Galnac cluster phosphoramidites and targeted therapeutic nucleosides

Abstract

The present invention provides oligonucleotide agents comprising one or more therapeutic oligonucleotides (e.g., siRNA) and one or more targeting conjugate compounds. In some embodiments, the conjugate compounds comprise one or more N-acetylgalactosamine, branching groups, and linker groups as targeting groups. By incorporating long carbon chains into the GalNAc cluster (rather than using multiple amide groups) to extend the chain length, a simplified synthesis is achieved by way of reducing the number of steps.

Description

[0001] This invention application is a divisional application of the patent application filed on March 8, 2022, with application number 202210230071.5 and invention title "GalNAc cluster phosphoramide and targeted therapeutic nucleoside". Technical Field

[0002] This invention relates to the field of delivering therapeutic agents using carbohydrate conjugates. In particular, the invention provides novel carbohydrate conjugates and iRNA reagents comprising these conjugates, which facilitate the in vivo delivery of these iRNA reagents, as well as iRNA compositions suitable for in vivo therapeutic use. Furthermore, the invention provides methods for preparing these compositions, and methods for introducing these iRNA reagents into cells using these compositions, for example, for treating various disease symptoms, including metabolic diseases or conditions such as liver disease or conditions. Background Technology

[0003] Hepatocellular-targeted delivery of therapeutic agents is a particularly attractive strategy for treating metabolic, cardiovascular, and other liver diseases. The desialylate glycoprotein receptor (ASGP-R) is highly expressed on hepatocytes but rarely found on extrahepatic cells, making it an ideal entry point for hepatocellular-targeted therapy. The carbohydrate-binding domain of ASGPR has been elucidated, which simplifies the design of effective binders (Bioconjugate Chem. 2017, 28, 283-295). Several multivalent ligands targeting ASGP-R have been developed, among which the well-defined multivalent N-acetyl-D-galactosamine (GalNAc) moiety exhibits high binding affinity (J Am Chem Soc. 2017, 139, 3528–3536). Recently, several gene delivery systems based on GalNAc ligands targeting ASGP-R have shown encouraging clinical results, and the FDA has approved GalNAc-conjugated siRNAs for liver diseases (Molecular Therapy, 2020, 28, 1759-1771).

[0004] Antisense oligonucleotides (ASOs) and siRNAs bind to complementary mRNAs and recruit factors to degrade target mRNAs to regulate protein expression, thereby generating a pharmacological response (Nucleic Acids Research, 2018, 46, 1584–1600). Second-generation ASOs are typically 20-nucleotide-long phosphate-thioester oligonucleotides containing a 10-nucleotide DNA “gap” and terminated with 2'-O-methyl, 2'-O-methoxyethyl (MOE), or locked nucleic acid (LNA) nucleotides (Drug DiscoveryToday, 2018, 23, 101–114). Several second-generation ASOs targeting various indications have entered clinical trials, many of which target mRNAs primarily expressed in hepatocytes, the liver. Recently, conjugation of ASOs and siRNAs with the tri-antennary GalNAc ligand has been shown to enhance hepatocyte potency (Molecular Therapy, 2019, 27, 1547–1555). GalNAc conjugations at the 3' and 5' ends of oligonucleotides have been evaluated, and they significantly enhanced potency in both cells and animals (Bioconjugate Chem. 2015, 26, 1451–1455).

[0005] Related existing technologies

[0006] WO2009 / 002944A1 describes an iRNA reagent conjugated to at least one (preferably dual- or triple-connector) carbohydrate ligand. The carbohydrate-conjugated iRNA reagent is specifically targeted at liver parenchymal cells.

[0007] WO2015 / 042447A1 describes a series of branching groups conjugated with therapeutic nucleoside agents and GalNAc ligands.

[0008] WO2017084987A1 describes GalNAc phosphoridamide derivatives that can be directly introduced as building blocks along with nucleoside building blocks in solid-phase oligonucleotide synthesis.

[0009] However, synthesizing suitable multivalent GalNAc ligands is not easy and typically requires more than 10 chemical reaction steps. Here, we improve GalNAc ligands by introducing long carbon chains to create new structures, enabling more efficient synthesis and longer durability of GalNAc conjugates. Attached Figure Description

[0010] Figure 1AThe plasma ApoB levels in mice treated with GalNAc cluster B001 (which has no spacer between GalNAc and oligonucleotides) and GalNAc cluster B005 (which has a spacer between GalNAc and oligonucleotides) were compared.

[0011] Figure 1B Plasma ApoB levels in mice treated with GalNAc cluster B003 (a spacer between GalNAc and oligonucleotides) and positive control GalNAc cluster B005.

[0012] Figure 1C The structure of GalNAc cluster B003 disclosed herein.

[0013] Figure 2A The present invention compares the ApoB levels of the GalNAc-ApoB antisense conjugate B006 (group 3 / 4) and the positive control GalNAc cluster B005 (group 1 / 2) at two dose levels.

[0014] Figure 2B Comparison of ApoB levels between the GalNAc-ApoB antisense conjugate B007 (group 5 / 6) disclosed herein and the positive control GalNAc cluster B005 (group 1 / 2) at two dose levels.

[0015] Figure 2C The present invention compares the ApoB levels of the GalNAc-ApoB antisense conjugate B008 (group 7 / 8) and the positive control GalNAc cluster B005 (group 1 / 2) at two dose levels.

[0016] Figure 2D The present invention compares the ApoB levels of the GalNAc-ApoB antisense conjugate B009 (group 9 / 10) and the positive control GalNAc cluster B005 (group 1 / 2) at two dose levels.

[0017] Figure 2E Comparison of ApoB levels at two dose levels between the GalNAc-ApoB antisense conjugate B011 (group 11 / 12) and the positive control GalNAc cluster B005 (group 1 / 2).

[0018] Figure 2F Comparison of ApoB levels at two dose levels between the GalNAc-ApoB antisense conjugate B013 (group 13 / 14) and the positive control GalNAc cluster B005 (group 1 / 2).

[0019] Figure 2GComparison of ApoB levels at two dose levels between the GalNAc-ApoB antisense conjugate B015 (group 15 / 16) and the positive control GalNAc cluster B005 (group 1 / 2).

[0020] Figure 3 Standard synthesis cycle for oligonucleotide synthesis using universal adapter solid support on a DNA / RNA synthesizer. Summary of the Invention

[0021] This invention relates to a series of conjugates, conjugated antisense oligonucleotide reagents (which can be used as therapeutic agents), methods for preparing conjugates and conjugated antisense oligonucleotide reagents, and methods including contacting cells with conjugated antisense reagents to reduce the amount or activity of nucleic acid transcripts in the cells.

[0022] In some embodiments, this disclosure relates to conjugates having the structure of formula (I):

[0023]

[0024] in,

[0025] T is a cell-targeting ligand;

[0026] L1 and L2 are each independently a tethering group;

[0027] C is a linking group;

[0028] B is a branching group;

[0029] D is a linking group;

[0030] E is an ester group;

[0031] A is the antisense sequence or messenger strand of the siRNA;

[0032] a is 0 or 1;

[0033] b is an integer between 1 and 5;

[0034] c is 1 or 2;

[0035] d is 0 or 1.

[0036] In some embodiments, this disclosure also relates to conjugates having di-antennary, tri-antennary, tetra-antennary, penta-antennary, or hexa-antennary cell-targeting ligands.

[0037] In some embodiments, this disclosure also relates to conjugated antisense oligonucleotide reagents (which can be used as therapeutic agents), RNA reagents or DNA reagents comprising conjugated compounds and antisense or siRNA oligonucleotides.

[0038] In some embodiments, this disclosure also relates to methods for preparing conjugates and methods for conjugating them with oligonucleotides.

[0039] Novel conjugates can be readily synthesized, and they readily facilitate the binding of cell-targeting ligands to increase liver delivery, or open new pathways to couple multiple ASOs onto a single molecule to improve delivery efficiency. Detailed Implementation

[0040] Conjugate structure

[0041] Some embodiments of the conjugate include compounds of formula (I):

[0042]

[0043] in,

[0044] T is a cell-targeting ligand;

[0045] L1 and L2 are each independently a tethering group;

[0046] C is a linking group;

[0047] B is a branching group;

[0048] D is a linking group;

[0049] E is an ester group;

[0050] A is the antisense sequence or messenger strand of the siRNA;

[0051] a is 0 or 1;

[0052] b is an integer between 1 and 5;

[0053] c is 1 or 2.

[0054] In some embodiments, T is selected from structures having an affinity for at least one type of receptor on target cells. In some embodiments, T is selected from structures having an affinity for at least one type of receptor on the surface of mammalian hepatocytes. In some embodiments, T is a carbohydrate, a carbohydrate derivative, a modified carbohydrate, a multivalent carbohydrate cluster, a polysaccharide, a modified polysaccharide, or a polysaccharide derivative. In some embodiments, each T is independently a carbohydrate, an amino sugar, or a thioglycolate. For example, a carbohydrate is selected from glucose, mannose, galactose, and fucose. For example, the amino sugar is selected from any number of compounds known in the art, such as glucosamine, sialic acid, α-D-galactosamine, N-acetylgalactosamine, 2-acetamido-2-deoxy-D-galactopyranose (GalNAc), 2-amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose (β-muramic acid), 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-carboxamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfano-D-glucopyranose, and N-sulfo-D-glucosamine and N-glycyl-α-neuraminic acid. For example, the thioglycolate can be selected from 5-thio-β-D-glucopyranose, 2,3,4-tri-O-acetyl-1-thio-6-O-triphenylmethyl-α-D-methylglucopyranoside, 4-thio-β-D-galactopyranose, and ethyl ethyl 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucopyranose. Preferably, T is 2-acetamido-2-deoxy-D-galactopyranose (GalNAc).

[0055] In some implementations, L1 and L2 are selected from C1-C 20 Alkylene, amide, (C1-C) 20 )alkylene-amide-(C1-C 20 Alkylene. In some embodiments, L1 and L2 are each independently selected from C1, C2, C3, C4, C5, C6, C7, C8, C9 ...9, C1, C1, C2, C3, C4, C5, C6, C7, C8, C9, C1, C1, C1, C1, C2, C1, C1, C1, C1, C1, C1, C1, 10 C 11 C 12 C 13 C 14 C 15 C 16 C 17 C 18 C 19 , or C 20 Alkylene, amide, C1, C2, C3, C4, C5, C6, C7, C8, C9, or C 10 Alkylene amides - C1, C2, C3, C4, C5, C6, C7, C8, C9, or C 10 Alkylene.

[0056] In some implementations, L1 and L2 are each independently selected from -(CH2). n -、-(CH2) m -CONH-(CH2) m -, or -(CH2) m -NHCO-(CH2) m -; m is an integer between 1 and 10; n is an integer between 5 and 20. In some implementations, m is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0057] In some implementations, C is selected from C1-C 20 Alkylene, amide, carbonyl, amide-(C1-C) 20 )alkylene, carbonyl-heterocyclic phosphate ester-(C1-C 10 Alkylene. In some embodiments, C is selected from C1, C2, C3, C4, C5, C6, C7, C8, C9, C6, C7, C8, C9, C9, C1, C1, C2, C3, C4, C5, C6, C7, C8, C9, C1 ... 10 C 11 C 12 C 13 C 14 C 15 C 16 C 17 C 18 C 19 , or C 20 Alkylenes, amides, amide-(C1,C2,C3,C4,C5,C6,C7,C8,C9,C) 10 C 11 C 12 C 13 C 14 C 15 C 16 C 17 C 18 C 19 , or C 20 )alkylene. In some embodiments, C is selected from carbonyl-heterocyclic-phosphate esters (C1, C2, C3, C4, C5, C6, C7, C8, C9, or C 10Alkylene, heterocyclic means 5- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocyclic, which is saturated, unsaturated, or aromatic, and contains one or two heteroatoms independently selected from nitrogen, oxygen, and sulfur, wherein the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatomium may optionally be quaternized, including bicyclic heterocycles in which any of the above heterocycles are fused to a benzene ring. Heterocyclic rings can be linked by any heteroatom or carbon atom. Heterocyclic rings include heteroaryl groups as defined below. Heterocyclic classes include morpholino, pyrrolidone, pyrrolylyl, piperidinyl, piperazine, hydantoin, valproamide, ethylene oxide, oxobutyric, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridyl, tetrahydropyrimidinyl, tetrahydrothiophene, tetrahydrothioranyl, tetrahydropyrimidinyl, tetrahydrothiophene, tetrahydrothioranyl, etc.

[0058] In some implementations, C is selected from:

[0059]

[0060] Where d is an integer between 0 and 5.

[0061] In some implementations, B is a two-joint branching group, a three-joint branching group, a four-joint branching group, a five-joint branching group, or a six-joint branching group.

[0062] In some implementation schemes, B is selected from:

[0063]

[0064] Where x is an integer between 1 and 5;

[0065] j is an integer between 0 and 5.

[0066] In some implementations, D is selected from C1-C of straight or branched chains. 20 alkylene, amide, carbonyl, (C1-C) 20 )alkylene-amide-(C1-C 20 ) Alkylene. In some embodiments, D is selected from C1, C2, C3, C4, C5, C6, C7, C8, C9, C 10 C 11 C 12 C 13 C 14 C 15 C 16 C 17 C 18 C 19 , or C 20 Alkylene, amide, carbonyl, (C1, C2, C3, C4, C5, C6, C7, C8, C9, C 10 C 11 C12 C 13 C 14 C 15 C 16 C 17 C 18 C 19 , or C 20) alkylene-amide group - (C1,C2,C3,C4,C5,C6,C7,C8,C9,C 10 C 11 C 12 C 13 C 14 C 15 C 16 C 17 C 18 C 19 , or C 20) Alkylene. In some embodiments, D is selected from -(CH2). k -、 -(C=O)-, -CONH, -NHCO-, where k is an integer between 0 and 5.

[0067] In some embodiments, E is a phosphate ester group, or a thiophosphate ester group, or a dithiophosphate ester group, or a borate ester group.

[0068] In some implementations, E is In some implementations, conjugates having the following structures are provided:

[0069]

[0070] L1 and L2 have the same definition as above.

[0071] In some implementations, conjugates having the following structures are provided:

[0072]

[0073]

[0074] L1 has the same definition as above.

[0075] Oligonucleotide agents

[0076] This disclosure relates to a series of oligonucleotide (RNA / DNA) agents, including conjugates and antisense oligonucleotides.

[0077] Exemplary oligonucleotide agents containing conjugate structures of this disclosure include those listed in the examples.

[0078] In some implementations, the antisense oligonucleotide is linked to the conjugate via an "E" group (e.g., a phosphate ester group).

[0079] In some implementations, the conjugate enhances the activity, cellular distribution, or cellular uptake of oligonucleotides through specific cell types (e.g., hepatocytes).

[0080] In some embodiments, the oligonucleotide sequence described herein is conjugated or modified at one or both ends by each conjugation motif of this disclosure. In some embodiments, the oligonucleotide chain includes a conjugation motif of this disclosure, which is conjugated to the 5' and / or 3' ends of the oligonucleotide via an "E" group (e.g., a phosphate ester group). In some embodiments, the conjugation motif of this disclosure is conjugated at the 3' end of the oligonucleotide chain. In some embodiments, the conjugation motif of this disclosure is conjugated at a nucleoside in the middle of the oligonucleotide chain.

[0081] In some embodiments, the conjugated antisense oligonucleotide agent (which can be used as a therapeutic agent) comprises an antisense oligonucleotide having any of the following nucleobase sequences disclosed in WO / 2013 / 003520 and the conjugate group described herein: SEQ ID NOs 321 / 485; 322 / 486; 324 / 488; 325 / 489; 326 / 490; 327 / 491; 328 / 492 and 350 / 514. In some embodiments, the conjugated antisense oligonucleotide agent (which can be used as a therapeutic agent) comprises an antisense oligonucleotide having any of the following nucleobase sequences disclosed in WO / 2019 / 079781 and the conjugate group described herein: SEQ ID NOs 3 / 5; 21 / 22 and HBV-219. In some embodiments, the conjugated antisense oligonucleotide agent (which can be used as a therapeutic agent) comprises an antisense oligonucleotide having any of the following nucleobase sequences disclosed in WO 2017 / 015175 and the conjugate group described herein: SEQ ID NOs 867-941. In some embodiments, the conjugated antisense oligonucleotide agent (which can be used as a therapeutic agent) comprises an antisense oligonucleotide having the (AC)n (n = 15-20) nucleobase sequence disclosed in WO2020 / 097342 and the conjugate group described herein. All siRNA or antisense oligonucleotide sequences of SEQ ID NOs mentioned above are incorporated herein by reference.

[0082] How to use

[0083] One aspect of this disclosure includes a method for treating a subject diagnosed with, suspected of having, or at risk of having any disease that can be alleviated by targeting the liver. An example is HBV infection and / or HBV-related disease. In therapeutic application, a composition of oligonucleotides conjugated with a targeting group (e.g., GalNAc) of this technology is administered to a subject suspected of or already suffering from such a disease (e.g., the presence of HBV surface antigens and envelope antigens (such as HBsAg and / or HBeAg) or elevated HBV DNA or HBV viral load levels in the subject's serum and / or liver), said dosage being sufficient to suppress or at least partially contain the symptoms of the disease, including its complications and intermediate pathological phenotypes in disease development.

[0084] In some embodiments, the oligonucleotide reagents disclosed herein are used to treat metabolic diseases or conditions, such as liver diseases or conditions; or to treat hepatitis, such as hepatitis B or hepatitis C.

[0085] Other examples include, but are not limited to, hereditary ATTR amyloidosis, acute hepatic porphyria, primary hyperoxaluria, hypercholesterolemia (PCSK9, ApoB), cardiovascular disease (Lpa, ANGPTL3, ApoCIII), ATTR amyloidosis, complement-mediated diseases (C3 and CFB), coagulation disorders (factor XI), NASH (PNPLA3 and DGAT2), alpha-1 antitrypsin deficiency, and ornithine transcarbamate deficiency.

[0086] Example

[0087] Synthesis method

[0088] Example 1: Synthesis of GalNAc components (for the synthesis of GalNAc phosphorous amide)

[0089] GalNAc constructs were designed and synthesized with each of the following reactive moieties for the expansion of (a) carboxylic acids, such as G001; G002 and G003, (b) amines, such as G004, G005, G006 and G012, (c) alcohols G007, (d) aldehydes G008, (e) alkenes G009, (f) alkynes G010, and (g) azides G011 (Table 1). These reactive moieties can react with appropriate counterparts to form 1,2-diol and 1,3-diol intermediates.

[0090] Table 1. GalNAc components with various reactive terminals

[0091]

[0092] Exemplary preparation methods and synthesis schemes are as follows:

[0093] Example 1-1 Synthesis of compound G001

[0094] Synthesis of Step 1.B

[0095]

[0096] TMSOTf (10.85 mL, 60.0 mmol) was added dropwise to a solution of aminoglycoside pentaacetate A (15.5 g, 39.85 mmol) in dichloroethane (90 mL). The mixture was heated to 50 °C for 1.5 hours and stirred overnight at ambient temperature. The reaction was quenched with a saturated aqueous solution of NaHCO3 and extracted with DCM (3 × 300 mL). The combined organic phases were washed with H2O, dried over Na2SO4, filtered, and evaporated under vacuum to give 10.5 g (~80%) of residue B, which required no further purification.

[0097] Synthesis of Step 2.C

[0098]

[0099] Dissolve B (4.28 g, 13.0 mmol) in anhydrous THF (40 mL) and react with [the solution] at ambient temperature. The molecular sieves were stirred together for 5 minutes, and then 1,8-octanediol (2.09 g, 14.3 mmol) was added. The mixture was stirred for 30 minutes and TMSOTf (1.18 mL, 6.5 mmol) was added dropwise. The resulting mixture was stirred overnight, the reaction was quenched with a saturated NaHCO3 solution, and extracted with DCM (3 × 100 mL). The combined organic phases were washed with H2O, dried over Na2SO4, filtered, and evaporated under vacuum to give the residue. The residue was purified on a silica gel column to give 4.01 g (65%) of C.

[0100] Step 3. Synthesis of D

[0101]

[0102] C (4 g, 8.42 mmol) was added to a 500 mL round-bottom flask. TEMPO (0.75 g, 4.8 mmol), 43 mL of acetonitrile, and 120 mL of 0.67 M sodium phosphate buffer were added with stirring, and the resulting mixture was heated to 35 °C. Sodium chlorite solution (32.5 mL, prepared by dissolving 9.14 g NaClO2 in 40 mL H2O) and sodium hypochlorite solution (16.25 mL, prepared by diluting household bleach (5.25% NaOCl, 1.06 mL, approximately 2.0 mol%) with 19 mL H2O) were added to the reaction mixture in five portions over 2 hours. The reaction was carried out with stirring at 35 °C for 16 hours, quenched with Na2S2O3, and acidified with saturated NH4Cl. The mixture was extracted with ethyl acetate (3 × 100 mL), and the combined organic phases were washed with water, dried over MgSO4, filtered, and evaporated under vacuum to obtain the residue. The residue was purified by silica gel column chromatography to give 3.75 g (91%) of D.

[0103] [M+H] + =489.6. 1 H NMR (400MHz, DMSO-d6) δ11.96(s,1H),7.80(d,J=9.2Hz,1H),5.21(d,J=3.4Hz,1H),4 .96(dd,J=11.2,3.5Hz,1H),4.48(d,J=8.5Hz,1H),4.02(m,3H),3.86(dt,J=11.2,8.8 Hz,1H),3.69(dt,J=9.9,6.2Hz,1H),3.41(dt,J=9.9,6.5Hz,1H),2.18(t,J=7.4Hz,2 H),2.10(s,3H),1.99(s,3H),1.89(s,3H),1.76(s,3H),1.47(m,5H),1.24(s,7H)ppm.

[0104] Synthesis of compound G004 in Examples 1-2

[0105]

[0106] Under an inert nitrogen atmosphere at 0 °C, TMSOTf (2.7 mL, 15.3 mmol) was added dropwise to 300 mL of a solution of B (10 g, 30.6 mmol) and tert-butyl (8-hydroxyoctyl)carbamate (9 g, 36.7 mmol) in 1,2-dichloroethane. The resulting solution was stirred at room temperature for 16 h. The reaction mixture was quenched by adding ice / water (100 mL) and then extracted with dichloromethane (200 mL × 2). The combined organic phases were washed with water (100 mL) and dried over anhydrous sodium sulfate. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, first eluting with PE / EA (1 / 2) and then by reversed-phase silica gel rapid chromatography (ACN / H2O = 5%-95%, 214 nm, 30 min) to give Boc-protected G004 (4 g, 23.5% yield) as a white solid. MS calculated value: 574.3; Measured value: 575.3 [M+H] + .1H NMR (400MHz, DMSO-d6): δ7.81(d,J=9.2Hz,1H),6.76-6.74(m,1H),5.21(d,J=3.2 Hz,1H),4.98-4.95(m,1H),4.48(d,J=8.8Hz,1H),4.04-4.00(m,3H),3.90-3.83( m,1H),3.72-3.66(m,1H),3.43-3.32(m,1H),2.90-2.85(m,2H),2.10(s,3H),2.0 0(s,3H),1.89(s,3H),1.77(s,3H),1.45-1.44(m,2H),1.37(s,11H),1.23(s,8H).

[0107] G004 was generated by treating Boc-protected G004 in a dichloromethane solution of 25% trifluoroacetic acid for 4 hours at room temperature, and removing volatile substances without further purification.

[0108] Synthesis of compound G007 in Examples 1-3

[0109]

[0110] TMSOTf (3.38 g, 15.19 mmol) was added dropwise to 100 mL of a DCE solution containing compound B (10 g, 30.37 mmol) and octane-1,8-diol (4.44 g, 30.37 mmol) under stirring at 0 °C. The resulting solution was stirred at room temperature for 16 hours. The reaction was quenched with water (100 mL) and extracted with DCM (100 mL × 3). The organic phase was concentrated, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by rapid chromatography on reversed-phase silica gel (ACN / H₂O = 5%-95%, 214 nm, 30 min) to give compound G007 (5.3 g, 37% yield) as a yellow solid. MS calculated value: 475; MS measured value: 476 [M+H] + .1HNMR(400MHz,DMSO-d6)δ:7.82(d,J=9.2Hz,1H),5.21(d,J=3.6Hz,1H),4.98-4.94(m,1H),4.98(d,J=8.4Hz,1H), 4.32(s,1H),4.05-4.01(m,1H),3.90-3.83(m,1H),3.72-3.67(m,3H),2.10(s,3H),2.00(s,3H),1.89(s,3H),1.77(s 3H),1.45-1.38(m,4H),1.24(br,8H).

[0111] Synthesis of compound G010 in Examples 1-4

[0112]

[0113] TMSOTf (3.38 g, 15.19 mmol) C was added dropwise to 100 mL of a DCM solution of compound B (5 g, 15.19 mmol) and 9-decyn-1-ol (3.41 g, 30.37 mmol) under stirring at 0 °C. The resulting solution was stirred at room temperature for 16 hours. The reaction was quenched with H₂O (100 mL) and extracted with DCM (100 mL × 3). The organic phase was concentrated. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by rapid chromatography on reversed-phase silica gel (ACN / H₂O = 5%-95%, 214 nm, 30 min) to give compound G010 (3.8 g, 83% yield) as a yellow solid. MS calculated value: 483; MS measured value: 484 [M+H] +.1H NMR(400MHz, DMSO-d6)δ:7.80(d,J=9.2Hz,1H),5.21(d,J=4.0Hz,1H),4.97(d,J= 7.6Hz,1H),4.48(d,J=8.4Hz,1H),4.04-4.01(m,3H),3.90-3.83(m,1H),3.72-3.6 7(m,1H),3.44-3.38(m,1H),2.71(t,J=2.8Hz,1H),2.16-2.10(m,2H),2.00(s,3H ),1.89(s,3H),1.77(s,3H),1.46-1.41(m,4H),1.35-1.32(m,2H),1.25(br,6H)).

[0114] Example 2: Method for coupling DBCO-GalNAc with azide oligonucleotides via click chemistry

[0115] Click chemistry is attractive for forming GalNAc oligonucleotide conjugates due to its simplicity and efficiency in two-part molecular bridging. Using click chemistry, the GalNAc moiety can be specifically incorporated into any position on the oligonucleotide site via azide substitution sites. Therefore, the aforementioned G010 and G011 GalNAc constructs can be conjugated with oligonucleotides having triazide or triterminal alkyne linkers under copper-mediated conditions to form tri-linked GalNAc oligonucleotide conjugates.

[0116]

[0117] Example 2-1 Synthesis of compound G010

[0118]

[0119] TMSOTf (3.38 g, 15.19 mmol) was added dropwise to 100 mL of DCM solution with compound B (5 g, 15.19 mmol) and 9-decyn-1-ol (3.41 g, 30.37 mmol) under stirring at 0 °C. The resulting solution was stirred at room temperature for 16 hours. The reaction was quenched with H₂O (100 mL) and extracted with DCM (100 mL × 3). The organic phase was concentrated. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by rapid chromatography on reversed-phase silica gel (ACN / H₂O = 5%-95%, 214 nm, 30 min) to give compound G010 (3.8 g, 83% yield) as a yellow solid. MS calculated value: 483; MS measured value: 484 [M+H] +.1H NMR(400MHz, DMSO-d6)δ:7.80(d,J=9.2Hz,1H),5.21(d,J=4.0Hz,1H),4.97(d,J= 7.6Hz,1H),4.48(d,J=8.4Hz,1H),4.04-4.01(m,3H),3.90-3.83(m,1H),3.72-3.6 7(m,1H),3.44-3.38(m,1H),2.71(t,J=2.8Hz,1H),2.16-2.10(m,2H),2.00(s,3H ),1.89(s,3H),1.77(s,3H),1.46-1.41(m,4H),1.35-1.32(m,2H),1.25(br,6H)).

[0120] Example 2-2 Synthesis of compound G01

[0121]

[0122] Trimethylsilyl trifluoromethanesulfonate (0.8 g, 3.6 mmol) was added dropwise to a solution of compound B (4 g, 12.1 mmol) and 8-azidooctyl-1-ol (3.1 g, 18.1 mmol) in dichloromethane (50 mL) at 0 °C and N2. The resulting solution was stirred at room temperature for 2 hours. The reaction was quenched by adding 100 mL of ice / water and extracted with DCM (100 mL × 3). The combined organic phases were washed with water and brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (DCM / MeOH = 100 / 1 to 20 / 1) to give compound G011 (2.5 g, 41.7%) as a pale yellow oil. LC-MS: Calculated value: 500.2; Found value: 501.1 [M+H + ].

[0123] Example 3: Synthesis of GalNAc phosphorous amide (can be used directly on an automated RNA / DNA synthesizer)

[0124] GalNAc building blocks are converted into GalNAc containing 1,2-diol and 1,3-diol using reactions disclosed in the literature, such as (a) amide coupling reaction, (b) nucleophilic substitution reaction, (c) reductive amidation reaction, (d) Heck reaction, and (e) click reaction. These can then be converted into reagents containing dimethoxytriphenylmethyl (DMTr-) and phosphorous amide (Scheme 1) in an oligonucleotide synthesizer (Table 2).

[0125]

[0126] Claim 1. Claims regarding triacetyl-protected GalNAc monomers for oligonucleotide synthesis.

[0127] Table 2. GalNAc phosphoramide monomers suitable for oligonucleotide synthesis

[0128]

[0129]

[0130]

[0131]

[0132] Example 3-1: Synthesis of L-005

[0133] Step 1: Synthesis of L005-diol

[0134]

[0135] In a 50 mL round-bottom flask, purged with argon and maintaining an inert atmosphere, 16-[[(2R,3R,4R,5R,6R)-4,5-di(acetoxy)-6-[(acetoxy)methyl]-3-acetaminooxa-2-yl]oxy]hexadecanoic acid G003 (6.00 g, 9.971 mmol, 1.00 equiv), dry DMF (60.00 mL), and HBTU (4.16 g, 10.968 mmol, 1.1 equiv) were added. Then, DIPEA (1.42 g, 10.968 mmol, 1.1 equiv) was added at room temperature. The resulting solution was stirred at room temperature for 1 hour. 3-Aminopropane-1,2-diol (1.09 g, 11.965 mmol, 1.2 equiv) was added at 25 °C. The resulting solution was stirred at room temperature for 2 hours. The reaction was then quenched by adding 100 mL of saturated NaHCO3. The resulting solution was extracted with ethyl acetate (2 × 100 mL), and the organic phases were combined. The mixture was washed with H₂O (4 × 100 mL) and brine. The mixture was dried over anhydrous sodium sulfate. The resulting mixture was concentrated. The product was precipitated by adding diethyl ether, filtration, and drying to give 6.2 g (purity ~90%) of methyl [(2R,3R,4R,5R,6R)-3,4-bis(acetoxy)-6-[(15-[[(2S)-2,3-dihydroxypropyl]-carbamoyl]pentadecanyl)oxy]-5-acetamidocyclohexane-2-yl]acetate, as a white solid. LC-MS: [M+H]+675.

[0136] Step 2 Synthesis of L005-OH

[0137]

[0138] In a 25 mL round-bottom flask, purged with argon and maintaining an inert atmosphere, methyl [(2R,3R,4R,5R,6R)-3,4-bis(acetoxy)-6-[(15-[[(2S)-2,3-dihydroxypropyl]carbamoyl]pentadecanyl)oxy]-5-acetamidocyclohexane-2-yl]acetate (1 g, 1.482 mmol, 1.00 equiv) and anhydrous pyridine (10 mL) were added. Then, 1-[chloro(4-methoxyphenyl)phenylmethyl]-4-methoxybenzene (903.78 mg, 2.667 mmol, 1.80 equiv) was added at 0 °C. The resulting solution was stirred at room temperature for 2 hours. The resulting mixture was concentrated. The reaction was then quenched by adding 100 mL of water. The resulting solution was extracted with ethyl acetate (3 × 100 mL), and the organic phases were combined and dried over anhydrous sodium sulfate. The solid was filtered off and the mixture was concentrated. The crude product was purified by flash-prep-HPLC under the following conditions (CombiFlash-1): column: C18 silica gel; mobile phase: ACN / H2O = 30 / 70 to ACN / H2O = 95 / 5 over 30 minutes. 634 mg (43.78%) of methyl [(2R,3R,4R,5R,6R)-3,4-bis(acetoxy)-6-[(15-[[(2S)-3-[bis(4)-methoxyphenyl)(phenyl)methoxy]-2-hydroxypropyl]carbamoyl]pentadecanyl)oxy]-5-acetamidooxa-2-yl]acetate was obtained as a white solid. 1 H NMR (300MHz, DMSO-d6) δ7.83(d,J=9.2Hz,1H),7.66(s,1H),7.42(d,J=7.7Hz,2H),7.37–7.17(m,7H),6.95– 6.85(m,4H),5.23(d,J=3.3Hz,1H),4.98(q,J=4.2Hz,2H),4.50(d,J=8.4Hz,1H),4.04(s,3H),3.88(d,J=9.7 Hz,1H),3.75(t,J=1.5Hz,8H),3.42(d,J=9.6Hz,1H),3.35–3.20(m,1H),3.08–2.78(m,3H),2.12(d,J=1.1H z,3H),2.08–1.96(m,5H),1.91(d,J=1.1Hz,3H),1.82–1.71(m,3H),1.44(s,4H),1.23(d,J=8.4Hz,22H)ppm.

[0139] Synthesis of L005 in step 3

[0140]

[0141] In a 50 mL round-bottom flask, purged with argon and maintaining an inert atmosphere, 3-(didiisopropylaminophosphoryl)propionitrile (771.12 mg, 2.558 mmol, 2.50 eq.) and dry DCM (2.00 mL) were added. Then, DCI (144.90 mg, 1.228 mmol, 1.20 equiv) was added at 0 °C. The resulting solution was stirred at 0 °C for 10 minutes. A solution of [(2R,3R,4R,5R,6R)-3,4-bis(acetoxy)-6-[(15-[[(2S)-3-[bis(4-methoxyphenyl))[(phenyl)methoxy]-2-hydroxypropyl]-carbamoyl]pentadecanyl)oxy]-5-acetamido-2-yl]acetate (1.00 g, 1.023 mmol, 1.00 equiv) in anhydrous DCM (4 mL) was added, and the mixture was stirred at 0 °C. The resulting solution was stirred at room temperature for 1 hour. The reaction was then quenched by adding 50 mL of NaHCO3 (saturated and cold). The resulting solution was extracted with dichloromethane (2 × 100 mL), and the organic phases were combined. The resulting mixture was washed with H2O and brine. The mixture was dried over anhydrous sodium sulfate. The solids were filtered off, and the mixture was concentrated. The crude product was purified by flash-prep-HPLC under the following conditions (CombiFlash-1): column: C18 silica gel; mobile phase: ACN / H2O (0.1% NH3·H2O) = 50 / 50 increased to ACN / H2O = 100 over 40 min, then maintained at ACN / H2O = 100 for 20 min; detector: 220 nm / 254 nm. 612 mg (50.79%, in argon atmosphere) was obtained. MS (stored at -70°C) [(2R,3R,4R,5R,6R)-3,4-bis(acetoxy)-6-[(15-[[(2S)-3-[bis(4-methoxyphenyl)(phenyl)methoxy]-2-[[(2-cyanoethoxy)-(diisopropylamino)phosphino]oxy]propyl]carbamoyl]pentadecanyl)oxy]-5-acetamido-acetamido-2-yl]acetaminooxy-acetamido-2-yl]acetaminooxy-acetaminooxy-2-yl] ... 1H NMR (300MHz, DMSO-d6) δ7.83(d,J=9.3Hz,1H),7.66(s,1H),7.43(d,J=7.6Hz,2H),7.28(qd,J=11.4,9.4,6.6Hz,7H),6.88(dd ,J=8.6,4.6Hz,4H),5.23(d,J=3.3Hz,1H),4.99(dd,J=11.3,3.3Hz,1H),4.50(d,J=8.5Hz,1H),4.04(s,4H),3.96–3.77(m,2H ),3.77–3.63(m,11H),3.43(dd,J=10.1,6.0Hz,1H),3.19(s,2H),3.03(d,J=6.2Hz,1H),2.79(t,J=6.0Hz,1H),2.65(t,J=5.9 Hz,1H),2.12(s,3H),2.01(s,6H),1.91(s,2H),1.78(s,3H),1.50–1.36(m,4H),1.28–1.10(m,31H),1.03(d,J=6.6Hz,3H)ppm. 31 P NMR (300MHz, DMSO-d6) δ148.41,147.94ppm.

[0142] Example 3-2: Synthesis of L045

[0143]

[0144] Step 1: Synthesis of compound K

[0145] Compound J (1.58 g, 3.61 mmol) was added to a t-BuOH (15 mL) solution of compound G011 (1.67 g, 3.33 mmol). A water (15 mL) solution of CuSO4·5H2O (164 mg, 0.66 mmol) and sodium ascorbate (328 mg, 1.66 mmol) was added to this stirred solution. After stirring at 35 °C for 4 hours, the reaction mixture was extracted with EtOAc (20 mL × 2). The organic phase was dried over Na2SO4, filtered, and concentrated to obtain a residue, which was purified by silica gel column chromatography (DCM / MeOH = 100 / 1 to 20 / 1) to give pure compound K (1.1 g, yield 33.3%) as a white solid. LC-MS: calculated m / z: 932.4; found: 955.4 [M+Na] +.1H NMR(DMSO-d6,400MHz), δ8.00(s,1H),7.80(d,J=9.2Hz,1H),7.38(d,J=7.6Hz,2H),7.30-7.18(m,7H),6.87(d,J=8.8Hz ,4H),5.21(d,J=2.8Hz,1H),4.96(dd,J=11.6Hz,3.6Hz,1H),4.88(d,J=5.6Hz,2H),4.50-4.46(m,3H),4.29(t,J=7.2Hz, 2H),4.03-4.01(m,3H),3.85(dd,J=20.4Hz,9.6Hz,1H),3.77-3.65(m,8H),3.52(dd,J=10.0Hz,4.4Hz,1H),3.45-3.36( m,2H),2.91(d,J=5.2Hz,2H),2.09(s,3H),1.98(s,3H),1.88(s,3H),1.77-1.75(m,5H),1.43-1.41(m,2H),1.21(s,6H).

[0146] Step 2: Synthesis of compound L045

[0147] In a 50 mL round-bottom flask, purged with argon and maintaining an inert atmosphere, 90 mg of 3-(didiisopropylaminophosphoryl)propionitrile (2.50 eq.) and 2.00 mL of dry DCM were added. Then, 78 mg of DCI (3.0 equiv) was added at 0 °C. The resulting solution was stirred at 0 °C for 10 min. While stirring at 0 °C, a solution of 1 mL of dry DCM containing compound K (186 mg, 1.0 equiv) was added dropwise. The resulting solution was stirred at room temperature for 1 h. The reaction mixture was concentrated and purified by silica gel column chromatography by elution with hexane / ethyl acetate and 1% triethylamine. 172 mg of L045 was given as a white semi-solid. 1H NMR(DMSO-d6,400MHz), δ7.95(d,J=9Hz,1H),7.80(d,J=9Hz,1H),7.d(m,2H),7.30-7.18(m,7H ),6.8(m,4H),5.21(d,J=3Hz,1H),4.96(dd,J=12Hz,4Hz,1H),4.50-4.46(m,3H),4.29(m,2H), 4.0(m,5H),3.85(m,1H),3.77-3.45(m,13H),3.45-3.36(m,2H),2.91(m,1H),2.75-2.55(m,2H ),2.09(s,3H),1.98(s,3H),1.88(s,3H),1.77(s,3H),1.43-1.41(m,2H),1.25-0.95(m,18H). 31 PNMR(300MHz,DMSO-d6)δ148.50,147.96ppm.

[0148] Example 4: Accelerating the synthesis of GalNAc monomers by simplifying the connector structure.

[0149] Certain phosphorusamide components (e.g., L035) can be synthesized in high yield from common intermediates via four direct steps. This process offers high yield and scalability for large-scale synthesis. Exemplary methods and synthetic schemes are as follows:

[0150] Example 4-1: Synthesis of L-035

[0151] Step 1: Synthesize olefin F using a method similar to that described for compound G009 in Example 1.

[0152] Step 2: Synthesis of L035-diol (G)

[0153]

[0154] Compound F (0.93 g, 1.72 mmol) was dissolved in THF / H₂O (12.23 mL / 1.58 mL) and cooled to -10 °C. Then, 4-methylmorpholine N-oxide hydrate (0.678 g, 5.02 mmol) and K₂O₄·2H₂O (0.027 g, 0.076 mmol) were added. The resulting mixture was stirred overnight at -10 °C, followed by the addition of Na₂S₂O₃ and further stirring for 30 min. The mixture was diluted with water and extracted with ethyl acetate (3 × 50 mL). The combined organic phases were washed with H₂O, dried over Na₂SO₄, filtered, and evaporated under vacuum to give the residue. The residue was purified on a silica gel column to give 0.741 g (75%) of G.

[0155] Step 3: Synthesis of L035-OH(H)

[0156]

[0157] 0.8 g (1.39 mmol) of compound G was dissolved in 7.5 mL of anhydrous pyridine and reacted with... The molecular sieve was stirred together. DMTTrCl (0.6 g, 1.77 mmol) was added in portions. The resulting mixture was stirred overnight and then diluted with DCM (30 mL). Pyridine was removed by repeated washing of the organic phase with saturated CuSO4, drying the organic phase with Na2SO4, filtering, and evaporating under vacuum. The residue was purified on a silica gel column to give 0.976 g (80%) H. [M+Na] + =900.2. 1 H NMR (400MHz, CDCl3) δ7.46–7.38(m,2H),7.36–7.16(m,7H),6.87–6.78(m,4H),5.42(d,J=8.6Hz,1H),5.39 –5.27(m,2H),4.71(d,J=8.3Hz,1H),4.22–4.07(m,2H),3.97–3.82(m,3H),3.79(s,6H),3.75(s,1H),3.47( dt,J=9.7,6.9Hz,1H),3.16(dd,J=9.3,3.3Hz,1H),3.00(dd,J=9.4,7.6Hz,1H),2.34(d,J=3.5Hz,1H),2.14 (s,3H),2.07–1.92(m,9H),1.61–1.51(m,1H),1.39(d,J=17.7Hz,3H),1.36–1.20(m,19H),1.11(s,1H)ppm.

[0158] Step 4: Synthesis of L035

[0159] L035 phosphorusamide was synthesized using a similar four-step synthesis method as described in Example 2.

[0160] Example 5: Automatic synthesis of a three-joint structure from GalNAc monomers

[0161]

[0162] The synthesis of triple-linker 5'-GalNAc conjugated oligonucleotides was performed on an ABI 394 or K&A-H8 DNA / RNA synthesizer. Synthesis was carried out on a NittoPhaseHL UnyLinker solid support at a scale of 1 μmole. A toluene solution of trichloroacetic acid (3% v) was used to cleave the 4,4'-dimethoxytriphenylmethyl (DMTr) group from the 5'-hydroxyl group of the nucleotide. 4,5-Dicyanimidazole was used as an activator in the coupling step in the presence of N-methylimidazole. In the coupling step, 10–50 mol equivalents of 0.05 M phosphorusamide solution (2'-deoxy, 2'-O-methoxyethyl, and locked nucleoside) and a 1:1 (v / v) solution of phosphorusamide and activator were used. The phosphorusamide and activator solutions were prepared using anhydrous acetonitrile (water content <30 ppm) and further dried by adding a molecular sieve pack. The thiophosphate bond was introduced by oxidizing the triphosphite with a pyridine solution of 0.05 M hydrogenated xanthanin. Iodine in a pyridine / water solution was used during the oxidation step to obtain the phosphodiester bond. Unreacted hydroxyl groups were capped by delivering N-methylimidazolium / pyridine / acetonitrile and acetic anhydride / acetonitrile at a 1:1 (v / v) flow ratio. At the end of the synthesis, the solid-support-bonded oligonucleotides were treated with a solution of triethylamine / acetonitrile (1:1, v / v) to remove acrylonitrile formed during the decyanoethylation of the triphosphite. Recommended reagent delivery rates and contact times from automated DNA / RNA instrument manufacturers are detailed in Table 3. Subsequently, the solid-support-bonded oligonucleotides were incubated with a concentrated ammonium hydroxide solution at 55 °C for approximately 15 hours to allow complete dissociation from the solid support, eliminating UnyLinker molecules to release the 3'-hydroxyl group of the oligonucleotide, as well as the protecting group of the deprotected nucleobase. After cooling the crude mixture to room temperature, it is filtered and the solid support is washed with purified water and collected. The crude product is concentrated and purified in an ammonia solution by gel electrophoresis and / or reversed-phase HPLC to obtain pure oligonucleotide-GalNAc conjugates. Typically, the purity of the conjugates exceeds 85% as determined by anion-exchange HPLC.

[0163] Table 3: Reaction parameters for 1 μmol-scale synthesis on the synthesizer

[0164]

[0165]

[0166] Example 6: Incorporation of the GalNAc conjugate into an antisense sequence

[0167] The oligonucleotide chosen for the GalNAc conjugation site can be a single-stranded antisense oligonucleotide or a double-stranded siRNA, where the multi-linker GalNAc can be coupled at the 3'- or 5'-end. For example, we have bound GalNAc to a 13-mer antisense oligonucleotide targeting the 5' end of ApoB100 mRNA and investigated target knockout in C57BL / 6 mice.

[0168] The following is the 13-mer gapmer sequence used in our study (Nucleic AcidsResearch, 2018, 46, 5366-5380): 5'-[L] n [Sp] m [+G]*[+mC]*[A]*[T]*[T]*[G]*[G]*[T]*[A]*[T]*[+T]*[+mC]*[+A]-3', where [L] is a GalNAc-containing ligand, n = 1-4; [Sp] is an optional spacer, which can be -(CH2). n - Chain, n = 3-12, or -(OCH2CH2) m O-, m = 1-3, between the GalNAc conjugate and the ApoB antisense sequence; m = 0-2; [+N] is a locked nucleic acid, and [N] is a deoxyribonucleoside, and * is a thiophosphate bond.

[0169] Methods: The oligonucleotide-GalNAc conjugate was prepared using a method similar to that described in Example 4. The crude oligo-GalNAc conjugate product was further purified by RP-HPLC or PAGE to obtain a pure product, and molecular integrity was confirmed by mass spectrometry. Endotoxin levels were examined prior to animal studies.

[0170] The following antisense oligonucleotide-GalNAc conjugates were synthesized using the general methods described in Examples 4 and 5. The structural and characterization data of each antisense oligonucleotide-GalNAc conjugate are shown in Table 4.

[0171] Table 4. Structure of ASO-GalNAc conjugates

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178] Example 7: Screening for GalNAc monomers using ApoB antisense oligonucleotides conjugated with GalNAc

[0179] The oligonucleotide-GalNAc conjugates for this study were prepared as described in Examples 5 and 6 and reconstituted in PBS prior to the study. Mice were grouped into groups of five mice each, based on their body weight on day -7. Mice were administered a single dose on day 0 at two different dose levels (high dose, 60 nmoles / kg and low dose, 20 nmoles / kg), followed by exsanguination to monitor plasma ApoB100 protein levels on days 3 and 6. The study was terminated on the last observation day or the humane endpoint, whichever came first. Approximately 50 μL / mouse / time point of blood was collected via tail or retro-orbital bleeding into EDTA-coated tubes. Samples were centrifuged at 1,000–2,000 x g for 10 minutes in a refrigerated centrifuge. After centrifugation, the resulting supernatant (plasma) was immediately transferred to clean, labeled polypropylene tubes and stored at -80°C until use.

[0180] Plasma ApoB levels were determined using a commercially available ELISA kit (AbCam#ab230932). The assay was performed according to the manufacturer's instructions. Two plasma samples were tested at a 5000-fold dilution. ApoB results were reported as μg / mL or as a percentage normalized to the initial level before oligonucleotide administration. Comparisons between compounds were used to elucidate structure-activity relationships (SARs), with comparison to the triple-linker positive control compound B005 serving as a standard compound.

[0181] B001 is a three-joint GalNAc gapmer, with no spacer between the GalNAc cluster and the gapmer. B003 has a 1,6-hexanediol spacer (e.g., C6) between the GalNAc and the gapmer via a phosphodiester bond. In vivo studies have shown that B003 exhibits superior activity to B001 at 100 nmol / kg and 20 nmol / kg levels, indicating the need for a spacer between the GalNAc moiety and the antisense moiety. Figure 1A-1C ).

[0182] Example 8: Using GalNAc monomers to form branched GalNAc clusters

[0183] By using a standard DNA / RNA synthesizer and branched adapter modules, such as doubler or trebler, monomeric GalNAc phosphonamides can be efficiently used to form various multi-adaptor GalNAc clusters. For example, in addition to the linear form of trebler GalNAc described in Example 6, we synthesized trebler trebler GalNAc oligonucleotides on the synthesizer.

[0184] The sequence 5'-[L]3[Trebler][+G]*[+mC]*[A]*[T]*[T]*[G]*[G]*[T]*[A]*[T]*[+T]*[+mC]*[+A]-3', where [L] is the GalNAc ligand:

[0185]

[0186] [Trebler] and [Trebler] are building blocks having the following chemical structures:

[0187]

[0188] [+N] represents a locked nucleic acid, [N] represents a deoxyribonucleoside, and * represents a phosphate thioester bond. The sequence was synthesized and evaluated in mice using the protocols described in Examples 4 and 6. The resulting compound exhibited superior plasma ApoB reduction compared to the positive control compound B005. To achieve multiplicity beyond the three-joint type, we can form a four-joint GalNAc cluster via doubler, thus providing multiple forms of GalNAc clusters for candidate leader selection:

[0189]

[0190] In both cases, the naked 5'-OH terminus generated by oligonucleotide synthesis can bind in other ways to modulate the properties of the oligonucleotide. These modalities include, but are not limited to, other antisense sequences, or small molecules that can modulate endosome escape agents to help the oligonucleotide enter the cytosol.

[0191] Figure 3 A standard synthesis cycle for oligonucleotide synthesis via a linker solid support is shown on a DNA / RNA synthesizer. After oligonucleotide synthesis, synthesized GalNAc phosphorous amide is used to bind to the oligonucleotide on the synthesizer.

[0192] All conjugates were purified by PAGE or anion-exchange HPLC. The purity of the final conjugates was found to be 85-95% by AE-HPLC. Molecular integrity was determined by mass spectrometry, and the results are shown in the table above. Prior to in vivo studies in mice, the conjugates were analyzed using Charles River... System and The LAL kit method was used to examine the endotoxin levels of all conjugates.

[0193] Example 9: Formation of GalNAc clusters using long carbon chains

[0194] Instead of using multiple amide groups to extend the chain length, we incorporated long carbon chains into the GalNAc cluster to simplify the synthesis by reducing the number of steps and to modulate the biophysical properties of the GalNAc-oligo conjugate to obtain the optimal pharmacokinetic profile scheme 2. (A, left) The GalNAc cluster in the published compound uses multiple amides and makes the compound overall hydrophilic (B, right). Long carbon chains in monomers and spacers are more easily formed than multiple amide bonds, which can balance the hydrophilicity of the compound.

[0195]

[0196] Option 2. Multiple amide groups and long carbon chains are incorporated into the structure of GalNAc.

[0197] Both the GalNAc moiety and the oligonucleotide moiety are known to be highly hydrophilic, and hydrophilic compounds are known to facilitate their renal clearance. Modulating the biophysical properties with hydrophobic carbon chains in the molecule may reduce renal clearance, thereby allowing the liver to take up more oligonucleotides.

[0198] Example 10 significantly reduced reaction steps by employing a monomeric GalNAc component.

[0199] By employing the monomeric GalNAc phosphorous amide, we significantly reduced the complexity of GalNAc cluster synthesis. A typical GalNAc cluster, exemplified by B005, requires at least 14 steps and is time-consuming to synthesize for use in oligonucleotide-conjugate synthesis (see below).

[0200]

[0201] Further detailed descriptions of the synthesis methods can be found in U.S. Patents 8,828,956 and 9,943,604, the disclosures of which are incorporated herein by reference.

[0202] In contrast, the monomer GalNAc phosphorous amide typically requires only 8 steps from commercially available starting materials, or only 4 steps from typical intermediates (such as G001) (as follows).

[0203]

[0204] The synthesis of the novel monomer GalNAc phosphorous amide can be completed in a short time in a typical chemical laboratory.

[0205] We also designed and synthesized the monomer GalNAc phosphoridamide by completely avoiding amide bonds or other typical linkers, thus simplifying the chemical synthesis. The diol moiety required for phosphoridamide synthesis can be efficiently constructed by dihydroxylation of a terminal olefin (such as G009). Subsequently, the diol is modified to dimethoxytriphenylmethyl protected (DMTr) and phosphoridamide, respectively, achieving high yields in just four steps (see below).

[0206]

[0207] Example 10-1 Synthesis of L009 and oligonucleotide conjugates.

[0208]

[0209] Step 1: Synthesis of L009-1

[0210] The starting material compound B crude product (3.3 g) and C 10 - Vinyl alcohol (1.7 g, 11 mmol) was dissolved in 20 mL of anhydrous THF. The mixture was degassed and purged with argon three times. Under argon protection, TMSOTf (1.1 g, 0.9 mmol) was added dropwise to the mixture. After addition, the mixture was stirred overnight at room temperature. When the reaction was complete, the mixture was poured into a cold 10% sodium bicarbonate solution (100 mL) and stirred for 10 minutes. 100 mL of ethyl acetate was added to the mixture, and the mixture was stirred for 10 minutes. The organic phase was separated, and the aqueous phase was extracted with ethyl acetate (50 mL × 2). The organic phases were combined, washed with brine, and then evaporated to a pale yellow liquid. The residue was purified by silica gel column chromatography (PE / EA = 0% to 80%) to give compound L009-1 as a colorless oil (2.6 g, two-step reaction yield 53.5%). MS theoretical value: 485.3; measured value: 486.3 [M+H] + . 1 H NMR (400MHz, CDCl3) δ7.8(m,1H),6.8(m,1H),5.2(d,J=8.0Hz,1H),5.10–4.90(m,3H),4.5(d,J=8.0Hz,1H),4.10–4.00( m,3H),3.90–3.60(m,2H),3.40-3.50(m,2H),2.14(s,3H),2.07–1.92(m,11H),1.6–1.5(m,2H),1.36–1.20(m,10H)ppm.

[0211] Step 2: Synthesis of L009-1,2-diol

[0212] L009-1 (2.6 g, 5.4 mmol) was dissolved in 30 mL of THF, and potassium osmium tetroxide dihydrate (18 mg, 0.05 mmol) was added to the mixture. 5 mL of water was added to the mixture until the potassium osmium tetroxide dissolved. The mixture was cooled to 0–10 °C in an ice bath, and 4-methylmorpholine N-oxide (937 mg, 8.0 mmol) was added in several batches. After addition, the ice bath was removed, and the mixture was stirred at room temperature for 16 hours. The mixture was poured into 50 mL of cold 10% sodium sulfite solution (50 mL), and ethyl acetate (50 mL) was added. The mixture was stirred for 10 minutes, and the organic phase was separated. The aqueous phase was extracted twice with 50 mL of ethyl acetate. The organic phases were combined, washed with brine, dried over anhydrous sodium sulfate, and evaporated to give a pale yellow oil. The crude product was purified by silica gel column chromatography (PE / EA = 0% to 100%) to give a pale yellow oil (2.6 g, 94.2%).

[0213] Step 3: Synthesis of L009-OH

[0214] To a 30 mL solution of anhydrous DCM containing L009-1,2-diol (2.6 g, 5.0 mmol) and TEA (1.5 g, 15.1 mmol), 10 mL of anhydrous DCM solution of DMTr-Cl (2.1 g, 6.1 mmol) was added dropwise. After addition, the mixture was stirred at room temperature for 16 hours. When the reaction was complete, 50 mL of DCM was added to dilute the mixture, followed by 50 mL of brine. The mixture was stirred for 10 minutes, and the organic phase was separated. The aqueous phase was extracted with DCM (50 mL). The organic phases were combined and evaporated to a yellow oil. The residue was purified by silica gel column chromatography (PE / EA = 0% to 80%) to obtain L009-OH (2.0 g, 48.4%) as a white bubbly solid. MS theoretical value: 822.0; Found value: 844.4 (M+Na) + ). 1 H NMR(400MHz, CDCl3)δ7.4(m,2H),7.4–7.2(m,7H),6.8(m,4H),5.6(m,1H),5.4(m,1H),4.7(m,1H),4.2–4.0(m,2H),4.0–3.8(m,3H),3.79(s,6H ),3.75(m,1H),3.50-3.45(m,1H),3.2(m,1H),3.00(m,1H),2.4(m,1H) ,2.14(s,3H),2.07–1.92(m,9H),1.6–1.5(m,2H),1.5-1.2(m,14H)ppm.

[0215] Step 4: Synthesis of L009 with oligonucleotide conjugates

[0216] L009 and L0009ApoB were synthesized according to the general method described in Examples 2, 3, and 5. L009, theoretical MS value: 1022.2; measured value: 1022.3 (M+H) + ).

[0217] L009-ApoB antisense conjugate, MS theoretical value: 5871.2; measured value: 5871.4 (M+H) + ).

[0218] Example 11: Application of multi-joint branched groups in the formation of GalNAc clusters

[0219] We designed and synthesized tri-linked GalNAc clusters and compared their in vivo efficacy with that of monomeric GalNAc. These novel clusters possess benzene rings or cycloalkene (azacrown ether) rings to construct multi-linked GalNAc clusters. The structures of the GalNAc phosphorous amide clusters are listed below.

[0220]

[0221]

[0222] Example 11-1: Synthesis of L016-OH

[0223]

[0224] Step 1: Synthesis of L016-3

[0225] To a DMF (60 mL) solution of 3,4,5-tris(2-((tert-butoxycarbonyl)amino)ethoxy)benzoic acid (2.45 g, 4.1 mmol), EDCI (1.0 g, 5.2 mmol), HOBT (0.70 g, 5.2 mmol), and DIPEA (1.5 mL, 8.6 mmol) were added. The resulting solution was stirred at room temperature for 10 min. Then, 6-aminohexyl-1-ol (0.45 g, 3.8 mmol) was added and the mixture was stirred for about 4 h. The reaction was quenched with H₂O (40 mL), then extracted with ethyl acetate (60 mL × 2), and dried over anhydrous Na₂SO₄. The residue was then purified on a silica gel column to give L016-3 as a white solid (2.50 g, 93%). MS theoretical value: 698.4; measured value: 721.5 (M + Na₂SO₄). + ).

[0226] Step 2: Synthesis of L016-4

[0227] Add 1.5 mL of trifluoroacetic acid to 8 mL of a dichloromethane solution of compound L016-3 (0.30 g), and then stir overnight at room temperature. Evaporate to obtain a heavy oil, which requires no further purification.

[0228] Step 3: Synthesis of L016-5

[0229] To a 30 mL solution of acid G003 (0.92 g, 1.53 mmol) in dichloromethane, DIPEA (3 mL) and pentafluorophenyl trifluoroacetate (1.5 mL) were added and stirred overnight at room temperature. The reaction was quenched with cold saturated NaHCO3 solution and extracted with DCM (30 mL × 2). The combined organic phases were washed with water, dried over Na2SO4, filtered, and evaporated to give 1.5 g of a brown oil. The residue was purified on a silica gel column to give L016-5, a colorless oil (1.0 g, 83%).

[0230] Step 4: Synthesis of L016-OH

[0231] To a 20 mL THF solution of compound L016-5 (1.0 g, 1.30 mmol), add 2 mL of DIPEA and a solution of compound L016-4 (0.17 g, 0.43 mmol) in 10 mL of THF. Stir at room temperature for 16 h. Quench the reaction with water and extract with ethyl acetate (30 mL × 2). Dry the combined organic phases with Na₂SO₄, filter, and evaporate under vacuum. Purify the residue on a silica gel column to give LO16-OH as a white solid (0.96 g, 96%). MS theoretical value: 2148.3; Observed value: 1076.40 [M / 2+H] + .1H NMR (400MHz, DMSO-d6): δ8.50(s,1H),8.1(m,2H),7.90(m,3H),7.20(S,2H),5.20(d,3H),4.90(m,3H),4.50(d,3H),4.34(t,1H),4.04(m,12H),3 .80(m,5H),3.70(m,3H),3.65-3.20(m,12H),3.0(m,6H),2.20(m,15H),2 .11(s,9H),2.00(s,9H),1.90(s,9H),1.77(m,16H),1.16-1.49(m,87H).

[0232] Step 5: Phosphoramide formation and oligonucleotide synthesis

[0233]

[0234] L016 and L016-ApoB were synthesized using the same general methods as described in Examples 2, 3 and 5.

[0235] L016, MS theoretical value: 2348.4; measured value: 1197.1 (M / 2+Na)+ ). 31 P-NMR (DMSO-d6), 147.6 ppm.

[0236] L016-ApoB, MS theoretical value: 6157.6; measured value: 6158.3.

[0237] Example 11-2 Synthesis of L017-OH

[0238]

[0239] L017-OH and L017 were synthesized using a method similar to that used for L016-OH and L016.

[0240] L017-OH,MS theoretical value: 1867.9; measured value: 935.6.0 [M / 2+H] + .1H NMR (400MHz, DMSO-d6): δ8.40(m,1H),8.1(m,2H),7.90(m,4H),7.20(S,2H) ,5.20(d,3H),4.90(m,3H),4.50(d,3H),4.34(t,1H),4.04(m,12H),3.90(m, 5H),3.70(m,3H),3.60-3.30(m,10H),3.20(m,3H),2.80(m,3H),2.20(m,15H ),2.11(s,9H),2.00(s,9H),1.90(s,9H),1.77(m,15H),1.16-1.49(m,36H).

[0241] L017, 31 P-NMR (DMSO-d6), 146.7 ppm

[0242] L017-ApoB conjugate, MS theoretical value: 5877.3; measured value: 5877.4.

[0243] Example 11-3 Synthesis of L031-OH

[0244]

[0245] Step 1: Synthesis of L031-1

[0246] DIPEA (775 mg, 6.0 mmol), EDCI (520 mg, 2.7 mmol), and HOBt (370 mg, 2.7 mmol) were added to 20 mL of anhydrous DMF solution of G003 (1.37 g, 2.3 mmol). The reaction was stirred at room temperature for 0.5 h, and cycloene (103 mg, 0.6 mmol) was added. The mixture was stirred at room temperature for more than 24 h. After the reaction was complete, 100 mL of ethyl acetate and 30 mL of brine were added to dilute the reaction mixture, and the mixture was stirred for 10 min. The organic phase was separated, and the aqueous phase was extracted with ethyl acetate (50 mL × 2). The organic phases were combined and dried over anhydrous sodium sulfate. The residue was purified by silica gel column chromatography (MeOH / EA = 0% to 5%) to give compound L031-1 (750 mg, 65.2% yield) as a white solid. MS theoretical value: 1754.0; Found value: 878.7 (M / 2 + H + )

[0247] Step 2: Synthesis of L031-2

[0248] Two drops of DMF were added to a solution of benzyl-protected 6-hydroxyhexanoic acid (130 mg, 0.59 mmol) in 3.0 mL of anhydrous THF. Oxaloyl chloride (123 mg, 0.98 mmol) was added dropwise to the reaction system with stirring. After reacting for 2 hours, the mixture was evaporated to dryness under vacuum. 5 mL of anhydrous THF was added, and the mixture was evaporated to dryness under vacuum. The residue was diluted with 4 mL of DCM and used directly as solution (L031-M1). DIPEA (504 mg, 3.9 mmol) was added to 10 mL of anhydrous DCM solution of L031-1 (750 mg, 0.39 mmol). The mixture was stirred in an ice bath until the temperature was below 5 °C. The L031-M1 solution was added dropwise with stirring at a temperature of 0–10 °C. After the addition, the ice bath was removed, and the reaction mixture was stirred for 1 hour. When the reaction was complete, ethyl acetate (50 mL) and brine (30 mL) were added to the system, and the mixture was stirred for 10 minutes. The organic phase was separated, and the aqueous phase was extracted with diethyl ether acetate (30 mL × 2). The organic phase was dried over anhydrous sodium sulfate and evaporated to a pale yellow oil. The residue was separated by silica gel column chromatography (MeOH / EA = 0% to 5%) to give compound L031-2 (400 mg, 53.3% yield) as a white solid. MS theoretical value: 1958.1; Found value: 980.8 (M / 2 + H + )

[0249] Step 3: Synthesis of L031-OH

[0250] L031-2 (400 mg, 0.19 mmol) was dissolved in 8 mL of methanol and Pd / C (120 mg) was added. The mixture was degassed and purged with argon three times. The mixture was then stirred at room temperature for 24 hours. After the reaction was complete, the system was filtered until the solution was clear. The clear solution was evaporated to dryness to obtain compound L031-OH (320 mg, 84.2% yield). MS theoretical value: 2036.2; Found value: 2038.0 [M+H] + .1H NMR (400MHz, DMSO-d6): δ7.81(d,J=9.2Hz,3H),5.22(d,J=4.4Hz,3H),4.97(dd,J=3.6Hz,11.2Hz,3H),4.48(d,J=8.4Hz,3H),4.34(t,J=4.8Hz,1H ),4.04(m,9H),3.87(q,J=8.8Hz,3H),3.37–3.50(m,24H),2.24(br,8H), 2.11(s,9H),2.00(s,3H),1.90(s,3H),1.77(s,3H),1.16-1.49(m,84H).

[0251] Examples 11-4: Synthesis of L032-OH

[0252]

[0253] L032-OH (440 mg, 78.5% yield) was synthesized using a similar method to L031-OH. MS theoretical value: 1868.1; measured value: 1887.0 [M+H2O+H] + .1H NMR (400MHz, DMSO-d6): δ7.81(d,J=9.2Hz,3H),5.22(d,J=3.6Hz,3H),4.97(dd,J=3.6Hz,11.2Hz,3H),4.49(d,J=8.0Hz,3H),4.34(t,J=5.2Hz,1H ),4.03(m,9H),3.87(q,J=9.2Hz,3H),3.36–3.72(m,24H),2.30(br,8H), 2.11(s,9H),2.00(s,3H),1.90(s,3H),1.77(s,3H),1.20-1.49(m,60H).

[0254] Example of effect

[0255] 1. Screening the efficacy of GalNAc clusters using ApoB reduction assays

[0256] The oligonucleotides used in the study were prepared as described in Examples 4 and 5 and formulated in PBS for the study. Mice were grouped according to body weight (BW) on day -4. The study lasted for 30 days to evaluate the persistence of target knockout achieved by each conjugate. Mice were administered once on day 0 at two dose levels (high dose, 60 nmoles / kg and low dose, 20 nmoles / kg), and blood was collected on days 3, 10, and 17 to monitor plasma ApoB protein levels. The study was terminated on the last observation day or the humane endpoint (whichever came first). Blood (~50 μL / mouse / time point) was collected via tail or retro-orbital hemorrhage into EDTA-coated tubes. Blood samples were centrifuged at 1,000–2,000 x g for 10 min in a refrigerated centrifuge. After centrifugation, the resulting supernatant (plasma) was immediately transferred to clean, labeled polypropylene tubes and stored at -80°C until use.

[0257] Plasma ApoB levels were determined using a commercial ELISA kit (AbCam#ab230932). The assay was performed according to the manufacturer's instructions. Two plasma samples were tested at a 10,000-fold dilution. ApoB results were reported as μg / mL or normalized to the initial ApoB level determined prior to administration of the oligo-GalNAc conjugate. Comparisons between compounds were used to elucidate the structure-activity relationship (SAR), and comparisons with a three-linker positive control were used to screen for active GalNAc moieties.

[0258] Surprisingly, most GalNAc clusters synthesized in this disclosure by repeated addition of monomers (including groups B006-3 / 4, B007-5 / 6, B008-7 / 8, B009-9 / 10, B011-11 / 12, B013-13 / 14, and B015-15 / 16) showed similar or better ApoB knockout durability compared to the positive control B005. Some GalNAc clusters in groups 11 / 12 and 13 / 14 (GalNAc clusters B011 and B013) actually showed higher efficiency from day 10 to day 17 (see appendix). Figure 2A-2G ).

[0259] Other specific implementation methods

[0260] It should be understood that although the invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate, and not limit, the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are also within the scope of the claims.

[0261] The various embodiments described above can be combined to provide further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned in and / or listed in the application data sheets are incorporated herein by reference in their entirety. Some aspects of the implementation may be modified, if desired, to incorporate concepts from various patents, applications, and publications to provide further embodiments.

[0262] These and other changes may be made to the embodiments based on the detailed description above. Generally, the terminology used in the claims should not be construed as limiting the claims to the specific embodiments disclosed in the specification and claims, but should be interpreted to include all possible embodiments and the full scope of such equivalents. Therefore, the claims are not limited to this specification.

Claims

1. A conjugate having the following structure: Where L1 is (C1-C 20 )alkylene-amide-(C1-C 20 )alkylene.

2. Conjugates, which have the following structures: Where L1 is (C1-C 20 )alkylene-amide-(C1-C 20 ) alkylene, and A is the antisense sequence of siRNA or the messenger strand.

3. The conjugate according to claim 1 or 2, having the following structure:

4. A pharmaceutical composition comprising the conjugate of claim 1 or 2, and a pharmaceutically acceptable carrier or diluent.

Images