Cationic lipid compound, composition containing cationic lipid compound, and use

Abstract

Provided in the invention are a cationic lipid compound, a composition containing same, and a use. The cationic lipid compound is represented by formula (I). Provided in the invention is a novel cationic lipid compound, which enriches the current types of cationic lipid compounds. The preparation method of the amino lipid compound has the advantages of easily available raw materials, mild reaction conditions, high product yield, low instrument and equipment requirements, and simple operations.

Description

Cationic lipid compound, composition containing same and application thereof Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a cationic lipid compound, a composition containing the same, and applications thereof. Background Art

[0002] Nucleic acids (RNA) have enormous potential for expressing proteins in vivo, exhibiting both preventative and therapeutic benefits depending on the protein's function. However, naked RNA has a short circulation time in the body, is inefficiently internalized by cells, and is easily cleared by the kidneys or degraded by RNases in the body, significantly reducing its effectiveness. To better deliver nucleic acids to their target sites and enhance their preventative and therapeutic effects, cationic lipid compounds and related formulations are key technologies.

[0003] LNP (lipid nanoparticles) is currently the mainstream delivery carrier. It can bind to mRNA to increase the in vivo circulation time of mRNA, control its escape from the body to improve its transfection rate, and increase protein expression. LNP is generally composed of four components, including: (1) cationic lipids, which provide charge to encapsulate mRNA molecules; (2) supporting phospholipids, which provide bilayer support and facilitate endosomal escape; (3) cholesterol, which enhances the stability of LNP and promotes membrane fusion; (4) PEG lipids, which reduce the particle size of LNP and increase its in vivo circulation time.

[0004] In vivo, cations, as one of the key components of LNP, can have a significant impact on the instability and efficacy of LNP preparations. The present invention provides a new class of highly effective and low-toxic cationic compounds. Summary of the Invention

[0005] One object of the present invention is to provide a cationic lipid compound;

[0006] Another object of the present invention is to provide a liposome preparation;

[0007] Another object of the present invention is to provide uses of the cationic lipid compound.

[0008] To achieve the above objectives, in one aspect, the present invention provides a cationic lipid compound represented by general formula (I) or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:

[0009] in,

[0010] Ring A is a substituted or unsubstituted 4-10 membered N-containing heterocyclic ring; the N-containing heterocyclic ring contains 0, 1 or 2 heteroatoms selected from N, O or S in addition to the N atom connected to L1; when substituted, the heterocyclic ring is substituted with 1, 2, 3, 4, 5 or 6 substituents selected from halogen, hydroxy, cyano, nitro, carboxyl, C1-C5 alkyl or C1-C5 alkoxy;

[0011] L1 is a C1-C6 straight chain alkylene group;

[0012] L2 and L3 are the same or different, and are each independently a C1-C12 alkylene group;

[0013] G1 and G2 are the same or different and are each independently -(C=O)O-, -O(C=O)-, -S(C=O)-, -O(C=S)- or -(C=O)S-;

[0014] R3 and R4 are the same or different, and are independently a C4-C17 straight-chain alkyl group, a C4-C17 straight-chain alkenyl group or a C8-C22 branched non-cyclic alkyl group.

[0015] According to some specific embodiments of the present invention, Ring A is a substituted or unsubstituted 4-10 membered N-containing heterocycle; the N-containing heterocycle contains 1-3 N atoms.

[0016] According to some specific embodiments of the present invention, Ring A is a substituted or unsubstituted 5-membered, 6-membered or 7-membered N-containing heterocycle.

[0017] According to some specific embodiments of the present invention, Ring A is a substituted or unsubstituted 5-membered, 6-membered or 7-membered N-containing heterocycle, and the heteroatoms in the N-containing heterocycle are all N atoms.

[0018] According to some specific embodiments of the present invention, L1 is a C2-C4 straight-chain alkylene group.

[0019] According to some specific embodiments of the present invention, at least one of L2 and L3 is a C1-C6 alkylene group.

[0020] According to some specific embodiments of the present invention, L2 and L3 are each independently a C1-C6 alkylene group.

[0021] According to some specific embodiments of the present invention, ring A is methylpiperazinyl, hydropyrrolyl, piperidinyl or cycloheximino.

[0022] According to some specific embodiments of the present invention, wherein Ring A is:

[0023] According to some specific embodiments of the present invention, G1 and G2 are -(C=O)O-.

[0024] According to some specific embodiments of the present invention, G1 and G2 are -(C=O)O-, and G1 and G2 are connected to R3 and R4 respectively through the -O- therein.

[0025] According to some specific embodiments of the present invention, wherein R3 and R4 are independently:

[0026] R5 and R6 are the same or different, and are each independently a C3-C11 straight-chain alkyl group.

[0027] It is understood that the sum of the carbon numbers of R5 and R6 should satisfy the previously defined range of C8-C22. For example, when one of R5 and R6 is a C3 straight-chain alkyl group, the other should be at least a C5 straight-chain alkyl group.

[0028] According to some specific embodiments of the present invention, R5 and R6 are the same or different, and are each independently a C4-C8 straight-chain alkyl group.

[0029] According to some specific embodiments of the present invention, wherein R3 and R4 are independently:

[0030] According to some specific embodiments of the present invention, the cationic lipid compound is selected from one or more of the following structures:

[0031] In another aspect, the present invention further provides a liposome formulation comprising the cationic lipid compound of any one of the present invention or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof and a prophylactic or therapeutic nucleic acid.

[0032] According to some specific embodiments of the present invention, the molar ratio of the cationic lipid compound to the nucleic acid is 20:1 to 1:1.

[0033] According to some specific embodiments of the present invention, the average particle size of the liposome preparation is 50 nm to 200 nm.

[0034] According to some specific embodiments of the present invention, the liposome preparation further comprises one or more other lipid components, wherein the other lipid components include neutral lipids, steroids and polymer-conjugated lipids.

[0035] According to some specific embodiments of the present invention, the steroid is β-sitosterol, soja sterol, ergosterol, cholesterol or dihydrocholesterol; cholesterol is preferred.

[0036] According to some specific embodiments of the present invention, the molar ratio of the steroid to the cationic lipid compound is (0.5-1):1.

[0037] According to some specific embodiments of the present invention, the polymer in the polymer-conjugated lipid is polyethylene glycol.

[0038] According to some specific embodiments of the present invention, the molar ratio of the cationic lipid compound to the polymer-conjugated lipid is 100:1 to 20:1.

[0039] According to some specific embodiments of the present invention, the polyethylene glycol-conjugated lipid is PEG2k-DSG, PEG2k-DMG, PEG2k-DPPE, PEG2k-DSPE, PEG2k-cer, PEG2k-DMG or ALC-0159; preferably PEG2k-DMG.

[0040] According to some specific embodiments of the present invention, the neutral lipid is selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-sodium phosphoglycerol (DPPG-Na), sphingomyelin (SM), ceramide and sterols. One or more combinations thereof.

[0041] According to some specific embodiments of the present invention, the molar ratio of the cationic lipid compound to the neutral lipid is 2:1 to 8:1.

[0042] According to some specific embodiments of the present invention, the nucleic acid is selected from mRNA, siRNA, miRNA or ASO; the nucleic acid is preferably mRNA.

[0043] In another aspect, the present invention further provides use of the cationic lipid compound of the present invention or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, or the liposome formulation of the present invention in the preparation of a medicament for inducing protein expression in a subject.

[0044] In summary, the present invention provides a cationic lipid compound, a composition comprising the same, and applications thereof. The cationic lipid compound of the present invention has the following advantages:

[0045] The present invention provides a new cationic lipid compound, enriching the types of current cationic lipid compounds. The preparation method of the amino lipid compound has the advantages of readily available raw materials, mild reaction conditions, high product yield, low instrumentation requirements, and simple operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 is the hydrogen spectrum of compound 1.

[0047] Figure 2 is the hydrogen spectrum of compound 2.

[0048] Figure 3 is the hydrogen spectrum of compound 3.

[0049] FIG4 is a hydrogen spectrum of compound 4.

[0050] Figure 5 is the hydrogen spectrum of compound 5.

[0051] Figure 6 is the hydrogen spectrum of compound 6.

[0052] FIG7 is a hydrogen spectrum of compound 7.

[0053] Figure 8 is the hydrogen spectrum of compound 8.

[0054] FIG9 is a hydrogen spectrum of compound 9.

[0055] FIG10 is a hydrogen spectrum of compound 10.

[0056] FIG11 is a hydrogen spectrum of compound 11.

[0057] FIG12 is a hydrogen spectrum of compound 12.

[0058] FIG13 is a hydrogen spectrum of compound 13.

[0059] FIG14 is a hydrogen spectrum of compound 14.

[0060] Figure 15 is the hydrogen spectrum of compound 15.

[0061] FIG16 is a diagram showing the effect of the nanolipid particle composition on the expression of erythropoietin (EPO) mRNA in mice. DETAILED DESCRIPTION

[0062] The following describes in detail the implementation process of the present invention and the beneficial effects produced by specific embodiments, which is intended to help readers better understand the essence and characteristics of the present invention and is not intended to limit the scope of implementation of this case.

[0063] Example 1

[0064] The synthetic route of compound 1 is as follows:

[0065] Step 1:

[0066] Compound 1-1 (5.0 g) was dissolved in DCM (200 ml) and stirred at room temperature. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 12.3 g), 4-dimethylaminopyridine (DMAP, 262 mg), triethylamine (6.51 g), and n-decanol (6.79 g) were added portionwise to the reaction system and stirred at room temperature for 16 h. A small amount of the reaction solution was diluted and spot-coated with a 1-1 standard sample (PE / EA = 10 / 1, phosphomolybdic acid and bromocresol green). New spots with reduced polarity were observed. The reaction solution was quenched with water, separated, and the organic phase was evaporated under reduced pressure. An appropriate amount of silica gel was added for sample mixing and purification (80 g normal phase column, PE / EA, 0-0% 5 min, 0-10% 20 min, 10-10% 10 min, flow rate 40 ml / min) was performed using a plate monitor. The pure product fractions were evaporated to give compound 1-2 (6.0 g, 54.5% yield) as a colorless oily liquid.

[0067] Step 2:

[0068] Trifluoroacetic acid (15 ml) was added to a solution of compound 1-2 (6.0 g) in dichloromethane (50 ml). The mixture was stirred at room temperature for 16 hours. TLC showed that compound 1-2 completely disappeared and a point with increased polarity was generated. The reaction solution was spin-dried and saturated aqueous sodium bicarbonate solution (100 ml) was added to quench the excess trifluoroacetic acid. The mixture was extracted with ethyl acetate (100 ml × 2). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and then added with an appropriate amount of silica gel and DCM to mix the sample and purify (80 g normal phase column, PE / EA, 0-0% 5 min, 0-50% 20 min, 50-50% min, flow rate 50 ml / min) to obtain white solid compound 1-3 (4.5 g, 93% yield).

[0069] Step 3:

[0070] Compound 1-3 (300 mg) was dissolved in ultra-dry dichloromethane (5 ml) and stirred in an ice bath. Ultra-dry pyridine (115 mg) and triphosgene (86 mg) were added sequentially, and the mixture was stirred in an ice bath for 0.5 h. A small amount of the reaction solution was plated with a standard control sample of 1-3 (PE / EA = 1 / 1, phosphomolybdic acid). New spots with reduced polarity were observed. The reaction solution was evaporated under reduced pressure, and the resulting solid was dissolved in ultra-dry dichloromethane (5 ml) and then added dropwise to a mixed solution of 1-(2-hydroxyethyl)-4-methylpiperazine (500 mg) and pyridine (10 mL). After the addition was complete, the reaction solution was stirred at 70°C for three hours. The solution was orange-yellow. TLC (DCM / MeOH / NH4OH = 10 / 1 / 0.1, phosphomolybdic acid) showed a new spot below the pyridine. An appropriate amount of silica gel and dichloromethane were added to the sample, and the sample was purified (10 g normal phase column, DCM:DCM / MeOH / NH4OH, 0-0% for 5 min, 0-70% for 20 min, 70-70% for 10 min, flow rate 20 ml / min) to afford Compound 1 (200 mg, 47% yield) as a pale yellow oily liquid. The hydrogen spectrum of Compound 1 is shown in Figure 1.

[0071] 1H NMR(400MHz,Chloroform-d)δ4.23(t,J=6.0Hz,2H),4.16–4.03(m,8H),2.75–2.30( m,10H),2.28(s,3H),1.67–1.57(m,8H),1.31–1.24(m,24H),0.88(t,J=8.0Hz,6H).

[0072] Example 2

[0073] The synthetic route of compound 2 is as follows:

[0074] Step 1:

[0075] Compound 1-3 (300 mg) was dissolved in dichloromethane (5 ml) and stirred on ice. Ultra-dry pyridine (115 mg) and triphosgene (86 mg) were added sequentially and stirred on ice for 0.5 h. A small amount of the reaction solution was plated with a standard sample of 1-3 (PE / EA = 1 / 1, phosphomolybdic acid). New spots with reduced polarity were observed. The reaction solution was evaporated under reduced pressure, and the resulting solid was dissolved in dichloromethane (5 ml) and then added dropwise to a mixed solution of 1-(3-hydroxypropyl)-4-methylpiperazine (500 mg) and pyridine (10 ml). After the addition was complete, the reaction solution was stirred at 70°C for three hours. The solution was orange-yellow. TLC (DCM / MeOH / NH4OH = 10 / 1 / 0.1, phosphomolybdic acid) showed a new spot below the pyridine. An appropriate amount of silica gel and dichloromethane were added to the sample, and the sample was purified (10 g normal phase column, DCM:DCM / MeOH / NH4OH, 0-0% over 5 min, 0-70% over 20 min, 70-70% over 10 min, flow rate 20 ml / min) to afford Compound 2 (214 mg, 47% yield) as a pale yellow oily liquid. The hydrogen spectrum of Compound 2 is shown in Figure 2.

[0076] 1 H NMR(400MHz,Chloroform-d)δ4.15–4.10(m,6H),4.06(s,2H),2.68–2.30(m,8H),2.28(s ,3H),1.83–1.76(m,2H),1.64–1.52(m,12H),1.36–1.13(m,24H),0.88(t,J=6.8Hz,6H).

[0077] Example 3

[0078] The synthetic route of compound 3 is as follows:

[0079] Step 1:

[0080] Compound 1-1 (5.0 g) was dissolved in DCM (200 ml) and stirred at room temperature. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 12.3 g), 4-dimethylaminopyridine (DMAP, 262 mg), triethylamine (6.51 g), and 6-undecanol (7.39 g) were added portionwise to the reaction system and stirred at room temperature for 16 h. A small amount of the reaction solution was diluted and spot-coated with a standard sample of 1-1 (PE / EA = 10 / 1, phosphomolybdic acid and bromocresol green). New spots with reduced polarity were observed. The reaction solution was quenched with water, separated, and the organic phase was evaporated under reduced pressure. An appropriate amount of silica gel was added to mix the sample and purified (80 g normal phase column, PE / EA, 0-0% 5 min, 0-10% 20 min, 10-10% 10 min, flow rate 40 ml / min). The plate was monitored and the pure product fraction was evaporated to obtain a colorless oily liquid compound 3-1 (8.0 g, 68.9% yield).

[0081] Step 2:

[0082] Trifluoroacetic acid (15 ml) was added to a solution of compound 3-1 (8.0 g) in dichloromethane (50 ml). The mixture was stirred at room temperature for 16 hours. TLC showed that compound 3-1 completely disappeared and a point with increased polarity was generated. The reaction solution was spin-dried and saturated aqueous sodium bicarbonate solution (100 ml) was added to quench the excess trifluoroacetic acid. The mixture was extracted with ethyl acetate (100 ml × 2). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and then added with an appropriate amount of silica gel and DCM to mix the sample and purify (80 g normal phase column, PE / EA, 0-0% 5 min, 0-40% 20 min, 40-40% min, flow rate 50 ml / min) to obtain a colorless oily liquid compound 3-2 (5.8 g, 89% yield).

[0083] Step 3:

[0084] Compound 3-2 (300 mg) was dissolved in ultra-dry dichloromethane (5 ml) and stirred in an ice bath. Ultra-dry pyridine (115 mg) and triphosgene (86 mg) were added sequentially, and the mixture was stirred in an ice bath for 0.5 h. A small amount of the reaction solution was plated with a 3-2 standard sample (PE / EA = 1 / 1, phosphomolybdic acid). New spots with reduced polarity were observed. The reaction solution was evaporated under reduced pressure, and the resulting solid was dissolved in ultra-dry dichloromethane (5 ml) and then added dropwise to a mixed solution of 1-(2-hydroxyethyl)-4-methylpiperazine (500 mg) and pyridine (10 ml). After the addition was complete, the reaction solution was stirred at 70°C for three hours. The solution was orange-yellow. TLC (DCM / MeOH / NH4OH = 10 / 1 / 0.1, phosphomolybdic acid) showed a new spot below the pyridine. An appropriate amount of silica gel and dichloromethane were added to the sample, and the sample was purified (10 g normal phase column, DCM:DCM / MeOH / NH4OH, 0-0% over 5 min, 0-70% over 20 min, 70-70% over 10 min, flow rate 20 ml / min) to afford Compound 3 (162 mg, 39% yield) as a pale yellow oily liquid. The hydrogen spectrum of Compound 3 is shown in Figure 3.

[0085] 1 H NMR(400MHz,Chloroform-d)δ4.96–4.86(m,2H),4.21(t,J=6.4Hz,2H),4.12(s,2H),4.04(s,2H ),2.71–2.32(m,10H),2.28(s,3H),1.61–1.51(m,10H),1.30–1.24(m,22H),0.90–0.85(m,12H).

[0086] Example 4

[0087] The synthetic route of compound 4 is as follows:

[0088] Step 1:

[0089] Compound 1-1 (1.5 g) was dissolved in DCM (50 ml) and stirred at room temperature. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 3.70 g), 4-dimethylaminopyridine (DMAP, 79 mg), triethylamine (1.95 g), and pentadecane-8-ol (2.94 g) were added portionwise to the reaction system and stirred at room temperature for 16 h. A small amount of the reaction solution was diluted and spot-coated with a standard sample of 1-1 (PE / EA = 10 / 1, phosphomolybdic acid and bromocresol green). New spots with reduced polarity were observed. The reaction solution was quenched with water, separated, and the organic phase was evaporated under reduced pressure. An appropriate amount of silica gel was added for sample mixing and purification (25 g normal phase column, PE / EA, 0-0% 5 min, 0-10% 20 min, 10-10% 10 min, flow rate 30 ml / min) was performed using a plate monitor. The pure product fraction was evaporated to obtain a colorless oily liquid compound 4-1 (2.5 g, 59.4% yield).

[0090] Step 2:

[0091] Trifluoroacetic acid (10 ml) was added to a solution of compound 4-1 (2.5 g) in dichloromethane (20 ml). The mixture was stirred at room temperature for 16 hours. TLC showed that compound 4-1 completely disappeared and a point with increased polarity was generated. The reaction solution was spin-dried and saturated aqueous sodium bicarbonate solution (50 ml) was added to quench the excess trifluoroacetic acid. The mixture was extracted with ethyl acetate (50 ml × 2). The organic phase was dried over anhydrous sodium sulfate and filtered. The crude product after concentration was added with an appropriate amount of silica gel and DCM, mixed and purified (25 g normal phase column, PE / EA, 0-0% 5 min, 0-50% 20 min, 50-50% 5 min, flow rate 30 ml / min) to obtain a colorless oily liquid compound 4-2 (1.8 g, 85% yield).

[0092] Step 3:

[0093] Compound 4-2 (200 mg) was dissolved in ultra-dry dichloromethane (5 ml) and stirred in an ice bath. Ultra-dry pyridine (58 mg) and triphosgene (43 mg) were added sequentially, and the mixture was stirred in an ice bath for 0.5 h. A small amount of the reaction solution was plated with a standard sample of 4-2 (PE / EA = 1 / 1, phosphomolybdic acid). New spots with reduced polarity were observed. The reaction solution was evaporated under reduced pressure, and the resulting solid was dissolved in ultra-dry dichloromethane (5 ml) and added dropwise to a mixed solution of 1-(3-hydroxypropyl)-4-methylpiperazine (500 mg) and pyridine (10 ml). After the addition was complete, the reaction solution was stirred at 70°C for three hours. The solution was orange-yellow. TLC (DCM / MeOH / NH4OH = 10 / 1 / 0.1, phosphomolybdic acid) showed a new spot below the pyridine. An appropriate amount of silica gel and dichloromethane were added to the sample, and the sample was purified (10 g normal phase column, DCM:DCM / MeOH / NH4OH, 0-0% for 5 min, 0-70% for 20 min, 70-70% for 10 min, flow rate 20 ml / min) to afford Compound 1 (130 mg, 51% yield) as a pale yellow oily liquid. The hydrogen spectrum of Compound 4 is shown in Figure 4.

[0094] 1 H NMR (400MHz, Chloroform-d) δ4.95–4.86 (m, 2H), 4.14 (t, J = 8.0Hz 2H),4.11–4.03(m,4H),2.72–2.31(m,10H),2.30(s,3H),1.84–1.77(m,2 H),1.56–1.48(m,8H),1.30–1.21(m,40H),0.90–0.85(t,J=6.8Hz,12H).

[0095] Example 5

[0096] The synthetic route of compound 5 is as follows:

[0097] Step 1:

[0098] Compound 4-2 (200 mg) was dissolved in ultra-dry dichloromethane (5 ml) and stirred in an ice bath. Ultra-dry pyridine (58 mg) and triphosgene (43 mg) were added sequentially, and the mixture was stirred in an ice bath for 0.5 h. A small amount of the reaction solution was plated with a standard sample of 4-2 (PE / EA = 1 / 1, phosphomolybdic acid). New spots with reduced polarity were observed. The reaction solution was evaporated under reduced pressure, and the resulting solid was dissolved in ultra-dry dichloromethane (5 ml) and added dropwise to a mixed solution of 1-(2-hydroxyethyl)-4-methylpiperazine (500 mg) and pyridine (10 mL). After the addition was complete, the reaction solution was stirred at 70°C for three hours. The solution was orange-yellow. TLC (DCM / MeOH / NH4OH = 10 / 1 / 0.1, phosphomolybdic acid) showed a new spot below the pyridine. An appropriate amount of silica gel and dichloromethane were added to the sample, and the sample was purified (10 g normal phase column, DCM:DCM / MeOH / NH4OH, 0-0% for 5 min, 0-70% for 20 min, 70-70% for 10 min, flow rate 20 ml / min) to obtain Compound 5 (130 mg, 50% yield) as a pale yellow oily liquid. The hydrogen spectrum of Compound 5 is shown in Figure 5.

[0099] 1H NMR(400MHz,Chloroform-d)δ4.79(s,2H),4.08(s,2H),3.87(s,4H),2.79(s,2H),2.54(s,4H),2.49(s,2H),2.44(s,2H),2.27(s,3 H),1.68(d,J=12.4Hz,4H),1.56(d,J=12.4Hz,4H),1.37(d,J=0.6Hz,8H),1.34(d,J=1.2Hz,8H),1.31-1.29(m,24H),0.90(s,12H).

[0100] Example 6

[0101] The synthetic route of compound 6 is as follows:

[0102] Step 1:

[0103] Compound 4-2 (200 mg) was dissolved in ultra-dry dichloromethane (5 ml) and stirred in an ice bath. Ultra-dry pyridine (58 mg) and triphosgene (43 mg) were added sequentially, and the mixture was stirred in an ice bath for 0.5 h. A small amount of the reaction solution was plated with a standard control sample 1-3 (PE / EA = 1 / 1, phosphomolybdic acid). New spots with reduced polarity were observed. The reaction solution was evaporated under reduced pressure, and the resulting solid was dissolved in ultra-dry dichloromethane (5 ml) and added dropwise to a mixed solution of compound 3-(1-pyrrolidinyl)-1-propanol (500 mg) and pyridine (10 ml). After the addition was complete, the reaction solution was stirred at 70°C for three hours. The solution turned red. TLC (DCM / MeOH / NH4OH = 10 / 1 / 0.1, phosphomolybdic acid) showed the formation of a new spot below the pyridine. An appropriate amount of silica gel and dichloromethane were added to the sample, and the sample was purified (10 g normal phase column, DCM:DCM / MeOH / NH4OH, 0-0% for 5 min, 0-70% for 20 min, 70-70% for 10 min, flow rate 20 ml / min) to obtain compound 6 (140 mg, 54.7% yield) as a pale yellow oily liquid. The hydrogen spectrum of compound 6 is shown in Figure 6.

[0104] 1H NMR(400MHz,Chloroform-d)δ4.79(s,2H),4.14(s,2H),3.87(s,4H),2.67(s,2H),2.56(s,4H),1.90(s,2H),1.75(s,4H),1.69(d,J= 12.4Hz, 4H), 1.56 (d, J = 12.4Hz, 4H), 1.37 (d, J = 0.6Hz, 8H), 1.33 (d, J = 1.2Hz, 8H), 1.31 (s, 8H), 1.28 (d, J = 4.6Hz, 16H), 0.90 (s, 12H).

[0105] Example 7

[0106] The synthetic route of compound 7 is as follows:

[0107] Step 1:

[0108] Compound 1-1 (5.0 g) was dissolved in DCM (200 ml) and stirred at room temperature. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 12.3 g), 4-dimethylaminopyridine (DMAP, 262 mg), triethylamine (6.51 g), and 1-tridecanol (8.59 g) were added portionwise to the reaction system and stirred at room temperature for 16 h. A small amount of the reaction solution was diluted and spot-coated with a 1-1 standard sample (PE / EA = 10 / 1, phosphomolybdic acid and bromocresol green). New spots with reduced polarity were observed. The reaction solution was quenched with water, separated, and the organic phase was evaporated under reduced pressure. An appropriate amount of silica gel was added to mix the sample and purified (80 g normal phase column, PE / EA, 0-0% 5 min, 0-10% 20 min, 10-10% 10 min, flow rate 40 ml / min). The plate was monitored and the pure product fraction was evaporated to obtain a colorless oily liquid compound 7-2 (8.5 g, 66.3% yield).

[0109] Step 2:

[0110] Trifluoroacetic acid (30 ml) was added to a solution of compound 7-2 (8.5 g) in dichloromethane (100 ml). The mixture was stirred at room temperature for 16 hours. TLC showed that compound 7-2 completely disappeared and a point with increased polarity was generated. The reaction solution was spin-dried and saturated aqueous sodium bicarbonate solution (150 ml) was added to quench the excess trifluoroacetic acid. The mixture was extracted with ethyl acetate (100 ml × 2). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and added with an appropriate amount of silica gel and DCM to mix the sample and purify (120 g normal phase column, PE / EA, 0-0% 5 min, 0-50% 20 min, 50-50% min, flow rate 60 ml / min) to obtain white solid compound 7-3 (6.3 g, 89% yield).

[0111] Step 3:

[0112] Compound 7-3 (300 mg) was dissolved in ultra-dry dichloromethane (5 ml) and stirred in an ice bath. Ultra-dry pyridine (96 mg) and triphosgene (72 mg) were added sequentially, and the mixture was stirred in an ice bath for 0.5 h. A small amount of the reaction solution was plated with a 7-3 standard sample (PE / EA = 1 / 1, phosphomolybdic acid). New spots with reduced polarity were observed. The reaction solution was evaporated under reduced pressure, and the resulting solid was dissolved in ultra-dry dichloromethane (5 ml) and then added dropwise to a mixed solution of compound 3-(1-pyrrolidinyl)-1-propanol (500 mg) and pyridine (10 mL). After the addition was complete, the reaction solution was stirred at 70°C for three hours. The solution turned red. TLC (DCM / MeOH / NH4OH = 10 / 1 / 0.1, phosphomolybdic acid) showed the formation of a new spot below the pyridine. An appropriate amount of silica gel and dichloromethane were added to the sample, and the sample was purified (10 g normal phase column, DCM:DCM / MeOH / NH4OH, 0-0% for 5 min, 0-70% for 20 min, 70-70% for 10 min, flow rate 20 ml / min) to obtain compound 7 (180 mg, 45.7% yield) as a yellow oily liquid. The hydrogen spectrum of compound 7 is shown in Figure 7.

[0113] 1H NMR(400MHz,Chloroform-d)δ4.14(s,2H),4.12(s,4H),3.88(s,4H),2.67(s,2H),2.56(s,4H),1.90( s,2H),1.75(s,4H),1.66(s,4H),1.38(s,4H),1.33(d,J=6.2Hz,8H),1.30–1.23(m,28H),0.90(s,6H).

[0114] Example 8

[0115] The synthetic route of compound 8 is as follows:

[0116] Step 1:

[0117] Compound 8-1 (2.0 g) was dissolved in DCM (30 ml) and stirred at room temperature. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 3.28 g), 4-dimethylaminopyridine (DMAP, 140 mg), triethylamine (1.73 g), and (8Z,11Z)-heptadeca-8,11-dien-1-ol (3.17 g) were added to the reaction system in batches and stirred at room temperature for 16 h. A small amount of the reaction solution was diluted and spot-coated with a standard sample of 8-1 (PE / EA = 10 / 1, potassium permanganate). New spots with reduced polarity were observed. The reaction solution was quenched with water, separated, and the organic phase was evaporated under reduced pressure. An appropriate amount of silica gel was added for sample mixing and purification (25 g normal phase column, PE / EA, 0-0% 5 min, 0-10% 20 min, 10-10% 10 min, flow rate 30 ml / min) was performed using a plate monitor. The pure product fractions were evaporated to obtain a colorless oily liquid compound 8-2 (3.0 g, 64% yield).

[0118] Step 2:

[0119] Trifluoroacetic acid (10 mL) was added to a solution of compound 8-2 (3.0 g) in dichloromethane (30 mL). The mixture was stirred at room temperature for 16 hours. TLC showed that compound 8-2 completely disappeared and a point with increased polarity was generated. The reaction solution was spin-dried and saturated aqueous sodium bicarbonate solution (150 mL) was added to quench the excess trifluoroacetic acid. The mixture was extracted with ethyl acetate (100 mL × 2). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and then added with an appropriate amount of silica gel and DCM for sample mixing and purification (120 g normal phase column, PE / EA, 0-0% 5 min, 0-50% 20 min, 50-50% min, flow rate 60 mL / min) to obtain compound 8-3 (1.9 g, 84% yield) as a light yellow oil.

[0120] Step 3:

[0121] Bromoacetic acid (2.0 g) was dissolved in DCM (40 ml) and stirred at room temperature. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 4.14 g), 4-dimethylaminopyridine (DMAP, 176 mg), triethylamine (2.18 g), and pentadecane-8-ol (3.62 g) were added portionwise to the reaction system and stirred at room temperature for 16 h. The reaction solution was quenched with water, separated, and the organic phase was evaporated under reduced pressure. An appropriate amount of silica gel was added for sample mixing and purification (25 g normal phase column, PE / EA, 0-0% 5 min, 0-10% 20 min, 10-10% 10 min, flow rate 30 ml / min) was performed using a spot plate monitor. The pure product fraction was evaporated to obtain compound 8-5 (3.6 g, 72% yield) as a colorless oily liquid.

[0122] Step 4:

[0123] 8-3 (1.5 g) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Potassium carbonate (1.34 g) and 8-5 (1.86 g) were weighed and added to the reaction system in batches. The reaction was stirred at 70°C for 3 h. The reaction solution was filtered, and the filter cake was washed with ethyl acetate. The organic phase was mixed with an appropriate amount of silica gel and purified (25 g normal phase column, PE / EA, 0-0% 5 min, 0-30% 20 min, 30-30% 10 min, flow rate 30 ml / min) with spot plate monitoring. The pure product fraction was evaporated to obtain compound 8-4 (800 mg, 28.6% yield) as a colorless oily liquid.

[0124] Step 5:

[0125] Compound 8-4 (300 mg) was dissolved in ultra-dry dichloromethane (5 ml) and stirred in an ice bath. Ultra-dry pyridine (83 mg) and triphosgene (62 mg) were added sequentially, and the mixture was stirred in an ice bath for 0.5 h. A small amount of the reaction solution was plated with a standard sample of 8-4 (PE / EA = 1 / 1, phosphomolybdic acid). New spots with reduced polarity were observed. The reaction solution was evaporated under reduced pressure, and the resulting solid was dissolved in ultra-dry dichloromethane (5 ml) and then added dropwise to a mixed solution of compound 3-(1-pyrrolidinyl)-1-propanol (500 mg) and pyridine (10 mL). After the addition was complete, the reaction solution was stirred at 70°C for three hours. The solution turned red, and TLC (DCM / MeOH / NH4OH = 10 / 1 / 0.1, phosphomolybdic acid) showed the formation of a new spot. An appropriate amount of silica gel and dichloromethane were added to the sample, and the sample was purified (10 g normal phase column, DCM:DCM / MeOH / NH4OH, 0-0% for 5 min, 0-70% for 20 min, 70-70% for 10 min, flow rate 20 ml / min) to obtain compound 8 (160 mg, 42% yield) as a yellow oily liquid. The hydrogen spectrum of compound 8 is shown in Figure 8.

[0126] 1H NMR(400MHz,Chloroform-d)δ5.35(d,J=16.0Hz,4H),4.79(s,1H),4.13(d,J=9.8Hz,4H),3.88(d,J=4.4Hz,4H),2.80–2.64(m,4H),2.56(s,4 H),2.08–2.00(dt,J=9.0,1.0Hz,4H),1.90(s,2H),1.75(s,4H),1.72–1.63(m,4H),1.55(d,J=12.6Hz,2H),1.38–1.25(m,34H),0.90(s,9H).

[0127] Example 9

[0128] The synthetic route of compound 9 is as follows:

[0129] Step 1:

[0130] Compound 8-1 (3.0 g) was dissolved in DCM (30 ml) and stirred at room temperature. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 4.92 g), 4-dimethylaminopyridine (DMAP, 210 mg), triethylamine (2.60 g), and undecanol (3.25 g) were weighed and added portionwise to the reaction system. The mixture was stirred at room temperature for 16 h. A small amount of the reaction solution was diluted and compared with a standard sample of 8-1 (PE / EA = 20 / 1, phosphomolybdic acid) and spot-blotted. New spots with similar polarity were observed. The reaction solution was quenched with water, separated, and the organic phase was evaporated under reduced pressure. An appropriate amount of silica gel was added for sample mixing and purification (40 g normal phase column, PE / EA, 0-0% 10 min, 0-6% 30 min, 6-6% 20 min, flow rate 30 ml / min). Spot-blot monitoring was performed, and the pure product fraction was evaporated to obtain compound 9-1 (3.2 g, 57% yield) as a white solid.

[0131] Step 2:

[0132] Trifluoroacetic acid (10 ml) was added to a solution of compound 9-1 (3.2 g) in dichloromethane (30 mL). The mixture was stirred at room temperature for 16 hours. TLC showed that compound 9-1 completely disappeared, and a point with increased polarity was formed. The reaction solution was spin-dried, and saturated sodium bicarbonate aqueous solution (150 ml) was added to quench the excess trifluoroacetic acid. The solution was extracted with ethyl acetate (100 ml × 2). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. An appropriate amount of silica gel and DCM were added to mix the sample and purified (120 g normal phase column, PE / EA, 0-0% 5 min, 0-40% 20 min, 40-40% 15 min, flow rate 30 mL / min) to obtain compound 9-2 (2.0 g, 90% yield) as a colorless oil.

[0133] Step 3:

[0134] Bromoacetic acid (2.0 g) was dissolved in DCM (40 ml) and stirred at room temperature. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 4.14 g), 4-dimethylaminopyridine (DMAP, 176 mg), triethylamine (2.18 g), and pentadecane-7-ol (3.62 g) were added portionwise to the reaction system and stirred at room temperature for 16 h. The reaction solution was quenched with water, separated, and the organic phase was evaporated under reduced pressure. An appropriate amount of silica gel was added for sample mixing and purification (25 g normal phase column, PE / EA, 0-0% for 5 min, 0-10% for 20 min, 10-10% for 10 min, flow rate 30 ml / min) was performed using a spot plate monitor. The pure product fraction was evaporated to obtain compound 9-4 (3.6 g, 70% yield) as a colorless oily liquid.

[0135] Step 4:

[0136] 9-2 (2.0 g) was dissolved in acetonitrile (20 ml) and stirred at room temperature. Potassium carbonate (2.41 g) and 9-4 (3.35 g) were weighed and added to the reaction system in batches. The reaction was stirred at 70°C for 3 h. The reaction solution was filtered, and the filter cake was washed with ethyl acetate. The organic phase was mixed with an appropriate amount of silica gel and purified (40 g normal phase column, PE / EA, 0-0% 5 min, 0-30% 20 min, 30-30% 20 min, flow rate 30 ml / min) with spot plate monitoring. The pure product fraction was evaporated to obtain compound 9-3 (900 mg, 20.7% yield) as a colorless oily liquid.

[0137] Step 5:

[0138] Compound 9-3 (300 mg) was dissolved in ultra-dry dichloromethane (5 ml) and stirred in an ice bath. Ultra-dry pyridine (96 mg) and triphosgene (72 mg) were added sequentially, and the mixture was stirred in an ice bath for 0.5 h. A small amount of the reaction solution was plated with a standard sample of 9-3 (PE / EA = 1 / 1, phosphomolybdic acid). New spots with reduced polarity were observed. The reaction solution was evaporated under reduced pressure, and the resulting solid was dissolved in ultra-dry dichloromethane (5 ml) and then added dropwise to a mixed solution of 1-(3-hydroxypropyl)-4-methylpiperazine (500 mg) and pyridine (10 ml). After the addition was complete, the reaction solution was stirred at 70°C for three hours. The solution turned red, and TLC (DCM / MeOH / NH4OH = 10 / 1 / 0.1, phosphomolybdic acid) showed the formation of a new spot. An appropriate amount of silica gel and dichloromethane were added to the sample, and the sample was purified (10 g normal phase column, DCM:DCM / MeOH / NH4OH, 0-0% over 5 min, 0-70% over 20 min, 70-70% over 10 min, flow rate 20 ml / min) to afford Compound 9 (180 mg, 43.9% yield) as a yellow oily liquid. The hydrogen spectrum of Compound 9 is shown in Figure 9.

[0139] 1H NMR (400MHz, Chloroform-d) δ4.79 (s, 1H), 4.14–4.11 (m, 4H), 3.87 (d, J = 4.4Hz, 4H), 2.66 (d, J = 1.6Hz, 2H), 2.50–2.44 (m, 8H), 2.27 (s, 3H), 2.01–1. 83(m,2H),1.73–1.63(m,4H),1.55(d,J=12.4Hz,2H),1.40–1.36(m,6H),1 .34(d,J=1.0Hz,2H),1.34–1.30(m,10H),1.30–1.25(m,18H),0.90(s,9H).

[0140] Example 10

[0141] Synthesis of compound 10

[0142] Step 1:

[0143] Compound 3-2 (300 mg) was dissolved in ultra-dry dichloromethane (5 ml) and stirred in an ice bath. Ultra-dry pyridine (115 mg) and triphosgene (86 mg) were added sequentially, and the mixture was stirred in an ice bath for 0.5 h. A small amount of the reaction solution was plated with a 3-2 standard sample (PE / EA = 1 / 1, phosphomolybdic acid). New spots with reduced polarity were observed. The reaction solution was evaporated under reduced pressure, and the resulting solid was dissolved in ultra-dry dichloromethane (5 ml) and then added dropwise to a mixed solution of N-(2-hydroxyethyl)hexamethylenediamine (500 mg) and pyridine (10 mL). After the addition was complete, the reaction solution was stirred at 70°C for three hours. The solution was orange-yellow. TLC (DCM / MeOH / NH4OH = 10 / 1 / 0.1, phosphomolybdic acid) showed a new spot below the pyridine. An appropriate amount of silica gel and dichloromethane were added to the sample, and the sample was purified (10 g normal phase column, DCM:DCM / MeOH / NH4OH, 0-0% for 5 min, 0-70% for 20 min, 70-70% for 10 min, flow rate 20 ml / min) to afford Compound 10 (170 mg, 41% yield) as a pale yellow oily liquid. The hydrogen spectrum of Compound 10 is shown in Figure 10.

[0144] 1 H NMR(400MHz,Chloroform-d)δ4.95–4.86(m,2H),4.27–4.22(m,2H),4.09(d,J=12.0Hz,4H),2.78 –2.70(m,2H),2.65–2.50(m,4H),1.80–1.30(m,16),1.28–1.10(m,24H),0.87(t,J=4.0Hz,12H).

[0145] Example 11

[0146] The synthetic route of compound 11 is as follows:

[0147] Step 1:

[0148] Compound 11-1 (3.0 g) was dissolved in DCM (30 ml) and stirred at room temperature. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 3.40 g), 4-dimethylaminopyridine (DMAP, 180 mg), triethylamine (2.24 g), and 5-undecanol (2.80 g) were weighed and added portionwise to the reaction system. The mixture was stirred at room temperature for 16 h. A small amount of the reaction solution was diluted with a control spot plate (PE / EA = 10 / 1, phosphomolybdic acid). New spots with decreasing polarity were observed. The reaction solution was quenched with water, separated, and the organic phase was evaporated under reduced pressure. An appropriate amount of silica gel was added for sample mixing and purification (40 g normal phase column, PE / EA, 0-0% 10 min, 0-6% 30 min, 6-6% 20 min, flow rate 30 ml / min). The fractions of pure product were evaporated to obtain compound 11-2 (4.0 g, 76% yield) as a white solid.

[0149] Step 2:

[0150] To a solution of compound 11-2 (4.0 g) in dichloromethane (30 ml) was added trifluoroacetic acid (10 ml). The mixture was stirred at room temperature for 16 hours. TLC showed that compound 11-2 completely disappeared and a point with increased polarity was generated. The reaction solution was spin-dried, and saturated aqueous sodium bicarbonate solution (150 ml) was added to quench the excess trifluoroacetic acid. The mixture was extracted with ethyl acetate (100 ml × 2). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and then added with an appropriate amount of silica gel and DCM to mix the sample and purify (25 g normal phase column, PE / EA, 0-0% 5 min, 0-40% 20 min, 40-40% 15 min, flow rate 30 ml / min) to obtain compound 11-3 (2.5 g, 87% yield) as a colorless oil.

[0151] Step 3:

[0152] 4-Bromobutyric acid (2.0 g) was dissolved in DCM (40 ml) and stirred at room temperature. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 3.44 g), 4-dimethylaminopyridine (DMAP, 147 mg), triethylamine (1.82 g), and 5-undecanol (2.27 g) were added portionwise to the reaction system and stirred at room temperature for 16 h. The reaction solution was quenched with water, separated, and the organic phase was evaporated under reduced pressure. An appropriate amount of silica gel was added for sample mixing and purification (25 g normal phase column, PE / EA, 0-0% 5 min, 0-10% 20 min, 10-10% 10 min, flow rate 30 ml / min) was performed using a plate monitor. The pure product fraction was evaporated to obtain compound 11-5 (3.0 g, 78% yield) as a colorless oily liquid.

[0153] Step 4:

[0154] 11-3 (2.0 g) was dissolved in acetonitrile (20 ml) and stirred at room temperature. Potassium carbonate (2.15 g) and 11-5 (2.75 g) were weighed and added to the reaction system in batches. The reaction was stirred at 70°C for 3 h. The reaction solution was filtered, and the filter cake was washed with ethyl acetate. The organic phase was mixed with an appropriate amount of silica gel and purified (40 g normal phase column, PE / EA, 0-0% 5 min, 0-40% 20 min, 40-40% 20 min, flow rate 30 ml / min) with spot plate monitoring. The pure product fraction was evaporated to obtain compound 11-4 (950 mg, 24.6% yield) as a colorless oily liquid.

[0155] Step 5:

[0156] Compound 11-4 (300 mg) was dissolved in ultra-dry dichloromethane (5 ml) and stirred in an ice bath. Ultra-dry pyridine (96 mg) and triphosgene (72 mg) were added sequentially, and the mixture was stirred in an ice bath for 0.5 h. A small amount of the reaction solution was plated with a standard sample of 11-4 (PE / EA = 1 / 1, phosphomolybdic acid). New spots with reduced polarity were observed. The reaction solution was evaporated under reduced pressure, and the resulting solid was dissolved in ultra-dry dichloromethane (5 ml) and then added dropwise to a mixed solution of N-(2-hydroxyethyl)-pyrrolidine (500 mg) and pyridine (10 ml). After the addition was complete, the reaction solution was stirred at 70°C for three hours. The solution turned red, and TLC (DCM / MeOH / NH4OH = 10 / 1 / 0.1, phosphomolybdic acid) showed the formation of a new spot. An appropriate amount of silica gel and dichloromethane were added to the sample, and the sample was purified (10 g normal phase column, DCM:DCM / MeOH / NH4OH, 0-0% for 5 min, 0-70% for 20 min, 70-70% for 10 min, flow rate 20 ml / min) to afford Compound 11 (160 mg, 41.5% yield) as a yellow oily liquid. The hydrogen spectrum of Compound 11 is shown in Figure 11.

[0157] 1H NMR(400MHz,Chloroform-d)δ4.77(s,2H),4.10(d,J=0.8Hz,2H),3.27(d,J= 0.8Hz,4H),2.85–2.70(m,6H),2.40–2.29(m,4H),2.28–2.16(m,4H),1.76(d, J=4.0Hz,4H),1.72–1.63(dd,J=12.4,4.0Hz,4H),1.61–1.51(dd,J=12.4,8. 4Hz,4H),1.42–1.32(m,16H),1.30(d,J=8.4Hz,8H),0.91(d,J=10.6Hz,12H).

[0158] Example 12

[0159] The synthetic route of compound 12 is as follows:

[0160] Step 1:

[0161] Compound 12-1 (3.00 g) was dissolved in DCM (30 ml) and stirred at room temperature. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 4.24 g), 4-dimethylaminopyridine (DMAP, 180 mg), triethylamine (2.24 g), and pentadecane-7-ol (3.71 g) were added portionwise to the reaction system and stirred at room temperature for 16 h. A small amount of the reaction solution was diluted onto a control spot plate (PE / EA = 10 / 1, phosphomolybdic acid), and new spots with reduced polarity were observed. The reaction solution was quenched with water, separated, and the organic phase was evaporated under reduced pressure. An appropriate amount of silica gel was added for sample mixing and purification (40 g normal phase column, PE / EA, 0-0% in 10 min, 0-6% in 30 min, 6-6% in 20 min, flow rate of 30 ml / min) was performed using a plate monitor. The pure product fractions were evaporated to give compound 12-2 (4.3 g, 70.4% yield) as a white solid.

[0162] Step 2:

[0163] Trifluoroacetic acid (10 ml) was added to a solution of compound 12-2 (4.3 g) in dichloromethane (30 ml). The mixture was stirred at room temperature for 16 hours. TLC showed that compound 12-2 completely disappeared and a point with increased polarity was generated. The reaction solution was spin-dried and saturated aqueous sodium bicarbonate solution (150 ml) was added to quench the excess trifluoroacetic acid. The mixture was extracted with ethyl acetate (100 ml × 2). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and then added with an appropriate amount of silica gel and DCM to mix the sample and purify (25 g normal phase column, PE / EA, 0-0% 5 min, 0-40% 20 min, 40-40% 15 min, flow rate 30 mL / min) to obtain compound 12-3 (2.9 g, 89% yield) as a colorless oil.

[0164] Step 3:

[0165] 4-Bromobutyric acid (2.0 g) was dissolved in DCM (40 ml) and stirred at room temperature. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 3.44 g), 4-dimethylaminopyridine (DMAP, 147 mg), triethylamine (1.82 g), and pentadecane-7-ol (3.01 g) were then added portionwise to the reaction system and stirred at room temperature for 16 h. The reaction mixture was quenched with water, separated, and the organic phase evaporated under reduced pressure. An appropriate amount of silica gel was added for sample mixing and purification (25 g normal phase column, PE / EA, 0-0% for 5 min, 0-10% for 20 min, 10-10% for 10 min, flow rate 30 ml / min) was performed using a plate-spotting monitor. The pure product fraction was evaporated to obtain compound 12-5 (3.3 g, 66.6% yield) as a colorless oily liquid.

[0166] Step 4:

[0167] 12-3 (2.0 g) was dissolved in acetonitrile (20 ml) and stirred at room temperature. Potassium carbonate (1.76 g) and 12-5 (2.65 g) were weighed and added to the reaction system in batches. The reaction was stirred at 70°C for 3 h. The reaction solution was filtered, and the filter cake was washed with ethyl acetate. The organic phase was mixed with an appropriate amount of silica gel and purified (40 g normal phase column, PE / EA, 0-0% 5 min, 0-40% 20 min, 40-40% 20 min, flow rate 30 ml / min) with spot plate monitoring. The pure product fraction was evaporated to obtain compound 12-4 (800 mg, 22.6% yield) as a colorless oily liquid.

[0168] Step 5:

[0169] Compound 12-4 (300 mg) was dissolved in ultra-dry dichloromethane (5 ml) and stirred in an ice bath. Ultra-dry pyridine (85 mg) and triphosgene (64 mg) were added sequentially, and the mixture was stirred in an ice bath for 0.5 h. A small amount of the reaction solution was plated with a standard sample of 12-4 (PE / EA = 1 / 1, phosphomolybdic acid). New spots with reduced polarity were observed. The reaction solution was evaporated under reduced pressure, and the resulting solid was dissolved in ultra-dry dichloromethane (5 ml) and then added dropwise to a mixed solution of 1-(3-hydroxypropyl)-4-methylpiperazine (500 mg) and pyridine (10 mL). After the addition was complete, the reaction solution was stirred at 70°C for three hours. The solution turned red, and TLC (DCM / MeOH / NH4OH = 10 / 1 / 0.1, phosphomolybdic acid) showed the formation of a new spot. An appropriate amount of silica gel and dichloromethane were added to the sample, and the sample was purified (10 g normal phase column, DCM:DCM / MeOH / NH4OH, 0-0% for 5 min, 0-70% for 20 min, 70-70% for 10 min, flow rate 20 ml / min) to afford Compound 12 (180 mg, 52.0% yield) as a yellow oily liquid. The hydrogen spectrum of Compound 12 is shown in Figure 12.

[0170] 1H NMR(400MHz,Chloroform-d)δ4.78(s,2H),4.15(d,J=0.8Hz,2H),3.26(d,J=0.8Hz,4H), 2.65(d,J=1.2Hz,2H),2.50(s,2H),2.47(s,2H),2.45(s,2H),2.44(s,2H),2.38–2.29(m, 4H),2.28–2.16(m,7H),1.96–1.84(m,2H),1.73–1.51(m,8H),1.41–1.36(m,8H),1.34(d ,J=1.0Hz,4H),1.31(d,J=6.8Hz,12H),1.30–1.25(dd,J=4.9,1.0Hz,16H),0.90(s,12H).

[0171] Example 13

[0172] The synthetic route of compound 13 is as follows:

[0173] Step 1:

[0174] The compound 4-(tert-Butoxycarbonylamino)butyric acid (3.00 g) was dissolved in DCM (30 ml) and stirred at room temperature. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 3.40 g), 4-dimethylaminopyridine (DMAP, 180 mg), triethylamine (2.24 g), and 1-nonanol (2.13 g) were added portionwise to the reaction system and stirred at room temperature for 16 hours. A small amount of the reaction solution was diluted onto a control spot plate (PE / EA = 10 / 1, phosphomolybdic acid), and new spots with reduced polarity were observed. The reaction solution was quenched with water, separated, and the organic phase was evaporated under reduced pressure. An appropriate amount of silica gel was added for sample mixing and purification (40 g normal phase column, PE / EA, 0-0% in 10 min, 0-6% in 30 min, 6-6% in 20 min, flow rate of 30 ml / min) was performed using a plate monitor. The pure product fraction was evaporated to obtain compound 13-1 (3.4 g, 69.9% yield) as a white solid.

[0175] Step 2:

[0176] Trifluoroacetic acid (10 ml) was added to a solution of compound 13-1 (3.4 g) in dichloromethane (30 ml). The mixture was stirred at room temperature for 16 hours. TLC showed that compound 13-1 completely disappeared, and a point with increased polarity was generated. The reaction solution was spin-dried, and saturated aqueous sodium bicarbonate solution (150 ml) was added to quench the excess trifluoroacetic acid. The solution was extracted with ethyl acetate (100 ml × 2). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. An appropriate amount of silica gel and DCM were added to mix the sample and purified (40 g normal phase column, PE / EA, 0-0% 5 min, 0-40% 20 min, 40-40% 15 min, flow rate 30 mL / min) to obtain compound 13-2 (2.1 g, 89% yield) as a colorless oil.

[0177] Step 3:

[0178] 13-2 (2.0 g) was dissolved in acetonitrile (20 ml) and stirred at room temperature. Potassium carbonate (1.76 g) and 12-5 (2.65 g) were weighed and added to the reaction system in batches. The reaction was stirred at 70°C for 3 h. The reaction solution was filtered, and the filter cake was washed with ethyl acetate. The organic phase was mixed with an appropriate amount of silica gel and purified (40 g normal phase column, PE / EA, 0-0% 5 min, 0-40% 20 min, 40-40% 20 min, flow rate 30 ml / min) with spot plate monitoring. The pure product fraction was evaporated to obtain compound 13-3 (800 mg, 22.6% yield) as a colorless oily liquid.

[0179] Step 4:

[0180] Compound 13-3 (300 mg) was dissolved in ultra-dry dichloromethane (5 ml) and stirred in an ice bath. Ultra-dry pyridine (90 mg) and triphosgene (68 mg) were added sequentially, and the mixture was stirred in an ice bath for 0.5 h. A small amount of the reaction solution was plated with a standard sample of 13-3 (PE / EA = 1 / 1, phosphomolybdic acid). New spots with reduced polarity were observed. The reaction solution was evaporated under reduced pressure, and the resulting solid was dissolved in ultra-dry dichloromethane (5 ml) and then added dropwise to a mixed solution of 1-(2-hydroxyethyl)-4-methylpiperazine (500 mg) and pyridine (10 mL). After the addition was complete, the reaction solution was stirred at 70°C for three hours. The solution was orange-red, and TLC (DCM / MeOH / NH4OH = 10 / 1 / 0.1, phosphomolybdic acid) showed the formation of a new spot. An appropriate amount of silica gel and dichloromethane were added to the sample, and the sample was purified (10 g normal phase column, DCM:DCM / MeOH / NH4OH, 0-0% for 5 min, 0-70% for 20 min, 70-70% for 10 min, flow rate 20 ml / min) to afford Compound 13 (200 mg, 50.4% yield) as a yellow oily liquid. The hydrogen spectrum of Compound 13 is shown in Figure 13.

[0181] 1H NMR(400MHz,Chloroform-d)δ4.78(s,1H),4.15–4.08(m,4H),3.27(d,J=0.8Hz,4H),2.79(d,J=1.6Hz ,2H),2.56(s,2H),2.52(s,2H),2.49(s,2H),2.44(s,2H),2.40–2.31(m,4H),2.30–2.24(m,6H),2.22– 2.20(m,1H),1.72–1.61(m,4H),1.55(d,J=12.4Hz,2H),1.39(d,J=2.4Hz,4H),1.36(d,J=0.6Hz,2H), 1.34(d,J=1.0Hz,2H),1.33(d,J=1.0Hz,4H),1.31(s,6H),1.30–1.27(m,14H),0.90(d,J=10.6Hz,9H).

[0182] Example 14

[0183] The synthetic route of compound 14 is as follows:

[0184] Step 1:

[0185] Compound 1-1 (1.0 g) was dissolved in DCM (20 ml) and stirred at room temperature. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 2.47 g), 4-dimethylaminopyridine (DMAP, 524 mg), triethylamine (1.30 g), and (8Z,11Z)-heptadeca-8,11-dien-1-ol (2.16 g) were added portionwise to the reaction system and stirred at room temperature for 16 h. A small amount of the reaction solution was diluted and spot-coated with a standard sample of 1-1 (PE / EA = 10 / 1, phosphomolybdic acid and bromocresol green). New spots with reduced polarity were observed. The reaction solution was quenched with water, separated, and the organic phase was evaporated under reduced pressure. An appropriate amount of silica gel was added for sample mixing and purification (80 g normal phase column, PE / EA, 0-0% 5 min, 0-10% 20 min, 10-10% 10 min, flow rate 40 ml / min) was performed using a plate monitor. The pure product fraction was evaporated to obtain a colorless oily liquid compound 14-1 (2.1 g, 69.8% yield).

[0186] Step 2:

[0187] Trifluoroacetic acid (5 ml) was added to a solution of compound 14-1 (2.1 g) in dichloromethane (20 ml). The mixture was stirred at room temperature for 16 hours. TLC showed that compound 14-1 completely disappeared, and a point with increased polarity was formed. The reaction solution was spin-dried, and saturated aqueous sodium bicarbonate solution (100 ml) was added to quench the excess trifluoroacetic acid. The solution was extracted with ethyl acetate (100 ml × 2). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. An appropriate amount of silica gel and DCM were added to mix the sample and purified (80 g normal phase column, PE / EA, 0-0% 5 min, 0-50% 20 min, 50-50% min, flow rate 50 ml / min) to obtain compound 14-2 (2.1 g, 88.9% yield) as a colorless oil.

[0188] Step 3:

[0189] Compound 14-2 (300 mg) was dissolved in ultra-dry dichloromethane (5 ml) and stirred in an ice bath. Ultra-dry pyridine (79 mg) and triphosgene (60 mg) were added sequentially, and the mixture was stirred in an ice bath for 0.5 h. A small amount of the reaction solution was plated with a standard sample of 14-2 (PE / EA = 1 / 1, phosphomolybdic acid). New spots with reduced polarity were observed. The reaction solution was evaporated under reduced pressure, and the resulting solid was dissolved in ultra-dry dichloromethane (5 ml) and then added dropwise to a mixed solution of 1-(2-hydroxyethyl)-4-methylpiperazine (500 mg) and pyridine (10 ml). After the addition was complete, the reaction solution was stirred at 70°C for three hours. The solution was orange-yellow, and TLC (DCM / MeOH / NH4OH = 10 / 1 / 0.1, phosphomolybdic acid) showed the formation of a new spot. An appropriate amount of silica gel and dichloromethane were added to the sample, and the sample was purified (10 g normal phase column, DCM:DCM / MeOH / NH4OH, 0-0% over 5 min, 0-70% over 20 min, 70-70% over 10 min, flow rate 20 ml / min) to afford Compound 14 (160 mg, 41.6% yield) as a pale yellow oily liquid. The hydrogen spectrum of Compound 14 is shown in Figure 14.

[0190] 1H NMR(400MHz,Chloroform-d)δ5.37(s,4H),5.33(s,4H),4.12(s,4H),4.08(s,2H),3.88(s,4H),2.79(s,2H),2.78–2.66(m,4H),2.54(s,4H ),2.49(s,2H),2.44(s,2H),2.27(s,3H),2.07–2.01(dt,J=9.0Hz,1.0Hz,8H),1.37(s,4H),1.35–1.30(m,20H),1.29(s,4H),0.90(s,6H).

[0191] Example 15

[0192] The synthetic route of compound 15 is as follows:

[0193] Step 1:

[0194] Compound 4-2 (200 mg) was dissolved in ultra-dry dichloromethane (5 ml) and stirred in an ice bath. Ultra-dry pyridine (58 mg) and triphosgene (43 mg) were added sequentially, and the mixture was stirred in an ice bath for 0.5 h. A small amount of the reaction solution was plated with a standard sample of 4-2 (PE / EA = 1 / 1, phosphomolybdic acid). New spots with reduced polarity were observed. The reaction solution was evaporated under reduced pressure, and the resulting solid was dissolved in ultra-dry dichloromethane (5 ml) and added dropwise to a mixed solution of 1-piperidinylpropanol (500 mg) and pyridine (10 ml). After the addition was complete, the reaction solution was stirred at 70°C for three hours. The solution turned red. TLC (DCM / MeOH / NH4OH = 10 / 1 / 0.1, phosphomolybdic acid) showed a new spot below the pyridine. An appropriate amount of silica gel and dichloromethane were added to the sample, and the sample was purified (10 g normal phase column, DCM:DCM / MeOH / NH4OH, 0-0% for 5 min, 0-70% for 20 min, 70-70% for 10 min, flow rate 20 ml / min) to obtain Compound 15 (164 mg, 62.8% yield) as a yellow oily liquid. The hydrogen spectrum of Compound 15 is shown in Figure 15.

[0195] 1H NMR(400MHz,Chloroform-d)δ4.79(s,2H),4.14(s,2H),3.87(s,4H),2.66(s,2H),2.47(s,4H),1.90(s,2H),1.69(d ,J=12.4Hz,4H),1.60–1.52(d,J=12.4Hz,8H),1.46(s,2H),1.37(d,J=0.6Hz,8H),1.35–1.25(m,32H),0.90(s,12H).

[0196] Example 16

[0197] Preparation and characterization of lipid nanoparticles

[0198] Prepare the aqueous phase: dilute mRNA (LUC-mRNA, the nucleotide sequence corresponding to LUC-mRNA is shown in SEQ ID NO: 1 of patent application 202210286081.0) in citric acid-sodium citrate buffer at a final concentration of 0.144 mg / mL.

[0199] Prepare the organic phase: mix the above cationic lipids, DSPC, cholesterol and PEG 2k-DMG was dissolved in ethanol at a total concentration of 10 mg / mL (the component ratio was cationic lipid: DSPC: cholesterol: PEG 2k -DMG=50:10:38.5:1.5).

[0200] Add 3ml of aqueous buffer and 1ml of lipid organic phase to a 15ml centrifuge tube, connect them to the A and B ends of the flow control respectively, install the chip into the microfluidic device, set a certain flow rate ratio, and conduct a preliminary experiment with pure water and pure ethanol. When the pressure and flow rate are stable, add the feed liquid. The feed liquid flows through the chip while observing the sample color at the chip outlet. Discard the first and last 3 to 5 drops of milky white droplets (about 100μL), collect the middle end sample into the EP tube, and then quickly transfer the sample to the dialysis bag. Dialyze in 20mM Tris-HCl buffer for 12-24h. After dialysis, transfer to a 4℃ refrigerator for storage.

[0201] The encapsulation efficiency of the sample was determined using the Ribogreen kit according to the operating instructions. The fluorescence of the sample was measured using a microplate reader at an excitation light of 485 nm and an emission light of 535 nm. The encapsulation efficiency of the sample was calculated based on the fluorescence value of the sample.

[0202] Particle size, PDI, and Zeta potential analysis were performed using a Malvern Zetasizer nano instrument using standard testing methods.

[0203] The test results of the particle size, PDI and encapsulation efficiency of the mRNA-loaded LNPs prepared in this example are shown in Table 1.

[0204] Table 1

[0205] Example 17

[0206] Expression and effect determination of erythropoietin (EPO) mRNA delivered by nanolipid particle composition in mice

[0207] Female Balb / c mice aged 6-8 weeks were injected with EPO-mRNA-lipid nanoparticles at 0.5 mg / kg through the tail vein (the nucleotide sequence corresponding to EPO-mRNA is shown in SEQ ID NO: 2 of patent application 202210286081.0). Five parallel mice were used for each formula, and mouse blood was collected at specific time points (6h and 12h). The basic characteristics of the mRNA used are ARCA cap structure, polyA tail length of 100-120nt, and complete replacement of pseudouracil. The obtained blood was centrifuged at 5000g for 10 minutes at 4°C to separate the serum, and ELISA analysis was performed according to commercially available kits. The test results are detailed in Table 2 and Figure 16.

[0208] Table 2

[0209] The cationic lipid compound control group SM102 of the present invention was purchased from Xiamen Sinobond (M2212000880009), and its structure is shown below:

[0210] The control group T13 was prepared according to CN114773217A (Compound 35), and the structure is shown below:

Claims

1. A cationic lipid compound represented by general formula (I) or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof: in, Ring A is a substituted or unsubstituted 4-10 membered N-containing heterocyclic ring; the N-containing heterocyclic ring contains 0, 1 or 2 heteroatoms selected from N, O or S in addition to the N atom connected to L1; when substituted, the N-containing heterocyclic ring is substituted with 1, 2, 3, 4, 5 or 6 substituents selected from halogen, hydroxyl, cyano, nitro, carboxyl, C1-C5 alkyl or C1-C5 alkoxy; L1 is a C1-C6 straight chain alkylene group; L2 and L3 are the same or different, and are each independently a C1-C12 alkylene group; G1 and G2 are the same or different and are each independently -(C=O)O-, -O(C=O)-, -S(C=O)-, -O(C=S)- or -(C=O)S-; R3 and R4 are the same or different, and are independently C4-C17 straight chain alkyl, C4-C17 straight chain alkenyl or C8-C22 branched non-cyclic alkyl.

2. The cationic lipid compound according to claim 1 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: Ring A is a substituted or unsubstituted 5-membered, 6-membered or 7-membered N-containing heterocycle; preferably, Ring A is a substituted or unsubstituted 5-membered, 6-membered or 7-membered N-containing heterocycle, and the heteroatoms in the N-containing heterocycle are all N atoms.

3. The cationic lipid compound according to claim 1 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: L1 is a C2-C4 straight chain alkylene group.

4. The cationic lipid compound according to claim 1 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: At least one of L2 and L3 is a C1-C6 alkylene group.

5. The cationic lipid compound according to claim 4 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: L2 and L3 are each independently a C1-C6 alkylene group.

6. The cationic lipid compound according to claim 1 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: Ring A is methylpiperazinyl, hydropyrrolyl, piperidinyl or cycloheximino.

7. The cationic lipid compound according to claim 1 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: G1 and G2 are -(C=O)O-; preferably, G1 and G2 are connected to R3 and R4 respectively through the -O- therein.

8. The cationic lipid compound according to claim 1 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: When either R3 or R4 is a C8-C22 branched non-cyclic alkyl group, R3 and R4 are independently: R5 and R6 are the same or different and are each independently a C3-C11 straight chain alkyl group.

9. The cationic lipid compound according to claim 1 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: R3 and R4 are independently:

10. The cationic lipid compound or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof according to claim 1, wherein: The cationic lipid compound is selected from one or more of the following structures:

11. A liposomal formulation comprising the cationic lipid compound of any one of claims 1 to 10 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof and a prophylactic or therapeutic nucleic acid.

12. The liposome preparation according to claim 11, wherein The molar ratio of the cationic lipid compound to the nucleic acid is 20:1 to 1:

1.

13. The liposome preparation according to claim 11, wherein The average particle size of the liposome preparation is 50 nm to 200 nm.

14. The liposome preparation according to claim 11, wherein The liposomal formulation may also contain one or more other lipid components including neutral lipids, steroids, and polymer-conjugated lipids.

15. The liposome preparation according to claim 14, wherein The steroid is β-sitosterol, soja sterol, ergosterol, cholesterol or dihydrocholesterol; cholesterol is preferred.

16. The liposome preparation according to claim 14, wherein The molar ratio of the steroid to the cationic lipid compound is (0.5-1):

1.

17. The liposome preparation according to claim 14, wherein The polymer in the polymer-conjugated lipid is polyethylene glycol.

18. The liposome preparation according to claim 14, wherein The molar ratio of the cationic lipid compound to the polymer-conjugated lipid is 100:1 to 20:

1.

19. The liposome preparation according to claim 17, wherein The polyethylene glycol-conjugated lipid is PEG2k-DSG, PEG2k-DMG, PEG2k-DPPE, PEG2k-DSPE, PEG2k-cer, PEG2k-DMG or ALC-0159; preferably PEG2k-DMG.

20. The liposome preparation according to claim 14, wherein The neutral lipid is selected from one or more combinations of 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-3-phosphoethanolamine, 1,2-dipalmitoyl-sn-sodium phosphoglycerol, sphingomyelin, ceramide and sterol.

21. The liposome preparation according to claim 14, wherein The molar ratio of the cationic lipid compound to the neutral lipid is 2:1 to 8:

1.

22. The liposome preparation according to claim 11, wherein The nucleic acid is selected from mRNA, siRNA, miRNA or antisense oligonucleotide; the nucleic acid is preferably mRNA.

23. Use of the cationic lipid compound of any one of claims 1 to 10 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof or the liposome formulation of any one of claims 11 to 22 in the preparation of a medicament for inducing protein expression in a subject.

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