Multivalent Ligand Clusters Containing Diamine Scaffolds for Targeted Delivery of Therapeutics

Abstract

Described is a multivalent ligand cluster having a diamine scaffold for targeted delivery of pharmaceuticals conjugated thereto. The multivalent ligand cluster may comprise one or more N-acetylgalactosamine (GalNAc) targeting ligands. The multivalent ligand cluster can be conjugated to one or more small interfering ribonucleic acids (siRNAs), where siRNA is an example of pharmaceuticals. Also described is a composition comprising the multivalent ligand cluster, and a method for making the multivalent ligand cluster.

Description

[Background technology]

[0001] Due to their high molecular weight and polyanionic nature, oligonucleotides generally have low cell membrane permeability.Therefore, targeting ligands are often conjugated to oligonucleotide compounds to enhance cellular uptake and improve the tissue specificity of in vivo delivery through the well-known mechanism of receptor-mediated cellular uptake.In some cases, multivalent ligand clusters have the advantage over single ligands in enhancing delivery to targeted tissues via specific receptors.Asialoglycoprotein receptor (ASGPR) is one such receptor.

[0002] It has been demonstrated that N-acetylgalactosamine (GalNAc), a ligand for ASGPR, can promote the delivery of oligonucleotide drugs to hepatocytes.It has also been demonstrated that multivalent GalNAc ligand clusters have higher binding affinity to ASGPR than individual GalNAc ligands, and therefore have higher efficiency in delivering therapeutic oligonucleotides to hepatocytes in the liver. Summary of the Invention

[0003] One aspect of the disclosure is a compound for targeted delivery of one or more pharmaceutical agents, the compound having the formula:

[0004] [ka] wherein each TL is an independently selected targeting ligand, m is an integer between 1 and 10, each n is an independently selected integer between 1 and 10, each linker A is an independently selected spacer, linker B is a spacer, and W is either one or more pharmaceutical agents or a functional group that can be linked to one or more pharmaceutical agents. In some embodiments, m is 1. In some embodiments, m is 2.

[0005] In some embodiments, n is 1. In some embodiments, n is 2.

[0006] In some embodiments, at least one of the independently selected TLs may bind to one or more cellular receptors, cellular channels, and cellular transporters that may facilitate cellular uptake. In some embodiments, at least one of the independently selected TLs comprises at least one small molecule ligand. In some embodiments, the at least one small molecule comprises at least one of N-acetylgalactosamine, galactose, galactosamine, N-formyl-galactosamine, N-propionylgalactosamine, N-butanoylgalactosamine, and N-iso-butanoylgalactosamine, macrocycles, folate 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 may bind to at least one asialoglycoprotein receptor (ASGPR). In some embodiments, at least one of the independently selected TLs may bind to at least one transferrin receptor. In some embodiments, at least one of the independently selected TLs may bind to at least one integrin receptor. In some embodiments, at least one of the independently selected TLs may bind to at least one folate receptor. In some embodiments, at least one of the independently selected TLs may bind to at least one G protein-coupled receptor (GPCR).

[0007] In some embodiments, at least one of the independently selected linkers A comprises at least one of polyethylene glycol, alkyl group, cycloalkyl group, alkenyl group, cycloalkenyl group, alkynyl group, aryl group, aralkyl group, aralkenyl group, and aralkynyl group. 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, 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 at least one of:

[0008] [ka] [wherein p is an integer between 0 and 12, pp is an integer between 0 and 12, q is an integer between 1 and 12, and qq is an integer between 1 and 12.]

[0009] In some embodiments, linker B comprises at least one of polyethylene glycol, alkyl group, cycloalkyl group, alkenyl group, cycloalkenyl group, alkynyl group, aryl group, aralkyl group, aralkenyl group, and aralkynyl group. 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 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, 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 nucleotide is an abasic deoxyribonucleic acid. In some embodiments, the inverted abasic nucleotide is an inverted abasic deoxyribonucleic acid. In some embodiments, the abasic nucleotide is an abasic ribonucleic acid. In some embodiments, the inverted abasic nucleotide is an inverted abasic ribonucleic acid. 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

[0010] [ka] [ka] [wherein j is an integer between 1 and 12, and k is an integer between 0 and 12.]

[0011] In some embodiments, the linker BW is

[0012] [ka] [In the formula, j is an integer between 0 and 12, and k is an integer between 0 and 12.] It is.

[0013] In some embodiments, W is a hydroxy group. In some embodiments, W is a protected hydroxy group. In some embodiments, the protected hydroxy group is protected using at least one of 4,4'-dimethoxytrityl (DMT), monomethoxytrityl (MMT), 9-(p-methoxyphenyl)xanthen-9-yl (Mox), and 9-phenylxanthen-9-yl (Px). In some embodiments, W is a group of the formula:

[0014] [ka] [In the formula, R a is a C1-C6 alkyl, C3-C6 cycloalkyl, isopropyl group, or R a is connected to R via the nitrogen atom b Together with this, it forms a ring, R b is a C1-C6 alkyl, C3-C6 cycloalkyl, isopropyl group, or R b is connected to R via the nitrogen atom a Together with this, it forms a ring, R c is a phosphite protecting group, a phosphate protecting group, or a 2-cyanoethyl group. is a phosphoramidite group having the formula:

[0015] In some embodiments, the phosphite protecting group comprises at least one of methyl, allyl, 2-cyanoethyl, 4-cyano-2-butenyl, 2-cyano-1,1-dimethylethyl, 2-(trimethylsilyl)ethyl, 2-(S-acetylthio)ethyl, 2-(S-pivaloylthio)ethyl, 2-(4-nitrophenyl)ethyl, 2,2,2-trichloroethyl, 2,2,2-trichloro-1,1-dimethylethyl, 1,1,1,3,3,3-hexafluoro-2-propyl, fluorenyl-9-methyl, 2-chlorophenyl, 4-chlorophenyl, and 2,4-dichlorophenyl. In some embodiments, the phosphate protecting group comprises at least one of methyl, allyl, 2-cyanoethyl, 4-cyano-2-butenyl, 2-cyano-1,1-dimethylethyl, 2-(trimethylsilyl)ethyl, 2-(S-acetylthio)ethyl, 2-(S-pivaloylthio)ethyl, 2-(4-nitrophenyl)ethyl, 2,2,2-trichloroethyl, 2,2,2-trichloro-1,1-dimethylethyl, 1,1,1,3,3,3-hexafluoro-2-propyl, fluorenyl-9-methyl, 2-chlorophenyl, 4-chlorophenyl, and 2,4-dichlorophenyl.

[0016] In some embodiments, W is a carboxyl group. In some embodiments, W is a group of the formula:

[0017] [ka] wherein X is a leaving group. In some embodiments, the leaving group is selected from the group consisting of carboxylate, sulfonate, chloride, phosphate, imidazole, hydroxybenzotriazole (HOBt), N-hydroxysuccinimide (NHS), tetrafluorophenol, pentafluorophenol, and para-nitrophenol.

[0018] In some embodiments, W is a Michael acceptor. In some embodiments, the Michael acceptor has the formula:

[0019] [ka] [In the formula, E is an electron-withdrawing group, and R d is hydrogen or a C1-C6 alkyl substituent on the olefin. In some embodiments, the electron withdrawing group is a carboxamide or an ester. In some embodiments, E and the carbon-carbon double bond are part of a maleimide.

[0020] In some embodiments, W is an oligonucleotide. In some embodiments, the oligonucleotide is a single-stranded oligonucleotide. In some embodiments, the oligonucleotide is a double-stranded oligonucleotide. In some embodiments, the oligonucleotide comprises at least 3 independently selected nucleotides. In some embodiments, the oligonucleotide comprises between 16 and 23 independently selected nucleotides. In some embodiments, the oligonucleotide comprises about 100 independently selected nucleotides. In some embodiments, the oligonucleotide comprises up to 14000 independently selected nucleotides.

[0021] In some embodiments, W is

[0022] [ka] [In the formula, Linker C is absent or is a spacer attached to the 3' or 5' end of the oligonucleotide; X is a methyl group, oxygen, sulfur, or amino group; Y is oxygen, sulfur, or an amino group. It is.

[0023] 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.

[0024] In some embodiments, W is

[0025] [ka] wherein the linker C is a spacer attached to the 3' or 5' end of the oligonucleotide. In some embodiments, the linker C comprises at least one of polyethylene glycol (PEG), an alkyl group, and a cycloalkyl group. In some embodiments, the 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, the 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, the linker C 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, the linker C comprises at least one amino acid. In some embodiments, the 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 an abasic deoxyribonucleic acid (DNA). In some embodiments, the inverted abasic nucleotide is an inverted abasic deoxyribonucleic acid (DNA). In some embodiments, the abasic nucleotide is an abasic ribonucleic acid (RNA). In some embodiments, the inverted abasic nucleotide is an inverted abasic ribonucleic acid (RNA). In some embodiments, the 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, the linker C comprises

[0026] [ka] [In the formula, j is an integer between 1 and 12, and k is an integer between 0 and 12.] Contains one or more of the following:

[0027] In some embodiments, W is

[0028] [ka] wherein the linker C is a spacer attached to the 3' or 5' end of the oligonucleotide. In some embodiments, the linker C comprises at least one of polyethylene glycol (PEG), an alkyl group, and a cycloalkyl group. In some embodiments, the linker C is

[0029] [ka] [In the formula, j is an integer between 1 and 12, and k is an integer between 0 and 12.] Contains one or more of the following:

[0030] In some embodiments, the compound is

[0031] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

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[0032] In some embodiments, the compound is a stereoisomer of one of compounds 1-75.

[0033] In some embodiments, W is one or more pharmaceutical agents. In some embodiments, the one or more pharmaceutical agents include at least one of small interfering RNA (siRNA), single-stranded siRNA, double-stranded siRNA, small activating RNA, RNAi, microRNA (miRNA), antisense oligonucleotide, short guide RNA (gRNA), single guide RNA (sgRNA), messenger RNA (mRNA), ribozyme, plasmid, immunostimulatory nucleic acid, antagomir, and aptamer. In some embodiments, the double-stranded siRNA includes at least one modified ribonucleotide. In some embodiments, each strand of the double-stranded siRNA is 19-23 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 ribonucleotide comprises 2'-O-methyl nucleotide, 2'-fluoro nucleotide, 2'-deoxy nucleotide, 2'3'-seco nucleotide mimic, locked nucleotide, 2'-F-arabino nucleotide, 2'-methoxyethyl nucleotide, abasic nucleotide, ribitol, inverted nucleotide, inverted abasic nucleotide, inverted 2'-OMe nucleotide, inverted 2' deoxy nucleotide, 2'-amino-modified nucleotide, 2'-alkyl-modified nucleotide, morpholino nucleotide, and 3'-OMe nucleotide, nucleotide comprising 5'-phosphorothioate group, or 5'-(E)-vinyl phosphonate nucleotide (antisense strand only), or terminal nucleotide linked to cholesteryl derivative or dodecanoic acid bisdecyl amide group, 2'-amino-modified nucleotide, 2'-alkyl-modified nucleotide, phosphoramidate, or non-natural base comprising nucleotide. In some embodiments, at least one strand of the double-stranded siRNA comprises at least one phosphorothioate bond. In some embodiments, at least one strand of the double-stranded siRNA comprises up to six 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 unlocked nucleic acid. In some embodiments, the double-stranded siRNA comprises at least one glycerol nucleic acid.

[0034] Another aspect of the present disclosure relates to a pharmaceutical composition comprising any one of the compounds detailed above. In some embodiments, the pharmaceutical composition comprises one or more pharmaceutical agents. In some embodiments, the pharmaceutical composition comprises one or more therapeutic agents. In some embodiments, the pharmaceutical composition comprises a pharma- ceutical acceptable carrier.

[0035] A further aspect of the present disclosure relates to a composition for targeted delivery of one or more pharmaceutical agents, comprising any one of the compounds described above, wherein W is one or more pharmaceutical agents. In some embodiments, the one or more pharmaceutical agents comprise at least one of small interfering RNA (siRNA), single-stranded siRNA, double-stranded siRNA, small activating RNA, microRNA (miRNA), antisense oligonucleotide, short guide RNA (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 19-23 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, modified ribonucleotides include 2'-O-methyl nucleotides, 2'-fluoro nucleotides, 2'-deoxy nucleotides, 2'3'-seconucleotide mimics, locked nucleotides, 2'-F-arabino nucleotides, 2'-methoxyethyl nucleotides, abasic nucleotides, ribitol, inverted nucleotides, inverted abasic nucleotides, inverted 2'-OMe nucleotides, inverted 2'deoxy nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, and 3'-OMe nucleotides, nucleotides containing a 5'-phosphorothioate group, or a 5'-(E)-vinyl phosphonate nucleotide (antisense strand only), or a terminal nucleotide linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, phosphoramidates, or non-natural base containing nucleotides. In some embodiments, at least one strand of the double-stranded siRNA contains at least one phosphorothioate bond.In some embodiments, at least one strand of the double-stranded siRNA comprises up to six phosphorothioate bonds.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 unlocked nucleic acid.In some embodiments, the double-stranded siRNA comprises at least one glycerol nucleic acid.

[0036] Another aspect of the present disclosure relates to a pharmaceutical composition comprising any one of the compounds described above.In some embodiments, the pharmaceutical composition comprises one or more therapeutic agents.In some embodiments, the pharmaceutical composition comprises a pharma- ceutical acceptable carrier.

[0037] A further aspect of the present disclosure is a method for making a compound for targeted delivery of one or more pharmaceutical 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; coupling a plurality of protected carboxylic acids with the first compound to produce a second compound, wherein the first nitrogen in the second compound 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 a protecting group and a third protected carboxylic acid; deprotecting the second nitrogen of the second compound such that the second nitrogen is a secondary amine comprising a third protected carboxylic acid, thereby producing a third compound; producing a fourth compound by attaching a hydroxy-containing moiety to a second nitrogen of a third compound such that the second nitrogen is a tertiary amine or amide comprising a third protected carboxylic acid and a hydroxy-containing moiety; producing a fifth compound by converting the protected carboxylic acid of the fourth compound to a carboxylic acid; and producing a sixth compound by performing an amide coupling reaction with the fifth compound, wherein the first nitrogen in the sixth compound is a tertiary amine comprising a first amide and a second amide, and the second nitrogen in the sixth compound is a tertiary amine comprising a hydroxy-containing moiety and a third amide, and wherein the first amide, second amide, and third amide are each coupled to an independently selected targeting ligand.

[0038] In some embodiments, the protecting group is selected from the group consisting of a benzyl group and a triphenylmethyl group. In some embodiments, producing the second compound comprises converting the first compound to S NIn some embodiments, producing the second compound comprises performing a reductive amination reaction using the first compound. In some embodiments, producing the second compound comprises performing a Michael addition reaction using the first compound. In some embodiments, the protecting group is a benzyl group and producing the third compound comprises performing a hydrogenation reaction using the second compound. In some embodiments, the protecting group is a triphenylmethyl group and producing the third compound comprises reacting the second component with at least one acid. In some embodiments, producing the fourth compound comprises using the third compound to produce S NIn some embodiments, producing the fourth compound comprises performing a reductive amination reaction using the third compound. In some embodiments, producing the fourth compound comprises performing a Michael addition reaction using the third compound. In some embodiments, producing the fourth compound comprises performing an amide coupling reaction using the third compound. In some embodiments, producing the fourth compound comprises performing a nucleophilic addition reaction using the third compound. In some embodiments, the moiety comprising a hydroxy group is attached to the second nitrogen using any of the linkers B described above. In some embodiments, producing the fifth compound comprises 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 using the fourth compound. In some embodiments, producing the fifth compound comprises performing a hydrolysis reaction using 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 of the independently selected linkers A described above. In some embodiments, the independently selected targeting ligand is an independently selected targeting ligand described above. In some embodiments, the method further comprises converting the hydroxy group to a phosphoramidite group using a phosphitylation reaction. In some embodiments, converting the hydroxy group to a phosphoramidite group is performed after performing an amide coupling reaction to produce the sixth compound.

[0039] Another aspect of the disclosure is a method for making a compound for targeted delivery of one or more pharmaceutical agents, comprising receiving a first compound comprising a diamine comprising a first nitrogen and a second nitrogen, where the first nitrogen is a secondary amine comprising a first protecting group and the second nitrogen is an amine comprising a second protecting group; coupling a first protected carboxylic acid to the first nitrogen of the first compound such that the first nitrogen becomes a tertiary amine, thereby producing a second compound; removing the first protecting group from the first nitrogen of the second compound. removing the second protecting group from the fourth compound to produce a third compound comprising a first nitrogen and a second nitrogen, the first nitrogen being a secondary amine comprising a first protected carboxylic acid and the second nitrogen being an amine comprising a second protecting group; coupling the second protected carboxylic acid to the first nitrogen of the third compound such that the first nitrogen becomes a tertiary amine, thereby producing a fourth compound; removing the second protecting group from the fourth compound to produce a fourth compound comprising a first nitrogen and a second nitrogen, the first nitrogen being a secondary amine comprising a first protected carboxylic acid and a second protected carboxylic acid; producing a fifth compound, the fifth compound being a tertiary amine containing a carboxylic acid and the second nitrogen being a primary amine; producing a sixth compound by coupling a third protected carboxylic acid to the second nitrogen of the fifth compound such that the second nitrogen is a secondary amine; producing a seventh compound by attaching a moiety containing a hydroxy group to the second nitrogen of the sixth compound such that the second nitrogen is a tertiary amine; producing an eighth compound by converting the third protected carboxylic acid of the seventh compound to a first carboxylic acid; producing a ninth compound by performing an amide coupling reaction using the eighth compound, the first nitrogen of the ninth compound comprising a first protected carboxylic acid and a second protected carboxylic acid, the second nitrogen of the ninth compound comprising a first amide having a first targeting ligand and a moiety containing a hydroxy group coupled thereto; producing a tenth compound by converting the second protected carboxylic acid of the ninth compound to a second carboxylic acid;the first nitrogen of the 11th compound includes a first protected carboxylic acid and a second amide having a second targeting ligand coupled thereto, and the second nitrogen of the 11th compound includes a moiety including a first amide having the first targeting ligand coupled thereto and a hydroxy group; the first protected carboxylic acid of the 11th compound is converted to a third carboxylic acid to produce a 12th compound; and the second nitrogen of the 13th compound includes a moiety including a first amide having the first targeting ligand coupled thereto and a hydroxy group by performing an amide coupling reaction using the 12th compound, and the first nitrogen of the 13th compound includes a second amide having the second targeting ligand coupled thereto and a third amide having a third targeting ligand coupled thereto, and the second nitrogen of the 13th compound includes a moiety including a first amide having the first targeting ligand coupled thereto and a hydroxy group;

[0040] In some embodiments, the first protecting group is a benzyl group and the second protecting group is a tert-butyloxycarbonyl (Boc) group. In some embodiments, producing the second compound comprises converting the first compound to S N In some embodiments, producing the second compound comprises performing a reductive amination reaction using the first compound. In some embodiments, producing the second compound comprises performing a Michael addition reaction using the first compound. In some embodiments, producing the third compound comprises performing a hydrogenation reaction using the second compound. In some embodiments, producing the fourth compound comprises performing a S2 substitution reaction using the third compound. NIn some embodiments, producing the fourth compound comprises performing a reductive amination reaction using the third compound. In some embodiments, producing the fourth compound comprises performing a Michael addition reaction using the third compound. In some embodiments, producing the fourth compound comprises performing an amide coupling reaction using the third compound. In some embodiments, producing the fourth compound comprises performing a nucleophilic addition reaction using the third compound. In some embodiments, producing the fifth compound comprises 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 reacting the fifth compound with S N In some embodiments, producing the sixth compound comprises performing a reductive amination reaction using the fifth compound. In some embodiments, producing the sixth compound comprises performing a Michael addition reaction using the fifth compound. In some embodiments, producing the seventh compound comprises performing a S2 substitution reaction using the sixth compound. NIn some embodiments, producing the seventh compound comprises performing a reductive amination reaction using the sixth compound. In some embodiments, producing the seventh compound comprises performing a Michael addition reaction using the sixth compound. In some embodiments, producing the seventh compound comprises performing an amide coupling reaction using the sixth compound. In some embodiments, producing the seventh compound comprises performing a nucleophilic addition reaction using 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 to be one or more of the targeting ligands as described above. In some embodiments, the hydroxy group is coupled to the second nitrogen using a linker B as described above. In some embodiments, the method further comprises converting the hydroxy group to a phosphoramidite group using a phosphitylation reaction. In some embodiments, converting the hydroxy group to a phosphoramidite group is performed after producing the thirteenth compound.

[0041] A further aspect of the present disclosure is a method for delivering a pharmaceutical agent to a subject, comprising administering to the subject (a) a compound as described above, wherein W is one or more pharmaceutical 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 compound is administered in a pharmaceutically acceptable carrier.

[0042] Another aspect of the present disclosure relates to a method for delivering a pharmaceutical agent to a subject, comprising administering to the subject a pharmaceutical composition as described above. In some embodiments, the subject is a vertebrate. In some embodiments, the subject is a mammal, and optionally the mammal is a human. In some embodiments, the one or more pharmaceutical agents include at least one of small interfering RNA (siRNA), single-stranded siRNA, double-stranded siRNA, small activating RNA, microRNA (miRNA), antisense oligonucleotide, short guide RNA (gRNA), single guide RNA (sgRNA), messenger RNA (mRNA), ribozyme, plasmid, immunostimulatory nucleic acid, antagomir, and aptamer. In some embodiments, the double-stranded siRNA includes 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 ribonucleotide comprises 2'-O-methyl nucleotide, 2'-fluoro nucleotide, 2'-deoxy nucleotide, 2'3'-seco nucleotide mimic, locked nucleotide, 2'-F-arabino nucleotide, 2'-methoxyethyl nucleotide, abasic nucleotide, ribitol, inverted nucleotide, inverted abasic nucleotide, inverted 2'-OMe nucleotide, inverted 2' deoxy nucleotide, 2'-amino-modified nucleotide, 2'-alkyl-modified nucleotide, morpholino nucleotide, and 3'-OMe nucleotide, nucleotide comprising 5'-phosphorothioate group, or 5'-(E)-vinyl phosphonate nucleotide (antisense strand only), or terminal nucleotide linked to cholesteryl derivative or dodecanoic acid bisdecyl amide group, 2'-amino-modified nucleotide, 2'-alkyl-modified nucleotide, phosphoramidate, or non-natural base comprising nucleotide. In some embodiments, at least one strand of the double-stranded siRNA comprises at least one phosphorothioate bond.In some embodiments, at least one strand of the double-stranded siRNA comprises up to six phosphorothioate bonds. 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 unlocked 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.

[0043] Another aspect of the present disclosure is a compound for use in delivering a pharmaceutical agent 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 compound is administered in a pharmaceutically acceptable carrier.

[0044] In another embodiment of the disclosure, the dsRNA drug includes 2'-fluoro modified nucleotides at positions 2, 7, 12, 14 and 16 of the antisense strand (counting from the first paired nucleotide from the 5' end of the antisense strand), and / or 2'-fluorine-modified nucleotides at positions 9, 11 and 13 of the sense strand (counting from the first paired nucleotide from the 3' end of the sense strand). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] Overview The present disclosure provides a multivalent ligand cluster having a diamine scaffold for targeted delivery of pharmaceuticals conjugated thereto. In some embodiments, the multivalent ligand cluster may comprise one or more N-acetylgalactosamine (GalNAc) targeting ligands. In some embodiments, the multivalent ligand cluster may be conjugated to one or more small interfering ribonucleic acids (siRNAs), where siRNA is an example of pharmaceuticals. The present disclosure also provides compositions comprising the multivalent ligand cluster of the present disclosure, as well as methods of making and using the multivalent ligand cluster of the present disclosure.

[0046] definition Before further describing the present invention, and in order that the invention may be more readily understood, certain terms are first defined and summarized here for convenience.

[0047] As used herein, the terms "treat," "treating," or "treatment" can include prophylaxis and mean ameliorating, alleviating, or eliminating the cause of symptoms, either temporarily or permanently, or preventing or delaying the appearance of symptoms of a named disorder or condition.

[0048] As used herein, the term "about" in reference to a measured quantity refers to normal variation in that measured quantity, as would be expected by one of ordinary skill in the art making the measurement and exercising a level of care commensurate with the purpose of the measurement and the precision of the measuring device.

[0049] As used herein, the term "conjugate" or "conjugate group" refers to an atom or group of atoms attached to an oligonucleotide or other oligomer. In general, conjugate groups modify one or more properties of the compound to which they are attached, including, but not limited to, pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge, and / or clearance properties.

[0050] As used herein, the term "linked" when referring to a connection between two molecules means that the two molecules are directly or indirectly joined together by a covalent bond, or that the two molecules are associated by a non-covalent bond (e.g., hydrogen bond or ionic bond). An example in which compound A is directly joined to compound B can be represented as AB. An example in which compound A is indirectly joined to compound B can be represented as ACB, where compound A is indirectly joined to compound B via compound C. It will be understood that in situations in which compounds are indirectly joined, there may be more than one intermediate compound.

[0051] As used herein, the term "nucleic acid" 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). Nucleic acids may include any combination of these elements in a single molecule. Nucleic acids may include natural nucleic acids, non-natural nucleic acids, or combinations of natural and non-natural nucleic acids. Nucleic acids may also be referred to herein as nucleotide sequences or as polynucleotides.

[0052] As used herein, the term "oligomer" refers to a nucleotide sequence that contains 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 nucleic acid base sequence that is at least partially complementary to the coding sequence in the target nucleic acid or target gene expressed in a cell. In some embodiments, the oligomer can inhibit the expression of a latent gene when delivered to a cell that expresses the gene. Gene expression can be inhibited in vitro or in vivo. Non-limiting examples of oligomers that can be included in the methods and complexes of the present invention are oligonucleotides, single-stranded oligonucleotides, single-stranded antisense oligonucleotides, small interfering RNA (siRNA), single-stranded siRNA, double-stranded RNA (dsRNA), microRNA (miRNA), small hairpin RNA (shRNA), ribozymes, interfering RNA molecules, and dicer substrates.

[0053] As used herein, the term "oligonucleotide" refers to a polymer of linked nucleotides, each of which may be independently modified or unmodified.

[0054] As used herein, the term "single-stranded oligonucleotide" refers to a single-stranded oligomer, and in certain embodiments, the single-stranded oligonucleotide can comprise a sequence that is at least partially complementary to target mRNA, and can hybridize with target mRNA through hydrogen bond under mammalian physiological conditions (or comparable conditions in vitro).In some embodiments, the single-stranded oligonucleotide is a single-stranded antisense oligonucleotide.

[0055] As used herein, the term "siRNA" refers to small interfering RNA or silencing RNA. siRNA is a class of double-stranded RNA molecules that can be 20-25 (or shorter) base pairs long, similar to microRNAs (miRNAs) that operate within the RNA interference (RNAi) pathway. siRNAs interfere with the expression of specific genes that contain nucleotide sequences complementary to the siRNA by degrading mRNA after transcription, preventing translation. siRNAs act in cells to silence gene expression by inducing the RNA-induced silencing complex (RISC) to cleave messenger RNA (mRNA).

[0056] As used herein, the term "effective amount", "therapeutically effective amount" or "effective dose" refers to an amount sufficient to induce the desired pharmacological or therapeutic effect, resulting in effective prevention or treatment of a disorder. Prevention of a disorder is represented by delaying the onset of symptoms of the disorder to a medically significant extent. Treatment of a disorder is represented by a reduction in symptoms associated with the disorder or amelioration of recurrence of symptoms of the disorder.

[0057] As used herein, the term "pharmaceutical composition" or "composition" refers to a mixture of substances suitable for administration to an individual.For example, and not intended to be limiting, a pharmaceutical composition may include one or more active agents and a pharmaceutical carrier (e.g., a sterile aqueous solution), also referred to herein as a "pharmaceutical acceptable carrier".In some embodiments, the pharmaceutical composition is sterile.

[0058] As used herein, the term “alkyl” when used alone or as part of another group refers to an alkyl group having 1 to 12 carbon atoms (i.e., C 1~12 alkyl) or a specified number of carbon atoms (i.e., C alkyl, such as methyl, C alkyl, such as ethyl, C alkyl, such as propyl or isopropyl, etc.). 1~10 Alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, iso-butyl, 3-pentyl, hexyl, heptyl, octyl, nonyl, decyl, and the like.

[0059] As used herein, the term "substituted alkyl," alone or as part of another group, means that an alkyl, as defined herein, is substituted with one or more (e.g., 1, 2, or 3) independently selected 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, sulfonamido, hydroxyl, (alkyl)sulfanyl, nitro, haloalkoxy, aryloxy, aralkyloxy, (alkyl)sulfonyl, (cycloalkyl)sulfonyl, (aryl)sulfonyl, cycloalkyl, sulfanyl, caboxamido, heterocyclyl, and (heterocyclyl)sulfonyl.

[0060] As used herein, the term “cycloalkyl,” alone or as part of another group, refers to an alkyl group having 3 to 12 carbon atoms (i.e., C 3~12Cycloalkyl refers to saturated and partially unsaturated (containing one or two double bonds) cyclic aliphatic hydrocarbons containing 1 to 3 rings having the specified number of carbons. Non-limiting exemplary cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, norbornyl, decalin, adamantyl, cyclohexenyl, cyclopentenyl, cyclohexenyl, and the like.

[0061] As used herein, the term "substituted cycloalkyl," either alone or as part of another group, means that cycloalkyl, 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, hydroxyl, amino, (alkyl)amino, (dialkyl)amino, haloalkyl, (hydroxyl)alkyl, (dihydroxy)alkyl, alkoxy, haloalkoxy, aryloxy, aralkyloxy, alkylthio, carboxamido, sulfonamido, (alkyl)carbonyl, (aryl)carbonyl, (alkyl)sulfonyl, arylsulfonyl, ureido, guanidino, carboxy, (carboxy)alkyl, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, (alkoxy)alkyl, (amino)alkyl, (hydroxyl)alkylamino, (alkylamino)alkyl, (dialkylamino)alkyl, (cyano)alkyl, (carboxamido)alkyl, (alkyl)sulfanyl, (heterocyclo)alkyl, (heteroaryl)alkyl, (alkoxy)carbonyl, and mercaptoalkyl.

[0062] As used herein, the term "alkenyl" alone or as part of another group refers to an alkyl group, as defined herein, containing one, two or three carbon-carbon double bonds. Non-limiting exemplary alkenyl groups include ethenyl, propenyl, isopropenyl, butenyl, sec-butenyl, pentenyl and hexenyl.

[0063] As used herein, the term "substituted alkenyl" means that an alkenyl, as defined herein, alone or as part of another group, is substituted with one, two, or three independently selected substituents. A non-limiting list of independently selected substituents includes halo, nitro, cyano, hydroxyl, amino, (alkyl)amino, (dialkyl)amino, haloalkyl, (hydroxy)alkyl, (dihydroxy)alkyl, alkoxy, haloalkoxy, aryloxy, aralkyloxy, (alkyl)sulfanyl, carboxamide, sulfonamide, (alkyl)carbonyl, (aryl)carbonyl, (alkyl)sulfonyl, (aryl)sulfonyl, ureido, guanidino, carboxy, (carboxy)alkyl, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and heterocyclyl.

[0064] As used herein, the term "cycloalkenyl," alone or as part of another group, refers to a non-aromatic cyclic alkyl group of 4 to 10 carbon atoms having a single or multiple cyclic rings and having at least one >C=C< ring unsaturation, preferably 1 to 2 sites of >C=C< ring unsaturation.

[0065] As used herein, the term "substituted cycloalkenyl," alone or as part of another group, refers to cycloalkenyl, as defined herein, having from 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, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, aryl, substituted aryl, aryloxy, substituted aryloxy, arylthio, substituted arylthio, azido, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, cyanate, cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substituted Examples include cycloalkyloxy, cycloalkylthio, substituted cycloalkylthio, cycloalkenyl, substituted cycloalkenyl, cycloalkenyloxy, substituted cycloalkenyloxy, cycloalkenylthio, substituted cycloalkenylthio, guanidino, substituted guanidino, halo, hydroxy, hydroxyamino, alkoxyamino, hydrazino, substituted hydrazino, heteroaryl, substituted heteroaryl, heteroaryloxy, substituted heteroaryloxy, heteroarylthio, substituted heteroarylthio, heterocyclic, substituted heterocyclic, heterocyclyloxy, substituted heterocyclyloxy, heterocyclylthio, substituted heterocyclylthio, nitro, SOH, substituted sulfonyl, sulfonyloxy, thioacyl, thiocyanate, thiol, alkylthio, and substituted alkylthio.

[0066] As used herein, the term "alkynyl" alone or as part of another group refers to an alkyl group, as defined herein, containing one to three carbon-carbon triple bonds. Non-limiting exemplary alkynyl groups include ethynyl, propynyl, butynyl, 2-butynyl, pentynyl, and hexynyl groups.

[0067] As used herein, the term "substituted alkynyl," alone or as part of another group, means that alkynyl, 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, hydroxyl, amino, alkylamino, dialkylamino, haloalkyl, (hydroxy)alkyl, (dihydroxy)alkyl, alkoxy, haloalkoxy, aryloxy, aralkyloxy, (alkyl)sulfanyl, carboxamide, sulfonamide, alkylcarbonyl, arylcarbonyl, alkylsulfonyl, arylsulfonyl, ureido, guanidino, carboxy, (carboxy)alkyl, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and heterocyclyl.

[0068] As used herein, the term "haloalkyl" refers to an alkyl group that is substituted with one or more fluorine, chlorine, bromine and / or iodine atoms, either alone or as part of another group. 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 groups.

[0069] 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 attached to a terminal oxygen atom.

[0070] As used herein, the term "haloalkoxy" alone or as part of another group refers to a haloalkyl attached to a terminal oxygen atom. Non-limiting exemplary haloalkoxy groups include fluoromethoxy, difluoromethoxy, trifluoromethoxy, and 2,2,2-trifluoroethoxy.

[0071] As used herein, the term “aryl,” alone or as part of another group, refers to an alkyl group having 6 to 14 carbon atoms (i.e., C 14 "aryl" refers to a monocyclic or bicyclic aromatic ring system having a ring atom (aryl). Non-limiting exemplary aryl groups include phenyl, naphthyl, phenanthryl, anthracyl, indenyl, azulenyl, biphenyl, biphenylenyl, and fluorenyl groups.

[0072] As used herein, the term "substituted aryl," alone or as part of another group, means that aryl, as defined herein, is substituted with 1 to 5 independently selected substituents. A non-limiting list of independently selected substituents includes halo, nitro, cyano, hydroxyl, amino, alkylamino, dialkylamino, haloalkyl, (hydroxy)alkyl, (dihydroxy)alkyl, alkoxy, haloalkoxy, aryloxy, heteroaryloxy, aralkyloxy, alkylthio, carboxamido, sulfonamido, (alkyl)carbonyl, (aryl)carbonyl, (alkyl)sulfonyl, (aryl)sulfonyl, ureido, guanidino, carboxy, carboxyalkyl, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclo, (alkoxy)alkyl, (amino)alkyl, [(hydroxyl)alkyl]amino, [(alkyl)amino]alkyl, [(dialkyl)amino)alkyl, (cyano)alkyl, (carboxamido)alkyl, mercaptoalkyl, (heterocyclo)alkyl, (cycloalkylamino)alkyl, (halo(C1-C4)alkoxy)alkyl, (heteroaryl)alkyl, and the like. 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 meant to include groups having fused substituted cycloalkyl and fused substituted heterocyclo rings.

[0073] As used herein, the term "aryloxy" alone or as part of another group refers to an aryl or substituted aryl attached to a terminal oxygen atom. A non-limiting exemplary aryloxy group is PhO-.

[0074] As used herein, the term "heteroaryloxy," alone or as part of another group, refers to a heteroaryl or substituted heteroaryl attached to a terminal oxygen atom.

[0075] As used herein, the term "aralkyloxy" alone or as part of another group refers to an aralkyl group attached to a terminal oxygen atom. A non-limiting exemplary aralkyloxy group is PhCHO-.

[0076] As used herein, the term "heteroaryl" refers to heteroaryls having 5 to 14 ring atoms (i.e., C5-C 14 Heteroaryl refers to monocyclic and bicyclic aromatic ring systems having one, two, three, or four heteroatoms independently selected from oxygen (O), nitrogen (N), and sulfur (S). Non-limiting exemplary heteroaryl groups include thienyl, benzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl, furyl, benzofuryl, pyranyl, isobenzofuranyl, benzoxazolyl, chromenyl, xanthenyl, 2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, isoindolyl, 3H-indolyl, indolyl, The term "heteroaryl" includes indazolyl, purinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, cinnolinyl, quinazolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl, phenazinyl, thiazolyl, isothiazolyl, phenothiazolyl, isoxazolyl, furazanyl, and phenoxazinyl. The term "heteroaryl" is also meant to include possible N-oxides. Exemplary N-oxides include pyridyl N-oxides and the like.

[0077] As used herein, the term "substituted heteroaryl," alone or as part of another group, means that heteroaryl, as defined herein, is substituted with from 1 to 4 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, aralkyloxy, alkylthio, carboxamido, sulfonamido, (alkyl)carbonyl, (aryl)carbonyl, (alkyl)sulfonyl, (aryl)sulfonyl, ureido, guanidino, carboxy, (carboxy)alkyl, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclo, (alkoxy)alkyl, (amino)alkyl, [(hydroxyl)alkyl]amino, [(alkyl)amino]alkyl, [(dialkyl)amino]alkyl, (cyano)alkyl, (carboxamido)alkyl, mercaptoalkyl, (heterocyclo)alkyl, and (heteroaryl)alkyl. Any available carbon or nitrogen atom may be substituted.

[0078] As used herein, the term "heterocyclo" or "heterocyclyl", alone or as part of another group, refers to saturated and partially unsaturated (e.g., containing one or two double bonds) cyclic groups containing 3 to 14 ring members (i.e., 3- to 14-membered heterocyclos) and one, two, or three rings having at least one heteroatom. Each heteroatom is independently selected. The term "heterocyclo" or "heterocyclyl" is meant to include cyclic ureido groups, such as 2-imidazolidinone, and cyclic amide groups, such as β-lactam, γ-lactam, δ-lactam, and ε-lactam. The term "heterocyclo" or "heterocyclyl" is also meant to include groups having fused aryl or substituted aryl groups, such as indolinyl. Heterocyclo or heterocyclyl may be linked to the remainder of the molecule through a carbon or nitrogen atom. Non-limiting exemplary heterocyclo (or heterocyclyl) groups include 2-oxopyrrolidin-3-yl, 2-imidazolidinone, piperidinyl, morpholinyl, piperazinyl, pyrrolidinyl, and indolinyl.

[0079] As used herein, the term "substituted heterocyclo" or "substituted heterocyclyl," alone or as part of another group, means that a heterocyclo or heterocyclyl group, as defined above, is substituted with one to four independently selected substituents. A non-limiting list of independently selected substituents includes halo, nitro, cyano, hydroxyl, amino, (alkyl)amino, (dialkyl)amino, haloalkyl, (hydroxy)alkyl, (dihydroxy)alkyl, alkoxy, haloalkoxy, aryloxy, aralkyloxy, alkylthio, carboxamido, sulfonamido, (alkyl)carbonyl, (aryl)carbonyl, (alkyl)sulfonyl, (aryl)sulfonyl, ureido, guanidino, carboxy, carboxyalkyl, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, alkoxyalkyl, (amino)alkyl, [(hydroxyl)alkyl]amino, [(alkyl)amino]alkyl, [(dialkyl)amino]alkyl, (cyano)alkyl, (carboxamido)alkyl, mercaptoalkyl, (heterocyclyl)alkyl, and (heteroaryl)alkyl. Substitution may occur on any available carbon or nitrogen atom and may form a spiro ring.

[0080] As used herein, the term "amino," alone or as part of another group, refers to --NH.sub.2.

[0081] As used herein, the terms "alkylamino" or "(alkyl)amino," alone or as part of another group, refers to -NHR, where R is alkyl.

[0082] As used herein, the term “dialkylamino” or “(dialkyl)amino” alone or as part of another group refers to —NR ’ R ” R ’ and R ” are each independently alkyl, or R ’ and R” taken together form a 3- to 8-membered heterocyclo or substituted heterocyclo.

[0083] As used herein, the term "cycloalkylamino" by itself or as part of another group refers to -NR ’ R ” R ’ is cycloalkyl or substituted cycloalkyl, R ” is hydrogen or alkyl.

[0084] As used herein, the term "(amino)alkyl" alone or as part of another group refers to an alkyl group substituted with an amino group. Non-limiting exemplary (amino)alkyl groups include -CH2CH2NH2, -CH2CH2CH2NH2, and -CH2CH2CH2CH2NH2.

[0085] As used herein, the terms "(alkylamino)alkyl" or "[(alkyl)amino]alkyl" alone or as part of another group refer to an alkyl group substituted with an alkylamino group. A non-limiting exemplary (alkylamino)alkyl group is -CH2CH2N(H)CH3.

[0086] As used herein, the term "(dialkylamino)alkyl" alone or as part of another group refers to an alkyl group substituted with a dialkylamino group. Non-limiting exemplary (dialkylamino)alkyl groups include -CHN(CH) and -CHCHN(CH-).

[0087] As used herein, the term "(cycloalkylamino)alkyl" alone or as part of another group refers to an alkyl group substituted with a cycloalkylamino group. Non-limiting exemplary (cycloalkylamino)alkyl groups include -CHN(H)cyclopropyl, -CHN(H)cyclobutyl, and -CHN(H)cyclohexyl.

[0088] As used herein, the term "carboxamide," alone or as part of another group, refers to a group of the formula -C(=O)NR ’ R ” R refers to the radical of ’ and R ” are each independently hydrogen, alkyl, substituted alkyl, aralkyl, substituted aralkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl, or R ’ and R ” together with the nitrogen to which they are attached form a 3- to 8-membered heterocyclo group. Non-limiting exemplary carboxamide groups include -CONH2, -CON(H)CH3, CON(CH3)2, and CON(H)Ph.

[0089] As used herein, the term "sulfonamide," alone or as part of another group, refers to a group having the formula -SONR ’ R ” R refers to the radical of ’ and R ” are each independently hydrogen, alkyl, substituted alkyl, aryl, or substituted aryl, or R ’ and R ” taken together with the nitrogen to which they are attached form a 3- to 8-membered heterocyclo group. Non-limiting exemplary sulfonamide groups include -SO2NH2, -SON2N(H)CH3, and -SON2N(H)Ph.

[0090] As used herein, the term "(alkyl)carbonyl" alone or as part of another group refers to a carbonyl group substituted with an alkyl group, i.e., -C(=O)-. A non-limiting exemplary alkylcarbonyl group is -COCH3.

[0091] As used herein, the term "(alkoxy)carbonyl" (or "ester"), alone or as part of another group, refers to a carbonyl group substituted with an alkoxy group, i.e., -C(=O)-. A non-limiting exemplary (alkoxy)carbonyl group is -C(O)OCH3.

[0092] As used herein, the term "(aryl)carbonyl" alone or as part of another group refers to a carbonyl group substituted with an aryl or substituted aryl group, i.e., -C(=O)-. A non-limiting exemplary arylcarbonyl group is -COPh.

[0093] As used herein, the term "sulfanyl," alone or as part of another group, refers to the --SH group.

[0094] As used herein, the term "(alkyl)sulfanyl" or "alkylthio" alone or as part of another group refers to a sulfur atom substituted with an alkyl or substituted alkyl group. Non-limiting exemplary alkylthio groups include -SCH3 and -SCH2CH3.

[0095] As used herein, the term "mercaptoalkyl," either alone or as part of another group, refers to an alkyl group substituted with an --SH group.

[0096] 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 group, i.e., -SO2-. A non-limiting exemplary alkylsulfonyl group is -SO2CH3.

[0097] 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 group, i.e., -SO2-. A non-limiting exemplary arylsulfonyl group is -SO2Ph.

[0098] As used herein, the term "carboxy," alone or as part of another group, refers to a radical of the formula --COOH.

[0099] As used herein, the term "(carboxy)alkyl" alone or as part of another group refers to an alkyl group substituted with -COOH. A non-limiting exemplary carboxyalkyl group is -CH2CO2H.

[0100] As used herein, the term "aralkyl," alone or as part of another group, refers to a residue in which an aryl moiety is attached to an alkyl residue. The aralkyl group can be attached to the parent structure at either the aryl or the alkyl residue.

[0101] As used herein, the term "substituted aralkyl," alone or as part of another group, refers to a residue in which an aryl moiety is bound to a substituted alkyl residue.

[0102] As used herein, the term “aralkenyl,” alone or as part of another group, refers to a radical of the formula —R d -R c R refers to the radical of d is an alkenylene chain, Rc is one or more aryl radicals.

[0103] As used herein, the term "substituted aralkenyl," alone or as part of another group, refers to an aralkenyl radical in which the alkenylene chain of the aralkenyl is an optionally substituted alkenylene chain and each aryl radical of the aralkenyl radical is an optionally substituted aryl radical.

[0104] As used herein, the term “aralkynyl” alone or as part of another group refers to a group of the formula —R e R c R refers to the radical of e is an alkynylene chain, R c is one or more aryl radicals.

[0105] As used herein, the term "substituted aralkynyl," alone or as part of another group, refers to an aralkynyl radical in which the alkynylene chain of the aralkynyl radical is an optionally substituted alkynylene chain and each aryl radical of the aralkynyl radical is an optionally substituted aryl radical.

[0106] 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 of the ring-forming atoms is a heteroatom.

[0107] As used herein, the term "heteroatom" refers to an atom inserted between a carbon atom and its parent molecule (i.e., between the points of attachment). Non-limiting exemplary heteroatoms include oxygen, nitrogen, sulfur (including sulfoxide and sulfone), and phosphorus (P).

[0108] As used herein, the term "sugar" refers to a single sugar moiety or monosaccharide unit, as well as combinations of two or more single sugar moieties or units covalently linked to form disaccharides, oligosaccharides, and polysaccharides. Polysaccharides may be linear or branched.

[0109] As used herein, the term "monosaccharide" refers to a single sugar residue in an oligosaccharide.

[0110] As used herein, the term "disaccharide" refers to a polysaccharide consisting of two monosaccharide units or moieties linked together by a glycosidic bond.

[0111] As used herein, "oligosaccharide" refers to a compound containing two or more monosaccharide units or moieties. Within the context of an oligosaccharide, each individual monomer unit or moiety is a monosaccharide that is or can be linked to another monosaccharide unit or moiety via a hydroxy 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 in vitro by enzymatic methods.

[0112] As used herein, the term "ureido" alone or as part of another group refers to a group of the formula -NR ’ -C(=O)-NR ” R ” R refers to the radical of ’ is hydrogen, alkyl, aryl, or substituted aryl; 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 a 4-8 membered heterocyclo group. Non-limiting exemplary ureido groups include -NH-C(=O)-NH2 and -NH-C(=O)-NHCH3.

[0113] As used herein, the term "guanidino," alone or as part of another group, refers to a group of the formula -NR ’ -C(=NR ” )-NR ”’ R ”” R refers to the radical of ’ , R ”’ , and R ”” are each independently hydrogen, alkyl, aryl, or substituted aryl; R ” is hydrogen, alkyl, cyano, alkylsulfonyl, alkylcarbonyl, carboxamide, or sulfonamide. Non-limiting exemplary guanidino groups include -NH-C(=NH)-NH2, -NH-C(=NCN)-NH2, and -NH-C(=NH)-NHCH3.

[0114] As used herein, the term "(heteroaryl)alkyl," alone or as part of another group, refers to an alkyl group that is substituted with one, two, or three heteroaryl or substituted heteroaryl groups.

[0115] As used herein, the term "heteroalkyl" alone or as part of another group refers to a stable straight or branched chain hydrocarbon radical containing at least one heteroatom, which may be the same or different. The heteroatom may be located at any internal or terminal position of the heteroalkyl group or at the position where the heteroalkyl group is attached to the remainder of the molecule. Non-limiting exemplary heteroalkyl groups include -CH2N(H)CH2CH2N(CH3), -CH2N(CH3)CH2CH2N(CH3), -CH2N(H)CH2CH2CH2N(CH3), -CH2N(H)CH2CH2OH, -CH2N(CH3)CH2CH2OH, -CH2OCH2CH2OCH3, -OCH2CH2OCH2CH2OCH3, -CH2NHCH2CH2OCH2, -OCH2CH2NH2, and -NHCH2CH2N(H)CH3.

[0116] As used herein, the term "(heterocyclo)alkyl" or "(heterocyclyl)alkyl," alone or as part of another group, refers to a heterocyclyl or substituted heterocyclyl group and an alkyl group that is optionally substituted with a hydroxy group.

[0117] As used herein, the term "(carboxamido)alkyl," alone or as part of another group, refers to an alkyl group that is substituted with a carboxamido group and optionally with a heterocyclo, amino, alkylamino, or dialkylamino group.

[0118] As used herein, the term "N-oxide" refers to an N + refers to a compound containing a functional group that is further linked to H and / or the remainder of the compound structure.

[0119] As used herein, the term "integer" refers to whole numbers including, but not limited to, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, etc.

[0120] compound General structure Multivalent ligand clusters having a diamine scaffold have the general structure of Formula 1:

[0121] [ka] [In the formula, Each TL is an independently selected target ligand; m is an integer between 1 and 10; each n is an independently selected integer between 1 and 10; Each linker A is an independently selected spacer, one end of which is attached to a TL and the other end of which is attached to a nitrogen of an alkylcarboxamide; Linker B is a spacer having one end attached to a drug or a functional group capable of linking to one or more drug substances and the other end attached to a diamine nitrogen; W is one or more pharmaceutical agents or a functional group that can be linked to one or more pharmaceutical agents. may have:

[0122] In some embodiments, m can be set based on the starting material used to synthesize the multivalent ligand cluster. For example, m can be 1 with ethylenediamine used as a starting material, m can be 2 with 1,3-propanediamine used as a starting material, m can be 3 with 1,4-butanediamine used as a starting material, etc.

[0123] As used herein, "spacer" refers to a compound or molecule that links other groups together. Examples of Linker A and Linker B spacers are described in detail herein below.

[0124] As used herein with respect to elements of the multivalent ligand cluster of the present invention, the term "independently selected" means that each of the elements of a given type may be different from one or more other of the same type of element in the multivalent ligand cluster. For example, a multivalent ligand cluster may contain more than one TL, where each may be selected to be different or the same as one or more other in the multivalent ligand cluster. In a further example, a multivalent ligand cluster may contain more than one "n", where each may be selected to be different or the same as one or more other "n" in the multivalent ligand cluster. In another example, a multivalent ligand cluster may contain more than one linker A, where each may be selected to be different or the same as one or more other linkers A in the multivalent ligand cluster.

[0125] Target Ligand As described above with respect to Formula 1, the multivalent ligand clusters of the present disclosure may include multiple (e.g., three) independently selected target ligands. In this context, the term "independently selected" means that each target ligand may be selected to be different or the same as one or more other target ligands in the same multivalent ligand cluster.

[0126] At least one of the independently selected targeting ligands of Formula 1 may bind to one or more cellular receptors, cellular channels, and / or cellular transporters that may facilitate cellular uptake.

[0127] In some embodiments, at least one of the independently selected targeting ligands of formula 1 may comprise at least one small molecule ligand. As used herein, "small molecule ligand" refers to a ligand smaller than protein. In some embodiments, at least one small molecule ligand may comprise at least one of N-acetylgalactosamine (GalNAc), galactose, galactosamine, N-formyl-galactosamine, N-propionylgalactosamine, N-butanoylgalactosamine, and N-iso-butanoylgalactosamine, macrocycle, folic acid molecule, fatty acid, bile acid, cholesterol, and its derivatives.

[0128] A macrocycle is a molecule or ion that contains 12 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.

[0129] Folic acid, also known as vitamin B9 and folacin, is used by the human body to make DNA and RNA and metabolize the amino acids required for cell division.Folic acid receptor binds to folic acid and reduced folic acid derivatives.Therefore, in some embodiments, at least one of the independently selected targeting ligands can include reduced folic acid derivatives.

[0130] Fatty acids are carboxylic acids that contain long aliphatic chains. In some embodiments, fatty acids may be saturated, meaning that the aliphatic chains all have a single carbon-carbon bond. In some embodiments, fatty acids may be unsaturated, meaning that the aliphatic chains contain at least one double or triple carbon-carbon bond. In some embodiments, fatty acids may contain branched chains. In some embodiments, fatty acids may contain ring structures. Fatty acids are known to aid in the uptake of pharmaceuticals 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 Conjugated 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.

[0131] Bile acids are steroid acids found in bile. Bile acids are known ligands for the farnesoid X receptor (FXR) and G protein-coupled bile acid receptor 1 (GPBAR1) (TGR5). In some embodiments, the bile acid may be a primary bile acid synthesized in the liver. In some embodiments, the bile acid may be a secondary bile acid resulting from bacterial action in the colon. Bile acids are known to be beneficial in inhibiting RNA translation. See Gonzalez-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.

[0132] 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. The present disclosure is not limited to any particular peptide. Both known and yet to be discovered peptides are within the scope of the present disclosure.

[0133] In some embodiments, at least one of the independently selected targeting ligands of formula 1 may include 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, the cyclic ring structure may be formed by linking one end of a peptide to the other with an amide bond or other chemically stable bond such as lactone, ether, thioether, disulfide, etc. In some embodiments, the cyclic peptide of the present disclosure may be a biologically active cyclic peptide in which head-to-tail (or NC) cyclization is formed by an amide bond between the amino and carboxyl termini. In some embodiments, the cyclic peptide of the present disclosure may be a biologically active cyclic peptide in which cyclization is formed by "click chemistry". See Rashad AA (2019) Click Chemistry for Cyclic Peptide Drug Design. In: Goetz G. (eds) Cyclic Peptide Design. Methods in Molecular Biology, vol 2001. Humana,ew 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.

[0134] In some embodiments, at least one of the independently selected target ligands of formula 1 may include at least one aptamer. 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 tend to form helices and single-stranded loops, and therefore assume a variety of shapes. The present disclosure is not limited to any particular aptamer. Known and yet to be discovered aptamers are within the scope of the present disclosure.

[0135] In some embodiments, at least one of the independently selected targeting ligands of formula 1 can bind to at least one asialoglycoprotein receptor (ASGPR). ASGPR is a lectin located on liver cells that binds to galactose residues. ASGPR has been demonstrated to be highly expressed on the surface of liver cells, human cancer cell lines, and liver cancer. ASGPR is also slightly expressed by glandular cells of the gallbladder and stomach.

[0136] In some embodiments, at least one of the independently selected targeting ligands of formula 1 can bind to at least one transferrin receptor. The transferrin receptor is a membrane glycoprotein that mediates cellular uptake of transferrin, a protein in the blood that binds iron and transports it throughout the body. The transferrin receptor-mediated cellular uptake 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 that can bind to at least one transferrin receptor. Known and yet to be discovered transferrin receptor ligands are within the scope of the present disclosure.

[0137] In some embodiments, at least one of the independently selected targeting ligands of formula 1 can bind to at least one integrin receptor. Integrin receptors are transmembrane receptors that promote cell-cell and cell-extracellular matrix (ECM) adhesion. Upon ligand binding, integrin receptors activate signaling pathways that mediate cell signals such as regulating cell cycle, organizing the intracellular cytoskeleton, and moving new receptors to the cell membrane. Integrin targeted delivery of gene therapy 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 that can bind to at least one integrin receptor. Known and yet to be discovered integrin receptor ligands are within the scope of the present disclosure.

[0138] In some embodiments, at least one of the independently selected targeting ligands of formula 1 can bind to at least one folate receptor. Folate receptors bind to folate and reduced folate derivatives to mediate the delivery of tetrahydrofolate to the inside of cells. Targeted drug delivery via 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 that can bind to at least one folate receptor. Known and yet to be discovered folate receptor ligands are within the scope of the present disclosure.

[0139] In some embodiments, at least one of the independently selected target ligands of formula 1 may bind to at least one G protein-coupled receptor (GPCR). GPCRs are cell surface receptors that bind to peptides, lipids, sugars, and proteins, among others. GPCRs interact with G proteins in the plasma membrane. When an external signaling molecule binds to the GPCR, this leads to a conformational change in the GPCR, initiating an interaction between the GPCR and a nearby G protein. GPCRs consist of a single polypeptide that is folded into a globular shape and embedded within the plasma membrane of a 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.0c12043.

[0140] Linker A As described above with respect to Formula 1, a multivalent ligand cluster of the present disclosure may include multiple (e.g., three) independently selected linkers A. In this context, the term "independently selected" means that each linker A may be selected to be different from or the same as one or more other linkers A in the same multivalent ligand cluster.

[0141] Each linker A is an independently selected spacer that is attached at one end to a targeting ligand (TL in Formula 1) and at the other end to an alkylcarboxamide nitrogen of a multivalent ligand cluster.

[0142] In some embodiments, at least one of the independently selected linkers A can include 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, P 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 more.

[0143] In some embodiments, at least one of the independently selected linkers A may include at least one alkyl group. In some embodiments, the alkyl group may 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.

[0144] In some embodiments, at least one of the independently selected linkers A may include at least one substituted alkyl group. In some embodiments, the substituted alkyl group may 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, the substituted alkyl group may include one or more of the following substituents: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.

[0145] In some embodiments, at least one of the independently selected linkers A may comprise at least one cycloalkyl group. In some embodiments, the cycloalkyl group may be a C3 cycloalkyl (i.e., cyclopropane), a C4 cycloalkyl (i.e., cyclobutene), a C5 cycloalkyl (i.e., cyclopentane), a C6 cycloalkyl (i.e., cyclohexane), a C7 cycloalkyl (i.e., cycloheptane), a C8 cycloalkyl (i.e., cyclooctane), a C9 cycloalkyl (i.e., cyclononane), or a C10 cycloalkyl (i.e., cyclodecane).

[0146] In some embodiments, at least one of the independently selected linkers A may comprise at least one substituted cycloalkyl group. In some embodiments, the substituted cycloalkyl group may 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, a C9 substituted cycloalkyl, or a C10 substituted cycloalkyl. In some embodiments, the substituted cycloalkyl group may comprise one or more of the following substituents: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.

[0147] In some embodiments, at least one of the independently selected linkers A may comprise at least one alkenyl group, in some embodiments, the alkenyl group may 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.

[0148] In some embodiments, at least one of the independently selected linkers A may include at least one substituted alkenyl group. In some embodiments, the alkenyl group may 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, the substituted alkenyl group may include one or more of the following substituents: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.

[0149] In some embodiments, at least one of the independently selected linkers A can include at least one cycloalkenyl group. In some embodiments, the cycloalkenyl group can be a C5 cycloalkenyl, a C6 cycloalkenyl, a C7 cycloalkenyl, a C8 cycloalkenyl, a C9 cycloalkenyl, or a C10 cycloalkenyl.

[0150] In some embodiments, at least one of the independently selected linkers A may comprise at least one substituted cycloalkenyl group. In some embodiments, the cycloalkenyl group may be a C5 cycloalkenyl, a C6 cycloalkenyl, a C7 cycloalkenyl, a C8 cycloalkenyl, a C9 cycloalkenyl, or a C10 cycloalkenyl. In some embodiments, the substituted cycloalkenyl group may comprise one or more of the following substituents: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.

[0151] In some embodiments, at least one of the independently selected linkers A may comprise at least one alkynyl group, in some embodiments, the alkynyl group may 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.

[0152] In some embodiments, at least one of the independently selected linkers A may include at least one substituted alkynyl group. In some embodiments, the substituted alkynyl group may 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, the substituted alkynyl group may include one or more of the following substituents: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.

[0153] In some embodiments, at least one of the independently selected linkers A may contain at least one aryl or heteroaryl group. Examples include phenyl, naphthyl, and pyridinyl, although it is noted that other aryl and heteroaryl groups falling within the definitions provided herein may also be used.

[0154] In some embodiments, at least one of the independently selected linkers A may include at least one substituted aryl group. In some embodiments, the substituted aryl group may include one or more of the following substituents: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.

[0155] In some embodiments, at least one of the independently selected linkers A may comprise at least one aralkyl group. Examples of aralkyl groups include, but are not limited to, phenylmethyl, phenylethyl, and phenylpropyl.

[0156] In some embodiments, at least one of the independently selected linkers A may include at least one substituted aralkyl group. In some embodiments, the substituted aralkyl group may include one or more of the following substituents: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.

[0157] In some embodiments, at least one of the independently selected linkers A may comprise at least one aralkenyl group. Examples of aralkenyl groups include, but are not limited to, ethenylbenzene and propenylbenzene.

[0158] In some embodiments, at least one of the independently selected linkers A may comprise at least one substituted aralkenyl group. In some embodiments, the substituted aralkenyl group may comprise one or more of the following substituents: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.

[0159] In some embodiments, at least one of the independently selected linkers A may comprise at least one aralkynyl group. Examples of aralkynyl groups include, but are not limited to, ethynylbenzene and propynylbenzene.

[0160] In some embodiments, at least one of the independently selected linkers A can include at least one substituted aralkynyl group. In some embodiments, the substituted aralkynyl group can include one or more of the following substituents: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.

[0161] In some embodiments, at least one of the independently selected linkers A may include at least one heteroatom. In some embodiments, at least one of the independently selected linkers A may include 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 may include at least one heteroalkyl group.

[0162] In some embodiments, at least one of the independently selected linkers A may include at least one aliphatic heterocycle. In some embodiments, at least one of the independently selected linkers A may include at least one of tetrahydrofuran (THF), tetrahydropyran (THP), morpholine, piperidine, piperazine, pyrrolidine, and / or azetidine.

[0163] In some embodiments, at least one of the independently selected linkers A may include at least one heteroaryl group. In some embodiments, at least one of the independently selected linkers A may include one or more of imidazole, pyrazole, pyridine, pyrimidine, triazole, and / or 1,2,3-triazole.

[0164] In some embodiments, at least one of the independently selected linkers A may comprise at least one substituted heteroaryl group. In some embodiments, the substituted heteroaryl group may comprise one or more of the following substituents: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.

[0165] In some embodiments, at least one of the independently selected linkers A may contain at least one amino acid. A variety of amino acids are known to those skilled in the art. The independently selected linkers A are not limited to containing one or more specific amino acids. For example, an 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 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 ​​(Sec) amino acids, one or more glycine (Gly) amino acids, one or more proline (Pro) amino acids, one or more alanine (Ala) amino acids, one or more valine (Val) amino acids, one or more isoleucine (Ile) amino acids, one or more leucine (Leu) amino acids, one or more methionine (Met) amino acids, one or more phenylalanine (Phe) amino acids, one or more tyrosine (Tyr) amino acids, and / or one or more tryptophan (Trp) amino acids.

[0166] In some embodiments, at least one of the independently selected linkers A may comprise at least one nucleotide. Various nucleotides are known to those skilled in the art. The independently selected linker A is not limited to comprising one or more specific nucleotides. For example, the independently selected linker A may comprise one or more nucleotides comprising guanine nucleobase, one or more nucleotides comprising adenine nucleobase, one or more nucleotides comprising cytosine nucleobase, one or more nucleotides comprising thymine nucleobase, and / or one or more nucleotides comprising uracil nucleobase.

[0167] In some embodiments, at least one independently selected linker A may comprise at least one abasic nucleotide. As known in the art, an abasic nucleotide is a nucleotide that has an abasic site that is a position that has neither a purine nor a pyrimidine base. For example, at least one independently selected linker A may comprise one or more abasic DNAs and / or one or more abasic RNAs. In some embodiments, at least one independently selected linker A may comprise at least one inverted abasic nucleotide. As known in the art, an inverted abasic nucleotide is an abasic nucleotide whose 5' end is connected to the 5' end of the next nucleotide and whose 3' end is connected to the 3' end of the next nucleotide. For example, at least one independently selected linker A may comprise one or more inverted abasic DNAs and / or one or more inverted abasic RNAs.

[0168] In some embodiments, at least one of the independently selected linkers A may 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 unit, and / or at least one glucosamine monosaccharide unit.

[0169] In some embodiments, at least one independently selected linker A is

[0170] [ka] [In the formula, p is an integer between 0 and 12, pp is an integer between 0 and 12, q is an integer between 1 and 12, qq is an integer between 1 and 12.] may include one or more of:

[0171] 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.

[0172] Linker B Linker B is a spacer that is attached at one end to a drug or a functional group that can be linked to one or more drugs and at the other end to a diamine nitrogen of the multivalent ligand cluster.

[0173] In some embodiments, the linker B may include at least one alkyl group. In some embodiments, the linker B may include at least one substituted alkyl group. In some embodiments, the alkyl group may 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.

[0174] In some embodiments, the linker B may comprise polyethylene glycol (PEG). The PEG may have any number of repeating O-CH2-CH2 units. For example, the PEG may 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, P PEG may be EG52, 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 more. In some embodiments, it may be advantageous for PEG to be PEG10 or less.

[0175] In some embodiments, the linker B may include at least one substituted alkyl group. In some embodiments, the substituted alkyl group may 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, the substituted alkyl group may include one or more of the following substituents: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.

[0176] In some embodiments, the linker B may comprise at least one cycloalkyl group. In some embodiments, the cycloalkyl group may be a C3 cycloalkyl (i.e., cyclopropane), a C4 cycloalkyl (i.e., cyclobutene), a C5 cycloalkyl (i.e., cyclopentane), a C6 cycloalkyl (i.e., cyclohexane), a C7 cycloalkyl (i.e., cycloheptane), a C8 cycloalkyl (i.e., cyclooctane), a C9 cycloalkyl (i.e., cyclononane), or a C10 cycloalkyl (i.e., cyclodecane).

[0177] In some embodiments, the linker B may include at least one substituted cycloalkyl group. In some embodiments, the substituted cycloalkyl group may 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, a C9 substituted cycloalkyl, or a C10 substituted cycloalkyl. In some embodiments, the substituted cycloalkyl group may include one or more of the following substituents: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.

[0178] In some embodiments, the linker B may include at least one alkenyl group, in some embodiments, the alkenyl group may 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.

[0179] In some embodiments, the linker B may include at least one substituted alkenyl group. In some embodiments, the alkenyl group may 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, the substituted alkenyl group may include one or more of the following substituents: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.

[0180] In some embodiments, linker B can include at least one cycloalkenyl group. In some embodiments, the cycloalkenyl group can be a C5 cycloalkenyl, a C6 cycloalkenyl, a C7 cycloalkenyl, a C8 cycloalkenyl, a C9 cycloalkenyl, or a C10 cycloalkenyl.

[0181] In some embodiments, the linker B may include at least one substituted cycloalkenyl group. In some embodiments, the cycloalkenyl group may be a C5 cycloalkenyl, a C6 cycloalkenyl, a C7 cycloalkenyl, a C8 cycloalkenyl, a C9 cycloalkenyl, or a C10 cycloalkenyl. In some embodiments, the substituted cycloalkenyl group may include one or more of the following substituents: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.

[0182] In some embodiments, the linker B may include at least one alkynyl group, in some embodiments, the alkynyl group may 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.

[0183] In some embodiments, the linker B may include at least one substituted alkynyl group. In some embodiments, the substituted alkynyl group may 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, the substituted alkynyl group may include one or more of the following substituents: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.

[0184] In some embodiments, the linker B may include at least one aryl or heteroaryl group. Examples include phenyl, naphthyl, and pyridinyl, although it is noted that other aryl and heteroaryl groups that fall within the definitions provided herein may also be used.

[0185] In some embodiments, the linker B may include at least one substituted aryl group. In some embodiments, the substituted aryl group may include one or more of the following substituents: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.

[0186] In some embodiments, the linker B may include at least one aralkyl group. Examples of aralkyl groups include, but are not limited to, phenylmethyl, phenylethyl, and phenylpropyl.

[0187] In some embodiments, the linker B may include at least one substituted aralkyl group. In some embodiments, the substituted aralkyl group may include one or more of the following substituents: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.

[0188] In some embodiments, the linker B may comprise at least one aralkenyl group. Examples of aralkenyl groups include, but are not limited to, ethenylbenzene and propenylbenzene.

[0189] In some embodiments, the linker B may include at least one substituted aralkenyl group. In some embodiments, the substituted aralkenyl group may include one or more of the following substituents: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.

[0190] In some embodiments, the linker B may comprise at least one aralkynyl group. Examples of aralkynyl groups include, but are not limited to, ethynylbenzene and propynylbenzene.

[0191] In some embodiments, the linker B can include at least one substituted aralkynyl group. In some embodiments, the substituted aralkynyl group can include one or more of the following substituents: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.

[0192] In some embodiments, linker B may include at least one heteroatom. In some embodiments, linker B may include 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 may include at least one heteroalkyl group.

[0193] In some embodiments, linker B may include at least one aliphatic heterocycle. In some embodiments, linker B may include at least one of tetrahydrofuran (THF), tetrahydropyran (THP), morpholine, piperidine, piperazine, pyrrolidine, and / or azetidine.

[0194] In some embodiments, linker B may include at least one heteroaryl group. In some embodiments, linker B may include one or more of imidazole, pyrazole, pyridine, pyrimidine, triazole, and / or 1,2,3-triazole.

[0195] In some embodiments, the linker B may include at least one substituted heteroaryl group. In some embodiments, the substituted heteroaryl group may include one or more of the following substituents: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.

[0196] In some embodiments, linker B may comprise at least one amino acid. A variety of amino acids are known to those of skill in the art. Linker B is not limited to comprising 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 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 ​​(Sec) amino acids, one or more glycine (Gly) amino acids, one or more proline (Pro) amino acids, one or more alanine (Ala) amino acids, one or more valine (Val) amino acids, one or more isoleucine (Ile) amino acids, one or more leucine (Leu) amino acids, one or more methionine (Met) amino acids, one or more phenylalanine (Phe) amino acids, one or more tyrosine (Tyr) amino acids, and / or one or more tryptophan (Trp) amino acids.

[0197] In some embodiments, linker B may comprise at least one nucleotide. Various nucleotides are known to those skilled in the art. Linker B is not limited to comprising one or more specific nucleotides. For example, linker B may comprise one or more nucleotides comprising guanine nucleobase, one or more nucleotides comprising adenine nucleobase, one or more nucleotides comprising cytosine nucleobase, one or more nucleotides comprising thymine nucleobase, and / or one or more nucleotides comprising uracil nucleobase. In some embodiments, linker B may comprise at least one abasic nucleotide. As known in the art, an abasic nucleotide is a nucleotide having an abasic site that is a position that has neither a purine nor a pyrimidine base. For example, linker B may comprise one or more abasic DNA and / or one or more abasic RNA. In some embodiments, linker B may comprise at least one inverted abasic nucleotide. For example, linker B may comprise one or more inverted abasic DNA and / or one or more inverted abasic RNA.

[0198] In some embodiments, linker B may 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 one glucosamine monosaccharide unit.

[0199] In some embodiments, the linker B is

[0200] [ka] [ka] [In the formula, j is an integer between 1 and 12, k is an integer between 0 and 12.] may include one or more of:

[0201] 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 some cases, the present invention has also found that when linker B contains a 6-membered ring fragment, particularly a 4-hydroxypiperidinyl group, when used as a targeted delivery of pharmaceuticals, it shows better in vivo stability and activity compared to a 5-membered ring, such as compound 75 of the present invention.

[0202] Pharmaceuticals In some embodiments, the pharmaceutical agent is a diagnostic or therapeutic drug, molecule, compound, or combination of drugs, molecules, or compounds, e.g., having properties that aid in the diagnosis, prevention, treatment, and / or alleviation of a disease or condition in a cell or subject. In certain embodiments, the pharmaceutical agent is a drug, molecule, compound, or combination of drugs, molecules, or compounds, e.g., having properties that aid in the enhancement of a desired condition in a cell or subject.

[0203] In some embodiments, the pharmaceutical agent may be an oligonucleotide. In some embodiments, the oligonucleotide may comprise siRNA. In some embodiments, the oligonucleotide may comprise double-stranded siRNA. In some embodiments, the double-stranded siRNA may comprise at least one modified ribonucleotide. In some embodiments of the compositions and methods of the invention, the at least one modified nucleotide comprises a 2'-O-methyl nucleotide, a 2'-fluoro nucleotide, a 2'-deoxy nucleotide, a 2'3'-seconucleotide mimic, a locked nucleotide, an unlocked nucleic acid nucleotide (UNA), a glycol nucleic acid nucleotide (GNA), a 2'-F-arabinonucleotide, a 2'-methoxyethyl nucleotide, an abasic nucleotide, a ribitol, an inverted nucleotide, an inverted abasic nucleotide, an inverted 2'-Ome nucleotide, an inverted 2'-deoxy nucleotide, a 2'-amino-modified nucleotide, a 2'-alkyl-modified nucleotide, a morpholino nucleotide, a 3'-Ome nucleotide, a nucleotide containing a 5'-phosphorothioate group, or a 5'-(E)-vinyl phosphonate nucleotide (antisense strand only), or a terminal nucleotide linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group, a 2'-amino-modified nucleotide, a phosphoramidate, or a nucleotide containing a non-natural base. In some embodiments, substantially all (ie, greater than 85%) of the ribonucleotides in the double-stranded siRNA may be modified.

[0204] In some embodiments, all ribonucleotides of the double-stranded siRNA may be modified. In some embodiments, at least one strand of the double-stranded siRNA may contain at least one phosphorothioate bond. In some embodiments, at least one strand of the double-stranded siRNA may contain up to six phosphorothioate bonds. In some embodiments, the double-stranded siRNA may contain 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 connecting the 2' oxygen and the 4' carbon, thereby locking the ribose in the 3'-endo conformation. LNA is known to increase stability against enzymatic degradation and improve specificity and affinity. In some embodiments, the double-stranded siRNA may contain 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 ribose ring of RNA. It is known to those of skill in the art that inclusion of a UNA at certain positions in the antisense strand of an siRNA can be permissive for activity and promote reduction of off-target activity.

[0205] In some embodiments, double-stranded siRNA may 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 similar to RNA, but differs in its sugar-phosphodiester backbone composition.It is known to those skilled in the art that the inclusion of GNA at certain positions in the antisense strand of siRNA can be tolerated for activity and promote the reduction of off-target activity.

[0206] In some embodiments, the oligonucleotide may comprise an siRNA that contains one or more modified nucleotides, including, but not limited to, 2'-modified nucleotides (e.g., F and MeO), abasic nucleotides, inverted abasic nucleotides, locked nucleotides, UNA unlocked nucleic acid (UNA), and glycerol nucleic acid (GNA).

[0207] In some embodiments, the oligonucleotide may comprise an siRNA that includes one or more phosphorothioate backbone linkages.

[0208] In some embodiments, the oligonucleotide may comprise a single-stranded siRNA. In some embodiments, the oligonucleotide may comprise a small activating RNA. In some embodiments, the oligonucleotide may comprise a microRNA (miRNA). In some embodiments, the oligonucleotide may comprise an antisense oligonucleotide. In some embodiments, the oligonucleotide may comprise a short guide RNA (gRNA). In some embodiments, the oligonucleotide may comprise a single guide RNA (sgRNA). In some embodiments, the oligonucleotide may comprise a messenger RNA (mRNA). In some embodiments, the oligonucleotide may comprise a ribozyme. In some embodiments, the oligonucleotide may comprise a plasmid. In some embodiments, the oligonucleotide may comprise an immunostimulatory nucleic acid. In some embodiments, the oligonucleotide may comprise an antagomir. In some embodiments, the oligonucleotide may comprise an aptamer. An aptamer is a short, single-stranded DNA or RNA molecule that can selectively bind to a specific target, such as a protein, peptide, carbohydrate, small molecule, toxin, or living cell. 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 yet to be discovered aptamers are within the scope of this disclosure.

[0209] In some embodiments, the oligonucleotide may comprise at least 3 independently selected nucleotides. In some embodiments, for example, when the oligonucleotide is an siRNA, the oligonucleotide may comprise between 16 and 23 independently selected nucleotides. In some embodiments, for example, when the oligonucleotide is an sgRNA, the oligonucleotide may comprise about 100 independently selected nucleotides. In some embodiments, for example, when the oligonucleotide is an mRNA, the oligonucleotide may comprise up to 14000 independently selected nucleotides.

[0210] A functional group that can be linked to one or more pharmaceutical agents As shown above, "W" in formula 1 may be a functional group that may be linked to one or more pharmaceutical agents.

[0211] In some embodiments, the functional group may be a hydroxy group (OH). In some embodiments, the functional group may be a protected hydroxy group. Those skilled in the art will recognize that various protecting groups may be used to protect the hydroxy group. Each of the various process groups is within the scope of the present disclosure. For example, but not limited to, the hydroxy group may be protected using at least one of 4,4'-dimethoxytrityl (DMT), monomethoxytrityl (MMT), 9-(p-methoxyphenyl)xanthen-9-yl (Mox), and 9-phenylxanthen-9-yl (Px).

[0212] In some embodiments, the functional group has the formula:

[0213] [ka] [In the formula, R a is a C1-C6 alkyl, C3-C6 cycloalkyl, isopropyl group, or R a is connected to R via the nitrogen atom b Together with R bis a C1-C6 alkyl, C3-C6 cycloalkyl, isopropyl group, or R b is connected to R via the nitrogen atom a Together with R c is a phosphite protecting group, a phosphate protecting group, or a 2-cyanoethyl group. It may be a phosphoramidite group having the formula:

[0214] R c may be one of a variety of phosphite protecting groups known to those skilled in the art. In some embodiments, the phosphite protecting group may include at least one of methyl, allyl, 2-cyanoethyl, 4-cyano-2-butenyl, 2-cyano-1,1-dimethylethyl, 2-(trimethylsilyl)ethyl, 2-(S-acetylthio)ethyl, 2-(S-pivaloylthio)ethyl, 2-(4-nitrophenyl)ethyl, 2,2,2-trichloroethyl, 2,2,2-trichloro-1,1-dimethylethyl, 1,1,1,3,3,3-hexafluoro-2-propyl, fluorenyl-9-methyl, 2-chlorophenyl, 4-chlorophenyl, and 2,4-dichlorophenyl.

[0215] R c may be one of a variety of phosphate protecting groups known to those skilled in the art. In some embodiments, the phosphate protecting group may include at least one of methyl, allyl, 2-cyanoethyl, 4-cyano-2-butenyl, 2-cyano-1,1-dimethylethyl, 2-(trimethylsilyl)ethyl, 2-(S-acetylthio)ethyl, 2-(S-pivaloylthio)ethyl, 2-(4-nitrophenyl)ethyl, 2,2,2-trichloroethyl, 2,2,2-trichloro-1,1-dimethylethyl, 1,1,1,3,3,3-hexafluoro-2-propyl, fluorenyl-9-methyl, 2-chlorophenyl, 4-chlorophenyl, and 2,4-dichlorophenyl.

[0216] In some embodiments, the functional group may be a carboxyl group (COH). In some embodiments, the functional group has the formula:

[0217] [ka] [In the formula, X is a leaving group.] It may be an activated carboxyl group having the formula:

[0218] A variety of activated carboxyl groups are known to those of skill in the art, all of which are within the scope of the present disclosure. In some embodiments, the leaving group (X) may be one of carboxylate, sulfonate, chloride, phosphate, imidazole, hydroxybenzotriazole (HOBt), N-hydroxysuccinimide (NHS), tetrafluorophenol, pentafluorophenol, para-nitrophenol.

[0219] In some embodiments, the functional group may be a Michael acceptor. In some embodiments, the Michael acceptor has the formula:

[0220] [ka] [In the formula, E is an electron withdrawing group; R d is hydrogen or C1-C6 alkyl substituents on the olefin (E and R d may be cis, trans, or iso about the carbon-carbon double bond. may have

[0221] 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) can be a carboxamide or ester. In some embodiments, the electron-withdrawing group (E) and carbon-carbon double bond of the Michael acceptor can be part of a maleimide, a cyclic dicarboximide in which the two carboacyl groups on the nitrogen form a 1H-pyrrole-2,5-dione structure together with the nitrogen itself.

[0222] In some embodiments, the functional group has the formula:

[0223] [ka] [In the formula, Linker C is absent or is a spacer attached to the 3' or 5' end of the oligonucleotide; X is a methyl group, oxygen, sulfur, or amino group; Y is oxygen, sulfur, or an amino group. may have

[0224] In some embodiments, the linker C of formula 5 may comprise at least one heterocyclic compound. In some embodiments, the heterocyclic compound may be an abasic nucleotide or an inverted abasic nucleotide.

[0225] In some embodiments, the functional group has the formula:

[0226] [ka] [wherein linker C is a spacer having one end attached to the nitrogen of the carboxamide and the other end attached to the 3' or 5' end of the oligonucleotide.] may have

[0227] In some embodiments, linker B is attached to the carbonyl of the carboxamide of formula 6.

[0228] In some embodiments, the linker C in formula 6 may include at least one PEG. The PEG may have any number of repeating O-CH2-CH2 units. For example, the PEG may 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, P 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 more.

[0229] In some embodiments, the linker C in formula 6 can include 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.

[0230] In some embodiments, the linker C in formula 6 can include at least one cycloalkyl group. In some embodiments, the cycloalkyl group can be a C3 cycloalkyl (i.e., cyclopropane), a C4 cycloalkyl (i.e., cyclobutene), a C5 cycloalkyl (i.e., cyclopentane), a C6 cycloalkyl (i.e., cyclohexane), a C7 cycloalkyl (i.e., cycloheptane), a C8 cycloalkyl (i.e., cyclooctane), a C9 cycloalkyl (i.e., cyclononane), or a C10 cycloalkyl (i.e., cyclodecane).

[0231] In some embodiments, the linker C in formula 6 can include at least one heteroatom. In some embodiments, the linker C can include 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.

[0232] In some embodiments, the linker C in formula 6 may include at least one aliphatic heterocycle. In some embodiments, the linker C may include at least one of tetrahydrofuran (THF), tetrahydropyran (THP), morpholine, piperidine, piperazine, pyrrolidine, and / or azetidine.

[0233] In some embodiments, the linker C in formula 6 may include at least one heteroaryl group. In some embodiments, the linker C may include one or more of imidazole, pyrazole, pyridine, pyrimidine, triazole, and / or 1,2,3-triazole.

[0234] In some embodiments, the linker C in formula 6 can include at least one substituted heteroaryl group. In some embodiments, the substituted heteroaryl group can include one or more of the following substituents: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.

[0235] In some embodiments, the linker C in formula 6 may include at least one amino acid. A variety of amino acids are known to those skilled in the art. The linker C is not limited to including one or more specific amino acids. For example, the 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 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 ​​(Sec) amino acids, one or more glycine (Gly) amino acids, one or more proline (Pro) amino acids, one or more alanine (Ala) amino acids, one or more valine (Val) amino acids, one or more isoleucine (Ile) amino acids, one or more leucine (Leu) amino acids, one or more methionine (Met) amino acids, one or more phenylalanine (Phe) amino acids, one or more tyrosine (Tyr) amino acids, and / or one or more tryptophan (Trp) amino acids.

[0236] In some embodiments, the linker C in formula 6 may comprise at least one nucleotide. Various nucleotides are known to those skilled in the art. The linker C is not limited to comprising one or more specific nucleotides. For example, the linker C 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, one or more nucleotides comprising a thymine nucleobase, and / or one or more nucleotides comprising a uracil nucleobase. In some embodiments, the linker C may comprise at least one abasic nucleotide. As known in the art, an abasic nucleotide is a nucleotide having an abasic site that is a position that has neither a purine nor a pyrimidine base. For example, the linker C may comprise one or more abasic DNAs and / or one or more abasic RNAs. In some embodiments, the linker C may comprise at least one inverted abasic nucleotide. For example, the linker C may comprise one or more inverted abasic DNAs and / or one or more inverted abasic RNAs.

[0237] In some embodiments, the linker C in formula 6 may comprise at least one sugar. In some embodiments, the 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 one glucosamine monosaccharide unit.

[0238] In some embodiments, the linker C in formula 6 is

[0239] [ka] [In the formula, j is an integer between 1 and 12, k is an integer between 0 and 12.] may include one or more of:

[0240] In some embodiments, the functional group has the formula:

[0241] [ka] [wherein linker C is a spacer having one end attached to one of the succinimide ring carbons via a thioether bond and the other end attached to the 3' or 5' end of the oligonucleotide.] may have

[0242] In some embodiments, linker B is attached to the succinimide nitrogen of Formula 7.

[0243] In some embodiments, the linker C in formula 7 may include at least one PEG. The PEG may have any number of repeating O-CH2-CH2 units. For example, the PEG may 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, P 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 more.

[0244] In some embodiments, the linker C in formula 7 can include 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.

[0245] In some embodiments, the linker C in formula 7 can include at least one cycloalkyl group. In some embodiments, the cycloalkyl group can be a C3 cycloalkyl (i.e., cyclopropane), a C4 cycloalkyl (i.e., cyclobutene), a C5 cycloalkyl (i.e., cyclopentane), a C6 cycloalkyl (i.e., cyclohexane), a C7 cycloalkyl (i.e., cycloheptane), a C8 cycloalkyl (i.e., cyclooctane), a C9 cycloalkyl (i.e., cyclononane), or a C10 cycloalkyl (i.e., cyclodecane).

[0246] In some embodiments, the linker C in formula 7 is

[0247] [ka] [In the formula, j is an integer between 1 and 12, k is an integer between 0 and 12.] may include one or more of:

[0248] Multivalent Ligand Clusters Containing GalNAc-Targeting Ligands In some embodiments, the multivalent ligand cluster of the present disclosure has the general structure of Formula 8:

[0249] [ka] [In the formula, m is an integer between 1 and 10; each n is an independently selected integer between 1 and 10; each linker A is an independently selected spacer attached at one end to TL and at the other end to a nitrogen of an alkylcarboxamide; Linker B is a spacer having one end attached to a drug or a functional group capable of linking to one or more drug substances and the other end attached to a diamine nitrogen; W is either one or more pharmaceutical agents or a functional group that can be linked to one or more pharmaceutical agents. may have

[0250] In some embodiments, the multivalent ligand cluster of the present disclosure has the general structure of Formula 9:

[0251] [ka] [In the formula, m is an integer between 1 and 10; each n is an independently selected integer between 1 and 10; Each linker A is an independently selected spacer, Linker B is a spacer, The linker C is a spacer or is absent, X is a methyl group, oxygen, sulfur, or amino group; Y is oxygen, sulfur, or an amino group. may have

[0252] In some embodiments, the multivalent ligand clusters of the present disclosure have the general structure of Formula 10:

[0253] [ka] [In the formula, m is an integer between 1 and 10; each n is an independently selected integer between 1 and 10; Each linker A is an independently selected spacer, Linker B is a spacer, The linker C is a spacer. may have

[0254] In some embodiments, the multivalent ligand clusters of the present disclosure have the general structure of Formula 11:

[0255] [ka] [In the formula, m is an integer between 1 and 10; each n is an independently selected integer between 1 and 10; Each linker A is an independently selected spacer, Linker B is a spacer, The linker C is a spacer. may have

[0256] The following are example formulas for multivalent ligand clusters that include a GalNAc or protected GalNAc targeting ligand, various "m"s from Formula 1, various "n"s from Formula 1, and various functional groups that can be linked to one or more pharmaceutical agents:

[0257] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0258] First Example of Preparation Method One method for preparing examples of compounds having general formula 1 is illustrated in Scheme 1 below. Starting materials and intermediates can be purchased from commercial sources, made from known procedures, or are otherwise exemplified. The order in which the steps of the reaction schemes are carried out can be varied.

[0259] [ka]

[0260] Scheme 1 Scheme 1 begins with a mono-protected diamine (Compound I). The mono-protected diamine contains a first nitrogen and a second nitrogen, where in Scheme 1 the first nitrogen is a primary amine and the second nitrogen is a secondary amine that contains a protecting group (PG).

[0261] A variety of protecting groups are known to those skilled in the art and can be used. In some embodiments, the protecting group can be a benzyl group. In some embodiments, the protecting group can be a triphenylmethyl group.

[0262] "m" in Scheme 1 can be any integer. In some embodiments, m in Scheme 1 can be an integer between 1 and 10.

[0263] Starting with compound I, the triester compound II can be synthesized in one step. In some embodiments, compound II is N Compound I can be synthesized using one or more suitable substrates via a 2-substitution reaction. In some embodiments, compound II can be synthesized using compound I and one or more suitable substrates via a reductive amination reaction. In some embodiments, compound II can be synthesized using compound I and one or more suitable substrates via a Michael addition reaction.

[0264] As illustrated 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 that contains a first protected carboxylic acid and a second protected carboxylic acid, and the second nitrogen is a tertiary amine that contains a protecting group and a third protected carboxylic acid.

[0265] Each "n" in Scheme 1 is an independently selected integer. In some embodiments, each n in Scheme 1 is an independently selected integer between 0 and 10.

[0266] Compound III is produced by deprotecting the second nitrogen of compound II such that the second nitrogen of compound III is a secondary amine containing a third protected carboxylic acid. In embodiments where the protecting group is a benzyl group, compound III can be produced by performing a hydrogenation reaction using compound II. In embodiments where the protecting group is a triphenylmethyl group, compound III can be produced by reacting the second compound with at least one acid. Examples of acids include, but are not limited to, hydrochloric acid (HCl) and trifluoroacetic acid (TFA).

[0267] 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 a tertiary amine or amide containing 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 linkers B described herein above.

[0268] In some embodiments, producing triester compound IV can be achieved by reacting compound III with one or more suitable substrates in a S NIn some embodiments, producing compound IV includes performing a reductive amination reaction using compound III and one or more suitable substrates. In some embodiments, producing compound IV includes performing a Michael addition reaction using compound III and one or more suitable substrates. In some embodiments, producing compound IV includes performing an amide coupling reaction using compound III and one or more suitable substrates. In some embodiments, producing compound IV includes performing a nucleophilic addition reaction using compound III and one or more suitable substrates. Examples of substrates include, but are not limited to, isocyanates and isothiocyanates.

[0269] The 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 (e.g., when R in Scheme 1 is an acid-sensitive group such as a tert-butyl group). 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 may include performing a hydrogenation reaction using compound IV (e.g., when R in Scheme 1 is a benzyl group). In some embodiments, producing compound V may include performing a hydrolysis reaction using compound IV.

[0270] Compound VI can be produced by carrying out an amide coupling reaction using compound V. In compound VI, the first nitrogen is a tertiary amine that includes a first amide and a second amide, and the second nitrogen is a tertiary amine that includes a moiety that includes a hydroxyl group and a third amide. Each of the first amide, the second amide, and the third amide can be coupled to an independently selected targeting ligand. The linker A in scheme 1 can be any linker A described herein. The TL in scheme 1 can be any TL described herein.

[0271] Compound VII is produced by converting the hydroxy group of compound VI (attached to linker B) to a phosphoramidite group using a phosphitylation reaction. As illustrated in Scheme 1, in some embodiments, the conversion of the hydroxy group to a phosphoramidite group can be performed after an amide coupling reaction to produce compound VI.

[0272] Second Example of Preparation Method Another method for preparing an example of a compound having general formula 1 is illustrated in scheme 2. Scheme 2 allows the compound having general formula 1 to have one or more different targeting ligands. Scheme 2 allows the targeting ligand to be introduced stepwise. Starting materials and intermediates can be purchased from commercial sources, made from known procedures, or otherwise exemplified. The order of carrying out the steps of the reaction scheme can be changed.

[0273] [ka] [ka]

[0274] Scheme 2 Scheme 2 begins with a doubly protected diamine (Compound I). The doubly protected diamine includes a first nitrogen and a second nitrogen, where the first nitrogen is protected by a first protecting group (PG 1 ) and the second nitrogen is a secondary amine containing a second protecting group (PG 2 ) is a primary or secondary amine.

[0275] The first and second protecting groups 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 tert-butyloxycarbonyl (Boc) group.

[0276] "m" in Scheme 2 can be any integer. In some embodiments, m in Scheme 2 can be an integer between 1 and 10.

[0277] 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. One of skill in the art will recognize that by distinguishing the protecting groups in compound I, the protecting groups can be strategically removed and replaced. 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-containing amine of compound I (but not the Boc-containing amine) can be subjected to S-coupling with one or more appropriate reagents. N It may undergo a disubstitution reaction, a reductive amination reaction, or a Michael addition reaction to form compound II.

[0278] Compound III can be produced by removing a first protecting group from compound II. In compound III, the first nitrogen is a secondary amine bearing a first protected carboxylic acid, and the second nitrogen is a primary or secondary amine bearing a second protecting group. In some embodiments, producing compound III can include performing a hydrogenation reaction (e.g., when the first protecting group is a benzyl group).

[0279] 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 be achieved by coupling compound III and one or more other suitable reagents to S N In some embodiments, producing compound IV may include performing a reductive amination reaction using compound III and one or more other suitable reagents. In some embodiments, producing compound IV may include performing a Michael addition reaction using compound III and one or more other suitable reagents. In some embodiments, producing compound IV may include performing an amide coupling reaction using compound III and one or more other suitable reagents. In some embodiments, producing compound IV may include performing a nucleophilic addition reaction using compound III and one or more other suitable reagents.

[0280] 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 containing a first protected carboxylic acid and a second protected carboxylic acid, and the second nitrogen of compound V is a primary amine. In some embodiments (e.g., when the second protecting group is a Boc group), compound V can be produced by reacting compound IV with at least one acid. Examples of 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 reacting compound V with S-alkylation using compound V and one or more other suitable reagents. N In some embodiments, compound VI can be produced by performing a reductive amination reaction using compound V and one or more other suitable reagents. In some embodiments, compound VI can be produced by performing a Michael addition reaction using compound V and one or more other suitable reagents.

[0281] Compound VII can be produced by attaching a moiety containing a hydroxy 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 moiety containing a hydroxy group can be attached to the second nitrogen using any of the linkers B described herein above. In some embodiments, compound VII can be produced by condensing S with compound VI and one or more other suitable reagents. NCompound VII can be produced by performing a 2-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.

[0282] In the example of Scheme 2, R a , R b , and R c may be sufficiently different to be able to selectively bind the target ligand, e.g., in one scenario, R a , R b , and R c can be methyl, benzyl and tert-butyl groups, respectively. Such selective binding of targeting ligands is now described.

[0283] Compound VIII can be 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 (e.g., R c is an acid sensitive group such as, for example, a tert-butyl group).

[0284] Compound IX can be produced by performing an amide coupling reaction using compound VIII, where the first nitrogen contains a first protected carboxylic acid and a second protected carboxylic acid, and the second nitrogen of compound IX contains a first amide having coupled thereto a first targeting ligand and a moiety that contains a hydroxy group.

[0285] 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 include performing a hydrogenation reaction using compound IX (e.g., R b is a benzyl group).

[0286] Compound XI can be produced by performing an amide coupling reaction using compound X. In compound XI, the first nitrogen contains a first protected carboxylic acid and a second amide having a second targeting ligand coupled thereto, and the second nitrogen of compound XI contains a first amide and a moiety that contains a hydroxy group and has a first targeting ligand coupled thereto.

[0287] 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 carrying out a hydrolysis reaction using compound XI (e.g., R a is a methyl group).

[0288] Compound XIII can be produced by performing an amide coupling reaction using compound XII, where the first nitrogen contains a second amide having a second targeting ligand coupled thereto and a third amide having a third targeting ligand coupled thereto, and the second nitrogen of compound XI contains a first amide having a first targeting ligand coupled thereto and a moiety containing a hydroxy group.

[0289] The first amide may be coupled to the first targeting ligand using any independently selected linker A described herein. The second amide may be coupled to the second targeting ligand using any independently selected linker A described herein. The third amide may be coupled to the third targeting ligand using any independently selected linker A described herein.

[0290] One or more of the first targeting ligand, the second targeting ligand, and the third targeting ligand may be independently selected to be one or more of the targeting ligands described herein.

[0291] The hydroxy group may be coupled to the second nitrogen using any linker B described herein.

[0292] In some embodiments, the hydroxy group can be converted to a phosphoramidite group using a phosphitylation reaction, hi some embodiments, the hydroxy group can be converted to a phosphoramidite group to produce compound XIV.

[0293] Each "n x ", "n y " or "n z " are independently selected integers. In some embodiments, each "n x ", "n y " or "n z " are independently selected integers between 0 and 10.

[0294] Some elements of preparation and use The multivalent ligand cluster embodiment of the present invention can be prepared and used to deliver oligonucleotide drugs to cells, tissues, and organs. Non-limiting examples of drugs that can be delivered include therapeutic drugs such as siRNA. The delivery method using the multivalent ligand cluster of the present invention can be used to deliver siRNA and other drugs conjugated to the targeting ligand cluster of the present invention to cells in vitro and in vivo. The multivalent ligand cluster of the present invention can be used as a delivery vehicle to deliver drugs, including but not limited to drugs that contain nucleic acids, to cells. As used herein, the term "multivalent ligand cluster / drug complex" refers to a multivalent ligand cluster as described herein linked to a drug as described herein. In some embodiments of the present invention, the drug is siRNA.

[0295] In another aspect of the disclosure, the dsRNA drug contains 2'-fluoro modified nucleotides at positions 2, 7, 12, 14, and 16 of the antisense strand (counting from the first paired nucleotide from the 5' end of the antisense strand), and / or 2'-fluorine-modified nucleotides at positions 9, 11, and 13 of the sense strand (counting from the first paired nucleotide from the 3' end of the sense strand). In some embodiments, no other positions of the dsRNA drug contain 2' fluorine-modifications. In some embodiments, all nucleotides of the antisense and / or sense strands of the dsRNA drug are modified nucleotides. In some embodiments, the dsRNA drug has 2'-fluoro modified nucleotides at positions 2, 7, 12, 14, and 16 of the antisense strand and / or 2' fluorine-modified nucleotides at positions 9, 11, and 13 of the sense strand, and other positions are 2'-O-methyl nucleotides, 2'-deoxy nucleotides, 2'3'-seconucleotide mimics, locked nucleotides, unlocked nucleotides (UNA), glycol nucleotides (GNA), 2'-F-arabino nucleotides, 2'-methoyxyethyl nucleotides, abasic nucleotides. The dsRNA agent contains modified nucleotides selected from: ribitol, inverted nucleotides, inverted abasic nucleotides, inverted 2'-Ome nucleotides, inverted 2'-deoxy nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, and 3'-OMe nucleotides, nucleotides containing a 5'-phosphorothioate group, or a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, 2'-amino-modified nucleotides, phosphoramidates, or non-natural base containing nucleotides. In some embodiments, the dsRNA agent contains an E-vinyl phosphonate nucleotide at the 5' end of the guide strand. In certain embodiments, the dsRNA agent contains at least one phosphorothioate internucleoside linkage. In certain embodiments, the sense strand contains 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 complementary region is between 16 and 23 nucleotides in length. In some embodiments, the complementary region is 19-21 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 30 or less nucleotides in length. In some embodiments, each strand is 25 nucleotides or less in length. In some embodiments, each strand is 23 nucleotides or less in length. In certain embodiments, the dsRNA drug comprises at least one modified nucleotide and further comprises one or more targeting groups 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 comprises N-acetyl-galactosamine (GalNAc). In some embodiments, the targeting group comprises the structure of GalNAc described above.

[0296] In some aspects of the present invention, the multivalent ligand cluster can be used to deliver pharmaceuticals to cells in a subject. Means of administering the multivalent ligand cluster / pharmaceutical complex to a subject can include methods known in the art. As a non-limiting example, the multivalent ligand cluster / pharmaceutical complex can be delivered locally in vivo by direct injection or by using an infusion pump. In some aspects of the present invention, the multivalent ligand cluster / pharmaceutical complex can be a pharmaceutical composition, which may also be referred to as a pharmaceutical. In some embodiments, the pharmaceutical of the present invention is administered to a subject in an amount effective to prevent, regulate, treat or alleviate the symptoms of a pathology in the subject.

[0297] Cells and subjects As used herein, subject is intended to mean a human or a vertebrate mammal, including, but not limited to, a dog, a cat, a horse, a goat, a cow, a sheep, a rodent, and a primate, such as a monkey. Thus, the present invention can be used to treat disease or conditions in human and non-human subjects. For example, the methods and compositions of the present invention can be used in veterinary applications, as well as in human prophylactic and treatment regimes. In some embodiments of the present invention, the vertebrate subject is a mammal.

[0298] In certain embodiments of the present invention, the multivalent ligand cluster / drug conjugate of the present invention is delivered to and contacted with a cell. In some embodiments of the present invention, the contacted cell is a cell in culture, and in other embodiments, the contacted cell is a cell in a subject. The types of cells that can be contacted with the multivalent ligand cluster / drug conjugate of the present invention include, but are not limited to, liver cells, muscle cells, cardiac cells, circulatory cells, neuronal cells, glial cells, fat cells, skin cells, hematopoietic cells, epithelial cells, sperm, eggs, muscle cells, adipocytes, kidney cells, liver cells, or pancreatic cells. In some embodiments, the cells that are contacted with the multivalent ligand cluster / drug conjugate of the present invention are liver cells.

[0299] Dosage Dosage levels of pharmaceuticals and pharmaceutical compositions that can be delivered using the multivalent ligand cluster / drug complexes of the present disclosure can be determined by one of ordinary skill in the art through routine experimentation. In at least some embodiments, a unit dose can contain between about 0.01 mg / kg to about 100 mg / kg of siRNA. Alternatively, the dose can be between 10 mg / kg to 25 mg / kg of body weight, or between 1 mg / kg to 10 mg / kg of body weight, or between 0.05 mg / kg to 5 mg / kg of body weight, or between 0.1 mg / kg to 5 mg / kg of body weight, or between 0.1 mg / kg to 1 mg / kg of body weight, or between 0.1 mg / kg to 0.5 mg / kg of body weight, or between 0.5 mg / kg to 1 mg / kg of body weight, or between 1 mg / kg to 3 mg / kg of body weight.

[0300] The pharmaceutical compositions may be in sterile injectable aqueous suspension or solution, or in lyophilized form. The pharmaceutical compositions and medicaments of the present disclosure may be administered to a subject in a pharma- ceutical effective dose.

[0301] Method of administration A variety of administrations are available for the polyvalent ligand cluster / drug complexes of the present invention. The particular delivery mode selected will depend on the particular condition being treated and the dosage required for therapeutic efficacy. The methods of the present invention can be carried out using any medically acceptable mode of administration, which generally means any mode that provides an effective level of treatment without causing clinically unacceptable adverse effects. In some embodiments of the present invention, the polyvalent ligand cluster / drug complexes of the present invention can be administered by oral, enteral, mucosal, transdermal, and / or parenteral routes. The term "parenteral" includes subcutaneous, intravenous, intramuscular, perivascular, and intracisternal injection or infusion techniques. Other routes include, but are not limited to, intranasal (e.g., nasogastric tube), dermal, vaginal, rectal, and sublingual. Delivery routes of the present invention can include intrathecal, intraventricular, or intracranial. In some embodiments of the present invention, the polyvalent ligand cluster / drug complexes of the present invention can be administered by placing them in a sustained release matrix and placing the matrix in the subject.

[0302] The polyvalent ligand cluster / drug complex of the present invention can be administered in a formulation, which can be administered in a pharma- ceutically acceptable solution that may routinely contain pharma- ceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients. In the method of the present invention, the polyvalent ligand cluster / drug complex can be administered in a pharmaceutical composition. In general, the pharmaceutical composition comprises the polyvalent ligand cluster / drug complex of the present invention and a pharma- ceutically acceptable carrier. Pharmaceutically acceptable carriers are well known to those skilled in the art and can be selected and utilized using routine methods. As used herein, a pharma- ceutically acceptable carrier refers to a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient (e.g., the ability of the nucleic acid, e.g., siRNA, delivered to prevent and / or treat the disease or condition targeted).

[0303] Pharmaceutically acceptable carriers may include diluents, bulking agents, salts, buffers, stabilizers, solubilizers, and other substances that are well known in the art. Exemplary pharma-ceutically acceptable carriers are described in, for example, U.S. Pat. No. 5,211,657, and are known to those skilled in the art. Such formulations may routinely contain salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents. When used in pharmaceutical products, salts should be pharma-ceutically acceptable, but pharma-ceutically unacceptable salts may be conveniently used to prepare pharma-ceutically acceptable salts and are not excluded from the scope of the present invention. Such pharmacologically and pharma-ceutically acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, succinic acid, and the like. Pharmaceutically acceptable salts may also be prepared as alkali metal or alkaline earth metal salts, such as sodium, potassium, or calcium salts.

[0304] In some embodiments of the present invention, the polyvalent ligand cluster / drug complex of the present invention can be administered directly to a tissue. Direct tissue administration can be achieved by direct injection or other means known in the art. The polyvalent ligand cluster / drug complex of the present invention can be administered in one dose, or alternatively, in multiple doses. When administered multiple times, the polyvalent ligand cluster / drug complex of the present invention can be administered by different routes. For example, the first (or first few) doses can be administered directly to the affected tissue or organ, while subsequent doses can be systemic.

[0305] When systemic administration is desired, the polyvalent ligand cluster / drug complex of the present invention can be formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion). Formulations for injection can be provided in unit dosage form (e.g., in ampoules or in multi-dose containers) with or without added preservatives. The pharmaceutical compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles and can contain formulatory agents such as suspending, stabilizing and / or dispersing agents.

[0306] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. 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 may also be present, such as, for example, antimicrobial agents, antioxidants, chelating agents, and inert gases and the like. Lower dosages should result from other forms of administration, such as intravenous administration. In the event that the response in the subject is inadequate at the initial dose administered, a higher dose (or a more effective dose via a different, more localized delivery route) may be used, as long as patient tolerance allows. Multiple doses per day may be used if necessary to achieve adequate systemic or local levels of one or more of the inventive multivalent ligand cluster / drug complexes to produce the desired level of drug, e.g., the desired level of siRNA.

[0307] Both non-biodegradable and biodegradable polymer matrices can be used to deliver one or more of the multivalent ligand cluster / drug complexes of the present invention to cells and / or subjects. In some embodiments, the matrix can be biodegradable. The matrix polymer can be a natural or synthetic polymer. The polymer can be selected based on the period over which release is desired, generally from a few hours to as long as a year or more. Typically, release over a period ranging between a few hours and 3-12 months can be used. The polymer is optionally in the form of a hydrogel that can absorb up to about 90% of its weight in water, and further, is optionally crosslinked with multivalent ions or other polymers.

[0308] In certain embodiments of the present invention, the polyvalent ligand cluster / drug complex of the present invention can be delivered by diffusion using a biodegradable implant or by degradation of the polymer matrix. Exemplary synthetic polymers for such use are well known in the art. Biodegradable and non-biodegradable polymers can be used to deliver one or more of the polyvalent ligand cluster / drug complexes of the present invention using methods known in the art. Such methods may be used to deliver one or more of the polyvalent ligand cluster / drug complexes of the present invention for treatment. Additional suitable delivery systems may include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administration of the polyvalent ligand cluster / drug complexes of the present invention, increasing convenience for subjects and health care providers. Many types of release delivery systems are available and known to those skilled in the art. [See, e.g., U.S. 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, some of which are adapted for implantation, can be used.

[0309] The use of long-term sustained release implants may be particularly suitable for the prophylactic treatment of subjects with siRNA delivered using the multivalent ligand cluster of the present invention, and for the prevention and / or treatment of subjects at risk of developing recurrent disease or pathology.Long-term release, as used herein, means that the implant is constructed and arranged to deliver therapeutic levels of active ingredient for at least 30 days, 60 days, 90 days or longer.Long-term sustained release implants are well known to those skilled in the art and include some of the release systems described above.

[0310] Therapeutic formulations of one or more of the polyvalent ligand cluster / drug conjugates of the present invention can be prepared by mixing the polyvalent ligand cluster / drug conjugates having a desired degree of purity with optional pharma- ceutically acceptable carriers, excipients, or stabilizers [Remington's Pharmaceutical Sciences 21 stedition, (2006)], lyophilized formulations or aqueous solutions can be prepared for storage. Acceptable carriers, additives, or stabilizers are non-toxic to recipients at the dosages and concentrations employed and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl 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; serum alginates; proteins such as albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0311] The polyvalent ligand cluster / drug complexes of the present disclosure can be formulated as pharmaceutical compositions. The pharmaceutical compositions can be used as medicines, alone or in combination with other drugs. The polyvalent ligand cluster / drug complexes of the present disclosure can also be administered in combination with other therapeutic compounds, either separately or synchronously (e.g., as a combined unit dose). In at least some embodiments, the present disclosure includes pharmaceutical compositions comprising one or more polyvalent ligand cluster / drug complexes according to the present disclosure in physiological / pharmaceutical acceptable additives, such as stabilizers, preservatives, diluents, buffers, and the like.

[0312] The pharmaceutical compositions of the present invention can be administered alone, in combination with each other, and / or in combination with other drug treatments or other treatment regimens administered to subjects with disease or pathology.The pharmaceutical compositions used in the embodiments of the present invention are preferably sterile and contain an effective amount of pharmaceutical agent, such as the multivalent ligand cluster / pharmaceutical agent complex, for preventing or treating the disease or pathology against which siRNA is directed.

[0313] The dose or doses of the pharmaceutical composition of the present invention that are sufficient to treat disease or pathology when administered to a subject can be selected according to various parameters, in particular according to the mode of administration used and the condition of the subject.Other factors can include the desired duration of treatment.In the event that the response in the subject is insufficient at the initial dose administered, a higher dose (or a more effective dose by a more localized different delivery route) can be used as long as patient tolerance allows.Some embodiments of the present invention use dosages that are determined using routine means, such as in clinical trials. EXAMPLES

[0314] [Example 1] Multivalent Ligand Clusters Containing GalNAc-Targeting Ligands In some embodiments of the present invention, the multivalent ligand cluster may comprise a GalNAc targeting ligand. The following is an example of a compound of a multivalent ligand cluster, which comprises an acetyl-protected GalNAc targeting ligand, a core C2 and C3 diamine, branched acetyl and propanoyl amide, PEG2 and PEG3 linkers A, various linkers B as described herein, and various functional groups that can be linked to one or more pharmaceutical agents as described herein. The acetyl-protecting group on the GalNAc ligand below can be easily removed after conjugation to generate the GalNAc targeting ligand is completed.

[0315] [ka]

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[0316] [Example 2] Preparation of intermediate compounds In Scheme 3 below, Intermediate-A was synthesized by treating commercially available pentaacetate galactosamine (compound I) with trimethylsilyl trifluoromethanesulfonate (TMSOTf) in dichloromethane (DCM). This was followed by glycosylation with Cbz-protected 2-(2-aminoethoxy)ethan-1-ol to give compound II. The Cbz-protecting group was removed by hydrogenation to give Intermediate-A as the trifluoroacetate (TFA) or HCl salt. Intermediate B was synthesized based on the same scheme, except that Cbz-protected 2-(2-(2-aminoethoxy)ethoxy)ethan-1-ol was used as the starting material. Scheme 3 allows access to variations in linker A and even variations in target ligands.

[0317] [ka]

[0318] Scheme 3 To a solution of compound I (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 h and then at 25° C. for 1 h. Cbz-protected 2-(2-aminoethoxy)ethan-1-ol (13.5 g, 56.5 mmol) in DCE (100 mL) dried over 4 Å molecular sieves (10 g) was added dropwise to the above reaction solution at 0° C. 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 NaHCO3 (200 mL), water (200 mL) and saturated brine (200 mL). The organic layer was dried over anhydrous Na2SO4, 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.

[0319] Into a dry, argon-purged hydrogenation bottle, 10% Pd / C (1.50 g) was carefully added, followed by THF (10 mL), then a solution of compound II (15.0 g, 26.4 mmol) in THF (300 mL) and TFA (3.00 g, 26.4 mmol). The resulting mixture was degassed and purged with H2 three times and stirred at 25 °C for 3 h under H2 (45 psi) atmosphere. TLC (DCM:MeOH = 10:1) showed that compound II was completely consumed. 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 three times to give intermediate-A (14.0 g, 96.5% yield) as a foamy white solid. 1H NMR (400 MHz DMSO-d6): δ ppm 7.90 (d, J = 9.29 Hz, 1 H), 7.78 (br s, 3 H), 5.23 (d, J = 3.26 Hz, 1 H), 4.98 (dd, J = 11.29, 3.26 Hz, 1 H), 4.56 (d, J = 8.53 Hz, 1 H), 3.98 - 4.07 (m, 3 H), 3.79 - 3.93 (m, 2 H), 3.55 - 3.66 (m, 5 H), 2.98 (br d, J = 4.77 Hz, 2 H), 2.11 (s, 3 H), 2.00 (s, 3 H), 1.90 (s, 3 H), 1.76 (s, 3 H).

[0320] Intermediate-B was synthesized using a similar procedure as for the synthesis of Intermediate-A. 1 H NMR (400 MHz DMSO-d6): δ ppm 7.90 (br d, J = 9.03 Hz, 4 H), 5.21 (d, J = 3.51 Hz, 1 H), 4.97 (dd, J = 11.1 Hz, 1 H), 4.54 (d, J = 8.53 Hz, 1 H), 3.98 - 4.06 (m, 3 H), 3.88 (dt, J = 10.9 Hz, 1 H), 3.76 - 3.83 (m, 1 H), 3.49 - 3.61 (m, 9 H), 2.97 (br s, 2 H), 2.10 (s, 3 H), 1.99 (s, 3 H), 1.88 (s, 3 H), 1.78 (s, 3 H).C 20 H 34 N2O 11 Calculated mass: 478.22; Found mass: 479.3 (M+H + ).

[0321] [Example 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(azanethryl))tetraacetic acid (compound I in Scheme 4) was converted to the dianhydride compound II. Upon treatment with 6-aminohexan-1-ol and subsequent hydrolysis, compound II was converted to the triacid compound III. Amide coupling between compound III and intermediate-A afforded compound IV. Compound IV was treated with 2-cyanoethyl N,N-diisopropylchlorophosphoramidite and a catalytic amount of 1H-tetrazole to afford the phosphoramidite compound 1.

[0322] [ka]

[0323] Scheme 4 To a stirred solution of Ac2O (8.83 g, 86.5 mmol) and pyridine (193 mg, 2.45 mmol) was added the tetraacid compound I (5.0 g, 16.3 mmol). After purging with N2 three times, 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 the volatiles were evaporated. This process was repeated three times to give compound II (2.20 g, 49.8% yield) as a yellow oil. 1 H NMR (400 MHz DMSO-d6): δ ppm 3.65 (s, 8 H), 2.46 (br t, J = 7.19 Hz, 4 H), 1.55 - 1.65 (m, 2 H).

[0324] To a mixture of compound II (1.80 g, 6.66 mmol) and imidazole (3.63 g, 53.2 mmol) in DMF (18 mL), 6-aminohexan-1-ol (624 mg, 5.33 mmol) and pyridine (263 mg, 3.33 mmol) were added in sequence. The mixture was kept stirring at 50° C. for 5 h under N2 atmosphere. The reaction mixture was concentrated under reduced pressure. The residue was purified by reverse phase prep-HPLC. Compound III was obtained (1.90 g, containing 8.4 wt % DMF and 63.2 wt % imidazole). 1 H NMR (400 MHz DMSO-d6): δ ppm 4.00 (br t, J = 6.50 Hz, 1 H), 3.44 - 3.56 (m, 6 H), 3.34 - 3.39 (m, 3 H), 3.20 - 3.27 (m, 2 H), 3.02 - 3.12 (m, 2 H), 2.80 - 2.86 (m, 1 H), 2.79 - 2.87 (m, 1 H), 2.64 - 2.70 (m, 2 H), 1.49 - 1.69 (m, 3 H), 1.33 - 1.73 (m, 4 H), 1.18 - 1.46 (m, 3 H).

[0325] To a solution of compound III (950 mg, 28.3% purity, 0.66 mmol) and intermediate-A (1.01 g, 2.32 mmol) in DMF (10 mL), DIEA (385 mg, 2.99 mmol), HOBt (358 mg, 2.65 mmol) and EDC (508 mg, 2.65 mmol) were added in sequence. The resulting reaction mixture was stirred at 25° C. for 3 h under N2 atmosphere. LC-MS showed the desired product. The reaction mixture was purified by reverse-phase prep-HPLC. The fractions containing the desired product were combined and concentrated to give compound IV (200 mg, 18.2% yield) as a white solid. 1H NMR (400 MHz DMSO-d6): δ ppm 8.03 - 8.11 (m, 3 H), 7.84 (d, J = 9.26 Hz, 3 H), 5.21 (d, J = 3.38 Hz, 3 H), 4.97 (dd, J = 11.19, 3.31 Hz, 3 H), 4.54 (d, J = 8.50 Hz, 3 H), 4.34 (br t, J = 4.88 Hz, 1 H), 4.03 (s, 9 H), 3.82 - 3.92 (m, 3 H), 3.73 - 3.81 (m, 3 H), 3.44 - 3.60 (m, 10 H), 3.38 - 3.43 (m, 8 H), 3.19 - 3.27 (m, 6 H), 3.01 - 3.07 (m, 8 H), 2.39 - 2.48 (m, 6 H), 2.10 (s, 9 H), 2.00 (s, 9 H), 1.89 (s, 9 H), 1.78 (s, 9 H), 1.55 (br s, 2 H), 1.33 - 1.44 (m, 4 H), 1.23 (br s, 4 H). LCMS: [M+2H + ] / 2, 828.0.

[0326] 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 of 3-bis(diisopropylamino)phosphanyloxypropanenitrile (145 mg, 483 umol) at 25° C. under N2. The reaction mixture was stirred at 25° C. for 2 h. LC-MS showed that compound IV was completely consumed. The reaction was quenched by adding a mixture of brine and saturated NaHCO3 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 organic layers were washed with brine / saturated aqueous NaHCO3 solution (1:1, 5 mL), dried over Na2SO4, 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 isolated by centrifugation. This process was repeated once more. The solid was then dissolved in anhydrous CH3CN and the volatiles were removed. This process was repeated three times to give compound 1 (103 mg, 45.9% yield) as a colorless oil. 1 H NMR (CDCl3): δ ppm 7.74 - 7.88 (m, 3 H), 6.70 - 7.02 (m, 3 H), 5.37 (br s, 3 H), 5.14 - 5.27 (m, 3 H), 4.77 (br d, J = 7.78 Hz, 3 H), 4.13 - 4.27 (m, 6 H), 3.95 (br s, 10 H), 3.71 - 3.82 (m, 4 H), 3.47 - 3.70 (m, 20 H), 3.42 (br s, 3 H), 3.13 - 3.29 (m, 10 H), 2.63 - 2.68 (m, 6 H), 2.15 - 2.23 (m, 9 H), 2.07 (s, 9 H), 2.02 (s, 9 H), 1.89 (s, 9 H), 1.53 - 1.77 (m, 6 H), 1.37 -1.18 (m, 16 H). 31 P NMR (CDCl3): δ ppm 147.14.

[0327] [Example 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 instead of intermediate A. 1 H NMR (CDCl3): δ ppm 8.01 - 8.09 (m, 1 H), 7.59 - 7.61 (m, 2 H), 7.21 - 7.23 (m, 1 H), 6.66 - 6.85 (m, 3 H), 5.35 (br s, 3 H), 5.06 - 5.25 (m, 3 H), 4.72 - 4.84 (m, 3 H), 4.05 - 4.25 (m, 10 H), 3.76 - 4.00 (m, 12 H), 3.46 - 3.62 (m, 32 H), 3.20 (br s, 10 H), 2.61 - 2.68 (m, 6 H), 2.16 - 2.18 (m, 9 H), 2.05 (s, 9 H), 1.96 - 2.02 (m, 18 H), 1.61 - 1.66 (m, 4 H), 1.52 (br s, 2 H), 1.36 (br s, 4 H), 1.17 - 1.19 (m, 12 H). 31 P NMR (CDCl3): δ ppm 147.07.

[0328] [Example 5] Preparation of compound 3 Scheme 5 below can be used to prepare compound 3 identified in Example 1 above.

[0329] [ka]

[0330] Scheme 5 Starting from tert-butyl (3-aminopropyl)carbamate, this was converted to benzyl-protected 2-bromoethanol (S N2 substitution) to give compound I. The Boc group can then be removed under acidic conditions to give compound II, which can be alkylated with tert-butyl 2-bromoacetate to give triester compound III. The tert-butyl protecting group can then be removed by treatment with formic acid to give triacid compound IV. Amide coupling with intermediate-A gives 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.

[0331] [Example 6] Preparation of compound 4 Scheme 6 below was used to prepare compound 4, identified in Example 1 above.

[0332] [ka]

[0333] Scheme 6 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 was removed by hydrogenation to give secondary amine compound II. Amide coupling with 6-hydroxyhexanoic acid gave compound III. The tert-butyl protecting group was then removed by treatment with HCl in dioxane to give triacid compound IV. Amide coupling between triacid compound IV and intermediate-A gave compound V. Phosphoramidite compound 4 was synthesized by phosphitylation of compound V with 2-cyanoethyl N,N-diisopropylchlorophosphoramidite and a catalytic amount of 1H-tetrazole.

[0334] N in DMF (100 mL) 1To a solution of tert-butyl 2-bromoacetate (23.7 g, 121 mmol) was added, followed by dropwise addition of DIEA (23.61 g, 182 mmol). The resulting reaction mixture was stirred at 25-30 °C for 16 h. LCMS showed N 1 The reaction mixture was diluted with H2O (500 mL) and extracted with EtOAc (500 mL x 2). The combined organic layers were washed with saturated brine (1 L), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give the crude product, which was purified by silica gel column chromatography (gradient: petroleum ether:ethyl acetate from 20:1 to 5:1). Compound I (12.1 g, 78.4% yield) was obtained as a colorless oil. 1 H NMR (400 MHz, CDCl3): δ ppm 7.26 - 7.40 (m, 5 H), 3.79 (s, 2 H), 3.43 (s, 4 H), 3.21 (s, 2 H), 2.72 (dt, J = 16.9, 7.34 Hz, 4 H), 1.70 (quintet, J = 7.2 Hz, 2 H), 1.44 - 1.50 (m, 27 H).

[0335] A dry hydrogenation bottle was purged with argon three times. Pd / C (200 mg, 10%) was added followed by MeOH (5 mL) and then a solution of compound I (1.00 g, 1.97 mmol) in MeOH (5 mL). The reaction mixture was degassed under vacuum and backfilled 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 that compound I was completely consumed. The reaction mixture was filtered under reduced pressure under an atmosphere of N2. The filtrate was concentrated under reduced pressure to give compound II (655 mg, 79.7% yield) as a yellow oil, which was used for the next step without further purification. 1H NMR (400 MHz, CDCl3): δ ppm 3.44 (s, 4 H), 3.31 (s, 2 H), 2.78 (t, J = 7.1 Hz, 2 H), 2.68 (t, J = 6.9 Hz, 2 H), 1.88 (br s, 1 H), 1.69 (quintet, J = 7.03 Hz, 2 H), 1.44 - 1.50 (s, 27 H).

[0336] A mixture of compound II (655 mg, 1.57 mmol), 6-hydroxyhexanoic acid (249 mg, 1.89 mmol), DIEA (1.02 g, 7.86 mmol), EDCI (904 mg, 4.72 mmol), and HOBt (637 mg, 4.72 mmol) in DMF (6 mL) was degassed and purged with N 3 times, then stirred at 25 °C for 3 h under N atmosphere. LCMS showed the desired product. The reaction mixture was diluted with H 2 O (10 mL) and extracted with 20 mL of EtOAc (10 mL × 2). The organic layers were combined, washed with saturated brine (20 mL), dried over anhydrous Na 2 SO 4 , filtered, and concentrated to give the crude product, which was purified by silica gel column chromatography (gradient: 5:1 to 1:1 petroleum ether:ethyl acetate) to give compound III (650 mg, 77.8% yield) as a yellow oil. 1 H NMR (400 MHz, CDCl3): δ ppm 3.90 - 3.95 (s, 2 H), 3.63 (t, J = 6.40 Hz, 2 H), 3.38 - 3.45 (m, 6 H), 2.72 (t, J = 6.65 Hz, 2 H), 2.40 (t, J = 7.28 Hz, 2 H), 1.55 - 1.75 (m, 8 H), 1.44 (s, 27 H).C 27 H 50 Calculated mass of N2O8: 530.36; Found: 531.3 (M+H + ).

[0337] 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 h. LCMS showed complete consumption of compound III. The reaction mixture was filtered, washed with EtOAc (50 mL), and dried under reduced pressure to give the crude product. It was dissolved in CH3CN (50 mL) and the volatiles were removed under vacuum. This process was repeated three times to give compound IV (2.05 g, 54.5% yield) as a white solid. 1 H NMR (400 MHz, D2O): δ ppm 4.21 (s, 1 H), 4.07 (d, J = 4.5 Hz, 4 H), 3.99 (s, 1 H), 3.45 - 3.52 (m, 3 H), 3.42 (t, J = 6.5 Hz, 1 H), 3.32 - 3.38 (m, 1 H), 3.24 - 3.31 (m, 1 H), 2.37 (t, J = 7.4 Hz, 1 H), 2.24 (t, J = 7.4 Hz, 1 H), 1.99 (dt, J = 15.5, 7.53 Hz, 1 H), 1.85 - 1.94 (m, 1 H), 1.85 - 1.94 (m, 1 H), 1.39 - 1.56 (m, 4 H), 1.19 - 1.31 (m, 2 H).

[0338] A mixture of compound IV (150 mg, 0.413 mmol), intermediate-B (693 mg, 1.45 mmol), DIEA (267 mg, 2.07 mmol), EDCI (277 mg, 1.45 mmol), and HOBt (195 mg, 1.45 mmol) in DMF (2.6 mL) was stirred at 25° C. for 3 h under N2 atmosphere. LCMS showed the desired product. The reaction mixture was purified by reverse-phase prep-HPLC to give compound V as a white solid after lyophilization (186 mg, 0.106 mmol, 25.7% yield). 1H NMR (400 MHz, CDCl3): ppm δ 7.91 - 8.13 (m, 1 H), 7.70 (br s, 1 H), 7.02 (br s, 1 H), 6.54 - 6.84 (m, 3 H), 5.27 (br d, J = 3.0 Hz, 2 H), 5.26 - 5.30 (m, 1 H), 4.99 - 5.15 (m, 3 H), 4.66 - 4.76 (m, 3 H), 3.98 - 4.17 (m, 10 H), 3.83 - 3.95 (m, 8 H), 3.63 - 3.76 (m, 4 H), 3.46 - 3.60 (m, 30 H), 3.40 (br s, 6 H), 3.12 - 3.18 (m, 4 H), 2.56 (br d, J = 7.2 Hz, 2 H), 2.22 - 2.39 (m, 2 H), 2.09 (s, 9 H), 1.98 (s, 9 H), 1.87 - 1.95 (m, 18 H), 1.69 (br d, J = 6.25 Hz, 2 H), 1.50 (br s, 2 H), 1.37 (br d, J = 7.0 Hz, 2 H).

[0339] 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)phosphanyloxypropanenitrile (124 mg, 0.412 mmol) at ambient temperature under N2. The reaction mixture was stirred at 20-25 °C for 2 h. LCMS showed that compound V was completely consumed. 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. The layers were separated. The organic layer was washed with brine / saturated aqueous NaHCO3 (1:1.5 mL), dried over Na2SO4, filtered and concentrated to a volume of approximately 1 mL. The residual solution was added dropwise to 20 mL of MTBE with stirring. This caused a white solid to precipitate. The mixture was centrifuged and the solid was collected. This process was repeated once more. The collected solid was dissolved in anhydrous CH3CN. The volatiles were removed. This process was repeated two more times to give compound 4 (106 mg, 52.8% yield) as a white solid. 1H NMR (400 MHz, CDCl3): ppm δ 7.94 - 8.18 (m, 1 H), 7.69 (br s, 1 H), 6.66 - 7.10 (m, 3 H), 5.35 (d, J = 3.5 Hz, 3 H), 5.07 - 5.25 (m, 3 H), 4.76 - 4.86 (m, 3 H), 4.01 - 4.31 (m, 10 H), 3.91 - 4.01 (m, 8 H), 3.74 - 3.86 (m, 4 H), 3.52 - 3.71 (m, 30 H), 3.42 - 3.50 (m, 6 H), 3.15 - 3.25 (m, 4H), 2.52 - 2.70 (m, 4 H), 2.22 - 2.45 (m, 2 H), 2.15 - 2.22 (s, 9 H), 2.06 (s, 9 H), 1.95 - 2.03 (m, 18 H), 1.77 (br s, 2 H), 1.58 - 1.66 (m, 4 H), 1.40 (m, 2 H), 1.08 - 1.24 (m, 12 H). 31 P NMR (CDCl3): ppm δ147.12.

[0340] [Example 7] Preparation of compound 5 Compound 5 was synthesized using the same procedure based on Scheme 6, except that intermediate A was used instead of intermediate B. 1H NMR (400 MHz, CDCl3): ppm δ 7.71 - 8.06 (m, 2 H), 7.36 - 7.49 (m, 0.5 H), 6.59 - 7.14 (m, 3 H), 6.34 - 6.43 (m, 0.5 H), 5.36 (br d, J=3.01 Hz, 3 H), 5.10 - 5.31 (m, 3 H), 4.57 - 4.85 (m, 3 H), 3.85 - 4.22 (m, 18 H), 3.29 - 3.81 (m, 30 H), 3.13 - 3.26 (m, 4 H), 2.61 - 2.68 (m, 4 H), 2.26 - 2.42 (m, 2 H), 2.13 - 2.19 (m, 9 H), 2.05 (s, 9 H), 1.97 - 2.01 (m, 9 H), 1.94 - 1.96 (m, 9 H), 1.63 (br s, 4 H), 1.35 - 1.46 (m, 2 H), 1.16 - 1.19 (m, 12 H). 31 P NMR (CDCl3): ppm δ 147.15.

[0341] [Example 8] Preparation of compound 6 Scheme 7 below can be used to prepare compound 6, identified in Example 1 above.

[0342] [ka]

[0343] Scheme 7 Starting from benzyl-protected propane-1,3-diamine (compound I), a Michael addition reaction with tert-butyl acrylate can be carried out to give triester compound II. Once compound II is synthesized, compound 6 can be synthesized by following the same procedure for synthesizing compound 4 in scheme 6 for the remaining steps.

[0344] [Example 9] Preparation of compound 7 Scheme 8 below can be used to prepare compound 7, identified in Example 1 above.

[0345] [ka]

[0346] Scheme 8 Starting from secondary amine compound I (compound II in scheme 6), Cbz protection can be used to obtain compound II. The tert-butyl group of compound II can be removed by treatment with acid to obtain triacid compound III. Compound III can be amide-coupled with intermediate-A to obtain compound IV. The Cbz protecting group of compound IV can be removed by hydrogenation to obtain secondary amine compound V, which can be reacted with glutaric anhydride to obtain carboxylic acid compound VI. The carboxylic acid of compound VI can be converted to a tetrafluorophenyl ester by standard procedures to obtain compound 7.

[0347] [Example 10] Preparation of compound 8 Scheme 9 below can be used to prepare compound 8, identified in Example 1 above.

[0348] [ka]

[0349] Scheme 9 Compound I (compound V in Scheme 8) can be reacted with the NHS-conjugated maleimide compound 2,5-dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoate to give compound 8.

[0350] [Example 11] Preparation of compound 74 Scheme 10 below can be used to prepare compound 74, identified in Example 1 above.

[0351] [ka]

[0352] Scheme 10 Starting from compound I (same as from Example 6, compound II in Scheme 6).

[0353] To a solution of compound I (275 g, 660 mmol, 1.00 equiv) in DCM (2.75 L), TEA (133 g, 1.32 mol, 2.00 equiv) was added, and then 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 h. LCMS showed that compound I was completely consumed, and one major peak with the desired mass was detected. The reaction mixture was diluted with NaHCO3 (800 mL) and extracted. The combined organic layers were washed with 500 mL of brine (500 mL x 1), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the crude product, which was purified by column chromatography (SiO2, PE / EA = 100 / 1 to 5 / 1) to give compound II (290 g, 527 mmol, 75.7% yield) as a colorless oil. 1 H NMR: 400 MHz, DMSO-d6δ ppm 7.23 - 7.40 (m, 5 H), 5.00 - 5.12 (m, 2 H), 3.86 - 3.95 (m, 2 H), 3.23 - 3.39 (m, 6 H), 2.55 - 2.67 (m, 2 H), 1.56 - 1.64 (m, 2 H), 1.31 - 1.46 (m, 27 H).

[0354] 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 h under air atmosphere. LCMS showed that compound III was completely consumed, with one major peak with the desired mass detected. The reaction was diluted with toluene and acetonitrile (ACN, 1500 mL each) and the mixture was concentrated in vacuo to azeotropically remove formic acid. 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 once more to give 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 give compound III (210 g, quantitative yield) as a white solid. 1 H NMR: 400 MHz, DMSO-d6δ ppm 7.26 - 7.40 (m, 5 H), 5.02 - 5.10 (m, 2 H), 3.89 - 4.00 (m, 2 H), 3.36 - 3.45 (m, 4 H), 3.24 - 3.34 (m, 2 H), 2.59 - 2.72 (m, 2 H), 1.40 (s, 2 H).

[0355] To a solution of compound III (100 g, 261 mmol, 1.00 equiv.), intermediate A (502 g, 915. mmol, 3.50 equiv., TFA) 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 h. LCMS showed that compound III was completely consumed, and one major peak with the desired mass was detected. The reaction mixture was added to H2O (4000 mL). The resulting mixture was extracted with MTBE (2000 mL x 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 x 2), saturated NaHCO3 (2000 mL x 2), 2000 mL of brine, dried over Na2SO4, filtered and concentrated under reduced pressure to give compound IV (260 g, 159 mmol, 60.9% yield) as a white solid. 1 H NMR: 400 MHz, DMSO-d6δ ppm 7.99 - 8.08 (m, 2 H), 7.93 (br d, J=5.50 Hz, 1 H), 7.79 - 7.86 (m, 3 H), 7.26 - 7.39 (m, 5 H), 5.22 (d, J=3.13 Hz, 3 H), 4.95 - 5.08 (m, 5 H), 4.54 (br d, J=8.38 Hz, 3 H), 4.03 (s, 9 H), 3.81 - 3.93 (m, 5 H), 3.76 (br d, J=4.88 Hz, 3 H), 3.44 - 3.62 (m, 10 H), 3.34 - 3.43 (m, 6 H), 3.24 (br d, J=6.13 Hz, 7 H), 3.02 - 3.09 (m, 4 H), 2.40 - 2.47 (m, 2 H), 2.10 (s, 9 H), 1.99 (s, 9 H), 1.89 (s, 9 H), 1.77 (s, 9 H), 1.57 - 1.68 (m, 2 H).

[0356] A 2.00 L hydrogenation bottle was purged with Ar three times and dry Pd / C (9 g) was carefully added. MeOH (50 mL) was then added to completely wet the Pd / C, followed by slow addition of a solution of compound IV (90 g, 55.1 mmol, 1.00 equiv.) and TFA (6.29 g, 55.1 mmol, 1.00 equiv.) in MeOH (850 mL) under Ar atmosphere. The resulting mixture was degassed and purged with H2 three times, and then the mixture was stirred at 25° C. for 10 h under H2 atmosphere. LCMS showed that compound IV was completely consumed and one main peak with the desired mass. The reaction mixture was carefully filtered under reduced pressure under N2 atmosphere. The filtrate was concentrated under reduced pressure to give compound V (160 g, 90.2% yield). 1H NMR: 400 MHz, DMSO-d6δ ppm 9.12 (br s, 2 H), 8.50 (br t, J=5.19 Hz, 1 H), 8.10 (br t, J=5.50 Hz, 2 H), 7.85 - 7.91 (m, 3 H), 5.22 (d, J=3.25 Hz, 3 H), 4.95 - 5.01 (m, 3 H), 4.52 - 4.58 (m, 3 H), 4.03 (s, 9 H), 3.84 - 3.93 (m, 3 H), 3.75 - 3.83 (m, 3 H), 3.39 - 3.61 (m, 17H), 3.23 - 3.32 (m, 7H), 3.15 - 3.18 (m, 3 H), 2.97 - 3.05 (m, 2 H), 2.54 - 2.61 (m, 2 H), 2.10 (s, 9 H), 2.00 (s, 9 H), 1.89 (s, 9 H), 1.77 - 1.80 (m, 9 H), 1.70 - 1.76 (m, 2 H).

[0357] 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., then 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 h. LC-MS showed that compound V was completely consumed and one major peak with the desired mass. The resulting reaction mixture was triturated with isopropyl ether twice (50 mL×2) and dried in vacuum to give compound VI (crude, 5.5 g) as a brown solid.

[0358] 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 equiv), TBTU (1.04 g, 3.23 mmol, 2.0 equiv) and DIEA (625.53 mg, 4.84 mmol, 843.03 uL, 3.0 equiv). The mixture was stirred at 25° C. for 1.0 h. LC-MS showed that compound int-D (protected (R)-3-aminopropane-1,2-diol) was completely consumed. The resulting reaction mixture was triturated with isopropyl ether (260 mL) to give the crude product. It was purified by column chromatography (SiO2, DCM / MeOH=100 / 1 to 10 / 1, 0.1% Et3N) to give compound 74 (900 mg, yield 28.0%) as a white solid.

[0359] compound 74 1H NMR: (400 MHz, DMSO-d6) δ ppm 8.06 (br d, J=6.00 Hz, 2 H), 7.83 (br d, J=8.50 Hz, 3 H), 7.66 (dt, J=10.22, 5.21 Hz, 1 H), 7.39 (br d, J=7.75 Hz, 2 H), 7.20 - 7.30 (m, 8 H), 6.87 (br d, J=8.63 Hz, 4 H), 5.21 (br d, J=3.00 Hz, 3 H), 4.98 (br dd, J=11.07, 2.81 Hz, 4 H), 4.49 - 4.59 (m, 3 H), 4.02 (br s, 9 H), 3.83 - 3.91 (m, 5 H), 3.75 - 3.80 (m, 3 H), 3.73 (s, 6 H), 3.54 - 3.59 (m, 5 H), 3.48 (br d, J=7.25 Hz, 8 H), 3.24 (br d, J=5.63 Hz, 9 H), 3.07 (br d, J=13.13 Hz, 4 H), 2.87 - 2.97 (m, 7 H), 2.43 (br d, J=7.38 Hz, 2 H), 2.30 (br d, J=6.38 Hz, 1 H), 2.16 (br d, J=7.50 Hz, 1 H), 2.09 (s, 9 H), 1.99 (s, 9 H), 1.89 (s, 9 H), 1.77 (s, 9 H), 1.65 (br dd, J=12.76, 6.25 Hz, 3 H), 1.50 - 1.57 (m, 1 H).

[0360] Compound 73 could be prepared by the procedure described for compound 74, except that protected (S)-3-aminopropane-1,2-diol was used instead of protected (R)-3-aminopropane-1,2-diol.

[0361] [Example 12] Preparation of compound 75 Scheme 11 below can be used to prepare compound 75, identified in Example 1 above.

[0362] [ka]

[0363] Scheme 11 Compound II was synthesized according to Scheme 11. Starting from compound I (compound VI in Scheme 10), compound II was obtained by coupling with piperidin-4-ol. Phosphoramidite compound 75 was synthesized by treating compound II with 2-cyanoethyl N,N-diisopropylchlorophosphoramidite and catalytic amount of 1H-tetrazole. compound 75 1 H NMR (400 MHz in DMSO-d6): δ ppm 8.05 (br d, J = 6.50 Hz, 2 H), 7.81 (br d, J=9.01 Hz, 3 H), 5.22 (d, J=3.25 Hz, 3 H), 4.98 (dd, J=11.26, 3.25 Hz, 3 H), 4.55 (br d, J=8.50 Hz, 3 H), 4.03 (s, 9 H), 3.64 - 3.97 (m, 12 H), 3.55 - 3.63 (m, 6 H), 3.50 (br s, 5 H), 3.40 (br d, J=6.13 Hz, 6 H), 3.17 - 3.30 (m, 9 H), 3.07 (br d, J=14.26 Hz, 4 H), 2.76 (t, J=5.82 Hz, 2 H), 2.18 - 2.47 (m, 6 H), 2.10 (s, 9 H), 1.99 (s, 9 H), 1.89 (s, 9 H), 1.78 (s, 9 H), 1.52 - 1.74 (m, 6 H), 1.12 - 1.19 (m, 12 H). 31P NMR (DMSO-d6): ppm δ 145.25.

[0364] [Example 13] A linker B attached to a functional group that can be linked to one or more pharmaceutical agents. It will be appreciated that the various linkers B of the present disclosure may be attached to various functional groups (W) that may be linked to one or more pharmaceutical agents. In particular, the linker B may contain a diol moiety where one alcohol is protected as a DMT ether while the other alcohol may be directly or indirectly linked to a solid phase synthesis solid support material. Removal of the DMT group produces a free alcohol that can be phosphitylated by reacting with a phosphoramidite to initiate oligonucleotide chain growth. Thus, the targeting ligand cluster of the present disclosure may be attached to the 3'-end of an oligonucleotide. A non-limiting list of linkers B of the present disclosure that may be attached to the 3'-end of an oligonucleotide includes the structures depicted below and their stereoisomers:

[0365] [ka] [In the formula, j is an integer between 0 and 12, k is an integer between 0 and 12.] Includes:

[0366] All of the target ligand clusters of the present disclosure may also bind to the 5'-end of an oligonucleotide, including but not limited to the 5'-end of the sense strand in a dsRNA, or the 5'-end of the antisense strand in a dsRNA.

[0367] [Example 14] Preparation of siRNA-conjugated ligand clusters The sense and antisense strand sequences of siRNA were synthesized on an oligonucleotide synthesizer using well-established solid-phase synthesis methods based on phosphoramidite chemistry. The growth of the oligonucleotide chain is achieved by a 4-step cycle: condensation, capping, oxidation, and deprotection steps for each nucleotide addition. Synthesis was performed on a solid support made of controlled pore glass (CPG, 1000 Å). Monomeric phosphoramidites were purchased from commercial sources. The phosphoramidite-linked ligand clusters were synthesized according to the procedures in Examples 3-12 herein. 5-Ethylthio-1H-tetrazole was used as the 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 solid support-linked oligomers were cleaved and the protecting groups were removed by treatment with a 1:1 volume solution of 40 wt% methylamine and 28% ammonium hydroxide solution in water. The crude single-stranded product was isolated by lyophilization and purified by ion-pairing reversed-phase HPLC (IP-RP-HPLC). The purified single-stranded oligonucleotide product from IP-RP-HPLC was converted to the sodium salt by dissolving in 1.0 M NaOAc and precipitating with the addition of ice-cold EtOH. Annealing of equimolar complementary sense and antisense strand oligonucleotides in water was performed to form the double-stranded siRNA product, which was lyophilized to give a fluffy white solid.

[0368] [Example 15] In vivo evaluation of GalNAc ligand clusters conjugated to siRNA To evaluate the delivery efficacy, GalNAc ligand clusters were conjugated to the 5'-end or 3'-end of the sense strand of a known active FXII siRNA from 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 are summarized in Table 1. Table 1 also shows six examples of GalNAc ligand clusters conjugated to FXII siRNA. The conjugation of GalNAc clusters to the 5'-end or 3'-end sense strand of FXII siRNA was performed as part of the solid-phase synthesis outlined in Example 14. Their structural diagrams are shown in Table 1 below. The masses of these six compounds and the positive control compound are summarized in Table 2.

[0369] These compounds were tested for their effectiveness in knocking down mouse FXII. FXII is a secreted protein that is mainly produced in hepatocytes. The reduction in FXII expression in plasma after siRNA treatment significantly correlates with the reduction in FXII mRNA in hepatocytes. Since these GalNAc ligand clusters are conjugated to the same FXII siRNA with known activity, the delivery efficacy can be evaluated and compared by measuring the degree of reduction in FXII expression in plasma for each conjugate.

[0370] Mice were given a single subcutaneous injection of siRNA compounds (see Table 1 below) or PBS at 0.5 or 1 mg / kg. 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 7, 14 and / or 28 days after dosing. Mouse FXII protein concentrations were measured by ELISA assay according to 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 as the percentage reduction of FXII protein in mouse plasma normalized to the PBS-treated group and is summarized in Table 3. FXII siRNA conjugated with GalNAc ligands GLS-1 and GLS-2 showed significant activity in knocking down mouse FXII protein expression in mouse plasma at both dosages on days 7, 14, and / or 28 post-dosing. This activity compared favorably with the positive control. The data confirm that GalNAc ligand clusters based on a diamine scaffold attached to the 5'-end of the sense strand are highly effective in delivering siRNA to hepatocytes in vivo.

[0371] [Table 1]

[0372] [Table 2]

[0373] [ka] [ka]

[0374] [Table 3]

[0375] [Example 16] In vivo evaluation of GalNAc ligand clusters conjugated to siRNA Mice were given a single subcutaneous injection of siRNA compounds at 1 mg / kg or PBS. Plasma samples were collected 14 days after dosing. Mouse FXII protein concentrations were measured by ELISA assay according to 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 as the percentage reduction of FXII protein in mouse plasma normalized to the PBS-treated group and is summarized in Table 4. FXII siRNA conjugated with GalNAc ligands GLS-1 and GLS-2 showed significant activity in knocking down mouse FXII protein expression in mouse plasma. The data confirm that GalNAc ligand clusters based on a diamine scaffold attached to the 5'-end of the sense strand are highly effective in delivering siRNA to hepatocytes in vivo.

[0376] It has also been found that when used for targeted delivery of pharmaceuticals, such as compounds AD00448, AD00449, the linker B exhibits better in vivo stability and activity when it contains a six-membered ring fragment, particularly a 4-hydroxypiperidinyl group.

[0377] [Table 4]

[0378] [Example 17] In vivo evaluation of GalNAc ligand clusters conjugated to siRNA AD00831 Mice were given a single subcutaneous injection of siRNA compounds or PBS at 2 mg / kg. Plasma samples were collected 7 and 14 days after dosing. Mouse FXII protein concentrations were measured by ELISA assay according to 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 as the percentage reduction of FXII protein in mouse plasma normalized to the PBS-treated group. The percentage of knockdown was 87% and 88% at 7 and 14 days after dosing. The data confirm that GalNAc ligand clusters based on diamine scaffolds attached to the 3'-end of the sense strand are highly effective in delivering siRNA to hepatocytes in vivo.

[0379] [Example 18] In vivo testing of ANGPTL3 siRNA duplexes 14 days before siRNA dosing, female C57BL / 6J mice were infected by intravenous administration of a solution of adeno-associated virus 8 (AAV8) vectors encoding human ANGPTL3 and luciferase genes. On day 0, mice were subcutaneously administered a single dose of AD00112-2 (Table 5) at 1, 3, or 10 mg / kg or PBS. Blood samples were collected before siRNA dosing on day 0 and at the end of day 7. Serum samples were isolated and luciferase activity of serum samples was measured by the manufacturer's recommended protocol. Since the expression level of human ANGPTL3 correlates with the expression level of luciferase, measuring luciferase activity is a surrogate for measuring ANGTPL3 expression. The remaining percent luciferase activity was calculated by comparing the luciferase activity in samples from each mouse before (day 0) and after (day 7) siRNA treatment, and normalized by the change in luciferase activity in samples from control-treated mice during the same period. The results are summarized in Table 6. AD00112-2 demonstrated dose-dependent activity in suppressing ANGPTL3 expression, again confirming that GalNAc ligand clusters based on the diamine scaffold are highly effective in delivering siRNA to hepatocytes in vivo.

[0380] [Table 5]

[0381] [Table 6]

[0382] Equivalent Although several embodiments of the invention have been described and illustrated herein, those skilled in the art will readily envision various other means and / or structures for performing the functions and / or obtaining one or more of the results and / or advantages described herein, and each of such variations and / or modifications is considered to be within the scope of the invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are intended to be exemplary, and that the actual parameters, dimensions, materials, and configurations will depend on the specific use or application in which the teachings of the invention are used. Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Thus, it should be understood that the above-described embodiments are provided 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 of such features, systems, articles, materials, and / or methods is included within the scope of the invention, if such features, systems, articles, materials, and / or methods are not mutually inconsistent.

[0383] All definitions and as 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.

[0384] The indefinite articles "a" and "an," as used in the specification and claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

[0385] The term "and / or" as used in this specification and claims should be understood to mean "either or both" of the elements so conjoined, i.e., conjunctively present in some cases and disjointly present in other cases. Other elements may optionally be present other than the elements specifically identified by the "and / or" clause, whether associated or unassociated with the specifically identified element, unless expressly indicated to the contrary.

[0386] All references, patents and patent applications, and publications cited or referred to in this application are hereby incorporated by reference in their entirety.

Claims

1. 1. A compound for targeted delivery of one or more pharmaceutical agents, comprising the formula: 【Chemical 1】 [In the formula, each TL is an independently selected target ligand; m is an integer between 1 and 10; each n is an independently selected integer between 1 and 10; Each linker A is an independently selected spacer, Linker B is a spacer, W is either one or more pharmaceutical agents or a functional group that can be linked to one or more pharmaceutical agents. A compound having the formula:

2. 2. The compound of claim 1, wherein m is 1 or 2.

3. The compound of claim 1 or 2, wherein at least one of the independently selected TLs is capable of binding to one or more cellular receptors, cellular channels, and cellular transporters that can promote cellular uptake.

4. At least one of the independently selected TLs is 1) at least one small molecule ligand; 2) at least one peptide, or 3) at least one aptamer 4. The compound of claim 3, comprising:

5. 5. The compound of claim 4, wherein the at least one small molecule comprises at least one of N-acetylgalactosamine, galactose, galactosamine, N-formyl-galactosamine, N-propionylgalactosamine, N-butanoylgalactosamine, and N-iso-butanoylgalactosamine, a macrocycle, a folate molecule, a fatty acid, a bile acid, and cholesterol.

6. 5. The compound of claim 4, wherein at least one of the independently selected TLs comprises at least one cyclic peptide.

7. 7. The compound of any one of claims 3 to 6, wherein at least one of the independently selected TLs is capable of binding to at least one asialoglycoprotein receptor (ASGPR), at least one transferrin receptor, at least one integrin receptor, at least one folate receptor, or at least one G protein-coupled receptor (GPCR).

8. 8. The compound of claim 1, wherein at least one independently selected linker A comprises at least one of polyethylene glycol, an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an alkynyl group, an aryl group, an aralkyl group, an aralkenyl group, and an aralkynyl group.

9. At least one of the independently selected linkers A is a) at least one heteroatom; b) at least one aliphatic heterocycle; c) at least one heteroaryl group; d) at least one amino acid; e) at least one nucleotide, and f) at least one sugar 9. The compound of claim 1, comprising one or more groups selected from the group consisting of:

10. a) the at least one heteroatom comprises at least one of oxygen, nitrogen, sulfur, or phosphorus; b) the at least one aliphatic heterocycle comprises at least one of tetrahydrofuran, tetrahydropyran, morpholine, piperidine, piperazine, pyrrolidine, and azetidine; c) at least one heteroaryl group comprises at least one of imidazole, pyrazole, pyridine, pyrimidine, triazole, and 1,2,3-triazole; or d) the at least one sugar comprises at least one of glucose, fructose, mannose, galactose, ribose, and glucosamine; The compound of claim 9.

11. At least one of the independently selected linkers A is 【Chemistry 2】 [In the formula, p is an integer between 0 and 12; pp is an integer between 0 and 12, q is an integer between 1 and 12; qq is an integer between 1 and 12.

2. The compound of claim 1, comprising one or more of:

12. 12. The compound of claim 1, wherein linker B comprises at least one of polyethylene glycol, an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an alkynyl group, an aryl group, an aralkyl group, an aralkenyl group, and an aralkynyl group.

13. Linker B is a) at least one heteroatom; b) at least one aliphatic heterocycle; c) at least one heteroaryl group; d) at least one amino acid; e) at least one nucleotide, and f) at least one sugar 13. The compound of any one of claims 1 to 12, comprising one or more groups selected from the group consisting of:

14. a) the at least one heteroatom comprises at least one of oxygen, nitrogen, sulfur, and phosphorus; b) the at least one aliphatic heterocycle comprises at least one of tetrahydrofuran, tetrahydropyran, morpholine, piperidine, piperazine, pyrrolidine, and azetidine; c) at least one heteroaryl group comprises at least one of imidazole, pyrazole, pyridine, pyrimidine, triazole, and 1,2,3-triazole; d) at least one nucleotide comprises at least one of an abasic nucleotide and an inverted abasic nucleotide; or e) the at least one sugar comprises at least one of glucose, fructose, mannose, galactose, ribose, and glucosamine; The compound of claim 13.

15. a) the abasic nucleotide is abasic deoxyribonucleic acid (DNA) or abasic ribonucleic acid (RNA), or b) the inverted abasic nucleotide is an inverted abasic deoxyribonucleic acid (DNA) or an inverted abasic ribonucleic acid (RNA); 15. The compound of claim 14.

16. Linker B is 【Chemistry 3-1】 【Chemistry 3-2】 [In the formula, j is an integer between 1 and 12; k is an integer between 0 and 12.

16. The compound of claim 1, comprising at least one of:

17. The linker B-W is 【Chemistry 4】 [In the formula, j is an integer between 0 and 12; k is an integer between 0 and 12.

17. The compound of any one of claims 1 to 16, wherein:

18. W is, a) a hydroxy group, b) a protected hydroxy group; c) Formula: 【Chemistry 5】 [In the formula, R ａ is a C1-C6 alkyl, C3-C6 cycloalkyl, isopropyl group, or R ａ is connected to R via a nitrogen atom ｂ Together with this, they form a ring, R ｂ is a C1-C6 alkyl, C3-C6 cycloalkyl, isopropyl group, or R ｂ is connected to R via a nitrogen atom ａ Together with this, they form a ring, R ｃ is a phosphite protecting group, a phosphate protecting group, or a 2-cyanoethyl group. a phosphoramidite group having the formula d) a carboxyl group, e) Formula: 【Chemistry 6】 [Wherein, X is a leaving group.] an activated carboxyl group having f) Michael acceptors, g) oligonucleotides, h) Formula: 【Chemistry 7】 [In the formula, Linker C is absent or is a spacer attached to the 3' or 5' end of the oligonucleotide; X is a methyl group, oxygen, sulfur, or amino group; Y is oxygen, sulfur, or an amino group. i) Formula: 【Chemistry 8】 wherein the linker C is a spacer attached to the 3' or 5' end of the oligonucleotide. j) Formula: 【Chemistry 9】 wherein the linker C is a spacer attached to the 3' or 5' end of the oligonucleotide; or k) one or more pharmaceutical products 18. The compound of any one of claims 1 to 17, wherein

19. 19. The compound of claim 18, wherein the protected hydroxy group is protected using at least one of 4,4'-dimethoxytrityl (DMT), monomethoxytrityl (MMT), 9-(p-methoxyphenyl)xanthen-9-yl (Mox), and 9-phenylxanthen-9-yl (Px).

20. the phosphite protecting group comprises at least one of methyl, allyl, 2-cyanoethyl, 4-cyano-2-butenyl, 2-cyano-1,1-dimethylethyl, 2-(trimethylsilyl)ethyl, 2-(S-acetylthio)ethyl, 2-(S-pivaloylthio)ethyl, 2-(4-nitrophenyl)ethyl, 2,2,2-trichloroethyl, 2,2,2-trichloro-1,1-dimethylethyl, 1,1,1,3,3,3-hexafluoro-2-propyl, fluorenyl-9-methyl, 2-chlorophenyl, 4-chlorophenyl, and 2,4-dichlorophenyl; or the phosphate protecting group comprises at least one of methyl, allyl, 2-cyanoethyl, 4-cyano-2-butenyl, 2-cyano-1,1-dimethylethyl, 2-(trimethylsilyl)ethyl, 2-(S-acetylthio)ethyl, 2-(S-pivaloylthio)ethyl, 2-(4-nitrophenyl)ethyl, 2,2,2-trichloroethyl, 2,2,2-trichloro-1,1-dimethylethyl, 1,1,1,3,3,3-hexafluoro-2-propyl, fluorenyl-9-methyl, 2-chlorophenyl, 4-chlorophenyl, and 2,4-dichlorophenyl; 19. The compound of claim 18.

21. 19. The compound of claim 18, wherein the leaving group is selected from the group consisting of carboxylate, sulfonate, chloride, phosphate, imidazole, hydroxybenzotriazole (HOBt), N-hydroxysuccinimide (NHS), tetrafluorophenol, pentafluorophenol, and para-nitrophenol.

22. The Michael acceptor has the formula: 【Chemistry 10】 [In the formula, E is an electron-withdrawing group; R ｄ is hydrogen or a C1-C6 alkyl substituent on the olefin.

19. The compound of claim 18, having the formula:

23. the electron-withdrawing group is a carboxamide or an ester, or E and the carbon-carbon double bond are part of a maleimide; 23. The compound of claim 22.

24. a) the oligonucleotide is a single-stranded oligonucleotide; b) the oligonucleotide is a double-stranded oligonucleotide; c) the oligonucleotide comprises at least three independently selected nucleotides; d) the oligonucleotide comprises between 16 and 23 independently selected nucleotides; e) the oligonucleotide comprises about 100 independently selected nucleotides; or f) the oligonucleotide comprises up to 14,000 independently selected nucleotides; 19. The compound of claim 18.

25. 25. The compound of claim 24, wherein the linker C of formula 5 comprises at least one heterocyclic compound.

26. 26. The compound of claim 25, wherein the heterocyclic compound is an abasic nucleotide or an inverted abasic nucleotide.

27. The linker C of formula 6 is a) at least one of polyethylene glycol (PEG), an alkyl group, and a cycloalkyl group; b) at least one heteroatom; c) at least one aliphatic heterocycle; d) at least one heteroaryl group; e) at least one amino acid, and f) at least one nucleotide 20. The compound of claim 18, comprising one or more groups selected from the group consisting of:

28. a) the at least one heteroatom comprises at least one of oxygen, nitrogen, sulfur, and phosphorus; b) the at least one aliphatic heterocycle comprises at least one of tetrahydrofuran, tetrahydropyran, morpholine, piperidine, piperazine, pyrrolidine, and azetidine; c) at least one heteroaryl group comprises at least one of imidazole, pyrazole, pyridine, pyrimidine, triazole, and 1,2,3-triazole; d) at least one nucleotide comprises at least one of an abasic nucleotide and an inverted abasic nucleotide; or e) the at least one sugar comprises at least one of glucose, fructose, mannose, galactose, ribose, and glucosamine; 28. The compound of claim 27.

29. the abasic nucleotide is abasic deoxyribonucleic acid (DNA) or abasic ribonucleic acid (RNA); or The inverted abasic nucleotide is an inverted abasic deoxyribonucleic acid (DNA) or an inverted abasic ribonucleic acid (RNA); 29. The compound of claim 28.

30. 20. The compound of claim 18, wherein the linker C of formula 7 comprises at least one of polyethylene glycol (PEG), an alkyl group, and a cycloalkyl group.

31. Linker C is 【Chemistry 11】 [In the formula, j is an integer between 1 and 12; k is an integer between 0 and 12.

31. The compound of claim 27 or 30, comprising one or more of: [Request 32] [Chemical 12-1] 【Chemistry 12-2】 【Chemistry 12-3】 【Chemistry 12-4】 【Chemistry 12-5】 【Chemistry 12-6】 【Chemistry 12-7】 【Chemistry 12-8】 【Chemistry 12-9】 【Chemistry 12-10】 【Chemistry 12-11】 【Chemistry 12-12】 【Chemistry 12-13】 【Chemistry 12-14】 【12-15】 【12-16】 【12-17】 【12-18】 【Chemistry 12-19】 【12-20】 【12-21】 【Chemistry 12-22】 【12-23】 【12-24】 【12-25】 32. The compound of any one of claims 1 to 31, selected from the group consisting of:

33. 32. The compound of claim 1, which is a stereoisomer of one of compounds 1-75 of claim 32.

34. 20. The compound of claim 18, wherein the one or more pharmaceutical agents comprise at least one of small interfering RNA (siRNA), single-stranded siRNA, double-stranded siRNA, small activating RNA, RNAi, microRNA (miRNA), antisense oligonucleotide, short guide RNA (gRNA), single guide RNA (sgRNA), messenger RNA (mRNA), ribozyme, plasmid, immunostimulatory nucleic acid, antagomir, and aptamer.

35. The double-stranded siRNA is a) the double-stranded siRNA comprises at least one modified ribonucleotide; b) substantially all ribonucleotides of the double-stranded siRNA are modified; c) all ribonucleotides of the double-stranded siRNA are modified; d) at least one strand of the double-stranded siRNA contains at least one phosphorothioate bond; e) at least one strand of the double-stranded siRNA contains up to six phosphorothioate linkages; f) the double-stranded siRNA comprises at least one locked nucleic acid; g) the double-stranded siRNA comprises at least one unlocked nucleic acid; and h) the double-stranded siRNA comprises at least one glycerol nucleic acid; 35. The compound of claim 34, having one or more features selected from the group consisting of:

36. 36. The compound of claim 34 or 35, wherein the modified ribonucleotide comprises a 2'-O-methyl nucleotide, a 2'-fluoro nucleotide, a 2'-deoxy nucleotide, a 2'3'-seconucleotide mimic, a locked nucleotide, a 2'-F-arabino nucleotide, a 2'-methoxyethyl nucleotide, an abasic nucleotide, ribitol, an inverted nucleotide, an inverted abasic nucleotide, an inverted 2'-OMe nucleotide, an inverted 2'-deoxy nucleotide, a 2'-amino-modified nucleotide, a 2'-alkyl-modified nucleotide, a morpholino nucleotide, and a 3'-OMe nucleotide, a nucleotide containing a 5'-phosphorothioate group, or a 5'-(E)-vinyl phosphonate nucleotide (antisense strand only), or a terminal nucleotide linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group, a 2'-amino-modified nucleotide, a 2'-alkyl-modified nucleotide, a phosphoramidate, or a non-natural base containing nucleotide.

37. 37. A pharmaceutical composition comprising a compound according to any one of claims 1 to 36.

38. 37. A method for making a compound of any one of claims 1 to 36 for targeted delivery of one or more pharmaceutical 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; coupling a plurality of protected carboxylic acids with a first compound to produce a second compound, wherein a first nitrogen in the second compound is a tertiary amine comprising a first protected carboxylic acid and a second protected carboxylic acid, and a second nitrogen of the second compound is a tertiary amine comprising a protecting group and a third protected carboxylic acid; deprotecting the second nitrogen of the second compound so that the second nitrogen becomes a secondary amine containing a third protected carboxylic acid, thereby producing a third compound; producing a fourth compound by attaching a hydroxy-containing moiety to a second nitrogen of the third compound such that the second nitrogen becomes a tertiary amine or amide containing a third protected carboxylic acid and a hydroxy-containing moiety; converting the protected carboxylic acid of the fourth compound to a carboxylic acid to produce a fifth compound; and performing an amide coupling reaction with the fifth compound to produce a sixth compound, wherein the first nitrogen in the sixth compound is a tertiary amine comprising a first amide and a second amide, and the second nitrogen in the sixth compound is a tertiary amine comprising a moiety comprising a hydroxy group and a third amide, and wherein the first amide, the second amide, and the third amide are each coupled to an independently selected target ligand. A method comprising:

39. 37. A method for making a compound of any one of claims 1 to 36 for targeted delivery of one or more pharmaceutical 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 and the second nitrogen being an amine comprising a second protecting group; coupling a first protected carboxylic acid to a first nitrogen of the first compound such that the first nitrogen becomes a tertiary amine, thereby producing a second compound; removing the first protecting group from the first nitrogen of the second compound to produce a third compound comprising a first nitrogen and a second nitrogen, wherein the first nitrogen is a secondary amine comprising a first protected carboxylic acid and the second nitrogen is an amine comprising a second protecting group; coupling a second protected carboxylic acid to the first nitrogen of the third compound such that the first nitrogen becomes a tertiary amine, thereby producing a fourth compound; removing the second protecting group from the fourth compound to produce a fifth compound comprising a first nitrogen and a second nitrogen, wherein 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 primary amine; coupling a third protected carboxylic acid to the second nitrogen of the fifth compound such that the second nitrogen becomes a secondary amine, thereby producing a sixth compound; producing a seventh compound by attaching a moiety containing a hydroxy group to a second nitrogen of the sixth compound such that the second nitrogen becomes a tertiary amine; converting the third protected carboxylic acid of the seventh compound to the first carboxylic acid to produce an eighth compound; performing an amide coupling reaction with the eighth compound to produce a ninth compound, wherein a first nitrogen of the ninth compound comprises a first protected carboxylic acid and a second protected carboxylic acid, and a second nitrogen of the ninth compound comprises a first amide having coupled thereto the first targeting ligand and a moiety comprising a hydroxy group; converting the second protected carboxylic acid of the ninth compound to a second carboxylic acid to produce a tenth compound; performing an amide coupling reaction with the tenth compound to produce an eleventh compound, wherein a first nitrogen of the eleventh compound comprises a first protected carboxylic acid and a second amide having a second targeting ligand coupled thereto, and a second nitrogen of the eleventh compound comprises a moiety comprising the first amide and a hydroxy group having the first targeting ligand coupled thereto; converting the first protected carboxylic acid of the eleventh compound to a third carboxylic acid to produce a twelfth compound; and performing an amide coupling reaction with the twelfth compound to produce a thirteenth compound, wherein the first nitrogen of the thirteenth compound comprises a second amide having a second targeting ligand coupled thereto and a third amide having a third targeting ligand coupled thereto, and the second nitrogen of the thirteenth compound comprises the first amide having the first targeting ligand coupled thereto and a moiety comprising a hydroxy group. A method comprising:

40. 38. The pharmaceutical composition of claim 37, which is used to deliver a pharmaceutical agent to a subject.

41. 2. The compound of claim 1, wherein n is 1 or 2.

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