Lipid compounds, compositions containing same and applications

Abstract

The present invention relates to lipid compounds, compositions containing the same, and uses thereof. The present invention provides compounds of formula (I) or pharmaceutically acceptable forms thereof. The compounds of the present invention can be used to deliver nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides, or protein drugs. #imgabs0#

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and gene therapy, and specifically relates to a class of lipid compounds and lipid carriers, nucleic acid lipid nanoparticle compositions and pharmaceutical preparations containing the same. Background Art

[0002] Gene therapy is a hot topic in biomedical research. Nucleic acid drugs can prevent cancer, infectious diseases, and treat genetic disorders. However, nucleic acid drugs are easily degraded and difficult to enter cells, requiring a delivery vehicle. Developing safe and efficient delivery vehicles is crucial.

[0003] Lipid nanoparticles (LNPs) are commonly used gene delivery vehicles, protecting nucleic acids from degradation, extending circulation time, and improving targeting. LNPs are composed of cationic lipids, structural lipids, phospholipids, and PEG lipids, with the cationic lipids being central to encapsulation and delivery efficiency.

[0004] In order to meet the treatment needs of different diseases, it is necessary to design diverse cationic lipid structures and combinations to achieve precise delivery. Summary of the Invention

[0005] The cationic lipid compound designed and synthesized in the present invention has lipid nanoparticles with advantages in terms of encapsulation efficiency, biosafety, transfection efficiency and protein expression, and is of great significance to the development of gene therapy products.

[0006] In one aspect, the present invention provides a compound of formula (I) or a pharmaceutically acceptable form thereof, wherein:

[0007]

[0008] G1, G2 and G3 are each independently C3-C 12 Alkylene, the C3-C 12 The alkylene group is optionally substituted with one or more of the following substituents: halogen, oxo, -OH or C 1-6 alkyl;

[0009] L1 and L2 are each independently selected from a bond, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -C(=S)O-, -OC(=S)-, -OC(=S)O-, -C(=O)S-, -SC(=O)-, -OC(=O)S-, -C(=S)S-, -SC(=S)-, -SC(=S)O-, -O- or -S-;

[0010] R1 and R2 are each independently selected from H, C 1-6 Alkoxy, C 1-30 Alkyl, C2-30 Alkenyl, C 2-30 Alkynyl, C 3-30 Cycloalkyl or C 3-30 Cycloalkenyl, the C 1-6 Alkoxy, C 1-30 Alkyl, C 2-30 Alkenyl, C 3-30 Cycloalkyl or C 3-30 Cycloalkenyl is optionally substituted with one or more of the following substituents: halogen, cyano, oxo, -OH or C 1-6 alkyl;

[0011] X1, X2, Y1 and Y2 are each independently a bond, -(CH2) 1-6 C(=O)-、C 1-6 Alkylene, the C 1-6 Alkylene is optionally substituted with one or more of the following substituents: halogen, oxo, -OH, C 1-6 Alkyl, C 1-6 Hydroxyalkyl, -(CH2) 1-6 OH or C 1-6 alkoxy;

[0012] Z is selected from a bond, O, -C(=O)O-, and S.

[0013] In certain embodiments, G1, G2, and G3 are each independently -(CH2)3-, -(CH2)4-, -(CH2)5-, -(CH2)6-, -(CH2)7-, -(CH2)8-, -(CH2)9-, or -(CH2) 10 -.

[0014] In certain embodiments, L1 and L2 are each independently selected from a bond, -OC(=O)-, -OC(=O)O-, -OC(=S)-, -OC(=S)O-, -SC(=O)-, -OC(=O)S-, -SC(=S)-, or -SC(=S)O-.

[0015] In certain embodiments, R1 and R2 are each independently selected from H, C 1-20 Alkyl, C 2-20 Alkenyl, C 3-20 Cycloalkyl or C 3-20 Cycloalkenyl, the C 1-20 Alkyl, C 2-20 Alkenyl, C 3-20 Cycloalkyl or C 3-20 Cycloalkenyl is optionally substituted with one or more of the following substituents: halogen, cyano, oxo, -OH or C 1-6 alkyl;

[0016] Optionally, R1 and R2 are each independently selected from

[0017] or .

[0018] In certain embodiments, X1, X2, Y1 and Y2 are each independently a bond, -CH2-, -(CH2)2-, -(CH2)3-, -(CH2)4-, -(CH2)2(C=O)-, -(CH2)CH(CH2OH)CH2- or -(CH2)CH(OH)CH2-.

[0019] In certain embodiments, said Z is selected from a bond, O, or -C(=O)O-.

[0020] In certain embodiments, the compound is selected from the following compounds:

[0021] (1);

[0022] (2);

[0023] (3);

[0024] (4);

[0025] (5);

[0026] (6);

[0027] (7);

[0028] (8); or

[0029] (9);

[0030] The pharmaceutically acceptable form is selected from pharmaceutically acceptable salts or stereoisomers.

[0031] In another aspect, the present invention provides a lipid carrier comprising the above-mentioned compound or a pharmaceutically acceptable form thereof.

[0032] In certain embodiments, the lipid carrier further comprises one or more excipients selected from the group consisting of neutral lipids, steroids, and polymer-conjugated lipids.

[0033] In another aspect, the present invention provides a nucleic acid lipid nanoparticle composition comprising the aforementioned compound or a pharmaceutically acceptable form thereof or the aforementioned lipid carrier, and a nucleic acid drug.

[0034] In certain embodiments, the nucleic acid drug is selected from RNA, DNA, antisense nucleic acid, aptamer, nucleic acid, immunostimulatory nucleic acid or peptide nucleic acid (PNA).

[0035] In another aspect, the present invention provides a pharmaceutical preparation comprising the aforementioned compound or a pharmaceutically acceptable form thereof, the aforementioned lipid carrier, or the aforementioned nucleic acid lipid nanoparticle composition, and a pharmaceutically acceptable excipient, carrier, or diluent.

[0036] In another aspect, the present invention provides the use of the above-mentioned compound or its pharmaceutically acceptable form or the above-mentioned lipid carrier or the above-mentioned nucleic acid lipid nanoparticle composition or the above-mentioned pharmaceutical preparation in the preparation of nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0038] Figure 1 The graph shows the percentage of TTR gene editing observed in mouse liver after administration of different nanolipid particles (comprising cationic lipid compounds 1-9 of the present invention, ACL-0315 as a positive control, and PBS as a negative control). DETAILED DESCRIPTION

[0039] For easier understanding of the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise explicitly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs.

[0040] In this specification, the use of “substantially” or “essentially” means that the standard deviation from a theoretical model or theoretical data is within a range of 5%, preferably 3%, and more preferably 1%.

[0041] In this specification, the use of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0042] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event occurs and instances where it does not.

[0043] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "embodiments," etc., mean that the specific elements (e.g., features, structures, properties, and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0044] Before the present invention is further described, it is to be understood that the present invention is not limited to the particular embodiments described herein; it is to be understood that the terminology used herein is for the purpose of describing only and is not intended to be limiting of the particular embodiments.

[0045] Definition of terms

[0046] Unless otherwise stated, the following terms have the following meanings:

[0047] The term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention that is substantially non-toxic to living organisms. Pharmaceutically acceptable salts generally include (but are not limited to) salts formed by reacting a compound of the present invention with a pharmaceutically acceptable inorganic / organic acid or inorganic / organic base. Such salts are also referred to as acid addition salts or base addition salts. Common inorganic acids include (but are not limited to) hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, etc.; common organic acids include (but are not limited to) trifluoroacetic acid, citric acid, maleic acid, fumaric acid, succinic acid, tartaric acid, lactic acid, pyruvic acid, oxalic acid, formic acid, acetic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc.; common inorganic bases include (but are not limited to) sodium hydroxide, potassium hydroxide, calcium hydroxide, barium hydroxide, etc.; common organic bases include (but are not limited to) diethylamine, triethylamine, ethambutol, etc.

[0048] The term "stereoisomer" (or "optical isomer") refers to a stable isomer that possesses at least one chiral element (including a chiral center, chiral axis, chiral plane, etc.) resulting in a perpendicular asymmetric plane, thereby rotating plane-polarized light. Because the compounds of the present invention contain asymmetric centers and other chemical structures that may lead to stereoisomerism, the present invention also encompasses these stereoisomers and mixtures thereof. Because the compounds of the present invention and their salts contain asymmetric carbon atoms, they can exist as single stereoisomers, racemates, enantiomers, and mixtures of diastereomers. Typically, these compounds can be prepared as racemic mixtures. However, if desired, such compounds can be prepared or isolated to obtain pure stereoisomers, i.e., single enantiomers or diastereomers, or mixtures enriched in a single stereoisomer (purity ≥98%, ≥95%, ≥93%, ≥90%, ≥88%, ≥85%, or ≥80%). Individual stereoisomers of a compound are synthesized from optically active starting materials containing the desired chiral center, or by preparing a mixture of enantiomeric products followed by separation or resolution, for example, by conversion to a mixture of diastereomers followed by separation or recrystallization, chromatography, use of a chiral resolving agent, or direct separation of the enantiomers on a chiral chromatographic column. Starting compounds of specific stereochemistry are either commercially available or prepared as described below and resolved by methods well known in the art.

[0049] The term "non-covalent complex" is formed by the interaction of a compound with another molecule, wherein no covalent bond is formed between the compound and the molecule. For example, complexation can occur through van der Waals interactions, hydrogen bonding, and electrostatic interactions (also known as ionic bonding).

[0050] The term "prodrug" refers to a derivative compound that, upon application to a patient, is capable of providing, directly or indirectly, a compound of the invention. Particularly preferred derivative compounds or prodrugs are compounds that, when administered to a patient, can increase the bioavailability of the compound of the invention (e.g., more readily absorbed into the bloodstream) or compounds that facilitate delivery of the parent compound to the site of action (e.g., the lymphatic system). Unless otherwise indicated, all prodrug forms of the compounds of the invention are within the scope of the invention, and various prodrug forms are well known in the art.

[0051] The term "each independently" means that at least two groups (or ring systems) present in a structure with the same or similar value ranges may have the same or different meanings in specific circumstances. For example, if substituent X and substituent Y are each independently hydrogen, halogen, hydroxyl, cyano, alkyl, or aryl, then when substituent X is hydrogen, substituent Y may be either hydrogen, or halogen, hydroxyl, cyano, alkyl, or aryl. Similarly, when substituent Y is hydrogen, substituent X may be either hydrogen, or halogen, hydroxyl, cyano, alkyl, or aryl.

[0052] The terms "including" and "comprising" are used in their open, non-limiting sense.

[0053] The term "alkyl" refers to a monovalent straight or branched alkane group consisting only of carbon and hydrogen atoms, containing no unsaturation, and connected to other moieties by a single bond, including (but not limited to) methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl and tert-butyl. For example, "C 1-30 "Alkyl" refers to a saturated monovalent straight or branched chain hydrocarbon group containing 1 to 30 carbon atoms.

[0054] The term "alkylene" refers to a divalent straight or branched alkane group consisting only of carbon atoms and hydrogen atoms, containing no saturation, and connected to other fragments by two single bonds, including (but not limited to) methylene, 1,1-ethylene and 1,2-ethylene. For example, "C 1-30 "Alkylene" refers to a saturated divalent straight or branched chain alkyl group containing from 1 to 30 carbon atoms.

[0055] The term "cycloalkyl" refers to a saturated, monocyclic or polycyclic (e.g., bicyclic, tricyclic or tetracyclic) non-aromatic hydrocarbon group consisting only of carbon atoms and hydrogen atoms. Cycloalkyl groups may include fused, bridged or spirocyclic ring systems. For example, the term "C 3-6 The term "cycloalkyl" refers to a cyclic alkyl group having 3 to 6 carbon atoms. For example, the cycloalkyl group may be cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or bicyclo[2.2.1]heptyl, etc.

[0056] The term "cycloalkylene" refers to a divalent group obtained by removing a hydrogen atom from a cycloalkyl group as defined above, including but not limited to cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, and cycloheptylene. For example, "C 3-30 The "cycloalkylene group" refers to a divalent group obtained by removing a hydrogen atom from a cycloalkyl group containing 3 to 30 carbon atoms.

[0057] The term "branched alkyl" refers to an alkane radical attached to a parent molecule and forming at least two branches.

[0058] The term "alkenyl" refers to a monovalent straight or branched alkane group consisting only of carbon atoms and hydrogen atoms, containing at least one double bond, and connected to other fragments by a single bond, including (but not limited to) vinyl, propenyl, allyl, isopropenyl, butenyl and isobutenyl groups. For example, "C 2-30 "Alkenyl" refers to a monovalent straight or branched chain hydrocarbon group containing 2 to 30 carbon atoms and having at least one carbon-carbon double bond (>C=C<).

[0059] The term "alkenylene" refers to a divalent straight or branched alkane group consisting only of carbon atoms and hydrogen atoms, containing at least one double bond, and connected to other fragments by two single bonds, including (but not limited to) vinylene, etc. For example, "C 2-30 "Alkenylene" refers to a divalent straight or branched chain hydrocarbon group containing 2 to 30 carbon atoms and having at least one carbon-carbon double bond (>C=C<).

[0060] The term "alkynyl" refers to a monovalent straight or branched alkane group consisting only of carbon atoms and hydrogen atoms, containing at least one carbon-carbon triple bond, and connected to other fragments by a single bond, including (but not limited to) ethynyl, propynyl, butynyl and pentynyl groups. For example, "C 2-30 "Alkynyl" refers to a monovalent straight or branched chain hydrocarbon radical containing from 2 to 30 carbon atoms and having at least one carbon-carbon triple bond.

[0061] The term "cycloalkenyl" refers to an unsaturated, monocyclic or polycyclic (e.g., bicyclic, tricyclic, or tetracyclic) non-aromatic hydrocarbon group consisting solely of carbon and hydrogen atoms. Cycloalkenyl groups may include cyclic, bridged, or spirocyclic ring systems. Examples include cyclopropenyl and cyclobutenyl.

[0062] The term "cycloalkenylene" refers to a divalent group obtained by removing a hydrogen atom from a cycloalkenyl group as defined above, including but not limited to cyclopropenylene and cyclobutenylene. For example, "C 3-30 The "cycloalkenylene group" refers to a divalent group obtained by removing a hydrogen atom from a cycloalkenyl group containing 3 to 30 carbon atoms.

[0063] The term "branched alkenyl" refers to an alkene radical attached to a parent molecule and forming at least two branches. For example

[0064] The term "heterocyclyl" refers to a saturated or partially saturated, monocyclic or polycyclic (such as a bicyclic, for example, fused, bridged or spirocyclic) non-aromatic group, the ring atoms of which are composed of carbon atoms and at least one heteroatom selected from N, O and S, wherein the S atom is optionally substituted to form S(=O), S(=O)2 or S(=O)(=NRx), and Rx is independently selected from H or C1-4 alkyl. If the valence requirements are met, the heterocyclyl can be connected to the rest of the molecule through any one of the ring atoms. For example, the term "3-8 membered heterocyclyl" as used in the present invention refers to a heterocyclyl having 3 to 8 ring atoms. For example, the heterocyclyl can be an oxirane, an aziridine, an azetidinyl, an oxetanyl, a tetrahydrofuranyl, a dioxolyl, a pyrrolidinyl, a pyrrolidonyl, an imidazolidinyl, a pyrazolidinyl, a tetrahydropyranyl, a piperidinyl, a piperazinyl, a morpholinyl, a thiomorpholinyl, a dithianyl or a trithianyl.

[0065] The term "aryl" refers to a monocyclic or fused polycyclic aromatic hydrocarbon group having a conjugated π electron system. For example, the term "C 6-10 The term "aryl" refers to an aromatic group having 6 to 10 carbon atoms. For example, the aromatic group may be phenyl, naphthyl, anthracenyl, phenanthrenyl, acenaphthenyl, azulenyl, fluorenyl, indenyl, pyrenyl, and the like.

[0066] The term "heteroaryl" refers to a monocyclic or fused polycyclic aromatic group having a conjugated π electron system, the ring atoms of which are composed of carbon atoms and at least one heteroatom selected from N, O and S. If the valence bond requirements are met, the heteroaryl group can be connected to the rest of the molecule through any one of the ring atoms. For example, the term "5-10 membered heteroaryl" as used in the present invention refers to a heteroaryl group having 5 to 10 ring atoms. For example, the heteroaryl group can be thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl and benzo derivatives thereof, pyrrolopyridinyl, pyrrolopyrazinyl, pyrazolopyridinyl, imidazopyridinyl, pyrrolopyrimidinyl, pyrazolopyrimidinyl, purinyl, etc.

[0067] The term "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).

[0068] The term "hydroxy" refers to -OH.

[0069] The term "cyano" refers to -CN.

[0070] The term "amino" refers to -NH2.

[0071] The term "nitro" refers to -NO2.

[0072] The term "oxo" refers to (=O).

[0073] "Steroids" are compounds containing the following carbon skeleton:

[0074] .

[0075] Non-limiting examples of steroids include one or more of cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, coprosterol, and corticosteroids.

[0076] The term "neutral lipid" refers to any of the many lipid substances present in the form of no charge or neutral zwitterions at a selected pH. At physiological pH, such lipids include, but are not limited to, phosphatidylcholine, such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), phosphatidylethanolamine such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelin (SM), ceramides, steroids such as sterols and derivatives thereof. Neutral lipids can be synthetic or naturally derived.

[0077] The term "polymer-conjugated lipid" refers to a molecule comprising a lipid portion and a polymer portion. An example of a polymer-conjugated lipid is a PEGylated lipid. The term "PEGylated lipid" refers to a molecule comprising a lipid portion and a polyethylene glycol portion. PEGylated lipids are known in the art and include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG) and the like.

[0078] Modification types are categorized as follows:

[0079] A. 2'-O-methyl modification

[0080] Modified sugars are thought to control the wrinkling of the nucleotide sugar ring, which affects the physical properties of the oligonucleotide's binding affinity for the complementary chain, duplex formation, and interaction with nucleases. Substitutions on the sugar ring can thus alter the alignment and wrinkling of these sugars. For example, 2'-O-methyl (2'-O-Me) modifications can increase the binding affinity and nuclease stability of oligonucleotides, although as shown in the examples, the effect of any modification at a given position in an oligonucleotide needs to be determined empirically.

[0081] The terms "mA," "mC," "mU," or "mG" are used to indicate a nucleotide that has been 2'-O-Me modified.

[0082] Modifications of ribonucleotides in the form of 2'-O-methyl ribonucleotides can be depicted as follows:

[0083]

[0084] B. Phosphorothioate Modification

[0085] A phosphorothioate (PS) linkage or bond refers to a sulfur-substituted phosphodiester linkage, such as a bond that replaces one of the non-bridging phosphate oxygens in the bond between nucleotide bases. When phosphorothioate is used to produce an oligonucleotide, the modified oligonucleotide may also be referred to as an S-oligonucleotide.

[0086] "*" is used to depict PS modification. In the present invention, the term A*, C*, U* or G* is used to indicate the nucleotide that is linked to the next (eg, 3') nucleotide with a PS bond.

[0087] In the present invention, the term "mA*", "mC*", "mU*" or "mG*" is used to indicate a nucleotide that has been substituted with 2'-O-Me and is linked to the next (e.g., 3') nucleotide having a PS bond. Similarly, the term "fA*", "fC*", "fU*" or "fG*" is used to indicate a nucleotide that has been substituted with 2'-F and is linked to the next (e.g., 3') nucleotide having a PS bond. The embodiments described herein encompass equivalents of PS linkages or bonds.

[0088] The following shows the substitution of an S- to a non-bridging phosphate oxygen, resulting in a PS bond in place of the phosphodiester bond:

[0089]

[0090] InDel (insertion / deletion): refers to an insertion / deletion mutation consisting of multiple nucleotides inserted or deleted at the double-strand break (DSB) site in the target nucleic acid.

[0091] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0092] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0093] Example 1: Synthesis of Compound 1

[0094] The synthetic route is as follows:

[0095]

[0096] 2-Hexyldecanoic acid (20 g, 78.1 mmol), DCC (24.1 g, 117.2 mmol), and DMAP (952 mg, 7.81 mmol) were added to a solution of 1,6-hexanediol (16 g, 87.75 mmol) in dichloromethane (500 mL). The solution was stirred at room temperature overnight and extracted with dichloromethane and water. The organic phase was dried over anhydrous sodium sulfate and purified by silica gel column chromatography to obtain intermediate H1-1 (16.5 g). MS m / z: [M+H] = 357.28

[0097] Intermediate H-1 (16.0 g, 44.9 mmol) and N,N-dimethylethylenediamine (11.6 g, 89.9 mmol) were dissolved in dichloromethane (200 mL). Methanesulfonic anhydride (7.8 g, 44.9 mmol) was added under ice-cooling and stirred for 16 hours. The mixture was extracted with dichloromethane (500 mL) and saturated NaHCO₃. The organic phase was dried over anhydrous sodium sulfate, the solvent was removed by vacuum drying, and finally purified by silica gel column chromatography to obtain intermediate H1-2 (13.6 g). MS m / z: [M+H]=435.3

[0098] Intermediate H-2 (13 g, 30 mmol), K2CO3 (8.2 g, 60 mmol), and KI (4.98 g, 30 mmol) were added to a solution of 4-amino-1-butanol (1.33 g, 15 mmol) in acetonitrile (200 mL), and the solution was stirred at 85°C overnight. The reaction mixture was filtered, the solvent was removed by vacuum drying, and the product was purified by silica gel chromatography to obtain intermediate H1-3 (9.2 g). MS m / z: [M+H]=766.7

[0099] Intermediate H-3 (9 g, 11.8 mmol) and N,N-dimethylethylenediamine (4.57 g, 35.4 mmol) were dissolved in dichloromethane (200 mL). Methanesulfonic anhydride (2.05 g, 11.8 mmol) was added under ice-cooling and stirred for 16 hours. The mixture was extracted with dichloromethane (500 mL) and saturated NaHCO₃. The organic phase was dried over anhydrous sodium sulfate, the solvent was removed by vacuum drying, and finally purified by silica gel column chromatography to obtain intermediate H1-4 (6.2 g). MS m / z: [M+H]: 844.7

[0100] Intermediate H1-4 (300 mg, 0.36 mmol), K2CO3 (120 mg, 0.72 mmol), KI (60 mg, 0.36 mmol), and starting material SM1 (49 mg, 0.36 mmol) were dissolved in acetonitrile (20 mL) and stirred at 85°C overnight. The reaction mixture was filtered, the solvent was removed by vacuum drying, and the product was purified by silica gel chromatography to afford compound 1 (120 mg).

[0101] MS m / z : [M+H]: 919.8

[0102] 1H NMR (400 MHz, Chloroform-d) δ 4.52 (dtd, J = 15.7, 12.1, 3.1 Hz,2H), 3.86 – 3.77 (m, 1H), 3.65 – 3.52 (m, 2H), 3.54 – 3.40 (m, 2H), 3.33(ddd, J = 12.5, 7.0, 5.5 Hz, 1H), 3.08 (td, J = 7.6, 4.2 Hz, 5H), 2.91 (dt, J= 12.5, 7.1 Hz, 1H), 2.74 (td, J = 12.3, 2.5 Hz, 1H), 2.63 (td, J = 12.4, 2.7Hz, 1H), 2.25 – 1.97 (m, 6H), 1.99 – 1.89 (m, 2H), 1.91 – 1.80 (m, 4H), 1.83 – 1.75 (m, 2H), 1.78 – 1.65 (m, 2H), 1.64 – 1.48 (m, 10H), 1.51 – 1.42 (m, 2H), 1.46 – 1.27 (m, 10H), 1.28 (s, 1H), 1.29 – 1.20 (m, 10H), 1.24 – 1.09(m, 10H), 0.94 – 0.85 (m, 12H).

[0103] Example 2: Synthesis of Compound 2

[0104] Intermediate H1-4 (300 mg, 0.36 mmol), K2CO3 (120 mg, 0.72 mmol), KI (60 mg, 0.36 mmol), and 2-oxazole-7-azaspiro[3.5]nonane (46 mg, 0.36 mmol) were dissolved in acetonitrile (20 mL) and stirred at 85°C overnight. The reaction mixture was filtered, the solvent was removed by vacuum drying, and the product was purified by silica gel chromatography to afford compound 2 (145 mg).

[0105] MS m / z : [M+H]: 875.8

[0106] 1H NMR (400 MHz, Chloroform-d) δ 4.66 – 4.53 (m, 6H), 3.44 (tt, J =12.1, 2.4 Hz, 2H), 3.08 (t, 2H), 2.96 (dtd, J = 29.9, 12.4, 2.7 Hz, 2H), 2.45(t, J = 5.6 Hz, 4H), 2.08 (p, J = 7.0 Hz, 2H), 1.92 – 1.74 (m, 7H), 1.60 –1.47 (m, 11H), 1.46 – 1.32 (m, 8H), 1.33 – 1.24 (m, 4H), 1.27 – 1.14 (m,46H), 1.01 (dtt, J = 13.0, 8.0, 2.6 Hz, 2H), 0.94 – 0.85 (m, 12H).

[0107] Example 3: Synthesis of Compound 3

[0108] Intermediate H1-4 (330 mg, 0.396 mmol), K2CO3 (132 mg, 0.79 mmol), KI (66 mg, 0.40 mmol), and 2-oxa-6-aza-spiro[3,3]heptane (40 mg, 0.396 mmol) were dissolved in acetonitrile (20 mL) and stirred at 85°C overnight. The reaction mixture was filtered, the solvent was removed by vacuum drying, and the product was purified by silica gel chromatography to afford compound 3 (109 mg).

[0109] MS m / z : [M+H]: 847.8

[0110] 1H NMR (400 MHz, Chloroform-d) δ 4.66 – 4.53 (m, 6H), 3.43 (tt, J =12.2, 1.9 Hz, 2H), 3.22 (s, 3H), 3.12 – 3.05 (m, 2H), 2.68 (td, J = 12.2, 2.8Hz, 2H), 2.45 – 2.38 (m, 2H), 2.13 – 1.97 (m, 4H), 1.92 – 1.73 (m, 6H), 1.59 – 1.48 (m, 8H), 1.45 – 1.12 (m, 49H), 1.07 – 0.96 (m, 2H), 0.94 – 0.85 (m,12H).

[0111] Example 4: Synthesis of Compound 4

[0112] Intermediate H1-4 (270 mg, 0.32 mmol), K2CO3 (120 mg, 0.72 mmol), KI (53 mg, 0.32 mmol), and 1-oxa-8-azaspiro[4.5]decan-3-ol (57 mg, 0.36 mmol) were dissolved in acetonitrile (20 mL) and stirred at 85°C overnight. The reaction mixture was filtered, the solvent was removed by vacuum drying, and the product was purified by silica gel chromatography to afford compound 4 (97 mg).

[0113] MS m / z : [M+H]: 905.8

[0114] 1H NMR (400 MHz, Chloroform-d) δ 4.61 (dtd, J = 15.6, 12.2, 3.2 Hz, 2H), 4.07 – 3.92 (m, 2H), 3.77 (t, J = 7.1 Hz, 1H), 3.43 (tt, J = 12.1, 3.6Hz, 2H), 3.12 – 2.99 (m, 5H), 2.91 (dt, J = 12.5, 7.1 Hz, 1H), 2.77 (td, J =12.3, 2.7 Hz, 1H), 2.67 (td, J = 12.3, 2.7 Hz, 1H), 2.57 (d, J = 4.9 Hz, 1H), 2.27 – 2.15 (m, 2H), 2.15 – 1.95 (m, 5H), 1.93 – 1.66 (m, 8H), 1.55 (ddt, J =13.5, 11.1, 6.6 Hz, 9H), 1.47 – 1.07 (m, 50H), 1.01 (dddd, J = 19.9, 14.0,7.2, 3.6 Hz, 2H), 0.93 – 0.85 (m, 12H).

[0115] Example 5: Synthesis of Compound 5

[0116] Intermediate H1-4 (243 mg, 0.29 mmol), K2CO3 (120 mg, 0.72 mmol), KI (48 mg, 0.29 mmol), and 8-azaspiro[4.5]decane (43 mg, 0.31 mmol) were dissolved in acetonitrile (10 mL) and stirred at 85°C overnight. The reaction mixture was filtered, the solvent was removed by vacuum drying, and the product was purified by silica gel chromatography to afford compound 5 (88 mg).

[0117] MS m / z : [M+H]:887.8

[0118] 1H NMR (400 MHz, Chloroform-d) δ 4.57 (dtd, J = 15.4, 12.2, 3.3 Hz, 2H), 3.41 (dtd, J = 20.6, 12.1, 2.8 Hz, 2H), 3.08 (td, J = 5.0, 2.4 Hz, 4H),2.80 – 2.68 (m, 1H), 2.62 (td, J = 12.4, 2.8 Hz, 1H), 2.45 (t, J = 5.6 Hz,4H), 2.13 – 1.97 (m, 5H), 1.91 – 1.74 (m, 4H), 1.67 (dddd, J = 15.1, 12.4,8.9, 2.9 Hz, 1H), 1.54 (dtd, J = 21.7, 6.8, 3.4 Hz, 12H), 1.45 – 1.10 (m,58H), 1.06 – 0.94 (m, 2H), 0.93 – 0.85 (m, 12H).

[0119] Example 6: Synthesis of Compound 6

[0120] Intermediate H1-4 (301 mg, 0.36 mmol), K2CO3 (120 mg, 0.72 mmol), KI (58 mg, 0.72 mmol), and 1-oxa-8-azaspiro[4.5]decan-2-one (65 mg, 0.42 mmol) were dissolved in acetonitrile (10 mL) and stirred at 85°C overnight. The reaction mixture was filtered, the solvent was removed by vacuum drying, and the product was purified by silica gel chromatography to afford compound 6 (110 mg).

[0121] MS m / z : [M+H]:903.8

[0122] 1H NMR (400 MHz, Chloroform-d) δ 4.64 – 4.54 (m, 2H), 3.44 (td, J =12.0, 2.9 Hz, 2H), 3.07 (dt, J = 11.9, 7.2 Hz, 3H), 2.95 (dt, J = 12.5, 7.0Hz, 1H), 2.77 – 2.62 (m, 4H), 2.30 (t, J = 7.2 Hz, 2H), 2.21 – 1.95 (m, 8H), 1.93 – 1.72 (m, 8H), 1.61 – 1.42 (m, 10H), 1.42 – 1.12 (m, 51H), 1.00 (dddd,J = 12.7, 10.6, 7.9, 3.9 Hz, 2H), 0.89 (t, J = 5.5 Hz, 12H).

[0123] Example 7: Synthesis of Compound 7

[0124] Intermediate H1-4 (299 mg, 0.35 mmol), K2CO3 (120 mg, 0.72 mmol), KI (58 mg, 0.35 mmol), and 1-oxa-8-azaspiro[4.5]decane (62 mg, 0.44 mmol) were dissolved in acetonitrile (10 mL) and stirred at 85°C overnight. The reaction mixture was filtered, the solvent was removed by vacuum drying, and the product was purified by silica gel chromatography to afford compound 7 (78 mg).

[0125] MS m / z : [M+H]:889.8

[0126] 1H NMR (400 MHz, Chloroform-d) δ 4.53 (dtd, J = 27.4, 12.2, 3.1 Hz, 2H), 3.75 (t, J = 6.1 Hz, 2H), 3.57 (td, J = 12.1, 2.8 Hz, 1H), 3.44 (td, J =12.1, 3.0 Hz, 1H), 3.12 – 3.05 (m, 4H), 2.98 (ddt, J = 16.3, 12.5, 7.0 Hz, 2H), 2.70 (dtd, J = 29.8, 12.4, 2.6 Hz, 2H), 2.21 – 2.09 (m, 3H), 2.12 – 2.03(m, 3H), 2.00 – 1.65 (m, 12H), 1.63 – 1.48 (m, 9H), 1.48 – 1.02 (m, 35H), 0.96 – 0.81 (m, 12H).

[0127] Example 8: Synthesis of Compound 8

[0128]

[0129] Intermediate H8-1 (250 mg, 0.31 mmol, synthesized using the same procedure as in Example 1), 1-oxa-8-azaspiro[4.5]decan-3-ol (57 mg, 0.36 mmol), KCO (120 mg, 0.72 mmol), and KI (51 mg, 0.31 mmol) were dissolved in acetonitrile (10 mL) and stirred at 85°C overnight. The reaction mixture was filtered, the solvent removed by vacuum drying, and purified by silica gel chromatography to afford compound 8 (78 mg).

[0130] MS m / z : [M+H]:889.8

[0131] 1H NMR (500 MHz, Chloroform-d) δ 4.61 (td, J = 12.0, 2.8 Hz, 1H), 4.12 – 4.02 (m, 2H), 3.75 (t, J = 6.1 Hz, 2H), 3.43 (td, J = 12.1, 3.1 Hz,1H), 3.12 – 3.00 (m, 5H), 2.93 (dt, J = 12.5, 7.1 Hz, 1H), 2.42 (t, J = 7.6Hz, 4H), 2.25 (p, J = 7.0 Hz, 2H), 2.09 (dtt, J = 12.6, 9.9, 6.3 Hz, 4H),1.96 (ttd, J = 12.4, 7.4, 2.3 Hz, 2H), 1.90 – 1.64 (m, 6H), 1.65 – 1.44 (m,14H), 1.48 – 1.33 (m, 3H), 1.37 – 1.14 (m, 34H), 1.10 (ttd, J = 11.9, 5.6,3.5 Hz, 4H), 0.89 (td, J = 7.8, 4.8 Hz, 12H).

[0132] Example 9: Synthesis of Compound 9

[0133] Intermediate H8-1 (250 mg, 0.31 mmol), K2CO3 (120 mg, 0.72 mmol), KI (58 mg, 0.35 mmol), and 1-oxa-8-azaspiro[4.5]decane (62 mg, 0.44 mmol) were dissolved in acetonitrile (10 mL) and stirred at 85°C overnight. The reaction mixture was filtered, the solvent was removed by vacuum drying, and the product was purified by silica gel chromatography to afford compound 9 (78 mg).

[0134] MS m / z : [M+H]:889.8

[0135] 1H NMR (400 MHz, Chloroform-d) δ 4.53 (dtd, J = 27.4, 12.2, 3.1 Hz, 2H), 3.75 (t, J = 6.1 Hz, 2H), 3.57 (td, J = 12.1, 2.8 Hz, 1H), 3.44 (td, J =12.1, 3.0 Hz, 1H), 3.12 – 3.05 (m, 4H), 2.98 (ddt, J = 16.3, 12.5, 7.0 Hz, 2H), 2.70 (dtd, J = 29.8, 12.4, 2.6 Hz, 2H), 2.21 – 2.09 (m, 3H), 2.12 – 2.03(m, 3H), 2.00 – 1.65 (m, 12H), 1.63 – 1.48 (m, 9H), 1.48 – 1.02 (m, 35H), 0.96 – 0.81 (m, 12H).

[0136] Example 10: Preparation of nucleic acid-lipid particles

[0137] (1) Preparation of Fluc mRNA-lipid particles

[0138] A cationic lipid or a cationic lipid of Compounds 1-9 of the present invention, cholesterol, DSPC, and PEG-DMG were dissolved in anhydrous ethanol at a molar ratio of 50 / 38.5 / 10 / 1.5, respectively, to prepare a mixed lipid ethanol solution. Compounds 1-9 of the present invention and ALC-0315 were dissolved in anhydrous ethanol with cholesterol, DSPC, and PEG-DMG at the aforementioned molar ratios, respectively, to prepare a mixed lipid ethanol solution.

[0139] Fluc-mRNA (Apexbio, Cat. No. R1005) was dissolved in 25 mM enzyme-free citrate buffer (pH 5) (mRNA concentration: 0.2 mg / mL) to prepare an mRNA citrate buffer. Using an Aitsen microfluidics device (Model: MPF-L2™), the mixed lipid ethanol solution and the mRNA citrate buffer were mixed at a flow rate of 1:3 (nitrogen-phosphorus ratio of cationic lipid to Fluc-mRNA: 6:1) at a total flow rate of 20 mL / min to prepare a mixed solution of the nanolipid particle composition. The mixed solution of the nanolipid particle composition was transferred to a 100 kDa dialysis bag and dialyzed overnight at 4°C against PBS (pH 7.4) with stirring. Finally, the lipid nanoparticles were filtered through a 0.22 μm sterile filter. The prepared nanolipid particle composition solution was stored at 4°C.

[0140] (2) Preparation of CRISPR Cas9 mRNA and sgRNA-lipid particles

[0141] A cationic lipid or a cationic lipid of Compounds 1-9 of the present invention, cholesterol, DSPC, and PEG-DMG were dissolved in anhydrous ethanol at a molar ratio of 50 / 38.5 / 10 / 1.5, respectively, to prepare a mixed lipid ethanol solution. Compounds 1-9 of the present invention and ALC-0315 were dissolved in anhydrous ethanol with cholesterol, DSPC, and PEG-DMG at the aforementioned molar ratios, respectively, to prepare a mixed lipid ethanol solution.

[0142] Cas9 mRNA and sgRNA (mRNA / sgRNA mass ratio of 1:1) were dissolved in 25 mM enzyme-free citrate buffer (mRNA concentration of 0.2 mg / mL) at pH 5 to prepare an mRNA and sgRNA citrate buffer. Using an Atson microfluidics device (model: MPF-L2™), the mixed lipid ethanol solution was mixed with the mRNA and sgRNA citrate buffer at a flow rate of 1:3 (nitrogen-to-phosphorus ratio of the cationic lipid compound to total RNA (including mRNA and sgRNA) of 6:1) at a total flow rate of 20 mL / min to prepare a mixed solution of the nanolipid particle composition. The mixed solution of the nanolipid particle composition was transferred to a 100 kDa dialysis bag and dialyzed against PBS (pH 7.4) at 4°C overnight with stirring. Finally, the lipid nanoparticles were filtered through a 0.22 μm sterile filter. The prepared nanolipid particle composition solution was stored at 4°C.

[0143] Example 11: Determination of encapsulation efficiency, particle size and polydispersity index (PDI) of nanolipid particles

[0144] The encapsulation efficiency of the lipid nanoparticle composition (prepared according to the preparation of Fluc mRNA-lipid particles in Example 10 (1)) was determined using the Quant-iT Ribogreen RNA Quantification Kit (Thermo Fisher Scientific, UK) according to the kit's instructions.

[0145] 10 μL of the nanolipid particle solution was diluted 100-fold with PBS and added to a microwell. The particle size and polydispersity index (PDI) were measured using a Malvern Zetasizer nano instrument (25°C). Each sample was measured three times, and the average of the three results was used. The results are shown in Table 1.

[0146] Table 1

[0147] Nanolipid particles Cationic lipids Particle size (nm) PDI Encapsulation efficiency LNP1 Compound 1 79.05 0.08 97.62% LNP2 Compound 2 78.20 0.02 98.93% LNP3 Compound 3 77.10 0.07 95.05% LNP4 Compound 4 79.30 0.10 95.73% LNP5 Compound 5 87.28 0.05 93.87% LNP6 Compound 6 86.22 0.04 94.57% LNP7 Compound 7 87.58 0.11 97.50% LNP8 Compound 8 68.57 0.08 95.51% LNP9 Compound 9 67.53 0.01 97.62% LNP10 ALC-0315 87.48 0.17 90.58%

[0148] As shown in Table 1, the nanolipid particles prepared using the cationic lipids of the present invention have a polydispersity index (PDI) of 0.01 to 0.15 and a particle size range of 65 to 90 nm. This indicates uniform size and excellent dispersion, making them suitable for mRNA delivery. Furthermore, the nanolipid particles exhibit a high encapsulation efficiency. Encapsulation efficiency, a key quality attribute of liposomes, reflects the degree of drug encapsulation. The high encapsulation efficiency of the nanolipid particles of the present invention facilitates efficient loading of active substances (such as siRNA and mRNA), thereby improving drug loading capacity.

[0149] Example 12: In vivo delivery experiment of mRNA-lipid nanoparticles

[0150] Female C57BL / 6 mice (purchased from Vital River) aged 6-8 weeks and weighing approximately 18-20 g were selected as experimental subjects.

[0151] According to the method of Example 10 (1) for preparing Fluc mRNA-lipid particles, nucleic acid lipid nanoparticles containing luciferase mRNA (Fluc mRNA) (Apexbio, Catalog No.: R1005) were prepared.

[0152] The resulting nucleic acid-lipid nanoparticles were administered via tail vein injection (at a dose of 0.2 mg / kg), and mice were sacrificed 4 hours after administration. The livers were collected in pre-weighed tubes, the weight was determined, and approximately 50 mg of the sample was cut. Luciferase expression in the mouse livers was measured (unit: ng / g (luciferase / mouse liver)). The expression results are shown in Table 2.

[0153] Table 2

[0154] Nanolipid particles Cationic lipids ng / g (luciferase / mouse liver) LNP1 Compound 1 20030±2300 LNP2 Compound 2 20177±2123 LNP3 Compound 3 21020±1979 LNP4 Compound 4 22003±2003 LNP5 Compound 5 22125±2391 LNP6 Compound 6 13530±1232 LNP7 Compound 7 20199±2358 LNP8 Compound 8 12921±989 LNP9 Compound 9 22012±1171 LNP10 ALC-0315 12897±1238

[0155] Example 13: Liposome stability experiment

[0156] After preparing the obtained lipid nanoparticles according to (1) Fluc mRNA-lipid particles in Example 10, liposomes LNP1, LNP4, LNP7 and LNP10 were stored at room temperature, -20 degrees Celsius, and -80 degrees Celsius for 14 days, and the stability was analyzed by particle size analysis, as shown in Table 3.

[0157] Table 3

[0158] lipid nanoparticles Cationic lipids Particle size (nm) PDI Encapsulation efficiency LNP1, room temperature Compound 1 79.25 0.08 92.62% LNP1, -20°C Compound 1 81.90 0.09 93.66% LNP1, -80°C Compound 1 82.55 0.08 92.21% LNP4, room temperature Compound 4 79.30 0.15 93.73% LNP4, -20°C Compound 4 81.30 0.10 94.63% LNP4, -80°C Compound 4 82.50 0.11 95.73% LNP10, room temperature ALC-0315 95.48 0.13 89.58% LNP10, -20°C ALC-0315 97.48 0.18 89.66% LNP10, -80°C ALC-0315 98.48 0.17 88.36%

[0159] Example 14: Liver-targeted mRNA and sgRNA delivery experiments

[0160] (1) Animal studies

[0161] Nanolipid particles containing compounds 1-9 of the present invention and nanolipid particles containing ACL-0315 (prepared according to the preparation of CRISPR Cas9 mRNA and sgRNA-lipid particles in Example 10 (2)) were injected into 6-8 week old female CD-1 mice weighing approximately 18-20 g (purchased from Weitong Lihua) at a dose of 0.15 mg / kg via the tail vein. In addition, PBS buffer was similarly injected into the tail vein of mice of the same age and gender as a negative control.

[0162] Table 4 Sequence Listing

[0163] SEQ ID NO: sequence Remark 1 AGGUCUAGAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGACCCCGGCGCCGCCACCAUGGACAAGAAGUACUCCAUCGGCCUGGACAUCGGCACCAACUCCGUGGGCUGGGCCGUGAUCACCGACGAGUACAAGGUGCCCUCCAAGAAGUUCAAGGUGCUGGGCAACACCGACCGGCACUCCAUCAAGAAGAACCUGAUCGGCGCCCUGCUGUUCGACUCCGGCGAGACCGCCGAGGCCACCCGGCUGAAGCGGACCGCCCGGCGGCGGUACACCCGGCGGAAGAACCGGAUCUGCUACCUGCAGGAGAUCUUCUCCAACGAGAUGGCCAAGGUGGACGACUCCUUCUUCCACCGGCUGGAGGAGUCCUUCCUGGUGGAGGAGGACAAGAAGCACGAGCGGCACCCCAUCUUCGGCAACAUCGUGGACGAGGUGGCCUACCACGAGAAGUACCCCACCAUCUACCACCUGCGGAAGAAGCUGGUGGACUCCACCGACAAGGCCGACCUGCGGCUGAUCUACCUGGCCCUGGCCCACAUGAUCAAGUUCCGGGGCCACUUCCUGAUCGAGGGCGACCUGAACCCCGACAACUCCGACGUGGACAAGCUGUUCAUCCAGCUGGUGCAGACCUACAACCAGCUGUUCGAGGAGAACCCCAUCAACGCCUCCGGCGUGGACGCCAAGGCCAUCCUGUCCGCCCGGCUGUCCAAGUCCCGGCGGCUGGAGAACCUGAUCGCCCAGCUGCCCGGCGAGAAGAAGAACGGCCUGUUCGGCAACCUGAUCGCCCUGUCCCUGGGCCUGACCCCCAACUUCAAGUCCAACUUCGACCUGGCCGAGGACGCCAAGCUGCAGCUGUCCAAGGACACCUACGACGACGACCUGGACAACCUGCUGGCCCAGAUCGGCGACCAGUACGCCGACCUGUUCCUGGCCGCCAAGAACCUGUCCGACGCCAUCCUGCUGUCCGACAUCCUGCGGGUGAACACCGAGAUCACCAAGGCCCCCCUGUCCGCCUCCAUGAUCAAGCGGUACGACGAGCACCACCAGGACCUGACCCUGCUGAAGGCCCUGGUGCGGCAGCAGCUGCCCGAGAAGUACAAGGAGAUCUUCUUCGACCAGUCCAAGAACGGCUACGCCGGCUACAUCGACGGCGGCGCCUCCCAGGAGGAGUUCUACAAGUUCAUCAAGCCCAUCCUGGAGAAGAUGGACGGCACCGAGGAGCUGCUGGUGAAGCUGAACCGGGAGGACCUGCUGCGGAAGCAGCGGACCUUCGACAACGGCUCCAUCCCCCACCAGAUCCACCUGGGCGAGCUGCACGCCAUCCUGCGGCGGCAGGAGGACUUCUACCCCUUCCUGAAGGACAACCGGGAGAAGAUCGAGAAGAUCCUGACCUUCCGGAUCCCCUACUACGUGGGCCCCCUGGCCCGGGGCAACUCCCGGUUCGCCUGGAUGACCCGGAAGUCCGAGGAGACCAUCACCCCCUGGAACUUCGAGGAGGUGGUGGACAAGGGCGCCUCCGCCCAGUCCUUCAUCGAGCGGAUGACCAACUUCGACAAGAACCUGCCCAACGAGAAGGUGCUGCCCAAGCACUCCCUGCUGUACGAGUACUUCACCGUGUACAACGAGCUGACCAAGGUGAAGUACGUGACCGAGGGCAUGCGGAAGCCCGCCUUCCUGUCCGGCGAGCAGAAGAAGGCCAUCGUGGACCUGCUGUUCAAGACCAACCGGAAGGUGACCGUGAAGCAGCUGAAGGAGGACUACUUCAAGAAGAUCGAGUGCUUCGACUCCGUGGAGAUCUCCGGCGUGGAGGACCGGUUCAACGCCUCCCUGGGCACCUACCACGACCUGCUGAAGAUCAUCAAGGACAAGGACUUCCUGGACAACGAGGAGAACGAGGACAUCCUGGAGGACAUCGUGCUGACCCUGACCCUGUUCGAGGACCGGGAGAUGAUCGAGGAGCGGCUGAAGACCUACGCCCACCUGUUCGACGACAAGGUGAUGAAGCAGCUGAAGCGGCGGCGGUACACCGGCUGGGGCCGGCUGUCCCGGAAGCUGAUCAACGGCAUCCGGGACAAGCAGUCCGGCAAGACCAUCCUGGACUUCCUGAAGUCCGACGGCUUCGCCAACCGGAACUUCAUGCAGCUGAUCCACGACGACUCCCUGACCUUCAAGGAGGACAUCCAGAAGGCCCAGGUGUCCGGCCAGGGCGACUCCCUGCACGAGCACAUCGCCAACCUGGCCGGCUCCCCCGCCAUCAAGAAGGGCAUCCUGCAGACCGUGAAGGUGGUGGACGAGCUGGUGAAGGUGAUGGGCCGGCACAAGCCCGAGAACAUCGUGAUCGAGAUGGCCCGGGAGAACCAGACCACCCAGAAGGGCCAGAAGAACUCCCGGGAGCGGAUGAAGCGGAUCGAGGAGGGCAUCAAGGAGCUGGGCUCCCAGAUCCUGAAGGAGCACCCCGUGGAGAACACCCAGCUGCAGAACGAGAAGCUGUACCUGUACUACCUGCAGAACGGCCGGGACAUGUACGUGGACCAGGAGCUGGACAUCAACCGGCUGUCCGACUACGACGUGGACCACAUCGUGCCCCAGUCCUUCCUGAAGGACGACUCCAUCGACAACAAGGUGCUGACCCGGUCCGACAAGAACCGGGGCAAGUCCGACAACGUGCCCUCCGAGGAGGUGGUGAAGAAGAUGAAGAACUACUGGCGGCAGCUGCUGAACGCCAAGCUGAUCACCCAGCGGAAGUUCGACAACCUGACCAAGGCCGAGCGGGGCGGCCUGUCCGAGCUGGACAAGGCCGGCUUCAUCAAGCGGCAGCUGGUGGAGACCCGGCAGAUCACCAAGCACGUGGCCCAGAUCCUGGACUCCCGGAUGAACACCAAGUACGACGAGAACGACAAGCUGAUCCGGGAGGUGAAGGUGAUCACCCUGAAGUCCAAGCUGGUGUCCGACUUCCGGAAGGACUUCCAGUUCUACAAGGUGCGGGAGAUCAACAACUACCACCACGCCCACGACGCCUACCUGAACGCCGUGGUGGGCACCGCCCUGAUCAAGAAGUACCCCAAGCUGGAGUCCGAGUUCGUGUACGGCGACUACAAGGUGUACGACGUGCGGAAGAUGAUCGCCAAGUCCGAGCAGGAGAUCGGCAAGGCCACCGCCAAGUACUUCUUCUACUCCAACAUCAUGAACUUCUUCAAGACCGAGAUCACCCUGGCCAACGGCGAGAUCCGGAAGCGGCCCCUGAUCGAGACCAACGGCGAGACCGGCGAGAUCGUGUGGGACAAGGGCCGGGACUUCGCCACCGUGCGGAAGGUGCUGUCCAUGCCCCAGGUGAACAUCGUGAAGAAGACCGAGGUGCAGACCGGCGGCUUCUCCAAGGAGUCCAUCCUGCCCAAGCGGAACUCCGACAAGCUGAUCGCCCGGAAGAAGGACUGGGACCCCAAGAAGUACGGCGGCUUCGACUCCCCCACCGUGGCCUACUCCGUGCUGGUGGUGGCCAAGGUGGAGAAGGGCAAGUCCAAGAAGCUGAAGUCCGUGAAGGAGCUGCUGGGCAUCACCAUCAUGGAGCGGUCCUCCUUCGAGAAGAACCCCAUCGACUUCCUGGAGGCCAAGGGCUACAAGGAGGUGAAGAAGGACCUGAUCAUCAAGCUGCCCAAGUACUCCCUGUUCGAGCUGGAGAACGGCCGGAAGCGGAUGCUGGCCUCCGCCGGCGAGCUGCAGAAGGGCAACGAGCUGGCCCUGCCCUCCAAGUACGUGAACUUCCUGUACCUGGCCUCCCACUACGAGAAGCUGAAGGGCUCCCCCGAGGACAACGAGCAGAAGCAGCUGUUCGUGGAGCAGCACAAGCACUACCUGGACGAGAUCAUCGAGCAGAUCUCCGAGUUCUCCAAGCGGGUGAUCCUGGCCGACGCCAACCUGGACAAGGUGCUGUCCGCCUACAACAAGCACCGGGACAAGCCCAUCCGGGAGCAGGCCGAGAACAUCAUCCACCUGUUCACCCUGACCAACCUGGGCGCCCCCGCCGCCUUCAAGUACUUCGACACCACCAUCGACCGGAAGCGGUACACCUCCACCAAGGAGGUGCUGGACGCCACCCUGAUCCACCAGUCCAUCACCGGCCUGUACGAGACCCGGAUCGACCUGUCCCAGCUGGGCGGCGACGGCGGCGGCUCCCCCAAGAAGAAGCGGAAGGUGUGAUAAUAGCGGCCGCGGCCCGAUCCAGUGGGCAGGCCAAGGCCUGCUGGGCCCCCGCGGACCCAGGUGCUCUGGGUCACGGUCCCUGUCCCCGCGCCCCCGCUUCUGUCUGCCCCAUUGUGGCUCCUCAGGCUCUCUCCCCUGCUCUCCCACCUCUACCUCCACCCCCACGGAUCCUCAGGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAUGCAUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA Cas9mRNA 2 mU*mU*mA*CAGCCACGUCUACAGCAGUUUUAGAmGmCmUmAmGmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU sgRNA

[0164] (2) Editing efficiency detection

[0165] The editing efficiency was tested one week after intravenous administration to the mice's tails. The mice were killed and their liver tissues were taken out. The genome was extracted after lysis and the editing efficiency was analyzed by second-generation sequencing.

[0166] A two-step nested PCR amplification and sequencing was performed, and sequencing adapters and indexes were added. PCR products were verified by gel electrophoresis to confirm the correct size of the amplified product, and the obtained PCR products were sent to BGI for sequencing. The deep sequencing results were processed using a custom Python-based tool for site-specific analysis and editing efficiency calculation. The calculation results are shown in the table. Figure 1 .

[0167] As shown in Table 1, Table 2, Table 3 and Figure 1 As shown, the cationic lipid nanoparticles used in the present invention have a good particle size distribution, high encapsulation efficiency, and excellent mRNA delivery performance. They can achieve efficient and saturated mRNA expression in mice, significantly better than the control lipid nanoparticles. The lipid storage stability and other aspects are superior to the positive control ALC-0315, meeting the requirements of in vivo delivery.

[0168] The descriptions presented in the above exemplary embodiments are only intended to illustrate the technical solutions of the present invention and are not intended to be exhaustive or to limit the present invention to the precise forms described. Obviously, it is possible for a person of ordinary skill in the art to make many changes and variations based on the above teachings. The exemplary embodiments are selected and described in order to explain the specific principles of the present invention and its practical applications, so that other persons skilled in the art can easily understand, implement and utilize the various exemplary embodiments of the present invention and its various selected forms and modified forms. The scope of protection of the present invention is intended to be defined by the appended claims and their equivalents.

[0169] In the description of this specification, the reference terms "one embodiment," "some embodiments," "example," "specific example," or "some examples" mean that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0170] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A compound represented by formula (I) or a pharmaceutically acceptable form thereof, wherein: ， G1 and G2 are each independently -(CH2)6- or -(CH2)9-; G3 is -(CH2)4-; L1 and L2 are each independently selected from -C(=O)O- or -OC(=O)-; R1 and R2 are each independently selected from or ; X1 and X2 are each independently -CH2- or -(CH2)2-; Y1 and Y2 are each independently a bond, -CH2-, -(CH2)2-, -(CH2)3-, -(CH2)CH(CH2OH)CH2-, -(CH2)CH(OH)CH2-; Z is selected from a bond, O or -C(=O)O-.

2. The compound according to claim 1 or a pharmaceutically acceptable form thereof, wherein The compound is selected from the following compounds: (1); (2); (3); (4); (5); (6); (7); (8); or (9)； The pharmaceutically acceptable form is selected from pharmaceutically acceptable salts or stereoisomers.

3. A lipid carrier comprising the compound according to claim 1 or 2 or a pharmaceutically acceptable form thereof.

4. The lipid carrier according to claim 3, further comprising one or more excipients selected from the group consisting of neutral lipids, steroids and polymer-conjugated lipids.

5. A nucleic acid lipid nanoparticle composition comprising the compound according to claim 1 or 2 or a pharmaceutically acceptable form thereof or the lipid carrier according to claim 3 or 4, and a nucleic acid drug.

6. The nucleic acid lipid nanoparticle composition according to claim 5, wherein the nucleic acid drug is selected from RNA, DNA, antisense nucleic acid, aptamer, nucleic acid, immunostimulatory nucleic acid or peptide nucleic acid.

7. A pharmaceutical preparation comprising a compound according to claim 1 or 2 or a pharmaceutically acceptable form thereof, or a lipid carrier according to claim 3 or 4, or a nucleic acid lipid nanoparticle composition according to claim 5 or 6, and a pharmaceutically acceptable excipient, carrier or diluent.

8. Use of the compound according to any one of claims 1 or 2, or a pharmaceutically acceptable form thereof, or the lipid carrier according to claim 3 or 4, or the nucleic acid lipid nanoparticle composition according to claim 5 or 6, or the pharmaceutical preparation according to claim 7 in the preparation of nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs.

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