Ligand compounds, conjugates and their applications

Abstract

The present disclosure features compositions comprising one or more chemical entities (e.g., compounds or pharmaceutically acceptable salts, and / or hydrates, and / or cocrystals, and / or pharmaceutical compositions of compounds) directed to an asialoglycoprotein receptor (ASGPR) ligand moiety. Exemplary chemical entities include oligonucleotides. The chemical entities are useful, for example, for targeted delivery of oligonucleotides to liver cells (e.g., hepatocytes). The chemical entities are useful, for example, for treating disorders or diseases caused by the expression (e.g., aberrant expression) of one or more genes in liver cells. The present disclosure also features compositions comprising the compositions, as well as methods of using and preparing the compositions.

Description

[Technical field]

[0001] The present disclosure relates to the technical field of nucleic acid delivery. Ligand compounds, oligonucleotide conjugates, and methods for preparing and using them are disclosed. [Background technology]

[0002] The asialoglycoprotein receptor (ASGPR) is an abundant hetero-oligomeric endocytic receptor that is present mainly on the surface of the plasma membrane of hepatocytes facing the sinusoids and is specific for carbohydrates. After removal of terminal sialic acids from glycoproteins by enzymatic or acid hydrolysis, the exposed secondary termini are galactose residues.

[0003] Thus, the sugar-binding specificity of ASGPR is actually on galactosyl groups, and it is also called the galactose-specific receptor. ASGPR is distributed mainly in hepatocytes, with low abundance in other cells. Thus, ASGPR provides a possible receptor for liver-directed transport.

[0004] Glycoproteins with terminal non-reducing galactose (Gal) or N-acetylgalactosamine (GalNAc) residues can be recognized by ASGPR, and the binding affinity of GalNAc to ASGPR is about 50 times higher than that of Gal (Lobst ST et al, J Biol Chem, 1996, 271(12):6686-6693). In vitro experiments have shown that the affinity of clustered sugar residues is much higher than that of non-clustered sugar residues by simultaneously occupying the binding sites of the receptor, and the order of affinity is tetraantennary>triantennary>biantennary>monantennary galactosides (Lee YC, et al, J Biol Chem, 1983, 258(1):199-202).

[0005] Liver-targeting oligonucleotides mediated by ASGPR receptors are a new breakthrough in the field of innovative drug research of nucleic acids. In 2012, Alnylam Pharmaceuticals covalently linked the previously studied triantennary GalNAc structure to small interfering RNA (siRNA) to achieve liver-targeting delivery of siRNA in vivo. Using this technology, researchers have developed drugs for diseases such as amyloidosis, hemophilia, hypercholesterolemia, hepatic porphyria, and hepatitis B.

[0006] The first GalNAc-siRNA drug was launched in 2019, and two drugs are currently under marketing application. More than a dozen drug candidates are in clinical trials (http: / / www.alnylam.com / product-pipeline / ). In 2014, the American pharmaceutical company ISIS covalently bound triantennary GalNAc to antisense nucleic acid to achieve liver-targeted drug delivery in vivo in animals, and the activity of the antisense nucleic acid after binding was increased 10-fold (Prakash TP et al, Nucleic Acids Res. 42, 8796-807). Summary of the Invention

[0007] The present disclosure features compositions that include one or more chemical entities (e.g., compounds or pharma- ceutically acceptable salts, and / or hydrates, and / or cocrystals, and / or pharmaceutical compositions of compounds) directed to an asialoglycoprotein receptor (ASGPR) ligand moiety. Exemplary chemical entities also include oligonucleotides. The chemical entities are useful, for example, for targeted delivery of oligonucleotides to hepatocytes (e.g., hepatocytes). The chemical entities are useful, for example, for treating disorders or diseases caused by the expression (e.g., aberrant expression) of one or more genes in hepatocytes. The present disclosure also features compositions that include the compositions, as well as methods of using and preparing the compositions.

[0008] In one aspect, the disclosure features a compound having formula (I) or a pharma- ceutically acceptable salt thereof: TIFF0007618778000001.tif41170, R X , R 3 ,c,R 4 , d and R 5 may be as defined anywhere herein.

[0009] In some embodiments, the compound of formula (I) is a conjugate compound having formula (II) or a pharma- ceutically acceptable salt thereof: TIFF0007618778000002.tif40170, R 5 teeth TIFF0007618778000003.tif16170, where Oligo is an oligonucleotide linked to L via the 5' end, 3' end or middle of the sequence of any strand by a phosphate bond, and -L-, R X , R 3 ,c,R 4 and d may be as defined for any of formula (I) herein.

[0010] In another aspect, the present disclosure provides a pharmaceutical composition comprising a chemical entity described herein (e.g., a compound having formula (II) or a pharma- ceutically acceptable salt thereof) and a pharma- ceutically acceptable excipient.

[0011] In another aspect, the present disclosure provides a method of treating and / or preventing a disorder or disease in a subject, wherein the pathological condition or disease is caused by expression of one or more genes in liver cells, the method comprising administering to the subject a chemical entity described herein (e.g., a compound having formula (II) or a pharma- ceutical acceptable salt thereof), or a pharmaceutical composition comprising a chemical entity described herein (e.g., a compound having formula (II) or a pharma- ceutical acceptable salt thereof) and a pharma- ceutical acceptable excipient.

[0012] In another aspect, the present disclosure provides a method of detecting or localizing RNA in the liver of a subject, the method comprising administering to the subject a chemical entity described herein (e.g., a compound having formula (II) or a pharma- ceutically acceptable salt thereof), or a pharmaceutical composition comprising a chemical entity described herein (e.g., a compound having formula (II) or a pharma- ceutically acceptable salt thereof) and a pharma- ceutically acceptable excipient.

[0013] Other embodiments include those described in the detailed description and / or claims.

[0014] Other definitions In order to facilitate understanding of the disclosure described herein, some additional terms are defined below. In general, the nomenclature used herein and the experimental procedures in organic chemistry, medicinal chemistry, and pharmacology described herein are those well known and commonly used in the art. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. All patents, applications, published applications, and other publications mentioned throughout this specification, as well as the accompanying appendices, are incorporated herein by reference in their entirety.

[0015] As used herein, the term "oligonucleotide" refers to an oligomeric compound comprising a plurality of linked nucleotides, either chemically modified or unmodified, that is less than about 100 nucleotides in length, e.g., 1-20 nucleotides, 20-40 nucleotides, 40-60 nucleotides, 60-80 nucleotides, 80-100 nucleotide acids, or 1-50 nucleotides in length. In certain embodiments, the oligonucleotide comprises a non-nucleic acid conjugate group.

[0016] In certain embodiments, the oligonucleotide comprises a ribonucleic acid (RNA), a deoxyribonucleic acid (DNA), or a polypeptide nucleic acid (PNA). In certain embodiments, the oligonucleotide is double-stranded or single-stranded. In certain embodiments, the oligonucleotide is an siRNA, an aptamer, an antisense nucleic acid, a sgRNA, a tracrRNA, or a crRNA.

[0017] As used herein, the term "conjugate" or "conjugate group" refers to an atom or group bond attached to an oligonucleotide. In certain cases, conjugate groups modify one or more properties of the oligonucleotide to which they are attached, including, but not limited to, pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge and / or clearance properties.

[0018] As used herein, the term "receptor" refers to a biopolymer consisting of a glycoprotein or lipoprotein, which is present in the cell membrane, cytoplasm or nucleus, has different receptors and has a specific structure and configuration. As used herein, the term "ligand" refers to a substance that can recognize and bind to a receptor.

[0019] In certain embodiments, the ligand is a ligand that has affinity for the asialoglycoprotein receptor (ASGPR), hi certain embodiments, the ligand is a sugar, such as a monosaccharide or polysaccharide, including, but not limited to, galactose, N-acetylgalactosamine, mannose, glucose, glucosamine, and fucose.

[0020] As used herein, the term "polysaccharide" refers to a polymer consisting of multiple monosaccharide groups linked by glycosidic bonds. As used herein, polysaccharide includes oligosaccharides and oligosaccharides. Typically, "oligosaccharide" refers to a polymer consisting of 2-10 monosaccharide groups linked by glycosidic bonds, and "oligosaccharide" refers to a polymer consisting of less than 20 monosaccharide groups linked by glycosidic bonds.

[0021] As used herein, "about" is understood by those of ordinary skill in the art and may vary to some extent depending on the context in which it is used. If the meaning of the term remains unclear to those of ordinary skill in the art based on the context in which it is used, "about" means a deviation of up to ±10% of the stated value or range.

[0022] As used herein, the term "prevention" refers to preventing or delaying the onset of a disease.

[0023] As used herein, the term "treatment" refers to curing or at least partially arresting the progression of a disease or alleviating a symptom of a disease.

[0024] As used herein, the term "effective amount" refers to an amount effective to achieve an intended purpose. For example, an amount effective to prevent a disease refers to an amount effective to prevent, inhibit, or delay the onset of the disease. Determination of such an effective amount is within the capabilities of one of ordinary skill in the art.

[0025] As used herein, the term "acceptable" in reference to a formulation, composition or ingredient means that there are no lasting adverse effects on the overall health of the subject receiving the treatment.

[0026] "API" refers to active pharmaceutical ingredients.

[0027] The term "excipient" or "pharmaceutically acceptable excipient" means a pharma- ceutically acceptable substance, ingredient, or carrier, such as a liquid or solid filler, diluent, carrier, solvent, or encapsulating material. In one embodiment, each component is "pharmaceutically acceptable" in the sense of being compatible with the other components of a pharmaceutical formulation, and suitable for contact with the tissues or organs of human beings and animals without undue toxicity, irritation, allergic response, immunogenicity, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0028] For example, Remington:The Science and Practice of Pharmacy,21st ed.;Lippincott Williams&Wilkins:Philadelphia,PA,2005;Handbook of Pharmaceutical Excipients,6th ed.;Rowe et al.,Eds.;The Pharmaceutical Press and the American Pharmaceutical Association:2009;Handbook of Pharmaceutical Additives,3rd ed.;Ash and Ash Eds.;Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd ed.; Gibson Ed.; CRC Press LLC: Boca Raton, FL, 2009.

[0029] The term "pharmaceutical acceptable salt" refers to a formulation of a compound that does not cause significant irritation to the organism to which it is administered and does not abolish the biological activity and properties of the compound. In certain cases, pharmaceutical acceptable salts are obtained by reacting the compounds described herein with an acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.

[0030] In certain cases, pharma- ceutically acceptable salts are formed by reacting a compound having an acidic group as described herein with a base to form a salt, e.g., an ammonium salt, an alkali metal salt such as a sodium salt or a potassium salt, an alkaline earth metal salt such as a calcium salt or a magnesium salt, an organic base salt such as dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine, and salts with amino acids such as arginine, lysine, or by other methods previously determined.

[0031] There is no particular limitation on the pharmaceutically acceptable salt as long as it can be used in medicine. Examples of the salt formed by the compound described in the present specification and a base include salts with inorganic bases such as sodium, potassium, magnesium, calcium, and aluminum; salts with organic bases such as methylamine, ethylamine, and ethanolamine; salts with basic amino acids such as lysine and ornithine, and ammonium salts.

[0032] The above salt may be an acid addition salt, specific examples of which include acid addition salts with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid; organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, and ethanesulfonic acid; and acidic amino acids such as aspartic acid and glutamic acid.

[0033] The term "pharmaceutical composition" refers to a mixture of a compound described herein with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, and / or thickening agents (collectively referred to herein as "excipients"). A pharmaceutical composition facilitates administration of a compound to a living organism. The art is replete with a variety of methods for administering a compound, including, but not limited to, rectal, oral, intravenous, aerosol, parenteral, ocular, pulmonary, and topical administration.

[0034] The term "subject" refers to an animal, including, but not limited to, a primate (e.g., a human), monkey, cow, pig, sheep, goat, horse, dog, cat, rabbit, rat, or mouse. The terms "subject" and "patient" are used interchangeably herein and refer, for example, to a mammalian subject, such as a human.

[0035] The term "gene-associated disease" refers to a disease caused by the abnormal expression of one or more genes and / or the abnormal activity of the proteins expressed by those genes. These genes are also referred to as disease-associated genes.

[0036] The term "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) or iodine (I).

[0037] The term "alkyl" refers to a hydrocarbon chain, which may be straight or branched, and which contains a specified number of carbon atoms. For example, C 1-10 means that the group can contain 1 to 10 (inclusive) carbon atoms. Non-limiting examples include methyl, ethyl, isopropyl, tert-butyl, and n-hexyl.

[0038] The term "haloalkyl" refers to an alkyl group in which one or more hydrogen atoms are replaced with independently selected halogens (e.g., -CF 3 )

[0039] The term "alkoxy" refers to an -O-alkyl group (e.g., -OCH 3 )

[0040] The term "alkylene" refers to a divalent alkyl group (e.g., -CH 2 -).

[0041] The term "alkenyl" refers to a straight or branched hydrocarbon chain that may have one or more carbon-carbon double bonds. The alkenyl moiety contains a specified number of carbon atoms. For example, C 2-6 means that the group can contain 2 to 6 (inclusive) carbon atoms.

[0042] The term "alkynyl" refers to a straight or branched hydrocarbon chain that may have one or more carbon-carbon triple bonds. The alkynyl moiety contains a specified number of carbon atoms. For example, C 2-6 means that the group can contain 2 to 6 (inclusive) carbon atoms.

[0043] The term "aryl" refers to a monocyclic, bicyclic, tricyclic, or polycyclic group of 6 to 20 carbons in which at least one ring in the system is aromatic (e.g., a 6 carbon monocyclic, 10 carbon bicyclic, or 14 carbon tricyclic aromatic ring system) and 0, 1, 2, 3, or 4 atoms of each ring may be substituted with a substituent. Examples of aryl groups include phenyl, naphthyl, tetrahydronaphthyl, and the like.

[0044] The term "cycloalkyl" as used herein includes non-aromatic cyclic hydrocarbon groups having 3 to 20 ring carbons, preferably 3 to 16 ring carbons, more preferably 3 to 12 ring carbons or 3 to 10 ring carbons or 3 to 6 ring carbons, where the cycloalkyl group may be optionally substituted. Cycloalkyl groups can have any degree of saturation, so long as none of the rings in the ring system are aromatic.

[0045] Thus, a cycloalkyl group can be fully saturated. Non-limiting examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Cycloalkyl groups may also include partially saturated cyclic hydrocarbon groups having 3 to 20 ring carbons, preferably 3 to 16 ring carbons, more preferably 3 to 12 ring carbons or 3 to 10 ring carbons or 3 to 6 ring carbons.

[0046] Non-limiting examples include cyclopentenyl, cyclohexenyl, cycloheptenyl, and cyclooctenyl. Cycloalkyl groups may contain multiple fused and / or bridged rings. Non-limiting examples of fused / bridged cycloalkyl groups include bicyclo[1.1.0]butane, bicyclo[2.1.0]pentane, bicyclo[1.1.1]pentane, bicyclo[3.1.0]hexane, bicyclo[2.1.1]hexane, bicyclo[3.2.0]heptane, bicyclo[4.1.0]heptane, bicyclo[2.2.1]heptane, bicyclo[3.1.1]heptane, bicyclo[4.2.0]octane, bicyclo[3.2.1]octane, bicyclo[2.2.2]octane, and the like.

[0047] Cycloalkyl groups also include spirocycles (e.g., spiro bicycles in which the two rings are joined by only one atom). Non-limiting examples of spirocyclic cycloalkyl groups include spiro[2.2]pentane, spiro[2.5]octane, spiro[3.5]nonane, spiro[3.5]nonane, spiro[3.5]nonane, spiro[4.4]nonane, spiro[2.6]nonane, spiro[4.5]decane, spiro[3.6]decane, spiro[5.5]undecane, and the like.

[0048] The term "heteroaryl," as used herein, refers to a monocyclic, bicyclic, tricyclic, or polycyclic group having 5 to 20 ring atoms, or 5, 6, 9, 10, or 14 ring atoms, and sharing 6, 10, or 14 pi electrons in a ring arrangement, where at least one ring in the system is an aromatic group (but not necessarily a ring containing a heteroatom, e.g., tetrahydroisoquinolyl, tetrahydroquinolyl), and where at least one ring in the system contains one or more heteroatom groups independently selected from the group consisting of N, O, and S.

[0049] Heteroaryl groups can be unsubstituted or substituted with one or more substituents. Examples of heteroaryl groups include thienyl, pyridyl, furyl, oxazolyl, oxadiazolyl, pyrrolyl, imidazolyl, triazolyl, thiadiazolyl, pyrazolyl, isoxazolyl, thiadiazolyl, pyranyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, thiazolylbenzothienyl, benzoxadiazolyl, benzofuranyl, benzimidazolyl, benzotriazolyl, cinnolinyl, indazolyl, indolyl, isoquinolinyl, isothiazolyl, naphthyridinyl, purinyl, thienopyridyl, pyrido[2,3 -d]pyrimidinyl, pyrrolo[2,3-b]pyridyl, quinazolinyl, quinolinyl, thieno[2,3-c]pyridyl, pyrazolo[3,4-b]pyridyl, pyrazolo[3,4-c]pyridyl, pyrazolo[4,3-c]pyridine, pyrazolo[4,3-b]pyridyl, tetrazolyl, chroman, 2,3-dihydrobenzo[b][1,4]dioxine, benzo[d][1,3]dioxole, 2,3-dihydrobenzofuran, tetrahydroquinoline, 2,3-dihydrobenzo[b]1,4]oxathiin, isoindoline, and others.

[0050] In some embodiments, heteroaryl is selected from the group consisting of thienyl, pyridyl, furyl, pyrazolyl, imidazolyl, isoindolinyl, pyranyl, pyrazinyl, and pyrimidinyl.

[0051] The term "heterocyclyl" refers to a monocyclic, bicyclic, tricyclic (e.g., a 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system) or polycyclic non-aromatic ring system having 3 to 16 ring atoms, with 1 to 3 heteroatoms in the monocyclic case, 1 to 6 heteroatoms in the bicyclic case, and 1 to 9 heteroatoms in the tricyclic or polycyclic case, selected from O, N, or S (e.g., 1 to 3, 1 to 6, or 1 to 9 carbon atoms and N, O, or S heteroatoms in the monocyclic, bicyclic, or tricyclic case, respectively), in which 0, 1, 2, or 3 atoms of each ring may be substituted by a substituent. Examples of heterocyclyl groups include piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, and the like. Heterocyclyl groups may contain multiple fused and bridged rings.

[0052] Non-limiting examples of fused / bridged heterocyclyl groups include 2-azabicyclo[1.1.0]butane, 2-azabicyclo[2.1.0]pentane, 2-azabicyclo[1.1.1]pentane, 3-azabicyclo[3.1.0]hexane, 5-azabicyclo[2.1.1]hexane, 3-azabicyclo[3.2.0]heptane, octahydrocyclopenta[c]pyrrole, 3-azabicyclo[4.1.0]heptane, 7-azabicyclo[2.2.1]heptane, 6-azabicyclo[3.1.1]heptane, 7-azabicyclo[4.2.0]octane, 2-azabicyclo[2.2.2]octane, 3-azabicyclo[4.1.0]hexane, 5-azabicyclo[2.1.1]hexane, 3-azabicyclo[4.1.0]hex ...5-azabicyclo[2.1.1]hexane, 3-azabicyclo[4.1.0]hexane, 5-azabicyclo[2.1.1]hexane, 3-azabicyclo[4.1.0]hexane, 5-azabicyclo[2.1.1]hexane, 3-azabicyclo[4.1.0]hexane, 5-azabicyclo[2.1.1]hexane, 3-azabicyclo[4.1.0]hexane, 5-azabicyclo[2. oxabicyclo[3.2.1]octane, 2-oxabicyclo[1.1.0]butane, 2-oxabicyclo[2.1.0]pentane, 2-oxabicyclo[1.1.1]pentane, 3-oxabicyclo[3.1.0]hexane, 5-oxabicyclo[2.1.1]hexane, 3-oxabicyclo[3.2.0]heptane, 3-oxabicyclo[4.1.0]heptane, 7-oxabicyclo[2.2.1]heptane, 6-oxabicyclo[3.1.1]heptane, 7-oxabicyclo[4.2.0]octane, 2-oxabicyclo[2.2.2]octane, 3-oxabicyclo[3.2.1]octane, and the like.

[0053] Heterocyclyl groups also include spirocycles (e.g., spiro bicycles in which the two rings are joined by only one atom). Non-limiting examples of spirocyclic heterocyclyl groups include 2-azaspiro[2.2]pentane, 4-azaspiro[2.5]octane, 1-azaspiro[3.5]nonane, 2-azaspiro[3.5]nonane, 7-azaspiro[3.5]nonane, 2-azaspiro[4.4]nonane, 6-azaspiro[2.6]nonane, 1,7-diazaspiro[4.5]decane, 7-azaspiro[4.5]decane, 2,5-diazaspiro[3.6]decane, 3-azaspiro[5.5]undecane, 2-oxaspiro[2. 1,2]pentane, 4-oxaspiro[2.5]octane, 1-oxaspiro[3.5]nonane, 2-oxaspiro[3.5]nonane, 7-oxaspiro[3.5]nonane, 2-oxaspiro[4.4]nonane, 6-oxaspiro[2.6]nonane, 1,7-dioxaspiro[4.5]decane, 2,5-dioxaspiro[3.6]decane, 1-oxaspiro[5.5]undecane, 3-oxaspiro[5.5]undecane, and 3-oxa-9-azaspiro[5.5]undecane.

[0054] Additionally, the atoms constituting the compounds of the present embodiments are intended to include all isotopic forms of those atoms. Isotopes as described herein include atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 29, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 112, 123, 134, 140, 141, 152, 163, 13 C and 14 Contains C.

[0055] Additionally, the compounds disclosed generically or specifically herein are intended to include all tautomeric forms. Thus, for example, the moieties Compounds containing TIFF0007618778000004.tif25170 contain the moiety TIFF0007618778000005.tif34170 are included. Similarly, a pyridyl or pyrimidinyl moiety that is depicted as optionally substituted with hydroxy includes the pyridone or pyrimidone tautomeric forms.

[0056] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features and advantages of the invention will become apparent from the description and drawings, and from the claims. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0057] The present disclosure features one or more chemical entities (e.g., compounds or pharma- ceutically acceptable salts, and / or hydrates, and / or cocrystals, and / or pharmaceutical compositions of compounds) directed to an asialoglycoprotein receptor (ASGPR) ligand moiety. Exemplary chemical entities also include oligonucleotides. The above chemical entities are useful, for example, for targeted delivery of oligonucleotides to liver cells (e.g., hepatocytes).

[0058] The chemical entities are useful, for example, for treating disorders or diseases caused by the expression (e.g., aberrant expression) of one or more genes in liver cells. The disclosure also features compositions comprising the compositions, as well as methods of using and preparing the compositions.

[0059] In one aspect, the disclosure provides a compound having formula (I) or a pharma- ceutically acceptable salt thereof: TIFF0007618778000006.tif40170 In the formula, each R X is, independently, ·H; -CH 2 OR X2 (where R X2 is H, C 1-6 alkyl, or hydroxyl protecting groups; and TIFF0007618778000007.tif28170, However, at least one R X is a radical of formula (A1), Each R 1 is an independently selected moiety capable of binding to the asialoglycoprotein receptor (ASGPR); Each R 2are independently -C(R 6 ) 2 -, -OC(R 6 ) 2 C(R 6 ) 2 O-, -C(R 6 )(OH)-C(R 6 )(OH)-, * -C(=O)NR 7 -, * -NR 7 C(=O)-, C 6-10 Arylene, C 2-6 Alkenylene, and C 2-6 alkynylene, Here, the above C 6-10 Arylene, C 2-6 Alkenylene, and C 2-6 Each alkynylene is independently selected from 1 to 4 R a , and the * above is represents the joining point to TIFF0007618778000008.tif20170, R 3 teeth, TIFF0007618778000009.tif43170 (where aa is TIFF0007618778000010.tif22170); -(CR 6 R 6 ) x -O-(CR 6 R 6 ) y - (wherein x and y are independently 0, 1, 2, or 3); and -L 3 -L 3C - (where L 3C is C 3-10 Cycloalkylene, C 6-10 arylene, 5- to 10-membered heteroarylene, and 4- to 10-membered heterocyclylene, each of which is independently selected from 1 to 4 R a and optionally substituted with -L 3 -C(R 6 )2 - and R 4 is -C(R 6 ) 2 -, -OC(R 6 ) 2 C(R 6 ) 2 O-, -C(R 6 )(OH)-C(R 6 )(OH)-, * -C(=O)NR 7 -, * -NR 7 C(=O)-, C 6-10 Arylene, C 3-10 selected from the group consisting of cycloalkylene, 5- to 10-membered heteroarylene, and 4- to 10-membered heterocyclylene; Here, C 6-10 Arylene, C 3-10 Cycloalkylene, 5- to 10-membered heteroarylene, and 4- to 10-membered heterocyclylene each represent 1 to 4 independently selected R a optionally replaced by The above* represents the attachment point to TIFF0007618778000011.tif45170, R 5 teeth, Hydroxyl; C(O)OH; TIFF0007618778000012.tif26170 (where Pg is a carboxyl activating group or a carboxyl protecting group) TIFF0007618778000013.tif49170 (where Z is a hydroxyl protector); TIFF0007618778000014.tif47170 (here Pg 2 is a hydroxyl protecting group); and TIFF0007618778000015.tif19170 (L is a bond or a divalent group selected from the group consisting of: ○ -R 2 -, -R 3 -, -R 4 -, -O-, -C(=O)-, -C(=O)O-, -OC(=O)-, TIFF0007618778000016.tif41170 (here Pg 3 is H or Pg 2 is) TIFF0007618778000017.tif46170 (where Z 2 is H or a hydroxyl protecting group. TIFF0007618778000018.tif43170, where bb represents a point of attachment to Oligo and Oligo is an oligonucleotide; Each R 6 are independent of each other, H, C 1-3 Alkyl, C 1-3 selected from the group consisting of haloalkyl, and halogen; Each R 7 are independently H and C 1-3 is selected from the group consisting of alkyl, each a and b is independently selected from an integer of 1 to 10; each c and d is independently selected from an integer from 0 to 10; Each R a are independently halogen, cyano, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy and C 1-6 haloalkoxy.

[0060] Variable R X In some embodiments, each R X teeth, H; and TIFF0007618778000019.tif28170.

[0061] In some embodiments, each R X teeth, -CH 2 OR X2 (where R X2 , H, C 1-6 alkyl, or hydroxy protecting group; and TIFF0007618778000020.tif28170.

[0062] In certain embodiments, the hydroxyl protecting group is selected from the group consisting of silyl protecting groups, 4-monomethoxytrityl (MMTR), 4,4'-dimethoxytrityl (DMTR), and trityl.

[0063] In certain embodiments, the silyl protecting group is selected from the group consisting of tert-butyldimethylsilyl (TBMDS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS).

[0064] In some embodiments, at least two R X are each independently a group of formula (A1).

[0065] In certain embodiments, each R X are independently a group of formula (A1).

[0066] In some embodiments, each R X is a group of formula (A1), and each R X is the same.

[0067] Variable R 1 In some embodiments, each R 1 are independently selected carbohydrate moieties. In certain embodiments, each R 1 is independently a monosaccharide or polysaccharide (e.g., a monosaccharide or a disaccharide). In certain embodiments, each R 1 is selected from the group consisting of galactose, N-acetylgalactosamine, mannose, glucose, glucosamine, and fucose. 1 is the same.

[0068] In certain embodiments, each R 1 are independently a group of formula (B1) or (B2), TIFF0007618778000021.tif40170TIFF0007618778000022.tif41170In the formula, R D is R C and TIFF0007618778000023.tif49170, Each R B are independently, -NR E R F -OR C is selected from the group consisting of Each R C are independently H and -C(=O)C 1-6 is selected from the group consisting of alkyl, Each R E are independently C(=O)C 1-6 is alkyl, Each R F are independently H and TIFF0007618778000024.tif32170, R G is C 1-6 is alkyl, Each q is independently selected from an integer of 1 to 10.

[0069] In certain embodiments, each R 1 is independently a group having the formula (B1-a), (B1-b) or (B2-a).

[0070] TIFF0007618778000025.tif33170TIFF0007618778000026.tif35170TIFF0007618778000027.tif51170

[0071] In certain embodiments of Formula (B1), (B2), (B1-a), (B1-b), or (B2-a), each R B is independently NR E R F In certain embodiments, each R E are independently -C(=O)C 1-6 It is an alkyl.

[0072] For example, each R E is -C(=O)CH 3 In certain embodiments, each R F is H. In certain embodiments, each R F teeth The file is TIFF0007618778000028.tif31170.

[0073] In certain embodiments of Formula (B1), (B2), (B1-a), (B1-b), or (B2-a), each R B is NHC(=O)CH 3 It is.

[0074] In certain embodiments of Formula (B1), (B2), (B1-a), (B1-b), or (B2-a), each R B teeth The file is TIFF0007618778000029.tif31170.

[0075] In certain embodiments of Formula (B1), (B2), (B1-a), (B1-b), or (B2-a), each R B are independent -OR C It is.

[0076] In certain embodiments of Formula (B1), (B2), (B1-a), (B1-b), or (B2-a), each R C are independently C(=O)C 1-6 For example, each R C is C(=O)CH 3 It is.

[0077] In certain embodiments of Formula (B1), (B2), (B1-a), (B1-b), or (B2-a), each R C is H.

[0078] In certain embodiments of Formula (B2) or (B2-a), each R G CH 3 It is.

[0079] In certain embodiments, each R1 is selected from the group consisting of:

[0080] TIFF0007618778000030.tif195170

[0081] In some embodiments, each R 1 is the same For example, each R 1 teeth TIFF0007618778000031.tif34170. As another non-limiting example, each R 1 teeth The file is TIFF0007618778000032.tif34170.

[0082] Variable R 2 , a and b In some embodiments, each R 2 is independent * -C(=O)NR 7 -or * -NR 7 In certain embodiments, each R 2 is independent * -C(=O)NR 7 In certain embodiments, each R 2 teeth * -C(=O)NH-.

[0083] In some embodiments, each R 2 is independently -C(R 6 ) 2 In certain embodiments, each R 2 Ha-CH 2 In some embodiments, each R 2 is the same.

[0084] In some embodiments, each a is independently 1, 2, 3, or 4. In some embodiments, each a is independently 5, 6, or 7. In some embodiments, each a is independently 8, 9, or 10.

[0085] In some embodiments, each a is an integer independently selected from 1 to 4. In certain embodiments, each a is independently 2 or 3. In some embodiments, each a is the same.

[0086] In some embodiments, each b is independently 1, 2, 3, or 4. In some embodiments, each b is independently 5, 6, or 7. In some embodiments, each b is independently 8, 9, or 10.

[0087] In some embodiments, each b is an integer independently selected from 1 to 4. In certain embodiments, each b is independently 2 or 3. In some embodiments, each b is the same.

[0088] In certain embodiments, each a is the same, each b is the same, and each R 2 is the same.

[0089] In certain embodiments, a is an integer selected from 1 to 4, b is an integer selected from 1 to 4, and R 2 teeth * -C(=O)NR 7 - For example, a is 3, b is 3, and R 2 teeth * -C(=O)NH.

[0090] In certain embodiments, (each a is the same, each b is the same, and each R 2 are the same), a is an integer selected from 1 to 4, b is an integer selected from 1 to 4, and R 2 -C(R 6 ) 2 - For example, R 2 Ha-CH 2 - and 3≦(a+b)≦5.

[0091] Variable R 3 In some embodiments, R 3 teeth The file is TIFF0007618778000033.tif37170.

[0092] In certain embodiments, R 3 teeth The file is TIFF0007618778000034.tif39170.

[0093] In certain embodiments, R 3 teeth The file is TIFF0007618778000035.tif33170.

[0094] In some embodiments, L 3 -C(R 6 ) 2 In a particular embodiment, L 3 Ha-CH 2 -It is.

[0095] Variable R 4 , c and d In some embodiments, c is independently 0 or 1. In some embodiments, c is independently 2, 3, or 4. In some embodiments, c is independently 5, 6, or 7. In some embodiments, c is independently 8, 9, or 10.

[0096] In some embodiments, c is an integer from 1 to 2.

[0097] In some embodiments, c is an integer from 2 to 5.

[0098] In some embodiments, c is an integer from 3 to 7.

[0099] In some embodiments, R 4 teeth * -C(=O)NR 7 In certain embodiments, R 4 teeth * -C(=O)NH-.

[0100] In some embodiments, R 4 -C(R 6 ) 2 In certain embodiments, R 4 Ha-CH2 -It is.

[0101] In some embodiments, d is independently 0 or 1. In some embodiments, d is independently 2, 3, or 4. In some embodiments, d is independently 5, 6, or 7. In some embodiments, d is independently 8, 9, or 10.

[0102] In some embodiments, d is an integer from 1 to 2.

[0103] In some embodiments, d is an integer from 3-7.

[0104] In certain embodiments, R 4 -C(R 6 ) 2 -, and each c and d is independently 1 or 2. In certain embodiments, R 4 Ha-CH 2 - and each c and d is 1.

[0105] In certain embodiments, R 4 -C(R 6 ) 2 - and 4≦(c+d)≦12. In certain embodiments, R 4 Ha-CH 2 - and 7≦(c+d)≦10.

[0106] In certain embodiments, R 4 teeth * -C(=O)NR 7 - and 5≦(c+d)≦10. In certain embodiments, R 4 teeth * -C(=O)NR 7 - and 7≦(c+d)≦9. In certain of the foregoing embodiments, c is 3.

[0107] Non-limiting combinations In certain embodiments, the compound of formula (I) is selected from the group consisting of: TIFF0007618778000036.tif69170TIFF0007618778000037.tif73170TIFF0007618778 000038.tif69170TIFF0007618778000039.tif75170TIFF0007618778000040.tif63170 TIFF0007618778000041.tif81170TIFF0007618778000042.tif96170TIFF0007618778 000043.tif73170TIFF0007618778000044.tif96170TIFF0007618778000045.tif78170

[0108] Variable R 5 In some embodiments, R 5 is C(O)OH or The file is TIFF0007618778000046.tif31170.

[0109] In certain embodiments, R 5 is C(O)OH.

[0110] In certain embodiments, R 5 teeth TIFF0007618778000047.tif28170. In certain embodiments, Pg is a carboxyl activating group.

[0111] A "carboxy activating group" as described herein is a chemical moiety that is covalently attached to a carboxyl oxygen atom and converts the oxygen atom into a leaving group. Correspondingly, when Pg is a carboxyl activating group: The "-O-Pg" group in TIFF0007618778000048.tif29170 is a leaving group. Non-limiting examples of carboxyl activating groups include N-centered heterocyclyl groups (e.g., succinimidyl) and electron-deficient heteroaryl and aryl groups (e.g., pentafluorophenyl).

[0112] In certain embodiments, Pg is TIFF0007618778000049.tif31170, where Ring D is a 5-10 membered heteroaryl or a 4-10 membered heterocyclyl, each of which is optionally substituted with 1-6 substituents, each of which is independently selected from halogen, oxygen, NO 2 , C(O)C 1-4 Alkyl, C(O)OC 1-4 Alkyl, S(O)C 1-4 Alkyl, C 1-6 Alkyl, C 1-6 haloalkyl, and -OH.

[0113] For example, Pg It could also be TIFF0007618778000050.tif48170.

[0114] As another non-limiting example, Pg TIFF0007618778000051.tif39170, which is optionally substituted with 1 to 6 substituents, each substituent being independently selected from halogen, NO 2 , C(O)C 1-4 Alkyl, C(O)OC 1-4 Alkyl, S(O)C 1-4 Alkyl, C 1-6 Alkyl, C 1-6 and -OH (eg, unsubstituted or substituted with 1 to 6 independently selected halogens).

[0115] In certain embodiments, Pg is C 6-10 aryl or 5-10 membered heteroaryl substituted with 1-6 substituents, each of which is independently -F, -Cl, -NO 2 , C(O)C 1-4 Alkyl, C(O)OC 1-4 Alkyl, and S(O)C 1-4 alkyl.

[0116] In certain embodiments, Pg is TIFF0007618778000052.tif27170 where p is an integer from 1 to 5, and each R p is -F, -Cl, or -NO 2 In certain embodiments, each R p As another non-limiting example, Pg is It could also be TIFF0007618778000053.tif46170.

[0117] In certain embodiments, R 5 teeth The file is TIFF0007618778000054.tif46170.

[0118] In certain embodiments, R 5 teeth The file is TIFF0007618778000055.tif35170.

[0119] In some embodiments, R 5 teeth The file is TIFF0007618778000056.tif57170.

[0120] In certain embodiments, R 5 teeth The file is TIFF0007618778000057.tif57170.

[0121] In some embodiments, each Pg 2 and Z are independently selected from the group consisting of silyl protecting groups, 4-monomethoxytrityl (MMTR), 4,4′-dimethoxytrityl (DMTR), and trityl.

[0122] In certain embodiments, the silyl protecting group is selected from the group consisting of tert-butyldimethylsilyl (TBMDS), tert-butyldiphenylsilyl (TBDPS) and triisopropylsilyl (TIPS).

[0123] Non-limiting exemplary compounds In certain embodiments, the compound is selected from compounds GalNAc-1 through GalNAc-12. For example, the compound is selected from compounds GalNAc-1 through GalNAc-10.

[0124] TIFF0007618778000058.tif52170TIFF0007618778000059.tif58170TIFF0007618778000060.tif57170TIFF0007618778 000061.tif67170TIFF0007618778000062.tif62170TIFF0007618778000063.tif56170TIFF0007618778000064.tif63170 TIFF0007618778000065.tif58170TIFF0007618778000066.tif68170TIFF0007618778000067.tif68170TIFF0007618778 000068.tif58170TIFF0007618778000069.tif58170TIFF0007618778000070.tif94170TIFF0007618778000071.tif93170

[0125] GalNAc-13 and GalNAc-14 are useful, for example, to prepare intermediates for compounds of formula (I).

[0126] Conjugates In some embodiments, the compound of Formula (I) is a conjugate of Formula (II) or a pharma- ceutically acceptable salt thereof: TIFF0007618778000072.tif40170, R 5 teeth TIFF0007618778000073.tif16170, where Oligo is oligonucleotide, -L-, R X (R 1 , R 2 , including a and b), R 3 ,c,R 4 and d may be as defined for any of formula (I) herein.

[0127] In some embodiments, Oligo is an oligonucleotide linked to L via the 5' end, the 3' end, or the middle of the sequence of any strand by a phosphate bond. Variable L In some embodiments, L is a bond.

[0128] In some embodiments, L is C(=O).

[0129] In some embodiments, L is --O--.

[0130] In some embodiments, L is TIFF0007618778000074.tif31170, where bb is the attachment point to the Oligo.

[0131] In some embodiments, L is TIFF0007618778000075.tif46170.

[0132] In certain embodiments, L is The file is TIFF0007618778000076.tif42170.

[0133] In certain embodiments, Pg 3 is H. In certain embodiments, Pg 3 is a hydroxyl protecting group.

[0134] In certain embodiments, L is The file is TIFF0007618778000077.tif45170.

[0135] In certain embodiments, Z 2 is H. In certain embodiments, Z 2 is a hydroxyl protecting group.

[0136] In certain embodiments, the hydroxyl protecting group is selected from the group consisting of silyl protecting groups, 4-monomethoxytrityl (MMTR), 4,4′-dimethoxytrityl (DMTR), and trityl.

[0137] In certain embodiments, the silyl protecting group is selected from the group consisting of tert-butyldimethylsilyl (TBMDS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl (TIPS).

[0138] Oligonucleotide moiety In some embodiments, the Oligo is an oligonucleotide, including a single stranded oligonucleotide and / or a double stranded oligonucleotide.

[0139] In certain embodiments, the Oligo is a single-stranded oligonucleotide. In certain embodiments, the Oligo is a double-stranded oligonucleotide.

[0140] In some embodiments, the oligonucleotide is selected from the group consisting of DNA, siRNA, miRNA, pre-miRNA, antagomir, mRNA, antisense oligonucleotide (ASO), aptamer, crRNA, tracRNA, and sgRNA.

[0141] The oligonucleotides may comprise unmodified and / or modified nucleotides.

[0142] Non-limiting examples of modified nucleotides include 2'-O-(2-methoxyethyl) modified nucleotides, 2'-O-alkyl modified nucleotides (e.g., 2'-O-methyl nucleotides), 2'-O-allyl modified nucleotides, 2'-C-allyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-deoxy modified nucleotides, 2'-hydroxyl modified nucleotides, locked nucleotide (LNA) modified nucleotides, hexitol nucleic acid (HNA) modified nucleotides, glycerol nucleic acid (GNA) modified nucleotides and non-locked nucleic acid (UNA) modified nucleotides.

[0143] In certain embodiments, the modified nucleotides are selected from the group consisting of 2'-O-alkyl modified nucleotides and 2'-fluorine modified nucleotides.

[0144] In some embodiments, the oligonucleotide comprises a modification group selected from the group consisting of cholesterol, polyethylene glycol, a fluorescent probe, biotin, a polypeptide, a vitamin, a tissue targeting molecule, and combinations thereof. In certain embodiments, the modification group is a terminal modification group.

[0145] Oligo can be linked to L via the 5' end, 3' end or middle of the sequence of any chain by a phosphate bond. In certain embodiments, the phosphate bond is a phosphodiester bond. In certain embodiments, the phosphate bond is a modified phosphate bond. In certain embodiments, the modified phosphate bond is selected from one or more of a thio-modified phosphate bond (e.g., phosphorothioate) and an amino-modified phosphate bond.

[0146] In some embodiments, an Oligo comprises one or more polypeptide nucleic acids and / or morpholino nucleic acids (eg, morpholino antisense oligonucleotides).

[0147] In some embodiments, the Oligo is an oligonucleotide containing 5 to 100 base pairs. For example, the Oligo can be an oligonucleotide having 5 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, or 90 to 100 base pairs.

[0148] In some embodiments, the compound of formula (II) is synthesized by solid phase synthesis or liquid phase synthesis. In certain embodiments, the compound of formula (II) is synthesized by solid phase synthesis. In certain embodiments, the compound of formula (II) is synthesized by liquid phase synthesis.

[0149] In some embodiments, the oligonucleotide may be as described in any of paragraphs

[0335] -

[0410] of U.S. Patent Application Publication No. 2015 / 0119444, or paragraphs

[0341] -

[0416] of U.S. Patent Application Publication No. 2015 / 0119445, each of which is incorporated herein by reference.

[0150] Non-Limiting Examples of Conjugates Non-limiting examples of compounds of formula (II) include the following, where n, m, and m′ are each independently an integer selected from 1 to 10:

[0151] TIFF0007618778000078.tif39170TIFF0007618778000079.tif41170TIFF0007618778000080.tif39170TIFF000 7618778000081.tif41170TIFF0007618778000082.tif38170TIFF0007618778000083.tif43170TIFF0007618778 000084.tif37170TIFF0007618778000085.tif34170TIFF0007618778000086.tif36170TIFF0007618778000087. tif31170TIFF0007618778000088.tif34170TIFF0007618778000089.tif34170TIFF0007618778000090.tif34170

[0152] Drug Composition and Administration Conventional In another aspect, the disclosure features a pharmaceutical composition that includes a chemical entity described herein (e.g., a compound of Formula (II) or a pharma- ceutically acceptable salt thereof) and a pharma- ceutically acceptable excipient.

[0153] In some embodiments, the chemical entity can be administered in combination with one or more conventional pharmaceutical excipients.

[0154] Pharmaceutically acceptable excipients include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-α-tocopherol polyethylene glycol 1000 succinate, surfactants for pharmaceutical dosage forms such as Tweens, poloxamers and other similar polymeric delivery matrices, serum proteins such as human serum globulin, buffer substances such as phosphates, Tris, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicon dioxide, magnesium trisilicate, polyvinylpyrrolidone, cellulosic materials, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, and lanolin.

[0155] Cyclodextrins, such as α-, β-, and γ-cyclodextrin, or chemically modified derivatives, such as hydroxyalkyl cyclodextrins, including 2- and 3-hydroxypropyl-β-cyclodextrin, or other soluble derivatives, can also be used to enhance delivery of the compounds described herein.

[0156] Dosage forms or compositions may be prepared that contain between 0.005% and 100% of the chemical entities described herein, with the balance being non-toxic excipients. Contemplated compositions may contain between 0.001% and 100%, in one embodiment between 0.1 and 95%, in another embodiment between 75 and 85%, and in another embodiment between 20 and 80% of the chemical entities described herein.

[0157] Actual methods for preparing such dosage forms are known or will be apparent to those skilled in the art. See, e.g., Remington: The Science and Practice of Pharmacy, 22 ndEdition (Pharmaceutical Press, London, UK. 2012).

[0158] In some embodiments, the pharmaceutical composition is in a dosage form selected from the group consisting of a powder, a tablet, a granule, a capsule, a solution, an emulsion, a suspension, an injection, a spray, an aerosol, a dry powder inhalant, or a microneedle patch.

[0159] In some embodiments, the pharmaceutical composition is suitable for administration intravenously, intramuscularly, subcutaneously, via a microneedle patch, orally, via an intrabuccal or intranasal spray, or topically to a subject in need thereof.

[0160] In some embodiments, the subject is a mammal, including bovine, equine, ovine, porcine, canine, feline, rodent, and primate. For example, the subject may be a human.

[0161] Dosage Dosage may vary depending on the patient's needs, the severity of the disorder being treated, and the specific compound being used.The appropriate dosage for a particular situation can be determined by those skilled in the medical field.The total daily dosage is divided and administered proportionately throughout the day, or administered by continuous delivery.

[0162] In some embodiments, a unit dose is less than 1.4 mg / kg of subject body weight, or less than 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001 mg / kg of subject body weight. In some embodiments, a unit dose is 0.00001-10 mg / kg of subject body weight. In some embodiments, a unit dose is 0.001-2 mg / kg of subject body weight.

[0163] plan The above doses can be administered daily (e.g., as a single dose or as two or more divided doses) or non-daily (e.g., every other day, every second day, every third day, weekly, twice weekly, biweekly, monthly, etc.).

[0164] Treatment method In another aspect, the disclosure features a method of treating and / or preventing a pathological condition or disease in a subject, where the pathological condition or disease is caused by expression of one or more genes in liver cells, the method includes administering to the subject a chemical entity described herein (e.g., a compound of Formula (II) or a pharma- ceutically acceptable salt thereof), or a pharmaceutical composition including a chemical entity described herein (e.g., a compound of Formula (II) or a pharma- ceutically acceptable salt thereof) and a pharma- ceutically acceptable excipient.

[0165] In some embodiments, the one or more genes are selected from the group consisting of HBV genome, HCV genome, PCSK9, xanthine oxidase expression gene, URAT1, APOB, liver fibrosis-related genes (AP3S2, AQP2, AZINl, DEGSl, STXBP5L, TLR4, TRPM5, etc.), non-alcoholic fatty liver-related genes (PNPLA3, FDFTl), and primary biliary cirrhosis-related genes (HLA-DQB1, IL-12, IL-12RB2, etc.).

[0166] In some embodiments, the disease or disorder is selected from the group consisting of hereditary angioedema, familial tyrosinemia type I, Alagille syndrome, alpha-1-antitrypsin deficiency, bile acid synthesis and metabolism disorders, biliary atresia, cystic fibrosis liver disease, idiopathic neonatal hepatitis, mitochondrial liver disease, progressive familial intrahepatic cholestasis, primary sclerosing cholangitis, transthyretin amyloidosis, hemophilia, homozygous familial hypercholesterolemia, hyperlipidemia, hepatitis B (HBV), hepatitis C (HCV), steatohepatitis, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), diseases associated with hyperglycemia or abnormally increased hepatic glucose production resembling type 2 diabetes, hepatitis, and hepatic porphyria.

[0167] In some embodiments, the subject is a mammal, including bovine, equine, ovine, porcine, canine, feline, rodent, and primate. For example, the subject may be a human.

[0168] In another aspect, the disclosure features a method of detecting or localizing RNA in the liver of a subject, the method includes administering to the subject a chemical entity described herein (e.g., a compound of Formula (II) or a pharma- ceutically acceptable salt thereof), or a pharmaceutical composition including a chemical entity described herein (e.g., a compound of Formula (II) or a pharma- ceutically acceptable salt thereof) and a pharma- ceutically acceptable excipient.

[0169] In some embodiments, the subject is diagnosed with one or more disorders or diseases caused by expression of one or more genes in liver cells.

[0170] In certain embodiments, the one or more genes are selected from the group consisting of HBV genome, HCV genome, PCSK9, xanthine oxidase expression gene, URAT1, APOB, liver fibrosis-related genes (AP3S2, AQP2, AZINl, DEGSl, STXBP5L, TLR4, TRPM5, etc.), non-alcoholic fatty liver-related genes (PNPLA3, FDFTl), and primary biliary cirrhosis-related genes (HLA-DQB1, IL-12, IL-12RB2, etc.).

[0171] In certain embodiments, the disease or disorder is selected from the group consisting of hereditary angioedema, familial tyrosinemia type I, Alagille syndrome, alpha-1-antitrypsin deficiency, bile acid synthesis and metabolism disorders, biliary atresia, cystic fibrosis liver disease, idiopathic neonatal hepatitis, mitochondrial liver disease, progressive familial intrahepatic cholestasis, primary sclerosing cholangitis, transthyretin amyloidosis, hemophilia, homozygous familial hypercholesterolemia, hyperlipidemia, hepatitis B (HBV), hepatitis C (HCV), steatohepatitis, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), diseases associated with hyperglycemia or abnormally increased hepatic glucose production resembling type 2 diabetes, hepatitis, and hepatic porphyria.

[0172] In some embodiments, the subject is a mammal, including bovine, equine, ovine, porcine, canine, feline, rodent, and primate. For example, the subject may be a human.

[0173] In some embodiments, a chemical entity described herein (e.g., a compound of Formula (II) or a pharma- ceutically acceptable salt thereof) is administered intravenously, intramuscularly, subcutaneously, via a microneedle patch, orally, via an intrabuccal or intranasal spray, or topically.

[0174] In another aspect, provided herein is a compound (e.g., a compound of Formula (II)), or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition thereof, for use in treating a disorder or disease caused by the expression of one or more genes in liver cells of a subject in need of such treatment.

[0175] In some embodiments, the disease or disorder is selected from the group consisting of hereditary angioedema, familial tyrosinemia type I, Alagille syndrome, alpha-1-antitrypsin deficiency, bile acid synthesis and metabolism disorders, biliary atresia, cystic fibrosis liver disease, idiopathic neonatal hepatitis, mitochondrial liver disease, progressive familial intrahepatic cholestasis, primary sclerosing cholangitis, transthyretin amyloidosis, hemophilia, homozygous familial hypercholesterolemia, hyperlipidemia, hepatitis B (HBV), hepatitis C (HCV), steatohepatitis, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), diseases associated with hyperglycemia or abnormally increased hepatic glucose production resembling type 2 diabetes, hepatitis, and hepatic porphyria.

[0176] The subject may be as defined anywhere herein. In some embodiments, the subject is a mammal (e.g., a human).

[0177] In another aspect, provided herein is a compound (e.g., a compound of formula (II)), or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition thereof, for use in detecting or localizing RNA in the liver of a subject. The subject may be as defined anywhere herein. In some embodiments, the subject is a mammal (e.g., a human).

[0178] In another aspect, provided herein is the use of a compound (e.g., a compound of Formula (II)), or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition thereof, in the treatment of a disorder or disease caused by the expression of one or more genes in liver cells of a subject in need of such treatment.

[0179] In some embodiments, the disease or disorder is selected from the group consisting of hereditary angioedema, familial tyrosinemia type I, Alagille syndrome, alpha-1-antitrypsin deficiency, bile acid synthesis and metabolism disorders, biliary atresia, cystic fibrosis liver disease, idiopathic neonatal hepatitis, mitochondrial liver disease, progressive familial intrahepatic cholestasis, primary sclerosing cholangitis, transthyretin amyloidosis, hemophilia, homozygous familial hypercholesterolemia, hyperlipidemia, hepatitis B (HBV), hepatitis C (HCV), steatohepatitis, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), diseases associated with hyperglycemia or abnormally increased hepatic glucose production resembling type 2 diabetes, hepatitis, and hepatic porphyria.

[0180] The subject may be as defined anywhere herein. In some embodiments, the subject is a mammal (e.g., a human).

[0181] In another aspect, provided herein is the use of a compound (e.g., a compound of formula (II)), or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition thereof, in detecting or localizing RNA in the liver of a subject. The subject may be as defined anywhere herein. In some embodiments, the subject is a mammal (e.g., a human).

[0182] In another aspect, provided herein is the use of a compound (e.g., a compound of Formula (II)), or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition thereof, in the manufacture of a medicament for treating a disorder or disease caused by the expression of one or more genes in liver cells of a subject in need of such treatment.

[0183] In some embodiments, the disease or disorder is selected from the group consisting of hereditary angioedema, familial tyrosinemia type I, Alagille syndrome, alpha-1-antitrypsin deficiency, bile acid synthesis and metabolism disorders, biliary atresia, cystic fibrosis liver disease, idiopathic neonatal hepatitis, mitochondrial liver disease, progressive familial intrahepatic cholestasis, primary sclerosing cholangitis, transthyretin amyloidosis, hemophilia, homozygous familial hypercholesterolemia, hyperlipidemia, hepatitis B (HBV), hepatitis C (HCV), steatohepatitis, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), diseases associated with hyperglycemia or abnormally increased hepatic glucose production resembling type 2 diabetes, hepatitis, and hepatic porphyria.

[0184] The subject may be as defined anywhere herein. In some embodiments, the subject is a mammal (e.g., a human).

[0185] In another aspect, provided herein is the use of a compound (e.g., a compound of formula (II)), or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition thereof, in the manufacture of a medicament for detecting or localizing RNA in the liver of a subject. The subject may be as defined anywhere herein. In some embodiments, the subject is a mammal (e.g., a human).

[0186] Preparation of compounds As will be appreciated by those of skill in the art, methods for synthesizing compounds represented by the general formulae herein will be apparent to those of ordinary skill in the art.

[0187] Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in the synthesis of the compounds described herein are known in the art and are described, for example, in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and R. G. M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995) and subsequent editions.

[0188] The starting materials used to prepare the compounds of the present invention are known, made by known methods, or commercially available. Those skilled in the art will understand that the conditions and reaction reagents described herein can be replaced with alternative equivalents recognized in the art. For example, in many reactions, triethylamine can be replaced with other bases, such as non-nucleophilic bases (e.g., diisopropylamine, 1,8-diazabicycloundecane-7-ene, 2,6-di-tert-butylpyridine, or tetrabutylphosphatase).

[0189] Those skilled in the art will understand, for example, 1 It is recognizable that a variety of analytical methods can be used to characterize the compounds described herein, including H NMR, heteronuclear NMR, mass spectrometry, liquid chromatography, and infrared spectroscopy. The above list is a subset of the characterization methods available to one of skill in the art and is not intended to be limiting.

[0190] To further illustrate the above, the following non-limiting exemplary synthetic routes are included. Variations of these examples within the scope of the appended claims are within the scope of the skilled artisan and are considered to be within the scope of the invention described and claimed herein. The reader will appreciate that the artisan and skilled artisan provided with the content of this disclosure can prepare and use the invention without the need for exhaustive examples.

[0191] Working Example The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. Any specific conditions not specified in the examples are conventional conditions or conditions recommended by the manufacturer. Any reagents or equipment for which the manufacturer is not specified are conventional products that are commercially available.

[0192] Example 1. Preparation of GalNAc-1 Synthetic Route TIFF0007618778000091.tif136170TIFF0007618778000092.tif65170TIFF0007618778000093.tif30170

[0193] Steps (1) To t-butyl acrylate (150 g), pentaerythritol (50 g), NaOH solution (6 mL, aqueous, 50%) and tetrabutylammonium hydroxide (9.5 g) were added, and then the reaction mixture was stirred at room temperature until the reaction was completed. After completion, ethyl acetate was added to extract the organic phase, which was then concentrated to obtain the crude product. The crude product was purified by column separation (PE (petroleum ether): EA (ethyl acetate) = 20: 1 to 7: 1) to obtain compound 1.

[0194] (2) Compound 1 (10 g) and TsCl (6.5 g) were dissolved in a pyridine solution (100 mL), and the reaction mixture was stirred at 70° C. After the reaction was completed, the reaction mixture was concentrated to remove pyridine. Ethyl acetate was added to the residue, and the mixture was concentrated to dryness. The crude product was purified by column separation (PE:EA=20:1 to 10:1) to obtain compound 2.

[0195] (3) Compound 2 (2 g) was dissolved in anhydrous DMF (50 mL), then sodium azide (0.53 g) was added, and the reaction mixture was heated to 100 °C. After the reaction was completed, the reaction mixture was cooled to room temperature and concentrated to remove DMF. Ethyl acetate was added to the residue and concentrated to dryness. The crude product was purified by column separation (PE:EA = 20:1 to 10:1) to obtain compound 3.

[0196] (4) Compound 3 (2 g) was dissolved in formic acid (10 mL), and the reaction mixture was stirred at room temperature. After the reaction was completed, the reaction mixture was concentrated to dryness to obtain compound 4.

[0197] (5) Compound 4 (1.6 g) was dissolved in dichloromethane (50 mL), and then HOBt (2.23 g), EDCl (3.0 g), DIEA (3.3 g), and N-Boc-1,3-diaminepropane (2.7 g) were added to the mixture. The reaction was stirred at room temperature until the reaction was completed. After the reaction was completed, the reaction was extracted with dichloromethane and water, and then the organic phase was dried and concentrated to dryness to obtain a crude product. The crude product was purified by column separation (dichloromethane:methanol = 2% to 5%) to obtain compound 5.

[0198] (6) Compound 5 (2.5 g) was dissolved in dichloromethane (50 mL), then trifluoroacetic acid (20 mL, 2N) was added and stirred at room temperature until the reaction was completed. After the reaction was completed, the reaction mixture was concentrated to give compound 6.

[0199] (7) Galactovaleric acid (11 g) was dissolved in dichloromethane (100 mL), then DCC (6.2 g) and pentafluorophenol (5.5 g) were added, and the reaction mixture was stirred at room temperature until the reaction was completed. After the reaction was completed, the reaction mixture was filtered, and the filtrate was washed with water, dried, and concentrated to obtain the crude product. The crude product was purified by column separation (PE:EA = 3:1 to 1:1) to obtain compound 7.

[0200] (8) Compound 6 (2.46 g) and compound 7 (10 g) were dissolved in dichloromethane (50 mL), then DIEA (8 mL) was added to the reaction mixture, and the mixture was stirred at room temperature until the reaction was completed. After the reaction was completed, the reaction mixture was extracted with dichloromethane and water, and then the organic phase was dried and concentrated to obtain a crude product. The crude product was purified by column separation (dichloromethane:methanol = 10% to 20%) to obtain compound 8.

[0201] (9) Glutaric anhydride (10 g) and propargylamine (4.82 g) were dissolved in tetrahydrofuran (50 mL) and stirred at room temperature until the reaction was completed. After the reaction was completed, the reaction mixture was concentrated to dryness to obtain compound 12.

[0202] (10) Compound 12 (3.7 g) was dissolved in dichloromethane (50 mL), and then DCC (5.41 g) and pentafluorophenol (4.83 g) were added to the reaction mixture and stirred at room temperature until the reaction was completed. After the reaction was completed, the reaction mixture was filtered, and the filtrate was washed with water, dried, and concentrated to obtain a crude product. The crude product was purified by column separation (PE:EA = 3:1 to 1:1) to obtain compound 13.

[0203] (11) Compound 13 (6.5 g) and aminocaproic acid (2.54 g) were dissolved in tetrahydrofuran (50 mL), and the reaction mixture was stirred at room temperature until the reaction was completed. After the reaction was completed, the reaction mixture was concentrated to dryness. The residue was extracted with dichloromethane and water. The organic phase was dried and then concentrated to obtain the crude product. The crude product was purified by column separation (dichloromethane:methanol = 3% to 8%) to obtain compound 14.

[0204] (12) Compound 14 (15 g) was dissolved in dichloromethane (100 mL), and then DCC (3.94 g) and pentafluorophenol (3.52) were added to the reaction mixture and stirred at room temperature until the reaction was completed. After the reaction was completed, the reaction mixture was filtered, and the filtrate was washed with water, then dried and concentrated to obtain the crude product. The crude product was purified by column separation (PE:EA = 3:1 to 1:1) to obtain compound 15.

[0205] (13) Compound 8 (0.34 g) and compound 15 (0.09 g) were dissolved in THF (5 mL), and then an aqueous solution (5 mL) of anhydrous copper sulfate (0.092 g) and sodium ascorbate (0.146 g) was added to the reaction mixture, and the mixture was stirred at room temperature until the reaction was completed. After the reaction was completed, THF was removed from the reaction mixture by concentration. The reaction residue was diluted with dichloromethane, decolorized with activated carbon, and dried over anhydrous sodium sulfate. The reaction solution was filtered to remove insoluble matter, and then concentrated to obtain the crude product. The crude product was purified by column separation (dichloromethane:methanol = 10-15%) to obtain GalNAc-1.

[0206] result The resulting GalNAc-1 was a white foamy solid (0.4 g). 1 H NMR (400 MHz, CDCl 3 )δ:ppm 7.69(s,1H),7.36(dd,4H),6.97(t,3H),6.81(d,1H),6.74(d,2H),6.40(s,1H),5.35(t,3H),5.19(dd,3H),4.61(d,3H),4.51(d,2H),4.3 2(s,2H),4.13(m,9H),3.92(dd,6H),3.65(d,6H),3.50(m,3H),3.27(m,18H),2.68(t,2H),2.44(t,6H),2.33(t,2H),2.21-1.26(m,61H). MS(ESI-TOF): m / z(M+H) + 2283.44, (M+Na) + 2305.41.

[0207] Example 2. Preparation of GalNAc-2 Synthetic Route TIFF0007618778000094.tif46170

[0208] The synthesis of compounds 8 and 13 is described in Example 1.

[0209] Steps GalNAc-2 was prepared using the synthetic method described in Example 1.

[0210] result The resulting GalNAc-2 was a white foamy solid (0.2 g). 1 HNMR (400MHz, CDCl 3 )δ:ppm7.73(s,1H),6.90-6.40(m,10H),5.36(d,3H),5.08(m,3H),4.60(s,3H),4.50(d,2H),4.35(s,2H),4.14(m,9H), 3.92(s,6H),3.66(s,6H),3.51(m,3H),3.27(m,18H),2.79(t,2H),2.45(m,6H),2.25-1.80(m,44H),1.80-1.26(m,20H). MS(ESI-TOF): m / z(M+Na) + 2191.38.

[0211] Example 3. Preparation of GalNAc-3 Synthetic Route TIFF0007618778000095.tif121170 The synthesis methods of compound 4 and compound 15 are described in Example 1.

[0212] Steps Compounds 9, 11 and GalNAc-3 were prepared using the synthetic methods described in Example 1 for compounds 7, 8 and GalNAc-1, respectively.

[0213] Cbz-hexylamine-galactose (5.5 g, purchased from Alading) was dissolved in ethyl acetate (100 mL), then palladium on carbon (10%, 1 g) was added to the reaction mixture, which was stirred at room temperature under hydrogen atmosphere until the reaction was completed. After completion of the hydrogenation reaction, the reaction mixture was filtered, and the filtrate was dried and concentrated to give compound 10.

[0214] result The resulting GalNAc-3 was a white foamy solid (0.2 g). 1 HNMR (400MHz, CDCl 3)δ:ppm.7.47-6.32(m,9H),5.36(s,3H),5.28(m,3H),4.69(s,3H),4.54(s,2H),4.36(m,2H),4.15(m,6H),4.05(m,3H),3 .94(dd,6H),3.66(s,6H),3.47(m,3H),3.25(m,12H),2.68(t,2H),2.44(m,6H),2.30-1.80(m,42H),1.80-1.26(m,32H). MS(ESI-TOF): m / z(M+Na) + 2133.43.

[0215] Example 4. Preparation of GalNAc-4 Synthetic Route TIFF0007618778000096.tif43170 Compounds 11 and 13 were prepared using the methods described in Examples 1 and 3, respectively.

[0216] Steps GalNAc-4 was prepared by reacting compound 11 with compound 13 using the method described in Example 1.

[0217] result The resulting GalNAc-4 was a white foamy solid (0.2 g). 1 HNMR (400MHz, CDCl 3 )δ:ppm.7.74-6.43(m,8H),5.36(d,3H),5.28(m,3H),4.68(d,3H),4.53(d,2H),4.31(s,2H),4.14(ddd,6H),4.05(dd,3H), 3.89(ddd,6H),3.67(m,6H),3.46(dd,3H),3.24(s,12H),2.78(t,2H),2.43(m,6H),2.30-1.80(m,38H),1.80-1.26(m,26H). MS(ESI-TOF): m / z(M+H) + 1998.43, (M+Na) + 2020.42.

[0218] Example 5. Preparation of GalNAc-5 Synthetic Route TIFF0007618778000097.tif91170 Compound 11 was the same as that described in Example 3.

[0219] Steps (1) Compound 16 (1.6 g) was dissolved in dichloromethane (50 mL), and then HOBt (2.23 g), EDCl (3.0 g), DIEA (3.3 g) and monopropargylamine (0.3 g) were added to the mixture. The reaction was stirred at room temperature until the reaction was completed. After the reaction was completed, the reaction mixture was extracted with dichloromethane and water, and then the organic phase was dried and concentrated to obtain the crude product. The crude product was purified by column separation (dichloromethane:methanol = 2% to 5%) to obtain compound 17.

[0220] (2) Compound 17 (2 g) was added to methanol (10 mL), and then Pd / C (10%, 0.5 g) was added to the reaction mixture, and the mixture was stirred at room temperature under a hydrogen atmosphere until the reaction was completed. After the hydrogenation reaction was completed, the reaction mixture was filtered, and the filtrate was dried and concentrated to give compound 18.

[0221] (3) PFP-laurate (4.5 g) was dissolved in dichloromethane (100 mL), and then DCC (3.94 g) and pentafluorophenol (3.53 g) were added to the reaction mixture and stirred at room temperature until the reaction was completed. After the reaction was completed, the reaction mixture was filtered, and the filtrate was washed with water, dried, and concentrated to obtain the crude product. The crude product was purified by column separation (PE:EA=3:1 to 1:1) to obtain compound 19.

[0222] (4) GalNAc-5 was prepared by reacting compound 11 with compound 19 using the method described in Example 1.

[0223] result The resulting GalNAc-5 was a white foamy solid (0.4 g). 1 HNMR (400MHz, CDCl 3)δ:ppm. 7.73-6.40(m,8H),5.35(d,3H),5.26(m,3H),4.67(d,3H),4.51(s,2H),4.33(s,2H),4.11(dd,6H),4.07(d,3H),3.89 (dt,6H),3.66(m,6H),3.46(dd,3H),3.24(s,12H),2.75(t,2H),2.41(m,6H),2.25-1.80(m,38H),1.80-1.26(m,40H). MS(ESI-TOF): m / z(M+H) + 2096.19, (M+Na) + 2119.20.

[0224] Example 6. Preparation of GalNAc-6 Synthetic Route TIFF0007618778000098.tif181170 Compound 8 was prepared using the method described in Example 1.

[0225] Steps (1) Aminocaproic acid (47.9 g) was dissolved in toluene (700 mL), and then benzyl alcohol (57 mL) and 4-toluenesulfonic acid (74 g) were added to the reaction mixture, which was then heated to 115° C. with stirring until the reaction was complete. After the reaction was complete, the reaction mixture was cooled to room temperature with stirring, and a large amount of solid precipitated. Methyl tert-butyl ether (500 mL) was added to the reaction mixture, which was then filtered. The solid was collected and dried to obtain compound 22.

[0226] (2) Compound 22 (19 g) was dissolved in dichloromethane (100 mL), and then a solution of glutaric anhydride (11 g) in dichloromethane (100 mL) was added to the reaction mixture and stirred at room temperature until the reaction was completed. After the reaction was completed, the reaction mixture was concentrated to give compound 23.

[0227] (3) Compound 23 (5 g) was dissolved in dichloromethane (100 mL), and then DCC (3.6 g) and pentafluorophenol (3.3 g) were added to the reaction mixture and stirred at room temperature until the reaction was completed. After the reaction was completed, the reaction mixture was filtered, and the filtrate was concentrated to obtain a crude product. The crude product was purified by column separation (PE:EA = 3:1 to 2:1) to obtain compound 24.

[0228] (4) Compound 8 (0.13 g) was dissolved in ethyl acetate (10 mL) and anhydrous methanol (10 mL), and then Pd / C (0.2 g, 10%) and 3 drops of acetic acid were added to the reaction mixture, and the mixture was stirred at room temperature under a hydrogen atmosphere until the reaction was completed. After the reaction was completed, the reaction mixture was filtered, and the filtrate was concentrated to obtain compound 25.

[0229] (5) Compound 25 (2.8 g) was dissolved in dichloromethane (50 mL), and then DIEA (0.5 g) and compound 24 (0.78 g) were added to the reaction mixture and stirred at room temperature until the reaction was completed. After the reaction was completed, the reaction mixture was concentrated to dryness. The crude product was purified by column separation (dichloromethane:methanol 15% to 25%) to obtain compound 26.

[0230] (6) Compound 26 (2.5 g) was dissolved in methanol (30 mL) and ethyl acetate (30 mL), and then Pd / C (1.3 g, 10%) was added to the reaction mixture, and the mixture was stirred at room temperature under a hydrogen atmosphere until the reaction was completed. After the reaction was completed, the reaction mixture was filtered, and the filtrate was concentrated to give compound 27.

[0231] (7) Compound 27 (0.8 g) was dissolved in dichloromethane, then DCC (0.12 g) and pentafluorophenol (0.11 g) were added to the reaction mixture, and the mixture was stirred at room temperature until the reaction was completed. After the reaction was completed, dichloromethane was added to the reaction mixture, which was then concentrated to obtain a crude product. The crude product was purified by column separation (dichloromethane:methanol = 10% to 15%) to obtain GalNAc-6.

[0232] result The resulting GalNAc-6 was a white foamy solid (0.7 g).1 HNMR(400MHz,d-DMSO)δ:ppm.7.75-7.26(m,11H),5.85(d,3H),5.59(d,3H),5.1 1(dd,3H),4.63(m,6H),4.50(d,6H),4.37(s,3H),4.17(d,6H),4.07(s,3H),3.90 3.70(m,18H),3.28(m,2H),2.92(d,6H),2.80-2.25(m,52H),2.25-1.26(m,24H). MS(ESI-TOF): m / z(M+H) + 2201.82, (M+Na) + 2223.86.

[0233] Example 7. Preparation of GalNAc-7 Synthetic Route TIFF0007618778000099.tif138170 The preparation of compound 25 is as described in Example 6.

[0234] Steps Compound 21 was prepared using the method for preparing compound 13 described in Example 1.

[0235] Compounds 29, 30 and GalNAc-7 were prepared using the methods for preparing compounds 26, 27 and GalNAc-6, respectively, described in Example 6.

[0236] result The resulting GalNAc-7 was a white foamy solid (0.5 g). 1 HNMR (400MHz, CDCl 3 )δ:ppm 7.32-6.63(m,10H),5.35(s,3H),5.20(s,3H),4.62(d,3H),4.13(ddd,9H),3.92(d,6H),3.66(dd, 6H),3.52(d,3H),3.33(m,20H),2.77(t,2H),2.44(s,6H),2.25-1.80(m,44H),1.80-1.26(m,20H). MS(ESI-TOF): m / z(M+H) + 2088.08, (M+Na)+ 2109.36.

[0237] Example 8. Preparation of GalNAc-8 Synthetic Route TIFF0007618778000100.tif252170 The preparation of compound 11 and compound 24 are as described in Example 3 and Example 6, respectively.

[0238] Steps Compounds 32, 36, 37 and GalNAc-8 were prepared using the methods for preparing Compounds 25, 26, 27 and GalNAc-6 described in Example 6, respectively.

[0239] result 1 HNMR (400MHz, CDCl 3 )δ:ppm.7.7.06-6.37(m,8H),5.36(d,3H),5.270(m,3H),4.69(t,3H),4.15(m,6H),4.00(m,3H),3.90(m,6H),3.65( t,6H),3.47(dt,3H),3.28(m,16H),2.68(t,2H),2.42(m,6H),2.29(t,4H),2.15-1.80(s,36H),1.80-1.26(m,32H). MS(ESI-TOF): m / z(M+H) + 2030.38, (M+Na) + 2052.41.

[0240] Example 9. Preparation of GalNAc-9 Synthetic Route TIFF0007618778000101.tif115170 The preparation of compound 11 is as described in Example 3.

[0241] Steps (1) Azide compound 11 (1.3 g) was dissolved in THF (15 mL), and then Pd / C (0.3 g) and glutaric anhydride (0.11 g) were added to the reaction mixture, and the mixture was stirred at room temperature under a hydrogen atmosphere until the hydrogenation reaction was completed. After the reaction was completed, the reaction mixture was filtered, the filtrate was collected, and the reaction residue was concentrated. The reaction residue was extracted with dichloromethane and water. The extracted organic phase was concentrated to obtain a crude product. The crude product was purified by column separation (dichloromethane:methanol = 10% to 15%) to obtain the product compound 34 (0.8 g). (2) GalNAc-9 was obtained by referring to the synthesis method of GalNAc-6 in Example 6.

[0242] result The resulting GalNAc-9 was a white foamy solid (0.8 g). 1 HNMR(400MHz,d-DMSO)δ:ppm.7.63(s,1H),7.42(s,3H),7.17(s,3H),5.85(d,3H),5.60(t,3H),5.10(dd,3H),4.63(td,6H),4.49(m,6H ),4.34(d,3H),4.16(m,6H),4.03(d,3H),3.84(d,6H),3.73(d,6H),3.35(m,2H),2.91(d,6H),2.70-2.40(m,42H),2.21-1.76(m,24H). MS(ESI-TOF): m / z(M+H) + 1917.93, (M+Na) + 1938.69.

[0243] Example 10. Preparation of GalNAc-10 Synthetic Route Compound 28 of this example was prepared similarly to the synthesis of compound 24 described in Example 6.

[0244] Steps (1) Amino compound 32 (3 g) was dissolved in dichloromethane (30 mL), then DIEA (1 mL) and compound 28 (1.31 g) were added to the reaction mixture, and the mixture was stirred at room temperature until the reaction was completed. After the reaction was completed, dichloromethane was added to the reaction mixture, and the reaction solution was concentrated to obtain a crude product. The crude product was purified by column separation (dichloromethane:methanol 10-15%) to obtain compound 38. (2) Compounds 39 and GalNAc-10 were prepared using the preparation methods of compound 27 and GalNAc-6 in Example 6, respectively.

[0245] result The resulting GalNAc-10 was a white foamy solid (0.45 g). 1 HNMR (400MHz, CDCl 3 )δ:ppm 7.7.06-6.40(m,7H),5.35(d,3H),5.28(m,3H),4.67(t,3H),4.14(m,6H),4.02(m,3H),3.93(m,6H),3.61(t,6H) ),3.48(dt,3H),3.29(m,14H),2.67(t,2H),2.43(m,6H),2.28(t,2H),2.15-1.80(s,36H),1.80-1.26(m,40H). MS(ESI-TOF): m / z(M+H) + 2015.11, (M+Na) + 2038.47.

[0246] Example 11. Preparation of GalNAc-11 Synthetic Route TIFF0007618778000103.tif226170 Compound GalNAc-4 was the same as that described in Example 4.

[0247] Steps (1) GalNAc-4 (3 g) was dissolved in dichloromethane (30 mL), then DIEA (0.3 mL) and 1-hydroxyhexanoic acid (0.18 g) were added to the reaction mixture, and the mixture was stirred at room temperature until the reaction was completed. After the reaction was completed, dichloromethane was added to the reaction mixture, which was then concentrated to obtain a crude product. The crude product was purified by column separation (dichloromethane:methanol 10-15%) to obtain compound 40.

[0248] (2) Compound 40 (1 g) was dissolved in anhydrous dichloromethane (10 mL), and then the reaction mixture was cooled to 0° C., followed by the addition of DIEA (0.2 mL) and phosphorus oxychloride (0.14 g). The reaction was stirred at 0° C. until the reaction was complete. After the reaction was completed, dichloromethane was added to the reaction mixture, which was then dried and concentrated to obtain the crude product. The crude product was purified by column separation (dichloromethane:methanol 10-15%) to obtain GalNAc-11.

[0249] result Compound 40: 1 HNMR(400MHz,CDCl3)δ:ppm.7.71-6.38(m,9H),5.36(d,3H),5.27(m,3H),4.68(d,3H),4.53(s,2H),4.31(s,2H),4.15(m,6H),4.01(dd,3H) ),3.89(ddd,6H),3.65(dd,8H),3.46(m,3H),3.24(m,14H),2.46(d,6H),2.31(d,2H),2.23(s,2H),2.16-1.80(m,36H),1.80-1.26(m,34H).

[0250] MS(ESI-TOF):m / z(MH) - 1929.81. The resulting GalNAc-11 was a white foamy solid (0.5 g). MS (ESI-TOF): m / z (M+Na) + 2153.62.

[0251] Example 12. Preparation of GalNAc-12 Synthetic Route TIFF0007618778000104.tif216170 Compound 42 was purchased from Alading, and compound GalNAc-5 was the same as that described in Example 5.

[0252] Steps (1) GalNAc-5 (e.g. 2 g) was dissolved in dichloromethane (about 20 mL), then DIEA (e.g. 0.3 mL), compound 42 (e.g. 0.44 g) were added to the reaction, and the mixture was stirred at room temperature until the reaction was almost complete. After the reaction was completed, dichloromethane was added to the reaction mixture, which was then dried and concentrated to obtain the crude product. The crude product was purified by column separation (appropriate gradient of dichloromethane:methanol, such as 10%-15% methanol) to obtain compound 41.

[0253] (2) Compound 41 (e.g., 1 g) was dissolved in anhydrous dichloromethane (about 10 mL), then the reaction mixture was cooled to 0° C., followed by the addition of DIEA (about 0.2 mL) and phosphorus oxychloride (about 0.12 g). The reaction was stirred at 0° C. until the reaction was complete. After completion of the reaction, dichloromethane was added to the reaction mixture, which was then dried and concentrated to obtain the crude product. The crude product was purified by column separation (appropriate gradient of dichloromethane:methanol, such as 10%-15% methanol) to obtain GalNAc-12.

[0254] Example 13. Preparation of modified single-stranded oligonucleotides (antisense strands) The sequence discussed in Examples 13, 16 to 18 is as follows: 5'-Cy5-mUmgmAfCmAmAmAfCmgmgmgfCmAmAfCmAfUmAfC-3' (SEQ ID NO: 1). In this example, the modified oligonucleotide was synthesized on a 1 μmol scale. The experiment was carried out as follows.

[0255] (1) 1 μmol of standard solid support controlled pore glass (CPG) or 3′-cholesterol modified CPG solid support (purchased from Chemgenes) or 3′-amino modified CPG solid support (purchased from Kinovite) was weighed out. 2′-O-TBDMS protected RNA phosphoramidite monomer, DNA monomer, 2′-methoxy monomer, 2′-fluoro monomer (purchased from Sigma Aldrich), and amino-C16-12-phosphoramidite (or phosphoramidite of fluorescent compound) used for the synthesis of 5′-modification were dissolved in anhydrous acetonitrile to prepare a solution with a concentration of 0.2 M.

[0256] For phosphate-backbone thio-modified oligonucleotides, 0.2M PADS solution was used as thio reagent, 0.25M solution of 5-ethylthio-1H-tetrazole (purchased from Chemgenes) in acetonitrile was prepared as activating agent, 0.02M iodine in pyridine / water solution was used as oxidizing agent, and 3% trichloroacetic acid in dichloromethane was used as deprotecting reagent, which were placed in the corresponding reagent designated position of the DNA / RNA automatic synthesizer (GE AKTA™ OP100).

[0257] (2) The base sequence of the designated oligonucleotide was input into the synthesis program, and the cycle of oligonucleotide synthesis was started. Each step of coupling took 6 minutes, and each step of galactose ligand monomer coupling took 10-20 minutes. After the automatic cycle, the solid-phase synthesis of the oligonucleotide was completed.

[0258] (3) The CPG was dried with dry nitrogen and transferred to a 5 mL EP tube. Aqueous ammonia / aqueous ethanol solution (3 / 1) (2 mL) was added to the EP tube and then heated at 55°C for 16-18 hours. The reaction solution was centrifuged at 10,000 rpm for 10 minutes. The supernatant was removed, and the aqueous ammonia / ethanol was dried under reduced pressure to obtain a white gel-like solid. This solid was dissolved in a TBAF solution (1 M in THF, 200 μL) and shaken at room temperature for 20 hours.

[0259] Tris-HCl buffer (pH 7.4, 1 M, 0.5 mL) was added to the reaction mixture. The mixture was shaken at room temperature for 15 min and centrifuged to 1 / 2 of the original volume. The reaction mixture was extracted twice with chloroform (0.5 mL) and treated with TEAA sample solution (1 mL, 0.1 M) to obtain a mixed solution, which was then injected into a solid-phase extraction column to remove excess salt from the solution.

[0260] (4) The concentration of the obtained oligonucleotide was measured by a micro-ultraviolet spectrophotometer (KO5500). Detection and analysis by oligonucleotide mass spectrometry were completed by an Oligo HTCS LC-MS system (Novatia). After the primary scan, the molecular weight was calculated by the normalization method using Promass software.

[0261] Example 14. Preparation of modified single-stranded oligonucleotides (sense strands) The sequence discussed in Examples 14 to 18 is as follows: 5'-mgfUmAfUmgfUfUmgfCfCfCmgfUfUfUmgfUfCmA-3' (SEQ ID NO: 2). In this example, the modified oligonucleotide was synthesized on a 1 μmol scale. The experiment was carried out as follows.

[0262] (1) 1 μmol of standard solid support controlled pore glass (CPG) or 3′-cholesterol-modified CPG solid support (purchased from Chemgenes), or 3′-amino-modified CPG solid support (purchased from Kinovite), DNA monomer, 2′-methoxy monomer, 2′-fluoro monomer (purchased from Sigma Aldrich), and amino-C16-12-phosphoramidite (or phosphoramidite of fluorescent compound) used in the synthesis of 5′-modification was dissolved in anhydrous acetonitrile to prepare a solution with a concentration of 0.2 M.

[0263] For phosphate-backbone thio-modified oligonucleotides, 0.2M PADS solution was used as thio reagent, 0.25M solution of 5-ethylthio-1H-tetrazole (purchased from Chemgenes) in acetonitrile was prepared as activating agent, 0.02M iodine in pyridine / water solution was used as oxidizing agent, and 3% trichloroacetic acid in dichloromethane was used as deprotecting agent, and placed in the corresponding reagent designated position on the DNA / RNA automatic synthesizer (GE AKTA™ OP100).

[0264] (2) The base sequence of the designated oligonucleotide was input into the synthesis program, and the cycle of oligonucleotide synthesis was started. Each step of coupling took 6 minutes, and each step of galactose ligand monomer coupling took 6-10 minutes. After the automatic cycle, the solid-phase synthesis of the oligonucleotide was completed.

[0265] (3) The CPG was dried with dry nitrogen and transferred to a 5 mL EP tube. Aqueous ammonia solution (2 mL) was added to the EP tube, and then heated at 55°C for 16 to 18 hours. The reaction solution was centrifuged at 10,000 rpm for 10 minutes. The supernatant was taken, and the ammonia water / ethanol was dried under reduced pressure to obtain a white or yellow gel-like solid. The TEAA sample solution (1 mL, 0.1 M) was added to this solid to obtain a mixed solution, which was then injected into a solid-phase extraction column to remove excess salts from the solution.

[0266] (4) The concentration of the obtained oligonucleotide was measured by a micro-ultraviolet spectrophotometer (KO5500). Detection and analysis by oligonucleotide mass spectrometry were completed by an Oligo HTCS LC-MS system (Novatia). After the primary scan, the molecular weight was calculated by the normalization method using Promass software.

[0267] Example 15. Preparation of GalNAc modified oligonucleotides. The amino-modified oligonucleotide (sense strand) prepared in Example 14 was dissolved in a buffer solution. To this amino-modified oligonucleotide solution, various GalNAc-pentafluorophenol esters (GalNAc selected from GalNAc-1 to GalNAc-10) dissolved in acetonitrile solvent were added, mixed uniformly, and reacted at room temperature for at least 3 hours.

[0268] After completion of the reaction, the acetyl protecting groups were removed and the reaction was purified by ion exchange chromatography (WATERS) using a linear gradient on a DNAPAc PA-100 ion exchange column, where mobile phase A was 20 nM NaOH and mobile phase B was a mixture of 20 nM NaOH + 2 M NaCl.

[0269] [Table 1]

[0270] The sequences of exemplary modified oligonucleotides and the corresponding molecular weights (MW) are shown in Table 1.

[0271] Example 16. Preparation of double-stranded oligonucleotides The experiment was carried out as follows: the 5'-Cy5-antisense strand oligonucleotide synthesized in Example 13 and the GalNAc-1-10 sense strand oligonucleotide prepared in Example 15 were mixed according to 1:1 UV absorption content, respectively. The mixture was incubated in a hot water bath at 95°C for 3 minutes, and then cooled to room temperature to form a double-stranded oligonucleotide.

[0272] [Table 2]

[0273] Example 17. Detection of the cell targeting ability of modified oligonucleotides. Modified oligonucleotides for animal studies were filtered through a 0.22 μm membrane before injection.

[0274] 1. Mouse Primary Hepatocyte Isolation Mice purchased from Beijing Weitong Lihua Laboratory Animal Co., Ltd. were anesthetized. The skin and muscle layers of the mice were incised to expose the liver. A perfusion catheter was inserted into the portal vein and a small opening was made in the inferior vena cava to prepare the liver for perfusion. Perfusion solution I (Hank's, 0.5 nM EGTA, pH 8) and perfusion solution II (low glucose DMEM, 100 U / mL type IV, pH 7.4) were preheated at 40°C.

[0275] The liver was perfused with perfusion solution I (37°C) along the portal vein catheter at a flow rate of 7 mL / min for 5 min until the liver turned grayish white. The liver was then perfused with perfusion solution II at 37°C at a flow rate of 7 mL / min for 7 min. After perfusion, the liver was removed and placed in solution III (10% FBS, low glucose DMEM, 4°C) to stop digestion. The liver capsule was cut with tweezers and gently shaken to release the hepatocytes. The hepatocytes were filtered through a 70 μm cell strainer and then centrifuged at 50 g for 2 min. After centrifugation, the supernatant was discarded.

[0276] The hepatocytes were then resuspended in solution IV (40% Percoll-low glucose DMEM, 4°C) and centrifuged at 100g for 2 min. The supernatant was discarded and 2% FBS-low glucose DMEM was added to resuspend the cells for subsequent experiments. Trypan blue staining was used to identify cell viability.

[0277] 2. Measurement of GalNAc binding curve and Kd value Freshly isolated mouse primary hepatocytes were cultured at 2 × 10 4 Cells / well (100 μL / well) were plated in a 96-well plate. A different GalNAc-siRNA was added to each well (see Table 2). The final concentration of each GalNAc-siRNA was 0.9 nM, 2.7 nM, 8.3 nM, 25 nM, 50 nM, or 100 nM. Mouse hepatocytes were incubated with GalNAc-siRNA at 4° C. for 2 hours and then centrifuged at 50 g for 2 minutes. The supernatant was discarded.

[0278] Hepatocytes were then resuspended in 10 μg / mL propidium iodide (PI), stained for 10 min, and centrifuged at 50 g for 2 min. Hepatocytes were then washed with cold PBS and centrifuged at 50 g for 2 min. The supernatant was discarded and hepatocytes were resuspended in PBS. Mean fluorescence intensity (MFI) of live cells was measured by flow cytometry (Beckman). GraphPad Prism5 software was used for nonlinear fitting and the dissociation constant K d The values ​​were calculated.

[0279] The results are shown in Table 3 below, indicating that GalNAc-siRNA can specifically target hepatocytes. The Kd values ​​of the tested GalNAc ligands binding to the cellular receptors ranged from 1.5 to 27.6 nM.

[0280] The inhibition constant Ki was calculated based on the function Ki=IC50 / (1+[S] / Km), where IC50 is the 50% inhibitory concentration, [S] is the substrate concentration, and Km is the Michaelis constant. Compared to the preferred galactose ligands disclosed in International Application PCT / US2014 / 046425 (Ki values ​​were determined to be 5.2-51.3 nM, see Example 44), the GalNAc ligands described herein showed higher binding affinity.

[0281] Furthermore, GalNAc-siRNAs with different ligand structures inhibited the binding ability of different receptors, for example, structures 101, 104, 108 and 109 showed relatively strong receptor-binding affinity (the smaller the Kd value, the higher the affinity).

[0282] [Table 3]

[0283] Example 18. In vivo liver targeting experiments Thirty 6-7 week-old SPF (specific-pathogen-free) grade male Balb / c-nu mice (purchased from Beijing Weitong Lihua Laboratory Animal Co., Ltd.) were used. The mice were randomly divided into six groups: blank control group, negative control group (or NC1, not conjugated to ligand), test group 1, test group 2, test group 3 and test group 4. Each group had five mice. The mice were administered a dose of approximately 10 mg / kg via tail vein injection (see Table 4 for the experimental design).

[0284] All animals underwent in vivo imaging, including white light imaging, before dosing and 15 min, 30 min, 1 h, 2 h, 4 h, and 6 h after dosing. Animals were euthanized 6 h after dosing. Brains, salivary glands, heart, spleen, lungs, liver, kidneys, and intestines were removed for in vitro organ imaging (Bruker Xtreme).

[0285] [Table 4]

[0286] The in vitro imaging results showed that sample numbers 100, 104, 107, 108, and 110 were mainly distributed in the liver, kidney, and gastrointestinal tract, and less distributed in tissues such as the brain, heart, lung, and spleen.

[0287] According to the statistical results of the average total photon number, compared with group 100 (negative control group), sample numbers 104, 107, 108 and 110 showed a certain liver targeting effect. Moreover, sample numbers 107 and 108 showed a statistically significant difference (P<0.001). Sample number 104 (P<0.01) and sample number 110 (P<0.05) both showed a statistically significant difference.

[0288] [Table 5]

Claims

1. A compound having the chemical formula (I) or a pharma- ceutically acceptable salt thereof: In the formula, each R Ｘ is, independently, · H; -CH ２ OR Ｘ２ (Here, R Ｘ２ is H, C １－６ alkyl, or hydroxyl protecting groups; and is selected from the group consisting of However, at least one R Ｘ is a group of formula (A1), Each R １ is an independently selected moiety capable of binding to the asialoglycoprotein receptor (ASGPR); Each R ２ are independently -C(R ６ ) ２ -, -OC(R ６ ) ２ C(R ６ ) ２ O-, -C(R ６ )(OH)-C(R ６ ) (OH)-, ＊ -C(=O)NR ７ --, ＊ -NR ７ C(=O)-, C ６－１０ Arylene, C ２－６ Alkenylene, and C ２－６ alkynylene, Here, the C ６－１０ Arylene, C ２－６ Alkenylene, and C ２－６ Each alkynylene is selected from 1 to 4 independently selected R ａ is optionally substituted with represents the attachment point to R ３ teeth, (Here, aa is represents the point of attachment to ・-(CR ６ R ６ ) ｘ -O-(CR ６ R ６ ) ｙ - (wherein x and y are independently 0, 1, 2, or 3); and -L ３ -L ３Ｃ - (where L ３Ｃ is C ３－１０ Cycloalkylene, C ６－１０ arylene, 5- to 10-membered heteroarylene, and 4- to 10-membered heterocyclylene, each of which is selected from 1 to 4 independently selected R ａ and optionally substituted with L ３ HA -C(R ６ ) ２ - and R ４ is -C(R ６ ) ２ -, -OC(R ６ ) ２ C(R ６ ) ２ O-, -C(R ６ )(OH)-C(R ６ ) (OH)-, ＊ -C(=O)NR ７ --, ＊ -NR ７ C(=O)-, C ６－１０ Arylene, C ３－１０ selected from the group consisting of cycloalkylene, 5- to 10-membered heteroarylene, and 4- to 10-membered heterocyclylene; Here, C ６－１０ Arylene, C ３－１０ Cycloalkylene, 5- to 10-membered heteroarylene, and 4- to 10-membered heterocyclylene each represent 1 to 4 independently selected R ａ optionally replaced by The above * is represents the attachment point to R ５ teeth, Hydroxyl; C(O)OH; (Here, Pg is wherein Ring D is a 5-10 membered heteroaryl or a 4-10 membered heterocyclyl, each of which is optionally substituted with 1 to 6 substituents, each of which is independently selected from the group consisting of halogen, oxygen, NO 2 , C(O)C 1-4 alkyl, C(O)OC 1-4 alkyl, S(O)C 1-4 alkyl, C 1-6 alkyl, C 1-6 haloalkyl, and —OH. (wherein Z is a hydroxyl protecting group); (Here, Pg ２ is a hydroxyl protecting group; and where L is a bond or a divalent group selected from the group consisting of: ○-R ２ -、-R ３ -、-R ４ -、-O-、-C(=O)-、-C(=O)O-、-OC(=O)-、 (Here, Pg ３ is H or Pg ２ (It is) (Here, Z ２ is H or a hydroxyl protecting group. wherein bb represents the point of attachment to Oligo, and Oligo is an oligonucleotide; Each R ６ are independently H, C １－３ Alkyl, C １－３ selected from the group consisting of haloalkyl, and halogen; Each R ７ are independently H and C １－３ is selected from the group consisting of alkyl, each a and b is independently selected from an integer from 1 to 10; each c and d is independently selected from an integer from 0 to 10; Each R ａ are independently halogen, cyano, C １－６ Alkyl, C １－６ Haloalkyl, C １－６ Alkoxy, and C １－６ A compound having the formula (I): or a pharma- ceutically acceptable salt thereof, selected from the group consisting of haloalkoxy.

2. At least two R Ｘ are each independently a group of formula (A1), Preferably, each R Ｘ The compound of claim 1 , wherein:

3. Each R １ are independently a group of formula (B1) or (B2), During the ceremony, R Ｄ is R Ｃ and is selected from the group consisting of Each R Ｂ are independently -NR Ｅ R Ｆ and -OR Ｃ is selected from the group consisting of Each R Ｃ are independently H and -C(=O)C １－６ is selected from the group consisting of alkyl, Each R Ｅ are independently C(=O)C １－６ is alkyl, Each R Ｆ are independently H and is selected from the group consisting of R Ｇ is C １－６ is alkyl, Each q is independently selected from an integer from 1 to 10; Preferably, each R １ are independently a group having the chemical formula (B1-a), (B1-b) or (B2-a); Preferably, each R Ｂ became independent and became NR Ｅ R Ｆ That is, Preferably, each R Ｅ are independently -C(=O)C １－６ is alkyl, More preferably, each R Ｅ -C(=O)CH ３ That is, Preferably, each R Ｆ is H, Preferably, each R Ｆ but That is, Preferably, each R Ｂ is NHC(=O)CH ３ That is, Preferably, each R Ｂ but That is, Preferably, each R Ｂ are independent -OR Ｃ That is, Preferably, each R Ｃ are independently C(=O)C １－６ is alkyl, More preferably, each R Ｃ is C(=O)CH ３ That is, More preferably, each R Ｃ is H, More preferably, each R Ｇ CH ３ 3. The compound according to claim 1 or 2,

4. Each R １ The compound according to claim 1 or 2, wherein is selected from the following group:

5. Each R １ are the same, Preferably, each R １ but That is, Preferably, each R １ but That is, Preferably, each R ２ Independent ＊ -C(=O)NR ７ -or- ＊ -NR ７ C(=O)-; More preferably, each R ２ Independent ＊ -C(=O)NR ７ - is, Preferably, each R ２ but ＊ -C(=O)NH-; Preferably, each R ２ is independently -C(R ６ ) ２ - is, Preferably, each R ２ Ga-CH ２ - is, More preferably, each R ２ are the same, Preferably, each a is an integer independently selected from 1 to 4. More preferably, each a is independently 2 or 3. Preferably, each a is the same. Preferably, each b is an integer independently selected from 1 to 4. More preferably, each b is independently 2 or 3. Preferably, the compound according to claim 1 or 2, wherein each b is the same.

6. Each a is the same, each b is the same, and each R ２ are the same, Preferably, a is an integer selected from 1 to 4, b is an integer selected from 1 to 4, and R ２ but ＊ -C(=O)NR ７ - is, More preferably, a is 3, b is 3, and R ２ but ＊ -C(=O)NH; Preferably, a is an integer selected from 1 to 4, b is an integer selected from 1 to 4, and R ２ -C(R ６ ) ２ - is, More preferably, R ２ Ga-CH ２ - and 3≦(a+b)≦5.

7. R ３ but or That is, Preferably, L ３ -C(R ６ ) ２ - is, Preferably, L ３ Ga-CH ２ - is, Preferably, c is an integer from 1 to 2. Preferably, c is an integer from 2 to 5. Preferably, c is an integer from 3 to 7. Preferably, R ４ but ＊ -C(=O)NR ７ - is, Preferably, R ４ but ＊ -C(=O)NH-; Preferably, R ４ -C(R ６ ) ２ - is, Preferably, R ４ Ga-CH ２ - is, Preferably, d is an integer from 1 to 2. Preferably, the compound according to claim 1 or 2, wherein d is an integer from 3 to 7.

8. R ４ -C(R ６ ) ２ - and c and d are each independently 1 or 2; Preferably, R ４ Ga-CH ２ - and c and d are each 1; Preferably, R ４ -C(R ６ ) ２ - and 4≦(c+d)≦12; Preferably, R ４ Ga-CH ２ - and 7≦(c+d)≦10; Preferably, R ４ but ＊ -C(=O)NR ７ - and 5≦(c+d)≦10; Preferably, R ４ but ＊ -C(=O)NR ７ - and 7≦(c+d)≦9; Preferably, the compound according to claim 1 or 2, wherein c is 3.

9. R ５ is C(O)OH or That is, Preferably, R ５ but That is, Preferably, Pg is or which is optionally substituted with 1 to 6 substituents, each of which is independently selected from halogen, NO ２ , C(O)C １－４ Alkyl, C(O)OC １－４ Alkyl, S(O)C １－４ Alkyl, C １－６ Alkyl, C １－６ selected from the group consisting of haloalkyl, and -OH; Preferably, Pg is C ６－１０ aryl or 5-10 membered heteroaryl substituted with 1 to 6 substituents, each of which is independently -F, -Cl, -NO ２ , C(O)C １－４ Alkyl, C(O)OC １－４ Alkyl, and S(O)C １－４ selected from the group consisting of alkyl, Preferably, Pg is where p is an integer from 1 to 5, and each R ｐ is -F, -Cl, or -NO ２ That is, Preferably, each R ｐ is -F, Preferably, Pg is That is, Preferably, R ５ but That is, Preferably, R ５ but That is, Preferably, R ５ but 3. The compound according to claim 1 or 2,

10. each hydroxyl protecting group is independently selected from the group consisting of silyl protecting groups, 4-monomethoxytrityl (MMTR), 4,4'-dimethoxytrityl (DMTR), and trityl; 3. The compound according to claim 1 or 2, wherein the silyl protecting group is preferably selected from the group consisting of tert-butyldimethylsilyl (TBMDS), tert-butyldiphenylsilyl (TBDPS) and triisopropylsilyl (TIPS).

11. The compound of claim 1, wherein the compound is selected from the group consisting of GalNAc-1 to GalNAc-12 compounds.

12. R ５ but where Oligo is an oligonucleotide linked to L via the 5' end, 3' end or middle of the sequence of any strand by a phosphoester bond; Preferably, L is a bond. Preferably, L is C(=O) or -O-. Preferably, L is That is, Preferably, L is Selected from the group consisting of: Preferably, the phosphate bond is a phosphodiester bond or a modified phosphate bond. Preferably, the modified phosphate bond is selected from one or more of a thio-modified phosphate bond and an amino-modified phosphate bond.

3. The compound according to claim 1 or 2, wherein the thio-modified phosphate bond is preferably a phosphorothioate bond.

13. The oligonucleotide comprises a single stranded oligonucleotide and / or a double stranded oligonucleotide; The compound according to claim 1 or 2, wherein the oligonucleotide is selected from the group consisting of DNA, siRNA, miRNA, pre-miRNA, antagomir, mRNA, antisense oligonucleotide (ASO), aptamer, crRNA, tracRNA and sgRNA.

14. the oligonucleotide comprises unmodified and / or modified nucleotides, Preferably, each modified nucleotide is independently selected from the group consisting of 2'-O-(2-methoxyethyl) modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-allyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-deoxy modified nucleotides, 2'-hydroxyl modified nucleotides, locked nucleotide (LNA) modified nucleotides, glycerol nucleic acid (GNA) modified nucleotides and non-locked nucleic acid (UNA) modified nucleotides. More preferably, the compound according to claim 1 or 2, wherein the 2'-O-alkyl modified nucleotide is a 2'-O-methyl nucleotide.

15. the oligonucleotide comprises a modification group, the modification group being selected from the group consisting of cholesterol, polyethylene glycol, a fluorescent probe, biotin, a polypeptide, a vitamin, a tissue targeting molecule, and combinations thereof; Preferably, the modifying group is a terminal modifying group. Preferably, the oligonucleotide comprises one or more polypeptide nucleic acids and / or morpholino nucleic acids. Preferably, the oligonucleotide is an oligonucleotide comprising 5 to 100 base pairs.

3. A compound according to claim 1 or 2.

16. A method for treating and / or preventing a disorder or disease in a subject, the disorder or disease being caused by the expression of one or more genes in liver cells, the method comprising administering to the subject (which is not a human subject) a compound according to any one of claims 12 to 15, or a pharmaceutical composition comprising a compound according to any one of claims 12 to 15 and a pharma- ceutical acceptable excipient.

17. A method of detecting or localizing RNA in the liver of a subject, the method comprising administering to the subject (which is not a human subject) a compound according to any one of claims 12 to 15, or a pharmaceutical composition comprising a compound according to any one of claims 12 to 15 and a pharma- ceutical acceptable excipient.

18. The one or more genes are selected from the group consisting of HBV genome, HCV genome, PCSK9, xanthine oxidase expression genes (e.g., XDH), URAT1, APOB, hepatic fibrosis-related genes, nonalcoholic fatty liver-related genes (e.g., PNPLA3, FDFT1), and primary biliary cirrhosis-related genes; Preferably, the disease or disorder is selected from the group consisting of hereditary angioedema, familial tyrosinemia type I, Alagille syndrome, alpha-1-antitrypsin deficiency, bile acid synthesis and metabolism disorders, biliary atresia, cystic fibrosis liver disease, idiopathic neonatal hepatitis, mitochondrial liver disease, progressive familial intrahepatic cholestasis, primary sclerosing cholangitis, transthyretin amyloidosis, hemophilia, homozygous familial hypercholesterolemia, hyperlipidemia, hepatitis B (HBV), hepatitis C (HCV), steatohepatitis, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), diseases associated with abnormally increased hepatic glucose production resembling hyperglycemia or type 2 diabetes, hepatitis and hepatic porphyria. Preferably, the compound or pharmaceutical composition is administered intravenously, intramuscularly, subcutaneously, via a microneedle patch, orally, via an intrabuccal or intranasal spray, or topically. Preferably, the subject is a mammal.

17. The method of claim 16, wherein the subject is selected from bovine, equine, ovine, porcine, canine, feline, rodent, and primate animals.

19. A pharmaceutical composition comprising a compound according to any one of claims 12 to 15 and a pharma- ceutically acceptable excipient.

20. The pharmaceutical composition is formulated into a dosage form selected from the group consisting of a powder, a tablet, a granule, a capsule, a solution, an emulsion, a suspension, an injection, a spray, an aerosol, a dry powder inhalant, or a microneedle patch; Preferably, the pharmaceutical composition is suitable for administration intravenously, intramuscularly, subcutaneously, via a microneedle patch, orally, via an intrabuccal or intranasal spray, or topically to a subject in need thereof. Preferably, the subject is a mammal. Preferably, the mammal is selected from bovine, equine, ovine, porcine, canine, feline, rodent, and primate. More preferably, the subject is a human.

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