EP4380624A1 - Agrégats de ligands multivalents avec échafaudage de diamine pour l'administration ciblée d'agents thérapeutiques - Google Patents

Agrégats de ligands multivalents avec échafaudage de diamine pour l'administration ciblée d'agents thérapeutiques

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Publication number
EP4380624A1
EP4380624A1 EP22872040.5A EP22872040A EP4380624A1 EP 4380624 A1 EP4380624 A1 EP 4380624A1 EP 22872040 A EP22872040 A EP 22872040A EP 4380624 A1 EP4380624 A1 EP 4380624A1
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EP
European Patent Office
Prior art keywords
compound
group
nucleotide
nitrogen
producing
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP22872040.5A
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German (de)
English (en)
Inventor
Dongxu Shu
Pengcheng Patrick Shao
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Argo Biopharmaceutical Co Ltd
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Shanghai Argo Biopharmaceutical Co Ltd
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Application filed by Shanghai Argo Biopharmaceutical Co Ltd filed Critical Shanghai Argo Biopharmaceutical Co Ltd
Publication of EP4380624A1 publication Critical patent/EP4380624A1/fr
Pending legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/66—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
    • C07H15/08—Polyoxyalkylene derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/18—Acyclic radicals, substituted by carbocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/26—Acyclic or carbocyclic radicals, substituted by hetero rings
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/322—2'-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • oligonucleotides Due to their high molecular weight and polyanionic nature, oligonucleotides generally have low cell membrane permeability. Thus, target ligands are often conjugated to oligonucleotide compounds to enhance cell uptake and improve tissue specificity of in vivo delivery, through a well-known mechanism of receptor-mediated endocytosis. In some cases, multivalent ligand clusters have an advantage over single ligands in enhancing delivery to targeted tissues via specific receptors. Asialoglycoprotein receptor (ASGPR) is one of such receptors.
  • ASGPR Asialoglycoprotein receptor
  • GalNAc N-acetylgalactosamine
  • One aspect of the present disclosure relates to a compound for targeted delivery of one or more pharmaceutical agents, where the compound has the formula:
  • each TL is an independently selected targeting ligand
  • m is an integral number between 1 and 10
  • each n is an independently selected integral number between 1 and 10
  • each linkerA is an independently selected spacer
  • linkerB is a spacer
  • W is either the one or more pharmaceutical agents or a functional group capable of linking to the one or more pharmaceutical agents.
  • m is 1. In some embodiments, m is 2.
  • n is 1. In some embodiments, n is 2.
  • 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. In some embodiments, at least one of the independently selected TLs comprises at least one small molecule ligand.
  • At least one small molecule comprises at least one of N-acetylgalactosamine, galactose, galactosamine, N-formyl-galactosamine, N-propionylgalactosamine, N-butanoylgalactosamine, and N-iso-butanoylgalactosamine, a macrocycle, a folate molecule, a fatty acid, a bile acid, and a cholesterol.
  • at least one of the independently selected TLs comprises at least one peptide.
  • at least one of the independently selected TLs comprises at least one cyclic peptide.
  • 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 to 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 to 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) .
  • GPCR G-protein-coupled receptor
  • At least one of the independently selected linkerAs comprises at least one of polyethylene glycol, an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an alkynyl group, an aryl group, an aralkyl group, an aralkenyl group, and an aralkynyl group.
  • at least one of the independently selected linkerAs comprises at least one heteroatom.
  • the at least one heteroatom comprises at least one of oxygen, nitrogen, sulfur, or phosphorous.
  • at least one of the independently selected linkerAs comprises at least one aliphatic heterocycle.
  • the at least one aliphatic heterocycle comprises at least one of tetrahydrofuran, tetrahydropyran, morpholine, piperidine, piperazine, pyrrolidine, and azetidine.
  • at least one of the independently selected linkerAs comprises at least one heteroaryl group.
  • the at least one heteroaryl group comprises at least one of imidazole, pyrazole, pyridine, pyrimidine, triazole, and 1, 2, 3-triazole.
  • at least one of the independently selected linkerAs comprises at least one amino acid.
  • at least one of the independently selected linkerAs comprises at least one nucleotide.
  • At least one of the independently selected linkerAs comprises at least one saccharide.
  • the at least one saccharide comprises at least one of glucose, fructose, mannose, galactose, ribose, and glucosamine.
  • at least one of the independently selected linkerAs comprises one or more of:
  • p is an integral number between 0 and 12
  • pp is an integral number between 0 and 12
  • q is an integral number between 1 and 12
  • qq is an integral number between 1 and 12.
  • linkerB comprises at least one of a polyethylene glycol, an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an alkynyl group, an aryl group, an aralkyl group, an aralkenyl group, and an aralkynyl group.
  • linkerB comprises at least one heteroatom.
  • the at least one heteroatom comprises at least one of oxygen, nitrogen, sulfur, and phosphorous.
  • linkerB comprises at least one aliphatic heterocycle.
  • the at least one aliphatic heterocycle comprises at least one of tetrahydrofuran, tetrahydropyran, morpholine, piperidine, piperazine, pyrrolidine, and azetidine.
  • linkerB comprises at least one heteroaryl group.
  • the at least one heteroaryl group comprises at least one of imidazole, pyrazole, pyridine, pyrimidine, triazole, and 1, 2, 3-triazole.
  • linkerB comprises at least one amino acid.
  • linkerB comprises at least one nucleotide.
  • the at least one nucleotide comprises at least one of an abasic nucleotide and an inverted abasic nucleotide.
  • the abasic nucleotide is an abasic deoxyribonucleic acid.
  • the inverted abasic nucleotide is an inverted abasic deoxyribonucleic acid.
  • the abasic nucleotide is an abasic ribonucleic acid.
  • the inverted abasic nucleotide is an inverted abasic ribonucleic acid.
  • linkerB comprises at least one saccharide.
  • the at least one saccharide comprises at least one of glucose, fructose, mannose, galactose, ribose, and glucosamine.
  • linkerB comprises at least one of:
  • j is an integral number between 1 and 12
  • k is an integral number between 0 and 12.
  • linkerB-W is:
  • j is an integral number between 0 and 12
  • k is an integral number between 0 and 12.
  • W is a hydroxy group. In some embodiments, W is a protected hydroxy group. In some embodiments, the protected hydroxy group is protected using at least one of 4, 4’-dimethoxytrityl (DMT) , monomethoxytrityl (MMT) , 9- (p-methoxyphenyl) xanthen-9-yl (Mox) , and 9-phenylxanthen-9-yl (Px) . In some embodiments, W is a phosphoramidite group having the formula:
  • R a is a C1 to C6 alkyl, C3 to C6 cycloalkyl, an isopropyl group, or R a joins with R b through a nitrogen atom to form a cycle,
  • R b is a C1 to C6 alkyl, C3 to C6 cycloalkyl, an isopropyl group, or R b joins with R a through a nitrogen atom to form a cycle, and
  • R c is a phosphite protecting group, phosphate protecting group, or a 2-cyanoethyl group.
  • the phosphite protecting group comprises at least one of methyl, allyl, 2-cyanoethyl, 4-cyano-2-butenyl, 2-cyano-1, 1-dimethylethyl, 2- (trimethylsilyl) ethyl, 2- (S-acetylthio) ethyl, 2- (S-pivaloylthio) ethyl, 2- (4-nitrophenyl) ethyl, 2, 2, 2-trichloroethyl, 2, 2, 2-trichloro-1, 1-dimethylethyl, 1, 1, 1, 3, 3, 3-hexafluoro-2-propyl, fluorenyl-9-methyl, 2-chlorophenyl, 4-chlorophenyl, and 2, 4-dichlorophenyl.
  • the phosphate protecting group comprises at least one of methyl, allyl, 2-cyanoethyl, 4-cyano-2-butenyl, 2-cyano-1, 1-dimethylethyl, 2- (trimethylsilyl) ethyl, 2- (S-acetylthio) ethyl, 2- (S-pivaloylthio) ethyl, 2- (4-nitrophenyl) ethyl, 2, 2, 2-trichloroethyl, 2, 2, 2-trichloro-1, 1-dimethylethyl, 1, 1, 1, 3, 3, 3-hexafluoro-2-propyl, fluorenyl-9-methyl, 2-chlorophenyl, 4-chlorophenyl, and 2, 4-dichlorophenyl.
  • W is a carboxyl group. In some embodiments, W is an activated carboxyl group having the formula:
  • X is a leaving group.
  • the leaving group is selected from the group consisting of carboxylate, sulfonate, chloride, phosphate, imidazole, hydroxybenzotriazole (HOBt) , N-hydroxysuccinimide (NHS) , tetrafluorophenol, pentafluorophenol, and para-nitrophenol.
  • W is a Michael acceptor.
  • the Michael acceptor has the formula:
  • E is an electron withdrawing group
  • R d is hydrogen or a C1-C6 alkyl substitution group on olefin.
  • the electron withdrawing group is carboxamide or an ester.
  • E and the carbon-carbon double bond are part of maleimide.
  • W is an oligonucleotide.
  • the oligonucleotide is a single-stranded oligonucleotide. In some embodiments, the oligonucleotide is a double-stranded oligonucleotide. In some embodiments, the oligonucleotide comprises at least 3 independently selected nucleotides. In some embodiments, the oligonucleotide comprises between 16 and 23 independently selected nucleotides. In some embodiments, the oligonucleotide comprises about 100 independently selected nucleotides. In some embodiments, the oligonucleotide comprises up to fourteen thousand independently selected nucleotides.
  • W is:
  • linkerC is absent or a spacer attached to a 3’ or 5’ end of an oligonucleotide
  • X is a methyl group, oxygen, sulfur, or an amino group
  • Y is oxygen, sulfur, or an amino group.
  • linkerC comprises at least a heterocyclic compound.
  • the heterocyclic compound is an abasic nucleotide or an inverted abasic nucleotide.
  • W is:
  • linkerC is a spacer attached to a 3’ or 5’ end of an oligonucleotide.
  • linkerC comprises at least one of polyethylene glycol (PEG) , an alkyl group, and a cycloalkyl group.
  • linkerC comprises at least one heteroatom.
  • the at least one heteroatom comprises at least one of oxygen, nitrogen, sulfur, and phosphorous.
  • linkerC comprises at least one aliphatic heterocycle.
  • the at least one aliphatic heterocycle comprises at least one of tetrahydrofuran, tetrahydropyran, morpholine, piperidine, piperazine, pyrrolidine, and azetidine.
  • linkerC comprises at least one heteroaryl group.
  • the at least one heteroaryl group comprises at least one of imidazole, pyrazole, pyridine, pyrimidine, triazole, and 1, 2, 3-triazole.
  • linkerC comprises at least one amino acid.
  • linkerC comprises at least one nucleotide.
  • the at least one nucleotide comprises at least one of an abasic nucleotide and an inverted abasic nucleotide.
  • the abasic nucleotide is an abasic deoxyribonucleic acid (DNA) .
  • the inverted abasic nucleotide is an inverted abasic deoxyribonucleic acid (DNA) .
  • the abasic nucleotide is an abasic ribonucleic acid (RNA) .
  • the inverted abasic nucleotide is an inverted abasic ribonucleic acid (RNA) .
  • linkerC comprises at least one saccharide.
  • the at least one saccharide comprises at least one of glucose, fructose, mannose, galactose, ribose, and glucosamine.
  • linkerC comprises one or more of:
  • j is an integral number between 1 and 12
  • k is an integral number between 0 and 12.
  • W is:
  • linkerC is a spacer attached to a 3’ or 5’ end of an oligonucleotide.
  • linkerC comprises at least one of polyethylene glycol (PEG) , an alkyl group, and a cycloalkyl group.
  • linkerC comprises one or more of:
  • j is an integral number between 1 and 12
  • k is an integral number between 0 and 12.
  • the compound is selected from the group consisting of:
  • the compound is a stereoisomer of one of Compound 1-75.
  • W is the one or more pharmaceutical agents.
  • the one or more pharmaceutical agents comprises at least one of a small interfering RNA (siRNA) , a single strand siRNA, a double stranded siRNA, a small activating RNA, an RNAi, a microRNA (miRNA) , an antisense oligonucleotide, a short guide RNA (gRNA) , a single guide RNA (sgRNA) , a messenger RNA (mRNA) , a ribozyme, a plasmid, an immune-stimulating nucleic acid, an antagomir, and an aptamer.
  • siRNA small interfering RNA
  • gRNA short guide RNA
  • sgRNA single guide RNA
  • mRNA messenger RNA
  • the double stranded siRNA comprises at least one modified ribonucleotide. In some embodiments, the double stranded siRNA each strand is 19-23 nucleotides in length. In some embodiments, substantially all ribonucleotides of the double stranded siRNA are modified. In some embodiments, all ribonucleotides of the double stranded siRNA are modified.
  • the modified ribonucleotide comprises a 2'-O-methyl nucleotide, 2'-Fluoro nucleotide, 2'-deoxy nucleotide, 2'3'-seco nucleotide mimic, locked nucleotide, 2'-F-Arabino nucleotide, 2'-methoyxyethyl 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, mopholino nucleotide, and 3'-OMe nucleotide, a nucleotide comprising a 5'-phosphorothioate group, or a 5'- (E)
  • At least one strand of the double-stranded siRNA comprises at least one phosphorothioate linkage. In some embodiments, at least one strand of the double-stranded siRNA comprises up to 6 phosphorothioate linkages. In some embodiments, the double-stranded siRNA comprises at least one locked nucleic acid. In some embodiments, the double-stranded siRNA comprises at least one unlocked nucleic acid. In some embodiments, the double-stranded siRNA comprises at least one glycerol nucleic acid.
  • compositions comprising the any one of the compounds detailed above.
  • the pharmaceutical composition comprises one or more pharmaceutical agents.
  • the pharmaceutical composition comprises one or more therapeutic agents.
  • the pharmaceutical composition comprises a pharmaceutically acceptable carrier.
  • a further aspect of the present disclosure relates to a composition for targeted delivery of one or more pharmaceutical agents, where the composition comprises any one of the compounds described above, and where W is the one or more pharmaceutical agents.
  • the one or more pharmaceutical agents comprises at least one of a small interfering RNA (siRNA) , a single strand siRNA, a double stranded siRNA, a small activating RNA, a microRNA (miRNA) , an antisense oligonucleotide, a short guide RNA (gRNA) , a single guide RNA (sgRNA) , a messenger RNA (mRNA) , a ribozyme, a plasmid, an immune stimulating nucleic acid, an antagomir, and an aptamer.
  • siRNA small interfering RNA
  • miRNA microRNA
  • gRNA short guide RNA
  • sgRNA single guide RNA
  • mRNA messenger RNA
  • ribozyme a plasmid
  • the double-stranded siRNA comprises at least one modified ribonucleotide in one or both strands of the siRNA.
  • the double stranded siRNA each strand is 19-23 nucleotides in length.
  • substantially all ribonucleotides of the double-stranded siRNA are modified.
  • all ribonucleotides of the double-stranded siRNA are modified.
  • the modified ribonucleotide comprises: a 2'-O-methyl nucleotide, 2'-Fluoro nucleotide, 2'-deoxy nucleotide, 2'3'-seco nucleotide mimic, locked nucleotide, 2'-F-Arabino nucleotide, 2'-methoyxyethyl 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, mopholino nucleotide, and 3'-OMe nucleotide, a nucleotide comprising a 5'-phosphorothioate group, or a 5'- (E)
  • At least one strand of the-double stranded siRNA comprises at least one phosphorothioate linkage. In some embodiments, at least one strand of the double-stranded siRNA comprises up to 6 phosphorothioate linkages. In some embodiments, the double-stranded siRNA comprises at least one locked nucleic acid. In some embodiments, the double-stranded siRNA comprises at least one unlocked nucleic acid. In some embodiments, the double-stranded siRNA comprises at least one glycerol nucleic acid.
  • compositions described above comprising any one of the compositions described above.
  • the pharmaceutical composition comprises one or more therapeutic agents.
  • the pharmaceutical composition comprises a pharmaceutically acceptable carrier.
  • a further aspect of the present disclosure relates to a method for making a compound for targeted delivery of one or more pharmaceutical agents, where the method comprises: receiving a first compound comprising a diamine, the diamine comprises a first nitrogen and a second nitrogen, the first nitrogen being a primary amine, the second nitrogen being a secondary amine comprising a protecting group; producing a second compound by coupling a plurality of protected carboxylic acids to the first compound, the first nitrogen in the second compound being a tertiary amine comprising a first protected carboxylic acid and a second protected carboxylic acid, the second nitrogen of the second compound being a tertiary amine comprising the protecting group and a third protected carboxylic acid; producing a third compound by deprotecting the second nitrogen of the second compound, resulting in the second nitrogen becoming a secondary amine comprising the third protected carboxylic acid; producing a fourth compound by attached a moiety comprising a hydroxy group to the second nitrogen of the third compound, resulting in the second nitrogen becoming a ter
  • the protecting group is selected from the group consisting of a benzyl group and a triphenylmethyl group.
  • producing the second compound comprises performing a S N 2 substitution reaction using the first compound.
  • producing the second compound comprises performing a reductive amination reaction using the first compound.
  • producing the second compound comprises performing a Michael addition reaction using the first compound.
  • the protecting group is a benzyl group, and producing the third compound comprises performing a hydrogenation reaction using the second compound.
  • the protecting group is a triphenylmethyl group, and producing the third compound comprises reacting the second component with at least one acid.
  • producing the fourth compound comprises performing a S N 2 substitution reaction using the third compound. In some embodiments, producing the fourth compound comprises performing a reductive amination reaction using the third compound. In some embodiments, producing the fourth compound comprises performing a Michael addition reaction using the third compound. In some embodiments, producing the fourth compound comprises performing an amide coupling reaction using the third compound. In some embodiments, producing the fourth compound comprises performing a nucleophilic addition reaction using the third compound. In some embodiments, the moiety comprising the hydroxy group is attached to the second nitrogen using any linkerB described above. In some embodiments, producing the fifth compound comprises reacting the fourth compound with at least one acid.
  • the at least one acid comprises at least one of hydrochloric acid, hydrobromic acid, trifluoroacetic acid, and formic acid.
  • producing the fifth compound comprises performing a hydrogenation reaction using the fourth compound.
  • producing the fifth compound comprises performing a hydrolysis reaction using the fourth compound.
  • the first amide, the second amide, and the third amide are each coupled to an independently selected targeting ligand using any independently selected linkerA described above.
  • the independently selected targeting ligand is an independently selected targeting ligand described above.
  • the method further comprises converting the hydroxy group to a phosphoramidite group using a phosphitylation reaction. In some embodiments, converting the hydroxy group to the phosphoramidite group is performed after performing the amide coupling reaction to produce the sixth compound.
  • Another aspect of the present disclosure relates to a method for making a compound for targeted delivery of one or more pharmaceutical agents, where the method comprises: receiving a first compound comprising a diamine, the diamine comprising a first nitrogen and a second nitrogen, the first nitrogen being a secondary amine comprising a first protecting group, the second nitrogen being an amine comprising a second protecting group; producing a second compound by coupling a first protected carboxylic acid to the first nitrogen of the first compound, resulting in the first nitrogen becoming a tertiary amine; removing the first protecting group from the first nitrogen of the second compound to produce a third compound comprising the first nitrogen and the second nitrogen, the first nitrogen being a secondary amine comprising the first protected carboxylic acid, the second nitrogen being an amine comprising the second protecting group; producing a fourth compound by coupling a second protected carboxylic acid to the first nitrogen of the third compound, resulting in the first nitrogen becoming a tertiary amine; removing the second protecting group from the fourth compound to produce a fifth compound
  • the first protecting group is a benzyl group and the second protecting group is a tert-butyloxycarbonyl (Boc) group.
  • producing the second compound comprises performing a S N 2 substitution reaction using the first compound.
  • producing the second compound comprises performing a reductive amination reaction using the first compound.
  • producing the second compound comprises performing a Michael addition reaction using the first compound.
  • producing the third compound comprises performing a hydrogenation reaction using the second compound.
  • producing the fourth compound comprises performing a S N 2 substitution reaction using the third compound.
  • producing the fourth compound comprises performing a reductive amination reaction using the third compound.
  • producing the fourth compound comprises performing a Michael addition reaction using the third compound. In some embodiments, producing the fourth compound comprises performing an amide coupling reaction using the third compound. In some embodiments, producing the fourth compound comprises performing a nucleophilic addition reaction using the third compound. In some embodiments, producing the fifth compound comprises reacting the fourth compound with at least one acid. In some embodiments, the at least one acid comprises at least one of hydrochloric acid and trifluoroacetic acid. In some embodiments, producing the sixth compound comprises performing a S N 2 substitution reaction using the fifth compound. In some embodiments, producing the sixth compound comprises performing a reductive amination reaction using the fifth compound. In some embodiments, producing the sixth compound comprises performing a Michael addition reaction using the fifth compound.
  • producing the seventh compound comprises performing a S N 2 substitution reaction using the sixth compound. In some embodiments, producing the seventh compound comprises performing a reductive amination reaction using the sixth compound. In some embodiments, producing the seventh compound comprises performing a Michael addition reaction using the sixth compound. In some embodiments, producing the seventh compound comprises performing an amide coupling reaction using the sixth compound. In some embodiments, producing the seventh compound comprises performing a nucleophilic addition reaction using the sixth compound.
  • the first amide is coupled to the first targeting ligand using an independently selected linkerA described above. In some embodiments, the second amide is coupled to the second targeting ligand using an independently selected linkerA described above.
  • the third amide is coupled to the third targeting ligand using an independently selected linkerA described above.
  • the first targeting ligand, the second targeting ligand, and the third targeting ligand are independently selected to be one or more of the targeting ligands described above.
  • the hydroxy group is coupled to the second nitrogen using a linkerB described above.
  • the method further comprises converting the hydroxy group to a phosphoramidite group using a phosphitylation reaction. In some embodiments, converting the hydroxy group to the phosphoramidite group is performed after producing the thirteenth compound.
  • a further aspect of the present disclosure relates to a method for delivering a pharmaceutical agent to a subject, the method comprising administering, to the subject, (a) a compound described above, where W is the one or more pharmaceutical agents, or (b) a composition described above.
  • the subject is a vertebrate.
  • the subject is a mammal.
  • the mammal is a human.
  • the compound is administered in a pharmaceutically acceptable carrier.
  • Another aspect of the present disclosure relates to a method for delivering a pharmaceutical agent to a subject, the method comprising administering, to the subject, a pharmaceutical composition described above.
  • the subject is a vertebrate.
  • the subject is a mammal, optionally the mammal is a human.
  • the one or more pharmaceutical agents comprises at least one of a small interfering RNA (siRNA) , a single strand siRNA, a double stranded siRNA, a small activating RNA, a microRNA (miRNA) , an antisense oligonucleotide, a short guide RNA (gRNA) , a single guide RNA (sgRNA) , a messenger RNA (mRNA) , a ribozyme, a plasmid, an immune stimulating nucleic acid, an antagomir, and an aptamer.
  • the double-stranded siRNA comprises at least one modified ribonucleotide in one or both strands of the siRNA.
  • substantially all ribonucleotides of the double-stranded siRNA are modified. In some embodiments, all ribonucleotides of the double-stranded siRNA are modified. In some embodiments, the modified ribonucleotide comprises: a 2'-O-methyl nucleotide, 2'-Fluoro nucleotide, 2'-deoxy nucleotide, 2'3'-seco nucleotide mimic, locked nucleotide, 2'-F-Arabino nucleotide, 2'-methoyxyethyl 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,
  • At least one strand of the-double stranded siRNA comprises at least one phosphorothioate linkage. In some embodiments, at least one strand of the double-stranded siRNA comprises up to 6 phosphorothioate linkages. In some embodiments, the double-stranded siRNA comprises at least one locked nucleic acid. In some embodiments, the double-stranded siRNA comprises at least one 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.
  • the compounds for use to deliver a pharmaceutical agent to a subject is a vertebrate.
  • the subject is a mammal.
  • the mammal is a human.
  • the compound is administered in a pharmaceutically acceptable carrier.
  • 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) .
  • the present disclosure provides multivalent ligand clusters, having a diamine scaffold, for targeted delivery of pharmaceutical agents conjugated thereto.
  • the multivalent ligand cluster may comprise one or more N-acetylgalactosamine (GalNAc) targeting ligands.
  • the multivalent ligand cluster may be conjugated to one or more small interfering ribonucleic acids (siRNAs) , with siRNA being an example of a pharmaceutical agent.
  • siRNAs small interfering ribonucleic acids
  • the present disclosure also provides compositions comprising the multivalent ligand clusters of the present disclosure, and methods of making and using the multivalent ligand clusters of the present disclosure.
  • treat, ” “treating, ” or “treatment” may include prophylaxis and means to ameliorate, alleviate symptoms, eliminate the causation of the symptoms either on a temporary or permanent basis, or to prevent or slow the appearance of symptoms of the named disorder or condition.
  • the term “about, ” in connection with a measured quantity refers to the normal variations in that measured quantity, as expected by the skilled artisan making the measurement and exercising a level of care commensurate with the objective of measurement and the precision of the measuring equipment.
  • conjugate means an atom or group of atoms bound to an oligonucleotide or other oligomer.
  • conjugate groups modify one or more properties of the compound to which they are attached, including, but not limited to pharmacodynamics, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge, and/or clearance properties.
  • the term “linked, ” when referring to the connection between two molecules, means the two molecules are joined, directly or indirectly, by a covalent bond or that the two molecules are associated via noncovalent bonds (e.g., hydrogen bonds or ionic bonds) .
  • An example of a Compound A being directly joined to a Compound B may be represented as A-B.
  • An example of a Compound A being indirectly joined to a Compound B may be represented as A-C-B, where Compound A is indirectly joined to Compound B through Compound C. It will be appreciated that more than one intermediary compound may be present in situations of indirect joining of compounds.
  • nucleic acid refers to molecules composed of monomeric nucleotides.
  • a nucleic acid includes ribonucleic acids (RNAs) , deoxyribonucleic acids (DNAs) , single-stranded nucleic acids (ssDNAs) , double-stranded nucleic acids (dsDNAs) , small interfering ribonucleic acids (siRNAs) and microRNAs (miRNAs) .
  • RNAs ribonucleic acids
  • DNAs deoxyribonucleic acids
  • ssDNAs single-stranded nucleic acids
  • dsDNAs double-stranded nucleic acids
  • miRNAs small interfering ribonucleic acids
  • a nucleic acid may also comprise any combination of these elements in a single molecule.
  • a nucleic acid may include natural nucleic acids, non-natural nucleic acids, or a combination of natural and non-natural nucleic acids.
  • oligomer refers to nucleotide sequence containing up to 5, up to 10, up to 15, up to 20, or more than 20 nucleotides or nucleotide base pairs.
  • an oligomer has a nucleobase sequence that is at least partially complementary to a coding sequence in an expressed target nucleic acid or target gene within a cell.
  • the oligomers upon delivery to a cell expressing a gene, are able to inhibit the expression of the underlying gene. The gene expression can be inhibited in vitro or in vivo.
  • Non-limiting examples of oligomers that may be included in methods and complexes of the invention are oligonucleotides, single-stranded oligonucleotides, single-stranded antisense oligonucleotides, short interfering RNAs (siRNAs) , single-stranded siRNA, double-strand RNAs (dsRNA) , micro RNAs (miRNAs) , short hairpin RNAs (shRNA) , ribozymes, interfering RNA molecules, and dicer substrates.
  • siRNAs short interfering RNAs
  • siRNA single-stranded siRNA
  • dsRNA double-strand RNAs
  • miRNAs micro RNAs
  • shRNA short hairpin RNAs
  • ribozymes interfering RNA molecules, and dicer substrates.
  • oligonucleotide refers to a polymer of linked nucleotides each of which can be independently modified or unmodified.
  • single-stranded oligonucleotide refers to a single-stranded oligomer and in certain embodiments a single-stranded oligonucleotide may comprise a sequence at least partially complementary to a target mRNA, that is capable of hybridizing to a target mRNA through hydrogen bonding under mammalian physiological conditions (or comparable conditions in vitro) .
  • a single-stranded oligonucleotide is a single stranded antisense oligonucleotide.
  • siRNA refers to a short interfering RNA or silencing RNA.
  • siRNAs are a class of double-stranded RNA molecules, that may be 20-25 (or shorter) base pairs in length, similar to microRNA (miRNA) that operate within the RNA interference (RNAi) pathway.
  • miRNA microRNA
  • RNAi RNA interference
  • siRNAs interfere with the expression of specific genes with complementary nucleotide sequences to the siRNA by degrading mRNA after transcription, preventing translation.
  • siRNAs act in cells to silence gene expression by inducing the RNA-induced silencing complex (RISC) to cleave messenger RNA (mRNA) .
  • RISC RNA-induced silencing complex
  • the term “effective amount, ” “therapeutically effective amount, ” or “effective dose” refers to an amount sufficient to elicit the desired pharmacological or therapeutic effects, thus resulting in effective prevention or treatment of the disorder.
  • Prevention of the disorder is manifested by delaying the onset of the symptoms of the disorder to a medically significant extent.
  • Treatment of the disorder is manifested by a decrease in the symptoms associated with the disorder or an amelioration of the reoccurrence of the symptoms of the disorder.
  • a pharmaceutical composition refers to a mixture of substances suitable for administering to an individual.
  • a pharmaceutical composition can comprise one or more active agents and a pharmaceutical carrier , also referred to herein as a “pharmaceutically acceptable carrier” (e.g. a sterile aqueous solution) .
  • a pharmaceutical composition is sterile.
  • alkyl refers to a straight-or branched-chain aliphatic hydrocarbon containing one to twelve carbon atoms (i.e., C 1-12 alkyl) or the number of carbon atoms designated (i.e., a C 1 alkyl such as methyl, a C 2 alkyl such as ethyl, a C 3 alkyl such as propyl or isopropyl, etc. ) .
  • Non-limiting illustrative C 1-10 alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, iso-butyl, 3-pentyl, hexyl, heptyl, octyl, nonyl, decyl, and the like.
  • substituted alkyl by itself or as part of another group means that the alkyl as defined herein is substituted with one or more (e.g., one, two, or three) independently selected substituents.
  • a non-limiting list of independently selected substituents includes amino, (alkyl) amino, (alkyl) carbonyl, (aryl) carbonyl, (alkoxy) carbonyl, [ (alkoxy) carbonyl] amino, carboxy, aryl, heteroaryl, ureido, guanidino, halogen, sulfonamido, hydroxyl, (alkyl) sulfanyl, nitro, haloalkoxy, aryloxy, aralkyloxy, (alkyl) sulfonyl, (cycloalkyl) sulfonyl, (aryl) sulfonyl, cycloalkyl, sulfanyl, caboxamido, hetero
  • cycloalkyl by itself or as part of another group refers to saturated and partially unsaturated (containing one or two double bonds) cyclic aliphatic hydrocarbons containing one to three rings having from three to twelve carbon atoms (i.e., C 3-12 cycloalkyl) or the number of carbons designated.
  • Non-limiting illustrative cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, norbornyl, decalin, adamantyl, cyclohexenyl, cyclopentenyl, cyclohexenyl, and the like.
  • substituted cycloalkyl by itself or as part of another group means that the cycloalkyl as defined herein is substituted with one, two, or three independently selected substituents.
  • independently selected substituents includes halo, nitro, cyano, hydroxyl, amino, (alkyl) amino, (dialkyl) amino, haloalkyl, (hydroxyl) alkyl, (dihydroxy) alkyl, alkoxy, haloalkoxy, aryloxy, aralkyloxy, alkylthio, carboxamido, sulfonamido, (alkyl) carbonyl, (aryl) carbonyl, (alkyl) sulfonyl, arylsulfonyl, ureido, guanidino, carboxy, (carboxy) alkyl, alkyl, cycloalkyl, alkenyl, alkynyl,
  • alkenyl by itself or as part of another group refers to an alkyl group as defined herein containing one, two or three carbon-to-carbon double bonds.
  • Non-limiting illustrative alkenyl groups include ethenyl, propenyl, isopropenyl, butenyl, sec-butenyl, pentenyl, and hexenyl.
  • substituted alkenyl by itself or as part of another group means the alkenyl as defined herein is substituted with one, two, or three independently selected substituents.
  • independently selected substituents includes 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, ureido, guanidino, carboxy, (carboxy) alkyl, alkyl, cycloalkyl, alkenyl, alkynyl
  • cycloalkenyl by itself or as part of another group refers to non-aromatic cyclic alkyl groups of from 4 to 10 carbon atoms having single or multiple cyclic rings and having at least one >C ⁇ C ⁇ ring unsaturation and preferably from 1 to 2 sites of >C ⁇ C ⁇ ring unsaturation.
  • substituted cycloalkenyl by itself or as part of another group 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, carboxy
  • alkynyl by itself or as part of another group refers to an alkyl group as defined herein containing one to three carbon-to-carbon triple bonds.
  • Non-limiting illustrative alkynyl groups include ethynyl, propynyl, butynyl, 2-butynyl, pentynyl, and hexynyl groups.
  • substituted alkynyl by itself or as part of another group means the alkynyl as defined herein is substituted with one, two, or three independently selected substituents.
  • independently selected substituents includes halo, nitro, cyano, hydroxyl, amino, alkylamino, dialkylamino, haloalkyl, (hydroxy) alkyl, (dihydroxy) alkyl, alkoxy, haloalkoxy, aryloxy, aralkyloxy, (alkyl) sulfanyl, carboxamido, sulfonamido, alkylcarbonyl, arylcarbonyl, alkylsulfonyl, arylsulfonyl, ureido, guanidino, carboxy, (carboxy) alkyl, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl
  • haloalkyl by itself or as part of another group refers to an alkyl group substituted by one or more fluorine, chlorine, bromine and/or iodine atoms.
  • Non-limiting illustrative haloalkyl groups include fluoromethyl, difluoromethyl, trifluoromethyl, pentafluoroethyl, 1, 1-difluoroethyl, 2, 2-difluoroethyl, 2, 2, 2-trifluoroethyl, 3, 3, 3-trifluoropropyl, 4, 4, 4-trifluorobutyl, and trichloromethyl groups.
  • alkoxy by itself or as part of another group refers to an alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, alkynyl, or substituted alkynyl attached to a terminal oxygen atom.
  • haloalkoxy by itself or as part of another group refers to a haloalkyl attached to a terminal oxygen atom.
  • Non-limiting illustrative haloalkoxy groups include fluoromethoxy, difluoromethoxy, trifluoromethoxy, and 2, 2, 2-trifluoroethoxy.
  • aryl by itself or as part of another group refers to a monocyclic or bicyclic aromatic ring system having from six to fourteen carbon atoms (i.e., C 6 -C 14 aryl) .
  • Non-limiting illustrative aryl groups include phenyl, naphthyl, phenanthryl, anthracyl, indenyl, azulenyl, biphenyl, biphenylenyl, and fluorenyl groups.
  • substituted aryl by itself or as part of another group means that the aryl as defined herein is substituted with one to five independently selected substituents.
  • a non-limiting list of independently selected substituents includes halo, nitro, cyano, hydroxyl, amino, alkylamino, dialkylamino, haloalkyl, (hydroxy) alkyl, (dihydroxy) alkyl, alkoxy, haloalkoxy, aryloxy, heteroaryloxy, aralkyloxy, alkylthio, carboxamido, sulfonamido, (alkyl) carbonyl, (aryl) carbonyl, (alkyl) sulfonyl, (aryl) sulfonyl, ureido, guanidino, carboxy, carboxyalkyl, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, hetero
  • Non-limiting illustrative substituted aryl groups include 2-methylphenyl, 2-methoxyphenyl, 2-fluorophenyl, 2-chlorophenyl, 2-bromophenyl, 3-methylphenyl, 3-methoxyphenyl, 3-fluorophenyl, 3-chlorophenyl, 4-methylphenyl, 4-ethylphenyl, 4-methoxyphenyl, 4-fluorophenyl, 4-chlorophenyl, 2, 6-di-fluorophenyl, 2, 6-di-chlorophenyl, 2-methyl, 3-methoxyphenyl, 2-ethyl, 3-methoxyphenyl, 3, 4-di-methoxyphenyl, 3, 5-di-fluorophenyl 3, 5-di-methylphenyl, 3, 5-dimethoxy, 4-methylphenyl, 2-fluoro-3-chlorophenyl, and 3-chloro-4-fluorophenyl.
  • substituted aryl is meant to
  • 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 illustrative aryloxy group is PhO-.
  • heteroaryloxy by itself or as part of another group refers to a heteroaryl or substituted heteroaryl attached to a terminal oxygen atom.
  • aralkyloxy by itself or as part of another group refers to an aralkyl group attached to a terminal oxygen atom.
  • a non-limiting illustrative aralkyloxy group is PhCH 2 O-.
  • heteroaryl refers to monocyclic and bicyclic aromatic ring systems having 5 to 14 ring atoms (i.e., C 5 -C 14 heteroaryl) and 1, 2, 3, or 4 heteroatoms independently chosen from oxygen (O) , nitrogen (N) , and sulfur (S) .
  • Non-limiting illustrative heteroaryl groups include thienyl, benzo [b] thienyl, naphtho [2, 3-b] thienyl, thianthrenyl, furyl, benzofuryl, pyranyl, isobenzofuranyl, benzooxazonyl, 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, ⁇ -
  • substituted heteroaryl by itself or as part of another group means that the heteroaryl as defined herein is substituted with one to four independently selected substituents.
  • a non-limiting list of independently selected substituents includes halo, nitro, cyano, hydroxy, amino, (alkyl) amino, (dialkyl) amino, haloalkyl, (hydroxy) alkyl, (dihydroxy) alkyl, alkoxy, haloalkoxy, aryloxy, aralkyloxy, alkylthio, carboxamido, sulfonamido, (alkyl) carbonyl, (aryl) carbonyl, (alkyl) sulfonyl, (aryl) sulfonyl, ureido, guanidino, carboxy, (carboxy) alkyl, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocycl
  • heterocyclo or “heterocyclyl” by itself or as part of another group refers to saturated and partially unsaturated (e.g., containing one or two double bonds) cyclic groups containing one, two, or three rings having from three to fourteen ring members (i.e., a 3-to 14-membered heterocyclo) and at least one heteroatom. Each heteroatom is independently selected.
  • the term “heterocyclo” or “heterocyclyl” is meant to include cyclic ureido groups, such as, 2-imidazolidinone, and cyclic amide groups, such as, ⁇ -lactam, ⁇ -lactam, ⁇ -lactam and ⁇ -lactam.
  • heterocyclo or “heterocyclyl” is also meant to include groups having fused aryl or substituted aryl groups, e.g., indolinyl.
  • the heterocyclo or heterocyclyl can be linked to the rest of the molecule through a carbon or nitrogen atom.
  • Non-limiting illustrative heterocyclo (or heterocyclyl) groups include 2-oxopyrrolidin-3-yl, 2-imidazolidinone, piperidinyl, morpholinyl, piperazinyl, pyrrolidinyl, and indolinyl.
  • substituted heterocyclo or “substituted heterocyclyl” by itself or part of another group means the heterocyclo or heterocyclyl group as defined above is substituted with one to four independently selected substituents.
  • a non-limiting list of independently selected substituents includes halo, nitro, cyano, hydroxyl, amino, (alkyl) amino, (dialkyl) amino, haloalkyl, (hydroxy) alkyl, (dihydroxy) alkyl, alkoxy, haloalkoxy, aryloxy, aralkyloxy, alkylthio, carboxamido, sulfonamido, (alkyl) carbonyl, (aryl) carbonyl, (alkyl) sulfonyl, (aryl) sulfonyl, ureido, guanidino, carboxy, carboxyalkyl, alkyl, cycloalkyl, alkenyl, alkyn
  • amino by itself or as part of another group refers to -NH 2 .
  • alkylamino or “ (alkyl) amino” by itself or as part of another group refers to -NHR, wherein R is alkyl.
  • dialkylamino or “ (dialkyl) amino” by itself or as part of another group refers to -NR’R”, wherein R’ and R” are each independently alkyl or R’ and R” are taken together to form a 3-to 8-membered heterocyclo or substituted heterocyclo.
  • cycloalkylamino by itself or as part of another group refers to -NR’R”, wherein R’ is cycloalkyl or substituted cycloalkyl, and R” is hydrogen or alkyl.
  • (amino) alkyl by itself or as part of another group refers to an alkyl group substituted with an amino group.
  • Non-limiting illustrative (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 .
  • alkylamino) alkyl or “ [ (alkyl) amino] alkyl” by itself or as part of another group refers to an alkyl group substituted with an alkylamino group.
  • a non-limiting illustrative (alkylamino) alkyl group is -CH 2 CH 2 N (H) CH 3 .
  • dialkylamino alkyl by itself or as part of another group refers to an alkyl group substituted by a dialkylamino group.
  • Non-limiting illustriative (dialkylamino) alkyl groups include -CH 2 N (CH 3 ) 2 and -CH 2 CH 2 N (CH- 3 ) 2 .
  • (cycloalkylamino) alkyl by itself or as part of another group refers to an alkyl group substituted by a cycloalkylamino group.
  • Non-limiting illustrative (cycloalkylamino) alkyl groups include -CH 2 N (H) cyclopropyl, -CH 2 N (H) cyclobutyl, and -CH 2 N (H) cyclohexyl.
  • carboxamido by itself or as part of another group refers to a radical of formula -C ( ⁇ O) NR’R”, where R’ and R” are each independently hydrogen, alkyl, substituted alkyl, aralkyl, substituted aralkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl, or R’ and R” taken together with the nitrogen to which they are attached form a 3-to 8-membered heterocyclo group.
  • Non-limiting illustrative carboxamido groups include -CONH 2 , -CON (H) CH 3 , CON (CH 3 ) 2 , and CON (H) Ph.
  • 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 alkyl, aryl, or substituted aryl, or R’ and R” taken together with the nitrogen to which they are attached form a 3-to 8-membered heterocyclo group.
  • Non-limiting illustrative sulfonamido groups include -SO 2 NH 2 , -SO 2 N (H) CH 3 , and -SO 2 N (H) Ph.
  • alkyl carbonyl by itself or as part of another group refers to a carbonyl group, i.e., -C ( ⁇ O) -, substituted by an alkyl group.
  • a non-limiting illustrative alkylcarbonyl group is -COCH 3 .
  • (alkoxy) carbonyl (or “ester” ) by itself or as part of another group refers to a carbonyl group, i.e., -C ( ⁇ O) -, substituted by an alkoxy group.
  • a non-limiting illustrative (alkoxy) carbonyl group is -C (O) OCH 3 .
  • (aryl) carbonyl by itself or as part of another group refers to a carbonyl group, i.e., -C ( ⁇ O) -, substituted by an aryl or substituted aryl group.
  • a non-limiting illustrative arylcarbonyl group is -COPh.
  • sulfanyl by itself or as part of another group refers to a -SH group.
  • alkyl sulfanyl or “alkylthio” by itself or as part of another group refers to a sulfur atom substituted by an alkyl or substituted alkyl group.
  • Non-limiting illustrative alkylthio groups include -SCH 3 , and -SCH 2 CH 3 .
  • mercaptoalkyl by itself or as part of another group refers to an alkyl group substituted by a -SH group.
  • alkylsulfonyl or “ (alkyl) sulfonyl” by itself or as part of another group refers to a sulfonyl group, i.e., -SO 2 -, substituted by an alkyl or substituted alkyl group.
  • a non-limiting illustrative alkylsulfonyl group is -SO 2 CH 3 .
  • arylsulfonyl or “ (aryl) sulfonyl” by itself or as part of another group refers to a sulfonyl group, i.e., -SO 2 -, substituted by an aryl or substituted aryl group.
  • a non-limiting illustrative arylsulfonyl group is -SO 2 Ph.
  • carboxy by itself or as part of another group refers to a radical of the formula -COOH.
  • (carboxy) alkyl by itself or as part of another group refers to an alkyl group substituted with a -COOH.
  • a non-limiting illustrative carboxyalkyl group is -CH 2 CO 2 H.
  • aralkyl by itself or as part of another group refers to a residue in which an aryl moiety is attached to an alkyl residue.
  • the aralkyl group may be attached to the parent structure at either the aryl or the alkyl residue.
  • substituted aralkyl by itself or as part of another group refers to a residue in which an aryl moiety is attached to a substituted alkyl residue.
  • aralkenyl by itself or as part of another group 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 aryl radicals.
  • substituted aralkenyl by itself or as part of another group refers to an aralkenyl radical where the alkenylene chain of the aralkenyl radical is an optionally substituted alkenylene chain, and each aryl radical of the aralkenyl radical is an optionally substituted aryl radical.
  • aralkynyl by itself or as part of another group refer to a radical of the formula -R e R c , where R e is an alkynylene chain and R c is one or more aryl radicals.
  • substituted aralkynyl by itself or as part of another group refers to an aralkynyl radical where the alkynylene chain of the aralkynyl radical is an optionally substituted alkynylene chain, and each aryl radical of the aralkynyl radical is an optionally substituted aryl radical.
  • aliphatic heterocycle by itself or as part of another group refers to a non-aromatic ring in which one or more of the ring-forming atoms is a heteroatom.
  • heteroatom refers to an atom inserted between a carbon atom and its parent molecule (i.e., between the points of attachment) .
  • Non-limiting illustrative heteroatoms include oxygen, nitrogen, sulfur (including sulfoxide and sulfone) , and phosphorous (P) .
  • saccharide refers to a single sugar moiety or monosaccharide unit as well as combinations of two or more single sugar moieties or monosaccharide units covalently linked to form disaccharides, oligosaccharides, and polysaccharides.
  • the polysaccharide may be linear or branched.
  • oligosaccharide refers to a single sugar residue in an oligosaccharide.
  • disaccharide refers to a polysaccharide composed of two monosaccharide units or moieties linked together by a glycosidic bond.
  • an “oligosaccharide” refers to a compound containing two or more monosaccharide units or moieties. Within the context of an oligosaccharide, an individual monomer unit or moiety is a monosaccharide which is, or can be, bound through a hydroxy group to another monosaccharide unit or moiety. Oligosaccharides can be prepared by either chemical synthesis from protected single residue sugars or by chemical degradation of biologically produced polysaccharides. Alternatively, oligosaccharides may be prepared by in vitro enzymatic methods.
  • ureido by itself or as part of another group refers to a radical of the formula -NR’-C ( ⁇ O) -NR”R”, wherein 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”’ taken together with the nitrogen to which they are attached form a 4-to 8-membered heterocyclo group.
  • Non-limiting illustrative ureido groups include -NH-C ( ⁇ O) -NH 2 and -NH-C ( ⁇ O) -NHCH 3 .
  • guanidino by itself or as part of another group refers to a radical of the formula -NR’-C ( ⁇ NR”) -NR”’R”” , wherein 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 illustrative guanidino groups include -NH-C ( ⁇ NH) -NH 2 , -NH-C ( ⁇ NCN) -NH 2 , and -NH-C ( ⁇ NH) -NHCH 3 .
  • heteroaryl alkyl by itself or as part of another group refers to an alkyl group substituted with one, two, or three heteroaryl or substituted heteroaryl groups.
  • heteroalkyl by itself or part of another group refers to a stable straight or branched chain hydrocarbon radical containing at least one heteroatom, which can be the same or different.
  • the heteroatom (s) can be placed at any interior position or terminal position of the heteroalkyl group, or at a position at which the heteroalkyl group is attached to the remainder of the molecule.
  • Non-limiting illustrative 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 .
  • heterocyclo alkyl or “ (heterocyclyl) alkyl” by itself or as part of another group refers to an alkyl group substituted with one heterocyclyl or substituted heterocyclyl group, and optionally one hydroxy group.
  • (carboxamido) alkyl by itself or as part of another group refers to an alkyl group substituted with one carboxamido group, and optionally one heterocyclo, amino, alkylamino, or dialkylamino group.
  • N-oxide refers to a compound that contains a functional group, wherein N + is further connected to H and/or the rest of the compound structure.
  • integral number refers to an integer including, but not limited to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, etc.
  • a multivalent ligand cluster, having a diamine scaffold may have the general structure of Formula 1:
  • each TL is an independently selected targeting ligand
  • n 1 and 10
  • each n is an independently selected integral number between 1 and 10,
  • each linkerA is an independently selected spacer, with one end attached to a TL and the other end attached to the nitrogen of an alkylcarboxamide,
  • linkerB is a spacer, with one end attached to a pharmaceutical agent or a functional group capable of linking to one or more pharmaceutical agents, and the other end attached to a diamine nitrogen, and
  • W is either one or more pharmaceutical agents, or a functional group capable of linking to one or more pharmaceutical agents.
  • m may be configured based on a starting material used to synthesize the multivalent ligand cluster.
  • m may be 1 due to ethylenediamine being used as a starting material
  • m may be 2 due to 1, 3-propanediamine being used as a starting material
  • m may be 3 due to 1, 4-butanediamine being used as a starting material, etc.
  • a “spacer” refers to a compound or molecule that links other groups together.
  • Example linkerA and linkerB spacers are described in detail herein below.
  • a multivalent ligand cluster may include more than one TLs, where each may be selected to be different from or the same as one or more other TLs in that multivalent ligand cluster.
  • a multivalent ligand cluster may include more than one “n, ” where each may be selected to be different from or the same as one or more other “n” in that multivalent ligand cluster.
  • a multivalent ligand cluster may include more than one linkerA, where each may be selected to be different from or the same as one or more other linkerAs in that multivalent ligand cluster.
  • a multivalent ligand cluster of the present disclosure may include multiple (e.g., three) independently selected targeting ligands.
  • independently selected means that each targeting ligand may be selected to be different from or the same as one or more other targeting ligands in the same multivalent ligand cluster.
  • At least one of the independently selected targeting ligands of Formula 1 may be capable of binding to one or more cell receptors, cell channels, and/or cell transporters capable of facilitating endocytosis.
  • At least one of the independently selected targeting ligands of Formula 1 may comprise at least one small molecule ligand.
  • a “small molecule ligand” refers to a ligand smaller than a protein.
  • at least one small molecular ligand may comprise at least one of N-acetylgalactosamine (GalNAc) , galactose, galactosamine, N-formyl-galactosamine, N-propionylgalactosamine, N-butanoylgalactosamine, and N-iso-butanoylgalactosamine, a macrocycle, a folate molecule, a fatty acid, a bile acid, a cholesterol, and derivatives thereof.
  • GalNAc N-acetylgalactosamine
  • a macrocycle is a molecule or ion containing a twelve or more membered ring.
  • the present disclosure is not limited to any particular macrocycle.
  • a non-limiting list of macrocycles within the scope of the present disclosure include terpenoid macrocycles, porphyrins, and cyclodextrins.
  • Folate also known as vitamin B9 and folacin, is used by the human body to make DNA and RNA, and metabolize amino acids necessary for cell division. Folate receptors bind folate, and reduced folic acid derivatives. Thus, in some embodiments, at least one of the independently selected targeting ligands may comprise a reduced folic acid derivative.
  • a fatty acid is a carboxylic acid with a long aliphatic chain.
  • the fatty acid may be saturated, meaning the aliphatic chain has all single carbon-to-carbon bonds.
  • the fatty acid may be unsaturated, meaning the aliphatic chain includes at least one double or triple carbon-to-carbon bond.
  • the fatty acid may include a branched chain.
  • the fatty acid may include a ring structure.
  • Fatty acids are known to assist in the update of pharmaceutical agents into cells. See Prakash et al. “Fatty acid conjugation enhances potency of antisense oligonucleotides in muscle. ” Nucleic Acids Res.
  • a bile acid is a steroidal acid found in bile.
  • Bile acid is a known ligand for the farnesoid X receptor (FXR) and G protein-coupled bile acid receptor 1 (GPBAR1) (TGR5) .
  • FXR farnesoid X receptor
  • GPBAR1 G protein-coupled bile acid receptor 1
  • the bile acid may be a primary bile acid synthesized in the liver.
  • the bile acid may be a secondary bile acid resulting from bacterial actions in the colon.
  • Bile acids are known to be beneficial in inhibiting RNA translation. See González-Carmona et al.
  • 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. Peptides known and not yet discovered are within the scope of the present disclosure.
  • At least one of the independently selected targeting ligands of Formula 1 may comprise at least one cyclic peptide.
  • a cyclic peptide is a polypeptide chain having a cyclic ring structure.
  • the cyclic ring structure may be formed by linking one end of the peptide to the other with an amide bond, or other chemically stable bond such as lactone, ether, thioether, disulfide, etc.
  • a cyclic peptide of the present disclosure may be a biologically active cyclic peptide where a head-to-tail (or N-to-C) cyclization is formed by an amide bond between amino and carboxyl termini.
  • a cyclic peptide of the present disclosure may be a biologically active cyclic peptide where cyclization is formed by “click chemistry. ” See Rashad A.A. (2019) Click Chemistry for Cyclic Peptide Drug Design. In: Goetz G. (eds) Cyclic Peptide Design. Methods in Molecular Biology, vol 2001. Humana, New York, NY. https: //doi. org/10.1007/978-1-4939-9504-2_8.
  • the present disclosure is not limited to any particular cyclic peptide.
  • a cyclic peptide of the present disclosure may be naturally occurring or synthetically produced.
  • At least one of the independently selected targeting ligands of Formula 1 may comprise at least one aptamer.
  • An aptamer is a short, single-stranded DNA or RNA molecule that can selectively bind to a specific target, such as a protein, peptide, carbohydrate, small molecule, toxin, or live cell. Aptamers assume a variety of shapes, as they tend to form helices and single-stranded loops. The present disclosure is not limited to any particular aptamer. Aptamers known and not yet discovered are within the scope of the present disclosure.
  • At least one of the independently selected targeting ligands of Formula 1 may be capable of binding to at least one Asialoglycoprotein receptor (ASGPR) .
  • ASGPRs are lectins, located on liver cells, that bind galactose residues. ASGPR has been demonstrated to have high expression on the surface of hepatocytes, human carcinoma cell lines, and liver cancers. ASGPR is also weakly expressed by glandular cells of the gallbladder and stomach.
  • At least one of the independently selected targeting ligands of Formula 1 may be capable of binding to at least one transferrin receptor.
  • a transferrin receptor is a membrane glycoprotein that mediates cellular uptake of transferrin, a protein in the blood that binds to iron and transports it through the body.
  • the transferrin receptor-mediated endocytosis pathway is known to those skilled in the art. See Qian et al. “Targeted drug delivery via the transferrin receptor-mediated endocytosis pathway. ” Pharmacol Rev. 2002 Dec; 54 (4) : 561-87. doi: 10.1124/pr. 54.4.561. PMID: 12429868.
  • the present disclosure is not limited to any particular ligand capable of binding to at least one transferring receptor. Transferrin receptor ligands known and not yet developed are within the scope of the present disclosure.
  • At least one of the independently selected targeting ligands of Formula 1 may be capable of binding to at least one integrin receptor.
  • An integrin receptor is a transmembrane receptor that facilitates cell-cell and cell-extracellular matrix (ECM) adhesion. Upon ligand binding, integrin receptors activate signal transduction pathways that mediate cellular signals such as regulation of the cell cycle, organization of the intracellular cytoskeleton, and movement of new receptors to the cell membrane.
  • Integrin targeted delivery of gene therapeutics is known to those skilled in the art. See Juliano et al. “Integrin targeted delivery of gene therapeutics. ” Theranostics vol. 1 211-9.2 Mar. 2011, doi: 10.7150/thno/v01p0211. The present disclosure is not limited to any particular ligand capable of binding to at least one integrin receptor. Integrin receptor ligands known and not yet developed are within the scope of the present disclosure.
  • At least one of the independently selected targeting ligands of Formula 1 may be capable of binding to at least one folate receptor.
  • a folate receptor binds folate and reduced folic acid derivatives, and mediates delivery of tetrahydrofolate to the interior of cells.
  • Targeted drug delivery via folate receptors is known to those skilled in the art. See Zhao et al. “Targeted drug delivery via folate receptors. ” Expert Opin Drug Deliv. 2008 Mar; 5 (3) : 309-19. doi: 10.1517/17425247.5.3.309. PMID: 18318652.
  • the present disclosure is not limited to any particular ligand capable of binding to at least one folate receptor. Folate receptor ligands known and not yet developed are within the scope of the present disclosure.
  • At least one of the independently selected targeting ligands of Formula 1 may be capable of binding to at least one G-protein-coupled receptor (GPCR) .
  • GPCRs are cell surface receptors that bind, among other things, peptides, lipids, sugars, and proteins. GPCRs interact with G proteins in the plasma membrane. When an external signaling molecule binds to a GPCR, it causes a conformational change in the GPCR, which triggers the interaction between the GPCR and a nearby G protein.
  • GPCRs consist of a single polypeptide that is folded into a globular shape and embedded in a cell's plasma membrane. GPCR targeted delivery of oligonucleotide therapeutics is known to those skilled in the art.
  • a multivalent ligand cluster of the present disclosure may include multiple (e.g., three) independently selected linkerAs.
  • independently selected means that each linkerA may be selected to be different from or the same as one or more other linkerAs in the same multivalent ligand cluster.
  • Each linkerA is an independently selected spacer, with one end attached to a targeting ligand (TL in Formula 1) and the other end attached to the nitrogen of an alkylcarboxamide of the multivalent ligand cluster.
  • the independently selected linkerAs may comprise polythethylene glycol (PEG) .
  • the PEG may have any number of repeating O-CH 2 -CH 2 units.
  • the PEG may be PEG1, PEG2, PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG10, PEG11, PEG12, PEG13, PEG14, PEG15, PEG16, PEG17, PEG18, PEG19, PEG20, PEG21, PEG22, PEG23, PEG24, PEG25, PEG26, PEG27, PEG28, PEG29, PEG30, PEG31, PEG32, PEG33, PEG34, PEG35, PEG36, PEG37, PEG38, PEG39, PEG40, PEG41, PEG42, PEG43, PEG44, PEG45, PEG46, PEG47, PEG48, PEG49, PEG50, PEG51, PEG40, PEG41,
  • At least one of the independently selected linkerAs may comprise at least one alkyl group.
  • an alkyl group may have 2 carbons, 3 carbons, 4 carbons, 5 carbons, 6 carbons, 7 carbons, 8 carbons, 9 carbons, 10 carbons, 11 carbons, 12 carbons, 13 carbons, 14 carbons, 15 carbons, 16 carbons, 17 carbons, 18 carbons, 19 carbons, or 20 carbons.
  • At least one of the independently selected linkerAs may comprise at least one substituted alkyl group.
  • a substituted alkyl group may have 2 carbons, 3 carbons, 4 carbons, 5 carbons, 6 carbons, 7 carbons, 8 carbons, 9 carbons, 10 carbons, 11 carbons, 12 carbons, 13 carbons, 14 carbons, 15 carbons, 16 carbons, 17 carbons, 18 carbons, 19 carbons, or 20 carbons.
  • a substituted alkyl group may include one or more of the following substitution groups: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.
  • a cycloalkyl group may be a C3 cycloalkyl (i.e., cyclopropane) , C4 cycloalkyl (i.e., cyclobutene) , C5 cycloalkyl (i.e., cyclopentane) , C6 cycloalkyl (i.e., cyclohexane) , C7 cycloalkyl (i.e., cycloheptane) , C8 cycloalkyl (i.e., cyclooctane) , C9 cycloalkyl (i.e., cyclononane) , or C10 cycloalkyl (i.e., cyclodecane) .
  • At least one of the independently selected linkerAs may comprise at least one substituted cycloalkyl group.
  • a substituted cycloalkyl group may be a C3 substituted cycloalkyl, C4 substituted cycloalkyl, C5 substituted cycloalkyl, C6 substituted cycloalkyl, C7 substituted cycloalkyl, C8 substituted cycloalkyl, C9 substituted cycloalkyl, or C10 substituted cycloalkyl.
  • a substituted cycloalkyl group may include one or more of the following substitution groups: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.
  • At least one of the independently selected linkerAs may comprise at least one alkenyl group.
  • an alkenyl group may have 4 carbons, 5 carbons, 6 carbons, 7 carbons, 8 carbons, 9 carbons, 10 carbons, 11 carbons, 12 carbons, 13 carbons, 14 carbons, 15 carbons, 16 carbons, 17 carbons, 18 carbons, 19 carbons, or 20 carbons.
  • At least one of the independently selected linkerAs may comprise at least one substituted alkenyl group.
  • an alkenyl group may have 4 carbons, 5 carbons, 6 carbons, 7 carbons, 8 carbons, 9 carbons, 10 carbons, 11 carbons, 12 carbons, 13 carbons, 14 carbons, 15 carbons, 16 carbons, 17 carbons, 18 carbons, 19 carbons, or 20 carbons.
  • a substituted alkenyl group may include one or more of the following substitution groups: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.
  • At least one of the independently selected linkerAs may comprise at least one cycloalkenyl group.
  • a cycloalkenyl group may be a C5 cycloalkenyl, C6 cycloalkenyl, C7 cycloalkenyl, C8 cycloalkenyl, C9 cycloalkenyl, or C10 cycloalkenyl.
  • At least one of the independently selected linkerAs may comprise at least one substituted cycloalkenyl group.
  • a cycloalkenyl group may be a C5 cycloalkenyl, C6 cycloalkenyl, C7 cycloalkenyl, C8 cycloalkenyl, C9 cycloalkenyl, or C10 cycloalkenyl.
  • a substituted cycloalkenyl group may include one or more of the following substitution groups: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.
  • At least one of the independently selected linkerAs may comprise at least one alkynyl group.
  • an alkynyl group may have 4 carbons, 5 carbons, 6 carbons, 7 carbons, 8 carbons, 9 carbons, 10 carbons, 11 carbons, 12 carbons, 13 carbons, 14 carbons, 15 carbons, 16 carbons, 17 carbons, 18 carbons, 19 carbons, or 20 carbons.
  • At least one of the independently selected linkerAs may comprise at least one substituted alkynyl group.
  • a substituted alkynyl group may have 4 carbons, 5 carbons, 6 carbons, 7 carbons, 8 carbons, 9 carbons, 10 carbons, 11 carbons, 12 carbons, 13 carbons, 14 carbons, 15 carbons, 16 carbons, 17 carbons, 18 carbons, 19 carbons, or 20 carbons.
  • a substituted alkynyl group may include one or more of the following substitution groups: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.
  • At least one of the independently selected linkerAs may comprise at least one aryl or heteroaryl group.
  • Examples include phenyl, naphthyl, and pyridinyl, although it is noted that other aryl and heteroaryl groups, that fall within the definitions provided herein, may be used.
  • At least one of the independently selected linkerAs may comprise at least one substituted aryl group.
  • a substituted aryl group may include one or more of the following substitution groups: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.
  • At least one of the independently selected linkerAs may comprise at least one aralkyl group.
  • Example aralkyl groups include, but are not limited to, phenylmethyl, phenylethyl, and phenylpropyl.
  • At least one of the independently selected linkerAs may comprise at least one substituted aralkyl group.
  • a substituted aralkyl group may include one or more of the following substitution groups: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.
  • At least one of the independently selected linkerAs may comprise at least one aralkenyl group.
  • Example aralkenyl groups include, but are not limited to ethenylbenzene and propenylbenzene.
  • At least one of the independently selected linkerAs may comprise at least one substituted aralkenyl group.
  • a substituted aralkenyl group may include one or more of the following substitution groups: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.
  • At least one of the independently selected linkerAs may comprise at least one aralkynyl group.
  • Example aralkynyl groups include, but are not limited to ethynylbenzene and propynylbenzene.
  • At least one of the independently selected linkerAs may comprise at least one substituted aralkynyl group.
  • a substituted aralkynyl group may include one or more of the following substitution groups: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.
  • At least one of the independently selected linkerAs may comprise at least one heteroatom. In some embodiments, at least one of the independently selected linkerAs may comprise one or more oxygen (O) heteroatoms, one or more nitrogen (N) heteroatoms, one or more sulfur (S) heteroatoms, and/or one or more phosphorous (P) heteroatoms. In some embodiments, at least one of the independently selected linkerAs may comprise at least one heteroalkyl group.
  • At least one of the independently selected linkerAs may comprise at least one aliphatic heterocycle. In some embodiments, at least one of the independently selected linkerAs may comprise at least one of tetrahydrofuran (THF) , tetrahydropyran (THP) , morpholine, piperidine, piperazine, pyrrolidine, and/or azetidine.
  • THF tetrahydrofuran
  • THP tetrahydropyran
  • morpholine morpholine
  • piperidine piperazine
  • pyrrolidine pyrrolidine
  • azetidine azetidine
  • At least one of the independently selected linkerAs may comprise at least one heteroaryl group. In some embodiments, at least one of the independently selected linkerAs may comprise one or more of imidazole, pyrazole, pyridine, pyrimidine, triazole, and. or 1, 2, 3-triazole.
  • At least one of the independently selected linkerAs may comprise at least one substituted heteroaryl group.
  • a substituted heteroaryl group may include one or more of the following substitution groups: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.
  • At least one of the independently selected linkerAs may comprise at least one amino acid.
  • Various amino acids are known to those skilled in the art.
  • An independently selected linkerA is not limited to including one or more specific amino acids.
  • an independently selected linkerA may comprise one or more arginine (Arg) amino acids, one or more histidine (His) amino acids, one or more lysine (Lys) amino acids, one or more aspartic acid (Asp) amino acids, one or more glutamic acid (Glu) amino acids, one or more serine (Ser) amino acids, one or more threonine (Thr) amino acids, one or more asparagine (Asn) amino acids, one or more glutamine (Gln) amino acids, one or more cysteine (Cys) amino acids, one or more selenocysteine (Sec) amino acids, one or more glycine (Gly) amino acids, one or more proline (Pro) amino acids, one or more alanine (A
  • At least one of the independently selected linkerAs may comprise at least one nucleotide.
  • Various nucleotides are known to those skilled in the art.
  • An independently selected linkerA is not limited to including one or more specific nucleotides.
  • an independently selected linkerA may comprise one or more nucleotides comprising a guanine nucleobase, one or more nucleotides comprising an adenine nucleobase, one or more nucleotides comprising a cytosine nucleobase, one or more nucleotides comprising a thymine nucleobase, and/or one or more nucleotides comprising a uracil nucleobase.
  • At least one independently selected linkerA may comprise at least one abasic nucleotide.
  • an abasic nucleotide is a nucleotide having an abasic site, which is a location that has neither a purine nor a pyrimidine base.
  • at least one independently selected linkerA may comprise one or more abasic DNAs and/or one or more abasic RNAs.
  • at least one independently selected linkerA may comprise at least one inverted abasis nucleotide.
  • an inverted abasis nucleotide is an abasic nucleotide whose 5’ end connects to a 5’ end of a next nucleotide, and whose 3’ end connects to a 3’ end of a next nucleotide.
  • at least one independently selected linkerA may comprise one or more inverted abasic DNAs and/or one or more inverted abasic RNAs.
  • At least one of the independently selected linkerAs may comprise at least one saccharide. In some embodiments, at least one of the independently selected linkerAs may comprise at least one glucose monosaccharide unit, at least one fructose monosaccharide unit, at least one mannose monosaccharide unit, at least one galactose monosaccharide unit, at least one ribose monosaccharide unit, and/or at least one glucosamine monosaccharide unit.
  • At least one of the independently selected linkerAs may comprise one or more of:
  • p is an integral number between 0 and 12
  • pp is an integral number between 0 and 12
  • q is an integral number between 1 and 12, and
  • qq is an integral number between 1 and 12.
  • p is an integral number independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12. In some embodiments, pp is an integral number independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12. In some embodiments, q is an integral number independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12. In some embodiments, qq is an integral number independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12.
  • LinkerB is a spacer, with one end attached to a pharmaceutical agent or a functional group capable of linking to one or more pharmaceutical agents, and the other end attached to a diamine nitrogen of the multivalent ligand cluster.
  • linkerB may comprise polythethylene glycol (PEG) .
  • the PEG may have any number of repeating O-CH 2 -CH 2 units.
  • the PEG may be PEG1, PEG2, PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG10, PEG11, PEG12, PEG13, PEG14, PEG15, PEG16, PEG17, PEG18, PEG19, PEG20, PEG21, PEG22, PEG23, PEG24, PEG25, PEG26, PEG27, PEG28, PEG29, PEG30, PEG31, PEG32, PEG33, PEG34, PEG35, PEG36, PEG37, PEG38, PEG39, PEG40, PEG41, PEG42, PEG43, PEG44, PEG45, PEG46, PEG47, PEG48, PEG49, PEG50, PEG51, PEG52, PEG53, PEG39,
  • linkerB may comprise at least one alkyl group. In some embodiments, linkerB may comprise at least one substituted alkyl group. In some embodiments, an alkyl group may have 2 carbons, 3 carbons, 4 carbons, 5 carbons, 6 carbons, 7 carbons, 8 carbons, 9 carbons, 10 carbons, 11 carbons, 12 carbons, 13 carbons, 14 carbons, 15 carbons, 16 carbons, 17 carbons, 18 carbons, 19 carbons, or 20 carbons.
  • linkerB may comprise at least one substituted alkyl group.
  • a substituted alkyl group may have 2 carbons, 3 carbons, 4 carbons, 5 carbons, 6 carbons, 7 carbons, 8 carbons, 9 carbons, 10 carbons, 11 carbons, 12 carbons, 13 carbons, 14 carbons, 15 carbons, 16 carbons, 17 carbons, 18 carbons, 19 carbons, or 20 carbons.
  • a substituted alkyl group may include one or more of the following substitution groups: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.
  • linkerB may comprise at least one cycloalkyl group.
  • a cycloalkyl group may be a C3 cycloalkyl (i.e., cyclopropane) , C4 cycloalkyl (i.e., cyclobutene) , C5 cycloalkyl (i.e., cyclopentane) , C6 cycloalkyl (i.e., cyclohexane) , C7 cycloalkyl (i.e., cycloheptane) , C8 cycloalkyl (i.e., cyclooctane) , C9 cycloalkyl (i.e., cyclononane) , or C10 cycloalkyl (i.e., cyclodecane) .
  • linkerB may comprise at least one at least one substituted cycloalkyl group.
  • a substituted cycloalkyl group may be a C3 substituted cycloalkyl, C4 substituted cycloalkyl, C5 substituted cycloalkyl, C6 substituted cycloalkyl, C7 substituted cycloalkyl, C8 substituted cycloalkyl, C9 substituted cycloalkyl, or C10 substituted cycloalkyl.
  • a substituted cycloalkyl group may include one or more of the following substitution groups: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.
  • linkerB may comprise at least one alkenyl group.
  • an alkenyl group may have 4 carbons, 5 carbons, 6 carbons, 7 carbons, 8 carbons, 9 carbons, 10 carbons, 11 carbons, 12 carbons, 13 carbons, 14 carbons, 15 carbons, 16 carbons, 17 carbons, 18 carbons, 19 carbons, or 20 carbons.
  • linkerB may comprise at least one substituted alkenyl group.
  • an alkenyl group may have 4 carbons, 5 carbons, 6 carbons, 7 carbons, 8 carbons, 9 carbons, 10 carbons, 11 carbons, 12 carbons, 13 carbons, 14 carbons, 15 carbons, 16 carbons, 17 carbons, 18 carbons, 19 carbons, or 20 carbons.
  • a substituted alkenyl group may include one or more of the following substitution groups: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.
  • linkerB may comprise at least one cycloalkenyl group.
  • a cycloalkenyl group may be a C5 cycloalkenyl, C6 cycloalkenyl, C7 cycloalkenyl, C8 cycloalkenyl, C9 cycloalkenyl, or C10 cycloalkenyl.
  • linkerB may comprise at least one substituted cycloalkenyl group.
  • a cycloalkenyl group may be a C5 cycloalkenyl, C6 cycloalkenyl, C7 cycloalkenyl, C8 cycloalkenyl, C9 cycloalkenyl, or C10 cycloalkenyl.
  • a substituted cycloalkenyl group may include one or more of the following substitution groups: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.
  • linkerB may comprise at least one alkynyl group.
  • an alkynyl group may have 4 carbons, 5 carbons, 6 carbons, 7 carbons, 8 carbons, 9 carbons, 10 carbons, 11 carbons, 12 carbons, 13 carbons, 14 carbons, 15 carbons, 16 carbons, 17 carbons, 18 carbons, 19 carbons, or 20 carbons.
  • linkerB may comprise at least one substituted alkynyl group.
  • a substituted alkynyl group may have 4 carbons, 5 carbons, 6 carbons, 7 carbons, 8 carbons, 9 carbons, 10 carbons, 11 carbons, 12 carbons, 13 carbons, 14 carbons, 15 carbons, 16 carbons, 17 carbons, 18 carbons, 19 carbons, or 20 carbons.
  • a substituted alkynyl group may include one or more of the following substitution groups: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.
  • linkerB may comprise at least one aryl group or heteroaryl group. Examples include phenyl, naphthyl, and pyridinyl, although it is noted that other aryl and heteroaryl groups, that fall within the definitions provided herein, may be used.
  • linkerB may comprise at least one substituted aryl group.
  • a substituted aryl group may include one or more of the following substitution groups: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.
  • linkerB may comprise at least one aralkyl group.
  • Example aralkyl groups include, but are not limited to, phenylmethyl, phenylethyl, and phenylpropyl.
  • linkerB may comprise at least one substituted aralkyl group.
  • a substituted aralkyl group may include one or more of the following substitution groups: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.
  • linkerB may comprise at least one aralkenyl group.
  • Example aralkenyl groups include, but are not limited to ethenylbenzene and propenylbenzene.
  • linkerB may comprise at least one substituted aralkenyl group.
  • a substituted aralkenyl group may include one or more of the following substitution groups: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.
  • linkerB may comprise at least one aralkynyl group.
  • Example aralkynyl groups include, but are not limited to ethynylbenzene and propynylbenzene.
  • linkerB may comprise at least one substituted aralkynyl group.
  • a substituted aralkynyl group may include one or more of the following substitution groups: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.
  • linkerB may comprise at least one heteroatom.
  • linkerB may comprise one or more oxygen (O) heteroatoms, one or more nitrogen (N) heteroatoms, one or more sulfur (S) heteroatoms, and/or one or more phosphorous (P) heteroatoms.
  • at least one of the independently selected linkerAs may comprise at least one heteroalkyl group.
  • linkerB may comprise at least one aliphatic heterocycle. In some embodiments, linkerB may comprise at least one of tetrahydrofuran (THF) , tetrahydropyran (THP) , morpholine, piperidine, piperazine, pyrrolidine, and/or azetidine.
  • THF tetrahydrofuran
  • TPP tetrahydropyran
  • morpholine morpholine
  • piperidine piperazine
  • pyrrolidine pyrrolidine
  • azetidine azetidine
  • linkerB may comprise at least one heteroaryl group. In some embodiments, linkerB may comprise one or more of imidazole, pyrazole, pyridine, pyrimidine, triazole, and. or 1, 2, 3-triazole.
  • linkerB may comprise at least one substituted heteroaryl group.
  • a substituted heteroaryl group may include one or more of the following substitution groups: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.
  • linkerB may comprise at least one amino acid.
  • Various amino acids are known to those skilled in the art.
  • LinkerB is not limited to including one or more specific amino acids.
  • linkerB may comprise one or more arginine (Arg) amino acids, one or more histidine (His) amino acids, one or more lysine (Lys) amino acids, one or more aspartic acid (Asp) amino acids, one or more glutamic acid (Glu) amino acids, one or more serine (Ser) amino acids, one or more threonine (Thr) amino acids, one or more asparagine (Asn) amino acids, one or more glutamine (Gln) amino acids, one or more cysteine (Cys) amino acids, one or more selenocysteine (Sec) amino acids, one or more glycine (Gly) amino acids, one or more proline (Pro) amino acids, one or more alanine (Ala) amino acids, one or more valine (Val) amino
  • linkerB may comprise at least one nucleotide.
  • Various nucleotides are known to those skilled in the art. LinkerB is not limited to including one or more specific nucleotides.
  • linkerB may comprise one or more nucleotides comprising a guanine nucleobase, one or more nucleotides comprising an adenine nucleobase, one or more nucleotides comprising a cytosine nucleobase, one or more nucleotides comprising a thymine nucleobase, and/or one or more nucleotides comprising a uracil nucleobase.
  • linkerB may comprise at least one abasic nucleotide.
  • an abasic nucleotide is a nucleotide having an abasic site, which is a location that has neither a purine nor a pyrimidine base.
  • linkerB may comprise one or more abasic DNAs and/or one or more abasic RNAs.
  • linkerB may comprise at least one inverted abasis nucleotide.
  • linkerB may comprise one or more inverted abasic DNAs and/or one or more inverted abasic RNAs.
  • linkerB may comprise at least one saccharide. In some embodiments, linkerB may comprise at least one glucose monosaccharide unit, at least one fructose monosaccharide unit, at least one mannose monosaccharide unit, at least one galactose monosaccharide unit, at least one ribose monosaccharide unit, and/or at least one glucosamine monosaccharide unit.
  • linkerB may comprise one or more of:
  • j is an integral number between 1 and 12, and
  • k is an integral number between 0 and 12.
  • j is an integral number independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12.
  • k is an integral number independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12.
  • linkerB contains a six-membered ring fragment, especially a 4-Hydroxypiperidinyl group, when it is used as targeted delivery of pharmaceutical agents, compared with a five-membered ring, it shows better in vivo stability and activity, such as the compound 75 of the present invention.
  • a pharmaceutical agent is a diagnostic or therapeutic drug, molecule, compound, or combination of drugs, molecules, or compounds that have a property of assisting in diagnosis, prevention, treatment, and/or mitigation of a disease or condition, for example in a cell or subject.
  • a pharmaceutical agent is a drug, molecule, compound or combination of drugs, molecules, or compounds that have a property of assisting in the enhancement of a desirable condition, for example in a cell or subject.
  • a pharmaceutical agent may be an oligonucleotide.
  • the oligonucleotide may comprise a siRNA.
  • the oligonucleotide may comprise a double stranded siRNA.
  • the double stranded siRNA may comprise at least one modified ribonucleotide.
  • the at least one modified nucleotide comprises: a 2'-O-methyl nucleotide, 2'-Fluoro nucleotide, 2'-deoxy nucleotide, 2'3'-seco nucleotide mimic, locked nucleotide, unlocked nucleic acid nucleotide (UNA) , glycol nucleic acid nucleotide (GNA) , 2'-F-Arabino nucleotide, 2'-methoyxyethyl 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, mopholino nucleotide
  • all ribonucleotides of the double stranded siRNA may be modified.
  • at least one strand of the double stranded siRNA may comprise at least one phosphorothioate linkage.
  • at least one strand of the double stranded siRNA may comprise up to 6 phosphorothioate linkages.
  • a double stranded siRNA may comprise at least one locked nucleic acid (LNA) .
  • a LNA (sometimes referred to as a bridged nucleic acid (BNA) or an inaccessible RNA) is a modified RNA molecule where the ribose moiety is modified with an extra bridge connecting the 2' oxygen and the 4' carbon, thereby locking the ribose in the 3’-endo conformation.
  • LNA is known to increase stability against enzymatic degradation, and improve specificity and affinity.
  • a double stranded siRNA may comprise at least one unlocked nucleic acid (UNA) .
  • UNA unlocked nucleic acid
  • a UNA is an acyclic derivative of RNA lacking a C2'-C3'-bond of the ribose ring of RNA. It is known to those skilled in the art that including a UNA in certain positions of an antisense strand of a siRNA is tolerated for activity, and may promote reduced off target activity.
  • a double stranded siRNA may comprise at least one glycerol nucleic acid (GNA) .
  • GNA glycerol nucleic acid
  • a GNA (sometimes referred to as a glycol nucleic acid) is a nucleic acid similar to RNA but differing in the composition of its sugar-phosphodiester backbone. It is known to those skilled in the art that including a GNA in certain positions of an antisense strand of a siRNA is tolerated for activity, and may promote reduced off target activity.
  • the oligonucleotide may comprise a siRNA including one or more modified nucleotides, including but not limited to a 2'-modified nucleotide (e.g. F and MeO) , an abasic nucleotide, an inverted abasic nucleotide, a locked nucleotide, UNA an unlocked nucleic acid (UNA) , and a glycerol nucleic acid (GNA) .
  • a 2'-modified nucleotide e.g. F and MeO
  • the oligonucleotide may comprise a siRNA including one or more phosphorothioate backbone linkages.
  • the oligonucleotide may comprise a single strand siRNA. In some embodiments, the oligonucleotide may comprise a small activating RNA. In some embodiments, the oligonucleotide may comprise a microRNA (miRNA) . In some embodiments, the oligonucleotide may comprise an antisense oligonucleotide. In some embodiments, the oligonucleotide may comprise a short guide RNA (gRNA) . In some embodiments, the oligonucleotide may comprise a single guide RNA (sgRNA) . In some embodiments, the oligonucleotide may comprise a messenger RNA (mRNA) .
  • miRNA microRNA
  • gRNA short guide RNA
  • gRNA short guide RNA
  • sgRNA single guide RNA
  • the oligonucleotide may comprise a messenger RNA (mRNA) .
  • the oligonucleotide may comprise a ribozyme. In some embodiments, the oligonucleotide may comprise a plasmid. In some embodiments, the oligonucleotide may comprise an immune stimulating nucleic acid. In some embodiments, the oligonucleotide may comprise an antagomir. In some embodiments, the oligonucleotide may comprise an aptamer.
  • An aptamer is a short, single-stranded DNA or RNA molecule that can selectively bind to a specific target, such as a protein, peptide, carbohydrate, small molecule, toxin, or live cell. Aptamers assume a variety of shapes, as they tend to form helices and single-stranded loops. The present disclosure is not limited to any particular aptamer. Aptamers known and not yet discovered are within the scope of the present disclosure.
  • the oligonucleotide may comprise at least 3 independently selected nucleotides. In some embodiments, the oligonucleotide may comprise between 16 and 23 independently selected nucleotides, for example when the oligonucleotide is a siRNA. In some embodiments, the oligonucleotide may comprise about 100 independently selected nucleotides, for example when the oligonucleotide is a sgRNA. In some embodiments, the oligonucleotide may comprise up to fourteen thousand independently selected nucleotides, for example when the oligonucleotide is an mRNA.
  • W in formula 1 may be a functional group capable of linking to one or more pharmaceutical agents.
  • the functional group may be a hydroxy group (OH) .
  • the functional group may be a protected hydroxy group.
  • various protecting groups may be used to protect a hydroxy group.
  • the hydroxy group may be protected using at least one of 4, 4’-dimethoxytrityl (DMT) , monomethoxytrityl (MMT) , 9- (p-methoxyphenyl) xanthen-9-yl (Mox) , and 9-phenylxanthen-9-yl (Px) .
  • the functional group may be a phosphoramidite group having the formula:
  • R a is a C1 to C6 alkyl, C3 to C6 cycloalkyl, an isopropyl group, or R a joins with R b through a nitrogen atom to form a cycle,
  • R b is a C1 to C6 alkyl, C3 to C6 cycloalkyl, an isopropyl group, or R b joins with R a through a nitrogen atom to form a cycle, and
  • R c is a phosphite protecting group, phosphate protecting group, or a 2-cyanoethyl group.
  • R c may be one of various phosphite protecting groups known to those skilled in the art.
  • the phosphite protecting group may comprise at least one of methyl, allyl, 2-cyanoethyl, 4-cyano-2-butenyl, 2-cyano-1, 1-dimethylethyl, 2- (trimethylsilyl) ethyl, 2- (S-acetylthio) ethyl, 2- (S-pivaloylthio) ethyl, 2- (4-nitrophenyl) ethyl, 2, 2, 2-trichloroethyl, 2, 2, 2-trichloro-1, 1-dimethylethyl, 1, 1, 1, 3, 3, 3-hexafluoro-2-propyl, fluorenyl-9-methyl, 2-chlorophenyl, 4-chlorophenyl, and 2, 4-dichlorophenyl.
  • R c may be one of various phosphate protecting groups known to those skilled in the art.
  • the phosphate protecting group may comprise at least one of methyl, allyl, 2-cyanoethyl, 4-cyano-2-butenyl, 2-cyano-1, 1-dimethylethyl, 2- (trimethylsilyl) ethyl, 2- (S-acetylthio) ethyl, 2- (S-pivaloylthio) ethyl, 2- (4-nitrophenyl) ethyl, 2, 2, 2-trichloroethyl, 2, 2, 2-trichloro-1, 1-dimethylethyl, 1, 1, 1, 3, 3, 3-hexafluoro-2-propyl, fluorenyl-9-methyl, 2-chlorophenyl, 4-chlorophenyl, and 2, 4-dichlorophenyl.
  • the functional group may be a carboxyl group (CO 2 H) . In some embodiments, the functional group may be an activated carboxyl group having the formula:
  • the leaving group (X) may be one of carboxylate, sulfonate, chloride, phosphate, imidazole, hydroxybenzotriazole (HOBt) , N-hydroxysuccinimide (NHS) , tetrafluorophenol, pentafluorophenol, of para-nitrophenol.
  • the functional group may be a Michael acceptor.
  • the Michael acceptor may have the formula:
  • E is an electron withdrawing group
  • R d is hydrogen or a C1-C6 alkyl substitution group on olefin (meaning E and R d may be cis, trans, or iso with respect to the carbon-carbon double bond) .
  • the electron withdrawing group (E) may be carboxamide or an ester.
  • the electron withdrawing group (E) and the carbon-carbon double bond of the Michael acceptor may be part of maleimide, a cyclic dicarboximide in which the two carboacyl groups on nitrogen together with the nitrogen itself form a 1H-pyrrole-2, 5-dione structure.
  • the functional group may have the formula:
  • linkerC is absent or a spacer attached to a 3’ or 5’ end of an oligonucleotide
  • X is a methyl group, oxygen, sulfur, or an amino group
  • Y is oxygen, sulfur, or an amino group.
  • linkerC of Formula 5 may comprise at least a heterocyclic compound.
  • the heterocyclic compound may be an abasic nucleotide or an inverted abasic nucleotide.
  • the functional group may have the formula:
  • linkerC is a spacer having one end attached to a nitrogen of a carboxamide and the other end attached to a 3’ or 5’ end of an oligonucleotide.
  • linkerB is attached to the carbonyl of the carboxyamide of Formula 6.
  • linkerC in Formula 6 may comprise at least one PEG.
  • the PEG may have any number of repeating O-CH 2 -CH 2 units.
  • the PEG may be PEG1, PEG2, PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG10, PEG11, PEG12, PEG13, PEG14, PEG15, PEG16, PEG17, PEG18, PEG19, PEG20, PEG21, PEG22, PEG23, PEG24, PEG25, PEG26, PEG27, PEG28, PEG29, PEG30, PEG31, PEG32, PEG33, PEG34, PEG35, PEG36, PEG37, PEG38, PEG39, PEG40, PEG41, PEG42, PEG43, PEG44, PEG45, PEG46, PEG47, PEG48, PEG49, PEG50, PEG51, PEG52, PEG53, PEG54
  • linkerC in Formula 6 may comprise at least one alkyl group.
  • an alkyl group may have 2 carbons, 3 carbons, 4 carbons, 5 carbons, 6 carbons, 7 carbons, 8 carbons, 9 carbons, 10 carbons, 11 carbons, 12 carbons, 13 carbons, 14 carbons, 15 carbons, 16 carbons, 17 carbons, 18 carbons, 19 carbons, or 20 carbons.
  • linkerC in Formula 6 may comprise at least one cycloalkyl group.
  • a cycloalkyl group may be a C3 cycloalkyl (i.e., cyclopropane) , C4 cycloalkyl (i.e., cyclobutene) , C5 cycloalkyl (i.e., cyclopentane) , C6 cycloalkyl (i.e., cyclohexane) , C7 cycloalkyl (i.e., cycloheptane) , C8 cycloalkyl (i.e., cyclooctane) , C9 cycloalkyl (i.e., cyclononane) , or C10 cycloalkyl (i.e., cyclodecane) .
  • linkerC in Formula 6 may comprise at least one heteroatom.
  • linkerC may comprise one or more oxygen (O) heteroatoms, one or more nitrogen (N) heteroatoms, one or more sulfur (S) heteroatoms, and/or one or more phosphorous (P) heteroatoms.
  • linkerC in Formula 6 may comprise at least one aliphatic heterocycle.
  • linkerC may comprise at least one of tetrahydrofuran (THF) , tetrahydropyran (THP) , morpholine, piperidine, piperazine, pyrrolidine, and/or azetidine.
  • linkerC in Formula 6 may comprise at least one heteroaryl group.
  • linkerC may comprise one or more of imidazole, pyrazole, pyridine, pyrimidine, triazole, and. or 1, 2, 3-triazole.
  • linkerC in Formula 6 may comprise at least one substituted heteroaryl group.
  • a substituted heteroaryl group may include one or more of the following substitution groups: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.
  • linkerC in Formula 6 may comprise at least one amino acid.
  • Various amino acids are known to those skilled in the art.
  • LinkerC is not limited to including one or more specific amino acids.
  • linkerC may comprise one or more arginine (Arg) amino acids, one or more histidine (His) amino acids, one or more lysine (Lys) amino acids, one or more aspartic acid (Asp) amino acids, one or more glutamic acid (Glu) amino acids, one or more serine (Ser) amino acids, one or more threonine (Thr) amino acids, one or more asparagine (Asn) amino acids, one or more glutamine (Gln) amino acids, one or more cysteine (Cys) amino acids, one or more selenocysteine (Sec) amino acids, one or more glycine (Gly) amino acids, one or more proline (Pro) amino acids, one or more alanine (Ala) amino acids, one or more valine (
  • linkerC in Formula 6 may comprise at least one nucleotide.
  • Various nucleotides are known to those skilled in the art. LinkerC is not limited to including one or more specific nucleotides.
  • linkerC may comprise one or more nucleotides comprising a guanine nucleobase, one or more nucleotides comprising an adenine nucleobase, one or more nucleotides comprising a cytosine nucleobase, one or more nucleotides comprising a thymine nucleobase, and/or one or more nucleotides comprising a uracil nucleobase.
  • linkerC may comprise at least one abasic nucleotide.
  • an abasic nucleotide is a nucleotide having an abasic site, which is a location that has neither a purine nor a pyrimidine base.
  • linkerC may comprise one or more abasic DNAs and/or one or more abasic RNAs.
  • linkerC may comprise at least one inverted abasis nucleotide.
  • linkerC may comprise one or more inverted abasic DNAs and/or one or more inverted abasic RNAs.
  • linkerC in Formula 6 may comprise at least one saccharide.
  • linkerC may comprise at least one glucose monosaccharide unit, at least one fructose monosaccharide unit, at least one mannose monosaccharide unit, at least one galactose monosaccharide unit, at least one ribose monosaccharide unit, and/or at least one glucosamine monosaccharide unit.
  • linkerC in Formula 6 may comprise one or more of:
  • j is an integral number between 1 and 12, and
  • k is an integral number between 0 and 12.
  • the functional group may have the formula:
  • linkerC is a spacer with one end attached to one of the succinimide ring carbons through a thioether bond and the other end attached to a 3’ or 5’ end of an oligonucleotide.
  • linkerB is attached to the succinimide nitrogen of Formula 7.
  • linkerC in Formula 7 may comprise at least one PEG.
  • the PEG may have any number of repeating O-CH 2 -CH 2 units.
  • the PEG may be PEG1, PEG2, PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG10, PEG11, PEG12, PEG13, PEG14, PEG15, PEG16, PEG17, PEG18, PEG19, PEG20, PEG21, PEG22, PEG23, PEG24, PEG25, PEG26, PEG27, PEG28, PEG29, PEG30, PEG31, PEG32, PEG33, PEG34, PEG35, PEG36, PEG37, PEG38, PEG39, PEG40, PEG41, PEG42, PEG43, PEG44, PEG45, PEG46, PEG47, PEG48, PEG49, PEG50, PEG51, PEG52, PEG53, PEG54
  • linkerC in Formula 7 may comprise at least one alkyl group.
  • an alkyl group may have 2 carbons, 3 carbons, 4 carbons, 5 carbons, 6 carbons, 7 carbons, 8 carbons, 9 carbons, 10 carbons, 11 carbons, 12 carbons, 13 carbons, 14 carbons, 15 carbons, 16 carbons, 17 carbons, 18 carbons, 19 carbons, or 20 carbons.
  • linkerC in Formula 7 may comprise at least one cycloalkyl group.
  • a cycloalkyl group may be a C3 cycloalkyl (i.e., cyclopropane) , C4 cycloalkyl (i.e., cyclobutene) , C5 cycloalkyl (i.e., cyclopentane) , C6 cycloalkyl (i.e., cyclohexane) , C7 cycloalkyl (i.e., cycloheptane) , C8 cycloalkyl (i.e., cyclooctane) , C9 cycloalkyl (i.e., cyclononane) , or C10 cycloalkyl (i.e., cyclodecane) .
  • linkerC in Formula 7 may comprise one or more of:
  • j is an integral number between 1 and 12, and
  • k is an integral number between 0 and 12.
  • Multivalent Ligand Clusters Comprising GalNAc Targeting Ligands
  • a multivalent ligand cluster of the present disclosure may have the general structure of Formula 8:
  • n 1 and 10
  • each n is an independently selected integral number between 1 and 10,
  • each linkerA is an independently selected spacer, with one end attached to a TL and the other end attached to the nitrogen of an alkylcarboxamide,
  • linkerB is a spacer, with one end attached to a pharmaceutical agent or a functional group capable of linking to one or more pharmaceutical agents, and the other end attached to a diamine nitrogen, and
  • W is either one or more pharmaceutical agents, or a functional group capable of linking to one or more pharmaceutical agents.
  • a multivalent ligand cluster of the present disclosure may have the general structure of Formula 9:
  • n 1 and 10
  • each n is an independently selected integral number between 1 and 10,
  • each linkerA is an independently selected spacer
  • linkerB is a spacer
  • linkerC is a spacer or absent
  • X is a methyl group, oxygen, sulfur, or an amino group
  • Y is oxygen, sulfur, or an amino group.
  • a multivalent ligand cluster of the present disclosure may have the general structure of Formula 10:
  • n 1 and 10
  • each n is an independently selected integral number between 1 and 10,
  • each linkerA is an independently selected spacer
  • linkerB is a spacer
  • linkerC is a spacer
  • a multivalent ligand cluster of the present disclosure may have the general structure of Formula 11:
  • n 1 and 10
  • each n is an independently selected integral number between 1 and 10,
  • each linkerA is an independently selected spacer
  • linkerB is a spacer
  • linkerC is a spacer
  • Scheme 1 starts with a mono-protected diamine (Compound I) .
  • the mono-protected diamine comprises a first nitrogen and a second nitrogen, where the first nitrogen is a primary amine, and the second nitrogen is a secondary amine comprising a protecting group (PG) in Scheme 1) .
  • the protecting group may be a benzyl group. In some embodiments, the protecting group may be a triphenylmethyl group.
  • m in Scheme 1 may be any integral number. In some embodiments, m in Scheme 1 may be an integral number between 1 and 10.
  • a triester Compound II can be synthesized in one step.
  • Compound II may be synthesized via a S N 2 substitution reaction using Compound I and one or more appropriate substrates.
  • Compound II may be synthesized via a reductive amination reaction using Compound I and one or more appropriate substrates.
  • Compound II may be synthesized via a Michael addition reaction using Compound I and one or more appropriate substrates.
  • the first nitrogen is a tertiary amine comprise a first protected carboxylic acid and a second protected carboxylic acid
  • the second nitrogen is a tertiary amine comprising the protecting group and a third protected carboxylic acid.
  • each “n” in Scheme 1 is an independently selected integral number. In some embodiments, each n in Scheme 1 is an independently selected integral number between 0 and 10.
  • Compound III is produced by deprotecting the second nitrogen of Compound II, resulting in the second nitrogen of Compound III being a secondary amine comprising the third protected carboxylic acid.
  • the protecting group is a benzyl group
  • Compound III may be produced by performing a hydrogenation reaction using Compound II.
  • the protecting group is a triphenylmethyl group
  • Compound III may be produced by reacting the second compound with at least one acid.
  • Example acids include, but are not limited to, hydrochloric acid (HCl) and trifluoroacetic acid (TFA) .
  • Compound IV is produced by attaching a moiety comprising a hydroxy group to the second nitrogen of Compound III, resulting in the second nitrogen of Compound IV being a tertiary amine or an amide comprising the third protected carboxylic acid and the moiety comprising the hydroxy group.
  • the moiety comprising the hydroxy group may be attached to the second nitrogen using any linkerB described herein above.
  • producing triester Compound IV comprises performing a S N 2 reaction using Compound III and one or more appropriate substrates. In some embodiments, producing Compound IV comprises performing a reductive amination reaction using Compound III and one or more appropriate substrates. In some embodiments, producing Compound IV comprises performing a Michael addition reaction using Compound III and one or more appropriate substrates. In some embodiments, producing Compound IV comprises performing an amide coupling reaction using Compound III and one or more appropriate substrates. In some embodiments, producing Compound IV comprises performing a nucleophilic addition reaction using Compound III and one or more appropriate substrates. Examples substrates include, but are not limited to, isocyaniate and isothiocyanate.
  • Triacid Compound V is produced by converting the protected carboxylic acids of Compound IV into carboxylic acids.
  • Compound V may be produced by reacting Compound IV with one or more acids (e.g., when R in Scheme 1 is an acid sensitive group, such as a tert-butyl group) .
  • the one or more acids may comprise hydrochloric acid (HCl) , hydrobromic acid (HBr) , trifluoroacetic acid (TFA) , and formic acid.
  • producing Compound V may comprise performing a hydrogenation reaction using Compound IV (e.g., when R in Scheme 1 is a benzyl group) .
  • producing Compound V may comprise performing a hydrolysis reaction using Compound IV.
  • Compound VI may be produced by perform an amide coupling reaction using the Compound V.
  • the first nitrogen is a tertiary amine comprising a first amide and a second amide
  • the second nitrogen is a tertiary amine comprising the moiety comprising the hydroxy group and a third amide.
  • the first amide, the second amide, and the third amide may each be coupled to an independently selected targeting ligand.
  • linkerA in Scheme 1 may be any linkerA described herein.
  • TL in Scheme 1 may be any TL described herein.
  • Compound VII is produced by converting the hydroxy group (attached to linkerB) of Compound VI to a phosphoramidite group using a phosphitylation reaction. As illustrated in Scheme 1, in some embodiments converting the hydroxy group to the phosphoramidite group may be performed after performing the amide coupling reaction to produce Compound VI.
  • Scheme 2 Another method for the preparation of examples of compounds with general Formula 1 is depicted in Scheme 2 below.
  • Scheme 2 allows for a compound with general Formula 1 to have one or more different targeting ligands.
  • Scheme 2 allows for stepwise introduction of targeting ligands.
  • Starting materials and intermediates may be purchased from commercial sources, made from known procedures, or are otherwise illustrated. The order of carrying out the steps of the reaction scheme may be varied.
  • Scheme 2 starts with a double-protected diamine (Compound I) .
  • the double-protected diamine comprises a first nitrogen and a second nitrogen, where the first nitrogen is a secondary amine comprising a first protecting group (PG 1 ) , and the second nitrogen is a primary or a secondary amine comprising a second protecting group (PG 2 ) .
  • the first protecting group and the second protecting group may be different, thereby allowing different linkerAs and targeting ligands to be attached to the diamine scaffold.
  • Various protecting groups are known to those skilled in the art, and may be used.
  • the first protecting group may be a benzyl group
  • the second protecting group may be a tert-butyloxycarbonyl (Boc) group.
  • m in Scheme 2 may be any integral number. In some embodiments, m in Scheme 2 may be an integral number between 1 and 10.
  • Compound II may be produced by coupling a first protected carboxylic acid to the first nitrogen of Compound I, resulting in the first nitrogen of Compound II being a tertiary amine.
  • the protecting groups may be strategically removed and replaced.
  • the first protecting group is a benzyl group and the second protecting group is a Boc group
  • the amine with the benzyl group (and not the amine with the Boc group) of Compound I may undergo a S N 2 substitution reaction, a reductive amination reaction, or a Michael addition reaction with one or more appropriate reagents to form Compound II.
  • Compound III may be produced by removing the first protecting group from Compound II.
  • the first nitrogen is a secondary amine having the first protected carboxylic acid
  • the second nitrogen is a primary or a secondary amine having the second protecting group.
  • producing Compound III may comprise performing a hydrogenation reaction (e.g., when the first protecting group is a benzyl group) .
  • Compound IV may be produced by coupling a second protected carboxylic acid to the first nitrogen of Compound III, resulting in the first nitrogen of Compound IV being a tertiary amine.
  • producing Compound IV may comprise performing a S N 2 substitution reaction using Compound III and one or more other appropriate reagents.
  • producing Compound IV may comprise performing a reductive amination reaction using Compound III and one or more other appropriate reagents.
  • producing Compound IV may comprise performing a Michael addition reaction using Compound III and one or more other appropriate reagents.
  • producing Compound IV may comprise performing an amide coupling reaction using Compound III and one or more other appropriate reagents.
  • producing Compound IV may comprise performing a nucleophilic addition reaction using Compound III and one or more other appropriate reagents.
  • Compound V may be produced by removing the second protecting group from Compound IV, resulting in the first nitrogen of Compound V being a tertiary amine comprising the first protected carboxylic acid and the second protected carboxylic acid, and the second nitrogen of Compound V being a primary amine.
  • Compound V may be produced by reacting Compound IV with at least one acid.
  • Example acids include, but are not limited to, hydrochloric acid (HCl) and trifluoroacetic acid (TFA) .
  • Compound VI may be produced by coupling a third protected carboxylic acid to the second nitrogen of Compound V, resulting in the second nitrogen of Compound VI being a secondary amine.
  • Compound VI may be produced by performing a S N 2 substitution reaction using Compound V and one or more other appropriate reagents. In some embodiments, Compound VI may be produced by performing a reductive amination reaction using Compound V and one or more other appropriate reagents. In some embodiments, Compound VI may be produced by performing a Michael addition reaction using Compound V and one or more other appropriate reagents.
  • Compound VII may be produced by attaching a moiety comprising a hydroxy group to the second nitrogen of Compound VI, resulting in the second nitrogen of Compound VII being a tertiary amine or an amide or a urea.
  • the moiety comprising the hydroxy group may be attached to the second nitrogen using any linkerB described herein above.
  • Compound VII may be produced by performing a S N 2 substitution reaction using Compound VI and one or more other appropriate reagents.
  • Compound VII may be produced by performing a reductive amination reaction using Compound VI and one or more other appropriate reagents.
  • Compound VII may be produced by performing a Michael addition reaction using Compound VI and one or more other appropriate reagents.
  • Compound VII may be produced by performing an amide coupling reaction using Compound VI and one or more other appropriate reagents. In some embodiments, Compound VII may be produced by performing a nucleophilic addition reaction using Compound VI and one or more other appropriate reagents.
  • R a , R b , and R c may be sufficiently different such that targeting ligands may be selectively attached, for example in one scenario R a , R b , and R c may be methyl, benzyl and tert-butyl groups, respectively.
  • R a , R b , and R c may be methyl, benzyl and tert-butyl groups, respectively. The following describes such selective attachment of targeting ligands.
  • Compound VIII is produced by converting the third protected carboxylic acid of Compound VII into a first carboxylic acid.
  • Compound VIII may be produced by reacting Compound VII with one or more acids (e.g., when R c in Scheme 2 is an acid sensitive group, for example a tert-butyl group) .
  • Compound IX may be produced by performing an amide coupling reaction using Compound VIII.
  • the first nitrogen comprises the first protected carboxylic acid and the second protected carboxylic acid
  • the second nitrogen of Compound IX comprises a first amide having a first targeting ligand coupled thereto and the moiety comprising the hydroxy group.
  • Compound X is produced by converting the second protected carboxylic acid of Compound IX into a second carboxylic acid.
  • producing Compound X may comprise performing a hydrogenation reaction using Compound IX (e.g., when R b in Scheme 2 is a benzyl group) .
  • Compound XI may be produced by performing an amide coupling reaction using Compound X.
  • the first nitrogen comprises the first protected carboxylic acid and a second amide having a second targeting ligand coupled thereto
  • the second nitrogen of Compound XI comprises the first amide having the first targeting ligand coupled thereto and the moiety comprising the hydroxy group.
  • Compound XII is produced by converting the first protected carboxylic acid of Compound XI into a third carboxylic acid.
  • producing Compound XII may comprise performing a hydrolysis reaction using Compound XI (e.g., when R a in Scheme 2 is a methyl group) .
  • Compound XIII may be produced by performing an amide coupling reaction using Compound XII.
  • the first nitrogen comprises the second amide having the second targeting ligand coupled thereto and a third amide having a third targeting ligand coupled thereto
  • the second nitrogen of Compound XI comprises a first amide having a first targeting ligand coupled thereto and the moiety comprising the hydroxy group.
  • the first amide may be coupled to the first targeting ligand using any independently selected linkerA described herein.
  • the second amide may be coupled to the second targeting ligand using any independently selected linkerA described herein.
  • the third amide may be coupled to the third targeting ligand using any independently selected linkerA described herein.
  • One or more of the first targeting ligand, the second targeting ligand, and the third targeting ligand may be independently selected to be one or more of the targeting ligands described herein.
  • the hydroxy group may be coupled to the second nitrogen using any linkerB described herein.
  • the hydroxy group may be converted to a phosphoramidite group using a phosphitylation reaction. In some embodiments, the hydroxy group may be converted to the phosphoramidite group producing Compound XIV.
  • each “n x ” , “n y ” or “n z ” in Scheme 2 is an independently selected integral number. In some embodiments, each “n x ” , “n y ” or “n z ” in Scheme 2 is an independently selected integral number between 0 and 10.
  • Embodiments of multivalent ligand clusters of the invention can be prepared and used to deliver oligonucleotide agents to cells, tissues, and organs.
  • agents that can be delivered include therapeutic agents such as siRNA.
  • Delivery methods using multivalent ligand clusters of the invention can be used to deliver siRNAs and other agents conjugated to a target ligand cluster of the invention to in vitro and in vivo cells.
  • Multivalent ligand clusters of the invention can be used as a delivery vehicle with which to deliver agents, such as but not limited to agents comprising nucleic acids, to a cell.
  • multivalent ligand cluster/pharmaceutical agent complex means a multivalent ligand cluster as described herein that is linked to a pharmaceutical agent as described herein.
  • the pharmaceutical agent is an siRNA.
  • the dsRNA agent comprises 2'-fluoro modified nucleotides at position 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 position 9, 11 and 13 of the sense strand (counting from the first paired nucleotide from the 3' end of the sense strand) .
  • no other positions of the dsRNA agent contain 2' fluorine-modified nucleotides.
  • nucleotides of the antisense strand and /or sense strand of the dsRNA agent are modified nucleotides.
  • the dsRNA agent has 2'-fluoro modified nucleotides at position 2, 7, 12, 14 and 16 of the antisense strand and/or 2' fluorine-modified nucleotide at positions 9, 11 and 13 of the sense strand, with other positions containing modified nucleotides selected from: 2’-O-methyl nucleotide, 2’-deoxy nucleotide, 2’3’-seco nucleotide mimic, locked nucleotide, unlocked nucleic acid nucleotide (UNA) , glycol nucleic acid nucleotide (GNA) , 2’-F-Arabino nucleotide, 2’-methoyxyethyl nucleotide, abasic nucleotide, ribitol, inverted nucleot
  • the dsRNA agent includes an E-vinylphosphonate nucleotide at the 5′ end of the guide strand. In certain embodiments, the dsRNA agent includes 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 includes at least one phosphorothioate internucleoside linkage. In some embodiments, the sense strand includes 1, 2, 3, 4, 5, or 6, phosphorothioate internucleoside linkages. In some embodiments, the antisense strand includes 1, 2, 3, 4, 5, or 6, phosphorothioate internucleoside linkages.
  • the sense strand is complementary or substantially complementary to the antisense strand, and the region of complementarity is between 16 and 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.
  • the dsRNA agent includes at least one modified nucleotide and further includes one or more targeting groups or linking groups.
  • the one or more targeting groups or linking groups are conjugated to the sense strand.
  • the targeting group comprises N-acetyl-galactosamine (GalNAc) .
  • the targeting group contains a structure of GalNAc described above.
  • a multivalent ligand cluster may be used to deliver a pharmaceutical agent to a cell in a subject.
  • Means of administering a multivalent ligand cluster/pharmaceutical agent complex to a subject may include art-known methods.
  • a multivalent ligand cluster/pharmaceutical agent complex may be locally delivered in vivo by direct injection or by use of an infusion pump.
  • a multivalent ligand cluster/pharmaceutical agent complex is in a pharmaceutical composition and may be referred to as a pharmaceutical agent.
  • a pharmaceutical agent of the invention is administered to a subject in an amount effective to prevent, modulate the occurrence, treat, or alleviate a symptom of a disease state in the subject.
  • a subject shall mean a human or vertebrate mammal including but not limited to a dog, cat, horse, goat, cow, sheep, rodent, and primate, e.g., monkey.
  • the invention can be used to treat diseases or conditions in human and non-human subjects.
  • methods and compositions of the invention can be used in veterinary applications as well as in human prevention and treatment regimens.
  • a vertebrate subject is a mammal.
  • a multivalent ligand cluster/pharmaceutical agent complex of the invention is delivered to and contacted with a cell.
  • a contacted cell is in culture, and in other embodiments a contacted cell is in a subject.
  • Types of cells that may be contacted with a multivalent ligand cluster/pharmaceutical agent complex of the invention include, but are not limited to, liver cells, muscle cells, cardiac cells, circulatory cells, neuronal cells, glial cells, fat cells, skin cells, hematopoietic cells, epithelial cells, sperm, oocytes, muscle cells, adipocytes, kidney cells, hepatocytes, or pancreas cells.
  • the cell contacted with a multivalent ligand cluster/pharmaceutical agent complex of the invention is a liver cell.
  • a unit dose may contain between about 0.01 mg/kg and about 100 mg/kg body weight of siRNA.
  • the dose can be from 10 mg/kg to 25 mg/kg body weight, or 1 mg/kg to 10 mg/kg body weight, or 0.05 mg/kg to 5 mg/kg body weight, or 0.1 mg/kg to 5 mg/kg body weight, or 0.1 mg/kg to l mg/kg body weight, or 0.1 mg/kg to 0.5 mg/kg body weight, or 0.5 mg/kg to 1 mg/kg body weight, or 1 mg/kg to 3 mg/kg body weight.
  • the pharmaceutical composition may be a sterile injectable aqueous suspension or solution, or in a lyophilized form.
  • the pharmaceutical compositions and medicaments of the present disclosure may be administered to a subject in a pharmaceutically effective dose.
  • a variety of administration routes for a multivalent ligand cluster/pharmaceutical agent complex of the invention are available.
  • the particular delivery mode selected will depend upon the particular condition being treated and the dosage required for therapeutic efficacy. Methods of this invention, generally speaking, may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of treatment without causing clinically unacceptable adverse effects.
  • a multivalent ligand cluster/pharmaceutical agent complex of the invention may be administered via an oral, enteral, mucosal, percutaneous, and/or parenteral route.
  • parenteral includes subcutaneous, intravenous, intramuscular, intraperitoneal, and intracisternal injection, or infusion techniques.
  • routes include but are not limited to nasal (e.g., via a gastro-nasal tube) , dermal, vaginal, rectal, and sublingual. Delivery routes of the invention may include intrathecal, intraventricular, or intracranial.
  • a multivalent ligand cluster/pharmaceutical agent complex of the invention may be placed within a slow release matrix and administered by placement of the matrix in the subject.
  • a multivalent ligand cluster/pharmaceutical agent complex of the invention may be administered in formulations, which may be administered in pharmaceutically acceptable solutions, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients.
  • the multivalent ligand cluster/pharmaceutical agent complex may be administered in a pharmaceutical composition.
  • a pharmaceutical composition comprises the multivalent ligand cluster/pharmaceutical agent complex of the invention and a pharmaceutically-acceptable carrier.
  • Pharmaceutically acceptable carriers are well known to the skilled artisan and may be selected and utilized using routine methods.
  • a pharmaceutically-acceptable carrier means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients (e.g., the ability of the delivered nucleic acid, for example the siRNA to prevent and/or treat a disease or condition to which it is directed) .
  • Pharmaceutically acceptable carriers may include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials that are well-known in the art. Illustrative pharmaceutically acceptable carriers are described in U.S. Pat. No. 5,211,657 and others are known by those skilled in the art. Such preparations may routinely contain salt, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. When used in medicine, the salts should 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 invention.
  • Such pharmacologically and pharmaceutically-acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like.
  • pharmaceutically-acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.
  • a multivalent ligand cluster/pharmaceutical agent complex of the invention maybe administered directly to a tissue.
  • Direct tissue administration may be achieved by direct injection, or other art-known means.
  • a multivalent ligand cluster/pharmaceutical agent complex of the invention may be administered once, or alternatively may be administered in a plurality of administrations. If administered multiple times, a multivalent ligand cluster/pharmaceutical agent complex of the invention may be administered via different routes. For example, the first (or the first few) administrations may be made directly into an affected tissue or organ while later administrations may be systemic.
  • a multivalent ligand cluster/pharmaceutical agent complex of the invention when it is desirable to have it administered systemically, may be formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion) .
  • Formulations for injection may be presented in unit dosage form (e.g., in ampoules or in multi-dose containers) , with or without an added preservative.
  • the pharmaceutical compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing, and/or dispersing agents.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions, or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose) , and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. Lower doses will result from other forms of administration, such as intravenous administration. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits.
  • 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 result in a desired level of the pharmaceutical agent, for example a desired level of the siRNA.
  • matrices can be used to deliver one or more multivalent ligand cluster/pharmaceutical agent complexes of the invention to a cell and/or subject.
  • a matrix may be biodegradable.
  • Matrix polymers may be natural or synthetic polymers.
  • a polymer can be selected based on the period of time over which release is desired, generally in the order of a few hours to a year or longer. Typically, release over a period ranging from between a few hours and three to twelve months can be used.
  • the polymer optionally is in the form of a hydrogel that can absorb up to about 90%of its weight in water and further, optionally is cross-linked with multivalent ions or other polymers.
  • a multivalent ligand cluster/pharmaceutical agent complex of the invention may be delivered using the bioerodible implant by way of diffusion, or by degradation of the polymeric matrix.
  • Illustrative synthetic polymers for such use are well known in the art.
  • Biodegradable polymers and non-biodegradable polymers can be used for delivery of one or more of a multivalent ligand cluster/pharmaceutical agent complex of the invention using art-known methods. Such methods may also be used to deliver one or more multivalent ligand cluster/pharmaceutical agent complexes of the invention for treatment.
  • Additional suitable delivery systems can include time-release, delayed release or sustained-release delivery systems.
  • Such systems can avoid repeated administrations of a multivalent ligand cluster/pharmaceutical agent complex of the invention, increasing convenience to the subject and the health-care provider.
  • Many types of release delivery systems are available and known to those of ordinary skill in the art. [See for example: U.S. Pat. Nos. 5,075,109; 4,452,775; 4,675,189; 5,736,152; 3,854,480; 5,133,974; and 5,407,686 (the teachings of each of which are incorporated herein by reference) ] .
  • pump-based hardware delivery systems can be used, some of which are adapted for implantation.
  • long-term sustained release implant 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 an siRNA delivered using a multivalent ligand cluster of the invention.
  • Long-term release means that the implant is constructed and arranged to delivery therapeutic levels of the active ingredient for at least 30 days, 60 days, 90 days, or longer.
  • Long-term sustained release implants are well-known to those of ordinary skill in the art and include some of the release systems described above.
  • Therapeutic formulations of one or more multivalent ligand cluster/pharmaceutical agent complexes of the invention may be prepared for storage by mixing the multivalent ligand cluster/pharmaceutical agent complex having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers [Remington's Pharmaceutical Sciences 21 st edition, (2006) ] , in the form of lyophilized formulations or aqueous solutions.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including 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 immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, as
  • the multivalent ligand cluster/pharmaceutical agent complexes of the present disclosure may be formulated as pharmaceutical compositions.
  • the pharmaceutical compositions may be used as medicaments, alone or in combination with other agents.
  • the multivalent ligand cluster/pharmaceutical agent complex of the present disclosure can also be administered in combination with other therapeutic compounds, either administrated separately or simultaneously (e.g., as a combined unit dose) .
  • the present disclosure includes a pharmaceutical composition comprising one or more multivalent ligand cluster/pharmaceutical agent complex according to the present disclosure in a physiologically/pharmaceutically acceptable excipient, such as a stabilizer, preservative, diluent, buffer, and the like.
  • a pharmaceutical composition of the invention may be administered alone, in combination with each other, and/or in combination with other drug therapies, or other treatment regimens that are administered to subjects with a disease or condition.
  • Pharmaceutical compositions used in the embodiments of the invention preferably are sterile and contain an effective amount of a multivalent ligand cluster/pharmaceutical agent complex to prevent or treat a disease or condition, to which the pharmaceutical agent, for example a siRNA, is directed.
  • the dose or doses of a pharmaceutical composition of the invention that are sufficient to treat a disease or condition when administered to a subject can be chosen in accordance with different parameters, in particular in accordance with the mode of administration used and the state of the subject. Other factors may include the desired period of treatment. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. In some embodiments of the invention, dosing is used that has been determined using routine means such as in clinical trials.
  • a multivalent ligand cluster may comprise GalNAc targeting ligands.
  • the following are example compounds of multivalent ligand clusters comprising acetyl group protected GalNAc targeting ligands, core C2 and C3 diamines, branching acetyl and propanoyl amides, PEG2 and PEG3 linkerAs, various linkerBs as described herein, and various functional groups capable of linking to one or more pharmaceutical agents, as described herein.
  • the acetyl protecting group on the below GalNAc ligands can be readily removed after conjugation is completed to generate GalNAc targeting ligands.
  • tert-butyl (3-aminopropyl) carbamate Starting from tert-butyl (3-aminopropyl) carbamate, it can be alkylated with benzyl protected 2-bromoethanol (S N 2 substitution) to give Compound I.
  • the Boc group can then be removed under acidic condition to afford Compound II, which can be alkylated with tert-butyl 2-bromoacetate to afford triester Compound III.
  • tert-Butyl protecting groups can then be removed upon treatment of formic acid to generate triacid Compound IV. Amide coupling with Intermediate-A affords Compound V.
  • the benzyl protecting group may then be removed by hydrogenation to afford 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.
  • Phosphoramidite Compound 4 was synthesized by phosphitylation of Compound V with 2-Cyanoethyl N, N-diisopropylchlorophosphoramidite and a catalytic amount of 1H-tetrazole.
  • Compound I (Compound V in Scheme 8) may be reacted with NHS conjugated maleimide compound 2, 5-dioxopyrrolidin-1-yl 3- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) propanoate to afford Compound 8.
  • Compound 73 could be prepared per the procedure described in compound 74, except protected (S) -3-aminopropane-1, 2-diol was used instead of the protected (R) -3-aminopropane-1, 2-diol.
  • linkerB may be attached to various functional groups (W) capable of linking to one or more pharmaceutical agents.
  • linkerB may comprise a diol moiety in which one of the alcohols is protected as a DMT ether, while the other alcohol may be linked directly or indirectly to a solid phase synthesis solid support material.
  • the free alcohol Upon removal of the DMT group, the free alcohol is generated, which can be phosphitylated by reacting with a phosphoramidite to initiate oligonucleotide chain growth.
  • target ligand clusters of the present disclosure can be attached at the 3’-end of an oligonucleotide.
  • a non-limiting list of linkerB’s of the present disclosure that may be attached to the 3’-end of an oligonucleotide includes the structures represented below and their stereoisomers:
  • j is an integral number between 0 and 12
  • k is an integral number between 0 and 12.
  • All target ligand clusters of the present disclosure can also be attached at the 5'-end of the oligonucleotide, including but not limited to the 5'-end of the sense strand in dsRNA, or the 5'-end of the antisense strand in dsRNA:
  • Sense and antisense strand sequences of siRNA were synthesized on oligonucleotide synthesizers using a well-established solid phase synthesis method based on phosphoramidite chemistry. Oligonucleotide chain propagation is achieved through 4-step cycles: a condensation, a capping, an oxidation, and a deprotection step for addition of each nucleotide. Syntheses were performed on a solid support made of controlled pore glass (CPG, ) . Monomer phosphoramidites were purchased from commercial sources. Ligand cluster attached phosphoramidites were synthesized according to the procedures of Examples 3-12 herein. 5-Ethylthio-1H-tetrazole was used as an activator.
  • Purified single strand oligonucleotide product from IP-RP-HPLC was converted to sodium salt by dissolving in 1.0 M NaOAc and precipitation by addition of ice cold EtOH. Annealing of equimolar complementary sense stand and antisense strand oligonucleotide in water was performed to form the double strand siRNA product, which was lyophilized to afford a fluffy white solid.
  • GalNAc ligand clusters were conjugated to the 5’ end or 3’ end of a sense strand of a known active FXII siRNA from literature. See Liu et al. “An investigational RNAi therapeutic targeting Factor XII (ALN-F12) for the treatment of hereditary angioedema” . RNA. 2019 Feb; 25 (2) : 255-263. doi: 10.1261/rna. 068916.118. Sequence and modification information of this FXII siRNA is summarized in Table 1. Shown in Table 1 are also six examples of GalNAc ligand clusters conjugated to FXII siRNA.
  • FXII is a secreted protein mainly produced in hepatocyte. Reduction of FXII expression in plasma after siRNA treatment correlates strongly with reduction of FXII mRNA in hepatocyte. Since these GalNAc ligand clusters are conjugated to the same FXII siRNA with known activity, delivery efficacy can be assessed and compared by measuring degree of reduction of FXII expression in plasma for each conjugate.
  • mice were given a single subcutaneous injection of siRNA compounds (see Table 1 below) at 0.5 or 1 mg/kg or PBS.
  • the literature compound (GalNAc ligand cluster conjugated to 3’-end of sense strand) was included in this study as the positive control.
  • Plasma samples were collected pre-dosing, and at day 7, 14 and /or 28 post-dosing.
  • Concentration of mouse FXII protein was measured by ELISA assay following literature procedure. See Liu et al. “An investigational RNAi therapeutic targeting Factor XII (ALN-F12) for the treatment of hereditary angioedema” . RNA. 2019 Feb; 25 (2) : 255-263. doi: 10.1261/rna. 068916.118) .
  • Knockdown activity was calculated for percent reduction of FXII protein in mouse plasma normalized to PBS treated group and is summarized in Table 3.
  • FXII siRNA conjugated with GalNAc ligand GLS-1 and GLS-2 showed significant activity knocking down mouse FXII protein expression in mouse plasma at both dosages at day 7, 14 and/or 28 post-dosing. This activity compares favorably to the positive control.
  • the data confirms GalNAc ligand clusters based on diamine scaffold attached to 5’-end of sense strand are highly efficacious in delivering siRNA into hepatocyte in vivo.
  • mice were given a single subcutaneous injection of siRNA compounds at 1 mg/kg or PBS. Plasma samples were collected at day 14 post-dosing. Concentration of mouse FXII protein was measured by ELISA assay following literature procedure. See Liu et al. “An investigational RNAi therapeutic targeting Factor XII (ALN-F12) for the treatment of hereditary angioedema” . RNA. 2019 Feb; 25 (2) : 255-263. doi: 10.1261/rna. 068916.118) . Knockdown activity was calculated for percent reduction of FXII protein in mouse plasma normalized to PBS treated group and is summarized in Table 4.
  • FXII siRNA conjugated with GalNAc ligand GLS-5 and GLS-15 showed significant activity knocking down mouse FXII protein expression in mouse plasma.
  • the data confirms GalNAc ligand clusters based on diamine scaffold attached to 5’-end of sense strand are highly efficacious in delivering siRNA into hepatocyte in vivo.
  • linkerB contains a six-membered ring fragment
  • linkerB contains a six-membered ring fragment
  • Table 4 Percent reduction of FXII protein in mouse plasma normalized to PBS treated group.
  • mice were given a single subcutaneous injection of siRNA compounds at 2 mg/kg or PBS. Plasma samples were collected at day 7 and 14 post-dosing. Concentration of mouse FXII protein was measured by ELISA assay following literature procedure. See Liu et al. “An investigational RNAi therapeutic targeting Factor XII (ALN-F12) for the treatment of hereditary angioedema” . RNA. 2019 Feb; 25 (2) : 255-263. doi: 10.1261/rna. 068916.118) . Knockdown activity was calculated for percent reduction of FXII protein in mouse plasma normalized to PBS treated group. The percent of knockdown at days 7 and 14 post dosing was 87%and 88%. The data confirms GalNAc ligand clusters based on diamine scaffold attached to 3’-end of sense strand are highly efficacious in delivering siRNA into hepatocyte in vivo.
  • mice were infected by intravenous administration of a solution of adeno-associated virus 8 (AAV8) vector encoding human ANGPTL3 and luciferase gene.
  • AAV8 adeno-associated virus 8
  • mice were subcutaneously administered a single dose of AD00112-2 (Table 5. ) at 1, 3 or 10 mg/kg or PBS.
  • Blood samples were collected at day 0, before dosing of siRNA and at day 7, at termination. Serum samples were isolated and luciferase activity of serum samples was measured per manufacturer’s recommended protocol. Since expression of human ANGPTL3 level correlates with expression level of luciferase, measurement of luciferase activity is the surrogate for measuring ANGTPL3 expression.
  • luciferase activity was calculated by comparing luciferase activity in samples from pre- (day 0) and post (day 7) treatment of siRNA for each mouse and normalized by the change of luciferase activity in the samples from the control treated mice during the same period of time. Result is summarized in Table 6.AD00112-2 demonstrated dose dependent activity suppressing expression of ANGPTL3, which confirms again GalNAc ligand clusters based on diamine scaffold are highly efficacious in delivering siRNA into hepatocyte in vivo.
  • ANGPTL3 siRNA compounds Upper case letters: 2′-deoxy-2′-fluoro (2′-F) ribonucleotide; lower case letters: 2′-O-methyl (2′-OMe) ribonucleotide; (*) indicates PS linkage.
  • Table 6 provides experimental results of in vivo studies (percent reduction of luciferase activity) .
  • the duplex sequence and modification of AD00112-2 is shown in Table 5.

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Abstract

L'invention concerne des agrégats de ligands multivalents, ayant un échafaudage de diamine, pour l'administration ciblée d'agents pharmaceutiques conjugués à ceux-ci. Un agrégat de ligands multivalents peut comprendre un ou plusieurs ligands de ciblage de N-acétylgalactosamine (GalNAc). Un agrégat de ligands multivalents peut être conjugué à un ou plusieurs petits acides ribonucléiques interférents (pARNi), le pARNi étant un exemple d'un agent pharmaceutique. L'invention concerne également des compositions comprenant un agrégat de ligands multivalents, et des procédés de fabrication d'un agrégat de ligands multivalents.
EP22872040.5A 2021-09-23 2022-09-22 Agrégats de ligands multivalents avec échafaudage de diamine pour l'administration ciblée d'agents thérapeutiques Pending EP4380624A1 (fr)

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TW202327658A (zh) 2023-07-16
KR20240082358A (ko) 2024-06-10
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