Novel long-acting and low-toxic cationic lipid compound and composition thereof

Abstract

The present invention provides a novel cationic lipid compound with long-acting and low toxicity, which is a compound of formula (I) or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer. Also provided are compositions comprising the aforementioned compounds and their use for delivering therapeutic agents or preventive agents.

Description

[0001] This application is a divisional application of the Chinese invention patent application (application number: 202310010912.6, application date: January 5, 2023, invention name: Novel long-acting and low-toxic cationic lipid compounds and their compositions). Technical Field

[0002] The present invention belongs to the field of medicine and specifically relates to a cationic lipid compound, a composition containing the same and its use. Background Art

[0003] The effective targeted delivery of bioactive substances such as small molecule drugs, peptides, proteins and nucleic acids, especially nucleic acids, is a persistent medical problem. Nucleic acid therapeutics face great challenges due to their low cell permeability and high sensitivity to degradation of certain nucleic acid molecules (including RNA).

[0004] Confirm that compositions, liposomes and liposome complexes (lipoplex) containing cationic lipids are used as transport vehicles to effectively transport bioactive substances such as small molecule drugs, polypeptides, proteins and nucleic acids to cells and / or intracellular compartments. These compositions generally include one or more "cationic" and / or amino (ionizable) lipids, including neutral lipids, structural lipids and polymer conjugated lipids. Cationic and / or ionizable lipids include amine-containing lipids that can be easily protonated. Although multiple such nanoparticle compositions containing lipids have been demonstrated, safety, efficacy and specificity are still to be improved. It should be noted that the increase of lipid nanoparticle (Lipid Nanoparticle, LNP) complexity complicates its production and may increase its toxicity, which is a major concern that may limit its clinical application. For example, LNP siRNA particles (such as patisiran) require the use of steroids and antihistamines in advance to eliminate unnecessary immune responses (T. Coelho, D. Adams, A. Silva, et al., Safety and efficacy of RNAi therapy for transthyretinamyloidosis, N Engl J Med, 369 (2013) 819-829.). Therefore, there is a need to develop improved cationic lipid compounds that help deliver therapeutic and / or preventive agents such as nucleic acids to cells, and the need for compositions comprising the same. Summary of the invention

[0005] On the one hand, the present invention provides a novel cationic lipid compound, which is a compound of formula (I)

[0006] or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0007] G1 is C 1～8 Alkylene;

[0008] G2 is C 2～8 Alkylene;

[0009] R1 is C 6～25 Straight or branched chain alkyl;

[0010] R2 is C 12～25 Straight or branched chain alkyl;

[0011] G3 is: HO(CH2)2N(R3)CH2CH(OH)CH2-, wherein R3 is -CH3 or -CH2CH3 or -CH2CH2OH.

[0012] For example, the compound of formula (I) has one of the following structures:

[0013]

[0014]

[0015]

[0016]

[0017]

[0018] Another aspect of the present invention provides a composition comprising a carrier, wherein the carrier comprises a cationic lipid, and the cationic lipid comprises the above-mentioned compound of formula (I) or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer.

[0019] Another aspect of the present invention provides the use of the above-mentioned compound of formula (I) or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer or the above-mentioned composition in the preparation of nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs.

[0020] Another aspect of the present invention provides the use of the above-mentioned compound of formula (I) or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer or the above-mentioned composition in the preparation of a medicament for treating a disease or condition in a mammal in need thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The results of cell transfection experiments showing different weight ratios of vector to mRNA used in preparing LNP preparations, a is vector:mRNA=5:1, b is vector:mRNA=15:1, c is vector:mRNA=35:1; d is a blank control.

[0022] Figure 2 The results of cell transfection experiments showing different molar ratios of cationic lipids to neutral lipid DSPC used in the preparation of LNP preparations, a is 3.5:1, b is 4.5:1, c is 4.9:1, and d is a blank control.

[0023] Figure 3 The results of cell transfection experiments are shown at different molar ratios of polymer-conjugated lipids to carriers when preparing LNP preparations, a is 1.5%, b is 10%, and c is a blank control.

[0024] Figure 4 The results of cell transfection experiments are shown in different ratios of the components of the carrier, cationic lipids, neutral lipids DSPC, structural lipids cholesterol and polymer conjugated lipids DMG-PEG2000, when preparing LNP preparations. a is 35:10:53.5:1.5, b is 45:10:43.5:1.5, c is 49:10:39.5:1.5, and d is a blank control.

[0025] Figure 5 The fluorescence absorption intensity of LNP preparations of Fluc-mRNA prepared with different cationic lipids is shown (a: YK-305; b: YK-310; c: YK-319; d: SM-102).

[0026] Figure 6 The fluorescence absorption intensity of LNP preparations of Fluc-mRNA prepared with different cationic lipids is shown (a: YK-301; b: YK-302; c: YK-304; d: compound 21).

[0027] Figure 7 The fluorescence absorption intensity of LNP preparations of Fluc-mRNA prepared with different cationic lipids is shown (a: YK-305; b: YK-310; c: YK-320; d: YK-321).

[0028] Figure 8 The LNP preparations of Fluc-mRNA prepared with different cationic lipids (YK-305, YK-310, YK-312, YK-319, YK-318, YK-009, SM-102, ALC-0315, compound 21, compound 23 and HHMA) and the Lipofectamine 3000 preparation containing Fluc-mRNA were added to the cell culture medium and the cell survival rate was shown after culturing for 24 hours.

[0029] Fig. 9The LNP preparations of Fluc-mRNA prepared with different cationic lipids (YK-305, YK-310, YK-312, YK-319, YK-318, YK-301, YK-302, YK-303, YK-304, YK-306, YK-307, SM-102, ALC-0315, compound 21, compound 23 and HHMA) and the Lipofectamine 3000 preparation containing Fluc-mRNA were added to the cell culture medium and the cell survival rate was shown after 24 hours of culture.

[0030] Fig.10 The LNP preparations of Fluc-mRNA prepared with different cationic lipids (YK-305, YK-310, YK-312, YK-319, YK-318, YK-308, YK-309, YK-311, YK-313, YK-314, YK-315, SM-102, ALC-0315, compound 21, compound 23 and HHMA) and the Lipofectamine 3000 preparation containing Fluc-mRNA were added to the cell culture medium and the cell survival rate was shown after 24 hours of culture.

[0031] Fig.11 The LNP preparations of Fluc-mRNA prepared with different cationic lipids (YK-305, YK-310, YK-312, YK-319, YK-318, YK-316, YK-317, YK-320, YK-321, SM-102, ALC-0315, compound 21, compound 23 and HHMA) and the Lipofectamine 3000 preparation containing Fluc-mRNA were added to the cell culture medium and the cell survival rate was shown after 24 hours of culture.

[0032] Fig.12 The results of in vivo mouse imaging experiments of LNP formulations of Fluc-mRNA prepared with different cationic lipids (YK-305, YK-312, YK-302, YK-313, SM-102, ALC-0315, Compound 21, Compound 23, and HHMA) are shown.

[0033] Fig.13 The results of in vivo mouse imaging experiments of LNP formulations of Fluc-mRNA prepared with different cationic lipids (YK-310, YK-318, YK-309, SM-102, ALC-0315, compound 21, compound 23, and HHMA) are shown.

[0034] Fig.14The results of in vivo mouse imaging experiments of LNP formulations of Fluc-mRNA prepared with different cationic lipids (YK-319, YK-321, YK-009, SM-102, ALC-0315, compound 21, compound 23, and HHMA) are shown.

[0035] Fig.15 The protein expression of Fluc-mRNA LNP formulations prepared with different cationic lipids (ALC-0315, SM-102, YK-305, YK-319, YK-310 and YK-313) in mice is shown. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solution and advantages of the embodiment of the present invention clearer, the technical solution of the embodiment of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiment of the present invention. Obviously, the described embodiment is a part of the embodiment of the present invention, not all of the embodiments. Based on the described embodiment of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] The present invention may be implemented in other specific forms without departing from the basic attributes of the present invention. It should be understood that, without conflict, any and all embodiments of the present invention may be combined with the technical features in any other embodiment or multiple other embodiments to obtain other embodiments. The present invention includes other embodiments obtained by such combination.

[0038] All publications and patents mentioned in the present invention are hereby incorporated into the present invention by reference in their entirety. If the purposes or terms used in any publications and patents incorporated by reference conflict with the purposes or terms used in the present invention, then the purposes and terms of the present invention shall prevail.

[0039] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0040] Unless otherwise specified, all technical and scientific terms used herein have the common meaning in the field to which the claimed subject matter belongs. If there are multiple definitions for a term, the definition herein shall prevail.

[0041] Except in the working examples or otherwise indicated, all numbers of quantitative properties such as dosage stated in the specification and claims should be understood to be modified by the term "about" in all cases. It should also be understood that any numerical range recited in the application is intended to include all subranges within the range and any combination of the respective endpoints of the range or subrange.

[0042] The words "include", "contain" or "comprises" and the like used in the present invention mean that the elements preceding the word include the elements listed after the word and their equivalents, without excluding unrecorded elements. The terms "contain" or "includes (comprising)" used herein may be open, semi-closed and closed. In other words, the term also includes "essentially consisting of" or "consisting of".

[0043] The term "pharmaceutically acceptable" as used herein means that the compound or composition is chemically and / or toxicologically compatible with the other ingredients constituting the formulation and / or with humans or mammals for the prevention or treatment of a disease or condition.

[0044] The term "subject" or "patient" in this application includes humans and mammals.

[0045] The term "treatment" as used herein refers to the administration of one or more pharmaceutical substances to a patient or subject suffering from a disease or having symptoms of the disease to cure, alleviate, mitigate, improve or affect the disease or the symptoms of the disease. In the context of this application, unless otherwise specifically stated, the term "treatment" may also include prevention.

[0046] The term "solvate" refers to a complex formed by combining a compound of formula (I) or a pharmaceutically acceptable salt thereof and a solvent (e.g., ethanol or water) in the present application. It should be understood that any solvate of a compound of formula (I) used in the treatment of a disease or condition, although it may provide different properties (including pharmacokinetic properties), will yield a compound of formula (I) once absorbed into a subject, such that the use of a compound of formula (I) encompasses the use of any solvate of a compound of formula (I) respectively.

[0047] The term "hydrate" refers to the case where the solvent in the above-mentioned term "solvate" is water.

[0048] It should be further understood that the compound of formula (I) or its pharmaceutically acceptable salt can be separated in the form of a solvate, and therefore any such solvate is included within the scope of the present invention. For example, the compound of formula (I) or its pharmaceutically acceptable salt can exist in an unsolvated form and in a solvated form formed with a pharmaceutically acceptable solvent (such as water, ethanol, etc.).

[0049] The term "pharmaceutically acceptable salt" refers to a relatively non-toxic, inorganic or organic acid addition salt of a compound of the invention. For example, see SM Berge et al. "Pharmaceutical Salts", J. Pharm. Sci. 1977, 66, 1-19. Among them, inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid or nitric acid, etc.; organic acids such as formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, hexanoic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)-benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectinic acid, 3-phenylpropionic acid, picric acid, Pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, aminosulfonic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheptonic acid, glycerophosphoric acid, aspartic acid, sulfosalicylic acid, etc. For example, HCl (or hydrochloric acid), HBr (or hydrobromic acid solution), methanesulfonic acid, sulfuric acid, tartaric acid or fumaric acid can be used to form a pharmaceutically acceptable salt with the compound shown in formula (I).

[0050] The nitrogen-containing compound of formula (I) of the present invention can be converted into N-oxide by treatment with an oxidant (e.g., meta-chloroperbenzoic acid, hydrogen peroxide, ozone). Therefore, under conditions where valence and structure permit, the compounds claimed in the present application include not only the nitrogen-containing compound shown in the structural formula, but also its N-oxide derivatives.

[0051] Certain compounds of the present invention may exist in the form of one or more stereoisomers. Stereoisomers include geometric isomers, diastereomers and enantiomers. Therefore, the compounds claimed in the present invention 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 other methods. 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 form salts with the compounds of the present invention, and the physical and chemical properties of the products, such as different solubility, can be used for separation. For example, when the raw material 1-amino-3-chloropropane-2-ol for synthesizing the cationic lipid is (R)-1-amino-3-chloropropane-2-ol or (S)-1-amino-3-chloropropane-2-ol, a single stereoisomer of the cationic lipid can be obtained; and when the raw material 1-amino-3-chloropropane-2-ol for synthesizing the cationic lipid is a racemic mixture, a racemic cationic lipid can be obtained.

[0052] The present invention also includes all suitable isotopic variations of the compounds of the present invention. An isotopic variation 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 compounds of the present invention include isotopes of hydrogen, carbon, nitrogen and oxygen, for example 2 H (deuterium), 3 H (tritium), 11 C. 13 C. 14 C. 15 N. 17 O and 18 O.

[0053] The term "alkyl" in the present invention refers to a branched and straight chain saturated aliphatic monovalent hydrocarbon group having a specified number of carbon atoms. The term "alkylene" in the present invention refers to a branched and straight chain saturated aliphatic divalent hydrocarbon group having a specified number of carbon atoms. n～m It refers to a group having n to m carbon atoms. 2～5 The alkylene group includes C2 alkylene group, C3 alkylene group, C4 alkylene group and C5 alkylene group.

[0054] The alkyl (or alkylene) group may be unsubstituted, or the alkyl (or alkylene) group may be substituted wherein at least one hydrogen is replaced with another chemical group.

[0055] A "therapeutically effective amount" is an amount of a therapeutic agent that ameliorates a disease or symptom when administered to a patient. A "prophylactically effective amount" is an amount of a prophylactic agent that prevents a disease or symptom when administered to a subject. 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" varies with the therapeutic agent / prophylactic agent, the disease state and its severity, the age, weight, etc. of the patient / subject to be treated / prevented. A person of ordinary skill in the art can routinely determine a therapeutically effective amount and a prophylactically effective amount based on his or her knowledge and the present invention.

[0056] In this application, when the name of a compound is inconsistent with the structural formula, the structural formula shall prevail.

[0057] It should be understood that the term "compounds of the present invention" used herein may include, depending on the context, compounds of formula (I), N-oxides thereof, solvates thereof, pharmaceutically acceptable salts thereof, stereoisomers thereof, and mixtures thereof.

[0058] As used herein, the term cationic lipid refers to a lipid that has a positive charge at a selected pH value.

[0059] Cationic liposomes easily bind to negatively charged nucleic acids, that is, they interact with negatively charged phosphate groups in nucleic acids through electrostatic forces to form lipid nanoparticles (LNPs). LNPs are currently one of the mainstream delivery vectors.

[0060] When screening a large number of compounds, the inventors found that it was very difficult to screen out suitable cationic lipid compounds that meet the following conditions: a huge difference in structure from representative cationic lipids in the prior art, extremely high transfection efficiency and extremely low cytotoxicity, and high and sustained expression in mice. The inventors found that some compounds, such as YK-305, YK-310, YK-312, YK-319 and YK-318, can deliver nucleic acids with significantly improved intracellular transfection efficiency, significantly reduced cytotoxicity, and significantly increased expression and duration in animals compared to cationic lipids with very different chemical structures in the prior art.

[0061] Briefly, the present invention is based on at least the following findings:

[0062] 1. The designed series of compounds, including YK-305, YK-310, YK-312, YK-319 and YK-318, have huge chemical structure differences from representative cationic lipids in the prior art, such as SM-102 (compound 25 disclosed in WO2017049245A2), ALC-0315 (compound 3 disclosed in CN108368028B), compounds 21 and 23 disclosed in WO2021055833A1, HHMA (compound 1 disclosed in CN112979483B) and compound YK-009 disclosed in CN114044741B. The G3 group is completely different, and other parts are also very different, so there will be great differences in polarity, acidity and alkalinity, and hydrophilicity.

[0063] Therefore, it is impossible to infer the cell transfection efficiency, cytotoxicity, and in vivo expression of the LNP preparations prepared from the cationic lipid compounds disclosed in the above-mentioned prior art based on this series of compounds.

[0064] The chemical structures of SM-102, ALC-0315, compound 21, compound 23 and HHMA are as follows:

[0065] (WO2017049245A2, specification page 29);

[0066] (CN108368028B, specification page 24); (WO2021055833A1, specification page 22);

[0067] (WO2021055833A1, specification page 22);

[0068] (CN112979483B, instruction manual page 12)

[0069] 2. Among this series of designed compounds, the LNP preparations prepared from YK-305, YK-310, YK-312, YK-319 and YK-318 have significantly improved cell transfection efficiency, significantly reduced cytotoxicity, significantly increased mRNA expression level and duration in mice, and reduced or no liver toxicity compared with representative cationic lipids in the prior art.

[0070] For example, the cell transfection efficiency of YK-305 can reach 17 times that of SM-102, 19 times that of compound 21, and 20 times that of compound 23; the cell survival rate of YK-305 and YK-310 can be 30% higher than that of ALC-0315, 12% higher than that of SM-102, and 15% higher than that of HHMA; the mRNA expression level in mice of YK-305 and YK-310 can reach 30 times that of SM-102, compound 21, and compound 23.

[0071] Among the series of compounds we designed with very small differences in chemical structure, the LNP preparations prepared from YK-305, YK-310, YK-312, YK-319 and YK-318 showed significantly improved cell transfection activity, significantly reduced cytotoxicity, and significantly increased mRNA expression level and duration in mice compared with other compounds.

[0072] The structures of this series of compounds are slightly different from those of YK-305, YK-310, YK-312, YK-319 and YK-318 in individual groups, but the cell transfection activity of YK-305 can reach 1,300 times that of YK-304 and 900 times that of YK-302; the cytotoxicity of YK-305 and YK-310 can be reduced by 65% ​​compared with YK-302; the mRNA expression level of YK-305 in mice can reach more than 1,000 times that of YK-302.

[0073] 3. There is no obvious correspondence between the structure of cationic lipid compounds and the intracellular transfection efficiency, toxicity to cells, and high and sustained expression of mRNA in LNP preparations prepared from them in animals. Compounds with small structural differences are very likely to have very large differences in transfection efficiency and / or toxicity to cells, and intracellular expression.

[0074] For example, compared with YK-305, YK-302 only has 2 fewer carbon atoms in the G1 group; the R1 group is a straight chain structure, while YK-305 is a branched structure; the R2 group has 1 more carbon in the single chain, and 2 fewer carbon atoms in each single chain of the double chain; the other structures are exactly the same, but the cell transfection efficiency of YK-305 is 900 times that of YK-302, and the toxicity of YK-305 to transfected cells is 65% lower than that of YK-302, and the mRNA expression of YK-305 in mice can reach 1000 times that of YK-302; compared with YK-310, YK-303 only has 1 less carbon atoms in the group connected to N in the G3 group; the other structures are exactly the same, but the cell transfection efficiency of YK-310 is 40 times that of YK-303, and the toxicity of YK-310 to transfected cells is 18% lower than that of YK-303.

[0075] Therefore, screening suitable cationic lipid compounds that can simultaneously have high transfection efficiency and low toxicity to cells, as well as high and sustained expression of mRNA in mice is a very difficult task, which requires a lot of creative work.

[0076] 4. Through unique design and extensive screening, the present invention has found some compounds, such as YK-305, YK-310, YK-312, YK-319 and YK-318, which can deliver nucleic acids with significantly improved cell transfection efficiency, significantly reduced cytotoxicity, and significantly increased expression amount and duration in animals, and have reduced or no toxicity to the liver, compared with other compounds in the prior art. Unexpected technical effects have been achieved.

[0077] In summary, the present invention has discovered some compounds, such as YK-305, YK-310, YK-312, YK-319 and YK-318, through unique design and extensive screening. These compounds have great differences in chemical structure from representative cationic lipids in the prior art. Compared with other compounds in the prior art, they can deliver nucleic acids with significantly improved cell transfection efficiency, significantly reduced cytotoxicity, and significantly improved expression amount and duration in animals, and have reduced or no toxicity to the liver. Unexpected technical effects have been achieved.

[0078] The details are as follows:

[0079] 1. Compared with the representative cationic lipids in the prior art, such as SM-102, ALC-0315, compound 21, compound 23, YK-009 and HHMA, the chemical structure is very different

[0080] Representative cationic lipids of the prior art, such as SM-102, ALC-0315, Compound 21, Compound 23, YK-009 and HHMA, are compared with this series of designed compounds:

[0081] a. The HHMA structure is the most different. From the chemical structure diagram, it can be seen that in the group connected to the central N atom of HHMA, only one side chain is similar to one side chain of this series of structures, and the other parts are completely different.

[0082] b. Other cationic lipids in the prior art, such as SM-102, ALC-0315, compound 21, compound 23 and YK-009, have completely different G3 groups. This series of compounds has one more tertiary amine group and 1-2 more hydroxyl groups in the G3 group, so there are also great differences in polarity, acidity and alkalinity, and hydrophilicity.

[0083] c. There are also huge differences in the G1, G2, R1 and R2 groups of SM-102, ALC-0315, Compound 21, Compound 23 and YK-009.

[0084] 2. The in vitro cell transfection efficiency is significantly improved compared to the representative cationic lipids and structurally similar compounds in the prior art

[0085] 1) The LNP preparations prepared from YK-305, YK-310, YK-312, YK-319 and YK-318 have the highest cell transfection efficiency, and their activity is significantly improved compared to the representative cationic lipids in the prior art. For example, YK-305 can reach 17.09 times that of SM-102, 19.14 times that of compound 21 and 20.21 times that of compound 23.

[0086] 2) Compared with compounds with similar structures and G3 groups of HO(CH2)2N(CH3)CH2CH(OH)CH2-, YK-305, YK-310, YK-312, YK-319 and YK-318 have the highest cell transfection efficiency. YK-305 is 1300 times higher than YK-304 and 900 times higher than YK-302.

[0087] 3) Compared with compounds with similar structures and G3 groups of HO(CH2)2N(CH2CH3)CH2CH(OH)CH2-, YK-305, YK-310, YK-312, YK-319 and YK-318 have the highest cell transfection efficiency. YK-305 and YK-310 are 200 times higher than YK-309.

[0088] 4) Compared with compounds with similar structures and G3 groups of (HO(CH2)2)2NCH2CH(OH)CH2-, YK-305, YK-310, YK-312, YK-319 and YK-318 have the highest cell transfection efficiency. For example, YK-305 can reach 20 times that of YK-321.

[0089] 5) There is no corresponding relationship between the structure of the compound and the transfection efficiency in the cell. Compounds with small structural differences are likely to have very large differences in transfection efficiency. Therefore, it is very difficult to screen out cationic lipid compounds with high transfection efficiency, which requires a lot of creative work.

[0090] 3. Cytotoxicity is significantly reduced compared to representative cationic lipids and structurally similar compounds in the prior art

[0091] 1) The LNP preparations prepared from YK-305, YK-310, YK-312, YK-319 and YK-318 have the lowest cytotoxicity and significantly improve cell survival rates compared to representative cationic lipids in the prior art. For example, the cell survival rates of YK-305 and YK-310 are 30% higher than ALC-0315, 12% higher than SM-102, and 15% higher than HHMA;

[0092] 2) Compared with compounds with similar structures and G3 groups of HO(CH2)2N(CH3)CH2CH(OH)CH2-, YK-305, YK-310, YK-312, YK-319 and YK-318 showed the lowest cytotoxicity and significantly improved cell survival rates. For example, YK-305 and YK-310 were both improved by 65% ​​compared with YK-302.

[0093] 3) Compared with compounds with similar structures and G3 groups of HO(CH2)2N(CH2CH3)CH2CH(OH)CH2-, YK-305, YK-310, YK-312, YK-319 and YK-318 have the lowest cytotoxicity and significantly improved cell survival rates. For example, YK-305 and YK-310 can improve cell survival rates by 50% compared to YK-302.

[0094] 4) Compared with compounds with similar structures and G3 groups of (HO(CH2)2)2NCH2CH(OH)CH2-, YK-305, YK-310, YK-312, YK-319 and YK-318 have the lowest cytotoxicity and significantly improve cell survival rates. For example, YK-305 and YK-310 can improve cell survival rates by 20% compared to YK-317.

[0095] 5) There is no correspondence between the structure and cytotoxicity of a compound. Even compounds with small structural differences are likely to have very large differences in cytotoxicity. Therefore, it is impossible to predict cytotoxicity based on chemical structure. Screening out cationic lipid compounds with low cytotoxicity is very difficult and requires a lot of creative work.

[0096] 4. The expression level and duration of mRNA in animals are significantly improved compared to representative cationic lipids and structurally similar compounds in the prior art, and the liver toxicity is reduced or non-toxic.

[0097] 1) The LNP preparations prepared by YK-305, YK-310, YK-312, YK-319 and YK-318 had the highest mRNA expression in mice and sustained expression, and the expression levels at 6h, 24h, 48h and 7d were significantly higher than those of representative cationic lipids in the prior art. For example, YK-305 and YK-310 were 30 times higher than those of SM-102, compound 21 and compound 23.

[0098] 2) Compared with compounds with similar structures and G3 groups of HO(CH2)2N(CH3)CH2CH(OH)CH2-, LNP preparations prepared from YK-305, YK-310, YK-312, YK-319 and YK-318 showed the highest mRNA expression intensity and the longest duration in mice. For example, YK-305 could reach more than 1000 times that of YK-302 at 48h and still more than 300 times at 7d.

[0099] 3) Compared with compounds with similar structures and G3 groups of HO(CH2)2N(CH2CH3)CH2CH(OH)CH2-, LNP preparations prepared from YK-305, YK-310, YK-312, YK-319 and YK-318 showed the highest mRNA expression intensity and the longest duration in mice. For example, YK-305 could reach 160 times that of YK-309 at 48h and 100 times at 7d.

[0100] 4) Compared with compounds with similar structures and G3 groups (HO(CH2)2)2NCH2CH(OH)CH2-, LNP preparations prepared from YK-305, YK-310, YK-312, YK-319 and YK-318 showed the highest mRNA expression intensity and the longest duration in mice. For example, YK-310 could reach 29 times that of YK-321 at 24h and 17 times at 7d.

[0101] 5) Compared with representative cationic lipids in the prior art, such as SM-102, ALC-0315, compound 21, compound 23 and HHMA, the liposomes prepared by the compounds designed by us have reduced expression of the target protein in the liver, or do not stay in the liver and express the target protein. Therefore, compared with cationic lipids in the prior art, the LNP preparations prepared by the compounds designed by us have reduced or no toxicity to the liver.

[0102] 6) There is no correspondence between the structure of cationic lipids and the high and sustained expression of delivered mRNA in mice. Even cationic lipid compounds with small structural differences are likely to have very different mRNA expression in animals in LNP preparations. It is impossible to predict whether mRNA is highly expressed and sustained in animals based on the chemical structure of cationic lipids. It is very difficult to screen out cationic lipid compounds with high and sustained mRNA expression, which requires a lot of creative work.

[0103] The present invention provides a novel cationic lipid compound for delivering a therapeutic agent or a preventive agent. The cationic lipid compound of the present invention can be used to deliver nucleic acid molecules, small molecules, polypeptides or proteins. Compared with known cationic lipid compounds, the cationic lipid compound of the present invention exhibits higher transfection efficiency and less cytotoxicity, thereby improving delivery efficiency and safety.

[0104] The present invention provides a cationic lipid, which is a compound of formula (I)

[0105] or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein

[0106] G1 is C 1～8 Alkylene, preferably unsubstituted C 3～7 Alkylene, more preferably unsubstituted C3 alkylene or unsubstituted C5 alkylene or unsubstituted C6 alkylene;

[0107] G2 is C 2～8 Alkylene, preferably unsubstituted C 3～7 Alkylene, more preferably unsubstituted C3 alkylene, C5 alkylene or C6 alkylene;

[0108] R1 is C 6～25 Straight or branched chain alkyl, preferably unsubstituted C 11 Straight chain alkyl or unsubstituted C 12～24 Branched alkyl, unsubstituted C 12～24 The branched alkyl group is preferably an unsubstituted C 18 Branched alkyl or C 15 Branched alkyl or C 24 Branched alkyl or C 17 Branched chain alkyl;

[0109] R2 is C 12～25 Straight or branched chain alkyl, preferably unsubstituted C 11 Straight chain alkyl or unsubstituted C 14～24 Branched alkyl, unsubstituted C 14～24 The branched alkyl group is preferably an unsubstituted C 18Branched alkyl or C 24 Branched alkyl or C 15 Branched alkyl or C 17 Branched chain alkyl;

[0110] G3 is: HO(CH2)2N(R3)CH2CH(OH)CH2-, wherein R3 is -CH3 or -CH2CH3 or -CH2CH2OH.

[0111] In one embodiment, G1 is unsubstituted C3 alkylene, for example, -(CH2)3-.

[0112] In one embodiment, G1 is unsubstituted C5 alkylene, for example, -(CH2)5-.

[0113] In one embodiment, G1 is unsubstituted C6 alkylene, for example, -(CH2)6-.

[0114] In one embodiment, G2 is unsubstituted C3 alkylene, for example, -(CH2)3-.

[0115] In one embodiment, G2 is unsubstituted C5 alkylene, for example, -(CH2)5-.

[0116] In one embodiment, G2 is unsubstituted C6 alkylene, for example, -(CH2)6-.

[0117] In one embodiment, G3 is HO(CH2)2N(CH3)CH2CH(OH)CH2-.

[0118] In one embodiment, G3 is HO(CH2)2N(CH2CH3)CH2CH(OH)CH2-.

[0119] In one embodiment, G3 is (HO(CH2)2)2NCH2CH(OH)CH2-.

[0120] In one embodiment, R1 is unsubstituted C 11 Straight chain alkyl, i.e. -(CH2) 10 CH3.

[0121] In one embodiment, R1 is unsubstituted C 18 Branched alkyl, C 15 Branched alkyl, C 24 Branched alkyl or C 17 Branched alkyl. For example, R1 is:

[0122] In one embodiment, R2 is unsubstituted C 11Straight chain alkyl, i.e. -(CH2) 10 CH3.

[0123] In one embodiment, R2 is unsubstituted C 18 Branched alkyl, C 24 Branched alkyl, C 15 Branched alkyl or C 17 Branched alkyl. For example, R2 is:

[0124] In one embodiment, G1 is -(CH2)5-, G2 is -(CH2)5-, G3 is HO(CH2)2N(CH3)CH2CH(OH)CH2-, and R1 is: R2 is:

[0125] In one embodiment, G1 is -(CH2)5-, G2 is -(CH2)3-, G3 is HO(CH2)2N(CH2CH3)CH2CH(OH)CH2-, R1 is -(CH2) 10 CH3, R2 is:

[0126] In one embodiment, G1 is -(CH2)5-, G2 is -(CH2)5-, G3 is HO(CH2)2N(CH2CH3)CH2CH(OH)CH2-, and R1 is: R2 is:

[0127] In one embodiment, G1 is -(CH2)6-, G2 is -(CH2)6-, G3 is (HO(CH2)2)2NCH2CH(OH)CH2-, and R1 is: R2 is:

[0128] In one embodiment, G1 is -(CH2)5-, G2 is -(CH2)5-, G3 is (HO(CH2)2)2NCH2CH(OH)CH2-, and R1 is: R2 is:

[0129] In an exemplary embodiment, the compound is selected from the following compounds or N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers thereof:

[0130]

[0131]

[0132]

[0133]

[0134]

[0135] Another aspect of the present invention provides a composition comprising a carrier, wherein the carrier comprises a cationic lipid, and the cationic lipid comprises the above-mentioned compound of formula (I) or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer.

[0136] In one embodiment, the composition is a nanoparticle preparation, the average size of the nanoparticle preparation is 10nm to 300nm, preferably 90nm to 280nm; the polydispersity coefficient of the nanoparticle preparation is ≤50%, preferably ≤45%, more preferably ≤40%.

[0137] Cationic lipids

[0138] In one embodiment of the composition / carrier of the present invention, the cationic lipid is selected from one or more of the above-mentioned formula (I) compound or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer. In one embodiment, the cationic lipid is selected from the above-mentioned formula (I) compound. For example, the cationic lipid is compound YK-301, YK-302, YK-303, YK-304, YK-305, YK-306, YK-307, YK-308, YK-309, YK-310, YK-311, YK-312, YK-313, YK-314, YK-315, YK-316, YK-317, YK-318, YK-319, YK-320 and YK-321. In one preferred embodiment, the cationic lipid is compound YK-305, in another preferred embodiment, the cationic lipid is compound YK-310, in another preferred embodiment, the cationic lipid is compound YK-312, in another preferred embodiment, the cationic lipid is compound YK-319, and in another preferred embodiment, the cationic lipid is compound YK-318.

[0139] In another embodiment of the composition / carrier of the present invention, the cationic lipid includes: (a) one or more selected from the above-mentioned compound of formula (I) or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer; (b) one or more other ionizable lipid compounds different from (a). (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 (Compound 25 in WO2017049245A2), or Compound 21 and Compound 23 in WO2021055833, or HHMA (Compound 1 in CN112979483B).

[0140] In one embodiment, the molar ratio of the cationic lipid to the carrier is 25% to 75%, such as 30%, 40%, 49%, 55%, 60%, 65%, or 70%.

[0141] The carrier can be used to deliver active ingredients such as therapeutic or prophylactic agents. The active ingredient can be encapsulated in the carrier or combined with the carrier.

[0142] For example, the therapeutic agent or preventive agent includes 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.

[0143] Neutral lipids

[0144] The carrier may include a neutral lipid. In the present invention, a neutral lipid refers to a lipid that is uncharged or exists in the form of a zwitterion at a selected pH value and plays an auxiliary role. The neutral lipid may adjust the fluidity of the nanoparticles to a lipid bilayer structure and improve efficiency by promoting lipid phase transition, and may also affect the specificity of the target organ.

[0145] In one embodiment, the molar ratio of the cationic lipid to the neutral lipid is about 1:1 to 15:1, such as about 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1 and 2:1. In a preferred embodiment, the molar ratio of the cationic lipid to the neutral lipid is about 4.5:1. In another preferred embodiment, the molar ratio of the cationic lipid to the neutral lipid is about 4.9:1.

[0146] For example, the neutral lipids may include one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterols, and derivatives thereof.

[0147] 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.

[0148] The neutral lipid part can be selected from the non-limiting group of the following composition: phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine and sphingomyelin. The fatty acid part can be selected from the non-limiting group of the 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 encompassed are non-natural species including natural species with modification and replacement, the modification and replacement include branching, oxidation, cyclization and alkynes. For example, phospholipids can be functionalized with one or more alkynes (e.g., one or more double bonds are replaced by triple bond alkenyl) or cross-linked with the 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).

[0149] The 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-Diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-Di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0Diether). 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 16.0PE), 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), palmitoyloleoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE) and mixtures thereof.

[0150] 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.

[0151] Structural lipids

[0152] The carrier of the composition comprising cationic lipids may further include one or more structural lipids. The structural lipids in the present invention refer to lipids that enhance the stability of nanoparticles by filling the gaps between lipids.

[0153] In one embodiment, the molar ratio of the cationic lipid to the structural lipid is about 0.6:1 to 3:1, for example, about 1.0:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2.0:1.

[0154] 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, alpha-tocopherol, corticosteroids, and mixtures thereof. In some embodiments, the structured lipid is cholesterol. In some embodiments, the structured lipid includes cholesterol and corticosteroids (such as prednisolone, dexamethasone, prednisone, and hydrocortisone) or a combination thereof.

[0155] Polymer-conjugated lipids

[0156] 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 LNP, regulates nanoparticle size by limiting lipid fusion, and increases the half-life of nanoparticles by reducing nonspecific interactions with macrophages.

[0157] 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 PEG-modified PEG is generally 350-5000Da.

[0158] 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).

[0159] In one embodiment of the composition / vector of the present invention, the polymer-conjugated lipid is DMG-PEG2000.

[0160] In one embodiment of the composition / carrier of the present invention, the carrier includes neutral lipids, structural lipids and polymer-conjugated lipids, and the molar ratio of the cationic lipids, the neutral lipids, the structural lipids, and the polymer-conjugated lipids is (25-75):(5-25):(15-65):(0.5-10), for example (35-49):(7.5-15):(35-55):(1-5).

[0161] In one embodiment of the composition / carrier of the present invention, the carrier comprises neutral lipids, structural lipids and polymer-conjugated lipids, and the molar ratio of the cationic lipids, the neutral lipids, the structural lipids and the polymer-conjugated lipids is 49:10:39.5:1.5 or 45:10:43.5:1.5 or 35:10:53.5:1.5.

[0162] Therapeutic and / or preventive agents

[0163] The composition may include one or more therapeutic and / or prophylactic agents. In one embodiment, the mass ratio of the carrier to the therapeutic or prophylactic agent is 10:1 to 30:1, for example, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1.

[0164] In one embodiment, the mass ratio of the carrier to the therapeutic agent or preventive agent is 12.5:1 to 20:1, preferably 15:1.

[0165] The therapeutic agent or preventive agent includes, but is not limited to, one or more of a nucleic acid molecule, a small molecule compound, a polypeptide or a protein.

[0166] For example, the therapeutic or prophylactic agent is a vaccine or a compound capable of eliciting an immune response.

[0167] The vectors of the present invention can deliver therapeutic agents and / or preventive agents to mammalian cells or organs. Therefore, the present invention also provides methods for treating diseases or disorders in mammals in need thereof, which methods include administering a composition including a therapeutic agent and / or preventive agent to the mammal and / or contacting mammalian cells with the composition.

[0168] Therapeutic and / or prophylactic agents include biologically active substances and are alternatively referred to as "active agents". Therapeutic and / or prophylactic agents can be substances that, after delivery to a cell or organ, cause a desired change in the 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, anti-neoplastic agents (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, and streptozotocin), anti-tumor agents (e.g., actinomycin D, vincristine, vinblastine, cytosine arabinoside, arabinoside), anthracyclines, alkylating agents, platinum compounds, antimetabolites, and nucleoside analogs, such as methotrexate and purine and pyrimidine analogs), anti-infectives, local anesthetics (e.g., dibucaine and chlorpromazine), beta-adrenergic blocking agents (e.g., propranolol, timolol, and labetalol), antihypertensives (e.g., clonidine and hydralazine), antidepressants (e.g., imipramine, amitriptyline, and doxepin), anticonvulsants (e.g., phenytoin), antihistamines

[0013] In some embodiments, the present invention includes but is not limited to: antibiotics (e.g., diphenhydramine, chlorpheniramine, and promethazine), antibiotics / antibacterials (e.g., gentamycin, ciprofloxacin, and cefoxitin), antifungals (e.g., miconazole, terconazole, econazole, isoconazole, butaconazole, clotrimazole, itraconazole, nystatin, naftifine, and amphotericin B), antiparasitics, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma agents, vitamins, sedatives, and imaging agents.

[0169] In some embodiments, the therapeutic and / or prophylactic agent is a cytotoxin, a radioactive ion, a chemotherapeutic agent, a vaccine, a compound that causes an immune response, and / or another therapeutic and / or prophylactic agent. Cytotoxins or cytotoxic agents include any agent that is harmful to cells. Examples include, but are not limited to, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracenedione, The invention relates to a radioactive ion, for example, anthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids such as maytansinol, rachelmycin (CC-1065), and analogs or homologues thereof. Radioactive ions include, but are not limited to, iodine (e.g., iodine-125 or iodine-131), strontium-89, phosphorus, palladium, cesium, iridium, phosphate, cobalt, yttrium-90, samarium-153, and praseodymium. Vaccines include compounds and preparations that can provide immunity for one or more conditions related to infectious diseases such as influenza, measles, human papillomavirus (HPV), rabies, meningitis, pertussis, tetanus, plague, hepatitis and tuberculosis, and can include mRNA encoding infectious disease-derived antigens and / or epitopes. Vaccines can also include compounds and preparations that guide the immune response for cancer cells and can include mRNA encoding tumor cell-derived antigens, epitopes and / or new epitopes. Compounds that cause immune response can include vaccines, corticosteroids (e.g., dexamethasone) and other species. In some embodiments, by including compounds (e.g., compounds 3, 18, 20, 25, 26, 29, 30, 60, 108-112 or 122) according to formula (I), (IA), (IB), (II), (IIa), (IIb), (IIc), (IId), (IIe), (IIg) or (III) intramuscular administration of a composition that can cause immune response vaccines and / or compounds.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, cyclosporine, cyclophosphamide ... The invention relates to antibiotics such as dactinomycin (formerly actinomycin), bleomycin, mithramycin and anthramycin (AMC), and antimitotic agents such as vincristine, vinblastine, taxol and maytansine.

[0170] In other embodiments, the therapeutic and / or prophylactic agent is a protein. The therapeutic proteins that can be used in the nanoparticles of the present invention 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.

[0171] In some embodiments, the therapeutic agent is a polynucleotide or nucleic acid (e.g., ribonucleic acid or deoxyribonucleic acid). The broadest meaning of the term "polynucleotide" includes any compound and / or substance that is an oligonucleotide chain or can be incorporated into an oligonucleotide chain. Exemplary polynucleotides used according to the present invention 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, the therapeutic agent and / or preventive agent is RNA. The RNA that can be used in the compositions and methods described herein can be selected from, but is not limited to, the group consisting of: shortmer, antagomir, antisense RNA, 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, RNA is mRNA.

[0172] In certain embodiments, therapeutic and / or preventive agents are mRNA. mRNA can encode any polypeptide of interest, including any natural or non-natural polypeptide or otherwise modified polypeptide. The polypeptide encoded by mRNA can have any size and can have any secondary structure or activity. In some embodiments, the polypeptide encoded by mRNA can have a therapeutic effect when expressed in a cell.

[0173] In other embodiments, the therapeutic and / or preventive agent is siRNA. siRNA can selectively reduce the expression of a gene of interest or down-regulate the expression of the gene. For example, the selection of siRNA can make the gene silencing relevant to a specific disease, disease or condition after the composition comprising the siRNA is administered to a subject in need. siRNA can include a sequence complementary to the mRNA sequence of a gene or protein of interest to encode. In some embodiments, siRNA can be an immunomodulatory siRNA.

[0174] In certain embodiments, the therapeutic and / or preventive agent is sgRNA and / or cas9 mRNA. 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 cell gene.

[0175] In some embodiments, the therapeutic and / or preventive agent is shRNA or its encoding vector or plasmid. shRNA can be produced inside the target cell after the appropriate construct is delivered to the nucleus. The construct and mechanism associated with shRNA are well-known in the relevant field.

[0176] Disease or condition

[0177] The composition / vector of the present invention can deliver a therapeutic agent or a preventive agent to a subject or patient. The therapeutic agent or preventive agent includes, but is not limited to, one or more of a nucleic acid molecule, a small molecule compound, a polypeptide or a protein. Therefore, the composition of the present invention can be used to prepare nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs. Due to the wide variety of the above-mentioned therapeutic agents or preventive agents, the composition of the present invention can be used to treat or prevent a variety of diseases or conditions.

[0178] In one embodiment, the disease or disorder is characterized by a malfunction or aberrant protein or polypeptide activity.

[0179] 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.

[0180] 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.

[0181] Other components

[0182] The composition may include one or more components other than 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.

[0183] The composition may also include one or more permeability enhancing molecules, carbohydrates, polymers, surface modifiers or other components. The permeability enhancing molecules may be, for example, molecules described in U.S. Patent Application Publication No. 2005 / 0222064. The carbohydrates may include simple sugars (e.g., glucose) and polysaccharides (e.g., glycogen and its derivatives and analogs).

[0184] 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), mucolytic agents (e.g., acetylcysteine, artemisia, bromelain, papain, clerodendrum, bromhexine, carbocisteine, eprazinone, , mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin β4, dornasealfa, 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).

[0185] The composition can also include one or more functionalized lipids. For example, lipids can be functionalized with alkynyl groups, which may undergo cycloaddition reactions when exposed to azides 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.

[0186] In addition to these components, the composition can include any substance that can be used in a 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, flavoring agents, coloring agents, etc. Excipients such as starch, lactose or dextrin. Pharmaceutically acceptable excipients are well known in the art (see, for example, Remington's The Science and Practice of Pharmacy, 21st edition, ARGennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006).

[0187] Examples of diluents may include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and / or combinations thereof.

[0188] In some embodiments, a composition comprising one or more lipids described herein may also include 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.

[0189] The composition of the present invention can be made into a preparation in the form of solid, semisolid, liquid or gas, such as tablets, capsules, ointments, elixirs, syrups, solutions, emulsions, suspensions, injections, aerosols. The composition of the present invention can be prepared by methods well known in the pharmaceutical field. For example, a sterile injection solution can be prepared by mixing the required amount of therapeutic agent or preventive agent with the required various other ingredients mentioned above into a suitable solvent such as sterile distilled water, and then filtering and sterilizing. Surfactants can also be added to promote the formation of uniform solutions or suspensions.

[0190] For example, the compositions of the invention can be administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation. In one embodiment, the composition is administered subcutaneously.

[0191] The compositions of the present invention 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 by the attending physician or clinician. For example, a therapeutic or preventive agent may be administered to a mammal (e.g., a human) at a dose of about 0.001 mg / kg to about 10 mg / kg.

[0192] The present invention includes but is not limited to the following embodiments:

[0193] 1. A compound of formula (I)

[0194] or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein

[0195] G1 is C 1～8 Alkylene;

[0196] G2 is C 2～8 Alkylene;

[0197] R1 is C 6～25 Straight or branched chain alkyl;

[0198] R2 is C 12～25 Straight or branched chain alkyl;

[0199] G3 is: HO(CH2)2N(R3)CH2CH(OH)CH2-, wherein R3 is -CH3 or -CH2CH3 or -CH2CH2OH.

[0200] 2. A compound of formula (I) according to Embodiment 1, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein G1 is an unsubstituted C 3～7 Alkylene.

[0201] 3. A compound of formula (I) according to Embodiment 1 or 2, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein G1 is unsubstituted C3 alkylene or C5 alkylene or C6 alkylene.

[0202] 4. A compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof according to any one of the preceding embodiments, wherein G2 is an unsubstituted C 3～7 Alkylene.

[0203] 5. A compound of formula (I) according to any one of the preceding embodiments, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein G2 is unsubstituted C3 alkylene or C5 alkylene or C6 alkylene.

[0204] 6. A compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof according to any one of the preceding embodiments, wherein R1 is an unsubstituted C 11 Straight chain alkyl or unsubstituted C 12～24 Branched chain alkyl.

[0205] 7. A compound of formula (I) according to Embodiment 6, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R1 is an unsubstituted C 18 Branched alkyl or C 15 Branched alkyl or C 24 Branched alkyl or C 17 Branched chain alkyl.

[0206] 8. A compound of formula (I) according to embodiment 7, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R1 is:

[0207] 9. A compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof according to any one of the preceding embodiments, wherein R2 is an unsubstituted C 11 Straight chain alkyl or unsubstituted C 14～24 Branched chain alkyl.

[0208] 10. A compound of formula (I) according to embodiment 9, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R2 is an unsubstituted C 18 Branched alkyl or C 24 Branched alkyl or C 15 Branched alkyl or C 17 Branched chain alkyl.

[0209] 11. A compound of formula (I) according to embodiment 10, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R2 is:

[0210] 12. A compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof according to any one of the preceding embodiments, wherein the compound of formula (I) has one of the following structures:

[0211]

[0212]

[0213]

[0214]

[0215]

[0216] 13. A compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof according to any one of the preceding embodiments, wherein the compound of formula (I) is compound YK-305 having the following structure:

[0217]

[0218] 14. A compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof according to any one of embodiments 1 to 12, wherein the compound of formula (I) is compound YK-310 having the following structure:

[0219]

[0220] 15. A compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof according to any one of embodiments 1 to 12, wherein the compound of formula (I) is compound YK-312 having the following structure:

[0221]

[0222] 16. A compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof according to any one of embodiments 1 to 12, wherein the compound of formula (I) is compound YK-319 having the following structure:

[0223]

[0224] 17. A compound of formula (I) or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof according to any one of embodiments 1 to 12, wherein the compound of formula (I) is compound YK-318 having the following structure:

[0225]

[0226] 18. A composition comprising a carrier, wherein the carrier comprises a cationic lipid, and the cationic lipid comprises a compound of formula (I) according to any one of the preceding embodiments, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof.

[0227] 19. The composition according to embodiment 18, wherein the molar ratio of the cationic lipid to the carrier is 25% to 75%.

[0228] 20. The composition of any one of embodiments 18-19, wherein the carrier further comprises a neutral lipid.

[0229] 21. A composition according to embodiment 20, wherein the molar ratio of the cationic lipid to the neutral lipid is 1:1 to 15:1, preferably 4.5:1.

[0230] 22. A composition according to any one of embodiments 20-21, wherein the neutral lipids include one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterols and their derivatives.

[0231] 23. A composition according to any one of embodiments 20-22, wherein the neutral lipid is selected from one or more of the following: 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-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0Diether 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 16.0PE), 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), palmitoyloleoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE) and mixtures thereof.

[0232] 24. A composition according to embodiment 23, wherein the neutral lipid is DOPE and / or DSPC.

[0233] 25. A composition according to any one of embodiments 18-24, wherein the carrier further comprises a structured lipid.

[0234] 26. A composition according to embodiment 25, wherein the molar ratio of the cationic lipid to the structural lipid is 0.6:1 to 3:1.

[0235] 27. A composition according to any one of embodiments 25-26, wherein the structured lipid is selected from one or more of the following: cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol, corticosteroids.

[0236] 28. A composition according to embodiment 27, wherein the structured lipid is cholesterol.

[0237] 29. A composition according to any one of embodiments 18-28, wherein the carrier further comprises a polymer-conjugated lipid.

[0238] 30. The composition according to embodiment 29, wherein the molar ratio of the polymer-conjugated lipid to the carrier is 0.5% to 10%, preferably 1.5%.

[0239] 31. A composition according to any of embodiments 29-30, wherein 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.

[0240] 32. A composition according to embodiment 31, wherein the polymer conjugated lipid is selected from one or more of the following: distearoylphosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), dimyristoylglycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG2000) and methoxypolyethylene glycol ditetradecanoyl acetamide (ALC-0159).

[0241] 33. A composition according to any one of embodiments 18-32, wherein the carrier comprises neutral lipids, structural lipids and polymer-conjugated lipids, and the molar ratio of the cationic lipids, the neutral lipids, the structural lipids, and the polymer-conjugated lipids is (25-75):(5-25):(15-65):(0.5-10).

[0242] 34. A composition according to embodiment 33, wherein the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer conjugated lipid is (35-49):(7.5-15):(35-55):(1-5).

[0243] 35. A composition according to embodiment 34, wherein the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is 45:10:43.5:1.5.

[0244] 36. A composition according to any one of embodiments 18-35, wherein the composition is a nanoparticle preparation, the average particle size of the nanoparticle preparation is 10nm to 300nm; and the polydispersity coefficient of the nanoparticle preparation is ≤50%.

[0245] 37. A composition according to embodiment 36, wherein the composition is a nanoparticle preparation, the average particle size of the nanoparticle preparation is 90nm to 280nm; and the polydispersity coefficient of the nanoparticle preparation is ≤45%.

[0246] 38. A composition according to any one of embodiments 18-37, wherein the cationic lipid further comprises one or more other ionizable lipid compounds.

[0247] 39. The composition of any one of embodiments 18-38, further comprising a therapeutic or prophylactic agent.

[0248] 40. The composition according to embodiment 39, wherein the mass ratio of the carrier to the therapeutic agent or preventive agent is 10:1 to 30:1.

[0249] 41. The composition of embodiment 40, wherein the mass ratio of the carrier to the therapeutic agent or preventive agent is 12.5:1 to 20:1.

[0250] 42. A composition according to embodiment 41, wherein the mass ratio of the carrier to the therapeutic agent or preventive agent is 15:1.

[0251] 43. A composition according to any one of embodiments 39-42, wherein the therapeutic agent or prophylactic agent comprises one or more of a nucleic acid molecule, a small molecule compound, a polypeptide or a protein.

[0252] 44. A composition according to any one of embodiments 39-42, wherein the therapeutic agent or prophylactic agent is a vaccine or a compound capable of eliciting an immune response.

[0253] 45. A composition according to any of the preceding embodiments 39-44, wherein the therapeutic or prophylactic agent is a nucleic acid.

[0254] 46. ​​A composition according to embodiment 45, wherein the therapeutic or prophylactic agent is ribonucleic acid (RNA).

[0255] 47. The composition of embodiment 45, wherein the therapeutic or prophylactic agent is deoxyribonucleic acid (DNA).

[0256] 48. A composition according to embodiment 46, wherein the RNA is selected from the group consisting of: small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), micro RNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA) and mixtures thereof.

[0257] 49. The composition of embodiment 48, wherein the RNA is mRNA.

[0258] 50. The composition of any one of embodiments 18-49, wherein the composition further comprises one or more pharmaceutically acceptable excipients or diluents.

[0259] 51. Use of a compound of formula (I) or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer according to any one of the preceding embodiments 1-17 or a composition according to any one of the preceding embodiments 18-50 in the preparation of nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs.

[0260] 52. Use of a compound of formula (I) or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer as described in any of the preceding embodiments 1-17 or a composition as described in any of the preceding embodiments 18-50 in the preparation of a medicament for treating a disease or condition in a mammal in need thereof.

[0261] 53. The use according to embodiment 52, wherein the disease or disorder is characterized by dysfunctional or abnormal protein or polypeptide activity.

[0262] 54. The use according to any one of embodiments 52-53, wherein the disease or condition is selected from the group consisting of infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.

[0263] 55. The use according to embodiment 54, wherein the infectious disease is selected from: a disease caused by a coronavirus, influenza virus or HIV virus, pediatric pneumonia, Rift Valley fever, yellow fever, rabies, or multiple herpes.

[0264] 56. The use according to any one of embodiments 52-55, wherein the mammal is a human.

[0265] 57. The use according to any one of embodiments 51-56, wherein the composition is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally or by inhalation.

[0266] 58. The use according to embodiment 57, wherein the composition is administered subcutaneously.

[0267] 59. The use of any one of embodiments 51-58, wherein a dose of about 0.001 mg / kg to about 10 mg / kg of the therapeutic or prophylactic agent is administered to the mammal.

[0268] Example

[0269] The present invention is further described below in conjunction with the examples. However, the present invention 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, and the implementation conditions not specified are conventional conditions in the industry. In the specific examples of the present invention, the raw materials used can be obtained commercially. Unless otherwise specified, the percentages in the context are weight percentages, and all temperatures are given in degrees Celsius. The technical features involved in each embodiment of the present invention can be combined with each other as long as they do not conflict with each other.

[0270] Example 1: Synthesis of cationic lipid compounds

[0271] 1. Synthesis of YK-301 and YK-305

[0272] The synthetic route is as follows:

[0273]

[0274] Step 1: Synthesis of (S)-tert-butyl (3-chloro-2-hydroxypropyl)carbamate (YK-301-PM1)

[0275] Dissolve (S)-1-amino-3-chloropropane-2-ol (5.0 g, 45.6 mmol) and triethylamine (5.1 g, 50.4 mmol) in dichloromethane (50 mL), dissolve di-tert-butyl carbonic anhydride (10.9 g, 49.9 mmol) in dichloromethane (20 mL), slowly drip into the above solution, and stir at room temperature for 5 hours. After the reaction is completed, add 100 mL of water to the reaction solution, then add dichloromethane (200 mL × 2) for extraction, combine the organic phases, wash with brine (50 mL × 3), dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under vacuum. The residue is purified by silica gel chromatography (ethyl acetate / n-hexane) to obtain YK-301-PM1 (7.0 g, 33.4 mmol, 73.2%). 16 ClNO3,MS(ES):m / z(M+H + )210.1.

[0276] Step 2: Synthesis of (S)-tert-butyl 2-hydroxy-3-(((2-hydroxyethyl)(methyl)amino)propyl)carbamate (YK-301-PM2)

[0277] YK-301-PM1 (300 mg, 1.43 mmol) and 2-(methylamino)ethanol (108 mg, 1.44 mmol) were dissolved in acetonitrile (3 mL), potassium carbonate (594 mg, 4.30 mmol) and potassium iodide (48 mg, 0.29 mmol) were added to the above system, and the mixture was heated to 70°C and stirred for 5 hours. After the reaction was completed, the reaction mixture was filtered, the filtrate was concentrated under vacuum, and the residue was purified by silica gel chromatography (methanol / dichloromethane) to obtain YK-301-PM2 (350 mg, 1.41 mmol, 98.6%). 11 H 24 N2O4,MS(ES):m / z(M+H + )249.2.

[0278] Step 3: Synthesis of (S)-1-amino-3-((2-hydroxyethyl)(methyl)amino)propan-2-ol (YK-301-PM3)

[0279] YK-301-PM2 (350 mg, 1.41 mmol) prepared above was added to 4M hydrochloric acid / 1,4-dioxane (2 mL) and stirred at room temperature for 10 hours. After the reaction was completed, the mixture was concentrated, 100 mL of dichloromethane and 100 mL of saturated sodium bicarbonate aqueous solution were added, the mixture was stirred and separated, and dichloromethane (100 mL × 2) was added for extraction. The organic phases were combined and then washed with brine (50 mL × 2), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under vacuum to obtain YK-301-PM3 (200 mg, 1.35 mmol, 95.7%). 16 N2O2,MS(ES):m / z(M+H + )149.1.

[0280] Step 4: Synthesis of (S)-6-(2-hydroxy-3-((2-hydroxyethyl)(methyl)amino)propyl)hexanoic acid-2-octyldecyl ester (YK-301-PM4) and (R)-bis(2-octyldecyl)-6,6'-((2-hydroxy-3-((2-hydroxyethyl)(methyl)amino)propyl)azadialkyl)dihexanoate (YK-305)

[0281] The YK-301-PM3 (200 mg, 1.35 mmol) prepared above and 6-bromohexanoic acid-2-octyldecyl ester (604 mg, 1.35 mmol) were dissolved in acetonitrile (2 mL), potassium carbonate (583 mg, 4.22 mmol) was added to the above system, heated to 70°C and stirred for 7 hours. After the reaction was completed, 20 mL of water was added to the reaction solution, and then ethyl acetate (20 mL×2) was added for extraction. The organic phases were combined and then washed with brine (20 mL×2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to remove the solvent. The residue was purified by silica gel chromatography (dichloromethane / methanol) to obtain YK-301-PM4 (210 mg, 0.41 mmol, 30.2%). 30 H 62 N2O4,MS(ES):m / z(M+H + )515.4; YK-305 (160 mg, 0.18 mmol, 13.4%) was obtained. 54 H 108 N2O6,MS(ES):m / z(M+H + )881.8.

[0282] Step 5: Synthesis of (R)-2-octyldecyl-6-((4-(decyloxy)-4-oxobutyl)(((2-hydroxy-3-(2-hydroxyethyl)(methyl)amino)propyl)amino)hexanoate (YK-301)

[0283] The YK-301-PM4 (210 mg, 0.41 mmol) and 4-bromobutyric acid-n-decyl ester (150 mg, 0.49 mmol) prepared above were dissolved in acetonitrile (2 mL), potassium carbonate (169 mg, 1.22 mmol) and potassium iodide (14 mg, 0.084 mmol) were added to the above system, and the mixture was heated to 70°C and stirred for 10 hours. After the reaction was completed, 20 mL of water was added to the reaction solution, and then ethyl acetate (20 mL×2) was added for extraction. The organic phases were combined and then washed with brine (20 mL×2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to remove the solvent. The residue was purified by silica gel chromatography (dichloromethane / methanol) to obtain the target compound (93 mg, 0.12 mmol, 30.2%). 44 H 88 N2O6,MS(ES):m / z(M+H + )741.6.

[0284] YK-301: 1 H NMR(400MHz,Chloroform-d)δ4.06(t,J=6.8Hz,2H),3.96(d,J

[0285] =5.8Hz,2H),3.90(s,1H),3.81–3.76(m,1H),3.70(t,J=4.7Hz,4H),2.76(s,2H),2.63–2.41(m,10H),2.31(q,J=7.3,6.3Hz, 4H),1.79(dp,J=13.5,6.9Hz,2H),1.63(q,J=7.2Hz,5H),1.54–1.43(m,2H),1.28(d,J=14.3Hz,44H),0.88(t,J=6.6Hz,9H).

[0286] YK-305: 1 H NMR (400MHz, Chloroform-d) δ4.07 (s, 2H), 3.96 (d, J = 5.8Hz,

[0287] 4H),3.76–3.69(m,1H),2.87–2.55(m,10H),2.51(s,3H),2.31(t,J=7.4Hz ,4H),1.62(dq,J=15.5,7.7Hz,10H),1.27(s,62H),0.88(t,J=6.7Hz,12H).

[0288] 2. Synthesis of YK-302 and YK-306

[0289] The synthetic route is as follows:

[0290]

[0291] Step 1: Synthesis of (S)-6-(2-hydroxy-3-((2-hydroxyethyl)(methyl)amino)propyl)hexanoic acid-3-hexylnonyl ester (YK-302-PM1) and (R)-bis(3-hexylnonyl)-6,6'-((2-hydroxy-3-((2-hydroxyethyl)(methyl)amino)propyl)azadialkyl)dihexanoate (YK-306)

[0292] YK-301-PM3 (221 mg, 0.99 mmol) and 6-bromohexanoic acid-3-hexylnonyl ester (400 mg, 1.49 mmol) were used as raw materials, and YK-302-PM1 (160 mg, 0.34 mmol, 35.2%) was obtained according to the method for synthesizing YK-301-PM4. 27 H 56 N2O4,MS(ES):m / z(M+H + )473.4; YK-306 (35 mg, 0.04 mmol, 4.4%) was obtained. 48 H 96 N2O6,MS(ES):m / z(M+H + )797.7.

[0293] Step 2: Synthesis of (R)-3-hexylnonyl-6-(4-(decyloxy)-4-oxobutyl)(((2-hydroxy-3-(2-hydroxyethyl)(methyl)amino)propyl)amino)hexanoate (YK-302)

[0294] YK-302-PM1 (160 mg, 0.34 mmol) and 4-bromobutyric acid-n-decyl ester (114 mg, 0.37 mmol) were used as raw materials, and YK-302 (120 mg, 0.17 mmol, 50.5%) was obtained according to the method for synthesizing YK-301. 41 H 82 N2O6,MS(ES):m / z(M+H + )699.6.

[0295] YK-302: 1 H NMR(400MHz,Chloroform-d)δ4.12–3.85(m,8H),3.75(t,J=

[0296] 4.9Hz,2H),2.86(t,J=4.8Hz,2H),2.73–2.50(m,9H),2.31(dt,J=15.1,7.2Hz,4H),1 .90–1.75(m,2H),1.65–1.54(m,6H),1.28(d,J=18.9Hz,40H),0.88(t,J=6.5Hz,9H).

[0297] YK-306: 1 H NMR (400MHz, Chloroform-d) δ4.37 (s, 1H), 4.08 (t, J = 7.1Hz,

[0298] 4H),3.82(t,J=4.5Hz,2H),3.48(s,4H),3.04–2.80(m,8H),2.64(s,3H),2.31(t,J=7.2Hz, 4H),1.67(dq,J=15.1,7.5Hz,7H),1.60–1.53(m,4H),1.25(s,48H),0.88(t,J=6.4Hz,12H).

[0299] 3. Synthesis of YK-303 and YK-304

[0300] The synthetic route is as follows:

[0301]

[0302] Step 1: Synthesis of (S)-6-(2-hydroxy-3-((2-hydroxyethyl)(methyl)amino)propyl)hexanoic acid-undecyl ester (YK-303-PM1) and (R)-bis(undecyl)-6,6'-((2-hydroxy-3-((2-hydroxyethyl)(methyl)amino)propyl)azadialkyl)dihexanoate (YK-304)

[0303] YK-301-PM3 (160 mg, 1.08 mmol) and 6-bromohexanoic acid-undecyl ester (251 mg, 0.72 mmol) were used as raw materials, and YK-303-PM1 (114 mg, 0.27 mmol, 38.0%) was obtained according to the method for synthesizing YK-301-PM4. 23 H 48 N2O4,MS(ES):m / z(M+H + )417.4; YK-304 (50 mg, 0.07 mmol, 10.1%) was obtained. 40 H 80 N2O6,MS(ES):m / z(M+H + )685.6.

[0304] Step 2: Synthesis of (R)-undecyl-6-(4-(4-decyltetradecyloxy)-4-oxobutyl)((2-hydroxy-3-((2-hydroxyethyl)(methyl)amino)propyl)amino)hexanoate (YK-303)

[0305] YK-303-PM1 (114 mg, 0.27 mmol) and 4-bromobutyric acid-4-decyltetradecyl ester (156 mg, 0.31 mmol) were used as raw materials, and YK-303 (75 mg, 0.09 mmol, 33.1%) was obtained according to the method for synthesizing YK-301. 51 H 102 N2O6,MS(ES):m / z(M+H + )839.8.

[0306] YK-303: 1 H NMR(400MHz,Chloroform-d)δ4.42(s,1H),4.08–4.00(m,4H),3.91(s,2H),3.49(s,1H),3.14(s,3H),2.82(d,J=10.5Hz,10H),2.39 (t,J=6.6Hz,2H),2.31(t,J=7.2Hz,2H),1.95(s,3H),1.63(dt,J=15.4,7.3Hz,8H),1.25(d,J=11.9Hz,62H),0.88(t,J=6.6Hz,9H).

[0307] YK-304: 1 H NMR (400 MHz, Chloroform-d) δ 5.30 (s, 1H), 4.80 (s, 2H), 4.16–4.02 (m, 4H), 3.75 (q, J = 7.6, 6.4 Hz, 2H), 3.02–2.61 (m, 9H), 2.56 (s, 3H), 2.31 (t, J = 7.4 Hz, 4H), 1.71–1.54 (m, 12H), 1.40–1.22 (m, 36H), 0.88 (t, J = 6.6 Hz, 6H). 4. Synthesis of YK-307

[0308] The synthetic route is as follows:

[0309]

[0310] Synthesis of (R)-bis(4-decyltetradecyl)-4,4'-(2-hydroxy-3-((2-hydroxyethyl)(methyl)amino)propyl)azadialkyl)dibutyrate (YK-307)

[0311] YK-301-PM3 (160 mg, 1.08 mmol) and 4-bromobutyric acid-4-decyltetradecyl ester (362 mg, 0.72 mmol) were used as raw materials, and YK-307 (75 mg, 0.08 mmol, 20.9%) was obtained according to the method for synthesizing YK-301-PM4. 62 H 124 N2O6,MS(ES):m / z(M+H + )993.9.

[0312] 1 H NMR(400MHz,Chloroform-d)δ5.30(s,1H),4.04(t,J=6.8Hz,4H),3.89–3.74(m,2H),3.03(d,J=4.1Hz,4H),2.60(dd,J=16. 6,9.7Hz,7H),2.32(t,J=6.8Hz,4H),1.87–1.74(m,4H),1.63–1.55(m,4H),1.25(d,J=12.2Hz,80H),0.88(t,J=6.7Hz,12H).

[0313] 5. Synthesis of YK-308 and YK-312

[0314] The synthetic route is as follows:

[0315]

[0316] Step 1: Synthesis of (S)-tert-butyl 2-hydroxy-3-(((2-hydroxyethyl)(ethyl)amino)propyl)carbamate (YK-308-PM1)

[0317] YK-301-PM1 (600 mg, 2.87 mmol) and 2-(ethylamino)ethanol (256 mg, 2.87 mmol) were used as raw materials, and YK-308-PM1 (300 mg, 1.14 mmol, 39.8%) was obtained according to the method for synthesizing YK-301-PM2. 12 H 26 N2O4,MS(ES):m / z(M+H + )263.2.

[0318] Step 2: Synthesis of (S)-1-amino-3-((2-hydroxyethyl)(ethyl)amino)propan-2-ol (YK-308-PM2)

[0319] YK-308-PM1 (200 mg, 0.76 mmol) was used as the raw material, and YK-308-PM2 (120 mg, 0.74 mmol, 97.3%) was obtained according to the method of synthesizing YK-301-PM3. 18 N2O2,MS(ES):m / z(M+H + )163.1.

[0320] Step 3: Synthesis of (S)-6-(2-hydroxy-3-(((2-hydroxyethyl)(ethyl)amino)propyl)hexanoic acid-2-octyldecyl ester (YK-308-PM3) and (S)-bis(2-octyldecyl)-6,6'-((2-hydroxy-3-((2-hydroxyethyl)(ethyl)amino)propyl)azadialkyl)dihexanoate (YK-312)

[0321] YK-308-PM2 (185 mg, 1.14 mmol) and 6-bromohexanoic acid-2-octyldecyl ester (349 mg, 1.14 mmol) were used as raw materials, and YK-308-PM3 (150 mg, 0.28 mmol, 24.9%) was obtained according to the method for synthesizing YK-301-PM4. 31 H 64 N2O4,MS(ES):m / z(M+H + )529.5; YK-312 (71 mg, 0.08 mmol, 7.0%) was obtained. 55 H 110 N2O6,MS(ES):m / z(M+H + )895.8.

[0322] Step 4: Synthesis of (S)-2-octyldecyl-6-(4-(decyloxy)-4-oxobutyl)(((2-hydroxy-3-(2-hydroxyethyl)(ethyl)amino)propyl)amino)hexanoate (YK-308)

[0323] YK-308-PM3 (150 mg, 0.28 mmol) and 4-bromobutyric acid-n-decyl ester (104 mg, 0.34 mmol) were used as raw materials, and YK-308 (90 mg, 0.12 mmol, 42.6%) was obtained according to the method for synthesizing YK-301. 45 H 90 N2O6,MS(ES):m / z(M+H + )755.7.

[0324] YK-308: 1 H NMR(400MHz,Chloroform-d)δ4.06(t,J=6.7Hz,2H),3.96(d,J

[0325] =5.8Hz,2H),3.74(s,1H),2.90(s,3H),2.84–2.38(m,10H),2.31(q,J=7.1,6.6Hz,4H ),1.80(s,2H),1.69–1.58(m,5H),1.49(s,2H),1.26(s,48H),0.88(t,J=6.6Hz,9H).

[0326] YK-312: 1 H NMR (400MHz, Chloroform-d) δ3.96 (d, J=5.8Hz, 4H), 3.84 (s,

[0327] 2H), 2.91 (d, J = 94.1Hz, 12H), 2.32 (t, J = 7.3Hz, 4H), 1.72–1.54 (m, 10H), 1.27 (s, 66H), 0.88 (t, J = 6.7Hz, 12H).

[0328] 6. Synthesis of YK-309 and YK-311

[0329] The synthetic route is as follows:

[0330]

[0331] Step 1: Synthesis of (S)-4-(2-hydroxy-3-((2-hydroxyethyl)(ethyl)amino)propyl)hexanoic acid-3-hexylnonyl ester (YK-309-PM1) and (S)-bis(3-hexylnonyl)-6,6'-((((2-hydroxyethyl)(ethyl)amino)propyl)azadialkyl)dihexanoate (YK-311)

[0332] YK-308-PM2 (247 mg, 1.52 mmol) and 6-bromohexanoic acid-3-hexylnonyl ester (680 mg, 1.67 mmol) were used as raw materials, and YK-309-PM1 (260 mg, 0.53 mmol, 35.1%) was obtained according to the method for synthesizing YK-301-PM4. 28 H 58 N2O4,MS(ES):m / z(M+H + )487.4; YK-311 (160 mg, 0.20 mmol, 13.0%) was obtained. 49 H 98 N2O6,MS(ES):m / z(M+H + )811.7.

[0333] Step 2: Synthesis of (S)-3-hexylnonyl-6-(4-(decyloxy)-4-oxobutyl)(2-hydroxy-3-(((2-hydroxyethyl)(ethyl)amino)propyl)amino)hexanoate (YK-309)

[0334] YK-309-PM1 (160 mg, 0.33 mmol) and 4-bromobutyric acid-3-decyl ester (101 mg, 0.33 mmol) were used as raw materials, and YK-309 (160 mg, 0.22 mmol, 68.0%) was obtained according to the method for synthesizing YK-301. 42 H 84 N2O6,MS(ES):m / z(M+H + )713.6.

[0335] YK-309: 1 H NMR (400MHz, Chloroform-d) δ4.07 (q, J=7.2Hz, 4H), 3.86

[0336] (dt,J=8.3,4.3Hz,1H),3.68(t,J=5.0Hz,2H),2.80(q,J=7.0Hz,4H),2.70–2.37(m,8H),2.34–2.27(m,3H),1.78(tt ,J=13.8,7.1Hz,2H),1.60(dp,J=20.9,7.2Hz,6H),1.51–1.23(m,40H),1.13(t,J=7.1Hz,3H),0.88(t,J=6.7Hz,9H).

[0337] YK-311: 1 H NMR (400MHz, Chloroform-d) δ4.08 (t, J = 7.1Hz, 4H), 3.87 (s,

[0338] 1H),3.66(t,J=5.1Hz,2H),2.82–2.71(m,4H),2.69–2.37(m,8H),2.29(t,J=7.4Hz,4H),1. 69–1.44(m,12H),1.39(s,2H),1.26(s,44H),1.11(t,J=7.1Hz,3H),0.88(t,J=6.7Hz,12H).

[0339] 7. Synthesis of YK-310 and YK-315

[0340] The synthetic route is as follows:

[0341]

[0342] Step 1: Synthesis of (S)-4-(2-hydroxy-3-((2-hydroxyethyl)(ethyl)amino)propyl)butyrate-4-decyltetradecyl ester (YK-310-PM1) and (S)-bis(4-decyltetradecyl)-4,4'-(((2-hydroxyethyl)(ethyl)amino)propyl)azadialkyl)dibutyrate (YK-315)

[0343] YK-308-PM2 (124 mg, 0.76 mmol) and 4-bromobutyric acid-4-decyltetradecyl ester (385 mg, 0.76 mmol) were used as raw materials, and YK-310-PM1 (142 mg, 0.24 mmol, 31.9%) was obtained according to the method for synthesizing YK-301-PM4. 35 H 72 N2O4,MS(ES):m / z(M+H + )585.6; YK-315 (30 mg, 0.03 mmol, 3.9%) was obtained. 63 H 126 N2O6,MS(ES):m / z(M+H + )1008.0.

[0344] Step 2: Synthesis of (S)-4-decyltetradecyl-4-(6-(undecyloxy)-6-oxohexyl)(2-hydroxy-3-(2-hydroxyethyl)(ethyl)amino)propyl)amino)butyrate (YK-310)

[0345] YK-310-PM1 (142 mg, 0.24 mmol) and 6-bromohexanoic acid undecyl ester (153 mg, 0.44 mmol) were used as raw materials, and YK-310 (90 mg, 0.11 mmol, 43.9%) was obtained according to the method for synthesizing YK-301. 52 H 104 N2O6,MS(ES):m / z(M+H + )853.8.

[0346] YK-310: 1 H NMR(400MHz,Chloroform-d)δ4.10–3.94(m,5H),3.75(q,J=

[0347] 6.2,5.6Hz,2H),2.95–2.80(m,4H),2.74–2.46(m,6H),2.36–2.23(m,4H),1.81(q,J=7.7Hz,2 H),1.68–1.56(m,6H),1.49(d,J=7.3Hz,2H),1.25(d,J=12.2Hz,62H),0.88(t,J=6.7Hz,9H).

[0348] YK-315: 1 H NMR(400MHz,Chloroform-d)δ4.39(s,1H),4.08–3.96(m,6H),3.27(d,J=7.1Hz,4H),2.72(s,5H),2.35(t,J=6.8H z,4H),1.85(d,J=6.9Hz,4H),1.59(s,4H),1.42(t,J=7.1Hz,4H),1.25(d,J=12.1Hz,80H),0.88(t,J=6.6Hz,12H).

[0349] 8. Synthesis of YK-313

[0350] The synthetic route is as follows:

[0351]

[0352] Synthesis of (S)-bis(heptadecan-9-yl)-6,6'-(((2-hydroxyethyl)(ethyl)amino)propyl)azadialkyl)dihexanoate (YK-313)

[0353] YK-308-PM2 (62 mg, 0.38 mmol) and heptadecan-9-yl-6-bromohexanoate (330 mg, 0.76 mmol) were used as raw materials, and YK-313 (170 mg, 0.20 mmol, 51.6%) was obtained according to the method for synthesizing YK-301-PM4. 53 H 106 N2O6,MS(ES):m / z(M+H + )867.8.

[0354] 1H NMR(400MHz,Chloroform-d)δ4.86(p,J=6.2Hz,2H),3.84(s,1H),3.65(t,J=5.1Hz,2H),2.80–2.69(m,4H),2.64–2.35(m,8H),2.28 (t,J=7.5Hz,4H),1.63(p,J=7.6Hz,4H),1.56–1.44(m,12H),1.28(d,J=15.2Hz,54H),1.09(t,J=7.1Hz,3H),0.88(t,J=6.8Hz,12H).

[0355] 9. Synthesis of YK-314

[0356] The synthetic route is as follows:

[0357]

[0358] Synthesis of (S)-bis(heptadecan-9-yl)-8,8'-(((2-hydroxyethyl)(ethyl)amino)propyl)azadialkyl)dioctanoate (YK-314)

[0359] YK-308-PM2 (60 mg, 0.37 mmol) and heptadecan-9-yl-8-bromooctanoate (512 mg, 1.11 mmol) were used as raw materials and YK-314 (120 mg, 0.13 mmol, 35.1%) was obtained according to the method for synthesizing YK-301-PM4. 57 H 114 N2O6,MS(ES):m / z(M+H + )923.9.

[0360] 1 H NMR(400MHz,Chloroform-d)δ4.86(p,J=6.1Hz,2H),3.87(s,1H),3.64(t,J=5.1Hz,2H),2.78–2.68(m,4H),2.66–2.35(m,8H), 2.27(t,J=7.5Hz,4H),1.65–1.57(m,4H),1.50(s,12H),1.29(d,J=22.9Hz,62H),1.09(t,J=7.0Hz,3H),0.88(t,J=6.6Hz,12H).

[0361] 10. Synthesis of YK-316

[0362] The synthetic route is as follows:

[0363]

[0364] Step 1: Synthesis of tert-butyl (S)-((3-(bis(2-hydroxyethyl)amino)-2-hydroxypropyl)carbamate (YK-316-PM1)

[0365] YK-301-PM1 (1.0 g, 4.78 mmol) and bis(hydroxyethyl)amine (452 ​​mg, 4.30 mmol) were used as raw materials, and YK-316-PM1 (550 mg, 1.98 mmol, 46.0%) was obtained according to the method for synthesizing YK-301-PM2. 12 H 26 N2O5,MS(ES):m / z(M+H + )279.2.

[0366] Step 2: Synthesis of (S)-1-amino-3-(bis(2-hydroxyethyl)amino)-2-propanol (YK-316-PM2)

[0367] YK-316-PM1 (200 mg, 0.72 mmol) was used as the raw material, and YK-316-PM2 (128 mg, 0.72 mmol, 100%) was obtained according to the method of synthesizing YK-301-PM3. 18 N2O3,MS(ES):m / z(M+H + )179.1.

[0368] Step 3: Synthesis of (S)-bis(2-octyldecyl)-6,6'-(((3-(bis(2-hydroxyethyl)amino)-2-hydroxypropyl)azadialkyl)dihexanoate (YK-316)

[0369] YK-316-PM2 (64 mg, 0.36 mmol) and 6-bromohexanoic acid-2-octyldecyl ester (160 mg, 0.36 mmol) were used as raw materials, and YK-316 (90 mg, 0.10 mmol, 27.4%) was obtained according to the method for synthesizing YK-301. 55 H 110 N2O7,MS(ES):m / z(M+H + )911.8.

[0370] 1 H NMR(400MHz,Chloroform-d)δ3.96(d,J=5.8Hz,4H),3.71(s,4H),3.09–2.77(m,10H),2. 33(t,J=7.2Hz,4H),1.66(dd,J=15.3,7.5Hz,6H),1.27(s,69H),0.88(t,J=6.8Hz,12H).

[0371] 11. Synthesis of YK-317

[0372] The synthetic route is as follows:

[0373]

[0374] Synthesis of (S)-bis(3-hexylnonyl)-6,6'-((3-(bis(2-hydroxyethyl)amino)-2-hydroxypropyl)azadialkyl)dihexanoate (YK-317)

[0375] YK-316-PM2 (105 mg, 0.59 mmol) and 6-bromohexanoic acid-3-hexylnonyl ester (500 mg, 1.23 mmol) were used as raw materials, and YK-317 (270 mg, 0.33 mmol, 55.3%) was obtained according to the method for synthesizing YK-301. 49 H 98 N2O7,MS(ES):m / z(M+H + )827.7.

[0376] 1 H NMR(400MHz,Chloroform-d)δ4.08(t,J=6.9Hz,4H),3.67(s,4H),3.48(s,2H),2.64(dd,J=34.4,20.4Hz,1 2H), 2.30 (t, J = 7.2Hz, 4H), 1.60 (dt, J = 31.5, 6.9Hz, 12H), 1.28 (d, J = 19.3Hz, 48H), 0.88 (t, J = 6.2Hz, 12H).

[0377] 12. Synthesis of YK-318

[0378] The synthetic route is as follows:

[0379]

[0380] Synthesis of (S)-bis(heptadecan-9-yl)-6,6'-((3-(bis(2-hydroxyethyl)amino)-2-hydroxypropyl)azadialkyl)dihexanoate (YK-318)

[0381] YK-316-PM2 (125 mg, 0.70 mmol) and heptadecan-9-yl-6-bromohexanoate (666 mg, 1.54 mmol) were used as raw materials and YK-318 (300 mg, 0.34 mmol, 48.5%) was obtained according to the method for synthesizing YK-301. 53 H 106 N2O7,MS(ES):m / z(M+H + )883.8.

[0382] 1 H NMR(400MHz,Chloroform-d)δ4.92–4.80(m,2H),3.90(s,4H),3.71–

[0383] 3.62(m,2H),3.62–3.53(m,2H),2.81–2.72(m,2H),2.72–2.48(m,9H),2.29(t,J=7.3 Hz, 4H), 1.68–1.60 (m, 4H), 1.59–1.44 (m, 12H), 1.26 (s, 53H), 0.88 (t, J = 5.5Hz, 12H).

[0384] 13. Synthesis of YK-319

[0385] The synthetic route is as follows:

[0386]

[0387] Synthesis of (S)-bis(3-hexylnonyl)-7,7'-(((3-(bis(2-hydroxyethyl)amino)-2-hydroxypropyl)azadialkyl)diheptanoate (YK-319)

[0388] YK-316-PM2 (34 mg, 0.19 mmol) and 3-hexylnonyl-7-bromoheptanoate (160 mg, 0.38 mmol) were used as raw materials and YK-319 (40 mg, 0.05 mmol, 24.6%) was obtained according to the method for synthesizing YK-301. 51 H 102 N2O7,MS(ES):m / z(M+H + )855.8.

[0389] 1 H NMR(400MHz,Chloroform-d)δ4.08(t,J=7.1Hz,4H),3.65(s,4H),2.76(t,J=45.6Hz,1 2H),2.29(t,J=7.3Hz,4H),1.74–1.51(m,12H),1.25(s,54H),0.88(t,J=6.4Hz,12H).

[0390] 14. Synthesis of YK-320

[0391] The synthetic route is as follows:

[0392]

[0393] Synthesis of (S)-bis(heptadecan-9-yl)-8,8'-(((3-(bis(2-hydroxyethyl)amino)-2-hydroxypropyl)azadialkyl) dioctanoate (YK-320)

[0394] YK-316-PM2 (41 mg, 0.23 mmol) and heptadecan-9-yl-8-bromooctanoate (244 mg, 0.53 mmol) were used as raw materials and YK-320 (150 mg, 0.16 mmol, 69.4%) was obtained according to the method for synthesizing YK-301. 57 H 114 N2O7,MS(ES):m / z(M+H + )939.9.

[0395] 1 H NMR(400MHz,Chloroform-d)δ5.30(s,1H),4.85(p,J=6.0Hz,2H),4.19(s,2H),3.76–3.55(m,5H),2.98–2.60(m,10H),2 .28(t,J=7.4Hz,4H),1.64(dd,J=15.8,7.4Hz,8H),1.55–1.46(m,8H),1.30(d,J=31.6Hz,60H),0.88(t,J=6.7Hz,12H).

[0396] 15. Synthesis of YK-321

[0397] The synthetic route is as follows:

[0398]

[0399] Synthesis of (S)-bis(4-decyltetradecyl)-4,4'-((3-(bis(2-hydroxyethyl)amino)-2-hydroxypropyl)azadialkyl)dibutyrate (YK-321)

[0400] YK-316-PM2 (41 mg, 0.23 mmol) and 4-bromobutyric acid-4-decyltetradecyl ester (266 mg, 0.53 mmol) were used as raw materials and YK-321 (85 mg, 0.08 mmol, 36.1%) was obtained according to the method for synthesizing YK-301. 63 H 126 N2O7,MS(ES):m / z(M+H + )1024.0.

[0401] 1H NMR(400MHz,Chloroform-d)δ5.30(s,1H),4.04(t,J=6.8Hz,4H),3.71(s,4H),2.72(s,10H),2.35 (t,J=6.8Hz,4H),1.89(s,4H),1.63–1.55(m,4H),1.25(d,J=12.2Hz,80H),0.88(t,J=6.6Hz,12H).

[0402] 16. Synthesis of YK-009

[0403] According to the method in CN114044741B, 105 mg of YK-009 was obtained.

[0404] 17. Synthesis of 9-heptadecyl-8-(8-((3-hexylnonyl)oxy)-8-oxooctyl)-((2-hydroxyethyl)amino)octanoate (Compound 21)

[0405] The synthetic route is as follows:

[0406]

[0407] Step 1: Synthesis of 8-bromooctanoic acid-9-heptadecanyl ester (Compound 21-PM1)

[0408] 9-heptadecanol (1.00 g, 3.90 mmol) and 8-bromooctanoic acid (1.04 g, 4.66 mmol) were dissolved in dichloromethane (10 mL), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.90 g, 4.68 mmol) and 4-dimethylaminopyridine (24 mg, 0.20 mmol) were added, and the mixture was stirred at 30-35°C for 8 hours. After the reaction was completed, the reaction solution was washed with saturated sodium carbonate and saturated brine, and dried over Na2SO4. The mixture was filtered, and the filtrate was concentrated under vacuum and purified by silica gel chromatography (ethyl acetate / n-hexane) to obtain 8-bromooctanoic acid-9-heptadecanyl ester (1.20 g, 2.60 mmol, 66.7%).

[0409] Step 2: Synthesis of 9-heptadecanyl-8-((2-hydroxyethyl)amino)octanoate (Compound 21-PM2)

[0410] 8-bromooctanoic acid-9-heptadecanyl ester (500 mg, 1.08 mmol) and ethanolamine (119 mg, 3.25 mmol) were dissolved in acetonitrile (5 mL), potassium carbonate (149 mg, 1.08 mmol) was added, and the mixture was heated to 70°C and stirred for 2 hours. After the reaction was completed, the reaction solution was cooled to room temperature and filtered, and the filtrate was concentrated under vacuum to remove the solvent. The residue was purified by silica gel chromatography (methanol / dichloromethane) to obtain 9-heptadecanyl-8-((2-hydroxyethyl)amino)octanoate (372 mg, 0.84 mmol, 78.0%), C 27 H 55 NO3,MS(ES):m / z(M+H + )442.3.

[0411] Step 3: Synthesis of 8-bromooctanoic acid-3-hexylnonyl ester (Compound 21-PM3)

[0412] Using 3-hexylnonanol (1.00 g, 4.38 mmol) and 8-bromooctanoic acid (1.17 g, 5.25 mmol) as raw materials, according to the method for preparing compound 21-PM1, the product was purified by silica gel chromatography (ethyl acetate / n-hexane) to obtain 8-bromooctanoic acid-3-hexylnonyl ester (1.62 g, 3.74 mmol, 85.3%).

[0413] Step 4: Synthesis of 9-heptadecyl-8-(8-((3-hexylnonyl)oxy)-8-oxooctyl)-((2-hydroxyethyl)amino)octanoate (Compound 21)

[0414] 9-Heptadecanyl-8-((2-hydroxyethyl)amino)octanoate (200 mg, 0.46 mmol) and 8-bromooctanoic acid-3-hexylnonyl ester (336 mg, 0.82 mmol) were dissolved in acetonitrile (6 mL), potassium carbonate (254 mg, 1.84 mmol) and potassium iodide (8.3 mg, 0.05 mmol) were added, and the mixture was heated to 70°C and stirred for 20 hours. The reaction solution was cooled to room temperature and filtered, and the filtrate was concentrated under vacuum to remove the solvent. The residue was purified by silica gel chromatography (ethyl acetate / n-hexane) to obtain the target compound (213 mg, 0.27 mmol, 58.4%). 50 H 99 NO5, MS (ES): m / z (M+H + )794.8.

[0415] 1H NMR(400MHz, CDCl3) δ4.90(p,J=6.3Hz,1H),4.21–4.02(m,2H),3.66(s,2H),2.73(s,2H),2.60(s,4H),2.43–2.20( m,4H),2.12–1.99(m,1H),1.75–1.49(m,13H),1.48–1.39(m,2H),1.42–1.15(m,56H),0.92(td,J=6.8,2.2Hz,12H).

[0416] 18. Synthesis of bis(3-hexylnonyl)-8,8'-((2-hydroxyethyl)azadialkyl) dioctanoate (Compound 23)

[0417] The synthetic route is as follows:

[0418]

[0419] 8-bromooctanoic acid-3-hexylnonyl ester (710 mg, 1.64 mmol) and ethanolamine (40 mg, 0.66 mmol) were dissolved in acetonitrile (10 mL), potassium carbonate (1.09 g, 7.92 mmol) and potassium iodide (66 mg, 0.39 mmol) were added to the above system, and the mixture was heated to 70 ° C and stirred for 20 hours. After the reaction was completed, the reaction solution was cooled to room temperature and filtered, and the filtrate was concentrated under vacuum to remove the solvent. The residue was purified by silica gel chromatography (methanol / dichloromethane) to obtain bis(3-hexylnonyl)-8,8'-((2-hydroxyethyl)azadialkyl) dioctanoate (150 mg, 0.20 mmol, 29.7%), C 48 H 95 NO5,MS(ES):m / z(M+H + )766.5.

[0420] 1 H NMR (400MHz, CDCl3) δ4.12(t,J=7.1Hz,4H),3.62(s,2H),2.68(s,2H),2.51(d,J=25.8Hz,4H),2.32 (t,J=7.5Hz,4H),1.72–1.57(m,8H),1.55–1.40(m,6H),1.40–1.17(m,55H),0.92(t,J=6.8Hz,12H).

[0421] Example 2: Optimization of preparation conditions of nanolipid particles (LNP preparation)

[0422] 1. Optimization of the ratio of carrier (liposome) to mRNA

[0423]

[0424] The cationic lipid compound YK-305 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 prepare an ethanol lipid solution. The ethanol lipid solution was quickly added to a citrate buffer (pH = 4-5) by an ethanol injection method and vortexed for 30 seconds for standby use. The eGFP-mRNA was diluted in a citrate buffer (pH = 4-5) to obtain an mRNA aqueous solution. A certain volume of liposome solution and an mRNA aqueous solution were prepared with a total lipid to mRNA weight ratio of 5:1, 10:1, 15:1, 20:1, 30:1 and 35:1 to prepare liposomes. Ultrasound at 25°C for 15 minutes (ultrasonic frequency 40kHz, ultrasonic power 800W). After the obtained liposomes were diluted to 10 times the volume with PBS, ethanol was removed by ultrafiltration using a 300KDa ultrafiltration tube. The mixture was then diluted to a certain volume with PBS to obtain an LNP preparation encapsulating eGFP-mRNA using cationic lipid YK-305 / DSPC / cholesterol / DMG-PEG2000 (molar percentage of 49:10:39.5:1.5).

[0425] The results of cell transfection experiments showed that the weight ratio of vector to mRNA in the range of 10:1 to 30:1 had good transfection effects, with the best transfection effect at 15:1. The ratios of 5:1 and 35:1 had poor transfection effects and should not be used to transport mRNA. Figure 1 )

[0426] The same results were obtained for LNP preparations prepared using YK-310, YK-312, YK-319 and YK-318, which are not shown in the figure.

[0427] 2. Optimization of the ratio of cationic lipids to neutral lipids

[0428] The LNP preparations encapsulating eGFP-mRNA were prepared according to the method in 1, wherein the molar ratios of cationic lipid YK-305 and neutral lipid DSPC were 1:1, 3:1, 3.5:1, 4:1, 4.5:1, 4.9:1, 10:1, 15:1 and 20:1, respectively.

[0429] Through the cell transfection experiment, it can be seen that the molar ratio of cationic lipid to neutral lipid is 1:1 to 15:1, and the transfection effect is good. Among them, the highest transfection efficiency is 4.5:1, and the ratios of 3.5:1 and 4.9:1 also have good transfection effects. Figure 2 )

[0430] The same results were obtained for LNP preparations prepared using YK-310, YK-312, YK-319 and YK-318, which are not shown in the figure.

[0431] 3. Optimization of the ratio of polymer-conjugated lipids to carrier (liposome)

[0432] According to the method in 1, LNP preparations encapsulating eGFP-mRNA were prepared, the cationic lipid in the carrier was YK-305, and the molar ratio of the polymer conjugated lipid DMG-PEG2000 to the carrier was 0.5%, 1.5%, 3.5%, 5%, 10% and 15%, respectively.

[0433] The results of cell transfection experiments showed that the polymer-conjugated lipid ratio in the carrier ranged from 0.5% to 10%, with the highest transfection efficiency at 1.5% and the lowest at 10%. Figure 3 )

[0434] The same results were obtained for LNP preparations prepared using YK-310, YK-312, YK-319 and YK-318, which are not shown in the figure.

[0435] 4. Optimization of the ratio of each component in the carrier (liposome)

[0436] The LNP preparation encapsulating eGFP-mRNA was prepared according to the method in 1, wherein the molar ratios of cationic lipid YK-305, neutral lipid DSPC, structural lipid cholesterol and polymer conjugated lipid DMG-PEG2000 were 75:5:15:5, 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.

[0437] Cell transfection experiments show that cationic lipids, neutral lipids, structural lipids and polymer conjugated lipids can be transfected at molar ratios of 75:5:15:5, 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. The ratio of (35-49):(7.5-15):(35-55):(1-5) has a good transfection effect, and the ratio of 45:10:43.5:1.5 has the best transfection effect. Figure 4) shows that the molar ratio of cationic lipids, neutral lipids, structural lipids and polymer conjugated lipids in the range of (25-75):(5-25):(15-65):(0.5-10) can be used to prepare LNP preparations, and the preferred ratio is (35-49):(7.5-15):(35-55):(1-5), among which the best ratio is 45:10:43.5:1.5.

[0438] The same results were obtained for LNP preparations prepared using YK-310, YK-312, YK-319 and YK-318, which are not shown in the figure.

[0439] Example 3: Cell transfection experiment of LNP preparation of eGFP-mRNA

[0440] Cell recovery and passaging: Recover 293T cells and culture them in culture dishes to the required cell number.

[0441] Seeding: Digest and count the cells in the culture dish, seed 10,000 cells per well in a 96-well plate, or 150,000 cells per well in a 12-well plate, and culture overnight until the cells adhere.

[0442] Cell transfection experiment: The LNP preparation containing 1.5 μg of the eGFP-mRNA prepared in Example 2 (the cationic lipid in the carrier is YK-305) and the Lipofectamin 3000 preparation of eGFP-mRNA were added to the cell culture medium of a 12-well plate, and the cells were cultured for 24 hours. The transfection efficiency of different samples was investigated by fluorescence microscopy based on the fluorescence intensity.

[0443] According to the experimental results, the preparation conditions of nano lipid particles (LNP preparations) were finally determined: the weight ratio of carrier to mRNA was 15:1; the molar ratio of cationic lipid to neutral lipid was 4.9:1; the polymer conjugated lipid accounted for 1.5% of the liposome molar ratio; the molar ratio of cationic lipid, neutral lipid, structural lipid and polymer conjugated lipid was 49:10:39.5:1.5. In this ratio, various cationic lipids designed in this application and cationic lipids used in the prior art all had better transfection effect (determined by Example 2, some experimental results are not shown), and the subsequent experiments used this condition to prepare nano lipid particles (LNP preparations).

[0444] Example 4: Preparation of Nanolipid Particles (LNP Preparation) (Optimal Ratio)

[0445] Table 1 Cationic lipid structures

[0446]

[0447]

[0448]

[0449]

[0450]

[0451]

[0452] The cationic lipids listed in Table 1 were 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 prepare an ethanol lipid solution, and the ethanol lipid solution was quickly added to a citrate buffer (pH = 4-5) by an ethanol injection method, and vortexed for 30 seconds for use. eGFP-mRNA (Shanghai Qifa Experimental Reagent Co., Ltd.) or Fluc-mRNA (Shanghai Qifa Experimental Reagent Co., Ltd.) was diluted in a citrate buffer (pH = 4-5) to obtain an mRNA aqueous solution. A certain volume of liposome solution and mRNA aqueous solution were mixed to prepare liposomes at a weight ratio of total lipid to mRNA of 15:1. Ultrasonication at 25°C for 15 minutes (ultrasonic frequency 40kHz, ultrasonic power 800W). The obtained liposomes were diluted to 10 times the volume with PBS, and then ultrafiltered to remove ethanol with a 300KDa ultrafiltration tube. The mixture was then diluted to a certain volume with PBS to obtain an LNP preparation encapsulating eGFP-mRNA or Fluc-mRNA using cationic lipid / DSPC / cholesterol / DMG-PEG2000 (molar percentage of 49:10:39.5:1.5).

[0453] Lipofectamine 3000 transfection reagent is currently widely used for cell transfection. It has very good transfection performance and excellent transfection efficiency, can improve cell activity, and is suitable for difficult-to-transfect cell types. We used Lipofectamine 3000 transfection reagent as a control and prepared Lipofectamine 3000 preparations of eGFP-mRNA or Fluc-mRNA according to the method in the instruction manual of Lipofectamine 3000 (Invitrogen (Shanghai) Trading Co., Ltd.).

[0454] Example 5: Determination of particle size and polydispersity index (PDI) of nanolipid particles

[0455] The particle size and polydispersity index (PDI) were determined using a Malvern laser particle size analyzer using dynamic light scattering.

[0456] Take 10 μL of liposome solution, dilute to 1 mL with RNase-free deionized water, add to the sample pool, and repeat the measurement 3 times for each sample. The measurement conditions are: 90° scattering angle, 25°C. The test results are as follows:

[0457] Table 2 Particle size and polydispersity index (PDI)

[0458]

[0459]

[0460] The particle size of the nanolipid particles prepared in Example 4 is between 140 and 280 nm, and can be used to deliver mRNA. The particle size prepared by compound 23 and YK-304 is the smallest, 147 nm and 149 nm, respectively, and the particle size prepared by YK-317 is the largest, 255 nm. The polydispersity coefficient of all nanolipid particles is between 5% and 45%, the smallest of which is YK-301, which is 7.0%, and the largest is YK-321, which is 39.1%.

[0461] Example 6: In vitro validation of the performance of LNP delivery vehicles

[0462] Cell recovery and passage: The method is the same as in Example 3.

[0463] Seed board: The method is the same as Example 3.

[0464] 1. Fluorescence detection of Fluc-mRNA

[0465] The LNP preparation containing 0.3 μg Fluc-mRNA (the LNP preparation carrier components are cationic lipids, neutral lipids, structural lipids and polymer conjugated lipids, with a molar ratio of 49:10:39.5:1.5, wherein the cationic lipids are the cationic lipids listed in Table 1) was added to the cell culture medium of the 96-well plate, and the culture was continued for 24 hours. The corresponding reagents were added according to the instructions of the Gaussia Luciferase AssayKit, and the fluorescence expression intensity of each well was detected by the IVIS fluorescence detection system. This experiment verified the transfection efficiency of the LNP preparation in the cell, and the specific test results are shown in Tables 4-7.

[0466] Experimental results:

[0467] (1) The compounds of the present application, including YK-305, YK-310, YK-312, YK-319 and YK-318, are very different from the cationic lipid chemical structures of the prior art.

[0468] The series of compounds designed in this application, including YK-305, YK-310, YK-312, YK-319 and YK-318, are very different from the prior art cationic lipid chemical structures. For example, these compounds are completely different from the HHMA structure; compared with SM-102, compound 21, compound 23 and YK-009, the G3 group is completely different, and the G1, G2, R1 and R2 groups are also very different. The specific structural comparison is shown in Table 3.

[0469] Table 3 Designed compounds and representative cationic lipids in the prior art

[0470]

[0471]

[0472] As can be seen from Table 3, this series of designed compounds, including YK-305, YK-310, YK-312, YK-319 and YK-318, are very different from the representative cationic lipid chemical structures of the prior art. YK-009 is disclosed in CN114044741B (claim 1), compounds 21 and 23 are disclosed in WO2021055833A1 (page 22 of the specification), SM-102 is compound 25 disclosed in WO2017049245A2 (page 29 of the specification), ALC-0315 is compound 3 disclosed in CN108368028B (page 24 of the specification), and HHMA is compound 1 disclosed in CN112979483B (page 12 of the specification).

[0473] Compared with this series of compounds:

[0474] a. The HHMA structure is the most different. From the chemical structure diagram, it can be seen that in the group connected to the central N atom of HHMA, only one side chain is similar to one side chain of this series of structures, and the other parts are completely different.

[0475] b. Other cationic lipids in the prior art, such as SM-102, ALC-0315, compound 21, compound 23 and YK-009, have completely different G3 groups. This series of compounds has one more tertiary amine group and 1-2 more hydroxyl groups in the G3 group, so there are also great differences in polarity, acidity and alkalinity, and hydrophilicity.

[0476] c. There are also huge differences in the G1, G2, R1 and R2 groups of SM-102, ALC-0315, Compound 21, Compound 23 and YK-009.

[0477] The details are as follows:

[0478] I.YK-305

[0479] YK-305 has a significant structural difference compared to prior art cationic lipids, such as SM-102, compound 21, compound 23, YK-009 and HHMA.

[0480] Compared with SM-102, the R1 group of YK-305 is a branched structure, while SM-102 is a straight chain structure; the G2 group has 2 fewer Cs; the R2 group single chain has 1 more C; the G3 group is completely different, HO(CH2)2N(CH3)CH2CH(OH)CH2-, while SM-102 is HO(CH2)2-.

[0481] Compared with ALC-0315, the G1 group of YK-305 has one less C; the R1 group has one more C in a single chain, and one more C in a double chain; the G2 group has one less C; the R2 group has one more C in a single chain, and one more C in a double chain; the G3 group is completely different, HO(CH2)2N(CH3)CH2CH(OH)CH2-, while ALC-0315 is HO(CH2)4-. In addition, the directions of the ester bonds between the G1 group and the R1 group, and between the G2 group and the R2 group, are also different in YK-305 and ALC-0315.

[0482] Compared with compound 21, the G1 group of YK-305 has 2 fewer Cs; the single chain of the R1 group has 1 less C, and each single chain in the double chain has 2 more Cs; the G2 group has 2 fewer Cs; the single chain of the R2 group has 1 more C; the G3 group is completely different, which is HO(CH2)2N(CH3)CH2CH(OH)CH2-, while compound 21 is HO(CH2)2-.

[0483] Compared with compound 23, the G1 group of YK-305 has 2 fewer Cs; the R1 group has 1 less C in a single chain, and each single chain in the double chain has 2 more Cs; the G2 group has 2 fewer Cs; the R2 group has 1 less C in a single chain, and each single chain in the double chain has 2 more Cs; the G3 group is completely different, being HO(CH2)2N(CH3)CH2CH(OH)CH2-, while compound 23 is HO(CH2)2-.

[0484] Compared with YK-009, the G1 group of YK-305 has 2 more Cs; the R1 group is a branched structure, while YK-009 is a straight chain structure; the G3 group is completely different, HO(CH2)2N(CH3)CH2CH(OH)CH2-, while YK-009 is HO(CH2)2-.

[0485] Compared with HHMA, the structure of YK-305 is completely different. Only one side chain connected to the N atom of HHMA is similar to the side chain structure of YK-305, and the other parts are very different.

[0486] II.YK-310

[0487] YK-310 has a significant structural difference compared to prior art cationic lipids, such as SM-102, compound 21, compound 23, YK-009 and HHMA.

[0488] Compared with SM-102, the G2 group of YK-310 has 4 fewer carbon atoms; the R2 group has 3 more carbon atoms in a single chain and 2 more carbon atoms in each single chain of the double chain; the G3 group is completely different, being HO(CH2)2N(CH2CH3)CH2CH(OH)CH2-, while SM-102 is HO(CH2)2-.

[0489] Compared with ALC-0315, the G1 group of YK-310 has one less C; the R1 group is a straight chain structure, while ALC-0315 is a branched structure; the G2 group has 3 less C; the R2 group has 3 more C in a single chain, 2 more C in one of the double chains, and 4 more C in the other; the G3 group is completely different, HO(CH2)2N(CH2CH3)CH2CH(OH)CH2-, while ALC-0315 is HO(CH2)4-. In addition, the directions of the ester bonds between the G1 group and the R1 group, and between the G2 group and the R2 group, are also different for YK-310 and ALC-0315.

[0490] Compared with compound 21, the G1 group of YK-310 has 2 fewer Cs; the R1 group is a straight chain structure, while compound 21 is a branched structure; the G2 group has 4 fewer Cs; the single chain of the R2 group has 3 more Cs, and each single chain in the double chain has 2 more Cs; the G3 group is completely different, which is HO(CH2)2N(CH2CH3)CH2CH(OH)CH2-, while compound 21 is HO(CH2)2-.

[0491] Compared with compound 23, the G1 group of YK-310 has 2 fewer Cs; the R1 group is a straight chain structure, while compound 23 is a branched structure; the G2 group has 4 fewer Cs; the single chain of the R2 group has 1 more C, and each single chain in the double chain has 4 more Cs; the G3 group is completely different, which is HO(CH2)2N(CH2CH3)CH2CH(OH)CH2-, while compound 23 is HO(CH2)2-.

[0492] Compared with YK-009, the G1 group of YK-310 has 2 more Cs; the R1 group has 1 more C; the G2 group has 2 fewer Cs; the R2 group has 2 more Cs in a single chain and 2 more Cs in each single chain of the double chain; the G3 group is completely different, HO(CH2)2N(CH2CH3)CH2CH(OH)CH2-, while YK-009 is HO(CH2)2-.

[0493] Compared with HHMA, the structure of YK-310 is completely different. Only one side chain connected to the N atom of HHMA is similar to the side chain structure of YK-310, and the other parts are very different.

[0494] III.YK-312

[0495] YK-312 has a significant structural difference compared to prior art cationic lipids, such as SM-102, compound 21, compound 23, YK-009 and HHMA.

[0496] Compared with SM-102, the R1 group of YK-312 is a branched structure, while SM-102 is a straight chain structure; the G2 group has 2 fewer Cs; the R2 group single chain has 1 more C; the G3 group is completely different, which is HO(CH2)2N(CH2CH3)CH2CH(OH)CH2-, while SM-102 is HO(CH2)2-.

[0497] Compared with ALC-0315, the G1 group of YK-312 has one less C; the R1 group has one more C in a single chain, and one more C in a double chain; the G2 group has one less C; the R2 group has one more C in a single chain, and one more C in a double chain; the G3 group is completely different, HO(CH2)2N(CH2CH3)CH2CH(OH)CH2-, while ALC-0315 is HO(CH2)4-. In addition, the directions of the ester bonds between the G1 group and the R1 group, and between the G2 group and the R2 group, are also different for YK-312 and ALC-0315.

[0498] Compared with compound 21, the G1 group of YK-312 has 2 fewer Cs; the single chain of the R1 group has 1 less C, and each single chain in the double chain has 2 more Cs; the G2 group has 2 fewer Cs; the single chain of the R2 group has 1 more C; the G3 group is completely different, which is HO(CH2)2N(CH2CH3)CH2CH(OH)CH2-, while compound 21 is HO(CH2)2-.

[0499] Compared with compound 23, the G1 group of YK-312 has 2 fewer Cs; the R1 group has 1 less C in a single chain, and each single chain in the double chain has 2 more Cs; the G2 group has 2 fewer Cs; the R2 group has 1 less C in a single chain, and each single chain in the double chain has 2 more Cs; the G3 group is completely different, being HO(CH2)2N(CH2CH3)CH2CH(OH)CH2-, while compound 23 is HO(CH2)2-.

[0500] Compared with YK-009, the G1 group of YK-312 has 2 more Cs; the R1 group is a branched structure, while YK-009 is a straight chain structure; the G3 group is completely different, HO(CH2)2N(CH2CH3)CH2CH(OH)CH2-, while YK-009 is HO(CH2)2-.

[0501] Compared with HHMA, the structure of YK-312 is completely different. Only one side chain connected to the N atom of HHMA is similar to the side chain structure of YK-312, and the other parts are very different.

[0502] IV.YK-319

[0503] YK-319 has a significant structural difference compared to prior art cationic lipids, such as SM-102, compound 21, compound 23, YK-009 and HHMA.

[0504] Compared with SM-102, the G1 group of YK-319 has one more C; the R1 group is a branched structure, while SM-102 is a straight chain structure; the G2 group has one less C; the single chain of the R2 group has 2 more Cs, and each single chain in the double chain has 2 less Cs; the G3 group is completely different, (HO(CH2)2)2NCH2CH(OH)CH2-, while SM-102 is HO(CH2)2-.

[0505] Compared with ALC-0315, the single chain of the R1 group of YK-319 has 2 more Cs, and one of the double chains has 2 less Cs; the single chain of the R2 group has 2 more Cs, and one of the double chains has 2 less Cs; the G3 group is completely different, (HO(CH2)2)2NCH2CH(OH)CH2-, while ALC-0315 is HO(CH2)4-. In addition, the directions of the ester bonds between the G1 group and the R1 group, and between the G2 group and the R2 group, are also different in YK-319 and ALC-0315.

[0506] Compared with compound 21, the G1 group of YK-319 has 1 less C; the G2 group has 1 less C; the R2 group has 2 more Cs in the single chain and 2 less Cs in each single chain of the double chain; the G3 group is completely different, which is (HO(CH2)2)2NCH2CH(OH)CH2-, while compound 21 is HO(CH2)2-.

[0507] Compared with compound 23, the G1 group of YK-319 has one less C; the G2 group has one less C; the G3 group is completely different, (HO(CH2)2)2NCH2CH(OH)CH2-, while compound 23 is HO(CH2)2-.

[0508] Compared with YK-009, the G1 group of YK-319 has 3 more Cs; the R1 group is a branched structure, while YK-009 is a straight chain structure; the G2 group has 1 more C; the single chain of the R2 group has 1 more C, and each single chain in the double chain has 2 less Cs; the G3 group is completely different, which is (HO(CH2)2)2NCH2CH(OH)CH2-, while YK-009 is HO(CH2)2-.

[0509] Compared with HHMA, the structure of YK-319 is completely different. Only one side chain connected to the N atom of HHMA is similar to the side chain structure of YK-319, and the other parts are very different.

[0510] V.YK-318

[0511] YK-318 has a significant structural difference compared to prior art cationic lipids, such as SM-102, compound 21, compound 23, YK-009 and HHMA.

[0512] Compared with SM-102, the R1 group of YK-318 is a branched structure, while SM-102 is a straight chain structure; the G2 group has 2 fewer Cs; the G3 group is completely different, (HO(CH2)2)2NCH2CH(OH)CH2-, while SM-102 is HO(CH2)2-.

[0513] Compared with ALC-0315, the G1 group of YK-318 has one less C; one single chain in the double chain of the R1 group has two more C; the G2 group has one less C; one single chain in the double chain of the R2 group has two more C; the G3 group is completely different, (HO(CH2)2)2NCH2CH(OH)CH2-, while ALC-0315 is HO(CH2)4-. In addition, the directions of the ester bonds between the G1 group and the R1 group, and between the G2 group and the R2 group, are also different in YK-318 and ALC-0315.

[0514] Compared with compound 21, the G1 group of YK-318 has 2 fewer Cs; the R1 group has 2 fewer Cs in the single chain and 2 more Cs in each single chain of the double chain; the G2 group has 2 fewer Cs; and the G3 group is completely different, being (HO(CH2)2)2NCH2CH(OH)CH2-, while compound 21 is HO(CH2)2-.

[0515] Compared with compound 23, the G1 group of YK-318 has 2 fewer Cs; the R1 group has 2 fewer Cs in a single chain, and 2 more Cs in each single chain of the double chain; the G2 group has 1 less C; the R2 group has 2 fewer Cs in a single chain, and 2 more Cs in each single chain of the double chain; the G3 group is completely different, which is (HO(CH2)2)2NCH2CH(OH)CH2-, while compound 23 is HO(CH2)2-.

[0516] Compared with YK-009, the G1 group of YK-318 has 2 more Cs; the R1 group is a branched structure, while YK-009 is a straight chain structure; the R2 group has 1 less C in a single chain; the G3 group is completely different, (HO(CH2)2)2NCH2CH(OH)CH2-, while YK-009 is HO(CH2)2-.

[0517] Compared with HHMA, the structure of YK-318 is completely different. Only one side chain connected to the N atom of HHMA is similar to the side chain structure of YK-318, and the other parts are very different.

[0518] From the above comparison, it can be seen that the designed series of compounds, including YK-305, YK-310, YK-312, YK-319 and YK-318, are very different in chemical structure from the prior art cationic lipid compounds, such as SM-102, ALC-0315, compound 21, compound 23, HHMA and YK-009. This series of compounds is completely different from the HHMA structure; it is completely different from the G3 group of SM-102, ALC-0315, compound 21, compound 23 and YK-009. The G3 group of this series of compounds has one more tertiary amine group and 1-2 more hydroxyl groups, and the G1, G2, R1 and R2 groups are also very different.

[0519] Due to the huge difference in chemical structure, the physicochemical properties of this series of compounds, such as polarity, acidity and alkalinity, and hydrophilicity, are also very different from those of SM-102, ALC-0315, compound 21, compound 23, HHMA, and YK-009. Therefore, it is impossible to infer the cell transfection efficiency, cytotoxicity, and in vivo expression of the LNP preparations prepared by this series of compounds based on the above cationic lipid compounds disclosed in the prior art.

[0520] (2) Among the designed series of compounds, the LNP preparations prepared by YK-305, YK-310, YK-312, YK-319 and YK-318 had the highest cell transfection efficiency, which was significantly improved compared with the representative cationic lipids in the prior art. For example, YK-305 could reach 17 times that of SM-102, 19 times that of compound 21 and 20 times that of compound 23.

[0521] Table 4 Chemical structures of designed compounds and representative cationic lipids in the prior art

[0522]

[0523]

[0524] Table 5 Fluorescence detection results of Fluc-mRNA-1

[0525]

[0526] Differences in cell transfection efficiency

[0527] Table 4 shows the chemical structure differences between the designed compounds and representative cationic lipids in the prior art.

[0528] Table 5 lists the fluorescence detection results of LNP preparations containing Fluc-mRNA prepared by different cationic lipids. Among them, YK-009 is disclosed in CN114044741B (claim 1), compound 21 and compound 23 are disclosed in WO2021055833A1 (page 22 of the specification), SM-102 is compound 25 disclosed in WO2017049245A2 (page 29 of the specification), ALC-0315 is compound 3 disclosed in CN108368028B (page 24 of the specification), and HHMA is compound 1 disclosed in CN112979483B (page 12 of the specification); Lipofectamine 3000 is a widely used cell transfection reagent. These cationic lipids are representative cationic lipids in the prior art and have good transfection performance.

[0529] From Table 5 and Figure 5 It can be seen that the LNP preparations containing Fluc-mRNA prepared by YK-305, YK-310, YK-312, YK-319 and YK-318 have the strongest fluorescence absorption, and the RLU values ​​are 27408734, 25797040, 20148450, 24467760 and 11190068, respectively.

[0530] The activity of YK-305 can reach 17.09 times that of SM-102, 13.48 times that of ALC-0315, 19.14 times that of compound 21, 20.21 times that of compound 23, 13.66 times that of HHMA, 23.12 times that of Lipofectamine 3000 and 5.35 times that of YK-009.

[0531] The activity of YK-310 can reach 16.08 times that of SM-102, 12.69 times that of ALC-0315, 18.01 times that of compound 21, 19.02 times that of compound 23, 12.86 times that of HHMA, 21.76 times that of Lipofectamine 3000 and 5.04 times that of YK-009.

[0532] The activity of YK-312 can reach 12.56 times that of SM-102, 9.91 times that of ALC-0315, 14.07 times that of compound 21, 14.86 times that of compound 23, 10.04 times that of HHMA, 16.99 times that of Lipofectamine 3000 and 3.94 times that of YK-009.

[0533] The activity of YK-319 can reach 15.25 times that of SM-102, 12.03 times that of ALC-0315, 17.08 times that of compound 21, 18.04 times that of compound 23, 12.20 times that of HHMA, 20.63 times that of Lipofectamine 3000 and 4.78 times that of YK-009.

[0534] The activity of YK-318 can reach 6.98 times that of SM-102, 5.50 times that of ALC-0315, 7.81 times that of compound 21, 8.25 times that of compound 23, 5.58 times that of HHMA, 9.44 times that of Lipofectamine 3000 and 2.19 times that of YK-009.

[0535] The data were analyzed using GraphPad Prism software. Any one of YK-305, YK-310, YK-312, YK-319 and YK-318 showed significant differences from SM-102, ALC-0315, compound 21, compound 23, HHMA, Lipofectamine 3000 and YK-009, and the transfection efficiency was significantly improved.

[0536] summary:

[0537] In terms of chemical structure, the designed series of compounds, including YK-305, YK-310, YK-312, YK-319 and YK-318, are very different from the representative cationic lipids in the prior art. For example, the structure is completely different from that of HHMA; compared with SM-102, ALC-0315, compound 21, compound 23 and YK-009, the G3 group is completely different, and the G1, G2, R1 and R2 groups are also very different.

[0538] The LNP preparations prepared from YK-305, YK-310, YK-312, YK-319 and YK-318 have the highest cell transfection efficiency and are significantly more active than representative cationic lipids in the prior art. For example, YK-305 can reach 17 times that of SM-102, 19 times that of compound 21 and 20 times that of compound 23.

[0539] At the same time, the present application designs for the first time a compound with a chemical structure greatly different from that of cationic lipids in the prior art, and the LNP preparation prepared therefrom has significantly improved transfection efficiency and significantly enhanced cell transfection activity.

[0540] (3) YK-305, YK-310, YK-312, YK-319, and YK-318 have the highest cell transfection efficiency compared to a series of compounds with similar structures and G3 groups of HO(CH2)2N(CH3)CH2CH(OH)CH2-. For example, YK-305 is 1300 times that of YK-304 and 900 times that of YK-302.

[0541] We compared a series of compounds with similar structures and G3 groups of HO(CH2)2N(CH3)CH2CH(OH)CH2- with YK-305, YK-310, YK-312, YK-319 and YK-318. The structural differences of these compounds are only slightly different in G1, G2, G3, R1 or R2 groups (Table 6). The results show that the activity of this series of compounds varies greatly, among which YK-305, YK-310, YK-312, YK-319 and YK-318 have the highest cell transfection efficiency, which can reach 1300 times, 1200 times, 900 times, 1100 times and 400 times of the lowest activity YK-304, respectively, and the transfection efficiency is significantly improved.

[0542] Table 6 Chemical structures of designed compounds

[0543]

[0544]

[0545] Table 7 Fluorescence detection results of Fluc-mRNA-2

[0546]

[0547] a. Differences in cell transfection efficiency

[0548] From Table 7 and Figure 6 It can be seen that the fluorescence absorption values ​​of the LNP preparations prepared from these compounds are very different from those of YK-305, YK-310, YK-312, YK-319 and YK-318.

[0549] The strength of YK-305 can reach 32.53 times that of YK-301, 986.71 times that of YK-302, 43.76 times that of YK-303, 1324.99 times that of YK-304, 12.47 times that of YK-306 and 6.01 times that of YK-307.

[0550] The strength of YK-310 can reach 30.62 times that of YK-301, 928.69 times that of YK-302, 41.19 times that of YK-303, 1247.08 times that of YK-304, 11.74 times that of YK-306 and 5.66 times that of YK-307.

[0551] The strength of YK-312 can reach 23.92 times that of YK-301, 725.34 times that of YK-302, 32.17 times that of YK-303, 974.01 times that of YK-304, 9.17 times that of YK-306 and 4.42 times that of YK-307.

[0552] The strength of YK-319 can reach 29.04 times that of YK-301, 880.83 times that of YK-302, 39.07 times that of YK-303, 1182.82 times that of YK-304, 11.14 times that of YK-306 and 5.37 times that of YK-307.

[0553] The strength of YK-318 can reach 13.28 times that of YK-301, 402.84 times that of YK-302, 17.87 times that of YK-303, 540.95 times that of YK-304, 5.09 times that of YK-306 and 2.46 times that of YK-307.

[0554] The activity differences among YK-301, YK-302, YK-303, YK-304, YK-306 and YK-307 are also large. The cell transfection efficiency of YK-306 and YK-307 is stronger than that of SM-102, which can reach 1.37 times and 2.84 times of SM-102 respectively; YK-301 and YK-303 are not much different from SM-102, but slightly lower, which are 0.53 times and 0.39 times respectively; YK-302 and YK-304 have the lowest cell transfection efficiency, which is only 0.017 times and 0.013 times of SM-102.

[0555] The data were analyzed using GraphPad Prism software. Any of YK-305, YK-310, YK-312, YK-319 and YK-318 showed significant differences compared with YK-301, YK-302, YK-303, YK-304, YK-306 and YK-307, and the transfection efficiency was significantly improved.

[0556] b. Differences in chemical structure

[0557] This series of compounds is very similar to YK-305, YK-310, YK-312, YK-319 and YK-318 in structure, with only slight differences in G1, G2, G3, R1 or R2 groups. This series of compounds is also very similar to each other (see Table 6).

[0558] I. Structural differences from YK-305

[0559] Compared with YK-305, only the R1 and R2 groups of YK-304 are straight-chain structures, while YK-305 is a branched structure; the other structures are exactly the same, but the cell transfection efficiency of YK-305 is 1324.99 times that of YK-304.

[0560] Compared with YK-305, the only difference between YK-302 and YK-305 is that the G1 group has 2 fewer carbon atoms; the R1 group is a straight chain structure, while YK-305 is a branched structure; the single chain of the R2 group has 1 more carbon atoms, and each single chain in the double chain has 2 fewer carbon atoms; the other structures are exactly the same, but the cell transfection efficiency of YK-305 is 986.71 times that of YK-302.

[0561] Compared with YK-305, the only difference between YK-303 and YK-305 is that the R1 group is a straight-chain structure, while YK-305 is a branched structure; the G2 group has 2 fewer carbon atoms; the R2 group has 2 more carbon atoms in the single chain, and each single chain in the double chain has 2 more carbon atoms; the other structures are exactly the same, but the cell transfection efficiency of YK-305 is 43.76 times that of YK-303.

[0562] II. Structural differences from YK-310

[0563] Compared with YK-310, YK-304 only has 2 more carbon atoms in the G2 group; the R2 group is a straight chain structure, while YK-310 is a branched structure; the group connected to N in the G3 group has 1 less carbon atom; the other structures are exactly the same, but the cell transfection efficiency of YK-310 is 1247.08 times that of YK-304.

[0564] Compared with YK-310, YK-302 only has 2 fewer Cs in the G1 group, 1 less C in the R1 group, 2 more Cs in the G2 group, 1 less C in the single chain of the R2 group, and 4 less Cs in each single chain of the double chain; and 1 less C in the group connected to N in the G3 group. The other structures are exactly the same, but the cell transfection efficiency of YK-310 is 928.69 times that of YK-302.

[0565] Compared with YK-310, the only difference in YK-303 is that the group connecting the G3 group and the N group has one less C; the other structures are exactly the same, but the cell transfection efficiency of YK-310 is 41.19 times that of YK-303.

[0566] III. Structural differences from YK-312

[0567] Compared with YK-312, YK-304 has only R1 and R2 groups that are straight-chain structures, while YK-312 has a branched structure; the group connected to N by G3 has one less C; the other structures are exactly the same, but the cell transfection efficiency of YK-312 is 974.01 times that of YK-304.

[0568] Compared with YK-312, the only difference in YK-302 is that the G1 group has 2 fewer carbon atoms; the R1 group is a straight chain structure, while YK-312 is a branched structure; the R2 group has 1 more carbon atom in the single chain, and 2 fewer carbon atoms in each single chain of the double chain; the group connected to N in the G3 group has 1 less carbon atom; the other structures are exactly the same, but the cell transfection efficiency of YK-312 is 725.34 times that of YK-302.

[0569] Compared with YK-312, the only difference in YK-303 is that the R1 group is a straight-chain structure, while YK-312 is a branched structure; the G2 group has 2 fewer Cs; the R2 group has 2 more Cs in the single chain, and 2 more Cs in each single chain of the double chain; the group connected to N in the G3 group has 1 less C; the other structures are exactly the same, but the cell transfection efficiency of YK-312 is 32.17 times that of YK-303.

[0570] IV. Structural differences from YK-319

[0571] Compared with YK-319, the only difference between YK-304 and YK-319 is that the G1 group and G2 group have one less C each; the R1 group and R2 group are straight-chain structures, while YK-319 has a branched structure; the group connected to N in the G3 group has one less hydroxymethyl group; the other structures are exactly the same, but the cell transfection efficiency of YK-319 is 1182.82 times that of YK-304.

[0572] Compared with YK-319, the only difference between YK-302 and YK-302 is that the G1 group has 3 fewer carbon atoms; the R1 group is a straight chain structure, while YK-319 is a branched structure; the G2 group has 1 fewer carbon atoms; the G3 group connected to N has 1 fewer hydroxymethyl group; the other structures are exactly the same, but the cell transfection efficiency of YK-319 is 880.83 times that of YK-302.

[0573] Compared with YK-319, YK-306 has only one less C in the G1 group and the G2 group; one less hydroxymethyl in the group connected to N in the G3 group; the other structures are exactly the same, but the cell transfection efficiency of YK-319 is 11.14 times that of YK-306.

[0574] V. Structural differences from YK-318

[0575] Compared with YK-318, YK-304 has only R1 and R2 groups that are straight-chain structures, while YK-318 has a branched structure; the group connected to N in the G3 group has one less hydroxymethyl group; the other structures are exactly the same, but the cell transfection efficiency of YK-318 is 540.95 times that of YK-304.

[0576] Compared with YK-318, YK-302 has only two fewer carbon atoms in the G1 group; the R1 group is a straight chain structure, while YK-318 is a branched structure; the G3 group connected to N has one less hydroxymethyl group; the other structures are exactly the same, but the cell transfection efficiency of YK-318 is 402.84 times that of YK-302.

[0577] Compared with YK-318, the only difference between YK-303 and YK-318 is that the R1 group is a straight-chain structure, while YK-318 is a branched structure; the G2 group has 2 fewer carbon atoms; the R2 group has 3 more carbon atoms in a single chain, and each single chain in a double chain has 2 more carbon atoms; the G3 group connected to N has 1 less hydroxymethyl group; the other structures are exactly the same, but the cell transfection efficiency of YK-318 is 17.87 times that of YK-303.

[0578] summary:

[0579] Among a series of compounds with very similar structures designed by us, YK-305, YK-310, YK-312, YK-319 and YK-318 have the highest cell transfection efficiency compared with compounds with similar structures and G3 groups of HO(CH2)2N(CH3)CH2CH(OH)CH2-. For example, YK-305 can increase the cell transfection efficiency by 1300 times compared with YK-304 and 900 times compared with YK-302.

[0580] At the same time, we found that there is no corresponding relationship between the structure of the compound and the intracellular transfection efficiency. Even a group of compounds with very similar structures are likely to have very different cell transfection efficiencies.

[0581] Therefore, it is very difficult to screen out cationic lipid compounds with high transfection efficiency from a series of compounds with very similar structures, and it requires a lot of creative work.

[0582] (4) YK-305, YK-310, YK-312, YK-319 and YK-318 have the highest cell transfection efficiency compared to a series of compounds with similar structures and G3 groups of HO(CH2)2N(CH2CH3)CH2CH(OH)CH2-. For example, YK-305 can reach more than 200 times that of YK-309.

[0583] We compared a series of compounds with similar structures and G3 groups of HO(CH2)2N(CH2CH3)CH2CH(OH)CH2- with YK-305, YK-310, YK-312, YK-319 and YK-318. The structural differences of these compounds are only that the G1, G2, G3, R1 or R2 groups are slightly different (see Table 8). The results show that the activity of this series of compounds varies greatly, among which YK-305, YK-310, YK-312, YK-319 and YK-318 have the highest cell transfection efficiency, which can reach 210 times, 200 times, 160 times, 190 times and 90 times of the lowest activity YK-309, respectively, and the transfection efficiency is significantly improved.

[0584] Table 8 Chemical structures of designed compounds

[0585]

[0586]

[0587]

[0588] Table 9 Fluorescence detection results of Fluc-mRNA-3

[0589]

[0590] a. Differences in cell transfection efficiency

[0591] Although the other compounds have only some minor differences in the G1, R1, G2, R2 or G3 groups compared with YK-305, YK-310, YK-312, YK-319 and YK-318 (see Table 8), their effects on cell transfection efficiency are very large, with a difference of more than 200 times.

[0592] Specifically, it can be seen from Table 9 that the fluorescence absorption value of the LNP preparation prepared by YK-309 is very different from that of YK-305, YK-310, YK-312, YK-319 and YK-318.

[0593] The content of YK-305, YK-310, YK-312, YK-319 and YK-318 can reach 218.62 times, 205.77 times, 160.71 times, 195.16 times and 89.26 times of that of YK-309 respectively.

[0594] The fluorescence absorption values ​​of LNP preparations prepared by YK-311, YK-313, YK-314, and YK-315 were significantly different from those of YK-305, YK-310, YK-312, YK-319, and YK-318 in terms of transfection efficiency.

[0595] YK-305 is 13.96 times that of YK-311, 31.63 times that of YK-313, 50.07 times that of YK-314, and 17.27 times that of YK-315.

[0596] YK-310 is 13.14 times that of YK-311, 29.77 times that of YK-313, 47.12 times that of YK-314, and 16.25 times that of YK-315.

[0597] YK-312 is 10.26 times that of YK-311, 23.25 times that of YK-313, 36.81 times that of YK-314, and 12.69 times that of YK-315.

[0598] YK-319 is 12.46 times that of YK-311, 28.24 times that of YK-313, 44.70 times that of YK-314, and 15.42 times that of YK-315.

[0599] YK-318 is 5.70 times that of YK-311, 12.91 times that of YK-313, 20.44 times that of YK-314, and 7.05 times that of YK-315.

[0600] The fluorescence absorption values ​​of the LNP preparations prepared by YK-308 were compared with those of YK-305, YK-310, YK-312, YK-319 and YK-318, and the transfection efficiency was also quite different.

[0601] The weights of YK-305, YK-310, YK-312, YK-318 and YK-319 are 5.44 times, 5.12 times, 4.00 times, 4.86 times and 2.22 times that of YK-308 respectively.

[0602] The data were analyzed using GraphPad Prism software. Any one of YK-305, YK-310, YK-312, YK-319 and YK-318 was significantly different from the other compounds, and the cell transfection efficiency was significantly improved.

[0603] b. Differences in chemical structure

[0604] This series of compounds is very similar to YK-305, YK-310, YK-312, YK-319 and YK-318 in structure, with only slight differences in G1, G2, G3, R1 or R2 groups. This series of compounds is also very similar to each other (see Table 8).

[0605] I. Structural differences from YK-305

[0606] Compared with YK-305, YK-309 only has 2 fewer carbon atoms in the G1 group; the R1 group is a straight chain structure, while YK-305 is a branched structure; the R2 group has 1 more carbon atom in the single chain, and 2 fewer carbon atoms in each single chain of the double chain; the G3 group connected to N has 1 more carbon atom; the other structures are exactly the same, but the cell transfection efficiency of YK-305 is 218.62 times that of YK-309.

[0607] Compared with YK-305, YK-314 has only two more Cs in G1 and G2 groups, one less C in the single chains of R1 and R2 groups, and one more C in the group connected to N of G3 group. The other structures are exactly the same, but the cell transfection efficiency of YK-305 is 50.07 times that of YK-314.

[0608] Compared with YK-305, the only difference in YK-313 is that the single chains of R1 and R2 groups have one less C each, and the group connected to N in G3 group has one more C. The other structures are exactly the same, but the cell transfection efficiency of YK-305 is 31.63 times that of YK-313.

[0609] II. Structural differences from YK-310

[0610] Compared with YK-310, YK-309 only has 2 fewer Cs in the G1 group, 1 less C in the R1 group, 2 more Cs in the G2 group, 1 less C in the single chain of the R2 group, and 4 fewer Cs in each single chain of the double chain; the other structures are exactly the same, but the cell transfection efficiency of YK-310 is 205.77 times that of YK-309.

[0611] Compared with YK-310, YK-314 only has 2 more carbon atoms in the G1 group; the R1 group is a branched structure, while YK-310 is a straight chain structure; the G2 group has 4 more carbon atoms; the R2 group has 3 fewer carbon atoms in the single chain, and each single chain in the double chain has 2 fewer carbon atoms; the other structures are exactly the same, but the cell transfection efficiency of YK-310 is 47.12 times that of YK-314.

[0612] Compared with YK-310, the only difference between YK-313 and YK-310 is that the R1 group is a branched structure, while YK-310 is a straight chain structure; the G2 group has 2 more carbon atoms; the R2 group has 3 fewer carbon atoms in a single chain, and each single chain in a double chain has 2 fewer carbon atoms; the other structures are exactly the same, but the cell transfection efficiency of YK-310 is 29.77 times that of YK-313.

[0613] III. Structural differences from YK-312

[0614] Compared with YK-312, the only difference between YK-309 and YK-312 is that the G1 group has 2 fewer carbon atoms; the R1 group is a straight chain structure, while YK-312 has a branched structure; the single chain of the R2 group has 1 more carbon atoms, and each single chain in the double chain has 2 fewer carbon atoms; the other structures are exactly the same, but the cell transfection efficiency of YK-312 is 160.71 times that of YK-309.

[0615] Compared with YK-312, YK-314 has only 2 more Cs in G1 and G2 groups and 1 less C in R1 and R2 single chains. The other structures are exactly the same, but the cell transfection efficiency of YK-312 is 36.81 times that of YK-314.

[0616] Compared with YK-312, the only difference between YK-313 and YK-313 is that the single chains of R1 and R2 groups have one less C each; the other structures are exactly the same, but the cell transfection efficiency of YK-312 is 23.25 times that of YK-313.

[0617] IV. Structural differences from YK-319

[0618] Compared with YK-319, YK-309 only has 3 fewer carbon atoms in the G1 group; the R1 group is a straight chain structure, while YK-319 is a branched structure; the G2 group has 1 less carbon atom; the group connected to N in the G3 group has 1 less hydroxyl group; the other structures are exactly the same, but the cell transfection efficiency of YK-319 is 195.16 times that of YK-309.

[0619] Compared with YK-319, YK-314 has only one more C in the G1 and G2 groups; two less C in the single chains of the R1 and R2 groups, and two more C in each single chain of the double chain; one less hydroxyl group in the group connected to N in the G3 group; the other structures are exactly the same, but the cell transfection efficiency of YK-319 is 44.70 times that of YK-314.

[0620] Compared with YK-319, YK-313 has only one less C in the G1 and G2 groups; two less C in the single chains of the R1 and R2 groups, and two more C in each single chain of the double chain; one less hydroxyl group in the group connected to N in the G3 group; the other structures are exactly the same, but the cell transfection efficiency of YK-319 is 28.24 times that of YK-313.

[0621] V. Structural differences from YK-318

[0622] Compared with YK-318, YK-309 only has 2 fewer carbon atoms in the G1 group; the R1 group is a straight chain structure, while YK-318 is a branched structure; the group connected to N in the G3 group has one less hydroxyl group; the other structures are exactly the same, but the cell transfection efficiency of YK-318 is 89.26 times that of YK-309.

[0623] Compared with YK-318, YK-314 has only two fewer carbon atoms in the G1 and G2 groups, and one less hydroxyl group in the group connected to N in the G3 group. The other structures are exactly the same, but the cell transfection efficiency of YK-318 is 20.44 times that of YK-314.

[0624] Compared with YK-318, the only difference in YK-313 is that the group connected to N in the G3 group has one less hydroxyl group; the other structures are exactly the same, but the cell transfection efficiency of YK-318 is 12.91 times that of YK-313.

[0625] summary:

[0626] Among a series of compounds that we designed with very similar structures, YK-305, YK-310, YK-312, YK-319, and YK-318 showed the highest cell transfection efficiency compared to compounds with similar structures but with the G3 group being HO(CH2)2N(CH2CH3)CH2CH(OH)CH2-. For example, YK-305 and YK-310 were 200 times more efficient than YK-309.

[0627] At the same time, we found that there is no corresponding relationship between the structure of the compound and the intracellular transfection efficiency. Even a group of compounds with very similar structures are likely to have very different cell transfection efficiencies.

[0628] Therefore, it is very difficult to screen out cationic lipid compounds with high transfection efficiency from a series of compounds with very similar structures, and it requires a lot of creative work.

[0629] (5) YK-305, YK-310, YK-312, YK-319, and YK-318 have the highest cell transfection efficiency compared to a series of compounds with similar structures and G3 groups of (HO(CH2)2)2NCH2CH(OH)CH2-. For example, YK-305 can reach 20 times that of YK-321.

[0630] Compared with the compounds with similar structures and G3 groups of (HO(CH2)2)2NCH2CH(OH)CH2- (see Table 10), YK-305, YK-310, YK-312, YK-319 and YK-318 have the highest cell transfection efficiency. For example, YK-305 can reach 20 times that of YK-321.

[0631] Table 10 Chemical structures of designed compounds

[0632]

[0633]

[0634]

[0635] Table 11 Fluorescence detection results of Fluc-mRNA-4

[0636]

[0637] a. Differences in cell transfection efficiency

[0638] Compared with compounds with similar structures and G3 groups of (HO(CH2)2)2NCH2CH(OH)CH2- (see Table 10), YK-305, YK-310, YK-312, YK-319 and YK-318 have the highest cell transfection efficiency. For example, YK-305 can reach 20 times that of YK-321.

[0639] The details are as follows:

[0640] As can be seen from Table 11, YK-321 is very different from YK-305, YK-310, YK-312, YK-318 and YK-319.

[0641] The weights of YK-305, YK-310, YK-312, YK-319 and YK-318 are 19.53 times, 18.38 times, 14.36 times, 17.43 times and 7.97 times that of YK-321 respectively.

[0642] In addition, YK-316, YK-317 and YK-320 are all quite different from YK-305, YK-310, YK-312, YK-319 and YK-318.

[0643] The strength of YK-305 is 5.70 times that of YK-316, 7.60 times that of YK-317 and 10.14 times that of YK-320.

[0644] The strength of YK-310 is 5.36 times that of YK-316, 7.16 times that of YK-317 and 9.54 times that of YK-320.

[0645] The strength of YK-312 is 4.19 times that of YK-316, 5.59 times that of YK-317 and 7.45 times that of YK-320.

[0646] The strength of YK-319 can reach 5.09 times that of YK-316, 6.79 times that of YK-317 and 9.05 times that of YK-320.

[0647] The strength of YK-318 is 2.33 times that of YK-316, 3.10 times that of YK-317 and 4.14 times that of YK-320.

[0648] Figure 7Shown are the fluorescence absorption images of LNP preparations prepared from YK-305, YK-310, YK-320 and YK-321. It can be seen that compared with YK-305 and YK-310, the fluorescence absorption of YK-320 and YK-321 is very weak.

[0649] The data were analyzed using GraphPad Prism software. Any one of YK-305, YK-310, YK-312, YK-319 and YK-318 was significantly different from the other compounds, and the transfection efficiency was significantly improved.

[0650] b. Differences in chemical structure

[0651] This series of compounds is very similar in structure to YK-305, YK-310, YK-312, YK-319 and YK-318, except for slight differences in G1, G2, G3, R1 or R2 groups. The structures of this series of compounds are also very similar. (See Table 10)

[0652] I. Structural differences from YK-305

[0653] Compared with YK-305, YK-321 has only 2 fewer carbon atoms in the G1 and G2 groups; 2 more carbon atoms in the single chains of the R1 and R2 groups, and 2 more carbon atoms in each single chain of the double chain; and 1 more hydroxymethyl group in the group connected to N of the G3 group. The other structures are exactly the same, but the cell transfection efficiency of YK-305 is 19.53 times that of YK-321.

[0654] Compared with YK-305, YK-320 has only two more carbon atoms in the G1 and G2 groups, one less carbon atoms in the single chains of the R1 and R2 groups, and one more hydroxymethyl group in the group connected to N of the G3 group. The other structures are exactly the same, but the cell transfection efficiency of YK-305 is 10.14 times that of YK-320.

[0655] Compared with YK-305, YK-316 only has one more hydroxymethyl group between the G3 group and the N group; the other structures are exactly the same, but the cell transfection efficiency of YK-305 is 5.70 times that of YK-316.

[0656] II. Structural differences from YK-310

[0657] Compared with YK-310, YK-321 has only two fewer carbon atoms in the G1 group; the R1 group is a branched structure, while YK-310 is a straight chain structure; the G3 group connected to the N group has one more hydroxyl group; the other structures are exactly the same, but the cell transfection efficiency of YK-310 is 18.38 times that of YK-321.

[0658] Compared with YK-310, YK-320 only has 2 more carbon atoms in the G1 group; the R1 group is a branched structure, while YK-310 is a straight chain structure; the G2 group has 4 more carbon atoms; the R2 group has 3 fewer carbon atoms in the single chain, and each single chain in the double chain has 2 fewer carbon atoms; the G3 group connected to N has 1 more hydroxyl group; the other structures are exactly the same, but the cell transfection efficiency of YK-310 is 9.54 times that of YK-320.

[0659] Compared with YK-310, the only difference between YK-317 and YK-310 is that the R1 group is a branched structure, while YK-310 is a straight chain structure; the G2 group has 2 more carbon atoms; the R2 group has 1 less carbon atom in the single chain, and 4 less carbon atoms in each single chain of the double chain; the G3 group connected to N has 1 more hydroxyl group; the other structures are exactly the same, but the cell transfection efficiency of YK-310 is 7.16 times that of YK-317.

[0660] III. Structural differences from YK-312

[0661] Compared with YK-312, YK-321 has only 2 fewer Cs in G1 and G2 groups; 2 more Cs in R1 and R2 groups in single chains, and 2 more Cs in each single chain in the double chain; and 1 more hydroxyl group in the group connected to N in G3 group; the other structures are exactly the same, but the cell transfection efficiency of YK-312 is 14.36 times that of YK-321.

[0662] Compared with YK-312, YK-320 has only two more carbon atoms in G1 and G1 groups, one less carbon atoms in the single chains of R1 and R2 groups, and one more hydroxyl group in the group connected to N of G3 group. The other structures are exactly the same, but the cell transfection efficiency of YK-312 is 7.45 times that of YK-320.

[0663] Compared with YK-312, YK-316 has only one more hydroxyl group between the G3 group and the N group; the other structures are exactly the same, but the cell transfection efficiency of YK-312 is 4.19 times that of YK-316.

[0664] IV. Structural differences from YK-319

[0665] Compared with YK-319, YK-321 has only 3 fewer Cs in the G1 and G1 groups, 1 more C in the single chains of the R1 and R2 groups, and 4 more Cs in each single chain of the double chain. The other structures are exactly the same, but the cell transfection efficiency of YK-319 is 17.43 times that of YK-321.

[0666] Compared with YK-319, YK-320 has only one more C in the G1 and G1 groups, two fewer C in the single chains of the R1 and R2 groups, and two more C in each single chain of the double chain. The other structures are exactly the same, but the cell transfection efficiency of YK-319 is 9.05 times that of YK-320.

[0667] Compared with YK-319, YK-317 only has one less C in the G1 and G1 groups; its structure is exactly the same, but the cell transfection efficiency of YK-319 is 6.79 times that of YK-317.

[0668] V. Structural differences from YK-318

[0669] Compared with YK-318, YK-321 has only 2 fewer Cs in the G1 and G2 groups, 3 more Cs in the single chains of the R1 and R2 groups, and 2 more Cs in each single chain of the double chain. The other structures are exactly the same, but the cell transfection efficiency of YK-318 is 7.97 times that of YK-321.

[0670] Compared with YK-318, YK-320 only has 2 more Cs in the G1 and G2 groups; the other structures are exactly the same, but the cell transfection efficiency of YK-318 is 4.14 times that of YK-320.

[0671] Compared with YK-318, the only difference between YK-316 and YK-318 is that the single chains of R1 and R2 groups have one more C each; the other structures are exactly the same, but the cell transfection efficiency of YK-318 is 2.33 times that of YK-316.

[0672] summary:

[0673] Among a series of compounds with very similar structures designed by us, YK-305, YK-310, YK-312, YK-319 and YK-318 have the highest cell transfection efficiency compared with compounds with similar structures and G3 groups of (HO(CH2)2)2NCH2CH(OH)CH2-. For example, YK-305 can reach 20 times that of YK-321.

[0674] At the same time, we found that it is impossible to infer the difference in cell transfection efficiency between different compounds (regardless of whether the structures are similar or very different) based on structural differences. Even a group of compounds with very small structural differences are very likely to have very large differences in cell transfection efficiency.

[0675] Therefore, it is very difficult to screen out cationic lipid compounds with high transfection efficiency from a series of compounds with similar chemical structures, and it requires a lot of creative work.

[0676] Summarize:

[0677] 1) Through multiple designs of compound structures and a lot of creative work, we designed and screened cationic lipid compounds with high cell transfection efficiency, such as YK-305, YK-310, YK-312, YK-319 and YK-318.

[0678] The designed series of compounds have great differences in chemical structure from representative cationic lipids in the prior art, such as SM-102, ALC-0315, compound 21, compound 23, HHMA and YK-009, with completely different G3 groups and differences in other parts, so there are also great differences in polarity, acidity and alkalinity, hydrophilicity, etc. It is impossible to infer the cell transfection efficiency, cytotoxicity, and in vivo expression of the LNP preparations prepared by this series of compounds based on the above cationic lipid compounds disclosed in the prior art.

[0679] 2) The LNP preparations prepared from YK-305, YK-310, YK-312, YK-319 and YK-318 have the highest cell transfection efficiency, and their activity is significantly improved compared to the representative cationic lipids in the prior art. For example, YK-305 can reach 17 times that of SM-102, 19 times that of compound 21, and 20 times that of compound 23.

[0680] Compared with compounds with similar structures and G3 groups of HO(CH2)2N(CH3)CH2CH(OH)CH2-, YK-305, YK-310, YK-312, YK-319 and YK-318 have the highest cell transfection efficiency. For example, YK-305 can be 1300 times higher than YK-304 and 900 times higher than YK-302.

[0681] Compared with compounds with similar structures and G3 groups of HO(CH2)2N(CH2CH3)CH2CH(OH)CH2-, YK-305, YK-310, YK-312, YK-319 and YK-318 have the highest cell transfection efficiency. For example, YK-305 and YK-310 can both be 200 times higher than YK-309.

[0682] Compared with compounds with similar structures and G3 groups of (HO(CH2)2)2NCH2CH(OH)CH2-, YK-305, YK-310, YK-312, YK-319 and YK-318 have the highest cell transfection efficiency. For example, YK-305 can reach 20 times that of YK-321.

[0683] 3) There is no corresponding relationship between the structure of the compound and the intracellular transfection efficiency. Compounds with small structural differences are likely to have very large differences in transfection efficiency. Therefore, screening cationic lipid compounds with high transfection efficiency requires multiple designs and a lot of creative work.

[0684] 2. Cell Viability Determination

[0685] The LNP preparation containing 1.5 μg Fluc-mRNA (the LNP preparation carrier components are cationic lipids, neutral lipids, structural lipids and polymer conjugated lipids, the molar ratio is 49:10:39.5:1.5, wherein the cationic lipids are the cationic lipids listed in Table 1) and the Lipofectamine 3000 preparation were added to the cell culture medium of the 96-well plate, and the culture was continued for 24 hours. Then, 10 μL of CCK-8 solution was added to each well, and the culture plate was incubated in the incubator for 1 hour, and the absorbance at 450 nm was measured by a microplate reader. The results are shown in Tables 12-15.

[0686] The cell survival rate can represent the toxicity of cationic lipids to cells. The higher the cell survival rate, the lower the toxicity to cells.

[0687] Experimental results:

[0688] (6) Among the designed series of compounds, the LNP preparations prepared from YK-305, YK-310, YK-312, YK-319 and YK-318 significantly reduced the cytotoxicity and significantly improved the cell survival rate compared to the representative cationic lipids in the prior art. For example, the cell survival rates of YK-305 and YK-310 were 12.73% and 12.65% higher than those of SM-102, and 15.71% and 15.63% higher than those of HHMA, respectively.

[0689] Table 12 Cell survival rate-1

[0690] Serial number Cationic lipids Cell survival rate (%) 1 YK-305 82.18 2 YK-310 82.10 3 YK-312 76.96 4 YK-319 75.21 5 YK-318 75.56 6 YK-009 72.55 7 SM-102 69.45 8 ALC-0315 51.03 9 Compound 21 70.21 10 Compound 23 71.50 11 HHMA 66.47 12 Lipofectamine 3000 25.01

[0691] a. Difference in cell survival rate

[0692] Table 12 lists the results of cytotoxicity tests of LNP preparations prepared from different cationic lipid compounds. Among them, YK-009 is disclosed in CN114044741B (claim 1), SM-102 is compound 25 disclosed in WO2017049245A2 (page 29 of the specification), ALC-0315 is compound 3 disclosed in CN108368028B (page 24 of the specification), compound 21 and compound 23 are disclosed in WO2021055833A1 (page 22 of the specification), and HHMA is compound 1 disclosed in CN112979483B (page 12 of the specification); Lipofectamine 3000 is a widely used cell transfection reagent with good transfection performance.

[0693] As shown in Table 12, the LNP preparations of Fluc-mRNA prepared by YK-305, YK-310, YK-312, YK-319 and YK-318 have the lowest cytotoxicity, as shown by the cell survival rates reaching 82.18%, 82.10%, 76.96%, 75.21% and 75.56%, respectively. Figure 8 )

[0694] YK-305 was 12.73% higher than SM-102, 31.15% higher than ALC-0315, 11.97% higher than compound 21, 10.68% higher than compound 23, 15.71% higher than HHMA, and 57.17% higher than Lipofectamine 3000.

[0695] YK-310 was 12.65% higher than SM-102, 31.07% higher than ALC-0315, 11.89% higher than compound 21, 10.60% higher than compound 23, 15.63% higher than HHMA, and 57.09% higher than Lipofectamine 3000.

[0696] YK-312 was 7.51% higher than SM-102, 25.93% higher than ALC-0315, 6.75% higher than compound 21, 5.46% higher than compound 23, 10.49% higher than HHMA, and 51.95% higher than Lipofectamine 3000.

[0697] YK-319 was 5.76% higher than SM-102, 24.18% higher than ALC-0315, 5.00% higher than compound 21, 3.71% higher than compound 23, 8.74% higher than HHMA, and 50.20% higher than Lipofectamine 3000.

[0698] YK-318 is 6.11% higher than SM-102, 24.53% higher than ALC-0315, 5.35% higher than compound 21, 4.06% higher than compound 23, 9.09% higher than HHMA, and 50.55% higher than Lipofectamine 3000.

[0699] The data were analyzed using GraphPad Prism software, and any one of YK-305, YK-310, YK-312, YK-319 and YK-318 had significant differences from SM-102, ALC-0315, compound 21, compound 23, HHMA and Lipofectamine 3000, and their cytotoxicity was significantly reduced.

[0700] b. Differences in chemical structure

[0701] Compared with the cationic lipids in the prior art, the chemical structures of YK-305, YK-310, YK-312, YK-319 and YK-318 are very different, among which the difference with HHMA structure is the greatest. It can be seen from the chemical structure diagram that among the groups connected to the central N atom of HHMA, only one side chain is similar to one side chain of this series of structures, and the other parts are completely different; compared with SM-102, ALC-0315, compound 21, compound 23 and YK-009, the G3 group is completely different, and the G1, R1, G2 and R2 groups are also very different.

[0702] summary:

[0703] Among the designed series of compounds, the LNP preparations prepared by YK-305, YK-310, YK-312, YK-319 and YK-318 have the lowest cytotoxicity and significantly improve the cell survival rate compared with the representative cationic lipids in the prior art. For example, the cell survival rates of YK-305 and YK-310 are 30% higher than ALC-0315, 12% higher than SM-102, and 15% higher than HHMA.

[0704] Compared with the representative cationic lipids in the prior art, YK-305, YK-310, YK-312, YK-319 and YK-318 have huge differences in chemical structure. The G3 groups are completely different, and the G1, R1, G2 and R2 groups are also very different.

[0705] The present application designs for the first time a compound with a chemical structure greatly different from that of the cationic lipids in the prior art. The LNP preparation prepared from the compound has significantly reduced cytotoxicity and significantly improved cell survival rate compared to the cationic lipids in the prior art.

[0706] (7) YK-305, YK-310, YK-312, YK-319, and YK-318 showed the lowest cytotoxicity and significantly improved cell survival compared to a series of compounds with similar structures and G3 groups of HO(CH2)2N(CH3)CH2CH(OH)CH2-. For example, compared with YK-302, the cell survival rates of YK-305 and YK-310 were both increased by 65%.

[0707] We compared YK-305, YK-310, YK-312, YK-319 and YK-318 with a series of compounds with similar structures and G3 groups of HO(CH2)2N(CH3)CH2CH(OH)CH2-. These compounds only differ slightly in G1, G2, G3, R1 or R2 groups.

[0708] The results showed that the cytotoxicity of this series of compounds was significantly different. Among them, YK-305, YK-310, YK-312, YK-319 and YK-318 had the highest cell survival rate. For example, YK-305 and YK-310 were both 65% higher than YK-302.

[0709] Table 13 Cell survival rate-2

[0710]

[0711]

[0712] a. Difference in cell survival rate

[0713] As shown in Table 13, the cytotoxicity of the LNP preparations prepared by these compounds varies greatly, among which YK-302 has the highest toxicity and the lowest cell survival rate, which is only 16.43%. Fig. 9 )

[0714] The cell survival rates of YK-305, YK-310, YK-312, YK-319 and YK-318 were increased by 65.75%, 65.67%, 60.53%, 58.78% and 59.13% respectively compared with YK-302.

[0715] The cell survival rates of other compounds were also significantly different from those of YK-305, YK-310, YK-312, YK-319 and YK-318.

[0716] The survival rates of YK-305 cells were 29.22% higher than those of YK-301, 18.86% higher than those of YK-303, 48.02% higher than those of YK-304, 39.99% higher than those of YK-306, and 21.04% higher than those of YK-307.

[0717] The survival rates of YK-310 cells were 29.14% higher than those of YK-301, 18.78% higher than those of YK-303, 47.94% higher than those of YK-304, 39.91% higher than those of YK-306, and 20.96% higher than those of YK-307.

[0718] The survival rates of YK-312 cells were 24.00% higher than those of YK-301, 13.64% higher than those of YK-303, 42.80% higher than those of YK-304, 34.77% higher than those of YK-306, and 15.82% higher than those of YK-307.

[0719] The survival rates of YK-319 cells were 22.25% higher than those of YK-301, 11.89% higher than those of YK-303, 41.05% higher than those of YK-304, 33.02% higher than those of YK-306, and 14.07% higher than those of YK-307.

[0720] The survival rates of YK-318 cells were 22.60% higher than those of YK-301, 12.24% higher than those of YK-303, 41.40% higher than those of YK-304, 33.37% higher than those of YK-306, and 14.42% higher than those of YK-307.

[0721] The data were analyzed using GraphPad Prism software, and any one of YK-305, YK-310, YK-312, YK-319 and YK-318 had significant differences in cytotoxicity compared with other compounds, with significantly reduced cytotoxicity and significantly increased cell survival rate.

[0722] b. Differences in chemical structure

[0723] The structures of this series of compounds are very similar, with only slight differences in individual groups. YK-305, YK-310, YK-312, YK-319 and YK-318 are very close to the structures of other compounds; the other compounds are also very similar to each other.

[0724] summary:

[0725] Among a series of compounds with very similar structures designed by us, YK-305, YK-310, YK-312, YK-319 and YK-318 showed the lowest cytotoxicity and significantly improved cell survival rate compared with compounds with similar structure and G3 group of HO(CH2)2N(CH3)CH2CH(OH)CH2-. For example, YK-305 and YK-310 both showed 65% improvement compared with YK-302.

[0726] At the same time, we found that there is no corresponding relationship between the structure of the compound and its cytotoxicity. Even for a group of compounds with the most similar structures, the cytotoxicity is likely to be very different.

[0727] Therefore, it is very difficult to screen out cationic lipid compounds with low cytotoxicity from a series of compounds with only slight differences in chemical structure, which requires a lot of creative work.

[0728] (8) Compared with a series of compounds with similar structures and G3 groups of HO(CH2)2N(CH2CH3)CH2CH(OH)CH2-, YK-305, YK-310, YK-312, YK-319 and YK-318 showed the lowest cytotoxicity and significantly improved cell survival rates. For example, the cell survival rates of YK-305 and YK-310 were both 50% higher than those of YK-309.

[0729] We compared YK-305, YK-310, YK-312, YK-319 and YK-318 with a series of compounds with similar structures and G3 groups of HO(CH2)2N(CH2CH3)CH2CH(OH)CH2-, which only slightly differ in G1, G2, G3, R1 or R2 groups.

[0730] The results showed that the cytotoxicity of this series of compounds was significantly different. Among them, YK-305, YK-310, YK-312, YK-319 and YK-318 had the highest cell survival rate. For example, YK-305 and YK-310 could both be 50% higher than YK-309.

[0731] Table 14 Cell survival rate-3

[0732]

[0733]

[0734] a. Difference in cell survival rate

[0735] Although the other compounds have only some minor differences in G1, G2, G3, R1 or R2 groups compared to YK-305, YK-310, YK-312, YK-319 and YK-318, the effect on cytotoxicity is very large. The cell viability of YK-305, YK-310, YK-312, YK-319 and YK-318 can be up to 50% higher than that of other compounds.

[0736] As can be seen from Table 14, among this series of compounds, the LNP preparation prepared from YK-309 has the highest cytotoxicity, with a cell survival rate of only 31.61%.

[0737] The cell survival rates of YK-305, YK-310, YK-312, YK-319 and YK-318 were increased by 50.57%, 50.49%, 45.35%, 43.60% and 43.95% respectively compared with YK-309.

[0738] The other compounds are also quite different from YK-305, YK-310, YK-312, YK-319 and YK-318.

[0739] The survival rates of YK-305 cells were 11.19% higher than those of YK-308, 12.12% higher than those of YK-311, 13.25% higher than those of YK-313, 14.30% higher than those of YK-314, and 10.90% higher than those of YK-315.

[0740] The cell survival rates of YK-310 were 11.11% higher than those of YK-308, 12.04% higher than those of YK-311, 13.17% higher than those of YK-313, 14.22% higher than those of YK-314, and 10.82% higher than those of YK-315.

[0741] The survival rates of YK-312 cells were 5.97% higher than those of YK-308, 6.90% higher than those of YK-311, 8.03% higher than those of YK-313, 9.08% higher than those of YK-314, and 5.68% higher than those of YK-315.

[0742] The survival rates of YK-319 cells were 4.22% higher than those of YK-308, 5.15% higher than those of YK-311, 6.28% higher than those of YK-313, 7.33% higher than those of YK-314, and 3.93% higher than those of YK-315.

[0743] The cell survival rates of YK-318 were 4.57% higher than those of YK-308, 5.50% higher than those of YK-311, 6.63% higher than those of YK-313, 7.68% higher than those of YK-314, and 4.28% higher than those of YK-315. Fig.10 )

[0744] The data were analyzed using GraphPad Prism software, and any one of YK-305, YK-310, YK-312, YK-319 and YK-318 had significant differences in cytotoxicity compared with YK-308, YK-309, YK-311, YK-313, YK-314 and YK-315, with significantly reduced cytotoxicity and significantly increased cell survival rate.

[0745] b. Differences in chemical structure

[0746] The structures of this series of compounds are very similar, with only slight differences in individual groups. YK-305, YK-310, YK-312, YK-319 and YK-318 are very close to the structures of other compounds; the other compounds are also very similar to each other.

[0747] summary:

[0748] Among a series of compounds with very similar structures designed by us, YK-305, YK-310, YK-312, YK-319 and YK-318 showed the lowest cytotoxicity and significantly improved cell survival rate compared with compounds with similar structure and G3 group of HO(CH2)2N(CH2CH3)CH2CH(OH)CH2-. For example, YK-305 and YK-310 can improve cell survival rate by 50% compared with YK-302.

[0749] At the same time, we found that there is no corresponding relationship between the structure of the compound and its cytotoxicity. Compounds with only slight differences in structure are likely to have very different cytotoxicity.

[0750] Therefore, it is very difficult to screen out cationic lipid compounds with low cytotoxicity from a series of compounds with only some minor differences in individual groups, which requires a lot of creative work.

[0751] (9) YK-305, YK-310, YK-312, YK-319, and YK-318 showed significantly lower cytotoxicity than a series of compounds with similar structures and G3 groups of (HO(CH2)2)2NCH2CH(OH)CH2-. For example, the cell survival rates of YK-305 and YK-310 were both 20% higher than those of YK-317.

[0752] We compared YK-305, YK-310, YK-312, YK-319 and YK-318 with a series of compounds with similar structures and G3 groups (HO(CH2)2)2NCH2CH(OH)CH2-, which only differ slightly in G1, G2, G3, R1 or R2 groups. The results showed that the cytotoxicity of this series of compounds was very different. Among them, YK-305, YK-310, YK-312, YK-319 and YK-318 had the highest cell survival rate. For example, YK-305 and YK-310 were 20% higher than YK-317.

[0753] Table 15 Cell survival rate-4

[0754]

[0755]

[0756] a. Difference in cell survival rate

[0757] Although the other compounds have only some minor differences in G1, G2, G3, R1 or R2 groups compared with YK-305, YK-310, YK-312, YK-319 and YK-318, the differences in cytotoxicity are very large. For example, the cell survival rate of YK-305 and YK-310 is 20% higher than that of YK-317.

[0758] It can be seen from Table 15 that among this series of compounds with similar structures, YK-317 has the greatest cytotoxicity, with a cell survival rate of only 59.18%.

[0759] The cell survival rates of YK-305, YK-310, YK-312, YK-319 and YK-318 were 23.00%, 22.92%, 17.78%, 16.03% and 16.38% higher than those of YK-317, respectively.

[0760] The other compounds are also quite different from YK-305, YK-310, YK-312, YK-319 and YK-318.

[0761] The survival rates of YK-305 cells were 11.65% higher than those of YK-316, 11.11% higher than those of YK-320, and 9.84% higher than those of YK-321.

[0762] The survival rate of YK-310 cells was 11.57% higher than that of YK-316, 11.03% higher than that of YK-320, and 9.76% higher than that of YK-321.

[0763] The survival rates of YK-312 cells were 6.43% higher than those of YK-316, 5.89% higher than those of YK-320, and 4.62% higher than those of YK-321.

[0764] The survival rates of YK-319 cells were 4.68% higher than those of YK-316, 4.14% higher than those of YK-320, and 2.87% higher than those of YK-321.

[0765] The cell survival rates of YK-318 were 5.03% higher than those of YK-316, 4.49% higher than those of YK-320, and 3.22% higher than those of YK-321. Fig.11 )

[0766] The data were analyzed using GraphPad Prism software, and any one of YK-305, YK-310, YK-312, YK-319 and YK-318 had significant differences from YK-316, YK-317, YK-320 and YK-321, with significantly reduced cytotoxicity and significantly increased cell survival rate.

[0767] b. Differences in chemical structure

[0768] The structures of this series of compounds are very similar, with only slight differences in individual groups. YK-305, YK-310, YK-312, YK-319 and YK-318 are very close to the structures of other compounds; the other compounds are also very similar to each other.

[0769] summary:

[0770] Among a series of compounds with very similar structures designed by us, YK-305, YK-310, YK-312, YK-319 and YK-318 showed the lowest cytotoxicity and significantly improved cell survival rate compared with compounds with similar structure and G3 group (HO(CH2)2)2NCH2CH(OH)CH2-. For example, YK-305 and YK-310 can improve cell survival rate by 20% compared with YK-317.

[0771] At the same time, we found that there is no corresponding relationship between the structure of the compound and its cytotoxicity. Even if there are only some differences in the structure of the G3 group, the cytotoxicity is likely to be very different.

[0772] Therefore, it is very difficult to screen cationic lipid compounds with low cytotoxicity from a series of compounds that have only some minor differences in individual groups, and this requires a lot of creative work.

[0773] Summarize:

[0774] 1) We performed cell viability assays on LNP preparations prepared from a series of designed compounds and screened out compounds that have significantly reduced cytotoxicity compared to cationic lipid compounds in the prior art, such as YK-305, YK-310, YK-312, YK-319 and YK-318.

[0775] This series of designed compounds have huge chemical structure differences from representative cationic lipids in the prior art, such as SM-102, compound 21, compound 23, HHMA and YK-009. The G3 group is completely different, and other parts are also different. Therefore, there are also great differences in polarity, acidity and alkalinity, and hydrophilicity.

[0776] 2) The LNP preparations prepared from YK-305, YK-310, YK-312, YK-319 and YK-318 have the lowest cytotoxicity and significantly improve the cell survival rate compared to the representative cationic lipids in the prior art. For example, the cell survival rates of YK-305 and YK-310 are 30% higher than ALC-0315, 12% higher than SM-102, and 15% higher than HHMA.

[0777] Compared with compounds with similar structures and G3 groups of HO(CH2)2N(CH3)CH2CH(OH)CH2-, YK-305, YK-310, YK-312, YK-319 and YK-318 showed the lowest cytotoxicity and significantly improved cell survival rates. For example, YK-305 and YK-310 can improve cell survival rates by 65% ​​compared to YK-302.

[0778] Compared with compounds with similar structures and G3 groups of HO(CH2)2N(CH2CH3)CH2CH(OH)CH2-, YK-305, YK-310, YK-312, YK-319 and YK-318 showed the lowest cytotoxicity and significantly improved cell survival rates. For example, YK-305 and YK-310 can improve cell survival rates by 50% compared to YK-302.

[0779] Compared with compounds with similar structures and G3 groups of (HO(CH2)2)2NCH2CH(OH)CH2-, YK-305, YK-310, YK-312, YK-319 and YK-318 showed the lowest cytotoxicity and significantly improved cell survival rates. For example, YK-305 and YK-310 can improve cell survival rates by 20% compared to YK-317.

[0780] 3) There is no correspondence between the structure and cytotoxicity of a compound. Even compounds with small structural differences are likely to have very different cytotoxicity. Therefore, it is impossible to predict their cytotoxicity based on their chemical structure. It is very difficult to screen out cationic lipid compounds with low cytotoxicity, which requires a lot of creative work.

[0781] Example 7: In vivo validation of the performance of cationic lipid delivery vehicles

[0782] In addition, we also verified the protein expression and duration of mRNA delivered by the designed cationic lipids in mice. In vivo experiments further proved that our LNP delivery vector can effectively deliver mRNA into the body and express it efficiently and continuously.

[0783] The LNP preparation containing 10 μg Fluc-mRNA was injected intramuscularly into female BALB / C mice aged 4-6 weeks and weighing 17-19 g. The mice were intraperitoneally injected with fluorescent imaging substrate at specific time points (6h, 24h, 48h and 7d) after administration. The mice moved freely for 5 minutes, and then the average radiation intensity (corresponding to the fluorescence expression intensity) of the protein expressed by the mRNA carried by the LNP in the mice was detected by IVIS Spectrum small animal in vivo imager.

[0784] Experimental results:

[0785] a. mRNA expression in mice

[0786] The average radiation intensity test results of the proteins expressed by mRNA in LNP preparations in mice are shown in Tables 16-19 and Figure 12-14 .

[0787] (1) Among the designed series of compounds, the LNP preparations prepared by YK-305, YK-310, YK-312, YK-319 and YK-318 expressed mRNA in mice at a very high level and continuously, which was significantly improved compared with the representative cationic lipids in the prior art. For example, YK-305 and YK-310 can reach 30 times that of SM-102, compound 21 and compound 23. The expression of mRNA in mice is consistent with the cell transfection activity.

[0788] Table 16 Mouse in vivo imaging experimental data-1

[0789]

[0790] a. Expression differences in mice

[0791] Table 16 lists the LNP preparations containing Fluc-mRNA prepared by different cationic lipids, and the expression intensity of mRNA at different times in mice. Among them, YK-009 is disclosed in CN114044741B (claim 1), SM-102 is compound 25 disclosed in WO2017049245A2 (page 29 of the specification), ALC-0315 is compound 3 disclosed in CN108368028B (page 24 of the specification), compound 21 and compound 23 are disclosed in WO2021055833A1 (page 22 of the specification), and HHMA is compound 1 disclosed in CN112979483B (page 12 of the specification). These cationic lipids can be used to prepare vectors for delivering mRNA.

[0792] As shown in Table 12, the Fluc-mRNA-containing LNP preparations prepared by YK-305, YK-310, YK-312, YK-319 and YK-318 expressed mRNA at a very high level in mice and the expression was sustained.

[0793] The average radiation intensity of YK-305 was 9239340 at 6 h, which was 13.18 times that of SM-102, 10.70 times that of ALC-0315, 15.14 times that of compound 21, 15.68 times that of compound 23, and 14.19 times that of HHMA; and 2823400 at 24 h, which was 23.44 times that of SM-102, 14.82 times that of ALC-0315, 25.70 times that of compound 21, 27.01 times that of compound 23, and 28.70 times that of HHMA. at 48h it was 1047720, which was 32.19 times that of SM-102, 26.32 times that of ALC-0315, 33.82 times that of compound 21, 31.63 times that of compound 23 and 32.18 times that of HHMA; at 7d it was 127015, which was 19.18 times that of SM-102, 18.02 times that of ALC-0315, 19.71 times that of compound 21, 20.35 times that of compound 23 and 21.63 times that of HHMA.

[0794] The average radiation intensity of YK-310 was 9125240 at 6 h, which was 13.01 times that of SM-102, 10.56 times that of ALC-0315, 14.95 times that of compound 21, 15.48 times that of compound 23, and 14.02 times that of HHMA; and 2952310 at 24 h, which was 24.51 times that of SM-102, 15.50 times that of ALC-0315, 26.88 times that of compound 21, 28.24 times that of compound 23, and 29.81 times that of HHMA. at 48h it was 981000, which was 30.14 times that of SM-102, 24.64 times that of ALC-0315, 31.67 times that of compound 21, 29.62 times that of compound 23 and 30.13 times that of HHMA; at 7d it was 117462, which was 17.74 times that of SM-102, 16.67 times that of ALC-0315, 18.23 times that of compound 21, 18.82 times that of compound 23 and 20.01 times that of HHMA.

[0795] The average radiation intensity of YK-312 was 8009850 at 6 h, which was 11.42 times that of SM-102, 9.27 times that of ALC-0315, 13.12 times that of compound 21, 13.59 times that of compound 23, and 12.30 times that of HHMA; and 2278520 at 24 h, which was 18.91 times that of SM-102, 11.96 times that of ALC-0315, 20.74 times that of compound 21, 21.80 times that of compound 23, and 22.9 times that of HHMA. at 48h it was 813200, which was 24.98 times that of SM-102, 20.43 times that of ALC-0315, 26.25 times that of compound 21, 24.55 times that of compound 23 and 24.98 times that of HHMA; at 7d it was 105500, which was 15.93 times that of SM-102, 14.97 times that of ALC-0315, 16.37 times that of compound 21, 16.91 times that of compound 23 and 17.97 times that of HHMA.

[0796] The average radiation intensity of YK-319 was 8230500 at 6 h, which was 11.74 times that of SM-102, 9.53 times that of ALC-0315, 13.49 times that of compound 21, 13.96 times that of compound 23, and 12.64 times that of HHMA; and 2433960 at 24 h, which was 20.20 times that of SM-102, 12.78 times that of ALC-0315, 22.16 times that of compound 21, 23.28 times that of compound 23, and 24.8 times that of HHMA. at 48h it was 887680, which were 27.27 times that of SM-102, 22.30 times that of ALC-0315, 28.65 times that of compound 21, 26.80 times that of compound 23 and 27.27 times that of HHMA; at 7d it was 104860, which were 15.84 times that of SM-102, 14.88 times that of ALC-0315, 16.27 times that of compound 21, 16.80 times that of compound 23 and 17.86 times that of HHMA.

[0797] The average radiation intensity of YK-318 was 3325680 at 6 h, which was 4.74 times that of SM-102, 3.85 times that of ALC-0315, 5.45 times that of compound 21, 5.64 times that of compound 23, and 5.11 times that of HHMA; and 968540 at 24 h, which was 8.04 times that of SM-102, 5.08 times that of ALC-0315, 8.82 times that of compound 21, 9.27 times that of compound 23, and at 48h it was 356810, which was 10.96 times that of SM-102, 8.96 times that of ALC-0315, 11.52 times that of compound 21, 10.77 times that of compound 23 and 10.96 times that of HHMA; at 7d it was 50659, which was 7.65 times that of SM-102, 7.19 times that of ALC-0315, 7.86 times that of compound 21, 8.12 times that of compound 23 and 8.63 times that of HHMA.

[0798] The data were analyzed using GraphPad Prism software. Any one of YK-305, YK-310, YK-312, YK-319 and YK-318 had significant differences compared with SM-102, ALC-0315, compound 21, compound 23, HHMA and YK-009 at each time, and the expression level and duration were significantly increased.

[0799] b. Differences in chemical structure

[0800] YK-305, YK-310, YK-312, YK-319 and YK-318 have very different chemical structures compared with cationic lipids in the prior art, such as SM-102, ALC-0315, compound 21, compound 23, HHMA and YK-009. Among them, the structural difference with HHMA is the largest. Except for one guillotine chain connected to the central N atom of HHMA, which is similar to one side chain of YK-305, YK-310, YK-312, YK-319 and YK-318, the other structures are completely different. Compared with SM-102, ALC-0315, compound 21, compound 23 and YK-009, the G3 groups of YK-305, YK-310, YK-312, YK-319 and YK-318 are completely different, and the G1, R1, G2 and R2 groups are also very different.

[0801] summary:

[0802] Among the designed series of compounds, the LNP preparations prepared by YK-305, YK-310, YK-312, YK-319 and YK-318 have the highest mRNA expression in mice, and the expression is sustained, and the expression levels at 6h, 24h, 48h and 7d are significantly higher than those of representative cationic lipids in the prior art. For example, YK-305 and YK-310 can reach 30 times that of SM-102, compound 21 and compound 23. The expression of mRNA in mice is consistent with the results of the cell transfection experiment in Example 6.

[0803] Furthermore, YK-305, YK-310, YK-312, YK-319 and YK-318 are very different from the representative cationic lipid structures in the prior art. The G3 group is completely different, and the G1, R1, G2 and R2 groups are also very different.

[0804] The present application unexpectedly discovered that the compound designed for the first time in the present application, which has a very different structure from the cationic lipids in the prior art, can express mRNA to a very high degree and continuously in the LNP preparation prepared therefrom.

[0805] (2) YK-305, YK-310, YK-312, YK-319, and YK-318 showed the highest mRNA expression in mice and the longest duration compared with a series of compounds with similar structures and G3 groups of HO(CH2)2N(CH3)CH2CH(OH)CH2-. For example, the expression level of YK-305 was more than 1000 times that of YK-302. The mRNA expression in mice was consistent with the cell transfection activity.

[0806] In order to compare the differences in the expression intensity and duration of mRNA delivered by delivery vectors prepared from compounds with very similar structures and only slightly different G1, G2, G3, R1 or R2 groups in mice, we compared a series of compounds with similar structures and G3 groups of HO(CH2)2N(CH3)CH2CH(OH)CH2- with YK-305, YK-310, YK-312, YK-319 and YK-318.

[0807] The results showed that the LNP preparations prepared from this series of compounds had very different mRNA expression in mice, among which YK-305, YK-310, YK-312, YK-319 and YK-318 had the highest expression levels and the longest duration. For example, the expression level of YK-305 could reach more than 1,000 times that of YK-302.

[0808] Table 17 Mouse in vivo imaging experimental data-2

[0809]

[0810] a. Expression differences in mice

[0811] It can be seen from Table 17 that compared with a series of compounds with similar structures and G3 groups of HO(CH2)2N(CH3)CH2CH(OH)CH2-, the LNP preparations prepared by YK-305, YK-310, YK-312, YK-319 and YK-318 have the highest mRNA expression level and duration in mice.

[0812] YK-305 can reach 633.70 times of YK-302 in 6 hours, 713.70 times in 24 hours, 1022.17 times in 48 hours and 330.77 times in 7 days.

[0813] YK-310 can reach 625.87 times of YK-302 in 6 hours, 746.29 times in 24 hours, 957.07 times in 48 hours and 305.89 times in 7 days.

[0814] YK-312 can reach 549.37 times of YK-302 in 6 hours, 575.97 times in 24 hours, 793.37 times in 48 hours and 274.74 times in 7 days.

[0815] YK-319 can reach 564.51 times of YK-302 in 6 hours, 615.26 times in 24 hours, 866.03 times in 48 hours, and 273.07 times in 7 days.

[0816] YK-318 can reach 228.10 times of YK-302 in 6 hours, 244.83 times in 24 hours, 348.11 times in 48 hours and 131.92 times in 7 days.

[0817] The data were analyzed using GraphPad Prism software. Any one of YK-305, YK-310, YK-312, YK-319 and YK-318 had significant differences from other compounds at all times, with both expression levels and duration significantly increased.

[0818] b. Differences in chemical structure

[0819] This series of compounds are very similar in structure to YK-305, YK-310, YK-312, YK-319 and YK-318, except for slight differences in the G1, G2, G3, R1 or R2 groups.

[0820] summary:

[0821] Compared with compounds with similar structures and G3 groups of HO(CH2)2N(CH3)CH2CH(OH)CH2-, LNP preparations prepared from YK-305, YK-310, YK-312, YK-319 and YK-318 have the highest mRNA expression intensity and the longest duration in mice. For example, YK-305 can reach more than 1000 times that of YK-302 in 48 hours, and can still reach more than 300 times in 7 days. The expression of mRNA in mice is consistent with the results of the cell transfection experiment in Example 6.

[0822] We also found that there was no correspondence between mRNA expression in mice and the structure of the cationic lipids, and that even LNP formulations made from a group of compounds with very similar structures, with only slight differences in the G1, G2, G3, R1, or R2 groups, were likely to have very different levels and durations of mRNA expression in mice.

[0823] Therefore, it is very difficult to screen out cationic lipid compounds that are expressed at extremely high levels and continuously in animals from a series of most structurally similar compounds, which requires a lot of creative work.

[0824] (3) YK-305, YK-310, YK-312, YK-319, and YK-318 showed the highest mRNA expression in mice and the longest duration compared with a series of compounds with similar structures and G3 groups of HO(CH2)2N(CH2CH3)CH2CH(OH)CH2-. For example, the expression level of YK-305 was 160 times that of YK-309. The mRNA expression in mice was consistent with the cell transfection activity.

[0825] We compared the differences in expression in mice of LNP formulations containing mRNA prepared from a series of compounds with similar structures and G3 groups of HO(CH2)2N(CH2CH3)CH2CH(OH)CH2- and YK-305, YK-310, YK-312, YK-319 and YK-318. The results showed that YK-305, YK-310, YK-312, YK-319 and YK-318 had the highest expression levels and the longest duration, which was significantly higher than other compounds. For example, YK-305 could reach 160 times that of YK-309.

[0826] Table 18 Mouse in vivo imaging experimental data-3

[0827]

[0828] a. Expression differences in mice

[0829] As can be seen from Table 18, among this series of compounds, the LNP preparations prepared from YK-305, YK-310, YK-312, YK-319 and YK-318 have the highest mRNA expression level and duration in mice.

[0830] The expression level of YK-305 was 140.67 times that of YK-309 and 20.89 times that of YK-313 at 6 hours, 167.86 times that of YK-309 and 27.04 times that of YK-313 at 24 hours, 159.93 times that of YK-309 and 40.99 times that of YK-313 at 48 hours, and 101.13 times that of YK-309 and 39.26 times that of YK-313 at 7 days.

[0831] The expression level of YK-310 was 138.93 times that of YK-309 and 20.64 times that of YK-313 at 6 hours, 175.52 times that of YK-309 and 28.27 times that of YK-313 at 24 hours, 149.75 times that of YK-309 and 38.38 times that of YK-313 at 48 hours, and 93.52 times that of YK-309 and 36.31 times that of YK-313 at 7 days.

[0832] The expression level of YK-312 was 121.95 times that of YK-309 and 18.11 times that of YK-313 at 6 hours, 135.46 times that of YK-309 and 21.82 times that of YK-313 at 24 hours, 124.13 times that of YK-309 and 31.82 times that of YK-313 at 48 hours, and 84.00 times that of YK-309 and 32.61 times that of YK-313 at 7 days.

[0833] The expression level of YK-319 was 125.31 times that of YK-309 and 18.61 times that of YK-313 at 6 hours, 144.71 times that of YK-309 and 23.31 times that of YK-313 at 24 hours, 135.50 times that of YK-309 and 34.73 times that of YK-313 at 48 hours, and 83.49 times that of YK-309 and 32.41 times that of YK-313 at 7 days.

[0834] The expression level of YK-319 was 50.63 times that of YK-309 and 7.52 times that of YK-313 at 6 hours, 57.58 times that of YK-309 and 9.27 times that of YK-313 at 24 hours, 54.47 times that of YK-309 and 13.96 times that of YK-313 at 48 hours, and 40.33 times that of YK-309 and 15.66 times that of YK-313 at 7 days.

[0835] The data were analyzed using GraphPad Prism software. Any one of YK-305, YK-310, YK-312, YK-319 and YK-318 had significant differences from other compounds at all times, with both expression levels and duration significantly increased.

[0836] b. Differences in chemical structure

[0837] This series of compounds are very similar in structure to YK-305, YK-310, YK-312, YK-319 and YK-318, except for slight differences in the G1, G2, G3, R1 or R2 groups.

[0838] summary:

[0839] Compared with compounds with similar structures and G3 groups of HO(CH2)2N(CH2CH3)CH2CH(OH)CH2-, LNP preparations prepared from YK-305, YK-310, YK-312, YK-319 and YK-318 have the highest mRNA expression intensity and the longest duration in mice. For example, YK-305 can reach 160 times that of YK-309 at 48h and still reach 100 times at 7d. The expression of mRNA in mice is consistent with the results of the cell transfection experiment in Example 6.

[0840] We also found that there was no correspondence between mRNA expression in mice and the structure of the cationic lipids, and that even LNP formulations made from a group of compounds with very similar structures, with only slight differences in the G1, G2, G3, R1, or R2 groups, were likely to have very different levels and durations of mRNA expression in mice.

[0841] Therefore, it is very difficult to screen out cationic lipid compounds that are expressed at extremely high levels and continuously in animals from a series of compounds with the most similar structures, and this requires a lot of creative work.

[0842] (4) YK-305, YK-310, YK-312, YK-319, and YK-318 showed the highest and longest-lasting mRNA expression in mice compared to a series of compounds with similar structures and G3 groups (HO(CH2)2)2NCH2CH(OH)CH2-. For example, YK-310 showed a 29-fold increase over YK-321. The mRNA expression in mice was consistent with the cell transfection activity.

[0843] We also compared the differences in expression in mice of LNP preparations containing mRNA prepared from a series of compounds with similar structures and G3 groups (HO(CH2)2)2NCH2CH(OH)CH2- and YK-305, YK-310, YK-312, YK-319 and YK-318. The results showed that YK-305, YK-310, YK-312, YK-319 and YK-318 had the highest expression levels and the longest duration, which was significantly higher than other compounds. For example, YK-310 could reach 29 times that of YK-321.

[0844] Table 19 Mouse in vivo imaging experimental data-4

[0845]

[0846] a. Expression differences in mice

[0847] As can be seen from Table 19, among this series of compounds, the LNP preparations prepared from YK-305, YK-310, YK-312, YK-319 and YK-318 have the highest mRNA expression level and duration in mice.

[0848] The expression level of YK-305 was 17.92 times that of YK-321 at 6h, 27.89 times at 24h, 28.65 times at 48h, and 19.29 times at 7d.

[0849] The expression level of YK-310 was 17.70 times that of YK-321 at 6 h, 29.16 times at 24 h, 26.83 times at 48 h, and 17.84 times at 7 d.

[0850] The expression level of YK-312 was 15.54 times that of YK-321 at 6h, 22.51 times at 24h, 22.24 times at 48h, and 16.02 times at 7d.

[0851] The expression level of YK-319 was 15.96 times that of YK-321 at 6h, 24.04 times at 24h, 24.28 times at 48h, and 15.92 times at 7d.

[0852] The expression level of YK-318 was 6.45 times that of YK-321 at 6 h, 9.57 times at 24 h, 9.76 times at 48 h, and 7.69 times at 7 d.

[0853] The data were analyzed using GraphPad Prism software. Any one of YK-305, YK-310, YK-312, YK-319 and YK-318 had significant differences from other compounds at all times, with both expression levels and duration significantly increased.

[0854] b. Differences in chemical structure

[0855] This series of compounds are very similar in structure to YK-305, YK-310, YK-312, YK-319 and YK-318, except for slight differences in the G1, G2, G3, R1 or R2 groups.

[0856] summary:

[0857] Compared with compounds with similar structures and G3 groups (HO(CH2)2)2NCH2CH(OH)CH2-, LNP preparations prepared from YK-305, YK-310, YK-312, YK-319 and YK-318 have the highest mRNA expression intensity and the longest duration in mice. For example, YK-310 can reach 29 times that of YK-321 in 24 hours and still reach 17 times in 7 days. The expression of mRNA in mice is consistent with the results of the cell transfection experiment in Example 6.

[0858] We also found that there was no correspondence between mRNA expression in mice and the structure of the cationic lipids, and that even LNP formulations made from a group of compounds with very similar structures, with only slight differences in the G1, G2, G3, R1, or R2 groups, were likely to have very different levels and durations of mRNA expression in mice.

[0859] Therefore, it is very difficult to screen out cationic lipid compounds that are highly and continuously expressed in animals from a series of compounds with the most similar structures, and this requires a lot of creative work.

[0860] b. Distribution of liposomes in mice

[0861] The results of in vivo imaging of mice showed that the distribution of liposomes prepared from different compounds in the mouse body was quite different. Some of them had protein expression in the liver, while some did not.

[0862] Specifically, at 6 hours, some cationic lipids, such as ALC-0315, SM-102, compound 21, compound 23, HHMA, YK-305 and YK-319, expressed proteins in the liver. Compared with ALC-0315 and SM-102, the expression of YK-305 and YK-319 was reduced; while some compounds, such as YK-310 and YK-313, had no protein expression in the liver. At 24 hours, the LNP preparations prepared by all compounds had been metabolized in the liver and had no protein expression. ( Fig.15 ) Compound 21, Compound 23 and HHMA are similar to SM-102, but the figure is not shown.

[0863] It can be seen that compared with cationic lipids in the prior art, the liposomes prepared by the compounds designed by us, after intramuscular injection, the liposomes express less target protein in the liver (YK-305 and YK-319), or do not stay in the liver and express the target protein (YK-310 and YK-313). The mRNA carried in the liposomes is expressed in the liver, and the expressed protein is metabolized by the liver, which will increase the burden on the liver. Therefore, some of the compounds designed by us will reduce the toxicity of liposomes in the liver compared to representative cationic lipids in the prior art.

[0864] summary:

[0865] Compared with representative cationic lipids in the prior art, such as SM-102, ALC-0315, compound 21, compound 23 and HHMA, the liposomes prepared by the compounds designed by us reduce the amount of target protein expressed in the liver, or do not stay in the liver and express the target protein. Therefore, compared with cationic lipids in the prior art, the LNP preparations prepared by the compounds designed by us have reduced or no toxicity to the liver.

[0866] Summarize:

[0867] 1) We conducted in vivo animal delivery experiments on LNP preparations prepared from a series of designed compounds and screened out cationic lipid compounds, such as YK-305, YK-310, YK-312, YK-319 and YK-318, whose mRNA was expressed at extremely high levels and continuously in mice.

[0868] This series of designed compounds have huge chemical structure differences from representative cationic lipids in the prior art, such as SM-102, ALC-0315, compound 21, compound 23, HHMA and YK-009. The G3 group is completely different, and other parts are also different. Therefore, there will be great differences in polarity, acidity and alkalinity, and hydrophilicity.

[0869] 2) The LNP preparations prepared from YK-305, YK-310, YK-312, YK-319 and YK-318 expressed mRNA in mice at a very high level and continuously, which was significantly higher than that of representative cationic lipids in the prior art. For example, YK-305 and YK-310 could reach 30 times that of SM-102, compound 21 and compound 23.

[0870] Compared with compounds with similar structures and G3 groups of HO(CH2)2N(CH3)CH2CH(OH)CH2-, LNP preparations prepared from YK-305, YK-310, YK-312, YK-319 and YK-318 showed the highest mRNA expression intensity and the longest duration in mice. For example, YK-305 could reach more than 1,000 times that of YK-302 at 48 hours and still more than 300 times at 7 days.

[0871] Compared with compounds with similar structures and G3 groups of HO(CH2)2N(CH2CH3)CH2CH(OH)CH2-, LNP preparations prepared from YK-305, YK-310, YK-312, YK-319 and YK-318 showed the highest mRNA expression intensity and the longest duration in mice. For example, YK-305 could reach 160 times that of YK-309 at 48h and 100 times at 7d.

[0872] Moreover, compared with representative cationic lipids in the prior art, such as SM-102, ALC-0315, compound 21, compound 23 and HHMA, the liposomes prepared by the compounds designed by us have reduced expression of the target protein in the liver, or do not stay in the liver and express the target protein. Therefore, compared with cationic lipids in the prior art, the LNP preparations prepared by the compounds designed by us have reduced or no toxicity to the liver.

[0873] 3) There is no correspondence between the structure of cationic lipids and the high and sustained expression of delivered mRNA in mice. Even cationic lipid compounds with small structural differences are likely to have very different mRNA expression in animals in LNP preparations. It is impossible to predict whether mRNA is highly expressed and sustained in animals based on the chemical structure of cationic lipids. It is very difficult to screen out cationic lipid compounds with extremely high and sustained mRNA expression, which requires a lot of creative work.

[0874] in conclusion:

[0875] 1. The designed series of compounds, including YK-305, YK-310, YK-312, YK-319 and YK-318, have huge chemical structure differences from the existing cationic lipids, such as SM-102, compound 21, compound 23, HHMA and YK-009. The G3 group is completely different, and other parts are also very different. Therefore, there will be great differences in polarity, acidity and alkalinity, and hydrophilicity.

[0876] Among the designed series of compounds, the LNP preparations prepared by YK-305, YK-310, YK-312, YK-319 and YK-318 have significantly improved cell transfection efficiency, significantly reduced cytotoxicity, significantly improved mRNA expression and duration in mice, reduced target protein expression in the liver, or will not stay in the liver and express the target protein, and reduced or no liver toxicity. For example, YK-305 can reach 17 times that of SM-102, 19 times that of compound 21, and 20 times that of compound 23; the cell survival rate of YK-305 and YK-310 can be 30% higher than ALC-0315, 12% higher than SM-102, and 15% higher than HHMA; the mRNA expression in mice, YK-305 and YK-310 can reach 30 times that of SM-102, compound 21 and compound 23.

[0877] Among the series of compounds we designed with very small differences in chemical structure, the LNP preparations prepared from YK-305, YK-310, YK-312, YK-319 and YK-318 significantly improved the cell transfection efficiency, significantly reduced the cytotoxicity, and significantly increased the expression level and duration of mRNA in mice compared with other compounds.

[0878] Structurally, this series of compounds differs slightly from YK-305, YK-310, YK-312, YK-319 and YK-318 only in the G1, G2, R1, R2 or G3 groups. However, the cell transfection efficiency of YK-305 can reach 1,300 times that of YK-304 and 900 times that of YK-302. The cytotoxicity can be reduced by 65% ​​compared with YK-302. The mRNA expression level in mice can reach 1,000 times that of YK-302.

[0879] 2. There is no obvious correspondence between the structure of cationic lipid compounds and the intracellular transfection efficiency, toxicity to cells, and high and sustained expression of mRNA in LNP preparations prepared from them in animals. Compounds with small structural differences are very likely to have very large differences in transfection efficiency and / or toxicity to cells, and intracellular expression.

[0880] For example, compared with YK-305, YK-302 only has 2 fewer carbon atoms in the G1 group; the R1 group is a straight chain structure, while YK-305 is a branched structure; the R2 group has 1 more carbon in the single chain, and 2 fewer carbon atoms in each single chain of the double chain; the other structures are exactly the same, but the cell transfection efficiency of YK-305 is 900 times that of YK-302, and the toxicity of YK-305 to transfected cells is 65% lower than that of YK-302, and the mRNA expression of YK-305 in mice can reach 1000 times that of YK-302; compared with YK-310, YK-303 only has 1 less carbon atoms in the group connected to N in the G3 group; the other structures are exactly the same, but the cell transfection efficiency of YK-310 is 40 times that of YK-303, and the toxicity of YK-310 to transfected cells is 18% lower than that of YK-303.

[0881] Therefore, screening suitable cationic lipid compounds that can simultaneously have high transfection efficiency and low toxicity to cells, as well as high and sustained expression of mRNA in mice is a very difficult task, which requires a lot of creative work.

[0882] 3. Through unique design and extensive screening, the present invention has discovered some compounds, such as YK-305, YK-310, YK-312, YK-319 and YK-318, which, compared with other compounds in the prior art, can deliver nucleic acids with significantly improved cell transfection efficiency, significantly reduced cytotoxicity, and significantly increased expression levels and duration in animals, achieving unexpected technical effects.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, wherein the compound has one of the following structures:

2. A composition comprising a carrier, wherein the carrier comprises a cationic lipid, and the cationic lipid comprises the compound according to claim 1 or a pharmaceutically acceptable salt thereof.

3. The composition according to claim 2, wherein the molar ratio of the cationic lipid to the carrier is 25% to 75%.

4. The composition of claim 2, wherein the carrier further comprises a neutral lipid. The composition according to claim 4 , wherein the molar ratio of the cationic lipid to the neutral lipid is 1:1 to 15:

1.

6. The composition of claim 5, wherein the molar ratio of the cationic lipid to the neutral lipid is 4.5:

1.

7. The composition according to claim 4, wherein the neutral lipid comprises one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol and derivatives thereof.

8. The composition according to claim 4, wherein the neutral lipid is selected from one or more of the following: 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-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0Diether 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 16.0PE), 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), palmitoyloleoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE) and mixtures thereof.

9. The composition of claim 8, wherein the neutral lipids are DOPE and DSPC.

10. The composition of claim 8, wherein the neutral lipid is DOPE or DSPC.

11. The composition of claim 2, wherein the carrier further comprises a structured lipid. 12 . The composition according to claim 11 , wherein the molar ratio of the cationic lipid to the structural lipid is 0.6:1 to 3:

1.

13. The composition according to claim 11, wherein the structured lipid is selected from one or more of the following: cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol, corticosteroids.

14. The composition of claim 13, wherein the structured lipid is cholesterol.

15. The composition of claim 2, wherein the carrier further comprises a polymer-conjugated lipid.

16. The composition according to claim 15, wherein the molar ratio of the polymer-conjugated lipid to the carrier is 0.5% to 10%. The composition according to claim 16 , wherein the molar ratio of the polymer-conjugated lipid to the carrier is 1.5%.

18. The composition of claim 15, wherein 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.

19. The composition according to claim 18, wherein 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).

20. The composition according to claim 2, wherein 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).

21. The composition according to claim 20, wherein the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is (35-49):(7.5-15):(35-55):(1-5).

22. The composition of claim 21, wherein the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is 45:10:43.5:1.

5.

23. The composition according to claim 2, wherein the composition is a nanoparticle preparation, the average particle size of the nanoparticle preparation is 10 nm to 300 nm; and the polydispersity coefficient of the nanoparticle preparation is ≤50%.

24. The composition according to claim 23, wherein the composition is a nanoparticle preparation, the average particle size of the nanoparticle preparation is 90nm to 280nm; and the polydispersity coefficient of the nanoparticle preparation is ≤45%.

25. The composition of claim 2, wherein the cationic lipid further comprises one or more other ionizable lipid compounds.

26. The composition of claim 2, further comprising a therapeutic or prophylactic agent.

27. The composition according to claim 26, wherein the mass ratio of the carrier to the therapeutic agent or preventive agent is 10:1 to 30:

1.

28. The composition according to claim 27, wherein the mass ratio of the carrier to the therapeutic agent or preventive agent is 12.5:1 to 20:

1.

29. The composition of claim 28, wherein the mass ratio of the carrier to the therapeutic or preventive agent is 15:

1.

30. The composition of claim 26, wherein the therapeutic agent or preventive agent is selected from one or more of a nucleic acid molecule, a small molecule compound, a polypeptide or a protein.

31. The composition of claim 26, wherein the therapeutic or prophylactic agent is a vaccine or a compound capable of eliciting an immune response.

32. The composition of claim 26, wherein the therapeutic or prophylactic agent is a nucleic acid.

33. The composition of claim 32, wherein the therapeutic or prophylactic agent is ribonucleic acid (RNA).

34. The composition of claim 32, wherein the therapeutic or prophylactic agent is deoxyribonucleic acid (DNA).

35. The composition of claim 33, wherein the RNA is selected from the group consisting of small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), micro RNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), and mixtures thereof.

36. The composition of claim 35, wherein the RNA is mRNA.

37. The composition of claim 2, wherein the composition further comprises one or more pharmaceutically acceptable excipients.

38. The composition of claim 2, wherein the composition further comprises one or more pharmaceutically acceptable diluents.

39. Use of the compound according to claim 1 or a pharmaceutically acceptable salt thereof or the composition according to any one of claims 2 to 38 in the preparation of nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs.

40. Use of a compound according to claim 1 or a pharmaceutically acceptable salt thereof or a composition according to any one of claims 2 to 38 in the preparation of a medicament for treating a disease or condition in a mammal.

41. The use of claim 40, wherein the disease or disorder is characterized by malfunctioning or aberrant protein or polypeptide activity.

42. The use according to claim 40, wherein the disease or condition is selected from the group consisting of infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases.

43. The use according to claim 42, wherein the infectious disease is selected from: a disease caused by a coronavirus, influenza virus or HIV virus, pediatric pneumonia, Rift Valley fever, yellow fever, rabies, or multiple herpes.

44. The use according to claim 40, wherein the mammal is a human.

45. The use according to claim 39 or 40, wherein the composition is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally or by inhalation.

46. ​​The use according to claim 45, wherein the composition is administered subcutaneously.

47. The use according to claim 40, wherein a dose of 0.001 mg / kg to 10 mg / kg of the drug is administered to the mammal.

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