Classifications

• • 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
• • 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
• • 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
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • 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
  • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
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  • 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
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  • 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 nucleic acids [NA]
• • 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
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/35—Nature of the modification
  • C12N2310/351—Conjugate

Definitions

• the invention relates to novel compositions and processes that can be used in conjugating carbohydrate ligands with oligonucleotides intended for biomedical applications.
• oligonucleotides have been the focus of many research and development efforts as these strings of nucleotides hold great promise for treating or preventing many diseases and for modulating physiological conditions.
• oligonucleotides include short/small interfering RNA (siRNA) , asymmetric short/small interfering RNA (aiRNA) , antisense oligonucleotide (ASO) , and micro-RNA (miRNA) .
• RNA interference works through short, double-stranded RNA (dsRNA) duplexes called siRNA in a gene-specific fashion in many organisms.
• the siRNAs have a well-defined structure of symmetric, short (usually 20-24 base pairs) dsRNA duplex having phosphorylated 5’ ends and hydroxylated 3’ ends that form two 3’ overhangs of equal lengths.
• Gene modulation is mediated through a multi-protein RNA-induced silencing complex (RISC) , which binds, unwinds, and incorporates the anti-sense siRNA strand from the siRNA duplex, and then recognizes and targets complementary messenger RNAs (mRNAs) for cleavage thereby reducing its gene expression in a post-transcriptional fashion.
• RISC multi-protein RNA-induced silencing complex
• aiRNA was developed to overcome off-target effects mediated by sense strand of the symmetrically configured canonical siRNA as well as other off-target mechanisms of siRNA (See PCT Patent Publication WO2009029688) .
• aiRNAs are designed to include short RNA duplex where the lengths of the two RNA strands are not equal, hence “asymmetric. ”
• an aiRNA can include a first strand that is 18-23 nucleotides long and a second that is 12-17 nucleotides long, forming a duplex where the first strand might have a 3’ overhang of 1-9 nucleotides and a 5’ overhang of 0-8 nucleotides.
• the aiRNA technology can be used in all areas where current siRNA or short-hairpin RNA (shRNA) are being applied including biology research, R&D research in biotechnology and pharmaceutical industry, and RNAi-based therapies.
• Antisense technology is a highly selective gene silencing technology based upon a concept originally proposed in 1978 (Zamecnik P.C. et al., 1978) .
• the principle behind the ASO technology is that an antisense oligonucleotide hybridizes to a target nucleic acid and modulates gene expression through post-transcriptional mechanisms.
• the mechanisms can be broadly categorized as: (1) occupancy only without promoting RNA degradation, in which the binding of the ASO leads to translational arrest, inhibition of splicing, or induction of alternatively spliced variants, or (2) occupancy-induced destabilization, in which the binding of the ASO promotes degradation of the RNA through endogenous enzymes, such as ribonuclease H1 (RNase H1) ; and (3) increased translation: ASO can block upstream open reading frames (uORFs) or other inhibitory elements in the 5’UTR, increasing translation efficiency (Stanley T. Crooke et al., 2008; C. Frank Bennett, 2010; Richard G. Lee, 2013; Stanley T. Crooke, 2017) .
• uORFs upstream open reading frames
• ASO a single-stranded deoxyribonucleotide sequence with sulfur chemistry modification, known as phosphorothioate.
• a miRNA molecule normally derives from noncoding regions of RNA transcripts that fold back onto themselves to form hairpins. After having been processed from its precursors through various cellular machineries, a mature miRNA is a small (about 22 nucleotides) RNA molecule found in plants, animal and some viruses that regulate gene expression through post-transcriptional silencing.
• Therapeutics based on these and other nucleic acids provide promising solutions to a variety of diseases, including non-druggable targets.
• oligonucleotides and oligonucleotide analogs as therapeutics, there continues to exist great needs for enhancing key pharmacological properties of these therapeutic oligonucleotides in areas such as serum stability, delivery to the intended organ or cell population, and uptake across cellular membranes.
• Preferred delivery of therapeutic oligonucleotides to cells in vivo requires specific targeting and protection from the extracellular environment inside the body including from proteins in the serum.
• a method that researchers have employed to achieve specific targeting is to conjugate a targeting moiety to the oligonucleotides to direct therapeutic oligonucleotides to the desired target site.
• One way to improve specificity in delivery is by taking advantage of receptor mediated endocytic activities that already exist in the body.
• the mechanism of uptake involves the movement of molecules bound to cell membrane receptors across the membrane and into the cell via invagination of the membrane structure or by fusion of the delivery system with the cell membrane. This process is initiated via activation of a cell-surface or membrane receptor following binding of a specific ligand to the receptor. Therefore, by conjugating a drug candidate to a targeting moiety that targets such cell surface receptor (s) , one can effectively borrow the innate endocytic pathways for drug delivery.
• ASGP-R Asialoglycoprotein receptor
• GalNAc N-Acetyl-D-Galactosylamine
• the invention relates to a compound, as a therapeutic agent, where an oligonucleotide is conjugated with at least one ligand, e.g., a carbohydrate ligand such as a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, polysaccharide or their derivatives, which can target the compound to receptor cells in the liver that can facilitate endocytic uptake as discussed above.
• a carbohydrate ligand such as a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, polysaccharide or their derivatives
• the compound includes more than one carbohydrate ligand, preferably two or three.
• the compound of the invention includes at least one (e.g., one, two or three or more) N-Acetyl-Galactosamine (GalNAc) , N-Ac-Glucosamine (GlcNAc) , galactose, lactose, or mannose (e.g., mannose-6-phosphate) .
• the compound of the invention includes at least one (e.g., one, two or three or more) ligand selected from the group consisting of GalNAc, cholesterol, tocopherol, biotin, cyanine dyes, folic acid, RGDp, transferrin, anisamide, lactobionic acid, cRGD, hyaluronic acid, low molecular weight protamine, lipid derivatives, peptides, cyclic peptides, and heterocycles.
• at least one e.g., one, two or three or more
• ligand selected from the group consisting of GalNAc, cholesterol, tocopherol, biotin, cyanine dyes, folic acid, RGDp, transferrin, anisamide, lactobionic acid, cRGD, hyaluronic acid, low molecular weight protamine, lipid derivatives, peptides, cyclic peptides, and heterocycles.
• the invention provides ligand-conjugated compounds having novel structure:
• R 205 , R 206 are each independently for each occurrence H, OH, a protecting group for OH, a phosphate group, a phosphodiester group, an activated phosphate group, an activated phosphite group, a phosphoramidite, a solid support, -OP (M') (M”) O-nucleoside, -OP (M') (M”) O-oligonucleotide, a lipid, a PEG, a steroid, a polymer, -O-nucleotide, a nucleoside, -OP (M') (M") O-R 201 -OP (M'") (M"”) O-oligonucleotide, -X-OP (M') (M”) O-oligonucleotide, -Z-OP (M') (M”) O-oligonucleotide or an oligonucleotide;
• M', M", M'" and M"" are each independently for each occurrence O or S;
• A1, A2 and A3 are each independently for each occurrence selected from (S-1H) or (S-1G) :
• R 202A is –R 202 -R 202L ;
• R 217A is –R 217 -R 217L ;
• R 202L , R 217L are independently for each occurrence one ligand capable of docking to a cell surface receptor
• R 1 , R 2 , R 204 , R 207 , R 208 , R 213 , R 214 , R 215 , R 216 , R 209 are independently for each occurrence selected one or more from the group consisting of H, alkyl, aryl, heteroaryl, haloalkyl, -O alkyl, -O alkylphenyl, -alkyl-OH, -O haloalkyl, -S alkyl, -S alkylphenyl, -alkyl-SH, -S haloalkyl, halo, -OH, -SH, -NH 2 , -alkyl-NH 2 , -N (alkyl) (alkyl) , -NH (alkyl) , -N (alkyl) (alkylphenyl) , -NH (alkylphenyl) , cyano, nitro, -CO 2 H, -C (
• n 201 , n 211 are each independently for each occurrence 1, 2, 3, 4, 5 or 6;
• J 201 , J 202 , J 211 , J 212 are each independently for each occurrence absent or a spacer;
• a, b and c are each independently for each occurrence integer 0-5, and the sum of a, b and c is an integer of 1-10;
• the oligonucleotide comprises naturally occurring or chemically modified nucleotides/nucleosides.
• Item 2 A compound of item 1, wherein the sum of a, b and c is an integer of 1 or 3.
• Item 3 A compound of item 1, wherein the compound having the structural formula (S-H1) :
• R 1 is selected one or more from the group consisting of H, C 1 -C 5 alkyl, aryl, heteroaryl, C 1 -C 5 haloalkyl, -C 1 -C 5 alkyl-OH, -C 1 -C 5 alkyl-SH, -C 1 -C 5 alkyl-NH 2 , -CO 2 H, -C (O) O alkyl, -CON (alkyl) (alkyl) , -CONH (alkyl) , -CONH 2 , -C (O) alkyl, -C (O) alkylphenyl, -C (O) haloalkyl, -SO 2 (alkyl) , -SO 2 (haloalkyl) , -SO 2 NH 2 , -SO 2 NH (alkyl) , and -SO 2 NH (phenyl) ;
• n 202 is selected from 1-10, preferably 1-3.
• J 202B is selected from a alkylene of 1 to 10 carbon atoms, optionally selected from a straight alkylene of 1 to 10 carbon atoms;
• R 205A is a group comprising a solid support or H
• the carbon marked with symbol “*” is chiral carbon atom.
• Item 5 The compound of item 3 or item 4, wherein n 202 is 1 or 3.
• Item 7 The compound of item 6, wherein the compound having the structural formula (S-H1-02) :
• Item 10 The compound of item 9, wherein n 201 is 1, n 202 is 1 or 3;
• A is O or S
• X 1 is independently selected from Table 2;
• J 202 , Z are independently for each occurrence selected from Table 3;
• R, R’a re each independently selected from the group consisting of a naturally occurring and/or chemically modified oligonucleotide, H and a protecting group for OH; at least one of R and R’ comprises an oligonucleotide formed by natural and/or chemically modified nucleotides/nucleosides;
• R 202 is selected from a straight alkylene of 3 to 15 carbon atoms, wherein one or more carbon atoms are optionally replaced with any one or more substituent of the group consisting of: C (O) , NH, O, S, OP (O) O, OP (S) O; and wherein R 202 is optionally not substituted or substituted by R 209 .
• R 202 is selected from -C 3 -C 8 straight alkylene-.
• Item 12 The compound of item 11, wherein the compound having the structural formula (S-H1-06) , (S-H1-07) , (S-H1-08) or (S-H2-01) :
• Item 13 The compound of item 11, the compound having the structural formula (S-H1-09) , (S-H1-10) , (S-H1-11) or (S-H2-02) :
• Item 14 The compound of item 11, wherein having the structural formula (S-H1-12) , (S-H1-13) , (S-H1-14) or (S-H2-03) :
• Item 15 The compound of item 1, the compound having the structural formula (S-H1-15) , (S-H1-16) or (S-H2-04) :
• R, R’ are each independently selected from the group consisting of a naturally occurring or chemically modified oligonucleotide, H and a protecting group for OH;
• each L independently comprises a ligand moiety capable of docking to a cell-surface receptor.
• Item 16 The compound of any one of items 11-15, wherein each A is O.
• n 212 is selected from 1-10, preferably 1-3.
• J 212B is selected from a alkylene of 1 to 10 carbon atoms; optionally selected from a straight alkylene of 1 to 10 carbon atoms;
• the carbon marked with symbol “*” is chiral carbon atom.
• Item 25 The compound of item 23 or item 24, wherein n 212 is selected from 1or 3.
• Item 26 The compound of any one of items 23-24, wherein R 206 comprises an oligonucleotide.
• Item 30 The compound of any of item 29, wherein n 211 is 1, n 212 is 1 or 3.
• Item 31 The compound of item 29, wherein the compound having the structural formula (S-G1-03) , (S-G1-04) , (S-G1-05) or (S-G2) :
• A is O or S
• each Z is independently selected from Table 3;
• Item 42 The compound of item 31, wherein the compound has the structure shown in formula GS-5, GS-13:
• Item 46 A compound of item 45, wherein the aiRNA comprising an antisense strand and a sense strand, wherein the antisense strand is longer than the sense strand, has a length of 19, 20, 21, 22, 23, 24, 25, 26 or 27 nucleotides and includes a 3'-overhang of 1-9nucleotides and a 5'-overhang of 0-8 nucleotides when duplexed with the sense strand;
• Item 51 A small interfering RNA (siRNA) agent duplex comprising a structural formula of any of items 1-50.
• siRNA small interfering RNA
• Item 52 An asymmetric interfering RNA (aiRNA) agent comprising a structural formula of any of items 1-50.
• aiRNA asymmetric interfering RNA
• Item 54 A micro-RNA (miRNA) agent comprising a structural formula of any of items 1-50.
• Item 55 A pharmaceutical composition comprising a compound of items 1-50 or agent of any one of items 51-54 and a pharmaceutically acceptable excipient, carrier, or diluent.
• Item 56 Use of a compound of any one of items 1-50 or agent of any one of items 51-54 in preparation of a medicament effective for treating a disease or condition.
• the invention features a compound comprising a carbohydrate ligand as provided in the second aspect above, and the presence of the carbohydrate ligand can increase delivery of the compound to the targeted organs, e.g. liver.
• a compound comprising a carbohydrate ligand can be useful for targeting a gene related to a disease or an undesired condition in the targeted organs.
• a compound of the invention comprising the carbohydrate ligand can target a nucleic acid expressed by a hepatitis virus.
• the invention provides compounds having novel structure:
• R 0 is selected one or more from the group consisting of H, C 1 -C 5 alkyl, aryl, heteroaryl, C 1 -C 5 haloalkyl, -C 1 -C 5 alkyl-OH, -C 1 -C 5 alkyl-SH, -C 1 -C 5 alkyl-NH 2 , -CO 2 H, -C (O) O alkyl, -CON (alkyl) (alkyl) , -CONH (alkyl) , -CONH 2 , -C (O) alkyl, -C (O) alkylphenyl, -C (O) haloalkyl, -SO 2 (alkyl) , -SO 2 (haloalkyl) , -SO 2 NH 2 , -SO 2 NH (alkyl) , and -SO 2 NH (phenyl) ;
• R 202A comprises at least one ligand capable of docking to a cell surface receptor
• R 204 , R 207 , R 208 are independently selected one or more from the group consisting of H, alkyl, aryl, heteroaryl, haloalkyl, -O alkyl, -O alkylphenyl, -alkyl-OH, -O haloalkyl, -S alkyl, -Salkylphenyl, -alkyl-SH, -S haloalkyl, halo, -OH, -SH, -NH 2 , -alkyl-NH 2 , -N (alkyl) (alkyl) , -NH (alkyl) , -N (alkyl) (alkylphenyl) , -NH (alkylphenyl) , cyano, nitro, -CO 2 H, -C (O) O alkyl, -CON (alkyl) (alkyl) , -CONH (alkyl) , -CONH
• n 201 is selected from 1, 2, 3, 4, 5 or 6;
• R 206 is selected from OH or a protecting group for OH
• J 201 , J 202 are each independently for each occurrence a spacer
• R 205B is a -C 2 -C 10 alkynylene-CN
• R 205C , R 205D are independently selected from -C 1 -C 6 -alkyl or R 205C and R 205D together form a five-or six-membered ring, optionally R 205C , R 205D are substituted, optionally R 205C , R 205D contain one further heteroatom selected from N and O.
• J 202B is selected from a alkylene of 1 to 10 carbon atoms.
• Item 60 A compound of item59, having the structural formula (G-P3) :
• Item 64 A compound of item 62, wherein the branching group is selected from the group consisting of:
• Item 66 A compound of item 61, having the structural formula (G-P5) :
• R 217A comprises at least one a ligand capable of docking to a cell surface receptor
• R 211C , R 211D are independently selected from -C 1 -C 6 -alkyl or R 211C and R 211D together form a five-or six-membered ring, optionally R 211C , R 211D are substituted, optionally R 211C , R 211D contain one further heteroatom selected from N and O.
• Item 70 A compound of item 68, having the structural formula (G-P8) :
• alkenyl refers to an unsaturated branched or straight-chain alkyl group having at least one carbon-carbon double bond derived by the removal of one molecule of hydrogen from adjacent carbon atoms of the parent alkyl.
• the group may be in either the cis or trans configuration about the double bond (s) .
• an alkenyl group has from 2 to 20 carbon atoms and in other embodiments, from 2 to 10, 2 to 8, or 2 to 6 carbon atoms.
• Alkenylene is a subset of alkenyl, referring to the same residues as alkenyl, but having two points of attachment.
• alkynyl refers to an unsaturated branched or straight-chain alkyl group having at least one carbon-carbon triple bond derived by the removal of two molecules of hydrogen from adjacent carbon atoms of the parent alkyl.
• Typical alkynyl groups include, but are not limited to, ethynyl; propynyls such as prop-1-yn-1-yl, prop-2-yn-1-yl; butynyls such as but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl; and the like.
• an alkynyl group has from 2 to 20 carbon atoms and in other embodiments, from 2 to 10, 2 to 8, or 2 to 6 carbon atoms.
• Alkynylene is a subset of alkynyl, referring to the same residues as alkynyl, but having two points of attachment.
• alkoxy refers to an alkyl group of the indicated number of carbon atoms attached through an oxygen bridge such as, for example, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, pentyloxy, 2-pentyloxy, isopentyloxy, neopentyloxy, hexyloxy, 2-hexyloxy, 3-hexyloxy, 3-methylpentyloxy, and the like. Alkoxy groups will usually have from 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms attached through the oxygen bridge.
• aryl refers to a radical derived from an aromatic monocyclic or multicyclic hydrocarbon ring system by removing a hydrogen atom from a ring carbon atom.
• the aromatic monocyclic or multicyclic hydrocarbon ring system contains only hydrogen and carbon from six to eighteen carbon atoms, where at least one of the rings in the ring system is fully unsaturated, i.e., it contains a cyclic, delocalized (4n+2) ⁇ –electron system in accordance with the Hückel theory.
• Aryl groups include, but are not limited to, groups such as phenyl, fluorenyl, and naphthyl.
• Arylene is a subset of aryl, referring to the same residues as aryl, but having two points of attachment.
• cycloalkyl refers to a non-aromatic carbocyclic ring, usually having from 3 to 7 ring carbon atoms. The ring may be saturated or have one or more carbon-carbon double bonds.
• cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, and cyclohexenyl, as well as bridged and caged ring groups such as norbornane.
• haloalkyl refers to alkyl as defined above having the specified number of carbon atoms, substituted with 1 or more halogen atoms, up to the maximum allowable number of halogen atoms.
• haloalkyl include, but are not limited to, trifluoromethyl, difluoromethyl, 2-fluoroethyl, and penta-fluoroethyl.
• Heterocyclyl refers to a stable 3-to 18-membered non-aromatic ring radical that comprises two to twelve carbon atoms and from one to six heteroatoms selected from nitrogen, oxygen and sulfur. Unless stated otherwise specifically in the specification, the heterocyclyl radical is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems. The heteroatoms in the heterocyclyl radical may be optionally oxidized. One or more nitrogen atoms, if present, are optionally quaternized. The heterocyclyl radical is partially or fully saturated. The heterocyclyl may be attached to the rest of the molecule through any atom of the ring (s) .
• heterocyclyl radicals include, but are not limited to, dioxolanyl, thienyl [1, 3] dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorph
• a solid support comprises the solid phase carrier for the synthesis of oligonucleotide, such as CPG.
• modified oligonucleotide means an oligonucleotide comprising at least one modified nucleotide.
• modified nucleotide means a nucleotide having at least one modified sugar moiety, modified internucleoside linkage, and/or modified nucleobase.
• modified nucleoside means a nucleoside having at least one modified sugar moiety, and/or modified nucleobase.
• natural sugar moiety means a sugar found in DNA (2-H) or RNA (2-OH) .
• modified sugar refers to a substitution or change from a natural sugar.
• a 2’-O-methoxyethyl modified sugar is a modified sugar.
• bicyclic sugar means a furosyl ring modified by the bridging of two non-geminal ring atoms.
• a bicyclic sugar is a modified sugar.
• modified nucleobase refers to any nucleobase other than adenine, cytosine, guanine, thymidine, or uracil.
• 5-methylcytosine is a modified nucleobase.
• An “unmodified nucleobase” means the purine bases adenine (A) and guanine (G) , and the pyrimidine bases thymine (T) , cytosine (C) , and uracil (U) .
• prevention and “preventing” are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results including but not limited to a prophylactic benefit.
• prophylactic benefit the conjugates or compositions may be administered to a patient at risk of developing a particular disease, or to a patient reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made.
• the term “effective amount” of an active agent refers to an amount sufficient to elicit the desired biological response.
• the effective amount of a compound of the invention may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the patient.
• treatment refers to a method of reducing, delaying or ameliorating such a condition before or after it has occurred. Treatment may be directed at one or more effects or symptoms of a disease and/or the underlying pathology.
• the treatment can be any reduction and can be, but is not limited to, the complete ablation of the disease or the symptoms of the disease. As compared with an equivalent untreated control, such reduction or degree of prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100%as measured by any standard technique.
• a "pharmaceutical composition” includes a pharmacologically effective amount of a dsRNA and a pharmaceutically acceptable carrier.
• pharmacologically effective amount “therapeutically effective amount” or simply “effective amount” refers to that amount of an RNA effective to produce the intended pharmacological, therapeutic or preventive result.
• a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 25%reduction in that parameter.
• pharmaceutically acceptable carrier refers to a carrier for administration of a therapeutic agent.
• Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
• the term specifically excludes cell culture medium.
• pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives.
• suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents.
• Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract. nce to a human subject.
• All stereoisomers of the compounds of the present invention are contemplated within the scope of this invention.
• Individual stereoisomers of the compounds of the invention may, for example, be substantially free of other isomers (e.g., as a pure or substantially pure optical isomer having a specified activity) , or may be admixed, for example, as racemates or with all other, or other selected, stereoisomers.
• the chiral centers of the present invention may have the S or R configuration as defined by the IUPAC 1974 Recommendations.
• racemic forms can be resolved by physical methods, such as, for example, fractional crystallization, separation or crystallization of diastereomeric derivatives or separation by chiral column chromatography.
• the individual optical isomers can be obtained from the racemates by any suitable method, including without limitation, conventional methods, such as, for example, salt formation with an optically active acid followed by crystallization.
• Amino acids contained within the peptides or polypeptides described herein will be understood to be in the L-or D-configuration.
• the present invention provides novel compounds with novel linker compositions for linking various components of the compound.
• the compound conjugates an oligonucleotide with one or more targeting ligands.
• the oligonucleotide can be naturally occurring (isolated from nature, or synthesized in a laboratory) or chemically modified in at least one subunit.
• the oligonucleotide is chemically modified oligonucleotide.
• chemically modified oligonucleotide comprises backbone modification (or internucleoside linkage modification, such as phosphate group modification) , ribose group modification, base modification.
• the oligonucleotide has at least one phosphorothioate internucleoside linkage, or at least one methylphosphonate internucleoside linkage, or at least one other modified internucleoside linkage such as:
• the oligonucleotide has at least one chemically modified nucleotide with ribose modification.
• the 2′position of the modified ribose moiety is replaced by a group selected from OR, R, halo, SH, SR, NH 2 , NHR, NR 2 , or CN, where each R is independently C 1 -C 6 alkyl, alkenyl or alkynyl, and halo is F, Cl, Br or I.
• the modified ribose moiety is selected from the group of 5’-vinyl, 5’-methyl (R or S) , 4’-S, 2’-F, 2’-OCH 3 , 2’-OCH 2 CH 3 , 2’-OCH 2 CH 2 F and 2’-O (CH2) 2 OCH 3 substituent groups.
• the modified ribose moiety is substituted by bicyclic sugar selected from the group of 4′- (CH 2 ) -O-2′ (LNA) ; 4′- (CH 2 ) -S-2; 4′- (CH 2 ) 2-O-2′ (ENA) ; 4′-CH (CH 3 ) -O-2′ (cEt) and 4′- CH (CH 2 OCH 3 ) -O-2′, 4′-C (CH 3 ) (CH 3 ) -O-2′, 4′-CH 2 -N (OCH 3 ) -2′, 4′-CH 2 -O-N (CH 3 ) -2′, 4′-CH 2 -N (R) -O-2′, where R is H, C1-C12 alkyl, or a protecting group, 4′-CH 2 -C (H) (CH 3 ) -2′, and 4′-CH 2 -C- ( ⁇ CH 2 ) -2′.
• R is H, C1-C
• the modified sugar moiety is selected from the group of 2’-O-methoxyethyl modified sugar (MOE) , a 4′- (CH 2 ) -O-2′bicyclic sugar (LNA) , 2’-deoxy-2’-fluoroarabinose (FANA) , and a methyl (methyleneoxy) (4′-CH (CH 3 ) -O-2) bicyclic sugar (cEt) .
• the oligonucleotide has a chemically modified nucleotide selected from the group consisting, 2'-methoxyethyl, 2'-OCH 3 and 2'-fluoro.
• the oligonucleotide has at least one chemically modified nucleobase.
• at least one chemically modified nucleobase is selected from the group of: 5-methylcytosine (5-Me-C) , inosine base, a tritylated base, 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C ⁇ C-CH 3 ) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil) , 4-thi
• any stabilizing modification known to a person skilled in the art can be used to improve the stability of the oligonucleotide molecules.
• chemical modifications can be introduced to the phosphate backbone (e.g., phosphorothioate linkages) , the sugar (e.g., locked nucleic acids, glycerol nucleic acid, cEt, 2’-MOE, 2’-fluorouridine, 2’-O-methyl) , and/or the base (e.g., 2’-fluoropyrimidines) .
• the oligonucleotide can be conjugated to the rest of the compound, or the “backbone” at the 5’ and/or 3’ end of the oligonucleotide.
• the conjugated oligonucleotide can be delivered as a single strand or hybridized to a substantially complementary oligonucleotide as part of a duplex.
• the substantially complementary oligonucleotide can be similarly conjugated or not.
• the conjugated oligonucleotide forms part of a siRNA duplex (either the sense or antisense strand, or both) .
• the conjugated oligonucleotide forms part of an aiRNA duplex (either the sense or antisense strand, or both) .
• the oligonucleotide that is conjugated according to principles of the invention is used as an antisense oligonucleotide (ASO) .
• the oligonucleotide that is conjugated according to principles of the invention is used as a micro-RNA (miRNA) molecule.
• the compound of the present invention has the structural formula as shown in (S-H1) , (S-H2) , (S-H1-01) ) to (S-H1-16) and (S-H2-01) to (S-H2-04) .
• the compound of the present invention has the structure as shown in Table 6.
• the configuration of the compound is R isomer, S isomer or racemate. In one embodiment, the configuration of the compound is a mixture of R isomer and S isomer. In a preferably embodiment, the configuration of the compound is R isomer. In one embodiment, the configuration of the compound means the isomer of the chiral carbon atom shown in the structural formula S-G1-01.
• Methyl (R) -3-hydroxybutanoate was replaced with methyl (S) -3-hydroxybutanoate. See M-3 synthesis in G-12 (Glu (R) -Sequential GalNAc) for the synthesis method.
• GS-13-9 was dissolved in anhydrous methanol, 10% (W/W) palladium carbon was added, and replaced air with hydrogen for three times, stirred at room temperature until the raw material was completely consumed, then the palladium carbon was removed by filtration, and the filtrate was dried and directly put into the next step.
• the synthesis method was the same as that of H-17, and the loading was 50 ⁇ mol/g.

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