EA043375B1 - TARGETTING LIGANDS - Google Patents

Description

Cross reference to related applications

This patent application claims the benefit of U.S. Provisional Patent Application Serial No. 62/383,221, filed September 2, 2016, and U.S. Provisional Patent Application No. 62/456,339, filed February 8, 2017, the contents of each of which are incorporated in their entirety in this document by reference.

State of the art

To obtain a therapeutic effect or be useful for diagnostic purposes, many compounds must be delivered to specific sites (eg, target cell(s)). This is often necessary when attempting to deliver a therapeutic compound in vivo. In addition, the ability to efficiently deliver a compound to a specific site may limit or largely eliminate off-target effects (such as side effects) that may be caused by administration of the compound. One method of achieving delivery of a compound, such as a therapeutic compound, to a desired site in vivo is based on binding or coupling of the compound to a targeting ligand.

One class of therapeutic compounds that can be targeted using targeting ligands are oligomeric compounds. Oligomeric compounds that include nucleotide sequences at least partially complementary to the target nucleotide sequence have been shown to alter the function and activity of the target both in vitro and in vivo. When delivered into a cell containing a target nucleotide sequence (such as mRNA), the oligomeric compounds have been shown to modulate the expression of the target, resulting in changes in the transcription or translation of the target nucleic acid. In some cases, the oligomeric compound may reduce gene expression by inhibiting the target nucleic acid and/or triggering the destruction of the target nucleic acid.

If the target nucleic acid is mRNA, one mechanism by which an expression inhibitory oligomeric compound can modulate the expression of the target mRNA is through RNA interference. RNA interference is a biological process that allows RNA or RNA-like molecules (such as chemically modified RNA molecules) to silence gene expression (silence) by destruction. Post-translational gene silencing is thought to be an evolutionarily conserved cell defense mechanism to prevent the expression of foreign genes.

Synthetic RNA molecules and RNA-like molecules have been shown to cause RNA interference in vivo. For example, Elbashir et al. (Nature 2000, 411, 494-98) describe RNAi performed by introducing duplexes of synthetic 21-nucleotide RNA molecules into cultured mammalian cells. The types of synthetic RNA molecules and RNA-like molecules that can trigger an RNAi response may include modified nucleotides and/or one or more linkages other than a phosphodiester linkage.

Additionally, single-stranded RNA molecules and RNA-like molecules, which may also include modified nucleotides and/or one or more linkages other than a phosphodiester linkage, can also alter the expression of a target nucleic acid, such as target mRNA.

Short description

Disclosed herein are targeting ligands that can enhance the delivery of therapeutic compounds to a specific site, for example, a specific organ or tissue in a subject, such as a human or animal patient. In some embodiments, the targeting ligands described herein can improve the targeting of expression inhibitory oligomeric compounds. In some embodiments, targeting ligands can improve delivery of expression inhibitory oligomeric compounds to the liver.

In some embodiments, the targeting ligands disclosed herein include, consist of, or essentially consist of one or more targeting moieties, one or more connecting moieties, one or more branch point groups, and one or more linkers. Linkers suitable for use in the targeting ligands disclosed herein include a rigid linker that can impart sufficient stability and rigidity to the entire targeting ligand to reduce potential interactions between one or more targeting moieties and the therapeutic compound to which it or they are attached. Additionally, rigid linkers suitable for use in the targeting ligands disclosed herein are useful for the efficient synthesis of targeting ligands such as phosphoramidite compounds (also referred to herein as phosphoramidite-containing compounds).

In some embodiments, the targeting ligands disclosed herein include, consist of, or essentially consist of one or more targeting moieties, one or more connecting moieties, and one or more branch point groups with a linker replacement moiety. The linker replacement moiety includes, consists of, or essentially consists of one or more substituted or unsubstituted cycloalkyl, cycloalkenyl, aryl, heteroaryl or heterocyclyl groups or covalently linked combinations thereof located within a branch point group. The presence of a linker replacement moiety within the branch point group imparts properties similar to those of the rigid linkers disclosed herein, providing sufficient stability and rigidity to the targeting ligand as a whole. Additionally, branch point groups with a linker replacement moiety suitable for use in targeting ligands are useful for the efficient synthesis of targeting ligands in the form of phosphoramidite compounds.

Disclosed herein are targeting ligands containing, consisting of, or consisting essentially of the structure of Formula I

targeting_connector fragment fragment _ branch point group

comprising a linker, a branch point group, one or more connecting moieties and one or more targeting moieties, where n is an integer from 1 to 4 (for example, 1, 2, 3 or 4), and wherein the linker is a structure selected group consisting of:

(Structure 4):

where 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 -C ₆ alkenyl, C ₂ -C ₆ alkynyl, substituted or unsubstituted amino, carboxyl, C1-C6 alkoxy, substituted C1-C6 alkyl, C1-C6 aminoalkyl, substituted C ₂ -C ₆ alkenyl, substituted C ₂ -C ₆ alkynyl , replaced by C1-C6 alcohox, replaced by C1-C6 aminoalkil, halogen (for example, F), hydroxilia, amido, replaced by amid, cyano, replaced or unnoticed, replaced or unnoticed alcohoxicarbonil, replaced or unnoticed, 2,043375 ariloxicarbonil, noticed or noticed or noticed or noticed or noticed or noticed or noticed. unsubstituted heteroaryloxycarbonyl and sulfhydryl (structure 7)

o where n is 0, 1, 2, 3, 4 (for example, 1, 2, 3 or 4), and, if present, each Z is independently selected from the group consisting of: C1-C6 alkyl, _C2 -C6 alkenyl, C ₂ -C6 alkynyl, C1-C6 alkoxy, substituted C1-C ₆ alkyl, C1-C ₆ aminoalkyl, substituted C ₂ -C ₆ alkenyl, substituted C ₂ -C ₆ alkynyl, substituted or unsubstituted amino, carboxyl, substituted C1-C6 alkoxy, substituted C1- _C6 aminoalkyl, halogen (e.g. F), hydroxyl, amido, substituted amide, cyano, substituted or unsubstituted keto, substituted or unsubstituted alkoxycarbonyl, substituted or unsubstituted aryloxycarbonyl, substituted or unsubstituted heteroaryloxycarbonyl and sulfhydroxycarbonyl silt ( structure 8); And

where V contains one or more groups such as substituted or unsubstituted cycloalkyl (for example, cyclohexyl, cyclopropyl, cyclobutyl, cyclopentyl, cycloheptyl, cyclooctyl, etc.), substituted or unsubstituted cycloalkenyl (for example, cyclohexenyl, cyclobutenyl, cyclopentenyl, cycloheptenyl, cyclooctenyl , cyclohexadienyl, cyclopentadienyl, cycloheptadienyl, cyclooctadienyl, etc.), substituted or unsubstituted aryl (e.g. phenyl, naphthyl, binaphthyl, anthracenyl, etc.), substituted or unsubstituted heteroaryl (e.g. pyridyl, pyrimidinyl, pyrrole, imidazole , furan, benzofuran, indole, etc.) or substituted or unsubstituted heterocyclyl (eg, tetrahydrofuran, tetrahydropyran, piperidine, pyrrolidine, etc.) or any covalently linked combinations thereof (structure 9).

In some embodiments, the targeting ligands include a branch point moiety with a linker replacement moiety.

Disclosed herein are targeting ligands containing, consisting of, or essentially consisting of the structure of Formula II:

comprising a branch point group with a linker replacement moiety, one or more connecting moieties, and one or more targeting moieties, where n is an integer from 1 to 4 (for example, 1, 2, 3 or 4), and wherein the moiety, substituent linker includes one or more of substituted or unsubstituted cycloalkyl (e.g., cyclohexyl, cyclopropyl, cyclobutyl, cyclopentyl, cycloheptyl, cyclooctyl, etc.), substituted or unsubstituted cycloalkenyl (e.g., cyclohexenyl, cyclobutenyl, cyclopentenyl, cycloheptenyl, cyclooctenyl, cyclohexadienyl, cyclopentadienyl, cycloheptadienyl, cyclooctadienyl, etc.), substituted or unsubstituted aryl (e.g. phenyl, naphthyl, binaphthyl, anthracenyl, etc.), substituted or unsubstituted heteroaryl (e.g. pyridyl, pyrimidinyl, pyrrole, imidazole, furan, benzofuran, indole, etc.) or substituted or unsubstituted heterocyclyl (eg, tetrahydrofuran, tetrahydropyran, piperidine, pyrrolidine, etc.) or any covalently linked combination thereof, and a branch point group is located.

The targeting ligands disclosed herein can be coupled, directly or indirectly, to a compound, such as a therapeutic compound, eg, an expression inhibitory oligomeric compound, eg, to the 3' or 5' end of the expression inhibitory oligomeric compound. In some embodiments, the expression inhibitory oligomeric compound includes one or more modified nucleotides. In some embodiments, the expression inhibitory oligomeric compound is an RNAi agent, such as a double-stranded RNAi agent. In some embodiments, the targeting ligands disclosed herein are coupled to the 5' end of the sense strand of the double-stranded RNAi agent. In some variants

- 3 043375 implementations of the targeting ligands disclosed herein are linked to the RNAi agent by a phosphate, phosphorothioate or phosphonate group at the 5' end of the sense strand of the double-stranded agent

RNAi.

The targeting ligands disclosed herein include one or more targeting moieties. In some embodiments, the targeting ligands disclosed herein include N-acetylgalactosamine as a targeting moiety.

In some embodiments, the targeting ligands disclosed herein have the structures shown below:

Disclosed herein are compositions comprising, consisting of, or essentially consisting of a targeting ligand and an expression inhibitory oligomeric compound. Disclosed herein are compositions containing a targeting ligand and an RNAi agent.

In some embodiments, compositions disclosed herein containing a targeting ligand and an RNAi agent have the structure shown below:

where R includes or consists of an expression inhibitory oligomeric compound (structure 1002a);

He

where R includes or consists of an expression inhibitory oligomeric compound (structure 1003a);

where R includes or consists of an expression inhibitory oligomeric compound (structure 1005a);

- 5 043375 he

where R includes or consists of an expression inhibitory oligomeric compound (structure 1008a);

where R includes or consists of an expression inhibitory oligomeric compound (structure 1012a); or

where R includes or consists of an expression inhibitory oligomeric compound (structure 1027a).

Disclosed herein are phosphoamidite compounds including targeting ligands.

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In some embodiments, the phosphoramidite compounds comprising the targeting ligands disclosed herein have the structure shown below:

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- 9 043375

- 10 043375

- 11 043375

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(Structure

- 13 043375

- 14 043375

(Structure 1027b).

Also disclosed are pharmaceutical compositions that include the targeting ligands disclosed herein.

Disclosed are methods of treating a disease or disorder that would benefit from administration of

- 15 043375 compounds, comprising administering to a subject a compound coupled to a targeting ligand disclosed herein.

Disclosed herein are methods of inhibiting the expression of a target nucleic acid in a subject, comprising administering a therapeutically effective amount of an expression inhibitory oligomeric compound coupled to targeting ligands disclosed herein.

Disclosed herein are methods of delivering an expression inhibitory oligomeric compound to the liver in vivo, comprising administering the expression inhibitory oligomeric compound bound to a targeting ligand disclosed herein to a subject.

Disclosed herein are processes or methods for making a phosphoramidite compound comprising a targeting ligand, comprising (i) covalently linking a linker to a branch point group, and (ii) linking the linker to a phosphoramidite atom by performing a phosphitylation reaction with a phosphoramidite-forming reagent to form a phosphoramidite compound .

As used herein, the term bonded, when applied to a connection between two molecules, means that the two molecules are connected by a covalent bond or that the two molecules are connected by non-covalent bonds (eg, hydrogen bonds or ionic bonds). In some examples where the term bound refers to the association of two molecules by non-covalent bonds, this association (association) of two different molecules is characterized by a K _D below 1x10 ^-4 M (for example, below 1x10 ^-5 M, below 1x10' ⁶ M, or below 1x10' ⁷ M) in a physiologically acceptable buffer (for example, phosphate buffer solution).

As used herein, the term bound directly refers to a first compound or group associated with a second compound or group without any intervening atoms or groups of atoms. As used herein, the term indirectly linked refers to a first compound linked to a second compound or group via an intermediate group, compound or molecule, such as, for example, a linker group. Unless otherwise specified, the term related herein includes both directly related and indirectly related as those terms are defined herein.

As used herein, an oligomeric compound is a nucleotide sequence containing about 10-50 nucleotides or nucleotide base pairs. In some embodiments, the oligomeric compound has a sequence of nucleotide bases that is at least partially complementary to a coding sequence in an expressed target nucleic acid or gene in the cell. In some embodiments, the oligomeric compounds, once delivered to a cell expressing a gene, are capable of inhibiting the expression of the corresponding gene and are referred to herein as expression-inhibiting oligomeric compounds. Gene expression can be inhibited in vitro or in vivo. Oligomeric compounds include, but are not limited to: oligonucleotides, single-stranded oligonucleotides, single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA). ), ribozymes, molecules mediating RNA interference and dicer substrates.

As used herein, the term oligonucleotide refers to a polymer of linked nucleosides, each of which may be modified or unmodified.

As used herein, the term single-stranded oligonucleotide refers to a single-stranded oligomeric compound having a sequence that is at least partially complementary to a target mRNA, which is capable of hybridizing to the target mRNA by hydrogen bonding under normal physiological conditions in a mammal (or under comparable in vitro conditions). In some embodiments, the single-stranded oligonucleotide is a single-stranded antisense oligonucleotide.

As used herein, an RNAi agent refers to an agent that contains an RNA or RNA-like oligonucleotide molecule (e.g., a chemically modified RNA oligonucleotide) that is capable of disrupting or inhibiting the translation of messenger RNA transcripts (mRNA) of target mRNA in a sequence-specific manner. As used herein, RNAi agents may act by RNA interference (i.e., causing RNA interference by interacting with components of the RNA interference pathway (RNA-induced gene silencing complex, or RISC) of mammalian cells), or by any alternative mechanism(s) or route(s). Although RNAi agents, as that term is used herein, are believed to act primarily through the mechanism of RNA interference, the disclosed RNAi agents are not associated or limited to any particular route or mechanism of action, RNAi agents include the following, but not limited to: single-stranded oligonucleotides, single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and dicer

- 16 043375 substrates. The RNAi agents described herein consist of an oligonucleotide whose strand is at least partially complementary to the mRNA being targeted. In some embodiments, the RNAi agents described herein are double-stranded, and consist of an antisense strand and a sense strand that is at least partially complementary to the sense strand, the RNAi agents may include modified nucleotides and/or one or more non-phosphodiester linkages communications. In some embodiments, the RNAi agents described herein are single-stranded.

As used herein, the terms silence, reduce, inhibit, suppress, or knockdown, as applied to the expression of a given gene, mean that the expression of that gene, as measured by the level of RNA transcribed from the gene, or the level of polypeptide, protein, or protein subunit translated from that gene The mRNA in a cell, group of cells, tissue, organ or body of a subject in which a given gene is transcribed is reduced when that cell, group of cells, organ or subject is treated with oligomeric compounds bound to the targeting ligands described herein compared to a second a cell, group of cells, tissue, organ or subject that has not been subjected to such treatment.

As used herein, the term sequence or nucleotide sequence refers to a sequence or order of nucleotide bases or nucleotides described by a sequence of letters using standard nomenclature for nucleotides.

As used herein, and unless otherwise indicated, the term complementary to the description of a first nucleotide sequence (e.g., the sense strand of an RNAi Agent or a target mRNA) with respect to a second nucleotide sequence (e.g., a single-stranded antisense oligonucleotide or the antisense strand of a double-stranded RNAi agent) refers to the ability of the oligonucleotide or a polynucleotide comprising said first nucleotide sequence, hybridize (form hydrogen bonds in a base pair under physiological conditions of a mammalian body (or under comparable in vitro conditions)) and form a duplex or double helix structure under specified conditions with an oligonucleotide or polynucleotide comprising a second nucleotide subsequence. Complementary sequences include Watson-Crick and non-Watson-Crick base pairs and include natural or modified nucleotides or nucleotide mimetics, at least as long as the hybridizability requirements above are met.

As used herein, absolutely complementary or fully complementary means that all (100%) of the nucleotides in the contiguous sequence of the first polynucleotide will hybridize to the same number of bases in the contiguous sequence of the second polynucleotide. The contiguous sequence may include all or part of the first or second nucleotide sequence.

As used herein, partially complementary means that in a hybridized pair of nucleotide base sequences, at least 70%, but not all, of the bases in the contiguous sequence of the first polynucleotide will hybridize to the same number of bases in the contiguous sequence of the second polynucleotide.

As used herein, substantially complementary means that in a hybridized pair of nucleotide base sequences, at least 85%, but not all, of the bases in the contiguous sequence of the first polynucleotide will hybridize to the same number of bases in the contiguous sequence of the second polynucleotide. The terms complementary, fully complementary, and substantially complementary as used herein may be used to refer to a match between a sense strand and an antisense strand of a double-stranded RNAi agent, between an antisense strand of a double-stranded RNAi agent and a target mRNA sequence, or between a single-stranded antisense oligonucleotide sequence and a target mRNA sequence.

As used herein, the terms treat, cure, and the like refer to methods and measures taken to ensure a reduction in the number, severity and/or frequency of one or more symptoms of a disease in a subject.

As used herein, the phrase cellular administration, when referring to an oligomeric compound, means the functional delivery of the oligomeric compound into a cell. The phrase functional delivery means the process of delivering an oligomeric compound into a cell in a manner that allows the oligomeric compound to exhibit the intended biological activity, for example, sequence-specific inhibition of gene expression.

Unless otherwise indicated, the use of a symbol herein indicates that any group or groups within the scope of the compound disclosed herein may be associated with it.

As used herein, the term isomers refers to compounds that have the same molecular formula but differ in the nature or sequence of bonds between atoms or the arrangement of atoms in space. Isomers that differ in the arrangement of atoms in space are called stereoisomers. Stereoisomers that are not mirror images of each other are called diastereoisomers, and stereoisomers that are incompatible mirror images are called enantiomers, or sometimes optical isomers. A carbon atom bonded to four non-identical substituents is called a chiral center.

In this text, unless an asymmetric center is specifically stated to have a specific conformation, for each structure in which asymmetric centers present may give rise to enantiomers, diastereomers, or other stereoisomeric configurations, each structure disclosed herein is intended to represent all such possible isomers, including their optically pure and racemic forms. For example, the structures disclosed herein are intended to cover mixtures of diastereomers as well as individual stereoisomers.

The term substituted (containing substituents) as used herein means that any one or more hydrogens on the designated atom, usually a carbon, oxygen or nitrogen atom, is replaced by any group designated herein, provided that the normal valency of the designated atom is not exceeded. atom, and that this substitution produces a stable compound. Non-limiting examples of substituents include _1-6 alkyl, C2- _C6 alkenyl, C2-C6 alkynyl, cyano, hydroxyl, oxo, carboxyl, cycloalkyl, cycloalkenyl, heterocyclyl, heteroaryl, aryl, keto, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, or halogen (eg , F, Cl, Br, I). If the substituent is keto or oxo (i.e. =O), then two (2) hydrogens on the indicated atom are replaced. Ring double bonds as used herein are double bonds formed between two adjacent atoms on the ring (eg, C=C, C=N, N=N, etc.).

Some of the compounds described herein may exist in a tautomeric form, which is also included within the scope of this description. Tautomers are compounds whose structures differ significantly in the arrangement of atoms, but which exist in an easily and quickly established equilibrium. It should be understood that the compounds described herein may be represented as various tautomers. It should also be understood that where compounds have tautomeric forms, all tautomeric forms are intended to be included within the scope of this specification, and the names assigned to the compounds do not exclude any tautomeric form.

The compounds and pharmaceutically acceptable salts disclosed herein may exist in one or more tautomeric forms, including ketone-enol, amide-nitrile, lactam-lactim, amide-imidic acid on heterocyelic rings (e.g., the nitrogenous bases guanine, thymine, and cytosine), amine-enamine and enamine-enamine, as well as in the form of geometric isomers and their mixtures. Ring tautomerism, exhibited by glucose and other sugars, results from the interaction of an aldehyde group (-CHO) on a sugar molecule with one of the hydroxy groups (-OH) on the same molecule, resulting in the formation of a cyclic (ring) form. All such tautomeric forms are included within the scope of the present invention. Tautomers exist as mixtures of a group of tautomers in solution. Even though a single tautomer may be described, the present invention includes all tautomers of the compound disclosed herein. The concept of tautomers that can change into each other through tautomerization is called tautomerism. In tautomerism, a simultaneous shift of electrons and a hydrogen atom occurs.

Various types of tauthymization are catalyzed by: Base: 1. deprotonation; 2. formation of a delocalized anion (for example, enolate); 3. protonation at various positions of the anion; Acid: 1. protonation; 2. formation of a delocalized cation; 3. deprotonation at various positions adjacent to the cation.

As used herein, the term alkyl refers to a saturated aliphatic hydrocarbon group, linear or branched, containing from 1 to 10 carbon atoms, unless otherwise noted. For example, C1-C6 alkyl includes alkyl groups containing 1, 2, 3, 4, 5 or 6 carbon atoms in a linear or branched configuration. As used herein, the term aminoalkyl refers to an alkyl group, as defined above, substituted at any position by one or more amino groups as permitted by the rules of valency. Amino groups can be unsubstituted, monosubstituted or disubstituted.

As used herein, the term cycloalkyl refers to a saturated or unsaturated non-aromatic hydrocarbon ring group containing from 3 to 14 carbon atoms, unless otherwise indicated. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, methylcyclopropyl, 2,2-dimethylcyclobutyl, 2-ethylcyclopentyl or cyclohexyl, etc. Cycloalkyls may contain multiple spiro or fused rings. Cycloalkyl groups are optionally mono-, di-, tri-, tetra- or penta-substituted at any position, as long as valence rules allow this.

As used herein, the term alkenyl refers to a non-aromatic hydrocarbon radical, which may be linear or branched, containing at least one carbon-carbon double bond and from 2 to 10 carbon atoms unless otherwise specified. Such groups can contain up to five carbon-carbon double bonds. For example, C ₂ -C ₆ is defined as an alkenyl radical containing from 2 to 6 carbon atoms. Examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl and cyclohexenyl. The linear, branched or cyclic moiety of an alkenyl group may contain double bonds and is optionally mono-, di-, tri-, tetra- or penta-substituted at any position as permitted by normal valence. The term cycloalkenyl refers to a monocyclic hydrocarbon group containing the specified number of carbon atoms and at least one carbon-carbon double bond.

As used herein, the term alkynyl refers to a carbohydrate radical, linear or branched, containing from 2 to 10 carbon atoms, unless specifically stated otherwise, and at least one carbon-carbon triple bond. Up to 5 carbon-carbon triple bonds may be present. Accordingly, C ₂ -C ₆ alkynyl denotes an alkynyl radical containing from 2 to 6 carbon atoms. Examples of alkynyl groups include, but are not limited to: ethynyl, 2propynyl and 2-butynyl. The linear or branched portion of an alkynyl group may contain triple bonds as permitted by conventional valency requirements, and may optionally be mono-, di-, tri-, tetra-, or penta-substituted at any position as permitted by conventional valency.

As used herein, alkoxy or alkoxy refers to an alkyl group as defined above, with the number of carbon atoms indicated, attached by an oxygen bridge. C1.6 alkoxy is intended to include C1, _C2 , _C3 , _C4 , _C5 and _C6 alkoxy groups. _{C1_8} alkoxy is intended to include C1, _C2 , _C3 , _C4 , _C5 , _C6 , _C7 and _C8 alkoxy groups. Examples of alkoxy include, but are not limited to: methoxy, ethoxy, n-propoxy, n-propoxy, n-butoxy, sec-butoxy, t-butoxy, n-pentoxy, sec-pentoxy, n-heptoxy and n-octoxy.

As used herein, keto refers to any alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclyl, heteroaryl or aryl group as defined herein, attached by a carbonyl bridge. Examples of a keto group include, but are not limited to: alkanoyl (e.g., acetyl, propionyl, butanoyl, pentanoyl, hexanoyl), aclenoyl (e.g., acryloyl), alkinoyl (e.g., ethinoyl, propinoyl, butinoyl, pentinoyl, hexanoyl), aryloyl ( eg benzoyl), heteroaryloyl (eg pyrroloyl, imidazoyl, quinolinoyl, pyridinoyl).

As used herein, alkoxycarbonyl refers to any alkoxy group as defined above attached by a carbonyl bridge (ie -C(O)O-alkyl). Examples of alkoxycarbonyl groups include, but are not limited to: methoxycarbonyl, ethoxycarbonyl, isopropoxycarbonyl, n-propoxycarbonyl, t-butoxycarbonyl, benzyloxycarbonyl, or npentoxycarbonyl.

As used herein, aryloxycarbonyl refers to any aryl group as defined herein attached via an oxycarbonyl bridge (ie -C(O)O-aryl). Examples of aryloxycarbonyl groups include, but are not limited to: phenoxycarbonyl and naphthyloxycarbonyl.

As used herein, heteroaryloxycarbonyl refers to any heteroaryl group as defined herein attached via an oxycarbonyl bridge (ie, -C(O)heteroaryl). Examples of heteroaryloxycarbonyl groups include, but are not limited to: 2-pyridyloxycarbonyl, 2-oxazolyloxycarbonyl, 4-thiazolyloxycarbonyl, or pyrimidinyloxycarbonyl.

As used herein, aryl or aromatic means any stable monocyclic or polycyclic carbohydrate ring containing up to 7 atoms on each ring, at least one ring being aromatic. Examples of aryl groups include, but are not limited to: phenyl, naphthyl, anthracenyl, tetrahydronaphthyl, indanyl and biphenyl. In the case where the aryl substituent is bicyclic and one ring is non-aromatic, the addition is assumed to occur via an aromatic ring. Aryl groups are not necessarily mono-, di-, tri-, tetra- or penta-substituted in any position as permitted by ordinary valency.

As used herein, the term heteroaryl represents a stable monocyclic or polycyclic ring containing up to 7 atoms on each ring, wherein at least one ring is aromatic and contains from 1 to 4 heteroatoms selected from the group consisting of O, N and S. Examples of heteroaryl groups include, but are not limited to, the following: acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrrazolyl, indolyl, benzotriazolyl, furanyl, thienyl, benzothienyl, benzofuranyl, benzimidazolonyl, benzoxazolonyl, quinolinyl, isoquinolinyl, dihydroisoindolinyl, imidazopyridinyl, isoindolinyl, indazolyl, oxazolyl, oxadiazolyl , isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, tetrahydroquinolinyl. Heteroaryl is also intended to include the N-oxide derivative of any nitrogen-containing heteroaryl. In cases where the heteroaryl substituent is bicyclic and one ring is non-aromatic or does not contain heteroatoms, the addition is assumed to occur through an aromatic ring or a ring containing a heteroatom. Heteroaryl groups are not necessarily mono-, di-, tri-, tetra- or penta-substituted in any position as permitted by normal valency.

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As used herein, the term heterocycle, heterocyclic or heterocyclyl refers to a 314-membered aromatic or non-aromatic heterocycle containing from 1 to 4 heteroatoms selected from the group consisting of O, N and S, including polycyclic groups. As used herein, the term heterocyclic is also considered synonymous with the terms heterocycle and heterocyclyl and is intended to be as defined herein. Heterocyclyl includes the above-mentioned heteroaryls, as well as their dihydro- and tetrahydro-analogues. Examples of heterocyclyl groups include, but are not limited to: azetidinyl, benzimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazolyl, indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isochy nolil , isothiazolyl, isoxazolyl, naftpyridinyl, oxadiazolyl, oxooxazolidinyl, oxazolyl, oxazoline, oxopiperazinyl, oxopyrrolidinyl, oxomorpholinyl, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyridine onyl, pyrimidyl, pyrimidinonyl, pyrrolyl, quinazolinyl, quinolyl , quinoxalinyl, tetrahydropyranyl, tetrahydrofuranyl, tetrahydrothiopyranyl, tetrahydroisoquinolinyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, 1,4-dioxanyl, hexahydroazepenyl, piperazinyl, piperidinyl, pyridin-2-onyl, pyrrolidinyl, morpholinyl, thiomorpholinyl, dihydrobenzimidazolyl, dihydrobenzofuranyl ,dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl, dihydroisoxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydroquinoline yl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, dioxidothiomorpholinyl, methylenedioxybenzoyl, tetrahydrofuranyl and tetrahydrothienyl and their N-oxides. The heterocyclyl substituent can be attached via a carbon atom or via a heteroatom. Heterocyclyl groups are not necessarily mono-, di-, tri-, tetra- or penta-substituted in any position as permitted by normal valency.

One of ordinary skill in the art will readily understand and appreciate that the compounds and compositions disclosed herein may contain some atoms (e.g., N, O, or S atoms) in a protonated or deprotonated state, depending on the environment in which the compound is placed or composition.

Accordingly, the structures disclosed herein contemplate that certain functional groups, such as, for example, OH, SH, or NH, may be protonated or deprotonated. The present disclosure is intended to cover the disclosed compounds and compositions regardless of their protonation status as determined by the pH of the environment, as one skilled in the art will readily understand.

As used herein, the expression consisting of excludes any elements, steps or ingredients not specified in the claim. When used in the claims of this document, the expression consisting essentially of limits the scope of the claim to specified materials or steps and materials or steps that do not significantly affect the essential and novel feature or characteristics of the claimed invention.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning usually given to them by one of ordinary skill in the art to which the invention belongs. Although methods and materials similar or equivalent to those described herein may be used to practice or study the present invention, suitable methods and materials are described below. The contents of all publications, patent applications, patents and other references noted herein are incorporated herein by reference in their entirety. In case of conflict, the present description, including definitions, controls. Moreover, the materials, methods, and examples are for illustrative purposes only and are not limiting.

Other features and advantages of the present invention will become apparent from the following detailed description and claims.

Brief description of drawings

Fig. 1 is the 1H NMR spectrum of compound 11 (described below in Example 1 and having chemical structure 1005b described herein).

Fig. 1A is a 1H NMR spectrum of structure 1004b disclosed herein (described below in Example 1).

Fig. 2 is a 31P-NMR spectrum of compound 19 (described below in Example 2 and having chemical structure 1008b described herein.).

Fig. 2A is the 1H NMR spectrum of compound 19.

Fig. 2B is the 1H NMR spectrum of compound 14 (described below in Example 2).

Fig. 2C is the 1H NMR spectrum of compound 15 (described below in Example 2).

Fig. 2D is the 1H NMR spectrum of compound 16 (described below in Example 2).

Fig. 2E is the 1H NMR spectrum of compound 17 (described below in Example 2).

Fig. 2F is the 1H NMR spectrum of compound 18 (described below in Example 2).

- 20 043375

Fig. 3 is the 'H NMR spectrum of compound 30 (described below in Example 3).

Fig. 4 is the 'H NMR spectrum of compound 38 (described below in Example 4).

Fig. 5 is the 'H NMR spectrum of compound 44 (described below in Example 5).

Fig. 6 is the 'H NMR spectrum of compound 47 (described below in Example 6).

Fig. 7 is a photograph of a phosphoramidite-containing compound with a PEG-linkerGalNAc in a vial (as described below in Example 7).

Fig. 8 is a photograph of a phosphoramidite-containing compound with structure '008b in a vial (as described below in Example 7).

Fig. 9 is ^{a 31} P-NMR spectrum of the PEG-linker-GalNAc structure (as described below in Example 8).

Fig. '0 is a graph illustrating normalized levels of mouse factor '2 (F'2) protein in wild-type mice (as described below in Example '').

Fig. '' is a graph illustrating normalized levels of mouse Factor '2 (F'2) protein in wild-type mice (as described below in Example '2).

Fig. '2 is a graph illustrating normalized levels of lipoprotein(a) particles (Lp(a)) in Lp(a) transgenic (Tg) mice (as described below in Example '3).

Fig. '3 is a graph illustrating normalized apo(a) levels in apo(a)transgenic (Tg) mice (as described below in Example '4).

Fig. '4 is a graph illustrating normalized levels of Lp(a) particles in Lp(a)transgenic Tg mice (as described below in Example '5).

Fig. '5 is a graph illustrating normalized F'2 protein levels in wild-type mice (as described below in Example '6).

Fig. '6 is a graph illustrating normalized levels of Lp(a) particles in Lp(a)transgenic Tg mice (as described below in Example '7).

Fig. '7 is a graph illustrating normalized apo(a) levels in apo(a) transgenic (Tg) mice (as described below in Example '8).

Fig. '8 is a graph illustrating normalized levels of Lp(a) particles in Lp(a)transgenic (Tg) mice (as described below in Example '9).

Fig. '9 is a graph illustrating normalized Lp(a) levels in cynomolgus monkeys (as described below in Example 20).

Fig. 20 is a graph illustrating normalized cF'2 protein levels in cynomolgus monkeys (as described below in Example 2').

Fig. 2' is a graph illustrating normalized AAT protein levels (Z-AAT) in PiZ transgenic mice (as described below in Example 22).

Detailed description

Described herein are targeting ligands that are associated with compounds, such as therapeutic or diagnostic compounds. In some embodiments, the compounds that are associated with the targeting ligands described herein include or consist of therapeutic compounds, such as expression inhibitory oligomeric compounds. Targeting ligands can be used to target therapeutic compounds to the desired position of a target nucleic acid or gene. Also disclosed herein are compositions including targeting ligands and therapeutic compounds, such as compositions including or consisting of targeting ligands and expression-inhibiting oligomeric compounds.

The new targeting ligands described herein provide several advantages over previously known targeting ligands to improve the delivery of therapeutic compounds. These advantages include, for example, increased ease and efficiency of manufacture, as well as providing efficient targeting or biodistribution, sufficient in vivo and/or in vitro stability, and/or other improvements desirable for the delivery of a therapeutic oligonucleotide product. The new targeting ligands are also particularly well suited to be synthesized in the form of phosphoramidite compounds, which reduces the cost and complexity of manufacture, and facilitates convenient attachment of the targeting ligand to compounds, particularly expression-inhibitory oligomeric compounds (such as RNAi agents), while providing close, or , in some cases, improved delivery and/or efficacy of the therapeutic compound.

Targeting ligands.

Targeting ligands consist of one or more targeting group(s) or targeting moiety(s) that can serve to improve the pharmacokinetics and biodistribution properties of the compound to which they are bound and improve the cell or tissue specific distribution and cell specific uptake of the conjugated composition. In general, a targeting ligand facilitates the targeted delivery of the therapeutic compound to which it is bound to the desired target site. In some cases, the targeting moiety can bind to a cell or cell

- 2' 043375 precise receptor and initiate endocytosis, facilitating the entry of the therapeutic compound into the cell. The targeting moiety may include compounds having affinity for cellular receptors or cell surface molecules or antibodies. Various targeting ligands, which contain targeting moieties, can be associated with therapeutic agents and other compounds to target the agents to cells and specific cellular receptors. Types of targeting moieties include carbohydrates, cholesterol and cholesterol groups, and steroids. Targeting moieties that can bind to cellular receptors include saccharides such as galactose, galactose derivatives (such as N-acetylgalactosamine), mannose and mannose derivatives; other carbohydrates; glycans; haptens, vitamins, folate; biotin, aptamers; and peptides such as RGD-containing peptides, insulin, EGF and transferrin.

Targeting moieties known to bind to the asialoglycoprotein receptor (ASGPR) are particularly useful for directing the delivery of oligomeric compounds to the liver. Asialoglycoprotein receptors are expressed in large quantities on liver cells, including hepatocytes. Targeting moieties for cellular receptors that target ASGPR include galactose and galactose derivatives. In particular, clusters of galactose derivatives, including clusters consisting of two, three or four N-acetylgalactosamines (GalNAc or NAG), may facilitate the uptake of certain compounds into liver cells. GalNAc clusters conjugated to oligomeric compounds serve to direct the composition to the liver, where N-acetylgalactosamine sugars can bind to asialoglycoprotein receptors on the surface of liver cells. Binding to the asialoglycoprotein receptor is thought to trigger receptor-mediated endocytosis, thereby facilitating entry of the compound into the cell.

The targeting ligands disclosed herein may include one, two, three, four, or more than four targeting moieties. In some embodiments, the targeting ligands disclosed herein may contain one, two, three, four, or more than four targeting moieties linked to a branch point group. In some embodiments, the targeting ligands disclosed herein may comprise one, two, three, four, or more than four targeting moieties linked to a branch point group, with each targeting moiety linked to the branch point group by a connecting moiety.

In some embodiments, the targeting ligands disclosed herein may comprise one, two, three, four, or more than four asialoglycoprotein receptor (ASGPR) targeting moieties linked to a branch point group. In some embodiments, the targeting ligands disclosed herein may comprise ASGPR) linked to a branch point group, with each targeting moiety for the ASGPR linked to the branch point group by a connecting moiety.

In some embodiments, the branch point group is associated with a linker. In some embodiments, the branch point group includes a linker replacement moiety, and the branch point group is linked to a therapeutic compound. In some embodiments, the branch point group is associated with an oligomeric compound. In some embodiments, the branch point group is linked to an expression inhibitory oligomeric compound.

In some embodiments, the targeting ligand is represented by Formula I:

where n is an integer from 1 to 4 (for example, 1, 2, 3 or 4) (Formula I).

In some embodiments, n in Formula I is an integer in the range 1-3, 1-2, 2-4, 2-3, or 3-4.

A linker of formula I is a group that includes one or more substituted or unsubstituted moieties selected from the following groups: cycloalkyl, cycloalkenyl, aryl, heteroaryl or heterocyclyl, or a covalently linked combination(s) thereof that links a branch point group at one end of the linker with a therapeutic compound (or a phosphoramidite phosphorus atom if the targeting ligand is synthesized as a phosphoramidite compound) at the other end of the linker. In some embodiments, one or more additional groups, such as cleavable moieties (such as a phosphate group or a disulfide bond-containing group) or phosphorothioate or phosphonate linkage(s), are inserted between the therapeutic compound and the linker. Linkers are rigid in the sense that they impart sufficient stability and rigidity to the targeting ligand as a whole, reducing the interaction between the targeting moiety(s) of Formula I and the therapeutic compound with which it

- 22 043375 connected. This, in turn, can improve the interaction of the targeting moiety with the target site. Additionally, linkers for use in targeting ligands disclosed herein are specifically designed to synthesize the targeting ligand(s) in the form of phosphoramidite compounds, thereby effectively linking the targeting ligand to the 5' end of the oligomeric compound.

A branch point group of Formula I is any group that allows the attachment of one or more targeting moieties (via one or more connecting moieties) to a linker.

In some embodiments, the targeting ligand is represented by Formula II:

where n is an integer from 1 to 4 (for example, 1, 2, 3 or 4). In some embodiments, n in Formula II is an integer from the range 1-3, 1-2, 2-4, 2-3, or 3-4.

In Formula II, a branch point group is any group that allows the attachment of one or more targeting moieties (via one or more connecting moieties) to the therapeutic compound (or to the phosphorus atom of the phosphoramidite if the targeting ligand is synthesized as a phosphoramidite compound) via the moiety, replacement linker. As used herein, a branch point group includes a moiety that replaces a linker if the branch point group includes one or more substituted or unsubstituted cycloalkyl, cycloalkenyl, aryl, heteroaryl or heterocyclyl groups, or combinations thereof (including fused ones within the branch point group that perform the same functions as the rigid linkers of Formula I disclosed herein.

The one or more connecting moieties of formula I and II are spacer groups that can further increase the flexibility and/or length of the bond between the targeting moiety and the branch point group. The connecting moiety is an effective means of connecting the targeting moiety to the branch point group. For the targeting ligands disclosed herein, there is at least one connecting moiety for each targeting moiety. In some embodiments, there are multiple (ie, two or more) connecting fragments between the branch point group and the targeting fragment.

The targeting moieties of formulas I and II are groups that serve to improve the pharmacokinetic or biodistribution properties of the therapeutic compound to which they are associated and to improve the cell or tissue specific distribution and cell specific absorption of the conjugated composition. The targeting moiety may include compounds having affinity for cellular receptors or cell surface molecules or antibodies. Types of targeting moieties include carbohydrates, cholesterol and cholesterol groups, and steroids. Targeting moieties that can bind to cellular receptors include saccharides such as galactose, galactose derivatives (such as N-acetylgalactosamine), mannose and mannose derivatives; other carbohydrates; glycans; haptens, vitamins, folate; biotin, aptamers; and peptides such as RGD-containing peptides, insulin, EGF and transferrin.

The targeting ligands disclosed herein may be associated with therapeutic compounds. In some embodiments, the targeting ligand is linked to the therapeutic compound by an additional linker and/or cleavable moiety, which in turn is linked to the therapeutic compound. In some embodiments, the targeting ligands are ligated to the therapeutic compound itself.

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.

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

- 23 043375 with the 5'-end or 3'-end of a single-stranded RNAi agent.

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

In some embodiments, the targeting ligand disclosed herein includes a cleavable moiety. In some embodiments, the cleavable moiety includes or consists of a phosphate or other internucleoside linking group that can be cleaved. In some embodiments, the targeting ligand is linked to the therapeutic compound by a cleavable moiety.

In some embodiments, the targeting ligand disclosed herein is linked to additional group or groups that include a cleavable moiety. In some embodiments, the targeting ligand is linked to a cleavable moiety, which in turn is linked to an expression inhibitory oligomeric compound.

In some embodiments, the binding ligand is a phosphoramidite-containing compound. The phosphoramidite compound comprising the targeting ligand described herein can be used to readily attach the targeting ligand to the therapeutic compound or other moieties using phosphoramidite synthesis methods well known in the art. In some embodiments, the phosphoramidite compound comprising the binding ligand is coupled to the expression inhibitory oligomeric compound using methods well known in the art. In some embodiments, the targeting ligand-containing phosphoramidite is linked to the 5' end of the sense strand of the double-stranded RNAi agent.

In some embodiments, the expression inhibitory oligomeric compound associated with the targeting ligand includes a single-stranded oligonucleotide. In some embodiments, the single-stranded oligonucleotide is a single-stranded antisense oligonucleotide. In some embodiments, the targeting ligand is linked directly to the single-stranded antisense oligonucleotide. In some embodiments, additional groups are inserted between the targeting ligand and the single-stranded oligonucleotide.

In some embodiments, the targeting ligand associated with the RNAi agent includes one or more N-acetylgalactosamine sugars as a targeting moiety or targeting moieties.

In some embodiments, the targeting ligand associated with the expression inhibitory oligomeric compound includes a connecting moiety that includes polyethylene glycol (PEG). In some embodiments, the connecting moiety consists of PEG. In some embodiments, the connecting moiety includes a PEG containing from 1 to 10 ethylene glycol units. In some embodiments, the connecting moiety includes a PEG containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ethylene glycol units.

In some embodiments, the expression inhibitory oligomeric compound associated with any of the targeting ligands disclosed herein includes an RNAi agent. In some embodiments, the targeting ligand disclosed herein is linked, directly or indirectly, to an RNAi agent.

In some embodiments, the targeting ligand disclosed herein is linked directly to the RNAi agent. In some embodiments, the targeting ligand disclosed herein is linked indirectly to the RNAi agent because an additional group or groups are embedded between the RNAi agent and the targeting ligand linker. In some embodiments, a second linker is included between the linker and the therapeutic compound.

Linkers.

The targeting ligands disclosed herein contain a linker as shown in Formula I, or, alternatively, the branch point group includes a linker replacement moiety as shown in Formula II.

A linker is a group of atoms linked to a branch point group at one end (or to a phosphoramidite phosphorus atom if the targeting ligand is synthesized as a phosphoramidite compound) at the other end. In some embodiments, the linker is linked to a branch point group at one end, and ligated at the other end to a group or groups that are further ligated to an expression inhibitory oligomeric compound. In some embodiments, the linker is directly linked to the oligomeric compound. In some embodiments, the linker is associated with a cleavable moiety, which in turn is associated with an oligomeric compound. Examples of cleavable moieties include, for example, phosphate groups, groups including a disulfide moiety, and/or other internucleotide linkages that can be cleaved. In some embodiments, the linker is not associated with the cleavable moiety. In some embodiments, the linker is linked to a phosphorothioate or phosphonate group.

For the targeting ligands disclosed herein, in accordance with Formula I,

- 24 043375 linker is a rigid linker. Rigid is a linking group that includes one or more substituted or unsubstituted cycloalkyl, cycloalkenyl, aryl, heteroaryl or heterocyclyl groups, or covalently linked combination(s) thereof.

In some embodiments, the targeting ligand of formula I includes or consists of a linker having the following structure:

(Structure 1).

In some embodiments, the targeting ligand of formula I includes or consists of a linker having the following structure:

O (Structure 2).

In some embodiments, the targeting ligand of Formula I has the following structure or consists of:

O (Structure 3).

In some embodiments, the targeting ligand of Formula I has the following structure or consists of:

O includes a linker having includes a linker having (Structure 4).

In some embodiments, the targeting ligand of Formula I has the following structure or consists of:

includes a linker having

(Structure 5).

In some embodiments, the targeting ligand of formula I includes or consists of a linker having the following structure:

(Structure 6a)

In some embodiments, the targeting ligand of formula I includes or consists of a linker having the following structure:

(Structure 6b).

In some embodiments, the targeting ligand of formula I includes or consists of a linker having the following structure:

- 25 043375

O (Structure 6c).

In some embodiments, the targeting ligand of formula I includes or consists of a linker having the following structure:

In some embodiments, the targeting ligand of formula I includes or consists of a linker having the following structure:

where n' is an integer from 0 to 10 (for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), and each Z' present 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 C ₂ -C ₆ alkenyl, substituted C ₂ -C ₆ alkynyl, substituted C ₁ -C ₆ alkoxy, substituted C ₁ -C ₆ aminoalkyl, halogen (for example, F), hydroxyl, amido, substituted amide, cyano, substituted or unsubstituted keto, substituted or unsubstituted alkoxycarbonyl, substituted or unsubstituted aryloxycarbonyl, substituted or unsubstituted heteroaryloxycarbonyl and sulfhydryl (structure 7).

In some embodiments, the targeting ligand of formula I includes or consists of a linker having the following structure:

where n is an integer from 0 to 4 (for example, 1, 2, 3 or 4), and each Z present is independently selected, and Z is independently selected from the group consisting of: C1-C6 alkyl, _C2 - _C6 alkenyl , C ₂ -C ₆ alkynyl, C1-C6 alkoxy, substituted C1-C6 alkyl, C1-C6 aminoalkyl, substituted C ₂ -C ₆ alkenyl, substituted C ₂ -C ₆ alkynyl, substituted or unsubstituted amino, carboxyl, substituted C1- C6 alkoxy, substituted C1-C6 aminoalkyl, halogen (e.g. F), hydroxyl, amido, 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 targeting ligand of formula I includes or consists of a linker having the following structure:

where V includes or consists of one or more groups such as substituted or unsubstituted cycloalkyl (for example, cyclohexyl, cyclopropyl, cyclobutyl, cyclopentyl, cycloheptyl, cyclooctyl, etc.), cycloalkenyl (for example, cyclohexenyl, cyclobutenyl, cyclopentenyl, cycloheptenyl, cyclooctenyl, cyclohexadienyl, cyclopentadienyl, cycloheptadienyl, cyclooctadienyl, etc.), aryl (e.g. phenyl, naphthyl, binaphthyl, anthracenyl, etc.), heteroaryl (e.g. pyridyl, pyrimidinyl, pyrrole, imidazole, furan, benzofuran, indole, etc.) or heterocyclyl (eg, tetrahydrofuran, tetrahydropyran, piperidine, pyrrolidine, etc.) or any covalently linked combination thereof (structure 9).

In some embodiments, linkers suitable for use in the targeting ligands disclosed herein are derived from a compound that includes a rigid structure with a terminal carboxylic acid moiety (or an activated ester thereof) 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 group

- 26 043375 feasting 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 coupling to the branch point group, and the alcohol moiety is suitable for coupling to the phosphorus atom of the phosphoramidite using a phosphitylation reaction with a phosphoramidite-forming reagent. An example of a phosphitylation reaction using phosphoramidite-forming reagents is described in the Examples section of this document. The linker structures disclosed herein are suitable for producing a phosphoramidite compound targeting ligand.

In some embodiments, the linker is coupled to an expression inhibitory oligomeric compound that is 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.

In some embodiments, the linker is associated with a cleavable moiety. In some embodiments, the terminal phosphate group of the expression inhibitory oligomeric compound may serve as a cleavable moiety. In some embodiments, an independently selected cleavable moiety is coupled to a linker. As used herein, a cleavable fragment is a group that is stable outside the cell but is cleaved once it enters the target cell. Cleavable fragments are cleaved under certain conditions, such as pH, or in the presence of certain cleavage agents, such as molecules that stimulate degradation or redox agents.

In some embodiments, the cleavable moiety may be pH sensitive. For example, it is known that endosomes and lysosomes in general have a more acidic pH (pH approximately 4.5 to 6.5) than human blood (pH approximately 7.35 to 7.45) and thus stimulate degradation of the cleaved fragment.

In some embodiments, the cleavable moiety is a phosphate group. Phosphate groups can be cleaved by agents known to resolve or hydrolyze phosphate groups.

In some embodiments, the targeting ligands disclosed herein contain a branch point group that includes a linker replacement group instead of a linker as shown in Formula II. If the linker is replaced with a linker replacement fragment, the linker replacement fragment is part of a branch point group.

In some embodiments, the linkers and linker replacement moieties disclosed herein allow for the insertion of only one isomer of the targeting ligand, which may provide additional benefits for oligonucleotide therapeutic products.

Branch point groups.

The targeting ligands disclosed herein contain at least one branch point group. In some embodiments, the branch point moiety of the targeting ligands disclosed herein is linked to a linker. In some embodiments, the branch point group is coupled to a linker at one end, and the branch point group is coupled to one or more connecting moieties at the other end(s). In some embodiments, the branch point group is linked to the expression inhibitory oligomeric compound through an additional group or groups. In some embodiments, the branch point group includes a linker replacement moiety and is linked to an expression inhibitory oligomeric compound.

Branch point groups disclosed herein can be any group that is capable of being attached to one or more targeting moieties and is further capable of being attached to a linker disclosed herein or, alternatively, where the branch point group contains a linker replacement moiety The branch point group may be any group that includes a linker replacement moiety that is capable of attachment to a therapeutic compound, such as an expression inhibitory oligomeric compound.

For the branch point groups of Formula I disclosed herein, prior to conjugation to the linker, the connection of the branch point group that serves to produce the branch point group contains one terminal amine for each required linker bond, and one terminal carboxylic acid moiety (or its activated ester) for each required linkage to the connecting moiety.

In some embodiments, the targeting ligand includes a branch point having a structure selected from the following:

- 27 043375

In some embodiments, the targeting ligand includes a branch point having the following structure:

where n is an integer from 1 to 20 (structure 209).

In some embodiments, the targeting ligand includes a branch point having the following structure:

where m represents 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 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 210).

In some embodiments, the targeting ligand includes a branch point having a structure represented by the following formula:

where m is an integer from 0 to 20 (for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,

- 28 043375

14, 15, 16, 17, 18, 19 or 20); n represents 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); x represents an integer from 1 to 10 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10); y is an integer from 1 to 10 (for example, 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 (for example, 1, 2, 3 or 4); and K is selected from the group consisting of substituted or unsubstituted cycloalkyl (e.g., cyclohexyl, cyclopropyl, cyclobutyl, cyclopentyl, cycloheptyl, cyclooctyl, etc.), substituted or unsubstituted cycloalkenyl (e.g., cyclohexenyl, cyclobutenyl, cyclopentenyl, cycloheptenyl, cyclooctenyl, cyclohexadienyl, cyclopentadienyl, cycloheptadienyl, cyclooctadienyl, etc.), substituted or unsubstituted aryl (for example, phenyl, naphthyl, binaphthyl, anthracene, etc.), substituted or unsubstituted heteroaryl (for example, pyridyl, pyrimidinyl, pyrrole, imidazole, furan, benzofuran, indole, etc.), and substituted or unsubstituted heterocyclyl (eg, tetrahydrofuran, tetrahydropyran, piperidine, pyrrolidine, etc.) or covalently linked combinations thereof (structure 211).

In some embodiments, the targeting ligand includes a branch point having the following structure:

where m represents 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); n represents 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); x represents an integer from 1 to 10 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10); and y is an integer from 1 to 10 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) (structure 212).

In some embodiments, the targeting ligand includes a branch point having the following structure:

where m represents 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); n represents 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); x is an integer between 1 and 10 (for example,

1, 2, 3, 4, 5, 6, 7, 8, 9 or 10); y is an integer from 1 to 10 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10); and G is selected from the group consisting of

or any substituted or unsubstituted cyclic or heterocyclic structure having a size of 5, 6, 7, 8 or 9 atoms, for example, such as substituted or unsubstituted cycloalkyl (for example, cyclohexyl, cyclopropyl, cyclobutyl, cyclopentyl, cycloheptyl, cyclooctyl, etc. ), substituted or unsubstituted cycloalkenyl (e.g. cyclohexenyl, cyclobutenyl, cyclopentenyl, cycloheptenyl, cyclooctenyl, cyclohexadienyl, cyclopentadienyl, cycloheptadienyl, cyclooctadienyl, etc.), substituted or unsubstituted aryl (e.g. phenyl, naphthyl, binaphthyl, anthracenyl, etc.) etc.), substituted or unsubstituted heteroaryl (for example, pyridyl, pyrimidinyl, pyrrole, imidazole, furan, benzofuran, indole, etc.) or substituted or unsubstituted heterocyclyl (for example, tetrahydrofuran, tetrahydropyran, piperidine, pyrrolidine, etc. ) (structure 213).

In some embodiments, the targeting ligand includes a branch point group having the following structure:

- 29 043375 where 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).

In some embodiments, the targeting ligand includes a branch point group having the following structure:

where 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 selected from the group consisting of:

In some embodiments, the targeting ligand includes a grouping structure of the following:

branch points have

(Structure 216).

In some embodiments, the targeting ligand includes a grouping structure of the following:

branch points have

(Structure 217).

In some embodiments, the targeting ligand includes a grouping structure of the following:

branch points have

/ (Structure 218).

In some embodiments, the targeting ligand includes a branch point group having the following structure:

- 30 043375

where n is an integer selected from 1 to 7 (eg, 1, 2, 3, 4, 5, 6, or 7) (structure 219). In some implementations, n in structure 219 is 1. In some implementations, n in structure 219 is 2. In some implementations, n in structure 219 is 3. In some implementations, n in structure 219 is 4. In some In 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 targeting ligand includes a branch point group that includes a linker replacement group.

In some embodiments, the targeting ligand includes a branch point group that includes a linker replacement moiety having the structure shown below:

O

I (Structure 220) or

(Structure 221).

Connecting fragments (Tethers).

The targeting ligands disclosed herein contain one or more additional moieties. The connecting fragment is positioned as a link between the branch point group and each targeting fragment. In some embodiments, the connecting moiety is linked directly to a targeting ligand at one end and directly to a branch point group at the other end. In some embodiments, the connecting moiety is linked directly to a targeting ligand at one end, and indirectly to a branch point group at the other end. In some embodiments, the connecting moiety is linked indirectly to a targeting ligand at one end and indirectly to a branch point group at the other end. In some embodiments, the targeting ligand described herein includes three connecting moieties and three targeting moieties. In some embodiments, the targeting ligand described herein includes four connecting moieties and four targeting moieties. In some embodiments, a targeting ligand described herein includes one connecting moiety and one targeting moiety. In some embodiments, a targeting ligand described herein includes a plurality of connecting moieties and a plurality of targeting moieties.

In some embodiments, additional connecting moieties and other groups are embedded between the connecting moiety and the targeting moiety of Formula I or Formula II. In some embodiments, the second connecting moiety is embedded between the connecting moiety and the targeting moiety of Formula I or Formula II. In some embodiments, the second connecting moiety and the third connecting moiety are embedded between the connecting moiety and the targeting moiety of Formula I or Formula II. In some embodiments, the second, third, and fourth connecting moieties are embedded between the connecting moiety and the targeting moiety of Formula I or Formula II. As disclosed herein, there is at least one connecting moiety per targeting moiety. In some embodiments, there is more than one connecting fragment per ca.

- 31 043375 each targeting fragment. The targeting ligands disclosed herein are intended to cover all such compositions.

In some embodiments, additional groups may be embedded between the connecting moiety and the branch point group of Formula I or Formula II.

As disclosed herein, the connecting moiety serves as a spacer that can further increase the flexibility and/or length of the bond between the targeting moiety and the branch point group, linker, and therapeutic compound. In some embodiments, the connecting moiety includes alkyl groups (including cycloalkyl groups), alkenyl groups (including cycloalkenyl groups), alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, or aralkynyl groups. In some embodiments, the connecting moiety includes one or more heteroatoms, heterocycles, heteroaryls, amino acids, nucleotides, or saccharides.

In some embodiments, the targeting ligand includes a connecting moiety having the following structure:

where n is an integer from 1 to 20 (for example, 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).

In some embodiments, the targeting ligand includes a connecting moiety having the following structure:

where X is O, S or NH (structure 302).

In some embodiments, the targeting ligand includes a connecting moiety having the following structure:

(Structure 302a).

In some embodiments, the targeting ligand includes a connecting moiety having the following structure:

where n is an integer from 1 to 20 (for example, 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).

In some embodiments, the targeting ligand includes a connecting moiety having the following structure:

ABOUT

where n is an integer from 1 to 20 (for example, 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).

In some embodiments, the targeting ligand includes a connecting moiety having the following structure:

where X is O, S or NH (structure 305).

In some embodiments, the targeting ligand includes a connecting moiety having the following structure:

where X is O, S or NH (structure 306).

In some embodiments, the targeting ligand includes more than one type of connecting moieties. In some embodiments, the connecting moiety acts as a flexible hydrophilic spacer (see, for example, US Pat. No. 5,885,968; and Biessen et al. J. Med. Chem. 1995, 39, 15381546, both of which are incorporated herein by reference in their entirety) and includes PEG-32 043375 spacer. In other embodiments, the PEG spacer contains from 1 to 20 ethylene units (PEG1 to _PEG20 ). For example, a PEG spacer contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or ethylene units.

Targeting fragments.

The targeting ligands disclosed herein may contain from one to four, or more than four targeting moieties.

In some embodiments, the targeting ligand may be a galactose cluster. As used herein, the galactose cluster includes a targeting ligand containing two to four galactose derivatives at the end. As used herein, the term galactose derivative includes both galactose and galactose derivatives having an affinity for the asialoglycoprotein receptor equal to or greater than that of galactose. The galactose derivative is a saccharide sugar, which is a type of targeting moiety. The terminal galactose derivative is linked to the connecting moiety through the C-1 carbon of the saccharide.

In some embodiments, the targeting ligand consists of three terminal galactosamines or galactosamine derivatives (such as N-acetylgalactosamine), each of which has affinity for the asialoglycoprotein receptor. In some embodiments, the targeting ligand includes three terminal N-acetylgalactosamines (GalNAc or NAG) as targeting moieties. For example, structures 1001, 1002, 1004 and 1008 are each targeting ligands containing three terminal N-acetylgalactosamines as targeting moieties.

In some embodiments, each targeting moiety includes a galactosamine derivative that is N-acetylgalactosamine. Other saccharides having affinity for the asialoglycoprotein receptor that can be used as targeting moieties can be selected from the list including: galactose, galactosamine, Nformylgalactosamine, N-propionylgalactosamine, N-n-butanoylgalactosamine and N-isobutanoylgalactosamine. The affinities of numerous galactose derivatives for the asialoglycoprotein receptor have been studied (see, e.g., Lobst, S.T., Drickamer, K.J.B.C. 1996, 277, 6686, which is incorporated herein by reference in its entirety) or can be readily determined using methods well known and widely used in this area.

In some embodiments, the targeting moiety is a cell targeting moiety.

In some embodiments, the targeting moiety includes N-acetylgalactosamine:

OH ^-OH

In some embodiments, the targeting ligand includes three targeting moieties. In some embodiments, the targeting ligand includes four targeting moieties. In some embodiments, the targeting ligand includes a single targeting moiety. In some embodiments, the targeting ligand includes two targeting moieties. In some embodiments, the targeting ligand includes four or more targeting moieties.

In some embodiments, the targeting moiety includes one or more of galactose, galactosamine, N-formylgalactosamine, N-acetylgalactosamine, N-propionylgalactosamine, N-nbutanoylgalactosamine, or N-iso-butanoylgalactosamine.

For example, in some embodiments, the N-acetylgalactosamine targeting moieties in any of structures 1001-1027 may be replaced by alternative targeting moieties. Such alternative targeting moieties include, for example, galactose, galactosamine, Nformylgalactosamine, N-acetylgalactosamine, N-propionylgalactosamine, N-n-butanoylgalactosamine, or N-iso-butanoylgalactosamine.

Additionally, in some embodiments, the targeting moieties in structures 10011027 may be replaced, for example, by other carbohydrates; glycans; haptens, vitamins, folate; biotin, aptamers; and/or peptides such as RGD-containing peptides, insulin, EGF and/or transferrin.

In some embodiments, the targeting ligand is in the form of a N-acetylgalactosamine trimer. In some embodiments, the targeting ligand is in the form of an N-acetylgalactosamine tetramer.

Representative structures of targeting ligands and phosphoramidite compounds comprising targeting ligands.

- 33 043375

The targeting ligands disclosed herein may consist of one or more targeting moieties, tethers, branch point groups, and linkers.

The targeting ligands disclosed herein may consist of one, two, three, four, or more than four targeting moieties.

In some embodiments, the targeting ligands disclosed herein are synthesized such that they are in the form of a phosphoramidite compound. Phosphoramidites are widely used in the chemical synthesis of RNA and DNA. In some embodiments, phosphoramidite-containing targeting ligands disclosed herein are coupled to the 5' end of the sense strand of a double-stranded RNAi agent. It may be particularly useful to provide the targeting ligand in the form of a phosphoamidite if the targeting ligand is coupled to the 5' end of the expression inhibitory oligomeric compound. Without intending to be limited by theory, it is believed that providing the targeting ligand as a phosphoramidite, where the targeting ligand is linked to the 5' end of an expression inhibitory oligomeric compound, allows the targeting ligand to be bound as the last component (which reduces the cost of manufacture), but also potentially allows the targeting ligand block sense strand loading into RISC if a targeting ligand is attached to the 5' end of the sense strand of a double-stranded RNAi agent. If the expression inhibitory oligomeric compound is a double-stranded RNAi agent, the targeting ligand can be provided in the form of a phosphoramidite compound, wherein the targeting ligand is coupled to the 5' end of the sense strand of the RNAi agent.

In some embodiments, the targeting ligand has the structure shown below:

° (Structure 1001).

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

where R includes or consists of an expression inhibitory oligomeric compound (structure 1001a).

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

- 34 043375

where R consists of or includes an expression inhibitory oligomeric compound; Y represents O or S; and Y' is O-, S' or NH- (structure 1001a(i)).

In some embodiments, the binding ligand is a phosphoramidite-containing compound having the structure shown below:

In some embodiments, the targeting ligand has the structure shown below:

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

where R includes or consists of an expression inhibitory oligomeric compound (structure 1002a).

- 35 043375

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

where R consists of or includes an expression inhibitory oligomeric compound; Y represents O or S; and Y' is O-, S- or NH- (structure 1002a(i)).

In some embodiments, the binding ligand is a phosphoramidite-containing compound having the structure shown below:

In some embodiments, the targeting ligand has the structure shown below:

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

where R includes or consists of an expression inhibitory oligomeric compound (structure 1003a).

In some embodiments, the expression inhibitory oligomeric compound is associated with

- 36 043375 targeting ligand and has the structure shown below: it

where R consists of or includes an expression inhibitory oligomeric compound; Y represents O or S; and Y' is O ^- , S ^- or NH ^- (structure 1003a(i)).

In some embodiments, the binding ligand is a phosphoramidite-containing compound having the structure shown below:

In some embodiments, the targeting ligand has the structure shown below:

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

where R includes or consists of an expression inhibitory oligomeric compound (structure

- 37 043375

1004a).

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

where R consists of or includes an expression inhibitory oligomeric compound; Y represents O or S; and Y' is O-, S' or NH- (structure 1004a(i)).

In some embodiments, the binding ligand is a phosphoramidite-containing compound having the structure shown below:

In some embodiments, the targeting ligand has the structure shown below:

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

where R includes or consists of an expression inhibitory oligomeric compound (structure 1005a).

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

- 38 043375

where R consists of or includes an expression inhibitory oligomeric compound; Y represents O or S; and Y' is O-, S' or NH- (structure 1005a(i)).

In some embodiments, the binding ligand is a phosphoramidite-containing compound having the structure shown below:

In some embodiments, the targeting ligand has the structure shown below:

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

where R includes or consists of an expression inhibitory oligomeric compound (structure 1006a).

In some embodiments, the expression inhibitory oligomeric compound is associated with

- 39 043375 targeting ligand and has the structure shown below:

where R consists of or includes an expression inhibitory oligomeric compound; Y represents O or S; and Y' is O-, S- or NH- (structure 1006a(i)).

In some embodiments, the binding ligand is a phosphoramidite-containing compound having the structure shown below:

(Structure

1006b).

In some embodiments, the targeting ligand has the structure shown below:

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

- 40 043375

where R includes or consists of an expression inhibitory oligomeric compound (structure 1007a).

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

where R consists of or includes an expression inhibitory oligomeric compound; Y represents O or S; and Y' is O ^- , S ^- or NH ^- (structure 1007a(i)).

In some embodiments, the binding ligand is a phosphoramidite-containing compound having the structure shown below:

(Structure 1007b).

In some embodiments, the targeting ligand has the structure shown below:

- 41 043375

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

where R includes or consists of an expression inhibitory oligomeric compound (structure 1008a).

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

where R consists of or includes an expression inhibitory oligomeric compound; Y represents O or S; and Y' is O ^- , S ^- or NH ^- (structure 1008a(i)).

In some embodiments, the binding ligand is a phosphoramidite-containing compound having the structure shown below:

- 42 043375

In some embodiments, the targeting ligand has the structure shown below:

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

where R includes or consists of an expression inhibitory oligomeric compound (structure 1009a).

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

where R consists of or includes an expression inhibitory oligomeric compound; Y represents O or S; and Y' is O-, S' or NH- (structure 1009a(i)).

In some embodiments, the binding ligand is a phosphoramidite containing compound having the structure shown below:

- 43 043375

(Structure 1009b).

In some embodiments, the targeting ligand has the structure shown below:

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

where R includes or consists of an expression inhibitory oligomeric compound (structure 1010a).

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

- 44 043375 where R consists of or includes an expression-inhibiting oligomeric compound; Y represents O or S; and Y' is ^O- , S- or NH- (structure 1010a(i)).

In some embodiments, the binding ligand is a phosphoramidite-containing compound having the structure shown below:

In some embodiments, the expression inhibitory oligomeric compound is linked to a targeting ligand and includes the structure shown below:

where J includes or consists of one or more substituted or unsubstituted cycloalkyl, cycloalkenyl, aryl, heteroaryl or heterocyclyl groups or covalently linked combinations thereof; Y represents O or S; R consists of or includes an expression inhibitory oligomeric compound; and Y' is O ^- , S' or NH - (structure 1011).

In some embodiments, the targeting ligand has the structure shown below:

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

- 45 043375

where R includes or consists of an expression inhibitory oligomeric compound (structure 1012a).

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

where R consists of or includes an expression inhibitory oligomeric compound; Y represents O or S; and Y' is O ^- , S ^- or NH ^- (structure 1012a(i)).

In some embodiments, the binding ligand is a phosphoramidite-containing compound having the structure shown below:

- 46 043375

In some embodiments, the targeting ligand has the structure shown below:

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

- 47 043375

where R includes or consists of an expression inhibitory oligomeric compound (structure 1013a).

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

where R consists of or includes an expression inhibitory oligomeric compound; Y represents O or S; and Y' is O ^- , S ^- or NH ^- (structure 1013a(i)).

In some embodiments, the binding ligand is a phosphoramidite-containing compound having the structure shown below:

- 48 043375

In some embodiments, the targeting ligand has the structure shown below:

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

where R includes or consists of an expression inhibitory oligomeric compound (structure 1014a).

- 49 043375

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

where R consists of or includes an expression inhibitory oligomeric compound; Y represents O or S; and Y' is O-, S- or NH- (structure 1014a(i)).

In some embodiments, the binding ligand is a phosphoramidite-containing compound having the structure shown below:

(Structure 1014b).

In some embodiments, the targeting ligand has the structure shown below:

- 50 043375

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

where R includes or consists of an expression inhibitory oligomeric compound (structure 1015a).

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

- 51 043375

where R consists of or includes an expression inhibitory oligomeric compound; Y represents O or S; and Y' is O ^- , S ^- or NH ^- (structure 1015a(i)).

In some embodiments, the binding ligand is a phosphoramidite-containing compound having the structure shown below:

(Structure 1015b).

In some embodiments, the targeting ligand has the structure shown below:

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

- 52 043375

where R includes or consists of an expression inhibitory oligomeric compound (structure 1016a).

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

where R consists of or includes an expression inhibitory oligomeric compound; Y represents O or S; and Y' is O ^- , S ^- or NH ^- (structure 1016a(i)).

In some embodiments, the binding ligand is a phosphoramidite-containing compound having the structure shown below:

O

ABOUT

Ν (Structure 1016b).

In some embodiments, the targeting ligand has the structure shown below:

- 53 043375

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

where R includes or consists of an expression inhibitory oligomeric compound (structure 1017a).

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

where R consists of or includes an expression inhibitory oligomeric compound; Y represents O or S; and Y' is O', S- or NH- (structure 1017a(i)).

In some embodiments, the binding ligand is a phosphoramiditO containing compound having the structure set forth below: O GE

N (Structure 1017b).

In some embodiments, the targeting ligand has the structure shown below:

- 54 043375 but oh

(Structure 1018).

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

where R includes or consists of an expression inhibitory oligomeric compound (structure 1018a).

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

where R consists of or includes an expression inhibitory oligomeric compound; Y represents O or S; and Y' is O-, S' or NH- (structure 1018a(i)).

In some embodiments, the binding ligand is a phosphoramidite-containing compound having the structure shown below:

- 55 043375

1018b).

In some embodiments, the targeting ligand has the structure shown below:

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

where R includes or consists of an expression inhibitory oligomeric compound (structure 1019a).

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

- 56 043375

where R consists of or includes an expression inhibitory oligomeric compound; Y represents O or S; and Y' is O- ^, S-, or NH- (structure 1019a(i)).

In some embodiments, the binding ligand is a phosphoramidite-containing compound having the structure shown below:

In some embodiments, the targeting ligand has the structure shown below:

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

- 57 043375

where R includes or consists of an expression inhibitory oligomeric compound (structure 1020a).

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

where R consists of or includes an expression inhibitory oligomeric compound; Y represents O or S; and Y' is O-, S- or NH- (structure 1020a(i)).

In some embodiments, the binding ligand is a phosphoramidite-containing compound having the structure shown below:

O

N (Structure

1020b).

In some embodiments, the targeting ligand has the structure shown below:

- 58 043375

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

where R includes or consists of an expression inhibitory oligomeric compound (structure 1021a).

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

where R consists of or includes an expression inhibitory oligomeric compound; Y represents O or S; and Y' is O ^- , S ^- or NH ^- (structure 1021a(i)).

In some embodiments, the binding ligand is a phosphoramidite-containing compound having the structure shown below:

- 59 043375

In some embodiments, the targeting ligand has the structure shown below:

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

where R includes or consists of an expression inhibitory oligomeric compound (structure 1022a).

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

- 60 043375

where R consists of or includes an expression inhibitory oligomeric compound; Y represents O or S; and Y' is O ^- , S ^- or NH ^- (structure 1022a(i)).

In some embodiments, the binding ligand is a phosphoramidite-containing compound having the structure shown below:

(Structure 1022b).

In some embodiments, the targeting ligand has the structure shown below: it

(Structure 1023).

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

- 61 043375

where R includes or consists of an expression inhibitory oligomeric compound (structure 1023a).

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

where R consists of or includes an expression inhibitory oligomeric compound; Y represents O or S; and Y' is O ^- , S ^- or NH ^- (structure 1023a(i)).

In some embodiments, the binding ligand is a phosphoramidite-containing compound having the structure shown below:

In some embodiments disclosed herein, the targeting ligand linker may be absent as long as the branch point group includes at least one aryl, cycloalkyl, and/or heterocyclic group. When present, one or more aryl, cycloalkyl, and/or heterocyclyl groups located within the branch point group serve as linker replacement groups. In some embodiments, one or more aryl, cycloalkyl, and/or heterocyclic groups within the branch point group are located between the central junction point(s) of the branch point group and the expression inhibitory oligomeric compound.

In some embodiments, the targeting ligand has the structure shown below:

- 62 043375

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

where R includes or consists of an expression inhibitory oligomeric compound (structure 1024a).

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

where R consists of or includes an expression inhibitory oligomeric compound; Y represents O or S; and Y' is O ^- , S ^- or NH ^- (structure 1024a(i)).

In some embodiments, the binding ligand is a phosphoramidite-containing compound having the structure shown below:

- 63 043375

(Structure 1024b).

In some embodiments, the targeting ligand has the structure shown below:

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

where R includes or consists of an expression inhibitory oligomeric compound (structure 1025a).

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

- 64 043375

where R consists of or includes an expression inhibitory oligomeric compound; Y represents O or S; and Y' is O ^- , S ^- or NH ^- (structure 1025a(i)).

In some embodiments, the binding ligand is a phosphoramidite-containing compound having the structure shown below:

In some embodiments, the targeting ligand has the structure shown below:

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

- 65 043375

where R includes or consists of an expression inhibitory oligomeric compound (structure 1026a).

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

where R consists of or includes an expression inhibitory oligomeric compound; Y represents O or S; and Y' is O ^- , S ^- or NH ^- (structure 1026a(i)).

In some embodiments, the binding ligand is a phosphoramidite-containing compound having the structure shown below:

1026b).

(Structure

In some embodiments, the targeting ligand has the structure shown below:

- 66 043375

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

where R includes or consists of an expression inhibitory oligomeric compound (structure 1027a).

In some embodiments, the expression inhibitory oligomeric compound is bound to a targeting ligand and has the structure shown below:

where R consists of or includes an expression inhibitory oligomeric compound; Y represents O or S; and Y' is O ^- , S ^- or NH ^- (structure 1027a(i)).

In some embodiments, the binding ligand is a phosphoramidite-containing compound having the structure shown below:

- 67 043375 (Structure 1027b).

In some embodiments, the targeting ligand is in the form of a galactose cluster. As used herein, the galactose cluster includes a targeting ligand containing two to four galactose derivatives at the end. As used herein, the term galactose derivative includes both galactose and galactose derivatives having an affinity for the asialoglycoprotein receptor equal to or greater than that of galactose. The galactose derivative is a saccharide sugar, which is a type of targeting moiety. The terminal galactose derivative can be linked to the connecting moiety via the C-1 carbon of the saccharide.

In some embodiments, the targeting ligand consists of three terminal galactosamines or galactosamine derivatives (such as N-acetylgalactosamine), each of which has affinity for the asialoglycoprotein receptor. In some embodiments, the targeting ligand includes three terminal N-acetylgalactosamines (GalNAc or NAG) as targeting moieties.

In some embodiments, the targeting ligand consists of four terminal galactosamines or galactosamine derivatives (such as N-acetylgalactosamine), each of which has affinity for the asialoglycoprotein receptor. In some embodiments, the targeting ligand includes four terminal N-acetylgalactosamines (GalNAc or NAG) as targeting moieties.

In some embodiments, each targeting moiety includes a galactosamine derivative that is N-acetylgalactosamine. Other saccharides having affinity for the asialoglycoprotein receptor that can be used as targeting moieties can be selected from the list including: galactose, galactosamine, N-formylgalactosamine, N-acetylgalactosamine, N-propionylgalactosamine, N-n-butanoylgalactosamine and N-iso-butanoylgalactosamine . The affinities of numerous galactose derivatives for the asialoglycoprotein receptor have been studied (see, e.g., Iobst, S.T., Drickamer, K.J.B.C. 1996, 271, 6686) or can be readily determined using methods well known and widely used in the art.

Terms commonly applied in the art to the three terminal N-acetylgalactosamines include triantennary, trivalent and trimeric (trimer).

Terms commonly used in the art to refer to the four terminal N acetylgalactosamines include tetraantennary, tetravalent and tetrameric.

Oligomeric compounds.

The targeting ligands disclosed herein may be associated with an oligomeric compound. 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. Expression inhibitory oligomeric compounds can be synthesized using methods commonly used in the art.

The expression inhibitory oligomeric compounds may include one or more modified nucleotides. A nucleotide base (or nucleobase) is a heterocyclic pyrimidine or purine compound found in all nucleic acids and includes adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U). As used herein, the term nucleotide may include a modified nucleotide or nucleotide mimetic, a region without a nitrogenous base, or a surrogate replacement fragment. As used herein, a modified nucleotide is a nucleotide, a nucleotide mimetic, a site without a nitrogenous base, or a surrogate replacement fragment other than a ribonucleotide (2'-hydroxynucleotide). In some embodiments, the modified nucleotide includes a 2'-modified nucleotide

- 68 043375 leotide (i.e. a nucleotide with a group other than a hydroxyl group at position 2' of a five-membered sugar ring). Modified nucleotides include, but are not limited to: 2'modified nucleotides, 2'-O-methylnucleotides (represented herein by the lowercase 'n' in the nucleotide sequence), 2'-deoxy-2'-fluoronucleotides (represented herein as Nf, also referred to herein as 2'-fluoronucleotide), 2'deoxynucleotides (represented herein as dN), 2'-methoxyethyl (2'-O-2methoxyethyl) nucleotides, (represented herein as NM or 2 '-MOE), 2'aminonucleotides, 2'-alkylnucleotides, 3'-3' linkage (inverted) nucleotides (represented herein as invdN, invN, invn, invX), non-natural bases including nucleotides, closed nucleotides, bridged nucleotides , peptide nucleic acids, 2',3'-seconucleotide mimetics (uncapped nucleobase analogues, referred to herein as N _una or NUNA), closed nucleotides (represented herein as N _lna or NLNA), 3'-O-methoxy ( with internucleotide linkage at position 2') nucleotide (represented herein as 3'-OMen), 2'-P-arabinonucleotides (represented herein as NfANA or Nf _ANA ), morpholinonucleotides, vinyl phosphonate deoxyribonucleotide (represented herein as vpdN) , vinylphosphonate nucleotides and nucleotides without a nucleoside base (represented herein as X or Ab). It is not necessary that all positions of a given connection be modified in the same way. Conversely, it is possible to introduce multiple modifications into a single expression-inhibitory oligomeric compound or even into a single nucleotide thereof. Expression inhibitory oligomeric compounds can be synthesized and/or modified by means known in the art. The modification of each nucleotide is independent of the modification of other nucleotides.

Modified nucleobases include synthetic and natural nucleobases such as 5-substituted pyrimidines, 6-azapyrimidines, N-2-, N-6-, and O-6-substituted purines (eg, 2-aminopropyladenine), 5-propynyluracil, 5- propynylcytosine, 5-methylcytosine (5-me-C), 5hydroxymethylcytosine, 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, 6-azouracil, 6-azocytosine, 6-azothimine, 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 (for example, 5-halo-uracils and cytosines (for example, 5-bromouracil and 5bromocytosine ), 5-trifluoromethyl uracil, 5-trifluoromethyl cytosine), 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine and 3-deazaadenine.

For the expression inhibitory oligomeric compounds described herein, any modified nucleotides may be linked by phosphate-containing or phosphate-free covalent bonds. Modified internucleoside linkages or scaffolds include, but are not limited to, the following: 5'-phosphorothioate group (referred to herein as a lowercase 's' before the nucleotide, as in sN, sn, sNf or sdN), chiral phosphorothioates, thiophosphate, phosphorodithioates, phosphotriesters , aminoalkyl phosphotriesters, methyl and other alkyl phosphonates, including 3'-alkylenephosphonates and chiral phosphonates, phosphinates, phosphoramidates, including 3'-aminophosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, morpholino bonds, boron nophosphates containing the usual 3'-5 '-linkages, 2'-5' linked analogues of boranophosphonates and boranophosphonates with inverted polarity, in which adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. In some embodiments, the modified internucleotide linkage or scaffold lacks a phosphorus atom. Modified internucleotide linkages without a phosphorus atom include, but are not limited to: short-chain alkyl or cycloalkyl sugar linkages, mixed heteroatom-sugar and alkyl or cycloalkyl linkages, or one or more short-chain heteroatom or cycloalkyl sugar linkages . In some embodiments, the modified internucleoside frameworks include, but are not limited to: siloxane frameworks, sulfide frameworks, sulfoxide frameworks, sulfone frameworks, formacetyl and thioformacetyl frameworks, methylene foracetyl and thioformacetyl frameworks, alkene-containing frameworks, sulfamate frameworks, methylene imine and methylene hydrazine frameworks , sulfonate and sulfonamide frameworks, amide frameworks, and other frameworks containing a mixture of N, O, S and CH ₂ components.

In some embodiments, the expression inhibitory oligomeric compound is a double-stranded RNAi agent and includes a sense strand and an antisense strand that are at least partially complementary (at least 70% complementary) to each other. The antisense strand contains a region having a sequence that is completely complementary (100% complementary) or at least substantially complementary (at least 85% complementary) to the sequence of the target mRNA. The length of each of the sense and antisense strands of a double-stranded RNAi agent can range from 16 to 30 nucleotides in length. Semantic and anti

- 69 043375 semantic threads can have the same length or different lengths. In some embodiments, the sense strand is about 19 nucleotides long and the sense strand is about 21 nucleotides long. In some embodiments, the sense strand is about 21 nucleotides in length and the sense strand is about 23 nucleotides in length. In other embodiments, the length of each of the sense and antisense strands is independently 17-21 nucleotides. 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 length of each of the sense and antisense strands is independently from 17 to 26 nucleotides. In some embodiments, the double-stranded RNAi agent comprises a duplex of about 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides in length. The length of the region of complete or substantially complete complementarity between the sense strand and the antisense strand is typically 15-25 (for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides) and is located 5' -end of the antisense strand or near it.

Expression inhibitory oligomeric compounds that are conjugated to the ligands disclosed herein optionally and additionally include 1, 2, 3, 4, 5, or 6 additional nucleotides (as an extension) at the 3' end, 5' end, or both. 3' and 5' ends of the main sequences. These additional nucleotides, if present, may or may not be complementary to the corresponding sequence in the target mRNA.

In some embodiments, when a double-stranded RNAi agent is conjugated to targeting ligands disclosed herein, the additional sense strand nucleotides, if present, may or may not be identical to the corresponding sequence in the target mRNA. Additional antisense strand nucleotides, if present, may be complementary or non-complementary to corresponding additional sense strand nucleotides, if present.

Double-stranded RNAi agents can be formed by combining an antisense strand with a sense strand.

In some embodiments, the targeting ligand is linked to the RNAi agent at the 3' or 5' end of either the sense or antisense strand of the RNAi agent. In some embodiments, the targeting ligand is associated with the 5' end of the sense strand. In some embodiments, the targeting ligand is associated with the 3' end of the sense strand. In some embodiments, the targeting ligand is linked to the RNAi agent by a labile, cleavable, or reversible bond. In some embodiments, the labile, cleavable, or reversible linkage is part of a cleavable moiety added between the RNAi agent and the targeting ligand.

In some embodiments, the expression inhibitory oligomeric compound is a single-stranded oligonucleotide. In some embodiments, the single-stranded oligonucleotide inhibits the expression of a target mRNA through an RNA interference mechanism. In some embodiments, single-stranded oligonucleotides reduce expression of a target nucleic acid by a mechanism other than RNA interference.

In some embodiments, the level of gene expression and/or the level of target mRNA in a subject who is administered a disclosed targeting ligand conjugated to an expression inhibitory oligomeric compound is reduced by at least about 5%, such as by 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 the subject not receiving the targeting ligand conjugate. The level of gene expression and/or the level of mRNA in a subject may decrease in a cell, group of cells and/or tissue of the subject. In some embodiments, the protein level of a subject administered a targeting ligand conjugated to an expression inhibitory oligomeric compound is reduced by at least about 5%, such as by 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 before administration of the targeting ligand conjugate or a subject not receiving the targeting ligand conjugate. A subject's protein level may decrease in a cell, group of cells, tissue, blood, and/or other fluid of the subject. Reductions in gene expression, mRNA or protein levels can be assessed by any methods known in the art. The decrease or decrease in the level of mRNA and/or protein level is collectively referred to as inhibition, reduction or decrease in the expression of the target gene.

Particular expression inhibitory oligomeric compounds that can be used with the disclosed targeting ligands are known in the art. In particular, numerous documents disclose expression inhibitory oligomeric compounds that can be conjugated to the targeting ligands disclosed herein to deliver the composition to the liver. Non-limiting examples include US Patent Application Serial No. 15/281,309 entitled Compositions and Methods for Inhibiting Gene Expression of LPA, which is incorporated herein by reference in its entirety, discloses various double-stranded expression inhibitory oligomeric compounds that target the human apolipoprotein(a) [LPA] gene (to inhibit the expression

- 70 043375 apo(a) protein, which is part of a lipoprotein (a) particle, and thus a lipoprotein (a) particle (Lp(a)) that is suitable for use with the targeting ligands disclosed herein. The apo(a) gene [LPA] is expressed in humans and non-human primates, primarily in the liver. Likewise, for example, U.S. Patent Application Serial No. 15/229,314 entitled RNAi Therapy for Hepatitis B Virus Infection, which is also incorporated herein by reference, discloses various double-stranded expression inhibitors oligomeric compounds that target the hepatitis B virus, which are suitable for use with the targeting ligands disclosed herein. Hepatitis B virus is a strictly hepatotrophic, double-stranded DNA virus and belongs to the hepadnaviruses belonging to the family Hepadnaviridae. Further, by way of further example, U.S. Patent Application Serial No. 15/229,314 entitled Compositions and Methods for Inhibiting Gene Expression of Factor XII, which is incorporated herein in its entirety by references, discloses various double-stranded expression inhibitory oligomeric compounds targeting the factor XII (or factor 12, F12) gene that are suitable for use with the targeting ligands disclosed herein. Factor XII is a serine protease expressed primarily in the liver and present in the blood. Additionally, as another example, in US patent application serial number 14/740,307 entitled Compositions and Methods for Inhibiting Gene Expression of Alpha-1 AntiTrypsin, which is included in its entirety Disclosed herein by reference are various double-stranded expression inhibitory oligomeric compounds targeting the alpha-1 antitrypsin (or AAT) gene that are suitable for use with the targeting ligands disclosed herein. AAT is a protease inhibitor belonging to the serpin superfamily, and normal AAT protein is synthesized mainly by liver hepatocytes and secreted into the blood. Further, WO 2016/01123 entitled Organic Compositions to Treat APOC3-Related Diseases, which is incorporated herein by reference in its entirety, discloses various double-stranded expression inhibitory oligomeric compounds targeting human apolipoprotein III (APOC3), which are suitable for use with the targeting ligands disclosed herein. Apolipoprotein C-III is a component of lipoproteins that is thought to inhibit the uptake of triglyceride-rich particles by the liver. Additional documents may also be found in the art that disclose various therapeutic compounds, including expression inhibitory oligomeric compounds, that may be suitable for use with the targeting ligands disclosed herein. These include, but are not limited to, compositions for which targeting to the liver would be desirable.

Pharmaceutical compositions and formulations.

The targeting ligands disclosed herein, when associated with an oligomeric compound, can be used to treat a subject (eg, human or mammal) with a disease or disorder that would benefit from administration of the compound. In some embodiments, the targeting ligands disclosed herein, linked to an expression inhibitory oligomeric compound, can be used to treat a subject (eg, a human) with a disease or disorder in which reduction or inhibition of target mRNA expression would be beneficial. The subject is administered a therapeutically effective amount of any one or more expression inhibitory oligomeric compounds, such as an RNAi agent, that is linked to a targeting ligand disclosed herein. The subject may be a human, a patient, or a human patient. The subject may be an adult, adolescent, child, or infant. The disclosed pharmaceutical compositions comprising a targeting ligand linked to an expression inhibitory oligomeric compound can be used to provide methods for the therapeutic treatment of diseases. Such methods include administering a pharmaceutical composition described herein to a human or animal.

The pharmaceutical compositions and methods disclosed herein can reduce the level of a target mRNA in a cell, group of cells, group of cells, tissue or body of a subject, including: administering to the subject a therapeutically effective amount of an expression inhibitory oligomeric compound described herein that is associated with a targeting ligand, thereby inhibiting expression of the target mRNA in the subject. In some embodiments, the subject has been previously identified as having a pathological upregulation of a target gene in the target cell or tissue.

In some embodiments, the pharmaceutical compositions include at least one expression inhibitory oligomeric compound linked to a targeting ligand. These pharmaceutical compositions are particularly useful in inhibiting the expression of target mRNA in a target cell, group of cells, tissue or organism. Pharmaceutical compositions can be used for le

- 71 043375 treatment of a subject with a disease or disorder that would benefit from reduction of target mRNA levels or inhibition of target gene expression. The pharmaceutical compositions can be used to treat a subject at risk of developing a disease or disorder that would benefit from reducing the level of a target mRNA or inhibiting the expression of a target gene. In one embodiment, the Method includes administering a composition comprising a targeting ligand described herein associated with an expression inhibitory oligomeric compound, such as an RNAi agent, to a subject being treated. In some embodiments, one or more pharmaceutically acceptable excipients (including bases, carriers, diluents, and/or delivery polymers) are added to pharmaceutical compositions comprising a targeting ligand linked to an expression inhibitory oligomeric compound, thereby producing pharmaceutical compositions. suitable for in vivo delivery to humans.

In some embodiments, the disclosed pharmaceutical compositions comprising a targeting ligand linked to an expression inhibitory oligomeric compound are used to treat or regulate clinical manifestations associated with expression of a target mRNA. In some embodiments, a therapeutically or prophylactically effective amount of one or more pharmaceutical compositions is administered to a subject in need of such treatment, prevention, or regulation. In some embodiments, administration of any of the conjugated ligands covalently linked to the oligomeric compound can be used to reduce the number, severity and/or frequency of disease symptoms in a subject.

The described pharmaceutical compositions comprising a targeting ligand linked to an expression inhibitory oligomeric compound can be used to treat at least one symptom in a subject with a disease that would benefit from reducing or inhibiting the expression of the target mRNA. In some embodiments, the subject is administered a therapeutically effective amount of one or more pharmaceutical compositions containing an expression inhibitory oligomeric compound, such as an RNAi agent, linked to a targeting ligand described herein, thereby treating the symptom. In other embodiments, a prophylactically effective amount of one or more expression inhibitory oligomeric compounds is administered to a subject, thereby preventing at least one symptom.

In some embodiments, the level of gene expression and/or the level of target mRNA in a subject administered the expression inhibitory oligomeric compound associated with the targeting is reduced by at least about 5%, such as by 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 receiving the pharmaceutical composition. The level of gene expression in a subject may decrease in a cell, group of cells, and/or tissue of the subject. In some embodiments, the level of mRNA is reduced. In other embodiments, the level of expressed protein is reduced. In some embodiments, the protein level of a subject administered an expression inhibitory oligomeric compound bound to a targeting ligand disclosed herein is reduced by at least about 5%, such as by 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 receiving the pharmaceutical composition. Reductions in expression, mRNA levels, or protein levels can be assessed by any methods known in the art. A decrease or reduction in the level of mRNA and/or protein level is generally referred to herein as inhibition, reduction or decrease in expression of the target gene.

The route of administration is the route by which the expression inhibitory oligomeric compound is brought into contact with the body. In general, methods of administering drugs and nucleic acids to treat a mammal are well known in the art and can be used to administer the compositions described herein. The expression inhibitory oligomeric compound bound to the targeting ligands described herein can be administered by any suitable route in a formulation specifically designed for a particular route. Accordingly, the pharmaceutical compositions described herein can be administered by injection, for example, intravenously, intramuscularly, intradermally, subcutaneously, intra-articularly or intraperitoneally (intraperitoneally). In some embodiments, pharmaceutical compositions are described and can be administered by inhalation.

Pharmaceutical compositions comprising an expression inhibitory oligomeric compound linked to a targeting ligand described herein can be delivered to a cell, group of cells, tissue or subject using oligonucleotide delivery techniques known in the art. In general, any suitable method for delivering a nucleic acid molecule (in vitro or in vivo) conventional in the art can be adapted for use with the compositions described herein. For example, delivery may be by local administration (e.g., direct injection, implantation, or topical administration), systemic administration, or subcutaneous, intravenous, oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal

- 72 043375 and intrathecal), intramuscular, transdermal, respiratory (in the form of an aerosol), intranasal, rectal or local (including buccal and sublingual) administration.

In some embodiments, the compositions are administered by subcutaneous or intravenous infusion or injection.

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

As used herein, a pharmaceutical composition or medicament includes a pharmacologically effective amount of at least one of the described therapeutic compounds and one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients (excipients) are substances other than the active pharmaceutical ingredient (API, therapeutic product, e.g., F12 RNAi agent) that are specifically included in a drug delivery system. Excipients do not or should not exhibit a therapeutic effect at the intended dosage. Excipients may serve to: a) improve the manufacturability of the drug delivery system during formulation, b) protect, maintain, or increase the stability, bioavailability or usability of the API in the patient, c) facilitate product identification, and/or d) improve one or more of the general safety, effectiveness or delivery of the API during storage or use. A pharmaceutically acceptable excipient may or may not be an inert substance.

Excipients include, but are not limited to: absorption enhancers, antiadhesives, antifoams, antioxidants, binders, buffers, carriers, coatings, colorants, delivery enhancers, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, bulking agents agents, fillers, flavoring agents, lubricants, humectants, lubricants, oils, polymers, preservatives, brine, salts, solvents, sugars, suspending agents, delayed release matrices, sweeteners, thickeners, tonicity regulators, bases, water repellents and wetting agents.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (for water-soluble substances) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Suitable vehicles for intravenous administration include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ, USA) or phosphate buffered saline (PBS). They must be stable under the conditions of manufacture and storage and must be protected from the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyhydric alcohol (eg, glycerin, propylene glycol and liquid polyethylene glycol) and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by the use of surfactants. In many cases, it is preferable that the composition includes isotonic agents, for example, sugars, polyhydric alcohols, such as mannitol, sorbitol, and sodium chloride. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

Sterile injection solutions can be prepared by incorporating the active compound into the required amount of a suitable diluent with one or a combination of the ingredients listed above, if necessary, followed by sterilization by filtration. Typically, dispersions are prepared by introducing the active compound into a sterile vehicle that contains the basic dispersion medium and other necessary ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the production method includes vacuum drying and freeze drying, which allows the powder of the active ingredient in combination with any additional desired ingredient to be obtained from a solution thereof previously subjected to sterile filtration.

Formulations suitable for intra-articular administration may be in the form of a sterile aqueous preparation of the drug, which may be in microcrystalline form, for example, in the form of an aqueous microcrystalline suspension. Lysosomal formulations or systems including a biodegradable polymer can also be used to prepare a drug for both intra-articular and ocular administration.

Forms suitable for topical administration, including eye treatments, include liquid or semi-liquid preparations such as liniments, lotions, gels, application forms, oil-in-water and water-in-oil emulsions such as creams, ointments or pastes; or solutions or suspensions, such as drops. Formulations for topical application to the surface of the skin can be prepared by dispersing the drug in a dermatologically acceptable carrier such as lotion, cream, ointment or soap. Suitable carriers are capable of forming a film or layer on the skin that allows localized application and reduces movement. For topical administration to internal tissue surfaces, the agent may be dispersed in a liquid tissue adhesive or other substance known to enhance adsorption to the tissue surface. For example, the use of hydroxypropylcellulose or fibrinogen/thrombin solutions may be useful. Alternatively, fabric coatings can be used in coatings, such as pectin-containing formulations.

For inhalation treatments, powder inhalation (self-propelling or spray formulations) can be used and can be dosed using an aerosol can, nebulizer or atomizer. Such formulations may be presented in fine powder form for pulmonary administration from a powder inhalation device or in self-propelling forms for powder administration. In the case of self-propelling solutions or spray forms. In the case of self-propelling solutions or spray forms, the desired effect can be achieved either by selecting a valve with the desired spray characteristics (i.e., capable of dispensing a spray of the desired particle size) or by introducing the active ingredient as a suspended powder into controlled particle sizes. For administration by inhalation, the compounds may also be administered in the form of an aerosol spray from a container or dispenser that contains a suitable propellant, for example a gas such as carbon dioxide, or a nebulizer.

Systemic administration may also be via mucosal or transdermal routes. For mucosal or transdermal administration, penetrants suitable for the barrier to be crossed are used in the form. Such penetrants are well known in the art and include, for example, mucous membrane penetrants, detergents and bile salts. Administration through mucous membranes can be accomplished using nasal sprays or suppositories. For transdermal administration, the active compounds are typically formulated into ointments, ointments, gels or creams, as is widely known in the art.

The active compounds can be combined with carriers that will protect the compound from rapid elimination from the body, such as sustained release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used. Methods for preparing such compositions will be apparent to those skilled in the art. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. They can be prepared in accordance with methods known to those skilled in the art, for example, as described in US Pat. No. 4,522,811.

Compositions for oral or parenteral use may be in dosage form for ease of administration and uniformity of dosage. Dosage form refers to physically discrete units that can be used as dosage units for a subject being treated; each unit contains a predetermined amount of active compound calculated to provide the desired therapeutic effect in combination with the required pharmaceutical carrier. The properties of the dosage forms of the present invention are dictated by and directly depend on the unique characteristics of the active compound and the therapeutic effect intended to be obtained, and the inherent limitations associated with combining such an active compound with other components for the treatment of individuals. In addition, administration may be accomplished by intermittent bolus injections, or may be more continuous using intravenous, intramuscular, or intraperitoneal administration of their external reservoir (eg, intravenous bag).

In combination with the methods of the present disclosure, pharmacogenomics (ie, the study of the relationship between an individual's genotype and the individual's response to a foreign compound or drug) can be considered. Differences in the metabolism of therapeutic agents can lead to severe toxic effects or treatment failure by altering the relationship between dose and plasma concentration of the pharmacologically active drug. Accordingly, the attending physician or clinician may consider using information obtained from appropriate pharmacogenomic studies to determine whether to administer a drug and to tailor the dosage and/or therapeutic regimen of the drug.

Pharmaceutical composition other additional components typically present in pharmaceutical compositions. Such additional components include, but are not limited to: antipruritics, astringents, local anesthetics or anti-inflammatory agents (eg, antihistamine, diphenhydramine, etc.). It is also contemplated that cells, tissues, or isolated organs that express or contain RNAi agents as defined herein can be used as pharmaceutical compositions. As used herein, a pharmacologically effective amount, a therapeutically effective amount, or

- 74 043375 simply an effective amount refers to that amount of an RNAi agent that provides the intended pharmacological, therapeutic or prophylactic result.

Typically, an effective amount of the active compound will range from about 0.1 to about 100 mg/kg body weight/day, for example from about 1.0 to about 50 mg/kg body weight/day. In some embodiments, the effective amount of the active compound will range from about 0.25 to about 5 mg/kg body weight per dose. In some embodiments, the effective amount of the active ingredient will range from about 0.5 to about 3 mg/kg body weight per dose. The amount administered will also likely depend on variables such as the general health of the patient, the relative biological effectiveness of the compound being delivered, the type of drug formulation, the presence and type of excipients in the formulation, and the route of administration. In addition, it should be understood that the initial dosage administered may be increased above an upper limit to rapidly achieve the target blood level or tissue level, or the initial dosage may be less than the optimal value.

To treat a disease or prepare a drug or composition for treating a disease, the pharmaceutical compositions described herein comprising an expression inhibitory oligomeric compound, such as an RNAi agent, linked to a targeting ligand, can be combined with an excipient or with a second therapeutic agent or treatment, including, but not limited to, a second or other expression inhibitory oligomeric compound, small molecule drug, antibody, antibody fragment, and/or vaccine.

The disclosed targeting ligands coupled to expression inhibitory oligomeric compounds and added to pharmaceutically acceptable excipients or adjuvants can be packaged in kits, containers, packages or dosing devices. The pharmaceutical compositions described herein may be packaged in pre-filled syringes or ampoules.

Below, the embodiments proposed above are described by way of non-limiting examples.

Examples

The following examples are not limiting and are intended to illustrate some of the embodiments disclosed herein.

Some of the abbreviations used in describing the experimental details of the synthesis of the example compounds described below are defined below: h = hour(s); min = minute(s); mole = mol(moles); mmol = millimole (millimoles); M = molar; µM = micromolar; g = gram(grams); mcg = microgram (micrograms); rt or RT = room temperature; l = liter(s); ml = milliliter (milliliters); wt. = mass; Et ₂ O = diethyl ether; THF = tetrahydrofuran; DMSO = dimethyl sulfoxide; EtOAc = ethyl acetate; Et ₃ N or TEA = triethylamine; i-Pr ₂ NEt, or DIPEA, or DIEA = diisopropylethylamine; CH ₂ Cl ₂ or DCM = methylene chloride; CHC13 = chloroform; CDCl ₃ = deuterated chloroform chloroform; CCl ₄ = carbon chloride; MeOH = methanol; EtOH = ethanol; DMF = dimethylformamide; BOC = tert-butoxycarbonyl; CBZ = benzyloxycarbonyl; TBS = t-butyldimethylsilyl; TBSCl = t-butyldimethylsilyl chloride; TFA = trifluoroacetic acid; DMAP = 4dimethylaminopyridine; NaN ₃ = sodium azide; Na ₂ SO ₄ = sodium sulfate; NaHCO ₃ = sodium bicarbonate; NaOH = sodium hydroxide; MgSO ₄ = magnesium sulfate; K2CO3 = potassium carbonate; KOH = potassium hydroxide; NH4OH = ammonium hydroxide; NH ₄ Cl = ammonium chloride; SiO ₂ = silicon oxide; Pd-C = palladium on carbon; HCl = hydrogen chloride or hydrochloric acid; NMM = N-methylmorpholine; H ₂ = hydrogen gas; KF = potassium fluoride; EDC-HCl = N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride; MTBE = methyl tert-butyl ether; MeOH = methanol; Ar = argon; RT = retention time.

Additionally, various tables in the Examples section below provide examples of expression inhibitory oligomeric compounds suitable for use with the targeting ligands disclosed herein. To designate modified nucleotides for the sequences shown in the tables below, the following designations are used:

N = 2'-OH (unmodified) ribonucleotide (capital letter without f or d);

n = 2'-OMe-modified nucleotide;

Nf = 2'-fluoro-modified nucleotide;

dN = 2'-deoxynucleotides;

N _una = 2',3'-seconucleotide mimetics (uncapped nucleoside analogues);

N _Lna = closed nucleotide;

Nf _ANA = 2'-F-arabinone nucleotide;

NM = 2'-methoxyethyl nucleotide;

X or Ab = ribose without nucleoside base;

R = ribitol;

(invdN) = inverted deoxyribonucleotide (3'-3'-linked nucleotide);

- 75 043375 (invAb) = inverted nucleotide without nucleoside base;

(invX) = inverted nucleotide without nucleoside base;

(invn) = inverted 2'-OMe nucleotide;

s = phosphorothioate-linked nucleotide;

vpdN = vinylphosphonate deoxyribonucleotide;

(3'OMen) = 3'-OMe nucleotide;

(5Me-Nf) = 5'-Me, 2'-fluoronucleotide;

cPrp = cyclopropyl phosphonate.

The compounds disclosed herein can be prepared using chemical techniques known to those skilled in the art.

Example 1 Synthesis of targeting ligands in the form of phosphoamidite-containing compounds 1005b, 1004b and 1002b.

Phosphoroamidite-containing compounds having structure 1005b, structure 1004b and structure 1002b were synthesized according to the following procedure, with the only difference being that 4-cis-hydroxycyclohexanecarboxylic acid (compound 8 herein) was used to synthesize compound 1005b. For the synthesis of the compound of structure 1004b, 4-trans-hydroxycyclohexanecarboxylic acid (compound 8a herein) was used, and for the synthesis of the compound of structure 1002b, a mixture of 4-cis-hydroxycyclohexanecarboxylic acid (compound 8 herein) and 4-trans-hydroxycyclohexanecarboxylic acid (compound 8a herein) was used. document).

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

Basic copper carbonate (1.67 grams (g), 7.59 mmol) was slowly added to a solution of compound 1 (7.00 g, 30.34 mmol) in water (100 ml). The resulting mixture was heated to 80°C until visible dissolution. The resulting dark blue solution was cooled to 25-30°C and then treated with sodium hydroxide (1.21 g, 30.34 mmol) as a solution in water (10 ml), which resulted in the precipitation of the amino acid-copper complex. The suspension was stirred for 1 h at ambient temperature and then treated dropwise with a solution of benzyl chloroformate (6.21 g, 36.41 mmol) in THF (20 ml) for 5 min. This mixture was stirred for 1-2 hours, then filtered. The crude filter residue was triturated in EtOAc and filtered again to remove water. The blue solids were then added to a flask containing 200 ml of water and treated with 10 ml of concentrated HCl. The suspension was stirred for 18 hours, then filtered and washed with water, resulting in 4.5 g of compound 2 in the form of a white solid (45% yield, apparent purity 95). R _T =5.8 min.

2) Preparation of a compound containing three acid groups (compound 3).

A suspension of compound 2 (6.00 g, 18.27 mmol) in 1.5 M NaOH (100 ml) was heated to 60°C, after which a solution was formed. This solution was then 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 hours (2=NMT 5% according to HPLC). After completion of the reaction, the solution was cooled to 10°C and 1M HCl was added until pH=1.7 was achieved. The suspension was allowed to stand for 2-3 hours and then filtered and washed with deionized water. The solids were dried under vacuum to obtain 3.01 g of triacid compound 3 (50%, apparent purity 94). RT=6.94 min.

3) Preparation of tetrafluorophenol ether (compound 4).

- 76 043375

A solution of compound 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 N-(3-dimethylaminopropyl)-N' -ethylcarbodiimide hydrochloride (4.66 g, 24.30 mmol) in portions over 5 minutes. The solution was then allowed to warm to ambient temperature over 20 min and stirred for 3 h. Once complete (apparent purity <10), the reaction mixture was washed with saturated sodium bicarbonate (20 mL) followed by brine (20 mL) and concentrated on a rotary evaporator. The resulting oil was purified by flash columns using a solvent gradient of 5-20% EtOAc/hexane to yield 2.6 g of compound 4 as a colorless oil (40%, apparent purity 94). R _T =12.99 min.

4) Preparation of aminotosylate (compound 5).

An appropriately sized pressure reactor was charged (10 volumes) with THF, followed by CbZ-protected amine (1.0 equiv, NAG-Z) and n-TsOH-H2O (1.0 equiv). The solution was degassed with nitrogen three times. 10% Pd/C (5.0 wt%) was loaded and then degassed with nitrogen three times. Degassed with nitrogen three times. Hydrogen was pumped to a pressure of 40 to 50 psi. inch. Stir at 20 to 30°C for three hours, then degass with nitrogen three times and take a sample for internal positive control (control value <0.5% NAG-Z, if not reached, stir under _H2 at 40 to 50 psi for 1 to 2 hours, after which the test was repeated). Filtered through diatomaceous earth to remove the catalyst, washed with THF (4 volumes). Concentrate the combined filtrates and wash under vacuum to approximately 2 volumes maintaining an internal temperature of <40°C. Dilute with dichloromethane (3.8 volumes) and then concentrate again to 2 volumes. Dilute again with DCM and re-concentrate, then dilute with dichloromethane (3.8 volume). Sample for Karl Fischer analysis (control value in CF analysis <0.05%, if this value is not achieved, concentration and dilution with dichloromethane must be repeated). After reaching the control value in the Karl Fischer assay, the solution was concentrated to obtain a white foamy solid. The yield without correction was 100%. It is also possible to carry out a similar reaction using trifluoroacetic acid instead of n-TsOH-H ₂ O, this reaction can also be used in this procedure instead of the one described.

5) Preparation of tri-NAG (compound 6).

Activated ester 4 (2.15 g, 2.41 mmol) and aminotosylate 5 (4.10 g, 6.75 mmol) were dissolved in dichloromethane (22 ml) and cooled to 10°C. The solution was treated dropwise with triethylamine (1.37 g, 13.54

- 77043375 mmol) for 5 min and then allowed to warm to ambient temperature for 2 h. The reaction mixture was washed with saturated sodium bicarbonate (10 ml) and then with brine (10 ml). The solution was dried with magnesium sulfate, filtered and concentrated by rotary evaporation to give a colorless oil. After treatment, HPLC showed the presence of ~10% deacylated compound. The impurity was re-acylated by stirring in pure acetic anhydride (90 ml) and triethylamine (6 ml) for 1 hour. The acetic anhydride was then removed under reduced pressure and the resulting oil was redissolved in dichloromethane and washed with aqueous sodium bicarbonate. The solution was concentrated to an oil and purified by flash chromatography using a gradient elution (2.5-25% MeOH/DCM) to provide 1.98 g of compound 6 as a white solid (47%, apparent purity 96). Rt=7.57 min; deacylated impurity =7.18 min.

6) Preparation of the amine salt (compound 7).

Protected amine 6 (1.98 g, 1.06 mmol) and p-toluenesulfonic acid monohydrate (202 mg, 1.06 mmol) were dissolved in absolute ethanol (30 ml) and placed under a nitrogen atmosphere. 5% palladium on carbon (198 mg, 0.106 mmol) was added to the flask, the flask was placed in a vacuum and refilled with hydrogen several times. After being placed under a nitrogen atmosphere, the mixture was left to stir at ambient temperature. The reaction was considered complete after 4 h or when HPLC no longer indicated the presence of starting material. The catalyst was filtered through a pad of celite and the filtrate was passed through a 0.2 μm membrane filter to remove fine particles. The solution was concentrated to dryness under reduced pressure to provide 2.01 g of compound 7 as a gray solid (100%, apparent purity 98). RT=5.82; p-toluenesulfonic acid RT=2.4 and 3.1 min.

7) Preparation of the activated linker (compound 9 and 9a).

Cis-4-hydroxy cyclohexylcarboxylic acid 8 (for the synthesis of structure 1005) (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 noted above, while cis-4-hydroxycyclohexylcarboxylic acid (compound 8) is used as a linker to obtain structure 1005, trans-4-hydroxycyclohexylcarboxylic acid (compound 8b) can be used in place of the cis isomer to obtain structure 1004b. in this case, for subsequent synthesis, one procedure is used:

EDC-HCl (6.38 g, 33.3 mmol) was added to this solution. The solution was allowed to warm to 22°C and stirred for 12 hours. The reaction was quenched with saturated aqueous NaHCO ₃ (50 ml) and the layers were separated. The organic layer was washed with brine (50 ml) and dried with Na ₂ SO ₄ . The drying agent was filtered and the solution was concentrated to a volume of approximately 20 ml, after which slow solidification occurred (seed crystals speed up the process). The solids were suspended in 5% MTBE/hexanes (50 mL) and filtered to provide 5.6 g of product 9 in 69% yield and 95% purity.

8) Linking with the linker (obtaining compound 10).

- 78 043375

NAG amine salt compound 7 (5.00 g, 2.88 mmol) and 2,3,5,6-tetrafluorophenyl-cis-4hydroxycyclohexanecarboxylate 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 allowed to warm to room temperature and stirred for 5 hours while being monitored by HPLC. The reaction was quenched with saturated aqueous NaHCO ₃ (35 ml) and the layers were separated. The organic layer was washed with brine (35 ml) and dried with Na ₂ SO ₄ . The drying agent was filtered and the solution was concentrated and purified by flash chromatography using a gradient elution (020% MeOH/DCM) which gave 3.90 g of compound 10 as a white solid (80%). R _T =6.16 min. Alternatively, direct linker attachment can be accomplished without the use of tetrafluorophenyl ether, as shown in Example 2 below.

9) Receiving connection 11.

Compound 10 (1.87 g, 1.11 mmol) was dissolved in 20 ml of dichloromethane and 2-cyanoethyl, N,N,N',N'-tetraisopropyl phosphoramidite (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 allowed to warm to room temperature and stirred for 1 hour. The degree of conversion was checked by HPLC (which indicated that ~2% of the starting material remained). Additional 2-cyanoethyl, N,N,N',N'-tetraisopropyl phosphoramidite (0.14 g, 0.46 mmol) was added and the reaction mixture was stirred for another 2.5 h (HPLC showed no significant change). The reaction was quenched with saturated aqueous NaHCO ₃ (20 ml) and the layers were separated. The organic layer was washed with aqueous NaHCO ₃ (20 ml) and brine (2x20 ml) and dried with Na ₂ SO ₄ . The drying agent was filtered and the solution was concentrated, resulting in 2.34 g of compound 11 in the form of a white solid.

100 mg of crude compound 11 was purified by flash column chromatography: first the gel-packed column was eluted with 2% triethylamine in dichloromethane for 30 min and then loaded onto the column. Crude compound 11 was purified using a gradient elution (020% (2%) triethylamine:methanol /2% triethylamine:dichloromethane). The final product, compound 11 (the chemical structure of which corresponded to structure 1005b as defined herein) was eluted in 2% triethylamine:dichloromethane (fraction 2) to yield 80 mg of a white solid.

In fig. 1 shows ^{the 1} H NMR spectrum for compound 11 (structure 1005b herein).

In fig. 1A shows the ¹ H NMR spectrum of the trans isomer of compound 11 (structure 1004b herein) obtained according to the alternative synthesis procedure of step 7 above.

Example 2. Synthesis of a phosphoramidite-containing compound with a targeting ligand of structure 1008b.

1) Preparation of tri-tert-butyl-N-[N-(benzyloxycarbonyl)-L-γ-glutamyl]-L-glutamate (compound 14)

- 79 043375

Compound 12 (10.00 g, 29.64 mmol) was added to a nitrogen-purged 250 mL three-neck round bottom flask equipped with a thermocouple, magnetic stirrer, nitrogen inlet, and powder funnel, followed by THF (100 mL, 10 vol). The resulting solution was stirred and Nmethylmorpholine (7.82 mL, 7.19 g, 71.15 mmol, 2.4 equivalents) was added (Karl Fischer test: 163 ppm).

The powder funnel was replaced with a rubber septum and the mixture was cooled using an ice bath to 0°C. Isobutyl chloroformate (iBuCOCl, 3.85 ml, 4.05 g, 29.64 mmol, 1.0 equivalents). The reaction mixture was added dropwise over 10 min using a syringe, maintaining the temperature in the vessel below 4.0°C. After addition, the mixture was stirred for another 40 minutes and the partition was replaced with a powder funnel. Compound 13 (8.767 g, 29.64 mmol, 1.0 equivalents) was added to the reaction mixture in portions over 15 min while maintaining the vessel temperature below 4.0°C (exothermic addition). After addition of compound 13, the ice bath and powder funnel were removed and the reaction mixture was allowed to warm to ambient temperature for the remaining steps. The clear, colorless solution was allowed to stand for 25 minutes after the addition of compound 13.

A sample of the reaction mixture was taken 40 min after the start of the addition of compound 13 and the percentage of conversion was examined by RP-HPLC. It was found that 23% of compound 12 remained, so after 60 min of reaction, more iBuCOCl (1.16 ml, 1.21 g, 30 mol%) was added sequentially and compound 13 (2.63 g, 30 mol%) was added. The solution was allowed to stand for an additional 60 min until HPLC indicated >99% conversion in the sample. The total reaction time was 2.5 h from the start of the first addition of compound 13.

The reaction solution was poured into a stirred solution of 0.5 M HCl( _bog .) (125 ml), cooled in an ice bath at 3°C and stirred for approximately 5 min. The neutralized reaction mixture was extracted with ethyl acetate (100 ml, 10 vol; the aqueous layer was checked for acidity to completely remove NMM), and the organic phase was washed with brine (100 ml, 10 vol), dried with Na ₂ SO ₄ , filtered through a funnel from large pore glass into a 500 ml round bottom flask and concentrated in vacuo to give a thick colorless oil. This oil was dissolved in MTBE (100 ml, 10 vol) and concentrated in vacuo to again give a thick, colorless oil.

Hexanes (100 ml, 10 vol.) were added to the stirred oil (~600 rpm). A white cloud appeared in the solution, which disappeared after stirring. The seed crystals were added and the mixture was left to stir for 40 min; During this time, crystals slowly formed.

Additional hexanes (50 ml, 5 vol.) were added over 20 min. After 40 min, the suspension was filtered using a coarse funnel, washed with 3x hexanes (~10 ml in each case) and air-dried in the funnel for 1 h, resulting in compound 14 as a fine white powder (15.64 g, 91%).

In fig. Figure 2B shows the γ-NMR spectrum of compound 14.

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

14 (72.57 g, 125.4 mmol) and formic acid (analytical grade, >95%, 1.45 L, 20 vol. equiv.) were added to a 3000 mL three-neck round bottom flask equipped with an overhead stirrer, powder funnel, thermocouple, and heating mantle. The powder funnel was replaced with a stopper with an N2 inlet, the resulting solution was heated to 45°C and stirred for 1 hour, monitoring the progress of the reaction by RP-HPLC. The reaction was considered complete when less than 2.0 area % of mono-Hbutyl esters remained.

- 80 043375

A sample of the reaction mixture was taken 60 min after the addition of formic acid, and the sample was examined by RP HPLC to determine the percentage of monoD-butyl esters remaining. Analysis showed that after 90 min 1.8% mono-t-Bu ester remained, and the reaction mixture was cooled to room temperature.

The reaction mixture was diluted with toluene and acetonitrile (1500 ml in each case), and the mixture was concentrated in vacuo. Formic acid was removed by azeotroping with a 1:1 mixture of acetonitrile:toluene (~600 ml) and twice with acetonitrile (~500 ml in each case). This material was dried under high vacuum overnight to yield a white foamed solid (54.3 g, quantitative yield, 96.8 area% at 254 nm).

In fig. 2C shows ^{the 1} H NMR spectrum for compound 15.

3) Preparation of tpu-NAG-6uc-G1u-NHZ (compound 16).

NAG-amine p-tosylate salt (5, 59.19 g, 97.6 mmol, 4.13 equiv.) and Z-bis-Glu-triacide (15, 10.01 g, 23.6 mmol, purity adjusted, 1.0) were added to a 1 L round bottom flask equiv.). The mixture was dissolved in acetonitrile (500 ml; Karl Fischer index = 1283 ppm) and concentrated in vacuo to azeotropically remove water. The residue was dissolved in fresh acetonitrile (400 ml) and transferred to a nitrogen-purged three-neck round bottom flask equipped with a stirrer and equipped with a thermocouple. Water content was measured by the Karl Fischer method (257 ppm).

TBTU (28.20 g, 87.8 mmol, 3.7 equiv.) was added to the stirred solution under nitrogen atmosphere through a powder funnel. DIPEA (34.0 mL, 25.2 g, 8.0 equiv.) was added dropwise via syringe over 20 min, maintaining the reaction temperature below 25°C (an exothermic effect corresponding to 5°C was observed during addition). The mixture was stirred for 2 hours from the start of the addition of DIPEA, monitoring by HPLC. At 78 min the analysis showed complete absorption of the starting material.

After 2 hours, the solvent was removed under vacuum. The resulting thick oil was dissolved in dichloromethane (1000 ml) and washed with 1.0 normal HCl ( _βоg .) (3x500 ml) and saturated NaHCO ₃ ( _aq .) (3x500 ml). The organic layer was dried with Na ₂ SO ₄ , filtered and concentrated in vacuo to give a whitish waxy solid (33.5 g).

Flash chromatography was carried out on an ISCO CombiFlash automated purification system using a 330 g ISCO RediSep Rf Gold silica gel column.

The crude material was loaded in the form of a solution in CHCl ₃ (~200 ml). Downward Gradient

Eluent A: CHCl ₃ ; Eluent B: MeOH was used and a total of 36 fractions were obtained (250-500 ml in each case). Fractions containing the product were concentrated to obtain 18.75 g (97.0% purity) of compound 16. From the mixed fractions, 12.2 g (78.8% purity) of compound 16 was obtained.

In fig. 2D shows the ¹ H NMR spectrum for compound 16.

4) Preparation of tpu-NAG-6uc-G1u-NH ₂ (compound 17).

Methanol (200 ml, 13 volume) was charged into a 3-mountain round-bottom flask equipped with a stirrer. Compound 16 (15.44 g, 9.02 mmol, adjusted for purity) was added to the stirred solvent, followed by additional methanol (200 mL, 13 vol) and trifluoroacetic acid (1.40 mL, 18.1 mmol, 2.0 equiv.). The mixture was stirred for approximately 10 minutes. To this mixture was added 10% Pd/C (50% wet weight, 1.547 g, 10% w/w). The headspace was purged with hydrogen gas (balloon) and the mixture was left to stir at ambient temperature for 2 hours while the progress of the reaction was monitored by RP-HPLC.

After 75 min, a sample of the reaction mixture (100 μl) was taken and mixed with a 1:1 mixture of acetonitrile:H ₂ O (900 μl) in a 1 ml syringe filter (10 mm, 0.1 μm hydrophilic polypropylene membrane). The HPLC chromatogram showed an area corresponding to greater than 96%

- 81 043375 clean, without residues of the original material. The reaction mixture was then purged with nitrogen and filtered through a pad of celite into a clean 1000 mL round bottom flask. The reaction vessel was rinsed with methanol (50 ml) and dichloromethane (50 ml), and the washouts were also filtered. The slightly cloudy filtrate was partially concentrated in vacuo. Additional washes were obtained from the celite layer in methanol (50 ml) and dichloromethane (50 ml); which were combined with the residue and filtered through a 0.2 µm hydrophilic polypropylene membrane filter into another clean 1000 ml round bottom flask. The membrane was rinsed with acetonitrile (50 ml) to azeotropically remove the toluene byproduct. The solution was concentrated in vacuo to provide 17 (14.15 g, 97.3% HPLC area based purity) as an off-white foamed solid.

In fig. 2E shows the ¹ H NMR spectrum for compound 17.

5) Preparation of tri-NAG-bis-Glu-NH linker (compound 18).

Compound 17 (93.7% purity, 20.00 g, 11.4 mmol) and dichloromethane (150 mL) were charged into a 500 mL three-neck round-bottom flask equipped with a magnetic stirrer, thermocouple, and nitrogen layer. Cis-4-hydroxycyclohexane-1-carboxylic acid (1.730 g, 12.0 mmol, 1.05 equiv.) was added to the stirred solution, followed by TBTU (2-(1H-benzotriazol-1-ul)-1,1,3,3- tetramethylamine tetrafluoroborate, 4.036 g, 12.6 mmol, 1.10 equiv.). The solution was cooled to -9°C using an ice-saline bath and DIPEA (6.97 mL, 5.17 g, 40.0 mmol, 3.5 equiv.) was added dropwise over 7 minutes, maintaining the internal temperature below -5°C. During the addition, a temperature increase of 1.7°C was observed. After addition of DIPEA was completed, the reaction mixture was stirred at -9°C for 90 min, after which HPLC analysis (Method B) showed complete absorbance of 17.

After 110 min, the reaction was quenched by adding saturated NH ₄ Cl( _{βоg_} ) (400 ml). The layers were separated and the aqueous layer was extracted with dichloromethane (2x200 ml).

The combined organic layers were washed with a 1:1 mixture of saturated NaHCO ₃ ( _{βоg_} ) and brine (400 ml), dried with Na ₂ SO ₄ , filtered and concentrated in vacuo to a volume of 125 ml. A small amount of methanol was used to ensure solubility. The resulting oil was added in a thin stream to a 3 mL round bottom flask containing MTBE (1600 mL), resulting in the formation of a white precipitate. Wastes from the flask containing the starting material, obtained using dichloromethane (~20 ml) and MTBE (~200 ml), were added to the suspension, then the mixture was allowed to stand for 1 hour, after which it was subjected to vacuum filtration using a 600-ml porous funnel glass The wet filter cake was re-diluted with MTBE (2x200 mL) in a funnel, filtered, and dried in a high-vacuum line until constant weight, resulting in crude compound 18 as a white powder (16.22 g, 86% unadjusted yield).

Crude compound 18 (17.16 g combined with the previous batch) was purified on an ISCO CombiFlash EZPrep automated purification system using a 330 g ISCO RediSep Rf Gold silica gel column. The crude material was loaded in the form of a solution in 8% MeOH/CH ₂ Cl ₂ (~160 ml). Eluent gradient A: CH ₂ Cl ₂ ; eluent B: 50% MeOH:CH ₂ Cl ₂ was used to obtain 33 fractions. Fractions containing the product were concentrated, resulting in 10.13 g (purity 98.1%, yield 59%) of compound 18. The mixed fractions were combined, resulting in another 6.52 g (purity 86.1%) of compound 18, which was further purified.

In fig. 2F shows the 1H NMR spectrum of compound 18.

6) Preparation of the targeting ligand phosphoramidite (compound 19)

- 82 043375

Compound 18 (13.0 g, 7.87 mmol) was dissolved in anhydrous dichloromethane (195 ml) and placed under nitrogen atmosphere. To this mixture were added DIPEA (4.11 mL, 23.61 mmol) and a solution of 2cyanoethyl-N,N-diisopronylchlorophosphorodiamidide (2.45 mL, 11.02 mmol) in anhydrous dichloromethane (5 mL) dropwise over 5 min. The reaction mixture was stirred at room temperature for 1 hour and monitored by HPLC (<1% starting material remained).

The reaction was quenched with saturated aqueous NaHCO ₃ (150 ml). The organic layer was separated, washed with saturated aqueous NaHCO ₃ (1x150 ml) and brine (1x150 ml), and dried with Na ₂ SO ₄ . The drying agent was filtered and the solution was concentrated and purified by flash chromatography; wherein the column was first treated with dichloromethane (+1% triethylamine) for 30 min and then the crude final product, compound 19 (which had a chemical structure corresponding to Structure 1008 herein) was loaded onto the column and purified using gradient elution (0- 20% MeOH (+1% TEA)/CH ₂ Cl ₂ (+1% TEA)) for 30 min. The result was 11.1 g of compound 19 in the form of a white solid (yield 76%, purity 96.6%).

In fig. 2 shows ^{the 31} P-NMR spectrum for compound 19. FIG. 2A shows the ¹ H NMR spectrum for compound 19. And FIG. 2, and fig. 2A correspond to the structure of compound 19 (structure 1008b herein).

Example 3. Synthesis of a phosphoramidite-containing compound with a targeting ligand of structure 1025.

1) Obtaining connection 21.

SOCl ₂ (52.5 g, 442 mmol, 32 ml, 2.00 equiv.) was added dropwise to a solution of compound 20 (40 g, 221 mmol, 1.00 eq.) in MeOH (350 ml) 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 absorption of the starting material, and LCMS (ET12452-6-P1A) showed the formation of product. This mixture was concentrated in vacuo to provide crude compound 21 (52.4 g, without purification) as a white solid.

1H NMR: (ET12452-6-р1с DMSO Bruker_B_400 MHz) δ 9.45 (s, 1H), 8.55 (br s, 3H), 7.00 (br d, J=8.0 Hz, 2H), 6.72 (d, J=8.0 Hz , 2H), 4.17 (br s, 1H), 3.67 (s, 3H), 3.01 (qd, J=14.2, 6.5 Hz, 2H).

2) Obtaining connection 22.

_TEA (68.7 g, 62.4 ml, 679

1.20 equiv.) dropwise at 0°C, the mixture was stirred at 0°C for 0.5 h, then stirred at 25°C for 16 h. TLC (petroleum ether/ESAAc = 1/1, Rf = 0.80) showed the formation new base stain and absorption of most of the original material. This mixture was concentrated then purified on a silica gel column (petroleum ether/EAE=1:1) to give compound 22 (57.4 g, 86% yield) as a white solid.

1H NMR: (ET12452-8-plg CDCl ₃ Bruker_B_400 MHz) δ 6.97 (d, J=8.5 Hz, 2H), 6.74 (br d, J=8.0 Hz, 2H), 5.65 (br s, 1H), 5.01 ( br d, J=8.0 Hz, 1H), 4.49-4.59 (m, 1H), 3.72 (s, 3H), 2.92-3.09 (m, 2H), 1.43 (s, 9H).

3) Obtaining connection 23.

Vos

MeOOS

He

BnBr, K ₂ CO ₃

Acetone

Vos

MeOOS

OBn

To a solution of compound 22 (35 g, 119 mmol, 1.00 eq.) dissolved in acetone (170 ml), K ₂ CO ₃ (21.3 g, 154 mmol, 1.30 eq.) and BnBr (24.3 g, 142 mmol, 16.9 ml, 1.20 eq.), The reaction mixture was refluxed (60°C) for 14 hours. TLC (petroleum ether/EtOAc=3/1, Rf=0.80) showed absorption of the starting material and the formation of a new spot. H ₂ O (500 ml) was added to this mixture at 5°C and stirred for 0.5 h, then filtered and washed with water (H2O, 80 ml * 3), dried under vacuum, resulting in compound 23 (43 g, yield 88%, purity 93%) in the form of a white solid.

1H NMR: (ET12452-9-p1a CDCl ₃ Bruker_B_400 MHz) δ 7.31-7.46 (m, 5H), 7.05 (d, J=8.5 Hz, 2H), 6.91 (d, J=9.0 Hz, 2H), 5.05 ( s, 2H), 4.97 (br d, J=8.0 Hz, 1H), 4.50-4.60 (m, 1H), 3.72 (s, 3H), 2.96-3.11 (m, 2H), 1.43 (s, 9H).

4) Receiving connection 24.

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HCl/EtOAc (4 M, 215 mL, 7.71 eq.) was added dropwise to a solution of compound 23 (43 g, 112 mmol, 1.00 eq.) in EtOAc (215 mL), and the mixture was stirred for 9 h at 25°C. TLC (Petroleum ether/EtOAc=3/1, Rf=0.10) showed absorption of the starting material and formation of a new product. This mixture was filtered and washed with ethyl acetate (30 ml*3), dried under vacuum, resulting in compound 24 (35 g, 97% yield, 99% purity) as a white solid.

1H NMR: (ET12452-12-p1a MeOD Varian_D_400 MHz) δ 7.40-7.45 (m, 2H), 7.34-7.39 (m, 2H), 7.297.33 (m, 1H), 7.17 (d, J=8.8 Hz , 2H), 7.00 (d, J=8.8 Hz, 2H), 5.09 (s, 2H), 4.26 (dd, J=7.3, 6.0 Hz, 1H), 3.81 (s, 3H), 3.07-3.23 (m, 2H).

5) Receiving connection 26.

Compound 24 (15.5 g, 48.2 mmol, 1.00 eq.) was dissolved in CH ₃ CN (40 ml) and NaOH (1.5 M, 70.6 ml, 2.20 eq.), then compound 25 (13.4 g, 96.3 mmol, 6.94 ml, 2.00 equiv.) at 15°C, pH check: ~2.5. Then 4N was added. NaOH to pH=13. The solution was heated at 70°C. After 30 min the pH dropped below 6 and was adjusted again with 4N. NaOH (pH 11-13). Additional compound 25 (6.69 g, 48.2 mmol, 3.47 mL, 1.00 eq) was added in portions (twice) and the pH was adjusted to 11-13 each time. This mixture was heated at 70°C for 14 hours. LCMS (ET12452-30-P1A, Rt=0.749 min) showed the formation of a product. This mixture was cooled to 15°C, then the pH was adjusted to 1 using 4N. HCl, filtered and washed with water (H2O, 80 ml * 2), dried. The residue was dissolved with tetrahydrofuran (600 ml) and then concentrated six times with batches ET12452-27, ET12452-19, then stirred with dichloromethane (500 ml) and filtered, the filter was dried, resulting in compound 26 (35.5 g, 87% yield , purity 97%) in the form of a white solid.

1H NMR: (ET12452-30-p1r MeOD Varian_D_400 MHz) δ 7.40-7.45 (m, 2H), 7.36 (t, J=7.4 Hz, 2H), 7.277.32 (m, 1H), 7.17 (d, J= 8.4 Hz, 2H), 6.91 (d, J=8.6 Hz, 2H), 5.49 (s, 1H), 5.05 (s, 2H), 3.71 (t, J=7.6 Hz, 1H), 3.61 (s, 4H) , 3.07 (dd, J=14.1, 7.5 Hz, 1H), 2.86-2.96 (m, 1H), 2.03 (s, 2H).

6) Receiving connection 27.

To a solution of compound 26 (15 g, 38.7 mmol, 1.00 eq.), compound 26A (25.7 g, 155 mmol, 4.00 eq.) in pyridine (250 ml) was added EDCl (29.7 g, 155 mmol, 4.00 eq.) at 5 °C. This mixture was stirred at 30°C for 12 hours. LCMS (ET12452-59-P1A, Rt=1.053 min) showed the predominance of the product. This mixture was concentrated, then dissolved with dichloromethane (200 ml), washed with saturated NaHCO ₃ solution (80 ml * 4), brine (80 ml ^x 2), dried with Na ₂ SO ₄ , filtered and concentrated. The residue was purified on a silica gel column (Petroleum ether/EtOAc=3:1, Rf=0.75) to give product 26A, which was dissolved with dichloromethane (200 ml), washed with saturated NaHCO ₃ solution (80 ml*4) and saline (80 ml*2), dried with Na ₂ SO ₄ , filtered and concentrated to give compound 27 (19.8 g, 61% yield) as an off-white gum.

1H NMR: (ET12452-59-p1g CDCl ₃ Bruker_B_400 MHz) δ 7.36-7.46 (m, 4H), 7.30-7.35 (m, 1H), 7.24 (d, J=8.7 Hz, 2H), 6.97-7.07 (m , 3H), 6.94 (d, J=8.7 Hz, 2H), 5.05 (s, 2H), 4.13-4.26 (m, 5H), 3.25 (d, J=7.5 Hz, 2H), 2.06 (s, 1H) , 1.25-1.29 (m, 1H).

7) Preparation of compound 27-2.

- 84 043375 compound 27-1 (230 g, 1.53 mol, 205 ml, 1.00 eq.) was dissolved in dry DCM (1.6 L) under N2. The solution was cooled to 0°C using an ice bath and TEA (232 g, 2.3 mol, 318 ml, 1.50 eq.) was added. TosCl (233 g, 1.22 mol, 0.80 eq) in DCM (500 mL) was then added to the cooled reaction mixture. After addition, the solution was allowed to warm to 20°C and stirred for 5 hours. TLC (petroleum ether/EAAc=1:1, Rf=0.15) showed absorbance of the starting material, and HPLC (ET1245215-P1L, Rt=1.71 min) showed 2 peak. The reaction mixture was neutralized by adding H2O (500 ml) at 0°C, and then the 2 reaction mixtures were extracted with dichloromethane (CH2C12, 800 ml). The combined organic layers were washed with water (H2O, 1 L) and brine (1 L), dried with Na ₂ SO ₄ , filtered and concentrated. The residue was purified by a silica gel column (petroleum ether/EtOAc=1:1) to give 27-2 (338 g, 36% yield) as a yellow oil.

1H NMR: (ET12452-15-plzl CDCl ₃ Bruker_B_400 MHz) δ 7.79 (d, J=8.0 Hz, 2H), 7.34 (d, J=8.5 Hz, 2H), 4.12-4.19 (m, 2H), 3.67- 3.72 (m, 4H), 3.60 (s, 4H), 3.55-3.58 (m, 2H), 2.44 (s, 3H), 2.32 (s, 1H).

8) Obtaining compound 27-3 A.

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 color of the reaction mixture changed to pink. This mixture was heated to 50°C and stirred for 1.5 hours. The reaction mixture was then cooled to 20°C and stirred for 14 hours. TLC (DCM/MeOH=20:1, Rf=0.6) showed absorption of the starting material. The mixture was poured into water. NaHCO ₃ (600 ml) at 0~5°C and stirred for 15 minutes. The color of the mixture changed to yellow. The mixture was extracted with dichloromethane (500 ml), washed with aqueous NaHCO ₃ (500 ml), water (500 ml*2) and brine (500 ml), dried with Na ₂ SO ₄ , filtered and concentrated to give the compound 27-3A (189 g, unpurified, 92% pure) as a brown oil.

1H NMR: (ET12452-28-plc CDCl ₃ Varian_D_400 MHz) δ 6.00 (d, J=6.6 Hz, 1H), 5.47 (t, J=3.0 Hz, 1H), 4.91 (dd, J=7.5, 3.3 Hz, 1H), 4.17-4.28 (m, 2H), 4.08-4.14 (m, 1H), 3.97-4.03 (m, 1H), 2.13 (s, 3H), 2.05-2.09 (m, 9H).

9) Obtaining a compound of 27-4 A.

TMSOTf ( 63.8 g, 287 mmol, 51.9 mL, 0.50 eq.) under N2, the mixture was stirred at 25°C for 8 hours. TLC (DCM/MeOH=20:1, Rf=0.46) showed absorbance of the starting material, and LCMS ( ET12452-35-P1A, R _t =0.76 min) showed the formation of a product. This mixture was filtered to remove sieves, then neutralized with cold aqueous NaHCO3 (1000 ml), extracted with dichloromethane (800 ml*2), the separated organic layers were washed with saturated NaHCO ₃ (800 ml), H2O (800 ml*2) and brine (800 ml), dried with Na ₂ SO ₄ , filtered and concentrated. Then purify on a silica gel column (DCM/MeOH=20:1) to give 27-4A (285 g, 73% yield) as a yellow oil.

1H NMR: (ET12452-35-plg CDCl ₃ Varian_D_400 MHz) δ 7.81 (d, J=8.4 Hz, 2H), 7.37 (d, J=8.2 Hz, 2H), 6.30 (brd, J=9.5 Hz, 1H) , 5.28-5.35 (m, 1H), 5.08 (dd, J=11.2, 3.3 Hz, 1H), 4.81 (d, J=8.6 Hz, 1H), 4.09-4.29 (m, 5H), 3.86-3.98 (m , 3H), 3.68-3.81 (m, 3H), 3.56-3.66 (m, 5H), 2.46 (s, 3H), 2.16 (s, 3H), 2.04 (s, 3H), 1.98 (s, 3H) , 1.95 (s, 3H)

10) Obtaining compound 27-4.

To a solution of compound 27-4A (285 g, 450 mmol, 1.00 eq.) in DMSO (1.4 L) was added NaN ₃ (38.1 g, 586 mmol, 1.30 eq.) at 10°C, the mixture was stirred at 60°C for 16 hours LCMS (ET12452-37-P1A, Rt=0.67 min) showed the formation of product, the starting material was absorbed. This mixture was poured into H2O (1500 ml), extracted with ethyl acetate (1 L * 5), washed with water (H2O 800 ml * 3) and brine (800 ml ^x 3), dried with Na2SO4, filtered and concentrated , resulting in compound 27-4 (168 g, without purification) as a red oil.

1H NMR: (ET12452-37-р1с CDC13 Bruker_В_400 MHz) δ 6.12 (br d, J=9.4 Hz, 1H), 5.32 (d, J=2.9 Hz,

1H), 5.06 (dd, J=11.3,3.4 Hz, 1H), 4.78 (d, J=8.7 Hz, 1H), 4.08-4.27 (m, 5H), 3.82 -3.94 (m, ZN), 3.61-3.77 (m,

10Н), 3.45-3.50 (m, 2Н), 2.16 (s, ЗН), 2.05 (d, J=1.5 Hz, 5Н), 1.99 (d, J=4.5 Hz, 6Н), 1.26 (t, J=7.2 Hz, 2H).

11) Obtaining compound 27 A.

OAc OAc

OAc OAc

27-4 27A

Pd(OH) ₂ /C (7.9 g) was added to a solution of compound 27-4 (79 g, 156 mmol, 1.00 eq.) in a mixture of EtOAc/MeOH (4:1) (640 ml), and the mixture was stirred at 15°C for 4 hours under _H2 (30 psi). TLC (DCM/MeOH=20:1) showed the absorption of the starting material, LCMS (ET12452-53-P1C, Rt=2.55 min) showed the formation of the product. The 2 reactions worked in parallel were filtered with celite and washed with dichloromethane (500 ml x 5) and MeOH (200 ml ^x 3) and concentrated to give 27A (140 g, crude) as a dark brown oil.

1H NMR: (ET12452-53-plc CDCI3 Varian_D_400 MHz) δ 7.02 (br d, J=9.3 Hz, 1H), 5.29-5.34 (m, 1H), 5.09 (dd, J=11.2, 3.3 Hz, 1H ), 4.80 (d, J=8.6 Hz, 1H), 4.09-4.24 (m, ZN), 3.82-3.95 (m, ZN), 3.52-3.70 (m, 10H),

2.91 (td, J=5.2, 2.8 Hz, 1H), 2.15 (s, ZN), 2.05 (s, 4H), 1.98 (d, J=6.4 Hz, 6H).

12) Receiving connection 28.

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). It was stirred at 30°C for 16 hours. LCMS (ET12452-64-P1A, Rt=1.21 min) showed the formation of a product. Dilute with dichloromethane (100 ml) and wash with a combination of saturated

NaHCO ₃ /saturated saline solution (1:1, 2x80 ml). The organic layer was dried with Na ₂ SO ₄ , filtered and concentrated to provide the crude product as a brown solid.

The crude product was dissolved in Ac ₂ O (42 ml), CH3CN (62.5 ml) and Ru (82.3 g, 1.04 mol, 84 ml, 23.96 eq.), the mixture was stirred at 25°C for 12 hours. HPLC (ET12452-65 -P1A, Rt=2.54 min) showed the predominance of the product. CH3CN was evaporated, then the mixture was diluted with dichloromethane (400 ml) and washed with saturated NaHCO3 solution (100 mlx4). The organic layer was separated and washed with a combination of 0.1 M HCl/brine (1:1, 100 mlx4), dried with Na ₂ SO ₄ , filtered and concentrated. The residue was purified on a silica gel column (DCM/MeOH=10:1, Rf=0.45), resulting in a product that was further purified by preparative HPLC (column: Phenomenex Gemini C18 250x50 mmx10 µm; mobile phase: [water (10 mM NH4CO3 )-ACN]; V%: 25-55%, 23 min), resulting in compound 28 (28.8 g, 58% yield, 98% purity) as a yellow solid.

1H NMR: (ET12452-65-p1j DMSO, Varian_D_400 MHz) δ 8.00-8.09 (m, ZN), 7.81 (d, J=9.0 Hz, ZN), 7.29-7.45 (m, 5H), 7.10 (d, J =8.6 Hz, 2 N), 6.89 (d, J=8.4 Hz, 2H), 5.21 (d, J=3.3 Hz, ZN), 5.04 (s, 2H), 4.97 (dd, J=11.2, Z.Z Hz, ZN), 4.54 (d, J=8.4 Hz, ZN), 4.02 (s, 9N), 3.83-3.92 (m, ZN), 3.73-3.81 (m, ZN), 3.53-3.61 (m, 4N) , 3.44-3.52 (m, 17Н), 3.42 (br d, J=4.4 Hz, 2Н), 3.35-3.40 (m, 6Н), 3.07-3.27 (m, 11Н), 2.74-2.87 (m, 2Н), 2.09 (s, 9H), 1.99 (s, 10H), 1.89 (s, 9H), 1.77 (s, 9H).

13) Receiving connection 29.

- 86 043375

Dry Pd/C (5.5 g, 5.48 mmol) was added to a solution of compound 28 (9.7 g, 5.48 mmol, 1.00 eq) in THF (250 mL), and the mixture was stirred at 40°C for 6.5 h under H2 (50 lb /sq. inch). TLC (DCM/MeOH=10:1, Rf=0.3) showed absorption of the starting material. 2 reaction mixtures, which were worked with in parallel, were filtered and washed with tetrahydrofuran (300 ml * 4) and DCM (200 ml * 3), concentrated. The residue was purified by preparative HPLC (column: Phenomenex luna C18 250*50 mm*10 µm; mobile phase: [water (0.1% TFA)-ACN]; B%: 15-45%, 20 min) with batch ET12452-78, in resulting in compound 29 (14 g, 63% yield) as a white solid.

1H NMR: (ET12452-80-plj DMSO, Varian_D_400 MHz) δ 9.19 (s, 1H), 7.99-8.10 (m, 3H), 7.83 (d,J=9.3 Hz, 3H), 6.95 (d, J=8.4 Hz, 2H), 6.62 (d, J=8.4 Hz, 2H), 5.76 (s, 2H), 5.21 (d, J=3.3 Hz, 3H), 4.97 (dd, J=11.2, 3.3 Hz, 3H), 4.54 (d, J=8.6 Hz, 3H), 4.03 (s, 9H), 3.83-3.92 (m, 3H), 3.73-3.81 (m, 3H), 3.53-3.61 (m, 4H), 3.443.52 ( m, 16H), 3.43 (br d, J=4.4 Hz, 3H), 3.36-3.39 (m, 3H), 3.26-3.33 (m, 4H), 3.05-3.24 (m, 9H), 2.65-2.82 (m , 2H), 2.10 (s, 9H), 2.00 (s, 9H), 1.89 (s, 9H), 1.77 (s, 9H).

14) Receiving connection 30.

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 (0.45M, 11.7 ml, 1.10 eq.) was added to this solution. The solution was allowed to warm to 15°C and stirred for 3.5 hours. TLC (DCM/MeOH=5:1, Rf=0.52) showed absorption of the starting material, and HPLC (ET12452-82P1A, Rt=2.69 min) showed the formation of product. Diluted with dichloromethane (50 ml), neutralized with NaHCO ₃ (30 ml), the aqueous vase was extracted with dichloromethane (30 ml * 2), the combined organic layers were washed with a saturated solution of NaHCO ₃ (30 ml * 2), H2O (30 ml) and saline solution (30 ml*2), dried with Na ₂ SO ₄ , filtered and concentrated. The residue was dissolved with dichloromethane (30 ml), then hexane (150 ml) was added dropwise at 0°C and stirred for 15 min, then cooled, the organic layer was drained and the oil was dissolved again with dichloromethane (30 ml) and hexane (150) was added dropwise ml) dropwise, this procedure was repeated 7 times, dried under vacuum, resulting in compound 30 (5.5 g, 55% yield) in the form of a white solid.

1H NMR: (ET12452-83-plb DMSO, Varian_D_400 MHz) δ 7.97-8.09 (m, 3H), 7.78 (d, J=9.3 Hz, 3H), 7.06 (d, J=8.2 Hz, 2H), 6.86 (d, J=8.2 Hz, 2H), 5.73 (s, 2H), 5.18 (d, J=3.3 Hz, 3H), 4.94 (dd, J=11.1, 3.4 Hz, 3H), 4.51 (d, J= 8.4 Hz, 3H), 3.99 (s, 9H), 3.79-3.89 (m, 4H), 3.70-3.78 (m, 4H), 3.59-3.69 (m, 2H), 3.49-3.58 (m, 4H), 3.44 (s, 16H), 3.40 (br d, J=4.2 Hz, 3H), 3.32-3.37 (m, 5H), 3.24-3.28 (m, 1H), 3.05-3.22 (m, 9H), 2.78 (br t , J=5.8 Hz, 4H), 2.07 (s, 9H), 1.96 (s, 9H), 1.86 (s, 9H), 1.74 (s, 9H), 1.15 (d, J=6.8 Hz, 6H), 1.09 (d,J=6.8 Hz, 6H).

In fig. 3 shows the 1H NMR spectrum for compound 30 (structure 1025b herein).

Example 4. Synthesis of a phosphoramidite-containing compound with a targeting ligand of structure 1014b.

1) Obtaining connection 32.

Solution of compound 31 (24.71 g, 87.85 mmol, 1.00 eq.), compound 31A, EDCI (39.07 g, 203.82

- 87 043375 mmol, 2.32 eq.), pyridine (19.39 g, 245.11 mmol, 19.79 ml, 2.79 eq.) in acetonitrile (260.00 ml) was stirred at 25°C for 2 hours. TLC (petroleum ether/ethyl acetate = 1/ 1, target product; Rf=0.7) showed the formation of the target product. This mixture was added to 300 ml EtOAc, washed with sodium hydrogen carbonate (NaHCO ₃ , 100 ml*2), 100 ml brine, dried with Na ₂ SO ₄ , filtered and concentrated to leave a residue. The crude product was purified by a silica gel column (petroleum ether/ethyl acetate=100/1~3/1) to give compound 32 (60.00 g, 79.25 mmol, 90.20% yield, 97.19% purity) as a yellow oil.

1H NMR: (ET12600-89-р1а DMSO,Varian_D_400 MHz) 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).

2) Obtaining connection 33.

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. LCMS (et12600-90-pla, MC=511) showed the formation of the desired product. The mixture was concentrated to leave a residue. The residue was washed with 1000 mL of dichloromethane to provide compound 33 (30.00 g, 54.71 mmol, 89.47% yield, 93.27% purity) as a white solid.

1H NMR: (ET12600-90-р1а DMSO Bruker_B_400 MHz) 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=5.6 Hz, 1H).

3) Obtaining connection 34.

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.), pyridine (11.60 g, 146.65 mmol, 11.84 ml, 5.00 eq.) in acetonitrile (90 ml ) EDCI (28.11 g, 146.65 mmol, 5.00 eq.) was added, then the mixture was stirred at 25°C for 1 hour. TLC (petroleum ether/ethyl acetate = 3/1) showed the formation of the desired product. This mixture was added with 500 ml of DCM, washed with sodium bicarbonate NaHCO ₃ (200 ml*2), 100 ml of brine, dried with Na ₂ SO ₄ , filtered and concentrated to leave a residue. Purify using a silica gel column (petroleum ether/ethyl acetate=4/1) to give compound 34 (28 g) as a yellow solid.

4) Obtaining connection 35.

- 88 043375

To a solution of compound 34 (16.57 g, 15.01 mmol, 1 eq.), compound 34A in DCM (140 ml) was added TEA (9.12 g, 90.08 mmol, 12.49 ml, 6.00 eq.), then the mixture was stirred at 25°C for 16 hours LCMS (et12600-98-plg) showed the formation of the target product. This mixture was poured into 200 ml DCM, washed with 100 ml sodium hydrogen carbonate (NaHCO ₃ ), 100 ml brine, dried with Na ₂ SO ₄ , filtered and concentrated to leave a residue. Purified by preparative HPLC (column: Phenomenex Gemini C18 250*50 10 μ; mobile phase: [water (10 mM NH ₄ HCO ₃ )-ACN]; B%: 15-45%, min), resulting in compound 5 ( 11 g, 4.65 mmol, yield 30.98%, purity 99.5%) as a yellow solid.

1H NMR: (ET12600-98-plal DMSO, Varian_D_400 MHz) 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).

5) Obtaining connection 36.

10% Pd(OH) ₂ /C (3.00 d), then the mixture was stirred at 20°C for 4 hours under an H2 atmosphere (50 psi). LCMS (et12600-107-p1a, Rt=2.195) indicated the formation of the desired product, the mixture was filtered and concentrated to give compound 36 (8 g, 3.60 mmol, 84.84% yield) as a yellow solid.

1H NMR: (ET12600-107-р1а DMSO, Varian_D_400 MHz) 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), 3.743.83 (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).

6) Obtaining connection 37.

- 89 043375

The batches were prepared in parallel. To a solution of compound 36 (2 g, 857.18 µmol, 1.00 eq., TFA), compound 36A (626.23 mg, 2.14 mmol, 2.50 eq.) in DCM (6 ml) was added TEA (312.26 mg, 3.09 mmol, 427.75 µl, 3.60 eq.), then the mixture was stirred at 25°C for 16 hours. LCMS showed the formation of the target product. All reaction mixtures were combined, dissolved in 200 ml DCM, poured into 30 ml NaHCO ₃ , washed with 30 ml brine, dried with Na ₂ SO ₄ , filtered and concentrated to leave a residue. Purified by preparative HPLC (column: Phenomenex Gemini C18 250*50 10 μ; mobile phase: [water (10 mM NH ₄ HCO ₃ )-ACN]; B%: 15-45%, 20 min), resulting in compound 37 (7.5 g, 3.20 mmol, 93.27% yield) as a white solid.

^1H NMR: (ET12600-111-р1а DMSO, Varian_D_400 MHz) 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=11.2 Hz, 4H).

8) Obtaining connection 38.

Combine 37 (4.4 g, 1.88 mmol, 1 eq) in DCM (26.4 mL) and 37A (1.13 g, 3.75 mmol, 1.19 mL, 2 eq). The resulting solution was cooled to 5°C. 2Htetrazole (0.45 M, 4.59 ml, 1.1 eq.) was added to this solution. The solution was allowed to warm to 20°C and stirred for 2 hours. This mixture was dissolved in 100 ml DCM, neutralized with 20 ml NaHCO ₃ , extracted with dichloromethane (50 ml x 2), washed with 20 ml NaHCO ₃ , 20 ml saline, dried with Na ₂ SO ₄ , filtered and concentrated to give a residue. The residue was dissolved with dichloromethane (25 ml, 0.2% TEA), then hexane (125 ml, 0.2% TEA) was added dropwise at 0°C and stirred for 15 min, then cooled, the organic layer was drained, the oil was dissolved with dichloromethane (30 ml) again and hexane (150 ml) was added dropwise, this procedure was repeated 3 times, and dried under vacuum. 20 mL of DCM was added to the white solid and dried under vacuum at 30°C to obtain 38 (4.8 g, 1.83 mmol, 67.34% yield, 97.15% purity) as a white solid. LCMS: [MiPr ₂ N] ⁺ /2, 1222.8.

¹ H NMR: (DMSO, Varian_400 MHz) 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 (brd, J=14.4Hz, 4H), 1.14 (d, J=6.4 Hz, 12H).

In fig. 4 shows ¹ H NMR spectrum for compound 38 (structure 1014b herein).

Example 5. Synthesis of a targeting ligand representing a forforamidite compound of structures 1006b and 1007b.

The phosphoramidite-containing compound of structure 1006b and structure 1007b were synthesized according to the same procedure, with one difference being that to synthesize the structure

1006b used 4-cis-hydroxycyclohexanecarboxylic acid (compound 8 herein), and 4-trans-hydroxycyclohexanecarboxylic acid (compound 8a herein) was used to synthesize structure 1007b.

1) Obtaining connection 41.

Solution of Z-Glu-(OtBu)-OH 39 (445 mg, 1.32 mmol), di-tert-butyliminodiacetate 40 (340 mg, 1.39 mmol), EDC (319 mg, 1.66 mmol, 1.23 equiv.) and Ru (3 equiv. ., 0.33 ml) in acetonitrile (3 ml) was stirred at room temperature. for 1 hour, diluted with ethyl acetate and washed with NaHCO3 (2x). The organic layer was dried with MgSO4 and evaporated. The crude product was then dissolved in DCM (5 ml) and TFA (5 ml) was added. This mixture was stirred at room temperature. for 16 hours and then evaporated. Ethyl acetate was added and evaporated 4x until foam/precipitate formed. Crude compound 41 was used without further treatment in the TFP (tetrafluorophenol) activation step. R _t =3.78 min, 90% purity. LCMS (electrospray, M/z): 379.0 [M+H]+.

2) Obtaining connection 42.

A solution of crude triacid 41 (~1.30 mmol), TFP (tetrafluorophenol, 7 equiv., 9.10 mmol, 1.51 g), TEA (4 equiv., 0.723 ml) and EDC (3.3 equiv., 4.29 mmol, 0.82 g) in acetonitrile (3.5 ml ) stirred at room temperature. for 1 hour, diluted with dichloromethane (250 ml) and washed with saturated NaHCO ₃ (2x100 ml). The organic phase was dried with Na ₂ SO ₄ , concentrated and poured onto a silica gel column. The resulting activated tri-tetrafluorophenol ester was eluted with ethyl acetate (AcOEt) in hexane (5-20%), resulting in 550 mg of product with trace amounts of TFP. R _t =7.06 min.

TEA (400 µl, 2.9 mmol) was added to a stirred solution containing tritetrafluorophenol ether (540 mg, 0.642 mmol) and GalNac-Peg ₃ -NH ₂ xTsOH (2.89 mmol, 1.88 g) in DCM (6 ml). It was stirred at room temperature. for 16 h, diluted with dichloromethane (200 ml) and washed with saturated NaHCO ₃ / brine (1:1, 2x150 ml). The organic layer was dried with Na ₂ SO ₄ and evaporated to give a white solid. The solid was dissolved in DCM and poured onto a silica gel column. Elution with matanol in DCM (0-10%) gave 748 mg at 95.4% purity and ~100 mg at 80% purity tri-GalNAc 42, 36% yield, after 2 steps. LCMS (electrospray, M/z): 1777.5 [M] ⁺ , R _t =4.67 min.

3) Obtaining connection 43.

10% Pd/C activated matrix (30 mg) was added to a solution of Cbz-protected amine 42 (715 mg, 0.402 mmol) and TsOH (74.5 mg, 0.402 mmol) in THF (4 ml) and TFE (4 ml). Then a nitrogen atmosphere (balloon) was created by pumping out the atmosphere and filling it with hydrogen. The mixture was stirred under a nitrogen atmosphere in

- 91 043375 for 24 hours, filtered through celite, washed with dichloromethane (2x10 ml) and evaporated, leaving alcohol C as a white solid as a residue. LCMS (electrospray, M/z): 1644.2

[M+H]+, Rt=4.67 min.

The unprotected intermediate (0.4 mmol) and 4-cishydroxycyclohexanecarboxylic acid TFP ester (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 room temperature. within 16 hours. It was then diluted with dichloromethane (100 ml), washed with a mixture of saturated NaHCO ₃ /saturated saline solution (1: 1, 100 ml x 2). The organic phase was dried using Na ₂ SO ₄ , concentrated and poured onto a column of silica gel. The product was eluted with methanol (MeOH) in DCM (2-20%), resulting in 430 mg of compound 43 with a purity of >95%, with a yield of 61%. Rt=4.20 min. LCMS: (electrospray, M/z): 1771.26 [M+H] ⁺ .

4) Obtaining connection 44.

2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidide (1.5 equiv., 110 µl, 0.343 mmol) was added at 0°C to a stirred solution of alcohol 43 (405 mg, 0.229 mmol, dried under vacuum) 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 room temperature. for 1 and additional tetrazole (0.125 ml) and 2-cyanoethylN,N,N',N'-tetraisopropylphosphorodiamidide (0.10 ml) were added. Stirring continued for 30 minutes, then the mixture was diluted with dichloromethane (200 ml) and washed with a combination of saturated NaHCO ₃ /saturated saline (1:1, 200 ml). The organic layer was dried with Na ₂ SO ₄ /MgSO ₄ , evaporated, then dissolved in anhydrous DCM and evaporated, the residue again leaving a white solid 44, 408 mg, HPLC, 92% purity, 83% yield. LCMS: (electrospray, M/z): 1870.4 [M-iPr ₂ N] ⁺ .

In fig. 5 shows the ¹ H NMR spectrum for compound 44 (structure 1007b herein).

Example 6. Synthesis of a phosphoramidite-containing compound with a targeting ligand of structure 1027b.

1) Obtaining connection 45.

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. Diluted with dichloromethane (100 ml) and washed with a combination of saturated NaHCO ₃ /saturated saline. The organic layer was dried with Na ₂ SO ₄ , filtered and concentrated to provide the crude product as a brown solid.

The crude product was dissolved in Ac ₂ O (3 ml), CH3CN (6 ml) and Ru (6 ml) and the mixture was stirred at 25°C for 16 hours. CH3CN was evaporated, then the mixture was diluted with dichloromethane and washed four times with saturated NaHCO ₃ solution. The organic layer was separated and washed with a combination of 0.1 M HCl/brine, dried with Na ₂ SO ₄ , filtered and concentrated. The residue was purified by a silica gel column (DCM/MeOH=10:1, Rf=0.45) to give product 45 (1.47 g, 68% yield, 96% purity) as a white solid.

4) Obtaining connection 46.

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To a solution of compound 45 (1.425 g, 0.871 mmol, 1.00 eq.) in THF/TFE (1:1, 5 ml) was added 10% Pd/C (24 mg) and the mixture was stirred at 40°C for 30 h in atmosphere H2. TLC (DCM/MeOH=10:1, Rf=0.3) showed absorption of the starting material. The mixture was filtered, washed with tetrahydrofuran (5 ml x 3), DCM (5 ml x 3) and concentrated. The residue was purified on a silica gel column. Elute with dichloromethane/MeOH to provide 46 (1.013 g, 75% yield, 95% purity) as a white solid. LCMS: (electrospray, M/z): 1547.5 [M+H]+.

5) Obtaining connection 47.

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 allowed to warm to 15°C and stirred for 2 hours. TLC (DCM/MeOH=5:1, Rf=0.52) showed uptake of starting material and HPLC showed formation of product. The mixture was diluted with dichloromethane (50 ml), washed with saturated NaHCO ₃ (30 ml), H2O (30 ml) and brine (30 ml), dried with Na ₂ SO ₄ , filtered and concentrated. The residue was dissolved with dichloromethane (2 ml) and added to hexane (120 ml). The white precipitate was filtered off to provide compound 47 (0.975 g, 93% purity, 82% yield) as a white solid. LCMS: (electrospray, M/z): 1747.5 [M+H] ⁺ .

In fig. 6 shows the ¹ H NMR spectrum for compound 47 (structure 1027b herein).

Example 7. Synthesis of a phosphoramidite-containing compound with a targeting ligand of structure 1026b.

1) Preparation of tri-acid 49.

Bromoacetic acid (8.17 g, 58.8 mmol) was added to a solution of 4-bromo-DL-phenylalanine hydrochloride (5.0 g, 17.8 mmol) in 1.5 M NaOH (100 ml). The solution was heated to 60°C for 1 hour, maintaining the pH above 12 by adding sodium hydroxide granules. After completion of the reaction, the reaction mixture was cooled to 15°C and the pH was adjusted to 1.75-2.00, the oil suspension was kept for 2 hours until the formation of a solid substance was observed, which could be filtered.

The solids were filtered and washed briefly with water to isolate a white solid (6.0 g, 93% yield).

2) Preparation of biaryl tri-acid 50.

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 dimethylformamide/water (168 ml) and degassed for 10 min. The solution was treated with potassium carbonate (8.0 g, 116.2 mmol) and PdCl2(dppf) (0.476 g, 0.6 mmol), the reaction vessel was placed under a nitrogen atmosphere and heated to 40°C for 5 h. After completion of the reaction, the pH was adjusted to 12 and aqueous the phase was washed with 2x (20 ml) ethyl acetate. Then the pH was adjusted to 1.75-2.00 and cooled to 15°C. Received

- 93 043375 the solids were filtered and washed with water several times to remove inorganic substances, resulting in 51 (4.8 g, 89% yield).

1) Preparation of tri-tetrafluorophenol ether 52.

A suspension of compound 51 containing three acid groups (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 (1-ethyl-3-(3dimethylaminopropyl)carbodiimide) hydrochloride (7.45 g, 38.8 mmol). The suspension was warmed to ambient temperature and stirred for 18 hours. After completion of the reaction, the reaction mixture was washed with water, the organic layer was concentrated to an oil and purified on a silica gel column to give tetrafluorophenol ester 52 (1.63 g, 16% yield).

A solution of tri-tetrafluorophenol ester and 52 (1.00 g, 1.10 mmol) NAG-amintosylate 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 allowed to warm to ambient temperature over 2 hours. After completion of the reaction, the reaction mixture was washed with water and concentrated to give an oil. The crude oil was dissolved in acetic anhydride (30 ml) and treated with a solution of 1 ml of triethylamine. After 3 h, the organics were removed under high vacuum to yield oil 54 (1.7 g, 85% yield).

3) Preparation of phenol-containing compound 55.

Benzyl-protected alcohol 54 (2.0 g, 1.08 mmol) was dissolved in ethanol (23 ml) and placed in a nitrogen atmosphere. 10% Pd/C (0.7 g, 30 mol%) was added to the solution. The suspension was stirred for 8 hours at ambient conditions and the catalyst was removed through a layer of celite. Organics were removed under high vacuum to provide white solid 55 (1.4 g, 74% yield).

4) Receiving connection 56.

A solution of phenol compound 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, and then allowed to warm to ambient temperature for 2 hours After completion of the reaction, the reaction mixture was washed with saturated sodium bicarbonate (10 ml) and then with water (10 ml), the organic layer was dried using sodium sulfate. The organics were concentrated under reduced pressure to give a white solid (1.4 g, 93% yield).

Compound 56 of Example 7 is structure 1026b herein.

Example 8. Physical properties of phosphoramidite-containing compounds containing targeting ligands.

Certain phosphoamidite compounds containing a GalNAc ligand, which optionally include the rigid linker structure disclosed herein, have been shown to gel in many common solvents. In the accompanying fig. 7 is a photograph illustrating the behavior of a GalNAc structure containing the same targeting moiety (Nacetylgalactosamine), connecting moiety, and branch point group as structure 1008b, but includes a PEG linker instead of the rigid linker of structure 1008b disclosed herein. The phosphoramidite compound containing the PEG linker-GalNAc was kept for 12 hours in a 3:1 acetonitrile:DMF mixture at 0.1 M dilution on molecular sieves. The PEG linker-GalNAc exhibits significant gelation in this highly polar solvent system. To maintain the solubility of this phosphoramidite compound containing PEG linker-GalNAc, it is necessary to use a mixture of acetonitrile:DMF with a certain ratio.

The proposed FIG. 8 is a photograph showing that the phosphoramidite compound of Structure 1008b was completely dissolved in 0.05 M acetonitrile and did not require highly polar solvents such as DMF. Unlike PEG-linkerGalNAc constructs, phosphoramidite compounds that incorporate a rigid linker according to structure 1008b are not in danger of requiring a highly polar solvent to maintain their solubility. Even though they dissolved at a lower concentration in the vial, this shows that the structures containing rigid linkers disclosed herein have better solubility in common solvents often used for oligonucleotide synthesis and do not require highly polar solvents to prevent gelation.

Example 9: Purity of Phosphoramidite-Containing Compounds Containing Targeting Ligands.

As noted above in Example 2, FIG. 2A shows the ³¹ P-NMR spectrum of the phosphoramidite compound Structure 1008b. In fig. Figure 2A shows a single peak showing the correct shift for phosphoramidite. There are no other peaks in the image, including peaks associated with hydrolysis, indicating high purity of the compound.

In fig. 9 shows ^{a 31} P NMR spectrum of a PEG linker-GalNAc structure which except includes the same branch point, connecting moiety and targeting moiety as structure 1008b. The chemical structure of phosphoramidite for which the spectrum shown in FIG. 9 is shown in FIG. 9. In FIG. 9, numerous impurity peaks are visible, which include impurities that appear to be by-products.

Example 10. Synthesis of an oligonucleotide composition.

A. Synthesis, RNAi agents were synthesized according to phosphoramidite theurdophase technology used in the synthesis of oligonucleotides. Depending on the scale, using either Mer

- 95 043375

Made96E® (Bioautomation) or MerMade12® (Bioautomation). The synthesis was carried out on a solid substrate made of glass with controlled pore size (CPG, 500 A or 600 A, obtained from

Prime Synthesis, Aston, PA, USA) All RNA and 2'-modified RNA phosphoramidites were purchased from

Thermo Fisher Scientific (Milwaukee, WI, USA). In particular, the following 2'-O-methyl phosphoramidites were used: (5'-O-dimethoxytrityl-N ⁶ -(benzoyl)-2'-O-methyl-adenosine-3'-O-(2-cyanoethyl-N,Ndiisopropylamino )phosphoramidite, 5'-O-dimethoxy-trityl-N ⁴ -(acetyl)-2'-O-methyl-cytidine-3'-O-(2cyanoethyl-N,N-diisopropylamino)phosphoramidite, (5'-O- dimethoxytrityl-N ² -(isobutyryl)-2'-Omethylguanosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite and 5'-O-dimethoxytrityl-2'-Omethyluridine-3'-O-( 2-cyanoethyl-N,N-diisopropylamino)phosphoramidite 2'-deoxy-2'-fluorophosphoramidites carried the same protecting groups as 2'-O-methyl-RNA amidites Targeting ligands containing phosphoramidites were dissolved in anhydrous dichloromethane or anhydrous acetonitrile (50 mM), and the other amidites were dissolved in anhydrous acetonitrile (50 mM) and molecular sieves (3A) were added.5-benzylthio-1H-tetrazole (BTT, 250 mM in acetonitrile) or 5-ethylthio-1H-tetrazole (ETT, 250 mM in acetonitrile) was used as the activating solution and the binding times were 10 min (RNA), 15 min (targeting ligand), 90 sec (2'OMe) and 60 sec (2'F). A 100 mM solution of 3-phenyl-1,2,4-dithiazolin-5-one (POS, obtained from PolyOrg, Inc., Leominster, MA, USA) in anhydrous acetonitrile was used to introduce phosphorothioate linkages.

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

C. Cleaning. Crude oligomers were purified by anion exchange HPLC using a TKSgel SuperQ-5PW 13μ column and a Shimadzu LC-8 system. Buffer A was 20 mM Tris, 5 mM EDTA, pH 9.0, and contained 20% acetonitrile, and buffer B was the same as buffer A but with the addition of 1.5 M sodium chloride. Residual UV was recorded at 260 nm. The appropriate fractions were pooled and then subjected to exclusion HPLC using a GE Healthcare XK 16/40 column packed with Sephadex G-25 running buffer of 100 mM ammonium bicarbonate, pH 6.7, and 20% acetonitrile.

D. Annealing. The complementary strands were mixed by combining equimolar solutions of RNA (sense and antisense) in 0.2x PBS (phosphate-buffered saline, 1x, Corning, Cellgro) to form RNAi agents. This solution was placed in a thermomixer at 70°C, heated to 95°C, kept at 95°C for 5 minutes and slowly cooled to room temperature. Some RNA agents were lyophilized and stored at -15 to -25°C. The concentration of duplexes was determined by measuring the absorbance of the solution using UV-Vis. spectrometer in 0.2x FBI. The absorbance of the solution at 260 nm was then multiplied by the conversion factor and the dilution factor, thereby determining the duplex concentration. Unless otherwise stated, the conversion factor was 0.037 mg/(ml-cm) in all cases. For some experiments, the conversion coefficient was calculated from the experimentally determined extinction coefficient.

Example 11 Comparison of 3' and 5' sense strand attachment sites for GalNAc targeting ligands using F12 expression inhibitory oligomeric compounds in wild type mice.

To evaluate differences in the attachment site of GalNAc ligands between the 3' and 5' ends of the sense strand, expression inhibitory oligomeric compounds (double-stranded RNAi agents) targeting F12 (referred to herein as F12 RNAi agents) were prepared with the sequences shown below in the table. 1.

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F12 expression inhibitory oligomeric compounds (duplex RNAi agents) from Example 11

Table 1

Duplex ID: AD02803 5' A 3' SEQ ID NO: Sequence of the semantic thread: (AM03628-SS) uAuAugscsccaagaAfaGfugaaagacca(NAG15) 1 Antisense strand sequence: (AM03157-AS) usGfsgucuuUfcAfcuuUfcuugggcsuscuAu 2 Duplex ID: AD02807 5’ -» 3’ SEQ ID NO: Sequence of the semantic thread: (AM03632-SS) (NAG18)uauaugscsccaagaAfaGfugaaagacc(invdA) 3 Antisense strand sequence: (AM03157-AS) usGfsgucuuUfcAfcuuUfcuugggcsuscuAu 4

In table 1 above the following notations are used:

Each strand of F12 RNAi agents was synthesized according to phosphoramidite theurdophase technology used in oligonucleotide synthesis using either MerMade96E® (Bioautomation) or MerMade12® (Bioautomation); complementary strands were mixed by combining equimolar solutions of RNA (sense and antisense) in 0.2x PBS (phosphate buffered saline, 1x, Corning, Cellgro) to form duplexes using methods generally described in Example 10 herein.

F12 RNAi agents bound to appropriate GalNAc ligands (namely (NAG15) or (NAG18)) were combined with a pharmaceutically acceptable subcutaneous (SC) injection buffer known in the art.

RNAi agents for F12 bound to the corresponding GalNAc ligands (namely (NAG15) or (NAG18) were delivered by SC injection. On day 1, SC injection was given into the fold of skin on the back between the shoulder blades with a 200 μg solution/20 g mouse body weight of a solution containing either saline or a 3 mg/kg (milligrams per kilogram - mpk) dose of one of two RNAi agents for F12 (AD02803 or AD02807) in buffered saline. Three (3) wild-type mice were used. per treatment group As shown above, AD02803 includes (NAG15) attached to the 3' end of the sense strand, and AD 2807 includes (NAG18) attached to the 5' end of the sense strand.

Serum samples from treated mice were collected on days 8, 15, 22 and 29 for monitoring

- 97 043375 knockdown. Knockdown was measured by quantifying circulating levels of mouse F12 protein (mF12) in serum using an in-house mF12 assay developed based on alphaLISA® (Perkin Elmer). Expression corresponding to a specific date of blood collection was normalized to the mean value corresponding to the same date for the saline control group.

In fig. 10 presents the results of this study. At trough levels (day 22), AD02803 demonstrated approximately a 70% reduction in circulating F12 levels, and AD02807 demonstrated a greater than 80% reduction. These data also indicate a difference in the duration of the knockdown effect, as at AD02803 day 29, AD02803-treated mice showed a more rapid return to basal levels compared to AD2807-treated mice. These data confirm that GalNAc ligand binding at the 5' end of the sense strand is superior to binding at the 3' end of the sense strand.

Example 12 Additional comparison of 3' and 5' sense strand attachment sites for GalNAc targeting ligands using F12 expression inhibitory oligomeric compounds in wild type mice.

To further evaluate the attachment site of GalNAc ligands at the 3' and 5' ends of the sense strand of double-stranded expression inhibitory oligomeric compounds (double-stranded RNAi agents), compositions targeting the F12 gene were prepared with the sequences presented in Table 1 below. 2.

table 2

F12 expression inhibitory oligomeric compounds (duplex RNAi agents) from Example 12

Duplex ID: AD02815 5' 3' SEQ ID NO: Sequence of the semantic thread: (AM03640-SS) (NAG20)uauaugscsccaagaAfaGfugaaagacc(invdA) 5 Antisense strand sequence: (AM03157-AS) usGfsgucuuUfcAfcuuUfcuugggcsuscuAu 6 Duplex ID: AD02816 5' A 3' SEQ ID NO: Sequence of the semantic thread: (AM03641-SS) uAuAugscsccaagaAfaGfugaaagacca(NAG20) 7 Antisense strand sequence: (AM03157-AS) usGfsgucuuUfcAfcuuUfcuugggcsuscuAu 8

In table 2 above the following notations are used: (NAG20)= he

Each strand of F12 RNAi agents was synthesized according to phosphoramidite theurdophase technology used in oligonucleotide synthesis using either MerMade96E® (Bioautomation) or MerMade12® (Bioautomation); complementary strands were mixed by combining equimolar solutions of RNA (sense and antisense) in 0.2x PBS (phosphate buffered saline, 1x, Corning, Cellgro) to form duplexes using methods generally described in Example 10 herein.

F12 RNAi agents bound to the appropriate GalNAc ligand (namely (NAG20)) were combined with a pharmaceutically acceptable subcutaneous (SC) injection buffer known in the art.

RNAi agents for F12 bound to the corresponding GalNAc ligand (i.e. (NAG20)) were delivered

- 98 043375 by subcutaneous injection. On day 1, a subcutaneous injection was given into the fold of skin on the back between the shoulder blades, injecting a 200 μg solution/20 g mouse body weight of a solution containing either saline or a 3 mg/kg (mpk) dose of one of two RNAi agents (AD02815 or AD02816) in saline. Three (3) wild-type mice per treatment group were used. As shown in the table above. 2, AD02815 includes (NAG20) attached to the 5' end of the sense strand, and AD02816 includes (NAG20) attached to the 3' end of the sense strand.

Serum samples from treated mice were collected on days 8, 15, 22, and 29 to monitor knockdown. Knockdown was measured by quantifying circulating levels of mouse F12 protein (mF12) in serum using an in-house mF12 assay developed based on alphaLISA® (Perkin Elmer). Expression corresponding to a specific date of blood collection was normalized to the mean value corresponding to the same date for the saline control group.

In fig. Figure 11 shows the results of this experiment. At trough levels (day 22), AD02816 showed approximately a 60% reduction in circulating F12 protein levels, and AD02815 showed a 79% reduction. These data also indicate differences in the duration of the knockdown effect. At day 29, AD02816-treated mice showed 40% knockdown, while AD02815-treated mice showed 71% knockdown relative to saline levels. These data confirm the binding of the GalNAc ligand at the 5' end of the sense strand.

Example 13. Lp(a) expression-inhibiting oligomeric compounds (double-stranded RNAi agents) bound to structure 1003 targeting ligands in transgenic (Tg) no Lp(a) mice.

Oligomeric compounds inhibiting the expression of Lp(a) (double-stranded RNAi agents for Lp(a)) were prepared with the sequences presented in Table 1 below. 3.

Table 3

Expression-inhibiting LP(a)oligomeric compounds (duplex RNAi agents) from Example 13

Duplex ID: AD03547 5' 3' SEQ ID NO: Sequence of semantic thread: (AM04498-SS) (NAG29)uauauaasuuaucgaGfGfcucauucucsa(invAb) 9 Antisense strand sequence: (AM04507-AS) usGfsasGfaAfuGfaGfccuCfgAfuAfausuAUAUA 10 Duplex ID: AD03549 5' 3' SEQ ID NO: Sequence of semantic thread: (AM04502-SS) (NAG25)uauauaasuuaucgaGfGfcucauucucsa(invAb) eleven Antisense strand sequence: (AM04507-AS) usGfsasGfaAfuGfaGfccuCfgAfuAfausuAUAUA 12

In table 3 above the following notations are used:

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(NAG29) has the chemical structure represented by structure 1003 herein.

Each strand of Lp(a) RNAi agents was synthesized according to phosphoramidite theurdphase technology used in oligonucleotide synthesis using either MerMade96E® (Bioautomation) or MerMade12® (Bioautomation); complementary strands were mixed by combining equimolar solutions of RNA (sense and antisense) in 0.2x PBS (phosphate buffered saline, 1x, Corning, Cellgro) to form duplexes using methods generally described in Example 10 herein.

Lp(a) transgenic (Tg) mice were used to evaluate the effectiveness of double-stranded RNAi agents with conjugated N-acetylgalactosamine ligands in vivo (Frazer KA et al. 1995, Nature Genetics 9:424-431). These YAC mice contain the complete LPA gene (encoding the apo(a) protein) with additional sequences in both the 5' and 3' directions, as well as human apoB-100, thereby producing humanized Lp(a) particles (Callow MJ et al. 1994, PNAS 91:2130-2134).

Lp(a) RNAi agents bound to appropriate GalNAc ligands (namely (NAG25) or (NAG29)) were combined with a pharmaceutically acceptable subcutaneous (SC) injection buffer known in the art.

RNAi agents for Lp(a) bound to the corresponding GalNAc ligands (namely (NAG25) or (NAG29)) at the 5′ end of the sense strand were delivered by SC injection. On day 1, a subcutaneous injection was made into the fold of skin on the back between the shoulder blades and a 200 μl solution/20 g mouse body weight of a solution containing either saline or a dose of 1 mg/kg (mpk) of the appropriate RNAi agent was injected (AD03547 or AD03549 ) in saline buffer solution. Four (4) Lp(a) Tg mice were used per treatment group.

Serum samples from treated mice were collected on days -1 (pre-dosing), 5, 11, 16, 22, 29 and 36. Knockdown was determined by calculating serum levels of circulating Lp(a) particles. Lp(a) particle levels were measured on a Cobas® Integra 400 (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-dosing expression level in that animal (in this case, the level for -1 day) to determine the expression ratio normalized to -1 day. Expression at a given time point was then normalized to the saline control group by dividing the -1 day normalized coefficient for an individual animal by the average of the -1 day normalized coefficient for all mice in the saline control group. In this way, the expression level at each time point was obtained, normalized to the expression level in the control group. The experimental error is presented as standard deviation.

The results are shown in Fig. 12. AD03549 (NAG25) showed 71% knockdown at trough values (day 16), and AD03547 (NAG29) showed 81% knockdown at trough values (day 11). Both triggers showed similar recovery curves from trough values, with less than 26% knockdown at day 36. These data confirm that the GalNAc ligands shown had both comparable initial knockdown activity and comparable knockdown duration in Lp(a) Tg mice when administered a single dose of 1 mg/kg.

Example 14. Lp(a) knockdown in Lp(a) transgenic (Tg) mice following administration of LP(a) expression-inhibiting oligomeric compounds (double-stranded RNAi agents) linked to targeting ligand structures 1002 and 1004.

Oligomeric compounds inhibiting Lp(a) expression (double-stranded Lp(a) RNAi agents) were prepared with the sequences presented in Table 1 below. 4.

Table 4

LP(a) expression inhibiting oligomeric compounds (RNAi agent duplexes) from Example 14

Duplex ID: AD03536 5' 3' SE QID NO: Sequence of semantic thread: (AM04496-SS) (NAG25)(invAb)GfcCfcCfuUfAfUfuGfuUfaUfaCfgausu(invA b) 13 Antisense strand sequence: (AM03972-AS) usCfsgsUfaUfaAfCfAfauaAfgGfgGfcusu 14 Duplex ID: AD03538 5’ -» 3’ SE QID NO: Sequence (NAG28)(invAb)GfcCfcCfuUfAfUfuGfuUfaUfaCfgausu(invA 15 ь semantic thread: (AM04499-SS) b) Antisense strand sequence: (AM03972-AS) usCfsgsUfaUfaAfCfAfauaAfgGfgGfcusu 16 Duplex ID: AD03540 5’ -» 3’ SE QID NO: Sequence of semantic thread: (AM04500-SS) (NAG30)(invAb)GfcCfcCfuUfAfUfuGfuUfaUfaCfgausu(invA b) 17 Antisense strand sequence: (AM03972-AS) usCfsgsUfaUfaAfCfAfauaAfgGfgGfcusu 18

In table 4 above the following notations are used:

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Additionally, (NAG25) is represented by the same structure as shown in Example 13 above.

(NAG28) has the chemical structure represented by structure 1002 herein (NAG30) has the chemical structure represented by structure 1004 herein (NAG28) includes a mixture of cis and trans isomers, and (NAG30) is represented exclusively by the trans isomer.

Each strand of Lp(a) RNAi agents was synthesized according to phosphoramidite theurdphase technology used in oligonucleotide synthesis using either MerMade96E® (Bioautomation) or MerMade12® (Bioautomation); complementary strands were mixed by combining equimolar solutions of RNA (sense and antisense) in 0.2x PBS (phosphate buffered saline, 1x, Corning, Cellgro) to form duplexes using methods generally described in Example 10 herein.

Lp(a) Tg mice were used to evaluate the effectiveness of double-stranded RNAi agents with N-acetylgalactosamine ligands conjugated in vivo. Apo(a) transgenic mice (Frazer KA et al. 1995, Nature Genetics 9:424-431) express human apo(a) with a YAC containing the complete LPA gene (encoding the apo(a) protein) with additional sequences as 5' , and 3' (hereinafter referred to as apo(a) Tg mice or apo(a) transgenic mice).

Lp(a) RNAi agents bound to appropriate GalNAc ligands (namely (NAG25), (NAG28) or (NAG30)) were combined with a pharmaceutically acceptable subcutaneous (SC) injection buffer known in the art.

RNAi agents for Lp(a) bound to the corresponding GalNAc ligands (namely (NAG25), (NAG28) or (NAG30)) at the 5′ end of the sense strand were delivered by SC injection. On day 1, a subcutaneous injection was made into the fold of skin on the back between the shoulder blades and a 200 μl solution/20 g mouse body weight of a solution containing either saline or a 0.5 mg/kg (mpk) dose of an RNAi agent (AD03536, AD03538 or AD03540) was injected. in saline buffer solution. Three (3) Lp(a) Tg mice were used per treatment group.

Serum samples from treated mice were collected on days -1 (pre-dosing), 8, 15, 22, 29, 36 and 43. Knockdown was determined by calculating serum levels of circulating Lp(a) particles. Lp(a) particle levels were measured on a Cobas® Integra 400 (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-dosing expression level in that animal (in this case, the -1 day level) to determine the expression ratio normalized to -1 day. Expression at a specific time point was then normalized to the saline control group by dividing the -1 day normalized coefficient for an individual animal by the average of the -1 day normalized coefficient for all mice in the saline control group. In this way, the expression level at each time point was obtained, normalized to the expression level in the control group. Experimental error is presented as standard mean error.

The results are shown in Fig. 13. The day with the lowest level was day 15 for everyone studied

- 102 043375 RNAi agents. On the trough day, AD03536 showed 74% apo(a) protein knockdown, AD03538 showed 74% apo(a) protein knockdown, and AD03540 showed 71% apo(a) protein knockdown. At day 29, all RNAi agents demonstrated >48% levels of apo(a) protein knockdown, with the exception of AD03536 (containing NAG25), which showed 16% knockdown. These data indicate that NAG-containing structures behave in terms of initial knockdown activity similar to RNAi agents containing NAG28 and NAG30 linker structures, which exhibit quantitatively greater knockdown at day 29.

Example 15. Lp Knockdown of Lp(a) in apo(a) transgenic (Tg) mice after administration of Lp(a) expression-inhibiting oligomeric compounds (double-stranded RNAi agents) bound to targeting ligand structures 1005 and 1008.

Oligomeric compounds inhibiting Lp(a) expression (double-stranded Lp(a) RNAi agents) were prepared with the sequences presented in Table 1 below. 5.

Table 5

LP(a) expression inhibiting oligomeric compounds (duplex RNAi agents) from Example 15

Duplex ID: AD03536 5' 3' SE QID NO: Sequence of semantic thread: (AM04496-SS) (NAG25)(invAb)GfcCfcCfuUfAfUfuGfuUfaUfaCfgausu(invA b) 19 Antisense strand sequence: (AM03972-AS) usCfsgsUfaUfaAfCfAfauaAfgGfgGfcusu 20 Duplex ID: AD03629 5' E 3' SE QID NO: Sequence of the semantic thread: (AM04611-SS) (NAG31)(invAb)GfcCfcCfuUfAfUfuGfuUfaUfaCfgausu(invA b) 21 Antisense strand sequence: (AM03972-AS) usCfsgsUfaUfaAfCfAfauaAfgGfgGfcusu 22 Duplex ID: AD04170 5' E 3' SE QID NO: Sequence of semantic thread: (AM05341-SS) (NAG37)(invAb)GfcCfcCfuUfAfUfuGfuUfaUfaCfgausu(invA b) 23 Antisense strand sequence: (AM03972-AS) usCfsgsUfaUfaAfCfAfauaAfgGfgGfcusu 24

In table 5 above the following notations are used:

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Additionally, (NAG25) is the same structure as shown in Example 13 above.

(NAG31) has the chemical structure represented by structure 1005 herein (NAG37) has the chemical structure represented by structure 1008 herein.

Each strand of Lp(a) RNAi agents was synthesized according to phosphoramidite theurdphase technology used in oligonucleotide synthesis using either MerMade96E® (Bioautomation) or MerMade12® (Bioautomation); complementary strands were mixed by combining equimolar solutions of RNA (sense and antisense) in 0.2x PBS (phosphate buffered saline, 1x, Corning, Cellgro) to form duplexes using methods generally described in Example 10 herein.

Lp(a) transgenic (Tg) mice were used to evaluate the in vivo efficacy of double-stranded RNAi agents with conjugated N-acetylgalactosamine ligands.

Lp(a) RNAi agents bound to appropriate GalNAc ligands (namely (NAG25), (NAG31) or (NAG37)) were combined with a pharmaceutically acceptable subcutaneous (SC) injection buffer known in the art.

RNAi agents for Lp(a) bound to the corresponding GalNAc ligands (namely, (NAG25), (NAG31), or (NAG37) were delivered by SC injection. On day 1, SC injection was given into the skin fold on back between the shoulder blades and injected 200 μl of a solution/20 g of mouse body weight of a solution containing either saline or a 3 mg/kg (mpk) dose of RNAi agent (AD03536, AD03629 or AD04170) in saline buffer solution. Four (4) Lp were used. (a) Tg mice per treatment group.

Serum samples from treated mice were collected on days -1 (pre-dosing), 8, 15, 22, 29, 36 and 43. Knockdown was determined by calculating serum levels of circulating Lp(a) particles. Lp(a) particle levels were measured on a Cobas® Integra 400 (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-dosing expression level in that animal (in this case, the -1 day level) to determine the expression ratio normalized to -1 day. Expression at a given time point was then normalized to the saline control group by dividing the -1 day normalized coefficient for an individual animal by the average of the -1 day normalized coefficient for all mice in the saline control group. In this way, the expression level at each time point was obtained, normalized to the expression level in the control group. The experimental error is presented as the standard mean error.

The obtained data are presented in Fig. 14. AD03536 demonstrated 95% knockdown of Lp(a) levels on trough day (day 15) and maintained 76% knockdown on day 36. AD03629 demonstrated 97% knockdown of Lp(a) levels on trough day (day 8) and maintained 90 % knockdown on day 36. AD04170 demonstrated 97% knockdown of Lp(a) levels on trough day (day 8) and maintained 78% knockdown on day 36.

Example 16 F12 knockdown following administration of F12 expression-inhibiting oligomeric compounds (double-stranded RNAi agents) bound to targeting ligands Structures 1005, 1008, 1025 and 1027 in wild-type mice.

Oligomeric compounds inhibiting F12 expression (double-stranded F12 RNAi agents) were prepared and conjugated at the 5' end with a phosphorothioate bond to GalNAc-targeting ligands (NAG25)s [AD04162]; (NAG37)s [AD04623]; (NAG31)s [AD04512]; (NAG33)s [AD04650] or (NAG38)s [AD04651]. Each of the double-stranded RNAi agents targeted F12.

The following notations were used for the structures of GalNAc-targeting ligands:

- 105 043375

(NAG31)s has the chemical structure represented by structure 1005 herein (NAG33)s has the chemical structure represented by structure 1025 herein (NAG37)s has the chemical structure represented by structure 1008 herein (NAG38)s has the chemical structure represented by structure 1027 in this document. The sequences and modification patterns were identical for AD04162, AD04623, AD04512, AD04650, and AD04651, with the only difference being the structure of the GalNAc-targeting ligand located at the 5' end of the sense strand of each F12 RNAi agent, as shown below.

- 106 043375

Each strand of F12 RNAi agents was synthesized according to phosphoramidite theurdophase technology used in oligonucleotide synthesis using either MerMade96E® (Bioautomation) or MerMade12® (Bioautomation); complementary strands were mixed by combining equimolar solutions of RNA (sense and antisense) in 0.2x PBS (phosphate buffered saline, 1x, Corning, Cellgro) to form duplexes using methods generally described in Example 10 herein.

F12 RNAi agents conjugated to example GalNAc-targeting ligands (i.e., (NAG25)s, (NAG31)s, (NAG33)s, (NAG37)s, or (NAG38)s) were combined with a pharmaceutically acceptable subcutaneous buffer ( SC) injections known in the art.

F12 RNAi agents bound to appropriate GalNAc ligands (namely, (NAG25)s, (NAG31)s, (NAG33)s, (NAG37)s, or (NAG38)s were delivered via SC injection. On Day 1 made a subcutaneous injection into the fold of skin on the back between the shoulder blades, thus injecting 200 μl of a solution/20 g of mouse body weight of a solution containing either a saline solution or a dose of 1 mg/kg (mpk) of one of five duplexes (AD04162, AD04623, AD04512 , AD04650 and AD04651) in buffered saline. Four (4) wild-type mice per treatment group were used. As shown above, AD04162 includes the (NAG25)s structure, AD04623 includes the (NAG37)s structure, AD04512 includes the (NAG31)s structure , AD04650 includes the (NAG33)s structure, and AD04651 includes the (NAG38)s structure. All GalNAc targeting ligands were attached at the 5' end of the sense strand of each example RNAi agent.

Serum samples from treated mice were collected at -1 (pre-dosing), 8, 15, and 22 to monitor knockdown. Knockdown was measured by quantifying circulating levels of mouse F12 protein (mF12) in serum using an in-house mF12 assay developed based on alphaLISA® (Perkin Elmer). mF12 levels for each animal at the corresponding time point were divided by the pre-treatment expression level in that animal to determine the expression ratio normalized to the pre-dosing level. Expression at a specific time point was then normalized to the saline control group by dividing the pre-dose day-normalized coefficient for an individual animal by the average pre-dose day-normalized coefficient for all mice in the saline control group. In this way, the expression level at each time point was obtained, normalized to the expression level in the control group. The experimental error is presented as standard deviation.

The results of this study are shown in Fig. 15. The day with minimum values was 8 for all RNAi agents studied. On trough day, AD04162 demonstrated 90% mF12 knockdown, AD04623 demonstrated 94% mF12 knockdown, AD04512 demonstrated 94% mF12 knockdown, AD04650 demonstrated 92% mF12 knockdown, and AD04651 demonstrated 87% mF12 knockdown. At day 2, all RNAi agents demonstrated >82% knockdown of mF12 levels, with the exception of AD04162 (containing NAG25), which showed only 74% knockdown. These data indicate that NAG-containing structures behave with respect to initial knockdown activity in a manner similar to RNAi agents containing rigid linker structures or linker replacement moieties disclosed herein (namely, NAG31, NAG33, NAG37, and NAG38). demonstrating a numerically higher mF12 knockdown on day 22.

Example 17. LP(a) expression inhibitory oligomeric compounds (double-stranded RNAi agents) bound to targeting ligands Structures 1004 and 1005 in Lp(a) transgenic (Tg) mice.

Oligomeric compounds inhibiting the expression of LP(a) (double-stranded Lp(a) RNAi agents) were prepared with the sequences presented in Table 1 below. 6.

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Table 6

LP(a) expression inhibiting oligomeric compounds (RNAi agent duplexes) from Example 17

Duplex ID: AD03629 5’ -» 3’ SEQ ID NO: Sequence of sense chain: (AM04611-SS) (NAG31 )(inv Ab)GfcCfcCfuUfAfUfuGfuUfaUfaCfgausu(inv Ab) 25 The sequence of the antisense chain is: (AM03972-AS) usCfsgsUfaUfaAfCfAfauaAfgGfgGfcusu 26 Duplex ID: AD03540 5' 3' SEQ ID NO: Sequence of sense chain: (AM04500-SS) (NAG30)(invAb)GfcCfcCfuUfAfUfuGfuUfaUfaCfgausu(invAb) 27 Antisense strand sequence: (AM03972-AS) usCfsgsUfaUfaAfCfAfauaAfgGfgGfcusu 28

In table 6, (NAG30) has the same chemical structure as shown in Example 14 above, and (NAG31) has the same chemical structure as shown in Example 15 above.

NAG30 has a chemical structure represented by structure 1004 herein. NAG31 has a chemical structure represented by structure 1005 herein.

Each strand of Lp(a) RNAi agents was synthesized according to phosphoramidite theurdphase technology used in oligonucleotide synthesis using either MerMade96E® (Bioautomation) or MerMade12® (Bioautomation); complementary strands were mixed by combining equimolar solutions of RNA (sense and antisense) in 0.2x PBS (phosphate buffered saline, 1x, Corning, Cellgro) to form duplexes using methods generally described in Example 10 herein.

Lp(a) transgenic (Tg) mice were used to evaluate the in vivo efficacy of double-stranded RNAi agents with conjugated N-acetylgalactosamine ligands.

Lp(a) RNAi agents bound to appropriate GalNAc ligands (namely NAG30 or NAG31) were combined with a pharmaceutically acceptable subcutaneous (SC) injection buffer known in the art.

RNAi agents for Lp(a) bound to the corresponding GalNAc ligands (namely NAG30 or NAG31) at the 5′ end of the sense strand were delivered by subcutaneous injection. On day 1, a subcutaneous injection was made into the dorsal skin fold between the shoulder blades and a 200 μl solution/20 g mouse body weight of a solution containing either saline or a 1 mg/kg (mpk) dose of Lp(a) RNAi agent (AD03629) was administered or AD03540) in saline. Four (4) Lp(a) transgenic mice were used per treatment group.

Serum samples from treated mice were collected on days -1 (pre-dosing), 8, 15, 22, 29, 36, and 43. Knockdown was determined by calculating serum levels of circulating Lp(a) particles. Lp(a) particle levels were measured on a Cobas® Integra 400 (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-dosing expression level in that animal (in this case, the level for -1 day) to determine the expression ratio normalized to -1 day. Expression at a given time point was then normalized to the saline control group by dividing the -1 day normalized coefficient for an individual animal by the average of the -1 day normalized coefficient for all mice in the saline control group. In this way, the expression level at each time point was obtained, normalized to the expression level in the control group. The experimental error is presented in the form of a standard

- 108 043375 cloning.

The results are shown in Fig. 16. The day with the lowest values was day 15 for both RNAi agents tested. AD03629 showed 89% knockdown of Lp(a) levels on the lowest day, and AD03540 showed 85% knockdown of Lp(a) levels on the lowest day. Both RNAi agents showed similar recovery curves up to day 36. But at day 43, while while AD03540 demonstrated a 16% knockdown of Lp(a) levels, AD03629 demonstrated a 55% knockdown of Lp(a) levels.

Example 18. Knockdown of apolipoprotein(a) (apo(a)) in apo(a) transgenic (Tg) mice after administration of LP(a) expression-inhibiting oligomeric compounds (double-stranded RNAi agents) bound to a targeting ligand of structure 1007, 1025 and 1026.

Oligomeric compounds inhibiting Lp(a) expression (double-stranded Lp(a) RNAi agents) were prepared with the sequences presented in Table 1 below. 7.

Table 7

LP(a) expression inhibiting oligomeric compounds (RNAi agent duplexes) from Example 18

Duplex ID: AD03721 5’ -» 3’ SE QID NO: Sequence of semantic thread: (AM04742-SS) (NAG33)(invAb)GfcCfcCfuUfAfUfuGfuUfaUfaCfgausu(invA b) 29 Antisense strand sequence: (AM03972-AS) usCfsgsUfaUfaAfCfAfauaAfgGfgGfcusu thirty Duplex ID: AD03722 5’ -» 3’ SE QID NO: Sequence of semantic thread: (AM04743-SS) (NAG34)(invAb)GfcCfcCfuUfAfUfuGfuUfaUfaCfgausu(invA b) 31 Antisense strand sequence: (AM03972-AS) usCfsgsUfaUfaAfCfAfauaAfgGfgGfcusu 32 Duplex ID: AD03723 5’ -» 3’ SE QID NO: Sequence of semantic thread: (AM04744-SS) (NAG35)(invAb)GfcCfcCfuUfAfUfuGfuUfaUfaCfgausu(invA b) 33 Antisense sequence threads: (AM03972-AS) usCfsgsUfaUfaAfCfAfauaAfgGfgGfcusu 34

In table 7 above the following notations are used:

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(NAG33) has the chemical structure represented by Structure 1025 herein (NAG34) has the chemical structure represented by Structure 1026 herein (NAG35) has the chemical structure represented by Structure 1007 herein.

Each strand of Lp(a) RNAi agents was synthesized according to phosphoramidite theurdphase technology used in oligonucleotide synthesis using either MerMade96E® (Bioautomation) or MerMade12® (Bioautomation); complementary strands were mixed by combining equimolar solutions of RNA (sense and antisense) in 0.2x PBS (phosphate buffered saline, 1x, Corning, Cellgro) to form duplexes using methods generally described in Example 10 herein.

Apo(a) transgenic (Tg) mice were used to evaluate the effectiveness of double-stranded RNAi agents with conjugated N-acetylgalactosamine ligands in vivo. Mouse Apo(a) Tg.

Lp(a) RNAi agents bound to appropriate GalNAc ligands (namely NAG33, NAG34 or NAG35) were combined with a pharmaceutically acceptable subcutaneous (SC) injection buffer known in the art.

RNAi agents for Lp(a) bound to the corresponding GalNAc ligands (namely NAG33, NAG34 or NAG35) were administered by subcutaneous injection. On day 1, a subcutaneous injection was made into the fold of skin on the back between the shoulder blades and a 200 μl solution/20 g mouse body weight of a solution containing either saline solution or a dose of 1 mg/kg (mpk) of the RNAi agent was injected and injected. AD03721, AD03722, or AD03723) in buffered saline. Three (3) Lp(a) Tg mice were used per treatment group.

Serum samples from treated mice were collected on days -1 (pre-dosing), 8, 15, 22, and 29. Knockdown was determined by monitoring circulating serum apo(a) protein levels using an apo(a) ELISA (Abcam). For normalization, the apo(a) level for each animal at a given time point was divided by the pre-treatment expression level of that animal (in this case, the level for -1 day) to determine the expression ratio normalized to -1 day. Expression at a specific time point was then normalized to the saline control group by dividing the -1 day normalized coefficient for an individual animal by the average of the -1 day normalized coefficient for all mice in the saline control group. In this way, the expression level at each time point was obtained, normalized to the expression level in the control group. Experimental error is presented as standard mean error.

The data obtained are presented in Figure 17. The day with the lowest values was day 15 for all RNAi agents tested. AD03721 demonstrated 91% knockdown of apo(a) protein levels at the lowest values, AD03722 demonstrated 81% knockdown of apo(a) protein levels at the lowest values, and AD03723 demonstrated 90% knockdown of apo(a) protein levels at the lowest values. Similar trajectories of recovery of apo(a) protein levels after treatment were observed for mice treated with AD03721 and AD03723, with virtually identical knockdown rates at each time point, and mice treated with AD03722 showed quantitatively lower knockdown at each time point examined. For example, at day 29, mice treated with AD03721 exhibited a 76% knockdown of apo(a) levels, mice treated with AD03723 exhibited an 83% knockdown of apo(a) levels, and mice treated with AD03722 exhibited a 61% knockdown of apo(a) levels. aro(a). These data indicate that the NAG33, NAG34, and NAG35 structures all exhibit knockdown activity, with RNAi agents containing the NAG33 and NAG35 structures exhibiting quantitatively higher knockdown at day 29.

Example 19. Dose-response study of LP(a) expression-inhibiting oligomeric compounds (double-stranded RNAi agents) bound to structure 1008 targeting ligands administered at doses of 1 mg/kg and 3 mg/kg to transgenic no Lp(a) mice.

Lp(a) transgenic mice described herein were used to evaluate the in vivo efficacy of double-stranded RNAi agents with conjugated N-acetylgalactosamine ligands. RNAi agents targeting Lp(a) were generated having duplex IDs: AD04170, shown above in Example 15. As shown above, duplex IDs for Lp(a): AD04170 includes a targeting ligand (NAG37) (structure 1008) attached to 5 ' - the end of the semantic thread.

Each strand of Lp(a) RNAi agents was synthesized according to phosphoramidite theurdphase technology used in oligonucleotide synthesis using either MerMade96E® (Bioautomation) or MerMade12® (Bioautomation); complementary strands were mixed by combining equimolar solutions of RNA (sense and antisense) in 0.2x PBS (phosphate buffered saline, 1x, Corning, Cellgro) to form duplexes using methods generally described in Example 10 herein.

Lp(a) RNAi agents bound to the targeting ligand of structure 1008 were combined with a pharmaceutically acceptable subcutaneous (SC) injection buffer known in the art.

RNAi agents for Lp(a) bound to ligand targeting structures 1008 were administered by subcutaneous (SC) injection. On day 1, a subcutaneous injection was made into the fold of skin on the back between the shoulder blades and 200 μl of a solution/20 g of mouse body weight was injected, containing a dose of saline, 1 mg/kg (mpk) RNAi agent in saline, or 3 mg /kg (mpk) of RNAi agent in saline.

Control serum samples (pre-treatment) were collected from mice on day -1 before dosing. Lp(a) particle levels were determined on a Cobas® Integra 400 (Roche Diagnostics) according to the manufacturer's recommendations. For normalization, Lp(a) levels for each animal at some time point were divided by the expression level in that animal (in this case, day 1 (pre-dosing)) to determine the expression ratio normalized to day -1 level. Expression at a particular time point was then normalized to the saline control group by dividing the day -1 normalized ratio for an individual animal by the average day -1 normalized ratio of all mice in the saline control group. The result was expression for each time point, normalized to expression in the control group.

The experimental error is presented as standard deviation.

The results are shown in Fig. 18. As shown in FIG. 18, there appears to be a dose-dependent relationship for the RNAi agent for Lp(a) across all time points.

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Example 20 LP(a) expression inhibitory oligomeric compounds (double-stranded RNAi agents) bound to targeting ligands having structures 1003 and 1004 in cynomolgus monkeys.

Oligomeric compounds (double-stranded RNAi agents) inhibiting the expression of Lp(a) were obtained, having the sequences presented below in the table. 8.

Table 8

Lp(a) expression inhibiting oligomeric compounds (duplex RNAi agents) from Example 20

Duplex ID: AD03668 5' Ά 3' SE QID NO: Sequence of sense chain: (AM04500-SS) (NAG30)(invAb)GfcCfcCfuUfAfUfuGfuUfaUfaCfgausu(invA b) 35 Antisense strand sequence: (AM04501-AS) cPrpTMsCfsgsUfaUfaAfCfAfauaAfgGfgGfcusu 36

The RNAi agent for Lp(a) AD03547 is the same as in Example 13, and is conjugated to (NAG29). The Lp(a) RNAi agent AD3668 was conjugated to (NAG30) (NAG30) had the chemical structure shown in Example 14 (NAG29), which is represented by structure 1003 herein (NAG30) is represented by structure 1004 herein.

Each strand of Lp(a) RNAi agents was synthesized according to phosphoramidite theurdphase technology used in oligonucleotide synthesis using either MerMade96E® (Bioautomation) or MerMade12® (Bioautomation); complementary strands were mixed by combining equimolar solutions of RNA (sense and antisense) in 0.2x PBS (phosphate buffered saline, 1x, Corning, Cellgro) to form duplexes using methods generally described in Example 10 herein.

Lp(a) RNAi agents conjugated to the targeting ligands disclosed herein, having structure 1003 or structure 1004, were prepared and combined with a pharmaceutically acceptable buffer in a manner known in the art for subcutaneous (SC) injection.

Control serum samples (pre-treatment) were collected from cynomolgus monkeys on pre-injection days 14, -7, and 1 (pre-dose). Lp(a) particle levels were determined on a Cobas® Integra 400 (Roche Diagnostics) according to the manufacturer's recommendations. For normalization, Lp(a) levels for each animal at a given time point were divided by the average of expression levels in that animal (in this case on days -14, -7 and day 1 (pre-dosing)) to determine the expression ratio normalized to pre-dosing levels. dosing. The experimental error is presented as standard deviation.

On Day 1, cynomolgus monkeys (Macaca fascicularis) were treated by subcutaneous injection with Lp(a) RNAi agents, the associated targeting ligands disclosed herein, at 3 mg/kg of either Lp(a) RNAi agent AD03668 or RNAi agent for Lp(a) AD03547. Two (2) monkeys per treatment group were used.

The results are shown in Fig. 19. RNAi triggers for Lp(a) conjugated to structure 1003 (AD03547) or structure 1004 (AD03668) showed knockdown in cynomolgus monkeys.

Example 21. F12 expression-inhibiting oligomeric compounds (Double-stranded RNAi agents) bound to a mating ligand of structure 1008 in cynomolgus monkeys.

F12 RNAi agents with different F12 targeting sequences and linked to the GalNAc targeting ligand 1008 structure [(NAG37)s] at the 5' end of the sense strand were prepared and mixed in a pharmaceutically acceptable subcutaneous (SC) injection buffer. known in the art (NAG37)s has the chemical structure shown in Example 16 above.

On Day 1, cynomolgus primates (Macaca fascicularis) were injected subcutaneously with 3 mg/kg of one of six (6) different Lp(a) RNAi agents having different structures and different modification patterns: AD04623, AD04624, AD04625, AD04626, AD04627 or AD04628. Compounds were administered to two (2) monkeys per treatment group.

Serum samples from treated cynomolgus monkeys were collected on days -7 and 1 (pre-dosing); and on days 8, 15, and 22 to monitor knockdown. Knockdown was measured by quantifying circulating cynomolgus F12 protein (cF12) levels in serum using a human F12 ELISA kit (Molecular Innovations). The cF12 levels for each animal at the corresponding time point were divided by the pre-treatment expression level of the indicated animal (average of day -7 and day 1) to determine the expression ratio normalized to the pre-dosing level. The experimental error is presented as standard deviation.

The results are shown in Fig. 20. Each of the RNA agents and for F12 bound by NAG37 (structure 1008) demonstrated knockdown in cynomolgus monkeys, with AD04625 AD04623 demonstrating

- 112 -

Claims (31)

maximum knockdown at all measurement points. Example 22. Alpha-1-antitrypsin expression inhibitory oligomeric compounds (double-stranded RNAi agents) bound to structure 1008 targeting ligands in PiZ transgenic mice. The PiZ transgenic mouse model (PiZ mice) was used to evaluate in vivo RNAi agents targeting the alpha-1 antitrypsin (AAT) gene. PiZ mice are carriers of the mutant human AAT allele PiZ and a model of human alpha-1 antitrypsin deficiency (AATD) (Carlson et al., Journal of Clinical Investigation 1989). Oligomeric compounds (double-stranded RNAi agents) inhibiting AAT expression were obtained with the sequences presented below in the table. 9. Table 9 AAT expression inhibiting oligomeric compounds (RNAi agent duplexes) from Example 22 Duplex ID: AD04663 5' 3' SEQ ID NO: Sense chain sequence: (AM05968-SS) (NAG37)s(invAb)sucaacaAfAfCfccuuugucuus(invAb) 37 Antisense strand sequence: (AM05969-AS) asAfsgsAfcAfaAfgGfgUfuUfgUfuGfausu 38 (NAG37)s has the chemical structure shown in Example 16 above. An RNAi agent for AAT was prepared in a pharmaceutically acceptable saline buffer and injected subcutaneously (s.c.) into the fold of skin on the back between the shoulder blades of PiZ mice with 200 μl of solution/20 g of mouse body weight to evaluate knockdown of AAT gene expression. Each mouse received one SC dose of 2 mg/kg (mpk) AD04454. Doses of the RNAi agent AAT were administered to three mice (n=3). Plasma samples were collected and analyzed for Z-AAT protein levels on days -1, 1 (pre-dosing), 8 and 15. AAT levels were normalized to plasma AAT levels on day 1 (pre-dosing). Protein levels were measured by quantifying circulating levels of human plasma Z-AAT using an ELISA kit. Mean normalized AAT levels (Z-AAT) are shown in FIG. 21. Knockdown was observed using an RNAi agent for AAT coupled to the targeting ligand of structure 1008 presented herein in PiZ transgenic mice. Other implementation options. It should be understood that although the present invention has been described in conjunction with a detailed description thereof, the above description is intended to be illustrative and not to limit the scope of the invention, which is defined by the scope of the appended claims. The scope of the following claims includes other aspects, advantages and modifications. CLAIM 1. A targeting ligand comprising a structure selected from the group consisting of: - 113 043375 - 114 043375 - 115 043375 - 116 043375 structure 1018): structure 1023): or its pharmaceutical salt. 2. The targeting ligand according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the targeting ligand is associated with an RNAi agent, wherein said RNAi agent is single-stranded or double-stranded. - 117 043375 3. The targeting ligand of claim 2, or a pharmaceutically acceptable salt thereof, wherein the RNAi agent is double-stranded, and said double-stranded RNAi agent is linked to a targeting ligand at the 5' end of the sense strand of the RNAi agent. 4. The targeting ligand according to claim 2 or 3 or a pharmaceutically acceptable salt thereof, wherein the RNAi agent is linked to the linker of the targeting ligand through a phosphate group, a phosphorothioate group or a phosphonate group. 5. The targeting ligand of claim 1 or a pharmaceutically acceptable salt thereof coupled to an RNAi agent for use as a drug. 6. The targeting ligand of claim 1 or a pharmaceutically acceptable salt thereof coupled to an RNAi agent for introducing the RNAi agent into a mammalian cell. 7. A pharmaceutical composition containing an RNAi agent associated with a targeting ligand according to claim 1, and one or more pharmaceutically acceptable excipients, where the structure of the RNAi agent associated with a targeting ligand is a structure selected from the group consisting of: (Structure 1004a); - 118 043375 - 119 043375 - 120 043375 or a pharmaceutically acceptable salt thereof, wherein R in each structure contains an RNAi agent. 8. The pharmaceutical composition according to claim 7, wherein said RNAi agent is single-stranded or double-stranded. 9. A pharmaceutical composition containing at least one RNAi agent associated with a targeting ligand or a pharmaceutically acceptable salt thereof according to claim 1. 10. The pharmaceutical composition according to claim 9, wherein the RNAi agent is single-stranded or double-stranded. 11. The pharmaceutical composition according to claim 10, wherein the RNAi agent is double-stranded, and said RNAi agent is linked to a targeting ligand at the 5' end of the sense strand of the RNAi agent. 12. The pharmaceutical composition according to claim 10 or 11, wherein the RNAi agent is connected to the linker of the targeting ligand through a phosphate group, phosphorothioate group or phosphonate group. 13. Pharmaceutical composition according to any one of claims 9 to 12, where the RNAi agent includes one or more modified nucleotides. 14. Pharmaceutical composition according to any one of claims 9-12 for the treatment and/or regulation of clinical manifestations associated with the expression of target mRNA. 15. A compound having a structure selected from the group consisting of: - 121 043375 - 122 043375 - 123 043375 - 124 043375 (Structure 1016b); - 125 043375 (Structure 1019b); - 126 043375 AcO (Structure 1023b); or a pharmaceutically acceptable salt thereof. 16. A compound having a structure selected from the group consisting of: - 127 043375 - 128 043375 - 129 043375 - 130 043375 (Structure 1019a(i)); - 131 043375 or a pharmaceutically acceptable salt thereof, wherein each structure R contains an RNAi agent; Y represents O or S; and Y' is O ^- , S ^- or NH ^- . 17. Use of a targeting ligand according to claim 1 or a pharmaceutically acceptable salt thereof coupled to an RNAi agent for the preparation of a medicament for the treatment of a disease or disorder. 18. The targeting ligand of claim 1, or a pharmaceutically acceptable salt thereof, coupled to an RNAi agent for inhibiting expression of a target nucleic acid in a subject. 19. The targeting ligand of claim 1, or a pharmaceutically acceptable salt thereof, coupled to an RNAi agent for the treatment of a disease or disorder that would benefit from administration of said RNAi agent. 20. The targeting ligand according to claim 1 or a pharmaceutically acceptable salt thereof containing structure 1003. 21. The targeting ligand according to claim 1 or a pharmaceutically acceptable salt thereof containing structure 1008. 22. The targeting ligand according to claim 1 or a pharmaceutically acceptable salt thereof containing structure 1023. 23. The pharmaceutical composition according to claim 7, wherein the structure of the RNAi agent bound to the targeting ligand is structure 1003a or a pharmaceutically acceptable salt thereof. 24. The pharmaceutical composition according to claim 7, wherein the structure of the RNAi agent associated with the targeting ligand is represented by structure 1008a or a pharmaceutically acceptable salt thereof. 25. The pharmaceutical composition according to claim 7, wherein the structure of the RNAi agent bound to the targeting ligand is structure 1023a or a pharmaceutically acceptable salt thereof. 26. The compound according to claim 15 or a pharmaceutically acceptable salt thereof having structure 1003b. 27. The compound according to claim 15 or a pharmaceutically acceptable salt thereof having structure 1008b. 28. The compound according to claim 15 or a pharmaceutically acceptable salt thereof having structure 1023b. 29. The compound according to claim 16 or a pharmaceutically acceptable salt thereof having structure 1003a(i). 30. A compound according to claim 16 or a pharmaceutically acceptable salt thereof having structure 1008a(i). 31. A compound according to claim 16 or a pharmaceutically acceptable salt thereof having structure 1023a(i). - 132 -