CONJUGATE GROUP AND CONJUGATE

Abstract

The present application relates to a novel conjugate group linkable to a compound (such as a therapeutic compound), for use in directing the compound to a target in the body. The conjugate group disclosed herein can enable an expression-inhibitory oligonucleotide (such as a RNAi reagent) to target liver cells to regulate gene expression. The conjugate group disclosed herein has a variety of applications when linked to expression-inhibitory oligonucleotides, comprising applications in therapy, diagnosis, target verification, and genome development. A composition comprising the conjugate group disclosed herein can mediate expression of target nucleic acid sequences in liver cells (such as hepatocytes) when linked to expression-inhibitory oligonucleotides, and can be used for treatment of diseases or disorders responding to the gene expression or activity of cells, tissues, or organisms.

Claims

1. A conjugate group, having a structure of formula (I): ##STR00016## wherein n is selected from an integer of 8 to 12.

2. The conjugate group according to claim 1, having the following structure: ##STR00017##

3. A conjugate, comprising the conjugate group according to claim 1, and a therapeutic agent linked to the conjugate group.

4. The conjugate according to claim 3, wherein the therapeutic agent is an expression-inhibitory oligonucleotide.

5. The conjugate according to claim 4, wherein the expression-inhibitory oligonucleotide is an RNAi reagent.

6. The conjugate according to claim 5, wherein the RNAi reagent includes one or more modified nucleotides.

7. The conjugate according to claim 5, wherein the RNAi reagent is a double-stranded siRNA containing a sense strand and an antisense strand.

8. The conjugate according to claim 7, wherein the double-stranded siRNA is linked to the conjugate group at the 5′ end of its sense strand.

9. The conjugate according to claim 4, wherein the expression-inhibitory oligonucleotide is linked to the conjugate group via a phosphate, phosphorothioate, or phosphonate group.

10. A compound, having a structure of formula (II) or formula (III) ##STR00018## wherein m is independently selected from an integer of 8 to 12, respectively.

11. The compound according to claim 10, having the following structure ##STR00019## ##STR00020##

12. A pharmaceutical composition, comprising the conjugate according to claim 3 and a pharmaceutically acceptable carrier or excipient.

13. A method for inhibiting the expression of a target nucleic acid in a subject in need thereof, comprising a step of administering to the subject the conjugate according to claim 3.

14. The method according to claim 13, wherein the target nucleic acid is a nucleic acid from a virus.

15. The method according to claim 14, wherein the target nucleic acid is a nucleic acid from the hepatitis B virus.

16. A method of treating a disease, comprising a step of administering to a subject the conjugate according to claim 3.

17. (canceled)

18. The method according to claim 16, wherein the disease is viral infection.

19. The method according to claim 16, wherein the disease is liver disease.

20. The method according to claim 16, wherein the disease is hepatitis B.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0068] The following examples describe the present disclosure in detail, but they are not meant to limit the present disclosure in any way. Compounds disclosed herein can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by the specific embodiments listed below combined with other chemical synthesis methods, and equivalent substitutions known to those skilled in the art. Alternative embodiments include, but are not limited to the examples disclosed herein. It will be obvious to those skilled in the art to make various changes and improvements to the specific embodiments of this disclosure without departing from the spirit and scope of this disclosure.

Example 1: Synthesis of D01

[0069] ##STR00006## ##STR00007## ##STR00008## ##STR00009##

[0070] Step A: 11-Dodecyne-1-ol (25 g, 137.14 mmol) and triethylamine (16.65 g, 164.56 mmol) were dissolved in dichloromethane (250 mL), and methanesulfonyl chloride (18.85 g, 164.56 mmol) was added at 0° C. The mixture solution was stirred at 0° C. for 2 hours. The reaction solution was diluted with water (400 mL) and extracted with 800 mL (400 mL×2) of dichloromethane. The combined organic phase was washed with 400 mL (200 mL×2) of water and saturated brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give 2-2.

[0071] Step B: The compound of formula 2-3 (20 g, 67.26 mmol) was dissolved in N,N-dimethylformamide (200 mL), and sodium hydride (60% purity, 4.04 g, 100.89 mmol) was added at 0° C., followed by the compound of formula 2-2 (19.27 g, 73.99 mmol). The mixture was stirred at 25° C. for 16 h. The reaction solution was quenched with water (1 L) and extracted with 1.6 L (800 mL×2) of dichloromethane. The combined organic phase was washed with 800 mL (800 mL×1) of saturated brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give 2-4. .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 7.63-6.89 (m, 10H), 5.64-5.52 (m, 2H), 4.27-4.01 (m, 2H), 3.98-3.77 (m, 2H), 3.72-3.18 (m, 4H), 2.23-2.14 (m, 2H), 1.98-1.92 (m, 1H), 1.54-1.23 (m, 16H).

[0072] Step C: The compound of formula 2-4 (48 g, 103.98 mmol) was dissolved in methanol (870 mL), and a solution of hydrogen chloride in methanol (4 mol/L, 400 mL, 1.6 mol) was added. The mixture solution was stirred at 30° C. for 2 h. To the reaction solution was added a solution of hydrogen chloride in methanol (4 mol/L, 350 mL, 1.4 mol). The mixture was stirred at 30° C. for 16 hours. The reaction solution was concentrated under reduced pressure, and 200 mL (100 mL×2) of trichloromethane was added. The mixture was concentrated under reduced pressure until a white solid appeared. Toluene (130 mL) and petroleum ether (130 mL) were added, and the mixture solution was stirred at 15° C. for 16 hours. The reaction solution was filtered through a Buchner funnel and the filter cake was collected and dried under vacuum to give a white solid. The white solid was dissolved in dichloromethane (50 mL), and aqueous solution (50 mL) of sodium hydroxide (6.59 g, 164.66 mmol) was added. The mixture was stirred at 20° C. for 1 hour. The reaction solution was diluted with water (500 mL) and extracted with 1 L (500 mL×2) of dichloromethane. The combined organic phase was dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give 2-5.

[0073] Step D: To a mixture solution of the compound of formula 2-5 (23 g, 80.58 mmol) and sodium hydroxide (322.31 mg, 8.06 mmol) in dimethyl sulfoxide (70 mL) and water (6 mL) was added tert-butyl acrylate (22.72 g, 177.28 mmol), and the mixture was stirred at 25° C. under nitrogen for 16 h. The reaction solution was diluted with water (500 mL) and extracted with 1 L of ethyl acetate (500 mL×2). The combined organic phase was dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give the crude product, which was purified by column chromatography (SiO.sub.2, petroleum ether/ethyl acetate/ethanol (with 0.1% ammonia)=36/3/1 to 16/3/1) to give 2-6. .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 3.60-3.54 (m, 4H), 3.32 (br s, 5H), 3.15 (s, 5H), 2.74-2.66 (m, 1H), 2.40 (t, J=6.0 Hz, 4H), 2.18-2.11 (m, 2H), 1.58-1.38 (m, 22H), 1.34-1.23 (m, 12H).

[0074] Step E: To a solution of the compound of formula 2-6 (24.5 g, 45.22 mmol) in dichloromethane (250 mL) were added triethylamine (9.15 g, 90.45 mmol) and succinic anhydride (6.79 g, 67.83 mmol), and the mixture was stirred at 20° C. for 16 hours. To the reaction solution were added dichloromethane (1 L) and hydrochloric acid (1 mol/L, 1 L), and the layers were separated. The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to give 2-7. .sup.1H NMR (400 MHz, CDCl.sub.3): δ 6.49-6.37 (m, 1H), 3.72 (s, 2H), 3.70-3.57 (m, 8H), 3.37 (t, J=6.7 Hz, 2H), 2.69-2.51 (m, 4H), 2.50-2.36 (m, 4H), 2.22-2.13 (m, 2H), 1.96-1.90 (m, 1H), 1.57-1.47 (m, 4H), 1.46-1.40 (m, 18H), 1.40-1.31 (m, 2H), 1.30-1.21 (m, 10H).

[0075] Step F: The compound of formula 2-7 (27.4 g, 42.69 mmol) was dissolved in formic acid (140 mL), and the mixture was stirred at 20° C. under nitrogen for 16 hours. The reaction solution was concentrated under reduced pressure. 300 mL (150 mL×2) of toluene was added. The mixture was concentrated under reduced pressure to give 2-8. .sup.1H NMR (400 MHz, CDCl.sub.3): δ 9.79-9.22 (m, 3H), 6.44-6.23 (m, 1H), 3.88-3.43 (m, 10H), 3.39-3.20 (m, 2H), 2.77-2.31 (m, 8H), 2.15-2.06 (m, 2H), 1.87 (t, J=2.6 Hz, 1H), 1.48-1.28 (m, 6H), 1.26-1.12 (m, 10H).

[0076] Step G: The compound of formula 2-8 (22.6 g, 42.67 mmol), N,N-diisopropylethylamine (33.09 g, 256.03 mmol) and O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (51.92 g, 136.55 mmol) were dissolved in N,N-dimethylformamide (250 mL), and tert-butyl N-(3-aminopropyl)carbamate (29.74 g, 170.69 mmol) was added. The mixture solution was stirred at 20° C. for 16 h. To the reaction solution were added dichloromethane (1 L) and hydrochloric acid (1 mol/L, 1 L), and the layers were separated. The organic phase was washed sequentially with 1 L of water (1 L x 1), 1 L of aqueous sodium bicarbonate (1 L x 1) and 1 L of saturated brine (1 L x 1), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give the crude product, which was purified by column chromatography (SiO.sub.2, petroleum ether/ethyl acetate/ethanol=40/3/1 to 10/3/1) to give 2-9. .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.22-6.79 (m, 3H), 6.77-6.44 (m, 1H), 5.45-5.00 (m, 3H), 3.86-3.73 (m, 2H), 3.72-3.63 (m, 4H), 3.62-3.45 (m, 4H), 3.41-3.32 (m, 2H), 3.32-3.20 (m, 6H), 3.19-3.03 (m, 6H), 2.56-2.47 (m, 4H), 2.47-2.39 (m, 4H), 2.21-2.12 (m, 2H), 1.95-1.90 (m, 1H), 1.70-1.57 (m, 6H), 1.56-1.47 (m, 4H), 1.46-1.38 (m, 29H), 1.30-1.25 (m, 10H).

[0077] Step H: The compound of formula 2-9 (15 g, 15.03 mmol) was dissolved in dichloromethane (114 mL), and trifluoroacetic acid (38 mL) was added. The mixture was stirred at 20° C. for 16 hours. The reaction solution was concentrated under reduced pressure, and 600 mL (250 mL×3) of a mixed solution of toluene and acetonitrile (toluene/acetonitrile=3/1) was added. The solution was concentrated under reduced pressure to give 2-10.

[0078] Step I: The compound of formula 2-11 (22.15 g, 49.50 mmol), N,N-diisopropylethylamine (7.75 g, 60.00 mmol), 1-hydroxy-7-azabenzotriazole (6.12 g, 45.00 mmol) and O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (20.53 g, 54.00 mmol) were dissolved in N,N-dimethylformamide (90 mL). To this mixture solution was added a solution of the compound of formula 2-10 (tris(trifluoroacetate), 15.6 g, 15.00 mmol) and N,N-diisopropylethylamine (21.32 g, 165.00 mmol) in N,N-dimethylformamide (120 mL). The mixture solution was stirred at 20° C. for 16 hours. To the reaction solution were added dichloromethane (1.2 L) and hydrochloric acid (1 mol/L, 1 L), and the layers were separated. The organic phase was washed sequentially with 1 L of water (1 L x 1), 1 L of aqueous sodium bicarbonate (1 L x 1) and 1 L of saturated brine (1 L x 1), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give the crude product, which was purified by column chromatography (SiO.sub.2, dichloromethane/methanol=100/1 to 10/1 to dichloromethane/ethanol=1/1) to give 2-12. .sup.1H NMR (400 MHz, DMSO-d6): δ 7.87-7.66 (m, 9H), 7.09 (s, 1H), 5.21 (d, J=3.4 Hz, 3H), 4.96 (dd, J=3.4, 11.3 Hz, 3H), 4.48 (d, J=8.5 Hz, 3H), 4.06-3.98 (m, 9H), 3.91-3.82 (m, 3H), 3.74-3.66 (m, 3H), 3.58-3.46 (m, 12H), 3.31 (br s, 3H), 3.07-2.98 (m, 12H), 2.71 (t, J=2.6 Hz, 1H), 2.33-2.22 (m, 8H), 2.16-2.12 (m, 2H), 2.10 (s, 9H), 2.04 (br t, J=7.1 Hz, 6H), 1.99 (s, 9H), 1.89 (s, 9H), 1.81-1.74 (m, 9H), 1.54-1.39 (m, 22H), 1.32 (br dd, J=4.5, 6.7 Hz, 2H), 1.24 (s, 10H).

[0079] Step J: The compound of formula 2-12 (1.00 g, 0.50 mmol) and N-methyl-N,N,N,N-tri-n-octylammonium chloride (20.35 mg, 50.35 μmol) were dissolved in a mixture of acetic acid (2.7 mL) and n-pentane (6.3 mL). To this mixture at 0° C. was added dropwise a solution of potassium permanganate (0.40 g, 2.52 mmol) in water (9 mL). The mixture solution was stirred at 0 to 15° C. for 2 hours. The reaction mixture was quenched with sodium bisulfite (1.27 g), and hydrochloric acid (2 mol/L, 5 mL) and water (30 mL) were added. The mixture was extracted with 120 mL (40 mL×3) of a mixed solution of trichloromethane and isopropanol (trichloromethane/isopropanol=3/1). The combined organic phase was dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and 180 mL (30 mL×6) of a mixed solution of toluene and acetonitrile (toluene/acetonitrile=1/1) was added. The solution was concentrated under reduced pressure to give 2-13. .sup.1H NMR (400 MHz, CD.sub.3OD): δ 5.34 (d, J=2.9 Hz, 3H), 5.06 (dd, J=3.3, 11.2 Hz, 3H), 4.56 (d, J=8.4 Hz, 3H), 4.19-4.06 (m, 9H), 4.04-3.98 (m, 3H), 3.87 (td, J=5.7, 9.9 Hz, 4H), 3.72-3.64 (m, 9H), 3.57-3.50 (m, 3H), 3.39 (br t, J=6.4 Hz, 2H), 3.22 (q, J=6.4 Hz, 12H), 2.51-2.40 (m, 9H), 2.21 (br t, J=7.3 Hz, 6H), 2.14 (s, 9H), 2.03 (s, 9H), 1.94 (d, J=7.9 Hz, 18H), 1.72-1.57 (m, 22H), 1.39 (br s, 12H).

[0080] Step K: To a solution of the compound of formula 2-13 (1.00 g, 0.50 mmol) in N,N-dimethylformamide (10 mL) were added N,N-diisopropylethylamine (0.26 g, 1.99 mmol) and O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (0.23 g, 0.60 mmol). The mixture was stirred and the compound of formula 2-14 (0.23 g, 0.55 mmol) was added. The mixture solution was stirred at 15° C. for 16 h. To the reaction solution were added dichloromethane (50 mL) and water (50 mL), and the layers were separated. The organic phase was washed sequentially with 50 mL of saturated aqueous sodium bicarbonate (50 mL×1), 50 mL of water (50 mL×1) and 50 mL of saturated brine (50 mL×1), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give the crude product, which was purified by column chromatography (SiO.sub.2, dichloromethane/methanol (with 0.1% triethylamine)=20/1 to 10/1) to give 2-15. .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 7.90-7.82 (m, 6H), 7.78 (br d, J=4.8 Hz, 3H), 7.40-7.26 (m, 10H), 6.91 (br dd, J=3.1, 9.0 Hz, 4H), 5.26 (d, J=3.4 Hz, 3H), 5.03-4.99 (m, 3H), 4.53 (d, J=8.4 Hz, 3H), 4.43 (br d, J=3.8 Hz, 1H), 4.23-4.14 (m, 1H), 4.12-4.02 (m, 9H), 3.92 (td, J=9.0, 11.0 Hz, 3H), 3.78 (s, 6H), 3.77-3.71 (m, 3H), 3.66-3.51 (m, 13H), 3.49-3.41 (m, 4H), 3.11-3.01 (m, 16H), 2.38-2.37 (m, 1H), 2.32 (br s, 9H), 2.14 (s, 9H), 2.08 (br t, J=6.9 Hz, 7H), 2.04 (s, 9H), 1.93 (s, 9H), 1.82 (s, 9H), 1.57-1.46 (m, 22H), 1.31-1.26 (m, 12H).

[0081] Step L: To a solution of the compound of formula 2-15 (0.80 g, 0.33 mmol) in dichloromethane (8 mL) were added sequentially triethylamine (67.24 mg, 0.64 mmol), 4-N,N-dimethylaminopyridine (0.12 g, 1.00 mmol) and succinic anhydride (83.13 mg, 0.83 mmol). The mixture solution was stirred at 10° C. for 16 hours. To the reaction solution were added dichloromethane (50 mL), water (30 mL) and saturated brine (30 mL), and the layers were separated. The organic phase was washed sequentially with 30 mL (30 mL×1) of water and 30 mL (30 mL×1) of saturated saline, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give the crude product, which was purified by p-HPLC (separation column Waters Xbridge C18 (specifications: 150 mm×50 mm, particle size: 10 μm); mobile phase: [water (10 mM ammonium bicarbonate)-acetonitrile]; elution gradient: 27%-57%, 11 min) to give D01. .sup.1H NMR (400 MHz, DMSO-d6): δ 7.96-7.69 (m, 9H), 7.33-7.09 (m, 10H), 6.90-6.78 (m, 4H), 5.21 (d, J=3.3 Hz, 3H), 4.97 (dd, J=3.3, 11.2 Hz, 3H), 4.49 (d, J=8.4 Hz, 3H), 4.06-3.97 (m, 9H), 3.91-3.83 (m, 3H), 3.79-3.66 (m, 11H), 3.63-3.45 (m, 18H), 3.02 (br d, J=4.6 Hz, 14H), 2.46-2.37 (m, 4H), 2.35-2.14 (m, 12H), 2.10 (s, 9H), 2.04 (br t, J=7.0 Hz, 6H), 1.99 (s, 9H), 1.88 (s, 9H), 1.77 (s, 9H), 1.57-1.37 (m, 22H), 1.22 (br s, 12H).

[0082] D is a residue of the small molecule fragment D01 after chemical reaction, which is bound to a nucleic acid through a covalent bond, and has a structure as shown in the following formula:

##STR00010##

Example 2: Synthesis of D02

[0083] ##STR00011## ##STR00012## ##STR00013## ##STR00014##

[0084] Step A: Compound 3-2 was prepared by referring to compound D01 of Example 1 and replacing 2-1 with 3-1.

[0085] Step B: The compound of formula 2-3 (35.70 g, 120.06 mmol) was dissolved in 2-methyltetrahydrofuran (285 mL), and potassium tert-butoxide (17.51 g, 156.07 mmol) was added. The mixture solution was stirred at 85° C. for 2 hours. Then the compound of formula 3-2 (28.40 g, 114.34 mmol) was added. The mixture solution was stirred at 85° C. for 12 h. To the reaction solution was added water (400 mL) and extracted with 800 mL (400 mL×2) of dichloromethane. The combined organic phase was dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give 3-3.

[0086] Step C: The compound of formula 3-3 (35 g, 77.84 mmol) was dissolved in methanol (525 mL), and a solution of hydrogen chloride in methanol (4 mol/L, 175 mL, 750 mmol) was added. The mixture was stirred at 50° C. for 12 hours. The reaction solution was poured into a mixed solution of potassium carbonate (80 g) and methanol (500 mL). The mixture was filtered through a Buchner funnel and the filtrate was concentrated under reduced pressure. The resulting crude product was dissolved in methanol (240 mL), and sodium acetate (12.77 g, 155.68 mmol) and hydroxylamine hydrochloride (5.41 g, 77.84 mmol) were added. The mixture was stirred at 25° C. for 0.5 h. The reaction solution was filtered through a Buchner funnel and the filtrate was concentrated under reduced pressure. The resulting crude product was added to aqueous sodium hydroxide (1 mol/L, 500 mL) and extracted with 500 mL (500 mL×1) of dichloromethane. The combined organic phase was washed sequentially with aqueous sodium hydroxide (1 mol/L, 500 mL) and 500 mL (500 mL×1) of saturated brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give 3-4. .sup.1H NMR (400 MHz, CDCl.sub.3): δ 5.81-5.66 (m, 1H), 4.98-4.79 (m, 2H), 3.50-3.37 (m, 4H), 3.36-3.30 (m, 2H), 3.24 (br s, 2H), 2.03-1.91 (m, 2H), 1.53-1.42 (m, 2H), 1.35-1.27 (m, 2H), 1.25-1.13 (m, 10H).

[0087] Steps D to I: Prepared by referring to each step of compound D01 of Example 1, respectively.

[0088] Step J: The compound of formula 3-10 (20 g, 10.13 mmol) and ruthenium trichloride trihydrate (52.98 mg, 202.61 μmol) were dissolved in a mixed solution of dichloromethane (60 mL), acetonitrile (60 mL) and water (90 mL). Sodium periodate (10.83 g, 50.65 mmol) was slowly added to this mixture solution. The mixture solution was stirred at 25° C. for 3.5 hours. To the reaction solution was added water (500 mL) and the mixture was extracted with 1 L (500 mL×2) of a mixed solution of dichloromethane and isopropanol (dichloromethane/isopropanol=3/1). The combined organic phase was washed with 150 mL (150 mL×1) of saturated aqueous sodium sulfite and concentrated under reduced pressure. The crude product was added to saturated aqueous sodium bicarbonate (500 mL), and the mixture was washed with 1 L (500 mL×2) of dichloromethane. Hydrochloric acid (1 mol/L) was added until the pH was adjusted to 3, and the mixture was extracted with 1.5 L (500 mL×3) of a mixed solution of dichloromethane and isopropanol (dichloromethane/isopropanol=3/1). The combined organic phase was washed with 500 mL of saturated brine (500 mL×1), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give 3-11. .sup.1H NMR (400 MHz, CD.sub.3OD): δ 5.37-5.29 (m, 3H), 5.06 (dd, J=3.4, 11.3 Hz, 3H), 4.61-4.51 (m, 3H), 4.20-3.96 (m, 12H), 3.92-3.82 (m, 3H), 3.70-3.63 (m, 9H), 3.57-3.49 (m, 3H), 3.43-3.36 (m, 2H), 3.28-3.14 (m, 12H), 2.50-2.38 (m, 8H), 2.33-2.24 (m, 3H), 2.24-2.17 (m, 6H), 2.17-2.11 (m, 9H), 2.03-1.99 (m, 9H), 1.98-1.90 (m, 18H), 1.75-1.51 (m, 22H), 1.38-1.27 (m, 10H).

[0089] Step K: To a solution of the compound of formula 3-11 (11 g, 5.52 mmol) in dichloromethane (110 mL) were added N,N-diisopropylethylamine (0.71 g, 5.52 mmol), 1-hydroxy-7-azabenzotriazole (1.50 g, 11.04 mmol), O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (2.52 g, 6.63 mmol) and the compound of formula 2-14 (2.43 g, 5.80 mmol). The mixture was stirred, and then N,N-diisopropylethylamine (2.14 g, 16.56 mmol) was added. The mixture solution was stirred at 25° C. for 12 h. To the reaction solution were added dichloromethane (1 L) and water (500 mL), and the layers were separated. The organic phase was washed sequentially with 500 mL of water (500 mL×1) and 500 mL of saturated brine (500 mL×1), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to give the crude product, which was purified by column chromatography (SiO.sub.2, dichloromethane/methanol (with 0.5% triethylamine)=30/1 to 10/1) to give 3-12. .sup.1H NMR (400 MHz, DMSO-d6): δ 7.92-7.73 (m, 9H), 7.38-7.16 (m, 10H), 6.94-6.83 (m, 4H), 5.22 (d, J=3.4 Hz, 3H), 4.97 (dd, J=3.4, 11.3 Hz, 3H), 4.53-4.44 (m, 3H), 4.44-4.35 (m, 1H), 4.21-4.09 (m, 1H), 4.03 (s, 8H), 3.94-3.83 (m, 3H), 3.77-3.68 (m, 9H), 3.58-3.48 (m, 10H), 3.43 (br s, 3H), 3.36-3.28 (m, 4H), 3.03 (br s, 14H), 2.33-2.19 (m, 10H), 2.11 (s, 9H), 2.04 (br t, J=6.9 Hz, 7H), 2.00 (s, 9H), 1.89 (s, 9H), 1.81-1.74 (m, 9H), 1.57-1.38 (m, 22H), 1.37-1.17 (m, 10H).

[0090] Step L: D02 was obtained with reference to the preparation of compound D01 of Example 1. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ=7.94-7.70 (m, 9H), 7.40-7.08 (m, 10H), 6.95-6.79 (m, 4H), 5.27-5.17 (m, 3H), 4.98 (dd, J=3.3, 11.2 Hz, 3H), 4.50 (d, J=8.4 Hz, 3H), 4.08-3.98 (m, 9H), 3.92-3.85 (m, 3H), 3.78-3.67 (m, 11H), 3.59-3.49 (m, 12H), 3.46-3.36 (m, 6H), 2.95 (br s, 14H), 2.48-2.38 (m, 4H), 2.16 (br s, 12H), 2.11 (s, 9H), 2.05 (br t, J=6.8 Hz, 6H), 2.00 (s, 9H), 1.89 (s, 9H), 1.83-1.72 (m, 9H), 1.57-1.36 (m, 22H), 1.31-1.12 (m, 10H).

Example 3: Synthesis of D-01-M

[0091] ##STR00015##

[0092] Step A: To a solution of D-01 (100 mg, 39.88 μmol) in acetonitrile (0.7 mL) was added ammonia (30%, 1.4 mL). The mixture solution was stirred at 50° C. for 12 h. The reaction solution was filtered and then purified by p-HPLC (separation column Phenomenex Gemini-NX C18 (specification: 75 mm×30 mm, particle size: 3 μm); mobile phase: [water (0.05% ammonia)-acetonitrile]; elution gradient: 14%-42%, 7 min) to give D-01-M. .sup.1H NMR (400 MHz, DMSO-d6): δ 7.90-7.57 (m, 9H), 7.38-7.10 (m, 10H), 6.93-6.80 (m, 4H), 4.67-4.52 (m, 6H), 4.52-4.44 (m, 3H), 4.22-4.19 (m, 3H), 3.77-3.62 (m, 15H), 3.58-3.47 (m, 16H), 3.46-3.43 (m, 2H), 3.32-3.25 (m, 6H), 3.12-2.91 (m, 14H), 2.36-2.16 (m, 10H), 2.11-1.99 (m, 7H), 1.85-1.73 (m, 9H), 1.59-1.35 (m, 22H), 1.32-1.13 (m, 12H).

Assay Example 1. Test of the Binding Affinity of the Compound to Human-Derived Anti-Asialoglycoprotein Receptors

[0093] Purpose of the Assay:

[0094] The binding affinity of the compound to human-derived anti-asialoglycoprotein receptors (ASGPR) was tested by surface plasmon resonance (SPR) technique, and the kinetic K.sub.D value of the compound was used as an indicator to evaluate the binding affinity of the compound to ASGPR, thereby reflecting the ability of the compound to specifically target hepatocytes for delivery of nucleic acid molecules.

[0095] Materials of the Assay:

[0096] 2.1 Protein:

[0097] Asialoglycoprotein Receptor Protein, Mouse, Recombinant (His Tag), Sino biological-50083-M07H-50 μg; Asialoglycoprotein Receptor Protein, Human, Recombinant (His Tag), Sino biological-10773-H07H-50 μg

[0098] 2.2 Reagent:

[0099] NiHC 1500M Chip (Xan Tec-SCNihc1500m0720); HEPES (SIGMA-V900477); NaCl (SIGMA-71376); Tween-20 (Aladin-T104863); CaCl2 (SIGMA-C3306-250G); EDTA (SIGMA-3609); NiCl.sub.2 (Energy-chemical-V830089); 10×PBS (Sangon-E607016-0500)

[0100] 2.3 Materials & Instruments:

[0101] Biacore 8 k; Series S CM5 chip (GE Healthcare—BR100530) 96-well plate-250 μL (Greiner-650201); 384-well plate-200 μL (Greiner-781270); 96-well plate-1 mL (Greiner-780201); 96 Microplate foils (GE Healthcare-28975816); 384 Microplate foils (GE Healthcare-BR100577)

[0102] 3. Steps and Methods of the Assay:

[0103] 3.1 The two proteins were respectively dissolved in 1×PBS to obtain a solution of 0.25 mg/mL, and the compound to be tested was dissolved in DMSO. The running buffer (10 mM HEPES, 150 mM NaCl, 0.05% Tween 20, 50 mM CaCl.sub.2, 50 μM EDTA) was prepared and filtered through a 0.22 μm membrane.

[0104] 3.2 The NiHC 1500M Chip was docked into a biacore, and the system was switched to a running buffer.

[0105] 3.3 Protein labeling: The chip was first treated with 350 mM EDTA at 30 μl/min for 5 min, rinsed with running buffer for 2 min, and then bound with 40 mM NiCl.sub.2 at 30 μl/min for 2 min. A protein diluted to 5 μg/mL with running buffer was then labeled on the chip at a flow rate of 10 μl/min for a final labeling amount of 1500-2000 RU.

[0106] 3.4 The instrument system was switched to a running buffer containing 2% DMSO.

[0107] 3.5 The compound with a starting concentration of 2 μM was serially diluted 2-fold with a running buffer containing 2% DMSO to obtain 9 concentrations. The final concentration of DMSO was 2%.

[0108] 3.6 Testing: For each cycle, the compound was bound for 60 s and dissociated for 180 s at a flow rate of 50 μl/min. The compound was tested in order of concentration from low to high. Before the gradient testing of the compound, 4 cycles of blank (running buffer with 2% DMSO) were introduced, and solvent correction was performed with 1.5%-3.5% DMSO (6 concentrations).

[0109] 3.7 Analysis of Data:

[0110] The data were analyzed with Biacore Insight Evaluation Software, kinetics was analyzed with a 1:1 model, and affinity was analyzed with a steady state affinity model.

TABLE-US-00002 Sample K.sub.D (mol/L) K.sub.a (L/(mol .Math. s)) K.sub.d (1/s) D-01-M 1.56 × 10.sup.−8 1.08 × 10.sup.6 1.68 × 10.sup.−2

[0111] Conclusions of the Assay:

[0112] In the present assay, the test D-01-M showed good binding affinity in the SPR assay.

[0113] The present disclosure demonstrates a delivery platform for oligonucleotide molecules with high efficiency and high hepatocyte targeting: it can bind to a specific, highly expressed ASGPR protein on the surface of hepatocytes, enter the cellular endosome via endocytosis, and work by release into the cytoplasm. The binding affinity constant of this delivery platform to ASGPR is superior to existing technologies. It shows good tissue distribution and metabolic stability in vivo, and is expected to achieve more efficient delivery and efficacy of liver-targeted molecules. The relevant conjugate in the present disclosure shows good activity for HBsAg reduction and long-term efficacy for HBsAg inhibition.