Ionizable lipid compound, lipid nanoparticles containing the same, pharmaceutical composition, pharmaceutical preparation and use thereof

Abstract

Provided are ionizable lipid compounds, lipid nanoparticles containing the same, pharmaceutical compositions, pharmaceutical preparations and applications thereof. Specifically provided are ionizable lipid compounds, N-oxides, pharmaceutically acceptable salts, prodrugs or stereoisomers thereof, wherein the compound structure is as follows: R1 is C 2‑10 Alkylene; R2 is C 2‑10 Alkylene or ‑CH2CH2‑N(CH2CH2OH)‑C n H 2n ‑, n is an integer from 2 to 10; L1, L2 are ‑O(C=O)‑, ‑(C=O)O‑, ‑NH(C=O)‑ or ‑(C=O)NH‑; R3 is a phenyl group substituted with an oxyalkyl group, wherein the oxyalkyl group is C 2‑20 Oxyalkyl; R4 is C 2‑25 A hydrocarbon group or a phenyl group substituted with an oxyalkyl group, wherein the oxyalkyl group is C 2‑20 Oxyalkyl; R5 is C 2‑6 The delivery system containing the compound has low toxicity, high delivery efficiency, and continuous and stable drug expression in vivo.

Description

Technical Field

[0001] The present application belongs to the field of biomedicine, and specifically relates to an ionizable lipid compound, lipid nanoparticles containing the same, a pharmaceutical composition, a pharmaceutical preparation and applications thereof. Background Art

[0002] The development of drug delivery technology has a history of more than 70 years. From the initial delivery of small molecule drugs to the current delivery of biomacromolecules, delivery technology has been continuously improving. The initial drug delivery system was mainly used to improve the physical and chemical properties of small molecule drugs, such as increasing the solubility of drugs, controlling drug release, and improving pharmacokinetics. With the rapid development of biopharmaceuticals, such as proteins, peptides, antibodies, and nucleic acids, their poor stability and difficulty in cellular entry have forced the continuous updating of delivery technology strategies.

[0003] In the process of development and application of delivery systems, liposomes have emerged and have been approved for the delivery of a variety of clinical drugs, such as the anticancer drug doxorubicin. Based on liposomes, lipid nanoparticles (LNP) with more stable structures and higher delivery efficiency have been developed for the delivery of nucleic acids. In 2018, the FDA approved the world's first siRNA drug Onpattro (also known as Patisiran) for the treatment of transthyretin amyloidosis. It is the first gene therapy drug with a non-viral delivery system. Since then, the two mRNA vaccines approved for marketing in 2020 by Pfizer and Moderna in the United States have used LNP as a delivery vector.

[0004] Among the four components that make up LNP (cationic or ionizable lipids, structural lipids, phospholipids and polymer-conjugated lipids), cationic or ionizable lipids are the core key components. Because it determines the encapsulation of LNP to mRNA and the efficiency of lysosomal escape after entering the cell. Therefore, researchers at home and abroad have conducted a lot of research on ionizable lipids to improve the in vivo delivery efficiency of mRNA. The ionizable lipids in the LNPs currently on the market are MC3, SM102 and ALC-0315. Although these three lipids have shown good delivery effects in vivo, their safety is also worrying. For example, with the large-scale use of mRNA vaccines in clinical practice, some side effects such as fever, allergies, fatigue, headaches, and local pain have occurred, which has made people begin to suspect that these are caused by LNP, because simple LNP can also cause the same adverse reactions. Therefore, it is still very necessary to develop ionizable lipids that are low in toxicity.

[0005] In addition, the present invention realizes that the existing research on ionizable lipids is mostly focused on improving the in vivo delivery activity, while ignoring the characteristics of in vivo delivery persistence. If a cationic lipid that can ensure the persistent expression of the delivered drug can be found in the research process, the frequency of medication can be reduced in actual medication, which can also reduce its side effects. Therefore, under the premise of ensuring delivery efficiency, it is still necessary to study how to reduce the toxicity of ionizable lipid compounds and maintain the persistence of gene expression. Summary of the invention

[0006] In order to solve the problems existing in the above-mentioned prior art, the present application provides an ionizable lipid compound, lipid nanoparticles containing the same, a pharmaceutical composition, a pharmaceutical preparation and applications thereof.

[0007] Specifically, this application provides:

[0008] (1) An ionizable lipid compound, its N-oxide, its pharmaceutically acceptable salt, prodrug or stereoisomer, wherein the ionizable lipid compound is represented by formula (I):

[0009]

[0010] in,

[0011] R1 is C 2-10 Alkylene,

[0012] R2 is C 2-10 Alkylene or -CH2CH2-N(CH2CH2OH)-C n H 2n -, wherein n is an integer from 2 to 10,

[0013] L1 and L2 are each independently -O(C=O)-, -(C=O)O-, -NH(C=O)- or -(C=O)NH-,

[0014] R3 is an alkoxy-substituted phenyl group, wherein the alkoxy group is C 2-20 Alkoxy,

[0015] R4 is C 2-25 A hydrocarbon group, or a phenyl group substituted with an alkoxy group, wherein the alkoxy group is C 2-20 Alkoxy, and

[0016] R5 is C 2-6 Alkylene.

[0017] (2) The ionizable lipid compound, its N-oxide, its pharmaceutically acceptable salt, prodrug or stereoisomer according to (1), wherein R1 and R2 are each independently C 2-10Alkylene, and preferably, R1 is C 3-9 Alkylene, R2 is C 5-9 Alkylene; or, wherein R1 is C 5-8 Alkylene, R2 is -CH2CH2-N(CH2CH2OH)-C n H 2n -, wherein n is an integer from 5 to 8.

[0018] (3) The ionizable lipid compound, N-oxide, pharmaceutically acceptable salt, prodrug or stereoisomer thereof according to (1), wherein R3 is a phenyl substituted with a para-alkoxy group or a phenyl substituted with a meta-dialkoxy group; preferably, the alkoxy group is C 2-12 Straight chain alkoxy, more preferably, the alkoxy is C 5-11 Straight chain alkoxy.

[0019] (4) The ionizable lipid compound, its N-oxide, its pharmaceutically acceptable salt, prodrug or stereoisomer according to (1), wherein

[0020] R4 is C 8-20 Preferably, R4 is C 13-19 A branched alkyl group, preferably -CH(C a H 2a+1 )(C b H 2b+1 ), wherein a and b are each independently an integer from 6 to 9, for example, R4 is -CH(C8H 17 )2; or R4 is substituted or unsubstituted C 8-10 Straight chain hydrocarbon group; or

[0021] R4 is an alkoxy-substituted phenyl group, wherein the alkoxy group is C 2-12 Straight chain alkoxy, preferably C 8-10 Straight chain alkoxy.

[0022] (5) The ionizable lipid compound, its N-oxide, its pharmaceutically acceptable salt, prodrug or stereoisomer according to (4), wherein the substituted or unsubstituted C 8-10 The straight chain hydrocarbon group is selected from substituted or unsubstituted C 8-10 Straight chain alkyl and unsubstituted C 8-10 Straight chain alkenyl; when R4 is substituted C 8-10 When the alkyl group is straight chain, the substituent is C 1-3 Alkyl or C 2-3 Alkenyl.

[0023] (6) The ionizable lipid compound, its N-oxide, its pharmaceutically acceptable salt, prodrug or stereoisomer according to (1), wherein

[0024] R1 and R2 are each independently C 5-9 Alkylene,

[0025] L1 and L2 are each independently -O(C=O)- or -(C=O)O-;

[0026] R3 is a phenyl group substituted with a para-alkoxy group, wherein the alkoxy group is C 5-11 A straight chain alkoxy group, and

[0027] R4 is C 13-19 A branched alkyl group, preferably -CH(C a H 2a+1 )(C b H 2b+1 ), wherein a and b are each independently an integer from 6 to 9.

[0028] (7) The ionizable lipid compound, its N-oxide, its pharmaceutically acceptable salt, prodrug or stereoisomer according to (1), wherein

[0029] R1 and R2 are each independently C 5-9 Alkylene,

[0030] L1 and L2 are each independently -O(C=O)- or -(C=O)O-;

[0031] R3 is a phenyl group substituted by a meta-dialkoxy group, wherein the alkoxy group is C 8-10 A straight chain alkoxy group, and

[0032] R4 is substituted or unsubstituted C 8-10 Straight chain hydrocarbon.

[0033] (8) The ionizable lipid compound, its N-oxide, its pharmaceutically acceptable salt, prodrug or stereoisomer according to (1), wherein

[0034] R1 and R2 are each independently C 5-7 Alkylene,

[0035] L1 and L2 are each independently -O(C=O)- or -(C=O)O-;

[0036] R3 is a phenyl group substituted by a meta-dialkoxy group, wherein the alkoxy group is C 8-10 A straight chain alkoxy group, and

[0037] R4 is a phenyl group substituted by a para-alkoxy group, wherein the alkoxy group is C 8-10 Straight chain alkoxy.

[0038] (9) The ionizable lipid compound, its N-oxide, its pharmaceutically acceptable salt, prodrug or stereoisomer according to (1), wherein

[0039] R1 is C 5-8 Alkylene,

[0040] R2 is -CH2CH2-N(CH2CH2OH)-C n H 2n -, wherein n is an integer from 5 to 8, and L1 and L2 are each independently -O(C=O)- or -(C=O)O-;

[0041] R3 is a phenyl group substituted with a para-alkoxy group, wherein the alkoxy group is C 8-11 A straight chain alkoxy group, and

[0042] R4 is C 13-19 A branched alkyl group, preferably -CH(C a H 2a+1 )(C b H 2b+1 ), wherein a and b are each independently an integer from 6 to 9.

[0043] (10) The ionizable lipid compound, its N-oxide, its pharmaceutically acceptable salt, prodrug or stereoisomer according to (1), wherein

[0044] R1 is C 5-8 Alkylene,

[0045] R2 is -CH2CH2-N(CH2CH2OH)-C n H 2n -, wherein n is an integer from 5 to 8, and L1 and L2 are each independently -O(C=O)- or -(C=O)O-;

[0046] R3 is a phenyl group substituted by a meta-dialkoxy group, wherein the alkoxy group is C 8-10 A straight chain alkoxy group, and

[0047] R4 is substituted or unsubstituted C 8-10 Straight chain alkyl.

[0048] (11) The ionizable lipid compound, its N-oxide, its pharmaceutically acceptable salt, prodrug or stereoisomer according to (1), wherein

[0049] R1 and R2 are each independently C 5-8 Alkylene,

[0050] L1 is -NH(C=O)- or -(C=O)NH-,

[0051] L2 is -O(C=O)- or -(C=O)O-,

[0052] R3 is a phenyl group substituted with a para-alkoxy group, wherein the alkoxy group is C 8-11 A straight chain alkoxy group, and

[0053] R4 is C 13-19 A branched alkyl group, preferably -CH(C a H 2a+1 )(C b H 2b+1 ), wherein a and b are each independently an integer from 6 to 9.

[0054] (12) Use of the ionizable lipid compound, its N-oxide, its pharmaceutically acceptable salt, prodrug or stereoisomer according to any one of (1) to (11) in the preparation of a drug for treating a disease or condition in a mammal; preferably, the drug is a nucleic acid drug, a small molecule drug, a polypeptide drug or a protein drug; preferably, the disease or condition includes infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, neurodegenerative diseases, cardiovascular and renal vascular diseases and metabolic diseases; preferably, the mammal is a human.

[0055] (13) A lipid nanoparticle comprising the ionizable lipid compound, its N-oxide, its pharmaceutically acceptable salt, prodrug or stereoisomer according to any one of (1) to (11).

[0056] (14) The lipid nanoparticle according to (13), wherein the molar ratio of the ionizable lipid compound, its N-oxide, its pharmaceutically acceptable salt, prodrug or stereoisomer is 40% to 65% of the lipid nanoparticle.

[0057] (15) The lipid nanoparticles according to (13) further comprise structured lipids; preferably, the structured lipids are selected from cholesterol, non-sterols, coprosterol, sitosterol, ergosterol, campesterol, brassicasterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, α-tocopherol, corticosteroids and mixtures thereof.

[0058] (16) The lipid nanoparticle according to (15), wherein the molar ratio of the structural lipid to the lipid nanoparticle is 35% to 55%.

[0059] (17) The lipid nanoparticles according to (13) further comprise a phospholipid; preferably, the phospholipid is selected from 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0-Diether-1-yl)-sn-glycero-3-phosphocholine (18:0-Diether-1-yl)-sn-glycero-3-phosphocholine (18:0-Diether-1-yl)-sn-glycero-3-phosphocholine (18:0 PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16:0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoyl Phosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE) and mixtures thereof.

[0060] (18) The lipid nanoparticle according to (17), wherein the molar ratio of the phospholipid to the lipid nanoparticle is 5% to 17.5%.

[0061] (19) The lipid nanoparticles according to (13) further comprise polymer-conjugated lipids; preferably, the polymer-conjugated lipids are selected from one or more of pegylated lipids, polylactic acid-conjugated lipids, polyamidated lipids, polysarcosinate lipids, polylactic acid-co-glycolic acid copolymer-conjugated lipids, polypeptide-conjugated lipids and poly-peptide-conjugated lipids.

[0062] (20) The lipid nanoparticle according to (19), wherein the molar ratio of the polymer-conjugated lipid to the lipid nanoparticle is 1.0% to 2.0%.

[0063] (21) The lipid nanoparticles according to (13) also contain structured lipids, phospholipids and polymer-conjugated lipids; preferably, the molar ratio of the ionizable lipid compound, its N-oxide, its pharmaceutically acceptable salt, prodrug or stereoisomer to the structured lipids, the phospholipids and the polymer-conjugated lipids is (40-65):(35-55):(5-17.5):(1.0-2.0).

[0064] (22) Use of the lipid nanoparticles according to any one of (13) to (21) in the preparation of a drug for treating a disease or condition in a mammal; preferably, the drug is a nucleic acid drug, a small molecule drug, a polypeptide drug or a protein drug; preferably, the disease or condition includes infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, neurodegenerative diseases, cardiovascular and renal vascular diseases and metabolic diseases; preferably, the mammal is a human.

[0065] (23) A pharmaceutical composition comprising the lipid nanoparticle according to any one of (13) to (21) and a therapeutic agent and / or a preventive agent.

[0066] (24) The pharmaceutical composition according to (23), wherein the therapeutic agent and / or preventive agent is selected from one or more of nucleic acids, small molecule compounds, polypeptides and proteins; preferably, the nucleic acid is selected from single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA, plasmid DNA, transfer RNA (tRNA), messenger RNA (mRNA), circular RNA, self-replicating mRNA, antisense nucleic acid molecules, small interfering RNA (siRNA), small activating RNA (saRNA), asymmetric interfering RNA (aiRNA), micro RNA (miRNA), small hairpin RNA (shRNA) and Dicer-substrate RNA (dsRNA).

[0067] (25) The pharmaceutical composition according to (23), wherein the pharmaceutical composition is in the form of nanoparticles, and the polydispersity coefficient of the nanoparticles is 0.20 or less.

[0068] (26) The pharmaceutical composition according to (23), wherein the therapeutic agent and / or preventive agent is encapsulated in the lipid nanoparticles; preferably, the encapsulation rate of the lipid nanoparticles for the therapeutic agent and / or preventive agent is greater than 90%.

[0069] (27) The pharmaceutical composition according to (23), wherein the therapeutic agent and / or preventive agent is a nucleic acid, preferably, the therapeutic agent and / or preventive agent is mRNA, and the nitrogen / phosphorus ratio of the ionizable lipid compound, its N-oxide, its pharmaceutically acceptable salt, prodrug or stereoisomer to the mRNA is 2:1 to 30:1.

[0070] (28) The pharmaceutical composition according to (23), wherein the pharmaceutical composition is configured for oral, intravenous, intramucosal, intraperitoneal, intramuscular, transdermal, intradermal, subcutaneous or intranasal administration, or for administration by inhalation.

[0071] (29) A pharmaceutical preparation comprising the pharmaceutical composition according to any one of (23) to (28) and a pharmaceutically acceptable excipient.

[0072] Compared with the prior art, this application has the following advantages and positive effects:

[0073] 1. The present invention improves the molecular structure of the ionizable lipid compound and introduces an alkoxy-substituted phenyl group through an ester bond or an amide bond, thereby improving the delivery efficiency while reducing the toxicity of the ionizable lipid compound, providing a basis for constructing a high-efficiency and low-toxic lipid nanoparticle delivery system.

[0074] 2. The ionizable lipid compound of the present invention can be prepared by esterification reaction and substitution reaction, and the reaction path is simple. In addition, the introduction of phenyl groups makes the compound have ultraviolet absorption, which reduces the difficulty of detection and purification. In addition, the raw materials are cheap and easy to obtain, the reaction conditions are mild, and it is easy to prepare and obtain, which is suitable for industrial application.

[0075] 3. The lipid nanoparticles constructed using the ionizable lipid compounds of the present invention can have an encapsulation rate of therapeutic agents and / or preventive agents of more than 90%, and the polydispersity coefficient of the particles is as low as below 0.2, or even as low as 0.03 (for example, compounds BM037, BM028, and BM043). They have good uniformity and show a cell survival rate of greater than 90% in a cell survival experiment, and can be used as a drug delivery system with good biocompatibility and safety.

[0076] 4. The lipid nanoparticles of the present invention have excellent delivery effects on therapeutic agents and / or preventive agents. The protein expression level of the delivered therapeutic agent and / or preventive agent in cells can reach more than 3 times that of the commercially available industry standard product SM102, and can even reach 9.12 times (compound BM043); the 4h in vivo expression level can reach up to 12.06 times that of SM102 (compound BM042); and it shows good in vivo delivery persistence, the delivered therapeutic agent and / or preventive agent can be continuously and stably expressed, and the sustained expression amount of 72h can reach up to 52.13 times that of SM102 (compound BM042). This has obvious advantages over the ionizable lipid compounds in the prior art. The ionizable lipid compounds and lipid nanoparticles of the present invention can continuously ensure the stable expression of the delivered drug in the delivery application of drugs (such as mRNA), thereby reducing the frequency of medication and improving the therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 The protein expression level of firefly luciferase mRNA delivered by the lipid nanoparticles of the present invention in 293T cells in Example 4 of the present application is shown, and the ordinate shows the relative light unit RLU / mg protein, which reflects the protein expression level.

[0078] Figure 2 The figure shows the multiple of the protein expression level of firefly luciferase mRNA delivered by the lipid nanoparticles of the present invention in 293T cells relative to that delivered by lipid nanoparticles prepared by SM102 in Example 4 of the present application.

[0079] Figure 3 The figure shows the survival rate of 293T cells transfected with lipid nanoparticles loaded with firefly luciferase mRNA in Example 6 of the present application.

[0080] Figure 4 The sustained protein expression level of firefly luciferase mRNA delivered by the lipid nanoparticles of the present invention in mice in Example 8 of the present application is shown, and the average fluorescence flux of each lipid nanoparticle at each time point is normalized by its average fluorescence flux at 4 hours. DETAILED DESCRIPTION

[0081] The present invention is further illustrated below through the description of specific implementation modes, but this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the present invention, but as long as they do not deviate from the basic idea of ​​the present invention, they are all within the scope of the present invention.

[0082] definition

[0083] As used herein, "hydrocarbyl" refers to a group consisting of carbon atoms and hydrogen atoms, including saturated and unsaturated hydrocarbon groups. In some embodiments, the hydrocarbon group has 2-25 carbon atoms, referred to as C 2-25 Hydrocarbon.

[0084] As used herein, "alkyl" refers to a saturated aliphatic hydrocarbon group, including straight chain alkyl and branched alkyl. In some embodiments, the alkyl group has 8-10 carbon atoms, referred to as C 8-10 In some embodiments, the alkyl group may be an unsubstituted alkyl group or may be substituted by other groups. The substituent may be a hydrocarbon group, such as C 1-3 Alkyl or C 2-3 Alkenyl groups, such as methyl, ethyl, propyl, vinyl, allyl, etc.

[0085] As used herein, "alkoxy" refers to -O-alkyl, and "straight chain alkoxy" refers to -O-straight chain alkyl. In some embodiments, alkoxy is an alkoxy group having 2-20 carbon atoms, referred to as C 2-20 In some embodiments, the alkoxy group is a straight chain alkoxy group having 8-10 carbon atoms, which is referred to as a C 8-10 Straight chain alkoxy.

[0086] As used herein, "alkylene" refers to an alkyl group with one hydrogen atom further substituted, for example, an alkylene group having 2 carbon atoms refers to -CH2CH2-. In some embodiments, an alkylene group has 2-10 carbon atoms and is referred to as C 2-10 Alkylene.

[0087] As used herein, "alkenyl" refers to an unsaturated aliphatic hydrocarbon group having at least one double bond. In some embodiments, the alkenyl group is a straight chain alkenyl group having 8-10 carbon atoms, referred to as a C 8-10 A straight-chain alkenyl group, wherein the double bond may be located at any position of the straight-chain alkenyl group.

[0088] As used herein, "N-oxide" refers to an N-oxide obtained by treating a nitrogen-containing compound with an oxidant (e.g., meta-chloroperbenzoic acid, hydrogen peroxide, ozone). Where valence and structure permit, the compounds claimed in the present application also include N-oxide derivatives of ionizable lipid compounds.

[0089] As used herein, "pharmaceutically acceptable salts" refer to salts of the ionizable lipid compounds of the present application that are pharmaceutically acceptable and have the desired pharmacological activity. Such salts include acid addition salts formed with the following acids: inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; or organic acids such as formic acid, acetic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4'-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid / acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acid, aromatic sulfuric acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid , carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, caproic acid, hydroxynaphthoic acid, lactic acid, lauryl sulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanoic acid, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tert-butylacetic acid, trimethylacetic acid, etc. Pharmaceutically acceptable salts also include base addition salts that can be formed when the acidic protons present are capable of reacting with inorganic or organic bases, inorganic bases include but are not limited to sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide; organic bases include but are not limited to ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, etc. It will be appreciated that the particular anion or cation that forms part of any salt described herein is not critical so long as the salt as a whole is pharmacologically acceptable.

[0090] As used herein, "prodrug" refers to a compound that can be converted into a biologically active compound under physiological conditions or by dissolution. Prodrugs are usually rapidly converted in vivo to produce the parent compound, such as by hydrolysis in the blood. Prodrug compounds usually have solubility, tissue compatibility or delayed release advantages in mammalian organisms. The term "prodrug" also means to include any covalently bonded carrier, which releases the active compound in vivo when the prodrug is administered to a mammalian subject. Prodrugs include compounds that hydroxyl, amino or sulfhydryl groups are combined with any group, and when the prodrug is administered to a mammalian subject, these groups are cleaved to form free hydroxyl, free amino or free sulfhydryl groups, respectively. Examples of prodrugs include, but are not limited to, amide derivatives of acetate, formate and benzoate derivatives or amine functional groups, etc.

[0091] As used herein, "stereoisomer" refers to an isomer of a given compound in which the same atoms are bonded to the same other atoms, but in which the three-dimensional configurations of those atoms are different. "Enantiomers" are stereoisomers of a given compound that are mirror images of each other. "Diastereomers" are stereoisomers of a given compound that are not enantiomers. A 50:50 mixture of enantiomers is called a racemic mixture. A mixture of enantiomers can be enriched so that one enantiomer is present in an amount greater than 50%. Typically, enantiomers and / or diastereomers can be split or separated using techniques known in the art. "Substantially free of other stereoisomers" means that the composition contains ≤15%, more preferably ≤10%, even more preferably ≤5%, or most preferably ≤1% of one or more other stereoisomers.

[0092] As used herein, "polydispersity" is a parameter that describes the homogeneity of the particle size distribution of a system. The smaller the polydispersity, the narrower the particle size distribution of the system. For example, a polydispersity of less than 0.30 indicates a narrower particle size distribution.

[0093] Ionizable lipid compounds

[0094] In one aspect, the present application provides an ionizable lipid compound, its N-oxide, its pharmaceutically acceptable salt, prodrug or stereoisomer. The ionizable lipid compound of the present application is shown in formula (I):

[0095]

[0096] Where R1 is C 2-10 Alkylene,

[0097] R2 is C 2-10 Alkylene or -CH2CH2-N(CH2CH2OH)-C n H 2n -, wherein n is an integer from 2 to 10,

[0098] L1 and L2 are each independently -O(C=O)-, -(C=O)O-, -NH(C=O)- or -(C=O)NH-,

[0099] R3 is an alkoxy-substituted phenyl group, wherein the alkoxy group is C 2-20 Alkoxy,

[0100] R4 is C 2-25 A hydrocarbon group, or a phenyl group substituted with an alkoxy group, wherein the alkoxy group is C 2-20 Alkoxy, and

[0101] R5 is C 2-6 Alkylene.

[0102] The ionizable lipid compound of the present invention introduces R3 and R4 groups respectively through ester bonds or amide bonds L1 and L2, thereby improving the structure and performance of the lipid molecule through organic coordination between the various groups.

[0103] In some embodiments, R1 and R2 are both linear alkylene groups, each of which is independently C 2-10 Alkylene, such as C 3-10 Alkylene, C 4-10 Alkylene, C 5-10 Alkylene, C 3-9 Alkylene or C 4-9 The range "2-10" includes the range formed by the endpoint values ​​and any values ​​therebetween, for example, the range "2-10" includes the range formed by any two values ​​of 2, 3, 4, 5, 6, 7, 8, 9, 10. In some embodiments, R1 and R2 are C 3-9 Preferably, R1 is C 3-9 Alkylene, R2 is C 5-9 Preferably, R1 and R2 are unsubstituted.

[0104] In other embodiments, R1 is C 2-10 Straight chain alkylene, R2 is -CH2CH2-N(CH2CH2OH)-C n H 2n -, wherein n can be an integer from 2 to 10. The range "2-10" includes the range formed by the endpoint values ​​and any values ​​therebetween, for example, the range "2-10" includes the range formed by any two values ​​of 2, 3, 4, 5, 6, 7, 8, 9, 10. Preferably, R1 is C 5-8 Straight chain alkylene, R2 is -CH2CH2-N(CH2CH2OH)-C n H 2n -, wherein n is an integer from 5 to 8. Preferably, R1 is unsubstituted.

[0105] Preferably, R3 is C 2-20 The range "2-20" includes the range formed by the endpoint values ​​and any values ​​therebetween, for example, the range "2-20" includes the range formed by any two values ​​of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20. In some preferred embodiments, R3 is a phenyl substituted with a para-alkoxy group, wherein the alkoxy group is C 2-12 Straight chain alkoxy, more preferably, the alkoxy is C 5-11 Straight chain alkoxy, such as C5 straight chain alkoxy, C6 straight chain alkoxy, C7 straight chain alkoxy, C8 straight chain alkoxy, C9 straight chain alkoxy, C10 Straight chain alkoxy, C 11 In some other preferred embodiments, R3 is a phenyl group substituted with a meta-dialkoxy group, and the two alkoxy groups may be the same or different. For example, the two alkoxy groups are each independently C 8-10 Straight chain alkoxy, such as C8 straight chain alkoxy, C9 straight chain alkoxy, C 10 More preferably, the two alkoxy groups are both C8 straight chain alkoxy groups. Preferably, the alkoxy group in R3 is unsubstituted.

[0106] In some embodiments, R4 is C 2-25 The range "2-25" includes the range formed by the endpoint values ​​and any values ​​therebetween, for example, the range "2-25" includes the range formed by any two values ​​of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25. In some embodiments, R4 is C 8-20 The hydrocarbon group may be an alkyl group or an alkenyl group. The range "8-20" includes a range formed by any two values ​​of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.

[0107] In some specific embodiments, R4 is C 13-19 Branched alkyl, preferably C 15-19 In a preferred embodiment, R4 is -CH(C a H 2a+1 )(C b H 2b+1 ), wherein a and b may be the same or different and may each independently be an integer from 6 to 9, preferably an integer from 7 to 9, for example, a and b may each independently be 7, 8 or 9; in some embodiments, a and b are the same integer, for example, R4 is -CH(C8H 17 )2.

[0108] In other specific embodiments, R4 is substituted or unsubstituted C 8-10 Straight chain hydrocarbon group, for example, R4 is an unsubstituted C 8-10 Straight chain alkyl, unsubstituted C 8-10 Straight chain alkenyl, unsubstituted C 9-10 Straight chain alkyl or unsubstituted C 9-10 Straight chain alkenyl; substituted C 8-10 Straight chain alkyl, substituted C 8-10 Straight chain alkenyl, substituted C 9-10 Straight chain alkyl or substituted C 9-10Preferably, the substituted or unsubstituted C 8-10 The straight chain hydrocarbon group is selected from substituted or unsubstituted C 8-10 Straight chain alkyl and unsubstituted C 8-10 Straight chain alkenyl. When R4 is substituted C 8-10 In the case of a straight chain alkyl group, the substituent may be C 1-3 Alkyl or C 2-3 Alkenyl, for example, methyl, ethyl, propyl, vinyl, allyl, the substituent is preferably located at C1. When R4 is a straight chain alkenyl, the position of the alkenyl is preferably between C2 and C3.

[0109] In other embodiments, R4 is alkoxy-substituted phenyl (preferably para-alkoxy-substituted phenyl), wherein the alkoxy is C 2-20 Alkoxy, such as C 2-20 Straight chain alkoxy. The alkoxy is preferably C 8-10 Preferably, the alkoxy group in R4 is unsubstituted.

[0110] In some embodiments, R5 is C 2-6 Alkylene, such as C 2-5 Alkylene, C 2-4 Alkylene or C 2-3 Alkylene.

[0111] In the ionizable lipid compound of the present application, a specific combination of R1-R5 groups can achieve a better drug delivery effect.

[0112] In a preferred embodiment of the ionizable lipid compound of the present application, R1 and R2 are each independently C 5-9 Alkylene, for example, R1 and R2 can be independently C5 alkylene, C6 alkylene, C7 alkylene, C8 alkylene, C9 alkylene; L1 and L2 can be independently -O(C=O)- or -(C=O)O-; R3 is a phenyl substituted with a para-alkoxy group, wherein the alkoxy group is C 5-11 Straight chain alkoxy; R4 is C 15-19 Branched alkyl, preferably C 15-19 Branched alkyl, preferably, R4 is -CH(C a H 2a+1 )(C b H 2b+1 ), wherein a and b may be the same or different and may each independently be an integer from 6 to 9, preferably an integer from 7 to 9, for example, a and b may each independently be 7, 8 or 9, in some embodiments, a and b are the same integer, for example, R4 is -CH(C8H 17 )2; and R5 is C 2-6 Alkylene, such as C 2-5Alkylene, C 2-4 Alkylene or C 2-3 Alkylene.

[0113] In some other preferred embodiments of the ionizable lipid compounds of the present application, R1 and R2 are each independently C 5-9 Alkylene, preferably C 5-8 Alkylene, for example, R1 and R2 can be independently C5 alkylene, C6 alkylene, C7 alkylene, C8 alkylene; L1 and L2 can be independently -O(C=O)- or -(C=O)O-; R3 is a phenyl substituted with a meta-dialkoxy group, wherein the alkoxy group is C 8-10 The two alkoxy groups may be the same or different. Preferably, the two alkoxy groups are both C8 straight chain alkoxy groups; R4 is a substituted or unsubstituted C 8-10 A straight chain hydrocarbon group, wherein the hydrocarbon group is preferably an alkyl group or an alkenyl group, for example, R4 is an unsubstituted C 8-10 Straight chain alkyl, unsubstituted C 8-10 Straight chain alkenyl, unsubstituted C 9-10 Straight chain alkyl or unsubstituted C 9-10 Straight chain alkenyl, or substituted C 8-10 Straight chain alkyl, substituted C 8-10 Straight chain alkenyl, substituted C 9-10 Straight chain alkyl or substituted C 9-10 Straight chain alkenyl, wherein the substituent may be C 1-3 Alkyl or C 2-3 alkenyl, for example, methyl, ethyl, propyl, vinyl, allyl, the position of the substituent is preferably C1; when R4 is a straight chain alkenyl, the position of the alkenyl is preferably between C2 and C3; and R5 is C 2-6 Alkylene, such as C 2-5 Alkylene, C 2-4 Alkylene or C 2-3 Alkylene.

[0114] In some other preferred embodiments of the ionizable lipid compounds of the present application, R1 and R2 are each independently C 5-7 Alkylene, for example, R1 and R2 can be independently C5 alkylene, C6 alkylene, C7 alkylene; L1 and L2 can be independently -O(C=O)- or -(C=O)O-; R3 is a phenyl substituted with a meta-dialkoxy group, wherein the alkoxy group is C 8-10 The two alkoxy groups may be the same or different. Preferably, the two alkoxy groups are both C8 straight chain alkoxy groups. R4 is a phenyl group substituted by a para-alkoxy group, wherein the alkoxy group is C 8-10 Straight chain alkoxy; and R5 is C 2-6 Alkylene, such as C2-5 Alkylene, C 2-4 Alkylene or C 2-3 Alkylene.

[0115] In some other preferred embodiments of the ionizable lipid compounds of the present application, R1 is C 5-8 Alkylene, for example, C5 alkylene, C6 alkylene, C7 alkylene, C8 alkylene; R2 is -CH2CH2-N(CH2CH2OH)-C n H 2n -, wherein n is an integer from 5 to 8, such as an integer from 5 to 7, an integer from 5 to 6, an integer from 6 to 8 or an integer from 6 to 7; R3 is a phenyl substituted with a para-alkoxy group, wherein the alkoxy group is C 8-11 Straight chain oxyalkyl, preferably C 8-10 Straight chain alkoxy; L1, L2 are each independently -O(C=O)- or -(C=O)O-; R4 is C 13-19 Branched alkyl, preferably C 15-19 Branched alkyl, preferably, R4 is -CH(C a H 2a+1 )(C b H 2b+1 ), wherein a and b may be the same or different and may each independently be an integer from 6 to 9, for example, a and b may each independently be 6, 7, 8 or 9, in some embodiments, a and b are the same integer, for example, R4 is -CH(C8H 17 )2; and R5 is C 2-6 Alkylene, such as C 2-5 Alkylene, C 2-4 Alkylene or C 2-3 Alkylene.

[0116] In some other preferred embodiments of the ionizable lipid compounds of the present application, R1 is C 5-8 Alkylene, for example, C5 alkylene, C6 alkylene, C7 alkylene, C8 alkylene; R2 is -CH2CH2-N(CH2CH2OH)-C n H 2n -, wherein n is an integer from 5 to 8, such as an integer from 5 to 7, an integer from 5 to 6, an integer from 6 to 8 or an integer from 6 to 7; L1 and L2 are each independently -O(C=O)- or -(C=O)O-; R3 is a phenyl substituted with a meta-dialkoxy group, wherein the alkoxy group is C 8-10 The two alkoxy groups may be the same or different. Preferably, the two alkoxy groups may be C8 straight chain alkoxy groups; R4 is a substituted or unsubstituted C 8-10 Straight chain alkyl, such as unsubstituted C 9-10 Straight chain alkyl, substituted C8-10 Straight chain alkyl or substituted C 9-10 Straight chain alkyl, where the substituent may be C 1-3 Alkyl or C 2-3 alkenyl, for example, methyl, ethyl, propyl, vinyl, allyl; and R5 is C 2-6 Alkylene, such as C 2-5 Alkylene, C 2-4 Alkylene or C 2-3 Alkylene.

[0117] In some other preferred embodiments of the ionizable lipid compounds of the present application, R1 and R2 are each independently C 5-8 Alkylene, for example, R1 and R2 can be independently C5 alkylene, C6 alkylene, C7 alkylene, C8 alkylene; L1 is -NH(C=O)- or -(C=O)NH-; L2 is -O(C=O)- or -(C=O)O-; R3 is a phenyl substituted with a para-alkoxy group, wherein the alkoxy group is C 8-11 Straight chain alkoxy, preferably C 8-10 Straight chain alkoxy; R4 is C 13-19 Branched alkyl, preferably C 15-19 Branched alkyl, preferably, R4 is -CH(C a H 2a+1 )(C b H 2b+1 ), wherein a and b may be the same or different and may each independently be an integer from 6 to 9, preferably an integer from 7 to 9, for example, a and b may each independently be 7, 8 or 9, in some embodiments, a and b are the same integer, for example, R4 is -CH(C8H 17 )2; and R5 is C 2-6 Alkylene, such as C 2-5 Alkylene, C 2-4 Alkylene or C 2-3 Alkylene.

[0118] The ionizable lipid compounds of the present application may also function through their N-oxides, their pharmaceutically acceptable salts, prodrugs or stereoisomers.

[0119] Table 1 below shows the structural formulas of exemplary ionizable lipid compounds of the present application, but it should be understood that these compounds are shown to enable those skilled in the art to better understand the technical solutions of the present application, rather than to limit the scope of the present application.

[0120] Table 1. Structural formulas of exemplary ionizable lipid compounds

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130] Lipid Nanoparticles

[0131] In another aspect, the present invention provides a lipid nanoparticle comprising an ionizable lipid compound, an N-oxide thereof, a pharmaceutically acceptable salt, a prodrug or a stereoisomer thereof according to the present invention. The lipid nanoparticle has a nanoscale size (e.g., 1 nm to 1000 nm) in at least one dimension.

[0132] In some embodiments, the molar ratio of the ionizable lipid compound, its N-oxide, its pharmaceutically acceptable salt, prodrug or stereoisomer is 40% to 65%, preferably 40% to 63%, and more preferably 46% to 50% of the lipid nanoparticle.

[0133] Lipid nanoparticles according to the present application may also include structured lipids. In some embodiments, the structured lipids are selected from cholesterol, non-sterols, coprosterol, sitosterol, ergosterol, campesterol, brassicasterol, stigmasterol, brassicasterol, tomatine, ursolic acid, alpha-tocopherol, corticosteroids and mixtures thereof. In a preferred embodiment, the structured lipid is cholesterol.

[0134] In some embodiments, the molar ratio of the structural lipid to the lipid nanoparticle is 35% to 55%, preferably 35% to 48.5%, and more preferably 38.5% to 45%.

[0135] The lipid nanoparticles according to the present application may also include phospholipids. In some embodiments, the phospholipids are selected from 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0Diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16:0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoyl Phosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE) and mixtures thereof. In a preferred embodiment, the phospholipid is DSPC.

[0136] In some embodiments, the molar proportion of phospholipids in the lipid nanoparticles is 5% to 17.5%.

[0137] The lipid nanoparticles according to the present application may also include polymer-conjugated lipids. In some embodiments, the polymer-conjugated lipids are selected from one or more of pegylated lipids, polylactic acid-conjugated lipids, polyamidated lipids, polysarcosinate lipids, polylactic-co-glycolic acid copolymerized lipids, polypeptide-conjugated lipids and poly-peptide-conjugated lipids. In a preferred embodiment, the polymer-conjugated lipid is a pegylated lipid, which can be selected from one or more of methoxypolyethylene glycol ditetradecyl acetamide (ALC-0159), dimyristoylglycerol-3-methoxypolyethylene glycol 1000 (PEG1000-DMG), dimyristoylglycerol-3-methoxypolyethylene glycol 5000 (PEG5000-DMG), dimyristoylglycerol-3-methoxypolyethylene glycol 2000 (PEG2000-DMG) and distearoylphosphatidylethanolamine polyethylene glycol 2000 (PEG2000-DSPE); preferably, the pegylated lipid is PEG2000-DMG.

[0138] In some embodiments, the molar ratio of polymer-conjugated lipid to lipid nanoparticles is 1.0% to 2.0%, preferably 1.5%.

[0139] In a preferred embodiment, the lipid nanoparticle comprises (i) an ionizable lipid compound of the present invention, an N-oxide thereof, a pharmaceutically acceptable salt, a prodrug or a stereoisomer thereof; (ii) a structured lipid; (iii) a phospholipid and (iv) a polymer-conjugated lipid.

[0140] In some embodiments, the molar ratio of the ionizable lipid compound of the present invention, its N-oxide, its pharmaceutically acceptable salt, prodrug or stereoisomer to the structured lipid, phospholipid and polymer-conjugated lipid is (35-65):(35-55):(5-17.5):(1.0-2.0), for example, the molar ratio may be 50:38.5:10:1.5; preferably, the molar ratio is (40-63):(35-48.5):(5-17.5):1.5.

[0141] In some embodiments, the average particle size of the lipid nanoparticles according to the present invention is 70 nm to 150 nm, such as 75 nm to 139 nm.

[0142] Preferably, the polydispersity index of the lipid nanoparticles according to the present invention is below 0.20, for example, from 0.01 to 0.20, from 0.02 to 0.20, from 0.03 to 0.20, from 0.03 to 0.15 or from 0.03 to 0.10.

[0143] The lipid nanoparticles according to the present application have good biocompatibility and biosafety. For example, in a cell survival rate experiment, a cell survival rate greater than 90% can be achieved.

[0144] Pharmaceutical composition

[0145] In another aspect, the present invention provides a pharmaceutical composition comprising a lipid nanoparticle according to the present invention and a therapeutic and / or preventive agent; preferably, the pharmaceutical composition is in the form of nanoparticles, which can be referred to as pharmaceutical composition nanoparticles; more preferably, the therapeutic and / or preventive agent is encapsulated in the lipid nanoparticles. The pharmaceutical composition nanoparticles have a nanoscale size (e.g., 1 nm to 1000 nm) in at least one dimension.

[0146] Therapeutic and / or preventive agents include, but are not limited to, one or more of nucleic acids, small molecule compounds, polypeptides, and proteins. Examples of therapeutic and / or preventive agents include, but are not limited to, anti-neoplastic agents (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, and streptozotocin), anti-tumor agents (e.g., actinomycin D, vincristine, vinblastine, cytosine arabinoside, anthracyclines, alkylating agents, platinum compounds, antimetabolites, and nucleoside analogs, such as methotrexate and purine and pyrimidine analogs), anti-infective agents, local anesthetics (e.g., dibucaine and chlorpromazine), beta-adrenergic blockers (e.g., propranolol, dimerolol, and labetalol), anti- Hypertensive agents (e.g., clonidine and hydralazine), antidepressants (e.g., imipramine, amitriptyline, and doxepin), anticonvulsants (e.g., phenytoin), antihistamines (e.g., diphenhydramine, chlorpheniramine, and promethazine), antibiotics / antibacterials (e.g., gentamicin, ciprofloxacin, and cefoxitin), antifungals (e.g., miconazole, terconazole, econazole, isoconazole, butoconazole, clotrimazole, itraconazole, nystatin, netifine, and amphotericin B), antiparasitics, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma agents, vitamins, sedatives, and imaging agents.

[0147] In some embodiments, the therapeutic and / or preventive agent can be selected from a cytotoxin, a radioactive ion, a chemotherapeutic agent, a vaccine, a compound that causes an immune response, and / or another therapeutic and / or preventive agent. Cytotoxins or cytotoxic agents include any agent that is harmful to cells. Examples include, but are not limited to, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraquinone, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansines such as maytansinol, razithromycin (CC-1065), and analogs or homologs thereof. Radioactive ions include, but are not limited to, iodine (e.g., iodine 125 or iodine 131), strontium 89, phosphorus, palladium, cesium, iridium, phosphate, cobalt, yttrium 90, samarium 153, and praseodymium. Vaccines include compounds and formulations capable of providing immunity against one or more conditions associated with infectious diseases such as influenza, measles, human papillomavirus (HPV), rabies, meningitis, pertussis, tetanus, plague, hepatitis, and tuberculosis.

[0148] In other embodiments, the therapeutic and / or preventive agent is a protein. The therapeutic proteins that can be used in the pharmaceutical composition of the present invention include, but are not limited to, gentamicin, amikacin, insulin, erythropoietin (EPO), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), factor VIR, luteinizing hormone releasing hormone (LHRH) analogs, interferon, heparin, hepatitis B surface antigen, typhoid vaccine, and cholera vaccine.

[0149] In a preferred embodiment, the therapeutic and / or preventive agent is a gene therapy drug, such as a nucleic acid having a therapeutic and / or preventive effect.

[0150] The nucleic acid includes any and all forms of nucleic acid molecules, including other forms of nucleic acid molecules known in the art or likely to be discovered / prepared in the future. In a preferred embodiment, the nucleic acid is selected from single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA, plasmid DNA, transfer RNA (tRNA), messenger RNA (mRNA), circular RNA, self-replicating mRNA, antisense nucleic acid molecules, small interfering RNA (siRNA), small activating RNA (saRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), small hairpin RNA (shRNA) and Dicer-substrate RNA (dsRNA, also known as small molecule double-stranded RNA). For example, a nucleic acid molecule may include mRNA encoding an infectious disease-derived antigen and / or epitope.

[0151] Preferably, the particle size of the nanoparticles of the pharmaceutical composition according to the present invention is 50nm to 200nm, for example, 70nm to 200nm, 50nm to 185nm, 75nm to 185nm, 60nm to 150nm, 70nm to 135nm or 80nm to 115nm.

[0152] In some embodiments, the polydispersity coefficient of the nanoparticles of the pharmaceutical composition according to the present invention is less than 0.20, for example, 0.01 to 0.20, 0.02 to 0.20, 0.03 to 0.20, 0.15 or less, 0.10 or less, 0.03 to 0.15 or 0.03 to 0.10. The polydispersity coefficients of the nanoparticles of the pharmaceutical composition of the present invention are all between 0.03 and 0.20, indicating that the uniformity of the nanoparticles is good.

[0153] Preferably, the encapsulation rate of the therapeutic agent and / or preventive agent by the lipid nanoparticles is 90% or more, for example, 95% or more, 96% or more, 97% or more, or 98% or more.

[0154] Herein, the term "encapsulation efficiency" refers to the ratio of RNA encapsulated inside lipid nanoparticles (LNPs) to the total RNA.

[0155] In some embodiments, the therapeutic agent and / or prophylactic agent in the pharmaceutical composition of the present invention is a nucleic acid, preferably, the therapeutic agent and / or prophylactic agent is mRNA, and the nitrogen / phosphorus ratio of the ionizable lipid compound, its N-oxide, its pharmaceutically acceptable salt, prodrug or stereoisomer to the mRNA is about 2:1 to 30:1.

[0156] Lipid nanoparticles encapsulating therapeutic agents and / or preventive agents can be prepared by methods known in the art, for example, using a microfluidic mixing system or an ethanol injection method, or other methods, as long as the pharmaceutical composition of the present application can be obtained.

[0157] The pharmaceutical compositions of the present invention may be formulated for oral, intravenous, intramucosal, intraperitoneal, intramuscular, transdermal, intradermal, subcutaneous or intranasal administration, or administration by inhalation.

[0158] Pharmaceutical preparations

[0159] In another aspect, the present invention provides a pharmaceutical preparation comprising the pharmaceutical composition according to the present invention and a pharmaceutically acceptable excipient.

[0160] "Pharmaceutically acceptable excipients" refer to excipients and additives used in the production of drugs and the preparation of prescriptions; they are substances that have been reasonably evaluated in terms of safety and are included in drug preparations in addition to active ingredients. In addition to excipients, acting as carriers, and improving stability, "pharmaceutically acceptable excipients" also have important functions such as solubilization, solubilization, and sustained and controlled release. They are important ingredients that may affect the quality, safety, and effectiveness of drugs. In some embodiments, pharmaceutically acceptable excipients may include solvents, propellants, solubilizers, solubilizers, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesive agents, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, etc.

[0161] application

[0162] In another aspect, the present invention also provides the use of the ionizable lipid compound, its N-oxide, its pharmaceutically acceptable salt, prodrug or stereoisomer, lipid nanoparticle, and pharmaceutical composition in the preparation of a drug for treating a disease or condition in a mammal. For example, the ionizable lipid compound, its N-oxide, its pharmaceutically acceptable salt, prodrug or stereoisomer, lipid nanoparticle, and pharmaceutical composition are used to prepare the pharmaceutical preparation according to the present invention for treating a disease or condition in a mammal.

[0163] In some embodiments, mammals include humans; domestic animals, such as laboratory animals and household pets, such as mice, rats, cats, dogs, pigs, cows, goats, sheep, monkeys, rabbits, horses, etc.; and non-domestic animals, such as wild animals, etc.

[0164] In some embodiments, the drug is a gene therapy drug, such as a nucleic acid with therapeutic and / or preventive effects, for example, single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA, plasmid DNA, transfer RNA (tRNA), messenger RNA (mRNA), circular RNA, self-replicating mRNA, antisense nucleic acid molecules, small interfering RNA (siRNA), small activating RNA (saRNA), asymmetric interfering RNA (aiRNA), micro RNA (miRNA) and small hairpin RNA (shRNA), Dicer-substrate RNA (dsRNA, also known as small molecule double-stranded RNA).

[0165] Such diseases, disorders and / or conditions include, but are not limited to, rare diseases, infectious diseases (drugs in the form of vaccines and therapeutics), cancer and proliferative diseases, genetic diseases (such as cystic fibrosis), autoimmune diseases, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases (e.g., diabetes).

[0166] In some embodiments, the infectious disease is selected from the group consisting of diseases caused by coronavirus, influenza virus, respiratory syncytial virus or HIV virus; pediatric pneumonia, Rift Valley fever, yellow fever, rabies, various herpes, etc.

[0167] In some embodiments, the pharmaceutical preparation of the present application is configured to be suitable for rapid absorption and distribution, so as to deliver active drugs to mammals. The delivery route is, for example, oral, intravenous, intramucosal (e.g., nose, vagina, etc.), intraperitoneal, intramuscular, transdermal, intradermal, subcutaneous, intranasal administration, or by inhalation administration. Preferably, the pharmaceutical preparation is formulated to be suitable for injection administration, for example, by intramuscular administration.

[0168] In some embodiments, after a pharmaceutical composition or pharmaceutical preparation comprising lipid nanoparticles encapsulating mRNA is injected and delivered into a mammal, an in vivo delivery effect much higher than that of a pharmaceutical composition or pharmaceutical preparation prepared using the commercially available product SM102 can be achieved. For example, the in vivo delivery effect can be more than 2 times, more than 3 times, more than 4 times, or more than 5 times that of SM102. Preferably, the in vivo delivery effect can be more than 10 times that of SM102.

[0169] In some embodiments, when the pharmaceutical composition or pharmaceutical preparation according to the present application is used to deliver mRNA in vivo, sustained and stable protein expression can be achieved for up to 72 hours, and the long-term sustained expression effect can be more than 3 times, more than 4 times, more than 5 times, or more than 6 times that of SM102. Preferably, the long-term sustained expression effect can be more than 10 times that of SM102, and more preferably, the long-term sustained expression effect can be more than 30 times that of SM102. Most preferably, the long-term sustained expression effect can be more than 50 times that of SM102.

[0170] The following further explains or illustrates the content of the present invention by way of examples, but these examples should not be construed as limiting the scope of protection of the present invention.

[0171] Example

[0172] The sources of materials and instruments used in the examples are as follows:

[0173] Unless otherwise stated, the raw materials used in the examples of the present application are commercially available.

[0174] Dynamic light scattering instrument: NANO-8S90, purchased from Malvern

[0175] Multi-function microplate reader: SpectraMax M5, purchased from Molecular Devices

[0176] Small animal in vivo imaging system: IVIS SPECTRUM, purchased from PerkinElmer

[0177] Example 1: Synthesis of compounds

[0178] In Example 1, the ionizable lipid compounds of the present application shown in Table 1 were synthesized by esterification reaction and substitution reaction. For comparison, the comparative compounds shown in Table 2 were also synthesized, which are not the ionizable lipid compounds of the present application.

[0179] Table 2. Structural formula of comparative compounds

[0180]

[0181] *SM102 is a commercially available product purchased from Xiamen Sinopang Biotechnology Co., Ltd.

[0182] 1. Synthesis of the intermediate 2-octyldecanoic acid-7-bromoheptyl ester

[0183] The synthetic route is as follows:

[0184]

[0185] To a solution of 2-octyldecanoic acid (2.5 g, 8.80 mmol, 1.0 eq) in dichloromethane (30 mL) were added 7-bromoheptanol (2.18 g, 11.18 mmol, 1.27 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.55 g, 13.20 mmol, 1.5 eq), N,N-diisopropylethylamine (3.41 g, 26.40 mmol, 3.0 eq) and 4-dimethylaminopyridine (0.21 g, 1.76 mmol, 0.2 eq) in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-10% ethyl acetate / hexane) to obtain an oily compound 2-octyldecanoic acid-7-bromoheptyl ester (1,818 mg, 43%).

[0186] 2. Synthesis of the intermediate 8-bromooctanoic acid heptadecan-9-ester

[0187] The synthetic route is as follows:

[0188]

[0189] 9-heptadecanol (1.88 g, 7.33 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.11 g, 10.10 mmol, 1.5 eq), N,N-diisopropylethylamine (2.84 g, 21.99 mmol, 3.0 eq), 4-dimethylaminopyridine (0.27 g, 2.2 mmol, 0.2 eq) were added to a solution of 8-bromooctanoic acid (2.5 g, 8.80 mmol, 1.2 eq) in dichloromethane (30 mL) in sequence, and the mixture was stirred overnight at room temperature. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution, the organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-10% ethyl acetate / hexane) to obtain an oily compound 8-bromooctanoic acid heptadecan-9-ester (2, 1.55 mg, 46%).

[0190] 3. Synthesis of ionizable lipid compound BM008

[0191] The synthetic route is as follows:

[0192]

[0193] (1) To a dichloromethane solution (30 mL) of 4-propoxybenzoic acid (2.50 g, 13.89 mmol, 1.0 eq) were added 5-bromo-1-pentanol (2.32 g, 13.89 mmol, 1.0 eq), N,N-diisopropylethylamine (5.38 g, 41.67 mmol, 3.0 eq), and 4-dimethylaminopyridine (0.39 g, 2.78 mmol, 0.2 eq) in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-10% ethyl acetate / hexane) to obtain an oily compound 5-bromopentyl-4-propoxybenzoate (2.99 g, 66%).

[0194] (2) 2-aminoethanol (5.0 mL, 83 mmol) was added to a solution of 5-bromopentyl-4-propoxybenzoate (1.0 g, 3.05 mmol, 1.0 eq) in ethanol (1.0 mL), and the mixture was stirred at 65° C. for 15 h. The reaction solution was concentrated under vacuum and diluted with ethyl acetate, washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound 5-((2-hydroxyethyl)amino)pentyl-4-propoxybenzoate (840 mg, 89%).

[0195] (3) 8-bromooctanoic acid heptadecan-9-yl ester (892 mg, 1.94 mmol, 1.2 eq) and N,N-diisopropylethylamine (504 mg, 3.88 mmol, 2.0 eq) were added to an ethanol solution (2.0 mL) of 5-((2-hydroxyethyl)amino)pentyl-4-propoxybenzoate (500 mg, 1.62 mmol, 1.0 eq), and the mixture was stirred at 65° C. for 17 h. The reaction solution was concentrated under reduced pressure, and the crude product was dissolved in ethyl acetate, washed with saturated sodium bicarbonate and saturated brine, respectively. The organic layer was separated and dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BM008 (525 mg, 47%).

[0196] BM008:C 42 H 75 NO6, ESI-MS m / z 690.57[M+H] + ; 1 H NMR (400MHz, CDCl3): δ7.90(dd,J=8.8,2.8Hz,2H), 6.84(dd,J=8.8,2.8Hz,2H), 4.79(m,J=6.4Hz,1H), 4.23(t,J=6.4Hz,2H), 3.91(t,J=6.4H z,2H), 3.78(t,J=5.2Hz,2H), 2.89-2.76(m,5H), 2.21(dt,J=7.6,1.6Hz,2H), 1.77-1.42(m,18H), 1.23-1.12(m,30H), 0.81(t,J=6.8Hz,9H).

[0197] 4. Synthesis of ionizable lipid compound BM009

[0198] The synthetic route is as follows:

[0199]

[0200] (1) To a dichloromethane solution (30 mL) of 4-n-pentyloxybenzoic acid (2.0 g, 9.60 mmol, 1.0 eq) were added 5-bromo-1-pentanol (1.60 g, 9.60 mmol, 1.0 eq), N,N-diisopropylethylamine (3.72 g, 28.80 mmol, 3.0 eq), and 4-dimethylaminopyridine (0.23 g, 1.92 mmol, 0.2 eq) in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-10% ethyl acetate / hexane) to obtain an oily compound 5-bromopentyl-4-(pentyloxy)benzoate (1.75 g, 51%).

[0201] (2) 2-aminoethanol (5.0 mL, 83 mmol) was added to a solution of 5-bromopentyl-4-(pentyloxy)benzoate (1.0 g, 2.80 mmol, 1.0 eq) in ethanol (1.0 mL), and the mixture was stirred at 65° C. for 15 h. The reaction solution was concentrated under vacuum and diluted with ethyl acetate, washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound 5-((2-hydroxyethyl)amino)pentyl-4-pentyloxybenzoate (830 mg, 88%).

[0202] (3) 8-bromooctanoic acid heptadecan-9-yl ester (836 mg, 1.78 mmol, 1.2 eq) and N,N-diisopropylethylamine (3.83 mg, 2.96 mmol, 2.0 eq) were added to an ethanol solution (2.0 mL) of 5-((2-hydroxyethyl)amino)pentyl-4-pentyloxybenzoate (500 mg, 1.48 mmol, 1.0 eq), and the mixture was stirred at 65° C. for 17 h. The reaction solution was concentrated under reduced pressure, and the crude product was dissolved in ethyl acetate, washed with saturated sodium bicarbonate and saturated brine, respectively. The organic layer was separated and dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BM009 (446 mg, 42%).

[0203] BM009:C 44 H 79 NO6, ESI-MS m / z 718.61[M+H] + ; 1 H NMR (400MHz, CDCl3): δ7.98 (dd, J=6.8, 2.0Hz, 2H), 6.92 (dd, J=6.8, 2.0Hz, 2H) , 4.87(m,J=6.4Hz,1H), 4.31(t,J=6.4Hz,2H), 4.02(t,J=6.4Hz,2H), 3.84(t,J= 5.2Hz,2H), 2.93-2.83(m,5H), 2.29(dt,J=7.2,1.6Hz,2H), 1.84-1.74(m,6H), 1 .67-1.57(m,4H), 1.55-1.47(m,8H), 1.36-1.26(m,34H), 0.89(t,J=6.8Hz,9H).

[0204] 5. Synthesis of ionizable lipid compound BM010

[0205] The synthetic route is as follows:

[0206]

[0207] (1) To a dichloromethane solution (30 mL) of 4-n-nonyloxybenzoic acid (2.5 g, 9.47 mmol, 1.0 eq), 5-bromo-1-pentanol (1.58 g, 9.47 mmol, 1.0 eq), N,N-diisopropylethylamine (3.66 g, 28.41 mmol, 3.0 eq), and 4-dimethylaminopyridine (0.23 g, 189 mmol, 0.2 eq) were added in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-10% ethyl acetate / hexane) to obtain an oily compound 5-bromopentyl-4-(nonyloxy)benzoate (1.88 g, 48%).

[0208] (2) 2-aminoethanol (5.0 mL, 83 mmol) was added to a solution of 5-bromopentyl-4-(nonyloxy)benzoate (1.0 g, 2.43 mmol, 1.0 eq) in ethanol (1.0 mL), and the mixture was stirred at 65° C. for 15 h. The reaction solution was concentrated under vacuum and diluted with ethyl acetate, washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound 5-((2-hydroxyethyl)amino)pentyl-4-nonyloxybenzoate (813 mg, 85%).

[0209] (3) 8-bromooctanoic acid heptadecan-9-yl ester (704 mg, 1.52 mmol, 1.2 eq) and N,N-diisopropylethylamine (325 mg, 2.54 mmol, 2.0 eq) were added to an ethanol solution (2.0 mL) of 5-((2-hydroxyethyl)amino)pentyl-4-nonyloxybenzoate (500 mg, 1.27 mmol, 1.0 eq), and the mixture was stirred at 65° C. for 17 h. The reaction solution was concentrated under reduced pressure, and the crude product was dissolved in ethyl acetate, washed with saturated sodium bicarbonate and saturated brine, respectively. The organic layer was separated and dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BM010 (432 mg, 44%).

[0210] BM010: C 48 H 87 NO6, ESI-MS m / z 774.66[M+H] + ; 1H NMR (400MHz, CDCl3): δ7.99 (dd, J=8.8, 2.0Hz, 2H), 6.92 (dd, J=8.8, 2.0Hz, 2H), 4.88 ( m,J=6.4Hz,1H), 4.31(t,J=6.4Hz,2H), 4.02(t,J=6.4Hz,2H), 3.74(t,J=5.2Hz,2H), 2 .81-2.69(m,5H), 2.30(dt,J=7.2,2.0Hz,2H), 1.81(m,J=6.8Hz,4H), 1.72-1.59(m,6H ), 1.54-1.46(m,8H), 1.35-1.27(m,42H), 0.90(t,J=6.8Hz,3H), 0.89(t,J=6.4Hz,6H).

[0211] 6. Synthesis of ionizable lipid compound BM011

[0212] The synthetic route is as follows:

[0213]

[0214] (1) To a dichloromethane solution (30 mL) of 4-n-octyloxybenzoic acid (2.5 g, 9.99 mmol, 1.0 eq) was added 5-bromo-1-pentanol (1.66 g, 9.99 mmol, 1.0 eq), N,N-diisopropylethylamine (3.87 g, 29.97 mmol, 3.0 eq), and 4-dimethylaminopyridine (0.24 g, 2.0 mmol, 0.2 eq) in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-10% ethyl acetate / hexane) to obtain an oily compound 5-bromopentyl-4-(octyloxy)benzoate (1.84 g, 46%).

[0215] (2) 2-aminoethanol (5.0 mL, 83 mmol) was added to a solution of 5-bromopentyl-4-(octyloxy)benzoate (1.0 g, 2.50 mmol, 1.0 eq) in ethanol (1.0 mL), and the mixture was stirred at 65° C. for 15 h. The reaction solution was concentrated under vacuum and diluted with ethyl acetate, washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound 5-((2-hydroxyethyl)amino)pentyl-4-nonyloxybenzoate (817 mg, 83%).

[0216] (3) 8-bromooctanoic acid heptadecan-9-yl ester (731 mg, 1.58 mmol, 1.2 eq) and N,N-diisopropylethylamine (335 mg, 2.64 mmol, 2.0 eq) were added to an ethanol solution (2.0 mL) of 5-((2-hydroxyethyl)amino)pentyl-4-nonyloxybenzoate (500 mg, 1.32 mmol, 1.0 eq), and the mixture was stirred at 65° C. for 17 h. The reaction solution was concentrated under reduced pressure, and the crude product was dissolved in ethyl acetate, washed with saturated sodium bicarbonate and saturated brine, respectively. The organic layer was separated and dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BM011 (441 mg, 44%).

[0217] BM011: C 47 H 85 NO6, ESI-MS m / z 760.64[M+H] + ; 1 H NMR (400MHz, CDCl3): δ7.97 (dd, J=8.8, 2.0Hz, 2H), 6.91 (dd, J=8.8, 2.0Hz, 2H), 4.86 (m,J=6.4Hz,1H), 4.30(t,J=6.4Hz,2H), 4.01(t,J=6.4Hz,2H), 3.76(t,J=4.8Hz,2H), 2.87-2.76(m,5H), 2.28(t,J=7.6Hz,2H), 1.80(m,J=6.8Hz,6H), 1.62(q,J=7.2Hz,4H) , 1.55-1.40(m,8H), 1.40-1.19(m,40H), 0.89(t,J=6.4Hz,3H), 0.88(t,J=6.8Hz,6H).

[0218] 7. Synthesis of ionizable lipid compound BM012

[0219] The synthetic route is as follows:

[0220]

[0221] (1) To a dichloromethane solution (30 mL) of 4-n-decyloxybenzoic acid (2.5 g, 8.98 mmol, 1.0 eq) were added 5-bromo-1-pentanol (1.50 g, 8.98 mmol, 1.0 eq), N,N-diisopropylethylamine (3.48 g, 26.94 mmol, 3.0 eq), and 4-dimethylaminopyridine (0.22 g, 1.80 mmol, 0.2 eq) in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-10% ethyl acetate / hexane) to obtain an oily compound 5-bromopentyl-4-(decyloxy)benzoate (1.57 g, 41%).

[0222] (2) 2-aminoethanol (5.0 mL, 83 mmol) was added to a solution of 5-bromopentyl-4-(decyloxy)benzoate (1.0 g, 2.33 mmol, 1.0 eq) in ethanol (1.0 mL), and the mixture was stirred at 65° C. for 15 h. The reaction solution was concentrated under vacuum and diluted with ethyl acetate, washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate. The mixture was concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound 5-((2-hydroxyethyl)amino)pentyl-4-decyloxybenzoate (788 mg, 83%).

[0223] (3) 8-bromooctanoic acid heptadecan-9-yl ester (681 mg, 1.48 mmol, 1.2 eq) and N,N-diisopropylethylamine (317 mg, 2.46 mmol, 2.0 eq) were added to an ethanol solution (2.0 mL) of 5-((2-hydroxyethyl)amino)pentyl-4-decyloxybenzoate (500 mg, 1.23 mmol, 1.0 eq), and the mixture was stirred at 65° C. for 17 h. The reaction solution was concentrated under reduced pressure, and the crude product was dissolved in ethyl acetate, washed with saturated sodium bicarbonate and saturated brine, respectively. The organic layer was separated and dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BM012 (388 mg, 40%).

[0224] BM012: C 49 H 89 NO6, ESI-MS m / z 788.68[M+H] + ; 1H NMR (400MHz, CDCl3): δ7.97 (dd, J=6.8, 2.0Hz, 2H), 6.91 (dd, J=6.8, 2.0Hz, 2H), 4.86 (m ,J=6.4Hz,1H), 4.30(t,J=6.4Hz,2H), 4.01(t,J=6.4Hz,2H), 3.79(t,J=5.2Hz,2H), 2.90 -2.80(m,5H), 2.28(t,J=7.2Hz,2H), 1.85-1.72(m,6H), 1.62(q,J=7.2Hz,4H), 1. 55-1.42(m,8H), 1.35-1.17(m,44H), 0.88(t,J=6.4Hz,3H), 0.87(t,J=6.4Hz,6H).

[0225] 8. Synthesis of ionizable lipid compound BM013

[0226] The synthetic route is as follows:

[0227]

[0228] (1) 3-bromo-1-propanol (1.32 g, 9.47 mmol, 1.0 eq), N,N-diisopropylethylamine (3.66 g, 28.41 mmol, 3.0 eq), and 4-dimethylaminopyridine (0.23 g, 1.89 mmol, 0.2 eq) were added to a dichloromethane solution (30 mL) of 4-n-nonyloxybenzoic acid (2.5 g, 9.47 mmol, 1.0 eq) in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-10% ethyl acetate / hexane) to obtain an oily compound 3-bromopropyl-4-(nonyloxy)benzoate (1.64 g, 45%).

[0229] (2) 2-aminoethanol (5.0 mL, 83 mmol) was added to a solution of 3-bromopropyl-4-(nonyloxy)benzoate (1.0 g, 2.60 mmol, 1.0 eq) in ethanol (1.0 mL), and the mixture was stirred at 65° C. for 15 h. The reaction solution was concentrated under vacuum and diluted with ethyl acetate, washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound 3-((2-hydroxyethyl)amino)propyl-4-nonyloxybenzoate (770 mg, 81%).

[0230] (3) 8-bromooctanoic acid heptadecan-9-yl ester (758 mg, 1.64 mmol, 1.2 eq) and N,N-diisopropylethylamine (353 mg, 2.74 mmol, 2.0 eq) were added to an ethanol solution (2.0 mL) of 3-((2-hydroxyethyl)amino)propyl-4-nonyloxybenzoate (500 mg, 1.37 mmol, 1.0 eq), and the mixture was stirred at 65° C. for 17 h. The reaction solution was concentrated under reduced pressure, and the crude product was dissolved in ethyl acetate, washed with saturated sodium bicarbonate and saturated brine, respectively. The organic layer was separated and dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BM013 (429 mg, 42%).

[0231] BM013: C 46 H 83 NO6, ESI-MS m / z 746.63[M+H] + ; 1 H NMR (400MHz, CDCl3): δ7.97 (dd, J=6.8, 2.0Hz, 2H), 6.91 (dd, J=6.8, 2.4Hz, 2H), 4.86 (m,J=6.4Hz,1H), 4.30(t,J=6.4Hz,2H), 4.01(t,J=6.4Hz,2H), 3.84(t,J=5.2Hz,2H) , 2.97-2.83(m,5H), 2.28(t,J=7.2Hz,2H), 1.87-1.74(m,6H), 1.61(q,J=7.2Hz,4H), 1.55-1.41(m,8H), 1.34-1.22(m,38H), 0.88(t,J=6.8Hz,3H), 0.87(t,J=6.4Hz,6H).

[0232] 9. Synthesis of ionizable lipid compound BM014

[0233] The synthetic route is as follows:

[0234]

[0235] (1) 7-bromo-1-heptanol (1.84 g, 9.47 mmol, 1.0 eq), N,N-diisopropylethylamine (3.66 g, 28.41 mmol, 3.0 eq), and 4-dimethylaminopyridine (0.23 g, 1.98 mmol, 0.2 eq) were added to a dichloromethane solution (30 mL) of 4-n-nonyloxybenzoic acid (2.5 g, 9.47 mmol, 1.0 eq) in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-10% ethyl acetate / hexane) to obtain an oily compound 7-bromoheptyl-4-(nonyloxy)benzoate (1.88 g, 45%).

[0236] (2) 2-aminoethanol (5.0 mL, 83 mmol) was added to a solution of 7-bromoheptyl-4-(nonyloxy)benzoate (1.0 g, 2.26 mmol, 1.0 eq) in ethanol (1.0 mL), and the mixture was stirred at 65° C. for 15 h. The reaction solution was concentrated under vacuum and diluted with ethyl acetate, washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound 7-((2-hydroxyethyl)amino)heptyl-4-nonyloxybenzoate (743 mg, 78%).

[0237] (3) 8-bromooctanoic acid heptadecan-9-yl ester (654 mg, 1.42 mmol, 1.2 eq) and N,N-diisopropylethylamine (304 mg, 2.36 mmol, 2.0 eq) were added to an ethanol solution (2.0 mL) of 7-((2-hydroxyethyl)amino)heptyl-4-nonyloxybenzoate (500 mg, 1.18 mmol, 1.0 eq), and the mixture was stirred at 65° C. for 17 h. The reaction solution was concentrated under reduced pressure, and the crude product was dissolved in ethyl acetate and washed with saturated sodium bicarbonate and saturated brine, respectively. The organic layer was separated and dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BM014 (369 mg, 39%).

[0238] BM014: C 50 H 91 NO6, ESI-MS m / z 802.70[M+H] + ; 1H NMR (400MHz, CDCl3): δ7.90(dd,J=8.8,2.0Hz,2H), 6.83(dd,J=9.2,2.4Hz,2H), 4.79(m,J=6.4Hz,1H), 4.22(t,J=6.4Hz,2H), 3.93(t,J=6.4Hz,2H), 3.4 9(t,J=5.2Hz,2H), 2.57-2.43(m,5H), 2.20(dt,J=7.6,2.0Hz,2H), 1.76-1.3 7(m,18H), 1.28-1.13(m,46H), 0.81(t,J=6.8Hz,3H), 0.80(t,J=6.8Hz,6H).

[0239] 10. Synthesis of ionizable lipid compound BM015

[0240] The synthetic route is as follows:

[0241]

[0242] (1) 7-bromo-1-heptanol (2.33 g, 12.00 mmol, 1.0 eq), N,N-diisopropylethylamine (4.64 g, 36.00 mmol, 3.0 eq) and 4-dimethylaminopyridine (0.29 g, 2.4 mmol, 0.2 eq) were added to a dichloromethane solution (30 mL) of 4-n-pentyloxybenzoic acid (2.5 g, 12.00 mmol, 1.0 eq) in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-10% ethyl acetate / hexane) to obtain an oily compound 7-bromoheptyl-4-(pentyloxy)benzoate (1.92 g, 46%).

[0243] (2) 2-aminoethanol (5.0 mL, 83 mmol) was added to a solution of 7-bromoheptyl-4-(pentyloxy)benzoate (1.0 g, 2.13 mmol, 1.0 eq) in ethanol (1.0 mL), and the mixture was stirred at 65° C. for 15 h. The reaction solution was concentrated under vacuum and diluted with ethyl acetate, washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound 7-((2-hydroxyethyl)amino)heptyl-4-pentyloxybenzoate (662 mg, 85%).

[0244] (3) 8-bromooctanoic acid heptadecan-9-yl ester (759 mg, 1.64 mmol, 1.2 eq) and N,N-diisopropylethylamine (353 mg, 2.74 mmol, 2.0 eq) were added to an ethanol solution (2.0 mL) of 7-((2-hydroxyethyl)amino)heptyl-4-pentyloxybenzoate (500 mg, 1.37 mmol, 1.0 eq), and the mixture was stirred at 65° C. for 17 h. The reaction solution was concentrated under reduced pressure, and the crude product was dissolved in ethyl acetate, washed with saturated sodium bicarbonate and saturated brine, respectively. The organic layer was separated and dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BM015 (419 mg, 41%).

[0245] BM015: C 46 H 83 NO6, ESI-MS m / z 746.63[M+H] + ; 1 H NMR (400MHz, CDCl3): δ7.97 (dd, J=8.8, 2.0Hz, 2H), 6.91 (dd, J=8.8, 2.0Hz, 2H), 4. 86(m,J=6.4Hz,1H), 4.28(t,J=6.4Hz,2H), 4.01(t,J=6.4Hz,2H), 3.83(brs,2H), 2 .95-2.85(m,5H), 2.28(t,J=7.6Hz,2H), 1.85-1.57(m,12H), 1.54-1.48(m,4H), 1. 46-1.37(m,8H), 1.35-1.22(m,32H), 0.94(t,J=6.8Hz,3H), 0.88(t,J=6.8Hz,6H).

[0246] 11. Synthesis of ionizable lipid compound BM016

[0247] The synthetic route is as follows:

[0248]

[0249] (1) To a solution of 5-bromo-1-pentanol (334 mg, 2.0 mmol, 1.0 eq) in dichloromethane (5.0 mL) were added 4-nonyloxybenzoic acid (634 mg, 2.4 mmol, 1.2 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (576 mg, 3.0 mmol, 1.5 eq), N,N-diisopropylethylamine (606 mg, 6.0 mmol, 3.0 eq), 4-dimethylaminopyridine (49 mg, 0.4 mmol, 0.2 eq), and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-10% ethyl acetate / hexane) to obtain an oily compound 5-bromopentyl-4-(nonyloxy)benzoate (340 mg, 41%).

[0250] (2) To a solution of 8-bromooctanoic acid (2.0 g, 8.96 mmol, 1.0 eq) in dichloromethane (25 mL) were added 7-pentadecanol (2.92 g, 11.38 mmol, 1.27 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.58 g, 13.44 mmol, 1.5 eq), N,N-diisopropylethylamine (3.47 g, 26.88 mmol, 3.0 eq), and 4-dimethylaminopyridine (0.22 g, 1.79 mmol, 0.2 eq), and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-10% ethyl acetate / hexane) to obtain an oily compound 8-bromooctanoic acid pentadecane-7-ester (1.9 g, 46%).

[0251] (3) 2-aminoethanol (5.0 mL, 83 mmol) and ethanol (1.0 mL) were added to 8-bromooctanoic acid pentadecane-7-ester (1.03 g, 2.25 mmol, 1.0 eq) in sequence, and the mixture was stirred at 65° C. for 15 h. The reaction solution was concentrated under vacuum and diluted with ethyl acetate, washed with water and saturated brine respectively, and then dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound 7-((2-hydroxyethyl)amino)octanoic acid pentadecane-7-ester (0.82 g, 87%).

[0252] (4) 5-bromopentyl-4-(nonyloxy)benzoate (496 mg, 1.2 mmol, 1.2 eq) and N,N-diisopropylethylamine (258 mg, 2.0 mmol, 2.0 eq) were added to a solution of 8-((2-hydroxyethyl)amino)octanoic acid pentadecane-7-ester (442 mg, 1.0 mmol, 1.0 eq) in ethanol (2.0 mL) in sequence, and the mixture was stirred at 65° C. for 17 h. The reaction solution was concentrated under reduced pressure, and the crude product was dissolved in ethyl acetate, washed with saturated sodium bicarbonate and saturated brine, respectively. The organic layer was separated and dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BM016 (325 mg, 42%).

[0253] BM016: C 46 H 83 NO6, ESI-MS m / z 746.7[M+H] + ; 1 H NMR (400MHz, CDCl3): δ7.97(dd,J=8.8,2.0Hz,2H), 6.91(dd,J=9.2,2.4Hz,2H), 4.86(quint,J=6.4Hz,1H), 4.28(t,J=6.4Hz,2H), 4.01(t,J=6.8Hz,2H) , 3.87(t,J=4.8Hz,2H), 3.01-2.87(m,5H), 2.28(t,J=7.2Hz,2H), 1.82-1.4 6(m,18H), 1.35-1.23(m,38H), 0.88(d,J=6.8Hz,3H), 0.87(d,J=6.8Hz,6H).

[0254] 12. Synthesis of ionizable lipid compound BM020

[0255] The synthetic route is as follows:

[0256]

[0257] (1) To a dichloromethane solution (30 mL) of 4-undecyloxybenzoic acid (2.5 g, 8.55 mmol, 1.0 eq) were added 5-bromo-1-pentanol (1.43 g, 8.55 mmol, 1.0 eq), N,N-diisopropylethylamine (3.31 g, 25.56 mmol, 3.0 eq), and 4-dimethylaminopyridine (0.21 g, 1.71 mmol, 0.2 eq) in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-10% ethyl acetate / hexane) to obtain an oily compound 5-bromopentyl-4-(undecyloxy)benzoate (1.43 g, 38%).

[0258] (2) 2-aminoethanol (5.0 mL, 83 mmol) was added to a solution of 5-bromopentyl-4-(undedecyloxy)benzoate (1.0 g, 2.26 mmol, 1.0 eq) in ethanol (1.0 mL), and the mixture was stirred at 65° C. for 15 h. The reaction solution was concentrated under vacuum and diluted with ethyl acetate, washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound 5-((2-hydroxyethyl)amino)pentyl-4-(undedecyloxy)benzoate (788 mg, 83%).

[0259] (3) 8-bromooctanoic acid heptadecan-9-yl ester (655 mg, 1.42 mmol, 1.2 eq) and N,N-diisopropylethylamine (304 mg, 2.36 mmol, 2.0 eq) were added to an ethanol solution (2.0 mL) of 5-((2-hydroxyethyl)amino)pentyl-4-(undecanyloxy)benzoate (500 mg, 1.18 mmol, 1.0 eq), and the mixture was stirred at 65° C. for 17 h. The reaction solution was concentrated under reduced pressure, and the crude product was dissolved in ethyl acetate, washed with saturated sodium bicarbonate and saturated brine, respectively. The organic layer was separated and dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BM020 (350 mg, 40%).

[0260] BM020: C 50 H 91 NO6, ESI-MS m / z 802.8[M+H] + ; 1H NMR (400MHz, CDCl3): δ8.01 (dd, J=8.8, 2.0Hz, 2H), 6.94 (dd, J=8.8, 2.4Hz, 2H) , 4.90(m,J=6.4Hz,1H), 4.32(t,J=6.4Hz,2H), 4.04(t,J=6.4Hz,2H), 3.61(t,J= 5.2Hz,2H), 2.67-2.56(m,6H), 2.31(t,J=7.6Hz,2H), 1.87-1.78(m,4H), 1.66- 1.45(m,14H), 1.37-1.29(m,45H), 0.92(t,J=6.8Hz,3H), 0.91(t,J=6.4Hz,6H).

[0261] 13. Synthesis of ionizable lipid compound BM021

[0262] The synthetic route is as follows:

[0263]

[0264] (1) To a dichloromethane solution (30 mL) of 4-dodecyloxybenzoic acid (2.5 g, 8.16 mmol, 1.0 eq) were added 5-bromo-1-pentanol (1.36 g, 8.16 mmol, 1.0 eq), N,N-diisopropylethylamine (3.16 g, 24.48 mmol, 3.0 eq), and 4-dimethylaminopyridine (0.20 g, 1.63 mmol, 0.2 eq) in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-10% ethyl acetate / hexane) to obtain an oily compound 5-bromopentyl-4-(dodecyloxy)benzoate (1.30 g, 35%).

[0265] (2) 2-aminoethanol (5.0 mL, 83 mmol) was added to a solution of 5-bromopentyl-4-(dodecyloxy)benzoate (1.0 g, 2.19 mmol, 1.0 eq) in ethanol (1.0 mL), and the mixture was stirred at 65° C. for 15 h. The reaction solution was concentrated under vacuum and diluted with ethyl acetate, washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound 5-((2-hydroxyethyl)amino)pentyl-4-(undedecyloxy)benzoate (715 mg, 75%).

[0266] (3) 8-bromooctanoic acid heptadecan-9-yl ester (637 mg, 1.38 mmol, 1.2 eq) and N,N-diisopropylethylamine (297 mg, 2.30 mmol, 2.0 eq) were added to an ethanol solution (2.0 mL) of 5-((2-hydroxyethyl)amino)pentyl-4-(dodecyloxy)benzoate (500 mg, 1.15 mmol, 1.0 eq), and the mixture was stirred at 65° C. for 17 h. The reaction solution was concentrated under reduced pressure, and the crude product was dissolved in ethyl acetate, washed with saturated sodium bicarbonate and saturated brine, respectively. The organic layer was separated and dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BM021 (338 mg, 36%).

[0267] BM021: C 51 H 93 NO6, ESI-MS m / z 816.71[M+H] + ; 1 H NMR (400MHz, CDCl3): δ7.97(dd,J=8.8,2.0Hz,2H), 6.91(dd,J=9.2,2.4Hz,2H), 4.86(m,J=6.4Hz,1H), 4.30(t,J=6.4Hz,2H), 4.01(t,J=6.6Hz,2H), 3 .84(t,J=5.2Hz,2H), 2.97-2.83(m,5H), 2.28(t,J=7.2Hz,2H), 1.85-1.44 (m,20H), 1.40-1.18(m,46H), 0.88(t,J=7.8Hz,3H), 0.87(t,J=7.8Hz,6H).

[0268] 14. Synthesis of ionizable lipid compound BM022

[0269] The synthetic route is as follows:

[0270]

[0271] (1) To a dichloromethane solution (30 mL) of 4-n-decyloxybenzoic acid (2.5 g, 8.98 mmol, 1.0 eq) were added 5-bromo-1-pentanol (1.50 g, 8.98 mmol, 1.0 eq), N,N-diisopropylethylamine (3.48 g, 26.94 mmol, 3.0 eq), and 4-dimethylaminopyridine (0.22 g, 1.80 mmol, 0.2 eq) in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-10% ethyl acetate / hexane) to obtain an oily compound 5-bromopentyl-4-(decyloxy)benzoate (1.57 g, 41%).

[0272] (2) 2-aminoethanol (5.0 mL, 83 mmol) was added to a solution of 5-bromopentyl-4-(decyloxy)benzoate (1.0 g, 2.33 mmol, 1.0 eq) in ethanol (1.0 mL), and the mixture was stirred at 65° C. for 15 h. The reaction solution was concentrated under vacuum and diluted with ethyl acetate, washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate. The mixture was concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound 5-((2-hydroxyethyl)amino)pentyl-4-decyloxybenzoate (788 mg, 83%).

[0273] (3) 8-bromooctanoic acid heptadecan-9-yl ester (681 mg, 1.48 mmol, 1.2 eq) and N,N-diisopropylethylamine (317 mg, 2.46 mmol, 2.0 eq) were added to an ethanol solution (2.0 mL) of 5-((2-hydroxyethyl)amino)pentyl-4-decyloxybenzoate (500 mg, 1.23 mmol, 1.0 eq), and the mixture was stirred at 65° C. for 17 h. The reaction solution was concentrated under reduced pressure, and the crude product was dissolved in ethyl acetate, washed with saturated sodium bicarbonate and saturated brine, respectively. The organic layer was separated and dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BM022 (388 mg, 40%).

[0274] BM022: C 50 H 91 NO6, ESI-MS m / z 802.70[M+H] + ; 1H NMR (400MHz, CDCl3): δ7.97 (dd, J=8.8, 2.0Hz, 2H), 6.91 (dd, J=9.2, 2.4Hz, 2H) , 4.86(m,J=6.4Hz,1H), 4.28(t,J=6.4Hz,2H), 4.01(t,J=6.4Hz,2H), 3.87(t,J= 4.8Hz,2H), 3.02-2.87(m,5H), 2.28(t,J=7.2Hz,2H), 1.83-1.68(m,8H), 1.65- 1.42(m,12H), 1.38-1.20(m,44H), 0.88(t,J=7.8Hz,3H), 0.87(t,J=7.8Hz,6H).

[0275] 15. Synthesis of ionizable lipid compound BM054

[0276] The synthetic route is as follows:

[0277]

[0278] (1) To a dichloromethane solution (30 mL) of 4-octyloxybenzoic acid (3.47 g, 13.89 mmol, 1.0 eq) were added 5-bromo-1-pentanol (2.32 g, 13.89 mmol, 1.0 eq), N,N-diisopropylethylamine (5.38 g, 41.67 mmol, 3.0 eq), and 4-dimethylaminopyridine (0.39 g, 2.78 mmol, 0.2 eq) in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-10% ethyl acetate / hexane) to obtain an oily compound 5-bromopentyl-4-octyloxybenzoate (3.65 g, 66%).

[0279] (2) To a solution of 7-bromoheptanol (1.72 g, 8.80 mmol, 1.2 eq) in dichloromethane (30 mL) were added 2-octyldecanoic acid (2.08 g, 7.33 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.11 g, 10.10 mmol, 1.5 eq), N,N-diisopropylethylamine (2.84 g, 21.99 mmol, 3.0 eq), and 4-dimethylaminopyridine (0.27 g, 2.2 mmol, 0.2 eq), and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-10% ethyl acetate / hexane) to obtain an oily compound 2-octyldecanoic acid-7-heptyl ester (1.87 g, 46%).

[0280] (3) 3-Aminopropanol (5.0 mL, 83 mmol) was added to a solution of the intermediate 2-octyldecanoic acid 7-heptyl ester (1.41 g, 3.05 mmol, 1.0 eq) in ethanol (1.0 mL), and the mixture was stirred at 65° C. for 15 h. The reaction solution was concentrated under vacuum and diluted with ethyl acetate, washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound 2-octyldecanoic acid-7-((3-hydroxypropyl)amino)heptyl ester (1.24 g, 89%).

[0281] (4) To an ethanol solution (2.0 mL) of 7-((3-hydroxypropyl)amino)heptyl 2-octyldecanoate (260 mg, 0.57 mmol, 1.0 eq), the intermediate 5-bromopentyl-4-octyloxybenzoate (543 mg, 0.68 mmol, 1.2 eq) and N,N-diisopropylethylamine (292 mg, 1.13 mmol, 2.0 eq) were added, and the mixture was stirred at 65° C. for 17 h. The reaction solution was concentrated under reduced pressure, and the crude product was dissolved in ethyl acetate and washed with saturated sodium bicarbonate and saturated brine, respectively. The organic layer was separated and dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BM054 (158 mg, 35%).

[0282] BM054: C 48 H 87 NO6, ESI-MS m / z 774.7[M+H] + ; 1 H NMR (400MHz, CDCl3): δ7.97(dd,J=8.8,2.4Hz,2H), 6.91(dd,J=8.8,2.0Hz,2H), 4.32(t,J=6.4Hz,2H), 4.05(t,J=6.4Hz,2H), 4.01(t,J=6.4Hz,2 H), 3.86(t,J=5.6Hz,2H), 3.26(t,J=6.8Hz,2H), 3.13-3.06(m,3H), 2.34 -2.27(m,1H), 1.94-1.78(m,8H), 1.64-1.25(m,52H), 0.90-0.86(m,9H).

[0283] 16. Synthesis of ionizable lipid compound BM023

[0284] The synthetic route is as follows:

[0285]

[0286] (1) 6-bromo-1-hexanol (1.63 g, 8.98 mmol, 1.0 eq), N,N-diisopropylethylamine (3.48 g, 26.94 mmol, 3.0 eq), and 4-dimethylaminopyridine (0.22 g, 1.80 mmol, 0.2 eq) were added to a dichloromethane solution (30 mL) of 4-n-decyloxybenzoic acid (2.5 g, 8.98 mmol, 1.0 eq) in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-10% ethyl acetate / hexane) to obtain an oily compound 6-bromohexyl-4-(decyloxy)benzoate (1.70 g, 43%).

[0287] (2) 2-aminoethanol (5.0 mL, 83 mmol) was added to a solution of 6-bromohexyl-4-(decyloxy)benzoate (1.0 g, 2.26 mmol, 1.0 eq) in ethanol (1.0 mL), and the mixture was stirred at 65° C. for 15 h. The reaction solution was concentrated under vacuum and diluted with ethyl acetate, washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate. The mixture was concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound 6-((2-hydroxyethyl)amino)hexyl-4-decyloxybenzoate (734 mg, 77%).

[0288] (3) 8-bromooctanoic acid heptadecan-9-yl ester (660 mg, 1.43 mmol, 1.2 eq) and N,N-diisopropylethylamine (307 mg, 2.38 mmol, 2.0 eq) were added to an ethanol solution (2.0 mL) of 6-((2-hydroxyethyl)amino)hexyl-4-decyloxybenzoate (500 mg, 1.19 mmol, 1.0 eq), and the mixture was stirred at 65° C. for 17 h. The reaction solution was concentrated under reduced pressure, and the crude product was dissolved in ethyl acetate, washed with saturated sodium bicarbonate and saturated brine, respectively. The organic layer was separated and dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BM023 (372 mg, 39%).

[0289] BM023: C 50 H 91 NO6, ESI-MS m / z 802.9[M+H] + ; 1H NMR (400MHz, CDCl3): δ8.01 (dd, J=8.8, 2.4Hz, 2H), 6.94 (dd, J=8.4, 2.0Hz, 2H), 4.9 0(m,J=6.4Hz,1H), 4.31(t,J=6.4Hz,2H), 4.04(t,J=6.4Hz,2H), 3.58(s,2H), 2.64(s ,2H), 2.52(s,4H), 2.31(t,J=7.2Hz,2H), 1.87-1.76(m,4H), 1.65(t,J=7.2Hz,2H), 1 .54-1.45(m,11H), 1.38-1.29(m,46H), 0.92(t,J=6.8Hz,3H), 0.91(t,J=6.4Hz,6H).

[0290] 17. Synthesis of ionizable lipid compound BM024

[0291] The synthetic route is as follows:

[0292]

[0293] (1) To a solution of 5-bromo-1-pentanol (334 mg, 2.0 mmol, 1.0 eq) in dichloromethane (5.0 mL) were added 4-nonyloxybenzoic acid (634 mg, 2.4 mmol, 1.2 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (576 mg, 3.0 mmol, 1.5 eq), N,N-diisopropylethylamine (606 mg, 6.0 mmol, 3.0 eq), 4-dimethylaminopyridine (49 mg, 0.4 mmol, 0.2 eq), and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-10% ethyl acetate / hexane) to obtain an oily compound 5-bromopentyl-4-(nonyloxy)benzoate (340 mg, 41%).

[0294] (2) To a solution of 9-bromononanoic acid (2.12 g, 8.96 mmol, 1.0 eq) in dichloromethane (25 mL) were added 9-heptadecanol (2.92 g, 11.38 mmol, 1.27 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.58 g, 13.44 mmol, 1.5 eq), N,N-diisopropylethylamine (3.47 g, 26.88 mmol, 3.0 eq), and 4-dimethylaminopyridine (0.22 g, 1.79 mmol, 0.2 eq), and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-10% ethyl acetate / hexane) to obtain an oily compound 9-bromononanoic acid heptadecane-9-ester (1.8 g, 43%).

[0295] (3) 2-Aminoethanol (5.0 mL, 83 mmol) and ethanol (1.0 mL) were added to 9-bromononanoic acid heptadecane-9-ester (1.07 g, 2.25 mmol, 1.0 eq) in sequence, and the mixture was stirred at 65° C. for 15 h. The reaction solution was concentrated under vacuum and diluted with ethyl acetate, washed with water and saturated brine respectively, and then dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound 9-((2-hydroxyethyl)amino)nonanoic acid heptadecane-9-ester (0.85 g, 83%).

[0296] (4) 5-bromopentyl-4-(nonyloxy)benzoate (496 mg, 1.2 mmol, 1.2 eq) and N,N-diisopropylethylamine (258 mg, 2.0 mmol, 2.0 eq) were added to a solution of 9-((2-hydroxyethyl)amino)nonanoic acid heptadecane-9-ester (456 mg, 1.0 mmol, 1.0 eq) in ethanol (2.0 mL) in sequence, and the mixture was stirred at 65° C. for 17 h. The reaction solution was concentrated under reduced pressure, and the crude product was dissolved in ethyl acetate, washed with saturated sodium bicarbonate and saturated brine, respectively. The organic layer was separated and dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BM024 (300 mg, 38%).

[0297] BM024: C 49 H 89 NO6, ESI-MS m / z 788.68[M+H] + ; 1H NMR (400MHz, CDCl3): δ7.90 (dd, J=8.8, 2.4Hz, 2H), 6.84 (dd, J=8.8, 2.0Hz, 2H), 4.7 9(m,J=6.4Hz,1H), 4.23(t,J=6.4Hz,2H), 3.94(t,J=6.4Hz,2H), 3.75(t,J=5.2Hz,2 H), 2.89-2.72(m,5H), 2.20(t,J=7.6Hz,2H), 1.78-1.66(m,6H), 1.64-1.50(m,4H), 1.46-1.35(m,8H), 1.33-1.11(m,44H), 0.82(t,J=6.4Hz,3H), 0.81(t,J=6.8Hz,6H).

[0298] 18. Synthesis of ionizable lipid compound BM025

[0299] The synthetic route is as follows:

[0300]

[0301] (1) To a solution of 5-bromo-1-pentanol (334 mg, 2.0 mmol, 1.0 eq) in dichloromethane (5.0 mL) were added 4-nonyloxybenzoic acid (634 mg, 2.4 mmol, 1.2 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (576 mg, 3.0 mmol, 1.5 eq), N,N-diisopropylethylamine (606 mg, 6.0 mmol, 3.0 eq), 4-dimethylaminopyridine (49 mg, 0.4 mmol, 0.2 eq), and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-10% ethyl acetate / hexane) to obtain an oily compound 5-bromopentyl-4-(nonyloxy)benzoate (340 mg, 41%).

[0302] (2) To a solution of 8-bromooctanoic acid (2.0 g, 8.96 mmol, 1.0 eq) in dichloromethane (25 mL) were added nonadecan-10-ol (3.23 g, 11.38 mmol, 1.27 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.58 g, 13.44 mmol, 1.5 eq), N,N-diisopropylethylamine (3.47 g, 26.88 mmol, 3.0 eq), and 4-dimethylaminopyridine (0.22 g, 1.79 mmol, 0.2 eq) in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-10% ethyl acetate / hexane) to obtain an oily compound 8-bromooctanoic acid nonadecan-10-ester (1.98 g, 45%).

[0303] (3) 2-aminoethanol (5.0 mL, 83 mmol) and ethanol (1.0 mL) were added to 8-bromooctanoic acid nonadecan-10-ester (1.1 g, 2.25 mmol, 1.0 eq) in sequence, and the mixture was stirred at 65° C. for 15 h. The reaction solution was concentrated under vacuum and diluted with ethyl acetate, washed with water and saturated brine respectively, and then dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound 8-((2-hydroxyethyl)amino)octanoic acid nonadecan-10-ester (0.89 g, 85%).

[0304] (4) 5-bromopentyl-4-(nonyloxy)benzoate (496 mg, 1.2 mmol, 1.2 eq) and N,N-diisopropylethylamine (258 mg, 2.0 mmol, 2.0 eq) were added to a solution of 8-((2-hydroxyethyl)amino)octanoic acid nonadecan-10-ester (470 mg, 1.0 mmol, 1.0 eq) in ethanol (2.0 mL) in sequence, and the mixture was stirred at 65° C. for 17 h. The reaction solution was concentrated under reduced pressure, and the crude product was dissolved in ethyl acetate and washed with saturated sodium bicarbonate and saturated brine, respectively. The organic layer was separated and dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BM025 (320 mg, 40%).

[0305] BM025: C 50 H 91 NO6, ESI-MS m / z 802.69[M+H] + ; 1H NMR (400MHz, CDCl3): δ7.90 (dd, J=8.8, 2.0Hz, 2H), 6.84 (dd, J=8.4, 2.0Hz, 2H), 4.79 (m,J=6.4Hz,1H), 4.23(t,J=6.4Hz,2H), 3.93(t,J=6.4Hz,2H), 3.67(d,J=6.4Hz,2H), 2.81-2.61(m,5H), 2.20(t,J=7.6Hz,2H), 1.73(m,J=7.2Hz,6H), 1.54(m,J=7.6Hz,4H) , 1.46-1.36(m,8H), 1.32-1.12(m,46H), 0.82(t,J=6.4Hz,3H), 0.81(t,J=6.4Hz,6H).

[0306] 19. Synthesis of ionizable lipid compound BM026

[0307] The synthetic route is as follows:

[0308]

[0309] (1) 8-bromo-1-octanol (1.98 g, 9.47 mmol, 1.0 eq), N,N-diisopropylethylamine (3.66 g, 28.41 mmol, 3.0 eq), and 4-dimethylaminopyridine (0.23 g, 1.98 mmol, 0.2 eq) were added to a dichloromethane solution (30 mL) of 4-n-nonyloxybenzoic acid (2.5 g, 9.47 mmol, 1.0 eq) in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-10% ethyl acetate / hexane) to obtain an oily compound 8-bromooctyl-4-(nonyloxy)benzoate (1.52 g, 36%).

[0310] (2) 2-aminoethanol (5.0 mL, 83 mmol) was added to a solution of 8-bromooctyl-4-(nonyloxy)benzoate (1.0 g, 2.20 mmol, 1.0 eq) in ethanol (1.0 mL), and the mixture was stirred at 65° C. for 15 h. The reaction solution was concentrated under vacuum and diluted with ethyl acetate, washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate. The mixture was concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound 8-((2-hydroxyethyl)amino)octyl-4-nonyloxybenzoate (738 mg, 77%).

[0311] (3) 8-((2-hydroxyethyl)amino)octyl-4-nonyloxybenzoate (500 mg, 1.15 mmol, 1.0 eq) in ethanol (2.0 mL) was added with 8-bromooctanoic acid heptadecan-9-ester (637 mg, 1.38 mmol, 1.2 eq) and N,N-diisopropylethylamine (297 mg, 2.30 mmol, 2.0 eq), and the mixture was stirred at 65° C. for 17 h. The reaction solution was concentrated under reduced pressure, and the crude product was dissolved in ethyl acetate, washed with saturated sodium bicarbonate and saturated brine, respectively. The organic layer was separated and dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BM026 (375 mg, 40%).

[0312] BM026: C 51 H 93 NO6, ESI-MS m / z 816.8[M+H] + ; 1 H NMR (400MHz, CDCl3): δ8.01 (dd, J=8.8, 2.4Hz, 2H), 6.93 (dd, J=8.4, 2.0Hz, 2H), 4. 90(m,J=6.4Hz,1H), 4.31(t,J=6.4Hz,2H), 4.04(t,J=6.4Hz,2H), 3.64(s,2H), 2.7 1-2.58(m,6H), 2.31(t,J=7.6Hz,2H), 1.87-1.74(m,4H), 1.65(t,J=7.6Hz,2H), 1. 53-1.45(m,11H), 1.36-1.29(m,47H), 0.92(t,J=6.4Hz,3H), 0.91(t,J=6.8Hz,6H).

[0313] 20. Synthesis of ionizable lipid compound BM049

[0314] The synthetic route is as follows:

[0315]

[0316] (1) To a dichloromethane solution (20 mL) of 8-bromooctanoic acid (1.0 g, 4.5 mmol, 1.0 eq), heptane-9-ol (1.15 g, 4.5 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.3 g, 6.8 mmol, 1.5 eq), and 4-dimethylaminopyridine (0.11 g, 0.9 mmol, 0.2 eq) were added in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-1% ethyl acetate / petroleum ether) to obtain an oily compound 9-bromooctanoic acid heptyl ester (1.7 g, 82.1%).

[0317] (2) 2-aminoethanol (2.3 g, 37.0 mmol, 10.0 eq) and potassium carbonate (0.76 g, 5.5 mmol, 1.5 eq) were added to a solution of 9-bromooctanoic acid heptyl ester (1.7 g, 3.7 mmol, 1.0 eq) in N,N-dimethylformamide (35 mL), and the mixture was stirred at room temperature overnight. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic phase was washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound 8-((2-hydroxyethyl)amino)octanoic acid heptadecan-9-ester (1.0 g, 61.3%).

[0318] (3) 8-bromooctan-1-ol (0.75 g, 3.6 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.04 g, 5.4 mmol, 1.5 eq), and 4-dimethylaminopyridine (88 mg, 0.72 mmol, 0.2 eq) were added to a dichloromethane solution (20 mL) of 4-(decyloxy)benzoic acid (1.0 g, 3.6 mmol, 1.0 eq) in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-1% ethyl acetate / petroleum ether) to obtain a solid compound 8-bromooctyl-4-(decyloxy)benzoate (1.4 g, 83.3%).

[0319] (4) To a solution of 8-bromooctyl-4-(decyloxy)benzoate (0.5 g, 1.1 mmol, 1.0 eq) in N,N-dimethylformamide (5.0 mL) were added 8-((2-hydroxyethyl)amino)octanoic acid heptadecan-9-ester (0.5 g, 1.1 mmol, 1.0 eq) and potassium carbonate (0.23 g, 1.65 mmol, 1.5 eq), and the mixture was stirred at 40° C. overnight. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic phase was washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BM049 (200 mg, 22.6%).

[0320] BM049:C 52 H 95 NO6, ESI-MS m / z 830.9[M+H] + ; 1 H NMR (400MHz, CDCl3): δ8.01 (dd, J=8.8, 2.0Hz, 2H), 6.94 (dd, J=8.8, 2.0Hz, 2 H), 4.89(m,J=6.0Hz,1H), 4.31(t,J=6.4Hz,2H), 4.04(t,J=6.4Hz,2H), 3.71( s,2H), 2.79-2.67(m,5H), 2.31(t,J=7.2Hz,2H), 1.87-1.75(m,4H), 1.66-1. 45(m,14H), 1.38-1.29(m,50H), 0.92(t,J=6.8Hz,3H), 0.91(t,J=6.8Hz,6H).

[0321] 21. Synthesis of ionizable lipid compound BM050

[0322] The synthetic route is as follows:

[0323]

[0324] (1) To a dichloromethane solution (20 mL) of 8-bromooctanoic acid (1.0 g, 4.5 mmol, 1.0 eq), heptane-9-ol (1.15 g, 4.5 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.3 g, 6.8 mmol, 1.5 eq), and 4-dimethylaminopyridine (0.11 g, 0.9 mmol, 0.2 eq) were added in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-1% ethyl acetate / petroleum ether) to obtain an oily compound 9-bromooctanoic acid heptyl ester (1.7 g, 82.1%).

[0325] (2) 2-aminoethanol (2.3 g, 37.0 mmol, 10.0 eq) and potassium carbonate (0.76 g, 5.5 mmol, 1.5 eq) were added to a solution of 9-bromooctanoic acid heptyl ester (1.7 g, 3.7 mmol, 1.0 eq) in N,N-dimethylformamide (35 mL), and the mixture was stirred at room temperature overnight. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic phase was washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound 8-((2-hydroxyethyl)amino)octanoic acid heptadecan-9-ester (1.0 g, 61.3%).

[0326] (3) 9-bromononan-1-ol (0.84 g, 3.8 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.09 g, 5.7 mmol, 1.5 eq), and 4-dimethylaminopyridine (93 mg, 0.76 mmol, 0.2 eq) were added to a dichloromethane solution (20 mL) of 4-(nonyloxy)benzoic acid (1.0 g, 3.8 mmol, 1.0 eq) in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-1% ethyl acetate / petroleum ether) to obtain a solid compound 9-bromononyl-4-(nonyloxy)benzoate (1.5 g, 84.7%).

[0327] (4) 8-((2-hydroxyethyl)amino)octanoic acid heptadecan-9-ester (0.5 g, 1.1 mmol, 1.0 eq) and potassium carbonate (0.23 g, 1.65 mmol, 1.5 eq) were added to a solution of 9-bromononyl-4-(nonyloxy)benzoate (0.5 g, 1.1 mmol, 1.0 eq) in N,N-dimethylformamide (5.0 mL), and the mixture was stirred at 40° C. overnight. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic phase was washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BM050 (210 mg, 23.7%).

[0328] BM050: C 52 H 95 NO6, ESI-MS m / z 831.0[M+H] + ; 1H NMR (400MHz, CDCl3): δ8.01 (dd, J=8.8, 2.0Hz, 2H), 6.94 (dd, J=9.2, 2.4Hz, 2 H), 4.90(m,J=6.4Hz,1H), 4.31(t,J=6.4Hz,2H), 4.04(t,J=6.4Hz,2H), 3.72( s,2H), 2.68-2.79(m,5H), 2.31(t,J=7.6Hz,2H), 1.87-1.75(m,4H), 1.67-1. 49(m,14H), 1.35-1.29(m,50H), 0.92(t,J=6.8Hz,3H), 0.91(t,J=6.8Hz,6H).

[0329] 22. Synthesis of ionizable lipid compound BM051

[0330] The synthetic route is as follows:

[0331]

[0332] (1) 8-bromooctan-1-ol (0.79 g, 3.8 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.09 g, 5.7 mmol, 1.5 eq), and 4-dimethylaminopyridine (93 mg, 0.76 mmol, 0.2 eq) were added to a dichloromethane solution (20 mL) of 4-(nonyloxy)benzoic acid (1.0 g, 3.8 mmol, 1.0 eq) in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-1% ethyl acetate / petroleum ether) to obtain a solid compound 8-bromooctyl-4-(nonyloxy)benzoate (1.5 g, 87.2%).

[0333] (2) 2-aminoethanol (2.0 g, 33.0 mmol, 10.0 eq) and potassium carbonate (0.68 g, 5.0 mmol, 1.5 eq) were added to a solution of 8-bromooctyl-4-(nonyloxy)benzoate (1.5 g, 3.3 mmol, 1.0 eq) in N,N-dimethylformamide (30 mL), and the mixture was stirred at room temperature overnight. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic phase was washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain a solid compound 8-((2-hydroxyethyl)amino)octyl-4-(nonyloxy)benzoate (1.1 g, 76.5%).

[0334] (3) To a dichloromethane solution (20 mL) of 9-bromononanoic acid (1.0 g, 4.2 mmol, 1.0 eq), heptadecan-9-ol (1.08 g, 4.2 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.2 g, 6.3 mmol, 1.5 eq), and 4-dimethylaminopyridine (102 mg, 0.84 mmol, 0.2 eq) were added in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-1% ethyl acetate / petroleum ether) to obtain an oily compound, 9-bromononanoic acid heptadecan-9-ester (1.7 g, 85%).

[0335] (4) 8-((2-hydroxyethyl)amino)octyl-4-(nonyloxy)benzoate (0.46 g, 1.05 mmol, 1.0 eq) and potassium carbonate (0.22 g, 1.58 mmol, 1.5 eq) were added to a solution of 9-bromononanoic acid heptadecane-9-ester (0.5 g, 1.05 mmol, 1.0 eq) in N,N-dimethylformamide (5.0 mL), and the mixture was stirred at 40° C. overnight. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic phase was washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BM051 (230 mg, 26.3%).

[0336] BM051:C 52 H 95 NO6, ESI-MS m / z 831.1[M+H] + ; 1 H NMR (400MHz, CDCl3): δ8.01 (dd, J=8.8, 2.0Hz, 2H), 6.94 (dd, J=8.8, 2.0Hz, 2 H), 4.90(m,J=6.4Hz,1H), 4.31(t,J=6.4Hz,2H), 4.04(t,J=6.4Hz,2H), 3.70( s,2H), 2.78-2.66(m,5H), 2.31(t,J=7.6Hz,2H), 1.87-1.75(m,4H), 1.65-1. 46(m,14H), 1.37-1.29(m,50H), 0.92(t,J=6.4Hz,3H), 0.91(t,J=6.8Hz,6H).

[0337] 23. Synthesis of ionizable lipid compound BM052

[0338] The synthetic route is as follows:

[0339]

[0340] (1) 8-bromooctan-1-ol (0.79 g, 3.8 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.09 g, 5.7 mmol, 1.5 eq), and 4-dimethylaminopyridine (93 mg, 0.76 mmol, 0.2 eq) were added to a dichloromethane solution (20 mL) of 4-(nonyloxy)benzoic acid (1.0 g, 3.8 mmol, 1.0 eq) in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-1% ethyl acetate / petroleum ether) to obtain a solid compound 8-bromooctyl-4-(nonyloxy)benzoate (1.5 g, 87.2%).

[0341] (2) 2-aminoethanol (2.0 g, 33.0 mmol, 10.0 eq) and potassium carbonate (0.68 g, 5.0 mmol, 1.5 eq) were added to a solution of 8-bromooctyl-4-(nonyloxy)benzoate (1.5 g, 3.3 mmol, 1.0 eq) in N,N-dimethylformamide (30 mL), and the mixture was stirred at room temperature overnight. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic phase was washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain a solid compound 8-((2-hydroxyethyl)amino)octyl-4-(nonyloxy)benzoate (1.1 g, 76.5%).

[0342] (3) To a dichloromethane solution (20 mL) of 10-bromodecanic acid (1.0 g, 4.0 mmol, 1.0 eq), heptadecan-9-ol (1.02 g, 4 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.2 g, 6 mmol, 1.5 eq), and 4-dimethylaminopyridine (98 mg, 0.8 mmol, 0.2 eq) were added in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-1% ethyl acetate / petroleum ether) to obtain an oily compound, 10-bromodecanic acid heptadecan-9-ester (1.8 g, 92.3%).

[0343] (4) 8-((2-hydroxyethyl)amino)octyl-4-(nonyloxy)benzoate (0.45 g, 1.02 mmol, 1.0 eq) and potassium carbonate (0.21 g, 1.53 mmol, 1.5 eq) were added to a solution of 10-bromodecane-9-yl (0.5 g, 1.02 mmol, 1.0 eq) in N,N-dimethylformamide (5.0 mL), and the mixture was stirred at 40° C. overnight. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic phase was washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BM052 (200 mg, 23.2%).

[0344] BM052: C 53 H 97 NO6, ESI-MS m / z 845.0[M+H] + ; 1 H NMR (400MHz, CDCl3): δ8.01 (dd, J=8.8, 2.4Hz, 2H), 6.94 (dd, J=8.8, 2.4Hz, 2 H), 4.90(m,J=6.4Hz,1H), 4.31(t,J=6.4Hz,2H), 4.04(t,J=6.4Hz,2H), 3.72( s,2H), 2.80-2.68(m,5H), 2.31(t,J=7.6Hz,2H), 1.87-1.75(m,4H), 1.66-1. 45(m,14H), 1.32-1.29(m,52H), 0.92(t,J=6.8Hz,3H), 0.91(t,J=6.8Hz,6H).

[0345] 24. Synthesis of ionizable lipid compound BM033

[0346] The synthetic route is as follows:

[0347]

[0348] (1) 6-bromo-1-hexanol (1.55 g, 8.55 mmol, 1.0 eq), N,N-diisopropylethylamine (3.31 g, 25.56 mmol, 3.0 eq), and 4-dimethylaminopyridine (0.21 g, 1.71 mmol, 0.2 eq) were added to a dichloromethane solution (30 mL) of 4-undecyloxybenzoic acid (2.5 g, 8.55 mmol, 1.0 eq) in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-10% ethyl acetate / hexane) to obtain an oily compound 6-bromohexyl-4-(undecyloxy)benzoate (1.52 g, 39%).

[0349] (2) 2-aminoethanol (5.0 mL, 83 mmol) was added to a solution of 6-bromohexyl-4-(undedecyloxy)benzoate (1.0 g, 2.20 mmol, 1.0 eq) in ethanol (1.0 mL), and the mixture was stirred at 65° C. for 15 h. The reaction solution was concentrated under vacuum and diluted with ethyl acetate, washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound 6-((2-hydroxyethyl)amino)hexyl-4-(undedecyloxy)benzoate (747 mg, 78%).

[0350] (3) 8-bromooctanoic acid heptadecan-9-yl ester (637 mg, 1.38 mmol, 1.2 eq) and N,N-diisopropylethylamine (297 mg, 2.30 mmol, 2.0 eq) were added to an ethanol solution (2.0 mL) of 6-((2-hydroxyethyl)amino)hexyl-4-(undecanyloxy)benzoate (500 mg, 1.15 mmol, 1.0 eq), and the mixture was stirred at 65° C. for 17 h. The reaction solution was concentrated under reduced pressure, and the crude product was dissolved in ethyl acetate, washed with saturated sodium bicarbonate and saturated brine, respectively. The organic layer was separated and dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BM033 (366 mg, 39%).

[0351] BM033:C 51 H 93 NO6, ESI-MS m / z 816.71[M+H] + ; 1H NMR (400MHz, CDCl3): δ7.98 (dd, J=8.8, 2.0Hz, 2H), 6.90 (dd, J=8.8, 2.0Hz, 2H), 4. 86(m,J=6.4Hz,1H), 4.28(t,J=6.4Hz,2H), 4.00(t,J=6.4Hz,2H), 3.55(s,2H), 2.60 -2.47(m,5H), 2.28(t,J=7.6Hz,2H), 1.83-1.72(m,4H), 1.61-1.60(m,6H), 1.51 -1.42(m,8H), 1.37-1.26(m,48H), 0.88(t,J=6.8Hz,3H), 0.87(t,J=6.8Hz,6H).

[0352] 25. Synthesis of ionizable lipid compound BM035

[0353] The synthetic route is as follows:

[0354]

[0355] (1) 7-bromo-1-heptanol (1.74 g, 8.98 mmol, 1.0 eq), N,N-diisopropylethylamine (3.48 g, 26.94 mmol, 3.0 eq), and 4-dimethylaminopyridine (0.22 g, 1.80 mmol, 0.2 eq) were added to a dichloromethane solution (30 mL) of 4-n-decyloxybenzoic acid (2.5 g, 8.98 mmol, 1.0 eq) in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-10% ethyl acetate / hexane) to obtain an oily compound 7-bromoheptyl-4-(decyloxy)benzoate (1.72 g, 42%).

[0356] (2) 2-aminoethanol (5.0 mL, 83 mmol) was added to a solution of 7-bromoheptyl-4-(decyloxy)benzoate (1.0 g, 2.20 mmol, 1.0 eq) in ethanol (1.0 mL), and the mixture was stirred at 65° C. for 15 h. The reaction solution was concentrated under vacuum and diluted with ethyl acetate, washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound 7-((2-hydroxyethyl)amino)heptyl-4-decyloxybenzoate (757 mg, 79%).

[0357] (3) 8-bromooctanoic acid-heptadecan-9-yl ester (609 mg, 1.32 mmol, 1.2 eq) and N,N-diisopropylethylamine (284 mg, 2.20 mmol, 2.0 eq) were added to an ethanol solution (2.0 mL) of 7-((2-hydroxyethyl)amino)heptyl-4-decyloxybenzoate (500 mg, 1.10 mmol, 1.0 eq), and the mixture was stirred at 65° C. for 17 h. The reaction solution was concentrated under reduced pressure, and the crude product was dissolved in ethyl acetate, washed with saturated sodium bicarbonate and saturated brine, respectively. The organic layer was separated and dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BM035 (323 mg, 36%).

[0358] BM035: C 51 H 93 NO6, ESI-MS m / z 817.0[M+H] + ; 1 H NMR (400MHz, CDCl3): δ8.01 (dd, J=8.8, 2.0Hz, 2H), 6.94 (dd, J=8.8, 2.0Hz, 2 H), 4.89(m,J=6.4Hz,1H), 4.31(t,J=6.8Hz,2H), 4.04(t,J=6.4Hz,2H), 3.67( s,2H), 2.75-2.63(m,5H), 2.31(t,J=7.6Hz,2H), 1.87-1.75(m,4H), 1.66-1. 44(m,14H), 1.35-1.29(m,48H), 0.92(t,J=6.8Hz,3H), 0.91(t,J=6.8Hz,6H).

[0359] 26. Synthesis of ionizable lipid compound BM005

[0360] The synthetic route is as follows:

[0361]

[0362] (1) To a dichloromethane solution (100 mL) of 4-n-nonyloxybenzoic acid (5.0 g, 18.9 mmol, 1.0 eq), 5-bromo-1-pentanol (3.2 g, 18.9 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (5.4 g, 28.4 mmol, 1.5 eq), and 4-dimethylaminopyridine (0.46 g, 3.78 mmol, 0.2 eq) were added in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-1% ethyl acetate / petroleum ether) to obtain an oily compound 5-bromopentyl-4-(nonyloxy)benzoate (5.5 g, 70%).

[0363] (2) 2-aminoethanol (7.4 g, 121 mmol, 10.0 eq) and potassium carbonate (2.5 g, 18.2 mmol, 1.5 eq) were added to a solution of 5-bromopentyl-4-(nonyloxy)benzoate (5.0 g, 12.1 mmol, 1.0 eq) in N,N-dimethylformamide (100 mL), and the mixture was stirred overnight at room temperature. The reaction solution was poured into water and extracted with ethyl acetate. The organic phases were combined, washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain 5-((2-hydroxyethyl)amino)pentyl-4-(nonyloxy)benzoate (3.0 g, 63%) as a white solid.

[0364] (3) 5-((2-hydroxyethyl)amino)pentyl-4-(nonyloxy)benzoate (425 mg, 1.08 mmol, 1.0 eq) and potassium carbonate (224 mg, 1.62 mmol, 1.5 eq) were added to a solution of 7-bromoheptyl 2-octyldecanoate (500 mg, 1.08 mmol, 1.0 eq) in N,N-dimethylformamide (10 mL), and the mixture was stirred at 40° C. for 17 h. The reaction solution was poured into water, extracted with ethyl acetate, and the organic phases were combined, washed with water and saturated brine, respectively. The organic layer was separated and dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (0-5% dichloromethane / methanol) to obtain oily compound BM005 (232 mg, 28%).

[0365] BM005:C 48 H 87 NO6, ESI-MS m / z 775.45[M+H] + ; 1H NMR (400MHz, CDCl3): δ8.01(dd,J=8.8,2.0Hz,2H), 6.94(dd,J=8.8,2.0Hz,2H), 4.32(t,J=6.4Hz,2H), 4.09(t,J=6.4Hz,2H), 4.04(t,J=6.4Hz,2H) , 3.62(t,J=5.2Hz,2H), 2.68-2.56(m,5H), 2.38-2.31(m,1H), 1.87-1.78 (m,5H), 1.53-1.29(m,55H), 0.92(t,J=6.4Hz,3H), 0.91(t,J=6.4Hz,6H).

[0366] 27. Synthesis of ionizable lipid compound BM006

[0367] The synthetic route is as follows:

[0368]

[0369] (1) 7-bromo-1-heptanol (0.76 g, 3.90 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.12 g, 5.85 mmol, 1.5 eq), and 4-dimethylaminopyridine (95 mg, 0.78 mmol, 0.2 eq) were added to a dichloromethane solution (20 mL) of 2-hexyldecanoic acid in sequence, and the mixture was stirred overnight at room temperature. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-1% ethyl acetate / petroleum ether) to obtain an oily compound 2-hexyldecanoic acid 7-bromoheptyl ester (1.5 g, 89%).

[0370] (2) To a solution of 7-bromoheptyl 2-hexyldecanoate (500 mg, 1.15 mmol, 1.0 eq) in N,N-dimethylformamide (10 mL) were added 5-((2-hydroxyethyl)amino)pentyl-4-(nonyloxy)benzoate (453 mg, 1.15 mmol, 1.0 eq) and potassium carbonate (238 mg, 1.73 mmol, 1.5 eq), and the mixture was stirred at 40° C. for 17 h. The reaction solution was poured into water, extracted with ethyl acetate, and the organic phases were combined, washed with water and saturated brine, respectively, and the organic layer was separated and dried over anhydrous sodium sulfate. After concentration, it was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BM006 (212 mg, 25%).

[0371] BM006:C 46 H 83 NO6, ESI-MS m / z 746.93[M+H]+ ; 1 H NMR (400MHz, CDCl3): δ8.01(dd,J=9.2,2.4Hz,2H), 6.94(dd,J=8.8,2.0Hz,2H), 4.32(t,J=6.4Hz,2H), 4.09(t,J=6.4Hz,2H), 4.04(t,J=6.4Hz ,2H), 3.63(s,2H), 2.70-2.59(m,5H), 2.38-2.31(m,1H), 1.87-1.78(m ,5H), 1.65-1.28(m,51H), 0.92(t,J=6.4Hz,3H), 0.91(t,J=6.8Hz,6H).

[0372] 28. Synthesis of ionizable lipid compound BM028

[0373] The synthetic route is as follows:

[0374]

[0375] (1) 5-bromo-1-pentanol (1.3 g, 8.0 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.3 g, 12 mmol, 1.5 eq), and 4-dimethylaminopyridine (195 mg, 1.6 mmol, 0.2 eq) were added to a dichloromethane solution (40 mL) of 4-(octyloxy)benzoic acid (2.0 g, 8.0 mmol, 1.0 eq) in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-1% ethyl acetate / petroleum ether) to obtain an oily compound 5-bromopentyl-4-(octyloxy)benzoate (1.6 g, 50%).

[0376] (2) To a solution of 5-bromopentyl-4-(octyloxy)benzoate (1.6 g, 4.0 mmol, 1.0 eq) in N,N-dimethylformamide (32 mL) were added 2-aminoethanol (2.4 g, 40.0 mmol, 10.0 eq) and potassium carbonate (0.83 g, 6.0 mmol, 1.5 eq), and the mixture was stirred at room temperature overnight. The reaction solution was poured into water and extracted with ethyl acetate. The organic phases were combined, washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain 5-((2-hydroxyethyl)amino)pentyl-4-(octyloxy)benzoate (0.9 g, 59%) as a white solid.

[0377] (3) 5-((2-hydroxyethyl)amino)pentyl-4-(octyloxy)benzoate (410 mg, 1.08 mmol, 1.0 eq) and potassium carbonate (224 mg, 1.62 mmol, 1.5 eq) were added to a solution of 2-octyldecanoic acid-7-bromoheptyl ester (500 mg, 1.08 mmol, 1.0 eq) in N,N-dimethylformamide (10 mL), and the mixture was stirred at 40° C. for 17 h. The reaction solution was poured into water, extracted with ethyl acetate, and the organic phases were combined, washed with water and saturated brine, respectively, and the organic layer was separated and dried over anhydrous sodium sulfate. After concentration, it was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BM028 (243 mg, 30%).

[0378] BM028: C 47 H 85 NO6, ESI-MS m / z 760.8[M+H] + ; 1 H NMR (400MHz, CDCl3): δ8.01(dd,J=8.8,2.0Hz,2H), 6.94(dd,J=8.8,2.0Hz,2H), 4.32(t,J=6.4Hz,2H), 4.09(t,J=6.4Hz,2H), 4.04(t,J=6.4Hz,2H), 3.6 3(s,2H), 2.70-2.58(m,5H), 2.38-2.31(m,1H), 1.87-1.78(m,4H), 1.67-1.4 4(m,15H), 1.38-1.28(m,39H), 0.92(t,J=6.4Hz,3H), 0.91(t,J=6.8Hz,6H).

[0379] 29. Synthesis of ionizable lipid compound BM029

[0380] The synthetic route is as follows:

[0381]

[0382] (1) 5-bromo-1-pentanol (1.2 g, 7.18 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.07 g, 10.8 mmol, 1.5 eq), and 4-dimethylaminopyridine (175 mg, 1.4 mmol, 0.2 eq) were added to a dichloromethane solution (40 mL) of 4-decyloxybenzoic acid (2.0 g, 7.18 mmol, 1.0 eq) in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-1% ethyl acetate / petroleum ether) to obtain an oily compound 5-bromopentyl-4-(decyloxy)benzoate (1.7 g, 55%).

[0383] (2) 2-aminoethanol (2.4 g, 39.8 mmol, 10.0 eq) and potassium carbonate (0.82 g, 5.97 mmol, 1.5 eq) were added to a solution of 5-bromopentyl-4-(decyloxy)benzoate (1.7 g, 3.98 mmol, 1.0 eq) in N,N-dimethylformamide (34 mL), and the mixture was stirred overnight at room temperature. The reaction solution was poured into water and extracted with ethyl acetate. The organic phases were combined, washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain 5-((2-hydroxyethyl)amino)pentyl-4-(decyloxy)benzoate (1.2 g, 74%) as a white solid.

[0384] (3) 5-((2-hydroxyethyl)amino)pentyl-4-(decyloxy)benzoate (440 mg, 1.08 mmol, 1.0 eq) and potassium carbonate (224 mg, 1.62 mmol, 1.5 eq) were added to a solution of 2-octyldecanoic acid-7-bromoheptyl ester (500 mg, 1.08 mmol, 1.0 eq) in N,N-dimethylformamide (10 mL), and the mixture was stirred at 40° C. for 17 h. The reaction solution was poured into water, extracted with ethyl acetate, and the organic phases were combined, washed with water and saturated brine, respectively, and the organic layer was separated and dried over anhydrous sodium sulfate. After concentration, it was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BM029 (233 mg, 27%).

[0385] BM029: C 49 H 89 NO6, ESI-MS m / z 788.96[M+H] + ; 1H NMR (400MHz, CDCl3): δ8.01 (dd, J=8.8, 2.0Hz, 2H), 6.94 (dd, J=9.2, 2.4Hz, 2H), 4.3 2(t,J=6.4Hz,2H), 4.09(t,J=6.4Hz,2H), 4.04(t,J=6.4Hz,2H), 3.56(t,J=5.2Hz,2H ), 2.62(t,J=5.2Hz,2H), 2.53-2.46(m,4H), 2.38-2.30(m,1H), 1.87-1.77(m,4H), 1 .67-1.43(m,15H), 1.36-1.28(m,42H), 0.92(t,J=6.4Hz,3H), 0.91(t,J=6.8Hz,6H).

[0386] 30. Synthesis of ionizable lipid compound BM031

[0387] The synthetic route is as follows:

[0388]

[0389] (1) 6-bromo-1-hexanol (1.63 g, 8.98 mmol, 1.0 eq), N,N-diisopropylethylamine (3.48 g, 26.94 mmol, 3.0 eq), and 4-dimethylaminopyridine (0.22 g, 1.80 mmol, 0.2 eq) were added to a dichloromethane solution (30 mL) of 4-n-decyloxybenzoic acid (2.5 g, 8.98 mmol, 1.0 eq) in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-10% ethyl acetate / hexane) to obtain an oily compound 6-bromohexyl-4-(decyloxy)benzoate (1.70 g, 43%).

[0390] (2) 2-Aminoethanol (1.98 g, 32.5 mmol, 10.0 eq) and potassium carbonate (0.67 g, 4.88 mmol, 1.5 eq) were added to a solution of 7-bromoheptyl 2-octyldecanoate (1.5 g, 3.25 mmol, 1.0 eq) in N,N-dimethylformamide (30 mL), and the mixture was stirred overnight at room temperature. The reaction solution was poured into water and extracted with ethyl acetate. The organic phases were combined, washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain 7-((2-hydroxyethyl)amino)-2-octyldecanoic acid heptyl ester (1.0 g, 70%) as a white solid.

[0391] (3) 7-((2-hydroxyethyl)amino)-2-octyldecanoate (500 mg, 1.10 mmol, 1.0 eq) and potassium carbonate (234 mg, 1.70 mmol, 1.5 eq) were added to a solution of 6-bromohexyl-4-(decyloxy)benzoate (500 mg, 1.13 mmol, 1.0 eq) in N,N-dimethylformamide (10 mL), and the mixture was stirred at 40° C. for 17 h. The reaction solution was poured into water, extracted with ethyl acetate, and the organic phases were combined, washed with water and saturated brine, respectively, and the organic layer was separated and dried over anhydrous sodium sulfate. After concentration, it was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BM031 (235 mg, 26%).

[0392] BM031:C 50 H 91 NO6, ESI-MS m / z 802.8[M+H] + ; 1 H NMR (400MHz, CDCl3): δ8.01(dd,J=8.4,2.0Hz,2H), 6.94(dd,J=8.8,2.0Hz,2H), 4.31(t,J=6.4Hz,2H), 4.09(t,J=6.4Hz,2H), 4.04(t,J=6.4Hz,2H), 3.6 3(s,2H), 2.69-2.57(m,5H), 2.36-2.30(m,1H), 1.87-1.76(m,5H), 1.67-1.4 8(m,14H), 1.39-1.28(m,45H), 0.92(t,J=6.4Hz,3H), 0.91(t,J=6.8Hz,6H).

[0393] 31. Synthesis of ionizable lipid compound BM032

[0394] The synthetic route is as follows:

[0395]

[0396] (1) To a dichloromethane solution (30 mL) of 4-undecyloxybenzoic acid (2.5 g, 8.55 mmol, 1.0 eq) were added 5-bromo-1-pentanol (1.43 g, 8.55 mmol, 1.0 eq), N,N-diisopropylethylamine (3.31 g, 25.56 mmol, 3.0 eq), and 4-dimethylaminopyridine (0.21 g, 1.71 mmol, 0.2 eq) in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-10% ethyl acetate / hexane) to obtain an oily compound 5-bromopentyl-4-(undecyloxy)benzoate (1.43 g, 38%).

[0397] (2) 2-aminoethanol (5.0 mL, 83 mmol) was added to a solution of 5-bromopentyl-4-(undedecyloxy)benzoate (1.0 g, 2.26 mmol, 1.0 eq) in ethanol (1.0 mL), and the mixture was stirred at 65° C. for 15 h. The reaction solution was concentrated under vacuum and diluted with ethyl acetate, washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound 5-((2-hydroxyethyl)amino)pentyl-4-(undedecyloxy)benzoate (788 mg, 83%).

[0398] (3) 2-octyldecanoic acid-7-bromoheptyl ester (655 mg, 1.42 mmol, 1.2 eq) and N,N-diisopropylethylamine (304 mg, 2.36 mmol, 2.0 eq) were added to an ethanol solution (2.0 mL) of 5-((2-hydroxyethyl)amino)pentyl-4-(undecanyloxy)benzoate (500 mg, 1.18 mmol, 1.0 eq), and the mixture was stirred at 65° C. for 17 h. The reaction solution was concentrated under reduced pressure, and the crude product was dissolved in ethyl acetate and washed with saturated sodium bicarbonate and saturated brine, respectively. The organic layer was separated and dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BM032 (376 mg, 43%).

[0399] BM032: C 50 H 91 NO6, ESI-MS m / z 802.69[M+H] + ; 1H NMR (400MHz, CDCl3): δ7.97 (dd, J=8.8, 2.0Hz, 2H), 6.90 (dd, J=8.8, 2.0Hz, 2H), 4.28(t,J=6.4Hz,2H), 4.06(t,J=6.8Hz,2H), 4.00(t,J=6.8Hz,2H), 3.55(s,2H), 2.61-2.49(m,5H), 2.34-2.27(m,1H), 1.83-1.74(m,4H), 1.63-1.57(m,12H), 1.4 7-1.39(m,8H), 1.37-1.25(m,40H), 0.88(t,J=6.8Hz,3H), 0.87(t,J=6.8Hz,6H).

[0400] 32. Synthesis of ionizable lipid compound BM037

[0401] The synthetic route is as follows:

[0402]

[0403] (1) 8-bromo-1-octanol (1.98 g, 9.47 mmol, 1.0 eq), N,N-diisopropylethylamine (3.66 g, 28.41 mmol, 3.0 eq), and 4-dimethylaminopyridine (0.23 g, 1.98 mmol, 0.2 eq) were added to a dichloromethane solution (30 mL) of 4-n-nonyloxybenzoic acid (2.5 g, 9.47 mmol, 1.0 eq) in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-10% ethyl acetate / hexane) to obtain an oily compound 8-bromooctyl-4-(nonyloxy)benzoate (1.52 g, 36%).

[0404] (2) To a solution of 8-bromooctyl-4-(nonyloxy)benzoate (500 mg, 1.10 mmol, 1.0 eq) in N,N-dimethylformamide (10 mL), 7-((2-hydroxyethyl)amino)-2-octyldecanoate (486 mg, 1.10 mmol, 1.0 eq) and potassium carbonate (228 mg, 1.65 mmol, 1.5 eq) were added, and the mixture was stirred at 40° C. for 17 h. The reaction solution was poured into water, extracted with ethyl acetate, and the organic phases were combined, washed with water and saturated brine, respectively, and the organic layer was separated and dried over anhydrous sodium sulfate. After concentration, it was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BM037 (245 mg, 27%).

[0405] BM037:C 51 H 93NO6, ESI-MS m / z 816.9[M+H] + ; 1 H NMR (400MHz, CDCl3): δ8.01(dd,J=8.8,2.0Hz,2H), 6.94(dd,J=8.8,2.4Hz,2H), 4.31(t,J=6.4Hz,2H), 4.09(t,J=6.4Hz,2H), 4.04(t,J=6.4Hz,2H), 3.6 3(s,2H), 2.70-2.57(m,5H), 2.38-2.30(m,1H), 1.87-1.75(m,4H), 1.67-1.4 7(m,14H), 1.39-1.28(m,48H), 0.93(t,J=6.4Hz,3H), 0.92(t,J=6.8Hz,6H).

[0406] 33. Synthesis of ionizable lipid compound BM038

[0407] The synthetic route is as follows:

[0408]

[0409] (1) 7-bromo-1-heptanol (1.74 g, 8.98 mmol, 1.0 eq), N,N-diisopropylethylamine (3.48 g, 26.94 mmol, 3.0 eq), and 4-dimethylaminopyridine (0.22 g, 1.80 mmol, 0.2 eq) were added to a dichloromethane solution (30 mL) of 4-n-decyloxybenzoic acid (2.5 g, 8.98 mmol, 1.0 eq) in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-10% ethyl acetate / hexane) to obtain an oily compound 7-bromoheptyl-4-(decyloxy)benzoate (1.72 g, 42%).

[0410] (2) 2-aminoethanol (5.0 mL, 83 mmol) was added to a solution of 7-bromoheptyl-4-(decyloxy)benzoate (1.0 g, 2.20 mmol, 1.0 eq) in ethanol (1.0 mL), and the mixture was stirred at 65° C. for 15 h. The reaction solution was concentrated under vacuum and diluted with ethyl acetate, washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to give an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to give an oily compound 7-((2-hydroxyethyl)amino)heptyl-4-decyloxybenzoate (757 mg, 79%).

[0411] (3) 2-Octyldecanoic acid-7-bromoheptyl ester (609 mg, 1.32 mmol, 1.2 eq) and N,N-diisopropylethylamine (284 mg, 2.20 mmol, 2.0 eq) were added to an ethanol solution (2.0 mL) of 7-((2-hydroxyethyl)amino)heptyl-4-decyloxybenzoate (500 mg, 1.10 mmol, 1.0 eq), and the mixture was stirred at 65° C. for 17 h. The reaction solution was concentrated under reduced pressure, and the crude product was dissolved in ethyl acetate and washed with saturated sodium bicarbonate and saturated brine, respectively. The organic layer was separated and dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BM038 (350 mg, 39%).

[0412] BM038:C 51 H 93 NO6, ESI-MS m / z 816.71[M+H] + ; 1 H NMR (400MHz, CDCl3): δ7.97(dd,J=8.8,2.0Hz,2H), 6.90(dd,J=8.8,2.0Hz,2H), 4.28(t,J=6.4Hz,2H), 4.06(t,J=6.4Hz,2H), 4.00(t,J=6.4Hz,2H), 3.7 2(s,2H), 2.82-2.71(m,5H), 2.34-2.27(m,1H), 1.83-1.75(m,4H), 1.64-1. 55(m,8H), 1.49-1.25(m,54H), 0.88(t,J=6.8Hz,3H), 0.87(t,J=6.8Hz,6H).

[0413] 34. Synthesis of ionizable lipid compound BM041

[0414] The synthetic route is as follows:

[0415]

[0416] (1) 6-bromo-1-hexanol (1.81 g, 9.99 mmol, 1.0 eq), N,N-diisopropylethylamine (3.87 g, 29.97 mmol, 3.0 eq) and 4-dimethylaminopyridine (0.24 g, 2.0 mmol, 0.2 eq) were added to a dichloromethane solution (30 mL) of 4-n-octyloxybenzoic acid (2.5 g, 9.99 mmol, 1.0 eq) in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-10% ethyl acetate / hexane) to obtain an oily compound 6-bromohexyl-4-(octyloxy)benzoate (1.82 g, 44%).

[0417] (2) 2-aminoethanol (5.0 mL, 83 mmol) was added to a solution of 6-bromohexyl-4-(octyloxy)benzoate (1.0 g, 2.42 mmol, 1.0 eq) in ethanol (1.0 mL), and the mixture was stirred at 65° C. for 15 h. The reaction solution was concentrated under vacuum and diluted with ethyl acetate, washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound 6-((2-hydroxyethyl)amino)hexyl-4-octyloxybenzoate (762 mg, 80%).

[0418] (3) 2-Octyldecanoic acid-7-bromoheptyl ester (671 mg, 1.45 mmol, 1.2 eq) and N,N-diisopropylethylamine (335 mg, 2.64 mmol, 2.0 eq) were added to an ethanol solution (2.0 mL) of 6-((2-hydroxyethyl)amino)hexyl-4-octyloxybenzoate (500 mg, 1.21 mmol, 1.0 eq), and the mixture was stirred at 65° C. for 17 h. The reaction solution was concentrated under reduced pressure, and the crude product was dissolved in ethyl acetate and washed with saturated sodium bicarbonate and saturated brine, respectively. The organic layer was separated and dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BM041 (393 mg, 42%).

[0419] BM041:C 48 H 87 NO6, ESI-MS m / z 774.66[M+H] + ; 1H NMR (400MHz, CDCl3): δ7.97 (dd, J=8.8, 2.0Hz, 2H), 6.91 (dd, J=8.8, 2.0Hz, 2H), 4. 30(t,J=6.4Hz,1H), 4.06(t,J=6.4Hz,2H), 4.00(t,J=6.4Hz,2H), 3.79(t,J=5.6Hz, 2H), 2.89-2.76(m,5H), 2.33-2.28(m,2H), 1.83-1.74(m,4H), 1.65-1.55(m,8H), 1 .49-1.43(m,6H), 1.39-1.23(m,42H), 0.89(t,J=6.4Hz,3H), 0.87(t,J=6.4Hz,6H).

[0420] 35. Synthesis of ionizable lipid compound BM053

[0421] The synthetic route is as follows:

[0422]

[0423] (1) 9-bromo-1-nonanol (2.1 g, 9.46 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.7 g, 14.2 mmol, 1.5 eq), and 4-dimethylaminopyridine (0.23 g, 1.89 mmol, 0.2 eq) were added to a dichloromethane solution (50 mL) of 4-nonyloxybenzoic acid (2.5 g, 9.46 mmol, 1.0 eq) in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-1% ethyl acetate / petroleum ether) to obtain an oily compound 9-bromononyl-4-(nonyloxy)benzoate (3.0 g, 68%).

[0424] (2) 2-aminoethanol (1.3 g, 21.2 mmol, 10.0 eq) and potassium carbonate (0.44 g, 3.18 mmol, 1.5 eq) were added to a solution of 9-bromononyl-4-(nonyloxy)benzoate (1.0 g, 2.12 mmol, 1.0 eq) in N,N-dimethylformamide (20 mL), and the mixture was stirred overnight at room temperature. The reaction solution was poured into water and extracted with ethyl acetate. The organic phases were combined, washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain 9-((2-hydroxyethyl)amino)nonyl-4-(nonyloxy)benzoate (0.8 g, 84%) as a white solid.

[0425] (3) 9-((2-hydroxyethyl)amino)nonyl-4-(nonyloxy)benzoate (495 mg, 1.10 mmol, 1.0 eq) and potassium carbonate (228 mg, 1.65 mmol, 1.5 eq) were added to a solution of 2-octyldecanoic acid-7-bromoheptyl ester (500 mg, 1.10 mmol, 1.0 eq) in N,N-dimethylformamide (10 mL), and the mixture was stirred at 40° C. for 17 h. The reaction solution was poured into water, extracted with ethyl acetate, and the organic phases were combined and washed with water and saturated brine, respectively. The organic layer was separated and dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (0-5% dichloromethane / methanol) to obtain oily compound BM053 (236 mg, 26%).

[0426] BM053:C 52 H 95 NO6, ESI-MS m / z 830.8[M+H] + ; 1 H NMR (400MHz, CDCl3): δ8.01(dd,J=8.4,2.0Hz,2H), 6.94(dd,J=8.4,2.0Hz,2H), 4.31(t,J=6.8Hz,2H), 4.10(t,J=6.8Hz,2H), 4.04(t,J=6.8Hz,2H), 3.6 9(s,2H), 2.77-2.65(m,5H), 2.38-2.31(m,1H), 1.87-1.75(m,4H), 1.67-1.4 4(m,14H), 1.40-1.29(m,49H), 0.92(t,J=6.4Hz,3H), 0.91(t,J=6.8Hz,6H).

[0427] 36. Synthesis of ionizable lipid compound BM036

[0428] The synthetic route is as follows:

[0429]

[0430] (1) 2-Decanol (0.71 g, 4.5 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.3 g, 6.8 mmol, 1.5 eq), and 4-dimethylaminopyridine (0.11 g, 0.9 mmol, 0.2 eq) were added to a dichloromethane solution (20 mL) of 8-bromooctanoic acid in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-1% ethyl acetate / petroleum ether) to obtain an oily compound 8-bromooctanoic acid-2-decyl ester (1.5 g, 92%).

[0431] (2) 2-aminoethanol (2.5 g, 41.0 mmol, 10.0 eq) and potassium carbonate (0.85 g, 6.2 mmol, 1.5 eq) were added to a solution of 8-bromooctanoic acid-2-decyl ester (1.5 g, 4.1 mmol, 1.0 eq) in N,N-dimethylformamide (30 mL), and the mixture was stirred at room temperature overnight. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic phase was washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound 8-((2-hydroxyethyl)amino)octanoic acid-2-decyl ester (1.2 g, 84.5%).

[0432] (3) 7-bromo-1-heptanol (0.51 g, 2.6 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.75 g, 3.9 mmol, 1.5 eq), and 4-dimethylaminopyridine (63 mg, 0.52 mmol, 0.2 eq) were added to a dichloromethane solution (20 mL) of 3,5-di(octyloxy)benzoic acid (1.0 g, 2.6 mmol, 1.0 eq) in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-1% ethyl acetate / petroleum ether) to obtain a solid compound 7-bromoheptyl-3,5-bis(octyloxy)benzoate (1.2 g, 82.2%).

[0433] (4) 8-((2-hydroxyethyl)amino)octanoic acid-2-decyl ester (0.31 g, 0.9 mmol, 1.0 eq) and potassium carbonate (0.19 g, 1.35 mmol, 1.5 eq) were added to a solution of 7-bromoheptyl-3,5-bis(octyloxy)benzoate (0.5 g, 0.9 mmol, 1.0 eq) in N,N-dimethylformamide (5.0 mL), and the mixture was stirred at 40° C. overnight. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic phase was washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BM036 (180 mg, 24.4%).

[0434] BM036:C 50 H 91 NO7, ESI-MS m / z 819.0[M+H] + ; 1H NMR(400MHz, CDCl3), δ7.19(d,J=2.4Hz,2H), 6.67(t,J=2.4Hz,1H), 4.93(m,J= 6.4Hz,1H), 4.32(t,J=6.8Hz,2H), 4.00(t,J=6.4Hz,4H), 3.65(s,2H), 2.72-2.6 0(m,5H), 2.30(t,J=7.6Hz,2H), 1.85-1.75(m,6H), 1.66-1.45(m,15H), 1.38-1. 22(m,39H), 1.23(d,J=6.4Hz,3H), 0.92(t,J=7.2Hz,6H), 0.91(t,J=6.8Hz,3H).

[0435] 37. Synthesis of ionizable lipid compound BM039

[0436] The synthetic route is as follows:

[0437]

[0438] (1) 7-bromo-1-heptanol (1.0 g, 5.3 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.5 g, 8.0 mmol, 1.5 eq), and 4-dimethylaminopyridine (0.13 g, 1.1 mmol, 0.2 eq) were added to a dichloromethane solution (40 mL) of 3,5-bis(octyloxy)benzoic acid (2.0 g, 5.3 mmol, 1.0 eq) in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-1% ethyl acetate / petroleum ether) to obtain an oily compound 7-bromoheptyl-3,5-bis(octyloxy)benzoate (1.7 g, 58.6%).

[0439] (2) 2-aminoethanol (1.9 g, 30.7 mmol, 10.0 eq) and potassium carbonate (0.64 g, 4.6 mmol, 1.5 eq) were added to a solution of 7-bromoheptyl-3,5-bis(octyloxy)benzoate (1.7 g, 3.07 mmol, 1.0 eq) in N,N-dimethylformamide (35 mL), and the mixture was stirred overnight at room temperature. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic phase was washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound 7-((2-hydroxyethyl)amino)-3,5-bis(octyloxy)benzoic acid heptyl ester (1.2 g, 75%).

[0440] (3) To a dichloromethane solution (20 mL) of 8-bromooctanoic acid (1.0 g, 4.5 mmol, 1.0 eq), trans-2-nonenol (0.64 g, 4.5 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.3 g, 6.8 mmol, 1.5 eq), and 4-dimethylaminopyridine (110 mg, 0.9 mmol, 0.2 eq) were added in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-1% ethyl acetate / petroleum ether) to obtain a solid compound 8-bromooctanoic acid-trans-2-nonenyl ester (1.3 g, 83.9%).

[0441] (4) To a solution of 8-bromooctanoic acid-trans-2-nonyl ester (0.5 g, 1.4 mmol, 1.0 eq) in N,N-dimethylformamide (5.0 mL) were added 7-((2-hydroxyethyl)amino)-3,5-bis(octyloxy)benzoic acid heptyl ester (0.77 g, 1.4 mmol, 1.0 eq) and potassium carbonate (0.29 g, 2.1 mmol, 1.5 eq), and the mixture was stirred at 40° C. overnight. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic phase was washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BM039 (150 mg, 13%).

[0442] BM039:C 49 H 87 NO7, ESI-MS m / z 802.83[M+H] + ; 1 H NMR (400MHz, CDCl3): δ7.18 (d, J = 2.4Hz, 2H), 6.66 (t, J = 2.3Hz, 1H), 5.83-5.76 (m, 1H), 5. 62-5.55(m,1H), 4.54(dd,J=6.4,1.2Hz,2H), 4.32(t,J=6.4Hz,2H), 4.00(t,J=6.4Hz,4H), 3.76(s,2H), 2.85-2.74(m,5H), 2.34(t,J=7.2Hz,2H), 2.08(q,J=7.2Hz,2H), 1.85-1.75(m ,6H), 1.66-1.65(m,5H), 1.52-1.30(m,43H), 0.92(t,J=7.2Hz,6H), 0.91(t,J=6.8Hz,3H).

[0443] 38. Synthesis of ionizable lipid compound BM040

[0444] The synthetic route is as follows:

[0445]

[0446] (1) 7-bromo-1-heptanol (1.0 g, 5.3 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.5 g, 8.0 mmol, 1.5 eq), and 4-dimethylaminopyridine (0.13 g, 1.1 mmol, 0.2 eq) were added to a dichloromethane solution (40 mL) of 3,5-bis(octyloxy)benzoic acid (2.0 g, 5.3 mmol, 1.0 eq) in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-1% ethyl acetate / petroleum ether) to obtain an oily compound 7-bromoheptyl-3,5-bis(octyloxy)benzoate (1.7 g, 58.6%).

[0447] (2) 2-aminoethanol (1.9 g, 30.7 mmol, 10.0 eq) and potassium carbonate (0.64 g, 4.6 mmol, 1.5 eq) were added to a solution of 7-bromoheptyl-3,5-bis(octyloxy)benzoate (1.7 g, 3.07 mmol, 1.0 eq) in N,N-dimethylformamide (35 mL), and the mixture was stirred overnight at room temperature. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic phase was washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound 7-((2-hydroxyethyl)amino)-3,5-bis(octyloxy)benzoic acid heptyl ester (1.2 g, 75%).

[0448] (3) 5-bromo-1-pentanol (0.6 g, 3.6 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.04 g, 5.4 mmol, 1.5 eq), and 4-dimethylaminopyridine (88 mg, 0.72 mmol, 0.2 eq) were added to a dichloromethane solution (20 mL) of 4-(decyloxy)benzoic acid (1.0 g, 3.6 mmol, 1.0 eq) in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-1% ethyl acetate / petroleum ether) to obtain a solid compound 5-bromopentyl-4-(decyloxy)benzoate (1.2 g, 78.4%).

[0449] (4) To a solution of 5-bromopentyl-4-(decyloxy)benzoate (0.5 g, 1.2 mmol, 1.0 eq) in N,N-dimethylformamide (5.0 mL) were added 7-((2-hydroxyethyl)amino)-3,5-bis(octyloxy)benzoic acid heptyl ester (0.63 g, 1.2 mmol, 1.0 eq) and potassium carbonate (0.25 g, 1.8 mmol, 1.5 eq), and the mixture was stirred at 40° C. overnight. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic phase was washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BM040 (200 mg, 19.4%).

[0450] BM040: C 54 H 91 NO8, ESI-MS m / z 882.49[M+H] + ; 1 H NMR (400MHz, CDCl3): δ8.00 (dd, J=9.2, 2.4Hz, 2H), 7.18 (d, J=2.0Hz, 2H), 6.9 3(dd,J=9.2,2.4Hz,2H), 6.66(t,J=2.4Hz,1H), 4.32(dt,J=6.4,2.4Hz,4H), 4. 02(q,J=6.4Hz,6H), 3.71(s,2H), 2.79-2.69(m,5H), 1.86-1.75(m,10H), 1.68- 1.61(m,4H), 1.51-1.29(m,44H), 0.92(t,J=6.8Hz,6H), 0.91(t,J=6.8Hz,3H).

[0451] 39. Synthesis of ionizable lipid compound BM042

[0452] The synthetic route is as follows:

[0453]

[0454] (1) 7-bromo-1-heptanol (1.0 g, 5.3 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.5 g, 8.0 mmol, 1.5 eq), and 4-dimethylaminopyridine (0.13 g, 1.1 mmol, 0.2 eq) were added to a dichloromethane solution (40 mL) of 3,5-bis(octyloxy)benzoic acid (2.0 g, 5.3 mmol, 1.0 eq) in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-1% ethyl acetate / petroleum ether) to obtain an oily compound 7-bromoheptyl-3,5-bis(octyloxy)benzoate (1.7 g, 58.6%).

[0455] (2) 2-aminoethanol (1.9 g, 30.7 mmol, 10.0 eq) and potassium carbonate (0.64 g, 4.6 mmol, 1.5 eq) were added to a solution of 7-bromoheptyl-3,5-bis(octyloxy)benzoate (1.7 g, 3.07 mmol, 1.0 eq) in N,N-dimethylformamide (35 mL), and the mixture was stirred overnight at room temperature. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic phase was washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound 7-((2-hydroxyethyl)amino)-3,5-bis(octyloxy)benzoic acid heptyl ester (1.2 g, 75%).

[0456] (3) 2-Decanol (0.76 g, 4.8 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.4 g, 7.2 mmol, 1.5 eq), and 4-dimethylaminopyridine (117 mg, 0.96 mmol, 0.2 eq) were added to a dichloromethane solution (20 mL) of 7-bromoheptanoic acid (1.0 g, 4.8 mmol, 1.0 eq) in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-1% ethyl acetate / petroleum ether) to obtain a solid compound 7-bromoheptanoic acid-2-decyl ester (1.3 g, 77.8%).

[0457] (4) To a solution of 2-decyl 7-bromoheptanoate (0.5 g, 1.4 mmol, 1.0 eq) in N,N-dimethylformamide (5.0 mL) was added heptyl 7-((2-hydroxyethyl)amino)-3,5-bis(octyloxy)benzoate (0.75 g, 1.4 mmol, 1.0 eq) and potassium carbonate (0.29 g, 2.1 mmol, 1.5 eq), and the mixture was stirred at 40° C. overnight. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic phase was washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BM042 (190 mg, 16.5%).

[0458] BM042: C 49 H 89 NO7, ESI-MS m / z 804.85[M+H] + ; 1 H NMR (400MHz, CDCl3): δ7.18 (d, J = 2.4Hz, 2H), 6.67 (t, J = 2.4Hz, 1H), 4.96-4.8 9(m,1H), 4.32(t,J=6.8Hz,2H), 4.00(t,J=6.4Hz,4H), 3.75(s,2H), 2.84-2.73 (m,5H), 2.30(t,J=7.6Hz,2H), 1.85-1.76(m,7H), 1.67-1.62(m,6H), 1.52-1.2 9(m,45H), 1.23(d,J=6.4Hz,3H), 0.92(t,J=6.8Hz,6H), 0.91(t,J=7.2Hz,3H).

[0459] 40. Synthesis of ionizable lipid compound BM043

[0460] The synthetic route is as follows:

[0461]

[0462] (1) 7-bromo-1-heptanol (1.0 g, 5.3 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.5 g, 8.0 mmol, 1.5 eq), and 4-dimethylaminopyridine (0.13 g, 1.1 mmol, 0.2 eq) were added to a dichloromethane solution (40 mL) of 3,5-bis(octyloxy)benzoic acid (2.0 g, 5.3 mmol, 1.0 eq) in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-1% ethyl acetate / petroleum ether) to obtain an oily compound 7-bromoheptyl-3,5-bis(octyloxy)benzoate (1.7 g, 58.6%).

[0463] (2) 2-aminoethanol (1.9 g, 30.7 mmol, 10.0 eq) and potassium carbonate (0.64 g, 4.6 mmol, 1.5 eq) were added to a solution of 7-bromoheptyl-3,5-bis(octyloxy)benzoate (1.7 g, 3.07 mmol, 1.0 eq) in N,N-dimethylformamide (35 mL), and the mixture was stirred overnight at room temperature. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic phase was washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound 7-((2-hydroxyethyl)amino)-3,5-bis(octyloxy)benzoic acid heptyl ester (1.2 g, 75%).

[0464] (3) To a dichloromethane solution (20 mL) of 8-bromooctanoic acid (1.0 g, 4.5 mmol, 1.0 eq), nonanol (0.65 g, 4.5 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.3 g, 6.75 mmol, 1.5 eq), and 4-dimethylaminopyridine (110 mg, 0.9 mmol, 0.2 eq) were added in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-1% ethyl acetate / petroleum ether) to obtain a solid compound 8-bromooctanoic acid nonyl ester (1.4 g, 89.2%).

[0465] (4) To a solution of 8-bromooctanoic acid nonyl ester (0.5 g, 1.4 mmol, 1.0 eq) in N,N-dimethylformamide (5.0 mL) was added 7-((2-hydroxyethyl)amino)-3,5-bis(octyloxy)benzoic acid heptyl ester (0.75 g, 1.4 mmol, 1.0 eq) and potassium carbonate (0.29 g, 2.1 mmol, 1.5 eq), and the mixture was stirred at 40° C. overnight. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic phase was washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BM043 (180 mg, 15.7%).

[0466] BM043:C 49 H 89 NO7, ESI-MS m / z 804.77[M+H] + ; 1 H NMR (400MHz, CDCl3): δ7.19 (d, J = 2.4Hz, 2H), 6.67 (t, J = 2.4Hz, 1H), 4.33 (t, J = 6.4Hz, 2H), 4.09 (t, J = 6.4Hz, 2H), 4.00 (t, J = 6.4Hz, 4H), 3.73 (s, 2H), 2.72(m,5H), 2.33(t,J=7.5Hz,2H), 1.85-1.76(m,6H), 1.67-1.63( m,5H), 1.49-1.31(m,49H), 0.93(t,J=6.8Hz,6H), 0.92(t,J=6.4Hz,3H).

[0467] 41. Synthesis of ionizable lipid compound BM034

[0468] The synthetic route is as follows:

[0469]

[0470] (1) 6-bromohexylamine (2.65 g, 12.70 mmol, 1.27 eq), Hatu (5.73 g, 15 mmol, 1.5 eq), and N,N-diisopropylethylamine (3.87 g, 30 mmol, 3.0 eq) were added to a solution of 4-(decyloxy)benzoic acid (2.78 g, 10 mmol, 1.0 eq) in dichloromethane (30 mL) in sequence, and the mixture was stirred at room temperature for 2 h. The reaction solution was diluted with dichloromethane, washed with water and saturated brine, the organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-10% ethyl acetate / hexane) to obtain an oily compound N-(6-bromohexyl)-4-(decyloxy)benzamide (3.5 g, 75%).

[0471] (2) 2-aminoethanol (10 mL, 165 mmol) and ethanol (2.0 mL) were added to N-(6-bromohexyl)-4-(decyloxy)benzamide (1.98 g, 4.5 mmol, 1.0 eq) in sequence, and the mixture was stirred at 65° C. for 15 h. The reaction solution was concentrated under vacuum and diluted with ethyl acetate, washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound 4-(decyloxy)-N-(6-((2-hydroxyethyl)amino)hexyl)benzamide (1.55 g, 82%).

[0472] (3) 4-(Decyloxy)-N-(6-((2-hydroxyethyl)amino)hexyl)benzamide (580 mg, 1.38 mmol, 1.2 eq) and N,N-diisopropylethylamine (297 mg, 2.3 mmol, 2.0 eq) were added to a solution of 8-bromooctanoic acid heptadecan-9-ester (500 mg, 1.15 mmol, 1.0 eq) in ethanol (2.0 mL) in sequence, and the mixture was stirred at 65° C. for 17 h. The reaction solution was concentrated under reduced pressure, and the crude product was dissolved in ethyl acetate and washed with saturated sodium bicarbonate and saturated brine, respectively. The organic layer was separated and dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BM034 (387 mg, 42%).

[0473] BM034: C 50 H 92 N2O5, ESI-MS m / z 801.71[M+H] + ; 1 H NMR (400MHz, CDCl3): δ7.76(dd,J=8.8,2.0Hz,2H), 6.91(dd,J=8.4,2.0Hz,2H), 6.38(t,J =6.0Hz,1H), 4.85(m,J=6.4Hz,1H), 3.98(t,J=6.4Hz,2H), 3.44(q,J=6.4Hz,2H), 3.19(t, J=4.8Hz,2H), 3.15-3.07(m,5H), 2.28(t,J=7.6Hz,2H), 1.91-1.75(m,6H), 1.67-1.57(m, 6H), 1.51-1.42(m,10H), 1.37-1.26(m,42H), 0.88(t,J=6.8Hz,3H), 0.87(t,J=6.8Hz,6H).

[0474] 42. Synthesis of ionizable lipid compound BM027

[0475] The synthetic route is as follows:

[0476]

[0477] (1) To a solution of 6-bromo-1-hexanoic acid (390 mg, 2.0 mmol, 1.0 eq) in N,N-dimethylformamide (5.0 mL) were added 4-octyloxyaniline (530 mg, 2.4 mmol, 1.2 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (576 mg, 3.0 mmol, 1.5 eq), N,N-diisopropylethylamine (606 mg, 6.0 mmol, 3.0 eq), and 4-dimethylaminopyridine (49 mg, 0.4 mmol, 0.2 eq), and the mixture was stirred at room temperature overnight. The reaction solution was diluted with ethyl acetate, washed with saturated water and sodium bicarbonate solution, the organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-20% ethyl acetate / hexane) to obtain an oily compound 6-bromo-N-(4-(octyloxy)phenyl)hexanamide (598 mg, 75%).

[0478] (2) To a solution of 8-bromooctanoic acid (2.0 g, 8.96 mmol, 1.0 eq) in dichloromethane (25 mL) were added 9-heptadecanol (2.92 g, 11.38 mmol, 1.27 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.58 g, 13.44 mmol, 1.5 eq), N,N-diisopropylethylamine (3.47 g, 26.88 mmol, 3.0 eq), and 4-dimethylaminopyridine (0.22 g, 1.79 mmol, 0.2 eq), and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-10% ethyl acetate / hexane) to obtain an oily compound 8-bromooctanoic acid heptadecan-9-ester (1.9 g, 46%).

[0479] (3) 2-aminoethanol (5.0 mL, 83 mmol) and ethanol (1.0 mL) were added to 8-bromooctanoic acid heptadecan-9-ester (1.03 g, 2.25 mmol, 1.0 eq) in sequence, and the mixture was stirred at 65° C. for 15 h. The reaction solution was concentrated under vacuum and diluted with ethyl acetate, washed with water and saturated brine respectively, and then dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound 8-((2-hydroxyethyl)amino)octanoic acid heptadecan-9-ester (0.82 g, 87%).

[0480] (4) 6-bromo-N-(4-(octyloxy)phenyl)hexanamide (478 mg, 1.2 mmol, 1.2 eq) and N,N-diisopropylethylamine (258 mg, 2.0 mmol, 2.0 eq) were added to a solution of 8-((2-hydroxyethyl)amino)octanoic acid heptadecan-9-ester (442 mg, 1.0 mmol, 1.0 eq) in ethanol (2.0 mL) in sequence, and the mixture was stirred at 65° C. for 17 h. The reaction solution was concentrated under reduced pressure, and the crude product was dissolved in ethyl acetate and washed with saturated sodium bicarbonate and saturated brine, respectively. The organic layer was separated and dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BM027 (297 mg, 39%).

[0481] BM027: C 47 H 86 N2O5, ESI-MS m / z 759.66[M+H] + ; 1 H NMR (400MHz, CDCl3): δ8.32(s,1H), 7.55(dd,J=9.2,2.4Hz,2H), 6.82(dd,J=8.8,2.0Hz,2H), 4.85(m,J=6.4Hz,1H), 4.00-3.98(m,2H), 3.91(t,J=6.4Hz ,2H), 3.21-3.18(m,2H), 3.12-3.07(m,3H), 2.46(t,J=7.6Hz,2H), 2.28(t,J =7.6Hz,2H), 1.98-1.92(m,2H), 1.81-1.71(m,6H), 1.62-1.41(m,12H), 1.36

[0482] -1.23(m,38H), 0.89(t,J=6.4Hz,3H), 0.88(t,J=6.4Hz,6H).

[0483] 43. Synthesis of ionizable lipid compound BM017

[0484] The synthetic route is as follows:

[0485]

[0486] (1) 2-aminoethanol (5.0 mL, 83 mmol) and ethanol (1.0 mL) were added to 8-bromooctanoic acid heptadecan-9-yl ester (1.03 g, 2.25 mmol, 1.0 eq) in sequence, and the mixture was stirred at 65° C. for 15 h. The reaction solution was concentrated under vacuum and diluted with ethyl acetate, washed with water and saturated brine respectively, and then dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound 8-((2-hydroxyethyl)amino)octanoic acid heptadecan-9-yl ester (0.82 g, 87%).

[0487] (2) 4-nonylphenol (1.1 g, 5.0 mmol, 1.0 eq), N,N-dimethylformamide (10 mL), and methyl chloroacetate (0.81 g, 7.5 mmol, 1.5 eq) were added to the reaction flask in sequence and reacted at 70°C for 5.0 h. The reaction solution was diluted with ethyl acetate, washed with water and saturated brine respectively, the organic layer was separated and dried over anhydrous sodium sulfate, concentrated and purified by column chromatography (0-10% ethyl acetate / hexane) to obtain an oily compound 2-(4-nonylphenoxy)acetic acid methyl ester (1.0 g, 72%).

[0488] (3) Add methyl 2-(4-nonylphenoxy)acetate (1.0 g, 3.4 mmol), tetrahydrofuran / methanol (8.0 mL, 1 / 1), and sodium hydroxide solution (2.0 M, 2.0 mL) to the reaction flask and react at room temperature overnight. Adjust the pH to 4.0-5.0 with hydrochloric acid solution (1.0 M), extract with ethyl acetate, combine the organic layers, wash with saturated brine, concentrate, and purify by column chromatography (0-30% ethyl acetate / hexane) to obtain an oily compound 2-(4-nonylphenoxy)acetic acid (0.75 g, 74%).

[0489] (4) To a solution of 2-(4-nonylphenoxy)acetic acid (0.75 g, 2.7 mmol, 1.2 eq) in dichloromethane (5.0 mL) were added 3-bromopropanol (320 mg, 2.3 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (675 mg, 3.5 mmol, 1.5 eq), N,N-diisopropylethylamine (697 mg, 6.9 mmol, 3.0 eq), and 4-dimethylaminopyridine (57 mg, 0.46 mmol, 0.2 eq), and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-10% ethyl acetate / hexane) to obtain an oily compound 2-(4-nonylphenoxy)acetic acid-3-bromopropyl ester (358 mg, 39%).

[0490] (5) 2-(4-nonylphenoxy)acetic acid 3-bromopropyl ester (480 mg, 1.2 mmol, 1.2 eq) and N,N-diisopropylethylamine (258 mg, 2.0 mmol, 2.0 eq) were added to a solution of 8-((2-hydroxyethyl)amino)octanoic acid heptadecan-9-ester (442 mg, 1.0 mmol, 1.0 eq) in ethanol (2.0 mL) in sequence, and the mixture was stirred at 65° C. for 17 h. The reaction solution was concentrated under reduced pressure, and the crude product was dissolved in ethyl acetate and washed with saturated sodium bicarbonate and saturated brine, respectively. The organic layer was separated and dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BM017 (288 mg, 38%).

[0491] BM017: C 50 H 91 NO6, ESI-MS m / z 802.69[M+H] + ; 1 H NMR (400MHz, CDCl3): δ7.25-7.15(m,2H), 6.86-6.84(m,2H), 4.88-4.83(quint,J=6.0Hz,1H), 4.70(s,2H), 3.78(q,J=4.8Hz,2H), 3.53(t,J=5.6Hz,2H) , 3.37(t,J=8.0Hz,2H), 3.25-3.12(m,1H), 2.28(t,J=7.2Hz,1H), 2.26(t,J =7.6Hz,1H), 1.69-1.48(m,12H), 1.32-1.18(m,48H), 0.88(t,J=6.4Hz,9H).

[0492] 44. Synthesis of ionizable lipid compound BM044

[0493] The synthetic route is as follows:

[0494]

[0495] (1) To a solution of 5-bromopentylamine hydrobromide (495 mg, 2.0 mmol, 1.0 eq) in N,N-dimethylformamide, 4-nonyloxybenzoic acid (550 mg, 2.2 mmol, 1.2 eq), N,N-diisopropylethylamine (516 mg, 4.0 mmol, 2.0 eq), and Hatu (1140 mg, 3.0 mmol, 1.5 eq) were added and reacted at room temperature for 2 h. The reaction solution was diluted with water and extracted with ethyl acetate. The organic layer was dried and concentrated, and purified by column chromatography (20% ethyl acetate / hexane) to obtain an oily compound N-(5-bromopentyl)-4-(nonyloxy)benzamide (676 mg, 85%).

[0496] (2) To a solution of 7-((2-hydroxyethyl)amino)-2-octyldecanoate (500 mg, 0.93 mmol, 1.0 eq) in ethanol (2.0 mL) were added N-(5-bromopentyl)-4-(nonyloxy)benzamide (446 mg, 1.12 mmol, 1.2 eq) and N,N-diisopropylethylamine (240 mg, 1.86 mmol, 2.0 eq) in sequence, and the mixture was stirred at 65° C. for 17 h. The reaction solution was concentrated under reduced pressure, and the crude product was dissolved in ethyl acetate, washed with saturated sodium bicarbonate and saturated brine, respectively. The organic layer was separated and dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BM044 (296 mg, 42%).

[0497] BM044:C 48 H 88 N2O5, ESI-MS m / z 773.68[M+H] + ; 1 H NMR (400MHz, CDCl3): δ7.77(dd,J=8.8,2.0Hz,2H), 6.91(dd,J=8.8,2.0Hz,2H), 6.39(t,J=6.0Hz,1H), 4.07-3.97(m,4H), 3. 74-3.70(m,2H), 3.44(q,J=6.8Hz,2H), 3.20(t,J=4.8Hz,2H), 3.17-3.07(m,5H), 2.34-2.24(m,1H), 1.68-1.55(m,18H), 1.40

[0498] -1.25(m,38H), 0.89(t,J=6.4Hz,3H), 0.88(t,J=6.4Hz,6H).

[0499] 45. Synthesis of ionizable lipid compound BM055

[0500] The synthetic route is as follows:

[0501]

[0502] (1) To a solution of 4-(octyloxy)benzoic acid (1.0 g, 4.00 mmol, 1.0 eq) in dichloromethane (20 mL) were added 5-amino-1-pentanol (0.49 g, 4.80 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.15 g, 6.00 mmol, 1.5 eq), 1-hydroxybenzotriazole (0.81 g, 6.00 mmol, 1.5 eq) and triethylamine (0.81 g, 8.00 mmol, 2.0 eq) in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography (0-1% ethyl acetate / petroleum ether) to give N-(5-hydroxypentyl)-4-(octyloxy)benzamide (1.2 g, 89%) as a white solid.

[0503] (2) Add triethylamine (0.72 g, 7.16 mmol, 2.0 eq) to a solution of N-(5-hydroxypentyl)-4-(octyloxy)benzamide (1.2 g, 3.58 mmol, 1.0 eq) in dichloromethane (24 mL), protect with nitrogen, and cool to 0°C in an ice bath. Add methylsulfonyl chloride (0.62 g, 5.37 mmol, 1.5 eq) dropwise, and continue to react in an ice bath for 2 h after the addition is complete. The reaction mixture is diluted with dichloromethane, washed with water and saturated brine, then dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain pentyl 5-(4-(octyloxy)benzamido)methylsulfonate (1.4 g, 94%) as a white solid.

[0504] (3) To a solution of pentyl 5-(4-(octyloxy)benzamido)methanesulfonate (1.4 g, 3.38 mmol, 1.0 eq) in N,N-dimethylformamide (28 mL) were added 2-aminoethanol (2.06 g, 33.8 mmol, 10.0 eq) and potassium carbonate (0.70 g, 5.07 mmol, 1.5 eq), and the mixture was heated to 60° C. for 8 h. The reaction solution was poured into water and extracted with ethyl acetate. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to give N-(5-((2-hydroxyethyl)amino)pentyl)-4-(octyloxy)benzamide (1.0 g, 78%) as a white solid.

[0505] (4) To a solution of 7-bromoheptyl 2-octyldecanoate (500 mg, 1.08 mmol, 1.0 eq) in N,N-dimethylformamide (10 mL), N-(5-((2-hydroxyethyl)amino)pentyl)-4-(octyloxy)benzamide (409 mg, 1.08 mmol, 1.0 eq) and potassium carbonate (224 mg, 1.62 mmol, 1.5 eq) were added, and the mixture was stirred at 40° C. for 17 h. The reaction solution was poured into water, extracted with ethyl acetate, and the organic phases were combined, washed with water and saturated brine, respectively, and the organic layer was separated and dried over anhydrous sodium sulfate. After concentration, it was purified by column chromatography (0-5% dichloromethane / methanol) to obtain oily compound BM055 (225 mg, 27%).

[0506] BM055:C 47 H 86 N2O5, ESI-MS m / z 759.86[M+H] + ; 1 H NMR (400MHz, CDCl3): δ7.79(dd,J=8.8,2.0Hz,2H), 6.94(dd,J=8.8,2.0Hz,2H), 6.42(s,1H), 4.09(t,J=6.4Hz,2H), 4.02(t,J=6.4Hz,2H ), 3.81(s,2H), 3.49(q,J=6.4Hz,2H), 2.94-2.83(m,5H), 2.37-2.30(m,1H), 1.86-1.58(m,12H), 1.52-1.28(m,46H), 0.94-0.89(m,9H).

[0507] 46. ​​Synthesis of ionizable lipid compound BM045

[0508] The synthetic route is as follows:

[0509]

[0510] (1) To a solution of 5-bromo-1-pentanol (334 mg, 2.0 mmol, 1.0 eq) in dichloromethane (5.0 mL) were added 4-nonylbenzoic acid (596 mg, 2.4 mmol, 1.2 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (576 mg, 3.0 mmol, 1.5 eq), N,N-diisopropylethylamine (606 mg, 6.0 mmol, 3.0 eq), and 4-dimethylaminopyridine (49 mg, 0.4 mmol, 0.2 eq), and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-10% ethyl acetate / hexane) to obtain an oily compound 5-bromopentyl-4-(nonyl)benzoate (316 mg, 39%).

[0511] (2) To a solution of 7-((2-hydroxyethyl)amino)-2-octyldecanoate (500 mg, 0.93 mmol, 1.0 eq) in ethanol (2.0 mL) were added 5-bromopentyl-4-(nonyl)benzoate (446 mg, 1.12 mmol, 1.2 eq) and N,N-diisopropylethylamine (240 mg, 1.86 mmol, 2.0 eq) in sequence, and the mixture was stirred at 65° C. for 17 h. The reaction solution was concentrated under reduced pressure, and the crude product was dissolved in ethyl acetate, washed with saturated sodium bicarbonate and saturated brine, respectively. The organic layer was separated and dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BM045 (282 mg, 40%).

[0512] BM045:C 48 H 87 NO5, ESI-MS m / z 758.67[M+H] + ; 1 H NMR (400MHz, CDCl3): δ7.94(d,J=7.6Hz,2H), 7.24(d,J=7.2Hz,2H), 4.32(t,J=6.4Hz,2H), 4.06(t,J=6.4Hz,2H), 3.75(d ,J=14.1Hz,2H), 2.81(s,7H), 2.30(dt,J=9.2,4.0Hz,1H), 1.89-1.44(m,18H), 1.43-1.08(m,42H), 0.87(t,J=6.7Hz,9H).

[0513] 47. Synthesis of ionizable lipid compound BMS020

[0514] The synthetic route is as follows:

[0515]

[0516] (1) 9-heptadecanol (1.15 g, 4.5 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.3 g, 6.8 mmol, 1.5 eq), and 4-dimethylaminopyridine (0.11 g, 0.9 mmol, 0.2 eq) were added to a dichloromethane solution (20 mL) of 8-bromooctanoic acid in sequence, and the mixture was stirred overnight at room temperature. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-1% ethyl acetate / petroleum ether) to obtain an oily compound 8-bromooctanoic acid heptadecan-9-ester (1.7 g, 82.1%).

[0517] (2) To a solution of 8-bromooctanoic acid heptadecan-9-yl ester (1.7 g, 3.7 mmol, 1.0 eq) in N,N-dimethylformamide (35 mL) were added N,N'-bis(2-hydroxyethyl)ethylenediamine (5.5 g, 37.0 mmol, 10.0 eq) and potassium carbonate (0.76 g, 5.5 mmol, 1.5 eq), and the mixture was stirred at room temperature overnight. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic phase was washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-10% dichloromethane / methanol) to obtain an oily compound 8-((2-hydroxyethyl)(2-((2-hydroxyethyl)amino)ethyl)amino)octanoic acid heptadecan-9-yl ester (0.8 g, 41%).

[0518] (3) To a dichloromethane solution (20 mL) of 4-undecyloxybenzoic acid (1.0 g, 3.4 mmol, 1.0 eq), 5-bromo-1-pentanol (0.57 g, 3.4 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.98 g, 5.1 mmol, 1.5 eq), and 4-dimethylaminopyridine (83 mg, 0.68 mmol, 0.2 eq) were added in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-1% ethyl acetate / petroleum ether) to obtain a solid compound 5-bromopentyl-4-(undecyloxy)benzoate (1.3 g, 86.7%).

[0519] (4) To a solution of 5-bromopentyl-4-(undecanyloxy)benzoate (0.5 g, 1.1 mmol, 1.0 eq) in N,N-dimethylformamide (5.0 mL) were added 8-((2-hydroxyethyl)(2-((2-hydroxyethyl)amino)ethyl)amino)octanoic acid heptadecan-9-ester (0.6 g, 1.1 mmol, 1.0 eq) and potassium carbonate (0.23 g, 1.65 mmol, 1.5 eq), and the mixture was stirred at 40° C. overnight. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic phase was washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BMS020 (220 mg, 22%).

[0520] BMS020:C 54 H 100 N2O7, ESI-MS m / z 889.4[M+H] + ; 1 H NMR (400MHz, CDCl3): δ8.01 (dd, J=9.2, 2.4Hz, 2H), 6.94 (dd, J=8.8, 2.0Hz, 2H), 4 .90(m,J=6.4Hz,1H), 4.32(t,J=6.4Hz,2H), 4.04(t,J=6.4Hz,2H), 3.69(s,4H), 2. 73-2.44(m,11H), 2.31(t,J=7.6Hz,2H), 1.87-1.78(m,4H), 1.66-1.63(m,4H), 1.4 9-1.47(m,10H), 1.35-1.29(m,46H), 0.93(t,J=6.4Hz,3H), 0.92(t,J=6.4Hz,6H).

[0521] 48. Synthesis of ionizable lipid compound BMS023

[0522] The synthetic route is as follows:

[0523]

[0524] (1) 9-heptadecanol (1.15 g, 4.5 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.3 g, 6.8 mmol, 1.5 eq), and 4-dimethylaminopyridine (0.11 g, 0.9 mmol, 0.2 eq) were added to a dichloromethane solution (20 mL) of 8-bromooctanoic acid in sequence, and the mixture was stirred overnight at room temperature. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-1% ethyl acetate / petroleum ether) to obtain an oily compound 8-bromooctanoic acid heptadecan-9-ester (1.7 g, 82.1%).

[0525] (2) To a solution of 8-bromooctanoic acid heptadecan-9-yl ester (1.7 g, 3.7 mmol, 1.0 eq) in N,N-dimethylformamide (35 mL) were added N,N'-bis(2-hydroxyethyl)ethylenediamine (5.5 g, 37.0 mmol, 10.0 eq) and potassium carbonate (0.76 g, 5.5 mmol, 1.5 eq), and the mixture was stirred at room temperature overnight. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic phase was washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-10% dichloromethane / methanol) to obtain an oily compound 8-((2-hydroxyethyl)(2-((2-hydroxyethyl)amino)ethyl)amino)octanoic acid heptadecan-9-yl ester (0.8 g, 41%).

[0526] (3) 6-bromo-1-hexanol (0.65 g, 3.6 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.04 g, 5.4 mmol, 1.5 eq), and 4-dimethylaminopyridine (88 mg, 0.72 mmol, 0.2 eq) were added to a dichloromethane solution (20 mL) of 4-decyloxybenzoic acid (1.0 g, 3.6 mmol, 1.0 eq) in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-1% ethyl acetate / petroleum ether) to obtain a solid compound 6-bromohexyl-4-(decyloxy)benzoate (1.35 g, 85.4%).

[0527] (4) To a solution of 6-bromohexyl-4-(decyloxy)benzoate (0.5 g, 1.1 mmol, 1.0 eq) in N,N-dimethylformamide (5.0 mL) were added 8-((2-hydroxyethyl)(2-((2-hydroxyethyl)amino)ethyl)amino)octanoic acid heptadecan-9-ester (0.6 g, 1.1 mmol, 1.0 eq) and potassium carbonate (0.23 g, 1.65 mmol, 1.5 eq), and the mixture was stirred at 40° C. overnight. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic phase was washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BMS023 (250 mg, 25%).

[0528] BMS023:C 54 H 100 N2O7, ESI-MS m / z 890.1[M+H] + ; 1 H NMR (400MHz, CDCl3): δ8.01 (dd, J=8.8, 2.0Hz, 2H), 6.94 (dd, J=8.8, 2.0Hz, 2 H), 4.90(m,J=6.4Hz,1H), 4.31(t,J=6.4Hz,2H), 4.04(t,J=6.4Hz,2H), 3.70( s,4H), 2.74-2.66(m,11H), 2.31(t,J=7.6Hz,2H), 1.87-1.76(m,4H), 1.66-1. 50(m,14H), 1.41-1.29(m,46H), 0.92(t,J=6.8Hz,3H), 0.91(t,J=6.4Hz,6H).

[0529] 49. Synthesis of ionizable lipid compound BMS026

[0530] The synthetic route is as follows:

[0531]

[0532] (1) 9-heptadecanol (1.15 g, 4.5 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.3 g, 6.8 mmol, 1.5 eq), and 4-dimethylaminopyridine (0.11 g, 0.9 mmol, 0.2 eq) were added to a dichloromethane solution (20 mL) of 8-bromooctanoic acid in sequence, and the mixture was stirred overnight at room temperature. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-1% ethyl acetate / petroleum ether) to obtain an oily compound 9-bromooctanoic acid heptadecan-9-ester (1.7 g, 82.1%).

[0533] (2) N,N'-bis(2-hydroxyethyl)ethylenediamine (5.5 g, 37.0 mmol, 10.0 eq) and potassium carbonate (0.76 g, 5.5 mmol, 1.5 eq) were added to a solution of 9-bromooctanoic acid heptadecan-9-ester (1.7 g, 3.7 mmol, 1.0 eq) in N,N-dimethylformamide (35 mL), and the mixture was stirred at room temperature overnight. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic phase was washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-10% dichloromethane / methanol) to obtain an oily compound 8-((2-hydroxyethyl)(2-((2-hydroxyethyl)amino)ethyl)amino)octanoic acid heptadecan-9-ester (0.8 g, 41%).

[0534] (3) 8-bromo-1-octanol (0.79 g, 3.8 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.09 g, 5.7 mmol, 1.5 eq), and 4-dimethylaminopyridine (93 mg, 0.76 mmol, 0.2 eq) were added to a dichloromethane solution (20 mL) of 4-nonyloxybenzoic acid (1.0 g, 3.8 mmol, 1.0 eq) in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-1% ethyl acetate / petroleum ether) to obtain a solid compound 8-bromooctyl-4-(nonyloxy)benzoate (1.5 g, 87.2%).

[0535] (4) To a solution of 8-bromooctyl-4-(nonyloxy)benzoate (0.5 g, 1.1 mmol, 1.0 eq) in N,N-dimethylformamide (5.0 mL) were added 8-((2-hydroxyethyl)(2-((2-hydroxyethyl)amino)ethyl)amino)octanoic acid heptadecan-9-ester (0.6 g, 1.1 mmol, 1.0 eq) and potassium carbonate (0.23 g, 1.65 mmol, 1.5 eq), and the mixture was stirred at 40° C. overnight. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic phase was washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BMS026 (250 mg, 25%).

[0536] BMS026:C 55 H 102 N2O7, ESI-MS m / z 904.6[M+H] + ; 1 H NMR (400MHz, CDCl3): δ8.01 (dd, J=8.8, 2.0Hz, 2H), 6.94 (dd, J=8.8, 2.4Hz, 2H), 4 .89(m,J=6.4Hz,1H), 4.31(t,J=6.8Hz,2H), 4.04(t,J=6.4Hz,2H), 3.69(s,4H), 2. 73-2.64(m,11H), 2.31(t,J=7.6Hz,2H), 1.87-1.74(m,4H), 1.66-1.62(m,2H), 1.5 4-1.47(m,12H), 1.35-1.29(m,49H), 0.92(t,J=6.4Hz,3H), 0.91(t,J=6.4Hz,6H).

[0537] 50. Synthesis of ionizable lipid compound BMS028

[0538] The synthetic route is as follows:

[0539]

[0540] (1) To a dichloromethane solution (30 mL) of 4-octyloxybenzoic acid (1.5 g, 6.00 mmol, 1.0 eq), 5-bromo-1-pentanol (1.00 g, 6.00 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.73 g, 9.00 mmol, 1.5 eq), and 4-dimethylaminopyridine (146 mg, 1.20 mmol, 0.2 eq) were added in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-1% ethyl acetate / petroleum ether) to obtain an oily compound 5-bromopentyl-4-(octyloxy)benzoate (1.4 g, 58%).

[0541] (2) To a solution of 5-bromopentyl-4-(octyloxy)benzoate (1.2 g, 3.00 mmol, 1.0 eq) in N,N-dimethylformamide (24 mL) were added N,N'-bis(2-hydroxyethyl)ethylenediamine (4.44 g, 30.0 mmol, 10.0 eq) and potassium carbonate (0.62 g, 4.50 mmol, 1.5 eq), and the mixture was stirred at room temperature overnight. The reaction solution was poured into water and extracted with ethyl acetate. The organic phases were combined, washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography (0-10% dichloromethane / methanol) to obtain 5-((2-hydroxyethyl)(2-((2-hydroxyethyl)amino)ethyl)amino)pentyl-4-(octyloxy)benzoate (0.6 g, 43%) as a white solid.

[0542] (3) To a solution of 7-bromoheptyl 2-octyldecanoate (500 mg, 1.10 mmol, 1.0 eq) in N,N-dimethylformamide (10 mL) were added 5-((2-hydroxyethyl)(2-((2-hydroxyethyl)amino)ethyl)amino)pentyl-4-(octyloxy)benzoate (513 mg, 1.10 mmol, 1.0 eq) and potassium carbonate (228 mg, 1.65 mmol, 1.5 eq), and the mixture was stirred at 40° C. for 17 h. The reaction solution was poured into water, extracted with ethyl acetate, and the organic phases were combined, washed with water and saturated brine, respectively. The organic layer was separated and dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BMS028 (241 mg, 26%).

[0543] BMS028:C 51 H 94 N2O7, ESI-MS m / z 847.6[M+H] + ; 1H NMR (400MHz, CDCl3): δ8.01(dd,J=8.8,2.0Hz,2H), 6.94(dd,J=8.8,2.0Hz,2H), 4.32(t,J=6.4Hz,2H), 4.09(t,J=6.8Hz,2H), 4.04(t,J=6.4Hz,2H) , 3.67(t,J=4.8Hz,4H), 2.70-2.63(m,11H), 2.37-2.30(m,1H), 1.87-1.78 (m,4H), 1.64-1.28(m,53H), 0.93(t,J=6.4Hz,3H), 0.91(t,J=6.4Hz,6H).

[0544] 51. Synthesis of ionizable lipid compound BMS031

[0545] The synthetic route is as follows:

[0546]

[0547] (1) 6-bromo-1-hexanol (1.63 g, 8.98 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.59 g, 13.5 mmol, 1.5 eq), and 4-dimethylaminopyridine (219 mg, 1.80 mmol, 0.2 eq) were added to a dichloromethane solution (50 mL) of 4-decyloxybenzoic acid (2.5 g, 8.98 mmol, 1.0 eq) in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-1% ethyl acetate / petroleum ether) to obtain an oily compound 6-bromohexyl-4-(decyloxy)benzoate (3.0 g, 76%).

[0548] (2) To a solution of 6-bromohexyl-4-(decyloxy)benzoate (1.5 g, 3.40 mmol, 1.0 eq) in N,N-dimethylformamide (30 mL) was added N,N'-bis(2-hydroxyethyl)ethylenediamine (5.03 g, 34.0 mmol, 10.0 eq) and potassium carbonate (0.70 g, 5.10 mmol, 1.5 eq), and the mixture was stirred at room temperature overnight. The reaction solution was poured into water and extracted with ethyl acetate. The organic phases were combined, washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography (0-10% dichloromethane / methanol) to obtain 6-((2-hydroxyethyl)(2-((2-hydroxyethyl)amino)ethyl)amino)hexyl-4-(decyloxy)benzoate (0.8 g, 46%) as a white solid.

[0549] (3) 6-((2-hydroxyethyl)(2-((2-hydroxyethyl)amino)ethyl)amino)hexyl-4-(decyloxy)benzoate (559 mg, 1.10 mmol, 1.0 eq) and potassium carbonate (228 mg, 1.65 mmol, 1.5 eq) were added to a solution of 2-octyldecanoic acid-7-bromoheptyl ester (500 mg, 1.10 mmol, 1.0 eq) in N,N-dimethylformamide (10 mL), and the mixture was stirred at 40° C. for 17 h. The reaction solution was poured into water, extracted with ethyl acetate, and the organic phases were combined, washed with water and saturated brine, respectively. The organic layer was separated and dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BMS031 (249 mg, 25%).

[0550] BMS031:C 54 H 100 N2O7, ESI-MS m / z 890.0[M+H] + ; 1 H NMR (400MHz, CDCl3): δ8.01(dd,J=8.8,2.0Hz,2H), 6.94(dd,J=8.8,2.0Hz,2H), 4.31(t,J=6.4Hz,2H), 4.09(t,J=6.8Hz,2H), 4.04(t,J=6.4Hz ,2H), 3.68(s,4H), 2.70-2.62(m,11H), 2.36-2.31(m,1H), 1.87-1.75(m ,4H), 1.66-1.28(m,59H), 0.92(t,J=6.4Hz,3H), 0.91(t,J=6.8Hz,6H).

[0551] 52. Synthesis of ionizable lipid compound BMS037

[0552] The synthetic route is as follows:

[0553]

[0554] (1) 8-bromo-1-octanol (1.98 g, 9.46 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.72 g, 14.2 mmol, 1.5 eq), and 4-dimethylaminopyridine (231 mg, 1.89 mmol, 0.2 eq) were added to a dichloromethane solution (50 mL) of 4-nonyloxybenzoic acid (2.5 g, 9.46 mmol, 1.0 eq) in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-1% ethyl acetate / petroleum ether) to obtain an oily compound 8-bromooctyl-4-(nonyloxy)benzoate (3.5 g, 81%).

[0555] (2) To a solution of 8-bromooctyl-4-(nonyloxy)benzoate (1.5 g, 3.29 mmol, 1.0 eq) in N,N-dimethylformamide (30 mL) were added N,N'-bis(2-hydroxyethyl)ethylenediamine (4.87 g, 32.9 mmol, 10.0 eq) and potassium carbonate (0.68 g, 4.94 mmol, 1.5 eq), and the mixture was stirred at room temperature overnight. The reaction solution was poured into water and extracted with ethyl acetate. The organic phases were combined, washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography (0-10% dichloromethane / methanol). 8-((2-hydroxyethyl)(2-((2-hydroxyethyl)amino)ethyl)amino)octyl-4-(nonyloxy)benzoate (0.9 g, 52%) was obtained as a white solid.

[0556] (3) 8-((2-hydroxyethyl)(2-((2-hydroxyethyl)amino)ethyl)amino)octyl-4-(nonyloxy)benzoate (575 mg, 1.10 mmol, 1.0 eq) and potassium carbonate (228 mg, 1.65 mmol, 1.5 eq) were added to a solution of 2-octyldecanoic acid-7-bromoheptyl ester (500 mg, 1.10 mmol, 1.0 eq) in N,N-dimethylformamide (10 mL), and the mixture was stirred at 40° C. for 17 h. The reaction solution was poured into water and extracted with ethyl acetate. The organic phases were combined, washed with water and saturated brine, respectively, and the organic layer was separated and dried over anhydrous sodium sulfate. After concentration, it was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BMS037 (258 mg, 26%).

[0557] BMS037:C 55 H 102 N2O7, ESI-MS m / z 903.9[M+H] + ; 1H NMR (400MHz, CDCl3): δ8.01(dd,J=8.8,2.0Hz,2H), 6.93(dd,J=8.8,2.0Hz,2H), 4.31(t,J=6.4Hz,2H), 4.09(t,J=6.8Hz,2H), 4.03(t,J=6.4Hz,2H) , 3.67(t,J=4.4Hz,2H), 2.71-2.61(m,11H), 2.37-2.30(m,1H), 1.86-1.74 (m,4H), 1.66-1.28(m,61H), 0.92(t,J=6.4Hz,3H), 0.91(t,J=6.8Hz,6H).

[0558] 53. Synthesis of ionizable lipid compound BMS001

[0559] The synthetic route is as follows:

[0560]

[0561] (1) 9-heptadecanol (1.15 g, 4.5 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.3 g, 6.8 mmol, 1.5 eq), and 4-dimethylaminopyridine (0.11 g, 0.9 mmol, 0.2 eq) were added to a dichloromethane solution (20 mL) of 8-bromooctanoic acid in sequence, and the mixture was stirred overnight at room temperature. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-1% ethyl acetate / petroleum ether) to obtain an oily compound 8-bromooctanoic acid heptadecan-9-ester (1.7 g, 82.1%).

[0562] (2) To a solution of 8-bromooctanoic acid heptadecan-9-yl ester (1.7 g, 3.7 mmol, 1.0 eq) in N,N-dimethylformamide (35 mL) were added N,N'-bis(2-hydroxyethyl)ethylenediamine (5.5 g, 37.0 mmol, 10.0 eq) and potassium carbonate (0.76 g, 5.5 mmol, 1.5 eq), and the mixture was stirred at room temperature overnight. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic phase was washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-10% dichloromethane / methanol) to obtain an oily compound 8-((2-hydroxyethyl)(2-((2-hydroxyethyl)amino)ethyl)amino)octanoic acid heptadecan-9-yl ester (0.8 g, 41%).

[0563] (3) 8-bromo-1-octanol (0.75 g, 3.6 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.04 g, 5.4 mmol, 1.5 eq), and 4-dimethylaminopyridine (88 mg, 0.72 mmol, 0.2 eq) were added to a dichloromethane solution (20 mL) of 4-decyloxybenzoic acid (1.0 g, 3.6 mmol, 1.0 eq) in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-1% ethyl acetate / petroleum ether) to obtain a solid compound 8-bromooctyl-4-(decyloxy)benzoate (1.4 g, 83.3%).

[0564] (4) To a solution of 8-bromooctyl-4-(decyloxy)benzoate (0.5 g, 1.1 mmol, 1.0 eq) in N,N-dimethylformamide (5.0 mL) were added 8-((2-hydroxyethyl)(2-((2-hydroxyethyl)amino)ethyl)amino)octanoic acid heptadecan-9-ester (0.6 g, 1.1 mmol, 1.0 eq) and potassium carbonate (0.23 g, 1.65 mmol, 1.5 eq), and the mixture was stirred at 40° C. overnight. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic phase was washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BMS001 (250 mg, 25.6%).

[0565] BMS001:C 55 H 102 N2O7, ESI-MS m / z 917.5[M+H] + ; 1 H NMR (400MHz, CDCl3): δ8.01(dd,J=8.8,2.0Hz,2H), 6.93(dd,J=8.8,2.0Hz,2H), 4.90(m,J=6.0Hz,1H), 4.31(t,J=6.4Hz,2H), 4.03(t,J=6.4Hz,2H), 3.7 6(s,4H), 2.83(m,12H), 2.31(t,J=7.2Hz,2H), 1.86-1.74(m,4H), 1.68-1.4 5(m,14H), 1.40-1.29(m,50H), 0.92(t,J=6.8Hz,3H), 0.91(t,J=6.4Hz,6H).

[0566] 54. Synthesis of ionizable lipid compound BMS036

[0567] The synthetic route is as follows:

[0568]

[0569] (1) 2-Decanol (0.7 g, 4.5 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.3 g, 6.8 mmol, 1.5 eq), and 4-dimethylaminopyridine (0.11 g, 0.9 mmol, 0.2 eq) were added to a dichloromethane solution (20 mL) of 8-bromooctanoic acid in sequence, and the mixture was stirred overnight at room temperature. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-1% ethyl acetate / petroleum ether) to obtain an oily compound 8-bromooctanoic acid-2-decyl ester (1.5 g, 92%).

[0570] (2) 2,2'-(ethane-1,2-diylbis(azetidinyl))bis(ethane-1-ol) (6.1 g, 41 mmol, 10.0 eq) and potassium carbonate (0.85 g, 6.2 mmol, 1.5 eq) were added to a solution of 8-bromooctanoic acid-2-decyl ester (1.5 g, 4.1 mmol, 1.0 eq) in N,N-dimethylformamide (30 mL), and the mixture was stirred at room temperature overnight. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic phase was washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-10% dichloromethane / methanol) to obtain an oily compound 2-decyl-8-((2-hydroxyethyl)(2-((2-hydroxyethyl)amino)ethyl)amino)octanoate (0.8 g, 44.9%).

[0571] (3) 7-bromo-1-heptanol (0.51 g, 2.6 mmol, 1.0 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.75 g, 3.9 mmol, 1.5 eq), and 4-dimethylaminopyridine (63 mg, 0.52 mmol, 0.2 eq) were added to a dichloromethane solution (20 mL) of 3,5-bis(octyloxy)benzoic acid (1.0 g, 2.6 mmol, 1.0 eq) in sequence, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with dichloromethane and washed with a saturated sodium bicarbonate solution. The organic layer was separated and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude oil, which was purified by column chromatography (0-1% ethyl acetate / petroleum ether) to obtain a solid compound 7-bromoheptyl-3,5-bis(octyloxy)benzoate (0.8 g, 54.8%).

[0572] (4) 2-Decyl-8-((2-hydroxyethyl)(2-((2-hydroxyethyl)amino)ethyl)amino)octanoate (0.39 g, 0.9 mmol, 1.0 eq) and potassium carbonate (0.19 g, 1.35 mmol, 1.5 eq) were added to a solution of 7-bromoheptyl-3,5-bis(octyloxy)benzoate (0.5 g, 0.9 mmol, 1.0 eq) in N,N-dimethylformamide (5.0 mL), and the mixture was stirred at 40° C. overnight. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic phase was washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily crude product, which was purified by column chromatography (0-5% dichloromethane / methanol) to obtain an oily compound BMS036 (180 mg, 22.1%).

[0573] BMS036:C 54 H 100 N2O8, ESI-MS m / z 906.04[M+H] + ; 1 H NMR (400MHz, CDCl3), δ7.18 (d, J = 2.4Hz, 2H), 6.67 (t, J = 2.4Hz, 1H), 4.96-4. 88(m,1H), 4.32(t,J=6.8Hz,2H), 4.00(t,J=6.4Hz,4H), 3.76(s,4H), 2.82(m, 12H), 2.30(t,J=7.6Hz,2H), 1.85-1.75(m,6H), 1.65-1.57(m,7H), 1.52-1.29 (m, 47H), 1.23 (d, J = 6.4Hz, 3H), 0.92 (t, J = 6.4Hz, 6H), 0.91 (t, J = 6.8Hz, 3H).

[0574] Example 2: Preparation of lipid nanoparticles (pharmaceutical composition nanoparticles) encapsulating mRNA

[0575] Materials used:

[0576] (1) The ionizable lipid compound of the present invention or the reference compound SM102.

[0577] (2) Structural lipids: cholesterol (purchased from Xiamen Sinobond Biotechnology Co., Ltd.).

[0578] (3) Phospholipids: DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine, Distearoylphosphatidylcholine), purchased from Xiamen Sinopang Biotechnology Co., Ltd.

[0579] (4) Polyethylene glycol lipids: PEG2000-DMG (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000) was purchased from Xiamen Sinopong Biotechnology Co., Ltd.

[0580] (5) mRNA (i.e., active ingredient): firefly luciferase mRNA (MR009), purchased from Suzhou Nearshore Protein Technology Co., Ltd.; or green fluorescent protein eGFP mRNA (MR008), purchased from Suzhou Nearshore Protein Technology Co., Ltd.; or SARS CoV-2 mRNA, the sequence of which is shown in SEQ ID NO: 1.

[0581] Microfluidic mixing system preparation method:

[0582] (1) dissolving an ionizable lipid compound, cholesterol, phospholipid and pegylated lipid in anhydrous ethanol to prepare a solution with a concentration of 1.0 mg / mL, and then taking corresponding volumes of the above four solutions in a molar ratio of 50:38.5:10:1.5 and mixing them to prepare an organic phase solution;

[0583] (2) dissolving the mRNA in 20 mM citric acid-sodium citrate buffer solution (pH = 4.5) to prepare an mRNA aqueous solution with a concentration of 33 μg / mL;

[0584] (3) using a microfluidic system to mix the organic phase containing the lipid mixture and the aqueous phase containing the mRNA component at a volume ratio and flow rate ratio of 1:3, respectively, with a total mixing flow rate of 20 mL / min;

[0585] (4) The prepared lipid nanoparticles encapsulating mRNA were diluted with 20 volumes of phosphate buffer solution (135 mM NaCl, 4.7 mM KCl, 10 mM Na2HPO4, 2.0 mM NaH2PO4, pH 7.3±0.1), and the lipid nanoparticle solution encapsulating mRNA was ultrafiltered to the original preparation volume using an ultrafiltration tube with a molecular weight cutoff of 30 kDa (purchased from Millipore).

[0586] Ethanol injection preparation method:

[0587] (1) Weigh a certain amount of ionizable lipid, DSPC, cholesterol, and PEG2000-DMG, dissolve them in anhydrous ethanol, and prepare 1.0 mg / mL anhydrous ethanol solutions of each substance. Pipette the corresponding volumes of each solution according to the molar ratio of 50:38.5:10:1.5, and mix them to prepare lipid ethanol solutions;

[0588] (2) Pipette 20 μL of mRNA stock solution (1.0 mg / mL) and add it to 580 μL of 20 mM (pH = 4.5) citric acid-sodium citrate buffer (pH = 4.5), and mix up and down with a pipette to prepare an mRNA aqueous solution;

[0589] (3) Use an insulin syringe to draw 200 μL of lipid ethanol solution and quickly inject it into the mRNA aqueous solution. Vortex gently for 30 seconds and incubate at room temperature for 10 minutes.

[0590] (4) Add 4.0 mL of phosphate buffer solution (135 mM NaCl, 4.7 mM KCl, 10 mM Na2HPO4, 2.0 mM NaH2PO4, pH 7.3 ± 0.1) (20 times the volume of the organic phase) to the above mRNA-encapsulated lipid nanoparticles. Add the above diluted liquid to a 30 kD ultrafiltration tube and centrifuge at 4000 rpm for 15 min. After the centrifugation, add another 4.0 mL of phosphate buffer solution, repeat once, and concentrate to 200 μL.

[0591] In the above two preparation methods, the nitrogen / phosphorus (N / P) ratio of the ionizable lipid compound to the mRNA is 2.5:1.

[0592] Example 3: Physical property characterization of pharmaceutical composition nanoparticles

[0593] (1) Characterization of particle size and polydispersity index: The particle size and polydispersity index (PDI) of the pharmaceutical composition nanoparticles containing firefly luciferase mRNA prepared in Example 2 were measured using a dynamic light scattering (DLS). The specific operation method is as follows: Take 100 μL of lipid nanoparticle solution and add it to a cuvette, being careful not to generate bubbles, and then measure at room temperature. Each sample is measured three times.

[0594] (2) Characterization of encapsulation efficiency: For lipid nanoparticles (drug composition nanoparticles) containing RNA, the fluorescence method can be used to evaluate the encapsulation of RNA by lipid nanoparticles and the mRNA content in the nanoparticles. The specific operation method is as follows: dilute the sample with TE buffer solution (10mM Tris-HCl, 1.0mM EDTA, pH 7.5) to a concentration of about 5.0μg / mL. Then, transfer 50μL of the TE-diluted sample to a 96-well plate (black frame, transparent flat bottom, LABSELECT), and add 50μL of TE buffer (to obtain an undamaged sample and keep the LNP intact) or 50μL of 2% Triton X-100 solution to each well (to obtain a damaged sample, the LNP is damaged to cause complete leakage of mRNA). Place the 96-well plate at 37°C and incubate for 15 minutes. Add RIBOGREEN reagent (Invitrogen TM , R11491) was diluted in TE buffer at a volume ratio of 1:200, and 100 μL of the diluted solution was added to each well. The fluorescence intensity was measured using a microplate reader (SpectraMax M5) at an excitation wavelength of 480 nm and an emission wavelength of 520 nm. The fluorescence value of the reagent blank (the sample solution was replaced with the same volume of TE buffer and 2% Triton X-100 solution, respectively, and other conditions were the same) was subtracted from the fluorescence value of each sample, and the percentage of free RNA was determined by dividing the fluorescence intensity of the undamaged sample (Triton X-100 not added) by the fluorescence value of the damaged sample (Triton X-100 added). RIBOGreen fluorescence method is an effective method for determining mRNA concentration, and its principle is based on the linear relationship between the fluorescence signal intensity generated by the binding of RIBOGreen fluorescent dye to mRNA and the mRNA concentration. The above-mentioned addition of Triton X-100 fully releases mRNA from lipid nanoparticles, and the fluorescence value corresponding to the well can be substituted into the standard curve to obtain the mRNA concentration.

[0595] Standard curve preparation method: Use sample mRNA as the standard, first prepare mRNA into solutions with different concentration gradients, and take 100 μL of each concentration solution into a 96-well plate. TM , R11491) was diluted in TE buffer at a volume ratio of 1:200, and 100 μL of the diluted reagent was added to each well. The fluorescence intensity was measured using an ELISA reader (SpectraMaxM5) at an excitation wavelength of 480 nm and an emission wavelength of 520 nm. A standard curve was drawn based on the concentration of the standard sample and the measured fluorescence value.

[0596] The measurement results are shown in Table 3 below.

[0597] Table 3. Particle size and PDI of nanoparticles of pharmaceutical composition

[0598]

[0599]

[0600]

[0601] *SM102 is a control compound, and BM017 and BM045 are comparative compounds.

[0602] As shown in Table 3, the particle size of the nanoparticles of the pharmaceutical composition of the present invention is in the range of 70 nm to 150 nm, which is suitable for the delivery of mRNA. The particle size of the nanoparticles prepared from the compound BMS028 is the smallest, which is 76 nm; the particle size of the nanoparticles prepared from the compound BM005 is the largest, which is 139 nm.

[0603] The polydispersity coefficients of the nanoparticles of the pharmaceutical composition are all in the range of 0.03 to 0.20, indicating that the uniformity of the nanoparticles of the pharmaceutical composition is good and suitable for use as a drug delivery system. The PDI of the nanoparticles prepared by the compound BM037 is the smallest, which is 0.03; the PDI of the nanoparticles prepared by the compound BMS023 is the largest, which is 0.18. The PDI of the nanoparticles prepared by the comparative compound BM017 is 0.19.

[0604] The encapsulation rate of the nanoparticles of the pharmaceutical composition of the present invention is greater than 90%, and most of them are above 95%, which has a high encapsulation rate. In comparison, the encapsulation rate of the nanoparticles prepared by the compound BM017 is only 4%.

[0605] Example 4: Evaluation of protein expression levels of mRNA in nanoparticles of pharmaceutical compositions

[0606] The pharmaceutical composition nanoparticles containing firefly luciferase mRNA prepared in Example 2 were transfected into cells to measure the expression of mRNA, wherein the pharmaceutical composition nanoparticles encapsulated firefly luciferase mRNA.

[0607] Cell passaging and plating:

[0608] 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM, SIGMA, D6429-500 mL) containing 10% fetal bovine serum and 1% penicillin-streptomycin, at 37°C and 5% CO2. When the cells grew to the required number, the culture medium was discarded, 2.0 mL of trypsin (gibco, 25200-056 100 mL) was added to the culture dish and placed in an incubator for digestion until all cells fell off, then 10 mL of culture medium containing 10% serum was added to terminate the digestion, and the cells were transferred to a 15 mL centrifuge tube and centrifuged at 1000 rpm for 5 min. The supernatant was discarded and 1.0 mL of culture medium was added to resuspend the cells and count them. 30,000 cells per well were plated in a 48-well plate and cultured overnight until the cells adhered to the wall. When the cell confluence was about 70%-90%, the subsequent transfection experiment could be performed.

[0609] Cell transfection experiments:

[0610] According to the preparation method in Example 2, that is, the lipid component molar ratio is 50:38.5:10:1.5, and the lipid and mRNA are at N / P=2.5, various pharmaceutical composition nanoparticles containing 1.0 μg luciferase-mRNA are prepared. The prepared pharmaceutical composition nanoparticles are diluted with DMEM culture medium to a final volume of 300 μL and administered to the above-mentioned 48-well cell culture plate, with three replicate wells set for each sample.

[0611] 4 hours after administration, 30 μL serum (Sijiqing, 11011-8611) was added to each well, and the culture was continued for 24 hours. The cell culture medium in the well plate was aspirated, and 100 μL cell lysis solution (Yeasen, 11401ES80-11401-A) was added to each well. The cell lysate was repeatedly blown with a pipette to ensure that the protein in the cell was fully released. After sufficient lysis, the cell lysate was collected into a centrifuge tube, centrifuged at 10,000-15,000 × g for 3-5 minutes, 20 μL supernatant was taken from each well and transferred to a 96-well white plate, 100 μL firefly luciferase detection reagent (Yeasen, 11401ES80-11401-B) was added to each well, and the detection was performed using a multifunctional microplate reader, and the measurement interval was set to 2s and the measurement time was set to 10s. The higher the intensity and relative light unit (RLU) of luciferase expression in cells, the higher the delivery efficiency of the nanoparticles of the drug composition at the cellular level.

[0612] The results are as follows Figure 1 As shown, the expression level of firefly luciferase in the nanoparticles of each pharmaceutical composition is 10 5 Up to 10 7The results show that the nanoparticles of each drug composition can achieve a certain degree of effective cell transfection. However, the transfection efficiency of the nanoparticles prepared by the comparative compound BM017 is low. R3 in the structure of the compound is an alkyl-substituted oxyphenyl group, not an alkoxy-substituted phenyl group, so efficient transfection cannot be achieved.

[0613] Figure 2 The expression of firefly luciferase of each pharmaceutical composition nanoparticle is shown relative to the multiple of the nanoparticle prepared by SM102. Compared with SM102, the relative fluorescence values ​​of the pharmaceutical composition nanoparticles prepared by the compounds BM023, BM024, BM025, BM028, BM029, BM032, BM036, BM038, BM039, BM040, BM041, BM042, BM043, BM049, BM050, BMS001, BMS026, BMS036 and BMS037 of the present application are higher, and the RLU values ​​are more than 3 times that of the positive control SM102; among them, the compound BM043 shows the highest fluorescence expression, and the "RLU / mg protein" reaches 9.12 times that of the positive control SM102.

[0614] It can be seen from the above in vitro transfection results that the nanoparticles encapsulating firefly luciferase mRNA prepared by the ionizable lipid compound of the present invention have a high delivery efficiency for firefly luciferase mRNA and can be used as a good nucleic acid drug delivery carrier.

[0615] Example 5: Physical property characterization and protein expression level evaluation of pharmaceutical composition nanoparticles

[0616] (1) Characterization of particle size, polydispersity index and encapsulation efficiency: The particle size, polydispersity index (PDI) and encapsulation efficiency of the pharmaceutical composition nanoparticles containing green fluorescent protein eGFP mRNA prepared in Example 2 were characterized using the same method as in Example 3. The results are shown in Table 4.

[0617] (2) Evaluation of protein expression level: The nanoparticles of the pharmaceutical composition containing eGFP mRNA were transfected into cells according to the method of Example 4, and the percentage of positive cells was detected using a flow cytometer (FACSCanto II flow cytometer). The specific operation was as follows:

[0618] The original cell culture medium was discarded, and the cells were washed once with phosphate buffer (PBS, 1x, pH 7.2-7.4). 50 μL of trypsin (gibco, 25200-056 100 mL) was added to each well to digest the cells for 5 min. 200 μL of DMEM medium (SIGMA, D6429-500 mL) containing 10% serum (Sijiqing, 11011-8611) was added to terminate trypsin digestion. The cell suspension was transferred to a 1.5 mL centrifuge tube, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, 200 μL of ice PBS was added to each centrifuge tube, and the cell suspension was obtained by repeated pipetting. The cell suspension was analyzed using a flow cytometer. The results are shown in Table 4.

[0619] Table 4. Particle size, PDI, encapsulation efficiency, and percentage of green fluorescent protein-positive cells of the pharmaceutical composition nanoparticles relative to the multiples of the nanoparticles prepared by SM102

[0620]

[0621]

[0622] *SM102 is a control compound

[0623] As can be seen from the results in Table 4, the lipid nanoparticles of the present invention are also suitable for the encapsulation and delivery of eGFP mRNA. The particle size of each pharmaceutical composition nanoparticle encapsulated with eGFP mRNA is in the range of 80nm to 150nm, which is suitable for the delivery of mRNA; PDI is in the range of 0.06 to 0.16, with good uniformity, suitable for use as a drug delivery system; the encapsulation rate of lipid nanoparticles is not less than 95%, with a high encapsulation rate. These pharmaceutical composition nanoparticles all showed a high cell transfection efficiency, reaching more than 2 times that of the positive control SM102, respectively, among which BM026, BM028, and BM031 all reached more than 3 times that of the positive control SM102. This example further proves that the lipid nanoparticles prepared by the ionizable lipid compound of the present invention have a high delivery efficiency for mRNA and can be used as a good nucleic acid drug delivery carrier.

[0624] Example 6: Evaluation of the safety of pharmaceutical composition nanoparticles

[0625] The pharmaceutical composition nanoparticles containing firefly luciferase mRNA prepared in Example 2 were used to conduct a cell survival rate experiment, and the cells used were 293T cells.

[0626] Cell survival assay:

[0627] For the 96-well plate cells treated with parallel transfection and drug administration, the cell viability was detected 24 hours after drug administration. After adding 10 μL of CCK-8 solution (Japanese Tongren, CK04) to each well, the cells were incubated in a cell culture incubator (37°C, CO2 concentration of 5%) for 2 hours. The absorbance value at 450nm was detected using a multifunctional microplate reader. The ratio of the absorbance value of the cell wells treated with the drug composition nanoparticles to the absorbance value of the negative control (blank control, the drug composition nanoparticles were replaced with PBS, and other conditions were the same as the experimental group) was used as the cell viability value.

[0628] The results are as follows Figure 3 As shown, compared with the blank control group, the survival rate of cells treated with the nanoparticles of the pharmaceutical composition of the present invention was greater than 90%, indicating that the nanoparticles of the pharmaceutical composition of the present invention have good biocompatibility and biosafety.

[0629] Example 7: Evaluation of in vivo delivery effects of nanoparticles of pharmaceutical compositions

[0630] The experimental mice were SPF-grade BALB / c female mice, 6-8 weeks old, weighing 16-18g, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. All animals were adaptively raised for at least 3 days before the experiment. During the experiment, they were free to eat and drink, and the light was alternating between light and dark for 12 / 12h. The indoor temperature was 20-26℃ and the humidity was 40-70%. The mice were randomly divided into groups, with 3 mice in each group.

[0631] An in vivo delivery experiment was conducted using the pharmaceutical composition nanoparticles containing firefly luciferase mRNA prepared in Example 2. The pharmaceutical composition nanoparticles were injected intramuscularly (inner thigh) into mice (mRNA dose of 5.0 μg / mouse), and the mice were subjected to in vivo bioluminescence detection using a small animal in vivo imaging system (IVIS SPECTRUM, purchased from PerkinElmer) at a specific time point (4 h) after administration.

[0632] The specific operation steps of the detection are as follows: prepare a 15 mg / mL D-luciferin solution with normal saline, and give each mouse 3.0 mg of the substrate (D-luciferin) by intraperitoneal injection. 10 minutes after the substrate is given, the mouse is placed in an anesthesia box and anesthetized with 2.5% isoflurane. The anesthetized mouse is placed in an imaging system for bioluminescent imaging, and data collection and data analysis are performed on the part where the light is concentrated. Table 5 below lists the expression intensity of mRNA in mice obtained by different drug preparations.

[0633] Table 5. Expression intensity of mRNA in mice obtained by different drug composition nanoparticles

[0634]

[0635]

[0636]

[0637] *SM102 is a control compound, and BM017 and BM045 are comparative compounds.

[0638] From the results in Table 5, it can be seen that the average fluorescence flux induced by the pharmaceutical composition nanoparticles containing ionizable lipid compounds BM009, BM011, BM015, BM024, BM025, BM032, BM035, BM036, BM037, BM038, BM039, BM040, BM041, BM042, BM043 and BM050 measured at 4 hours is more than 4 times the average fluorescence flux induced by the preparation containing SM102, among which BM042 achieves the highest average fluorescence flux, which can reach 12.06 times that of SM102.

[0639] In addition, the average fluorescence flux induced by the pharmaceutical composition nanoparticles containing compounds BM005, BM006, BM008, BM010, BM012, BM013, BM014, BM016, BM021, BM022, BM028, BM029, BM031, BM034, BM044, BM049, BM053 and BM054 is more than twice the average fluorescence flux induced by the formulation containing SM102. The average fluorescence flux achieved by the pharmaceutical composition nanoparticles containing compounds BM020, BM023, BM026, BM027, BM033, BM051, BM052, BM055, BMS001, BMS036 and BMS037 is also higher than that of the formulation containing SM102.

[0640] Among the compounds of the present invention, the 4h average fluorescence flux achieved by the pharmaceutical composition nanoparticles containing other compounds of the present invention is higher than the 4h average fluorescence flux achieved by the pharmaceutical composition nanoparticles containing compounds BMS020, BMS023, BMS026, BMS028, and BMS031. Therefore, relative to compounds BMS020, BMS023, BMS026, BMS028, and BMS031, other compounds of the present invention are more preferred.

[0641] The 4-h average fluorescence flux of the pharmaceutical preparation containing the comparative compound BM017 was 0, and the 4-h average fluorescence flux of the pharmaceutical preparation containing the comparative compound BM045 was lower than that of SM102.

[0642] Example 8: Evaluation of sustained and stable expression of nanoparticles of pharmaceutical compositions in vivo

[0643] The pharmaceutical composition nanoparticles with a higher average fluorescence flux in vivo for 4 hours in Example 7 were selected for continuous expression monitoring, and specific time points (4 hours, 24 hours, 48 ​​hours and 72 hours) after administration were selected to perform in vivo bioluminescence detection on mice using a small animal in vivo imaging system (IVISSPECTRUM, purchased from PerkinElmer). The experimental mice, feeding and experimental conditions, and detection procedures were the same as those in Example 7. Table 6 lists the continuous expression of mRNA in mice obtained from different pharmaceutical preparations.

[0644] Table 6. Sustained mRNA expression in vivo of different drug composition nanoparticles (average fluorescence flux monitored at different time points [p / s / cm 2 / sr])

[0645]

[0646]

[0647] *SM102 is a control compound, and BM045 is a comparative compound.

[0648] Table 6 lists the sustained expression activity of firefly luciferase at different time points in mice using different pharmaceutical composition nanoparticles of the present invention. In long-term continuous monitoring, it was found that the average fluorescence intensity of SM102, BM009, BM015, BM023, BM028, BM033, BM034, BM036, BM037, BM039, BM040, BM042, BM043, BM044, BM045, BM050 and BMS036 groups showed the highest value at 24h, and the average fluorescence intensity basically began to weaken after 24h; the average fluorescence intensity of BM032 and BM035 groups still showed the highest value at 48h, and the average fluorescence intensity began to weaken after 48h. After observation for 72 hours, the average fluorescence values ​​of the BM009, BM015, BM023, BM028, BM032, BM033, BM034, BM035, BM036, BM037, BM039, BM040, BM042, BM043, BM044, BM050 and BMS036 groups were still above 1.0E+07, maintaining a high expression activity.

[0649] It can be seen from Table 6 that as time goes by, the average fluorescence intensity values ​​of the SM102 and BM045 groups decrease rapidly, and the advantage of the average fluorescence intensity of the nanoparticles of the pharmaceutical composition of the present invention gradually increases.

[0650] Table 7 lists the average fluorescence flux of different pharmaceutical composition nanoparticles of the present invention at different time points in mice relative to SM102. At 72 hours, the average fluorescence intensity values ​​of the pharmaceutical composition nanoparticles containing BM015, BM032, BM036 and BM042 were more than 30 times that of SM102, and the BM042 group was as high as 52.13 times that of SM102. The comparison compound BM045 group was only 1.16 times that of SM102 at 72 hours.

[0651] Table 7. In vivo sustained expression of mRNA of different drug composition nanoparticles (average fluorescence flux monitored at different time points relative to SM102 multiples)

[0652]

[0653]

[0654] *SM102 is a control compound, and BM045 is a comparative compound.

[0655] In order to compare the sustained expression ability of the drug preparations containing each ionizable lipid compound, the average fluorescence intensity of each sample at different time points was normalized with its own 4 h average fluorescence intensity. The results are shown in Table 8 and Figure 4 As shown in the results, the control compound SM102 retained 62% of its fluorescence intensity at 48h, and its fluorescence intensity at 72h rapidly decayed to 19%. In contrast, the compounds BM009, BM015, BM023, BM028, BM032, BM033, BM034, BM035, BM036, BM037, BM039, BM040, BM042, BM043, BM044, BM050 and BMS036 of the present application maintained a fluorescence intensity of more than 50% at 72h; among them, the fluorescence intensity of compounds BM033, BM035 and BM037 maintained at more than 50% at 72h. The fluorescence intensity of compounds BM009, BM023, BM028, BM034, BM042 and BMS036 were all stable at 80-90% at 72h; the fluorescence intensity of compounds BM015, BM028, BM032, BM034 and BMS036 was still as high as over 90% at 72h; the fluorescence intensity change trend of compound BM045 at the four time points was similar to that of SM102, and the fluorescence intensity decayed rapidly with time.

[0656] Table 8. In vivo sustained expression of mRNA of different pharmaceutical composition nanoparticles (average fluorescence flux monitored at different time points of each sample relative to the multiple of each 4h)

[0657]

[0658] *SM102 is a control compound, and BM045 is a comparative compound.

[0659] In summary, compounds BM009, BM015, BM023, BM028, BM032, BM033, BM034, BM035, BM036, BM037, BM039, BM040, BM042, BM043, BM044, BM050 and BMS036 not only show efficient mRNA delivery expression levels in vivo, but also can achieve sustained and stable expression of the delivered mRNA. Compared with the representative cationic lipid SM102 in the prior art, the expression stability has obvious advantages. When applied to mRNA vaccines, it can continuously and stably express antigens to continuously produce effective stimulation, and when applied to mRNA therapeutic drugs, it can also show sustained and stable protein expression.

[0660] Example 9: Evaluation of the encapsulation efficiency and in vivo transfection efficiency of nanoparticles of pharmaceutical compositions with different formulations

[0661] Pharmaceutical composition nanoparticles (containing firefly luciferase mRNA) comprising BM023, BM028, BM031, BM043, BMS001 and BMS036 were prepared by the same method as in Example 2, and the proportions of the components are shown in Table 9. The particle size, polydispersity coefficient, and encapsulation efficiency of the pharmaceutical composition nanoparticles were evaluated by the same method as in Example 3, and the in vivo transfection efficiency was evaluated by the same method as in Example 7.

[0662] Table 9. Pharmaceutical composition nanoparticle formulation and properties

[0663]

[0664]

[0665] As shown in Table 9, for the pharmaceutical composition nanoparticles of different formulations prepared using compound BM023, when the molar ratio of the ionizable lipid compound is higher than 40%, the particle size of the nanoparticles is in the range of 80 nm to 90 nm, and the PDI is less than 0.10; as the proportion of compound BM023 decreases, the particle size of the nanoparticles reaches below 80 nm, indicating that the particle size of the pharmaceutical composition nanoparticles containing BM023 can be compressed to a smaller size as the content of ionizable lipid compounds decreases; when the compound BM023 accounts for 40%, the increase in phospholipid content will make the particle size of the nanoparticles uneven and unstable. Compared with the classic formula (ionizable lipid compound / DSPC / cholesterol / PEG2000-DMG=50 / 10 / 38.5 / 1.5), the ionizable lipid compound / DSPC / cholesterol / PEG2000-DMG adjusted to 48.5 / 10 / 40 / 1.5 and 62.3 / 10 / 38.5 / 1.5 can obtain relatively high in vivo transfection activity; as the proportion of ionizable lipid compounds gradually decreases, the transfection activity gradually decreases.

[0666] Compound BM028 refines the ratio adjustment between phospholipids and cholesterol after reducing ionizable lipid compounds in the formulation adjustment. The results show that for the same BM028 ratio, the increase in phospholipids will lead to a decrease in in vivo transfection efficiency. Under different formulations studied in the present invention, the particle size of the nanoparticles of the pharmaceutical composition prepared by compound BM028 is mostly in the range of 80nm to 90nm, and the PDI is less than 0.10.

[0667] The pharmaceutical composition nanoparticles prepared from compounds BM031, BM043, BMS001 and BMS036 according to different prescriptions showed similar rules. In general, the novel ionizable lipid compounds designed by the present invention have good adaptability to changes in prescriptions, that is, no significant changes were found in particle size and activity, and were less affected by the content of the prescription; at the same time, the novel ionizable lipid compounds showed higher in vivo transfection efficiency in a prescription lower than the classic prescription.

[0668] Example 10: Physical property characterization of pharmaceutical composition nanoparticles (containing SARS CoV-2 mRNA) and mice Immune effect evaluation

[0669] Compounds that were less than 2 times more effective than SM102 in delivering firefly luciferase mRNA were further evaluated for their effectiveness in delivering SARS CoV-2 mRNA.

[0670] SARS CoV-2mRNA is the active ingredient of the new coronavirus mRNA vaccine.

[0671] (1) Characterization of particle size, polydispersity coefficient and encapsulation efficiency: The pharmaceutical composition nanoparticles encapsulating SARS CoV-2 mRNA were prepared according to the preparation method in Example 2, and the particle size, polydispersity coefficient and encapsulation efficiency of the obtained pharmaceutical composition nanoparticles were characterized according to the method of Example 3. The results are shown in Table 10.

[0672] Table 10. Particle size, PDI and encapsulation efficiency of nanoparticles of pharmaceutical compositions encapsulating SARS CoV-2 mRNA

[0673]

[0674]

[0675] *SM102 is a control compound

[0676] (2) Mouse immunization test and neutralizing antibody test

[0677] The experimental mice were SPF-grade BALB / c mice, female, 6-8 weeks old, weighing 16-18g, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. All animals were adaptively raised for at least 3 days before the experiment, and were free to eat and drink during the experiment. The light was alternating between light and dark for 12 / 12h, the indoor temperature was 20-26°C, and the humidity was 40-70%. The mice were randomly divided into groups, with 5 mice in each group. They were inoculated with the above-mentioned pharmaceutical composition nanoparticles prepared by SM102, BM020, BM023, BM026, BM028, BM033 or BM035, or PBS. Immunization was performed by intramuscular injection into the inner thigh on day 0 and day 14, with a dose of 6μg (125μL) per mouse.

[0678] 24 days after the first immunization, the mouse serum was extracted and the neutralizing antibody test of the immunized serum was performed using a virus microneutralization test (cytopathic method). The serum was serially diluted (starting from 1:10, 2-fold gradient serial dilution, and the diluent was M199 medium) and mixed with 100CCID50 / 0.05ml of SARS CoV-2BA.1 virus strain (Sinopharm Zhongsheng Biotechnology Research Institute Co., Ltd.) in equal proportions, incubated in a 5% CO2, 37°C incubator for 2 hours, and then 1×10 5 Up to 2.5×10 5 / mL of Vero cell suspension was cultured in a 5% CO2, 37°C incubator for 4 days. At the same time, a positive serum control group (positive quality control serum control, which has good neutralization against the BA.1 strain), a virus control group (the virus control is 100CCID50 / 0.05ml of SARS CoV-2BA.1 virus liquid, which does not contain serum) and a normal cell control group were set up. In this experiment, the test results can only be determined when the positive control, the virus control, and the normal cell control are established. If any one of them does not meet the requirements, the test fails; the cell pathological changes are observed after 4 days. When all controls are established, the neutralization endpoint is calculated by the Kaber method, that is, the highest dilution of serum that can protect 50% of cells from virus attack is used as the neutralizing antibody titer of the serum. All virus operations are completed in the BSL-3 laboratory. Table 11 shows the neutralizing antibody titers produced by mice immunized with nanoparticles of the pharmaceutical composition encapsulated with SARS CoV-2mRNA.

[0679] Table 11. Neutralizing antibody titers produced by mice immunized with nanoparticles of the pharmaceutical composition encapsulating SARS CoV-2 mRNA

[0680]

[0681]

[0682] *SM102 is a control compound

[0683] From the results of Table 10 and Table 11, it can be seen that the lipid nanoparticles of the present invention are also suitable for the encapsulation and delivery of SARS CoV-2 mRNA. The particle size of each pharmaceutical composition nanoparticle encapsulating SARS CoV-2 mRNA is in the range of 100nm to 150nm, which is suitable for the delivery of mRNA; the PDI is in the range of 0.06 to 0.16, with good uniformity, and is suitable for use as a drug delivery system; the encapsulation rate of the lipid nanoparticles is greater than 90%, with a high encapsulation rate.

[0684] The neutralizing antibody titers produced after the pharmaceutical preparation of the present invention (containing SARS CoV-2mRNA) immunized mice were better than the commercially available product SM102. In addition, it can be seen that the effect of lipid nanoparticles containing BM020, BM023, BM026, and BM033 in delivering firefly luciferase mRNA is less than 2 times that of SM102, but after long-term drug expression in this experiment, the neutralizing antibody results finally presented are significantly improved than SM102, and BM028 and BM035 still maintain a higher advantage than SM102. This once again verifies the superiority of the ionizable lipid compound of the present invention in the delivery of vaccines.

Claims

1. An ionizable lipid compound, a pharmaceutically acceptable salt or a stereoisomer thereof, wherein the ionizable lipid compound is represented by formula (I): in, R1 is C 2-10 Alkylene, R2 is C 2-10 Alkylene or -CH2CH2-N(CH2CH2OH)-C n H 2n -, wherein n is an integer from 2 to 10, L1 and L2 are each independently -O(C=O)-, -(C=O)O-, -NH(C=O)- or -(C=O)NH-, R3 is a para-alkoxy-substituted phenyl group or a meta-dialkoxy-substituted phenyl group, wherein the alkoxy group is C 2-20 Alkoxy, R4 is C 13-19 branched alkyl; or R4 is selected from substituted or unsubstituted C 8-10 Straight chain alkyl and unsubstituted C 8-10 Straight chain alkenyl; when R4 is substituted C 8-10 When the alkyl group is straight chain, the substituent is C 1-3 Alkyl or C 2-3 or R4 is phenyl substituted by an alkoxy group, wherein the alkoxy group is C 2-20 Alkoxy, and R5 is C 2-6 Alkylene.

2. The ionizable lipid compound, pharmaceutically acceptable salt or stereoisomer thereof according to claim 1, wherein R1 and R2 are each independently C 2-10 Alkylene.

3. The ionizable lipid compound according to claim 1, its pharmaceutically acceptable salt or stereoisomer, wherein R1 is C 3-9 Alkylene, R2 is C 5-9 Alkylene; or, wherein R1 is C 5-8 Alkylene, R2 is -CH2CH2-N(CH2CH2OH)-C n H 2n -, wherein n is an integer from 5 to 8.

4. The ionizable lipid compound, pharmaceutically acceptable salt or stereoisomer thereof according to claim 1, wherein the alkoxy group in R3 is C 2-12 Straight chain alkoxy.

5. The ionizable lipid compound, pharmaceutically acceptable salt or stereoisomer thereof according to claim 1, wherein the alkoxy group in R3 is C 5-11 Straight chain alkoxy.

6. The ionizable lipid compound, pharmaceutically acceptable salt or stereoisomer thereof according to claim 1, wherein R4 is -CH(C a H 2a+1 )(C b H 2b+1 ), wherein a and b are each independently an integer from 6 to 9; or R4 is an alkoxy-substituted phenyl group, wherein the alkoxy group is C 2-12 Straight chain alkoxy.

7. The ionizable lipid compound, pharmaceutically acceptable salt or stereoisomer thereof according to claim 1, wherein R4 is -CH(C8H 17 )2; or R4 is phenyl substituted by an alkoxy group, wherein the alkoxy group is C 8-10 Straight chain alkoxy.

8. The ionizable lipid compound, pharmaceutically acceptable salt or stereoisomer thereof according to claim 1, wherein R1 and R2 are each independently C 5-9 Alkylene, L1 and L2 are each independently -O(C=O)- or -(C=O)O-; R3 is a phenyl group substituted with a para-alkoxy group, wherein the alkoxy group is C 5-11 A straight chain alkoxy group, and R4 is C 13-19 Branched alkyl.

9. The ionizable lipid compound, pharmaceutically acceptable salt or stereoisomer thereof according to claim 8, wherein R4 is -CH(C a H 2a+1 )(C b H 2b+1 ), wherein a and b are each independently an integer from 6 to 9.

10. The ionizable lipid compound, pharmaceutically acceptable salt or stereoisomer thereof according to claim 1, wherein R1 and R2 are each independently C 5-9 Alkylene, L1 and L2 are each independently -O(C=O)- or -(C=O)O-; R3 is a phenyl group substituted by a meta-dialkoxy group, wherein the alkoxy group is C 8-10 A straight chain alkoxy group, and R4 is selected from substituted or unsubstituted C 8-10 Straight chain alkyl and unsubstituted C 8-10 Straight chain alkenyl; when R4 is substituted C 8-10 When the alkyl group is straight chain, the substituent is C 1-3 Alkyl or C 2-3 Alkenyl.

11. The ionizable lipid compound, pharmaceutically acceptable salt or stereoisomer thereof according to claim 1, wherein R1 and R2 are each independently C 5-7 Alkylene, L1 and L2 are each independently -O(C=O)- or -(C=O)O-; R3 is a phenyl group substituted by a meta-dialkoxy group, wherein the alkoxy group is C 8-10 A straight chain alkoxy group, and R4 is a phenyl group substituted by a para-alkoxy group, wherein the alkoxy group is C 8-10 Straight chain alkoxy.

12. The ionizable lipid compound, pharmaceutically acceptable salt or stereoisomer thereof according to claim 1, wherein R1 is C 5-8 Alkylene, R2 is -CH2CH2-N(CH2CH2OH)-C n H 2n -, wherein n is an integer from 5 to 8, L1 and L2 are each independently -O(C=O)- or -(C=O)O-; R3 is a phenyl group substituted with a para-alkoxy group, wherein the alkoxy group is C 8-11 A straight chain alkoxy group, and R4 is C 13-19 Branched alkyl.

13. The ionizable lipid compound, pharmaceutically acceptable salt or stereoisomer thereof according to claim 12, wherein R4 is -CH(C a H 2a+1 )(C b H 2b+1 ), wherein a and b are each independently an integer from 6 to 9.

14. The ionizable lipid compound, pharmaceutically acceptable salt or stereoisomer thereof according to claim 1, wherein R1 is C 5-8 Alkylene, R2 is -CH2CH2-N(CH2CH2OH)-C n H 2n -, wherein n is an integer from 5 to 8, L1 and L2 are each independently -O(C=O)- or -(C=O)O-; R3 is a phenyl group substituted by a meta-dialkoxy group, wherein the alkoxy group is C 8-10 A straight chain alkoxy group, and R4 is substituted or unsubstituted C 8-10 Straight chain alkyl; when R4 is substituted C 8-10 When the alkyl group is straight chain, the substituent is C 1-3 Alkyl or C 2-3 Alkenyl.

15. The ionizable lipid compound, pharmaceutically acceptable salt or stereoisomer thereof according to claim 1, wherein R1 and R2 are each independently C 5-8 Alkylene, L1 is -NH(C=O)- or -(C=O)NH-, L2 is -O(C=O)- or -(C=O)O-, R3 is a phenyl group substituted with a para-alkoxy group, wherein the alkoxy group is C 8-11 A straight chain alkoxy group, and R4 is C 13-19 Branched alkyl.

16. The ionizable lipid compound, pharmaceutically acceptable salt or stereoisomer thereof according to claim 15, wherein R4 is -CH(C a H 2a+1 )(C b H 2b+1 ), wherein a and b are each independently an integer from 6 to 9.

17. Use of the ionizable lipid compound, a pharmaceutically acceptable salt or a stereoisomer thereof according to any one of claims 1 to 16 in the preparation of a drug for treating a disease or condition in a mammal; wherein the drug is a nucleic acid drug.

18. The use according to claim 17, wherein the disease or condition comprises infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, neurodegenerative diseases, cardiovascular and renal vascular diseases and metabolic diseases.

19. The use according to claim 17, wherein the mammal is a human.

20. A lipid nanoparticle comprising the ionizable lipid compound, a pharmaceutically acceptable salt or a stereoisomer thereof according to any one of claims 1 to 16.

21. The lipid nanoparticle of claim 20, wherein the ionizable lipid compound, a pharmaceutically acceptable salt or a stereoisomer thereof accounts for 40% to 50% by molar proportion of the lipid nanoparticle.

22. The lipid nanoparticle of claim 20, further comprising cholesterol.

23. The lipid nanoparticle of claim 22, wherein the cholesterol accounts for 35% to 48.5% by molar ratio of the lipid nanoparticle.

24. The lipid nanoparticle of claim 20, further comprising a phospholipid.

25. The lipid nanoparticle of claim 24, wherein the phospholipid is selected from 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-diondecanoyl-sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn- Glycerol-3-phosphocholine, 1,2-di-O-octadecenyl-sn-glycerol-3-phosphocholine, 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycerol-3-phosphocholine, 1-hexadecyl-sn-glycerol-3-phosphocholine, 1,2-dialinolenoyl-sn-glycerol-3-phosphocholine, 1,2-diarachidonoyl-sn-glycerol-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycerol-3-phosphocholine, 1,2-dioleoyl-sn-glycerol-3-phosphocholine Acid ethanolamine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1 -glycerol) sodium salt, dipalmitoylphosphatidylglycerol, palmitoyloleoylphosphatidylethanolamine, distearoyl-phosphatidyl-ethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphoethanolamine, 1-stearoyl-2-oleoyl-stearoylethanolamine, 1-stearoyl-2-oleoyl-phosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine and mixtures thereof.

26. The lipid nanoparticle of claim 24, wherein the phospholipid accounts for 5% to 17.5% by molar ratio of the lipid nanoparticle.

27. The lipid nanoparticle of claim 20, further comprising a polymer-conjugated lipid.

28. The lipid nanoparticle according to claim 27, wherein The polymer-conjugated lipid is selected from one or more of pegylated lipids, polylactic acid-conjugated lipids, polyamidated lipids, polysarcosinate lipids, polylactic-co-glycolic acid copolymer-conjugated lipids, polypeptide-conjugated lipids and poly-peptide-conjugated lipids.

29. The lipid nanoparticle of claim 27, wherein the polymer-conjugated lipid accounts for 1.0% to 2.0% of the lipid nanoparticle in molar proportion.

30. The lipid nanoparticle of claim 20, further comprising cholesterol, phospholipids, and polymer-conjugated lipids.

31. The lipid nanoparticle of claim 30, wherein the molar ratio of the ionizable lipid compound, its pharmaceutically acceptable salt or stereoisomer to the cholesterol, the phospholipid and the polymer-conjugated lipid is (40-50):(35-48.5):(5-17.5):(1.0-2.0).

32. Use of the lipid nanoparticle according to any one of claims 20-31 in the preparation of a drug for treating a disease or condition in a mammal; wherein the drug is a nucleic acid drug.

33. Use according to claim 32, wherein the disease or condition comprises infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, neurodegenerative diseases, cardiovascular and renal vascular diseases and metabolic diseases.

34. The use according to claim 32, wherein the mammal is a human.

35. A pharmaceutical composition comprising a lipid nanoparticle according to any one of claims 20-31 and a therapeutic and / or prophylactic agent, wherein the therapeutic and / or prophylactic agent is a nucleic acid.

36. The pharmaceutical composition of claim 35, wherein the nucleic acid is selected from single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA, plasmid DNA, transfer RNA, messenger RNA, circular RNA, self-replicating mRNA, antisense nucleic acid molecules, small interfering RNA, small activating RNA, asymmetric interfering RNA, microRNA, small hairpin RNA, and Dicer-substrate RNA.

37. The pharmaceutical composition of claim 35, wherein the pharmaceutical composition is in the form of nanoparticles, and the polydispersity coefficient of the nanoparticles is less than 0.

20.

38. The pharmaceutical composition of claim 35, wherein the therapeutic and / or prophylactic agent is encapsulated in the lipid nanoparticles.

39. The pharmaceutical composition according to claim 38, wherein the encapsulation efficiency of the lipid nanoparticles for the therapeutic agent and / or preventive agent is greater than 90%.

40. The pharmaceutical composition of claim 35, wherein the therapeutic agent and / or preventive agent is mRNA, and the nitrogen / phosphorus ratio of the ionizable lipid compound, a pharmaceutically acceptable salt or stereoisomer thereof to the mRNA is 2:1 to 30:

1.

41. The pharmaceutical composition of claim 35, wherein the pharmaceutical composition is configured for oral, intravenous, intramucosal, intraperitoneal, intramuscular, transdermal, intradermal, subcutaneous or intranasal administration, or for administration by inhalation.

42. A pharmaceutical preparation comprising the pharmaceutical composition according to any one of claims 35-41 and a pharmaceutically acceptable excipient.

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