Lipids and Compositions for Delivery

Abstract

Lipids and compositions for delivery. Specifically, the compound represented by formula I or a salt thereof, wherein R1 to R3, H1 to H3, L1, and L2 are as defined in the specification. JPEG2025501967000066.jpg2443

Description

[Technical field]

[0001] The present disclosure is in the pharmaceutical field and relates to lipids and compositions for delivery. [Background technology]

[0002] Nucleic acid-based drugs, such as messenger RNA (mRNA), antisense oligonucleotides, small interfering RNA (siRNA), and plasmids, have the potential for a wide range of applications, but how to safely and efficiently deliver them to target organs and cells in the body remains a challenging problem that limits the development of this technology.

[0003] Currently, nucleic acid drug delivery systems are broadly divided into two types: viral vector systems and non-viral systems, and nucleic acid drug delivery via lipid nanoparticles is the main method belonging to the non-viral delivery system.

[0004] In gene therapy and vaccine applications, lipid nanoparticles have been proven to be excellent vectors of nucleic acids to treat various diseases. Lipid nanoparticles formed from cationic lipids and other helper lipids, such as cholesterol, phospholipids and PEGylated lipids, can encapsulate nucleic acids, protect them from degradation, and promote cellular uptake and reduce immune responses. Lipid nanoparticles also have other advantages, such as good targeting, fewer side effects, good stability, and high transfection efficiency, by carrying out cellular delivery of bioactive components.

[0005] Due to the rapid development of the field of nucleic acid-based therapeutics, the demand for delivery of nucleic acid drugs is increasing, which requires the development of efficient and safe nucleic acid delivery vectors. Summary of the Invention

[0006] The disclosure provides a compound according to formula I, or a salt thereof: [ka] Among them, L 1 and L 2 are each independently -C(O)O-, -OC(O)-, -C(O)-, -OC(O)O-, -O-, -S(O) x -, -SS-, -C(O)S-, -SC(O)-, -NR a C(O)-, -C(O)NR a -, -NR a C(O)NR a -, -NR a C(O)O-, -OC(O)NR a - or a bond; R a is hydrogen or C 1-6 Alkyl group or C 2-6 alkenyl groups, H 1 and H 2 are each independently C 1-12 Heteroalkylene group, C 1-12 Alkylene group, C 2-12 alkenylene groups, and H 1 and H 2 At least one of the following is C 1-12 is a heteroalkylene group, H 3 is C 1-24 Alkylene group, C 2-24 Alkenylene group, C 3-8 Cycloalkylene group or C 3-8 cycloalkenylene groups, R 1 and R 2 are each independently C 1-24 Alkyl group or C 2-24 alkenyl groups, R 3 is hydrogen, -CN, -C(O)OR 4 , -OC(O)R 4 , -OR 5 or -NR 5 C(O)R 4 Selected from R 4 is C 1-6 Alkyl group or C 2-6 alkenyl groups, R 5 is hydrogen, C 1-6 Alkyl group or C 2-6alkenyl groups, x is selected from 0, 1 or 2.

[0007] In some embodiments, H in the compound of formula I or a salt thereof. 1 and H 2 At least one of the above is a heteroalkylene group, and the heteroalkylene group contains at least one heteroatom selected from O, N and S.

[0008] In some embodiments, H in the compound of formula I or a salt thereof. 1 is C 1-12 Heteroalkylene groups, preferably C 2-9 In some embodiments, the heteroalkylene group is a heteroalkylene group containing at least one oxygen atom. In some embodiments, the heteroalkylene group is a heteroalkylene group containing two oxygen atoms. In some embodiments, the heteroalkylene group is a heteroalkylene group containing at least one nitrogen atom. In some embodiments, the heteroalkylene group is a heteroalkylene group containing at least one oxygen atom.

[0009] In some embodiments, L in the compound of formula I or a salt thereof is 1 and L 2 are each independently selected from -C(O)O-, -OC(O)-, or a bond.

[0010] In some embodiments, L in the compound of formula I or a salt thereof is 1 and L 2 are each independently selected from -C(O)-, -OC(O)O-, -O-, or a bond.

[0011] In some embodiments, L in the compound of formula I or a salt thereof is 1 and L 2 are each independently -S(O) x It is selected from -, -SS-, -C(O)S-, -SC(O)- or a bond.

[0012] In some embodiments, L in the compound of formula I or a salt thereof is 1 and L 2 are each independently -NR a C(O)-, -C(O)NR a -, -NR a C(O)NR a -, -NR a C(O)O- or a bond.

[0013] In some embodiments, L in the compound of formula I or a salt thereof is 1 and L 2 are each independently -NR a C(O)NR a -, -NR a C(O)O-, -OC(O)NR a -or a combination.

[0014] In another aspect, in some embodiments, R in the compound of formula I or a salt thereof is 1 and R 2 are each independently C 2-24 Alkyl group (C2 alkyl group, C3 alkyl group, C4 alkyl group, C5 alkyl group, C6 alkyl group, C7 alkyl group, C8 alkyl group, C9 alkyl group, C 10 Alkyl group, C 11 Alkyl group, C 12 Alkyl group, C 13 Alkyl group, C 14 Alkyl group, C 15 Alkyl group, C 16 Alkyl group, C 17 Alkyl group, C 18 Alkyl group, C 19 Alkyl group, C 20 Alkyl group, C 21 In some further embodiments, R in the compound of formula I or a salt thereof is selected from the group consisting of, but not limited to, alkyl. 1 and R 2 are each independently C 4-18 The alkyl group is selected from the group consisting of alkyl groups.

[0015] In some embodiments, R in the compound of formula I or a salt thereof 1 and R 2 are each independently a branched chain C 4-18 The alkyl group is selected from the group consisting of alkyl groups.

[0016] In some embodiments, R in the compound of formula I or a salt thereof 1 and R 2 are each independently linear C 4-18 The alkyl group is selected from the group consisting of alkyl groups.

[0017] In some embodiments, R in the compound of formula I or a salt thereof 1 is a linear C 4-18 alkyl groups, R 2 is a branched chain C 4-18 The alkyl group is selected from the group consisting of alkyl groups.

[0018] In some embodiments, R in the compound of formula I or a salt thereof 1 is a branched chain C 4-18 alkyl groups, R 2 is a branched chain C 4-18 The alkyl group is selected from the group consisting of alkyl groups.

[0019] In some embodiments, compounds of formula I or salts thereof are provided, where R 1 and R 2 are each independently C 2-24 Alkenyl group (C2 alkenyl group, C3 alkenyl group, C4 alkenyl group, C5 alkenyl group, C6 alkenyl group, C7 alkenyl group, C8 alkenyl group, C9 alkenyl group, C 10 Alkenyl group, C 11 Alkenyl group, C 12 Alkenyl group, C 13 Alkenyl group, C 14 Alkenyl group, C 15 Alkenyl group, C 16 Alkenyl group, C 17 Alkenyl group, C 18 Alkenyl group, C19 Alkenyl group, C 20 Alkenyl group, C 21 In some embodiments, R in the compound of formula I or a salt thereof is selected from the group consisting of aryl, ... 1 and R 2 are each independently C 4-18 alkenyl groups.

[0020] In some embodiments, R in the compound of formula I or a salt thereof 1 and R 2 are each independently a branched chain C 4-18 alkenyl groups.

[0021] In some embodiments, R in the compound of formula I or a salt thereof 1 and R 2 are each independently linear C 4-18 alkenyl groups.

[0022] In some embodiments, R in the compound of formula I or a salt thereof 1 is a linear C 4-18 alkenyl groups, R 2 is a branched chain C 4-18 alkenyl groups.

[0023] In some embodiments, R in the compound of formula I or a salt thereof 1 is a branched chain C 4-18 alkenyl groups, R 2 is a branched chain C 4-18 alkenyl groups.

[0024] Additionally, some embodiments provide compounds according to formula I, [ka] Of these, H 1 , H 2 , H 3 , R 1 , R2 and R 3 is as defined above.

[0025] In some embodiments, H in the compound of formula I or IIa or IIb or a salt thereof 3 is C 2-24 Alkenylene group, C 3-8 Cycloalkylene group or C 3-8 cycloalkenylene groups, or H 3 is C 1-24 In some other embodiments, the H in the compound of formula I or IIa or IIb or a salt thereof is selected from the group consisting of alkylene groups. 3 is C 3-8 It is selected from cycloalkylene groups.

[0026] In some embodiments, C 2-24 The alkenylene group is a C2 alkenylene group, a C3 alkenylene group, a C4 alkenylene group, a C5 alkenylene group, a C6 alkenylene group, a C7 alkenylene group, a C8 alkenylene group, a C9 alkenylene group, a C 10 Alkenylene group, C 11 Alkenylene group, C 12 Alkenylene group, C 13 Alkenylene group, C 14 Alkenylene group, C 15 Alkenylene group, C 16 Alkenylene group, C 17 Alkenylene group, C 18 Alkenylene group, C 19 Alkenylene group, C 20 Alkenylene group, C 21 Alkenylene group, C 22 Alkenylene group or C 23 This includes, but is not limited to, alkenylene groups.

[0027] In some embodiments, C 2-24 The alkylene group is a C4 alkylene group, a C5 alkylene group, a C6 alkylene group, a C7 alkylene group, a C8 alkylene group, a C9 alkylene group, a C 10 Alkylene group, C 11Alkylene group, C 12 Alkylene group, C 13 Alkylene group, C 14 Alkylene group, C 15 Alkylene group, C 16 Alkylene group, C 17 Alkylene group, C 18 Alkylene group, C 19 Alkylene group, C 20 Alkylene group, C 21 Alkylene group, C 22 Alkylene group or C 23 This includes, but is not limited to, alkylene groups.

[0028] According to some further embodiments, the compound of formula I or IIa or IIb is [ka] Of these, R 6 are each independently a hydrogen atom, a hydroxyl group, or C 1-6 Alkyl group or C 2-6 alkenyl groups, n is selected from an integer between 1 and 12, including, but not limited to, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12; H 1 , H 2 , R 1 , R 2 and R 3 is as defined for compounds of formula I.

[0029] In some embodiments, n is 2, 3, 4, 5, or 6 in the compound of formula IIIa or IIIb, or a salt thereof.

[0030] In some embodiments, R in the compound of Formula I or Formula IIIa or IIIb or a salt thereof 3 In some embodiments, R in the compound of Formula I or Formula IIIa or IIIb or a salt thereof is selected from -CN or a hydroxy group. 3 is -C(O)OR 4 , -OC(O)R 4or -NHC(O)R 4 Selected from R 4 is as defined above.

[0031] In some embodiments, R in the compound of formula I or a salt thereof 4 C includes, but is not limited to, a methyl group, an ethyl group, or a propyl group. 1-6 The alkyl group is selected from the group consisting of alkyl groups.

[0032] In some embodiments, the compound of formula I is [ka] In this case, X is -N(R 7 )- or -O-, o is selected from an integer between 1 and 11, p is selected from an integer between 1 and 5, and o+p is 12 or less; R 7 is hydrogen, C 1-6 Alkyl group or C 2-6 alkenyl groups, R 8 , R 9 , R 10 , R 11 are each independently a hydrogen atom, a hydroxyl group, or C 1-6 Alkyl group or C 2-6 alkenyl groups; L 1 , L 2 , H 2 , H 3 , R 1 , R 2 and R 3 is as defined for compounds of formula I.

[0033] In some embodiments, p in the compound of formula IIc or IId, or a salt thereof, is selected from 1 or 2.

[0034] In some embodiments, o in the compound of formula IIc or IId, or a salt thereof, is selected from 4, 5, 6, 7, or 8.

[0035] In some embodiments, X in the compound of formula IIc or IId, or a salt thereof, is --O--.

[0036] In some embodiments, R in the compound of formula IIc or IId or a salt thereof is 8 , R 9 , R 10 , R 11 are each independently selected from hydrogen.

[0037] In some embodiments, R in the compound of formula IIc or IId or a salt thereof is 10 , R 11 are each independently C 1-6 It is selected from alkyl groups.

[0038] In some embodiments, R in the compound of formula IIc or IId or a salt thereof is 3 is selected from hydroxy groups.

[0039] In some other embodiments, the compound of formula I is [ka] and H 2 , R 1 , R 2 and R 3 is as defined for compounds of formula I; R 8 , R 9 , R 10 , R 11 , X, o and p are as defined for the compound of formula IIc.

[0040] In some embodiments, H in the compound of formula I or IIIc or IIId or a salt thereof. 2 is C 3-7 Alkylene group or C 3-7 alkenylene groups.

[0041] Some embodiments of the compound of formula I include [ka] Of these, H 2 , R 1 , R 2 and R 3 is as defined for compounds of formula I; R 8 , R 9 , R 10 , R 11 , X, o and p are as defined for the compound of formula IIc; R 6 and n is as defined above.

[0042] In another aspect, in some embodiments, R in the compound of formula I or a salt thereof is 1 , R 2 have the following structure: [ka] Among them, R 12a , R 12b , R 12c are each independently hydrogen, C 1-12 Alkyl group or C 2-12 alkenyl groups, and k is an integer between 2 and 12.

[0043] In some embodiments, R in the compound of formula I or a salt thereof 12c is selected from hydrogen.

[0044] In some embodiments, R in the compound of formula I or a salt thereof 1 teeth, [ka] Selected from.

[0045] Exemplary compounds of formula I, or pharma- ceutically acceptable salts thereof, are [ka] [ka] Including, but not limited to:

[0046] The present disclosure further provides isotopic substitutions of the above compounds or salts thereof, preferably, the isotopic substitutions are deuterium atom substitutions.

[0047] The present disclosure further provides a lipid particle comprising the compound or a salt thereof. In some embodiments, the lipid particle further comprises an active agent.

[0048] In some embodiments, the active agent is selected from a polynucleotide or a nucleic acid (eg, a ribonucleic acid or a deoxyribonucleic acid).

[0049] In some embodiments, the active agent is selected from mRNA. The present disclosure also provides a pharmaceutical composition comprising the lipid particle and a pharma- ceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises 0.01%-99.99% of a pharma- ceutically acceptable excipient, based on the total weight of the composition. In some embodiments, the pharmaceutical composition comprises 0.1%-99.9% of a pharma- ceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises 0.5%-99.5% of a pharma- ceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises 1%-99% of a pharma- ceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises 2%-98% of a pharma- ceutical acceptable excipient.

[0050] The present disclosure further provides the use of the above compound or a salt thereof, or an isotopic substitution thereof, or the above lipid particle, or the above pharmaceutical composition in the preparation of a medicament for preventing and / or treating a disease or condition that elicits an immune response in a subject.

[0051] The present disclosure further provides the use of the above compound or a salt thereof, or an isotopic substitution thereof, or the above lipid particle, or the above pharmaceutical composition in the preparation of a medicament for preventing and / or treating a disease or condition associated with overexpression of a polypeptide.

[0052] The present disclosure further provides the use of the above compound or a salt thereof, or an isotopic substitution thereof, or the above lipid particle, or the above pharmaceutical composition in the preparation of a medicament for preventing and / or treating a disease or condition associated with insufficient expression of a polypeptide.

[0053] In some embodiments, the disease or condition includes, but is not limited to, cancer, infectious disease, autoimmune disease, neurodegenerative disease, and inflammation.

[0054] The present disclosure further provides a method for preventing and / or treating a disease or condition that induces an immune response in a subject, comprising administering to the patient the compound or salt thereof, or the lipid particle, or the pharmaceutical composition.

[0055] The present disclosure further provides a method for preventing and / or treating a disease or condition associated with overexpression of a polypeptide, comprising administering to the patient the compound or a salt thereof, or the lipid particle, or the pharmaceutical composition.

[0056] The present disclosure further provides a method for preventing and / or treating a disease or condition associated with insufficient expression of a polypeptide, comprising administering to the patient the compound or a salt thereof, or the lipid particle, or the pharmaceutical composition.

[0057] In another aspect, the present disclosure further provides the above compound or a salt thereof, or the above lipid particle, or the above pharmaceutical composition for preventing and / or treating an immune response-inducing disease or condition in a subject.

[0058] The present disclosure further provides the above compound or a salt thereof, or the above lipid particle, or the above pharmaceutical composition for preventing and / or treating a disease or condition associated with overexpression of a polypeptide.

[0059] The present disclosure further provides the above compound or a salt thereof, or the above lipid particle, or the above pharmaceutical composition for preventing and / or treating a disease or condition associated with insufficient expression of a polypeptide.

[0060] The present disclosure further provides the use of the above compound or a salt thereof, or an isotopic substitution thereof, or the above lipid particle, or the above pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of cancer, an infectious disease, an autoimmune disease, a neurodegenerative disease and inflammation.

[0061] The present disclosure further provides a method for preventing and / or treating cancer, infectious diseases, autoimmune diseases, neurodegenerative diseases and inflammation, comprising administering to the patient the compound or a salt thereof, or the lipid particle, or the pharmaceutical composition.

[0062] The compound salts described in this disclosure include "acid" addition salts and "base" addition salts. For example, salts formed by acid-base reaction of basic groups (amino groups), the acid including organic acids or inorganic acids. The compound salts also include salts formed by quaternization of basic groups (amino groups), the quaternizing agents including linear or branched chlorinated hydrocarbons.

[0063] The compounds of the present disclosure may be in particular geometric or stereoisomeric forms. The present disclosure includes cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, and all such compounds are intended to be within the scope of the present disclosure. Substituents such as alkyl groups may have other asymmetric carbon atoms. All such isomers and mixtures thereof are within the scope of the present disclosure. Compounds containing asymmetric carbon atoms of the present disclosure can be isolated in optically pure or racemic forms. Optically pure forms may be resolved from racemic mixtures or synthesized by using chiral starting materials or chiral reagents.

[0064] Optically active (R)- and (S)-isomers and D- and L-isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. One enantiomer of a compound of the present disclosure may be prepared by asymmetric synthesis or derivatization with chiral auxiliaries, in which the resulting diastereomeric mixture is isolated and the resulting diastereomeric mixture is cleaved by assisted group splitting to provide the desired enantiomer in pure form. Alternatively, if the molecule contains a basic (e.g., amino) or acidic (e.g., carboxy) functional group, the diastereomeric salt is formed with an appropriate optically active acid or base, and the diastereomeric separation is carried out by conventional methods well known in the art, followed by recovery to obtain the enantiomer in pure form. Note that the separation of enantiomers and diastereomers is generally accomplished by the use of chromatography, which employs a chiral stationary phase and is optionally combined with a chemical derivatization method (e.g., forming a carbamate from an amine).

[0065] In the chemical structures of the compounds described in this disclosure, [ka] indicates that the configuration is not specified, i.e., if chiral isomers are present in the chemical structure, [ka] The bond [ka] or [ka] In the chemical structure of the compound described in the present disclosure, [ka] The bond "Z" is not specified, i.e., it may be in the Z or E configuration, or it may include the two configurations simultaneously.

[0066] The compounds and intermediates of the present disclosure may also exist in different tautomeric forms, and all such forms are included within the scope of the present disclosure. The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that are interconvertible by a low energy barrier. For example, proton tautomers (also called protolytic tautomers) include interconversions by protolysis, such as keto-enol and imine-enamine, lactam-lactim isomerization, etc. An example of lactam-lactim equilibrium is between A and B as shown below.

[0067] [ka] All of the compounds in this disclosure can be depicted in Form A or Form B. All tautomeric forms are within the scope of this disclosure. The naming of a compound does not exclude any tautomeric forms.

[0068] The present disclosure further includes certain isotopically labeled compounds of the present disclosure that are the same as those described herein, but in which one or more atoms are replaced with an atom having an atomic mass or mass number different from the atomic mass or mass number normally found in nature. Illustrative examples of isotopes that can be attached to the compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, e.g., 2 H, 3 H, 11 C. 13 C. 14 C. 13 N, 15 N, 15 O. 17 O. 18 O. 31 P, 32 P, 35 S, 18 F, 123 I, 125 I and 36 Cl, etc.

[0069] Unless otherwise stated, when a position is specifically designated as deuterium (D), it should be understood that the position is deuterium (i.e., at least 10% deuterium incorporated) with an abundance at least 1000 times higher than the natural abundance of deuterium (which is 0.015%). In the example compounds, having an abundance higher than the natural abundance of deuterium may be at least 1000 times more abundant deuterium, at least 2000 times more abundant deuterium, at least 3000 times more abundant deuterium, at least 4000 times more abundant deuterium, at least 5000 times more abundant deuterium, at least 6000 times more abundant deuterium, or more abundant deuterium. The present disclosure further includes various deuterated forms of the compound of formula (I). Each available hydrogen atom connected to a carbon atom may be independently replaced with a deuterium atom. Those skilled in the art can synthesize deuterated forms of the compound of formula (I) by referring to the relevant literature. Deuterated forms of the compounds of formula (I) may be prepared using commercially available deuterated starting materials or may be synthesized by conventional techniques with deuterated reagents including, but not limited to, borane deuteride, borane tritide in tetrahydrofuran, lithium aluminum deuteride, deuterated iodoethane, deuterated iodomethane, and the like.

[0070] "Optionally" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes cases where the event or circumstance occurs and cases where it does not. For example, "C optionally substituted with a halogen or cyano group" 1-6 By "alkyl group" is meant that a halogen or cyano group may or may not be present, and this description includes cases where the alkyl group is substituted with a halogen or cyano group and cases where the alkyl group is not substituted with a halogen or cyano group. Explanation of terms:

[0071] A "pharmaceutical composition" refers to a mixture of one or more compounds described herein or physiologically acceptable salts or prodrugs thereof with other chemical components, and other components such as physiologically acceptable vectors and excipients. The pharmaceutical composition is intended to facilitate administration to a living body and contribute to the absorption of the active ingredient to further exert biological activity.

[0072] A "pharmaceutically acceptable excipient" includes, but is not limited to, any auxiliary, vector, filler, glidant, sweetener, diluent, preservative, dye / colorant, flavoring agent, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonicity agent, solvent, or emulsifier approved by the U.S. Food and Drug Administration as acceptable for use in humans or domestic animals.

[0073] An "effective amount" or "therapeutic effective amount" as described in this disclosure includes an amount sufficient to ameliorate or prevent the symptoms or condition of a medical condition. An effective amount also refers to an amount sufficient to permit or facilitate diagnosis. The effective amount used in a particular patient or veterinary subject can vary depending on factors such as, for example, the condition being treated, the overall health of the patient, the route and dose of administration, and the severity of side effects. An effective amount may be the maximum dose or dosing regimen in which significant side effects or toxic effects are avoided.

[0074] The term "nucleic acid" refers to a polymer of nucleotides, such as deoxyribonucleotides (DNA) or ribonucleotides (RNA).

[0075] The term "oligonucleotide" refers to a single- or double-stranded nucleotide multimer having a length of 2 to 100 nucleotides. A "polynucleotide" refers to a single- or double-stranded polymer composed of nucleotide monomers. In some embodiments, a polynucleotide is 100 or more nucleotides in length.

[0076] In some alternative embodiments, exemplary polynucleotides include, but are not limited to, deoxyribonucleotides (DNA), ribonucleic acids (RNA, including messenger RNA), RNAi-inducing agents, shRNA, siRNA, miRNA, antisense RNA, and analogs.

[0077] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogues of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.

[0078] The term "white blood cells" refers to white blood cells (WBCs) that are produced and derived from pluripotent hematopoietic stem cells in the bone marrow. White blood cells have a cell nucleus, and based on functional or physical properties, white blood cell types can be classified into five major types, including neutrophils, eosinophils, basophils, lymphocytes, and monocytes.

[0079] "Alkyl group" refers to a saturated aliphatic hydrocarbon group, including straight-chain and branched-chain alkyl groups. In some embodiments, an alkyl group has 1 to 4 carbons, and is preferably C 1-4 Also referred to as an alkyl group. In some embodiments, the alkyl group has 10 to 22 carbons, C 10-22 Also referred to as an alkyl group. In some embodiments, the alkyl group has 4 to 22 carbons, C 4-22 Also called an alkyl group.

[0080] The alkyl group may be unsubstituted or substituted with one or more groups selected from halogen, hydroxy group, amino group, oxo, alkyl group, alkenyl group, alkoxycarbonyl group, amido group, alkylamido group, dialkylamido group, nitro group, amino group, alkylamino group, dialkylamino group, carboxy group, thio and thioalkyl group.

[0081] A "heteroalkyl group" refers to a straight or branched chain alkyl group preferably having 1 to 14 carbons in the chain, more preferably 2 to 10 carbons in which one or more carbons are replaced with a heteroatom selected from S, O and N. Exemplary heteroalkyl groups include alkyl ethers, secondary and tertiary alkyl amines, amides, alkyl sulfides, and the like.

[0082] The heteroalkyl group may be unsubstituted or substituted with one or more groups selected from halogen, hydroxy, alkyl, amino, oxo, alkenyl, alkoxycarbonyl, amido, alkylamido, dialkylamido, nitro, amino, alkylamino, dialkylamino, carboxy, thio, and thioalkyl groups.

[0083] "Alkenyl group" refers to an unsaturated aliphatic hydrocarbon group, including straight-chain and branched-chain alkenyl groups. In some embodiments, an alkenyl group has 1 to 4 carbons, and is 1-4 Also referred to as an alkenyl group. In some embodiments, an alkenyl group has 10 to 22 carbons, such as C 10-22 Also referred to as an alkenyl group. In some embodiments, an alkenyl group has 4 to 22 carbons, such as C 4-22 Also referred to as an alkenyl group. Exemplary alkenyl groups include vinyl, propenyl, n-butenyl, isobutenyl, 3-methylbut-2-enyl, n-pentenyl, heptenyl, octenyl, cyclohexyl-butenyl, and decenyl groups.

[0084] The alkenyl group may be unsubstituted or substituted with one or more groups selected from halogen, hydroxy, amino, oxo, alkyl, alkenyl, alkoxycarbonyl, amido, alkylamido, dialkylamido, nitro, amino, alkylamino, dialkylamino, carboxy, thio, and thioalkyl groups.

[0085] A "monovalent group" is an atom or group that has one valency removed "in form" from a compound. A "subunit" is an atom or group of atoms that has two valencies or one divalent removed "in form" from a compound.

[0086] The term "alkylene group" refers to the portion remaining after removing two hydrogen atoms from an alkane molecule. In some embodiments, an alkylene group has 1 to 4 carbons, such as C 1-4 Also referred to as an alkylene group. In some embodiments, the alkyl group has 10 to 22 carbons, C 10-22 Also referred to as an alkylene group. In some embodiments, the alkyl group has 4 to 22 carbons, such as C 4-22 Also called an alkylene group.

[0087] Similarly, the definitions of "alkyleneoxy group", "alkenylene group", "alkenyleneoxy group", "cycloalkylene group", and "heterocycloalkylene group" are the same as those of "alkylene group".

[0088] The term "hydroxy" refers to an -OH group.

[0089] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0090] The term "cyano" refers to -NH2.

[0091] The term "oxo" refers to a ═O substituent.

[0092] "Substituted" refers to one or more hydrogen atoms in a group, preferably up to 5, more preferably 1 to 3 hydrogen atoms, being independently replaced with a corresponding number of substituents. Of course, the substituents are located only at their chemically possible sites, and a person skilled in the art can determine possible or impossible substitutions (by experiment or theory) without much effort. [Brief description of the drawings]

[0093] [Figure 1] Protein expression levels in mice after intramuscular injection of hepatocyte growth factor mRNA lipid nanoparticles [Diagram 2] Fluorescence intensity detected by intravenous injection of luciferase mRNA lipid nanoparticles into mice [Diagram 3] Cationic lipid concentrations in the liver of mice injected intravenously with mRNA lipid nanoparticles [Figure 4] Cationic lipid concentrations in the spleen of mice injected intravenously with mRNA lipid nanoparticles [Diagram 5] Cationic lipid concentrations in plasma of mice after intravenous injection of mRNA-lipid nanoparticles [Figure 6] Detection of interleukin-6 (IL-6) concentrations in serum of mice injected intravenously with mRNA lipid nanoparticles. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0094] The present disclosure will be further described below in conjunction with examples, but these examples are not intended to limit the scope of the present disclosure.

[0095] Experimental methods for which specific conditions are not specified in the examples of this disclosure generally follow conventional conditions or conditions recommended by the raw material or product manufacturers. Reagents for which specific sources are not specified are conventional reagents that are commercially available.

[0096] The structures of the compounds are determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). The NMR shifts (δ) are within the range of 10 -6 The units are shown in ppm. A Bruker AVANCE-400 nuclear magnetic resonance spectrometer was used for the NMR measurements, and the measurement solvents were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (Methanol-d4), and the internal standard was tetramethylsilane (TMS).

[0097] For HPLC measurements, an Agilent 1100 high pressure liquid chromatograph, a GAS15B DAD ultraviolet detector, and a Water Vbridge C18 150 × 4.6 mm 5 μm column were used.

[0098] MS measurements were performed using an Agilent 6120 triple quadrupole mass spectrometer, G1315D DAD detector, and a Waters Xbridge C18 4.6 × 50 mm, 5 μm column, scanned in positive / negative ion mode with a mass scan range of 80–1200.

[0099] Yantai Huanghai HSGF254 silica gel plate was used as the silica gel plate for thin layer chromatography. The specification for the silica gel plate used in thin layer chromatography (TLC) is 0.2 mm ± 0.03 mm, and the specification for separation and purification of products by thin layer chromatography is 0.4-0.5 mm.

[0100] The flash column purification system used was Combiflash Rf150 (TELEDYNE ISCO) or Isolara one (Biotage).

[0101] For normal phase column chromatography, Yantai Huanghai silica gel 200-300 mesh or 300-400 mesh silica gel is generally used as the vector, or Changzhou Santai Yo-packed ultra-high purity normal phase silica gel column (40-63 μm, 60 g, 24 g, 40 g, 120 g or other specifications) is used.

[0102] Known starting materials in this disclosure may be synthesized by or according to methods known in the art, or may be purchased from companies such as Shanghai Taitan Technology, ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Shaoyuan Chemical Technology (Accela ChemBio Inc.), BiDe Pharmaceutical, etc.

[0103] Unless otherwise specified in the examples, all reactions can be carried out in a nitrogen atmosphere.

[0104] Nitrogen atmosphere refers to a nitrogen balloon of approximately 1 L volume connected to the reaction flask.

[0105] Hydrogen atmosphere refers to a hydrogen balloon of approximately 1 L volume connected to the reaction flask.

[0106] Hydrogen gas is produced by Shanghai Quanpu Scientific Equipment Co., Ltd.'s QPH-1L hydrogen generator.

[0107] The nitrogen or hydrogen atmosphere is usually created by repeating the process of evacuation and introducing nitrogen gas or hydrogen gas three times.

[0108] In the examples, unless otherwise specified, the solution refers to an aqueous solution.

[0109] In the examples, unless otherwise specified, the reaction temperature is room temperature, 20 to 30°C.

[0110] In the examples, thin layer chromatography (TLC) was employed to monitor the progress of the reaction. The volume ratio of the developing solvent used in the reaction, the eluent system of column chromatography for purifying the compound, and the developing solvent system of thin layer chromatography was adjusted according to the polarity of the compound, and may be adjusted by adding a small amount of a basic or acidic reagent such as triethylamine and acetic acid. EXAMPLES

[0111] Example 1 [ka]

[0112] Step 1) [ka] 6-Aminocaproic acid (21.5 g, 100 mmol) was weighed and dissolved in a mixture of ethanol and water (EtOH / HO, v / v=2:1, 200 mL), CsOH·HO (4.0 g, 100 mmol) was added, and benzyloxybromoethane (13.1 g, 100 mmol) was added. The mixture was reacted at room temperature for 40 h. Most of the ethanol was removed by concentration, and the residue was directly purified by reverse phase column chromatography (acetonitrile / water / 0.1% trifluoroacetic acid) and dried by suction to obtain 5.6 g of a white solid, with a yield of 21%. MS:266.2[M+H] + . 1 H NMR (400 MHz, D2O) δ 7.36 (s, 5H), 4.53 (s, 2H), 3.72 - 3.67 (m, 2H), 3.20 - 3.14 (m, 2H), 2.95 - 2.88 (m, 2H), 2.10 (t, J = 7.3 Hz, 2H), 1.57 (dd, J = 15.3, 7.7 Hz, 2H), 1.47 (dd, J = 15.0, 7.5 Hz, 2H), 1.30 - 1.22 (m, 2H).

[0113] Step 2) [ka] 6-((2-(benzyloxy)ethyl)amino)hexanoic acid (5.6 g, 21.1 mmol) was weighed into a flask, 1,4-dioxane (80 mL), aqueous sodium hydroxide (42.2 mL, 1.0 M) was added, Boc2O (5.5 g, 25.2 mmol) was added dropwise, and the mixture was reacted at room temperature for 1 h. LC-MS showed the reaction was complete, and the dioxane was concentrated, the aqueous phase was adjusted to pH 5 with dilute hydrochloric acid, and extracted with ethyl acetate (50 mL x 3). The organic phases were combined, dried, and concentrated to obtain 7.2 g of a nearly colorless gel, with a yield of 93%. The mixture was used as is in the next reaction. MS:366.2[M+H] + .

[0114] Step 3) [ka] 6-((2-(benzyloxy)ethyl)(tert-butoxycarbonyl)amino)hexanoic acid (6.1g, 16.7mmol) was weighed into a flask, dichloromethane (150mL) was added, and the mixture was cooled in an ice bath. Undecanol (2.73g, 15.9mmol), DMAP (408mg, 3.34mmol) and DIPEA (4.3g, 33.4mmol) were added in sequence, EDCI (3.84g, 20.0mmol) was added, the mixture was stirred for 10 minutes, the ice bath was removed, and the mixture was reacted at room temperature overnight. The reaction was monitored by TLC, and the reaction was completed. Water (100mL) was added to the reaction solution, and the mixture was separated. The organic phase was washed with dilute hydrochloric acid, water, and sodium bicarbonate in sequence, dried, concentrated, and purified by column chromatography (PE: EtOAc = 20: 1 to 10: 1). 6.8g of a nearly colorless gel was obtained, and the yield was 82%. MS:520.4[M+H] + . 1 H NMR (400 MHz, CDCl3) δ 7.30 - 7.20 (m, 5H), 4.44 (s, 2H), 3.98 (t, J = 6.7 Hz, 2H), 3.51 (d, J = 18.6 Hz, 2H), 3.32 (d, J = 21.5 Hz, 2H), 3.16 (s, 2H), 2.21 (t, J = 7.5 Hz, 2H), 1.55 (tt, J = 13.3, 6.8 Hz, 6H), 1.36 (d, J = 8.4 Hz, 9H), 1.21 (d, J = 15.5 Hz, 18H), 0.81 (t, J = 6.8 Hz, 3H).

[0115] Step 4) [ka] 6-((2-(benzyloxy)ethyl)(tert-butoxycarbonyl)amino)undecyl hexanoate (7.6 g, 14.6 mmol) was weighed and dissolved in dichloromethane, TFA (20 mL) was added, the mixture was reacted at room temperature for 2 h, and the mixture was concentrated as it was. The residue was dissolved in dichloromethane (100 mL), saturated sodium bicarbonate (100 mL) was added, the mixture was separated, the organic phase was dried, and the mixture was concentrated to obtain a gel-like substance. The mixture was again dissolved in dichloromethane, HCl / dioxane (4.0 M, 4 mL) was added, and the mixture was concentrated as it was to obtain 5.0 g of a white solid, with a yield of 81%. MS:420.3[M+H] + . 1 H NMR (400 MHz, CDCl3) δ 9.51 (s, 2H), 7.39 - 7.28 (m, 5H), 4.57 (s, 2H), 4.03 (t, J = 6.8 Hz, 2H), 3.88 (t, J = 4.8 Hz, 2H), 3.18 (s, 2H), 3.03 (d, J = 3.4 Hz, 2H), 2.28 (t, J = 7.4 Hz, 2H), 2.02 (s, 1H), 1.93 - 1.84 (m, 2H), 1.61 (dd, J = 13.8, 6.7 Hz, 4H), 1.42 - 1.17 (m, 18H), 0.88 (t, J = 6.8 Hz, 3H).

[0116] Step 5) [ka] 2-((5-(benzyloxy)pentyl)oxy)acetic acid (prepared with reference to Chemical and Pharmaceutical Bulletin, 1992, vol. 40, #3, p. 617-623) (7.6 g, 30.12 mmol) was weighed into a flask, dichloromethane (150 mL) was added, and the mixture was placed in an ice bath. Heptadec-9-ol (prepared with reference to WO2020 / 219876) (6.18 g, 24.1 mmol) and DMAP (3.68 g, 30.12 mmol) were added in sequence, and EDCI (6.93 g, 36.14 mmol) was added, stirred for 10 minutes, the ice bath was removed, and the reaction was allowed to proceed at room temperature overnight. The reaction was monitored by TLC to determine whether the reaction was complete. Water (100 mL) was added to the reaction solution, the liquid was separated, and the organic phase was washed successively with dilute hydrochloric acid, water, and sodium bicarbonate, dried, concentrated, and purified by column chromatography (PE: EtOAc = 50: 1 to 10: 1) to obtain 10.9 g of a nearly colorless gel-like product, with a yield of 92%. 1 H NMR (400 MHz, CDCl3) δ 7.36 - 7.31 (m, 4H), 7.30 - 7.26 (m, 1H), 4.96 (p, J = 6.3 Hz, 1H), 4.50 (s, 2H), 4.04 (s, 2H), 3.52 (t, J = 6.6 Hz, 2H), 3.47 (t, J = 6.6 Hz, 2H), 1.69 - 1.61 (m, 4H), 1.56 (d, J = 16.5 Hz, 6H), 1.25 (s, 24H), 0.88 (t, J = 6.8 Hz, 6H).

[0117] Step 6) [ka] Weigh out 4.0 g (8.15 mmol) of heptadecan-9-yl 2-((5-(benzyloxy)pentyl)oxy)acetate, dissolve it in 100 mL of THF, add 400 mg (20%) of Pd(OH)2 / C, and flush with hydrogen gas. The reaction was carried out at room temperature for 2 h. The reaction was monitored by TLC and completed. The reaction was filtered and concentrated. 3.2 g of a colorless gel was obtained, with a yield of 98%. The product was used in the next reaction as it was. 1 H NMR (400 MHz, CDCl3) δ 4.97 (dd, J = 12.4, 6.1 Hz, 1H), 4.04 (s, 2H), 3.66 (t, J = 6.5 Hz, 2H), 3.54 (t, J =6.5 Hz, 2H), 1.71 - 1.57 (m, 4H), 1.56 - 1.42 (m, 6H), 1.25 (s, 24H), 0.88 (t, J = 6.8 Hz, 6H).

[0118] Step 7) [ka] Heptadecan-9-yl 2-((5-hydroxypentyl)oxy)acetate (2.4 g, 6.0 mmol) was weighed and dissolved in dichloromethane (100 mL). DMP (3.82 g, 9.0 mmol) was added in an ice bath and the mixture was reacted at room temperature for 1.5 h. A saturated aqueous solution of sodium thiosulfate (100 mL) was added, the mixture was stirred for 30 minutes, and the liquids were separated. The organic phase was washed with a saturated aqueous solution of sodium bicarbonate (100 mL x 2), dried, and concentrated to obtain 2.5 g of a pale yellow gel. This was used as is in the next reaction. 1H NMR (400 MHz, CDCl3) δ 9.78 (s, 1H), 4.96 (p, J = 6.3 Hz, 1H), 4.04 (s, 2H), 3.55 (t, J = 6.1 Hz, 2H), 2.49 (td, J = 7.2, 1.5 Hz, 2H), 1.81 - 1.72 (m, 2H), 1.68 (d, J = 7.6 Hz, 2H), 1.60 - 1.48 (m, 4H), 1.26 (s, 24H), 0.88 (t, J = 6.8 Hz, 6H).

[0119] Step 8) [ka] Weigh out heptadecan-9-yl 2-((5-oxopentyl)oxy)acetate (2.4g, 6mmol) and dissolve it in dichloromethane (50mL). Add 6-((2-(benzyloxy)ethyl)amino)hexanoic acid undecyl hydrochloride (2.46g, 5.4mmol), add DIPEA (1.16g, 9.0mmol), acetic acid (1.08g, 18.0mmol), stir to clear, ice bath, add NaBH(OAc)3 (3.18g, 15.0mmol), react at room temperature overnight, monitor by TLC, complete the reaction, add water (100mL), separate, wash the organic phase with saturated sodium bicarbonate aqueous solution (50mL×1), dry, concentrate, and obtain 4.1g of colorless liquid by column chromatography (PE:EtOAc=5:1-2:1), yield was 85%. 1H NMR (400 MHz, CDCl3) δ 7.33 (d, J = 4.5 Hz, 4H), 7.30 - 7.26 (m, 1H), 4.99 - 4.91 (m, 1H), 4.52 (s, 2H), 4.08 - 4.02 (m, 4H), 3.57 - 3.47 (m, 4H), 2.68 (t, J = 5.8 Hz, 2H), 2.45 (s, 4H), 2.28 (t, J = 7.5 Hz, 2H), 1.67 - 1.57 (m, 6H), 1.48 (dd, J = 37.5, 6.0 Hz, 6H), 1.38 - 1.18 (m, 46H), 0.88 (t, J = 6.8 Hz, 9H).

[0120] Step 9) [ka] Undecyl 6-((2-(benzyloxy)ethyl)(5-(2-(heptadecan-9-yloxy)-2-oxoethoxy)pentyl)amino)hexanoate (1g, 1.25mmol) was weighed into a flask, dissolved in ethanol, and Pd(OH)2 / C (500mg, 20%) was added. Hydrogen gas was poured in and the reaction was carried out at room temperature overnight. The reaction was monitored by TLC and completed. The reaction was filtered and concentrated. A colorless gel was obtained, and 450mg of a colorless gel was obtained by column chromatography (CH2Cl2:MeOH=10:1), with a yield of 51%. 1 H NMR (400 MHz, CDCl3) δ 5.00 - 4.89 (m, 1H), 4.09 - 4.01 (m, 4H), 3.61 (s, 2H), 3.52 (t, J = 6.4 Hz, 2H), 2.68 (s, 2H), 2.57 (s, 4H), 2.30 (t, J = 7.4 Hz, 2H), 1.70 - 1.59 (m, 6H), 1.53 (s, 8H), 1.43 - 1.18 (m, 44H), 0.88 (t, J = 6.7 Hz, 9H).

[0121] Example 2 [ka]

[0122] Step 1) [ka] Compound 2-2 (50.4 g, 331 mmol) was weighed and dissolved in THF (400 mL), n-BuLi (206 mL, 1.6 M) was gradually added dropwise under a dry ice ethanol bath, and stirred at constant temperature for 1 hour. Compound 2-1 (20.0 g, 110 mmol) was dissolved in THF (100 mL), gradually added dropwise to the reaction system under a dry ice ethanol bath, and stirred at constant temperature for 1 hour, and then stirred at room temperature overnight. The sample was detected by LC-MS, and the starting materials were completely reacted. 1 L of water was added, and the mixture was extracted with 1 L of ethyl acetate x 3. The organic phase was washed with saturated saline, then dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by normal phase column chromatography (PE:EA = 5:1) to obtain 9.00 g of yellow oil, with a yield of 32.3%. MS:253.1[M+H] + . 1 H NMR (400 MHz, CDCl3) δ 7.40-7.24 (m, 5H), 4.57 (s, 2H), 3.65-3.57 (m, 4H), 3.49 (t, J = 6.0 Hz, 2H), 2.39 (t, J = 7.2 Hz, 2H), 1.77-1.61 (m, 4H).

[0123] Step 2) [ka] Compound 2-3 (1.00 g, 3.96 mmol) was dissolved in DCM (20 mL), and heptan-9-ol (914 mg, 3.56 mmol), DMAP (96.0 mg, 786 μmol), DIEA (2.04 g, 15.8 mmol), and EDCI (1.14 g, 9.95 mmol) were added in sequence under an ice-water bath, and the mixture was stirred at 40 ° C overnight. The sample was detected by LCMS and TLC (PE: EA = 10: 1), and the starting materials were completely reacted. 100 mL of water was added, and the mixture was extracted with 100 mL × 3 of ethyl acetate. The organic layer was washed with saturated saline, then dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by normal phase column chromatography (PE: EA = 20: 1) to obtain 1.50 g of a colorless oil, with a yield of 77.1%. MS:513.3 [M+Na] + . 1 H NMR (400 MHz, CDCl3) δ 7.38-7.24 (m, 5H), 4.90-4.82 (m, 1H), 4.57 (s, 2H), 3.64-3.58 (m, 4H), 3.48 (t, J = 6.4 Hz, 2H), 2.31 (t, J = 7.2 Hz, 2H), 1.75-1.59 (m, 4H), 1.55-1.45 (m, 4H), 1.34-1.19 (m, 24H), 0.87 (t, J = 6.8 Hz, 6H).

[0124] Step 3) [ka] Compound 2-4 (1.50 g, 3.06 mmol) was weighed and dissolved in THF (30 mL), Pd(OH)2 / C (300 mg, 20 wt%) was added, hydrogen gas was purged, and the mixture was stirred at room temperature overnight. The sample was detected by TLC (PE:EA=20:1) and the starting material was completely reacted. After filtration, the filtrate was concentrated and purified by normal phase column chromatography (PE:EA=20:1) to obtain 1.10 g of a colorless oil, with a yield of 89.8%. MS:423.3 [M+Na] + . 1H NMR (400 MHz, CDCl3) δ 4.91-4.82 (m, 1H), 3.76-3.69 (m, 2H), 3.55-3.46 (m, 4H), 2.32 (t, J = 7.2 Hz, 2H), 1.95 (s, 1H), 1.76-1.58 (m, 4H), 1.57-1.44 (m, 4H), 1.36-1.18 (m, 24H), 0.87 (t, J = 6.8 Hz, 6H).

[0125] Step 4) [ka] Compound 2-5 (120 mg, 300 μmol) was dissolved in DCM (4 mL), DMP (190 mg, 448 μmol) was added under ice water bath, and the mixture was stirred at room temperature for 2 h. The sample was detected by TLC (PE:EA = 5:1), and the starting materials were completely reacted. 1 mL of saturated aqueous sodium thiosulfate solution and 20 mL of saturated aqueous sodium bicarbonate solution were added to the reaction solution, and the mixture was extracted with 20 mL x 3 of ethyl acetate. The organic layer was washed with saturated saline, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 80 mg of a pale yellow oil, which was used directly in the next reaction without purification. 1 H NMR (400 MHz, CDCl3) δ 9.73 (s, 1H), 4.91-4.81 (m, 1H), 4.09 (s, 1H), 4.06 (s, 1H), 3.65-3.44 (m, 2H), 2.33 (t, J = 7.2 Hz, 2H), 1.79-1.59 (m, 4H), 1.57-1.43 (m, 4H), 1.36-1.16 (m, 24H), 0.87 (t, J = 6.8 Hz, 6H).

[0126] Step 5) [ka] Compound 2-6 (1.09 g, 2.73 mmol) was dissolved in DCM (20 mL), and 2-7 (1.12 g, 2.46 mmol), DIEA (530 mg, 4.10 mmol), and AcOH (492 mg, 8.19 mmol) were added in sequence. After stirring for 10 min, NaBH(OAc)3 (1.45 g, 6.84 mmol) was added and stirred at room temperature overnight. The sample was detected by TLC (pure EA), and the starting materials were completely reacted. 100 mL of saturated aqueous sodium bicarbonate solution was added to the reaction solution, and extracted with 100 mL x 3 of ethyl acetate. The organic phase was washed with saturated saline, then dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by normal phase column chromatography (PE:EA = 2:1) to obtain 290 mg of a pale yellow oil, with a yield of 13.2%. MS:802.4 [M+H] + . 1 H NMR (400 MHz, CDCl3) δ 7.39-7.26 (m, 5H), 4.89-4.82 (m, 1H), 4.51 (s, 2H), 4.05 (t, J = 6.8 Hz, 2H), 3.67-3.44 (m, 4H), 3.41 (t, J = 6.4 Hz, 2H), 2.87-2.37 (m, 6H), 2.33-2.24 (m, 4H), 1.79-1.55 (m, 8H), 1.55-1.43 (m, 6H), 1.38-1.15 (m, 42H), 0.87 (t, J = 6.8 Hz, 9H).

[0127] Step 6) [ka] Compound 2-8 (390 mg, 486 μmol) was weighed and dissolved in EtOH (15 mL), Pd(OH)2 / C (390 mg, 20 wt%) was added, hydrogen gas was purged, and the mixture was stirred at room temperature overnight. The sample was detected by TLC (DCM:MeOH=10:1) and the starting material was completely reacted. After filtration, the filtrate was concentrated and purified by normal phase column chromatography (DCM:MeOH=20:1) to obtain 110 mg of a colorless oil, with a yield of 31.8%. MS: 712.2[M+H] + . 1 H NMR (400 MHz, CDCl3) δ 4.89-4.81 (m, 1H), 4.05 (t, J = 6.8 Hz, 2H), 3.55 (t, J = 5.2 Hz, 2H), 3.48 (t, J = 5.6 Hz, 2H), 3.43 (t, J = 6.4 Hz, 2H), 2.71 (t, J = 5.6 Hz, 2H), 2.67 (t, J = 5.2 Hz, 2H), 2.59-2.52 (m, 2H), 2.34-2.26 (m, 4H), 1.72-1.56 (m, 8H), 1.53-1.44 (m, 6H), 1.36-1.21 (m, 42H), 0.89-0.85 (m, 9H).

[0128] Example 3 [ka]

[0129] Step 1) [ka] 3-2 (5.90 g, 50.0 mmol) was weighed and dissolved in DMF (250 mL), and NaH (2.4 g, 60 mmol) was added in several portions under an ice water bath, and the mixture was stirred at constant temperature for 1 hour. 3-1 (12.9 g, 50.0 mmol) was added in several portions under an ice water bath, and the mixture was stirred at room temperature overnight. 300 mL of water was added, and the mixture was extracted with 300 mL x 2 of ethyl acetate. The organic phase was washed with saturated saline, then dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by normal phase column chromatography (PE:EA = 10:1) to obtain 6.20 g of a pale yellow oil, with a yield of 42.2%. MS: 295.2 [M+H]. 1H NMR (400 MHz, CDCl3) δ 7.34-7.31 (m, 4H), 7.30-7.26 (m, 1H), 4.50 (s, 2H), 4.25-4.15 (m, 2H), 3.92 (q, J = 6.8 Hz, 1H), 3.59-3.53 (m, 1H), 3.47 (t, J = 6.4 Hz, 2H), 3.39-3.33 (m, 1H), 1.68-1.59 (m, 4H), 1.49-1.42 (m, 2H), 1.39 (d, J = 6.8 Hz, 3H), 1.28 (t, J = 6.4Hz, 3H).

[0130] Step 2) [ka] Compound 3-3 (7.40 g, 25.0 mmol) was dissolved in MeOH (150 mL), and a solution of NaOH (4.00 g, 100 mmol) in HO (40 mL) was gradually added under ice water bath, and the mixture was stirred at room temperature overnight. The temperature was lowered in an ice water bath, and the pH was adjusted to about 3 with concentrated hydrochloric acid, and extracted with DCM (200 mL x 3). The organic phase was washed with saturated saline, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 5.50 g of a pale yellow oil, with a yield of 82.6%. MS:267.2 [M+H]. 1 H NMR (400 MHz, CDCl3) δ 7.37-7.33 (m, 4H), 7.31-7.26 (m, 1H), 4.51 (s, 2H), 3.99 (q, J = 6.8 Hz, 1H), 3.60-3.40 (m, 4H), 1.69-1.61 (m, 4H), 1.50-1.44 (m, 5H).

[0131] Step 3) [ka] 3-4 (5.30g, 20.0mmol) and 3-5 (5.10g, 20.0mmol) were weighed and dissolved in DCM (160mL), DMAP (4.90g, 40.0mmol) and EDCI (4.60g, 24.0mmol) were weighed and added to the reaction solution in order, purged with hydrogen gas, and stirred at room temperature overnight. Normal phase column chromatography (PE:EA=10:1) gave 8.60g of a pale yellow oil, with a yield of 85.9%. 1 H NMR (400 MHz, CDCl3) δ 7.34-7.31 (m, 4H), 7.31-7.27 (m, 1H), 4.96-4.91 (m, 1H), 4.50 (s, 2H), 3.91 (q, J = 6.8 Hz, 1H), 3.59-3.53 (m, 1H), 3.47 (t, J = 6.4 Hz, 2H), 3.38-3.32 (m, 1H), 1.68-1.60 (m, 4H), 1.54-1.52 (m, 4H), 1.49-1.43 (m, 2H), 1.39 (d, J = 6.8 Hz, 3H), 1.35-1.18 (m, 24H), 0.87 (t, J = 6.8 Hz, 6H).

[0132] Step 4) [ka] Compound 3-6 (5.00 g, 9.90 mmol) was dissolved in THF (100 mL), Pd(OH)2 / C (500 mg) was added under nitrogen gas protection, the mixture was replaced with hydrogen gas, and hydrogenated by stirring at room temperature overnight. Pd(OH)2 / C was removed by filtration and collected, and the filtrate was concentrated and dried under reduced pressure. A colorless oily product (4.00 g) was obtained by normal phase column chromatography (PE:EA=5:1) in a yield of 96.4%. 1H NMR (400 MHz, CDCl3) δ 4.96-4.92 (m, 1H), 3.91 (q, J = 6.8 Hz, 1H), 3.65 (t, J = 6.8 Hz, 2H), 3.59-3.53 (m, 1H), 3.40-3.36 (m, 1H), 1.68-1.56 (m, 4H), 1.54-1.52 (m, 4H), 1.49-1.41 (m, 2H), 1.39 (d, J = 6.8 Hz, 3H), 1.35-1.19 (m, 24H), 0.87 (t, J = 6.8 Hz, 6H).

[0133] Step 5) [ka] Compound 3-7 (4.10 g, 10.0 mmol) was dissolved in DCM (80 mL), cooled in an ice-water bath, and under nitrogen gas protection, CBr4 (5.00 g, 15 mmol) and PPh3 (3.90 g, 15.0 mmol) were added in sequence, stirred at constant temperature for 30 min, and then stirred at room temperature overnight. The sample was detected by TLC (PE:EA = 5:1), and the starting materials were completely reacted. Normal phase column chromatography (PE:EA = 10:1) gave 4.30 g of a pale yellow oil, with a yield of 90.0%. 1 H NMR (400 MHz, CDCl3) δ 4.97-4.90 (m, 1H), 3.91 (q, J = 6.8 Hz, 1H), 3.59-3.53 (m, 1H), 3.43-3.33 (m, 3H), 1.93-1.85 (m, 2H), 1.67-1.59 (m, 2H), 1.57-1.48 (m, 6H), 1.39 (d, J = 6.8 Hz, 3H), 1.35-1.19 (m, 24H), 0.87 (t, J = 6.8 Hz, 6H).

[0134] Step 6) [ka] 3-8 (4.30 g, 9.00 mmol) was weighed and dissolved in anhydrous EtOH (85 mL), and under nitrogen gas protection, ethanolamine (16.5 g, 270 mmol) was added and stirred at 30 °C overnight. The sample was detected by LC-MS, and the starting materials were completely reacted. The solvent was concentrated and dried under reduced pressure, and EA (200 mL) was added to the residue, washed twice with water (50 mL × 2), washed twice with saturated saline (50 mL × 2), then dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by normal phase column chromatography (DCM:MeOH = 10:1) to obtain 3.30 g of a colorless oil, the yield was 80.1%. MS: 458.4 [M+H]. 1 H NMR (400 MHz, CDCl3) δ 4.96-4.88 (m, 1H), 3.90 (q, J = 6.8 Hz, 1H), 3.64 (t, J = 5.2 Hz, 2H), 3.57-3.51 (m, 1H), 3.38-3.31 (m, 1H), 2.77 (t, J = 4.8 Hz, 2H), 2.63 (t, J = 7.2 Hz, 2H), 1.65-1.57 (m, 2H), 1.56-1.48 (m, 6H), 1.44-1.27 (m, 5H), 1.26-1.19 (m, 24H), 0.86 (t, J = 6.8 Hz, 6H).

[0135] Step 7) [ka] 3-9 (2.30 g, 5.00 mmol) was weighed and dissolved in anhydrous EtOH (46 mL). Under nitrogen gas protection, DIEA (1.90 g, 15.0 mmol) and 3-10 (3.50 g, 10 mmol) were added and stirred at 80 °C overnight. The sample was detected by LC-MS, and the starting material was almost completely reacted. The solvent was concentrated and dried under reduced pressure, and EA (100 mL) was added to the residue, washed twice with water (50 mL × 2), washed twice with saturated saline (50 mL × 2), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by normal phase column chromatography (DCM:MeOH = 15:1) to obtain 2.10 g of a colorless oil, the yield was 57.9%. MS: 726.6[M+H]. 1 H NMR (400 MHz, CDCl3) δ 4.94-4.88 (m, 1H), 4.05 (t, J = 6.8 Hz, 2H), 3.90 (q, J = 6.8 Hz, 1H), 3.69-3.65 (m, 2H), 3.57-3.50 (m, 1H), 3.39-3.33 (m, 1H), 2.80-2.70 (m, 2H), 2.70-2.60 (m, 4H), 2.30 (t, J = 7.6 Hz, 2H), 1.65-1.20 (m, 61H), 0.89-0.85 (m, 9H).

[0136] Example 4 [ka]

[0137] Step 1) [ka] NaH (3.63 g, 0.091 mol) was weighed and dissolved in DMF (200 mL), protected with argon gas, and cooled to 0°C. 4-2 (10 g, 0.076 mol) was added to the system in several portions and stirred at room temperature. 4-1 (19.5 g, 0.076 mol) was added to the reaction and stirred at room temperature overnight. 100 mL of water was added, and the mixture was extracted with 0.5 L x 2 of ethyl acetate. The organic phase was washed with saturated saline, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by normal phase column chromatography (PE:EA = 6:1) to obtain 5.30 g of a colorless oil, with a yield of 22.3%. MS: 309.2 [M+H]. 1 H NMR (400 MHz, CDCl3) δ 7.40-7.25 (m, 5H), 4.50 (s, 2H), 4.18 (q, J = 7.2 Hz, 2H), 3.47 (t, J = 6.4 Hz, 2H), 3.36 (t, J = 6.8 Hz, 2H), 1.69-1.55 (m, 4H), 1.48-1.40 (m, 2H), 1.41 (s, 6H), 1.27 (t, J = 6.8 Hz, 3H).

[0138] Step 2) [ka] Compound 4-3 (5.20 g, 0.017 mol) was dissolved in MeOH (50 mL), and a solution of NaOH (2.69 g, 0.067 mol) in water (10 mL) was added under ice-water bath, and the mixture was stirred at room temperature overnight. Methanol was evaporated under reduced pressure, 100 mL of water was added, and the pH value (3-4) was adjusted with 1N hydrochloric acid. Extraction was performed with 400 mL x 2 of ethyl acetate, and the organic phase was washed with saturated saline, then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 4.80 g of a colorless oil, with a yield of 100%. MS:281.2 [M+H]. 1H NMR (400 MHz, CDCl3) δ 7.35-7.25 (m, 5H), 4.51 (s, 2H), 3.50-3.42 (m, 4H), 1.70-1.50 (m, 4H), 1.50-1.40 (m, 8H).

[0139] Step 3) [ka] 4-4 (3.29 g, 0.012 mol) was weighed and dissolved in DCM (100 mL), 4-5 (2.70 g, 0.011 mol), EDCI (2.70 g, 0.014 mol), DMAP (2.86 g, 0.023 mol), argon gas was substituted, and the mixture was stirred at room temperature overnight. The reaction solution was concentrated to dryness under reduced pressure, and the crude product was purified by normal phase column chromatography (PE:EA = 50:1) to obtain 4.70 g of a colorless oil in 85.7% yield. 1 H NMR (400 MHz, CDCl3) δ 7.36-7.20 (m, 5H), 4.93-4.80 (m, 1H), 4.50 (s, 2H), 3.47 (t, J = 6.8 Hz, 2H), 3.37 (t, J = 6.8 Hz, 2H), 1.70-1.50 (m, 8H), 1.47-1.39 (m, 8H), 1.30-1.15 (m, 24H), 0.87 (t, J = 6.8 Hz, 6H).

[0140] Step 4) [ka] Compound 4-6 (4.50 g, 8.67 mmol) was dissolved in THF (90 mL), Pd(OH)2 / C (0.90 g, 20 wt%) was added, and the mixture was replaced with a hydrogen gas balloon and stirred at room temperature for 5 h. The reaction solution was concentrated to dryness under reduced pressure, and the crude product was purified by normal phase column chromatography (PE:EA = 10:1) to obtain 3.19 g of a colorless oil in 85.7% yield. 1H NMR (400 MHz, CDCl3) δ 4.93-4.86 (m, 1H), 3.65 (t, J = 6.4 Hz, 2H), 3.38 (t, J = 6.4 Hz, 2H), 1.70-1.50 (m, 8H), 1.48-1.40 (m, 8H), 1.32-1.25 (m, 24H), 0.87 (t, J = 6.8 Hz, 6H).

[0141] Step 5) [ka] Compound 4-7 (1.50 g, 3.49 mmol) was dissolved in THF (30 mL), CBr4 (1.16 g, 3.49 mmol) and PPh3 (0.92 g, 3.49 mmol) were added in sequence, and the mixture was stirred at room temperature overnight under argon gas protection. The sample was detected by TLC (PE:EA = 6:1) and the starting material was completely reacted. After filtration, the reaction solution was concentrated to dryness under reduced pressure, and the crude product was purified by normal phase column chromatography (PE:EA = 50:1) to obtain 1.50 g of a colorless oil, with a yield of 87.2%. 1 H NMR (400 MHz, CDCl3) δ 4.92-4.80 (m, 1H), 3.43-3.35 (m, 4H), 1.93-1.80 (m, 2H), 1.65-1.50 (m, 8H), 1.41 (s, 6H), 1.36-1.24 (m, 24H), 0.87 (t, J = 6.8 Hz, 6H).

[0142] Step 6) [ka] 4-8 (1.25 g, 2.55 mmol) was weighed and dissolved in EtOH (15 mL), ethanolamine (4.67 g, 76.5 mmol) was added, and the mixture was protected with argon gas and stirred overnight at 25° C. Ethyl acetate (200 mL) was added, washed with saturated sodium chloride solution (100 mL×2), dried over sodium sulfate, filtered, and the filtrate was concentrated and purified by normal phase column chromatography (DCM:MeOH=10:1) to give 0.84 g of a colorless oil, with a yield of 71.3%. MS Calc:471.4,Found: 472.4[M+H]. 1 H NMR (400 MHz, CDCl3) δ 4.91-4.80 (m, 1H), 3.68 (t, J = 4.8 Hz, 2H), 3.37 (t, J = 6.4 Hz, 2H), 2.83 (t, J = 5.2 Hz, 2H), 2.69 (t, J = 7.2 Hz, 2H), 1.62-1.52 (m, 8H), 1.45-1.36 (m, 8H), 1.55-1.24 (m, 24H), 0.87 (t, J = 6.8 Hz, 6H).

[0143] Step 7) [ka] 4-9 (0.84 g, 1.78 mmol) was weighed and dissolved in EtOH (15 mL), 4-10 (0.81 g, 2.32 mmol) and DIPEA (0.46 g, 3.56 mmol) were added, and the mixture was protected with argon gas and stirred overnight at 80° C. The reaction solution was concentrated under reduced pressure and purified by normal phase column chromatography (DCM:MeOH=20:1) to obtain 0.61 g of a colorless oil, with a yield of 46.2%. MS: 740.7 [M+H]. 1H NMR (400 MHz, CDCl3) δ 4.90-4.86 (m, 1H), 4.05 (t, J = 6.8 Hz, 2H), 3.77 (s, 2H), 3.38 (t, J = 6.4 Hz, 2H), 2.90-2.70 (m, 6H), 2.31 (t, J = 7.2 Hz, 2H), 1.70-1.20 (m, 64H), 0.90-0.85 (m, 9H).

[0144] Comparative Example 1 [ka]

[0145] It was prepared with reference to the method in WO2017049245.

[0146] Test Example 1: Delivery Ability of Lipid Particle Composition 1.1 Preparation method Compound 1, compound 2, and comparative compound 1 were each dissolved in ethanol solution, and mixed with DSPC, cholesterol, and DMG-PEG solutions dissolved in ethanol at a molar ratio of 50:10:38.5:1.5 to prepare an ethanol lipid solution. An mRNA encoding human growth factor was dissolved in a citrate buffer to prepare an aqueous mRNA solution. Liposomes were prepared by mixing an ethanol lipid solution and an aqueous mRNA solution in a microfluidic system so that the weight ratio of total lipid to mRNA was about 20:1. Ethanol was removed by dialysis against a PBS solution to obtain a lipipid nanoparticle composition encapsulating mRNA.

[0147] 1.2 Characterization of lipid particle composition Characterization Method Dynamic light scattering was used to detect the nanosize and polydispersity index PDI of lipid nanoparticles using a Malvern Zetasizer Nano ZS in 173° backscattering detection mode.

[0148] The liposome encapsulation rate was detected using the Quant-iT RiboGreen RNA Assay Kit RNA quantitative detection kit.

[0149] The pKa of the cations in the lipid nanoparticles was detected using a fluorescence assay based on 6-(p-toluidine)-2-naphthalenesulfonic acid sodium salt (TNS). Buffer solutions of 150 mM NaCl, 10 mM sodium phosphate, 10 mM sodium citrate, 10 mM sodium borate, and different pHs from pH 3 to 11.5 were prepared. A 300 μM TNS solution was prepared and added to the buffer solution. After adding the lipid nanoparticles to the buffer solutions of different pHs and mixing thoroughly, the fluorescence intensity was detected at room temperature with an excitation wavelength of 325 nm and an emission wavelength of 435 nm using a fluorescence plate reader. A fitting analysis was performed on the fluorescence data, and the pKa is the pH that produces half-maximal fluorescence intensity. The correlation data is shown in Table 1.

[0150] [Table 1]

[0151] 1.3 Evaluation of in vivo delivery ability of lipid particle compositions by detecting protein expression levels To evaluate lipid nanoparticles that efficiently deliver mRNA in the body and express the corresponding encoded protein, lipid nanoparticles encapsulated with mRNA expressing hepatocyte growth factor were injected into the thigh muscle of 6-8 week-old female BALB / c mice at a dose of 0.05 mg / kg. After 24 hours, the muscle at the injection site was excised and ground and dissolved, and the hepatocyte growth factor protein expression level (pg / mg), i.e., the hepatocyte growth factor protein amount corresponding to the total protein amount of unit muscle tissue, was detected using an ELISA kit. The calculation of the average protein concentration was repeated for at least three sets of lipid nanoparticles corresponding to each compound.

[0152] The lipid nanoparticles corresponding to the comparative compound 1 were used as a control to detect the mRNA delivery efficiency by intramuscular injection of the lipid nanoparticles corresponding to the compounds 1 and 2. The correlation data are shown in FIG. 1 and Table 2.

[0153] In FIG. 1, that is, statistically 0.01 < P < 0.05 indicates that there is a statistically significant difference between groups, ** that is, statistically P < 0.01, *** that is, statistically P < 0.001 indicates that there is an extremely significant difference.

[0154]

Table 2

[0155] Conclusion: As shown in the protein expression levels in Table 2 and FIG. 1, it can be seen that the mRNA delivery effect by intramuscular injection in vivo of lipid nanoparticles corresponding to Compound 1 is significantly superior to that of Comparative Compound 1. Compound 1 is similarly significantly superior to Compound 2. It was suggested that the lipid nanoparticles corresponding to Compound 1 can efficiently deliver mRNA to the muscle site and express proteins.

[0156] Test Example 2: Evaluation of mRNA Delivery Efficiency by Intravenous Injection of Lipid Particle Composition in Vivo To evaluate lipid nanoparticles that can efficiently deliver mRNA in vivo and express the corresponding encoded protein, lipid nanoparticles encapsulated with mRNA expressing luciferase were injected into the tail vein of 6 - 8 week - old female BALB / c at a dose of 0.5 mg / kg. Six hours later, luciferase substrate was injected intraperitoneally into each mouse, and fluorescence images of the mice were taken using an IVIS small - animal optical bioimaging device (PerkinElme), and the fluorescence intensity of the whole body of the mice was statistically analyzed. The height of the fluorescence intensity indicates the height of the expression level of luciferase protein, that is, it reflects the mRNA delivery efficiency of lipid nanoparticles in vivo. The average fluorescence intensity was calculated by biological replicates for at least 3 sets of lipid nanoparticles corresponding to each compound, and the data are shown in Table 3 and FIG. 2. The fluorescence intensity in FIG. 2 is the fluorescence intensity of the whole body of the mice photographed and statistically analyzed by the IVIS small - animal optical bioimaging device. The height of the fluorescence intensity indicates the height of the luciferase protein expression level, that is, the high mRNA delivery efficiency of lipid nanoparticles in vivo.

[0157] Using the lipid nanoparticles corresponding to Comparative Compound 1 as a control, the mRNA delivery efficiency of the lipid nanoparticles corresponding to Compound 1, Compound 3, and Compound 4 was detected by intravenous injection into the tail veins of mice.

[0158] In Figure 2, * that is, statistically 0.01 < P < 0.05 indicates that there is a statistically significant difference between groups, ** that is, statistically P < 0.01, *** that is, statistically P < 0.001 indicates that there is a highly significant difference.

[0159]

Table 3

[0160] Conclusion: As shown by the fluorescence intensity in Table 3 and Figure 2, compared with Comparative Compound 1, the mRNA delivery efficiency by intravenous injection of the lipid nanoparticles corresponding to Compound 1 was significantly superior, and Compound 1 was 2.1 times the protein expression level of the lipid nanoparticles corresponding to Comparative Compound 1. At the same time, the lipid nanoparticles corresponding to Compound 1 were significantly superior to Comparative Compound 3, and the lipid nanoparticles corresponding to Compound 4 were significantly superior to Comparative Compound 1.

[0161] Test Example 3: Evaluation of tissue target-directedness in in vivo delivery of lipid nanoparticles To evaluate the target-directedness in in vivo delivery of lipid nanoparticles, lipid nanoparticles encapsulating mRNA expressing hepatocyte growth factor were injected into the tail veins of 6-8-week-old female BALB / c mice at a dose of 0.5 mg / kg. Tissue samples were collected at 1 hour, 2 hours, 4 hours, 6 hours, 24 hours, and 48 hours after lipid nanoparticle injection, and to evaluate the distribution of lipid nanoparticles in different tissues, the concentration of cationic lipid molecules in the liver and spleen at each time point was detected using LC-MS mass spectrometry. The average cationic lipid concentration in the tissue was calculated for at least 3 sets of lipid nanoparticles corresponding to each compound for biological replicates.

[0162] Conclusion: As shown in Figure 3, the concentration of Compound 1 in the samples taken at various time points in the liver is significantly higher than that of Comparative Compound 1. As shown in Figure 4, the concentration of Compound 1 in the spleen is significantly lower than that of Comparative Compound 1. It was suggested that the lipid nanoparticles corresponding to Compound 1 were more distributed in the liver and less distributed in the spleen.

[0163] From the pharmacokinetic parameters, the total concentration of the cationic lipid in the tissue 48 hours after administration was calculated to evaluate the distribution of the lipid nanoparticles in the liver and spleen. The table shows the average values calculated for multiple sets. As shown in Table 4, the concentration of Compound 1 is significantly higher in the liver than that of Comparative Compound 1, but significantly lower in the spleen than that of Comparative Compound 1.

[0164] Therefore, the lipid nanoparticles corresponding to Compound 1 can deliver nucleic acids with better targeting of liver tissue compared to Comparative Compound 1.

[0165] In Figures 3 and 4, * that is, statistically 0.01 < P < 0.05 indicates that there is a statistically significant difference between groups, ** that is, statistically P < 0.01, *** that is, statistically P < 0.001 indicates that there is a highly significant difference.

[0166]

Table 4

[0167] Test Example 4: Evaluation of the In Vivo Pharmacokinetics of Lipid Nanoparticles To evaluate the targeting ability of lipid nanoparticles in vivo delivery, lipid nanoparticles encapsulating mRNA expressing hepatocyte growth factor were injected into the tail vein of 6-8 week-old BALB / c female mice at a dose of 0.5 mg / kg. Plasma, liver and spleen samples were collected at 1, 2, 4, 6, 24 and 48 hours after lipid nanoparticle injection, and the concentration of cationic lipid molecules in plasma was detected at each time point using LC-MS mass spectrometry to evaluate the metabolic rate and clearance rate of lipid nanoparticles in the body. The average cationic lipid concentration in tissues was calculated for at least three sets of lipid nanoparticles corresponding to each compound in biological replicates.

[0168] Conclusion: As shown in Figure 5, the concentration of compound 1 in plasma at 1 hour was 1163ng / mL, which was significantly lower than that of comparative compound 1, whose concentration was 6170ng / mL, and the remaining concentration of comparative compound 1 was 5.3 times that of compound 1, indicating that the lipid nanoparticles corresponding to compound 1 can be rapidly distributed to target tissues, thereby reducing retention in the blood system. Compound 1 was completely eliminated at 4 hours, while the concentration of comparative compound 1 could still be detected at 6 hours. Compound 1 showed faster clearance in the blood system and better metabolism in the body.

[0169] From the pharmacokinetic parameters, the half-life of cationic lipid in tissue was calculated, and the average half-life of compound 1 in the liver was 34.2 hours, which is significantly lower than the half-life of comparative compound 1, 61.1 hours. Similarly, the half-life of compound 1 in the spleen was 31.9 hours, which is significantly lower than the half-life of comparative compound 1, 37.9 hours. It was suggested that compound 1 is better decomposed and metabolized in tissue. Meanwhile, the side effects of lipid nanoparticles on the human body, such as inflammation, are mainly derived from cationic lipid. Compound 1 allows for faster metabolic decomposition and has better biological safety than comparative compound 1.

[0170] In Figure 5, that is, statistically 0.01 < P < 0.05 indicates that there is a statistically significant difference between groups, and **that is, statistically P < 0.01, ***that is, statistically P < 0.001 indicates that there is a very significant difference.

[0171]

Table 5

[0172] Test Example 5: Evaluation of the in vivo immunogenicity of lipid nanoparticles The entry of foreign substances into the body of mammals triggers an innate immune response and promotes the production of cytokines. These foreign substances are likely to cause an inflammatory reaction when taken into the body, leading to adverse reactions in the body such as fever and edema. Therefore, the immunogenicity of lipid nanoparticles in the body is evaluated by assessing the blood cytokine concentration after injection of lipid nanoparticles in mice, for example, interleukin 6 (IL-6). Lower cytokine concentrations indicate that lipid nanoparticles have lower immunogenicity, that is, better biosafety.

[0173] Lipid nanoparticles encapsulating mRNA expressing the luciferase reporter gene were injected into the tail vein of 6 - 8-week-old female BALB / c mice at a dose of 0.5 mg / kg, and blood was collected 6 hours later to separate serum. The IL-6 concentration in the serum was detected using a mouse IL-6 ELISA kit. The IL-6 concentration in the serum was biologically repeatedly detected for at least 3 sets of lipid nanoparticles corresponding to each compound, and the average value was calculated.

[0174] Conclusion: As shown in Table 6 and Figure 6, it can be seen that the IL-6 concentrations in the mouse sera injected with lipid nanoparticles corresponding to Compound 1 and Compound 4 are significantly lower than that of Comparative Compound 1. It is suggested that the lipid nanoparticles corresponding to Compound 1 and 4 have lower immunogenicity and better biological safety in the body compared to the lipid nanoparticles corresponding to Comparative Compound 1.

[0175] In Fig. 6, that is, statistically 0.01 < P < 0.05 indicates that there is a statistically significant difference between groups, and that is, statistically P < 0.01, and that is, statistically P < 0.001 indicates that there is an extremely significant difference.

[0176]

Table 6

Claims

1. A compound of formula I or a salt thereof, 【Chemistry 1】 Here, L １ and L ２ are each independently —C(O)O—, —OC(O)—, —C(O)—, —OC(O)O—, —O—, or —S(O) ｘ -, -S-S-, -C(O)S-, -SC(O)-, -NR ａ C(O)-, -C(O)NR ａ -, -NR ａ C(O)NR ａ -, -NR ａ C(O)O-, -OC(O)NR ａ - or a bond, R ａ is hydrogen or C １－６ Alkyl group or C ２－６ alkenyl groups, H １ and H ２ are each independently C １－１２ Heteroalkylene group, C １－１２ Alkylene group, C ２－１２ alkenylene groups, and H １ and H ２ At least one of １－１２ is a heteroalkylene group, H ３ is C １－２４ Alkylene group, C ２－２４ Alkenylene group, C ３－８ Cycloalkylene group or C ３－８ cycloalkenylene groups, R １ and R ２ are each independently C １－２４ Alkyl group or C ２－２４ alkenyl groups, R ３ represents hydrogen, —CN, —C(O)OR ４ , -OC(O)R ４ , -OR ５ or -NR ５ C(O)R ４ Selected from R ４ is C １－６ Alkyl group or C ２－６ alkenyl groups, R ５ is hydrogen, C １－６ Alkyl group or C ２－６ alkenyl groups, x is selected from 0, 1 or 2; A compound or a salt thereof.

2. H １ But C １－１２ heteroalkylene groups, The compound or salt thereof according to claim 1.

3. L １ and L ２ are each independently selected from —C(O)O—, —OC(O)—, or a bond; The compound or salt thereof according to claim 1 or 2.

4. R １ and R ２ However, each independently C ２－２４ alkyl groups or R １ and R ２ However, each independently C ２－２４ selected from alkenyl groups, The compound or salt thereof according to claim 1.

5. The compound of formula I is 【Chemistry 2】 where H １ , H ２ , H ３ , R １ , R ２ and R ３ is as defined in claim 1 The compound or salt thereof according to claim 1.

6. H ３ But C ２－２４ Alkenylene group, C ３－８ Cycloalkylene group or C ３－８ cycloalkenylene groups, or H ３ But C ２－２４ selected from alkylene groups, The compound or salt thereof according to claim 1.

7. The compound of formula I is 【Transformation 3】 where R ６ are each independently hydrogen, a hydroxy group, or C １－６ Alkyl group or C ２－６ alkenyl groups, n is an integer between 1 and 12, for example, 2, 3, 4, 5, or 6; １ , H ２ , R １ , R ２ and R ３ is as defined in claim 1 The compound or salt thereof according to claim 1.

8. R ３ is selected from —CN or a hydroxy group, or R ３ is -C(O)OR ４ , -OC(O)R ４ or -NHC(O)R ４ Selected from R ４ is as defined in claim 1 The compound or salt thereof according to claim 1.

9. The compound of formula I is 【Chemistry 4】 where X is —N(R ７ )- or -O-, o is selected from an integer between 1 and 11, p is selected from an integer between 1 and 5, and o+p is 12 or less; R ７ is hydrogen, C １－６ Alkyl group or C ２－６ alkenyl groups, R ８ , R ９ , R １０ , R １１ are each independently hydrogen, a hydroxy group, or C １－６ Alkyl group or C ２－６ alkenyl groups, and L １ , L ２ , H ２ , H ３ , R １ , R ２ and R ３ is as defined in claim 1 The compound or salt thereof according to claim 1 or 2.

10. p is selected from 1 or 2; The compound or salt thereof according to claim 9.

11. H ２ But C ３－７ Alkylene group or C ３－７ selected from alkenylene groups, The compound or salt thereof according to claim 1.

12. The compound of formula I is 【Transformation 5】 where H ２ , R １ , R ２ and R ３ is as defined in claim 1, and R ６ and n is as defined in claim 7, R ８ , R ９ , R １０ , R １１ , X, o and p are as defined in claim 9; The compound or salt thereof according to claim 1.

13. R １ teeth, 【Transformation 6】 Selected from The compound or salt thereof according to claim 1.

14. The compound of formula I is 【Transformation 7】 【Transformation 8】 Selected from The compound or salt thereof according to claim 1.

15. An isotopic substitution of the compound of claim 1, wherein the isotopic substitution is a deuterium atom substitution. Isotopic substitution.

16. A lipid particle comprising the compound of claim 1 or a salt thereof. lipid particles.

17. The lipid particle of claim 16, further comprising an active agent, wherein the active agent is a polynucleotide or a nucleic acid.

18. The lipid particle described in claim 17, wherein the active agent is mRNA.

19. 17. A lipid particle comprising the lipid particle of claim 16 and a pharmaceutically acceptable excipient. Pharmaceutical compositions.

20. The pharmaceutical composition of claim 19 for preventing and / or treating cancer, infectious diseases, autoimmune diseases, neurodegenerative diseases and inflammation.

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