Ionizable lipids and compositions thereof for nucleic acid delivery

Abstract

Ionizable lipids and compositions thereof for nucleic acid delivery are provided. The present invention relates to compounds of formula (I) or pharma- ceutically acceptable salts thereof, and their use in lipid particle delivery systems for delivering pharma- ceutical active ingredients, such as nucleic acids. [Formula 1] JPEG2024516574000051.jpg56170

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims priority to and the benefit of Chinese Patent Application No. 202110396368.4, filed on April 13, 2021, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to ionizable lipids and compositions thereof for nucleic acid delivery and is in the field of medicinal chemistry. [Background technology]

[0003] Nucleic acid drugs include DNA, antisense oligonucleotides (ASO), small interfering RNA (siRNA), microRNA (miRNA), miRNA mimics, anti-miRs, ribozymes, mRNA, nucleic acid aptamers, plasmids, CRISPR RNA, etc. The use of nucleic acid drugs is limited due to their chemical instability, and they are easily degraded into single nucleotides by nucleases in vitro and in vivo, resulting in a loss of efficacy.

[0004] The use of nucleic acid medicines often requires specialized delivery carriers, including viral and non-viral vectors. Common viral vectors include retroviruses, lentiviruses, and adeno-associated viruses. Viral vectors, due to their natural cell infection activity, easily enter cells and have high delivery efficiency. However, their clinical application is limited by factors such as immunogenicity, limited loading capacity, and complex production processes. Non-viral vectors are currently being widely studied as gene delivery carriers with promising applications. They primarily carry mRNA through the adsorption of cations formed in the delivery material with mRNA phosphate ions, forming structures such as liposomes or nanoparticles to prevent nuclease degradation and alteration of the mRNA's cell entry pathway. Their advantages include relatively easy availability, low immunogenicity, and high safety.

[0005] Most conventional non-viral nucleic acid delivery materials are cationic lipids or cationic polymers, which, due to their high electropositivity, are prone to adsorption by plasma proteins and capture by the reticuloendothelial system in vivo, resulting in the destruction of the loaded nucleic acid drug. The most studied non-viral vectors are lipid nanoparticles based on ionizable lipids. Nanoparticles prepared with ionizable lipid materials exhibit electropositivity in an acidic environment in vitro, enabling nucleic acid drug loading through electrostatic adsorption. In vivo, they exhibit electroneutrality, avoiding plasma protein adsorption and reticuloendothelial system capture. Based on this, ionizable lipid nanoparticles hold great promise in the field of nucleic acid delivery.

[0006] However, clinical applications of ionizable lipid nanoparticles remain relatively limited, and the development of safe and efficient ionizable lipids remains a key challenge. Therefore, the development of ionizable lipid nucleic acid delivery materials with higher delivery efficiency and greater safety is crucial for the widespread application of nucleic acid medicine and gene therapy. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention provides compounds of formula (1) and pharmaceutically acceptable salts that can be used to form nucleic acid-lipid nanoparticles. The ionizable lipids provided by the present invention and nanoparticles prepared therefrom have high mRNA encapsulation rates and strong transfection capabilities, and the encapsulated mRNA has strong expression and anti-tumor immune effects in vivo.

[0008] In one aspect, the present invention provides a compound of Formula I or a pharmaceutically acceptable salt thereof, wherein the variables of Formula (I) are defined herein. Pharmaceutical compositions of nucleic acid medicines comprising the compound of Formula (I) and pharmaceutically acceptable salts thereof are also provided. [ka]

[0009] The present invention aims to solve at least one of the problems of the prior art and to provide ionizable lipids for nucleic acid delivery. [Means for solving the problem]

[0010] In a first embodiment, the present application provides a compound of formula (I) or a pharmaceutically acceptable salt thereof: [ka] During the ceremony, m1, m2, m3, and m4 are each independently selected from 1, 2, 3, 4, and 5; L1, L2, L3, and L4 each independently represent -CH(OH)-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)S-, -SC(=O)-, or -C(=O)NR a -, -NR a C(=O)-, -NR a C(=O)O-, -OC(=O)NR a -, -O-, -OO-, -S-, -SS-, -SSS-, -CH(OH)CHO-, -CH(OH)CHS-, or absent; a is independently selected from —H or optionally substituted C1-C6 alkyl; R1, R2, R3, and R4 each independently represent an optionally substituted C6-C 30 Alkyl, optionally substituted C-C 30 Alkenyl, optionally substituted C-C 30 alkynyl, G1, G2, G3, and G4 are each independently -R c -, -R c CH(OH)R d -, -R c C(=O)R d -, -R c C(=O)OR d -, -R c OC(=O)R d -, -R c C(=O)SR d -, -Rc SC(=O)R d -, -R c C(=O)N(R b )R d -, -R c N(R b )C(=O)R d -, -R c N(R b )C(=O)OR d -, -R c OC(=O)N(R b )R d -, -R c OR d -, -R c -OOR d -, -R c SR d -, -R c -SSR d -, -R c -SSSR d - selected from, or absent, each R b are each independently selected from —H or optionally substituted C1-C6 alkyl; and each R c , R d are each independently -(CH2) n and n is 0, 1, 2, 3, or 4; R5 and R6 are each independently selected from -H, -OH, or optionally substituted C1-C6 alkyl.

[0011] In a second embodiment, the present application provides a compound of formula (1) or a pharmaceutically acceptable salt thereof, wherein G1, G2 are each independently -R c G3 and G4 are each independently selected from -R c -, -R c C(=O)R d -, -R c C(=O)OR d -, -R c OC(=O)R d -, -R c C(=O)N(R b )R d -, -R c N(R b )C(=O)Rd -, -R c N(R b )C(=O)OR d -, -R c OC(=O)N(R b )R d - selected from or absent, Each R b are each independently selected from —H or C1-C6 alkyl; Each R c , R d are each independently -(CH2) n -, where n is 0, 1, 2, 3, or 4. Definitions for the remaining variables are provided in the first embodiment.

[0012] In a third embodiment, the present invention provides a compound or pharmaceutically acceptable salt according to the second embodiment, wherein G1, G2 are each independently -R c G3 and G4 are each independently selected from -R c -, -R c C(=O)OR d -, -R c OC(=O)R d -, -R c C(=O)N(R b )R d -, -R c N(R b )C(=O)R d - selected from, or absent, each R b are each independently selected from —H or C1-C2 alkyl; Each R c and R d are each independently -(CH2) n - or absent, and n is 0, 1, or 2. Definitions for the remaining variables are provided in the first embodiment.

[0013] In a fourth embodiment, the present invention provides a compound or pharmaceutically acceptable salt described in the third embodiment, wherein G and G are absent, and G, G are each independently selected from -CH-, -CHC(=O)OCH-, -CHOC(=O)CH-, -CHC(=O)NHCH-, -CHNHC(=O)CH-, or absent, with definitions for the remaining variables as provided in the first embodiment.

[0014] In a fifth embodiment, the present invention provides a compound or pharmaceutically acceptable salt according to the first embodiment, wherein G3, G4 are each independently -R c G1 and G2 are each independently selected from -R c -, -R c C(=O)R d -, -R c C(=O)OR d -, -R c OC(=O)R d -, -R c C(=O)N(R b )R d -, -R c N(R b )C(=O)R d -, -R c N(R b )C(=O)OR d -, -R c OC(=O)N(R b )R d - selected from, or absent, each R b are each independently —H or C1-C6 alkyl; Each R c and R d are independently -(CH2) n -, where n is 0, 1, 2, 3, or 4. Definitions for the remaining variables are provided in the first embodiment.

[0015] In a sixth embodiment, the present invention provides a compound or pharmaceutically acceptable salt according to the fifth embodiment, wherein G3 and G4 are each independently -R c -, and G1 and G2 each independently represent -R c-, -R c C(=O)OR d -, -R c OC(=O)R d -, -R c C(=O)N(R b )R d -, -R c N(R b )C(=O)R d - selected from or absent, Each R b are independently —H or C1-C2 alkyl; Each R c and R d are independently -(CH2) n - or absent, and n is 0, 1, or 2. Definitions for the remaining variables are provided in the first embodiment.

[0016] In a seventh embodiment, the present invention provides a compound or a pharmaceutically acceptable salt according to the sixth embodiment, wherein G3 and G4 are absent, and G1 and G2 are each independently -CH2-, -CH2C(=O)O(CH2) 1または2 -, -(CH2) 1または2 OC(=O)CH2-, -CH2C(=O)N(R b )CH2-, -CH2N(R b )C(=O)CH2- or absent; Each R b is independently —H or C1-C2 alkyl. The definitions of the remaining variables are provided in the first embodiment.

[0017] In an eighth embodiment, the present invention provides a compound or a pharmaceutically acceptable salt according to any one of the first to seventh embodiments, wherein L1, L2, L3, and L4 are each independently -CH(OH)-, -C(=O)-, -C(=O)O-, -OC(=O)-, or -C(=O)NR a -, -NR a C(=O)-, -NR a C(=O)O-, -OC(=O)NR a-, -O-, -S-, -CH(OH)CHO-, -CH(OH)CHS-, or absent. The definitions of the remaining variables are provided in any one of the first to seventh embodiments.

[0018] In a ninth embodiment, the present invention provides a compound or a pharmaceutically acceptable salt according to any one of the first to seventh embodiments, wherein L1, L2, L3, and L4 are each independently -CH(OH)-, -C(=O)-, -C(=O)O-, -OC(=O)-, or -C(=O)NR a -, -NR a -C(=O)-, -O-, -S-, or absent; a are each independently -H or C1-C2 alkyl. The definitions for the remaining variables are provided in any one of the first through seventh embodiments.

[0019] In a tenth embodiment, the present invention provides a compound or a pharmaceutically acceptable salt according to any one of the first to ninth embodiments, wherein m1, m2, m3, and m4 are each independently selected from 1 or 2. The definitions of the remaining variables are as provided in any one of the first to ninth embodiments.

[0020] In an eleventh embodiment, the present invention provides a compound or pharmaceutically acceptable salt according to any one of the first to tenth embodiments, wherein R1, R2, R3, and R4 are each independently selected from the group consisting of C6-C 18 Alkyl, C6-C 18 Alkenyl or C6-C 18 alkynyl, wherein C-C 18 Alkyl, C6-C 18 Alkenyl or C6-C 18 The alkynyl is optionally substituted with 1 to 3 substituents selected from halogen, OH, or ═O. The definitions of the remaining variables are provided in any one of the first to tenth embodiments.

[0021] In a twelfth embodiment, the present invention provides a compound or pharmaceutically acceptable salt according to any one of the first to tenth embodiments, wherein R1, R2, R3, and R4 are each independently selected from the group consisting of C6-C 18 alkyl. The definitions for the remaining variables are provided in any one of the first through tenth embodiments.

[0022] In a thirteenth embodiment, the present invention provides a compound or pharmaceutically acceptable salt according to any one of the first to twelfth embodiments, wherein R5, R6 are each independently selected from -H, -OH, or C1-C4 alkyl optionally substituted with -OH, with the remaining variables having the definitions provided in any one of the first to twelfth embodiments.

[0023] In a fourteenth embodiment, the present invention provides a compound of formula (I-1) or a pharmaceutically acceptable salt thereof: [ka] wherein m1, m2, m3, and m4 are selected from the same number and each is 1, 2, 3, 4, or 5; L1, L2, L3, and L4 are selected from the same group, and each L1, L2, L3, and L4 is -CH(OH)-, -C(=O)-, ^-C(=O)O-^^, ^-OC(=O)-^^, ^-C(=O)S-^^, ^-SC(=O)-^^, ^-C(=O)NR a -^^,^-NR a C(=O)-^^, ^-NR a C(=O)O-^^, ^-OC(=O)NR a -^^, -O-, -OO-, -S-, -SS-, -SSS-, ^-CH(OH)CH2O-^^, ^-CH(OH)CH2S-^^, or absent, ^- is selected from R 1-4 represents the site that can be linked to -^^ is -(CH2) m1-m4 - represents a site that can be linked to each R a are independently —H or optionally substituted C1-C6 alkyl; R1, R2, R3, and R4 are selected from the same group, and each R1, R2, R3, and R4 is an optionally substituted C6-C30 Alkyl, optionally substituted C-C 30 Alkenyl or optionally substituted C-C 30 alkynyl, G1 and G2 are selected from the same group, G3 and G4 are selected from the same group, and G1 and G2 are -R c -If G3, G4 are -R c -, *-R c CH(OH)R d -**, *-R c C(=O)R d -**, *-R c C(=O)OR d -**, *-R c OC(=O)R d -**, *-R c C(=O)SR d -**, *-R c SC(=O)R d -**, *-R c C(=O)N(R b )R d -**, *-R c N(R b )C(=O)R d -**, *-R c N(R b )C(=O)OR d -**, *-R c OC(=O)N(R b )R d -**, *-R c OR d -**, *-R c -OOR d -**, *-R c SR d -**, **-R c -SSR d -**, *-R c -SSSR d -** or absent, where *- represents the linking site to the -CH2- group adjacent to the terminal tertiary amine atom in formula (I-1), and -** represents the linking site to the -CH2- group adjacent to the middle tertiary amine atom in formula (I-1); G3 and G4 are -R c- When it is, G1 and G2 are -R c - # -R c CH(OH)R d - ## 、 # -R c C(=O)R d - ## 、 # -R c C(=O)OR d - ## 、 # -R c OC(=O)R d - ## 、 # -R c C(=O)SR d - ## 、 # -R c SC(=O)R d - ## 、<00002​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​-SSSR d - ## or absent, # represents the linking site to the -CH2- group adjacent to the intermediate quaternary amine in the compound of formula (I-1), ## represents the site connecting G1 and G2, and each R b are independently —H or optionally substituted C1-C6 alkyl; Each R c and R d are independently -(CH2) n and n is 0, 1, 2, 3 or 4; R5 and R6 are selected from the same group, each selected from -H, -OH, or optionally substituted C1-C6 alkyl.

[0024] In a fifteenth embodiment, the present invention provides a compound or a pharmaceutically acceptable salt according to embodiment 14, wherein G1, G2 are -R c - and G3 and G4 are -R c -, *-R c C(=O)R d -**, *-R c C(=O)OR d -**, *-R c OC(=O)R d -**, *-R c C(=O)N(R b )R d -**, *-R c N(R b )C(=O)R d -**, *-R c N(R b )C(=O)OR d -**, *-R c OC(=O)N(R b )R d -Selected from ** or absent, Each R b are independently —H or C1-C6 alkyl; Each R c and R d are independently -(CH2) n- and n is 0, 1, 2, 3, or 4. Definitions of the remaining variables are provided in embodiment 14.

[0025] In a sixteenth embodiment, the present invention provides a compound or pharmaceutically acceptable salt according to embodiment 15, wherein G1 and G2 are -R c - and G3 and G4 are *-R c C(=O)OR d -**, *-R c OC(=O)R d -**, *-R c C(=O)N(R b )R d -**, *-R c N(R b )C(=O)R d -Selected from ** or absent, Each R b are independently —H or C1-C2 alkyl; Each R c and R d are independently -(CH2) n - or absent, and n is 0, 1, or 2. Definitions of the remaining variables are provided in embodiment 14.

[0026] In a seventeenth embodiment, the present invention provides a compound or a pharmaceutically acceptable salt according to embodiment 16, wherein G1, G2 are absent, and G3, G4 are selected from -CH2-, *-CH2C(=O)OCH2-**, *-CH2OC(=O)CH2-**, *-CH2C(=O)NHCH2-**, *-CH2NHC(=O)CH2-**, or absent. The definitions of the remaining variables are provided in embodiment 16.

[0027] In an eighteenth embodiment, the present invention provides a compound or pharmaceutically acceptable salt of claim 14, wherein G3, G4 are -R c - and G1 and G2 are -R c -, # -R c C(=O)R d - ## , # -R c C(=O)ORd - ## , # -R c OC(=O)R d - ## , # -R c C(=O)N(R b )R d - ## , # -R c N(R b )C(=O)R d - ## , # -R c N(R b )C(=O)OR d - ## , # -R c OC(=O)N(R b )R d - ## or absent, Each R b are independently —H or C1-C6 alkyl; Each R c and R d are independently -(CH2) n - and n is 0, 1, 2, 3, or 4. Definitions of the remaining variables are provided in embodiment 14.

[0028] In a nineteenth embodiment, the present invention provides a compound or pharmaceutically acceptable salt of embodiment eighteen, wherein G3, G4 are -R c - and G1 and G2 are -R c -, # -R c C(=O)OR d - ## , # -R c OC(=O)R d - ## , # -R c C(=O)N(R b )R d - ## , # -R c N(R b )C(=O)R d -## or absent, Each R b are independently —H or C1-C2 alkyl; Each R c , R d are independently -(CH2) n - or absent, and n is 0, 1, or 2. Definitions of the remaining variables are provided in embodiment 14.

[0029] In embodiment 20, the present invention provides a compound or a pharmaceutically acceptable salt according to embodiment 19, wherein G3 and G4 are absent, and G1 and G2 are -CH2-, # -CH2C(=O)O(CH2) 1または2 - ## , # -(CH2) 1または2 OC(=O)CH2- ## , # -CH2C(=O)N(R b )CH2- ## , # -CH2N(R b )C(=O)CH2- ## or absent, Each R b is independently —H or C1-C2 alkyl. Definitions of the remaining variables are provided in embodiment 14.

[0030] In embodiment 21, the present invention provides a compound or pharmaceutically acceptable salt according to any one of embodiments 14 to 20, wherein L1, L2, L3, and L4 are the same, and each L1, L2, L3, and L4 is selected from the group consisting of -CH(OH)-, -C(=O)-, -C(=O)O-^^, -OC(=O)-^^, and -C(=O)NR a -^^,^-NR a C(=O)-^^, ^-NR a C(=O)O-^^, ^-OC(=O)NR a -^^, -O-, -S-, ^-CH(OH)CHO-^^, ^-CH(OH)CHS-^^, or absent. The definitions of the remaining variables are provided in any one of embodiments 14-20.

[0031] In embodiment 22, the present invention provides a compound or a pharmaceutically acceptable salt according to any one of embodiments 14 to 20, wherein L1, L2, L3, and L4 are selected from the same group, and each L1, L2, L3, and L4 is selected from the group consisting of -CH(OH)-, -C(=O)-, -C(=O)O-^^, -OC(=O)-^^, and -C(=O)NR a -^^,^-NR a C(=O)-^^, -O-, -S-, or absent, and each R a is independently —H or C1-C2 alkyl. The definitions of the remaining variables are provided in any one of Embodiments 14-20.

[0032] In embodiment 23, the present invention provides a compound or a pharmaceutically acceptable salt according to embodiments 14-22, wherein m1, m2, m3, and m4 are selected from the same number, and each m1, m2, m3, and m4 is 1 or 2. Definitions for the remaining variables are provided in any one of embodiments 14-22.

[0033] In embodiment 24, the present invention provides a compound or pharmaceutically acceptable salt according to any one of embodiments 14 to 23, wherein R1, R2, R3, and R4 are selected from the same group, and each R1, R2, R3, and R4 is selected from the group consisting of C6-C 18 Alkyl, C6-C 18 Alkenyl or C6-C 18 alkynyl, wherein C-C 18 Alkyl, C6-C 18 Alkenyl or C6-C 18 The alkynyl is optionally substituted with 1 to 3 substituents selected from halogen, OH, or ═O. Definitions for the remaining variables are provided in any one of embodiments 14 to 23.

[0034] In embodiment 25, the present invention provides a compound or pharmaceutically acceptable salt according to any one of embodiments 14 to 23, wherein R1, R2, R3, and R4 are selected from the same group, and each R1, R2, R3, and R4 is selected from the group consisting of C6-C 18alkyl. The definitions for the remaining variables are provided in any one of embodiments 14 to 23.

[0035] In embodiment 26, the present invention provides a compound or pharmaceutically acceptable salt according to any one of embodiments 14 to 25, wherein R5 and R6 are the same and are selected from —H, —OH, or C1-C4 alkyl optionally substituted with —OH. The definitions of the remaining variables are provided in any one of embodiments 14 to 25.

[0036] In one embodiment, the present invention provides a compound or a pharmaceutically acceptable salt shown in Table 1 below. [Table 1] JPEG0007752186000005.jpg231170JPEG0007752186000006.jpg225170JPEG000 7752186000007.jpg232170JPEG0007752186000008.jpg227170JPEG00077521860 00009.jpg235170JPEG0007752186000010.jpg226170JPEG0007752186000011.j pg239170JPEG0007752186000012.jpg228170JPEG0007752186000013.jpg176170

[0037] The present invention provides a compound of formula I, or a pharmaceutically acceptable salt, isomer, deuterated salt, or prodrug thereof: [ka] In the formula, L1, L2, L3, and L4 are independently -R k CH(OH)-, -R k C(=O)-, -R k C(=O)O-, -R k OC(=O)-, -R k C(=O)S-, -R k SC(=O)-, -R k C(=O)NRa -, -R k NR a C(=O)-, -R k NR a C(=O)O-, -R k OC(=O)NR a -, -R k O-, -R k -OO-, -R k S-, -R k -SS-, -R k -SSS-, -R k CH(OH)CHO-, -R k CH(OH)CHS- or absent, R k is -(CH2) k - or absent, k is an integer equal to or greater than 1, and R a -H, substituted or unsubstituted alkyl; R1, R2, R3, and R4 are independently C1-C 30 Straight chain alkyl, C1-C 30 Branched alkyl, C2-C 30 Straight chain alkenyl, C2-C 30 Branched alkenyl, C2-C 30 Straight chain alkynyl or C2-C 30 branched alkynyl; G1, G2, G3, and G4 are independently -R c -, -R c CH(OH)R d -, -R c C(=O)R d -, -R c C(=O)OR d -, -R c OC(=O)R d -, -R c C(=O)SR d -, -R c SC(=O)R d -, -R c C(=O)N(R b )R d -, -R c N(R b )C(=O)R d -, -R c N(R b )C(=O)OR d-, -R c OC(=O)N(R b )R d -, -R c OR d -, -R c -OOR d -, -R c SR d -, -R c -SSR d -, -R c -SSSR d - selected from, or absent, R b -H, substituted or unsubstituted alkyl, and R c , R d are independently -(CH2) n or absent, and n is an integer greater than or equal to 1; R5, R6 are independently selected from -OH, -H, and substituted or unsubstituted alkyl.

[0038] Furthermore, k is an integer from 1 to 10.

[0039] Furthermore, k is 1.

[0040] Furthermore, L1, L2, L3, and L4 are independently -CH(OH)-, -C(=O)-, -CH2C(=O)O-, -C(=O)O-, -OC(=O)-, -C(=O)S-, -SC(=O)-, or -CH2C(=O)NR a -, -C(=O)NR a -, -NR a C(=O)-, -NR a C(=O)O-, -OC(=O)NR a -, -CHO-, -O-, -CH-OO-, -CHS-, -S-, -CH-SS-, -CH-SSS-, -CH(OH)CHO-, -CH(OH)CHS-, or absent; R a is —H, substituted or unsubstituted alkyl.

[0041] Furthermore, L1, L2, L3, and L4 are independently -C(=O)-, -C(=O)NR a -, -CH2C(=O)NRa -, -NR a selected from C(=O)-, -C(=O)O-, -CHC(=O)O-, -OC(=O)-, -CHO-, -O-, -CHS-, -S-, -CH(OH)-, -CH(OH)CHO-, -CH(OH)CHS-, or absent; R a is —H or unsubstituted alkyl.

[0042] Furthermore, R a is —H or unsubstituted C1-C6 alkyl.

[0043] Furthermore, R a is -H.

[0044] Furthermore, L1, L2, L3, and L4 are independently selected from -C(=O)-, -C(=O)NH-, -CHC(=O)NH-, -C(=O)O-, -CHC(=O)O-, -CHO-, -CHS-, -CH(OH)-, and -CH(OH)CHO-, or are absent; preferably, L1, L2, L3, and L4 are independently selected from -C(=O)NH-, -C(=O)O-, -CH(OH)-, and -CH(OH)CHO-, or are absent.

[0045] Furthermore, L1 and L2 are selected from the same group, and L3 and L4 are selected from the same group.

[0046] Furthermore, L1, L2, L3 and L4 are selected from the same group.

[0047] Furthermore, R1, R2, R3, and R4 are independently C1-C 30 Straight chain alkyl, C2-C 30 Straight chain alkenyl, C2-C 30 straight chain alkynyl.

[0048] Further, R1, R2, R3, and R4 are independently unsubstituted C1-C 30 straight chain alkyl.

[0049] Further, R1, R2, R3, and R4 are independently unsubstituted C8-C 18 straight chain alkyl.

[0050] Additionally, R1, R2, R3 and R4 may independently be unsubstituted C 10 -C 14 straight chain alkyl.

[0051] Furthermore, R1, R2, R3 and R4 are selected from the same group.

[0052] Furthermore, G3 [ka] -(CH2) n3 - selected from, or absent, R f is —H or unsubstituted alkyl, and n3 is an integer from 1 to 10.

[0053] Preferably, G3 is [ka] -(CH2) n3 - selected from, or absent, R f is —H or unsubstituted alkyl, and n3 is an integer from 1 to 10.

[0054] Furthermore, R f is —H or unsubstituted C1-C6 alkyl.

[0055] Furthermore, R f is —H, methyl, ethyl or propyl.

[0056] Furthermore, n3 is 1 or 2.

[0057] Preferably, n3 is 1.

[0058] Furthermore, G1-G2 or G2-G1 [ka] -(CH2) n2 - selected from, or absent, R g , R h , R i is independently selected from —H or unsubstituted alkyl, and n2 is an integer from 1 to 10.

[0059] Furthermore, R g , R h , R i is independently selected from —H or unsubstituted C1-C6 alkyl.

[0060] Furthermore, R g , R h , R i is independently selected from —H, methyl, or ethyl.

[0061] Furthermore, n2 is 1 or 2.

[0062] Furthermore, G4 [ka] -(CH2) n4 - selected from, or absent, R j is —H or unsubstituted alkyl, and n4 is an integer from 1 to 10.

[0063] Furthermore, R j is —H or unsubstituted C1-C6 alkyl.

[0064] Furthermore, R j is —H, methyl, ethyl or propyl.

[0065] Furthermore, n4 is 1 or 2.

[0066] Further, R5 and R6 are independently selected from -OH, -H, unsubstituted C1-C6 alkyl, and C1-C6 alkyl substituted with -OH.

[0067] Further, R5 and R6 are independently selected from -OH, -H, methyl, ethyl, propyl, hydroxymethyl, hydroxyethyl, and hydroxypropyl.

[0068] Furthermore, R5 and R6 are selected from the same group.

[0069] The order in which the bonds L1, L2, L3, and L4 defined above are written corresponds to the proximal nitrogen terminus to the distal nitrogen terminus from left to right.

[0070] The order in which the bonds G1, G2, G3, and G4 defined above are written is from left to right, corresponding to the direction of the main chain in Formula I.

[0071] The present invention provides the use of the compound, or a pharmaceutically acceptable salt, isomer, deuterated product or prodrug thereof, as a carrier for nucleic acid delivery.

[0072] The present invention provides a pharmaceutical composition comprising the compound, or a pharmaceutically acceptable salt, isomer, deuterated product or prodrug thereof, and a nucleic acid drug.

[0073] Additionally, the pharmaceutical composition further comprises at least one excipient selected from the group consisting of neutral phospholipids, steroids, and polyethylene glycol lipids.

[0074] In some embodiments, the neutral phospholipid is selected from 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), dioleoyl lecithin (DOPC), distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylcholine (DMPC), 1,2-bis(dimethylphosphino)ethane (DMPE), dipalmitoylphosphatidylcholine (DPPC), 1,2-bis(diphenylphosphino)ethane (DPPE), diethylpyrocarbonate (DEPC), hydrogenated soybean lecithin (HSPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), or a combination thereof.

[0075] In some embodiments, the neutral phospholipid is DOPE.

[0076] In some embodiments, the molar ratio of a compound described herein to neutral phospholipid is between 1:1 and 5:1.

[0077] In some embodiments, the steroid is selected from cholesterol, sitosterol, soy sterol, lanosterol, ergosterol, or compositions thereof.

[0078] In some embodiments, the steroid is cholesterol.

[0079] In some embodiments, the molar ratio of the compound of the invention to the steroid is 1:2 to 2:1.

[0080] In some embodiments, the polyethylene glycol lipid is selected from dimyristoylglycerol-polyethylene glycol (DMG-PEG), methoxypolyethylene glycol phospholipid (DSPE-PEG), or a combination thereof.

[0081] More preferably, the polyethylene glycol lipid is DMG-PEG2000.

[0082] In some embodiments, the molar ratio of the compound of the invention to the polyethylene glycol lipid is between 5:1 and 100:1.

[0083] More preferably, the ratio of the compound according to the present invention to the polyethylene glycol lipid is 10:1 to 20:1.

[0084] In some embodiments, the nucleic acid drug is selected from DNA, ASO, siRNA, miRNA, mRNA, ribozyme, nucleic acid aptamer, or a combination thereof, and is further mRNA.

[0085] In some embodiments, the pharmaceutical composition is formulated into a lipid nanoparticle LNP.

[0086] The lipid nanoparticles described above can be used to deliver nucleic acid drugs such as mRNA in vivo to achieve upregulation or downregulation of corresponding genes, or to deliver antigens that are expressed by mRNA in vivo to achieve immunotherapy, or to deliver antibodies encoded by mRNA to be expressed in vivo.

[0087] Definitions of terms: The compounds and derivatives provided herein may be named according to the IUPAC (International Union of Pure and Applied Chemistry) or CAS (Chemical Abstracts Service, Columbus, OH) nomenclature systems.

[0088] The term "alkyl" refers to a straight-chain or branched-chain saturated hydrocarbon radical with the structure -C n H (2n+1)The C1-C6 alkyl group includes, but is not limited to, methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), isobutyl (C4), n-pentyl (C5), 3-pentyl (C5), pentyl (C5), neopentyl (C5), 3-methyl-2-butyl (C5), tert-pentyl (C5), and n-hexyl (C6). As used herein, the term "C6-C 30 (or C6-C 18 "C6-C 6-C alkyl" refers to a group having 6 to 30 (or 6 to 18) carbon atoms arranged in a linear or branched chain. In some embodiments, "C6-C 30 (or C6-C 18 The term "alkyl" refers to a group having 6 to 30 (or 6 to 18) carbon atoms arranged in a branched chain.

[0089] The term "alkenyl" includes, but is not limited to, vinyl, propenyl, butyl 1-alkenyl, 2-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, hex-1-enyl, hex-2-enyl, hex-3-enyl, and hex-4-enyl. 30 (or C6-C 18 A "C6-C" alkenyl group refers to a group having 6 to 30 (or 6 to 18) carbon atoms arranged in a straight or branched chain. In some embodiments, a "C6-C" alkenyl group refers to a group having 6 to 30 (or 6 to 18) carbon atoms arranged in a straight or branched chain. 30 (or C6-C 18 An "alkenyl" group refers to a group having 6 to 30 (or 6 to 18) carbon atoms in a linear arrangement. In some embodiments, "C6-C 30 (or C6-C 18 An "alkenyl" group refers to a group having 6 to 30 (or 6 to 18) carbon atoms arranged in a straight line.

[0090] The term "alkynyl" refers to a straight or branched chain hydrocarbon group containing at least one triple bond. Acetyl groups include, but are not limited to, ethyl, propynyl, butyl-1-ynyl, butyl-2-ynyl, pentyl-1-ynyl, pentyl-2-ynyl, pentyl-3-ynyl, hex-1-ynyl, hex-2-ynyl, hex-3-ynyl, and hex-4-ynyl. As used herein, "C-C 30 (or C6-C 18 A "C6-C" alkynyl group refers to a group having 6 to 30 (or 6 to 18) carbon atoms arranged in a straight or branched chain. In some embodiments, a "C6-C" alkynyl group refers to a group having 6 to 30 (or 6 to 18) carbon atoms arranged in a straight or branched chain. 30 (or C6-C 18 A "C6-C" alkynyl group refers to a group of 6 to 30 (or 6 to 18) carbon atoms in a linear arrangement. 30 (or C6-C 18 An "alkynyl" group refers to a group having 6 to 30 (or 6 to 18) carbon atoms arranged in a branched chain.

[0091] If suitable substituents are not specifically listed, exemplary substituents include C 1-5 Alkyl, C 1-5 Haloalkyl, C 1-5 Hydroxyalkyl, C 1-5 Alkoxy, C 1-5 Haloalkoxy, halogen, hydroxy, amino, -CN, -NO2, -OR c1 , -NR a1 R b1 , -S(O) i R a1 , -NR a1 S(O) i R b1 , S(O) i NR a1 R b1 , -C(=O)OR a1 , -OC(=O)OR a1 , -C(=S)OR a1 , -O(C=S)R a1 , -C(=O)NR a1 R b1 , -NR a1 C(=O)Rb1 , -C(=S)NR a1 R b1 , -C(=O)R a1 , -C(=S)R a1 , N.R. a1 C(=S)R b1 , -O(C=O)NR a1 R b1 , -NR a1 (C=S)OR b1 , -O(C=S)NR a1 R b1 , -NR a1 (C=O)NR a1 R b1 , -NR a1 (C=S)NR a1 R b1 , phenyl, or 5-6 membered heteroaryl. a1 and each R b1 are independently -H, C 1-5 C optionally substituted with alkyl or hydroxyl 1-3 alkoxy; R c1 -H, C 1-5 Haloalkyl or C 1-5 alkyl, C 1-5 The alkyl may be substituted with a hydroxy or C1-C3 alkoxy substituent.

[0092] The term "pharmaceutically acceptable" means that a carrier, excipient, salt, etc. is chemically or physically compatible with other ingredients that generally constitute a pharmaceutical dosage form and physiologically compatible with the recipient. The term "pharmaceutically acceptable salt" refers to acidic and / or basic salts formed by the compounds of the present invention with inorganic and / or organic acids and bases, including zwitterionic salts (internal salts) and further including quaternary ammonium salts, such as alkylammonium salts. These salts can be obtained directly during the final isolation and purification of the compounds. They can also be obtained by appropriately mixing the compounds with a certain amount (e.g., equivalent amount) of acid or base. These salts can form precipitates in solution and be collected by filtration, or recovered after evaporation of the solvent, or can be obtained by reaction in an aqueous medium followed by lyophilization. Salts according to the present invention can be the hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate salts of the compound.

[0093] The present invention provides novel ionizable lipids, whose hydrophilic center is composed of four available tertiary amine nitrogens and whose hydrophobic tail is composed of four saturated or unsaturated fatty chains. The novel ionizable lipids provided by the present invention are positively charged in an acidic environment and almost uncharged in a neutral environment. Utilizing this property, nucleic acid drugs can be loaded in an acidic buffer system. After loading of the nucleic acid drug is completed, the system can be adjusted to neutral, resulting in electropositive lipid nanoparticles that avoid adsorption by plasma proteins, achieving higher delivery efficiency and safety.

[0094] The solution of the present invention will be described below with reference to examples. Those skilled in the art will understand that the following examples are used only to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific techniques or conditions are shown in the examples, they shall be in accordance with the techniques, conditions or product specifications described in the literature in this field. Unless the manufacturer is specified, the reagents or equipment used are all common commercially available products. [Brief explanation of the drawings]

[0095] [Figure 1] II-1 Gel retardation results of LNP@mRNA. [Figure 2] II-1 Particle size, PDI, and potential of LNP@mRNA. [Figure 3] II-1, II-5, III-5, and VI-1 are microscopic morphologies of LNP@Luc mRNA. [Figure 4] Transfection of DC2.4 cells with MC3, II-13, III-9, and IV-4 LNP@GFP mRNA. [Figure 5] In vivo expression and distribution of II-11, III-6, V-2 and MC3 LNP@Luc mRNA. [Figure 6] II-9, III-8, II-22, and VI-4 are immunoantitumor effects of LNP@OVA mRNA. [Figure 7] Comparison of the safety of intramuscular injection of MC3, III-3, and VI-2 LNP@OVA mRNA. [Figure 8] Bioluminescence images of LNP@Luc mRNA expression after intramuscular injection. [Figure 9] RBD-specific IgG titers in immunized mice. [Figure 10] Alanine aminotransferase (ALT) levels in immunized mice. [Figure 11] Aspartate transaminase (AST) levels in immunized mice. [Figure 12] Creatinine (CRE) levels in immunized mice. DETAILED DESCRIPTION OF THE INVENTION

[0096] Abbreviation DCM 1,2-dichloromethane DIPEA N-ethyl-N-isopropylpropylamine-2-amino EA Ethyl acetate eq equivalent EtOH ethanol MeOH Methanol PE Petroleum Ether TEA Triethylamine TFA trifluoroacetic acid

[0097] Example 1 Synthesis of Compound II-1 [ka] (1) Synthesis of Compound 3: N-Boc-1,2-ethylenediamine (1.0 eq) and TEA (2.0 eq) were added to a single-neck flask and dissolved in an appropriate amount of anhydrous DCM. The mixture was stirred uniformly in an ice-water bath. Acryloyl chloride (1.2 eq) was dissolved in an appropriate amount of anhydrous DCM and added to a constant-pressure dropping funnel. The flow rate was controlled and the mixture was added dropwise to the single-neck flask. The reaction mixture was then allowed to react in an ice-water bath for 6 h. The reaction solvent was then spun down, and the crude product was purified by silica gel column chromatography (DCM:MeOH = 50:1) and concentrated to dryness to give a white solid, 3, in 90.5% yield. (2) Synthesis of Compound 5: N,N'-bis(2-hydroxyethyl)ethylenediamine (1.0 eq) and compound 3 (2.0 eq) were added to a single-necked flask, dissolved in an appropriate amount of absolute ethanol, and the flask was attached to a condenser and refluxed in an oil bath at 80 °C for 12 h. The reaction solvent was evaporated, and the crude product was purified by silica gel column chromatography (DCM:MeOH = 15:1, 0.5% aqueous ammonia), concentrated, and dried to give a pale yellow solid, 5, in an 85.3% yield. (3) Synthesis of II-1: Compound 5 was dissolved in DCM, and a sufficient amount of TFA was added with stirring. The mixture was allowed to react at room temperature for 6 hours. The TFA / DCM was then rotary evaporated to give a yellow oil. The oil was dissolved in an appropriate amount of acetonitrile, and a sufficient amount of anhydrous potassium carbonate was added with stirring. The mixture was stirred at room temperature until the reaction mixture became alkaline. 1-Bromo-2-hexadecanone (6.0 eq) was added to the reaction mixture, and the reaction mixture was refluxed in a 90 °C oil bath for 36 hours with a condenser attached. The reaction mixture was then rotary evaporated, and the crude product was purified by silica gel column chromatography (DCM:MeOH = 20:1, 0.5% aqueous ammonia) and concentrated to dryness to give yellow oil II-1 in 76.8% yield.

[0098] Example 2 Synthesis of Compound III-1 [ka] (1) Synthesis of Compound 9: Dodecanol (1.0 eq) and TEA (2.0 eq) were added to a single-neck flask and dissolved in an appropriate amount of anhydrous DCM. The mixture was stirred uniformly in an ice-water bath. Bromoacetyl bromide (1.2 eq) was dissolved in an appropriate amount of anhydrous DCM and added to a constant-pressure dropping funnel. The flow rate was controlled and the mixture was added dropwise to the single-neck flask. The reaction mixture was allowed to react in an ice-water bath for 6 h. The reaction solvent was evaporated, and the crude product was purified by silica gel column chromatography (PE:DCM = 1:1) and concentrated to dryness to give a colorless liquid, 9, in 86.1% yield. (2) Synthesis of compound 11: N,N'-Dimethylethylenediamine (1.0 eq) and TEA (3.0 eq) were added to a single-neck flask and dissolved in an appropriate amount of anhydrous DCM. The mixture was stirred uniformly in an ice-water bath. Acryloyl chloride (2.5 eq) was dissolved in an appropriate amount of anhydrous DCM and added to a constant-pressure dropping funnel. The flow rate was controlled and the mixture was added dropwise to the single-neck flask. The reaction mixture was then allowed to react in an ice-water bath for 6 h. The reaction solvent was then evaporated, and the crude product was purified by silica gel column chromatography (DCM:MeOH = 60:1) and concentrated to dryness to give a white solid, 11, in 92.5% yield. (3) Synthesis of compound 13: Compound 11 (1.0 eq) and tert-butyl 2-(methylamino)ethylcarbamate (2.0 eq) were added to a single-necked flask and dissolved in an appropriate amount of absolute ethanol. The flask was equipped with a condenser and refluxed in an oil bath at 80 °C for 12 h. The reaction solvent was evaporated, and the crude product was purified by silica gel column chromatography (DCM:MeOH = 20:1, 0.5% aqueous ammonia), concentrated, and dried to give yellow semi-solid 13 in 89.1% yield. (4) Synthesis of III-1: Compound 13 was dissolved in DCM, and a sufficient amount of TFA was added with stirring. The mixture was allowed to react at room temperature for 6 hours. The TFA / DCM was then rotary evaporated to give a yellow oil. The oil was dissolved in an appropriate amount of acetonitrile, and a sufficient amount of anhydrous potassium carbonate was added with stirring. The mixture was stirred at room temperature until the reaction mixture became alkaline. Compound 9 (6.0 eq) was added to the reaction mixture, and the reaction mixture was refluxed in a 90 °C oil bath for 36 hours with a condenser attached. The reaction mixture was then rotary evaporated, and the crude product was purified by silica gel column chromatography (DCM:MeOH = 20:1, 0.5% aqueous ammonia) and concentrated to dryness to give yellow oil III-1 in a 70.8% yield.

[0099] Example 3 Synthesis of Compound III-2 [ka] Compound 13 was dissolved in DCM, and a sufficient amount of TFA was added with stirring. The reaction was allowed to proceed at room temperature for 6 hours. The TFA / DCM solution was then rotary evaporated to give a yellow oil. The oil was dissolved in an appropriate amount of isopropanol, and a sufficient amount of anhydrous potassium carbonate was added with stirring. The mixture was stirred at room temperature until the reaction mixture became alkaline. 1,2-Epoxytetradecane (6.0 eq) was added to the reaction mixture, and the reaction mixture was refluxed in a 90 °C oil bath for 36 hours with a condenser attached. The reaction mixture was then rotary evaporated, and the crude product was purified by silica gel column chromatography (DCM:MeOH = 15:1, 0.5% aqueous ammonia) and concentrated to dryness to give yellow oil III-2 in 75.1% yield.

[0100] Example 4 Synthesis of Compound III-3 [ka] (1) Synthesis of compound 16: Ethylene glycol (1.0 eq) and TEA (3.0 eq) were added to a single-neck flask and dissolved in an appropriate amount of anhydrous DCM. The mixture was stirred uniformly in an ice-water bath. Acryloyl chloride (2.5 eq) was dissolved in an appropriate amount of anhydrous DCM and added to a constant-pressure dropping funnel. The flow rate was controlled and the mixture was added dropwise to the single-neck flask. The reaction mixture was then allowed to react in an ice-water bath for 6 h. The reaction solvent was then evaporated, and the crude product was purified by silica gel column chromatography (DCM:MeOH = 80:1) and concentrated to dryness to give a colorless liquid, 16, in an 87.5% yield. (2) Synthesis of compound 17: Compound 16 (1.0 eq) and tert-butyl 2-(methylamino)ethylcarbamate (2.0 eq) were added to a single-necked flask and dissolved in an appropriate amount of absolute ethanol. The flask was equipped with a condenser and refluxed in an oil bath at 80 °C for 12 h. The reaction solvent was evaporated, and the crude product was purified by silica gel column chromatography (DCM:MeOH = 25:1, 0.5% aqueous ammonia) and concentrated to dryness to give pale yellow oil 17 in 84.4% yield. (3) Synthesis of III-3: Compound 17 was dissolved in DCM, and an appropriate amount of TFA was added with stirring. The mixture was allowed to react at room temperature for 6 hours. The TFA / DCM mixture was then rotary evaporated to give a yellow oil. The oil was dissolved in an appropriate amount of isopropanol, and a sufficient amount of anhydrous potassium carbonate was added with stirring. The mixture was stirred at room temperature until the reaction mixture became alkaline. 1,2-Epoxytetradecane (6.0 eq) was added to the reaction mixture, and the reaction mixture was refluxed in a 90 °C oil bath for 36 hours with a condenser attached. The reaction mixture was then rotary evaporated, and the crude product was purified by silica gel column chromatography (DCM:MeOH = 25:1, 0.5% aqueous ammonia) and concentrated to dryness to give a pale yellow oil, III-3, in 67.8% yield.

[0101] Example 5 Synthesis of Compound VI-1 [ka] (1) Synthesis of Compound 19: N,N'-Dimethylethylenediamine (1.0 eq) and N-Boc-3-aminopropyl bromide (2.5 eq) were added to a single-necked flask and dissolved in an appropriate amount of acetonitrile. The flask was equipped with a condenser and stirred under reflux in an oil bath at 90°C for 12 h. The reaction solvent was evaporated, and the crude product was purified by silica gel column chromatography (DCM:MeOH = 25:1, 0.5% aqueous ammonia) and concentrated to dryness to give pale yellow oil 17 in 78.8% yield. (2) Synthesis of VI-1: Compound 19 was dissolved in DCM, and an appropriate amount of TFA was added with stirring. The mixture was allowed to react at room temperature for 6 hours. The TFA / DCM mixture was then rotary evaporated to give a yellow oil. The oil was dissolved in an appropriate amount of isopropanol, and a sufficient amount of anhydrous potassium carbonate was added with stirring. The mixture was stirred at room temperature until the reaction mixture became alkaline. 1,2-Epoxytetradecane (6.0 eq) was added to the reaction mixture, and the reaction mixture was refluxed in a 90 °C oil bath for 36 hours with a condenser attached. The reaction mixture was then rotary evaporated, and the crude product was purified by silica gel column chromatography (DCM:MeOH = 15:1, 0.5% aqueous ammonia) and concentrated to dryness to give yellow oil VI-1 in 70.8% yield.

[0102] Example 6 Synthesis of Compound II-7 [ka] (1) Synthesis of compound 20: A single-neck flask was charged with tert-butyl 2-(methylamino)ethylcarbamate (1.0 eq) and TEA (3.0 eq), dissolved in an appropriate amount of anhydrous DCM, and stirred uniformly in an ice-water bath. Acryloyl chloride (1.2 eq) was dissolved in an appropriate amount of anhydrous DCM and added to a constant-pressure dropping funnel. The flow rate was controlled and the mixture was added dropwise to the single-neck flask. The reaction mixture was then allowed to react in an ice-water bath for 6 h. The reaction solvent was then spun down, and the crude product was purified by silica gel column chromatography (DCM:MeOH = 60:1) and concentrated to dryness to give a white semi-solid 20 in 89.5% yield. (2) Synthesis of compound 21: N,N'-dimethylethylenediamine (1.0 eq) and compound 20 (2.0 eq) were added to a single-necked flask and dissolved in an appropriate amount of absolute ethanol. The flask was equipped with a condenser and refluxed in an oil bath at 80 °C for 12 h. The reaction solvent was evaporated, and the crude product was purified by silica gel column chromatography (DCM:MeOH = 20:1, 0.5% aqueous ammonia) and concentrated to dryness to give yellow oil 21 in 84.0% yield. (3) Synthesis of II-7: Compound 21 was dissolved in DCM, and an appropriate amount of TFA was added with stirring. The mixture was allowed to react at room temperature for 6 hours. The TFA / DCM mixture was then rotary evaporated to give a yellow oil. The oil was dissolved in an appropriate amount of isopropanol, and a sufficient amount of anhydrous potassium carbonate was added with stirring. The mixture was stirred at room temperature until the reaction mixture became alkaline. 1,2-Epoxytetradecane (6.0 eq) was added to the reaction mixture, and the reaction mixture was refluxed in a 90 °C oil bath for 36 hours with a condenser attached. The reaction mixture was then rotary evaporated, and the crude product was purified by silica gel column chromatography (DCM:MeOH = 20:1, 0.5% aqueous ammonia) and concentrated to dryness to give yellow oil II-7 in 69.1% yield.

[0103] Example 7 Synthesis of Compound VI-2 [ka] (1) Synthesis of compound 23: Undecanol (1.0 eq) and TEA (2.0 eq) were added to a single-neck flask and dissolved in an appropriate amount of anhydrous DCM. The mixture was stirred uniformly in an ice-water bath. Acryloyl chloride (1.2 eq) was dissolved in an appropriate amount of anhydrous DCM and added to a constant-pressure dropping funnel. The flow rate was controlled and the mixture was added dropwise to the single-neck flask. The reaction mixture was allowed to react in an ice-water bath for 6 h. The reaction solvent was evaporated, and the crude product was purified by silica gel column chromatography (PE:EA = 2:1) and concentrated to dryness to give 23, a colorless liquid, in 90.0% yield. (2) Synthesis of VI-2: Compound 19 was dissolved in DCM, and an appropriate amount of TFA was added with stirring. The mixture was allowed to react at room temperature for 6 hours. The TFA / DCM was then rotary evaporated to give a yellow oil. The oil was dissolved in an appropriate amount of isopropanol, and a sufficient amount of anhydrous potassium carbonate was added with stirring. The mixture was stirred at room temperature until the reaction mixture became alkaline. Compound 23 (6.0 eq) was added to the reaction mixture, and the reaction mixture was refluxed in a 90 °C oil bath for 36 hours with a condenser attached. The reaction mixture was then rotary evaporated, and the crude product was purified by silica gel column chromatography (DCM:MeOH = 25:1, 0.5% aqueous ammonia) and concentrated to dryness to give pale yellow oil VI-2 in 74.1% yield.

[0104] Example 8 Synthesis of Compound VI-3 [ka] (1) Synthesis of compound 24: N,N'-bis(2-hydroxyethyl)ethylenediamine (1.0 eq) and N-Boc-3-aminopropyl bromide (2.5 eq) were added to a single-necked flask and dissolved in an appropriate amount of acetonitrile. The flask was equipped with a condenser and stirred under reflux in an oil bath at 90 °C for 12 h. The reaction solvent was evaporated, and the crude product was purified by silica gel column chromatography (DCM:MeOH = 20:1, 0.5% aqueous ammonia) and concentrated to dryness to give 24 as a pale yellow oil in 71.2% yield. (2) Synthesis of VI-3: Compound 24 was dissolved in DCM, and an appropriate amount of TFA was added with stirring. The mixture was allowed to react at room temperature for 6 hours. The TFA / DCM was then rotary evaporated to give a yellow oil. The oil was dissolved in an appropriate amount of isopropanol, and a sufficient amount of anhydrous potassium carbonate was added with stirring. The mixture was stirred at room temperature until the reaction mixture became alkaline. Compound 23 (6.0 eq) was added to the reaction mixture, and the reaction mixture was refluxed in a 90 °C oil bath for 36 hours with a condenser attached. The reaction mixture was then rotary evaporated, and the crude product was purified by silica gel column chromatography (DCM:MeOH = 15:1, 0.5% aqueous ammonia) and concentrated to dryness to give pale yellow oil VI-3 in 69.5% yield.

[0105] Example 9 Synthesis of Compound II-11 [ka] (1) Synthesis of compound 26: N-Boc-ethanolamine (1.0 eq) and TEA (2.0 eq) were added to a single-neck flask and dissolved in an appropriate amount of anhydrous DCM. The mixture was stirred uniformly in an ice-water bath. Acryloyl chloride (1.2 eq) was dissolved in an appropriate amount of anhydrous DCM and added to a constant-pressure dropping funnel. The flow rate was controlled and the mixture was added dropwise to the single-neck flask. The reaction mixture was then allowed to react in an ice-water bath for 6 h. The reaction solvent was then evaporated, and the crude product was purified by silica gel column chromatography (DCM:MeOH = 70:1) and concentrated to dryness to give a white semi-solid 26 in 85.4% yield. (2) Synthesis of compound 27: N,N'-dimethylethylenediamine (1.0 eq) and compound 26 (2.0 eq) were added to a single-necked flask and dissolved in an appropriate amount of absolute ethanol. The flask was equipped with a condenser and refluxed in an oil bath at 80 °C for 12 h with stirring. The reaction solvent was evaporated, and the crude product was purified by silica gel column chromatography (DCM:MeOH = 25:1, 0.5% aqueous ammonia), concentrated, and dried to give pale yellow semi-solid 27 in 82.4% yield. (3) Synthesis of II-11: Compound 27 was dissolved in DCM, and a sufficient amount of TFA was added with stirring. The mixture was allowed to react at room temperature for 6 hours. The TFA / DCM was then rotary evaporated to give a yellow oil. The oil was dissolved in an appropriate amount of acetonitrile, and a sufficient amount of anhydrous potassium carbonate was added with stirring. The mixture was stirred at room temperature until the reaction mixture became alkaline. Compound 9 (6.0 eq) was added to the reaction mixture, and the single-neck flask was equipped with a condenser and stirred under reflux in a 90 °C oil bath for 36 hours. The reaction mixture was then rotary evaporated, and the crude product was purified by silica gel column chromatography (DCM:MeOH = 30:1, 0.5% aqueous ammonia) and concentrated to dryness to give pale yellow oil II-11 in 72.1% yield.

[0106] Example 10 Synthesis of Compound II-13 [ka] (1) Synthesis of compound 28: N,N'-dimethylethylenediamine (1.0 eq) and compound 3 (2.0 eq) were added to a single-necked flask and dissolved in an appropriate amount of absolute ethanol. The flask was equipped with a condenser and stirred under reflux in an oil bath at 80 °C for 12 h. The reaction solvent was evaporated, and the crude product was purified by silica gel column chromatography (DCM:MeOH = 20:1, 0.5% aqueous ammonia), concentrated, and dried to give a pale yellow solid 28 in 85.9% yield. (2) Synthesis of II-13: Compound 28 was dissolved in DCM, and a sufficient amount of TFA was added with stirring. The mixture was allowed to react at room temperature for 6 hours. The TFA / DCM solution was rotary evaporated to give a yellow oil. The oil was dissolved in an appropriate amount of acetonitrile, and a sufficient amount of anhydrous potassium carbonate was added with stirring. The mixture was stirred at room temperature until the reaction mixture became alkaline. 1-Bromotetradecane (6.0 eq) was added to the reaction mixture, and the single-neck flask was equipped with a condenser and stirred under reflux in an oil bath at 90 °C for 36 hours. The reaction mixture was rotary evaporated, and the crude product was purified by silica gel column chromatography (DCM:MeOH = 25:1, 0.5% aqueous ammonia) and concentrated to dryness to give pale yellow semi-solid II-13 in 75.5% yield.

[0107] Example 11 Synthesis of Compound II-22 [ka] (1) Synthesis of Compound 31: Dodecanol (1.0 eq), epichlorohydrin (2.0 eq), sodium hydroxide (2.0 eq), tetrabutylammonium bromide, water, and cyclohexane were added to a single-neck flask to form a white emulsion. The mixture was stirred at room temperature for 4 h. After the reaction was complete, the mixture was filtered under suction, the filter cake was washed with dichloromethane two to three times, the filtrate was dried over anhydrous sodium sulfate, and the solvent was evaporated. The crude product was purified by silica gel column chromatography (PE / EA = 8:1-4:1) to obtain a colorless liquid 31. (2) Synthesis of II-22: Compound 28 was dissolved in DCM, and a sufficient amount of TFA was added with stirring. The mixture was allowed to react at room temperature for 6 hours. The TFA / DCM mixture was then rotary evaporated to give a yellow oil. The oil was dissolved in an appropriate amount of isopropanol, and a sufficient amount of anhydrous potassium carbonate was added with stirring. The mixture was stirred at room temperature until the reaction mixture became alkaline. Compound 31 (6.0 eq) was added to the reaction mixture, and the reaction mixture was refluxed in a 90 °C oil bath for 36 hours with a condenser attached. The reaction mixture was then rotary evaporated, and the crude product was purified by silica gel column chromatography (DCM:MeOH = 25:1, 0.5% aqueous ammonia) and concentrated to dryness to give II-22 as a pale yellow oil in 69.5% yield.

[0108] Example 12 Synthesis of Compound II-5 [ka] Synthesis of II-5: Compound 28 was dissolved in DCM, and a sufficient amount of TFA was added with stirring. The mixture was allowed to react at room temperature for 6 hours. The TFA / DCM was then rotary evaporated to give a yellow oil. The oil was dissolved in an appropriate amount of isopropanol, and a sufficient amount of anhydrous potassium carbonate was added with stirring. The mixture was stirred at room temperature until the reaction mixture became alkaline. Compound 14 (6.0 eq) was added to the reaction mixture, and the reaction mixture was refluxed in a 90 °C oil bath for 36 hours with a condenser attached. The reaction mixture was then rotary evaporated, and the crude product was purified by silica gel column chromatography (DCM:MeOH = 15:1, 0.5% aqueous ammonia) and concentrated to dryness to give pale yellow oil II-5 in 52.0% yield.

[0109] Example 13 Synthesis of Compound II-18 [ka] Synthesis of II-18: Compound 27 was dissolved in DCM, and a sufficient amount of TFA was added with stirring. The mixture was allowed to react at room temperature for 6 h. The TFA / DCM was then rotary evaporated to give a yellow oil. The oil was dissolved in an appropriate amount of isopropanol, and a sufficient amount of anhydrous potassium carbonate was added with stirring. The mixture was stirred at room temperature until the reaction mixture became alkaline. Compound 32 (6.0 eq) was added to the reaction mixture, and the reaction mixture was refluxed in a 90 °C oil bath for 36 h. The reaction mixture was then rotary evaporated, and the crude product was purified by silica gel column chromatography (DCM:MeOH = 15:1, 0.5% aqueous ammonia) and concentrated to dryness to give pale yellow oil II-18 in 61.7% yield.

[0110] Example 14 Synthesis of Compound II-24 [ka] (1) Synthesis of compound 34: Dodecanol (1.0 eq) and TEA (2.0 eq) were added to a single-neck flask and dissolved in an appropriate amount of anhydrous DCM. The mixture was stirred uniformly in an ice-water bath. Acryloyl chloride (1.2 eq) was dissolved in an appropriate amount of anhydrous DCM and added to a constant-pressure dropping funnel. The flow rate was controlled and the mixture was added dropwise to the single-neck flask. The reaction mixture was allowed to react in an ice-water bath for 6 h. The reaction solvent was evaporated, and the crude product was purified by silica gel column chromatography (PE:EA = 2:1) and concentrated to dryness to give a colorless liquid, 34, in a 91.0% yield. (2) Synthesis of II-24: Compound 27 was dissolved in DCM, and an appropriate amount of TFA was added with stirring. The mixture was allowed to react at room temperature for 6 hours. The TFA / DCM was then rotary evaporated to give a yellow oil. The oil was dissolved in an appropriate amount of isopropanol, and a sufficient amount of anhydrous potassium carbonate was added with stirring. The mixture was stirred at room temperature until the reaction mixture became alkaline. Compound 34 (6.0 eq) was added to the reaction mixture, and the reaction mixture was refluxed in a 90 °C oil bath for 36 hours with a condenser attached. The reaction mixture was then rotary evaporated, and the crude product was purified by silica gel column chromatography (DCM:MeOH = 25:1, 0.5% aqueous ammonia) and concentrated to dryness to give II-24 as a pale yellow oil in 70.2% yield.

[0111] Example 15 Synthesis of Compound II-25 [ka] Synthesis of II-25: Compound 5 was dissolved in DCM, and an appropriate amount of TFA was added with stirring. The mixture was allowed to react at room temperature for 6 hours. The TFA / DCM was then rotary evaporated to give a yellow oil. The oil was dissolved in an appropriate amount of acetonitrile, and a sufficient amount of anhydrous potassium carbonate was added with stirring. The mixture was stirred at room temperature until the reaction mixture became alkaline. Compound 29 (6.0 eq) was added to the reaction mixture, and the reaction mixture was refluxed in a 90 °C oil bath for 36 hours with a condenser attached. The reaction mixture was then rotary evaporated, and the crude product was purified by silica gel column chromatography (DCM:MeOH = 20:1, 0.5% aqueous ammonia) and concentrated to dryness to give pale yellow oil II-25 in 65.3% yield.

[0112] Example 16 Synthesis of Compound III-11 [ka] (1) Synthesis of compound 36: Ethylenediamine (1.0 eq) and TEA (3.0 eq) were added to a single-neck flask and dissolved in an appropriate amount of anhydrous DCM. The mixture was stirred uniformly in an ice-water bath. Acryloyl chloride (2.5 eq) was dissolved in an appropriate amount of anhydrous DCM and added to a constant-pressure dropping funnel. The flow rate was controlled and the mixture was added dropwise to the single-neck flask. The reaction mixture was then allowed to react in an ice-water bath for 6 h. The reaction solvent was then spun down, and the crude product was purified by silica gel column chromatography (DCM:MeOH = 80:1) and concentrated to dryness to give a white solid, 36, in 89.6% yield. (2) Synthesis of compound 37: Compound 36 (1.0 eq) and Boc-ethylenediamine (2.5 eq) were added to a single-necked flask and dissolved in an appropriate amount of absolute ethanol. The flask was equipped with a condenser and stirred under reflux in an oil bath at 80 °C for 12 h. The reaction solvent was evaporated, and the crude product was purified by silica gel column chromatography (DCM:MeOH = 15:1, 0.5% aqueous ammonia), concentrated, and dried to give pale yellow oil 17 in 80.4% yield. (3) Synthesis of compound 38: Compound 37 (1.0 eq) and iodoethanol (2.5 eq) were added to a single-neck flask, dissolved in an appropriate amount of anhydrous acetonitrile, and then an appropriate amount of potassium carbonate was added and stirred overnight at room temperature. The reaction solvent was evaporated, and the crude product was purified by silica gel column chromatography (DCM:MeOH = 10:1, 1% aqueous ammonia), concentrated, and dried to give pale yellow oil 38 in 65.4% yield. (4) Synthesis of III-11 Compound 38 was dissolved in DCM, and an appropriate amount of TFA was added with stirring. The mixture was allowed to react at room temperature for 6 hours. The TFA / DCM was then rotary evaporated to give a yellow oil. The oil was dissolved in an appropriate amount of acetonitrile, and a sufficient amount of anhydrous potassium carbonate was added with stirring. The mixture was stirred at room temperature until the reaction mixture became alkaline. Compound 29 (6.0 eq) was added to the reaction mixture, and the reaction mixture was refluxed in a 90 °C oil bath for 36 hours with a condenser attached. The reaction mixture was then rotary evaporated, and the crude product was purified by silica gel column chromatography (DCM:MeOH = 15:1, 1% aqueous ammonia) and concentrated to dryness to give a pale yellow oil, III-11, in a 57.2% yield.

[0113] Example 17 Synthesis of compounds of other examples Other compounds were synthesized using different starting materials with reference to the synthesis procedures of Examples 1-16. [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0114] Example 18 Preparation of lipid nanoparticle solution of II-1 compound by thin film hydration method Film formation: The lipid compound II-1, DOPE, cholesterol, and DMG-PEG2000 prepared in Example 1 were prepared in absolute ethanol at concentrations of 20 mg / mL, 10 mg / mL, 20 mg / mL, and 10 mg / mL, respectively, and refrigerated for use. Using the above-mentioned stock solution, the four materials were prepared into approximately 3 mL of solution at a ratio of 40:10:47.5:2.5 (mol / mol), and the concentration of lipid compound II-1 was approximately 5 mg / mL. The solution was placed in an appropriate size eggplant-shaped flask, and the solvent was removed by rotary evaporation at 37 ° C. in a rotary evaporator to form a film. Hydration: 3 mL of 10 mM citrate buffer solution was added to the membrane-formed eggplant-shaped flask and rotated on a rotary evaporator at 60 °C to hydrate the membrane. The hydrated solution was transferred to an appropriate container and homogenized by sonication using a probe sonicator to obtain a homogenous and transparent lipid nanoparticle (LNP) solution with a lipid compound II-1 concentration of 3 mg / mL.

[0115] Example 19 II-1 Preparation of LNP@mRNA The basic structural units of nucleic acid molecules such as DNA, siRNA, and mRNA are deoxynucleotides or ribonucleotides, and the phosphate groups in the nucleotides dissociate into phosphate ions, making the nucleic acid molecules negatively charged. In Example 18, in the citrate buffer system of the LNP solution, the ionizable lipid II-1 in the lipid nanoparticles ionized into cations in an acidic environment, making the nanoparticles electropositive, i.e., enabling the adsorption of negatively charged nucleic acid drugs through electrostatic interaction. Using II-1 LNP and Luciferase mRNA as an example, LNP@mRNA was prepared using the following specific method. II-1 LNP (II-1 concentration: 3 mg / mL) in Example 18 was prepared by incubation to obtain II-1 LNP@mRNA. To prepare LNP@mRNA with a mass ratio of ionizable lipid to mRNA of 10:1, 33 μL of II-1 LNP was labeled as A (II-1 content: 100 μg), 10 μg of Luc mRNA was added to RNase-free water, and mixed uniformly to obtain B (total volume: 67 μL). B was added to A, and the mixture was mixed uniformly by pipetting up and down with the tip of a pipette. The mixture was then incubated at room temperature for 10 minutes to obtain an LNP@mRNA solution with a weight ratio of ionizable lipid to mRNA of 10:1, and the mRNA concentration was 0.1 mg / mL. Similarly, when II-1 LNP@mRNA with a mass ratio of 15:1 was prepared, the volume of phase A was 50 μL (II-1 content 100 μg) and the volume of phase B was 50 μL. This method was also applied to LNP@mRNA with other different mass ratios. The above experimental method can be scaled up at the same ratio to prepare a larger volume of LNP@mRNA solution.

[0116] Example 20 II-1 Examination of nucleic acid carrying capacity of LNP In Example 19, II-1 LNP@mRNA was prepared, and the loading capacity of different ionizable lipid nanoparticles for nucleic acid molecules was investigated, i.e., the ratio of ionizable lipid to nucleic acid molecules was investigated. Using II-1 LNP and mRNA as an example, the loading capacity of ionizable lipid nanoparticles for nucleic acid molecules was investigated. (1) Preparation of denatured agarose gel 36 mL of RNase-free water and 0.4 g of agarose were placed in an Erlenmeyer flask and heated in a microwave oven for 2 minutes. After cooling to approximately 60°C, 4 mL of 10x MOPS was added and mixed evenly. 7.5 mL of 37% formaldehyde was added and mixed evenly. The mixture was poured into a gel tank, the thickness of which was controlled to approximately 0.5 cm. A comb was inserted and removed after solidification. The gel was then placed in an electrophoresis tank, and freshly prepared 1x MOPS electrophoresis buffer was added to the electrophoresis tank to allow the gel to flow over the tank. (2) Preparation of electrophoresis samples 0.5 μL of mRNA (0.5 μg) was homogeneously mixed with 4.5 μL of RNase-free water, or 5.0 μL of II-1 LNP@mRNA (0.5 μg of mRNA) prepared in Example 19 at different mass ratios was added to 5 μL of formaldehyde loading buffer containing ethidium bromide, heated at 70°C for 5 min, and then immediately centrifuged at 4°C to obtain the LNPs. (3) Gel electrophoresis The sample was added to the gel well with a loading volume of 10 μL, and the electrophoresis conditions were set to 200 V (current 300 mA, power 60 W). When the indicator tip reached 2 / 3 of the way through the gel (approximately 25 min), the electrophoresis was stopped, the gel was removed, and the gel was placed in a gel imager for observation. The results are shown in Figure 1. Figure 1 shows that in the II-1 LNP@mRNA solution, mRNA can be completely encapsulated when the mass ratio of ionizable lipid II-1 to Luc mRNA is 10:1.

[0117] Example 21 II-1 Pharmaceutical Characterization of LNP@mRNA An appropriate amount of the LNP solution prepared in Example 19 was diluted 100 times with purified water, and the particle size, particle size distribution (PDI), and zeta potential of the LNP solution were measured using a laser particle size analyzer. The results showed that the particle size of II-1LNP@mRNA was 102.3 nm, PDI was 0.195, and potential was 31.2 mV, indicating that the formulation properties were stable. The particle size results and the zeta potential test results are shown in FIG.

[0118] Example 22 Morphological Characterization of II-1 LNP@mRNA Sample preparation: The LNP solution prepared in Example 19 was diluted with purified water to a total lipid concentration of approximately 1 mg / mL, dropped onto a dedicated copper grid, and left to stand for 3 minutes. After that, excess LNP solution was aspirated using filter paper, and 2% phosphotungstic acid staining solution was added to perform negative staining for 5 minutes. After that, excess staining solution was aspirated using filter paper, and the grid was left to dry naturally. Photography: After air drying, the morphology of II-1 LNPs was observed and photographed under a transmission electron microscope. According to the methods described in Examples 18 and 19, II-5 LNP@mRNA, III-5 LNP@mRNA, and VI-1 LNP@mRNA were prepared, and the mass ratio of ionizable lipid to mRNA was all 10:1. According to the above methods, the samples were prepared and photographed. The microscopic morphology of II-1 LNP@mRNA, II-5 LNP@mRNA, III-5 LNP@mRNA, and VI-1 LNP@mRNA is shown in Figure 3.

[0119] Example 23 Preparation of mRNA-loaded ionizable lipid nanoparticle solutions by a microfluidic one-step method Using II-7 LNP@mRNA as an example, we prepared LNP@mRNA using microfluidic technology. The formulation consisted of II-7, DOPE, Chol, DMG-PEG2000, and mRNA. The organic and aqueous phases were prepared at a 10:1 mass ratio of II-7 to mRNA. II-7, DOPE, Chol, and DMG-PEG2000 were dissolved in absolute ethanol to form a fixed volume organic phase at a molar ratio of 40:10:47.5:2.5. Simultaneously, Luc mRNA was added to a fixed volume aqueous phase using RNase-free water. The volume ratio of the aqueous phase to the organic phase was 3:1. The mRNA-loaded II-7 LNP formulation was prepared in one step using a microfluidic nanoformulation system. The system parameters were as follows: the aqueous-to-organic phase ratio was fixed at 3:1, and the flow rate was fixed at 9 mL / min. The microfluidic initial formulation was ultrafiltered with phosphate-buffered saline (PBS) to remove ethanol, and the mRNA concentration in the final formulation was controlled to 0.1 mg / mL to obtain II-7 LNP@mRNA.

[0120] Example 24 Quant-iT TM RiboGreen TM Kit detection of encapsulation efficiency Quant-iT TM RiboGreen TM An RNA detection kit was used to measure the mRNA encapsulation rates of the LNP formulations of Examples 22 and 23. The results showed that the mRNA encapsulation rates in the II-1 LNP@mRNA, II-5 LNP@mRNA, III-5 LNP@mRNA, VI-1 LNP@mRNA, and II-7 LNP@mRNA formulations were 86.1%, 82.8%, 85.0%, 78.6%, and 90.5%, respectively. This demonstrates that the ionizable lipids provided by the present invention have good encapsulation rates for mRNA in different formulation methods.

[0121] Example 25 Preparation of MC3 LNP@mRNA DLin-MC3-DMA (MC3) is a cationic lipid used in the commercially available siRNA drug Patisiran (Onpattro) and is often used as a positive control for nucleic acid-loading materials. According to the method of Example 23, MC3 LNP@mRNA was prepared using MC3 as a nucleic acid-loading material. The formulation was MC3:DSPC:Chol:DMG-PEG2000 = 50:10:37.5:2.5, with a mass ratio of MC3 to mRNA of 10:1 and a concentration of mRNA in the formulation of 0.1 mg / mL. MC3 LNP@mRNA was used as a positive control for nucleic acid-loaded ionizable lipid nanoparticles in the present invention.

[0122] Example 26 Transfection of LNP@GFP mRNA into DC2.4 cells The above examples verified the formulation properties of the ionizable lipid nanoparticles provided by the present invention, and further verified the in vitro effects of the LNP@mRNA provided by the present invention. II-13 LNP@GFP mRNA, III-9 LNP@GFP mRNA, and IV-4 LNP@GFP mRNA were prepared according to the method described in Example 23. The mass ratio of ionizable lipid to GFP mRNA was all 10:1, and the mRNA concentration in the formulation was all 0.1 mg / mL. MC3 LNP@GFP mRNA was prepared according to Example 25. DC2.4 cells in logarithmic growth phase were harvested, resuspended in medium, and counted to a cell density of 20 x 10 4 To each well of a 6-well plate, 0.5 mL of complete medium was added, followed by 0.5 mL of the cell suspension, to adjust the cell density to 10 × 10 4 The solution was diluted to 1 mL / well, mixed evenly, and cultured in an incubator for 18 to 24 hours before use. After overnight incubation, 1 mL of the medium was replaced with complete medium. MC3 LNP@GFP mRNA, II-13 LNP@GFP mRNA, III-9 LNP@GFP mRNA, and IV-4 LNP@GFP mRNA containing 1 μg of GFP mRNA were added to each well of a 6-well plate. Each formulation was added in triplicate. 24 h after administration, the transfection effects of the different formulations were observed and detected using an inverted fluorescence microscope and a flow cytometer. The results are shown in Figure 4. The average GFP positivity rates of DC2.4 cells in the MC3 LNP@GFP mRNA group, II-13 LNP@GFP mRNA group, III-9 LNP@GFP mRNA group, and IV-4 LNP@GFP mRNA group were 39.15%, 80.24%, 67.95%, and 90.33%, respectively. The results showed that the ionizable lipids and nanoparticles thereof provided by the present invention have greater transfection ability to cells than MC3.

[0123] Example 27 In vivo expression and distribution of LNP@Luc mRNA The ability of the ionizable lipid compounds and nanoparticles provided by the present invention to deliver mRNA was further validated in vivo.

[0124] II-11 LNP@Luc mRNA, III-6 LNP@Luc mRNA, and V-2 LNP@Luc mRNA were prepared according to the method described in Example 23. The mass ratio of ionizable lipid to Luc mRNA was all 10:1, and the mRNA concentration in the formulation was all 0.1 mg / mL. MC3 LNP@Luc mRNA was prepared according to Example 25. mRNA expression and distribution experiments were performed using Balb / C male mice. The four formulations were administered via the tail vein at 10 μg / 100 μL per mouse (100 μL of saline was administered to the saline group), with three mice per group allowed to eat and drink ad libitum. Six hours after administration, 200 μL of luciferin substrate was injected intraperitoneally. As shown in Figure 5, 15 minutes after luciferin substrate injection, the mice were euthanized, and organs such as the liver, spleen, and lungs were isolated. The in vivo expression and distribution of Luc mRNA were monitored using a small animal bioimaging system. The results showed that the ionizable lipid nanoparticles provided by the present invention have a stronger total fluorescence than MC3, i.e., a stronger ability to express mRNA in vivo than MC3.

[0125] Example 28: Antitumor immune effect of LNP@OVA mRNA vaccine Based on the results of Examples 26 and 27, the ionic lipids and nanoparticles thereof provided by the present invention can be used as a delivery system for mRNA vaccines. Furthermore, OVA mRNA loaded on the ionizable lipid nanoparticles provided by the present invention can be used for immune anti-tumor therapy in E.G7 model mice. LNP@OVA mRNA (II-9), LNP@OVA mRNA (III-8), LNP@OVA mRNA (II-22), and LNP@OVA mRNA (VI-4) were prepared according to the method described in Example 23. The mass ratio of ionizable lipid to OVA mRNA was 10:1, and the mRNA concentration in the formulation was 0.1 mg / mL. MC3 LNP@OVA mRNA was prepared according to Example 25. Newly purchased female C57BL / 6 mice were labeled and randomly divided into groups (10 mice per group): saline group, MC3 LNP@OVA mRNA group, II-9 LNP@OVA mRNA group, III-8 LNP@OVA mRNA group, II-22 LNP@OVA mRNA group, and VI-4 LNP@OVA mRNA group. After one week of feeding, the mice were inoculated with tumors. The tumor inoculation process was as follows: When E.G7 cells were in the logarithmic growth phase, the cells were harvested, washed with sterile PBS, centrifuged to remove the supernatant, and resuspended in sterile PBS to a cell density of 10 × 10. 6 The concentration of E.G7 cells was adjusted to 1 / mL. Approximately 6-week-old female C57BL / 6 mice were subcutaneously inoculated with 100 μg of E.G7 cells into the right flank. The mice's growth and subcutaneous tumor size were observed. Seven days after inoculation, clear tumor nodules formed subcutaneously in the mice. There were no significant differences in mental status, activity, appetite, or bowel movements between tumor-bearing mice and control mice. The day of tumor inoculation was recorded as Day 0. The appearance of a tumor mass indicated successful modeling. On Day 8, 10 μg of mRNA vaccine was injected into the tail vein of each mouse (100 μL of saline was injected into the saline group). Subsequently, the mice were immunized once every other week for a total of three doses. From Day 8 onward, tumor volume was measured every two days. First, the maximum diameter of the tumor (a) was measured, and then the longest diameter line perpendicular to the maximum diameter line (b) was measured. The unit is mm, and the formula V (mm 3 )=ab 2 Tumor volume was calculated according to the 2000 stanford standard deviation (SDD) method. Growth curves of individual tumors and the average tumor volume of each group were recorded. On day 24, the mice were euthanized, and tumors and organs were isolated, weighed, and recorded. The results are shown in Figure 6. The results showed that the ionizable lipids and nanoparticles thereof provided by the present invention have stronger immune anti-tumor ability than MC3, and have good application prospects in the field of mRNA vaccine delivery.

[0126] Example 29 Safety Evaluation of Intramuscularly Injected LNP@mRNA Furthermore, the injection site reactions of ionizable lipids and their nanoparticles upon intramuscular administration were investigated. II-5 LNP@OVA mRNA and VI-2 LNP@OVA mRNA were prepared according to the method described in Example 23. The mass ratio of ionizable lipid to OVA mRNA was 10:1 in all formulations, and the mRNA concentration in all formulations was 0.5 mg / mL. MC3 LNP@OVA mRNA was prepared according to Example 25. Newly purchased SD rats were randomly divided into four groups (three rats per group): saline, III-3 LNP@OVA mRNA, VI-2 LNP@OVA mRNA, and MC3 LNP@OVA mRNA. After one week of adaptation incubation, the rats' hind paws were shaved and 500 μL of a 0.5 mg / mL mRNA formulation was intramuscularly injected into each side (i.e., a dose of 0.5 mg per rat). The saline group received 500 μL of saline intramuscularly on both sides, with the administration day recorded as D0. A second administration of the same dose was administered on D8. After the first administration, changes at the injection site were observed every other day and recorded. The results are shown in Figure 7. The results showed that the MC3 group had severe inflammatory reactions at the injection site, while the III-3, VI-2, and saline groups had comparable results, with essentially no inflammatory reactions. This indicates that the ionizable lipids and nanoparticles thereof provided by the present invention are safer than MC3 and have a good prospect for clinical application.

[0127] Example 30 Preparation of Luc mRNA-loaded LNP solution by microfluidic method Using II-5 LNP@Luc mRNA as an example, we prepared LNP@mRNA using microfluidic technology. The formulation consisted of II-5, DSPC, Chol, DMG-PEG2000, and mRNA. The organic and aqueous phases were prepared at a 10:1 mass ratio of II-5 to mRNA. II-5, DSPC, Chol, and DMG-PEG2000 were dissolved in absolute ethanol and prepared into a fixed volume organic phase at a molar ratio of 50:10:38.5:1.5. Simultaneously, Luc mRNA was prepared in a fixed volume aqueous phase using RNase-free water. The aqueous-to-organic volume ratio was 3:1. The mRNA-loaded II-7 LNP formulation was prepared in one step using a microfluidic nanoformulation system. The system parameters were as follows: the aqueous-to-organic phase ratio was fixed at 3:1, and the flow rate was fixed at 9 mL / min. The microfluidic initial formulation was ultrafiltered with phosphate-buffered saline (PBS) to remove ethanol, and the mRNA concentration in the final formulation was controlled to 0.1 mg / mL to obtain II-5 LNP@mRNA.

[0128] Example 31 Luciferase Expression of Intramuscularly Injected LNP@Luc mRNA The ability of the ionizable lipid compounds and nanoparticles provided by the present invention to deliver mRNA was further validated in vivo. II-5 LNP@Luc mRNA, II-18 LNP@Luc mRNA, II-24 LNP@Luc mRNA, II-25 LNP@Luc mRNA, III-3 LNP@Luc mRNA, and III-11 LNP@Luc mRNA were prepared according to the method described in Example 30. The mass ratio of ionizable lipid to Luc mRNA was all 15:1, and the mRNA concentration in the formulation was all 0.1 mg / mL. MC3 LNP@Luc mRNA was prepared according to Example 25. mRNA expression and distribution were examined using BALB / C male mice. The four formulations were intramuscularly injected at 20 μg / 100 μL per mouse (100 μL of saline was injected in the saline group), with three mice per group. After the injection, the mice were allowed to eat and drink ad libitum. Eight hours after administration, 3 mg of luciferin substrate was intraperitoneally injected. As shown in Figure 8, 15 minutes after injection of the luciferin substrate, in vivo expression of Luc mRNA was monitored using a small animal bioimaging system. The results indicated that the ionizable lipid nanoparticles provided by the present invention had stronger total fluorescence than MC3, i.e., had a stronger ability to express mRNA in vivo than MC3.

[0129] Example 32 Quant-iT TM RiboGreen TM Detection of inclusion rate by kit Quant-iT TM RiboGreen TMAn RNA detection kit was used to measure the mRNA encapsulation rate of the LNP formulation of Example 31. The results showed that the mRNA encapsulation rates in the II-5 LNP@Luc mRNA, II-18 LNP@Luc mRNA, II-24 LNP@Luc mRNA, II-25 LNP@Luc mRNA, III-3 LNP@Luc mRNA, and III-11 LNP@Luc mRNA formulations were 88.5%, 89.4%, 85.7%, 90.5%, 88.0%, and 87.9%, respectively. This demonstrates that the LNPs prepared with the ionizable lipids provided by the present invention have good encapsulation rates for mRNA.

[0130] Example 33 Preparation of LNP@SmRNA by microfluidic method Furthermore, the ionizable lipids provided by the present invention were applied to a novel coronavirus mRNA vaccine, and mRNA encoding the S protein of ARS-CoV-2 was designed. II-18 LNP@S mRNA, II-24 LNP@S mRNA, II-25 LNP@S mRNA, III-3 LNP@S mRNA, and III-11 LNP@S mRNA were prepared according to the method described in Example 30. The mass ratio of ionizable lipid to Luc mRNA was 15:1 in all formulations, and the mRNA concentration in all formulations was 0.1 mg / mL.

[0131] Example 34 SARS-CoV-2 mRNA Vaccine Immunization Protocol (1) Mouse grouping and administration: Male BALB / c mice were randomly divided into groups according to the formulation. At each immunization, mice received a 100 μL tail vein injection, i.e., 10 μg S mRNA per mouse. PBS was used as a negative control. The formulation groups were II-5 LNP@S mRNA, II-18 LNP@S mRNA, II-24 LNP@S mRNA, II-25 LNP@S mRNA, III-3 LNP@S mRNA, and III-11 LNP@S mRNA. (2) Second immunization and sample collection: The first immunization was recorded as Day 0, and two weeks later, blood was collected for the second immunization, and then blood was collected every two weeks.

[0132] Example 35 Detection of specific antibody titers by enzyme-linked immunosorbent assay (1) Antigen coating: RBD protein (Delta, WT, or Omicron) was prepared as an antigen protein solution in 1x coating working solution, and 100 μL was added to each well. Avoiding the presence of air bubbles, the plate was left overnight in a refrigerator at 4°C. (2) BSA blocking: After overnight antigen coating, the liquid in the coated ELISA plate was shaken off, and 300 μL of 1x washing solution was added to each well. The liquid in the ELISA plate was shaken off and the plate was washed four times. BSA was prepared as a blocking solution using the 1x washing solution. After washing the plate was complete, 100 μL of blocking solution was added to each well and incubated at 25°C for 4 hours. (3) Serum dilution and sample addition: Orbital blood from immunized mice was centrifuged, and the serum was aspirated and inactivated by incubation at 60°C for 30 minutes. Starting with a 100x dilution, serial 2-fold dilutions were made using antibody diluent. After blocking, the plate was washed four times. 100µL of serum sample dilution was added to each well of the washed ELISA plate. The plate was then incubated overnight in a 4°C refrigerator. (4) Antibody incubation After overnight incubation with serum, the plate was washed four times. The HRP-labeled antibody was diluted to the corresponding dilution ratio using antibody diluent, and 100 μL of antibody diluent was added to each well and incubated at 25°C for 2 hours. (5) Color development and detection: After antibody incubation, the plate was washed four times. 100 μL of color development solution was added to each well and incubated at 25°C in the dark for 30 minutes. 100 μL of sulfuric acid stop solution was added to each well. Immediately after adding the stop solution, absorbance values ​​at 450 nm and 630 nm were read using a microplate reader. The data were analyzed, and the dilution endpoint was determined based on the difference in absorbance values ​​from the control group. The endpoint dilution factor, i.e., titer, for the immunized group was determined. Figure 9 shows the RBD-specific IgG titers for the II-5 LNP@S mRNA, II-18 LNP@S mRNA, II-24 LNP@S mRNA, II-25 LNP@S mRNA, III-3 LNP@S mRNA, and III-11 LNP@S mRNA groups. The results demonstrate that the ionizable lipids provided by the present invention can induce a strong humoral immune response when used in a novel coronavirus SARS-CoV-2 mRNA vaccine.

[0133] Example 36 Safety evaluation of LNP@S mRNA vaccine (1) Preparation and administration of the formulation: II-5 LNP@S mRNA, II-18 LNP@S mRNA, II-24 LNP@S mRNA, II-25 LNP@S mRNA, III-3 LNP@S mRNA, and III-11 LNP@S mRNA formulations were prepared according to Example 33. The mRNA concentration of the formulations was adjusted to 0.1 mg / mL, and 200 μL, i.e., 20 μg of S mRNA per mouse, was intramuscularly injected into blank BALB / c mice, with an equal volume of PBS used as a control. (2) Blood collection and detection: 24 hours after administration, orbital blood was collected from the mice, centrifuged, and serum was aspirated. The main biochemical indicators, such as ALT and CRE, were detected using a biochemical analyzer. As shown in Figures 10 to 12, the results showed that the ionizable lipids provided by the present invention have good safety when used with SARS-CoV-2 mRNA.

[0134] It should be noted that particular features, structures, materials, or characteristics described herein may be combined in any suitable manner in any one or more examples. Furthermore, those skilled in the art may combine features of different examples and embodiments described herein without conflict.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof: 【Chemical 1】 Formula (I) (In the formula, m1, m2, m3, and m4 are each independently selected from 1, 2, 3, 4, and 5; L １ and L ２ are the same group and are —CH(OH)—, —C(═O)—, —OC(═O)—, —NHC(═O)—, —O—, —S—, —CH(OH)CH ２ O- or absent; L ３ and L ４ are the same group and are —CH(OH)—, —C(═O)—, —C(═O)O—, —C(═O)NH—, —O—, —S—, —CH(OH)CH ２ O- or absent; G ３ , G ４ are the same group and are -CH ２ CH ２ C(=O)CH ２ -, -CH ２ C(=O)OCH ２ -, -CH ２ -, -CH ２ C(=O)NHCH ２ -, -CH ２ C(=O)N(C ２ H ５ ) CH ２ -, -CH ２ C(=O)N(CH ３ ) CH ２ - or absent, G １ and G ２ are the same group and are -CH ２ C(=O)N(CH ３ ) CH ２ -, -CH ２ C(=O)OCH ２ -, -CH ２ -, -CH ２ C(=O)NHCH ２ -, -CH ２ C(=O)N(C ２ H ５ ) CH ２ -, -CH ２ C(=O)OCH ２ CH ２ -, -CH ２ C(=O)NHCH ２ CH ２ -, or G １ and G ２ Let's get together -CH ２ NHC(=O)OCH ２ - or -CH ２ CH ２ - constitutes or does not exist, G １ , G ２ , G ３ , G ４ may not exist at the same time, G １ , G ２ , G ３ , G ４ When both exist simultaneously, G １ and G ２ Let's get together -CH ２ NHC(=O)OCH ２ - constitutes G ３ and G ４ are the same group and -CH ２ C(=O)OCH ２ -, -CH ２ C(=O)CH ２ CH ２ -, -CH ２ -, -CH ２ C(=O)N(C ２ H ５ ) CH ２ -, or G １ and G ２ Let's get together -CH ２ CH ２ - constitutes G ３ and G ４ are the same group and -CH ２ C(=O)OCH ２ - and G ３ and G ４ If there is no G １ and G ２ are the same group and are -CH ２ C(=O)N(CH ３ ) CH ２ -, -CH ２ C(=O)OCH ２ -, -CH ２ C(=O)NHCH ２ -, -CH ２ C(=O)N(C ２ H ５ ) CH ２ -, -CH ２ C(=O)OCH ２ CH ２ -, -CH ２ C(=O)NHCH ２ CH ２ - is selected from G １ and G ２ If there is no G ３ and G ４ are the same group and are -CH ２ CH ２ C(=O)CH ２ -, -CH ２ C(=O)OCH ２ -, -CH ２ C(=O)N(CH ３ ) CH ２ -, -CH ２ C(=O)N(C ２ H ５ ) CH ２ -, -CH ２ C(=O)NHCH ２ - is selected from R １ , R ２ , R ３ , R ４ are the same group and C ８ -C １８ selected from alkyl or alkenyl; R ５ and R ６ are the same group, -H, -OH, or C optionally substituted with -OH. １ -C ３ alkyl.)

2. In formula (I), L １ , L ２ , L ３ , L ４ are selected from the same group, and each L １ , L ２ , L ３ , L ４ is -CH(OH)-, -C(=O)-, ^-C(=O)O-^^, ^-OC(=O)-^^, ^-NHC(=O)-^^, ^-C(=O)NH-^^, -O-, -S-, -CH(OH)CH ２ O-, ^-OCH ２ CH(OH)-^ or absent, ^- is selected from R １－４ represents a site that can be linked to -^^, and -^^ represents -(CH ２ ) ｍ１－ｍ４ represents a site that can be linked to -, G ３ , G ４ are the same group and *-CH ２ CH ２ C(=O)CH ２ -**, *-CH ２ C(=O)OCH ２ -**, *-CH ２ OC(=O)CH ２ -**, -CH ２ -, *-CH ２ C(=O)NHCH ２ -**, *-CH ２ NHC(=O)CH ２ -**, *-CH ２ C(=O)N(C ２ H ５ ) CH ２ -**, *-CH ２ C(=O)N(CH ３ ) CH ２ -** or absent, *- is a —CH adjacent to the terminal tertiary amine atom in formula (I). ２ - represents the site of connection to the - group, and -** represents the -CH group adjacent to the middle tertiary amino atom in formula (I). ２ represents the site of attachment to the - group, G １ and G ２ are the same group ＃ -CH ２ C(=O)N(CH ３ ) CH ２ - ＃＃ , ＃ -CH ２ C(=O)OCH ２ - ＃＃ , ＃ -CH ２ OC(=O)CH ２ - ＃＃ , -CH ２ -, ＃ -CH ２ C(=O)NHCH ２ - ＃＃ , ＃ -CH ２ NHC(=O)CH ２ - ＃＃ , ＃ -CH ２ C(=O)N(C ２ H5) CH ２ - ＃＃ , ＃ -CH ２ C(=O)OCH ２ CH ２ - ＃＃ , ＃ -CH ２ C(=O)NHCH ２ CH ２ - ＃＃ or G １ and G ２ Let's get together -CH ２ NHC(=O)OCH ２ - or -CH ２ CH ２ - constitutes or does not exist, ＃ - represents the -CH adjacent to the middle tertiary amine atom in the compound of formula (I). ２ represents the site of attachment to the - group, ＃＃ is G １ and G ２ represents a portion that connects G １ , G ２ , G ３ , G ４ may not exist at the same time, G １ , G ２ , G ３ , G ４ When both exist simultaneously, G １ and G ２ Let's get together -CH ２ NHC(=O)OCH ２ - constitutes G ３ and G ４ are the same group and *-CH ２ C(=O)OCH ２ -**, *-CH ２ OC(=O)CH ２ -**, -CH ２ -, *-CH ２ C(=O)N(C ２ H ５ ) CH ２ -**, or G １ and G ２ Let's get together -CH ２ CH ２ - constitutes G ３ and G ４ are the same group and *-CH ２ C(=O)OCH ２ -**, *-CH ２ OC(=O)CH ２ - is selected from **, G ３ and G ４ If there is no G １ and G ２ are the same group ＃ -CH ２ C(=O)N(CH ３ ) CH ２ - ＃＃ , ＃ -CH ２ C(=O)OCH ２ - ＃＃ , ＃ -CH ２ OC(=O)CH ２ - ＃＃ , ＃ -CH ２ C(=O)NHCH ２ - ＃＃ , ＃ -CH ２ NHC(=O)CH ２ - ＃＃ , ＃ -CH ２ C(=O)N(C ２ H ５ ) CH ２ - ＃＃ , ＃ -CH ２ C(=O)OCH ２ CH ２ - ＃＃ , ＃ -CH ２ C(=O)NHCH ２ CH ２ - ＃＃ is selected from G １ and G ２ If there is no G ３ and G ４ are the same group and *-CH ２ CH ２ C(=O)CH ２ -**, *-CH ２ C(=O)OCH ２ -**, *-CH ２ OC(=O)CH ２ -**, *-CH ２ C(=O)N(CH ３ ) CH ２ -**, *-CH ２ C(=O)N(C ２ H ５ ) CH ２ -**, *-CH ２ C(=O)NHCH ２ -**, *-CH ２ NHC(=O)CH ２ 2. The compound or pharmaceutically acceptable salt thereof according to claim 1, which is selected from -**.

3. 3. The compound or pharmaceutically acceptable salt according to claim 1 or 2, wherein m1, m2, m3, and m4 are selected from the same number, and each of m1, m2, m3, and m4 is 1 or 2.

4. 3. The compound of claim 1 or 2, or a pharmaceutically acceptable salt thereof, selected from the following compounds: 【Table 1】

5. 3. The compound of claim 1 or 2, wherein the compound is present as a pharmaceutically acceptable salt.

6. A pharmaceutical composition comprising the compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof and a nucleic acid drug.

7. 7. The pharmaceutical composition of claim 6, wherein the composition comprises at least one excipient selected from neutral phospholipids, steroids, and polyethylene glycol lipids.

8. (i) the neutral phospholipid is selected from DOPE, DSPC, DOPC, DSPE, DMPC, DMPE, DPPC, DPPE, DEPC, HSPC, POPC, or a combination thereof; or (ii) The pharmaceutical composition of claim 7, wherein the neutral phospholipid is DOPE.

9. 8. The pharmaceutical composition according to claim 7, wherein (i) the molar ratio of the compound to the neutral phospholipid is 1:1 to 5:1, or (ii) the molar ratio of the compound to the steroid is 1:2 to 2:

1.

10. (i) the steroid is selected from cholesterol, sitosterol, soy sterol, lanosterol, ergosterol, or a combination thereof; or (ii) The pharmaceutical composition according to claim 7, wherein the steroid is cholesterol.

11. (i) the polyethylene glycol lipid is selected from DMG-PEG, DSPE-PEG, or a combination thereof; or (ii) The pharmaceutical composition of claim 7, wherein the polyethylene glycol lipid is DMG-PEG2000.

12. (i) the molar ratio of compound:polyethylene glycol lipid is between 5:1 and 100:1; or (ii) The pharmaceutical composition of claim 7, wherein the ratio of compound to polyethylene glycol lipid is 10:1 to 20:

1.

13. (i) the nucleic acid drug is selected from DNA, ASO, siRNA, miRNA, mRNA, ribozyme, nucleic acid aptamer, or a combination thereof; or (ii) The pharmaceutical composition of claim 7, wherein the pharmaceutical composition is formulated into a lipid nanoparticle (LNP).

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