WO2020259497A1 - 一种新化合物及其应用 - Google Patents

一种新化合物及其应用 Download PDF

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WO2020259497A1
WO2020259497A1 PCT/CN2020/097732 CN2020097732W WO2020259497A1 WO 2020259497 A1 WO2020259497 A1 WO 2020259497A1 CN 2020097732 W CN2020097732 W CN 2020097732W WO 2020259497 A1 WO2020259497 A1 WO 2020259497A1
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synthesis
add
liver
gbl
compound
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PCT/CN2020/097732
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French (fr)
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卢雪琴
穆卓
王圣军
杜艳春
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厦门甘宝利生物医药有限公司
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Priority to JP2021577836A priority Critical patent/JP7383308B2/ja
Priority to KR1020227003111A priority patent/KR20220035398A/ko
Priority to AU2020305793A priority patent/AU2020305793B2/en
Priority to EP20830838.7A priority patent/EP3992291A4/en
Priority to US17/623,569 priority patent/US20230374513A1/en
Publication of WO2020259497A1 publication Critical patent/WO2020259497A1/zh
Priority to US17/807,731 priority patent/US11840691B2/en

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Definitions

  • the invention relates to a new compound and its application to the inhibition of HBV gene expression.
  • the structure of the compound contains interfering nucleic acid that inhibits HBV gene expression, a transfer point and its terminal modified chain. By modifying the end of the chain, 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. Amine, the total number of introduced N-acetylgalactosamine is four. The efficacy experiments of HepG 2 cells and transgenic mice proved that this new compound can continue to inhibit the expression of HBV HBsAg, HBeAg and HBV DNA.
  • RNAi RNA interference
  • dsRNA double-stranded RNA
  • RNAi an endonuclease called “Dicer” cuts or “cuts” long-stranded dsRNA. "D” into small fragments 21-25 nucleotides long. These small fragments are called small interfering RNA (siRNA), and the antisense strand (Guide Strand) is loaded onto the Argonaute protein (AGO2).
  • siRNA small interfering RNA
  • AGO2 loading occurs in the RISC-loading complex, which is a ternary complex composed of Argonaute protein, Dicer, and dsRNA binding protein (referred to as TRBP).
  • TRBP dsRNA binding protein
  • AGO2 uses the antisense strand to bind to the mRNA containing the fully complementary sequence, and then catalyzes the cleavage of these mRNAs, causing the mRNA to split and lose the role of translation template, thereby preventing the synthesis of related proteins. After cleavage, the cleaved mRNA is released, and the RISC-loading complex loaded with the antisense strand is recycled for another round of cleavage.
  • RNAi technology treats diseases at the mRNA level, and has higher efficiency than chemical small molecule drugs and biological macromolecular drugs at the protein level.
  • the sense strand and antisense strand sequences of siRNA with high specificity and good inhibitory effect can be designed according to specific gene sequences. These single-stranded sequences are synthesized through solid phase, and then the sense strand and antisense strand are annealed in a specific manner. The buffer is paired into siRNA according to the base-pairing principle, and finally delivered to the corresponding target in the body through the carrier system to degrade the target mRNA and destroy the function of the target mRNA as a translation template, thereby preventing the synthesis of related proteins.
  • siRNA is unstable in blood and tissues and is easily degraded by nucleases.
  • siRNA backbone can be modified, but these chemical modifications only provide limited protection from nuclease degradation and may eventually affect siRNA ⁇ activity. Therefore, a corresponding delivery system is also needed to ensure the safe and efficient passage of siRNA through the cell membrane. Because siRNA has a large molecular weight, a large amount of negative charge, and high water solubility, it cannot smoothly cross the cell membrane to reach the cell.
  • liposomes The basic structure of liposomes is composed of a hydrophilic core and a phospholipid bilayer. It has a phospholipid bilayer similar to a biological membrane and has high biocompatibility. Therefore, liposomes were once the most popular and most widely used.
  • siRNA carrier Liposome-mediated siRNA delivery mainly encapsulates siRNA into liposomes, protects siRNA from degradation by nucleases, improves the efficiency of siRNA through cell membrane barriers, and promotes cell absorption. For example, anionic liposomes, pH-sensitive liposomes, immunoliposomes, fusogenic liposomes and cationic lipids, etc. Although certain progress has been made, liposomes themselves are prone to trigger inflammation.
  • the asialoglycoprotein receptor (ASGPR) in the liver is a receptor specifically expressed by hepatocytes and a highly efficient endocytic receptor.
  • ASGPR asialoglycoprotein receptor
  • the sugar specifically bound by ASGPR is galactosyl, so it is also called galactose specific receptor .
  • Monosaccharide and polysaccharide molecules such as galactose, galactosamine, and N-acetylgalactosamine have high affinity for ASGPR.
  • ASGPR The main physiological function of ASGPR is to mediate the elimination of asialoglycoprotein, lipoprotein and other substances in the blood, and is closely related to the occurrence and development of liver diseases such as viral hepatitis, cirrhosis, and liver cancer.
  • liver diseases such as viral hepatitis, cirrhosis, and liver cancer.
  • the discovery of this characteristic of ASGPR plays an important role in the diagnosis and treatment of liver-derived diseases (Ashwell G, Harford J, Carbohydrate specific Receptors of the Liver, Ann Rev Biochem 1982 51:531-554).
  • the therapeutic drugs for liver-derived diseases containing galactose or galactosamine and its derivatives in the structure can specifically affinity with ASGPR, so that it has active liver targeting and does not require other carrier systems for delivery.
  • the invention relates to a new compound and its application to the inhibition of HBV gene expression.
  • the structure of the compound contains interfering nucleic acid that inhibits HBV gene expression, a transfer point and its terminal modified chain. The terminal modified strand and the interfering nucleic acid are connected through a transfer point.
  • two or three N-acetylgalactosamine can be introduced at the 3'end of the antisense strand of this siRNA, and two or one N-acetylgalactose can be introduced correspondingly at the 5'end of the sense strand.
  • Amine a total of four N-acetylgalactosamine, is a brand-new introduction method. Through in vivo and in vitro pharmacodynamic experiments, this new compound can continue to efficiently express HBV HBsAg, HBeAg and HBV DNA. inhibition.
  • a compound containing an interfering nucleic acid that inhibits HBV gene expression, a transfer point, and a terminal modified chain thereof in a structure is provided.
  • the terminal modified chain is composed of a connecting chain D, a linker B, a branched chain L, and a liver.
  • the targeting specific ligand X is composed of, the terminal modified strand and the interfering nucleic acid are connected by a transfer point R 1 /R 2 and the structure is shown in the general formula (I):
  • n When n is 1, m is 3; when n is 2, m is also 2;
  • R 1 is -NH(CH 2 ) x CH 2 -, where x can be an integer of 3-10;
  • R 2 is -NHCH 2 CH(OH)CH 2 (OH)-, or other nitrogen-containing structure with both primary and secondary alcohols or a single primary alcohol.
  • This structure can be a straight chain or a straight chain with The branched chain may also have a cyclic structure.
  • R 2 can be a pyrrole ring or a piperidine ring with primary and secondary alcohols, specifically selected from the following structures:
  • the liver targeting specific ligand X is selected from galactose, galactosamine and N-acetylgalactosamine;
  • the branched chain L is a C3-C18 straight chain, and the straight chain contains a carbonyl group, an amide group, a phosphoryl group, an oxygen atom or a combination of these groups;
  • the linker B is selected from the following structural formulas:
  • a 2 is C, O, S, amino, carbonyl, amide, phosphoryl or sulfur Phosphoryl;
  • the linking chain D contains C5-C20, which contains an amino group, a carbonyl group, an amide group, an oxygen atom, a sulfur atom, a thiophosphoryl group, a phosphoryl group, a cyclic structure or a combination of these groups.
  • the interfering nucleic acid includes but is not limited to siRNA, miRNA or Agomir, preferably siRNA, more preferably siRNA against hepatitis B virus.
  • the sequence design of the 19mer of the siRNA used for HBV RNAi is designed according to the HBV cDNA target sequence (GenBank Accession#AF100309.1). These target sequences include the core region of 19mer de1 and the corresponding DNA sequences that are mainly expanded or displaced based on these core regions. Its purpose is to find the best effective sequence through the basic target site, and these sequences can be partially or fully adapted to the target site and can be used to treat chronic hepatitis B.
  • the 19mer nucleotide sequence of the target includes two strands, the sense strand (S) and the antisense strand (AS).
  • the 19th nucleotide (5′ ⁇ 3′) of the sense strand can be formed with the first nucleotide (5′ ⁇ 3′) of the antisense strand according to the Watson-Crick principle Base pairs.
  • the 1-19 bases of the sense strand (5' ⁇ 3') can pair with the 19-1 bases of the antisense strand (5' ⁇ 3') and its corresponding bases to form a double strand.
  • One to three unpaired bases can be tolerated in the terminal position in the double strand.
  • the basic sequence of HBV siRNA can be screened according to actual applications. Shift the 3'or 5'end according to the basic sequence to screen out more effective and specific sequences.
  • the best choice for the single-stranded protruding part at the 3'end of the sense strand or the antisense strand is TT, UU, AU, or UA to obtain the changed sequence.
  • Any sense strand can be used to form a double strand with the antisense strand, and the two strands must maintain a continuous base pairing of at least 16, 17, 18, 19, 20, 21, 22, or 23. Some of the listed sequences may be 1, 2, or 3 different from the target.
  • the last base at the 3'end of the sense strand can be U, A, or T.
  • the last position at the 3'end of the antisense strand can be U, A, or T.
  • the present invention screens out the following candidate sequences:
  • each monomer of siRNA is modified without affecting or even enhancing the activity.
  • one, two or three incompletely matched bases can be allowed.
  • the nucleotides can have different modification groups, and they can be whole chain or part modified. There can be one, multiple, or even all thio-containing bases in each chain.
  • the modified sense strand and antisense strand are selected from the following sequences:
  • mG 2'-O-methylguanylic acid
  • mA 2'-O-methyladenosine
  • mU 2'-O-methyluronic acid
  • mC 2'-O-methylcytidine acid
  • mGs 2'-O-methyl-3'-thioguanylic acid
  • mAs 2'-O-methyl-3'-thioadenosine
  • mUs 2'-O-methyl-3'- Thioururic acid
  • mCs 2'-O-methyl-3'-thiocytidylic acid
  • fG 2'-fluoroguanylic acid
  • fA 2'-fluoroadenylic acid
  • fU 2'-fluorouridine acid
  • fC 2'-fluorocytidine acid
  • fGs 2'-fluoro-3'-thioguanylic acid
  • fAs 2'-fluoro-3'-thioadenylic acid
  • fUs 2'-fluoro-3'-thiouridine acid
  • fCs 2'-Fluoro-3'-thiocytidylic acid.
  • the modified sense strand and antisense strand are selected from the following sequences:
  • the terminal modified chain is composed of a connecting chain D, a linker B, a branched chain L containing a sterically stable structure, and a liver targeting specific ligand X.
  • the general formula (II) of the terminal modified chain is:
  • the liver targeting specific ligand X may be one or more polysaccharides, polysaccharide derivatives, or monosaccharides and monosaccharide derivatives.
  • the general formula (III) of the liver targeting specific ligand X is:
  • R 1 , R 2 and R 3 are respectively hydrogen or a hydroxyl protecting group.
  • the liver targeting specific ligand X is selected from one or more of galactose, galactosamine, N-acetylgalactosamine or the following structures:
  • R 1 is selected from one or two of OH, NHCOH or NHCOCH 3 .
  • the branched chain L containing a sterically hindered stable structure is a C3-C18 straight chain, which contains one or more carbonyl groups, amide groups, phosphoryl groups, oxygen atoms or a combination of these groups, which can be selected from the following structures One or more of:
  • r1 is a positive integer of 1-12
  • r2 is an integer of 0-20
  • Z is H or CH 3 .
  • the linker B is selected from the following structural formulas:
  • the linker B is selected from the following structural formulas:
  • r1, r3, r4, and r5 are positive integers from 1-15, respectively; r6 is a positive integer from 1-20, r7 is a positive integer from 2-6, and r8 is a positive integer from 1-3.
  • linker B is selected from the following structures:
  • the linking chain D contains C5-C20, and may contain an amino group, a carbonyl group, an amide group, an oxygen atom, a sulfur atom, a thiophosphoryl group, a phosphoryl group, a cyclic structure or a combination of these groups.
  • the connecting chain D is selected from one of the following structures:
  • each n is a positive integer from 1-20, and each n is the same or different positive integer.
  • p is a positive integer of 1-6;
  • s is a positive integer of 2-13;
  • R 1 and R 2 are the same or different substituent groups, and their structural formula is one of the following structures: -H, -CH 3 ,- CH-(CH 3 ) 2 , -CH 2 -CH(CH 3 ) 2 , -CH(CH 3 )-CH 2 -CH 3 , -CH 2 -C 6 H 5 , -C 8 NH 6 , -CH 2 -C 6 H 4 -OH, -CH 2 -COOH, -CH 2 -CONH 2 , -(CH 2 ) 2 -COOH, -(CH 2 ) 4 -NH 2 , -(CH 2 ) 2 -CONH 2 , -(CH 2 )-S-CH 3 , -CH 2 -OH, -CH(CH
  • connecting chain D is selected from one of the following structures:
  • the modified chain at the 5'end of the sense chain has one N-acetylgalactosamine or two N-acetylgalactosamine, selected from one of the following structures:
  • the modified chain at the 5'end of the sense chain of the compound is selected from the structural formulas in the following table:
  • the modified chain at the 3'end of the antisense strand of the compound has two or three N-acetylgalactosamines, and the modified chain One of the following structures:
  • the modified chain at the 3'end of the antisense strand of the compound is preferably selected from the structural formulas in the following table:
  • the combination of the 5'end modified strand of the sense strand and the 3'end modified strand of the antisense strand in the compound is preferably one of the structures shown in the following table:
  • the compound of the present invention includes the 5'end of the sense strand and the 3'end of the antisense strand with access to the modified chain as shown in the following table:
  • the structure of the compound of the present invention is shown in the following table:
  • liver-related diseases include acute and chronic hepatitis, liver cancer, inherited liver-derived diseases, liver Cirrhosis, fatty liver, diabetes.
  • Another aspect of the present invention provides the application of the compound of the present invention in the preparation of a medicament for the treatment of HBV infection related diseases, wherein the HBV infection includes chronic hepatitis B virus infection and acute hepatitis B virus infection .
  • the liver-targeting specific ligand X is specific for the asialoglycoprotein receptor (ASGPR) in the liver, the related disease of HBV infection is chronic hepatitis B, and the compound can continuously make HBV The expression of HBsAg, HBeAg and HBV DNA is suppressed.
  • ASGPR asialoglycoprotein receptor
  • Another aspect of the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the compound of the present invention and pharmaceutically acceptable auxiliary materials, and the preferred dosage form is a subcutaneous injection.
  • liposome-mediated siRNA delivery mainly involves liposomes encapsulating siRNA into liposomes, protecting siRNA from degradation by nucleases, and improving the barriers of siRNA passing through cell membranes. The efficiency, thereby promoting cell absorption.
  • Liposomes such as anionic liposomes, pH-sensitive liposomes, immunoliposomes, fusogenic liposomes and cationic lipids, etc.
  • liposomes are easy to Inflammation is triggered, a variety of antihistamines and hormones such as ceritizine and dexamethasone must be used before administration to reduce the possible acute inflammation, so it is not suitable for all therapeutic areas in actual clinical applications Especially for diseases with long treatment cycles like chronic hepatitis B, the accumulated toxicity produced by long-term use may be a potential safety hazard.
  • the siRNA drugs currently in phase I/II for the treatment of chronic hepatitis B include ARO-HBV and ALN-HBV02.
  • ARO-HBV is at the 5'end of the sense strand of siARNA, and three N-acetylgalactosamine is introduced through the linking strand;
  • ALN-HBV02 is at the 3'end of the siRNA sense strand, and three N-acetyl halves are introduced through the linking strand Lactosamine.
  • the above-mentioned drugs introduce galactosamine in the sense chain, and all three N-acetylgalactosamines are introduced.
  • the 5'end of the siRNA sense strand and the 3'end of the antisense strand simultaneously introduce different or the same amount of N-acetylgalactosamine.
  • N-acetylgalactosamine at the 5'end and antisense strand of the sense strand.
  • the introduction of the 3'end of the chain at the same time, especially the introduction of three N-acetylgalactosamines at the 3'of the antisense strand is a brand-new introduction method. It is demonstrated through the demonstration that this introduction method enables siRNA to effectively inhibit HBV The effect of genes.
  • Figure 1 is a high-resolution mass spectrum of 5’YICd-01-c4;
  • Figure 2 is a high-resolution mass spectrum of 5’YICc-01-c7;
  • Figure 3 is a high-resolution mass spectrum of 5’ERCd-01-c7;
  • Figure 4 is a high-resolution mass spectrum of 5’ERCc-01-c4;
  • Figure 5 is a high-resolution mass spectrum of 3’SANCd-01-c6;
  • Figure 6 is a bar graph showing the inhibitory effect on HBsAg in HepG2.215 cells
  • Figure 7 is a bar graph showing the inhibitory effect on HBeAg in HepG2.215 cells
  • Figure 8 is a bar graph showing the inhibitory effect on HBVDNA in HepG2.215 cells
  • Figure 9 is a bar graph showing the inhibitory effect of HBV gene on transgenic mouse model
  • Figure 10 shows the effect of GBL-0401 on inhibiting HBV and HBsAg in vivo.
  • the Chinese name of DMF is N,N-dimethylformamide
  • HBTU O-benzotriazole-tetramethylurea hexafluorophosphate
  • DIPEA N,N-diisopropylethylamine
  • the Chinese name of DCM is dichloromethane
  • the Chinese name of DMAP is 4-dimethylaminopyridine
  • DMT-CL 4,4'-dimethoxytriphenylchloromethane
  • THF tetrahydrofuran
  • the Chinese name of TBTU is O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate;
  • the Chinese name of DBU is 1,8-diazabicycloundec-7-ene
  • HOBt 1-hydroxybenzotriazole
  • the Chinese name of DCC is dicyclohexylcarbodiimide
  • the Chinese name of Pd-C is Palladium Carbon Catalyst
  • the Chinese name is solid phase carrier, such as resin (Resin).
  • 5'YICd-01-c4 2.2g (3.2mmol) was dissolved in 30mL methanol, and 1.0g of 10% Pd-C (wet Degussa type E101NE/W) was added. Then hydrogenate under normal pressure and react overnight. The reaction solution was filtered with Celite, and the filtrate was evaporated to dryness under reduced pressure to obtain 1.70 g of white foam.
  • Pd-C wet Degussa type E101NE/W
  • mG is the starting monomer
  • the C6NH phosphoramidite monomer is the terminal monomer.
  • Different phosphoramidite monomers are introduced through solid phase phosphoramidite coupling.
  • the basic steps of the solid-phase phosphoramidite method include: 1) deprotection: remove the protective group (DMT) on the oxygen atom on the solid support; 2) coupling: add the first nucleotide monomer, pass 3' Coupling reaction occurs in the 5'direction; 3) Oxidation: the resulting nucleoside phosphite is oxidized to a more stable nucleoside phosphate (ie trivalent phosphorus is oxidized to pentavalent phosphorus); 4) Blocking: the unreacted In the previous step, the nucleotide monomer 5'-OH is added and sealed so that it will no longer participate in the reaction; repeat the above steps until the desired sequence is completed.
  • methylamine ethanol solution and ammonia are used to cleave the ester bond between the connecting compound and the initial nucleoside on the solid support, and each base on the oligonucleotide is combined with the protective group cyanoethyl (P ), benzoyl (mA, fA), acetyl (mC, fC), isobutyryl (mG, fG) and 4-methoxytrityl (C6NH) are removed.
  • P cyanoethyl
  • benzoyl mA, fA
  • acetyl mC, fC
  • isobutyryl mG, fG
  • 4-methoxytrityl C6NH
  • 3'SANCd-01-c1 (5.480g, 0.030mol) was dissolved in pyridine (30mL), cooled, DMT-CL (10.423g, 0.031mol) was added in batches, and reacted overnight in the dark, and the pyridine was removed by rotary evaporation. The residue was dissolved in CH 2 Cl 2 (50 mL), washed with saturated brine (50 mL), the organic phase was dried over anhydrous sodium sulfate, filtered, rotary evaporated, and passed through the column. Get the product 3'SANCd-01-c2 (10.805g).
  • 3'SANCd-01-c2 (10.805g, 0.022mol) was dissolved in methanol (60mL) and THF (30mL), cooled, and KOH (5.69g) aqueous solution (24mL) was added dropwise, reacted at room temperature for 2h, and the methanol and THF. The residue was added with water (50 mL), extracted with EtOAc (30 mL*3), washed with saturated brine (50 mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and evaporated. Pass the column with the eluent containing 1% triethylamine to obtain the product 3'SANCd-01-c3 (8.286g).
  • 3'SANCd-01-c3 (2.890g, 0.007mol) was dissolved in CH 2 Cl 2 (20mL), cooled, DCC (1.680g) in CH 2 Cl 2 solution (10mL) was added dropwise, stirred for 20 minutes, and then added Diacid monomethyl ester (1.522g) in CH 2 Cl 2 solution (10 mL), react at room temperature overnight, quench with 5% NaHCO 3 (20 mL), extract with CH 2 Cl 2 (20 mL*2), saturated brine (10 mL) Washing, drying with anhydrous sodium sulfate, filtering, rotary evaporation, and passing the column with an eluent containing 1% triethylamine to obtain the product 3'SANCd-01-c4 (3.193g).
  • 3'SANCd-01-c4 (2.193g, 0.004mol) was dissolved in THF (10mL), cooled, and an aqueous solution (4.5g) of LiOH (0.645g) was added dropwise. TLC showed no raw material after 2h reaction. The reaction solution was rotary evaporated to remove the solvent, the residue was neutralized with saturated ammonium chloride, extracted with CH2Cl2 (20 mL*2), and washed with saturated brine (10 mL). Drying with anhydrous sodium sulfate, filtering, rotary evaporation, and passing the column with an eluent containing 1% triethylamine to obtain the product 3'SANCd-01-c5 (1.979g).
  • 3'SANCd-01-c5 (0.389g, 0.004mol), dissolved in DMF (2mL), cool, add DIPEA (0.15mL), TBTU (0.183g), stir for 10 minutes, then add dlSANC-c12 (0.756g, 0.0005mol) in DMF (2mL) solution at room temperature overnight. Quench with water (20mL), extract with CH 2 Cl 2 (20mL*2), wash with saturated brine (10mL), dry with anhydrous sodium sulfate, filter, rotary evaporate, and pass the column with eluent containing 5% triethylamine , The product 3'SANCd-01-c6 (0.803g) was obtained, and its high-resolution mass spectrum is shown in Figure 5.
  • phosphoramidite monomers are introduced through solid-phase phosphoramidite coupling, mU is the starting monomer, and mU is the terminal monomer.
  • the basic steps of the solid-phase phosphoramidite method include: 1) deprotection: remove the protective group (DMT) on the oxygen atom on the 3'SANCd-01 resin; 2) coupling: add the first nucleotide monomer, The coupling reaction occurs through the 3'to 5'direction; 3) Oxidation: the resulting nucleoside phosphite is oxidized to a more stable nucleoside phosphate (ie trivalent phosphorus is oxidized to pentavalent phosphorus); 4) blocked: The unreacted nucleoside monomer 5'-OH in the previous step is added and sealed so that it will no longer participate in the reaction; repeat the above steps until the desired sequence is completed.
  • methylamine ethanol solution and ammonia are used to cleave the ester bond between the connecting compound and the initial nucleoside on the solid support, and each base on the oligonucleotide is combined with the protective group cyanoethyl (P ), benzoyl (mA, fA), acetyl (mC, fC) and isobutyryl (mG, fG) are removed.
  • P cyanoethyl
  • benzoyl mA, fA
  • acetyl mC, fC
  • isobutyryl mG, fG
  • mG is the starting monomer, and the C6NH phosphoramidite monomer is the terminal monomer.
  • Different phosphoramidite monomers are introduced through solid phase phosphoramidite coupling. The synthesis steps are the same as in Example 1.2 solid phase synthesis.
  • 3'SANCc-01 resin The synthesis route and process steps of 3'SANCc-01 resin are the same as 3'SANCd-01 resin except for the synthesis of 3'SANCc-01-c6.
  • 3'SANCd-01-c5 (0.295g), dissolved in DMF (2mL), cooled, added DIPEA (0.14mL), TBTU (0.177g), stirred for 10 minutes, then added SANC-c12 (0.756g) DMF ( 2mL) solution, react overnight at room temperature. Quench with water (50mL), extract with CH 2 Cl 2 (20mL*2), wash with saturated brine (10mL), dry with anhydrous sodium sulfate, filter, rotary evaporate, and pass the column with eluent containing 5% triethylamine , Get the product 3'SANCc-01-c6 (0.815g).
  • mU is the starting monomer, and mU is the terminal monomer.
  • Different phosphoramidite monomers are introduced through solid-phase phosphoramidite coupling. The synthesis steps are the same as in Example 2.2. Solid-phase synthesis of Kyas-01.
  • mG is the starting monomer, and the C6NH phosphoramidite monomer is the terminal monomer.
  • Different phosphoramidite monomers are introduced through solid phase phosphoramidite coupling. The synthesis steps are the same as in Example 1.2 solid phase synthesis.
  • mA is the starting monomer
  • T is the terminal monomer
  • different phosphoramidite monomers are introduced through solid phase phosphoramidite coupling.
  • the synthesis steps are the same as in Example 2.2. Solid-phase synthesis of Kyas-01.
  • mA is the starting monomer
  • C6NH phosphoramidite monomer is the terminal monomer.
  • Different phosphoramidite monomers are introduced through solid-phase phosphoramidite coupling. The synthesis steps are the same as in Example 1.2 solid phase synthesis.
  • mG is the starting monomer
  • mU is the terminal monomer
  • different phosphoramidite monomers are introduced through solid-phase phosphoramidite coupling.
  • the synthesis steps are the same as in Example 2.2. Solid-phase synthesis of Kyas-01.
  • mU is the starting monomer, and mU is the terminal monomer.
  • Different phosphoramidite monomers are introduced through solid-phase phosphoramidite coupling. The synthesis steps are the same as in Example 2.2. Solid-phase synthesis of Kyas-01.
  • mU is the starting monomer
  • C9NH phosphoramidite monomer is the terminal monomer.
  • Different phosphoramidite monomers are introduced through solid phase phosphoramidite coupling.
  • the synthesis steps are the same as in Example 1.2 for solid-phase synthesis of C6NH-S-01.
  • Example 7 GBL0405 ⁇ GBL0408 and GBL0411 ⁇ GBL0418 refer to the synthesis of GBL-0401 ⁇ GBL0404.
  • Blank control group Add DMEM medium containing 2% FBS and incubate for 72 hours.
  • Test product group add test product diluents with a concentration of 5nM, 0.5nM or 0.05nM, 3 replicate wells for each concentration, and culture for 72h in a 37°C 5% CO 2 incubator.
  • HepG2.2.15 cells were cultured in a 96-well cell culture plate, and the fresh medium was changed every three days. On the 6th day, the above-mentioned drug-containing medium of different concentrations was added, and the culture was continued to the 9th day. The supernatant is collected, and the detection kit detects the content of HBsAg, HBeAg and HBV DNA in the cell supernatant. The OD value was compared with the control non-administered group, and the effectiveness was determined according to the ratio.
  • mice of appropriate age requiring significant HBsAg expression
  • pick out 90 mice weighing about 25g and randomly divide them into 18 groups, 5 mice in each group, and give them by subcutaneous injection on Day0: 3mg/kg; administration volume is 100-200 ⁇ L.
  • do mouse blood HBsAg determination try to keep the average level of HBsAg in each group consistent.
  • GBL-0405 500 ⁇ g 93.3% 5 GBL-0406 500 ⁇ g 91.3% 6 GBL-0407 500 ⁇ g 88.3% 7 GBL-0408 500 ⁇ g 94.4% 8 GBL-0409 500 ⁇ g 92.3% 9 GBL-0410 500 ⁇ g 93.6% 10 GBL-0411 500 ⁇ g 90.5% 11 GBL-0412 500 ⁇ g 89.5% 12 GBL-0413 500 ⁇ g 94.8% 13 GBL-0414 500 ⁇ g 92.5% 14 GBL-0414 500 ⁇ g 90.6% 15 GBL-0415 500 ⁇ g 91.5% 16 GBL-0416 500 ⁇ g 93.4% 17 GBL-0417 500 ⁇ g 91.7% 18 GBL-0418 500 ⁇ g 92.5% 19 Normal saline 500ml/bottle 0.9%
  • Example 10 Continuous study of the inhibitory effect of GBL-0401 on the expression of HBsAg in the transgenic mouse model HBV
  • mice choose age-appropriate male HBV transgenic mice (requiring significant HBsAg expression) for experimental evaluation, pick out 10 mice weighing about 25g, and randomly divide them into 2 groups, 5 in each group, which are the control group and the administration group. On Day 0, 3 mg/kg was administered by subcutaneous injection; the administration volume was 100-200 ⁇ L. Before medication, blood was collected to determine HBsAg, try to keep the HBsAg level in each group consistent. Whole blood was collected from the mouse orbital venous plexus. The blood sampling time points were before administration (day 0), after administration-week 1, week 2, week 3, week 4, week 5, and week 6. Detect HBsAg to investigate the persistence of GBL-0401's inhibition of HBV gene expression.
  • GBL-0401 reached the best inhibitory effect 99.08% in the first week, and the trend declined from the second to the third week, but still showed a high inhibition rate of about 90%, and the fourth to sixth week showed a downward trend. , But still maintain the inhibitory effect at about 75%.
  • GBL-0401 has a continuous inhibitory effect on the expression of HBV and HBsAg, and can stably inhibit the expression for about 6 weeks.
  • Figure 10 shows the effect of GBL-0401 on inhibiting HBV and HBsAg in vivo.

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Abstract

涉及一种新的化合物及其对HBV基因表达抑制的应用。该化合物结构中含有抑制HBV基因表达的干扰核酸、转接点及其末端修饰链。通过末端修饰链，可以在这种siRNA的反义链3'端引入两个或三个的N-乙酰基半乳糖胺，在正义链5'端相应引入两个或一个N-乙酰基半乳糖胺，引入的N-乙酰基半乳糖胺总数量为四。通过体内外药效实验证明，这种新的化合物可以持续高效的抑制HBV基因表达。

Description

一种新化合物及其应用 技术领域
本发明涉及一种新的化合物及其对HBV基因表达抑制的应用。该化合物结构中含有抑制HBV基因表达的干扰核酸、转接点及其末端修饰链。通过末端修饰链，可以在这种siRNA的反义链3’端引入两个到三个的N-乙酰基半乳糖胺，在正义链5’端相应引入两个到一个N-乙酰基半乳糖胺，引入的N-乙酰基半乳糖胺的总数量为四个。通过HepG 2细胞和转基因小鼠的药效实验证明，这种新的化合物可以持续使HBV的HBsAg、HBeAg和HBV DNA的表达得到抑制。
背景技术
RNAi
RNAi(RNA干扰)于1998年，由安德鲁·法厄(Andrew Z.Fire)等在秀丽隐杆线虫中进行反义RNA抑制实验时发现，并将这一过程称为RNAi。这一发现被《Science》杂志评为2001年的十大科学进展之一，并名列2002年十大科学进展之首。自此以后，以RNAi为作用机理的siRNA作为潜在的基因治疗药物得到人们广泛的关注，2006年，安德鲁·法厄与克雷格·梅洛(Craig C.Mello)由于在RNAi机制研究中的贡献获得诺贝尔生理或医学奖。RNAi是在许多生物中，包括动物、植物和真菌，都可由双链RNA(dsRNA)触发的，在RNAi过程中，一种称为“Dicer”的核酸内切酶将长链dsRNA切割或“切丁”成21～25个核苷酸长的小片段。这些小片段，被称为小干扰RNA(siRNA)，其中的反义链(Guide strand)被加载到Argonaute蛋白(AGO2)上。AGO2加载发生在RISC-loading复合物中，这是一个三元复合物，由Argonaute蛋白、Dicer和dsRNA结合蛋白(简称为TRBP)组成。在装载过程中，正义链(Passenger strand)链被AGO2裂解并排出。然后，AGO2使用反义链与包含完全互补序列的mRNA结合，然后催化这些mRNA的切割，致使mRNA分裂丧失翻译模板的作用，进而阻止相关蛋白质的合成。切割后，被切割的mRNA被释放，加载着反义链的RISC-loading复合物被循环用于另一轮的切割。
据统计，在人体内的疾病相关蛋白中，大约超过80％的蛋白质不能被目前常规的小分子药物以及生物大分子制剂所靶向，属于不可成药蛋白。旨在通过基因的表达、沉默等功能治疗疾病的基因治疗被业界认为是继化学小分子药物、生物大分子药物之后的第三代治疗药物，这种疗法在基因水平上实现对疾病的治疗，不受不可成药蛋白的制约。作为基因治疗中RNAi技术最主流的类型，RNAi技术是从mRNA的水平对疾病进行治疗，相比化学小分子药物及生物大分子药物在蛋白质水平的治疗具有更高的效率。利用RNAi技术，可以根据特定基因序列，设计出特异性高、抑制效果好的siRNA的正义链和反义链序列，通过固相合成这些单链序列，然后正义链与反义链在特定的退火缓冲液中按照碱基配对原则配对成siRNA，最后通过载体系统输送到体内相应靶点，降解目标mRNA，破坏目标mRNA作为翻译模板的功能，从而阻止相关蛋白的合成。
siRNA的递送系统
siRNA在血液和组织中不稳定，容易被核酸酶降解，为了提高siRNA的稳定性，可以通过对siRNA骨架修饰，但这些化学修饰只提供有限的免受核酸酶降解的保护作用并且可能最终影响siRNA的活性。因此，还需要相应的传递系统来保障siRNA安全高效的穿过细胞膜。由于siRNA分子质量较大，且带有大量负电荷，而且具有高水溶解性，所以自身无法顺利穿越细胞膜到达细胞内。
脂质体基本结构是由亲水核和磷脂双分子层构成，具备类似生物膜的磷脂双分子层，拥有很高的生物相容性，所以脂质体一度成为最受欢迎、应用最广泛的siRNA载体。脂质体 介导的siRNA递送主要将siRNA包裹到脂质体内，保护siRNA不被核酸酶降解，提高siRNA的通过细胞膜障碍的效率，从而促进细胞的吸收。例如阴离子脂质体、pH敏感性脂质体、免疫脂质体、膜融合脂质体(fusogenic liposome)和阳离子脂质等等，尽管取得了一定的进展，但脂质体本身容易引发炎症反应，给药前必须使用多种抗组胺和激素类如西利替嗪和地塞米松类等药物，以减少可能发生的急性炎症反应，因此在实际临床应用中并不适合所有治疗领域，尤其像慢性乙肝这一类治疗周期长的疾病，长期使用可能产生的积蓄毒性是潜在的安全隐患。
去唾液酸糖蛋白受体(ASGPR)
肝脏中去唾液酸糖蛋白受体(ASGPR)，是肝细胞特异性表达的受体，是一种高效的内吞型受体。由于体内生理情况下各种糖蛋白在酶或酸水解唾液酸后，暴露出的次末端是半乳糖残基，所以ASGPR特异性结合的糖为半乳糖基，故又称半乳糖特异性受体。半乳糖、半乳糖胺、N-乙酰半乳糖胺等单糖和多糖分子对ASGPR有高亲和性。ASGPR主要生理功能是介导血液中去唾液酸糖蛋白、脂蛋白等物质的清除，且与病毒性肝炎、肝硬化、肝癌等肝脏疾病的发生发展有着密切联系。ASGPR这一特性的发现，对肝源性疾病的诊断及治疗起着重要作用(Ashwell G、Harford J，Carbohydrate specific Receptors of the Liver，Ann Rev Biochem 1982 51:531-554)。结构中含有半乳糖或半乳糖胺及其衍生物的肝源性疾病治疗药物可以特异性地与ASGPR亲和，从而具有主动肝靶向性，不需要其它的载体系统来输送。
发明内容
本发明涉及一种新的化合物及其对HBV基因表达抑制的应用。该化合物结构中含有抑制HBV基因表达的干扰核酸、转接点及其末端修饰链。末端修饰链与干扰核酸之间通过转接点连接。通过末端修饰链，可以在这种siRNA的反义链3’端引入两个或三个的N-乙酰基半乳糖胺，在正义链5’端相应引入两个或一个N-乙酰基半乳糖胺，总数四个N-乙酰基半乳糖胺，是一种全新的引入方式，通过体内外药效实验证明，这种新的化合物可以持续使HBV的HBsAg、HBeAg和HBV DNA的表达得到高效抑制。
本发明一个方面，提供了一种结构中包含有抑制HBV基因表达的干扰核酸、转接点及其末端修饰链的化合物，所述末端修饰链由连接链D、接头B、支链L和肝靶向特异性配体X组成，所述末端修饰链与所述干扰核酸之间通过转接点R 1/R 2连接，其结构为通式(I)所示：
Figure PCTCN2020097732-appb-000001
其中：
n为1时，m为3；n为2时，m也为2；
R 1为-NH(CH 2) xCH 2-，其中x可以是3-10的整数；
R 2为-NHCH 2CH(OH)CH 2(OH)-，或其它同时带有伯醇和仲醇或单独的伯醇的含氮的结构，这种结构可以是一条直链或直链带有支链，也可以是环状结构。在一些优选实施方案中，R 2可以是带有伯醇和仲醇的吡咯环或哌啶环，具体选自以下结构：
Figure PCTCN2020097732-appb-000002
所述肝靶向特异性配体X选自半乳糖、半乳糖胺和N-乙酰半乳糖胺；
所述支链L是C3-C18的直链，该直链中含有羰基、酰胺基、磷酰基、氧原子或这些基团的组合；
所述接头B选自以下结构式：
Figure PCTCN2020097732-appb-000003
其中，A 1是C、O、S或NH；r1为1-15的正整数，r2为0-5的整数；A 2是C、O、S、氨基、羰基、酰胺基、磷酰基或硫代磷酰基；
所述连接链D含有C5-C20，其含有氨基、羰基、酰胺基、氧原子、硫原子、硫代磷酰基、磷酰基、环状结构或这些基团的组合。
在上述技术方案中，所述干扰核酸包括但不限于siRNA、miRNA或Agomir，优选为siRNA，进一步优选为抗乙肝病毒的siRNA。
所述的siRNA用于HBV RNAi的19mer的序列设计是依照HBV cDNA靶序列(GenBank Accession#AF100309.1)设计的。这些靶点序列包括19mer de1核心区域和以这些核心区域为主的扩展或位移的相应的DNA序列。其目的是通过基本的靶位点找到最佳有效序列，而这些序列可以部分或者全部与靶点位置相适应，能够应用于治疗慢性乙型肝炎。靶点的19mer核苷酸序列包括两条链，正义链(S)和反义链(AS)。其中正义链的第19个核苷酸(5′→3′)可以和反义链的第1个核苷酸(5′→3′)按照华生-克里克(Watson-Crick)原则形成碱基对。按照此原则，正义链(5′→3′)的1-19个碱基可以和反义链(5′→3′)的19-1个碱基与其相对应的碱基配对形成双链。在双链中末端位置可以容许一到三个不配对的碱基存在。在本发明中可以根据实际应用，筛选出HBV siRNA基本序列。按照基本序列进行3’或5’端位移，筛选出更为有效和特异性的序列。按照siRNA结构的研究结果，正义链或反义链3'端的单链突出部分的最优选择是TT、UU、AU或者UA，得到变化序列。任一条正义链可以用来和反义链形成双链，两条链必须保持一个连续的至少16、17、18、 19、20、21、22或23个碱基配对。有些所列出来的序列可能和靶点有1、2或3个不同，在正义链的3′端的最后一个碱基可以是U、A或T。在反义链的3′端的最后一个位置可以是U、A或T。按照上述原则，本发明筛选出以下候选序列：
Figure PCTCN2020097732-appb-000004
Figure PCTCN2020097732-appb-000005
为了siRNA在组织中的稳定性，siRNA的每个单体在没有影响活性甚至增强活性的情况下，进行修饰。在正义链和反义链中可以容许有1个、2个或3个不完全配对的碱基存在。其中的核苷酸可以带有不同的修饰基团，可以整条链或者部分修饰。其中的每一条链中可以有一个、多个甚至全部带有硫代的碱基。
本发明所述的化合物中，修饰后的正义链和反义链选自下列序列：
Figure PCTCN2020097732-appb-000006
Figure PCTCN2020097732-appb-000007
其中：
mG、mA、mC和mU为2′-甲氧基(2′-OMe)修饰的核苷酸；fG、fA、fC和fU为2′-氟修饰的核苷酸；s为硫代磷酸核苷间键，其余的核苷酸单体通过磷酸二酯键连接。详述如下：
G＝鸟苷酸、A＝腺苷酸、U＝尿苷酸、C＝胞苷酸、dT或T＝2′-脱氧胸腺嘧啶核苷酸；
Gs＝3′-硫代鸟苷酸、As＝3′-硫代腺苷酸、Us＝3′-硫代尿苷酸、Cs＝3′-硫代胞苷酸、dTs或Ts＝2′-脱氧-3′-硫代胸腺嘧啶核苷酸；
mG＝2'-O-甲基鸟苷酸、mA＝2'-O-甲基腺甘酸、mU＝2'-O-甲基尿甘酸、mC＝2'-O-甲基胞苷酸；
mGs＝2'-O-甲基-3’-硫代鸟苷酸、mAs＝2'-O-甲基-3'-硫代腺甘酸、mUs＝2'-O-甲基-3'-硫代尿甘酸、mCs＝2'-O-甲基-3'-硫代胞苷酸；
fG＝2'-氟鸟苷酸、fA＝2'-氟腺苷酸、fU＝2'-氟尿苷酸、fC＝2'-氟胞苷酸；
fGs＝2'-氟-3'-硫代鸟苷酸、fAs＝2'-氟-3'-硫代腺苷酸、fUs＝2'-氟-3'-硫代尿苷酸、fCs＝2'-氟-3'-硫代胞苷酸。
进一步优选地，在一些优选实施例方案中，在所述化合物中，修饰后的正义链和反义链选自下列序列：
Figure PCTCN2020097732-appb-000008
所述末端修饰链是由连接链D、接头B、含有位阻稳定结构的支链L和肝靶向特异性配体X组成，该末端修饰链的通式(II)为：
Figure PCTCN2020097732-appb-000009
当n＝1时，结构通式(II)为：
Figure PCTCN2020097732-appb-000010
当n或m＝2时，结构通式(II)为
Figure PCTCN2020097732-appb-000011
当m＝3时，结构通式(II)为：
Figure PCTCN2020097732-appb-000012
所述肝靶向特异性配体X可以是一种或多种多糖、多糖衍生物或单糖和单糖衍生物。
优选地，所述肝靶向特异性配体X的通式(III)为：
Figure PCTCN2020097732-appb-000013
其中，R 1、R 2和R 3分别为氢或羟基保护基。
进一步优选地，所述肝靶向特异性配体X选自半乳糖、半乳糖胺、N-乙酰半乳糖胺或以下结构中的一种或多种：
Figure PCTCN2020097732-appb-000014
其中，R 1选自OH、NHCOH或NHCOCH 3中的一种或两种。
所述含有位阻稳定结构的支链L是C3-C18的直链，该直链中含有一个或多个羰基、酰胺基、磷酰基、氧原子或这些基团的组合，可以选自如下结构中的一种或多种：
Figure PCTCN2020097732-appb-000015
其中，r1是1-12的正整数，r2为0-20的整数，Z为H或CH 3。
所述接头B选自以下结构式：
Figure PCTCN2020097732-appb-000016
其中，A 1是C、O、S或NH；r1为1-15的正整数，r2为0-5的整数；A 2是C、O、S、NH、羰基、酰胺基、磷酰基或硫代磷酰基。
优选地，所述接头B选自以下结构式：
Figure PCTCN2020097732-appb-000017
Figure PCTCN2020097732-appb-000018
Figure PCTCN2020097732-appb-000019
其中，r1、r3、r4、r5分别为1-15的正整数；r6为1-20的正整数，r7为2-6的正整数，r8为1-3的正整数。
进一步优选地，所述接头B选自以下结构：
Figure PCTCN2020097732-appb-000020
Figure PCTCN2020097732-appb-000021
所述连接链D含有C5-C20，可以含有氨基、羰基、酰胺基、氧原子、硫原子、硫代磷酰基、磷酰基、环状结构或这些基团的组合。
优选地，所述连接链D选自以下结构中的一种：
Figure PCTCN2020097732-appb-000022
Figure PCTCN2020097732-appb-000023
Figure PCTCN2020097732-appb-000024
Figure PCTCN2020097732-appb-000025
其中，每个n为1-20的正整数，且每个n为相同或不同的正整数。p为1-6的正整数；s为2-13的正整数；R 1和R 2为相同或者不同的取代基团，其结构式为以下结构中的一种：-H、-CH 3、-CH-(CH 3) 2、-CH 2-CH(CH 3) 2、-CH(CH 3)-CH 2-CH 3、-CH 2-C 6H 5、-C 8NH 6、-CH 2-C 6H 4-OH、-CH 2-COOH、-CH 2-CONH 2、-(CH 2) 2-COOH、-(CH 2) 4-NH 2、-(CH 2) 2-CONH 2、-(CH 2)-S-CH 3、-CH 2-OH、-CH(CH 3)-OH、-CH 2-SH、-CH 2-C 3H 3N 2、-(CH 2) 3NHC(NH)NH 2。
进一步优选地，所述连接链D选自以下结构中的一种：
Figure PCTCN2020097732-appb-000026
Figure PCTCN2020097732-appb-000027
Figure PCTCN2020097732-appb-000028
Figure PCTCN2020097732-appb-000029
所述的化合物，正义链5’端的修饰链带有一个N-乙酰半乳糖胺或两个N-乙酰半乳糖胺，选自以下结构中的一种：
Figure PCTCN2020097732-appb-000030
Figure PCTCN2020097732-appb-000031
Figure PCTCN2020097732-appb-000032
在一些优选实施例中，所述的化合物的正义链5’端的修饰链选自下表的结构式：
Figure PCTCN2020097732-appb-000033
Figure PCTCN2020097732-appb-000034
Figure PCTCN2020097732-appb-000035
所述的化合物的反义链3’端的修饰链带有两个或三个N-乙酰半乳糖胺，修饰链中的
Figure PCTCN2020097732-appb-000036
选自以下结构中的一种：
Figure PCTCN2020097732-appb-000037
Figure PCTCN2020097732-appb-000038
Figure PCTCN2020097732-appb-000039
Figure PCTCN2020097732-appb-000040
在一些优选实施例中，所述的化合物反义链3’端的修饰链优选选自下表的结构式：
Figure PCTCN2020097732-appb-000041
Figure PCTCN2020097732-appb-000042
Figure PCTCN2020097732-appb-000043
在一些优选实施例中，所述的化合物中正义链5’端末端修饰链与反义链3’末端修饰链组合优先为下表所示结构中的一种：
Figure PCTCN2020097732-appb-000044
Figure PCTCN2020097732-appb-000045
Figure PCTCN2020097732-appb-000046
Figure PCTCN2020097732-appb-000047
在一些优选实施例中，本发明所述的化合物包含正义链5’端和反义链3’端接入修饰链如下表所示：
Figure PCTCN2020097732-appb-000048
Figure PCTCN2020097732-appb-000049
在一些优选实施例中，本发明所述的化合物的结构如下表所示：
Figure PCTCN2020097732-appb-000050
Figure PCTCN2020097732-appb-000051
本发明的另一方面提供了本发明所述的化合物在制备用于治疗肝脏相关疾病的药物中的应用，其中，所述肝脏相关疾病包括急慢肝炎、肝癌、遗传性肝源性疾病、肝硬化、脂肪肝、糖尿病。
本发明的又一方面提供了本发明所述的化合物在制备用于治疗HBV感染的相关疾病的药物中的应用，其中，所述HBV感染包括慢性乙型肝炎病毒感染、急性乙型肝炎病毒感染。
其中，所述肝靶向特异性配体X是针对肝脏中去唾液酸糖蛋白受体(ASGPR)特异性的，所述HBV感染的相关疾病为慢性乙型肝炎，所述化合物可以持续使HBV的HBsAg、HBeAg和HBV DNA的表达得到抑制。
本发明的再一方面提供了一种药物组合物，该药物组合物包含本发明所述的化合物和药学上可接受的辅料，优选剂型为皮下注射剂。
与现有技术相比，本发明的有益效果为：
(1)与脂质体介导的siRNA递送相比：脂质体介导的siRNA递送主要是脂质体将siRNA包裹 到脂质体内，保护siRNA不被核酸酶降解，提高siRNA的通过细胞膜障碍的效率，从而促进细胞的吸收。脂质体例如阴离子脂质体、pH敏感性脂质体、免疫脂质体、膜融合脂质体(fusogenic liposome)和阳离子脂质等等，尽管取得了一定的进展，但脂质体本身容易引发炎症反应，给药前必须使用多种抗组胺和激素类如西利替嗪和地塞米松类等药物，以减少可能发生的急性炎症反应，因此在实际临床应用中并不适合所有治疗领域，尤其像慢性乙肝这一类治疗周期长的疾病，长期使用产生的积蓄毒性可能是潜在的安全隐患。
(2)全新的N-乙酰半乳糖胺引入方式：
与带有三个N-半乳糖胺结构的siRNA在抑制HBV的HBsAg效果进行比较：目前处于I/II期的治疗慢性乙型肝炎的siRNA药物有ARO-HBV和ALN-HBV02。ARO-HBV是在siARNA的正义链的5’末端，通过连接链引入三个N-乙酰半乳糖胺；ALN-HBV02是在siRNA正义链的3’末端，通过连接链引入三个N-乙酰半乳糖胺。上述药物引入半乳糖胺的部位都在正义链上，而且引入的都是三个N-乙酰半乳糖胺。本发明提供的化合物中，是siRNA正义链5’末端和反义链的3’末端同时引入数量不同的或相同的N-乙酰半乳糖胺，目前未见有在正义链5’末端和反义链的3’末端同时引入的报道，尤其是反义链的3'引入三个N-乙酰半乳糖胺，是一种全新的引入方式，通过实施例证实，该引入方式使siRNA具有高效抑制HBV基因的效果。
附图说明
为了使本发明的目的、技术方案和有益效果更加清楚，本发明提供如下附图说明：
图1为5’YICd-01-c4的高分辨率质谱图；
图2为5’YICc-01-c7的高分辨率质谱图；
图3为5’ERCd-01-c7的高分辨率质谱图；
图4为5’ERCc-01-c4的高分辨率质谱图；
图5为3’SANCd-01-c6的高分辨率质谱图；
图6为对HepG2.215细胞中HBsAg的抑制效果柱状图；
图7为对HepG2.215细胞中HBeAg的抑制效果柱状图；
图8为对HepG2.215细胞中HBVDNA的抑制效果柱状图；
图9为对转基因小鼠模型HBV基因的抑制效果柱状图；
图10为GBL-0401体内抑制HBV HBsAg效果图。
具体实施方式
以下实施例说明了本发明公开的一些实施方案，但并不局限于这些。此外，在提供具体实施方案时，发明人预期了那些具体实施方案的应用。例如具有具体同类或类似化学结构的化合物，用于不同的肝源性疾病的治疗。
说明：
DMF的中文名称是N,N-二甲基甲酰胺；
HBTU的中文名称为O-苯并三氮唑-四甲基脲六氟磷酸酯；
DIPEA(DIEA)的中文名称为N,N-二异丙基乙胺；
DCM的中文名称为二氯甲烷；
DMAP的中文名称为4-二甲氨基吡啶；
DMT-CL的中文名称为4,4'-二甲氧基三苯基氯甲烷；
THF的中文名称为四氢呋喃；
TBTU的中文名称为O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸；
DBU的中文名称为1,8-二氮杂二环十一碳-7-烯；
HOBt的中文名称为1-羟基苯并三唑；
DCC的中文名称为二环己基碳二亚胺；
Pd-C的中文名称为钯碳催化剂；
Figure PCTCN2020097732-appb-000052
的中文名称为固相载体，如树脂(Resin)。
实施例一、GBL-0401的合成
1、Kys-01的合成
1.1. 5’YICd-01结构的化合物：5'YICd-01-PFP的合成
1.1.1. 5'YICd-01-c1的合成
Figure PCTCN2020097732-appb-000053
量取2-羟基乙胺5.0g(81.9mmol)加入二甲基亚砜50mL，氢氧化钠溶液5mL(浓度1g/mL)，滴加丙烯酸叔丁酯12mL(81.9mmol)1小时加完，室温反应24h，加石油醚100mL，饱和食盐水洗2次，有机层干燥。过柱得无色油状物7.5g。
1.1.2. 5'YICd-01-c2的合成
Figure PCTCN2020097732-appb-000054
称取5'YICd-01-c1 7.5g(39.7mmol)，加DCM50mL，碳酸钠溶液(25％)23mL，室温滴加氯甲酸苄酯7.7g(45.0mmol),室温反应过夜，饱和食盐水洗涤2次，无水硫酸钠干燥，蒸干溶剂，过层析柱(乙酸乙酯：石油醚＝15％-30％)得油状物11.3g。
1.1.3. 5'YICd-01-c3的合成
Figure PCTCN2020097732-appb-000055
取5'YICd-01-c2 11.3g(35.0mmol)加甲酸20mL，室温反应过夜，减压蒸干溶剂，得5'YICd-01-c3 9.2g。
1.1.4. 5'YICd-01-c4的合成
Figure PCTCN2020097732-appb-000056
将化合物5'YICd-01-c3 1.0g(3.73mmol)和dlSANC-c4 2.0g(4.48mmol)加到DMF30mL中，然后加入HOBt 0.38g和HBTU 2.30g，然后缓慢加入DIEA 1.0mL。再加入水20mL，DCM40mL萃取。饱和食盐水100mL洗。用无水硫酸钠干燥，减压蒸干，用硅胶柱层析纯化(洗脱液：1-15％甲醇in DCM)。得白色泡状固体2.2g，其高分辨率质谱图如图1所示。
1.1.5. 5'YICd-01-c5的合成
Figure PCTCN2020097732-appb-000057
5'YICd-01-c4 2.2g(3.2mmol)用甲醇30mL溶解，加入10％Pd-C 1.0g(湿式Degussa型E101NE/W)。然后常压下加氢，反应过夜。反应液用硅藻土过滤，滤液减压蒸干，得白色泡状物1.70g。
1.1.6. 5'YICd-01-c6的合成
Figure PCTCN2020097732-appb-000058
取戊二酸单苄酯0.80g(3.60mmol)，用DMF 2mL溶解，然后加入TBTU1.28g,和DIEA 2.0mL，搅拌反应5分钟，加5'YICd-01-c5 1.70g(3.0mmol)，室温搅拌反应过夜。减压蒸干反应液，加DCM 50mL，水50mL，搅拌5分钟。分层，有机层用无水硫酸钠干燥。过层析柱(洗脱剂：DCM：甲醇＝1％-10％)，减压蒸干溶剂，得白色产品2.1g。
1.1.7. 5'YICd-01-c7的合成
Figure PCTCN2020097732-appb-000059
100mL单口瓶中投5'YICd-01-c6 2.1g(2.7mmol)，加钯炭0.2g，水泵抽真空，补氢气，重复三次，氢气加压反应过夜，第二日，TLC反应完全。铺硅藻土过滤钯炭，减压蒸干滤液，得产品1.8g。
1.1.8. 5'YICd-01-PFP的合成
Figure PCTCN2020097732-appb-000060
100mL单口瓶中投5'YICd-01-c7 1.8g(2.66mmol),加DCM20mL,滴加三氟甲磺酸五氟苯酯1.1g(4.0mmol)，室温反应1小时，加水40mL洗涤，加饱和亚硫酸氢钠10mL洗涤，有机层用无水硫酸钠干燥，有机层减压蒸干得产品2.3g。
1.2.固相合成C6NH-S-01
mG为起始单体，C6NH亚磷酰胺单体为终端单体，通过固相亚磷酰胺法偶联引入不同的亚磷酰胺单体。固相亚磷酰胺法基本步骤包括：1)脱保护：脱掉固相载体上氧原子上的保护基(DMT)；2)偶联：加上第一个核苷酸单体，通过3’至5’方向发生偶联反应；3)氧化：将所得的核苷亚磷酸酯氧化成更稳定的核苷磷酸酯(即三价磷氧化成五价磷)；4)封闭：将没有反应的前一步核苷酸单体5’-OH加冒封死，使其不再进一步参与反应；重复上述步骤，直至完成所需的序列。合成后，用甲胺乙醇溶液和氨水来裂解固相载体上连接化合物与初始核苷间的酯键，并将寡聚核苷酸上的各个碱基与磷酸上的保护基氰乙基(P)、苯甲酰基(mA、fA)、乙酰基(mC、fC)、异丁酰基(mG、fG)和4-甲氧基三苯甲基(C6NH)脱掉。经HPLC分离纯化后，过滤除菌，冻干。
1.3.液相合成Kys-01
1.3.1.Kys-01-c1的合成
Figure PCTCN2020097732-appb-000061
称取纯化冻干后的化合物C6NH-S-01(12.5mg)加入硼酸钠缓冲液(650μL，0.06mol/L)完全溶解，称取5'YICd-01-PFP(10.3mg)溶解于二甲基亚砜(100μL)，将溶液加入化合物C6NH-S-01中，混合均匀后加入N-甲基吗啡啉(5μL)，在室温下超声3h，HPLC检测反应完全后使用C18柱进行纯化。
1.3.2.Kys-01的合成
Figure PCTCN2020097732-appb-000062
取纯化后的化合物Kys-01-c1(32mL,5mg)加入25％水合肼(16mL)，混合均匀后室温下超声10min，HPLC检测反应完全使用C18柱进行纯化，冻干后得到白色冻干粉化合物Kys-01(2mg)。
2、Kyas-01的合成
2.1. 3’SANCd-01结构的化合物：3’SANCd-01树脂的合成
2.1.1. 3’SANCd-01-c1的合成
Figure PCTCN2020097732-appb-000063
称量3-氨基丙二醇(9.114g,0.100mol)，溶于THF中(50mL)，冷却，滴加三氟乙酸乙酯(15.62g,0.110mol)，室温反应1h，反应液旋蒸，得3’SANCd-01-c1粗品(18.871g)。
2.1.2. 3’SANCd-01-c2的合成
Figure PCTCN2020097732-appb-000064
3’SANCd-01-c1(5.480g，0.030mol)溶于吡啶中(30mL)，冷却，分批加入DMT-CL(10.423g,0.031mol)，避光反应过夜，旋蒸除去吡啶。残留物溶于CH 2Cl 2(50mL)，饱和食盐水(50mL)洗涤，有机相无水硫酸钠干燥，过滤，旋蒸，过柱。得产品3’SANCd-01-c2(10.805g)。
2.1.3. 3’SANCd-01-c3的合成
Figure PCTCN2020097732-appb-000065
3’SANCd-01-c2(10.805g,0.022mol)溶于甲醇(60mL)和THF(30mL)，冷却，滴加KOH(5.69g)的水溶液(24mL)，室温反应2h，旋蒸除去甲醇和THF。残留物加水(50mL)，EtOAc萃取(30mL*3)，饱和食盐水(50mL)洗涤，有机相无水硫酸钠干燥，过滤，旋蒸。用含1％三乙胺洗脱液过柱，得产品3’SANCd-01-c3(8.286g)。
2.1.4. 3’SANCd-01-c4的合成
Figure PCTCN2020097732-appb-000066
3’SANCd-01-c3(2.890g,0.007mol)溶于CH 2Cl 2(20mL)，冷却，滴加DCC(1.680g)的CH 2Cl 2溶液(10mL)，搅拌20分钟后再加入辛二酸单甲酯(1.522g)的CH 2Cl 2溶液(10mL)，室温反应过夜，5％NaHCO 3(20mL)淬灭，CH 2Cl 2萃取(20mL*2)，饱和食盐水(10mL)洗涤，无水硫酸钠干燥，过滤，旋蒸，用含1％三乙胺的洗脱液过柱，得产品3’SANCd-01-c4(3.193g)。
2.1.5. 3’SANCd-01-c5的合成
Figure PCTCN2020097732-appb-000067
3’SANCd-01-c4(2.193g，0.004mol)溶于THF(10mL)中，冷却，滴加LiOH(0.645g)的水溶液(4.5g)，反应2h后TLC显示无原料。反应液旋蒸除去溶剂，用饱和氯化铵中和残留物，CH2Cl2萃取(20mL*2)，饱和食盐水(10mL)洗涤。无水硫酸钠干燥，过滤，旋蒸，用含1％三乙胺的洗脱液过柱，得产品3’SANCd-01-c5(1.979g)。
2.1.6. 3’SANCd-01-c6的合成
Figure PCTCN2020097732-appb-000068
3’SANCd-01-c5(0.389g，0.004mol)，溶于DMF(2mL)，冷却，加DIPEA(0.15mL)，TBTU(0.183g)，搅拌10分钟，再加入dlSANC-c12(0.756g,0.0005mol)的DMF(2mL)溶液，室温反应过夜。水(20mL)淬灭，CH 2Cl 2萃取(20mL*2)，饱和食盐水(10mL)洗涤，无水硫酸钠干燥，过滤，旋蒸，用含5％三乙胺的洗脱液过柱，得产品3’SANCd-01-c6(0.803g)，其高分辨率质谱图如图5所示。
2.1.7. 3’SANCd-01-c7的合成
Figure PCTCN2020097732-appb-000069
向反应瓶内依次加入3’SANCd-01-c6(2.15g 0.001mol)和22mL的DCM，室温搅拌溶解，再依次加入DBU(0.156g)和丁二酸酐(0.3g 0.003mmol)，室温搅拌反应，TLC分析， 反应合格浓缩掉DCM，加水，用DCM萃取，有机相再用饱和食盐水洗涤，有机相经无水硫酸钠干燥、过滤、浓缩，最后过硅胶柱进行纯化，得到3’SANCd-01-c7为2.03g。
2.1.8. 3’SANCd-01树脂的合成
Figure PCTCN2020097732-appb-000070
向反应瓶内依次加入3’SANCd-01-c7(1.13g,0.0005mmol)和12mL的DMF，室温搅拌溶解，再依次加入HBTU(0.11g)、DIPEA(0.104g)和GE树脂(1.80g)，35℃摇床摇动24h，将物料转移到合成管中，过滤，在氮气鼓泡下树脂用DMF冲洗4次，然后加入CAP A+CAP B在氮气鼓泡下进行封端反应半小时，取少量树脂进行kaiser test至测试液呈现黄色，封端完毕，滤饼分别用甲醇、DCM和甲醇进行冲洗；真空拉干；得3’SANCd-01树脂为2.48g，取代度150μmol/g。
2.2.固相合成Kyas-01
通过固相亚磷酰胺法偶联引入不同的亚磷酰胺单体，mU为起始单体，mU为终端单体。固相亚磷酰胺法基本步骤包括：1)脱保护：脱掉3’SANCd-01树脂上氧原子上的保护基(DMT)；2)偶联：加上第一个核苷酸单体，通过3’至5’方向发生偶联反应；3)氧化：将所得的核苷亚磷酸酯氧化成更稳定的核苷磷酸酯(即三价磷氧化成五价磷)；4)封闭：将没有反应的前一步核苷酸单体5’-OH加冒封死，使其不再进一步参与反应；重复上述步骤，直至完成所需的序列。合成后，用甲胺乙醇溶液和氨水来裂解固相载体上连接化合物与初始核苷间的酯键，并将寡聚核苷酸上的各个碱基与磷酸上的保护基氰乙基(P)、苯甲酰基(mA、fA)、乙酰基(mC、fC)和异丁酰基(mG、fG)脱掉。经HPLC分离纯化后，过滤除菌，冻干得Kyas-01。
3、GBL-0401合成
精确测定Kys-01与Kyas-01溶液的浓度，按等摩尔浓度进行混合后，加入该体积1/20体积的1M PBS溶液再次混匀，将混合体系加热至95℃，持续5min，然后自然降温3h至40℃或者室温时，进行HPLC检测，若单链残余＜5％即可认为反应完成。
实施例二、GBL-0402的合成
1、Kys-02的合成
1.1. 5’YICc-01结构的化合物：5'YICc-01-PFP的合成
1.1.1. 5’YICc-01-c1的合成
Figure PCTCN2020097732-appb-000071
取SANC-c8 7.0g(40.0mmol)，5’YICd-01-c3 9.2g(34.4mmol)，加DMF 25mL溶解，加TBTU 9.0g，降温至10℃，加DIEA 2ml，室温反应过夜。加水30mL，二氯甲烷50mL，有机层用饱和食盐水洗三次。有机层干燥，减压蒸干。过层析柱(洗脱剂：二氯甲烷：甲醇＝1％-10％)，得黄色粘稠固体10.0g。
1.1.2. 5’YICc-01-c2的合成
Figure PCTCN2020097732-appb-000072
取5’YICc-01-c1 10.0g，加15ml浓盐酸，室温反应过夜。减压蒸干，得7.3g。
1.1.3. 5’YICc-01-c3的合成
Figure PCTCN2020097732-appb-000073
将化合物5’YICc-01-c2 7.3g(22.6mmol)和SANC-c4 12.1g(27.1mmol)加到DMF60mL中，然后加入HOBt 3.8g和HBTU 12.4g，然后缓慢加入DIEA 5.0ml。反应液室温下搅拌反应过夜。然后加入水50mL，二氯甲烷100mL萃取。饱和食盐水100mL洗。用无水Na 2SO 4干燥，减压蒸干，用硅胶柱层析纯化(洗脱液：3-15％MeOH in DCM)。得白色泡状固体8.3g。
1.1.4. 5’YICc-01-c7的合成
合成步骤同实施例一1.1.5，其高分辨率质谱图如图2所示。
1.1.5. 5’YICc-01-c8的合成
合成步骤同实施例一1.1.6。
1.1.6. 5’YICc-01-c9的合成
合成步骤同实施例一1.1.7。
1.1.7. 5’YICc-01-PFP的合成
合成步骤同实施例一1.1.8。
1.2.固相合成C6NH-S-02
mG为起始单体，C6NH亚磷酰胺单体为终端单体，通过固相亚磷酰胺法偶联引入不同的亚磷酰胺单体。合成步骤同实施例一1.2固相合成。
1.3.液相合成得Kys-02
1.3.1.Kys-02-c1的合成
Figure PCTCN2020097732-appb-000074
合成步骤同实施例一1.3.1。
1.3.2.Kys-02的合成
Figure PCTCN2020097732-appb-000075
合成步骤同实施例一1.3.2。
2、Kyas-02的合成
2.1. 3’SANCc-01结构的化合物：3’SANCc-01树脂的合成
3’SANCc-01树脂的合成路线和工艺步骤除3’SANCc-01-c6的合成，其它与3’SANCd-01树脂一致。
2.1.1. 3’SANCc-01-c1的合成
Figure PCTCN2020097732-appb-000076
3’SANCd-01-c5(0.295g)，溶于DMF(2mL)，冷却，加DIPEA(0.14mL)，TBTU(0.177g)，搅拌10分钟，再加入SANC-c12(0.756g)的DMF(2mL)溶液，室温反应过夜。水(50mL)淬灭，CH 2Cl 2萃取(20mL*2)，饱和食盐水(10mL)洗涤，无水硫酸钠干燥，过滤，旋蒸，用含5％三乙胺的洗脱液过柱，得产品3’SANCc-01-c6(0.815g)。
2.2.固相合成Kyas-02
mU为起始单体，mU为终端单体，通过固相亚磷酰胺法偶联引入不同的亚磷酰胺单体。合成步骤同实施例一2.2固相合成Kyas-01。
3、GBL-0402的合成
精确测定Kys-02与Kyas-02溶液的浓度，合成步骤同实施例一3、GBL-0401的合成。
实施例三、GBL-0403的合成
1、Kys-03的合成
1.1. 5’ERCd-01结构的化合物：5’ERCd-01-PFP的合成
1.1.1. 5’ERCd-01-c1的合成
Figure PCTCN2020097732-appb-000077
量取2-氨基-1,3-丙二醇5.0g(54.9mmol)加入DMSO 50mL，氢氧化钠溶液5mL(浓度1g/mL)，降温到0℃，滴加丙烯酸叔丁酯20mL(137.8mol)2小时加完，室温反应48h，加石油醚100mL，饱和食盐水洗2次，有机层干燥。过层析柱(洗脱液：乙酸乙酯：石油醚＝25％-75％)，上柱加0.05％的三乙胺，得无色油状物6.2g。
1.1.2. 5’ERCd-01-c2的合成
Figure PCTCN2020097732-appb-000078
称取5’ERCd-01-c1 6.2g(17.9mmol)，加二氯甲烷50mL，碳酸钠溶液(25％)23mL，室温滴加氯甲酸苄酯8.2mL(57.4mmol)，2小时滴加完，室温反应过夜，饱和食盐水洗涤3次，无水硫酸钠干燥，蒸干溶剂，过层析柱(乙酸乙酯：石油醚＝5％-30％)得油状物4.0g。
1.1.3. 5’ERCd-01-c3的合成
Figure PCTCN2020097732-appb-000079
取5’ERCd-01-c2 4.0g(8.3mmol)加甲酸12mL，室温反应过夜，减压蒸干溶剂，得5’ERCd-01-c3 2.8g。
1.1.4. 5’ERCd-01-c4的合成
Figure PCTCN2020097732-appb-000080
将化合物5’ERCd-01-c3 1.11g(3.0mmol)和dlSANC-c4 3.6g(8.04mmol)加到DMF60mL中，然后加入HOBt 2.24g和HBTU 3.36g，然后缓慢加入DIEA 4.16mL。反应液室温下搅拌反应3小时。然后加入水，水层用二氯甲烷萃取2x 10mL。合并有机层，然后依次用饱和NaHCO 3 80mL,水(2x 60mL),饱和食盐水(60mL)洗。用无水Na 2SO 4干燥，减压蒸干，用硅胶柱层析纯化(洗脱液：3-15％MeOH in DCM)。得淡黄色固3.24g。
1.1.5. 5’ERCd-01-c5的合成
Figure PCTCN2020097732-appb-000081
5’ERCd-01-c4 3.24g(2.6mmol)用甲醇60mL溶解，加入10％Pd-C 0.3g，湿式Degussa型E101NE/W)，乙酸2.0mL。然后常压下加氢，反应过夜。反应液用硅藻土过滤，滤液减压蒸干，得油状物2.9g。
1.1.6. 5’ERCd-01-c6的合成
Figure PCTCN2020097732-appb-000082
取戊二酸单苄酯0.21g(0.001mol)，用DMF 2mL溶解，然后加入TBTU 0.36g和DIEA 0.4mL，搅拌反应5分钟，加5’ERCd-01-c5 0.50g(溶于10ml DMF中)，室温搅拌反应过夜。减压蒸干反应液，蒸干后，加二氯甲烷40mL，水20mL，搅拌5分钟。分层，有机层用无水硫酸钠干燥。过层析柱，(洗脱剂：二氯甲烷：甲醇＝1％-10％)，减压蒸干溶剂，得白色产品0.51g。
1.1.7. 5’ERCd-01-c7的合成
Figure PCTCN2020097732-appb-000083
100mL单口瓶中投5’ERCd-01-c6 0.51g(0.42mmol)，加钯炭127mg,水泵抽真空，补氢气，重复三次，氢气加压反应过夜，第二日，TLC反应完全。铺硅藻土过滤钯炭，减压蒸干滤液，得产品0.40g，其高分辨率质谱图如图3所示。
1.1.8. 5’ERCd-01-PFP的合成
Figure PCTCN2020097732-appb-000084
50mL单口瓶中投5’ERCd-01-c7 0.40g(0.33mmol)，加二氯甲烷10mL，滴加三氟甲磺酸五氟苯酯0.19g(0.6mmol)，10分钟滴完，室温反应2小时，加水10mL洗涤两次，加饱和亚硫酸氢钠5mL洗涤一次，有机层用无水硫酸钠干燥10分钟，有机层减压蒸干得产品0.5g。
1.2.固相合成C6NH-S-03
mG为起始单体，C6NH亚磷酰胺单体为终端单体，通过固相亚磷酰胺法偶联引入不同的亚磷酰胺单体。合成步骤同实施例一1.2固相合成。
1.3.液相合成Kys-03
1.3.1.Kys-03-c1的合成
Figure PCTCN2020097732-appb-000085
合成步骤同实施例一1.3.1。
1.3.2.Kys-03的合成
Figure PCTCN2020097732-appb-000086
合成步骤同实施例一1.3.2。
2、Kyas-03的合成
2.1. 3’ERCd-01结构的化合物：3’ERCd-01树脂的合成2.1.1. 3’ERCd-01-c1的合成
Figure PCTCN2020097732-appb-000087
向反应瓶内依次加入3’SANCd-01-c5(0.824g，0.0015mol)和10mL的DMF，室温搅拌溶解，再依次加入TBTU(0.563g)和DIPEA(0.517g)，室温搅拌溶解，最后加入dlERC-c12(1.09g，0.001mol)室温搅拌反应过夜，TLC分析，反应合格浓缩掉DMF，加水，用DCM萃取，有机相再用饱和氯化钠水溶液洗涤，有机相经无水硫酸钠干燥、过滤、浓缩，最后过硅胶柱进行纯化，得到类白色泡状固体1.3g。
2.1.2. 3’ERCd-01-c2的合成
Figure PCTCN2020097732-appb-000088
合成步骤同实施例一2.1.7。
2.1.3. 3’ERCd-01树脂的合成
Figure PCTCN2020097732-appb-000089
合成步骤同实施例一2.1.8。
2.2.固相合成Kyas-03
mA为起始单体，T为终端单体，通过固相亚磷酰胺法偶联引入不同的亚磷酰胺单体。合成步骤同实施例一2.2固相合成Kyas-01。
3、GBL-0403的合成
精确测定Kys-03与Kyas-03溶液的浓度，合成步骤同实施例一3、GBL-0401的合成。
实施例四、GBL-0404的合成
1、Kys-04的合成
1.1. 5’ERCc-01结构的化合物：5’ERCc-01-PFP的合成
1.1.1. 5’ERCc-01-c1的合成
Figure PCTCN2020097732-appb-000090
取N-叔丁氧羰基-1,3-丙二胺5.0g(28.7mmol)，5’ERCd-01-c3 2.8g(7.6mmol)，加DMF 25mL溶解，加TBTU 9.0g，加DIEA 2mL，室温反应过夜。加水30mL，DCM50mL，有机层用饱和食盐水洗涤，减压蒸干。过层析柱，用石油醚装柱，用1L石油醚冲洗柱子(洗脱剂：DCM：甲醇＝5％-10％)，得黄色粘稠固体2.9g。
1.1.2. 5’ERCc-01-c2的合成
Figure PCTCN2020097732-appb-000091
取5’ERCc-01-c1 2.9g，加9mL浓盐酸，室温反应过夜。减压蒸干，得2.7g。
1.1.3. 5’ERCc-01-c3的合成
Figure PCTCN2020097732-appb-000092
将化合物5’ERCc-01-c2 1.56g(2.44mmol)和Sanc-c4 3.6g(8.04mmol)加到DMF60mL中，然后加入HOBt 2.24g和HBTU 3.36g，然后缓慢加入DIEA 4.16mL。反应液室温下搅拌反应1小时。然后加入水，水层用DCM萃取2x10mL。合并有机层，然后依次用饱和碳酸氢钠80mL,、水40mL、饱和食盐水60mL洗。用无水硫酸钠干燥，减压蒸干，用硅胶柱层析纯化(洗脱液：3-15％甲醇in DCM)，得淡黄色固2.36g。
1.1.4. 5’ERCc-01-c4的合成
Figure PCTCN2020097732-appb-000093
5’ERCc-01-c3 2.36g(1.2mmol)用甲醇120mL溶解，加入10％Pd-C 1.0g，湿式Degussa型E101NE/W)。然后常压下加氢，反应过夜。反应液用硅藻土过滤，滤液减压蒸干,得油状物1.8g，其高分辨率质谱图如图4所示。
1.1.5. 5’ERCc-01-c5的合成
Figure PCTCN2020097732-appb-000094
取戊二酸单苄酯0.21g(0.001mol)，用DMF 2mL溶解，然后加入TBTU 0.36g,和DIEA 0.4mL，搅拌反应5分钟，加5’ERCc-01-c4 1.09g，室温搅拌反应过夜。减压蒸干反应液，蒸干后，加DCM40mL，水20mL，搅拌5分钟。分层，有机层用无水硫酸钠干燥。过层析柱(洗脱剂：DCM：甲醇＝1％-10％)，减压蒸干溶剂，得白色产品0.85g。
1.1.6. 5’ERCc-01-c6的合成
Figure PCTCN2020097732-appb-000095
100mL单口瓶中投5’ERCc-01-c5 0.85g(0.43mmol)，加钯炭127mg，水泵抽真空，补氢气，重复三次，氢气加压反应过夜，第二日，TLC反应完全。铺硅藻土过滤钯炭，减压蒸干滤液，得产品0.76g。
1.1.7. 5’ERCc-01-PFP的合成
Figure PCTCN2020097732-appb-000096
50mL单口瓶中投5’ERCc-01-c6 0.76g(0.40mmol)，加DCM10mL，滴加三氟甲磺酸五氟苯酯0.19g(0.6mmol)，室温反应1小时，加水10mL洗涤，加饱和亚硫酸氢钠5mL洗涤，有机层用无水硫酸钠干燥10分钟，有机层减压蒸干得产品0.8g。
1.2.固相合成C6NH-S-04
mA为起始单体，C6NH亚磷酰胺单体为终端单体，通过固相亚磷酰胺法偶联引入不同的亚磷酰胺单体。合成步骤同实施例一1.2固相合成。
1.3.液相合成Kys-04
1.3.1.Kys-04-c1的合成
Figure PCTCN2020097732-appb-000097
合成步骤同实施例一1.3.1。
1.3.2.Kys-04的合成
Figure PCTCN2020097732-appb-000098
合成步骤同实施例一1.3.2。
2、Kyas-04的合成
2.1. 3’ERCc-01结构的化合物：3’ERCc-01树脂的合成
2.1.1. 3’ERCc-01-c1的合成
Figure PCTCN2020097732-appb-000099
向反应瓶内依次加入3’SANCd-01-c5(0.824g 0.0015mol)和10mL的DMF，室温搅拌溶解，再依次加入TBTU(0.563g)和DIPEA(0.517g)，室温搅拌溶解，最后加入ERC-c12(1.21g，0.001mol)室温搅拌反应过夜，TLC分析，反应合格浓缩掉DMF，加水，用DCM萃取，有机相再用饱和氯化钠水溶液洗涤，有机相经无水硫酸钠干燥、过滤、浓缩，最后过硅胶柱进行纯化，得到白色泡状固体1.4g。
2.1.2. 3’ERCc-01-c2的合成
Figure PCTCN2020097732-appb-000100
合成步骤同实施例一2.1.7。
2.1.3. 3’ERCc-01树脂的合成
Figure PCTCN2020097732-appb-000101
合成步骤同实施例一2.1.8。
2.2.固相合成Kyas-04
mG为起始单体，mU为终端单体，通过固相亚磷酰胺法偶联引入不同的亚磷酰胺单体。合成步骤同实施例一2.2固相合成Kyas-01。
3、GBL-0404的合成
精确测定Kys-04与Kyas-04溶液的浓度，合成步骤同实施例一3、GBL-0401的合成。
实施例五、GBL-0409的合成
1、Kyas-09的合成
1.1. 3’qfSANCd-01结构的化合物：3’qfSANCd-01树脂的合成
1.1.1. 3’qfSANCd-01-c1的合成
Figure PCTCN2020097732-appb-000102
向反应瓶内依次加入羟脯氨醇盐酸盐(1.53g 0.01mol)和15mL的DMF，室温搅拌溶解，再依次加入辛二酸单甲酯(1.98g 0.0105mol)、HBTU(4.55g)和DIPEA(3.88g)，室温搅拌反应过夜，TLC分析，反应合格浓缩掉DMF，加水，用DCM萃取，有机相再用饱和氯化钠水溶液洗涤，有机相经无水硫酸钠干燥、过滤、浓缩，最后过硅胶柱进行纯化，得到黄色粘稠液体2.38g。
1.1.2. 3’qfSANCd-01-c2的合成
Figure PCTCN2020097732-appb-000103
向反应瓶内依次加入3’qfSANCd-01-c1(2.87g 0.01mol)和30ml吡啶，室温搅拌溶解，再依次加入DMAP(0.61g)和DMT-CL(4.06g 0.012mol)，室温搅拌反应过夜，TLC分析，反 应合格浓缩掉吡啶，加水，用DCM萃取，有机相再用饱和氯化钠水溶液洗涤，有机相经无水硫酸钠干燥、过滤、浓缩，最后过硅胶柱进行纯化，得到黄色粘稠液体4.13g(收率70％)
1.1.3. 3’qfSANCd-01-c3的合成
Figure PCTCN2020097732-appb-000104
向反应瓶内依次加入3’qfSANCd-01-c2(5.89g 0.01mol)和60mL溶剂(THF/水/甲醇＝1:1:4)，室温搅拌溶解，再加入LiOH(1.26g)，室温搅拌反应2h，TLC分析，反应合格浓缩掉溶剂，加水，用DCM萃取，有机相再用饱和氯化钠水溶液洗涤，有机相经无水硫酸钠干燥、过滤、浓缩，最后过硅胶柱进行纯化，得到黄色粘稠液体4.5g。
1.1.4. 3’qfSANCd-01-c4的合成
Figure PCTCN2020097732-appb-000105
向反应瓶内依次加入3’qfSANCd-01-c3(0.863g 1.5mmol)和10mL的DMF，室温搅拌溶解，再依次加入TBTU(0.963g)和DIPEA(0.517g)，室温搅拌溶解，最后加入dlSANC-c12(1.62g 1mmol)室温搅拌反应过夜，TLC分析，反应合格浓缩掉DMF，加水，用DCM萃取，有机相再用饱和氯化钠水溶液洗涤，有机相经无水硫酸钠干燥、过滤、浓缩，最后过硅胶柱进行纯化，得到黄色粘稠液体1.743g。
1.1.5. 3’qfSANCd-01-c5的合成
Figure PCTCN2020097732-appb-000106
向反应瓶内依次加入3’qfSANCd-01-c4(2.18g，0.001mol)和10mL的DCM，室温搅拌溶解，再依次加入DBU(0.256g)和丁二酸酐(0.3g，0.003mmol)，室温搅拌反应，TLC分析，反应合格浓缩掉DCM，加水，用DCM萃取，有机相再用饱和氯化钠水溶液洗涤，有机相经无水硫酸钠干燥、过滤、浓缩，最后过硅胶柱进行纯化，得到3’qfSANCd-01-c5为2.05g。
1.1.6. 3’qfSANCd-01-c6的合成
Figure PCTCN2020097732-appb-000107
向反应瓶内依次加入3’qfSANCd-01-c5(1.14g，0.0005mmol)和12mL的DMF，室温搅拌溶解，再依次加入HBTU(0.19g)、DIPEA(0.194g)和GE树脂(1.83g)，35℃摇床摇动4h，将物料转移到合成管中，过滤，在氮气鼓泡下树脂用DMF冲洗4次，然后加入CAP A+CAP B在氮气鼓泡下进行封端反应半小时，取少量树脂进行kaiser test至测试液呈现 黄色，封端完毕，滤饼分别用甲醇、DCM进行冲洗；真空拉干；得3’qfSANCd-01为2.5g，取代度140μmol/g。
1.2.固相合成Kyas-09
mU为起始单体，mU为终端单体，通过固相亚磷酰胺法偶联引入不同的亚磷酰胺单体。合成步骤同实施例一2.2固相合成Kyas-01。
2、GBL-0409的合成
精确测定Kys-01与Kyas-09溶液的浓度，合成步骤同实施例一3、GBL-0401的合成。
实施例六、GBL-0410的合成
1、Kys-10的合成
1.1.固相合成C9NH-S-01
mU为起始单体，C9NH亚磷酰胺单体为终端单体，通过固相亚磷酰胺法偶联引入不同的亚磷酰胺单体。合成步骤同实施例一1.2固相合成C6NH-S-01。
1.2.液相合成Kys-10
1.2.1.Kys-10-c1的合成
Figure PCTCN2020097732-appb-000108
合成步骤同实施例一1.3.3。
1.2.2.Kys-01的合成
Figure PCTCN2020097732-appb-000109
合成步骤同实施例一1.3.4。
2、Kyas-10的合成
2.1. 3’pdSANCd-01结构的化合物：3’pdSANCd-01树脂的合成
2.1.1. 3’pdSANCd-01-c1的合成
Figure PCTCN2020097732-appb-000110
向反应瓶内依次加入4,4-哌啶二基二甲醇(1.59g，0.01mol)和20mL的DMF，室温搅拌溶解，再依次加入辛二酸单甲酯(1.98g，0.0105mol)、HBTU(4.55g)和DIPEA(3.88g)，室温搅拌反应过夜，TLC分析，反应合格浓缩掉DMF，加水，用DCM萃取，有机相再用饱和氯化钠水溶液洗涤，有机相经无水硫酸钠干燥、过滤、浓缩，最后过硅胶柱进行纯化，得到黄色粘稠液体2.65g。
2.1.2. 3’pdSANCd-01-c2的合成
Figure PCTCN2020097732-appb-000111
向反应瓶内依次加入3’pdSANCd-01-c1(3.29g，0.01mol)和33mL吡啶，室温搅拌溶解，再依次加入DMAP(0.61g)和DMT-CL(4.06g，0.012mol)，室温搅拌反应过夜，TLC分析，反应合格浓缩掉吡啶，加水，用DCM萃取，有机相再用饱和氯化钠水溶液洗涤，有机相经无水硫酸钠干燥、过滤、浓缩，最后过硅胶柱进行纯化，得到黄色粘稠液体4.74g。
2.1.3. 3’pdSANCd-01-c3的合成
Figure PCTCN2020097732-appb-000112
向反应瓶内依次加入3’pdSANCd-01-c2(3.16g，5mmol)和32mL溶剂(THF/水/甲醇＝1:1:4)，室温搅拌溶解，再加入LiOH(0.63g)，室温搅拌反应2h，TLC分析，反应合格浓缩掉溶剂，加水，用DCM萃取，有机相再用饱和氯化钠水溶液洗涤，有机相经无水硫酸钠干燥、过滤、浓缩，最后过硅胶柱进行纯化，得到黄色粘稠液体2.78g。
2.1.4. 3’pdSANCd-01-c4的合成
Figure PCTCN2020097732-appb-000113
向反应瓶内依次加入3’pdSANCd-01-c3(0.93g，1.5mmol)和10mL的DMF，室温搅拌溶解，再依次加入TBTU(0.963g)和DIPEA(0.517g)，室温搅拌溶解，最后加入dlSANC-c12(0.562g，1mmol)室温搅拌反应过夜，TLC分析，反应合格浓缩掉DMF，加水，用DCM萃取，有机相再用饱和氯化钠水溶液洗涤，有机相经无水硫酸钠干燥、过滤、浓缩，最后过硅胶柱进行纯化，得到黄色粘稠液体1.688g。
2.1.5. 3’pdSANCd-01-c5的合成
Figure PCTCN2020097732-appb-000114
向反应瓶内依次加入3’pdSANCd-01-c4(2.22g，0.001mol)和22mL的DCM，室温搅拌溶解，再依次加入DBU(0.256g)和丁二酸酐(0.3g，0.003mmol)，室温搅拌反应，TLC分析，反应合格浓缩掉DCM，加水，用DCM萃取，有机相再用饱和氯化钠水溶液洗涤，有机相经无水硫酸钠干燥、过滤、浓缩，最后过硅胶柱进行纯化，得到3’pdSANCd-01-c5为2.11g。
2.1.6. 3’pdSANCd-01的合成
Figure PCTCN2020097732-appb-000115
向反应瓶内依次加入3’pdSANCd-01-c5(1.16g 0.0005mmol)和12mL的DMF，室温搅拌溶解，再依次加入HBTU(0.19g)、DIPEA(0.194g)和GE树脂(1.85g)，35℃摇床摇动4h，将物料转移到合成管中，过滤，在氮气鼓泡下树脂用DMF冲洗4次，然后加入CAP A+CAP B在氮气鼓泡下进行封端反应半小时，取少量树脂进行kaiser test至测试液呈现黄色，封端完毕，滤饼分别用甲醇、DCM进行冲洗；真空拉干；得3’pdSANCd-01为2.6g，取代度145μmol/g。
2、固相合成Kyas-10
通过固相亚磷酰胺法偶联引入不同的亚磷酰胺单体，mU为起始单体，mU为终端单体。合成步骤同实施例一2.2固相合成Kyas-01。
3、GBL-0410的合成
精确测定Kys-10与Kyas-10溶液的浓度，合成步骤同实施例一3、GBL-0401的合成。
实施例七、GBL0405～GBL0408以及GBL0411～GBL0418参照GBL-0401～GBL0404的合成。
实施例八、HepG2.215细胞实验考察化合物对HBV基因的体外抑制效果
1、实验分组
空白对照组：加入含2％FBS的DMEM培养基孵育72h。
供试品组：分别加入浓度为5nM、0.5nM或0.05nM的供试品稀释液，每个浓度3个复孔，37℃5％CO 2培养箱中培养72h。
2、实验材料
HepG2.2.15细胞
3、实验试剂
名称 品牌 批号
DMEM高糖培养基 Gibco 8119164
胎牛血清 Gibco 20190907
PBS Solarbio 20190624
Trypsin-EDTA溶液 Gibco 2062475
双抗(青霉素/链霉素)溶液 Gibco 2029632
HBsAg、HBeAg试剂盒 上海科华 201812381
4、实验仪器
名称 品牌 型号
生物安全柜 海尔 HR40-IIA2
二氧化碳培养箱 ASTEC SCA-165DS
普通光学显微镜 尼康 TS2-S-SM
低速离心机 飞鸽 KA-1000
多门冰箱 美菱 BCD-318WTPZM(E)
5、供试品：
序号 新化合物代码 重量 纯度
1 GBL-0401 13.8μg 92.3％
2 GBL-0402 12.9μg 86.4％
3 GBL-0403 13.4μg 89.3％
4 GBL-0404 14.0μg 93.3％
5 GBL-0405 13.7μg 91.3％
6 GBL-0406 20.5μg 88.3％
7 GBL-0407 20.1μg 94.4％
8 GBL-0408 20.3μg 92.3％
9 GBL-0409 20.4μg 93.6％
10 GBL-0410 20.2μg 90.5％
11 GBL-0411 20.0μg 89.5％
12 GBL-0412 15.1μg 94.8％
13 GBL-0413 15.2μg 92.5％
14 GBL-0414 15.5μg 90.6％
15 GBL-0415 15.7μg 91.5％
16 GBL-0416 16.0μg 93.4％
17 GBL-0417 15.9μg 91.7％
18 GBL-0418 15.5μg 92.5％
6、试验过程
HepG2.2.15细胞在96孔细胞培养板中培养，每三天更换一次新鲜培养基，第6天加入上述所配不同浓度含药培养液，继续培养到第9天。收集上清液，检测试剂盒检测细胞上清中HBsAg、HBeAg及HBV DNA的含量。并与对照不给药组进行OD值对比，按照比值确定有效性。
7.实验结果
7.1对HepG2.2.15细胞中HBsAg的抑制效果：见附图6
7.2对HepG2.2.15细胞上清中HBeAg的抑制效果：见附图7
7.3对HepG2.2.15细胞上清中HBV DNA的抑制效果：见附图8
实施例九、新化合物对转基因小鼠模型HBV基因的抑制效果考察
1.实验方案
取适龄的雄性HBV转基因小鼠(要求HBsAg表达显著)用于实验评估，挑出90只体重处于25g左右的小鼠，随机分成18组，每组5只，在Day0分别通过皮下注射给药：3mg/kg；给药体积为100-200μL。用药前，做小鼠血HBsAg测定，尽量保持各组的HBsAg的平均水平一致。
2.供试品与试剂
序号 新化合物代码 规格 纯度/含量
1 GBL-0401 500μg 92.3％
2 GBL-0402 500μg 86.4％
3 GBL-0403 500μg 89.3％
序号 新化合物代码 规格 纯度/含量
4 GBL-0405 500μg 93.3％
5 GBL-0406 500μg 91.3％
6 GBL-0407 500μg 88.3％
7 GBL-0408 500μg 94.4％
8 GBL-0409 500μg 92.3％
9 GBL-0410 500μg 93.6％
10 GBL-0411 500μg 90.5％
11 GBL-0412 500μg 89.5％
12 GBL-0413 500μg 94.8％
13 GBL-0414 500μg 92.5％
14 GBL-0414 500μg 90.6％
15 GBL-0415 500μg 91.5％
16 GBL-0416 500μg 93.4％
17 GBL-0417 500μg 91.7％
18 GBL-0418 500μg 92.5％
19 生理盐水 500ml/瓶 0.9％
3.试剂盒
Figure PCTCN2020097732-appb-000116
4.实验仪器
名称 型号 厂家
旋涡混匀器 MIX-28 大龙兴创
离心机 S1010E THERMO
全自动化学发光分析仪 602 德国罗氏
5.实验结果
抑制效果见图9。
实施例十、GBL-0401对转基因小鼠模型HBV的HBsAg表达的抑制效果的持续性研究
1.实验方案
取适龄的雄性HBV转基因小鼠(要求HBsAg表达显著)用于实验评估，挑出10只体重处于25g左右的小鼠，随机分成2组，每组5只，分别为对照组和给药组，在Day0分别通过皮下注射给药3mg/kg；给药体积为100-200μL。用药前，采血测定HBsAg，尽量保持各组的HBsAg的水平一致。通过小鼠眼眶静脉丛采集全血，采血时间点为给药前(day 0)、给药后-第1周、第2周、第3周、第4周、第5周和第6周，检测HBsAg，考察GBL-0401对HBV基因表达抑制的持续性。
具体给药信息如下表所示：
序号 试验药物 给药剂量 小鼠数量/组 溶媒 给药途径
1 空白溶剂 - 5只 生理盐水 皮下注射
2 GBL-0401 3mg/kg 5只 生理盐水 皮下注射
2.样品与试剂
序号 名称 规格 纯度/含量
1 GBL-0401 500μg/瓶*1瓶 92.3％
2 生理盐水 500ml/瓶 0.生9％理盐水
3.试剂盒
Figure PCTCN2020097732-appb-000117
4.实验仪器
名称 型号 厂家
旋涡混匀器 MIX-28 大龙兴创
离心机 S1010E THERMO
全自动化学发光分析仪 602 德国罗氏
5.试验结果
结果表明，GBL-0401在第1周即达到最佳抑制效果99.08％，第2周至第3周趋势有所下降，但仍呈现高的抑制率90％左右，第4到第6周呈下降趋势，但依然维持抑制效果在75％左右。GBL-0401对HBV HBsAg表达有持续的抑制效果，可稳定抑制表达在6周左右。GBL-0401体内抑制HBV HBsAg效果图见图10。

Claims (12)

  1. 一种结构中包含有抑制HBV基因表达的干扰核酸、转接点及其末端修饰链的化合物，所述末端修饰链由连接链D、接头B、支链L和肝靶向特异性配体X组成，所述末端修饰链与所述干扰核酸之间通过转接点R 1/R 2连接，其结构为通式(I)所示：
    Figure PCTCN2020097732-appb-100001
    其中：
    n为1时，m为3；n为2时，m也为2；
    R 1为-NH(CH 2) xCH 2-，其中x为3-10的整数；
    R 2为-NHCH 2CH(OH)CH 2(OH)-，或带有伯醇和仲醇的吡咯环或哌啶环；
    所述肝靶向特异性配体X选自半乳糖、半乳糖胺和N-乙酰半乳糖胺；
    所述支链L是C3-C18的直链，该直链中含有羰基、酰胺基、磷酰基、氧原子或这些基团的组合；
    所述接头B选自以下结构式：
    Figure PCTCN2020097732-appb-100002
    其中，A 1是C、O、S或NH；r1为1-15的正整数，r2为0-5的整数；A 2是C、O、S、氨基、羰基、酰胺基、磷酰基或硫代磷酰基；
    所述连接链D含有C5-C20，其含有氨基、羰基、酰胺基、氧原子、硫原子、硫代磷酰基、磷酰基、环状结构或这些基团的组合。
  2. 根据权利要求1所述的化合物，其中，所述干扰核酸包括但不限于siRNA、miRNA或Agomir。
  3. 根据权利要求2所述的化合物，其中，所述干扰核酸为siRNA。
  4. 根据权利要求3所述的化合物，其中，所述干扰核酸为抗乙肝病毒的siRNA。
  5. 根据权利要求4所述的化合物，其中，所述的抗乙肝病毒的siRNA正义链5’端的末端修饰链和所述的反义链3’端的末端修饰链组合选自GBL-01～GBL-16。
  6. 根据权利要求5所述的化合物，其中，所述筛选得到的siRNA的正义链的相对于HBV DNA的起始位点为208、210、1522、1525、1575、1576、1578、1580。
  7. 根据权利要求6所述的化合物，其中，所述的siRNA序列选自：
    1)正义链SEQ ID NO.1和反义链SEQ ID NO.2的组合序列；或
    2)正义链SEQ ID NO.5和反义链SEQ ID NO.6的组合序列；或
    3)正义链SEQ ID NO.7和反义链SEQ ID NO.8的组合序列；或
    4)正义链SEQ ID NO.9和反义链SEQ ID NO.10的组合序列；或
    5)正义链SEQ ID NO.13和反义链SEQ ID NO.14的组合序列；或
    6)正义链SEQ ID NO.17和反义链SEQ ID NO.18的组合序列；或
    7)正义链SEQ ID NO.27和反义链SEQ ID NO.28的组合序列；或
    8)正义链SEQ ID NO.31和反义链SEQ ID NO.32的组合序列。
  8. 根据权利要求1-7中任一项所述的化合物，其选自GBL-0401～GBL-0418。
  9. 如权利要求1-3中任一项所述的化合物在制备用于治疗肝脏相关疾病的药物中的应用，其中，所述肝脏相关疾病包括但不限于急慢肝炎、肝癌、遗传性肝源性疾病、肝硬化、脂肪肝、糖尿病。
  10. 如权利要求1-8中任一项所述的化合物在制备用于治疗HBV感染的相关疾病的药物中的应用，其中，所述HBV感染包括慢性乙型肝炎病毒感染、急性乙型肝炎病毒感染。
  11. 根据权利要求9或10所述的应用，其特征在于：所述肝靶向特异性配体X是针对肝脏中去唾液酸糖蛋白受体(ASGPR)特异性的，所述HBV感染的相关疾病包括慢性乙型肝炎，所述化合物可以持续使HBV的HBsAg、HBeAg和HBV DNA的表达得到抑制。
  12. 一种药物组合物，该药物组合物包含权利要求1-8中任一项所述的化合物和药学上可接受的辅料，优选剂型为皮下注射剂。
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