TW202327658A - Multivalent ligand clusters with diamine scaffold for targeted delivery of therapeutic agent - Google Patents

Abstract

Multivalent ligand clusters, having a diamine scaffold, for targeted delivery of pharmaceutical agents conjugated thereto are described. A multivalent ligand cluster may comprise one or more N-acetylgalactosamine (GalNAc) targeting ligands. A multivalent ligand cluster may be conjugated to one or more small interfering ribonucleic acids (siRNAs), with siRNA being an example of a pharmaceutical agent. Compositions comprising a multivalent ligand cluster, and methods of making a multivalent ligand cluster, are also described.

Description

Multivalent ligand clusters with diamine scaffolds for targeted delivery of therapeutic agents

Multivalent ligand clusters with diamine scaffolds for targeted delivery of agents conjugated thereto are described. A multivalent ligand cluster may comprise one or more N-acetylgalactosamine (GalNAc) targeting ligands. Multivalent ligand clusters can be conjugated to one or more small interfering ribonucleic acids (siRNAs), where one example of such agents is siRNA. Compositions comprising multivalent ligand clusters, and methods of making multivalent ligand clusters are also described.

Oligonucleotides generally have low cell membrane permeability due to their high molecular weight and polyanionic nature. Thus, through the well-known mechanism of receptor-mediated endocytosis, target ligands are often conjugated to oligonucleotide compounds to enhance cellular uptake and improve tissue specificity of in vivo delivery. In some cases, clusters of multivalent ligands have advantages over single ligands in enhancing delivery to target tissues through specific receptors. The asialoglycoprotein receptor (ASGPR) is one such receptor.

The ligand of ASGPR, N-acetylgalactosamine (GalNAc), has been shown to facilitate the delivery of oligonucleotide drugs to hepatocytes. It has also been demonstrated that clusters of multivalent GalNAc ligands have a higher binding affinity for ASGPR than individual GalNAc ligands and are therefore more efficient in delivering therapeutic oligonucleotides into hepatic hepatocytes.

One aspect of the present disclosure pertains to compounds for targeted delivery of one or more agents, wherein the compounds have the formula:

wherein each TL is an independently selected targeting ligand, m is an integer from 1 to 10, each n is an independently selected integer from 1 to 10, each linker A is an independently selected spacer, and linker B is a spacer , and W is one or more agents or a functional group capable of linking to one or more agents. In some embodiments, m is 1. In some embodiments, m is 2.

In some embodiments, n is 1. In some embodiments, n is 2.

In some embodiments, at least one of the independently selected TLs is capable of binding to one or more cellular receptors, cellular channels, and cellular transporters capable of promoting endocytosis. In some embodiments, at least one of the independently selected TLs comprises at least one small molecule ligand. In some embodiments, at least one small molecule comprises N-acetylgalactosamine, galactose, galactosamine, N-formyl-galactosamine, N-propionylgalactosamine, N-butyrylgalactose Amines and at least one of N-isobutyrylgalactosamine, macrocycles, folic acid molecules, fatty acids, bile acids and cholesterol. In some embodiments, at least one of the independently selected TLs comprises at least one peptide. In some embodiments, at least one of the independently selected TLs comprises at least one cyclic peptide. In some embodiments, at least one of the independently selected TLs comprises at least one aptamer. In some embodiments, at least one of the independently selected TLs is capable of binding at least one asialoglycoprotein receptor (ASGPR). In some embodiments, at least one of the independently selected TLs is capable of binding at least one transferrin receptor. In some embodiments, at least one of the independently selected TLs is capable of binding at least one integrin receptor. In some embodiments, at least one of the independently selected TLs is capable of binding at least one folate receptor. In some embodiments, at least one of the independently selected TLs is capable of binding to at least one G-protein-coupled receptor (GPCR).

In some embodiments, at least one of the independently selected linkers A comprises polyethylene glycol, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, aralkyl, aralkenyl, and aryl at least one of the alkynyl groups. In some embodiments, at least one of the independently selected linkers A comprises at least one heteroatom. In some embodiments, the at least one heteroatom comprises at least one of oxygen, nitrogen, sulfur, or phosphorus. In some embodiments, at least one of the independently selected linkers A comprises at least one aliphatic heterocycle. In some embodiments, the at least one aliphatic heterocycle comprises at least one of tetrahydrofuran, tetrahydropyran, morpholine, piperidine, piperazine, pyrrolidine, and azetidine. In some embodiments, at least one of the independently selected linkers A comprises at least one heteroaryl group. In some embodiments, the at least one heteroaryl group comprises at least one of imidazole, pyrazole, pyridine, pyrimidine, triazole, and 1,2,3-triazole. In some embodiments, at least one of the independently selected linkers A comprises at least one amino acid. In some embodiments, at least one of the independently selected linkers A comprises at least one nucleotide. In some embodiments, at least one of the independently selected linkers A comprises at least one sugar. In some embodiments, the at least one sugar comprises at least one of glucose, fructose, mannose, galactose, ribose, and glucosamine. In some embodiments, at least one of the independently selected linkers A comprises one or more of the following: wherein p is an integer of 0 to 12, pp is an integer of 0 to 12, q is an integer of 1 to 12, and qq is an integer of 1 to 12.

In some embodiments, linker B comprises at least one of polyethylene glycol, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, aralkyl, aralkenyl, and aralkynyl. In some embodiments, linker B comprises at least one heteroatom. In some embodiments, the at least one heteroatom comprises at least one of oxygen, nitrogen, sulfur, and phosphorus. In some embodiments, linker B comprises at least one aliphatic heterocycle. In some embodiments, the at least one aliphatic heterocycle comprises at least one of tetrahydrofuran, tetrahydropyran, morpholine, piperidine, piperazine, pyrrolidine, and azetidine. In some embodiments, linker B comprises at least one heteroaryl. In some embodiments, the at least one heteroaryl group comprises at least one of imidazole, pyrazole, pyridine, pyrimidine, triazole, and 1,2,3-triazole. In some embodiments, linker B comprises at least one amino acid. In some embodiments, linker B comprises at least one nucleotide. In some embodiments, the at least one nucleotide comprises at least one of an abasic nucleotide and an inverted abasic nucleotide. In some embodiments, the abasic nucleotides are abasic deoxyribonucleic acid. In some embodiments, the inverted abasic nucleotides are inverted abasic deoxyribonucleic acids. In some embodiments, the abasic nucleotide is an abasic ribonucleic acid. In some embodiments, the inverted abasic nucleotides are inverted abasic ribonucleic acids. In some embodiments, linker B comprises at least one sugar. In some embodiments, the at least one sugar comprises at least one of glucose, fructose, mannose, galactose, ribose, and glucosamine. In some embodiments, linker B comprises at least one of the following: wherein j is an integer of 1 to 12, and k is an integer of 0 to 12.

In some embodiments, the linker BW is: wherein j is an integer of 0 to 12, and k is an integer of 0 to 12.

In some embodiments, W is hydroxyl. In some embodiments, W is a protected hydroxy group. In some embodiments, the protected hydroxyl group is 4,4'-dimethoxytrityl (DMT), monomethoxytrityl (MMT), 9-(p-methoxybenzene group) at least one of xanthen-9-yl (Mox) and 9-phenylxanthene-9-yl (Px) to protect. In some embodiments, W is a phosphoramidite group having the formula: , wherein: R ^a is C1 to C6 alkyl, C3 to C6 cycloalkyl, isopropyl, or R ^a is connected to R ^b through a nitrogen atom to form a ring, R ^b is C1 to C6 alkyl, C3 to C6 ring Alkyl, isopropyl, or ^Rb is linked to Ra through a nitrogen atom to form a ring, and ^Rc is a phosphite protecting group ^, a phosphate protecting group or 2-cyanoethyl.

In some embodiments, the phosphite protecting group comprises methyl, allyl, 2-cyanoethyl, 4-cyano-2-butenyl, 2-cyano-1,1-dimethyl Ethyl, 2-(trimethylsilyl)ethyl, 2-(S-acetylthio)ethyl, 2-(S-pivalylthio)ethyl, 2-(4-nitro phenyl)ethyl, 2,2,2-trichloroethyl, 2,2,2-trichloro-1,1-dimethylethyl, 1,1,1,3,3,3-hexafluoro -at least one of 2-propyl, fluorenyl-9-methyl, 2-chlorophenyl, 4-chlorophenyl and 2,4-dichlorophenyl. In some embodiments, the phosphate protecting group comprises methyl, allyl, 2-cyanoethyl, 4-cyano-2-butenyl, 2-cyano-1,1-dimethylethyl 2-(trimethylsilyl)ethyl, 2-(S-acetylthio)ethyl, 2-(S-pivalylthio)ethyl, 2-(4-nitrobenzene base) ethyl, 2,2,2-trichloroethyl, 2,2,2-trichloro-1,1-dimethylethyl, 1,1,1,3,3,3-hexafluoro- At least one of 2-propyl, fluorenyl-9-methyl, 2-chlorophenyl, 4-chlorophenyl and 2,4-dichlorophenyl.

In some embodiments, W is carboxy. In some embodiments, W is an activated carboxyl group having the formula: , where X is a leaving group. In some embodiments, the leaving group is selected from carboxylate, sulfonate, chloride, phosphate, imidazole, hydroxybenzotriazole (hydroxybenzotriazole, HOBt), N-hydroxysuccinimide (N-hydroxysuccinimide , NHS), tetrafluorophenol, pentafluorophenol and p-nitrophenol.

In some embodiments, W is a Michael acceptor. In some embodiments, the Michael acceptor has the formula: , where E is an electron-withdrawing group, and ^Rd is hydrogen or a C1-C6 alkyl substituent on the alkene. In some embodiments, the electron withdrawing group is formamide or ester. In some embodiments, E and the carbon-carbon double bond are part of a maleimide.

In some embodiments, W is an oligonucleotide. In some embodiments, the oligonucleotides are single-stranded oligonucleotides. In some embodiments, the oligonucleotides are double-stranded oligonucleotides. In some embodiments, the oligonucleotide comprises at least 3 independently selected nucleotides. In some embodiments, the oligonucleotide comprises 16 to 23 independently selected nucleotides. In some embodiments, the oligonucleotide comprises about 100 independently selected nucleotides. In some embodiments, the oligonucleotides comprise up to fourteen thousand independently selected nucleotides.

In some embodiments, W is: , wherein: Linker C is absent or is a spacer attached to the 3' or 5' end of the oligonucleotide, X is methyl, oxygen, sulfur or amino, and Y is oxygen, sulfur or amino.

In some embodiments, linker C comprises at least one heterocyclic compound. In some embodiments, the heterocyclic compound is an abasic nucleotide or an inverted abasic nucleotide.

In some embodiments, W is: , wherein linker C is a spacer attached to the 3' end or 5' end of the oligonucleotide. In some embodiments, linker C comprises at least one of polyethylene glycol (polyethylene glycol, PEG), alkyl and cycloalkyl. In some embodiments, linker C comprises at least one heteroatom. In some embodiments, the at least one heteroatom comprises at least one of oxygen, nitrogen, sulfur, and phosphorus. In some embodiments, linker C comprises at least one aliphatic heterocycle. In some embodiments, the at least one aliphatic heterocycle comprises at least one of tetrahydrofuran, tetrahydropyran, morpholine, piperidine, piperazine, pyrrolidine, and azetidine. In some embodiments, linker C comprises at least one heteroaryl. In some embodiments, the at least one heteroaryl group comprises at least one of imidazole, pyrazole, pyridine, pyrimidine, triazole, and 1,2,3-triazole. In some embodiments, Linker C comprises at least one amino acid. In some embodiments, linker C comprises at least one nucleotide. In some embodiments, the at least one nucleotide comprises at least one of an abasic nucleotide and an inverted abasic nucleotide. In some embodiments, the abasic nucleotide is abasic deoxyribonucleic acid (DNA). In some embodiments, the inverted abasic nucleotide is inverted abasic deoxyribonucleic acid (DNA). In some embodiments, the abasic nucleotide is abasic ribonucleic acid (RNA). In some embodiments, the inverted abasic nucleotide is an inverted abasic ribonucleic acid (RNA). In some embodiments, linker C comprises at least one sugar. In some embodiments, the at least one sugar comprises at least one of glucose, fructose, mannose, galactose, ribose, and glucosamine. In some embodiments, linker C comprises one or more of the following: wherein j is an integer of 1 to 12, and k is an integer of 0 to 12.

In some embodiments, W is: , wherein linker C is a spacer attached to the 3' end or 5' end of the oligonucleotide. In some embodiments, linker C comprises at least one of polyethylene glycol (PEG), alkyl, and cycloalkyl. In some embodiments, linker C comprises one or more of the following: wherein j is an integer of 1 to 12, and k is an integer of 0 to 12.

In some embodiments, the compound is selected from: Compound 28; Compound 29; Compound 30; Compound 31; Compound 32; Compound 33; Compound 34; Compound 35; Compound 36; .

In some embodiments, the compound is a stereoisomer of one of Compounds 1-75.

In some embodiments, W is one or more pharmaceutical agents. In some embodiments, the one or more agents comprise small interfering RNA (small interfering RNA, siRNA), single-stranded siRNA, double-stranded siRNA, small activating RNA, RNAi, microRNA (microRNA, miRNA), antisense Oligonucleotides, short guide RNA (guide RNA, gRNA), single guide RNA (single guide RNA, sgRNA), messenger RNA (messenger RNA, mRNA), ribozymes, plasmids, immunostimulatory nucleic acids, antagomirs and aptamers at least one of . In some embodiments, the double-stranded siRNA comprises at least one modified ribonucleotide. In some embodiments, each strand of the double-stranded siRNA is 19 to 23 nucleotides in length. In some embodiments, substantially all ribonucleotides of the double-stranded siRNA are modified. In some embodiments, all ribonucleotides of the double-stranded siRNA are modified. In some embodiments, the modified ribonucleotides comprise 2'-O-methyl nucleotides, 2'-fluoro nucleotides, 2'-deoxy nucleotides, 2'3'-seco cores Nucleotide mimetics, locked nucleotides, 2'-F-arabinonucleotides, 2'-methoxyethyl nucleotides, abasic nucleotides, ribitol, reverse nucleotides, reverse Abasic Nucleotide, Inverted 2'-OMe Nucleotide, Inverted 2'Deoxy Nucleotide, 2'-Amino Modified Nucleotide, 2'-Alkyl Modified Nucleotide, Morpholine Nucleotides and 3'-OMe nucleotides, nucleotides containing 5'-phosphorothioate groups, or 5'-(E)-vinylphosphonate nucleotides (antisense strand only) , or terminal nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, phosphoramidates, or Nucleotides containing unnatural bases. In some embodiments, at least one strand of the double-stranded siRNA comprises at least one phosphorothioate linkage. In some embodiments, at least one strand of the double-stranded siRNA comprises up to 6 phosphorothioate linkages. In some embodiments, the double-stranded siRNA comprises at least one locked nucleic acid. In some embodiments, the double-stranded siRNA comprises at least one unlocking nucleic acid. In some embodiments, the double-stranded siRNA comprises at least one glycerol nucleic acid.

Another aspect of the present disclosure pertains to pharmaceutical compositions comprising any one of the compounds detailed above. In some embodiments, the pharmaceutical composition comprises one or more agents. In some embodiments, the pharmaceutical composition comprises one or more therapeutic agents. In some embodiments, the pharmaceutical composition includes a pharmaceutically acceptable carrier.

Another aspect of the present disclosure relates to compositions for targeted delivery of one or more agents, wherein the composition comprises any one of the compounds described above, and wherein W is one or more agents. In some embodiments, the one or more agents comprise small interfering RNA (siRNA), single-stranded siRNA, double-stranded siRNA, small activating RNA, microRNA (miRNA), antisense oligonucleotides, short guide RNA At least one of (gRNA), single guide RNA (sgRNA), messenger RNA (mRNA), ribozyme, plasmid, immunostimulatory nucleic acid, antagomir and aptamer. In some embodiments, the double-stranded siRNA comprises at least one modified ribonucleotide in one or both strands of the siRNA. In some embodiments, each strand of the double-stranded siRNA is 19 to 23 nucleotides in length. In some embodiments, substantially all ribonucleotides of the double-stranded siRNA are modified. In some embodiments, all ribonucleotides of the double-stranded siRNA are modified. In some embodiments, the modified ribonucleotides comprise: 2'-O-methyl nucleotides, 2'-fluoro nucleotides, 2'-deoxy nucleotides, 2'3'-seco Nucleotide mimetics, locked nucleotides, 2'-F-arabinonucleotides, 2'-methoxyethyl nucleotides, abasic nucleotides, ribitol, inverted nucleotides, Inverted abasic nucleotides, inverted 2'-OMe nucleotides, inverted 2'deoxynucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, Phylino and 3'-OMe nucleotides, nucleotides containing a 5'-phosphorothioate group, or 5'-(E)-vinylphosphonate nucleotides (antisense strand only ), or terminal nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, phosphoramidates, or nucleotides containing unnatural bases. In some embodiments, at least one strand of the double-stranded siRNA comprises at least one phosphorothioate linkage. In some embodiments, at least one strand of the double-stranded siRNA comprises up to 6 phosphorothioate linkages. In some embodiments, the double-stranded siRNA comprises at least one locked nucleic acid. In some embodiments, the double-stranded siRNA comprises at least one unlocking nucleic acid. In some embodiments, the double-stranded siRNA comprises at least one glycerol nucleic acid.

Another aspect of the present disclosure pertains to pharmaceutical compositions comprising any one of the aforementioned compositions. In some embodiments, the pharmaceutical composition comprises one or more therapeutic agents. In some embodiments, the pharmaceutical composition includes a pharmaceutically acceptable carrier.

Another aspect of the present disclosure relates to a method for preparing a compound for targeted delivery of one or more agents, wherein the method comprises: receiving a first compound comprising a diamine comprising a first nitrogen and a second nitrogen, the first nitrogen being a primary amine, the second nitrogen being a secondary amine comprising a protecting group; by coupling a plurality of protected carboxylic acids with the first compound to produce a second compound, the second compound The first nitrogen in is a tertiary amine comprising a first protected carboxylic acid and a second protected carboxylic acid, and the second nitrogen of the second compound is a tertiary amine comprising said protecting group and a third protected carboxylic acid; deprotection of the second nitrogen of the second compound to produce a third compound resulting in said second nitrogen becoming a secondary amine comprising said third protected carboxylic acid; by attaching a hydroxyl comprising moiety to the second nitrogen of the third compound to produce a fourth compound resulting in said second nitrogen becoming a tertiary amine or amide comprising said third protected carboxylic acid and said hydroxyl-containing moiety; by converting said protected carboxylic acid of the fourth compound to Carboxylic acid to produce the fifth compound; and by using the fifth compound to carry out amide coupling reaction to produce the sixth compound, the first nitrogen in the sixth compound is a tertiary amine comprising a first amide and a second amide, the sixth compound The second nitrogen in the six compounds is a tertiary amine comprising the hydroxyl-containing moiety and a third amide, wherein the first amide, the second amide, and the third amide are each coupled to an independently selected targeting ligand .

In some embodiments, the protecting group is selected from benzyl and triphenylmethyl. In some embodiments, producing the second compound comprises performing a _SN2 substitution reaction with the first compound. In some embodiments, producing the second compound comprises performing a reductive amination reaction with the first compound. In some embodiments, producing the second compound comprises performing a Michael addition reaction with the first compound. In some embodiments, the protecting group is benzyl, and producing the third compound comprises hydrogenating with the second compound. In some embodiments, the protecting group is tritylmethyl, and generating the third compound comprises reacting the second component with at least one acid. In some embodiments, producing the fourth compound comprises performing an _SN2 substitution reaction with the third compound. In some embodiments, producing the fourth compound comprises performing a reductive amination reaction with the third compound. In some embodiments, producing the fourth compound comprises performing a Michael addition reaction with the third compound. In some embodiments, producing the fourth compound comprises performing an amide coupling reaction with the third compound. In some embodiments, producing the fourth compound comprises performing a nucleophilic addition reaction with the third compound. In some embodiments, the hydroxyl-containing moiety is attached to the second nitrogen using any of the Linker B described above. In some embodiments, producing the fifth compound includes reacting the fourth compound with at least one acid. In some embodiments, the at least one acid comprises at least one of hydrochloric acid, hydrobromic acid, trifluoroacetic acid, and formic acid. In some embodiments, producing the fifth compound comprises performing a hydrogenation reaction with the fourth compound. In some embodiments, producing the fifth compound comprises performing a hydrolysis reaction with the fourth compound. In some embodiments, the first amide, the second amide, and the third amide are each coupled to an independently selected targeting ligand using any independently selected linker A described above. In some embodiments, the independently selected targeting ligand is the independently selected targeting ligand described above. In some embodiments, the method further comprises converting the hydroxyl group to a phosphoramidite group using a phosphoritylation reaction. In some embodiments, converting a hydroxyl group to a phosphoramidite group is performed after performing the amide coupling reaction to produce the sixth compound.

Another aspect of the present disclosure relates to a method for preparing a compound for targeted delivery of one or more agents, wherein the method comprises: receiving a first compound comprising a diamine comprising a first nitrogen and a second nitrogen, the first nitrogen being a secondary amine comprising a first protecting group, the second nitrogen being an amine comprising a second protecting group; by combining the first protected carboxylic acid with the first nitrogen of the first compound coupling to produce a second compound resulting in the first nitrogen becoming a tertiary amine; removing the first protecting group from the first nitrogen of the second compound to produce a third compound comprising the first nitrogen and a second nitrogen, the The first nitrogen is a secondary amine comprising the first protected carboxylic acid and the second nitrogen is a carboxylic acid comprising the second protecting group; by combining the second protected carboxylic acid with the first nitrogen of the third compound coupling to produce a fourth compound resulting in the first nitrogen becoming a tertiary amine; removing the second protecting group from the fourth compound to produce a fifth compound comprising a first nitrogen and a second nitrogen, the first nitrogen being a tertiary amine comprising said first protected carboxylic acid and said second protected carboxylic acid, the second nitrogen being a primary amine; generating the second nitrogen by coupling a third protected carboxylic acid with a second nitrogen of a fifth compound a sixth compound, causing the second nitrogen to become a secondary amine; generating a seventh compound by linking a moiety comprising a hydroxyl group to the second nitrogen of the sixth compound, causing the second nitrogen to become a tertiary amine; Conversion of the third protected carboxylic acid to the first carboxylic acid to produce an eighth compound; a ninth compound is produced by performing an amide coupling reaction using the eighth compound, the first nitrogen of the ninth compound comprising said first protected carboxylic acid acid and the second protected carboxylic acid, the second nitrogen of the ninth compound comprises a first amide having a first targeting ligand coupled thereto and the moiety comprising a hydroxyl group; Conversion of a diprotected carboxylic acid to a second carboxylic acid produces a tenth compound; an amide coupling reaction using the tenth compound produces an eleventh compound, the first nitrogen of the eleventh compound comprising said first protected A carboxylic acid and a second amide having a second targeting ligand coupled thereto, the second nitrogen of the eleventh compound comprises a first amide having a first targeting ligand coupled thereto and said hydroxyl the twelfth compound is produced by converting the first protected carboxylic acid of the eleventh compound to a third carboxylic acid; and the thirteenth compound is produced by using the twelfth compound for an amide coupling reaction, the thirteenth compound, The first nitrogen of the thirteenth compound comprises a second amide with a second targeting ligand coupled thereto and a third amide with a third targeting ligand coupled thereto, the second nitrogen of the thirteenth compound comprising a first amide having a first targeting ligand coupled thereto and the hydroxyl-containing moiety.

In some embodiments, the first protecting group is benzyl and the second protecting group is t-butoxycarbonyl (Boc). In some embodiments, producing the second compound comprises performing a _SN2 substitution reaction with the first compound. In some embodiments, producing the second compound comprises performing a reductive amination reaction with the first compound. In some embodiments, producing the second compound comprises performing a Michael addition reaction with the first compound. In some embodiments, producing the third compound comprises performing a hydrogenation reaction with the second compound. In some embodiments, producing the fourth compound comprises performing an _SN2 substitution reaction with the third compound. In some embodiments, producing the fourth compound comprises performing a reductive amination reaction with the third compound. In some embodiments, producing the fourth compound comprises performing a Michael addition reaction with the third compound. In some embodiments, producing the fourth compound comprises performing an amide coupling reaction with the third compound. In some embodiments, producing the fourth compound comprises performing a nucleophilic addition reaction with the third compound. In some embodiments, producing the fifth compound includes reacting the fourth compound with at least one acid. In some embodiments, the at least one acid comprises at least one of hydrochloric acid and trifluoroacetic acid. In some embodiments, producing the sixth compound comprises performing an _SN2 substitution reaction with the fifth compound. In some embodiments, producing the sixth compound comprises performing a reductive amination reaction with the fifth compound. In some embodiments, producing the sixth compound comprises performing a Michael addition reaction with the fifth compound. In some embodiments, producing the seventh compound comprises performing an _SN2 substitution reaction with the sixth compound. In some embodiments, producing the seventh compound comprises performing a reductive amination reaction with the sixth compound. In some embodiments, producing the seventh compound comprises performing a Michael addition reaction with the sixth compound. In some embodiments, producing the seventh compound comprises performing an amide coupling reaction with the sixth compound. In some embodiments, producing the seventh compound comprises performing a nucleophilic addition reaction with the sixth compound. In some embodiments, the first amide is coupled to the first targeting ligand using an independently selected linker A as described above. In some embodiments, the second amide is coupled to the second targeting ligand using an independently selected linker A as described above. In some embodiments, the third amide is coupled to the third targeting ligand using an independently selected linker A as described above. In some embodiments, the first targeting ligand, the second targeting ligand, and the third targeting ligand are independently selected as one or more of the targeting ligands described above. In some embodiments, the hydroxyl group is coupled to the second nitrogen using Linker B described above. In some embodiments, the method further comprises converting the hydroxyl group to a phosphoramidite group using a phosphoritylation reaction. In some embodiments, converting a hydroxyl group to a phosphoramidite group is performed after generating the thirteenth compound.

Another aspect of the present disclosure pertains to a method for delivering an agent to a subject, the method comprising administering to the subject (a) a compound as described above, wherein W is one or more agents, or (b) a composition as described above. In some embodiments, the subject is a vertebrate. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the compounds are administered in a pharmaceutically acceptable carrier.

Another aspect of the present disclosure relates to a method for delivering a medicament to a subject, the method comprising administering to the subject the pharmaceutical composition described above. In some embodiments, the subject is a vertebrate. In some embodiments, the subject is a mammal, optionally, the mammal is a human. In some embodiments, the one or more agents comprise small interfering RNA (siRNA), single-stranded siRNA, double-stranded siRNA, small activating RNA, microRNA (miRNA), antisense oligonucleotides, short guide RNA At least one of (gRNA), single guide RNA (sgRNA), messenger RNA (mRNA), ribozyme, plasmid, immunostimulatory nucleic acid, antagomir and aptamer. In some embodiments, the double-stranded siRNA comprises at least one modified ribonucleotide in one or both strands of the siRNA. In some embodiments, substantially all ribonucleotides of the double-stranded siRNA are modified. In some embodiments, all ribonucleotides of the double-stranded siRNA are modified. In some embodiments, the modified ribonucleotides comprise: 2'-O-methyl nucleotides, 2'-fluoro nucleotides, 2'-deoxy nucleotides, 2'3'-seco Nucleotide mimetics, locked nucleotides, 2'-F-arabinonucleotides, 2'-methoxyethyl nucleotides, abasic nucleotides, ribitol, inverted nucleotides, Inverted abasic nucleotides, inverted 2'-OMe nucleotides, inverted 2'deoxynucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, Phylino and 3'-OMe nucleotides, nucleotides containing a 5'-phosphorothioate group, or 5'-(E)-vinylphosphonate nucleotides (antisense strand only ), or terminal nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, phosphoramidates, or nucleotides containing unnatural bases. In some embodiments, at least one strand of the double-stranded siRNA comprises at least one phosphorothioate linkage. In some embodiments, at least one strand of the double-stranded siRNA comprises up to 6 phosphorothioate linkages. In some embodiments, the double-stranded siRNA comprises at least one locked nucleic acid. In some embodiments, the double-stranded siRNA comprises at least one unlocking nucleic acid. In some embodiments, the double-stranded siRNA comprises at least one glycerol nucleic acid. In some embodiments, the pharmaceutical composition further comprises one or more therapeutic agents.

Another aspect of the disclosure is a compound for use in delivering a medicament to a subject. In some embodiments, the subject is a vertebrate. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the compounds are administered in a pharmaceutically acceptable carrier.

In another aspect of the disclosure, the dsRNA reagent comprises a 2'- Fluoromodified nucleotides, and/or contain 2'-fluoromodified nucleotides at positions 9, 11, and 13 of the sense strand (counting from the first pair of nucleotides at the 3' end of the sense strand) Modified nucleotides.

overview

The present disclosure provides multivalent ligand clusters with diamine scaffolds for targeted delivery of agents conjugated thereto. In some embodiments, the multivalent ligand cluster may comprise one or more N-acetylgalactosamine (GalNAc) targeting ligands. In some embodiments, the cluster of multivalent ligands can be conjugated to one or more small interfering ribonucleic acids (siRNAs), wherein an example of an agent is siRNA. The present disclosure also provides compositions comprising the multivalent ligand clusters of the present disclosure, as well as methods of making and using the multivalent ligand clusters of the present disclosure. definition

Before further describing the present invention, and in order that the present invention may be more easily understood, some terms are firstly defined and summarized herein for convenience.

As used herein, the term "treatment" and variations thereof may include prophylaxis and means to ameliorate symptoms, alleviate symptoms, eliminate the cause of symptoms, or prevent or slow down the specified disorder on a temporary or permanent basis or symptoms of disease.

The term "about" as used herein in relation to a measurement refers to the normal variation in that measurement as would be expected by one of skill in the art when making the measurement and with a level of care commensurate with the purpose of the measurement and the precision of the measuring equipment.

The term "conjugate" or "conjugate group" as used herein means an atom or group of atoms bound to an oligonucleotide or other oligomer. In general, a conjugate group modifies one or more properties of the compound to which it is attached, including but not limited to pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge and/or Clear properties.

As used herein, the term "connected" when referring to a link between two molecules means that the two molecules are directly or indirectly connected by a covalent bond, or that two molecules are connected by a non-covalent bond (for example, a hydrogen bond or an ionic bond). ) association. An example of a direct link of Compound A to Compound B can be denoted as A-B. An example of the indirect linkage of Compound A to Compound B can be represented as A-C-B, wherein Compound A is indirectly linked to Compound B via Compound C. It is understood that where compounds are linked indirectly there may be more than one intermediate compound.

The term "nucleic acid" as used herein refers to a molecule composed of monomeric nucleotides. Nucleic acids include ribonucleic acid (RNA), deoxyribonucleic acid (DNA), single-stranded nucleic acid (ssDNA), double-stranded nucleic acid (dsDNA), small interfering ribonucleic acid (siRNA) and microRNA (miRNA). A nucleic acid can also contain any combination of these elements in a single molecule. Nucleic acids can include natural nucleic acids, non-natural nucleic acids, or a combination of natural and non-natural nucleic acids. A nucleic acid may also be referred to herein as a nucleotide sequence or polynucleotide.

The term "oligomer" as used herein refers to a nucleotide sequence comprising up to 5, up to 10, up to 15, up to 20 or more than 20 nucleotides or nucleotide base pairs. In some embodiments, the oligomer has a nucleobase sequence that is at least partially complementary to a coding sequence in a target nucleic acid or target gene expressed in the cell. In some embodiments, the oligomer is capable of inhibiting the expression of the underlying gene upon delivery to a gene-expressing cell. Gene expression can be inhibited in vitro or in vivo. Some non-limiting examples of oligomers that can be included in the methods and complexes of the invention are: oligonucleotides, single stranded oligonucleotides, single stranded antisense oligonucleotides, short interfering RNA (short interfering RNA, siRNA), single-stranded siRNA, double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (short hairpin RNA, shRNA), ribozymes, interfering RNA molecules, and dicer substrates .

The term "oligonucleotide" as used herein refers to a polymer of linked nucleotides, each of which may independently be modified or unmodified.

As used herein, the term "single-stranded oligonucleotide" refers to a single-stranded oligomer, and in certain embodiments, a single-stranded oligonucleotide may comprise a sequence that is at least partially complementary to a target mRNA that is capable of Hybridizes to target mRNA via hydrogen bonding under physiological animal conditions (or similar in vitro conditions). In some embodiments, the single-stranded oligonucleotide is a single-stranded antisense oligonucleotide.

The term "siRNA" as used herein refers to short interfering RNA or silencing RNA. siRNAs are a class of double-stranded RNA molecules that can be 20 to 25 (or shorter) base pairs in length, similar to microRNAs (miRNAs) that function in the RNA interference (RNAi) pathway. siRNA interferes with the expression of a specific gene having a nucleotide sequence complementary to siRNA by degrading mRNA after transcription, thereby preventing translation. siRNA silences gene expression in cells by inducing the RNA-induced silencing complex (RISC) to cleave messenger RNA (mRNA).

As used herein, the terms "effective amount", "therapeutically effective amount" or "effective dose" refer to an amount sufficient to cause a desired pharmacological or therapeutic effect, resulting in effective prevention or treatment of a condition. Prevention of a disorder is manifested by delaying the onset of symptoms of the disorder to a medically significant degree. Treatment of a disorder is manifested by reducing symptoms associated with the disorder or ameliorating recurrence of disease symptoms.

The term "pharmaceutical composition" or "composition" as used herein refers to a mixture of substances suitable for administration to an individual. For example, although not intended to be limiting, a pharmaceutical composition can comprise one or more active agents and a pharmaceutically acceptable carrier, also referred to herein as a "pharmaceutically acceptable carrier" (eg, a sterile aqueous solution). In some embodiments, pharmaceutical compositions are sterile.

As used herein, the term "alkyl" when used (alone or as part of another group) means containing one to twelve carbon atoms (i.e., C _1-12 alkyl) or a specified number of carbon atoms (i.e. , C ₁ alkyl such as methyl, C ₂ alkyl such as ethyl, C ₃ alkyl such as propyl or isopropyl, etc.) linear or branched aliphatic hydrocarbons. Some non-limiting exemplary C _1-10 alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, t-butyl, isobutyl, 3-pentyl, hexyl, heptyl , octyl, nonyl, decyl, etc.

The term "substituted alkyl" (alone or as part of another group) as used herein means that an alkyl group as defined herein is independently selected by one or more (eg, one, two or three) of substituents. A non-limiting list of independently selected substituents includes amino, (alkyl)amino, (alkyl)carbonyl, (aryl)carbonyl, (alkoxy)carbonyl, [(alkoxy)carbonyl]amino, carboxy , aryl, heteroaryl, ureido, guanidino, halogen, sulfonamide, hydroxyl, (alkyl)sulfanyl, nitro, haloalkoxy, aryloxy, aralkyloxy, (alk (yl)sulfonyl, (cycloalkyl)sulfonyl, (aryl)sulfonyl, cycloalkyl, sulfanyl, carboxamido, heterocyclyl and (heterocyclyl)sulfonyl base.

The term "cycloalkyl" (alone or as part of another group) as used herein refers to saturated and partially unsaturated (containing one or two double bonds) cyclic aliphatic hydrocarbons containing one to three Rings of three to twelve carbon atoms (ie, C _3-12 cycloalkyl) or the specified number of carbons. Some non-limiting exemplary cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, norbornyl, decalin, adamantyl, Cyclohexenyl, cyclopentenyl, cyclohexenyl, and the like.

The term "substituted cycloalkyl" (alone or as part of another group) as used herein means that a cycloalkyl group as defined herein is substituted with one, two or three independently selected substituents. A non-limiting list of independently selected substituents includes halo, nitro, cyano, hydroxy, amino, (alkyl)amino, (dialkyl)amino, haloalkyl, (hydroxy)alkyl, (dihydroxy) Alkyl, alkoxy, haloalkoxy, aryloxy, aralkoxy, alkylthio, formamido, sulfonamide, (alkyl)carbonyl, (aryl)carbonyl, (alkyl) Sulfonyl, arylsulfonyl, ureido, guanidino, carboxyl, (carboxy)alkyl, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, (alk Oxy)alkyl, (amino)alkyl, (hydroxy)alkylamino, (alkylamino)alkyl, (dialkylamino)alkyl, (cyano)alkyl, (formamido)alkane (alkyl)sulfanyl, (heterocyclo)alkyl, (heteroaryl)alkyl, (alkoxy)carbonyl and mercaptoalkyl.

The term "alkenyl" (alone or as part of another group) as used herein refers to an alkyl group as defined herein containing one, two or three carbon-carbon double bonds. Some non-limiting exemplary alkenyl groups include ethenyl, propenyl, isopropenyl, butenyl, sec-butenyl, pentenyl, and hexenyl.

The term "substituted alkenyl" (alone or as part of another group) as used herein means that an alkenyl group as defined herein is substituted with one, two or three independently selected substituents. A non-limiting list of independently selected substituents includes halo, nitro, cyano, hydroxy, amino, (alkyl)amino, (dialkyl)amino, haloalkyl, (hydroxy)alkyl, (dihydroxy) Alkyl, alkoxy, haloalkoxy, aryloxy, aralkoxy, (alkyl)sulfanyl, formamido, sulfonamido, (alkyl)carbonyl, (aryl)carbonyl, (Alkyl)sulfonyl, (aryl)sulfonyl, ureido, guanidino, carboxy, (carboxy)alkyl, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl and heterocyclyl.

As used herein, the term "cycloalkenyl" (alone or as part of another group) refers to a non-aromatic cycloalkyl group having from 4 to 10 carbon atoms having single or multiple cyclic ring and have at least one >C=C< ring unsaturation and preferably >C=C< 1 to 2 sites of ring unsaturation.

The term "substituted cycloalkenyl" (alone or as part of another group) as used herein refers to a cycloalkenyl group as defined herein having 1 to 5 independently selected substituents. A non-limiting list of independently selected substituents includes oxo, thione, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy Amyl, Acyl, Amino, Acyloxy, Amino, Substituted Amino, Aminocarbonyl, Aminothiocarbonyl, Aminocarbonylamino, Aminothiocarbonylamino, Aminocarbonyloxy, Aminosulfonyl, Aminosulfonyl Oxy, aminosulfonylamino, amidino, aryl, substituted aryl, aryloxy, substituted aryloxy, arylthio, substituted arylthio, azido, carboxyl, carboxyl ester, ( Carboxyl ester) amino, (carboxy ester) oxy, cyano, cyanate, cycloalkyl, substituted cycloalkyl, cycloalkoxy, substituted cycloalkoxy, cycloalkylthio, substituted cycloalkane Thio, cycloalkenyl, substituted cycloalkenyl, cycloalkenyloxy, substituted cycloalkenyloxy, cycloalkenylthio, substituted cycloalkenylthio, guanidino, substituted guanidino, halogen, hydroxy, hydroxy Amino, alkoxyamino, hydrazino, substituted hydrazino, heteroaryl, substituted heteroaryl, heteroaryloxy, substituted heteroaryloxy, heteroarylthio, substituted heteroarylthio, heteroaryl Cyclic, substituted heterocyclyl, heterocyclyloxy, substituted heterocyclyloxy, heterocyclylthio, substituted heterocyclylthio, nitro, _SO3H , substituted sulfonyl, Sulfonyloxy, thioacyl, thiocyanate, thiol, alkylthio and substituted alkylthio.

The term "alkynyl" (alone or as part of another group) as used herein refers to an alkyl group as defined herein comprising one to three carbon-carbon triple bonds. Some non-limiting exemplary alkynyl groups include ethynyl, propynyl, butynyl, 2-butynyl, pentynyl, and hexynyl.

The term "substituted alkynyl" (alone or as part of another group) as used herein means that an alkynyl group as defined herein is substituted with one, two or three independently selected substituents. A non-limiting list of independently selected substituents includes halo, nitro, cyano, hydroxy, amino, alkylamino, dialkylamino, haloalkyl, (hydroxy)alkyl, (dihydroxy)alkyl, alkane Oxy, haloalkoxy, aryloxy, aralkoxy, (alkyl)sulfanyl, formamido, sulfonamide, alkylcarbonyl, arylcarbonyl, alkylsulfonyl, aryl Sulfonyl, ureido, guanidino, carboxy, (carboxy)alkyl, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl and heterocyclyl.

As used herein, the term "haloalkyl" (alone or as part of another group) refers to an alkyl group substituted with one or more fluorine, chlorine, bromine and/or iodine atoms. Some non-limiting exemplary haloalkyl groups include fluoromethyl, difluoromethyl, trifluoromethyl, pentafluoroethyl, 1,1-difluoroethyl, 2,2-difluoroethyl, 2,2, 2-trifluoroethyl, 3,3,3-trifluoropropyl, 4,4,4-trifluorobutyl and trichloromethyl.

As used herein, the term "alkoxy" (alone or as part of another group) refers to an alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, Substituted alkenyl, alkynyl or substituted alkynyl.

The term "haloalkoxy" (alone or as part of another group) as used herein refers to a haloalkyl group attached to a terminal oxygen atom. Some non-limiting exemplary haloalkoxy groups include fluoromethoxy, difluoromethoxy, trifluoromethoxy, and 2,2,2-trifluoroethoxy.

As used herein, the term "aryl" (alone or as part of another group) refers to a monocyclic or bicyclic aromatic ring system having six to fourteen carbon atoms (ie, C ₆ -C ₁₄ aryl). Some non-limiting exemplary aryl groups include phenyl, naphthyl, phenanthrenyl, anthracenyl, indenyl, azulenyl, biphenyl, biphenylenyl, and fluorenyl.

The term "substituted aryl" (alone or as part of another group) as used herein means an aryl group as defined herein substituted with one to five independently selected substituents. A non-limiting list of independently selected substituents includes halo, nitro, cyano, hydroxy, amino, alkylamino, dialkylamino, haloalkyl, (hydroxy)alkyl, (dihydroxy)alkyl, alkane Oxygen, haloalkoxy, aryloxy, heteroaryloxy, aralkoxy, alkylthio, formamido, sulfonamide, (alkyl)carbonyl, (aryl)carbonyl, (alkyl )sulfonyl, (aryl)sulfonyl, ureido, guanidine, carboxy, carboxyalkyl, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, ( Alkoxy)alkyl, (amino)alkyl, [(hydroxy)alkyl]amino, [(alkyl)amino]alkyl, [(dialkyl)amino)alkyl, (cyano)alkyl, (formamido)alkyl, mercaptoalkyl, (heterocyclo)alkyl, (cycloalkylamino)alkyl, (halo(C ₁ -C ₄ )alkoxy)alkyl, (heteroaryl ) alkyl, etc. Some non-limiting exemplary substituted aryl groups include 2-methylphenyl, 2-methoxyphenyl, 2-fluorophenyl, 2-chlorophenyl, 2-bromophenyl, 3-methylphenyl , 3-methoxyphenyl, 3-fluorophenyl, 3-chlorophenyl, 4-methylphenyl, 4-ethylphenyl, 4-methoxyphenyl, 4-fluorophenyl, 4 -Chlorophenyl, 2,6-di-fluorophenyl, 2,6-di-chlorophenyl, 2-methyl, 3-methoxyphenyl, 2-ethyl, 3-methoxyphenyl , 3,4-di-methoxyphenyl, 3,5-di-fluorophenyl, 3,5-di-methylphenyl, 3,5-dimethoxy, 4-methylphenyl, 2-fluoro-3-chlorophenyl and 3-chloro-4-fluorophenyl. The term substituted aryl is intended to include groups having fused substituted cycloalkyl groups and fused substituted heterocyclic rings.

As used herein, the term "aryloxy" (alone or as part of another group) refers to an aryl or substituted aryl group attached to a terminal oxygen atom. A non-limiting exemplary aryloxy group is PhO-.

As used herein, the term "heteroaryloxy" (alone or as part of another group) refers to a heteroaryl or substituted heteroaryl group attached to a terminal oxygen atom.

As used herein, the term "aralkoxy" (alone or as part of another group) refers to an aralkyl group attached to a terminal oxygen atom. A non-limiting exemplary aralkoxy group is _PhCH2O- .

As used herein, the term "heteroaryl" means a group having 5 to 14 ring atoms (ie, C ₅ -C ₁₄ heteroaryl) and independently selected from oxygen (O), nitrogen (N) and sulfur (S) Monocyclic and bicyclic aromatic ring systems of 1, 2, 3 or 4 heteroatoms. Some non-limiting exemplary heteroaryl groups include thienyl, benzo[b]thienyl, naphtho[2,3-b]thienyl, thienthyl, furyl, benzofuryl, pyranyl, iso Benzofuryl, benzooxazolyl, chromenyl, xanthenyl, 2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, Pyrimidinyl (pyrimidinyl), pyridazinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl, isoquinolyl, quinolinyl, phthalazinyl (phthalazinyl), naphthyridine Naphthyridinyl, cinnolinyl, quinazolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl, β-carbolinyl , phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl, phenazinyl, thiazolyl, isothiazolyl, phenothiazinyl (phenothiazolyl), isoxazolyl (isoxazolyl), furazanyl (furazanyl) and phenoxazinyl (phenoxazinyl). The term "heteroaryl" is also meant to include possible N-oxides. Exemplary N-oxides include pyridyl N-oxides, and the like.

The term "substituted heteroaryl" (alone or as part of another group) as used herein means a heteroaryl group as defined herein substituted with one to four independently selected substituents. A non-limiting list of independently selected substituents includes halo, nitro, cyano, hydroxy, amino, (alkyl)amino, (dialkyl)amino, haloalkyl, (hydroxy)alkyl, (dihydroxy) Alkyl, alkoxy, haloalkoxy, aryloxy, aralkoxy, alkylthio, formamido, sulfonamide, (alkyl)carbonyl, (aryl)carbonyl, (alkyl) Sulfonyl, (aryl)sulfonyl, ureido, guanidino, carboxy, (carboxy)alkyl, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocycle, ( Alkoxy)alkyl, (amino)alkyl, [(hydroxy)alkyl]amino, [(alkyl)amino]alkyl, [(dialkyl)amino]alkyl, (cyano)alkyl, (formamido)alkyl, mereaptoalkyl, (heterocyclo)alkyl and (heteroaryl)alkyl. Any available carbon or nitrogen atoms may be substituted.

As used herein, the term "heterocycle" or "heterocyclyl" (alone or as part of another group) means a group comprising one, two or three ring members having three to fourteen (ie, 3- to 14-membered heterocycles) and saturated and partially unsaturated (eg, containing one or two double bonds) ring groups of at least one heteroatom. Each heteroatom is independently selected. The term "heterocycle" or "heterocyclyl" is intended to include cyclic urea groups (e.g., 2-imidazolidinone) and cyclic amide groups (e.g., β-lactam, γ-lactam, δ- lactam and ε-lactam). The terms "heterocycle" or "heterocyclyl" are also intended to include groups having fused or substituted aryl groups, such as indolinyl. A heterocycle or heterocyclyl group can be attached to the rest of the molecule through a carbon or nitrogen atom. Some non-limiting exemplary heterocyclic (or heterocyclyl) groups include 2-oxopyrrolidin-3-yl, 2-imidazolidinone, piperidinyl, morpholinyl, piperazinyl, pyrrolidinyl, and Indolinyl.

The term "substituted heterocycle" or "substituted heterocyclyl" (alone or as part of another group) as used herein means a heterocycle or heterocyclyl as defined above replaced by one to four independently selected substituents replace. A non-limiting list of independently selected substituents includes halo, nitro, cyano, hydroxy, amino, (alkyl)amino, (dialkyl)amino, haloalkyl, (hydroxy)alkyl, (dihydroxy) Alkyl, alkoxy, haloalkoxy, aryloxy, aralkoxy, alkylthio, formamido, sulfonamide, (alkyl)carbonyl, (aryl)carbonyl, (alkyl) Sulfonyl, (aryl)sulfonyl, ureido, guanidino, carboxy, carboxyalkyl, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, alkoxy ylalkyl, (amino)alkyl, [(hydroxy)alkyl]amino, [(alkyl)amino]alkyl, [(dialkyl)amino]alkyl, (cyano)alkyl, (formyl Amino)alkyl, mercaptoalkyl, (heterocyclyl)alkyl and (heteroaryl)alkyl. Substitution can occur at any available carbon or nitrogen atom, and spirocycles can be formed.

As used herein, the term "amino" (alone or as part of another group) refers to _-NH2 .

The term "alkylamino" or "(alkyl)amino" (alone or as part of another group) as used herein refers to -NHR, where R is alkyl.

The term "dialkylamino" or "(dialkyl)amino" (alone or as part of another group) as used herein refers to -NR'R'', where R' and R'' are each independently is alkyl or R' and R'' together form a 3- to 8-membered heterocyclic or substituted heterocyclic ring.

As used herein, the term "cycloalkylamino" (alone or as part of another group) refers to -NR'R'', where R' is cycloalkyl or substituted cycloalkyl, and R'' is hydrogen or alkyl.

The term "(amino)alkyl" (alone or as part of another group) as used herein refers to an alkyl group substituted with an amino group. _Some non- _limiting exemplary ₍ amino) _{alkyl groups include -CH2CH2NH2 , -CH2CH2CH2NH2 ,} and _{-CH2CH2CH2CH2NH2 .}

The term "(alkylamino)alkyl" or "[(alkyl)amino]alkyl" (alone or as part of another group) as used herein refers to an alkyl group substituted with an alkylamino group. A non-limiting exemplary ( _alkylamino )alkyl group is _-CH2CH2N (H) _CH3 .

The term "(dialkylamino)alkyl" (alone or as part of another group) as used herein refers to an alkyl group substituted with a dialkylamino group. Some non-limiting exemplary (dialkylamino)alkyl groups include -CH ₂ N(CH ₃ ) ₂ and -CH ₂ CH ₂ N(CH- ₃ ) ₂ .

The term "(cycloalkylamino)alkyl" (alone or as part of another group) as used herein refers to an alkyl group substituted with a cycloalkylamino group. Some non-limiting exemplary (cycloalkylamino)alkyl groups include _-CH2N (H)cyclopropyl, _-CH2N (H)cyclobutyl, and _-CH2N (H)cyclohexyl.

As used herein, the term "formamido" (alone or as part of another group) refers to a group of formula -C(=O)NR'R'', wherein R' and R'' are each independently is hydrogen, alkyl, substituted alkyl, aralkyl, substituted aralkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl, or to which R' and R'' are attached The nitrogens together form a 3- to 8-membered heterocyclic group. Some non-limiting exemplary formamide groups include _-CONH2 , -CON(H) _CH3 , CON( _CH3 ) ₂ , and CON(H)Ph.

As used herein, the term "sulfonylamino" (alone or as part of another group) refers to a group of formula -SO ₂ NR'R'', wherein R' and R'' are each independently hydrogen, Alkyl, substituted alkyl, aryl, or substituted aryl, or R' and R'' together with the nitrogen to which they are attached form a 3- to 8-membered heterocyclic group. Some non-limiting exemplary sulfonamide groups include -SO ₂ NH ₂ , -SO ₂ N(H)CH ₃ , and -SO ₂ N(H)Ph.

The term "(alkyl)carbonyl" (alone or as part of another group) as used herein refers to a carbonyl group substituted with an alkyl group, ie -C(=O)-. A non-limiting exemplary alkylcarbonyl group is -COCH ₃ .

The term "(alkoxy)carbonyl" (or "ester") as used herein, alone or as part of another group, refers to a carbonyl group substituted with an alkoxy group, ie -C(=O)-. A non-limiting exemplary (alkoxy)carbonyl group is -C(O) _OCH3 .

The term "(aryl)carbonyl" (alone or as part of another group) as used herein refers to a carbonyl group substituted with an aryl or substituted aryl, ie -C(=O)-. A non-limiting exemplary arylcarbonyl group is -COPh.

As used herein, the term "sulfanyl" (alone or as part of another group) refers to a -SH group.

The term "(alkyl)sulfanyl" or "alkylthio" (alone or as part of another group) as used herein refers to a sulfur atom substituted with an alkyl or substituted alkyl. Some non- _limiting exemplary alkylthio groups include _-SCH3 and _-SCH2CH3 .

The term "mercaptoalkyl" (alone or as part of another group) as used herein refers to an alkyl group substituted with a -SH group.

As used herein, the term "alkylsulfonyl" or "(alkyl)sulfonyl" (alone or as part of another group) refers to a sulfonyl group substituted with an alkyl or substituted alkyl, i.e. -SO ₂ -. A non-limiting exemplary alkylsulfonyl group is -SO ₂ CH ₃ .

As used herein, the term "arylsulfonyl" or "(aryl)sulfonyl" (alone or as part of another group) refers to a sulfonyl group substituted with an aryl or substituted aryl, i.e. -SO ₂ -. A non-limiting exemplary arylsulfonyl group is -SO ₂ Ph.

As used herein, the term "carboxy" (alone or as part of another group) refers to a group of formula -COOH.

The term "(carboxy)alkyl" (alone or as part of another group) as used herein refers to an alkyl group substituted with -COOH. A non-limiting exemplary carboxyalkyl is _{-CH2CO2H .}

The term "aralkyl" (alone or as part of another group) as used herein refers to a residue in which the aryl moiety is attached to an alkyl residue. Aralkyl groups can be attached to the parent structure at either aryl or alkyl residues.

The term "substituted aralkyl" (alone or as part of another group) as used herein refers to a residue wherein the aryl moiety is attached to a substituted alkyl residue.

As used herein, the term "arylalkenyl" (alone or as part of another group) refers to a group of formula _-Rd - _Rc , where _Rd is an alkenylene chain and _Rc is one or more an aryl group.

As used herein, the term "substituted aralkenyl" (alone or as part of another group) refers to an aralkenyl group in which the alkenylene chain of the aralkenyl group is optionally substituted an alkenylene chain, and each aryl group in the aralkenyl group is an optionally substituted aryl group.

As used herein, the term "aralkynyl" (alone or as part of another group) refers to a group of formula -R _e R _c , where R _e is an alkynylene chain and R _c is one or more aryl group.

As used herein, the term "substituted aralkynyl" (alone or as part of another group) refers to an aralkynyl group in which the alkynylene chain of the aralkynyl group is optionally substituted an alkynylene chain, and each aryl group in the aralkynyl group is an optionally substituted aryl group.

As used herein, the term "aliphatic heterocycle" (alone or as part of another group) refers to a non-aromatic ring in which one or more atoms forming the ring are heteroatoms.

The term "heteroatom" as used herein refers to an atom inserted between a carbon atom and its parent molecule (ie, between the points of attachment). Some non-limiting exemplary heteroatoms include oxygen, nitrogen, sulfur (including phosphonium and phosphonium), and phosphorus (P).

The term "sugar" as used herein refers to a single sugar moiety or monosaccharide unit as well as combinations of two or more single sugar moieties or monosaccharide units covalently linked to form disaccharides, oligosaccharides and polysaccharides. Polysaccharides can be linear or branched.

The term "monosaccharide" as used herein refers to a single sugar residue in an oligosaccharide.

The term "disaccharide" as used herein refers to a polysaccharide consisting of two monosaccharide units or moieties linked together by glycosidic bonds.

"Oligosaccharide" as used herein refers to a compound comprising two or more monosaccharide units or moieties. In the case of oligosaccharides, a single monomeric unit or moiety is a monosaccharide which is or can be bound to another monosaccharide unit or moiety via a hydroxyl group. Oligosaccharides can be prepared by chemical synthesis from protected single residue sugars or by chemical degradation of biologically produced polysaccharides. Alternatively, oligosaccharides can be prepared by in vitro enzymatic methods.

As used herein, the term "ureido" (alone or as part of another group) refers to a group of formula -NR'-C(=O)-NR''R'', where R' is hydrogen, alkane radical, aryl, or substituted aryl, and R" and R"" are each independently hydrogen, alkyl, aryl, or substituted aryl, or R" and R"" together with the nitrogen to which they are attached form 4 - to 8-membered heterocyclyl. Some non-limiting exemplary ureido groups include -NH-C(=O) _-NH2 and -NH-C(=O) _-NHCH3 .

The term "guanidino" (alone or as part of another group) as used herein refers to a group of formula -NR'-C(=NR'')-NR'''R'''', where R ', R''', and R'''' are each independently hydrogen, alkyl, aryl, or substituted aryl, and R'' is hydrogen, alkyl, cyano, alkylsulfonyl, alkyl Carbonyl, formamido or sulfonamide. Some non-limiting exemplary guanidine groups include -NH-C(=NH) _-NH2 , -NH-C(=NCN) _-NH2 , and -NH-C(=NH) _-NHCH3 .

The term "(heteroaryl)alkyl" (alone or as part of another group) as used herein refers to an alkyl group substituted with one, two or three heteroaryl or substituted heteroaryl groups.

The term "heteroalkyl" (alone or as part of another group) as used herein refers to a stable straight or branched chain hydrocarbyl group comprising at least one heteroatom which may be the same or different. The heteroatom can be located at any internal or terminal position of the heteroalkyl group, or at the position where the heteroalkyl group is attached to the rest of the molecule. Some non-limiting exemplary heteroalkyl groups include -CH ₂ N(H)CH ₂ CH ₂ N(CH ₃ ) ₂ , -CH ₂ N(CH ₃ )CH ₂ CH ₂ N(CH ₃ ) ₂ , -CH ₂ N(H)CH ₂ CH ₂ CH ₂ N(CH ₃ ) ₂ , -CH ₂ N(H)CH ₂ CH ₂ OH, -CH ₂ N(CH ₃ )CH ₂ CH ₂ OH, -CH ₂ OCH ₂ CH ₂ OCH ₃ , -OCH ₂ CH ₂ OCH ₂ CH ₂ OCH ₃ , -CH ₂ NHCH ₂ CH ₂ OCH ₂ , -OCH ₂ CH ₂ NH ₂ , and -NHCH ₂ CH ₂ N(H)CH ₃ .

The term "(heterocyclyl)alkyl" or "(heterocyclyl)alkyl" (alone or as part of another group) as used herein means a heterocyclyl or substituted heterocyclyl and optionally An alkyl group substituted with a hydroxy group.

As used herein, the term "(formylamino)alkyl" (alone or as part of another group) refers to a group consisting of a formylamino group and optionally a heterocyclyl, amino, alkylamino or dialkyl group. Amino substituted alkyl.

The term "N-oxide" as used herein refers to compounds containing functional groups where N ⁺ is also attached to H and/or the rest of the compound structure.

As used herein, the term "integer" refers to integers including, but not limited to, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and the like. Compound General Structure

Multivalent ligand clusters with diamine scaffolds can have the general structure of formula 1: Formula 1 wherein: each TL is an independently selected targeting ligand, m is an integer from 1 to 10, each n is an independently selected integer from 1 to 10, each linker A is an independently selected spacer, one end is linked to TL and the other end is linked to the nitrogen of an alkylformamide, linker B is a spacer, one end is linked to an agent or a functional group capable of linking one or more agents, and the other end is linked to a diamine nitrogen, and W is one or more agents, or is a functional group capable of linking to one or more agents.

In some embodiments, m can be configured based on the starting materials used to synthesize the multivalent ligand cluster. For example, m can be 1 due to the use of ethylenediamine as the starting material; m can be 2 due to the use of 1,3-propylenediamine as the starting material; m can be 2 due to the use of 1,4-butanediamine as the starting material, m can be 3.

As used herein, a "spacer" refers to a compound or molecule that links other groups together. Exemplary Linker A spacers and Linker B spacers are described in detail herein below.

The term "independently selected" is used herein when referring to elements of a multivalent ligand cluster of the invention to mean that each of a given type of element may be associated with a different other elements of the same type. For example, a cluster of multivalent ligands may comprise more than one TL, wherein each TL may be selected to be different or the same as one or more other TLs in the cluster of multivalent ligands. As another example, a multivalent ligand cluster may comprise more than one "n", wherein each "n" may be selected to be different or the same as one or more other "n" in the multivalent ligand cluster. In another example, a cluster of multivalent ligands may comprise more than one linker A, wherein each linker A may be selected to be different or the same as one or more other linkers A in the cluster of multivalent ligands. targeting ligand

As mentioned above with respect to Formula 1, a multivalent ligand cluster of the present disclosure may comprise a plurality (eg, three) of independently selected targeting ligands. In this context, the term "independently selected" means that each targeting ligand may be selected to be different or identical to one or more other targeting ligands in the same cluster of multivalent ligands.

At least one of the independently selected targeting ligands of Formula 1 may be capable of binding to one or more cellular receptors, cellular channels, and/or cellular transporters capable of promoting endocytosis.

In some embodiments, at least one of the independently selected targeting ligands of Formula 1 can comprise at least one small molecule ligand. As used herein, "small molecule ligand" refers to a ligand that is smaller than a protein. In some embodiments, the at least one small molecule ligand may comprise N-acetylgalactosamine (GalNAc), galactose, galactosamine, N-formyl-galactosamine, N-acrylgalactosamine , N-butyrylgalactosamine and N-isobutyrylgalactosamine, macrocycles, folic acid molecules, fatty acids, bile acids, cholesterol and derivatives thereof.

Macrocycles are molecules or ions that contain twelve or more membered rings. The present disclosure is not limited to any particular macrocycle. A non-limiting list of macrocycles within the scope of the present disclosure includes terpenoid macrocycles, porphyrins, and cyclodextrins.

Folate, also known as vitamin B9 and folic acid (folacin), is used by the body to produce DNA and RNA and metabolize amino acids necessary for cell division. Folate receptors bind folate and reduced folate derivatives. Thus, in some embodiments, at least one of the independently selected targeting ligands may comprise a reduced folate derivative.

Fatty acids are carboxylic acids with long fatty chains. In some embodiments, fatty acids may be saturated, meaning that the fatty chains all have carbon-carbon single bonds. In some embodiments, the fatty acid may be unsaturated, meaning that the fatty chain contains at least one carbon-carbon double bond or carbon-carbon triple bond. In some embodiments, fatty acids may contain branched chains. In some embodiments, the fatty acid may comprise a ring structure. Fatty acids are known to aid in the renewal of agents into cells. See Prakash et al . “Fatty acid conjugation enhances potency of antisense oligonucleotides in muscle.” Nucleic Acids Res. 2019;47(12):6029-6044. doi:10.1093/nar/gkz354; Raouane et al . “Lipid Conj ugated Oligonucleotides: A Useful Strategy for Delivery.” Bioconjugate Chemistry 2012 23 (6), 1091-1104, DOI: 10.1021/bc200422w; and Osborn et al . “Improving siRNA Delivery In Vivo Through Lipid Conjugation.” Nucleic Acid Ther. 2018;28(3 ):128-136. doi: 10.1089/nat.2018.0725.

Bile acids are steroidal acids present in bile. Bile acids are known ligands of farnesoid X receptor (FXR) and G protein-coupled bile acid receptor 1 (GPBAR1) (TGR5). In some embodiments, the bile acid can be a primary bile acid synthesized in the liver. In some embodiments, the bile acids may be secondary bile acids produced by bacterial action in the colon. Bile acids are known to be beneficial in inhibiting RNA translation. See González-Carmona et al . “Inhibition of hepatitis C virus RNA translation by antisense bile acid conjugated phosphorothioate modified oligodeoxynucleotides (ODN).” Antiviral Res. 2013, 97, 49–59. doi:10.1089/nat .2018.0725.

In some embodiments, at least one of the independently selected targeting ligands of Formula 1 may comprise at least one peptide. A variety of peptides and corresponding peptide receptors are known to those skilled in the art. This disclosure is not limited to any particular peptide. Known and as yet undiscovered peptides are within the scope of this disclosure.

In some embodiments, at least one of the independently selected targeting ligands of Formula 1 may comprise at least one cyclic peptide. As known to those skilled in the art, a cyclic peptide is a polypeptide chain having a cyclic ring structure. In some embodiments, cyclic ring structures can be formed by linking one end of the peptide to the other with an amide bond or some other chemically stable bond such as a lactone, ether, thioether, disulfide bond, or the like. In some embodiments, the cyclic peptides of the present disclosure may be biologically active cyclic peptides in which head-to-tail (or N-to-C) cyclization is formed through an amide bond between the amino-terminus and carboxy-terminus. In some embodiments, the cyclic peptides of the present disclosure may be biologically active cyclic peptides in which cyclization is formed by "click chemistry." See Rashad A.A. (2019) Click Chemistry for Cyclic Peptide Drug Design. In: Goetz G. (eds) Cyclic Peptide Design. Methods in Molecular Biology, vol 2001. Humana, New York, NY. https://doi.org/10.1007 /978-1-4939-9504-2_8. The present disclosure is not limited to any particular cyclic peptide. The cyclic peptides of the present disclosure may be naturally occurring or synthetically produced.

In some embodiments, at least one of the independently selected targeting ligands of Formula 1 can comprise at least one aptamer. Aptamers are short, single-stranded DNA or RNA molecules that selectively bind to specific targets, such as proteins, peptides, carbohydrates, small molecules, toxins, or living cells. Aptamers assume a variety of shapes due to their tendency to form helices and single-stranded loops. The present disclosure is not limited to any particular aptamer. Known and as yet undiscovered aptamers are within the scope of the present disclosure.

In some embodiments, at least one of the independently selected targeting ligands of Formula 1 may be capable of binding at least one asialoglycoprotein receptor (ASGPR). ASGPR is a lectin located on hepatocytes that binds galactose residues. ASGPR has been shown to be highly expressed on the surface of hepatocytes, human cancer cell lines and liver cancer. Glandular cells of the gallbladder and stomach also weakly express ASGPR.

In some embodiments, at least one of the independently selected targeting ligands of Formula 1 may be capable of binding to at least one transferrin receptor. The transferrin receptor is a membrane glycoprotein that mediates the cellular uptake of transferrin, a protein in the blood that binds iron and transports it throughout the body. The transferrin receptor-mediated endocytosis pathway is known to those skilled in the art. See Qian et al . "Targeted drug delivery via the transferrin receptor-mediated endocytosis pathway." Pharmacol Rev. 2002 Dec; 54(4):561-87. doi: 10.1124/pr.54.4.561. PMID: 12429868. The present disclosure is not limited to any particular ligand capable of binding at least one metastatic receptor. Known and as yet unexplored transferrin receptor ligands are within the scope of the present disclosure.

In some embodiments, at least one of the independently selected targeting ligands of Formula 1 may be capable of binding to at least one integrin receptor. Integrin receptors are transmembrane receptors that promote cell-cell and cell-extracellular matrix (ECM) adhesion. Following ligand binding, integrin receptors activate signal transduction pathways that mediate cellular signals, such as regulation of the cell cycle, organization of the intracellular cytoskeleton, and movement of new receptors to the cell membrane. Integrin-targeted delivery of gene therapy agents is known to those skilled in the art. See Juliano et al . “Integrin targeted delivery of gene therapeutics.” Theranostics vol. 1 211-9. 2 Mar. 2011, doi:10.7150/thno/v01p0211. The present disclosure is not limited to any particular ligand capable of binding at least one integrin receptor. Known and as yet unexplored integrin receptor ligands are within the scope of the present disclosure.

In some embodiments, at least one of the independently selected targeting ligands of Formula 1 may be capable of binding at least one folate receptor. Folate receptors bind folate and reduced folate derivatives and mediate the delivery of tetrahydrofolate to the interior of the cell. Targeted drug delivery through folate receptors is known to those skilled in the art. See Zhao et al . "Targeted drug delivery via folate receptors." Expert Opin Drug Deliv. 2008 Mar; 5(3):309-19. doi: 10.1517/17425247.5.3.309. PMID: 18318652. The present disclosure is not limited to any particular ligand capable of binding at least one folate receptor. Known and as yet unexplored folate receptor ligands are within the scope of the present disclosure.

In some embodiments, at least one of the independently selected targeting ligands of Formula 1 may be capable of binding to at least one G protein-coupled receptor (GPCR). GPCRs are cell surface receptors that bind peptides, lipids, sugars and proteins, among others. GPCRs interact with G proteins in the plasma membrane. When an external signaling molecule binds to a GPCR, it causes a conformational change in the GPCR, which triggers the interaction between the GPCR and a neighboring G protein. GPCRs consist of a single polypeptide folded into a spherical shape and embedded in the plasma membrane of the cell. GPCR-targeted delivery of oligonucleotide therapeutics is known to those skilled in the art. See Knerr et al. "Glucagon Like Peptide 1 Receptor Agonists for Targeted Delivery of Antisense Oligonucleotides to Pancreatic Beta Cell" J. Am. Chem. Soc., 2021 143 (9), 3416-3429. DOI: 10.1021/jacs.0c1204 3. Connector A

As mentioned above with respect to Formula 1, multivalent ligand clusters of the present disclosure may comprise a plurality (eg, three) of independently selected linkers A. In this context, the term "independently selected" means that each linker A can be chosen to be different or identical to one or more other linkers A in the same cluster of multivalent ligands.

Each linker A is an independently selected spacer with one end attached to the targeting ligand (TL in Formula 1) and the other end attached to the nitrogen of the alkylformamide of the multivalent ligand cluster.

In some embodiments, at least one of the independently selected linkers A may comprise polyethylene glycol (PEG). PEG can have any number of repeating O- _CH2 - _CH2 units. For example, PEG can be PEG1, PEG2, PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG10, PEG11, PEG12, PEG13, PEG14, PEG15, PEG16, PEG17, PEG18, PEG19, PEG20, PEG21, PEG22, PEG23 , PEG24, PEG25, PEG26, PEG27, PEG28, PEG29, PEG30, PEG31, PEG32, PEG33, PEG34, PEG35, PEG36, PEG37, PEG38, PEG39, PEG40, PEG41, PEG42, PEG43, PEG44, PEG45, PEG46, PEG47, PEG48 , PEG49, PEG50, PEG51, PEG52, PEG53, PEG54, PEG55, PEG56, PEG57, PEG58, PEG59, PEG60, PEG61, PEG62, PEG63, PEG64, PEG65, PEG66, PEG67, PEG68, PEG69, PEG70, PEG71, PEG72, PEG73 , PEG74, PEG75, PEG76, PEG77, PEG78, PEG79, PEG80, PEG81, PEG82, PEG83, PEG84, PEG85, PEG86, PEG87, PEG88, PEG89, PEG90, PEG91, PEG92, PEG93, PEG94, PEG95, PEG96, PEG97, PEG98 , PEG99, PEG100 or greater.

In some embodiments, at least one of the independently selected linkers A can comprise at least one alkyl group. In some embodiments, the alkyl group can have 2 carbons, 3 carbons, 4 carbons, 5 carbons, 6 carbons, 7 carbons, 8 carbons, 9 carbons, 10 carbons, 11 carbons, 12 carbons, 13 carbons, 14 carbons, 15 carbons, 16 carbons, 17 carbons, 18 carbons, 19 carbons or 20 carbons.

In some embodiments, at least one of the independently selected linkers A can comprise at least one substituted alkyl group. In some embodiments, a substituted alkyl can have 2 carbons, 3 carbons, 4 carbons, 5 carbons, 6 carbons, 7 carbons, 8 carbons, 9 carbons, 10 carbons, 11 carbons Carbon, 12 carbons, 13 carbons, 14 carbons, 15 carbons, 16 carbons, 17 carbons, 18 carbons, 19 carbons or 20 carbons. In some embodiments, a substituted alkyl group may comprise one or more of the following substituent groups: alkyl, cycloalkyl, hydroxyl, alkoxide, carboxyl, amine, amide, halide, sulfonate Acyl and sulfonamides.

In some embodiments, at least one of the independently selected linkers A can comprise at least one cycloalkyl group. In some embodiments, the cycloalkyl can be C3 cycloalkyl (i.e., cyclopropane), C4 cycloalkyl (i.e., cyclobutene), C5 cycloalkyl (i.e., cyclopentane), C6 cycloalkyl (i.e. cyclohexane), C7 cycloalkyl (i.e. cycloheptane), C8 cycloalkyl (i.e. cyclooctane), C9 cycloalkyl (i.e. cyclononane) or C10 cycloalkyl (i.e. , cyclodecane).

In some embodiments, at least one of the independently selected linkers A can comprise at least one substituted cycloalkyl. In some embodiments, the substituted cycloalkyl can be a C3 substituted cycloalkyl, a C4 substituted cycloalkyl, a C5 substituted cycloalkyl, a C6 substituted cycloalkyl, a C7 substituted cycloalkyl, a C8 substituted cycloalkyl, Substituted cycloalkyl, C9 substituted cycloalkyl, or C10 substituted cycloalkyl. In some embodiments, a substituted cycloalkyl group can include one or more of the following substituent groups: alkyl, cycloalkyl, hydroxyl, alcoholate, carboxyl, amine, amide, halide, sulfonyl and sulfonamides.

In some embodiments, at least one of the independently selected linkers A can comprise at least one alkenyl group. In some embodiments, an alkenyl group can have 4 carbons, 5 carbons, 6 carbons, 7 carbons, 8 carbons, 9 carbons, 10 carbons, 11 carbons, 12 carbons, 13 carbons, 14 carbons, 15 carbons, 16 carbons, 17 carbons, 18 carbons, 19 carbons or 20 carbons.

In some embodiments, at least one of the independently selected linkers A can comprise at least one substituted alkenyl group. In some embodiments, an alkenyl group can have 4 carbons, 5 carbons, 6 carbons, 7 carbons, 8 carbons, 9 carbons, 10 carbons, 11 carbons, 12 carbons, 13 carbons, 14 carbons, 15 carbons, 16 carbons, 17 carbons, 18 carbons, 19 carbons or 20 carbons. In some embodiments, a substituted alkenyl can include one or more of the following substituent groups: alkyl, cycloalkyl, hydroxyl, alcoholate, carboxyl, amine, amide, halide, sulfonyl, and Sulfonamide.

In some embodiments, at least one of the independently selected linkers A can comprise at least one cycloalkenyl group. In some embodiments, the cycloalkenyl group can be C5 cycloalkenyl, C6 cycloalkenyl, C7 cycloalkenyl, C8 cycloalkenyl, C9 cycloalkenyl, or C10 cycloalkenyl.

In some embodiments, at least one of the independently selected linkers A can comprise at least one substituted cycloalkenyl. In some embodiments, the cycloalkenyl group can be C5 cycloalkenyl, C6 cycloalkenyl, C7 cycloalkenyl, C8 cycloalkenyl, C9 cycloalkenyl, or C10 cycloalkenyl. In some embodiments, a substituted cycloalkenyl can include one or more of the following substituent groups: alkyl, cycloalkyl, hydroxyl, alcoholate, carboxyl, amine, amide, halide, sulfonyl and sulfonamides.

In some embodiments, at least one of the independently selected linkers A can comprise at least one alkynyl group. In some embodiments, the alkynyl group can have 4 carbons, 5 carbons, 6 carbons, 7 carbons, 8 carbons, 9 carbons, 10 carbons, 11 carbons, 12 carbons, 13 carbons, 14 carbons, 15 carbons, 16 carbons, 17 carbons, 18 carbons, 19 carbons or 20 carbons.

In some embodiments, at least one of the independently selected linkers A can comprise at least one substituted alkynyl group. In some embodiments, a substituted alkynyl can have 4 carbons, 5 carbons, 6 carbons, 7 carbons, 8 carbons, 9 carbons, 10 carbons, 11 carbons, 12 carbons, 13 carbons carbon, 14 carbons, 15 carbons, 16 carbons, 17 carbons, 18 carbons, 19 carbons or 20 carbons. In some embodiments, a substituted alkynyl can include one or more of the following substituent groups: alkyl, cycloalkyl, hydroxyl, alcoholate, carboxyl, amine, amide, halide, sulfonyl, and Sulfonamide.

In some embodiments, at least one of the independently selected linkers A can comprise at least one aryl or heteroaryl group. Some examples include phenyl, naphthyl, and pyridyl, although it is noted that other aryl and heteroaryl groups may be used that fall within the definitions provided herein.

In some embodiments, at least one of the independently selected linkers A can comprise at least one substituted aryl group. In some embodiments, a substituted aryl can include one or more of the following substituent groups: alkyl, cycloalkyl, hydroxyl, alcoholate, carboxyl, amine, amide, halide, sulfonyl, and Sulfonamide.

In some embodiments, at least one of the independently selected linkers A can comprise at least one aralkyl group. Exemplary aralkyl groups include, but are not limited to, benzyl, phenethyl, and phenylpropyl.

In some embodiments, at least one of the independently selected linkers A can comprise at least one substituted aralkyl group. In some embodiments, a substituted aralkyl group may include one or more of the following substituent groups: alkyl, cycloalkyl, hydroxyl, alcoholate, carboxyl, amine, amide, halide, sulfonyl and sulfonamides.

In some embodiments, at least one of the independently selected linkers A can comprise at least one aralkenyl group. Exemplary aralkenyl groups include, but are not limited to, vinylbenzene and propenylbenzene.

In some embodiments, at least one of the independently selected linkers A can comprise at least one substituted aralkenyl group. In some embodiments, a substituted aralkenyl group may include one or more of the following substituent groups: alkyl, cycloalkyl, hydroxyl, alcoholate, carboxyl, amine, amide, halide, sulfonyl and sulfonamides.

In some embodiments, at least one of the independently selected linkers A can comprise at least one aralkynyl group. Exemplary aralkynyl groups include, but are not limited to, ethynylbenzene and propynylbenzene.

In some embodiments, at least one of the independently selected linkers A can comprise at least one substituted aralkynyl group. In some embodiments, a substituted aralkynyl group may include one or more of the following substituent groups: alkyl, cycloalkyl, hydroxyl, alcoholate, carboxyl, amine, amide, halide, sulfonyl and sulfonamides.

In some embodiments, at least one of the independently selected linkers A can comprise at least one heteroatom. In some embodiments, at least one of the independently selected linkers A may comprise one or more oxygen (O) heteroatoms, one or more nitrogen (N) heteroatoms, one or more sulfur (S) heteroatoms and/or one or more phosphorus (P) heteroatoms. In some embodiments, at least one of the independently selected linkers A can comprise at least one heteroalkyl group.

In some embodiments, at least one of the independently selected linkers A can comprise at least one aliphatic heterocycle. In some embodiments, at least one of the independently selected linkers A may comprise tetrahydrofuran (THF), tetrahydropyran (THP), morpholine, piperidine, piperazine, pyrrolidine, and/or azetidine. at least one of .

In some embodiments, at least one of the independently selected linkers A can comprise at least one heteroaryl group. In some embodiments, at least one of the independently selected linkers A may comprise one or more of imidazole, pyrazole, pyridine, pyrimidine, triazole, and/or 1,2,3-triazole.

In some embodiments, at least one of the independently selected linkers A can comprise at least one substituted heteroaryl. In some embodiments, a substituted heteroaryl can include one or more of the following substituent groups: alkyl, cycloalkyl, hydroxyl, alcoholate, carboxyl, amine, amide, halide, sulfonyl and sulfonamides.

In some embodiments, at least one of the independently selected linkers A can comprise at least one amino acid. A variety of amino acids are known to those skilled in the art. An independently selected linker A is not limited to including one or more specific amino acids. For example, independently selected linker A may comprise one or more arginine (Arg) amino acids, one or more histidine (His) amino acids, one or more lysine (Lys) amino acids, one or more more aspartic acid (Asp) amino acids, one or more glutamic acid (Glu) amino acids, one or more serine (Ser) amino acids, one or more threonine (Thr) amino acids, one or more asparagine (Asn) amino acids, one or more glutamine (Gln) amino acids, one or more cysteine (Cys) amino acids, one or more selenocysteine amino acid (Sec), one or more amino acids glycine (Gly), one or more amino acids proline (Pro), one or more amino acids alanine (Ala), one or more amino acids valine Amino acid (Val) amino acid, one or more isoleucine (Ile) amino acid, one or more leucine (Leu) amino acid, one or more methionine (Met) amino acid, one or More phenylalanine (Phe) amino acids, one or more tyrosine (Tyr) amino acids, and/or one or more tryptophan (Trp) amino acids.

In some embodiments, at least one of the independently selected linkers A can comprise at least one nucleotide. A variety of nucleotides are known to those skilled in the art. Independently selected linkers A are not limited to including one or more specific nucleotides. For example, independently selected linker A may comprise one or more nucleotides comprising a guanine nucleobase, one or more nucleotides comprising an adenine nucleobase, one or more nucleotides comprising a cytosine nucleobase, base, one or more nucleotides comprising a thymine nucleobase, and/or one or more nucleotides comprising a uracil nucleobase.

In some embodiments, at least one of the independently selected linkers A can comprise at least one abasic nucleotide. As known in the art, an abasic nucleotide is a nucleotide that has an abasic site, which is a position that has neither a purine nor a pyrimidine base. For example, at least one of the independently selected adapters A can comprise one or more abasic DNAs and/or one or more abasic RNAs. In some embodiments, at least one of the independently selected adapters A can comprise at least one inverted abasic nucleotide. As known in the art, an inverted abasic nucleotide is an abasic nucleotide whose 5' end is joined to the 5' end of the next nucleotide and whose 3' end is joined to the 3' end of the next nucleotide base nucleotides. For example, at least one of the independently selected adapters A can comprise one or more inverted abasic DNAs and/or one or more inverted abasic RNAs.

In some embodiments, at least one of the independently selected linkers A can comprise at least one sugar. In some embodiments, at least one of the independently selected linkers A may comprise at least one glucose monosaccharide unit, at least one fructose monosaccharide unit, at least one mannose monosaccharide unit, at least one galactose monosaccharide unit, at least one ribose monosaccharide units, and/or at least one glucosamine monosaccharide unit.

In some embodiments, at least one of the independently selected linkers A may comprise one or more of the following: wherein: p is an integer of 0 to 12, pp is an integer of 0 to 12, q is an integer of 1 to 12, and qq is an integer of 1 to 12.

In some embodiments, p is an integer independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. In some embodiments, pp is an integer independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. In some embodiments, q is an integer independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. In some embodiments, qq is an integer independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. Connector B

Linker B is a spacer with one end attached to the agent or a functional group capable of linking to one or more agents and the other end attached to the diamine nitrogen of the multivalent ligand cluster.

In some embodiments, Linker B may comprise polyethylene glycol (PEG). PEG can have any number of repeating O- _CH2 - _CH2 units. For example, PEG can be PEG1, PEG2, PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG10, PEG11, PEG12, PEG13, PEG14, PEG15, PEG16, PEG17, PEG18, PEG19, PEG20, PEG21, PEG22, PEG23 , PEG24, PEG25, PEG26, PEG27, PEG28, PEG29, PEG30, PEG31, PEG32, PEG33, PEG34, PEG35, PEG36, PEG37, PEG38, PEG39, PEG40, PEG41, PEG42, PEG43, PEG44, PEG45, PEG46, PEG47, PEG48 , PEG49, PEG50, PEG51, PEG52, PEG53, PEG54, PEG55, PEG56, PEG57, PEG58, PEG59, PEG60, PEG61, PEG62, PEG63, PEG64, PEG65, PEG66, PEG67, PEG68, PEG69, PEG70, PEG71, PEG72, PEG73 , PEG74, PEG75, PEG76, PEG77, PEG78, PEG79, PEG80, PEG81, PEG82, PEG83, PEG84, PEG85, PEG86, PEG87, PEG88, PEG89, PEG90, PEG91, PEG92, PEG93, PEG94, PEG95, PEG96, PEG97, PEG98 , PEG99, PEG100 or greater. In some embodiments, it may be beneficial for the PEG to be PEG10 or less.

In some embodiments, linker B can comprise at least one alkyl group. In some embodiments, linker B can comprise at least one substituted alkyl group. In some embodiments, the alkyl group can have 2 carbons, 3 carbons, 4 carbons, 5 carbons, 6 carbons, 7 carbons, 8 carbons, 9 carbons, 10 carbons, 11 carbons, 12 carbons, 13 carbons, 14 carbons, 15 carbons, 16 carbons, 17 carbons, 18 carbons, 19 carbons, or 20 carbons.

In some embodiments, linker B can comprise at least one substituted alkyl group. In some embodiments, a substituted alkyl group can have 2 carbons, 3 carbons, 4 carbons, 5 carbons, 6 carbons, 7 carbons, 8 carbons, 9 carbons, 10 carbons, 11 carbons, 12 carbons, 13 carbons, 14 carbons, 15 carbons, 16 carbons, 17 carbons, 18 carbons, 19 carbons, or 20 carbons. In some embodiments, a substituted alkyl group may include one or more of the following substituents: alkyl, cycloalkyl, hydroxyl, alcoholate, carboxyl, amine, amide, halide, sulfonyl, and Sulfonamide.

In some embodiments, linker B can comprise at least one cycloalkyl group. In some embodiments, the cycloalkyl can be C3 cycloalkyl (i.e. cyclopropane), C4 cycloalkyl (i.e. cyclobutene), C5 cycloalkyl (i.e. cyclopentane), C6 cycloalkyl (i.e. Hexane), C7 cycloalkyl (i.e. cycloheptane), C8 cycloalkyl (i.e. cyclooctane), C9 cycloalkyl (i.e. cyclononane) or C10 cycloalkyl (i.e. cyclodecane).

In some embodiments, linker B can comprise at least one at least one substituted cycloalkyl group. In some embodiments, the substituted cycloalkyl can be C3 substituted cycloalkyl, C4 substituted cycloalkyl, C5 substituted cycloalkyl, C6 substituted cycloalkyl, C7 substituted Cycloalkyl, C8 substituted cycloalkyl, C9 substituted cycloalkyl, or C10 substituted cycloalkyl. In some embodiments, substituted cycloalkyl groups may include one or more of the following substituents: alkyl, cycloalkyl, hydroxyl, alcoholate, carboxyl, amine, amide, halide, sulfonyl and sulfonamides.

In some embodiments, linker B can comprise at least one alkenyl group. In some embodiments, an alkenyl group can have 4 carbons, 5 carbons, 6 carbons, 7 carbons, 8 carbons, 9 carbons, 10 carbons, 11 carbons, 12 carbons, 13 carbons, 14 carbons, 15 carbons, 16 carbons, 17 carbons, 18 carbons, 19 carbons, or 20 carbons.

In some embodiments, linker B can comprise at least one substituted alkenyl group. In some embodiments, an alkenyl group can have 4 carbons, 5 carbons, 6 carbons, 7 carbons, 8 carbons, 9 carbons, 10 carbons, 11 carbons, 12 carbons, 13 carbons, 14 carbons, 15 carbons, 16 carbons, 17 carbons, 18 carbons, 19 carbons, or 20 carbons. In some embodiments, a substituted alkenyl can include one or more of the following substituents: alkyl, cycloalkyl, hydroxyl, alcoholate, carboxyl, amine, amide, halide, sulfonyl, and Sulfonamide.

In some embodiments, linker B can comprise at least one cycloalkenyl group. In some embodiments, the cycloalkenyl group can be C5 cycloalkenyl, C6 cycloalkenyl, C7 cycloalkenyl, C8 cycloalkenyl, C9 cycloalkenyl, or C10 cycloalkenyl.

In some embodiments, linker B can comprise at least one substituted cycloalkenyl. In some embodiments, the cycloalkenyl group can be C5 cycloalkenyl, C6 cycloalkenyl, C7 cycloalkenyl, C8 cycloalkenyl, C9 cycloalkenyl, or C10 cycloalkenyl. In some embodiments, a substituted cycloalkenyl group may include one or more of the following substituents: alkyl, cycloalkyl, hydroxyl, alcoholate, carboxyl, amine, amide, halide, sulfonyl and sulfonamides.

In some embodiments, linker B can comprise at least one alkynyl group. In some embodiments, the alkynyl group can have 4 carbons, 5 carbons, 6 carbons, 7 carbons, 8 carbons, 9 carbons, 10 carbons, 11 carbons, 12 carbons, 13 carbons, 14 carbons, 15 carbons, 16 carbons, 17 carbons, 18 carbons, 19 carbons or 20 carbons.

In some embodiments, linker B can comprise at least one substituted alkynyl group. In some embodiments, a substituted alkynyl can have 4 carbons, 5 carbons, 6 carbons, 7 carbons, 8 carbons, 9 carbons, 10 carbons, 11 carbons, 12 carbons, 13 carbons carbons, 14 carbons, 15 carbons, 16 carbons, 17 carbons, 18 carbons, 19 carbons, or 20 carbons. In some embodiments, a substituted alkynyl can include one or more of the following substituents: alkyl, cycloalkyl, hydroxyl, alcoholate, carboxyl, amine, amide, halide, sulfonyl, and Sulfonamide.

In some embodiments, linker B can comprise at least one aryl or heteroaryl group. Examples include phenyl, naphthyl and pyridyl, although it should be noted that other aryl and heteroaryl groups falling within the definitions provided herein may be used.

In some embodiments, linker B can comprise at least one substituted aryl group. In some embodiments, substituted aryl groups may include one or more of the following substituents: alkyl, cycloalkyl, hydroxyl, alcoholate, carboxyl, amine, amide, halide, sulfonyl, and Sulfonamide.

In some embodiments, linker B can comprise at least one aralkyl group. Exemplary aralkyl groups include, but are not limited to, phenylmethyl, phenylethyl, and phenylpropyl.

In some embodiments, linker B can comprise at least one substituted aralkyl group. In some embodiments, substituted aralkyl groups may include one or more of the following substituents: alkyl, cycloalkyl, hydroxyl, alcoholate, carboxyl, amine, amide, halide, sulfonyl and sulfonamides.

In some embodiments, linker B can comprise at least one aralkenyl group. Exemplary aralkenyl groups include, but are not limited to, vinylbenzene and propenylbenzene.

In some embodiments, linker B can comprise at least one substituted aralkenyl group. In some embodiments, a substituted aralkenyl group may include one or more of the following substituents: alkyl, cycloalkyl, hydroxyl, alcoholate, carboxyl, amine, amide, halide, sulfonyl and sulfonamides.

In some embodiments, linker B can comprise at least one aralkynyl group. Exemplary aralkynyl groups include, but are not limited to, ethynylbenzene and propynylbenzene.

In some embodiments, linker B can comprise at least one substituted aralkynyl group. In some embodiments, a substituted aralkynyl group may include one or more of the following substituents: alkyl, cycloalkyl, hydroxyl, alcoholate, carboxyl, amine, amide, halide, sulfonyl and sulfonamides.

In some embodiments, Linker B may comprise at least one heteroatom. In some embodiments, linker B may comprise one or more oxygen (O) heteroatoms, one or more nitrogen (N) heteroatoms, one or more sulfur (S) heteroatoms, and/or one or More phosphorus (P) heteroatoms. In some embodiments, at least one independently selected linker A can comprise at least one heteroalkyl group.

In some embodiments, linker B can comprise at least one aliphatic heterocycle. In some embodiments, Linker B may comprise at least one of tetrahydrofuran (THF), tetrahydropyran (THP), morpholine, piperidine, piperazine, pyrrolidine, and/or azetidine.

In some embodiments, linker B can comprise at least one heteroaryl group. In some embodiments, Linker B may comprise one or more of imidazole, pyrazole, pyridine, pyrimidine, triazole, and/or 1,2,3-triazole.

In some embodiments, linker B can comprise at least one substituted heteroaryl. In some embodiments, a substituted heteroaryl group may include one or more of the following substituents: alkyl, cycloalkyl, hydroxyl, alcoholate, carboxyl, amine, amide, halide, sulfonyl and sulfonamides.

In some embodiments, linker B can comprise at least one amino acid. A variety of amino acids are known to those skilled in the art. Linker B is not limited to including one or more specific amino acids. For example, linker B may comprise one or more arginine (Arg) amino acids, one or more histidine (His) amino acids, one or more lysine (Lys) amino acids, one or more Aspartic acid (Asp) amino acid, one or more glutamic acid (Glu) amino acid, one or more serine (Ser) amino acid, one or more threonine (Thr) amino acid, one or more one asparagine (Asn) amino acid, one or more glutamine (Gln) amino acid, one or more cysteine (Cys) amino acid, one or more selenocysteine ( Sec) amino acid, one or more glycine (Gly) amino acid, one or more proline (Pro) amino acid, one or more alanine (Ala) amino acid, one or more valine ( Val) amino acid, one or more isoleucine (Ile) amino acid, one or more leucine (Leu) amino acid, one or more methionine (Met) amino acid, one or more Phenylalanine (Phe) amino acid, one or more tyrosine (Tyr) amino acids, and/or one or more tryptophan (Trp) amino acids.

In some embodiments, linker B can comprise at least one nucleotide. Various nucleotides are known to those skilled in the art. Linker B is not limited to including one or more specific nucleotides. For example, Linker B may contain one or more nucleotides comprising a guanine nucleobase, one or more nucleotides comprising an adenine nucleobase, one or more nucleotides comprising a cytosine nucleobase Nucleotides, one or more nucleotides comprising a thymine nucleobase and/or one or more nucleotides comprising a uracil nucleobase. In some embodiments, linker B can comprise at least one abasic nucleotide. As known in the art, an abasic nucleotide is a nucleotide that has an abasic site, which is a position that has neither a purine nor a pyrimidine base. For example, linker B can comprise one or more abasic DNAs and/or one or more abasic RNAs. In some embodiments, linker B can comprise at least one inverted abasic nucleotide. For example, linker B can comprise one or more inverted abasic DNAs and/or one or more inverted abasic RNAs.

In some embodiments, linker B can comprise at least one sugar. In some embodiments, linker B may comprise at least one glucose monosaccharide unit, at least one fructose monosaccharide unit, at least one mannose monosaccharide unit, at least one galactose monosaccharide unit, at least one ribose monosaccharide unit and/or at least A glucosamine monosaccharide unit.

In some embodiments, Linker B may comprise one or more of the following: Where: j is an integer from 1 to 12, and k is an integer from 0 to 12.

In some embodiments, j is an integer independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. In some embodiments, k is an integer independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. In certain cases, the present invention has also found that when linkerB contains a six-membered ring fragment, especially a 4-hydroxypiperidinyl group, it exhibits better performance when used as a targeted delivery agent than a five-membered ring. Good in vivo stability and activity, such as compound 75 of the present invention. potion

In some embodiments, the medicament is a diagnostic or therapeutic drug, molecule, compound or combination of drugs, molecules or compounds that have properties that aid in the diagnosis, prevention, treatment and/or amelioration of a disease or condition, for example in a cell or object middle. In certain embodiments, an agent is a drug, molecule, compound or combination of drugs, molecules or compounds that have properties that help enhance a desired condition, eg, in a cell or a subject.

In some embodiments, the agent can be an oligonucleotide. In some embodiments, oligonucleotides may comprise siRNA. In some embodiments, oligonucleotides may comprise double-stranded siRNA. In some embodiments, the double-stranded siRNA can comprise at least one modified ribonucleotide. In some embodiments of the compositions and methods of the invention, the at least one modified nucleotide comprises: 2'-O-methyl nucleotides, 2'-fluoro nucleotides, 2'-deoxynucleotides Nucleotides, 2'3'-seco nucleotide mimics, locked nucleotides, unlocked nucleic acid nucleotides (UNA), glycol nucleic acid nucleotides (GNA), 2'-F-arabinonucleotides, 2'-Methoxyethyl Nucleotide, Abasic Nucleotide, Ribitol, Reverse Nucleotide, Reverse Abasic Nucleotide, Reverse 2'-Ome Nucleotide, Reverse 2 '-deoxynucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, 3'-Ome nucleotides, containing 5'-phosphorothioate Nucleotides with ester groups or 5'-(E)-vinylphosphonate nucleotides (antisense strand only), or terminal cores linked to cholesterol derivatives or dodecanoic acid didecylamide groups nucleotides, 2'-amino-modified nucleotides, phosphoramidates, or nucleotides containing unnatural bases. In some embodiments, substantially all (ie, greater than 85%) of the ribonucleotides of the double-stranded siRNA can be modified.

In some embodiments, all ribonucleotides of the double-stranded siRNA can be modified. In some embodiments, at least one strand of the double-stranded siRNA can comprise at least one phosphorothioate linkage. In some embodiments, at least one strand of the double-stranded siRNA can comprise up to 6 phosphorothioate linkages. In some embodiments, the double-stranded siRNA can comprise at least one locked nucleic acid (LNA). As known in the art, LNA (sometimes called bridged nucleic acid (BNA) or inaccessible RNA) is a modified RNA molecule in which the ribose moiety is modified with an additional bridge linking the 2' oxygen to the 4' carbon , thereby locking the ribose in the 3'-endo conformation. LNAs are known to increase stability against enzymatic degradation, and to increase specificity and affinity. In some embodiments, the double-stranded siRNA can comprise at least one unlocked nucleic acid (UNA). As known in the art, UNA is an acyclic derivative of RNA that lacks the C2'-C3'-bond of the RNA ribose ring. It is known to those skilled in the art that the inclusion of UNA at certain positions on the antisense strand of an siRNA is resistant to activity and can contribute to reduced off-target activity.

In some embodiments, the double-stranded siRNA can comprise at least one glycerol nucleic acid (GNA). As known in the art, GNA (sometimes referred to as glycol nucleic acid) is a nucleic acid that is similar to RNA but differs in the composition of its sugar-phosphodiester backbone. It is known to those skilled in the art that inclusion of GNAs at certain positions on the antisense strand of siRNAs is permissive for activity and can contribute to reduced off-target activity.

In some embodiments, an oligonucleotide may comprise an siRNA comprising one or more modified nucleotides, including but not limited to 2'-modified nucleotides (such as F and MeO), abasic Nucleotides, Inverted Abasic Nucleotides, Locked Nucleotides, UNA and Unlocked Nucleic Acids (UNA) and Glycerol Nucleic Acids (GNA). [157] In some embodiments, an oligonucleotide may comprise an siRNA comprising one or more phosphorothioate backbone linkages.

In some embodiments, oligonucleotides may comprise single-stranded siRNA. In some embodiments, oligonucleotides may comprise small activating RNAs. In some embodiments, oligonucleotides may comprise microRNAs (miRNAs). In some embodiments, oligonucleotides may comprise antisense oligonucleotides. In some embodiments, the oligonucleotide may comprise a short guide RNA (gRNA). In some embodiments, an oligonucleotide may comprise a single guide RNA (sgRNA). In some embodiments, the oligonucleotide may comprise messenger RNA (mRNA). In some embodiments, an oligonucleotide may comprise a ribozyme. In some embodiments, an oligonucleotide may comprise a plasmid. In some embodiments, an oligonucleotide may comprise an immunostimulatory nucleic acid. In some embodiments, an oligonucleotide may comprise an antagomir. In some embodiments, oligonucleotides may comprise aptamers. Aptamers are short, single-stranded DNA or RNA molecules that can selectively bind to specific targets such as proteins, peptides, carbohydrates, small molecules, toxins, or living cells. Aptamers take on a variety of shapes because of their tendency to form helices and single-stranded loops. This disclosure is not limited to any particular aptamer. Known and as yet undiscovered aptamers are within the scope of this disclosure.

In some embodiments, an oligonucleotide may comprise at least 3 independently selected nucleotides. In some embodiments, an oligonucleotide may comprise 16 to 23 independently selected nucleotides, such as when the oligonucleotide is an siRNA. In some embodiments, an oligonucleotide may comprise about 100 independently selected nucleotides, such as when the oligonucleotide is a sgRNA. In some embodiments, an oligonucleotide may comprise up to fourteen thousand independently selected nucleotides, such as when the oligonucleotide is mRNA. A functional group capable of linking to one or more agents

As noted above, "W" in Formula 1 may be a functional group capable of linking one or more agents.

In some embodiments, the functional group can be hydroxyl (OH). In some embodiments, the functional group can be a protected hydroxyl group. Those skilled in the art will appreciate that a variety of protecting groups can be used to protect hydroxyl groups. Each of the plurality of treatment groups is within the scope of the present disclosure. For example, but not limited to, 4,4'-dimethoxytrityl (DMT), monomethoxytrityl (MMT), 9-(p-methoxyphenyl)xanthene-9 At least one of -group (Mox) and 9-phenylxanthene-9-yl (Px) protects the hydroxyl group.

In some embodiments, the functional group may be a phosphoramidite group having the formula: Formula 2 wherein: R ^a is C1 to C6 alkyl, C3 to C6 cycloalkyl, isopropyl, or R ^a is connected to R ^b through a nitrogen atom to form a ring, R ^b is C1 to C6 alkyl, C3 to C6 ring Alkyl, isopropyl, or R ^b is connected to Ra through a nitrogen atom to form a ring, and ^{R c is} a phosphite protecting group, a phosphate protecting group or 2-cyanoethyl. ^Rc can be one of a variety of phosphite protecting groups known to those skilled in the art. In some embodiments, the phosphite protecting group may comprise methyl, allyl, 2-cyanoethyl, 4-cyano-2-butenyl, 2-cyano-1,1-dimethyl Ethyl, 2-(trimethylsilyl)ethyl, 2-(S-acetylthio)ethyl, 2-(S-pivalylthio)ethyl, 2-(4-nitro phenyl) ethyl, 2,2,2-trichloroethyl, 2,2,2-trichloro-1,1-dimethylethyl, 1,1,1,3,3,3-hexa At least one of fluoro-2-propyl, fluorenyl-9-methyl, 2-chlorophenyl, 4-chlorophenyl and 2,4-dichlorophenyl. ^Rc can be one of a variety of phosphate protecting groups known to those skilled in the art. In some embodiments, the phosphate protecting group may comprise methyl, allyl, 2-cyanoethyl, 4-cyano-2-butenyl, 2-cyano-1,1-dimethyl Ethyl, 2-(trimethylsilyl)ethyl, 2-(S-acetylthio)ethyl, 2-(S-pivalylthio)ethyl, 2-(4-nitro phenyl)ethyl, 2,2,2-trichloroethyl, 2,2,2-trichloro-1,1-dimethylethyl, 1,1,1,3,3,3-hexafluoro -at least one of 2-propyl, fluorenyl-9-methyl, 2-chlorophenyl, 4-chlorophenyl and 2,4-dichlorophenyl.

In some embodiments, the functional group may be a carboxyl group (CO ₂ H). In some embodiments, the functional group may be an activated carboxyl group having the formula: Formula 3 wherein X is a leaving group.

A variety of activated carboxyl groups are known to those skilled in the art, all of which are within the scope of the present disclosure. In some embodiments, the leaving group (X) can be carboxylate, sulfonate, chlorine, phosphate, imidazole, hydroxybenzotriazole (HOBt), N-hydroxysuccinimide (NHS), tetrafluoro One of phenol, pentafluorophenol, p-nitrophenol.

In some embodiments, the functional group can be a Michael acceptor. In some embodiments, a Michael acceptor can have the formula: Formula 4 where: E is an electron-withdrawing group; and ^Rd is hydrogen or a C1-C6 alkyl substituent on the alkene (meaning that E and ^Rd can be cis, trans, or iso with respect to the carbon-carbon double bond formula ( iso )).

A variety of electron withdrawing groups are known to those skilled in the art, all of which are within the scope of the present disclosure. In some embodiments, the electron withdrawing group (E) may be a formamide or an ester. In some embodiments, the electron-withdrawing group (E) and the carbon-carbon double bond of the Michael acceptor may be part of a maleimide, which is a cyclic dimethylimide in which The two carbonyl groups together with the nitrogen itself form a 1H-pyrrole-2,5-dione structure.

In some embodiments, a functional group can have the following formula: Formula 5 wherein: Linker C is absent or a spacer is attached to the 3' or 5' end of the oligonucleotide, X is methyl, oxygen, sulfur or amino, and Y is oxygen, sulfur or amino.

In some embodiments, Linker C of Formula 5 may comprise at least one heterocyclic compound. In some embodiments, the heterocyclic compound can be an abasic nucleotide or an inverted abasic nucleotide.

In some embodiments, a functional group can have the following formula: Formula 6 [158] wherein linker C is a spacer with one end attached to the nitrogen of formamide and the other end attached to the 3' or 5' end of the oligonucleotide.

In some embodiments, Linker B is attached to the carbonyl of the carboxamide of Formula 6.

In some embodiments, Linker C in Formula 6 may comprise at least one PEG. PEG can have any number of repeating O- _CH2 - _CH2 units. For example, PEG can be PEG1, PEG2, PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG10, PEG11, PEG12, PEG13, PEG14, PEG15, PEG16, PEG17, PEG18, PEG19, PEG20, PEG21, PEG22, PEG23 , PEG24, PEG25, PEG26, PEG27, PEG28, PEG29, PEG30, PEG31, PEG32, PEG33, PEG34, PEG35, PEG36, PEG37, PEG38, PEG39, PEG40, PEG41, PEG42, PEG43, PEG44, PEG45, PEG46, PEG47, PEG48 , PEG49, PEG50, PEG51, PEG52, PEG53, PEG54, PEG55, PEG56, PEG57, PEG58, PEG59, PEG60, PEG61, PEG62, PEG63, PEG64, PEG65, PEG66, PEG67, PEG68, PEG69, PEG70, PEG71, PEG72, PEG73 , PEG74, PEG75, PEG76, PEG77, PEG78, PEG79, PEG80, PEG81, PEG82, PEG83, PEG84, PEG85, PEG86, PEG87, PEG88, PEG89, PEG90, PEG91, PEG92, PEG93, PEG94, PEG95, PEG96, PEG97, PEG98 , PEG99, PEG100 or greater.

In some embodiments, Linker C in Formula 6 may comprise at least one alkyl group. In some embodiments, the alkyl group can have 2 carbons, 3 carbons, 4 carbons, 5 carbons, 6 carbons, 7 carbons, 8 carbons, 9 carbons, 10 carbons, 11 carbons, 12 carbons, 13 carbons, 14 carbons, 15 carbons, 16 carbons, 17 carbons, 18 carbons, 19 carbons or 20 carbons.

In some embodiments, Linker C in Formula 6 can comprise at least one cycloalkyl group. In some embodiments, the cycloalkyl can be C3 cycloalkyl (i.e. cyclopropane), C4 cycloalkyl (i.e. cyclobutene), C5 cycloalkyl (i.e. cyclopentane), C6 cycloalkyl (i.e. Hexane), C7 cycloalkyl (i.e. cycloheptane), C8 cycloalkyl (i.e. cyclooctane), C9 cycloalkyl (i.e. cyclononane) or C10 cycloalkyl (i.e. cyclodecane).

In some embodiments, Linker C in Formula 6 may comprise at least one heteroatom. In some embodiments, linker C may comprise one or more oxygen (O) heteroatoms, one or more nitrogen (N) heteroatoms, one or more sulfur (S) heteroatoms, and/or one or More phosphorus (P) heteroatoms.

In some embodiments, Linker C in Formula 6 may comprise at least one aliphatic heterocycle. In some embodiments, linker C may comprise at least one of tetrahydrofuran (THF), tetrahydropyran (THP), morpholine, piperidine, piperazine, pyrrolidine, and/or azetidine.

In some embodiments, linker C in Formula 6 can comprise at least one heteroaryl group. In some embodiments, linker C may comprise one or more of imidazole, pyrazole, pyridine, pyrimidine, triazole, and/or 1,2,3-triazole.

In some embodiments, linker C in Formula 6 can comprise at least one substituted heteroaryl. In some embodiments, a substituted heteroaryl group may include one or more of the following substituents: alkyl, cycloalkyl, hydroxyl, alcoholate, carboxyl, amine, amide, halide, sulfonyl and sulfonamides.

In some embodiments, Linker C in Formula 6 may comprise at least one amino acid. A variety of amino acids are known to those skilled in the art. Linker C is not limited to including one or more specific amino acids. For example, linker C may comprise one or more arginine (Arg) amino acids, one or more histidine (His) amino acids, one or more lysine (Lys) amino acids, one or more Aspartic acid (Asp) amino acid, one or more glutamic acid (Glu) amino acid, one or more serine (Ser) amino acid, one or more threonine (Thr) amino acid, one or more one asparagine (Asn) amino acid, one or more glutamine (Gln) amino acid, one or more cysteine (Cys) amino acid, one or more selenocysteine ( Sec) amino acid, one or more glycine (Gly) amino acid, one or more proline (Pro) amino acid, one or more alanine (Ala) amino acid, one or more valine ( Val) amino acid, one or more isoleucine (Ile) amino acid, one or more leucine (Leu) amino acid, one or more methionine (Met) amino acid, one or more Phenylalanine (Phe) amino acid, one or more tyrosine (Tyr) amino acids, and/or one or more tryptophan (Trp) amino acids.

In some embodiments, Linker C in Formula 6 may comprise at least one nucleotide. Various nucleotides are known to those skilled in the art. Linker C is not limited to including one or more specific nucleotides. For example, Linker C may contain one or more nucleotides comprising a guanine nucleobase, one or more nucleotides comprising an adenine nucleobase, one or more nucleotides comprising a cytosine nucleobase, Nucleotides, one or more nucleotides comprising a thymine nucleobase and/or one or more nucleotides comprising a uracil nucleobase. In some embodiments, linker C can comprise at least one abasic nucleotide. As known in the art, an abasic nucleotide is a nucleotide that has an abasic site, which is a position that has neither a purine nor a pyrimidine base. For example, linker C can comprise one or more abasic DNAs and/or one or more abasic RNAs. In some embodiments, linker C can comprise at least one inverted abasic nucleotide. For example, linker C can comprise one or more inverted abasic DNAs and/or one or more inverted abasic RNAs.

In some embodiments, Linker C in Formula 6 may comprise at least one sugar. In some embodiments, linker C may comprise at least one glucose monosaccharide unit, at least one fructose monosaccharide unit, at least one mannose monosaccharide unit, at least one galactose monosaccharide unit, at least one ribose monosaccharide unit and/or at least A glucosamine monosaccharide unit.

In some embodiments, Linker C in Formula 6 may comprise one or more of the following: Where: j is an integer from 1 to 12, and k is an integer from 0 to 12.

In some embodiments, a functional group can have the following formula: Formula 7 wherein linker C is a spacer, one end of which is linked to one of the succinimide ring carbons through a thioether bond, and the other end is linked to the 3' or 5' end of the oligonucleotide.

In some embodiments, Linker B is linked to the succinimide nitrogen of Formula 7.

In some embodiments, Linker C in Formula 7 may comprise at least one PEG. PEG can have any number of repeating O- _CH2 - _CH2 units. For example, PEG can be PEG1, PEG2, PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG10, PEG11, PEG12, PEG13, PEG14, PEG15, PEG16, PEG17, PEG18, PEG19, PEG20, PEG21, PEG22, PEG23 , PEG24, PEG25, PEG26, PEG27, PEG28, PEG29, PEG30, PEG31, PEG32, PEG33, PEG34, PEG35, PEG36, PEG37, PEG38, PEG39, PEG40, PEG41, PEG42, PEG43, PEG44, PEG45, PEG46, PEG47, PEG48 , PEG49, PEG50, PEG51, PEG52, PEG53, PEG54, PEG55, PEG56, PEG57, PEG58, PEG59, PEG60, PEG61, PEG62, PEG63, PEG64, PEG65, PEG66, PEG67, PEG68, PEG69, PEG70, PEG71, PEG72, PEG73 , PEG74, PEG75, PEG76, PEG77, PEG78, PEG79, PEG80, PEG81, PEG82, PEG83, PEG84, PEG85, PEG86, PEG87, PEG88, PEG89, PEG90, PEG91, PEG92, PEG93, PEG94, PEG95, PEG96, PEG97, PEG98 , PEG99, PEG100 or greater.

In some embodiments, Linker C in Formula 7 may comprise at least one alkyl group. In some embodiments, the alkyl group can have 2 carbons, 3 carbons, 4 carbons, 5 carbons, 6 carbons, 7 carbons, 8 carbons, 9 carbons, 10 carbons, 11 carbons, 12 carbons, 13 carbons, 14 carbons, 15 carbons, 16 carbons, 17 carbons, 18 carbons, 19 carbons or 20 carbons.

In some embodiments, Linker C in Formula 7 may comprise at least one cycloalkyl group. In some embodiments, the cycloalkyl can be C3 cycloalkyl (i.e. cyclopropane), C4 cycloalkyl (i.e. cyclobutene), C5 cycloalkyl (i.e. cyclopentane), C6 cycloalkyl (i.e. Hexane), C7 cycloalkyl (i.e. cycloheptane), C8 cycloalkyl (i.e. cyclooctane), C9 cycloalkyl (i.e. cyclononane) or C10 cycloalkyl (i.e. cyclodecane).

In some embodiments, Linker C in Formula 7 may comprise one or more of the following: Where: j is an integer from 1 to 12, and k is an integer from 0 to 12. Multivalent Ligand Clusters Containing GalNAc Targeting Ligands

In some embodiments, multivalent ligand clusters of the present disclosure may have the general structure of Formula 8: Formula 8 wherein: m is an integer from 1 to 10, each n is an independently selected integer from 1 to 10, each linker A is an independently selected spacer having one end attached to TL and the other end attached to an alkylformyl The nitrogen of the amine, the linker B is a spacer, one end of which is attached to the agent or a functional group capable of being attached to one or more agents, and the other end is attached to the diamine nitrogen, and W is one or more agents, or is capable of being attached to one or more agents A functional group to which one or more agents are attached.

In some embodiments, multivalent ligand clusters of the present disclosure may have the general structure of Formula 9: Formula 9 wherein: m is an integer from 1 to 10, each n is an independently selected integer from 1 to 10, each linker A is an independently selected spacer, linker B is a spacer, linker C is a spacer or does not exist , X is methyl, oxygen, sulfur or amino, and Y is oxygen, sulfur or amino.

In some embodiments, multivalent ligand clusters of the present disclosure may have the general structure of Formula 10: Formula 10 wherein: m is an integer from 1 to 10, each n is an independently selected integer from 1 to 10, each linker A is an independently selected spacer, linker B is a spacer, and linker C is a spacer.

In some embodiments, multivalent ligand clusters of the present disclosure may have the general structure of Formula 11: Formula 11 wherein: m is an integer from 1 to 10, each n is an independently selected integer from 1 to 10, each linker A is an independently selected spacer, linker B is a spacer, and linker C is a spacer.

The following are multivalent ligands comprising GalNAc or protected GalNAc targeting ligands, multiple "m"s from Formula 1, multiple "n"s from Formula 1, and multiple functional groups capable of linking to one or more agents An example formula for a volume cluster: Preparation method of the first example

One method of preparing examples of compounds of general formula 1 is depicted in Scheme 1 below. Starting materials and intermediates can be purchased from commercial sources, prepared from known procedures or otherwise illustrated. The order in which the steps of the reaction schemes are performed can be varied. plan 1

Scheme 1 starts with a monoprotected diamine (compound I). The mono-protected diamine comprises a first nitrogen and a second nitrogen, wherein the first nitrogen is a primary amine and the second nitrogen is a secondary amine comprising the protecting group (PG) in scheme 1).

A variety of protecting groups are known to those skilled in the art and can be used. In some embodiments, the protecting group may be benzyl. In some embodiments, the protecting group may be triphenylmethyl.

"m" in Scheme 1 can be any integer. In some embodiments, m in Scheme 1 can be an integer from 1 to 10.

Starting from compound I, triester compound II can be synthesized in one step. In some embodiments, compound II can be synthesized by a _SN2 substitution reaction using compound I and one or more suitable substrates. In some embodiments, Compound II can be synthesized by reductive amination using Compound I and one or more suitable substrates. In some embodiments, compound II can be synthesized by Michael addition reaction using compound I and one or more suitable substrates.

As shown in Scheme 1, Compound II results from the coupling of various protected carboxylic acids to Compound I. In compound II, the first nitrogen is a tertiary amine comprising a first protected carboxylic acid and a second protected carboxylic acid, and the second nitrogen is a tertiary amine comprising a protecting group and a third protected carboxylic acid.

Each "n" in Scheme 1 is an independently chosen integer. In some embodiments, each n in Scheme 1 is an independently selected integer from 0 to 10.

Compound III is produced by deprotecting the second nitrogen of compound II such that the second nitrogen of compound III is a secondary amine comprising a third protected carboxylic acid. In embodiments where the protecting group is benzyl, compound III can be produced by hydrogenation using compound II. In embodiments where the protecting group is triphenylmethyl, compound III can be produced by reacting a second compound with at least one acid. Example acids include, but are not limited to, hydrochloric acid (HCl) and trifluoroacetic acid (TFA).

Compound IV is produced by attaching a hydroxyl-containing moiety to the second nitrogen of compound III such that the second nitrogen of compound IV is an amide or tertiary amine comprising a third protected carboxylic acid and a hydroxyl-containing moiety. The hydroxyl-containing moiety can be attached to the second nitrogen using any of the linker B described above.

In some embodiments, producing triester Compound IV comprises performing a _SN2 reaction using Compound III and one or more suitable substrates. In some embodiments, producing Compound IV comprises performing a reductive amination reaction using Compound III and one or more suitable substrates. In some embodiments, producing Compound IV comprises performing a Michael addition reaction using Compound III and one or more suitable substrates. In some embodiments, producing Compound IV comprises performing an amide coupling reaction using Compound III and one or more suitable substrates. In some embodiments, producing Compound IV comprises performing a nucleophilic addition reaction using Compound III and one or more suitable substrates. Exemplary substrates include, but are not limited to, isocyanates and isothiocyanates.

Triacid compound V is produced by converting the protected carboxylic acid of compound IV to a carboxylic acid. In some embodiments, Compound V can be produced by reacting Compound IV with one or more acids (eg, when R in Scheme 1 is an acid sensitive group, such as tert-butyl). In some embodiments, the one or more acids may include hydrochloric acid (HCl), hydrobromic acid (HBr), trifluoroacetic acid (TFA), and formic acid. In some embodiments, producing compound V can include hydrogenation using compound IV (eg, when R in Scheme 1 is benzyl). In some embodiments, producing Compound V can include performing a hydrolysis reaction with Compound IV.

Compound VI can be produced by using compound V for amide coupling reaction. In compound VI, the first nitrogen is a tertiary amine comprising a first amide and a second amide, and the second nitrogen is a tertiary amine comprising a hydroxyl-containing moiety and a third amide. The first amide, the second amide, and the third amide can each be coupled to an independently selected targeting ligand. Linker A in Scheme 1 can be any linker A described herein. The TL in scheme 1 can be any TL described herein.

Compound VII was produced by converting the hydroxyl group of compound VI (attached to Linker B) to a phosphoramidite group using a phosphoritylation reaction. As shown in Scheme 1, in some embodiments, conversion of a hydroxyl group to a phosphoramidite group can be performed after an amide coupling reaction to produce compound VI. Preparation method of the second example

Another method for preparing examples of compounds of general formula 1 is described in Scheme 2 below. Scheme 2 allows compounds of general formula 1 to have one or more different targeting ligands. Protocol 2 allows for the gradual introduction of targeting ligands. Starting materials and intermediates can be purchased from commercial sources, prepared by known procedures or otherwise illustrated. The order in which the steps of the reaction schemes are performed can be varied. Scenario 2

Scheme 2 starts with a doubly protected diamine (compound I). Doubly protected diamines contain a first nitrogen and a second nitrogen, where the first nitrogen is a secondary amine containing a first protecting group (PG ¹ ) and the second nitrogen is a primary amine containing a second protecting group (PG ² ) or secondary amines.

The first protecting group and the second protecting group can be different, allowing different linkers A and targeting ligands to be attached to the diamine scaffold. A variety of protecting groups are known to those skilled in the art and can be used. In some embodiments, the first protecting group can be a benzyl group and the second protecting group can be a t-butoxycarbonyl (Boc) group.

"m" in Scheme 2 can be any integer. In some embodiments, m in Scheme 2 can be an integer from 1 to 10.

Compound II can be produced by coupling a first protected carboxylic acid to the first nitrogen of Compound I such that the first nitrogen of Compound II is a tertiary amine. Those skilled in the art will understand that by differentiating the protecting groups in Compound I, the protecting groups can be removed and replaced strategically. For example, in an embodiment where the first protecting group is a benzyl group and the second protecting group is a Boc group, the benzyl-bearing amine of Compound I (rather than the Boc-bearing amine) can be subjected to one or more suitable reagents _SN2 substitution reaction, reductive amination reaction or Michael addition reaction to form compound II.

Compound III can be produced by removing the first protecting group from compound II. In compound III, the first nitrogen is a secondary amine with a first protected carboxylic acid, and the second nitrogen is a primary or secondary amine with a second protecting group. In some embodiments, producing compound III can include performing a hydrogenation reaction (eg, when the first protecting group is benzyl).

Compound IV can be produced by coupling a second protected carboxylic acid to the first nitrogen of Compound III such that the first nitrogen of Compound IV is a tertiary amine. In some embodiments, producing Compound IV can comprise performing a _SN2 substitution reaction using Compound III and one or more other suitable reagents. In some embodiments, producing Compound IV can include reductive amination using Compound III and one or more other suitable reagents. In some embodiments, producing Compound IV can comprise performing a Michael addition reaction using Compound III and one or more other suitable reagents. In some embodiments, producing Compound IV can include performing an amide coupling reaction using Compound III and one or more other suitable reagents. In some embodiments, producing Compound IV can include nucleophilic addition reactions using Compound III and one or more other suitable reagents.

Compound V can be produced by removing the second protecting group from Compound IV such that the first nitrogen of Compound V is a tertiary amine comprising the first protected carboxylic acid and the second protected carboxylic acid, and the second nitrogen of Compound V is a primary amine. In some embodiments (eg, when the second protecting group is a Boc group), compound V can be produced by reacting compound IV with at least one acid. Example acids include, but are not limited to, hydrochloric acid (HCl) and trifluoroacetic acid (TFA). Compound VI can be produced by coupling a third protected carboxylic acid to the second nitrogen of Compound V such that the second nitrogen of Compound VI is a secondary amine. In some embodiments, Compound VI can be produced by performing a _SN2 substitution reaction using Compound V and one or more other suitable reagents. In some embodiments, Compound VI can be produced by reductive amination using Compound V and one or more other suitable reagents. In some embodiments, Compound VI can be produced by Michael addition reaction using Compound V and one or more other suitable reagents.

Compound VII can be produced by attaching a moiety comprising a hydroxyl group to the second nitrogen of compound VI such that the second nitrogen of compound VII is a tertiary amine or amide or urea. The hydroxyl-containing moiety can be attached to the second nitrogen using any of the linker B described above. In some embodiments, Compound VII can be produced by performing a _SN2 substitution reaction using Compound VI and one or more other suitable reagents. In some embodiments, Compound VII can be produced by performing a reductive amination reaction using Compound VI and one or more other suitable reagents. In some embodiments, Compound VII can be produced by performing a Michael addition reaction using Compound VI and one or more other suitable reagents. In some embodiments, Compound VII can be produced by performing an amide coupling reaction using Compound VI and one or more other suitable reagents. In some embodiments, Compound VII can be produced by performing a nucleophilic addition reaction using Compound VI and one or more other suitable reagents.

In an example of Scheme 2, R ^a , R ^b and R ^c can be sufficiently different that the targeting ligand can be selectively attached, for example in one case R ^a , R ^b and R ^c can be methyl, Benzyl and tert-butyl. Such selective attachment of targeting ligands is described below.

Compound VIII is produced by converting the third protected carboxylic acid of compound VII to the first carboxylic acid. In some embodiments, Compound VIII can be produced by reacting Compound VII with one or more acids (eg, when R ^c in Scheme 2 is an acid sensitive group such as t-butyl).

Compound IX can be produced by amide coupling reaction using compound VIII. In Compound IX, the first nitrogen comprises a first protected carboxylic acid and a second protected carboxylic acid, and the second nitrogen of Compound IX comprises a first amide having a first targeting ligand coupled thereto and comprising a hydroxyl part.

Compound X is produced by converting the second protected carboxylic acid of Compound IX to a second carboxylic acid. In some embodiments, producing compound X can comprise hydrogenation using compound IX (eg, when R ^b in Scheme 2 is benzyl).

Compound XI can be produced by using compound X for amide coupling reaction. In compound XI, the first nitrogen comprises a first protected carboxylic acid and a second amide having a second targeting ligand coupled thereto, and the second nitrogen of compound XI comprises a first targeting ligand coupled thereto. The first amide of the ligand and the hydroxyl containing moiety.

Compound XII is produced by converting the first protected carboxylic acid of Compound XI to a third carboxylic acid. In some embodiments, producing compound XII can include performing a hydrolysis reaction using compound XI (eg, when ^Ra in Scheme 2 is methyl).

Compound XIII can be produced by amide coupling reaction using compound XII. In compound XIII, the first nitrogen comprises a second amide with a second targeting ligand coupled thereto and a third amide with a third targeting ligand coupled thereto, and the second nitrogen of compound XI A first amide having a first targeting ligand coupled thereto and a hydroxyl-containing moiety are included.

The first amide can be coupled to the first targeting ligand using any independently selected linker A described herein. The second amide can be coupled to the second targeting ligand using any independently selected linker A described herein. The third amide can be coupled to the third targeting ligand using any independently selected linker A described herein.

One or more of the first targeting ligand, the second targeting ligand, and the third targeting ligand can be independently selected as one or more targeting ligands described herein.

The hydroxyl group can be coupled to the second nitrogen using any linker B described herein.

In some embodiments, a phosphoritylation reaction can be used to convert a hydroxyl group to a phosphoramidite group. In some embodiments, a hydroxyl group can be converted to a phosphoramidite group, resulting in compound XIV.

Each " ^nx ", " ^ny " or " ^nz " in scheme 2 is an independently selected integer. In some embodiments, each " ^nx ", " ^ny ", or " ^nz " in Scheme 2 is an independently selected integer from 0 to 10. Certain elements of preparation and use

Embodiments of the multivalent ligand clusters of the invention can be prepared and used to deliver oligonucleotide agents to cells, tissues and organs. Some non-limiting examples of agents that can be delivered include therapeutic agents such as siRNA. Delivery methods using the multivalent ligand clusters of the invention can be used to deliver siRNA and other agents conjugated to the targeting ligand clusters of the invention to cells in vitro and in vivo. The multivalent ligand clusters of the invention are useful as delivery vehicles for delivering agents, such as, but not limited to, nucleic acid-containing agents, to cells. The term "multivalent ligand cluster/agent complex" as used herein means a multivalent ligand cluster as described herein linked to an agent described herein. In some embodiments of the invention, the agent is siRNA.

In another aspect of the disclosure, the dsRNA agent comprises 2'-fluoro-modified nucleotides at positions 2, 7, 12, 14, and 16 of the antisense strand (from the first pair of nucleosides at the 5' end of the antisense strand acid), and/or 2'-fluoro-modified nucleotides at positions 9, 11, and 13 of the sense strand (counting from the first pair of nucleotides at the 3' end of the sense strand). In some embodiments, other positions of the dsRNA agent do not contain 2' fluoro-modified nucleotides. In some embodiments, all nucleotides of the antisense strand and/or the sense strand of a dsRNA agent are modified nucleotides. In some embodiments, the dsRNA agent has 2'-fluoro-modified nucleotides at positions 2, 7, 12, 14, and 16 of the antisense strand and/or at positions 9, 11, and 13 of the sense strand. 2'-fluoro-modified nucleotides, other positions contain modified nucleotides selected from the group consisting of 2'-O-methyl nucleotides, 2'-deoxy nucleotides, 2'3'-seco nucleosides Acid mimetics, locked nucleotides, unlocked nucleic acid nucleotides (UNA), glycol nucleic acid nucleotides (GNA), 2'-F-arabinonucleotides, 2'-methoxyethyl nucleotides , Abasic Nucleotide, Ribitol, Reverse Nucleotide, Reverse Abasic Nucleotide, Reverse 2'-Ome Nucleotide, Reverse 2'-Deoxy Nucleotide, 2'-Amino - modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides and 3'-OMe nucleotides, nucleotides comprising a 5'-phosphorothioate group, or Terminal nucleotides linked to cholesterol derivatives or dodecanoic acid didecylamide groups, 2'-amino-modified nucleotides, phosphoramidates or nucleotides containing unnatural bases. In some embodiments, the dsRNA agent comprises E-vinylphosphonate nucleotides at the 5' end of the guide strand. In certain embodiments, the dsRNA agent comprises at least one phosphorothioate internucleoside linkage. In certain embodiments, the sense strand comprises at least one phosphorothioate internucleoside linkage. In some embodiments, the antisense strand comprises at least one phosphorothioate internucleoside linkage. In some embodiments, the sense strand comprises 1, 2, 3, 4, 5, or 6 phosphorothioate internucleoside linkages. In some embodiments, the antisense strand comprises 1, 2, 3, 4, 5, or 6 phosphorothioate internucleoside linkages. In certain embodiments, the sense strand is complementary or substantially complementary to the antisense strand, and the region of complementarity is 16 to 23 nucleotides in length. In some embodiments, the complementary region is 19 to 21 nucleotides in length. In certain embodiments, the complementary region is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. In some embodiments, each strand is no more than 30 nucleotides in length. In some embodiments, each strand is no more than 25 nucleotides in length. In some embodiments, each strand is no more than 23 nucleotides in length. In certain embodiments, a dsRNA agent comprises at least one modified nucleotide and further comprises one or more targeting or linking groups. In some embodiments, one or more targeting groups or linking groups are conjugated to the sense strand. In some embodiments, the targeting group includes N-acetyl-galactosamine (GalNAc). In some embodiments, the targeting group comprises the structure of GalNAc described above.

In some aspects of the invention, clusters of multivalent ligands can be used to deliver agents to cells of a subject. Methods of administering the multivalent ligand cluster/agent complex to a subject can include methods known in the art. As a non-limiting example, multivalent ligand cluster/agent complexes can be delivered locally in vivo by direct injection or by use of an infusion pump. In some aspects of the invention, the multivalent ligand cluster/agent complex is in a pharmaceutical composition and may be referred to as an agent. In some embodiments, an agent of the invention is administered to a subject in an amount effective to prevent the disease state, modulate the onset of the disease state, treat the disease state, or alleviate the symptoms of the disease state in the subject. cells and objects

As used herein, subject shall mean a human or a vertebrate mammal including, but not limited to, dogs, cats, horses, goats, cattle, sheep, rodents, and primates such as monkeys. Accordingly, the invention can be used to treat diseases or conditions in both human and non-human subjects. For example, the methods and compositions of the invention are useful in veterinary applications as well as prophylactic and therapeutic regimens in humans. In some embodiments of the invention, the vertebrate subject is a mammal.

In certain embodiments of the invention, multivalent ligand cluster/agent complexes of the invention are delivered to and contacted with cells. In some embodiments of the invention, the cells contacted are in culture, while in other embodiments, the cells contacted are in a subject. Cell types that can be contacted with the multivalent ligand cluster/agent complexes of the invention include, but are not limited to, hepatocytes, myocytes, cardiomyocytes, circulating cells, neuronal cells, glial cells, adipocytes, skin cells, hematopoietic cells , epithelial cells, sperm, oocytes, muscle cells, fat cells, kidney cells, liver cells or pancreatic cells. In some embodiments, the cells contacted with the multivalent ligand cluster/agent complexes of the invention are hepatocytes. dose

Dosage levels of drugs and pharmaceutical compositions that can be delivered using the multivalent ligand cluster/agent complexes of the disclosure can be determined by one skilled in the art by routine experimentation. In at least some embodiments, a unit dose may comprise from about 0.01 mg/kg to about 100 mg/kg body weight of the siRNA. Alternatively, the dosage may be 10 mg/kg to 25 mg/kg body weight, or 1 mg/kg to 10 mg/kg body weight, or 0.05 mg/kg to 5 mg/kg body weight, or 0.1 mg/kg to 5 mg/kg Body weight, or 0.1 mg/kg to 1 mg/kg body weight, or 0.1 mg/kg to 0.5 mg/kg body weight, or 0.5 mg/kg to 1 mg/kg body weight, or 1 mg/kg to 3 mg/kg body weight.

The pharmaceutical composition can be a sterile injectable aqueous suspension or solution, or in lyophilized form. The pharmaceutical compositions and medicaments of the present disclosure can be administered to a subject in a pharmaceutically effective dosage. Application method

Various routes of administration of the multivalent ligand cluster/agent complexes of the invention are available. The particular mode of delivery chosen will depend upon the particular condition being treated and the dosage required for therapeutic efficacy. In general, the methods of the invention may be practiced using any mode of administration that is medically acceptable (meaning any mode that produces therapeutically effective levels without causing clinically unacceptable adverse effects). In some embodiments of the invention, the multivalent ligand cluster/agent complexes of the invention may be administered by oral, enteral, transmucosal, transdermal and/or parenteral routes. The term "parenteral" includes subcutaneous, intravenous, intramuscular, intraperitoneal and intracisternal injection or infusion techniques. Other routes include, but are not limited to, nasal (eg, via a gastro-nasal tube), transdermal, vaginal, rectal, and sublingual. The delivery routes of the present invention may include intrathecal, intraventricular or intracranial. In some embodiments of the invention, the multivalent ligand cluster/agent complexes of the invention can be placed in a slow release matrix and administered by placing the matrix in a subject.

The multivalent ligand cluster/agent complexes of the invention may be administered in formulations which may be administered in pharmaceutically acceptable solutions which may routinely contain pharmaceutically acceptable concentrations of salts, buffers, preservatives, compatible Sexual carriers, excipients and optionally other therapeutic ingredients. According to the methods of the invention, multivalent ligand cluster/agent complexes can be administered as pharmaceutical compositions. In general, pharmaceutical compositions comprise a multivalent ligand cluster/agent complex of the invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known to those skilled in the art and can be selected and used using routine methods. As used herein, a pharmaceutically acceptable carrier means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient (eg, the ability to deliver a nucleic acid such as siRNA to prevent and/or treat the disease or condition for which it is directed).

Pharmaceutically acceptable carriers may include diluents, fillers, salts, buffers, stabilizers, solubilizers and other substances known in the art. Illustrative pharmaceutically acceptable carriers are described in US Patent No. 5,211,657 and others are known to those of skill in the art. Such formulations generally may contain salts, buffers, preservatives, compatible carriers and optionally other therapeutic agents. When used in medicine, the salt should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts are conveniently used to prepare pharmaceutically acceptable salts thereof and are not excluded from the scope of the present invention. Such pharmacologically acceptable and pharmaceutically acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, Formic acid, malonic acid, succinic acid, etc. In addition, pharmaceutically acceptable salts can be prepared as alkali metal or alkaline earth metal salts, such as sodium, potassium or calcium salts.

In some embodiments of the invention, the multivalent ligand cluster/agent complexes of the invention can be administered directly to tissue. Direct tissue administration can be achieved by direct injection or other means known in the art. The multivalent ligand cluster/agent complexes of the invention can be administered once, or alternatively can be administered in multiple administrations. If administered multiple times, the multivalent ligand cluster/agent complexes of the invention may be administered by different routes. For example, the first (or first few) administrations may be directed into the affected tissue or organ, while subsequent administrations may be systemic.

When systemic administration is desired, the multivalent ligand cluster/agent complexes of the invention can be formulated for parenteral administration by injection, eg, by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, eg, in ampoules or in multi-dose containers, with or without an added preservative. The pharmaceutical compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Some examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents, and inert gases, etc., may also be present. Lower doses will result from other forms of administration, eg intravenous administration. In cases where the response in the subject is insufficient when the initial dose is administered, higher doses (or effectively higher doses by a different, more local route of delivery) may be employed to the extent patient tolerance allows. Multiple doses per day can be used as needed to achieve appropriate systemic or local levels of one or more multivalent ligand cluster/agent complexes of the invention to produce the desired levels of the agent, eg, the desired siRNA levels.

Both non-biodegradable and biodegradable polymer matrices can be used to deliver one or more multivalent ligand cluster/agent complexes of the invention to cells and/or subjects. In some embodiments, the matrix can be biodegradable. Matrix polymers can be natural or synthetic polymers. Polymers can be selected based on the desired time period of release (typically on the order of hours to a year or more). Typically, release over a period of a few hours to three to twelve months can be used. The polymer is optionally in the form of a hydrogel, which can absorb up to about 90% of its weight in water, and is also optionally crosslinked with multivalent ions or other polymers.

In certain embodiments of the invention, the multivalent ligand cluster/agent complexes of the invention can be delivered by means of diffusion using bioerodible implants or by degradation of the polymer matrix. Exemplary synthetic polymers for such use are well known in the art. Biodegradable polymers and non-biodegradable polymers can be used to deliver one or more multivalent ligand cluster/agent complexes of the invention using methods known in the art. Such methods can also be used to deliver one or more multivalent ligand cluster/agent complexes of the invention for therapy. Additional suitable delivery systems may include time-release, delayed-release or sustained-release delivery systems. Such a system would avoid repeated administration of the multivalent ligand cluster/agent complexes of the invention, increasing convenience for both the subject and the health care provider. Many types of release delivery systems are available and known to those of ordinary skill in the art. [See eg: US Patent Nos. 5,075,109; 4,452,775; 4,675,189; 5,736,152; 3,854,480; 5,133,974; and 5,407,686 (the teachings of each of which are incorporated herein by reference)]. Additionally, pump-based hardware delivery systems are available, some of which are suitable for implantation.

The use of long-term sustained-release implants may be particularly suitable for the prophylactic treatment of subjects and subjects at risk of developing recurrent diseases or conditions for which siRNAs delivered using the multivalent ligand clusters of the invention are to be treated. Prophylaxis and/or treatment. Long-term release as used herein means that the implant is constructed and arranged to deliver therapeutic levels of the active ingredient for at least 30 days, 60 days, 90 days or longer. Long term sustained release implants are well known to those of ordinary skill in the art and include some of the release systems described above.

Therapeutic formulations of one or more multivalent ligand cluster/agent complexes of the invention in the form of lyophilized formulations or aqueous solutions can be obtained by combining said multivalent ligand cluster/agent complexes of the desired purity with any Optionally, it may be prepared in admixture with a pharmaceutically acceptable carrier, excipient or stabilizer for storage [Remington's Pharmaceutical Sciences 21st Edition, (2006)]. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers, such as phosphates, citrates, and other organic acids; antioxidants, Includes ascorbic acid and methionine; preservatives (eg, octadecyldimethylbenzyl ammonium chloride; hexaquaternium chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol, or benzyl alcohol ; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histamine Acids, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol ; a salt-forming counterion such as sodium; a metal complex (eg, a Zn-protein complex); and/or a nonionic surfactant such as TWEEN ^® , PLURONICS ^® or polyethylene glycol (PEG).

The multivalent ligand cluster/agent complexes of the present disclosure can be formulated into pharmaceutical compositions. A pharmaceutical composition can be used as a medicine alone or in combination with other agents. The multivalent ligand cluster/agent complexes of the disclosure can also be administered in combination with other therapeutic compounds, either alone or simultaneously (eg, as a combined unit dose). In at least some embodiments, the present disclosure encompasses pharmaceutical compositions comprising in physiologically acceptable/pharmaceutically acceptable excipients (e.g., stabilizers, preservatives, diluents, buffers, etc.) One or more multivalent ligand cluster/agent complexes according to the present disclosure.

The pharmaceutical compositions of the invention may be administered alone, in combination with each other, and/or in combination with other drug treatments or other therapeutic regimens administered to a subject suffering from a disease or condition. Pharmaceutical compositions used in embodiments of the present invention are preferably sterile and comprise an effective amount of a multivalent ligand cluster/agent complex to prevent or treat the disease or condition to which the agent, eg, siRNA, is directed.

The dosage of the pharmaceutical composition of the invention sufficient to treat a disease or condition when administered to a subject can be selected according to different parameters, in particular according to the mode of administration used and the state of the subject. Other factors may include the desired duration of treatment. In cases where the response in the subject is insufficient when the initial dose is administered, higher doses (or effectively higher doses by a different, more local route of delivery) may be employed to the extent patient tolerance allows. In some embodiments of the invention, dosages which have been determined using routine methods, eg, in clinical trials, are used. EXAMPLES Example 1. Multivalent Ligand Clusters Comprising GalNAc Targeting Ligands

In some embodiments of the invention, multivalent ligand clusters may comprise GalNAc targeting ligands. The following are example compounds of multivalent ligand clusters comprising acetyl-protected GalNAc targeting ligands, core C2 and C3 diamines, branched acetyl and acrylamides, PEG2 and PEG3 Linker A, various linkers B described herein, and various functional groups capable of linking to one or more agents are as described herein. The acetyl protecting group on the following GalNAc ligands can be easily removed after conjugation to generate a GalNAc targeting ligand. Embodiment 2. Preparation of intermediate compound

In Scheme 3 below, intermediate A was obtained by using commercially available galactosamine pentaacetate (compound I) with trimethylsilyl triflate in dichloromethane (DCM). (TMSOTf) processing to synthesize. Subsequent glycosylation with Cbz protected 2-(2-aminoethoxy)ethan-1-ol affords compound II. The Cbz protecting group was removed by hydrogenation to give intermediate A as trifluoroacetate (TFA) or HCl salt. Intermediate B was synthesized based on the same protocol except that Cbz protected 2-(2-(2-aminoethoxy)ethoxy)ethan-1-ol was used as starting material. Scheme 3 allows variants of the access linker A as well as variants of the targeting ligand. Option 3

To a solution of Compound 1 (20.0 g, 51.4 mmol) in DCE (100 mL) was added TMSOTf (17.1 g, 77.2 mmol). The resulting reaction solution was stirred at 60°C for 2 hours, and then at 25°C for 1 hour. Cbz-protected 2-(2-aminoethoxy)ethan-1-ol (13.5 g, 56.5 mmol) in DCE (100 mL) dried over 4 Å powdered molecular sieves (10 g) was incubated at 0 °C Add dropwise to the above reaction solution under _N2 atmosphere. The resulting reaction mixture was stirred at 25 °C for 16 h under _N2 atmosphere. The reaction mixture was filtered and washed with saturated NaHCO ₃ (200 mL), water (200 mL) and saturated brine (200 mL). The organic layer was dried over _anhydrous NaSO, filtered and concentrated under reduced pressure to give the crude product, which was triturated _with 2-Me-THF/heptane (5/3, v/v, 1.80 L) for 2 h, filtered and dried to give compound II (15.0 g, 50.3% yield) as a white solid.

To a dry and argon-purged hydrogenation bottle, 10% Pd/C (1.50 g) was carefully added, followed by THF (10 mL), and then 10% Pd/C in THF (300 mL) and TFA (3.00 g, 26.4 mmol). Solution of compound II (15.0 g, 26.4 mmol). The resulting mixture was degassed and purged three times with _H2 and stirred at 25 °C for 3 h under an atmosphere of _H2 (45 psi). TLC (DCM:MeOH=10:1) showed complete consumption of Compound II. The reaction mixture was filtered and concentrated under reduced pressure. The residue was dissolved in anhydrous DCM (500 mL) and concentrated. This process was repeated 3 times to afford Intermediate A (14.0 g, 96.5% yield) as a foamy white solid.

Intermediate B was synthesized using a similar procedure to that used for the synthesis of Intermediate A. Mass calcd. for C ₂₀ H ₃₄ N ₂ O ₁₁ : 478.22; found: 479.3 (M+H ⁺ ). Embodiment 3. Preparation of compound 1

Scheme 4 below was used to prepare Compound 1 identified in Example 1 above. Commercially available 2,2',2'',2'''-(propane-1,3-diylbis(azanetriyl))tetraacetic acid (compound I in Scheme 4) was converted to di Anhydride compound II. Following work-up of 6-aminohexan-1-ol followed by hydrolysis, compound II is converted to the triacid compound III. Amide coupling between compound III and intermediate A affords compound IV. Treatment of compound IV with 2-cyanoethyl N,N-diisopropylphosphoramidite chloride and a catalytic amount of 1H-tetrazole affords phosphoramidite compound 1. Option 4

To a stirred solution of Ac ₂ O (8.83 g, 86.5 mmol) and pyridine (193 mg, 2.45 mmol) was added tetraacid Compound I (5.0 g, 16.3 mmol). After purging 3 times with _N2 , the reaction mixture was stirred at 65 °C for 12 h under _N2 atmosphere. After cooling, the reaction mixture was filtered to remove insoluble solids. The filtrate was concentrated in vacuo. Toluene was added to the residue and volatiles were evaporated. This process was repeated 3 times to obtain Compound II (2.20 g, 49.8% yield) as a yellow oil.

To a mixture of imidazole (3.63 g, 53.2 mmol) and compound II (1.80 g, 6.66 mmol) in DMF (18 mL) was added sequentially 6-aminohexan-1-ol (624 mg, 5.33 mmol) and pyridine (263 mg, 3.33 mmol). The mixture was stirred at 50 °C for 5 h under _N2 atmosphere. The reaction mixture was concentrated under reduced pressure. The residue was purified by reverse phase preparative HPLC. Compound III (1.90 g, containing 8.4% by weight of DMF and 63.2% by weight of imidazole) was obtained.

To a solution of Intermediate A (1.01 g, 2.32 mmol) and compound III (950 mg, purity 28.3% 0.66 mmol) in DMF (10 mL) was added DIEA (385 mg, 2.99 mmol), HOBt (358 mg, 2.65 mmol) and EDC (508 mg, 2.65 mmol). The resulting reaction mixture was stirred at 25 °C for 3 h under _N2 atmosphere. LC-MS indicated the desired product. The reaction mixture was purified by reverse phase preparative HPLC. Fractions containing the desired product were combined and concentrated to give Compound IV (200 mg, 18.2% yield) as a white solid.

To a solution of compound IV (200 mg, 120 umol) in anhydrous DCM (2.0 mL) was added diisopropylammonium tetrazolide (22.9 mg, 132 umol) followed by dropwise addition at 25 °C under _N 3-Bis(diisopropylamino)phosphinooxypropionitrile (145 mg, 483 umol). The reaction mixture was stirred at 25°C for 2 hours. LC-MS indicated complete consumption of Compound IV. The reaction was quenched by adding a mixture of brine and saturated _NaHCO solution (1:1, 5 mL) at −20 °C, and the resulting mixture was stirred at 0 °C for 1 min. The layers were separated. The aqueous phase was extracted with additional DCM (5 mL). The combined organics were washed with brine/saturated aqueous NaHCO ₃ (1:1, 5 mL), dried over Na ₂ SO ₄ , filtered and concentrated to a volume of approximately 1 mL. This solution was added dropwise to MTBE (20 mL) with stirring. This resulted in the formation of a white solid which was separated by centrifugation. The process was repeated one more time. The solid was then dissolved in anhydrous _CH3CN and the volatiles were removed. This process was repeated 3 times to obtain Compound 1 (103 mg, 45.9% yield) as a colorless oil. Embodiment 4. Preparation of compound 2

Compound 2 (in Example 1) was synthesized using the same procedure based on Scheme 4 above, except that Intermediate B was used in place of Intermediate A. Embodiment 5. Preparation of compound 3

Scheme 5 below can be used to prepare compound 3 identified in Example 1 above. Option 5

Starting from tert-butyl (3-aminopropyl)carbamate, it can be alkylated with benzyl-protected 2-bromoethanol ( _SN2 substitution) to give compound I. Removal of the Boc group under acidic conditions then gives compound II, which can be alkylated with tert-butyl 2-bromoacetate to give triester compound III. The t-butyl protecting group can then be removed following formic acid treatment to yield the triacid compound IV. Coupling of amide with intermediate A affords compound V. The benzyl protecting group can then be removed by hydrogenation to give compound VI. Phosphoramidite compound 3 can be synthesized by treating compound VI with 2-cyanoethyl N,N-diisopropylchlorophosphoramidite and a catalytic amount of 1H-tetrazole. Embodiment 6. Preparation of Compound 4

Scheme 6 below was used to prepare compound 4 identified in Example 1 above. Option six

Starting from benzyl-protected propane-1,3-diamine, it was alkylated with tert-butyl 2-bromoacetate to give triester compound I. The benzyl protecting group is removed by hydrogenation to give the secondary amine compound II. Coupling of amide with 6-hydroxyhexanoic acid affords compound III. The tert-butyl protecting group is then removed after treatment with HCl in dioxane to yield the triacid compound IV. Amide coupling between triacid compound IV and intermediate A affords compound V. Phosphoramidite Compound 4 was synthesized by phosphorylating Compound V with 2-cyanoethyl N,N-diisopropylchlorophosphoramidite and a catalytic amount of 1H-tetrazole.

To a solution of ^N1 -benzylpropane-1,3-diamine (5.00 g, 30.4 mmol) in DMF (100 mL) was added tert-butyl 2-bromoacetate (23.7 g, 121 mmol) and then dropwise DIEA (23.61 g, 182 mmol) was added. The resulting reaction mixture was stirred at 25 to 30°C for 16 hours. LCMS showed complete consumption of ^N1 -benzylpropane-1,3-diamine. The reaction mixture was diluted with H ₂ O (500 mL) and extracted with EtOAc (500 mL×2). The combined organics were washed with saturated brine (1 L), dried over _anhydrous NaSO, _filtered , and concentrated under reduced pressure to give the crude product, which was purified by silica gel column chromatography (gradient: petroleum ether:ethyl acetate at 20 :1 to 5:1). Compound I (12.1 g, 78.4% yield) was obtained as a colorless oil.

The dry hydrogenation bottle was purged three times with argon. Pd/C (200 mg, 10%) was added, followed by MeOH (5 mL), and then a solution of Compound 1 (1.00 g, 1.97 mmol) in MeOH (5 mL). The reaction mixture was degassed under vacuum and refilled with _H2 . This process was repeated three times. The mixture was stirred at 25 °C for 12 h under an atmosphere of _H2 (15 psi). LCMS showed complete consumption of compound I. The reaction mixture was filtered under reduced pressure under _N2 atmosphere. The filtrate was concentrated under reduced pressure to give Compound II (655 mg, 79.7% yield) as a yellow oil, which was used in the next step without further purification.

HOBt (637 mg, 4.72 mmol), EDCI (904 mg, 4.72 mmol), DIEA (1.02 g, 7.86 mmol), 6-hydroxyhexanoic acid (249 mg, 1.89 mmol) and compound The mixture of II (655 mg, 1.57 mmol) was degassed and purged with _N2 3 times, and then stirred at 25 °C under _N2 atmosphere for 3 h. LCMS indicated the desired product. The reaction mixture was diluted with H ₂ O (10 mL) and extracted with EtOAc 20 mL (10 mL×2). The organics were combined and washed with saturated brine (20 mL), dried over _{anhydrous NaSO} , filtered, and concentrated to give the crude product, which was purified by silica gel column chromatography (gradient: petroleum ether: ethyl acetate 5:1 to 1:1) to give compound III (650 mg, 77.8% yield) as a yellow oil. Mass calcd. for _C27H50N2O8 : _530.36 ; found: _531.3 ( _M +H ⁺ ).

A mixture of compound III (5.5 g, 10.3 mmol) in HCl/dioxane (2 M, 55 mL) was stirred at 25 °C for 3 hours. LCMS showed complete consumption of compound III. The reaction mixture was filtered, washed with EtOAc (50 mL), and dried under reduced pressure to give crude product. It was dissolved in _CH3CN (50 mL) and the volatiles were removed in vacuo. This process was repeated 3 times to obtain Compound IV (2.05 g, 54.5% yield) as a white solid.

HOBt (195 mg, 1.45 mmol), EDCI (277 mg, 1.45 mmol), DIEA (267 mg, 2.07 mmol), Intermediate B (693 mg, 1.45 mmol) and compound IV ( 150 mg, 0.413 mmol) was stirred at 25 °C for 3 h under _N2 atmosphere. LCMS indicated the desired product. The reaction mixture was purified by reverse phase preparative HPLC to afford Compound V, (186 mg, 0.106 mmol, 25.7% yield) as a white solid after lyophilization.

To a solution of compound V (180 mg, 0.103 mmol) in anhydrous DCM (3.6 mL) was added diisopropylammonium tetrazolide (19.44 mg, 0.114 mmol) followed by dropwise addition of ₃ - Bis(diisopropylamino)phosphinooxypropionitrile (124 mg, 0.412 mmol). The reaction mixture was stirred at 20 to 25°C for 2 hours. LCMS indicated complete consumption of compound V. After cooling to -20 °C, the reaction mixture was added to a stirred solution of brine/saturated aqueous _NaHCO3 (1:1, 5 mL) at 0 °C. After stirring for 1 min, DCM (5 mL) was added. Separate layers. The organics were washed with brine/saturated aqueous NaHCO ₃ (1:1.5 mL), dried over Na ₂ SO ₄ , filtered and concentrated to a volume of about 1 mL. The residue solution was added dropwise to 20 mL MTBE with stirring. This resulted in the precipitation of a white solid. The mixture was centrifuged and the solid collected. The process was repeated one more time. The collected solid was dissolved in anhydrous _CH3CN . Remove volatiles. This process was repeated two more times to obtain compound 4 (106 mg, 52.8% yield) as a white solid. Embodiment 7. Preparation of compound 5

Compound 5 was synthesized using the same procedure based on Scheme 6, except that Intermediate A was used in place of Intermediate B. Embodiment 8. Preparation of compound 6

Scheme 7 below can be used to prepare compound 6 identified in Example 1 above. Option 7

Starting from benzyl-protected propane-1,3-diamine (compound I), a Michael addition reaction with tert-butyl acrylate can be performed to afford the triester compound II. Once Compound II is synthesized, for the remaining steps to synthesize Compound 6, the same procedure used for the synthesis of Compound 4 in Scheme 6 can be followed. Embodiment 9. Preparation of Compound 7

Scheme 8 below can be used to prepare compound 7 identified in Example 1 above. Option 8

Starting from the secondary amine compound I (compound II in Scheme 6), Cbz protection can be used to give compound II. The tert-butyl group of compound II can be removed by treatment with acid to give triacid compound III. Amide coupling of compound III with intermediate A can give compound IV. The Cbz protecting group of compound IV can be removed by hydrogenation to give secondary amine compound V, which can be reacted with glutaric anhydride to give carboxylic acid compound VI. The carboxylic acid of compound VI can be converted to the tetrafluorophenyl ester to provide compound 7 by standard procedures. Embodiment 10. Preparation of Compound 8

Scheme 9 below can be used to prepare compound 8 identified in Example 1 above. Option 9

Compound I (compound V in Scheme 8) can be conjugated with NHS maleimide compound 2,5-dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro -1H-pyrrol-1-yl)propionate was reacted to give compound 8. Example 11. Preparation of Compound 74

Scheme 10 below can be used to prepare compound 74 identified in Example 1 above. Scheme 10

Start with compound I (same as Example 6 compound II in scheme 6).

To a solution of compound I (275 g, 660 mmol, 1.00 equiv) in DCM (2.75 L) was added TEA (133 g, 1.32 mol, 2.00 equiv) followed by Cbz-Cl (169 g, 990 mmol, 1.50 equiv ) was added dropwise to the reaction mixture. The mixture was stirred at 25°C for 2 hours. LCMS showed complete consumption of Compound I and one main peak was detected with the expected mass. The reaction mixture was diluted with NaHCO ₃ (800 mL) and extracted. The combined organic layers were washed with brine 500 mL (500 mL × 1), dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give the crude product, which was purified by column chromatography (SiO ₂ , PE/EA=100 /1 to 5/1) to obtain compound II (290 g, 527 mmol, 75.7% yield) as a colorless oil.

To a solution of compound II (145 g, 263 mmol, 1.00 equiv) in HCOOH (2.9 L). The mixture was stirred at 60° C. for 12 hours under an air atmosphere. LCMS showed complete consumption of compound III and one main peak was detected with the expected mass. The reaction was diluted with toluene and acetonitrile (ACN, 1500 mL each), and the mixture was concentrated in vacuo to remove formic acid azeotropically. The residue was diluted with 1:1 ACN:toluene (ca. 750 mL) and concentrated. The residue was diluted with ACN (1000 mL) and concentrated. This process was repeated one more time to obtain the crude product as a solid. The crude product was triturated with ACN (700 mL) at 60 °C for 2 h, filtered and dried to afford Compound III (210 g, quantitative yield) as a white solid.

To a solution of Intermediate A (502 g, 915. mmol, 3.50 equiv, TFA), compound III (100 g, 261 mmol, 1.00 equiv) in DMF (1.00 L) was added TBTU (327 g, 1.02 mol, 3.90 equiv), TEA (212 g, 2.09 mol, 291 mL, 8.00 equiv). The mixture was stirred at 25°C for 1 hour. LCMS showed complete consumption of compound III and one main peak was detected with the expected mass. The reaction mixture was added to H ₂ O (4000 mL). The resulting mixture was extracted with MTBE (2000 mL × 2) to remove impurities. The remaining aqueous portion was extracted with DCM (3000 mL x 2). The combined DCM extracts were washed with 10% citric acid (2000 mL × 2), saturated NaHCO ₃ (2000 mL × 2), brine 2000 mL, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give Compound IV as a solid (260 g, 159 mmol, 60.9% yield).

A 2.00 L hydrogenation bottle was purged 3 times with Ar and dry Pd/C (9 g) was carefully added. MeOH (50 mL) was then added to completely wet the Pd/C, then TFA (6.29 g, 55.1 mmol, 1.00 equiv) and compound IV (90 g, 55.1 mmol) in MeOH (850 mL) were added slowly under an Ar atmosphere. , 1.00 eq) solution. The resulting mixture was degassed and purged 3 times with _H2 , and then the mixture was stirred at 25 °C under _H2 atmosphere for 10 h. LCMS showed complete consumption of compound IV and one main peak with expected mass. The reaction mixture was carefully filtered under reduced pressure under _N2 atmosphere. The filtrate was concentrated under reduced pressure to obtain compound V (160 g, 90.2% yield).

To a solution of compound V (5.0 g, 3.10 mmol, 1.0 equiv, TFA salt) in DCM (50 mL) was added glutaric anhydride compound 5A, 531 mg, 4.65 mmol, 1.5 equiv) at 25 °C, followed by TEA (1.26 g, 12.4 mmol, 1.73 mL, 4.0 equiv) was added dropwise to the mixture. The mixture was stirred at 25°C for 1.0 hour. LC-MS showed complete consumption of compound V and a main peak with the expected product mass. The resulting reaction mixture was triturated twice with isopropyl ether (50 mL x 2), dried in vacuo to give Compound VI (crude, 5.5 g) as a brown solid.

To a solution of compound VI (2.6 g, 1.61 mmol, 1.0 equiv) in DMF (26 mL) was added int-D (protected (R)-3-aminopropane-1,2-diol) (952 mg , 2.42 mmol, 1.5 eq), TBTU (1.04 g, 3.23 mmol, 2.0 eq) and DIEA (625.53 mg, 4.84 mmol, 843.03 uL, 3.0 eq). The mixture was stirred at 25°C for 1.0 hour. LC-MS showed complete consumption of compound int-D (protected (R)-3-aminopropane-1,2-diol). The resulting reaction mixture was triturated with isopropyl ether (260 mL) to give crude product. It was purified by column chromatography (SiO ₂ , DCM/MeOH = 100/1 to 10/1, 0.1% Et ₃ N) to give compound 74 (900 mg, 28.0% yield) as a white solid. Compound 74

Compound 73 can be prepared according to the procedure described in Compound 74, except that protected (S)-3-aminopropane-1,2-diol is used instead of protected (R)-3-aminopropane-1, 2-diol. Example 12. Preparation of Compound 75

Scheme 11 below can be used to prepare compound 75 identified in Example 1 above. Scheme 11

Compound II was synthesized based on Scheme 11. Starting from compound I (compound VI in Scheme 10), coupling with piperidin-4-ol affords compound II. Phosphoramidite compound 75 was synthesized by treating compound II with 2-cyanoethyl N,N diisopropylphosphoramidite chloride and a catalytic amount of 1H-tetrazole. [159] Compound 75 Example 13. Linker B linked to a functional group capable of linking to one or more agents

It should be understood that the various linkers B of the present disclosure can be attached to various functional groups (W) capable of linking to one or more agents. In particular, Linker B may comprise a diol moiety, where one alcohol is protected as a DMT ether and the other alcohol may be directly or indirectly attached to the solid phase synthesis solid support material. After removal of the DMT group, a free alcohol is produced, which can be phosphorylated by reaction with phosphoramidite, thereby initiating the growth of the oligonucleotide chain. Thus, the target ligand clusters of the present disclosure can be attached at the 3' end of the oligonucleotide. A non-limiting list of Linker B of the present disclosure that can be ligated to the 3' end of an oligonucleotide includes the structures shown below and stereoisomers thereof: Where: j is an integer from 0 to 12, and k is an integer from 0 to 12.

All target ligand clusters of the present disclosure can also be attached at the 5' end of the oligonucleotide, including but not limited to the 5' end of the sense strand in dsRNA, or the 5' end of the antisense strand in dsRNA: Example 14. Preparation of ligand cluster conjugated siRNA

The sense and antisense strand sequences of the siRNA were synthesized on an oligonucleotide synthesizer using a well-established solid-phase synthesis method based on phosphoramidite chemistry. Oligonucleotide chain growth is achieved through a 4-step cycle: for each nucleotide added, condensation, capping, oxidation and deprotection steps. Synthesis was performed on a solid support made of controlled pore glass (CPG, 1000 Å). Monomeric phosphoramidites were purchased from commercial sources. Ligand cluster linked phosphoramidites were synthesized according to the procedures of Examples 3 to 12 herein. 5-Ethylthio-1H-tetrazole was used as activator. _I2 in THF/Py/ _H2O and phenylacetyl disulfide (PADS) in pyridine/MeCN were used for the oxidation and sulfurization reactions, respectively. After the final solid phase synthesis step, the protecting groups were removed and the solid support bound oligomer was cleaved by treatment with a 1 : 1 volume solution of 40 wt% methylamine and 28% ammonium hydroxide in water. The crude single-stranded product was isolated by lyophilization and purified by ion-pair reverse-phase HPLC (IP-RP-HPLC). Purified single-stranded oligonucleotide products from IP-RP-HPLC were converted to the sodium salt by dissolving in 1.0 M NaOAc and precipitating by addition of ice-cold EtOH. Annealing of equimolar complementary sense and antisense strand oligonucleotides in water was performed to form a double-stranded siRNA product which yielded a fluffy white solid after lyophilization. Example 15. In vivo evaluation of siRNA conjugated to the GalNAc ligand cluster

To evaluate delivery efficacy, the GalNAc ligand cluster was conjugated to the 5' or 3' end of the sense strand of an active FXII siRNA known in the literature. See Liu et al . "An investigational RNAi therapeutic targeting Factor XII (ALN-F12) for the treatment of hereditary angioedema". RNA. 2019 Feb;25(2):255-263. doi: 10.1261/rna.068916.118. The sequence and modification information of this FXII siRNA is summarized in Table 1. Also shown in Table 1 are six examples of GalNAc ligand clusters conjugated to FXII siRNA. Conjugation of the GalNAc cluster to the 5' or 3' sense strand of FXII siRNA was performed as part of the solid phase synthesis outlined in Example 14. Their structures are shown in Table 1 below. The molecular weights of these six compounds and the positive control compound are summarized in Table 2.

These compounds were tested for their efficacy in knocking down FXII in mice. FXII is a secreted protein produced mainly in hepatocytes. The reduction of FXII expression in plasma after siRNA treatment was closely correlated with the reduction of FXII mRNA in hepatocytes. Since these GalNAc ligand clusters were conjugated to the same FXII siRNA with known activity, delivery efficacy could be assessed and compared by measuring the degree of reduction in FXII expression in plasma for each conjugate.

Mice were given a single subcutaneous injection of 0.5 or 1 mg/kg of siRNA compound (see Table 1 below) or PBS. A literature compound (GalNAc ligand cluster conjugated to the 3′ end of the sense strand) was included in this study as a positive control. Plasma samples were collected before dosing and on days 7, 14 and/or 28 after dosing. The concentration of mouse FXII protein was measured by ELISA assay following literature procedures. In Liu et al . “An investigational RNAi therapeutic targeting Factor XII (ALN-F12) for the treatment of hereditary angioedema”. RNA. 2019 Feb;25(2):255-263. doi: 10.1261/rna.068916.118). Knockdown activity was calculated for the percent reduction of FXII protein in mouse plasma normalized to the PBS-treated group and is summarized in Table 3. FXII siRNA conjugated to GalNAc ligands GLS-1 and GLS-2 showed significant activity in knocking down mouse FXII protein expression in mouse plasma at both doses at 7, 14 and/or 28 days after administration . This activity was superior to the positive control. The data confirm that a GalNAc ligand cluster based on a diamine scaffold attached to the 5' end of the sense strand is very effective in delivering siRNA into hepatocytes in vivo. Table 1. FXII siRNA compounds. Upper case letters: 2'-deoxy-2'-fluoro (2'-F) ribonucleotides; lower case letters: 2'-O-methyl (2'-OMe) ribonucleotides; (*) indicate PS bonds couplet. L96 is a trivalent GalNAc ligand cluster in the literature. (GalNAc3 as in Jayaprakash, et al., (2014) J. Am. Chem. Soc., 136, 16958−16961) compound Sense sequence 5'->3' serial number Antisense sequence 5'->3'' serial number AD00127 Positive Control a*a*cucaauAaAgUgcuuuga*a-L96 1 u*U*caaaGcacuUuAuUgag*u*u 8 AD00130 GLS-1-*a*a*cucaauAaAgUgcuuuga*a 2 AD00131 GLS-2-*a*a*cucaauAaAgUgcuuuga*a 3 AD00197 GLS-5-*a*a*cucaauAaAgUgcuuuga*a 4 AD00448 GLS-15-*(Invab)*aacucaauAaAgUgcuuugaa*(Invab) 5 AD00449 GLS-15-*a*acucaauAaAgUgcuuuga*a 6 AD00831 a*a*cucaauAaAgUgcuuuga*a-GLS-14 7 Table 2. Quality of FXII siRNA compounds compound Calculation quality observation quality sense strand antisense strand sense strand antisense strand positive control 8784.68 6918.66 8785.31 6918.95 AD00130 8482.28 8482.74 AD00131 8350.12 8350.60 AD00197 8307.05 6918.66 8307.84 6918.89 AD00448 8734.265 6918.66 8734.45 6918.76 AD00449 8374.075 6918.66 8374.49 6918.76 AD00831 8364.04 6918.66 8364.70 6918.44 Table 3. Percentage reduction of FXII protein in mouse plasma normalized to PBS-treated group Compound ID Dose (mg/kg) day 7 day 14 day 28 knock down STD knock down STD knock down STD Positive control AD00127 1 74% 0.011 81% 0.011 67% 0.011 AD00130 0.5 67% 0.044 62% 0.041 45% 0.041 1 82% 0.030 82% 0.016 71% 0.016 AD00131 0.5 61% 0.075 72% 0.034 51% 0.067 1 84% 0.048 80% 0.045 66% 0.020 Example 16. In vivo evaluation of siRNA conjugated to the GalNAc ligand cluster

Mice were given a single subcutaneous injection of 1 mg/kg of siRNA compound or PBS. Plasma samples were collected on day 14 after dosing. The concentration of mouse FXII protein was measured by ELISA assay following literature procedures. See Liu et al . "An investigational RNAi therapeutic targeting Factor XII (ALN-F12) for the treatment of hereditary angioedema". RNA. 2019 Feb;25(2):255-263. doi: 10.1261/rna.068916.118). Knockdown activity was calculated for the percent reduction of FXII protein in mouse plasma normalized to the PBS-treated group and is summarized in Table 4. FXII siRNA conjugated to GalNAc ligands GLS-5 and GLS-15 showed significant activity knocking down mouse FXII protein expression in mouse plasma. The data confirm that a GalNAc ligand cluster based on a diamine scaffold attached to the 5' end of the sense strand is very effective in delivering siRNA into hepatocytes in vivo.

It was also found that when linkerB contains a six-membered ring fragment, it exhibits better stability and activity in vivo when it is used for the targeted delivery of agents, especially 4-hydroxypiperidinyl, such as compounds AD00448 and AD00449. Table 4. Percent reduction of FXII protein in mouse plasma normalized to PBS-treated group. Compound ID Dose (mg/kg) day 14 knock down STD AD00197 1 83.1% 0.015 AD00448 1 86.0% 0.013 AD00449 1 80.0% 0.031 Example 17. In vivo evaluation of GalNAc ligand cluster-conjugated siRN AD00831

Mice were given a single subcutaneous injection of 2 mg/kg of siRNA compound or PBS. Plasma samples were collected on days 7 and 14 after dosing. The concentration of mouse FXII protein was measured by ELISA assay following literature procedures. See Liu et al . "An investigational RNAi therapeutic targeting Factor XII (ALN-F12) for the treatment of hereditary angioedema". RNA. 2019 Feb;25(2):255-263. doi: 10.1261/rna.068916.118). Knockdown activity was calculated for the percent reduction of FXII protein in mouse plasma normalized to the PBS-treated group. The knockdown percentages were 87% and 88% on day 7 and day 14 after administration, respectively. The data confirm that a GalNAc ligand cluster based on a diamine scaffold attached to the 3' end of the sense strand is very efficient in delivering siRNA into hepatocytes in vivo. Example 18 In vivo testing of ANGPTL3 siRNA duplexes

Fourteen days before siRNA administration, female C57BL/6J mice were infected by intravenous administration of adeno-associated virus 8 (AAV8) vector solution encoding human ANGPTL3 and luciferase genes. On day 0, mice were administered a single dose of AD00112-2 (Table 5) or PBS subcutaneously at 1, 3 or 10 mg/kg. Blood samples were collected on day 0, prior to siRNA administration and on day 7, at termination. Serum samples were isolated and luciferase activity of serum samples was measured according to the manufacturer's recommended protocol. Since the expression of human ANGPTL3 levels correlates with that of luciferase, measurement of luciferase activity is a surrogate for measuring ANGPTL3 expression. By comparing the luciferase activity in samples from each mouse before (day 0) and after (day 7) siRNA treatment, and by comparing the luciferase activity in samples from control-treated mice during the same period Changes were normalized to calculate the percent remaining luciferase activity. The results are summarized in Table 6. AD00112-2 demonstrated dose-dependent activity in inhibiting ANGPTL3 expression, which reaffirmed that diamine scaffold-based GalNAc ligand clusters are highly effective in delivering siRNA into hepatocytes in vivo. Table 5. ANGPTL3 siRNA compounds. Upper case letters: 2'-deoxy-2'-fluoro (2'-F) ribonucleotides; lower case letters: 2'-O-methyl (2'-OMe) ribonucleotides; (*) indicate PS bonds couplet. compound Sense sequence 5'->3' serial number Antisense sequence 5'->3' serial number AD00112-2 (GLS-15)*(Invab)* gaauggaaGgUuAuacucuaa*(Invab) 9 u*U*agagUauaaCcUuCcau*u*c 10 Table 6 provides the experimental results of the in vivo study (percent reduction in luciferase activity). The duplex sequence and modifications of AD00112-2 are shown in Table 5. Duplex AD# Dose (mg/kg) Day 7, relative to PBS (mean ± SD) AD00112-2 1 0.33 ± 0.06 AD00112-2 3 0.20±0.09 AD00112-2 10 0.11±0.03 equivalent scheme

While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision numerous other ways of performing the functions and/or obtaining the results and/or one or more advantages described herein and/or structure, and each such variation and/or modification are considered to be within the scope of the present invention. More generally, those skilled in the art will readily understand that all parameters, dimensions, materials and configurations described herein are meant to be exemplary and actual parameters, dimensions, materials and/or configurations will depend on the A specific application or applications of the teachings. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to some of the specific embodiments of the invention described herein. It is therefore to be understood that the foregoing embodiments are given by way of example only, and that, within the scope of the appended claims and their equivalents, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials and/or methods is encompassed within the scope of the present invention provided that such features, systems, articles, materials and/or methods are not mutually inconsistent. .

All definitions, as defined and used herein, should be understood to take precedence over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

As used herein in the specification and claims, nouns modified by a quantifier are to be understood to mean "at least one" unless expressly stated to the contrary.

As used herein in the specification and claims, the phrase "and/or" should be understood to mean "one or both" of the elements so conjoined, that is, the elements co-exist in some instances and not in others. In some cases elements exist separately. Unless expressly stated to the contrary, other elements may optionally be present other than the elements expressly identified by the "and/or" clause, whether related or unrelated to those elements expressly identified

All references, patents and patent applications, and publications cited or referred to in this application are hereby incorporated by reference in their entirety.

none

Claims (188)

Compounds for targeted delivery of one or more agents having the formula: , wherein: each TL is an independently selected targeting ligand, m is an integer from 1 to 10, each n is an independently selected integer from 1 to 10, each linker A is an independently selected spacer, linker B is A spacer, and W is the one or more agents or a functional group capable of linking to the one or more agents. The compound of claim 1, wherein m is 1. The compound of claim 1, wherein m is 2. The compound of any one of claims 1 to 3, wherein at least one of the independently selected TLs is capable of binding to one or more cellular receptors, cellular channels, and cellular transporters capable of promoting endocytosis. 4. The compound of claim 4, wherein at least one of the independently selected TLs comprises at least one small molecule ligand. The compound of claim 5, wherein at least one small molecule comprises N-acetylgalactosamine, galactose, galactosamine, N-formyl-galactosamine, N-acrylgalactosamine, N- At least one of butyrylgalactosamine and N-isobutyrylgalactosamine, macrocycles, folic acid molecules, fatty acids, bile acids, and cholesterol. The compound of claim 4, wherein at least one of the independently selected TLs comprises at least one peptide. The compound of claim 7, wherein at least one of the independently selected TLs comprises at least one cyclic peptide. The compound of claim 4, wherein at least one of the independently selected TLs comprises at least one aptamer. The compound of any one of claims 4 to 9, wherein at least one of the independently selected TLs is capable of binding at least one asialoglycoprotein receptor (ASGPR). The compound of any one of claims 4 to 9, wherein at least one of the independently selected TLs is capable of binding at least one transferrin receptor. The compound of any one of claims 4 to 9, wherein at least one of the independently selected TLs is capable of binding at least one integrin receptor. 9. The compound of any one of claims 4 to 9, wherein at least one of the independently selected TLs is capable of binding to at least one folate receptor. The compound of any one of claims 4 to 9, wherein at least one of the independently selected TLs is capable of binding to at least one G protein-coupled receptor (GPCR). The compound of any one of claims 1 to 14, wherein at least one of the independently selected linkers A comprises polyethylene glycol, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl At least one of radical, aralkyl, aralkenyl and aralkynyl. The compound of any one of claims 1 to 15, wherein at least one of the independently selected linkers A comprises at least one heteroatom. The compound of claim 16, wherein the at least one heteroatom comprises at least one of oxygen, nitrogen, sulfur, or phosphorus. The compound of any one of claims 1 to 17, wherein at least one of the independently selected linkers A comprises at least one aliphatic heterocycle. The compound of claim 18, wherein the at least one aliphatic heterocycle comprises at least one of tetrahydrofuran, tetrahydropyran, morpholine, piperidine, piperazine, pyrrolidine, and azetidine. The compound of any one of claims 1 to 19, wherein at least one of the independently selected linkers A comprises at least one heteroaryl. The compound of claim 20, wherein the at least one heteroaryl group comprises at least one of imidazole, pyrazole, pyridine, pyrimidine, triazole, and 1,2,3-triazole. The compound of any one of claims 1 to 21, wherein at least one of the independently selected linkers A comprises at least one amino acid. The compound of any one of claims 1 to 22, wherein at least one of the independently selected linkers A comprises at least one nucleotide. The compound of any one of claims 1 to 23, wherein at least one of the independently selected linkers A comprises at least one sugar. The compound of claim 24, wherein the at least one sugar comprises at least one of glucose, fructose, mannose, galactose, ribose, and glucosamine. The compound of any one of claims 1 to 25, wherein at least one of the independently selected linkers A comprises one or more of the following: wherein: p is an integer of 0 to 12, pp is an integer of 0 to 12, q is an integer of 1 to 12, and qq is an integer of 1 to 12. The compound according to any one of claims 1 to 26, wherein linker B comprises polyethylene glycol, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, aralkyl, aralkenyl and at least one of aralkynyl. The compound of any one of claims 1 to 27, wherein linker B comprises at least one heteroatom. The compound of claim 28, wherein the at least one heteroatom comprises at least one of oxygen, nitrogen, sulfur, and phosphorus. The compound of any one of claims 1 to 29, wherein linker B comprises at least one aliphatic heterocycle. The compound of claim 30, wherein the at least one aliphatic heterocycle comprises at least one of tetrahydrofuran, tetrahydropyran, morpholine, piperidine, piperazine, pyrrolidine, and azetidine. The compound of any one of claims 1 to 31, wherein linker B comprises at least one heteroaryl. The compound of claim 32, wherein the at least one heteroaryl group comprises at least one of imidazole, pyrazole, pyridine, pyrimidine, triazole, and 1,2,3-triazole. The compound of any one of claims 1 to 33, wherein linker B comprises at least one amino acid. The compound of any one of claims 1 to 34, wherein linker B comprises at least one nucleotide. The compound of claim 35, wherein the at least one nucleotide comprises at least one of an abasic nucleotide and an inverted abasic nucleotide. The compound of claim 36, wherein the abasic nucleotide is abasic deoxyribonucleic acid. The compound of claim 36, wherein the inverted abasic nucleotide is an inverted abasic deoxyribonucleic acid. The compound of claim 36, wherein the abasic nucleotide is an abasic ribonucleic acid. The compound of claim 36, wherein the inverted abasic nucleotide is an inverted abasic ribonucleic acid. The compound of any one of claims 1 to 40, wherein linker B comprises at least one sugar. The compound of claim 41, wherein the at least one sugar comprises at least one of glucose, fructose, mannose, galactose, ribose, and glucosamine. The compound of any one of claims 1 to 42, wherein linker B comprises at least one of the following: Where: j is an integer from 1 to 12, and k is an integer from 0 to 12. The compound of any one of claims 1 to 26, wherein the linker BW is: Where: j is an integer from 0 to 12, and k is an integer from 0 to 12. The compound of any one of claims 1 to 43, wherein W is hydroxy. The compound of any one of claims 1 to 43, wherein W is a protected hydroxy group. The compound of claim 46, wherein the protected hydroxyl group is 4,4'-dimethoxytrityl (DMT), monomethoxytrityl (MMT), 9-(p-methyl Oxyphenyl) xanthen-9-yl (Mox) and 9-phenyl xanthen-9-yl (Px) to protect at least one. The compound of any one of claims 1 to 43, wherein W is a phosphoramidite group having the formula: , wherein: R ^a is C1 to C6 alkyl, C3 to C6 cycloalkyl, isopropyl, or R ^a is connected to R ^b through a nitrogen atom to form a ring, R ^b is C1 to C6 alkyl, C3 to C6 ring Alkyl, isopropyl, or ^Rb is linked to Ra through a nitrogen atom to form a ring, and ^Rc is a phosphite protecting group ^, a phosphate protecting group or 2-cyanoethyl. The compound of claim 48, wherein the phosphite protecting group comprises methyl, allyl, 2-cyanoethyl, 4-cyano-2-butenyl, 2-cyano-1,1- Dimethylethyl, 2-(trimethylsilyl)ethyl, 2-(S-acetylthio)ethyl, 2-(S-pivalylthio)ethyl, 2-(4 -nitrophenyl)ethyl, 2,2,2-trichloroethyl, 2,2,2-trichloro-1,1-dimethylethyl, 1,1,1,3,3,3 - at least one of hexafluoro-2-propyl, fluorenyl-9-methyl, 2-chlorophenyl, 4-chlorophenyl and 2,4-dichlorophenyl. The compound of claim 48, wherein the phosphate protecting group comprises methyl, allyl, 2-cyanoethyl, 4-cyano-2-butenyl, 2-cyano-1,1-di Methylethyl, 2-(trimethylsilyl)ethyl, 2-(S-acetylthio)ethyl, 2-(S-pivalylthio)ethyl, 2-(4- Nitrophenyl) ethyl, 2,2,2-trichloroethyl, 2,2,2-trichloro-1,1-dimethylethyl, 1,1,1,3,3,3- At least one of hexafluoro-2-propyl, fluorenyl-9-methyl, 2-chlorophenyl, 4-chlorophenyl and 2,4-dichlorophenyl. The compound of any one of claims 1 to 43, wherein W is carboxy. The compound of claim 51, wherein W is an activated carboxyl group of the formula: , where X is a leaving group. The compound of claim 52, wherein the leaving group is selected from carboxylate, sulfonate, chloride, phosphate, imidazole, hydroxybenzotriazole (HOBt), N-hydroxysuccinimide (NHS) , tetrafluorophenol, pentafluorophenol and p-nitrophenol. The compound of any one of claims 1 to 43, wherein W is a Michael acceptor. The compound of claim 54, wherein the Michael acceptor has the formula: , wherein: E is an electron-withdrawing group; and R ^d is hydrogen or a C1-C6 alkyl substituent on an alkene. The compound of claim 55, wherein the electron withdrawing group is formamide or ester. The compound of claim 55 or 56, wherein E and the carbon-carbon double bond are part of a maleimide. The compound of any one of claims 1 to 43, wherein W is an oligonucleotide. The compound of claim 58, wherein the oligonucleotide is a single stranded oligonucleotide. The compound of claim 58, wherein the oligonucleotide is a double stranded oligonucleotide. The compound of claim 58, wherein the oligonucleotide comprises at least 3 independently selected nucleotides. The compound of claim 61, wherein said oligonucleotide comprises 16 to 23 independently selected nucleotides. The compound of claim 61, wherein said oligonucleotide comprises about 100 independently selected nucleotides. The compound of claim 61, wherein said oligonucleotide comprises up to fourteen thousand independently selected nucleotides. The compound of any one of claims 1 to 43, wherein W is: , wherein: Linker C is absent or is a spacer attached to the 3' or 5' end of the oligonucleotide, X is methyl, oxygen, sulfur or amino, and Y is oxygen, sulfur or amino. The compound of claim 65, wherein linker C comprises at least one heterocyclic compound. The compound of claim 66, wherein the heterocyclic compound is an abasic nucleotide or an inverted abasic nucleotide. The compound of any one of claims 1 to 43, wherein W is: , wherein linker C is a spacer attached to the 3' end or 5' end of the oligonucleotide. The compound of claim 68, wherein linker C comprises at least one of polyethylene glycol (PEG), alkyl, and cycloalkyl. The compound of claim 68 or 69, wherein linker C comprises at least one heteroatom. The compound of claim 70, wherein the at least one heteroatom comprises at least one of oxygen, nitrogen, sulfur, and phosphorus. The compound of any one of claims 68 to 71, wherein linker C comprises at least one aliphatic heterocycle. The compound of claim 72, wherein the at least one aliphatic heterocycle comprises at least one of tetrahydrofuran, tetrahydropyran, morpholine, piperidine, piperazine, pyrrolidine, and azetidine. The compound of any one of claims 68 to 73, wherein linker C comprises at least one heteroaryl. The compound of claim 74, wherein the at least one heteroaryl group comprises at least one of imidazole, pyrazole, pyridine, pyrimidine, triazole, and 1,2,3-triazole. The compound of any one of claims 68 to 75, wherein linker C comprises at least one amino acid. The compound of any one of claims 68 to 76, wherein linker C comprises at least one nucleotide. The compound of claim 77, wherein the at least one nucleotide comprises at least one of an abasic nucleotide and an inverted abasic nucleotide. The compound of claim 78, wherein the abasic nucleotide is abasic deoxyribonucleic acid (DNA). The compound of claim 78, wherein the inverted abasic nucleotide is inverted abasic deoxyribonucleic acid (DNA). The compound of claim 78, wherein the abasic nucleotide is abasic ribonucleic acid (RNA). The compound of claim 78, wherein the inverted abasic nucleotide is an inverted abasic ribonucleic acid (RNA). The compound of any one of claims 68 to 82, wherein linker C comprises at least one sugar. The compound of claim 83, wherein the at least one sugar comprises at least one of glucose, fructose, mannose, galactose, ribose, and glucosamine. The compound of any one of claims 68 to 84, wherein linker C comprises one or more of the following: Where: j is an integer from 1 to 12, and k is an integer from 0 to 12. The compound of any one of claims 1 to 43, wherein W is: , wherein linker C is a spacer attached to the 3' end or 5' end of the oligonucleotide. The compound of claim 86, wherein linker C comprises at least one of polyethylene glycol (PEG), alkyl, and cycloalkyl. The compound of claim 86 or 87, wherein linker C comprises one or more of the following: Where: j is an integer from 1 to 12, and k is an integer from 0 to 12. The compound of any one of claims 1 to 88, wherein the compound is selected from the group consisting of: . The compound of claim 89, wherein the compound is a stereoisomer of one of compounds 1-75. The compound of any one of claims 1-90, wherein W is one or more agents. The compound of claim 91, wherein the one or more agents comprise small interfering RNA (siRNA), single-stranded siRNA, double-stranded siRNA, small activating RNA, RNAi, microRNA (miRNA), antisense oligonucleotides At least one of acid, short guide RNA (gRNA), single guide RNA (sgRNA), messenger RNA (mRNA), ribozyme, plasmid, immunostimulatory nucleic acid, antagomir and aptamer. The compound of claim 92, wherein the double-stranded siRNA comprises at least one modified ribonucleotide. The compound of claim 92, wherein substantially all ribonucleotides of the double-stranded siRNA are modified. The compound of claim 92, wherein all ribonucleotides of the double-stranded siRNA are modified. The compound of any one of claims 92 to 95, wherein the modified ribonucleotides comprise 2'-O-methyl nucleotides, 2'-fluoro nucleotides, 2'-deoxynucleosides acid, 2'3'-seco nucleotide mimetic, locked nucleotide, 2'-F-arabinonucleotide, 2'-methoxyethyl nucleotide, abasic nucleotide, ribose alcohol, inverted nucleotide, inverted abasic nucleotide, inverted 2'-OMe nucleotide, inverted 2'deoxynucleotide, 2'-amino modified nucleotide, 2'-alkane Modified nucleotides, morpholino and 3'-OMe nucleotides, nucleotides containing 5'-phosphorothioate groups, or 5'-(E)-vinylphosphonate Nucleotides (antisense strand only), or terminal nucleotides linked to cholesterol derivatives or dodecanoic acid didecylamide groups, 2'-amino modified nucleotides, 2'-alkyl modified Nucleotides, phosphoramidates, or nucleotides containing unnatural bases. The compound of any one of claims 92 to 96, wherein at least one strand of the double stranded siRNA comprises at least one phosphorothioate linkage. The compound of any one of claims 92 to 97, wherein at least one strand of the double stranded siRNA comprises up to 6 phosphorothioate linkages. The compound of any one of claims 92 to 98, wherein the double-stranded siRNA comprises at least one locked nucleic acid. The compound of any one of claims 92 to 99, wherein the double-stranded siRNA comprises at least one unlocking nucleic acid. The compound of any one of claims 92 to 100, wherein the double-stranded siRNA comprises at least one glycerol nucleic acid. A pharmaceutical composition comprising the compound of any one of claims 1-101. The pharmaceutical composition of claim 102, wherein W is one or more pharmaceutical agents. The pharmaceutical composition of claim 103, further comprising one or more therapeutic agents. The pharmaceutical composition of claim 103 or 104, further comprising a pharmaceutically acceptable carrier. A composition for targeted delivery of one or more agents comprising the compound of any one of claims 1 to 90, wherein W is the one or more agents. The composition of claim 106, wherein the one or more agents comprise small interfering RNA (siRNA), single-stranded siRNA, double-stranded siRNA, small activating RNA, microRNA (miRNA), antisense oligonucleotides At least one of short guide RNA (gRNA), single guide RNA (sgRNA), messenger RNA (mRNA), ribozyme, plasmid, immunostimulatory nucleic acid, antagomir and aptamer. The composition of claim 107, wherein the double-stranded siRNA comprises at least one modified ribonucleotide in one or both strands of the siRNA. The composition of claim 108, wherein substantially all ribonucleotides of the double-stranded siRNA are modified. The composition of claim 108, wherein all ribonucleotides of the double-stranded siRNA are modified. The composition of any one of claims 108 to 110, wherein the modified ribonucleotides comprise: 2'-O-methyl nucleotides, 2'-fluoro nucleotides, 2'-deoxy Nucleotides, 2'3'-seco nucleotide mimics, locked nucleotides, 2'-F-arabinonucleotides, 2'-methoxyethyl nucleotides, abasic nucleotides , ribitol, inverted nucleotides, inverted abasic nucleotides, inverted 2'-OMe nucleotides, inverted 2'deoxynucleotides, 2'-amino modified nucleotides, 2' -Alkyl modified nucleotides, morpholino and 3'-OMe nucleotides, nucleotides containing 5'-phosphorothioate groups, or 5'-(E)-vinylphosphine ester nucleotides (antisense strand only), or terminal nucleotides linked to cholesterol derivatives or dodecanoic acid didecylamide groups, 2'-amino modified nucleotides, 2'-alkyl Modified nucleotides, phosphoramidates, or nucleotides comprising unnatural bases. The composition of any one of claims 107 to 111, wherein at least one strand of the double-stranded siRNA comprises at least one phosphorothioate linkage. The composition of claim 112, wherein at least one strand of the double-stranded siRNA comprises up to 6 phosphorothioate linkages. The composition of any one of claims 107 to 113, wherein the double-stranded siRNA comprises at least one locked nucleic acid. The composition of any one of claims 107 to 114, wherein the double-stranded siRNA comprises at least one unlocking nucleic acid. The compound of any one of claims 107-115, wherein the double-stranded siRNA comprises at least one glycerol nucleic acid. A pharmaceutical composition comprising the composition of any one of claims 106-116. The pharmaceutical composition of claim 117, further comprising one or more therapeutic agents. The pharmaceutical composition of claim 117 or 118, further comprising a pharmaceutically acceptable carrier. A method for the preparation of a compound for targeted delivery of one or more agents comprising: receiving a first compound comprising a diamine comprising a first nitrogen and a second nitrogen, the first nitrogen being a primary amine and the second nitrogen being a secondary amine comprising a protecting group; A second compound is produced by coupling a plurality of protected carboxylic acids with the first compound, the first nitrogen in the second compound is comprising the first protected carboxylic acid and the second protected carboxylic acid tertiary amine, the second nitrogen of the second compound is a tertiary amine comprising the protecting group and a third protected carboxylic acid; producing a third compound by deprotecting a second nitrogen of the second compound, resulting in the second nitrogen becoming a secondary amine comprising the third protected carboxylic acid; A fourth compound is produced by linking a hydroxyl-containing moiety to a second nitrogen of said third compound, resulting in said second nitrogen becoming a tertiary amine comprising said third protected carboxylic acid and said hydroxyl-containing moiety or amide; producing a fifth compound by converting the protected carboxylic acid of the fourth compound to a carboxylic acid; and A sixth compound is produced by using the fifth compound to perform an amide coupling reaction, the first nitrogen in the sixth compound is a tertiary amine comprising a first amide and a second amide, and in the sixth compound The second nitrogen is a tertiary amine comprising the hydroxyl-containing moiety and a third amide, wherein the first amide, the second amide, and the third amide are each associated with an independently selected targeting ligand body coupling. The method of claim 120, wherein the protecting group is selected from benzyl and triphenylmethyl. The method of claim 120 or 121, wherein producing the second compound comprises performing a _SN2 substitution reaction with the first compound. The method of claim 120 or 121, wherein producing the second compound comprises performing a reductive amination reaction with the first compound. The method of claim 120 or 121, wherein producing the second compound comprises performing a Michael addition reaction with the first compound. The method of any one of claims 120 to 124, wherein: The protecting group is benzyl; and Producing the third compound includes performing a hydrogenation reaction with the second compound. The method of any one of claims 120 to 124, wherein: The protecting group is triphenylmethyl; and Producing the third compound includes reacting the second component with at least one acid. The method of any one of claims 120 to 126, wherein producing the fourth compound comprises performing a _SN2 substitution reaction with the third compound. The method of any one of claims 120 to 126, wherein producing the fourth compound comprises performing a reductive amination reaction with the third compound. The method of any one of claims 120 to 126, wherein producing the fourth compound comprises performing a Michael addition reaction with the third compound. The method of any one of claims 120 to 126, wherein producing the fourth compound comprises performing an amide coupling reaction with the third compound. The method of any one of claims 120 to 126, wherein producing the fourth compound comprises performing a nucleophilic addition reaction with the third compound. The method of any one of claims 120-131, wherein the hydroxyl-containing moiety is attached to the second nitrogen using the linker B of any one of claims 24-40. The method of any one of claims 120 to 132, wherein producing the fifth compound comprises reacting the fourth compound with at least one acid. The method of claim 133, wherein the at least one acid comprises at least one of hydrochloric acid, hydrobromic acid, trifluoroacetic acid, and formic acid. The method of any one of claims 120 to 134, wherein producing the fifth compound comprises performing a hydrogenation reaction with the fourth compound. The method of any one of claims 120 to 134, wherein producing the fifth compound comprises performing a hydrolysis reaction with the fourth compound. The method of any one of claims 120 to 136, wherein the first amide, the second amide, and the Each tertiary amide is coupled to said independently selected targeting ligand. The method of any one of claims 120-137, wherein the independently selected targeting ligand is independently selected as the targeting ligand of any one of claims 4-11. The method of any one of claims 120 to 138, further comprising converting the hydroxyl group to a phosphoramidite group using a phosphoritylation reaction. The method of claim 139, wherein converting the hydroxyl group to the phosphoramidite group is performed after performing the amide coupling reaction to produce the sixth compound. A method for the preparation of a compound for targeted delivery of one or more agents comprising: receiving a first compound comprising a diamine comprising a first nitrogen and a second nitrogen, the first nitrogen being a secondary amine comprising a first protecting group, the second nitrogen being an amine comprising a second protecting group ; producing a second compound by coupling a first protected carboxylic acid to a first nitrogen of said first compound, causing said first nitrogen to become a tertiary amine; Removal of the first protecting group from a first nitrogen of the second compound to yield a third compound comprising a first nitrogen and a second nitrogen, the first nitrogen being a secondary amine comprising the first protected carboxylic acid , the second nitrogen is an amine comprising the second protecting group; producing a fourth compound by coupling a second protected carboxylic acid to a first nitrogen of said third compound, causing said first nitrogen to become a tertiary amine; Removal of the second protecting group from the fourth compound yields a fifth compound comprising a first nitrogen and a second nitrogen, the first nitrogen comprising the first protected carboxylic acid and the second protected a tertiary amine of a carboxylic acid, the second nitrogen being a primary amine; producing a sixth compound by coupling a third protected carboxylic acid with a second nitrogen of said fifth compound, causing said second nitrogen to become a secondary amine; producing a seventh compound by linking a moiety comprising a hydroxyl group to a second nitrogen of said sixth compound, causing said second nitrogen to become a tertiary amine; producing an eighth compound by converting the third protected carboxylic acid of the seventh compound into the first carboxylic acid; A ninth compound is produced by performing an amide coupling reaction using the eighth compound, the first nitrogen of the ninth compound comprising the first protected carboxylic acid and the second protected carboxylic acid, the The second nitrogen of said ninth compound comprises a first amide having a first targeting ligand coupled thereto and said hydroxyl-containing moiety; producing a tenth compound by converting the second protected carboxylic acid of the ninth compound into a second carboxylic acid; An eleventh compound is produced by performing an amide coupling reaction using the tenth compound, the first nitrogen of the eleventh compound comprising a first protected carboxylic acid and having a second targeting ligand coupled thereto a second amide, the second nitrogen of the eleventh compound comprising a first amide having a first targeting ligand coupled thereto and the moiety comprising the hydroxyl group; producing a twelfth compound by converting the first protected carboxylic acid of the eleventh compound into a third carboxylic acid; and A thirteenth compound is produced by performing an amide coupling reaction using the twelfth compound, the first nitrogen of the thirteenth compound comprising a second amide having a second targeting ligand coupled thereto and having A third amide of a third targeting ligand coupled thereto, the second nitrogen of the thirteenth compound comprising a first amide having a first targeting ligand coupled thereto and the moiety comprising a hydroxyl group . The method of claim 141, wherein: said first protecting group is benzyl; and The second protecting group is t-butoxycarbonyl (Boc). The method of claim 141 or 142, wherein producing the second compound comprises performing a _SN2 substitution reaction with the first compound. The method of claim 141 or 142, wherein producing the second compound comprises performing a reductive amination reaction with the first compound. The method of claim 141 or 142, wherein producing the second compound comprises performing a Michael addition reaction with the first compound. The method of any one of claims 141 to 145, wherein producing the third compound comprises performing a hydrogenation reaction with the second compound. The method of any one of claims 141 to 146, wherein producing the fourth compound comprises performing a _SN2 substitution reaction with the third compound. The method of any one of claims 141 to 146, wherein producing the fourth compound comprises performing a reductive amination reaction with the third compound. The method of any one of claims 141 to 146, wherein producing the fourth compound comprises performing a Michael addition reaction with the third compound. The method of any one of claims 141 to 146, wherein producing the fourth compound comprises performing an amide coupling reaction with the third compound. The method of any one of claims 141 to 146, wherein producing the fourth compound comprises performing a nucleophilic addition reaction with the third compound. The method of any one of claims 141 to 151, wherein producing the fifth compound comprises reacting the fourth compound with at least one acid. The method of claim 152, wherein the at least one acid comprises at least one of hydrochloric acid and trifluoroacetic acid. The method of any one of claims 141 to 153, wherein producing the sixth compound comprises performing an SN2 substitution reaction with the fifth compound. The method of any one of claims 141 to 153, wherein producing the sixth compound comprises performing a reductive amination reaction with the fifth compound. The method of any one of claims 141 to 153, wherein producing the sixth compound comprises performing a Michael addition reaction with the fifth compound. The method of any one of claims 141 to 156, wherein producing the seventh compound comprises performing an SN2 substitution reaction with the sixth compound. The method of any one of claims 141 to 156, wherein producing the seventh compound comprises performing a reductive amination reaction with the sixth compound. The method of any one of claims 141 to 156, wherein producing the seventh compound comprises performing a Michael addition reaction with the sixth compound. The method of any one of claims 141 to 156, wherein producing the seventh compound comprises performing an amide coupling reaction with the sixth compound. The method of any one of claims 141 to 156, wherein producing the seventh compound comprises performing a nucleophilic addition reaction with the sixth compound. The method of any one of claims 141 to 161, wherein the first amide is coupled to the first targeting ligand using the independently selected linker A of any one of claims 12 to 23 couplet. The method of any one of claims 141 to 162, wherein the second amide is coupled to the second targeting ligand using the independently selected linker A of any one of claims 12 to 23 couplet. The method of any one of claims 141 to 163, wherein the third amide is coupled to the third targeting ligand using the independently selected linker A of any one of claims 12 to 23 couplet. The method of any one of claims 141 to 164, wherein the first targeting ligand, the second targeting ligand, and the third targeting ligand are independently selected as claimed in claims 4 to 164. One or more of the targeting ligands of any one of 11. The method of any one of claims 141-165, wherein the hydroxyl group is coupled to the second nitrogen using the linker B of any one of claims 24-40. The method of any one of claims 141 to 166, further comprising converting the hydroxyl group to a phosphoramidite group using a phosphoritylation reaction. The method of claim 167, wherein converting the hydroxyl group to the phosphoramidite group is performed after producing the thirteenth compound. A method for delivering a medicament to a subject, the method comprising: Administering to the subject: (a) the compound of any one of claims 1 to 90, wherein W is one or more agents, or (b) A composition as claimed in any one of claims 106 to 116. The method of claim 169, wherein said subject is a vertebrate. The method of claim 169, wherein said subject is a mammal. The method of claim 169, wherein said mammal is a human. The method of any one of claims 169 to 172, wherein the compound is administered in a pharmaceutically acceptable carrier. A method for delivering a medicament to a subject, the method comprising: Administering the pharmaceutical composition of any one of claims 102, 103, 104, 105, 116, 117, 118 or 119 to the subject. The method of claim 174, wherein said subject is a vertebrate. The method of claim 174, wherein said subject is a mammal, optionally said mammal is a human. The method of claim 174, wherein the one or more agents comprise small interfering RNA (siRNA), single-stranded siRNA, double-stranded siRNA, small activating RNA, microRNA (miRNA), antisense oligonucleotides, At least one of short guide RNA (gRNA), single guide RNA (sgRNA), messenger RNA (mRNA), ribozyme, plasmid, immunostimulatory nucleic acid, antagomir and aptamer. The method of claim 177, wherein the double-stranded siRNA comprises at least one modified ribonucleotide in one or both strands of the siRNA. The method of claim 178, wherein substantially all ribonucleotides of the double-stranded siRNA are modified. The method of claim 178, wherein all ribonucleotides of the double-stranded siRNA are modified. The method of any one of claims 178 to 180, wherein the modified ribonucleotides comprise: 2'-O-methyl nucleotides, 2'-fluoro nucleotides, 2'-deoxynucleosides nucleotides, 2'3'-seco nucleotide mimics, locked nucleotides, 2'-F-arabinonucleotides, 2'-methoxyethyl nucleotides, abasic nucleotides, Ribitol, inverted nucleotides, inverted abasic nucleotides, inverted 2'-OMe nucleotides, inverted 2'deoxynucleotides, 2'-amino modified nucleotides, 2'- Alkyl modified nucleotides, morpholino and 3'-OMe nucleotides, nucleotides containing a 5'-phosphorothioate group, or 5'-(E)-vinylphosphonic acid ester nucleotides (antisense strand only), or terminal nucleotides linked to cholesterol derivatives or dodecanoic acid didecylamide groups, 2'-amino modified nucleotides, 2'-alkyl Modified nucleotides, phosphoramidates, or nucleotides containing unnatural bases. The method of any one of claims 177-181, wherein at least one strand of the double-stranded siRNA comprises at least one phosphorothioate linkage. The method of claim 182, wherein at least one strand of the double-stranded siRNA comprises up to 6 phosphorothioate linkages. The method of any one of claims 177-183, wherein the double-stranded siRNA comprises at least one locked nucleic acid. The method of any one of claims 177-184, wherein the double-stranded siRNA comprises at least one unlocking nucleic acid. The method of any one of claims 177-185, wherein the double-stranded siRNA comprises at least one glycerol nucleic acid. The method of any one of claims 174-186, wherein the pharmaceutical composition further comprises one or more therapeutic agents. The compound of claim 1, wherein n is 1 or 2.