Lipid compound, composition containing lipid compound and application

Abstract

The invention relates to a lipid compound, a composition containing the same and application. The present invention provides a compound of formula (I) or a pharmaceutically acceptable form thereof. The compounds of the present invention can be used for delivering nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and gene therapy technology, 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 diseases. However, nucleic acid drugs are easily degraded and difficult to enter cells, so they need to rely on delivery vectors. Developing safe and efficient delivery vectors is the key.

[0003] Lipid nanoparticles (LNP) are commonly used gene delivery vectors that can protect nucleic acids from degradation, prolong circulation time and improve targeting. LNPs are composed of cationic lipids, structural lipids, phospholipids and PEG lipids, among which cationic lipids are the core that determines 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 by the present invention has lipid nanoparticles with advantages in terms of encapsulation rate, 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 having formula (I) or a pharmaceutically acceptable form thereof, wherein:

[0007]

[0008] G 1 and G 2 and G 3 Each independently is C 3 -C 12 Alkylene, the C 3 -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] L 1 and L 2each 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] R 1 and R 2 Each independently selected from H, C 1-6 Alkoxy, C 1-30 Alkyl, C 2-30 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 The cycloalkenyl group is optionally substituted with one or more of the following substituents: halogen, cyano, oxo, -OH or C 1-6 alkyl;

[0011] X 1 , X 2 , Y 1 and Y 2 are each independently a bond, -(CH 2 ) 1-6 C(=O)-、C 1-6 Alkylene, the C 1-6 The alkylene group is optionally substituted with one or more of the following substituents: halogen, oxo, -OH, C 1-6 Alkyl, C 1-6 Hydroxyalkyl, -(CH 2 ) 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, the G 1 and G 2 and G 3 Each is independently -(CH 2 ) 3 -、-(CH 2 ) 4 -、-(CH 2 ) 5 -、-(CH 2 ) 6 -、-(CH 2 )7 -、-(CH 2 ) 8 -、-(CH 2 ) 9 -or-(CH 2 ) 10 -.

[0014] In certain embodiments, the L 1 and L 2 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, the R 1 and R 2 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 The cycloalkenyl group is optionally substituted with one or more of the following substituents: halogen, cyano, oxo, -OH or C 1-6 alkyl;

[0016] Optionally, R 1 and R 2 Each independently selected from

[0017] or .

[0018] In certain embodiments, the X 1 , X 2 , Y 1 and Y 2 Each is independently a bond, -CH 2 -、-(CH 2 ) 2 -、-(CH 2 ) 3 -、-(CH 2 ) 4 -、-(CH 2 ) 2 (C=O)-、-(CH 2 )CH(CH 2 OH)CH 2 -or-(CH 2 )CH(OH)CH 2-.

[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, which comprises the above-mentioned compound or a pharmaceutically acceptable form thereof or the above-mentioned 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 above-mentioned compound or a pharmaceutically acceptable form thereof or the above-mentioned lipid carrier or the above-mentioned 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 easily understood from the description of the embodiments in conjunction with the following drawings, in which:

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

[0039] For easier understanding of the present invention, some technical and scientific terms are specifically defined below. Unless otherwise specifically 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 the present 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 word "may" means both performing a certain process and not performing a certain process.

[0042] In the present specification, "optional" or "optionally" means that the event or situation described below may or may not occur, and the description includes cases where the event occurs and cases where it does not occur.

[0043] In this specification, the 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 exist 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 also to be understood that the terminology used herein is for the purpose of description 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 the compound of the present invention that is substantially non-toxic to an organism. Pharmaceutically acceptable salts generally include (but are not limited to) salts formed by reacting the compound of the present invention with a pharmaceutically acceptable inorganic / organic acid or inorganic / organic base, and 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., and 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 has a vertical asymmetric plane due to at least one chiral factor (including chiral center, chiral axis, chiral plane, etc.), thereby being able to rotate plane polarized light. Since there are asymmetric centers and other chemical structures that may cause stereoisomerism in the compounds of the present invention, the present invention also includes these stereoisomers and mixtures thereof. Since the compounds of the present invention and their salts include asymmetric carbon atoms, they can exist in the form of a single stereoisomer, a racemate, an enantiomer, and a mixture of diastereomers. Generally, these compounds can be prepared in the form of a racemic mixture. However, if desired, such compounds can be prepared or separated to obtain pure stereoisomers, i.e., single enantiomers or diastereomers, or single stereoisomer-enriched (purity ≥98%, ≥95%, ≥93%, ≥90%, ≥88%, ≥85% or ≥80%) mixtures. A single stereoisomer of a compound is prepared synthetically from an optically active starting material containing the desired chiral center, or by preparing a mixture of enantiomeric products followed by separation or resolution, such as 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 particular 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 can directly or indirectly provide a compound of the present invention after being applied to a patient. Particularly preferred derivative compounds or prodrugs are compounds that can increase the bioavailability of the compound of the present invention when administered to a patient (e.g., more easily absorbed into the blood), or compounds that promote the 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 present invention are within the scope of the present 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) with the same or similar value ranges in the structure may have the same or different meanings under certain circumstances. For example, substituent X and substituent Y are each independently hydrogen, halogen, hydroxyl, cyano, alkyl or aryl. When substituent X is hydrogen, substituent Y may be hydrogen, halogen, hydroxyl, cyano, alkyl or aryl; similarly, when substituent Y is hydrogen, substituent X may be hydrogen, halogen, hydroxyl, cyano, alkyl or aryl.

[0052] The terms "comprising" and "including" 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 atoms and hydrogen atoms, containing no unsaturation, and connected to other fragments by a single bond, including (but not limited to) methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl and tert-butyl, etc. 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 systems. For example, the term "C 3-6 The term "cycloalkyl" refers to a cycloalkyl 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, etc. 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. 2-30 "Alkynyl" refers to a monovalent straight or branched chain hydrocarbon group 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 only of carbon atoms and hydrogen atoms. Cycloalkenyl may include cyclic, bridged or spirocyclic systems. For example, cyclopropenyl and cyclobutenyl, etc.

[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, etc. 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 branched structures. For example

[0064] The term "heterocyclyl" refers to a saturated or partially saturated, monocyclic or polycyclic (such as bicyclic, for example, fused, bridged or spiro) 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), Rx is independently selected from H or C1-4 alkyl. If the valence bond requirements are met, the heterocyclic group can be connected to the rest of the molecule through any one of the ring atoms. For example, the term "3-8 membered heterocyclic group" as used in the present invention refers to a heterocyclic group having 3 to 8 ring atoms. For example, the heterocyclic group can be an oxirane group, an aziridine group, an azetidine group, an oxetane group, a tetrahydrofuranyl group, a dioxolyl group, a pyrrolidinyl group, a pyrrolidonyl group, an imidazolidinyl group, a pyrazolidinyl group, a tetrahydropyranyl group, a piperidinyl group, a piperazinyl group, a morpholinyl group, a thiomorpholinyl group, a dithianyl group or a trithianyl group.

[0065] The term "aryl" refers to a monocyclic or condensed polycyclic aromatic hydrocarbon group having a conjugated π electron system. 6-10 The term "aryl" refers to an aromatic group having 6 to 10 carbon atoms. For example, the aryl 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 can be connected to the rest of the molecule through any one of the ring atoms. For example, the term "5-10 membered heteroaryl" used in the present invention refers to a heteroaryl having 5 to 10 ring atoms. For example, the heteroaryl 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 -NO 2 .

[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, tomatine, 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-glyceryl-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glyceryl-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glyceryl-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glyceryl-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glyceryl-3-phosphocholine (DOPC), phosphatidylethanolamine such as 1,2-dioleoyl-sn-glyceryl-3-phosphoethanolamine (DOPE), sphingomyelin (SM), ceramide, 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 rugosity of the nucleotide sugar ring, which affects the physical properties of the oligonucleotide's binding affinity for the complementary strand, duplex formation, and interaction with nucleases. Substitutions on the sugar ring can thus alter the alignment and rugosity of these sugars. For example, 2'-O-methyl (2'-O-Me) modifications can increase the binding affinity and nuclease stability of an oligonucleotide, 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] The modification 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 between nucleotide bases that replaces one of the non-bridging phosphate oxygens. When phosphorothioates are used to produce oligonucleotides, 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 with 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 with a PS bond. The embodiments described herein encompass equivalents of PS connections or bonds.

[0088] The following shows the substitution of an S- to a non-bridging phosphate oxygen, creating 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 construed as limiting the present invention.

[0092] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[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 dichloromethane (500 mL) solution of 1,6-hexanediol (16 g, 87.75 mmol), and the solution was stirred at room temperature overnight, washed with dichloromethane and water, and the organic phase was dried over anhydrous sodium sulfate and purified by silica gel 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), and methylsulfonic anhydride (7.8 g, 44.9 mmol) was added under ice bath and stirred for 16 hours. 3 Extraction, drying of the organic phase with anhydrous sodium sulfate, removal of the solvent by vacuum drying, and finally purification 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), K 2 CO 3 (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 intermediate H1-3 (9.2 g) was purified by silica gel chromatography. 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), and methylsulfonic anhydride (2.05 g, 11.8 mmol) was added under ice bath and stirred for 16 hours. 3 Extraction, drying of the organic phase with anhydrous sodium sulfate, removal of the solvent by vacuum drying, and finally purification by silica gel 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), K 2 CO 3 (120 mg, 0.72 mmol), KI (60 mg, 0.36 mmol) and 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 purified by silica gel chromatography to give 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), K 2 CO 3 (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 the solution was stirred at 85° C. overnight. The reaction mixture was filtered, the solvent was removed by vacuum drying, and purified by silica gel chromatography to give 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), K 2 CO 3 (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 the solution was stirred at 85° C. overnight. The reaction mixture was filtered, the solvent was removed by vacuum drying, and purified by silica gel chromatography to give 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), K 2 CO 3 (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 the solution was stirred at 85° C. overnight. The reaction mixture was filtered, the solvent was removed by vacuum drying, and the compound 4 (97 mg) was purified by silica gel chromatography.

[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), K 2 CO 3(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 the solution was stirred at 85° C. overnight. The reaction mixture was filtered, the solvent was removed by vacuum drying, and the compound 5 (88 mg) was purified by silica gel chromatography.

[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), K 2 CO 3 (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 the solution was stirred at 85° C. overnight. The reaction mixture was filtered, the solvent was removed by vacuum drying, and the compound 6 (110 mg) was purified by silica gel chromatography.

[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), K 2 CO 3 (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 the solution was stirred at 85° C. overnight. The reaction mixture was filtered, the solvent was removed by vacuum drying, and purified by silica gel chromatography to give 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, the synthesis steps are the same as those in Example 1), 1-oxa-8-azaspiro[4.5]decan-3-ol (57 mg, 0.36 mmol), K 2 CO 3 (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 was removed by vacuum drying, and purified by silica gel chromatography to give 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), K 2 CO 3 (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 the solution was stirred at 85° C. overnight. The reaction mixture was filtered, the solvent was removed by vacuum drying, and purified by silica gel chromatography to give 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] The cationic lipid or the compound 1-9 of the present invention, the cationic lipid, cholesterol, DSPC and PEG-DMG were dissolved in anhydrous ethanol at a molar ratio of 50 / 38.5 / 10 / 1.5 to prepare a mixed lipid ethanol solution. The compound 1-9 of the present invention and ALC-0315 were dissolved in anhydrous ethanol with cholesterol, DSPC and PEG-DMG according to the above molar ratios to prepare a mixed lipid ethanol solution.

[0139] Fluc-mRNA (Apexbio, catalog number: R1005) was dissolved in 25mM enzyme-free citrate buffer at pH 5 (mRNA concentration was 0.2mg / mL) to obtain mRNA citrate buffer. The mixed lipid ethanol solution was mixed with the mRNA citrate buffer at a flow rate ratio of 1:3 (nitrogen-phosphorus ratio of cationic lipid to Fluc-mRNA 6:1) using an Atson microfluidic device (model: MPF-L2™), and the total flow rate was 20mL / min to prepare a mixed solution of the nanolipid particle composition. The mixed solution of the above nanolipid particle composition was transferred to a 100 kDa dialysis bag and dialyzed overnight at 4°C in PBS at pH 7.4. Finally, the lipid nanoparticles were filtered through a 0.22μm sterile filter. The prepared nanolipid particle composition solution was stored in a 4°C environment.

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

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

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

[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 (1) of Example 10) was determined using the Quant-iT Ribogreen RNA Quantification Kit (Thermo Fisher Scientific, UK) according to the instructions of the kit.

[0145] 10 μL of the nanolipid particle composition solution was diluted 100 times with PBS and added to a micro sample pool. The particle size and polydispersity index (PDI) were measured using a Zetasizer nano instrument (25°C) from Malvern. Each sample was measured three times, and the average of the three results was taken. 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 can be seen from Table 1, the nanolipid particles prepared by 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, indicating that the nanolipid particles are uniform in size and have excellent dispersibility, and are suitable for mRNA delivery. In addition, the encapsulation efficiency of the nanolipid particles is high. Encapsulation efficiency, as an important quality attribute of liposomes, reflects the degree of encapsulation of drugs. The high encapsulation efficiency nanolipid particles of the present invention help to efficiently load active substances (such as siRNA, mRNA), thereby improving drug loading capacity.

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

[0150] Female C57BL / 6 mice (purchased from Vitallife) 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 obtained nucleic acid lipid nanoparticles were administered by tail vein injection (dose of 0.2 mg / kg), and the mice were killed 4 hours after administration. The liver was collected in a pre-weighed tube, the weight was determined, and about 50 mg was cut to measure the luciferase expression in the mouse liver (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 the lipid nanoparticles obtained were prepared 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 experiment

[0160] (1) Animal studies

[0161] The nanolipid particles containing the mRNA (SEQ ID NO: 1) and sgRNA (SEQ ID NO: 2, targeting the TTR gene, synthesized by Nanjing GenScript Co., Ltd.) of CRISPR Cas9, the nanolipid particles containing the compounds 1-9 of the present invention, and the 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 weeks old, female CD-1 mice weighing about 18-20 g (purchased from Vital River) at a dose of 0.15 mg / kg through 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 liver tissues were obtained. The genome was extracted after lysis, and the editing efficiency was analyzed by next-generation sequencing.

[0166] After two steps of nested PCR amplification and sequencing, sequencing adapters and indexes were added. The PCR products were verified by gel electrophoresis to confirm that the amplified product size was correct, and the obtained PCR products were sent to BGI for sequencing. The deep sequencing results were processed with a custom Python-based tool for specific site analysis and editing efficiency calculation. The calculation results can be found in Figure 1 .

[0167] As shown in Table 1, Table 2, Table 3 and Figure 1 As shown, the cationic lipid compound lipid nanoparticles used in the present invention have good particle size distribution, high encapsulation efficiency, and good mRNA delivery performance. It can make mRNA highly efficiently expressed in mice to reach a saturated state, which is significantly better than the comparative lipid nanoparticles, and the lipid storage stability and other aspects are better than the positive control ALC-0315, which can meet the needs of in vivo delivery.

[0168] The description presented in the above exemplary embodiments is only used to illustrate the technical solution of the present invention, and is not intended to be exhaustive, nor is it intended to limit the present invention to the precise form 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 to explain the specific principles of the present invention and its practical application, so that other technicians in the field can easily understand, implement and use 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 attached claims and their equivalent forms.

[0169] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means 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 may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0170] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary 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, 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; 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-; R1 and R2 are each independently selected from H, C 1-6 Alkoxy, C 1-30 Alkyl, C 2-30 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 The cycloalkenyl group is optionally substituted with one or more of the following substituents: halogen, cyano, oxo, -OH or C 1-6 alkyl; X1, X2, Y1 and Y2 are each independently a bond, -(CH2) 1-6 C(=O)-、C 1-6 Alkylene, the C 1-6 The alkylene group 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; Z is selected from a bond, O, -C(=O)O-, and S.

2. The compound according to claim 1 or a pharmaceutically acceptable form thereof, wherein: The G1, G2 and G3 are each independently -(CH2)3-, -(CH2)4-, -(CH2)5-, -(CH2)6-, -(CH2)7-, -(CH2)8-, -(CH2)9- or -(CH2) 10 -.

3. A compound according to claim 1 or 2 or a pharmaceutically acceptable form thereof, wherein: The 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-.

4. The compound according to claim 1 or a pharmaceutically acceptable form thereof, wherein: The 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 The cycloalkenyl group is optionally substituted with one or more of the following substituents: halogen, cyano, oxo, -OH or C 1-6 alkyl; Optionally, said R1 and R2 are each independently selected from or .

5. The compound according to claim 1 or a pharmaceutically acceptable form thereof, wherein: The 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-.

6. The compound according to claim 1 or a pharmaceutically acceptable form thereof, wherein: The Z is selected from a bond, O or -C(=O)O-.

7. 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 a pharmaceutically acceptable salt or a stereoisomer.

8. A lipid carrier comprising the compound according to any one of claims 1 to 7 or a pharmaceutically acceptable form thereof.

9. The lipid carrier according to claim 8, further comprising one or more excipients selected from the group consisting of neutral lipids, steroids and polymer-conjugated lipids.

10. A nucleic acid lipid nanoparticle composition comprising the compound according to any one of claims 1 to 7 or a pharmaceutically acceptable form thereof or the lipid carrier according to claim 8 or 9, and a nucleic acid drug.

11. According to the nucleic acid lipid nanoparticle composition described in claim 10, the nucleic acid drug is selected from RNA, DNA, antisense nucleic acid, aptamer, nucleic acid, immunostimulatory nucleic acid or peptide nucleic acid.

12. A pharmaceutical preparation comprising a compound according to any one of claims 1 to 7 or a pharmaceutically acceptable form thereof, or a lipid carrier according to claim 8 or 9, or a nucleic acid lipid nanoparticle composition according to claim 10 or 11, and a pharmaceutically acceptable excipient, carrier or diluent.

13. Use of a compound according to any one of claims 1 to 7 or a pharmaceutically acceptable form thereof, or a lipid carrier according to claim 8 or 9, or a nucleic acid lipid nanoparticle composition according to claim 10 or 11, or a pharmaceutical preparation according to claim 12 in the preparation of nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs.

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