KR20240082358A - Multivalent Ligand Clusters with Diamine Scaffolds for Targeted Delivery of Therapeutics - Google Patents

Abstract

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

Description

Multivalent Ligand Clusters with Diamine Scaffolds for Targeted Delivery of Therapeutics

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

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

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

where each TL is an independently selected targeting ligand, m is an independently selected integer from 1 to 10, each n is an independently selected integer from 1 to 10, each LinkerA is an independently selected spacer, and LinkerB is a spacer. and W is 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.

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

In some embodiments, at least one of the independently selected TLs is capable of binding to one or more cell receptors, cell channels, and cell transporters that can facilitate endocytosis. 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 is N-acetylgalactosamine, galactose, galactosamine, N-formyl-galactosamine, N-propionylgalactosamine, N-butanoylgalactosamine, and It includes at least one of N-iso-butanoylgalactosamine, macrocycle, folate molecule, fatty acid, bile acid, and cholesterol. In some embodiments, at least one of the independently selected TLs comprises at least one peptide. In some embodiments, at least one of the independently selected TLs comprises at least one cyclic peptide. In some embodiments, at least one of the independently selected TLs comprises at least one aptamer. In some embodiments, at least one of the independently selected TLs is capable of binding to at least one asialoglycoprotein receptor (ASGPR). In some embodiments, at least one of the independently selected TLs is capable of binding at least one transferrin receptor. In some embodiments, at least one of the independently selected TLs is capable of binding to at least one integrin receptor. In some embodiments, at least one of the independently selected TLs is capable of binding at least one folate receptor. In some embodiments, at least one of the independently selected TLs is capable of binding to at least one G-protein-coupled receptor (GPCR).

In some embodiments, at least one of the independently selected Linkers A is 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 It contains at least one type of 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 includes 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 includes at least one of tetrahydrofuran, tetrahydropyran, morpholine, piperidine, piperazine, pyrrolidine, and azetidine. In some embodiments, at least one of the independently selected Linkers A comprises at least one heteroaryl group. In some embodiments, the at least one heteroaryl group includes 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 saccharide. In some embodiments, the at least one saccharide includes at least one of glucose, fructose, mannose, galactose, ribose, and glucosamine. In some embodiments, at least one of the independently selected Linkers A comprises one or more of the following:

Here, p is an integer from 0 to 12, pp is an integer from 0 to 12, q is an integer from 1 to 12, and qq is an integer from 1 to 12.

In some embodiments, Linker B is polyethylene glycol, at least one of 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. Includes species. In some embodiments, Linker B includes at least one heteroatom. In some embodiments, the at least one heteroatom includes 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 includes at least one of tetrahydrofuran, tetrahydropyran, morpholine, piperidine, piperazine, pyrrolidine, and azetidine. In some embodiments, Linker B includes at least one heteroaryl group. In some embodiments, the at least one heteroaryl group includes 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 saccharide. In some embodiments, the at least one saccharide includes at least one of glucose, fructose, mannose, galactose, ribose, and glucosamine. In some embodiments, Linker B comprises at least one of:

Here, j is an integer from 1 to 12, and k is an integer from 0 to 12.

In some embodiments, LinkerB-W is:

Here, j is an integer from 0 to 12, and k is an integer from 0 to 12.

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 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 phosphoramidite group having the formula:

here:

R ^a is C1 to C6 alkyl, C3 to C6 cycloalkyl, isopropyl group, or R ^a is connected to R ^b through a nitrogen atom to form a cycle,

R ^b is C1 to C6 alkyl, C3 to C6 cycloalkyl, isopropyl group, or R ^b is connected to R ^a through a nitrogen atom to form a cycle,

R ^c is a phosphite protecting group, a phosphate protecting group, or a 2-cyanoethyl group.

In some embodiments, the phosphite protecting group is 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-dichloro Contains at least one type of phenyl. In some embodiments, the phosphate protecting group is 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 Includes at least one of the following.

In some embodiments, W is a carboxyl group. In some embodiments, W is an activated carboxyl group having the formula:

Here, X is a leaving group. In some embodiments, the leaving group is carboxylate, sulfonate, chloride, phosphate, imidazole, hydroxybenzotriazole (HOBt), N-hydroxysuccinimide (NHS), tetrafluorophenol, pentafluorophenol and para-nitrophenol.

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

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

In some embodiments, W is an oligonucleotide. In some embodiments, the oligonucleotide is a single-stranded oligonucleotide. In some embodiments, the oligonucleotide is a double-stranded oligonucleotide. In some embodiments, the oligonucleotide comprises at least three independently selected nucleotides. In some embodiments, the oligonucleotide comprises 16 to 23 independently selected nucleotides. In some embodiments, the oligonucleotide comprises about 100 independently selected nucleotides. In some embodiments, oligonucleotides comprise up to 14,000 independently selected nucleotides.

In some embodiments, W is:

here:

Linker C is a spacer that is absent or 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 amino group.

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

In some embodiments, W is:

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

Here, j is an integer from 1 to 12, and k is an integer from 0 to 12.

In some embodiments, W is:

Here, Linker C is a spacer attached to the 3' or 5' end of the oligonucleotide. In some embodiments, Linker C includes at least one of polyethylene glycol (PEG), an alkyl group, and a cycloalkyl group. In some embodiments, Linker C comprises one or more of the following:

Here, j is an integer from 1 to 12, and k is an integer from 0 to 12.

In some embodiments, the compound is selected from the group consisting of:

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

In some embodiments, W is one or more pharmaceutical agents. In some embodiments, one or more pharmaceutical agents include small interfering RNA (siRNA), single-stranded siRNA, double-stranded siRNA, small activating RNA, RNAi, microRNA (miRNA), antisense oligonucleotide, short guide RNA (gRNA), single It includes at least one of guide RNA (sgRNA), messenger RNA (mRNA), ribozyme, plasmid, immune-stimulating nucleic acid, antagomir, and aptamer. In some embodiments, the double-stranded siRNA includes at least one modified ribonucleotide. In some embodiments, each strand of 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 a double-stranded siRNA are modified. In some embodiments, the modified ribonucleotide is 2'-O-methyl nucleotide, 2'-fluoro nucleotide, 2'-deoxy nucleotide, 2'3'-seco nucleotide mimetic, lock 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 nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, and 3'-OMe nucleotides, nucleotides containing a 5'-phosphorothioate group, or 5'-(E)-vinyl phosphonate nucleotides ( antisense strand alone), or a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, a 2'-amino-modified nucleotide, a 2'-alkyl-modified nucleotide, a phosphoramidate, or a non-natural base. Contains nucleotides. In some embodiments, at least one strand of the double-stranded siRNA comprises at least one phosphorothioate linkage. In some embodiments, at least one strand of the double-stranded siRNA comprises up to 6 phosphorothioate linkages. In some embodiments, the double-stranded siRNA includes at least one locked nucleic acid. In some embodiments, the double-stranded siRNA includes at least one unlocked nucleic acid. In some embodiments, the double-stranded siRNA comprises at least one glycerol nucleic acid.

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

A further aspect of the disclosure relates to compositions for targeted delivery of one or more pharmaceutical agents, wherein the composition comprises any of the compounds described above, and wherein W is one or more pharmaceutical agents. In some embodiments, one or more pharmaceutical agents include 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, a double-stranded siRNA includes at least one modified ribonucleotide on one or both strands of the siRNA. In some embodiments, each strand of double-stranded siRNA is 19-23 nucleotides long. In some embodiments, substantially all ribonucleotides of the double-stranded siRNA are modified. In some embodiments, all ribonucleotides of a double-stranded siRNA are modified. In some embodiments, the modified ribonucleotide is 2'-O-methyl nucleotide, 2'-fluoro nucleotide, 2'-deoxy nucleotide, 2'3'-seco nucleotide mimetic, lock 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 nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, and 3'-OMe nucleotides, nucleotides containing a 5'-phosphorothioate group, or 5'-(E)-vinyl phosphonate nucleotides ( antisense strand alone), or a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, a 2'-amino-modified nucleotide, a 2'-alkyl-modified nucleotide, a phosphoramidate, or a non-natural base. Contains nucleotides. In some embodiments, at least one strand of the double-stranded siRNA comprises at least one phosphorothioate linkage. In some embodiments, at least one strand of the double-stranded siRNA comprises up to 6 phosphorothioate linkages. In some embodiments, the double-stranded siRNA includes 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.

Another aspect of the disclosure relates to pharmaceutical compositions comprising any of the compositions described above. In some embodiments, the pharmaceutical composition includes one or more therapeutic agents. In some embodiments, the pharmaceutical composition includes a pharmaceutically acceptable carrier.

A further aspect of the disclosure relates to a method of preparing a compound for targeted delivery of one or more pharmaceutical agents, wherein the method comprises: receiving a first compound comprising a diamine, wherein the diamine A step comprising a first nitrogen and a second nitrogen, wherein the first nitrogen is a primary amine and the second nitrogen is a secondary amine containing a protecting group; preparing a second compound by coupling a plurality of protected carboxylic acids to the first compound, wherein the first nitrogen in the second compound comprises the first protected carboxylic acid and the second protected carboxylic acid. is a tertiary amine, and the second nitrogen in the second compound is a tertiary amine comprising a protecting group and a third protected carboxylic acid; preparing a third compound by deprotecting the second nitrogen of the second compound such that the second nitrogen becomes a secondary amine comprising a third protected carboxylic acid; A fourth compound is formed by attaching a moiety containing a hydroxy group to the second nitrogen of a third compound such that the second nitrogen is a tertiary amine or amide containing a third protected carboxylic acid and a moiety containing a hydroxy group. Preparing a compound; preparing a fifth compound by converting the protected carboxylic acid of the fourth compound to a carboxylic acid; and preparing a sixth compound by performing an amide coupling reaction using 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 sixth compound The second nitrogen in is a tertiary amine comprising a moiety comprising a hydroxy group and a third amide, wherein each of the first amide, second amide and third amide is coupled to an independently selected targeting ligand. step.

In some embodiments, the protecting group is selected from the group consisting of a benzyl group and a triphenylmethyl group. In some embodiments, preparing the second compound includes performing a S _N 2 substitution reaction using the first compound. In some embodiments, preparing the second compound includes performing a reductive amination reaction using the first compound. In some embodiments, preparing the second compound includes performing a Michael addition reaction using the first compound. In some embodiments, the protecting group is a benzyl group, and preparing the third compound includes performing a hydrogenation reaction using the second compound. In some embodiments, the protecting group is a triphenylmethyl group, and preparing the third compound includes reacting the second component with at least one acid. In some embodiments, preparing the fourth compound includes performing an S _N 2 substitution reaction using the third compound. In some embodiments, preparing the fourth compound includes performing a reductive amination reaction using the third compound. In some embodiments, preparing the fourth compound includes performing a Michael addition reaction using the third compound. In some embodiments, preparing the fourth compound includes performing an amide coupling reaction using the third compound. In some embodiments, preparing the fourth compound includes 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, preparing the fifth compound includes reacting the fourth compound with at least one acid. In some embodiments, the at least one acid includes at least one of hydrochloric acid, hydrobromide acid, trifluoroacetic acid, and formic acid. In some embodiments, preparing the fifth compound includes performing a hydrogenation reaction using the fourth compound. In some embodiments, preparing the fifth compound includes performing a hydrolysis reaction using the fourth compound. In some embodiments, the first amide, second amide, and third amide are each coupled to an independently selected targeting ligand using any of the independently selected LinkerA 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 a hydroxy group to a phosphoramidite group using a phosphorylation reaction. In some embodiments, converting the hydroxy group to a phosphoramidite group is performed after performing an amide coupling reaction to prepare the sixth compound.

Another aspect of the disclosure relates to a method of preparing a compound for targeted delivery of one or more pharmaceutical agents, wherein the method comprises: receiving a first compound comprising a diamine, wherein the diamine includes a first nitrogen and a second nitrogen, the first nitrogen is a secondary amine containing a first protecting group, and the second nitrogen is an amine containing a second protecting group; preparing a second compound by coupling the first protected carboxylic acid to the first nitrogen of the first compound such that the first nitrogen is a tertiary amine; removing the first protecting group from the first nitrogen of the second compound to prepare 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 containing a second protecting group; preparing a fourth compound by coupling the second protected carboxylic acid to the first nitrogen of the third compound such that the first nitrogen is a tertiary amine; removing the second protecting group from the fourth compound to produce a fifth compound comprising a first nitrogen and a second nitrogen, wherein the first nitrogen comprises a first protected carboxylic acid and a second protected carboxylic acid. is a tertiary amine, and the second nitrogen is a primary amine; preparing a sixth compound by coupling the third protected carboxylic acid to the second nitrogen of the fifth compound such that the second nitrogen is a secondary amine; preparing a seventh compound by attaching a moiety comprising a hydroxy group to the second nitrogen of the sixth compound such that the second nitrogen is a tertiary amine; preparing an eighth compound by converting the third protected carboxylic acid of the seventh compound to a first carboxylic acid; Preparing a ninth compound by performing an amide coupling reaction using the eighth compound, wherein the first nitrogen of the ninth compound includes a first protected carboxylic acid and a second protected carboxylic acid, wherein the second nitrogen of compound 9 comprises a moiety comprising a first amide and hydroxy group with a first targeting ligand coupled thereto; preparing a tenth compound by converting the second protected carboxylic acid of the ninth compound to a second carboxylic acid; Preparing an eleventh compound by performing an amide coupling reaction using the tenth compound, wherein the first nitrogen of the eleventh compound is a second nitrogen having a first protected carboxylic acid and a second targeting ligand coupled thereto. comprising an amide, wherein the second nitrogen of the eleventh compound comprises a moiety comprising a hydroxy group and a first amide having a first targeting ligand coupled thereto; preparing a twelfth compound by converting the first protected carboxylic acid of the eleventh compound to a third carboxylic acid; and preparing a thirteenth compound by performing an amide coupling reaction using the twelfth compound, wherein the first nitrogen of the thirteenth compound is a second amide having a second targeting ligand coupled thereto and a second amide coupled thereto. 3 comprising a third amide having a targeting ligand, and wherein the second nitrogen of the thirteenth compound comprises a moiety comprising a hydroxy group and a first amide having a first targeting ligand coupled thereto.

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, preparing the second compound includes performing a S _N 2 substitution reaction using the first compound. In some embodiments, preparing the second compound includes performing a reductive amination reaction using the first compound. In some embodiments, preparing the second compound includes performing a Michael addition reaction using the first compound. In some embodiments, preparing the third compound includes performing a hydrogenation reaction using the second compound. In some embodiments, preparing the fourth compound includes performing an S _N 2 substitution reaction using the third compound. In some embodiments, preparing the fourth compound includes performing a reductive amination reaction using the third compound. In some embodiments, preparing the fourth compound includes performing a Michael addition reaction using the third compound. In some embodiments, preparing the fourth compound includes performing an amide coupling reaction using the third compound. In some embodiments, preparing the fourth compound includes performing a nucleophilic addition reaction using the third compound. In some embodiments, preparing the fifth compound includes reacting the fourth compound with at least one acid. In some embodiments, the at least one acid includes at least one of hydrochloric acid and trifluoroacetic acid. In some embodiments, preparing the sixth compound includes performing a S _N 2 substitution reaction using the fifth compound. In some embodiments, preparing the sixth compound includes performing a reductive amination reaction using the fifth compound. In some embodiments, preparing the sixth compound includes performing a Michael addition reaction using the fifth compound. In some embodiments, preparing the seventh compound includes performing a S _N 2 substitution reaction using the sixth compound. In some embodiments, preparing the seventh compound includes performing a reductive amination reaction using the sixth compound. In some embodiments, preparing the seventh compound includes performing a Michael addition reaction using the sixth compound. In some embodiments, preparing the seventh compound includes performing an amide coupling reaction using the sixth compound. In some embodiments, preparing the seventh compound includes 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 described above. In some embodiments, the second amide is coupled to the second targeting ligand using an independently selected linker A described above. In some embodiments, the third amide is coupled to the third targeting ligand using an independently selected linker A described above. In some embodiments, the first targeting ligand, second targeting ligand and third targeting ligand are independently selected to be one or more of the targeting ligands described above. In some embodiments, the hydroxy group is coupled to the second nitrogen using Linker B described above. In some embodiments, the method further comprises converting a hydroxy group to a phosphoramidite group using a phosphorylation reaction. In some embodiments, converting the hydroxy group to a phosphoramidite group is performed after preparing the thirteenth compound.

Additional aspects of the disclosure relate to methods of 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.

Another aspect of the disclosure relates to a method of 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, one or more pharmaceutical agents include 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, a double-stranded siRNA includes at least one modified ribonucleotide on 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 a double-stranded siRNA are modified. In some embodiments, the modified ribonucleotide is 2'-O-methyl nucleotide, 2'-fluoro nucleotide, 2'-deoxy nucleotide, 2'3'-seco nucleotide mimetic, lock 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 nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, and 3'-OMe nucleotides, nucleotides containing a 5'-phosphorothioate group, or 5'-(E)-vinyl phosphonate nucleotides ( antisense strand alone), or a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, a 2'-amino-modified nucleotide, a 2'-alkyl-modified nucleotide, a phosphoramidate, or a non-natural base. Contains nucleotides. In some embodiments, at least one strand of the double-stranded siRNA comprises at least one phosphorothioate linkage. In some embodiments, at least one strand of the double-stranded siRNA comprises up to 6 phosphorothioate linkages. In some embodiments, the double-stranded siRNA includes 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.

Another aspect of the present disclosure is compounds for use in delivering pharmaceutical agents 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.

In another aspect of the disclosure, the dsRNA agent comprises 2'-fluoro modified nucleotides at positions 2, 7, 12, 14, and 16 of the antisense strand (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 (counted from the first paired nucleotide from the 3' end of the sense strand).

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The present disclosure provides multivalent ligand clusters with diamine scaffolds for targeted delivery of pharmaceutical agents conjugated thereto. In some embodiments, a multivalent ligand cluster may include one or more N-acetylgalactosamine (GalNAc) targeting ligands. In some embodiments, a multivalent ligand cluster can be conjugated to one or more small interfering ribonucleic acids (siRNAs), where siRNAs are examples of pharmaceutical agents. The disclosure also provides compositions comprising the multivalent ligand clusters of the disclosure, and methods of making and using the multivalent ligand clusters of the disclosure.

Justice

Before further description of the invention, and to enable the invention to be more readily understood, certain terms are first defined and collected herein for convenience.

As used herein, the terms “treat,” “treating,” or “treatment” may include prevention, improving, alleviating, eliminating the cause of symptoms on a temporary or permanent basis, or naming. It is meant to prevent or delay the appearance of symptoms of a disorder or condition.

As used herein with respect to a measured quantity, the term "about" means about that measured quantity as would be expected by a person 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 equipment. Refers to normal fluctuations.

As used herein, the term “conjugate” or “conjugate group” means an atom or group of atoms bound to an oligonucleotide or other oligomer. Typically, 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.

As used herein, the term “linked” refers to a connection between two molecules, either directly or indirectly, when the two molecules are connected by a covalent bond or when the two molecules are linked by a non-covalent bond (e.g. refers to being associated through a hydrogen bond or ionic bond. An example of compound A directly linked to compound B can be represented as A-B. An example of compound A indirectly linked to compound B can be represented as A-C-B, where compound A is indirectly linked to compound B through compound C. It will be appreciated that more than one intermediate compound may be present in the indirect linkage context of the compounds.

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 can also contain any combination of these elements within a single molecule. Nucleic acids may include natural nucleic acids, non-natural nucleic acids, or a combination of natural and non-natural nucleic acids. Nucleic acids may also be referred to herein as nucleotide sequences, or as polynucleotides.

As used herein, the term “oligomer” refers to a nucleotide sequence containing 5 or fewer, 10 or fewer, 15 or fewer, 20 or fewer, or more than 20 nucleotides or nucleotide base pairs. In some embodiments, the oligomer has a nucleobase sequence that is at least partially complementary to a coding sequence in a target gene or expressed target nucleic acid in a cell. In some embodiments, oligomers can inhibit expression of an underlying gene when delivered to a cell expressing 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 include oligonucleotides, single-stranded oligonucleotides, single-stranded antisense oligonucleotides, short interfering RNA (siRNA), single-stranded siRNA, double-stranded RNA (dsRNA). ), micro RNA (miRNA), short hairpin RNA (shRNA), ribozyme, interfering RNA molecule and Dicer substrate.

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

As used herein, the term “single-stranded oligonucleotide” refers to a single-stranded oligomer, and in certain embodiments, a single-stranded oligonucleotide targets its target via hydrogen bonding under mammalian physiological conditions (or equivalent conditions in vitro). It may comprise a sequence that is at least partially complementary to the target mRNA, capable of hybridizing to the mRNA. In some embodiments, the single-stranded oligonucleotide is a single-stranded antisense oligonucleotide.

As used herein, the term “siRNA” refers to short interfering RNA or silencing RNA. siRNAs are a class of double-stranded RNA molecules that can be 20-25 base pairs long (or shorter), similar to microRNAs (miRNAs) that operate within the RNA interference (RNAi) pathway. siRNA degrades mRNA after transcription and prevents translation, thereby interfering with the expression of a specific gene with a nucleotide sequence complementary to the siRNA. siRNA acts to silence gene expression by inducing the RNA-induced silencing complex (RISC) in cells to cleave messenger RNA (mRNA).

As used herein, the term “effective amount,” “therapeutically effective amount,” or “effective dose” refers to an amount sufficient to elicit the desired pharmacological or therapeutic effect and result in effective prevention or treatment of a disorder. Prevention of a disorder occurs by delaying the onset of symptoms of the disorder to a medically significant extent. Treatment of a disorder is indicated by reduction of symptoms associated with the disorder or amelioration of recurrence of symptoms of the disorder.

As used herein, the term “pharmaceutical composition” or “composition” refers to a mixture of substances suitable for administration to a subject. For example, a pharmaceutical composition may include, but is not limited to, one or more active agents and a pharmaceutical carrier, also referred to herein as a “pharmaceutically acceptable carrier” (e.g., a sterile aqueous solution). In some embodiments, the pharmaceutical composition is sterile.

As used herein, the term “alkyl,” by itself or as part of another group, refers to a group consisting of 1 to 12 carbon atoms (i.e., C _1-12 alkyl) or the specified number of carbon atoms (i.e., C ₁ refers to a straight or branched chain aliphatic hydrocarbon containing an alkyl such as methyl, C ₂ alkyl such as ethyl, C ₃ alkyl such as propyl or isopropyl, etc. Non-limiting exemplary C _1-10 alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, iso-butyl, 3-pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc. .

As used herein, by itself or as part of another group, the term "substituted alkyl" means that alkyl, as defined herein, has one or more (e.g., 1, 2 or 3) independently selected substituents. It means being replaced with . 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) Includes sulfonyl, cycloalkyl, sulfanyl, carboxamido, heterocyclyl and (heterocyclyl)sulfonyl.

As used herein, the term “cycloalkyl,” by itself or as part of another group, contains a ring having 3 to 12 carbon atoms (i.e., C _3-12 cycloalkyl) or 1 to 3 rings having the indicated number of carbons. refers to saturated and partially unsaturated (containing one or two double bonds) cyclic aliphatic hydrocarbons. Non-limiting exemplary cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, norbornyl, decalin, adamantyl, cyclohexenyl, cyclopentenyl, cyclohexenyl, etc. .

As used herein, by itself or as part of another group, the term “substituted cycloalkyl” means that cycloalkyl, as defined herein, is substituted with 1, 2 or 3 independently selected substituents. A non-limiting list of independently selected substituents include 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. Includes.

As used herein, by itself or as part of another group, the term “alkenyl” refers to an alkyl group as defined herein containing 1, 2 or 3 carbon-to-carbon double bonds. Non-limiting exemplary alkenyl groups include ethenyl, propenyl, isopropenyl, butenyl, sec-butenyl, pentenyl, and hexenyl.

As used herein, by itself or as part of another group, the term "substituted alkenyl" means that alkenyl, as defined herein, is substituted with 1, 2 or 3 independently selected substituents. A non-limiting list of independently selected substituents include halo, nitro, cyano, hydroxyl, amino, (alkyl)amino, (dialkyl)amino, haloalkyl, (hydroxy)alkyl, (dihydroxy)alkyl, alkoxy, Haloalkoxy, aryloxy, aralkyloxy, (alkyl)sulfanyl, carboxamido, sulfonamido, (alkyl)carbonyl, (aryl)carbonyl, (alkyl)sulfonyl, (aryl)sulfonyl, urei , guanidino, carboxy, (carboxy)alkyl, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and heterocyclyl.

As used herein, the term "cycloalkenyl", by itself or as part of another group, has a single or multiple cyclic ring and has at least one >C=C< ring unsaturation and preferably one to two >C =C<refers to a non-aromatic cyclic alkyl group of 4 to 10 carbon atoms with a site of ring unsaturation.

As used herein, by itself or as part of another group, the term “substituted cycloalkenyl” refers to a 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, cyclo Alkyl, substituted cycloalkyl, cycloalkyloxy, substituted cycloalkyloxy, cycloalkylthio, substituted cycloalkylthio, cycloalkenyl, substituted cycloalkenyl, cycloalkenyloxy, substituted cycloalkenyloxy, cyclo Alkenylthio, substituted cycloalkenylthio, guanidino, substituted guanidino, halo, hydroxy, hydroxyamino, alkoxyamino, hydrazino, substituted hydrazino, heteroaryl, substituted heteroaryl, hetero Aryloxy, substituted heteroaryloxy, heteroarylthio, substituted heteroarylthio, heterocyclic, substituted heterocyclic, heterocyclyloxy, substituted heterocyclyloxy, heterocyclylthio, substituted heterocycle Includes lylthio, nitro, SO ₃ H, substituted sulfonyl, sulfonyloxy, thioacyl, thiocyanate, thiol, alkylthio and substituted alkylthio.

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

As used herein, by itself or as part of another group, the term “substituted alkynyl” means that an alkynyl, as defined herein, is substituted with 1, 2 or 3 independently selected substituents. A non-limiting list of independently selected substituents include halo, nitro, cyano, hydroxyl, amino, alkylamino, dialkylamino, haloalkyl, (hydroxy)alkyl, (dihydroxy)alkyl, alkoxy, haloalkoxy, aryl Oxy, aralkyloxy, (alkyl)sulfanyl, carboxamido, sulfonamido, alkylcarbonyl, arylcarbonyl, alkylsulfonyl, arylsulfonyl, ureido, guanidino, carboxy, (carboxy)alkyl , alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and heterocyclyl.

As used herein, by itself or as part of another group, the term “haloalkyl” refers to an alkyl group substituted by one or more fluorine, chlorine, bromine and/or iodine atoms. Non-limiting exemplary haloalkyl groups include fluoromethyl, difluoromethyl, trifluoromethyl, pentafluoroethyl, 1,1-difluoroethyl, 2,2-difluoroethyl, 2,2,2- It includes trifluoroethyl, 3,3,3-trifluoropropyl, 4,4,4-trifluorobutyl and trichloromethyl groups.

As used herein, the term "alkoxy" by itself or as part of another group means alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, alkynyl or Refers to substituted alkynyl.

As used herein, by itself or as part of another group, the term “haloalkoxy” refers to a haloalkyl attached to a terminal oxygen atom. Non-limiting exemplary haloalkoxy groups include fluoromethoxy, difluoromethoxy, trifluoromethoxy, and 2,2,2-trifluoroethoxy.

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

As used herein, by itself or as part of another group, the term “substituted aryl” means that an aryl as defined herein is substituted with 1 to 5 independently selected substituents. A non-limiting list of independently selected substituents include halo, nitro, cyano, hydroxyl, amino, alkylamino, dialkylamino, haloalkyl, (hydroxy)alkyl, (dihydroxy)alkyl, alkoxy, haloalkoxy, aryl Oxy, heteroaryloxy, aralkyloxy, alkylthio, carboxamido, sulfonamido, (alkyl)carbonyl, (aryl)carbonyl, (alkyl)sulfonyl, (aryl)sulfonyl, ureido, anidino, 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(C ₁ - C ₄ )Alkoxy)alkyl, (heteroaryl)alkyl, etc. 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, Includes 5-dimethoxy, 4-methylphenyl, 2-fluoro-3-chlorophenyl and 3-chloro-4-fluorophenyl. The term substituted aryl is intended to include groups having fused substituted cycloalkyl and fused substituted heterocyclo rings.

As used herein, the term “aryloxy,” by itself 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-.

As used herein, by itself or as part of another group, the term “heteroaryloxy” refers to a heteroaryl or substituted heteroaryl attached to a terminal oxygen atom.

As used herein, by itself or as part of another group, the term “aralkyloxy” refers to an aralkyl group attached to a terminal oxygen atom. A non-limiting exemplary aralkyloxy group is PhCH ₂ O-.

As used herein, the term “heteroaryl” refers to a ring atom having 5 to 14 ring atoms (i.e., C ₅ -C ₁₄ heteroaryl) and 1, 2, Refers to monocyclic and bicyclic aromatic ring systems having 3 or 4 heteroatoms. Non-limiting exemplary heteroaryl groups include thienyl, benzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl, furyl, benzofuryl, pyranyl, isobenzofuranyl, benzoxazonyl, Chromenyl, xanthenyl, 2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, isoindolyl, 3H-indolyl, indolyl, 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 intended to include possible N-oxides. Exemplary N-oxides include pyridyl N-oxide and the like.

As used herein, by itself or as part of another group, the term "substituted heteroaryl" means that heteroaryl as defined herein is substituted with 1 to 4 independently selected substituents. A non-limiting list of independently selected substituents include 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, anidino, carboxy, (carboxy)alkyl, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclo, (alkoxy)alkyl, (amino)alkyl, [(hydroxyl)alkyl]amino, [( Includes alkyl)amino]alkyl, [(dialkyl)amino]alkyl, (cyano)alkyl, (carboxamido)alkyl, mereapthoalkyl, (heterocyclo)alkyl and (heteroaryl)alkyl. Any available carbon or nitrogen atom may be substituted.

As used herein, by itself or as part of another group, the term "heterocyclo" or "heterocyclyl" refers to a group having 3 to 14 ring members (i.e., 3- to 14-membered heterocyclo) and at least one refers to saturated and partially unsaturated (e.g., containing 1 or 2 double bonds) cyclic groups containing 1, 2, or 3 rings with heteroatoms. Each heteroatom is selected independently. The term “heterocyclo” or “heterocyclyl” includes cyclic ureido groups such as 2-imidazolidinone, and cyclic amide groups such as β-lactam, γ-lactam, δ-lactam and ε-lactam. It is intended to. The term “heterocyclo” or “heterocyclyl” is also intended to include groups having fused aryl or substituted aryl groups, such as indolinyl. Heterocyclo or heterocyclyl can be connected to the rest 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. Includes.

As used herein, by itself or as part of another group, the term "substituted heterocyclo" or "substituted heterocyclyl" means that the heterocyclo or heterocyclyl group as defined above is one to four independently selected It means being substituted with a substituent. A non-limiting list of independently selected substituents include 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, anidino, 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. Substitutions can occur on any available carbon or nitrogen atom and can form a spirocycle.

As used herein, by itself or as part of another group, the term “amino” refers to -NH ₂ .

As used herein, by itself or as part of another group, the term “alkylamino” or “(alkyl)amino” refers to -NHR where R is alkyl.

As used herein, by itself or as part of another group, the term "dialkylamino" or "(dialkyl)amino" refers to -NR ^' R ^" , where R ^' and R ^" are each independently alkyl or R ^' and R ^" together form a 3- to 8-membered heterocyclo or substituted heterocyclo.

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

As used herein, by itself or as part of another group, the term “(amino)alkyl” refers to an alkyl group substituted with an amino group. Non-limiting exemplary (amino)alkyl groups include -CH ₂ CH ₂ NH ₂ , -CH ₂ CH ₂ CH ₂ NH ₂ , and -CH ₂ CH ₂ CH ₂ CH ₂ NH ₂ .

As used herein, by itself or as part of another group, the term “(alkylamino)alkyl” or “[(alkyl)amino]alkyl” refers to an alkyl group substituted with an alkylamino group. A non-limiting exemplary (alkylamino)alkyl group is -CH ₂ CH ₂ N(H)CH ₃ .

As used herein, by itself or as part of another group, the term “(dialkylamino)alkyl” refers to an alkyl group substituted by a dialkylamino group. Non-limiting exemplary (dialkylamino)alkyl groups include -CH ₂ N(CH ₃ ) ₂ and -CH ₂ CH ₂ N(CH- ₃ ) ₂ .

As used herein, by itself or as part of another group, the term “(cycloalkylamino)alkyl” refers to an alkyl group substituted by a cycloalkylamino group. Non-limiting exemplary (cycloalkylamino)alkyl groups include -CH ₂ N(H)cyclopropyl, -CH ₂ N(H)cyclobutyl, and -CH ₂ N(H)cyclohexyl.

As used herein, by itself or as part of another group, the term "carboxamido" refers to a radical of the formula -C(=O)NR ^' R ^" , where R ^' and R ^" are each independently is 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, are 3- to 8 Non-limiting exemplary carboxamido groups include -CONH ₂ , -CON(H)CH ₃ , CON(CH ₃ ) ₂ , and CON(H)Ph.

As used herein, the term "sulfonamido", by itself or as part of another group, refers to a radical of the formula -SO ₂ NR ^' R ^" , where R ^' and R ^" are each independently hydrogen, alkyl, substituted is an alkyl, aryl or substituted aryl, or R ^' and R ^" , together with the nitrogen to which they are attached, form a 3- to 8-membered heterocyclo group. Non-limiting exemplary sulfonamido groups include -SO ₂ NH ₂ , -SO ₂ N(H)CH ₃ and -SO ₂ N(H)Ph.

As used herein, by itself or as part of another group, the term "(alkyl)carbonyl" refers to a carbonyl group substituted by an alkyl group, i.e. -C(=O)-. A non-limiting exemplary alkylcarbonyl group is -COCH ₃ .

As used herein, by itself or as part of another group, the term "(alkoxy)carbonyl" (or "ester") refers to a carbonyl group substituted by an alkoxy group, i.e. -C(=O)- . A non-limiting exemplary (alkoxy)carbonyl group is -C(O)OCH ₃ .

As used herein, by itself or as part of another group, the term “(aryl)carbonyl” refers to a carbonyl group substituted by an aryl or substituted aryl group, i.e., -C(=O)-. A non-limiting exemplary arylcarbonyl group is -COPh.

As used herein, by itself or as part of another group, the term “sulfanyl” refers to the group -SH.

As used herein, by itself or as part of another group, the term “(alkyl)sulfanyl” or “alkylthio” refers to a sulfur atom substituted by an alkyl or substituted alkyl group. Non-limiting exemplary alkylthio groups include -SCH ₃ and -SCH ₂ CH ₃ .

As used herein, by itself or as part of another group, the term “mercaptoalkyl” refers to an alkyl group substituted by an -SH group.

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

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

As used herein, by itself or as part of another group, the term “carboxy” refers to a radical of the formula -COOH.

As used herein, by itself or as part of another group, the term “(carboxy)alkyl” refers to an alkyl group substituted with -COOH. A non-limiting exemplary carboxyalkyl group is -CH ₂ CO ₂ H.

As used herein, by itself or as part of another group, the term “aralkyl” refers to the moiety to which an aryl moiety is attached to an alkyl moiety. An aralkyl group can be attached to the parent structure at an aryl or alkyl moiety.

As used herein, by itself or as part of another group, the term “substituted aralkyl” refers to the moiety to which an aryl moiety is attached to a substituted alkyl moiety.

As used herein, by itself or as part of another group, the term "araalkenyl" refers to a radical of the formula -R _d -R _c , where R _d is an alkenylene chain and R _c is one or more It is an aryl radical.

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

As used herein, by itself or as part of another group, the term "aralkynyl" refers to a radical of the formula -R _e R _c , wherein R _e is an alkynylene chain and R _c is one or more aryl groups. It is a radical.

As used herein, by itself or as part of another group, the term "substituted aralkynyl" means an alkynylene chain in which the alkynylene chain of the aralkynyl radical is optionally substituted, and each aryl radical of the aralkynyl radical is This optionally substituted aryl radical refers to an aralkynyl radical.

As used herein, by itself or as part of another group, the term “aliphatic heterocycle” refers to a non-aromatic ring in which one or more of the ring-forming atoms is a heteroatom.

As used herein, the term “heteroatom” refers to an atom inserted between a carbon atom and its parent molecule (i.e., between the point of attachment). Non-limiting exemplary heteroatoms include oxygen, nitrogen, sulfur (including sulfoxides and sulfones), and phosphorus (P).

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

As used herein, the term “monosaccharide” refers to a single sugar residue within an oligosaccharide.

As used herein, the term “disaccharide” refers to a polysaccharide composed of two monosaccharide units or moieties linked together by a glycosidic bond.

As used herein, “oligosaccharide” refers to a compound containing two or more monosaccharide units or moieties. With regard to oligosaccharides, the individual monomer units or moieties are monosaccharides that are or can be linked to another monosaccharide unit or moiety through a hydroxy group. Oligosaccharides can be prepared by chemical synthesis from protected single residue sugars or by chemical breakdown of biologically produced polysaccharides. Alternatively, oligosaccharides can be prepared by in vitro enzymatic methods.

As used herein, by itself or as part of another group, the term "ureido" refers to a radical of the formula -NR ^' -C(=O)-NR ^" R ^" , where R ^' is hydrogen, alkyl, aryl or substituted aryl, and R ^" and R ^"' are each independently hydrogen, alkyl, aryl or substituted aryl, or R ^" and R ^"' , together with the nitrogen to which they are attached, are 4- to 8-membered It forms a heterocyclyl group. Non-limiting exemplary ureido groups include -NH-C(=O)-NH ₂ and -NH-C(=O)-NHCH ₃ .

As used herein, the term "guanidino", by itself or as part of another group, refers to a radical of the formula -NR ^' -C(=NR ^" ) -NR ^"' R ^"" , where R ^' , R ^"' and R ^"" are each independently hydrogen, alkyl, aryl, or substituted aryl, and R ^" is hydrogen, alkyl, cyano, alkylsulfonyl, alkylcarbonyl, carboxamido, or sulfonamido. Non-limiting exemplary guanidino groups include -NH-C(=NH)-NH ₂ , -NH-C(=NCN)-NH ₂ , and -NH-C(=NH)-NHCH ₃ .

As used herein, by itself or as part of another group, the term “(heteroaryl)alkyl” refers to an alkyl group substituted with 1, 2 or 3 heteroaryl or substituted heteroaryl groups.

As used herein, by itself or as part of another group, the term “heteroalkyl” refers to a stable straight-chain or branched-chain hydrocarbon radical containing at least one heteroatom, which may be the same or different. The heteroatom(s) may be located at any internal or terminal position of the heteroalkyl group, or at the position at which the heteroalkyl group is attached to the remainder of the molecule. Non-limiting exemplary heteroalkyl groups include -CH ₂ N(H)CH ₂ CH ₂ N(CH ₃ ) ₂ , -CH ₂ N(CH ₃ )CH ₂ CH ₂ N(CH ₃ ) ₂ , -CH ₂ N(H )CH ₂ CH ₂ CH ₂ N(CH ₃ ) ₂ , -CH ₂ N(H)CH ₂ CH ₂ OH, -CH ₂ N(CH ₃ )CH ₂ CH ₂ OH, -CH ₂ OCH ₂ CH ₂ OCH ₃ , -OCH ₂ CH ₂ OCH ₂ CH ₂ OCH ₃ , -CH ₂ NHCH ₂ CH ₂ OCH ₂ , -OCH ₂ CH ₂ NH ₂ , and -NHCH ₂ CH ₂ N(H)CH ₃ .

As used herein, by itself or as part of another group, the term “(heterocyclo)alkyl” or “(heterocyclyl)alkyl” refers to one heterocyclyl or substituted heterocyclyl group, and optionally one Refers to an alkyl group substituted with a hydroxy group.

As used herein, by itself or as part of another group, the term "(carboxamido)alkyl" means one carboxamido group, and optionally one heterocyclo, amino, alkylamino or dialkylamino group. Refers to a substituted alkyl group.

As used herein, the term “N-oxide” refers to a compound containing a functional group, wherein N ⁺ is further connected to H and/or the remainder of the compound structure.

As used herein, the term “integer” refers to an integer including, but not limited to, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, etc.

compound

general structure

A multivalent ligand cluster with a diamine scaffold may have the structure of Formula 1:

here:

Each TL is an independently selected targeting ligand,

m is an integer from 1 to 10,

Each n is an independently selected integer from 1 to 10,

Each Linker A is an independently selected spacer with one end attached to TL and the other end attached to the nitrogen of an alkylcarboxamide,

Linker B is a spacer having one end attached to a pharmaceutical agent or a functional group capable of being linked to one or more pharmaceutical agents 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.

In some embodiments, m can be constructed based on the starting materials used to synthesize the multivalent ligand cluster. For example, m may be 1 due to ethylenediamine used as starting material, m may be 2 due to 1,3-propanediamine used as starting material, and m may be 2 due to 1,4-butane used as starting material. Due to the diamine, m may be 3.

As used herein, “spacer” refers to a compound or molecule that links different groups together. Examples of Linker A and Linker B spacers are described in detail herein below.

As used herein in relation to elements of a multivalent ligand cluster of the invention, the term "independently selected" means that each given type of element may differ from one or more others of the same type of element within the multivalent ligand cluster. . For example, a multivalent ligand cluster may include more than one type of TL, where each may be selected to be different or the same as one or more other TLs within the multivalent ligand cluster. For a further example, a multivalent ligand cluster may include more than one “n”, where each may be selected to be different or the same as one or more other “n”s within the multivalent ligand cluster. In another example, a multivalent ligand cluster may include more than one Linker A, where each may be selected to be different or the same as one or more other Linker A's within the multivalent ligand cluster.

targeting ligand

As noted above with respect to Formula 1, multivalent ligand clusters of the present disclosure may include multiple (e.g., three) independently selected targeting ligands. In this context, the term “independently selected” means that each targeting ligand may be selected to be different from or identical to one or more other targeting ligands within the same multivalent ligand cluster.

At least one of the independently selected targeting ligands of Formula 1 is capable of binding to one or more cell receptors, cell channels and/or cell transporters that can facilitate endocytosis.

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 that is smaller than a protein. In some embodiments, the at least one small molecule ligand is N-acetylgalactosamine (GalNAc), galactose, galactosamine, N-formyl-galactosamine, N-propionylgalactosamine, N-butanoylgal. It may include at least one of lactosamine, N-iso-butanoylgalactosamine, macrocycle, folate molecule, fatty acid, bile acid, cholesterol, and derivatives thereof.

A macrocycle is a molecule or ion containing a ring of 12 or more members. The present disclosure is not limited to any particular macrocycle. A non-limiting list of macrocycles within the scope of this disclosure includes terpenoid macrocycles, porphyrins, and cyclodextrins.

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

Fatty acids are carboxylic acids with long aliphatic chains. In some embodiments, fatty acids may be saturated, meaning that the aliphatic chains all have a single carbon-to-carbon bond. In some embodiments, the fatty acid may be unsaturated, meaning that the aliphatic chain contains at least one double or triple carbon-to-carbon bond. In some embodiments, fatty acids may comprise branched chains. In some embodiments, fatty acids may comprise ring structures. Fatty acids are known to assist in the updating of pharmaceutical agents into cells. 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].

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 acids may be secondary bile acids produced from bacterial action in the colon. Bile acids are known to be beneficial in inhibiting RNA translation. 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].

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

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

In some embodiments, at least one of the independently selected targeting ligands of Formula 1 may comprise 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 take on a variety of shapes because they tend to form helices and single-stranded loops. The present disclosure is not limited to any particular aptamer. Known and as yet undiscovered aptamers are within the scope of this disclosure.

In some embodiments, at least one of the independently selected targeting ligands of Formula 1 is capable of binding to at least one asialoglycoprotein receptor (ASGPR). ASGPR is a lectin located on liver cells that binds galactose residues. ASGPR has been demonstrated to have high expression on the surface of hepatocytes, human carcinoma cell lines, and liver cancer. ASGPR is also weakly expressed by glandular cells of the gallbladder and stomach.

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

In some embodiments, at least one of the independently selected targeting ligands of Formula 1 is capable of binding to at least one integrin receptor. Integrin receptors are transmembrane receptors that facilitate cell-cell and cell-extracellular matrix (ECM) adhesion. Upon ligand binding, integrin receptors activate signaling pathways that mediate cell signaling, such as regulation of the cell cycle, organization of the intracellular cytoskeleton, and trafficking of new receptors to the cell membrane. Integrin targeted delivery of gene therapeutics is known to those skilled in the art. In 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 specific ligand capable of binding to at least one integrin receptor. Known and as yet undeveloped integrin receptor ligands are within the scope of this disclosure.

In some embodiments, at least one of the independently selected targeting ligands of Formula 1 is capable of binding to at least one folate receptor. The folate receptor binds folate and reduced folic acid derivatives and mediates the transport of tetrahydrofolate into the interior of the cell. Targeted drug delivery through folate receptors is known to those skilled in the art. 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 specific ligand capable of binding to at least one folate receptor. Known and as yet undeveloped folate receptor ligands are within the scope of this disclosure.

In some embodiments, at least one of the independently selected targeting ligands of Formula 1 is capable of binding 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 a GPCR, it causes a conformational change in the GPCR, which triggers an interaction between the GPCR and a nearby G protein. GPCRs consist of a single polypeptide that folds into a spherical shape and is embedded in the cell's plasma membrane. GPCR targeted delivery of oligonucleotide therapeutics is known to those skilled in the art. 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].

Linker A

As noted above with respect to Formula 1, the multivalent ligand clusters of the present disclosure may include multiple (e.g., three) independently selected LinkerAs. In this context, the term “independently selected” means that each Linker A may be selected to be different from or identical to one or more other Linker A's within the same multivalent ligand cluster.

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

In some embodiments, at least one of the independently selected Linkers A may comprise polyethylene glycol (PEG). PEG may have any number of repeating O-CH ₂ -CH ₂ units. For example, PEG is 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, 7, PEG48, PEG49, PEG50, PEG51, PEG52, PEG53, PEG54, PEG55, PEG56, PEG57, PEG58, PEG59, PEG60, PEG61, PEG62, PEG63, PEG64, PEG65, PEG66, PEG67, PEG68, PEG69, PEG70, PEG71, 2, PEG73, PEG74, PEG75, PEG76, PEG77, PEG78, PEG79, PEG80, PEG81, PEG82, PEG83, PEG84, PEG85, PEG86, PEG87, PEG88, PEG89, PEG90, PEG91, PEG92, PEG93, PEG94, PEG95, PEG96, 7, It may be PEG98, PEG99, PEG100, or higher.

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

In some embodiments, at least one of the independently selected Linkers A can include at least one substituted alkyl group. In some embodiments, the substituted alkyl group has 2 carbon, 3 carbon, 4 carbon, 5 carbon, 6 carbon, 7 carbon, 8 carbon, 9 carbon, 10 carbon, 11 carbon, 12 It can have 2 carbons, 13 carbons, 14 carbons, 15 carbons, 16 carbons, 17 carbons, 18 carbons, 19 carbons, or 20 carbons. In some embodiments, a substituted alkyl group can include one or more of the following substituents: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.

In some embodiments, at least one of the independently selected Linkers A may include at least one cycloalkyl group. In some embodiments, a cycloalkyl group is C3 cycloalkyl (i.e., cyclopropane), C4 cycloalkyl (i.e., cyclobutene), C5 cycloalkyl (i.e., cyclopentane), C6 cycloalkyl (i.e., cyclohexane), C7 cycloalkyl It may be alkyl (i.e. cycloheptane), C8 cycloalkyl (i.e. cyclooctane), C9 cycloalkyl (i.e. cyclononane), or C10 cycloalkyl (i.e. cyclodecane).

In some embodiments, at least one of the independently selected Linkers A can include at least one substituted cycloalkyl group. In some embodiments, a substituted cycloalkyl group is C3 substituted cycloalkyl, C4 substituted cycloalkyl, C5 substituted cycloalkyl, C6 substituted cycloalkyl, C7 substituted cycloalkyl, C8 substituted cycloalkyl, C9 substituted cycloalkyl. It may be alkyl, or C10 substituted cycloalkyl. In some embodiments, a substituted cycloalkyl group can include one or more of the following substituents: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.

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

In some embodiments, at least one of the independently selected Linkers A can include at least one substituted alkenyl group. In some embodiments, an alkenyl group has 4 carbons, 5 carbons, 6 carbons, 7 carbons, 8 carbons, 9 carbons, 10 carbons, 11 carbons, 12 carbons, 13 carbons, 14 carbons, It can have 15 carbons, 16 carbons, 17 carbons, 18 carbons, 19 carbons, or 20 carbons. In some embodiments, a substituted alkenyl group can include one or more of the following substituents: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.

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

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

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

In some embodiments, at least one of the independently selected Linkers A can include at least one substituted alkynyl group. In some embodiments, a substituted alkynyl group has 4 carbons, 5 carbons, 6 carbons, 7 carbons, 8 carbons, 9 carbons, 10 carbons, 11 carbons, 12 carbons, 13 carbons, It can have 14 carbons, 15 carbons, 16 carbons, 17 carbons, 18 carbons, 19 carbons, or 20 carbons. In some embodiments, substituted alkynyl groups can include one or more of the following substituents: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.

In some embodiments, at least one of the independently selected Linkers A may include at least one aryl or heteroaryl group. Examples include phenyl, naphthyl, and pyridinyl, but note that other aryl and heteroaryl groups may be used that fall within the definitions provided herein.

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

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.

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

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.

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

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.

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

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

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

In some embodiments, at least one of the independently selected Linkers A may comprise 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.

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

In some embodiments, at least one of the independently selected Linkers A may comprise at least one amino acid. A variety of amino acids are known to those skilled in the art. The independently selected Linker A is not limited to containing one or more specific amino acids. For example, an independently selected linker A may include 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, and one or more amino acids. Glutamic acid (Glu) amino acid, 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 , 1 or more selenocysteine (Sec) amino acids, 1 or more glycine (Gly) amino acids, 1 or more proline (Pro) amino acids, 1 or more alanine (Ala) amino acids, 1 or more valine (Val) amino acids, 1 At least one isoleucine (Ile) amino acid, at least one leucine (Leu) amino acid, at least one methionine (Met) amino acid, at least one phenylalanine (Phe) amino acid, at least one tyrosine (Tyr) amino acid, and/or at least one tryptophan (Trp) may contain amino acids.

In some embodiments, at least one of the independently selected Linkers A may comprise at least one nucleotide. A variety of 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 can be selected from one or more nucleotides containing a guanine nucleobase, one or more nucleotides containing an adenine nucleobase, one or more nucleotides containing a cytosine nucleobase, or one or more nucleotides containing a thymine nucleobase. It may comprise more than one type of nucleotide, and/or one or more types of nucleotide containing a uracil nucleobase.

In some embodiments, at least one independently selected Linker A may comprise at least one abasic nucleotide. As is known in the art, an abasic nucleotide is a nucleotide that has an abasic site, which is a position that has neither a purine base nor a pyrimidine base. For example, at least one independently selected Linker A may comprise one or more types of abasic DNA and/or one or more types of abasic RNA. In some embodiments, at least one independently selected Linker A may comprise at least one inverted basic nucleotide. As is known in the art, an inverted abasic nucleotide is an abasic nucleotide whose 5' end is joined to the 5' end of the next nucleotide and its 3' end is joined to the 3' end of the next nucleotide. For example, at least one independently selected Linker A may comprise one or more inverted abasic DNA and/or one or more inverted abasic RNA.

In some embodiments, at least one of the independently selected Linkers A may comprise at least one saccharide. In some embodiments, at least one of the independently selected Linkers A has 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 species of ribose monosaccharide units, and/or at least one glucosamine monosaccharide unit.

In some embodiments, at least one of the independently selected Linkers A may include one or more of the following:

here:

p is an integer from 0 to 12,

pp is an integer from 0 to 12,

q is an integer from 1 to 12,

qq is an integer from 1 to 12.

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

Linker B

Linker B is a spacer with one end attached to a pharmaceutical agent or a functional group that can be linked to one or more pharmaceutical agents and the other end attached to the diamine nitrogen of the multivalent ligand cluster.

In some embodiments, Linker B may comprise polyethylene glycol (PEG). PEG may have any number of repeating O-CH ₂ -CH ₂ units. For example, PEG is 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, 7, PEG48, PEG49, PEG50, PEG51, PEG52, PEG53, PEG54, PEG55, PEG56, PEG57, PEG58, PEG59, PEG60, PEG61, PEG62, PEG63, PEG64, PEG65, PEG66, PEG67, PEG68, PEG69, PEG70, PEG71, 2, PEG73, PEG74, PEG75, PEG76, PEG77, PEG78, PEG79, PEG80, PEG81, PEG82, PEG83, PEG84, PEG85, PEG86, PEG87, PEG88, PEG89, PEG90, PEG91, PEG92, PEG93, PEG94, PEG95, PEG96, 7, It may be PEG98, PEG99, PEG100, or higher. In some embodiments, it may be advantageous for the PEG to be PEG10 or less.

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

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

In some embodiments, Linker B may include at least one cycloalkyl group. In some embodiments, a cycloalkyl group is C3 cycloalkyl (i.e., cyclopropane), C4 cycloalkyl (i.e., cyclobutene), C5 cycloalkyl (i.e., cyclopentane), C6 cycloalkyl (i.e., cyclohexane), C7 cycloalkyl It may be alkyl (i.e. cycloheptane), C8 cycloalkyl (i.e. cyclooctane), C9 cycloalkyl (i.e. cyclononane), or C10 cycloalkyl (i.e. cyclodecane).

In some embodiments, Linker B can include at least one substituted cycloalkyl group. In some embodiments, a substituted cycloalkyl group is C3 substituted cycloalkyl, C4 substituted cycloalkyl, C5 substituted cycloalkyl, C6 substituted cycloalkyl, C7 substituted cycloalkyl, C8 substituted cycloalkyl, C9 substituted cycloalkyl. It may be alkyl, or C10 substituted cycloalkyl. In some embodiments, a substituted cycloalkyl group can include one or more of the following substituents: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.

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

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

In some embodiments, Linker B may include at least one cycloalkenyl group. In some embodiments, a cycloalkenyl group can be C5 cycloalkenyl, C6 cycloalkenyl, C7 cycloalkenyl, C8 cycloalkenyl, C9 cycloalkenyl, or C10 cycloalkenyl.

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

In some embodiments, Linker B may include at least one alkynyl group. In some embodiments, an alkynyl group has 4 carbons, 5 carbons, 6 carbons, 7 carbons, 8 carbons, 9 carbons, 10 carbons, 11 carbons, 12 carbons, 13 carbons, 14 carbons, It can have 15 carbons, 16 carbons, 17 carbons, 18 carbons, 19 carbons, or 20 carbons.

In some embodiments, Linker B may include at least one substituted alkynyl group. In some embodiments, a substituted alkynyl group has 4 carbons, 5 carbons, 6 carbons, 7 carbons, 8 carbons, 9 carbons, 10 carbons, 11 carbons, 12 carbons, 13 carbons, It can have 14 carbons, 15 carbons, 16 carbons, 17 carbons, 18 carbons, 19 carbons, or 20 carbons. In some embodiments, substituted alkynyl groups can include one or more of the following substituents: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.

In some embodiments, Linker B may include at least one aryl group or heteroaryl group. Examples include phenyl, naphthyl, and pyridinyl, but note that other aryl and heteroaryl groups may be used that fall within the definitions provided herein.

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

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

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

In some embodiments, Linker B may include at least one aralkenyl group. Examples of aralkenyl groups include, but are not limited to, ethenylbenzene and propenylbenzene.

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

In some embodiments, Linker B may include at least one aralkynyl group. Examples of aralkynyl groups include, but are not limited to, ethynylbenzene and propynylbenzene.

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

In some embodiments, Linker B may include at least one heteroatom. In some embodiments, Linker B comprises 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. It can be included. In some embodiments, at least one of the independently selected Linkers A may comprise at least one heteroalkyl group.

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

In some embodiments, Linker B 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.

In some embodiments, Linker B can include at least one substituted heteroaryl group. In some embodiments, substituted heteroaryl groups can include one or more of the following substituents: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.

In some embodiments, Linker B may include at least one amino acid. A variety of amino acids are known to those skilled in the art. Linker B is not limited to containing one or more specific amino acids. For example, linker B contains 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, and one or more glutamic acids (Glu). ) amino acids, 1 or more serine (Ser) amino acids, 1 or more threonine (Thr) amino acids, 1 or more asparagine (Asn) amino acids, 1 or more glutamine (Gln) amino acids, 1 or more cysteine (Cys) amino acids, 1 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 acid, 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) May contain amino acids.

In some embodiments, Linker B may comprise at least one nucleotide. A variety of nucleotides are known to those skilled in the art. Linker B is not limited to containing one or more specific nucleotides. For example, linker B is one or more nucleotides containing a guanine nucleobase, one or more nucleotides containing an adenine nucleobase, one or more nucleotides containing a cytosine nucleobase, and one or more nucleotides containing a thymine nucleobase. , and/or may include one or more nucleotides containing a uracil nucleobase. In some embodiments, Linker B may comprise at least one abasic nucleotide. As is known in the art, an abasic nucleotide is a nucleotide that has an abasic site, which is a position that has neither a purine base nor a pyrimidine base. For example, Linker B may comprise one or more types of abasic DNA and/or one or more types of abasic RNA. In some embodiments, Linker B may comprise at least one inverted basic nucleotide. For example, Linker B may comprise one or more inverted abasic DNA and/or one or more inverted abasic RNA.

In some embodiments, Linker B may comprise at least one saccharide. In some embodiments, Linker B comprises 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 may contain at least one type of glucosamine monosaccharide unit.

In some embodiments, Linker B may include one or more of the following:

here:

j is an integer from 1 to 12,

k is an integer from 0 to 12.

In some embodiments, j is an integer independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. In some embodiments, k is an integer independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. In some cases, the invention also provides that when Linker B contains a 6-membered ring fragment, particularly a 4-hydroxypiperidinyl group, when used for targeted delivery of a pharmaceutical agent compared to a 5-membered ring, the invention It was found that it exhibits superior in vivo stability and activity, similar to Compound 75.

pharmaceutical agent

In some embodiments, the pharmaceutical agent is, for example, a diagnostic or therapeutic drug, molecule, compound, or a group of drugs, molecules, or compounds that have properties that assist in the diagnosis, prevention, treatment, and/or alleviation of a disease or condition in a cell or subject. It is a combination. In certain embodiments, the pharmaceutical agent is a drug, molecule, compound, or combination of drugs, molecules, or compounds that has properties that assist, for example, in promoting a desired state in a cell or subject.

In some embodiments, the pharmaceutical agent may be an oligonucleotide. In some embodiments, oligonucleotides may include siRNA. In some embodiments, oligonucleotides may comprise double-stranded siRNA. In some embodiments, a double-stranded siRNA may include at least one modified ribonucleotide. In some embodiments of the compositions and methods of the invention, the at least one modified nucleotide is a 2'-O-methyl nucleotide, a 2'-fluoro nucleotide, a 2'-deoxy nucleotide, a 2'3'-seco nucleotide mimic. sieve, locked nucleotide, unlocked nucleic acid nucleotide (UNA), glycol nucleic acid nucleotide (GNA), 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, 3'-Ome nucleotide, A nucleotide containing a 5'-phosphorothioate group, or a 5'-(E)-vinyl phosphonate nucleotide (antisense strand alone), or a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, 2'-amino -comprises modified nucleotides, phosphoramidates, or nucleotides containing non-natural bases. In some embodiments, substantially all (i.e., greater than 85%) ribonucleotides of a double-stranded siRNA may be modified.

In some embodiments, all ribonucleotides of a double-stranded siRNA may be modified. In some embodiments, at least one strand of a double-stranded siRNA may comprise at least one phosphorothioate linkage. In some embodiments, at least one strand of a double-stranded siRNA may include up to 6 phosphorothioate linkages. In some embodiments, a double-stranded siRNA may include at least one locked nucleic acid (LNA). As is known in the art, LNA (sometimes referred to as cross-linked nucleic acid (BNA) or inaccessible RNA) is a ribose moiety modified with an extra bridge connecting the 2' oxygen and 4' carbon to form a ribose 3 '-endo is a modified RNA molecule that is locked into its conformation. LNAs are known to increase stability against enzymatic degradation and improve specificity and affinity. In some embodiments, a double-stranded siRNA may include at least one unlocked nucleic acid (UNA). As is known in the art, UNA is a non-cyclic derivative of RNA that lacks the C2'-C3'-linkage of the ribose ring of RNA. It is known to those skilled in the art that inclusion of UNA at specific positions in the antisense strand of siRNA is permissive for activity and can promote reduced off-target activity.

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

In some embodiments, oligonucleotides are 2'-modified nucleotides (e.g. F and MeO), abasic nucleotides, reversed abasic nucleotides, locked nucleotides, UNA unlocked nucleic acids (UNA), and glycerol nucleic acids (GNA). It may include siRNA containing one or more modified nucleotides, including but not limited to.

In some embodiments, oligonucleotides may comprise siRNA comprising one or more phosphorothioate backbone linkages.

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

In some embodiments, an oligonucleotide may comprise at least three independently selected nucleotides. In some embodiments, for example when the oligonucleotide is siRNA, the oligonucleotide may comprise 16 to 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 if the oligonucleotide is mRNA, the oligonucleotide may comprise up to 14,000 independently selected nucleotides.

A functional group that can be linked to one or more pharmaceutical agents

As indicated above, “W” in Formula 1 may be a functional group that can be linked to one or more pharmaceutical agents.

In some embodiments, the functional group can be a hydroxy group (OH). In some embodiments, the functional group can be a protected hydroxy group. Those skilled in the art will recognize that a variety of protecting groups can be used to protect hydroxy groups. Each of the various treatment groups is within the scope of this disclosure. For example, and without limitation, the hydroxy group is 4,4'-dimethoxytrityl (DMT), monomethoxytrityl (MMT), 9-(p-methoxyphenyl)xanthen-9-yl ( Mox), and 9-phenylxanthen-9-yl (Px).

In some embodiments, the functional group can be a phosphoramidite group having the formula:

here:

R ^a is C1 to C6 alkyl, C3 to C6 cycloalkyl, isopropyl group, or R ^a is connected to R ^b through a nitrogen atom to form a cycle,

R ^b is C1 to C6 alkyl, C3 to C6 cycloalkyl, isopropyl group, or R ^b is connected to R ^a through a nitrogen atom to form a cycle,

R ^c is a phosphite protecting group, a phosphate protecting group, or a 2-cyanoethyl group.

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 is 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-dichloro It may contain at least one type of phenyl.

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 is 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 It may include at least one of the following.

In some embodiments, the functional group can be a carboxyl group (CO ₂ H). In some embodiments, the functional group can be an activated carboxyl group having the formula:

Here, X is a leaving group.

A variety of activated carboxyl groups are known to those skilled in the art, all of which are within the scope of this disclosure. In some embodiments, leaving group (X) is carboxylate, sulfonate, chloride, phosphate, imidazole, hydroxybenzotriazole (HOBt), N-hydroxysuccinimide (NHS), tetrafluorophenol, It may be either pentafluorophenol, or para-nitrophenol.

In some embodiments, the functional group may be a Michael acceptor. In some embodiments, the Michael acceptor can have the formula:

here:

E is an electron withdrawing group;

R ^d is hydrogen or a C1-C6 alkyl substituent on the olefin (meaning that E and R ^d may be cis, trans or iso to the carbon-carbon double bond).

A variety of electron withdrawing groups are known to those skilled in the art, all of which are within the scope of this disclosure. In some embodiments, the electron withdrawing group (E) can be a carboxamide or ester. In some embodiments, the carbon-carbon double bond of the electron withdrawing group (E) and the Michael acceptor is a cyclic dicarboxyl group such that the two carboacyl groups on the nitrogen together with the nitrogen itself form a 1H-pyrrole-2,5-dione structure. It may be part of maleimide, which is a voximide.

In some embodiments, the functional group can have the formula:

here:

Linker C is a spacer that is absent or 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 amino group.

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

In some embodiments, the functional group can have the formula:

Here, Linker C is a spacer with one end attached to the nitrogen of the carboxamide and the other end attached to the 3' or 5' end of the oligonucleotide.

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

In some embodiments, Linker C in Formula 6 may include at least one PEG. PEG may have any number of repeating O-CH ₂ -CH ₂ units. For example, PEG is 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, 7, PEG48, PEG49, PEG50, PEG51, PEG52, PEG53, PEG54, PEG55, PEG56, PEG57, PEG58, PEG59, PEG60, PEG61, PEG62, PEG63, PEG64, PEG65, PEG66, PEG67, PEG68, PEG69, PEG70, PEG71, 2, PEG73, PEG74, PEG75, PEG76, PEG77, PEG78, PEG79, PEG80, PEG81, PEG82, PEG83, PEG84, PEG85, PEG86, PEG87, PEG88, PEG89, PEG90, PEG91, PEG92, PEG93, PEG94, PEG95, PEG96, 7, It may be PEG98, PEG99, PEG100, or higher.

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

In some embodiments, Linker C in Formula 6 can include at least one cycloalkyl group. In some embodiments, a cycloalkyl group is C3 cycloalkyl (i.e., cyclopropane), C4 cycloalkyl (i.e., cyclobutene), C5 cycloalkyl (i.e., cyclopentane), C6 cycloalkyl (i.e., cyclohexane), C7 cycloalkyl It may be alkyl (i.e. cycloheptane), C8 cycloalkyl (i.e. cyclooctane), C9 cycloalkyl (i.e. cyclononane), or C10 cycloalkyl (i.e. cyclodecane).

In some embodiments, Linker C in Formula 6 may include at least one heteroatom. In some embodiments, Linker C has 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. It can be included.

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

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

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

In some embodiments, 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. Linker C is not limited to containing one or more specific amino acids. For example, linker C contains 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, and one or more glutamic acids (Glu). ) amino acids, 1 or more serine (Ser) amino acids, 1 or more threonine (Thr) amino acids, 1 or more asparagine (Asn) amino acids, 1 or more glutamine (Gln) amino acids, 1 or more cysteine (Cys) amino acids, 1 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 acid, 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) May contain amino acids.

In some embodiments, Linker C in Formula 6 may comprise at least one nucleotide. A variety of nucleotides are known to those skilled in the art. Linker C is not limited to containing one or more specific nucleotides. For example, linker C is one or more nucleotides containing a guanine nucleobase, one or more nucleotides containing an adenine nucleobase, one or more nucleotides containing a cytosine nucleobase, and one or more nucleotides containing a thymine nucleobase. , and/or may include one or more nucleotides containing a uracil nucleobase. In some embodiments, Linker C may include at least one abasic nucleotide. As is known in the art, an abasic nucleotide is a nucleotide that has an abasic site, which is a position that has neither a purine base nor a pyrimidine base. For example, Linker C may include one or more types of abasic DNA and/or one or more types of abasic RNA. In some embodiments, Linker C may include at least one inverted basic nucleotide. For example, Linker C may comprise one or more inverted abasic DNA and/or one or more inverted abasic RNA.

In some embodiments, Linker C in Formula 6 may comprise at least one saccharide. In some embodiments, Linker C comprises 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 may comprise at least one glucosamine monosaccharide unit.

In some embodiments, Linker C in Formula 6 may include one or more of the following:

here:

j is an integer from 1 to 12,

k is an integer from 0 to 12.

In some embodiments, the functional group can have the formula:

Here Linker C is a spacer with 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.

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

In some embodiments, Linker C in Formula 7 may include at least one PEG. PEG may have any number of repeating O-CH ₂ -CH ₂ units. For example, PEG is 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, 7, PEG48, PEG49, PEG50, PEG51, PEG52, PEG53, PEG54, PEG55, PEG56, PEG57, PEG58, PEG59, PEG60, PEG61, PEG62, PEG63, PEG64, PEG65, PEG66, PEG67, PEG68, PEG69, PEG70, PEG71, 2, PEG73, PEG74, PEG75, PEG76, PEG77, PEG78, PEG79, PEG80, PEG81, PEG82, PEG83, PEG84, PEG85, PEG86, PEG87, PEG88, PEG89, PEG90, PEG91, PEG92, PEG93, PEG94, PEG95, PEG96, 7, It may be PEG98, PEG99, PEG100, or higher.

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

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

In some embodiments, Linker C in Formula 7 may include one or more of the following:

here:

j is an integer from 1 to 12,

k is an integer from 0 to 12.

Multivalent ligand cluster containing GalNAc targeting ligand

In some embodiments, multivalent ligand clusters of the present disclosure can have the general structure of Formula 8:

here:

m is an integer from 1 to 10,

Each n is an independently selected integer from 1 to 10,

Each Linker A is an independently selected spacer with one end attached to TL and the other end attached to the nitrogen of an alkylcarboxamide,

Linker B is a spacer having one end attached to a pharmaceutical agent or a functional group capable of being linked to one or more pharmaceutical agents 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.

In some embodiments, multivalent ligand clusters of the present disclosure can have the general structure of Formula 9:

here:

m is an integer from 1 to 10,

Each n is an independently selected integer from 1 to 10,

Each linker A is an independently selected spacer,

Linker B is a spacer,

Linker C is a spacer or absent,

X is a methyl group, oxygen, sulfur or amino group,

Y is oxygen, sulfur or amino group.

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

here:

m is an integer from 1 to 10,

Each n is an independently selected integer from 1 to 10,

Each linker A is an independently selected spacer,

Linker B is a spacer,

Linker C is a spacer.

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

here:

m is an integer from 1 to 10,

Each n is an independently selected integer from 1 to 10,

Each linker A is an independently selected spacer,

Linker B is a spacer,

Linker C is a spacer.

Below are examples of multivalent ligand clusters comprising GalNAc or protected GalNAc targeting ligands, various "m" from Formula 1, various "n" from Formula 1, and various functional groups that can be linked to one or more pharmaceutical agents. The chemical formula is:

First Example Manufacturing Method

One method for preparing an example of a compound having Formula 1 is shown in Scheme 1 below. Starting materials and intermediates can be purchased from commercial sources, prepared from known procedures, or otherwise exemplified. The order in which the steps in the reaction scheme are performed may vary.

Scheme 1

Scheme 1 starts with a mono-protected diamine (Compound I). A single-protected diamine includes a first nitrogen and a second nitrogen, where the first nitrogen is a primary amine and the second nitrogen is a secondary amine that includes a protecting group (PG) in Scheme 1.

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

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

Starting with compound I, triester compound II can be synthesized in one step. In some embodiments, Compound II can be synthesized via a S _N 2 substitution reaction using Compound I and one or more suitable substrates. In some embodiments, Compound II can be synthesized via a reductive amination reaction using Compound I and one or more suitable substrates. In some embodiments, Compound II can be synthesized via a Michael addition reaction using Compound I and one or more suitable substrates.

As illustrated in Scheme 1, compound II is generated from various protected carboxylic acids coupled to compound I. In compound II, the first nitrogen is a tertiary amine comprising a first protected carboxylic acid and a second protected carboxylic acid, and the second nitrogen is a tertiary amine comprising a protecting group and a third protected carboxylic acid. am.

In Scheme 1, each “n” is an independently selected integer. In some embodiments, each n in Scheme 1 is an independently selected integer from 0 to 10.

Compound III is prepared by deprotecting the secondary nitrogen of Compound II such that the secondary nitrogen of Compound III becomes a secondary amine containing a third protected carboxylic acid. In embodiments where the protecting group is a benzyl group, compound III can be prepared by performing a hydrogenation reaction using compound II. In embodiments where the protecting group is a triphenylmethyl group, compound III can be prepared 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).

Compound IV attaches a moiety comprising a hydroxy group to the second nitrogen of Compound III such that the second nitrogen of Compound IV is a tertiary amine comprising a third protected carboxylic acid and a moiety comprising a hydroxy group. It is prepared by turning it into an amide. The moiety comprising a hydroxy group can be attached to the second nitrogen using any of the linkers B described herein above.

In some embodiments, preparing triester compound IV includes carrying out a S _N 2 reaction using compound III and one or more suitable substrates. In some embodiments, preparing Compound IV includes performing a reductive amination reaction using Compound III and one or more suitable substrates. In some embodiments, preparing Compound IV includes performing a Michael addition reaction using Compound III and one or more suitable substrates. In some embodiments, preparing Compound IV includes performing an amide coupling reaction using Compound III and one or more appropriate substrates. In some embodiments, preparing 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.

Triic acid compound V is prepared by converting the protected carboxylic acid of compound IV to a carboxylic acid. In some embodiments, Compound V can be prepared 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, one or more acids may include hydrochloric acid (HCl), hydrobromic acid (HBr), trifluoroacetic acid (TFA), and formic acid. In some embodiments, preparing 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, preparing Compound V may include performing a hydrolysis reaction using Compound IV.

Compound VI can be prepared by performing an amide coupling reaction using compound V. In compound VI, the first nitrogen is a tertiary amine comprising a first amide and a second amide, and the second nitrogen is a tertiary amine comprising a moiety comprising a hydroxy group and a tertiary amide. Each of the first amide, second amide and third amide can independently couple to a selected targeting ligand. Linker A in Scheme 1 may be any Linker A described herein. TL in Scheme 1 may be any TL described herein.

Compound VII is prepared 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 conversion of a hydroxy group to a phosphoramidite group can be performed after performing an amide coupling reaction to prepare Compound VI.

Second Example Manufacturing Method

Another method for preparing an example of a compound having Formula 1 is shown in Scheme 2 below. Scheme 2 allows compounds of Formula 1 to have one or more different targeting ligands. Scheme 2 allows stepwise introduction of targeting ligands. Starting materials and intermediates can be purchased from commercial sources, prepared from known procedures, or otherwise exemplified. The order in which the steps in the reaction scheme are performed may vary.

Scheme 2

Scheme 2 starts with a double-protected diamine (Compound I). A double-protected diamine comprises a first nitrogen and a second nitrogen, where the first nitrogen is a secondary amine (PG ¹ ) comprising a first protecting group and the second nitrogen is a primary or secondary amine comprising a second protecting group. It is a secondary amine (PG ² ).

The first and second protecting groups can be different, thereby allowing different Linker A and targeting ligands to attach to the diamine scaffold. A variety of protecting groups are known to those skilled in the art and may 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.

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

Compound II can be prepared by coupling a first protected carboxylic acid to the first nitrogen of Compound I such that the first nitrogen of Compound II is a tertiary amine. Those skilled in the art will recognize that by distinguishing the protecting groups in Compound I, protecting groups can be strategically removed and replaced. For example, in embodiments where the first protecting group is a benzyl group and the second protecting group is a Boc group, the benzyl group-bearing amine (but not the Boc group-bearing amine) of Compound I can be subjected to a _SN 2 substitution reaction with one or more suitable reagents, reducing Compound II can be formed through an amination reaction or a Michael addition reaction.

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

Compound IV can be prepared by coupling a second protected carboxylic acid to the first nitrogen of compound III to prepare the first nitrogen of compound IV, which is a tertiary amine. In some embodiments, preparing Compound IV may include performing a S _N 2 substitution reaction using Compound III and one or more other suitable reagents. In some embodiments, preparing Compound IV may include performing a reductive amination reaction using Compound III and one or more other suitable reagents. In some embodiments, preparing Compound IV may include performing a Michael addition reaction using Compound III and one or more other suitable reagents. In some embodiments, preparing Compound IV may include performing an amide coupling reaction using Compound III and one or more other suitable reagents. In some embodiments, preparing Compound IV may include performing a nucleophilic addition reaction using Compound III and one or more other suitable reagents.

Compound V is obtained by removing the second protecting group from Compound IV such that the first nitrogen of Compound V is a tertiary amine comprising a first protected carboxylic acid and a second protected carboxylic acid, and the second nitrogen of Compound V is a tertiary amine comprising a first protected carboxylic acid and a second protected carboxylic acid. It can be prepared by making it a primary amine. In some embodiments (e.g., when the second protecting group is a Boc group), compound V can be prepared 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 prepared by coupling a third protected carboxylic acid to the second nitrogen of Compound V to prepare the second nitrogen of Compound VI, which is a secondary amine. In some embodiments, Compound VI can be produced by carrying out a S _N 2 substitution reaction using Compound V and one or more other suitable reagents. 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 prepared by performing a Michael addition reaction using Compound V and one or more other suitable reagents.

Compound VII can be prepared 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 comprising 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 prepared by carrying out a S _N 2 substitution reaction using Compound VI and one or more other suitable reagents. In some embodiments, Compound VII can be prepared by performing a reductive amination reaction using Compound VI and one or more other suitable reagents. In some embodiments, Compound VII can be prepared by performing a Michael addition reaction using Compound VI and one or more other suitable reagents. In some embodiments, Compound VII can be prepared by performing an amide coupling reaction using Compound VI and one or more other suitable reagents. In some embodiments, Compound VII can be prepared by performing a nucleophilic addition reaction using Compound VI and one or more other suitable reagents.

In the example of Scheme 2, R ^a , R ^b and R ^c may be different enough to allow the targeting ligand to attach selectively, for example, in one scenario R ^a , R ^b and R ^c are methyl, benzyl and It may be a tert-butyl group. The following describes this selective attachment of targeting ligands.

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

Compound IX can be prepared by performing an amide coupling reaction using compound VIII. In compound IX, the first nitrogen comprises a first protected carboxylic acid and a second protected carboxylic acid, and the second nitrogen in compound IX comprises a first amide and a hydroxyl group with a first targeting ligand coupled thereto. Includes a moiety containing a group.

Compound X is prepared by converting the second protected carboxylic acid of Compound IX to a second carboxylic acid. In some embodiments, preparing ^Compound

Compound XI can be prepared by performing an amide coupling reaction using compound X. In Compound XI, the first nitrogen comprises a first protected carboxylic acid and a second amide with a second targeting ligand coupled thereto, and the second nitrogen in Compound and a moiety comprising a first amide and a hydroxy group.

Compound XII is prepared by converting the first protected carboxylic acid of Compound XI to a third carboxylic acid. In some embodiments, preparing Compound XII may include performing a hydrolysis reaction using Compound XI (e.g., when R ^a in Scheme 2 is a methyl group).

Compound XIII can be prepared by performing an amide coupling reaction using compound XII. In compound and a moiety comprising a first amide and hydroxy group with a first targeting ligand.

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

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

The hydroxy group can be coupled to the second nitrogen using any of the linkers B described herein.

In some embodiments, a hydroxy group can be converted to a phosphoramidite group using a phosphitylation reaction. In some embodiments, a hydroxy group can be converted to a phosphoramidite group to prepare Compound XIV.

In Scheme 2, each “n ^x ”, “n ^y ” or “n ^z ” is an independently selected integer. In some embodiments, each “n ^x ”, “n ^y ” or “n ^z ” in Scheme 2 is an independently selected integer from 0 to 10.

Specific manufacturing and use factors

Embodiments of multivalent ligand clusters of the invention can be prepared and used to deliver oligonucleotide agents to cells, tissues, and organs. Non-limiting examples of agents that can be delivered include therapeutic agents such as siRNA. Delivery methods using the multivalent ligand clusters of the invention can be used to deliver siRNAs and other agents conjugated to targeting ligand clusters of the invention to cells in vitro and in vivo. The multivalent ligand clusters of the invention can be used as a delivery vehicle to deliver agents, including but not limited to nucleic acids, to cells. As used herein, the term “multivalent ligand cluster/pharmaceutical agent complex” means a multivalent ligand cluster as described herein linked to a pharmaceutical agent as described herein. In some embodiments of the invention, the pharmaceutical agent is siRNA.

In another aspect of the disclosure, the dsRNA agent comprises 2'-fluoro modified nucleotides at positions 2, 7, 12, 14, and 16 of the antisense strand (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, other positions of the dsRNA agent do not contain 2' fluorine-modified nucleotides. In some embodiments, all nucleotides in the antisense strand and/or sense strand of the dsRNA agent are modified nucleotides. In some embodiments, the dsRNA agent contains a 2'-fluorine-modified nucleotide at positions 2, 7, 12, 14, and 16 of the antisense strand and/or a 2'-fluorine-modified nucleotide at positions 9, 11, and 13 of the sense strand. nucleotides, and other positions include 2'-O-methyl nucleotide, 2'-deoxy nucleotide, 2'3'-seco nucleotide mimetic, locked nucleotide, unlocked nucleic acid nucleotide (UNA), glycol nucleic acid nucleotide (GNA), 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 nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, and 3'-OMe nucleotides, nucleotides containing a 5'-phosphorothioate group, or cholesteryl derivatives or dodecanoic acid. It contains a modified nucleotide selected from a terminal nucleotide linked to a bisdecylamide group, a 2'-amino-modified nucleotide, a phosphoramidate, or a non-natural base containing nucleotide. In some embodiments, the dsRNA agent comprises an E-vinylphosphonate nucleotide at the 5' end of the guide strand. In certain embodiments, the dsRNA agent comprises at least one phosphorothioate internucleoside linkage. In certain embodiments, the sense strand includes at least one phosphorothioate internucleoside linkage. In some embodiments, the antisense strand comprises at least one phosphorothioate internucleoside linkage. In some embodiments, the sense strand comprises 1, 2, 3, 4, 5, or 6 phosphorothioate internucleoside linkages. In some embodiments, the antisense strand comprises 1, 2, 3, 4, 5, or 6 phosphorothioate internucleoside linkages. In certain embodiments, the sense strand is complementary or substantially complementary to the antisense strand, and the region of complementarity is 16 to 23 nucleotides in length. In some embodiments, the region of complementarity is 19-21 nucleotides in length. In certain embodiments, the region of complementarity is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, each strand is no more than 30 nucleotides in length. In some embodiments, each strand is no more than 25 nucleotides in length. In some embodiments, each strand is no more than 23 nucleotides in length. In certain embodiments, the dsRNA agent comprises at least one modified nucleotide and further comprises one or more targeting or linking groups. In some embodiments, one or more targeting groups or linking groups are conjugated to the sense strand. In some embodiments, the targeting group comprises N-acetyl-galactosamine (GalNAc). In some embodiments, the targeting group contains the structure of GalNAc described above.

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

cells and objects

As used herein, subject shall mean a human or vertebrate mammal, including but not limited to dogs, cats, horses, goats, cattle, sheep, rodents and primates, such as monkeys. Accordingly, the present invention can be used to treat diseases 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 human prophylactic and therapeutic regimens. In some embodiments of the invention, the vertebrate subject is a mammal.

In certain embodiments of the invention, the multivalent ligand cluster/pharmaceutical agent complex of the invention is delivered to and contacted with a cell. In some embodiments of the invention, the contacted cells are in culture, and in other embodiments, the contacted cells are in a subject. The types of cells that can be contacted with the multivalent ligand cluster/pharmaceutical agent complex of the invention include liver cells, muscle cells, cardiac cells, circulating cells, neuronal cells, glial cells, adipocytes, skin cells, hematopoietic cells, epithelial cells, and spermatozoa. , oocytes, muscle cells, adipocytes, kidney cells, hepatocytes, or pancreatic cells. In some embodiments, the cells contacted with the multivalent ligand cluster/pharmaceutical agent complex of the invention are liver cells.

dosage

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

Pharmaceutical compositions may be in sterile injectable aqueous suspension or solution, or in lyophilized form. Pharmaceutical compositions and medicaments of the present disclosure can be administered to subjects in pharmaceutically effective doses.

Method of administration

A variety of routes of administration are available for the multivalent ligand cluster/pharmaceutical agent complexes of the invention. The particular mode of delivery chosen will depend on the particular condition being treated and the dosage required for therapeutic efficacy. In general, the methods of the present invention can be practiced using any medically acceptable mode of administration, meaning any mode that produces an effective level of treatment without causing clinically unacceptable side effects. In some embodiments of the invention, the multivalent ligand cluster/pharmaceutical agent complexes of the invention may be administered via oral, enteral, mucosal, transdermal and/or parenteral routes. The term “parenteral” includes subcutaneous, intravenous, intramuscular, intraperitoneal and intracisternal injection, or infusion techniques. Other routes include, but are not limited to, the nasal cavity (e.g., via the gastro-nasal tube), cutaneous, vaginal, rectal, and sublingual. The route of delivery of the invention may include intrathecally, intracerebroventricularly, or intracranially. In some embodiments of the invention, the multivalent ligand cluster/pharmaceutical agent complex of the invention can be placed in a sustained release matrix and administered by placement of the matrix in a subject.

The multivalent ligand cluster/pharmaceutical agent complex of the invention may be administered as a pharmaceutically acceptable solution, which may routinely contain pharmaceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients. It can be administered as a preparation that can be administered. According to the methods of the invention, the multivalent ligand cluster/pharmaceutical agent complex can be administered as a pharmaceutical composition. Generally, pharmaceutical compositions comprise the multivalent ligand cluster/pharmaceutical agent complex of the invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known to those skilled in the art and can be selected and used using commercial methods. As used herein, a pharmaceutically acceptable carrier refers to the effectiveness of the biological activity of the active ingredient (e.g., the ability to prevent and/or treat the disease or condition for which the delivered nucleic acid, e.g., siRNA, is directed). means a non-toxic substance that does not interfere with

Pharmaceutically acceptable carriers may include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other substances well known in the art. Exemplary pharmaceutically acceptable carriers are described in U.S. Pat. No. 5,211,657, and others are known to those skilled in the art. These preparations typically may contain salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents. When used in medicine, the salts must be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof and are not excluded from the scope of the present invention. Such pharmacologically and pharmaceutically 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, etc. It doesn't work. Pharmaceutically acceptable salts can also be prepared as alkali metal or alkaline earth metal salts, such as sodium, potassium or calcium salts.

In some embodiments of the invention, the multivalent ligand cluster/pharmaceutical agent complexes of the invention may be administered directly to tissues. Direct tissue administration can be accomplished by direct injection, or other means known in the art. The multivalent ligand cluster/pharmaceutical agent complex of the invention may be administered in a single administration, or alternatively, may be administered in multiple administrations. When administered multiple times, the multivalent ligand cluster/pharmaceutical agent complex of the invention may be administered via different routes. For example, the first (or first several) administrations may be directly to the affected tissue or organ, whereas later administrations may be systemic.

The multivalent ligand cluster/pharmaceutical agent complexes of the invention may be formulated for parenteral administration by injection (e.g., bolus injection or continuous infusion), when it is desirable to administer systemically. Formulations for injection may be presented in unit dosage form (e.g., ampoules or multi-dose containers) with or without added preservatives. Pharmaceutical compositions may take the form of suspensions, solutions or emulsions in oily or aqueous vehicles and may contain formulation agents such as suspending agents, stabilizing agents and/or dispersing agents.

Formulations 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 such as antimicrobial agents, antioxidants, chelating agents and inert gases may also be present. Lower doses will result from other forms of administration, such as intravenous administration. If the response in the subject is insufficient at the initial dose applied, higher doses (or effectively higher doses by a different, more localized route of delivery) may be used to the extent patient tolerance allows. Multiple doses per day may be used, as needed, to achieve appropriate systemic or local levels of one or more multivalent ligand cluster/pharmaceutical agent complexes of the invention to produce desired levels of pharmaceutical agent, e.g., desired levels of siRNA. You can.

Both non-biodegradable and biodegradable polymer matrices can be used to deliver one or more multivalent ligand cluster/pharmaceutical agent complexes of the invention to cells and/or subjects. In some embodiments, the matrix may be biodegradable. The matrix polymer may be a natural or synthetic polymer. The polymer may be selected based on the time period for which release is desired, typically on the order of hours to a year or more. Typically, release over a period ranging from a few hours to 3 to 12 months may be used. The polymer is optionally in the form of a hydrogel capable of absorbing up to about 90% of its weight in water, and is further optionally crosslinked with multivalent ions or other polymers.

In certain embodiments of the invention, the multivalent ligand cluster/pharmaceutical agent complexes of the invention can be delivered by diffusion using bioerodible implants, or by degradation of the polymeric matrix. Exemplary synthetic polymers for this use are well known in the art. Biodegradable and non-biodegradable polymers can be used for delivery of one or more multivalent ligand cluster/pharmaceutical agent complexes of the invention using methods known in the art. These methods can also be used to deliver one or more multivalent ligand cluster/pharmaceutical agent complexes of the invention for treatment. Additional suitable delivery systems may include time-release, delayed-release or sustained-release delivery systems. Such a system can avoid repeated administration of the multivalent ligand cluster/pharmaceutical agent complex of the invention, increasing convenience for subjects and health care providers. Many types of emission delivery systems are available and known to those skilled in the art. [For example, U.S. Patent No. 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 can be used, some of which are suitable for implantation.

The use of long-term sustained release implants may be particularly suitable for prophylactic treatment of subjects, and for subjects at risk of developing a recurrent disease or condition to be prevented and/or treated with siRNA delivered using the multivalent ligand clusters of the invention. . As used herein, prolonged release 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 more. Long-term sustained release implants are well known to those skilled in the art and include some of the release systems described above.

Therapeutic formulations of one or more multivalent ligand cluster/pharmaceutical agent complexes of the present invention can be prepared by combining the multivalent ligand cluster/pharmaceutical agent complex of the desired degree of purity with an optional pharmaceutically acceptable carrier, excipient or stabilizer [Remington's Pharmaceutical Sciences 21 ^st. edition, (2006)] and can be prepared for storage in the form of a lyophilized preparation or an aqueous solution. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate and other organic acids; Antioxidants such as ascorbic acid and methionine; Preservatives (such as octadecyldimethylbenzyl ammonium 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; Proteins such as serum albumin, gelatin or immunoglobulin; Hydrophilic polymers such as polyvinylpyrrolidone; Amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates such as glucose, mannose or dextrins; Chelating agents such as EDTA; Sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (eg, Zn-protein complexes); and/or non-ionic surfactants such as TWEEN®, PLURONICS® or polyethylene glycol (PEG).

The multivalent ligand cluster/pharmaceutical agent complexes of the present disclosure can be formulated as pharmaceutical compositions. Pharmaceutical compositions can be used as medicines alone or in combination with other agents. The multivalent ligand cluster/pharmaceutical agent complexes of the present disclosure can also be combined with other therapeutic compounds and administered separately or simultaneously (e.g., as combined unit doses). In at least some embodiments, the present disclosure provides pharmaceutical compositions comprising one or more multivalent ligand cluster/pharmaceutical agent complexes according to the present disclosure in physiologically/pharmaceutically acceptable excipients such as stabilizers, preservatives, diluents, buffers, etc. Includes.

The pharmaceutical compositions of the present invention can be administered alone, in combination with each other, and/or in combination with other drug therapies or other treatment regimens administered to a subject with a disease or condition. Pharmaceutical compositions used in embodiments of the invention are preferably sterile and contain an effective amount of a multivalent ligand cluster/pharmaceutical agent complex to prevent or treat the disease or condition for which the pharmaceutical agent, e.g., siRNA, is directed.

The dose or doses of the pharmaceutical composition of the invention sufficient to treat the disease or condition when administered to a subject may be selected according to different parameters, especially depending on the mode of administration used and the condition of the subject. Other factors may include the desired treatment duration. If the response in the subject is insufficient at the initial dose applied, higher doses (or effectively higher doses by a different, more localized route of delivery) may be used to the extent patient tolerance allows. In some embodiments of the invention, dosing determined using commercial means, such as in clinical trials, is used.

Example

Example 1. Multivalent Ligand Cluster Containing GalNAc Targeting Ligand

In some embodiments of the invention, the multivalent ligand cluster may comprise a GalNAc targeting ligand. The following is an acetyl group protected GalNAc targeting ligand, core C2 and C3 diamines, branched acetyl and propanoyl amides, PEG2 and PEG3 linker A, various linker B as described herein, and one or more pharmaceuticals as described herein. This is an example compound of a multivalent ligand cluster containing various functional groups that can be linked to an agonist. The acetyl protecting group on the following GalNAc ligand can be easily removed after conjugation is complete to generate a GalNAc targeting ligand.

Example 2. Preparation of intermediate compounds

In Scheme 3 below, Intermediate-A was synthesized by treating commercially available galactosamine pentaacetate (Compound I) with trimethylsilyl trifluoromethanesulfonate (TMSOTf) in dichloromethane (DCM). Subsequent glycosylation with Cbz protected 2-(2-aminoethoxy)ethane-1-ol afforded compound II. The Cbz protecting group was removed by hydrogenation to give Intermediate-A as trifluoroacetate (TFA) or HCl salt. Intermediate B was synthesized based on the same scheme except that Cbz protected 2-(2-(2-aminoethoxy)ethoxy)ethan-1-ol was used as starting material. Scheme 3 allows access to modifications of linker A as well as modifications of the targeting ligand.

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 hours and then at 25°C for 1 hour. Cbz-protected 2-(2-aminoethoxy)ethan-1-ol (13.5 g, 56.5 mmol) in DCE (100 mL) dried over 4 Å powder molecular sieves (10 g) was added to the above-mentioned reaction solution with N It was added dropwise at 0°C under ₂ atmosphere. The resulting reaction mixture was stirred at 25°C for 16 hours under N ₂ atmosphere. The reaction mixture was filtered and washed with saturated NaHCO ₃ (200 mL), water (200 mL), and saturated brine (200 mL). The organic layer was dried over anhydrous Na ₂ SO ₄ , 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. Processing, filtering, and drying gave Compound II (15.0 g, 50.3% yield) as a white solid.

Compound II (15.0 g, 26.4 mmol) in 10% Pd/C (1.50 g) followed by THF (10 mL) followed by THF (300 mL) and TFA (3.00 g, 26.4 mmol) in a dried and argon purged hydrogenation bottle. The solution was carefully added. The resulting mixture was degassed, purged three times with H ₂ and stirred at 25° C. for 3 hours under an H ₂ (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 dry DCM (500 mL) and concentrated. This process was repeated three times to obtain Intermediate-A (14.0 g, 96.5% yield) as a foamy white solid. ¹ H NMR (400 MHz DMSO-d ₆ ): 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).

Intermediate-B was synthesized using a procedure similar to the synthesis of Intermediate-A. ¹ H NMR (400 MHz DMSO-d ₆ ): 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) . Calculated mass C ₂₀ H ₃₄ N ₂ O ₁₁ : 478.22; Actual value: 479.3 (M+H ⁺ ).

Example 3. Preparation of Compound 1

Compound 1 identified in Example 1 was prepared using Scheme 4 below. Commercially available 2,2',2",2"'-(propane-1,3-diylbis(azantriyl))tetraacetic acid (Compound I in Scheme 4) was converted to the dianhydride Compound II. Upon treatment with 6-aminohexan-1-ol followed by hydrolysis, compound II was converted to 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 give phosphoramidite compound 1.

Scheme 4

To a stirred solution of Ac ₂ O (8.83 g, 86.5 mmol) and pyridine (193 mg, 2.45 mmol) was added tetrahydrate I (5.0 g, 16.3 mmol). After purging with N ₂ three times, the reaction mixture was stirred at 65° C. for 12 hours under N ₂ atmosphere. After cooling, the reaction mixture was filtered to remove insoluble solids. The filtrate was concentrated under vacuum. Toluene was added to the residue and volatiles were evaporated. This process was repeated three times to obtain compound II (2.20 g, 49.8% yield) as a yellow oil. ¹ H NMR (400 MHz DMSO-d ₆ ): δ ppm 3.65 (s, 8 H), 2.46 (br t, J = 7.19 Hz, 4 H), 1.55 - 1.65 (m, 2 H).

6-aminohexan-1-ol (624 mg, 5.33 mmol) and pyridine (263 mg, 3.33 mmol) in a mixture of compound II (1.80 g, 6.66 mmol) and imidazole (3.63 g, 53.2 mmol) in DMF (18 mL) mmol) were added sequentially. The mixture was stirred at 50° C. for 5 hours under N ₂ atmosphere. The reaction mixture was concentrated under reduced pressure. The residue was purified by reverse phase preparative HPLC. Compound III was obtained (1.90 g, containing 8.4% DMF and 63.2% imidazole by weight). ¹ H NMR (400 MHz DMSO-d ₆ ): 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).

DIEA (385 mg, 2.99 mmol), HOBt (358 mg, 2.65 mmol) and EDC (508 mg, 2.65 mmol) was added sequentially. The resulting reaction mixture was stirred at 25°C for 3 hours under N ₂ atmosphere. LC-MS showed the desired product. The reaction mixture was purified by reverse phase preparative HPLC. Fractions containing the desired product were combined and concentrated to give compound IV (200 mg, 18.2% yield) as a white solid. ¹ H NMR (400 MHz DMSO-d ₆ ): 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.

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 3-bis(diisopropylamino)phosphanyloxy. Propannitrile (145 mg, 483 umol) was added dropwise at 25°C under N ₂ . The reaction mixture was stirred at 25°C for 2 hours. LC-MS showed that Compound IV was completely consumed. The reaction was quenched by addition of a mixture of brine and saturated NaHCO ₃ 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 portion was washed with brine/saturated aqueous NaHCO ₃ solution (1:1, 5 mL), dried over Na ₂ SO ₄ , filtered, and concentrated to a volume of ~1 mL. This solution was added dropwise to MTBE (20 mL) with stirring. This formed a white solid, which was isolated by centrifugation. This process was repeated one more time. The solid was then dissolved in anhydrous CH ₃ CN and volatiles were removed. This process was repeated three times to obtain compound 1 (103 mg, 45.9% yield) as a colorless oil. ¹ H NMR (CDCl ₃ ): 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 (CDCl ₃ ): δ ppm 147.14.

Example 4 Preparation of Compound 2

Compound 2 (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 (CDCl ₃ ): 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 (CDCl ₃ ): δ ppm 147.07.

Example 5 Preparation of Compound 3

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

Scheme 5

Starting from tert-butyl (3-aminopropyl)carbamate, compound I can be obtained by alkylation (S _N 2 substitution) with benzyl protected 2-bromoethanol. 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 the triester compound III. The tert-butyl protecting group can then be removed upon treatment with formic acid to yield triacid compound IV. Amide coupling with intermediate-A affords compound V. The benzyl protecting group can then be removed by hydrogenation to yield 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.

Example 6. Preparation of Compound 4

Compound 4 identified in Example 1 was prepared using Scheme 6 below.

Scheme 6

Starting from benzyl protected propane-1,3-diamine, this was alkylated with tert-butyl 2-bromoacetate to give the 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 upon treatment with HCl in dioxane to yield triacid compound IV. Amide coupling between triacid compound IV and intermediate-A was performed to obtain compound V. Phosphoramidite compound 4 was synthesized by phosphitylation of compound V using 2-cyanoethyl N,N-diisopropylchlorophosphoramidite and a catalytic amount of 1H-tetrazole.

To a solution of N ¹ -benzylpropane-1,3-diamine (5.00 g, 30.4 mmol) in DMF (100 mL) was added tert-butyl 2-bromoacetate (23.7 g, 121 mmol), followed by DIEA (23.61 g, 182 mmol) was added dropwise. The resulting reaction mixture was stirred at 25-30°C for 16 hours. LCMS showed complete consumption of N ¹ -benzylpropane-1,3-diamine. The reaction mixture was diluted with H ₂ O (500 mL) and extracted with EtOAc (500 mL x 2). The combined organics were washed with saturated brine (1 L), dried over anhydrous Na ₂ SO ₄ , filtered, and concentrated under reduced pressure to give the crude product, which was purified by silica gel column chromatography (gradient: petroleum ether:ethyl acetate, 20:1 to 5:1). Compound I (12.1 g, 78.4% yield) was obtained as a colorless oil. ¹ H NMR (400 MHz, CDCl ₃ ): 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 (quin, J = 7.2 Hz, 2 H), 1.44 - 1.50 (m, 27 H).

The dried hydrogenation bottle was purged three times with argon. Pd/C (200 mg, 10%) was added, followed by MeOH (5 mL) followed by a solution of Compound I (1.00 g, 1.97 mmol) in MeOH (5 mL). The reaction mixture was degassed under vacuum and recharged with H ₂ . This process was repeated three times. The mixture was stirred at 25° C. for 12 hours under H ₂ (15 psi) atmosphere. LCMS showed that Compound I was completely consumed. The reaction mixture was filtered under N ₂ atmosphere under reduced pressure. The filtrate was concentrated under reduced pressure to give compound II (655 mg, 79.7% yield) as a yellow oil, which was used in the next step without further purification. ¹ H NMR (400 MHz, CDCl ₃ ): 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 (quin, J = 7.03 Hz, 2 H), 1.44 - 1.50 (s, 27 H).

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 ( The mixture (637 mg, 4.72 mmol) was degassed, purged three times with N ₂ , and then stirred at 25° C. for 3 hours under N ₂ 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 x 2). The organics 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: petroleum ether:ethyl acetate, 5 :1 to 1:1) to give compound III (650 mg, 77.8% yield) as a yellow oil. ¹ H NMR (400 MHz, CDCl ₃ ): 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). Calculated mass C ₂₇ H ₅₀ N ₂ O ₈ : 530.36; Actual value: 531.3 (M+H ⁺ ).

A mixture of Compound III (5.5 g, 10.3 mmol) in HCl/dioxane (2M, 55 mL) was stirred at 25°C for 3 hours. LCMS showed complete consumption of Compound III. The reaction mixture was filtered, washed with EtOAc (50 mL) and dried under reduced pressure to give the crude product. This was dissolved in CH ₃ CN (50 mL) and volatiles were removed under vacuum. This process was repeated three times to obtain compound IV (2.05 g, 54.5% yield) as a white solid. ¹ H NMR (400 MHz, D ₂ O): 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).

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) in DMF (2.6 mL) , 1.45 mmol) was stirred at 25°C for 3 hours under N ₂ atmosphere. LCMS showed the desired product. The reaction mixture was purified by reverse-phase preparative HPLC to obtain compound V as a white solid (186 mg, 0.106 mmol, 25.7% yield) after lyophilization. ¹ H NMR (400 MHz, CDCl ₃ ): 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).

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 3-bis(diisopropylamino)phosphanyloxy. Propannitrile (124 mg, 0.412 mmol) was added dropwise under N ₂ at ambient temperature. The reaction mixture was stirred at 20-25°C for 2 hours. 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 NaHCO ₃ (1:1, 5 mL) at 0°C. After stirring for 1 minute, DCM (5 mL) was added. The layers were separated. The organic portion was washed with brine/saturated aqueous NaHCO ₃ solution (1:1.5 mL), dried over Na ₂ SO ₄ , filtered, and concentrated to a volume of ˜1 mL. The residue solution was added dropwise to 20 mL MTBE with stirring. This caused a white solid to precipitate. The mixture was centrifuged and the solid collected. This process was repeated one more time. The collected solid was dissolved in anhydrous CH ₃ CN. Volatile substances were removed. This process was repeated two more times to obtain compound 4 (106 mg, 52.8% yield) as a white solid. ¹ H NMR (400 MHz, CDCl ₃ ): 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, 4 H), 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 (CDCl ₃ ): ppm δ 147.12.

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. ¹ H NMR (400 MHz, CDCl ₃ ): 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, 12H). ³¹ P NMR (CDCl ₃ ): ppm δ 147.15.

Example 8. Preparation of Compound 6

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

Scheme 7

Starting from benzyl protected propane-1,3-diamine (compound I), the triester compound II can be obtained by performing a Michael addition reaction with tert-butylacrylate. Once Compound II is synthesized, the same procedure for the synthesis of Compound 4 in Scheme 6 can be followed for the remaining steps to synthesize Compound 6.

Example 9. Preparation of Compound 7

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

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 give triacid compound III. Amide coupling of compound III with intermediate-A can yield 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 give carboxylic acid compound VI. The carboxylic acid of compound VI can be converted to the tetrafluorophenyl ester by standard procedures to give compound 7.

Example 10. Preparation of Compound 8

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

Scheme 9

Compound I (compound V in Scheme 8) was coupled to the NHS conjugated maleimide compound 2,5-dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro-1H-pyrrole- Compound 8 can be obtained by reacting with 1-yl)propanoate.

Example 11. Preparation of Compound 74

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

Scheme 10

Starting from Compound I (same as Example 6 in Scheme 6, Compound II).

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

¹ H NMR: 400 MHz, DMSO-d ₆ δ 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).

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

¹ H NMR: 400 MHz, DMSO-d ₆ δ 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).

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

¹ H NMR: 400 MHz, DMSO-d ₆ δ 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).

The 2.00 L hydrogenation bottle was purged three times with Ar and dry Pd/C (9 g) was carefully added. Then, MeOH (50 mL) was added to thoroughly wet the Pd/C, followed by compound IV (90 g, 55.1 mmol, 1.00 eq) and TFA (6.29 g, 55.1 mmol, 1.00 equivalent) of the solution was added slowly. The resulting mixture was degassed and purged three times with H ₂ and then the mixture was stirred at 25° C. for 10 hours under H ₂ atmosphere. LCMS showed that Compound IV was completely consumed and one major peak had the target mass. The reaction mixture was carefully filtered under N ₂ atmosphere under reduced pressure. The filtrate was concentrated under reduced pressure to obtain compound V (160 g, 90.2% yield).

¹ H NMR: 400 MHz, DMSO-d ₆ δ 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).

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 equivalent) 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 the main peak had the desired product mass. The resulting reaction mixture was triturated with isopropyl ether twice (50 mL * 2) and vacuum dried to obtain Compound VI (crude, 5.5 g) as a brown solid.

int-D (protected (R)-3-aminopropane-1,2-diol) (952 mg, 2.42 mmol, 1.5 eq) in a solution of Compound VI (2.6 g, 1.61 mmol, 1.0 eq) in DMF (26 mL) eq.), TBTU (1.04 g, 3.23 mmol, 2.0 eq.) and DIEA (625.53 mg, 4.84 mmol, 843.03 uL, 3.0 eq.) were added. 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. This was purified by column chromatography (SiO ₂ , DCM/MeOH = 100/1 to 10/1, 0.1% Et ₃ N) to obtain compound 74 (900 mg, 28.0% yield) as a white solid.

Compound 74 ¹ H NMR: (400 MHz, DMSO-d ₆ ) 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).

Compound 73 can be prepared following the procedure described for Compound 74, except that protected (S)-3-aminopropane-1,2-diol is used instead of protected (R)-3-aminopropane-1,2-diol. was used.

Example 12. Preparation of Compound 75

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

Scheme 11

Compound II was synthesized based on Scheme 11. Starting from compound I (compound VI in Scheme 10), coupling with piperidin-4-ol gave compound II. Phosphoramidite compound 75 was synthesized by treating compound II with 2-cyanoethyl N,N diisopropylchlorophosphoramidite and a catalytic amount of 1H-tetrazole.

Compound 75 ¹ H NMR (in 400 MHz 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.

Example 13. Linker B attached to a functional group capable of being 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, Linker B may comprise a diol moiety, where one of the alcohols may be protected as a DMT ether while the other alcohol may be linked directly or indirectly to a solid phase synthesis solid support material. Upon removal of the DMT group, a free alcohol is generated, which can be phosphitylated by reaction with phosphoramidite to initiate oligonucleotide chain growth. Accordingly, the targeting ligand cluster of the present disclosure can be attached to the 3'-end of an oligonucleotide. A non-limiting list of Linker Bs of the present disclosure that can be attached to the 3'-end of an oligonucleotide includes the structures shown below and their stereoisomers:

here:

j is an integer from 0 to 12,

k is an integer from 0 to 12.

All targeting ligand clusters of the present disclosure can also be attached 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. :

Example 14. Preparation of Ligand Cluster Conjugated siRNA

Sense and antisense strand sequences of siRNA were synthesized on an oligonucleotide synthesizer using a well-established solid-phase synthesis method based on phosphoramidite chemistry. Oligonucleotide chain propagation is achieved through a four-step cycle: condensation for addition of each nucleotide, capping, oxidation and deprotection steps. The synthesis was performed on a solid support made of controlled pore glass (CPG, 1000 Å). Monomeric phosphoramidite was purchased from commercial sources. Ligand cluster-attached phosphoramidite was synthesized according to the procedure in Examples 3-12 herein. 5-Ethylthio-1H-tetrazole was used as an activator. I ₂ in THF/Py/H ₂ O and phenylacetyl disulfide (PADS) in pyridine/MeCN were used for the oxidation and sulfidation reactions, respectively. After the final solid phase synthesis step, the solid support bound oligomer was 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 reverse 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 by adding ice-cold EtOH. Annealing of equimolar complementary sense and antisense strand oligonucleotides in water was performed to form a double-stranded siRNA product, which was lyophilized to give a fluffy white solid.

Example 15. In vivo evaluation of GalNAc ligand cluster conjugated siRNA

To assess delivery efficacy, the GalNAc ligand cluster was conjugated to the 5' or 3' end of the sense strand of an active FXII siRNA known from the literature. 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 these FXII siRNAs are summarized in Table 1. Six examples of GalNAc ligand clusters conjugated to FXII siRNA are also presented in Table 1. Conjugation of the GalNAc cluster 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. Its structural diagram is presented in Table 1 below. The masses of these six compounds and the positive control compound are summarized in Table 2.

These compounds were tested for their efficacy in knocking down mouse FXII. FXII is a secreted protein produced primarily in hepatocytes. The decrease in FXII expression in plasma after siRNA treatment strongly correlates with the decrease in FXII mRNA in hepatocytes. Because these GalNAc ligand clusters are conjugated to the same FXII siRNA with known activity, delivery efficacy can be assessed and compared by measuring the degree of reduction in FXII expression in plasma for each conjugate.

Mice were given a single subcutaneous injection of 0.5 or 1 mg/kg of siRNA compound (see Table 1 below) or PBS. A literature compound (GalNAc ligand cluster conjugated to the 3'-end of the sense strand) was included in this study as a positive control. Plasma samples were collected pre-dose and on days 7, 14, and/or 28 post-dose. The concentration of mouse FXII protein was measured by ELISA assay according to literature procedures. Liu et al. “An investigational RNAi therapeutic targeting Factor XII (ALN-F12) for the treatment of hereditary angioedema”. RNA. 2019 Feb;25(2):255-263. doi: 10.1261/rna.068916.118]. Knockdown activity was calculated for percent reduction of FXII protein in mouse plasma normalized to the PBS treatment group and is summarized in Table 3. FXII siRNA conjugated with GalNAc ligands GLS-1 and GLS-2 had significant activity in knocking down mouse FXII protein expression in mouse plasma at both doses on days 7, 14, and/or 28 post administration. indicated. This activity compares favorably with the positive control. The data confirm that GalNAc ligand clusters based on diamine scaffolds attached to the 5'-end of the sense strand are highly effective for delivering siRNA into hepatocytes in vivo.

Table 1. FXII siRNA compounds. Capital letters: 2'-deoxy-2'-fluoro (2'-F) ribonucleotide; Lowercase: 2'-O-methyl (2'-OMe) ribonucleotide; (*) indicates PS connection. L96 is a trivalent GalNAc ligand cluster from the literature (GalNAc3 as in Jayaprakash, et al., (2014) J. Am. Chem. Soc., 136, 16958-16961).

Table 2. Mass of FXII siRNA compounds.

Table 3. Percentage reduction of FXII protein in mouse plasma normalized to PBS treatment group.

Example 16. In vivo evaluation of GalNAc ligand cluster conjugated siRNA

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

Here also, when Linker B is used as targeted delivery of pharmaceutical agents, it contains a 6-membered ring fragment, especially a 4-hydroxypiperidinyl group, such as in the case of compounds AD00448, AD00449, which leads to better in vivo stability and It was found to be active.

Table 4. Percentage reduction of FXII protein in mouse plasma normalized to PBS treatment group.

Example 17. In vivo evaluation of GalNAc ligand cluster conjugated siRNA AD00831

Mice were given a single subcutaneous injection of 2 mg/kg of siRNA compound or PBS. Plasma samples were collected on days 7 and 14 post-dose. The concentration of mouse FXII protein was measured by ELISA assay according to literature procedures. Liu et al. “An investigational RNAi therapeutic targeting Factor XII (ALN-F12) for the treatment of hereditary angioedema”. RNA. 2019 Feb;25(2):255-263. doi: 10.1261/rna.068916.118]. Knockdown activity was calculated for the percent reduction of FXII protein in mouse plasma normalized to the PBS treatment group. The percent knockdown on days 7 and 14 post-dose was 87% and 88%. The data confirm that GalNAc ligand clusters based on diamine scaffolds attached to the 3'-end of the sense strand are highly effective for delivering siRNA into hepatocytes in vivo.

Example 18. In vivo testing of ANGPTL3 siRNA duplex

Fourteen days prior to siRNA administration, female C57BL/6J mice were infected by intravenous administration of a solution of adeno-associated virus 8 (AAV8) vector encoding human ANGPTL3 and luciferase genes. On day 0, mice were administered subcutaneously a single dose of AD00112-2 (Table 5) or 1, 3, or 10 mg/kg in PBS. Blood samples were collected on day 0 prior to administration of siRNA and on day 7 upon termination. Serum samples were isolated, and their luciferase activity was measured according to the manufacturer's recommended protocol. Because expression of human ANGPTL3 levels correlates with the expression level of luciferase, measurement of luciferase activity is a surrogate for measuring ANGTPL3 expression. The residual percent of luciferase activity was calculated by comparing the luciferase activity in samples from before (day 0) and after (day 7) treatment of siRNA for each mouse, and control treated for the same period of time. Normalized by change in luciferase activity in samples from mice. The results are summarized in Table 6. AD00112-2 demonstrated dose-dependent activity to inhibit the expression of ANGPTL3, again confirming that diamine scaffold-based GalNAc ligand clusters are highly effective in delivering siRNA into hepatocytes in vivo.

Table 5. ANGPTL3 siRNA compounds. Capital letters: 2'-deoxy-2'-fluoro (2'-F) ribonucleotide; Lowercase: 2'-O-methyl (2'-OMe) ribonucleotide; (*) indicates PS connection.

Table 6 provides experimental results (percent reduction in luciferase activity) of the in vivo study. The duplex sequence and modifications of AD00112-2 are shown in Table 5.

equivalent

Although several embodiments of the invention have been described and illustrated herein, those skilled in the art will recognize various other means and/or structures for performing the functions and/or obtaining the results and/or one or more advantages described herein. As will be readily contemplated, each such change and/or modification is considered to be within the scope of the present invention. More generally, those skilled in the art will understand that all parameters, dimensions, materials and/or configurations set forth herein are intended to be exemplary and that actual parameters, dimensions, materials and/or configurations are not intended to reflect the specific application in which the teachings of the present invention are used. Alternatively, it will be readily appreciated that it will vary depending on the applications. 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. Accordingly, it is to be understood that the above embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention relates to each individual feature, system, article, material and/or method described herein. Additionally, any combination of two or more such features, systems, articles, materials and/or methods is included within the scope of the present invention provided that such features, systems, articles, materials and/or methods are not mutually contradictory. .

All definitions defined and used herein are to be understood to supersede dictionary definitions, definitions in documents incorporated by reference, and/or the ordinary meaning of the defined term.

As used herein in the specification and claims, the singular forms “a,” “an,” and “an” should be understood to mean “at least one,” unless clearly indicated otherwise.

As used herein in the specification and claims, the phrase "and/or" refers to "either or both" of the elements so combined, i.e., elements that exist in combination in some instances and separately in other instances. It should be understood as Unless explicitly indicated otherwise, elements other than those specifically identified by the “and/or” clause may optionally be present, whether or not related to the specifically identified element.

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

Sequence list electronic file attached

Claims (188)

A compound for targeted delivery of one or more pharmaceutical agents, having the formula:

here:
Each TL is an independently selected targeting ligand,
m is an integer from 1 to 10,
Each n is an independently selected integer from 1 to 10,
Each linker A is an independently selected spacer,
Linker B is a spacer,
W is one or more pharmaceutical agents or a functional group that can be linked to one or more pharmaceutical agents. The compound of claim 1, wherein m is 1. The compound according to claim 1, wherein m is 2. The method of any one of claims 1 to 3, wherein at least one of the independently selected TLs is capable of binding to one or more cell receptors, cell channels, and cell transporters capable of facilitating endocytosis. Phosphorus compounds. 5. The compound of claim 4, wherein at least one of the independently selected TLs comprises at least one small molecule ligand. 6. The method of claim 5, wherein the at least one small molecule is N-acetylgalactosamine, galactose, galactosamine, N-formyl-galactosamine, N-propionylgalactosamine, N-butanoylgalactosamine and A compound comprising at least one of N-iso-butanoylgalactosamine, macrocycle, folate molecule, fatty acid, bile acid, and cholesterol. 5. The compound of claim 4, wherein at least one of the independently selected TLs comprises at least one peptide. 8. The compound of claim 7, wherein at least one of the independently selected TLs comprises at least one cyclic peptide. 5. The compound of claim 4, wherein at least one of the independently selected TLs comprises at least one aptamer. 10. The compound according to any one of claims 4 to 9, wherein at least one of the independently selected TLs is capable of binding to at least one asialoglycoprotein receptor (ASGPR). 10. The compound according to any one of claims 4 to 9, wherein at least one of the independently selected TLs is capable of binding to at least one type of transferrin receptor. The compound according to any one of claims 4 to 9, wherein at least one of the independently selected TLs is capable of binding to at least one integrin receptor. 10. The compound according to any one of claims 4 to 9, wherein at least one of the independently selected TLs is capable of binding to at least one type of folate receptor. 10. The compound according to any one of claims 4 to 9, wherein at least one of the independently selected TLs is capable of binding to at least one G-protein-coupled receptor (GPCR). 15. The method of any one of claims 1 to 14, wherein at least one of the independently selected linkers A is polyethylene glycol, an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an alkynyl group, an aryl group, an ar A compound comprising at least one of an alkyl group, an aralkenyl group, and an aralkynyl group. 16. The compound according to any one of claims 1 to 15, wherein at least one of the independently selected linkers A comprises at least one heteroatom. 17. The compound of claim 16, wherein at least one heteroatom contains at least one of oxygen, nitrogen, sulfur, or phosphorus. 18. The compound of any one of claims 1 to 17, wherein at least one of the independently selected linkers A comprises at least one aliphatic heterocycle. 19. The compound of claim 18, wherein the at least one aliphatic heterocycle comprises at least one of tetrahydrofuran, tetrahydropyran, morpholine, piperidine, piperazine, pyrrolidine, and azetidine. 20. The compound of any one of claims 1 to 19, wherein at least one of the independently selected linkers A comprises at least one heteroaryl group. 21. The compound of claim 20, wherein the at least one heteroaryl group comprises at least one of imidazole, pyrazole, pyridine, pyrimidine, triazole, and 1,2,3-triazole. 22. The compound according to any one of claims 1 to 21, wherein at least one of the independently selected linkers A comprises at least one amino acid. 23. The compound according to any one of claims 1 to 22, wherein at least one of the independently selected linkers A comprises at least one nucleotide. 24. The compound according to any one of claims 1 to 23, wherein at least one of the independently selected linkers A comprises at least one saccharide. 25. The compound of claim 24, wherein the at least one saccharide comprises at least one of glucose, fructose, mannose, galactose, ribose, and glucosamine. 26. The compound according to any one of claims 1 to 25, wherein at least one of the independently selected linkers A comprises one or more of the following.

here:
p is an integer from 0 to 12,
pp is an integer from 0 to 12,
q is an integer from 1 to 12,
qq is an integer from 1 to 12. 27. The method of any one of claims 1 to 26, wherein Linker B is 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. A compound comprising at least one of a group and an aralkynyl group. 28. The compound according to any one of claims 1 to 27, wherein linker B contains at least one heteroatom. 29. The compound of claim 28, wherein the at least one heteroatom comprises at least one of oxygen, nitrogen, sulfur, and phosphorus. 30. The compound according to any one of claims 1 to 29, wherein linker B comprises at least one aliphatic heterocycle. 31. The compound of claim 30, wherein the at least one aliphatic heterocycle comprises at least one of tetrahydrofuran, tetrahydropyran, morpholine, piperidine, piperazine, pyrrolidine, and azetidine. 32. The compound according to any one of claims 1 to 31, wherein linker B comprises at least one heteroaryl group. 33. The compound of claim 32, wherein the at least one heteroaryl group comprises at least one of imidazole, pyrazole, pyridine, pyrimidine, triazole, and 1,2,3-triazole. 34. The compound according to any one of claims 1 to 33, wherein linker B contains at least one amino acid. 35. The compound according to any one of claims 1 to 34, wherein linker B comprises at least one nucleotide. 36. The compound of claim 35, wherein the at least one nucleotide comprises at least one of an abasic nucleotide and an inverted abasic nucleotide. 37. The compound of claim 36, wherein the abasic nucleotide is an abasic deoxyribonucleic acid. 37. The compound of claim 36, wherein the inverted abasic nucleotide is an inverted abasic deoxyribonucleic acid. 37. The compound of claim 36, wherein the abasic nucleotide is an abasic ribonucleic acid. 37. The compound of claim 36, wherein the inverted abasic nucleotide is an inverted abasic ribonucleic acid. 41. The compound according to any one of claims 1 to 40, wherein linker B comprises at least one saccharide. 42. The compound of claim 41, wherein the at least one saccharide comprises at least one of glucose, fructose, mannose, galactose, ribose, and glucosamine. The compound according to any one of claims 1 to 42, wherein linker B contains at least one of the following.

here:
j is an integer from 1 to 12,
k is an integer from 0 to 12. 27. The compound according to any one of claims 1 to 26, wherein the linker BW is:

here:
j is an integer from 0 to 12,
k is an integer from 0 to 12. 44. The compound according to any one of claims 1 to 43, wherein W is a hydroxy group. 44. The compound according to any one of claims 1 to 43, wherein W is a protected hydroxy group. 47. The method of claim 46, wherein the protected hydroxy group is 4,4'-dimethoxytrityl (DMT), monomethoxytrityl (MMT), 9-(p-methoxyphenyl)xanthen-9-yl (Mox ) and 9-phenylxanthen-9-yl (Px). 44. The compound according to any one of claims 1 to 43, wherein W is a phosphoramidite group having the formula:

here:
R ^a is C1 to C6 alkyl, C3 to C6 cycloalkyl, isopropyl group, or R ^a is connected to R ^b through a nitrogen atom to form a cycle,
R ^b is C1 to C6 alkyl, C3 to C6 cycloalkyl, isopropyl group, or R ^b is connected to R ^a through a nitrogen atom to form a cycle,
R ^c is a phosphite protecting group, a phosphate protecting group, or a 2-cyanoethyl group. The method of claim 48, wherein the phosphite protecting group is 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- A compound containing at least one type of dichlorophenyl. 49. The method of claim 48, wherein the phosphate protecting group is 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-dichloro A compound containing at least one type of phenyl. 44. The compound according to any one of claims 1 to 43, wherein W is a carboxyl group. 52. The compound of claim 51, wherein W is an activated carboxyl group having the formula:

Here, X is a leaving group. 53. The method of claim 52, wherein the leaving group is carboxylate, sulfonate, chloride, phosphate, imidazole, hydroxybenzotriazole (HOBt), N-hydroxysuccinimide (NHS), tetrafluorophenol, pentafluoro A compound selected from the group consisting of phenol and para-nitrophenol. 44. The compound of any one of claims 1 to 43, wherein W is a Michael acceptor. 55. The compound of claim 54, wherein the Michael acceptor has the formula:

here:
E is an electron withdrawing group;
R ^d is hydrogen or a C1-C6 alkyl substituent on the olefin. 56. The compound of claim 55, wherein the electron withdrawing group is a carboxamide or an ester. 57. The compound of claim 55 or 56, wherein E and the carbon-carbon double bond are part of a maleimide. 44. The compound of any one of claims 1 to 43, wherein W is an oligonucleotide. 59. The compound of claim 58, wherein the oligonucleotide is a single-stranded oligonucleotide. 59. The compound of claim 58, wherein the oligonucleotide is a double-stranded oligonucleotide. 59. The compound of claim 58, wherein the oligonucleotide comprises at least three independently selected nucleotides. 62. The compound of claim 61, wherein the oligonucleotide comprises 16 to 23 independently selected nucleotides. 62. The compound of claim 61, wherein the oligonucleotide comprises about 100 independently selected nucleotides. 62. The compound of claim 61, wherein the oligonucleotide comprises up to 14,000 independently selected nucleotides. 44. The compound according to any one of claims 1 to 43, wherein W is:

here:
Linker C is a spacer that is absent or 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 amino group. 66. The compound of claim 65, wherein Linker C comprises at least one heterocyclic compound. 67. The compound of claim 66, wherein the heterocyclic compound is an abasic nucleotide or an inverted abasic nucleotide. 44. The compound according to any one of claims 1 to 43, wherein W is:

Here, Linker C is a spacer attached to the 3' or 5' end of the oligonucleotide. 69. The compound of claim 68, wherein Linker C comprises at least one of polyethylene glycol (PEG), an alkyl group, and a cycloalkyl group. 70. The compound according to claim 68 or 69, wherein linker C contains at least one heteroatom. 71. The compound of claim 70, wherein the at least one heteroatom comprises at least one of oxygen, nitrogen, sulfur, and phosphorus. 72. The compound according to any one of claims 68 to 71, wherein linker C comprises at least one aliphatic heterocycle. 73. The compound of claim 72, wherein the at least one aliphatic heterocycle comprises at least one of tetrahydrofuran, tetrahydropyran, morpholine, piperidine, piperazine, pyrrolidine, and azetidine. 74. The compound of any one of claims 68-73, wherein linker C comprises at least one heteroaryl group. 75. The compound of claim 74, wherein the at least one heteroaryl group comprises at least one of imidazole, pyrazole, pyridine, pyrimidine, triazole, and 1,2,3-triazole. 76. The compound according to any one of claims 68 to 75, wherein linker C contains at least one amino acid. 77. The compound according to any one of claims 68 to 76, wherein linker C comprises at least one nucleotide. 78. The compound of claim 77, wherein the at least one nucleotide comprises at least one of an abasic nucleotide and an inverted abasic nucleotide. 79. The compound of claim 78, wherein the abasic nucleotide is an abasic deoxyribonucleic acid (DNA). 79. The compound of claim 78, wherein the inverted abasic nucleotide is inverted abasic deoxyribonucleic acid (DNA). 79. The compound of claim 78, wherein the abasic nucleotide is an abasic ribonucleic acid (RNA). 79. The compound of claim 78, wherein the inverted abasic nucleotide is an inverted abasic ribonucleic acid (RNA). 83. The compound according to any one of claims 68 to 82, wherein linker C comprises at least one saccharide. 84. The compound of claim 83, wherein the at least one saccharide comprises at least one of glucose, fructose, mannose, galactose, ribose, and glucosamine. The compound according to any one of claims 68 to 84, wherein linker C comprises one or more of the following.

here:
j is an integer from 1 to 12,
k is an integer from 0 to 12. 44. The compound according to any one of claims 1 to 43, wherein W is:

Here, Linker C is a spacer attached to the 3' or 5' end of the oligonucleotide. 87. The compound of claim 86, wherein Linker C comprises at least one of polyethylene glycol (PEG), an alkyl group, and a cycloalkyl group. The compound according to claim 86 or 87, wherein linker C comprises one or more of the following.

here:
j is an integer from 1 to 12,
k is an integer from 0 to 12. 89. The compound according to any one of claims 1 to 88, selected from the group consisting of:

90. The compound of claim 89, which is a stereoisomer of one of compounds 1-75. 91. The compound according to any one of claims 1 to 90, wherein W is one or more pharmaceutical agents. 92. The method of claim 91, wherein the one or more pharmaceutical agents are selected from the group consisting of small interfering RNA (siRNA), single-stranded siRNA, double-stranded siRNA, small activating RNA, RNAi, microRNA (miRNA), antisense oligonucleotide, short guide RNA (gRNA), A compound comprising at least one of a single guide RNA (sgRNA), messenger RNA (mRNA), ribozyme, plasmid, immune-stimulating nucleic acid, antagomir, and aptamer. 93. The compound of claim 92, wherein the double-stranded siRNA comprises at least one modified ribonucleotide. 93. The compound of claim 92, wherein substantially all ribonucleotides of the double-stranded siRNA are modified. 93. The compound of claim 92, wherein all ribonucleotides of the double-stranded siRNA are modified. 95. The method of any one of claims 92 to 95, wherein the modified ribonucleotide is a 2'-O-methyl nucleotide, a 2'-fluoro nucleotide, a 2'-deoxy nucleotide, a 2'3'-seco nucleotide mimetic. , locking nucleotide, 2'-F-arabino nucleotide, 2'-methoxyethyl nucleotide, abasic nucleotide, ribitol, inverted nucleotide, inverted abasic nucleotide, inverted 2'-OMe nucleotide, 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 5'-( E) -vinyl phosphonate nucleotides (antisense strand alone), or terminal nucleotides linked to cholesteryl derivatives or dodecanoic acid bisdecylamide groups, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, phospho A compound comprising amidate, or a nucleotide containing a non-natural base. 97. The compound of any one of claims 92-96, wherein at least one strand of the double-stranded siRNA comprises at least one phosphorothioate linkage. 98. The compound of any one of claims 92-97, wherein at least one strand of the double-stranded siRNA comprises up to 6 phosphorothioate linkages. 99. The compound of any one of claims 92-98, wherein the double-stranded siRNA comprises at least one locked nucleic acid. 100. The compound of any one of claims 92-99, wherein the double-stranded siRNA comprises at least one unlocked nucleic acid. 101. The compound of any one of claims 92-100, wherein the double-stranded siRNA comprises at least one glycerol nucleic acid. A pharmaceutical composition comprising the compound of any one of claims 1 to 101. 103. The pharmaceutical composition of claim 102, wherein W is one or more pharmaceutical agents. 104. The pharmaceutical composition of claim 103, further comprising one or more therapeutic agents. 105. The pharmaceutical composition of claim 103 or 104, further comprising a pharmaceutically acceptable carrier. A composition for targeted delivery of one or more pharmaceutical agents, comprising the compound of any one of claims 1 to 90, wherein W is one or more pharmaceutical agents. 107. The method of claim 106, wherein the one or more pharmaceutical agents are small interfering RNAs (siRNAs), single-stranded siRNAs, double-stranded siRNAs, small activating RNAs, microRNAs (miRNAs), antisense oligonucleotides, short guide RNAs (gRNAs), single guides. A composition comprising at least one of RNA (sgRNA), messenger RNA (mRNA), ribozyme, plasmid, immunostimulatory nucleic acid, antagomir, and aptamer. 108. The composition of claim 107, wherein the double-stranded siRNA comprises at least one modified ribonucleotide on one or both strands of the siRNA. 109. The composition of claim 108, wherein substantially all ribonucleotides of the double-stranded siRNA are modified. 109. The composition of claim 108, wherein all ribonucleotides of the double-stranded siRNA are modified. 111. The method of any one of claims 108 to 110, wherein the modified ribonucleotide is a 2'-O-methyl nucleotide, a 2'-fluoro nucleotide, a 2'-deoxy nucleotide, a 2'3'-seco nucleotide mimetic. , locking nucleotide, 2'-F-arabino nucleotide, 2'-methoxyethyl nucleotide, abasic nucleotide, ribitol, inverted nucleotide, inverted abasic nucleotide, inverted 2'-OMe nucleotide, 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 5'-( E) -vinyl phosphonate nucleotides (antisense strand alone), or terminal nucleotides linked to cholesteryl derivatives or dodecanoic acid bisdecylamide groups, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, phospho A composition comprising amidate, or a nucleotide containing a non-natural base. 112. The composition of any one of claims 107-111, wherein at least one strand of the double-stranded siRNA comprises at least one phosphorothioate linkage. 113. The composition of claim 112, wherein at least one strand of the double-stranded siRNA comprises up to 6 phosphorothioate linkages. 114. The composition of any one of claims 107-113, wherein the double-stranded siRNA comprises at least one locked nucleic acid. 115. The composition of any one of claims 107-114, wherein the double-stranded siRNA comprises at least one unlocked nucleic acid. 116. The compound of any one of claims 107-115, wherein the double-stranded siRNA comprises at least one glycerol nucleic acid. A pharmaceutical composition comprising the composition of any one of claims 106 to 116. 118. The pharmaceutical composition of claim 117, further comprising one or more therapeutic agents. 119. The pharmaceutical composition of claim 117 or 118, further comprising a pharmaceutically acceptable carrier. As a method of preparing a compound for targeted delivery of one or more pharmaceutical agents:
Receiving a first compound comprising a diamine, the 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;
preparing a second compound by coupling a plurality of protected carboxylic acids to the first compound, wherein the first nitrogen in the second compound comprises the first protected carboxylic acid and the second protected carboxylic acid. is a tertiary amine, and the second nitrogen in the second compound is a tertiary amine comprising a protecting group and a third protected carboxylic acid;
preparing a third compound by deprotecting the second nitrogen of the second compound such that the second nitrogen becomes a secondary amine comprising a third protected carboxylic acid;
A fourth compound is formed by attaching a moiety containing a hydroxy group to the second nitrogen of a third compound such that the second nitrogen is a tertiary amine or amide containing a third protected carboxylic acid and a moiety containing a hydroxy group. Preparing a compound;
preparing a fifth compound by converting the protected carboxylic acid of the fourth compound to a carboxylic acid; and
Preparing a sixth compound by performing an amide coupling reaction using the fifth compound, wherein the first nitrogen in the sixth compound is a tertiary amine comprising a first amide and a second amide, and in the sixth compound The second nitrogen of is a tertiary amine comprising a moiety comprising a hydroxy group and a third amide, wherein each of the first amide, second amide, and third amide is coupled to an independently selected targeting ligand. step
How to include . 121. The method of claim 120, wherein the protecting group is selected from the group consisting of benzyl group and triphenylmethyl group. 122. The method of claim 120 or 121, wherein preparing the second compound comprises performing a S _N 2 substitution reaction using the first compound. 122. The method of claim 120 or 121, wherein preparing the second compound comprises performing a reductive amination reaction using the first compound. 122. The method of claim 120 or 121, wherein preparing the second compound comprises performing a Michael addition reaction using the first compound. The method of any one of claims 120 to 124,
The protecting group is a benzyl group;
wherein the step of preparing the third compound includes performing a hydrogenation reaction using the second compound.
method. The method of any one of claims 120 to 124,
The protecting group is a triphenylmethyl group;
wherein the step of preparing the third compound includes reacting the second component with at least one acid.
method. 127. The method of any one of claims 120-126, wherein preparing the fourth compound comprises performing a S _N 2 substitution reaction using the third compound. 127. The method of any one of claims 120-126, wherein preparing the fourth compound comprises performing a reductive amination reaction using the third compound. 127. The method of any one of claims 120-126, wherein preparing the fourth compound comprises performing a Michael addition reaction using the third compound. 127. The method of any one of claims 120-126, wherein preparing the fourth compound comprises performing an amide coupling reaction using the third compound. 127. The method of any one of claims 120-126, wherein preparing the fourth compound comprises performing a nucleophilic addition reaction using the third compound. 132. The method of any one of claims 120-131, wherein the moiety comprising a hydroxy group is attached to the second nitrogen using the linker B of any of claims 24-40. 133. The method of any one of claims 120-132, wherein preparing the fifth compound comprises reacting the fourth compound with at least one acid. 134. The method of claim 133, wherein the at least one acid comprises at least one of hydrochloric acid, hydrobromide acid, trifluoroacetic acid, and formic acid. 135. The method of any one of claims 120-134, wherein preparing the fifth compound comprises performing a hydrogenation reaction using the fourth compound. 135. The method of any one of claims 120-134, wherein preparing the fifth compound comprises performing a hydrolysis reaction using the fourth compound. 137. The method of any one of claims 120 to 136, wherein the first amide, the second amide and the third amide are each independently selected using the independently selected linker A of any one of claims 12 to 23. A method of coupling to a ligand. 138. The method of any one of claims 120-137, wherein the independently selected targeting ligand is independently selected to be the targeting ligand of any one of claims 4-11. 139. The method of any one of claims 120-138, further comprising converting the hydroxy group to a phosphoramidite group using a phosphitylation reaction. 139. The method of claim 139, wherein converting the hydroxy group to a phosphoramidite group is performed after performing an amide coupling reaction to prepare the sixth compound. As a method of preparing a compound for targeted delivery of one or more pharmaceutical agents:
Receiving a first compound comprising a diamine, wherein the diamine comprises a first nitrogen and a second nitrogen, the first nitrogen being a secondary amine comprising a first protecting group, and the second nitrogen comprising a second protecting group. An amine step;
preparing a second compound by coupling the first protected carboxylic acid to the first nitrogen of the first compound such that the first nitrogen is a tertiary amine;
removing the first protecting group from the first nitrogen in the second compound to prepare a third compound comprising the first nitrogen and the second nitrogen, wherein the first nitrogen is a secondary compound comprising the first protected carboxylic acid. is an amine, and the second nitrogen is an amine comprising a second protecting group;
preparing a fourth compound by coupling the second protected carboxylic acid to the first nitrogen of the third compound such that the first nitrogen is a tertiary amine;
removing the second protecting group from the fourth compound to produce a fifth compound comprising a first nitrogen and a second nitrogen, wherein the first nitrogen comprises a first protected carboxylic acid and a second protected carboxylic acid. is a tertiary amine, and the second nitrogen is a primary amine;
preparing a sixth compound by coupling the third protected carboxylic acid to the second nitrogen of the fifth compound such that the second nitrogen is a secondary amine;
preparing a seventh compound by attaching a moiety comprising a hydroxy group to the second nitrogen of the sixth compound such that the second nitrogen is a tertiary amine;
preparing an eighth compound by converting the third protected carboxylic acid of the seventh compound to a first carboxylic acid;
Preparing a ninth compound by performing an amide coupling reaction using the eighth compound, wherein the first nitrogen of the ninth compound includes a first protected carboxylic acid and a second protected carboxylic acid, wherein the second nitrogen of compound 9 comprises a moiety comprising a first amide and hydroxy group with a first targeting ligand coupled thereto;
preparing a tenth compound by converting the second protected carboxylic acid of the ninth compound to a second carboxylic acid;
Preparing an eleventh compound by performing an amide coupling reaction using the tenth compound, wherein the first nitrogen of the eleventh compound is a second nitrogen having a first protected carboxylic acid and a second targeting ligand coupled thereto. comprising an amide, wherein the second nitrogen of the eleventh compound comprises a moiety comprising a hydroxy group and a first amide having a first targeting ligand coupled thereto;
preparing a twelfth compound by converting the first protected carboxylic acid of the eleventh compound to a third carboxylic acid; and
preparing a thirteenth compound by performing an amide coupling reaction using the twelfth compound, wherein the first nitrogen of the thirteenth compound is a second amide having a second targeting ligand coupled thereto and a third amide coupled thereto. comprising a third amide having a targeting ligand, wherein the second nitrogen of the thirteenth compound comprises a moiety comprising a hydroxy group and a first amide having a first targeting ligand coupled thereto.
How to include . According to clause 141,
the first protecting group is a benzyl group;
The second protecting group is a tert-butyloxycarbonyl (Boc) group
method. 143. The method of claim 141 or 142, wherein preparing the second compound comprises performing a S _N 2 substitution reaction using the first compound. 143. The method of claim 141 or 142, wherein preparing the second compound comprises performing a reductive amination reaction using the first compound. 143. The method of claim 141 or 142, wherein preparing the second compound comprises performing a Michael addition reaction using the first compound. 146. The method of any one of claims 141-145, wherein preparing the third compound comprises performing a hydrogenation reaction using the second compound. 147. The method of any one of claims 141-146, wherein preparing the fourth compound comprises performing a S _N 2 substitution reaction using the third compound. 147. The method of any one of claims 141-146, wherein preparing the fourth compound comprises performing a reductive amination reaction using the third compound. 147. The method of any one of claims 141-146, wherein preparing the fourth compound comprises performing a Michael addition reaction using the third compound. 147. The method of any one of claims 141-146, wherein preparing the fourth compound comprises performing an amide coupling reaction using the third compound. 147. The method of any one of claims 141-146, wherein preparing the fourth compound comprises performing a nucleophilic addition reaction using the third compound. 152. The method of any one of claims 141-151, wherein preparing the fifth compound comprises reacting the fourth compound with at least one acid. 153. The method of claim 152, wherein the at least one acid comprises at least one of hydrochloric acid and trifluoroacetic acid. 154. The method of any one of claims 141-153, wherein preparing the sixth compound comprises performing a S _N 2 substitution reaction using the fifth compound. 154. The method of any one of claims 141-153, wherein preparing the sixth compound comprises performing a reductive amination reaction using the fifth compound. 154. The method of any one of claims 141-153, wherein preparing the sixth compound comprises performing a Michael addition reaction using the fifth compound. 157. The method of any one of claims 141-156, wherein preparing the seventh compound comprises performing a S _N 2 substitution reaction using the sixth compound. 157. The method of any one of claims 141-156, wherein preparing the seventh compound comprises performing a reductive amination reaction using the sixth compound. 157. The method of any one of claims 141-156, wherein preparing the seventh compound comprises performing a Michael addition reaction using the sixth compound. 157. The method of any one of claims 141-156, wherein preparing the seventh compound comprises performing an amide coupling reaction using the sixth compound. 157. The method of any one of claims 141-156, wherein preparing the seventh compound comprises performing a nucleophilic addition reaction using the sixth compound. 162. The method of any one of claims 141-161, wherein the first amide is coupled to the first targeting ligand using the independently selected linker A of any of claims 12-23. 163. The method of any one of claims 141-162, wherein the second amide is coupled to the second targeting ligand using the independently selected linker A of any of claims 12-23. 164. The method of any one of claims 141-163, wherein the third amide is coupled to the third targeting ligand using the independently selected linker A of any of claims 12-23. 165. The method of any one of claims 141 to 164, wherein the first targeting ligand, the second targeting ligand and the third targeting ligand are independently one or more of the targeting ligands of any one of claims 4 to 11. How to be chosen. 166. The method of any one of claims 141-165, wherein the hydroxy group is coupled to the second nitrogen using the linker B of any of claims 24-40. 167. The method of any one of claims 141-166, further comprising converting the hydroxy group to a phosphoramidite group using a phosphitylation reaction. 168. The method of claim 167, wherein converting the hydroxy group to a phosphoramidite group is performed after preparing the thirteenth compound. to the object
(a) the compound of any one of claims 1 to 90, wherein W is one or more pharmaceutical agents, or
(b) the composition of any one of claims 106 to 116
A method of delivering a pharmaceutical agent to a subject, comprising administering. 169. The method of claim 169, wherein the subject is a vertebrate. 169. The method of claim 169, wherein the subject is a mammal. 169. The method of claim 169, wherein the mammal is a human. 173. The method of any one of claims 169-172, wherein the compound is administered in a pharmaceutically acceptable carrier. A method of delivering a pharmaceutical agent to a subject comprising administering to the subject the pharmaceutical composition of any one of claims 102-105 and 116-119. 175. The method of claim 174, wherein the subject is a vertebrate. 175. The method of claim 174, wherein the subject is a mammal, and optionally the mammal is a human. The method of claim 174, wherein the one or more pharmaceutical agents are small interfering RNAs (siRNAs), single-stranded siRNAs, double-stranded siRNAs, small activating RNAs, microRNAs (miRNAs), antisense oligonucleotides, short guide RNAs (gRNAs), single guides. A method comprising at least one of RNA (sgRNA), messenger RNA (mRNA), ribozyme, plasmid, immunostimulatory nucleic acid, antagomir, and aptamer. 178. The method of claim 177, wherein the double-stranded siRNA comprises at least one modified ribonucleotide on one or both strands of the siRNA. 179. The method of claim 178, wherein substantially all ribonucleotides of the double-stranded siRNA are modified. 179. The method of claim 178, wherein all ribonucleotides of the double-stranded siRNA are modified. 181. The method of any one of claims 178 to 180, wherein the modified ribonucleotide is a 2'-O-methyl nucleotide, a 2'-fluoro nucleotide, a 2'-deoxy nucleotide, a 2'3'-seco nucleotide mimetic. , locking nucleotide, 2'-F-arabino nucleotide, 2'-methoxyethyl nucleotide, abasic nucleotide, ribitol, inverted nucleotide, inverted abasic nucleotide, inverted 2'-OMe nucleotide, 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 5'-( E) -vinyl phosphonate nucleotides (antisense strand alone), or terminal nucleotides linked to cholesteryl derivatives or dodecanoic acid bisdecylamide groups, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, phospho A method comprising amidate, or a nucleotide containing a non-natural base. 182. The method of any one of claims 177-181, wherein at least one strand of the double-stranded siRNA comprises at least one phosphorothioate linkage. 183. The method of claim 182, wherein at least one strand of the double-stranded siRNA comprises up to 6 phosphorothioate linkages. 184. The method of any one of claims 177-183, wherein the double-stranded siRNA comprises at least one locked nucleic acid. 185. The method of any one of claims 177-184, wherein the double-stranded siRNA comprises at least one unlocked nucleic acid. 186. The method of any one of claims 177-185, wherein the double-stranded siRNA comprises at least one glycerol nucleic acid. 187. The method of any one of claims 174-186, wherein the pharmaceutical composition further comprises one or more therapeutic agents. The compound according to claim 1, wherein n is 1 or 2.