Linkages for coupling functional ligands to macromolecules

Abstract

This article provides a functional portion comprising at least one β-amino acid within an amino acid cluster for conjugation with one or more ligands. This functional portion can be used to link a macromolecule to the one or more functional ligands, which can facilitate the delivery of the macromolecule to a site in the body of a subject and enhance the in vitro and / or in vivo stability of the macromolecule (e.g., an oligonucleotide).

Description

Related applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 602,246, filed November 22, 2023, the entire contents of which are incorporated herein by reference.

[0002] sequence list This application contains a sequence list submitted electronically in XML format, the entire contents of which are incorporated herein by reference. The XML copy was created on November 22, 2024, and is named 0817444_00338_SL.xml, with a size of 45,729 bytes. Background Technology

[0003] Short interfering RNA (siRNA)-induced RNAi responses hold great potential for treating a wide range of human diseases, from cancer and pandemic viral outbreaks to Parkinson's disease. However, after administration, naked siRNA is difficult for target cells to take up in therapeutically relevant amounts. One solution to this problem of delivery to target cells is to use... N -Acetylgalactosamine (GalNAc) modifies siRNA to form siRNA conjugates for delivery to the liver. Tri-GalNAc binds to the desialyl glycoprotein receptor, which is highly expressed on hepatocytes and several human cancer cell lines, leading to rapid endocytosis. Although the exact mechanism of escape across the endosome lipid bilayer is not fully understood, sufficient amounts of siRNA can enter the cytoplasm to induce a strong, target-selective RNAi response in vivo. Several clinical trials of GalNAc-siRNA conjugates have been initiated, including two phase III trials, for the treatment of various diseases, and three commercially available GalNAc-siRNAs are available. GalNAc-siRNA conjugates represent an option for addressing the problem of siRNA delivery to hepatocytes and demonstrate a forward direction for targeting other tissue types in the field of RNAi (and antisense oligonucleotides).

[0004] Besides GalNAc, there are several other well-known ligands, such as mannose / N - Acetylglucosamine (GlcNAc or GluNac) is delivered to macrophages via mannose receptors. Furthermore, conjugation with lipophilic molecules such as cholesterol, bile acids, and fatty acids increases the binding affinity of siRNA to plasma proteins, thereby improving siRNA delivery through passive targeting or active targeting by blocking endogenous lipid transport pathways.

[0005] The conjugation of these ligands has been resolved previously, but the in vivo stability of the conjugation link between the ligand and siRNA remains an issue. Summary of the Invention

[0006] This article provides adapters for coupling ligands to macromolecules (e.g., oligonucleotides, proteins, peptides, etc.). Attached Figure Description

[0007] Figure 1 In vitro stability data of the compounds described herein under rat tritosome conditions are described.

[0008] Figure 2 In vitro stability data of the compounds described herein in mouse liver homogenate conditions are described.

[0009] Figure 3 In vitro factor IX potency data for oligonucleotide 1 conjugate with tri-GalNAc are described.

[0010] Figure 4 In vitro factor VII potency data for oligonucleotide 2 conjugate with tri-GalNAc are described.

[0011] Figure 5 In vivo factor IX potency data for oligonucleotide 1 conjugate with tri-GalNAc are described.

[0012] Figure 6 In vivo factor VII potency data for oligonucleotide 2 conjugate with tri-GalNAc are described.

[0013] Figure 7 In vitro stability data of the compounds described herein under monkey liver homogenate conditions are described.

[0014] Figure 8 Example synthetic schemes for preparing the phosphoramidides compounds of formula (A) and the like are described herein.

[0015] Figure 9 A general synthetic scheme for preparing the phosphoramidides compounds of formula (A) and the like is described herein.

[0016] Figure 10 Example synthetic schemes for preparing compounds such as those of formula (A) that are bound to the solid support described herein are described.

[0017] Figure 11 General synthetic schemes for preparing compounds such as those of formula (A) that are bound to the solid support described herein are described.

[0018] Figure 12 Oligonucleotide 1 and oligonucleotide 2, which are double-stranded pairs of SEQ ID NO:1 and SEQ ID NO:2 and SEQ ID NO:3 and SEQ ID NO:4, are described in Table 4. Detailed Implementation

[0019] definition Certain terms, whether used alone or as part of a phrase or another term, are defined below.

[0020] The articles “a” and “an” refer to one or more grammatical objects of the article.

[0021] Numerical values ​​related to measurements are subject to measurement errors, which impose limitations on their accuracy. Therefore, unless otherwise stated, all numerical values ​​provided herein should be understood as being modified by the term "approximately". Thus, the last decimal place of the numerical values ​​provided herein indicates their degree of accuracy. Unless otherwise specified, the maximum tolerance is determined by applying rounding rules to the last decimal place, or, when no decimal place exists in the given numerical value.

[0022] The term "amelioration" refers to a reduction in the severity of at least one indicator of a condition or disease, such as a delay or slowing of the progression of one or more indicators of the condition or disease. The severity of the indicator can be determined by subjective or objective measures known to those skilled in the art.

[0023] The term "composition" refers to a mixture of at least two or more components.

[0024] The terms "effective amount" and "therapeutic effective amount" refer to the amount of a therapeutic compound, combination of compounds, or composition that effectively produces the desired therapeutic effect, whether as a single dose or part of a series of doses. Generally, the therapeutic effective amount can initially be estimated in cell culture assays or in mammalian models, such as in non-human primates, mice, rabbits, dogs, or pigs. Animal models can also be used to determine appropriate concentration ranges and routes of administration. This information can then be used to determine the available dose and route of administration in non-human and human subjects.

[0025] The term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or carrier, such as a liquid filler, solid filler, stabilizer, dispersant, suspension, diluent, excipient, thickener, solvent, or encapsulating material, relating to carrying or transporting at least one compound described herein into or to a patient so that the compound can perform its intended function. A given carrier must be "acceptable," meaning it is compatible with other components of a particular formulation, including the compounds described herein, and is harmless to the patient. Other components that may be included in the pharmaceutical compositions described herein are well known in the art and are described, for example, in "Remington's Pharmaceutical Sciences" (edited. Genaro, Mack Publishing, 1985), the entire contents of which are incorporated herein by reference.

[0026] The term "pharmaceutical composition" refers to a mixture of at least one compound described herein with a pharmaceutically acceptable carrier. Pharmaceutical compositions facilitate the administration of compounds or combinations thereof to a patient or subject. Various techniques exist for administering compounds, combinations, or compositions, including but not limited to intravenous, oral, aerosol, parenteral, ocular, pulmonary, and topical administration. For example, in some cases, the administration of therapeutic proteins, peptides, oligosaccharides, or oligonucleotides is carried out via oral, inhalation, or injection routes.

[0027] The terms "treatment" or "treating" refer to the application of one or more specific procedures to improve a disease. "Preventive" treatment refers to slowing the progression of a disease or condition, delaying its onset, or reducing its severity.

[0028] The numerical ranges described herein are intended solely as a shorthand for individually referring to each individual value falling within that range. Unless otherwise stated herein, each individual value is incorporated into the description as if it were described separately herein. Therefore, in order to describe the numerical ranges herein, every intermediate number having the same degree of precision is explicitly included. For example, for the range of 6–9, the numbers 7 and 8 are included in addition to 6 and 9, and for the range of 6.0–7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly included.

[0029] All methods described herein may be performed in any suitable order unless otherwise stated herein or clearly contradicted by the context. The use of any and all instances or exemplary language (e.g., “such as”) provided herein is intended only to better illustrate the described subject matter and not to limit the scope of additionally claimed subject matter. No language in this specification should be construed as indicating any unclaimed element that is essential to the subject matter described in practice.

[0030] The grouping of alternative elements or embodiments in this disclosure should not be construed as limiting. Each member of a group may be mentioned and claimed individually or in any combination with other members of that group or other elements found herein. Furthermore, for convenience or patentability reasons, members of a recited group may be included in or excluded from another recited group. When any such inclusion or exclusion occurs, this specification shall be deemed to include the modified group, thereby satisfying the written description of all Markush groups used in the appended claims.

[0031] This specification incorporates references to patents and print publications throughout, the entire contents of which are incorporated herein by reference individually.

[0032] It should be understood that the embodiments of this disclosure are illustrative. Therefore, this disclosure is not limited to exactly as shown and described.

[0033] Compounds / Conjugates Oligonucleotides modified with three GalNAc moieties, such as those in formula (A) (e.g., formula (II)), containing at least one β-amino acid (e.g., D- or L- or racemic), such as β-lysine or β-glutamic acid, have been found to have higher in vitro and in vivo stability (e.g., resistance to enzymatic degradation) compared to corresponding moieties having L- or D-α-amino acids instead of at least one β-amino acid (e.g., L- or D-α-lysine instead of β-lysine, and L- or D-α-glutamic acid instead of β-glutamic acid). Furthermore, formula (II) (e.g., where R...) has been found to... 2(These are oligonucleotides) that are at least as stable as those of formula (V). In other words, compounds of formula (A) described herein have been found to improve the synthesis of commercially available solid supports or to improve in vitro / in vivo stability and target site delivery (e.g., cellular uptake by target cells). This is important because the preparation of formula (A) (e.g., formula (II)) requires fewer synthetic steps and is more atomically efficient than that of formula (V), thus being more cost-effective, and also results in less foreign matter being delivered to the subject (e.g., a lower dose compared to therapeutic siRNAs conjugated with tri-GalNAc as in formula (V)). Compounds of formula (I) would similarly benefit from this synthetic efficiency. Corresponding phosphoridamide compounds as described herein can be used to prepare such conjugates via solid-phase synthesis. Therefore, as described herein, compounds such as those of formula (A) have been found to be useful, for example, in relation to medical applications, for improving stability or enhancing delivery to target sites in subjects (e.g., uptake by target cells), whether in the form of phosphorus amides for the synthesis of macromolecular conjugates with ligands (e.g., GalNAC, GlcNAC, fatty acids, etc.) using atomically efficient solid supports, or as intermediates or products of such synthesis. Intermediates described herein for the preparation of such phosphorus amides are also provided herein.

[0034] Therefore, in some embodiments, this document provides compounds comprising formula (A): (A) Or its salts (e.g., pharmaceutically acceptable salts). in R 1 It is hydrogen (H), -R 7 - (Solid support) (e.g., wherein the solid support is selected from, but not limited to, silica gel, controlled-porosity glass (CPG) (e.g., long-chain alkyl-amine CPG) or resin (e.g., polystyrene)) or phosphoramide (e.g., 3-((diisopropylamino)phosphono)oxy)propionitrile moiety (abbreviated as DIPA CEP): ); R 2This includes H, oligonucleotides (e.g., having or not having a protecting group from a solid support synthesized from a standard cyanoethylphosphoramide oligonucleotide (e.g., DNA or RNA, optionally single-stranded or double-stranded, including optionally nucleotide analogs therein), peptides (i.e., having or not having 2 to 49 amino acid residues from a solid support synthesized from a standard FMOC amide), proteins (i.e., having or not having 50 or more amino acid residues from a solid support synthesized from a standard FMOC amide), or acid-labile protecting groups (e.g., triphenylmethyl, monomethoxytriphenylmethyl, dimethoxytriphenylmethyl (also abbreviated as DMTr), trimethoxytriphenylmethyl, monomethyltriphenylmethyl, dimethyltriphenylmethyl, trimethyltriphenylmethyl, monochlorotriphenylmethyl, dichlorotriphenylmethyl, trichlorotriphenylmethyl, methylsulfonyltriphenylmethyl, monomethoxymethylsulfonyltriphenylmethyl, dimethoxymethylsulfonyltriphenylmethyl, monomethoxydimethylsulfonyltriphenylmethyl, or trimethylsulfonyltriphenylmethyl). Or R 1 It is an acid-instable protecting group (e.g., triphenylmethyl, monomethoxytriphenylmethyl, dimethoxytriphenylmethyl (also abbreviated as DMTr), trimethoxytriphenylmethyl, monomethyltriphenylmethyl, dimethyltriphenylmethyl, trimethyltriphenylmethyl, monochlorotriphenylmethyl, dichlorotriphenylmethyl, trichlorotriphenylmethyl, methylsulfonyltriphenylmethyl, monomethoxymethylsulfonyltriphenylmethyl, dimethoxymethylsulfonyltriphenylmethyl, monomethoxydimethylsulfonyltriphenylmethyl or trimethylsulfonyltriphenylmethyl), and R 2 This includes oligonucleotides (e.g., those with or without protecting groups synthesized from a standard cyanoethylphosphoramide oligonucleotide (e.g., DNA or RNA, optionally single-stranded or double-stranded) solid support), peptides (i.e., 2 to 49 amino acid residues with or without protecting groups synthesized from a standard FMOC amide solid support), proteins (i.e., 50 or more amino acid residues with or without protecting groups synthesized from a standard FMOC amide solid support), and those covalently linked to a solid support (e.g., directly or through a linker, as described above R). 1 As shown, for example, -R 7 - (solid support) oligonucleotides, peptides or proteins; R 3 R 4 and R 5 Independently selected from hydrogen (H), , , , , , , , , , , , , , , , , , , , , , , , , , , , or ; Each R 6 The group is independently selected from H, acyl groups (including but not limited to acetyl, chloroacetyl, trichloroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, 4-isopropylphenoxyacetyl or neopentanoyl), silyl groups (including but not limited to trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBDMS), triphenylsilyl (TPS or tert-butyldiphenylsilyl (TBDPS)), carbonate groups (including but not limited to 2-cyanoethylcarbonyl, 2-(2,4-dinitrophenyl)ethoxycarbonyl, 2-(p-nitrophenyl)ethoxycarbonyl or fluorenylmethoxycarbonyl), or benzyl groups (including but not limited to benzyl, 2-nitrobenzyl or 4-nitrobenzyl). R 7 yes ; R 8 It is O or NH (i.e., R7 and R8 together with the atoms to which they are attached form a moiety, which includes an ester and an ester at each end of the moiety or an ester and an amide at one of the ends of the moiety); and n is 2, 3, 4, 5, or 6 or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20).

[0035] In some embodiments, the oligonucleotides referred to herein include one or more nucleotide analogs. In some embodiments, the nucleotide analogs are independently selected from 2'-O-methyl nucleotides (e.g., mA, mC, mG, mU, mT), 2'-deoxy-2'-fluoro nucleotides (e.g., fA, fC, fG, fU, fT), or vinylphosphonate nucleotides (e.g., 5'-(E)-vinylphosphonate dA, dC, dG, dU, dT, mA, mC, mG, mU, mT, fA, fC, fG, fU, or fT). In some embodiments, the oligonucleotides referred to herein include single-stranded or double-stranded oligonucleotides. In some embodiments, the oligonucleotide is a double-stranded oligonucleotide, each strand independently having a length of about 15-30 nucleotides. In some embodiments, each oligonucleotide has the same or different nucleotide lengths. In some embodiments, the difference in nucleotide length is 1, 2, 3, 4, or 5 nucleotides. In some embodiments, the length of the oligonucleotide is independently about 15-30 nucleotides, for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the length of the oligonucleotide is independently about 18–25 nucleotides. In some embodiments, the length of the oligonucleotide is independently about 20–25 nucleotides. In some embodiments, the length of the oligonucleotide is independently about 21 nucleotides.

[0036] In some embodiments of the formula described herein, R 1 Yes -R 7 - (Solid support) (e.g., a solid support selected from, but not limited to, silica gel, controlled porosity glass (CPG), or resin (e.g., polystyrene), R 2 It is an acid-instable protecting group (e.g., triphenylmethyl, monomethoxytriphenylmethyl, dimethoxytriphenylmethyl (also abbreviated as DMTr), trimethoxytriphenylmethyl, monomethyltriphenylmethyl, dimethyltriphenylmethyl, trimethyltriphenylmethyl, monochlorotriphenylmethyl, dichlorotriphenylmethyl, trichlorotriphenylmethyl, methylsulfonyltriphenylmethyl, monomethoxymethylsulfonyltriphenylmethyl, dimethoxymethylsulfonyltriphenylmethyl, monomethoxydimethylsulfonyltriphenylmethyl, or trimethylsulfonyltriphenylmethyl), and each R 6The group is independently selected from acyl groups (including but not limited to acetyl, chloroacetyl, trichloroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, 4-isopropylphenoxyacetyl, or neopentanoyl), silyl groups (including but not limited to trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBDMS), triphenylsilyl (TPS), or tert-butyldiphenylsilyl (TBDPS)), carbonate groups (including but not limited to 2-cyanoethylcarbonyl, 2-(2,4-dinitrophenyl)ethoxycarbonyl, 2-(p-nitrophenyl)ethoxycarbonyl, or fluorenylmethoxycarbonyl), or benzyl groups (including but not limited to benzyl, 2-nitrobenzyl, or 4-nitrobenzyl). In some embodiments of the formula herein, R 1 Yes -R 7 - (Solid support) (e.g., a solid support selected from, but not limited to, silica gel, controlled porosity glass (CPG), or resin (e.g., polystyrene), R 2 It is DMTr and R 6 It is an acetyl group.

[0037] In some embodiments of the formula described herein, R 1 It is an acid-instable protecting group (e.g., triphenylmethyl, monomethoxytriphenylmethyl, dimethoxytriphenylmethyl (also abbreviated as DMTr), trimethoxytriphenylmethyl, monomethyltriphenylmethyl, dimethyltriphenylmethyl, trimethyltriphenylmethyl, monochlorotriphenylmethyl, dichlorotriphenylmethyl, trichlorotriphenylmethyl, methylsulfonyltriphenylmethyl, monomethoxymethylsulfonyltriphenylmethyl, dimethoxymethylsulfonyltriphenylmethyl, monomethoxydimethylsulfonyltriphenylmethyl or trimethylsulfonyltriphenylmethyl), and R 2 This includes oligonucleotides (e.g., those with or without protecting groups synthesized from a standard cyanoethylphosphoramide oligonucleotide (e.g., DNA or RNA, optionally single-stranded or double-stranded) solid support), peptides (i.e., 2 to 49 amino acid residues with or without protecting groups synthesized from a standard FMOC amide solid support), proteins (i.e., 50 or more amino acid residues with or without protecting groups synthesized from a standard FMOC amide solid support), oligonucleotides, peptides, or proteins covalently linked to a solid support, and each R 6The group is independently selected from acyl groups (including but not limited to acetyl, chloroacetyl, trichloroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, 4-isopropylphenoxyacetyl, or neopentanoyl), silyl groups (including but not limited to trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBDMS), triphenylsilyl (TPS), or tert-butyldiphenylsilyl (TBDPS)), carbonate groups (including but not limited to 2-cyanoethylcarbonyl, 2-(2,4-dinitrophenyl)ethoxycarbonyl, 2-(p-nitrophenyl)ethoxycarbonyl, or fluorenylmethoxycarbonyl), or benzyl groups (including but not limited to benzyl, 2-nitrobenzyl, or 4-nitrobenzyl). In some embodiments of the formula herein, R 1 It's DMTr, R 2 It is covalently linked to a solid support (e.g., -R) 7 - (solid support) oligonucleotides, peptides or proteins, and R 6 It is an acetyl group. In some embodiments of the formula herein, R 1 It's DMTr, R 2 It is covalently linked to a solid support (e.g., -R) 7 - (solid support) oligonucleotides, peptides or proteins, and R 6 It is H. In some embodiments of the formula in this paper, R 1 It is H, R 2 It is covalently linked to a solid support (e.g., -R) 7 - (solid support) oligonucleotides, peptides or proteins, and R 6 It is an acetyl group. In some embodiments of the formula herein, R 1 It is H, R 2 It is covalently linked to a solid support (e.g., -R) 7 - (solid support) oligonucleotides, peptides or proteins, and R 6 It's H.

[0038] In some embodiments of the formula described herein, R 1 It is H, R 2 It is H and R 6 It's H.

[0039] In some embodiments of the formula described herein, R 1 It is H, R 2 It is H and R 6 It is an acetyl group.

[0040] In some embodiments of the formula described herein, R 1 It is H, R 2 It is H and each R6 The group is independently selected from acyl groups (including but not limited to acetyl, chloroacetyl, trichloroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, 4-isopropylphenoxyacetyl or neopentanoyl), silyl groups (including but not limited to trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBDMS), triphenylsilyl (TPS or tert-butyldiphenylsilyl (TBDPS)), carbonate groups (including but not limited to 2-cyanoethylcarbonyl, 2-(2,4-dinitrophenyl)ethoxycarbonyl, 2-(p-nitrophenyl)ethoxycarbonyl or fluorenylmethoxycarbonyl) or benzyl groups (including but not limited to benzyl, 2-nitrobenzyl or 4-nitrobenzyl).

[0041] In some embodiments of the formula described herein, R 1 It is H and R 2 It is an acid-instable protecting group (e.g., triphenylmethyl, monomethoxytriphenylmethyl, dimethoxytriphenylmethyl (also abbreviated as DMTr), trimethoxytriphenylmethyl, monomethyltriphenylmethyl, dimethyltriphenylmethyl, trimethyltriphenylmethyl, monochlorotriphenylmethyl, dichlorotriphenylmethyl, trichlorotriphenylmethyl, methylsulfonyltriphenylmethyl, monomethoxymethylsulfonyltriphenylmethyl, dimethoxymethylsulfonyltriphenylmethyl, monomethoxydimethylsulfonyltriphenylmethyl, or trimethylsulfonyltriphenylmethyl). In some embodiments of the formula herein, R 1 It is H and R 2 It is DMTr. In some implementations of the formula in this paper, R 1 It is H, R 2 It is DMTr and each R 6 The group is independently selected from acyl groups (including but not limited to acetyl, chloroacetyl, trichloroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, 4-isopropylphenoxyacetyl, or neopentanoyl), silyl groups (including but not limited to trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBDMS), triphenylsilyl (TPS), or tert-butyldiphenylsilyl (TBDPS)), carbonate groups (including but not limited to 2-cyanoethylcarbonyl, 2-(2,4-dinitrophenyl)ethoxycarbonyl, 2-(p-nitrophenyl)ethoxycarbonyl, or fluorenylmethoxycarbonyl), or benzyl groups (including but not limited to benzyl, 2-nitrobenzyl, or 4-nitrobenzyl). In some embodiments of the formula herein, R 1 It is H, R 2 It is DMTr and R 6 It is an acetyl group.

[0042] In some embodiments of the formula described herein, R 1 It is DIPA CEP, R 2 It is an acid-instable protecting group (e.g., triphenylmethyl, monomethoxytriphenylmethyl, dimethoxytriphenylmethyl (also abbreviated as DMTr), trimethoxytriphenylmethyl, monomethyltriphenylmethyl, dimethyltriphenylmethyl, trimethyltriphenylmethyl, monochlorotriphenylmethyl, dichlorotriphenylmethyl, trichlorotriphenylmethyl, methylsulfonyltriphenylmethyl, monomethoxymethylsulfonyltriphenylmethyl, dimethoxymethylsulfonyltriphenylmethyl, monomethoxydimethylsulfonyltriphenylmethyl, or trimethylsulfonyltriphenylmethyl) and each R 6 The group is independently selected from acyl groups (including but not limited to acetyl, chloroacetyl, trichloroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, 4-isopropylphenoxyacetyl, or neopentanoyl), silyl groups (including but not limited to trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBDMS), triphenylsilyl (TPS), or tert-butyldiphenylsilyl (TBDPS)), carbonate groups (including but not limited to 2-cyanoethylcarbonyl, 2-(2,4-dinitrophenyl)ethoxycarbonyl, 2-(p-nitrophenyl)ethoxycarbonyl, or fluorenylmethoxycarbonyl), or benzyl groups (including but not limited to benzyl, 2-nitrobenzyl, or 4-nitrobenzyl). In some embodiments of the formula herein, R 1 It is DIPA CEP, R 2 It is DMTr and R 6 It is an acetyl group.

[0043] In some embodiments, this document provides compounds comprising formula (I): (I) Or its pharmaceutically acceptable salt. in R 1 It is hydrogen (H); R 2 This includes either oligonucleotides or peptides (i.e., 2 to 49 amino acid residues) or proteins (i.e., 50 or more amino acid residues); and R 3 R 4 and R 5Independently selected from, but not limited to, the following ligands: hydrogen (H), ETA, CPA, GalNAc, GluNAc, PGA, CA, UDA, DDA, DDA 12-OH, TDA, MA, PDA, PA, HDA, SA, SA 18-OH, SA 12-OH, SA 2-OH, ACA, BA, DHA, ARA, EPA, ALA, GLA, RA, OA, EA, or LA. Compounds with the above abbreviations are described in Table 1 below.

[0044] In some implementations, R 3 R 4 and R 5 It is independently linked to a ligand via a bond selected from, but not limited to, direct bonds, -O-, -NH-, -N(CH3)-, -SS-, -C(O)-O-, -OC(O)-, -C(O)-S-, -SC(O)-, -C(O)-N(H)-, -N(H)-C(O)-, -C(O)-N(CH3)-, -N(CH3)-C(O)-, -OC(O)-O-, -OC(O)-O-, -OC(O)-N(H)-, -N(H)-C(O)-O-, -OC(O)-N(CH3)-, -N(CH3)-C(O)-O-, -N(H)-C(O)-N(H)- and N(CH3)-C(O)-N(CH3)-.

[0045] In some implementations, R 3 R 4 and R 5 They are the same.

[0046] In some implementations, R 3 R 4 and R 5 It's GalNAc.

[0047] In some implementations, R 3 R 4 and R 5 It is GluNAc.

[0048] Table 1: Chemical names of some abbreviations used in this article

[0049] In some implementations, R 2 It is an oligonucleotide, and R 3 R 4 and R 5 It's GalNAc: .

[0050] In some implementations, R 2 It is an oligonucleotide, and R 3 R 4 and R 5 It is GluNAc: .

[0051] In some embodiments, the present invention provides the compounds listed in Table 2.

[0052] Table 2: Compounds related to formula (A) (e.g., formula (I)) (H = hydrogen)

[0053] In some embodiments, formula (I) is further conjugated with an oligonucleotide or peptide. In some embodiments, formula (I) is further conjugated with a ribonucleic acid, for example, formula (I) includes dsRNA.

[0054] In some embodiments, compounds of formula (II) are provided herein: (II) Or its pharmaceutically acceptable salt. in R 1 It is hydrogen (H), and R 2 This includes either oligonucleotides or peptides (i.e., 2 to 49 amino acid residues) or proteins (i.e., 50 or more amino acid residues).

[0055] In some implementations, R 2 It is an oligonucleotide. In some implementations, the oligonucleotide is siRNA.

[0056] This article also provides compounds containing formula (III): (III), in R 1 It is as defined above -R 7 -(Solid support), the solid support being selected from, but not limited to, silica gel, controlled porosity glass (CPG), or resin, such as polystyrene (PS), or R... 1 It is a phosphoramide, such as the 3-((diisopropylamino)phosphono)oxy)propionitrile moiety (abbreviated as DIPA CEP): , R 2 It is triphenylmethyl, monomethoxytriphenylmethyl, dimethoxytriphenylmethyl (also abbreviated as DMTr), trimethoxytriphenylmethyl, monomethyltriphenylmethyl, dimethyltriphenylmethyl, trimethyltriphenylmethyl, monochlorotriphenylmethyl, dichlorotriphenylmethyl, trichlorotriphenylmethyl, methylsulfonyltriphenylmethyl, monomethoxymethylsulfonyltriphenylmethyl, dimethoxymethylsulfonyltriphenylmethyl, monomethoxydimethylsulfonyltriphenylmethyl, or trimethylsulfonyltriphenylmethyl; and R 3 R 4 and R 5 Independently selected from, but not limited to, the following ligands: hydrogen (H), ETA, CPA, GalNAc, GluNAc, PGA, CA, UDA, DDA, DDA 12-OH, TDA, MA, PDA, PA, HDA, SA, SA 18-OH, SA 12-OH, SA 2-OH, ACA, BA, DHA, ARA, EPA, ALA, GLA, RA, OA, EA, or LA (see Table 1), wherein each hydroxyl moiety of GalNAc, GluNAc, DDA 12-OH, SA 18-OH, SA 12-OH, and SA 2-OH is acetylated. In some embodiments, R 3 R 4 and R 5Independently linked to a ligand via a bond selected from, but not limited to, -O-, -NH-, -N(CH3)-, -SS-, -C(O)-O-, -OC(O)-, -C(O)-S-, -SC(O)-, -C(O)-N(H)-, -N(H)-C(O)-, -C(O)-N(CH3)-, -N(CH3)-C(O)-, -OC(O)-O-, -OC(O)-O-, -OC(O)-N(H)-, -N(H)-C(O)-O-, -OC(O)-N(CH3)-, -N(CH3)-C(O)-O-, -N(H)-C(O)-N(H)- and N(CH3)-C(O)-N(CH3)-. In some embodiments, R 3 R 4 and R 5 The GalNAc or GluNAc moiety is independently linked by an oxygen bond on the anomeric carbon of the galactosamine or glucosamine ring. In some embodiments, R 3 R 4 and R 5 They are the same. In some implementations, R 3 R 4 and R 5 It is GalNAc, where each hydroxyl moiety of GalNAc is protected with a base-unstable protecting group, for example, acetylated, such as GalNAc(OAc)3, or as shown in the figure below. .

[0057] In some implementations, R 3 R 4 and R 5 They are the same. In some implementations, R 3 R 4 and R 5 It is GluNAc, wherein each hydroxyl moiety of GluNAc is protected with a base-unstable protecting group, for example, acetylated, such as GluNAc(OAc)3 or as shown in the figure below. .

[0058] In some implementations, R 3 R 4 and R 5 They are the same. In some implementations, R 3 R 4 and R 5These are GluNAc, GalNAc, DDA 12-OH, SA 18-OH, SA 12-OH, or SA 2-OH. In some embodiments, the free hydroxyl group of each GluNAc, GalNAc, DDA 12-OH, SA 18-OH, SA 12-OH, or SA 2-OH is protected with a base-sensitive group (e.g., R). 6 It can be represented as -OR 6 Protection. Any suitable base-sensitive protecting group can be selected. In some embodiments, the base-sensitive protecting group is the acetyl moiety as described above. In other embodiments, the base-sensitive protecting group can be another acyl group (including but not limited to chloroacetyl, trichloroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, 4-isopropylphenoxyacetyl and neopentanoyl), silyl group (including but not limited to TMS, TES, TIPS, TBDMS, TPS and TBDPS), carbonate group (including but not limited to 2-cyanoethylcarbonyl, 2-(2,4-dinitrophenyl)ethoxycarbonyl, 2-(p-nitrophenyl)ethoxycarbonyl and fluorenylmethoxycarbonyl), and benzyl group (including but not limited to benzyl, 2-nitrobenzyl and 4-nitrobenzyl).

[0059] In some embodiments, the present invention provides the compounds listed in Table 3.

[0060] Table 3: Compounds related to formula (A) (e.g., formula (III)).

[0061] In some embodiments, compounds of formula (IV) are provided herein: (IV).

[0062] In some embodiments, compounds of formula (V) are provided herein: (V) Or its pharmaceutically acceptable salt. in R 1 It is hydrogen (H); and R 2 It is an oligonucleotide.

[0063] In some embodiments, compounds of formula (VI) are provided herein: (VI) Or its pharmaceutically acceptable salt. in R 1 It is hydrogen (H); and R 2 It is an oligonucleotide.

[0064] This document also provides compositions comprising the compounds described herein and a carrier. In some embodiments, the composition is a pharmaceutical composition, for example, comprising a pharmaceutically acceptable carrier.

[0065] The abbreviations used in this article include those in Table 1. In the context, the abbreviations may refer to the “yl” or “di-yl” of the reference compound, or the corresponding “ate” or “amide” or “amidyl”. For example, GalNAc, which refers to the 2-(acetylamino)-2-deoxy-D-galactose parent compound, may also refer to the 2-(acetylamino)-2-deoxy-D-galactosyl moiety, and CA, which refers to decanoic acid, may also refer to decanoyl or decanoic acid ester or decanoic acid amide.

[0066] In some embodiments, this document provides compositions comprising one or more compounds provided herein. The composition may comprise one or more carriers, including but not limited to one or more solvents. In some embodiments, this document provides pharmaceutical compositions comprising one or more compounds provided herein and at least one pharmaceutically acceptable carrier. In some embodiments, the composition is a solid composition. In some embodiments, the composition is an implantable composition. In some embodiments, the composition is an inhalable composition. In some embodiments, the composition is an orally ingested composition. In some embodiments, the composition is an injectable composition. In some embodiments, the composition is a flowable powder composition. In some embodiments, the composition is a liquid composition, including but not limited to suspensions or emulsions in which the compounds are contained. In some embodiments, the composition is a gel, cream, or ointment containing the compound.

[0067] method In addition to the corresponding phosphoramidite compounds, the amino acid clusters described herein can be used as components for therapeutic applications. Therefore, it is understood that such compounds can be administered in combination with a treatment method for a subject in need. Thus, this document provides at least one method of administering the compounds to a subject. The route of administration can be any suitable method for delivering the compounds described herein to a subject, including those described herein.

[0068] Reagent test kit In some embodiments, this document provides packaging forms, packaging compositions, or packaging pharmaceutical compositions of the compounds provided herein, comprising a container holding a therapeutically effective amount of the compounds described herein, and instructions for using the compounds according to one or more methods provided herein.

[0069] The compounds and related materials of the present invention can be made into commercial products using conventional steps performed in the art, such as appropriate sterilization and packaging steps. For example, both electron beams and gamma rays can effectively sterilize drugs at doses of 25-35 kGy. Alternatively, the materials can be treated by UV / vis irradiation (200-500 nm), for example using photoinitiators with different absorption wavelengths (e.g., Irgacure 184, 2959), preferably water-soluble initiators (e.g., Irgacure 2959). Such irradiation typically lasts for 1-60 min, but longer irradiation times may be applied depending on the specific method. The materials according to this disclosure can be finally aseptically wrapped to maintain sterility until use and packaged (e.g., by adding a specific product information leaflet) into suitable containers (boxes, etc.). The compounds can also be packaged under inert conditions (e.g., a deoxygenating or dehydrating atmosphere, such as nitrogen or argon) to preserve the compounds from degradation.

[0070] According to further embodiments, the compounds of the present invention may also be provided in kit form in combination with other components, including but not limited to those components necessary for using the material in a synthetic method or for administering the material to a patient. For example, the disclosed kits (such as those for treatment) may also include, for example, an administration material.

[0071] The compounds or compositions provided herein can be prepared and placed in containers for storage at ambient or elevated temperatures. When the compounds or compositions are stored in polyolefin plastic containers, discoloration is reduced compared to, for example, polyvinyl chloride plastic containers, whether suspended in a liquid composition (e.g., an aqueous solution or an organic liquid solution) or in solid form. Not wishing to be bound by theory, containers can reduce the exposure of the container contents to electromagnetic radiation, whether visible light (e.g., with wavelengths of about 380–780 nm) or ultraviolet (UV) light (e.g., with wavelengths of about 190–320 nm (UV B light) or about 320–380 nm (UV A light)). Some containers also include the ability to reduce the exposure of the container contents to infrared light, or a second component having this ability. Some containers also include the ability to reduce the exposure of the container contents to heat or humidity. Containers that can be used include those made of polyolefins, such as polyethylene, polypropylene, polyethylene terephthalate, polycarbonate, polymethylpentene, polybutene, or combinations thereof, especially polyethylene, polypropylene, or combinations thereof. In some embodiments, the container is a glass container, including but not limited to amber glass containers. The container may also be placed within a second container, such as a paper container, cardboard container, stiff cardboard container, metal film container, or aluminum foil container, or combinations thereof, to further reduce the exposure of the container contents to UV, visible, or infrared light. Articles that benefit from reduced discoloration, decomposition, or both during storage contain the phosphorus amides or dosage forms described herein, which contain the compounds or compositions described herein. The compounds or compositions provided herein may require storage for up to or longer than three months; in some cases, up to or longer than one year. The container may be any form suitable for containing the contents—for example, a bag, bottle, or box, or any combination thereof.

[0072] Implementation Implementation Method 1. A compound having the formula (I): (I) Or a pharmaceutically acceptable salt thereof, wherein R 1 It is hydrogen (H); R 2 This includes either oligonucleotides or peptides (i.e., 2 to 49 amino acid residues) or proteins (i.e., 50 or more amino acid residues); and R 3 R 4 and R 5Independently selected from, but not limited to, the following ligands: hydrogen (H), ETA, CPA, GalNAc, GluNAc, PGA, CA, UDA, DDA, DDA 12-OH, TDA, MA, PDA, PA, HDA, SA, SA 18-OH, SA 12-OH, SA 2-OH, ACA, BA, DHA, ARA, EPA, ALA, GLA, RA, OA, EA, or LA (see Table 1).

[0073] R 3 R 4 and R 5 It is independently linked to a ligand via a bond selected from, but not limited to, -O-, -NH-, -N(CH3)-, -SS-, -C(O)-O-, -OC(O)-, -C(O)-S-, -SC(O)-, -C(O)-N(H)-, -N(H)-C(O)-, -C(O)-N(CH3)-, -N(CH3)-C(O)-, -OC(O)-O-, -OC(O)-O-, -OC(O)-N(H)-, -N(H)-C(O)-O-, -OC(O)-N(CH3)-, -N(CH3)-C(O)-O-, -N(H)-C(O)-N(H)- and N(CH3)-C(O)-N(CH3)-.

[0074] Embodiment 2. The compound as described in Embodiment 1, wherein R 3 R 4 and R 5 They are the same.

[0075] Embodiment 3. The compound as described in Embodiment 1, wherein R 3 R 4 and R 5 They are different.

[0076] Embodiment 4. The compound as described in Embodiment 1, wherein R 3 R 4 and R 5 It is GalNAc, connected via -O-.

[0077] Embodiment 5. The compound as described in Embodiment 1, wherein R 2 It is an oligonucleotide, and R 3 R 4 and R 5 It is GalNAc, connected via -O-.

[0078] Implementation Method 6. The compound as described in Implementation Method 5, wherein the oligonucleotide is ribonucleic acid (e.g., siRNA).

[0079] Implementation Method 7. A compound having the formula (III): (III), Where R 1 It is a solid support material selected from, but not limited to, silica gel, controlled-porosity glass (CPG), or resins such as polystyrene (PS), or phosphoramides such as the 3-((diisopropylamino)phosphono)oxy)propionitrile moiety (abbreviated as DIPA CEP): ;R 2 It is triphenylmethyl, monomethoxytriphenylmethyl, dimethoxytriphenylmethyl, trimethoxytriphenylmethyl, monomethyltriphenylmethyl, dimethyltriphenylmethyl, trimethyltriphenylmethyl, monochlorotriphenylmethyl, dichlorotriphenylmethyl, trichlorotriphenylmethyl, methylsulfonyltriphenylmethyl, monomethoxymethylsulfonyltriphenylmethyl, dimethoxymethylsulfonyltriphenylmethyl, monomethoxydimethylsulfonyltriphenylmethyl, or trimethylsulfonyltriphenylmethyl; and R 3 R 4 and R 5 Independently selected from, but not limited to, the following ligands: hydrogen (H), ETA, CPA, GalNAc, GluNAc, PGA, CA, UDA, DDA, DDA12-OH, TDA, MA, PDA, PA, HDA, SA, SA18-OH, SA12-OH, SA2-OH, ACA, BA, DHA, ARA, EPA, ALA, GLA, RA, OA, EA, or LA (see Table 1), wherein each hydroxyl moiety of GalNAc, GluNAc, DDA12-OH, SA18-OH, SA12-OH, and SA2-OH is acetylated; R 3 R 4 and R 5 It is independently linked to a ligand via a bond selected from, but not limited to, -O-, -NH-, -N(CH3)-, -SS-, -C(O)-O-, -OC(O)-, -C(O)-S-, -SC(O)-, -C(O)-N(H)-, -N(H)-C(O)-, -C(O)-N(CH3)-, -N(CH3)-C(O)-, -OC(O)-O-, -OC(O)-O-, -OC(O)-N(H)-, -N(H)-C(O)-O-, -OC(O)-N(CH3)-, -N(CH3)-C(O)-O-, -N(H)-C(O)-N(H)- and N(CH3)-C(O)-N(CH3)-.

[0080] Embodiment 8. The compound as described in Embodiment 7, wherein R3 R 4 and R 5 They are the same.

[0081] Embodiment 9. The compound as described in Embodiment 7, wherein R 3 R 4 and R 5 They are different.

[0082] Embodiment 10. The compound as described in Embodiment 7, wherein R 3 R 4 and R 5 It is GalNAc, linked by -O-, where each hydroxyl moiety of GalNAc is acetylated.

[0083] Embodiment 11. A composition comprising a compound as described in any one of Embodiments 1-10 and a carrier, optionally wherein the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

[0084] Implementation 12. A method comprising administering to a subject in need a compound as described in any one of Implementations 1-10 (e.g., in the form of a pharmaceutical compound, i.e., not phosphoramidite) or a composition as described in Implementation 11.

[0085] Embodiment 13. An article comprising the compound as described in any one of claims 1-10 or the composition as described in Embodiment 11 and its instructions for use.

[0086] The compounds and methods described herein will be better understood by referring to the following examples, which are intended to illustrate the purpose of this specification and not to limit its scope.

[0087] Example The following selected examples describe certain techniques for specific and general synthetic methods used to produce pharmaceutically stable functional moieties and their siRNA conjugates as described herein, as well as certain analyses of the stability and activity of some of the compounds described herein. The synthesis and results of these examples are described in Figure 1-12 middle.

[0088] Example 1: A general method for synthesizing oligonucleotides containing multivalent ligands A. A general method for synthesizing multivalent ligand solid supports from Fmoc or ivDde AmC7 (DMT) CPG (controlled porosity glass) or PS (polystyrene) or CPSG (controlled porosity silica gel) or Nitto phase. Under DCM containing DMAP and DIPEA, Fmoc-β-high-lysine (Boc)-OH was reacted with Cbz-Cl to obtain a Cbz-protected intermediate, and the Fmoc protecting group was removed with TEA in isopropanol. Under EtOAc containing DIPEA, the free amine of the backbone was extended using Boc-D-lysine (Boc)-OPfp. After global deprotection of the Boc protecting group using 1,4-dioxane containing 4M HCl, the GalNAc-C5 acid was conjugated using an activated ester. After removing the Cbz protecting group by hydrogenation, the extension was performed using the AmC7 moiety under DCM / DMF containing BOP and DIPEA. Under DMF containing TEA, the free hydroxyl group was reacted with succinic anhydride to obtain a carboxylic acid form for supporting a solid support. The resulting acid was loaded onto an amino-functionalized CPG with HBTU, DMPA, and DIEA, and then capped with acetic anhydride. The loading capacity was measured by DMT quantification. Other compounds of formula (A) can be prepared similarly. See also Figure 8 , Figure 9 , Figure 10 and Figure 11 .

[0089] Alternatively, Fmoc- or ivDde-protected AmC7 (DMT) CPG is placed in a solid-phase reactor and rinsed with DCM and DMF. The Fmoc protecting group is removed using DMF containing 20% ​​4-methylpiperidine, and the ivDde protecting group is removed using DMF containing 4% hydrazine. The first β-amino acid is coupled in DMF containing HATU and DIPEA. Subsequent amino acids are then sequentially coupled to the backbone and / or side chains by repeating the N-terminal deprotection of the Fmoc or ivDde protecting group and coupling in DMF containing HATU and DIPEA until a targeted multivalent ligand is obtained. Loading capacity is measured by DMT quantification.

[0090] B. A general method for synthesizing oligonucleotides using multivalent ligand solid supports. Functionalized oligonucleotides (e.g., with a length of approximately 10 to 30 NTs) were synthesized on a multivalent ligand solid support using an automated oligonucleotide solid-phase synthesizer. Oligonucleotides containing multivalent ligands were synthesized using standard methods on the multivalent ligand solid support using phosphoramide technology. Depending on scale, MerMade 12 (Bioautomation), Dr. Oligonucleotide48 (Biolytic), or OligoPilot 100 (Cytiva) were used. All phosphoramides were purchased from, but not limited to, ChemGenes and Glen Research. All phosphoramides were dissolved in anhydrous acetonitrile and / or DMF and / or DCM at sufficient concentrations. The deblocking solution was selected from, but not limited to, inert solvents containing acetic acid, chloroacetic acid, dichloroacetic acid, trichloroacetic acid, or trifluoroacetic acid, such as DCM or toluene. The activator solution was selected from, but not limited to, acidic azole catalysts, including 1 H -Tetrazolium, 5-Ethylthio-1 H -Tetraazole (ETT) and 2-Benzylthio-1 H -Tetraazole (BTT) or 4,5-dicyanimidazole (DCI) or several similar compounds, dissolved in anhydrous acetonitrile at a sufficient concentration. The capping solution is selected from, but is not limited to, THF containing acetic anhydride and pyridine, and other compounds containing... N A mixture of methylimidazolium and acetonitrile. The oxidizing solution is selected from, but is not limited to, iodine-containing water, pyridine, and THF, as well as tert-butyl hydroperoxide and (1S)-(+)-(10-camphorsulfonyl)-oxazolidinium (CSO). The sulfiding solution is selected from, but is not limited to, 3-(dimethylaminomethylene)amino-3- H -1,2,4-Dithiazolyl-3-thione (DDTT), 3 H -1,2-benzodithiol-3-one 1,1-dioxide (Beaucage reagent), hydroflavin or N,N,N',N' -Tetraethylthiuram disulfide (TETD).

[0091] C. A general method for synthesizing polyvalent ligand phosphoramidites Synthesis of multivalent ligand phosphorusamides using solid-phase synthesis method 1: An AmC7 (DMT) solid support protected with Fmoc or ivDde was placed in a solid-phase reactor and rinsed with DCM and DMF. The Fmoc protecting group was removed using DMF containing 20% ​​4-methylpiperidine, and the ivDde protecting group was removed using DMF containing 4% hydrazine. The first β-amino acid was coupled in DMF containing HATU and DIPEA. Subsequent amino acids were then sequentially coupled to the backbone and / or side chains by repeating the N-terminal deprotection of the Fmoc or ivDde protecting group and the coupling reaction in DMF containing HATU and DIPEA until the targeted multivalent ligand was obtained. The solid support was then removed using ammonium hydroxide solution, and the resulting alcohol compound was converted to the multivalent ligand phosphorusamide via phosphorylation.

[0092] Method 2 for the synthesis of multivalent ligand phosphoramids via stepwise organic synthesis (see...) Figure 8 Fmoc-β-high-lysine (Boc)-OH was reacted with Cbz-Cl in DCM containing DMAP and DIPEA to obtain a Cbz-protected intermediate, and the Fmoc protecting group was removed with TEA in isopropanol. The free amine of the skeleton was extended using Boc-D-lysine (Boc)-OPfp in EtOAc containing DIPEA. After global deprotection of the Boc protecting group using 1,4-dioxane containing 4M HCl, the GalNAc-C5 acid was conjugated using an activated ester. After removing the Cbz protecting group by hydrogenation, the AmC7 moiety was extended in DCM / DMF containing BOP and DIPEA to obtain a substrate for phosphorylation. The final product, tri-GalNAc phosphorylamide, was obtained after reaction with ETT and 3-((bis(diisopropylamino)phosphono)oxy)propionitrile in DCM / DMF. Subsequently, the phosphoramidol corresponding to formula (IV) is used for oligonucleotide synthesis, wherein the oligonucleotide corresponds to formula (II). Other compounds of formula (A) are prepared similarly using corresponding alternative intermediates, for example, the acetyl group can be replaced by a base-instable protecting group, and DMTr can be replaced by an acid-instable protecting group.

[0093] D. A general method for synthesizing oligonucleotides using multivalent ligand phosphoramidite. UnyLinker CPG was placed in a synthesis column, and functionalized oligonucleotides were synthesized on a solid support using an automated oligonucleotide solid-phase synthesizer. The multivalent ligand phosphorous amide was dissolved in anhydrous acetonitrile and / or DCM and / or DMF at a sufficient concentration. Oligonucleotide synthesis was performed according to the general method for oligonucleotide synthesis shown in B.

[0094] E. A general method for the reverse synthesis of oligonucleotides followed by multivalent ligand synthesis. Functionalized oligonucleotides were reverse synthesized using an automated oligonucleotide solid-phase synthesizer, followed by post-synthesis using stepwise conjugation with β-amino acids, amino acids, and ligands under HATU, DIPEA, and DMF conditions. Oligonucleotides were reverse synthesized using standard methods on a UnyLinker solid support via phosphoramidite technology. Depending on scale, MerMade 12 (Bioautomation), Dr. Oligonucleotide48 (Biolytic), or OligoPilot 100 (Cytiva) were used. All reverse phosphoramidites were purchased from, but not limited to, ChemGenes and Glen Research. All reverse phosphoramidites were dissolved in anhydrous acetonitrile and / or DMF and / or DCM at sufficient concentrations. Deblocking solutions were selected from, but not limited to, inert solvents containing acetic acid, chloroacetic acid, dichloroacetic acid, trichloroacetic acid, or trifluoroacetic acid, such as DCM or toluene. Activator solutions were selected from, but not limited to, acidic azole catalysts, including 1 H -Tetrazolium, 5-Ethylthio-1 H -Tetraazole (ETT) and 2-Benzylthio-1 H -Tetraazole (BTT) or 4,5-dicyanimidazole (DCI) or several similar compounds, dissolved in anhydrous acetonitrile at a sufficient concentration. The capping solution is selected from, but is not limited to, THF containing acetic anhydride and pyridine, and other compounds containing... N A mixture of methylimidazolium and acetonitrile. The oxidizing solution is selected from, but is not limited to, iodine-containing water, pyridine, and THF, as well as tert-butyl hydroperoxide and (1S)-(+)-(10-camphorsulfonyl)-oxazolidinium (CSO). The sulfiding solution is selected from, but is not limited to, 3-(dimethylaminomethylene)amino-3- H -1,2,4-Dithiazolyl-3-thione (DDTT), 3 H -1,2-benzodithiol-3-one 1,1-dioxide (Beaucage reagent), hydroflavin or N,N,N', N’ -Tetraethylthiuram disulfide (TETD).

[0095] F. Double stranding of single-stranded RNA Carefully mix the sense and antisense chains in equimolar amounts and vortex for at least 30 seconds. After quantifying the sense and antisense chains in the method analysis, adjust the sense or antisense chain to ensure no residual single-chain material remains. Heat the double-chain solution to 85 °C for 3 minutes, gradually cool to room temperature, and then lyophilize.

[0096] Example 2: Stability test under protein digestion conditions 1 The stability of oligonucleotides containing tri-GalNAc conjugates was tested under protein digestion conditions. Test materials were prepared by doubling the sense and antisense strands. Test materials were prepared using 1X PBS (Gibco, 10010-023). 10 μL of 10 μM diluted test material was added to a mixture of 32.5 μL of lysis buffer (LGC Biosearch Technologies #MTC096H) and 2.5 μL of proteinase K (50 mg / mL), and the mixture was incubated at 37 °C for 1 hour, approximately 5 days, or approximately 7 days. After adding 2.5 μL of 3 M KCl, the sample was thoroughly mixed and vortexed, followed by incubation on ice for 10 minutes to precipitate SDS. After centrifugation at 10000g for 10 minutes at 4 °C, the supernatant (40 μL) was transferred to a clean, pre-chilled tube. Then, 10 μL of the mixture was mixed with 2 μL of 6x loading dye (Promega, G190A). A total of 12 μL was loaded onto a 12% non-denaturing PAGE and kept at a constant voltage of 120V for 30 minutes, followed by staining with GelRed (Biotuum, 41003) for 15 minutes.

[0097] All oligonucleotide samples containing β-amino acid conjugated ligands showed better stability under protein digestion conditions than oligonucleotide samples containing only D- or L-α-amino acid moieties (data not shown).

[0098] The stability of oligonucleotides containing tri-GalNAc conjugates was tested under mouse plasma, mouse serum, and rat triton bodies conditions. Six-week-old C57BL / 6 mice were purchased from KOATECH (Pyeongtaek, South Korea). After four weeks, mice were sacrificed and plasma and serum were separated. To prepare mouse plasma, blood was centrifuged at 2500 g for 15 minutes at RT using EDTA. To prepare mouse serum, blood was centrifuged at 2500 g for 15 minutes at RT. Each fraction was carefully separated from the centrifuged blood sample. Test materials were prepared by double-stranding of the sense and antisense strands. Test materials were prepared using 1X PBS (Gibco, 10010-023). 1 μL of 10 μM diluted test material was added to 9 μL of mouse plasma or mouse serum, and the mixture was incubated at 37 °C for 17 hours. For the rat tripton body assay, 1 μL of 10 μM diluted test material was added to a mixture of 5 μL rat tripton bodies (0.5 mg / mL), 1 μL catabolic buffer (10X), and 3 μL UPW, and the mixture was incubated at 37 °C for 5 days. Then, 10 μL of the mixture was mixed with 2 μL of 6x loading dye (Promega, G190A). A total of 12 μL was loaded onto a 12% non-denaturing PAGE, incubated at a constant voltage of 120 V for 30 minutes, and then stained with GelRed (Biotuum, 41003) for 15 minutes.

[0099] All oligonucleotide samples containing β-amino acid conjugated ligands showed better stability in mouse plasma, mouse serum, and rat triton bodies than oligonucleotide samples containing only D- or L-α amino acid moieties. Figure 1 This is a representative in vitro stability data under rat triponite conditions. The sequence of formula (VI), containing a tri-GalNAc conjugate with α-lysine, shows lower stability than sequences of formula (II) or (V). Sequences of formula (II) and (V) show similar in vitro stability data, with formula (V) containing an additional extension via γ-butyric acid (GABA). The synthetically efficient formula (II) has proven comparable to formula (V).

[0100] Example 5: In vitro test under mouse liver homogenate conditions 3 The stability of oligonucleotides containing tri-GalNAc conjugates was tested under mouse liver homogenate conditions. Six-week-old C57BL / 6 mice were purchased from KOATECH (Pyeongtaek, South Korea). After three weeks, mice were sacrificed and the entire liver (approximately 2.5 g) was isolated. To prepare the liver homogenate, the entire liver was completely homogenized and placed in 50 mL polycarbonate centrifuge tubes containing 10 mL of homogenization buffer (100 mM Tris, 1 mM magnesium acetate, pH 8.0). The liver homogenate was pre-incubated at 37 °C for 72 hours, followed by the addition of test materials. Test materials were prepared by double-stranding of the sense and antisense strands. Test materials were prepared using 1X PBS (Gibco, 10010-023). 1 μL of 10 μM diluted test material was added to 9 μL of liver homogenate, and the mixture was incubated at 37 °C for 24, 48, 72, and 96 hours. After incubation, the homogenate sample was mixed with 6x loading dye (Promega, G190A) and heated at 65 °C for 10 minutes. 3 μL of the sample was loaded onto a 10% non-denaturing PAGE and stained at a constant voltage of 100 V for 30 minutes, followed by staining with GelRed (Biotuum, 41003) for 5 minutes.

[0101] All oligonucleotide samples containing β-amino acid conjugated ligands showed better stability under mouse liver homogenization conditions than oligonucleotide samples containing only D- or L-α-amino acid moieties. Figure 2 This is a representative in vitro stability data under mouse liver homogenate conditions. The sequence of formula (VI), containing a tri-GalNAc conjugate with α-lysine, shows lower stability than sequences of formula (II) or (V). Sequences of formula (II) and (V) show similar in vitro stability data, with formula (V) containing an additional extension via γ-butyric acid (GABA). The synthetically efficient formula (II) has proven comparable to formula (V).

[0102] Example 8: In vitro KD targeting efficacy test of tri-GalNAc-conjugated oligonucleotide duplexes After treating primary human hepatocytes with each of the tri-GalNAc-conjugated oligonucleotide duplexes, qRT-PCR was performed on them to measure the expression levels of factor IX mRNA or factor VII mRNA. Specifically, primary human hepatocytes were treated at 3.0 × 10⁻⁶ mRNAs. 4Cells / well were seeded in 96-well plates and treated with factor IX or factor VII tri-GalNAc-conjugated oligonucleotide duplexes at 2, 20, and 200 nM. After 24 hours of treatment, cell lysates were prepared using the SuperPrep™ Cell Lysis & RT Kit II for qPCR (TOYOBO, SCQ-401), and cDNA was synthesized by reverse transcription using the mRNA contained in the lysates as a template. The synthesized cDNA was then used as a template for quantitative PCR, performed using the THUNDERBIRD probe qPCR MIX (TOYOBO, QPS-101), factor IX or factor VII probes, and RNA18S5 probe (Thermofisher, Hs03928985_g1). The expression levels of factor IX or factor VII mRNA were then identified using the CFX ligation real-time PCR system (Bio-Rad). In this example, the untreated group (NT) served as a control. Data showed that... Figure 3 and Figure 4 middle.

[0103] Example 6: In vivo testing of tri-GalNAc-conjugated oligonucleotide duplexes 1 Six-week-old C57BL / 6 mice were purchased from KOATECH (Pyeongtaek, South Korea). Each test group had n=3 mice. After a one-week acclimatization period, oligonucleotide duplexes were administered subcutaneously on day 0 at a single injection dose of 3 mg / kg. The oligonucleotide duplexes were prepared using 1X PBS (Gibco, 10010-023). Mouse plasma was collected from facial veins using an animal lancet (Medipoint, GR-5). Immediately after collection, the blood was mixed with 0.109 M trisodium citrate solution (Sigma, S1804) at a 9:1 ratio. The anticoagulated blood was centrifuged at 2,500 g for 15 min at room temperature. Mouse plasma was collected from the supernatant and stored at -80°C. Mouse plasma was collected on days 0 (before oligonucleotide duplex injection), 7, 14, 21, 28, 35, 42, 49, 56, 63, and 70. Factor IX levels in mouse plasma were analyzed using Biophen Factor IX (HYPHEN BioMed, 221806-RUO) according to the manufacturer's instructions. Factor IX levels for each mouse at different time points were normalized to the factor IX level on day 0 of the same individual.

[0104] All oligonucleotide samples containing β-amino acid conjugated ligands showed better stability and potency than oligonucleotide samples containing only D- or L-α-amino acid moieties. Figure 5The in vivo factor IX potency of oligonucleotide 1 with tri-GalNAc conjugate was demonstrated.

[0105] Example 7: In vivo testing of tri-GalNAc-conjugated oligonucleotide duplexes 2 Six-week-old C57BL / 6 mice were purchased from KOATECH (Pyeongtaek, South Korea). Each test group had n=3 mice. After a one-week acclimatization period, oligonucleotide duplexes were injected once daily (SC) at a dose of 3 mg / kg on day 0. The oligonucleotide duplexes were prepared using 1X PBS (Gibco, 10010-023). Mouse plasma was collected from facial veins using an animal lancet (Medipoint, GR-5). Immediately after collection, the blood was mixed with 0.109 M trisodium citrate solution (Sigma, S1804) at a 9:1 ratio. The anticoagulated blood was centrifuged at 2,500 g for 15 min at room temperature. Mouse plasma was collected from the supernatant and stored at -80 °C. Mouse plasma was collected on days 0 (before oligonucleotide duplex injection), 7, 14, 21, 28, 35, 42, 49, 56, 63, and 70. Factor VII levels in mouse plasma were analyzed using Biophen Factor VII (HYPHEN BioMed, 221304-RUO) according to the manufacturer's instructions. Factor VII levels for each mouse at different time points were normalized to the factor VII level on day 0 of the same individual.

[0106] All oligonucleotide samples containing β-amino acid conjugated ligands showed better stability and potency than oligonucleotide samples containing only D- or L-α-amino acid moieties. Figure 6 The in vivo factor VII potency of oligonucleotide 2 with tri-GalNAc conjugate was demonstrated.

[0107] Example 8: In vitro test under monkey liver homogenate conditions 4 The stability of oligonucleotides containing tri-GalNAc conjugates was tested under homogenized monkey liver conditions. Male cynomolgus monkeys aged 2–4 years were purchased from KITOX (Daejeon, South Korea). The monkeys were euthanized after 3 weeks. To prepare the liver homogenate, the left lobe of the liver was completely homogenized and placed in a 50 mL polycarbonate centrifuge tube containing homogenization buffer (100 mM Tris-acetate, pH 8.0; NBB-2414, Novatein Bio. / 1 mM magnesium acetate, pH 8.0; 100 mg / mL monkey liver homogenate). The homogenate was aliquoted into 400 μL units in 1.5 mL microcentrifuge tubes. Before using the homogenate for stability testing, the monkey liver homogenate was pre-incubated at 37 °C for 72 h, and the supernatant was transferred to a new 1.5 mL tube, followed by the addition of the test material. The test material was prepared using 1X PBS (Gibco, 10010-024). Test material was prepared by doubling the sense and antisense strands. 1 μL of a 10 μM diluted test material was added to 9 μL of monkey liver homogenate, and the mixture was incubated at 37 °C for 1, 2, 3, and 7 days. After incubation, the homogenate sample was mixed with 2 μL of 6X loading dye (Promega, G190A) to form a 10 μL resuspended sample. 3 μL of the sample was loaded onto a 12% non-denaturing PAGE gel and incubated at a constant voltage of 100 V for 40 min, followed by staining with GelRed (Biotuum, 41003) for 5 min. The gel was imaged using ChemiDoc XRS+.

[0108] All oligonucleotide samples containing β-amino acid conjugated ligands showed better stability under monkey liver homogenization conditions than oligonucleotide samples containing only D- or L-α-amino acid moieties. Figure 7 This is a representative in vitro stability data under monkey liver homogenate conditions. The sequence of formula (VI), containing the tri-GalNAc conjugate with α-lysine, shows lower stability than sequences of formula (II) or (V). Sequences of formula (II) and (V) show similar in vitro stability data, with formula (V) containing an additional extension via γ-butyric acid (GABA). The synthetically efficient formula (II) has proven comparable to formula (V).

[0109] Comparative Example 1: Synthesis of phosphoramids containing tri-GalNAc conjugates of β-lysine and GABA (VII) Phosphoramide (VII) containing a tri-GalNAc conjugate of β-lysine and GABA was synthesized via a similar synthetic pathway using β-lysine and γ-aminobutyric acid extension. This phosphoramide was then used for oligonucleotide synthesis, wherein the oligonucleotide contained formula (V).

[0110] The discovery will be as follows Figure 8 The phosphoramidite compound (e.g., formula (VIII) below) prepared in the preparation of oligonucleotides conjugated thereto will yield oligonucleotide conjugates that are at least as stable or more stable (e.g., have comparable or enhanced resistance to enzymatic degradation in vitro / in vivo) and at least as effective or more efficient (e.g., have comparable or enhanced delivery, e.g., cellular delivery) as oligonucleotide conjugates prepared by formula (VII).

[0111] Comparative Example 2: Synthesis of phosphoramids from tri-GalNAc conjugates containing D-α-lysine (VIII) Phosphoramide (VIII) containing a tri-GalNAc conjugate with D-α-lysine was synthesized via a similar synthetic pathway in place of β-lysine. This phosphoramide was then used for the synthesis of oligonucleotides containing formula (VI).

[0112] Table 4 provides sequence information for some implementations of the oligonucleotides referred to herein. Figure 12 Oligonucleotide 1 and oligonucleotide 2 are described as double-stranded pairs of SEQ ID NO:1 and SEQ ID NO:2, and SEQ ID NO:3 and SEQ ID NO:4. The abbreviations refer to the following: =Thiophosphate ester / empty = phosphate ester; (mA), (mC), (mG), (mU) = 2'-O-methyl nucleotide; (fA), (fC), (fG), (fU) = 2'-deoxy-2'-fluoronucleotide; (Conjugate) = a tri-GalNAc conjugate derived from a selected formula (e.g., formula (A), formula (II), formula (V), or formula (VI), etc.); and (EVP-mU) = 5'-(E)-vinylphosphonate 2'-O-methyluridine nucleotide with the following structure: .

[0113] Table 4: Sequence Information

Claims

1. A compound having the following formula: (A) or its salt, in R 1 It is a phosphoramide (e.g., 3-((diisopropylamino)phosphono)oxy)propionitrile moiety (abbreviated as DIPA CEP): ）、 -R 7 -(solid support) (e.g., said solid support is selected from, but not limited to, silica gel, controlled porosity glass (CPG) (e.g., long-chain alkyl-amine CPG) or resin (e.g., polystyrene)) or hydrogen (H); R 2 This includes, or acid-labile protecting groups (e.g., triphenylmethyl, monomethoxytriphenylmethyl, dimethoxytriphenylmethyl (also abbreviated as DMTr), trimethoxytriphenylmethyl, monomethyltriphenylmethyl, dimethyltriphenylmethyl, trimethyltriphenylmethyl, monochlorotriphenylmethyl, dichlorotriphenylmethyl, trichlorotriphenylmethyl, methylsulfonyltriphenylmethyl, monomethoxymethylsulfonyltriphenylmethyl, dimethoxymethylsulfonyltriphenylmethyl, monomethoxydimethylsulfonyltriphenylmethyl or trimethylsulfonyltriphenylmethyl), oligonucleotides (e.g., having or not having protecting groups from a standard cyanoethylphosphoramide oligonucleotide (e.g., DNA or RNA, optionally single-stranded or double-stranded) solid support), peptides (i.e., having or not having 2 to 49 amino acid residues from a standard FMOC amide solid support), proteins (i.e., having or not having 50 or more amino acid residues from a standard FMOC amide solid support), or H; Or R 1 It is an acid-instable protecting group (e.g., triphenylmethyl, monomethoxytriphenylmethyl, dimethoxytriphenylmethyl (also abbreviated as DMTr), trimethoxytriphenylmethyl, monomethyltriphenylmethyl, dimethyltriphenylmethyl, trimethyltriphenylmethyl, monochlorotriphenylmethyl, dichlorotriphenylmethyl, trichlorotriphenylmethyl, methylsulfonyltriphenylmethyl, monomethoxymethylsulfonyltriphenylmethyl, dimethoxymethylsulfonyltriphenylmethyl, monomethoxydimethylsulfonyltriphenylmethyl or trimethylsulfonyltriphenylmethyl), and R 2 This includes oligonucleotides (e.g., those with or without protecting groups synthesized from a standard cyanoethylphosphoramide oligonucleotide (e.g., DNA or RNA, optionally single-stranded or double-stranded) solid support), peptides (i.e., 2 to 49 amino acid residues with or without protecting groups synthesized from a standard FMOC amide solid support), proteins (i.e., 50 or more amino acid residues with or without protecting groups synthesized from a standard FMOC amide solid support), and those covalently linked to a solid support (e.g., directly or through a linker, as described above R). 1 As shown, for example, -R 7 - (solid support) oligonucleotides, peptides or proteins; R 3 R 4 and R 5 Selected independently , , , , , , , , , , , , , , , , , , , , , , , , , , , , Or H; Each R 6 The group is independently selected from H, acyl groups (including but not limited to acetyl, chloroacetyl, trichloroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, 4-isopropylphenoxyacetyl or neopentanoyl), silyl groups (including but not limited to trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBDMS), triphenylsilyl (TPS or tert-butyldiphenylsilyl (TBDPS)), carbonate groups (including but not limited to 2-cyanoethylcarbonyl, 2-(2,4-dinitrophenyl)ethoxycarbonyl, 2-(p-nitrophenyl)ethoxycarbonyl or fluorenylmethoxycarbonyl), or benzyl groups (including but not limited to benzyl, 2-nitrobenzyl or 4-nitrobenzyl). R 7 yes ； R 8 It is O or NH (i.e., R7 and R8 together with the atoms to which they are attached form a moiety, said moiety comprising an ester and an ester at each end of said moiety, or an ester and an amide at one of the ends of said moiety); and n is 2, 3, 4, 5, or 6 or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20).

2. The compound of claim 1, wherein: R 1 Yes -R 7 -(Solid support) (e.g., a solid support selected from silica gel, controlled porosity glass (CPG), or resin); R 2 It is dimethoxytriphenylmethyl (DMTr), triphenylmethyl, monomethoxytriphenylmethyl, trimethoxytriphenylmethyl, monomethyltriphenylmethyl, dimethyltriphenylmethyl, trimethyltriphenylmethyl, monochlorotriphenylmethyl, dichlorotriphenylmethyl, trichlorotriphenylmethyl, methylsulfonyltriphenylmethyl, monomethoxymethylsulfonyltriphenylmethyl, dimethoxymethylsulfonyltriphenylmethyl, monomethoxydimethylsulfonyltriphenylmethyl, or trimethylsulfonyltriphenylmethyl; and Each R 6 It is independently selected from acetyl, chloroacetyl, trichloroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, 4-isopropylphenoxyacetyl, neopentanoyl, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBDMS), triphenylsilyl (TPS), tert-butyldiphenylsilyl (TBDPS), 2-cyanoethylcarbonyl, 2-(2,4-dinitrophenyl)ethoxycarbonyl, 2-(p-nitrophenyl)ethoxycarbonyl, fluorenylmethoxycarbonyl, benzyl, 2-nitrobenzyl, or 4-nitrobenzyl.

3. The compound of claim 1, wherein: R 1 Yes -R 7 -(Solid support) (e.g., a solid support selected from silica gel, controlled porosity glass (CPG), or resin); R 2 It is DMTr; and R 6 It is an acetyl group.

4. The compound of claim 1, wherein: R 1 It is dimethoxytriphenylmethyl (DMTr), triphenylmethyl, monomethoxytriphenylmethyl, trimethoxytriphenylmethyl, monomethyltriphenylmethyl, dimethyltriphenylmethyl, trimethyltriphenylmethyl, monochlorotriphenylmethyl, dichlorotriphenylmethyl, trichlorotriphenylmethyl, methylsulfonyltriphenylmethyl, monomethoxymethylsulfonyltriphenylmethyl, dimethoxymethylsulfonyltriphenylmethyl, monomethoxydimethylsulfonyltriphenylmethyl, or trimethylsulfonyltriphenylmethyl; and R 2 This includes oligonucleotides, peptides, or proteins, which are covalently linked to a solid support (e.g., -R). 7 -(solid support)); and Each R 6 It is independently selected from acetyl, chloroacetyl, trichloroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, 4-isopropylphenoxyacetyl, neopentanoyl, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBDMS), triphenylsilyl (TPS), tert-butyldiphenylsilyl (TBDPS), 2-cyanoethylcarbonyl, 2-(2,4-dinitrophenyl)ethoxycarbonyl, 2-(p-nitrophenyl)ethoxycarbonyl, fluorenylmethoxycarbonyl, benzyl, 2-nitrobenzyl, or 4-nitrobenzyl.

5. The compound of claim 1, wherein: R 1 It is DMTr; R 2 It is covalently linked to a solid support (e.g., -R) 7 Oligonucleotides, peptides, or proteins (with solid support); and R 6 It is an acetyl group.

6. The compound of claim 1, wherein: R 1 It is DMTr; R 2 It is covalently linked to a solid support (e.g., -R) 7 Oligonucleotides, peptides, or proteins (with solid support); and R 6 It is H.

7. The compound of claim 1, wherein: R 1 It is H; R 2 It is covalently linked to a solid support (e.g., -R) 7 Oligonucleotides, peptides, or proteins (with solid support); and R 6 It is an acetyl group.

8. The compound of claim 1, wherein: R 1 It is H; R 2 It is covalently linked to a solid support (e.g., -R) 7 Oligonucleotides, peptides, or proteins (with solid support); and R 6 It is H.

9. The compound of claim 1, wherein: R 1 It is H; R 2 It is H; and R 6 It is H.

10. The compound of claim 1, wherein: R 1 It is H; R 2 It is H; and R 6 It is an acetyl group.

11. The compound of claim 1, wherein: R 1 It is H; R 2 It is H; and Each R 6 It is independently selected from acetyl, chloroacetyl, trichloroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, 4-isopropylphenoxyacetyl, neopentanoyl, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBDMS), triphenylsilyl (TPS), tert-butyldiphenylsilyl (TBDPS), 2-cyanoethylcarbonyl, 2-(2,4-dinitrophenyl)ethoxycarbonyl, 2-(p-nitrophenyl)ethoxycarbonyl, fluorenylmethoxycarbonyl, benzyl, 2-nitrobenzyl, or 4-nitrobenzyl.

12. The compound of claim 1, wherein: R 1 It is H; R 2 It is dimethoxytriphenylmethyl (DMTr), triphenylmethyl, monomethoxytriphenylmethyl, trimethoxytriphenylmethyl, monomethyltriphenylmethyl, dimethyltriphenylmethyl, trimethyltriphenylmethyl, monochlorotriphenylmethyl, dichlorotriphenylmethyl, trichlorotriphenylmethyl, methylsulfonyltriphenylmethyl, monomethoxymethylsulfonyltriphenylmethyl, dimethoxymethylsulfonyltriphenylmethyl, monomethoxydimethylsulfonyltriphenylmethyl, or trimethylsulfonyltriphenylmethyl.

13. The compound of claim 1, wherein: R 1 It is H; and R 2 It is DMTr.

14. The compound of claim 1, wherein: R 1 It is H; R 2 It is DMTr; and Each R 6 It is independently selected from acetyl, chloroacetyl, trichloroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, 4-isopropylphenoxyacetyl, neopentanoyl, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBDMS), triphenylsilyl (TPS), tert-butyldiphenylsilyl (TBDPS), 2-cyanoethylcarbonyl, 2-(2,4-dinitrophenyl)ethoxycarbonyl, 2-(p-nitrophenyl)ethoxycarbonyl, fluorenylmethoxycarbonyl, benzyl, 2-nitrobenzyl, or 4-nitrobenzyl.

15. The compound of claim 1, wherein: R 1 It is H; R 2 It is DMTr; and R 6 It is an acetyl group.

16. The compound of claim 1, wherein: R 1 It is DIPA CEP; R 2 It is triphenylmethyl, monomethoxytriphenylmethyl, dimethoxytriphenylmethyl (also abbreviated as DMTr), trimethoxytriphenylmethyl, monomethyltriphenylmethyl, dimethyltriphenylmethyl, trimethyltriphenylmethyl, monochlorotriphenylmethyl, dichlorotriphenylmethyl, trichlorotriphenylmethyl, methylsulfonyltriphenylmethyl, monomethoxymethylsulfonyltriphenylmethyl, dimethoxymethylsulfonyltriphenylmethyl, monomethoxydimethylsulfonyltriphenylmethyl, or trimethylsulfonyltriphenylmethyl; and Each R 6 It is independently selected from acetyl, chloroacetyl, trichloroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, 4-isopropylphenoxyacetyl, neopentanoyl, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBDMS), triphenylsilyl (TPS), tert-butyldiphenylsilyl (TBDPS), 2-cyanoethylcarbonyl, 2-(2,4-dinitrophenyl)ethoxycarbonyl, 2-(p-nitrophenyl)ethoxycarbonyl, fluorenylmethoxycarbonyl, benzyl, 2-nitrobenzyl, or 4-nitrobenzyl.

17. The compound of claim 1, wherein: R 1 It is DIPA CEP; R 2 It is DMTr; and R 6 It is an acetyl group.

18. A composition comprising a compound and a carrier as described in any one of claims 1 to 17, wherein the composition is optionally a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

19. An article comprising a compound as claimed in any one of claims 1 to 17 or a composition as claimed in claim 18 and its instructions for use, optionally wherein the article is for therapeutic purposes or for use in the synthesis of solid support.

20. Use of the compound, composition or article of any one of claims 1 to 19 in the preparation of a medicament optionally used as an oligonucleotide therapy or in the synthesis of a solid support.

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