Highly efficient, low-toxicity, stably expressed cationic lipid compounds and compositions thereof

Abstract

The present invention provides highly efficient, low-toxicity, stably expressed cationic lipid compounds that are compounds of formula (I) or N-oxides, solvates, pharmaceutically acceptable salts, or stereoisomers thereof, as well as compositions comprising such compounds and their use in the delivery of therapeutic or prophylactic agents. JPEG0007764663000087.jpg2335

Description

[Technical Field]

[0001] This application claims priority from Chinese Patent Application No. 202310010915.X, filed on January 5, 2023, the entire disclosure of which is incorporated herein by reference.

[0002] The present invention is in the field of medicine, and specifically relates to cationic lipid compounds, compositions containing same, and uses. [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, their safety, functionality, and specificity remain to be improved. Furthermore, the increased complexity of lipid nanoparticles (LNPs) complicates their production and potentially increases 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 which are compounds of formula (I) or N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers thereof: [ka] (however, G1 is C 1-6 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 6-15 straight chain alkyl, or R3-S-R4, R5-SS-R6; R2 is C 12-25 is a branched alkyl; G3 is HO-R7- or (CH3)2N(CH2)3C(O)O(CH2)2-, where R3 is C 1-7 alkylene, and R4 is C 1-7 is alkyl, R5 is C 1-7 alkylene, and R6 is C 1-7 is alkyl, R7 is C 2-8 Alkylene, C 5-6 Cycloalkylene, -CH2CH(OH)CH2-, -(CH2CH2O) m -(CH)-, or -(CH)O-(CH)-, where m and n are each independently 1, 2, 3, or 4. For example, compounds of formula (I) have one of the following structures: [ka] JPEG0007764663000004.jpg250101 JPEG0007764663000005.jpg242100 JPEG0007764663000006.jpg242122 JPEG0007764663000007.jpg153116

[0006] 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.

[0007] 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.

[0008] 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]

[0009] [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, 3:1; 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] 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 are shown below: a) 30:10:58.5:1.5, 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-101, b: YK-107, c: YK-108, d: SM-102) is shown. [Figure 6] Fluorescence absorption intensities of LNP formulations of Fluc-mRNA prepared with various cationic lipids (a: YK-102, b: YK-104, c: YK-106, d: Compound 21) are shown. [Figure 7] Fluorescence absorption intensity of LNP formulations of Fluc-mRNA prepared with various cationic lipids (a: YK-101, b: YK-108, c: YK-112, d: YK-119) is shown. [Figure 8] LNP formulations of Fluc-mRNA prepared with various cationic lipids (YK-101, YK-107, YK-108, 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 9] LNP formulations of Fluc-mRNA prepared with various cationic lipids (YK-101, YK-107, YK-108, YK-102, YK-103, YK-104, YK-105, YK-106, YK-109, 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] Fluc-mRNA LNP formulations prepared with various cationic lipids (YK-101, YK-107, YK-108, YK-110, YK-111, YK-112, YK-113, YK-114, YK-115, YK-116, YK-117, YK-118, YK-119, YK-120, 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 11]LNP formulations of Fluc-mRNA prepared with various cationic lipids (YK-101, YK-107, YK-108, YK-121, YK-122, YK-123, YK-124, YK-125, YK-126, 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 12] Shown are the results of in vivo imaging experiments in mice of LNP formulations of Fluc-mRNA prepared with various cationic lipids (YK-101, YK-102, YK-109, YK-120, SM-102, ALC-0315, Compound 21, Compound 23, and HHMA). [Figure 13] Shown are the results of in vivo imaging experiments in mice of LNP formulations of Fluc-mRNA prepared with various cationic lipids (YK-106, YK-107, YK-111, YK-123, SM-102, ALC-0315, Compound 21, Compound 23, and HHMA). [Figure 14] We show the results of in vivo imaging experiments in mice of LNP formulations of Fluc-mRNA prepared with various cationic lipids (YK-104, YK-108, YK-114, YK-126, YK-009, SM-102, ALC-0315, Compound 21, Compound 23, and HHMA). DETAILED DESCRIPTION OF THE INVENTION

[0010] 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.

[0011] 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 another embodiment. The present invention includes the other embodiments obtained by such combinations.

[0012] 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.

[0013] The section titles used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0014] 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.

[0015] 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.

[0016] 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, or closed. In other words, the terms also include "consisting essentially of" or "consisting of."

[0017] 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.

[0018] As used herein, the term "subject" or "patient" refers to mammals, including but not limited to humans, non-human primates, farm animals such as cows, horses, sheep, goats, and pigs, domestic animals such as rabbits, dogs, and cats, laboratory animals including rodents such as rats, mice, and guinea pigs, etc. In some embodiments, the subject or patient is human.

[0019] 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.

[0020] The term "solvate," as used herein, 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 upon absorption by a subject, will be the compound of Formula (I); thus, each use of a compound of Formula (I) should be understood to encompass the use of all solvates of the compound of Formula (I).

[0021] The term "hydrate" refers to the above term "solvate" where the solvent is water.

[0022] 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.).

[0023] 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-hydroxybenzoylbenzoic acid, 2-methyl-3-methylpropional, 2-methyl-4-methylpropional, 2-methyl-5-methylpropional, 2-methyl-6-methylpropional, 2-methyl-7-methylpropional, 2-methyl-8-methylpropional, 2-methyl-9-methylpropional, 2-methyl-1 ... 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).

[0024] 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.

[0025] Certain compounds of the present invention may exist in one or more stereoisomeric forms. Stereoisomers include geometric isomers, diastereomers, and enantiomers. Therefore, compounds protected by the present invention include racemic mixtures, single stereoisomers, and optically active mixtures. Those skilled in the art will appreciate that certain stereoisomers may have superior efficacy and / or fewer side effects than 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.

[0026] 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.

[0027] 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 2-8Alkylene includes C2 alkylene, C3 alkylene, C4 alkylene, C5 alkylene, C6 alkylene, C7 alkylene, and C8 alkylene. 5-6 Cycloalkylene refers to cyclopentylene or cyclohexylene.

[0028] An alkyl (or alkylene or cycloalkylene) can be unsubstituted, or an alkyl (or alkylene or cycloalkylene) can be substituted, where at least one hydrogen is replaced with another chemical group, such as hydroxy, halogen, etc. For example, C alkylene includes -(CH)-, -CHCH(OH)CH-, -CH(CH)CH-, etc. C alkylene includes -(CH)-, -CHCH(CH)CH-, -CH(CH)CHCH-, etc.

[0029] 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.

[0030] In this application, when the name and structural formula of a compound are inconsistent, the structural formula shall prevail.

[0031] 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.

[0032] The term cationic lipid as used herein refers to lipids that are positively charged at selected pH values. 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).

[0033] The present inventors have found that when screening many compounds, it is very difficult to find a suitable cationic lipid compound that simultaneously has high transfection efficiency, extremely low cytotoxicity, and ultra-high expression and sustained expression in the body of mice.The present inventors have found that some compounds, such as YK-101, YK-107, and YK-108, can deliver nucleic acids with significantly improved intracellular transfection efficiency, significantly reduced cytotoxicity, and significantly improved expression and sustained expression in the body of animals compared with prior art compounds.

[0034] Here, SM-102 (compound 25 described in WO2017049245A2) was selected for comparison with the designed compounds. SM-102 is currently widely used for mRNA delivery.

[0035] Chemical structure of SM-102: [ka] The compounds of the present invention were also compared with ALC-0315 (compound 3 disclosed in CN108368028B), cationic lipid compounds 21 and 23 disclosed in WO2021055833A1, and HHMA (compound 1 disclosed in CN112979483B).

[0036] Chemical structure of ALC-0315: [ka] Chemical structure of compound 21: [ka] Chemical structure of compound 23: [ka] Chemical structure of HHMA: [ka] Among the designed series of compounds, the LNP formulations prepared with YK-101, YK-107, and YK-108 have significantly improved cell transfection activity, significantly reduced cytotoxicity, and significantly improved mRNA expression levels and duration in mice compared with similar cationic lipids or cationic lipids used in the prior art. For example, YK-101, YK-107, and YK-108 have cell transfection activity 16 times higher than that of SM-102 (compound 25 disclosed in WO2017049245A2) and 19 times higher than that of compound 23 (compound 23 disclosed in WO2021055833A1). Their cell viability is 39% higher than that of ALC-0315 (compound 3 disclosed in CN108368028B) and 22% higher than that of SM-102 and HHMA (compound 1 disclosed in CN112979483B). Their mRNA expression levels in mice are 11 times higher than that of SM-102 and 12 times higher than that of compound 21 (compound 21 disclosed in WO2021055833A1) and compound 23.

[0037] Even compounds with minimal structural differences can have significant differences in transfection efficiency, cytotoxicity, and intracellular expression. For example, in the case of compounds YK-108 and YK-109 of the present application, YK-108 has a 34-fold higher cell transfection efficiency than YK-109, a 20% higher cell viability than YK-109, and a 15-fold higher mRNA expression in mice than YK-109. YK-101, YK-107, and YK-108 all have a 10,000-fold higher cell transfection activity than YK-112, a 80% higher cell viability than YK-112, and a 62-fold higher mRNA expression in animals than YK-126.

[0038] The compounds of the present invention have been found to have significant improvements in transfection efficiency and / or cell toxicity. For example, YK-101, YK-107, and YK-108 have cell transfection efficiency 16 times higher than SM-102 and 19 times higher than compound 23, cell viability 39% higher than ALC-0315 (compound 3 disclosed in CN108368028B), and 22% higher than SM-102 and HHMA (compound 1 disclosed in CN112979483B), and mRNA expression levels in mice are 11 times higher than SM-102 and 12 times higher than compound 21 and compound 23.

[0039] It was also found that there is no clear correlation between the structure of cationic lipid compounds and the intracellular transfection efficiency, cellular toxicity, or high and sustained mRNA expression in animal bodies in LNP formulations prepared with cationic lipid compounds. Even compounds with minimal structural differences can have significant differences in transfection efficiency, cellular toxicity, and high intracellular expression. For example, in the case of the compounds YK-108 and YK-109 of the present application, YK-108 exhibited a 34-fold higher cell transfection efficiency than YK-109, a 20% higher cell viability than YK-10, and 15-fold higher mRNA expression in mice than YK-109. YK-101, YK-107, and YK-108 all have cell transfection activity more than 10,000 times higher than YK-112, and cell viability is 80% higher than YK-112. Compound YK-107 expresses mRNA in vivo 62 times more efficiently than YK-126. Therefore, it is extremely difficult to find a suitable cationic lipid compound that simultaneously exhibits high transfection efficiency, low toxicity to cells, and high and sustained mRNA expression in mice.

[0040] Through unique design and extensive screening, the present invention has discovered several compounds, such as YK-101, YK-107, YK-108, YK-103, YK-104, YK-105, and YK-106, which can deliver nucleic acids with significantly improved cell transfection efficiency, significantly reduced cytotoxicity, and significantly improved expression and sustained expression in animal bodies, compared with other compounds in the prior art, achieving unexpected technical effects.

[0041] 1. The cell transfection efficiency is significantly improved compared to the activity of cationic lipids in the prior art and the designed cationic lipids.

[0042] 1) Through extensive design of compound structures and a large amount of creative work, we screened cationic lipid compounds with high cell transfection efficiency, such as YK-101, YK-107, and YK-108.

[0043] The LNP formulations prepared with YK-101, YK-107, and YK-108 exhibited the highest cell transfection activity, significantly improving it compared with similar cationic lipids or cationic lipids used in the prior art. For example, YK-108 exhibited cell transfection activity 16-fold higher than that of SM-102 and 19-fold higher than that of compound 23.

[0044] Among a series of compounds with the most similar designed structures, YK-101, YK-107, and YK-108 have the highest cell transfection activity, reaching 34-fold that of other compounds such as YK-109.

[0045] YK-101, YK-107, and YK-108 have the highest transfection activity compared to compounds with minor structural differences only in the G1, G3, or R1 groups, up to 10,000-fold higher than other compounds such as YK-112.

[0046] YK-101, YK-107, and YK-108 have the highest transfection activity compared to compounds in which an ether bond is simply introduced into the G3 group or the G3 group is modified to 4-(dimethylamino)butyryl, for example, up to 300-fold higher than that of YK-124.

[0047] 2) There is no correlation between compound structure and intracellular transfection efficiency. Even compounds with very small structural differences are likely to have very large differences in transfection efficiency.

[0048] 3) Changing the same group in different compounds to another identical group does not necessarily have the same effect on activity, and may even have the opposite effect. In other words, it is impossible to predict the change in activity depending on the change in chemical structure. Therefore, screening for cationic lipid compounds with high transfection efficiency requires a lot of design and creative work.

[0049] 2. Cytotoxicity is significantly reduced compared to prior art cationic lipids and designed cationic lipids.

[0050] 1) We measured cell viability for LNP formulations prepared with a series of designed compounds and screened cationic lipid compounds with low cytotoxicity, such as YK-101, YK-107, and YK-108.

[0051] LNP formulations prepared with YK-101, YK-107, and YK-108 exhibited the lowest cytotoxicity, significantly reduced compared to similar or previously used cationic lipids. For example, these three compounds exhibited cell viability 39% higher than that of ALC-0315 (compound 3 disclosed in CN108368028B) and 22% higher than that of SM-102 and HHMA (compound 1 disclosed in CN112979483B).

[0052] YK-101, YK-107, and YK-108 are the least cytotoxic compounds among the most structurally similar compounds designed, i.e., compounds differing by only 1–2 carbon atoms in individual groups such as G1, G2, and R1. These three compounds have 45% higher cell viability than other compounds, such as YK-103.

[0053] YK-101, YK-107, and YK-108 have the lowest cytotoxicity compared to compounds with only minor structural differences in the G1, G3, or R1 groups. These three compounds are 80% more effective than other compounds, such as YK-112, in terms of cell viability.

[0054] YK-101, YK-107, and YK-108 have the lowest cytotoxicity compared to compounds with an ether bond simply introduced into the G3 group or with the G3 group modified to 4-(dimethylamino)butyryl. For example, these three compounds showed a 60% increase in cell viability compared to YK-126.

[0055] 2) Furthermore, there is no correspondence between the structure of a compound and its cytotoxicity. Even compounds with very small structural differences are likely to have very large differences in their cytotoxicity.

[0056] 3) It is impossible to predict cytotoxicity from chemical structure. Screening for compounds with high transfection efficiency and low cytotoxicity is extremely difficult and requires a great deal of creative work.

[0057] 3. The expression level and duration in the animal body are significantly improved compared to conventional cationic lipids and designed cationic lipids.

[0058] 1) We conducted experiments to evaluate the in vivo performance of LNP formulations prepared from a series of designed compounds as delivery vehicles in animals, and screened cationic lipid compounds such as YK-101, YK-107, and YK-108, which induced high and sustained mRNA expression in mice.

[0059] The LNP formulations prepared with YK-101, YK-107, and YK-108 showed high and sustained mRNA expression in mice, significantly improving upon similar or previously used cationic lipids. For example, YK-108 was 11-fold higher than SM-102 and 12-fold higher than Compounds 21 and 23.

[0060] YK-101, YK-107, and YK-108 are the most structurally similar compounds, i.e., differing by only one or two carbon atoms in individual groups such as G1, G2, and R1. YK-108 exhibited the highest mRNA expression levels and the longest duration of expression in mice. The expression level of YK-108 was 14-fold higher than that of other compounds, such as YK-109.

[0061] YK-101, YK-107, and YK-108 showed the highest mRNA expression levels and durations in mice compared with compounds with only slight structural differences in the G1, G3, or R1 groups. For example, YK-108's expression level was 15 times higher than that of YK-114.

[0062] YK-101, YK-107, and YK-108 have the highest mRNA expression levels and the longest duration in mice compared with compounds in which an ether bond is simply introduced into the G3 group or the G3 group is changed to 4-(dimethylamino)butyryl. For example, YK-108 can achieve up to 67-fold higher expression levels than YK-126.

[0063] 2) There is no correlation between the structure of cationic lipids and high and sustained mRNA expression in mice. In other words, even if the structural differences between cationic lipid compounds are minimal, the mRNA in LNP formulations prepared with these compounds is likely to be significantly different in vivo.

[0064] 3) It is impossible to predict from the chemical structure of a cationic lipid whether it will result in high and sustained mRNA expression in the animal body. Screening for cationic lipid compounds that result in high and sustained mRNA expression is extremely difficult and requires a great deal of creative work.

[0065] 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 can be 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.

[0066] 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] (however, G1 is C 1-6 Alkylene, preferably unsubstituted C 2-5 alkylene, more preferably unsubstituted C3, C5 or C4 alkylene, even more preferably unsubstituted C3, C5 or C4 linear alkylene; G2 is C 2-8 Alkylene, preferably unsubstituted C 4-6 alkylene, more preferably unsubstituted C5, C6 or C4 alkylene, even more preferably unsubstituted C5, C6 or C4 linear alkylene; G3 is HO-R7-, or (CH3)2N(CH2)3C(O)O(CH2)2-, where R7 is C 2-8 Alkylene, C 5-6 Cycloalkylene, -CH2CH(OH)CH2-, -(CH2CH2O) m -(CH2) n - or -(CH2) m -O-(CH2) n -, where m and n are each independently 1, 2, 3, or 4; R7 is, for example, -(CH2)2-, -(CH2)3-, -CH2CH(OH)CH2-, -CH(CH3)CH2-, -CH(CH3)(CH2)2-, -C(CH3)2(CH2)2-, -(CH2)2O(CH2)2O(CH2)2-, -(CH2)2O(CH2)2-, or [ka] and G3 is preferably HO(CH3)2- or HO(CH3)3-, L1 is -C(O)O- or -OC(O)-; L2 is -C(O)O- or -OC(O)-; R1 is C 6-15 straight chain alkyl, R3-S-R4, or R5-SS-R6, where R3 is C 1-7 alkylene, preferably -CH2CH2-, and R4 is C 1-7 alkyl, preferably C4 linear alkyl, and R5 is C 1-7 alkylene, preferably -CH2CH2CH2-, and R6 is C 1-7 alkyl, preferably a C5 linear alkyl; R1 is preferably unsubstituted C 8-12 Straight chain alkyl, more preferably unsubstituted C 10 , C 11 , C9 or C8 linear alkyl; R2 is C 12-25 Branched alkyl, preferably unsubstituted C 14-22 Branched alkyl, more preferably unsubstituted C 15 , C 18 , and C 14 It is a branched alkyl.

[0067] In some embodiments, G1 is an unsubstituted C3 alkylene, for example, -(CH2)3-.

[0068] In some embodiments, G1 is an unsubstituted C5 alkylene, for example, -(CH2)5-.

[0069] In some embodiments, G1 is unsubstituted C4 alkylene, for example, -(CH2)4-.

[0070] In some embodiments, G2 is an unsubstituted C5 alkylene, for example, -(CH2)5-.

[0071] In some embodiments, G2 is an unsubstituted C6 alkylene, such as -(CH2)6-.

[0072] In some embodiments, G2 is unsubstituted C4 alkylene, for example, -(CH2)4-.

[0073] In some embodiments, G3 is HO(CH2)2-.

[0074] In some embodiments, G3 is HO(CH2)3-.

[0075] In some embodiments, G3 is HOCH2CH(OH)CH2-.

[0076] In some embodiments, G3 is HOCH(CH3)CH2-.

[0077] In some embodiments, G3 is HOCH(CH3)(CH2)2-.

[0078] In some embodiments, G3 is HOC(CH3)2(CH2)2-.

[0079] In some embodiments, G3 is HO(CH2)2O(CH2)2O(CH2)2-.

[0080] In some embodiments, G3 is HO(CH2)2O(CH2)2-.

[0081] In some embodiments, G3 is [ka] is.

[0082] In some embodiments, L1 is -C(O)O-.

[0083] In some embodiments, L1 is -OC(O)-.

[0084] In some embodiments, L2 is -C(O)O-.

[0085] In some embodiments, L2 is -OC(O)-.

[0086] In some embodiments, R is unsubstituted C 8-12 Straight chain alkyl, preferably unsubstituted C 10 It is a straight chain alkyl, i.e., -(CH2)9CH3.

[0087] In some embodiments, R is unsubstituted C 8-12 Straight chain alkyl, preferably unsubstituted C 11 Straight chain alkyl, i.e., -(CH2) 10 It is CH3.

[0088] In some embodiments, R2 is an unsubstituted C 14-22 Branched alkyl, preferably unsubstituted C 14 , C 15 or C 18 For example, R2 is [ka] is.

[0089] In some embodiments, G1 is —(CH2)3—, G2 is —(CH2)5—, G3 is HO(CH2)2—, L1 is —C(O)O—, L2 is —C(O)O—, R1 is —(CH2)9CH3, and R2 is [ka] is.

[0090] In some embodiments, G1 is —(CH2)3—, G2 is —(CH2)5—, G3 is HO(CH2)3—, L1 is —C(O)O—, L2 is —C(O)O—, R1 is —(CH2)9CH3, and R2 is [ka] is.

[0091] In some embodiments, G1 is —(CH2)3—, G2 is —(CH2)6—, G3 is HO(CH2)2—, L1 is —C(O)O—, L2 is —C(O)O—, and R1 is —(CH2) 10 CH3 and R2 is [ka] is.

[0092] In an exemplary embodiment, the invention provides a compound of Formula (I), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, selected from: [ka] JPEG0007764663000021.jpg250103 JPEG0007764663000022.jpg24295 JPEG0007764663000023.jpg235114 JPEG0007764663000024.jpg200122

[0093] 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.

[0094] In some embodiments, the composition is a nanoparticle formulation, wherein the nanoparticle formulation has an average size of 10 nm to 300 nm, preferably 90 nm to 260 nm, more preferably 90 nm to 200 nm, and a polydispersity of 50% or less, preferably 40% or less, more preferably 30% or less.

[0095] 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 some embodiments, the cationic lipid is selected from the compounds of formula (I) above. For example, the cationic lipid is compound YK-101, YK-102, YK-103, YK-104, YK-105, YK-106, YK-107, YK-108, YK-109, YK-110, YK-111, YK-112, YK-113, YK-114, YK-115, YK-116, YK-117, YK-118, YK-119, YK-120, YK-121, YK-122, YK-123, YK-124, YK-125, or YK-126. In one preferred embodiment, the cationic lipid is compound YK-101, in another preferred embodiment, the cationic lipid is compound YK-107, and in another preferred embodiment, the cationic lipid is compound YK-108.

[0096] 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 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), ALC-0315 (compound 3 described in CN108368028B), compound 21 and compound 23 described in WO2021055833, or HHMA (compound 1 described in CN112979483B).

[0097] In some embodiments, the molar ratio of the cationic lipid to the carrier is about 25% to 75%, for example, about 30%, 40%, 49%, 55%, 60%, 65%, or 70%.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] In some embodiments, the molar ratio of the cationic lipid to the neutral lipid is about 1:1 to 15:1, e.g., 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 4.9:1. In another preferred embodiment, the molar ratio of the cationic lipid to the neutral lipid is about 4:1.

[0102] For example, the neutral lipid may comprise one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol, and derivatives thereof.

[0103] 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.

[0104] 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 penetration or cellular recognition, or to conjugate the composition with useful components such as targeting or imaging moieties (e.g., dyes).

[0105] 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.

[0106] 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.

[0107] 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.

[0108] In some embodiments, the molar ratio of the cationic lipid to the structural lipid is about 0.6:1 to 3:1, e.g., about 0.6:1, 0.7:1, 0.8:1, 0.9:1, 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.

[0109] 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.

[0110] 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.

[0111] In some embodiments, the molar ratio of the polymer-conjugated lipid to the carrier is 0.5% to 10%, preferably 1.5%.

[0112] In some embodiments, 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.

[0113] 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).

[0114] In one embodiment of the composition / carrier of the present invention, the polymer-conjugated lipid is DMG-PEG2000.

[0115] 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, (30-49):(7.5-15):(35-55):(1-5).

[0116] 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 40:10:48.5:1.5, or 49:10:39.5:1.5, preferably 40:10:48.5:1.5.

[0117] Therapeutic and / or prophylactic agents The composition may comprise one or more therapeutic and / or prophylactic agents. In one embodiment, the weight ratio of the carrier to the therapeutic or prophylactic agent is 10:1 to 30:1, for example, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, or 25:1.

[0118] 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.

[0119] 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.

[0120] For example, the therapeutic or prophylactic agent is a vaccine or a compound capable of eliciting an immune response.

[0121] 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.

[0122] Therapeutic and / or prophylactic agents include biologically active substances, alternatively 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 that can be used to treat specific diseases, disorders, or conditions. 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.

[0123] In some embodiments, the therapeutic and / or prophylactic agent is a cytotoxin, a radioactive ion, a chemotherapeutic agent, a vaccine, a compound that elicits an immune response, and / or another therapeutic and / or prophylactic agent. A cytotoxin or cytotoxic agent includes any 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 pulmonary tuberculosis, and may include mRNA encoding antigens and / or epitopes from infectious diseases. Vaccines may 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), and the like. , streptozotocin, mitomycin C, and 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).

[0124] 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.

[0125] In some embodiments, the therapeutic and / or prophylactic 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 strand. Exemplary polynucleotides for use in the present invention include, but are not limited to, one or more of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) (including messenger mRNA (mRNA) and hybrids thereof), RNAi inducers, RNAi factors, siRNA, shRNA, miRNA, antisense RNA, ribozymes, catalytic DNA, RNA that induces triple helix formation, aptamers, and the like. 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.

[0126] 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.

[0127] In other embodiments, the therapeutic and / or prophylactic agent is an siRNA. The siRNA can selectively reduce or downregulate the expression of a gene of interest. 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.

[0128] 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.

[0129] In some embodiments, therapeutic and / or prophylactic agent is shRNA, or the vector or plasmid that encodes it.shRNA can be produced in target cells after appropriate construct is delivered into the nucleus.The construct and mechanism related to shRNA are well known in the relevant art.

[0130] 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.

[0131] In one embodiment, the disease or disorder is characterized by dysfunctional or abnormal protein or polypeptide activity.

[0132] 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.

[0133] In one embodiment, 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.

[0134] Other ingredients The composition may include one or more other ingredients in addition to those described above. For example, the composition may include one or more hydrophobic small molecules such as vitamins (e.g., vitamin A or vitamin E) or sterols.

[0135] 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).

[0136] 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, Surface modifiers may include, but are not limited to, romhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin β4, dornase alfa, neltenexin, and erdosteine, and DNA enzymes (e.g., rhDNA enzyme). Surface modifiers 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).

[0137] 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 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 that can be used for target cell delivery, imaging, and membrane permeation are well known in the art.

[0138] In addition to these ingredients, the composition may contain any substance that can be used in pharmaceutical compositions.For example, the composition may contain one or more pharmaceutically acceptable excipients or auxiliary ingredients, 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).

[0139] 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.

[0140] 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.

[0141] The compositions of the present invention may be prepared in the form of solid, semi-solid, liquid, or gaseous preparations, 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.

[0142] 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.

[0143] The compositions of the present invention are administered in a therapeutically effective amount, which may vary depending not only on the particular agent 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 may 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.

[0144] The present invention includes, but is not limited to, the following embodiments.

[0145] 1. A compound of formula (I), or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof. [ka] (however, G1 is C 1-6 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 6-15straight chain alkyl, R3-S-R4, or R5-SS-R6; R2 is C 12-25 is a branched alkyl; G3 is HO-R7- or (CH3)2N(CH2)3C(O)O(CH2)2-, where R3 is C 1-7 alkylene, and R4 is C 1-7 is alkyl, R5 is C 1-7 alkylene, and R6 is C 1-7 is alkyl, R7 is C 2-8 Alkylene, C 5-6 Cycloalkylene, -CH2CH(OH)CH2-, -(CH2CH2O) m -(CH2) n - or -(CH2) m -O-(CH2) n -, where m and n are each independently 1, 2, 3, or 4. 2. Compounds of formula (I) according to embodiment 1, or N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers thereof, wherein G1 is an unsubstituted C 2-5 It is alkylene. 3. Compounds of formula (I) according to embodiment 2, or N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers thereof, wherein G1 is unsubstituted C3 alkylene. 4. Compounds of formula (I) according to embodiment 2, or N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers thereof, wherein G1 is unsubstituted C5 alkylene. 5. Compounds of formula (I) according to embodiment 2, or N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers thereof, wherein G1 is unsubstituted C4 alkylene. 6. Compounds of formula (I) according to any one of the preceding embodiments, or N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers thereof, wherein G2 is an unsubstituted C 4-6 It is alkylene. 7. The compound of formula (I) according to embodiment 6, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein G2 is unsubstituted C5 alkylene. 8. The compound of formula (I) according to embodiment 6, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein G2 is unsubstituted C6 alkylene. 9. The compound of formula (I) according to embodiment 6, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein G2 is unsubstituted C4 alkylene. 10. Compounds of formula (I) according to any one of embodiments 1 to 9, or N-oxides, solvates, pharmaceutically acceptable salts, or stereoisomers thereof, wherein L1 is -C(O)O-. 11. Compounds of formula (I) according to any one of embodiments 1 to 9, or N-oxides, solvates, pharmaceutically acceptable salts, or stereoisomers thereof, wherein L1 is -OC(O)-. 12. Compounds of formula (I) according to any one of embodiments 1 to 11, or N-oxides, solvates, pharmaceutically acceptable salts, or stereoisomers thereof, wherein L2 is -C(O)O-. 13. Compounds of formula (I) according to any one of embodiments 1 to 11, or N-oxides, solvates, pharmaceutically acceptable salts, or stereoisomers thereof, wherein L2 is -OC(O)-. 14. Compounds of formula (I) according to any one of embodiments 1 to 13, or N-oxides, solvates, pharmaceutically acceptable salts, or stereoisomers thereof, wherein R1 is unsubstituted C 8-12 It is a straight chain alkyl. 15. Compounds of formula (I) according to embodiment 14, or N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers thereof, wherein R1 is unsubstituted C 10 It is a straight chain alkyl. 16. Compounds of formula (I) according to embodiment 14, or N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers thereof, wherein R1 is unsubstituted C 11 It is a straight chain alkyl. 17. The compound of formula (I) according to embodiment 14, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R1 is unsubstituted C9 straight chain alkyl. 18. The compound of formula (I) according to embodiment 14, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R1 is unsubstituted C8 straight chain alkyl. 19. Compounds of formula (I) according to any one of embodiments 1 to 13, or N-oxides, solvates, pharmaceutically acceptable salts, or stereoisomers thereof, wherein R3 is -CH2CH2-. 20. Compounds of formula (I) according to embodiment 19, or N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers thereof, wherein R4 is C4 straight chain alkyl. 21. Compounds of formula (I) according to any one of embodiments 1 to 13, or N-oxides, solvates, pharmaceutically acceptable salts, or stereoisomers thereof, wherein R5 is -CH2CH2CH2-. 22. The compound of formula (I) according to embodiment 21, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R6 is C5 straight chain alkyl. 23. Compounds of formula (I) according to any one of the preceding embodiments, or N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers thereof, wherein R2 is an unsubstituted C 14-22 It is a branched alkyl. 24. Compounds of formula (I) according to embodiment 23, or N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers thereof, wherein R2 is unsubstituted C 15 It is a branched alkyl. 25. Compounds of formula (I) according to embodiment 23, or N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers thereof, wherein R2 is unsubstituted C 18 It is a branched alkyl. 26. Compounds of formula (I) according to embodiment 23, or N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers thereof, wherein R2 is unsubstituted C 14 It is a branched alkyl. 27. Compounds of formula (I) according to embodiment 23, or N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers thereof, wherein R2 is [ka] is. 28. Compounds of formula (I) according to any one of the preceding embodiments, or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein R7 is -(CH2)2-, -(CH2)3-, -CH2CH(OH)CH2-, -CH(CH3)CH2-, -CH(CH3)(CH2)2-, -C(CH3)2(CH2)2-, -(CH2)2O(CH2)2O(CH2)2-, -(CH2)2O(CH2)2-, or [ka] is. 29. Compounds of formula (I) according to embodiment 28, or N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers thereof, wherein R7 is -(CH2)2-. 30. Compounds of formula (I) according to embodiment 28, or N-oxides, solvates, pharmaceutically acceptable salts or stereoisomers thereof, wherein R7 is -(CH2)3-. 31. The compound of formula (I), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, according to embodiment 1, wherein said compound of formula (I) has one of the following structures: [ka] JPEG0007764663000029.jpg25086 JPEG0007764663000030.jpg23593 JPEG0007764663000031.jpg242121 JPEG0007764663000032.jpg14510832. A compound of formula (I) according to embodiment 31, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound of formula (I) is compound YK-101 having the following structure: [ka] 33. The compound of formula (I) according to embodiment 31, or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein said compound of formula (I) is compound YK-107, having the following structure: [ka] 34. The compound of formula (I) according to embodiment 31, or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein said compound of formula (I) is compound YK-108, having the following structure: [ka] 35. A composition comprising a carrier, wherein the carrier comprises a cationic lipid, and the cationic lipid comprises a compound of formula (I) according to any one of the preceding embodiments, or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof. 36. The composition according to embodiment 35, wherein the molar ratio of the cationic lipid to the carrier is 25% to 75%. 37. The composition according to any one of embodiments 35-36, wherein the carrier further comprises a neutral lipid. 38. The composition according to embodiment 37, wherein the molar ratio of the cationic lipid to the neutral lipid is from 1:1 to 15:1, preferably 4:1. 39. The composition according to any one of embodiments 37-38, wherein the neutral lipid comprises one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol, and derivatives thereof. 40. The composition of any one of embodiments 37 to 39, wherein the neutral lipid is selected from the group consisting of 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 one or more selected from the group consisting of 1-stearoyl-2-oleoyl-phosphatidylethanolamine (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. 41. The composition according to embodiment 40, wherein the neutral lipid is DOPE and / or DSPC. 42. The composition of any one of embodiments 35-41, wherein the carrier further comprises a structured lipid. 43. The composition of embodiment 42, wherein the molar ratio of the cationic lipid to the structural lipid is from 0.6:1 to 3:1. 44. The composition according to any one of embodiments 42-43, wherein the structural lipid is one or more selected from cholesterol, nonsterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, α-tocopherol, and corticosteroids. 45. The composition of any one of embodiments 42-44, wherein the structural lipid is cholesterol. 46. ​​The composition of any one of embodiments 35-45, wherein the carrier further comprises a polymer-conjugated lipid. 47. The composition according to embodiment 46, wherein the molar ratio of the polymer-conjugated lipid to the carrier is 0.5% to 10%, preferably 1.5%. 48. The composition of any one of embodiments 46 to 47, wherein the polymer-conjugated lipid is one or more selected from PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. 49. The composition of any one of embodiments 46 to 48, 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). 50. The composition of any one of embodiments 35-49, 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). 51. The composition of embodiment 50, wherein the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is (30-49):(7.5-15):(35-55):(1-5). 52. The composition of any one of embodiments 50-51, wherein the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is 40:10:48.5:1.5. 53. The composition according to any one of embodiments 35 to 52, 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. 54. The composition of embodiment 53, wherein the nanoparticle formulation has an average particle size of 90 nm to 260 nm and a polydispersity of 40% or less. 55. The composition of any one of embodiments 35-54, wherein the cationic lipid further comprises one or more other ionizable lipid compounds. 56. The composition of any one of embodiments 35-55, further comprising a therapeutic or prophylactic agent. 57. The composition according to embodiment 56, wherein the mass ratio of the carrier to the therapeutic or prophylactic agent is 10:1 to 30:1. 58. The composition of embodiment 57, wherein the mass ratio of the carrier to the therapeutic or prophylactic agent is 12.5:1 to 20:1. 59. The composition of embodiment 58, wherein the mass ratio of the carrier to the therapeutic or prophylactic agent is 15:1. 60. The composition of any one of embodiments 56-59, wherein the therapeutic or prophylactic agent comprises one or more of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein. 61. The composition according to any one of embodiments 56 to 60, wherein the therapeutic or prophylactic agent is a vaccine or a compound capable of eliciting an immune response. 62. The composition according to any one of embodiments 56 to 61, wherein the therapeutic or prophylactic agent is a nucleic acid. 63. The composition according to any one of embodiments 56 to 62, wherein the therapeutic or prophylactic agent is a ribonucleic acid (RNA). 64. The composition according to any one of embodiments 56 to 62, wherein the therapeutic or prophylactic agent is deoxyribonucleic acid (DNA). 65. The composition of embodiment 63, 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. 66. The composition according to embodiment 65, wherein the RNA is mRNA. 67. The composition according to any one of embodiments 35-66, wherein the composition further comprises one or more pharmaceutically acceptable excipients or diluents. 68. Use of a compound of formula (I) according to any one of embodiments 1 to 34, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, or a composition according to any one of embodiments 35 to 67, in the preparation of a nucleic acid drug, a gene vaccine, a small molecule drug, a polypeptide, or a protein drug. 69. Use of a compound of formula (I), as defined in any one of embodiments 1 to 34, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, or a composition as defined in any one of embodiments 35 to 67, in the preparation of a medicament for treating a disease or disorder in a mammal in need thereof. 70. The use according to embodiment 69, wherein the disease or disorder is characterized by dysfunctional or abnormal protein or polypeptide activity. 71. The use according to any one of embodiments 69 to 70, 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. 72. The use according to embodiment 71, 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. 73. The use according to any one of embodiments 69 to 72, wherein the mammal is a human. 74. The use according to any one of embodiments 69 to 73, wherein the composition is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally or by inhalation. 75. The use according to embodiment 74, wherein the composition is administered subcutaneously. 76. The use according to any one of embodiments 69 to 75, wherein the therapeutic or prophylactic agent is administered to the mammal at a dose of about 0.001 mg / kg to about 10 mg / kg. [Example]

[0146] 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.

[0147] The following abbreviations represent the following reagents:

[0148] DCM: dichloromethane; EDCI: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; DMAP: 4-dimethylaminopyridine; DCC: N,N'-dicyclohexylcarbodiimide

[0149] Example 1: Synthesis of cationic lipid compounds 1. Intermediate 1 (INT-1): Synthesis of n-decyl 4-bromobutyrate Synthesis scheme: [ka] 4-Bromobutyric acid (15.00 g, 89.82 mmol) and 1-decanol (12.90 g, 81.50 mmol) were dissolved in DCM (100 mL). EDCI (18.70 g, 97.55 mmol) and DMAP (500 mg, 4.09 mmol) were added to the above solution and 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. The residue was purified by silica gel chromatography (ethyl acetate / n-hexane) to obtain n-decyl 4-bromobutyrate (INT-1) (10.52 g, 34.24 mmol, 42.01%). 1 H NMR (400 MHz, CDCl3) δ 4.12 (t, J = 6.8 Hz, 2H), 3.51 (t, J = 6.5 Hz, 2H), 2.54 (t, J = 7.2 Hz, 2H), 2.22 (p, J = 6.8 Hz, 2H), 1.66 (dd, J = 14.5, 7.3 Hz, 2H), 1.35 (dd, J = 19.1, 15.8 Hz, 14H), 0.92 (t, J = 6.9 Hz, 3H).

[0150] 2. Intermediate 2 (INT-2): Synthesis of 3-hexylnonyl 6-bromohexanoate Synthesis scheme: [ka] 6-Bromohexanoic acid (5.85 g, 30.00 mmol) and 3-hexylnonanol (6.24 g, 27.30 mmol) were dissolved in DCM (60 mL). EDCI (5.41 g, 35.50 mmol) and DMAP (333 mg, 2.73 mmol) were added to the above solution and stirred at 30-35 °C for 9 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. The residue was purified by silica gel chromatography (ethyl acetate / n-hexane) to obtain 3-hexylnonyl 6-bromohexanoate (INT-2) (7.89 g, 19.46 mmol, 71.3%). 1 H NMR (400 MHz, CDCl3) δ 4.13 (t, J = 7.1 Hz, 2H), 3.45 (t, J = 6.8 Hz, 2H), 2.35 (t, J = 7.4 Hz, 2H), 1.98- 1.87 (m, 2H), 1.70 (dt, J = 20.6, 7.4 Hz, 2H), 1.65 - 1.57 (m, 2H), 1.52 (ddd, J = 8.6, 6.9, 4.2 Hz, 2H), 1.44 (s, 1H), 1.37- 1.24 (m, 20H), 0.93 (t, J = 6.8 Hz, 6H).

[0151] 3. Synthesis of 3-hexylnonyl-6-(4-(decyloxy)-4-oxobutyl)((2-hydroxyethyl)amino)hexanoate (YK-101) Synthesis scheme: [ka]

[0152] Step 1: Synthesis of 4-(2-hydroxyethylamino) n-decyl butyrate (YK-101-PM1) n-Decyl 4-bromobutyrate (4.30 g, 14.00 mmol) and ethanolamine (2.56 g, 42.00 mmol) were dissolved in acetonitrile (150 mL), and potassium carbonate (1.94 g, 14.00 mmol) was added to the above system. The mixture was heated to 70°C and stirred for 2 hours. After the reaction was completed, the reaction mixture was cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel chromatography (methanol / dichloromethane) to obtain 4-((2-hydroxyethyl)amino)n-decyl butyrate (2.01 g, 6.97 mmol, 49.72%). 16 H 33 NO3, MS(ES): m / z (M+H + )288.3.

[0153] Step 2: Synthesis of 3-hexylnonyl-6-(4-(decyloxy)-4-oxobutyl)((2-hydroxyethyl)amino)hexanoate (YK-101) 6-Bromohexanoic acid-3-hexylnonyl (1.15 g, 2.83 mmol) and 4-((2-hydroxyethyl)amino)n-decyl butyrate (1.72 g, 6 mmol) were dissolved in acetonitrile (40 mL). Potassium carbonate (1.56 g, 11.32 mmol) and potassium iodide (46 mg, 0.28 mmol) were added to the above system, 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 (1.21 g, 1.98 mmol, 70.0%). 37 H 73 In the case of NO5, MS(ES): m / z (M+H + )612.8. 1H NMR (400 MHz, CDCl3) δ 4.13 - 3.89 (m, 4H), 3.77 (dd, J = 10.2, 6.0 Hz, 2H), 3.53 - 3.30 (m, 2H), 2.97 - 2.81 (m, 1H), 2.77 (dd, J = 15.4, 7.3 Hz, 3H), 2.43 (t, J = 8.1 Hz, 1H), 2.31 (dd, J = 16.7, 9.4 Hz, 3H), 2.14 - 1.94 (m, 1H), 1.76 - 1.46 (m, 9H), 1.46 - 1.08 (m, 38H), 0.87 (t, J = 6.8 Hz, 9H).

[0154] 4. Synthesis of 3-hexylnonyl-6-(4-(decyloxy)-4-oxobutyl)((3-hydroxypropyl)amino)hexanoate (YK-102) Synthesis scheme: [ka]

[0155] Step 1: Synthesis of 3-hexylnonyl-6-((3-hydroxypropyl)amino)hexanoate (YK-102-PM1) INT-2 (500 mg, 1.23 mmol) and 3-aminopropan-1-ol (282 mg, 3.69 mmol) were used as raw materials, and purified by silica gel chromatography (methanol / dichloromethane) according to the preparation method of YK-101-PM2 to obtain 3-hexylnonyl-6-((3-hydroxypropyl)amino)hexanoate (405 mg, 1.01 mmol, 82.4%). 24 H 49 NO3, MS(ES): m / z (M+H + )400.3.

[0156] Step 2: Synthesis of 3-hexylnonyl-6-(4-(decyloxy)-4-oxobutyl)((3-hydroxypropyl)amino)hexanoate (YK-102) The 3-hexylnonyl-6-((3-hydroxypropyl)amino)hexanoate (200 mg, 0.50 mmol) and INT-1 (199 mg, 0.65 mmol) obtained by the purification above were used as raw materials, and purified by silica gel chromatography (ethyl acetate / n-hexane) according to the preparation method of YK-101 to obtain the target compound (70 mg, 0.11 mmol, 22.4%). 38 H 75 NO5, MS(ES): m / z (M+H + )626.7. 1 H NMR (400 MHz, CDCl3) δ 4.11 (q, J = 6.9 Hz, 4H), 3.88 - 3.73 (m, 2H), 2.78 (t, J = 5.8 Hz, 2H), 2.67 - 2.48 (m, 4H), 2.42 - 2.24 (m, 4H), 1.89 (dt, J = 14.9, 7.3 Hz, 2H), 1.78 (dt, J = 10.7, 5.5 Hz, 2H), 1.75 - 1.49 (m, 8H), 1.46 - 1.18 (m, 37H), 0.92 (t, J = 6.7 Hz, 9H).

[0157] 5. Synthesis of 3-hexylnonyl-6-((6-(decyloxy)-6-oxohexyl)(2-hydroxyethyl)amino)hexanoate (YK-103) Synthesis scheme: [ka]

[0158] Step 1: Synthesis of n-decyl 6-bromohexanoate (YK-103-PM1) 6-Bromohexanoic acid (1.12 g, 10.00 mmol) and n-decanol (1.45 g, 9.16 mmol) were used as raw materials, and purified by silica gel chromatography (ethyl acetate / n-hexane) according to the preparation method of INT-1 to obtain n-decyl 6-bromohexanoate (2.8 g, 8.35 mmol, 91.2%).

[0159] Step 2: Synthesis of 6-((2-hydroxyethyl)amino) n-decylhexanoate (YK-103-PM2) The n-decyl 6-bromohexanoate (1.12 g, 3.47 mmol) and ethanolamine (3.18 g, 52.05 mmol) obtained by the purification process above were dissolved in ethanol (15 mL) and stirred at room temperature for 16 hours. The reaction mixture was concentrated under reduced pressure to remove the solvent, then diluted with ethyl acetate (80 mL) and washed three times with brine (50 mL). The organic phase was concentrated under reduced pressure and purified by silica gel chromatography (methanol / dichloromethane) to obtain 6-((2-hydroxyethyl)amino)n-decylhexanoate (0.81 g, 2.56 mmol, 74.0%). 18 H 37 NO3, MS(ES): m / z (M+H + )316.4.

[0160] Step 3: Synthesis of 3-hexylnonyl-6-((6-(decyloxy)-6-oxohexyl)(2-hydroxyethyl)amino)hexanoate (YK-103) Using 6-((2-hydroxyethyl)amino) n-decylhexanoate (159 mg, 0.51 mmol) and INT-2 (202 mg, 0.50 mmol) as starting materials, the target compound (81 mg, 0.13 mmol, 25.3%) was obtained according to the preparation method for YK-101. 39 H 77 NO5, MS(ES): m / z (M+H + )640.8. 1 H NMR (400 MHz, CDCl3) δ 4.08 (dt, J = 9.3, 7.0 Hz, 4H), 3.66 (t, J = 5.1 Hz, 2H), 2.74 (t, J = 4.8 Hz, 2H), 2.68 - 2.44 (m, 4H), 2.31 (td, J = 7.4, 2.9 Hz, 4H), 1.76 - 1.45 (m, 11H), 1.49 - 0.99 (m, 40H), 0.89 (t, J = 6.8 Hz, 9H).

[0161] 6. Synthesis of 3-hexylnonyl-5-((4-(decyloxy)-4-oxybutyl)((2-hydroxyethyl)amino)valerate (YK-104) Synthesis scheme: [ka]

[0162] Step 1: Synthesis of 5-bromovalerate-3-hexylnonyl ester (YK-104-PM1) 3-Hexylnonanol (1.14 g, 5.00 mmol) and 5-bromovaleric acid (0.91 g, 5.03 mmol) were used as raw materials, and purified by silica gel chromatography (ethyl acetate / n-hexane) according to the preparation method of INT-2 to obtain 5-bromovalerate-3-hexylnonyl ester (1.40 g, 3.58 mmol, 71.5%).

[0163] Step 2: Synthesis of 3-hexylnonyl-5-((4-(decyloxy)-4-oxybutyl)((2-hydroxyethyl)amino)valerate (YK-104) Using 5-bromovalerate-3-hexylnonyl (196 mg, 0.50 mmol) and 4-((2-hydroxyethyl)amino)n-decyl butyrate (145 mg, 0.51 mmol) as starting materials, the product was purified by silica gel chromatography (ethyl acetate / n-hexane) according to the preparation method for YK-101 to obtain the target compound (178 mg, 0.30 mmol, 59.5%). 36 H 71 NO5, MS(ES): m / z (M+H + )598.3. 1H NMR (400 MHz, CDCl3) δ 4.06 (dt, J = 8.5, 7.0 Hz, 4H), 3.71 (d, J = 7.0 Hz, 1H), 3.56 (t, J = 5.2 Hz, 2H), 2.62 (t, J = 5.2 Hz, 2H), 2.52 (dd, J = 14.9, 7.7 Hz, 4H), 2.31 (td, J = 7.3, 5.5 Hz, 4H), 1.85 - 1.73 (m, 2H), 1.66 - 1.41 (m, 6H), 1.43 - 1.11 (m, 37H), 0.87 (dd, J = 7.2, 6.4 Hz, 9H).

[0164] 7. Synthesis of hexyl 6-(((4-(decyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)-3-hexylnonanoate (YK-105) Synthesis scheme: [ka]

[0165] Step 1: Preparation of 3-hexylnonanoic acid-6-bromohexyl ester (YK-105-PM1) 6-Bromohexanol (412 mg, 2.18 mmol) and 3-hexylnonanoic acid (460 mg, 1.90 mmol) were used as raw materials and purified by silica gel chromatography (ethyl acetate / n-hexane) according to the preparation method of INT-1 to obtain 3-hexylnonanoic acid-6-bromohexyl ester (393 mg, 0.97 mmol, 51.0%).

[0166] Step 2: Synthesis of 3-hexylnonanoic acid-6-(2-hydroxyethyl-amino)hexyl ester (YK-105-PM2) Using 3-hexylnonanoic acid-6-bromohexyl ester (393 mg, 0.97 mmol) and ethanolamine (178 mg, 2.91 mmol) as starting materials, 3-hexylnonanoic acid-6-(2-hydroxyethyl-amino)hexyl ester (312 mg, 0.81 mmol, 83.4%) was obtained according to the synthesis method of YK-101-PM2. 23 H 47 NO3, MS(ES): m / z (M+H + )386.6.

[0167] Step 3: Synthesis of hexyl 6-(((4-(decyloxy)-4-oxobutyl)(2-hydroxyethyl)amino)-3-hexylnonanoate (YK-105) Using 3-hexylnonanoic acid-6-(2-hydroxyethyl-amino)hexyl ester (312 mg, 0.85 mmol) and INT-1 (447 mg, 1.54 mmol) as starting materials, the target compound (329 mg, 0.54 mmol, 63.2%) was obtained according to the preparation method of YK-101. 37 H 73 NO5, MS(ES): m / z (M+H + )612.3. 1 H NMR (400 MHz, CDCl3) δ 5.34 (s, 1H), 4.10 (td, J = 6.7, 5.1 Hz, 4H), 3.62 (d, J = 5.5 Hz, 2H), 2.62 (d, J = 44.1 Hz, 5H), 2.37 (t, J = 7.2 Hz, 2H), 2.26 (d, J = 6.9 Hz, 2H), 1.97 - 1.76 (m, 3H), 1.74 - 1.59 (m, 4H), 1.53 (s, 2H), 1.48 - 1.06 (m, 39H), 0.92 (t, J = 6.7 Hz, 9H).

[0168] 8. Synthesis of 3-hexylnonyl-6-((10-oxodecyl-10-oxy-4-butyl)((2-hydroxyethyl)amino)hexanoate (YK-106) Synthesis scheme: [ka]

[0169] Step 1: Synthesis of 4-bromobutyl n-decanoate (YK-106-PM1) 4-Bromobutanol (888 mg, 5.80 mmol) and n-decanoic acid (1.00 g, 5.81 mmol) were dissolved in DCM (20 mL). EDCI (1.46 g, 7.62 mmol) and DMAP (71 mg, 0.58 mmol) were added to the solution and stirred at 30–35°C for 7 hours. After completion of the reaction, the reaction mixture was washed once with saturated sodium carbonate and once with saturated brine. The organic phase was dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography (ethyl acetate / n-hexane) to give 4-bromobutyl n-decanoate (600 mg, 1.95 mmol, 33.7%).

[0170] Step 2: Synthesis of 2-octyldecyl-6-((2-hydroxyethyl)amino)hexanoate (YK-106-PM2) INT-2 (200 mg, 0.45 mmol) and 2-aminoethanol (81.8 mg, 1.34 mmol) were used as raw materials, and purified by silica gel chromatography (ethyl acetate / n-hexane) according to the synthesis method for YK-101-PM2 to obtain 2-octyldecyl-6-((2-hydroxyethyl)amino)hexanoate (106 mg, 0.25 mmol, 55.1%). 26 H 53 NO3, MS(ES): m / z (M+H + )428.5.

[0171] Step 3: Synthesis of 2-octyldecyl-6-((10-oxodecyl-10-oxy-4-butyl)((2-hydroxyethyl)amino)hexanoate (YK-106) Using 2-octyldecyl-6-((2-hydroxyethyl)amino)hexanoate (140 mg, 0.33 mmol) and n-decanoic acid-4-bromobutyl (181 mg, 0.59 mmol) as starting materials, the target compound (60 mg, 0.09 mmol, 27.8%) was obtained according to the preparation method of YK-101. 40 H 79 NO5, MS(ES): m / z (M+H + )654.5. 1 H NMR (400 MHz, CDCl3) δ 4.13 - 3.93 (m, 2H), 3.90 (d, J = 5.8 Hz, 2H), 3.76 - 3.56 (m, 2H), 2.88 - 2.53 (m, 6H), 2.24 (q, J = 7.4 Hz, 4H), 1.58 (dddd, J = 23.9, 21.6, 10.9, 5.4 Hz, 13H), 1.41 - 1.01 (m, 40H), 0.81 (td, J = 6.9, 1.3 Hz, 9H).

[0172] 9. Synthesis of 2-octyldecyl-6-(4-(decyloxy)-4-oxobutyl)((3-hydroxypropyl)amino)hexanoate (YK-107) Synthesis scheme: [ka]

[0173] Step 1: Synthesis of 2-octyldecyl 6-bromohexanoate (YK-107-PM1) Using 2-octyldecanol (3.00 g, 11.09 mmol) and 6-bromohexanoic acid (2.60 g, 13.33 mmol) as raw materials, 2-octyldecyl 6-bromohexanoate (3.05 g, 6.82 mmol, 61.50%) was obtained according to the preparation method of INT-1.

[0174] Step 2: Synthesis of 2-octyldecyl-6-((3-hydroxypropyl)amino)hexanoate (YK-107-PM2) Starting from 2-octyldecyl 6-bromohexanoate (200 mg, 0.45 mmol) and 3-aminopropan-1-ol (102 mg, 1.34 mmol), 2-octyldecyl-6-((3-hydroxypropyl)amino)hexanoate (126 mg, 0.29 mmol, 63.4%) was obtained according to the method for YK-101-PM2. 27 H 55 NO3, MS(ES): m / z (M+H + )442.3.

[0175] Step 3: Synthesis of 2-octyldecyl-6-(4-(decyloxy)-4-oxobutyl)((3-hydroxypropyl)amino)hexanoate (YK-107) Using 2-octyldecyl-6-((3-hydroxypropyl)amino)hexanoate (106 mg, 0.24 mmol) and INT-1 (118 mg, 0.38 mmol) as starting materials, the target compound (88 mg, 0.13 mmol, 54.9%) was obtained according to the preparation method of YK-101. 41 H 81 NO5, MS(ES): m / z (M+H + )668.8. 1 H NMR (400 MHz, CDCl3) δ 4.89 (t, J = 6.2 Hz, 1H), 4.72 (s, 2H), 4.08 (t, J = 6.8 Hz, 2H), 3.57 (s, 2H), 2.44 (s, 3H), 2.28 (td, J = 7.6, 3.3 Hz, 4H), 1.81 - 1.41 (m, 15H), 1.30 (d, J = 4.1 Hz, 41H), 0.92 (td, J = 6.8, 1.6 Hz, 9H).

[0176] 10. Synthesis of 3-hexylnonyl-7-(4-(undecyloxy)-4-oxobutyl)((2-hydroxyethyl)amino)enanthate (YK-108) Synthesis scheme: [ka]

[0177] Step 1: Synthesis of 7-bromoheptanoic acid-3-hexylnonyl ester (YK-108-PM1) 7-Bromoheptanoic acid (4.48 g, 21.43 mmol) and 3-hexylnonanol (5.00 g, 21.89 mmol) were dissolved in DCM (150 mL). EDCI (4.86 g, 25.35 mmol) and DMAP (261 mg, 2.14 mmol) were added to the solution and stirred at 30-35 °C for 10 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. The residue was purified by silica gel chromatography (ethyl acetate / n-hexane) to obtain 7-bromoheptanoic acid-3-hexylnonyl ester (5.98 g, 14.26 mmol, 66.5%).

[0178] Step 2: Synthesis of 3-hexylnonyl-7-(4-(undecyloxy)-4-oxobutyl)((2-hydroxyethyl)amino)enanthate (YK-108) 4-(2-Hydroxyethylamino)n-undecylbutyrate (2.01 g, 6.67 mmol) and 7-bromoheptanoic acid-3-hexylnonyl ester (3.06 g, 7.29 mmol) were dissolved in acetonitrile (100 mL). Potassium carbonate (3.65 g, 26.41 mmol) and potassium iodide (110 mg, 0.66 mmol) were added to the above system, 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 (1.52 g, 2.37 mmol, 35.5%). 39 H 77 NO5, MS(ES): m / z (M+H + )640.8. 1H NMR (400 MHz, CDCl3) δ 4.08 (q, J = 7.1 Hz, 4H), 3.55 (t, J = 5.3 Hz, 2H), 2.60 (t, J = 5.2 Hz, 2H), 2.57 - 2.41 (m, 4H), 2.31 (dt, J = 14.7, 7.4 Hz, 4H), 1.79 (p, J = 7.2 Hz, 2H), 1.61 (ddt, J = 20.9, 13.8, 7.0 Hz, 5H), 1.51 - 1.37 (m, 3H), 1.40 - 1.10 (m, 42H), 0.99 - 0.78 (m, 9H).

[0179] 11. Synthesis of 3-hexylnonyl-6-((10-oxodecyl-10-oxy-4-butyl)((2-hydroxyethyl)amino)hexanoate (YK-109) Synthesis scheme: [ka]

[0180] Step 1: Synthesis of 4-bromobutyl n-decanoate (YK-109-PM1) 4-Bromobutanol (888 mg, 5.80 mmol) and n-decanoic acid (1.00 g, 5.81 mmol) were dissolved in DCM (20 mL). EDCI (1.46 g, 7.62 mmol) and DMAP (71 mg, 0.58 mmol) were added to the solution and stirred at 30–35°C for 7 hours. After completion of the reaction, the reaction mixture was washed once with saturated sodium carbonate and once with saturated brine. The organic phase was dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography (ethyl acetate / n-hexane) to give 4-bromobutyl n-decanoate (600 mg, 1.95 mmol, 33.7%).

[0181] Step 2: Synthesis of n-decanoic acid-4-(2-hydroxyethyl-amino)butyl ester (YK-109-PM2) The n-decanoic acid-4-bromobutyl ester (600 mg, 1.96 mmol) and ethanolamine (360 mg, 5.88 mmol) obtained by the purification process above were dissolved in acetonitrile (6 mL). Potassium carbonate (813 mg, 5.88 mmol) was added to the above system, and the mixture was heated to 70°C and stirred for 2 hours. After the reaction was completed, the reaction mixture was cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel chromatography (methanol / dichloromethane) to obtain n-decanoic acid-4-(2-hydroxyethylamino)butyl ester (250 mg, 0.87 mmol, 44.4%). 16 H 33 NO3, MS(ES): m / z (M+H + )288.4.

[0182] Step 3: Synthesis of 3-hexylnonyl-6-((10-oxodecyl-10-oxy-4-butyl)((2-hydroxyethyl)amino)hexanoate (YK-109) The purified n-decanoic acid-4-(2-hydroxyethylamino)butyl ester (107 mg, 0.35 mmol) and 6-bromohexanoic acid-3-hexylnonyl (300 mg, 0.75 mmol) were dissolved in acetonitrile (5 mL). Potassium carbonate (193 mg, 1.40 mmol) and potassium iodide (6 mg, 0.04 mmol) were added to the system, 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 (150 mg, 0.25 mmol, 70.0%). 37 H 73 NO5, MS(ES): m / z (M+H + )612.8. 1H NMR (400 MHz, CDCl3) δ 4.09 (dt, J = 12.9, 7.0 Hz, 4H), 3.96 (d, J = 5.8 Hz, 1H), 3.63 (t, J = 5.2 Hz, 2H), 2.89 - 2.45 (m, 6H), 2.43 - 2.18 (m, 4H), 1.79 - 1.43 (m, 11H), 1.45 - 1.05 (m, 36H), 0.87 (td, J = 6.7, 1.6 Hz, 9H).

[0183] 12. Synthesis of 3-hexylnonyl-6-(6-(2-(butylthio)ethoxy)-6-oxohexyl)((2-hydroxyethyl)amino)hexanoate (YK-110) Synthesis scheme: [ka]

[0184] Step 1: Synthesis of 3-(5-pentanyl-dithio)propan-1-ol (YK-110-PM1) 3-Mercaptopropan-1-ol (1.00 g, 10.85 mmol), n-pentyl mercaptan (1.13 g, 10.85 mmol), and pyridine (1.71 g, 21.62 mmol) were added sequentially to a mixture of DCM (50 mL) and methanol (50 mL). Under nitrogen protection, iodine particles (2.74 g, 10.85 mmol) were added twice to the solution, and the mixture was stirred at room temperature for 20 hours. After the reaction was complete, 50 mL of DCM was added, washed sequentially with saturated sodium carbonate and saturated brine, and dried over Na2SO4. 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 give 3-(5-pentanyl-dithio)propan-1-ol (0.66 g, 3.40 mmol, 31.3%).

[0185] Step 2: Synthesis of 4-bromobutyric acid-3-(5-pentanyl-dithio)propyl ester (YK-110-PM2) 4-Bromobutyric acid (119 mg, 0.61 mmol) and 3-(5-pentanyl-dithio)propan-1-ol (100 mg, 0.51 mmol) were used as raw materials and purified by silica gel chromatography (ethyl acetate / n-hexane) according to the preparation method of INT-1 to obtain 4-bromobutyric acid-3-(5-pentanyl-dithio)propyl ester (140 mg, 0.41 mmol, 80.0%).

[0186] Step 3: Synthesis of 3-hexylnonyl-6-((2-hydroxyethyl)amino)hexanoate (YK-110-PM3) INT-2 (500 mg, 1.23 mmol) and 2-aminoethanol (226 mg, 3.70 mmol) were used as raw materials and purified by silica gel chromatography (ethyl acetate / n-hexane) according to the method for YK-101-PM2 to obtain 3-hexylnonyl-6-((2-hydroxyethyl)amino)hexanoate (248 mg, 0.64 mmol, 52.3%). 23 H 47 NO3, MS(ES): m / z (M+H + )386.5.

[0187] Step 4: Synthesis of 3-hexylnonyl-6-(4-(2-(butylthio)ethoxy)-4-oxobutyl)((2-hydroxyethyl)amino)hexanoate (YK-110) Using 3-hexylnonyl-6-((2-hydroxyethyl)amino)hexanoate (200 mg, 0.52 mmol) and 4-bromobutyric acid-3-(5-pentanyl-dithio)propyl ester (349 mg, 0.94 mmol) as starting materials, the target compound (110 mg, 0.16 mmol, 31.3%) was obtained according to the preparation method of YK-101. 35 H 69 NO5S, MS(ES): m / z (M+H + )676.4. 1H NMR (400 MHz, CDCl3) δ 4.21 (t, J = 6.3 Hz, 2H), 4.12 (t, J = 7.1 Hz, 2H), 3.70 (s, 2H), 2.86 - 2.52 (m, 4H), 2.45 - 2.21 (m, 4H), 2.08 (dt, J = 13.3, 6.5 Hz, 2H), 1.77 - 1.50 (m, 15H), 1.50 - 1.18 (m, 32H), 0.94 (dt, J = 9.3, 7.0 Hz, 9H).

[0188] 13. Synthesis of 2-octyldecyl-6-(6-(2-(butylthio)ethoxy)-6-oxohexyl)((2-hydroxyethyl)amino)hexanoate (YK-111) Synthesis scheme: [ka]

[0189] Step 1: Synthesis of 2-octyldecyl-6-((2-hydroxyethyl)amino)hexanoate (YK-111-PM1) INT-2 (200 mg, 0.45 mmol) and 2-aminoethanol (81.8 mg, 1.34 mmol) were used as raw materials, and purified by silica gel chromatography (ethyl acetate / n-hexane) according to the synthesis method for YK-101-PM2 to obtain 2-octyldecyl-6-((2-hydroxyethyl)amino)hexanoate (106 mg, 0.25 mmol, 55.1%). 26 H 53 NO3, MS(ES): m / z (M+H + )428.5.

[0190] Step 2: Synthesis of 2-octyldecyl-6-(4-(2-(butylthio)ethoxy)-4-oxobutyl)((2-hydroxyethyl)amino)hexanoate (YK-111) Using 2-octyldecyl-6-((2-hydroxyethyl)amino)hexanoate (130 mg, 0.30 mmol) and 4-bromobutyric acid-3-(5-pentanyl-dithio)propyl ester (200 mg, 0.54 mmol) as starting materials, the target compound (60 mg, 0.08 mmol, 27.8%) was obtained according to the preparation method of YK-101. 40 H 79 NO5S2, MS(ES): m / z (M+H + )718.2. 1 H NMR (400 MHz, CDCl3) δ 4.10 (td, J = 6.3, 2.1 Hz, 3H), 3.90 (d, J = 5.8 Hz, 2H), 3.55 (d, J = 5.3 Hz, 2H), 2.75 - 2.56 (m, 6H), 2.50 (d, J = 7.4 Hz, 4H), 2.25 (td, J = 7.3, 1.4 Hz, 6H), 2.06 - 1.91 (m, 2H), 1.70 - 1.37 (m, 9H), 1.37 - 0.97 (m, 36H), 0.83 (dt, J = 10.4, 7.0 Hz, 9H).

[0191] 14. Synthesis of 3-hexylnonyl-6-(6-(2-(butylthio)ethoxy)-6-oxohexyl)((2-hydroxyethyl)amino)hexanoate (YK-112) Synthesis scheme: [ka]

[0192] Step 1: Synthesis of 6-bromohexanoic acid-2-(butylthio)ethyl ester (YK-112-PM1): 6-Bromohexanoic acid (308 mg, 1.58 mmol) and 2-(butylthio)ethan-1-ol (200 mg, 1.49 mmol) were used as raw materials and purified by silica gel chromatography (ethyl acetate / n-hexane) according to the preparation method of INT-1 to obtain 6-bromohexanoic acid-2-(butylthio)ethyl ester (0.35 g, 1.12 mmol, 75.5%).

[0193] Step 2: Synthesis of 3-hexylnonyl-6-(6-(2-(butylthio)ethoxy)-6-oxohexyl)((2-hydroxyethyl)amino)hexanoate (YK-112) Using 3-hexylnonyl-6-((2-hydroxyethyl)amino)hexanoate (200 mg, 0.52 mmol) and 6-bromohexanoic acid-2-(butylthio)ethyl ester (291 mg, 0.93 mmol) as starting materials, the target compound (67 mg, 0.11 mmol, 20.9%) was obtained according to the preparation method of YK-101. 35 H 69 NO5S, MS(ES): m / z (M+H + )616.5. 1 H NMR (400 MHz, CDCl3) δ 4.26 (t, J = 7.0 Hz, 2H), 4.12 (t, J = 7.1 Hz, 2H), 3.62 (t, J = 5.1 Hz, 2H), 2.77 (t, J = 7.0 Hz, 2H), 2.68 (t, J = 4.9 Hz, 2H), 2.64 - 2.47 (m, 6H), 2.35 (dt, J = 12.7, 7.5 Hz, 4H), 1.79 - 1.49 (m, 12H), 1.49 - 1.13 (m, 28H), 1.04 - 0.81 (m, 9H).

[0194] 15. Synthesis of 2-octyldecyl-6-(6-(2-(butylthio)ethoxy)-6-oxohexyl)((2-hydroxyethyl)amino)hexanoate (YK-113) Synthesis scheme: [ka] Using 2-octyldecyl-6-((2-hydroxyethyl)amino)hexanoate (200 mg, 0.52 mmol) and 6-bromohexanoic acid-2-(butylthio)ethyl ester (287 mg, 0.93 mmol) as starting materials, the target compound (214 mg, 0.33 mmol, 62.5%) was obtained according to the preparation method of YK-101. 38 H 75 NO5S, MS(ES): m / z (M+H + ) 658.6. 1 H NMR (400 MHz, CDCl3) δ 4.15 (t, J = 7.0 Hz, 2H), 3.90 (d, J = 5.8 Hz, 2H), 3.53 (t, J = 5.2 Hz, 2H), 2.66 (t, J = 7.0 Hz, 2H), 2.60 (t, J = 5.3 Hz, 2H), 2.49 (ddt, J = 9.5, 7.8, 2.6 Hz, 6H), 2.25 (q, J = 7.3 Hz, 4H), 1.68 - 1.40 (m, 9H), 1.39 - 1.05 (m, 37H), 0.91 - 0.69 (m, 9H).

[0195] 16. Synthesis of 3-hexylnonyl-6-(4-(decyloxy)-4-oxobutyl)((2,3-dihydroxypropyl)amino)hexanoate (YK-114) Synthesis scheme: [ka]

[0196] Step 1: Synthesis of 3-hexylnonyl-6-((2,3-dihydroxypropyl)amino)hexanoate (YK-114-PM1) Using INT-2 (1014 mg, 2.50 mmol) and 3-aminopropane-1,2-diol (273 mg, 3.00 mmol) as starting materials, 3-hexylnonyl-6-((2,3-dihydroxypropyl)amino)hexanoate (400 mg, 0.96 mmol, 38.5%) was obtained according to the synthesis method of YK-101-PM2. 24 H 49 NO4, MS(ES): m / z (M+H + )416.4.

[0197] Step 2: Synthesis of 3-hexylnonyl-6-(4-(decyloxy)-4-oxobutyl)((-2,3-dihydroxypropyl)amino)hexanoate (YK-114) Using 3-hexylnonyl-6-((2,3-dihydroxypropyl)amino)hexanoate (400 mg, 0.96 mmol) and INT-1 (554 mg, 1.93 mmol) as starting materials, the target compound (350 mg, 0.55 mmol, 56.8%) was obtained according to the preparation method for YK-101. 38 H 75 NO6, MS(ES): m / z (M+H + )642.5. 1 H NMR (400 MHz, CDCl3) δ 4.17 - 4.04 (m, 4H), 3.89 (s, 1H), 3.78 (dd, J = 11.4, 3.9 Hz, 1H), 3.68 (s, 1H), 3.55 (dd, J = 11.4, 4.4 Hz, 1H), 2.88 - 2.50 (m, 6H), 2.45 - 2.22 (m, 4H), 1.91 (d, J = 7.8 Hz, 2H), 1.75 - 1.50 (m, 8H), 1.48 - 1.10 (m, 38H), 1.00 - 0.80 (m, 9H).

[0198] 17. Synthesis of 3-hexylnonyl-6-(4-(decyloxy)-4-oxobutyl)((R)-2,3-dihydroxypropyl)amino)hexanoate (YK-115) Synthesis scheme: [ka]

[0199] Step 1: Synthesis of 3-hexylnonyl-6-(((R)2,3-dihydroxypropyl)amino)hexanoate (YK-115-PM1) Using INT-2 (890 mg, 2.19 mmol) and (R)-3-aminopropane-1,2-diol (200 mg, 2.19 mmol) as starting materials, 3-hexylnonyl-6-(((R)2,3-dihydroxypropyl)amino)hexanoate (400 mg, 0.96 mmol, 43.9%) was obtained according to the synthesis method of YK-101-PM2. 24 H 49 NO4, MS(ES): m / z (M+H + )416.4.

[0200] Step 2: Synthesis of 3-hexylnonyl-6-(4-(decyloxy)-4-oxobutyl)((R)-2,3-dihydroxypropyl)amino)hexanoate (YK-115) Using 3-hexylnonyl-6-(((R)2,3-dihydroxypropyl)amino)hexanoate (200 mg, 0.48 mmol) and INT-1 (295 mg, 0.96 mmol) as starting materials, the target compound (125 mg, 0.19 mmol, 40.6%) was obtained according to the preparation method for YK-101. 38 H 75 NO6, MS(ES): m / z (M+H + )642.5. 1 H NMR (400 MHz, CDCl3) δ 4.07 (q, J = 7.0 Hz, 4H), 3.88 - 3.64 (m, 2H), 3.48 (dd, J = 8.1, 2.6 Hz, 1H), 2.84 - 2.36 (m, 7H), 2.40 - 2.20 (m, 4H), 1.93 - 1.71 (m, 2H), 1.72 - 1.43 (m, 8H), 1.43 - 1.04 (m, 38H), 0.88 (t, J = 6.7 Hz, 9H).

[0201] 18. Synthesis of 3-hexylnonyl-6-(4-(decyloxy)-4-oxobutyl)(((S)-2,3-dihydroxypropyl)amino)hexanoate (YK-116) Synthesis scheme: [ka]

[0202] Step 1: Synthesis of 3-hexylnonyl-6-(((S)2,3-dihydroxypropyl)amino)hexanoate (YK-116-PM1) Using INT-2 (890 mg, 2.19 mmol) and (S)-3-aminopropane-1,2-diol (200 mg, 2.19 mmol) as starting materials, 3-hexylnonyl-6-(((S)2,3-dihydroxypropyl)amino)hexanoate (250 mg, 0.60 mmol, 27.5%) was obtained according to the synthesis method for YK-101-PM2. 24 H 49 NO4, MS(ES): m / z (M+H + )416.5.

[0203] Step 2: Synthesis of 3-hexylnonyl-6-(4-(decyloxy)-4-oxobutyl)(((S)-2,3-dihydroxypropyl)amino)hexanoate (YK-116) Using 3-hexylnonyl-6-(((S)2,3-dihydroxypropyl)amino)hexanoate (200 mg, 0.48 mmol) and INT-1 (278 mg, 0.97 mmol) as starting materials, the target compound (205 mg, 0.32 mmol, 66.5%) was obtained according to the preparation method for YK-101. 38 H 75 NO6, MS(ES): m / z (M+H + )642.5. 1H NMR (400 MHz, CDCl3) δ 4.11 - 3.90 (m, 4H), 3.89 (d, J = 7.3 Hz, 1H), 3.78 (d, J = 7.3 Hz, 1H), 3.67 (dd, J = 11.4, 3.9 Hz, 1H), 3.51 - 3.34 (m, 1H), 2.55 (ddd, J = 52.8, 24.9, 9.9 Hz, 6H), 2.36 - 2.15 (m, 4H), 1.80 (d, J = 8.7 Hz, 2H), 1.66 - 1.40 (m, 8H), 1.38 - 1.06 (m, 38H), 0.91 - 0.71 (m, 9H).

[0204] 19. Synthesis of 3-hexylnonyl-6-(4-(decyloxy)-4-oxobutyl)(((S)2-hydroxypropyl)amino)hexanoate (YK-117) Synthesis scheme: [ka]

[0205] Step 1: Synthesis of 4-(((S)2-hydroxypropyl)amino)decyl butyrate (YK-117-PM1) Using INT-1 (1.03 g, 3.37 mmol) and (S)-1-aminopropan-2-ol (253 mg, 3.37 mmol), 4-((S)2-hydroxypropyl)amino)decyl butyrate (300 mg, 1.00 mmol, 29.5%) was obtained according to the synthesis method of YK-101-PM2. 18 H 37 NO3, MS(ES): m / z (M+H + )302.4.

[0206] Step 2: Synthesis of 3-hexylnonyl-6-(4-(decyloxy)-4-oxobutyl)(((S) 2-hydroxypropyl)amino)hexanoate (YK-117) Using 4-(((S)2-hydroxypropyl)amino)decyl butyrate (151 mg, 0.5 mmol) and INT-2 (205 mg, 0.51 mmol) as starting materials, the target compound (50 mg, 0.08 mmol, 16.0%) was obtained according to the preparation method for YK-101. 38 H 75 NO5, MS(ES): m / z (M+H + )626.6. 1 H NMR (400 MHz, CDCl3) δ 4.12 - 4.01 (m, 4H), 3.75 - 3.60 (m, 1H), 2.89 - 2.45 (m, 6H), 2.40 - 2.22 (m, 4H), 1.99 - 1.79 (m, 2H), 1.71 - 1.48 (m, 8H), 1.44 - 1.20 (m, 38H), 1.13 (dd, J = 18.6, 7.4 Hz, 3H), 0.87 (t, J = 6.7 Hz, 9H).

[0207] 20. Synthesis of 3-hexylnonyl-6-(4-(decyloxy)-4-oxobutyl)((3-hydroxybutyl)amino)hexanoate (YK-118) Synthesis scheme: [ka]

[0208] Step 1: Synthesis of 4-((3-hydroxybutyl)amino)decyl butyrate (YK-118-PM1) Using INT-1 (300 mg, 0.98 mmol) and 4-aminobutan-2-ol (96.2 mg, 1.08 mmol), 4-((3-hydroxybutyl)amino)decyl butyrate (225 mg, 0.71 mmol, 72.8%) was obtained according to the synthesis method of YK-101-PM2. 18 H 37 NO3, MS(ES): m / z (M+H + )316.4.

[0209] Step 2: Synthesis of 3-hexylnonyl-6-(4-(decyloxy)-4-oxobutyl)((3-hydroxybutyl)amino)hexanoate (YK-118) Using 4-((3-hydroxybutyl)amino)decyl butyrate (200 mg, 0.63 mmol) and 6-bromohexanoic acid-3-hexylnonyl (458 mg, 1.13 mmol) as starting materials, the target compound (80 mg, 0.12 mmol, 19.8%) was obtained according to the preparation method of YK-101. 39 H 77 NO5, MS(ES): m / z (M+H + )640.5. 1 H NMR (400 MHz, CDCl3) δ 4.08 (td, J = 7.0, 3.2 Hz, 4H), 4.02 - 3.86 (m, 2H), 3.49 (s, 2H), 3.14 (ddd, J = 11.0, 8.1, 2.4 Hz, 2H), 3.07 - 2.87 (m, 4H), 2.41 (t, J = 6.7 Hz, 2H), 2.31 (t, J = 7.3 Hz, 2H), 2.10 - 1.92 (m, 2H), 1.89 - 1.52 (m, 8H), 1.47 - 1.08 (m, 40H), 0.88 (t, J = 6.8 Hz, 9H).

[0210] 21. Synthesis of 3-hexylnonyl-6-(4-(decyloxy)-4-oxobutyl)((3-hydroxy-3-methylbutyl)amino)hexanoate (YK-119) Synthesis scheme: [ka]

[0211] Step 1: Synthesis of 4-((3-hydroxy-3-methylbutyl)amino)decyl butyrate (YK-119-PM1) Using INT-1 (596 mg, 1.94 mmol) and 4-amino-2-methylbutanol (200 mg, 1.94 mmol), 4-((3-hydroxy-3-methylbutyl)amino)decyl butyrate (317 mg, 0.96 mmol, 49.6%) was obtained according to the synthesis method for YK-101-PM2. 19 H 39 NO3, MS(ES): m / z (M+H + )330.7.

[0212] Step 2: Synthesis of 3-hexylnonyl-6-(4-(decyloxy)-4-oxobutyl)((3-hydroxy-3-methylbutyl)amino)hexanoate (YK-119) Using 4-((3-hydroxy-3-methylbutyl)amino)decyl butyrate (200 mg, 0.61 mmol) and 6-bromohexanoic acid-3-hexylnonyl (492 mg, 1.21 mmol) as starting materials, the target compound (200 mg, 0.31 mmol, 50.1%) was obtained according to the preparation method of YK-101. 40 H 79 NO5, MS(ES): m / z (M+H + )654.4. 1 H NMR (400 MHz, CDCl3) δ 4.08 (q, J = 7.1 Hz, 4H), 2.76 (s, 2H), 2.54 (s, 3H), 2.32 (dt, J = 15.0, 7.4 Hz, 4H), 1.85 (s, 2H), 1.72 - 1.45 (m, 10H), 1.45 - 1.02 (m, 44H), 0.89 (t, J = 6.8 Hz, 9H).

[0213] 22. Synthesis of 3-hexylnonyl-6-(4-(decyloxy)-4-oxobutyl)((2-hydroxycyclopentyl)amino)hexanoate (YK-120) Synthesis scheme: [ka]

[0214] Step 1: Synthesis of 4-((2-hydroxycyclopentyl)amino)decyl butyrate (YK-120-PM1) Using INT-1 (909 mg, 2.96 mmol) and 2-aminocyclopentan-1-ol (300 mg, 2.97 mmol), 4-((2-hydroxycyclopentyl)amino)decyl butyrate (580 mg, 1.77 mmol, 59.8%) was obtained according to the synthesis method for YK-101-PM2. 19 H 37 NO3, MS(ES): m / z (M+H + )328.3.

[0215] Step 2: Synthesis of 3-hexylnonyl-6-(4-(decyloxy)-4-oxobutyl)((2-hydroxycyclopentyl)amino)hexanoate (YK-120) Using 4-((2-hydroxycyclopentyl)amino)decyl butyrate (150 mg, 0.46 mmol) and 6-bromohexanoic acid-3-hexylnonyl (186 mg, 0.46 mmol) as starting materials, the target compound (160 mg, 0.24 mmol, 53.34%) was obtained according to the preparation method of YK-101. 40 H 77 NO5, MS(ES): m / z (M+H + )652.8. 1 H NMR (400 MHz, CDCl3) δ 4.30 - 4.15 (m, 1H), 4.15 - 3.86 (m, 4H), 3.61 - 3.30 (m, 1H), 3.10 (s, 1H), 2.70 (d, J = 37.2 Hz, 4H), 2.51 - 2.18 (m, 4H), 2.18 - 1.82 (m, 4H), 1.81 - 1.50 (m, 12H), 1.49 - 1.02 (m, 37H), 1.01 - 0.74 (m, 9H).

[0216] 23. Synthesis of 3-hexylnonyl-6-(4-(decyloxy)-4-oxobutyl)(2-(2-(2-hydroxyethoxy)ethoxy-ethyl)amino)hexanoate (YK-121) Synthesis scheme: [ka]

[0217] Step 1: Synthesis of 3-hexylnonyl-6-(2-(2-(2-hydroxyethoxy)ethoxy-ethyl)amino)hexanoate (YK-121-PM1) Using INT-2 (500 mg, 1.23 mmol) and 2-(2-(2-aminoethoxy-ethyl)ethoxy)ethan-1-ol (552 mg, 3.70 mmol) as starting materials, 3-hexylnonyl-6-(2-(2-(2-hydroxyethoxy)ethoxy-ethyl)amino)hexanoate (519 mg, 1.10 mmol, 89.1%) was obtained according to the synthesis method for YK-101-PM2. 27 H 55 NO5, MS(ES): m / z (M+H + )474.7.

[0218] Step 2: Synthesis of 3-hexylnonyl-6-(4-(decyloxy)-4-oxobutyl)(2-(2-(2-hydroxyethoxy)ethoxy-ethyl)amino)hexanoate (YK-121) Using 3-hexylnonyl-6-(2-(2-(2-hydroxyethoxy)ethoxy-ethyl)amino)hexanoate (100 mg, 0.21 mmol) and INT-1 (117 mg, 0.38 mmol) as starting materials, the target compound (72 mg, 0.10 mmol, 49.0%) was obtained according to the preparation method of YK-101. 41 H 81 NO7, MS(ES): m / z (M+H + )700.5. 1H NMR (400 MHz, CDCl3) δ 5.34 (s, 1H), 4.11 (td, J = 7.0, 2.7 Hz, 4H), 3.87 - 3.75 (m, 2H), 3.77 - 3.66 (m, 4H), 3.67 - 3.54 (m, 2H), 3.39 - 2.89 (m, 6H), 2.48 (t, J = 6.6 Hz, 2H), 2.35 (t, J = 7.3 Hz, 2H), 2.15 (s, 2H), 1.90 (s, 3H), 1.80 - 1.51 (m, 7H), 1.51 - 1.15 (m, 37H), 0.92 (t, J = 6.8 Hz, 9H).

[0219] 24. Synthesis of 2-octyldecyl-6-(4-(decyloxy)-4-oxobutyl)(2-(2-(2-hydroxyethoxy)ethoxy-ethyl)amino)hexanoate (YK-122) Synthesis scheme: [ka]

[0220] Step 1: Synthesis of 2-octyldecyl-6-(2-(2-(2-hydroxyethoxy)ethoxy-ethyl)amino)hexanoate (YK-122-PM1) Using YK-107-PM1 (500 mg, 1.12 mmol) and 2-(2-(2-aminoethoxy-ethyl)ethoxy)ethan-1-ol (500 mg, 3.35 mmol) as starting materials, 2-octyldecyl-6-(2-(2-(2-hydroxyethoxy)ethoxy-ethyl)amino)hexanoate (452 ​​mg, 0.88 mmol, 78.2%) was obtained according to the synthesis method for YK-101-PM2. 30 H 61 NO5, MS(ES): m / z (M+H + )516.7.

[0221] Step 2: Synthesis of 2-octyldecyl-6-(4-(decyloxy)-4-oxobutyl)(2-(2-(2-hydroxyethoxy)ethoxy-ethyl)amino)hexanoate (YK-122) Using 2-octyldecyl-6-(2-(2-(2-hydroxyethoxy)ethoxy-ethyl)amino)hexanoate (200 mg, 0.39 mmol) and INT-1 (214 mg, 0.70 mmol) as starting materials, the target compound (105 mg, 0.14 mmol, 36.3%) was obtained according to the preparation method of YK-101. 44 H 87 NO7, MS(ES): m / z (M+H + )742.6. 1 H NMR (400 MHz, CDCl3) δ 4.10 (t, J = 6.8 Hz, 2H), 4.00 (d, J = 5.8 Hz, 2H), 3.84 - 3.73 (m, 2H), 3.73 - 3.55 (m, 6H), 2.74 (d, J = 58.3 Hz, 4H), 2.37 (dt, J = 17.7, 7.3 Hz, 5H), 2.05 (s, 2H), 1.88 (s, 4H), 1.76 - 1.51 (m, 7H), 1.31 (d, J = 6.8 Hz, 44H), 1.00 - 0.81 (m, 9H).

[0222] 25. Synthesis of 3-hexylnonyl-6-(4-(decyloxy)-4-oxobutyl)(2-(2-hydroxyethoxy-ethyl)amino)hexanoate (YK-123) Synthesis scheme: [ka]

[0223] Step 1: Synthesis of 3-hexylnonyl-6-(2-(2-hydroxyethoxy-ethyl)amino)hexanoate (YK-123-PM1) Using INT-2 (500 mg, 1.23 mmol) and 2-(2-aminoethoxy)ethan-1-ol (338 mg, 3.69 mmol) as starting materials, 3-hexylnonyl-6-(2-(2-hydroxyethoxy-ethyl)amino)hexanoate (474 ​​mg, 1.10 mmol, 89.7%) was obtained according to the synthesis method for YK-101-PM2. 25 H 51 NO4, MS(ES): m / z (M+H + )430.6.

[0224] Step 2: Synthesis of 3-hexylnonyl-6-(4-(decyloxy)-4-oxobutyl)(2-(2-hydroxyethoxy-ethyl)amino)hexanoate (YK-123) Using 3-hexylnonyl-6-(2-(2-hydroxyethoxy-ethyl)amino)hexanoate (200 mg, 0.47 mmol) and INT-1 (289 mg, 0.94 mmol) as starting materials, the target compound (240 mg, 0.37 mmol, 77.8%) was obtained according to the preparation method of YK-101. 39 H 77 NO6, MS(ES): m / z (M+H + )656.5. 1 H NMR (400 MHz, CDCl3) δ 5.34 (s, 1H), 4.11 (dd, J = 14.7, 7.1 Hz, 4H), 3.74 (dd, J = 5.2, 3.6 Hz, 2H), 3.69 - 3.58 (m, 3H), 2.67 (d, J = 59.1 Hz, 5H), 2.35 (dt, J = 16.7, 7.4 Hz, 4H), 1.86 (s, 2H), 1.73 - 1.50 (m, 8H), 1.46 - 1.17 (m, 39H), 0.92 (t, J = 6.8 Hz, 9H).

[0225] 26. Synthesis of 2-octyldecyl-6-(4-(decyloxy)-4-oxobutyl)(2-(2-hydroxyethoxy-ethyl)amino)hexanoate (YK-124) Synthesis scheme: [ka]

[0226] Step 1: Synthesis of 2-octyldecyl-6-(2-(2-hydroxyethoxy-ethyl)amino)hexanoate (YK-124-PM1) Using YK-107-PM1 (260 mg, 0.58 mmol) and 2-(2-aminoethoxy)ethan-1-ol (183 mg, 1.74 mmol) as starting materials, 2-octyldecyl-6-(2-(2-hydroxyethoxy-ethyl)amino)hexanoate (132 mg, 0.28 mmol, 48.2%) was obtained according to the synthesis method for YK-101-PM2. 28 H 57 NO4, MS(ES): m / z (M+H + )472.3.

[0227] Step 2: Synthesis of 2-octyldecyl-6-(4-(decyloxy)-4-oxobutyl)(2-(2-hydroxyethoxy-ethyl)amino)hexanoate (YK-124) Using 2-octyldecyl-6-(2-(2-hydroxyethoxy-ethyl)amino)hexanoate (130 mg, 0.28 mmol) and INT-1 (129 mg, 0.42 mmol) as starting materials, the target compound (109 mg, 0.16 mmol, 55.8%) was obtained according to the preparation method of YK-101. 42 H 83 NO6, MS(ES): m / z (M+H + )698.5. 1H NMR (400 MHz, CDCl3) δ 3.99 (t, J = 6.8 Hz, 2H), 3.89 (d, J = 5.8 Hz, 2H), 3.72 - 3.59 (m, 2H), 3.61 - 3.45 (m, 4H), 2.64 (s, 2H), 2.49 (s, 4H), 2.25 (dt, J = 11.6, 7.4 Hz, 4H), 1.75 (p, J = 7.5 Hz, 2H), 1.67 - 1.34 (m, 7H), 1.34 - 0.98 (m, 45H), 0.90 - 0.68 (m, 9H).

[0228] 27. Synthesis of 3-hexylnonyl-6-(4-(decyloxy)-4-oxobutyl)(2-((4-(dimethylamino)butyryl)oxy)ethyl))amino)hexanoate (YK-125) Synthesis scheme: [ka] 3-Hexylnonyl-6-(4-(decyloxy)-4-oxobutyl)((2-hydroxyethyl)amino)hexanoate (100 mg, 0.16 mmol) and 4-(dimethylamino)butyric acid (39 mg, 0.29 mmol) were dissolved in DCM (2 mL). EDCI (98 mg, 0.51 mmol) and DMAP (20 mg, 0.16 mmol) were added to the above solution and stirred at 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. The residue was purified by silica gel chromatography (ethyl acetate / n-hexane) to give 3-hexylnonyl-6-(4-(decyloxy)-4-oxobutyl)(2-((4-(dimethylamino)butyryl)oxy)ethyl))amino)hexanoate (84 mg, 0.11 mmol, 72.4%). 43 H 84 N2O6, MS(ES): m / z (M+H + )725.5. 1H NMR (400 MHz, CDCl3) δ 4.23 - 3.90 (m, 6H), 2.70 (t, J = 6.3 Hz, 2H), 2.59 - 2.43 (m, 4H), 2.44 - 2.17 (m, 14H), 1.93 - 1.71 (m, 4H), 1.72 - 1.51 (m, 6H), 1.53 - 1.39 (m, 3H), 1.42 - 1.17 (m, 36H), 1.00 - 0.81 (m, 9H).

[0229] 28. Synthesis of 2-octyldecyl-6-(4-(decyloxy)-4-oxobutyl)(2-((4-(dimethylamino)butyryl)oxy)ethyl))amino)hexanoate (YK-126) Synthesis scheme: [ka] 270 mg of YK-009 was prepared according to the method described in CN114044741B. Using YK-009 (120 mg, 0.18 mmol) and 4-(dimethylamino)butyric acid (29 mg, 0.22 mmol) as starting materials, the target compound (72 mg, 0.09 mmol, 52.1%) was obtained according to the preparation method for YK-125. 46 H 90 N2O6, MS(ES): m / z (M+H + )767.5. 1 H NMR (400 MHz, CDCl3) δ 4.00 (dt, J = 18.8, 6.5 Hz, 4H), 3.89 (d, J = 5.9 Hz, 2H), 2.60 (t, J = 6.3 Hz, 2H), 2.46 - 2.33 (m, 4H), 2.32 - 2.09 (m, 13H), 1.70 (dp, J = 29.3, 7.4 Hz, 4H), 1.55 (ddd, J = 10.1, 7.5, 4.2 Hz, 6H), 1.35 (ddd, J = 9.1, 7.2, 5.2 Hz, 3H), 1.31 - 1.00 (m, 45H), 0.91 - 0.68 (m, 9H).

[0230] 29. Synthesis of 9-heptadecyl-8-(8-((3-hexylnonyl)oxy)-8-oxooctyl)-((2-hydroxyethyl)amino)caprylate (Compound 21) Synthesis scheme: [ka]

[0231] Step 1: Synthesis of 8-bromooctanoic acid-9-heptadecyl ester (compound 21-PM1) 9-Heptadecanol (1.00 g, 3.90 mmol) and 8-bromooctanoic acid (1.04 g, 4.66 mmol) were used as raw materials and purified by silica gel chromatography (ethyl acetate / n-hexane) according to the preparation method of INT-1 to obtain 9-heptadecyl 8-bromooctanoate (1.20 g, 2.60 mmol, 66.7%).

[0232] Step 2: Synthesis of 9-heptadecyl-8-((2-hydroxyethyl)amino)caprylate (compound 21-PM2) Using 9-heptadecyl 8-bromooctanoate (500 mg, 1.08 mmol) and ethanolamine (119 mg, 3.25 mmol), 9-heptadecyl-8-((2-hydroxyethyl)amino)caprylate (372 mg, 0.84 mmol, 78.0%) was obtained according to the synthesis method for YK-101-PM2. 27 H 55 NO3, MS(ES): m / z (M+H + )442.3.

[0233] 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 INT-1 to obtain 8-bromooctanoate-3-hexylnonyl ester (1.62 g, 3.74 mmol, 85.3%).

[0234] Step 4: Synthesis of 9-heptadecyl-8-(8-((3-hexylnonyl)oxy)-8-oxooctyl)-((2-hydroxyethyl)amino)caprylate (Compound 21) Using 9-heptadecyl-8-((2-hydroxyethyl)amino)caprylate (200 mg, 0.46 mmol) and 8-bromooctanoic acid-3-hexylnonyl ester (336 mg, 0.82 mmol) as starting materials, the target compound (213 mg, 0.27 mmol, 58.4%) was obtained according to the preparation method of YK-101. 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).

[0235] 30. Synthesis of bis(3-hexylnonyl)-8,8'-((2-hydroxyethyl)azadialkyl) dicaprylate (compound 23) Synthesis scheme: [ka] 3-Hexylnonyl 8-bromooctanoate (710 mg, 1.64 mmol) and ethanolamine (40 mg, 0.66 mmol) were dissolved in acetonitrile (10 mL). Potassium carbonate (1.09 g, 7.92 mmol) and potassium iodide (66 mg, 0.39 mmol) were added to the above system, and the mixture was heated to 70 °C and stirred for 20 hours. After the reaction was completed, the reaction mixture was cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel chromatography (methanol / dichloromethane) to obtain bis(3-hexylnonyl)-8,8'-((2-hydroxyethyl)azadialkyl) dicaprylate (150 mg, 0.20 mmol, 29.7%). 48 H 95 NO5, MS(ES): m / z (M+H + )766.5. 1 H 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).

[0236] Example 2: Optimization of preparation conditions for lipid nanoparticles (LNP formulations) 1. Optimization of the ratio of carrier (liposome) to mRNA [ka] The cationic lipid compound YK-108 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 obtain 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, respectively. 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 an LNP formulation encapsulating eGFP-mRNA using the cationic lipids YK-108 / DSPC / cholesterol / DMG-PEG2000 (mol percentage 49:10:39.5:1.5). Cell transfection experiments showed that carrier-to-mRNA weight ratios ranging from 10:1 to 30:1 produced good transfection results, with the highest transfection efficiency at 15:1. Ratios of 5:1 and 35:1 produced poor transfection results and should not be used for mRNA delivery (Figure 1). The same results were obtained with LNP formulations prepared with YK-101 and YK-107, but these were not shown in the figures.

[0237] 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-108 to the neutral lipid DSPC were 1:1, 3:1, 4:1, 4.9:1, 10:1, 15:1, and 20:1, respectively. Cell transfection experiments showed that a molar ratio of cationic lipid to neutral lipid of 1:1 to 15:1 was effective for transfection, with the highest transfection efficiency at 4:1. Ratios of 3:1 and 4.9:1 also showed good transfection efficacy (Figure 2). The same results were obtained with LNP formulations prepared with YK-101 and YK-107, but these were not shown in the figures.

[0238] 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-108, 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%, the transfection efficiency was high, when it was 1.5%, the transfection efficiency was highest, and when it was 10%, the transfection efficiency was lowest (Figure 3). The same results were obtained with LNP formulations prepared with YK-101 and YK-107, but these were not shown in the figures.

[0239] 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-108, 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, 45:17.5:37:0.5, 40:10:48.5:1.5, 30:10:58.5:1.5, and 25:8:65:2, respectively. Cell transfection experiments showed 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, 45:17.5:37:0.5, 40:10:48.5:1.5, 30:10:58.5:1.5, and 25:8:65:2, with the transfection effect being the best at a ratio of 40:10:48.5:1.5. 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 optimal ratio was 40:10:48.5:1.5. The same results were obtained with LNP formulations prepared with YK-101 and YK-107, but these were not shown in the figures.

[0240] Example 3: Cell transfection experiments of LNP formulations of eGFP-mRNA Cell resuscitation and passage: 293T cells were resuscitated, cultured in a culture dish to the required cell number, and passaged. 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. Cell transfection experiment: The LNP formulation (the cationic lipid in the carrier was YK-108) 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. Based on the experimental results, the final preparation conditions for nanolipid particles (LNP preparation) were determined. The carrier to mRNA ratio was 15:1, the molar ratio of cationic lipid to neutral lipid was 4.9:1, the polymer-conjugated lipid accounted for 1.5% of the liposome, 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, the various cationic lipids designed in this application and those 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.

[0241] Example 4: Preparation of nanolipid particles (LNP formulation) [Table 1] JPEG0007764663000068.jpg222164 JPEG0007764663000069.jpg238164 JPEG0007764663000070.jpg215164 JPEG0007764663000071.jpg215164 JPEG0007764663000072.jpg70164

[0242] 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).

[0243] 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 activity and making it suitable for difficult-to-transfect cell types. Lipofectamine 3000 transfection reagent was used as a control, and Lipofectamine 3000 formulations of eGFP-mRNA or Fluc-mRNA were prepared according to the Lipofectamine 3000 (Lipofectamine Shanghai) Trading Co., Ltd. (Lipofectamine Shanghai) instruction manual.

[0244] 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.

[0245] 10 μL of 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 were shown in the following table.

[0246] [Table 2]

[0247] The nanolipid particles prepared in Example 4, all with particle diameters between 90 and 260 nm, were suitable for mRNA delivery. Among these, the particles prepared with YK-107 and YK-119 had the smallest particle diameters at 96 and 98 nm, respectively, while the particles prepared with YK-123 and YK-126 had the largest particle diameters at 246 and 260 nm, respectively. The polydispersity of all nanolipid particles ranged from 7.7% to 41.3%, with compounds 23 and YK-122 having the smallest polydispersity (both 7.7%) and YK-119 having the largest polydispersity (41.3%).

[0248] Example 6: In vitro validation of LNP delivery vehicle performance Cell resuscitation and passaging: The method was the same as in Example 3.

[0249] Seeding: The method was the same as in Example 3.

[0250] 1. Fluorescent detection of Fluc-mRNA 0.3 µg of an LNP formulation containing Fluc-mRNA (the LNP formulation carrier components 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 in 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 3–6.

[0251] Test Results: (1) Among the designed compounds, LNP formulations prepared with YK-101, YK-107, and YK-108 exhibited the highest cell transfection activity, significantly improving the activity of similar or previously used cationic lipids. For example, the activity was up to 16-fold higher than that of SM-102 and 19-fold higher than that of compound 23.

[0252] [Table 3]

[0253] a. Differences in cell transfection activity Table 3 shows the fluorescence detection results of LNP formulations containing Fluc-mRNA prepared with various cationic lipids. Among these, 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). All LNPs prepared with these cationic lipids exhibited excellent cell transfection efficacy. Lipofectamine 3000 is a widely used cell transfection reagent with excellent transfection performance. As can be seen from Table 3 and Figure 5, the LNP formulations containing Fluc-mRNA prepared with YK-101, YK-107, and YK-108 exhibited the strongest fluorescence absorption, with RLU values ​​of 20,335,193, 22,361,210, and 24,660,507, respectively, which were 13.25-fold, 14.58-fold, and 16.07-fold higher than those of SM-102, 9.89-fold, 10.87-fold, and 11.99-fold higher than those of ALC-0315, 14.64-fold, 16.10-fold, and 17.75-fold higher than those of compound 21, 15.75-fold, 17.32-fold, and 19.10-fold higher than those of compound 23, 9.93-fold, 10.92-fold, and 12.04-fold higher than those of HHMA, and Lipofectamine, respectively. It can reach 18.94x, 20.83x, and 22.97x for 3000, and 4.01x, 4.41x, and 4.86x for YK-009. Data analysis using GraphPad Prism software showed that YK-101, YK-107, and YK-108 significantly improved transfection efficiency compared with SM-102, ALC-0315, compound 21, compound 23, HHMA, Lipofectamine 3000, and YK-009.

[0254] b.Differences in chemical structure YK-101, YK-107, and YK-108 are similar in chemical structure to cationic lipids in the prior art, with only one or two C differences in the G1, R1, G2, or R2 groups. For example, compared to the SM-102 chemical structure, in YK-101, the G1 group had two fewer Cs, the R1 group had one fewer C, the G2 group had two fewer Cs, and the R2 group had two more Cs in one strand, with two fewer Cs in each strand of the duplex, but the rest of the structure was completely identical. In YK-107, the G1 group had two fewer Cs, the R1 group had one fewer C, the G2 group had two fewer Cs, the R2 group had one more C in one strand, and the G3 group had one more C, but the rest of the structure was completely identical. In YK-108, the G1 group had two fewer Cs, the G2 group had one fewer C, and the R2 group had two more Cs in one strand, with two fewer Cs in each strand of the duplex, but the rest of the structure was completely identical.

[0255] Interim summary Among the designed compounds, LNP formulations prepared with YK-101, YK-107, and YK-108 exhibited the highest cell transfection activity, significantly improving it compared to similar cationic lipids or cationic lipids used in the prior art. For example, the activity was up to 16-fold higher than that of SM-102 and 19-fold higher than that of compound 23. Therefore, compounds with similar chemical structures do not necessarily have similar cell transfection activity, and there was a strong possibility that significant differences could exist.

[0256] (2) YK-101, YK-107, and YK-108 have the highest cell transfection activity among the most structurally similar designed compounds, reaching 34-fold higher than other compounds such as YK-109.

[0257] To verify whether structurally similar compounds have relatively large differences in activity, we compared compounds with structures most similar to YK-101, YK-107, and YK-108. The results showed that there were significant differences in activity among this series of compounds, with YK-101, YK-107, and YK-108 exhibiting the highest cell transfection activity, 28-fold, 31-fold, and 34-fold higher than that of YK-109, the least active compound, respectively, resulting in significantly improved transfection efficiency.

[0258] [Table 4]

[0259] a. Differences in cell transfection activity As can be seen from Table 4 and Figure 6, the fluorescence absorption values ​​of the LNP formulations prepared with these compounds were significantly different from those of YK-101, YK-107, and YK-108. YK-101, YK-107, and YK-108 were 13.89-fold, 15.27-fold, and 16.84-fold lower than YK-102, and 16.84-fold lower than YK-103, respectively. The ATP levels were 8.79-fold, 9.67-fold, and 10.66-fold for YK-104, 12.01-fold, 13.20-fold, and 14.56-fold for YK-104, 12.76-fold, 14.03-fold, and 15.47-fold for YK-105, 12.49-fold, 13.73-fold, and 15.15-fold for YK-106, and 28.34-fold, 31.17-fold, and 34.37-fold for YK-109. The activity differences were also significant among compounds YK-102, YK-103, YK-104, YK-105, YK-106, and YK-109. The fluorescence absorbance values ​​of LNP formulations prepared with YK-103, YK-104, YK-105, and YK-106 were 2,312,883, 1,693,707, 1,593,805, and 1,628,063, respectively, all of which were slightly higher than those of SM-102, being 1.51-fold, 1.10-fold, 1.04-fold, and 1.06-fold, respectively. The fluorescence absorbance value of YK-102 was 1,464,032, which was not significantly different from that of SM-102, being 0.95-fold. The fluorescence absorbance value of YK-109 was 717431, which was only 0.47 times that of SM-102. Among this series of compounds, the most active was YK-103, which was 3.2 times more active than the least active YK-109. Data analysis using GraphPad Prism software showed that YK-102, YK-103, YK-104, YK-105, YK-106, and YK-109 were not significantly different from SM-102, whereas YK-101, YK-107, and YK-108 were significantly different from all of YK-102, YK-103, YK-104, YK-105, YK-106, and YK-109, indicating significantly improved transfection efficiency.

[0260] b.Difference in chemical structure This series of compounds are very similar in structure, differing only by 1-2 carbon atoms in individual groups. YK-101, YK-107, and YK-108 are very close in structure to the other compounds, and the remaining compounds are also very similar. For example, compared with YK-101, YK-104 has the same structure except for one less C in the G2 group, but the cell transfection activity of YK-101 was 12-fold higher. Compared with YK-107, YK-102 has the same structure except for one more C in the R2 group and two fewer Cs in each strand of the double strand. However, the cell transfection activity of YK-107 was 15-fold higher. Compared with YK-108, YK-109 has the same structure except for one more C in the G1 group, two fewer Cs in the R1 group, and a different L1 group (-OC(O)-). However, the cell transfection activity of YK-108 was 34-fold higher than that of YK-109. Compared with YK-109, YK-103 has exactly the same structure except that G1 and R1 each have one more C and L1 is different and is -C(O)O-. However, the cell transfection activity of YK-103 was 3.2 times higher than that of YK-109.

[0261] Interim summary Among the designed series of compounds with very similar structures, YK-101, YK-107, and YK-108 had the highest cell transfection activity, 28-fold, 31-fold, and 34-fold higher than that of YK-109, respectively. There was no correlation between compound structure and intracellular transfection efficiency. Even within a set of compounds with the most similar structures, there was a high likelihood of significant differences in cell transfection efficiency. Therefore, screening for cationic lipid compounds with high transfection efficiency from a series of compounds with very similar structures was not easy, and multiple designs were required.

[0262] (3) Screening for compounds with high cell transfection activity is very difficult and requires a large amount of creative work.

[0263] I. YK-101, YK-107, and YK-108 had the highest transfection activity compared to compounds with slight structural differences only in the G1, G3, or R1 groups, up to more than 10,000 times higher than other compounds such as YK-112.

[0264] Furthermore, when the cell transfection activity of YK-101, YK-107, and YK-108 was compared with other compounds with similar structures, this series of compounds only differed slightly in the G1, G3, or R1 groups (e.g., G1 has two more C atoms, G3 has a side chain group introduced, or R1 has an -S- or -SS- group introduced). As a result, there were significant differences in activity between this series of compounds and YK-101, YK-107, and YK-108, and the cell transfection efficiencies of YK-101, YK-107, and YK-108 were significantly higher than those of the other compounds, up to 10,000 times higher.

[0265] [Table 5]

[0266] a. Differences in cell transfection activity Compared with YK-101, YK-107, and YK-108, the other compounds only had slight differences in the G1, G3, or R1 groups, but these differences had a significant impact on the cell transfection activity, with the cell transfection activity varying up to 10,000-fold or more. Specifically, as can be seen from Table 5, the LNP formulations prepared with YK-111, YK-114, YK-115, YK-116, and YK-120 were close to SM-102 in terms of fluorescence absorption values, respectively: 0.87-fold, 0.92-fold, 0.93-fold, 0.86-fold, and 0.91-fold for SM-102; 0.96-fold, 1.02-fold, 1.03-fold, 0.95-fold, and 1.01-fold for compound 21; 1.03-fold, 1.10-fold, 1.11-fold, 1.02-fold, and 1.09-fold for compound 23; 0.65-fold, 0.69-fold, 0.70-fold, 0.65-fold, and 0.69-fold for HHMA; and Lipofectamine. For 3000, the values ​​were 1.24, 1.32, 1.33, 1.23, and 1.31. The activity of the above five compounds was relatively different from that of YK-101, YK-107, and YK-108, which were 15.31-fold, 16.83-fold, and 18.57-fold stronger than that of YK-111, 14.33-fold, 15.76-fold, and 17.38-fold stronger than that of YK-114, 14.25-fold, 15.66-fold, and 17.28-fold stronger than that of YK-115, 15.39-fold, 16.92-fold, and 18.66-fold stronger than that of YK-116, and 14.49-fold, 15.93-fold, and 17.57-fold stronger than that of YK-120, respectively. The activities of YK-110, YK-113, and YK-117 were significantly different from that of SM-102: 0.18-fold, 0.10-fold, and 0.45-fold against SM-102, 0.20-fold, 0.11-fold, and 0.50-fold against compound 21, 0.21-fold, 0.12-fold, and 0.54-fold against compound 23, 0.13-fold, 0.07-fold, and 0.34-fold against HHMA, and 0.25-fold, 0.14-fold, and 0.65-fold against Lipofectamine 3000, respectively. The activity of the above three compounds was significantly different from that of YK-101, YK-107, and YK-108, which were 74.82-fold, 82.28-fold, and 90.74-fold more potent than YK-110, 134.05-fold, 147.41-fold, and 162.57-fold more potent than YK-113, and 29.23-fold, 32.14-fold, and 35.45-fold more potent than YK-117, respectively. YK-112, YK-118, and YK-119 showed significantly reduced activity compared to SM-102, with only 0.0013-fold, 0.0139-fold, and 0.0056-fold reductions against SM-102, 0.0014-fold, 0.0153-fold, and 0.0062-fold reductions against compound 21, 0.0016-fold, 0.0165-fold, and 0.0066-fold reductions against compound 23, 0.0010-fold, 0.0104-fold, and 0.0042-fold reductions against HHMA, and 0.0019-fold, 0.0199-fold, and 0.0080-fold reductions against Lipofectamine 3000. The intracellular transfection efficiencies of YK-112, YK-118, and YK-119 were only 1‰ to 1% of those of SM-102. The activity of the above three compounds is significantly different from that of YK-101, YK-107, and YK-108, which are 10,106.95-fold, 11,113.92-fold, and 12,256.71-fold more potent than YK-23, 954.26-fold, 1049.33-fold, and 1157.23-fold more potent than YK-118, and 2,376.44-fold, 2613.21-fold, and 2,881.91-fold more potent than YK-119, respectively. Data were analyzed using GraphPad Prism software, and it was found that YK-110, YK-112, YK-113, YK-118, and YK-119 were significantly different from SM-102, and YK-101, YK-107, and YK-108 were significantly different from all compounds, significantly improving cell transfection efficiency.

[0267] b.Differences in chemical structure This series of compounds differs slightly from YK-101, YK-107, and YK-108 only in the G1, G3, or R1 groups, e.g., G1 has two more C atoms, G3 has a side chain group introduced, or R1 has an -S- or -SS- group introduced. The structures of these compounds also differ slightly. For example, compared with YK-101, YK-112 has two more Cs in G1, four fewer Cs in R1, and one more S, yet the cell transfection activity of YK-101 is more than 10,000 times that of YK-112. YK-118 differs only in the G3 group, with one more branched methyl, yet the cell transfection activity of YK-101 is 900 times that of YK-118. Compared with YK-107, YK-113 has two more Cs in G1, four fewer Cs in R1, and one more S, while the cell transfection activity of YK-107 is 140 times that of YK-113. Compared with YK-108, YK-119 only has one less C in R1, one less C in G2, and one more -C(CH3)2- group in G3, but the cell transfection activity of YK019 can reach 2800 times that of YK-119. Figure 7 shows the fluorescence absorption diagrams of the LNP formulations prepared with YK-101, YK-108, YK-112, and YK-119. Compared with YK-101 and YK-108, YK-112 and YK-119 showed much weaker fluorescence absorption. Among these compounds, YK-110 has only a slight difference in the R2 group compared with YK-111; a single strand of YK-110 has one more carbon atom than a single strand of YK-111Y, and two fewer carbon atoms per strand of the double strand than YK-111. However, the cell transfection efficiency of YK-111 was five times that of YK-110. YK-112 and YK-113 also have only a slight difference in the R2 group; a single strand of YK-112 has one more carbon atom than a single strand of YK-113, and two fewer carbon atoms per strand of the double strand than YK-113. However, the cell transfection activity of YK-113 is 75 times that of YK-112. There was only a slight difference in the G3 group among YK-117, YK-118, and YK-119, but the cell transfection efficiency of YK-117 was 33 times higher than that of YK-118 and 80 times higher than that of YK-119.

[0268] Interim summary Compared with compounds G1 (containing two more Cs), G3 (containing a side chain group), or R1 (containing an -S- or -SS- group), YK-101, YK-107, and YK-108 had the highest cell transfection activity. For example, YK-101, YK-107, and YK-108 were all more than 10,000-fold more active than YK-112 and more than 2,000-fold more active than YK-119. Furthermore, even among compounds with similar structures, the activity varied significantly; for example, YK-117 was 80-fold more active than YK-119. As can be seen, even slight structural differences can significantly affect transfection activity, making screening for compounds with high cell transfection activity extremely challenging and requiring a great deal of creative effort.

[0269] II. YK-101, YK-107, and YK-108 showed the highest transfection activity compared to compounds in which an ether bond was simply introduced into the G3 group or the G3 group was modified to 4-(dimethylamino)butyryl, for example, up to 300-fold higher than that of YK-124.

[0270] The compounds listed in Table 6 differ from YK-101, YK-107, and YK-108 only in that the G3 group is either introduced with an ether bond or changed to 4-(dimethylamino)butyryl. The results of cell transfection activity clearly show that among this series of structurally similar compounds, YK-101, YK-107, and YK-108 had higher cell transfection activity than the other compounds, being 240-, 270-, and 300-fold higher than YK-124, respectively.

[0271] [Table 6]

[0272] a. Differences in cell transfection activity When an ether bond was introduced into the G3 group of YK-101, YK-107, and YK-108 or the G3 group was changed to 4-(dimethylamino)butyryl, the cell transfection activity was significantly reduced, and the activities of YK-101, YK-107, and YK-108 were 240-, 270-, and 300-fold lower than that of YK-124, respectively. Specifically, it is as follows: As can be seen from Table 6, the LNP formulations prepared with YK-122 and YK-123 were close to SM-102 in terms of fluorescence absorption, being 0.92-fold and 0.53-fold, 1.02-fold and 0.58-fold, respectively, compared to SM-102, 1.10-fold and 0.63-fold, respectively, compared to compound 21, 0.69-fold and 0.39-fold, respectively, compared to HHMA, and 1.32-fold and 0.75-fold, respectively, compared to Lipofectamine 3000. The above two compounds were relatively different from YK-101, YK-107, and YK-108, whose fluorescence absorption values ​​were 14.33-fold, 15.76-fold, and 17.38-fold higher than those of YK-122, and 25.18-fold, 27.68-fold, and 30.53-fold higher than those of YK-123, respectively. YK-121, YK-125, and YK-126 showed relatively large differences from SM-102: 0.28-fold, 0.11-fold, and 0.14-fold against SM-102, 0.31-fold, 0.12-fold, and 0.16-fold against compound 21, 0.33-fold, 0.13-fold, and 0.17-fold against compound 23, 0.21-fold, 0.08-fold, and 0.11-fold against HHMA, and 0.40-fold, 0.16-fold, and 0.20-fold against Lipofectamine 3000, respectively. The above three compounds were significantly different from YK-101, YK-107, and YK-108, whose fluorescence absorption values ​​were 47.56-fold, 52.29-fold, and 57.67-fold lower than YK-121, 119.74-fold, 131.67-fold, and 145.21-fold lower than YK-125, and 92.44-fold, 101.65-fold, and 112.10-fold lower than YK-126, respectively. The compound with the lowest fluorescence absorption was YK-124, whose fluorescence absorption was only 0.05 times that of SM-102, 0.06 times that of compound 21, 0.06 times that of compound 23, 0.04 times that of HHMA, and 0.08 times that of Lipofectamine 3000. The fluorescence absorption values ​​of YK-101, YK-107, and YK-108 could reach 249.22 times, 274.05 times, and 302.23 times that of YK-124, respectively. Data analysis using GraphPad Prism software showed that YK-122 and YK-123 were not significantly different from SM-102, while YK-121, YK-125, and YK-126 were significantly different from YK-124. YK-101, YK-107, and YK-108 were significantly different from the other compounds, significantly improving transfection efficiency.

[0273] b.Difference in chemical structure This series of compounds had very small structural differences from YK-101, YK-107, and YK-108, with only the introduction of an ether bond to the G3 group or the modification of the G3 group to 4-(dimethylamino)butyryl, and even among these compounds, the structural differences were all very small. For example, three compounds, YK-121, YK-123, and YK-125, differ from YK-101 only in the G3 group, and from YK-108, the G3 group is different and the R1 and G2 groups each have one less C, but the rest of the structures are completely identical. However, there were significant differences in cell transfection activity: YK-101 was 47-, 25-, and 120-fold more potent than YK-121, YK-123, and YK-125, respectively, and YK-108 was 58-, 31-, and 145-fold more potent than YK-121, YK-123, and YK-125, respectively. Comparing YK-121 and YK-122, there was only a slight difference in the R2 group; the single strand of YK-121 had one more carbon atom than the single strand of YK-122 and two fewer carbon atoms per strand of the double strand than YK-122. However, the cell transfection efficiency of YK-122 was 3.3 times higher than that of YK-121. Comparing YK-123 and YK-124, there was only a slight difference in the R2 group; the single strand of YK-123 had one more carbon atom than the single strand of YK-124 and two fewer carbon atoms per strand of the double strand than YK-124. However, the cell transfection efficiency of YK-123 was much higher than that of YK-124, 10 times higher. The structural differences between the two compounds, YK-125 and YK-126, are similar to those between YK-121 and YK-122; that is, there is only a slight difference in the R2 group; the single strand of YK-125 has one more carbon atom than the single strand of YK-126, and each single strand of the double strand has two fewer carbon atoms than YK-126. However, in terms of cell transfection activity, YK-125 and YK-126 are very similar, with YK-126 only 1.3 times stronger than YK-125. A comparison of the activities of three pairs of compounds, YK-121 / YK-122, YK-123 / YK-124, and YK-125 / YK-126, revealed that although the structural changes in the R2 group were the same in YK-121, YK-123, and YK-125 (i.e., one additional C in the single-stranded complex and two fewer Cs in each of the double-stranded complexes), the changes in activity were quite different: one activity improved, the other activity significantly decreased, and the activity of the remaining one remained almost unchanged. Therefore, changing one group to another does not necessarily have the same effect on activity, meaning that it is impossible to predict changes in transfection activity depending on the change in chemical structure.

[0274] Interim summary As can be seen from the above, compared to a series of compounds in which an ether bond was introduced into the G3 group or the G3 group was modified to 4-(dimethylamino)butyryl, YK-101, YK-107, and YK-108 had the highest cell transfection activity, reaching 240-fold, 270-fold, and 300-fold higher than that of YK-124, respectively. Furthermore, even among compounds with similar structures, differences in cell transfection activity cannot be inferred from structural differences. Therefore, it is impossible to obtain cationic lipids with high transfection activity based on simple chemical structure combinations or group substitutions. Screening for compounds with high transfection efficiency is extremely difficult and requires a great deal of creative effort.

[0275] summary 1) Through extensive design of compound structures and a large amount of creative work, we screened cationic lipid compounds with high cell transfection efficiency, such as YK-101, YK-107, and YK-108. The LNP formulations prepared with YK-101, YK-107, and YK-108 exhibited the highest cell transfection activity, significantly improving the activity of similar cationic lipids or cationic lipids used in the prior art, reaching, for example, up to 16-fold that of SM-102 and 19-fold that of compound 23. Among the series of compounds with the most similar structures designed, YK-101, YK-107, and YK-108 have the highest cell transfection activity, which can reach 34-fold higher than others such as YK-109. YK-101, YK-107, and YK-108 had the highest transfection activity compared to compounds with only minor structural differences in the G1, G3, or R1 groups, and were up to 10,000-fold more potent than other compounds, such as YK-112. YK-101, YK-107, and YK-108 showed the highest transfection activity compared to compounds with an ether bond simply introduced into the G3 group or with the G3 group modified to 4-(dimethylamino)butyryl, for example, up to 300-fold higher than that of YK-124. 2) There was no correlation between the structure of the compound and the intracellular transfection efficiency. Even compounds with very small structural differences could have very large differences in transfection efficiency. 3) Changing the same group in different compounds to another identical group does not necessarily have the same effect on activity, and in some cases, the opposite effect may occur. In other words, it is impossible to predict the change in activity depending on the change in chemical structure. Therefore, screening for cationic lipid compounds with high transfection efficiency required multiple designs and a large amount of creative work.

[0276] 2. Measuring Cell Viability An LNP formulation containing 1.5 μg of Fluc-mRNA (the carrier components of the LNP formulation are 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 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 7-10.

[0277] Test Results: (1) Among the designed compounds, LNP formulations prepared with YK-101, YK-107, and YK-108 exhibited the lowest cytotoxicity and significantly improved cell viability compared to similar or previously used cationic lipids. For example, YK-107 demonstrated a 39.43% higher cell viability than ALC-0315, a 22.28% higher cell viability than SM-102, and a 22.68% higher cell viability than HHMA.

[0278] [Table 7]

[0279] a. Differences in cell viability Table 7 shows the cytotoxicity detection results of LNP formulations prepared with various cationic lipid compounds. Among these, YK-009 is disclosed in CN114044741B (claim 1), SM-102 is compound 25 disclosed in WO2017049245A2 (page 29), ALC-0315 is compound 3 disclosed in CN108368028B (page 24), compounds 21 and 23 are disclosed in WO2021055833A1 (page 22), and HHMA is compound 1 disclosed in CN112979483B (page 12). Liposomes prepared with these cationic lipids all exhibited excellent cell transfection efficacy. Lipofectamine 3000 is a widely used cell transfection reagent with excellent transfection performance. As can be seen from Table 7, the LNP formulations of Fluc-mRNA prepared with YK-101, YK-107, and YK-108 had the highest cell viability, reaching 90.12%, 90.45%, and 89.86%, respectively, which were 21.95%, 22.28%, and 21.69% higher than SM-102, 39.10%, 39.43%, and 38.84% higher than ALC-0315, 21.59%, 21.92%, and 21.33% higher than compound 21, 19.21%, 19.54%, and 18.95% higher than compound 23, 22.35%, 22.68%, and 22.09% higher than HHMA, and Lipofectamine. 68.21%, 68.54%, and 67.95% higher than 3000, and 16.55%, 16.88%, and 16.29% higher than YK-009 (Fig. 8). Data analysis using GraphPad Prism software showed that YK-101, YK-107, and YK-108 had significantly reduced cytotoxicity compared to SM-102, ALC-0315, compound 21, compound 23, Lipofectamine 3000, and YK-009.

[0280] b.Differences in chemical structure YK-101, YK-107, and YK-108 are similar in chemical structure to cationic lipids in the prior art, with only one or two C differences in the G1, R1, G2, or R2 groups.

[0281] Interim summary Among the designed compounds, LNP formulations prepared with YK-101, YK-107, and YK-108 exhibited the lowest cytotoxicity and significantly improved cell viability compared to similar cationic lipids or cationic lipids used in the prior art. For example, YK-107 exhibited cytotoxicity 39.43% higher than ALC-0315, 22.28% higher than SM-102, and 22.68% higher than HHMA. Furthermore, there was no correlation between the structure of the cationic lipid compounds and their cytotoxicity. Compounds with minimal structural differences were not necessarily associated with differences in cytotoxicity, but rather were highly likely to exhibit significant differences.

[0282] (2) Among the designed compounds with the most similar structures, i.e., differences of only 1–2 C in individual groups such as G1, G2, and R1, YK-101, YK-107, and YK-108 had the lowest cytotoxicity and cell viability 45% higher than that of other compounds such as YK-103.

[0283] To compare the cytotoxicity of structurally similar compounds, we compared YK-101, YK-107, and YK-108 with the compounds with the closest structures. The results showed that this series of compounds showed significant differences in cytotoxicity, with cell viabilities ranging from 45% to 91%. The cell viabilities of YK-101, YK-107, and YK-108 were the highest, reaching 90.12%, 90.45%, and 89.86%, respectively, 45% higher than those of other compounds such as YK-103.

[0284] [Table 8]

[0285] a. Differences in cell viability As can be seen from Table 8, the LNP formulations prepared with these compounds showed significant differences in cytotoxicity. Among them, YK-101, YK-107, and YK-108 had the lowest toxicity and the highest cell viability. YK-103 and YK-102 had the lowest cell viability, at only 45.25% and 51.82%, respectively. YK-101, YK-107, and YK-108 had cell viability that was 44.87%, 45.20%, and 44.61% higher than YK-103, and 38.30%, 38.63%, and 38.04%, respectively, higher than YK-102 (Figure 9). The cell viabilities of YK-106 and YK-109 were slightly higher, at 59.59% and 69.45%, respectively. Compared with YK-101, YK-107, and YK-108, YK-106 was 30.53%, 30.86%, and 30.27%, respectively, and YK-109 was 20.67%, 21.00%, and 20.41%, respectively. YK-104 and YK-105 had relatively high cell viability, 73.55% and 78.58%, respectively. However, compared with YK-101, YK-107, and YK-108, YK-104 had a lower viability by 16.57%, 16.90%, and 16.31%, respectively, and YK-105 had a lower viability by 11.54%, 11.87%, and 11.28%, respectively. Data were analyzed using GraphPad Prism software, and YK-101, YK-107, and YK-108 all showed significant differences in cytotoxicity compared to the other compounds, with significantly reduced cytotoxicity.

[0286] b.Differences in chemical structure This series of compounds has some very small differences from YK-101, YK-107, and YK-108 in individual groups, for example, G1, G2, and R1 differ by 1-2 C. These compounds are also very similar to each other.

[0287] Interim summary Among this series of closely related compounds, YK-101, YK-107, and YK-108 had the highest cell viability, 44.87%, 45.20%, and 44.61%, respectively, compared with YK-103. There was no correlation between compound structure and cytotoxicity, and even within a pair of compounds with the most similar structures, there was likely to be significant differences in cytotoxicity. Therefore, screening for compounds with high transfection efficiency and low cytotoxicity from a series of closely related compounds was extremely difficult.

[0288] (3) Screening for compounds with low cytotoxicity is extremely difficult and requires a great deal of creative work.

[0289] I. YK-101, YK-107, and YK-108 had the highest cell viability compared to compounds with slight structural differences only in the G1, G3, or R1 groups, and were up to 80% higher than other compounds such as YK-112.

[0290] A comparison of cell viability between YK-101, YK-107, and YK-108 and other compounds with similar structures revealed that this series of compounds differed only slightly in the G1, G3, or R1 groups (e.g., G1 has two more C atoms, G3 has a side chain group, or R1 has an -S- or -SS- group). As a result, there were significant differences in the cytotoxicity of this series of compounds, with YK-101, YK-107, and YK-108 exhibiting the highest cell viability, 80% higher than that of other compounds such as YK-112.

[0291] [Table 9]

[0292] a. Differences in cell viability Compared with YK-101, YK-107, and YK-108, the other compounds only had slight differences in the G1, G3, or R1 groups, but these differences had a significant impact on cytotoxicity, with cell viability being up to 80% lower than that of YK-101, YK-107, and YK-108. As can be seen from Table 9, YK-111, YK-114, YK-116, YK-119, and YK-120 had relatively large differences in cell viability compared to YK-101, YK-107, and YK-108. YK-111 was 14.36%, 14.69%, and 14.10%, respectively; YK-114 was 21.79%, 22.12%, and 21.53%, respectively; YK-116 was 22.16%, 22.49%, and 21.90%, respectively; YK-119 was 9.85%, 10.18%, and 9.59%, respectively; and YK-120 was 16.69%, 17.02%, and 16.43%, respectively (Figure 10). The cell viabilities of YK-113, YK-115, YK-117, and YK-118 were 43.05%, 59.07%, 57.93%, and 63.33%, respectively. Compared with YK-101, YK-107, and YK-108, YK-113 was 47.07%, 47.40%, and 46.81% lower, YK-115 was 31.05%, 31.38%, and 30.79%, respectively, YK-117 was 32.19%, 32.52%, and 31.93%, respectively, and YK-118 was 26.79%, 27.12%, and 26.53% lower, respectively. Although YK-112 had the highest cytotoxicity, the cell viability was only 9.82%, which was 80.30%, 80.63%, and 80.04% lower than those of YK-101, YK-107, and YK-108, respectively. Data analysis using GraphPad Prism software showed that YK-101, YK-107, and YK-108 all showed significant differences in cytotoxicity compared with other compounds, with significantly reduced cytotoxicity.

[0293] b.Difference in chemical structure This series of compounds differs slightly from YK-101, YK-107, and YK-108 only in the G1, G3, or R1 groups, e.g., G1 has two more C atoms, G3 has a side chain group introduced, or R1 has an -S- or -SS- group introduced. There are also small differences in the structures of these compounds.

[0294] Interim summary Compared with compounds with two more Cs (G1), side chain groups (G3), or -S- or -SS- groups (R1), YK-101, YK-107, and YK-108 had the highest cell viability. For example, the cell viability of YK-101, YK-107, and YK-108 was 80% higher than that of YK-112 and 40% higher than that of YK-110 and YK-113. Furthermore, because even very small structural differences can have a significant impact on cytotoxicity, screening for compounds with low cytotoxicity is extremely difficult and requires a great deal of creative effort.

[0295] II. YK-101, YK-107, and YK-108 exhibited the lowest cytotoxicity compared to compounds with an ether bond simply introduced into the G3 group or with the G3 group modified to 4-(dimethylamino)butyryl. For example, cell viability was up to 60% higher than that of YK-126.

[0296] The cytotoxicity results clearly showed that the only structural difference between YK-101, YK-107, and YK-108 was in the G3 group. Compounds in which an ether bond was introduced into the G3 group or the G3 group was changed to 4-(dimethylamino)butyryl showed improved cytotoxicity compared to YK-101, YK-107, and YK-108. For example, the cell viability of YK-126 was reduced by 60%.

[0297] [Table 10]

[0298] a. Differences in cell viability As can be seen from Table 10, when an ether bond was introduced into G3 of YK-101, YK-107, and YK-108 or when G3 was changed to 4-(dimethylamino)butyryl, there were significant differences in cytotoxicity. In terms of cell viability, some compounds were not significantly different from YK-101, YK-107, and YK-108, while others, such as YK-126, were 60% lower than YK-101, YK-107, and YK-108 (Figure 11). Specifically, YK-121, YK-122, YK-123, and YK-124 had cell viabilities of 73.11%, 79.15%, 76.68%, and 77.56%, respectively. Compared with YK-101, YK-107, and YK-108, YK-121 had cell viabilities that were 17.01%, 17.34%, and 16.75%, respectively, YK-122 had cell viabilities that were 10.97%, 11.30%, and 10.71%, respectively, YK-123 had cell viabilities that were 13.44%, 13.77%, and 13.18%, respectively, and YK-124 had cell viabilities that were 12.56%, 12.89%, and 12.30%, respectively. The cell viabilities of YK-125 and YK-126 were only 35.39% and 25.03%, respectively. Compared with YK-101, YK-107, and YK-108, YK-125 had a cell viability that was 54.73%, 55.06%, and 54.47%, respectively, lower, and YK-126 had a cell viability that was 65.09%, 65.42%, and 64.83%, lower, respectively. Compared with YK-122 and YK-124, the cell viability of YK-126 was 54.12% and 52.53% lower, respectively. Data analysis using GraphPad Prism software showed that YK-101, YK-107, and YK-108 were significantly different from YK-122, YK-123, YK-124, YK-125, and YK-126, and showed significantly reduced cytotoxicity.

[0299] b.Differences in chemical structure This series of compounds had very little structural difference from YK-101, YK-107, and YK-108, with only the introduction of an ether bond to the G3 group or the modification of the G3 group to 4-(dimethylamino)butyryl; the structural differences among these compounds were also very small.

[0300] Interim summary Compared with a series of compounds in which an ether bond was introduced into the G3 group or the G3 group was modified to 4-(dimethylamino)butyryl, YK-101, YK-107, and YK-108 exhibited the lowest cytotoxicity. For example, cell viability was 54.73%, 55.06%, and 54.47%, respectively, higher than that of YK-125, and 65.09%, 65.42%, and 64.83%, respectively, higher than that of YK-126. Even within this series of compounds, there were significant differences in cytotoxicity; for example, the cell viability of YK-126 was 54.12% and 52.53%, respectively, lower than that of YK-122 and YK-124. Therefore, it is impossible to predict cytotoxicity from chemical structure. Screening for compounds with high transfection efficiency and low cytotoxicity is extremely difficult and requires a great deal of creative effort.

[0301] summary 1) Cell viability was measured for LNP formulations prepared with a series of designed compounds. Cationic lipid compounds with low cytotoxicity, such as YK-101, YK-107, and YK-108, were screened. LNP formulations prepared with YK-101, YK-107, and YK-108 exhibited the lowest cytotoxicity, significantly reducing cytotoxicity compared to similar or conventionally used cationic lipids. For example, cell viability was 39.43% higher than that of ALC-0315, 22.28% higher than that of SM-102, and 22.68% higher than that of HHMA. Among the compounds with the most similar structures, i.e., differences of only 1–2 carbon atoms in individual groups such as G1, G2, and R1, YK-101, YK-107, and YK-108 exhibited the lowest cytotoxicity. These three compounds showed 45% higher cell viability than other compounds, such as YK-103. YK-101, YK-107, and YK-108 were the least cytotoxic compared to compounds with minor structural differences only in the G1, G3, or R1 groups, and these three compounds were 80% more cytotoxic than other compounds, such as YK-112, in terms of cell viability. YK-101, YK-107, and YK-108 exhibited the lowest cytotoxicity compared to compounds with an ether bond or a 4-(dimethylamino)butyryl group in the G3 group. For example, compared to YK-126, these three compounds exhibited 60% higher cell viability. 2) Furthermore, there was no correlation between the structure of the compounds and their cytotoxicity. Even compounds with very small structural differences could have very large differences in their cytotoxicity. 3) It is impossible to predict cytotoxicity from chemical structure. Screening for compounds with high transfection efficiency and low cytotoxicity is extremely difficult and requires a great deal of creative work.

[0302] Example 7: In vivo validation of the performance of cationic lipid delivery vehicles We also verified the protein expression and duration of expression in mice after delivery of mRNA using the designed cationic lipid delivery vehicle. Furthermore, in vivo experiments demonstrated that the LNP delivery vehicle effectively delivered mRNA into the body, resulting in efficient and continuous expression.

[0303] An LNP formulation containing 10 μg of Fluc-mRNA was intramuscularly injected into female BALB / C mice, 4–6 weeks old and weighing 17–19 g. At specific time points (6 h, 24 h, 48 h, and 7 d) after administration, a fluorescent imaging substrate was injected intraperitoneally into the mice. The mice were allowed to move freely for 5 min, after which the mean emission intensity (corresponding to the fluorescence intensity) of the protein expressed in the mice by the LNP-containing mRNA was detected using an IVIS Spectrum small animal in vivo imaging system. The detection results are shown in Tables 11–14 and Figures 12–14.

[0304] Test Results: (1) Among the designed compounds, LNP formulations prepared with YK-101, YK-107, and YK-108 demonstrated high and sustained mRNA expression in mice, significantly improving the expression level compared to similar cationic lipids or cationic lipids used in the prior art. For example, YK-108 achieved expression levels 11-fold higher than SM-102 and 12-fold higher than compounds 21 and 23. The mRNA expression in mice was consistent with cell transfection activity.

[0305] [Table 11]

[0306] a. Differences in expression in the mouse body Table 11 shows the mRNA expression intensity in mice at different time points for LNP formulations containing Fluc-mRNA prepared with various cationic lipids. Among these, 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. As can be seen from Table 11, the LNP formulations containing Fluc-mRNA prepared in YK-101, YK-107, and YK-108 resulted in high and sustained mRNA expression in the mice. The average radiant intensities of YK-101, YK-107, and YK-108 at 6 h were 1,496,240, 1,601,250, and 2,017,280, respectively, which were 2.2-fold, 2.3-fold, and 2.9-fold higher than those of SM-102, 1.8-fold, 1.9-fold, and 2.4-fold higher than those of ALC-0315, 2.5-fold, 2.6-fold, and 3.3-fold higher than those of compound 21, 2.5-fold, 2.7-fold, and 3.4-fold higher than those of compound 23, and 2.3-fold, 2.5-fold, and 3.2-fold higher than those of HHMA, respectively. At 24 h, the concentrations were 1,056,210, 1,302,640, and 1,406,010, respectively, which were 9.0-fold, 11.1-fold, and 11.9-fold higher than SM-102, 5.9-fold, 7.2-fold, and 7.8-fold higher than ALC-0315, 9.6-fold, 11.8-fold, and 12.8-fold higher than compound 21, 9.4-fold, 11.6-fold, and 12.5-fold higher than compound 23, and 9.1-fold, 11.3-fold, and 12.2-fold higher than HHMA, respectively. At 48 h, the concentrations were 185,905, 226,170, and 230,360, respectively, which were 5.8-fold, 7.1-fold, and 7.2-fold higher than SM-102, 4.8-fold, 5.8-fold, and 5.9-fold higher than ALC-0315, 6.0-fold, 7.3-fold, and 7.4-fold higher than compound 21, 5.6-fold, 6.8-fold, and 7.0-fold higher than compound 23, and 6.0-fold, 7.3-fold, and 7.5-fold higher than HHMA. For 7d, the values ​​were 12165, 13288, and 14194, respectively, which were 2.1-fold, 2.3-fold, and 2.4-fold higher than SM-102, 1.8-fold, 2.0-fold, and 2.1-fold higher than ALC-0315, 2.0-fold, 2.2-fold, and 2.4-fold higher than compound 21, 2.1-fold, 2.3-fold, and 2.4-fold higher than compound 23, and 2.3-fold, 2.5-fold, and 2.7-fold higher than HHMA, respectively. Data analysis using GraphPad Prism software showed that YK-101, YK-107, and YK-108 were significantly different from SM-102, ALC-0315, compound 21, compound 23, HHMA, and YK-009 at various time points, and both the expression level and duration of expression were significantly improved.

[0307] b.Differences in chemical structure YK-101, YK-107, and YK-108 are similar in chemical structure to cationic lipids in the prior art, with only one or two C differences in the G1, R1, G2, or R2 groups.

[0308] Interim summary Among the designed compounds, LNP formulations prepared with YK-101, YK-107, and YK-108 showed the highest and most sustained mRNA expression in mice. At 6 h, 24 h, 48 h, and 7 d, the expression levels were significantly higher than those of similar cationic lipids or cationic lipids used in the prior art. For example, YK-108 achieved an expression level 11-fold higher than that of SM-102 and 12-fold higher than that of compounds 21 and 23. The mRNA expression in mice was consistent with the results of the cell transfection experiment in Example 6. Furthermore, there was no correlation between the structure of the cationic lipid compounds and the mRNA expression in mice. LNP formulations prepared with cationic lipids with minimal structural differences were unlikely to result in similar mRNA expression in mice; rather, there was a very high possibility of significant differences.

[0309] (2) Among the compounds with the most similar designed structures, i.e., with only one or two carbon atoms differing in individual groups such as G1, G2, and R1, LNP formulations prepared with YK-101, YK-107, and YK-108 showed the highest mRNA expression levels and the longest duration in mice. The expression level of YK-108 was 14-fold higher than that of other compounds, such as YK-109. The mRNA expression in mice was consistent with cell transfection activity.

[0310] To compare the in vivo expression intensity and duration of mRNA delivered by delivery vehicles prepared with the most structurally similar cationic lipids, YK-101, YK-107, and YK-108 were compared with the structurally most similar compounds YK-102, YK-104, YK-106, and YK-109. The results showed that the LNP formulations prepared with this series of compounds exhibited significantly different mRNA expression in mice, with YK-101, YK-107, and YK-108 demonstrating the highest expression levels and longest durations, reaching levels 11-fold, 13-fold, and 14-fold higher than those of YK-109, respectively.

[0311] [Table 12]

[0312] a. Differences in expression in the mouse body As can be seen from Table 12, among this series of compounds that are closest in structure and differ only by 1–2 C in individual groups, the LNP formulations prepared with YK-101, YK-107, and YK-108 had the highest mRNA expression levels and expression durations in mice. At 6 h, the mean radiant intensities of YK-101, YK-107, and YK-108 were 2.3-, 2.5-, and 3.1-fold higher than those of YK-102, 2.0-, 2.1-, and 2.7-fold higher than those of YK-104, 2.2-, 2.3-, and 2.9-fold higher than those of YK-106, and 2.4-, 2.6-, and 3.2-fold higher than those of YK-109, respectively. At 24 h, the concentrations were 10.5-, 13.0-, and 14.0-fold for YK-102, 9.3-, 11.4-, and 12.3-fold for YK-104, 10.0-, 12.3-, and 13.3-fold for YK-106, and 11.2-, 13.8-, and 14.8-fold for YK-109, respectively. At 48 h, the efflux was 6.1-, 7.4-, and 7.6-fold against YK-102, 5.6-, 6.8-, and 7.0-fold against YK-104, 5.6-, 6.9-, and 7.0-fold against YK-106, and 10.1-, 12.2-, and 12.5-fold against YK-109, respectively. At 7d, the efflux was 1.9-fold, 2.1-fold, and 2.2-fold against YK-102, 1.5-fold, 1.7-fold, and 1.8-fold against YK-104, 1.7-fold, 1.9-fold, and 2.0-fold against YK-106, and 2.5-fold, 2.7-fold, and 2.9-fold against YK-109, respectively. Data were analyzed using GraphPad Prism software, and YK-101, YK-107, and YK-108 were significantly different from other compounds at various time points, and both the expression intensity and expression duration were significantly improved.

[0313] b.Difference in chemical structure The structures of this series of compounds are very small compared to YK-101, YK-107, and YK-108, and only individual groups differ, for example, G1, G2, and R1, which differ by 1-2 C.

[0314] Interim summary Among this series of compounds with very similar structures, LNP formulations prepared with YK-101, YK-107, and YK-108 exhibited the highest mRNA expression intensity and the longest duration of expression in mice. For example, at 24 hours, they were 11.2-fold, 13.8-fold, and 14.8-fold higher than YK-109, respectively, and at 7 days, they were 2.5-fold, 2.7-fold, and 2.9-fold higher than YK-109, respectively. The mRNA expression in mice was consistent with the results of the cell transfection experiment in Example 6. Furthermore, there was no corresponding relationship between mRNA expression in mice and the structure of the cationic lipids. This means that even LNP formulations prepared with a group of compounds with the most similar structures were likely to exhibit significant differences in the level and duration of mRNA expression in mice. Therefore, it is extremely difficult to screen for compounds with high and sustained mRNA expression in animals from a series of structurally similar cationic lipid compounds.

[0315] (3) Screening for cationic lipid compounds that induce high and sustained mRNA expression in animals is extremely difficult and requires a great deal of creative work.

[0316] I. YK-101, YK-107, and YK-108 showed the highest mRNA expression levels and duration of expression in mice compared to compounds with slight structural differences only in the G1, G3, or R1 groups. For example, the expression level of YK-108 was 15-fold higher than that of YK-114. The mRNA expression in mice was consistent with cell transfection activity.

[0317] Furthermore, we compared the mRNA expression in mice between LNP formulations prepared with YK-101, YK-107, and YK-108 and those prepared with other structurally similar compounds. This series of compounds differed only slightly in the G1, G3, or R1 groups. For example, G1 had two additional Cs, G3 had a side chain group, or R1 had an -S- or -SS- group. As a result, there were significant differences in mRNA expression in mice. YK-101, YK-107, and YK-108 all exhibited the highest and sustained expression levels, with YK-108 expression levels 15-fold higher than those of other compounds, such as YK-114.

[0318] [Table 13]

[0319] a. Differences in expression in the mouse body As can be seen from Table 13, among this series of compounds with slight differences in chemical structure only in the G1, G3, or R1 groups, the LNP formulations prepared with YK-101, YK-107, and YK-108 had the highest mRNA expression levels and expression durations in mice. At 6 h, the mean radiant intensities of YK-101, YK-107, and YK-108 were 2.3-, 2.4-, and 3.0-fold higher than those of YK-111, 2.8-, 3.0-, and 3.8-fold higher than those of YK-114, and 2.7-, 2.9-, and 3.6-fold higher than those of YK-120, respectively. At 24 h, the concentrations were 10.6-, 13.1-, and 14.1-fold for YK-111, 11.3-, 13.9-, and 15.0-fold for YK-114, and 11.1-, 13.7-, and 14.8-fold for YK-120, respectively. At 48 h, the levels were 9.2-, 11.1-, and 11.3-fold higher than YK-111, 9.8-, 11.9-, and 12.1-fold higher than YK-114, and 8.9-, 10.9-, and 11.1-fold higher than YK-120, respectively. At 7d, the efflux was 2.4-fold, 2.6-fold, and 2.8-fold against YK-111, 2.2-fold, 2.4-fold, and 2.6-fold against YK-114, and 2.4-fold, 2.6-fold, and 2.8-fold against YK-120, respectively. Data were analyzed using GraphPad Prism software, and YK-101, YK-107, and YK-108 were significantly different from other compounds at various time points, and both the onset effect and duration were significantly improved.

[0320] b.Differences in chemical structure This series of compounds differs slightly from YK-101, YK-107, and YK-108 only in the G1, G3, or R1 groups; for example, G1 has two more C atoms, G3 has an introduced side chain group, or R1 has an introduced -SS- group.

[0321] Interim summary Compared with compounds with only slight structural differences, such as G1 with two additional Cs, G3 with a side chain group, or R1 with an -SS- group, LNP formulations prepared with YK-101, YK-107, and YK-108 showed the highest mRNA expression levels and the longest duration of expression in mice. For example, YK-101, YK-107, and YK-108 showed the highest mRNA expression levels, 11-fold, 13-fold, and 15-fold higher than YK-114, respectively. The mRNA expression in mice was consistent with the results of the cell transfection experiment in Example 6. Even very small structural differences can have a significant impact on mRNA expression in mice. Therefore, screening for cationic lipid compounds that produce high and sustained mRNA expression in mice is extremely difficult and requires a great deal of creative effort.

[0322] II. Compared with compounds in which an ether bond is simply introduced into the G3 group or the G3 group is changed to 4-(dimethylamino)butyryl, YK-101, YK-107, and YK-108 exhibited the highest mRNA expression levels and the longest duration of expression in mice. For example, compared with YK-126, the expression level of YK-108 was 67-fold higher. The mRNA expression in mice was consistent with cell transfection activity.

[0323] [Table 14]

[0324] a. Differences in expression in the mouse body As can be seen from Table 14, compared with compounds with only minor structural differences, i.e., compounds in which an ether bond was simply introduced into the G3 group or the G3 group was changed to 4-(dimethylamino)butyryl, the LNP formulations prepared with YK-101, YK-107, and YK-108 had the highest degree of mRNA expression and duration of expression in mice. At 6 h, the mean radiant intensities of YK-101, YK-107, and YK-108 were 4.7-, 5.0-, and 6.4-fold higher than those of YK-123, and 9.6-, 10.3-, and 13.0-fold higher than those of YK-126, respectively. At 24 h, the increases were 18.0-fold, 22.2-fold, and 23.9-fold for YK-123 and 50.4-fold, 62.2-fold, and 67.1-fold for YK-126, respectively. At 48 h, the levels were 19.9-, 24.2-, and 24.7-fold higher than those of YK-123, and 27.9-, 34.0-, and 34.6-fold higher than those of YK-126, respectively. At 7d, the efflux was 8.6-fold, 9.4-fold, and 10.0-fold against YK-123, and 13.9-fold, 15.2-fold, and 16.2-fold against YK-126, respectively. Data analysis using GraphPadPrism software showed that YK-101, YK-107, and YK-108 were significantly different from YK-123 and YK-126 at various time points, and both the expression effect and duration of expression were significantly improved.

[0325] b.Difference in chemical structure YK-123 and YK-126 have slight differences compared to YK-101, YK-107, and YK-108 only in the G3 group, for example, an ether bond is introduced into G3 or G3 is changed to 4-(dimethylamino)butyryl.

[0326] Interim summary Compared with compounds with only slight structural differences, such as an ether bond introduced into G3 or a 4-(dimethylamino)butyryl group, LNP formulations prepared with YK-101, YK-107, and YK-108 showed the highest and most sustained mRNA expression in mice, with these three compounds being 50-, 62-, and 67-fold higher than YK-126, respectively. The mRNA expression in mice was consistent with the results of the cell transfection experiment in Example 6. It is impossible to predict the mRNA expression effect in mice from chemical structure alone, and screening for cationic lipid compounds that result in high and sustained mRNA expression in mice is extremely difficult and requires a great deal of creative effort.

[0327] summary 1) We conducted experiments to evaluate the in vivo delivery performance of LNP formulations prepared with a series of designed compounds, and screened cationic lipid compounds such as YK-101, YK-107, and YK-108, which induced high and sustained mRNA expression in mice. LNP formulations prepared with YK-101, YK-107, and YK-108 demonstrated high and sustained mRNA expression in mice, significantly improved compared to similar or previously used cationic lipids (e.g., YK-108 was 11-fold more potent than SM-102, and 12-fold more potent than Compounds 21 and 23). Among the compounds with the most similar structures (i.e., differences of 1-2 carbon atoms in only the G1, G2, and R1 groups), YK-101, YK-107, and YK-108 showed the highest mRNA expression levels and the longest duration in mice. YK-108 was 14-fold higher than other compounds, such as YK-109. Compared with compounds with slight structural differences only in the G1, G3, or R1 groups, YK-101, YK-107, and YK-108 showed the highest mRNA expression levels and durations in mice, with YK-108's expression level reaching 15-fold that of YK-114. Compared with compounds in which an ether bond is simply introduced into the G3 group or the G3 group is changed to 4-(dimethylamino)butyryl, YK-101, YK-107, and YK-108 showed the highest mRNA expression levels and the longest duration of expression in mice. For example, the expression level of YK-108 was 67-fold higher than that of YK-126. 2) Furthermore, there was no correlation between the structure of the cationic lipid and the high and sustained expression of mRNA in the mouse body. Even if the structural differences between cationic lipid compounds were very small, LNP formulations prepared with them were likely to have very large differences in mRNA expression in the animal's body. 3) It is impossible to predict from the chemical structure of cationic lipids whether mRNA will be expressed at a high and sustained level in the animal's body. Screening for cationic lipid compounds that result in high and sustained mRNA expression is extremely difficult and requires a great deal of creative work.

[0328] conclusion 1. Among the designed compounds, LNP formulations prepared with YK-101, YK-107, and YK-108 showed significantly improved cell transfection activity, significantly reduced cytotoxicity, and significantly improved mRNA expression levels and expression duration in mice compared with similar cationic lipids or cationic lipids used in the prior art. For example, YK-108 achieved 16-fold higher cell transfection activity than SM-102 and 19-fold higher than compound 23. These three compounds also demonstrated 39% higher cell viability than ALC-0315 and 22% higher than both SM-102 and HHMA. YK-108 achieved 11-fold higher mRNA expression levels than SM-102 and 12-fold higher than compounds 21 and 23 in mice. Among a series of designed compounds with very small differences in chemical structure, LNP formulations prepared with YK-101, YK-107, and YK-108 showed significantly improved cell transfection activity, significantly reduced cytotoxicity, and significantly improved mRNA expression levels and duration in mice compared with other compounds. This series of compounds has structural differences of only one or two carbon atoms in each group, slight differences in the G1, G3, and R1 groups, the introduction of an ether bond to the G3 group, or the modification of the G3 group to 4-(dimethylamino)butyryl. However, the cell transfection activity of YK-101, YK-107, and YK-108 is more than 10,000 times that of YK-112, the cell viability of YK-101, YK-107, and YK-108 is 80% higher than that of YK-112, and the mRNA expression level in mice is 67 times higher than that of YK-126.

[0329] 2. There was 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 animal tissues in LNP formulations prepared with them. Compounds with minimal structural differences can have significant differences in transfection efficiency and / or cellular toxicity, or high intracellular expression. For example, in the present application, compounds YK-108 and YK-109 showed 34-fold higher cell transfection efficiency than YK-109, 20% lower toxicity to transfected cells than YK-109, and 15-fold higher mRNA expression in mice than YK-109. YK-101, YK-107, and YK-108 all showed cell transfection activity more than 10,000 times higher than YK-112. The mRNA expression of compound YK-107 in vivo was 62 times higher than that of YK-126. Therefore, it is very difficult to screen for a suitable cationic lipid compound that simultaneously has high transfection efficiency, low toxicity to cells, and high and sustained mRNA expression in mice.

[0330] 3. Through original design and extensive screening, the present invention has discovered several compounds, including YK-101, YK-107, YK-108, YK-103, YK-104, YK-105, and YK-106, which, compared with other compounds in the prior art, can deliver nucleic acids with high cell transfection efficiency, low toxicity to cells, and high and sustained expression in animal bodies, 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】

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

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

4. The composition of claim 2 , wherein the carrier further comprises a neutral lipid.

5. 5. The composition according to claim 4, wherein the molar ratio of the cationic lipid to the neutral lipid is 1:1 to 15:

1.

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

7. The neutral lipids include 1,2-dilinoleoyl-sn-glycerol-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-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphorylethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphorylethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphorylethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphorylethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphorylethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphorylethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphorylethanolamine, 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 6, 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.

8. The composition of claim 2 , wherein the carrier further comprises a structured lipid.

9. 9. The composition of claim 8, wherein the molar ratio of said cationic lipid to said structural lipid is from 0.6:1 to 3:

1.

10. 9. The composition of claim 8, 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.

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

12. The composition according to claim 11, wherein the molar ratio of the polymer-conjugated lipid to the carrier is 0.5% to 10%.

13. The composition of claim 11, 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.

14. The composition of claim 13, 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).

15. 3. The composition of claim 2, wherein the carrier comprises a neutral lipid, a structural lipid, and a polymer-conjugated lipid, and the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is (25-75):(5-25):(15-65):(0.5-10).

16. The composition according to claim 2, wherein the composition is a nanoparticle formulation, and the nanoparticle formulation has an average particle size of 10 nm to 300 nm and a polydispersity of 50% or less.

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

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

19. 19. The composition according to claim 18, wherein the mass ratio of the carrier to the therapeutic or prophylactic agent is 10:1 to 30:

1.

20. The therapeutic or prophylactic agent comprises one or more of a nucleic acid molecule, a small molecule compound, a polypeptide, or a protein; or The composition of claim 18, wherein the therapeutic or prophylactic agent is a vaccine or a compound capable of eliciting an immune response.

21. The composition of claim 18 , wherein the therapeutic or prophylactic agent is a nucleic acid.

22. 22. The composition of claim 21, wherein the therapeutic or prophylactic agent is ribonucleic acid (RNA) or deoxyribonucleic acid (DNA).

23. 23. The composition of claim 22, 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.

24. Use of the compound of claim 1, or a pharmaceutically acceptable salt thereof, or the composition of any one of claims 2 to 23, in the preparation of a nucleic acid drug, a gene vaccine, a small molecule drug, a polypeptide or protein drug.

25. 24. Use of a compound of claim 1, or a pharmaceutically acceptable salt thereof, or a composition of any one of claims 2 to 23, in the preparation of a medicament for treating a disease or disorder in a mammal in need thereof.

26. 26. The use of claim 25, 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.

27. 27. The use according to claim 26, 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.

28. The use according to any one of claims 25 to 27, wherein the composition is administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation.

29. The use of any one of claims 25 to 27, wherein the drug is administered to the mammal in a dose of 0.001 mg / kg to 10 mg / kg.

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