Highly efficient and low-toxic cationic lipid compounds for extrahepatic targeting and compositions thereof

Abstract

The present disclosure provides a compound of formula (I), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, which is a highly efficient and low-toxicity cationic lipid compound with extrahepatic targeting. Compositions comprising the aforementioned compounds and their use in delivering therapeutic or prophylactic agents are also provided. JPEG0007761816000094.jpg3940

Description

[Technical Field]

[0001] This application claims priority from Chinese Patent Application No. 202310077355.X, filed on February 8, 2023, the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to the pharmaceutical field, and more particularly to cationic lipid compounds, compositions containing the same, and uses thereof. [Background technology]

[0003] The targeted and effective delivery of biologically active agents, such as small molecule drugs, polypeptides, proteins, and nucleic acids, particularly nucleic acids, has long been a medical challenge. Nucleic acid therapeutics face significant challenges due to poor cell permeability and the high susceptibility of certain nucleic acid molecules (including RNA) to degradation.

[0004] Compositions, liposomes, and lipoplexes containing cationic lipids have been shown to effectively deliver biologically active substances, such as small molecule drugs, polypeptides, proteins, and nucleic acids, to cells and / or intracellular compartments. These compositions generally contain one or more "cationic" and / or amino (ionizable) lipids, and may include neutral lipids, structural lipids, and polymer-conjugated lipids. Cationic and / or ionizable lipids include, for example, amine-containing lipids that can be easily protonated. While various such lipid-containing nanoparticle compositions have been demonstrated, there is still room for improvement in safety, functionality, and specificity. Furthermore, the increased complexity of lipid nanoparticles (LNPs) can complicate their production and potentially increase their toxicity, which are major concerns limiting their clinical application. For example, LNP siRNA particles (e.g., patisiran) require prior administration of steroids or antihistamines to eliminate unwanted immune responses (T. Coelho, D. Adams, A. Silva, et al., Safety and efficacy of RNAi therapy for transthyretin amyloidosis, N Engl J Med, 369 (2013) 819-829.). Thus, there remains a need for the development of improved cationic lipid compounds and compositions containing same that facilitate the delivery of therapeutic and / or prophylactic agents, such as nucleic acids, into cells. Summary of the Invention

[0005] One aspect of the present invention provides novel cationic lipid compounds that are compounds of formula (I), or N-oxides, solvates, pharmaceutically acceptable salts, or stereoisomers thereof: [ka] (where, G1 is C 2-8 is alkylene, G2 is C 2-8 is alkylene, L1 is -C(O)O- or -OC(O)-; L2 is -C(O)O- or -OC(O)-; R1 is C 6-25 is a straight-chain or branched alkyl; R2 is C 6-25 is a straight-chain or branched alkyl; G3 is HO(CH2)2- or HO(CH2)3-, G4 is HO(CH2)2- or HO(CH2)3-, L is -(CH2)2-, -(CH2)3-, or -(CH2)4-.

[0006] For example, compounds of formula (I) have one of the following structures: [ka] JPEG0007761816000004.jpg22183 JPEG0007761816000005.jpg23279 JPEG0007761816000006.jpg22775

[0007] A further aspect of the present invention provides a composition comprising a carrier, said carrier comprising a cationic lipid, said cationic lipid comprising a compound of formula (I) above, or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof. In one embodiment, the composition further comprises a therapeutic or prophylactic agent.

[0008] A further aspect of the present invention provides the above cationic lipid or composition for delivery of a therapeutic or prophylactic agent to a patient in need thereof.

[0009] A further aspect of the present invention provides a method for treating or preventing a disease or disorder, comprising administering to a patient or subject in need thereof a therapeutically or prophylactically effective amount of the above-described composition.

[0010] A further aspect of the present invention provides the use of the compound of formula (I) above, or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, or the composition above, in the preparation of a nucleic acid drug, a genetic vaccine, a small molecule drug, a polypeptide, or a protein drug.

[0011] A further aspect of the present invention provides the use of a compound of formula (I) as defined above, or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, or a composition as defined above, in the preparation of a medicament for treating a disease or disorder in a mammal in need thereof. [Brief explanation of the drawings]

[0012] [Figure 1] Figure 1 shows the results of cell transfection experiments with various weight ratios of carrier to mRNA used in the preparation of LNP formulations; a, carrier:mRNA = 5:1; b, carrier:mRNA = 15:1; c, carrier:mRNA = 35:1; d, blank control. [Figure 2] Figure 1 shows the results of cell transfection experiments with various molar ratios of cationic lipid to neutral lipid DSPC used in the preparation of LNP formulations: a, 3.5:1; b, 4:1; c, 4.9:1; d, blank control. [Figure 3] Figure 1 shows the results of cell transfection experiments with various molar ratios of polymer-conjugated lipid to carrier in the preparation of LNP formulations: a, 1.5%, b, 10%, c, blank control. [Figure 4] Figure 1 shows the results of cell transfection experiments using various ratios of the carrier components (cationic lipid, neutral lipid DSPC, structural lipid cholesterol, and polymer-conjugated lipid DMG-PEG2000) in the preparation of LNP formulations: a: 35:10:53.5:1.5, b: 40:10:48.5:1.5, c: 49:10:39.5:1.5, and d: blank control. [Figure 5]Fluorescence absorption intensity of LNP formulations of Fluc-mRNA prepared with various cationic lipids (a: YK-407, b: YK-401, c: YK-423, d: SM-102) is shown. [Figure 6] Fluorescence absorption intensity of LNP formulations of Fluc-mRNA prepared with various cationic lipids (a: YK-404, b: YK-406, c: YK-411, d: SM-102) is shown. [Figure 7] LNP formulations of Fluc-mRNA prepared with various cationic lipids (YK-407, YK-401, YK-402, YK-403, YK-422, YK-423, YK-009, SM-102, ALC-0315, Compound 21, Compound 23, and HHMA) and Lipofectamine 3000 formulations containing Fluc-mRNA were added to cell culture medium, and the cell viability after 24 hours of culture is shown. [Figure 8] LNP formulations of Fluc-mRNA prepared with various cationic lipids (YK-407, YK-401, YK-402, YK-403, YK-422, YK-423, YK-404, YK-405, YK-406, YK-408, YK-409, YK-410, YK-411, SM-102, ALC-0315, Compound 21, Compound 23, and HHMA) and Lipofectamine 3000 formulations containing Fluc-mRNA were added to cell culture medium, and the cell viability after 24 hours of culture is shown. [Figure 9] LNP formulations of Fluc-mRNA prepared with various cationic lipids (YK-407, YK-401, YK-402, YK-403, YK-422, YK-423, YK-412, YK-413, YK-414, YK-415, YK-416, YK-417, YK-418, YK-419, YK-420, YK-421, YK-424, SM-102, ALC-0315, Compound 21, Compound 23, and HHMA) and Lipofectamine 3000 formulations containing Fluc-mRNA were added to cell culture medium, and the cell viability after 24 hours of culture is shown. [Figure 10]Shown are the results of in vivo imaging experiments in mice of LNP formulations of Fluc-mRNA prepared with various cationic lipids (YK-407, YK-401, YK-415, SM-102, ALC-0315, compound 21, compound 23, and HHMA). [Figure 11] Shown are the results of in vivo imaging experiments in mice of LNP formulations of Fluc-mRNA prepared with various cationic lipids (YK-402, YK-411, YK-009, SM-102, ALC-0315, Compound 21, Compound 23, and HHMA). [Figure 12] Figure 1 shows the results of in vivo imaging experiments in mice of LNP formulations of Fluc-mRNA prepared with various cationic lipids (YK-403, YK-422, SM-102, ALC-0315, Compound 21, Compound 23, and HHMA). [Figure 13] Results of in vivo imaging experiments in mice are shown for LNP formulations of Fluc-mRNA prepared with various cationic lipids (YK-423, YK-417, SM-102, ALC-0315, Compound 21, Compound 23, and HHMA). [Figure 14] Figure 1 shows in vivo protein expression in mice using LNP formulations of Fluc-mRNA prepared with various cationic lipids (SM-102, YK-402, YK-407, YK-411, YK-418, YK-419, and YK-424). [Figure 15] Expression in the liver, spleen, lung, heart, and kidney of mice by LNP formulations of Fluc-mRNA prepared with various cationic lipids (SM-102, YK-402, YK-407, YK-411, YK-418, YK-419, and YK-424) is shown. DETAILED DESCRIPTION OF THE INVENTION

[0013] In order to clarify the objectives, technical solutions and advantages of the embodiments of the present invention, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the drawings of the embodiments of the present invention. It is clear that the described embodiments are only a part of the embodiments of the present invention, and are not all of the embodiments. Based on the described embodiments of the present invention, all other embodiments that can be obtained by those skilled in the art without requiring creative efforts fall within the protection scope of the present invention.

[0014] The present invention may be embodied in other specific forms without departing from the essential attributes of the present invention. It should be understood that any and all embodiments of the present invention may be combined with technical features of any other embodiment or multiple other embodiments, unless inconsistent, to obtain additional embodiments. The present invention includes the additional embodiments obtained by such combinations.

[0015] All publications and patents mentioned in this invention are hereby incorporated by reference in their entirety. If the terms or usage used in the publications and patents incorporated by reference conflict with the terms or usage used in this invention, the terms and usage in this invention shall control.

[0016] The section titles used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0017] Unless otherwise defined, all technical and scientific terms used herein have their ordinary meaning within the field to which the claimed subject matter belongs. In the event that there are multiple definitions for a term, those herein prevail.

[0018] All numbers, including quantitative properties such as dosages, set forth in the specification and claims, except in the examples or where otherwise indicated, should be understood to be modified in all instances by the term "about." Also, any numerical range recited herein should be understood to be intended to include all subranges within that range, and any combination of the individual endpoints of that range or subrange.

[0019] As used herein, the terms "comprise," "contain," "include," and similar terms such as "comprise," mean that the elements appearing before the term include the elements listed after the term and their equivalents, and do not exclude elements not listed. The terms "comprise" or "comprise" used herein may be open, semi-closed, and closed. In other words, the terms also include "consisting essentially of" or "consisting of."

[0020] As used herein, the term "pharmaceutically acceptable" means that a compound or composition is chemically and / or toxicologically compatible with the other ingredients that make up the formulation and / or with humans or mammals for preventing or treating a disease or disorder.

[0021] As used herein, the term "subject" or "patient" includes humans and mammals.

[0022] 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 said disease in order to cure, alleviate, relieve, improve or affect the disease or the symptoms of said disease. In the context of this application, the term "treatment" may also include prophylaxis, unless specifically stated to the contrary.

[0023] As used herein, the term "solvate" refers to a complex formed by combining a compound of Formula (I) or a pharmaceutically acceptable salt thereof with a solvent (e.g., ethanol or water). All solvates of a compound of Formula (I) used to treat a disease or disorder may provide different properties (including pharmacokinetic properties), but will be the compound of Formula (I) when absorbed by a subject, and therefore, references to the use of a compound of Formula (I) should be understood to encompass the use of all solvates of the compound of Formula (I).

[0024] The term "hydrate" refers to the above term "solvate" where the solvent is water.

[0025] Furthermore, it should be understood that the compounds of formula (I) or pharmaceutically acceptable salts thereof may be isolated in the form of solvates, and therefore, any and all such solvates are included within the scope of the present invention. For example, the compounds of formula (I) or pharmaceutically acceptable salts thereof may exist in unsolvated or solvated forms with pharmaceutically acceptable solvents (e.g., water, ethanol, etc.).

[0026] The term "pharmaceutically acceptable salt" refers to a relatively non-toxic inorganic or organic acid addition salt of the compound of the present invention. See, for example, SM Berge et al., "Pharmaceutical Salts, J. Pharm. Sci. 1977, 66, 1-19." Among these, inorganic acids include, for example, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and nitric acid. Organic acids include, for example, 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-hydroxybenzoylbenzo ... 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, glycerophosphate, aspartic acid, sulfosalicylic acid, etc. For example, HCl (or hydrochloric acid), HBr (or hydrobromic acid solution), methanesulfonic acid, sulfuric acid, tartaric acid, or fumaric acid can be used to form a pharmaceutically acceptable salt with a compound of Formula (I).

[0027] The nitrogen-containing compounds of formula (I) of the present invention can be converted to N-oxides by treatment with an oxidizing agent (e.g., metachloroperbenzoic acid, hydrogen peroxide, ozone). Thus, where valence and structure permit, the compounds protected herein include not only the nitrogen-containing compounds represented by the structural formula, but also their N-oxide derivatives.

[0028] Certain compounds of the present invention may exist in one or more stereoisomeric forms. Stereoisomers include geometric isomers, diastereomers, and enantiomers. Accordingly, compounds protected by the present invention include racemic mixtures, single stereoisomers, and optically active mixtures. Those skilled in the art should understand that certain stereoisomers may possess the potency and / or fewer side effects of other stereoisomers. Single stereoisomers and optically active mixtures can be obtained by methods such as chiral pool synthesis, chiral catalysis, and chiral resolution. Racemic compounds can be chirally resolved by chromatographic or chemical resolution. For example, chiral acid resolving agents, such as chiral tartaric acid or chiral malic acid, can be added to form salts with the compounds of the present invention, and the salts can then be separated based on the physical and chemical properties of the resulting products, such as differences in solubility.

[0029] The present invention also includes any suitable isotopic variant of the compound of the present invention. An isotopic variant is defined as a compound in which at least one atom has been replaced with an atom having the same atomic number but an atomic mass different from the atomic mass that is usually or predominantly found in nature. Examples of isotopes that can be introduced into the compound of the present invention include isotopes of hydrogen, carbon, nitrogen, and oxygen, such as isotopes of hydrogen, carbon, nitrogen, and oxygen, respectively. 2 H (deuterium), 3 H (tritium), 11 C. 13 C. 14 C. 15 N, 17 O, and 18 Includes O.

[0030] As used herein, the term "alkyl" refers to both branched and straight-chain saturated aliphatic monovalent hydrocarbon radicals having the specified number of carbon atoms. As used herein, the term "alkylene" is intended to include both branched and straight-chain saturated aliphatic divalent hydrocarbon radicals having the specified number of carbon atoms. n-m refers to a group having n to m carbon atoms. For example, C 2-5Alkylene includes C2 alkylene, C3 alkylene, C4 alkylene, and C5 alkylene.

[0031] An alkyl (or alkylene) can be unsubstituted, or an alkyl (or alkylene) can be substituted, where at least one hydrogen has been replaced with another chemical group.

[0032] A "therapeutically effective amount" is the amount of a therapeutic agent that ameliorates a disease or symptom when administered to a patient. A "prophylactically effective amount" is the 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 depending on the therapeutic / prophylactic agent, the disease state and its severity, and the age, weight, etc. of the patient / subject to be treated / prevented. Those skilled in the art can routinely determine therapeutically effective amounts and prophylactically effective amounts based on their own knowledge and the present invention.

[0033] In this application, when the name and structural formula of a compound are inconsistent, the structural formula shall prevail.

[0034] It should be understood that the term "compounds of the invention" as used herein may include compounds of formula (I), N-oxides thereof, solvates thereof, pharmaceutically acceptable salts thereof, stereoisomers thereof, and mixtures thereof, depending on the context.

[0035] As used herein, the term cationic lipid refers to a lipid that is positively charged at a selected pH value.

[0036] Cationic liposomes readily bind to negatively charged nucleic acids, i.e., they interact with the negatively charged phosphate groups present in nucleic acids through electrostatic forces, forming lipid nanoparticles (LNPs).

[0037] The present inventors have found that screening many compounds has a significant difference in structure compared to typical cationic lipids of the prior art, and it is extremely difficult to find a suitable cationic lipid compound that simultaneously has a very high transfection efficiency, extremely low cytotoxicity, and high and sustained expression in mice. The present inventors have found that some compounds, such as YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423, can deliver nucleic acids with significantly improved intracellular transfection efficiency, significantly reduced cytotoxicity, and significantly improved expression levels and sustained expression in animals, compared to cationic lipids of the prior art that have a very significant difference in chemical structure.

[0038] Briefly, the present invention is based on at least the following discoveries.

[0039] 1. The designed series of compounds, including YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423, were compared with representative cationic lipids of 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 23 disclosed in CN112979483B), and compound YK-009 disclosed in CN114044741B. Therefore, there are significant differences in their chemical structures, with significantly different head groups. The head groups of this series of designed compounds contain two tertiary amine groups, an L group connecting two tertiary amine nitrogen atoms, and a G3 group and a G4 group connected to the two tertiary amine nitrogen atoms, respectively. However, the head groups of SM-102, ALC-0315, Compound 21, Compound 23, and YK-009 contain only one tertiary amine group and an HO(CH2)2- group connected to the tertiary amine nitrogen atom, with all other moieties being relatively different. Therefore, there are also significant differences in terms of polarity, acidity, alkalinity, and hydrophilicity.

[0040] Therefore, it is impossible to predict the cell transfection efficiency, cytotoxicity, and animal expression of LNP formulations prepared with this series of compounds from the cationic lipid compounds disclosed in the prior art.

[0041] The chemical structures of SM-102, ALC-0315, Compound 21, Compound 23, and HHMA are as follows: [ka] (WO2017049245A2, page 29 of the specification) [ka] (CN108368028B, page 24 of the specification) [ka] (WO2021055833A1, page 22 of the specification) [ka] (WO2021055833A1, page 22 of the specification) [ka] (CN112979483B, page 12 of the specification)

[0042] 2. Among this series of compounds, the LNP formulations prepared with YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 showed significantly improved encapsulation rates, drug loading concentrations, and total RNA concentrations, significantly improved cell transfection efficiencies, significantly reduced cytotoxicity, significantly increased mRNA expression levels and duration in mice, reduced or no liver toxicity, and the ability to deliver mRNA directly to the spleen, compared with representative cationic lipids of the prior art.

[0043] For example, YK-407 exhibited a 29.0% higher encapsulation rate than Compound 23, a 1.78-fold higher drug loading concentration than Compound 23, a 1.41-fold higher total RNA concentration than Compound 21, a 12-fold higher cell transfection efficiency than SM-102, a 13-fold higher drug loading concentration than Compound 21, and a 15-fold higher cell transfection efficiency than Compound 23. YK-401 also exhibited a 28.00% higher cell viability than ALC-0315, a 7.31% higher cell viability than SM-102, and a 10.94% higher cell viability than HHMA. LNP formulations prepared with YK-407 exhibited 27-fold higher mRNA expression levels than SM-102, 22-fold higher drug loading concentration than ALC-0315, 28-fold higher drug loading concentration than Compound 21, 27-fold higher drug loading concentration than Compound 23, and 27-fold higher drug loading concentration than Compound 23. LNP formulations prepared with the compounds designed in this application have reduced or no liver toxicity because they either express less of the target protein in the liver (YK-402) or remain in the liver and do not express the target protein (YK-407, YK-411, YK-418, YK-419, and YK-424). YK-407, YK-419, and YK-424 can deliver mRNA directly to the spleen and are not expressed in other organs such as the liver, lungs, heart, and kidneys, significantly improving the protective effect without changing the vaccine components.

[0044] Among the series of compounds designed in this application that have very small differences in chemical structure, LNP formulations prepared with YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 have significantly improved cell transfection efficiency, significantly reduced cytotoxicity, and significantly improved both the amount and duration of mRNA expression in mice compared to other compounds. This series of compounds differs only by one or two carbon atoms in individual groups, such as G3, G4, or L groups, and other structures are slightly different. However, the cell transfection efficiency of YK-407 is 2500 times higher than that of YK-404 and YK-411. The cytotoxicity of YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 is 50% lower than that of YK-411. The mRNA expression level in mice was 1000 times higher in LNP formulations prepared with YK-407 than that of YK-411.

[0045] 3. There is no clear correlation between the structure of cationic lipid compounds and the intracellular transfection efficiency, cellular toxicity, or high and sustained expression of mRNA in animals using LNP formulations prepared with those cationic lipid compounds. Even compounds with very small structural differences are likely to result in very large differences in transfection efficiency, cellular toxicity, and intracellular expression.

[0046] For example, YK-411 has a very similar structure to YK-407. Compared with YK-407, YK-411 simply has two more Cs in the G1 group, one less C in the R1 group, and two more Cs in the R2 group per strand and two less Cs in each strand of the double strand. However, the cell transfection efficiency of YK-407 is 2500 times higher than that of YK-411, the toxicity of YK-407 to transfected cells is 55% lower than that of YK-411, and the mRNA expression in mice is 1000 times higher than that of YK-411.

[0047] Therefore, screening for suitable cationic lipid compounds that simultaneously have high transfection efficiency, low toxicity to cells, and high and sustained expression of mRNA in mice is extremely challenging and requires a great deal of creative work.

[0048] 4. Through unique design and extensive screening, the present invention has discovered several compounds, such as YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423, which, compared with other compounds in the prior art, can deliver nucleic acids with significantly improved encapsulation rates, drug loading concentrations, and total RNA concentrations, significantly improved cell transfection efficiency, significantly reduced cytotoxicity, significantly improved expression levels and durations in animal bodies, reduced or no liver toxicity, and can deliver mRNA directly to the spleen without expression in other organs such as the liver, lungs, heart, and kidneys, thereby achieving unexpected technical effects.

[0049] In summary, through unique design and extensive screening, the present invention has discovered several compounds, such as YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423, which have significant differences in chemical structure from typical cationic lipids of the prior art. Compared with other compounds of the prior art, these compounds can deliver nucleic acids with significantly improved cell transfection efficiency, significantly reduced cytotoxicity, significantly improved expression levels and expression duration in animals, and reduced or no liver toxicity, achieving unexpected technical effects.

[0050] Specifically, it is as follows:

[0051] 1. There are huge differences in chemical structure compared to representative cationic lipids of the prior art such as SM-102, ALC-0315, Compound 21, Compound 23, YK-009, and HHMA.

[0052] In comparison to representative cationic lipids of the prior art, this series of compounds designed is as follows:

[0053] 1) The structural difference from HHMA is the greatest. From the chemical structure diagram, it was found that in HHMA, only one side chain of the group connected to the central N atom is similar to one side chain of this series of compounds, while the other parts are significantly different.

[0054] 2) The head structures of these compounds are significantly different from those of SM-102, ALC-0315, Compound 21, Compound 23, and YK-009. The head structures of these compounds contain two tertiary amine groups, an L group connecting two tertiary amine nitrogen atoms, and G3 and G4 groups connected to the two tertiary amine nitrogen atoms, respectively. However, the head structures of SM-102, ALC-0315, Compound 21, Compound 23, and YK-009 contain only one tertiary amine group and HO(CH2)2- connected to the tertiary amine nitrogen atom. Furthermore, the G1, L1, R1, G2, and R2 groups of these compounds are also significantly different from those of SM-102, ALC-0315, Compound 21, Compound 23, and YK-009.

[0055] 3) Due to the large structural differences, this series of compounds also differs significantly from SM-102, ALC-0315, Compound 21, Compound 23, HHMA, and YK-009 in terms of polarity, acid-alkali property, and hydrophilicity.

[0056] 2. Compared with typical cationic lipids of the prior art and compounds with similar structures, the encapsulation rate, drug loading concentration, and total RNA concentration are all significantly improved.

[0057] LNP formulations prepared with several compounds designed herein, including YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423, all exhibited significantly improved encapsulation efficiency, drug loading concentration, and total RNA concentration compared to prior art cationic lipids such as SM-102, ALC-0315, compound 21, compound 23, HHMA, and YK-009. For example, YK-407 exhibited a 29.0% improvement in encapsulation efficiency over compound 23, a 1.78-fold increase in drug loading concentration over compound 23, and a 1.41-fold increase in total RNA concentration over compound 21.

[0058] 3. Significantly improved in vitro cell transfection efficiency compared to representative cationic lipids of the prior art and multiple compounds of similar structure designed in this application.

[0059] 1) LNP formulations prepared with YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 exhibited the highest cell transfection efficiency and significantly improved activity compared with representative cationic lipids of the prior art. For example, YK-407 was 12-fold more potent than SM-102, 13-fold more potent than compound 21, and 15-fold more potent than compound 23.

[0060] 2) Compared with similar compounds in which the G3 and G4 groups are HO(CH2)2- and the L group is -(CH2)2-, YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 have the highest cell transfection efficiency. For example, YK-407 can reach 2500 times that of YK-404 and YK-411, while YK-401, YK-402, YK-403, YK-422, and YK-423 can all reach more than 600 times that of YK-404 and YK-411.

[0061] 3) Compared with a series of similar compounds in which G3 and G4 are HO(CH2)2- or HO(CH2)3- and L is -(CH2)2-, -(CH2)3-, or -(CH2)4-, YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 have the highest cell transfection efficiency. For example, YK-407 is 170-fold higher than YK-417 and 180-fold higher than YK-418.

[0062] 4) There is no correlation between the structure of a compound and its intracellular transfection efficiency. Whether the structural difference between compounds is very small or relatively large, it is likely to result in a very large difference in transfection efficiency. Therefore, screening for cationic lipid compounds with high transfection efficiency is very difficult and requires a large amount of creative work.

[0063] 4. Cytotoxicity is significantly reduced compared to representative cationic lipids of the prior art and multiple compounds with similar structures designed in this application.

[0064] 1) LNP formulations prepared with YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 exhibited the lowest cytotoxicity and significantly improved cell viability compared to representative cationic lipids of the prior art. For example, YK-401 demonstrated cell viability 28.00% higher than ALC-0315, 7.31% higher than SM-102, and 10.94% higher than HHMA.

[0065] 2) Compared with a series of structurally similar compounds in which G3 and G4 are HO(CH2)2- and L is -(CH2)2-, YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 exhibited the lowest cytotoxicity and the highest cell viability. For example, cell viability of YK-401 was 58.88% higher than that of YK-411 and 50.25% higher than that of YK-406, while that of YK-407 was 55.04% higher than that of YK-411 and 46.41% higher than that of YK-406.

[0066] 3) Compared with a series of structurally similar compounds in which G3 and G4 are HO(CH2)2- or HO(CH2)3- and L is -(CH2)2-, -(CH2)3-, or -(CH2)4-, YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 have the lowest cytotoxicity. For example, YK-401 is 53.87% and 54.16% higher than YK-417 and YK-418, respectively, and YK-407 is 50.03% and 50.32% higher than YK-417 and YK-418, respectively.

[0067] 4) There is no correspondence between the structure of a compound and its cytotoxicity. Whether the structural difference in a compound is very small or relatively large, it is likely to result in a very large difference in cytotoxicity. Therefore, it is impossible to predict cytotoxicity from chemical structure. Screening for cationic lipid compounds with low cytotoxicity is extremely difficult and requires a great deal of creative work.

[0068] 5. Compared with representative cationic lipids of the prior art and multiple compounds with similar structures designed in this application, the amount and duration of mRNA expression in the animal's body are significantly improved, there is reduced or no liver toxicity, and only the mRNA is delivered directly to the spleen.

[0069] 1) LNP formulations prepared with YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 showed high and sustained mRNA expression in mice, significantly improving expression compared to representative cationic lipids of the prior art. For example, YK-407 showed 27-fold higher mRNA expression than SM-102, 22-fold higher mRNA expression than ALC-0315, 28-fold higher mRNA expression than Compound 21, 27-fold higher mRNA expression than Compound 23, and 27-fold higher mRNA expression than HHMA.

[0070] 2) Compared with similar compounds with G3 and G4 being HO(CH2)2- and L being -(CH2)2-, LNP formulations prepared with YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 showed the highest mRNA expression intensity and longest duration in mice. For example, YK-407 was more than 600-fold higher than YK-411 at 6 h, 1,000-fold higher at 24 h, and more than 100-fold higher at 7 d.

[0071] 3) Compared with compounds with similar structures but with G3 and G4 groups of HO(CH2)2- or HO(CH2)3-, L groups of -(CH2)2-, -(CH2)3-, or -(CH2)4-, and other minor differences, LNP formulations prepared with YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 exhibited the highest mRNA expression intensity and longest duration in mice. For example, YK-407 reached 120-fold higher expression levels than YK-417 at 48 hours and 20-fold higher expression levels at 7 days.

[0072] 4) Compared with representative cationic lipids of the prior art, such as SM-102, ALC-0315, Compound 21, Compound 23, and HHMA, liposomes prepared with compounds designed herein, such as YK-402, YK-407, YK-411, YK-418, YK-419, and YK-424, show reduced target protein expression in the liver or do not remain in the liver and express target proteins. Therefore, compared with cationic lipids of the prior art, LNP formulations prepared with compounds designed herein have reduced or no liver toxicity. Furthermore, some compounds designed herein, such as YK-407, YK-419, and YK-424, can deliver mRNA directly to the spleen, where it is expressed only in the spleen and not in other organs, such as the liver, lungs, heart, and kidneys. This significantly improves the preventive effect without changing the vaccine's components, which is of great clinical significance.

[0073] 5) There is no correlation between the structure of cationic lipids and the high and sustained expression of delivered mRNA in mice. Regardless of whether the structural differences between cationic lipid compounds are very small or relatively large, the mRNA in LNP formulations prepared with them is likely to be significantly different in vivo. It is impossible to predict from the chemical structure of a cationic lipid whether high and sustained mRNA expression will occur in an animal. Screening for cationic lipid compounds that result in high and sustained mRNA expression is extremely difficult and requires a great deal of creative effort.

[0074] One aspect of the present invention provides novel cationic lipid compounds for delivering therapeutic or prophylactic agents. The cationic lipid compounds of the present invention are used to deliver nucleic acid molecules, small molecule compounds, polypeptides, or proteins. Compared to known cationic lipid compounds, the cationic lipid compounds of the present invention exhibit high transfection efficiency and low cytotoxicity, thereby improving delivery efficiency and safety.

[0075] The present invention provides a cationic lipid that is a compound of formula (I), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof: [ka] (where, G1 is C 2-8 alkylene, preferably unsubstituted C 3-8 is alkylene, G2 is C 2-8 alkylene, preferably unsubstituted C 3-8 is alkylene, L1 is -C(O)O- or -OC(O)-; L2 is -C(O)O- or -OC(O)-; R1 is C 6-25 A straight-chain or branched alkyl, preferably an unsubstituted C 11 Straight chain alkyl or unsubstituted C 10 Straight chain alkyl or unsubstituted C8-18 is a branched alkyl; R2 is C 6-25 A straight-chain or branched alkyl, preferably an unsubstituted C 11 Straight chain alkyl or unsubstituted C 10 Straight chain alkyl or unsubstituted C 8-18 is a branched alkyl; G3 is HO(CH2)2- or HO(CH2)3-, G4 is HO(CH2)2- or HO(CH2)3-, L is -(CH2)2-, -(CH2)3-, or -(CH2)4-, preferably -(CH2)2- or -(CH2)3-.

[0076] In one embodiment, G1 is an unsubstituted C3 alkylene, for example, -(CH2)3-.

[0077] In one embodiment, G1 is an unsubstituted C5 alkylene, for example, -(CH2)5-.

[0078] In one embodiment, G1 is an unsubstituted C6 alkylene, for example, -(CH2)6-.

[0079] In one embodiment, G1 is an unsubstituted C7 alkylene, for example, -(CH2)7-.

[0080] In one embodiment, G1 is unsubstituted C8 alkylene, for example, -(CH2)8-.

[0081] In one embodiment, G2 is an unsubstituted C3 alkylene, for example, -(CH2)3-.

[0082] In one embodiment, G2 is an unsubstituted C5 alkylene, for example, -(CH2)5-.

[0083] In one embodiment, G2 is an unsubstituted C6 alkylene, for example, -(CH2)6-.

[0084] In one embodiment, G2 is an unsubstituted C7 alkylene, for example, -(CH2)7-.

[0085] In one embodiment, G2 is an unsubstituted C8 alkylene, for example, -(CH2)8-.

[0086] In one embodiment, L1 is -C(O)O-.

[0087] In one embodiment, L1 is -OC(O)-.

[0088] In one embodiment, L2 is -C(O)O-. For example, L1 and L2 are all -C(O)O-.

[0089] In one embodiment, L2 is -OC(O)-. For example, L1 and L2 are all -OC(O)-.

[0090] In one embodiment, R1 is unsubstituted C 11 Straight chain alkyl, i.e., -(CH2) 10 It is CH3.

[0091] In one embodiment, R1 is unsubstituted C 10 It is a straight chain alkyl, i.e., -(CH2)9CH3.

[0092] In one embodiment, R1 is unsubstituted C 8-18 In one preferred embodiment, R is an unsubstituted C 17 Branched alkyl, C 18 Branched alkyl, C 15 Branched alkyl, or C 14 Branched alkyl C8 branched alkyl. For example, R1 is [ka] In another preferred embodiment, R is unsubstituted C 17 Branched alkyl, C 18 For example, R1 is: [ka] is.

[0093] In one embodiment, R2 is unsubstituted C 8-18 In one preferred embodiment, R2 is an unsubstituted C 17 Branched alkyl, C 18 Branched alkyl, C 15 Branched alkyl, or C 14 Branched alkyl C8 branched alkyl. For example, R2 is [ka] is.

[0094] In one embodiment, L is —(CH 2 ) 2 —.

[0095] In one embodiment, L is —(CH 2 ) 3 —.

[0096] In one embodiment, L is —(CH 2 ) 4 —.

[0097] In one embodiment, G1 is —(CH2)3—, G2 is —(CH2)5—, L1 is —C(O)O—, L2 is —C(O)O—, and R1 is —(CH2) 10 CH3 and R2 is [ka] wherein G3 is HO(CH2)2-, G4 is HO(CH2)2-, and L is -(CH2)2-.

[0098] In one embodiment, G1 is —(CH2)5—, G2 is —(CH2)5—, L1 is —C(O)O—, L2 is —C(O)O—, and R1 is [ka] and R2 is [ka] wherein G3 is HO(CH2)2-, G4 is HO(CH2)2-, and L is -(CH2)2-.

[0099] In one embodiment, G1 is —(CH2)6—, G2 is —(CH2)6—, L1 is —OC(O)—, L2 is —OC(O)—, and R1 is [ka] and R2 is [ka] wherein G3 is HO(CH2)2-, G4 is HO(CH2)2-, and L is -(CH2)2-.

[0100] In one embodiment, G1 is —(CH2)7—, G2 is —(CH2)7—, L1 is —C(O)O—, L2 is —C(O)O—, and R1 is [ka] and R2 is [ka] wherein G3 is HO(CH2)2-, G4 is HO(CH2)2-, and L is -(CH2)2-.

[0101] In one embodiment, G1 is —(CH2)5—, G2 is —(CH2)5—, L1 is —C(O)O—, L2 is —C(O)O—, and R1 is [ka] and R2 is [ka] wherein G3 is HO(CH2)3-, G4 is HO(CH2)3-, and L is -(CH2)2-.

[0102] In one embodiment, G1 is —(CH2)5—, G2 is —(CH2)5—, L1 is —C(O)O—, L2 is —C(O)O—, and R1 is [ka] and R2 is [ka] wherein G3 is HO(CH2)3-, G4 is HO(CH2)3-, and L is -(CH2)3-.

[0103] In exemplary embodiments, the compound is selected from the following compounds, or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof: [ka] JPEG0007761816000028.jpg23590 JPEG0007761816000029.jpg24283 JPEG0007761816000030.jpg23580 JPEG0007761816000031.jpg8775

[0104] A further aspect of the present invention provides a composition comprising a carrier, said carrier comprising a cationic lipid, said cationic lipid comprising a compound of formula (I) above, or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof.

[0105] In one embodiment, the composition is a nanoparticle formulation, the average size of the nanoparticle formulation is between 10 nm and 300 nm, preferably between 90 nm and 280 nm, and the polydispersity of the nanoparticle formulation is 50% or less, preferably 40% or less, more preferably 30% or less.

[0106] cationic lipids In one embodiment of the composition / carrier of the present invention, the cationic lipid is one or more selected from the compounds of formula (I) above, or N-oxides, solvates, pharmaceutically acceptable salts, or stereoisomers thereof. In one embodiment, the cationic lipid is selected from the compounds of formula (I) above. For example, cationic lipids include the compounds YK-401, YK-402, YK-403, YK-404, YK-405, YK-406, YK-407, YK-408, YK-409, YK-410, YK-411, They are YK-412, YK-413, YK-414, YK-415, YK-416, YK-417, YK-418, YK-419, YK-420, YK-421, YK-422, YK-423, and YK-424. In one preferred embodiment, the cationic lipid is compound YK-407, in another preferred embodiment, the cationic lipid is compound YK-401, in another preferred embodiment, the cationic lipid is compound YK-402, in another preferred embodiment, the cationic lipid is compound YK-403, in another preferred embodiment, the cationic lipid is compound YK-422, and in another preferred embodiment, the cationic lipid is compound YK-423.

[0107] In another embodiment of the composition / carrier of the present invention, the cationic lipid comprises (a) one or more compounds selected from the group consisting of the compound of formula (I) above, or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer, and (b) one or more other ionizable lipid compounds other than (a). The (b) cationic lipid compound may be a commercially available cationic lipid or a cationic lipid compound reported in the literature. For example, the (b) cationic lipid compound may be SM-102 (compound 25 described in WO2017049245A2), compound 21 and compound 23 described in WO2021055833, or HHMA (compound 1 described in CN112979483B).

[0108] In one embodiment, the molar ratio of the cationic lipid in the carrier is about 25% to 75%, for example, about 30%, 40%, 49%, 55%, 60%, 65%, or 70%.

[0109] The carrier may be used to deliver an active ingredient, such as a therapeutic or prophylactic agent, which may be encapsulated within the carrier or bound to the carrier.

[0110] For example, the therapeutic or prophylactic agent comprises one or more of nucleic acid molecules, small molecule compounds, polypeptides, or proteins.The nucleic acid comprises, but is not limited to, single-stranded DNA, double-stranded DNA, and RNA.Suitable RNA comprises, but is not limited to, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), short hairpin RNA (shRNA), messenger RNA (mRNA), and mixtures thereof.

[0111] neutral lipid The carrier may comprise a neutral lipid. In the present invention, the neutral lipid refers to a lipid that is uncharged or exists in zwitterionic form at a selected pH value and plays an auxiliary role. This neutral lipid can promote the phase transition of lipids, thereby regulating the flow of nanoparticles into the lipid bilayer structure, improving efficiency, and may also affect the specificity of target organs.

[0112] 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 one preferred embodiment, the molar ratio of the cationic lipid to the neutral lipid is about 4:1. In another preferred embodiment, the molar ratio of the cationic lipid to the neutral lipid is about 4.9:1.

[0113] For example, the neutral lipid may comprise one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol, and derivatives thereof.

[0114] The carrier component of the composition containing cationic lipid may include one or more neutral lipids-phospholipids, for example, one or more (poly)unsaturated lipids.Phospholipids may be organized into one or more lipid bilayers.Generally, phospholipids may include a phospholipid moiety and one or more fatty acid moieties.

[0115] The neutral lipid moiety may be selected from the non-limiting group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin. The fatty acid moiety may be selected from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Also included are non-naturally occurring species, including naturally occurring species with modifications and substitutions such as branching, oxidation, cyclization, and alkynes. For example, the phospholipid may be functionalized with one or more alkynes (e.g., alkenyl with one or more double bonds replaced with triple bonds) or crosslinked with one or more alkynes. Under appropriate reaction conditions, alkynyl groups can undergo copper-catalyzed cycloaddition reactions when exposed to azides. These reactions can be used to functionalize the lipid bilayer of the composition to facilitate membrane permeation or cellular recognition, or to conjugate the composition with useful components such as targeting or imaging moieties (e.g., dyes).

[0116] The neutral lipids used in these compositions were 1,2-dilinolenoyl-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 (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16).0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-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), palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl- ... The phosphatidylcholine may be selected from the non-limiting group consisting of 1-stearoyl-2-oleoyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof.

[0117] In some embodiments, the neutral lipid comprises DSPC. In some embodiments, the neutral lipid comprises DOPE. In some embodiments, the neutral lipid comprises both DSPC and DOPE.

[0118] structural lipids The carrier of the composition containing the cationic lipid may also contain one or more structural lipids, which in the present invention refer to lipids that enhance the stability of the nanoparticles by filling the gaps between the lipids.

[0119] In one embodiment, the molar ratio of the cationic lipid to the structural lipid is about 0.6:1 to 3:1, e.g., about 1.0:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2.0:1.

[0120] The structural lipid may be selected from the group consisting of, but not limited to, cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, α-tocopherol, corticosteroids, and mixtures thereof. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid comprises cholesterol, a corticosteroid (e.g., prednisolone, dexamethasone, prednisone, and hydrocortisone), or a combination thereof.

[0121] Polymer-conjugated lipids The carrier of the composition containing the cationic lipid may also contain one or more polymer-conjugated lipids. Polymer-conjugated lipids mainly refer to polyethylene glycol (PEG)-modified lipids. Hydrophilic PEG stabilizes LNPs, regulates nanoparticle size by limiting lipid fusion, and extends the half-life of nanoparticles by reducing nonspecific interactions with macrophages.

[0122] In one embodiment, the polymer-conjugated lipid is one or more selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. The molecular weight of the PEG-modified PEG is typically 350 to 5,000 Da.

[0123] For example, the polymer-conjugated lipid is one or more selected from distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), dimyristoylglycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG2000), and methoxypolyethylene glycol ditetradecylacetamide (ALC-0159).

[0124] In one embodiment of the composition / carrier of the present invention, the polymer-conjugated lipid is DMG-PEG2000.

[0125] In one embodiment of the composition / carrier of the present invention, the carrier comprises a neutral lipid, a structural lipid, and a polymer-conjugated lipid, and the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is (25-75):(5-25):(15-65):(0.5-10), for example, (35-49):(7.5-15):(35-55):(1-5).

[0126] In one embodiment of the composition / carrier of the present invention, the carrier comprises a neutral lipid, a structural lipid, and a polymer-conjugated lipid, wherein the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is 49:10:39.5:1.5, or 40:10:48.5:1.5, or 35:10:53.5:1.5.

[0127] Therapeutic and / or prophylactic agents The composition may comprise one or more therapeutic and / or prophylactic agents. In one embodiment, the weight ratio of carrier to therapeutic or prophylactic agent is 10:1 to 30:1, e.g., 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.

[0128] In one embodiment, the mass ratio of the carrier to the therapeutic or prophylactic agent is 12.5:1 to 20:1, preferably 15:1.

[0129] The therapeutic or prophylactic agent may include, but is not limited to, one or more of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein.

[0130] For example, the therapeutic or prophylactic agent is a vaccine or a compound capable of eliciting an immune response.

[0131] The carriers of the present invention are capable of delivering therapeutic and / or prophylactic agents to mammalian cells or organs. Accordingly, the present invention also provides methods for treating a disease or disorder in a mammal in need thereof, comprising administering to the mammal a composition comprising a therapeutic and / or prophylactic agent and / or contacting mammalian cells with the composition.

[0132] Therapeutic and / or prophylactic agents include biologically active substances and are also referred to as "active agents." Therapeutic and / or prophylactic agents are substances that, when delivered to a cell or organ, can induce a desired change in that cell or organ, or other body tissue or system. Such substances may be used to treat one or more diseases, disorders, or conditions. In some embodiments, therapeutic and / or prophylactic agents are small molecule drugs useful for treating a particular disease, disorder, or condition. Examples of drugs that can be used in the composition include anti-tumorigenic agents (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, and streptozotocin), anti-tumor agents (e.g., actinomycin D, vincristine, vinblastine, cytosine arabinoside,arabinosides, anthracyclines, alkylating agents, platinum-based compounds, antimetabolites, and nucleoside analogues, such as methotrexate, and purine and pyrimidine analogues), anti-infectives, local anesthetics (e.g., dibucaine and chlorpromazine), β-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), anti-inflammatory drugs (e.g., steroids, steroid drugs, steroid agonists ... Histamines (e.g., diphenhydramine, chlorpheniramine, and promethazine), antibiotics / antibacterial agents (e.g., gentamycin, ciprofloxacin, and cefoxitin), antifungals (e.g., miconazole, terconazole, econazole, isoconazole, butaconazole, clotrimazole, itraconazole, nystatin, naftifine, and amphotericin B), B)) including, but not limited to, anthelmintics, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma drugs, vitamins, sedatives, and contrast media.

[0133] In some embodiments, the therapeutic and / or prophylactic agent is a cytotoxin, a radioactive ion, a chemotherapeutic agent, a vaccine, a compound that elicits an immune response, and / or another therapeutic and / or prophylactic agent. Cytotoxins or cytotoxic agents include any substance that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, and the like. 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 against one or more pathologies associated with infectious diseases such as influenza, measles, human papillomavirus (HPV), rabies, meningitis, whooping cough, tetanus, plague, hepatitis, and tuberculosis, and may include mRNA encoding antigens and / or epitopes from infectious diseases. Vaccines may also include compounds and preparations that induce an immune response against cancer cells, and may include mRNA encoding antigens, epitopes, and / or neoepitopes from tumor cells.Compounds that elicit an immune response may include vaccines, corticosteroids (e.g., dexamethasone), and other substances. In some embodiments, vaccines and / or compounds that can elicit an immune response by intramuscular administration of a composition comprising a compound according to Formula (I), (IA), (IB), (II), (IIa), (IIb), (IIc), (IId), (IIe), (IIf), (IIg), or (III) (e.g., compounds 3, 18, 20, 25, 26, 29, 30, 60, 108-112, or 122). Other therapeutic and / or prophylactic agents include antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, and 5-dacarbazine), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, rachelmycin (CC-1065), melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, bromomannitol, Antibiotics include, but are not limited to, cis-dichlorodiamineplatinum(II) (DDP), cisplatin, anthracyclines (e.g., daunorubicin (formerly known as daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly known as actinomycin), bleomycin, mithramycin, and azithromycin (anthramycin, AMC)), and antimitotic agents (e.g., vincristine, vinblastine, taxol, and maytansinoids).

[0134] In other embodiments, the therapeutic and / or prophylactic agent is a protein. 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.

[0135] In some embodiments, the therapeutic agent is a polynucleotide or nucleic acid (e.g., ribonucleic acid or deoxyribonucleic acid). The term "polynucleotide," in its broadest sense, includes any compound and / or substance that can be presented as or incorporated into an oligonucleotide chain. Exemplary polynucleotides used in the present invention include, but are not limited to, one or more of deoxyribonucleic acid (DNA); ribonucleic acid (RNA), such as messenger mRNA (mRNA), hybrids thereof; RNAi inducers; RNAi factors; siRNA; shRNA; miRNA; antisense RNA; ribozymes; catalytic DNA; RNA that induces triple helix formation; aptamers, etc. In some embodiments, the therapeutic and / or prophylactic agent is RNA. RNA that can be used in the compositions and methods described herein can be selected from the group consisting of shortmers, antagomirs, antisense RNA, ribozymes, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), short hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), and mixtures thereof. In some embodiments, the RNA is mRNA.

[0136] In some embodiments, the therapeutic and / or prophylactic agent is an mRNA. The mRNA can encode any polypeptide of interest, including naturally occurring or non-naturally occurring or otherwise modified polypeptides. The polypeptide encoded by the mRNA can be of any size and can have any secondary structure or activity. In some embodiments, the polypeptide encoded by the mRNA can exert a therapeutic effect when expressed in a cell.

[0137] In other embodiments, the therapeutic and / or prophylactic agent is an siRNA. The siRNA can selectively reduce the expression of a gene of interest or downregulate the expression of the gene. For example, the siRNA can be selected to silence a gene associated with a particular disease, disorder, or condition when a composition containing the siRNA is administered to a subject in need thereof. The siRNA may comprise a sequence complementary to the mRNA sequence encoding the gene or protein of interest. In some embodiments, the siRNA may be an immunomodulatory siRNA.

[0138] In some embodiments, the therapeutic and / or prophylactic agent is sgRNA and / or cas9 mRNA. sgRNA and / or cas9 mRNA can be used as a gene editing tool. For example, sgRNA-cas9 complexes can affect mRNA translation of cellular genes.

[0139] In some embodiments, therapeutic and / or prophylactic agent is shRNA, or its encoding carrier or plasmid.shRNA can be produced in target cell after appropriate construct is delivered into nucleus.The construct and mechanism related to shRNA are well known in the relevant art.

[0140] Disease or disorder The compositions / carriers of the present invention may deliver therapeutic or prophylactic agents to subjects or patients. The therapeutic or prophylactic agents include, but are not limited to, one or more of nucleic acid molecules, small molecule compounds, polypeptides, or proteins. Therefore, the compositions of the present invention can be used to prepare nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides, or protein drugs. Since the above therapeutic or prophylactic agents are diverse, the compositions of the present invention can be used to treat or prevent many diseases or disorders.

[0141] In some embodiments, the disease or disorder is characterized by dysfunctional or abnormal protein or polypeptide activity.

[0142] 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 renovascular diseases, and metabolic diseases.

[0143] In some embodiments, the infectious disease is selected from the group consisting of diseases caused by coronavirus, influenza virus, or HIV virus, childhood pneumonia, Rift Valley fever, yellow fever, rabies, and several types of herpes.

[0144] Other ingredients The composition may include one or more other ingredients in addition to those described in the previous section. For example, the composition may include one or more hydrophobic small molecules such as vitamins (e.g., vitamin A or vitamin E) or sterols.

[0145] The composition may also include one or more permeability-enhancing molecules, carbohydrates, polymers, surface modifiers, or other components. Permeability-enhancing molecules may be, for example, molecules described in U.S. Patent Application Publication No. 2005 / 0222064. Carbohydrates may include monosaccharides (e.g., glucose) and polysaccharides (e.g., glycogen and its derivatives and analogs).

[0146] Surface modifiers include anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants, e.g., dimethyldioctadecylammonium bromide), sugars or sugar derivatives (e.g., cyclodextrins), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytics (e.g., acetylcysteine, mugwort, bromelain, papain, clerodendrum, bromhexine, The surface modifier may include, but is not limited to, romhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin β4, dornase alfa, neltenexine, and erdosteine, and DNA enzymes (e.g., rhDNA enzyme). The surface modifier may be disposed within and / or on the surface of the nanoparticles of the composition (e.g., by coating, adsorption, covalent bonding, or other methods).

[0147] The composition may also comprise one or more functionalized lipids. For example, the lipid may be functionalized with an alkynyl, which can undergo cycloaddition when exposed to an azide compound under suitable reaction conditions. Specifically, the lipid bilayer may be functionalized with one or more groups that are effective for promoting membrane permeation, cell recognition, or imaging. The surface of the composition may be bound to one or more useful antibodies. Functional groups and conjugates useful for target cell delivery, imaging, and membrane permeation are well known in the art.

[0148] In addition to these ingredients, the composition may contain any substance useful in pharmaceutical compositions.For example, the composition may contain one or more pharmaceutically acceptable excipients or auxiliary components, such as one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, granulation aids, disintegrants, fillers, glidants, liquid media, binders, surfactants, isotonicity agents, thickeners or emulsifiers, buffers, lubricants, oils, preservatives, flavoring agents, coloring agents, etc.Excipients are, for example, starch, lactose, or dextrin.Pharmaceutically acceptable excipients are well known in the art (see, for example, Remington's The Science and Practice of Pharmacy, 21st Edition, ARGennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006).

[0149] Examples of diluents 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.

[0150] In some embodiments, compositions comprising one or more lipids described herein may further comprise one or more adjuvants such as glucopyranosyl lipid adjuvant (GLA), CpG oligodeoxyribonucleotides (e.g., class A or class B), poly(I:C), aluminum hydroxide, and Pam3CSK4.

[0151] The compositions of the present invention can be prepared in the form of solid, semi-solid, liquid, or gaseous formulations such as tablets, capsules, ointments, elixirs, syrups, solutions, emulsions, suspensions, injections, and aerosols. The compositions 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 a therapeutic or preventive agent and the other necessary ingredients listed above in an appropriate solvent such as sterile distilled water, followed by filtration sterilization. A surfactant can also be added to promote the formation of a homogeneous solution or suspension.

[0152] For example, the compositions of the present invention may be administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation. In one embodiment, the composition is administered subcutaneously.

[0153] The compositions of the present invention are administered in a therapeutically effective amount, which may vary depending not only on the specific reagent selected but also on the route of administration, the nature of the disease being treated, and the age and condition of the patient, and which can ultimately be determined by the attending physician or clinician. For example, a therapeutic or prophylactic agent may be administered to a mammal (e.g., a human) in a dose of about 0.001 mg / kg to about 10 mg / kg. [Example]

[0154] The present invention will be further described below with reference to examples. However, the present invention is not limited to the following examples. The operating conditions used in the examples can be further adjusted according to different requirements of specific applications, and operating conditions not specified are conventional conditions in the industry. In the specific examples of the present invention, all raw materials used are commercially available. Unless otherwise specified, the percentages above and below are percentages by weight, and all temperatures are in °C. The technical features included in various embodiments of the present invention can be combined with each other unless they are mutually inconsistent.

[0155] Example 1: Synthesis of cationic lipid compounds 1. Synthesis of YK-401 Synthesis scheme: [ka] Synthesis of 6,6'-(ethane-1,2-diyl-bis((2-hydroxyethyl)azanediyl))dihexanoic acid di(heptadecan-9-yl) ester (YK-401) N,N'-bis(hydroxyethyl)ethylenediamine (28 mg, 0.19 mmol) and 6-bromohexanoic acid heptadecan-9-yl ester (200 mg, 0.48 mmol) were dissolved in acetonitrile (3 mL). Potassium carbonate (79 mg, 0.57 mmol) was added to the above system, heated to 75°C, and stirred for 5 hours. After the reaction was completed, 20 mL of water was added to the reaction solution, followed by extraction with ethyl acetate (20 mL x 2). The combined organic phases were washed with brine (20 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel chromatography (dichloromethane / methanol) to obtain the target compound (62 mg, 0.07 mmol, 38.2%). 52 H 104 N2O6, MS(ES): m / z (M+H + )853.8. 1 H NMR (400 MHz, chloroform-d) δ 4.86 (p, J = 6.1 Hz, 2H), 3.66 (s, 4H), 2.71 (s, 12H), 2.29 (t, J = 7.4 Hz, 4H), 1.67 - 1.46 (m, 16H), 1.26 (s, 52H), 0.88 (t, J = 6.7 Hz, 12H).

[0156] 2. Synthesis of YK-402 Synthesis scheme: [ka] Step 1: Synthesis of 2-octyldecanoic acid 6-bromohexyl ester (YK-402-PM1) 2-Octyldecanoic acid (300 mg, 1.05 mmol) and 6-bromohexanol (202.5 mg, 1.12 mmol) were dissolved in dichloromethane (5 mL). 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (404.4 mg, 2.11 mmol) and 4-dimethylaminopyridine (64.4 mg, 0.53 mmol) were added to the solution and stirred at 30-35 °C for 5 hours. After completion of the reaction, the reaction solution was washed with saturated sodium bicarbonate, washed with saturated brine, and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography (ethyl acetate / n-hexane) to obtain YK-402-PM1 (478.2 mg, 1.06 mmol, 100%). Step 2: Synthesis of (ethane-1,2-diyl-bis((2-hydroxyethyl)azanediyl))bis(hexane-6,1-diyl)bis(2-octyldecanoate) (YK-402) Using YK-402-PM1 (447.5 mg, 0.67 mmol) and N,N'-bis(hydroxyethyl)ethylenediamine (39.7 mg, 0.27 mmol) as starting materials, YK-402 (53.10 mg, 0.06 mmol, 22.3%) was obtained according to the synthesis method for YK-401. 54 H 108 N2O6, MS(ES): m / z (M+H + )881.8. 1 H NMR (400 MHz, chloroform-d) δ 4.06 (t, J = 6.7 Hz, 4H), 3.68 (s, 4H), 2.74 (s, 12H), 2.31 (tt, J = 8.8, 5.3 Hz, 2H), 1.59 (dq, J = 22.3, 6.8 Hz, 12H), 1.25 (s, 60H), 0.88 (t, J = 6.7 Hz, 12H).

[0157] 3. Synthesis of YK-403 Synthesis scheme: [ka] Step 1: Synthesis of 8-bromooctanoic acid heptadecan-9-yl ester (YK-403-PM1) Using 9-heptadecanol (3.73 g, 14.53 mmol) and 8-bromooctanoic acid (2.70 g, 12.11 mmol) as raw materials, YK-403-PM1 (4.43 g, 9.60 mmol, 79.3%) was obtained according to the synthesis method of YK-402-PM1. Step 2: Synthesis of 8,8'-(ethane-1,2-diyl-bis((2-hydroxyethyl)azanediyl))dioctanoic acid di(heptadecan-9-yl) ester (YK-403) Using YK-403-PM1 (233.6 mg, 0.51 mmol) and N,N'-bis(hydroxyethyl)ethylenediamine (30.0 mg, 0.20 mmol) as starting materials, YK-403 (98.7 mg, 0.11 mmol, 54.3%) was obtained according to the synthesis method for YK-401. 56 H 112 N2O6, MS(ES): m / z (M+H + )909.8. 1 H NMR (400 MHz, chloroform-d) δ 4.86 (p, J = 6.2 Hz, 2H), 3.64 - 3.57 (m, 4H), 2.61 (dd, J = 9.9, 5.1 Hz, 8H), 2.55 - 2.47 (m, 4H), 2.27 (t, J = 7.5 Hz, 4H), 1.65 - 1.57 (m, 4H), 1.55 - 1.44 (m, 12H), 1.28 (d, J = 16.0 Hz, 62H), 0.88 (t, J = 6.8 Hz, 12H).

[0158] 4. Synthesis of YK-404 Synthesis scheme: [ka] Step 1: Synthesis of 9-bromononanoic acid octan-3-yl ester (YK-404-PM1) Using octan-3-ol (65.9 mg, 0.51 mmol) and 9-bromononanoic acid (100 mg, 0.42 mmol) as raw materials, YK-404-PM1 (114.9 mg, 0.33 mmol, 78.3%) was obtained according to the synthesis method of YK-402-PM1. Step 2: Synthesis of 9,9'-(ethane-1,2-diyl-bis((2-hydroxyethyl)azanediyl))dinonanoic acid di(octan-3-yl) ester (YK-404) YK-404-PM1 (114.9 mg, 0.33 mmol) and N,N'-bis(hydroxyethyl)ethylenediamine (19.5 mg, 0.13 mmol) were used as starting materials, and YK-404 (30.9 mg, 0.05 mmol, 34.7%) was obtained according to the synthesis method for YK-401. 40 H 80 N2O6, MS(ES): m / z (M+H + )685.6. 1 H NMR (400 MHz, chloroform-d) δ 4.85 (p, J = 6.5 Hz, 2H), 3.79 (s, 4H), 2.87 (s, 12H), 2.32 (t, J = 7.5 Hz, 4H), 1.73 - 1.53 (m, 16H), 1.35 - 1.29 (m, 28H), 0.94 - 0.89 (m, 12H).

[0159] 5. Synthesis of YK-405 Synthesis scheme: [ka] Step 1: Synthesis of decyl 4-((2-hydroxyethyl)(2-((2-hydroxyethyl)amino)ethyl)amino)butyrate (YK-405-PM1) N,N'-bis(hydroxyethyl)ethylenediamine (434.1 mg, 2.93 mmol) and 4-bromobutyric acid-n-decyl ester (300 mg, 0.98 mmol) were dissolved in acetonitrile (4 mL). Potassium carbonate (404.8 mg, 2.93 mmol) was added to the above system, heated to 70 °C, and stirred for 4 hours. After the reaction was completed, 20 mL of water was added to the reaction solution, followed by extraction with dichloromethane (20 mL x 2). The combined organic phases were then washed with saturated aqueous sodium bicarbonate (20 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel chromatography (dichloromethane / methanol (2% triethylamine)) to obtain YK-405-PM1 (179.5 mg, 0.48 mmol, 48.9%). 20 H 42 N2O4, MS(ES): m / z (M+H + )375.3. Step 2: Synthesis of 6-((2-hydroxyethyl)(2-((2-hydroxyethyl)(4-(decyloxy)-4-oxobutyl)amino)ethyl)amino)hexanoic acid heptadecan-9-yl ester (YK-405) YK-405-PM1 (160 mg, 0.43 mmol) and 6-bromohexanoic acid-heptadecan-9-yl ester (215.0 mg, 0.52 mmol) were used as starting materials, and YK-405 (42.5 mg, 0.06 mmol, 13.6%) was obtained according to the synthesis method for YK-401. 43 H 86 N2O6, MS(ES): m / z (M+H + )727.6. 1H NMR (400 MHz, chloroform-d) δ 4.96 - 4.69 (m, 1H), 4.06 (t, J = 6.8 Hz, 2H), 3.78 - 3.72 (m, 2H), 3.72 - 3.65 (m, 2H), 3.45 (s, 4H), 2.88 - 2.79 (m, 6H), 2.78 - 2.64 (m, 4H), 2.32 (dt, J = 19.1, 7.2 Hz, 4H), 1.84 (p, J = 7.1 Hz, 2H), 1.63 (dq, J = 13.9, 7.4 Hz, 6H), 1.53 - 1.44 (m, 4H), 1.26 (s, 40H), 0.88 (t, J = 6.6 Hz, 9H).

[0160] 6.Synthesis of YK-406 Synthesis scheme: [ka] Step 1: Synthesis of 3-hexylnonanoic acid-6-(2-hydroxyethyl)(2-((2-hydroxyethyl)amino)ethyl)amino)hexyl ester (YK-406-PM1) Using N,N'-bis(hydroxyethyl)ethylenediamine (183.7 mg, 1.24 mmol) and 3-hexylnonanoic acid-6-bromohexyl ester (200 mg, 0.49 mmol) as starting materials, YK-406-PM1 (134.7 mg, 0.28 mmol, 58.1%) was obtained according to the synthesis method for YK-405-PM1. 27 H 56 N2O4, MS(ES): m / z (M+H + )473.4. Step 2: Synthesis of 3-hexylnonanoic acid 6-((2-hydroxyethyl)(2-((2-hydroxyethyl)(4-(undecyloxy)-4-oxobutyl)amino)ethyl)amino)-hexyl ester (YK-406) YK-406-PM1 (134.7 mg, 0.28 mmol) and 4-bromobutyric acid-n-decyl ester (149.9 mg, 0.49 mmol) were used as starting materials, and YK-406 (41.5 mg, 0.06 mmol, 20.8%) was obtained according to the synthesis method for YK-401. 42 H 84 N2O6, MS(ES): m / z (M+H + )713.6. 1 H NMR (400 MHz, chloroform-d) δ 4.05 (q, J = 6.5 Hz, 4H), 3.68 - 3.50 (m, 4H), 2.76 - 2.48 (m, 12H), 2.32 (t, J = 7.1 Hz, 2H), 2.22 (d, J = 6.9 Hz, 2H), 1.81 (dt, J = 15.0, 7.2 Hz, 3H), 1.68 - 1.56 (m, 4H), 1.50 (d, J = 7.3 Hz, 2H), 1.28 (d, J = 15.8 Hz, 40H), 0.88 (t, J = 6.6 Hz, 9H).

[0161] 7.Synthesis of YK-407 Synthesis scheme: [ka] Step 1: Synthesis of undecyl 4-((2-hydroxyethyl)(2-((2-hydroxyethyl)amino)ethyl)amino)butyrate (YK-407-PM1) Using N,N'-bis(hydroxyethyl)ethylenediamine (691.9 mg, 4.67 mmol) and 4-bromobutyric acid undecyl ester (500 mg, 1.56 mmol) as starting materials, YK-407-PM1 (271 mg, 0.70 mmol, 44.7%) was obtained according to the synthesis method for YK-405-PM1. 21 H 44 N2O4, MS(ES): m / z (M+H + )389.4. Step 2: Synthesis of 6-((2-hydroxyethyl)(2-(2-hydroxyethyl)(4-oxo-4-(undecyloxy)butyl)amino)ethyl)amino)hexanoic acid heptadecan-9-yl ester (YK-407) YK-407-PM1 (271 mg, 0.70 mmol) and 6-bromohexanoic acid heptadecan-9-yl ester (453.5 mg, 1.05 mmol) were used as starting materials, and YK-407 (129 mg, 0.17 mmol, 24.3%) was obtained according to the synthesis method for YK-401. 44 H 88 N2O6, MS(ES): m / z (M+H + )741.7. 1 H NMR (400 MHz, CDCl3) δ 4.93 - 4.84 (m, 1H), 4.09 (t, J = 6.8 Hz, 2H), 3.74 (s, 4H), 2.80 (s, 10H), 2.39 - 2.30 (m, 4H), 1.89 (s, 2H), 1.67 (dt, J = 14.9, 7.6 Hz, 6H), 1.54 (d, J = 5.3 Hz, 4H), 1.29 (s, 44H), 0.92 (d, J = 6.5 Hz, 9H).

[0162] 8.Synthesis of YK-408 Synthesis scheme: [ka] Synthesis of 4-((2-hydroxyethyl)(2-((2-hydroxyethyl)(4-(decyloxy)-4-oxobutyl)amino)ethyl)amino)butyric acid heptadecan-9-yl ester (YK-408) Using YK-405-PM1 (276 mg, 0.74 mmol) and 4-bromobutyric acid heptadecan-9-yl ester (537.8 mg, 1.33 mmol) as starting materials, YK-408 (80 mg, 0.11 mmol, 15.5%) was obtained according to the synthesis method for YK-401. 41 H 82 N2O6, MS(ES): m / z (M+H + )699.6. 1 H NMR (400 MHz, chloroform-d) δ 5.89 (s, 2H), 5.57 - 5.34 (m, 1H), 4.65 (t, J = 6.7 Hz, 2H), 4.19 (t, J = 4.4 Hz, 4H), 3.20 (td, J = 15.1, 13.0, 6.2 Hz, 12H), 2.90 (q, J = 7.0 Hz, 4H), 2.40 (p, J = 7.1 Hz, 4H), 2.25 - 2.17 (m, 2H), 2.15 - 2.06 (m, 4H), 1.85 (s, 36H), 1.47 (t, J = 6.4 Hz, 9H).

[0163] 9.Synthesis of YK-409 Synthesis scheme: [ka] Step 1: Synthesis of 6-(2-hydroxyethyl)(2-((2-hydroxyethyl)amino)ethyl)amino)hexanoic acid 2-octyldecyl ester (YK-409-PM1) Using N,N'-bis(hydroxyethyl)ethylenediamine (298.0 mg, 2.01 mmol) and 6-bromohexanoic acid-2-octyldecyl ester (300 mg, 0.67 mmol) as starting materials, YK-409-PM1 (144.7 mg, 0.28 mmol, 41.9%) was obtained according to the synthesis method for YK-405-PM1. 30 H 62 N2O4, MS(ES): m / z (M+H + )515.4. Step 2: Synthesis of 6-((2-hydroxyethyl)(2-((2-hydroxyethyl)(4-(undecyloxy)-4-oxobutyl)amino)ethyl)amino)hexanoic acid 2-octyldecyl ester (YK-409) Using YK-409-PM1 (144.7 mg, 0.28 mmol) and 4-bromobutyric acid-undecyl ester (108.4 mg, 0.34 mmol) as starting materials, YK-409 (54.6 mg, 0.07 mmol, 25.8%) was obtained according to the synthesis method for YK-401. 45 H 90 N2O6, MS(ES): m / z (M+H + )755.7. 1 H NMR (400 MHz, chloroform-d) δ 4.06 (t, J = 6.8 Hz, 2H), 3.96 (d, J = 5.8 Hz, 2H), 3.65 - 3.56 (m, 4H), 2.70 - 2.46 (m, 12H), 2.31 (td, J = 7.3, 3.7 Hz, 4H), 1.81 (p, J = 7.3 Hz, 2H), 1.70 - 1.56 (m, 5H), 1.51 (p, J = 7.7 Hz, 2H), 1.28 (d, J = 15.2 Hz, 46H), 0.88 (t, J = 6.7 Hz, 9H).

[0164] 10.Synthesis of YK-410 Synthesis scheme: [ka] Synthesis of 6-((2-hydroxyethyl)(2-((2-hydroxyethyl)(6-(decyloxy)-6-oxohexyl)amino)ethyl)amino)hexanoic acid 2-octyldecyl ester (YK-410) Using YK-409-PM1 (195.0 mg, 0.38 mmol) and n-decyl 6-bromohexanoate (190.5 mg, 0.57 mmol) as starting materials, YK-410 (141 mg, 0.18 mmol, 48.2%) was obtained according to the synthesis method for YK-401. 46 H 92 N2O6, MS(ES): m / z (M+H + )769.7. 1H NMR (400 MHz, chloroform-d) δ 4.05 (t, J = 6.4 Hz, 2H), 3.96 (d, J = 5.4 Hz, 2H), 3.61 (s, 4H), 2.63 (d, J = 9.0 Hz, 8H), 2.58 - 2.50 (m, 4H), 2.30 (s, 4H), 1.77 - 1.57 (m, 8H), 1.55 - 1.46 (m, 4H), 1.26 (s, 44H), 0.87 (d, J = 6.3 Hz, 9H).

[0165] 11.Synthesis of YK-411 Synthesis scheme: [ka] Step 1: Synthesis of 6-(2-hydroxyethyl)(2-((2-hydroxyethyl)amino)ethyl)amino)hexanoic acid 3-hexylnonyl ester (YK-411-PM1) Using N,N'-bis(hydroxyethyl)ethylenediamine (329.0 mg, 2.22 mmol) and 6-bromohexanoic acid-3-hexylnonyl ester (300 mg, 0.74 mmol) as starting materials, YK-411-PM1 (103.1 mg, 0.22 mmol, 29.4%) was obtained according to the synthesis method for YK-405-PM1. 27 H 56 N2O4, MS(ES): m / z (M+H + )473.4. Step 2: Synthesis of 6-((2-hydroxyethyl)(2-((2-hydroxyethyl)(6-(decyloxy)-6-oxobutyl)amino)ethyl)amino)hexanoic acid 3-hexylnonyl ester (YK-411) Using YK-411-PM1 (103.1 mg, 0.22 mmol) and n-decyl 6-bromohexanoate (76.6 mg, 0.23 mmol) as starting materials, YK-411 (68.6 mg, 0.09 mmol, 42.9%) was obtained according to the synthesis method for YK-401. 43 H 86 N2O6, MS(ES): m / z (M+H + )727.7. 1 H NMR (400 MHz, chloroform-d) δ 4.07 (q, J = 7.0 Hz, 4H), 3.64 (t, J = 6.3 Hz, 4H), 2.69 (s, 8H), 2.30 (t, J = 7.0 Hz, 4H), 1.58 (ddp, J = 32.0, 15.4, 7.2 Hz, 13H), 1.28 (d, J = 15.0 Hz, 42H), 0.88 (t, J = 6.4 Hz, 9H).

[0166] 12.Synthesis of YK-412 Synthesis scheme: [ka] Step 1: Synthesis of 2-octyldecanoic acid-6-(2-hydroxyethyl-amino)hexyl ester (YK-412-PM1) YK-412-PM1 (165 mg, 0.39 mmol, 68.9%) was obtained using 2-octyldecanoic acid-6-bromohexyl ester (250.0 mg, 0.56 mmol) and ethanolamine (136.5 mg, 2.23 mmol) according to the synthesis method for YK-401. 26 H 53 NO3, MS(ES): m / z (M+H + )428.4. Step 2: Synthesis of (propane-1,3-diyl-bis((2-hydroxyethyl)azanediyl))-bis(hexane-6,1-diyl)bis(2-octyldecanoate) (YK-412) Using YK-412-PM1 (165 mg, 0.39 mmol) and 1,3-dibromopropane (42.8 mg, 0.21 mmol) as starting materials, YK-412 (94 mg, 0.10 mmol, 53.8%) was obtained according to the synthesis method for YK-401. 55 H 110 N2O6, MS(ES): m / z (M+H + )895.8. 1H NMR (400 MHz, chloroform-d) δ 4.06 (t, J = 6.6 Hz, 4H), 3.61 (s, 4H), 2.60 (d, J = 15.9 Hz, 8H), 2.51 (s, 4H), 2.32 (dt, J = 9.9, 5.1 Hz, 2H), 1.61 (tt, J = 14.4, 7.0 Hz, 12H), 1.25 (s, 62H), 0.88 (t, J = 6.6 Hz, 12H).

[0167] 13.Synthesis of YK-413 Synthesis scheme: [ka] Step 1: Synthesis of undecyl 4-((3-(tert-butyloxycarbonyl)amino)propyl)aminobutyrate (YK-413-PM1) Using tert-butyl (3-aminopropyl)carbamate (4.88 g, 28.01 mmol) and undecyl 4-bromobutyrate (3.00 g, 9.34 mmol) as starting materials, YK-413-PM1 (2.00 g, 4.81 mmol, 51.5%) was obtained according to the synthesis method for YK-401. 23 H 46 N2O4, MS(ES): m / z (M+H + )415.5. Step 2: Synthesis of undecyl 4-((3-(tert-butyloxycarbonyl)amino)propyl)(2-hydroxyethyl)amino)butyrate (YK-413-PM2) Using YK-413-PM1 (2.00 g, 4.81 mmol) and bromoethanol (1.80 g, 14.40 mmol) as raw materials, YK-413-PM2 (2.20 g, 4.80 mmol, 99.8%) was obtained according to the synthesis method for YK-401. 25 H 50 N2O5, MS(ES): m / z (M+H + )459.4. Step 3: Synthesis of undecyl 4-(3-aminopropyl)((2-hydroxyethyl)amino)butyrate (YK-413-PM3) YK-413-PM2 (2.20 g, 4.80 mmol) was dissolved in tetrahydrofuran (10 mL), the system temperature was controlled at 0°C, and 4 M hydrogen chloride dioxane solution (12 mL) was slowly added dropwise. After the addition was complete, the temperature was slowly raised to room temperature and the reaction was allowed to proceed for 2 hours. After the reaction was complete, saturated sodium bicarbonate solution was added dropwise to adjust the pH to 7-8. The aqueous phase was washed with dichloromethane (20 mL x 2), the layers were separated, the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent, yielding YK-413-PM3 (1.09 g, 3.05 mmol, 63.5%). 20 H 42 N2O3, MS(ES): m / z (M+H + )359.3. Step 4: Synthesis of 6-((3-(2-hydroxyethyl)(4-oxo-4-(undecyloxy)butyl)amino)propyl)amino)hexanoic acid heptadecan-9-yl ester (YK-413-PM4) YK-413-PM4 (398.9 mg, 0.56 mmol, 41.8%) was obtained using YK-413-PM3 (480 mg, 1.34 mmol) and 6-bromohexanoic acid heptadecan-9-yl ester (580.3 mg, 1.34 mmol) according to the synthesis method for YK-401. 43 H 86 N2O5, MS(ES): m / z (M+H + )711.7. Step 5: Synthesis of 6-((2-hydroxyethyl)(3-((2-hydroxyethyl)(4-oxo-4-(undecyloxy)butyl)amino)propyl)amino)hexanoic acid heptadecan-9-yl ester (YK-413) YK-413-PM4 (398.9 mg, 0.56 mmol) and bromoethanol (310.2 mg, 2.48 mmol) were used as starting materials, and YK-413 (104.8 mg, 0.14 mmol, 24.8%) was obtained according to the synthesis method for YK-401. 45 H 90 N2O6, MS(ES): m / z (M+H + )755.8. 1H NMR (400 MHz, CDCl3) δ 4.90 - 4.81 (m, 1H), 4.06 (t, J = 6.8 Hz, 2H), 3.67 (s, 4H), 2.70 (s, 12H), 2.37 - 2.28 (m, 4H), 1.85 - 1.79 (m, 2H), 1.68 - 1.58 (m, 6H), 1.50 (d, J = 5.3 Hz, 4H), 1.26 (s, 44H), 0.88 (t, J = 6.6 Hz, 9H).

[0168] 14.Synthesis of YK-414 Synthesis scheme: [ka] Step 1: Synthesis of decyl 4-((3-(tert-butyloxycarbonyl)amino)propyl)aminobutyrate (YK-414-PM1) Using tert-butyl (3-aminopropyl)carbamate (577.8 mg, 3.32 mmol) and n-decyl 4-bromobutyrate (1.00 g, 3.25 mmol) as starting materials, YK-414-PM1 (760 mg, 1.90 mmol, 58.4%) was obtained according to the synthesis method for YK-401. 22 H 44 N2O4, MS(ES): m / z (M+H + )401.4. Step 2: Synthesis of decyl 4-((3-(tert-butyloxycarbonyl)amino)propyl)(2-hydroxyethyl)amino)butyrate (YK-414-PM2) Using YK-414-PM1 (760 mg, 1.90 mmol) and bromoethanol (284.0 mg, 2.27 mmol) as starting materials, YK-414-PM2 (600 mg, 1.35 mmol, 71.0%) was obtained according to the synthesis method for YK-401. 24 H 48 N2O5, MS(ES): m / z (M+H + )445.3. Step 3: Synthesis of decyl 4-((3-aminopropyl)(2-hydroxyethyl)amino)butyrate (YK-414-PM3) Using YK-414-PM2 (600 mg, 1.35 mmol) as a starting material, YK-414-PM3 (465 mg, 1.35 mmol, 100%) was obtained according to the synthesis method for YK-413-PM3. 19 H 40 N2O3, MS(ES): m / z (M+H + )345.3. Step 4: Synthesis of decyl 4-((3-(heptadecan-9-yloxy)-4-oxobutyl)amino)propyl)(2-hydroxyethyl)amino)butyrate (YK-414-PM4) YK-414-PM4 (308.0 mg, 0.46 mmol, 46.0%) was obtained using YK-414-PM3 (344.5 mg, 1.00 mmol) and 4-bromobutyric acid-heptadecan-9-yl ester (405.5 mg, 1.00 mmol) as starting materials according to the synthesis method for YK-401. 40 H 80 N2O5, MS(ES): m / z (M+H + )669.6. Step 5: Synthesis of decyl 4-(3-((4-(heptadecan-9-yloxy)-4-oxobutyl)(2-hydroxyethyl)amino)propyl)(2-hydroxyethyl)aminobutyrate (YK-414) YK-414-PM4 (308.0 mg, 0.46 mmol) and bromoethanol (172.4 mg, 1.38 mmol) were used as starting materials, and YK-414 (80.0 mg, 0.11 mmol, 23.9%) was obtained according to the synthesis method for YK-401. 42 H 84 N2O6, MS(ES): m / z (M+H + )713.6. 1H NMR (400 MHz, CDCl3) δ 4.94 - 4.84 (m, 1H), 4.09 (t, J = 6.8 Hz, 2H), 3.74 (s, 4H), 2.79 (s, 12H), 2.38 (d, J = 6.5 Hz, 4H), 1.88 (s, 4H), 1.71 - 1.60 (m, 4H), 1.54 (s, 4H), 1.31 (d, J = 16.1 Hz, 38H), 0.91 (t, J = 6.6 Hz, 9H).

[0169] 15.Synthesis of YK-415 Synthesis scheme: [ka] Step 1: Synthesis of 6-((3-(2-hydroxyethyl)(4-oxo-4-(undecyloxy)butyl)amino)propyl)amino)hexanoic acid-2-octyldecyl ester (YK-415-PM1) Using YK-413-PM3 (300 mg, 0.84 mmol) and 6-bromohexanoic acid-2-octyldecyl ester (374.0 mg, 0.84 mmol) as starting materials, YK-415-PM1 (320.0 mg, 0.44 mmol, 52.4%) was obtained according to the synthesis method for YK-401. 44 H 88 N2O5, MS(ES): m / z (M+H + )725.7. Step 2: Synthesis of 6-((2-hydroxyethyl)(3-((2-hydroxyethyl)(4-oxo-4-(undecyloxy)butyl)amino)propyl)amino)hexanoic acid-2-octyldecyl ester (YK-415) Using YK-415-PM1 (320.0 mg, 0.44 mmol) and bromoethanol (200.0 mg, 1.60 mmol) as starting materials, YK-415 (37.0 mg, 0.05 mmol, 10.9%) was obtained according to the synthesis method for YK-401. 46 H 92 N2O6, MS(ES): m / z (M+H + )769.8. 1 H NMR (400 MHz, CDCl3) δ 4.07 (t, J = 6.5 Hz, 4H), 3.96 (d, J = 5.6 Hz, 2H), 3.30 (s, 4H), 3.16 (s, 2H), 2.48 (s, 2H), 2.33 (t, J = 7.0 Hz, 2H), 2.25 - 2.18 (m, 2H), 2.02 (s, 2H), 1.90 (s, 3H), 1.62 (s, 8H), 1.43 (s, 4H), 1.28 (d, J = 15.2 Hz, 48H), 0.87 (d, J = 7.0 Hz, 9H).

[0170] 16.Synthesis of YK-416 Synthesis scheme: [ka] Step 1: Synthesis of 6-(3-(tert-butyloxycarbonyl)amino)propyl)aminohexanoic acid decyl ester (YK-416-PM1) Using tert-butyl (3-aminopropyl)carbamate (1.14 g, 6.54 mmol) and n-decyl 6-bromohexanoate (2.20 g, 6.56 mmol) as starting materials, YK-416-PM1 (754 mg, 1.76 mmol, 26.9%) was obtained according to the synthesis method for YK-401. 24 H 48 N2O4, MS(ES): m / z (M+H + )429.3. Step 2: Synthesis of 6-((3-((tert-butyloxycarbonyl)amino)propyl)(2-hydroxyethyl)amino)hexanoic acid decyl ester (YK-416-PM2) Using YK-416-PM1 (754 mg, 1.76 mmol) and bromoethanol (284.0 mg, 2.27 mmol) as starting materials, YK-416-PM2 (620 mg, 1.31 mmol, 74.5%) was obtained according to the synthesis method for YK-401. 26 H 52 N2O5, MS(ES): m / z (M+H + )473.4. Step 3: Synthesis of 6-((3-aminopropyl)(2-hydroxyethyl)amino)hexanoic acid decyl ester (YK-416-PM3) Using YK-416-PM2 (620 mg, 1.31 mmol) as a starting material, YK-416-PM3 (488 mg, 1.31 mmol, 100%) was obtained according to the synthesis method for YK-413-PM3. 21 H 44 N2O3, MS(ES): m / z (M+H + )373.3. Step 4: Synthesis of 6-((2-hydroxyethyl)(3-((6-((2-octyldecyl)oxy)-6-oxohexyl)amino)propyl)amino)hexanoic acid decyl ester (YK-416-PM4) YK-416-PM4 (58 mg, 0.08 mmol, 12.6%) was obtained using YK-416-PM3 (244 mg, 0.65 mmol) and 6-bromohexanoic acid-2-octyldecyl ester (278.5 mg, 0.62 mmol) as starting materials according to the synthesis method for YK-401. 45 H 90 N2O5, MS(ES): m / z (M+H + )739.7. Step 5: Synthesis of 6-((2-hydroxyethyl)(3-((2-hydroxyethyl)(6-((2-octyldecyl)oxy)-6-oxohexyl)amino)propyl)amino)hexanoic acid decyl ester (YK-416) YK-416-PM4 (58 mg, 0.08 mmol) and bromoethanol (19.6 mg, 0.16 mmol) were used as starting materials, and YK-416 (30 mg, 0.04 mmol, 48.8%) was obtained according to the synthesis method for YK-401. 47 H 94 N2O6, MS(ES): m / z (M+H + )783.7. 1H NMR (400 MHz, CDCl3) δ 4.05 (t, J = 6.8 Hz, 2H), 3.96 (d, J = 5.8 Hz, 2H), 3.90 (s, 2H), 3.14 (s, 2H), 3.06 (s, 2H), 2.95 (s, 2H), 2.32 (t, J = 7.3 Hz, 4H), 2.26 - 2.16 (m, 2H), 2.02 (s, 1H), 1.73 (s, 4H), 1.69 - 1.59 (m, 8H), 1.44 - 1.36 (m, 4H), 1.28 (d, J = 14.6 Hz, 48H), 0.88 (t, J = 6.7 Hz, 9H).

[0171] 17.Synthesis of YK-417 Synthesis scheme: [ka] Step 1: Synthesis of 6-((3-((6-((3-hexyl)oxy)-6-oxohexyl)amino)propyl)(2-hydroxyethyl)amino)hexanoic acid decyl ester (YK-417-PM1) YK-416-PM3 (81.1 mg, 0.22 mmol) and 6-bromohexanoic acid 3-hexylnonyl ester (83.9 mg, 0.21 mmol) were used as starting materials, and YK-417-PM1 (44.3 mg, 0.06 mmol, 30.3%) was obtained according to the synthesis method for YK-401. 42 H 84 N2O5, MS(ES): m / z (M+H + )697.6. Step 2: Synthesis of 6-((3-((6-((3-hexyl)oxy)-6-oxohexyl)(2-hydroxyethyl)amino)propyl)(2-hydroxyethyl)amino)hexanoic acid decyl ester (YK-417) Using YK-417-PM1 (44.3 mg, 0.06 mmol) and bromoethanol (15.9 mg, 0.13 mmol) as starting materials, YK-417 (26.3 mg, 0.04 mmol, 59.1%) was obtained according to the synthesis method for YK-401. 44 H88 N2O6, MS(ES): m / z (M+H + )741.7. 1 H NMR (400 MHz, CDCl3) δ 4.06 (dd, J = 15.3, 8.1 Hz, 4H), 3.82 (s, 2H), 2.94 (d, J = 17.7 Hz, 6H), 2.82 (s, 2H), 2.36 - 2.26 (m, 4H), 2.03 (d, J = 11.7 Hz, 4H), 1.61 (ddd, J = 21.2, 14.6, 7.3 Hz, 12H), 1.43 - 1.20 (m, 43H), 0.88 (t, J = 6.4 Hz, 9H).

[0172] 18.Synthesis of YK-418 Synthesis scheme: [ka] Step 1: Synthesis of 3-hexylnonanoic acid 6-((3-((4-(decyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)propyl)amino)hexyl ester (YK-418-PM1) Using YK-414-PM3 (231.7 mg, 0.67 mmol) and 3-hexylnonanoic acid 6-bromohexyl ester (258.4 mg, 0.64 mmol) as starting materials, YK-418-PM1 (111.5 mg, 0.17 mmol, 26.0%) was obtained according to the synthesis method for YK-401. 40 H 80 N2O5, MS(ES): m / z (M+H + )669.6. Step 2: Synthesis of 3-hexylnonanoic acid 6-((3-((4-(decyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)propyl)(2-hydroxyethyl)amino)hexyl ester (YK-418) Using YK-418-PM1 (111.5 mg, 0.17 mmol) and bromoethanol (25.0 mg, 0.20 mmol) as starting materials, YK-418 (26.3 mg, 0.04 mmol, 21.7%) was obtained according to the synthesis method for YK-401. 42 H 84 N2O6, MS(ES): m / z (M+H + )713.6. 1 H NMR (400 MHz, chloroform-d) δ 4.05 (q, J = 6.5 Hz, 4H), 3.68 - 3.50 (m, 4H), 2.76 - 2.48 (m, 12H), 2.32 (t, J = 7.1 Hz, 2H), 2.22 (d, J = 6.9 Hz, 2H), 1.81 (dt, J = 15.0, 7.2 Hz, 3H), 1.68 - 1.56 (m, 6H), 1.50 (d, J = 7.3 Hz, 2H), 1.28 (d, J = 15.8 Hz, 38H), 0.88 (t, J = 6.6 Hz, 9H).

[0173] 19.Synthesis of YK-419 Synthesis scheme: [ka] Step 1: Synthesis of 6-((2-hydroxyethyl)amino)hexanoic acid heptadecan-9-yl ester (YK-419-PM1) YK-419-PM1 (459.1 mg, 1.11 mmol, 93.3%) was obtained using 6-bromohexanoic acid heptadecanoic-9-ester (500 mg, 1.19 mmol) and ethanolamine (291.2 mg, 4.77 mmol) according to the synthesis method for YK-401. 25 H 51 NO3, MS(ES): m / z (M+H + )414.4. Step 2: Synthesis of 6,6'-(propane-1,3-diyl-bis((2-hydroxyethyl)azanediyl))dihexanoic acid di(heptadecan-9-yl) ester (YK-419) YK-419-PM1 (300.0 mg, 0.73 mmol) and potassium carbonate (302.7 mg, 2.19 mmol) were dissolved in acetonitrile (3 mL). 1,3-Dibromopropane (73.2 mg, 0.36 mmol) was slowly added to the mixture, which was then heated to 50°C and stirred for 6 hours. After the reaction was complete, 20 mL of saturated aqueous sodium bicarbonate solution was added to the reaction mixture, followed by extraction with dichloromethane (20 mL x 2). The combined organic phases were washed with brine (20 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel chromatography (dichloromethane / methanol) to obtain YK-419 (47.5 mg, 0.05 mmol, 15.2%). 53 H 106 N2O6, MS(ES): m / z (M+H + )867.8. 1 H NMR (400 MHz, chloroform-d) δ 4.86 (p, J = 6.2 Hz, 2H), 3.69 (s, 4H), 2.73 (s, 8H), 2.67 - 2.60 (m, 4H), 2.29 (t, J = 7.3 Hz, 4H), 1.84 - 1.75 (m, 2H), 1.64 (q, J = 7.6 Hz, 4H), 1.55 - 1.47 (m, 10H), 1.26 (s, 56H), 0.88 (t, J = 6.6 Hz, 12H).

[0174] 20.Synthesis of YK-420 Synthesis scheme: [ka] Step 1: Synthesis of (4-aminobutyl)-tert-butylcarbamate (YK-420-PM1) 1,4-Butanediamine (10.00 g, 113.44 mmol) was dissolved in dichloromethane (100 mL), potassium carbonate (15.67 g, 113.44 mmol) was added, and the system temperature was controlled at 15°C. Di-tert-butyl dicarbonate (6.20 g, 28.36 mmol) was slowly added dropwise. After the addition was completed, the mixture was slowly warmed to room temperature and reacted for 2 hours. After the reaction was completed, the mixture was filtered with suction through a pad of diatomaceous earth. The mother liquor was washed twice with saturated brine (100 mL x 2), separated, and the organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to remove the solvent. YK-420-PM1 (4616.4 mg, 24.52 mmol, 21.6%) was obtained. 20 N2O2, MS(ES): m / z (M+H + )189.2. Step 2: Synthesis of 6-((4-(tert-butyloxycarbonyl)amino)butyl)amino)hexanoic acid heptadecan-9-yl ester (YK-420-PM2) Using YK-420-PM1 (1.04 g, 5.52 mmol) and 6-bromohexanoic acid-heptadecan-9-yl ester (2.00 g, 4.77 mmol) as starting materials, YK-420-PM2 (1.26 g, 2.33 mmol, 52.1%) was obtained according to the synthesis method for YK-401. 32 H 64 N2O4, MS(ES): m / z (M+H + )541.5. Step 3: Synthesis of 6-(((4-(tert-butyloxycarbonyl)amino)butyl)(2-hydroxyethyl)amino)hexanoic acid heptadecan-9-yl ester (YK-420-PM3) YK-420-PM3 (980.4 mg, 1.68 mmol, 72.1%) was obtained using YK-420-PM2 (1.26 g, 2.33 mmol) and bromoethanol (0.35 g, 2.80 mmol) according to the synthesis method for YK-401. 34 H 68 N2O5, MS(ES): m / z (M+H + )585.5. Step 4: Synthesis of 6-((4-aminobutyl)(2-hydroxyethyl)amino)hexanoic acid heptadecan-9-yl ester (YK-420-PM4) Using YK-420-PM3 (980.4 mg, 1.68 mmol) as a starting material, YK-420-PM4 (814.5 mg, 1.68 mmol, 100%) was obtained according to the synthesis method for YK-413-PM3. 29 H 60 N2O3, MS(ES): m / z (M+H + )485.5. Step 5: Synthesis of 6-((4-((6-(heptadecan-9-yloxy)-6-oxohexyl)(2-hydroxyethyl)amino)butyl)amino)hexanoic acid heptadecan-9-yl ester (YK-420-PM5) YK-420-PM5 (306.0 mg, 0.37 mmol, 22.0%) was obtained using YK-420-PM4 (814.5 mg, 1.68 mmol) and 6-bromohexanoic acid-heptadecan-9-yl ester (740.4 mg, 1.76 mmol) as starting materials according to the synthesis method for YK-401. 52 H 104 N2O5, MS(ES): m / z (M+H + )837.9. Step 6: Synthesis of 6,6'-(butane-1,4-diylbis((2-hydroxyethyl)azanediyl))dihexanoic acid di(heptadecan-9-yl) ester (YK-420) YK-420-PM5 (306.0 mg, 0.37 mmol) and bromoethanol (54.8 mg, 0.44 mmol) were used as starting materials, and YK-420 (130.5 mg, 0.15 mmol, 40.5%) was obtained according to the synthesis method for YK-401. 54 H 108 N2O6, MS(ES): m / z (M+H + )881.8. 1H NMR (400 MHz, CDCl3) δ 4.93 - 4.83 (m, 2H), 3.84 (s, 4H), 2.94 (d, J = 24.3 Hz, 9H), 2.33 (t, J = 7.4 Hz, 4H), 1.82 (s, 4H), 1.68 (dd, J = 15.3, 7.6 Hz, 9H), 1.53 (d, J = 5.5 Hz, 8H), 1.43 - 1.37 (m, 5H), 1.29 (s, 51H), 0.91 (t, J = 6.7 Hz, 12H).

[0175] 21.Synthesis of YK-421 Synthesis scheme: [ka] Step 1: Synthesis of decyl 4-((4-((tert-butyloxycarbonyl)amino)butyl)amino)butyrate (YK-421-PM1) Using YK-420-PM1 (2.00 g, 10.62 mmol) and decyl 4-bromobutyrate (3.20 g, 10.41 mmol) as starting materials, YK-421-PM1 (1300 mg, 3.14 mmol, 30.1%) was obtained according to the synthesis method for YK-401. 23 H 46 N2O4, MS(ES): m / z (M+H + )415.4. Step 2: Synthesis of decyl 4-((4-((tert-butyloxycarbonyl)amino)butyl)(2-hydroxyethyl)amino)butyrate (YK-421-PM2) Using YK-421-PM1 (1.30 g, 3.14 mmol) and bromoethanol (469 mg, 3.75 mmol) as starting materials, YK-421-PM2 (1.20 g, 2.62 mmol, 83.3%) was obtained according to the synthesis method for YK-401. 25 H 50 N2O5, MS(ES): m / z (M+H + )459.4. Step 3: Synthesis of decyl 4-((4-aminobutyl)(2-hydroxyethyl)amino)butyrate (YK-421-PM3) Using YK-421-PM2 (1.20 g, 2.62 mmol) as a starting material, YK-421-PM3 (720 mg, 2.01 mmol, 76.6%) was obtained according to the synthesis method for YK-413-PM3. 20 H 42 N2O3, MS(ES): m / z (M+H + )359.3. Step 4: Synthesis of 6-((4-((4-(decyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)butyl)amino)hexanoic acid heptadecan-9-yl ester (YK-421-PM4) YK-421-PM4 (106 mg, 0.15 mmol, 9.7%) was obtained using YK-421-PM3 (580 mg, 1.62 mmol) and 6-bromohexanoic acid-heptadecan-9-yl ester (664.2 mg, 1.53 mmol) as starting materials according to the synthesis method for YK-401. 43 H 86 N2O5, MS(ES): m / z (M+H + )711.7. Step 5: Synthesis of 6-((4-((4-(decyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)butyl)(2-hydroxyethyl)amino)hexanoic acid heptadecan-9-yl ester (YK-421) Using YK-421-PM4 (106 mg, 0.15 mmol) and bromoethanol (37.5 mg, 0.30 mmol) as starting materials, YK-421 (23.1 mg, 0.03 mmol, 20.4%) was obtained according to the synthesis method for YK-401. 45 H 90 N2O6, MS(ES): m / z (M+H + )755.7. 1H NMR (400 MHz, CDCl3) δ 4.95 - 4.72 (m, 1H), 4.07 (t, J = 6.8 Hz, 2H), 3.92 (d, J = 24.6 Hz, 2H), 3.08 (d, J = 46.2 Hz, 6H), 2.42 (t, J = 6.3 Hz, 2H), 2.37 - 2.25 (m, 2H), 2.26 - 2.16 (m, 2H), 2.02 (s, 4H), 1.96 - 1.88 (m, 2H), 1.83 (s, 2H), 1.65 (dd, J = 14.3, 7.6 Hz, 8H), 1.50 (s, 4H), 1.26 (s, 42H), 0.88 (t, J = 6.6 Hz, 9H).

[0176] 22.Synthesis of YK-422 Synthesis scheme: [ka] Step 1: Synthesis of 6-((3-hydroxypropyl)amino)hexanoic acid 2-octyldecyl ester (YK-422-PM1) YK-422-PM1 (91 mg, 0.21 mmol, 46.1%) was obtained using 6-bromohexanoic acid-2-octyldecyl ester (200 mg, 0.46 mmol) and 3-aminopropan-1-ol (174.6 mg, 2.24 mmol) as starting materials according to the synthesis method for YK-401. 27 H 55 NO3, MS(ES): m / z (M+H + )442.4. Step 2: Synthesis of 6,6'-(ethane-1,2-diyl-bis((3-hydroxypropyl)azanediyl))dihexanoic acid di(2-octyldecyl ester) (YK-422) Using YK-422-PM1 (71 mg, 0.16 mmol) and 1,2-dibromoethane (15.1 mg, 0.08 mmol) as starting materials, YK-422 (25.1 mg, 0.03 mmol, 34.5%) was obtained according to the synthesis method for YK-419. 56 H 112N2O6, MS(ES): m / z (M+H + )909.8. 1 H NMR (400 MHz, chloroform-d) δ 3.96 (d, J = 5.8 Hz, 4H), 3.76 (t, J = 5.1 Hz, 4H), 3.54 (q, J = 7.2 Hz, 2H), 3.45 (s, 4H), 3.19 (t, J = 6.7 Hz, 4H), 3.03 - 2.93 (m, 4H), 2.32 (t, J = 7.2 Hz, 4H), 1.98 - 1.87 (m, 4H), 1.80 - 1.54 (m, 12H), 1.26 (s, 58H), 0.87 (d, J = 7.0 Hz, 12H).

[0177] 23.Synthesis of YK-423 Synthesis scheme: [ka] Synthesis of 6,6'-(propane-1,3-diyl-bis((3-hydroxypropyl)azanediyl))dihexanoic acid di(2-octyldecyl ester) (YK-423) Using YK-422-PM1 (200 mg, 0.45 mmol) and 1,3-dibromopropane (45.6 mg, 0.23 mmol) as starting materials, YK-423 (59.9 mg, 0.06 mmol, 28.2%) was obtained according to the synthesis method for YK-419. 57 H 114 N2O6, MS(ES): m / z (M+H + )923.8. 1 H NMR (400 MHz, chloroform-d) δ 3.96 (d, J = 5.7 Hz, 4H), 3.78 (t, J = 5.1 Hz, 4H), 2.75 (s, 4H), 2.57 (s, 8H), 2.31 (t, J = 7.4 Hz, 4H), 1.78 (d, J = 22.1 Hz, 4H), 1.66 - 1.55 (m, 8H), 1.27 (s, 64H), 0.88 (t, J = 6.7 Hz, 12H).

[0178] 24.Synthesis of YK-424 Synthesis scheme: [ka] Step 1: Synthesis of 6-(((4-(tert-butyloxycarbonyl)amino)butyl)amino)hexanoic acid-2-octyldecyl ester (YK-424-PM1) Using YK-420-PM1 (200 mg, 1.06 mmol) and 6-bromohexanoic acid-2-octyldecyl ester (475.5 mg, 1.06 mmol) as starting materials, YK-424-PM1 (141.8 mg, 0.26 mmol, 24.5%) was obtained according to the synthesis method for YK-401. 33 H 66 N2O4, MS(ES): m / z (M+H + )555.6. Step 2: Synthesis of 6-((4-(tert-butyloxycarbonyl)amino)butyl)(3-hydroxypropyl)aminohexanoic acid 2-octyldecyl ester (YK-424-PM2) YK-424-PM2 (137.1 mg, 0.22 mmol, 84.6%) was obtained using YK-424-PM1 (141.8 mg, 0.26 mmol) and 3-bromo-1-propanol (78.1 mg, 0.56 mmol) according to the synthesis method for YK-401. 36 H 72 N2O5, MS(ES): m / z (M+H + )613.5. Step 3: Synthesis of 6-((4-aminobutyl)(3-hydroxypropyl)amino)hexanoic acid-2-octyldecyl ester (YK-424-PM3) Using YK-424-PM2 (137.1 mg, 0.22 mmol) as a starting material, YK-424-PM3 (103.3 mg, 0.20 mmol, 90.9%) was obtained according to the synthesis method for YK-413-PM3. 31 H 64 N2O3, MS(ES): m / z (M+H + ) 513.4. Step 4: Synthesis of 6-((3-hydroxypropyl)(4-((6-(2-octyldecyl)oxy)-6-oxohexyl)amino)butyl)amino)hexanoic acid-2-octyldecyl ester (YK-424-PM4) Using YK-424-PM3 (103.3 mg, 0.20 mmol) and 6-bromohexanoic acid-2-octyldecyl ester (85.5 mg, 0.19 mmol) as starting materials, YK-424-PM4 (46.7 mg, 0.05 mmol, 27.9%) was obtained according to the synthesis method for YK-401. 55 H 110 N2O5, MS(ES): m / z (M+H + )879.9. Step 5: Synthesis of 6,6'-(butane-1,4-diylbis((3-hydroxypropyl)azanediyl))dihexanoic acid bis(2-octyldecyl ester) (YK-424) YK-424-PM4 (46.7 mg, 0.05 mmol) and 3-bromo-1-propanol (7.2 mg, 0.05 mmol) were used as starting materials, and YK-424 (23.6 mg, 0.03 mmol, 50.0%) was obtained according to the synthesis method for YK-401. 58 H 116 NO MS (ES): m / z (M + H + )937.9. 1 H NMR (400 MHz, chloroform-d) δ 3.96 (d, J = 5.8 Hz, 4H), 3.76 (t, J = 5.1 Hz, 4H), 3.54 (q, J = 7.2 Hz, 2H), 3.45 (s, 4H), 3.19 (t, J = 6.7 Hz, 4H), 3.03 - 2.93 (m, 4H), 2.32 (t, J = 7.2 Hz, 4H), 1.98 - 1.87 (m, 4H), 1.80 - 1.54 (m, 16H), 1.26 (s, 58H), 0.87 (d, J = 7.0 Hz, 12H).

[0179] 25.Synthesis of YK-009 According to the method described in CN114044741B, 162 mg of YK-009 was obtained.

[0180] 26. Synthesis of 8-(8-((3-hexylnonyl)oxy)-8-oxooctyl)-((2-hydroxyethyl)amino)octanoic acid heptadecan-9-yl ester (Compound 21) Synthesis scheme: [ka] Step 1: Synthesis of 8-bromooctanoic acid heptadecan-9-yl ester (compound 21-PM1) 9-Heptadecanol (1.00 g, 3.90 mmol) and 8-bromooctanoic acid (1.04 g, 4.66 mmol) were dissolved in dichloromethane (10 mL), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.90 g, 4.68 mmol) and 4-dimethylaminopyridine (24 mg, 0.20 mmol) were added. The mixture was stirred at 30-35 °C for 8 hours. After completion of the reaction, the reaction solution was washed with saturated sodium carbonate, washed with saturated brine, and dried over Na2SO4. The mixture was filtered, and the filtrate was concentrated under reduced pressure and purified by silica gel chromatography (ethyl acetate / n-hexane) to give 8-bromooctanoic acid-9-heptadecanyl ester (1.28 g, 2.77 mmol, 71.0%). Step 2: Synthesis of 8-((2-hydroxyethyl)amino)octanoic acid heptadecan-9-yl ester (compound 21-PM2) 8-Bromooctanoic acid heptadecan-9-yl 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 complete, the reaction mixture was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel chromatography (methanol / dichloromethane) to obtain 9-heptadecyl-8-((2-hydroxyethyl)amino)octanoic acid ester (365 mg, 0.83 mmol, 76.9%). 27 H 55 NO3, MS(ES): m / z (M+H + )442.3. Step 3: Synthesis of 8-bromooctanoic acid 3-hexylnonyl ester (compound 21-PM3) 3-Hexylnonanol (1.00 g, 4.38 mmol) and 8-bromooctanoic acid (1.17 g, 5.25 mmol) were used as raw materials and purified by silica gel chromatography (ethyl acetate / n-hexane) according to the preparation method of compound 21-PM1 to obtain 8-bromooctanoic acid-3-hexylnonyl ester (1.57 g, 3.62 mmol, 82.6%). Step 4: Synthesis of 8-(8-((3-hexylnonyl)oxy)-8-oxooctyl)-((2-hydroxyethyl)amino)octanoic acid heptadecan-9-yl ester (compound 21) 8-((2-hydroxyethyl)amino)octanoic acid heptadecan-9-yl ester (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 mixture was cooled to room temperature and then filtered. The filtrate was concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel chromatography (ethyl acetate / n-hexane) to obtain the target compound (220 mg, 0.28 mmol, 60.9%).50 H 99 NO5, MS(ES): m / z (M+H + )794.8. 1 H NMR (400 MHz, CDCl3) δ 4.90 (p, J = 6.3 Hz, 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, 2.2 Hz, 12H).

[0181] 27. Synthesis of 8,8'-((2-hydroxyethyl)azanediyl)dioctanoic acid bis(3-hexylnonyl) ester (compound 23) Synthesis scheme: [ka] 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 completion of the reaction, the reaction mixture was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel chromatography (methanol / dichloromethane) to obtain 8,8'-((2-hydroxyethyl)azanediyl)dioctanoic acid bis(3-hexylnonyl) ester (160 mg, 0.21 mmol, 31.8%). 48 H 95 NO5, MS(ES): m / z (M+H + )766.5. 1H NMR (400 MHz, CDCl3) δ 4.12 (t, J = 7.1 Hz, 4H), 3.62 (s, 2H), 2.68 (s, 2H), 2.51 (d, J = 25.8 Hz, 4H), 2.32 (t, J = 7.5 Hz, 4H), 1.72 - 1.57 (m, 8H), 1.55 - 1.40 (m, 6H), 1.40 - 1.17 (m, 55H), 0.92 (t, J = 6.8 Hz, 12H).

[0182] Example 2: Optimization of preparation conditions for lipid nanoparticles (LNP formulations) 1. Optimization of the carrier (liposome) to mRNA ratio [ka] The cationic lipid compound YK-407 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 in a molar ratio of 49:10:39.5:1.5 to prepare an ethanol lipid solution. The ethanol lipid solution was quickly added to citrate buffer (pH = 4-5) via ethanol injection and vortexed for 30 s before use. eGFP-mRNA was diluted with citrate buffer (pH = 4-5) to obtain an mRNA aqueous solution. Liposomes were prepared using a fixed volume of liposome solution and mRNA aqueous solution at total lipid to mRNA weight ratios of 5:1, 10:1, 15:1, 20:1, 30:1, and 35:1. The solution was sonicated at 25°C for 15 min (ultrasonic frequency 40 kHz, ultrasonic power 800 W). The resulting liposomes were diluted 10-fold with PBS, then ultrafiltered using a 300 kDa ultrafiltration tube to remove ethanol, and then adjusted to a constant volume with PBS to obtain an LNP formulation encapsulating eGFP-mRNA using the cationic lipids YK-407 / DSPC / cholesterol / DMG-PEG2000 (mol percentage 49:10:39.5:1.5). The results of cell transfection experiments showed that when the weight ratio of carrier to mRNA was in the range of 10:1 to 30:1, the transfection effect was good, with the highest transfection effect at 15:1. Ratios of 5:1 and 35:1 showed poor transfection effects and should not be used for mRNA delivery (Figure 1). LNP formulations prepared with YK-401, YK-402, YK-403, YK422, and YK-423 could obtain the same results but were not shown in the figures.

[0183] 2. Optimizing the ratio of cationic lipids to neutral lipids LNP formulations encapsulating eGFP-mRNA were prepared according to the method described in 1, where the molar ratios of the cationic lipid YK-407 to the 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. Through cell transfection experiments, it was found that transfection was effective when the molar ratio of cationic lipid to neutral lipid was between 1:1 and 15:1, with the highest transfection efficiency at 4:1. Ratios of 3.5:1 and 4.9:1 also showed good transfection effects (Figure 2). The same results could also be obtained with LNP formulations prepared with YK-401, YK-402, YK-403, YK422, and YK-423, but these were not shown in the figures.

[0184] 3. Optimization of the ratio of polymer-conjugated lipid to carrier (liposome) LNP formulations encapsulating eGFP-mRNA were prepared according to the method described in 1. The cationic lipid in the carrier was YK-407, and the molar ratios of the polymer-conjugated lipid DMG-PEG2000 in the carrier were 0.5%, 1.5%, 3.5%, 5%, 10%, and 15%, respectively. The results of cell transfection experiments showed that when the molar ratio of polymer-conjugated lipid to carrier was in the range of 0.5% to 10%, transfection efficiency was highest when it was 1.5%, and lowest when it was 10% (Figure 3). The same results could also be obtained with LNP formulations prepared with YK-401, YK-402, YK-403, YK422, and YK-423, but these were not shown in the figures.

[0185] 4. Optimization of the ratio of each component in the carrier (liposome) LNP formulations encapsulating eGFP-mRNA were prepared according to the method described in 1, where the molar ratios of the cationic lipid YK-407, the neutral lipid DSPC, the structural lipid cholesterol, and the 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. Cell transfection experiments demonstrated that transfection was possible when the molar ratios of cationic lipids, neutral lipids, structural lipids, and polymer-conjugated lipids 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. A ratio of (35-49):(7.5-15):(35-55):(1-5) yielded good transfection efficacy, with a ratio of 40:10:48.5:1.5 showing the best transfection efficacy. 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 applied to the preparation of LNP formulations, with the preferred ratio being (35-49):(7.5-15):(35-55):(1-5), of which the preferred ratio was 40:10:48.5:1.5. The same results could also be obtained with LNP formulations prepared with YK-401, YK-402, YK-403, YK422, and YK-423, but these were not shown in the figures.

[0186] Example 3: Transfection experiment of cells containing LNP formulation of eGFP-mRNA Cell resuscitation and passage: 293T cells were resuscitated, cultured in a culture dish to the required cell number, and passaged.

[0187] Seeding: Cells in culture dishes were digested and counted, and 10,000 cells per well were seeded in 96-well plates, and 150,000 cells per well in 12-well plates, and cultured overnight until cells adhered.

[0188] Cell transfection experiment: The LNP formulation (carrier: YK-407 cationic lipid) containing 1.5 μg of eGFP-mRNA prepared in Example 2 and the Lipofectamin 3000 formulation of eGFP-mRNA were added to cell culture medium in a 12-well plate and cultured for 24 hours. The transfection efficiency of various samples was examined based on the fluorescence intensity under a fluorescent microscope.

[0189] Based on the experimental results, we finally determined the conditions for preparing nanolipid particles (LNP formulations). The weight ratio of carrier to mRNA was 15:1, the molar ratio of cationic lipid to neutral lipid was 4.9:1, the molar ratio of polymer-conjugated lipids in the liposome was 1.5%, and the molar ratio of cationic lipid, neutral lipid, structural lipid, and polymer-conjugated lipid was 49:10:39.5:1.5. At these ratios, various cationic lipids designed in this application and cationic lipids used in prior art all exhibited excellent transfection efficacy (as determined in Example 2; some experimental results are not shown). In the following experiments, nanolipid particles (LNP formulations) were prepared under these conditions.

[0190] Example 4: Preparation of nanolipid particles (LNP formulation) [Table 1] JPEG0007761816000060.jpg210164 JPEG0007761816000061.jpg238164 JPEG0007761816000062.jpg193164 JPEG0007761816000063.jpg204164 JPEG0007761816000064.jpg221164 JPEG0007761816000065.jpg37164

[0191] 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 in a molar ratio of 49:10:39.5:1.5 to obtain an ethanolic lipid solution. The ethanolic lipid solution was quickly added to citrate buffer (pH = 4-5) via the ethanol injection method and vortexed for 30 s before use. eGFP-mRNA (Shanghai Origin Experimental Reagents Co., Ltd.) or Fluc-mRNA (Shanghai Origin Experimental Reagents Co., Ltd.) was diluted with citrate buffer (pH = 4-5) to obtain an mRNA aqueous solution. Liposomes were prepared by combining a fixed volume of liposome solution and an mRNA aqueous solution at a total lipid to mRNA weight ratio of 15:1. The solution was sonicated at 25 °C for 15 min (ultrasonic frequency 40 kHz, ultrasonic power 800 W). The resulting liposomes were diluted 10 times with PBS, then ultrafiltered using a 300 kDa ultrafiltration tube to remove ethanol, and then adjusted to a constant volume with PBS to obtain LNP formulations encapsulating eGFP-mRNA or Fluc-mRNA using a cationic lipid / DSPC / cholesterol / DMG-PEG2000 (mol percentage 49:10:39.5:1.5).

[0192] Lipofectamine 3000 transfection reagent is currently widely used for cell transfection, boasting excellent transfection performance and efficiency, as well as the ability to improve cell viability and making it suitable for difficult-to-transfect cell types. Lipofectamine 3000 transfection reagent was used as a control. Lipofectamine 3000 formulations of eGFP-mRNA or Fluc-mRNA were prepared according to the Lipofectamine 3000 (Invitrogen (Shanghai) Trading Co., Ltd.) instruction manual.

[0193] Example 5: Measurement of particle size and polydispersity index (PDI) of nanolipid particles Particle size and polydispersity index (PDI) were measured by dynamic light scattering using a Malvern Laser Particle Size Analyzer.

[0194] 10 μL of the liposome solution was diluted to 1 mL with RNase-free deionized water and added to the sample pool. Measurements were repeated three times for each sample. The measurement conditions were a scattering angle of 90° and 25°C. The detection results are shown in the following table.

[0195] [Table 2]

[0196] The nanolipid particles prepared in Example 4, all of which had particle sizes between 120 and 210 nm, could be used for mRNA delivery. Among these, the particles prepared with YK-417 had the smallest particle size at 128 nm, while the particles prepared with ALC-0315 had the largest particle size at 205 nm. The polydispersity of all nanolipid particles ranged from 4% to 25%, with the smallest being 4.2% for YK-404 and the largest being 23.9% for YK-414.

[0197] Example 6: Detection of encapsulation rate, drug loading concentration, and total RNA concentration The encapsulation rate is an important quality characteristic of liposomes. It refers to the percentage of drug content encapsulated in the lipid bilayer relative to the total drug content, and can reflect the degree of drug encapsulation in liposomes. The Chinese Pharmacopoeia stipulates that the encapsulation rate should generally be 80% or higher.

[0198] The drug loading capacity is the ratio of the amount of drug in the liposome to the total amount of drug and carrier in the liposome. The drug loading capacity directly affects the clinical dose of the drug, so a higher drug loading capacity can meet clinical needs. The drug loading concentration is proportional to the drug loading capacity, and the relative ratio of the drug loading concentration can represent the relative ratio of the drug loading capacity. The relative ratio of the total RNA concentration can represent the relative ratio of the amount of mRNA contained in the LNP formulation.

[0199] Reagent preparation: Stock solutions of 1×TE buffer, 0.1% Triton X-100 buffer, RiboGreen reagent (1:200), and mRNA standards were prepared.

[0200] Sample detection: An appropriate amount of sample was taken and added to an appropriate amount of 1x TE buffer, and diluted to approximately 2.8 μg per mL. The sample was then added to a 96-well plate, and 50 μL of diluted test article or mRNA standard stock solution was added per well, followed by 1x TE buffer and 0.1% Triton X-100 buffer. The sample was incubated at 37°C for 10 minutes, and 100 μL of RiboGreen reagent (1:200) was added to each test article well of the 96-well plate. The plate was centrifuged, read using a microplate reader, and the data was processed. Specific data are shown in Tables 3 and 4.

[0201] Test Results: (1) The LNP formulations prepared with YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 all showed significantly improved encapsulation efficiency, drug loading concentration, and total RNA concentration compared to conventional cationic lipids. For example, YK-407 showed a 29.0% improvement in encapsulation efficiency over compound 23, a 1.78-fold increase in drug loading concentration over compound 23, and a 1.41-fold increase in total RNA concentration over compound 21.

[0202] [Table 3]

[0203] As can be seen from Table 3, the LNP formulations prepared with various compounds showed significant differences in encapsulation efficiency. YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 showed significantly improved encapsulation efficiency compared to SM-102, ALC-0315, Compound 21, Compound 23, HHMA, and YK-009.

[0204] The encapsulation rate of YK-407 was 85.2%, which was 22.3% higher than that of SM-102, 21.9% higher than that of ALC-0315, 27.2% higher than that of compound 21, 29.0% higher than that of compound 23, 25.0% higher than that of HHMA, and 21.8% higher than that of YK-009.

[0205] The encapsulation rate of YK-401 was 83.6%, which was 20.7% higher than that of SM-102, 20.3% higher than that of ALC-0315, 25.6% higher than that of compound 21, 27.4% higher than that of compound 23, 23.4% higher than that of HHMA, and 20.2% higher than that of YK-009.

[0206] The encapsulation rate of YK-402 was 91.0%, which was 28.1% higher than that of SM-102, 27.7% higher than that of ALC-0315, 33.0% higher than that of compound 21, 34.8% higher than that of compound 23, 30.8% higher than that of HHMA, and 27.6% higher than that of YK-009.

[0207] The encapsulation rate of YK-403 was 87.9%, which was 25.0% higher than that of SM-102, 24.6% higher than that of ALC-0315, 29.9% higher than that of compound 21, 31.7% higher than that of compound 23, 27.7% higher than that of HHMA, and 24.4% higher than that of YK-009.

[0208] The encapsulation rate of YK-422 was 92.4%, which was 29.5% higher than that of SM-102, 29.1% higher than that of ALC-0315, 34.4% higher than that of compound 21, 36.2% higher than that of compound 23, 32.2% higher than that of HHMA, and 29.0% higher than that of YK-009.

[0209] The encapsulation rate of YK-423 was 89.7%, which was 26.8% higher than that of SM-102, 26.4% higher than that of ALC-0315, 31.7% higher than that of compound 21, 33.5% higher than that of compound 23, 29.5% higher than that of HHMA, and 26.3% higher than that of YK-009.

[0210] Furthermore, there were significant differences in drug loading and total RNA concentrations among LNP formulations prepared with various compounds. YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 all showed significantly improved drug loading and total RNA concentrations compared with SM-102, ALC-0315, compound 21, compound 23, and YK-009.

[0211] For YK-407, the drug loading concentration was 35.67 μg / mL, and the total RNA concentration was 40.77 μg / mL, which were 1.34-fold and 1.13-fold higher than those of SM-102, 1.33-fold and 1.08-fold higher than those of ALC-0315, 1.62-fold and 1.41-fold higher than those of compound 21, 1.78-fold and 1.31-fold higher than those of compound 23, 1.09-fold and 1.12-fold higher than those of HHMA, and 1.30-fold and 1.06-fold higher than those of YK-009, respectively.

[0212] For YK-401, the drug loading concentration was 37.16 μg / mL, and the total RNA concentration was 41.71 μg / mL, which were 1.40-fold and 1.16-fold higher than those of SM-102, 1.38-fold and 1.10-fold higher than those of ALC-0315, 1.69-fold and 1.44-fold higher than those of compound 21, 1.86-fold and 1.34-fold higher than those of compound 23, 1.14-fold and 1.14-fold higher than those of HHMA, and 1.35-fold and 1.08-fold higher than those of YK-009, respectively.

[0213] For YK-402, the drug loading concentration was 37.67 μg / mL, and the total RNA concentration was 40.09 μg / mL, which were 1.42-fold and 1.11-fold higher than those of SM-102, 1.40-fold and 1.06-fold higher than those of ALC-0315, 1.71-fold and 1.39-fold higher than those of compound 21, 1.88-fold and 1.29-fold higher than those of compound 23, 1.15-fold and 1.10-fold higher than those of HHMA, and 1.37-fold and 1.04-fold higher than those of YK-009, respectively.

[0214] For YK-403, the drug loading concentration was 34.21 μg / mL, and the total RNA concentration was 40.53 μg / mL, which were 1.29-fold and 1.12-fold higher than those of SM-102, 1.27-fold and 1.07-fold higher than those of ALC-0315, 1.56-fold and 1.40-fold higher than those of compound 21, 1.71-fold and 1.31-fold higher than those of compound 23, 1.05-fold and 1.11-fold higher than those of HHMA, and 1.24-fold and 1.05-fold higher than those of YK-009, respectively.

[0215] For YK-422, the drug loading concentration was 45.05 μg / mL, and the total RNA concentration was 45.76 μg / mL, which were 1.70-fold and 1.27-fold higher than those of SM-102, 1.68-fold and 1.21-fold higher than those of ALC-0315, 2.05-fold and 1.58-fold higher than those of compound 21, 2.25-fold and 1.47-fold higher than those of compound 23, 1.38-fold and 1.26-fold higher than those of HHMA, and 1.64-fold and 1.19-fold higher than those of YK-009, respectively.

[0216] For YK-423, the drug loading concentration was 37.47 μg / mL, and the total RNA concentration was 41.00 μg / mL, which were 1.41-fold and 1.14-fold higher than those of SM-102, 1.39-fold and 1.08-fold higher than those of ALC-0315, 1.71-fold and 1.42-fold higher than those of compound 21, 1.87-fold and 1.32-fold higher than those of compound 23, 1.15-fold and 1.13-fold higher than those of HHMA, and 1.36-fold and 1.06-fold higher than those of YK-009, respectively.

[0217] Data analysis using GraphPad Prism software showed that YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 all significantly improved the encapsulation efficiency, drug loading concentration, and total RNA concentration compared with SM-102, ALC-0315, compound 21, compound 23, HHMA, and YK-009.

[0218] Interim summary LNP formulations prepared with several compounds designed herein, including YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423, all exhibited significantly improved encapsulation efficiency, drug loading concentration, and total RNA concentration compared to prior art cationic lipids such as SM-102, ALC-0315, compound 21, compound 23, HHMA, and YK-009. For example, YK-407 exhibited a 29.0% improvement in encapsulation efficiency over compound 23, a 1.78-fold increase in drug loading concentration over compound 23, and a 1.41-fold increase in total RNA concentration over compound 21.

[0219] (2) There were significant differences in the encapsulation efficiency and drug loading amount among the LNP formulations prepared with various designed compounds. For various compounds, the encapsulation efficiency ranged from 70% to 95%, the drug loading concentration ranged from 20 to 50 μg / mL, and the total RNA concentration ranged from 25 to 55 μg / mL.

[0220] [Table 4]

[0221] As can be seen from Table 4, for this series of designed compounds, the encapsulation efficiency ranged from 70% to 95%, the drug loading concentration ranged from 20 to 50 μg / mL, and the total RNA concentration ranged from 25 to 55 μg / mL. There were significant differences among the various compounds. The encapsulation efficiency was highest for YK-420 (94.9%), while that for YK-417 (74.8%) was lowest. The drug loading concentration was highest for YK-408 (45.42 μg / mL), while that for YK-413 (only 23.63 μg / mL) was lowest. The total RNA concentration was highest for YK-408 (52.46 μg / mL), while that for YK-413 (only 28.36 μg / mL) was lowest.

[0222] Interim summary LNP formulations prepared with various designed compounds showed significant differences in encapsulation efficiency and drug loading. For various compounds, the encapsulation efficiency ranged from 70% to 95%, the drug loading concentration ranged from 20 to 50 μg / mL, and the total RNA concentration ranged from 25 to 55 μg / mL. This indicates that the encapsulation efficiency and drug loading are not similar when LNP formulations are prepared with compounds with similar structures.

[0223] summary LNP formulations prepared with several compounds designed herein, including YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423, all exhibited significantly improved encapsulation efficiency, drug loading concentration, and total RNA concentration compared to prior art cationic lipids, including SM-102, ALC-0315, compound 21, compound 23, HHMA, and YK-009. For example, YK-407 exhibited a 29.0% improvement in encapsulation efficiency over compound 23, a drug loading concentration 1.78-fold higher than that of compound 23, and a total RNA concentration 1.41-fold higher than that of compound 21.

[0224] In conclusion, the mRNA delivery carriers prepared with YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 all showed significantly improved encapsulation rates and drug loadings, thus significantly reducing the amount of LNPs used and providing LNP-mRNA formulations with more uniform distribution and more controllable quality in the application of multivalent mRNA vaccines.

[0225] Among this series of compounds designed, LNP formulations prepared with various compounds showed significant differences in encapsulation efficiency and drug loading. The encapsulation efficiency ranged from 70% to 95%, the drug loading concentration ranged from 20 to 50 μg / mL, and the total RNA concentration ranged from 25 to 55 μg / mL. This indicates that LNP formulations prepared with compounds with similar structures do not necessarily have similar encapsulation efficiency and drug loading. Rather, significant differences in encapsulation efficiency and drug loading are likely to occur. Therefore, it is impossible to predict the encapsulation efficiency and drug loading of LNP formulations prepared with a given compound from its structure.

[0226] Example 7: In vitro validation of LNP delivery vehicle performance Cell resuscitation and passaging: The method was the same as in Example 3.

[0227] Seeding: The method was the same as in Example 3.

[0228] 1. Fluorescent detection of Fluc-mRNA 0.3 µg of an LNP formulation containing Fluc-mRNA (the carrier components of the LNP formulation are cationic lipids, neutral lipids, structural lipids, and polymer-conjugated lipids, with a molar ratio of 49:10:39.5:1.5; the cationic lipids are listed in Table 1) was added to the cell culture medium in a 96-well plate and cultured for 24 hours. The corresponding reagents were then added according to the Gaussia Luciferase Assay Kit's instruction manual, and the fluorescence intensity of each well was detected using an IVIS fluorescence detection system. This experiment verified the intracellular transfection efficiency of the LNP formulation, and the specific detection results are shown in Tables 6–9.

[0229] Test Results: (1) The compounds of the present invention, including YK-407, YK-401, YK-402, YK-403, 422, and YK-423, have chemical structures that are significantly different from those of prior art cationic lipids.

[0230] The series of compounds designed in this application, including YK-407, YK-401, YK-402, YK-403, 422, and YK-423, have significantly different chemical structures from prior art cationic lipids. For example, these compounds have significantly different structures compared to HHMA, and compared to SM-102, ALC-0315, Compound 21, Compound 23, and YK-009, the head groups, including G3, G4, and L, are significantly different, and the G1, L1, R1, G2, L2, and R2 groups are also significantly different. Specific structural comparisons are shown in Table 5.

[0231] [Table 5] JPEG0007761816000070.jpg240147 JPEG0007761816000071.jpg111147

[0232] As can be seen from Table 5, this series of designed compounds, including YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423, have significant differences in chemical structure from typical cationic lipids of the prior art. 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).

[0233] In comparison with typical cationic lipids of the prior art, the series of compounds designed in this application are as follows:

[0234] 1) The structural difference from HHMA is the greatest. From the chemical structure diagram, it was found that in HHMA, only one side chain of the group connected to the central N atom is similar to one side chain of this series of compounds, while the other parts are significantly different, and the structural difference is very large.

[0235] 2) The head structures of these compounds are significantly different from those of SM-102, ALC-0315, Compound 21, Compound 23, and YK-009. The head structures of these compounds contain two tertiary amine groups, an L group connecting two tertiary amine nitrogen atoms, and G3 and G4 groups connected to the two tertiary amine nitrogen atoms, respectively. However, the head structures of SM-102, ALC-0315, Compound 21, Compound 23, and YK-009 contain only one tertiary amine group and HO(CH2)2- connected to the tertiary amine nitrogen atom. Furthermore, the G1, L1, R1, G2, L2, and R2 groups of these compounds are also significantly different from those of SM-102, ALC-0315, Compound 21, Compound 23, and YK-009. Due to the significant differences in head structure, this series of compounds also differs significantly from SM-102, ALC-0315, compound 21, compound 23, HHMA, and YK-009 in terms of polarity, acid-alkali property, and hydrophilicity.

[0236] Specifically, it is as follows:

[0237] I.YK-407 YK-407 has significant structural differences compared to prior art cationic lipids, including SM-102, ALC-0315, Compound 21, Compound 23, YK-009, and HHMA.

[0238] YK-407 differs significantly from SM-102 in its headgroup: the YK-407 headgroup has two tertiary amine groups, and the L group connecting the two tertiary amine nitrogen atoms is -(CH2)2-, and the G3 and G4 groups are both HO(CH2)2-, whereas the SM-102 headgroup has one tertiary amine group, and the L group connecting the tertiary amine nitrogen atom is HO(CH2)2-. Other differences exist: in YK-407, the G1 group has two C's and the G2 group has two fewer C's compared to SM-102.

[0239] YK-407 has a significantly different head group compared to ALC-0315: the YK-407 head group has two tertiary amine groups, the L group connecting the two tertiary amine nitrogen atoms is -(CH2)2-, and the G3 and G4 groups are both HO(CH2)2-, while the ALC-0315 head group has one tertiary amine group, and the tertiary amine nitrogen atom is connected to an HO(CH2)4- group. In YK-407, compared to ALC-0315, the G1 group has three fewer Cs, the L1 group is -C(O)O-, and in ALC-0315 it is -OC(O)-, the R1 group is a linear structure and in ALC-0315 it is a branched structure, the L2 group is -C(O)O-, and in ALC-0315 it is -OC(O)-, the G2 group has one fewer C, and the R2 group has two fewer Cs per strand of the duplex.

[0240] YK-407 has a significantly different head group compared to compound 21. The YK-407 head group has two tertiary amine groups, and the L group connecting the two tertiary amine nitrogen atoms is -(CH2)2-, and both G3 and G4 groups are HO(CH2)2-. Compound 21 has a single tertiary amine group connected to the tertiary amine nitrogen atom by HO(CH2)2-. Other differences exist: In YK-407, the G1 group has four fewer carbon atoms, the R1 group is linear, and in compound 21, it is branched, and the G2 group has two fewer carbon atoms.

[0241] YK-407 has a significantly different head group compared to compound 23. The YK-407 head group has two tertiary amine groups, and the L group connecting the two tertiary amine nitrogen atoms is -(CH2)2-, and both G3 and G4 groups are HO(CH2)2-. Meanwhile, compound 23 has a single tertiary amine group connected to the tertiary amine nitrogen atom by HO(CH2)2-. Other differences exist: In YK-407, the G1 group has four fewer carbon atoms, and the R1 group is linear, whereas in compound 23, the G2 group has two fewer carbon atoms, and the R2 group has two fewer carbon atoms per strand and two more carbon atoms per strand of the duplex.

[0242] YK-407 differs significantly from YK-009 in its headgroup: the YK-407 headgroup contains two tertiary amine groups, with the L group connecting the two tertiary amine nitrogen atoms being -(CH2)2-, and both G3 and G4 groups being HO(CH2)2-. Meanwhile, the YK-009 headgroup contains a single tertiary amine group, with the tertiary amine nitrogen atom being connected to HO(CH2)2-. Other differences exist: in YK-407, the R1 group has one more C and the R2 group has one less C per chain compared to YK-009.

[0243] YK-407 has a significantly different head group compared to HHMA, which has only one side chain linked to the N atom that is similar to one side chain structure of YK-401, with other significant differences.

[0244] II.YK-401 YK-401 has significant structural differences compared to prior art cationic lipids such as SM-102, ALC-0315, Compound 21, Compound 23, YK-009, and HHMA.

[0245] YK-401 differs significantly from SM-102 in its headgroup: the YK-401 headgroup contains two tertiary amine groups, the L group connecting the two tertiary amine nitrogen atoms is -(CH2)2-, and both G3 and G4 groups are HO(CH2)2-, whereas the SM-102 headgroup contains one tertiary amine group, connected to the tertiary amine nitrogen atom by HO(CH2)2-. Other differences exist: the R1 group in YK-401 is branched, whereas in SM-102 it is linear, and the G2 group has two fewer Cs.

[0246] YK-401 has a significantly different head group compared to ALC-0315: the YK-401 head group has two tertiary amine groups, the L group connecting the two tertiary amine nitrogen atoms is -(CH2)2-, and the G3 and G4 groups are both HO(CH2)2-, while the ALC-0315 head group has one tertiary amine group, and the tertiary amine nitrogen atom is connected to an HO(CH2)4- group. In YK-401, compared to ALC-0315, the G1 group has one less C, the L1 group is -C(O)O-, and in ALC-0315 it is -OC(O)-, the R1 group has two less Cs per strand of the duplex, the G2 group has one less C, the L2 group is -C(O)O-, and in ALC-0315 it is -OC(O)-, and the R2 group has two less Cs per strand of the duplex.

[0247] YK-401 has a significantly different head group compared to compound 21. The YK-401 head group has two tertiary amine groups, and the L group connecting the two tertiary amine nitrogen atoms is -(CH2)2-, and both G3 and G4 groups are HO(CH2)2-. Compound 21 has a single tertiary amine group connected to the tertiary amine nitrogen atom by HO(CH2)2-. Other differences exist: In YK-401, the G1 group has two fewer C atoms, the R1 group has two fewer C atoms per strand, and two more C atoms per strand of the duplex, and the G2 group has two fewer C atoms.

[0248] YK-401 has a significantly different headgroup compared to compound 23. The YK-401 headgroup has two tertiary amine groups, and the L group connecting the two tertiary amine nitrogen atoms is -(CH2)2-, and both G3 and G4 groups are HO(CH2)2-. Meanwhile, compound 23 has a single tertiary amine group connected to the tertiary amine nitrogen atom by HO(CH2)2-. Other differences exist: In YK-401, the G1 group has two fewer Cs, the R1 group has two fewer Cs per strand, and two more Cs per strand of the duplex compared to compound 23. The G2 group has two fewer Cs, and the R2 group has two fewer Cs per strand, and two more Cs per strand of the duplex.

[0249] YK-401 differs significantly from YK-009 in its head group. The YK-401 head group contains two tertiary amine groups, with the L group connecting the two tertiary amine nitrogen atoms being -(CH2)2-, and both the G3 and G4 groups being HO(CH2)2-. Meanwhile, the YK-009 head group contains one tertiary amine group, with the tertiary amine nitrogen atom being connected to HO(CH2)2-. Other differences exist as well. In YK-401, the G1 group has two more carbon atoms, and the R1 group is branched, whereas in YK-009, the R2 group is linear and has one less carbon atom.

[0250] YK-401 has a significantly different head group compared to HHMA, and HHMA has only one side chain linked to the N atom that is similar to the structure of one side chain of YK-401, with other significant differences.

[0251] III.YK-402 YK-402 has significant structural differences compared to prior art cationic lipids such as SM-102, ALC-0315, Compound 21, Compound 23, YK-009, and HHMA.

[0252] YK-402 differs significantly from SM-102 in its headgroup: the YK-402 headgroup has two tertiary amine groups, and the L group connecting the two tertiary amine nitrogen atoms is —(CH)—, and both G and G groups are HO(CH)—. SM-102 has a single tertiary amine group, and the L group connected to the tertiary amine nitrogen atom is HO(CH). Other differences exist: the G group in YK-402 has one more carbon atom, the L group is —OC(O)—, whereas in SM-102 it is —C(O)O—; the R group is branched, whereas in SM-102 it is linear; the G group has one less carbon atom, and the L group is —OC(O)—, whereas in SM-102 it is —C(O)O—.

[0253] YK-402 has a significantly different headgroup compared to ALC-0315: the YK-402 headgroup has two tertiary amine groups, and the L group connecting the two tertiary amine nitrogen atoms is -(CH2)2-, and both G3 and G4 groups are HO(CH2)2-, whereas the ALC-0315 headgroup has one tertiary amine group, and the tertiary amine nitrogen atom is connected to an HO(CH2)4- group. Other differences exist: the R1 group in YK-402 has two fewer carbon atoms per strand of the duplex, and the R2 group has two fewer carbon atoms per strand of the duplex, compared to ALC-0315.

[0254] YK-402 has a significantly different head group compared to compound 21. The YK-402 head group has two tertiary amine groups, and the L group connecting the two tertiary amine nitrogen atoms is —(CH)—. The G and G groups are both HO(CH)—. Compound 21 has a single tertiary amine group, and the L group connected to the tertiary amine nitrogen atom is HO(CH). Other differences exist: In YK-402, compared to compound 21, the G group has one less C and the L group is —OC(O)—, whereas in compound 21 it is —C(O)O—. The R group has two fewer Cs in one strand and two more Cs per strand of the duplex. The G group has one less C and the L group is —OC(O)—, whereas in compound 21 it is —C(O)O—.

[0255] YK-402 has a significantly different head group compared to compound 23: the head group of YK-402 has two tertiary amine groups, the L group connecting the two tertiary amine nitrogen atoms is -(CH2)2-, and the G3 and G4 groups are both HO(CH2)2-, while the head group of compound 23 has one tertiary amine group, and the one connected to the tertiary amine nitrogen atom is HO(CH2)2-. There are other differences as well. In YK-402, compared to compound 23, the G1 group has one less C and the L1 group is -OC(O)-; in compound 23, it is -C(O)O-; the R1 group has two fewer Cs in one strand and two more Cs per strand of the duplex; the G2 group has one less C and the L2 group is -OC(O)-; in compound 23, it is -C(O)O-; the R2 group has two fewer Cs in one strand and two more Cs per strand of the duplex.

[0256] YK-402 has a significantly different head group compared to YK-009: the head group of YK-402 has two tertiary amine groups, the L group connecting the two tertiary amine nitrogen atoms is -(CH2)2-, and the G3 and G4 groups are both HO(CH2)2-, while the head group of YK-009 has one tertiary amine group, and the one connected to the tertiary amine nitrogen atom is HO(CH2)2-. There are other differences as well. In YK-402, compared with YK-009, the G1 group has three more carbon atoms, the L1 group is -OC(O)-, and in YK-009, it is -C(O)O-, and the R1 group is a branched structure, while in YK-009, it is a linear structure, the G1 group has one more carbon atom, the L2 group is -OC(O)-, and in YK-009, it is -C(O)O-, and the R2 group has one less carbon atom in a single chain.

[0257] YK-402 has a significantly different head group compared to HHMA, and HHMA has only one side chain linked to the N atom that is similar to the structure of one side chain of YK-402, with other significant differences.

[0258] IV.YK-403 YK-403 has significant structural differences compared to prior art cationic lipids such as SM-102, ALC-0315, Compound 21, Compound 23, YK-009, and HHMA.

[0259] YK-403 differs significantly from SM-102 in its headgroup: the YK-403 headgroup contains two tertiary amine groups, the L group connecting the two tertiary amine nitrogen atoms is -(CH2)2-, and both G3 and G4 groups are HO(CH2)2-, whereas the SM-102 headgroup contains one tertiary amine group, connected to the tertiary amine nitrogen atom by HO(CH2)2-. Other differences exist: the G1 group in YK-403 has two more C's, and the R1 group is branched, whereas in SM-102 it is linear.

[0260] YK-403 has a significantly different head group compared to ALC-0315: the YK-403 head group has two tertiary amine groups, the L group connecting the two tertiary amine nitrogen atoms is -(CH2)2-, and the G3 and G4 groups are both HO(CH2)2-, while the ALC-0315 head group has one tertiary amine group, and the tertiary amine nitrogen atom is connected to an HO(CH2)4- group. In YK-403, compared to ALC-0315, the G1 group has one more C and the L1 group is -C(O)O-, which in ALC-0315 is -OC(O)-; the R1 group has two fewer Cs per strand of the duplex; the G2 group has one more C and the L2 group is -C(O)O-, which in ALC-0315 is -OC(O)-.

[0261] YK-403 has a significantly different head group compared to compound 21. The head group of YK-403 has two tertiary amine groups, and the L group connecting the two tertiary amine nitrogen atoms is -(CH2)2-, and the G3 and G4 groups are both HO(CH2)2-. Meanwhile, the head group of compound 21 has one tertiary amine group, and the one connected to the tertiary amine nitrogen atom is HO(CH2)2-. There are other differences as well. In YK-403, the R1 group has two fewer Cs in one strand and two more Cs in each strand of the duplex compared to compound 21.

[0262] YK-403 has a significantly different headgroup compared to compound 23. The YK-403 headgroup has two tertiary amine groups, and the L group connecting the two tertiary amine nitrogen atoms is -(CH2)2-, and both G3 and G4 groups are HO(CH2)2-. Meanwhile, compound 23 has a single tertiary amine group connected to the tertiary amine nitrogen atom by HO(CH2)2-. Other differences exist: In YK-403, the R1 group has two fewer Cs per strand and two more Cs per strand of the duplex compared to compound 23; the R2 group has two fewer Cs per strand and two more Cs per strand of the duplex.

[0263] YK-403 differs significantly from YK-009 in its head group. The YK-403 head group has two tertiary amine groups, and the L group connecting the two tertiary amine nitrogen atoms is -(CH2)2-. The G3 and G4 groups are both HO(CH2)2-. Meanwhile, the YK-009 head group has one tertiary amine group, and the R1 group is a branched structure. Other differences exist: In YK-403, the G1 group has four more carbon atoms, and the R1 group is a branched structure, whereas in YK-009, the G2 group has two more carbon atoms, and the R2 group is a single chain with one less carbon atom.

[0264] YK-403 has a significantly different head group compared to HHMA, and HHMA has only one side chain linked to the N atom that is similar to one side chain structure of YK-403, with significant differences elsewhere.

[0265] V.YK-422 YK-422 has significant structural differences compared to prior art cationic lipids such as SM-102, ALC-0315, Compound 21, Compound 23, YK-009, and HHMA.

[0266] YK-422 differs significantly from SM-102 in its headgroup: the YK-422 headgroup contains two tertiary amine groups, with the L group connecting the two tertiary amine nitrogen atoms being -(CH2)2-, and both the G3 and G4 groups being HO(CH2)3-. SM-102's headgroup contains a single tertiary amine group, with the tertiary amine nitrogen atom being connected to HO(CH2)2-. Other differences exist: the R1 group in YK-422 is branched, whereas the R1 group in SM-102 is linear, with the G2 group having two fewer carbon atoms and the R2 group having one more carbon atom in a single chain.

[0267] YK-422 has a significantly different head group compared to ALC-0315: the YK-422 head group has two tertiary amine groups, the L group connecting the two tertiary amine nitrogen atoms is -(CH2)2-, and the G3 and G4 groups are both HO(CH2)3-, while the ALC-0315 head group has one tertiary amine group, and the one connected to the tertiary amine nitrogen atom is HO(CH2)2-. Other differences exist as well. In YK-422, compared to ALC-0315, the G1 group has one less C, the L1 group is -C(O)O-, and in ALC-0315 it is -OC(O)-, the R1 group has two less Cs per strand of the duplex, the G2 group has one less C, the L2 group is -C(O)O-, and in ALC-0315 it is -OC(O)-, and the R2 group has two less Cs per strand of the duplex.

[0268] YK-422 has a significantly different head group compared to compound 21. The YK-422 head group has two tertiary amine groups, and the L group connecting the two tertiary amine nitrogen atoms is -(CH2)2-, and both G3 and G4 groups are HO(CH2)3-. Meanwhile, compound 21 has a single tertiary amine group connected to the tertiary amine nitrogen atom by HO(CH2)2-. Other differences exist: In YK-422, the G1 group has two fewer Cs, the R1 group has one fewer C per strand, and two more Cs per strand of the duplex compared to compound 21. The G2 group has two fewer Cs, and the R2 group has one more C per strand.

[0269] YK-422 has a significantly different headgroup compared to compound 23. The YK-422 headgroup has two tertiary amine groups, and the L group connecting the two tertiary amine nitrogen atoms is -(CH2)2-, and both G3 and G4 groups are HO(CH2)3-. Meanwhile, compound 23 has a single tertiary amine group connected to the tertiary amine nitrogen atom by HO(CH2)2-. Other differences exist: In YK-422, the G1 group has two fewer Cs, the R1 group has one fewer C per strand and two more Cs per strand of the duplex compared to compound 23, the G2 group has two fewer Cs, and the R2 group has one fewer C per strand and two more Cs per strand of the duplex.

[0270] YK-422 differs significantly from YK-009 in its head group: the YK-422 head group contains two tertiary amine groups, and the L group connecting the two tertiary amine nitrogen atoms is -(CH2)2-, and both G3 and G4 groups are HO(CH2)3-. Meanwhile, the YK-009 head group contains one tertiary amine group, and the R1 group connected to the tertiary amine nitrogen atom is HO(CH2)2-. Other differences exist: the G1 group in YK-422 has two more C atoms than the YK-009 group, and the R1 group is branched, whereas the R1 group in YK-009 is linear.

[0271] YK-422 has a significantly different head group compared to HHMA, and HHMA has only one side chain linked to the N atom that is similar to the structure of one side chain of YK-422, with other significant differences.

[0272] VI.YK-423 YK-423 has significant structural differences compared to prior art cationic lipids such as SM-102, ALC-0315, Compound 21, Compound 23, YK-009, and HHMA.

[0273] YK-423 differs significantly from SM-102 in its headgroup: the YK-423 headgroup contains two tertiary amine groups, with the L group connecting the two tertiary amine nitrogen atoms being -(CH2)3-, and both the G3 and G4 groups being HO(CH2)3-. SM-102's headgroup contains a single tertiary amine group, with the tertiary amine nitrogen atom being connected to HO(CH2)2-. Other differences exist: the R1 group in YK-423 is branched, whereas the R1 group in SM-102 is linear, with the G2 group having two fewer carbon atoms and the R2 group having one more carbon atom in a single chain.

[0274] YK-423 has a significantly different head group compared to ALC-0315: the YK-423 head group has two tertiary amine groups, the L group connecting the two tertiary amine nitrogen atoms is -(CH2)3-, and both the G3 and G4 groups are HO(CH2)3-, while the ALC-0315 head group has one tertiary amine group, and the one connected to the tertiary amine nitrogen atom is HO(CH2)2-. In YK-423, compared to ALC-0315, the G1 group has one less C, the L1 group is -C(O)O-, and in ALC-0315 it is -OC(O)-, the R1 group has one more C in one strand and two more Cs in each of the two strands, the G2 group has one more C, the L2 group is -C(O)O-, and in ALC-0315 it is -OC(O)-, and the R2 group has one more C in one strand and two more Cs in each of the two strands.

[0275] YK-423 has a significantly different head group compared to compound 21. The YK-423 head group has two tertiary amine groups, and the L group connecting the two tertiary amine nitrogen atoms is -(CH2)3-, and both G3 and G4 groups are HO(CH2)3-. Meanwhile, compound 21 has a single tertiary amine group connected to the tertiary amine nitrogen atom by HO(CH2)2-. Other differences exist: In YK-423, the G1 group has two fewer Cs, the R1 group has one fewer C per strand, and two more Cs per strand of the duplex compared to compound 21. The G2 group has two fewer Cs, and the R2 group has one more C per strand.

[0276] YK-423 has a significantly different headgroup compared to compound 23. The YK-423 headgroup has two tertiary amine groups, and the L group connecting the two tertiary amine nitrogen atoms is -(CH2)3-. The G3 and G4 groups are both HO(CH2)3-. Meanwhile, compound 23 has a single tertiary amine group connected to the tertiary amine nitrogen atom by HO(CH2)2-. Other differences exist: In YK-423, the G1 group has two fewer Cs, the R1 group has one fewer C per strand and two more Cs per strand of the duplex compared to compound 23, the G2 group has two fewer Cs, and the R2 group has one fewer C per strand and two more Cs per strand of the duplex.

[0277] YK-423 differs significantly from YK-009 in its head group: the YK-423 head group contains two tertiary amine groups, and the L group connecting the two tertiary amine nitrogen atoms is -(CH2)3-, and both G3 and G4 groups are HO(CH2)3-. Meanwhile, the YK-009 head group contains one tertiary amine group, and the R1 group connected to the tertiary amine nitrogen atom is HO(CH2)2-. Other differences exist: the G1 group in YK-423 has two more C atoms, and the R1 group is branched, whereas the R1 group in YK-009 is linear.

[0278] YK-423 has a significantly different head group compared to HHMA, and HHMA has only one side chain linked to the N atom that is similar to the structure of one side chain of YK-423, with other significant differences.

[0279] As can be seen from the above comparison, this series of designed compounds, including YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423, has significantly different chemical structures compared to prior art cationic lipid compounds such as SM-102, ALC-0315, Compound 21, Compound 23, HHMA, and YK-009. This series of compounds has significantly different head groups compared to HHMA, and significantly different head groups including G3, G4, and L groups compared to SM-102, ALC-0315, Compound 21, Compound 23, and YK-009, as well as significant differences in G1, L1, R1, G2, L2, and R2 groups.

[0280] Due to the significant differences in their chemical structures, this series of compounds also has significant differences in their physicochemical properties, such as polarity, acid-alkali resistance, and hydrophilicity, compared to SM-102, ALC-0315, compound 21, compound 23, HHMA, and YK-009. Therefore, it is impossible to predict the cell transfection efficiency, cytotoxicity, and animal expression of LNP formulations prepared with this series of compounds from the above cationic lipid compounds disclosed in the prior art.

[0281] (2) Among these compounds, LNP formulations prepared with YK-407, YK-401, YK-402, YK-422, and YK-423 exhibited the highest cell transfection efficiency, significantly improved compared to representative cationic lipids of the prior art. For example, YK-407 was 12-fold higher than SM-102, 13-fold higher than compound 21, and 15-fold higher than compound 23.

[0282] [Table 6]

[0283] Differences in cell transfection efficiency Table 6 shows the fluorescence detection results of LNP formulations containing Fluc-mRNA prepared with various cationic lipids. Among them, 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), HHMA is compound 1 disclosed in CN112979483B (page 12 of the specification), and Lipofectamine 3000 is a widely used cell transfection reagent with excellent transfection performance.

[0284] As can be seen from Table 6 and Figure 5 , the LNP formulations containing Fluc-mRNA prepared with YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 had the strongest fluorescence absorption, with RLU values ​​of 20385506, 5609550, 5044102, 5249304, 6432971, and 6227115, respectively.

[0285] YK-407 can reach 11.96 times that of SM-102, 9.31 times that of ALC-0315, 13.49 times that of compound 21, 14.53 times that of compound 23, 9.81 times that of HHMA, 16.98 times that of Lipofectamine 3000, and 3.96 times that of YK-009.

[0286] YK-401 can reach 3.29 times that of SM-102, 2.56 times that of ALC-0315, 3.71 times that of compound 21, 4.00 times that of compound 23, 2.70 times that of HHMA, 4.67 times that of Lipofectamine 3000, and 1.09 times that of YK-009.

[0287] YK-402 can reach 2.96 times that of SM-102, 2.30 times that of ALC-0315, 3.34 times that of compound 21, 3.60 times that of compound 23, 2.43 times that of HHMA, 4.20 times that of Lipofectamine 3000, and 0.98 times that of YK-009.

[0288] YK-403 can reach 3.08 times that of SM-102, 2.40 times that of ALC-0315, 3.47 times that of compound 21, 3.74 times that of compound 23, 2.52 times that of HHMA, 4.37 times that of Lipofectamine 3000, and 1.02 times that of YK-009.

[0289] YK-422 can reach 3.77 times that of SM-102, 2.94 times that of ALC-0315, 4.26 times that of compound 21, 4.59 times that of compound 23, 3.09 times that of HHMA, 5.36 times that of Lipofectamine 3000, and 1.25 times that of YK-009.

[0290] YK-423 can reach 3.65 times that of SM-102, 2.84 times that of ALC-0315, 4.12 times that of compound 21, 4.44 times that of compound 23, 3.00 times that of HHMA, 5.19 times that of Lipofectamine 3000, and 1.21 times that of YK-009.

[0291] Data analysis using GraphPad Prism software showed that YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 all showed significant differences from SM-102, ALC-0315, Compound 21, Compound 23, HHMA, and Lipofectamine 3000, while YK-407 showed significant differences from YK-009, significantly improving transfection efficiency.

[0292] Interim summary In terms of chemical structure, the designed series of compounds, including YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423, are significantly different from prior art cationic lipids. For example, this series of designed compounds has significantly different head structures containing G3, G4, and L groups compared to SM-102, ALC-0315, Compound 21, Compound 23, and YK-009. The head structures of this series of compounds have two tertiary amine groups, and the two tertiary amine nitrogen atoms are linked by the L group. The head structures also have two hydroxy-containing groups G3 and G4, which are HO(CH2)2- or HO(CH2)3-, respectively, linked to the two tertiary amine nitrogen atoms, whereas the head groups of SM-102, ALC-0315, Compound 21, Compound 23, and YK-009 contain only one tertiary amine group and one hydroxy-containing group, HO(CH2)2-. Additionally, there are significant differences in the G1, L1, R1, G2, L2, and R2 groups in this series compared to SM-102, ALC-0315, Compound 21, Compound 23, and YK-009. This series also has significantly different head groups, which is a huge difference compared to HHMA.

[0293] LNP formulations prepared with YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 exhibited the highest cell transfection efficiency and significantly improved activity compared with representative cationic lipids of the prior art. For example, YK-407 was 12-fold more potent than SM-102, 13-fold more potent than compound 21, and 15-fold more potent than compound 23.

[0294] We found that not only compounds with similar chemical structures to conventional cationic lipids have cell transfection activity, but rather, LNP formulations prepared with compounds with significantly different structures may have significantly improved transfection efficiency and very high cell transfection activity.

[0295] (3) YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 have the highest cell transfection efficiency compared to the similar compounds designed in this application, in which the G3 and G4 groups are both HO(CH2)2- and the L group is -(CH2)2-. For example, YK-407 has a transfection efficiency 2500 times higher than that of YK-404 and YK-411.

[0296] To determine whether structurally similar compounds have similar transfection efficiencies, compounds designed herein in which both G3 and G4 groups are HO(CH2)2-, L is -(CH2)2-, and the remaining groups are slightly different were compared with YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423. As a result, this series of compounds exhibits very large differences in activity. Among them, YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 have the highest cell transfection efficiency, with YK-407 reaching 2,500 times that of YK-404 and YK-411, and YK-401, YK-402, YK-403, YK-422, and YK-423 all reaching more than 600 times that of YK-404 and YK-411, significantly improving transfection efficiency.

[0297] [Table 7] JPEG0007761816000074.jpg223147 JPEG0007761816000075.jpg127147

[0298] [Table 8]

[0299] a. Differences in cell transfection efficiency As can be seen from Table 8 and Figure 6, the LNP formulations prepared with these compounds are significantly different from YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 in terms of RLU values.

[0300] Specifically, YK-404 and YK-411 had the lowest RLU values, 7543 and 7976, respectively, which are significantly different from YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423.

[0301] YK-407 can reach 2702.57 times and 2555.78 times the strength of YK-404 and YK-411.

[0302] YK-401 can reach 743.68 times and 703.28 times that of YK-404 and YK-411.

[0303] YK-402 can reach 668.71 times and 632.39 times that of YK-404 and YK-411.

[0304] YK-403 can reach 695.92 times and 658.12 times the strength of YK-404 and YK-411.

[0305] YK-422 can reach 852.84 times and 806.52 times the potency of YK-404 and YK-411.

[0306] YK-423 can reach 825.55 times and 780.71 times the potency of YK-404 and YK-411.

[0307] The RLU value of YK-406 is 78251, which is significantly different from those of YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423.

[0308] YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 are 260.51, 71.69, 64.46, 67.08, 82.21, and 79.58 times the potency of YK-406, respectively.

[0309] The RLU values ​​of YK-405, YK-408, YK-409, and YK-410 were 3,197,780, 1,474,136, 669,807, and 407,868, respectively, which were relatively different from those of YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423.

[0310] YK-407 can reach 6.37 times that of YK-405, 13.83 times that of YK-408, 30.43 times that of YK-409, and 49.98 times that of YK-410.

[0311] YK-401 can reach 1.75 times that of YK-405, 3.81 times that of YK-408, 8.37 times that of YK-409, and 13.75 times that of YK-410.

[0312] YK-402 can reach 1.58 times that of YK-405, 3.42 times that of YK-408, 7.53 times that of YK-409, and 12.37 times that of YK-410.

[0313] YK-403 can reach 1.64 times that of YK-405, 3.56 times that of YK-408, 7.84 times that of YK-409, and 12.87 times that of YK-410.

[0314] YK-422 can reach 2.01 times that of YK-405, 4.36 times that of YK-408, 9.60 times that of YK-409, and 15.77 times that of YK-410.

[0315] YK-423 can reach 1.95 times that of YK-405, 4.22 times that of YK-408, 9.30 times that of YK-409, and 15.27 times that of YK-410.

[0316] There is also a relatively large difference in activity among YK-404, YK-405, YK-406, YK-408, YK-409, YK-410, and YK-411. In terms of cell transfection efficiency, YK-405 is higher than SM-102, being 1.88-fold higher, YK-408 is not significantly different from SM-102, being 0.87-fold lower, YK-406 is only 0.05-fold lower, and YK-404 and YK-411 are the lowest, being only 0.004-fold and 0.005-fold lower, respectively.

[0317] Data were analyzed using GraphPad Prism software, and it was found that YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 all showed significant differences from YK-404, YK-405, YK-406, YK-408, YK-409, YK-410, and YK-411, significantly improving the transfection efficiency.

[0318] b.Difference in chemical structure This series of compounds are very similar in structure, with only minor variations in individual groups: YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 are structurally very similar to the other compounds, and to each other.

[0319] I. Structural differences from YK-407 Compared with YK-407, YK-404 has five more carbon atoms in the G1 group and a branched structure in the R1 group, while YK-407 has a linear structure, three more carbon atoms in the G2 group, and three fewer carbon atoms in each of the two strands and six fewer carbon atoms in each of the two strands in the R2 group. The other structures are completely similar, but the cell transfection efficiency of YK-407 is 2702.57 times higher than that of YK-404.

[0320] In YK-411, the G1 group has two more Cs, the R1 group has one less C, and the R2 group has two more Cs in one strand and two less Cs in each strand of the double strand. The other structures are completely similar, but the cell transfection efficiency of YK-407 is 2555.78 times higher than that of YK-411.

[0321] In YK-406, the G2 group has one more C and the L2 group is -OC(O)-, while in YK-407 it is -C(O)O-, and the R2 group has one more C in one strand and two less C in each strand of the double strand. Except for this, the other structures are completely similar, but the cell transfection efficiency of YK-407 was 260.51 times higher than that of YK-406.

[0322] II. Structural differences from YK-401 In YK-404, compared with YK-401, the G1 and G2 groups each have three more carbon atoms, and the R1 and R2 groups each have three fewer carbon atoms in each of the two strands, and six fewer carbon atoms in each of the two strands. The other structures are completely similar, but the cell transfection efficiency of YK-401 is 743.68 times higher than that of YK-404.

[0323] In YK-411, the R1 group has a linear structure, while in YK-401, it has a branched structure. The R2 group has two more carbon atoms in one strand and two fewer carbon atoms in each strand of the double strand. The other structures are completely similar, but the cell transfection efficiency of YK-401 is 703.28 times higher than that of YK-411.

[0324] In YK-410, the R1 group has a linear structure, while in YK-401, it has a branched structure. The R2 group has one more C in the single chain, but the other structures are completely similar. However, the cell transfection efficiency of YK-401 was 13.75 times higher than that of YK-410.

[0325] III. Structural differences from YK-402 Compared with YK-402, the G1 and G2 groups of YK-404 each have two more carbon atoms, the L1 and L2 groups are -C(O)O-, and the R1 and R2 groups of YK-402 are -OC(O)-, and the R1 and R2 groups have three fewer carbon atoms in each of the two strands and six fewer carbon atoms in each of the two strands. The other structures are completely similar, but the cell transfection efficiency of YK-402 is 668.71 times higher than that of YK-404.

[0326] In YK-411, the G1 and G2 groups each have one less carbon atom, the R1 group has a linear structure, while in YK-402, it has a branched structure, the R2 group has two more carbon atoms in one strand and two less carbon atoms in each strand of the double strand, and the L1 and L2 groups are both -C(O)O-, while in YK-402 they are -OC(O)-. Other than these, the structures are completely similar, but the cell transfection efficiency of YK-402 was 632.39 times higher than that of YK-411.

[0327] In YK-406, the G1 group has three fewer carbon atoms, the L1 group is -C(O)O-, in YK-402 it is -OC(O)-, the R1 group has a linear structure, while in YK-402 it has a branched structure, and the R2 group has one more carbon atom in one strand and two fewer carbon atoms in each strand of the double strand. The other structures are completely similar, but the cell transfection efficiency of YK-402 was 64.46 times higher than that of YK-406.

[0328] IV. Structural differences from YK-403 In YK-404, the G1 and G2 groups each have one more C, and the R1 and R2 groups each have three fewer Cs in each of the two strands and six fewer Cs in each of the two strands, respectively, compared with YK-403. However, the cell transfection efficiency of YK-403 was 695.92 times higher than that of YK-404.

[0329] In YK-411, the G1 and G2 groups each have two fewer carbon atoms, and the R1 group has a linear structure. In YK-403, the R2 group has a branched structure, and the single strand of the R2 group has two more carbon atoms, and each strand of the double strand has two fewer carbon atoms. The other structures are completely similar, but the cell transfection efficiency of YK-403 was 658.12 times higher than that of YK-411.

[0330] In YK-406, the G1 group has four fewer carbon atoms, and the R1 group has a linear structure; in YK-403, it has a branched structure, the G2 group has one fewer carbon atom, and the L2 group is -OC(O)-; in YK-403, it is -C(O)O-. The R2 group has one more carbon atom in one strand and two fewer carbon atoms in each strand of the double strand. The other structures are completely similar, but the cell transfection efficiency of YK-403 was 67.08 times higher than that of YK-406.

[0331] Structural differences from V.YK-422 In YK-404, compared with YK-422, the G1 and G2 groups each have three more Cs, the G3 and G4 groups each have one less C, and the R1 and R2 groups each have one less C in one strand, three less Cs in each of the two strands, and six less Cs in one strand. The other structures are completely similar, but the cell transfection efficiency of YK-422 is 852.84 times higher than that of YK-404.

[0332] In YK-411, the G3 and G4 groups each have one less carbon atom, and the R1 group has a linear structure. In YK-422, the R2 group has a branched structure, and the single strand of the R2 group has one more carbon atom, and each strand of the double strand has two fewer carbon atoms. The other structures are completely similar, but the cell transfection efficiency of YK-422 is 806.52 times higher than that of YK-411.

[0333] In YK-406, the G1 group has two fewer carbon atoms, the G3 and G4 groups each have one fewer carbon atom, and the R1 group has a linear structure. In YK-422, it has a branched structure, the G2 group has one more carbon atom, the L2 group is -OC(O)-, and in YK-422, it is -C(O)O-. The R2 group has two fewer carbon atoms in each strand of the double strand, but the other structures are completely similar. However, the cell transfection efficiency of YK-422 was 82.21 times higher than that of YK-406.

[0334] Structural differences from VI.YK-423 In YK-404, compared with YK-423, the G1 and G2 groups each have three more Cs, the G3 and G4 groups each have one less C, the R1 and R2 groups each have one less C in one strand, three less Cs in each of the two strands, and six less Cs in one strand. The other structures are completely similar, but the cell transfection efficiency of YK-423 is 825.55 times higher than that of YK-404.

[0335] In YK-411, the G3 and G4 groups each have one less carbon atom, and the R1 group has a linear structure. In YK-423, the R2 group has a branched structure, and the R2 group has one more carbon atom in one strand and two fewer carbon atoms in each strand of the double strand. The other structures are completely similar, but the cell transfection efficiency of YK-423 was 780.71 times higher than that of YK-411.

[0336] In YK-406, the G1 group has two fewer carbon atoms, the G3 and G4 groups each have one fewer carbon atom, and the R1 group has a linear structure. In YK-422, it has a branched structure, the G2 group has one more carbon atom, the L2 group is -OC(O)-, and in YK-423, it is -C(O)O-. The R2 group has two fewer carbon atoms in each strand of the double strand, but the other structures are completely similar. However, the cell transfection efficiency of YK-423 was 79.58 times higher than that of YK-406.

[0337] Interim summary Compared with similar compounds in which the G3 and G4 groups are HO(CH2)2- and the L group is -(CH2)2-, YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 have the highest cell transfection efficiencies. For example, YK-407 is 2500 times more efficient than YK-404 and YK-411, while YK-401, YK-402, YK-403, YK-422, and YK-423 are all more than 600 times more efficient than YK-404 and YK-411, significantly improving transfection efficiencies.

[0338] We also found that there is no correspondence between compound structure and intracellular transfection efficiency, and that even a pair of compounds with very similar structures is likely to have very large differences in cell transfection efficiency.

[0339] Therefore, screening for cationic lipid compounds with high transfection efficiency from a series of compounds with very similar structures is extremely difficult and requires a great deal of creative work.

[0340] (4) YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 have the highest cell transfection efficiency compared to similar compounds designed herein, in which the G3 and G4 groups are HO(CH2)2- or HO(CH2)3-, and the L group is -(CH2)2-, -(CH2)3-, or -(CH2)4-. For example, YK-407 can achieve a transfection efficiency 170 times higher than that of YK-417 and 180 times higher than that of YK-418.

[0341] Furthermore, we compared structurally similar compounds, in which G3 and G4 are HO(CH2)2- or HO(CH2)3-, and L is -(CH2)2-, -(CH2)3-, or -(CH2)4-, with YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423. The results showed that this series of compounds exhibited significantly different activities, with YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 exhibiting the highest cell transfection efficiencies. For example, YK-407 was 170-fold more potent than YK-417 and 180-fold more potent than YK-418, significantly improving transfection efficiency.

[0342] [Table 9] JPEG0007761816000078.jpg244147 JPEG0007761816000079.jpg245147 JPEG0007761816000080.jpg217147

[0343] [Table 10]

[0344] a. Differences in cell transfection efficiency Compared with YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423, the other compounds have relatively small differences in their individual groups, but these differences have a significant impact on cell transfection efficiency, and the difference in cell transfection efficiency can reach 180-fold.

[0345] Specifically, as can be seen from Table 10, YK-417 and YK-418 are significantly different from YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423.

[0346] YK-407 can reach 173.81 times that of YK-417 and 183.24 times that of YK-418.

[0347] YK-401 can reach 47.83 times that of YK-417 and 50.42 times that of YK-418.

[0348] YK-402 can reach 43.01 times that of YK-417 and 45.34 times that of YK-418.

[0349] YK-403 can reach 44.76 times that of YK-417 and 47.18 times that of YK-418.

[0350] YK-422 can reach 54.85 times the potency of YK-417 and 57.82 times that of YK-418.

[0351] YK-423 can reach 53.09 times that of YK-417 and 55.97 times that of YK-418.

[0352] YK-413, YK-414, YK-415, and YK-421 also have significantly different activities compared to YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423.

[0353] YK-407 can reach 14.80 times that of YK-413, 45.90 times that of YK-414, 68.40 times that of YK-415, and 43.69 times that of YK-421.

[0354] YK-401 can reach 4.07 times that of YK-413, 12.63 times that of YK-414, 18.82 times that of YK-415, and 12.02 times that of YK-421.

[0355] YK-402 can reach 3.66 times that of YK-413, 11.36 times that of YK-414, 16.93 times that of YK-415, and 10.81 times that of YK-421.

[0356] YK-403 can reach 3.81 times that of YK-413, 11.82 times that of YK-414, 17.61 times that of YK-415, and 11.25 times that of YK-421.

[0357] YK-422 can reach 4.67 times that of YK-413, 14.49 times that of YK-414, 21.59 times that of YK-415, and 13.79 times that of YK-421.

[0358] YK-423 can reach 4.52 times that of YK-413, 14.02 times that of YK-414, 20.89 times that of YK-415, and 13.35 times that of YK-421.

[0359] Five compounds, YK-412, YK-416, YK-419, YK-420, and YK-424, also have very large differences in activity compared with YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423.

[0360] YK-407 can reach 6.05 times that of YK-412, 5.13 times that of YK-416, 6.44 times that of YK-419, 5.07 times that of YK-420, and 7.07 times that of YK-424.

[0361] YK-401 can reach 1.66 times that of YK-412, 1.41 times that of YK-416, 1.77 times that of YK-419, 1.40 times that of YK-420, and 1.95 times that of YK-424.

[0362] YK-402 can reach 1.50 times that of YK-412, 1.27 times that of YK-416, 1.59 times that of YK-419, 1.25 times that of YK-420, and 1.75 times that of YK-424.

[0363] YK-403 can reach 1.56 times that of YK-412, 1.32 times that of YK-416, 1.66 times that of YK-419, 1.31 times that of YK-420, and 1.82 times that of YK-424.

[0364] YK-422 can reach 1.91 times that of YK-412, 1.62 times that of YK-416, 2.03 times that of YK-419, 1.60 times that of YK-420, and 2.23 times that of YK-424.

[0365] YK-423 can reach 1.85 times that of YK-412, 1.57 times that of YK-416, 1.97 times that of YK-419, 1.55 times that of YK-420, and 2.16 times that of YK-424.

[0366] Data analysis using GraphPad Prism software showed that YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 all significantly improved cell transfection efficiency, with significant differences from other compounds.

[0367] b.Difference in chemical structure Compared to YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423, this series of compounds has slight differences in individual groups. YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 are very similar in structure to the other compounds, and to each other.

[0368] I. Structural differences from YK-407 Compared with YK-407, YK-417 has one more C in the L group, two more C in the G1 group, one less C in the R1 group, and two more C in the R2 group per strand and two less C in each strand of the double strand, but the other structures are completely similar. However, the cell transfection efficiency of YK-407 was 173.81 times higher than that of YK-417.

[0369] In YK-418, the L group has one more C, the R1 group has one less C, the G2 group has one more C, and the L2 group is -OC(O)-. In YK-407, the L2 group is -C(O)O-. The R2 group has one more C in one strand and two less Cs in each strand of the double strand. The other structures are completely similar, but the cell transfection efficiency of YK-407 was 183.24 times higher than that of YK-418.

[0370] In YK-415, the L group has one more C and the R2 group has one more C in the single chain, but the other structures are completely similar. However, the cell transfection efficiency of YK-407 was 68.40 times higher than that of YK-415.

[0371] II. Structural differences from YK-401 Compared with YK-401, YK-417 has one more C in the L group and a linear R1 group, while YK-401 has a branched structure and the R2 group has two more Cs in one strand and two fewer Cs in each strand of the double strand. The other structures are completely similar, but the cell transfection efficiency of YK-401 is 47.83 times higher than that of YK-417.

[0372] In YK-418, the L group has one more C, the G1 group has two fewer Cs, and the R1 group has a linear structure; in YK-401, it has a branched structure, the G2 group has one more C, the L2 group is -OC(O)-, and in YK-401, it is -C(O)O-. The R2 group has one more C in the single strand and two fewer Cs in each strand of the double strand, but the other structures are completely similar. However, the cell transfection efficiency of YK-401 was 50.42 times higher than that of YK-418.

[0373] In YK-415, the L group has one more C, the G1 group has two less C, and the R1 group has a linear structure, while in YK-401, it has a branched structure and the R2 group has one more C in the single chain, but the other structures are completely similar. However, the cell transfection efficiency of YK-401 was 18.82 times higher than that of YK-415.

[0374] III. Structural differences from YK-402 Compared with YK-402, the L group in YK-417 has one more carbon atom, the G1 and G2 groups each have one less carbon atom, the L1 and L2 groups are -C(O)O-, while in YK-402 they are -OC(O)-, the R1 group has a linear structure, while in YK-402 they have a branched structure, and the R2 group has two more carbon atoms in one strand and two fewer carbon atoms in each strand of the double strand. The other structures are completely similar, but the cell transfection efficiency of YK-402 was 43.01 times higher than that of YK-417.

[0375] In YK-418, the L group has one more C, the G group has three fewer C, and the L group is -C(O)O-; in YK-402, it is -OC(O)-; the R group has a linear structure, while in YK-402, it has a branched structure; and the R group has one more C in one strand and two fewer Cs in each strand of the double strand, except that the other structures are completely similar. However, the cell transfection efficiency of YK-402 was 45.34 times higher than that of YK-418.

[0376] In YK-415, the L group has one more C, the G group has three fewer C, and the L and L groups are -C(O)O-. In YK-402, it is -OC(O)-. The R group has a linear structure, while in YK-402 it is a branched structure. The R group has one more C in the single chain, but the other structures are completely similar. However, the cell transfection efficiency of YK-402 was 16.93 times higher than that of YK-415.

[0377] IV. Structural differences from YK-403 Compared with YK-403, YK-417 has one more C in the L group, two fewer Cs in the G1 and G2 groups, and a linear R1 group, while YK-403 has a branched structure and two more Cs in the R2 group per strand and two fewer Cs in each strand of the double strand. The other structures are completely similar, but the cell transfection efficiency of YK-403 was 44.76 times higher than that of YK-417.

[0378] In YK-418, the L group has one more carbon atom, the G1 group has four fewer carbon atoms, and the R1 group has a linear structure; in YK-403, it has a branched structure, the G2 group has one fewer carbon atom, the L2 group is -OC(O)-, and in YK-403, it is -C(O)O-. The R2 group has one more carbon atom in one strand and two fewer carbon atoms in each strand of the double strand. The other structures are completely similar, but the cell transfection efficiency of YK-403 was 47.18 times higher than that of YK-418.

[0379] In YK-414, the L group has one more C, the G1 and G2 groups each have four fewer C, and the R1 group has a linear structure, while in YK-401 it has a branched structure. The other structures are completely similar, but the cell transfection efficiency of YK-403 was 11.82 times higher than that of YK-414.

[0380] Structural differences from V.YK-422 Compared with YK-422, YK-417 has one more C in the L group, one less C in each of the G3 and G4 groups, and the R1 group has a linear structure, while YK-422 has a branched structure, and the R2 group has one more C in each of the single strands and two less Cs in each of the double strands. The other structures are completely similar, but the cell transfection efficiency of YK-422 is 54.85 times higher than that of YK-417.

[0381] In YK-418, the L group has one more carbon atom, the G3 and G4 groups each have one less carbon atom, the G1 group has two less carbon atoms, and the R1 group has a linear structure. In YK-422, the G2 group has one more carbon atom, the L2 group is -OC(O)-, and in YK-422, the R2 group has two less carbon atoms per strand of the double strand. The other structures are completely similar, but the cell transfection efficiency of YK-422 was 57.82 times higher than that of YK-418.

[0382] In YK-414, the L group has one more C, the G3 and G4 groups each have one less C, the G1 and G2 groups each have two less C, and the R1 group has a linear structure. In YK-422, it has a branched structure, and the R2 group has one less C in a single chain, but the other structures are completely similar. However, the cell transfection efficiency of YK-422 was 14.49 times higher than that of YK-414.

[0383] Structural differences from VI.YK-423 Compared with YK-423, YK-417 has one less C in the G3 and G4 groups, and the R1 group has a linear structure, while YK-423 has a branched structure, and the R2 group has one more C in one strand and two less C in each strand of the double strand. The other structures are completely similar, but the cell transfection efficiency of YK-423 was 53.09 times higher than that of YK-417.

[0384] In YK-418, the G3 and G4 groups each have one less carbon atom, the G1 group has two less carbon atoms, and the R1 group has a linear structure. In YK-423, the G2 group has one more carbon atom, the L2 group is -OC(O)-, and in YK-423, the R2 group has two less carbon atoms per strand of the double strand. The structures of the two are completely similar, but the cell transfection efficiency of YK-423 was 55.97 times higher than that of YK-418.

[0385] In YK-415, the G3 and G4 groups each have one less C, the G1 group has two less C, and the R1 group has a linear structure, while in YK-423 it has a branched structure. The other structures are completely similar, but the cell transfection efficiency of YK-423 was 20.89 times higher than that of YK-415.

[0386] Furthermore, even compounds with very small structural differences within this series can have large differences in cell transfection efficiency. For example, YK-416 has the same structure as YK-417, except that the R2 group has one less C in the single strand and two more C in each strand of the double strand. However, the cell transfection efficiency of YK-416 was 34 times higher than that of YK-417.

[0387] Interim summary Compared with a series of compounds with similar structures in which G3 and G4 are HO(CH2)2- or HO(CH2)3- and L is -(CH2)2-, -(CH2)3-, or -(CH2)4-, YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 have the highest cell transfection efficiencies. For example, the transfection efficiency of YK-407 is 170-fold higher than that of YK417 and 180-fold higher than that of YK-418, significantly improving the transfection efficiency.

[0388] Furthermore, there is no correspondence between compound structure and intracellular transfection efficiency, and even a pair of compounds with very small structural differences can likely result in very large differences in intracellular transfection efficiency.

[0389] Therefore, screening for cationic lipid compounds with high transfection efficiency from a series of compounds with only slight differences in chemical structure is extremely difficult and requires a great deal of creative work.

[0390] summary 1) Through multiple designs of compound structures and a large amount of creative work, we designed and screened cationic lipid compounds with high cell transfection efficiency, including YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423.

[0391] This series of compounds has significant differences in chemical structure compared to representative cationic lipids of the prior art, such as SM-102, ALC-0315, Compound 21, Compound 23, HHMA, and YK-009. The most significant difference is with HHMA, which shares only one side chain linked to the central N atom, while the other parts are significantly different. This series of compounds differs significantly in their head structures compared to SM-102, ALC-0315, Compound 21, Compound 23, and YK-009, which contain two tertiary amine groups, an L group connecting the two tertiary amine nitrogen atoms, and G3 and G4 groups, both of which contain hydroxyl groups. However, the head groups of SM-102, ALC-0315, Compound 21, Compound 23, and YK-009 contain only one tertiary amine group and HO(CH2)2- linked to the tertiary amine nitrogen atom. Due to the large structural differences, this series of compounds also differs significantly from M-102, ALC-0315, Compound 21, Compound 23, HHMA, and YK-009 in terms of polarity, acid-alkali property, and hydrophilicity.

[0392] 2) LNP formulations prepared with YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 exhibited the highest cell transfection efficiency and significantly improved activity compared to representative cationic lipids of the prior art. For example, YK-407 was 12-fold more potent than SM-102, 13-fold more potent than compound 21, and 15-fold more potent than compound 23.

[0393] Compared with similar compounds in which the G3 and G4 groups are HO(CH2)2- and the L group is -(CH2)2-, YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 have the highest cell transfection efficiencies. For example, YK-407 is 2500 times more efficient than YK-404 and YK-411, while YK-401, YK-402, YK-403, YK-422, and YK-423 are all more than 600 times more efficient than YK-404 and YK-411, significantly improving transfection efficiencies.

[0394] Compared with a series of compounds with similar structures in which G3 and G4 are HO(CH2)2- or HO(CH2)3- and L is -(CH2)2-, -(CH2)3-, or -(CH2)4-, YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 have the highest cell transfection efficiencies. For example, the transfection efficiency of YK-407 is 170-fold higher than that of YK-417 and 180-fold higher than that of YK-418, demonstrating significant improvements in transfection efficiency.

[0395] 3) There is no correlation between the structure of a compound and its intracellular transfection efficiency, and even compounds with very small structural differences are likely to have large differences in transfection efficiency. Therefore, screening for cationic lipid compounds with high transfection efficiency requires multiple designs and a large amount of creative work.

[0396] 2. Measuring Cell Viability An LNP formulation containing 1.5 μg of Fluc-mRNA (prepared according to Example 4; the carrier components of the LNP formulation were cationic lipids, neutral lipids, structural lipids, and polymer-conjugated lipids in a molar ratio of 49:10:39.5:1.5, with the cationic lipids listed in Table 1) and a Lipofectamine 3000 formulation were added to cell culture medium in a 96-well plate. After 24 hours of incubation, 10 μL of CCK-8 solution was added to each well. The culture plate was incubated in an incubator for 1 hour, and the absorbance at 450 nm was measured using a microplate reader. The results are shown in Tables 13 and 14.

[0397] Test Results: (1) Among these compounds, LNP formulations prepared with YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 exhibit significantly reduced cytotoxicity compared to representative cationic lipids of the prior art. For example, YK-401 significantly increased cell viability by 28.00% compared to ALC-0315, 4.31% compared to SM-102, and 10.94% compared to HHMA.

[0398] [Table 11]

[0399] a. Differences in cell survival Table 11 shows the cytotoxicity detection results of LNP formulations prepared with various 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), HHMA is compound 1 disclosed in CN112979483B (page 12 of the specification), and Lipofectamine 3000 is a widely used cell transfection reagent with excellent transfection performance.

[0400] As can be seen from Table 11, the LNP formulations of Fluc-mRNA prepared with YK-407, YK-401, YK402, YK-403, YK-422, and YK-423 have the lowest cytotoxicity, and the cell viability can reach 71.37%, 75.21%, 72.70%, 70.76%, 70.48%, and 71.14%, respectively.

[0401] YK-407 is 3.47% higher than SM-102, 24.16% higher than ALC-0315, 3.53% higher than compound 21, 2.26% higher than compound 23, 7.10% higher than HHMA, and 49.71% higher than Lipofectamine 3000.

[0402] YK-401 is 7.31% higher than SM-102, 28.00% higher than ALC-0315, 7.37% higher than compound 21, 6.10% higher than compound 23, 10.94% higher than HHMA, and 53.55% higher than Lipofectamine 3000.

[0403] YK-402 is 4.80% higher than SM-102, 25.49% higher than ALC-0315, 4.86% higher than compound 21, 3.59% higher than compound 23, 8.43% higher than HHMA, and 51.04% higher than Lipofectamine 3000.

[0404] YK-403 is 2.86% higher than SM-102, 23.55% higher than ALC-0315, 2.92% higher than compound 21, 1.65% higher than compound 23, 6.49% higher than HHMA, and 49.10% higher than Lipofectamine 3000.

[0405] YK-422 is 2.58% higher than SM-102, 23.27% higher than ALC-0315, 2.64% higher than compound 21, 1.37% higher than compound 23, 6.21% higher than HHMA, and 48.82% higher than Lipofectamine 3000.

[0406] YK-423 was 3.24% higher than SM-102, 23.93% higher than ALC-0315, 3.30% higher than compound 21, 2.03% higher than compound 23, 6.87% higher than HHMA, and 49.48% higher than Lipofectamine 3000 (Figure 7).

[0407] Data analysis using GraphPad Prism software showed that YK-407, YK-401, YK402, YK-403, YK-422, and YK-423 all exhibited significantly reduced cytotoxicity compared to SM-102, Compound 21, Compound 23, ALC-0315, HHMA, and Lipofectamine 3000.

[0408] b.Difference in chemical structure YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 have very different chemical structures from prior art cationic lipids, with the greatest structural difference from HHMA. The chemical structure diagram shows that in HHMA, only one side chain linked to the central N atom is similar to one side chain in this series of structures, while the other parts are significantly different.

[0409] This series of compounds differs significantly from SM-102, ALC-0315, Compound 21, Compound 23, and YK-009 in their head structures, including the G3, G4, and L groups. The head structures of these compounds contain two tertiary amine groups, an L group connecting two tertiary amine nitrogen atoms, and hydroxyl-containing G3 and G4 groups. However, the head groups of SM-102, ALC-0315, Compound 21, Compound 23, and YK-009 contain only one tertiary amine group and a HO(CH2)2- group connected to the tertiary amine nitrogen atom. Furthermore, the G1, L1, R1, G2, L2, and R2 groups in this series are also significantly different from those of SM-102, ALC-0315, Compound 21, Compound 23, and YK-009.

[0410] Interim summary Among this series of compounds, LNP formulations prepared with YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 exhibited the lowest cytotoxicity and significantly improved cell viability compared to representative cationic lipids of the prior art. For example, YK-401 demonstrated cell viability 28.00% higher than ALC-0315, 7.31% higher than SM-102, and 10.94% higher than HHMA.

[0411] YK-407, YK-401, YK402, YK-403, YK-422, and YK-423 have significant differences in chemical structure compared to representative cationic lipids of the prior art, including significantly different head groups, as well as significant differences in the G1, L1, R1, G2, L2, and R2 groups.

[0412] Therefore, it is not the case that only LNP formulations prepared with compounds that are structurally similar to prior art cationic lipids have low cytotoxicity. Rather, LNP formulations prepared with compounds that have significant structural differences are likely to have significantly reduced cytotoxicity.

[0413] (2) YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 have the lowest cytotoxicity compared to similar compounds designed herein, in which the G3 and G4 groups are HO(CH2)2- and the L group is -(CH2)2-. For example, in terms of cell viability, YK-401 is 58.88% higher than YK-411 and 50.25% higher than YK-406, while YK-407 is 55.04% higher than YK-411 and 46.41% higher than YK-406.

[0414] To compare the differences in cytotoxicity among structurally similar compounds, we compared the cell viability of a structurally similar compound in which G3 and G4 are HO(CH2)2- and L is -(CH2)2- with YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423. The results showed significant differences in cytotoxicity among this series of compounds. YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 had the highest cell viability, with YK-401 being 58.88% higher than YK-411 and 50.25% higher than YK-406, and YK-407 being 55.04% higher than YK-411 and 46.41% higher than YK-406.

[0415] [Table 12]

[0416] a. Differences in cell viability As can be seen from Table 12, the LNP formulations prepared with these compounds showed significant differences in cytotoxicity, with YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 showing the lowest toxicity and highest cell viability, while YK-411 and YK-406 showed the lowest cell viability, at only 16.33% and 24.96%, respectively.

[0417] YK-407 is 55.04% higher than YK-411 and 46.41% higher than YK-406.

[0418] YK-401 is 58.88% higher than YK-411 and 50.25% higher than YK-406.

[0419] YK-402 is 56.37% higher than YK-411 and 47.74% higher than YK-406.

[0420] YK-403 is 54.43% higher than YK-411 and 45.80% higher than YK-406.

[0421] YK-422 is 54.15% higher than YK-411 and 45.52% higher than YK-406.

[0422] YK-423 is 54.81% higher than YK-411 and 46.18% higher than YK-406.

[0423] The cell viabilities of YK-404, YK-405, YK-408, YK409, and YK-410 are 42.12%, 41.86%, 42.31%, 46.11%, and 44.92%, respectively.

[0424] YK-407 is 29.25% higher than YK-404, 29.51% higher than YK-405, 29.06% higher than YK-408, 25.26% higher than YK-409, and 26.45% higher than YK-410.

[0425] YK-401 is 33.09% higher than YK-404, 33.35% higher than YK-405, 32.90% higher than YK-408, 29.10% higher than YK-409, and 30.29% higher than YK-410.

[0426] YK-402 is 30.58% higher than YK-404, 30.84% ​​higher than YK-405, 30.39% higher than YK-408, 26.59% higher than YK-409, and 27.78% higher than YK-410.

[0427] YK-403 is 28.64% higher than YK-404, 28.90% higher than YK-405, 28.45% higher than YK-408, 24.65% higher than YK-409, and 25.84% higher than YK-410.

[0428] YK-422 is 28.36% higher than YK-404, 28.62% higher than YK-405, 28.17% higher than YK-408, 24.37% higher than YK-409, and 25.56% higher than YK-410.

[0429] YK-423 was 29.02% higher than YK-404, 29.28% higher than YK-405, 28.83% higher than YK-408, 25.03% higher than YK-409, and 26.22% higher than YK-410 (Figure 8).

[0430] Data analysis using GraphPad Prism software showed that YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 all had significant differences in cytotoxicity compared to the other compounds, with significantly reduced cytotoxicity.

[0431] b.Difference in chemical structure This series of compounds are very similar in structure, with only minor variations in individual groups: YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 are structurally very similar to the other compounds, and to each other.

[0432] Interim summary Compared with a series of structurally similar compounds in which G3 and G4 are HO(CH2)2- and L is -(CH2)2-, YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 exhibited the lowest cytotoxicity and the highest cell viability. For example, cell viability of YK-401 was 58.88% higher than that of YK-411 and 50.25% higher than that of YK-406, while that of YK-407 was 55.04% higher than that of YK-411 and 46.41% higher than that of YK-406.

[0433] They also found that there is no correspondence between compound structure and cytotoxicity, and that even a pair of compounds with the most similar structures is likely to have very large differences in cytotoxicity.

[0434] Therefore, screening for cationic lipid compounds with low cytotoxicity from a series of compounds with only slight differences in chemical structure is extremely difficult and requires a great deal of creative work.

[0435] (3) YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 have the lowest cytotoxicity compared to similar compounds designed herein in which the G3 and G4 groups are HO(CH2)2- or HO(CH2)3- and the L group is -(CH2)2-, -(CH2)3-, or -(CH2)4-. For example, YK-401 has a cytotoxicity 53.87% higher than YK-417 and 54.16% higher than YK-418, and YK-407 has a cytotoxicity 50.03% higher than YK-417 and 50.32% higher than YK-418.

[0436] Furthermore, we compared YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 with other structurally similar compounds to determine their cytotoxicity. In this series of compounds, G3 and G4 are either HO(CH2)2- or HO(CH2)3-, L is -(CH2)2-, -(CH2)3-, or -(CH2)4-, with only minor variations in other groups. As a result, this series of compounds exhibited significant differences in cytotoxicity, with YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 exhibiting the highest cell viability. For example, YK-401 is 53.87% higher than YK-417 and 54.16% higher than YK-418, and YK-407 is 50.03% higher than YK-417 and 50.32% higher than YK-418.

[0437] [Table 13]

[0438] a. Differences in cell viability As can be seen from Table 13, the cell viabilities of YK-414, YK-417, YK-418, and YK-424 were the lowest, at 28.75%, 21.34%, 21.05%, and 27.93%, respectively, all of which were below 30%.

[0439] YK-407 is 42.62% higher than YK-414, 50.03% higher than YK-417, 50.32% higher than YK-418, and 43.44% higher than YK-424.

[0440] YK-401 is 46.46% higher than YK-414, 53.87% higher than YK-417, 54.16% higher than YK-418, and 47.28% higher than YK-424.

[0441] YK-402 is 43.95% higher than YK-414, 51.36% higher than YK-417, 51.65% higher than YK-418, and 44.77% higher than YK-424.

[0442] YK-403 is 42.01% higher than YK-414, 49.42% higher than YK-417, 49.71% higher than YK-418, and 42.83% higher than YK-424.

[0443] YK-422 is 41.73% higher than YK-414, 49.14% higher than YK-417, 49.43% higher than YK-418, and 42.55% higher than YK-424.

[0444] YK-423 is 42.39% higher than YK-414, 49.80% higher than YK-417, 50.09% higher than YK-418, and 43.21% higher than YK-424.

[0445] The cell viabilities of YK-412, YK-413, YK-415, YK-416, YK-419, YK-420, and YK-421 are 54.14%, 47.20%, 61.16%, 40.97%, 38.92%, 35.59%, and 35.71%, respectively.

[0446] YK-407 is 17.23% higher than YK-412, 24.17% higher than YK-413, 10.21% higher than YK-415, 30.40% higher than YK-416, 32.45% higher than YK-419, 35.78% higher than YK-420, and 35.66% higher than YK-421.

[0447] YK-401 is 21.07% higher than YK-412, 28.01% higher than YK-413, 14.05% higher than YK-415, 34.24% higher than YK-416, 36.29% higher than YK-419, 39.62% higher than YK-420, and 39.50% higher than YK-421.

[0448] YK-402 is 18.56% higher than YK-412, 25.50% higher than YK-413, 11.54% higher than YK-415, 31.73% higher than YK-416, 33.78% higher than YK-419, 37.11% higher than YK-420, and 36.99% higher than YK-421.

[0449] YK-403 is 16.62% higher than YK-412, 23.56% higher than YK-413, 9.60% higher than YK-415, 29.79% higher than YK-416, 31.84% higher than YK-419, 35.17% higher than YK-420, and 35.05% higher than YK-421.

[0450] YK-422 is 16.34% higher than YK-412, 23.28% higher than YK-413, 9.32% higher than YK-415, 29.51% higher than YK-416, 31.56% higher than YK-419, 34.89% higher than YK-420, and 34.77% higher than YK-421.

[0451] YK-423 was 17.00% higher than YK-412, 23.94% higher than YK-413, 9.98% higher than YK-415, 30.17% higher than YK-416, 32.22% higher than YK-419, 35.55% higher than YK-420, and 35.43% higher than YK-421 (Figure 9).

[0452] Data analysis using GraphPad Prism software showed that YK-412, YK-413, YK-415, YK-416, YK-419, YK-420, and YK-421 all had significant differences in cytotoxicity compared to the other compounds, with significantly reduced cytotoxicity.

[0453] b.Difference in chemical structure This series of compounds are very similar in structure, with only minor variations in individual groups: YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 are structurally very similar to the other compounds, and to each other.

[0454] Interim summary Compared with a series of structurally similar compounds in which G3 and G4 are HO(CH2)2- or HO(CH2)3- and L is -(CH2)2-, -(CH2)3-, or -(CH2)4-, YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 have the lowest cytotoxicity. For example, YK-401 has a cytotoxicity 53.87% higher than YK-417 and 54.16% higher than YK-418, while YK-407 has a cytotoxicity 50.03% higher than YK-417 and 50.32% higher than YK-418.

[0455] They also found that there is no corresponding relationship between the structure of a compound and its cytotoxicity, and that even slight differences in structure are likely to result in very large differences in cytotoxicity.

[0456] Therefore, screening for cationic lipid compounds with low cytotoxicity from a series of compounds with only slight differences in the individual groups is extremely difficult and requires a great deal of creative work.

[0457] summary 1) We measured cell viability for LNP formulations prepared with a series of designed compounds and screened cationic lipid compounds with low cytotoxicity, including YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423.

[0458] This series of designed compounds has significant differences in chemical structure compared to representative cationic lipids of the prior art, such as SM-102, ALC-0315, Compound 21, Compound 23, HHMA, and YK-009. The greatest difference is with HHMA, in which only one side chain of the group linked to the central N atom is close to one side chain in this series of structures, while the remaining parts are significantly different. Compared to SM-102, ALC-0315, Compound 21, Compound 23, and YK-009, the head structure is significantly different, and all other groups are also different. Due to the significant differences in structure, there are also significant differences in terms of polarity, acid-alkali resistance, and hydrophilicity.

[0459] 2) LNP formulations prepared with YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 exhibited the lowest cytotoxicity and significantly improved cell viability compared to representative cationic lipids of the prior art. For example, YK-401 demonstrated cell viability 28.00% higher than ALC-0315, 7.31% higher than SM-102, and 10.94% higher than HHMA.

[0460] Compared with a series of structurally similar compounds in which G3 and G4 are HO(CH2)2- and L is -(CH2)2-, YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 exhibited the lowest cytotoxicity and the highest cell viability. For example, cell viability of YK-401 was 58.88% higher than that of YK-411 and 50.25% higher than that of YK-406, while that of YK-407 was 55.04% higher than that of YK-411 and 46.41% higher than that of YK-406.

[0461] Compared with a series of structurally similar compounds in which G3 and G4 are HO(CH2)2- or HO(CH2)3- and L is -(CH2)2-, -(CH2)3-, or -(CH2)4-, YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 have the lowest cytotoxicity. For example, in terms of cell viability, YK-401 is 53.87% higher than YK-417 and 54.16% higher than YK-418, while YK-407 is 50.03% higher than YK-417 and 50.32% higher than YK-418.

[0462] 3) There is no correlation between compound structure and cytotoxicity, and even compounds with very small structural differences are likely to have very large differences in cytotoxicity. Therefore, it is impossible to predict cytotoxicity from chemical structure, and screening for cationic lipid compounds with low cytotoxicity is extremely difficult and requires a large amount of creative work.

[0463] Example 8: In vivo validation of the performance of cationic lipid delivery vehicles We also verified the protein expression and duration of expression of mRNA delivered by the designed cationic lipids in mice.Furthermore, in vivo experiments demonstrated that the LNP delivery vehicle can effectively deliver mRNA into the body and achieve efficient and continuous expression.

[0464] LNP formulations (prepared according to Example 4) containing 10 μg of Fluc-mRNA were intramuscularly injected into female BALB / C mice aged 4-6 weeks and weighing 17-19 g. At specific time points (6 h, 24 h, 48 h, and 7 d) after administration, fluorescent imaging substrates were injected intraperitoneally. The mice were allowed to move freely for 5 minutes, after which the mean emission intensity (corresponding to the fluorescence intensity) of proteins expressed in the mice by the mRNA contained in the LNPs was detected using an IVIS Spectrum small animal in vivo imaging system.

[0465] Experimental results a. Expression of mRNA in mice The results of detecting the average radiation intensity of proteins expressed by mRNA in the LNP formulations in mice are shown in Tables 14-16 and Figures 10-13. Of this series of compounds, LNP formulations prepared with YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 achieved high and sustained mRNA expression in mice, significantly improving expression levels compared to representative cationic lipids of the prior art. For example, YK-407 achieved 27-fold higher levels than SM-102, 22-fold higher levels than ALC-0315, 28-fold higher levels than Compound 21, 27-fold higher levels than Compound 23, and 27-fold higher levels than HHMA. The mRNA expression in mice was consistent with cell transfection activity.

[0466] [Table 14]

[0467] a. Differences in expression in mice Table 14 shows the mRNA expression intensity in mice at different times for LNP formulations containing Fluc-mRNA prepared with various cationic lipids. 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 carriers for mRNA delivery.

[0468] As can be seen from Table 14, the LNP formulations containing Fluc-mRNA prepared in YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 showed high and sustained mRNA expression in mice.

[0469] The average emission intensity of YK-407 was 4,001,500 at 6 h, which was 5.79 times that of SM-102, 4.76 times that of ALC-0315, 6.66 times that of compound 21, 6.88 times that of compound 23, and 6.05 times that of HHMA, and at 24 h, it was 2,015,870, which was 16.34 times that of SM-102, 10.62 times that of ALC-0315, 18.15 times that of compound 21, 19.97 times that of compound 23, and 10.62 times that of HHMA. At 48h, it was 885288, 26.93-fold higher than SM-102, 22.06-fold higher than ALC-0315, 28.07-fold higher than Compound 21, 26.89-fold higher than Compound 23, and 26.55-fold higher than HHMA; at 7d, it was 48410, 7.20-fold higher than SM-102, 6.90-fold higher than ALC-0315, 7.59-fold higher than Compound 21, 7.93-fold higher than Compound 23, and 8.13-fold higher than HHMA.

[0470] The average emission intensity of YK-401 was 1557640 at 6 h, which was 2.25 times that of SM-102, 1.85 times that of ALC-0315, 2.59 times that of compound 21, 2.68 times that of compound 23, and 2.36 times that of HHMA. At 24 h, it was 806240, which was 6.53 times that of SM-102, 4.25 times that of ALC-0315, 7.26 times that of compound 21, 7.98 times that of compound 23, and 6.7 times that of HHMA. At 48 h, it was 393020, 11.96-fold higher than SM-102, 9.79-fold higher than ALC-0315, 12.46-fold higher than Compound 21, 11.94-fold higher than Compound 23, and 11.79-fold higher than HHMA; at 7 d, it was 10512, 1.56-fold higher than SM-102, 1.50-fold higher than ALC-0315, 1.65-fold higher than Compound 21, 1.72-fold higher than Compound 23, and 1.76-fold higher than HHMA.

[0471] The average radiation intensity of YK-402 at 6 h was 1,470,100, which was 2.13 times that of SM-102, 1.75 times that of ALC-0315, 2.45 times that of compound 21, 2.53 times that of compound 23, and 2.22 times that of HHMA, and at 24 h it was 798,408, which was 6.47 times that of SM-102, 4.21 times that of ALC-0315, 7.19 times that of compound 21, 7.91 times that of compound 23, and 6.0 times that of HHMA. At 48 h, it was 345,740, 10.52 times that of SM-102, 8.62 times that of ALC-0315, 10.96 times that of compound 21, 10.50 times that of compound 23, and 10.37 times that of HHMA; at 7 d, it was 9,940, 1.48 times that of SM-102, 1.42 times that of ALC-0315, 1.56 times that of compound 21, 1.63 times that of compound 23, and 1.67 times that of HHMA.

[0472] The average emission intensity of YK-403 was 1588400 at 6 h, which was 2.30 times that of SM-102, 1.89 times that of ALC-0315, 2.65 times that of compound 21, 2.73 times that of compound 23, and 2.40 times that of HHMA, and 855204 at 24 h, which was 6.93 times that of SM-102, 4.51 times that of ALC-0315, 7.70 times that of compound 21, 8.47 times that of compound 23, and 1.00 times that of HHMA. At 48 h, it was 308540, 9.39 times that of SM-102, 7.69 times that of ALC-0315, 9.78 times that of compound 21, 9.37 times that of compound 23, and 9.25 times that of HHMA; at 7 d, it was 9857, 1.47 times that of SM-102, 1.41 times that of ALC-0315, 1.55 times that of compound 21, 1.61 times that of compound 23, and 1.65 times that of HHMA.

[0473] The average emission intensity of YK-422 at 6 h was 1329410, which was 1.92 times that of SM-102, 1.58 times that of ALC-0315, 2.21 times that of compound 21, 2.29 times that of compound 23, and 2.01 times that of HHMA, and at 24 h it was 708120, which was 5.74 times that of SM-102, 3.73 times that of ALC-0315, 6.38 times that of compound 21, 7.01 times that of compound 23, and 1.01 times that of HHMA. At 48 h, it was 271372, 8.26-fold higher than SM-102, 6.76-fold higher than ALC-0315, 8.60-fold higher than compound 21, 8.24-fold higher than compound 23, and 8.14-fold higher than HHMA; at 7 d, it was 9476, 1.41-fold higher than SM-102, 1.35-fold higher than ALC-0315, 1.49-fold higher than compound 21, 1.55-fold higher than compound 23, and 1.59-fold higher than HHMA.

[0474] The average radiation intensity of YK-423 at 6 h was 1,627,420, 2.35 times that of SM-102, 1.94 times that of ALC-0315, 2.71 times that of compound 21, 2.80 times that of compound 23, and 2.46 times that of HHMA. At 24 h, it was 756,472, 6.13 times that of SM-102, 3.99 times that of ALC-0315, 6.81 times that of compound 21, 7.49 times that of compound 23, and 6 times that of HHMA. At 30 h, it was 350120, which was 10.65 times that of SM-102, 8.73 times that of ALC-0315, 11.10 times that of compound 21, 10.64 times that of compound 23, and 10.50 times that of HHMA, and at 7 d, it was 10093, which was 1.50 times that of SM-102, 1.44 times that of ALC-0315, 1.58 times that of compound 21, 1.65 times that of compound 23, and 1.69 times that of HHMA.

[0475] Data analysis using GraphPad Prism software showed that YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 were significantly different from SM-102, ALC-0315, Compound 21, Compound 23, and HHMA at each time point, and both the expression level and duration of expression were significantly improved.

[0476] b.Difference in chemical structure YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 have significantly different chemical structures compared to conventional cationic lipids, with the greatest structural difference from HHMA. From the chemical structure diagram, it was found that in HHMA, only one side chain of the group linked to the central N atom is similar to one side chain in this series of structures, while the other parts are significantly different. This series of compounds has significantly different head structures compared to SM-102, ALC-0315, compound 21, compound 23, and YK-009. The head groups of this series of compounds contain two tertiary amine groups, an L group connecting two tertiary amine nitrogen atoms, and both hydroxy-containing G3 and G4 groups, whereas the head groups of SM-102, ALC-0315, Compound 21, Compound 23, and YK-009 contain only one tertiary amine group and HO(CH2)2- linked to the tertiary amine nitrogen atom. Furthermore, the G1, L1, R1, G2, L2, and R2 groups of this series of compounds also differ significantly from those of SM-102, ALC-0315, Compound 21, Compound 23, and YK-009.

[0477] Interim summary Among these compounds, LNP formulations prepared with YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 demonstrated the highest mRNA expression levels in mice, with sustained expression levels at 6 h, 24 h, 48 h, and 7 days, significantly higher than those of representative cationic lipids in the prior art. For example, YK-407 was 27-fold higher than SM-102, 22-fold higher than ALC-0315, 28-fold higher than Compound 21, 27-fold higher than Compound 23, and 27-fold higher than HHMA. The mRNA expression levels in mice were consistent with the results of the cell transfection experiments in Example 7.

[0478] Furthermore, YK-407, YK-401, YK402, YK-403, YK-422, and YK-423 have significant differences in chemical structure compared to representative cationic lipids of the prior art, including significantly different head groups including G3, G4, and L groups, as well as significant differences in G1, L1, R1, G2, L2, and R2 groups.

[0479] Therefore, it is not the case that only LNP formulations prepared with compounds structurally similar to the cationic lipids of the prior art exhibit high expression in the mouse body; rather, LNP formulations prepared with compounds with significantly different structures are also likely to exhibit high and sustained mRNA expression.

[0480] (1) Compared with similar compounds designed herein, in which the G3 and G4 groups are HO(CH2)2- and the L group is -(CH2)2-, YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 have the highest mRNA expression levels and the longest duration of expression in mice. In terms of expression level, YK-407 can reach 1,000 times that of other compounds, such as YK-411. The mRNA expression in mice was consistent with cell transfection activity.

[0481] To compare the expression intensity and duration of mRNA delivered by delivery vehicles formulated with structurally similar compounds, we compared cell viability with compounds designed with G3 and G4 as HO(CH2)2-, L as -(CH2)2-, and other slight variations in other groups, YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423. The results showed significant differences in cytotoxicity among this series of compounds. LNP formulations formulated with various compounds showed significant differences in mRNA expression in mice. Among them, YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 had the highest expression levels and the longest expression durations, with YK-407 expressing 1,000-fold more mRNA than YK-411.

[0482] [Table 15]

[0483] a. Differences in expression in mice As can be seen from Table 15, compared to the compounds with similar structures in which G3 and G4 are HO(CH2)2- and L is -(CH2)2-, the LNP formulations prepared with YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 had both the highest mRNA expression levels and expression durations in mice.

[0484] YK-407 can reach 684.02 times that of YK-411 in 6 hours, 996.97 times in 24 hours, 907.99 times in 48 hours, and 100.85 times in 7 days.

[0485] YK-401 can reach 266.26 times that of YK-411 in 6 hours, 398.73 times in 24 hours, 403.10 times in 48 hours, and 21.90 times in 7 days.

[0486] YK-402 can reach 251.30 times that of YK-411 in 6 hours, 394.86 times in 24 hours, 354.61 times in 48 hours, and 20.71 times in 7 days.

[0487] YK-403 can reach 271.52 times that of YK-411 in 6 hours, 422.95 times in 24 hours, 316.45 times in 48 hours, and 20.54 times in 7 days.

[0488] YK-422 can reach 227.25 times that of YK-411 in 6 hours, 350.21 times in 24 hours, 278.33 times in 48 hours, and 19.74 times in 7 days.

[0489] YK-423 can reach 278.19 times that of YK-411 in 6 hours, 374.12 times in 24 hours, 359.10 times in 48 hours, and 21.03 times in 7 days.

[0490] Data analysis using GraphPad Prism software showed that YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 all showed significant differences from the other compounds at each time point, significantly improving both the expression level and duration of expression.

[0491] b.Difference in chemical structure This series of compounds is very similar in structure to YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423, with G3 and G4 being HO(CH2)2-, L being -(CH2)2-, and other groups being slightly different.

[0492] Interim summary Compared with compounds in which G3 and G4 are HO(CH2)2-, L is -(CH2)2-, and other groups are slightly different, LNP formulations prepared with YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 showed the highest mRNA expression intensity and longest expression duration in mice. For example, YK-407 was more than 600 times more potent than YK-411 at 6 hours, 1,000 times more potent at 24 hours, and 100 times more potent at 7 days. The mRNA expression in mice was consistent with the results of the cell transfection experiment in Example 7.

[0493] Furthermore, we found that there is no correspondence between mRNA expression in mice and the structure of the cationic lipids, and that even when LNP formulations are prepared using a set of compounds that are very similar in structure, i.e., the G3, G4, and L groups differ by one or two carbon atoms, and the other groups are slightly different, there is a high possibility that the degree and duration of mRNA expression in mice will also be very different.

[0494] Therefore, screening a series of compounds with the most similar structures for cationic lipid compounds with high and sustained expression in the body of an animal is extremely difficult and requires a great deal of creative work.

[0495] (2) YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 showed the highest mRNA expression levels and longest duration in mice compared with similar compounds designed herein, in which the G3 and G4 groups are HO(CH2)2- or HO(CH2)3- and the L group is -(CH2)2-, -(CH2)3-, or -(CH2)4-. For example, the expression level of YK-407 was 120-fold higher than that of YK-417. The mRNA expression in mice was consistent with cell transfection activity.

[0496] Furthermore, to examine differences in expression in mice, LNP formulations containing mRNA prepared with YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 were compared with similar compounds in which the G3 and G4 groups are HO(CH2)2- or HO(CH2)3-, and the L group is -(CH2)2-, -(CH2)3-, or -(CH2)4-. Within this series of compounds, there are slight differences in the individual groups, e.g., the G3, G4, or L groups differ by one or two carbon atoms, while the remaining structures are slightly different. As a result, YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 exhibited the highest expression levels and the longest expression durations, significantly higher than the other compounds. For example, the expression level of YK-407 can reach 120 times that of YK-417.

[0497] [Table 16]

[0498] a. Differences in expression in mice As can be seen from Table 19, among this series of compounds, LNP formulations prepared with YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 showed both the highest mRNA expression levels and duration of expression in mice.

[0499] In terms of expression level, YK-407 was 33.96 times higher than YK-415 and 66.16 times higher than YK-417 at 6 hours, 31.25 times higher than YK-415 and 100.86 times higher than YK-417 at 24 hours, 56.07 times higher than YK-415 and 125.66 times higher than YK-417 at 48 hours, and 11.98 times higher than YK-415 and 21.20 times higher than YK-417 at 7 days.

[0500] In terms of expression level, YK-401 was 13.22 times higher than YK-415 and 25.75 times higher than YK-417 at 6 hours, 12.50 times higher than YK-415 and 40.34 times higher than YK-417 at 24 hours, 24.89 times higher than YK-415 and 55.79 times higher than YK-417 at 48 hours, and 2.60 times higher than YK-415 and 4.60 times higher than YK-417 at 7 days.

[0501] In terms of expression level, YK-402 was 12.48 times higher than YK-415 and 24.31 times higher than YK-417 at 6 hours, 12.38 times higher than YK-415 and 39.95 times higher than YK-417 at 24 hours, 21.90 times higher than YK-415 and 49.08 times higher than YK-417 at 48 hours, and 2.46 times higher than YK-415 and 4.35 times higher than YK-417 at 7 days.

[0502] In terms of expression level, YK-403 was 13.48 times higher than YK-415 and 26.26 times higher than YK-417 at 6 h, 13.26 times higher than YK-415 and 42.79 times higher than YK-417 at 24 h, 19.54 times higher than YK-415 and 43.80 times higher than YK-417 at 48 h, and 2.44 times higher than YK-415 and 4.32 times higher than YK-417 at 7 d.

[0503] In terms of expression level, YK-422 was 11.28 times higher than YK-415 and 21.98 times higher than YK-417 at 6 hours, 10.98 times higher than YK-415 and 35.43 times higher than YK-417 at 24 hours, 17.19 times higher than YK-415 and 38.52 times higher than YK-417 at 48 hours, and 2.34 times higher than YK-415 and 4.15 times higher than YK-417 at 7 days.

[0504] In terms of expression level, YK-423 was 13.81 times higher than YK-415 and 26.91 times higher than YK-417 at 6 h, 11.73 times higher than YK-415 and 37.85 times higher than YK-417 at 24 h, 22.17 times higher than YK-415 and 49.70 times higher than YK-417 at 48 h, and 2.50 times higher than YK-415 and 4.42 times higher than YK-417 at 7 d.

[0505] Data analysis using GraphPad Prism software showed that YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 all showed significant differences from the other compounds at each time point, significantly improving both the expression level and duration of expression.

[0506] b.Difference in chemical structure In this series of compounds, only the individual groups are slightly different, for example, the G3, G4 or L groups have one or two carbon atoms different, and the other structures are slightly different. However, in terms of mRNA expression level, YK-407 can reach 120 times that of YK-417.

[0507] Interim summary Compared with similar compounds in which the G3 and G4 groups are HO(CH2)2- or HO(CH2)3- and the L group is -(CH2)2-, -(CH2)3-, or -(CH2)4-, LNP formulations prepared with YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 exhibited the highest mRNA expression intensity and longest expression duration in mice. For example, YK-407 was 120-fold more potent than YK-417 at 48 hours and 20-fold more potent at 7 days. The mRNA expression in mice was consistent with the results of the cell transfection experiment in Example 7.

[0508] Furthermore, we found that there was no correspondence between mRNA expression in the mouse body and the structure of the cationic lipid; that is, only the individual groups differed slightly; for example, the G3, G4, or L groups differed by one or two carbon atoms, and the other structures differed slightly. We also found that LNP formulations prepared with these compounds were likely to have very large differences in the degree and duration of mRNA expression in the mouse body.

[0509] Therefore, screening for cationic lipid compounds with high persistence and sustained expression in the animal body from a series of compounds with only slight differences in individual groups is extremely difficult and requires a great deal of creative work.

[0510] b. Distribution of liposomes in the mouse body The results of in vivo imaging in mice showed that there were relatively large differences in the distribution of liposomes prepared with various compounds within the mouse body, some of which showed protein expression in the liver, while some of which showed no protein expression in the liver but showed protein expression in the spleen.

[0511] Specifically, at 6 h, several cationic lipids, including SM-102, ALC-0315, compound 21, compound 23, HHMA, and YK-402, showed protein expression in the liver, whereas the expression level of YK-402 was reduced compared to SM-102. Meanwhile, several compounds, including YK-407, YK-411, YK-418, YK-419, and YK-424, showed no protein expression in the liver (Figure 14). SM-102, YK-402, YK-407, YK-419, and YK-424 showed protein expression in the spleen, but YK-407, YK-419, and YK-424 were only expressed in the spleen and could deliver mRNA directly to the spleen, but were not expressed in any other organs, such as the liver, lung, heart, or kidney (Figure 15). YK-411 and YK-418 showed no protein expression in the liver, spleen, lung, heart, or kidney, suggesting that these two compounds were expressed in muscle, whereas current mRNA vaccines typically show protein expression. ALC-0315, Compound 21, Compound 23, and HHMA are similar to SM-102 and are not shown in the figure.

[0512] From the above, it was found that, compared to cationic lipids of the prior art, liposomes prepared with some compounds designed in this application, such as YK-407, YK-411, YK-418, YK-419, and YK-424, do not remain in the liver and express target proteins after intramuscular injection. If mRNA carried in the liposomes is expressed in the liver, the expressed protein will be metabolized in the liver, increasing the burden on the liver. Therefore, compared to representative cationic lipids of the prior art, the compounds designed in this application have reduced liposomal toxicity in the liver, or some even have no liver toxicity at all.

[0513] Furthermore, liposomes prepared with several compounds designed in this study, such as YK-407, YK-419, and YK-424, can deliver mRNA directly to the spleen, without expression in other organs such as the liver, lungs, heart, and kidneys. As the largest secondary lymphoid organ in the body, the spleen is capable of rapidly inducing immune responses and producing antibodies. This significantly improves the preventive effect without changing the vaccine's components, which is of great clinical significance. This compound offers excellent targeting effects for the development of treatments for diseases caused by splenic damage or abnormalities, such as lymphoma and leukemia.

[0514] Interim summary Compared with representative cationic lipids of the prior art, such as SM-102, ALC-0315, Compound 21, Compound 23, and HHMA, liposomes prepared with compounds designed in this application either express a reduced amount of target protein in the liver (YK-402) or remain in the liver without expressing the target protein (YK-407, YK-411, YK-418, YK-419, and YK-424), resulting in reduced or no liver toxicity. Furthermore, liposomes prepared with some compounds designed in this application, such as YK-407, YK-419, and YK-424, can deliver mRNA directly to the spleen, without expression in other organs such as the liver, lungs, heart, and kidneys. This significantly improves the preventive effect without changing the vaccine's components, which is of great clinical significance.

[0515] summary 1) In vivo animal delivery experiments were conducted on LNP formulations prepared with a series of designed compounds, and cationic lipid compounds such as YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 were screened for their ability to induce high and sustained mRNA expression in mice.

[0516] This series of designed compounds has significant differences in chemical structure compared to representative cationic lipids of the prior art, such as SM-102, ALC-0315, compound 21, compound 23, HHMA, and YK-009. The head structures including G3, G4, and L groups are significantly different, and all other moieties are also different. Therefore, there are also significant differences in terms of polarity, acid-alkali property, and hydrophilicity.

[0517] 2) LNP formulations prepared with YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 demonstrated high and sustained mRNA expression in mice, significantly improving the expression levels compared to representative cationic lipids of the prior art. For example, YK-407 demonstrated 27-fold higher expression levels than SM-102, 22-fold higher expression levels than ALC-0315, 28-fold higher expression levels than Compound 21, 27-fold higher expression levels than Compound 23, and 27-fold higher expression levels than HHMA.

[0518] Compared with similar compounds with G3 and G4 being HO(CH2)2- and L being -(CH2)2-, LNP formulations prepared with YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 showed the highest mRNA expression intensity and longest expression duration in mice. For example, YK-407 was more than 600-fold more potent than YK-411 at 6 h, 1,000-fold more potent at 24 h, and 100-fold more potent at 7 d.

[0519] Compared with similar compounds with G3 and G4 groups of HO(CH2)2- or HO(CH2)3- and L groups of -(CH2)2-, -(CH2)3-, or -(CH2)4-, LNP formulations prepared with YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 showed the highest mRNA expression intensity and longest duration in mice. For example, YK-407 was 120-fold stronger than YK-417 at 48 hours and 20-fold stronger at 7 days.

[0520] Compared with representative cationic lipids of the prior art, such as SM-102, ALC-0315, Compound 21, Compound 23, and HHMA, liposomes prepared with compounds designed herein either expressed reduced amounts of target proteins in the liver (YK-402) or remained in the liver without expressing target proteins (YK-407, YK-411, YK-418, YK-419, and YK-424). Therefore, compared with cationic lipids of the prior art, LNP formulations prepared with compounds designed herein exhibit reduced or no liver toxicity. Furthermore, some compounds designed herein, such as YK-407, YK-419, and YK-424, can deliver mRNA directly to the spleen, without expression in other organs, such as the liver, lungs, heart, and kidneys. This significantly improves the preventive effect without changing the vaccine's components, which is of great clinical significance.

[0521] 3) There is no correlation between the structure of cationic lipids and the high and sustained expression of delivered mRNA in mice. Regardless of whether the structural differences in the compounds are very small or relatively large, the mRNA expression of LNP formulations prepared with them is likely to vary significantly in the animal's body. It is impossible to predict whether high and sustained mRNA expression will occur in the animal's body from the chemical structure of the cationic lipid. Screening for cationic lipid compounds that result in high and sustained mRNA expression is extremely difficult and requires a great deal of creative work.

[0522] conclusion 1. The designed series of compounds, including YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423, have significant differences in chemical structure compared with prior art cationic lipids such as SM-102, ALC-0315, compound 21, compound 23, HHMA, and YK-009. These differences include significant differences in the head groups including G3, G4, and L groups, as well as in other moieties. Consequently, they also have significant differences in terms of polarity, acidity-alkali property, and hydrophilicity.

[0523] Among the compounds designed in this study, the LNP formulations prepared with YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 showed significantly improved encapsulation rates, drug loading concentrations, and total RNA concentrations, significantly improved cell transfection efficiencies, significantly reduced cytotoxicity, and significantly improved mRNA expression levels and duration in mice compared with representative cationic lipids of the prior art. For example, YK-407 showed a 29.0% improvement in encapsulation rate over compound 23, a drug loading concentration 1.78-fold higher than that of compound 23, and a total RNA concentration 1.41-fold higher than that of compound 21. YK-407's cell transfection efficiency was 12 times higher than that of SM-102, 13 times higher than that of compound 21, and 15 times higher than that of compound 23. YK-401's cell viability was 28.00% higher than that of ALC-0315, 7.31% higher than that of SM-102, and 10.94% higher than that of HHMA. LNP formulations prepared with YK-407 were able to express 27 times more mRNA in vivo than that of SM-102, 22 times higher than that of ALC-0315, 28 times higher than that of compound 21, 27 times higher than that of compound 23, and 27 times higher than that of HHMA.

[0524] Furthermore, compared with typical cationic lipids of the prior art, LNP formulations prepared with the compounds designed herein either express a reduced amount of target protein in the liver (YK-402) or remain in the liver without expressing the target protein (YK-407, YK-411, YK-418, YK-419, and YK-424), resulting in reduced or no liver toxicity. YK-407, YK-419, and YK-424 can deliver mRNA directly to the spleen, without expression in other organs such as the liver, lungs, heart, and kidneys, significantly improving the preventive effect without changing the vaccine's components.

[0525] Among the designed compounds, LNP formulations prepared with YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 showed significantly improved cell transfection efficiency, significantly reduced cytotoxicity, and significantly improved both the amount and duration of mRNA expression in mice compared to other compounds.

[0526] Compared with other compounds, YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423, the structures are similar and only differ in individual groups (e.g., the G3, G4, or L groups differ by one or two carbon atoms). Other structures are slightly different, but the activity is significantly different. For example, YK-407 has a cell transfection efficiency 2,500 times higher than that of YK-404 and YK-411. YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423 all have cytotoxicity 50% lower than that of YK-411. LNP formulations prepared with YK-407 can express 1,000 times more mRNA in mice than YK-411.

[0527] 2. There is no clear correlation between the structure of cationic lipid compounds and their intracellular transfection efficiency, cellular toxicity, or the high and sustained expression of mRNA in vivo in animals using LNP formulations prepared with them. Compounds with very small structural differences are likely to have very large differences in transfection efficiency, cellular toxicity, and intracellular expression.

[0528] For example, YK-411 has a very similar structure to YK-407. Although YK-411 simply has two more Cs in the G1 group, one less C in the R1 group, and two more Cs in the R2 group per strand and two less Cs in each strand of the double strand, the cell transfection efficiency of YK-407 is 2500 times higher than that of YK-411, the toxicity of YK-407 to transfected cells is 55% lower than that of YK-411, and the mRNA expression in mice is 1000 times higher than that of YK-411.

[0529] Therefore, screening for suitable cationic lipid compounds that simultaneously have high transfection efficiency, low toxicity to cells, and high and sustained expression of mRNA in mice is extremely challenging and requires a great deal of creative work.

[0530] 3. Through unique design and extensive screening, the present invention has discovered several compounds, including YK-407, YK-401, YK-402, YK-403, YK-422, and YK-423, which can deliver nucleic acids with significantly improved encapsulation rates, drug loading concentrations, and total RNA concentrations, significantly improved cell transfection efficiencies, significantly reduced cytotoxicity, and significantly improved expression levels and durations in animal bodies, compared with other conventional compounds, achieving unexpected technical effects.

Claims

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

2. A compound which is Compound YK-407, Compound YK-401, Compound YK-402, Compound YK-403, Compound YK-422 or Compound YK-423 having the following structure, or a pharmaceutically acceptable salt thereof. 【Chemistry 2】

3. 10. A composition comprising a carrier, said carrier comprising a cationic lipid, said cationic lipid comprising the compound of claim 1 or 2, or a pharmaceutically acceptable salt thereof.

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

5. The composition of claim 3 , wherein the carrier further comprises a neutral lipid.

6. 6. The composition of claim 5, wherein the molar ratio of the cationic lipid to the neutral lipid is from 1:1 to 15:

1.

7. 6. The composition of claim 5, wherein the neutral lipid comprises one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol, and derivatives thereof.

8. The neutral lipids include 1,2-dilinolenoyl-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 (18:0 Diether). PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-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), palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-phosphatidyl The composition according to claim 7, wherein the phosphatidylcholine is one or more selected from the group consisting of 1-stearoyl-2-oleoyl-phosphatidylcholine (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 3 , wherein the carrier further comprises a structured lipid.

10. 10. The composition of claim 9, wherein the molar ratio of said cationic lipid to said structural lipid is from 0.6:1 to 3:

1.

11. 10. The composition of claim 9, wherein the structural lipid is one or more selected from cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, α-tocopherol, and corticosteroids.

12. The composition of claim 3 , wherein the carrier further comprises a polymer-conjugated lipid.

13. The composition of claim 12, wherein the molar ratio of the polymer-conjugated lipid to the carrier is 0.5% to 10%.

14. The composition of claim 12, wherein the polymer-conjugated lipid is one or more selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

15. 15. The composition of claim 14, wherein the polymer-conjugated lipid is one or more selected from distearoylphosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), dimyristoylglycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG2000), and methoxypolyethylene glycol ditetradecylacetamide (ALC-0159).

16. 4. The composition of claim 3, 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).

17. The composition according to any one of claims 3 to 16, wherein the composition is a nanoparticle formulation, the mean particle size of the nanoparticle formulation is 10 nm to 300 nm, and the polydispersity of the nanoparticle formulation is 50% or less.

18. 17. The composition of any one of claims 3 to 16, wherein the cationic lipid further comprises one or more other ionizable lipid compounds.

19. The composition of any one of claims 3 to 16, further comprising a therapeutic or prophylactic agent.

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

1.

21. the therapeutic or prophylactic agent comprises one or more of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein; or 20. The composition of claim 19, wherein the therapeutic or prophylactic agent is a vaccine or compound capable of eliciting an immune response.

22. 20. The composition of claim 19, wherein the therapeutic or prophylactic agent is a nucleic acid.

23. 23. The composition of claim 22, wherein the therapeutic or prophylactic agent is a ribonucleic acid (RNA).

24. 23. The composition of claim 22, wherein the therapeutic or prophylactic agent is deoxyribonucleic acid (DNA).

25. 24. The composition of claim 23, wherein the RNA is selected from the group consisting of small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), short hairpin RNA (shRNA), messenger RNA (mRNA), and mixtures thereof.

26. 26. Use of a compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, or a composition according to any one of claims 3 to 25, in the preparation of a medicament for treating a disease or disorder in a mammal in need thereof.

27. 27. The use of claim 26, wherein 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.

28. 28. The use according to claim 27, wherein the infectious disease is selected from diseases caused by coronavirus, influenza virus or HIV virus, childhood pneumonia, Rift Valley fever, yellow fever, rabies, or multiple types of herpes.

29. The use according to any one of claims 26 to 28, wherein the composition is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation.

30. The use according to any one of claims 26 to 28, wherein the drug is administered to the mammal at a dose of 0.001 mg / kg to 10 mg / kg.

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