WO2024212958A1 - Ionizable lipid compound, lipid carrier comprising same, and application - Google Patents

Abstract

The present invention relates to an ionizable lipid compound, a lipid carrier comprising same, and an application of the ionizable lipid compound. The present invention provides a series of compounds as shown in formula (1), a lipid carrier comprising same as an ionizable lipid, a nucleic acid lipid nanoparticle composition, and a preparation thereof. Lipid nanoparticles formed by the ionizable lipid can deliver nucleic acid molecules into cells, thus increasing the transfer rate of the nucleic acid molecules, and thereby improving the treatment effect of a nucleic acid drug.

Description

Ionizable lipid compound, lipid carrier containing the same and application thereof Technical Field

The invention belongs to the field of biomedicine, and specifically relates to an ionizable lipid compound and a lipid carrier, a nucleic acid lipid nanoparticle composition and a pharmaceutical preparation containing the same.

Background Art

Lipid nanoparticles are widely used in the field of drug delivery. Among them, ionizable lipids are not only excellent protein/peptide antigen carriers, but also a new type of immune adjuvant that can directly activate antigen-presenting cells and enhance vaccine-induced immune responses. Therefore, ionizable lipids are widely used in the vaccine field to encapsulate and transport nucleic acid molecules. Ionizable lipids are the most critical link in the targeting and delivery of lipid nanoparticles. They bind to negatively charged nucleic acids, which helps to escape from endosomal endosomes and transfect nucleic acid molecules in vivo. They have many characteristics such as pH sensitivity. Ionizable lipids determine the delivery efficiency and transfection efficiency of lipid nanoparticle delivery systems. Therefore, designing ionizable lipids with good targeting and delivery properties is an essential key element of lipid nanoparticles.

At present, the most commonly used DLin-MC3-DMA (hereinafter referred to as MC3) on the market is a new type of ionizable lipid. It has the advantages of "low toxicity and high efficiency" and is the world's first ionizable lipid used in siRNA liposome products (Onpattro). With the launch of Onpattro in the United States, MC3 and new ionizable lipids have attracted more attention from scientists. Compared with common ionizable lipids such as DOTAP, DOTMA, DC-CHOL, new ionizable lipids such as MC3 are less toxic, and their drug loading and safety are significantly improved.

Currently, there are 4 siRNA and mRNA drugs (including emergency use authorization) certified by the FDA, all of which use new ionizable lipids. Therefore, new ionizable lipids have broad application prospects in nucleic acid drug delivery.

Summary of the invention

The present invention provides a series of compounds represented by formula (1), lipid carriers containing the compounds as ionizable lipids, nucleic acid lipid nanoparticle compositions and preparations thereof. The lipid nanoparticles formed by the ionizable lipids can deliver nucleic acid molecules into cells, thereby improving the transport rate of nucleic acid molecules and thus improving the therapeutic effect of nucleic acid molecules.

In a first aspect, the present invention provides a compound represented by formula (1) or a pharmaceutically acceptable salt thereof:

in,

A is selected from -O-CO-NH- or -NH-CO-O-;

^G1 and ^G2 are independently _C1-4 alkylene;

^R1 and ^R2 are independently selected from ^-G3- OC(=O) ^-R5 , -G3 ^- C(=O) ^-OR5 , ^-G3- NH-C(=O) ^-OR5 , -G3 ^- NH-OC(=O) ^-R5 , ^-G3- NH-C(=O) ^-R5 or ^-G3- OC(=O)-NH- ^R5 ;

G ³ is independently C _2-10 alkylene;

R ⁵ is selected from C _2-30 alkyl or C _2-30 alkenyl, wherein the alkyl or alkenyl is optionally substituted with one or more -OC _1-30 alkyl, -C(=O)-OC _1-30 alkyl, -OC(=O)-C _1-30 alkyl, -OC _2-30 alkenyl, -C(=O)-OC _2-30 alkenyl or -OC(=O)-C _2-30 alkenyl;

^R3 and ^R4 are independently _C1-8 alkyl.

In some embodiments, ^G1 and ^G2 are independently selected from _{-CH2- , -CH2CH2- , -CH2CH2CH2- , or -CH2CH2CH2CH2-} ; preferably ^, _G1 ^and _{G2 are independently} selected _{from -CH2CH2-} or _-CH2CH2CH2- .

In some embodiments, R ¹ and R ² are independently selected from -G ³ -OC(=O)-R ⁵ , -G ³ -C(=O)-OR ⁵ , -G ³ -NH-C(=O)-OR ⁵ , or -G ³ -OC(=O)-NH-R ⁵ .

In some embodiments, G ³ is independently C _2-8 alkylene; preferably, G ³ is independently selected from

In some embodiments, R ⁵ is selected from C _2-20 alkyl or C _2-20 alkenyl, which is optionally substituted with one or more -OC _1-20 alkyl, -C(＝O)-OC _1-20 alkyl, -OC(＝O)-C _{1-20 alkyl, -OC 2-20 alkenyl} , -C(＝O)-OC _2-20 alkenyl, or -OC(＝O)-C _2-20 alkenyl.

In some embodiments, R ⁵ is selected from C _2-20 alkyl or C _2-20 alkenyl, which is optionally substituted with one or more -OC _1-20 alkyl, -C(=O)-OC _1-20 alkyl, or -C(=O)-OC _2-20 alkenyl.

In some embodiments, ^R5 is selected from

In some embodiments, ^R1 and ^R2 are independently selected from

In some embodiments, R ³ and R ⁴ are independently C _1-6 alkyl; preferably, R ³ and R ⁴ are independently C _1-4 alkyl; preferably, R ³ and R ⁴ are independently selected from methyl or ethyl.

The present invention also provides the following compounds or pharmaceutically acceptable salts thereof:

In a second aspect, the present invention provides a lipid carrier comprising a compound represented by formula (1) or a pharmaceutically acceptable salt thereof as an ionizable lipid.

In some embodiments, the lipid carrier comprises a compound represented by formula (1) or a pharmaceutically acceptable salt thereof, an auxiliary lipid, a structural lipid and a polymer-bound lipid.

In some embodiments, the helper lipid is selected from 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine DOPE, 1,2-distearoyl-sn-glycero-3-phosphatidylcholine DSPC, dioleoylphosphatidylserine DOPS, distearoylphosphatidylserine DSPS, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine DSPE, dipalmitoylphosphatidylserine DPPS, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine DPPC, 1 , at least one of 2-dioleoyl-sn-glycerol-3-phosphatidylcholine DOPC, dipalmitoylphosphatidylglycerol DPPG, oleoylphosphatidylcholine POPC, 1-palmitoyl-2-oleoylphosphatidylethanolamine POPE, 1,2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine DPPE, 1,2-dimyristoyl-sn-glycerol-3-phosphoethanolamine DMPE, distearoylphosphatidylethanolamine DSPE and 1-stearoyl-2-oleoylphosphatidylethanolamine SOPE.

In some embodiments, the structured lipid is at least one selected from cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol, coproposterol and corticosteroids.

In some embodiments, the polymer-bound lipid is selected from 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (DMG-PEG2000), DMG-PEG2000-mannose, cholesterol-PEG2000, 1,2-dimyristoyl-sn-glyceromethoxy-polyethylene glycol PEG-DMG, dimyristoylglycerol-polyethylene glycol PEG-c-DMG, polyethylene glycol-dimyristoylglycerol PEG-C14, PEG-1,2-dimyristoyloxypropyl-3-amine PEG-c-DMA, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)]PEG-DSPE, PEGylated phosphatidylethanolamine PEG-PE, P At least one of EG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, Tween-20, Tween-80, 1,2-dipalmityl-sn-glycerol-methoxypolyethylene glycol PEG-DPG, 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)succinate PEG-s-DMG, PEG-dialkoxypropyl PEG-DAA, mPEG2000-1,2-di-O-alkyl-sn3-carbamoylglyceride PEG-c-DOMG and N-acetylgalactosamine ((R)-2,3-bis(octadecyloxy)propyl-1-(methoxypoly(ethylene glycol) 2000)propylcarbamate))GalNAc-PEG-DSG.

In some embodiments, in the lipid carrier, the molar ratio of ionizable lipids, auxiliary lipids, structural lipids and polymer-bound lipids is (20-75):(2-25):(15-55):(0-15); illustratively, the molar ratio of ionizable lipids, auxiliary lipids, structural lipids and polymer-bound lipids can be 45:10:42:3, 30:25:30:10, 46:15:40:3, 50:10:38.5:1.5, 50:10:37:3, 50:9:38:3, etc.

In a third aspect, the present invention provides a nucleic acid lipid nanoparticle composition, which comprises a compound represented by formula (1) or a pharmaceutically acceptable salt thereof or the above-mentioned lipid carrier, and a nucleic acid.

In some embodiments, the nucleic acid is selected from DNA, RNA, a complex containing DNA or RNA (such as a complex of DNA and RNA, a complex of DNA and polypeptide/protein, a complex of RNA and polypeptide/protein), a modified At least one of a modified DNA, a modified RNA, and a modified complex containing DNA or RNA.

In some embodiments, the RNA is selected from mRNA, siRNA, dsRNA, rRNA, circRNA, saRNA, tRNA, snRNA or shRNA, preferably mRNA.

In some embodiments, the mass ratio of the nucleic acid to any one of the compounds or pharmaceutically acceptable salts thereof is 1:(3-40).

In some embodiments, the mass ratio of the nucleic acid to the lipid carrier is 1:(3-40).

Exemplarily, the above mass ratio is 1:3, 1:5, 1:10, 1:15, 1:20, 1:30, etc.

In a fourth aspect, the present invention provides a pharmaceutical preparation comprising any one of the above compounds or a pharmaceutically acceptable salt thereof, or the above lipid carrier, or the above nucleic acid lipid nanoparticle composition, and a pharmaceutically acceptable carrier.

In some embodiments, the particle size of the pharmaceutical preparation is 30 to 500 nm. Exemplarily, the particle size can be 30 nm, 50 nm, 60 nm, 80 nm, 100 nm, 120 nm, 150 nm, 250 nm, 350 nm, 500 nm, etc.

In some embodiments, the encapsulation rate of nucleic acid in the pharmaceutical preparation is greater than 50%. Exemplarily, the encapsulation rate can be 55%, 60%, 65%, 70%, 75%, 79%, 80%, 85%, 89%, 90%, 93%, 95%, 97%, etc.

In a fifth aspect, the present invention also provides use of the above-mentioned compound or its pharmaceutically acceptable salt 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 or gene vaccines.

The present invention also provides a method for in vivo delivery of nucleic acid drugs or gene vaccines, comprising administering the nucleic acid lipid nanoparticle composition or the pharmaceutical preparation to a subject in need thereof.

In some embodiments, the nucleic acid lipid nanoparticle composition or the pharmaceutical formulation is administered by one of the following routes of administration: oral, intranasal, intravenous, intraperitoneal, intramuscular, intraarticular, intralesional, intratracheal, subcutaneous, and intradermal. In some embodiments, the nucleic acid lipid nanoparticle composition or the pharmaceutical formulation is administered, for example, via an enteral or parenteral administration route. In some embodiments, a dose of about 0.001 mg/kg to about 10 mg/kg of the nucleic acid lipid nanoparticle composition or pharmaceutical formulation is administered to the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: H NMR spectrum of compound 1.

Figure 2: H NMR spectrum of compound 11.

Figure 3: H NMR spectrum of compound 12.

Figure 4: H NMR spectrum of compound 15.

Figure 5: H NMR spectrum of compound 16.

Figure 6: H NMR spectrum of compound 17.

Figure 7: H NMR spectrum of compound 18.

Figure 8: H NMR spectrum of compound 2.

DETAILED DESCRIPTION

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.

In this specification, the numerical range represented by "value A to value B" refers to a range including the endpoint values A and B. When the lower limit and upper limit of a numerical range are disclosed, any value or any sub-range falling within the range is indicated to be specifically disclosed. In particular, each numerical range of a parameter disclosed herein (for example, in the form of "about a to b", or equivalently "about a to b", or equivalently "about ab") should be understood to include each numerical value and sub-range therein. For example, "C _1-4 " should be understood to include any sub-range and each point value therein, such as C _2-4 , C _3-4 , C _1-2 , C _1-3 , C _1-4 , etc., as well as C ₁ , C ₂ , C ₃ , C ₄ , etc.

The terms "comprises," "including," "having," or "containing" or any other variation thereof are intended to cover a non-exclusive or open-ended inclusion. For example, a composition, method, or apparatus comprising a list of elements is not necessarily limited to only the elements expressly listed but may also include other elements not expressly listed or inherent to such composition, method, or apparatus.

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.

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.

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.

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.

The term "pharmaceutically acceptable carrier" refers to an excipient that is administered together with the above-mentioned nucleic acid lipid nanoparticle composition or the above-mentioned pharmaceutical preparation, and is suitable for contacting the tissues of humans and/or other animals without excessive toxicity, irritation, allergic reaction or other problems or complications corresponding to a reasonable benefit/risk ratio within the scope of reasonable medical judgment. Pharmaceutically acceptable carriers that can be used in the present invention include, but are not limited to: a) diluents; b) lubricants; c) binders; d) disintegrants; e) absorbents, colorants, flavorings and/or sweeteners; f) emulsifiers or dispersants; and/or g) substances that enhance the absorption of compounds, etc.

The term "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 in specific 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.

The term "alkyl" refers to a linear or branched monovalent saturated aliphatic hydrocarbon group. For example, "C _1-30 alkyl" refers to a linear or branched monovalent saturated aliphatic hydrocarbon group containing 1 to 30 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl or tert-butyl.

The term "alkylene" refers to a straight or branched divalent saturated aliphatic hydrocarbon group, and the two groups (or fragments) connected thereto may be connected to the same carbon atom or to different carbon atoms. For example, the term "C _1-4 alkylene" used herein refers to an alkylene group having 1 to 4 carbon atoms (such as methylene, 1,1-ethylene, 1,2-ethylene, 1,2-propylene, 1,3-butylene, etc.).

The term "alkenyl" refers to a straight or branched unsaturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms and having at least one double bond. For example, " _C2-30 alkenyl" refers to a monovalent straight or branched hydrocarbon group containing 2 to 30 carbon atoms and having at least one carbon-carbon double bond. Non-limiting examples of alkenyl include, but are not limited to, vinyl, 1-propenyl, 2-propenyl, 1-butenyl, isobutenyl, 1,3-butadienyl, etc.

In order to make the purpose and technical solution of the present invention clearer, the embodiments of the present invention are described in detail below in conjunction with examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as To limit the scope of the present invention.

The reagents or instruments used in the examples are all conventional products that can be obtained commercially. If no specific conditions are specified, they are carried out according to conventional conditions or conditions recommended by the manufacturer. The term "room temperature" used in the present invention refers to 20°C ± 5°C. When used to modify a certain value or numerical range, the term "about" used in the present invention refers to an acceptable error range for those skilled in the art including the value or numerical range and the value or numerical range, for example, the error range is ± 10%, ± 5%, ± 4%, ± 3%, ± 2%, ± 1%, ± 0.5%, etc.

The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

In the following examples, all solvents and drugs used were analytically pure or chemically pure; the solvents were redistilled before use; and the anhydrous solvents were treated according to standard methods or literature methods.

The abbreviations used herein have the following meanings:

The compounds of the present invention are separated and purified by preparative TLC, silica gel column chromatography, Prep-HPLC and/or flash column chromatography, and their structures are confirmed by ¹ H NMR and/or MS. Reaction monitoring is performed by TLC or LC-MS.

¹ H NMR spectroscopy was performed using a Bruker superconducting nuclear magnetic resonance spectrometer (model AVACE III HD 400 MHz).

LC/MS uses Aglient 1260Infinity/Aglient 6120Quadrupole.

TLC used silica gel GF 254 as the stationary phase.

Column chromatography generally uses 200-300 mesh silica gel (Qingdao Ocean) as the stationary phase.

Flash column chromatography was performed using a Biotage flash column chromatograph.

Prep-HPLC used Agilent 1260 and Waters 2489.

In the following examples, unless otherwise specified, the reaction temperature is room temperature (15-30°C).

Synthesis Example 1: Preparation of Intermediate 1

The raw material 1 (20 g, 89.64 mmol, 1 eq) and the raw material 2 (26.51 g, 183.77 mmol, 2.05 eq) were dissolved in DCM (400 mL), EDCI (17.18 g, 89.64 mmol, 1 eq) and DMAP (2.19 g, 17.93 mmol, 0.2 eq) were added, and the mixture was stirred at 15°C for 18 hours. The reaction mixture was diluted with 200 mL H ₂ O and extracted with 400 mL DCM, the organic layers were combined, washed, dried, filtered, and concentrated under vacuum, and the crude oil was purified by flash column chromatography to obtain a colorless liquid, which was the target product intermediate 1.

Synthesis Example 2: Preparation of Intermediate 2

The raw material 2 (14.2 g, 25.72 mmol, 1 eq) and the intermediate 1 prepared in Synthesis Example 1 (8.98 g, 25.72 mmol, 1 eq) were dissolved in ACN (250 mL), K ₂ CO ₃ (14.22 g, 102.87 mmol, 4 eq) and KI (4.70 g, 28.29 mmol, 1.1 eq) were added, and the mixture was diluted with cyclopentyl methyl ether (60 mL), heated to 90° C. and stirred under N ₂ for 16 hours. The reaction mixture was filtered, the filtrate was diluted with 100 mL H ₂ O and extracted with 200 mL DCM, the organic layers were combined, washed, dried, filtered, and concentrated under vacuum, and purified by flash column chromatography to obtain a yellow liquid, which was the intermediate 2.

Synthesis Example 3: Preparation of Raw Material 4

The raw material 3 (300 mg, 422.44 umol, 1 eq) was dissolved in DCM (4 mL), DIEA (272.99 mg, 2.11 mmol, 367.91 uL, 5 eq) and CDI (205.49 mg, 1.27 mmol, 3 eq) were added, and the mixture was stirred at 20°C for 16 hours. The reaction mixture was diluted with 20 mL H ₂ O and extracted with 40 mL DCM, and the organic layers were combined, washed, dried, filtered and concentrated under reduced pressure to obtain a yellow jelly, which was the raw material 4.

Example 1: Preparation of Compound 18

The intermediate 2 (370 mg) prepared in Synthesis Example 2 was dissolved in ACN (4 mL), TEA (325.88 mg) and raw material 4 (187.12 mg) were added, and the mixture was stirred at 20°C for 16 hours. The mixture was concentrated under reduced pressure, and the residue was purified by preparative HPLC to obtain a red oily substance, which was compound 18 (126.9 mg). The H NMR spectrum data of compound 18 are shown in Figure 7.

Example 2: Preparation of Compound 15

Intermediate 2 (2 g) was dissolved in DCE (40 mL), DIEA (1.82 g) and CDI (2.74 g) were added, and the mixture was stirred at 50°C for 16 hours. The reactant was diluted with 50 mL H ₂ O and extracted with 100 mL DCM. The organic layers were combined, washed, dried, filtered and concentrated under reduced pressure to obtain a yellow oil, which was raw material 1 (2.49 g).

The raw material 1 (1 g) was dissolved in ACN (5 mL), and TEA (629.11 mg), DMAP (75.95 mg) and raw material 2 (635.25 mg) were added. The reaction was stirred at 50°C for 16 hours. The mixture was concentrated under reduced pressure, and the obtained residue was purified by preparative HPLC to obtain a red oily substance, which was compound 15. The H NMR spectrum data of compound 15 are shown in As shown in Figure 4.

Example 3: Preparation of Compound 16

The raw material 1 (3 g) was dissolved in ACN (20 mL), and TEA (1.89 g), DMAP (227.86 mg) and raw material 2 (1.64 g) were added, and the mixture was stirred at 50° C. for 16 hours. The mixture was concentrated under reduced pressure, and the obtained residue was purified by preparative HPLC to obtain a red oily compound, which was compound 16. The H NMR spectrum data of compound 16 are shown in FIG5 .

Example 4: Preparation of Compound 17

The raw material 1 (3 g) was dissolved in ACN (20 mL), and TEA (1.89 g), DMAP (227.86 mg) and raw material 2 (1.91 g) were added, and the mixture was stirred at 50° C. for 16 hours. The mixture was concentrated under reduced pressure, and the obtained residue was purified by preparative HPLC to obtain a red oily substance, which was compound 17. The H NMR spectrum data of compound 17 are shown in FIG6 .

Example 5: Preparation of Compound 1

The raw material 1 (5 g) was dissolved in DCM (25 mL), and EDCI (4.49 g), DMAP (2.38 g) and raw material 2 (3.53 g, 19.50 mmol, 2.56 mL, 1 eq) were added, and the reaction was stirred at 20° C. for 16 hours. The reaction mixture was concentrated under reduced pressure, and the obtained residue was purified by flash silica gel chromatography to obtain a colorless oily substance, which was raw material 3.

The raw material 3 (2 g) was dissolved in ethanol (1 mL), and the raw material 4 (8.74 g) was added. The reaction was stirred at 50°C for 18 hours. The reaction mixture was concentrated under reduced pressure to remove the solvent, and the residue was diluted with 80 mL of H ₂ O and extracted with 200 mL of ethyl acetate. The organic layers were combined, washed, dried, filtered and concentrated under reduced pressure to obtain a yellow liquid, which was the raw material 5.

The raw material 5 (890 mg) and the raw material 3 (1.12 g) were dissolved in ACN (4 mL), K ₂ CO ₃ (1.23 g) and KI (406.65 mg) were added, and the mixture was diluted with cyclopentyl methyl ether (1 mL), heated to 90°C and stirred for 16 hours under N _2. The reaction mixture was concentrated under reduced pressure to remove the solvent, the residue was diluted with 80 mL H ₂ O and extracted with 200 mL ethyl acetate, the organic layer was washed with 100 mL brine, dried, filtered, and concentrated under reduced pressure, and the residue was purified by flash silica gel chromatography to obtain a colorless liquid, which was the raw material 6.

DIEA (1.34 g) and CDI (1.01 g) were added to a solution of raw material 6 (900 mg) in DCE (20 mL), and the mixture was stirred at 50°C for 16 hours. The reaction mixture was concentrated under reduced pressure to remove the solvent, the residue was diluted with 30 mL of H ₂ O and extracted with 30 mL of dichloromethane, the organic layer was washed with 30 mL of brine, dried, filtered and concentrated under reduced pressure to obtain a yellow oily substance, which was raw material 7.

The raw material 7 (600 mg) was dissolved in ACN (5 mL), TEA (510.64 mg), DMAP (61.65 mg) and raw material 8 (586.41 mg) were added, and the mixture was stirred at 50° C. for 16 hours. The mixture was concentrated under reduced pressure, and the obtained residue was purified by preparative HPLC to obtain a red oily substance, which was compound 1. The H NMR spectrum data of compound 1 are shown in FIG1 .

Example 6: Preparation of Compound 12

The raw material 2 (1 g) and the raw material 1 (1.28 g) were dissolved in DCM (20 mL), EDCI (839.43 mg) and DMAP (106.99 mg) were added, and the mixture was stirred at 15°C for 18 h. The reaction mixture was diluted with 100 mL of H ₂ O and extracted with 200 mL of DCM, the organic layer was washed with 100 mL of brine, dried, filtered and concentrated under reduced pressure, and the residue was purified by flash silica gel chromatography to obtain a colorless liquid, which was the raw material 3.

The starting material 4 (5 g) and the starting material 1 (5.71 g) were dissolved in DCM (100 mL), EDCI (3.74 g) and DMAP (476.44 mg) were added, and the mixture was stirred at 15°C for 18 hours. The reaction mixture was diluted with 100 mL of H ₂ O and washed with 200 mL of The organic layer was washed with 100 mL of brine, dried, filtered and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography to obtain a colorless liquid as starting material 5.

The raw material 5 (7.06 g) was dissolved in ethanol (6 mL), and the raw material 6 (29.84 g) was added, and the mixture was stirred at 50°C for 18 hours. The reaction mixture was diluted with 200 mL of H ₂ O and extracted with 300 mL (150 mL*2) of ethyl acetate. The organic layers were combined, washed with 300 mL of brine, dried, filtered and concentrated under reduced pressure to obtain a colorless liquid, which was the raw material BB5.

The raw material BB5 (1 g) and the raw material 3 (980.12 mg) were dissolved in cyclopentyl methyl ether (10 mL), K ₂ CO ₃ (1.34 g) and KI (441.41 mg) were added, and the mixture was diluted with ACN (40 mL). The resulting mixture was heated to 90°C and stirred under N ₂ for 28 hours. The reaction mixture was cooled to room temperature, filtered and concentrated under reduced pressure, and the obtained residue was purified by flash silica gel chromatography to obtain a yellow oily substance, which was the raw material 7.

The raw material 7 (800 mg) was dissolved in DCM (8 mL), DIEA (630.26 mg) and CDI (474.44 mg) were added, and the mixture was stirred at 50°C for 16 hours. The reaction mixture was diluted with 15 mL H ₂ O and extracted with 30 mL DCM, and the organic layer was washed with 15 mL brine, dried, filtered, and concentrated under reduced pressure to obtain a colorless oil, which was the raw material 8.

The raw material 8 (1.16 g) was dissolved in ACN (10 mL), and TEA (705.17 mg), DMAP (85.14 mg) and raw material 9 (809.80 mg) were added. The mixture was stirred at 50° C. for 16 hours. The mixture was concentrated under reduced pressure, and the residue was purified by preparative HPLC to obtain a yellow oily substance, which was compound 12. The H NMR spectrum data of compound 12 are shown in FIG3 .

Example 7: Preparation of Compound 11

The raw material 1 (4.23 g) and the raw material 2 (2 g) were dissolved in DCM (50 mL), EDCI (2.42 g) and DMAP (308.74 mg) were added, and the reaction was stirred at 15°C for 18 hours. The reaction mixture was diluted with 60 mL of H ₂ O and extracted with 100 mL of DCM, and the organic layer was washed, dried, filtered and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography to obtain a colorless liquid, which was the raw material 3.

The raw material 3 (1 g) and the raw material BB5 (878.40 mg) obtained in Example 6 were dissolved in cyclopentyl methyl ether (10 mL), K ₂ CO ₃ (1.34 g) and KI (441.41 mg) were added, and diluted with ACN (40 mL), heated to 90°C and stirred under N ₂ for 28 hours. The reaction mixture was cooled to room temperature, filtered and concentrated under reduced pressure, and the obtained residue was purified by flash silica gel chromatography to obtain a yellow oily substance, which was the raw material 4.

The raw material 4 (700 mg) was dissolved in DCM (10 mL), DIEA (584.81 mg) and CDI (440.23 mg) were added, and the mixture was stirred at 50°C for 16 hours. The reaction mixture was diluted with 50 mL H ₂ O and extracted with 120 mL DCM. The organic layers were combined, washed, dried, filtered and concentrated under reduced pressure to obtain a colorless oil, which was the raw material 5.

The raw material 5 (950 mg) was dissolved in ACN (10 mL), and TEA (608.26 mg), DMAP (73.44 mg) and raw material 6 (698.51 mg) were added, and the mixture was stirred at 50° C. for 16 hours. The mixture was concentrated under reduced pressure, and the obtained residue was purified by preparative HPLC to obtain a yellow oily substance, which was compound 11. The H NMR spectrum data of compound 11 is shown in FIG2 .

Example 8: Preparation of Compound 2:

The raw material 7 was obtained in the same manner as in Example 5. The raw material 7 (600 mg) was dissolved in ACN (7.9 mL), TEA (510.64 mg), DMAP (61.6 mg) and raw material 8 (515 mg) were added, and the mixture was stirred at 50° C. for 16 hours. The mixture was concentrated under reduced pressure, and the obtained residue was purified by preparative HPLC to obtain a red oily substance, which was compound 2. The H NMR spectrum data of compound 2 is shown in FIG8 .

Example 9: Preparation and characterization of lipid nanoparticles

1. Preparation of lipid nanoparticles (LNP) (using Miana instrument)

The preparation of lipid nanoparticles includes mixing and dissolving the above-mentioned ionizable lipids and auxiliary lipids (such as DSPC, DOPE, DOPC, etc.), polymer-bound lipids (such as DMG-PEG2000) and structural lipids (such as cholesterol, steroids) in anhydrous ethanol according to a certain molar ratio so that the total lipid phase concentration is 0.5-20 mg/mL. The nucleic acid is dissolved in a buffer solution with a pH lower than 7 (such as 3-6.5), and the preparation is achieved by rapid mixing with the ethanol solution of the lipid.

In this embodiment, the total lipid phase concentration was prepared to 1 mg/mL with anhydrous ethanol according to the molar ratio of ionizable lipid (Compound 1, Compound 11 or Compound 12): auxiliary lipid (DSPC or DOPE): cholesterol: DMG-PEG2000 of 50%: 10%: 38.5%: 1.5%. The aqueous phase buffer was 50 mM citric acid buffer at pH 4.0 to dissolve the corresponding concentration of Luc-eGFP mRNA. The mass ratio of mRNA to total lipid was 1:30.

LNP was prepared using the Myana prescription screening chip, with a total flow rate of 12 ml/min and a lipid phase to water phase flow rate ratio of 1:3. The collected lipid nanoparticles were dialyzed with PBS at 4°C overnight to remove ethanol and acidic buffer salts. The dialysate volume was more than 400 times the sample volume. The dialyzed sample was filtered using a 0.2 micron filter membrane. In addition to using microfluidic chips, lipid nanoparticles can also be prepared using other methods such as injection mixing. After dialysis filtration, the sample was concentrated using a 10kDa cellulose ultrafiltration membrane. The centrifugation parameters were 1000g to 2000g, and the centrifugation time was 30 minutes to 1 hour. The centrifugation temperature was 4°C.

2. Particle size determination: The particle size and polydispersity index (PDI) of the prepared lipid nanoparticles were measured using a Malvern particle size analyzer.

3. Determination of mRNA encapsulation efficiency:

For lipid nanoparticles encapsulating mRNA, Quant-iT ^TM RNA kit detection method. The specific operation is as follows: Use TE buffer (10mM Tris-HCl, 1mM EDTA, pH 7.5) to dilute the sample to 10-20 times. Add an equal volume of demulsifier (2% Triton X-100) to the sample to be tested and dilute it again 10-20 times. The reagents were diluted 100-fold in TE buffer and 100 μl was added to a 96-well plate. 100 μl of the sample to be tested was added to a 96-well plate. A standard curve was prepared using mRNA standards. The fluorescence value at 520 nm excited by 480 nm excitation light was read in an ELISA reader. The concentration of the encapsulated and free mRNA was calculated using the standard curve.

Table 1: Particle size and nucleic acid encapsulation efficiency of lipid nanoparticles containing different ionizable lipids and helper lipids

Example 10: Luciferase mRNA delivery experiment in animals

The nucleic acid delivery efficiency of lipid nanoparticles was characterized by observing the fluorescence expression in mice. The lipid nanoparticles encapsulating Luc-eGFP mRNA prepared in Example 9 were injected into mice through the tail vein at a certain dose. Female C57/BL6 mice weighing 18-22g were taken and randomly divided into groups, with 2 mice in each group. After adaptive feeding, LNPs encapsulating Luc-eGFP mRNA (in sterile PBS solution) were injected into the tail vein at a dose of 0.5mg/kg (0.5mg here refers to the dose of mRNA). In order to observe the expression of luciferase, the luciferase substrate was dissolved in a sterile PBS solution to prepare a solution with a concentration of 30mg/ml.

Six hours after LNP injection, each mouse was intraperitoneally injected with 150ul of luciferase substrate solution. The mice were left alone for 5 minutes, then anesthetized in a carbon dioxide chamber for 3 minutes. After anesthesia, they were placed in a small animal in vivo imaging device for imaging.

Table 2: Chemiluminescence intensity detection of Luc-eGFP mRNA delivered by lipid nanoparticles

Example 11: Mouse Animal Experiment—Subretinal Injection

The efficiency of nucleic acid delivery of lipid nanoparticles was characterized by observing the fluorescence expression in mice. Lipid nanoparticles were prepared according to the molar ratio of ionizable lipid (compound 17, compound 15, compound 18, compound 2 or MC3): DSPC: cholesterol: DSPE-PEG2000 of 50%: 10%: 39.5%: 0.5%. Female C57/BL6 mice weighing 18-22g were taken. After adaptive feeding, lipid nanoparticles encapsulating Luc-eGFP mRNA were injected subretinaally into mice at a dose of 0.9μg (±10%) mRNA (1 microliter in sterile PBS solution) per eye (both eyes were injected). In order to observe the expression of luciferase, the luciferase substrate was dissolved in a sterile PBS solution to prepare a solution with a concentration of 30mg/ml.

Mouse imaging: 18 hours after administration, each mouse was intraperitoneally injected with 150 μl of luciferase substrate solution. The mouse was left alone for 5 minutes, then anesthetized in a carbon dioxide box for 3 minutes. After anesthesia, the mouse was placed in a small animal in vivo imaging instrument for imaging.

Table 3: Particle size and nucleic acid encapsulation efficiency of lipid nanoparticles containing different ionizable lipids

Table 4: Chemiluminescence intensity detection of lipid nanoparticles delivering Luc-eGFP mRNA subretinally

Claims (12)

1. A compound represented by formula (1) or a pharmaceutically acceptable salt thereof:
   

   in,

   A is selected from -O-CO-NH- or -NH-CO-O-;

   ^G1 and ^G2 are independently _C1-4 alkylene;

   ^R1 and ^R2 are independently selected from ^-G3- OC(=O) ^-R5 , -G3 ^- C(=O) ^-OR5 , ^-G3- NH-C(=O) ^-OR5 , -G3 ^- NH-OC(=O) ^-R5 , ^-G3- NH-C(=O) ^-R5 or ^-G3- OC(=O)-NH- ^R5 ;

   G ³ is independently C _2-10 alkylene;

   R ⁵ is selected from C _2-30 alkyl or C _2-30 alkenyl, wherein the alkyl or alkenyl is optionally substituted with one or more -OC _1-30 alkyl, -C(═O)-OC _1-30 alkyl, -OC(═O)-C _1-30 alkyl, -OC _2-30 alkenyl, -C(═O)-OC _2-30 alkenyl or -OC(═O)-C _2-30 alkenyl;

   ^R3 and ^R4 are independently _C1-8 alkyl.
2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein

   ^G1 and ^G2 are independently selected from _-CH2- , _{-CH2CH2- , -CH2CH2CH2-} or _{-CH2CH2CH2CH2-} ; preferably _{, G1} ^and _G2 ^are _{independently selected from -CH2CH2- or -CH2CH2CH2- .}
3. The compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein

   ^R1 and ^R2 are independently selected from ^-G3- OC(=O) ^-R5 , ^-G3- C(=O) ^-OR5 , ^-G3- NH-C(=O) ^-OR5 or ^-G3- OC(=O)-NH- ^R5 ;

   G ³ is independently C _2-8 alkylene; preferably, G ³ is independently selected from

   R ⁵ is selected from C _2-20 alkyl or C _2-20 alkenyl, wherein the alkyl or alkenyl is optionally substituted with one or more -OC _1-20 alkyl, -C(=O)-OC _1-20 alkyl, -OC(=O)-C _1-20 alkyl, -OC _2-20 alkenyl, -C(=O)-OC _2-20 alkenyl or -OC(=O)-C _2-20 alkenyl;

   Preferably, R ⁵ is selected from C _2-20 alkyl or C _2-20 alkenyl, wherein the alkyl or alkenyl is optionally substituted with one or more -OC _1-20 alkyl, -C(=O)-OC _1-20 alkyl or -C(=O)-OC _2-20 alkenyl;

   Preferably, ^R5 is selected from
4. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein

   ^R1 and ^R2 are independently selected from
5. The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein

   R ³ and R ⁴ are independently C _1-6 alkyl; preferably, R ³ and R ⁴ are independently C _1-4 alkyl; preferably, R ³ and R ⁴ are independently selected from methyl or ethyl.
6. A compound or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the following compounds:
   
   
   
7. A lipid carrier comprising the compound according to any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof as an ionizable lipid.
8. The lipid carrier according to claim 7, further comprising a helper lipid, a structural lipid and a polymer-bound lipid.
9. A nucleic acid lipid nanoparticle composition, comprising the compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 6 or the lipid carrier according to claim 7 or 8, and a nucleic acid.
10. The nucleic acid-lipid nanoparticle composition according to claim 9, wherein the nucleic acid is at least one selected from DNA, RNA, a complex containing DNA or RNA, a modified DNA, a modified RNA, and a modified complex containing DNA or RNA.
11. A pharmaceutical preparation comprising the compound according to any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof, the lipid carrier according to claim 7 or 8, or the nucleic acid lipid nanoparticle composition according to claim 9 or 10, and a pharmaceutically acceptable carrier.
12. Use of the compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, the lipid carrier according to claim 7 or 8, the nucleic acid lipid nanoparticle composition according to claim 9 or 10, or the pharmaceutical preparation according to claim 11 in the preparation of nucleic acid drugs or gene vaccines.