A new compound and its application

Abstract

The present invention relates to a novel compound. The structure of the compound contains an interfering nucleic acid, a transition point and a modified chain at its end. Said terminal modification chain can introduce two or three N-acetyl galactosamines at the 3' end of the antisense strand of the interfering nucleic acid, and two or one N-acetylgalactosamines can be introduced at the 5' end of the sense strand correspondingly. N-acetylgalactosamine, the total number of N-acetylgalactosamine introduced is four. In vivo and in vitro pharmacodynamic experiments have proved that this new compound can continuously and efficiently inhibit HBV gene expression.

Description

technical field

[0001] The present invention relates to a novel compound. The structure of the compound contains an interfering nucleic acid, a transition point and a modified chain at its end. By modifying the end of the strand, two to three N-acetylgalactosamines can be introduced at the 3' end of the antisense strand of this siRNA, and two to one N-acetylgalactose can be introduced at the 5' end of the sense strand amines, the total number of N-acetylgalactosamines introduced was four. The drug efficacy experiments on HepG 2 cells and transgenic mice prove that this new compound can continuously inhibit the expression of HBsAg, HBeAg and HBV DNA of HBV. Background technique

[0002] RNAi

[0003] RNAi (RNA interference) was discovered in 1998 by Andrew Z. Fire et al. when conducting antisense RNA inhibition experiments in Caenorhabditis elegans, and this process was called RNAi. This discovery was rated as one of the top ten scientific advances in 2001 by the "Science...

Examples

Embodiment 1

[0146] Embodiment 1, the synthesis of GBL-0401

[0147] 1. Synthesis of Kys-01

[0148] 1.1. Compounds with 5'YICd-01 structure: Synthesis of 5'YICd-01-PFP

[0149] 1.1.1. Synthesis of 5'YICd-01-c1

[0150]

[0151] Measure 5.0 g (81.9 mmol) of 2-hydroxyethylamine, add 50 mL of dimethyl sulfoxide, 5 mL of sodium hydroxide solution (concentration 1 g / mL), add 12 mL (81.9 mmol) of tert-butyl acrylate dropwise for 1 hour, and After reacting for 24 hours, 100 mL of petroleum ether was added, washed twice with saturated brine, and the organic layer was dried. After passing through the column, 7.5 g of a colorless oily substance was obtained.

[0152] 1.1.2. Synthesis of 5'YICd-01-c2

[0153]

[0154] Weigh 7.5g (39.7mmol) of 5'YICd-01-c1, add 50mL of DCM, 23mL of sodium carbonate solution (25%), add 7.7g (45.0mmol) of benzyl chloroformate dropwise at room temperature, react overnight at room temperature, wash with saturated saline Twice, dried over anhydrous sodium sulfate...

Embodiment 2

[0212] Embodiment two, the synthesis of GBL-0402

[0213] 1. Synthesis of Kys-02

[0214] 1.1. Compounds with 5'YICc-01 structure: Synthesis of 5'YICc-01-PFP

[0215] 1.1.1. Synthesis of 5’YICc-01-c1

[0216]

[0217] Take 7.0g (40.0mmol) of SANC-c8 and 9.2g (34.4mmol) of 5’YICd-01-c3, add 25mL of DMF to dissolve, add 9.0g of TBTU, cool down to 10°C, add 2ml of DIEA, and react overnight at room temperature. Add 30 mL of water and 50 mL of dichloromethane, and wash the organic layer three times with saturated brine. The organic layer was dried and evaporated to dryness under reduced pressure. After passing through a column chromatography (eluent: dichloromethane: methanol = 1%-10%), 10.0 g of a yellow viscous solid was obtained.

[0218] 1.1.2. Synthesis of 5'YICc-01-c2

[0219]

[0220] Take 10.0g of 5'YICc-01-c1, add 15ml of concentrated hydrochloric acid, and react overnight at room temperature. Evaporate to dryness under reduced pressure to obtain 7.3g.

[0221...

Embodiment 3

[0251] Embodiment three, the synthesis of GBL-0403

[0252] 1. Synthesis of Kys-03

[0253]1.1. Compounds with 5’ERCd-01 structure: Synthesis of 5’ERCd-01-PFP

[0254] 1.1.1. Synthesis of 5’ERCd-01-c1

[0255]

[0256] Measure 5.0g (54.9mmol) of 2-amino-1,3-propanediol, add 50mL of DMSO, 5mL of sodium hydroxide solution (concentration: 1g / mL), cool down to 0°C, add dropwise 20mL (137.8mol) of tert-butyl acrylate2 After the addition was completed in 1 hour, react at room temperature for 48 hours, add 100 mL of petroleum ether, wash with saturated brine twice, and dry the organic layer. After passing through a chromatography column (eluent: ethyl acetate: petroleum ether = 25%-75%), add 0.05% triethylamine to the column to obtain 6.2 g of a colorless oil.

[0257] 1.1.2. Synthesis of 5’ERCd-01-c2

[0258]

[0259] Weigh 6.2g (17.9mmol) of 5'ERCd-01-c1, add 50mL of dichloromethane, 23mL of sodium carbonate solution (25%), add 8.2mL of benzyl chloroformate (57.4mmol) dro...

technical field

[0001] The present invention relates to a novel compound. The structure of the compound contains an interfering nucleic acid, a transition point and a modified chain at its end. By modifying the end of the strand, two to three N-acetylgalactosamines can be introduced at the 3' end of the antisense strand of this siRNA, and two to one N-acetylgalactose can be introduced at the 5' end of the sense strand amines, the total number of N-acetylgalactosamines introduced was four. The drug efficacy experiments on HepG 2 cells and transgenic mice prove that this new compound can continuously inhibit the expression of HBsAg, HBeAg and HBV DNA of HBV. Background technique

[0002] RNAi

[0003] RNAi (RNA interference) was discovered in 1998 by Andrew Z. Fire et al. when conducting antisense RNA inhibition experiments in Caenorhabditis elegans, and this process was called RNAi. This discovery was rated as one of the top ten scientific advances in 2001 by the "Science...

Examples

Embodiment 1

[0146] Embodiment 1, the synthesis of GBL-0401

[0147] 1. Synthesis of Kys-01

[0148] 1.1. Compounds with 5'YICd-01 structure: Synthesis of 5'YICd-01-PFP

[0149] 1.1.1. Synthesis of 5'YICd-01-c1

[0150]

[0151] Measure 5.0 g (81.9 mmol) of 2-hydroxyethylamine, add 50 mL of dimethyl sulfoxide, 5 mL of sodium hydroxide solution (concentration 1 g / mL), add 12 mL (81.9 mmol) of tert-butyl acrylate dropwise for 1 hour, and After reacting for 24 hours, 100 mL of petroleum ether was added, washed twice with saturated brine, and the organic layer was dried. After passing through the column, 7.5 g of a colorless oily substance was obtained.

[0152] 1.1.2. Synthesis of 5'YICd-01-c2

[0153]

[0154] Weigh 7.5g (39.7mmol) of 5'YICd-01-c1, add 50mL of DCM, 23mL of sodium carbonate solution (25%), add 7.7g (45.0mmol) of benzyl chloroformate dropwise at room temperature, react overnight at room temperature, wash with saturated saline Twice, dried over anhydrous sodium sulfate...

Embodiment 2

[0212] Embodiment two, the synthesis of GBL-0402

[0213] 1. Synthesis of Kys-02

[0214] 1.1. Compounds with 5'YICc-01 structure: Synthesis of 5'YICc-01-PFP

[0215] 1.1.1. Synthesis of 5’YICc-01-c1

[0216]

[0217] Take 7.0g (40.0mmol) of SANC-c8 and 9.2g (34.4mmol) of 5’YICd-01-c3, add 25mL of DMF to dissolve, add 9.0g of TBTU, cool down to 10°C, add 2ml of DIEA, and react overnight at room temperature. Add 30 mL of water and 50 mL of dichloromethane, and wash the organic layer three times with saturated brine. The organic layer was dried and evaporated to dryness under reduced pressure. After passing through a column chromatography (eluent: dichloromethane: methanol = 1%-10%), 10.0 g of a yellow viscous solid was obtained.

[0218] 1.1.2. Synthesis of 5'YICc-01-c2

[0219]

[0220] Take 10.0g of 5'YICc-01-c1, add 15ml of concentrated hydrochloric acid, and react overnight at room temperature. Evaporate to dryness under reduced pressure to obtain 7.3g.

[0221...

Embodiment 3

[0251] Embodiment three, the synthesis of GBL-0403

[0252] 1. Synthesis of Kys-03

[0253]1.1. Compounds with 5’ERCd-01 structure: Synthesis of 5’ERCd-01-PFP

[0254] 1.1.1. Synthesis of 5’ERCd-01-c1

[0255]

[0256] Measure 5.0g (54.9mmol) of 2-amino-1,3-propanediol, add 50mL of DMSO, 5mL of sodium hydroxide solution (concentration: 1g / mL), cool down to 0°C, add dropwise 20mL (137.8mol) of tert-butyl acrylate2 After the addition was completed in 1 hour, react at room temperature for 48 hours, add 100 mL of petroleum ether, wash with saturated brine twice, and dry the organic layer. After passing through a chromatography column (eluent: ethyl acetate: petroleum ether = 25%-75%), add 0.05% triethylamine to the column to obtain 6.2 g of a colorless oil.

[0257] 1.1.2. Synthesis of 5’ERCd-01-c2

[0258]

[0259] Weigh 6.2g (17.9mmol) of 5'ERCd-01-c1, add 50mL of dichloromethane, 23mL of sodium carbonate solution (25%), add 8.2mL of benzyl chloroformate (57.4mmol) dro...

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