CN117186172A - Conjugate group, conjugate, preparation method and application thereof - Google Patents

Abstract

The invention provides a conjugate group, a conjugate and a preparation method and application thereof, and belongs to the technical field of medicines. The conjugate group has a structure shown in a formula (I) or a structure shown in a formula (II),the conjugate comprises a conjugate group and a therapeutic molecule linked to the conjugate group, when the conjugate is linked to an expression-inhibiting oligonucleotideNucleotide linkages, when attached, are capable of mediating expression of a target nucleic acid sequence in a liver cell (e.g., a hepatocyte), which can be used to treat diseases or conditions responsive to cellular, tissue or organism gene expression or activity.

Description

Conjugate group, conjugate, preparation method and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a conjugate group, a conjugate, a preparation method and application thereof.

Background

RNA interference (RNAi) is a powerful gene silencing tool that utilizes small interfering RNA (siRNA) of 19-25 nucleotides to degrade specific mRNAs in a highly sequence-specific manner. siRNA is considered a major participant in gene therapy and has shown broad potential for the treatment of a variety of gene-facilitated diseases including viral infections, cardiovascular diseases and cancers. Compared with small molecular drugs and antibodies, the novel drug is a novel drug which takes direct targeting mRNA as a distinction and can inhibit the expression of pathogenic proteins.

Despite the great therapeutic potential of siRNA, its clinical use remains challenging, mainly due to the lack of an effective delivery system. siRNA has been developed clinically for about twenty years, and only one siRNA-based drug (Patisiran) has been approved by the us Food and Drug Administration (FDA) as an orphan drug state from the beginning.

Up to now, in preclinical studies, siRNA delivery strategies have been freed from complex lipid or polymer nanoparticles, in which multicomponent well-defined siRNA bioconjugates comprising chemical functions have a more uniform, stable and biocompatible structure. Since siRNA became a new class of gene therapies for the treatment of various diseases, siRNA bioconjugates, one of the main delivery strategies, offer the property of siRNA with the potential to enhance and broaden pharmacology while minimizing heterogeneity and stability-related toxicology.

Conjugates of N-acetylgalactosamine have become a major clinical strategy for delivering oligonucleotides to hepatocytes. Such conjugates recognize and internalize a glycoprotein galactose (Gal) or N-acetylgalactosamine (GalNAc) moiety containing a terminus through binding to an effective internalizing asialoglycoprotein receptor (ASGPR). Trivalent and tetravalent GalNAc clusters have a higher affinity for ASGPR than monovalent and divalent sugar clusters.

Disclosure of Invention

The present invention provides a conjugate group, a conjugate, and methods of making and using the same, wherein the conjugate group composition, when linked to an expression-inhibiting oligonucleotide, is capable of mediating expression of a target nucleic acid sequence in a liver cell (e.g., a hepatocyte), which is useful in treating a disease or condition responsive to gene expression or activity in a cell, tissue or organism.

The invention provides a conjugate group which has a structure shown in a formula (I),

wherein,

L ₁ 、L ₂ 、L ₃ independently selected from galactose, galactosamine, N-formyl galactosamine, N-acetyl galactosamine, N-propionyl galactosamine, N-N-butyryl galactosamine or N-isobutyryl galactosamine;

A ₁ 、A ₂ 、A ₃ are respectively and independently selected from

K ₁ Independently selected from

Or has a structure shown in a formula (II),

wherein L is ₅ 、L ₆ 、L ₇ 、L ₈ Independently selected from galactose, galactosamine, N-formyl galactosamine, N-acetyl galactosamine, N-propionyl galactosamine, N-N-butyryl galactosamine or N-isobutyryl galactosamine;

A ₅ 、A ₆ 、A ₇ 、A ₈ are respectively and independently selected from

K ₂ Independently selected from

Further, L ₁ 、L ₂ 、L ₃ Independently selected from N-acetylgalactosamine

Further, L ₅ 、L ₆ 、L ₇ 、L ₈ Independently selected from N-acetylgalactosamine

Further, the conjugate group has a structure represented by the formula KAK-1, KAK-2, KAK-3, KAK-4, KAK-5, KAK-6, EEE-1, EEE-2 or EEE-3, specifically as follows:

the present invention contemplates a conjugate comprising any of the above-described conjugate groups and a therapeutic molecule attached to the conjugate groups.

Further, the therapeutic molecule is selected from a functional oligonucleotide, an antibiotic or an antibody.

Further, the functional oligonucleotide is a single-stranded oligonucleotide or a double-stranded oligonucleotide.

Further, the double-stranded oligonucleotide comprises a sense strand and an antisense strand, one end of the conjugate being attached to the 3 '-end or the 5' -end of one of the strands of the double-stranded oligonucleotide; wherein, sense strand: agaccuugufuufufufugcuuugsu b; antisense strand: ascfsaaaaaaafgcaaaaacafgcfucusasg;

preferably, the double-stranded oligonucleotide is an siRNA, each nucleotide in the siRNA being independently a modified and an unmodified nucleotide;

more preferably, the target point corresponding to the siRNA is PCSK9.

The invention also proposes the use of a conjugate as defined in any of the above for the manufacture of a medicament for the treatment and/or prophylaxis of a pathological condition or disease caused by the expression of a specific gene in hepatocytes.

Further, the specific gene is an endogenous gene expressed in the liver, or a pathogen gene propagated in the liver;

preferably, the specific gene is ApoB, apoC, ANGPTL3, PCSK9, SCD1, TIMP-1, col1A1, FVII, STAT3, p53, HBV or HCV.

The invention has the following advantages:

the present invention provides a novel conjugate group that can be attached to a compound (e.g., a therapeutic agent) that can be used to direct the compound to a target in vivo. The disclosed conjugate groups can target expression-inhibiting oligonucleotides (e.g., RNAi agents) to liver cells to modulate gene expression.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention. In the drawings:

FIG. 1 is a schematic diagram showing the results of the free uptake experiment of the human primary cells of test example 1 of the present invention.

Detailed Description

It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other.

Embodiments of the present invention provide a novel conjugate group that can be attached to a compound (e.g., a therapeutic agent) that can be used to direct the compound to a target in vivo. The disclosed conjugate groups can target expression-inhibiting oligonucleotides (e.g., RNAi agents) to liver cells to modulate gene expression.

The conjugate groups disclosed in the examples of the present invention, when attached to expression-inhibiting oligonucleotides, can be used for a variety of purposes, including for therapeutic, diagnostic, target validation, and genomic development purposes. Compositions comprising the disclosed conjugate groups, when linked to expression-inhibiting oligonucleotides, are capable of mediating expression of a target nucleic acid sequence in a liver cell (e.g., a hepatocyte), which can be used to treat diseases or conditions responsive to cellular, tissue or organism gene expression or activity.

An embodiment of the invention provides a conjugate group having a structure represented by formula (I),

wherein,

L ₁ 、L ₂ 、L ₃ each independently selected from galactose (Gal), galactosamine, N-formyl galactosamine, N-acetyl galactosamine (GalNAc), N-propionyl galactosamine, N-N-butyryl galactosamine or N-isobutyryl galactosamine;

A ₁ 、A ₂ 、A ₃ are respectively and independently selected from

K ₁ Independently selected from

Or has a structure shown in a formula (II),

wherein L is ₅ 、L ₆ 、L ₇ 、L ₈ Each independently selected from galactose (Gal), galactosamine, N-formyl galactosamine, N-acetyl galactosamine (GalNAc), N-propionyl galactosamine, N-N-butyryl galactosamine or N-isobutyryl galactosamine;

A ₅ 、A ₆ 、A ₇ 、A ₈ are respectively and independently selected from

K ₂ Independently selected from

It is to be noted that,representing the site of attachment of the group by covalent bonds.

Preferably, L ₁ 、L ₂ 、L ₃ Independently selected from N-acetylgalactosamine

Preferably, L ₅ 、L ₆ 、L ₇ 、L ₈ Independently selected from N-acetylgalactosamine

In the embodiment of the invention, L ₁ 、L ₂ 、L ₃ 、L ₅ 、L ₆ 、L ₇ 、L ₈ Is a targeting ligand; k (K) ₁ 、K ₂ Is a group for attachment of active drugs.

In a preferred embodiment of the present invention, the conjugate group has a structure represented by the formulas KAK-1, KAK-2, KAK-3, KAK-4, KAK-5, KAK-6, EEE-1, EEE-2 or EEE-3, specifically as follows:

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an embodiment of the present invention also provides a conjugate comprising the above-described conjugate group and a therapeutic molecule attached to the conjugate group.

Preferably, the therapeutic molecule is selected from a functional oligonucleotide, an antibiotic or an antibody.

Further, the functional oligonucleotide is a single-stranded oligonucleotide or a double-stranded oligonucleotide.

Specifically, the double-stranded oligonucleotide comprises a sense strand and an antisense strand, one end of the conjugate being attached to the 3 '-end or the 5' -end of one of the strands of the double-stranded oligonucleotide, wherein the sense strand: agaccuugufuufufufugcuuugsu b; antisense strand: ascfsaaaaaaafgcaaaaacafgcfucusasg.

Note that: the letter C, G, U, A denotes the base composition of a nucleotide, wherein the lower case letter is a 2 '-methoxy modified nucleotide, the upper case letter is a 2' -fluoro modified nucleotide, and s denotes that the linkage between two nucleotides adjacent to the left and right of the letter is phosphorothioate.

More specifically, the double-stranded oligonucleotide is an siRNA, each nucleotide in the siRNA being independently a modified and an unmodified nucleotide. Specifically, the target point corresponding to the siRNA is PCSK9.

The conjugate containing any therapeutic molecule and the conjugate group can effectively improve the delivery efficiency of the therapeutic molecule, thereby improving the therapeutic effect of the conjugate and the pharmaceutical composition thereof.

Use of any of the conjugates described above in accordance with an embodiment of the present invention for the manufacture of a medicament for the treatment and/or prevention of a pathological condition or disease caused by the expression of a specific gene in hepatocytes.

Preferably, the specific gene is an endogenous gene expressed in the liver, or a pathogen gene propagated in the liver.

More preferably, the specific gene is ApoB, apoC, ANGPTL, PCSK9, SCD1, TIMP-1, col1A1, FVII, STAT3, p53, HBV or HCV.

In the examples of the present invention, the proposed conjugates are mainly directed against cell surface receptors specific for hepatocytes, in particular the asialoglycoprotein receptor (asialoglycoprotein receptors, ASGPR) on the liver surface. The conjugates presented in the examples of the present invention specifically bind to specific receptors of a specific tissue, thereby achieving tissue-specific targeting.

The invention will be described in detail with reference to examples.

Example 1 preparation of intermediates

Preparation of intermediate 2:

to a 250mL three-necked flask, the compound (2S, 3R,4R,5R, 6R) -3-acetamido-6- (acetoxymethyl) tetrahydro-2H-pyran-2, 4, 5-triacetate (12.5 g,32.1 mmol) and anhydrous methylene chloride (50 mL) were added, the mixture was replaced with argon, the ice water bath was controlled at about 0deg.C, and trimethylsilyl triflate (7.5 mL) was added dropwise. After the completion of the dropwise addition, the mixture was stirred at room temperature overnight. TLC detects the disappearance of the starting material spot, work up. The reaction solution was poured into saturated sodium bicarbonate solution, and extracted twice with dichloromethane. The organic phases were combined, washed with saturated brine and dried over anhydrous sodium sulfate. Further filtration and concentration gave 10.9g of crude compound 2 (3 aR,5R,6R,7 aR) -5- (acetoxymethyl) -2-methyl-3 a,6,7 a-tetrahydro-5H-pyrano [3,2-d ] oxazol-6, 7-diacetate, which was used directly in the next step.

Preparation of intermediate GT-2:

in a 250mL three-necked flask, compound 2 (10 g,30.4 mmol) and benzyl 6-hydroxycaproate (8.0 g,35.96 mmol) were dissolved in 150mL anhydrous dichloromethane, replaced with argon, the ice water bath was controlled at about 0deg.C, and trimethylsilyl triflate (2.8 mL) was added dropwise. After the completion of the dropwise addition, the mixture was stirred at room temperature overnight. TLC detection reaction was complete and work up. The reaction solution was poured into saturated sodium bicarbonate solution, and extracted twice with dichloromethane. The organic phases were combined, washed with saturated brine and dried over anhydrous sodium sulfate. The crude product was filtered again and concentrated to give 13.9g of the product compound 4 (2R, 3R,4R,5R, 6R) -5-acetamido-2- (acetoxymethyl) -6- ((6- (benzyloxy) -6-oxohexyl) oxy) tetrahydro-2H-pyran-3, 4-diacetic acid diester in 92% yield. LC-MS: m/z 581.2[ M+H ]] ⁺ .

In a 250mL single vial, compound 4 (10.0 g,18.1 mmol) and compound p-toluenesulfonic acid monohydrate (3.5 g,18.4 mmol) were dissolved in 150mL methanol and replaced with 10% palladium on carbon (1 g) under argon. The reaction mixture was replaced with hydrogen atmosphere and stirred at room temperature overnight. TLC detection reaction was complete and work up. The reaction solution was filtered through celite to remove palladium on carbon. The filtrate was concentrated to give 7.0g of the product intermediate GT-2 6- (((2R, 3R,4R,5R, 6R) -3-acetamido-4, 5-diacetoxy-6- (acetoxymethyl) tetrahydro-2H-pyran-2-yl) oxy) hexanoic acid in 83% yield. LC-MS: m/z 447.2[ M+H ]] ⁺ .

Preparation of intermediate GT-3:

intermediate GT-2 (5.0 g,10.8 mmol) was dissolved in anhydrous 40mL DCM, pentafluorophenol (3.0 g,16.3 mmol) and EDCi (3.2 g,16.7 mmol) were added, the mixture was stirred and replaced with argon for 12 hours at room temperature, and the condensation reaction was completed. 100mL of saturated sodium bicarbonate solution was added, extracted twice with 50mL of ethyl acetate, and the organic phases were combined. Dried over anhydrous sodium sulfate, filtered, the filtrate concentrated to give crude product, which is finally separated by column chromatography to give 4.2g of product intermediate GT-3 (2R, 3R,4R,5R, 6R) -5-acetamido-2- (acetoxymethyl) -6- ((6-oxo-6- (perfluorophenoxy) hexyl) oxy) tetrahydro-2H-pyran-3, 4-diacetate diester in 61.8% yield. LC-MS: m/z 628.2[ M+H ]] ⁺ .

Example 2 preparation of KAK-4:

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the synthesis steps comprise:

step 1: 2-Cl-trityl resin (0.4-3.0 mmol/g,7.0 g) was weighed into the reaction tube and 50mL DMF/DCM was added to swell the resin for 2 hours. Compound 6Fmoc- (NCbz) -Lys (L) -OH (21.1 g,42 mmol) was weighed into 10mL DMF and DIEA (10.8 g,84 mmol) was added, mixed well and added to the swollen resin and reacted at room temperature for 12 hours. After the reaction was completed, 40mL of methanol was added. After the reaction was continued for 30min, the resin was drained, washed with DMF and DCM, and finally the resin was shrunk with methanol to give the dried intermediate 7Fmoc- (NCbz) -Lys (L) -2-Cl-trityl resin, which gave a resin substitution value of 0.7mmol/g.

Step 2: the above intermediate 7 (0.7 mmol/g, crop, 5 mmol) was placed in the reaction tube and 20mL DMF/DCM was added to swell the resin for 1 hour. The resin was drained and Fmoc removal was performed by adding 60mL of 20% diethyl amine/DMF. The removal reaction was carried out twice for 60 minutes each. The reacted resin was washed with DMF and DCM, respectively.

Fmoc-Ala-OH (3.1 g,10 mmol), EDCi (1.9 g,10 mmol) and HOBT (1.4 g,10 mmol) were weighed into 50mL DMF, and after mixing, the washed resin was added and reacted at room temperature for 12 hours to effect amino condensation. After the reaction was completed, the resin was drained and the reacted resin was washed with DMF and DCM, respectively. Finally, the detection reagent is used for determining that no amino group remains on the resin.

Step 3: the above intermediate 8 (0.7 mmol/g, crop, 5 mmol) was placed in the reaction tube and 20mL DMF/DCM was added to swell the resin for 1 hour. The resin was drained and Fmoc removal was performed by adding 60mL of 20% diethyl amine/DMF. The removal reaction was carried out twice for 60 minutes each. The reacted resin was washed with DMF and DCM, respectively.

Cbz- (NCbz) -Lys (L) -OH (5.0 g,10 mmol), EDCi (1.9 g,10 mmol) and HOBT (1.4 g,4 mmol) were weighed into 50mL DMF, and after mixing, the above-mentioned washed resin was added and reacted at room temperature for 12 hours to effect the condensation of amino groups. After the reaction was completed, the resin was drained and the reacted resin was washed with DMF and DCM, respectively. Finally, the detection reagent is used for determining that no amino group remains on the resin.

Step 4: the above intermediate 9 (0.7 mmol/g, crude,5 mmol) was reacted in a reaction tube with 30mL of 20% trifluoroethanol/DCM solution at room temperature for 2 hours. And (5) carrying out suction filtration and collecting the full-protection cutting fluid. Repeating the cutting operation once, and combining the two cutting liquids. After the solvent was removed by rotary evaporation of the cutting fluid, KAK 2.5.5 g of crude product was obtained.

LC-MS:m/z 748.4[M+H] ⁺ .

¹ H NMR(400MHz,DMSO-d ₆ )δ8.12–7.92(m,3H),7.88(d,J＝7.6Hz,2H),7.71(dd,J＝7.2,4.1Hz,2H),7.50–7.26(m,14H),7.21(t,J＝5.3Hz,2H),5.02(d,J＝18.0Hz,4H),4.37–4.07(m,5H),3.97(d,J＝4.0Hz,1H),1.61(m,4H),1.46–1.14(m,11H).

Step 5: compound KAK (1.5 g,2.01 mmol) was dissolved in anhydrous 40mL DCM, added (S) -3-pyrrolidinol (348.5 mg,4 mmol), EDCi (766.8 mg,4 mmol) and HOBt (540.5 mg,4 mmol) were mixed and replaced with argon for 12 hours at room temperature, and the condensation reaction was completed. 100mL of NaHCO3 saturated solution was added, extracted twice with 50mL of dichloromethane, and the organic phases were combined. Dried over anhydrous sodium sulfate, filtered, the filtrate concentrated to give crude product, which was separated by column chromatography to give 1.1g of product 10. The yield thereof was found to be 67.1%.

LC-MS:m/z 817.4[M+H] ⁺ .

Step 6: in a 100mL single vial, compound 10 (1.1 g,1.35 mmol) and p-toluenesulfonic acid monohydrate (819.5 mg,4.3 mmol) were dissolved in 30mL of methanol and replaced with 10% palladium on carbon (0.3 g) under argon. The reaction mixture was replaced with hydrogen atmosphere and stirred at room temperature overnight. TLC detection reaction was complete and work up. The reaction solution was filtered through celite to remove palladium on carbon. The filtrate was concentrated to give 1.1g of crude 11 in 99.0% yield.

Step 7: in a 100mL single vial, compound 11 (1.1 g,1.35 mmol) and compound GalNAc active ester GT-3 (4.24 g,6.75 mmol) were dissolved in acetonitrile (5 mL) solvent and 10% NaHCO3 (5 mL) was added with magnetic stirring. The reaction was then stirred at room temperature overnight. TLC detection reaction was complete and work up. The reaction was added to 100mL of saturated NaHCO3 solution, extracted twice with 50mL of dichloromethane, and the organic phases were combined. Drying by anhydrous sodium sulfate, filtering, concentrating the filtrate to obtain crude product, and separating by column chromatography to obtain 2.1g of product 12 with the yield of 89.1%.

LC-MS:m/z 1745.9[M+H] ⁺ .

Step 8: in a 100mL single vial, compound 12 (400 mg,0.23 mmol) and 10mL of anhydrous acetonitrile were replaced under argon, the ice water temperature was controlled at about 0deg.C, and 5-ethylsulfanyl-1H-tetrazole (60 mg,0.46 mmol) and 2-cyanoethyl-bis (diisopropyl) phosphoramidite (139 mg,0.46 mmol) were added. Stirred at room temperature for 2 hours. LCMS detected complete reaction, work-up. And washed with saturated sodium bicarbonate solution and extracted twice with dichloromethane. The dichloromethane phases were combined, washed with saturated sodium chloride solution and dried over anhydrous sodium sulfate. Finally, filtering and concentrating, separating the crude product by column chromatography to obtain 160mg of compound KAK-4 with the yield of 35.9%.

PNMR：146.5ppm，MS：1808.6；1864.0[M-N(iPr) ₂ +OH ⁺ ].

Example 3 preparation of KAK-6:

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the synthesis steps comprise:

step 1: in a 500mL three-necked flask, 13N2, N6-bis (t-butoxycarbonyl) -L-lysine (25.0 g,72.17 mmol) and N, N-dimethylformamide (250 mL) were added and replaced with argon, and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (16.6 g,86.6 mmol) and N, N-diisopropylethylamine (15.1 mL,86.6 mmol) were added. After the addition was completed, the mixture was stirred at room temperature overnight. TLC detects the disappearance of the starting material spot, work up. The reaction solution was poured into saturated sodium bicarbonate solution, and extracted twice with dichloromethane. The organic phases were combined, washed with saturated brine and dried over anhydrous sodium sulfate. And then filtering and concentrating to obtain crude product, and separating by column chromatography to obtain 20.0g of compound 14 2, 5-dioxopyrrolidin-1-yl N2, N6-bis (tert-butoxycarbonyl) -L-lysine with yield of 62%.

Step 2: in a 250mL three-necked flask, the compound 14, 5-dioxapyrrolidin-1-yl N2, N6-bis (t-butoxycarbonyl) -L-lysine (16.0 g,46.19 mmol) and N, N-dimethylformamide (160 mL) were added and replaced with L-alanine (4.1 g,46.19 mmol) under argon. After the addition, the mixture was stirred at 80℃overnight. TLC detects substantial disappearance of starting material spot, work-up. The reaction solution was poured into saturated sodium bicarbonate solution, and extracted twice with dichloromethane. The organic phases were combined, washed with saturated brine and dried over anhydrous sodium sulfate. The crude product is obtained by filtration and concentration, and 14.0g of the compound 15N2, N6-bis (tert-butoxycarbonyl) -L-lysyl-L-alanine is obtained by column chromatography separation, and the yield is 92%.

Step 3: in a 250mL three-necked flask, 15N2, N6-bis (t-butoxycarbonyl) -L-lysyl-L-alanine (9.0 g,21.56 mmol) and N, N-dimethylformamide (90 mL) were added and replaced with argon, N6- (t-butoxycarbonyl) -L-lysine benzyl ester hydrochloride (9.7 g,25.87 mmol), 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate (9.8 g,25.87 mmol) and N, N-diisopropylethylamine (11.3 mL,64.67 mmol) were added. After the addition, the mixture was stirred at 80℃overnight. TLC detects substantial disappearance of starting material spot, work-up. The reaction solution was poured into saturated sodium bicarbonate solution, and extracted twice with dichloromethane. The organic phases were combined, washed with saturated brine and dried over anhydrous sodium sulfate. The crude product is obtained by filtration and concentration, and 7.0g of compound 16N2-N2, N6-bis (tert-butoxycarbonyl) -L-lysyl-L-alanyl-N6- (tert-butoxycarbonyl) -L-lysine benzyl ester is obtained by column chromatography separation, and the yield is 44%.

Step 4: in a 250mL three-necked flask, the compound 16N2-N2, N6-bis (t-butoxycarbonyl) -L-lysyl-L-alanyl-N6- (t-butoxycarbonyl) -L-lysine benzyl ester (7 g,9.51 mmol) and formic acid (70 mL) were added. After the addition was completed, the mixture was stirred at 40℃overnight. TLC detects substantial disappearance of starting material spot, work-up. The reaction solution was concentrated to obtain a crude product, which was separated by column chromatography to obtain 5.6g of L-lysyl-L-alanyl-L-lysine benzyl formate Int3 in 99% yield.

Step 5: in a 250mL three-necked flask, compound L-lysyl-L-alanyl-L-lysine benzyl formate Int3 (1.0 g,2.30 mmol) and N, N-dimethylformamide (30 mL) were added, replaced with argon, and 6- (((2R, 3R,4R,5R, 6R) -3-acetamido-4, 5-diacetoxy-6- (acetoxymethyl) tetrahydro-2H-pyran-2-yl) oxy) hexanoic acid GT-2 (3.7 g,8.04 mmol), O-benzotriazol-N, N, N ', N' -tetramethylurea tetrafluoroborate (2.70 g,8.50 mmol), 1-hydroxybenzotriazole (1.1 g,8.50 mmol) and N, N-diisopropylethylamine (6.4 mL,36.73 mmol) were added. After the addition was completed, the mixture was stirred at room temperature overnight. TLC detects substantial disappearance of starting material spot, work-up. The reaction solution was poured into saturated sodium bicarbonate solution, and extracted twice with dichloromethane. The organic phases were combined, washed with saturated brine and dried over anhydrous sodium sulfate. Filtering and concentrating to obtain crude product, separating by column chromatography to obtain KAK-6-001 with yield of 24%.

Step 6: in a 100mL single-necked flask, compound KAK-4-001 (1.0 g,0.49 mmol) was dissolved in 30mL of methanol, replaced with argon, and 10% palladium on carbon (0.3 g) was added. The reaction mixture was replaced with hydrogen atmosphere and stirred at room temperature overnight. TLC detection reaction was complete and work up. The reaction solution was filtered through celite to remove palladium on carbon. The filtrate was concentrated to give 0.7g of Int-5 as a product in 73% yield.

Step 7: in a 50mL three-necked flask, compound Int-5 (950 mg,0.57 mmol) and N, N-dimethylformamide (10 mL) were added and replaced with argon, 2- (piperazin-1-yl) ethan-1-ol (148 mg,1.13 mmol), O-benzotriazol-N, N, N ', N' -tetramethylurea tetrafluoroborate (406 mg,1.70 mmol), 1-hydroxybenzotriazole (230 mg,1.70 mmol) and N, N-diisopropylethylamine (220 mg,1.70 mmol) were added. After the addition was completed, the mixture was stirred at room temperature overnight. TLC detects substantial disappearance of starting material spot, work-up. The reaction solution was poured into saturated sodium bicarbonate solution, and extracted twice with dichloromethane. The organic phases were combined, washed with saturated brine and dried over anhydrous sodium sulfate. Filtering and concentrating to obtain crude product, and preparing liquid phase separation by Prep-HPLC to obtain 660mg of Int-6 with 65% yield.

Step 8: in a 100mL single-necked flask, compound Int-6 (300 mg,0.17 mmol) and 20mL of anhydrous methylene chloride were replaced with argon, the temperature in the ice water area was controlled at about 0deg.C, and diisopropylammonium tetrazole (29 mg,0.17 mmol) and 2-cyanoethyl-bis (diisopropyl) phosphoramidite (76 mg,0.25 mmol) were added. After the addition, stirring was carried out at 0℃for 3 hours. LCMS detected complete reaction, work-up. And washed with saturated sodium bicarbonate solution and extracted twice with dichloromethane. The dichloromethane phases were combined, washed with saturated sodium chloride solution and dried over anhydrous sodium sulfate. Finally, the crude product is filtered and concentrated, 185mg of compound KAK-6 is obtained by column chromatography separation, and the yield is 55%. PNMR:146.9ppm, MS:1851.5;1905.0[ M-N (iPr) ₂ +OH ⁺ ]。

Example 4 preparation of conjugates

1. Synthesis of nucleic acid sequences:

adopts a phosphoramidite nucleic acid solid phase synthesis method, uses a general solid phase carrier (CPG general column of Universal linker) to start a solid phase, the nucleotide monomers are linked one by one in the 3'-5' direction according to the sense (SS) or Antisense (AS) strand sequence order of the sequence. Each nucleoside monomer connected comprises four reaction cycles of deprotection, coupling, capping and oxidation. The synthesis conditions set parameters according to the instrument.

The synthesis procedure included the following unit cycles:

1) Deprotection (De-blocking): ribonucleotides with DMT (di-p-methoxytrityl) protecting groups at the 5 '-OH end are removed from the solid phase carrier and ribonucleotides using trichloroacetic acid (TCA) in the first step of synthesis, so that the 5' -OH of the naked ribonucleotide is coupled with a new base;

2) Coupling (Coupling): firstly, pumping an activating reagent Activator into the CPG column, then pumping the next phosphoramidite nucleotide monomer, and mixing in the column to form a phosphoramidite tetrazole active intermediate;

when the activated phosphoramidite tetrazole contacts CPG-Oligo, nucleophilic reaction is carried out with 5' hydroxyl, coupling is carried out, tetrazole is removed, and one nucleotide is extended;

3) Capping (Capping): because the coupling efficiency can not reach 100%, in order to prevent the uncoupling of the CPG-Oligo from continuing to enter the next coupling step, the 5' hydroxyl cap of the CPG-Oligo is blocked by using CapA and CapB reagents;

4) Oxidation (Oxidation) or thio: after the coupling reaction, the nucleotide is connected with the oligonucleotide on CPG through a phosphite ester bond (trivalent phosphorus), the phosphite ester bond is unstable and is easy to be subjected to acid and alkaline hydrolysis, and the trivalent phosphorus is oxidized into pentavalent phosphorus oxide through an oxidizing reagent. Thio refers to the reaction of trivalent phosphorus of a phosphorous ester linkage to form a pentavalent phosphorus sulfide linkage by a thio reagent under weakly alkaline conditions.

2. Preparation of conjugate 1

Complete sequences were synthesized from the sense/antisense strand of PCSK9 via solid phase, and finally the 5' -end of the sense strand was conjugated with KAK-4 (see Anna)Sandra M Ocampo, ricardo Lucas, mol. Divers.2011,15,751-757, supra).

Coupling KAK-4 of CPG carrier by the previous solid phase synthesis (1 umol) was added to 1ml ammonia water 25wt% and reacted at 55℃for 16 hours, and the liquid was removed by centrifugation to dryness. LC-MS showed the end of the reaction, purification using preparative reverse phase chromatography purification column, purification of Sense Strand (SS) and Antisense Strand (AS) was completed.

Annealing the Sense Strand (SS) and the Antisense Strand (AS) to obtain conjugate 1;

3. preparation of conjugate 2

As above, the complete sequence was synthesized from the sense strand/antisense strand of PCSK9 via solid phase, and finally the 5' -end of the sense strand was ligated to KAK-6 (see Anna)Sandra M Ocampo, ricardo Lucas, mol. Divers.2011,15, 751-757.). Annealing the sense strand and the antisense strand to obtain a conjugate 2;

see in particular table 2:

TABLE 2

Note that: the letter C, G, U, A denotes the base composition of a nucleotide, wherein the lower case letter is a 2 '-methoxy modified nucleotide, the upper case letter is a 2' -fluoro modified nucleotide, and s denotes that the linkage between two nucleotides adjacent to the left and right of the letter is phosphorothioate.

Test example 1 biological assay

Free uptake assay of human Primary cells

Human primary hepatocytes (supplier: BIOIVT) (cryopreservation) and at 37℃and 5% CO ₂ The culture is carried out in a resuscitating culture medium in a humidifying incubator under atmosphere. After resuscitation, cells were plated in collagen-coated 96-well plates at a density of 6X 10 ⁵ After 24 hours of cell/ml exchange, siRNA with the final concentration of 250 nM, 100nM and 10nM is added into the culture medium, the supernatant is collected for 48 hours to detect the concentration of PCSK9, and RNA is extracted for RT-qPCR detection. The test results are shown in FIG. 1.

As can be seen from fig. 1, the knockdown levels of PCSK9 by conjugate 1 and conjugate 2 were detected with nearly 50% knockdown at 100nM levels. KAK4 and KAK can achieve the aim of siRNA liver targeting.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

Claims (10)

1. A conjugate group is characterized by having a structure represented by formula (I),

wherein,

L ₁ 、L ₂ 、L ₃ independently selected from galactose, galactosamine, N-formyl galactosamine, N-acetyl galactosamine, N-propionyl galactosamine, N-N-butyryl galactosamine or N-isobutyryl galactosamine;

A ₁ 、A ₂ 、A ₃ are respectively and independently selected fromK ₁ Independently selected from

Or has a structure shown in a formula (II),

wherein L is ₅ 、L ₆ 、L ₇ 、L ₈ Independently selected from galactose, galactosamine, N-formyl galactosamine, N-acetyl galactosamine, N-propionyl galactosamine, N-N-butyryl galactosamine or N-isobutyryl galactosamine;

A ₅ 、A ₆ 、A ₇ 、A ₈ are respectively and independently selected from

K ₂ Independently selected from

2. The conjugate group according to claim 1, wherein,

L ₁ 、L ₂ 、L ₃ independently selected from N-acetylgalactosamine

3. The conjugate group according to claim 1, wherein,

L ₅ 、L ₆ 、L ₇ 、L ₈ independently selected from N-acetylgalactosamine

4. A conjugate group according to claim 2 or 3, characterized in that,

the conjugate group has a structure represented by the formula KAK-1, KAK-2, KAK-3, KAK-4, KAK-5, KAK-6, EEE-1, EEE-2 or EEE-3, and is specifically as follows:

5. a conjugate comprising a conjugate group according to any one of claims 1 to 4 and a therapeutic molecule attached to the conjugate group.

6. The conjugate of claim 5, wherein the therapeutic molecule is selected from a functional oligonucleotide, an antibiotic, or an antibody.

7. The conjugate of claim 6, wherein the functional oligonucleotide is a single-stranded oligonucleotide or a double-stranded oligonucleotide.

8. The conjugate of claim 7, wherein the double-stranded oligonucleotide comprises a sense strand and an antisense strand, one end of the conjugate being attached to the 3 '-terminus or the 5' -terminus of one of the strands of the double-stranded oligonucleotide; wherein, sense strand: agaccuugufuufufufugcuuugsu b; antisense strand: ascfsaaaaaaafgcaaaaacafgcfucusasg;

preferably, the double-stranded oligonucleotide is an siRNA, each nucleotide in the siRNA being independently a modified and an unmodified nucleotide;

more preferably, the target point corresponding to the siRNA is PCSK9.

9. Use of a conjugate according to any one of claims 5 to 8 in the manufacture of a medicament for the treatment and/or prophylaxis of a pathological condition or disease caused by the expression of a specific gene in hepatocytes.

10. The use according to claim 9, wherein the specific gene is an endogenous gene expressed in the liver, or a pathogen gene propagated in the liver;

preferably, the specific gene is ApoB, apoC, ANGPTL3, PCSK9, SCD1, TIMP-1, col1A1, FVII, STAT3, p53, HBV or HCV.