of cationic lipid compounds

[0211] 1.1 Synthesis of intermediate INT-1

[0212]

[0213]

[0214] Step 1: Synthesis of INT-1-PM1

[0215] (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, followed by the slow dropwise addition of di-tert-butyl dicarbonate (55.30 g, 253.40 mmol). The temperature was raised to 40°C for 24 h, and the reaction was monitored by TLC until the reaction was complete. Heating was discontinued, and saturated aqueous sodium bicarbonate was added to the reaction system to quench the reaction. The layers were separated, and the aqueous phase was extracted twice with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness under reduced pressure. The resulting residue was purified by silica gel column chromatography. The product was collected and concentrated to obtain INT-I-PM1 (9.10 g, 43.40 mmol, 63.3%). 16 ClNO3, MS(ES):m / z(M+H + )210.1.

[0216] Step 2: Synthesis of INT-1-PM2

[0217] Dissolve INT-I-PM1 (2.81 g, 13.40 mmol) in acetonitrile (40 mL), then add tetrahydropyrrole (1.14 g, 16.08 mmol), potassium carbonate (5.55 g, 40.20 mmol), and potassium iodide (0.44 g, 2.68 mmol) in that order. Heat to 70°C and allow to react for 8 h. Monitor the reaction by TLC until the reaction is complete. Remove from heat, filter, and concentrate the filtrate under reduced pressure. Purify the resulting residue by silica gel column chromatography. Collect and concentrate the product to obtain INT-I-PM2 (2.43 g, 9.94 mmol, 74.2%). 12 H 24 N2O3, MS(ES):m / z(M+H + )245.2.

[0218] Step 3: Synthesis of INT-1

[0219] INT-I-PM2 (2.43 g, 9.94 mmol) and a solution of hydrochloric acid in 1,4-dioxane (24 mL) were added and allowed to react at room temperature for 2 h. The reaction was monitored by LCMS until the reaction of the starting material was complete. The reaction solution was concentrated under reduced pressure, and the resulting residue was dissolved in methanol and concentrated under reduced pressure again. This was repeated three times to obtain INT-1 (2.05 g, crude product). 16 N2O, MS(ES):m / z(M+H + )145.2.

[0220] 1.2 Synthesis of intermediate INT-2

[0221]

[0222] Step 1: Synthesis of INT-2

[0223] 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 reaction is complete. Remove the heat and add saturated aqueous sodium bicarbonate to the reaction system to quench the reaction. Separate the layers, extract the aqueous phase once with n-hexane, 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%). 22 H 43 BrO2, MS(ES):m / z(M+H + )419.2.

[0224] 1.3 Synthesis of intermediate INT-3

[0225]

[0226] Step 1: Synthesis of INT-3

[0227] 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 reaction is complete. Remove heating and add saturated aqueous sodium bicarbonate to the reaction system to quench the reaction. Separate the layers, extract the aqueous phase once with n-hexane, 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%). 14 H 27 BrO2, MS(ES):m / z(M+H + )307.1.

[0228] 1.4 Synthesis of intermediate INT-4

[0229]

[0230] Step 1: Synthesis of INT-4

[0231] Dissolve heptadecan-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). The reaction was allowed to react at room temperature for 16 h, monitored by TLC until the starting material had reacted completely. Saturated aqueous sodium bicarbonate was added to the reaction system to quench the reaction. The layers were separated, and the aqueous phase was extracted once with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography. The product was collected and concentrated to yield INT-4 (8.00 g, 18.45 mmol, 94.6%). 23 H 45 BrO2, MS(ES):m / z(M+H + )433.2.

[0232] 1.5 Synthesis of intermediate INT-5

[0233]

[0234] Step 1: Synthesis of INT-5

[0235] 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 and react at 110°C for 8 h. Monitor the reaction by TLC until the reaction is complete. Remove heating and add saturated aqueous sodium bicarbonate to the reaction system to quench the reaction. Separate the layers, extract the aqueous phase once with n-hexane, combine the organic phases, dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain INT-5 (40.19 g, 89.80 mmol, 97.1%). 24 H 47 BrO2, MS(ES):m / z(M+H + )447.2.

[0236] 1.6 Synthesis of intermediate INT-6

[0237]

[0238] Step 1: Synthesis of INT-6

[0239] Under nitrogen protection, hexadecylamine (10.0 g, 41.41 mmol) and TEA (12.57 g, 124.24 mmol) were dissolved in DCM (100 ml) in sequence. The temperature was lowered to 0°C and a solution of acryloyl chloride (4.49 g, 49.69 mmol) in dichloromethane (40 ml) was added dropwise. After the addition was complete, the mixture was allowed to react at room temperature. TLC was used to monitor the reaction. After the reaction was complete, the system was cooled to 5°C and aqueous NaHCO₃ was added to quench the reaction. The aqueous phase was extracted twice with DCM, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness under reduced pressure. The resulting residue was purified by silica gel column chromatography. The product was collected and concentrated to obtain INT-6 (9.78 g, 33.09 mmol, 79.9%). 19 H 37 NO, MS(ES):m / z(M+H + )296.3.

[0240] 1.7 Synthesis of intermediate INT-7

[0241]

[0242]

[0243] Step 1: Synthesis of INT-7-PM1

[0244] 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 stirred overnight at room temperature. After completion of the reaction, saturated sodium bicarbonate aqueous solution was added, and the mixture was extracted with dichloromethane. The combined organic phases were washed with pure water and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure and purified by silica gel chromatography (0% to 50% ethyl acetate / n-hexane) to obtain the product INT-7-PM1 (44.00 g, 0.21 mol, 48.8%). 16 ClNO3, MS (ES): m / z (M + H + ) = 210.70.

[0245] Step 2: Synthesis of INT-7-PM2

[0246] 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. The reaction was stirred at 75°C and monitored by TLC. After completion, the reaction was diluted with purified water and extracted with ethyl acetate. The combined organic phases were washed with saturated sodium chloride solution and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure and purified by silica gel chromatography (0% to 20% methanol / dichloromethane) to obtain the product INT-7-PM2 (5.05 g, 19.78 mmol, 32.5%). 12 H 21 N3O3, MS (ES): m / z (M + H + ) = 256.05.

[0247] Step 3: Synthesis of INT-7

[0248] Dissolve INT-7-PM2 (5.00 g, 19.58 mmol) in a solution of hydrochloric acid in 1,4-dioxane (100 mL) and stir at room temperature overnight. After the reaction, the solution was spin-dried to dryness to obtain crude INT-7 (5.00 g) as a hydrochloride salt, which was used directly in the next step.

[0249] 1.8 Synthesis of YK-1501

[0250]

[0251]

[0252]

[0253] Step 1: Synthesis of YK-1501-PM1

[0254] Dissolve didecylamine (20.00 g, 67.21 mmol) in DMF (100 mL), add Boc-β-alanine (12.71 g, 67.21 mmol), HATU (38.33 g, 100.81 mmol), and DIEA (26.05 g, 201.63 mmol), and allow to react at room temperature for 3 h. Monitor the reaction by LCMS until the reaction is complete. Quench the reaction with water, extract twice with ethyl acetate, combine the organic phases, wash three times with saturated brine, dry over anhydrous sodium sulfate, filter, and decompress the filtrate into dryness. The resulting residue is purified by silica gel column chromatography. The product is collected and concentrated to obtain YK-1501-PM1 (23.40 g, 49.91 mmol, 74.2%). 28H 56 N2O3, MS(ES):m / z(M+H + )469.4.

[0255] Step 2: Synthesis of YK-1501-PM2

[0256] YK-1501-PM1 (23.40 g, 49.91 mol) and a solution of hydrochloric acid in 1,4-dioxane (120 mL) were added and allowed to react at room temperature for 2 h. The reaction was monitored by LCMS until the reaction of the starting material was complete. The reaction solution was concentrated under reduced pressure, and the resulting residue was dissolved in methanol and then concentrated under reduced pressure again. This was repeated three times to obtain YK-1501-PM2 (18.40 g, crude product). 23 H 48 N2O, MS(ES):m / z(M+H + )369.4.

[0257] Step 3: Synthesis of YK-1501-PM3

[0258] Under nitrogen, YK-1501-PM2 (7.70 g, 20.89 mmol) and TEA (6.34 g, 62.66 mmol) were dissolved in DCM (80 ml) in sequence. The temperature was lowered to 0°C and a solution of acryloyl chloride (2.27 g, 25.06 mmol) in dichloromethane (20 ml) was added dropwise. After the addition was complete, the mixture was allowed to react at room temperature. TLC was used to monitor the reaction. After the reaction was complete, the system was cooled to 5°C and aqueous NaHCO₃ was added to quench the reaction. The aqueous phase was extracted twice with DCM, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness under reduced pressure. The resulting residue was purified by silica gel column chromatography. The product was collected and concentrated to obtain YK-1501-PM3 (5.88 g, 13.91 mmol, 66.6%). 26 H 50 N2O2,MS(ES):m / z(M+H + )423.4.

[0259] Step 4: Synthesis of YK-1501

[0260] INT-1 (90 mg, 0.62 mmol) was dissolved in xylene (3 mL), followed by the addition of YK-1501-PM3 (659 mg, 1.56 mmol) and potassium carbonate (258 mg, 1.87 mmol). The reaction was heated to 90°C for 3 days, monitored by LCMS, until the reaction was complete. Heating was stopped, the mixture was filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography. The product was collected and concentrated to yield YK-1501 (35 mg, 0.03 mmol, 5.6%).

[0261] C 59 H116 N6O5, MS(ES):m / z(M+H + )989.6. 1 H 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).

[0262] 1.9 Synthesis of YK-1502

[0263]

[0264]

[0265] Step 1: Synthesis of YK-1502-PM1

[0266] Dissolve INT-1 (90 mg, 0.62 mmol) in xylene (3 mL), then add YK-1501-PM3 (237 mg, 0.56 mmol) and potassium carbonate (258 mg, 1.87 mmol) in that order. Heat to 90°C for 3 h. Monitor the reaction by LCMS until the starting materials have reacted completely. Remove from heat, filter, and concentrate the filtrate under reduced pressure. The resulting residue is purified by silica gel column chromatography. The product is collected and concentrated to yield YK-1502-PM1 (150 mg, 0.26 mmol, 42.3%). 33 H 66 N4O3, MS(ES):m / z(M+H + )567.5.

[0267] Step 2: Synthesis of YK-1502

[0268] YK-1502-PM1 (150 mg, 0.26 mmol) was dissolved in xylene (3 mL), followed by the addition of INT-6 (93 mg, 0.31 mmol) and potassium carbonate (109 mg, 0.79 mmol). The reaction was heated to 90°C for 3 days, monitored by LCMS, until the reaction was complete. Heating was stopped, the mixture was filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography. The product was collected and concentrated to yield YK-1502 (30 mg, 0.03 mmol, 13.1%).

[0269] C52 H 103 N5O4, MS(ES):m / z(M+H + )862.8. 1 H 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).

[0270] 1.10 Synthesis of YK-1503

[0271]

[0272]

[0273] Step 1: Synthesis of YK-1503-PM1

[0274] INT-1 (0.71 g, 4.92 mmol) was dissolved in acetonitrile (25 mL), followed by the addition of 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). The reaction was heated to 70°C for 7 h. LCMS was used to monitor the reaction until the starting materials had reacted completely. Heating was stopped, the mixture was filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography. The product was collected and concentrated to yield YK-1503-PM1 (0.70 g, 1.44 mmol, 29.4%). 29 H 58 N2O3, MS(ES):m / z(M+H + )483.4.

[0275] Step 2: Synthesis of YK-1503

[0276] YK-1503-PM1 (650 mg, 1.34 mmol) was dissolved in acetonitrile (10 mL). INT-3 (413 mg, 1.34 mmol), potassium carbonate (558 mg, 4.03 mmol), and potassium iodide (44 mg, 0.26 mmol) were added sequentially. The reaction was heated to 70°C for 8 h. The reaction was monitored by LCMS until the starting material had reacted completely. Heating was stopped, the mixture was filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography. The product was collected and concentrated to yield YK-1503 (390 mg, 0.54 mmol, 40.8%).

[0277] C 43 H 84 N2O3, MS(ES):m / z(M+H + )709.6. 1 H 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).

[0278] 1.11 Synthesis of YK-1504

[0279]

[0280] Step 1: Synthesis of YK-1504

[0281] INT-1 (0.71 g, 4.92 mmol) was dissolved in acetonitrile (25 mL), followed by the addition of 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). The reaction was heated to 70°C for 7 h. LCMS monitored the reaction until the starting material had reacted completely. Heating was stopped, the mixture was filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography. The product was collected and concentrated to yield YK-1504 (0.26 g, 0.31 mmol, 6.4%).

[0282] C 51 H 100 N2O5, MS(ES):m / z(M+H + )821.7.1 H 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).

[0283] 1.12 Synthesis of YK-1505

[0284]

[0285] Step 1: Synthesis of YK-1505

[0286] INT-1 (80 mg, 0.55 mmol) was dissolved in acetonitrile (7 mL), followed by the addition of INT-4 (480 mg, 1.10 mmol), potassium carbonate (229 mg, 1.66 mmol), and potassium iodide (18 mg, 0.11 mmol). The reaction was heated to 70°C for 8 h. LCMS was used to monitor the reaction until the starting material had reacted completely. Heating was stopped, the mixture was filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography. The product was collected and concentrated to yield YK-1505 (19 mg, 0.02 mmol, 4.0%).

[0287] 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).

[0288] 1.13 Synthesis of YK-1506

[0289]

[0290]

[0291] Step 1: Synthesis of YK-1506-PM1

[0292] INT-1 (0.96 g, 6.65 mmol) was dissolved in DMF (20 mL), followed by the addition of INT-5 (1.48 g, 3.32 mmol) and potassium carbonate (1.84 g, 13.31 mmol). The reaction was heated to 70°C for 7 h, monitored by LCMS until the reaction was complete. Heating was stopped, the mixture was filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography. The product was collected and concentrated to yield YK-1506-PM1 (0.24 g, 0.46 mmol, 7.0%). 31 H 62 N2O3, MS(ES):m / z(M+H + )511.4.

[0293] Step 2: Synthesis of YK-1506

[0294] YK-1506-PM1 (200 mg, 0.39 mmol) was dissolved in DMF (5 mL). INT-3 (180 mg, 0.58 mmol), potassium carbonate (162 mg, 1.17 mmol), and potassium iodide (64 mg, 0.39 mmol) were added sequentially. The reaction was heated to 70°C for 8 h. The reaction was monitored by LCMS until the starting material had reacted completely. Heating was stopped, the mixture was filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography. The product was collected and concentrated to yield YK-1506 (40 mg, 0.05 mmol, 13.8%).

[0295] C 45 H 88 N2O5, MS(ES):m / z(M+H + )737.6. 1H 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).

[0296] 1.14 Synthesis of YK-1507

[0297]

[0298] Step 1: Synthesis of YK-1507

[0299] INT-I (0.96 g, 6.65 mmol) was dissolved in DMF (20 mL), followed by the addition of INT-5 (1.48 g, 3.32 mmol) and potassium carbonate (1.84 g, 13.31 mmol). The reaction was heated to 70°C for 7 h, monitored by LCMS until the reaction of the starting materials was complete. Heating was stopped, the mixture was filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography. The product was collected and concentrated to yield YK-1507 (0.12 g, 0.13 mmol, 2.0%). 55 H 108 N2O5, 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).

[0300] 1.15 Synthesis of YK-1508

[0301]

[0302] Step 1: Synthesis of YK-1508

[0303] Dissolve INT-I (200 mg, 1.38 mmol) in DMF (20 mL), then add bromolinolene (456 mg, 1.38 mmol), potassium carbonate (383 mg, 2.77 mmol), and potassium iodide (46 mg, 0.27 mmol) in that order. Heat to 70°C and allow to react for 8 hours. Monitor the reaction by LCMS until the starting materials have reacted completely. Remove from heat, filter, and concentrate the filtrate under reduced pressure. The resulting residue is purified by silica gel column chromatography. The product is collected and concentrated to yield YK-1508 (80 mg, 0.12 mmol, 8.9%). 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 = 8.3Hz, 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).

[0304] 1.16 Synthesis of YK-1509

[0305]

[0306] Step 1: Synthesis of YK-1509-PM1

[0307] INT-7 (1.00 g, crude) and INT-2 (1.60 g, 3.90 mmol) were dissolved in acetonitrile (10 mL). Potassium carbonate (1.60 g, 11.70 mmol) and potassium iodide (130 mg, 0.80 mmol) were added at room temperature. The reaction was stirred at 75°C and monitored by LCMS. After completion, the reaction was filtered, and the filter cake was washed with a small amount of acetonitrile. The filtrate was dried and purified by silica gel chromatography (0% to 50% methanol / dichloromethane containing 10% aqueous ammonia) to obtain the product YK-1509-PM1 (200 mg, 0.41 mmol, two-step yield 10.5%). 29 H 55 N3O3, MS (ES): m / z (M + H +) = 494.80.

[0308] Step 2: Synthesis of YK-1509

[0309] YK-1509-PM1 (100 mg, 0.20 mmol) and INT-3 (75 mg, 0.24 mmol) were dissolved in acetonitrile (2 mL). Potassium carbonate (84 mg, 0.60 mmol) and potassium iodide (7 mg, 0.040 mmol) were added at room temperature. The reaction was stirred at 75°C and monitored by LCMS. After completion, the reaction was filtered, and the filter cake was washed with a small amount of acetonitrile. The filtrate was dried and purified by silica gel chromatography (0% to 50% methanol / dichloromethane containing 10% aqueous ammonia) to obtain the product YK-1509 (35 mg, 0.049 mmol, 24.3%). 43 H 81 N3O5, MS (ES): m / z (M + H + ) = 721.15.

[0310] 1 H 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).

[0311] 1.17 Synthesis of YK-1510

[0312]

[0313] Step 1: Synthesis of YK-1509-PM1

[0314] INT-7 (1.28 g, crude) and INT-2 (4.20 g, 10.02 mmol) were dissolved in acetonitrile (10 mL). Potassium carbonate (3.60 g, 26.05 mmol) and potassium iodide (172 mg, 1.04 mmol) were added at room temperature. The reaction was stirred at 75°C and monitored by LCMS. After completion, the reaction was filtered, and the filter cake was washed with a small amount of acetonitrile. The filtrate was dried and purified by silica gel chromatography (0% to 50% methanol / dichloromethane containing 10% aqueous ammonia) to obtain the product YK-1510 (550 mg, 0.66 mmol, two-step yield 13.2%). 51 H 97 N3O5, MS (ES): m / z (M + H + ) = 833.35.

[0315] 1 H NMR (400 MHz, CDCl3) δ 7.56 (d, 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).

[0316] 1.18 Synthesis of YK-1511

[0317]

[0318] Step 1: Synthesis of YK-1511

[0319] INT-7 (150 mg, crude) and INT-4 (512 mg, 1.18 mmol) were dissolved in acetonitrile (2 mL). Potassium carbonate (361 mg, 2.61 mmol) and potassium iodide (17.3 mg, 0.10 mmol) were added at room temperature. The reaction was stirred at 75°C and monitored by LCMS. After completion, the reaction was filtered, and the filter cake was washed with a small amount of acetonitrile. The filtrate was dried and purified by silica gel chromatography (0% to 50% methanol / dichloromethane containing 10% aqueous ammonia) to obtain the product YK-1511 (8 mg, 0.044 mmol, two-step yield 7.5%). 53 H 101N3O5, MS (ES): m / z (M + H + ) = 861.40.

[0320] 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).

[0321] 1.19 Synthesis of YK-1512

[0322]

[0323] Step 1: Synthesis of YK-1512-PM1

[0324] INT-7 (1.00 g, crude) and INT-5 (1.75 g, 3.92 mmol) were dissolved in DMF (10 mL). Potassium carbonate (1.60 g, 11.70 mmol) and potassium iodide (130 mg, 0.80 mmol) were added at room temperature. The reaction was stirred at 75°C and monitored by LCMS. After completion, the reaction was diluted with purified water and extracted with ethyl acetate. The combined organic phases were washed with saturated sodium chloride solution and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure and purified by silica gel chromatography (0% to 50% methanol / dichloromethane containing 10% aqueous ammonia) to obtain the product YK-1512-PM1 (300 mg, 0.57 mmol, two-step yield 14.5%). 31 H 59 N3O3, MS (ES): m / z (M + H + ) = 523.03.

[0325] Step 2: Synthesis of YK-1512

[0326] YK-1512-PM1 (150 mg, 0.29 mmol) and INT-3 (89 mg, 0.29 mmol) were dissolved in DMF (2 mL). Potassium carbonate (120 mg, 0.87 mmol) and potassium iodide (7 mg, 0.040 mmol) were added at room temperature. The reaction was stirred at 75°C and monitored by LCMS. After completion, the reaction was diluted with purified water and extracted with ethyl acetate. The combined organic phases were washed with saturated sodium chloride solution and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure and purified by silica gel chromatography (0% to 50% methanol / dichloromethane containing 10% aqueous ammonia) to yield the product YK-1512 (60 mg, 0.080 mmol, 27.6%). 45 H 85 N3O5,MS(ES): m / z (M + H + ) = 749.46.

[0327] 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).

[0328] 1.20 Synthesis of YK-1513

[0329]

[0330] Step 1: Synthesis of YK-1513

[0331] INT-7 (500 mg, crude) and INT-5 (1.75 g, 3.92 mmol) were dissolved in DMF (10 mL). Potassium carbonate (1.60 g, 11.70 mmol) and potassium iodide (130 mg, 0.80 mmol) were added at room temperature. The reaction was stirred at 75°C and monitored by LCMS. After completion, the reaction was diluted with purified water and extracted with ethyl acetate. The combined organic phases were washed with saturated sodium chloride solution and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure and purified by silica gel chromatography (0% to 50% methanol / dichloromethane containing 10% aqueous ammonia) to afford the product YK-1513 (100 mg, 0.11 mmol, 5.6% yield over two steps). 55 H105 N3O5,MS(ES): m / z (M + H + ) = 889.41.

[0332] 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).

[0333] 1.21 Synthesis of YK-1514

[0334]

[0335] Step 1: Synthesis of YK-1514

[0336] 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. The reaction was stirred at 75°C and monitored by LCMS. After completion, the reaction was diluted with purified water and extracted with ethyl acetate. The combined organic phases were washed with saturated sodium chloride solution and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure and purified by silica gel chromatography (0% to 50% methanol / dichloromethane containing 10% aqueous ammonia) to obtain the product YK-1514 (20 mg, 0.14 mmol, two-step yield 3.1%). 43 H 77 N3O,MS (ES): m / z(M + H + ) = 653.03.

[0337] 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).

[0338] 1.22 Synthesis of YK-1515

[0339]

[0340]

[0341]

[0342] Step 1: Synthesis of YK-1515-PM1

[0343] (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) were dissolved in dichloromethane (50.0 mL) and reacted at 40°C for 24 hours until the reaction was complete. The reaction solution was slowly added to saturated sodium bicarbonate aqueous solution and stirred for 10 minutes. The mixture was separated and extracted twice with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and dried by rotary evaporation. Wet-loading with n-hexane / ethyl acetate (0-18%) was performed and the product was dried by rotary evaporation to obtain YK-1515-PM1 (9.1 g, 43.40 mmol, 63.3%).

[0344] Step 2: Synthesis of YK-1515-PM2

[0345] 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) were dissolved in acetonitrile (40.0 mL) and heated to 70°C for 12 hours. The reaction was complete on a plate. The reaction was stopped, filtered, washed with dichloromethane, and dried. A small amount of dichloromethane was added for wet loading, with a dichloromethane / methanol ratio of 0-15%, and dried to yield YK-1515-PM2 (3.28 g, 12.00 mmol, 81.1%). 13 H 27 N3O3, MS(ES): m / z(M+H+ )274.2.

[0346] Step 3: Synthesis of YK-1515-PM3

[0347] Weigh YK-1515-PM2 (3.28 g, 12.00 mmol) and slowly add a 1,4-dioxane solution of hydrochloric acid (30 ml) at room temperature. Let the mixture react for 12 hours until the reaction is complete. Spin dry to obtain the crude product YK-1515-PM3 (3.05 g, crude product). 19 N3O, MS(ES): m / z(M+H + ) 174.2.

[0348] Step 4: Synthesis of YK-1515-PM4 and YK-1515

[0349] 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) were dissolved in acetonitrile (20.0 mL) and heated to 70°C for 12 hours. The reaction was complete on a plate. The reaction was stopped, filtered, washed with dichloromethane, and dried. A small amount of dichloromethane was added for wet loading (dichloromethane / methanol = 0-16%), and dried to yield YK-1515-PM4 (582 mg, 1.14 mmol, 28.7%). 30 H 61 N3O3, MS(ES): m / z(M+H + )512.2. Dichloromethane / methanol = 0-8%, spin-dried to give YK-1515 (370 mg, 0.44 mmol, 10.9%). C 52 H 103 N3O5, MS(ES): m / z(M+H + )850.4.

[0350] 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).

[0351] 1.23 Synthesis of YK-1516

[0352]

[0353] Step 1: Synthesis of YK-1516

[0354] 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) were dissolved in acetonitrile (5.0 mL) and heated to 70°C for 12 hours. The reaction was complete on a plate. The reaction was stopped, filtered, washed with dichloromethane, and dried. A small amount of dichloromethane was added for wet loading (dichloromethane / methanol = 0-39%), and dried to yield YK-1516 (55 mg, 0.06 mmol, 13.1%). 54 H 107 N3O5,MS(ES): m / z(M+H + )879.2.

[0355] 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).

[0356] 1.24 Synthesis of YK-1517

[0357]

[0358] Step 1: Synthesis of YK-1517

[0359] 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) were dissolved in acetonitrile (10.0 mL) and heated to 70°C for 8 hours. The reaction was complete on a plate. The reaction was stopped, filtered, washed with dichloromethane, and dried. A small amount of dichloromethane was added for wet loading (dichloromethane / methanol = 0-16%), and dried to yield YK-1517 (133 mg, 0.18 mmol, 18.0%). 44 H 87 N3O5,MS(ES): m / z(M+H + )738.2.

[0360] 1 H NMR (400 MHz, CDCl3) δ 4.08 (t, J = 6.8 Hz, 2H), 3.99 (d, J = 5.8 Hz,2H), 2.81 - 2.38 (m, 18H), 2.38 - 2.28 (m, 4H), 1.80 (m, J = 12.5, 5.4 Hz, 2H),1.72 - 1.58 (m, 5H), 1.51 (m, J = 13.4, 6.5 Hz, 3H), 1.31 (d, J = 14.3 Hz, 41H),0.91 (t, J = 6.6 Hz, 9H).

[0361] 1.25 Synthesis of YK-1518

[0362]

[0363] Step 1: Synthesis of YK-1518-PM1

[0364] 2-Hexyl-n-decanol (5.00 g, 20.62 mmol), N-Boc-6-aminocaproic acid (5.70 g, 24.64 mmol), EDCI (5.00 g, 26.60 mmol), and DMAP (756 mg, 6.22 mmol) were dissolved in dichloromethane (50.0 mL) and allowed to react at room temperature for 12 hours until the reaction was complete. Wet-cook the sample using a 0-50% hexane / ethyl acetate ratio and spin dry to obtain YK-1518-PM1 (8.6 g, 18.87 mmol, 91.5%). 27 H 53 NO4, MS(ES): m / z(M+H + ) 456.2.

[0365] Step 2: Synthesis of YK-1518-PM2

[0366] Weigh YK-1518-PM1 (8.60 g, 18.87 mmol) and slowly add HCl / dioxane (50 ml) at room temperature. Allow to react for 12 hours until the reaction is complete. Spin dry to obtain crude YK-1518-PM2 (6.50 g, 18.28 mmol, 96.8%). 22 H 45 NO2, MS(ES): m / z(M+H + ) 356.2.

[0367] Step 3: Synthesis of YK-1518-PM3

[0368] 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) were dissolved in acetonitrile (20.0 mL) and heated to 70°C for 12 hours. The reaction was complete on a plate. The reaction was stopped, filtered, washed with dichloromethane, and dried. A small amount of dichloromethane was added for wet loading (dichloromethane / methanol = 0-10%), and dried to yield YK-1518-PM3 (1.6 g, 2.31 mmol, 40.9%). 45 H 89 NO4, MS(ES): m / z(M+H + )694.3.

[0369] Step 4: Synthesis of YK-1518-PM4

[0370] Weigh CDI (555 mg, 3.44 mmol) as starting material and dissolve it in dichloromethane (10.0 mL). Slowly add YK-1518-PM3 (600 mg, 0.98 mmol) at 0°C. Return to room temperature and stir for 5 hours until the reaction is complete. Stop the reaction, wash three times with water, extract with dichloromethane, dry, filter, and spin dry. This yields YK-1518-PM4 (350 mg, 0.50 mmol, 51.4%). 48 H 89 N3O5, MS(ES): m / z(M+H + ) 788.6.

[0371] Step 5: Synthesis of YK-1518

[0372] YK-1518-PM4 (350 mg, 0.50 mmol), YK-1515-PM3 (182 mg, 1.24 mmol), and K2CO3 (207 mg, 1.50 mmol) were dissolved in acetonitrile (5.0 mL) and heated to 70°C for 12 hours. The reaction was complete on a plate. The sample was then filtered, washed with dichloromethane, and dried. A small amount of dichloromethane was added for wet loading, with a dichloromethane / methanol (10% ammonia solution) ratio of 0-10%, and dried to yield YK-1518 (15 mg, 0.02 mmol, 3.8%). 53 H 104 N4O6, MS(ES):m / z(M+H + ) 893.7.

[0373] 1 H NMR (400 MHz, CDCl3) δ 6.53 (s, 1H), 4.7 (d, J = 5.8 Hz, 4H), 3.23(s, 11H), 2.75 (s, 3H), 2.71 (d, J = 7.6 Hz, 2H), 2.43 (t, J = 7.3 Hz, 4H), 1.83(s, 5H), 1.75 (m, J = 24.2, 8.2 Hz, 8H), 1.42 (m, J = 14.6, 7.1 Hz, 4H), 1.25-1.34 (m, 49H), 0.92 (t, J = 6.7 Hz, 12H).

[0374] 1.26 Synthesis of C16

[0375] The synthesis procedure of C16 in CN 202380010167.3 was followed to obtain 44 mg of C16.

[0376] 1.27 Synthesis of 9322-O17S

[0377] According to the synthesis 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, 55 mg of 9322-O17S was synthesized.

[0378] 1.28. Synthesis of 76-017Se

[0379] According to the 76-O17Se synthesis method in In Vitro Engineering Chimeric Antigen Receptor Macrophages and TCells by Lipid Nanoparticle-Mediated mRNA Delivery, Zhongfei Ye et al., ACS Biomater Sci Eng., 2022 Feb 14;8(2):722-733, 38 mg of 76-O17Se was synthesized.

[0380] Example 2: mRNA-LNP formulation optimization

[0381] The specific operations of the cell transfection experiment used in this example include:

[0382] Step 1: Cell recovery and passaging: Recover Jurkat cells and culture them in culture dishes to the desired cell number.

[0383] Step 2: Plating: Digest and count the cells in the culture dish, plate 150,000 cells per well in a 12-well plate, and culture overnight until the cells adhere.

[0384] Step 3: Cell transfection: mRNA-LNP preparations encapsulated with eGFP-mRNA using different vectors were added to the cell culture medium of a 12-well plate (1.5 μg of mRNA-LNP preparation was added to each well). After continued culture for 24 hours, the transfection efficiency was assessed based on the fluorescence intensity under a fluorescence microscope.

[0385] 2.1 Optimization of the ratio of vector (liposome) to mRNA

[0386] Step 1: The cationic lipids YK-1503, YK-1504, YK-1505, and YK-1507 synthesized in Example 1 were dissolved in ethanol with DSPC (Aiweituo (Shanghai) Pharmaceutical Technology Co., Ltd.), cholesterol (Aiweituo (Shanghai) Pharmaceutical Technology Co., Ltd.), and DMG-PEG2000, respectively, at a molar ratio of cationic lipid:DSPC:cholesterol:DMG-PEG2000 of 49:10:39.5:1.5 to obtain solution A. Solution A was quickly added to citrate buffer (pH = 4.5 ± 0.5) by ethanol injection and vortexed for 30 seconds to obtain an ethanol lipid solution.

[0387] Step 2: Dilute eGFP-mRNA (Shanghai Qifa Experimental Reagent Co., Ltd.) in citrate buffer (pH = 4.5 ± 0.5) to obtain an eGFP-mRNA aqueous solution.

[0388] Step 3: Using a microfluidic device, the ethanolic lipid solution prepared in Step 1 was mixed with the mRNA aqueous solution prepared in Step 2 at a flow rate of 10 mL / min at a vector:mRNA mass ratio of 5:1, 10:1, 15:1, 20:1, 30:1, and 35:1 to prepare the corresponding liposome solutions. The liposome solutions were diluted to 10 times their volume with PBS and then ultrafiltered using a 300 kDa ultrafiltration tube to remove the ethanol. The liposome solutions were then adjusted to the appropriate volume with PBS and filtered through a 0.2 μm sterile filter to obtain mRNA-LNP formulations encapsulating eGFP-mRNA at a molar ratio of 49:10:39.5:1.5 for cationic lipid (YK-1503, YK-1504, YK-1505, or YK-1507), DSPC, cholesterol, and DMG-PEG2000.

[0389] The results of cell transfection experiments showed that when the mass ratio of vector to mRNA was in the range of 10:1-30:1, the corresponding mRNA-LNP compositions had good transfection effects, among which the best transfection effect was 15:1, while no appropriate transfection effect could be obtained when the mass ratio was 5:1 and 35:1.

[0390] 2.2 Optimization of the ratio of cationic lipids to neutral lipids

[0391] mRNA-LNP compositions encapsulating eGFP-mRNA were prepared similarly to the method in 2.1, wherein the molar ratios of cationic lipid (YK-1503, YK-1504, YK-1505, or YK-1507) to 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.

[0392] Cell transfection experiments showed that when the molar ratio of cationic lipid to neutral lipid was in the range of 1:1-15:1, the corresponding mRNA-LNP compositions were able to transfect cells, among which the highest transfection efficiency was 4.9:1.

[0393] 2.3 Optimization of the ratio of polymer-conjugated lipid to carrier

[0394] mRNA-LNP compositions encapsulating eGFP-mRNA were prepared according to a method similar to that in 2.1, wherein the cationic lipid was 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.

[0395] The results of cell transfection experiments showed that when the molar percentage of polymer-conjugated lipids in the carrier was in the range of 0.5%-10%, the corresponding mRNA-LNP compositions were able to transfect cells, with the highest transfection efficiency at 1.5% and the lowest at 10%.

[0396] 2.4 Optimization of the ratio of each component in the carrier

[0397] The mRNA-LNP formulation encapsulating eGFP-mRNA was prepared according to a method similar to that in 2.1. In step 1, the molar ratios of cationic lipid (YK-1503, YK-1504, YK-1505, or YK-1507), neutral lipid DSPC, structural lipid cholesterol, and 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.

[0398] Cell transfection experiments showed that cationic lipids, neutral lipids, structural lipids and polymer conjugated lipids can be transfected at molar ratios of 75:5:15:5, 65:8:25:2, 49:10:39.5:1.5, 45:10:43.5:1.5, 45:25:20:10, 40:10:48.5:1.5, 35:10:53.5:1.5 and 25:5:65:5. Good transfection effects were observed within the ratio range of (35-49):(7.5-15):(35-55):(1-5), and the best transfection effect was achieved when the molar ratio was 49:10:39.5:1.5.

[0399] Example 3: Cell transfection with mRNA-LNP formulation encapsulating eGFP-mRNA

[0400] The specific operations of cell transfection used in this embodiment include:

[0401] Step 1: Cell recovery and passaging: Recover Jurkat cells and culture them in culture dishes to the desired cell number.

[0402] Step 2: Plating: Digest and count the cells in the culture dish, plate 150,000 cells per well in a 12-well plate, and culture overnight until the cells adhere.

[0403] Step 3: Cell transfection: 1.5 μg of the mRNA-LNP preparation encapsulating eGFP-mRNA prepared in Example 2 (wherein the cationic lipids were YK-1503, YK-1504, YK-1505, or YK-1507, respectively) was added to the cell culture medium of a 12-well plate. After further culture for 24 hours, the transfection efficiency was assessed by fluorescence intensity under a fluorescence microscope.

[0404] Based on the transfection efficiency results, an mRNA-LNP formulation with the following ratios was selected for the following examples: the mass ratio of vector to mRNA was 15:1; the molar ratio of cationic lipid to neutral lipid was 4.9:1; the molar ratio of polymer-conjugated lipid to liposome was 1.5%; and the molar ratio of cationic lipid, neutral lipid, structural lipid, and polymer-conjugated lipid was 49:10:39.5:1.5.

[0405] Example 4: Preparation of mRNA-LNP formulation

[0406] Table 1 Cationic lipid compounds

[0407] 4.1

[0409] The corresponding ethanol lipid solutions of the cationic lipids in Table 1 were prepared according to the method in step 1 of Example 2.1. 4.2

[0411] eGFP-mRNA (Shanghai Qifa Experimental Reagent Co., Ltd.) or Fluc-mRNA (Shanghai Qifa Experimental Reagent Co., Ltd.) was diluted in citrate buffer (pH = 4.5 ± 0.5) to obtain the corresponding mRNA aqueous solution. 4.3

[0413] Using a microfluidic device, mix the ethanolic lipid solution obtained in 4.1 with the aqueous eGFP-mRNA or Fluc mRNA solution obtained in 4.2 at a volume ratio of 1:3 at a flow rate of 10 mL / min. Prepare the corresponding liposome solution with a mass ratio of carrier (liposome) to mRNA of approximately 15:1. Dilute the liposome solution to 10 times its volume with PBS and ultrafilter using a 300 kDa ultrafiltration tube to remove the ethanol. Then, adjust the volume to the appropriate volume with PBS and filter through a 0.2 μm sterile filter to obtain mRNA-LNP formulations encapsulating eGFP-mRNA or Fluc-mRNA, respectively, with a molar ratio of cationic lipid: DSPC: cholesterol: DMG-PEG2000 of 49:10:39.5:1.5.

[0414] Example 5: Determination of mRNA-LNP Particle Size, Polydispersity Index (PDI) and Encapsulation Efficiency

[0415] The particle size and polydispersity index (PDI) were determined using a Malvern laser particle size analyzer using dynamic light scattering.

[0416] Take 10 μL of the mRNA-LNP solution prepared in Example 4, dilute it to 1 mL with RNase-free deionized water, and add it to the sample tank. Repeat the measurement three times for each sample. The measurement conditions are: 90° scattering angle, 25°C;

[0417] The LNP encapsulation efficiency was determined using the Quant-iT™ RiboGreen® RNA Quantification Assay Kit (ThermoFisher Scientific, UK) according to the manufacturer's instructions. The results are shown in Table 2:

[0418] Table 2 Particle size, polydispersity index (PDI), and encapsulation efficiency of mRNA-LNP

[0419]

[0420] As shown in Table 2, the nanolipid particles prepared in Example 4 had a particle size between 70 and 90 nm, making them suitable for mRNA delivery. The polydispersity coefficients were all less than 0.15, indicating good particle size uniformity. Furthermore, they exhibited high encapsulation efficiencies, exceeding 90%.

[0421] Example 6: In vitro ( in vitro ) Delivery performance and toxicity

[0422] The cell recovery, passaging and plating methods refer to steps 1 and 2 of Example 3.

[0423] The 96-well plate containing Jurkat cells obtained in step 2 was supplemented with an appropriate volume of Jurkat cell culture medium. The mRNA-LNP formulation (prepared in Example 4) containing 0.3 μg of Fluc-mRNA was then added to the 96-well plate. After a further 24 hours of incubation, the corresponding reagents were added according to the instructions of the Gaussia Luciferase Assay Kit (Thermo Fisher). The relative fluorescence intensity of each well was measured using an IVIS fluorescence detection system. Finally, 10 μL of CCK-8 solution was added to each well of the plate after 24 hours of incubation. The plate was incubated in an incubator for 1 hour, and the absorbance at 450 nm was measured using a microplate reader to determine cell viability. The results of relative fluorescence intensity and cell viability are shown in Table 3.

[0424] Table 3 Fluorescence detection results of Fluc-mRNA

[0425]

[0426] The relative fluorescence intensities of the mRNA-LNP compositions (which can reflect the translation efficiency of mRNA) are significantly different. The relative fluorescence intensities of 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 are significantly higher than those of the mRNA-LNP compositions prepared from SM-102, MC3, 9322-O17S, 76-017Se, and C16. Specifically:

[0427] 1. The mRNA-LNP compositions prepared using YK-1503, YK-1504, YK-1505, YK-1507, YK-1510, YK-1511, YK-1513, YK-1515, and YK-1516 significantly improve cell transfection efficiency compared to representative cationic lipids used in the prior art. For example, the cell transfection efficiency of YK-1507 is 2.8 times that of SM-102 and 6.0 times that of MC3.

[0428] 2. The mRNA-LNP compositions prepared with YK-1503, YK-1504, YK-1505, and YK-1507 showed significantly improved cell transfection efficiency compared to the 76-017Se cationic lipid, which also has a tetrahydropyrrole head structure. For example, the cell transfection efficiency of YK-1507 was 4.9 times that of 76-017Se.

[0429] 3. The cell transfection efficiency of mRNA-LNP compositions prepared with YK-1510, YK-1511, and YK-1513 was significantly improved compared to the 9322-O17S cationic lipid, which also has a 2-methylimidazole head structure. For example, the cell transfection efficiency of YK-1513 was 4.6 times that of 9322-O17S.

[0430] 4. The cell transfection efficiency of mRNA-LNP compositions prepared with YK-1515 and YK-1516 was significantly improved compared to C16 cationic lipids, which also have a piperazine head structure. For example, the cell transfection efficiency of YK-1516 was 1.8 times that of C16.

[0431] Example 7: In vivo ( in vivo ) Delivery performance

[0432] The Fluc-mRNA-LNP composition prepared in Example 4 was injected into female BALB / c albino mice aged 4-6 weeks and weighing 17-19 g via the tail vein (at a dosage of approximately 5 μg Fluc-mRNA / mouse), and 6 hours after administration, the fluorescent 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 in the mice by the mRNA carried by the mRNA-LNP composition was detected by IVIS Spectrum small animal in vivo imaging (corresponding to the fluorescent protein expression intensity, i.e., protein expression amount). After sampling, the mice were killed by cervical dislocation and dissected to accurately separate the liver and spleen of the mice. The total radiation intensity of the protein expressed by Fluc-mRNA in various organs of the mice was detected by IVIS Spectrum small animal in vivo imaging (corresponding to the fluorescent protein expression intensity, i.e., protein expression amount). The results of mouse in vivo imaging and protein expression detection in the liver and spleen are shown in Tables 4 and Figure 1-3 .

[0433] Table 4 Mouse in vivo and organ imaging experimental data

[0434]

[0435] 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 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 by the prior art ionizable cationic lipids (SM-102, MC3, 9322-O17S, 76-017Se, C16 and YK-301), the mRNA-LNP compositions prepared by the present disclosure YK-1503, YK-1504, YK-1505, YK-1507, YK-1510, YK-1511, YK-1513, YK-1515 and YK-1516 have significantly enhanced spleen total radiation intensity and in vivo total radiation intensity, for example For example, the mRNA-LNP composition prepared by YK-1507 had a total radiation intensity in vivo that was 1.8 times, 2.2 times, 2.2 times, 2.4 times and 1.9 times that of the mRNA-LNP compositions prepared by SM-102, MC3, 9322-O17S, 76-017Se and C16, respectively, and the total radiation intensity in the spleen was 4.0 times, 5.1 times, 2.6 times, 2.7 times and 2.7 times that of the mRNA-LNP compositions prepared by SM-102, MC3, 9322-O17S, 76-017Se and C16, respectively.

[0436] Example 8: Targeting of mRNA-LNP Composition to Mouse Splenocytes

[0437] 1. The eGFP-mRNA compositions containing different cationic lipids prepared in Example 4 were injected into female C57BL / 6 mice aged 4-6 weeks and weighing 17-19 g via the tail vein (approximately 20 μg eGFP-mRNA per mouse). 24 hours after administration, the mice were killed by cervical dislocation and then dissected to accurately separate the spleens.

[0438] 2. Preparation of single cells

[0439] 1) Grind the separated spleen tissue to make it single-celled and pass it through a cell sieve.

[0440] 2) Add 10 volumes (approximately 4 mL) of red blood cell lysis buffer to lyse and remove red blood cells from the tissue.

[0441] 3) Count the cells and take 5×10 6 Transfer cells to flow cytometry tubes (ensure that the number of cells taken for each sample is consistent).

[0442] 3. Detection of immune cells in spleen tissue

[0443] 1) Add 100 μL of surface antibody MIX (see Table 5 for surface antibody MIX components) to each single-cell suspension and incubate at room temperature in the dark for 15 minutes (one negative control).

[0444] Table 5 Reagents and sources of surface antibody MIX used in mouse spleen cell flow cytometry experiments

[0445]

[0446] 2) Add 2 mL of PBS, centrifuge at 500 g for 5 minutes, and discard the supernatant.

[0447] 3) Resuspend the cells in 200 μL of PBS (filter through a 200-mesh nylon mesh), and analyze them on a Cytoflex S flow cytometer. Analyze the percentage of GFP expression in each cell line. The assay sequence for each cell line is as follows:

[0448] T cell GFP ratio:CD45 + →CD3 + →GFP +

[0449] B cell GFP ratio:CD45 + →CD3 - CD19 + →GFP +

[0450] DC cell GFP ratio:CD45 + →CD11c + →GFP +

[0451] Macrophage GFP ratio:CD45 + →F4 / 80 + →GFP +

[0452] Mice injected with an equal volume of 0.9% sodium chloride solution were set as the blank control group.

[0453] The percentage of eGFP-positive cells in mouse spleen cells is shown in Tables 6 and Figure 4 .

[0454] Table 6 Percentage of eGFP-positive cells in mouse spleen cells (%)

[0455]

[0456] From the data in Table 6 and Figure 4It can be seen that the mRNA-LNP compositions prepared with the cationic lipids disclosed herein can significantly increase the ratio of immune cells (T cells, B cells, DC cells, and macrophages) expressing antigens in the spleen, compared to mRNA-LNP compositions prepared with SM-102 and MC3. For example, the mRNA-LNPs prepared with YK-1507 increased the ratio of eGFP-positive T cells, B cells, DC cells, and macrophages in the spleen by 1.8-fold, 1.5-fold, 2.4-fold, and 5.0-fold, respectively, compared to the mRNA-LNPs prepared with MC3.

[0457] The preferred embodiments of the present disclosure are described in detail above, but the present disclosure is not limited thereto. Within the technical concept of the present disclosure, various simple variations of the technical solution of the present disclosure may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as the contents of the present disclosure and fall within the scope of protection of the present disclosure.

Claims

1. A cationic lipid compound or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the cationic lipid compound has any one of the following structures: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 and 。 2. A carrier, wherein The carrier comprises a cationic lipid, wherein the cationic lipid comprises the cationic lipid compound according to claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof.

3. The carrier according to claim 2, wherein The molar percentage of the cationic lipid in the carrier is 25%-75%.

4. The carrier according to claim 2, wherein The carrier further comprises a neutral lipid; and / or, the carrier further comprises a structured lipid; And / or, the carrier further comprises a polymer-conjugated lipid.

5. The carrier according to claim 4, 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 structured lipid is selected from any one or a combination of at least two of the group consisting of: cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol and corticosteroids; 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.

6. The carrier according to claim 5, 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-diondecanoyl-sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, n-Glycerol-3-phosphocholine, 1,2-di-O-octadecenyl-sn-glycerol-3-phosphocholine, 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycerol-3-phosphocholine, 1-hexadecyl-sn-glycerol-3-phosphocholine, 1,2-dialinolenoyl-sn-glycerol-3-phosphocholine, 1,2-diarachidonoyl-sn-glycerol-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycerol-3-phosphocholine, 1,2-dioleoyl-sn-glycerol -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-bisdocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine rac-(1-glycerol) sodium salt, dipalmitoylphosphatidylglycerol, palmitoyloleoylphosphatidylethanolamine, distearoyl-phosphatidyl-ethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphoethanolamine, 1-stearoyl-2-oleoyl-stearoylethanolamine, 1-stearoyl-2-oleoyl-phosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine.

7. The carrier according to claim 5, wherein The neutral lipid is selected from 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine and / or 1,2-distearoyl-sn-glycero-3-phosphocholine.

8. The carrier according to claim 5, wherein The structural lipid is cholesterol.

9. The carrier according to claim 5, wherein The polymer-conjugated lipid is selected from any one or a combination of at least two of the group consisting of: distearoylphosphatidylethanolamine polyethylene glycol 2000, dimyristoylglycerol-3-methoxy polyethylene glycol 2000 and methoxy polyethylene glycol ditetradecanoyl acetamide.

10. The carrier according to claim 4, 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%.

11. The carrier according to claim 10, wherein In the carrier, the molar ratio of cationic lipid to 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.

12. The vector according to any one of claims 2 to 11, 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).

13. The carrier according to claim 12, 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):(35-55):(1-5).

14. The carrier according to claim 13, 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.

15. The carrier according to claim 2, wherein The cationic lipids may also include one or more other cationic lipid compounds.

16. A composition, wherein The composition comprises an active ingredient and a carrier, and the carrier is the carrier according to any one of claims 2 to 15.

17. The composition according to claim 16, wherein The composition is a nanoparticle preparation, the average particle size of the nanoparticle preparation is 10nm-300nm; and the polydispersity coefficient of the nanoparticle preparation is ≤0.

5.

18. The composition according to claim 17, wherein The average particle size of the nanoparticle preparation is 40 nm-240 nm; and the polydispersity coefficient of the nanoparticle preparation is ≤0.

4.

19. The composition according to claim 16, wherein The active ingredient comprises a therapeutic or prophylactic agent.

20. The composition according to claim 19, wherein The mass ratio of the carrier to the therapeutic agent or preventive agent is 10:1-30:

1.

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

1.

22. The composition according to claim 21, wherein The mass ratio of the carrier to the therapeutic agent or preventive agent is 13:1-17:

1.

23. The composition of any one of claims 19-22, wherein the therapeutic or prophylactic agent is a vaccine or a compound capable of eliciting an immune response.

24. The composition according to claim 23, wherein The therapeutic agent or preventive agent is selected from any one of the group consisting of nucleic acids, small molecule compounds, polypeptides or proteins, or a combination of at least two of them.

25. The composition according to claim 24, wherein The therapeutic or preventive agent is a nucleic acid.

26. The composition according to claim 25, wherein The therapeutic agent or preventive agent is ribonucleic acid.

27. The composition according to claim 26, wherein The ribonucleic acid is selected from any one or a combination of at least two of the following groups: small interfering RNA, asymmetric interfering RNA, microRNA, Dicer-substrate RNA, small hairpin RNA, and messenger RNA.

28. The composition according to claim 27, wherein The ribonucleic acid is messenger RNA.

29. The composition according to claim 16, wherein The composition further comprises a pharmaceutically acceptable excipient and / or diluent.

30. Use of the compound of claim 1 or a pharmaceutically acceptable salt or stereoisomer thereof, or the carrier of any one of claims 2 to 15, or the composition of any one of claims 16 to 29 in the preparation of a medicament.

31. The use according to claim 30, wherein The active component of the drug is selected from any one of the group consisting of nucleic acids, small molecule compounds, polypeptides or proteins, or a combination of at least two of them.

32. Use of the compound of claim 1 or a pharmaceutically acceptable salt or stereoisomer thereof, or the vector of any one of claims 2 to 15, in improving cell transfection efficiency and / or reducing cytotoxicity, wherein the use is non-therapeutic and non-diagnostic.

33. The use according to claim 32, wherein The use is to improve cell transfection efficiency and / or reduce cytotoxicity in vitro.

34. Use of the compound of claim 1 or its pharmaceutically acceptable salt or stereoisomer, or the vector of any one of claims 2 to 15, in improving the targeting of a nucleic acid to a target, and / or in increasing the expression level of a nucleic acid in a 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, and the use is non-therapeutic and non-diagnostic.

35. The use according to claim 34, 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 cells, NK cells, DC cells, T cells and macrophages.

36. The use according to claim 35, 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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