Nitrogen-containing chain compound, preparation method, composition containing nitrogen-containing chain compound and application

Abstract

The invention discloses a nitrogen-containing chain compound, a preparation method, a composition containing the nitrogen-containing chain compound and application, and particularly provides the nitrogen-containing chain compound as shown in the formula I or pharmaceutically acceptable salt of the nitrogen-containing chain compound. An LNP preparation prepared from the nitrogen-containing chain compound is relatively uniform in nanoparticle size, high in encapsulation efficiency and good in stability. The in-vivo expression activity is relatively high. # imgabs0 #

Description

[0001] This application claims the priority of Chinese patent application CN202410868083X, filed on June 28, 2024. This application cites the full text of the above Chinese patent application. Technical Field

[0002] The invention relates to a nitrogen-containing chain compound, a preparation method, a composition containing the same and application. Background Art

[0003] Nucleic acid drugs are an important direction of basic and applied research today. Nucleic acid drugs can be used to prevent and / or treat viral and bacterial infectious diseases, tumors, metabolic diseases, etc. Their production costs are lower and the cycle is shorter, which is conducive to the rapid development of personalized drugs. However, nucleic acids are negatively charged macromolecules that are difficult to penetrate cell membranes. At the same time, nucleic acids are not stable. By developing various nucleic acid packaging and delivery systems, the instability of nucleic acid drugs can be overcome to a certain extent and their delivery efficiency can be improved.

[0004] Lipid nanoparticles have been shown to be useful as carriers for delivering bioactive substances (such as small molecule drugs, proteins, and nucleic acids) into cells and / or intracellular compartments. Optimizing the nucleic acid drug delivery system by designing and optimizing the types and amounts of each component in the lipid nanoparticles is of great significance for improving the efficacy of nucleic acid drugs for prevention and treatment, especially lipid compounds that can be used to deliver RNA preventive and / or therapeutic agents and related methods and compositions. Summary of the invention

[0005] In order to overcome the problem that the types of ionizable lipid compounds and nucleic acid preventive and / or therapeutic agent delivery carriers in the prior art are few and the effects are poor, the present invention provides a nitrogen-containing chain compound, a preparation method, a composition comprising the same and an application thereof. The composition of the present invention can be used for efficient delivery of nucleic acid drugs. The LNP preparation prepared by using the nitrogen-containing chain compound of the present invention has one or more of the following advantages: (1) high encapsulation efficiency (2) uniform particle size (3) high in vivo expression activity.

[0006] The present invention solves the above technical problems through the following technical solutions.

[0007] The present invention provides a compound I or a pharmaceutically acceptable salt thereof:

[0008]

[0009] Among them, R 1 is a C substituted with one or more hydroxyl groups 1-6 alkyl;

[0010] X and Y are independently C 3-12 Alkylene;

[0011] Z 1 and Z 2 Independently

[0012] W 1 For chemical bonds, * indicates the same as Z 1 connect;

[0013] W 2 for * indicates the same as Z 2 connect;

[0014] R w11 , R w12 , R w21 and R w22 Independently for C 1-5 Straight chain alkylene;

[0015] R 2 C 1-24 alkyl;

[0016] R 3 C 1-24 alkyl.

[0017] In certain preferred embodiments of the present invention, certain groups of the compound I or a pharmaceutically acceptable salt thereof are defined as follows, and the unmentioned groups are the same as those described in any embodiment of the present invention (referred to as "in a certain embodiment of the present invention").

[0018] In one embodiment of the present invention, R 1 C is substituted by a hydroxyl group 1-6 alkyl.

[0019] In one embodiment of the present invention, R 1 In the case of a hydroxyl group, only the terminal carbon not attached to the nitrogen atom is replaced by a hydroxyl group.

[0020] In one embodiment of the present invention, R 1 for

[0021] In one embodiment of the present invention, X and Y are independently C 3-7 Alkylene, preferably C 3-6 Alkylene.

[0022] In one embodiment of the present invention, X and Y are independently straight chain alkylene groups.

[0023] In one embodiment of the present invention, X and Y are independently

[0024] In one embodiment of the present invention, Z 1 for *Denotes the same as W 1 connect.

[0025] In one embodiment of the present invention, Z 2 for *Denotes the same as W 2 connect.

[0026] In one embodiment of the present invention, R w11 , R w12 , R w21 and R w22 C 2-4 Straight chain alkylene.

[0027] In one embodiment of the present invention, W 1 For chemical bonds, * indicates the same as Z 1 connect.

[0028] In one embodiment of the present invention, W 2 for Represents Z 2 connect.

[0029] In one embodiment of the present invention, R 2 A straight chain or a branched C 4-18 Alkyl; preferably C 5-11 Straight chain alkyl or a branched C 15-17 alkyl.

[0030] In one embodiment of the present invention, R 2 for

[0031] In one embodiment of the present invention, R 3 for R 31 and R 32 C 3-10 Alkyl groups, such as C 6-9 Straight chain alkyl group.

[0032] In one embodiment of the present invention, R 31 and R 32 Independently

[0033] In one embodiment of the present invention, R 3 for

[0034] In one embodiment of the present invention, for

[0035] In one embodiment of the present invention, for

[0036] In one embodiment of the present invention, the compound I has a structure as shown in IA, IB or IC:

[0037]

[0038] Among them, R 1 , X, Y, Z 1 , W 1 , R 2 , R w21 , R w22 and R 3 The definition of is as described in any of the previous schemes. In one embodiment of the present invention, the compound I is any of the following structures:

[0039]

[0040]

[0041]

[0042] The present invention also provides a use of the compound I or a pharmaceutically acceptable salt thereof in the preparation of a nucleic acid preventive agent and / or therapeutic agent delivery vector;

[0043] The nucleic acid therapeutic agent and / or preventive agent is preferably one or more of single-stranded deoxyribonucleic acid (DNA), double-stranded DNA, small interfering RNA (siRNA), asymmetric double-stranded small interfering RNA (aiRNA), microRNA (miRNA), small hairpin RNA (shRNA), circular RNA (circRNA), transfer RNA (tRNA) or messenger RNA (mRNA), more preferably mRNA, such as firefly luciferase (Fluc) mRNA, SARS-CoV-2 spike protein (Spike) mRNA, herpes zoster virus (VZV) mRNA, respiratory syncytial virus (RSV) mRNA or influenza virus (Flu) mRNA.

[0044] The present invention also provides a composition comprising a substance Z, wherein the substance Z is the compound I as described above or a pharmaceutically acceptable salt thereof.

[0045] The present invention also provides a lipid carrier, which includes a substance Z, wherein the substance Z is the compound I as described above or a pharmaceutically acceptable salt thereof.

[0046] In one embodiment of the present invention, the lipid carrier further comprises a diluent, which can be a phosphate buffer, a sodium acetate buffer or a Tris-acetate buffer.

[0047] In one embodiment of the present invention, the lipid carrier further comprises phospholipids.

[0048] In a certain embodiment of the present invention, the phospholipid may be a conventional phospholipid in the art, which is an amphiphilic auxiliary molecule that helps the fusion of lipid particles and cell membranes. The phospholipid may be a phospholipid molecule having an electrically charged polar end and a non-polar end of an aliphatic chain, such as distearoylphosphatidylcholine (DSPC), dimyristoylphosphocholine (DMPC), dioleoylphosphocholine (DOPC), palmitoylphosphocholine (DPPC), heneicosanoylphosphocholine (DUPC) or palmitoylphosphocholine (POPC), etc., preferably distearoylphosphatidylcholine.

[0049] In a certain embodiment of the present invention, the lipid carrier further comprises PEG lipid (lipid modified with polyethylene glycol).

[0050] In a certain embodiment of the present invention, the PEG lipid may be a lipid molecule modified with a polyethylene glycol hydrophilic end. The PEG lipid is preferably selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol and PEG-modified dialkylglycerol, such as PEG-modified dimyristoylglycerol (DMG-PEG2000).

[0051] In one embodiment of the present invention, the lipid carrier further comprises sterol.

[0052] In a certain embodiment of the present invention, the sterol can be a conventional sterol in the art, and the sterol includes animal, plant or fungal sterols. The sterol is selected from one or more of cholesterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid and α-tocopherol, such as cholesterol.

[0053] In a certain embodiment of the present invention, in the lipid carrier, the molar ratio of the substance Z to sterol is (0.5-5):1, preferably (0.5-3):1, for example 1.3:1.

[0054] In one embodiment of the present invention, in the lipid carrier, the molar ratio of the substance Z to the phospholipid is (1-15):1, preferably (2-8):1, for example (3-6):1.

[0055] In one embodiment of the present invention, in the lipid carrier, the molar ratio of the substance Z to the phospholipid is (1-15):1, preferably (1-

[0056] 10):1, for example 5:1.

[0057] In one embodiment of the present invention, in the lipid carrier, the molar ratio of the substance Z to the PEG lipid is (10-100):1, preferably It is (10-50):1, for example 33.3:1.

[0058] In the present invention, the molar content means the percentage of a substance in the total mass of the lipid carrier, and the sum of the molar contents of the components in the lipid carrier shall not exceed 100 mol%.

[0059] In one embodiment of the present invention, the molar content of the substance Z is 30 mol% to 70 mol%, for example, 50 mol%.

[0060] In one embodiment of the present invention, the molar content of the phospholipid is 5 mol% to 20 mol%, for example, 10 mol%.

[0061] In one embodiment of the present invention, the molar content of the sterol is 20 mol% to 60 mol%, for example 38.5 mol%.

[0062] In one embodiment of the present invention, the molar content of the PEG lipid is about 0.2 mol% to 5 mol%, for example, 1.5% mol.

[0063] In a certain embodiment of the present invention, the lipid carrier is composed of the substance Z, the diluent, the phospholipid, the PEG lipid and the sterol.

[0064] The present invention also provides a use of the lipid carrier in preparing a nucleic acid preventive agent and / or therapeutic agent delivery carrier;

[0065] The nucleic acid therapeutic agent and / or preventive agent is preferably one or more of single-stranded deoxyribonucleic acid (DNA), double-stranded DNA, small interfering RNA (siRNA), asymmetric double-stranded small interfering RNA (aiRNA), microRNA (miRNA), small hairpin RNA (shRNA), circular RNA (circRNA), transfer RNA (tRNA) or messenger RNA (mRNA), preferably mRNA, such as firefly luciferase (Fluc) mRNA, SARS-CoV-2 spike protein (Spike) mRNA, herpes zoster virus (VZV) mRNA, respiratory syncytial virus (RSV) mRNA or influenza virus (Flu) mRNA.

[0066] The present invention also provides a lipid nanoparticle, which comprises a nucleic acid preventive agent and / or a therapeutic agent and the aforementioned lipid carrier;

[0067] The nucleic acid therapeutic agent and / or preventive agent is preferably one or more of single-stranded deoxyribonucleic acid (DNA), double-stranded DNA, small interfering RNA (siRNA), asymmetric double-stranded small interfering RNA (aiRNA), microRNA (miRNA), small hairpin RNA (shRNA), circular RNA (circRNA), transfer RNA (tRNA) or messenger RNA (mRNA), preferably mRNA, such as firefly luciferase (Fluc) mRNA, SARS-CoV-2 spike protein (Spike) mRNA, herpes zoster virus (VZV) mRNA, respiratory syncytial virus (RSV) mRNA or influenza virus (Flu) mRNA.

[0068] In a certain embodiment of the present invention, the nitrogen-phosphorus ratio in the lipid nanoparticles is 2-30:1; preferably 3-16:1; for example 6:1; the nitrogen-phosphorus ratio refers to the ratio of the molar number of ionizable nitrogen atoms in the substance Z to the molar number of phosphate groups in the RNA in the lipid nanoparticles.

[0069] In a certain embodiment of the present invention, the particle size (average particle size) of the lipid nanoparticles is 10-200nm, preferably 40-150nm, for example 50.32nm, 53.71nm, 59.16nm, 61.74nm, 67.40nm, 84.31nm, 63.89nm, 95.80, 100.27, 73.07, 62.60, 97.72, 131.87 or 131.40; for example 84.31nm, 63.89nm, 95.80, 100.27, 73.07, 62.60 or 131.40.

[0070] In one embodiment of the present invention, the polydispersity index of the lipid nanoparticles is 0.001-0.3; for example, 0.135, 0.323, 0.107, 0.089, 0.106, 0.188, 0.201, 0.129, 0.079, 0.159, 0.298, 0.235 or 0.073, for example, 0.129,

[0071] 0.079, 0.159, 0.298, 0.235 or 0.073.

[0072] In one embodiment of the present invention, the encapsulation efficiency of the lipid nanoparticles is 84%-100%, for example, 84.5%, 92.5%, 93.4%, 96.3%, 94.9%, 94.1%, 92.9%, 96.4%, 96.7%, 92.7%, 93.5%, 96.2%, 96.1% or 86.8%.

[0073] In one embodiment of the present invention, the encapsulation efficiency of the lipid nanoparticles is 85%-100%, for example, 96.40% or 96.66%, 92.74%, 93.51%, 96.17%, 96.10% or 86.82%.

[0074] In one embodiment of the present invention, in the lipid nanoparticles, the lipid carrier encapsulates the nucleic acid preventive agent and / or therapeutic agent.

[0075] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0076] The reagents and raw materials used in the present invention are commercially available.

[0077] The positive and progressive effects of the present invention are that the LNP preparation prepared by using the nitrogen-containing chain compound of the present invention has relatively uniform nanoparticle size, high encapsulation efficiency and high in vivo expression activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 The graph of the change of chemiluminescence intensity over time in the liver area of ​​mice after intravenous administration of compound 2 and compound 3;

[0079] Figure 2 The graph of the change of chemiluminescence intensity over time at the administration site of mice after intramuscular administration of compound 2 and compound 3;

[0080] Figure 3 The graph of the change of chemiluminescence intensity over time in the liver area of ​​mice after intramuscular administration of compound 2 and compound 3;

[0081] Figure 4 The graph of the change of chemiluminescence intensity over time in the liver area of ​​mice after intravenous administration of compound 4 and compound 5;

[0082] Figure 5 The graph of the change of chemiluminescence intensity over time in the liver area of ​​mice after intravenous administration of compound 4 and compound 5;

[0083] Figure 6 This is a graph showing the change in chemiluminescence intensity over time in the liver area of ​​mice after intravenous administration of compound 1.

[0084] Figure 7This is a graph showing the change in chemiluminescence intensity over time in the liver area of ​​mice after intravenous administration of H21, H23, compound 10, compound 11, compound 12, compound 14 and compound 17.

[0085] Figure 8 This is a graph showing the change in chemiluminescence intensity over time in the liver area of ​​mice after intravenous administration of compound 6 and compound 8. DETAILED DESCRIPTION

[0086] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples.

[0087] Preparation Example 1 Preparation of Compound 2

[0088] Step 1: Preparation of 2-1

[0089] Reaction:

[0090]

[0091] Material ratio:

[0092] Material Name Molecular weight Feed ratio Feeding amount mmol Succinic anhydride 100 4eq 8g 80 8-Pentadecanol 228 1 eq 4.6g 20 DMAP 122 1 2.5g 20 Triethylamine 101 0.5 1g 10 DCM - - 100mL -

[0093] Operation process:

[0094] Add succinic anhydride, 8-pentadecanol, DMAP, triethylamine and DCM to the reaction flask and stir the reaction at room temperature for 16 hours. The product Rf value is 0.6, TLC (DCM: MeOH = 10: 1). The reaction solution is washed once with 100 mL of water, the organic phase is dried over anhydrous sodium sulfate, filtered and concentrated, and purified by column chromatography to obtain 6.5 g of colorless oil.

[0095] Step 2: Preparation of 2-2

[0096] Reaction:

[0097]

[0098] Material ratio:

[0099] Material Name Molecular weight Feed ratio Feeding amount mmol 2-1 328.49 1.1eq 5.3g 16.1 6-Bromohexanol 181 1 eq 2.6g 14.6 EDCI 192 2 eq 5.6g 29.2 DMAP 122 0.2 eq 350mg 2.9 DCM - - 80mL -

[0100] Operation process:

[0101] Add 2-1,6-bromohexanol, EDCI, DMAP and DCM to the reaction flask and react at room temperature for 16 hours. The product Rf value is 0.5, TLC (PE:EA = 10:1). The reaction solution is washed once with 100 mL of water, and the organic phase is dried over anhydrous sodium sulfate, filtered and concentrated, and purified by column chromatography to obtain 5.6 g of colorless oil.

[0102] Step 3: Preparation of compound 2

[0103] Reaction:

[0104]

[0105] Material ratio:

[0106] Material Name Molecular weight Feed ratio Feeding amount mmol 2-2 491.55 1.1eq 2g 4 SM102-3 329.53 1 eq 1.2g 3.7 Potassium carbonate 138 2eq 1g 7.4 Potassium iodide 166 2 eq 1.2g 7.4 Acetonitrile - - 40mL

[0107] Operation process:

[0108] Add 2-2, SM102-3, potassium carbonate, potassium iodide and acetonitrile to the reaction flask, stir at 65°C for 16h. The product Rf value is 0.5, TLC (DCM: MeOH = 10: 1). The reaction solution is concentrated, then diluted with 100 mL of ethyl acetate, washed once with 100 mL of water, the organic phase is dried over anhydrous sodium sulfate, filtered and concentrated, and purified by column chromatography to obtain 1.6 g of colorless oil.

[0109] 1 H NMR(600MHz,Chloroform-d)δ5.08(qd,J=7.0,3.2Hz,1H),4.22(dd,J=11.8,3.3Hz,1H),4.07–3.99(m,3H),3.53(t,J=5.3Hz,2H),2.57(t, J=5.4Hz,2H),2.46(q,J=8.3Hz,4H),2.33–2.27(m,6H),1.65–1.53(m,10H),1.50–1.43(m,4H),1.34–1.22(m,38H),0.87(t,J=6.8Hz,9H).

[0110] MS (ES+) m / z): 740.6 (M+H) + .

[0111] Preparation Example 2 Preparation of Compound 3

[0112] Step 1: Preparation of compound 3

[0113] Reaction:

[0114]

[0115] Material ratio:

[0116] Material Name Molecular weight Feed ratio Feeding amount mmol 2-2 491.55 2.2eq 3.8g 7.73 Ethanolamine 61 1 eq 214mg 3.5 Potassium carbonate 138 3 eq 1.5g 10.5 Potassium iodide 166 3 eq 1.7g 10.5 Acetonitrile - - 40mL -

[0117] Operation process:

[0118] Add 2-2, ethanolamine, potassium carbonate, potassium iodide and acetonitrile to the reaction flask, stir at 65°C for 16h. The product Rf value is 0.5, TLC (DCM: MeOH = 10: 1). The reaction solution is concentrated, then diluted with 100 mL of ethyl acetate, washed once with 100 mL of water, the organic phase is dried over anhydrous sodium sulfate, filtered and concentrated, and purified by column chromatography to obtain 1.6 g of colorless oil.

[0119] 1 H NMR(600MHz,Chloroform-d)δ4.89–4.84(m,2H),4.11(q,J=7.1Hz,1H),4.07(t,J=6.7Hz,4H),3.55(t,J=5.3Hz,2H),2.6 1(s,10H),2.48(t,J=7.6Hz,4H),1.62(p,J=6.9Hz,4H),1.54–1.44(m,12H),1.37–1.22(m,48H),0.87(t,J=7.0Hz,12H).

[0120] MS (ES+) m / z): 882.7 (M+H) + .

[0121] Preparation Example 3 Preparation of Compound 4

[0122] Step 1: Synthesis of 4-(heptadecan-9-oxy)-4-oxybutyric acid

[0123] Reaction:

[0124]

[0125] Material ratio:

[0126] Material Name Molecular weight Feed ratio Feeding amount mmol Heptadecan-9-ol 256 1.0 eq 3.00g 11.7mmol Succinic anhydride 100 1.5eq 1.76g 17.5mmol 4-Dimethylaminopyridine 122 1.0eq 1.43g 11.7mmol Pyridine 79 1.5eq 1.39g 17.5mmol Tetrahydrofuran - - 30.0mL -

[0127] Operation process:

[0128] Tetrahydrofuran (30.0 mL), heptadecan-9-ol (3.00 g, 11.7 mmol), succinic anhydride (1.76 g, 17.5 mmol), 4-dimethylaminopyridine (1.43 g, 11.7 mmol), pyridine (1.39 g, 17.5 mmol) were added to the reaction bottle in sequence, and the mixture was reacted at 60°C for 12 hours under nitrogen protection. The reaction was completed by TLC monitoring, and the reaction solution was concentrated and purified by column chromatography to obtain a colorless liquid 4-(heptadecan-9-oxy)-4-oxylylidenebutanoic acid (3.74 g).

[0129] Step 2: Synthesis of 1-(6-bromohexyl)-4-heptadecan-9-ylbutanediol

[0130] Reaction:

[0131]

[0132] Material ratio:

[0133]

[0134] Operation process:

[0135] Dissolve 4-(heptadecan-9-oxy)-4-oxydecanedioic acid (2.00 g, 5.61 mmol) in dichloromethane (20.0 mL), add 6-bromohexane-1-ol (1.22 g, 6.73 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.61 g, 8.41 mmol), 4-dimethylaminopyridine (68.5 mg, 560 μmol) in sequence, and finally react at 25 ° C for 12 hours under nitrogen protection. The reaction is completed by TLC monitoring, and the reaction solution is concentrated. The crude product is subjected to column chromatography to obtain colorless liquid 1-(6-bromohexyl) 4-heptadecan-9-yl butanediol (2.50 g).

[0136] Step 3: Synthesis of 1-heptadecan-9-yl 4-{6-[(2-hydroxyethyl)[6-oxyylidene-6-(undecyloxy)hexyl]amino]hexyl}butanediol

[0137] Reaction:

[0138]

[0139] Material ratio:

[0140]

[0141] Operation process:

[0142] Dissolve undecyl 6-[(2-hydroxyethyl)amino]hexyl ester (600 mg, 1.82 mmol) in acetonitrile (6.00 mL), add 1-(6-bromohexyl) 4-heptadecan-9-yl butanediol (1.23 g, 2.37 mmol), potassium carbonate (880 mg, 6.37 mmol), potassium iodide (362 mg, 2.19 mmol), tetrahydrofuran (6.00 mL) in sequence, and finally react at 80°C for 12 hours under nitrogen protection. After TLC monitoring, the reaction is completed, filtered, and concentrated. Purification by column chromatography gives a colorless liquid 1-heptadecan-9-yl 4-{6-[(2-hydroxyethyl)[6-oxyylidene-6-(undecyloxy)hexyl]amino]hexyl}butanediol (715 mg).

[0143] 1H NMR (400MHz, CHLOROFORM-d) δ = 4.88 (t, J = 6.2Hz, 1H), 4.07 (q, J = 6.8Hz, 4H), 3.53 (t, J = 5.2Hz, 2H), 2.62 (s, 4H), 2.58 (t ,J=5.2Hz,2H),2.49-2.41(m,4H),2.31(t,J=7.4Hz,2H),1.63-1.42(m,14H),1.39-1.18(m,46H),0.99-0.83(m,9H)ppm.

[0144] LCMS: RT = 2.015, m / z 767 [M+H] +

[0145] Preparation Example 4 Preparation of Compound 5

[0146] Step 1: Synthesis of 6-(heptadecan-9-oxy)-6-oxyhexanoic acid

[0147] Reaction:

[0148]

[0149] Material ratio:

[0150] Material Name Molecular weight Feed ratio Feeding amount mmol Heptadecan-9-ol 256 1.0 eq 3.00g 11.7mmol Oxepin-2,7-dione 128 1.5eq 2.25g 17.5mmol 4-Dimethylaminopyridine 122 1.0eq 1.43g 11.7mmol Pyridine 79 1.5eq 1.39g 17.5mmol Tetrahydrofuran - - 30mL -

[0151] Operation process:

[0152] In a reaction flask, heptadecan-9-ol (3.00 g, 11.7 mmol) was dissolved in tetrahydrofuran (30 mL), and oxepane-2,7-dione (2.25 g, 17.5 mmol), 4-dimethylaminopyridine (1.43 g, 11.7 mmol), and pyridine (1.39 g, 17.5 mmol) were added in sequence, and the mixture was reacted at 60° C. for 16 hours. The reaction was monitored by TLC, and the reaction solution was concentrated to remove pyridine, and purified by column chromatography to obtain yellow liquid 6-(heptadecan-9-oxy)-6-oxyylidene hexanoic acid (2.00 g).

[0153] Step 2: Synthesis of 1-(6-bromohexyl)-6-heptadecan-9-yl hexanediol

[0154] Reaction:

[0155]

[0156] Material ratio:

[0157]

[0158] Operation process:

[0159] In a reaction flask, 6-(heptadecan-9-oxy)-6-oxyidene hexanoic acid (1.90 g, 4.94 mmol) was dissolved in tetrahydrofuran (20 mL), and 6-bromohexane-1-ol (1.79 g, 9.88 mmol), 4-dimethylaminopyridine (120 mg, 988 μmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.42 g, 7.41 mmol) were added in sequence, and the reaction was finally carried out at 40° C. for 16 hours. The reaction was monitored by TLC, extracted three times with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product, which was purified by column chromatography to obtain a colorless liquid 1-(6-bromohexyl) 6-heptadecan-9-yl hexanediol (2.30 g).

[0160] Step 3: Synthesis of 1-heptadecan-9-yl 6-{6-[(2-hydroxyethyl)[6-oxyylidene-6-(undecyloxy)hexyl]amino]hexyl}hexanediol

[0161] Reaction:

[0162]

[0163] Material ratio:

[0164]

[0165] Operation process:

[0166] In a reaction flask, 1-(6-bromohexyl) 6-heptadecan-9-yl hexanediol (1.00 g, 1.83 mmol) was dissolved in acetonitrile (10 mL), and then undecyl 6-[(2-hydroxyethyl)amino]hexyl ester (661 mg, 2.01 mmol), potassium carbonate (757 mg, 5.48 mmol), potassium iodide (363 mg, 2.19 mmol), and tetrahydrofuran (10 mL) were added in sequence. Finally, the mixture was reacted at 80° C. for 12 hours under nitrogen protection. The reaction was completed after monitoring by TLC. The mixture was concentrated to obtain a crude product, which was purified by column chromatography to obtain a colorless liquid 1-heptadecan-9-yl 6-{6-[(2-hydroxyethyl)[6-oxyylidene-6-(undecyloxy)hexyl]amino]hexyl}hexanediol (1.10 g).

[0167] 1H NMR (400MHz, CHLOROFORM-d) δ = 4.86 (quin, J = 6.2Hz, 1H), 4.06 (t, J = 6.6Hz, 4H), 3.74 (br s, 2H), 2.83 (br s, 2H), 2.72 (br s,4H),2.36-2.29(m,6H),1.71-1.58(m,14H),1.51(br d,J=5.4Hz,4H),1.41-1.23(m,46H),0.88(br t,J=6.6Hz,9H).

[0168] LCMS: RT=2.054,m / z 796.7[M+H] +

[0169] Preparation Example 5 Preparation of Compound 7

[0170] Step 1: Synthesis of 1-(4-bromobutyl)-6-heptadecan-9-yl hexanediol

[0171] Reaction:

[0172]

[0173] Material ratio:

[0174]

[0175] Operation process:

[0176] In a reaction flask, 6-(heptadecan-9-oxy)-6-oxyylidene hexanoic acid (1.10 g, 2.86 mmol) was dissolved in tetrahydrofuran (12 mL), and 4-bromobutane-1-ol (875 mg, 5.72 mmol), 4-dimethylaminopyridine (69.8 mg, 572 μmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (822 mg, 4.29 mmol) were added in sequence, and the mixture was reacted at 40° C. for 16 hours. The reaction was monitored by TLC, and the mixture was extracted three times with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product, which was purified by column chromatography to obtain a yellow liquid 1-(4-bromobutyl)6-heptadecan-9-yl hexanediol (800 mg).

[0177] Step 2: Synthesis reaction formula of 1-heptadecan-9-yl 6-{4-[(2-hydroxyethyl)[6-oxyylidene-6-(undecyloxy)hexyl]amino]butyl}hexanediol:

[0178]

[0179] Material ratio:

[0180]

[0181]

[0182] Operation process:

[0183] In a reaction flask, 1-(4-bromobutyl)6-heptadecan-9-yl hexanediol (800 mg, 1.54 mmol) was dissolved in acetonitrile (5 mL), and then undecyl 6-[(2-hydroxyethyl)amino]hexyl ester (558 mg, 1.69 mmol), potassium carbonate (638 mg, 4.62 mmol), potassium iodide (306 mg, 1.85 mmol), and tetrahydrofuran (5 mL) were added in sequence. Finally, the mixture was reacted at 80° C. for 12 hours under nitrogen protection. The reaction was completed after TLC monitoring. The mixture was concentrated to obtain a crude product, which was purified by column chromatography to obtain a yellow liquid 1-heptadecan-9-yl 6-{4-[(2-hydroxyethyl)[6-oxyylidene-6-(undecyloxy)hexyl]amino]butyl}hexanediol (500 mg).

[0184] 1 H NMR (400MHz, CHLOROFORM-d) δ = 4.87 (quin, J = 6.4Hz, 1H), 4.08 (td, J1 = 6.6, J2 = 10.2Hz, 4H), 3.62 (br s,2H),2.73-2.53(m,6H),2.36-2.29(m,6H),1.69-1.48(m,20H),1.26(br s,40H),0.89(t,J=6.8Hz,9H).

[0185] LCMS: RT=2.033,m / z 768.6[M+H] +

[0186] Preparation Example 6 Preparation of Compound 9

[0187] Step 1: Preparation of 9-1

[0188] Reaction:

[0189]

[0190] Material ratio:

[0191] Material Name Molecular weight Feed ratio Feeding amount mmol Succinic anhydride 100 4eq 8g 80 8-Heptadecanol 256.47 1 eq 5.1g 20 DMAP 122 1 2.5g 20 Triethylamine 101 0.5 1g 10 DCM - - 100mL -

[0192] Operation process:

[0193] Add succinic anhydride, 8-heptadecanol, DMAP, triethylamine and DCM to the reaction flask and stir the reaction at room temperature for 16 hours. The product Rf value is 0.6, TLC (DCM: MeOH = 10: 1). The reaction solution is washed once with 100 mL of water, the organic phase is dried over anhydrous sodium sulfate, filtered and concentrated, and 6 g of colorless oil is obtained after column chromatography purification.

[0194] Step 2: Preparation of 9-2

[0195] Reaction:

[0196]

[0197] Material ratio:

[0198] Material Name Molecular weight Feed ratio Feeding amount mmol 9-1 356.55 1.1eq 5.7g 16.1 6-Bromohexanol 181 1 eq 2.6g 14.6 EDCI 192 2 eq 5.6g 29.2 DMAP 122 0.2 eq 350mg 2.9 DCM - - 80mL -

[0199] Operation process:

[0200] Add 9-1, 6-bromohexanol, EDCI, DMAP and DCM to the reaction flask and react at room temperature for 16 hours. The product Rf value is 0.5, TLC (PE:EA = 10:1). The reaction solution is washed once with 100 mL of water, and the organic phase is dried over anhydrous sodium sulfate, filtered and concentrated, and purified by column chromatography to obtain 5.6 g of colorless oil.

[0201] Step 3: Preparation of compound 9

[0202] Reaction:

[0203]

[0204] Material ratio:

[0205] Material Name Molecular weight Feed ratio Feeding amount mmol 9-2 519.61 2.2eq 4g 7.73 Ethanolamine 61 1eq 214mg 3.5 Potassium carbonate 138 3eq 1.5g 10.5 Potassium iodide 166 3eq 1.7g 10.5 Acetonitrile - - 40mL -

[0206] Operation process:

[0207] Add 9-2, ethanolamine, potassium carbonate, potassium iodide and acetonitrile to the reaction flask, stir at 65°C for 16h. The product Rf value is 0.5, TLC (DCM: MeOH = 10: 1). The reaction solution is concentrated, then diluted with 100 mL of ethyl acetate, washed once with 100 mL of water, the organic phase is dried over anhydrous sodium sulfate, filtered and concentrated, and purified by column chromatography to obtain 2 g of colorless oil.

[0208] 1H NMR(600MHz,Chloroform-d)δ4.89–4.84(m,2H),4.11(q,J=7.1Hz,1H),4.07(t,J=6.7Hz,4H),3.55(t,J=5.3Hz,2H),2.6 1(s,10H),2.48(t,J=7.6Hz,4H),1.62(p,J=6.9Hz,4H),1.54–1.44(m,12H),1.37–1.22(m,56H),0.87(t,J=7.0Hz,12H).

[0209] MS (ES+) m / z): 938.7 (M+H) + .

[0210] Preparation Example 7 Preparation of Compound 1

[0211] Step 1: Preparation of 1-1

[0212] Reaction:

[0213]

[0214] Material ratio:

[0215] Material Name Molecular weight Feed ratio Feeding amount mmol 1,4-Butanediol 90 1eq 9g 100 Hexanoic acid 116 0.9eq 10.44g 90 EDCI 192 1.2 23g 120 DMAP 122 0.12 1.5g 12 DCM - - 200mL -

[0216] Operation process:

[0217] Add 1,4-butanediol, n-hexanoic acid, EDCI, DMAP and DCM to the reaction flask and stir at room temperature for 16 hours. The product Rf value is 0.4, TLC (PE:EA = 10:1). The reaction solution is washed once with 200 mL of water, and the organic phase is dried over anhydrous sodium sulfate, concentrated and purified by column chromatography to obtain 12 g of oil.

[0218] Step 2: Preparation of 1-2

[0219] Reaction:

[0220]

[0221] Material ratio:

[0222] Material Name Molecular weight Feed ratio Feeding amount mmol 1-1 188 1eq 5g 26.6 6-Bromohexanoic acid 195 1.1 eq 5.7g 29 EDCI 192 1.5eq 7.6g 40 DMAP 122 0.15 eq 480mg 4 DCM - - 150mL -

[0223] Operation process:

[0224] Add 1-1,6-bromohexanoic acid, EDCI, DMAP and DCM to the reaction flask and react at room temperature for 16 hours. The product Rf value is 0.5, TLC (PE:EA = 20:1). The reaction solution is washed once with 150 mL of water, and the organic phase is dried over anhydrous sodium sulfate, concentrated and purified by column chromatography to obtain 6 g of oil.

[0225] Step 3: Preparation of 1-3

[0226] Reaction:

[0227]

[0228] Material ratio:

[0229] Material Name Molecular weight Feed ratio Feeding amount mmol Succinic anhydride 100 4eq 8g 80 7-Tridecyl alcohol 200 1eq 4g 20 DMAP 122 1 2.5g 20 Triethylamine 101 0.5 1g 10 DCM - - 100mL -

[0230] Operation process:

[0231] Add succinic anhydride, 7-tridecanol, DMAP, triethylamine and DCM to the reaction flask and stir the reaction at room temperature for 16 hours. The reaction solution was concentrated and purified by column chromatography to obtain 6 g of colorless oil. The product Rf value was 0.6, TLC (DCM: MeOH = 10: 1). The reaction solution was concentrated and purified by column chromatography to obtain 6 g of white solid.

[0232] Step 4: Preparation of compound 1-4

[0233] Reaction:

[0234]

[0235] Material ratio:

[0236] Material Name Molecular weight Feed ratio Feeding amount mmol 1-3 300.44 1.1 eq 4.83g 16.1 6-Bromohexanol 181 1 eq 2.6g 14.6 EDCI 192 2 eq 5.6g 29.2 DMAP 122 0.2 eq 350mg 2.9 DCM - - 100mL -

[0237] Operation process:

[0238] Add 1-3, 6-bromohexanol, EDCI, DMAP and DCM to the reaction flask and react at room temperature for 16 hours. The product Rf value is 0.6, TLC (PE:EA = 20:1). The reaction solution is washed once with 100 mL of water, and the organic phase is dried over anhydrous sodium sulfate, concentrated and purified by column chromatography to obtain 4 g of oil.

[0239] Step 5: Preparation of 1-5

[0240] Reaction:

[0241]

[0242] Material ratio:

[0243] Material Name Molecular weight Feed ratio Feeding amount mmol 1-4 463.50 1eq 3g 6.47 Ethanolamine 61 10eq 4g 65 Acetonitrile - - 40mL -

[0244] Operation process:

[0245] Add 1-4, ethanolamine and acetonitrile to the reaction flask, stir and react at room temperature for 16 hours. The product Rf value is 0.2, TLC (DCM: MeOH = 10: 1). After the reaction solution is concentrated, it is diluted with 100 mL of ethyl acetate, and then washed twice with 100 mL of water. The organic phase is dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain 2 g of oil.

[0246] Step 6: Preparation of Compound 1

[0247] Reaction:

[0248]

[0249] Material ratio:

[0250] Material Name Molecular weight Feed ratio Feeding amount mmol 1-2 365.31 1.1eq 1.8g 5 1-5 443.67 1eq 2g 4.5 Potassium carbonate 138 1.5eq 1g 6.8 Potassium iodide 166 1 eq 750mg 4.5 Acetonitrile - - 50mL -

[0251] Operation process:

[0252] Add 1-2, 1-5, potassium carbonate, potassium iodide and acetonitrile to the reaction flask, stir at 65°C for 16h. The product Rf value is 0.6, TLC (DCM: MeOH = 10: 1). After the reaction solution is concentrated, it is diluted with 100 mL of ethyl acetate, washed with 100 mL of water, and the organic phase is dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain 1.5 g of an oily substance.

[0253] 1 H NMR(600MHz,Chloroform-d)δ4.84(h,J=5.7Hz,1H),4.09–4.02(m,6H),3.58(t ,J=5.3Hz,2H),2.65(t,J=5.3Hz,2H),2.59(d,J=2.2Hz,4H),2.56–2.50(m,4H) ,2.28(dt,J=10.2,7.5Hz,4H),1.67(h,J=3.0Hz,4H),1.64–1.57(m,6H),1.49( td,J=10.9,7.9,3.4Hz,8H),1.35–1.19(m,26H),0.86(dt,J=12.5,6.9Hz,9H).

[0254] MS (ES+) m / z): 728.6 (M+H) + .

[0255] Preparation Example 8 Preparation of Compound 10

[0256] Step 1: Preparation of 10-1

[0257] Reaction:

[0258]

[0259] Material ratio:

[0260] Material Name Molecular weight Feed ratio Feeding amount mmol Succinic anhydride 100 4eq 8g 80 8-Pentadecanol 228 1 eq 4.6g 20 DMAP 122 1 2.5g 20 Triethylamine 101 0.5 1g 10 DCM - - 100mL -

[0261] Operation process:

[0262] Succinic anhydride, 8-pentadecanol, DMAP, triethylamine and DCM were added to the reaction flask and stirred at room temperature for 16 hours. The product rf value was 0.6, TLC (DCM: MeOH = 10: 1). The reaction solution was concentrated and purified by column chromatography to obtain 6 g of colorless oil.

[0263] Step 2: Preparation of 10-2

[0264] Reaction:

[0265]

[0266] Material ratio:

[0267]

[0268]

[0269] Operation process:

[0270] Add 10-1, 3-bromopropanol, EDCI, DMAP and DCM to the reaction flask and react at room temperature for 2 hours. The product Rf value is 0.5, TLC (PE:EA = 10:1). The reaction solution is washed once with 80 mL of water, and the organic phase is dried over anhydrous sodium sulfate, concentrated and purified by column chromatography to obtain 3.8 g of oil.

[0271] Step 3: Preparation of compound 10

[0272] Reaction:

[0273]

[0274] Material ratio:

[0275] Material Name Molecular weight Feed ratio Feeding amount mmol 10-2 449.47 2.2eq 3.8g 8.45 Ethanolamine 61 1 eq 230mg 3.84 Potassium carbonate 138 3 eq 1.6g 11.5 Potassium iodide 166 1 eq 650mg 3.84 Acetonitrile - - 40mL -

[0276] Operation process:

[0277] Add 10-2, ethanolamine, potassium carbonate, potassium iodide and acetonitrile to the reaction flask, heat to 65°C and stir to react for 16 hours. TLC (DCM: MeOH = 10: 1) shows that the product Rf value is 0.5. Dilute the reaction solution with 100 mL of ethyl acetate, wash once with 100 mL of water, take the organic phase, dry it with anhydrous sodium sulfate, filter and concentrate, and purify it by column chromatography to obtain 2 g of colorless oil.

[0278] 1 H NMR(400MHz,Chloroform-d)δ4.86(p,J=6.2Hz,2H),4.13(t,J=6.3Hz,4H),3.55(t,J=5.3Hz,2H),2.58(d,J =25.4Hz, 14H), 1.78 (p, J = 6.6Hz, 4H), 1.50 (q, J = 6.2Hz, 8H), 1.26 (d, J = 6.1Hz, 40H), 0.87 (t, J = 6.6Hz, 12H).

[0279] MS (ES+) m / z): 798.6 (M) + .

[0280] Preparation Example 9 Preparation of Compound 11

[0281] Step 1: Preparation of 11-1

[0282] Reaction:

[0283]

[0284] Material ratio:

[0285] Material Name Molecular weight Feed ratio Feeding amount mmol 8-Pentadecanol 228 1eq 4.5g 20 1,6-Hexanedioic acid 146 1eq 3g 20 EDCI 192 1.5eq 6g 30 DMAP 122 0.15 eq 400mg 3 DCM - - 100mL -

[0286] Operation process:

[0287] Add 1,6-hexanediol, 8-pentadecanol, EDCI, DMAP and DCM to the reaction flask and stir at room temperature for 2 hours. The product rf value is 0.6, TLC (DCM: MeOH = 10: 1). The reaction solution is concentrated and purified by column chromatography to obtain 6 g of colorless oil.

[0288] Step 2: Preparation of 11-2

[0289] Reaction:

[0290]

[0291] Material ratio:

[0292] Material Name Molecular weight Feed ratio Feeding amount mmol 11-1 356 1 eq 3.56g 10 4-Bromobutanol 153 1eq 1.53g 10 EDCI 192 1.5eq 3g 15 DMAP 122 0.15 eq 200mg 1.5 DCM - - 80mL -

[0293] Operation process:

[0294] Add 11-1, 4-bromobutanol, EDCI, DMAP and DCM to the reaction flask and react at room temperature for 2 hours. The product Rf value is 0.5, TLC (PE:EA = 10:1). The reaction solution is washed once with 80 mL of water, and the organic phase is dried over anhydrous sodium sulfate, concentrated and purified by column chromatography to obtain 3.8 g of oil.

[0295] Step 3: Preparation of compound 11

[0296] Reaction:

[0297]

[0298] Material ratio:

[0299] Material Name Molecular weight Feed ratio Feeding amount mmol 11-2 491 2.2eq 3.5g 7.2 Ethanolamine 61 1 eq 200mg 3.27 Potassium carbonate 138 3 eq 1.6g 11.5 Potassium iodide 166 1 eq 650mg 3.84 Acetonitrile - - 40mL -

[0300] Operation process:

[0301] Add 11-2, ethanolamine, potassium carbonate, potassium iodide and acetonitrile to the reaction flask, heat to 65°C and stir to react for 16 hours. TLC (DCM: MeOH = 10: 1) shows that the product Rf value is 0.5. Dilute the reaction solution with 100 mL of ethyl acetate, wash once with 100 mL of water, take the organic phase, dry it with anhydrous sodium sulfate, filter and concentrate, and purify it by column chromatography to obtain 2 g of colorless oil.

[0302] 1 H NMR(400MHz,Chloroform-d)δ4.86(p,J=6.3Hz,2H),4.07(t,J=6.6Hz,4H),3.54(t,J=5.4Hz,2H),2.58(t,J=5.3Hz,2H),2 .48(t,J=7.3Hz,4H),2.36–2.27(m,8H),1.67–1.60(m,12H),1.50(d,J=6.9Hz,12H),1.25(s,40H),0.87(t,J=6.7Hz,12H).

[0303] MS (ES+) m / z): 883.0 (M+H) + .

[0304] Preparation Example 10 Preparation of Compound 12

[0305] Step 1: Preparation of 12-1

[0306] Reaction:

[0307]

[0308] Material ratio:

[0309] Material Name Molecular weight Feed ratio Feeding amount mmol 11-1 356 1 eq 3.56g 10 6-Bromohexanol 181 1eq 1.81g 10 EDCI 192 1.5eq 3g 15 DMAP 122 0.15 eq 200mg 1.5 DCM - - 80mL -

[0310] Operation process:

[0311] Add 11-1, 6-bromohexanol, EDCI, DMAP and DCM to the reaction flask and react at room temperature for 2 hours. The product Rf value is 0.5, TLC (PE:EA = 10:1). The reaction solution is washed once with 80 mL of water, and the organic phase is dried over anhydrous sodium sulfate, concentrated and purified by column chromatography to obtain 3.8 g of oil.

[0312] Step 2: Preparation of compound 12

[0313] Reaction:

[0314]

[0315] Material ratio:

[0316] Material Name Molecular weight Feed ratio Feeding amount mmol 12-1 519 2.2eq 3.7g 7.2 Ethanolamine 61 1 eq 200mg 3.27 Potassium carbonate 138 3 eq 1.6g 11.5 Potassium iodide 166 1 eq 650mg 3.84 Acetonitrile - - 40mL -

[0317] Operation process:

[0318] Add 12-1, ethanolamine, potassium carbonate, potassium iodide and acetonitrile to the reaction flask, heat to 65°C and stir to react for 16 hours. TLC (DCM: MeOH = 10: 1) shows that the product Rf value is 0.5. Dilute the reaction solution with 100 mL of ethyl acetate, wash once with 100 mL of water, take the organic phase, dry it with anhydrous sodium sulfate, filter and concentrate, and purify it by column chromatography to obtain 2 g of colorless oil.

[0319] 1 H NMR(400MHz,Chloroform-d)δ4.86(p,J=6.2Hz,2H),4.05(t,J=6.7Hz,4H),3.56(t,J=5.3Hz,2H),2.62(t,J=5.3Hz,2H),2.49(t,J =7.5Hz, 4H), 2.31 (q, J = 6.5Hz, 8H), 1.68–1.61 (m, 12H), 1.47 (dd, J = 16.4, 9.3Hz, 12H), 1.35–1.22 (m, 48H), 0.87 (t, J = 6.7Hz, 12H).

[0320] MS (ES+) m / z): 938.7 (M) + .

[0321] Preparation Example 11 Preparation of Compound 14

[0322] Step 1: Synthesis of 3-(1-heptyloctaoxycarbonyloxy)propyl benzyl ester

[0323] Reaction:

[0324]

[0325] Material ratio:

[0326] Material Name Molecular weight Feed ratio Feeding amount mmol Benzyl 3-hydroxypropyl ester 180 1.00 eq 3.00g 16.7 (4-Nitrophenyl) chloride 201 1.05eq 3.52g 17.5 Pentadecane-8-ol 228 1.00eq 3.80g 16.7 4-Dimethylaminopyridine 122 2.20eq 4.47g 36.63 Dichloromethane - - 30.0mL -

[0327] Operation process:

[0328] Benzyl 3-hydroxypropyl ester (3.00 g, 16.7 mmol) and (4-nitrophenyl) chloride (3.52 g, 17.5 mmol l), 4-dimethyl 4.47 g, 36.6 mmol) was dissolved in dichloromethane (58.0 mL). React at 20℃ for 3 hours under protection, and continue Pentadecane-8-ol (3.80 g, 16.7 mmol) was added to the reaction solution, and the reaction was completed by TLC monitoring. The reaction solution was concentrated. The residue was diluted with ethyl acetate, and the mixed organic layer was washed with 60 mL (20 mL*3) of brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue. The crude product was subjected to column chromatography to obtain a colorless liquid benzyl 3-(1-heptyloctaoxycarbonyloxy)propyl ester (4.6 g, 10.58 mmol).

[0329] Step 2: Synthesis of 3-(1-heptyloctaoxycarbonyloxy)propionic acid

[0330] Reaction:

[0331]

[0332] Material ratio:

[0333]

[0334] Operation process:

[0335] Dissolve benzyl 3-(1-heptyloctaoxycarbonyloxy)propyl ester (4.60 g, 10.58 mmol) in methanol (10.0 mL), add palladium / carbon (1.13 g, 1.06 mmol) under argon atmosphere, and finally react at 35°C for 12 hours under hydrogen (40 Psi) atmosphere. The reaction is completed by TLC monitoring, and the reaction liquid is filtered and concentrated. The crude product is spin-dried and subjected to column chromatography to obtain colorless liquid 3-(1-heptyloctaoxycarbonyloxy)propionic acid (2.50 g).

[0336] Step 3: Synthesis of 6-bromohexyl 3-(1-heptyloctaoxycarbonyloxy)propyl ester

[0337] Reaction:

[0338]

[0339] Material ratio:

[0340] Material Name Molecular weight Feed ratio Feeding amount mmol 3-(1-Heptyloctaoxycarbonyloxy)propanoic acid 344 1.00 eq 1.00g 2.90 6-Bromohexan-1-ol 181 1.30eq 1.42g 3.77 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride 191 1.5eq 0.83g 4.35 4-Dimethylaminopyridine 122 0.1eq 35.5mg 0.29 Dichloromethane - - 10.0mL -

[0341] Operation process:

[0342] Dissolve 3-(1-heptyloctaoxycarbonyloxy)propionic acid (1.00 g, 2.90 mmol) in dichloromethane (10.0 mL), add 6-bromohexane-1-ol (1.42 g, 3.77 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.83 g, 4.35 mmol), 4-dimethylaminopyridine (35.5 mg, 290 μmol) in sequence, and finally react at 25 ° C for 12 hours under nitrogen protection. The reaction is completed by TLC monitoring, and the reaction solution is concentrated. The crude product is subjected to column chromatography to obtain colorless liquid 1-[(6-bromohexanoyl)oxy]dodec-3-yloctyl ester ((600 mg).

[0343] Step 4: 6-[(2-hydroxyethyl)({6-[(3-{[(pentadecan-8-oxy)carbonyl]oxy}propionyl)oxy]hexyl})amino]hexyl 3-

[0344] Synthesis of {[(pentadecan-8-oxy)carbonyl]oxy}propyl ester

[0345] Reaction:

[0346]

[0347] Material ratio:

[0348] Material Name Molecular weight Feed ratio Feeding amount mmol 6-Bromohexyl 3-(1-heptyloctaoxycarbonyloxy)propyl ester 507 2.00eq 500mg 0.99 Ethanolamine 61 1.00eq 30.1mg 0.492 Potassium carbonate 138 6.00eq 0.409g 2.96 Potassium iodide 166 2.30eq 188mg 1.13 Acetonitrile - - 10.0mL - Tetrahydrofuran - - 5.00mL -

[0349] Operation process:

[0350] Dissolve 6-bromohexyl 3-(1-heptyloctaoxycarbonyloxy)propyl ester (500 mg, 0.99 mmol) in acetonitrile (10.0 mL), add ethanolamine (30.1 mg, 492 μmol), potassium carbonate (408 mg, 2.96 mmol), potassium iodide (188 mg, 1.13 mmol), tetrahydrofuran (5.00 mL), acetonitrile (10.0 mL) in sequence, and react at 75 ° C for 12 hours under nitrogen protection. The reaction is completed by TLC monitoring, filtered, and concentrated. Purification by column chromatography gives a colorless liquid 6-[6-[3-(1-heptyloctaoxycarbonyloxy)propyl oxo]hexyl-(2-hydroxyethyl)amino]hexyl 3-(1-heptyloctaoxycarbonyloxy)propyl ester (70.0 mg).

[0351] 1 H NMR (400MHz, CHLOROFORM-d) δ = 4.72-4.66 (m, 2H), 4.41-4.38 (t, J = 12Hz, 4H), 4.12-4.09 (t ,J=12Hz,4H),3.82(s,2H),2.96-2.89(m,2H),2.87(s,3H),2.72-2.69(t,J=12Hz,4H)ppm.

[0352] LCMS:RT=2.463,m / z 915.2[M+H] + .

[0353] Preparation Example 12 Preparation of Compound 17

[0354] Step 1: Synthesis of 6-bromohexyl 4-nitrophenyl carbonate

[0355] Reaction:

[0356]

[0357] Material ratio:

[0358] Material Name Molecular weight Feed ratio Feeding amount mmol 6-Bromohexan-1-ol 180 1.0 eq 3.00g 16.5 Chlorinated ester 4-nitrophenyl 200 1.2eq 4.01g 19.8 4-Dimethylaminopyridine 122 2.0eq 4.05g 33.1 Dichloromethane - - 20mL - Dichloromethane - - 10mL -

[0359] Operation process:

[0360] Dissolve 6-bromohexane-1-ol (3.00 g, 16.5 mmol) and 4-dimethylaminopyridine (4.05 g, 33.1 mmol) in dichloromethane (20 mL), cool to 0°C, dissolve chlorinated ester 4-nitrophenyl (4.01 g, 19.8 mmol) in dichloromethane (10 mL) and add to the reaction solution at 0°C, and finally react at 25°C under nitrogen protection for 4 hours. TLC monitoring shows that new spots are generated in the reaction, and the reaction solution is directly used in the next step.

[0361] Step 2: Synthesis of Benzyl 5-({[(6-bromohexyl)oxy]carbonyl}oxy)pentyl ester

[0362] Reaction:

[0363]

[0364] Material ratio:

[0365] Material Name Molecular weight Feed ratio Feeding amount mmol 6-Bromohexyl 4-nitrophenyl carbonate 345 1.0 eq 5.74g 16.5 Benzyl 5-hydroxypentyl ester 208 1.0eq 3.45g 16.5 Dichloromethane - - 30mL -

[0366] Operation process:

[0367] Benzyl 5-hydroxypentyl ester (3.45 g, 16.5 mmol) was directly added to the reaction solution of the previous step, and the reaction was carried out at 25°C for 12 hours. After the reaction was completed by TLC monitoring, 50 mL of water was added, and dichloromethane was extracted twice, each time with 50 mL. The organic phase was dried and concentrated. Benzyl 5-({[(6-bromohexyl)oxy]carbonyl}oxy)pentyl ester (1.30 g) was obtained by column chromatography purification as a yellow liquid.

[0368] Step 3: Synthesis of 5-({[(6-bromohexyl)oxy]carbonyl}oxy)pentanoic acid

[0369] Reaction:

[0370]

[0371] Material ratio:

[0372]

[0373]

[0374] Operation process:

[0375] Dissolve benzyl 5-({[(6-bromohexyl)oxy]carbonyl}oxy)pentyl ester (1.00 g, 2.41 mmol) in methanol (10 mL), add palladium carbon (512 mg, 0.481 mmol), react at 25°C, 40 Psi for 12 hours under hydrogen protection, monitor the completion of the reaction by TLC, filter, and concentrate. Purify by column chromatography to obtain yellow liquid 5-({[(6-bromohexyl)oxy]carbonyl}oxy)pentanoic acid (530 mg).

[0376] Step 4: Synthesis of Pentadecane-8-yl 5-({[(6-bromohexyl)oxy]carbonyl}oxy)pentyl ester

[0377] Reaction:

[0378]

[0379] Material ratio:

[0380]

[0381] Operation process:

[0382] 5-({[(6-bromohexyl)oxy]carbonyl}oxy)pentanoic acid (530 mg, 1.63 mmol) and pentadecane-8-ol (409 mg, 1.79 mmol) were dissolved in dichloromethane (6 mL), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (156 mg, 0.814 mmol) and 4-dimethylaminopyridine (19.9 mg, 0.162 mmol) were added in sequence. Finally, the mixture was reacted at 25° C. for 12 hours under nitrogen protection. New spots were generated during the reaction under TLC monitoring. The reaction solution was concentrated. The crude product was purified by column chromatography to obtain colorless liquid pentadecane-8-yl 5-({[(6-bromohexyl)oxy]carbonyl}oxy)pentyl ester (830 mg).

[0383] Step 5: Synthesis of pentadecan-8-yl 5-{[({6-[(2-hydroxyethyl)({6-[({[5-oxyylidene-5-(pentadecan-8-oxy)pentyl]oxy}carbonyl)oxy]hexyl})amino]hexyl}oxy)carbonyl]oxy}pentyl ester

[0384] Reaction:

[0385]

[0386] materials Ratio:

[0387]

[0388]

[0389] Operation process:

[0390] Dissolve pentadecan-8-yl 5-({[(6-bromohexyl)oxy]carbonyl}oxy)pentyl ester (600 mg, 1.12 mmol) in acetonitrile (4 mL), add 2-aminoethane-1-ol (30.7 mg, 0.504 mmol), potassium carbonate (464 mg, 3.36 mmol), potassium iodide (278 mg, 1.68 mmol), tetrahydrofuran (2 mL) in sequence, and finally react at 75°C for 12 hours under nitrogen protection. After the reaction is completed by TLC monitoring, the mixture is filtered and concentrated. Purification by column chromatography gives yellow liquid pentadecan-8-yl 5-{[({6-[(2-hydroxyethyl)({6-[({[5-oxyylidene-5-(pentadecan-8-oxy)pentyl]oxy}carbonyl)oxy]hexyl})amino]hexyl}oxy)carbonyl]oxy}pentyl ester (40 mg).

[0391] 1 H NMR (400MHz, CHLOROFORM-d) δ = 4.87 (t, J = 6.3Hz, 2H), 4.17-4.10 (m, 8H), 3.85 (br d,J=1.4Hz,1H),3.01-2.80(m,5H),2.38-2.29(m,4H),1.79-1.67(m,18H),1.65 -1.48(m,36H),1.46-1.35(m,10H),1.35-1.20(m,44H),0.88(t,J=6.8Hz,12H).

[0392] LCMS:RT=2.174,m / z 970.7[M+H] + .

[0393] Preparation Example 13 Preparation of Compound 6

[0394] Step 1: Synthesis of Undecyl 6-bromohexyl Ester

[0395] Reaction:

[0396]

[0397] Material ratio:

[0398]

[0399] Operation process:

[0400] Undecane-1-ol (3.00 g, 17.4 mmol) was dissolved in dichloromethane (10.0 mL), and 6-bromohexanoic acid (5.09 g, 26.1 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (5.01 g, 26.1 mmol), and 4-dimethylaminopyridine (213 mg, 1.74 mmol) were added in sequence. Finally, the mixture was reacted at 25°C for 12 hours under nitrogen protection. The reaction was completed after TLC monitoring. The reaction solution was concentrated. The crude product was subjected to column chromatography to obtain colorless liquid undecyl 6-bromohexyl ester (6.00 g).

[0401] Step 2: Synthesis of O1-[4-[2-hydroxyethyl-(6-oxyylidene-6-undecyloxy-hexyl)amino]butyl]O4-(1-octylnonyl)butanediol

[0402] Reaction:

[0403]

[0404] Material ratio:

[0405]

[0406] Operation process:

[0407] Dissolve O1-[4-(2-hydroxyethylamino)butyl]O4-(1-octylnonyl)butanediol (1.00g, 2.12mmol) in acetonitrile (9.00mL), add undecyl 6-bromohexyl ester (889mg, 2.54mmol), potassium carbonate (1.03mg, 7.42mmol), potassium iodide (422mg, 2.54mmol), tetrahydrofuran (3.00mL) in sequence, and finally react at 75℃ for 12 hours under nitrogen protection. After TLC monitoring, the reaction is completed, filtered, and concentrated. Purify by column chromatography to obtain colorless liquid O1-[4-[2-hydroxyethyl-(6-oxyylidene-6-undecyloxy-hexyl)amino]butyl]O4-(1-octylnonyl)butanediol (630mg).

[0408] 1H NMR(400MHz,CHLOROFORM-d)δppm 4.86(t,J=6.2Hz,1H)4.03-4.14(m,4H)3.68(br t,J=5.0Hz,2H)2.73-2.80(m,2H)2.58-2.72(m,8H)2.31(t,J=7.4Hz,3H)1.58-1.70(m,10H)1.51(br d,J=6.0Hz,4H)1.22-1.36(m,42H)0.80-0.98(m,9H)ppm.

[0409] LCMS: RT=2.350, m / z=741.0[M+H] + .

[0410] Preparation Example 14 Preparation of Compound 8

[0411] Step 1: Synthesis of 4-hexaoxo-4-oxy-butyric acid

[0412] Reaction:

[0413]

[0414] Material ratio:

[0415] Material Name Molecular weight Feed ratio Feeding amount mmol Hexane-1-ol 102 1.00 eq 3.00g 29.4 Tetrahydrofuran-2,5-dione 100 1.50eq 4.41g 44.0 Pyridine 101 1.50eq 3.48g 44.0 4-Dimethylaminopyridine 122 1.00eq 3.59g 29.4 Tetrahydrofuran - - 30.0mL -

[0416] Operation process:

[0417] Dissolve hexane-1-ol (3.00 g, 29.4 mmol) in tetrahydrofuran (30 mL), and add tetrahydrofuran-2,5-dione (4.41 g, 44.0 mmol), 4-dimethylaminopyridine (3.59 g, 29.4 mmol), and pyridine (3.48 g, 44.0 mmol) in sequence. Finally, react at 60 ° C for 12 hours under nitrogen protection, and monitor the completion of the reaction by TLC. Concentrate. The crude product is subjected to column chromatography to obtain colorless liquid 4-hexaoxo-4-oxyylidene-butyric acid (5.00 g).

[0418] Step 2: Synthesis of O4-(6-bromohexyl)O1-hexylbutanediol

[0419] Reaction:

[0420]

[0421] Material ratio:

[0422]

[0423] Operation process:

[0424] Dissolve 4-hexaoxo-4-oxy-butyric acid (1.00 g, 4.90 mmol) in dichloromethane (10.0 mL), add 6-bromohexane-1-ol (1.34 g, 7.42 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.42 g, 7.42 mmol), 4-dimethylaminopyridine (66.0 mg, 500 μmol) in sequence, and finally react at 25 ° C for 12 hours under nitrogen protection. The reaction is completed by TLC monitoring, and the reaction solution is concentrated. The crude product is subjected to column chromatography to obtain colorless liquid O4-(6-bromohexyl)O1-hexyl butanediol (1.50 g).

[0425] Step 3: Synthesis of 4-(1-octylnonaoxo)-4-oxydebutyric acid

[0426] Reaction:

[0427]

[0428] Material ratio:

[0429] Material Name Molecular weight Feed ratio Feeding amount mmol Heptadecan-9-ol 256 1.00 eq 3.00g 11.7 Tetrahydrofuran-2,5-dione 100 1.50eq 1.76g 17.6 Pyridine 101 1.50eq 1.39g 17.6 4-Dimethylaminopyridine 122 1.00eq 1.43g 11.7 Tetrahydrofuran - - 30.0mL -

[0430] Operation process:

[0431] Dissolve heptadecan-9-ol (3.00 g, 11.7 mmol) in tetrahydrofuran (30 mL), and add tetrahydrofuran-2,5-dione (1.76 g, 17.6 mmol), 4-dimethylaminopyridine (1.43 g, 11.7 mmol), and pyridine (1.39 g, 17.6 mmol) in sequence. Finally, react at 60 ° C for 12 hours under nitrogen protection, and monitor the completion of the reaction by TLC. Concentrate. The crude product is subjected to column chromatography to obtain a colorless liquid 4-(1-octyl nonaoxo)-4-oxyylidene-butyric acid (3.00 g).

[0432] Step 4: Synthesis of 4-bromobutyl 4-(1-octylnonaoxo)butyl ester

[0433] Reaction:

[0434]

[0435] Material ratio:

[0436]

[0437] Operation process:

[0438] Dissolve 4-(1-octyl nonaoxo)-4-oxy-butyric acid (1.00 g, 2.80 mmol) in dichloromethane (10.0 mL), add 4-bromobutane-1-ol (0.64 g, 3.37 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.55 g, 4.21 mmol), 4-dimethylaminopyridine (34.0 mg, 280 μmol) in sequence, and finally react at 25 ° C for 12 hours under nitrogen protection. The reaction is completed by TLC monitoring, and the reaction solution is concentrated. The crude product is subjected to column chromatography to obtain a colorless liquid 4-bromobutyl 4-(1-octyl nonaoxo)butyl ester (1.10 g).

[0439] Step 5: Synthesis of 3-octaoxododecyl 6-[2-hydroxyethyl-[6-(3-octaoxododecyloxy)-6-oxyylidene-hexyl]amino]hexyl ester

[0440] Reaction:

[0441]

[0442] Material ratio:

[0443] Material Name Molecular weight Feed ratio Feeding amount mmol 4-Bromobutyl 4-(1-octylnonaoxo)butyl ester 477 1.00 eq 3.00g 6.28 Ethanolamine 61 12.0eq 4.60g 75.4 Acetonitrile - - 10.0mL -

[0444] Operation process:

[0445] Dissolve 4-bromobutyl 4-(1-octyl nonaoxo)butyl ester (3.00 g, 6.28 mmol) in acetonitrile (10.0 mL), add ethanolamine (4.60 g, 75.4 mmol) in turn, and react at 25°C for 12 hours under nitrogen protection. Monitor the reaction by TLC and concentrate. Purify by column chromatography to obtain colorless liquid O1-[4-(2-hydroxyethylamino)butyl]O4-(1-octylnonyl)butanediol (2.40 g).

[0446] Step 6: Synthesis of O1-hexyl O4-[6-[2-hydroxyethyl-[4-[4-(1-octyl nonaoxo)-4-oxyylidene-butyryl]oxybutyl]amino]hexyl]butanediol

[0447] Reaction:

[0448]

[0449] Material ratio:

[0450]

[0451] Operation process:

[0452] Dissolve O1-[4-(2-hydroxyethylamino)butyl]O4-(1-octylnonyl)butanediol (1.00g, 2.12mmol) in acetonitrile (9.00mL), add O4-(6-bromohexyl)O1-hexylbutanediol (930mg, 2.54mmol), potassium carbonate (1.03mg, 7.42mmol), potassium iodide (422mg, 2.54mmol), tetrahydrofuran (3.00mL) in sequence, and finally react at 75°C for 12 hours under nitrogen protection. After TLC monitoring, the reaction is completed, filtered, and concentrated. Purify by column chromatography to obtain colorless liquid O1-hexyl O4-[6-[2-hydroxyethyl-[4-[4-(1-octylnonaoxo)-4-oxyylidene-butyryl]oxybutyl]amino]hexyl]butanediol (880mg).

[0453] 1 H NMR(400MHz,CHLOROFORM-d)δppm 4.86(t,J=6.2Hz,1H)3.95-4.17(m,6H)3.67(br t,J=4.8Hz,2H)2.70-2.80(m,2H)2.50-2.70(m,12H)1.45-1.74(m,14H)1.16-1.42(m,34H)0.59-0.97(m,9H)ppm.

[0454] LCMS: RT=2.450, m / z=756.5.[M+H] + .

[0455] Example 1 Preparation and Detection of Lipid Nanoparticles (LNP)

[0456] The lipid nanoparticle (LNP) preparation prepared by the ionizable lipid compound disclosed in the present application can effectively encapsulate mRNA and maintain the structural integrity of mRNA. The prepared ionizable lipid compound, distearoylphosphatidylcholine (DSPC, purchased from Nippon Seika Co., Ltd., item number: S01005), cholesterol (purchased from Nippon Seika Co., Ltd., item number: O01001) and dimyristoylglycerol-polyethylene glycol 2000 (DMG-PEG2000, purchased from Guobang Pharmaceutical, item number: O02005) were dissolved in ethanol (manufacturer: Nanjing Chemical Reagent Co., Ltd., purity 99.6%) solution, and then mixed according to a certain molar ratio to prepare an ethanol solution of mixed lipids, wherein the total lipid concentration is 12.5mM (the measurement unit "M" in the present application refers to mol / L). The homemade firefly luciferase (Fluc) mRNA was diluted in a 25mM sodium acetate buffer at pH 5.0 to obtain an mRNA solution. By using a microfluidic device, the flow rate was controlled to be 12mL / min, the volume ratio of the ethanol solution of the mixed lipids to the mRNA solution prepared in the previous step was controlled to be 1:3, and lipid nanoparticles were prepared according to the nitrogen-phosphorus ratio of the ionizable lipids to the mRNA of 3 to 15:1. The ethanol was removed by dialysis in 20mM Tris acetate for 12 to 24 hours. Finally, the LNP solution was filtered through a sterile filter with a pore size of 0.22μm (manufacturer: Millex, item number: SLGPR33RB), and ultrafiltration concentration (manufacturer: Amicon-Ultra, molecular weight cutoff: 10kDa) obtained by encapsulating Fluc mRNA with the ionizable lipids described in this application and DSPC, cholesterol and DMG-PEG2000. The particle size and polydispersity index (PDI) of each LNP preparation were determined by dynamic light scattering using a Malvern Zetasizer Ultra instrument (manufacturer: Malvern); the encapsulation efficiency of LNP was determined using the Quant-it Ribogreen RNA quantification kit (manufacturer: ThermoFisher Scientific, catalog number: R11490).

[0457] Table 1

[0458]

[0459] In the art, a PDI of less than 0.3 indicates that the nanoparticle size in the LNP preparation is relatively uniform; an encapsulation rate is used to indicate whether the LNP can effectively encapsulate mRNA, and an encapsulation rate of more than 70% indicates that the LNP can effectively encapsulate mRNA. The particle size of the LNP prepared by the present application is maintained at 60 to 100 nm, the PDI is less than 0.15, and the encapsulation rate is higher than 90%.

[0460] Example 2 In vivo animal study of LNP preparations

[0461] In this embodiment, the LNP prepared in Example 1 was injected into female Balb / C mice (Weitong Lihua) aged 6 to 8 weeks at a dose of 5 μg / mouse by tail vein injection or lower limb muscle injection (n=3, i.e., 3 mice were injected and tested in each group, and the data results presented are the mean values ​​of the determinations of each group), and D-luciferin potassium salt was injected intraperitoneally at a specific time point after administration (4 hours, 24 hours, and 48 hours in this embodiment), and then the luminescence was detected by IVIS Spectrum small animal in vivo imager (manufacturer: PerkinElmer), and the total luminescence intensity of the in vivo expression site of the mouse (e.g., liver, lower limb administration site, etc.) was counted. The higher the luminescence intensity, the higher the luciferase expression, i.e., the better the expression of the corresponding LNP preparation in the mouse. The total luminescence intensity is measured by bioluminescence imaging, and the luminescence intensity data of the luminescent site is obtained 6 to 15 minutes (min) after the intraperitoneal injection of D-luciferin potassium salt. The total luminescence intensity of the in vivo expression area was counted by Living Image software (manufacturer: PerkinElmer). Normally, the total luminescence intensity reading of mice not treated with drugs is on the order of 10 5 .

[0462] Referring to the above-mentioned in vivo mouse experimental method, the LNP preparation in Example 1 was injected into 6- to 8-week-old female Balb / C mice via the tail vein at a dose of 5 μg / mouse, and the total luminescence intensity of the liver area was counted. The test results are shown in Table 2 and Figure 1 The LNP preparations tested in this example all showed strong expression in mice, with an AUC range of 10 9 ~10 12 This indicates that the LNP preparations corresponding to the ionizable lipids described in the preparation examples can effectively deliver mRNA into the body and express it. In terms of the overall expression trend, compound 3>compound 2.

[0463] Table 2 Area under the expression kinetic curve (AUC) in the liver region from 4 to 48 hours

[0464] Group Liver AUC (p / s*hour) Compound 2 9.86E+11 Compound 3 6.39E+12

[0465] Similarly, the LNP preparation prepared in Example 1 was injected intramuscularly (right lower limb of the calf) into female Balb / C mice aged 6 to 8 weeks at a dose of 5 μg / mouse. The total luminescence intensity of the liver region and the administration muscle site under this administration method was counted and the corresponding 4-48h expression kinetic curve area (AUC) was calculated. The test results are shown in Table 3 and Figures 2-3It can be seen that the expression in the liver area and administration site after intramuscular injection is: Compound 3>Compound 2.

[0466] Table 3 Area under the expression kinetic curve (AUC) of liver region and administration site at 4 to 48 h

[0467] Group Local AUC after administration (p / s×hour) Liver AUC (p / s×hour) Compound 2 1.65E+11 3.62E+10 Compound 3 1.68E+11 1.45E+11

[0468] Although the specific embodiments of the present invention are described above, it should be understood by those skilled in the art that this is only for illustration and the protection scope of the present invention is defined by the appended claims. Those skilled in the art may make various changes or modifications to these embodiments without departing from the principles and essence of the present invention, but these changes and modifications all fall within the protection scope of the present invention.

[0469] Example 3 Preparation and Detection of Lipid Nanoparticles (LNP)

[0470] This embodiment selects compound 4 and compound 5 as ionizable lipids, and prepares LNP preparations (encapsulating Fluc mRNA) according to the molar ratio and nitrogen-phosphorus ratio in Table 4, with reference to Example 1. The mRNA of this embodiment is diluted in a 25mM sodium acetate solution with a pH of 5.0, and a 20mM Tris-acetic acid solution with a pH of 7.5 is used for dialysis. The particle size, PDI and encapsulation efficiency of all LNP preparations in this embodiment are measured. As shown in Table 4, the particle size of the LNP preparations prepared in this embodiment is between 60-110nm, the PDI is less than 0.3, and the encapsulation efficiency is higher than 90%.

[0471] Table 4

[0472]

[0473] Example 4 In vivo animal study of LNP preparations

[0474] Referring to the in vivo test method of Example 2, the LNP reagent prepared in Example 3 was injected into female Balb / C mice aged 6 to 8 weeks through the tail vein at a dose of 5 μg / mouse, and the total luminescence intensity of the liver of the mice was counted. The test results are shown in Table 5 and Figure 4 The LNP preparations prepared in this example were strongly expressed in mice.

[0475] Table 5 Area under the expression kinetic curve (AUC) in the liver region from 4 to 48 hours

[0476] Group Liver AUC (p / s*hour) Compound 4 2.67E+12 Compound 5 2.12E+11

[0477] Example 5 Preparation and Detection of Lipid Nanoparticles (LNP)

[0478] This embodiment selects compound 7 and compound 9 as ionizable lipids, and prepares LNP preparations (encapsulating Fluc mRNA) according to the molar ratio and nitrogen-phosphorus ratio in Table 6, with reference to Example 1. The mRNA of this embodiment is diluted in a 25mM sodium acetate solution with a pH of 5.0, and a 20mM Tris-acetic acid solution with a pH of 7.5 is used for dialysis. The particle size, PDI and encapsulation efficiency of all LNP preparations in this embodiment are measured. As shown in Table 6, the particle size of the LNP preparations prepared in this embodiment is between 60-100nm, the PDI is less than 0.25, and the encapsulation efficiency is higher than 90%.

[0479] Table 6

[0480]

[0481] Example 6 In vivo animal study of LNP preparations

[0482] Referring to the in vivo test method of Example 2, the LNP reagent prepared in Example 5 was injected into female Balb / C mice aged 6 to 8 weeks through the tail vein at a dose of 5 μg / mouse, and the total luminescence intensity of the liver of the mice was counted. The test results are shown in Table 7 and Figure 5 The LNP preparations prepared in this example were strongly expressed in mice.

[0483] Table 7 Area under the expression kinetic curve (AUC) in the liver region from 4 to 48 hours

[0484] Group Liver AUC (p / s*hour) Compound 7 1.03E+12 Compound 9 2.40E+12

[0485] Example 7 Preparation and Detection of Lipid Nanoparticles (LNP)

[0486] This embodiment selects compound 1 as an ionizable lipid, and prepares LNP preparations (encapsulating Fluc mRNA) according to the molar ratio and nitrogen-phosphorus ratio in Table 8, with reference to Example 1. The mRNA of this embodiment is diluted in a 25mM sodium acetate solution with a pH of 5.0, and a 20mM Tris-acetic acid solution with a pH of 7.5 is used for dialysis. The particle size, PDI and encapsulation efficiency of all LNP preparations in this embodiment are measured. As shown in Table 8, the particle size of the LNP preparations prepared in this embodiment is between 60-140nm, the PDI is less than 0.25, and the encapsulation efficiency is higher than 85%.

[0487] Table 8

[0488]

[0489] Example 8 In vivo animal studies of LNP preparations

[0490] Referring to the in vivo test method of Example 2, the LNP reagent prepared in Example 7 was injected into female Balb / C mice aged 6 to 8 weeks through the tail vein at a dose of 5 μg / mouse, and the total luminescence intensity of the liver of the mice was counted. The test results are shown in Table 9 and Figure 6 The LNP preparations prepared in this example were strongly expressed in mice.

[0491] Table 9 Area under the expression kinetic curve (AUC) in the liver region from 4 to 48 hours

[0492] Group Liver AUC (p / s*hour) Compound 1 3.10E+08

[0493] Example 9 Preparation and Detection of Lipid Nanoparticles (LNP)

[0494] This embodiment selects compound 10, compound 11, compound 12, compound 14 and compound 17 as ionizable lipids, and prepares LNP preparations (encapsulating Fluc mRNA) in the manner of reference Example 1 according to the molar ratio and nitrogen-phosphorus ratio in Table 10. The mRNA of this embodiment is diluted in a 25mM sodium acetate solution with a pH of 5.0, and a 20mM Tris-acetic acid solution with a pH of 7.5 is used for dialysis. The particle size, PDI and encapsulation efficiency of all LNP preparations in this embodiment are measured. As shown in Table 10, the particle size of the LNP preparations prepared in this embodiment is between 60-140nm, the PDI is less than 0.25, and the encapsulation efficiency is higher than 85%.

[0495]

[0496] H23 and H21 were cited from international application WO2023186041A1 and were synthesized using the same preparation method as WO2023186041A1.

[0497] Table 10

[0498]

[0499] Example 10 In vivo animal study of LNP preparations

[0500] Referring to the in vivo test method of Example 2, the LNP reagent prepared in Example 7 was injected into female Balb / C mice aged 6 to 8 weeks through the tail vein at a dose of 5 μg / mouse, and the total luminescence intensity of the liver of the mice was counted. The test results are shown in Table 11 and Figure 7 The LNP preparations prepared in this example were strongly expressed in mice.

[0501] Table 11 Area under the expression kinetic curve (AUC) in the liver region from 4 to 48 hours

[0502] Group Liver AUC (p / s*hour) H21 1.50E+06 H23 1.08E+06 Compound 10 3.01E+10 Compound 11 6.99E+11 Compound 12 1.41E+12 Compound 14 1.37E+12 Compound 17 8.05E+11

[0503] Example 11 Preparation and Detection of Lipid Nanoparticles (LNP)

[0504] This embodiment selects compound 6 and compound 8 as ionizable lipids, and prepares LNP preparations (encapsulating Fluc mRNA) according to the molar ratio and nitrogen-phosphorus ratio in Table 12, and the method of reference Example 1. The mRNA of this embodiment is diluted in 25mM sodium acetate solution with a pH of 5.0, and a 20mM Tris-acetic acid solution with a pH of 7.5 is used for dialysis. The particle size, PDI and encapsulation efficiency of all LNP preparations in this embodiment are measured. As shown in Table 12, the particle size of the LNP preparations prepared in this embodiment is between 60-140nm, the PDI is less than 0.35, and the encapsulation efficiency is higher than 84%.

[0505] Table 12

[0506]

[0507] Example 12 In vivo animal study of LNP preparations

[0508] Referring to the in vivo test method of Example 2, the LNP reagent prepared in Example 11 was injected into female Balb / C mice aged 6 to 8 weeks through the tail vein at a dose of 5 μg / mouse, and the total luminescence intensity of the liver of the mice was counted. The test results are shown in Table 13 and Figure 8 The LNP preparations prepared in this example were strongly expressed in mice.

[0509] Table 13 Area under the expression kinetic curve (AUC) in the liver region from 4 to 48 hours

[0510]

[0511]

Claims

1. A compound I or a pharmaceutically acceptable salt thereof: in, R 1 is a C substituted with one or more hydroxyl groups 1-6 alkyl; X and Y are independently C 3-12 Alkylene; Z 1 and Z 2 Independently W 1 For chemical bonds, * indicates the same as Z 1 connect; W 2 for * indicates the same as Z 2 connect; R w11 , R w12 , R w21 and R w22 Independently for C 1-5 Straight chain alkylene; R 2 C 1-24 alkyl; R 3 C 1-24 alkyl.

2. The compound I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: It meets one or more of the following conditions: (1)R 1 C is substituted by a hydroxyl group 1-6 alkyl; (2)R 1 In the case of a hydroxyl group, only the terminal carbon not connected to the nitrogen atom is replaced by a hydroxyl group. (3) X and Y are independently C 3-7 Alkylene; (4) X and Y are independently straight chain alkylene groups; (5) Z 1 for *Denotes the same as W 1 connect; (6) Z 2 for *Denotes the same as W 2 connect; (7)R w11 , R w12 , R w21 and R w22 C 2-4 Straight chain alkylene; (8)R 2 A straight chain or a branched C 4-18 alkyl; and (9)R 3 for R 31 and R 32 C 3-10 alkyl; Preferably, the compound I or a pharmaceutically acceptable salt thereof satisfies one or more of the following conditions: (1) X and Y are independently C 3-6 Alkylene; (2)R 2 C 5-11 Straight chain alkyl or a branched C 15-17 alkyl; and (3) R 31 and R 32 C 6-9 Straight chain alkyl groups; e.g.

3. The compound I or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that: It meets one or more of the following conditions: (1)R 1 for (2) X and Y are independently (3)W 1 For chemical bonds, Represents Z 1 connect; (4)W 2 for * indicates the same as Z 2 connect; (5)R 2 for and (6)R 3 for Preferably, the compound I or a pharmaceutically acceptable salt thereof satisfies one or more of the following conditions: (1) for and (2) for 4. Compound I or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, characterized in that: The compound I is a structure as shown in IA, IB or IC: Among them, R 1 , X, Y, Z 1 , W 1 , R 2 , R w21 , R w22 and R 3 The definition as described in any one of claims 1 to 3.

5. The compound I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The compound I is any of the following structures:

6. A composition, characterized in that It includes substance Z, which is compound I or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 5.

7. The composition according to claim 6, characterized in that The composition is a lipid carrier; Preferably, the lipid carrier satisfies one or more of the following conditions: (1) The lipid carrier further comprises a diluent, which may be a phosphate buffer, a sodium acetate buffer or a Tris-acetate buffer; (2) The lipid carrier further comprises a phospholipid, which may be a phospholipid molecule having an electrically charged polar end and a non-polar fatty chain end, preferably distearoylphosphatidylcholine, dimyristoylphosphocholine, dioleoylphosphocholine, palmitoylphosphocholine, heneicosanoylphosphocholine or palmitoylphosphocholine, such as distearoylphosphatidylcholine; (3) The lipid carrier further comprises PEG lipid, which may be a lipid molecule having a polyethylene glycol hydrophilic end modified; preferably one or more selected from PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol and PEG-modified dialkylglycerol, such as PEG-modified dimyristoylglycerol; and (4) the lipid carrier further comprises a sterol, which may be an animal, plant or fungal sterol, preferably one or more selected from cholesterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid and α-tocopherol, such as cholesterol; More preferably, the lipid carrier satisfies one or more of the following conditions: (1) The molar ratio of the substance Z to the sterol is 0.5-5:1, preferably 0.5-3:1, for example 1.3:1; (2) the molar ratio of the substance Z to the phospholipid is 1-15:1, preferably 2-8:1, for example 3-6:1; (3) The molar ratio of the substance Z to the phospholipid is 1-15:1, preferably 1-10:1, for example 5:1; (4) The molar ratio of substance Z to PEG lipid is 10-100:1, preferably 10-50:1, for example 33.3:1; (5) The molar content of the substance Z is 30 mol% to 70 mol%, for example 50 mol%; (6) The molar content of the phospholipid is 5 mol% to 20 mol%, for example 10 mol%; (7) The molar content of the sterol is 20 mol% to 60 mol%, for example 38.5 mol%; (8) the molar content of the PEG lipid is about 0.2 mol% to 5 mol%, for example 1.5% mol; and (9) the lipid carrier consists of the substance Z, the diluent, the phospholipid, the PEG lipid and the sterol.

8. Use of the compound I or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5 or the composition according to any one of claims 6 to 7 in the preparation of a nucleic acid preventive agent and / or therapeutic agent delivery vector; The nucleic acid preventive and / or therapeutic agent is preferably one or more of single-stranded deoxyribonucleic acid, double-stranded DNA, small interfering RNA, asymmetric double-stranded small interfering RNA, microRNA, small hairpin RNA, circular RNA, transfer RNA or messenger RNA, and is further preferably mRNA, such as firefly luciferase mRNA, SARS-CoV-2 spike protein mRNA, herpes zoster virus mRNA, respiratory syncytial virus mRNA or influenza virus mRNA.

9. A lipid nanoparticle, characterized in that: It includes a nucleic acid preventive and / or therapeutic agent and a composition as described in any one of claims 6-7; The nucleic acid preventive and / or therapeutic agent is preferably one or more of single-stranded deoxyribonucleic acid, double-stranded DNA, small interfering RNA, asymmetric double-stranded small interfering RNA, microRNA, small hairpin RNA, circular RNA, transfer RNA or messenger RNA, and is further preferably mRNA, such as firefly luciferase mRNA, SARS-CoV-2 spike protein mRNA, herpes zoster virus mRNA, respiratory syncytial virus mRNA or influenza virus mRNA.

10. The lipid nanoparticle according to claim 9, characterized in that It meets one or more of the following conditions: (1) The nitrogen-to-phosphorus ratio in the lipid nanoparticles is (2-30):1, preferably, the nitrogen-to-phosphorus ratio is (3-16):1, for example 6:1; (2) The particle size of the lipid nanoparticles is 10-200 nm, preferably 40-150 nm, such as 50.32 nm, 53.71 nm, 59.16 nm, 61.74 nm, 67.40 nm, 84.31 nm, 63.89 nm, 95.80, 100.27, 73.07, 62.60, 97.72, 131.87 or 131.40; and for example 84.31 nm, 63.89 nm, 95.80, 100.27, 73.07, 62.60 or 131.40; (3) The polydispersity index of the lipid nanoparticles is 0.001-0.3; for example, 0.135, 0.323, 0.107, 0.089, 0.106, 0.188, 0.201, 0.129, 0.079, 0.159, 0.298, 0.235 or 0.073, for example, 0.129, 0.079, 0.159, 0.298, 0.235 or 0.073; (4) The encapsulation efficiency of the lipid nanoparticles is 84%-100%, for example, 84.5%, 92.5%, 93.4%, 96.3%, 94.9%, 94.1%, 92.9%, 96.4%, 96.7%, 92.7%, 93.5%, 96.2%, 96.1% or 86.8%; preferably, the encapsulation efficiency of the lipid nanoparticles is 85%-100%, for example, 96.40% or 96.66%, 92.74%, 93.51%, 96.17%, 96.10% or 86.82%; and (5) the composition encapsulates the nucleic acid preventive and / or therapeutic agent.

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