Poly tertiary amine multi-tailed chain ionizable cationic lipids, compositions comprising the same, and uses thereof

Abstract

The present disclosure belongs to the field of medicine, more specifically, relates to the technical field of lipid delivery, and discloses a multi-tertiary amine multi-tail chain ionizable cationic lipid, a composition containing the same and use. The multi-tertiary amine multi-tail chain ionizable cationic lipid provided by the present disclosure comprises a structure as shown in formula (I), can be used for nucleic acid targeted delivery, and can significantly enhance the off-target prevention effect of an mRNA vaccine or drug.

Description

Technical Field

[0001] The present disclosure belongs to the field of medicine, and more specifically, to the field of lipid delivery technology, and in particular to poly-tertiary amine poly-tail chain ionizable cationic lipids, compositions containing the same, and uses thereof. Background Art

[0002] 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).

[0003] Lipid nanoparticles (LNPs) typically contain four main components: ionizable lipids, helper lipids, PEG-lipids, and cholesterol. As transport vehicles, LNPs can effectively deliver bioactive substances such as small molecule drugs, peptides, proteins, and nucleic acids to cells and / or intracellular compartments. Among the four components, 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, their safety, efficacy, and specificity remain to be improved. Furthermore, many LNPs tend to accumulate in specific organs, particularly the liver, raising concerns about their potential hepatotoxicity.

[0004] Recent studies have designed ionizable lipids for muscle-selective mRNA delivery, thereby reducing off-target accumulation in organs such as the liver. Despite this, existing muscle-selective ionizable lipids still exhibit high levels of off-target organ accumulation. Therefore, research is needed to develop new lipid compounds with improved muscle selectivity and reduced off-target organ accumulation, thereby improving the efficacy and safety of LNPs prepared from them and meeting the stringent standards required for medical applications. Summary of the Invention

[0005] The present disclosure provides polytertiary amine and polytail ionizable cationic lipids, compositions containing the same, and uses thereof. The ionizable cationic lipids provided herein can be used to deliver therapeutic and / or preventive agents such as nucleic acid molecules, small molecule compounds, polypeptides, or proteins. The preparation method is simple, the targeting is strong, the off-target accumulation in organs is low, the active pharmaceutical ingredient can be carried to transfect cells with high transfection efficiency, and the lipids have low cytotoxicity, which can improve delivery efficiency and safety.

[0006] The technical solutions provided by the present disclosure may include, for example:

[0007] [1] An (ionizable cationic lipid) compound or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, wherein the compound has the structure represented by formula (I):

[0008] ,

[0009] in:

[0010] R1 is a substituted or unsubstituted N-containing heterocycle, or a disubstituted amino group;

[0011] R2 is substituted or unsubstituted C 5-20 Straight-chain or branched olefins, or substituted or unsubstituted C 5-20 straight-chain or branched-chain alkanes;

[0012] R3 is substituted or unsubstituted C 5-20 Straight-chain or branched olefins, or substituted or unsubstituted C 5-20 Straight-chain or branched alkane, or H, or omitted;

[0013] R4 is substituted or unsubstituted C 5-20 straight-chain or branched-chain alkanes;

[0014] L1 is unsubstituted C 2-10 Straight chain alkylene, or -C(O)(CH2) n -, n is an integer from 2 to 10;

[0015] L2 and L3 are each independently unsubstituted C 2-10 Straight chain alkylene, or absent;

[0016] M1 is -CH=CH-, -C(O)O- or -OC(O)-;

[0017] M2 is -CH=CH-, -C(O)O-, or is absent;

[0018] M3 is -OC(O)-;

[0019] M4 is -OC(O)-, or is omitted;

[0020] When L2 and M2 are both defaulted, L3 and / or M4 are also defaulted.

[0021] The technical solution [1] provided in the present disclosure includes a compound of formula (I) and any N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof.

[0022] In some preferred embodiments, the stereoisomers of the compound of formula (I) do not include the compound having the structure shown in formula (II):

[0023]

[0024] The inventors have found that although LNPs prepared from the (completely) achiral compound of formula (II) also have good targeting, low off-target accumulation in organs, are not prone to organ toxicity, and have low cytotoxicity, the expression level of the nucleic acid they carry at the target location (e.g., in the muscle tissue at the injection site) is low.

[0025] In the compounds of formula (I) provided herein, "default" means that the corresponding group does not exist at that position. For example, if M4 is absent, L3 is directly connected to R4.

[0026] The "substituted groups" (e.g., substituted N-containing heterocycles, substituted C 5-20 Straight-chain or branched olefins, substituted C 5-20 Straight-chain or branched alkanes, etc.) refer to groups formed by replacing H in a group with other elements or groups (commonly referred to as "substituents"). When a substituted group is used at a certain position, the substituent contained in the group can be selected from any one or a combination of at least two of the following groups: halogen, hydroxyl, amino, mercapto, etc. The present disclosure does not specifically limit the number and position of substituents in the substituted group. For example, if R2 is substituted C 10 The straight-chain alkyl group means that in the compound of formula (I), R2 may be a group obtained by replacing at least one H on any one or more C groups with the above-mentioned substituents.

[0027] In the compound of formula (I) provided by the present disclosure, R1 may be a substituted N-containing heterocycle, an unsubstituted N-containing heterocycle, or a disubstituted amino group.

[0028] An "N-containing heterocycle" refers to a cyclic structural group whose backbone is composed of carbon atoms and non-carbon atoms (also known as "heteroatoms," such as N, S, O, and P), wherein at least one heteroatom is nitrogen. For example, the N-containing heterocycle may contain one, two, or three nitrogen atoms. Preferably, the N-containing heterocycle contains no heteroatoms other than nitrogen. Preferably, the total number of carbon atoms and heteroatoms in the backbone structure of the N-containing heterocycle may be 3-8, for example, 3, 4, 5, 6, 7, or 8.

[0029] "Disubstituted amino" means that both H in the amino group (-NH2) are replaced by a substituent. For example, a disubstituted amino group can be formed by replacing both H in the amino group by any one selected from the group consisting of halogen, C 1-5 Straight or branched chain alkyl, C 1-5 In a disubstituted amino group, the two substituents may be the same or different.

[0030] In the compounds of formula (I) provided herein, the number of carbon atoms of R2 can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or can be a range consisting of any two of the above values, or any integer intermediate value in the range.

[0031] In the compounds of formula (I) provided herein, when R3 is not H or is not absent, the number of carbon atoms in R3 can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or can be a range consisting of any two of the above values, or any integer intermediate value in the range.

[0032] In the compounds of formula (I) provided herein, the number of carbon atoms of R4 can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or can be a range consisting of any two of the above values, or any integer intermediate value in the range.

[0033] In the compounds of formula (I) provided by the present disclosure, R2, R3 and R4 may be the same or different.

[0034] In the compound of formula (I) provided by the present disclosure, if L1 is unsubstituted C 2-10 The number of carbon atoms in the straight-chain alkylene group can be 2, 3, 4, 5, 6, 7, 8, 9, or 10, or can be a range consisting of any two of the above values, or any integer intermediate value in the range.

[0035] In the compound of formula (I) provided by the present disclosure, if L1 is -C(O)(CH2) n -, n can be 2, 3, 4, 5, 6, 7, 8, 9, 10, or a range consisting of any two of the above values, or any integer intermediate value in the range.

[0036] In the compounds of formula (I) provided by the present disclosure, if L2 and / or L3 are not missing, the number of carbon atoms thereof can be independently 2, 3, 4, 5, 6, 7, 8, 9, 10, or can be a range consisting of any two of the above values, or any integer intermediate value in the range.

[0037] In the compounds of formula (I) provided by the present disclosure, L1, L2 and L3 may be the same or different.

[0038] In the compounds of formula (I) provided by the present disclosure, M1, M2, M3 and M4 may be the same or different.

[0039] [2] The compound according to [1], or its N-oxide compound, solvate, pharmaceutically acceptable salt or stereoisomer, wherein R2 is unsubstituted C 5-15 Straight-chain alkanes, or unsubstituted C 10-20 Branched alkyl, or unsubstituted C 5-10 straight-chain alkenyl;

[0040] and / or, R3 is unsubstituted C 5-20 Straight-chain alkanes, or unsubstituted C 15-20 Branched alkyl, or unsubstituted C 5-10 straight-chain alkenyl, or H;

[0041] and / or, R4 is unsubstituted C 5-15 Straight-chain alkanes, or unsubstituted C 10-20 Branched chain alkyl.

[0042] [3] The compound according to [1] or [2], or its N-oxide compound, solvate, pharmaceutically acceptable salt or stereoisomer, wherein R1 is 、 ,or ;

[0043] and / or, R2 is 、 , or unsubstituted C 8-11 straight-chain alkanes;

[0044] and / or, R3 is 、 、 , unsubstituted C 8-11 straight-chain alkanes, or H;

[0045] and / or, R4 is , or unsubstituted C 8-10 straight-chain alkanes;

[0046] and / or, L1 is -C(O)(CH2)5-, -(CH2)3-, or -(CH2)8-;

[0047] and / or, L2 is -(CH2)5-, -(CH2)8-, or is absent;

[0048] And / or, L3 is -(CH2)5-, or is omitted.

[0049] [4] The compound according to any one of [1] to [3], or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound has the structure of any one of the following (YK-1601 to YK-1618):

[0050] 、

[0051] 、

[0052] 、

[0053] 、

[0054] 、

[0055] 、

[0056] 、

[0057] 、

[0058] 、

[0059] 、

[0060] 、

[0061] 、

[0062] 、

[0063] 、

[0064] 、

[0065] 、

[0066] and

[0067] .

[0068] In some particularly preferred embodiments, the compound has the structure of any one of YK-1603, YK-1604, YK-1605, YK-1606, YK-1610, YK-1612, YK-1613, YK-1615, and YK-1618.

[0069] [5] A carrier comprising a cationic lipid provided by the compound of any one of [1] to [4], or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof.

[0070] [6] The vector according to [5], wherein the vector further comprises a neutral lipid;

[0071] and / or, the carrier further comprises a structured lipid;

[0072] and / or, the carrier further comprises a polymer-conjugated lipid;

[0073] And / or, the carrier further comprises one or more other ionizable lipid compounds.

[0074] In the carriers provided herein, "other ionizable lipid compounds" refers to any type of ionizable lipid compound other than the cationic lipid provided by the compound described in any one of [1] to [4], or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer. Any ionizable lipid compound that can be used in lipid delivery systems in the art can be used in the carriers provided herein. The present disclosure does not particularly limit the source of other ionizable lipid compounds, which can be obtained, for example, from commercial sources or prepared according to existing techniques.

[0075] In some preferred embodiments, the other ionizable lipid compound may be an ionizable cationic lipid compound and / or an ionizable anionic lipid compound.

[0076] [7] The carrier according to [5] or [6], wherein the molar percentage of the cationic lipid in the carrier is 25%-75%;

[0077] and / or, in the carrier, the molar percentage of neutral lipid in the carrier is 5%-25%;

[0078] and / or, in the carrier, the molar percentage of the structural lipid in the carrier is 15%-65%;

[0079] And / or, in the carrier, the molar percentage of the polymer-conjugated lipid in the carrier is 0.5%-10%.

[0080] In the present disclosure, the molar percentage of a lipid in a carrier refers to the percentage of the molar number of the lipid in the carrier, based on the molar number of all lipids in the carrier. For example, if the total molar number of lipids in the carrier is 100 mol and the content of the cationic lipid is 30 mol, the molar percentage of the cationic lipid in the carrier is 30%.

[0081] For example, the molar percentage of the cationic lipid in the carrier can be 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or a range consisting of any two of the above values, or any intermediate value in the range.

[0082] For example, the molar percentage of neutral lipids in the carrier can be 5%, 8%, 10%, 12%, 15%, 18%, 20%, 22%, 25%, or a range consisting of any two of the above values, or any intermediate value in the range.

[0083] For example, in the carrier, the molar percentage of the structural lipid can be 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or a range consisting of any two of the above values, or any intermediate value in the range.

[0084] For example, in the carrier, the molar percentage of the polymer-conjugated lipid can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.5%, 3%, 3.5%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or it can be a range consisting of any two of the above values, or any intermediate value in the range.

[0085] In some preferred embodiments, the molar percentage of the polymer-conjugated lipid in the carrier may be 0.5-2.5%.

[0086] In a particularly preferred embodiment, the molar percentage of the polymer-conjugated lipid in the carrier may be 1.5%.

[0087] [8] The carrier according to any one of [5] to [7], wherein the molar ratio of the cationic lipid to the neutral lipid in the carrier is 1:1-15:1;

[0088] and / or, in the carrier, the molar ratio of the cationic lipid to the structural lipid is 0.5:1-3:1;

[0089] And / or, in the carrier, the molar ratio of the cationic lipid to the polymer-conjugated lipid is 4:1-35:1.

[0090] For example, in the carrier, the molar ratio of the cationic lipid to the neutral lipid can be 1:1, 2:1, 2.5:1, 3:1, 3.2:1, 3.4:1, 3.6:1, 3.8:1, 4:1, 4.2:1, 4.4:1, 4.6:1, 4.8:1, 5:1, 5.5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, or a range consisting of any two of the above ratios, or any intermediate ratio within the range. Preferably, the molar ratio of the cationic lipid to the neutral lipid is 4-5:1.

[0091] For example, in the carrier, the molar ratio of the cationic lipid to the structural lipid can be 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.2:1, 1.4:1, 1.6:1, 1.8:1, 2:1, 2.5:1, 3:1, or a range consisting of any two of the above ratios, or any intermediate ratio within the range.

[0092] For example, in the carrier, the molar ratio of the cationic lipid to the polymer conjugated lipid can be 4:1, 4.1:1, 4.2:1, 4.3:1, 4.4:1, 4.5:1, 4.6:1, 4.7:1, 4.8:1, 4.9:1, 5:1, 5.5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1 1, 31:1, 32:1, 32.1:1, 32.2:1, 32.3:1, 32.4:1, 32.5:1, 32.6:1, 32.7:1, 32.8:1, 32.9:1, 33:1, 33.5:1, 34:1, 34.5:1, 35:1, or a range consisting of any two of the above ratios, or any intermediate ratio in the range.

[0093] [9] The carrier according to any one of [5] to [8], 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).

[0094]

[10] The carrier according to any one of [5] to [9], wherein the molar ratio of cationic lipid, neutral lipid, structural lipid and polymer conjugated lipid in the carrier is (35-49):(7.5-15):(35-55):(1-5).

[0095] In a particularly preferred embodiment, 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.

[0096]

[11] The carrier according to any one of [5] to

[10] , wherein the neutral lipid is any one selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol and their derivatives, or a combination of at least two thereof;

[0097] 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;

[0098] 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.

[0099]

[12] The carrier according to any one of [5] to

[11] , 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, Choline, 1-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-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2 -Dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-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;

[0100] and / or, the structured lipid is cholesterol;

[0101] And / or, 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.

[0102]

[13] The carrier according to any one of [5] to

[12] , wherein the neutral lipid is selected from 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine and / or 1,2-distearoyl-sn-glycero-3-phosphocholine.

[0103]

[14] A composition comprising a carrier and an active ingredient, wherein the carrier includes the carrier described in any one of [5] to

[13] .

[0104]

[15] The composition according to

[14] , wherein the composition is a nanoparticle preparation, the average particle size of the nanoparticle preparation is 10 nm-300 nm; and the polydispersity index (PDI) of the nanoparticle preparation is ≤0.5.

[0105] For example, the average particle size of the nanoparticle preparation can be 10nm, 15nm, 20nm, 25nm, 30nm, 35nm, 40nm, 45nm, 50nm, 60nm, 70nm, 80nm, 81nm, 82nm, 83nm, 84nm, 85nm, 86nm, 87nm, 88nm, 89nm, 90nm, 91nm, 92nm, 93nm, 94nm, 95nm, 96nm, 97nm, 98nm, 99nm, 100nm, 110nm, 120nm, 130nm, 140nm, 150nm, 160nm, 170nm, 180nm, 190nm, 200nm, 250nm, 300nm, or a range consisting of any two of the above values, or any intermediate value in the range.

[0106] For example, the PDI of the nanoparticle formulation can be 0.0001, 0.001, 0.01, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5, or can be a range consisting of any two of the above values, or any intermediate value in the range.

[0107] In the present disclosure, parameters such as the average particle size and polydispersity index of the nanoparticle preparation have the general meanings in the art. For example, the average particle size refers to the average value of the particle sizes of all nanoparticles in a certain amount of nanoparticle preparation; the polydispersity index (PDI) refers to the ratio of the standard deviation of the particle sizes of all nanoparticles in a certain amount of nanoparticle preparation to the average particle size, which is used to characterize the uniformity of the size distribution of the nanoparticles. The smaller the PDI, the narrower the size distribution of the nanoparticles.

[0108] In the present disclosure, parameters such as the average particle size and polydispersity index of nanoparticle preparations can be measured using conventional methods in the art. For example, a laser particle size analyzer (diluted to a concentration of approximately 0.02 mg / mL of the active ingredient (e.g., mRNA)) can be used to analyze the nanoparticle preparation, and the corresponding parameter test results can be directly read using the instrument or its supporting software. Testing conditions can refer to those commonly used in the art, such as room temperature (e.g., 25°C) and a scattering angle of 90°.

[0109]

[16] The composition according to

[14] or

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

[0110]

[17] The composition according to any one of

[14] to

[16] , wherein the active ingredient comprises a therapeutic agent and / or a preventive agent.

[0111]

[18] The composition according to any one of

[14] to

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

[0112]

[19] The composition according to any one of

[14] to

[18] , wherein the therapeutic agent and / or preventive agent is a vaccine or compound capable of eliciting an immune response.

[0113]

[20] The composition according to any one of

[14] to

[19] , wherein the therapeutic agent and / or preventive agent is a nucleic acid.

[0114]

[21] The composition according to any one of

[14] to

[20] , wherein the therapeutic agent and / or preventive agent is RNA.

[0115]

[22] The composition according to any one of

[14] -

[21] , wherein the RNA 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.

[0116]

[23] The composition according to any one of

[14] -

[22] , wherein the RNA is messenger RNA.

[0117]

[24] The composition according to any one of

[14] to

[23] , wherein the mass ratio of the carrier to the active ingredient in the composition is 10:1-30:1.

[0118] For example, in the composition, the mass ratio of the carrier to the active ingredient can be 10:1, 10.5:1, 11:1, 11.5:1, 12:1, 12.5:1, 13:1, 13.5:1, 14:1, 14.5:1, 15:1, 15.5:1, 16:1, 16.5:1, 17:1, 17.5:1, 18:1, 18.5:1, 19:1, 19.5:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, or 30:1, or it can be a range consisting of any two of the above ratios, or any intermediate ratio in the range.

[0119]

[25] The composition according to any one of

[14] to

[24] , wherein the mass ratio of the carrier to the active ingredient in the composition is 12.5:1-20:1.

[0120]

[26] The composition according to any one of

[14] -

[25] , wherein the mass ratio of the carrier to the active ingredient in the composition is 13:1-17:1.

[0121]

[27] The composition according to any one of

[14] to

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

[0122]

[28] Use of the compound described in any one of [1] to [4] or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, or the vector described in any one of [5] to

[13] , or the composition described in any one of

[14] to

[27] in improving cell transfection efficiency.

[0123] Transfection efficiency refers to the ratio of the number of cells transfected with the desired substance (e.g., active ingredient) to the total number of cells during cell transfection. In other words, the use of the disclosed compounds, or their N-oxides, solvates, pharmaceutically acceptable salts, or stereoisomers; or carriers; or compositions, enables the active ingredient (e.g., those described in the aforementioned technical solutions) to enter a greater number of cells.

[0124]

[29] The use according to

[28] , wherein the use is for improving cell transfection efficiency in vitro.

[0125] For example, the use may include increasing the proportion of transfected cells in all experimental cells in an in vitro cell transfection experiment.

[0126]

[30] Use of the compound or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer described in any one of [1] to [4], or the vector described in any one of [5] to

[13] , or the composition described in any one of

[14] to

[27] for improving the targeting of a drug to 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.

[0127] In the present disclosure, improving the targeting of a drug to a target means increasing the amount of the administered drug that enters the target, that is, increasing the ratio of the amount of the drug that enters the target to the amount of the drug that enters non-target locations, or decreasing the ratio of the amount of the drug that enters non-target locations to the amount of the drug that enters the target. Preferably, the drug includes at least the active ingredient in the aforementioned composition.

[0128]

[31] The use according to

[30] , wherein the target tissue is selected from muscle;

[0129] And / or, the target cells are selected from muscle cells.

[0130] The inventors have discovered that when a lipid composition is used to encapsulate a pharmaceutically active ingredient to form a pharmaceutical composition, if the compound of the present disclosure is introduced into the lipid composition, the targeting of the pharmaceutical composition to muscle tissue can be effectively improved, so that the pharmaceutical composition is retained more at the administration site (such as the injection site), while the amount of the pharmaceutical composition that enters the internal organs (such as the heart, liver, spleen, lungs, kidneys, etc., especially the liver and spleen) is greatly reduced, thereby effectively avoiding the side effects or organ toxicity of the drug.

[0131]

[32] Use of the compound or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer described in any one of [1] to [4], or the vector described in any one of [5] to

[13] , or the composition described in any one of

[14] to

[27] for increasing the ratio of the expression level of a nucleic acid in a target to the expression level in a non-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.

[0132] In this disclosure, increasing the ratio of nucleic acid expression in a target to non-target sites refers to the ratio of the amount of nucleic acid that enters and is expressed at the target to the amount of nucleic acid that enters and is expressed at non-target sites. Non-target sites are any locations other than the target site (e.g., any other tissues, organs, etc.) after administration of the nucleic acid to a subject. Preferred non-target sites include any one of the group consisting of the heart, liver, spleen, lungs, and kidneys, or a combination of at least two. More preferably, they include the liver and / or spleen.

[0133]

[33] The use according to

[32] , wherein the target tissue is selected from muscle;

[0134] And / or, the target cells are selected from muscle cells.

[0135]

[34] Use of the compound of any one of [1] to [4] or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, or the carrier of any one of [5] to

[13] , or the composition of any one of

[14] to

[27] in reducing the organ toxicity of a drug.

[0136] In the present disclosure, reducing the organ toxicity of a drug refers to reducing the adverse effects on non-target organs caused by the drug's off-target accumulation in non-target organs. Preferably, the drug comprises at least the active ingredient in the aforementioned composition.

[0137] Preferably, the organ toxicity of the drug includes cardiotoxicity, hepatotoxicity, splenoretoxicity, pulmonary toxicity and nephrotoxicity, etc., that is, the side effects caused by the drug entering the above organs off-target.

[0138] Preferably, the organ toxicity of the drug is selected from hepatotoxicity and / or splenoretoxicity, more preferably hepatotoxicity.

[0139]

[35] Use of the compound of any one of [1] to [4] or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, or the carrier of any one of [5] to

[13] , or the composition of any one of

[14] to

[27] in the preparation of a medicament.

[0140]

[36] The use according to

[35] , 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.

[0141]

[37] The use according to

[35] or

[36] , wherein accumulation of the active ingredient of the drug in an organ leads to organ toxicity.

[0142]

[38] The use according to any one of

[35] to

[37] , wherein the organ is selected from any one or a combination of at least two of the group consisting of heart, liver, spleen, lung and kidney.

[0143]

[39] Use of a compound or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer as described in any one of [1] to [4], or a vector as described in any one of [5] to

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

[14] to

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

[0144]

[40] The use according to

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

[0145] That is, the disease or disorder may be caused by malfunction or abnormality of a protein or polypeptide.

[0146]

[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, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.

[0147]

[42] The use according to any one of

[39] -

[41] , wherein the subject is a mammal.

[0148] For example, the subject can be a human, a companion animal (such as a cat, a dog, etc.), a livestock animal (such as a cow, a horse, a pig, etc.), an experimental animal (such as a rat, a mouse, a rabbit, a monkey, etc.), etc.

[0149] Preferably, the subject is a human.

[0150]

[43] The use according to any one of

[39] to

[42] , wherein the drug is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally or by inhalation, preferably intramuscularly and / or subcutaneously. For example, the drug can be administered by intramuscular / subcutaneous injection.

[0151]

[44] The use according to any one of

[39] to

[43] , wherein the amount of the drug administered is such that a dose of the active ingredient (such as a therapeutic agent and / or prophylactic agent) of about 0.001 mg / kg body weight to about 10 mg / kg body weight is administered to the subject.

[0152] For example, the amount of drug administered is such that the active ingredient is administered to a subject at a dose of about 0.001 mg / kg body weight, about 0.005 mg / kg body weight, about 0.008 mg / kg body weight, about 0.01 mg / kg body weight, about 0.05 mg / kg body weight, about 0.08 mg / kg body weight, about 0.1 mg / kg body weight, about 0.5 mg / kg body weight, about 1 mg / kg body weight, about 1.5 mg / kg body weight, about 2 mg / kg body weight, about 2.5 mg / kg body weight, about 3 mg / kg body weight , about 3.5 mg / kg body weight, about 4 mg / kg body weight, about 4.5 mg / kg body weight, about 5 mg / kg body weight, about 5.5 mg / kg body weight, about 6 mg / kg body weight, about 6.5 mg / kg body weight, about 7 mg / kg body weight, about 7.5 mg / kg body weight, about 8 mg / kg body weight, about 8.5 mg / kg body weight, about 9 mg / kg body weight, about 9.5 mg / kg body weight, about 10 mg / kg body weight, or it can be a range consisting of any two of the above values, or any intermediate value in the range.

[0153] Furthermore, the present disclosure also provides the following technical solutions:

[0154]

[45] A method for improving cell transfection efficiency, the method comprising:

[0155] i. encapsulating the substance to be transfected with a carrier comprising the compound described in any one of [1] to [4] or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer and then performing cell transfection, or

[0156] ii. using the vector described in any one of [5]-

[13] to encapsulate the substance to be transfected and then perform cell transfection, or

[0157] iii. Using the composition described in any one of

[14] -

[27] for cell transfection.

[0158] In the methods provided herein, the substance to be transfected is the substance to be transfected and introduced into cells, and can be any active ingredient known in the art for cell transfection. For example, it can be a nucleic acid, a protein, a peptide, a small molecule compound, etc. The selection of the substance to be transfected can also refer to the active component of the composition described above and will not be repeated here.

[0159]

[46] The method according to

[45] , wherein the method is a method for improving cell transfection efficiency in vitro.

[0160]

[47] A method for cell transfection, comprising:

[0161] i. encapsulating the substance to be transfected using a carrier comprising the compound described in any one of [1]-[4] or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer; or

[0162] ii. using a vector as described in any one of [5]-

[13] to encapsulate the substance to be transfected, or

[0163] iii. Provide the composition described in any one of

[14] -

[27] .

[0164]

[48] ​​The method according to

[47] , wherein the method further comprises a step of performing cell transfection after the step of encapsulating the substance to be transfected or providing the composition. In the method disclosed herein, any cell transfection method using liposome transduction known in the art can be used.

[0165]

[49] The method according to

[47] or

[48] , wherein the method is an in vitro cell transfection method.

[0166]

[50] A method for improving the targeting of a drug to 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;

[0167] The method includes:

[0168] i. encapsulating the active ingredient of the drug using a carrier comprising the compound described in any one of [1] to [4] or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, or

[0169] ii. using a carrier encapsulated with the active ingredient of the drug described in any one of [5]-

[13] , or

[0170] iii. Provide the composition of any one of

[14] -

[27] , wherein the active ingredient in the composition comprises the active ingredient of the drug.

[0171]

[51] The method according to

[50] , wherein the target tissue is selected from muscle;

[0172] And / or, the target cells are selected from muscle cells.

[0173]

[52] A method for increasing the expression of a drug at a target, wherein the target is selected from any one of the group consisting of a target organ, a target tissue, and a target cell, or a combination of at least two thereof;

[0174] The method includes:

[0175] i. encapsulating the active ingredient of the drug using a carrier comprising the compound described in any one of [1] to [4] or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, or

[0176] ii. using a carrier encapsulated with the active ingredient of the drug described in any one of [5]-

[13] , or

[0177] iii. Provide the composition of any one of

[14] -

[27] , wherein the active ingredient in the composition comprises the active ingredient of the drug.

[0178]

[53] The method according to

[51] , wherein the target tissue is selected from muscle;

[0179] And / or, the target cells are selected from muscle cells.

[0180]

[54] A method for reducing organ toxicity / side effects of a drug, the method comprising:

[0181] i. encapsulating the active ingredient of the drug using a carrier comprising the compound described in any one of [1] to [4] or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, or

[0182] ii. using a carrier encapsulated with the active ingredient of the drug described in any one of [5]-

[13] , or

[0183] iii. Provide the composition of any one of

[14] -

[27] , wherein the active ingredient in the composition comprises the active ingredient of the drug.

[0184] Preferably, the medicine comprises at least the active ingredient in the aforementioned composition.

[0185] Preferably, the organ toxicity of the drug includes cardiotoxicity, hepatotoxicity, splenoretoxicity, pulmonary toxicity and nephrotoxicity, etc., that is, the side effects caused by the drug entering the above organs off-target.

[0186] Preferably, the organ toxicity of the drug is selected from hepatotoxicity and / or splenoretoxicity, more preferably hepatotoxicity.

[0187]

[55] A method of treating a disease or condition in a subject in need thereof, the method comprising:

[0188] (1) Provide medicines, including:

[0189] i. encapsulating the active ingredient of the drug using a carrier comprising the compound described in any one of [1] to [4] or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, or

[0190] ii. using a carrier encapsulated with the active ingredient of the drug described in any one of [5]-

[13] , or

[0191] iii. providing a composition according to any one of

[14] -

[27] , wherein the active ingredient in the composition comprises an active ingredient of the drug;

[0192] (2) administering the drug to a subject in a therapeutically effective amount.

[0193]

[56] The method according to

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

[0194] That is, the disease or disorder may be caused by malfunction or abnormality of a protein or polypeptide.

[0195]

[57] The method according to

[55] or

[56] , wherein the disease or condition is selected from any one or a combination of at least two of the following groups: infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.

[0196]

[58] The method according to any one of

[55] -

[57] , wherein the subject is a mammal.

[0197] For example, the subject can be a human, a companion animal (such as a cat, a dog, etc.), a livestock animal (such as a cow, a horse, a pig, etc.), an experimental animal (such as a rat, a mouse, a rabbit, a monkey, etc.), etc.

[0198] Preferably, the subject is a human.

[0199]

[59] The method according to any one of

[55] to

[58] , wherein the drug is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally or by inhalation, preferably intramuscularly and / or subcutaneously. For example, the drug can be administered by intramuscular injection.

[0200]

[60] The method of any one of

[55] -

[59] , wherein the amount of the drug administered is such that a dose of the active ingredient (e.g., therapeutic and / or prophylactic agent) of about 0.001 mg / kg body weight to about 10 mg / kg body weight is administered to the subject.

[0201] For example, the amount of drug administered is such that the active ingredient is administered to a subject at a dose of about 0.001 mg / kg body weight, about 0.005 mg / kg body weight, about 0.008 mg / kg body weight, about 0.01 mg / kg body weight, about 0.05 mg / kg body weight, about 0.08 mg / kg body weight, about 0.1 mg / kg body weight, about 0.5 mg / kg body weight, about 1 mg / kg body weight, about 1.5 mg / kg body weight, about 2 mg / kg body weight, about 2.5 mg / kg body weight, about 3 mg / kg body weight , about 3.5 mg / kg body weight, about 4 mg / kg body weight, about 4.5 mg / kg body weight, about 5 mg / kg body weight, about 5.5 mg / kg body weight, about 6 mg / kg body weight, about 6.5 mg / kg body weight, about 7 mg / kg body weight, about 7.5 mg / kg body weight, about 8 mg / kg body weight, about 8.5 mg / kg body weight, about 9 mg / kg body weight, about 9.5 mg / kg body weight, about 10 mg / kg body weight, or it can be a range consisting of any two of the above values, or any intermediate value in the range.

[0202] It should be understood that the above-mentioned uses and methods provided by the present disclosure may include both therapeutic and diagnostic uses or methods, as well as non-therapeutic and non-diagnostic uses or methods. For example, therapeutic aspects may include using the compounds provided by the present disclosure, or their N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers, or using the lipid compositions (i.e., carriers) provided by the present disclosure to package (drug) active ingredients and deliver them to target organs / tissues / cells, or using the compositions provided by the present disclosure to deliver the active ingredients contained therein to target organs / tissues / cells, thereby achieving the effects of treating diseases, improving symptoms, regulating physiological activities in the body, etc.; diagnostic uses may include packaging active ingredients for disease diagnosis in the compounds provided by the present disclosure, or their N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers, or using the lipid compositions (i.e., carriers) provided by the present disclosure to package the active ingredients (drugs) and deliver them ... etc. The active ingredient can be delivered to the target organ / tissue / cell by encapsulating the compound provided by the present disclosure, or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, or lipid composition, so that the active ingredient is delivered to the target organ / tissue / cell, thereby achieving the purpose of disease diagnosis; the non-therapeutic / non-diagnostic aspects can include using the compound provided by the present disclosure, or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, or using the lipid composition provided by the present disclosure to encapsulate the active ingredient, thereby delivering it to the target organ / tissue / cell, for scientific research, detection, and other non-therapeutic, non-diagnostic work (such as disease mechanism research, drug action mechanism research, new drug development, drug screening, etc.).

[0203] The beneficial effects of the present disclosure include at least:

[0204] The cationic lipid compound and lipid composition carrier comprising the same disclosed herein can be used to encapsulate active pharmaceutical ingredients such as nucleic acids (such as mRNA, etc.), with an encapsulation efficiency reaching a high level (e.g., above 90%).

[0205] The mRNA-LNP composition prepared using the cationic lipid disclosed herein has at least the following advantages: it can significantly increase protein expression in vivo (such as protein expression in mice); it also has significant muscle targeting; it has less off-target accumulation in organs, especially in the liver, which is significantly lower than that of the prior art, thereby avoiding or mitigating drug hepatotoxicity; it can significantly increase ( in vivo ) and in vitro ( intro ) protein expression level. BRIEF DESCRIPTION OF THE DRAWINGS

[0206] 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.

[0207] Figure 1 The relative fluorescence intensity of the mRNA-LNP compositions encapsulating Fluc-mRNA prepared based on YK-1601 to YK-1618, SM-102, MC3, lipid-028, E10-1, E24-1, E7-1, compound 92-12, compound 5, compound 9 and control 1 after transfection of HEK293T cells is shown.

[0208] Figure 2 The cell survival rate of the mRNA-LNP compositions encapsulating Fluc-mRNA prepared based on YK-1601 to YK-1618, SM-102, MC3, lipid-028, E10-1, E24-1, E7-1, compound 92-12, compound 5, compound 9 and control 1 after transfection of HEK293T cells is shown.

[0209] Figure 3 The figure shows the relative fluorescence intensity of the mouse liver relative to the injection site (i.e., the ratio of the fluorescence intensity in the liver to the fluorescence intensity at the injection site) 6 h after intramuscular injection of mRNA-LNP compositions encapsulating Fluc-mRNA prepared based on YK-1603, YK-1604, YK-1605, YK-1606, YK-1608, YK-1610, YK-1612, YK-1613, YK-1614, YK-1615 and YK-1618, SM-102, MC3, lipid-028, E10-1, E24-1, E7-1, compound 92-12, compound 5, compound 9, and control 1 into mice.

[0210] Figure 4 Fluorescence imaging of the mouse body and its heart, liver, spleen, lung, and kidney 6 hours after intramuscular injection of the mRNA-LNP composition encapsulating Fluc-mRNA prepared based on YK-1605 and YK-1606. DETAILED DESCRIPTION

[0211] 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.

[0212] All publications and patents mentioned in this disclosure are hereby incorporated into the 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, the purposes and terms of this disclosure shall prevail.

[0213] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0214] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly used in the art to which the claimed subject matter belongs. If there are multiple definitions for a term, the definition herein shall prevail.

[0215] Except in the working examples or otherwise noted, all numbers stating quantitative properties such as dosages 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 herein, the above range is considered 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 herein should be understood to include any and all subranges included therein.

[0216] As used in this disclosure, words such as "include," "comprising," or "including" mean that the elements preceding the word include the elements listed after the word and their equivalents, without excluding unrecited elements. The terms "include," "comprising," or "including" as used herein may be open, semi-closed, or closed. In other words, the above terms also encompass "consisting essentially of" or "consisting of."

[0217] 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.

[0218] The term "subject" or "patient" in the present disclosure includes humans and mammals.

[0219] As used herein, the term "treatment" refers to the administration of one or more pharmaceutical substances to a patient or subject suffering from a disease or symptoms of the disease 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.

[0220] 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).

[0221] The term "hydrate" refers to the above-mentioned term "solvate" in which the solvent is water.

[0222] 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.).

[0223] 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).

[0224] 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.

[0225] 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.

[0226] 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.

[0227] 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.

[0228] An alkyl (or alkylene) group can be unsubstituted, or an alkyl (or alkylene) group can be substituted where at least one hydrogen is replaced with another chemical group.

[0229] 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.

[0230] In the present disclosure, when the name of a compound is inconsistent with the structural formula, the structural formula shall prevail.

[0231] 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.

[0232] As used herein, the term "cationic lipid" refers to a lipid that is positively charged at a selected pH value or range.

[0233] 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).

[0234] While screening a large number of compounds, the inventors discovered that it was extremely difficult to identify suitable cationic lipid compounds that met the following criteria: structural differences from prior art cationic lipids, high transfection efficiency, low cytotoxicity, and high and sustained expression in mice (particularly at the target site). Through extensive research, the inventors discovered several compounds, such as YK-1603, YK-1604, YK-1605, YK-1606, YK-1608, YK-1610, YK-1612, YK-1613, YK-1614, YK-1615, and YK-1618. Compared to prior art cationic lipids, these compounds were capable of delivering biologically active molecules, such as nucleic acids, with significantly improved intracellular transfection efficiency, lower levels of cytotoxicity, significantly increased expression in animals (particularly at the target site), and muscle targeting.

[0235] This disclosure is based on at least the following findings:

[0236] The cationic lipid compounds disclosed herein can be used to deliver nucleic acid molecules, small molecules, polypeptides, or proteins. Compared to known cationic lipid compounds, the cationic lipid compounds disclosed herein exhibit higher transfection efficiency and lower cytotoxicity, significantly increase expression in animal muscle, and improve delivery efficiency.

[0237] The present disclosure also provides a carrier comprising a cationic lipid provided by the compound of formula (I) of the present disclosure, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof.

[0238] The present disclosure further provides a composition comprising the above-mentioned carrier.

[0239] 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.

[0240] The present disclosure further provides uses of the above-mentioned cationic lipid compounds, carriers, or compositions and related methods.

[0241] Cationic lipids

[0242] In one embodiment of the compounds / carriers / compositions of the present disclosure, the cationic lipid includes one or more selected from the compounds of formula (I) of the present disclosure or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer. In one embodiment, the cationic lipid is selected from the above-mentioned compounds of formula (I). For example, the cationic lipid is a compound of formula (I). In a preferred embodiment, the cationic lipid is any one of compounds YK-1601 to YK-1618. In a more preferred embodiment, the cationic lipid is any one of compounds YK-1603, YK-1604, YK-1605, YK-1606, YK-1608, YK-1610, YK-1612, YK-1613, YK-1614, YK-1615 and YK-1618.

[0243] In another embodiment of the carrier / composition disclosed herein, the cationic lipid includes: (a) one or more compounds selected from the above-mentioned formula (I) compound or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer; and, optionally (b) one or more other ionizable lipid compounds different from (a). (b) The cationic lipid compound can be a commercially available cationic lipid, or a cationic lipid compound reported in the literature. For example, (b) the cationic lipid compound can be SM-102 in CN201880017979.X, or MC3 in CN201080026228.8, or E10-1 in CN202380014467.9.

[0244] 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%.

[0245] The carrier can be used to deliver 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.

[0246] For example, examples of the therapeutic agent or the 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.

[0247] neutral lipids

[0248] 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.

[0249] 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.

[0250] For example, the neutral lipids may include one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterols, and derivatives thereof.

[0251] 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.

[0252] 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).

[0253] 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.

[0254] 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.

[0255] Structured lipids

[0256] 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.

[0257] 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.

[0258] 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 (e.g., prednisolone, dexamethasone, prednisone, and hydrocortisone), or a combination thereof.

[0259] Polymer-conjugated lipids

[0260] 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.

[0261] 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.

[0262] 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).

[0263] In one embodiment of the composition / vector of the present disclosure, the polymer-conjugated lipid is DMG-PEG2000.

[0264] In one embodiment of the composition / carrier of the present disclosure, the carrier 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 (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 sum of the molar ratios of the cationic lipid, the neutral lipid, the structural lipid and the polymer conjugated lipid is 100.

[0265] 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.

[0266] Therapeutic and / or preventive agents

[0267] The composition may include one or more therapeutic and / or prophylactic agents (as active ingredients). In one embodiment, the mass ratio of the carrier to the therapeutic and / 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.

[0268] In one embodiment, the mass ratio of the carrier to the therapeutic agent and / or preventive agent is 12.5:1-20:1, preferably 13-17:1, and more preferably 15:1.

[0269] The therapeutic and / or preventive agents include, but are not limited to, one or more of nucleic acid molecules, small molecule compounds, polypeptides or proteins.

[0270] For example, the therapeutic and / or prophylactic agent is a vaccine or a compound capable of eliciting an immune response.

[0271] 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.

[0272] 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.

[0273] Therapeutic and / or prophylactic agents include biologically active substances and may alternatively be referred to as "active agents," "active ingredients," "active components," etc. Therapeutic and / or prophylactic agents can be substances that, upon delivery to a cell or organ, cause 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, the therapeutic and / or prophylactic agent is a small molecule drug that can be used to treat a specific disease, disorder, or condition.Examples of drugs 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), D), vincristine, vinblastine, cytosine 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., dapoxetine), and nausea and vomiting agents. Examples include phenytoin), antihistamines (e.g., diphenhydramine, chlorpheniramine, and promethazine), antibiotics / antibacterials (e.g., gentamycin, ciprofloxacin, and cefoxitin), antifungals (e.g., miconazole, terconazole, econazole, isoconazole, butaconazole, clotrimazole, itraconazole, nystatin, naftifine, and amphotericin B), antiparasitics, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma medications, vitamins, sedatives, and imaging agents.

[0274] 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.

[0275] 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.

[0276] In some embodiments, therapeutic and / or preventive agents are polynucleotides or nucleic acids (e.g., ribonucleic acids or deoxyribonucleic acids). The broadest meaning of the term "polynucleotide" includes any compound and / or substance that is an oligonucleotide chain or can be incorporated into an oligonucleotide chain. Exemplary polynucleotides used in accordance with the present disclosure include, but are not limited to, one or more of the following: deoxyribonucleic acid (DNA); ribonucleic acid (RNA), including messenger mRNA (mRNA), its 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, therapeutic and / or preventive agents are 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.

[0277] 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 / preventive effect when expressed in a cell.

[0278] 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.

[0279] 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.

[0280] 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.

[0281] Disease or condition

[0282] The compounds / vectors / compositions disclosed herein can deliver therapeutic or prophylactic agents to a subject or patient. The therapeutic and / or prophylactic agents include, but are not limited to, one or more of nucleic acid molecules, small molecule compounds, polypeptides, or proteins. Therefore, the compounds / vectors / 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 and / or prophylactic agents described above, the compositions disclosed herein can be used to treat and / or prevent a variety of diseases or conditions.

[0283] In one embodiment, the disease or disorder is characterized by a malfunction or aberrant protein or polypeptide activity.

[0284] 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.

[0285] 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.

[0286] Other components

[0287] 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.

[0288] 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).

[0289] 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).

[0290] 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.

[0291] 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).

[0292] 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.

[0293] 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.

[0294] 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.

[0295] For example, the compositions of the present disclosure can be administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation. In some embodiments, the compositions are administered intramuscularly and / or subcutaneously.

[0296] 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 dose of about 0.001 mg / kg to about 10 mg / kg of the therapeutic and / or prophylactic agent may be administered to a subject (preferably a mammal, such as a human).

[0297] Example

[0298] 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.

[0299] In the following embodiments, the abbreviations have the following meanings:

[0300] 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.

[0301] Example 1: Synthesis of cationic lipid compounds

[0302] 1.1 Synthesis of intermediate INT-1

[0303]

[0304]

[0305] Step 1: Synthesis of INT-1-PM1

[0306] (S)-1-Amino-3-chloro-2-propanol hydrochloride (10.00 g, 68.49 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 (59.75 g, 273.95 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 stopped, 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 (0-18% ethyl acetate / n-hexane). The product was collected and concentrated to yield INT-1-PM1 (9.10 g, 43.40 mmol, 63.4%). 16 ClNO3,MS(ES):m / z(M+H + )210.1.

[0307] Step 2: Synthesis of INT-1-PM2

[0308] Dissolve INT-1-PM1 (6.00 g, 28.62 mmol) in acetonitrile (60 mL), then add dimethylamine hydrochloride (2.32 g, 28.62 mmol), potassium carbonate (11.86 g, 85.85 mmol), and potassium iodide (0.95 g, 5.72 mmol) in that order. Heat to 70°C and allow to react for 24 hours. Monitor the reaction by TLC until the starting material has reacted completely. Discontinue heating, filter, and concentrate the filtrate under reduced pressure. The resulting residue is purified by silica gel column chromatography (0-12% methanol / dichloromethane). The product is collected and concentrated to yield INT-1-PM2 (5.20 g, 23.82 mmol, 83.2%). 10 H 22 N2O3,MS(ES):m / z(M+H + )219.2.

[0309] Step 3: Synthesis of INT-1

[0310] INT-1-PM2 (3.00 g, 13.74 mmol) and a 1,4-dioxane solution of hydrochloric acid (30 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 twice to obtain INT-1 (1.60 g, 13.54 mmol, 98.5%). 14 N2O,MS(ES):m / z(M+H + )119.1.

[0311] 1.2 Synthesis of intermediate INT-2

[0312]

[0313]

[0314] Step 1: Synthesis of INT-2-PM1

[0315] Using INT-1-PM1 (4.50 g, 21.46 mmol) as the starting material, INT-2-PM1 (3.00 g, 10.97 mmol, 51.1%) was obtained according to the synthetic method of INT-1-PM2. 13 H 27 N3O3,MS(ES):m / z(M+H + )274.2.

[0316] Step 2: Synthesis of INT-2

[0317] INT-2-PM1 (3.00 g, 10.97 mmol) was used as the starting material and INT-2 (1.90 g, 10.97 mmol, 99.9%) was obtained according to the synthetic method of INT-1. 19 N3O,MS(ES):m / z(M+H + )174.1.

[0318] 1.3 Synthesis of intermediate INT-3

[0319]

[0320]

[0321] Step 1: Synthesis of INT-3-PM1

[0322] Using INT-1-PM1 (4.50 g, 21.46 mmol) as the starting material, INT-3-PM1 (2.50 g, 10.15 mmol, 47.3%) was obtained according to the synthetic method of INT-1-PM2. 12 H 26 N2O3,MS(ES):m / z(M+H + )247.2.

[0323] Step 2: Synthesis of INT-3

[0324] INT-3-PM1 (2.20 g, 8.93 mmol) was used as the starting material and INT-3 (1.20 g, 8.21 mmol, 91.9%) was obtained according to the synthetic method of INT-1. 18 N2O,MS(ES):m / z(M+H + )147.2.

[0325] 1.4 Synthesis of intermediate INT-4

[0326]

[0327] 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 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-4 (41.42 g, 98.74 mmol, 98.7%). 22 H 43 BrO2,MS(ES):m / z(M+H + )419.2.

[0328] 1.5 Synthesis of intermediate INT-5

[0329]

[0330] Using n-decanol (25.00 g, 157.95 mmol) as the starting material, INT-5 (47.31 g, 153.97 mmol, 97.5%) was obtained according to the synthesis method of INT-4. 14 H 27 BrO2,MS(ES):m / z(M+H + )307.1.

[0331] 1.6 Synthesis of intermediate INT-6

[0332]

[0333] Dissolve heptadecan-9-ol (5.00 g, 19.50 mmol) in dichloromethane (60 mL), then add 6-bromohexanoic acid (4.18 g, 21.45 mmol), EDCI (5.61 g, 29.24 mmol), and DMAP (0.48 g, 3.90 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 (0-10% ethyl acetate / n-hexane). The product was collected and concentrated to yield INT-6 (8.00 g, 18.45 mmol, 94.6%).23 H 45 BrO2,MS(ES):m / z(M+H + )433.2.

[0334] 1.7 Synthesis of intermediate INT-7

[0335]

[0336] Step 1: Synthesis of INT-7-PM1

[0337] Using tert-butyl 6-hydroxyhexanoate (2.00 g, 10.62 mmol) as raw material, INT-7-PM1 (4.40 g, 10.31 mmol, 97.1%) was obtained according to the synthesis method of INT-6. 26 H 50 O4,MS(ES):m / z(M+H + )427.3.

[0338] Step 2: Synthesis of INT-7

[0339] Dissolve INT-7-PM1 (2.00 g, 4.69 mmol) in dichloromethane (20 mL) and add trifluoroacetic acid (4 mL). Allow to react at room temperature for 5 h. Monitor the reaction by TLC until the reaction is complete. Concentrate the reaction mixture under reduced pressure, dissolve the resulting residue in dichloromethane, and concentrate again under reduced pressure to yield INT-7 (1.70 g, 4.59 mmol, 97.9%). 22 H 42 O4,MS(ES):m / z(M+H + )371.3.

[0340] 1.8 Synthesis of intermediate INT-8

[0341]

[0342] Step 1: Synthesis of INT-8-PM1

[0343] Using tert-butyl 6-hydroxyhexanoate (400 mg, 2.12 mmol) as the starting material, INT-8-PM1 (750 mg, 2.19 mmol, 103.0%) was obtained according to the synthesis method of INT-6. 20 H 38 O4,MS(ES):m / z(M+H + )343.3.

[0344] Step 2: Synthesis of INT-8

[0345] INT-8-PM1 (300 mg, 0.88 mmol) was used as the starting material and INT-8 (245 mg, 0.85 mmol, 97.6%) was obtained according to the synthesis method of INT-7. 16 H 30 O4,MS(ES):m / z(M+H + )287.2.

[0346] 1.9 Synthesis of intermediate INT-9

[0347]

[0348] Using undecanol (5.00 g, 29.02 mmol) as the starting material, INT-9 (7.10 g, 22.10 mmol, 76.4%) was obtained according to the synthesis method of INT-6. 15 H 29 BrO2,MS(ES):m / z(M+H + )321.2.

[0349] 1.10 Synthesis of YK-1601

[0350]

[0351]

[0352]

[0353] Step 1: Synthesis of YK-1601-PM1

[0354] INT-1 (300 mg, 2.54 mmol) was dissolved in acetonitrile (5 mL), followed by the addition of INT-4 (1.06 g, 2.54 mmol), potassium carbonate (1.05 g, 7.61 mmol), and potassium iodide (42 mg, 0.25 mmol). The reaction was heated to 70°C for 7 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 (0-10% methanol / dichloromethane). The product was collected and concentrated to yield YK-1601-PM1 (260 mg, 0.57 mmol, 22.4%). 27 H 56 N2O3,MS(ES):m / z(M+H + )457.5.

[0355] Step 2: Synthesis of YK-1601-PM2

[0356] Dissolve YK-1601-PM1 (260 mg, 0.57 mmol) in acetonitrile (3 mL), then add INT-5 (175 mg, 0.57 mmol), potassium carbonate (236 mg, 1.71 mmol), and potassium iodide (9 mg, 0.06 mmol) in that order. Heat to 70°C and allow to react for 8 hours. Monitor the reaction by LCMS until the starting material has reacted completely. Remove from heat, filter, and concentrate the filtrate under reduced pressure. The resulting residue is purified by silica gel column chromatography (0-10% methanol / dichloromethane). The product is collected and concentrated to yield YK-1601-PM2 (90 mg, 0.13 mmol, 23.1%). 41 H 82 N2O5,MS(ES):m / z(M+H + )683.6.

[0357] Step 3: Synthesis of YK-1601

[0358] YK-1601-PM2 (90 mg, 0.13 mmol) was dissolved in dichloromethane (2 mL), and INT-7 (49 mg, 0.13 mmol), EDCI (38 mg, 0.20 mmol), and DMAP (2 mg, 0.01 mmol) were added. The reaction was allowed to react at room temperature for 15 h, monitored by TLC until the reaction of the starting materials was complete. 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 (0-8% methanol / dichloromethane). The product was collected and concentrated to obtain YK-1601 (90 mg, 0.08 mmol, 65.9%). 63 H 122 N2O8,MS(ES):m / z(M+H + )1036.1. 1 H NMR (400 MHz, CDCl3) δ 5.09 (dd, J = 11.9, 6.0 Hz, 1H),4.08 (q, J = 6.6 Hz, 4H), 3.99 (d, J = 5.7 Hz, 2H), 2.61 – 2.40 (m, 12H), 1.79 –1.56 (m, 14H), 1.50 – 1.26 (m, 74H), 0.91 (t, J = 6.8 Hz, 15H).

[0359] 1.11 Synthesis of YK-1602

[0360]

[0361] Step 1: Synthesis of YK-1602-PM1

[0362] Using INT-1 (240 mg, 2.03 mmol) as the starting material, and following the synthetic method of YK-1601-PM1, YK-1602-PM1 (270 mg, 0.57 mmol, 28.2%) was obtained. 28 H 58 N2O3,MS(ES):m / z(M+H + )471.5.

[0363] Step 2: Synthesis of YK-1602-PM2

[0364] YK-1602-PM1 (270 mg, 0.57 mmol) was used as the starting material and YK-1601-PM2 was synthesized according to the method described for YK-1601-PM2 to obtain YK-1602-PM2 (90 mg, 0.13 mmol, 22.1%). 43 H 86 N2O5,MS(ES):m / z(M+H + )711.6.

[0365] Step 3: Synthesis of YK-1602

[0366] YK-1602-PM2 (90 mg, 0.13 mmol) was used as the starting material and YK-1602 (25 mg, 0.02 mmol, 18.6%) was obtained according to the synthetic method of YK-1601. 65 H 126 N2O8,MS(ES):m / z(M+H + )1064.1. 1 H NMR (400 MHz, CDCl3)δ 4.22 (t, J = 5.1 Hz, 1H), 4.06 (d, J = 7.7 Hz, 4H), 3.52 (d, J = 5.4 Hz, 1H), 2.44 (s, 2H), 2.38 – 2.21 (m, 10H), 2.00 (s, 2H), 1.68 – 1.58 (m, 10H), 1.48(d, J = 12.5 Hz, 8H), 1.45 – 1.37 (m, 7H), 1.28 (d, J = 15.7 Hz, 58H), 1.17 (d,J =10.0 Hz, 2H), 0.92 – 0.84 (t, 15H).

[0367] 1.12 Synthesis of YK-1603

[0368]

[0369] Step 1: Synthesis of YK-1603-PM1

[0370] Using INT-1 (141 mg, 1.19 mmol) as the starting material, following the synthetic method of YK-1601-PM1, YK-1603-PM1 (82 mg, 0.13 mmol, 11.1%) was obtained. 41 H 82 N2O, MS(ES):m / z(M+H + )619.6.

[0371] Step 2: Synthesis of YK-1603

[0372] Using YK-1603-PM1 (82 mg, 0.13 mmol) as the starting material, following the synthetic method of YK-1601, YK-1603 (74 mg, 0.07 mmol, 56.8%) was obtained. 63 H 122 N2O4,MS(ES):m / z(M+H + )972.1. 1 H NMR (400 MHz, CDCl3)δ 5.36 – 5.32 (m, 4H), 4.08 – 4.28 (dd, J = 12.9, 6.4 Hz, 3H), 2.48 – 2.41 (m,3H), 2.33 – 2.22 (m, 9H), 2.09 – 1.93 (m, 7H), 1.72 – 1.52 (m, 8H), 1.46 –1.37 (m, 8H), 1.34 – 1.25 (m, 63H), 0.89 – 0.85 (t, 12H).

[0373] 1.13 Synthesis of YK-1604

[0374]

[0375] Step 1: Synthesis of YK-1604-PM1

[0376] Using INT-1 (141 mg, 1.19 mmol) as the starting material, following the synthetic method of YK-1601-PM1, YK-1604-PM1 (102 mg, 0.16 mmol, 13.9%) was obtained. 41 H 78 N2O,MS(ES):m / z(M+H + )615.6.

[0377] Step 2: Synthesis of YK-1604

[0378] YK-1604-PM1 (102 mg, 0.16 mmol) was used as the starting material and YK-1604 (55 mg, 0.06 mmol, 34.3%) was obtained according to the synthetic method of YK-1601. 63 H 118 N2O4,MS(ES):m / z(M+H + )968.1. 1 H NMR (400 MHz, CDCl3)δ 4.11 – 4.03 (m, 4H), 2.40 – 2.30 (m, 6H), 2.30 – 2.20 (m, 3H), 1.75 – 1.51(m, 20H), 1.43 (ddd, J = 15.8, 10.9, 6.1 Hz, 11H), 1.30 (dd, J = 14.7, 7.4 Hz, 56H), 1.25 (s, 6H), 0.91 – 0.84 (t, 12H).

[0379] 1.14 Synthesis of YK-1605

[0380]

[0381] Step 1: Synthesis of YK-1605-PM1

[0382] Using INT-2 (317 mg, 1.83 mmol) as the starting material, and following the synthetic method of YK-1601-PM1, YK-1605-PM1 (294 mg, 0.57 mmol, 31.4%) was obtained. 30 H 61 N3O3,MS(ES):m / z(M+H + )512.5.

[0383] Step 2: Synthesis of YK-1605PM2

[0384] YK-1605-PM1 (294 mg, 0.57 mmol) was used as the starting material and YK-1601-PM2 was synthesized according to the method described for YK-1601-PM2 to obtain YK-1605-PM2 (160 mg, 0.22 mmol, 37.7%). 44 H 87 N3O5, MS(ES):m / z(M+H + )738.6.

[0385] Step 3: Synthesis of YK-1605

[0386] YK-1605-PM2 (160 mg, 0.22 mmol) was used as the starting material and YK-1605 (51 mg, 0.05 mmol, 21.6%) was obtained according to the synthetic method of YK-1601. 66 H 127 N3O8,MS(ES):m / z(M+H + )1091.1. 1 H NMR (400 MHz, CDCl3)δ 5.05 – 5.02 (m, 1H), 4.09 – 4.03 (m, 4H), 3.96 (d, J = 5.7 Hz, 2H), 2.48(tdd, J = 18.1, 12.2, 6.0 Hz, 7H), 2.34 – 2.14 (m, 12H), 1.70 (ddd, J = 15.0,11.4, 5.2 Hz, 9H), 1.67 – 1.57 (m, 5H), 1.46 – 1.37 (m, 9H), 1.33 – 1.25 (m,62H), 0.90 – 0.86 (t, 15H).

[0387] 1.15 Synthesis of YK-1606

[0388]

[0389] Step 1: Synthesis of YK-1606-PM1

[0390] Using INT-2 (380 mg, 2.20 mmol) as the starting material, following the synthetic method of YK-1601-PM1, YK-1606-PM1 (360 mg, 0.68 mmol, 31.2%) was obtained. 31 H 63 N3O3,MS(ES):m / z(M+H + )526.5.

[0391] Step 2: Synthesis of YK-1606-PM2

[0392] YK-1606-PM1 (360 mg, 0.68 mmol) was used as the starting material and the synthetic method of YK-1601-PM2 was followed to obtain YK-1606-PM2 (210 mg, 0.27 mmol, 40.0%). 46 H 91 N3O5,MS(ES):m / z(M+H + )766.7.

[0393] Step 3: Synthesis of YK-1606

[0394] YK-1606-PM2 (90 mg, 0.12 mmol) was used as the starting material and YK-1606 (103 mg, 0.09 mmol, 78.4%) was obtained according to the synthetic method of YK-1601. 68 H 131 N3O8,MS(ES):m / z(M+H + )1119.1. 1 H NMR (400 MHz, CDCl3) δ 5.19 – 4.99 (m, 1H), 4.89 (p, J = 6.3 Hz, 1H), 4.09 (q, J = 6.8 Hz, 4H), 2.67 (s, 2H), 2.59 – 2.44 (m, 10H), 2.37 – 2.27 (m, 8H), 1.80 – 1.62 (m, 11H), 1.57 – 1.39 (m, 11H), 1.35 (dd, J = 7.8, 3.6 Hz, 12H), 1.30 (s, 48H), 0.92 (t, J = 6.7 Hz, 15H).

[0395] 1.16 Synthesis of YK-1607

[0396]

[0397] Using YK-1606-PM2 (90 mg, 0.12 mmol) as the starting material, following the synthetic method of YK-1601, YK-1607 (105 mg, 0.10 mmol, 89.0%) was obtained. 62 H 121 N3O6,MS(ES):m / z(M+H + )1005.1.1 H NMR (400 MHz, CDCl3) δ 5.05 (dd, J = 11.9, 6.1 Hz, 1H), 4.86 (dd, J = 12.5, 6.3 Hz, 1H), 4.06(d, J = 6.8 Hz, 2H), 2.60 (s, 2H), 2.50 (d, J = 7.1 Hz, 4H), 2.41 (d, J = 6.1 Hz,5H), 2.34 – 2.21 (m, 6H), 1.66 – 1.54 (m, 7H), 1.53 – 1.46 (m, 5H), 1.45 –1.37 (m, 5H), 1.34 – 1.23 (m, 66H), 0.92 – 0.84 (t, 15H).

[0398] 1.17 Synthesis of YK-1608

[0399]

[0400] Step 1: Synthesis of YK-1608-PM1

[0401] Using INT-2 (126 mg, 0.73 mmol) as the starting material, following the synthetic method of YK-1601-PM1, YK-1608-PM1 (140 mg, 0.21 mmol, 28.6%) was obtained. 44 H 87 N3O,MS(ES):m / z(M+H + )674.7.

[0402] Step 2: Synthesis of YK-1608

[0403] Using YK-1608-PM1 (70 mg, 0.10 mmol) as the starting material, following the synthetic method of YK-1601, YK-1608 (53 mg, 0.05 mmol, 49.7%) was obtained. 66 H 127 N3O4,MS(ES):m / z(M+H + )1027.1. 1 H NMR (400 MHz, CDCl3)δ 5.38 (m, 4H), 5.07 (d, J= 5.8 Hz, 1H), 4.05 (t, 2H), 2.57 – 2.36 (m, 12H), 2.03 (d, J = 5.8 Hz, 7H), 1.72 – 1.52 (m, 7H), 1.46 – 1.35 (m, 10H), 1.34 –1.18 (m, 69H), 0.92 – 0.84 (t, 12H).

[0404] 1.18 Synthesis of YK-1609

[0405]

[0406] YK-1608-PM1 (70 mg, 0.10 mmol) was used as the starting material and YK-1609 (33 mg, 0.04 mmol, 34.8%) was obtained according to the synthetic method of YK-1601. 60 H 117 N3O2,MS(ES):m / z(M+H + )913.1. 1 H NMR (400 MHz, CDCl3)δ 5.36 (dt, J = 9.9, 4.3 Hz, 3H), 5.15 – 5.04 (m, 1H), 2.56 – 2.36 (m, 11H), 2.29 (d, J = 6.5 Hz, 3H), 2.06 – 2.00 (m, 5H), 2.00 – 1.92 (m, 2H), 1.69 – 1.52(m, 4H), 1.44 (d, J = 8.4 Hz, 2H), 1.38 (dd, J = 15.8, 8.0 Hz, 8H), 1.34 – 1.25 (m, 60H), 1.25 – 1.23 (m, 3H), 0.92 – 0.81 (s, 12H).

[0407] 1.19 Synthesis of YK-1610

[0408]

[0409] Step 1: Synthesis of YK-1610-PM1

[0410] Using INT-2 (126 mg, 0.73 mmol) as the starting material, following the synthetic method of YK-1601-PM1, YK-1610-PM1 (150 mg, 0.22 mmol, 30.8%) was obtained. 44 H 83 N3O,MS(ES):m / z(M+H + )670.7.

[0411] Step 2: Synthesis of YK-1610

[0412] Using YK-1610-PM1 (70 mg, 0.10 mmol) as the starting material, following the synthetic method of YK-1601, YK-1610 (48 mg, 0.05 mmol, 44.9%) was obtained. 66 H 123 N3O4,MS(ES):m / z(M+H + )1023.1. 1 H NMR (400 MHz, CDCl3)δ 5.44 – 5.27 (m, 8H), 4.10 – 4.02 (t, 2H), 2.81 – 2.73 (t, 4H), 2.53 (dd, J =13.1, 4.5 Hz, 5H), 2.50 – 2.38 (m, 8H), 2.35 – 2.25 (m, 6H), 2.10 – 2.00 (m,8H), 1.71 – 1.60 (m, 5H), 1.60 – 1.51 (m, 2H), 1.47 – 1.33 (m, 15H), 1.33 –1.23 (m, 47H), 0.93 – 0.84 (m, 12H).

[0413] 1.20 Synthesis of YK-1611

[0414]

[0415] Using YK-1610-PM1 (70 mg, 0.10 mmol) as the starting material, following the synthetic method of YK-1601, YK-1611 (50 mg, 0.05 mmol, 52.7%) was obtained. 60 H 113 N3O2,MS(ES):m / z(M+H + )909.1. 1H NMR (400 MHz, CDCl3)δ 5.45 – 5.32 (m, 8H), 2.84 – 2.73 (m, 7H), 2.63 – 2.53 (m, 6H), 2.29 (ddd, J =25.1, 14.3, 5.7 Hz, 3H), 2.08 (q, J = 7.0 Hz, 8H), 1.61 (dd, J = 15.0, 7.9 Hz,4H), 1.48 (d, J = 7.5 Hz, 3H), 1.43 – 1.27 (m, 59H), 0.92 (dd, J = 11.5, 6.0 Hz,12H).

[0416] 1.21 Synthesis of YK-1612

[0417]

[0418]

[0419] Step 1: Synthesis of YK-1612-PM1

[0420] Using INT-1-PM2 (600 mg, 2.19 mmol) as the starting material, following the synthetic method of YK-1601, YK-1612-PM1 (840 mg, 1.64 mmol, 74.8%) was obtained. 29 H 57 N3O4,MS(ES):m / z(M+H + )512.4.

[0421] Step 2: Synthesis of YK-1612-PM2

[0422] YK-1612-PM1 (120 mg, 0.23 mmol) was used as the starting material and YK-1612-PM2 (95 mg, 0.23 mmol, 98.4%) was obtained according to the synthesis method of INT-1. 24 H 49 N3O2,MS(ES):m / z(M+H + )412.4.

[0423] Step 3: Synthesis of YK-1612

[0424] Using YK-1612-PM2 (95 mg, 0.23 mmol) as the starting material, following the synthetic method of YK-1601, YK-1612 (55 mg, 0.07 mmol, 31.2%) was obtained. 46 H 89 N3O5,MS(ES):m / z(M+H + )764.7. 1 H NMR (400 MHz, CDCl3) δ6.12 (d, J = 5.8 Hz, 1H), 5.04 (t, J = 5.8 Hz, 1H), 4.06 (t, J = 6.7 Hz, 2H), 3.58– 3.39 (m, 2H), 2.71 – 2.55 (m, 5H), 2.55 – 2.43 (m, 4H), 2.31 (p, J = 5.4 Hz,2H), 2.16 (t, 2H), 1.65 (ddd, J = 53.1, 30.1, 23.9 Hz, 8H), 1.49 – 1.34 (m,7H), 1.32 – 1.17 (m, 43H), 0.92 – 0.82 (t, 12H).

[0425] 1.22 Synthesis of YK-1613

[0426]

[0427] Step 1: Synthesis of YK-1613-PM1

[0428] Using INT-3 (500 mg, 3.42 mmol) as the starting material, and following the synthetic method of YK-1601-PM1, YK-1613-PM1 (420 mg, 0.87 mmol, 25.3%) was obtained. 29 H 60 N2O3,MS(ES):m / z(M+H + )485.5.

[0429] Step 2: Synthesis of YK-1613-PM2

[0430] YK-1613-PM1 (420 mg, 0.87 mmol) was used as the starting material and YK-1601-PM2 was synthesized according to the method described for YK-1601-PM2 to obtain YK-1613-PM2 (420 mg, 0.59 mmol, 68.2%). 43 H 86N2O5,MS(ES):m / z(M+H + )711.6.

[0431] Step 3: Synthesis of YK-1613

[0432] Using YK-1613-PM2 (80 mg, 0.11 mmol) as the starting material, following the synthetic method of YK-1601, YK-1613 (50 mg, 0.05 mmol, 41.8%) was obtained. 65 H 126 N2O8,MS(ES):m / z(M+H + )1064.1. 1 H NMR (400 MHz, CDCl3)δ 4.10 – 4.01 (m, 4H), 3.96 (d, J = 5.8 Hz, 2H), 2.60 – 2.38 (m, 11H), 2.34 –2.25 (m, 7H), 1.71 – 1.53 (m, 12H), 1.47 – 1.35 (m, 7H), 1.33 – 1.23 (m,59H), 1.03 – 0.95 (t, 6H), 0.92 – 0.81 (t, 15H).

[0433] 1.23 Synthesis of YK-1614

[0434]

[0435] Step 1: Synthesis of YK-1614-PM1

[0436] Using INT-3 (100 mg, 0.68 mmol) as the starting material, following the synthetic method of YK-1601-PM1, YK-1614-PM1 (120 mg, 0.15 mmol, 21.3%) was obtained. 51 H 102 N2O5,MS(ES):m / z(M+H + )823.8.

[0437] Step 2: Synthesis of YK-1614

[0438] Using YK-1614-PM1 (120 mg, 0.15 mmol) as the starting material, following the synthetic method of YK-1601, YK-1614 (100 mg, 0.10 mmol, 70.2%) was obtained. 61 H 120N2O6,MS(ES):m / z(M+H + )978.1. 1 H NMR (400 MHz, CDCl3)δ 4.08 – 4.00 (m, 3H), 3.96 (d, J = 5.7 Hz, 3H), 3.63 – 3.28 (m, 3H), 2.55 (s,10H), 2.35 – 2.25 (m, 7H), 1.71 – 1.55 (m, 12H), 1.29 (dd, J = 14.5, 7.3 Hz, 60H), 1.06 – 0.98 (m, 6H), 0.92 – 0.84 (t, 15H).

[0439] 1.24 Synthesis of YK-1615

[0440]

[0441] Step 1: Synthesis of YK-1615-PM1

[0442] Using INT-3 (300 mg, 2.05 mmol) as the starting material, and following the synthetic method of YK-1601-PM1, YK-1615-PM1 (200 mg, 0.40 mmol, 19.5%) was obtained. 30 H 62 N2O3,MS(ES):m / z(M+H + )499.5.

[0443] Step 2: Synthesis of YK-1615-PM2

[0444] Using YK-1615-PM1 (200 mg, 0.40 mmol) as the starting material, following the synthetic method of YK-1601-PM2, YK-1615-PM2 (156 mg, 0.21 mmol, 52.6%) was obtained. 45 H 90 N2O5,MS(ES):m / z(M+H + )739.7.

[0445] Step 3: Synthesis of YK-1615

[0446] Using YK-1615-PM2 (80 mg, 0.11 mmol) as the starting material, following the synthetic method of YK-1601, YK-1615 (100 mg, 0.09 mmol, 84.6%) was obtained. 67 H 130N2O8,MS(ES):m / z(M+H + )1092.1. 1 H NMR (400 MHz, CDCl3) δ 5.01 (d, J = 6.0 Hz, 1H), 4.88 (d, J = 6.2 Hz, 1H), 4.10 – 4.01 (m, 4H), 2.60 – 2.38 (m, 11H), 2.36 – 2.23 (m, 7H), 1.78 – 1.67 (m, 3H), 1.67 – 1.61(m, 7H), 1.60 (d, J = 4.0 Hz, 2H), 1.50 (dd, J = 12.4, 6.3 Hz, 4H), 1.47 – 1.35 (m, 8H), 1.33 – 1.28 (m, 60H), 1.03 – 0.95 (t, 6H), 0.92 – 0.84 (t, 15H).

[0447] 1.25 Synthesis of YK-1616

[0448]

[0449] Step 1: Synthesis of YK-1616-PM1

[0450] Using INT-3 (118 mg, 0.81 mmol) as the starting material, and following the synthetic method of YK-1601-PM1, YK-1616-PM1 (238 mg, 0.37 mmol, 45.6%) was obtained. 43 H 86 N2O,MS(ES):m / z(M+H + )647.7.

[0451] Step 2: Synthesis of YK-1616

[0452] YK-1616-PM1 (80 mg, 0.12 mmol) was used as the starting material and YK-1616 (45 mg, 0.05 mmol, 36.4%) was obtained according to the synthetic method of YK-1601. 65 H 126 N2O4,MS(ES):m / z(M+H + )1000.1. 1H NMR (400 MHz, CDCl3)δ 5.41 – 5.31 (m, 3H), 4.10 – 4.02 (m, 3H), 2.55 – 2.47 (m, 5H), 2.40 (d, J =7.3 Hz, 3H), 2.35 – 2.24 (m, 3H), 2.05 (d, J = 4.9 Hz, 4H), 2.01 (dd, J = 12.5,6.4 Hz, 6H), 1.70 – 1.60 (m, 6H), 1.60 – 1.51 (m, 2H), 1.47 – 1.35 (m, 10H), 1.32 – 1.23 (m, 60H), 1.03 – 0.95 (m, 6H), 0.92 – 0.84 (t, 12H).

[0453] 1.26 Synthesis of YK-1617

[0454]

[0455] Step 1: Synthesis of YK-1617-PM1

[0456] Using INT-3 (120 mg, 0.81 mmol) as the starting material, following the synthetic method of YK-1601-PM1, YK-1617-PM1 (200 mg, 0.31 mmol, 37.9%) was obtained. 43 H 82 N2O,MS(ES):m / z(M+H + )643.7.

[0457] Step 2: Synthesis of YK-1617

[0458] YK-1617-PM1 (80 mg, 0.12 mmol) was used as the starting material and YK-1617 (52 mg, 0.05 mmol, 42.0%) was obtained according to the synthetic method of YK-1601. 65 H 122 N2O4,MS(ES):m / z(M+H + )996.1. 1 H NMR (400 MHz, CDCl3)δ 5.41 – 5.27 (m, 8H), 4.17 – 3.85 (m, 2H), 2.81 – 2.73 (m, 4H), 2.47 (d, J=49.9 Hz, 9H), 2.35 – 2.21 (m, 2H), 2.05 (dd, J = 13.8, 6.9 Hz, 8H), 1.71 – 1.51(m, 8H), 1.49 – 1.36 (m, 10H), 1.33 (dd, J = 14.4, 7.6 Hz, 14H), 1.28 (dd, J =9.9, 7.1 Hz, 39H), 1.02 (d, J = 7.3 Hz, 6H), 0.88 (dd, J = 13.0, 6.5 Hz, 12H).

[0459] 1.27 Synthesis of YK-1618

[0460]

[0461] Using YK-1613-PM2 (74 mg, 0.10 mmol) as the starting material, following the synthetic method of YK-1601, YK-1618 (100 mg, 0.10 mmol, 98.1%) was obtained. 59 H 114 N2O8,MS(ES):m / z(M+H + )980.1. 1 H NMR (400 MHz, CDCl3)δ 4.09 – 4.01 (t, 4H), 3.96 (d, J = 5.8 Hz, 2H), 2.60 – 2.47 (m, 7H), 2.43 (dd, J = 17.2, 8.2 Hz, 4H), 2.34 – 2.26 (m, 8H), 1.73 – 1.55 (m, 14H), 1.45 – 1.37(m, 4H), 1.29 (m, 53H), 1.03 – 0.95 (t, 6H), 0.92 – 0.84 (t, 12H).

[0462] 1.28 Synthesis of Lipid-028

[0463] Follow In vivo genome editing of human hematopoietic stem cells for treatment of blood

[0464] Disorders by mRNA delivery, Saijuan Xu et al., bioRxiv preprint, doi: https: / / doi.org / 10.1101 / 2024.10.28.620445, synthesis route of lipid-028 (Lipid-028), 76 mg of lipid-028 (Lipid-028) was synthesized.

[0465] 1.29 Synthesis of E10-1

[0466] According to the E10-1 synthesis method in CN202380014467.9, 43 mg of E10-1 was synthesized.

[0467] 1.30 Synthesis of E24-1

[0468] According to the E24-1 synthesis method in CN202380014467.9, 55 mg of E24-1 was synthesized.

[0469] 1.31 Synthesis of E7-1

[0470] According to the E7-1 synthesis method in PCT / CN2023 / 098155, 36 mg of E7-1 was synthesized.

[0471] 1.32 Synthesis of Compound 92-12

[0472] According to the synthesis method of compound 92-12 in PCT / CN2023 / 098155, 23 mg of compound 92-12 was synthesized.

[0473] 1.33 Synthesis of Compound 5

[0474] According to the synthesis method of compound 5 in CN202410487810.8, 107 mg of compound 5 was synthesized.

[0475] 1.34 Synthesis of Compound 9

[0476] According to the synthesis method of compound 9 in CN202410487810.8, 45 mg of compound 9 was synthesized.

[0477] 1.35 Synthesis of Reference Material 1

[0478] The synthesis method of YK-1606 in Example 1 was followed, except that the raw materials for preparing INT-1 were Replace with , 110 mg of reference substance 1 was synthesized.

[0479] Example 2: mRNA-LNP formulation optimization

[0480] 2.1 Optimization of the ratio of vector (liposome) to mRNA

[0481] Step 1: The cationic lipid YK-1602 or YK-1605 synthesized in Example 1 was dissolved in ethanol with DSPC (Aiweituo (Shanghai) Pharmaceutical Technology Co., Ltd.), cholesterol (Aiweituo (Shanghai) Pharmaceutical Technology Co., Ltd.), and DMG-PEG2000 at a molar ratio 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.

[0482] 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.

[0483] 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-1602 or YK-1605), DSPC, cholesterol, and DMG-PEG2000.

[0484] 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.

[0485] 2.2 Optimization of the ratio of cationic lipids to neutral lipids

[0486] mRNA-LNP compositions encapsulating eGFP-mRNA were prepared similarly to the method in 2.1, wherein the molar ratios of cationic lipid (YK-1602 or YK-1605) 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.

[0487] 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.

[0488] 2.3 Optimization of the ratio of polymer-conjugated lipid to carrier

[0489] mRNA-LNP compositions encapsulating eGFP-mRNA were prepared according to a method similar to that in 2.1, wherein the cationic lipid was YK-1602, YK-1603, YK-1604, or YK-1607, 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.

[0490] 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%.

[0491] 2.4 Optimization of the ratio of components in the carrier

[0492] 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-1602 or YK-1605), 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.

[0493] 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.

[0494] Example 3: Cell transfection with mRNA-LNP formulation encapsulating eGFP-mRNA

[0495] Step 1: Cell recovery and passaging: Recover HEK293T cells and culture them in culture dishes to the desired cell number.

[0496] Step 2: Plating: Digest and count the cells in the culture dish, plate 10,000 cells per well in a 96-well plate, and culture overnight until the cells adhere.

[0497] Step 3: Cell transfection: 1.5 μg of the mRNA-LNP preparation encapsulating eGFP-mRNA prepared in Example 2 (wherein the cationic lipid was YK-1602 or YK-1605, respectively) was added to the cell culture medium of a 96-well plate. After further culture for 24 hours, the transfection efficiency was assessed by fluorescence intensity under a fluorescence microscope.

[0498] 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.

[0499] Example 4: Preparation of mRNA-LNP formulation

[0500] Table 1 Cationic lipid compounds

[0501]

[0502] 4.1 Prepare the corresponding ethanol lipid solutions of the cationic lipids listed in Table 1 according to the method in step 1 of Example 2.1.

[0503] 4.2 Dilute eGFP-mRNA (Shanghai Qifa Experimental Reagent Co., Ltd.) or Fluc-mRNA (Shanghai Qifa Experimental Reagent Co., Ltd.) in citrate buffer (pH = 4.5 ± 0.5) to obtain the corresponding mRNA aqueous solution.

[0504] 4.3 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 (in the form of dispersions, containing approximately 0.1 mg / mL of mRNA) with a molar ratio of cationic lipid: DSPC: cholesterol: DMG-PEG2000 of 49:10:39.5:1.5.

[0505] Example 5: Determination of mRNA-LNP Particle Size, Polydispersity Index (PDI) and Encapsulation Efficiency

[0506] The particle size and polydispersity index (PDI) were determined using a Malvern laser particle size analyzer using dynamic light scattering.

[0507] 25 μL of the mRNA-LNP preparation prepared in Example 4 was diluted to 125 μL with RNase-free deionized water and added to the sample well. Each sample was measured three times, and the average value was taken as the test result. The measurement conditions were: 90° scattering angle, 25°C; the LNP encapsulation efficiency was determined using the Quant it Ribogreen RNA Quantification Assay Kit (Thermo Fisher Scientific, UK) according to the manufacturer's instructions. The test results are shown in Table 2:

[0508] Table 2 Particle size, polydispersity index (PDI), and encapsulation efficiency of mRNA-LNP

[0509]

[0510] As shown in Table 2, the nanolipid particles prepared in Example 4 had a particle size range of 80-110 nm, making them suitable for mRNA delivery. The polydispersity coefficients were all less than 0.15, indicating good particle size uniformity. Furthermore, the nanolipid particles exhibited high encapsulation efficiencies, exceeding 90%.

[0511] Example 6: In vitro delivery performance and toxicity of LNPs

[0512] The cell recovery, passaging and plating methods refer to steps 1 and 2 of Example 3.

[0513] The 96-well plate containing HEK293T cells obtained in step 2 was supplemented with an appropriate volume of HEK293T cell culture medium, and the mRNA-LNP preparation containing 0.3 μg Fluc-mRNA (prepared in Example 4) was added to the 96-well plate. After continued cultivation for 24 hours, the corresponding reagents were added according to the instructions of the Gaussia Luciferase Assay Kit, and the relative fluorescence intensity of each well was detected by the IVIS fluorescence detection system. Finally, 10 μL of CCK-8 solution was added to each well of the well plate after 24 hours of culture. After the culture plate was incubated in the incubator for 1 hour, the absorbance at 450 nm was measured by a microplate reader to detect the cell viability. The results of relative fluorescence intensity and cell viability are shown in Tables 3 and Figure 1-2 .

[0514] Table 3 Fluorescence detection results of Fluc-mRNA

[0515]

[0516] The relative fluorescence intensities (corresponding to the translation efficiency of mRNA) of the above mRNA-LNP compositions were significantly different. The relative fluorescence intensities of the mRNA-LNP compositions prepared from YK-1603, YK-1604, YK-1605, YK-1606, YK-1608, YK-1610, YK-1612, and YK-1614 were significantly higher than those of the mRNA-LNP compositions prepared from SM-102, MC3, lipid-028, E10-1, E24-1, E7-1, compound 92-12, compound 5, compound 9, and control 1. Specifically:

[0517] 1. The mRNA-LNP compositions prepared using YK-1603, YK-1604, YK-1605, YK-1606, YK-1608, YK-1610, YK-1612, and YK-1614 all significantly improved their cell transfection efficiency compared to representative cationic lipids used in the prior art. For example, the cell transfection efficiency of YK-1606 was 1.37 times that of SM-102 and 2.85 times that of MC3.

[0518] 2. The cell transfection efficiency of mRNA-LNP compositions prepared with YK-1603 and YK-1604 was significantly improved compared to cationic lipids E10-1, E7-1, Compound 5, and Compound 9, which also have a dimethylamino head structure. For example, the cell transfection efficiency of YK-1603 was 1.88-fold, 2.47-fold, 1.56-fold, and 1.77-fold that of E10-1, E7-1, Compound 5, and Compound 9, respectively.

[0519] 3. The cell transfection efficiency of mRNA-LNP compositions prepared with YK-1605, YK-1606, YK-1608, YK-1610, and YK-1612 was significantly improved compared to cationic lipids E24-1 and Compound 92-12, which also have a 4-methylpiperazine head structure. For example, the cell transfection efficiency of YK-1606 was 2.49 times and 3.19 times that of E24-1 and Compound 92-12, respectively.

[0520] 4. The cell transfection efficiency of mRNA-LNP compositions prepared with YK-1614 was significantly improved compared to Lipid-028, a cationic lipid with the same diethylamino head structure. For example, the cell transfection efficiency of YK-1614 was 1.80 times that of Lipid-028.

[0521] 5. The cell transfection efficiency of the mRNA-LNP composition prepared with YK-1606 was significantly improved compared to the cationic lipid Reference 1, which was structurally identical at all other positions but differed in chirality. For example, the cell transfection efficiency of YK-1606 was 2.58 times that of Reference 1.

[0522] Example 7: In vivo delivery performance of LNPs

[0523] The Fluc-mRNA-LNP composition prepared in Example 4 was injected intramuscularly into female BALB / c albino mice, 4-6 weeks old and weighing 17-19 g (approximately 5 μg Fluc-mRNA / mouse). Six hours after administration, the fluorescent imaging substrate was injected intraperitoneally into the mice. The mice were allowed to move freely for 5 minutes. The total radiant intensity of the protein expressed in the mice by the mRNA-LNP composition (corresponding to the fluorescent protein expression intensity, i.e., protein expression level) was then measured using an IVIS Spectrum Small Animal Live Imager. After sampling, the mice were sacrificed by cervical dislocation and dissected. The heart, liver, spleen, lungs, and kidneys were precisely isolated. The total radiant intensity of the protein expressed by the Fluc-mRNA at the injection site and in each organ was measured using an IVIS Spectrum Small Animal Live Imager (corresponding to the fluorescent protein expression intensity, i.e., protein expression level). Based on the test results, the relative fluorescence intensity of the heart, liver, spleen, lungs, and kidneys relative to the injection site was calculated (see Formula 1 for the calculation formula). The test results are shown in Table 4 and Figure 3 .

[0524] Relative fluorescence intensity relative to the injection site = total radiation intensity of the protein expressed in the organ / total radiation intensity of the protein expressed at the injection site - Formula 1

[0525] Table 4 Mouse in vivo and organ imaging experimental data

[0526]

[0527] Figure 4 Figure 2 exemplifies fluorescence imaging of the mouse body and its heart, liver, spleen, lung, and kidney 6 hours after intramuscular injection of an mRNA-LNP composition encapsulating Fluc-mRNA prepared based on YK-1605 and YK-1606. It can be seen from the figure that there is strong fluorescence at the injection site of the mouse, while no obvious fluorescence is found in organs such as the heart, liver, spleen, lung, and kidney.

[0528] The mRNA-LNP composition prepared from the cationic lipid compound of the present disclosure can efficiently target mRNA to muscle, and the delivery effect and targeting are significantly enhanced compared with SM-102, MC3, lipid-028, E10-1, E24-1, E7-1, compound 92-12, compound 5, compound 9 and control 1. Specifically:

[0529] 1. Except for the injection site, the mRNA-LNP compositions prepared by the compounds of the present invention had no obvious expression in the heart, liver, spleen, lung and kidney, while the mRNA-LNP compositions prepared by SM-102, MC3, lipid-028, E10-1, E24-1, E7-1, compound 92-12, compound 5 and compound 9 were significantly expressed in organs, especially the liver and spleen. For example, the total radiation intensity in the liver of the mRNA-LNP composition prepared by YK-1606 was 3%, 5%, 11%, 11%, 12%, 13%, 20%, 50% and 40% of the total radiation intensity in the liver of mice injected with SM-102, MC3, lipid-028, E10-1, E24-1, E7-1 and the mRNA-LNP compositions prepared by compound 92-12, compound 5 and compound 9, respectively. After injection of the mRNA-LNP compositions prepared with the compounds disclosed herein, the ratio of the total radiation intensity of each organ of the mouse to the total radiation intensity at the injection site was less than 0.1. This indicates that these mRNA-LNP compositions have extremely high targeting to the muscles, and the amount of off-target radiation that enters the heart, liver, spleen, lungs, and kidneys is extremely small, indicating that the possibility of causing organ toxicity is greatly reduced.

[0530] Although the ratio of the total radiation intensity of each organ to the total radiation intensity at the injection site of the mRNA-LNP compositions prepared with Compound 5 and Compound 9 is also at a lower level compared to mRNA-LNP compositions prepared with other prior art lipids, their total radiation intensity at the injection site is still lower. For example, the total radiation intensity at the injection site of the mRNA-LNP composition prepared using YK-1612 was 1.5 times and 2.7 times the total radiation intensity at the injection site of the mouse mRNA-LNP composition prepared with Compound 5 and Compound 9, respectively. Moreover, compared with the compounds of the present disclosure, the ratio of the total radiation intensity of each organ to the total radiation intensity at the injection site of the mRNA-LNP compositions prepared with Compound 5 and Compound 9 is still higher (i.e., the possibility of organ toxicity is greater). For example, compared with the mRNA-LNP composition prepared by YK-1612, the relative fluorescence intensity of the mRNA-LNP compositions prepared using Compound 5 and Compound 9 in the heart increased by 88% and 106%, respectively; the relative fluorescence intensity in the liver increased by 120% and 403%, respectively; the relative fluorescence intensity in the spleen increased by 18% and 168%, respectively; the relative fluorescence intensity in the lung increased by 375% and 1075%, respectively; and the relative fluorescence intensity in the kidney increased by 225% and 500%, respectively (Note: increase in relative fluorescence intensity = (relative fluorescence intensity of the mRNA-LNP composition prepared by Compound 5 or Compound 9 - relative fluorescence intensity of the mRNA-LNP composition prepared by YK-1612) / relative fluorescence intensity of the mRNA-LNP composition prepared by YK-1612 × 100%).

[0531] 2. In addition to the extremely low ratio of the total radiation intensity in each organ to the total radiation intensity at the injection site, the mRNA-LNP composition prepared from the disclosed compounds also has the advantage of achieving a higher level of expression at the injection site (muscle).

[0532] For example, the total radiation intensity of the mRNA-LNP compositions prepared by YK-1603, YK-1604, YK-1605, YK-1606, YK-1608, YK-1610, YK-1612, YK-1613, YK-1614, YK-1615, and YK-1618 at the injection site reached 2.63 × 10 8 p / s, 3.77×10 8 p / s, 4.66×10 8 p / s, 4.89×10 8 p / s, 1.99×10 8 p / s, 2.83×10 8 p / s, 2.3×10 8 p / s, 2.79×10 8 p / s, 1.23×108 p / s, 3.05×10 8 p / s, 3.56×10 8 p / s, reaching a comparable level or even exceeding several times that of SM-102, the control compound with the highest expression at the injection site. The control compound SM-102 accumulated significantly in the liver and spleen (ratios to the total radiation intensity at the injection site were 0.958 and 0.458, respectively), significantly higher than the mRNA-LNP compositions prepared using the compounds of the present disclosure.

[0533] For another example, the total radiation intensity in the muscle area of ​​the mRNA-LNP composition prepared by YK-1606 was 2.3 times, 4.1 times, 5.9 times, 6.4 times, 7.3 times, 6.9 times, 8.4 times, 3.2 times and 5.8 times that of the mRNA-LNP compositions prepared by SM-102, MC3, lipid-028, E10-1, E24-1, E7-1, compound 92-12, compound 5 and compound 9, respectively.

[0534] 3. In addition, the test results also show that compared with the mRNA-LNP composition prepared from the control sample 1, which has the same structure at other positions but no chirality, the total radiation intensity at the muscle site of the mRNA-LNP composition prepared from the YK-1606 disclosed in the present invention is significantly enhanced. The total radiation intensity at the muscle site of the mRNA-LNP composition prepared from the control sample 1 is only 1.03×10 8 p / s, while the total radiation intensity of the mRNA-LNP composition prepared by YK-1606 in the muscle area was 4.7 times that of the control 1.

[0535] Furthermore, the expression level of the mRNA-LNP composition prepared with YK-1606 in various organs was also lower compared to the control 1. For example, compared to the mRNA-LNP composition prepared with YK-1606, the relative fluorescence intensity of the mRNA-LNP composition prepared with the control 1 in the heart, liver, spleen, lung, and kidney increased by 217%, 142%, 138%, 150%, and 217%, respectively (Note: relative fluorescence intensity increase = (relative fluorescence intensity of the mRNA-LNP composition prepared with the control 1 - relative fluorescence intensity of the mRNA-LNP composition prepared with YK-1606) / relative fluorescence intensity of the mRNA-LNP composition prepared with YK-1606 × 100%).

Claims

1. A compound or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound has a structure represented by formula (I): ， in: R1 is 、 ,or ; R2 is 、 , or unsubstituted C 8-11 straight-chain alkanes; R3 is 、 、 , unsubstituted C 8-11 straight-chain alkanes, or H; R4 is , or unsubstituted C 8-10 straight-chain alkanes; L1 is -C(O)(CH2)5-, -(CH2)3-, or -(CH2)8-; L2 is -(CH2)5-, -(CH2)8-, or is absent; L3 is -(CH2)5-, or is omitted; M1 is -CH=CH-, -C(O)O- or -OC(O)-; M2 is -CH=CH-, -C(O)O-, or is absent; M3 is -OC(O)-; M4 is -OC(O)-, or is omitted; When L2 and M2 are both defaulted, L3 and / or M4 are also defaulted.

2. The compound according to claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein The compound has any of the following structures: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 and 。 3. A carrier comprising a cationic lipid provided by the compound according to claim 1 or 2, or a pharmaceutically acceptable salt or stereoisomer thereof.

4. The carrier according to claim 3, 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; And / or, the carrier further comprises one or more other ionizable lipid compounds.

5. The carrier according to claim 3 or 4, wherein In the carrier, the molar percentage of cationic lipid is 25%-75%; and / or, in the carrier, the molar percentage of neutral lipid in the carrier is 5%-25%; and / or, in the carrier, the molar percentage of the structural lipid in the carrier is 15%-65%; And / or, in the carrier, the molar percentage of the polymer-conjugated lipid in the carrier is 0.5%-10%.

6. The carrier according to claim 5, wherein The molar ratio of cationic lipid to neutral lipid in the carrier is 1:1-15:1; and / or, in the carrier, the molar ratio of the cationic lipid to the structural lipid is 0.5:1-3:1; And / or, in the carrier, the molar ratio of the cationic lipid to the polymer-conjugated lipid is 4:1-35:

1.

7. The carrier according to claim 6, wherein In the carrier, the molar ratio of cationic lipid, neutral lipid, structural lipid and polymer conjugated lipid is (25-75):(5-25):(15-65):(0.5-10).

8. The carrier according to claim 7, wherein In the carrier, the molar ratio of cationic lipid, neutral lipid, structural lipid and polymer conjugated lipid is (35-49):(7.5-15):(35-55):(1-5).

9. 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.

10. The carrier according to claim 9, 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, 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-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero- 3-Phosphoethanolamine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-r ac-(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; and / or, the structured lipid is cholesterol; And / or, 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.

11. The carrier according to claim 10, wherein The neutral lipid is selected from 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine and / or 1,2-distearoyl-sn-glycero-3-phosphocholine.

12. A composition comprising a carrier and an active ingredient, wherein: The vector comprises the vector according to any one of claims 3 to 11.

13. The composition according to claim 12, 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.

14. The composition according to claim 13, 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.

15. The composition according to claim 12, wherein The active ingredient comprises a therapeutic agent and / or a prophylactic agent.

16. The composition according to claim 15, wherein The therapeutic agent and / or preventive agent is selected from any one of the group consisting of nucleic acid molecules, small molecule compounds, polypeptides or proteins, or a combination of at least two of them.

17. The composition according to claim 15, wherein The therapeutic and / or preventive agent is a vaccine or a compound capable of eliciting an immune response.

18. The composition according to claim 15, wherein The therapeutic and / or prophylactic agent is a nucleic acid.

19. The composition according to claim 18, wherein The therapeutic and / or prophylactic agent is RNA.

20. The composition according to claim 19, wherein The RNA is selected from any one or a combination of at least two of the group consisting of small interfering RNA, asymmetric interfering RNA, microRNA, Dicer-substrate RNA, small hairpin RNA, and messenger RNA.

21. The composition according to claim 20, wherein The RNA is messenger RNA.

22. The composition according to claim 12, wherein In the composition, the mass ratio of the carrier to the active component is 10:1-30:

1.

23. The composition according to claim 22, wherein In the composition, the mass ratio of the carrier to the active component is 12.5:1-20:

1.

24. The composition according to claim 23, wherein In the composition, the mass ratio of the carrier to the active component is 13:1-17:

1.

25. The composition according to any one of claims 12 to 24, wherein The composition further comprises a pharmaceutically acceptable excipient and / or diluent.

26. Use of the compound of claim 1 or 2 or a pharmaceutically acceptable salt or stereoisomer thereof, or the vector of any one of claims 3 to 11, or the composition of any one of claims 12 to 25 for improving cell transfection efficiency, wherein the use is non-therapeutic, non-diagnostic.

27. The use according to claim 26, wherein The use is to improve cell transfection efficiency in vitro.

28. Use of the compound according to claim 1 or 2, or a pharmaceutically acceptable salt or stereoisomer thereof, or the vector according to any one of claims 3 to 11, or the composition according to any one of claims 12 to 25, in improving the targeting of a drug to 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.

29. The use according to claim 28, wherein The target tissue is selected from muscle; and / or the target cell is selected from muscle cell.

30. Use of the compound of claim 1 or 2 or a pharmaceutically acceptable salt or stereoisomer thereof, or the vector of any one of claims 3 to 11, or the composition of any one of claims 12 to 25 for increasing the ratio of the expression amount of a nucleic acid in a target to the expression amount in a non-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.

31. The use according to claim 30, wherein The target tissue is selected from muscle; and / or the target cell is selected from muscle cell.

32. Use of the compound according to claim 1 or 2, or a pharmaceutically acceptable salt or stereoisomer thereof, or the vector according to any one of claims 3 to 11, or the composition according to any one of claims 12 to 25, in reducing organ toxicity of a drug, wherein the use is non-therapeutic, non-diagnostic.

33. Use of the compound according to claim 1 or 2 or a pharmaceutically acceptable salt or stereoisomer thereof, or the carrier according to any one of claims 3 to 11, or the composition according to any one of claims 12 to 25 in the preparation of a medicament.

34. The use according to claim 33, 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.

35. The use according to claim 33 or 34, wherein Accumulation of the active ingredient of the drug in organs can lead to organ toxicity.

36. The use according to claim 35, wherein The organ is selected from any one or a combination of at least two of the group consisting of heart, liver, spleen, lung and kidney.

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