Targeted ligand

Abstract

The document describes compounds and novel targeted ligands that can be linked to therapeutic compounds useful for directing the compounds to their in vivo targets. [Solution] The targeted ligands disclosed herein are useful for targeting expression inhibitory oligomeric compounds, such as RNAi agents, to liver cells in order to regulate gene expression. When bound to therapeutic compounds, the targeted ligands disclosed herein can be used in a variety of applications, including therapeutic, diagnostic, target validation, and genome discovery applications. Compositions comprising the targeted ligands disclosed herein, when bound to expression inhibitory oligomeric compounds, can mediate the expression of target nucleic acid sequences in liver cells, such as hepatocytes, and may be useful in treating diseases or disorders that respond to inhibition of gene expression or activity in cells, tissues, or organisms.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 383,221, filed on 2 September 2016, and U.S. Provisional Patent Application No. 62 / 456,339, filed on 8 February 2017, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] background Many compounds need to be delivered to a specific location (e.g., to a desired number of cells) in order to have a therapeutic effect or be useful for diagnostic purposes. This is frequently the case when attempting to deliver therapeutic compounds in vivo. Furthermore, the ability to efficiently deliver a compound to a specific location limits or potentially eliminates unintended consequences (such as off-target effects) that may be caused by the administration of the compound. One way to facilitate the delivery of compounds, such as therapeutic compounds, to a desired location in vivo is to ligate or attach the compound to a targeted ligand.

[0003] Oligomers are a class of therapeutic compounds that can be targeted using targeted ligands. Oligomers containing nucleotide sequences at least partially complementary to a target nucleic acid have been shown to alter the function and activity of the target both in vitro and in vivo. When delivered to cells containing a target nucleic acid (such as mRNA), oligomers have been shown to modulate the expression of the target, resulting in altered transcription or translation of the target nucleic acid. In certain cases, the oligomers may reduce gene expression by inhibiting the nucleic acid target and / or causing degradation of the target nucleic acid.

[0004] If the target nucleic acid is mRNA, one mechanism by which expression-inhibiting oligomer compounds can regulate the expression of mRNA targets is through RNA interference. RNA interference is a biological process in which RNA or RNA-like molecules (such as chemically modified RNA molecules) can suppress gene expression through degradation. Post-transcriptional gene silencing is considered an evolutionarily conserved cellular defense mechanism used to prevent the expression of foreign genes.

[0005] Synthetic RNA and RNA-like molecules have been shown to induce RNA interference in vivo. For example, Elbashir et al. (Nature 2000, 411, 494-98) described RNAi induced by the introduction of a double-stranded 21-nucleotide synthetic RNA molecule in cultured mammalian cells. The type of synthetic RNA or RNA-like molecule that may trigger an RNAi response mechanism may consist of modified nucleotides and / or one or more non-phosphodiester bonds.

[0006] Furthermore, single-stranded RNA and RNA-like molecules (which may contain modified nucleotides and have one or more non-phosphodiester bonds) may also alter the expression of target nucleic acids, such as target mRNA. [Overview of the project]

[0007] overview This specification discloses targeted ligands that may facilitate the delivery of therapeutic compounds to specific organs or tissues within the body of a subject, such as a human patient or an animal, for example, to a specific target site. In some embodiments, the targeted ligands described herein may facilitate the targeted delivery of an expression inhibitory oligomer compound. In some embodiments, the targeted ligand facilitates the delivery of an expression inhibitory oligomer compound to the liver.

[0008] In some embodiments, the targeted ligands disclosed herein include, consist of, or substantially consist of, one or more targeted moieties, one or more tethers, one or more branching groups, and one or more linkers. Linkers suitable for use in the targeted ligands disclosed herein include “rigid” linkers, which provide sufficient stability and rigidity to the entire targeted ligand to reduce potential interactions between the one or more targeted moieties and the therapeutic compound to which they are linked. In addition, “rigid” linkers suitable for use in the targeted ligands disclosed herein are useful for efficiently synthesizing the targeted ligands as phosphoramidite compounds (also referred to herein as “phosphoramidite-containing compounds”).

[0009] In some embodiments, the targeted ligands disclosed herein include, consist of, or substantially consist of, one or more branched groups having one or more targeted moieties, one or more tethers, and linker-substituted moieties. The linker-substituted moieties are located within the branched group and include, consist of, or substantially consist of, one or more substituted or unsubstituted cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl groups, or combinations thereof covalently linked. Having linker-substituted moieties within the branched group provides sufficient stability and rigidity to the entire targeted ligand, thereby giving it properties similar to those of the “rigid” linkers disclosed herein. In addition, branched groups having linker-substituted moieties suitable for use in targeted ligands are useful for efficiently synthesizing targeted ligands as phosphoramidite compounds.

[0010] Disclosed herein are targeted ligands comprising a linker, a branching point, one or more tethers, and one or more targeting moieties, comprising, consisting of, or substantially consisting of the structure of Formula I:

[0011] [ka] {wherein n is an integer between 1 and 4 (for example, 1, 2, 3, or 4), and the linker is a structure selected from the following group:

[0012] [ka]

[0013] [ka]

[0014] (wherein n' is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and if present, each Z' is independently selected from the group consisting of: C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted or unsubstituted amino, carboxyl, C1-C6 alkoxy, substituted C1-C6 alkyl, C1-C6 aminoalkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl, substituted C1-C6 alkoxy, substituted C1-C6 aminoalkyl, halogen (e.g., F), hydroxyl, amide, substituted amide, cyano, substituted or unsubstituted keto, substituted or unsubstituted alkoxycarbonyl, substituted or unsubstituted aryloxycarbonyl, substituted or unsubstituted heteroaryloxycarbonyl, or sulfhydryl) (Structure 7);

[0015] [ka]

[0016] (wherein n'' is 0, 1, 2, 3, 4 (e.g., 1, 2, 3, or 4), and if present, each Z'' is independently selected from the group consisting of: C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, substituted C1-C6 alkyl, C1-C6 aminoalkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl, substituted or unsubstituted amino, carboxyl, substituted C1-C6 alkoxy, substituted C1-C6 aminoalkyl, halogen (e.g., F), hydroxyl, amide, substituted amide, cyano, substituted or unsubstituted keto, substituted or unsubstituted alkoxycarbonyl, substituted or unsubstituted aryloxycarbonyl, substituted or unsubstituted heteroaryloxycarbonyl, or sulfhydryl) (Structure 8); and

[0017] [ka]

[0018] (wherein V is one or more substituted or unsubstituted cycloalkyls (e.g., cyclohexyl, cyclopropyl, cyclobutyl, cyclopentyl, cycloheptyl, cyclooctyl, etc.), substituted or unsubstituted cycloalkenyls (e.g., cyclohexenyl, cyclobutenyl, cyclopentenyl, cycloheptenyl, cyclooctenyl, cyclohexadienyl, cyclopentadienyl, cycloheptadienyl, cyclooctadienyl, etc.), substituted or unsubstituted aryls (e.g., phenyl, naphthyl, binaphthyl, anthracenyl, etc.), substituted or unsubstituted heteroaryls (e.g., pyridyl, pyrimidinyl, pyrrole, imidazole, furan, benzofuran, indole, etc.), or substituted or unsubstituted heterocyclyls (e.g., tetrahydrofuran, tetrahydropyran, piperidine, pyrrolidine, etc.), or any combination thereof linked by covalent bonds) (Structure 9)

[0019] In some embodiments, the targeted ligand includes a branching group having a linker substitution moiety. This specification discloses targeted ligands comprising, consisting of, or substantially consisting of a structure of Formula II, comprising a branching point group having a linker substitution moiety, one or more tethers, and one or more targeting moieties:

[0020] [ka]

[0021] {wherein n is an integer from 1 to 4 (e.g., 1, 2, 3, or 4), and in the formula one or more substituted or unsubstituted cycloalkyls (e.g., cyclohexyl, cyclopropyl, cyclobutyl, cyclopentyl, cycloheptyl, cyclooctyl, etc.), substituted or unsubstituted cycloalkenyls (e.g., cyclohexenyl, cyclobutenyl, cyclopentenyl, cycloheptenyl, cyclooctenyl, cyclohexadienyl, cyclopentadienyl, cycloheptadienyl, cyclo} The linker substitution moiety, including looctadinyl, substituted or unsubstituted aryls (e.g., phenyl, naphthyl, binaphthyl, anthracenyl), substituted or unsubstituted heteroaryls (e.g., pyridyl, pyrimidinyl, pyrrole, imidazole, furan, benzofuran, indole), or substituted or unsubstituted heterocyclyls (e.g., tetrahydrofuran, tetrahydropyran, piperidine, pyrrolidine), or any combination thereof, is located within the branching point group.

[0022] The targeted ligands disclosed herein may be directly or indirectly ligated to compounds such as therapeutic compounds, for example, to the 3' or 5' end of an expression inhibitory oligomer compound. In some embodiments, the expression inhibitory oligomer compound may be one or more modified nucleotides. In some embodiments, the expression inhibitory oligomer compound may be an RNAi agent, such as a double-stranded RNAi agent. In some embodiments, the targeted ligands disclosed herein are ligated to the 5' end of the sense strand of the double-stranded RNAi agent. In some embodiments, the targeted ligands disclosed herein are ligated to the RNAi agent at the 5' end of the sense strand of the double-stranded RNAi agent via a phosphate group, a phosphorothioate group, or a phosphonate group.

[0023] The targeted ligands disclosed herein comprise one or more targeted moieties. In some embodiments, the targeted ligands disclosed herein comprise N-acetylgalactosamine as the targeted moiety. In some embodiments, the targeted ligands disclosed herein have a structure represented by the following structural formula:

[0024] [ka] (Structure 1003);

[0025] [ka] (Structure 1008);

[0026] [ka] (Structure 1023); or

[0027] [ka] (Structure 1027).

[0028] This specification discloses compositions comprising, or substantially comprising, targeted ligands and expression inhibitory oligomer compounds. This specification also discloses compositions comprising targeted ligands and RNAi agents.

[0029] In some embodiments, the compositions disclosed herein, comprising a targeted ligand and an RNAi agent, have a structure represented by the following structural formula:

[0030] [ka] {wherein R comprises or consists of an expression-inhibiting oligomer compound} (structure 1002a);

[0031] [ka] {In the formula, R comprises or consists of an expression-inhibiting oligomer compound} (Structure 1003a);

[0032] [ka] {wherein R comprises or consists of an expression-inhibiting oligomer compound} (structure 1005a);

[0033] [ka] {wherein R comprises or consists of an expression-inhibiting oligomer compound} (Structure 1008a);

[0034] [ka] {wherein R comprises or consists of an expression inhibitory oligomer compound} (structure 1012a); or

[0035] [ka] {In the formula, R comprises or consists of an expression-inhibiting oligomer compound} (structure 1027a).

[0036] Phosphoramidite compounds containing targeted ligands are disclosed herein. In some embodiments, the phosphoramidite compounds containing the targeted ligands disclosed herein have a structure represented by the following structural formula:

[0037] [ka] (Structure 1001b);

[0038] [ka] (Structure 1002b);

[0039] [ka] (Structure 1003b);

[0040] [ka] (Structure 1004b);

[0041] [ka] (Structure 1005b);

[0042] [ka] (Structure 1006b);

[0043] [ka] (Structure 1007b);

[0044] [ka] (Construct 1008b);

[0045]

change

[0046]

change

[0047]

change

[0048]

change

[0049]

change

[0050]

change

[0051]

change

[0052]

change

[0053]

change

[0054] [ka] (Structure 1019b);

[0055] [ka] (Structure 1020b);

[0056] [ka] (Structure 1021b);

[0057] [ka] (Structure 1022b);

[0058] [ka] (Structure 1023b);

[0059] [ka] (Structure 1024b);

[0060] [ka] (Structure 1025b);

[0061] [ka] (structure 1026b); or

[0062] [ka] (Structure 1027b).

[0063] Pharmaceutical compositions comprising the targeted ligands disclosed herein are also disclosed. Disclosed is a method for treating a disease or disorder that would benefit from the administration of a compound, the method comprising administering a compound linked to a targeted ligand as disclosed herein. A method for inhibiting the expression of a target nucleic acid in a subject is disclosed herein, comprising administering a therapeutic dose of an expression-inhibiting oligomer compound linked to a targeted ligand as disclosed herein. A method for delivering an expression inhibitory oligomer compound to the liver in vivo is disclosed herein, the method comprising administering the expression inhibitory oligomer compound linked to a targeted ligand disclosed herein.

[0064] Disclosed herein is a process or method for producing a phosphoramidite compound containing a targeted ligand, comprising (i) covalently linking a linker to a branching group, and (ii) linking the linker to a phosphorus atom of a phosphoramidite by a phosphytylation reaction using a phosphoramidite-forming reagent, thereby forming a phosphoramidite compound.

[0065] Where used herein, the term “linked” refers to a connection between two molecules, meaning that the two molecules are joined by a covalent bond or that the two molecules are joined by a non-covalent bond (e.g., a hydrogen bond or an ionic bond). In some examples where the term “linked” refers to a connection between two molecules via a non-covalent bond, the connection between the two different molecules is 1 × 10⁻¹⁶ in a physiologically acceptable buffer (e.g., phosphate-buffered saline). -4 Less than M (for example, 1 × 10) -5 Less than M, 1 x 10 -6 Less than M, or 1 × 10 -7 K (less than M) D It has.

[0066] As used herein, the term “directly linked” refers to a first compound or group linked to a second compound or group without the interposition of any atoms or groups of atoms. As used herein, the term “indirectly linked” refers to a first compound linked to a second compound or group by an intermediary group, compound, or molecule, such as a linking group. Unless otherwise specified, as used herein, the term “linked” includes both “directly linked” and “indirectly linked” as defined herein.

[0067] As used herein, “oligomer compound” is a nucleotide sequence containing approximately 10 to 50 nucleotides or nucleotide base pairs. In some embodiments, the oligomer compound has a nucleic acid base sequence that is at least partially complementary to the coding sequence in a target nucleic acid or target gene expressed in a cell. In some embodiments, upon delivery to a gene-expressing cell, the oligomer compound can inhibit the expression of the causative gene and is therefore referred to herein as an “expression-inhibiting oligomer compound.” Gene expression can be inhibited in vitro or in vivo. Examples of “oligomer compounds” include, but are not limited to, oligonucleotides, single-stranded oligonucleotides, single-stranded antisense oligonucleotides, small interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), small hairpin RNA (shRNA), ribozymes, interfering RNA molecules, and Dicer substrates.

[0068] As used herein, the term “oligonucleotide” means a polymer of linked nucleosides, each of which may be independently modified or unmodified. As used herein, the term “single-stranded oligonucleotide” means a single-stranded oligomeric compound having a sequence that is at least partially complementary to the target mRNA, i.e., capable of hybridizing to the target mRNA by hydrogen bond formation under mammalian physiological conditions (or comparable in vitro conditions). In some embodiments, the single-stranded oligonucleotide is a single-stranded antisense oligonucleotide.

[0069] As used herein, “RNAi agent” means an agent comprising an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule capable of reducing or inhibiting the translation of messenger RNA (mRNA) transcripts of target mRNA in a sequence-specific manner. As used herein, RNAi agents function through RNA interference mechanisms (i.e., by inducing RNA interference through interaction with the RNA interference pathway mechanism (RNA-induced silencing complex or RISC) in mammalian cells) or by any (one or more) alternative mechanisms or pathways. While the term is generally understood to function primarily through RNA interference mechanisms when used herein, the disclosed RNAi agents are not bound or limited to any particular pathway or mechanism of action. Examples of RNAi agents, but not limited to, include: single-stranded oligonucleotides, single-stranded antisense oligonucleotides, small interfering RNAs (siRNAs), double-stranded RNAs (dsRNAs), microRNAs (miRNAs), small hairpin RNAs (shRNAs), and Dicer substrates. The RNAi agents described herein consist of oligonucleotides having a chain at least partially complementary to the targeted mRNA. In some embodiments, the RNAi agents described herein are double-stranded and consist of an antisense strand and a sense strand at least partially complementary to the antisense strand. The RNAi agent may consist of a modified nucleotide and / or one or more non-phosphodiester bonds. In some embodiments, the RNAi agents described herein are single-stranded.

[0070] As used herein, the terms “suppression,” “reduction,” “inhibition,” “downregulation,” or “knockdown” refer to the expression of a given gene, in which the gene expression, as measured by the level of RNA transcribed from the gene or the level of polypeptides, proteins, or protein subunits translated from mRNA in the cells, cell populations, tissues, organs, or subjects on which the gene is transcribed, is reduced compared to a second set of cells, cell populations, tissues, organs, or subjects that have not received such treatment, when those cells, cell populations, tissues, organs, or subjects are treated with an oligomeric compound linked to a targeted ligand as described herein. As used herein, the terms “sequence” or “nucleotide sequence” mean a sequence or order of nucleic acid bases or nucleotides written using a sequence of letters in accordance with standard nucleotide nomenclature.

[0071] Where used herein, and unless otherwise indicated, the term “complementary” means the ability of the oligonucleotide or polynucleotide containing the first nucleotide sequence to hybridize with the oligonucleotide or polynucleotide containing the second nucleotide sequence under certain conditions (by forming base-pair hydrogen bonds under mammalian physiological conditions (or comparable in vitro conditions)) and to form a double-stranded or double-helical structure. The complementary sequence includes Watson-Crick base pairs or non-Watson-Crick base pairs and includes native or modified nucleotides, or nucleotide mimetic to the extent that it fulfills the above requirements regarding the ability to hybridize.

[0072] As used herein, “perfectly complementary” or “completely complementary” means that all (100%) of the bases in the sequence of the first polynucleotide hybridize to the same number of bases in the sequence of the second polynucleotide. The sequence may comprise all or part of the first or second nucleotide sequence. As used herein, “partially complementary” means that in a pair of hybridized nucleic acid sequences, at least 70% of the bases of the first polynucleotide sequence, rather than all of them, hybridize to the same number of bases of the second polynucleotide sequence.

[0073] As used herein, “substantially complementary” means that in a pair of hybridized nucleic acid sequences, at least 85% of the bases in the first polynucleotide sequence, rather than all of them, hybridize to the same number of bases in the second polynucleotide sequence. The terms “complementary,” “fully complementary,” and “substantially complementary” as used herein may be used with respect to matching bases between the sense strand and antisense strand of a double-stranded RNAi agent, between the antisense strand of a double-stranded RNAi agent and the sequence of the target mRNA, or between a single-stranded antisense oligonucleotide and the sequence of the target mRNA.

[0074] Where used herein, terms such as “treat” and “cure” mean methods or steps taken to provide relief from or alleviate one or more symptoms, severity, and / or frequency of the disease in question. As used herein, when referring to an oligomeric compound, the phrase "introduced into the cell" means functionally delivering the oligomeric compound into the cell. The phrase "functionally delivered" means delivering the oligomeric compound to the cell in a manner that allows the oligomeric compound to have the expected biological activity, for example, sequence-specific inhibition of gene expression.

[0075] Unless otherwise specified, the symbols used in this specification [ka] The use of means that any (one or more) groups may be linked to the scope of the invention as described herein.

[0076] As used herein, the term "isomer" refers to compounds that have the same molecular formula but differ in the properties of their atoms, the arrangement of their bonds, or the spatial arrangement of their atoms. Isomers that differ in the spatial arrangement of their atoms are called "stereoisomers." Stereoiomers that are not mirror images of each other are called "diastereoisomers," and stereoisomers that are mirror images that cannot be superimposed are called "enantiomers," or sometimes optical isomers. A carbon atom attached to four non-identical substituents is called a "chiral center." When used herein, each structure disclosed herein is intended to represent all such isomers, including their optically pure and racemic forms, without the structure being specifically identified as having a particular stereostructure with respect to each structure that exhibits an asymmetric center and thereby the configuration of an enantiomer, diastereomer, or other stereoisomer. For example, the structures disclosed herein are intended to cover mixtures of diastereomers, as well as a single stereoisomer.

[0077] The term “substituted,” as used herein, means that any one or more hydrogens on the indicated atom, usually a carbon, oxygen, or nitrogen atom, are substituted with any group as defined herein, provided that the valence does not exceed the standard ionic value of the indicated atom, and that such substitution results in a stable compound. Examples of substituents that are not limited include C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, cyano, hydroxyl, oxo, carboxyl, cycloalkyl, cycloalkenyl, heterocyclyl, heteroaryl, aryl, keto, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, or halo (e.g., F, Cl, Br, I). When the substituent is keto or oxo (i.e., =O), two (2) hydrogens on the atom are substituted. A ring double bond, as used herein, is a double bond formed between two adjacent ring atoms (e.g., C=C, C=N, N=N, etc.).

[0078] Some of the compounds in this disclosure may exist in tautomers, which are also intended to be included within the scope of this disclosure. A “tautomer” is a compound whose structure differs significantly in the arrangement of atoms, but which can easily and rapidly exist in equilibrium. It should be understood that the compounds in this disclosure may be shown as different tautomers. It should also be understood that when a compound has tautomers, all tautomers are within the scope of this disclosure, and that the names of the compounds are intended not to exclude any tautomers.

[0079] The compounds and pharmaceutically acceptable salts of this disclosure may exist in one or more tautomers, including ketone-enols, amide-nitriles, lactam-lactims, amide-imido acid tautomers of heterocyclic rings (e.g., guanine, thymine, and cytosine nucleic acid bases), amine-enamines and enamine-enamines, and geometric isomers, as well as mixtures thereof. Cyclic chain tautomerism, as demonstrated by glucose and other sugars, arises as a result of an aldehyde group (-CHO) in the sugar chain molecule, which reacts with an intramolecular hydroxyl group (-OH) to produce a ring (ring-shaped) form. All such tautomers are included within the scope of this disclosure. Tautomers exist in solution as a mixture of tautomer sets. In solids, typically one tautomer is dominant. Even if only one tautomer is described, this disclosure includes all tautomers of the compounds disclosed herein. The concept of tautomers that are interconvertible by tautomerization is called tautomerism. In tautomerism, simultaneous shifts of electrons and hydrogen atoms occur.

[0080] Tautomerism is catalyzed by: bases: 1. deprotonation; 2. formation of a delocalized anion (e.g., an enolate); 3. protonation of the anion at a different position; and acids: 1. protonation; 2. formation of a delocalized cation; 3. deprotonation at a different position adjacent to the cation.

[0081] As used herein, the term "alkyl" refers to a linear or branched aliphatic saturated hydrocarbon group having 1 to 10 carbon atoms, unless otherwise specified. For example, "C1-C6 alkyl" includes alkyl groups having 1, 2, 3, 4, 5, or 6 carbon atoms in a linear or branched configuration. As used herein, the term "aminoalkyl" refers to the previously defined alkyl groups substituted at any position with one or more amino groups, as permitted by standard ion valency. The amino groups may be unsubstituted, monosubstituted, or disubstituted.

[0082] As used herein, the term "cycloalkyl" means, unless otherwise specified, a saturated or unsaturated non-aromatic hydrocarbon ring group having 3 to 14 carbon atoms. Examples of cycloalkyls, but not limited to, include cyclopropyl, methylcyclopropyl, 2,2-dimethylcyclobutyl, 2-ethylcyclopentyl, and cyclohexyl. Cycloalkyls may contain multiple spiro rings or fusion rings. Cycloalkyl groups may be optionally monosubstituted, disubstituted, trisubstituted, tetrasubstituted, or pentasubstituted at any position permitted by standard ion valency.

[0083] As used herein, the term “alkenyl” means, unless otherwise specified, a linear or branched non-aromatic hydrocarbon group having 2 to 10 carbon atoms and containing at least one carbon-carbon double bond. Up to five carbon-carbon double bonds may be present in such group. For example, a “C2-C6” alkenyl is defined as an alkenyl radical having 2 to 6 carbon atoms. Examples of alkenyls, but not limited to, include ethenyl, propenyl, butenyl, and cyclohexenyl. The linear, branched, or cyclic portion of the alkenyl group may contain double bonds and may be monosubstituted, disubstituted, trisubstituted, tetrasubstituted, or pentasubstituted at any position permitted by standard ionic valency. The term “cycloalkenyl” means a monocyclic hydrocarbon group having a specific number of carbon atoms and at least one carbon-carbon double bond.

[0084] As used herein, the term “alkynyl” means, unless otherwise specified, a linear or branched hydrocarbon group comprising 2 to 10 carbon atoms and at least one carbon-carbon triple bond. Up to 5 carbon-carbon triple bonds may be present. Thus, “C2-C6 alkynyl” means an alkynyl radical having 2 to 6 carbon atoms. Examples of alkynyl groups, but not limited to, include ethynyl, 2-propynyl, and 2-butynyl. The linear or branched portion of the alkynyl group may contain triple bonds as permitted by standard ionic valence, and may be optionally monosubstituted, disubstituted, trisubstituted, tetrasubstituted, or pentasubstituted at any position permitted by standard ionic valence, or pentasubstituted.

[0085] As used herein, “alkoxyl” or “alkoxy” refers to the previously defined alkyl group, which has an indicated number of carbon atoms attached by oxygen crosslinking. 1-6 Alkoxy groups are intended to include C1, C2, C3, C4, C5, and C6 alkoxy groups. 1-8Alkoxy groups are intended to include C1, C2, C3, C4, C5, C6, C7, and C8 alkoxy groups. Examples of alkoxy groups, but not limited to, include methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy, t-butoxy, n-pentoxy, s-pentoxy, n-heptoxy, and n-octoxy. As used herein, “keto” refers to any alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclyl, heteroaryl, or aryl group attached by carbonyl crosslinking as defined herein. Examples of keto groups, though not limited to these, include alkanoyl (e.g., acetyl, propionyl, butanoyl, pentanoyl, hexanoyl), alkenoyl (e.g., acryloyl), alkinoyl (e.g., ethinoyl, propinoyl, butinoyl, pentinoyl, hexinoyl), allyloyl (e.g., benzoyl), and heteroallyloyl (e.g., imidazoloyl, quinolinoyl, pyridinoyl, pyrroloyl).

[0086] As used herein, “alkoxycarbonyl” refers to any of the previously defined alkoxy groups attached by a carbonyl crosslink (i.e., -C(O)O-alkyl-). Examples of alkoxycarbonyl groups, but not limited to, include methoxycarbonyl, ethoxycarbonyl, isopropoxycarbonyl, n-propoxycarbonyl, t-butoxycarbonyl, benzyloxycarbonyl, or n-pentoxycarbonyl.

[0087] As used herein, “aryloxycarbonyl” refers to any aryl group as defined herein, attached by an oxycarbonyl crosslink (i.e., -C(O)O-aryl-). Examples of aryloxycarbonyl groups, but not limited to these, include phenoxycarbonyl and naphthyloxycarbonyl.

[0088] As used herein, “heteroaryloxycarbonyl” refers to any heteroaryl group as defined herein, attached by an oxycarbonyl crosslink (i.e., -C(O)O-heteroaryl-). Examples of heteroaryloxycarbonyl groups, but not limited to, include 2-pyridyloxycarbonyl, 2-oxazolyloxycarbonyl, 4-thiazolyloxycarbonyl, or pyrimidinyloxycarbonyl.

[0089] As used herein, “aryl” or “aromatic” means any stable monocyclic or polycyclic carbocyclic ring consisting of up to seven atoms in each ring, with at least one of the rings being aromatic. Examples of aryl groups, but not limited to, include phenyl, naphthyl, anthracenyl, tetrahydronaphthyl, indanyl, and biphenyl. When the aryl substituent is bicyclic and one ring is non-aromatic, it is understood that the attachment is via an aromatic ring. The aryl group may be optionally monosubstituted, disubstituted, trisubstituted, tetrasubstituted, or pentasubstituted at any position permitted by the standard ionic valency.

[0090] As used herein, the term “heteroaryl” means a stable monocyclic or polycyclic ring having up to seven atoms in each ring, with at least one ring being aromatic and containing 1 to 4 heteroatoms selected from the group consisting of O, N, and S. Examples of heteroaryl groups, but not limited to, include acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrorazolyl, indolyl, benzotriazolyl, furanyl, thienyl, benzothienyl, benzofuranyl, benzimidazolonyl, benzoxazolonyl, quinolinyl, isoquinolinyl, dihydroisoindonyl, imidazopyridinyl, isoindonyl, indazolyl, oxazolyl, oxadiazolyl, isoxazolyl, indolyl, pyrazinyl, pyridadinyl, pyridinyl, pyrimidinyl, pyrrolyl, and tetrahydroquinoline. “Heteroaryl” is also understood to include N-oxide derivatives of any nitrogen-containing heteroaryl. When a heteroaryl substituent is bicyclic and one ring is non-aromatic or does not contain a heteroatom, attachment is understood to occur via an aromatic ring or a heteroatom-containing ring. The heteroaryl group may be optionally monosubstituted, disubstituted, trisubstituted, tetrasubstituted, or pentasubstituted at any position permitted by the standard ionic valency.

[0091] As used herein, the terms “heterocyclic compound,” “heterocyclic formula,” or “heterocyclyl” mean a 3- to 14-membered aromatic or non-aromatic heterocycle containing 1 to 4 heteroatoms selected from the group consisting of O, N, and S, including polycyclic groups. As used herein, the term “heterocyclic formula” is also considered synonymous with the terms “heterocyclic compound” and “heterocyclyl” and is understood to have the same definitions as set forth herein. “Heterocyclyl” includes not only the heteroaryls described above, but also their dihydro and tetrahydro analogs.Examples of heterocyclyl groups are not limited to these, but include azetidinyl, benzimidazolyl, benzofuranil, benzoflazanil, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbonil, cinnolinil, furanil, imidazolyl, indolinyl, indolyl, indazyl, indazolyl, isobenzofuranil, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, and naphthopyridinyl. Oxadiazolyl, oxoxazolidinil, oxazolyl, oxazoline, oxopiperazinil, oxopiperidinil, oxomorpholinil, isoxazoline, oxetanil, pyranil, pyrazinil, pyrazolyl, pyridadinil, pyridopyridinil, pyridadinil, pyridyl, pyridinonil, pyrimidyl, pyrimidinonil, pyrrolyl, quinazolinil, quinolyl, quinoxalinil, tetrahydropyranil, tetrahydrofuranil, tetrahydrothiopyranil, tetrahydroisoquinolinil Tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, 1,4-dioxanyl, hexahydroazepinyl, piperazinyl, piperidinyl, pyridine-2-onyl, pyrrolidinyl, morpholinyl, thiomorpholinyl, dihydrobenzimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl, dihydroisoxazolyl, dihydroisoth Examples include azolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrolyl, dihydroquinolinyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, dioxidethiomorpholinyl, methylenedioxybenzoyl, tetrahydrofuranyl, tetrahydrothienyl, and N-oxides. The attachment of heterocyclyl substituents may occur via carbon atoms or via heteroatoms. The heterocyclyl group may be optionally monosubstituted, disubstituted, trisubstituted, tetrasubstituted, or pentasubstituted at any position permitted by the standard ionic valency.

[0092] Those skilled in the art will readily understand or recognize that the compounds and compositions disclosed herein have certain atoms (e.g., N, O, or S atoms) in a protonated or deprotonated state depending on the environment in which the compound or composition is placed. Therefore, when used herein, the structures disclosed herein are assumed to have certain functional groups, such as OH, SH, or NH, that can be protonated or deprotonated. The disclosures herein are intended to cover the disclosed compounds and compositions regardless of the protonation status based on the pH of the environment, as will be readily understood by those skilled in the art.

[0093] When used in the claims herein, the phrase "consists of" excludes any components, steps, or elements not specified in the claims. When used in the claims of this specification, the phrase “substantially consisting of” means that the scope of the claim is limited to the specified materials or steps and that do not materially affect the (one or more) fundamental and novel features of the invention described in the claim.

[0094] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in the field to which the present invention pertains. Methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the present invention, but preferred methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. In the event of any conflict, this specification, including definitions, shall prevail. In addition, materials, methods and examples are merely illustrative and not intended to be limiting. Other features and advantages of the present invention will become apparent from the following detailed description and claims. [Brief explanation of the drawing]

[0095] [Figure 1] Figure 1 shows the 1H NMR spectrum of compound 11 (described below in Example 1 and having the chemical structure of structure 1005b as specified herein). [Figure 1A] Figure 1A shows the 1H NMR spectrum of structure 1004b (described below in Example 1) as used herein. [Figure 2] Figure 2 shows the 31P NMR spectrum of compound 19 (described below in Example 2 and having the chemical structure of structure 1008b as specified herein). [Figure 2A] Figure 2A shows the 1H NMR spectrum of compound 19. [Figure 2B] Figure 2B shows the 1H NMR spectrum of compound 14 (described below in Example 2). [Figure 2C] Figure 2C shows the 1H NMR spectrum of compound 15 (described below in Example 2). [Figure 2D] Figure 2D shows the 1H NMR spectrum of compound 16 (described below in Example 2). [Figure 2E] Figure 2E shows the 1H NMR spectrum of compound 17 (described below in Example 2). [Figure 2F] Figure 2F shows the 1H NMR spectrum of compound 18 (described below in Example 2). [Figure 3] Figure 3 shows the 1H NMR spectrum of compound 30 (described below in Example 3). [Figure 4] Figure 4 shows the 1H NMR spectrum of compound 38 (described below in Example 4). [Figure 5] Figure 5 shows the 1H NMR spectrum of compound 44 (described below in Example 5). [Figure 6] Figure 6 shows the 1H NMR spectrum of compound 47 (described below in Example 6). [Figure 7]Figure 7 is a photograph of the PEG linker-GalNAc phosphoramidite-containing compound (described below in Example 7) in the bottle. [Figure 8] Figure 8 is a photograph of the phosphoramidite-containing compound structure 1008b inside the bottle (described below in Example 7). [Figure 9] Figure 9 shows the 31P NMR spectrum of the PEG linker-GalNAc structure (described below in Example 8). [Figure 10] Figure 10 is a graph illustrating the normalized mouse factor 12 (mF12) protein levels in wild-type mice (described below in Example 11). [Figure 11] Figure 11 is a graph illustrating the normalized mouse factor 12 (F12) protein levels in wild-type mice (described below in Example 12). [Figure 12] Figure 12 is a graph illustrating the normalized lipoprotein (a) (Lp(a)) particle level in Lp(a) transgenic (Tg) mice (described below in Example 13). [Figure 13] Figure 13 is a graph illustrating the normalized apo(a) levels (described below in Example 14) in apo(a) transgenic (Tg) mice. [Figure 14] Figure 14 is a graph illustrating the normalized Lp(a) particle levels in Lp(a)Tg mice (described below in Example 15). [Figure 15] Figure 15 is a graph illustrating the normalized mouse F12 protein levels in wild-type mice (described below in Example 16). [Figure 16] Figure 16 is a graph illustrating the normalized Lp(a) particle levels in Lp(a)Tg mice (described below in Example 17). [Figure 17] Figure 17 is a graph illustrating the normalized apo(a) level in apo(a)Tg mice (described below in Example 18). [Figure 18]Figure 18 is a graph illustrating the normalized Lp(a) particle levels in Lp(a)Tg mice (described below in Example 19). [Figure 19] Figure 19 is a graph illustrating the normalized Lp(a) particle levels in cynomolgus monkeys (described below in Example 20). [Figure 20] Figure 20 is a graph illustrating the normalized cF12 protein levels in cynomolgus monkeys (described below in Example 21). [Figure 21] Figure 21 is a graph illustrating the normalized AAT (Z-AAT) protein levels in PiZ transgenic mice (described below in Example 22). [Modes for carrying out the invention]

[0096] Detailed explanation Targeting ligands linked to compounds such as therapeutic or diagnostic compounds are described herein. In some embodiments, the compounds linked to the targeting ligands described herein include or consist of therapeutic compounds such as expression inhibitory oligomer compounds. The targeting ligands can be used to target the therapeutic compounds to a target nucleic acid or target gene at a desired location. Compositions containing targeting ligands and therapeutic compounds, such as compositions containing or consisting of targeting ligands and expression inhibitory oligomer compounds, are also described herein.

[0097] The novel targeted ligands described herein offer advantages over previously known targeted ligands for facilitating the delivery of therapeutic compounds. These advantages include, for example, improved ease and efficiency of manufacture, while also providing effective targeting or biodistribution, sufficient stability in vivo and / or in vitro, and / or other desirable improvements for oligonucleotide therapeutic delivery. The novel targeted ligands are also particularly suitable for synthesis as phosphoramidite compounds, which reduces manufacturing costs and burdens and simplifies the attachment of targeted ligands to compounds, particularly expression inhibitory oligomer compounds (such as RNAi agents), while providing similar, or possibly improved, delivery and / or efficacy of therapeutic compounds.

[0098] Targeted ligand A targeted ligand consists of one or more targeted groups or moieties, which may help enhance the pharmacokinetic or biodistribution properties of the compound to which they are linked, and improve the cell or tissue-specific distribution or cell-specific uptake of the complex composition. Generally, targeted ligands help induce the delivery of the therapeutic compound to which they are linked to a desired target site. In some cases, the targeted moiety may bind to a cell or cell receptor and initiate endocytosis to facilitate the entry of the therapeutic compound into the cell. Examples of targeted moieties include compounds that have affinity for cell receptors, cell surface molecules, or antibodies. A variety of targeted ligands containing targeted moieties can be linked to therapeutics and other compounds to target agents to cells and specific cell receptors. Types of targeted moieties include carbohydrates, cholesterol and cholesteryl groups, as well as steroids. Targeting moieties that can bind to cell receptors include saccharides such as galactose, galactose derivatives (such as N-acetylgalactosamine), mannose, and mannose derivatives; other carbohydrates; glycans; haptens; vitamins; phorates; biotin; aptamers; and peptides such as RGD-containing peptides, insulin, EGF, and transferrin.

[0099] Targeting moieties known to bind to the asialoglycoprotein receptor (ASGPR) are particularly useful in inducing the delivery of oligomeric compounds to the liver. The asialoglycoprotein receptor is abundantly expressed in liver cells, including hepatocytes. Examples of cell receptor targeting moieties that target ASGPR include galactose and galactose derivatives. In particular, clusters of galactose derivatives containing clusters of 2, 3, or 4 N-acetyl-galactosamines (GalNAc or NAG) can facilitate the uptake of specific compounds into liver cells. GalNAc clusters bound to oligomeric compounds help guide the composition to the liver, where the N-acetyl-galactosamine sugar can bind to the asialoglycoprotein receptor on the surface of liver cells. Binding to the asialoglycoprotein receptor is thought to initiate receptor-mediated endocytosis, thereby facilitating the entry of the compound into the cell.

[0100] The targeted ligands disclosed herein may comprise one, two, three, four, or more targeted moieties. In some embodiments, the targeted ligands disclosed herein may comprise one, two, three, four, or more targeted moieties linked to a branching group. In some embodiments, the targeted ligands disclosed herein may comprise one, two, three, four, or more targeted moieties linked to a branching group, where each targeted moiety is linked to the branching group via a tether.

[0101] In some embodiments, the targeted ligands disclosed herein may comprise one, two, three, four, or more asial glycoprotein receptor (ASGPR) targeting moieties linked to a branching group.

[0102] In some embodiments, the branching group is linked to a linker. In some embodiments, the branching group includes a linker substitution portion and is linked to a therapeutic compound. In some embodiments, the branching group is linked to an oligomeric compound. In some embodiments, the branching group is linked to an expression-inhibiting oligomeric compound.

[0103] In some embodiments, the targeted ligand is represented by the following formula I: [ka] {wherein n is an integer between 1 and 4 (e.g., 1, 2, 3, or 4)} (Equation I). In some embodiments, n in Equation I is an integer between 1 and 3, 1 and 2, 2 and 4, 2 and 3, or 3 and 4.

[0104] The linker of formula I is a group containing one or more substituted or unsubstituted moieties selected from (one or more) cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heterocyclyl groups, or combinations thereof covalently linked, which connect the branching point group at one end of the linker to the therapeutic compound at the other end of the linker (or to the phosphorus atom of the phosphoramidite when the targeted ligand is synthesized as a phosphoramidite compound). In some embodiments, one or more additional groups, such as cleavable moieties (e.g., groups containing phosphate or disulfide bonds) or groups that form (one or more) phosphorothioate or phosphonate linkages, are inserted between the therapeutic compound and the linker. The linker is "rigid" in the sense that it gives the entire targeted ligand sufficient stability and rigidity to reduce the interaction between the one or more targeted moieties of formula I and the therapeutic compound to which it is linked. This, in turn, can improve the interaction between the target site and the targeted moiety. In addition, the linker for use in targeted ligands disclosed herein is specifically designed to synthesize the targeted ligand as a phosphoramidite compound, thereby enabling effective linking of the targeted ligand to the 5' end of the oligomeric compound.

[0105] The branch point base in Equation I is any base that allows one or more targeted portions to be attached to the linker (via one or more tethers). In some embodiments, the targeted ligand is represented by the following formula II:

[0106] [ka] {wherein n is an integer between 1 and 4 (e.g., 1, 2, 3, or 4)}. In some embodiments, n in formula II is an integer between 1 and 3, 1 and 2, 2 and 4, 2 and 3, or 3 and 4.

[0107] In Formula II, the branching group is any group that enables the attachment of one or more targeting moieties (via one or more tethers) to a therapeutic compound (or, when the targeted ligand is synthesized as a phosphoramidite compound, to the phosphorus atom of the phosphoramidite). As used herein, the branching group includes a linker-substituted moiety when the branching group includes one or more substituted or unsubstituted cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heterocyclyl groups, or a combination thereof (fused within the branching group, satisfying the same function as the rigid linker of Formula I disclosed herein).

[0108] One or more tethers of Formulas I and II are groups that serve as spacers, which can further add flexibility and / or length to the linkage between the targeting moiety and the branching group. The tethers provide an effective method for linking the targeting moiety to the branching group. With respect to the targeting ligands disclosed herein, there is at least one tether for each targeting moiety. In some embodiments, there are multiple (i.e., two or more) tethers between the branching group and the targeting moiety.

[0109] The targeting moieties of formulas I and II are groups that help enhance the pharmacokinetic or biodistribution properties of the therapeutic compound to which they are linked, and that help improve cell or tissue-specific distribution and cell-specific uptake of the complex composition. The targeting moieties may include compounds that have affinity for cell receptors or cell surface molecules or antibodies. Types of targeting moieties include carbohydrates, cholesterol and cholesteryl groups, and steroids. Targeting moieties that can bind to cell receptors include saccharides such as galactose, galactose derivatives (such as N-acetylgalactosamine), mannose, and mannose derivatives; other carbohydrates; glycans; haptens; vitamins; phorates; biotin; aptamers; and peptides such as RGD-containing peptides, insulin, EGF, and transferrin.

[0110] The targeted ligands disclosed herein can be linked to therapeutic compounds. In some embodiments, the targeted ligand is linked to the therapeutic compound via an additional linker and / or cleavable moiety, and then it is linked to the therapeutic compound. In some embodiments, the targeted ligand is bound to the therapeutic compound itself.

[0111] In some embodiments, the therapeutic compound is an oligomeric compound. In some embodiments, the therapeutic compound is an expression inhibitory oligomeric compound. In some embodiments, the expression inhibitory oligomeric compound is an RNAi agent. In some embodiments, the expression inhibitory oligomeric compound is a double-stranded RNAi agent.

[0112] In some embodiments, the targeting ligand is directly or indirectly ligated to the 5' end of the sense strand of the double-stranded RNAi agent. In some embodiments, the targeting ligand is directly or indirectly ligated to the 3' end of the sense strand of the double-stranded RNAi agent. In some embodiments, the targeting ligand is directly or indirectly ligated to the 5' or 3' end of the antisense strand of the double-stranded RNAi agent. In some embodiments, the targeting ligand is directly or indirectly ligated to the 5' or 3' end of the single-stranded RNAi agent.

[0113] In some embodiments, the targeting ligand is ligated to the double-stranded RNAi agent at the 5' end of the terminal nucleoside of the sense strand of the double-stranded RNAi agent via a phosphate group, a phosphonate group, a phosphorothioate group, or other internucleoside linking group.

[0114] In some embodiments, the targeted ligands disclosed herein include cleavable moieties. In some embodiments, the cleavable moieties include or consist of phosphate groups or other cleavable internucleoside linking groups. In some embodiments, the targeted ligand is linked to a therapeutic compound via the cleavable moieties.

[0115] In some embodiments, the targeted ligands disclosed herein are linked to a group containing an additional group or a cleavable moiety. In some embodiments, the targeted ligand is linked to a cleavable moiety, which is then linked to an expression inhibitory oligomer compound.

[0116] In some embodiments, the targeted ligand is a phosphoramidite compound. Phosphoramidite compounds containing the targeted ligand described herein may be useful for readily attaching the targeted ligand to a therapeutic compound or other group using methods commonly known in the art for phosphoramidite synthesis. In some embodiments, the phosphoramidite compound containing the targeted ligand is ligated to an expression inhibitory oligomer compound using methods commonly known in the art. In some embodiments, the targeted ligand-containing phosphoramidite is ligated to the 5' end of the sense strand of a double-stranded RNAi agent.

[0117] In some embodiments, the expression inhibitory oligomer compound linked to the targeted ligand includes a single-stranded oligonucleotide. In some embodiments, the single-stranded oligonucleotide is a single-stranded antisense oligonucleotide. In some embodiments, the targeted ligand is directly linked to the single-stranded antisense oligonucleotide. In some embodiments, an additional group is inserted between the targeted ligand and the single-stranded oligonucleotide. In some embodiments, the targeted ligand linked to the RNAi agent comprises one or more N-acetyl-galactosamine sugars as the targeted moiety or as the targeted moiety.

[0118] In some embodiments, the targeted ligand linked to the expression-inhibiting oligomer compound includes a tether containing polyethylene glycol (PEG). In some embodiments, the tether consists of PEG. In some embodiments, the tether contains PEG having 1 to 10 ethylene glycol units. In some embodiments, the tether contains PEG having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ethylene glycol units.

[0119] In some embodiments, the expression inhibitory oligomer compound linked to any of the targeted ligands disclosed herein includes an RNAi agent. In some embodiments, the targeted ligands disclosed herein are linked directly or indirectly to the RNAi agent.

[0120] In some embodiments, the targeted ligands disclosed herein are directly linked to the RNAi agent. In some embodiments, the targeted ligands disclosed herein are indirectly linked to the RNAi agent when one or more additional groups are inserted between the RNAi agent and the linker of the targeted ligand. In some embodiments, a second linker is included between the linker and the therapeutic compound.

[0121] Linker The targeted ligands disclosed herein include a linker as shown in Formula I, or the branching point group includes a linker substitution moiety as shown in Formula II. The linker is a group of atoms that are linked at one end to a branching group and at the other end to a therapeutic compound (or, when the targeted ligand is synthesized as a phosphoramidite compound, to the phosphorus atom of the phosphoramidite). In some embodiments, the linker is linked at one end to a branching group and at the other end to one or more groups that are then bound to the expression inhibitory oligomer compound. In some embodiments, the linker is directly linked to the oligomer compound. In some embodiments, the linker is linked to a cleavable moiety, which is then linked to the oligomer compound. Examples of cleavable moieties, but not limited to these, include phosphate groups, groups including disulfide moieties, and / or other cleavable internucleoside bonds. In some embodiments, the linker is not linked to a cleavable moiety. In some embodiments, the linker is linked to a phosphorothioate group or a phosphonate group.

[0122] With respect to the targeted ligands disclosed herein by formula I, the linker is a “rigid” linker. A rigid linker is a linking group comprising one or more substituted or unsubstituted cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heterocyclyl groups, or combinations thereof linked by covalent bonds.

[0123] In some embodiments, the targeted ligand of formula I includes or consists of linkers having the following structural formula:

[0124] [ka] (Structure 1).

[0125] In some embodiments, the targeted ligand of formula I includes or consists of linkers having the following structural formula:

[0126] [ka] (Structure 2).

[0127] In some embodiments, the targeted ligand of formula I includes or consists of linkers having the following structural formula:

[0128] [ka] (Structure 3).

[0129] In some embodiments, the targeted ligand of formula I includes or consists of linkers having the following structural formula:

[0130] [ka] (Structure 4).

[0131] In some embodiments, the targeting ligand of Formula I comprises or consists of a linker having the following structural formula:

[0132]

Chemical Structure

[0133] In some embodiments, the targeting ligand of Formula I comprises or consists of a linker having the following structural formula:

[0134]

Chemical Structure

[0135] In some embodiments, the targeting ligand of Formula I comprises or consists of a linker having the following structural formula:

[0136]

Chemical Structure

[0137] In some embodiments, the targeting ligand of Formula I comprises or consists of a linker having the following structural formula:

[0138]

Chemical Structure

[0139] In some embodiments, the targeting ligand of Formula I comprises or consists of a linker having the following structural formula:

[0140]

Chemical Structure

[0141] In some embodiments, the targeted ligand of formula I includes or consists of linkers having the following structural formula:

[0142] [ka]

[0143] {wherein n' is an integer between 0 and 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), and for each Z' present, Z' is independently selected and Z' is independently selected from the group consisting of: C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted or unsubstituted amino, carboxyl, C1-C6 alkoxy, substituted C1-C6 alkyl, C1-C6 aminoalkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl, substituted C1-C6 alkoxy, substituted C1-C6 aminoalkyl, halogen (e.g., F), hydroxyl, amide, substituted amide, cyano, substituted or unsubstituted keto, substituted or unsubstituted alkoxycarbonyl, substituted or unsubstituted aryloxycarbonyl, substituted or unsubstituted heteroaryloxycarbonyl, and sulfhydryl} (Structure 7).

[0144] In some embodiments, the targeted ligand of formula I includes or consists of linkers having the following structural formula:

[0145] [ka]

[0146] {wherein n'' is an integer between 0 and 4 (e.g., 1, 2, 3, or 4), and for each Z'' present, Z'' is independently selected and Z'' is independently selected from the group consisting of: C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, substituted C1-C6 alkyl, C1-C6 aminoalkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl, substituted or unsubstituted amino, carboxyl, substituted C1-C6 alkoxy, substituted C1-C6 aminoalkyl, halogen (e.g., F), hydroxyl, amide, substituted amide, cyano, substituted or unsubstituted keto, substituted or unsubstituted alkoxycarbonyl, substituted or unsubstituted aryloxycarbonyl, substituted or unsubstituted heteroaryloxycarbonyl, and sulfhydryl} (Structure 8). In some embodiments, the targeted ligand of formula I includes or consists of linkers having the following structural formula:

[0147] [ka]

[0148] {wherein V includes or consists of one or more substituted or unsubstituted cycloalkyls (e.g., cyclohexyl, cyclopropyl, cyclobutyl, cyclopentyl, cycloheptyl, cyclooctyl, etc.), cycloalkenyls (e.g., cyclohexenyl, cyclobutenyl, cyclopentenyl, cycloheptenyl, cyclooctenyl, cyclohexadienyl, cyclopentadienyl, cycloheptadienyl, cyclooctadienyl, etc.), aryls (e.g., phenyl, naphthyl, binaphthyl, anthracenyl, etc.), heteroaryls (e.g., pyridyl, pyrimidinyl, pyrrole, imidazole, furan, benzofuran, indole, etc.), or heterocyclyls (e.g., tetrahydrofuran, tetrahydropyran, piperidine, pyrrolidine, etc.), or any combination thereof linked by covalent bonds} (Structure 9).

[0149] In some embodiments, linkers suitable for use in the targeted ligands disclosed herein are derived from compounds comprising a rigid structure having a terminal carboxylic acid moiety (or its activated ester) at one end and a terminal alcohol moiety at the other end. In some embodiments, the alcohol moiety is a secondary alcohol. In some embodiments, the alcohol moiety is a tertiary alcohol. In some embodiments, the alcohol moiety is a primary alcohol. The carboxylic acid moiety (or its activated ester) is suitable for attachment to a branching point, while the alcohol moiety is suitable for attachment to the phosphorus atom of a phosphoramidite by a phosphytylation reaction using a phosphoramidite-forming reagent. Examples of phosphytylation reactions using phosphoramidite-forming reagents are described in the examples herein. The linker structures disclosed herein are suitable for the preparation of targeted ligands as phosphoramidite compounds.

[0150] In some embodiments, the linker is linked to an expression inhibitory oligomer compound, i.e., a double-stranded RNAi agent. In some embodiments, the linker is linked to the 5' end of the sense strand of the double-stranded RNAi agent. In some embodiments, the linker is linked to the 3' end of the sense strand of the double-stranded RNAi agent. In some embodiments, the linker is linked to the 3' end of the antisense strand of the double-stranded RNAi agent. In some embodiments, the linker is linked to the 5' end of the antisense strand of the double-stranded RNAi agent.

[0151] In some embodiments, the linker is linked to a cleavable moiety. In some embodiments, the terminal phosphate group of the expression inhibitory oligomer compound can serve as a cleavable moiety. In some embodiments, an independently selected cleavable moiety is linked to the linker. As used herein, a cleavable moiety is a group that is stable outside the cell but is cleaved upon entering the target cell. The cleavable moiety is susceptible to cleavage under specific conditions, such as pH, or by specific cleavage agents, such as degradation-promoting molecules or redox agents.

[0152] In some embodiments, the cleavable portion may be susceptible to pH. For example, endosomes and lysosomes are generally known to have a more acidic pH (approximately 4.5–6.5) than human blood (approximately 7.35–7.45), which may facilitate the cleavage of the cleavable portion. In some embodiments, the cleavable portion is a phosphate group. The phosphate group can be cleaved by an agent known to decompose or hydrolyze phosphate groups.

[0153] In some embodiments, the targeted ligands disclosed herein include a branching point group containing a linker substituent instead of a linker, as shown in Formula II. When the linker is substituted with a linker substitution portion, the linker substitution portion is part of the branching point group. In some embodiments, the linker-to-linker substitution moieties disclosed herein allow for the incorporation of only a single isomer of the targeted ligand, which may provide additional advantages for oligonucleotide therapeutics.

[0154] Branch point base The targeting ligands disclosed herein include at least one branching point group. In some embodiments, the branching point group of the targeting ligands disclosed herein is linked to a linker. In some embodiments, the branching point group of the targeting ligands disclosed herein is linked to a linker at one end and the branching point group is linked to one or more tethers at the other end(s). In some embodiments, the branching point group is linked to an expression-inhibiting oligomeric compound via one or more additional groups. In some embodiments, the branching point group includes a linker substitution moiety and is linked to an expression-inhibiting oligomeric compound.

[0155] The branching point groups disclosed herein are any groups that allow attachment of one or more targeting moieties and further allow attachment to the linkers disclosed herein, or, if the branching point group includes a linker substitution moiety, the branching point group is any group that includes a linker substitution moiety that allows attachment to a therapeutic compound such as, for example, an expression-inhibiting oligomeric compound.

[0156] With respect to the branching point groups of Formula I disclosed herein, prior to complex formation with a linker, the branching point groups useful for creating the branching point groups have an amine at one end for each desired attachment to a linker and a carboxylic acid moiety (or its activated ester) at one end for each desired attachment to a tether.

[0157] In some embodiments, examples of targeting ligands include branching points having structures selected from the following structural formulas:

[0158]

Chemical formula

[0159] In some embodiments, the targeting ligand includes a branching point having the following structural formula:

Chemical formula

[0160] In some embodiments, the targeted ligand includes a branching point having the following structural formula: [ka]

[0161] {wherein m is an integer between 0 and 20 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20), and n is an integer between 0 and 20 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20)} (structure 210).

[0162] In some embodiments, the targeted ligand includes a branching point having a structure represented by the following structural formula: [ka]

[0163] {wherein m is an integer between 0 and 20 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20); n is an integer between 0 and 20 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20); x is an integer between 1 and 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10); y is an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20); z is an integer from 1 to 4 (e.g., 1, 2, 3 or 4); and K is a substituted or unsubstituted cycloalkyl (e.g., cyclohexyl, cyclopropyl, cyclobutyl, cyclo) Selected from the group consisting of lopentyl, cycloheptyl, cyclooctyl, etc., substituted or unsubstituted cycloalkenyls (e.g., cyclohexenyl, cyclobutenyl, cyclopentenyl, cycloheptenyl, cyclooctenyl, cyclohexadienyl, cyclopentadienyl, cycloheptadienyl, cyclooctadienyl, etc.), substituted or unsubstituted aryls (e.g., phenyl, naphthyl, binaphthyl, anthracenyl, etc.), substituted or unsubstituted heteroaryls (e.g., pyridyl, pyrimidinyl, pyrrole, imidazole, furan, benzofuran, indole, etc.), and substituted or unsubstituted heterocyclyls (e.g., tetrahydrofuran, tetrahydropyran, piperidine, pyrrolidine, etc.), or combinations thereof linked by covalent bonds (structure 211).

[0164] In some embodiments, the targeted ligand includes a branching point having the following structural formula: [ka]

[0165] {wherein m is an integer between 0 and 20 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20); n is an integer between 0 and 20 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20); x is an integer between 1 and 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10); and y is an integer between 1 and 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10)} (structure 212).

[0166] In some embodiments, the targeted ligand includes a branching point having the following structural formula: [ka]

[0167] {wherein m is an integer between 0 and 20 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20); n is an integer between 0 and 20 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20); x is an integer between 1 and 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10); y is an integer between 1 and 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10); and G is,

[0168] [ka]

[0169] ;or any substituted or unsubstituted ring or heterocyclic structure having a ring size of 5, 6, 7, 8, or 9 atoms, selected from the group consisting of substituted or unsubstituted cycloalkyls (e.g., cyclohexyl, cyclopropyl, cyclobutyl, cyclopentyl, cycloheptyl, cyclooctyl, etc.), substituted or unsubstituted cycloalkenyls (e.g., cyclohexenyl, cyclobutenyl, cyclopentenyl, cycloheptenyl, cyclooctenyl, cyclohexadienyl, cyclopentadienyl, cycloheptadienyl, cyclooctadienyl, etc.), substituted or unsubstituted aryls (e.g., phenyl, naphthyl, binaphthyl, anthracenyl, etc.), substituted or unsubstituted heteroaryls (e.g., pyridyl, pyrimidinyl, pyrrole, imidazole, furan, benzofuran, indole, etc.), or substituted or unsubstituted heterocyclyls (e.g., tetrahydrofuran, tetrahydropyran, piperidine, pyrrolidine, etc.) (structure 213).

[0170] In some embodiments, the targeted ligand includes a branching point group having the following structure: [ka] {wherein n is an integer from 0 to 20 (for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20)} (structure 214).

[0171] In some embodiments, the targeted ligand includes a branching point group having the following structure: [ka] {In the expression, n is an integer from 0 to 20 (for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20), and Q is:

[0172] [ka] Selected from the group consisting of (structure 215).

[0173] In some embodiments, the targeted ligand includes a branching point group having the following structure: [ka] (Structure 216).

[0174] In some embodiments, the targeted ligand includes a branching point group having the following structure: [ka] (Structure 217).

[0175] In some embodiments, the targeted ligand includes a branching point group having the following structure: [ka] (Structure 218).

[0176] In some embodiments, the targeted ligand includes a branching point group having the following structure: [ka]

[0177] {wherein n is an integer selected from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7)} (structure 219). In some embodiments, n in structure 219 is 1. In some embodiments, n in structure 219 is 2. In some embodiments, n in structure 219 is 3. In some embodiments, n in structure 219 is 4. In some embodiments, n in structure 219 is 5. In some embodiments, n in structure 219 is 6. In some embodiments, n in structure 219 is 7. In some embodiments, the targeted ligand includes a branched group containing a linker substituent.

[0178] In some embodiments, the targeted ligand includes a branching point group comprising a linker-substituted moiety having a structure represented by the following structural formula: [ka] (Structure 220), or

[0179] [ka] (Structure 221).

[0180] Tether The targeted ligands disclosed herein include one or more tethers. The tethers are coupled between a branching base and each targeted portion. In some embodiments, the tether is coupled directly to the targeted ligand at one end and directly to the branching base at the other end. In some embodiments, the tether is coupled directly to the targeted ligand at one end and indirectly to the branching base at the other end. In some embodiments, the tether is coupled indirectly to the targeted ligand at one end and indirectly to the branching base at the other end. In some embodiments, the targeted ligands described herein include three tethers and three targeted portions. In some embodiments, the targeted ligands described herein include four tethers and four targeted portions. In some embodiments, the targeted ligands described herein include one tether and one targeted portion. In some embodiments, the targeted ligands described herein include multiple tethers and multiple targeted portions.

[0181] In some embodiments, an additional tether or other group is inserted between the tether of Formula I or Formula II and the targeting portion. In some embodiments, a second tether is inserted between the tether of Formula I or Formula II and the targeting portion. In some embodiments, a second and a third tether are inserted between the tether of Formula I or Formula II and the targeting portion. In some embodiments, second, third, and fourth tethers are inserted between the tether of Formula I or Formula II and the targeting portion. As disclosed herein, there is at least one tether with respect to each targeting portion. In some embodiments, there are two or more tethers with respect to each targeting portion. The targeting ligands disclosed herein are intended to encompass such compositions.

[0182] In some embodiments, an additional group may be inserted between the tether of formula I or formula II and the branching point group. Where disclosed herein, the tether serves as a spacer that can add further flexibility and / or length to the linkage between the targeting moiety and the branching group, linker, and therapeutic compound. In some embodiments, the tether includes alkyl groups (including cycloalkyl groups), alkenyl groups (including cycloalkenyl groups), alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, or aralkylyl groups. In some embodiments, the tether includes one or more heteroatoms, heterocyclic compounds, heteroaryls, amino acids, nucleotides, or saccharides.

[0183] In some embodiments, the targeted ligand includes a tether having the following structure: [ka] {wherein n is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20), and X is O, S, or NH} (structure 301).

[0184] In some embodiments, the targeted ligand includes a tether having the following structure: [ka] {wherein X is O, S, or NH} (Structure 302).

[0185] In some embodiments, the targeted ligand includes a tether having the following structure: [ka] (Structure 302a).

[0186] In some embodiments, the targeted ligand includes a tether having the following structure: [ka] {wherein n is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20), and X is O, S, or NH} (structure 303).

[0187] In some embodiments, the targeted ligand includes a tether having the following structure: [ka] {wherein n is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20), and X is O, S, or NH} (structure 304).

[0188] In some embodiments, the targeted ligand includes a tether having the following structure: [ka] {wherein X is O, S, or NH} (Structure 305).

[0189] In some embodiments, the targeted ligand includes a tether having the following structure: [ka] {wherein X is O, S, or NH} (structure 306).

[0190] In some embodiments, the targeted ligand comprises two or more types of tethers. In some embodiments, the tether functions as a flexible hydrophilic spacer (e.g., US5,885,968; and Biessen et al. J. Med. Chem. 1995, 39, 1538-1546, both of which are incorporated herein by reference as a whole) and includes a PEG spacer. In other embodiments, the PEG spacer comprises 1 to 20 ethylene units (PEG1 to PEG1). 20 ) has. For example, the PEG spacer has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ethylene units.

[0191] Targeting part: The targeted ligands disclosed herein may comprise one to four, or more than four, targeted moieties. In some embodiments, the targeting ligand may be a galactose cluster. As used herein, galactose clustering comprises a targeting ligand having 2 to 4 terminal galactose derivatives. As used herein, the term galactose derivative includes both galactose and galactose derivatives having an affinity for asialocryprotein receptors equivalent to or greater than that of galactose. The galactose derivative is a saccharide sugar, which is a type of targeting moiety. The terminal galactose derivatives are tethered through the C-1 carbon of the saccharide.

[0192] In some embodiments, the targeted ligand comprises three terminal galactosamines or galactosamine derivatives (such as N-acetyl-galactosamine) each having affinity for an asial glycoprotein receptor. In some embodiments, the targeted ligand includes three terminal N-acetyl-galactosamines (GalNAc or NAG) as the targeting moiety. For example, structures 1001, 1002, 1004, and 1008 are each targeted ligands having three terminal N-acetyl-galactosamines as the targeting moiety.

[0193] In some embodiments, the targeting moiety comprises a galactosamine derivative, namely N-acetyl-galactosamine. Other saccharides that have affinity for asialocryprotein receptors and can be used as targeting moieties may be selected from a list including: galactose, galactosamine, N-formyl-galactosamine, N-propionyl-galactosamine, Nn-butanoylgalactosamine, and N-iso-butanoylgalactosamine. The affinity of many galactose derivatives for asialocryprotein receptors has been studied (see, e.g., Iobst, ST and Drickamer, KJBC 1996, 271, 6686, which is incorporated herein by reference in whole) or can be readily measured using commonly used methods well known in the art.

[0194] In some embodiments, the targeting portion is the portion that targets cells. In some embodiments, the targeted portion comprises N-acetyl-galactosamine: [ka] .

[0195] In some embodiments, the targeted ligand includes three targeted moieties. In some embodiments, the targeted ligand includes four targeted moieties. In some embodiments, the targeted ligand includes one targeted moiety. In some embodiments, the targeted ligand includes two targeted moieties. In some embodiments, the targeted ligand includes four or more targeted moieties.

[0196] In some embodiments, the targeted portion comprises one or more of galactose, galactosamine, N-formyl-galactosamine, N-acetyl-galactosamine, N-propionyl-galactosamine, Nn-butanoylgalactosamine, or N-iso-butanoylgalactosamine.

[0197] For example, in some embodiments, the N-acetyl-galactosamine targeting moiety of any of structures 1001 to 1027 may be replaced with an alternative targeting moiety. Examples of such alternative targeting moieties include galactose, galactosamine, N-formyl-galactosamine, N-acetyl-galactosamine, N-propionyl-galactosamine, Nn-butanoylgalactosamine, or N-iso-butanoylgalactosamine.

[0198] Furthermore, in some embodiments, the targeted moieties of structures 1001-1027 may be replaced with, for example, other carbohydrates; glycans; haptens; vitamins; phorates; biotin; aptamers; and / or peptides such as RGD-containing peptides, insulin, EGF, and / or transferrin.

[0199] In some embodiments, the targeted ligand is in the form of an N-acetyl-galactosamine trimer. In some embodiments, the targeted ligand is in the form of an N-acetyl-galactosamine tetramer.

[0200] Typical targeting ligand structures and phosphoramidite compounds containing targeting ligands The targeted ligands disclosed herein may consist of one or more targeted moieties, tethers, branching points, and linkers. The targeted ligands disclosed herein may consist of one, two, three, four, or more than four targeted moieties.

[0201] In some embodiments, the targeted ligands disclosed herein are synthesized in the form of phosphoramidite compounds. Phosphoramidites are widely used in the chemical synthesis of RNA and DNA. In some embodiments, the phosphoramidite-containing targeted ligands disclosed herein are added to the 5' end of the sense strand of a double-stranded RNAi agent. Preparing the targeted ligand as a phosphoramidite may be particularly advantageous when the targeted ligand is to be ligated to the 5' end of an expression-inhibiting oligomer compound. While we do not wish to be constrained by theory, it is understood that preparing the targeted ligand as a phosphoramidite allows for the ligation of the targeted ligand as the last component (thus reducing manufacturing costs) when the targeted ligand is ligated to the 5' end of an expression-inhibiting oligomer compound, and allows the targeted ligand to potentially block the addition of the sense strand into the RISC when the targeted ligand is attached to the 5' end of the sense strand of a double-stranded RNAi agent. When the expression-inhibiting oligomer compound is a double-stranded RNAi agent, the targeting ligand can be prepared as a phosphoramidite compound, provided that the targeting ligand is to be ligated to the 5' end of the sense strand of the RNAi agent.

[0202] In some embodiments, the targeted ligand has a structure represented by the following structural formula: [ka] (Structure 1001).

[0203] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {wherein, R comprises or consists of an expression-inhibiting oligomeric compound} (Structure 1001a).

[0204] In some embodiments, the expression-inhibiting oligomeric compound is linked to a targeting ligand and has a structure represented by the following structural formula:

Chemical formula

[0205] In some embodiments, the targeting ligand is a phosphoramidite-containing compound having a structure represented by the following structural formula:

Chemical formula

[0206] In some embodiments, the targeting ligand has a structure represented by the following structural formula:

Chemical formula

[0207] In some embodiments, the expression-inhibiting oligomeric compound is linked to a targeting ligand and has a structure represented by the following structural formula:

Chemical formula

[0208] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {In the formula, R consists of or contains expression inhibitory oligomer compounds; Y is O or S; and Y' is O - S - , or NH - (Structure 1002a(i)).

[0209] In some embodiments, the targeted ligand is a phosphoramidite-containing compound having a structure represented by the following structural formula: [ka] (Structure 1002b).

[0210] In some embodiments, the targeted ligand has a structure represented by the following structural formula: [ka] (Structure 1003).

[0211] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {In the formula, R includes or consists of an expression-inhibiting oligomer compound} (structure 1003a).

[0212] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {In the formula, R consists of or contains expression inhibitory oligomer compounds; Y is O or S; and Y' is O - S - , or NH - (Structure 1003a(i)).

[0213] In some embodiments, the targeted ligand is a phosphoramidite-containing compound having a structure represented by the following structural formula: [ka] (Structure 1003b).

[0214] In some embodiments, the targeted ligand has a structure represented by the following structural formula: [ka] (Structure 1004).

[0215] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {In the formula, R includes or consists of an expression-inhibiting oligomer compound} (structure 1004a).

[0216] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {In the formula, R consists of or contains expression inhibitory oligomer compounds; Y is O or S; and Y' is O - S - , or NH - (Structure 1004a(i)).

[0217] In some embodiments, the targeted ligand is a phosphoramidite-containing compound having a structure represented by the following structural formula: [ka] (Structure 1004b).

[0218] In some embodiments, the targeted ligand has a structure represented by the following structural formula: [ka] (Structure 1005).

[0219] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {In the formula, R includes or consists of an expression-inhibiting oligomer compound} (structure 1005a).

[0220] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {In the formula, R consists of or contains expression inhibitory oligomer compounds; Y is O or S; and Y' is O - S - , or NH - (Structure 1005a(i)).

[0221] In some embodiments, the targeted ligand is a phosphoramidite-containing compound having a structure represented by the following structural formula: [ka] (Structure 1005b).

[0222] In some embodiments, the targeted ligand has a structure represented by the following structural formula: [ka] (Structure 1006).

[0223] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {In the formula, R includes or consists of an expression-inhibiting oligomer compound} (structure 1006a).

[0224] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {In the formula, R consists of or contains expression inhibitory oligomer compounds; Y is O or S; and Y' is O - S - , or NH - (Structure 1006a(i)).

[0225] In some embodiments, the targeted ligand is a phosphoramidite-containing compound having a structure represented by the following structural formula: [ka] (Structure 1006b).

[0226] In some embodiments, the targeted ligand has a structure represented by the following structural formula: [ka] (Structure 1007).

[0227] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {In the formula, R includes or consists of an expression-inhibiting oligomer compound} (structure 1007a).

[0228] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {wherein R consists of or contains expression inhibitory oligomer compounds; Y is O or S; and Y' is O - S - , or NH - (Structure 1007a(i)).

[0229] In some embodiments, the targeted ligand is a phosphoramidite-containing compound having a structure represented by the following structural formula: [ka] (Structure 1007b).

[0230] In some embodiments, the targeted ligand has a structure represented by the following structural formula: [ka] (Structure 1008).

[0231] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {wherein R comprises or consists of an expression-inhibiting oligomer compound} (structure 1008a).

[0232] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {wherein R consists of or contains expression inhibitory oligomer compounds; Y is O or S; and Y' is O - S - , or NH - (Structure 1008a(i)).

[0233] In some embodiments, the targeted ligand is a phosphoramidite-containing compound having a structure represented by the following structural formula: [ka] (Structure 1008b).

[0234] In some embodiments, the targeted ligand has a structure represented by the following structural formula: [ka] (Structure 1009).

[0235] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {In the formula, R includes or consists of an expression-inhibiting oligomer compound} (structure 1009a).

[0236] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {wherein R consists of or contains expression inhibitory oligomer compounds; Y is O or S; and Y' is O - S - , or NH - (Structure 1009a(i)).

[0237] In some embodiments, the targeted ligand is a phosphoramidite-containing compound having a structure represented by the following structural formula: [ka] (Structure 1009b).

[0238] In some embodiments, the targeted ligand has a structure represented by the following structural formula: [ka] (Structure 1010).

[0239] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {wherein R comprises or consists of an expression-inhibiting oligomer compound} (structure 1010a).

[0240] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {wherein R consists of or contains expression inhibitory oligomer compounds; Y is O or S; and Y' is O - S - , or NH -(Structure 1010a(i)).

[0241] In some embodiments, the targeted ligand is a phosphoramidite-containing compound having a structure represented by the following structural formula: [ka] (Structure 1010b).

[0242] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and comprises a structure represented by the following structural formula: [ka] {wherein J comprises or consists of one or more substituted or unsubstituted cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heterocyclyl groups, or combinations thereof linked by covalent bonds; Y is O or S; and R comprises or consists of an expression inhibitory oligomer compound; and Y' is O - S - , or NH - (Structure 1011).

[0243] In some embodiments, the targeted ligand has a structure represented by the following structural formula: [ka] (Structure 1012).

[0244] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {In the formula, R includes or consists of an expression-inhibiting oligomer compound} (structure 1012a).

[0245] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {wherein R consists of or contains expression inhibitory oligomer compounds; Y is O or S; and Y' is O - S - , or NH - (Structure 1012a(i)).

[0246] In some embodiments, the targeted ligand is a phosphoramidite-containing compound having a structure represented by the following structural formula: [ka] (Structure 1012b).

[0247] In some embodiments, the targeted ligand has a structure represented by the following structural formula: [ka] (Structure 1013).

[0248] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {In the formula, R includes or consists of an expression-inhibiting oligomer compound} (structure 1013a).

[0249] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {wherein R consists of or contains expression inhibitory oligomer compounds; Y is O or S; and Y' is O - S - , or NH - (Structure 1013a(i)).

[0250] In some embodiments, the targeted ligand is a phosphoramidite-containing compound having a structure represented by the following structural formula: [ka] (Structure 1013b).

[0251] In some embodiments, the targeted ligand has a structure represented by the following structural formula: [ka] (Structure 1014).

[0252] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {In the formula, R includes or consists of an expression-inhibiting oligomer compound} (structure 1014a).

[0253] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {wherein R consists of or contains expression inhibitory oligomer compounds; Y is O or S; and Y' is O - S - , or NH - (Structure 1014a(i)).

[0254] In some embodiments, the targeted ligand is a phosphoramidite-containing compound having a structure represented by the following structural formula: [ka] (Structure 1014b).

[0255] In some embodiments, the targeted ligand has a structure represented by the following structural formula: [ka] (Structure 1015).

[0256] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {In the formula, R includes or consists of an expression-inhibiting oligomer compound} (structure 1015a).

[0257] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {wherein R consists of or contains expression inhibitory oligomer compounds; Y is O or S; and Y' is O - S - , or NH - (Structure 1015a(i)).

[0258] In some embodiments, the targeted ligand is a phosphoramidite-containing compound having a structure represented by the following structural formula: [ka] (Structure 1015b).

[0259] In some embodiments, the targeted ligand has a structure represented by the following structural formula: [ka] (Structure 1016).

[0260] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {wherein R comprises or consists of an expression-inhibiting oligomer compound} (structure 1016a).

[0261] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {wherein R consists of or contains expression inhibitory oligomer compounds; Y is O or S; and Y' is O - S - , or NH - (Structure 1016a(i)).

[0262] In some embodiments, the targeted ligand is a phosphoramidite-containing compound having a structure represented by the following structural formula: [ka] (Structure 1016b).

[0263] In some embodiments, the targeted ligand has a structure represented by the following structural formula: [ka] (Structure 1017).

[0264] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {In the formula, R includes or consists of an expression-inhibiting oligomer compound} (structure 1017a).

[0265] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {wherein R consists of or contains expression inhibitory oligomer compounds; Y is O or S; and Y' is O - S - , or NH - (Structure 1017a(i)).

[0266] In some embodiments, the targeted ligand is a phosphoramidite-containing compound having a structure represented by the following structural formula: [ka] (Structure 1017b).

[0267] In some embodiments, the targeted ligand has a structure represented by the following structural formula: [ka] (Structure 1018).

[0268] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {In the formula, R includes or consists of an expression-inhibiting oligomer compound} (structure 1018a).

[0269] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {wherein R consists of or contains expression inhibitory oligomer compounds; Y is O or S; and Y' is O - S - , or NH - (Structure 1018a(i)).

[0270] In some embodiments, the targeted ligand is a phosphoramidite-containing compound having a structure represented by the following structural formula: [ka] (Structure 1018b).

[0271] In some embodiments, the targeted ligand has a structure represented by the following structural formula: [ka] (Structure 1019).

[0272] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {In the formula, R includes or consists of an expression-inhibiting oligomer compound} (structure 1019a).

[0273] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {wherein R consists of or contains expression inhibitory oligomer compounds; Y is O or S; and Y' is O - S - , or NH - (Structure 1019a(i)).

[0274] In some embodiments, the targeted ligand is a phosphoramidite-containing compound having a structure represented by the following structural formula: [ka] (Structure 1019b).

[0275] In some embodiments, the targeted ligand has a structure represented by the following structural formula: [ka] (Structure 1020).

[0276] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {In the formula, R includes or consists of an expression-inhibiting oligomer compound} (structure 1020a).

[0277] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {wherein R consists of or contains expression inhibitory oligomer compounds; Y is O or S; and Y' is O - S - , or NH -(Structure 1020a(i)).

[0278] In some embodiments, the targeted ligand is a phosphoramidite-containing compound having a structure represented by the following structural formula: [ka] (Structure 1020b).

[0279] In some embodiments, the targeted ligand has a structure represented by the following structural formula: [ka] (Structure 1021).

[0280] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {wherein R comprises or consists of an expression-inhibiting oligomer compound} (structure 1021a).

[0281] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {wherein R consists of or contains expression inhibitory oligomer compounds; Y is O or S; and Y' is O - S - , or NH - (Structure 1021a(i)).

[0282] In some embodiments, the targeted ligand is a phosphoramidite-containing compound having a structure represented by the following structural formula: [ka] (Structure 1021b).

[0283] In some embodiments, the targeted ligand has a structure represented by the following structural formula: [ka] (Structure 1022).

[0284] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {In the formula, R includes or consists of an expression-inhibiting oligomer compound} (structure 1022a).

[0285] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {wherein R consists of or contains expression inhibitory oligomer compounds; Y is O or S; and Y' is O - S - , or NH - (Structure 1022a(i)).

[0286] In some embodiments, the targeted ligand is a phosphoramidite-containing compound having a structure represented by the following structural formula: [ka] (Structure 1022b).

[0287] In some embodiments, the targeted ligand has a structure represented by the following structural formula: [ka] (Structure 1023).

[0288] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {In the formula, R includes or consists of an expression-inhibiting oligomer compound} (structure 1023a).

[0289] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {wherein R consists of or contains expression inhibitory oligomer compounds; Y is O or S; and Y' is O - S - , or NH - (Structure 1023a(i)).

[0290] In some embodiments, the targeted ligand is a phosphoramidite-containing compound having a structure represented by the following structural formula: [ka] (Structure 1023b).

[0291] In some embodiments, as disclosed herein, a linker of the targeted ligand may be absent if the branching group contains at least one aryl, cycloalkyl, and / or heterocyclic group. Having one or more aryl, cycloalkyl, and / or heterocyclic groups located within the branching group serves as a linker substituent. In some embodiments, one or more aryl, cycloalkyl, and / or heterocyclic groups within the branching group are positioned between one or more central junctions within the branching group and the expression-inhibiting oligomer compound.

[0292] In some embodiments, the targeted ligand has a structure represented by the following structural formula: [ka] (Structure 1024).

[0293] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {In the formula, R includes or consists of an expression-inhibiting oligomer compound} (structure 1024a).

[0294] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {wherein R consists of or contains expression inhibitory oligomer compounds; Y is O or S; and Y' is O - S - , or NH - (Structure 1024a(i)).

[0295] In some embodiments, the targeted ligand is a phosphoramidite-containing compound having a structure represented by the following structural formula: [ka] (Structure 1024b).

[0296] In some embodiments, the targeted ligand has a structure represented by the following structural formula: [ka] (Structure 1025).

[0297] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {In the formula, R includes or consists of an expression-inhibiting oligomer compound} (structure 1025a).

[0298] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {wherein R consists of or contains expression inhibitory oligomer compounds; Y is O or S; and Y' is O - S - , or NH - (Structure 1025a(i)).

[0299] In some embodiments, the targeted ligand is a phosphoramidite-containing compound having a structure represented by the following structural formula: [ka] (Structure 1025b).

[0300] In some embodiments, the targeted ligand has a structure represented by the following structural formula: [ka] (Structure 1026).

[0301] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {In the formula, R includes or consists of an expression-inhibiting oligomer compound} (structure 1026a).

[0302] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {wherein R consists of or contains expression inhibitory oligomer compounds; Y is O or S; and Y' is O - S - , or NH - (Structure 1026a(i)).

[0303] In some embodiments, the targeted ligand is a phosphoramidite-containing compound having a structure represented by the following structural formula: [ka] (Structure 1026b).

[0304] In some embodiments, the targeted ligand has a structure represented by the following structural formula: [ka] (Structure 1027).

[0305] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {In the formula, R comprises or consists of an expression-inhibiting oligomer compound} (structure 1027a).

[0306] In some embodiments, the expression inhibitory oligomer compound is linked to a targeted ligand and has a structure represented by the following structural formula: [ka] {wherein R consists of or contains expression inhibitory oligomer compounds; Y is O or S; and Y' is O - S - , or NH - (Structure 1027a(i)).

[0307] In some embodiments, the targeted ligand is a phosphoramidite-containing compound having a structure represented by the following structural formula: [ka] (Structure 1027b).

[0308] In some embodiments, the targeted ligand is in the form of a galactose cluster. As used herein, galactose clustering comprises a targeted ligand having 2 to 4 terminal galactose derivatives. As used herein, the term galactose derivative includes both galactose and galactose derivatives having an affinity for asialoclycoprotein receptors equivalent to or greater than that of galactose. The galactose derivative is a saccharide sugar, which is a type of targeted moiety. The terminal galactose derivative may be tethered through the C-1 carbon of the saccharide.

[0309] In some embodiments, the targeted ligand comprises three terminal galactosamines or galactosamine derivatives (such as N-acetyl-galactosamine) each having affinity for an asial glycoprotein receptor. In some embodiments, the targeted ligand includes three terminal N-acetyl-galactosamines (GalNAc or NAG) as the targeting moiety.

[0310] In some embodiments, the targeted ligand comprises four terminal galactosamines or galactosamine derivatives (such as N-acetyl-galactosamine) each having affinity for an asialoclycoprotein receptor. In some embodiments, the targeted ligand comprises four terminal N-acetyl-galactosamines (GalNAc or NAG) as the targeting moiety.

[0311] In some embodiments, the targeting moiety comprises a galactosamine derivative, namely N-acetyl-galactosamine. Other saccharides that have affinity for asialocryprotein receptors and can be used as targeting moieties may be selected from a list including: galactose, galactosamine, N-formyl-galactosamine, N-acetyl-galactosamine, N-propionyl-galactosamine, Nn-butanoylgalactosamine, and N-iso-butanoylgalactosamine. The affinity of many galactose derivatives for asialocryprotein receptors has been studied (see, e.g., Iobst, ST and Drickamer, KJBC 1996, 271, 6686) or can be readily measured using commonly used methods well known in the art.

[0312] When referring to the three terminal N-acetylgalactosamines, commonly used terms in this field include tri-antennary, trivalent, and trimer. When referring to four-terminal N-acetylgalactosamines, commonly used terms in the field include tetra-antennary, tetravalent, and tetramer.

[0313] Oligomer compounds The targeted ligands disclosed herein can be linked to oligomeric compounds. In some embodiments, the oligomeric compound is an expression inhibitory oligomeric compound. In some embodiments, the expression inhibitory oligomeric compound is an RNAi agent. In some embodiments, the expression inhibitory oligomeric compound is a double-stranded RNAi agent. In some embodiments, the expression inhibitory oligomeric compound is a single-stranded oligonucleotide. The expression inhibitory oligomeric compounds can be synthesized using methods commonly used in the art.

[0314] The expression-inhibiting oligomeric compound may contain one or more modified nucleotides. Nucleotide bases (or nucleobases) are components of all nucleic acids and are heterocyclic pyrimidine or purine compounds including adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U). As used herein, the term "nucleotide" may include modified nucleotides or nucleotide mimics, abasic sites, or surrogate substitution moieties. As used herein, a "modified nucleotide" is a nucleotide other than a ribonucleotide (2'-hydroxyl nucleotide), a nucleotide mimic, an abasic site, or a surrogate substitution moiety. In some embodiments, modified nucleotides include 2'-modified nucleotides (i.e., nucleotides having a group other than a hydroxyl group at the 2' position of the five-membered sugar ring). Modified nucleotides include, but are not limited to: 2'-modified nucleotides, 2'-O-methyl nucleotides (also represented herein as the lowercase "n" within a nucleotide sequence), 2'-deoxy-2'-fluoro nucleotides (represented herein as Nf and also represented herein as 2'-fluoro nucleotides), 2'-deoxy nucleotides (also represented herein as dN), 2'-methoxyethyl (2'-O-2-methoxyl ethyl) nucleotides (also represented herein as NM or 2'-MOE), 2'-amino nucleotides, 2'-alkyl nucleotides, 3'-3' linked (inverted) nucleotides (also represented herein as invdN, invN, invn, invX), unnatural bases containing nucleotides, locked nucleotides, cross-linked nucleotides, peptide nucleic acids, 2',3'-seco nucleotide mimics (unlocked nucleic acid base analogs, also represented herein as N UNA or also represented as NUNA), locked nucleotides (also represented herein as N LNA or also represented as NLNA), 3'-O-methoxy (2'-nucleotide internucleoside linkage) nucleotides (also represented herein as 3'-OMen), 2'-F-arabinonucleotides (also represented herein as NfANA or Nf ANAExamples include morpholinonucleotides (also represented as vpdN), vinylphosphonate deoxyribonucleotides (also represented as vpdN), vinylphosphonate nucleotides, and debasalized nucleotides (also represented as X or Ab). Uniform modification is not required at all positions of a given compound. Conversely, two or more modifications can be incorporated into just one expression inhibitory oligomer compound, or even into its single nucleotide. The expression inhibitory oligomer compounds can be synthesized and / or modified by methods known in the art. Modifications of each nucleotide are independent of modifications of other nucleotides.

[0315] Modified nucleic acid bases include synthetic and native nucleic acid bases, such as 5-substituted pyrimidines, 6-azapyrimidines, N-2-, N-6-, O-6-substituted purines (e.g., 2-aminopropyladenine), 5-propynyluracil, 5-propynylcytosine, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil, 5-halocytosine, 5-propynyluracil, 5-propynylcytosine Examples include tosine, 6-azo-uracil, 6-azo-cytosine, 6-azo-thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines, 5-substituted uracils and cytosines (e.g., 5-halouracil and cytosine (e.g., 5-bromouracil and 5-bromocytosine)), 5-trifluoromethyluracil, 5-trifluoromethylcytosine), 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-azaguanine, 3-deazaguanine, and 3-azaguanine.

[0316] In the expression inhibitory oligomeric compounds described herein, any modified nucleotides may be linked by phosphate-containing or non-phosphate-containing covalent nucleoside bonds. Modified nucleoside bonds or skeletons include, but are not limited to, 5'-phosphorothioates (also represented herein by a lowercase "s" before the nucleotide, and also by sN, sn, sNf, or sdN), chiral phosphorothioates, phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, 3'-alkylene phosphonates, and methyl and other alkyl phosphonates including chiral phosphonates, phosphinates, and 3'-alkylene phosphonates. Examples include phosphoramidates, thionophosphoramidates, thionoalkyl-phosphonates, thionoalkyl-phosphoriesters, morpholino linkages, boranophosphonates typically having 3'-5' linkages, 2'-5' linked analogues of boranophosphonates, and boranophosphonates having opposite polarity, where adjacent pairs of nucleoside units are linked from 3'-5' to 5'-3' or from 2'-5' to 5'-2'. In some embodiments, the modified internucleoside linkage or skeleton lacks a phosphorus atom. Examples of modified internucleoside linkages lacking a phosphorus atom include, but are not limited to, short-chain alkyl or cycloalkyl sugar linkages, mixed heteroatom-alkyl or cycloalkyl sugar linkages, or one or more short-chain heteroatom or heterocyclic sugar linkages. In some embodiments, the modified internucleoside skeletons include, but are not limited to, siloxane skeletons, sulfide skeletons, sulfoxide skeletons, sulfone skeletons, formacetyl and thioformacetyl skeletons, methyleneformacetyl and thioformacetyl skeletons, alkene-containing skeletons, sulfamate skeletons, methyleneimino and methylenehydrazino skeletons, sulfonate and sulfonamide skeletons, amide skeletons, and other skeletons having hybridized N, O, S, and CH2 components.

[0317] In some embodiments, the expression inhibitory oligomer compound is a double-stranded RNAi agent comprising a sense strand and an antisense strand that are at least partially complementary (at least 70% complementary). The antisense strand includes a region having a sequence that is either perfectly complementary (100% complementary) or at least substantially complementary (at least 85% complementary) to the sequence of the target mRNA. The length of the sense strand and antisense strand of the double-stranded RNAi agent may be 16 to 30 nucleotides each. The sense and antisense strands may be the same length or they may be of different lengths. In some embodiments, the sense strand is about 19 nucleotides long, while the antisense strand is about 21 nucleotides long. In some embodiments, the sense strand is about 21 nucleotides long, while the antisense strand is about 23 nucleotides long. In other embodiments, the sense and antisense strands are independently 17 to 21 nucleotides long. In some embodiments, both the sense and antisense strands are 21–26 nucleotides in length. In some embodiments, both the sense and antisense strands are 26 nucleotides in length. In some embodiments, the sense and antisense strands are independently 17–26 nucleotides in length. In some embodiments, the double-stranded RNAi agent has a double-stranded length of approximately 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides. This fully or substantially complementary region between the sense and antisense strands is typically 15–25 nucleotides in length (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides) and occurs at or near the 5' end of the antisense strand.

[0318] The ligand-binding inhibitory oligomeric compounds disclosed herein optionally and independently include an additional 1, 2, 3, 4, 5, or 6 nucleotides (as extensions) at the 3', 5', or both 3' and 5' ends of the core sequence. These additional nucleotides, if present, may or may not be complementary to the corresponding sequence of the targeted mRNA.

[0319] In some embodiments, when a double-stranded RNAi agent is bound to a targeted ligand disclosed herein, additional sense strand append-nucleotides, if present, may be identical to or different from the corresponding sequence of the targeted mRNA. If present, additional antisense strand append-nucleotides, if present, may be complementary to or different from the corresponding append-nucleotide of the sense strand. Double-stranded RNAi agents can be formed by annealing the antisense strand with the sense strand.

[0320] In some embodiments, the targeted ligand is ligated to the RNAi agent at the 3' or 5' end of either the sense strand or antisense strand of the RNAi agent. In some embodiments, the targeted ligand is ligated to the 5' end of the sense strand. In some embodiments, the targeted ligand is ligated to the 3' end of the sense strand. In some embodiments, the targeted ligand is ligated to the RNAi agent via an unstable, cleavable, or reversible bond. In some embodiments, the unstable, cleavable, or reversible bond is contained in a cleavable portion added between the RNAi agent and the targeted ligand.

[0321] In some embodiments, the expression-inhibiting oligomer compound is a single-stranded oligonucleotide. In some embodiments, the single-stranded oligonucleotide utilizes an RNA interference mechanism to inhibit the expression of a target mRNA. In some embodiments, the single-stranded oligonucleotide is active in reducing the expression of a target nucleic acid through a mechanism other than RNA interference.

[0322] In some embodiments, the gene expression level and / or mRNA level of the target in a subject to which the aforementioned targeted ligand conjugated to an expression-inhibiting oligomer compound is administered is reduced by at least about 5%, for example, at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% compared to the subject before administration or to a subject not given the targeted ligand complex. The gene expression level and / or mRNA level of the subject may be reduced in the cells, cell populations, and / or tissues of the subject. In some embodiments, the protein levels of a subject administered with the aforementioned targeted ligand conjugated to an expression-inhibiting oligomer compound are reduced by at least about 5%, for example, at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% compared to the subject before administration of the targeted ligand complex or to a subject not administered with the targeted ligand complex. The protein levels of the subject may be reduced in the cells, cell populations, tissues, blood, and / or other bodily fluids of the subject. Reductions in gene expression, mRNA, or protein levels can also be assessed by any method known in the art. Reductions or decreases in mRNA levels and / or protein levels are also considered herein as inhibiting, reducing, or decreasing the expression of the targeted gene.

[0323] Specific expression inhibitory oligomeric compounds used in conjunction with the disclosed targeted ligands are known in the art. In particular, numerous reference documents disclose expression inhibitory oligomeric compounds that can be bound to the targeted ligands disclosed herein for delivery of compositions to the liver. A non-limiting example is U.S. Patent Application No. 15 / 281,309, entitled Compositions and Methods for Inhibiting Gene Expression of LPA (which is incorporated herein by reference in its entirety), which discloses a variety of double-strand expression inhibitory oligomeric compounds that target the human apolipoprotein(a) gene [LPA] (for inhibiting the expression of the apo(a) protein, which is a portion of the lipoprotein(a) particle, and the lipoprotein(a) particle (Lp(a)) thereon), which are suitable for use in conjunction with the targeted ligands disclosed herein. The apo(a) gene [LPA] is primarily expressed in the livers of humans and non-human primates. Similarly, for example, U.S. Patent Application No. 15 / 229,314, entitled "RNAi Therapy for Hepatitis B Virus Infection" (the above-mentioned document is also incorporated herein by reference in its entirety), discloses a variety of double-strand expression inhibitory oligomer compounds that target hepatitis B virus and are suitable for use with the targeted ligands disclosed herein. The hepatitis B virus is a strictly liver-targeting, double-stranded DNA-containing virus and is classified as a member of the hepadnavirus family, belonging to the Hepadnaviridae family. Furthermore, as another example, U.S. Patent Application No. 15 / 229,314, entitled "Compositions and Methods for Inhibiting Gene Expression of Factor XII" (the above-mentioned document is also incorporated herein by reference in its entirety), discloses a variety of double-strand expression inhibitory oligomer compounds that target the factor XII (or factor XII, F12) gene and are suitable for use with the targeted ligands disclosed herein. Factor XII is a serine protease that is primarily expressed in the liver and can be found in blood.Furthermore, as another example, U.S. Patent Application No. 14 / 740,307, entitled "Compositions and Methods for Inhibiting Gene Expression of Alpha-1 AntiTrypsin" (the above-mentioned document is incorporated herein by reference in its entirety), discloses various double-strand expression inhibitory oligomeric compounds that target the α-1 antitrypsin (or AAT) gene and are suitable for use with the targeted ligands disclosed herein. AAT is a protease inhibitor belonging to the serpine superfamily, and the AAT protein is typically synthesized primarily in the liver by hepatocytes and then secreted into the bloodstream. Furthermore, WO2016 / 01123, entitled "Organic Compositions to Treat APOC3-Related Diseases" (the above-mentioned document is incorporated herein by reference in its entirety), discloses various double-strand expression inhibitory oligomeric compounds that target human apolipoprotein III (APOC3) and are suitable for use with the targeted ligands disclosed herein. Apolipoprotein C-III is a lipoprotein component thought to inhibit the hepatic uptake of triglyceride-rich particles. Further references disclosing various therapeutic compounds, including expression inhibitory oligomeric compounds, which may be suitable for use with the targeted ligands disclosed herein, can also be found in the art. These include, but are not limited to, compositions that are preferably targeted to the liver.

[0324] Pharmaceutical compositions and formulations The targeted ligands disclosed herein, when linked to an oligomeric compound, can be used to treat subjects (e.g., humans or mammals) suffering from a disease or disorder that would benefit from administration of the compound. In some embodiments, the targeted ligands disclosed herein, when linked to an expression-inhibiting oligomeric compound, can be used to treat subjects (e.g., humans) suffering from a disease or disorder that would benefit from reduction or inhibition of the expression of a target mRNA. The subject is administered a therapeutically effective amount of any one or more expression-inhibiting oligomeric compounds, such as RNAi agents, linked to the targeted ligand disclosed herein. The subject may be a human, a patient, or a human patient. The subject may be an adult, an adolescent, a child, or an infant. The aforementioned pharmaceutical compositions comprising a targeted ligand linked to an expression-inhibiting oligomeric compound can be used to provide a method for the therapeutic treatment of a disease. Such a method includes the administration of the pharmaceutical compositions described herein to a human or animal.

[0325] The pharmaceutical compositions and methods disclosed herein may reduce the level of target mRNA in cells, cell populations, tissues, or subjects: by administering a therapeutically effective amount of the expression inhibitory oligomer compound described herein, ligated to a targeting ligand, to the subject, thereby inhibiting the expression of the target mRNA. In some embodiments, the subject has been previously identified as having upregulation of the pathogenicity of a target gene in the target cells or tissues.

[0326] In some embodiments, the pharmaceutical composition comprises at least one expression inhibitory oligomer compound linked to a targeted ligand. These pharmaceutical compositions are particularly useful in inhibiting the expression of the target mRNA in target cells, cell populations, tissues, or organisms. The pharmaceutical composition is used to treat subjects suffering from a disease or disorder that would benefit from a reduction in the level of the target mRNA or inhibition of the expression of the target gene. The pharmaceutical composition can be used to treat subjects at risk of developing a disease or disorder that would benefit from a reduction in the level of the target mRNA or inhibition of the expression of the target gene. In one embodiment, the method comprises administering to a subject to be treated a composition comprising the targeted ligand described herein, linked to an expression inhibitory oligomer compound such as an RNAi agent. In some embodiments, one or more pharmaceutically acceptable excipients (including vehicles, carriers, diluents, and / or delivery polymers) are added to the pharmaceutical composition comprising the targeted ligand linked to the expression inhibitory oligomer compound to form a pharmaceutical formulation suitable for in vivo delivery to humans.

[0327] In some embodiments, the aforementioned pharmaceutical compositions comprising a targeted ligand linked to an expression-inhibiting oligomeric compound are used to treat or manage clinical features associated with the expression of a target mRNA. Alternatively, in some embodiments, therapeutic or prophylactic effective doses of one or more pharmaceutical compositions are administered to subjects in need of such treatment, prevention, or management. In some embodiments, administration of any complexing ligand covalently linked to an oligomeric compound can be used to reduce the number, severity, and / or frequency of symptoms of a disease in question.

[0328] The aforementioned pharmaceutical compositions, comprising a targeted ligand linked to an expression-inhibiting oligomer compound, can be used to treat at least one symptom in a subject suffering from a disease or disorder that would benefit from the reduction or inhibition of the expression of a target mRNA. In some embodiments, the subject is administered a therapeutically effective dose of one or more pharmaceutical compositions comprising an expression-inhibiting oligomer compound, such as an RNAi agent linked to a targeted ligand as described herein, thereby treating the symptom. In other embodiments, the subject is administered a prophylactically effective dose of one or more expression-inhibiting oligomer compounds, thereby preventing at least one symptom.

[0329] In some embodiments, the expression or level of target mRNA in subjects administered with the expression inhibitory oligomer compound linked to the targeted ligand disclosed herein is reduced by at least about 5%, for example, at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% compared to subjects not given the pharmaceutical composition. The gene expression level in the subject may be reduced in the cells, cell populations, and / or tissues of the subject. In some embodiments, mRNA levels are reduced. In other embodiments, protein expression levels are reduced. In some embodiments, the level of a target protein administered with an expression-inhibiting oligomeric compound linked to a targeted ligand disclosed herein is reduced by at least about 5%, for example, at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% compared to a subject not given the pharmaceutical composition. The reduction in expression, mRNA level, or protein level can be assessed by any method known in the art. The reduction or decrease in mRNA level and / or protein level is referred to herein as the reduction or decrease of target RNA, or the inhibition or reduction of target mRNA expression.

[0330] The route of administration is a route through which the expression inhibitory oligomer compound is brought into contact with the body. In general, methods for administering drugs and nucleic acids for the treatment of mammals are well known in the art and can be applied to the administration of the compositions described herein. The expression inhibitory oligomer compounds linked to the targeted ligands described herein can be administered via any suitable route by preparations appropriately adapted to specific routes. Thus, the pharmaceutical compositions described herein can be administered, for example, by injection intravenously, intramuscularly, intradermally, subcutaneously, intra-articularly, or intraperitoneally. In some embodiments, the pharmaceutical compositions described herein are administered by inhalation.

[0331] Pharmaceutical compositions comprising an expression inhibitory oligomer compound linked to a targeted ligand as described herein can be delivered to cells, cell populations, tumors, tissues, or subjects using oligonucleotide delivery technologies known in the art. Generally, any preferred method recognized in the art for delivering nucleic acid molecules (in vitro or in vivo) can be adapted for use with the compositions described herein. For example, delivery may be by topical administration (e.g., direct injection, implantation, or local administration), systemic administration, or by subcutaneous, intravenous, peritoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, and subarachnoid), intramuscular, percutaneous, airway (aerosol), nasal, oral, rectal, or topical (including buccal and sublingual) administration. In certain embodiments, the compositions are administered by subcutaneous or intravenous infusion or injection.

[0332] Accordingly, in some embodiments, the pharmaceutical compositions described herein may contain one or more pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical compositions described herein can be formulated for administration to a subject.

[0333] When used herein, a pharmaceutical composition or drug comprises a pharmacologically effective amount of one or more of the aforementioned therapeutic compounds and at least one pharmaceutically acceptable excipient. A pharmaceutically acceptable excipient (excipient) is a substance other than the active pharmaceutical ingredient (API, therapeutic agent, e.g., F12 RNAi agent) that is intentionally included in a drug delivery system. Excipients do not exert, or are not intended to exert, a therapeutic effect at the intended dosage. Excipients may function to a) assist in the processing of the drug delivery system during manufacturing, b) protect, assist, or enhance the stability, bioavailability, or patient acceptability of the API, c) assist in product identification, and / or d) enhance the overall safety, efficacy, or other properties of the delivery of the API during storage or use. A pharmaceutically acceptable excipient may or may not be an inert substance.

[0334] Excipients, though not limited to these, include: absorption enhancers, anticaking agents, antifoaming agents, antioxidants, binders, buffering agents, carriers, coatings, colorants, delivery enhancers, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, chain extenders, bulking agents, flavorings, flow enhancers, wetting agents, lubricants, oils, polymers, preservatives, physiological saline, salts, solvents, sugars, suspending agents, sustained-release matrices, sweeteners, thickeners, isotonic agents, vehicles, water repellents, and wetting agents.

[0335] Suitable pharmaceutical compositions for injection include sterile aqueous solutions (if soluble in water) or dispersions, and sterile powders for the immediate preparation of sterile injection solutions or dispersions. For intravenous administration, suitable carriers include physiological saline solutions, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). These must be stable under manufacturing and storage conditions and protected from contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by the use of coatings such as lecithin, in the case of dispersions, by maintaining the required particle size, and by the use of surfactants. In many cases, it is desirable for the composition to contain isotonic agents, such as sugars, polyalcohols such as mannitol and sorbitol, and sodium chloride. Sustained absorption of an injectable composition can be achieved by including absorption-delaying agents in the composition, such as aluminum monosteaphosphate and gelatin.

[0336] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound in a suitable solvent, along with one or a combination of the components listed above, as needed, and then sterilizing by filtration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other necessary components from the components listed above. In the case of sterile powders for preparing sterile injectable solutions, the preparation method involves vacuum drying and freeze-drying, thereby obtaining powders of the active compound and any further desired components from the pre-sterilized filtered solution.

[0337] Formulations suitable for intra-articular administration may be in the form of a sterile aqueous formulation of the drug, which may exist in a microcrystalline form, such as a microcrystalline aqueous suspension. Liposome formulations or biodegradable polymer systems can also be used to deliver the drug for both intra-articular and ocular administration.

[0338] Suitable formulations for topical administration, including treatment of the eyes, include liquid or semi-liquid formulations, such as ointments, lotions, gels, topical medications, oil-in-water or water-in-oil emulsions (e.g., creams, ointments, or pastes); or solutions or suspensions such as eye drops. Formulations for topical administration to the skin surface can be prepared by dispersing the drug in a dermatologically acceptable carrier, such as a lotion, cream, ointment, or soap. Carriers that can form a film or layer covering the skin to localize the application and prevent removal are effective. With regard to topical administration to the surface of internal tissues, the agent can be dispersed in a liquid tissue adhesive or other substance known to promote adsorption to the tissue surface. For example, hydroxypropyl cellulose or fibrinogen / thrombin solution may be used as beneficial. Alternatively, tissue coating solutions such as pectin-containing formulations can be used.

[0339] With regard to inhalation therapy, inhalation of powders (self-propelled or spray formulations) dispensed using spray cans, nebulizers, or atomizers can be used. Such formulations may be in the form of fine powders for intrapulmonary administration from powder inhalation devices or self-propelled powder dispensing formulations. In the case of self-propelled solutions and spray formulations, the effect can be achieved by selecting a valve that has the desired spray characteristics (i.e., a spray with the desired particle size can be produced) or by incorporating the active ingredient as a powder suspended to a controlled particle size. With regard to administration by inhalation, the compound can also be delivered in the form of an aerosol spray from a pressurized container or dispenser containing a suitable propellant, such as a gas such as carbon dioxide, or from a nebulizer. Systemic administration is also possible by transmucosal or transdermal means. With regard to transmucosal or transdermal administration, a suitable wetting agent for the barrier to be permeated can be used in the formulation. Such wetting agents are generally known in the art and include, for example, lavage agents and bile salts for transmucosal administration. Transmucosal administration can be achieved by using nasal sprays or suppositories. With regard to transdermal administration, the active compound is typically formulated in the form of ointments, gels, or creams commonly known in the art.

[0340] The active compound can be prepared using a carrier that protects the compound from rapid elimination from the body, such as a sustained-release formulation containing a graft and a microencapsulated delivery system. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoester, and polylactic acid may be used. Methods for preparing such formulations will be apparent to those skilled in the art. Liposome suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared, for example, according to methods known to those skilled in the art, such as those described in U.S. Patent No. 4,522,811.

[0341] Oral or parenteral compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to a physically separated unit suitable as a unit dose for the subject being treated, each unit containing a predetermined amount of the active compound calculated to produce the desired therapeutic effect in relation to the required pharmaceutical carrier. Details regarding the dosage unit form of this disclosure are determined and directly depend on the inherent characteristics of the active compound, the therapeutic effect to be achieved, and the inherent limitations in the art to synthesize such active compound for the treatment of an individual. Furthermore, administration can be performed by periodic injection of bolus, or more continuously by intravenous, intramuscular, or intraperitoneal administration from an external reservoir (e.g., an intravenous bag).

[0342] In connection with the methods described herein, pharmacogenomics (i.e., studies of the correlation between an individual's genotype and their response to an exogenous compound or drug) may be considered. Differences in the metabolism of therapeutic agents can alter the relationship between the dose and blood concentration of pharmacologically active drugs, potentially leading to severe toxicity or treatment failure. Therefore, physicians or clinicians may consider applying knowledge gained from relevant pharmacogenomics studies when deciding whether to administer a drug and when adjusting the dosage and / or treatment plan for treatment using such drug.

[0343] A pharmaceutical composition may include other additional components commonly found in pharmaceutical compositions. Such additional components may include, but are not limited to, antipruritic agents, astringents, local anesthetics, or anti-inflammatory agents (e.g., antihistamines, diphenhydramines, etc.). It is also conceivable that cells, tissues, or excised organs expressing or containing the RNAi agents defined herein may be used as a “pharmaceutical composition.” As used herein, “pharmacologically effective amount,” “therapeutically effective amount,” or simply “effective amount” refers to the amount of RNAi agent that produces a pharmacological, therapeutic, or prophylactic effect.

[0344] Generally, the effective dose of the active compound is in the range of approximately 0.1 to 100 mg / kg body weight / day, for example, approximately 1.0 to 50 mg / kg body weight / day. In some embodiments, the effective dose of the active compound is in the range of approximately 0.25 to 5 mg / kg body weight per dose. In some embodiments, the effective dose of the active ingredient is in the range of approximately 0.5 to 3 mg / kg body weight per dose. The amount administered may also depend on variables such as the patient's overall health, the relative biological efficiency of the compound being delivered, the drug formulation, the presence and type of excipients in the formulation, and the route of administration. It should also be understood that the initial dose administered may be increased beyond high levels to rapidly achieve the desired blood concentration or tissue level, or the initial dose may be less than the optimal level.

[0345] With regard to the treatment of a disease or the formation of a drug or composition for the treatment of a disease, the pharmaceutical compositions described herein, which include expression inhibitory oligomeric compounds such as RNAi agents linked to a targeted ligand, may be combined with excipients, or, but are not limited to, a second therapeutic agent or treatment including a second or other expression inhibitory oligomeric compound, a small molecule drug, an antibody, an antibody fragment, and / or a vaccine.

[0346] The aforementioned targeted ligands can be packaged in kits, containers, packs, or dispensers when linked to an expression-inhibiting oligomer compound and when added to a pharmaceutically acceptable excipient or adjuvant. The pharmaceutical compositions described herein can be packaged in pre-filled syringes or vials. The embodiments provided previously are illustrated here by the following unrestricted examples. [Examples]

[0347] The following embodiments are intended to be illustrative and not to limit the specific embodiments disclosed herein. Some of the abbreviations used in the experimental details of the synthesis of the following examples are defined below: h or hour = hour; min = minute; mol = mole; mmol = millimoles; M = mole; μM = micromoles; g = grams; μg = micrograms; rt or RT = room temperature; L = liters; mL = milliliters; wt = weight; Et2O = diethyl ether; THF = tetrahydrofuran; DMSO = dimethyl sulfoxide; Depositphotos = ethyl acetate; Et3N or Tea = triethylamine; i-Pr2NEt, DIPEA or DIEA = diisopropylethylamine; CH2Cl2 or DCM = methylene chloride; CHCl3 = chloroform; CDCl3 = deuterated chloroform; CCl4 = carbon tetrachloride; MeOH = methanol; EtOH = ethanol; DMF = dimethylformamide; BOC = t-butoxycarbonyl; CBZ = benzyloxycarbonyl TBS = t-butyldimethylsilyl; TBSCl = t-butyldimethylsilyl chloride; TFA = trifluoroacetic acid; DMAP = 4-dimethylaminopyridine; NaN3 = sodium azide; Na2SO4 = sodium sulfate; NaHCO3 = sodium bicarbonate; NaOH = sodium hydroxide; MgSO4 = magnesium sulfate; K2CO3 = potassium carbonate; KOH = potassium hydroxide; NH4OH = ammonium hydroxide; NH4Cl = ammonium chloride; SiO2 = silica; Pd-C = palladium carbon; HCl = hydrogen chloride or hydrochloric acid; NMM = N-methylmorpholine; H2 = hydrogen gas; KF = potassium fluoride; EDC-HCl = N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride; MTBE = methyl-tert-butyl ether; MeOH = methanol; Ar = argon; SiO2 = silica; RT = retention time.

[0348] Furthermore, examples of expression-inhibiting oligomer compounds suitable for use with the targeted ligands disclosed herein are listed in various tables within the following examples. The following notation is used to indicate modified nucleotides with respect to the sequences listed in the tables disclosed herein:

[0349] N = 2'-OH (unmodified) ribonucleotide (uppercase letter without f or d designation) n = 2'-OMe modified nucleotide nf = 2'-fluoromodified nucleotide dN = 2'-deoxynucleotide N UNA = 2',3'-Seconucleotide mimetic (unlocked nucleic acid base analog) N LNA = Locked nucleotides Nf ANA = 2'-F-arabinonucleotide NM = 2'-methoxyethylnucleotide X or Ab = Debase-ribose R = Livitol (invdN) = Inverted deoxyribonucleotide (3'-3' linked nucleotide) (invAb) = Inverted debasalized nucleotide (invX) = Inverted debased nucleotide (invn) = inverted 2'-OMenucleotide s = phosphorothioate-bonded nucleotide vpdN = vinylphosphonate deoxyribonucleotide (3'OMen) = 3'-OMenucleotide (5Me-Nf) = 5'-Me,2'-fluoronucleotide cPrp = Cyclopropylphosphonate The compounds of this disclosure can be prepared using synthetic chemical techniques known to those skilled in the art.

[0350] Example 1. Synthesis of targeted ligands, phosphoramidite compounds, structures 1005b, 1004b, and 1002b The phosphoramidite-containing compounds of structures 1005b, 1004b, and 1002b were synthesized according to the following procedure, with the only difference being that 4-cis-hydroxycyclohexanecarboxylic acid (compound 8 as used herein) was used to synthesize compound 1005b, 4-trans-hydroxycyclohexanecarboxylic acid (compound 8a as used herein) was used to synthesize compound 1004b, and a mixture of 4-cis-hydroxycyclohexanecarboxylic acid (compound 8 as used herein) and 4-trans-hydroxycyclohexanecarboxylic acid (compound 8a as used herein) was used to synthesize compound 1002b.

[0351] 1) Preparation of 2-amino-3-[4-({[(benzyloxy)carbonyl]amino}methyl)phenyl]propanoic acid (compound 2) [ka]

[0352] Copper carbonate base (1.67 g, 7.59 mmol) was slowly added to a solution of 1 g (7.00 g, 30.34 mmol) in water (100 mL). The resulting mixture was heated to 80°C until dissolution was observed. The resulting dark blue solution was cooled to 25-30°C and then treated with sodium hydroxide (1.21 g, 30.34 mmol) in water (10 mL), which yielded a precipitate of amino acid-copper complexes. The suspension was stirred at ambient temperature for 1 hour, and then treated with a solution of benzyl chloroformate (6.21 g, 36.41 mmol) in THF (20 mL) dropwise over 5 minutes. The mixture was stirred for 1-2 hours and then filtered. The wet cake was crushed in phosphate and filtered again to aid in dehydration. The blue solid was then added to a flask containing 200 mL of water and treated with 10 mL of concentrated HCl. The slurry was stirred for 18 hours, then filtered and washed with water to obtain 4.5 g of compound 2 as a white solid (45% yield, 95 AP). T =5.8 minutes.

[0353] 2) Preparation of Triacid (Compound 3)

Chem

[0354] A slurry of 2 (6.00 g, 18.27 mmol) in 1.5 M NaOH (100 mL) was heated to 60 °C at which point a solution formed. Next, the solution was treated with a solution of bromoacetic acid (10.15 g, 73.20 mmol) dissolved in 1.5 M NaOH (20 mL). The solution was stirred at 60 °C for 2 h (2 = NMT 5% by HPLC). When the reaction was complete, the solution was cooled to 10 °C and 1 M HCl was added until pH = 1.7 was reached. The slurry was allowed to stand for 2 - 3 h, then filtered and washed with deionized water. The solid was dried in vacuo to give 3.01 g of triacid 3 (50%, 94 AP). R T = 6.94 min.

[0355] 3) Preparation of TFP Ester (Compound 4)

Chem

[0356] A solution of 3 (3.00 g, 6.75 mmol) and 2,3,5,6 - tetrafluorophenol (3.99 g, 24.30 mmol) in DCM (50 mL) was cooled to 10 °C and treated with divided N-(3 - diethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (4.66 g, 24.30 mmol) over 5 min. Next, the solution was warmed to ambient temperature over 20 min and stirred for 3 h. After completion (3 < 10 AP), the reaction mixture was washed with saturated sodium bicarbonate (20 mL), followed by brine (20 mL), and concentrated by rotary evaporator. The resulting oil was purified by flash column using a 5 - 20% EtOAc / hexane solvent gradient to give 2.6 g of 4 as a colorless oil (40%, 94 AP). R T = 12.99 min

[0357] 4) Preparation of amine tosylate (compound 5) [ka]

[0358] Pack a pressure reactor of appropriate size with THF, followed by CbZ-protecting amine (1.0 eq, NAGZ) and p-TsOH-H2O (1.0 equivalent) (10 times the volume). Degas the solution three times with nitrogen. Pack with 10% Pd / C (5.0 wt%), then degas three times with nitrogen. Degas three times with hydrogen. Charge with hydrogen to a pressure of 40-50 psi. Stir at 20-30°C for 3 hours, then degas three times with nitrogen and extract the sample for IPC assay (specify ≤ 0.5% NAG-Z; if the specification is not met, stir under H2 at 40-50 psi for 1-2 hours, then re-assay). Remove the catalyst by filtration through diatomaceous earth and wash with THF (4 times the volume). Concentrate the combined filtrate and wash under reduced pressure to approximately 2 times the volume while maintaining Ti ≤ 40°C. Dilute with DCM (3.8 times the volume), then concentrate again to 2 times the volume. Repeat the DCM dilution and reconcentration, then dilute with DCM (3.8 times the volume). Extract a sample for KF analysis (spec KF ≤ 0.05%; if the KF specification is not met, repeat the concentration and dilution with DCM). After the KF specification is met, concentrate the solution to a white foamy solid. 100% uncorrected yield. A similar reaction using trifluoroacetic acid instead of p-TsOH-H2O may also be carried out and is interchangeable.

[0359] 5) Preparation of tri-NAG (compound 6) [ka]

[0360] Activated ester 4 (2.15 g, 2.41 mmol) and amine silate 5 (4.10 g, 6.75 mmol) were dissolved in dichloromethane (22 mL) and cooled to 10°C. The solution was then treated with triethylamine (1.37 g, 13.54 mmol) dropwise over 5 minutes, followed by heating to ambient temperature and holding for 2 hours. The reaction mixture was washed with saturated sodium bicarbonate (10 mL) and then with brine (10 mL). The solution was dried over magnesium sulfate, filtered, and concentrated using a rotary evaporator to obtain a colorless oil. After testing, ~10% desacyl impurities were detected by HPLC. The impurities were re-acylated by stirring in undiluted acetic anhydride (90 mL) and triethylamine (6 mL) for 1 hour. Next, the acetic anhydride was removed under reduced pressure, and the resulting oil was redissolved in dichloromethane and washed with an aqueous sodium bicarbonate solution. The solution was concentrated to the oil and purified by flash chromatography using gradient elution (2.5-25% MeOH / DCM) to obtain 1.98 g of 6 as a white solid (47%, 96 AP). T =7.57 min; Desacyl impurity = 7.18 min

[0361] 6) Preparation of the amine salt (compound 7) [ka]

[0362] Protected amine 6 (1.98 g, 1.06 mmol) and p-toluenesulfonic acid monohydrate (202 mg, 1.06 mmol) were dissolved in anhydrous ethanol (30 mL) and placed under a nitrogen atmosphere. 5% palladium-carbon (198 mg, 0.106 mmol) was added to the flask, and the flask was placed under vacuum and back-filled several times with hydrogen. It was found that once the reaction was stirred at ambient temperature under a hydrogen atmosphere, it would complete within 4 hours or until the initiating substance was no longer detectable by HPLC. The catalyst was filtered through a Celite bed, and the filtrate was passed through a 0.2 micron membrane filter to remove particulate matter. The solution was concentrated to dryness under reduced pressure to obtain 2.01 g of 7 as a gray solid (100%, 98 AP). T =5.82; p-toluenesulfonic acid R T = 2.4 and 3.1 minutes

[0363] 7) Preparation of activated linkers (compounds 9 and 9a) [ka]

[0364] cis-4-hydroxycyclohexylcarboxylic acid 8 (structure 1005 for synthesis) (4.00 g, 27.7 mmol) and 2,3,5,6-tetrafluorophenol (5.53 g, 33.3 mmol) were dissolved in 24 mL of dichloromethane and cooled to 0°C. [As previously stated, cis-4-hydroxycyclohexylcarboxylic acid (compound 8) is used as a linker to formulate structure 1005, while trans-4-hydroxycyclohexylcarboxylic acid (compound 8b) may be substituted with a cis-isomer, which leads to the synthesis of structure 1004b, following the same procedure for the rest of the synthesis:]

[0365] [ka] ].

[0366] To this solution, EDC-HCl (6.38 g, 33.3 mmol) was added. The solution was heated to 22°C and stirred for 12 hours. The reaction was quenched with saturated NaHCO3 aqueous solution (50 mL), and the layers were separated. The organic layer was washed with saturated brine (50 mL) and dried over Na2SO4. The drying agent was filtered, and the solution was concentrated to about 20 mL, which solidified slowly (accelerated by seed crystals). The solid was slurryed with 5% MTBE / hexane (50 mL) and filtered to obtain 5.6 g of product 9 in 69% yield and 95% purity.

[0367] 8) Linker linking (Preparation of compound 10) [ka]

[0368] NAGamine salt 7 (5.00 g, 2.88 mmol) and 2,3,5,6-tetrafluorophenyl cis-4-hydroxycyclohexane carboxylate 9 (1.68 g, 5.77 mmol) were dissolved in 25 mL of dichloromethane and cooled to 0°C. Triethylamine (1.60 mL, 11.55 mmol) was added to this solution. The solution was warmed to room temperature and stirred for 5 hours while being observed by HPLC. The reaction was quenched with saturated NaHCO3 aqueous solution (35 mL), and the layers were separated. The organic layer was washed with saturated brine (35 mL) and dried over Na2SO4. The drying agent was filtered, and the solution was concentrated and purified by flash chromatography using gradient elution (0-20% MeOH / DCM) to give 3.90 g of compound 10 as a white solid (80%). T = 6.16 minutes. Alternatively, direct linking of the linker can be carried out without using TFP ester, as shown in Example 2 below.

[0369] 9) Preparation of compound 11 [ka]

[0370] Compound 10 (1.87 g, 1.11 mmol) was dissolved in 20 mL of dichloromethane and 2-cyanoethyl, and N,N,N',N'-tetraisopropylphosphoramidite (0.84 g, 2.77 mmol) was added. The resulting solution was cooled to 5°C. 4,5-dicyanoimidiazole (0.026 g, 0.22 mmol) was added to this solution. The solution was heated to room temperature and stirred for 1 hour. Next, the degree of conversion was checked by HPLC (showing a residue of 2% of the starting material). Additional 2-cyanoethyl, N,N,N',N'-tetraisopropylphosphoramidite (0.14 g, 0.46 mmol) was added, and the reaction was stirred for a further 2.5 hours (no significant changes were observed by HPLC). The reaction was quenched with saturated NaHCO3 aqueous solution (20 mL), and the layers were separated. The organic layer was washed with an aqueous solution of NaHCO3 (20 mL) and saturated brine (2 × 20 mL), and then dried over Na2SO4. The drying agent was filtered, and the solution was concentrated to obtain 2.34 g of compound 11 as a white solid.

[0371] 100 mg of unpurified compound 11 was purified by flash column chromatography by first eluting a silica gel packed column with 2% triethylamine in dichloromethane for 30 minutes, followed by adding unpurified compound 11 to the column and purifying it using gradient elution (0-20% 2% triethylamine:methanol / 2% triethylamine:dichloromethane). The final product, compound 11 (having the chemical structure of structure 1005b as defined herein), was eluted in 2% triethylamine:dichloromethane (fraction 2) to obtain 80 mg of a white solid.

[0372] Figure 1 shows compound 11 (structure 1005b in this specification). 1 The 1H NMR spectrum is shown. Figure 1A shows the trans isomer (structure 1004b in this specification) of compound 11, following the alternative synthesis described in step 7 above. 1 The 1H NMR spectrum is shown.

[0373] Example 2. Synthesis of a targeted ligand, a phosphoramidite-containing compound, and structure 1008b. 1) Preparation of tri-tert-butyl N-[N-(benzyloxycarbonyl)-L-γ-glutamyl]-L-glutamic acid (compound 14)

[0374] [ka]

[0375] In a 250 mL three-necked flat-bottom flask, flushed with nitrogen and equipped with a thermocouple, magnetic stirring bar, nitrogen inlet, and powder funnel, 12 (10.00 g, 29.64 mmol) was added, followed by THF (100 mL, 10 vol.). The resulting solution was stirred, and then N-methylmorpholine (7.82 mL, 71.15 mmol, 2.4 equivalents) was added (KF of reaction mixture: 163 ppm).

[0376] The powder funnel was replaced with a rubber partition, and the mixture was cooled to 0°C using an ice bath. While maintaining the pot temperature below 4.0°C, isobutyl chloroformate (iBuCOCl, 3.85 mL, 4.05 g, 29.64 mmol, 1.0 equivalent) was added dropwise to the reaction mixture using a syringe over 10 minutes. Following the addition, the mixture was stirred for a further 40 minutes, and the partition was replaced with a powder funnel. To the reaction mixture, while maintaining the pot temperature below 4.0°C, 13 (8.767 g, 29.64 mmol, 1.0 equivalent) was added in portions over 15 minutes (exothermic addition). Following the addition of 13, the ice bath and powder funnel were removed, and the reactants were warmed to ambient temperature during the remaining steps. Following the addition of 13, the clear, colorless solution was allowed to stand for 25 minutes.

[0377] A sample of the reaction (98 μL diluted in 5.0 mL of ACN in a 5 mL volumetric flask) was taken 40 minutes after the start of the addition of 13 and analyzed for conversion percentage by RP-HPLC. Since 23% of 12 remained, additional iBuCOCl (1.16 mL, 1.21 g, 30 mol%) and 13 (2.63 g, 30 mol%) were added consecutively 60 minutes after the reaction. The solution was allowed to stand for another 60 minutes until the sample showed over 99% conversion by HPLC. The total reaction time was 2.5 hours from the start of the initial addition of 13.

[0378] The reaction solution was cooled to 3°C in an ice bath with 0.5M HCl. (aq) The mixture was poured into 125 mL of a stirring solution and stirred for approximately 5 minutes. The quenched reaction mixture was extracted with ethyl acetate (100 mL, 10 vol.; ensure the aqueous layer is acidic to ensure complete removal of NMM), the layers were separated, the organic phase was washed with brine (100 mL, 10 vol.), dried over Na₂SO₄, filtered through a coarse frit funnel into a 500 mL flat-bottom flask, and concentrated under vacuum to obtain a high-viscosity, colorless oil. The oil was dissolved in MTBE (100 mL, 10 vol.) and concentrated under vacuum to obtain a high-viscosity, colorless oil.

[0379] Hexane (100 mL, 10 vol.) was added to stirred oil (~600 rpm). A white haze appeared in the solution, which then disappeared with further stirring. Seed crystals were added, and the mixture was stirred for 40 minutes, during which time white crystals slowly formed.

[0380] Within 20 minutes, the slurry was thick enough to hinder stirring, and additional hexane (50 mL, 5 vol.) was added. After 40 minutes, the slurry was filtered through a coarse frit funnel, washed three times with hexane (~10 mL each), and air-dried in the funnel for 1 hour to obtain compound 14 as a fine white powder (15.64 g, 91%). Figure 2B shows the compound 14. 1 The 1H NMR spectrum is shown.

[0381] 1) Preparation of N-[N-(benzyloxycarbonyl)-L-γ-glutamyl]-L-glutamic acid (15)

[0382] [ka]

[0383] In a 3000 mL three-necked flat-bottom flask equipped with an overhead stirrer, powder funnel, thermocouple, and heating mantle, 3 (72.57 g, 125.4 mmol) and formic acid (reagent grade, >95%, 1.45 L, 20 volume equivalents) were added. The powder funnel was replaced with a stopper equipped with an N2 inlet, and the resulting solution was heated to 45°C and stirred for 1 hour while being observed by RP-HPLC. The reaction was considered complete when less than 2.0 area percent of mono-t-butyl ester remained.

[0384] A sample of the reaction product was taken 60 minutes after the addition of formic acid, and the sample was analyzed by RP-HPLC for the percentage of remaining mono-t-butyl ester. The analysis showed that 1.8% of mono-t-Bu ester remained after 90 minutes, so the reaction product was allowed to cool to room temperature.

[0385] The reaction products were diluted with toluene and acetonitrile (ACN, 1500 mL each), and the mixture was concentrated under vacuum. Formic acid was removed by azeotropy twice, first with 1:1 ACN:toluene (~600 mL) and then with ACN (~500 mL each). The substance was dried overnight under high vacuum to obtain a white, foamy solid compound (54.3 g, quantitative yield, 96.8 area %) at 254 nm. Figure 2C shows the results for compound 15. 1 The 1H NMR spectrum is shown.

[0386] 3) Preparation of tri-NAG-bis-Glu-NHZ (compound 16) [ka]

[0387] In a 1-liter flat-bottom flask, NAG-amine p-tosylate salt (5, 59.19 g, 97.6 mmol, 4.13 equivalents) and Z-bis-Glu trioxide base (15, 10.01 g, 23.6 mmol corrected purity, 1.0 equivalent) were added. The mixture was dissolved in acetonitrile (500 mL; KF of solution = 1283 ppm), concentrated under vacuum, and water was removed by azeotrope. The residue was dissolved in fresh acetonitrile (400 mL), a stirring bar was added, and the mixture was transferred to a nitrogen-flashed 1-liter three-necked flat-bottom flask equipped with a thermocouple. The water content was measured by KF (257 ppm).

[0388] TBTU (28.20 g, 87.8 mmol, 3.7 equivalents) was added to a solution stirred under nitrogen using a powder funnel. While maintaining the reaction temperature below 25°C, DIPEA (34.0 mL, 25.2 g, 8.0 equivalents) was added dropwise over 20 minutes using a syringe (a 5°C exothermic reaction was observed during the addition). The mixture was stirred for 2 hours from the start of DIPEA addition while being monitored by HPLC. Analysis at 78 minutes showed complete consumption of the starting material.

[0389] After 2 hours, the solvent was removed under vacuum. The resulting high-viscosity oil was dissolved in dichloromethane (1000 mL) and HCl was added. (aq) Wash with (3 x 500 mL) and NaHCO3 3(aq) The solution was saturated with (3 × 500 mL). The organic layer was dried over Na₂SO₄, filtered, and concentrated under vacuum to obtain an off-white waxy solid (33.5 g).

[0390] Flash column chromatography was performed using an ISCO CombiFlash automated purification system with a 330g ISCO RediSep Rf Gold silica column. The starting material was added as a solution in CHCl3 (~200mL). A gradient consisting of eluent A: CHCl3 and eluent B: MeOH was used to collect a total of 36 fractions (250-500mL each). The product-containing fraction was concentrated to obtain 18.75g of ¹⁶ (97.0% purity). The mixed fraction yielded 12.2g of ¹⁶ (78.8% purity). Figure 2D shows the relationship between compound 16 and 1 The 1H NMR spectrum is shown.

[0391] 4) Preparation of tri-NAG-bis-Glu-NH2 (compound 17) [ka]

[0392] A 1000 mL three-necked flat-bottom flask containing a stirring bar was filled with methanol (200 mL, 13 vol.). Compound 16 (15.44 g, 9.02 mmol corrected purity) was added to the stirring solvent, followed by additional methanol (200 mL, 13 vol.) and trifluoroacetic acid (1.40 mL, 18.1 mmol, 2.0 equivalents). The mixture was stirred for approximately 10 minutes. 10% Pd / C (50% wet basis, 1.547 g, 10% w / w) was added to the mixture. The headspace was flushed with hydrogen gas (balloon), and the mixture was stirred at ambient temperature for 2 hours while being observed by RP-HPLC.

[0393] After 75 minutes, a sample (100 μL) was extracted from the reaction and mixed with 1:1 acetonitrile:H2O (900 μL) in a 1 mL syringe filter (10 mm, 0.1 μm GHP membrane). The HPLC chromatogram showed a purity of over 96 area % with no residue of the starting material. The reaction mixture was then flushed with nitrogen and filtered through a Celite bed into a clean 1000 mL flat-bottom flask. The reactor was rinsed with methanol (50 mL) and dichloromethane (50 mL), and the rinse was also filtered. The slightly cloudy filtrate was partially concentrated under vacuum. An additional rinse of the Celite bed was performed using methanol (50 mL) and dichloromethane (50 mL); these, combined with the residue, were filtered through a 0.2 μm GHP membrane filter into another clean 1000 mL flat-bottom flask. The membrane was rinsed with acetonitrile (50 mL) to allow toluene byproducts to be removed by azeotrope. The solution was concentrated under vacuum to obtain 17 (14.15 g, 97.3 area % purity by HPLC) as an off-white foamy solid. Figure 2E shows the relationship between compound 17 and 1 The 1H NMR spectrum is shown.

[0394] 5) Preparation of tri-NAG-bis-Glu-NH-linker (compound 18) [ka]

[0395] A 500 mL three-necked flat-bottom flask equipped with magnetic stirring, a thermocouple, and a nitrogen blanket was packed with 17 (93.7% purity, 20.00 g, 11.4 mmol) and dichloromethane (150 mL). To the stirred solution, cis-4-hydroxycyclohexane-1-carboxylic acid (1.730 g, 12.0 mmol, 1.05 equivalents) was added, followed by TBTU (4.036 g, 12.6 mmol, 1.10 equivalents). The solution was cooled to -9°C using an ice-salt bath, and DIPEA (6.97 mL, 5.17 g, 40.0 mmol, 3.5 equivalents) was added dropwise over 7 minutes while maintaining the internal temperature below -5°C. An exothermic reaction of 1.7°C was observed during the addition. Once the addition of DIPEA was complete, the reaction mixture was stirred at -9°C for 90 minutes, at which point HPLC analysis (Method B) showed a complete consumption of 17.

[0396] After 110 minutes, the reaction was stopped with saturated NH4Cl (aq) Quenched by adding (400 mL). The layers were separated, and the aqueous layer was extracted with dichloromethane (2 x 200 mL). The combined organic layers were saturated with NaHCO3. 3(aq) The oil was washed with a 1:1 mixture of saline solution (400 mL), dried over Na₂SO₄, filtered, and concentrated to approximately 125 mL under vacuum. A small amount of methanol was used to ensure solubility. The resulting oil was added in a small stream to a 3 L flat-bottom flask containing stirred MTBE (1600 mL) to form a white precipitate. The rinse solution from the origin flask with dichloromethane (~20 mL) and MTBE (~200 mL) was added to the slurry, then allowed to stand for 1 hour (age), and then vacuum filtered through a 600 mL coarse glass frit funnel. The wet cake was re-slurried in MTBE (2 × 200 mL) in the funnel, filtered, and dried to a constant weight on a high vacuum line to obtain unpurified 18 as a white powder (16.22 g, 86% uncorrected yield).

[0397] Unpurified 18 (17.16 g, combined with previous lots) was purified using an ISCO CombiFlash EZPrep automated purification system with a 330 g ISCO RediSep Rf Gold silica column. The starting material was added as a solution in 8% MeOH / CH2Cl2 (~160 mL). Elution A: CH2Cl2; Elution B: A gradient of 50% MeOH:CH2Cl2 was used to produce fraction 33. The product-containing fraction was concentrated to obtain 10.13 g (98.1% purity, 59% recovery) of 18. The mixed fraction was stored to obtain an additional 6.52 g (86.1% purity) of 18, which could then be re-purified. Figure 2F shows the relationship between compound 18 and 1 The 1H NMR spectrum is shown.

[0398] 6) Preparation of targeted ligand and phosphoramidite (compound 19) [ka]

[0399] Compound 18 (13.0 g, 7.87 mmol) was dissolved in anhydrous dichloromethane (195 mL) and placed under a nitrogen atmosphere. To this mixture, DIPEA (4.11 mL, 23.61 mmol) and a solution of 2-cyanoethyl-N,N-diisopropyl chlorophosphodiamidite (2.45 mL, 11.02 mmol) in anhydrous dichloromethane (5 mL) were added dropwise over 5 minutes. The reaction mixture was stirred at room temperature for 1 hour while being monitored by HPLC (<1% SM residue). The reaction was quenched with saturated NaHCO3 aqueous solution (150 mL). The organic layer was separated, washed with saturated NaHCO3 aqueous solution (1 × 150 mL) and brine (1 × 150 mL), and then dried over Na2SO4. The solution was filtered with a drying agent and concentrated via flash chromatography by first treating a silica column with dichloromethane (+1% triethylamine) for 30 minutes, then adding the unpurified final product, compound 19 (having the chemical structure of structure 1008 herein), to the same column, and purifying it using gradient elution (0-20% MeOH (+1% TEA) / CH2Cl2 (+1% TEA)) over 30 minutes to obtain 11.1 g of compound 19 as a white solid (76% yield, 96.6% purity).

[0400] Figure 2 shows the relationship between compound 19 and 31 Figure 2A shows the 1P NMR spectrum for compound 19. 1 The 1H NMR spectra are shown. Both Figure 2 and Figure 2A are consistent with the structure of compound 19 (structure 1008b as used herein).

[0401] Example 3. Synthesis of a targeted ligand, a phosphoramidite-containing compound, and structure 1025. 1) Preparation of compound 21 [ka]

[0402] To a solution of compound 20 (40 g, 221 mmol, 1.00 eq) in MeOH (350 mL), SOCl2 (52.5 g, 442 mmol, 32 mL, 2.00 eq) was added dropwise at 0-5°C. The solution was heated to 60°C and stirred for 16 hours. TLC (DCM / MeOH = 5 / 1 with 5 drops of HOAc, Rf = 0.43) showed the consumed starting material, and LCMS (ET12452-6-P1A) showed the formed product. The mixture was concentrated under reduced pressure to obtain unpurified compound 21 (52.4 g, unpurified) as a white solid.

[0403] 1 H NMR: (ET12452-6-p1c DMSO Bruker_B_400MHz) δ 9.45 (s, 1 H), 8.55 (br s, 3 H), 7.00 (br d, J = 8.0 Hz, 2 H), 6.72 (d, J = 8.0 Hz, 2 H), 4.17 (br s, 1 H), 3.67 (s, 3 H), 3.01 (qd, J = 14.2, 6.5 Hz, 2 H)

[0404] 2) Preparation of compound 22 [ka]

[0405] To a solution of compound 21 (52.4 g, 226 mmol, 1.00 eq) in MeOH (230 mL), TEA (68.7 g, 679 mmol, 94 mL, 3.00 eq) and Boc2O (59.2 g, 271 mmol, 62.4 mL, 1.20 eq) were added dropwise at 0°C. The mixture was stirred at 0°C for 0.5 hours, and then stirred at 25°C for 16 hours. TLC (petroleum ether / siRNA = 1 / 1, Rf = 0.80) showed a new major spot, indicating that most of the starting material had been consumed. The mixture was concentrated and then purified by silica column (petroleum ether / siRNA = 1:1) to obtain compound 22 (57.4 g, 86% yield) as a white solid.

[0406] 1 H NMR: (ET12452-8-p1g CDCl3 Bruker_B_400MHz) δ 6.97 (d, J = 8.5 Hz, 2 H), 6.74 (br d, J = 8.0 Hz, 2 H), 5.65 (br s, 1 H), 5.01 (br d, J = 8.0 Hz, 1 H), 4.49 - 4.59 (m, 1 H), 3.72 (s, 3 H), 2.92 - 3.09 (m, 2 H), 1.43 (s, 9 H).

[0407] 3) Preparation of compound 23 [ka]

[0408] To a solution of compound 22 (35 g, 119 mmol, 1.00 eq) dissolved in acetone (170 mL), K2CO3 (21.3 g, 154 mmol, 1.30 eq) and BnBr (24.3 g, 142 mmol, 16.9 mL, 1.20 eq) were added, and the reaction mixture was heated at reflux temperature (60 °C) for 14 hours. TLC (petroleum ether / SiO₂ = 3 / 1, Rf = 0.80) showed that the starting material had been consumed and new spots had formed. H₂O (500 mL) was added to the mixture at 5 °C, stirred for 0.5 hours, then filtered, and H₂O (80 mL) was added. * 3) The mixture was washed and dried under reduced pressure to obtain compound 23 (43 g, 88% yield, 93% purity) as a white solid.

[0409] 1H NMR: (ET12452-9-p1a CDCl3 Bruker_B_400MHz) δ 7.31 - 7.46 (m, 5 H), 7.05 (d, J = 8.5 Hz, 2 H), 6.91 (d, J = 9.0 Hz, 2 H), 5.05 (s, 2 H), 4.97 (br d, J = 8.0 Hz, 1 H), 4.50 - 4.60 (m, 1 H), 3.72 (s, 3 H), 2.96 - 3.11 (m, 2 H), 1.43 (s, 9 H)

[0410] 4) Preparation of compound 24 [ka]

[0411] To a solution of compound 23 (43 g, 112 mmol, 1.00 eq) in HCl (215 mL), HCl / HCl (4 M, 215 mL, 7.71 eq) was added dropwise, and the mixture was stirred at 25°C for 9 hours. TLC (petroleum ether / HCl = 3 / 1, Rf = 0.10) indicated that the starting material had been consumed and a new pot had formed. The mixture was filtered and HCl (30 mL) was added. * 3) The mixture was washed and dried under reduced pressure to obtain compound 24 (35 g, 97% yield, 99% purity) as a white solid.

[0412] 1 H NMR: (ET12452-12-p1a MeOD Varian_D_400MHz) δ 7.40 - 7.45 (m, 2 H), 7.34 - 7.39 (m, 2 H), 7.29 - 7.33 (m, 1 H), 7.17 (d, J = 8.8 Hz, 2 H), 7.00 (d, J = 8.8 Hz, 2 H), 5.09 (s, 2 H), 4.26 (dd, J = 7.3, 6.0 Hz, 1 H), 3.81 (s, 3 H), 3.07 - 3.23 (m, 2 H)

[0413] 5) Preparation of compound 26 [ka]

[0414] Compound 24 (15.5g, 48.2 mmol, 1.00eq) was dissolved in CH3CN (40mL) and NaOH (1.5M, 70.6mL, 2.20eq). Then, Compound 25 (13.4g, 96.3 mmol, 6.94mL, 2.00eq) was added at 15°C, pH check: ~2.5. Next, 4N NaOH was added until pH=13. The solution was heated to 70°C. After 30 minutes, the pH dropped to less than 6, and was readjusted with 4N NaOH (pH 11~13). Additional Compound 25 (6.69g, 48.2 mmol, 3.47mL, 1.00eq) was added in two portions, adjusting the pH to 11~13 each time. The mixture was heated at 70°C for 14 hours. LC-MS (ET12452-30-P1A, Rt=0.749 min) showed that the product had formed. The mixture was cooled to 15°C, then adjusted to pH 1 with 4N HCl, filtered, and then H2O (80 mL) was added. * The residue was washed and dried. The residue was dissolved in THF (600 mL), then concentrated to 1 / 6 with batches of ET12452-27 and ET12452-19, then stirred with DCM (500 mL), and then filtered. The filter was dried to obtain compound 26 (35.5 g, 87% yield, 97% purity) as a white solid.

[0415] 1H NMR: (ET12452-30-p1r MeOD Varian_D_400MHz) δ 7.40 - 7.45 (m, 2 H), 7.36 (t, J = 7.4 Hz, 2 H), 7.27 - 7.32 (m, 1 H), 7.17 (d, J = 8.4 Hz, 2 H), 6.91 (d, J = 8.6 Hz, 2 H), 5.49 (s, 1 H), 5.05 (s, 2 H), 3.71 (t, J = 7.6 Hz, 1 H), 3.61 (s, 4 H), 3.07 (dd, J = 14.1, 7.5 Hz, 1 H), 2.86 - 2.96 (m, 1H), 2.03 (s, 2H)

[0416] 6) Preparation of compound 27 [ka]

[0417] To a solution of compound 26 (15 g, 38.7 mmol, 1.00 eq) and compound 26A (25.7 g, 155 mmol, 4.00 eq) in pyridine (250 mL), EDCI (29.7 g, 155 mmol, 4.00 eq) was added at 5°C. The mixture was stirred at 30°C for 12 hours. LC-MS (ET12452-59-P1A, Rt=1.053 min) showed mainly the product. The mixture was concentrated, then dissolved in DCM (200 mL), and dissolved in saturated NaHCO3 (80 mL). * 4) Saltwater (80mL) * 2) Washed, dried over Na2SO4, filtered, and concentrated. The residue was purified by silica column (petroleum ether / dimethyl = 3:1, Rf = 0.75) to obtain the product with compound 26A, which was then dissolved in DCM (200 mL) and saturated NaHCO3 (80 mL). * 4) and saline solution (80 mL) * The mixture was washed (2), dried over Na2SO4, filtered, and concentrated to obtain compound 27 (19.8 g, 61% yield) as an off-white rubber.

[0418] 1H NMR: (ET12452-59-p1g CDCl3 Bruker_B_400MHz) δ 7.36 - 7.46 (m, 4 H), 7.30 - 7.35 (m, 1 H), 7.24 (d, J = 8.7 Hz, 2 H), 6.97 - 7.07 (m, 3 H), 6.94 (d, J = 8.7 Hz, 2 H), 5.05 (s, 2 H), 4.13 - 4.26 (m, 5 H), 3.25 (d, J = 7.5 Hz, 2 H), 2.06 (s, 1 H), 1.25 - 1.29 (m, 1 H)

[0419] 7) Preparation of compound 27-2 [ka]

[0420] Compound 27-1 (230 g, 1.53 mol, 205 mL, 1.00 eq) was dissolved in dry DCM (1.6 L) under an N2 atmosphere. The solution was cooled to 0°C in an ice bath, and TEA (232 g, 2.3 mol, 318 mL, 1.50 eq) was added. Subsequently, TosCl (233 g, 1.22 mol, 0.80 eq) in DCM (500 mL) was added to the cooled reaction mixture. After the addition, the solution was heated to 20°C and stirred for 5 hours. TLC (petroleum ether / dimethyl = 1:1, Rf = 0.15) showed that the starting material had been consumed, and HPLC (ET12452-15-P1L, Rt = 1.71 min) showed two peaks. The reaction mixture was quenched at 0°C by adding H2O (500 mL), and the two reaction products were then extracted with CH2Cl2 (800 mL). The combined organic layers were washed with H2O (1 L) and brine (1 L), dried over Na2SO4, filtered, and concentrated. The residue was purified by silica column (petroleum ether / alkyl = 1:1) to obtain compound 27-2 (338 g, 36% yield) as a yellow oil.

[0421] 1H NMR: (ET12452-15-p1z1 CDCl3 Bruker_B_400MHz) δ 7.79 (d, J = 8.0 Hz, 2 H), 7.34 (d, J = 8.5 Hz, 2 H), 4.12 - 4.19 (m, 2 H), 3.67 - 3.72 (m, 4 H), 3.60 (s, 4 H), 3.55 - 3.58 (m, 2 H), 2.44 (s, 3 H), 2.32 (s, 1 H)

[0422] 8) Preparation of compound 27-3A [ka]

[0423] Compound 27-3-1 (230 g, 591 mmol, 1.00 eq) was suspended in DCM (700 mL) at 20°C, and TMSOTf (197 g, 886 mmol, 160 mL, 1.50 eq) was added under N2. The mixture changed color to pink. The mixture was heated to 50°C and stirred for 1.5 hours. Next, the reaction mixture was cooled to 20°C and stirred for 14 hours. TLC (DCM / MeOH = 20:1, Rf = 0.6) indicated that the starting material had been consumed. The mixture was poured into an aqueous NaHCO3 solution (600 mL) at 0-5°C and stirred for 15 minutes. The mixture changed color to yellow. The mixture was extracted with DCM (500 mL), and then diluted with an aqueous NaHCO3 solution (500 mL) and water (500 mL). * 2) Washed with brine (500 mL), dried over Na2SO4, filtered, and concentrated to obtain compound 27-3A (189 g, 92% unrefined purity) as a brown oil.

[0424] 1H NMR: (ET12452-28-p1c CDCl3 Varian_D_400MHz) δ 6.00 (d, J = 6.6 Hz, 1 H), 5.47 (t, J = 3.0 Hz, 1 H), 4.91 (dd, J = 7.5, 3.3 Hz, 1 H), 4.17 - 4.28 (m, 2 H), 4.08 - 4.14 (m, 1 H), 3.97 - 4.03 (m, 1 H), 2.13 (s, 3 H), 2.05 - 2.09 (m, 9 H)

[0425] 9) Preparation of compound 27-4A [ka]

[0426] A mixture of compound 27-3A (189 g, 574 mmol, 1.00 eq), compound 7-2 (140 g, 460 mmol, 0.80 eq), and 4A molecular sieve (150 g) in DCM (1.5 L) was mixed with TMSOTf (63.8 g, 287 mmol, 51.9 mL, 0.50 eq) under an N2 atmosphere, and the mixture was stirred at 25°C for 8 hours. TLC (DCM / MeOH = 20:1, Rf = 0.46) showed that the starting material had been consumed, and LCMS (ET12452-35-P1A, Rt = 0.76 min) showed that the product had been formed. The mixture was filtered to remove the sieve, then quenched with cold NaHCO3 aqueous solution (1000 mL), extracted with DCM (800 mL). * 2) The separated organic layer is saturated with NaHCO3 (800 mL) and H2O (800 mL). * 2) Washed with brine (800 mL), dried over Na2SO4, filtered, and concentrated. Next, purified by silica column (DCM / MeOH = 20:1) to obtain compound 27-4A (285 g, 73% yield) as a yellow oil.

[0427] 1H NMR: (ET12452-35-p1g CDCl3 Varian_D_400MHz) δ 7.81 (d, J = 8.4 Hz, 2 H), 7.37 (d, J = 8.2 Hz, 2 H), 6.30 (br d, J = 9.5 Hz, 1 H), 5.28 - 5.35 (m, 1 H), 5.08 (dd, J = 11.2, 3.3 Hz, 1 H), 4.81 (d, J = 8.6 Hz, 1 H), 4.09 - 4.29 (m, 5 H), 3.86 - 3.98 (m, 3 H), 3.68 - 3.81 (m, 3 H), 3.56 - 3.66 (m, 5 H), 2.46 (s, 3 H), 2.16 (s, 3 H), 2.04 (s, 3 H), 1.98 (s, 3 H), 1.95 (s, 3 H)

[0428] 10) Preparation of compound 27-4 [ka]

[0429] To a solution of compound 27-4A (285 g, 450 mmol, 1.00 eq) in DMSO (1.4 L), NaN3 (38.1 g, 586 mmol, 1.30 eq) was added at 10°C, and the mixture was stirred at 60°C for 16 hours. LC-MS (ET12452-37-P1A, Rt=0.67 min) showed that the product had formed and the starting material had been consumed. The mixture was poured into H2O (1500 mL) and SiO2O (1 L * 5) Extract and add H2O (800mL) * 3) and saltwater ( * 800 mL was washed with 3), dried over Na2SO4, filtered, and concentrated to obtain compound 27-4 (168 g, unrefined) as a red oil.

[0430] 1H NMR: (ET12452-37-p1c CDCl3 Bruker_B_400MHz) δ 6.12 (br d, J = 9.4 Hz, 1 H), 5.32 (d, J = 2.9 Hz, 1 H), 5.06 (dd, J = 11.3, 3.4 Hz, 1 H), 4.78 (d, J = 8.7 Hz, 1 H), 4.08 - 4.27 (m, 5 H), 3.82 - 3.94 (m, 3 H), 3.61 - 3.77 (m, 10 H), 3.45 - 3.50 (m, 2 H), 2.16 (s, 3 H), 2.05 (d, J = 1.5 Hz, 5 H), 1.99 (d, J = 4.5 Hz, 6 H), 1.26 (t, J = 7.2 Hz, 2 H)

[0431] 11) Preparation of compound 27A [ka]

[0432] To a solution of compound 27-4 (79 g, 156 mmol, 1.00 eq) in SiO / MeOH (4:1) (640 mL), Pd(OH)2 / C (7.9 g) was added, and the mixture was stirred at 15°C for 4 hours under an H2 (30 psi) atmosphere. TLC (DCM / MeOH = 20:1) showed that the starting material had been consumed, and LCMS (ET12452-53-P1C, Rt = 2.55 min) showed that the product had been formed. The two parallel reactions were filtered through Celite, and DCM (500 mL) was used to analyze the mixture. * 5) and MeOH (200 mL) * 3) The mixture was washed and concentrated to obtain compound 27A (140 g, unrefined) as a dark brown oil.

[0433] 1H NMR: (ET12452-53-p1c CDCl3 Varian_D_400MHz) δ 7.02 (br d, J = 9.3 Hz, 1 H), 5.29 - 5.34 (m, 1 H), 5.09 (dd, J = 11.2, 3.3 Hz, 1 H), 4.80 (d, J = 8.6 Hz, 1 H), 4.09 - 4.24 (m, 3 H), 3.82 - 3.95 (m, 3 H), 3.52 - 3.70 (m, 10 H), 2.91 (td, J = 5.2, 2.8 Hz, 1 H), 2.15 (s, 3 H), 2.05 (s, 4 H), 1.98 (d, J = 6.4 Hz, 6 H)

[0434] 12) Preparation of compound 28 [ka]

[0435] TEA (12.1 g, 119 mmol, 16.5 mL, 5.00 eq) was added to a stirred solution containing compound 27 (19.8 g, 23.8 mmol, 1.00 eq) and compound 27A (57 g, 119 mmol, 5.00 eq) in DCM (160 mL). The mixture was stirred at 30°C for 16 hours. LC-MS (ET12452-64-P1A, Rt=1.21 min) showed that a product had formed. The mixture was diluted in DCM (100 mL) and washed with saturated NaHCO3 / saturated brine (1:1, 2 × 80 mL). The organic layer was dried over Na2SO4, filtered, and concentrated to obtain the unpurified product as a brown solid.

[0436] The unpurified product was dissolved in Ac2O (42 mL), CH3CN (62.5 mL), and Py (82.3 g, 1.04 mol, 84 mL, 23.96 eq), and the mixture was stirred at 25°C for 12 hours. HPLC (ET12452-65-P1A, Rt=2.54 min) showed almost no product. CH3CN was removed by distillation, then diluted with DCM (400 mL), and finally saturated with NaHCO3 (100 mL). *4) Washed. The organic layer was separated and then rinsed in 0.1 M HCl / saturated brine (1:1, 100 mL). * 4) Washed, dried over Na2SO4, filtered, and concentrated. The residue was purified by silica column (DCM / MeOH=10:1, Rf=0.45) to obtain the product, and then p-HPLC (column: Phenomenex Gemini C18 250) * 50mm * Further purification was performed using a 10 μm mobile phase (water (10 mM NH4HCO3)-ACN); B%: 25%-55%, 23 min) to obtain compound 28 as a yellow solid (28.8 g, 58% yield, 98% purity).

[0437] 1 H NMR: (ET12452-65-p1j DMSO Varian_D_400MHz) δ 8.00 - 8.09 (m, 3 H), 7.81 (d, J = 9.0 Hz, 3 H), 7.29 - 7.45 (m, 5 H), 7.10 (d, J = 8.6 Hz, 2 H), 6.89 (d, J = 8.4 Hz, 2 H), 5.21 (d, J = 3.3 Hz, 3 H), 5.04 (s, 2 H), 4.97 (dd, J = 11.2, 3.3 Hz, 3 H), 4.54 (d, J = 8.4 Hz, 3 H), 4.02 (s, 9 H), 3.83 - 3.92 (m, 3 H), 3.73 - 3.81 (m, 3 H), 3.53 - 3.61 (m, 4 H), 3.44 - 3.52 (m, 17 H), 3.42 (br d, J = 4.4 Hz, 2 H), 3.35 - 3.40 (m, 6 H), 3.07 - 3.27 (m, 11 H), 2.74 - 2.87 (m, 2 H), 2.09 (s, 9 H), 1.99 (s, 10 H), 1.89 (s, 9 H), 1.77 (s, 9 H)

[0438] 13) Preparation of compound 29 [ka]

[0439] To a solution of compound 28 (9.7 g, 5.48 mmol, 1.00 eq) in THF (250 mL), dry Pd / C (5.5 g, 5.48 mmol) was added, and the mixture was stirred at 40°C for 6.5 hours under an H2 atmosphere (50 psi). TLC (DCM / MeOH = 10:1, Rf = 0.3) showed that the starting material had been consumed. The two parallel reaction products were filtered and collected in THF (300 mL). * 4) and DCM (200 mL) * 3) Washed and concentrated. The residue was subjected to p-HPLC using batch ET12452-78 (column: Phenomenex Gemini C18 250). * 50mm * The compound was purified using a 10 μm mobile phase (water (0.1% TFA)-ACN); B%: 15%-45%, 20 mins) to obtain compound 29 (14 g, 63% yield) as a white solid.

[0440] 1H NMR: (ET12452-80-p1j DMSO Varian_D_400MHz) δ 9.19 (s, 1 H), 7.99 - 8.10 (m, 3 H), 7.83 (d, J = 9.3 Hz, 3 H), 6.95 (d, J = 8.4 Hz, 2 H), 6.62 (d, J = 8.4 Hz, 2 H), 5.76 (s, 2 H), 5.21 (d, J = 3.3 Hz, 3 H), 4.97 (dd, J = 11.2, 3.3 Hz, 3 H), 4.54 (d, J = 8.6 Hz, 3 H), 4.03 (s, 9 H), 3.83 - 3.92 (m, 3 H), 3.73 - 3.81 (m, 3 H), 3.53 - 3.61 (m, 4 H), 3.44 - 3.52 (m, 16 H), 3.43 (br d, J = 4.4 Hz, 3 H), 3.36 - 3.39 (m, 3 H), 3.26 - 3.33 (m, 4 H), 3.05 - 3.24 (m, 9 H), 2.65 - 2.82 (m, 2 H), 2.10 (s, 9 H), 2.00 (s, 9 H), 1.89 (s, 9 H), 1.77 (s, 9 H)

[0441] 14) Preparation of compound 30

change

[0442] Compound 29 (8 g, 4.77 mmol, 1.00 eq) was dissolved in DCM (65 mL), and compound 29A (2.88 g, 9.54 mmol, 3 mL, 2.00 eq) was added. The resulting solution was cooled to 5°C. 2H-tetrazole (0.45 M, 11.7 mL, 1.10 eq) was added to this solution. The solution was heated to 15°C and stirred for 3.5 hours. TLC (DCM / MeOH=5:1, Rf=0.52) showed that the starting material had been consumed, and HPLC (ET12452-82-P1A, Rt=2.69 min) showed that the product had formed. The solution was diluted with DCM (50 mL), quenched with NaHCO3 (30 mL), and the aqueous solution was extracted with DCM (30 mL). * 2) Combine the organic layers and add saturated NaHCO3 (30 mL) * 2) H2O (30 mL) and saline solution (30 mL) * The mixture was washed in 2), dried over Na2SO4, filtered, and concentrated. The residue was dissolved in DCM (30 mL), then hexane (150 mL) was added dropwise at 0°C, stirred for 15 minutes, then cooled, the organic layer was poured out, and the oil was dissolved again in DCM (30 mL), hexane (150 mL) was added dropwise. This procedure was repeated 7 times, and the mixture was dried under reduced pressure to obtain compound 30 (5.5 g, 55% yield) as a white solid.

[0443] 1H NMR: (ET12452-83-p1b DMSO Varian_D_400MHz) δ 7.97 - 8.09 (m, 3 H), 7.78 (d, J = 9.3 Hz, 3 H), 7.06 (d, J = 8.2 Hz, 2 H), 6.86 (d, J = 8.2 Hz, 2 H), 5.73 (s, 2 H), 5.18 (d, J = 3.3 Hz, 3 H), 4.94 (dd, J = 11.1, 3.4 Hz, 3 H), 4.51 (d, J = 8.4 Hz, 3 H), 3.99 (s, 9 H), 3.79 - 3.89 (m, 4 H), 3.70 - 3.78 (m, 4 H), 3.59 - 3.69 (m, 2 H), 3.49 - 3.58 (m, 4 H), 3.44 (s, 16 H), 3.40 (br d, J = 4.2 Hz, 3 H), 3.32 - 3.37 (m, 5 H), 3.24 - 3.28 (m, 1 H), 3.05 - 3.22 (m, 9 H), 2.78 (br t, J = 5.8 Hz, 4 H), 2.07 (s, 9 H), 1.96 (s, 9 H), 1.86 (s, 9 H), 1.74 (s, 9 H), 1.15 (d, J = 6.8 Hz, 6 H), 1.09 (d, J = 6.8Hz, 6H) Figure 3 relates to compound 30 (structure 1025b in this specification). 1 The 1H NMR spectrum is shown. Example 4: Synthesis of a targeted ligand, a phosphoramidite-containing compound, and structure 1014b

[0444] 1) Preparation of compound 32 [ka]

[0445] A solution of compound 31 (24.71 g, 87.85 mmol, 1.00 eq), compound 31A, EDCI (39.07 g, 203.82 mmol, 2.32 eq), and pyridine (19.39 g, 245.11 mmol, 19.79 mL, 2.79 eq) in ACN (260.00 mL) was stirred at 25°C for 2 hours. TLC (petroleum ether / ethyl acetate = 1 / 1, desired product; Rf = 0.7) showed that the desired product was formed. The mixture was added to 300 mL of siRNA and NaHCO3 (100 mL) * 2) The product was washed with 100 mL of brine, dried using Na2SO4, filtered, and concentrated to obtain the residue. The unpurified product was purified using a silica column (petroleum ether / ethyl acetate = 100 / 1 to 3 / 1) to obtain compound 32 (60.00 g, 79.25 mmol, 90.20% yield, 97.19% purity) as a yellow oil.

[0446] 1 H NMR: (ET12600-89-p1a DMSO Varian_D_400MHz) δ ppm 7.52 (d, J = 8.4 Hz, 1H), 7.27-7.38 (m, 5H), 4.99 (s, 2H), 4.26-4.42 (m, 3H), 3.80-4.15 (m, 8H), 2.27 (br s, 2H), 1.78-1.88 (m, 1H), 1.66 (br dd, J = 14.4, 7.2 Hz, 1H), 1.37-1.41 (m, 35H)

[0447] 2) Preparation of compound 33 [ka]

[0448] A solution of compound 32 (45.00 g, 61.15 mmol, 1.00 eq) in formic acid (800.00 mL) was stirred at 45°C for 6 hours. LC-MS (et12600-90-p1a, MS=511) showed that the desired product was formed. The mixture was concentrated to obtain a residue. The residue was washed with 1000 mL of DCM to obtain compound 33 (30.00 g, 54.71 mmol, 89.47% yield, 93.27% purity) as a white solid.

[0449] 1 H NMR: (ET12600-90-p1a DMSO Bruker_B_400MHz) δ 12.75 (br s, 3H), 7.53 (br d, J = 8.4 Hz, 1H), 7.29-7.38 (m, 5H), 4.99 (d, J = 3.6 Hz, 2H), 4.27-4.38 (m, 2H), 4.12 (br s, 2H), 3.84-4.07 (m, 6H), 2.30 (br t, J = 7.2 Hz, 2H), 2.07 (s, 1H), 1.59-1.88 (m, 2H), 1.39 (t, J = 5.6Hz, 1H)

[0450] 3) Preparation of compound 34 [ka]

[0451] To a solution of compound 33 (15 g, 29.33 mmol, 1.00 eq), compound 33A (29.22 g, 175.98 mmol, 6.00 eq), and pyridine (11.60 g, 146.65 mmol, 11.84 mL, 5.00 eq) in ACN (90 mL), EDCI (28.11 g, 146.65 mmol, 5.00 eq) was added, and the mixture was stirred at 25°C for 1 hour. TLC (petroleum ether / ethyl acetate = 3 / 1) showed that the desired product was formed. 500 mL of DCM was added to the mixture, and NaHCO3 (200 mL) was added. *2) The solution was washed with 100 mL of saline solution, dried over Na2SO4, filtered, and concentrated to obtain the residue. It was purified using a silica column (petroleum ether / ethyl acetate = 4 / 1) to obtain compound 34 (28 g) as a yellow solid.

[0452] 4) Preparation of compound 35 [ka]

[0453] To a solution of compound 34 (16.57 g, 15.01 mmol, 1 eq) and compound 34A in DCM (140 mL), TEA (9.12 g, 90.08 mmol, 12.49 mL, 6.00 eq) was added, and the mixture was stirred at 25°C for 16 hours. LC-MS (et12600-98-p1g) showed that the desired product had formed. The mixture was poured into 200 mL of DCM, washed with 100 mL of NaHCO3 and 100 mL of brine, dried with Na2SO4, filtered, and concentrated to obtain the residue. HPLC for preparation (column: Phenomenex Gemini C18 250) * The compound was purified with 50 u (mobile phase: [water (10 mM NH4HCO3)-ACN]; B%: 15%-45%, 20 min) to obtain compound 5 (11 g, 4.65 mmol, 30.98% yield, 99.5% purity) as a yellow solid.

[0454] 1H NMR: (ET12600-98-p1a1 DMSO Varian_D_400MHz) δ 8.65-8.71 (m, 1H), 8.51 (br s, 1H), 8.18-8.25 (m, 1H), 8.11 (br s, 1H), 7.80 (d, J = 8.8 Hz, 4H), 7.47 (br d, J = 7.6 Hz, 1H), 7.28-7.40 (m, 5H), 5.75 (s, 4H), 5.22 (d, J = 3.2 Hz, 4H), 4.95-5.03 (m, 6H), 4.55 (d, J = 8.4 Hz, 4H), 3.98-4.06 (m, 15H), 3.88 (dt, J = 11.2, 8.8 Hz, 7H), 3.78 (dt, J=10, 5.2 Hz, 5H), 3.54-3.62 (m, 6H), 3.46-3.53 (m, 25H), 3.41 (q, J = 5.6 Hz, 9H), 3.23 (br dd, J = 11.6, 5.6 Hz, 8H), 2.10 (s, 12H), 2.00 (s, 12H), 1.89 (s, 12H), 1.77 (s, 12H)

[0455] 5) Preparation of compound 36 [ka]

[0456] To a solution of compound 35 (10 g, 4.25 mmol, 1 eq) in MeOH (10 mL) and TFA (484.52 mg, 4.25 mmol, 314.62 μL, 1 eq), 10% Pd(OH)2 / C (3.00 g) was added, and the mixture was stirred at 20°C for 4 hours under H2 (50 Psi). LC-MS (et12600-107-p1a, Rt=2.195) showed that the desired product had formed. The mixture was filtered and concentrated to obtain compound 36 (3.60 mmol, 8 g, 84.84% yield) as a yellow solid.

[0457] 1H NMR: (ET12600-107-p1a DMSO Varian_D_400MHz) δ 8.68 (br t, J = 5.2 Hz, 1H), 8.46 (br t, J = 5.2 Hz, 1H), 8.21-8.27 (m, 1H), 8.15 (br d, J = 5.6 Hz, 2H), 7.84 (br d, J = 9.2 Hz, 4H), 5.22 (d, J = 3.2 Hz, 4H), 4.98 (dd, J = 11.2, 3.2 Hz, 4H), 4.56 (d, J = 8.4 Hz, 4H), 4.24 (br s, 1H), 3.99-4.14 (m, 23H), 3.84-3.94 (m, 7H), 3.74-3.83 (m, 5H), 3.55-3.62 (m, 5H), 3.51 (s, 25H), 3.38-3.46 (m, 9H), 3.20-3.30 (m, 9H), 3.17 (d, J = 5.2 Hz, 14H), 2.11 (s, 12H), 2.00 (s, 13H), 1.89 (s, 12H), 1.78 (s, 12H)

[0458] 6) Preparation of compound 37 [ka]

[0459] The batches were parallel. To a solution of compound 36 (2 g, 857.18 μmol, 1.00 eq, TFA) and compound 36A (626.23 mg, 2.14 mmol, 2.50 eq) in DCM (6 mL), TEA (312.26 mg, 3.09 mmol, 427.75 μL, 3.60 eq) was added, and the mixture was stirred at 25°C for 16 hours. LC-MS showed that the desired product was formed. All reaction mixtures were combined, dissolved in 200 mL of DCM, poured over 30 mL of NaHCO3, washed with 30 mL of brine, dried over Na2SO4, filtered, and concentrated to obtain the residue. HPLC for preparation (column: Phenomenex Gemini C18 250) *The compound was purified with 50 u (mobile phase: [water (10 mM NH4HCO3)-ACN]; B%: 15%-45%, 20 min) to obtain compound 37 (3.20 mmol, 7.5 g, 93.27% yield) as a white solid.

[0460] 1 H NMR: (ET12600-111-p1a DMSO Varian_D_400MHz) δ 8.66 (s, 1H), 8.51 (br s, 1H), 8.20 (s, 1H), 8.08 (s, 1H), 7.91 (br d, J = 7.2 Hz, 1H), 7.80 (d, J=9.2 Hz, 4H), 5.22 (d, J =3.2 Hz, 4H), 4.98 (dd, J=11.2, 3.2 Hz, 4H), 4.55 (d, J = 8.4 Hz, 4H), 4.47 (br s, 1H), 4.30 (s, 1H), 4.25 (d, J = 3.2 Hz, 1H), 4.03 (s, 11H), 3.97 (br s, 2H), 3.84-3.92 (m, 7H), 3.73-3.82 (m, 6H), 3.55-3.62 (m, 5H), 3.47-3.54 (m, 24H), 3.41 (q, J = 5.6 Hz, 9H), 3.23 (br dd, J = 11.2, 5.6 Hz, 8H), 2.19 (br s, 1H), 2.10 (s, 12H), 2.00 (s, 13H), 1.89 (s, 12H), 1.77 (s, 13H), 1.61 (br s, 3H), 1.40 (br d, J=11.2Hz, 4H)

[0461] 8) Preparation of compound 38 [ka]

[0462] Compound 37 (4.4 g, 1.88 mmol, 1 eq) and Compound 37A (1.13 g, 3.75 mmol, 1.19 mL, 2 eq) were added to DCM (26.4 mL). The resulting solution was cooled to 5°C. 2H-tetrazole (0.45 M, 4.59 mL, 1.1 eq) was added to this solution. The solution was heated to 20°C and stirred for 2 hours. The mixture was dissolved in 100 mL of DCM, quenched with 20 mL of NaHCO3, and then dissolved in DCM (50 mL). * 2) Extraction was performed, washed with 20 mL of NaHCO3 and 20 mL of brine, dried with Na2SO4, filtered, and concentrated to obtain the residue. The residue was dissolved in DCM (25 mL, 0.2% TEA), then hexane (125 mL, 0.2% TEA) was added dropwise at 0°C, stirred for 15 minutes, then cooled, the organic layer was poured out, and the oil was dissolved again in DCM (30 mL), hexane (150 mL) was added dropwise. The procedure was repeated three times and dried under reduced pressure. 20 mL of DCM was added to the white solid, and it was dried under reduced pressure at 30°C to obtain 38 (4.8 g, 1.83 mmol, 67.34% yield, 97.15% purity) as a white solid. LCMS:[M-iPr2N] + / 2, 1222.8.

[0463] 1H NMR: (DMSO, Varian_400MHz) δ 8.67 (br s, 1H), 8.52 (br s, 1H), 8.20 (br s, 1H), 8.08 (br s, 1H), 7.98 (br d, J = 7.6 Hz, 1H), 7.79 (br d, J=9.2 Hz, 4H), 5.21 (d, J=3.2 Hz, 4H), 4.98 (dd, J = 11.2, 3.2 Hz, 4H), 4.55 (d, J = 8.4 Hz, 4H), 4.47 (br s, 1H), 4.29 (br d, J = 17.6 Hz, 1H), 3.94-4.11 (m, 16H), 3.83-3.94 (m, 8H), 3.78 (br dd, J = 10.4, 5.2 Hz, 6H), 3.64-3.74 (m, 3H), 3.54-3.63 (m, 8H), 3.50 (br s, 26H), 3.36-3.44 (m, 9H), 3.14-3.29 (m, 9H), 2.75 (t, J = 5.6 Hz, 2H), 2.15-2.27 (m, 4H), 2.10 (s, 13H), 2.00 (s, 13H), 1.82-1.95 (m, 15H), 1.77 (s, 14H), 1.59-1.73 (m, 4H), 1.45 (br d, J = 14.4Hz, 4H), 1.14 (d, J = 6.4Hz, 12H)

[0464] Figure 4 relates to compound 38 (structure 1014b in this specification). 1 The 1H NMR spectrum is shown.

[0465] Example 5: Synthesis of targeted ligands, phosphoramidite compounds, structures 1006b and 1007b The phosphoramidite-containing compounds of structure 1006b and structure 1007b were synthesized according to the same procedure below, with the only difference being that 4-cis-hydroxycyclohexanecarboxylic acid (compound 8 as used herein) was used to synthesize structure 1006b, and 4-trans-hydroxycyclohexanecarboxylic acid (compound 8a as used herein) was used to synthesize structure 1007b.

[0466] 1) Preparation of compound 41 [ka]

[0467] A solution of Z-Glu-(OtBu)-OH39 (445 mg, 1.32 mmol), di-tert-butyliminodiaacetate 40 (340 mg, 1.39 mmol), EDC (319 mg, 1.66 mmol, 1.23 eq), and Py (3 eq, 0.33 mL) in ACN (3 mL) was stirred at RT for 1 hour, diluted with ethyl acetate, and washed with NaHCO3 (2 ×). The organic layer was dried over MgSO4 and then removed by distillation. Next, the unpurified product was dissolved in DCM (5 mL) and TFA (5 mL) was added. This was stirred at RT for 16 hours and then removed by distillation. Ethyl acetate was added until foam / precipitate formed, and then removed by distillation (4 ×). The unpurified 41 was used directly in the TFP activation step. Rt = 3.78 min, 90% purity. LCMS(ES, M / z):379.0[M+H] + .

[0468] 2) Preparation of compound 42 [ka]

[0469] A solution of unpurified tri-acid 41 (~1.30 mmol), TFP (7 eq, 9.10 mmol, 1.51 g), TEA (4 eq, 0.723 mL), and EDC (3.3 eq, 4.29 mmol, 0.82 g) in ACN (3.5 mL) was stirred at RT for 1 hour, diluted with DCM (250 mL), and washed with saturated NaHCO3 (2 × 100 mL). The organic phase was dried over Na2SO4, concentrated, and purified by silica column. The product, activated tri-TFP ester, was eluted with AcOEt in hex (5-20%) to obtain 550 mg of the product along with a trace amount of TFP. Rt = 7.06 min.

[0470] TEA (400 μL, 2.9 mmol) is mixed with tri-TFP ester (540 mg, 0.642 mmol) and GalNac-Peg3-NH in DCM (6 mL). 2X The solution was added to a stirred solution containing TsOH (2.89 mmol, 1.88 g). This solution was stirred at RT for 16 hours, diluted with DCM (200 mL), and washed with saturated NaHCO3 / saturated brine (1:1, 2 × 150 mL). The organic layer was dried over Na2SO4 and removed by distillation, leaving a white solid. The solid was dissolved in DCM and purified by silica column chromatography. Elution with MeOH in DCM (0-10%) yielded 748 mg of tri-GalNAc42 with 95.4% purity and ~100 mg of tri-GalNAc42 with 80% purity, in 36% yield, 2 steps. LCMS (ES, M / z): 1777.5 [M] + , Rt=4.67 minutes.

[0471] 3) Preparation of compound 43 [ka]

[0472] A 10% Pd / C activated matrix (30 mg) was added to a solution of Cbz-protecting amine 42 (715 mg, 0.402 mmol) and TsOH (74.5 mg, 0.402 mmol) in THF (4 mL) and TFE (4 mL). A hydrogen atmosphere (balloon) was then constructed by hydrogen pulling vacuum and back filling. The mixture was stirred under the hydrogen atmosphere for 24 hours, filtered through Celite, washed with DCM (2 × 10 mL), and then distilled off, leaving alcohol C as a white solid. LCMS (ES, M / z): 1644.2 [M + H] + , Rt=4.67 minutes.

[0473] The deprotection intermediate (0.4 mmol) and the TFP ester of 4-cis-hydroxycyclohexanecarboxylic acid (350 mg, 1.20 mmol) were dissolved in DCM (2.5 mL), and TEA (3.5 eq, 0.195 mL) was added. The mixture was stirred at RT for 16 hours. Next, it was diluted with DCM (100 mL) and washed with saturated NaHCO3 / saturated brine (1:1, 100 mL x 2). The organic phase was dried over Na2SO4, concentrated, and purified by silica column. The product was eluted with MeOH (2-20%) in DCM to obtain 43% and 61% yields of 430 mg with >95% purity. Rt = 4.20 min. LCMS: (ES, M / z): 1771.26 [M+H] + .

[0474] 4) Preparation of compound 44 [ka]

[0475] 1.5 eq of 2-cyanoethyl N,N,N',N'-tetraisopropyl phosphorodiamidite (110 μL, 0.343 mmol) was added at 0°C to a stirred solution of alcohol 43 (405 mg, 0.229 mmol, vacuum-dried) and tetrazole (0.50 eq, 0.25 mL, 0.112 mmol, 0.45 M in ACN) in anhydrous DCM (2.4 mL). The mixture was stirred at RT for 1 hour, and then an additional 0.125 mL of tetrazole and 0.10 mL of 2-cyanoethyl N,N,N',N'-tetraisopropyl phosphorodiamidite were added. The mixture was stirred for 30 minutes, then diluted with DCM (200 mL), and washed with saturated NaHCO3 / saturated brine (1:1, 200 mL). The organic layer was dried over Na2SO4 / MgSO4, removed by distillation, then dissolved in anhydrous DCM, and removed again by distillation, leaving 44 and 408 mg of white solids. HPLC purity was 92% and yield was 83%. LCMS:(ES, M / z):1870.4[M-iPr2N] + . Figure 5 shows the relationship between compound 44 and 1 The 1H NMR spectrum is shown (structure 1007b in this specification).

[0476] Example 6: Synthesis of a targeted ligand, a phosphoramidite-containing compound, and structure 1027b 1) Preparation of compound 45 [ka]

[0477] TEA (5.3 mmol, 0.735 mL, 4.00 eq) was added to a stirred solution containing compound 27 (1.1 g, 1.32 mmol, 1.00 eq) and compound 45A (3.20 g, 5.29 mmol, 4.00 eq) in DCM (9 mL). The mixture was stirred at 30°C for 16 hours. LC-MS (ET12452-64-P1A, Rt=1.21 min) showed that a product had formed. The mixture was diluted with DCM (100 mL) and washed with saturated NaHCO3 / saturated brine (1:1, 2 × 80 mL). The organic layer was dried over Na2SO4, filtered, and concentrated to obtain the unpurified product as a brown solid.

[0478] The unpurified product was dissolved in Ac2O (3 mL), CH3CN (6 mL), and Py (6 mL), and the mixture was stirred at 25°C for 16 hours. CH3CN was removed by distillation, and the mixture was then diluted with DCM and washed four times with saturated NaHCO3. The organic layer was separated, washed with 0.1 M HCl / saturated brine, dried over Na2SO4, filtered, and concentrated. The residue was purified by silica column (DCM / MeOH = 10:1, Rf = 0.45) to obtain product 45 (1.47 g, 68% yield, 96% purity) as a white solid.

[0479] 4) Preparation of compound 46 [ka]

[0480] To a solution of compound 45 (1.425 g, 0.871 mmol, 1.00 eq) in THF / TFE (1:1, 5 mL), 10% Pd / C (24 mg) was added, and the mixture was stirred at 40°C for 30 hours under an H2 atmosphere. TLC (10:1, DCM / MeOH = Rf = 0.3) showed that the starting material had been consumed. The mixture was filtered, washed with THF (5 mL × 3) and DCM (5 mL × 3), and concentrated. The residue was purified by silica column chromatography. Elution with DCM / MeOH yielded compound 46 (1.013 g, 75% yield, 95% purity) as a white solid. LCMS: (ES, M / z): 1547.5 [M + H] + .

[0481] 5) Preparation of compound 47 [ka]

[0482] Compound 46 (970 mg, 0.627 mmol, 1.00 eq) was dissolved in DCM (4.2 mL), and compound 46A (0.941 mmol, 0.298 mL, 1.5 eq) was added. The resulting solution was cooled to 5°C, and dicyanoimidazole (DCI) (23.1 mg, 0.188 mmol, 0.3 eq) was added. The solution was heated to 15°C and stirred for 2 hours. TLC (5:1, DCM / MeOH = Rf = 0.52) showed that the starting material had been consumed, and HPLC showed that the product had been formed. It was diluted with DCM (50 mL), washed with saturated NaHCO3 (30 mL), H2O (30 mL), and brine (30 mL), dried over Na2SO4, filtered, and concentrated. The residue was dissolved in DCM (2 mL) and added to hexane (120 mL). The white precipitate was filtered to obtain compound 47 (0.975 g, 93% yield, 82% yield) as a white solid. LCMS:(ES, M / z):1747.5[M+H] + . Figure 6 shows the relationship between compound 47 and 1 The 1H NMR spectrum is shown (structure 1027b in this specification).

[0483] Example 7. Synthesis of a targeted ligand, a phosphoramidite-containing compound, and structure 1026b. 1) Preparation of triacid 49 [ka]

[0484] To a solution of 4-bromo-ahiru lysozyme phenylalanine hydrochloride (5.0 g, 17.8 mmol) in 1.5 M NaOH (100 mL), bromoacetic acid (8.17 g, 58.8 mmol) was added. The solution was heated to 60°C for 1 hour, and the pH was maintained above 12 by adding a sodium hydroxide pellet. Upon completion, the reaction mixture was cooled to 15°C, the pH was adjusted to 1.75–2.00, and the oily suspension was allowed to stand for 2 hours until a filterable solid was observed. The solid was filtered and washed several times with water to separate a white solid (6.0 g, 93% yield).

[0485] 2) Preparation of biaryl triacide 50 [ka]

[0486] Aryl bromide 49 (4.2 g, 11.6 mmol) and boronic acid 50 (2.8 g, 12.2 mmol) were dissolved in a 1:1 mixture of DMF / water (168 mL) and degassed for 10 minutes. The solution was treated with potassium carbonate (8.0 g, 116.2 mmol) and PdCl2 (dppf) (0.476 g, 0.6 mmol), and the reactor was heated at 40°C for 5 hours under a nitrogen atmosphere. At completion, the pH was adjusted to 12, and the aqueous phase was washed with 2 × (20 mL) ethyl acetate. The pH was then adjusted to 1.75–2.00, and the mixture was cooled to 15°C. The resulting solid was filtered and washed several times with water to remove all inorganic matter to obtain 51 (4.8 g, 89% yield).

[0487] 1) Preparation of Tri-TFP Ester 52 [ka]

[0488] A slurry of triacid 51 (5.0 g, 10.7 mmol) and tetrafluorophenol (6.5 g, 38.8 mmol) in dichloromethane (50 mL) was cooled to 0°C and treated with EDC hydrochloride (7.45 g, 38.8 mmol). The slurry was warmed to ambient temperature and stirred for 18 hours. Upon completion of the reaction, the reaction products were washed with water, and the organic layer was concentrated to the oil and purified by silica column chromatography to obtain TFP ester 52 (1.63 g, 16% yield).

[0489] 2) Preparation of Tri-NAG Protective Alcohol 54 [ka]

[0490] A solution of tri-TFP ester 52 (1.00 g, 1.10 mmol) and NAG amine sylate 53 (2.15 g, 3.33 mmol) in dichloromethane (5 mL) was cooled to 0°C and treated with triethylamine (0.66 g, 6.6 mmol). The solution was heated to ambient temperature for 2 hours. Upon completion, the reaction mixture was washed with water and concentrated to the oil. The unrefined oil was dissolved in acetic anhydride (30 mL), and the solution was treated with 1 mL of triethylamine. After 3 hours, the organic layer was removed under high vacuum to obtain oil 54 (1.7 g, 85% yield).

[0491] 3) Preparation of phenol 55 [ka]

[0492] Benzyl protected alcohol 54 (2.0 g, 1.08 mmol) was dissolved in ethanol (23 mL) and placed under a nitrogen atmosphere. 10% Pd / C (0.7 g, 30 mol%) was added to the solution. The slurry was stirred at ambient temperature for 8 hours, and the catalyst was removed using a Celite pad. The organic layer was removed under high vacuum to obtain a white solid 55 (1.4 g, 74% yield).

[0493] 4) Preparation of compound 56 [ka]

[0494] A solution of phenol 55 (1.3 g, 0.74 mmol) and phosphoramidite reagent (0.364 mg, 1.11 mmol) in dichloromethane (10 mL) was cooled to 0°C and treated with 4,5-dicyanoimidazole, then heated to ambient temperature for 2 hours. Upon completion, the reaction mixture was washed with saturated sodium bicarbonate (10 mL), followed by water (10 mL), and the organic layer was dried over sodium sulfate. The organic layer was concentrated under reduced pressure to obtain a white solid (1.4 g, 93% yield).

[0495] Compound 56 of Example 7 is structure 1026b as defined herein. Example 8. Physical properties of targeted ligand and phosphoramidite-containing compounds Certain GalNAc ligand phosphoramidite compounds that do not have the rigid linker structure disclosed herein showed a tendency to gel in many common solvents.

[0496] Figure 7 shows a photograph illustrating the behavior of a GalNAc structure having the same targeting moiety (N-acetyl-galactosamine), tether, and branching group as structure 1008b, but with a PEG linker instead of the rigid linker of structure 1008b disclosed herein. The PEG linker-GalNAc phosphoramidite compound was held on a molecular sieve in a 3:1 mixture of ACN:DMF at a 0.1 M dilution for 12 hours. The PEG linker-GalNAc exhibits significant gelation in this highly polar solvent system. To maintain solubility of this PEG linker-GalNAc phosphoramidite compound, the ACN:DMF mixture A mixture of up to 1:1 ratio is required.

[0497] Figure 8 shows a photograph of a phosphoramidite compound, structure 1008b, which is completely soluble in acetonitrile at 0.05 M without the need for highly polar solvents such as DMF. Unlike PEG-linker-GalNAc constructs, the phosphoramidite compound containing the rigid linker of structure 1008b does not pose the risk of or require highly polar solvents to maintain solubility. Despite being soluble in the bottle at lower concentrations, this illustrates that the rigid linker-containing structures disclosed herein are more soluble in common solvents typically used in oligonucleotide synthesis and do not require the addition of highly polar solvents to prevent gelation.

[0498] Example 9. Purity of the targeted ligand and phosphoramidite-containing compound As previously described in Example 2, Figure 2A shows the phosphoramidite compound of structure 1008b. 31 The 1P NMR spectrum is shown. Figure 2A shows a single peak representing the correct shift of the phosphoramidite. No other peaks, including hydrolysis peaks, are shown, indicating a highly pure compound.

[0499] Figure 9 shows the PEG linker-GalNAc structure. 31The 1P NMR spectrum is shown, and it contains, in other words, the same branching point, tether, and targeting region as structure 1008b. The chemistry structure of the phosphoramidite, from which the spectrum in Figure 9 was obtained, is shown in Figure 9. Figure 9 shows multiple impurity peaks, including one that appears to indicate the presence of hydrolyzed byproducts.

[0500] Example 10. Synthesis of oligonucleotide composition A.Synthesis RNAi agents were synthesized according to solid-phase phosphoramidite techniques used in oligonucleotide synthesis. Depending on the scale, MerMade96E (Bioautomation) or MerMadel2 (Bioautomation) was used. Synthesis was performed on solid supports consisting of controlled-pore glass (CPG, 500 Å or 600 Å, obtained from Prime Synthesis, Aston, PA, USA). All RNA, 2'-modified RNA, and UNA phosphoramidites were purchased from Thermo Fisher Scientific (Milwaukee, WI, USA). Specifically, the following 2'-O-methylphosphoramidites were used: (5'-O-dimethoxytrityl-N6-(vegizoyl)-2'-O-methyl-adenosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite, 5'-O-dimethoxytrityl-N4-(acetyl)-2'-O-methylcytidine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite, (5'-O-dimethoxytrityl-N2-(isobutyryl)-2'-O-methyl-guanosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite, and 5'-O-dimethoxytrityl-2'-O-methyl-uridine-3'-O-(2-cyanoethyl-N,N-diisopropyl Amino)phosphoramidites. Targeted ligand-containing phosphoramidites were dissolved in anhydrous dichloromethane or anhydrous acetonitrile (50 mM), while all other amidites were dissolved in anhydrous acetonitrile (50 mM), and molecular sieves (3 Å) were added. 5-benzylthio-1H-tetrazole (BTT, 250 mM in acetonitrile) or 5-ethylthio-1H-tetrazole (ETT, 250 mM in acetonitrile) were used as activator solutions. Coupling times were 10 min (RNA), 15 min (targeted ligand), 90 sec (2'OMe), and 60 sec (2'F). A 100 mM solution of 3-phenyl1,2,4-dithiazolin-5-one (POS, obtained from Poly Org, Inc., Leominster, MA, USA) in anhydrous acetonitrile was used to introduce phosphorothioate binding.

[0501] B. Cleavage and deprotection of oligomers bound to the support. After the completion of solid-phase synthesis, the dried solid support was treated with a 1:1 solution of 40 wt% methylamine and 28% ammonium hydroxide (Aldrich) in water at 30°C for 2 hours. The solution was evaporated, and the solid residue was restored with water (see below).

[0502] C. Purification The unpurified oligomer was purified by anion exchange HPLC using a TKSgel SuperQ-5PW1 3u column and a Shimadzu LC-8 system. Buffer A consisted of 20 mM Tris, 5 mM EDTA, pH 9.0, and 20% acetonitrile, while Buffer B was the same as Buffer A but with the addition of 1.5 M sodium chloride. UV traces were recorded at 260 nm. Appropriate fractions were collected and then run under size exclusion HPLC using a GE Healthcare XK16 / 40 column packed with Sephadex G-25 medium, along with an electrophoresis buffer consisting of 100 mM ammonium bicarbonate, pH 6.7, and 20% acetonitrile.

[0503] D. Annealing RNAi agents were prepared by mixing complementary strands of equimolar RNA solutions (sense and antisense) in 0.2× PBS (phosphate-buffered saline, 1×, Corning, Cellgro). This solution was placed in a 70°C thermomix, heated to 95°C, held at 95°C for 5 minutes, and slowly cooled to room temperature. Some of the RNAi agents were lyophilized and stored at -15 to -25°C. Double-stranded concentrations were determined by measuring the absorbance of the solution in 0.2× PBS using a UV-Vis spectrometer. The double-stranded concentration was then determined by multiplying the absorbance of the solution at 260 nm by the conversion factor and dilution ratio. Unless otherwise noted, all conversion factors were 0.037 mg / (mL·cm). In some experiments, the conversion factor was calculated from experimentally determined extinction coefficients.

[0504] Example 11. Comparison of 3' and 5' sense strand attachment sites of GalNAc-targeting ligands using F12 expression inhibitory oligomer compounds in wild-type mice. To evaluate the difference in GalNAc ligand attachment sites between the 3' and 5' ends of the sense strand, we prepared expression inhibitory oligomer compounds (double-stranded RNAi agents) directed towards F12 (also referred to herein as F12 RNAi agents) having the sequences shown in Table 1 below:

[0505] Table 1. F12 expression inhibitory oligomer compounds (RNAi double-stranded agents) of Example 11 [Table 1]

[0506] In Table 1 above, the following notation is used:

[0507] (NAG15) [ka]

[0508] (NAG18) [ka]

[0509] Each strand of the F12 RNAi agent was synthesized according to the solid-phase phosphoramidite technique used for oligonucleotide synthesis using either MerMade96E® (Bioautomation) or MerMade12® (Bioautomation), and the complementary strand was mixed with equimolar RNA solutions (sense and antisense) in 0.2× PBS (phosphorus-buffered saline, 1×, Corning, Cellgro) to form a double helix, generally according to the method described in Example 10 herein.

[0510] Each GalNAc ligand (i.e., (NAG15) or (NAG18)) was linked to an F12 RNAi agent, which was then combined with a pharmaceutically acceptable buffer known in the art for subcutaneous (SC) injection.

[0511] Each GalNAc ligand (i.e., (NAG15) or (NAG18)) was linked to an F12 RNAi agent, which was delivered by SC injection. On day 1, 200 μl of a 20g mouse SC injection containing either one of the two F12 RNAi agents (AD02803 or AD02807) at a dose of 3 mg / kg (mpk) in either physiological saline or buffered physiological saline was administered into the loose skin between the shoulders on the back. There were three (3) wild-type mice per treatment group. As previously shown, AD02803 contains (NAG15) attached to the 3' end of the sense strand, while AD2807 contains (NAG18) attached to the 5' end of the sense strand.

[0512] To observe knockdown, serum samples from treated mice were collected on days 8, 15, 22, and 29. Knockdown was measured by quantifying circulating mouse F12 protein (mF12) levels in the serum using internally expressed mF12 alphaLISA® (Perkin Elmer). Expression levels on specific blood collection days were normalized to the mean of the saline control group on the same date.

[0513] Figure 10 shows the results of this study. At its lowest point (day 22), AD02803 showed a reduction of approximately 70% in circulating F12 levels, compared to AD02807, which showed a reduction of over 80%. On day 29, AD02803-treated mice showed a faster return to baseline compared to AD2807-treated mice, so the data also indicates a difference in the length of the knockdown effect. These data support the conclusion that ligation of GalNAc ligand at the 5' end of the sense strand is superior to ligation at the 3' sense strand.

[0514] Example 12. Further comparison of 3' and 5' sense strand attachment sites of GalNAc-targeting ligands using F12 expression inhibitory oligomer compounds in wild-type mice. To further evaluate the GalNAc ligand attachment sites at the 3' and 5' ends of the sense strand of double-stranded RNAi oligomeric compounds (double-stranded RNAi agents), compositions directed to F12 were prepared, having the sequences shown in Table 3 below:

[0515] Table 2. F12 expression inhibitory oligomer compounds (RNAi double-stranded agents) of Example 12 [Table 2]

[0516] In Table 2 above, the following notation is used:

[0517] (NAG20) [ka]

[0518] Each strand of the F12 RNAi agent was synthesized according to the solid-phase phosphoramidite technique used for oligonucleotide synthesis using either MerMade96E® (Bioautomation) or MerMade12® (Bioautomation), and the complementary strand was mixed with equimolar RNA solutions (sense and antisense) in 0.2× PBS (phosphorus-buffered saline, 1×, Corning, Cellgro) to form a double helix, generally according to the method described in Example 10 herein.

[0519] Each GalNAc ligand (i.e., (NAG20))-linked F12 RNAi agent was combined with a pharmaceutically acceptable buffer known in the art for subcutaneous (SC) injection.

[0520] Each GalNAc ligand (i.e., (NAG20))-linked F12 RNAi agents were delivered by SC injection. On day 1, 200 μl of a 20g mouse SC injection containing either one of the two F12 RNAi agents (AD02815 or AD02816) at a dose of 3 mg / kg (mpk) in either physiological saline or buffered physiological saline was administered into the loose skin between the shoulders on the back. There were three (3) wild-type mice per treatment group. As previously shown in Table 2, AD2815 contains (NAG20) attached to the 5' end of the sense strand, while AD02816 contains (NAG20) attached to the 3' end of the sense strand.

[0521] To observe knockdown, serum samples from treated mice were collected on days 8, 15, 22, and 29. Knockdown was measured by quantifying circulating mouse F12 protein (mF12) levels in the serum using internally expressed mF12 alphaLISA® (Perkin Elmer). Expression levels on specific blood collection days were normalized to the mean of the saline control group on the same date.

[0522] Figure 11 shows the results of this experiment. At its lowest point (day 22), AD02816 showed a reduction of approximately 60% in circulating F12 protein levels, compared to a 79% reduction for AD02815. On day 29, AD02815-treated mice showed a 71% knockdown from saline levels, compared to a 40% knockdown for AD02816-treated mice. These data support the linkage of GalNAc ligand at the 5' end of the sense strand.

[0523] Example 13. Lp(a) expression inhibitory oligomer compound (double-stranded RNAi agent) linked to the targeted ligand of structure 1003 in Lp(a) transgenic (Tg) mice. Lp(a) expression inhibitory oligomer compounds (double-stranded Lp(a)RNAi agents) having the sequences shown in Table 3 below were prepared:

[0524] Table 3. LP(a) expression inhibitory oligomer compounds (double-stranded RNA inhibitors) of Example 13 [Table 3]

[0525] In Table 3 above, the following notation is used:

[0526] (NAG25) [ka]

[0527] (NAG29) [ka]

[0528] (NAG29) has the chemical structure represented by structure 1003 in this specification. Each strand of the Lp(a) RNAi agent was synthesized according to the solid-phase phosphoramidite technique used for oligonucleotide synthesis using either MerMade96E® (Bioautomation) or MerMade12® (Bioautomation), and the complementary strand was mixed with equimolar RNA solutions (sense and antisense) in 0.2× PBS (phosphorus-buffered saline, 1×, Corning, Cellgro) to form a double helix, generally according to the method described in Example 10 herein.

[0529] Lp(a) transgenic (Tg) mice (Frazer KA et al 1995, Nature Genetics 9:424-431) were used to evaluate the efficacy of double-stranded RNAi agents with a complexed N-acetyl-galactosamine ligand in vivo. These mice express human apo(a) and human apoB-100 from a YAC containing the complete LPA gene (encoding the apo(a) protein) with additional 5' and 3' sequences, thereby producing humanized Lp(a) particles (hereinafter also referred to as "Lp(a)Tg mice") (Callow MJ et al 1994, PNAS 91:2130-2134).

[0530] Lp(a)RNAi agents linked to each GalNAc ligand (i.e., (NAG25) or (NAG29)) were combined with pharmaceutically acceptable buffers known in the art for subcutaneous (SC) injection.

[0531] Lp(a)RNAi agents, ligated to their respective GalNAc ligands (i.e., (NAG25) or (NAG29)) at the 5' end of the sense strand, were delivered by SC injection. On day 1, 200 μl of a 20g mouse SC injection containing either 1 mg / kg (mpk) of one of the Lp(a)RNAi agents (AD03547 or AD03549) in either physiological saline or buffered physiological saline was administered into the loose skin between the shoulders on the back. There were four (4) Lp(a)Tg mice per treatment group.

[0532] Serum samples were collected from treated mice on days -1 (pre-administration), 5, 11, 16, 22, 29, and 36. Knockdown was measured by calculating the circulating Lp(a) particle level in the serum. Lp(a) particle levels were measured using a Cobas® Integra400 (Roche Diagnostics) according to the manufacturer's recommendations. For normalization, the Lp(a) level for each animal at a given time point was divided by the pre-administration expression level for that animal (in this case, on day -1) to determine the "normalized to day -1" expression rate. Next, the expression at a particular time point was normalized to the saline control group by dividing the "normalized to day -1" ratio for each individual animal by the mean "normalized to day -1" ratio for all mice in the saline control group. This resulted in expression at each time point normalized to the expression in the control group. Experimental error is shown as the standard deviation.

[0533] The results are shown in Figure 12. AD03549 (NAG25) showed a minimum knockdown of 71% (day 16), and AD03547 (NAG29) showed a minimum knockdown of 81% (day 11). Both triggers showed similar recovery curves, with less than 26% knockdown at day 36 after the minimum point. These data support the idea that the GalNAc ligands shown in Example 13 have commonalities in both initial knockdown activity and duration of knockdown in Lp(a)Tg mice using a single dose of 1 mg / kg.

[0534] Example 14. Apo(a) knockdown in apo(a) transgenic (Tg) mice after administration of Lp(a) expression inhibitory oligomer compounds (double-stranded RNAi agents) linked to targeted ligands of structures 1002 and 1004. Lp(a) expression inhibitory oligomer compounds (double-stranded Lp(a)RNAi agents) having the sequences shown in Table 4 below were prepared:

[0535] Table 4. LP(a) expression inhibitory oligomer compounds (double-stranded RNAi agents) of Example 14 [Table 4]

[0536] In Table 4 above, the following notation is used:

[0537] (NAG28) [ka]

[0538] (NAG30) [ka]

[0539] Furthermore, (NAG25) has the same structure as shown in Example 13. (NAG28) has the chemical structure represented in structure 1002 as described herein. (NAG30) has the chemical structure represented in structure 1004 as described herein. (NAG28) includes a mixture of cis- and trans-isomers, while (NAG30) is exclusive to the trans-isomer.

[0540] Each strand of the Lp(a) RNAi agent was synthesized according to the solid-phase phosphoramidite technique used for oligonucleotide synthesis using either MerMade96E® (Bioautomation) or MerMade12® (Bioautomation), and the complementary strand was mixed with equimolar RNA solutions (sense and antisense) in 0.2× PBS (phosphorus-buffered saline, 1×, Corning, Cellgro) to form a double helix, as generally described in Example 14 herein.

[0541] Apo(a) transgenic (Tg) mice were used to evaluate the efficacy of double-stranded RNAi agents with a complexed N-acetyl-galactosamine ligand in vivo. Apo(a)Tg mice (Frazer KA et al 1995, Nature Genetics 9:424-431) produce human apo(a) (hereinafter also referred to as "apo(a)Tg mice") from YAC containing the complete LPA gene (encoding the apo(a) protein) with both 5' and 3' additional sequences.

[0542] Each GalNAc ligand (i.e., (NAG25), (NAG28), or (NAG30)) was linked to an Lp(a)RNAi agent, which was then combined with a pharmaceutically acceptable buffer known in the art for subcutaneous (SC) injection. Lp(a)RNAi agents, ligated to their respective GalNAc ligands (i.e., (NAG25), (NAG28), or (NAG30)) at the 5' end of the sense strand, were delivered by SC injection. On day 1, SC injection consisted of 200 μl solution / 20g mice containing either 0.5 mg / kg (mpk) of the RNAi agent (AD03536, AD03538, or AD03540) in either physiological saline or buffered physiological saline, administered into the loose skin between the shoulders on the back. There were three (3) apo(a)Tg mice per treatment group.

[0543] Serum samples from treated mice were collected on days -1 (pre-administration), 8, 15, 22, and 29. Knockdown was measured by assaying the serum from the mice using apo(a) ELISA (Abcam). Lp(a) particle levels were measured using a Cobas® Integra400 (Roche Diagnostics) according to the manufacturer's recommendations. For normalization, the Lp(a) level for each animal at a given time point was divided by the pre-administration expression level of that animal (in this case, at day -1) to determine the "normalized to day -1" expression rate. Next, the expression at a particular time point was normalized to the saline control group by dividing the "normalized to day -1" ratio for each individual animal by the mean "normalized to day -1" ratio for all mice in the saline control group. This resulted in expression at each time point normalized to the expression in the control group. Experimental error is shown as the standard error of the mean.

[0544] The results are shown in Figure 13. The lowest values ​​were observed at day 15 for all RNAi tested. At the lowest values, AD03536 showed a 74% knockdown of the apo(a) protein, AD03538 showed a 74% knockdown of the apo(a) protein, and AD03540 showed a 71% knockdown of the apo(a) protein. At day 29, all RNAi agents showed a knockdown of >48% of the apo(a) protein level, with the exception of AD03536 (containing NAG25), which showed only a 16% knockdown. These data support the idea that the NAG structure functions similarly to RNAi agents containing linker structures NAG28 and NAG30, which show numerically superior knockdown at day 29 in terms of initial knockdown activity.

[0545] Example 15. Lp(a) knockdown in Lp(a) transgenic (Tg) mice after administration of Lp(a) expression inhibitory oligomer compounds (double-stranded RNAi agents) linked to targeted ligands of structures 1005 and 1008. Lp(a) expression inhibitory oligomer compounds (double-stranded Lp(a)RNAi agents) having the sequences shown in Table 5 below were prepared:

[0546] Table 5. LP(a) expression inhibitory oligomer compounds (double-stranded RNA inhibitors) of Example 15 [Table 5]

[0547] In Table 5 above, the following notation is used:

[0548] (NAG31) [ka]

[0549] (NAG37) [ka]

[0550] Furthermore, (NAG25) has the same structure as the one shown in Example 13. (NAG31) has the chemical structure shown in structure 1005 of this specification. (NAG37) has the chemical structure shown in structure 1008 of this specification. Each strand of the Lp(a) RNAi agent was synthesized according to the solid-phase phosphoramidite technique used for oligonucleotide synthesis using either MerMade96E® (Bioautomation) or MerMade12® (Bioautomation), and the complementary strand was mixed with equimolar RNA solutions (sense and antisense) in 0.2× PBS (phosphorus-buffered saline, 1×, Corning, Cellgro) to form a double helix, generally according to the method described in Example 10 herein.

[0551] Lp(a)Tg mice were used to evaluate the efficacy of double-stranded RNAi agents with a complexed N-acetyl-galactosamine ligand in vivo. Each GalNAc ligand (i.e., (NAG25), (NAG31), or (NAG37)) was linked to an Lp(a)RNAi agent, which was then combined with a pharmaceutically acceptable buffer known in the art for subcutaneous (SC) injection. Lp(a)RNAi agents linked to each GalNAc ligand (i.e., (NAG25), (NAG31), or (NAG37)) were delivered by SC injection. On day 1, SC injections were administered to 200 μl of 20g mice in either physiological saline or buffered physiological saline at a dose of 3 mg / kg (mpk) of the RNAi agent (AD03536, AD03629, or AD04170) into the loose skin between the shoulders on the back. There were four (4) Lp(a)Tg mice in each treatment group.

[0552] Serum samples were collected from treated mice on days -1 (pre-administration), 8, 15, 22, 29, and 36. Knockdown was measured by calculating the circulating Lp(a) particle level in the serum. Lp(a) particle levels were measured using a Cobas® Integra400 (Roche Diagnostics) according to the manufacturer's recommendations. For normalization, the Lp(a) level for each animal at a given time point was divided by the pre-administration expression level for that animal (in this case, on day -1) to determine the "normalized to day -1" expression rate. Next, the expression at a particular time point was normalized to the saline control group by dividing the "normalized to day -1" ratio for each individual animal by the mean "normalized to day -1" ratio for all mice in the saline control group. This resulted in expression at each time point normalized to the expression in the control group. Experimental error is shown as the standard deviation.

[0553] The obtained data are shown in Figure 14. AD03536 showed a 95% knockdown of Lp(a) levels at its lowest point (day 15) and maintained a 76% knockdown at day 36. AD03629 showed a 97% knockdown of Lp(a) levels at its lowest point (day 8) and maintained a 90% knockdown at day 36. AD04170 showed a 97% knockdown of Lp(a) levels at its lowest point (day 8) and maintained a 78% knockdown at day 36.

[0554] Example 16: F12 knockdown in wild-type mice after administration of F12 expression inhibitory oligomer compounds (double-stranded RNAi agents) linked to target ligands of structures 1005, 1008, 1025, and 1027 and structure 1003. F12 expression inhibitory oligomer compounds (double-stranded F12 RNAi agents) were prepared by ligating GalNAc-targeting ligands (NAG25)[AD04162]; (NAG37)[AD04623]; (NAG31)[AD04512]; (NAG33)[AD04650] or (NAG38)[AD04651] at their 5' ends via phosphorothioate linkage. Each double-stranded RNAi agent targeted F12. The following notation is used for GalNAc-targeting ligand structures:

[0555] (NAG25) [ka]

[0556] (NAG31) [ka]

[0557] (NAG33) = [ka]

[0558] (NAG37) [ka]

[0559] (NAG38) [ka]

[0560] (NAG31) has the chemical structure represented by structure 1005 in this specification. (NAG33) has the chemical structure represented by structure 1025 in this specification. (NAG37) has the chemical structure represented by structure 1008 in this specification. (NAG38) has the chemical structure represented by structure 1027 in this specification. As previously shown, the sequences and modification patterns were identical for AD04162, AD04623, AD04512, AD04650, and AD04651, with the only difference being the composition present in the GalNAc-targeting ligand structure located at the 5' end of the sense strand of each F12 RNAi agent.

[0561] Each strand of the F12 RNAi agent was synthesized according to the solid-phase phosphoramidite technique used for oligonucleotide synthesis using either MerMade96E® (Bioautomation) or MerMade12® (Bioautomation), and the complementary strand was mixed with equimolar RNA solutions (sense and antisense) in 0.2× PBS (phosphorus-buffered saline, 1×, Corning, Cellgro) to form a double helix, as generally described in Example 16 herein.

[0562] Each GalNAc-targeting ligand (i.e., (NAG25), (NAG31), (NAG33), (NAG37), or (NAG38)) conjugated to an F12 RNAi agent was combined with a pharmaceutically acceptable buffer known in the art for subcutaneous (SC) injection.

[0563] Each GalNAc ligand (i.e., (NAG25), (NAG31), (NAG33), (NAG37), or (NAG38)) was ligated to an F12 RNAi agent via SC injection. On day 1, 200 μl of solution / 20 g mouse SC injection containing either 1 mg / kg (mpk) of one of five double-stranded agents (AD04162, AD04623, AD04512, AD04650, and AD04651) in either physiological saline or buffered physiological saline was administered into the loose skin between the shoulders on the back. There were four (4) wild-type mice per treatment group. As previously shown, AD04162 contains structure (NAG25), AD04623 contains structure (NAG37), AD04512 contains structure (NAG31), AD04650 contains structure (NAG33), and AD04651 contains structure (NAG38). All GalNAc-targeting ligands are attached to the 5' end of the sense strand of their respective individual RNAi agents.

[0564] To observe knockdown, serum samples from treated mice were collected on days -1 (pre-administration), 8, 15, and 22. Knockdown was measured by quantifying circulating mouse F12 protein (mF12) levels in the serum using internally expressed mF12 alphaLISA® (Perkin Elmer). The mF12 level of each animal at each time point was divided by the pre-treatment expression level in that animal to determine the "normalized to pre-administration" expression rate. Next, the "normalized to pre-administration" ratio for each individual animal was divided by the average "normalized to pre-administration" ratio for all mice in the saline control group to normalize the expression at a particular time point relative to the saline control group. This resulted in expression at each time point that was normalized relative to the control group expression. Experimental error is shown as the standard deviation.

[0565] The results from this study are shown in Figure 15. The lowest levels were observed on day 8 for all RNAi agents tested. At their lowest levels, AD04162 showed a 90% knockdown of mF12, AD04623 showed a 94% knockdown, AD04512 showed a 94% knockdown, AD04650 showed a 92% knockdown, and AD04651 showed an 87% knockdown. On day 22, all RNAi agents showed a knockdown of >82% mF12 levels, with the exception of AD04162 (containing NAG25), which showed only a 74% knockdown. These data support the idea that the NAG structure functions similarly to RNAi agents containing rigid linker structures or linker substitution moieties (i.e., NAG31, NAG33, NAG37, and NAG38) disclosed herein, exhibiting numerically superior mF12 knockdown at day 22 in terms of initial knockdown activity.

[0566] Example 17. Lp(a) expression inhibitory oligomer compounds (double-stranded RNAi agents) linked to targeting ligands of structures 1004 and 1005 in Lp(a)Tg mice. Lp(a) expression inhibitory oligomer compounds (double-stranded Lp(a)RNAi agents) having the sequences shown in Table 6 below were prepared:

[0567] Table 6. LP(a) expression inhibitory oligomer compounds (double-stranded RNAi agents) of Example 17 [Table 6]

[0568] In Table 6, (NAG30) has the same chemical structure as shown in Example 14, and (NAG31) has the same chemical structure as shown in Example 15. NAG30 has the chemical structure represented by structure 1004 as specified herein. NAG31 has the chemical structure represented by structure 1005 as specified herein.

[0569] Each strand of the Lp(a) RNAi agent was synthesized according to the solid-phase phosphoramidite technique used for oligonucleotide synthesis using either MerMade96E® (Bioautomation) or MerMade12® (Bioautomation), and the complementary strand was mixed with equimolar RNA solutions (sense and antisense) in 0.2× PBS (phosphorus-buffered saline, 1×, Corning, Cellgro) to form a double helix, generally according to the method described in Example 10 herein.

[0570] Lp(a)Tg mice were used to evaluate the efficacy of double-stranded RNAi agents with a complexed N-acetyl-galactosamine ligand in vivo.

[0571] Each GalNAc ligand (i.e., NAG30 or NAG31) was linked to an Lp(a)RNAi agent, which was then combined with a pharmaceutically acceptable buffer known in the art for subcutaneous (SC) injection.

[0572] Lp(a)RNAi agents, ligated to their respective GalNAc ligands (i.e., NAG30 or NAG31) at the 5' end of the sense strand, were delivered by SC injection. On day 1, 200 μl of a 20g mouse SC injection containing either 1 mg / kg (mpk) of one of the Lp(a)RNAi agents (AD03629 or AD03540) in either physiological saline or buffered physiological saline was administered into the loose skin between the shoulders on the back. There were four (4) Lp(a)Tg mice per treatment group.

[0573] Serum samples were collected from treated mice on days -1 (pre-administration), 8, 15, 22, 29, 36, and 43. Knockdown was measured by calculating the circulating Lp(a) particle level in the serum. Lp(a) particle levels were measured using a Cobas® Integra400 (Roche Diagnostics) according to the manufacturer's recommendations. For normalization, the Lp(a) level for each animal at a given time point was divided by the pre-administration expression level for that animal (in this case, on day -1) to determine the "normalized to day -1" expression rate. Next, the expression at a particular time point was normalized to the saline control group by dividing the "normalized to day -1" ratio for each individual animal by the mean "normalized to day -1" ratio for all mice in the saline control group. This resulted in expression at each time point normalized to the expression in the control group. Experimental error is shown as the standard deviation.

[0574] The results are shown in Figure 16. The lowest values ​​were observed on day 15 for both RNAi agents tested. AD03629 showed an 89% knockdown of Lp(a) levels at its lowest point, while AD03540 showed an 85% knockdown of Lp(a) levels at its lowest point. Both RNAi agents showed similar recovery curves by day 36. However, by day 43, AD03540 showed a 16% knockdown of Lp(a) levels, compared to a 55% knockdown of Lp(a) levels for AD03629.

[0575] Example 18. Apo(a) knockdown in apo(a)Tg mice after administration of Lp(a) expression inhibitory oligomer compounds linked to targeted ligands of structures 1007, 1025, and 1026. Lp(a) expression inhibitory oligomer compounds (double-stranded RNAi agents) having the sequences shown in Table 7 below were prepared:

[0576] Table 7. LP(a) expression inhibitory oligomer compounds (double-stranded RNAi agents) of Example 18 [Table 7]

[0577] In Table 7 above, the following notation is used:

[0578] (NAG33) = [ka]

[0579] (NAG34) [ka]

[0580] (NAG35) [ka]

[0581] (NAG33) has the chemical structure represented by structure 1025 in this specification. (NAG34) has the chemical structure represented by structure 1026 in this specification. (NAG35) has the chemical structure represented by structure 1007 in this specification.

[0582] Each strand of the Lp(a) RNAi agent was synthesized according to the solid-phase phosphoramidite technique used for oligonucleotide synthesis using either MerMade96E® (Bioautomation) or MerMade12® (Bioautomation), and the complementary strand was mixed with equimolar RNA solutions (sense and antisense) in 0.2× PBS (phosphorus-buffered saline, 1×, Corning, Cellgro) to form a double helix, generally according to the method described in Example 18 herein.

[0583] Apo(a) transgenic (Tg) mice were used to evaluate the efficacy of double-stranded RNAi agents with a complexed N-acetyl-galactosamine ligand in vivo.

[0584] Each GalNAc ligand (i.e., NAG33, NAG34, or NAG35) was linked to an Lp(a)RNAi agent, which was then combined with a pharmaceutically acceptable buffer known in the art for subcutaneous (SC) injection. Lp(a)RNAi agents linked to each GalNAc-targeting ligand (i.e., NAG33, NAG34, or NAG35) were administered by SC injection. On day 1, SC injection consisted of 200 μl solution / 20g mouse containing either 1 mg / kg (mpk) of the RNAi agent (AD03721, AD03722, or AD03723) in either physiological saline or buffered physiological saline, administered into the loose skin between the shoulders on the back. There were three (3) apo(a)Tg mice per treatment group.

[0585] Serum samples were collected from treated mice on days -1 (pre-administration), 8, 15, 22, and 29. Knockdown was measured by assaying circulating apo(a) protein levels in serum. Human apo(a) protein levels in serum were observed by assaying serum from mice using apo(a) ELISA (Abcam). Lp(a) particle levels were measured using Cobas® Integra400 (Roche Diagnostics) according to the manufacturer's recommendations. For normalization, the Lp(a) level for each animal at a given time point was divided by the pre-administration expression level of that animal (in this case, on day -1) to determine the "normalized to day -1" expression rate. Then, the expression at a specific time point was normalized to the saline control group by dividing the "normalized to day -1" ratio for each individual animal by the mean "normalized to day -1" ratio for all mice in the saline control group. Experimental error is shown as the standard error of the mean.

[0586] The obtained data are shown in Figure 17. The lowest point was at day 15 for all RNAi agents tested. AD03721 showed a 91% knockdown of apo(a) protein levels at its lowest point, AD03722 showed an 81% knockdown of apo(a) protein levels at its lowest point, while AD03723 showed a 90% knockdown of apo(a) protein levels at its lowest point. Both AD03721 and AD03722-treated mice showed nearly identical knockdown at each time point, while AD03723-treated mice showed numerically lower knockdown at each time point tested, and the recovery of apo(a) protein levels after treatment followed a similar trajectory. For example, on day 29, AD03721-treated mice showed a 76% knockdown of apo(a) levels, AD03723-treated mice showed an 83% knockdown of apo(a) levels, while AD03722-treated mice showed a 61% knockdown of apo(a) levels. These data support the fact that RNAi agents containing structures NAG33 and NAG35 showed numerically superior knockdown on day 29, indicating that all NAG33, NAG34, and NAG35 structures exhibit knockdown activity.

[0587] Example 19. Dose response of LP(a) expression inhibitory oligomer compound (double-stranded RNAi agent) linked to the targeted ligand of structure 1008 in Lp(a)Tg mice, administered at 1 mg / kg and 3 mg / kg. The Lp(a) transgenic mice described herein were used to evaluate the efficacy of double-stranded RNAi agents conjugated with N-acetyl-galactosamine ligands in vivo. An RNAi agent for Lp(a) having double-stranded ID:AD04170, as previously described in Example 15, was prepared. As previously described, Lp(a) double-stranded ID:AD04170 contains a (NAG37) targeted ligand (structure 1008) attached to the 5' end of the sense strand.

[0588] Each strand of the Lp(a) RNAi agent was synthesized according to the solid-phase phosphoramidite technique used for oligonucleotide synthesis using either MerMade96E® (Bioautomation) or MerMade12® (Bioautomation), and the complementary strand was mixed with equimolar RNA solutions (sense and antisense) in 0.2× PBS (phosphorus-buffered saline, 1×, Corning, Cellgro) to form a double helix, generally according to the method described in Example 10 herein.

[0589] An Lp(a)RNAi agent linked to the targeted ligand structure 1008 was combined with a pharmaceutically acceptable buffer known in the art for subcutaneous (SC) injection.

[0590] An Lp(a)RNAi agent linked to the targeted ligand structure 1008 was administered by subcutaneous (SC) injection. On day 1, 200 μl of a solution containing either 1 mg / kg (mpk) of the RNAi agent in physiological saline or buffered physiological saline, or 3 mg / kg (mpk) of the RNAi agent in buffered physiological saline, was administered via SC injection into the loose skin between the shoulders on the back of 20 g mice.

[0591] Control serum (pre-treatment) samples were collected from mice before injection on day 1. Lp(a) particle levels were determined using Cobas® Integra400 (Roche Diagnostics) according to the manufacturer's recommendations. For normalization, the Lp(a) level for each animal at a given time point was divided by the pre-treatment expression level of that animal (in this case, on day -1) to determine the "normalized to day -1" expression rate. Next, the expression at a specific time point was normalized to the saline control group by dividing the "normalized to day -1" ratio for each individual animal by the mean "normalized to day -1" ratio for all mice in the saline control group. This resulted in expression at each time point normalized to the expression in the control group. Experimental error is shown as the standard deviation.

[0592] The results are shown in Figure 18. As shown in Figure 18, dose-dependent correlations are evident for Lp(a)RNAi agents across all time points.

[0593] Example 20: LP(a) expression inhibitory oligomer compounds (double-stranded RNAi agents) linked to targeting ligands of structures 1003 and 1004 in cynomolgus monkeys. Lp(a) expression inhibitory oligomer compounds (double-stranded RNAi agents) having the sequences shown in Table 8 below were prepared:

[0594] Table 8. Lp(a) expression inhibitory oligomer compounds (RNAi double-stranded agents) of Example 20 [Table 8]

[0595] The Lp(a)RNAi agent, AD03547, is the same as that shown in Example 13 and is bound to (NAG29). The Lp(a)RNAi agent, AD3668, was bound to (NAG30). (NAG30) has the chemical structure shown in Example 14. (NAG29) is represented by structure 1003 as described herein. (NAG30) is represented by structure 1004 as described herein.

[0596] Each strand of the Lp(a) RNAi agent was synthesized according to the solid-phase phosphoramidite technique used for oligonucleotide synthesis using either MerMade96E® (Bioautomation) or MerMade12® (Bioautomation), and the complementary strand was mixed with equimolar RNA solutions (sense and antisense) in 0.2× PBS (phosphorus-buffered saline, 1×, Corning, Cellgro) to form a double helix, generally according to the method described in Example 10 herein.

[0597] Lp(a)RNAi agents conjugated to the targeted ligands disclosed herein having structure 1003 or structure 1004 were prepared and combined with pharmaceutically acceptable buffers known in the art for subcutaneous (SC) injection.

[0598] Control serum (pre-treatment) samples were collected from cynomolgus monkeys before injection on days 14, 7, and 1 (pre-administration). Lp(a) particle levels were determined using Cobas® Integra400 (Roche Diagnostics) according to the manufacturer's recommendations. For normalization, the Lp(a) level for each animal at a given time point was divided by the mean of the pre-treatment expression level of that animal (in this case, on days 14, 7, and 1 (pre-administration)) to determine the "normalized to baseline" expression rate.

[0599] On day 1, cynomolgus macaques were subcutaneously injected with either 3 mg / kg of Lp(a)RNAi agent AD03668 or Lp(a)RNAi agent AD03547, using an Lp(a)RNAi agent linked to a targeted ligand disclosed herein. Two (2) monkeys were administered per treatment group.

[0600] The results are reported in Figure 19. Lp(a)RNAi triggers bound to either structure 1003 (AD03547) or structure 1004 (AD03668) showed knockdown in cynomolgus monkeys.

[0601] Example 21: F12 expression inhibitory oligomer compound (double-stranded RNAi agent) linked to a targeted ligand of structure 1008 in cynomolgus monkeys F12 RNAi agents were prepared, each having a different sequence to match F12 and linked to the GalNAc-targeting ligand structure 1008 [(NAG37)] at the 5' end of the sense strand, and then combined with pharmaceutically acceptable buffers known in the art for subcutaneous (SC) injection.

[0602] On day 1, cynomolgus macaques (crab-eating macaques) were subcutaneously injected at a dose of 3 mg / kg of one of six different Lp(a)RNAi agents having different sequence structures and modification patterns: AD04623, AD04624, AD04625, AD04626, AD04627, or AD04628. Two monkeys were administered per treatment group.

[0603] To observe knockdown, serum samples were collected from treated cynomolgus monkeys on day -7 and day 1 (pre-administration), as well as on days 8, 15, and 22. Knockdown was measured by quantifying circulating cyno F12 protein (cF12) levels in serum using a human F12 ELISA kit (Molecular Innovations). The cF12 level of each animal at each time point was divided by the pre-treatment expression level in that animal (average of day -7 and day 1) to determine the "normalized to pre-administration" expression rate. Experimental error is shown as the standard deviation.

[0604] The results are shown in Figure 20. AD04625 and AD04623 showed the greatest knockdown across all time points measured, and each F12 RNAi agent linked to NAG37 (structure 1008) showed knockdown in cynomolgus monkeys.

[0605] Example 22: α-1 antitrypsin expression inhibitory oligomer compound (double-stranded RNAi agent) linked to the targeting ligand of structure 1008 in PiZ transgenic mice. To evaluate RNAi agents directed at the α-1 antitrypsin (AAT) gene in vivo, we used a transgenic PiZ mouse model (PiZ mouse). The PiZ mouse carries the human PiZ AAT mutant allele and the model human AATD (Carlson et al., Journal of Clinical Investigation 1989).

[0606] AAT expression inhibitory oligomer compounds (double-stranded RNAi agents) having the sequences shown in Table 9 below were prepared:

[0607] Table 9. AAT expression inhibitory oligomer compounds (RNAi double-stranded agents) of Example 22 [Table 9]

[0608] (NAG37) has the chemical structure shown in Example 16 above. AAT RNAi agents were prepared in pharmaceutically acceptable physiological salt buffer, and administered to PiZ mice by subcutaneous (SC) injection of 200 μl of the solution into the loose skin between the shoulders on the back of the mouse to evaluate knockdown of AAT gene expression. Each mouse received a single SC dose of 3 mg / kg (mpk) of AD04463. Three mice were administered the AAT RNAi agent (n=3).

[0609] Plasma samples were plotted and analyzed for AAT (Z-AAT) protein levels on day 1, day 1 (pre-administration), day 8, and day 15. AAT levels were normalized to the AAT plasma level on day 1 (pre-administration). Protein levels were measured by quantifying circulating human Z-AAT levels in plasma using an ELISA kit.

[0610] The average normalized AAT (Z-AAT) levels are shown in Figure 21. The AAT RNAi agent linked to the targeting ligand of structure 1008 herein showed knockdown in PiZ transgenic mice.

[0611] Other Embodiments Although the present invention has been described in conjunction with its detailed description, it should be understood that the foregoing description is illustrative and does not limit the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A targeted ligand comprising a linker, a branching group, one or more tethers, and one or more targeting moieties, having the structure of formula (I), or a pharmaceutically acceptable salt thereof: 【Chemistry 1】 {In the formula, n is an integer from 1 to 4, and in the formula, (a) The linker is a structure selected from the group consisting of: 【Chemistry 2】 (Structure 1); 【Transformation 3】 (Structure 6a); 【Chemistry 4】 (Structure 6b); 【Transformation 5】 (Structure 6c); and 【Transformation 6】 (Structure 6d); (b) The branching point base is a structure selected from the group consisting of: 【Transformation 7】 (Structure 201); 【Transformation 8】 (Structure 202); 【Chemistry 9】 (Structure 203); 【Chemistry 10】 (Structure 205); 【Chemistry 11】 (Structure 206); 【Chemistry 12】 (Structure 207); 【Chemistry 13】 (Structure 208); 【Chemistry 14】 (Structure 216); 【Chemistry 15】 (Structure 217); and 【Chemistry 16】 (Structure 218); (c) Each tether is independently selected from the following group: 【Chemistry 17】 (Structure 301) (wherein n in structure 301 is an integer between 1 and 20, and X in structure 301 is O, S, or NH); [Chemistry 18] (Structure 303) (wherein the formula, n in structure 303 is an integer between 1 and 20, and X in structure 303 is O, S, or NH); 【Chemistry 19】 (Structure 304) (In the formula, n in structure 304 is an integer between 1 and 20, and X in structure 304 is O, S, or NH); 【Chemistry 20】 (Structure 305) (In the formula, X in structure 305 is O, S, or NH); and 【Chemistry 21】 (Structure 306) (In the formula, X in structure 306 is O, S, or NH); and (d) Each targeting segment is independently selected from the following group: N-acetylgalactosamine, galactose, galactosamine, N-formyl-galactosamine, N-propionyl-galactosamine, N-n-butanoylgalactosamine, and N-iso-butanoylgalactosamine. However, a targeted ligand containing the structure of formula (I) is not a targeted ligand containing a structure selected from the group consisting of the following, or a pharmaceutically acceptable salt thereof: 【Chemistry 22】 (Structure 1001); 【Chemistry 23】 (Structure 1002); 【Chemistry 24】 (Structure 1003); 【Chemistry 25】 (Structure 1004); 【Chemistry 26】 (Structure 1005); 【Chemistry 27】 (Structure 1006); 【Chemistry 28】 (Structure 1007); 【Chemistry 29】 (Structure 1008); 【Transformation 30】 (Structure 1009); 【Chemistry 31】 (Structure 1012); 【Chemistry 32】 (Structure 1014); 【Transformation 33】 (Structure 1016); 【Transformation 34】 (Structure 1018); 【Chemistry 35】 (Structure 1019); 【Transformation 36】 (Structure 1021); and 【Chemistry 37】 (Structure 1023).

2. The linker is the targeted ligand or a pharmaceutically acceptable salt thereof according to claim 1, wherein the linker is as follows: 【Transformation 38】

3. The targeting ligand or a pharmaceutically acceptable salt thereof according to claim 1, wherein the branching group is as follows: 【Chemistry 39】 (Structure 205) or 【Chemistry 40】 (Structure 216).

4. The targeted ligand or a pharmaceutically acceptable salt thereof according to claim 1, wherein the targeted portion is N-acetylgalactosamine.

5. The targeted ligand according to claim 1, or a pharmaceutically acceptable salt thereof, wherein n in formula (I) is 3.

6. The targeted ligand according to claim 1, or a pharmaceutically acceptable salt thereof, wherein n in formula (I) is 4.

7. The targeted ligand according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the targeted ligand is linked to an RNAi agent.

8. The targeted ligand or a pharmaceutically acceptable salt thereof according to claim 7, wherein the RNAi agent is single-stranded or double-stranded.

9. The targeted ligand or a pharmaceutically acceptable salt thereof according to claim 8, wherein the RNAi agent is double-stranded.

10. A pharmaceutical composition for inhibiting the expression of a target nucleic acid in a subject, comprising an RNAi agent linked to any of the targeting ligands described in claim 1.

11. A pharmaceutical composition for introducing an RNAi agent into mammalian cells, comprising a targeted ligand according to claim 1, which is linked to the RNAi agent.

12. The pharmaceutical composition according to claim 11, wherein the cells are present in the body of the subject.

13. The pharmaceutical composition according to claim 12, wherein the subject is a human.

14. The pharmaceutical composition according to claim 11, wherein the RNAi agent is a double-stranded RNAi agent.

15. A method for producing a phosphoramidite compound comprising a targeted ligand or a pharmaceutically acceptable salt thereof as described in claim 1, the method being: (i) The carboxylic acid portion (or activated ester) of the linker is covalently linked to the terminal amine located on the branching point group, and then, (ii) A method comprising linking a linker to a phosphorus atom of a phosphoramidite by a phosphytylation reaction using a phosphoramidite-forming reagent; thereby forming a phosphoramidite compound containing a targeted ligand.

16. The method according to claim 15, wherein the phosphoramidite-forming reagent is selected from the following: 【Chemistry 41】 【Chemistry 42】

17. A targeted ligand according to claim 1, linked to an RNAi agent for use as a pharmaceutical agent.

18. The targeted ligand according to claim 17, wherein the RNAi agent is a double-stranded RNAi agent.

Images