ARNATAR Compounds and Methods for Enhancing Cellular Uptake

Abstract

The present invention relates generally to the field of compounds and methods for enhancing cellular uptake. Specifically, the present invention relates to N-acetylgalactosamine compounds and conjugates thereof. Also provided are methods for the preparation and use of these molecules, particularly in medicine.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This PCT application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 433,730, filed December 19, 2022, U.S. Provisional Patent Application No. 63 / 533,273, filed August 17, 2023, and U.S. Provisional Patent Application No. 63 / 602,245, filed November 22, 2023, the entire contents of each of which are hereby incorporated by reference in their entirety.

[0002] Incorporation by Reference of Electronically Submitted Articles A computer-readable nucleotide / amino acid sequence listing is incorporated by reference in its entirety, and is identified as the text file entitled "GalNac_sequence_listing", filed concurrently herewith and created on December 18, 2023.

[0003] Throughout this application, various publications are referenced. All publications, gene transcript identifiers, patents, and patent applications mentioned in this document are herein incorporated by reference to the same extent as if each individual publication, gene transcript identifier, patent, and patent application was specifically and individually indicated to be incorporated by reference.

[0004] The present invention relates generally to the field of compounds and methods for enhancing cellular uptake. Specifically, the present invention relates to N-acetylgalactosamine compounds and conjugates thereof. Also provided are methods for the preparation and use of these molecules, particularly in medicine. [Background technology]

[0005] Pharmaceutical agents such as oligomeric compounds (e.g., oligonucleotides) need to enter target cells to be active.Various modalities have been used to make oligomeric compounds enter target cells, including viral delivery vectors, lipid-based delivery, polymer-based delivery, and conjugate-based delivery (Paunovska et al., Drug Delivery Systems for RNA Therapeutics, 2022, Nature Reviews Genetics, 23(5):265-280; Chen et al., 2022, Molecular Therapy, Nucleic Acids, 29:150-160).

[0006] Conjugation of oligomeric compounds with N-acetylgalactosamine (GalNAc) compounds is becoming a major delivery strategy for delivering oligomeric compounds into cells such as hepatocytes. Various GalNAc compound conjugates have been described in the literature, including Sharma et al., 2018, Bioconjugate Chem, 29:2478-2488; Nair et al., 2014, J. Am. Chem. S°C. 136(49):16958-16961; Keam, 2022, Drugs, 82:1419-1425; US Patent 10,087,208; Prakash et al., 2014, Nucleic Acids Res, 42(13):8796-807; Debacker et al., 2020, Molecular Therapy, 28(8):1759-1771; Huang et al., 2017, Bioconjugate Chem, 28:283-295; Nair et al. al., 2017, Nucleic Acids Res, 45(19):10969-10977, U.S. Patent No. 11,110,174, U.S. Patent No. 9,796,756, U.S. Patent No. 9,181,549, U.S. Patent No. 10,344,275, U.S. Patent No. 10,570,169, U.S. Patent No. 9,506,030, U.S. Patent No. 7,582,744, U.S. Patent No. 8,106,022, U.S. Patent No. 11,692,001, WO2022162161, WO2022162154, WO2022136466, WO2021257917, and WO2021257916, all of which are incorporated herein by reference.

[0007] Several oligomeric compounds bearing various types of GalNAc compound conjugates have been approved by the U.S. Food and Drug Administration (FDA) (Moumne et al., Oligonucleotide Therapeutics: From Discovery and Development to Patentability, Pharmaceutics, 2022, 14(2):260; Friedrich and Aigner, Therapeutic siRNA: State-of-the-Art and Future Perspectives, 2022, BioDrugs, 36(5):549-571; Hu et al., Therapeutic siRNA: State of the Art, Signal Transduction and Targeted Therapy, 2020, 5:101). However, improved GalNAc compounds for conjugation to pharmaceutical agents remain awaited. Summary of the Invention

[0008] The present invention relates generally to novel GalNAc compounds for conjugation to pharmaceutical agents, GalNAc compound conjugates, and processes for their production and use.

[0009] In one aspect, the present invention relates to a compound having the structure of formula (0): [ka] During the ceremony, k is an integer from 1 to 5, m is an integer from 0 to 11, n is an integer from 0 to 5, X is a hydroxyl protecting group or an H atom; Y is a hydroxyl protecting group, an H atom, a payload, or a solid support, the payload or solid support optionally being linked via a spacer; R 1 are independently selected from a hydroxyl protecting group and an H atom.

[0010] In one aspect, the present invention relates to a process for the preparation of a compound having the structure of formula (0): [ka] During the ceremony, k is an integer from 1 to 5, m is an integer from 0 to 11, n is an integer from 0 to 5, X is a hydroxyl protecting group or an H atom; Y is a hydroxyl protecting group, an H atom, a payload, or a solid support, the payload or solid support optionally being linked via a spacer; R 1 are independently selected from a hydroxyl protecting group and an H atom; The process comprises reacting a compound having the structure of formula (III): [ka] During the ceremony, k is an integer from 1 to 5, preferably 3; R 2 represents an amino protecting group or an H atom, R 3 represents a solid support, a hydroxyl protecting group, or an H atom, the solid support being optionally linked via a spacer; R 6 represents a hydroxyl protecting group, preferably dimethoxytriyl (DMT) or monomethoxytrityl (MMT), more preferably MMT.

[0011] In another aspect, the present invention relates to a compound having the structure of formula (III): [ka] During the ceremony, k is an integer from 1 to 5, R 2 represents an amino protecting group or an H atom, R 3 represents a solid support, a hydroxyl protecting group, or an H atom, the solid support being optionally linked via a spacer; R 6 represents a hydroxyl protecting group, preferably DMT or MMT, more preferably MMT. This compound is used in the preparation of a compound of formula (0) in an embodiment of the present invention.

[0012] In yet another aspect, the present invention relates to compounds having the structure of Formula (0) for use in medicine. [ka] During the ceremony, k is an integer from 1 to 5, m is an integer from 0 to 11, n is an integer from 0 to 5, X is a H atom, Y is a payload, optionally linked via a spacer; R 1 is a H atom.

[0013] In a related aspect, the invention relates to the above-described compound for use in a method of treating a subject with a payload, wherein the subject comprises cells expressing an asialoglycoprotein receptor (ASGPR).

[0014] In one aspect, the invention relates to a method for delivering a payload into a cell expressing an asialoglycoprotein receptor (ASGPR), the method comprising administering a compound having a structure of formula (0): [ka] During the ceremony, k is an integer from 1 to 5, m is an integer from 0 to 11, n is an integer from 0 to 5, X is a H atom, Y is a payload, optionally linked via a spacer; R 1 is a H atom, The payload is delivered into cells expressing the asialoglycoprotein receptor (ASGPR) in the subject in an amount sufficient to treat the subject with the payload.

[0015] In another aspect, the present invention relates to a pharmaceutical composition comprising a compound having the structure of Formula (0) and a pharmaceutically acceptable carrier, excipient, and / or diluent: [ka] During the ceremony, k is an integer from 1 to 5, m is an integer from 0 to 11, n is an integer from 0 to 5, preferably 3; X is a H atom, Y is a payload, optionally linked via a spacer; R 1 is a H atom.

[0016] Kits and pharmaceutical compositions comprising the compounds of the invention, including methods for making and using, are further contemplated. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 shows LMNA mRNA levels in human primary hepatocytes (hPH) after 51 hours of free-uptake of GalNAc-conjugated siRNA at different doses (μM). [Figure 2] FIG. 1 shows LMNA mRNA levels in human primary hepatocytes (hPH) over time after free uptake of GalNAc-conjugated siRNA (5 μM). [Figure 3] FIG. 1 shows LMNA mRNA levels in mouse primary hepatocytes (mPH) 60 hours after free uptake of GalNAc-conjugated siRNA at different doses (μM). [Figure 4]FIG. 1 shows PCSK9 mRNA levels in human primary hepatocytes (HPH) 4 hours after free uptake of GalNAc-conjugated siRNA at different doses (μM). Detailed Description

[0018] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and do not limit the invention as defined by the claims. As used herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of "or" means "and / or" unless specifically stated otherwise. Furthermore, the use of the term "comprising" as well as other forms such as "comprises" and "includes" is not limiting. Also, terms such as "element" or "component" encompass both elements and components that include one unit and elements and components that include multiple subunits, unless specifically stated otherwise.

[0019] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents or portions of documents cited in this application, including but not limited to patents, patent applications, articles, books, and papers, are hereby expressly incorporated by reference in their entirety and for the portions of the documents discussed herein.

[0020] definition Unless specific definitions are provided, the nomenclature, procedures, and techniques utilized in connection with analytical chemistry, synthetic organic chemistry, medicinal chemistry, and pharmaceutical chemistry described herein are well known and commonly used in the art. Standard techniques may be used for chemical synthesis and chemical analysis. Where permitted, all patents, applications, published applications, and other publications referenced throughout this disclosure, GENBANK accession numbers and related sequence information available through databases such as the National Center for Biotechnology Information (NCBI), and other data are incorporated by reference in their entirety and for the portions of the documents discussed herein.

[0021] Unless otherwise indicated, the following terms have the following meanings.

[0022] "2'-O-Methoxyethyl" (also 2'-MOE and 2'-O(CH2)2-OCH3) refers to an O-methoxy-ethyl modification at the 2' position of the furanose ring. A 2'-O-methoxyethyl modified sugar is a modified sugar.

[0023] "2'-MOE nucleoside" (also 2'-O-methoxyethyl nucleoside) refers to a nucleoside containing a 2'-MOE modified sugar moiety. "2'-MOE nucleotide" (also 2'-O-methoxyethyl nucleotide) refers to a nucleotide containing a 2'-MOE modified sugar moiety.

[0024] "2'-O-methyl" (also 2'-OCH3 and 2'-OMe) refers to an O-methyl modification at the 2' position of the furanose ring. A 2'-O-methyl modified sugar is a modified sugar.

[0025] "2'-OMe nucleoside" (also 2'-O-methyl nucleoside) means a nucleoside that includes a 2'-OMe modified sugar moiety. "2'-OMe nucleotide" (also 2'-O-methyl nucleotide) means a nucleotide that includes a 2'-OMe modified sugar moiety. "2'-substituted nucleoside" means a nucleoside that includes a substituent other than H or OH at the 2'-position of the furanosyl ring. In certain embodiments, 2'-substituted nucleosides include nucleosides with a fluoro (2'-F) modification, an O-methyl (2'-OMe) modification, an O-methoxyethyl (2'-MOE) modification, or a bicyclic sugar modification.

[0026] "5-methylcytosine" means a cytosine modified with a methyl group added to position 5. 5-methylcytosine is a modified nucleobase.

[0027] "About" means within ±7% of a value. For example, stating that "the compound affected at least about 70% inhibition of mRNA" implies that mRNA levels are inhibited within a range of 63% to 77%.

[0028] "Animal" refers to a human or non-human animal, including but not limited to mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including but not limited to monkeys and chimpanzees.

[0029] "Antibody" refers to a molecule having the characteristic of reacting specifically with an antigen in some manner, where the antibody and antigen are each defined relative to the other. An antibody may refer to an intact antibody molecule or any antigen-binding fragment or region thereof. Examples of such antigen-binding fragments or regions include the heavy chain, light chain, F chain, and F chain of an antibody. ab Area, and F c Areas include:

[0030] "Antisense oligonucleotide" or "ASO" refers to a single-stranded oligonucleotide having a nucleic acid base sequence that allows hybridization to the corresponding region or segment of target nucleic acid. In certain embodiments, antisense oligonucleotides contain one or more ribonucleosides (RNA nucleosides) and / or deoxyribonucleosides (DNA nucleosides). Antisense oligonucleotides can be modified, and examples of such modifications include 5-methylcytosine and 2'-MOE.

[0031] " Base complementarity " refers to the ability of the nucleic acid base of an oligonucleotide to form base pairs with the corresponding nucleic acid base in target nucleic acid (i.e., hybridization), and is mediated by Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonds between corresponding nucleic acid bases. Base complementarity also refers to canonical (e.g., A:U, A:T or C:G) or non-canonical base pairing (e.g., A:G, A:U, G:U, I:U, I:A or I:C).

[0032] "Bicyclic sugar" means a furanose ring modified by bridging two non-geminal carbon atoms. A bicyclic sugar is a modified sugar.

[0033] "Cap structure" or "end-capping moiety" means a chemical modification incorporated at either end of an oligomeric compound.

[0034] "Chemical modification" refers to the modification of molecular structure or elements from natural molecules. For example, siRNA compounds are composed of linked ribonucleosides (sometimes referred to herein as RNA), and therefore, the substitution of ribonucleosides with deoxyribonucleosides (sometimes referred to herein as DNA) is considered a chemical modification of siRNA compounds.

[0035] A "chemically distinct region" refers to a region of an oligomeric compound that is chemically distinct in some way from another region of the same oligomeric compound, e.g., a region having 2'-OMe nucleotides is chemically distinct from a region having nucleotides without 2'-OMe modifications.

[0036] " Chimeric oligomeric compound " refers to an oligomeric compound that has at least two chemically distinct regions, and each position has multiple subunits.For example, as disclosed herein, siRNA can comprise a peripheral region and a central region.The peripheral region comprises motifs with various modified or unmodified nucleobases to increase stability, specificity, safety and potency, while the central region comprises various modified or unmodified nucleobases to serve as the substrate for RISC-mediated degradation.

[0037] "Complementary" means the capacity for pairing between nucleobases of a first nucleic acid and a second nucleic acid.

[0038] "Adherence" means that an individual adheres to a recommended treatment.

[0039] "Comprise" and "comprising" shall be understood to imply the inclusion of a stated step or component or group of steps or components, but excluding any other step or component or group of steps or components.

[0040] "Contiguous nucleobases" means nucleobases immediately adjacent to each other.

[0041] "Deoxyribonucleoside" refers to a nucleoside having a hydrogen atom at the 2' position of the sugar moiety of the nucleoside. Deoxyribonucleosides are sometimes referred to herein as DNA nucleosides, "D," or "d." Deoxyribonucleosides may be modified with any of a variety of substituents or linked by a covalent bond other than the natural phosphodiester, such as phosphorothioate.

[0042] "Deoxyribonucleotide" refers to a nucleotide having a hydrogen atom at the 2' position of the sugar moiety of the nucleotide. Deoxyribonucleotides are sometimes referred to herein as DNA nucleotides, "D" or "d". Deoxyribonucleotides may be modified with any of a variety of substituents and may be linked by covalent bonds other than natural phosphodiesters, such as phosphorothioates.

[0043] "Designing" or "design" refers to the process of engineering oligomeric compounds that specifically hybridize to target nucleic acid molecules.

[0044] "Efficacy" means the ability to produce a desired effect.

[0045] "Expression" includes all functions that convert a gene's coded information into structures present and operating in the cell, including, but not limited to, the products of transcription and translation.

[0046] "Fully complementary" or "100% complementary" means that each nucleobase of a first nucleic acid has a complementary nucleobase in a second nucleic acid. In certain embodiments, the first nucleic acid is an oligomeric compound and the target nucleic acid is the second nucleic acid.

[0047] A "fully modified motif" refers to an oligomeric compound that contains a contiguous sequence of nucleosides, essentially each nucleoside bearing a chemical modification.

[0048] "GalNAc" means "N-acetylgalactosamine." Thus, a "GalNAc compound" refers to a compound that contains one or more N-acetylgalactosamine units.

[0049] " Hybridization " refers to the annealing of complementary nucleic acid molecules. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, oligomeric compounds and nucleic acid targets. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, siRNA and nucleic acid targets.

[0050] "Immediately adjacent" means that there are no intervening elements between the immediately adjacent elements.

[0051] "Individual" means a human or non-human animal selected for treatment or therapy.

[0052] "Induce," "inhibit," "enhance," "enhance," "increase," "decrease," and the like generally refer to a quantitative difference between two states.

[0053] "Inhibiting expression or activity" refers to the reduction, blocking of expression or activity, and does not necessarily indicate the total elimination of expression or activity.

[0054] "Internucleoside linkage" refers to the chemical bond between nucleosides.

[0055] "Linked nucleosides" means adjacent nucleosides (e.g., A, G, C, T, or U) linked together by an internucleoside bond. Examples of linked nucleosides include deoxyribonucleosides (sometimes referred to herein as DNA nucleosides) or ribonucleosides (sometimes referred to herein as RNA nucleosides).

[0056] A "mismatch" or "non-complementary nucleobase" refers to when a nucleobase of a first nucleic acid cannot pair with the corresponding nucleobase of a second or target nucleic acid through Watson-Crick base pairing (e.g., A:T, A:U, or C:G).

[0057] A "modified internucleoside linkage" refers to a substitution or any change from a naturally occurring internucleoside linkage (ie, a phosphodiester internucleoside linkage).

[0058] "Modified nucleobase" means any nucleobase other than adenine, cytosine, guanine, thymidine, or uracil. "Unmodified nucleobase" means the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).

[0059] "Modified nucleoside" refers to a nucleoside having, independently, a modified sugar moiety and / or a modified nucleobase. As used herein, when an oligomeric compound is RNA-based, the substitution of a ribonucleoside with a deoxyribonucleoside (sometimes referred to herein as a DNA nucleoside) is considered a modification of the oligomeric compound. Also, when an oligomeric compound is DNA-based, the substitution of a deoxyribonucleoside with a ribonucleoside (sometimes referred to herein as an RNA nucleoside) is considered a modification of the oligomeric compound.

[0060] "Modified nucleotide" means a nucleotide having, independently, a modified sugar moiety, a modified internucleoside linkage, a ribonucleoside (sometimes referred to herein as an RNA nucleoside) to a deoxyribonucleoside (sometimes referred to herein as a DNA nucleoside), a deoxyribonucleoside (sometimes referred to herein as a DNA nucleoside) to a ribonucleoside (sometimes referred to herein as an RNA nucleoside), and / or a modified nucleobase.

[0061] "Modified oligonucleotide" means an oligonucleotide that includes at least one modified internucleoside linkage, a modified sugar, a ribonucleoside (sometimes referred to herein as RNA nucleosides) to deoxyribonucleosides (sometimes referred to herein as DNA nucleosides), a deoxyribonucleoside (sometimes referred to herein as DNA nucleosides) to ribonucleosides (sometimes referred to herein as RNA nucleosides), and / or a modified nucleobase.

[0062] By "modified sugar" is meant substitutions and / or any changes derived from a naturally occurring sugar moiety.

[0063] "Moiety" means one of the subdivisions of something, i.e., a portion or component of something. For example, the sugar portion of a nucleotide is the sugar component of the nucleotide.

[0064] "Monomer" refers to a single unit of an oligomer. Monomers include, but are not limited to, nucleosides and nucleotides, whether natural or modified.

[0065] "Motif" refers to a pattern of modifications in an oligomeric compound. For example, as disclosed herein, ARNATAR has designed oligomeric compounds containing motifs with various modified nucleobases and modified internucleoside linkages to improve compound delivery, stability, specificity, safety, and efficacy.

[0066] By "natural sugar moiety" is meant a sugar moiety found in DNA (2'-H) or RNA (2'-OH).

[0067] "Natural internucleoside linkage" means a 3' to 5' phosphodiester linkage.

[0068] "Non-complementary nucleobases" refers to a pair of nucleobases that do not form hydrogen bonds or otherwise support hybridization with one another.

[0069] "Nucleic acid" refers to a molecule composed of monomeric nucleotides. Nucleic acids include, but are not limited to, ribonucleic acid (RNA), messenger RNA (mRNA), deoxyribonucleic acid (DNA), single-stranded nucleic acid, double-stranded nucleic acid, small interfering ribonucleic acid (siRNA), and microRNA (miRNA).

[0070] "Nucleobase" means a heterocyclic moiety capable of pairing with a base of another nucleic acid.

[0071] "Nucleobase complementarity" refers to a nucleobase that can base pair (also known as complementary) with another nucleobase. If a nucleobase at a particular position in an oligomeric compound can hydrogen bond with a nucleobase at a particular position in a target nucleic acid, the hydrogen bonding positions between the oligomeric compound and the target nucleic acid are considered complementary in that nucleobase pair. For example, in DNA, adenine (A) is complementary to thymine (T), in RNA, adenine (A) is complementary to uracil (U), and in both DNA and RNA, guanine (G) is complementary to cytosine (C). Base pairs or complementary nucleobases are usually canonical Watson-Crick base pairs (C:G, A:U, or A:T), but also include non-canonical base pairs such as Hoogsteen base pairs (e.g., A:G, or A:U), wobble base pairs (e.g., G:U, I:U, I:A, or I:C, where I is hypoxanthine), etc. Nucleobase complementarity promotes hybridization of the oligomeric compounds described herein with their target nucleic acids.

[0072] By "nucleobase sequence" is meant the order of contiguous nucleobases independent of any sugar, linkage, and / or nucleobase modifications.

[0073] "Nucleoside" means a nucleobase linked to a sugar.

[0074] "Nucleoside mimetics" include structures used to replace sugars or sugars and bases, not necessarily at one or more positions in an oligomeric compound, such as morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclo, or tricyclo sugar mimetics, e.g., nucleoside mimetics having non-furanose sugar units. Nucleotide mimetics include structures used to replace nucleosides, not necessarily at one or more positions in an oligomeric compound, such as peptide nucleic acids or morpholinos (morpholino or other non-phosphodiester linkages linked by -N(H)-C(=O)-O-). Sugar surrogates overlap with the slightly broader term nucleoside mimetics, but are not intended to indicate replacement of only the sugar unit (furanose ring). The tetrahydropyranyl ring provided herein illustrates one example of a sugar surrogate, in which the furanose sugar group is replaced with a tetrahydropyranyl ring system. "Mimetic" refers to groups substituted in place of the sugar, nucleobase, and / or internucleoside linkage. Generally, the mimetic is substituted for the sugar or sugar-internucleoside linkage combination, and the nucleobase is maintained for hybridization with a selected target.

[0075] "Nucleotide" refers to a nucleoside having a linking group (e.g., a phosphate (p) group or a phosphorothioate (PS) group) covalently attached to the sugar portion of the nucleoside. Nucleotides include ribonucleotides and deoxyribonucleotides. Ribonucleotides are the linked nucleotide units that form RNA. Deoxyribonucleotides are the linked nucleotide units that form DNA.

[0076] "Off-target effect" refers to unwanted or adverse biological effects associated with the modulation of RNA or protein expression of a gene other than the intended target nucleic acid.

[0077] "Oligomeric activity" refers to any detectable or measurable activity of an oligomeric compound that is conducive to hybridization with its target nucleic acid. In certain embodiments, the oligomeric activity is a decrease in the amount or expression of the target nucleic acid or the protein encoded by the target nucleic acid. The oligomeric activity can be regulated by an oligomeric compound such as siRNA.

[0078] "Oligomeric compound" refers to a sequence of linked monomeric subunits that can hybridize to at least a region of a target nucleic acid via hydrogen bonding. The monomeric subunits can be modified or unmodified nucleotides or nucleosides. The oligomeric compound acts as a template for RISC to recognize complementary messenger RNA (mRNA) transcripts, targeting specific mRNA transcripts for cleavage. Cleavage of the target mRNA blocks translation of the target mRNA and silences the target gene. Examples of oligomeric compounds include single-stranded and double-stranded compounds, such as antisense oligonucleotides, ssRNA, siRNA, shRNA, and miRNA.

[0079] "Oligomeric inhibition" means that in the presence of an oligomeric compound complementary to a target nucleic acid, the level of the target nucleic acid is reduced compared to the level of the target nucleic acid in the absence of the oligomeric compound.

[0080] "Oligomeric machinery" includes machinery associated with RISC or RNase H that involves hybridization of an oligomeric compound with a target nucleic acid, the result or effect of which is targeted degradation and inhibition of gene expression.

[0081] As used herein, "oligonucleotide" refers to a sequence of linked nucleosides, each of which can be independently modified or unmodified. Oligonucleotides can have linking groups other than phosphate groups (e.g., phosphorothioate = thiophosphate groups) used as linking groups between nucleosides. In certain embodiments, oligonucleotides comprise one or more ribonucleosides (RNA nucleosides) and / or deoxyribonucleosides (DNA nucleosides).

[0082] As used herein, "payload" refers to any substance that is transported into a cell via a vehicle, where the vehicle is a GalNAc compound. In the sense of the present invention, "payload" includes pharmaceutical agents, i.e., agents that provide a biological effect in a cell, such as leading to the therapeutic treatment of a disease or disorder or the diagnosis of a disease or disorder. Suitable representatives of payloads are further described below.

[0083] As used herein, a "peptide" is a compound containing at least two amino acids linked in a chain, typically by a peptide bond. Peptides may also contain modified amino acids.

[0084] As used herein, a "pharmaceutical agent" is any compound that has pharmaceutical activity, ie, a compound that is used in therapeutic and / or diagnostic methods.

[0085] "Phosphorothioate linkage" or "PS" refers to an internucleoside linkage in which the phosphodiester bond has been modified by replacing one of the non-bridging oxygen atoms with a sulfur atom. A phosphorothioate linkage is a modified internucleoside linkage.

[0086] By "portion" is meant a defined number of contiguous (i.e., linked) nucleobases of a nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of a target nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of an oligomeric compound.

[0087] A "region" is defined as a portion of a target nucleic acid that has at least one distinguishable structure, function, or property.

[0088] "RNA" or "ribonucleic acid" consists of ribose nucleotides or ribonucleotides (nitrogenous bases attached to a ribose sugar) linked by phosphodiester bonds to form chains of various lengths. The nitrogenous bases in RNA are adenine, guanine, cytosine, and uracil.

[0089] "Ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2'-position of the sugar moiety of the nucleotide. Ribonucleotides can be modified with any of a variety of substituents and can be linked by covalent bonds other than natural phosphodiester, such as phosphorothioate. Ribonucleotides are sometimes referred to herein as RNA.

[0090] A "segment" is defined as a smaller portion or subportion of a region within a target nucleic acid.

[0091] A "site," as used herein, is defined as a unique nucleobase position within a target nucleic acid.

[0092] "Specifically hybridizable" refers to an oligomeric compound having a sufficient degree of complementarity between the oligomeric compound (e.g., siRNA) and the target nucleic acid to induce a desired effect while having minimal or no effect on non-target nucleic acids under the conditions for which specific binding is desired, i.e., physiological conditions in in vivo assays and therapeutic treatments.

[0093] "Stringent hybridization conditions" or "stringent conditions" refer to conditions under which an oligomeric compound will hybridize to its target sequence, but to a minimal number of other sequences.

[0094] "Subject" means a human or non-human animal selected for treatment, diagnosis, or therapy.

[0095] "Target" refers to a protein or nucleic acid sequence (eg, mRNA) whose modulation is desired.

[0096] "Target gene" refers to a gene that encodes a target.

[0097] "Targeting" refers to the process of designing and selecting oligomeric compounds that will specifically hybridize to a target nucleic acid and induce a desired effect.

[0098] "Target nucleic acid," "target RNA," "target RNA transcript," and "nucleic acid target" all refer to a nucleic acid that can be targeted by an oligomeric compound.

[0099] "Target region" means a portion of a target nucleic acid to which one or more oligomeric compounds are targeted.

[0100] "Target segment" means a sequence of nucleotides of a target nucleic acid that is targeted by an oligomeric compound. A "5' target site" is the 5'-most nucleotide of a target segment. A "3' target site" is the 3'-most nucleotide of a target segment. In one embodiment, a target segment is at least a 12-nucleobase portion (i.e., at least 12 conserved nucleobases) of a target region that is targeted by an oligomeric compound.

[0101] "Therapeutic efficacy" refers to the effectiveness of a therapeutic compound, such as an oligomeric compound. Therapeutic efficacy can be increased by improving the delivery, stability, specificity, safety, and efficacy of the therapeutic compound.

[0102] "Unmodified" RNA nucleobase refers to the purine bases adenine (A) and guanine (G), and the pyrimidine bases cytosine (C) and uracil (U). "Unmodified" DNA nucleobase refers to the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T) and cytosine (C). In certain embodiments, an unmodified RNA nucleobase is considered to be modified when a DNA nucleobase is substituted for an RNA nucleobase. In certain embodiments, an unmodified DNA nucleobase is considered to be modified when an RNA nucleobase is substituted for a DNA nucleobase.

[0103] "Unmodified nucleotide" means a nucleotide composed of naturally occurring nucleobases, sugar moieties, and internucleoside linkages. In certain embodiments, the unmodified nucleotide is an RNA nucleotide or a DNA nucleotide. Subject of the Invention

[0104] The present invention provides novel N-acetylgalactosamine-containing compounds (i.e., GalNAc compounds) that have enhanced cellular uptake. Interestingly, and quite unexpectedly, it has been found that—when conjugated to a payload such as an oligonucleotide—the novel GalNAc compounds provide enhanced activity in cells compared to prior art GalNAc compounds that are highly closed.

[0105] The present invention also provides novel methods for preparing the novel GalNAc compounds, as well as novel methods for preparing intermediate compounds well suited for the novel methods. Furthermore, the present invention also provides potential uses and methods for the novel GalNAc compounds, particularly in medicine.

[0106] GalNAc compounds In one aspect, the present invention relates to a compound having the structure of formula (0): [ka] During the ceremony, k is an integer from 1 to 5, preferably 2 or 3, more preferably 3; m is an integer from 0 to 11, preferably from 7 to 9, more preferably 7; n is an integer from 0 to 5, preferably 2 or 3, more preferably 3; X is a hydroxyl protecting group or an H atom; Y is a hydroxyl protecting group, an H atom, a payload, or a solid support, the payload or solid support optionally being linked via a spacer; R 1 are independently selected from a hydroxyl protecting group and an H atom.

[0107] Compound (0) comprises a trimer unit containing an N-acetylgalactosamine (GalNAc) unit. In the compounds of the present invention, the cyclic sugar moiety is based on D-galactose, i.e., (2R, 3S, 4S, 5R)-2,3,4,5,6-pentahydroxyhexanal. Residues X, Y, and R 1 Depending on the selection of, compound (0) may be either a GalNAc compound conjugate having a payload (such as compound (I)) or an intermediate GalNAc compound for synthesizing a GalNAc compound conjugate (such as compound (II)). As used herein, the term "GalNAc compound" encompasses both a GalNAc compound conjugate having a payload and an intermediate GalNAc compound.

[0108] The GalNAc units are linked via a linker comprising three variable regions, which may contain different numbers of repeating -CH2- or -OCH2- groups, designated as integers k, m, or n, respectively. Here, k is an integer from 1 to 5, preferably 2 or 3, more preferably 3; m is an integer from 0 to 11, preferably 7 to 9, more preferably 7; and n is an integer from 0 to 5, preferably 2 or 3, more preferably 3. In one embodiment, the sum of k and m is an integer from 8 to 12, preferably 10. Linkers of such lengths have been found to be particularly useful in GalNAc compounds. In a preferred embodiment, k is 3, m is 7, and n is 3, and the compound has the structure of formula (0a). [ka]

[0109] In compounds (0) and / or (0a), residues X, Y, and R 1 may represent a hydroxyl protecting group or an H (hydrogen) atom. These residues may be independently selected from all possible hydroxyl protecting groups and H atoms. Suitable hydroxyl protecting groups include acetyl, benzoyl, phenoxyacetyl, pivaloyl, monomethoxytrityl (MMT), dimethoxytrityl (DMT), isobutyryl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl, and isopropyldimethylsilyl. Suitable hydroxyl protecting groups include residues R 1and / or monomethoxytrityl (MMT) or dimethoxytrityl (DMT) for residue X. These groups have been found to be particularly useful in the synthesis of GalNAc compounds of the present invention and in coupling payloads to intermediate GalNAc compounds. Specifically, MMT is useful in the synthesis of oligonucleotides. Due to the symmetry of the -CH2OX and -CH2OY groups, the formula includes these groups regardless of their preparation process. However, it should be understood that when a solid support is present as residue Y, the oligonucleotide will be attached to the other residue, i.e., residue X.

[0110] Residues X and R 1 Each of the X residues and R may represent an H atom. In one embodiment of a GalNAc compound conjugate with a payload, 1 Preferably, each of the residues is an H atom.

[0111] Residue Y may also be a payload or a solid support, which may optionally be linked via a spacer.

[0112] In one embodiment, residue Y is a payload. In this embodiment, the GalNAc compound can be the final conjugate for intracellular introduction or therapeutic use. As defined above, a payload is any substance transported into cells via a vehicle, where the vehicle is a GalNAc compound. In the sense of the present invention, a payload includes a pharmaceutical agent, i.e., an agent that provides a biological effect in cells, leading to, for example, the therapeutic treatment of a disease or disorder or the diagnosis of a disease or disorder. Suitable payloads in the present invention include any oligonucleotide (i.e., modified or unmodified), peptide, antibody, antibody fragment, or oligomeric compound, including chemical compounds, that have pharmaceutical activity. It should be understood that each of these compounds can be conjugated to the (intermediate) GalNAc compound either directly or via a spacer suitable for the conjugation of each compound. Therefore, a payload within the meaning of the present invention may include a spacer, even if not explicitly stated.

[0113] In some embodiments, the payload comprises an oligomeric compound, preferably an oligonucleotide.Oligomeric compounds include but are not limited to single-stranded oligomeric compounds, such as microRNA (miRNA), single-stranded RNA (ssRNA) and antisense oligonucleotide (ASO); and double-stranded oligomeric compounds, such as short hairpin RNA (shRNA) and small interfering RNA (siRNA).Oligomeric compounds can be "antisense" or can comprise the "antisense strand" of target nucleic acid, which means that they can hybridize with target nucleic acid through hydrogen bond.

[0114] In certain embodiments, oligomeric compound has nucleobase sequence, when written in 5 ' to 3 ' direction, comprises the reverse complement of the target segment of the targeted target nucleic acid.For example, in certain such embodiments, siRNA comprises antisense strand, when written in 5 ' to 3 ' direction, comprises the reverse complement of the target segment of the targeted target nucleic acid.

[0115] In certain embodiments, the oligomeric compound is 12 to 30 subunits (e.g., nucleotides or nucleosides) in length. In certain embodiments, the oligomeric compound is 18 to 30 subunits in length. In certain embodiments, the oligomeric compound is 12 to 22 subunits in length. In some embodiments, the oligomeric compound is an siRNA.

[0116] It is possible to increase or decrease the length of an oligomeric compound such as siRNA and / or introduce base mismatches without eliminating activity (U.S. Patent No. 7,772,203, incorporated herein by reference).For example, it is possible to introduce non-canonical base pairs (e.g., A:G, A:C, G:U, I:U, I:A, or I:C) into an oligomeric compound without eliminating activity.In certain embodiments, the activity of an oligomeric compound is enhanced by designing an oligomeric compound with one or more non-canonical base pairs, i.e., mismatches.

[0117] Oligomeric compounds can contain mismatches with the target, mismatches between oligomer strands within the duplex, or a combination thereof. Mismatches can occur throughout the siRNA, such as in the overhang region or the duplex region.

[0118] In some embodiments, the oligomeric compounds are single-stranded (e.g., single-stranded oligonucleotides, single-stranded RNA (ssRNA)) or double-stranded (e.g., shRNA and siRNA) and modified. Single-stranded oligomeric compounds include a sense strand or an antisense strand. Double-stranded oligomeric compounds include a sense strand and an antisense strand. The antisense strand can be fully or partially complementary to the target nucleic acid.

[0119] Oligomeric compounds, preferably oligonucleotides, can generally be modified and / or unmodified compounds. Nucleoside is a base-sugar combination. The nucleobase (also known as base) portion of nucleoside is usually a heterocyclic base portion. Nucleotide is a nucleoside that further comprises a covalent bond (e.g., a phosphate group or a chemically modified bond as described below) with the sugar portion of nucleoside. Oligonucleotides are formed through the covalent bond between adjacent nucleotides to form linear polymeric oligonucleotides. Within the oligonucleotide structure, linking groups are generally considered to form the internucleoside bond of the oligonucleotide. Oligomeric compounds are composed of one oligonucleotide (e.g., ssRNA, antisense oligonucleotide or miRNA) or multiple oligonucleotides (e.g., siRNA or shRNA).

[0120] Modifications to oligomeric compounds include substitutions or changes to the nucleobase, internucleoside linkage, or sugar moiety. Modified oligomeric compounds are often preferred over native or unmodified forms because they possess desirable properties, such as enhanced delivery (e.g., increased cellular uptake), increased specificity or affinity for a nucleic acid target, increased stability in the presence of nucleases, enhanced safety (e.g., reduced side effects following administration of the compound to a subject), or increased potency (e.g., inhibitory activity).

[0121] Modified oligomeric compounds, preferably modified oligonucleotides, preferably contain modified nucleobases or modified internucleoside linkages. In some embodiments, oligomeric compounds, preferably oligonucleotides, are modified to resist degradation, reduce toxicity, and / or enhance activity.

[0122] In certain embodiments, the oligomeric compounds disclosed herein have chemically modified subunits arranged in motifs to confer beneficial properties to the oligomeric compounds, including, but not limited to, enhanced inhibitory activity to increase potency, increased binding affinity to the target nucleic acid to limit off-target effects and increase safety, or enhanced resistance to in vivo nuclease degradation to increase stability and durability. In certain embodiments, the oligomeric compounds are chimeric, and the peripheral nucleobases of the oligomeric compounds contain motifs with various modified or unmodified nucleobases to increase stability, specificity, safety, and potency, while the central region of the compound contains various modified or unmodified nucleobases that serve as substrates for RISC-mediated degradation. Each distinct region may contain uniform, modified, or alternative sugar moieties. Each region may contain diverse patterns of phosphate and phosphorothioate linkages.

[0123] In certain embodiments, the sense strand contains one or more phosphorothioate internucleotide (PS) linkages between two nucleosides. In yet another embodiment, the sense strand contains phosphorothioate internucleotide (PS) linkages adjacent to a deoxyribonucleoside (D) or ribonucleoside (R). The PS linkages can be adjacent to the deoxyribonucleoside (D) or ribonucleoside (R) on the 5' side, the 3' side, or both sides. The PS linkages can also be adjacent to two nucleosides at the 5' end of the strand and / or adjacent to two nucleosides at the 3' end of the strand. In one embodiment, the oligomeric compound is ssRNA or siRNA.

[0124] The oligomeric compound, preferably an oligonucleotide, may be attached to the GalNAc compound via the 3'-end or the 5'-end. In some embodiments, the oligomeric compound, preferably an oligonucleotide, is attached to the GalNAc compound via the 3'-end of the sense strand. In some embodiments, the oligomeric compound, preferably an oligonucleotide, is attached to the GalNAc compound via the 5'-end of the sense strand.

[0125] In several embodiments, the oligomeric compound is a modified and / or unmodified oligonucleotide. In some embodiments, the oligonucleotide is a modified oligonucleotide modified to resist degradation, reduce toxicity, and / or enhance activity. Preferably, the modified and / or unmodified oligonucleotide binds to a target nucleotide sequence and alters expression of a gene encoded by the target nucleotide sequence. In some embodiments, the modified and / or unmodified oligonucleotide inhibits expression of a gene encoded by the target nucleotide sequence, preferably by inhibiting transcription or translation of the target nucleotide sequence. In some embodiments, the target nucleotide sequence of the modified and / or unmodified oligonucleotide is associated with a liver disorder, preferably a metabolic liver disorder, e.g., the target nucleotide sequence of the modified and / or unmodified oligonucleotide encodes a gene associated with a disease expressed in the liver or a gene associated with a liver disorder, preferably a metabolic liver disorder.

[0126] In certain embodiments, the oligomeric compounds, preferably oligonucleotides, described herein inhibit expression of a target nucleic acid by at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%.

[0127] Some embodiments are directed to modulating gene expression by inhibition with oligomeric compounds. In some embodiments, the target nucleotide sequence is messenger RNA (mRNA).

[0128] In certain embodiments, it is contemplated to inhibit gene expression of laminin (LMNA) in cells, which involves administering to the cells an oligomeric compound targeted to the LMNA mRNA transcript.

[0129] In certain embodiments, it is intended to inhibit apolipoprotein C3 (ApoC3) gene expression in cells, which involves administering to the cells an oligomeric compound targeted to the ApoC3 mRNA transcript.

[0130] In certain embodiments, it is contemplated to inhibit nucleolin (NCL) gene expression in a cell, which involves administering to the cell an oligomeric compound targeted to the mRNA transcript of NCL.

[0131] In some embodiments, hybridization occurs between the oligomeric compound and mRNA. The most common mechanism of hybridization involves hydrogen bonding (e.g., Watson-Crick hydrogen bonding, Hoogsteen hydrogen bonding, or reversed Hoogsteen hydrogen bonding) between complementary nucleobases of nucleic acid molecules.

[0132] The GalNAc-payload conjugates according to the present invention may be specifically designed for binding to the asialoglycoprotein receptor (ASGPR). Thus, according to some embodiments, the GalNAc-payload conjugates according to the present invention have a strong affinity for ASGPR. In the context of the present invention, "strong affinity" refers to a high affinity for the ASGPR, such as an IC 50 IC refers to an affinity characterized by a value of less than 50 nM, preferably 25 nM or less, more preferably 10 nM or less, more preferably 5 nM or less. 50 is the concentration of GalNAc-payload conjugate that inhibits 50% of labeled ligand binding to ASGPR. IC 50 can be determined by the method described in Rensen et al., 2001, Journal of Biological Chemistry, Vol. 276, pp. 37577. Briefly, hepatocytes (primary or cultured) are incubated with the ligand GalNAc in the presence of increasing amounts of the GalNAc-payload conjugate under investigation. 125The antibody is incubated with I-labeled nonsialylated orosomucoid (ASOR) at one concentration (e.g., 5 nM) for 2 hours at 4°C. The concentration can be increased from 0.2 nM to 200 nM. Binding of the labeled ASOR is followed in the presence of the GalNAc-payload conjugate being studied. Nonspecific binding can be determined in the presence of 100 mM GalNAc. Displacement binding data can be analyzed using a single-site binding model, and IC 50 Calculate.

[0133] The asialoglycoprotein receptor (ASGPR) is generally expressed on hepatocytes. Therefore, the GalNAc-payload conjugates of the present invention are particularly suitable for delivery to hepatocytes. Therefore, GalNAc-payload conjugates can be specifically designed by selecting the respective payloads for use in the prevention, treatment, or diagnosis of liver diseases or disorders, or for metabolism that occurs in the liver. Specifically, payloads can be designed for the treatment of diseases such as hepatitis (including viral hepatitis such as HBV and HCV), fatty liver (including metabolic disorders), atherosclerosis, hyperlipidemia, hypercholesterolemia, familial hypercholesterolemia, such as functional mutations in apolipoprotein B, HDL / LDL cholesterol imbalance, dyslipidemia, such as familial hyperlipidemia (e.g., familial combined hyperlipidemia (FCH), familial hypercholesterolemia (FH)), acquired hyperlipidemia, statin-resistant hypercholesterolemia, coronary artery disease (CAD), coronary heart disease (CHD), acute coronary syndrome (ACS), liver fibrosis (or diseases associated with liver fibrosis), cirrhosis, and cancer. Thus, in some embodiments, GalNAc-payload conjugates are designed for and / or use in the treatment of liver disorders, preferably metabolic liver disorders.

[0134] The asialoglycoprotein receptor (ASGPR) is also expressed on testicular cells, and therefore, in some embodiments, GalNAc-payload conjugates are designed for and / or for use in the treatment of testicular disorders.

[0135] The asialoglycoprotein receptor (ASGPR) is expressed on cells other than the liver and testis. In some embodiments, the GalNAc-payload conjugate is designed to deliver the GalNAc-payload conjugate into cells that express the ASGPR.

[0136] In one preferred embodiment of a GalNAc compound conjugate with a payload, the GalNAc compound has the structure of formula (I): [ka] In the formula, oligo represents the modified and / or unmodified oligonucleotides described above.

[0137] In an even more preferred embodiment of the GalNAc compound conjugate with a payload, the GalNAc compound has the structure of Formula (Ia): [ka]

[0138] Embodiments of GalNAc compound conjugates comprising a payload having the structure of Formula (I) or Formula (Ia) may be prepared with any of the above features and in any of the above embodiments.

[0139] In addition to oligomeric compounds, payloads can also include peptides, antibodies, antibody fragments, or other chemical compounds with pharmaceutical activity.Any molecule can be considered to have pharmaceutical activity according to the general definition of payload herein.Some drugs based on peptides, antibodies or antibody fragments, or other chemical compounds are known in the art.Generally, the GalNAc compounds of the present invention can be used to conjugate with these molecules for intracellular transport, and these molecules can then exhibit pharmaceutical activity.

[0140] In one embodiment, the payload comprises a peptide. Preferably, the peptide comprises a sequence of at least two amino acids, for example, at least three amino acids, or at least four amino acids or more. The amino acids may include natural amino acids and modified amino acids. Modified amino acids are known in the art. In a preferred embodiment, the peptide is the amino acid sequence of a specific protein. As used herein, a peptide may include a portion of a specific protein or the entire amino acid sequence of a specific protein, in which case the amino acids may be modified and / or unmodified. Therapeutic peptides are known in the art and are described, for example, in Wang et al., Therapeutic peptides: current applications and future directions. Sig Transduct Target Ther 7, 48 (2022), which is incorporated herein by reference. Examples of therapeutic peptides for the treatment of liver disease are PGPIPN and FFW, as disclosed in Qi et al., Therapeutic hexapeptide (PGPIPN) prevents and cures alcoholic fatty liver disease by affecting the expressions of genes related with lipid metabolism and oxidative stress. Oncotarget. 2017 Sep 30;8(50):88079-88093 or National University of Singapore. "Scientists develop novel drug that could potentially treat liver cancer more effectively: Peptide drug FFW shows promise in reducing tumor growth and slowing down spread of cancer cells." ScienceDaily, 2 August 2018. www.sciencedaily.com / releases / 2018 / 08 / 180802102347.htm.Peptide compounds with potential for use in the treatment of liver fibrosis are disclosed in Xun et al., Peptide mediated therapy in fibrosis: Mechanisms, advances and prospects, Biomedicine & Pharmacotherapy, Volume 157, 2023, 113978. In one embodiment, the payload comprises an antibody or antibody fragment. The antibody fragment is preferably an F that can be generated from the variable region of an antibody (e.g., IgG and / or IgM). (ab’)2 , F ab , F ab’ , and F v It is an antigen-binding fragment. In one embodiment, the payload comprises a chemical compound having pharmaceutical activity. Preferably, the chemical compound differs from other molecules described as payloads in that it is a non-peptide compound, a non-oligonucleotide compound, and does not contain any antibody or antibody fragment. Typically, the chemical compound is an organic molecule having biological activity, such as a so-called "small molecule" used as a medicine. Compounds suitable for treating liver diseases are described, for example, in Muriel P, Rivera-Espinoza Y. Beneficial drugs for liver diseases. J Appl Toxicol. 2008 Mar;28(2):93-103. doi: 10.1002 / jat.1310. PMID: 17966118. Suitable examples include colchicine, corticosteroids, curcumin, glycyrrhizin, interferon, resveratrol, sulfoadenosylmethionine, and thalidomide.

[0141] In one embodiment of the compound of formula (0), residue Y is a solid support, optionally including a spacer. In this embodiment, each GalNAc compound may be used in the synthesis of a GalNAc compound conjugate. In principle, any solid support known in the art for attaching compounds may be used in the specific attachment and solid-phase synthesis of nucleotide sequences. In certain embodiments, the solid support is a CPG solid support, such as an LCAA (log chain alkylamine) spacer CPG support. In certain embodiments, the solid support is a polymer support for solid-phase synthesis known in the art, such as a polystyrene solid support, such as an aminomethyl polystyrene support. Synthesis on solid supports is well known in the art and is described, for example, in Current Protocols in Nucleic Acid Chemistry (2000) 3.1.1-3.1.28.

[0142] In a preferred embodiment, the GalNAc compound is linked to the solid support via a spacer. The spacer may include spacer (2) and / or spacer (1). Spacer (2) is generally a spacer specific to the specific solid support being used. In many cases, the solid support is purchased with spacer (2) already attached. For example, it may be an LCAA (long-chain alkylamine) spacer (when used with CPG supports) or a spacer specific to aminomethyl polystyrene supports (often an amine-containing spacer). Spacer (1) may contain a dicarboxylic acid-derived moiety or a dicarboxylic acid-derived moiety. The dicarboxylic acid-derived moiety may contain two carboxyl groups, one or both of which may be modified to an amide group. In a specific embodiment, spacer (1) contains at least one carboxyl group, which is reacted with an amine-containing group of spacer (2) to form an amide group. Preferably, the dicarboxylic acid-derived moiety has 3 to 10 carbon atoms, i.e., 1 to 8 carbon atoms between the carboxyl and / or amide groups. The 1 to 8 carbon atoms can be a linear or branched alkylene having 1 to 8 carbon atoms (optionally, one or more carbon atoms are substituted with O, S, NH, and / or N—(C1-C3 alkyl) (i.e., N-methyl, N-ethyl, and / or N-propyl)) and / or a cycloalkylene having 5 to 8 carbon atoms. Preferably, the 1 to 8 carbon atoms is ethylene, 1,1 dimethylethylene, propylene, n-butylene, or 1,2 cyclohexylene. Preferably, the dicarboxylic acid-derived moiety is derived from succinic acid, 2,2 dimethylsuccinic acid, glutaric acid, adipic acid, 1,2 cyclohexanedicarboxylic acid, dimethylene oxide, dimethylene sulfide, and / or dimethylenemethylamine. The spacer (1) may be prepared by using the anhydride of each dicarboxylic acid.

[0143] Generally, the spacer of residue Y can be any moiety suitable for its intended function of providing spacing between two or more units. Preferably, the spacer is an alkylene moiety, such as C 2-10 It comprises an alkylene group, and such moieties are optionally substituted with heteroatoms to facilitate removal of the spacer after reaction of the GalNAc compound with a payload.

[0144] Residue Y may also be an H atom or a hydroxyl protecting group such as those defined above. In one embodiment, residue Y is an H atom. In another embodiment, residue Y is a hydroxyl protecting group, preferably selected from any of the hydroxyl protecting groups defined above.

[0145] In one preferred embodiment, the GalNAc compound used in the synthesis of the GalNAc compound conjugate has the structure of formula (II): [ka] During the ceremony, k, m, and n are as defined above; R 1 are independently selected from a hydroxyl protecting group as defined above and an H atom, preferably an acetyl group; R 6 represents a hydroxyl-protecting group, preferably DMT or MMT, more preferably MMT; R 7 represents the moiety of formula (VI), [ka] During the ceremony, R represents a solid support, an amino protecting group, a hydroxyl protecting group, or an H atom, the solid support optionally being linked via a spacer; Z represents NH or O; R 8represents a straight or branched alkylene having from 1 to 8 carbon atoms, optionally in which one or more carbon atoms are replaced by O, S, NH and / or N—(C1-C3 alkyl) (e.g., N-methyl, N-ethyl and / or N-propyl), and / or R 8 is cycloalkylene having from 5 to 8 carbon atoms, preferably ethylene, 1,1 dimethylethylene, propylene, n-butylene, 1,2 cyclohexylene, dimethylene oxide, dimethylene sulfide and / or dimethylenemethylamine.

[0146] In some preferred embodiments, R 7 represents any one moiety of formula (VIa1) to formula (VIa8). [ka] , [ka] , [ka] , [ka] , [ka] , [ka] , [ka] , and [ka] .

[0147] In some preferred embodiments, R 7represents a moiety of any one of formula (VIa3) or formula (VIa4).

[0148] In a further preferred embodiment, the GalNAc compound used in the synthesis of the GalNAc compound conjugate has the structure of Formula (IIa): [ka] In the formula, R 1 , R 6 , and R 7 is as defined above.

[0149] Part R 7 In some embodiments of compounds of formula (II) or (IIa) where Z represents an NH group, the residue R represents an amino-protecting group, preferably including 9-fluorenylmethylcarbamate (Fmoc), t-butylcarbamate (Boc), benzylcarbamate, acetamide, trifluoroacetamide, benzylamine, triphenylmethylamine (tritylamine), p-toluenesulfonamide, and / or tosylamide. Although the above terminology is used, those skilled in the art will recognize that the residue R, in combination with the N unit, constitutes the above-listed group. The use of amino-protecting groups leading to the formation of phthalimides and benzylideneamines by elimination of an H atom from the N atom is also possible.

[0150] Part R 7 In some embodiments of compounds of formula (II) or (IIa) in which Z represents an O atom, the residue R represents a hydroxyl protecting group, which is preferably selected from acetyl, benzoyl, phenoxy-acetyl, pivaloyl, dimethoxytrityl (DMT), monomethoxytrityl (MMT), isobutyryl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl, and isopropyldimethylsilyl.

[0151] In specific embodiments, the moiety R in the structures of formulas (II) and (IIa) 7Residue R is a solid support, optionally linked via a spacer, specifically a solid support as defined above. Each (intermediate) GalNAc compound is particularly suitable for the addition of nucleotides and therefore for the preparation of GalNAc compound conjugates with oligonucleotides, such as those depicted in the structures of formulas (I) and (Ia), respectively.

[0152] Compounds used in the preparation of GalNAc compounds In one aspect, the present invention relates to compounds, i.e., intermediate compounds, used in the preparation of the GalNAc compounds of the invention. In one embodiment, the intermediate compound has the structure of formula (III): [ka] During the ceremony, k is an integer as defined above, preferably 3; R 2 represents an amino protecting group or an H atom, R 3 represents a solid support, a hydroxyl protecting group, or an H atom, the solid support being optionally linked via a spacer; R 6 represents a hydroxyl-protecting group, preferably DMT or MMT, more preferably MMT.

[0153] In some embodiments of the compound of Formula (III), the residue R 2 represents an amino-protecting group, which preferably includes 9-fluorenylmethylcarbamate (Fmoc), t-butylcarbamate (Boc), benzylcarbamate, acetamide, trifluoroacetamide, benzylamine, triphenylmethylamine (tritylamine), p-toluenesulfonamide, and / or tosylamide. Although the above terminology is used, those skilled in the art will recognize that the residue R 2 This recognizes that the N unit can be combined with the groups listed above to form the N-unit. It also allows for the use of amino protecting groups, which lead to the formation of phthalimides and benzylideneamines by removal of an H atom from the N atom.

[0154] In some embodiments of the compound of Formula (III), the residue R 3 represents a solid support or a hydroxyl protecting group, and the solid support is optionally linked via a spacer.

[0155] In some embodiments of the compound of Formula (III), the residue R 3 represents a hydroxyl-protecting group, and the hydroxyl-protecting group is preferably a hydroxyl-protecting group selected from the above X residue, Y residue, and R 1 The hydroxyl protecting group is defined for the residue.

[0156] In some specific embodiments of the compound of formula (III), the residue R 2 represents an amino protecting group, preferably 9-fluorenylmethylcarbamate (Fmoc), and / or a residue R 3 represents an H atom. In one specific embodiment, the compound of formula (III) has the structure of formula (IIIa): [ka]

[0157] In some specific embodiments of the compound of formula (III), the residue R 2 represents an H atom and / or a residue R 3 represents a solid support, which is optionally linked via a spacer. In some more specific embodiments, the compound of formula (III) has the structure of any one of formulas (IIIb1) to (IIIb5). [ka] , [ka] , [ka] , [ka] , [ka] During the ceremony, R represents a solid support, an amino protecting group, or an H atom, the solid support optionally being linked via a spacer; R 6 represents a hydroxyl-protecting group, preferably DMT or MMT, more preferably MMT. In some specific embodiments, the compound of formula (III) has the structure of any one of formulas (IIIb3) or (IIIb4) defined above. In specific embodiments of compounds of Formulae (IIIb1)-(IIIb5), the R residue is a solid support, optionally linked via a spacer as defined above for solid supports (e.g., as defined for the Y residue).

[0158] Processes used to prepare GalNAc compounds In one aspect, the invention relates to a process for preparing a compound having the structure of formula (0) in any of the above embodiments, such as compound (0a). In some embodiments, the process is for preparing a compound having the structure of formula (I) and / or (II) in any of the above embodiments, such as compound (Ia) and / or (IIa).

[0159] The process comprises reacting a compound having the structure of formula (III) as defined above, which is highly suitable as an intermediate compound for preparing the GalNAc compounds of the present invention.

[0160] In some embodiments of the process, the process comprises reacting a compound having a structure of formula (IV): [ka] During the ceremony, m is an integer as defined above, R 4 teeth, [ka] represents R 5 represents a hydroxyl-protecting group, preferably a benzyl group.

[0161] The compound of formula (IV) has been found to be highly suitable as yet another intermediate compound for preparing the GalNAc compounds of the present invention, providing high yields of the GalNAc compounds.

[0162] In some embodiments, the process comprises: a) providing a compound having the structure of formula (III), [ka] During the ceremony, k is an integer as defined above, R 2 represents a H atom, R 3 is a solid support, a hydroxyl protecting group, or an H atom, the solid support optionally being linked via a spacer; R 6 represents a hydroxyl protecting group, preferably DMT or MMT, more preferably MMT; b) providing a compound having the structure of formula (V), [ka] During the ceremony, m and n are integers as defined above; R 1 is independently a hydroxyl protecting group, e.g., selected from those defined above, preferably an acetyl group; c) reacting a compound of formula (III) as shown in step a) with a compound of formula (V) as shown in step b) to obtain a compound of formula (0) or a compound of formula (II) in any of the above-defined embodiments, wherein Y is an H atom, a hydroxyl protecting group, or a solid support, optionally linked via a spacer, and X is a hydroxyl protecting group, preferably DMT or MMT, more preferably MMT. Includes:

[0163] In some embodiments, the process further comprises the step of: d) reacting the compound obtained in step c) to obtain a compound of formula (0), or to obtain a compound of formula (I) in any of the embodiments defined above, wherein Y is a payload, and X and R 1 are each an H atom. This step may involve further reaction of the compound obtained in step c) for direct addition of a payload or payload precursor, and / or addition of an optional linker and solid support. The payload precursor may be any compound that leads to the addition of a payload to the (intermediate) GalNAc compound. Due to the symmetry of the -CH2OX and -CH2OY groups, the formula includes these groups regardless of their preparation process. However, it should be understood that when a solid support is present as the Y residue, the payload will be added to another residue, i.e., the X residue.

[0164] In these embodiments, the residue R 3 is preferably as defined above, including the definitions of solid support and hydroxyl protecting groups for residue Y above. Preferably, residue R of compound of formula (0) 3 is the residue R of the intermediate compound (III) 3 or is an H atom. More preferably, R 3 is R 7 and represents a moiety of any one of formulas (VIa1) to (VIa5).

[0165] Preferably, compound (V) is prepared by using compound (IV), which is further subjected to reaction with a compound of formula (III) as shown in step a) of the process.

[0166] In specific embodiments, a compound of formula (II) or (IIa) is prepared in step c) and may be further reacted to a payload-containing compound, such as a compound of formula (I) or (Ia).

[0167] In some embodiments of the process, the compound of formula (III) comprises a solid support, i.e., the residue R 3 represents a solid support, which is optionally linked via a spacer. Thus, compound (V) can be directly reacted with the solid support-containing compound (III), and a payload can be added in step d) by directly reacting with compound (III) in step c). In particular, this embodiment of the process is well suited for preparing GalNAc compound conjugates with oligonucleotides, such as those of formula (I) or (Ia).

[0168] In a preferred embodiment, in step d) of the process, the compound of formula (I) is formed by adding modified and / or unmodified nucleotides using standard phosphoramidite chemistry and cleaving the GalNAc compound conjugate from the solid support (as described in the Examples section herein), thus readily providing a GalNAc compound conjugate bearing an oligonucleotide.

[0169] In some embodiments, in step d) of the process, a compound of formula (0) wherein Y is a hydroxyl protecting group, an H atom, or a solid support, and wherein the solid support is optionally linked via a spacer, is reacted with a payload or payload precursor to obtain a compound of formula (0) wherein Y is a payload and X is an H atom.

[0170] Applications of GalNAc compound conjugates In one aspect, the present invention relates to a compound having the structure of formula (0) as defined above, wherein residues X and R are each independently a substituted or unsubstituted aryl group, for use in a method of treating a subject having a disease or disorder or diagnosing a subject. 1 are each an H atom, residue Y is a payload, and the subject comprises cells expressing an asialoglycoprotein receptor (ASGPR). The payload can be any of the payloads of the above embodiments.

[0171] In some embodiments, the compound for use has the structure of Formula (I). In some preferred embodiments, the compound for use has the structure of Formula (Ia). In one embodiment, the modified and / or unmodified oligonucleotide in Formula (I) or (Ia) binds to a target nucleotide sequence in a hepatocyte and modifies the expression of the gene encoded by the target nucleotide sequence. Modulation of expression is herein understood to alter expression by increasing or decreasing the expression rate. In some preferred embodiments, the modified and / or unmodified oligonucleotide inhibits the expression of the gene encoded by the target nucleotide sequence.

[0172] In one aspect, the present invention relates to a method for delivering a payload into a cell expressing an asialoglycoprotein receptor (ASGPR), the method comprising administering a compound having the structure of formula (0) as defined above, wherein residues X and R are fused to each other. 1 are H atoms, residue Y is a payload, and the payload is delivered into cells expressing asialoglycoprotein receptor (ASGPR) in the subject in an amount sufficient to treat the subject with the payload. The payload is any of the payloads of the above embodiments.

[0173] In some embodiments of the method, the compound has a structure of Formula (I). In some preferred embodiments of the method, the compound has a structure of Formula (Ia). In some embodiments of the method, the modified and / or unmodified oligonucleotide of Formula (I) or (Ia) binds to a target nucleotide sequence in a hepatocyte and modifies the expression of a gene encoded by the target nucleotide sequence. Modification of expression is herein understood to mean altering expression by increasing or decreasing the expression rate. In some preferred embodiments of the method, the modified and / or unmodified oligonucleotide inhibits the expression of a gene encoded by the target nucleotide sequence. The compound may be administered in any manner suitable for administration to a subject. In some preferred embodiments of the method, the compound is administered to a subject subcutaneously or intravenously.

[0174] In one aspect, the present invention relates to a pharmaceutical composition comprising a compound having the structure of formula (0) as defined above, and a pharmaceutically acceptable carrier, excipient, and / or diluent, wherein residues X and R 1 are H atoms and residue Y is a payload. The payload can be any of the payloads in the above embodiments.

[0175] In some embodiments of the pharmaceutical composition, the compound has a structure of Formula (I). In some preferred embodiments of the pharmaceutical composition, the compound has a structure of Formula (Ia). In some embodiments of the pharmaceutical composition, the modified and / or unmodified oligonucleotide in Formula (I) or (Ia) binds to a target nucleotide sequence in a hepatocyte and modifies the expression of a gene encoded by the target nucleotide sequence. Modulation of expression is herein understood to mean altering expression by increasing or decreasing the expression rate. In some preferred embodiments of the pharmaceutical composition, the modified and / or unmodified oligonucleotide inhibits the expression of a gene encoded by the target nucleotide sequence. The pharmaceutical composition may be administered in any form suitable for administration to a subject. In some preferred embodiments of the pharmaceutical composition, the pharmaceutical composition is suitable for subcutaneous or intravenous administration to a subject. kit

[0176] In some embodiments, the compounds and pharmaceutical compositions of the present disclosure are provided in kits. In various aspects, the kits include the compounds as unit doses. For purposes herein, a "unit dose" refers to a discrete amount dispersed in a suitable carrier. In various aspects, a unit dose is an amount sufficient to provide a desired effect in a subject, e.g., reduction of target gene expression. Thus, kits are provided herein that include the compounds of the present disclosure, optionally provided in unit doses. In various aspects, the kits include several unit doses, e.g., a weekly or monthly supply, optionally each individually packaged or otherwise divided into other unit doses. In some embodiments, the components of the kit / unit dose are packaged with instructions for administration to a patient. In some embodiments, the kits include one or more devices for administration to a patient, such as a needle and / or a syringe. In some aspects, the compounds of the present disclosure, or pharmaceutically acceptable salts thereof, are pre-packaged in a ready-to-use form, e.g., a syringe and / or an intravenous bag. In some embodiments, the kit further comprises other therapeutic or diagnostic agents, including those described herein, or a pharmaceutically acceptable carrier (e.g., a solvent, buffer, and / or diluent, etc.) In specific embodiments, the kit comprises a compound of the present disclosure along with an agent, e.g., a therapeutic agent.

[0177] Specific Embodiments Embodiment 1 of the present invention includes compounds having the structure of formula (0): [ka] During the ceremony, k is an integer from 1 to 5, m is an integer from 0 to 11, n is an integer from 0 to 5, X is a hydroxyl protecting group or an H atom; Y is a hydroxyl protecting group, an H atom, a payload, or a solid support, the payload or solid support optionally being linked via a spacer; R 1 are independently selected from a hydroxyl protecting group and an H atom.

[0178] Embodiment 2 of the present invention includes compounds according to embodiment 1, wherein Y is a payload, which comprises an oligomeric compound such as an oligonucleotide, a peptide, an antibody, an antibody fragment, or a chemical compound having pharmaceutical activity.

[0179] Embodiment 3 of the present invention includes compounds according to embodiment 1 or 2, wherein X and R 1 represents a H atom.

[0180] Embodiment 4 of the invention includes a compound according to any one of the preceding embodiments, wherein the compound has the structure of formula (I): [ka] During the ceremony, k is an integer from 1 to 5, m is an integer from 0 to 11, n is an integer from 0 to 5, Oligo refers to modified and / or unmodified oligonucleotides.

[0181] Embodiment 5 of the invention comprises compounds according to embodiment 4, wherein the modified and / or unmodified oligonucleotide is a single-stranded or double-stranded modified and / or unmodified oligonucleotide.

[0182] Embodiment 6 of the invention includes compounds according to embodiment 4, wherein the modified and / or unmodified oligonucleotide is a single-stranded oligonucleotide, a microRNA (miRNA), or a single-stranded RNA (ssRNA).

[0183] Embodiment 7 of the present invention includes compounds according to embodiment 4, wherein the modified and / or unmodified oligonucleotide is a double-stranded oligonucleotide, and is a short hairpin RNA (shRNA) or a small interfering RNA (siRNA).

[0184] Embodiment 8 of the invention includes compounds according to any one of embodiments 4 to 7, wherein the modified and / or unmodified oligonucleotide is attached via the 3' end of the sense strand.

[0185] Embodiment 9 of the present invention includes compounds according to any one of embodiments 4 to 8, wherein the modified and / or unmodified oligonucleotide binds to a target nucleotide sequence and alters expression of a gene encoded by the target nucleotide sequence.

[0186] Embodiment 10 of the invention includes compounds according to embodiment 9, wherein the modified and / or unmodified oligonucleotide inhibits expression of a gene encoded by a target nucleotide sequence, preferably by inhibiting transcription or translation of the target nucleotide sequence.

[0187] Embodiment 11 of the invention includes compounds according to embodiment 10, wherein the modified and / or unmodified oligonucleotide inhibits expression of the gene by at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%.

[0188] Embodiment 12 of the present invention includes compounds according to any one of embodiments 4 to 11, wherein the target nucleotide sequence of the modified and / or unmodified oligonucleotide is messenger RNA (mRNA).

[0189] Embodiment 13 of the invention includes compounds according to any one of embodiments 4 to 12, wherein the target nucleotide sequence of the modified and / or unmodified oligonucleotide encodes a gene expressed in the liver that is associated with a disease or a gene associated with a liver disorder, preferably a metabolic liver disorder.

[0190] Embodiment 14 of the invention includes compounds according to any one of embodiments 4 to 13, wherein the oligonucleotide is a modified oligonucleotide modified to make it resistant to degradation, to reduce toxicity, and / or to enhance activity.

[0191] Embodiment 15 of the present invention includes compounds according to embodiment 1 having the structure of formula (II): [ka] During the ceremony, k is an integer from 1 to 5, m is an integer from 0 to 11, n is an integer from 0 to 5, R 1 are independently selected from a hydroxyl protecting group and an H atom, preferably an acetyl group; R 6 represents a hydroxyl-protecting group, preferably DMT or MMT, more preferably MMT; R 7 represents the moiety of formula (VI), [ka] During the ceremony, R represents a solid support, an amino protecting group, a hydroxyl protecting group, or an H atom, the solid support optionally being linked via a spacer; Z represents NH or O; R 8 represents a straight or branched alkylene having from 1 to 8 carbon atoms, optionally in which one or more carbon atoms are replaced by O, S, NH and / or N—(C1-C3 alkyl), and / or R 8 is a cycloalkylene having from 5 to 8 carbon atoms, preferably ethylene, 1,1 dimethylethylene, propylene, n-butylene, or 1,2 cyclohexylene.

[0192] Embodiment 16 of the invention includes a compound according to any one of the preceding embodiments, During the ceremony, k is 2 or 3; m is 7, 8, or 9; n is 2 or 3.

[0193] Embodiment 17 of the invention includes compounds according to any one of the preceding embodiments, wherein the sum of k and m is an integer from 8 to 12, preferably 10. Embodiment 18 of the invention includes compounds according to any one of the preceding embodiments, wherein the compound has the structure of formula (0a), (Ia), or (IIa): [ka] [ka] [ka] In the formula, X, Y, oligo, R 1 , R 6 , and R 7 is as defined in the previous embodiment.

[0194] Embodiment 19 of the present invention includes a compound of the structure of formula (III): [ka] During the ceremony, k is an integer from 1 to 5, R 2 represents an amino protecting group or an H atom, R 3 represents a solid support, a hydroxyl protecting group, or an H atom, the solid support being optionally linked via a spacer; R 6 represents a hydroxyl-protecting group, preferably DMT or MMT, more preferably MMT.

[0195] Embodiment 20 of the present invention includes compounds according to embodiment 19, wherein k is 2 or 3, preferably 3.

[0196] Embodiment 21 of the present invention includes compounds according to embodiment 19 or 20, wherein R 2 represents an amino protecting group, preferably a fluorenylmethoxycarbonyl group (Fmoc), and / or R 3 represents a H atom.

[0197] Embodiment 22 of the present invention includes compounds according to any one of embodiments 19-21, wherein the compound has the structure of formula (IIIa): [ka] During the ceremony, R 6 represents a hydroxyl-protecting group, preferably DMT or MMT, more preferably MMT.

[0198] Embodiment 23 of the present invention refers to a compound according to embodiment 19 or 20, wherein R 2 represents a H atom and / or R 3 represents a solid support, which is optionally linked via a spacer.

[0199] Embodiment 24 of the present invention refers to a compound according to any one of embodiments 19, 20, and 23, having the structure of any one of formulas (IIIb1)-(IIIb5): [ka] , [ka] , [ka] , [ka] , and [ka] During the ceremony, R represents a solid support, an amino protecting group, or an H atom, the solid support optionally being linked via a spacer; R 6 represents a hydroxyl-protecting group, preferably DMT or MMT, more preferably MMT.

[0200] Embodiment 25 of the present invention includes a process for preparing a compound having the structure of formula (0): [ka] During the ceremony, k is an integer from 1 to 5, m is an integer from 0 to 11, n is an integer from 0 to 5, X is a hydroxyl protecting group or an H atom; Y is a hydroxyl protecting group, an H atom, a payload, or a solid support, the payload or solid support optionally being linked via a spacer; R 1 are independently selected from a hydroxyl protecting group and an H atom; The method comprises reacting a compound having the structure of formula (III) to compound (V), for example, by standard amide coupling: [ka] During the ceremony, k is an integer from 1 to 5, R 2 represents an amino protecting group or an H atom, R 3 represents a solid support, a hydroxyl protecting group, or an H atom, said solid support being optionally linked via a spacer; R 6 represents a hydroxyl protecting group, preferably dimethoxytriyl (DMT) or monomethoxytrityl (MMT), more preferably MMT.

[0201] Embodiment 26 of the invention includes the process according to embodiment 25, wherein the compound of formula (0) has the structure of formula (I): [ka] During the ceremony, k is an integer from 1 to 5, m is an integer from 0 to 11, n is an integer from 0 to 5, Oligos are modified and / or unmodified oligonucleotides.

[0202] Embodiment 27 of the invention includes the process according to embodiment 25, wherein the compound of formula (0) has the structure of formula (II): [ka] During the ceremony, k is an integer from 1 to 5, m is an integer from 0 to 11, n is an integer from 0 to 5, R 1 are independently selected from a hydroxyl protecting group and an H atom, preferably an acetyl group; R 6 represents a hydroxyl protecting group, preferably MMT; R 7 represents the moiety of formula (VI), [ka] During the ceremony, R represents a solid support, an amino protecting group, a hydroxyl protecting group, or an H atom, the solid support optionally being linked via a spacer; Z represents NH or O; R 8represents a straight or branched alkylene having from 1 to 8 carbon atoms, optionally in which one or more carbon atoms are replaced by O, S, NH and / or N—(C1-C3 alkyl), and / or R 8 is a cycloalkylene having from 5 to 8 carbon atoms, preferably ethylene, 1,1 dimethylethylene, propylene, n-butylene, or 1,2 cyclohexylene.

[0203] Embodiment 28 of the present invention includes processes according to embodiments 25-27, comprising reacting a compound having the structure of formula (IV): [ka] During the ceremony, m is an integer from 0 to 11, preferably 7; R 4 teeth, [ka] represents R 5 represents a hydroxyl-protecting group, preferably a benzyl group.

[0204] Embodiment 29 of the present invention includes the process according to any one of embodiments 25 to 28, further comprising: a) providing a compound having the structure of formula (III), [ka] During the ceremony, k is an integer from 1 to 5, preferably 3; R 2 represents a H atom, R 3 represents a solid support, a hydroxyl protecting group, or an H atom, the solid support being optionally linked via a spacer; R 6 represents a hydroxyl protecting group, preferably DMT or MMT, more preferably MMT; b) providing a compound having the structure of formula (V), [ka] During the ceremony, m is an integer from 0 to 11, preferably 7; n is an integer from 0 to 5, preferably 3; R 1 are independently selected from hydroxyl protecting groups, preferably acetyl groups; c) reacting a compound of formula (III) as shown in step a) with a compound of formula (V) as shown in step b) to obtain a compound of formula (0) or to obtain a compound of formula (II), wherein Y is an H atom, a hydroxyl protecting group, or a solid support, optionally linked via a spacer, and X is a hydroxyl protecting group, preferably DMT or MMT, more preferably MMT; Includes.

[0205] Embodiment 30 of the present invention includes the process according to embodiment 29, further comprising: d) reacting the compound obtained in step c) to obtain a compound of formula (0) or to obtain a compound of formula (I), wherein Y is a payload, which is optionally linked via a spacer, and X and R 1 are H atoms, respectively, Further includes:

[0206] Embodiment 31 of the present invention includes the process according to any one of embodiments 25 to 30, wherein in step d) of the process, the compound of formula (II) is reacted with at least two modified and / or unmodified nucleotides and / or nucleosides to obtain the compound of formula (I).

[0207] Embodiment 32 of the present invention comprises the process according to any one of embodiments 25 to 30, wherein in step d) of the process, the compound of formula (0), wherein Y is a hydroxyl protecting group, an H atom, or a solid support, and wherein the solid support is optionally linked via a spacer, is further reacted with a payload or payload-precursor to obtain a compound of formula (0), wherein Y is a payload, and wherein the payload is optionally linked via a spacer.

[0208] Embodiment 33 of the present invention comprises the process according to embodiment 32, wherein the payload comprises an oligomeric compound, such as an oligonucleotide, a peptide, an antibody, an antibody fragment, or a chemical compound having pharmaceutical activity.

[0209] Embodiment 34 of the present invention includes the process according to any one of embodiments 25 to 33, wherein step b) of the process comprises reacting a compound having the structure of formula (IV): [ka] During the ceremony, m is an integer from 0 to 11, preferably 7; R 4 teeth, [ka] represents R 5 represents a hydroxyl-protecting group, preferably a benzyl group.

[0210] Embodiment 35 of the present invention includes a method for preparing a compound having the structure of formula (0): [ka] During the ceremony, k is an integer from 1 to 5, m is an integer from 0 to 11, n is an integer from 0 to 5, X is a hydroxyl protecting group or an H atom; Y is a hydroxyl protecting group, an H atom, a payload, or a solid support, wherein the payload or solid support is optionally linked via a spacer; R 1 are independently selected from a hydroxyl protecting group and an H atom; The method includes reacting a compound having the structure of formula (III) with a compound having the structure of formula (V), for example, by amide coupling: [ka] During the ceremony, k is an integer from 1 to 5, R 2 represents an amino protecting group or an H atom, R 3 represents a solid support, a hydroxyl protecting group, or an H atom, the solid support being optionally linked via a spacer; R 6 represents a hydroxyl-protecting group, preferably DMT or MMT, more preferably MMT; wherein the compound having the structure of formula (V) is [ka] is.

[0211] Embodiment 36 of the invention includes the method according to embodiment 35, wherein the compound of formula (0) has the structure of formula (I): [ka] During the ceremony, k is an integer from 1 to 5, m is an integer from 0 to 11, n is an integer from 0 to 5, Oligos are modified and / or unmodified oligonucleotides.

[0212] Embodiment 37 of the invention includes the method according to embodiment 35, wherein the compound of formula (0) has the structure of formula (II): [ka] During the ceremony, k is an integer from 1 to 5, m is an integer from 0 to 11, n is an integer from 0 to 5, R 1 are independently selected from a hydroxyl protecting group and an H atom, preferably an acetyl group; R 6 represents a hydroxyl protecting group, preferably MMT; R 7 represents the moiety of formula (VI), [ka] During the ceremony, R represents a solid support, an amino protecting group, a hydroxyl protecting group, or an H atom, the solid support optionally being linked via a spacer; Z represents NH or O; R 8 represents a straight or branched alkylene having from 1 to 8 carbon atoms, optionally in which one or more carbon atoms are replaced by O, S, NH and / or N—(C1-C3 alkyl), and / or R 8 is a cycloalkylene having from 5 to 8 carbon atoms, preferably ethylene, 1,1 dimethylethylene, propylene, n-butylene, or 1,2 cyclohexylene.

[0213] Embodiment 38 of the present invention includes the method according to any one of embodiments 35-37, comprising reacting a compound having the structure of formula (IV): [ka] During the ceremony, m is an integer from 0 to 11, preferably 7; R 4 teeth, [ka] represents R 5 represents a hydroxyl-protecting group, preferably a benzyl group.

[0214] Embodiment 39 of the present invention includes the method according to any one of embodiments 35 to 38, further comprising: a) providing a compound having the structure of formula (III), [ka] During the ceremony, k is an integer from 1 to 5, preferably 3; R 2 is a H atom, R 3 represents a solid support, a hydroxyl protecting group, or an H atom, the solid support being optionally linked via a spacer; R 6 represents a hydroxyl protecting group, preferably DMT or MMT, more preferably MMT; b) providing a compound having the structure of formula (V), [ka] During the ceremony, m is an integer from 0 to 11, preferably 7; n is an integer from 0 to 5, preferably 3; R 1 are independently selected from hydroxyl protecting groups, preferably acetyl groups; c) reacting a compound of formula (III) as shown in step a) with a compound of formula (V) as shown in step b) to obtain a compound of formula (0) or to obtain a compound of formula (II), wherein Y is an H atom, a hydroxyl protecting group, or a solid support, optionally linked via a spacer, and X is a hydroxyl protecting group, preferably DMT or MMT, more preferably MMT; Includes.

[0215] Embodiment 40 of the present invention includes the method according to embodiment 39, further comprising: d) reacting the compound obtained in step c) to obtain a compound of formula (0) or to obtain a compound of formula (I), wherein Y is a payload, the payload optionally being linked via a spacer, and X and R 1 are H atoms, and steps Further includes:

[0216] Embodiment 41 of the invention includes the method according to any one of embodiments 35 to 40, wherein in step d) of the process, the compound of formula (II) is reacted with at least two modified and / or unmodified nucleotides and / or nucleosides to obtain the compound of formula (I).

[0217] Embodiment 42 of the present invention comprises the method according to any one of embodiments 35 to 40, wherein in step d) of the process, the compound of formula (0), wherein Y is a hydroxyl protecting group, an H atom, or a solid support, and wherein the solid support is optionally linked via a spacer, is further reacted with a payload or payload-precursor to obtain a compound of formula (0), wherein Y is a payload, and wherein the payload is optionally linked via a spacer.

[0218] Embodiment 43 of the present invention includes the method according to embodiment 42, wherein the payload comprises an oligomeric compound, such as an oligonucleotide, a peptide, an antibody, an antibody fragment, or a chemical compound having pharmaceutical activity.

[0219] Embodiment 44 of the invention includes methods according to any one of embodiments 35-43, wherein step b) of the process comprises reacting a compound having the structure of formula (IV): [ka] During the ceremony, m is an integer from 0 to 11, preferably 7; R4 teeth, [ka] represents R 5 represents a hydroxyl-protecting group, preferably a benzyl group.

[0220] Embodiment 45 of the invention includes a compound having the structure of Formula (0) for use in a method of treating a subject having a disease or disorder or diagnosing a subject, [ka] During the ceremony, k is an integer from 1 to 5, preferably 3; m is an integer from 0 to 11, preferably 7; n is an integer from 0 to 5, preferably 3; X is a H atom, Y is a payload, optionally linked via a spacer; R 1 is a H atom, The subject comprises a cell expressing an asialoglycoprotein receptor (ASGPR).

[0221] Embodiment 46 of the invention includes compounds for use according to embodiment 45, wherein the payload comprises an oligomeric compound, such as an oligonucleotide, a peptide, an antibody, an antibody fragment, or a chemical compound having pharmaceutical activity.

[0222] Embodiment 47 of the invention includes a compound for use according to embodiment 45 or 46, wherein the compound has the structure of formula (I): [ka] During the ceremony, k is an integer from 1 to 5, m is an integer from 0 to 11, n is an integer from 0 to 5, Oligos are modified and / or unmodified oligonucleotides.

[0223] Embodiment 48 of the invention comprises compounds for use according to embodiment 47, wherein the modified and / or unmodified oligonucleotide is a single-stranded or double-stranded modified and / or unmodified oligonucleotide.

[0224] Embodiment 49 of the invention includes compounds for use according to embodiment 47, wherein the modified and / or unmodified oligonucleotide is a single-stranded oligonucleotide, a microRNA (miRNA), or a single-stranded RNA (ssRNA).

[0225] Embodiment 50 of the present invention includes a compound for use according to embodiment 47, wherein the modified and / or unmodified oligonucleotide is a double-stranded oligonucleotide, and is a short hairpin RNA (shRNA) or a small interfering RNA (siRNA).

[0226] Embodiment 51 of the invention includes compounds for use according to any one of embodiments 47 to 50, wherein the modified and / or unmodified oligonucleotide is attached via the 3' end of the sense strand.

[0227] Embodiment 52 of the invention includes a compound for use according to any one of embodiments 47 to 51, wherein the modified and / or unmodified oligonucleotide binds to a target nucleotide sequence and alters expression of a gene encoded by the target nucleotide sequence.

[0228] Embodiment 53 of the invention includes compounds for use according to embodiment 52, wherein the modified and / or unmodified oligonucleotide inhibits expression of a gene encoded by a target nucleotide sequence, preferably by inhibiting transcription or translation of the target nucleotide sequence.

[0229] Embodiment 54 of the invention includes compounds for use according to embodiment 53, wherein the modified and / or unmodified oligonucleotide inhibits expression of the gene by at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%.

[0230] Embodiment 55 of the invention includes compounds for use according to any one of embodiments 47 to 54, wherein the target nucleotide sequence of the modified and / or unmodified oligonucleotide is messenger RNA (mRNA).

[0231] Embodiment 56 of the invention includes compounds for use according to any one of embodiments 47 to 55, wherein the target nucleotide sequence of the modified and / or unmodified oligonucleotide encodes a gene associated with a disease expressed in the liver or a gene associated with a liver disorder, preferably a metabolic liver disorder.

[0232] Embodiment 57 of the invention includes a compound for use according to any one of embodiments 47 to 56, wherein the oligonucleotide is a modified oligonucleotide modified to resist degradation, reduce toxicity and / or enhance activity.

[0233] Embodiment 58 of the present invention includes compounds for use according to any one of embodiments 47 to 57, wherein: k is 2 or 3; m is 7, 8, or 9; n is 2 or 3.

[0234] Embodiment 59 of the present invention includes compounds according to any one of embodiments 47-58, wherein the sum of k and m is an integer from 8 to 12, preferably 10.

[0235] Embodiment 60 of the invention includes a compound for use according to any one of embodiments 47-59, wherein the compound has the structure of Formula (Ia). [ka]

[0236] Embodiment 61 of the present invention includes a method for delivering a payload into a cell expressing an asialoglycoprotein receptor (ASGPR), the method comprising administering a compound having the structure of formula (0): [ka] During the ceremony, k is an integer from 1 to 5, m is an integer from 0 to 11, n is an integer from 0 to 5, X is a H atom, Y is a payload, optionally linked via a spacer; R 1 is a H atom, wherein the payload is delivered into cells expressing asialoglycoprotein receptor (ASGPR) in the subject in an amount sufficient to treat the subject with the payload.

[0237] Embodiment 62 of the present invention includes the method according to embodiment 61, wherein the payload comprises an oligomeric compound, such as an oligonucleotide, a peptide, an antibody, an antibody fragment, or a chemical compound having pharmaceutical activity.

[0238] Embodiment 63 of the invention includes methods according to embodiment 61 or 62, wherein the compound comprises the structure of formula (I): [ka] During the ceremony, k is an integer from 1 to 5, m is an integer from 0 to 11, n is an integer from 0 to 5, Oligos are modified and / or unmodified oligonucleotides.

[0239] Embodiment 64 of the present invention comprises the method according to embodiment 63, wherein the modified and / or unmodified oligonucleotide is a single-stranded or double-stranded modified and / or unmodified oligonucleotide.

[0240] Embodiment 65 of the present invention comprises the method according to embodiment 63, wherein the modified and / or unmodified oligonucleotide is a single-stranded oligonucleotide, a microRNA (miRNA), or a single-stranded RNA (ssRNA).

[0241] Embodiment 66 of the present invention includes the method according to embodiment 63, wherein the modified and / or unmodified oligonucleotide is a double-stranded oligonucleotide, and is a short hairpin RNA (shRNA) or a small interfering RNA (siRNA).

[0242] Embodiment 67 of the present invention comprises the method according to any one of embodiments 63 to 66, wherein the modified and / or unmodified oligonucleotide is attached via the 3' end of the sense strand.

[0243] Embodiment 68 of the present invention includes methods according to any one of embodiments 63 to 67, wherein the modified and / or unmodified oligonucleotide binds to a target nucleotide sequence and alters expression of a gene encoded by the target nucleotide sequence.

[0244] Embodiment 69 of the present invention includes methods according to embodiment 68, wherein the modified and / or unmodified oligonucleotide inhibits expression of a gene encoded by the target nucleotide sequence, preferably by inhibiting transcription or translation of the target nucleotide sequence.

[0245] Embodiment 70 of the invention includes the method according to embodiment 69, wherein the modified and / or unmodified oligonucleotide inhibits expression of the gene by at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%.

[0246] Embodiment 71 of the present invention comprises the method according to any one of embodiments 63 to 70, wherein the target nucleotide sequence of the modified and / or unmodified oligonucleotide is messenger RNA (mRNA).

[0247] Embodiment 72 of the present invention includes methods according to any one of embodiments 63 to 71, wherein the target nucleotide sequence of the modified and / or unmodified oligonucleotide encodes a gene associated with a disease expressed in the liver or a gene associated with a liver disorder, preferably a metabolic liver disorder.

[0248] Embodiment 73 of the invention includes methods according to any one of embodiments 63 to 72, wherein the oligonucleotide is a modified oligonucleotide modified to make it resistant to degradation, to reduce toxicity, and / or to enhance activity.

[0249] Embodiment 74 of the present invention includes the method according to any one of embodiments 63 to 73, wherein: k is 2 or 3; m is 7, 8, or 9; n is 2 or 3.

[0250] Embodiment 75 of the present invention includes the method according to any one of embodiments 63-74, wherein the sum of k and m is an integer from 8 to 12, preferably 10.

[0251] Embodiment 76 of the present invention includes the method according to any one of embodiments 63 to 75, wherein the compound has the structure of Formula (Ia). [ka]

[0252] Embodiment 77 of the present invention includes a compound for use according to any one of embodiments 45 to 60 or for a method according to any one of embodiments 61 to 76, wherein the compound is administered to a subject subcutaneously or intravenously.

[0253] Embodiment 78 of the present invention includes a pharmaceutical composition comprising a compound having the structure of Formula (0) and a pharmaceutically acceptable carrier, excipient, and / or diluent: [ka] During the ceremony, k is an integer from 1 to 5, preferably 3; m is an integer from 0 to 11, preferably 7; n is an integer from 0 to 5, preferably 3; X is a H atom, Y is linked to the payload optionally via a spacer; R 1 is a H atom.

[0254] Embodiment 79 of the invention comprises a pharmaceutical composition according to embodiment 78, wherein the payload comprises an oligomeric compound, such as an oligonucleotide, a peptide, an antibody, an antibody fragment, or a chemical compound having pharmaceutical activity.

[0255] Embodiment 80 of the invention includes a pharmaceutical composition according to embodiment 78 or 79, wherein the compound comprises the structure of formula (I): [ka] During the ceremony, k is an integer from 1 to 5, m is an integer from 0 to 11, n is an integer from 0 to 5, Oligos are modified and / or unmodified oligonucleotides.

[0256] Embodiment 81 of the present invention comprises a pharmaceutical composition according to embodiment 80, wherein the modified and / or unmodified oligonucleotide is a single-stranded or double-stranded modified and / or unmodified oligonucleotide.

[0257] Embodiment 82 of the present invention comprises a pharmaceutical composition according to embodiment 80, wherein the modified and / or unmodified oligonucleotide is a single-stranded oligonucleotide, a microRNA (miRNA), or a single-stranded RNA (ssRNA).

[0258] Embodiment 83 of the present invention comprises a pharmaceutical composition according to embodiment 80, wherein the modified and / or unmodified oligonucleotide is a double-stranded oligonucleotide, and is a short hairpin RNA (shRNA) or a small interfering RNA (siRNA).

[0259] Embodiment 84 of the present invention comprises a pharmaceutical composition according to any one of embodiments 80 to 83, wherein the modified and / or unmodified oligonucleotide is attached via the 3' end of the sense strand.

[0260] Embodiment 85 of the present invention comprises a pharmaceutical composition according to any one of embodiments 80 to 84, wherein the modified and / or unmodified oligonucleotide binds to a target nucleotide sequence and alters expression of a gene encoded by the target nucleotide sequence.

[0261] Embodiment 86 of the invention includes compounds according to embodiment 85, wherein the modified and / or unmodified oligonucleotide inhibits expression of a gene encoded by the target nucleotide sequence, preferably by inhibiting transcription or translation of the target nucleotide sequence.

[0262] Embodiment 87 of the present invention comprises a pharmaceutical composition according to embodiment 86, wherein the modified and / or unmodified oligonucleotide inhibits expression of the gene by at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%.

[0263] Embodiment 88 of the present invention comprises a pharmaceutical composition according to any one of embodiments 80 to 87, wherein the target nucleotide sequence of the modified and / or unmodified oligonucleotide is messenger RNA (mRNA).

[0264] Embodiment 89 of the present invention comprises a pharmaceutical composition according to any one of embodiments 80 to 88, wherein the target nucleotide sequence of the modified and / or unmodified oligonucleotide encodes a gene associated with a disease expressed in the liver or a gene associated with a liver disorder, preferably a metabolic liver disorder.

[0265] Embodiment 90 of the invention comprises a pharmaceutical composition according to any one of embodiments 80 to 89, wherein the oligonucleotide is a modified oligonucleotide modified to make it resistant to degradation, to reduce toxicity, and / or to enhance activity.

[0266] Embodiment 91 of the present invention comprises a pharmaceutical composition according to any one of embodiments 80 to 90, wherein: k is 2 or 3; m is 7, 8, or 9; n is 2 or 3.

[0267] Embodiment 92 of the present invention includes compounds according to any one of embodiments 80-91, wherein the sum of k and m is an integer from 8 to 12, preferably 10.

[0268] Embodiment 93 of the present invention includes a pharmaceutical composition according to any one of embodiments 80-92, wherein the compound has the structure of Formula (Ia). [ka]

[0269] Embodiment 94 of the present invention includes a pharmaceutical composition according to any one of embodiments 80 to 93, wherein the pharmaceutical composition is suitable for subcutaneous or intravenous administration to a subject.

[0270] Example Non-limiting disclosure and incorporation by reference While certain compounds, compositions, and methods described herein have been described with specificity according to certain embodiments, the following examples are intended only to help illustrate, but not to limit, the compounds described herein. Each reference cited in this application is incorporated herein by reference in its entirety.

[0271] Example 1: Preparation of GalNAc Compounds with Solid Support Procedure for the synthesis of ARNATAR GalNAc-linker-solid support Two synthetic procedures for producing trivalent GalNAc solid supports are shown below in Reaction Scheme 1 and Reaction Scheme 2.

[0272] Reaction Scheme 1 [ka] 10-(Benzyloxy)-10-oxodecanoic acid (CAS number: 67852-88-4, compound (2)) (4.80 g / 16.00 mmol) was reacted with tris[[2-(tert-butoxycarbonyl)ethoxy]methyl]methylamine (CAS number: 175724-30-8, compound (1)) (8.00 g / 16.00 mmol) in the presence of DIEA (diisopropylethylamine) and HBTU (hexafluorophosphate benzothiazole tetramethyluronium) in DCM (dichloromethane) for 16 hours at room temperature. Compound (3) was obtained in a 70.4% yield. [ka]

[0273] Compound 3 (9.20 g / 11.80 mmol) was treated with formic acid at 25° C. for 24 hours to remove the tert-butyl protecting group, resulting in an 86.1% yield of compound 4. This tricarboxylate compound 4 (6.90 g / 11.30 mmol) was dissolved in dichloromethane containing EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), DIEA, and HOAT (1-hydroxy-7-azabenzotriazole). 20.00 g / 38.30 mmol) [5-acetamido-3,4-diacetyloxy-6-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]oxan-2-yl]methyl acetate 5 (preparation of compound 5 is shown below—compound 9 in Reaction Scheme 2)—was added to the DCM mixture and stirred at 25° C. for 16 hours. Compound (6) was obtained in 70% yield with a purity of 97.2% by LCMS. [ka]

[0274] Compound (6) was dissolved in THF in the presence of Pd / C catalyst under hydrogen gas (15 psi) for 20 hours to remove the benzyl protecting group, and the crude material (7) was purified by preparative HPLC to give compound (7). [ka]

[0275] Compound 7 (0.5 g / 0.25 mmol) was dissolved in DCM containing DIEA and HBTU. To this mixture was added compound 8 (150.0 mg / 0.30 mmol) (Wuxi AppTec, Shanghai, China) (Dunetz et al., Org. Process Res. Dev. 2016, 20, 2, 140-177). The reaction mixture was stirred at 25 °C for 16 hours. After preparative HPLC purification, compound 9 was obtained with a purity of 96.3% and a yield of 41%. [ka]

[0276] Compound (10) was made by dissolving compound (9) (380 mg / 0.26 mmol) in DCM with the addition of DMAP and an excess of succinic anhydride to provide complete reaction. [ka]

[0277] Compound 10 was coupled to both amino-modified polystyrene beads using HBTU, DMAP, and DIEA in THF. The second step of the reaction was capping any unreacted sites using acetic anhydride with pyridine. The resulting yield was approximately 150 μmol / g. In an alternative step, compound 10 was attached to CPG as a solid support.

[0278] Alternatives in Reaction Scheme 1: The above reaction can be used with slight modifications to arrive at similar results.

[0279] Specifically, for example, compound (8) may contain MMT(r) instead of DMT(r) as the hydroxyl-protecting group, which allows for highly efficient coupling of nucleotides with MMT as the hydroxyl-protecting group.

[0280] Furthermore, other dicarboxylic acid anhydrides may be employed to prepare spacers for connecting solid supports. Examples of very useful dicarboxylic acid anhydrides, such as succinic anhydride, 2,2-dimethylsuccinic anhydride, glutaric anhydride, adipic anhydride, 1,2-cyclohexanedicarboxylic anhydride, acetic ether anhydride, acetic thioether anhydride, and methyliminodiacetic anhydride, as well as their reactions with MMT-containing compound (9) are illustrated below. The resulting variants of compound 10 (compounds 10a-10h) are further reacted with modified compound 11 (not shown) in the same manner as illustrated above. [ka] [ka]

[0281] Reaction Scheme 2 Step 1: Synthesis of activated triester (5) [ka]

[0282] 10-(Benzyloxy)-10-oxodecanoic acid (CAS No.: 67852-88-4, Compound (1)) is reacted with tris[[2-(tert-butoxycarbonyl)ethoxy]methyl]methylamine (CAS No.: 175724-30-8, Compound (2)) to give Compound (3). Compound (4) is then reacted with pentafluorophenyl trifluoroacetate (CAS No.: 14533-84-7) to give the activated trivalent ester (5).

[0283] Step 2: Synthesis of the amine GalNAc arm (9) [ka]

[0284] 1-Azido-3,6,9-trioxaundecan-11-ol (CAS number: 86770-67-4, compound (6)) is reacted with (2S,3R,4R,5R,6R)-3-acetamido-6-(acetoxymethyl)tetrahydro-2H-pyran-2,4,5-triyl triacetate (CAS number: 3006-60-8, compound (7)) to give compound (8), followed by compound (9).

[0285] Step 3: Coupling of (5) with (9) to generate the triGalNAc carboxylic acid moiety followed by reduction of the benzyl group: [ka] The compounds obtained in steps 1 and 2 were reacted in a 3:1 ratio of compound (5):compound (9) to give compound (10), which was then treated as above to give compound (11).

[0286] Step 4: Synthesis of linker support for conjugation with (11): [ka] Compound 12 (Wuxi AppTec, Shanghai, China) was reacted with succinic anhydride to give compound 13, which contains a succinic acid spacer. Compound 13 is then attached to a solid support, which can be any solid support known in the art for coupling reactions, such as CPG (optionally containing an LCAA spacer) and polystyrene solid supports.

[0287] Step 5: Coupling reaction of trivalent GalNAc (11) with linker-modified support (14) [ka] The compounds obtained in steps 3 and 4 were reacted in a 1:1 ratio of compound (11):compound (14) to obtain compound (15).

[0288] Alternatives in Reaction Scheme 2: The above reaction can be used with slight modifications to arrive at similar results.

[0289] Specifically, for example, compound (12) may contain MMT(r) instead of DMT(r) as the hydroxyl-protecting group, and highly efficient coupling of nucleotides with MMT as the hydroxyl-protecting group can be achieved.

[0290] Furthermore, other dicarboxylic acid anhydrides may be employed to prepare spacers for connecting solid supports. Examples of very useful dicarboxylic acid anhydrides include succinic anhydride, 2,2 dimethylsuccinic anhydride, glutaric anhydride, adipic anhydride, 1,2 cyclohexanedicarboxylic anhydride, acetic ether anhydride, acetic thioether anhydride, and methyliminodiacetic anhydride, and their reaction with MMT-containing compounds (12) leads to modified compounds (15) containing spacers derived from these acids, respectively.

[0291] Example 2: Preparation of GalNAc Compound Conjugates Synthesis of oligomeric compounds on ARNATAR GalNAc supports Oligonucleotides were added to the ARNATAR GalNAc compound having the structure (11), obtained as described in Reaction Scheme 1 and equipped with a CPG(500 Å)-LCAA solid support.

[0292] As a comparative example, the GalNAc compound described by Sharma et al. (2018, Bioconjugate Chem, 29: 2478-2488, compound 8 in Scheme 1, Gene Link, Elmsford, NY, USA or Primetech, ALC., min. k, Belarus, as "GalNAc TEG CPG" (1000 Å)) was conjugated to the same oligonucleotide.

[0293] Solid-phase synthesis of oligonucleotides was performed on a MerMade™ 48x synthesizer (BioAutomation, LGC, Biosearch Technologies, Hoddesdon, UK), which can generate up to 48 1- or 5-µmole-scale oligonucleotides per run using standard phosphoramidite chemistry. The solid support was either controlled pore glass (500A, WuxiAppTec, Shanghai, China) loaded with 3'-GalNAc conjugates or a general-purpose solid support (AM Chemicals, Vista, CA, USA). Accompanying synthesis reagents and standard 2′-cyanoethyl phosphoramidite monomers (2′-fluoro, 2′- O -methyl, RNA, DNA) were obtained from various sources (Hongene Biotech, Shanghai, China; Sigma-Aldrich, St. Louis, MO, USA; Glen Research, Stirling, VA, USA; ThermoFisher Scientific, Waltham, MA, USA; LGC Biosearch Technologies, Hoddesdon, UK).

[0294] Phosphoramidite mixtures were prepared in anhydrous acetonitrile or 30% DMF:acetonitrile and coupled with 0.25 M 4,5-dicyanoimidazole (DCI) (Sigma-Aldrich, St. Louis, MO, USA) for coupling times ranging from 120 to 360 s. Standard phosphodiester coupling was achieved using a mixture of tetrahydrofuran (THF), pyridine, and 0.02 M iodine in water.

[0295] Phosphothioate bonds were generated using 0.05 M sulfurization reagent II (3-((dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-3-thione, DDTT) (40:60, pyridine / acetonitrile) (LGC Biosearch Technologies, Hoddesdon, UK) with an oxidation time of 6 min. The dimethoxytrityl (DMT) protecting groups were removed to synthesize the entire sequence.

[0296] Upon completion of solid-phase synthesis, the oligonucleotides were cleaved from the solid support and deprotected from base-labile groups by incubation in ammonium hydroxide at 55°C for 6 hours. The ammonium hydroxide was removed using a centrifugal vacuum concentrator and the mixture was dried at room temperature. For sequences containing natural ribonucleotides (2'-OH) protected with tert-butyldimethylsilyl (TBDMS), a second deprotection was performed using triethylamine:trihydrofluoride (TEA:3HF). For each TBDMS-protected oligonucleotide, 100 μL of DMSO and 125 μL of TEA:3HF were added and incubated at 65°C for 2.5 hours. After incubation, 25 μL of 3 M sodium acetate was added to the solution, followed by precipitation in butanol at -20°C for 30 minutes. The cloudy solution was centrifuged to form a cake, and the supernatant was carefully decanted by pipette. A standard precipitation process was then completed using 75% ethanol:water as the supernatant solution, followed by 100% ethanol. The oligonucleotide cake was dried in a centrifugal vacuum concentrator for 30 minutes.

[0297] After precipitation with 3 M sodium acetate and subsequent elution on a G25 Sephadex® column (Sigma-Aldrich, St. Louis, MO, USA), desalting without HPLC purification was performed. Oligonucleotide purification was achieved by anion-exchange chromatography on a Gilson GX271 preparative HPLC system (Middleton, WI, USA) using BioWorks Q40 resin (Uppsala, Sweden). Final desalting was performed on a Sephadex® G25 column. All oligonucleotides were analyzed for purity by ion-pairing reversed-phase HPLC using an Agilent 1200 analytical HPLC (Santa Clara, CA, USA), for intact mass by negative ion mass spectrometry on an Agilent 6130 single quadrupole mass spectrometer (Santa Clara, CA, USA), and by UV / visible A260 quantification on a Tecan Infinite® M Plex plate reader (Zurich, Switzerland).

[0298] GalNAc-conjugated oligonucleotides can be used alone as single-stranded compounds (e.g., antisense oligonucleotides (ASOs), ssRNAs, or miRNAs, e.g., Example 3), or duplexed to form double-stranded compounds (e.g., siRNAs and / or shRNAs). In some examples (e.g., Examples 4 and 5), GalNAc is conjugated to the sense strand of the double-stranded compound.

[0299] Duplex formation of double-stranded oligomeric compounds Generally, for double-stranded oligomeric compounds, such as siRNA compounds, sense and antisense oligonucleotides are annealed together to form a duplex. Formation of a 50-300 mM duplex can be achieved by heating the sample in a block heater in 1× phosphate-buffered saline at 94°C for 4 minutes, then removing the heating block containing the sample from the block heater and gradually cooling to room temperature over the course of 1 hour.

[0300] Example 3: Stability of ARNATAR GalNAc conjugates ARNATAR GalNAc compound conjugates and Sharma GalNAc compound conjugates were prepared as described in Example 2 and contained the oligonucleotides shown in Table 1. They were evaluated for stability and durability.

[0301] Four oligonucleotides were synthesized on a 1 μmol scale. Two 24-mer poly-T oligonucleotides were synthesized by standard phosphodiester synthesis using Sharma GalNAc or ARNATAR GalNAc supports. Two 24-mer poly-T oligonucleotides were synthesized by phosphorothioate (PS) internucleoside chemistry (denoted by an internucleoside *) using Sharma GalNAc or ARNATAR GalNAc supports. All four oligonucleotides were purified using standard synthesis methods and ion-exchange purification. Analytical HPLC showed that all four oligonucleotides had greater than 90% purity after purification and desalting.

[0302] Table 1: GalNAc-conjugated oligonucleotide constructs [Table 1]

[0303] Samples (approximately 500 μM) of each GalNAc-conjugated oligonucleotide construct in water were combined with either AMA (ammonium hydroxide / 40% aqueous methylamine 1:1 v / v) or 300 mM NaAc (sodium acetate) buffer, pH 4.5. Table 2 shows a comparison of the stability of the two GalNAc-oligonucleotide conjugates. Here, columns 2 and 3 show the results obtained for stability measurements of the linkage between each GalNAc compound and the oligonucleotide, while columns 4 and 5 show the results obtained for stability measurements within the oligonucleotide.

[0304] Table 2: GalNAc stability [Table 2]

[0305] Reactions were analyzed by mass spectrometry to determine whether cleavage products were present. In all cases where degradation was observed, phosphodiester cleavage and phosphorothioate linkages were the major cleavage products. Analysis with mild degradation results showed some N-1 and N-2 products. Analysis with degradation results showed that the majority of the full-length product was reduced to monomers. Under both acidic and basic conditions, the oligonucleotide phosphate linkage began to degrade before any GalNAc degradation products were observed. ARNATAR GalNAc was found to be stable under stress conditions.

[0306] Example 4: Evaluation of ARNATAR GalNAc in human primary hepatocytes ARNATAR GalNAc compound conjugates and Sharma GalNAc compound conjugates were prepared as described in Example 2, except that the GalNAc compound was conjugated to the same siRNA compound (ATsi103) targeting laminin (LMNA) on the sense strand of the duplex shown in Table 3.

[0307] Table 3: LMNA siRNAs [Table 3] (5p) = 5'-phosphate d (or no notation before the nucleotide) = deoxyribonucleotide substituted for a ribonucleotide f=2'-F m=2'-OMe * = phosphorothioate (PS) bond GalNAc-AN = ARNATAR GalNAc GalNAc-GL = Sharma GalNAc GalNAc-conjugated LMNA siRNA was incubated with human primary hepatocytes to allow free intracellular uptake of the GalNAc-siRNA compounds (i.e., cell entry without transfection). Using knockdown of LMNA expression as a marker, the ability of each GalNAc type to transport LMNA-targeting siRNA into hepatocytes was assessed.

[0308] Varying doses of each LMNA siRNA (final concentrations of 0 μM, 0.008 μM, 0.04 μM, 0.2 μM, 1 μM, or 5 μM) were added to human primary hepatocytes (HPH) (Xenotech, Kansas City, KS, USA) and incubated at 37°C and 5% CO for 16, 31, or 51 hours. siRNA activity was determined by measuring target mRNA levels via qRT-PCR using the LMNA primer-probe set listed in Table 4. qRT-PCR was performed using AgPath-ID™ One-Step RT-PCR reagents on a QS3 Real-Time PCR System (ThermoFisher Scientific, Waltham, MA, USA). Target RNA levels detected in the qRT-PCR assay were normalized to either total RNA levels measured by Ribogreen™ (ThermoFisher Scientific, Waltham, MA, USA) or GAPDH mRNA levels detected in an aliquot of the RNA sample using qRT-PCR.

[0309] The percent of LMNA mRNA 51 hours after free uptake of GalNAc-conjugated siRNA by HPH is shown in Figure 1. The time course of LMNA mRNA levels is shown in Figure 2. Mock PBS treatment was used as a control.

[0310] Table 4: Human LMNA primer-probe sets [Table 4]

[0311] Table 5: Activity of GalNAc-conjugated siRNA targeting LMNA in human primary hepatocytes [Table 5]

[0312] The ARNATAR GalNAc compound is a novel GalNAc compound that offers enhanced activity over the GalNAc compound previously disclosed by Sharma et al. As shown in this study, the ARNATAR GalNAc conjugate performed better than the Sharma GalNAc conjugate in human primary hepatocytes at all doses and time periods evaluated. Given the subtle structural differences, it was unexpected that the ARNATAR GalNAc conjugate performed so much better than the Sharma GalNAc conjugate.

[0313] Example 5: Evaluation of ARNATAR GalNAc conjugates in mouse hepatocytes Because the GalNAc-conjugated LMNA siRNA disclosed in Example 4 targets both human and mouse LMNA mRNA, a study was performed to confirm the ability of ARNATAR GalNAc to deliver siRNA into mouse hepatocytes.

[0314] In this study, GalNAc-conjugated LMNA siRNA was incubated with mouse hepatocytes to allow free intracellular uptake of the GalNAc-siRNA compound (i.e., cell entry without transfection). As in Example 4, knockdown of LMNA expression was used as a marker to assess the ability of each GalNAc type to deliver LMNA-targeting siRNA into hepatocytes.

[0315] Varying doses of each LMNA siRNA (final concentrations of 0 μM, 0.008 μM, 0.04 μM, 0.2 μM, 1 μM, or 5 μM) were added to mouse hepatocytes (mPH) (Xenotech, Kansas City, KS, USA) and incubated at 37°C and 5% CO for 60 hours. siRNA activity was determined by measuring target mRNA levels via qRT-PCR using the LMNA primer-probe set listed in Table 6. qRT-PCR was performed using AgPath-ID™ One-Step RT-PCR Reagents on a QS3 Real-Time PCR System (ThermoFisher Scientific, Waltham, MA, USA). Target RNA levels detected in the qRT-PCR assay were normalized to either total RNA levels measured by Ribogreen™ (ThermoFisher Scientific, Waltham, MA, USA) or GAPDH mRNA levels detected in an aliquot of the RNA sample using qRT-PCR.

[0316] The percent of LMNA mRNA 60 hours after free uptake of GalNAc-conjugated siRNA by HPH is shown in Figure 3 .

[0317] Table 6: Mouse LMNA primer-probe sets [Table 6] *56-FAM, ZEN, and / 3IABkFQ are dyes used in the oligos.

[0318] Example 6: Evaluation of ARNATAR GalNAc in vitro The ARNATAR GalNAc compound conjugate and the Sharma GalNAc compound conjugate were prepared as described in Example 2, except that the GalNAc compound was conjugated to the same siRNA compound targeting proprotein convertase subtilisin / kexin type 9 (PCSK9) on the sense strand of the duplex shown in Table 7.

[0319] Table 7: PCSK9 siRNA [Table 7] (5p) = 5'-phosphate d (or no notation before the nucleotide) = deoxyribonucleotide substituted for a ribonucleotide f=2'-F m=2'-OMe * = phosphorothioate (PS) bond GalNAc-AN = ARNATAR GalNAc GalNAc-GL = Sharma GalNAc

[0320] Table 8: Human PCSK9 primer-probe set [Table 8] *56-FAM, ZEN, and / 3IABkFQ are dyes used in the oligos.

[0321] In vitro evaluation in human primary hepatocytes GalNAc-conjugated PCSK9 siRNA was incubated with human primary hepatocytes to allow free intracellular uptake of the GalNAc-siRNA compounds (i.e., cell entry without transfection). Using knockdown of PCSK9 expression as a marker, the ability of each GalNAc type to transport siRNA targeting PCSK9 into hepatocytes was assessed.

[0322] Various doses of each PCSK9 siRNA (final concentrations of 0 μM, 0.001 μM, 0.01 μM, 0.1 μM, 1 μM, or 10 μM) were added to human primary hepatocytes (HPH) (Xenotech, Kansas City, KS, USA) and incubated at 37°C and 5% CO for 4 days. siRNA activity was determined by measuring target mRNA levels via qRT-PCR using the PCSK9 primer-probe set listed in Table 8. qRT-PCR was performed using AgPath-ID™ One-Step RT-PCR reagents on a QS3 Real-Time PCR System (ThermoFisher Scientific, Waltham, MA, USA). Target RNA levels detected in the qRT-PCR assay were normalized to either total RNA levels measured by Ribogreen™ (ThermoFisher Scientific, Waltham, MA, USA) or GAPDH mRNA levels detected in an aliquot of the RNA sample using qRT-PCR.

[0323] The percent of PCSK9 mRNA at day 4 after free uptake of GalNAc-conjugated siRNA by HPH is shown in Figure 4. Mock PBS treatment was used as a control.

[0324] conclusion The ARNATAR GalNAc compound is a novel GalNAc compound that offers enhanced activity over the GalNAc compound previously disclosed by Sharma et al. As shown in this study, the ARNATAR GalNAc conjugate performed better than Sharma's GalNAc conjugate in human primary hepatocytes at all doses and time periods evaluated. Given the subtle structural differences, it was unexpected that the ARNATAR GalNAc conjugate performed so much better than the Sharma GalNAc conjugate.

Claims

1. having the structure of formula (0): 【Chemistry 1】 During the ceremony, k is an integer from 1 to 5, m is an integer from 0 to 11; n is an integer from 0 to 5, X is a hydroxyl protecting group or an H atom; Y is a hydroxyl protecting group, an H atom, a payload, or a solid support, said payload or said solid support being optionally linked via a spacer; R １ are independently selected from a hydroxyl protecting group and an H atom; compound.

2. 2. The compound of claim 1, wherein Y is a payload, said payload comprising an oligomeric compound such as an oligonucleotide, a peptide, an antibody, an antibody fragment, or a chemical compound having pharmaceutical activity.

3. X and R １ 3. The compound according to claim 1 or 2, wherein each represents a H atom.

4. having the structure of formula (I): 【Chemistry 2】 During the ceremony, k is an integer from 1 to 5, m is an integer from 0 to 11; n is an integer from 0 to 5, Oligo refers to modified and / or unmodified oligonucleotides. A compound according to any one of the preceding claims.

5. The compound of claim 4, wherein the modified and / or unmodified oligonucleotide is a single-stranded or double-stranded modified and / or unmodified oligonucleotide.

6. The compound according to any one of claims 4 to 5, wherein the modified and / or unmodified oligonucleotide is attached via the 3' end of the sense strand.

7. The compound according to any one of claims 4 to 6, wherein the modified and / or unmodified oligonucleotide binds to a target nucleotide sequence and modifies the expression of a gene encoded by the target nucleotide sequence.

8. having the structure of formula (II): 【Transformation 3】 During the ceremony, k is an integer from 1 to 5, m is an integer from 0 to 11; n is an integer from 0 to 5, R １ are independently selected from a hydroxyl protecting group and an H atom, preferably an acetyl group; R ６ represents a hydroxyl-protecting group, preferably DMT or MMT, more preferably MMT; R ７ represents a moiety of formula (VI), 【Chemistry 4】 During the ceremony, R represents a solid support, an amino protecting group, a hydroxyl protecting group, or an H atom, the solid support being optionally linked via a spacer; Z represents NH or O; R ８ represents a straight or branched alkylene having from 1 to 8 carbon atoms, optionally wherein one or more carbon atoms are selected from O, S, NH and / or N—(C １ -C ３ alkyl), and / or R ８ is a cycloalkylene having from 5 to 8 carbon atoms, preferably ethylene, 1,1 dimethylethylene, propylene, n-butylene, or 1,2 cyclohexylene; The compound of claim 1.

9. During the ceremony, k is 2 or 3; m is 7, 8, or 9; n is 2 or 3; A compound according to any one of the preceding claims.

10. the sum of k and m is an integer from 8 to 12, preferably 10; A compound according to any one of the preceding claims.

11. The compound has a structure of formula (0a), formula (Ia), or formula (IIa): 【Transformation 5】 【Transformation 6】 【Transformation 7】 In the formula, X, Y, oligo, R １ , R ６ , and R ７ is as defined in the preceding claims, A compound according to any one of the preceding claims.

12. having the structure of formula (III): 【Transformation 8】 During the ceremony, k is an integer from 1 to 5, R ２ represents an amino protecting group or an H atom, R ３ represents a solid support, a hydroxyl protecting group, or an H atom, said solid support being optionally linked via a spacer; R ６ represents a hydroxyl protecting group, preferably DMT or MMT, more preferably MMT; compound.

13. 13. The compound of claim 12, wherein k is 2 or 3, preferably 3.

14. R ２ represents an amino protecting group, preferably a fluorenylmethoxycarbonyl group (Fmoc), and / or R ３ 14. The compound according to claim 12 or 13, wherein represents an H atom.

15. The compound has the structure of formula (IIIa): 【Chemistry 9】 During the ceremony, R ６ represents a hydroxyl protecting group, preferably DMT or MMT, more preferably MMT; The compound according to any one of claims 12 to 14.

16. R ２ represents a H atom, and / or R ３ 14. The compound of claim 12 or 13, wherein represents a solid support, said solid support being optionally linked via a spacer.

17. The compound has a structure according to any one of formulas (IIIb1) to (IIIb5): 【Chemistry 10】 、 【Chemistry 11】 、 【Chemistry 12】 、 【Chemistry 13】 、 【Chemistry 14】 During the ceremony, R represents a solid support, an amino protecting group, or an H atom, said solid support being optionally linked via a spacer; R ６ represents a hydroxyl protecting group, preferably DMT or MMT, more preferably MMT; 17. A compound according to any one of claims 12, 13 and 16.

18. A method for preparing a compound having the structure of formula (0): 【Chemistry 15】 During the ceremony, k is an integer from 1 to 5, m is an integer from 0 to 11; n is an integer from 0 to 5, X is a hydroxyl protecting group or an H atom; Y is a hydroxyl protecting group, an H atom, a payload, or a solid support, said payload or said solid support being optionally linked via a spacer; R １ are independently selected from a hydroxyl protecting group and an H atom; The method includes reacting a compound having the structure of formula (III) with a compound having the structure of formula (V): 【Chemistry 16】 During the ceremony, k is an integer from 1 to 5, R ２ represents an amino protecting group or an H atom, R ３ represents a solid support, a hydroxyl protecting group, or an H atom, said solid support being optionally linked via a spacer; R ６ represents a hydroxyl-protecting group, preferably DMT or MMT, more preferably MMT; The compound comprising the structure of formula (V) is 【Chemistry 17】 That's the method.

19. The compound of formula (0) has the structure of formula (I): [Chemistry 18] During the ceremony, k is an integer from 1 to 5, m is an integer from 0 to 11; n is an integer from 0 to 5, Oligo refers to modified and / or unmodified oligonucleotides.

20. The method of claim 18.

20. The compound of formula (0) has the structure of formula (II): 【Chemistry 19】 During the ceremony, k is an integer from 1 to 5, m is an integer from 0 to 11; n is an integer from 0 to 5, R １ are independently selected from a hydroxyl protecting group and an H atom, preferably an acetyl group; R ６ represents a hydroxyl-protecting group, preferably MMT; R ７ represents a moiety of formula (VI), 【Chemistry 20】 During the ceremony, R represents a solid support, an amino protecting group, a hydroxyl protecting group, or an H atom, said solid support being optionally linked via a spacer; Z represents NH or O; R ８ represents a straight or branched alkylene having from 1 to 8 carbon atoms, optionally wherein one or more carbon atoms are selected from O, S, NH and / or N—(C １ -C ３ alkyl), and / or R ８ is a cycloalkylene having from 5 to 8 carbon atoms, preferably ethylene, 1,1 dimethylethylene, propylene, n-butylene, or 1,2 cyclohexylene; 20. The method of claim 19.

21. A pharmaceutical composition comprising a compound having the structure of formula (0) and a pharmaceutically acceptable carrier, excipient, and / or diluent, 【Chemistry 21】 During the ceremony, k is an integer from 1 to 5, preferably 3; m is an integer from 0 to 11, preferably 7; n is an integer from 0 to 5, preferably 3; X is a H atom, Y is a payload, optionally linked via a spacer; R １ is a H atom, Pharmaceutical compositions.

22. 22. The pharmaceutical composition of claim 21, wherein the payload comprises an oligomeric compound such as an oligonucleotide, a peptide, an antibody, an antibody fragment, or a chemical compound having pharmaceutical activity.

23. The compound has the structure of formula (I): 【Chemistry 22】 During the ceremony, k is an integer from 1 to 5, m is an integer from 0 to 11; n is an integer from 0 to 5, Oligo refers to modified and / or unmodified oligonucleotides.

23. The pharmaceutical composition of claim 21 or 22.

Images