Detailed Description
The present invention will be described in detail with reference to the accompanying drawings and specific embodiments.
EXAMPLE 1 Synthesis of lipid Compound L-1
Compound 1-1 (10 mmol) was dissolved in 10ml DCM in a round bottom flask, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI, 3.35 g), 4-dimethylaminopyridine (DMAP, 1.64 g) and compound 1-2 (11 mmol) were added to the reaction system in sequence, reacted for 2 hours at room temperature, purified by silica gel column and compound 1-3 was obtained by rotary evaporation.
In a round bottom flask, compound 1-4 (10 mmol) was dissolved in 10ml DCM, EDCI (3.35 g), DMAP (1.64 g) and Compound 1-5 (11 mmol) were added sequentially to the reaction system, reacted for 2 hours at room temperature, purified by silica gel column and Compound 1-6 was obtained by rotary evaporation.
Compounds 1-7 (12 mmol), and compounds 1-6 (10 mmol) were added to a solution of acetonitrile (100 mL) and ground potassium carbonate (4.5 g) was added. The mixture was stirred at 70 ℃ for 3 hours, after which time the product was filtered to remove potassium carbonate, purified by a silica gel column and the solvent was removed by a rotary evaporator to give compounds 1-8.
Compounds 1 to 8 (1 mmol) were dissolved in acetonitrile (10 ml) and stirred at room temperature. Then sequentially weighing NaI (170 mg), K2 CO3 (470 mg) and Compound 1-3 (1.24 mmol) were added in portions to the above reaction system, heated under reflux at 85℃under stirring for 2 hours, purified by a silica gel column, and then the solvent was removed by a rotary evaporator to give lipid Compound L-1 (600 mg, yield 76.9%).1 H NMR(400MHz,Chloroform-d)δ8.45-8.59(m,1H),7.66(m,1H),7.16-7.22(m,2H),4.45-4.49(m,1H),4.10-1.15(m,2H),2.69-2.72(m,2H),2.36-2.40(m,6H),2.30-2.34(m,4H),1.60-1.66(m,8H),1.47-1.49(m,6H),1.26-1.33(s,50H),0.89(d,9H)。
EXAMPLE 2 Synthesis of lipid Compound L-2
Compound 2-1 (12 mmol), and compound 1-6 (10 mmol) were added to a solution of acetonitrile (100 mL), and ground potassium carbonate (4.5 g) was added. The mixture was stirred at 70 ℃ for 3 hours, after which time the product was filtered to remove potassium carbonate, purified by a silica gel column and the solvent was removed by a rotary evaporator to give compound 2-2.
Compound 2-2 (1 mmol) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then sequentially weighing NaI (170 mg), K2 CO3 (470 mg) and the compound 1-3 (1.24 mmol) were added in portions to the above reaction system, heated under reflux at 85℃under stirring for 2 hours, purified by a silica gel column, and then the solvent was removed by a rotary evaporator to give the lipid compound L-2 (550 mg, yield 70.1%).1 H NMR(400MHz,Chloroform-d)δ8.41-8.44(m,2H),7.46-7.50(m,1H),7.16-7.18(m,1H),4.45-4.49(m,1H),4.10-1.15(m,2H),2.69-2.72(m,2H),2.36-2.40(m,6H),2.30-2.34(m,4H),1.60-1.66(m,8H),1.47-1.49(m,6H),1.26-1.33(s,50H),0.89(d,9H)。
EXAMPLE 3 Synthesis of lipid Compound L-3
Compound 3-1 (12 mmol), and compound 1-6 (10 mmol) were added to a solution of acetonitrile (100 mL), and ground potassium carbonate (4.5 g) was added. The mixture was stirred at 70 ℃ for 3 hours, after which time the product was filtered to remove potassium carbonate, purified by a silica gel column and the solvent was removed by a rotary evaporator to give compound 3-2.
Compound 3-2 (1 mmol) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then sequentially weighing NaI (170 mg), K2 CO3 (470 mg) and Compound 1-3 (1.24 mmol) were added in portions to the above reaction system, heated under reflux at 85℃under stirring for 2 hours, purified by a silica gel column, and then the solvent was removed by a rotary evaporator to give lipid Compound L-3 (500 mg, yield 63.7%).1 H NMR(400MHz,Chloroform-d)δ8.42-8.46(d,2H),7.06-7.08(d,2H),4.45-4.49(m,1H),4.10-1.15(m,2H),2.69-2.72(m,2H),2.36-2.40(m,6H),2.30-2.34(m,4H),1.60-1.66(m,8H),1.47-1.49(m,6H),1.26-1.33(s,50H),0.89(d,9H)。
EXAMPLE 4 Synthesis of lipid Compound L-4
Compound 4-1 (12 mmol), and compound 1-6 (10 mmol) were added to a solution of acetonitrile (100 mL), and ground potassium carbonate (4.5 g) was added. The mixture was stirred at 70 ℃ for 3 hours, after which time the product was filtered to remove potassium carbonate, purified by a silica gel column and the solvent was removed by a rotary evaporator to give compound 4-2.
Compound 4-2 (1 mmol) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then sequentially weighing NaI (170 mg), K2 CO3 (470 mg) and the compounds 1 to 3 (1.24 mmol) were added in portions to the above-mentioned reaction system at 85 ℃CAfter purification by silica gel column under reflux with heating and stirring for 2 hours, the solvent was removed by rotary evaporator to give lipid compound L-4 (516 mg, yield 65.8%).1 H NMR(400MHz,Chloroform-d)δ7.21-7.25(s,2H),4.45-4.49(m,1H),4.10-1.15(m,2H),3.40-3.42(m,2H),2.66-2.70(s,6H),2.40-2.42(m,6H),2.30-2.34(m,4H),1.60-1.66(m,8H),1.47-1.49(m,6H),1.26-1.33(s,50H),0.89(d,9H)。
EXAMPLE 5 Synthesis of lipid Compound L-5
Compound 5-1 (12 mmol), and compound 1-6 (10 mmol) were added to a solution of acetonitrile (100 mL), and ground potassium carbonate (4.5 g) was added. The mixture was stirred at 70 ℃ for 3 hours, after which time the product was filtered to remove potassium carbonate, purified by a silica gel column and the solvent was removed by a rotary evaporator to give compound 5-2.
Compound 5-2 (1 mmol) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then sequentially weighing NaI (170 mg), K2 CO3 (470 mg) and the compounds 1 to 3 (1.24 mmol) were added in portions to the above reaction system, heated under reflux at 85℃under stirring for 2 hours, purified by a silica gel column, and then the solvent was removed by a rotary evaporator to give the lipid compound L-5 (534 mg, yield 64.0%).1 H NMR(400MHz,Chloroform-d)δ8.08-8.12(d,1H),7.99-8.02(d,1H),7.79-7.83(t,1H),7.62-7.66(t,1H),7.47-7.51(t,1H),7.28-7.32(d,1H),4.45-4.49(m,1H),4.10-1.15(m,2H),3.40-3.42(m,2H),2.66-2.70(s,6H),2.40-2.42(m,6H),2.30-2.34(m,4H),1.60-1.66(m,8H),1.47-1.49(m,6H),1.26-1.33(s,50H),0.89(d,9H)。
EXAMPLE 6 Synthesis of lipid Compound L-6
Compound 6-1 (12 mmol), and compound 1-6 (10 mmol) were added to a solution of acetonitrile (100 mL), and ground potassium carbonate (4.5 g) was added. The mixture was stirred at 70℃for 3 hours, after which time the product was filtered to remove potassium carbonate, purified by column chromatography on silica gel and the solvent was removed by rotary evaporator to give compound 6-2.
Compound 6-2 (1 mmol) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then sequentially weighing NaI (170 mg), K2 CO3 (470 mg) and the compounds 1 to 3 (1.24 mmol) were added in portions to the above reaction system, heated under reflux at 85℃under stirring for 2 hours, purified by a silica gel column, and then the solvent was removed by a rotary evaporator to give the lipid compound L-6 (610 mg, yield 80%).1 H NMR(400MHz,Chloroform-d)δ8.54-8.56(d,2H),7.21-7.23(d,2H),4.45-4.49(m,1H),4.10-1.15(m,2H),3.43-3.47(m,2H),3.00-3.02(m,4H),2.69-2.71(t,2H),2.30-2.34(m,4H),1.60-1.66(m,8H),1.47-1.49(m,6H),1.26-1.33(s,50H),0.89(d,9H)。
EXAMPLE 7 Synthesis of lipid Compound L-7
Compound 7-1 (12 mmol), and compound 1-6 (10 mmol) were added to a solution of acetonitrile (100 mL), and ground potassium carbonate (4.5 g) was added. The mixture was stirred at 70℃for 3 hours, after which time the product was filtered to remove potassium carbonate, purified by column chromatography on silica gel and the solvent was removed by rotary evaporator to give compound 7-2.
Compound 7-2 (1 mmol) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then sequentially weighing NaI (170 mg), K2 CO3 (470 mg) and Compound 1-3 (1.24 mmol) were added in portions to the above reaction system, heated under reflux at 85℃under stirring for 2 hours, purified by a silica gel column, and then the solvent was removed by a rotary evaporator to give lipid Compound L-7 (555 mg, yield 71.9%).1 H NMR(400MHz,Chloroform-d)δ8.54-8.56(d,2H),7.21-7.23(d,2H),4.45-4.49(m,1H),4.10-1.15(m,2H),2.75-2.79(m,2H),2.41-2.43(m,6H),2.30-2.34(m,4H),1.60-1.66(m,8H),1.47-1.49(m,6H),1.26-1.33(s,50H),1.21-1.23(m,3H),0.89(d,9H)。
EXAMPLE 8 Synthesis of lipid Compound L-8
Compound 8-1 (12 mmol), and compound 1-6 (10 mmol) were added to a solution of acetonitrile (100 mL), and ground potassium carbonate (4.5 g) was added. The mixture was stirred at 70℃for 3 hours, after which time the product was filtered to remove potassium carbonate, purified by column chromatography on silica gel and the solvent was removed by rotary evaporator to give compound 8-2.
Compound 8-2 (1 mmol) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then sequentially weighing NaI (170 mg), K2 CO3 (470 mg) and the compound 1-3 (1.24 mmol) were added in portions to the above reaction system, heated under reflux at 85℃under stirring for 2 hours, purified by a silica gel column, and then the solvent was removed by a rotary evaporator to give the lipid compound L-8 (480 mg, yield 59.0%).1 H NMR(400MHz,Chloroform-d)δ8.54-8.56(d,2H),7.21-7.23(d,2H),4.45-4.49(m,1H),4.10-1.15(m,2H),2.75-2.79(m,2H),2.41-2.43(m,6H),2.30-2.34(m,4H),1.60-1.66(m,8H),1.47-1.49(m,6H),1.26-1.33(s,50H),0.88-0.89(d,18H)。
EXAMPLE 9 Synthesis of lipid Compound L-9
Compound 9-1 (12 mmol), and compound 1-6 (10 mmol) were added to a solution of acetonitrile (100 mL), and ground potassium carbonate (4.5 g) was added. The mixture was stirred at 70℃for 3 hours, after which time the product was filtered to remove potassium carbonate, purified by column chromatography on silica gel and the solvent was removed by rotary evaporator to give compound 9-2.
Compound 9-2 (1 mmol) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then sequentially weighing NaI (170 mg), K2 CO3 (470 mg) and the compounds 1 to 3 (1.24 mmol) were added in portions to the above reaction system, heated under reflux at 85℃under stirring for 2 hours, purified by a silica gel column, and then the solvent was removed by a rotary evaporator to give lipid compound L-9 (510 mg, yield 66%).1 H NMR(400MHz,Chloroform-d)δ8.54-8.56(d,2H),7.21-7.23(d,2H),4.45-4.49(m,1H),4.10-1.15(m,2H),3.39-3.43(m,2H),2.99-3.03(m,4H),2.44-2.48(m,2H),2.30-2.34(m,4H),1.93-1.96(m,2H),1.60-1.66(m,6H),1.26-1.49(m,54H),0.88-0.89(d,9H)。
EXAMPLE 10 Synthesis of lipid Compound L-10
Compound 10-1 (12 mmol), and compound 1-6 (10 mmol) were added to a solution of acetonitrile (100 mL), and ground potassium carbonate (4.5 g) was added. The mixture was stirred at 70℃for 3 hours, after which time the product was filtered to remove potassium carbonate, purified by column chromatography on silica gel and the solvent was removed by rotary evaporator to give compound 10-2.
Compound 10-2 (1 mmol) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then sequentially weighing NaI (170 mg), K2 CO3 (470 mg) and the compounds 1 to 3 (1.24 mmol) were added in portions to the above reaction system, heated under reflux at 85℃under stirring for 2 hours, purified by a silica gel column, and then the solvent was removed by a rotary evaporator to give lipid compound L-10 (560 mg, yield 71.4%).1 H NMR(400MHz,Chloroform-d)δ8.54-8.56(d,2H),7.21-7.23(d,2H),4.45-4.49(m,1H),4.10-1.15(m,2H),2.99-3.03(m,4H),2.53-2.57(m,1H),2.41-2.45(m,2H),2.30-2.34(m,4H),1.66-1.69(m,8H),1.26-1.49(m,54H),0.88-0.89(d,9H)。
EXAMPLE 11 Synthesis of lipid Compound L-11
Compound 11-1 (12 mmol), and compound 1-6 (10 mmol) were added to a solution of acetonitrile (100 mL), and ground potassium carbonate (4.5 g) was added. The mixture was stirred at 70℃for 3 hours, after which time the product was filtered to remove potassium carbonate, purified by column chromatography on silica gel and the solvent was removed by rotary evaporator to give compound 11-2.
Compound 11-2 (1 mmol) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then sequentially weighing NaI (170 mg), K2 CO3 (470 mg) and the compounds 1 to 3 (1.24 mmol) were added in portions to the above reaction system and heated back at 85 ℃After the mixture was stirred for 2 hours and purified by a silica gel column, the solvent was removed by a rotary evaporator to give lipid compound L-11 (540 mg, yield 72.6%).1 H NMR(400MHz,Chloroform-d)δ8.54-8.56(d,2H),7.21-7.23(d,2H),4.43-4.45(m,2H),4.10-1.15(m,2H),2.46-2.48(m,4H),2.30-2.34(m,4H),1.60-1.66(m,6H),1.26-1.43(m,54H),0.88-0.89(d,9H)。
EXAMPLE 12 Synthesis of lipid Compound L-12
Compound 12-1 (12 mmol), and compound 1-6 (10 mmol) were added to a solution of acetonitrile (100 mL), and ground potassium carbonate (4.5 g) was added. The mixture was stirred at 70℃for 3 hours, after which time the product was filtered to remove potassium carbonate, purified by a silica gel column and the solvent was removed by a rotary evaporator to give compound 12-1.
Compound 12-1 (1 mmol) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then sequentially weighing NaI (170 mg), K2 CO3 (470 mg) and the compounds 1 to 3 (1.24 mmol) were added in portions to the above reaction system, heated under reflux at 85℃under stirring for 2 hours, purified by a silica gel column, and then the solvent was removed by a rotary evaporator to give the lipid compound L-12 (500 mg, yield 62.5%).1 H NMR(400MHz,Chloroform-d)δ8.54-8.56(d,2H),7.21-7.23(d,2H),4.43-4.45(m,1H),4.10-1.15(m,2H),2.52-2.72(m,3H),1.97-1.99(m,1H),1.60-1.66(m,6H),1.26-1.43(m,54H),0.88-0.89(d,9H)。
EXAMPLE 13 Synthesis of lipid Compound L-13
Compound 13-1 (10 mmol) was dissolved in 10ml DCM in a round bottom flask, EDCI (3.35 g), DMAP (1.64 g) and compound 13-2 (11 mmol) were added sequentially to the reaction system, reacted for 2 hours at room temperature, purified by silica gel column and compound 13-3 was obtained by rotary evaporation.
Compound 6-1 (12 mmol), and compound 13-3 (10 mmol) were added to a solution of acetonitrile (100 mL), and ground potassium carbonate (4.5 g) was added. The mixture was stirred at 70℃for 3 hours, after which time the product was filtered to remove potassium carbonate, purified by column chromatography on silica gel and the solvent was removed by rotary evaporator to give compound 13-4.
Compound 13-4 (1 mmol) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then sequentially weighing NaI (170 mg), K2 CO3 (470 mg) and the compounds 1 to 3 (1.24 mmol) were added in portions to the above reaction system, heated under reflux at 85℃under stirring for 2 hours, purified by a silica gel column, and then the solvent was removed by a rotary evaporator to give the lipid compound L-13 (600 mg, yield 75.0%).1 H NMR(400MHz,Chloroform-d)δ8.42-8.46(d,2H),7.08-7.10(d,2H),4.45-4.49(m,1H),4.10-1.15(m,2H),3.43-3.47(m,2H),2.69-2.72(m,2H),2.36-2.40(m,4H),2.30-2.34(m,4H),1.58-1.6(m,6H),1.47-1.49(m,6H),1.26-1.33(m,54H),0.89(d,9H)。
EXAMPLE 14 Synthesis of lipid Compound L-14
Compound 1-4 (10 mmol) was dissolved in 10ml DCM in a round bottom flask, EDCI (3.35 g), DMAP (1.64 g) and compound 14-1 (11 mmol) were added sequentially to the reaction system, reacted for 2 hours at room temperature, purified by silica gel column and compound 14-2 was obtained by rotary evaporation.
Compound 6-1 (12 mmol) and compound 14-2 (10 mmol) were added to a solution of acetonitrile (100 mL) and ground potassium carbonate (4.5 g) was added. The mixture was stirred at 70℃for 3 hours, after which time the product was filtered to remove potassium carbonate, purified by column chromatography on silica gel and the solvent was removed by rotary evaporator to give compound 14-3.
Compound 14-3 (1 mmol) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then sequentially weighing NaI (170 mg), K2 CO3 (470 mg) and the compounds 1 to 3 (1.24 mmol) were added in portions to the above reaction system, heated under reflux at 85℃under stirring for 2 hours, purified by a silica gel column, and then the solvent was removed by a rotary evaporator to give lipid compound L-14 (575 mg, yield 73.3%).1 H NMR(400MHz,Chloroform-d)δ8.42-8.46(d,2H),7.08-7.10(d,2H),4.45-4.49(m,1H),4.10-1.15(m,2H),3.43-3.47(m,2H),2.69-2.72(m,2H),2.36-2.40(m,4H),2.30-2.34(m,4H),1.58-1.6(m,6H),1.47-1.49(m,6H),1.26-1.33(m,52H),0.89(d,9H)。
EXAMPLE 15 Synthesis of lipid Compound L-15
Compound 1-1 (10 mmol) was dissolved in 10ml DCM in a round bottom flask, EDCI (3.35 g), DMAP (1.64 g) and compound 1-5 (11 mmol) were added sequentially to the reaction system, reacted for 2 hours at room temperature, purified by silica gel column and compound 15-1 was obtained by rotary evaporation.
Compound 6-1 (12 mmol) and compound 15-1 (10 mmol) were added to a solution of acetonitrile (100 mL) and ground potassium carbonate (4.5 g) was added. The mixture was stirred at 70℃for 3 hours, after which time the product was filtered to remove potassium carbonate, purified by column chromatography on silica gel and the solvent was removed by rotary evaporator to give compound 15-2.
Compound 15-2 (1 mmol) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then sequentially weighing NaI (170 mg), K2 CO3 (470 mg) and the compounds 1 to 3 (1.24 mmol) were added in portions to the above reaction system, heated under reflux at 85℃under stirring for 2 hours, purified by a silica gel column, and then the solvent was removed by a rotary evaporator to give the lipid compound L-15 (600 mg, yield 75.0%).1 H NMR(400MHz,Chloroform-d)δ8.42-8.46(d,2H),7.08-7.10(d,2H),4.45-4.49(m,1H),4.10-1.15(m,2H),3.43-3.47(m,2H),2.69-2.72(m,2H),2.36-2.40(m,4H),2.30-2.34(m,4H),1.58-1.6(m,6H),1.47-1.49(m,6H),1.26-1.33(m,50H),0.89(d,9H)。
EXAMPLE 16 Synthesis of lipid Compound L-16
Compound 16-1 (10 mmol) was dissolved in 10ml DCM in a round bottom flask, EDCI (3.35 g), DMAP (1.64 g) and compound 16-2 (11 mmol) were added sequentially to the reaction system, reacted for 2 hours at room temperature, purified by silica gel column and compound 16-3 was obtained by rotary evaporation.
Compound 6-1 (12 mmol), and compound 16-3 (10 mmol) were added to a solution of acetonitrile (100 mL), and ground potassium carbonate (4.5 g) was added. The mixture was stirred at 70℃for 3 hours, after which time the product was filtered to remove potassium carbonate, purified by column chromatography on silica gel and the solvent was removed by rotary evaporator to give compound 16-4.
Compound 16-4 (1 mmol) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then sequentially weighing NaI (170 mg), K2 CO3 (470 mg) and the compounds 1 to 3 (1.24 mmol) were added in portions to the above reaction system, heated under reflux at 85℃under stirring for 2 hours, purified by a silica gel column, and then the solvent was removed by a rotary evaporator to give the lipid compound L-16 (566 mg, yield 72.5%).1 H NMR(400MHz,Chloroform-d)δ8.42-8.46(d,2H),7.08-7.10(d,2H),4.45-4.49(m,1H),4.10-1.15(m,2H),3.43-3.47(m,2H),2.69-2.72(m,2H),2.36-2.40(m,4H),2.30-2.34(m,4H),1.58-1.6(m,6H),1.47-1.49(m,6H),1.26-1.33(m,46H),0.89(d,9H)。
EXAMPLE 17 Synthesis of lipid Compound L-17
Compound 13-1 (10 mmol) was dissolved in 10ml DCM in a round bottom flask, EDCI (3.35 g), DMAP (1.64 g) and compound 17-1 (11 mmol) were added sequentially to the reaction system, reacted for 2 hours at room temperature, purified by silica gel column and compound 17-2 was obtained by rotary evaporation.
Compound 15-2 (1 mmol) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then sequentially weighing NaI (170 mg), K2 CO3 (470 mg) and Compound 17-2 (1.24 mmol) were added in portions to the above reaction system, heated under reflux at 85℃under stirring for 2 hours, purified by a silica gel column, and then the solvent was removed by a rotary evaporator to give lipid Compound L-17 (520 mg, yield 67.4%).1 H NMR(400MHz,Chloroform-d)δ8.42-8.46(d,2H),7.08-7.10(d,2H),4.45-4.49(m,1H),4.10-1.15(m,2H),3.43-3.47(m,2H),2.69-2.72(m,2H),2.36-2.40(m,4H),2.30-2.34(m,4H),1.58-1.6(m,6H),1.47-1.49(m,6H),1.26-1.33(m,50H),0.89(d,9H)。
EXAMPLE 18 Synthesis of lipid Compound L-18
Compound 16-1 (10 mmol) was dissolved in 10ml DCM in a round bottom flask, EDCI (3.35 g), DMAP (1.64 g) and compound 18-1 (11 mmol) were added sequentially to the reaction system, reacted for 2 hours at room temperature, purified by silica gel column and compound 18-2 was obtained by rotary evaporation.
Compound 15-2 (1 mmol) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then sequentially weighing NaI (170 mg), K2 CO3 (470 mg) and Compound 18-2 (1.24 mmol) were added in portions to the above reaction system, and after heating and stirring at 85℃for 2 hours, purification by silica gel column, the solvent was removed by rotary evaporator to give lipid Compound L-18 (490 mg, yield 63.5%).1 H NMR(400MHz,Chloroform-d)δ8.42-8.46(d,2H),7.08-7.10(d,2H),4.45-4.49(m,1H),4.10-1.15(m,2H),3.43-3.47(m,2H),2.69-2.72(m,2H),2.36-2.40(m,4H),2.30-2.34(m,4H),1.58-1.6(m,6H),1.47-1.49(m,6H),1.26-1.33(m,50H),0.89(d,9H)。
EXAMPLE 19 Synthesis of lipid Compound L-19
In a round bottom flask, compound 19-1 (10 mmol) was dissolved in 10ml DCM, EDCI (3.35 g), DMAP (1.64 g) and compound 19-2 (11 mmol) were added sequentially to the reaction system, reacted at room temperature for 2 hours, purified by silica gel column and compound 19-3 was obtained by rotary evaporation.
Compound 15-2 (1 mmol) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then sequentially weighing NaI (170 mg), K2 CO3 (470 mg) and Compound 19-2 (1.24 mmol) were added in portions to the above reaction system, heated under reflux at 85deg.C under stirring for 2 hours, purified by silica gel column, and the solvent was removed by rotary evaporator to give lipid Compound L-19 (500 mg, collected)Yield 64.5%).1 H NMR(400MHz,Chloroform-d)δ8.42-8.46(d,2H),7.08-7.10(d,2H),4.45-4.49(m,1H),4.10-1.15(m,2H),3.43-3.47(m,2H),2.69-2.72(m,2H),2.36-2.40(m,4H),2.30-2.34(m,4H),1.58-1.6(m,6H),1.47-1.49(m,6H),1.26-1.33(m,50H),0.89(d,9H)。
EXAMPLE 20 Synthesis of lipid Compound L-20
Compound 15-2 (1 mmol) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then sequentially weighing NaI (170 mg), K2 CO3 (470 mg) and Compound 13-3 (1.24 mmol) were added in portions to the above reaction system, heated under reflux at 85℃under stirring for 2 hours, purified by a silica gel column, and then the solvent was removed by a rotary evaporator to give lipid Compound L-20 (510 mg, yield 55.9%).1 H NMR(400MHz,Chloroform-d)δ8.42-8.46(d,2H),7.08-7.10(d,2H),4.45-4.49(m,2H),3.43-3.47(m,2H),2.69-2.72(m,2H),2.36-2.40(m,4H),2.30-2.34(m,4H),1.58-1.68(m,4H),1.47-1.49(m,8H),1.26-1.33(m,68H),0.89(d,9H)。
EXAMPLE 21 Synthesis of lipid Compound L-21
Compound 15-2 (1 mmol) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then sequentially weighing NaI (170 mg), K2 CO3 (470 mg) and Compound 15-1 (1.24 mmol) were added in portions to the above reaction system, and after heating and stirring at 85℃for 2 hours, purification by silica gel column, the solvent was removed by rotary evaporator to give lipid Compound L-21 (523 mg, yield 57.4%).1 H NMR(400MHz,Chloroform-d)δ8.42-8.46(d,2H),7.08-7.10(d,2H),4.45-4.49(m,2H),3.43-3.47(m,2H),2.69-2.72(m,2H),2.36-2.40(m,4H),2.30-2.34(m,4H),1.58-1.68(m,4H),1.47-1.49(m,8H),1.26-1.33(m,56H),0.89(d,9H)。
EXAMPLE 22 Synthesis of lipid Compound L-22
Compound 15-2 (1 mmol) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then sequentially weighing NaI (170 mg), K2 CO3 (470 mg) and Compound 16-3 (1.24 mmol) were added in portions to the above reaction system, heated under reflux at 85℃under stirring for 2 hours, purified by a silica gel column, and then the solvent was removed by a rotary evaporator to give lipid Compound L-21 (545 mg, yield 63.7%).1 H NMR(400MHz,Chloroform-d)δ8.42-8.46(d,2H),7.08-7.10(d,2H),4.45-4.49(m,2H),3.43-3.47(m,2H),2.69-2.72(m,2H),2.36-2.40(m,4H),2.30-2.34(m,4H),1.58-1.68(m,4H),1.47-1.49(m,8H),1.26-1.33(m,56H),0.89(d,9H)。
EXAMPLE 23 Synthesis of lipid Compound L-23
Compound 6-1 (12 mmol), and compound 1-3 (10 mmol) were added to a solution of acetonitrile (100 mL), and ground potassium carbonate (4.5 g) was added. The mixture was stirred at 70℃for 3 hours, after which time the product was filtered to remove potassium carbonate, purified by a silica gel column and the solvent was removed by a rotary evaporator to give compound 23-1.
Compound 23-1 (1 mmol) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then sequentially weighing NaI (170 mg), K2 CO3 (470 mg) and the compounds 1 to 3 (1.24 mmol) were added in portions to the above reaction system, heated under reflux at 85℃under stirring for 2 hours, purified by a silica gel column, and then the solvent was removed by a rotary evaporator to give the lipid compound L-24 (450 mg, yield 69.7%).1 H NMR(400MHz,Chloroform-d)δ8.42-8.46(d,2H),7.08-7.10(d,2H),4.13-4.15(m,4H),3.43-3.47(m,2H),2.69-2.72(m,2H),2.33-2.38(m,8H),1.60-1.66(m,8H),1.47-1.49(m,4H),1.26-1.33(m,34H),0.89(d,6H)。
EXAMPLE 24 Synthesis of lipid Compound L-24
Compound 23-1 (1 mmol) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then sequentially weighing NaI (170 mg), K2 CO3 (470 mg) and Compound 18-2 (1.24 mmol) were added in portions to the above reaction system, and after heating and stirring at 85℃for 2 hours, purification by silica gel column, the solvent was removed by rotary evaporator to give lipid Compound L-24 (450 mg, yield 69.7%).1 H NMR(400MHz,Chloroform-d)δ8.42-8.46(d,2H),7.08-7.10(d,2H),4.13-4.15(m,4H),3.43-3.47(m,2H),2.69-2.72(m,2H),2.33-2.38(m,8H),1.60-1.66(m,8H),1.47-1.49(m,4H),1.26-1.33(m,34H),0.89(d,6H)。
EXAMPLE 25 Synthesis of lipid Compound L-25
Compound 23-1 (1 mmol) was dissolved in acetonitrile (10 ml) and stirred at room temperature. Then sequentially weighing NaI (170 mg), K2 CO3 (470 mg) and compound 17-2 (1.24 mmol) were added in portions to the above reaction system, heated under reflux at 85℃under stirring for 2 hours, purified by a silica gel column, and then the solvent was removed by a rotary evaporator to give lipid compound L-25 (430 mg, yield 66.6%).1 H NMR(400MHz,Chloroform-d)δ8.42-8.46(d,2H),7.08-7.10(d,2H),4.13-4.15(m,4H),3.43-3.47(m,2H),2.69-2.72(m,2H),2.33-2.38(m,8H),1.60-1.66(m,8H),1.47-1.49(m,4H),1.26-1.33(m,34H),0.89(d,6H)。
EXAMPLE 26 Synthesis of lipid Compound L-26
1mmol of compound 3-1,2.4mmol of compound 26-1 were taken and put into a polytetrafluoroethylene-lined glass screw cap vial, and after adding 2ml of ACN solvent and mixing uniformly, they were subjected to hydrothermal reaction at 70℃for 48 hours. Purifying with silica gel column, and removing solvent with rotary evaporator to obtain lipid compound L-26 (510 mg, 75.6% yield).1 H NMR(400MHz,Chloroform-d)δ8.42-8.46(m,2H),7.08-7.10(m,2H),3.44-3.46(m,4H),2.37-2.43(m,6H),2.14-2.15(s,2H),1.60-1.66(m,2H),1.38-1.41(m,4H),1.26-1.28(m,44H),0.89(d,6H)。
EXAMPLE 27 Synthesis of lipid Compound L-27
1mmol of compound 3-1,2.4mmol of compound 27-1 were taken and put into a polytetrafluoroethylene-lined glass screw cap vial, and after adding 2ml of ACN solvent and mixing uniformly, they were subjected to hydrothermal reaction at 70℃for 48 hours. After purification on a silica gel column, the solvent was removed by rotary evaporator to give lipid compound L-27 (412 mg, yield 65.4%).1 H NMR(400MHz,Chloroform-d)δ8.42-8.46(m,2H),7.08-7.10(m,2H),3.44-3.46(m,4H),2.37-2.43(m,6H),2.14-2.15(s,2H),1.60-1.66(m,2H),1.38-1.41(m,4H),1.26-1.28(m,50H),0.89(d,6H)。
EXAMPLE 28 Synthesis of lipid Compound L-28
1mmol of compound 3-1,2.4mmol of compound 28-1 were taken and put into a polytetrafluoroethylene-lined glass screw cap vial, and after adding 2ml of ACN solvent and mixing uniformly, they were subjected to hydrothermal reaction at 70℃for 48 hours. After purification on a silica gel column, the solvent was removed by rotary evaporator to give lipid compound L-28 (412 mg, yield 60%).1 H NMR(400MHz,Chloroform-d)δ8.42-8.46(m,2H),7.08-7.10(m,2H),3.44-3.46(m,4H),2.37-2.43(m,6H),2.14-2.15(s,2H),1.60-1.66(m,2H),1.38-1.41(m,4H),1.26-1.28(m,58H),0.89(d,6H)。
EXAMPLE 29 Synthesis of lipid Compound L-29
1mmol of compound 3-1,2.4mmol of compound 29-1 were taken, added to a polytetrafluoroethylene-lined glass screw cap vial, and after adding 2ml of ACN solvent and mixing uniformly, the mixture was subjected to hydrothermal reaction at 70℃for 48 hours. After purification on a silica gel column, the solvent was removed by rotary evaporator to give lipid compound L-29 (464 mg, yield 65%).1 H NMR(400MHz,Chloroform-d)δ8.42-8.46(m,2H),7.08-7.10(m,2H),3.44-3.46(m,4H),2.37-2.43(m,6H),2.14-2.15(s,2H),1.60-1.66(m,2H),1.38-1.41(m,4H),1.26-1.28(m,62H),0.89(d,6H)。
EXAMPLE 30 Synthesis of lipid Compound L-30
1mmol of compound 1-7 and 2.4mmol of compound 28-1 are taken, added into a polytetrafluoroethylene-lined glass spiral cover small bottle, and after being added with 2ml of ACN solvent and uniformly mixed, the mixture is subjected to hydrothermal reaction at 70 ℃ for 48 hours. After purification on a silica gel column, the solvent was removed by rotary evaporator to give lipid compound L-30 (510 mg, yield 74.2%).1 H NMR(400MHz,Chloroform-d)δ8.42-8.46(m,1H),7.65-7.68(m,1H),7.16-7.22(m,2H),3.44-3.46(m,2H),2.69-2.72(m,2H),2.37-2.43(m,6H),2.14-2.15(s,2H),1.60-1.66(m,2H),1.38-1.41(m,4H),1.26-1.28(m,58H),0.89(d,6H)。
EXAMPLE 31 Synthesis of lipid Compound L-31
1mmol of compound 2-1 and 2.4mmol of compound 28-1 are taken, added into a polytetrafluoroethylene-lined glass spiral cover small bottle, and after being added with 2ml of ACN solvent and uniformly mixed, the mixture is subjected to hydrothermal reaction at 70 ℃ for 48 hours. After purification on a silica gel column, the solvent was removed by rotary evaporator to give lipid compound L-31 (500 mg, yield 72.7%).1 H NMR(400MHz,Chloroform-d)δ8.42-8.46(m,2H),7.45-7.47(m,1H),7.15-7.17(m,1H),3.44-3.46(m,2H),2.69-2.72(m,2H),2.37-2.43(m,4H),2.14-2.15(s,2H),1.60-1.66(m,2H),1.38-1.41(m,4H),1.26-1.28(m,58H),0.89(d,6H)。
EXAMPLE 32 Synthesis of lipid Compound L-32
1mmol of compound 4-1,2.4mmol of compound 28-1 were taken and put into a polytetrafluoroethylene-lined glass screw cap vial, and after adding 2ml of ACN solvent and mixing uniformly, they were subjected to hydrothermal reaction at 70℃for 48 hours. After purification on a silica gel column, the solvent was removed by rotary evaporator to give lipid compound L-32 (490 mg, yield 71.3%).1 H NMR(400MHz,Chloroform-d)δ7.21-7.23(s,2H),3.42-3.46(m,4H),2.67-2.69(m,6H),2.37-2.62(m,4H),2.14-2.15(s,2H),1.60-1.66(m,2H),1.38-1.41(m,4H),1.26-1.28(m,58H),0.89(d,6H)。
EXAMPLE 33 Synthesis of lipid Compound L-33
1mmol of compound 5-1 and 2.4mmol of compound 28-1 are taken, added into a polytetrafluoroethylene-lined glass spiral cover small bottle, and after being added with 2ml of ACN solvent and uniformly mixed, the mixture is subjected to hydrothermal reaction at 70 ℃ for 48 hours. After purification on a silica gel column, the solvent was removed by rotary evaporator to give lipid compound L-33 (440 mg, yield 59.7%).1 H NMR(400MHz,Chloroform-d)δ8.09-8.11(m,1H),8.00-8.02(m,1H),7.80-7.82(m,1H),7.61-7.64(m,1H),7.47-7.51(m,1H),7.29-7.31(m,1H),3.42-3.46(m,2H),2.69-2.71(m,2H),2.37-2.42(m,6H),2.14-2.15(s,2H),1.60-1.66(m,2H),1.38-1.41(m,4H),1.26-1.28(m,58H),0.89(d,6H)。
Example 34 preparation of LNP lipid nanoparticles
Ionizable lipids L-X (x=1 to 33) or SM102, DSPC, cholesterol and DMG-PEG2000 were mixed in a molar ratio of 50:10:38.5:1.5 dissolved in ethanol solution, total lipid concentration 12mM. mRNA containing firefly luciferase (luciferases) or green fluorescent protein (eGFP) reporter gene was dissolved in a citrate-ammonium citrate buffer at ph=4.5 according to N: p=6:1 (mass ratio of ionizable lipid to mRNA is 6:1). Ethanol lipid solution and mRNA buffer were mixed in a volume ratio of 1:3 mixing. The product obtained was placed in a dialysis bag (molecular weight cut-off 100 kDa) and dialyzed against PBS at pH=7.4 for 12 hours, followed by concentration of mRNA-LNP with an ultrafiltration tube and volume fixation of mRNA to 150. Mu.g/ml with 1XPBS and cryopreservation.
Test example:
experiment one
Experiment name: characterization and determination of particle size encapsulation efficiency of L-X-LNP and SM102 reference substances
Experimental materials: example 34 preparation of eGFP mRNA-LNP
The experimental process comprises the following steps: 1) Particle size analysis and test: the size and distribution of the particle size of the sample solution are tested by DLS using a quartz cuvette with the specification of 10mm×10mm×40mm as a sample cuvette, the test is continuously performed for 2 times at intervals of 30s, and finally, the average value of the two tests is taken as final data. 3) mRNA encapsulation efficiency assay: using Quant-itTM RiboGreen RNA Assay Kit kit the encapsulation efficiency was determined. a. The prepared eGFP mRNA-LNP suspension and PBS (negative control, equal volume of TE buffer) were diluted to 4 ng/. Mu.l with TE buffer in the kit to obtain an eGFP mRNA-LNP working solution. b. The eGFP mRNA-LNP working solution was further diluted with TE buffer (or TE buffer containing 2% Triton-X100) in equal volume and left to stand at 37℃for 10min after mixing (TE buffer without Triton-X100 was used to determine unencapsulated free mRNA, while TE buffer containing 2% Triton-X100 was used to determine Total mRNA in the eGFP mRNA-LNP working solution, including free mRNA and mRNA encapsulated in lipid nanoparticles). c. After calibrating the fluorescence intensity with the standard: after concentration standard curve, a proper amount of Quant-it is sucked according to the instruction of the kitTM RiboGreen RNA reagent nucleic acid dye is added into each group of samples, the samples are dyed for 5min, each group of dyed samples are transferred into an enzyme-labeled instrument for detection, and the mRNA in the samples is accurately quantified by using software. d. The mRNA encapsulation efficiency in lipid nanoparticles was calculated using the following formula Encapsulation efficiency (EE%) = [1-m (free mRNA): m (total mRNA)]×100％]。
Experimental results: as shown in tables 2 to 6, the L-X-LNP (x=1 to 33) prepared in the present invention has a narrower particle size distribution, and the size satisfies the ideal nanovaccine size. Meanwhile, mRNA can be well loaded by virtue of the ionizable lipid in an acidic buffer solution, so that the prepared eGFP mRNA-LNP has higher mRNA encapsulation efficiency. Meanwhile, taking L-9-LNP as an example, as shown in FIG. 1, the prepared mRNA/L-9-LNP has regular particle morphology.
Table 1
| Numbering device | Ionizable lipids | Encapsulation efficiency (%) | Particle size (nm) | PDI | Potential mV |
| 1 | L-1 | 91.3 | 87.5 | 0.08 | -0.15 |
| 2 | L-2 | 90.4 | 86.5 | 0.10 | -0.56 |
| 3 | L-3 | 98.7 | 85.0 | 0.05 | -1.2 |
| 4 | L-4 | 91.2 | 96.1 | 0.12 | 1.2 |
| 5 | L-5 | 88.2 | 102.5 | 0.15 | 3.0 |
Table 2
TABLE 3
| Numbering device | Ionizable lipids | Encapsulation efficiency (%) | Particle size (nm) | PDI | Potential mV |
| 13 | L-13 | 95.6 | 78.5 | 0.11 | 0.22 |
| 14 | L-14 | 96.5 | 96.5 | 0.12 | 1.52 |
| 15 | L-15 | 93.5 | 88.5 | 0.08 | -2.3 |
| 16 | L-16 | 92.6 | 91.4 | 0.11 | -3.0 |
| 17 | L-17 | 95.6 | 85.4 | 0.15 | 0.54 |
| 18 | L-18 | 94.3 | 86.5 | 0.08 | -0.53 |
| 19 | L-19 | 92.8 | 91.5 | 0.17 | 1.25 |
Table 4
| Numbering device | Ionizable lipids | Encapsulation efficiency (%) | Particle size (nm) | PDI | Potential mV |
| 20 | L-20 | 94.5 | 81.2 | 0.12 | -1.9 |
| 21 | L-21 | 93.4 | 78.9 | 0.05 | 2.1 |
| 22 | L-22 | 91.7 | 85.5 | 0.07 | -1.6 |
| 23 | L-23 | 92.1 | 91.3 | 0.15 | -1.5 |
| 24 | L-24 | 96.5 | 88.6 | 0.11 | 2.4 |
| 25 | L-25 | 94.3 | 78.9 | 0.09 | 1.25 |
Table 5
| Numbering device | Ionizable lipids | Encapsulation efficiency (%) | Particle size (nm) | PDI | Potential mV |
| 26 | L-26 | 93.5 | 88.2 | 0.15 | 1.32 |
| 27 | L-27 | 94.6 | 86.2 | 0.16 | 1.22 |
| 28 | L-28 | 96.3 | 96.4 | 0.08 | -1.54 |
| 29 | L-29 | 95.2 | 94.1 | 0.11 | -0.36 |
| 30 | L-30 | 94.6 | 82.1 | 0.12 | -0.45 |
| 31 | L-31 | 93.5 | 91.5 | 0.10 | 0.69 |
| 32 | L-32 | 97.5 | 90.6 | 0.11 | -0.11 |
| 33 | L-33 | 90.7 | 89.8 | 0.12 | 0.15 |
| 34 | SM102 | 90.3 | 86.5 | 0.20 | 2.6 |
Experiment two
Experiment name: in vitro mRNA translational expression assessment experiments
Experimental materials: example 34 preparation of eGFP mRNA-LNP
The experimental process comprises the following steps: DC2.4 cells were cultured to log phase with RPMI 1640 medium supplemented with 10% (V/V) fetal bovine serum FBS (Gibco), 1% (V/V) P/S antibody, respectively. Cells were seeded in 24-well plates at 100000 per well and transfected with L-X-LNP containing 2. Mu.g of eGFP mRNA. After 24 hours of transfection, the cell culture medium was aspirated, washed slowly and gently once with PBS, and then the positive cell ratios of each group were analyzed with a flow cytometer.
Experimental results: first, we consider the effect of the position of the pyridine substituent on transfection efficiency, as shown in FIG. 2, with the highest transfection efficiency for para-substitution. Whereas transfection of the clinical substitution is relatively inefficient, possibly due to steric hindrance. Next we further studied the effect of the linker L1, and the side chain substitution of the linker as shown in FIG. 3 reduced transfection efficiency. We then further studied the effect of the hydrophobic tails of different structures on the transfection efficiency of cells in vitro, as shown in fig. 4, the effect of the length of the hydrophobic segment before and after the ester linkage on the transfection efficiency of cells was not great. Meanwhile, we also consider the effect of different numbers of hydrophobic tails on transfection efficiency, as shown in fig. 5, LNP transfection efficiency of 4 hydrophobic tails is significantly higher than that of 2 hydrophobic tails, which may be due to the increased hydrophobic fragments that can increase the interaction force of LNP with cell membrane. Compared with commercial SM102-LNP, the LNP containing the novel pyridyl ionizable lipid provided by the invention has the advantage that the transfection efficiency of cells is remarkably improved.
Experiment III:
experiment name: expression evaluation experiment of firefly luciferase mRNA in mice
Experimental materials: EXAMPLE 34 preparation of luc mRNA-LNP
The experimental process comprises the following steps: 20 μg of Luc-mRNA from different delivery vehicles was injected subcutaneously in the root of the tail of BALB/c mice over 4-6 weeks, respectively. 8 hours after administration, a volume of 100. Mu.L of luciferin at a concentration of 15mg/ml was injected intraperitoneally. After 10 minutes, the distribution of the fluorescent signal was observed with an IVIS mouse living imager (PerkinElmer).
Experimental results: to directly assess the translational expression efficiency of different L-X-LNP delivery systems in mice, we used different delivery systems to package Luc-mRNA. As shown in fig. 6, compared with commercial SM102-LNP, the LNP containing the novel pyridyl ionizable lipid provided by the invention of the patent can significantly promote the efficient expression of mRNA encoding antigen in vivo. Meanwhile, the LNP containing the novel pyridyl ionizable lipid has insignificant toxic and side effects, and the mice have good survival state and good safety.
In summary, the present invention effectively overcomes the disadvantages of the prior art and has high industrial utility value.
The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.