Targeted nucleic acid conjugate compositions

Abstract

To provide compositions and methods that can be used to deliver (e.g. guide) therapeutic nucleic acids in living subjects.SOLUTION: The present invention discloses conjugates that comprise a targeting moiety, a nucleic acid, and arbitrary linking groups, as well as synthetic intermediates and synthetic methods useful for preparing the conjugates.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims priority to U.S. Provisional Patent Application No. 62 / 321,034, filed April 11, 2016, U.S. Provisional Patent Application No. 62 / 417,156, filed November 3, 2016, and U.S. Provisional Patent Application No. 62 / 438,310, filed December 22, 2016, which applications are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. Said ASCII copy, created on April 7, 2017, is named 08155_056WOl_SL.txt and is 7,824 bytes in size. [Background technology]

[0003] Many diseases are liver specific (e.g., Hepatitis B and nonalcoholic steatohepatitis (NASH)). Therefore, it would be beneficial to have a therapeutic composition that can primarily target the liver, kidney, heart, pancreas or other organs of a living subject.

[0004] Nucleic acids, including siRNAs, are useful as therapeutic agents. Summary of the Invention [Problem to be solved by the invention]

[0005] Currently, there is a need for compositions and methods that can be used to deliver (eg, direct) therapeutic nucleic acids in living subjects. [Means for solving the problem]

[0006] The present invention provides compounds, compositions and methods that can be used to target therapeutic nucleic acids (eg, to the liver).

[0007] Thus, in one aspect, the present invention provides a compound of formula I: [ka] During the ceremony, R 1 is the targeting ligand; L 1 is absent or is a linking group, L 2 is absent or is a linking group, R 2 is a nucleic acid, Ring A is absent, 3- to 20-membered cycloalkyl, 5- to 20-membered aryl, 5- to 20-membered heteroaryl or 3- to 20-membered heterocycloalkyl; Each R A are independently hydrogen, hydroxy, CN, F, Cl, Br, I, -C 1-2 Alkyl-OR B , C 1-10 Alkyl C 2-10 Alkenyl and C 2-10 alkynyl, wherein C 1-10 Alkyl C 2-10 Alkenyl and C 2-10 Alkynyl is optionally selected from halo, hydroxy and C 1-3 substituted with one or more groups independently selected from alkoxy; R B is hydrogen, a protecting group, a covalent bond to a solid support, or a bond to a linking group attached to a solid support; Compounds in which n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; or a salt thereof.

[0008] Diseases and conditions that can be treated with the siRNA molecules (including combinations of siRNA molecules) include liver diseases, such as liver diseases associated with or caused by fat accumulation (such as hepatic steatosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), fatty liver transplantation, hepatitis (e.g., type B or type D) and / or acute liver failure (including fulminant and subfulminant hepatic failure), and hypertriglyceridemia).

[0009] The present invention also provides synthetic intermediates and processes disclosed herein that are useful in the preparation of compounds of formula I.

[0010] Other objects, features and advantages of the present invention will become apparent to those skilled in the art from the following detailed description and drawings. [Brief description of the drawings]

[0011] [Figure 1] 1 shows an intermediate compound of formula Ie in which the targeting ligand / linker is attached to a solid support and Pg1 is the protecting group DMTr. [Diagram 2] A representative compound of formula Id is shown in which a targeting ligand is attached to a solid support along with a covalently attached nucleic acid. [Diagram 3] A representative compound of formula Id is shown in which the targeting ligand-nucleic acid conjugate is cleaved from the solid support and deprotected to provide a compound of formula I. [Figure 4] 1 shows a TTR mRNA gene knockdown experiment following transfection of various doses of GalNAc conjugates into C57 primary hepatocytes for 24 hours. [Diagram 5] Shown is knockdown of TTR protein following a single subcutaneous administration of 1 mg / kg TTR siRNA-GalNAc conjugate to C57 mice (n=4). [Figure 6]Shows knockdown of TTR mRNA in the liver following a single 1 mg / kg subcutaneous administration of TTR siRNA-GalNAc conjugate to C57 mice (n=4). [Figure 7] Data obtained in Example 25 for representative compounds are shown. [Figure 8] Data obtained in Example 25 for representative compounds are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0013] The term "small interfering RNA" or "siRNA" as used herein refers to a double-stranded RNA (i.e., duplex RNA) that can reduce or inhibit expression of a target gene or sequence (e.g., by mediating degradation of an mRNA that is complementary to the siRNA sequence or by inhibiting translation of that mRNA) when the siRNA is in the same cell as the target gene or sequence. The siRNA may have substantial or complete identity to the target gene or sequence, or may contain a mismatch region (i.e., a mismatch motif). In certain embodiments, the siRNA may be about 19-25 (duplex) nucleotides in length, preferably about 20-24, 21-22, or 21-23 (duplex) nucleotides in length. The siRNA duplex may include a 3' overhang of about 1 to about 4 nucleotides or about 2 to about 3 nucleotides and a 5' phosphate terminus. Examples of siRNA include, but are not limited to, double-stranded polynucleotide molecules assembled from two separate stranded molecules, one strand being the sense strand and the other being the complementary antisense strand.

[0014] In certain embodiments, the 5' and / or 3' overhangs of one or both strands of the siRNA comprise from 1 to 4 (e.g., 1, 2, 3, or 4) modified and / or unmodified deoxythymidine (t or dT) nucleotides, from 1 to 4 (e.g., 1, 2, 3, or 4) modified (e.g., 2'OMe) and / or unmodified uridine (U) ribonucleotides, and / or from 1 to 4 (e.g., 1, 2, 3, or 4) modified (e.g., 2'OMe) and / or unmodified ribonucleotides or deoxyribonucleotides that are complementary to a target sequence (e.g., the 3' overhang of the antisense strand) or to its complementary strand (e.g., the 3' overhang of the sense strand).

[0015] Preferably, the siRNA is chemically synthesized. siRNA can also be generated by cleaving a dsRNA longer than the siRNA (e.g., a dsRNA longer than about 25 nucleotides) with E. coli RNAase III or Dicer. These enzymes process dsRNA into biologically active siRNAs (see, e.g., Yang et al., Proc. Natl. Acad. Sci. USA, 99:9942-9947 (2002); Calegari et al., Proc. Natl. Acad. Sci. USA, 99:14236 (2002); Byrom et al., Ambion TechNotes, 10(1):4-6 (2003); Kawasaki et al., Nucleic Acids Res., 31:981-987 (2003); Knight et al., Science, 293:2269-2271 (2001); and Robertson et al., J. Biol. Chem., 243:82 (1968)). Preferably, dsRNA is at least 50 nucleotides long, about 100 nucleotides long, 200 nucleotides long, 300 nucleotides long, 400 nucleotides long or 500 nucleotides long.dsRNA can be as long as 1000 nucleotides long, 1500 nucleotides long, 2000 nucleotides long, 5000 nucleotides long or longer.dsRNA can code for whole gene transcript or partial gene transcript.In some cases, siRNA can be encoded by plasmid (e.g., transcribed as sequence that automatically folds into double strand with hairpin loop).

[0016] The phrase "inhibits the expression of a target gene" refers to the ability of the siRNA of the present invention to silence, reduce or inhibit the expression of a target gene (e.g., the expression of DGAT2 and, optionally, the expression of ANGPTL3). To determine the degree of gene silencing, a test sample (e.g., a biological sample obtained from an organism of interest expressing the target gene, or a sample of cultured cells expressing the target gene) is contacted with an siRNA that silences, reduces or inhibits the expression of the target gene. The expression of the target gene in the test sample is compared with the expression of the target gene in a control sample (e.g., a biological sample obtained from an organism of interest expressing the target gene, or a sample of cultured cells expressing the target gene) that is not contacted with the siRNA. The value of the control sample (e.g., the sample expressing the target gene) may be 100%. In particular embodiments, expression of a target gene is silenced, inhibited or reduced when the value of the test sample relative to a control sample (e.g., buffer only, an siRNA sequence targeting a different gene, a scrambled siRNA sequence, etc.) is about 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or 0%. Suitable assays include, but are not limited to, assays that examine protein or mRNA levels using techniques known to those of skill in the art (e.g., dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, etc.) and phenotypic assays known to those of skill in the art.

[0017] The term "synthetic activating group" refers to a group that can be attached to an atom to activate the atom and allow the atom to form a covalent bond with another reactive group. It is understood that the nature of the synthetic activating group can depend on the atom that is activated. For example, when the synthetic activating group is attached to an oxygen atom, the synthetic activating group is a group that activates the oxygen atom to form a bond (e.g., an ester bond, a carbamate bond, or an ether bond) with another reactive group. Such synthetic activating groups are known. Examples of synthetic activating groups that can be attached to an oxygen atom include, but are not limited to, acetate, succinate, triflate, and mesylate. When the synthetic activating group is attached to an oxygen atom of a carboxylic acid, the synthetic activating group can be a group that is derivable from a known coupling reagent (e.g., a known amide coupling reagent). Such coupling reagents are known. Examples of such coupling reagents include, but are not limited to, N,N'-dicyclohexylcarbodiimide (DCC), hydroxybenzotriazole (HOBt), N-(3-dimethylaminopropyl)-N'-ethyl carbonate (EDC), (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) or O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU).

[0018] An "effective amount" or "therapeutically effective amount" of a therapeutic nucleic acid (such as a siRNA) is an amount sufficient to produce a desired effect, e.g., inhibit expression of a target sequence relative to normal expression levels detected in the absence of the siRNA. In particular embodiments, inhibition of expression of a target gene or target sequence occurs when the value obtained by the siRNA is about 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or 0% compared to a control (e.g., buffer only, an siRNA sequence targeting a different gene, a scrambled siRNA sequence, etc.). Suitable assays for measuring expression of a target gene or sequence include, but are not limited to, assays that examine protein or mRNA levels using techniques known to those of skill in the art (e.g., dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, etc.) and phenotypic assays known to those of skill in the art.

[0019] The term "nucleic acid" as used herein refers to a polymer containing at least two nucleotides (i.e., deoxyribonucleotides or ribonucleotides) in either single- or double-stranded form, including DNA and RNA. A "nucleotide" contains the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate group. "Base" includes purines and pyrimidines, which further include the natural compounds of purines and pyrimidines adenine, thymine, guanine, cytosine, uracil, inosine, natural analogs, and synthetic derivatives, including but not limited to modifications that place new reactive groups, including but not limited to amines, alcohols, thiols, carboxylates, and alkyl halides. Nucleic acids include synthetic, natural, and non-natural nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, and which have similar binding properties as their reference nucleic acid. Examples of such analogs and / or modified residues include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2'-O-methyl ribonucleotides, and peptide nucleic acids (PNAs). In addition, the nucleic acid can contain one or more UNA moieties.

[0020] The term "nucleic acid" includes any oligonucleotide or polynucleotide, with fragments containing up to 60 nucleotides generally referred to as oligonucleotides, and longer fragments referred to as polynucleotides. Deoxyribooligonucleotides are composed of a 5-carbon sugar called deoxyribose covalently linked to phosphate at the 5' and 3' carbons of the sugar to form an alternating, unbranched polymer. DNA can be in the form of, for example, antisense molecules, plasmid DNA, precondensed DNA, PCR products, vectors, expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups. Ribooligonucleotides are composed of similar repeating structures in which the 5-carbon sugar is ribose. RNA can be in the form of, for example, small interfering RNA (siRNA), dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, tRNA, viral RNA (vRNA), and combinations thereof. Thus, in the context of the present invention, the terms "polynucleotide" and "oligonucleotide" refer to a polymer or oligomer of nucleotide or nucleoside monomers that consist of naturally occurring bases, sugars, and intersugar (backbone) linkages. The terms "polynucleotide" and "oligonucleotide" also include polymers or oligomers that contain non-naturally occurring monomers or portions thereof that function similarly. Such modified or substituted oligonucleotides are often preferred over native forms due to properties such as, for example, enhanced cellular uptake, reduced immunogenicity, and increased stability in the presence of nucleases.

[0021] Unless otherwise specified, a particular nucleic acid sequence also implicitly includes its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be made by creating sequences in which the third position of one or more given (or all) codons is replaced with mixed base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)).

[0022] In one embodiment, the nucleic acid can be an siRNA. Suitable siRNAs and methods and intermediates useful for their preparation are reported in WO 2016 / 054421. In one embodiment, the siRNA can be selected from the siRNAs shown in the table below. [Table 1A] [Table 1B]

[0023] The term "gene" refers to a nucleic acid (eg, DNA or RNA) sequence that comprises a partial or full-length coding sequence necessary for the production of a polypeptide or a precursor polypeptide.

[0024] "Gene product," as used herein, refers to a gene product such as an RNA transcript or a polypeptide.

[0025] As used herein, the term “alkyl,” by itself or as part of another substituent, has the number of carbon atoms indicated, unless otherwise stated (i.e., C 1-8means 1 to 8 carbons) straight or branched chain hydrocarbon groups. Examples of alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, t-butyl, iso-butyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. The term "alkenyl" refers to unsaturated alkyl groups having one or more double bonds. Similarly, the term "alkynyl" refers to unsaturated alkyl groups having one or more triple bonds. Examples of such unsaturated alkyl groups include vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1-propynyl, 3-propynyl, 3-butynyl, and higher homologs and isomers.

[0026] The term "alkylene," alone or as part of another substituent, means a divalent group derived from an alkane, including straight-chain and branched alkanes, as exemplified by -CH2CH2CH2CH2 and -CH(CH3)CH2CH2-.

[0027] The terms "cycloalkyl," "carbocyclic," or "carbocycle" refer to a hydrocarbon ring system having a total of 3 to 20 ring atoms (e.g., a 3-20 membered cycloalkyl is a cycloalkyl having 3 to 20 ring atoms, or C 3-20 Cycloalkyl is a cycloalkyl having 3 to 20 carbon ring atoms, 3- to 5-membered cycloalkyls are fully saturated or have only one double bond between the ring vertices, and 6 or more-membered cycloalkyls are fully saturated or have no more than two double bonds between the ring vertices. As used herein, "cycloalkyl", "carbocyclic" or "carbocycle" refers to bicyclic, polycyclic and spirocyclic hydrocarbon ring systems (e.g., bicyclo[2.2.1]heptane, pinane, bicyclo[2.2.2]octane, adamantane, norbornene, spirocyclic C 5-12As used herein, the terms "alkenyl," "alkynyl," "cycloalkyl," "carbocycle," and "carbocyclic" are also intended to include mono- and poly-halogenated variants thereof.

[0028] The terms "heterocycloalkyl", "heterocyclic" or "heterocycle" refer to a saturated or partially unsaturated ring system having a total of 3 to 20 ring atoms (e.g., a 3-20 membered heterocycloalkyl is a heterocycloalkyl group having 3 to 20 ring atoms, C 2-19Heterocycloalkyl refers to a group having 3 to 10 ring atoms, with 2 to 19 ring atoms being carbon (heterocycloalkyl being heterocycloalkyl), containing 1 to 10 heteroatoms selected from N, O and S as ring atoms, the nitrogen and sulfur atoms of which are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. Unless otherwise specified, a "heterocycloalkyl", "heterocyclic" or "heterocycle" ring can be a monocyclic, bicyclic, spirocyclic or polycyclic ring system. Non-limiting examples of "heterocycloalkyl", "heterocyclic" or "heterocycle" rings include pyrrolidine, piperidine, N-methylpiperidine, imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin, dioxolane, phthalimide, piperidine, pyrimidine-2,4(1H,3H)-dione, 1,4-dioxane, morpholine, thiomorpholine, thiomorpholine-S-oxide, thiomo Examples of heterocyclic rings include fluoroline-S,S-oxide, piperazine, pyran, pyridone, 3-pyrroline, thiopyran, pyrone, tetrahydrofuran, tetrahydrothiophene, quinuclidine, tropane, 2-azaspiro[3.3]heptane, (1R,5S)-3-azabicyclo[3.2.1]octane, (1s,4s)-2-azabicyclo[2.2.2]octane, (1R,4R)-2-oxa-5-azabicyclo[2.2.2]octane, and the like. A "heterocycloalkyl", "heterocyclic" or "heterocycle" group can be attached to the remainder of the molecule through one or more ring carbons or heteroatoms. A "heterocycloalkyl", "heterocyclic" or "heterocycle" can include mono- and poly-halogenated variants thereof.

[0029] The terms "alkoxy" and "alkylthio" are used in their conventional sense to refer to an alkyl group attached to the remainder of the molecule through either an oxygen atom ("oxy") or a thio group, and these terms can further include mono- and polyhalogenated variants thereof.

[0030] The terms "halo" or "halogen," by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. The term "(halo)alkyl" is intended to include both "alkyl" and "haloalkyl" substituents. In addition, the term "haloalkyl" is intended to include monohaloalkyl and polyhaloalkyl. For example, "C 1-4 The term "haloalkyl" is meant to include trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, difluoromethyl and the like.

[0031] The term "aryl" means a carbocyclic aromatic group having 6 to 14 carbon atoms, whether or not fused to one or more groups. Unless otherwise specified, examples of aryl groups include phenyl, naphthyl, biphenyl, and the like.

[0032] The term "heteroaryl" refers to an aryl ring(s) containing from 1 to 5 heteroatoms selected from N, O, and S, in which the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom(s) are optionally quaternized. A heteroaryl group can be attached to the remainder of the molecule through a heteroatom. Examples of heteroaryl groups include pyridyl, pyridazinyl, pyrazinyl, pyrimidinyl, triazinyl, quinolinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, benzotriazinyl, purinyl, benzimidazolyl, benzopyrazolyl, benzotriazolyl, benzisoxazolyl, isobenzofuryl, isoindolyl, indolizinyl, benzotriazinyl, thienopyridinyl, thienopyrimidinyl, pyrazolopyrimidinyl, imidazopyridine, benzothiazolyl, benzofuranyl, benzothienyl, indolyl, quinolyl, isoquinolyl, isothiazolyl, pyrazolyl, indazolyl, pteridinyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiadiazolyl, pyrrolyl, thiazolyl, furyl, thienyl, and the like.

[0033] The term sugar includes monosaccharides, disaccharides and trisaccharides. It includes glucose, sucrose, fructose, galactose and ribose, as well as deoxysugars (such as deoxyribose) and aminosugars (such as galactosamine). Sugar derivatives can be conveniently prepared as described in WO 96 / 34005 and WO 97 / 03995. The sugar can be conveniently linked to the remainder of the compound of formula I through an ether bond, a thioether bond (e.g. S-glycosides), an amine nitrogen (e.g. N-glycosides) or a carbon-carbon bond (e.g. C-glycosides). In one embodiment, the sugar can be conveniently linked to the remainder of the compound of formula I through an ether bond. In one embodiment, the term sugar includes groups of the formula: [ka] During the ceremony, X is NR 3 and Y is -(C=O)R 4 , -SO2R 5 and -(C=O)NR 6 R 7 or X is -(C=O)- and Y is NR 8 R 9 and R 3 is hydrogen or (C1-C4) alkyl, R 4 , R 5 , R 6 , R 7 , R 8 and R 9 are each independently selected from the group consisting of hydrogen, (C1-C8)alkyl, (C1-C8)haloalkyl, (C1-C8)alkoxy, and (C3-C6)cycloalkyl optionally substituted with one or more groups independently selected from the group consisting of halo, (C1-C4)alkyl, (C1-C4)haloalkyl, (C1-C4)alkoxy, and (C1-C4)haloalkoxy; R 10 -OH, -NR 8 R9 or -F, R 11 -OH, -NR 8 R 9 , -F, or a 5-membered heterocycle optionally substituted with one or more groups independently selected from the group consisting of halo, hydroxyl, carboxyl, amino, (C1-C4) alkyl, (C1-C4) haloalkyl, (C1-C4) alkoxy, and (C1-C4) haloalkoxy. [ka] The compound can be selected from the group consisting of:

[0034] In another embodiment, the sugar is [ka] It can be.

[0035] The term "animal" includes mammalian species (humans, mice, rats, dogs, cats, hamsters, guinea pigs, rabbits, farm animals, etc.).

[0036] Creation of siRNA molecules siRNA can be provided in several forms, including, for example, as one or more isolated small interfering RNA (siRNA) duplexes, as longer double-stranded RNA (dsRNA), or as siRNA or dsRNA transcribed from a transcription cassette in a DNA plasmid. In some embodiments, siRNA can be made enzymatically or by partial / full organic synthesis, and modified ribonucleotides can be introduced by in vitro enzymatic or organic synthesis. In certain cases, each strand is chemically prepared. Methods for synthesizing RNA molecules are known in the art, for example, chemical synthesis methods as described in Verma and Eckstein (1998) or as described herein.

[0037] Methods for isolating RNA, synthesizing RNA, hybridizing nucleic acids, making and screening cDNA libraries, and performing PCR are well known in the art (see, e.g., Gubler and Hoffman, Gene, 25:263-269 (1983); Sambrook et al., supra; Ausubel et al., supra), and PCR methods are well known (see U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., eds, 1990)). Expression libraries are also well known to those of skill in the art. Further basic texts disclosing general methods useful in the present invention include Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989), Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990), and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994). The disclosures of these references are incorporated herein by reference in their entireties for all purposes.

[0038] Typically, siRNA is chemically synthesized. Oligonucleotides, including siRNA molecules of the present invention, can be synthesized using any of a variety of techniques known in the art, such as those described in Usman et al., J. Am. Chem. Soc., 109:7845 (1987), Scaringe et al., Nucl. Acids Res., 18:5433 (1990), Wincott et al., Nucl. Acids Res., 23:2677-2684 (1995) and Wincott et al., Methods Mol. Bio., 74:59 (1997). Synthesis of oligonucleotides utilizes common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5' end and phosphoramidite at the 3' end. As a non-limiting example, small scale synthesis can be performed using a 0.2 μmole scale protocol on an Applied Biosystems synthesizer. Alternatively, 0.2 μmole scale syntheses can be performed on a 96-well plate synthesizer from Protogene (Palo Alto, Calif.). However, larger and smaller scale syntheses are also within the scope of the invention. Reagents suitable for the synthesis of oligonucleotides, methods for deprotecting RNA, and methods for purifying RNA are known to those of skill in the art.

[0039] The siRNA molecule can be assembled from two separate oligonucleotides, where one oligonucleotide comprises the sense strand and the other comprises the antisense strand of the siRNA, e.g., each strand can be synthesized separately and joined together by hybridization or ligation after synthesis and / or deprotection.

[0040] EMBODIMENTS OF THE PRESENT DISCLOSURE One aspect of the invention is a compound of Formula I, or a salt thereof, as set forth in the Summary of the Invention.

[0041] In one embodiment of the compound of formula I, R 1 is a targeting ligand, L 1is absent or a linking group, L 2 is absent or a linking group, R 2 is a nucleic acid, Ring A is absent, a 3- to 20-membered cycloalkyl, a 5- to 20-membered aryl, a 5- to 20-membered heteroaryl or a 3- to 20-membered heterocycloalkyl; Each R A are independently hydrogen, hydroxy, CN, F, Cl, Br, I, -C 1-2 Alkyl-OR B , as well as halo, hydroxy and C 1-3 C optionally substituted with one or more groups independently selected from alkoxy 1-8 is selected from the group consisting of alkyl, R B is hydrogen, a protecting group, a covalent bond to a solid support, or a bond to a linking group attached to a solid support; n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0042] In one embodiment, R 1 is -C(H) (3-p) (L 3 -sugar) p and each L 3 is independently a linking group, p is 1, 2 or 3, and the sugar is a monosaccharide or a disaccharide.

[0043] In one embodiment, the sugar is [ka] And, During the ceremony, X is NR 3 and Y is -(C=O)R 4 , -SO2R 5 and -(C=O)NR 6 R 7 or X is -(C=O) and Y is NR 8 R 9 and R3 is hydrogen or (C1-C4) alkyl; R 4 , R 5 , R 6 , R 7 , R 8 and R 9 are each independently selected from the group consisting of hydrogen, (C1-C8)alkyl, (C1-C8)haloalkyl, (C1-C8)alkoxy, and (C3-C6)cycloalkyl optionally substituted with one or more groups independently selected from the group consisting of halo, (C1-C4)alkyl, (C1-C4)haloalkyl, (C1-C4)alkoxy, and (C1-C4)haloalkoxy; R 10 -OH, -NR 8 R 9 or -F, R 11 -OH, -NR 8 R 9 , -F, or a 5-membered heterocyclic ring optionally substituted with one or more groups independently selected from the group consisting of halo, hydroxyl, carboxyl, amino, (C1-C4)alkyl, (C1-C4)haloalkyl, (C1-C4)alkoxy, and (C1-C4)haloalkoxy; or a salt thereof.

[0044] In one embodiment, the sugar is [ka] and salts thereof.

[0045] In one embodiment, the sugar is [ka] It is.

[0046] In one embodiment, each L 3is independently a divalent branched or unbranched, saturated or unsaturated hydrocarbon chain having 0 to 50 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms of the hydrocarbon chain are optionally -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X - or -S-, where R X is hydrogen or (C1-C6)alkyl, where the hydrocarbon chain is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0047] In one embodiment, each L 3 are independently a divalent branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 20 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms of the hydrocarbon chain are optionally -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X - or -S-, where R X is hydrogen or (C1-C6)alkyl, where the hydrocarbon chain is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0048] In one embodiment, L 3 teeth, [ka] or a salt thereof.

[0049] In one embodiment, R 1 teeth, [ka] or a salt thereof.

[0050] In one embodiment, R 1 teeth, [ka] And, In the formula, G is -NH- or -O-; R C is hydrogen, (C1-C8) alkyl, (C1-C8) haloalkyl, (C1-C8) alkoxy, (C1-C6) alkanoyl, (C3-C 20 )Cycloalkyl, (C3-C 20 ) a heterocycle, an aryl, a heteroaryl, a monosaccharide, a disaccharide, or a trisaccharide, wherein the cycloalkyl, the heterocycle, the aryl, the heteroaryl, and the sugar are optionally substituted with one or more groups independently selected from the group consisting of halo, carboxyl, hydroxyl, amino, (C1-C4) alkyl, (C1-C4) haloalkyl, (C1-C4) alkoxy, and (C1-C4) haloalkoxy; or a salt thereof.

[0051] In one embodiment, R C teeth, [ka] It is.

[0052] In one embodiment, R 1 teeth, [ka] It is.

[0053] In one embodiment, R C teeth [ka] It is.

[0054] In one embodiment, G is -NH-.

[0055] In one embodiment, R 1 teeth, [ka] It is.

[0056] In one embodiment, R 1 teeth, [ka] and In the formula, each R D is hydrogen, (C1-C6) alkyl, (C9-C 20 ) alkylsilyl, (R W )3Si-, (C2-C6)alkenyl, tetrahydropyranyl, (C1-C6)alkanoyl, benzoyl, aryl(C1-C3)alkyl, TMTr (trimethoxytrityl), DMTr (dimethoxytrityl), MMTr (monomethoxytrityl) and Tr (trityl); Each R W is independently selected from the group consisting of (C1-C4) alkyl and aryl.

[0057] In one embodiment, the linking group L 1 and L 2 are independently a divalent branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 50 carbon atoms, in which one or more (e.g., 1, 2, 3, or 4) of the carbon atoms of the hydrocarbon chain are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X- or -S-, where R is optionally substituted X is hydrogen or (C1-C6)alkyl, where the hydrocarbon chain is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0058] In one embodiment, L 1 and L 2 are independently a divalent branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 20 carbon atoms, in which one or more (e.g., 1, 2, 3, or 4) of the carbon atoms of the hydrocarbon chain are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X - or -S-, where R is optionally substituted X is hydrogen or (C1-C6)alkyl, where the hydrocarbon chain is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0059] In one embodiment, L 1 and L 2 are independently a divalent branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 14 carbon atoms, in which one or more (e.g., 1, 2, 3, or 4) of the carbon atoms of the hydrocarbon chain are -O-, -NR X -, -NR X-C(=O)-, -C(=O)-NR X - or -S-, where R is optionally substituted X is hydrogen or (C1-C6)alkyl, where the hydrocarbon chain is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0060] In one embodiment, L 1 is R through -NH-, -O-, -S-, -(C=O)-, -(C=O)-NH-, -NH-(C=O)-, -(C=O)-O-, -NH-(C=O)-NH- or -NH-(SO2)- 1 is linked to.

[0061] In one embodiment, L 2 through -O-, R 2 is linked to.

[0062] In one embodiment, L 1 teeth, [ka] is selected from the group consisting of:

[0063] In one embodiment, L 1 teeth, [ka] and salts thereof.

[0064] In one embodiment, L 2 is -CH2-O- or -CH2-CH2-O-.

[0065] In one embodiment, the compound of formula I is a compound having formula Ia: [ka] During the ceremony, Each D is [ka] and -N=, or a salt thereof.

[0066] In one embodiment, the compound of formula Ia is [ka] wherein Q 1 is hydrogen, and Q 2 But R 2 or Q 1 But R 2 And Q 2 is hydrogen, Z is -L 1 -R 1 A compound, and their salts is selected from the group consisting of:

[0067] In one embodiment, the compound of formula I is a compound having formula Ib: [ka] During the ceremony, Each D is independent, [ka] and -N=; compounds in which each m is independently 1 or 2; or a salt thereof.

[0068] In one embodiment, the compound of formula Ib is [ka] And, During the ceremony, Q 1 is hydrogen, and Q 2 But R 2 or Q 1 But R 2 And Q 2 is hydrogen, Z is -L 1 -R 1 A compound, and their salts is selected from the group consisting of:

[0069] In one embodiment, the compound of formula I is a compound of formula (Ic): [ka] In the formula, E is -O- or -CH2-; n is selected from the group consisting of 0, 1, 2, 3 and 4; A compound in which n1 and n2 are each independently selected from the group consisting of 0, 1, 2 and 3; or a salt thereof.

[0070] In certain embodiments, the compound of formula (Ic) is [ka] And, In the formula, Z is -L 1 -R 1 A compound, and their salts is selected from the group consisting of:

[0071] In one embodiment, -AL 2 -R 2 The part is [ka] And, During the ceremony, Q1 is hydrogen, and Q 2 But R 2 or Q 1 But R 2 And Q 2 is hydrogen, a moiety in which each q is independently 0, 1, 2, 3, 4, or 5; or a salt thereof.

[0072] In one embodiment, R 2 is an oligonucleotide.

[0073] In one embodiment, R 2 is an siRNA.

[0074] In one embodiment, the compound of formula (I) is [ka] [ka] and their salts is selected from the group consisting of:

[0075] In one embodiment, R 1 teeth, [ka] is selected from the group consisting of In the formula, R S teeth [ka] and n is 2, 3 or 4; x is 1 or 2.

[0076] In one embodiment, L 1 teeth, [ka] is selected from the group consisting of:

[0077] In one embodiment, L 1 teeth, [ka] is selected from the group consisting of:

[0078] In one embodiment, A is absent, phenyl, pyrrolidinyl, or cyclopentyl.

[0079] In one embodiment, L 2 is optionally substituted with hydroxy 1-4 It is alkylene-O-.

[0080] In one embodiment, L 2 is -CH2O-, -CH2CH2O- or -CH(OH)CH2O-.

[0081] In one embodiment, each R A C is optionally substituted with hydroxy or hydroxyl. 1-8 It is an alkyl.

[0082] In one embodiment, each R A is independently selected from the group consisting of hydroxy, methyl, and -CHOH.

[0083] In one embodiment, the compound of formula I has formula (Ig): [ka] In the formula, B is -N- or -CH-; L 1 is absent or is -NH-, L 2 is optionally substituted with hydroxyl or halo 1-4 alkylene-O-; Compounds in which n is 0, 1 or 2; or a salt thereof.

[0084] In one embodiment, the compound of formula I has formula (Ig): [ka] In the formula, B is -N- or -CH-; L 1 is absent or is -NH-, L 2 is optionally substituted with hydroxyl or halo 1-4 alkylene-O-; Compounds in which n is 0, 1, 2, 3, 4, 5, 6 or 7; or a salt thereof.

[0085] In one embodiment, the compound of formula I has formula (Ig): [ka] In the formula, B is -N- or -CH-; L 1 is absent or is -NH-, L 2 is optionally substituted with hydroxyl or halo 1-4 alkylene-O-; Compounds in which n is 0, 1, 2, 3 or 4; or a salt thereof.

[0086] In one embodiment, the compound of formula Ig is [ka] And, In the formula, R' is C 1-9 Alkyl, C 2-9 Alkenyl or C 2-9 Alkynyl, and its C 1-9 Alkyl, C 2-9 Alkenyl or C 2-9 compounds wherein alkynyl is optionally substituted with halo or hydroxyl; and their salts is selected from the group consisting of:

[0087] In one embodiment, the compound of formula I is [ka] and their salts is selected from the group consisting of:

[0088] In one embodiment, the compound of formula I or a salt thereof is [ka] [ka] [ka] [ka] or a pharma- ceutically acceptable salt thereof. is selected from the group consisting of:

[0089] One aspect of the invention is a pharmaceutical composition comprising a compound of Formula I and a pharma- ceutically acceptable carrier.

[0090] Another aspect of the invention is a method for delivering a nucleic acid to the liver of an animal comprising administering to the animal a compound of formula I, or a pharma- ceutically acceptable salt thereof.

[0091] In one embodiment, the compound of formula I has the formula (Id): [ka] During the ceremony, R 1d teeth, [ka] It is selected from X dis C 2-10 is alkylene, N d is 0 or 1, R 2d is a nucleic acid, R 3d is H, a protecting group, a covalent bond to a solid support, or a bond to a linking group attached to a solid support.

[0092] In one embodiment, R 3d contains a linking group that connects the remainder of the compound of formula Id to a solid support. 2d When is a suitable intermediate for the preparation of a compound of formula Id which is a nucleic acid, the nature of the linking group is not critical.

[0093] In one embodiment, R 3d The linker has a molecular weight of about 20 daltons to about 1,000 daltons.

[0094] In one embodiment, R 3d The linker has a molecular weight of about 20 daltons to about 500 daltons.

[0095] In one embodiment, R 3d separates the solid support from the remainder of the compound of Formula I by a length of at least about 5 angstroms and no more than about 40 angstroms.

[0096] In one embodiment, R 3dis a divalent branched or unbranched, saturated or unsaturated hydrocarbon chain having 2 to 15 carbon atoms, one or more (e.g., 1, 2, 3, or 4) of which carbon atoms are optionally substituted by (-O-) or (-N(H)-), and the chain is optionally substituted on carbon with one or more (e.g., 1, 2, 3, or 4) substituents selected from (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0097] In one embodiment, R 3d is a divalent branched or unbranched, saturated or unsaturated hydrocarbon chain having 2 to 10 carbon atoms, one or more (e.g., 1, 2, 3, or 4) of which carbon atoms are optionally substituted by (-O-) or (-N(H)-), and the chain is optionally substituted on carbon with one or more (e.g., 1, 2, 3, or 4) substituents selected from (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0098] In one embodiment, R 3d The linker is -C(=O)CH2CH2C(=O)N(H)-.

[0099] In one embodiment, R 1d teeth, [ka] It is.

[0100] In one embodiment, R 1d teeth, [ka] It is.

[0101] In one embodiment, X d is a C8 alkylene.

[0102] In one embodiment, n d is 0.

[0103] In one embodiment, R 2d is an siRNA.

[0104] In one embodiment, R 3d is H.

[0105] In another embodiment, the compound of formula (Id) or a salt thereof is [ka] and their salts is selected from the group consisting of:

[0106] One aspect of the invention is a pharmaceutical composition comprising a compound of formula (Id) and a pharma- ceutically acceptable carrier.

[0107] One aspect of the invention is a method for administering to the liver of an animal comprising administering to the animal a compound of formula (Id) or a pharma- ceutically acceptable salt thereof.

[0108] The present invention also provides synthetic intermediates and methods disclosed herein that are useful for preparing compounds of formula (Id). For example, the present invention provides intermediate compounds of formula Ie: [ka] or a salt thereof, wherein R 1d teeth, [ka] It is selected from X d is C 2-8 is alkylene, n d is either 0 or 1, Pg 1 is H or a suitable protecting group, R 3d is H, a protecting group, a covalent bond to a solid support, or a bond to a linking group attached to a solid support. FIG. 1 shows a representative intermediate compound of formula (Ie), in which the targeting ligand / linker is attached to a solid support and Pg 1 is the protecting group DMTr.

[0109] In one embodiment, Pg 1 is TMTr (trimethoxytrityl), DMTr (dimethoxytrityl), MMTr (monomethoxytrityl) or Tr (trityl).

[0110] The present invention relates to a process for the preparation of a compound of formula (Id) as described herein, comprising the corresponding compound of formula (Ie) [ka] During the ceremony, X d But, C 2-8 is alkylene, n d is 0 or 1, Pg 1 But H, R 3d is a covalent bond to a solid support or a bond to a linking group attached to a solid support, is subjected to solid phase nucleic acid synthesis conditions to obtain a corresponding compound of formula Id, 2d providing a compound which is a nucleic acid.

[0111] In one embodiment, the method comprises cleaving the compound from the solid support to produce the corresponding compound of formula Id, R 3d is H.

[0112] In one embodiment, the compound is [ka] isn't it.

[0113] In one embodiment, the compound is a compound of formula Id: [ka] or a salt thereof, wherein R 1d but, [ka] It is selected from X d But, C 2-10 is alkylene, N d is 0 or 1, R 2d is a nucleic acid, R 3d is H, a protecting group, a covalent bond to a solid support, or a bond to a linking group attached to a solid support. It is not a compound or its salt.

[0114] In one embodiment, the compound is a compound of formula Ie: [ka] or a salt thereof, wherein R 1d but, [ka] It is selected from X d But, C 2-8 is alkylene, n d is 0 or 1, Pg 1 is H or a suitable protecting group; R 3d is H, a protecting group, a covalent bond to a solid support, or a bond to a linking group attached to a solid support. It is not a compound or its salt.

[0115] In one embodiment, R 3d is H.

[0116] In one embodiment, R 3d is a covalent bond to the solid support.

[0117] In one embodiment, R 3d is a bond to a linking group attached to a solid support, the linking group being a divalent branched or unbranched, saturated or unsaturated hydrocarbon chain having from 2 to 15 carbon atoms, one or more (e.g., 1, 2, 3, or 4) of which carbon atoms are optionally substituted by (-O-) or (-N(H)-), and the chain is optionally substituted on carbon with one or more (e.g., 1, 2, 3, or 4) substituents selected from (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0118] In one embodiment, R 3dis a bond to a linking group attached to a solid support, the linking group being a divalent branched or unbranched, saturated or unsaturated hydrocarbon chain having from 2 to 10 carbon atoms, one or more (e.g., 1, 2, 3, or 4) of which carbon atoms are optionally substituted by (-O-) or (-N(H)-), the chain optionally being substituted on carbon with one or more (e.g., 1, 2, 3, or 4) substituents selected from (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0119] In one embodiment, R 3d is the bond to a linking group that is attached to a solid support, the linking group being -C(=O)CH2CH2C(=O)N(H)-.

[0120] In one embodiment, the present invention provides a compound of formula (I): [ka] During the ceremony, R 1 is H or a synthetic activating group, L 1 is absent or is a linking group, L 2 is absent or is a linking group, R 2 is a nucleic acid, Ring A is absent, 3- to 20-membered cycloalkyl, 5- to 20-membered aryl, 5- to 20-membered heteroaryl or 3- to 20-membered heterocycloalkyl; Each R A are independently hydrogen, hydroxy, CN, F, Cl, Br, I, -C 1-2 Alkyl-OR B , C 1-10 Alkyl C 2-10Alkenyl and C 2-10 alkynyl, wherein C 1-10 Alkyl C 2-10 Alkenyl and C 2-10 Alkynyl is optionally selected from halo, hydroxy and C 1-3 substituted with one or more groups independently selected from alkoxy; R B is hydrogen, a protecting group, a covalent bond to a solid support, or a bond to a linking group attached to a solid support; Compounds in which n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; or its salt to provide.

[0121] In one embodiment, the present invention provides a compound of formula (I): [ka] During the ceremony, R 1 is the targeting ligand; L 1 is absent or is a linking group, L 2 is absent or is a linking group, R 2 is H or a synthetic activating group, Ring A is absent, 3- to 20-membered cycloalkyl, 5- to 20-membered aryl, 5- to 20-membered heteroaryl or 3- to 20-membered heterocycloalkyl; Each R A are independently hydrogen, hydroxy, CN, F, Cl, Br, I, -C 1-2 Alkyl-OR B , C 1-10 Alkyl C 2-10 Alkenyl and C 2-10 alkynyl, wherein C 1-10 Alkyl C 2-10 Alkenyl and C 2-10 Alkynyl is optionally selected from halo, hydroxy and C 1-3substituted with one or more groups independently selected from alkoxy; R B is hydrogen, a protecting group, a covalent bond to a solid support, or a bond to a linking group attached to a solid support; Compounds in which n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; or its salt to provide.

[0122] In one embodiment, the present invention provides a compound of formula (Ig): [ka] During the ceremony, B is -N- or -CH-; L 2 is optionally substituted with hydroxyl or halo 1-4 alkylene-O-; Compounds in which n is 0, 1, 2, 3, 4, 5, 6 or 7; or its salt to provide.

[0123] In one embodiment, the present invention provides [ka] A compound selected from the group consisting of During the ceremony, Q is -L 1 -R 1 and R' is C 1-9 Alkyl, C 2-9 Alkenyl or C 2-9 Alkynyl, and its C 1-9 Alkyl, C 2-9 Alkenyl or C 2-9 compounds in which the alkynyl is optionally substituted with halo or hydroxyl; The salt to provide.

[0124] In one embodiment, the present invention provides [ka] A compound selected from the group consisting of In the formula, Q is -L 1 -R 1 and salts thereof.

[0125] In one embodiment, the present invention provides a compound of formula (Ig): [ka] During the ceremony, B is -N- or -CH-; L 1 is absent or is a linking group, L 2 is optionally substituted with hydroxyl or halo 1-4 alkylene-O-; n is 0, 1, 2, 3, 4, 5, 6 or 7; R 1 is H or a synthetic activating group, R 2 is H or a synthetic activating group; or its salt to provide.

[0126] In one embodiment, the present invention provides [ka] A compound selected from the group consisting of In the formula, Q is -L 1 -R 1 and L 1 is absent or is a linking group, R' is C 1-9 Alkyl, C 2-9 Alkenyl or C 2-9 Alkynyl, and its C 1-9 Alkyl, C 2-9 Alkenyl or C 2-9alkynyl is optionally substituted with halo or hydroxyl; R 1 is H or a synthetic activating group, R 2 is H or a synthetic activating group; or its salt to provide.

[0127] In one embodiment, the present invention provides [ka] A compound selected from the group consisting of: During the ceremony, Q is -L 1 -R 1 and L 1 is absent or is a linking group, R 1 is H or a synthetic activating group, R 2 is H or a synthetic activating group; or its salt to provide.

[0128] In one embodiment, R 1 is H or a synthetic activating group derivable from DCC, HOBt, EDC, BOP, PyBOP or HBTU.

[0129] In one embodiment, R 2 is H, acetate, triflate, mesylate or succinate.

[0130] In one embodiment, R 1 is a synthetic activating group derivable from DCC, HOBt, EDC, BOP, PyBOP or HBTU.

[0131] In one embodiment, R 2 is acetate, triflate, mesylate or succinate.

[0132] In one embodiment, L 1 is a divalent branched or unbranched, saturated or unsaturated hydrocarbon chain having 5 to 20 carbon atoms in which one or more (e.g., 1, 2, 3, or 4) of the carbon atoms are optionally substituted by -O-, -NH-, -NH-C(=O)-, -C(=O)-NH-, or -S-.

[0133] In one embodiment, the present invention provides a compound of formula (XX): [ka] During the ceremony, R 1 is the targeting ligand; L 1 is absent or is a linking group, L 2 is absent or is a linking group, R 2 is a nucleic acid, B is divalent and [ka] is selected from the group consisting of During the ceremony, Each R' is independently C 1-9 Alkyl, C 2-9 Alkenyl or C 2-9 Alkynyl, and its C 1-9 Alkyl, C 2-9 Alkenyl or C 2-9 alkynyl is optionally substituted with halo or hydroxyl; * The bond marked with L 1 or L 1 If does not exist, R 1 is bound to ** The bond marked with L 2 or L 2 If does not exist, R 2 Compounds that are bound to or its salt to provide.

[0134] In one embodiment, R 1 contains 2 to 8 sugars.

[0135] In one embodiment, R 1 contains 2 to 6 sugars.

[0136] In one embodiment, R 1 contains 2 to 4 sugars.

[0137] In one embodiment, R 1 contains 3 to 8 sugars.

[0138] In one embodiment, R 1 contains 3 to 6 sugars.

[0139] In one embodiment, R 1 contains 3 to 4 sugars.

[0140] In one embodiment, R 1 contains three sugars.

[0141] In one embodiment, R 1 contains four sugars.

[0142] In one embodiment, R 1 is the following formula: [ka] B 1 is a trivalent group containing about 1 to about 20 atoms, and L 1 , T 1 and T 2 is covalently bonded to B 2 is a trivalent group containing about 1 to about 20 atoms, T 1 , T 3 and T 4 is covalently bonded to B 3 is a trivalent group containing about 1 to about 20 atoms, T2 , T 5 and T 6 is covalently bonded to T 1 is absent or is a linking group, T 2 is absent or is a linking group, T 3 is absent or is a linking group, T 4 is absent or is a linking group, T 5 is absent or is a linking group, T 6 is absent or is a linking group having the formula:

[0143] In one embodiment, each sugar is independently [ka] It is selected from During the ceremony, X is NR 3 and Y is -(C=O)R 4 , -SO2R 5 and -(C=O)NR 6 R 7 or X is -(C=O)- and Y is NR 8 R 9 and R 3 is hydrogen or (C1-C4) alkyl, R 4 , R 5 , R 6 , R 7 , R 8 and R 9are each independently selected from the group consisting of hydrogen, (C1-C8)alkyl, (C1-C8)haloalkyl, (C1-C8)alkoxy, and (C3-C6)cycloalkyl optionally substituted with one or more groups independently selected from the group consisting of halo, (C1-C4)alkyl, (C1-C4)haloalkyl, (C1-C4)alkoxy, and (C1-C4)haloalkoxy; R 10 -OH, -NR 8 R 9 or -F, R 11 -OH, -NR 8 R 9 , -F, or a 5-membered heterocycle optionally substituted with one or more groups independently selected from the group consisting of halo, hydroxyl, carboxyl, amino, (C1-C4)alkyl, (C1-C4)haloalkyl, (C1-C4)alkoxy, and (C1-C4)haloalkoxy.

[0144] In one embodiment, each sugar is independently [ka] is selected from the group consisting of:

[0145] In one embodiment, each sugar is independently [ka] It is.

[0146] In one embodiment, T 1 and T 2 One of them does not exist.

[0147] In one embodiment, T 1 and T 2 both do not exist.

[0148] In one embodiment, T 1 , T 2 , T 3 , T 4 , T 5and T 6 each independently is absent or a branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 50 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms of the hydrocarbon chain are optionally selected from -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X - or -S-, where R X is hydrogen or (C1-C6)alkyl, where the hydrocarbon chain is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0149] In one embodiment, T 1 , T 2 , T 3 , T 4 , T 5 and T 6 are each independently absent or a branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 20 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms of the hydrocarbon chain are optionally selected from -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X - or -S-, where R Xis hydrogen or (C1-C6)alkyl, where the hydrocarbon chain is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0150] In one embodiment, T 1 , T 2 , T 3 , T 4 , T 5 and T 6 is independently absent or a branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 50 carbon atoms, or a salt thereof, in which one or more (e.g., 1, 2, 3, or 4) of the carbon atoms of the hydrocarbon chain are optionally -O- or -NR X - is replaced by R X is hydrogen or (C1-C6)alkyl, where the hydrocarbon chain is optionally substituted with one or more (e.g., 1, 2, 3 or 4) substituents selected from halo, hydroxy, and oxo (=O).

[0151] In one embodiment, T 1 , T 2 , T 3 , T 4 , T 5 and T 6 each is independently zero or a branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 20 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms of the hydrocarbon chain are optionally substituted with -O-, and the hydrocarbon chain is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from halo, hydroxy, and oxo (=O).

[0152] In one embodiment, T 1 , T 2 , T 3 , T 4 , T 5 and T 6 each is independently zero or a branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 20 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms of the hydrocarbon chain are optionally substituted with -O-, and the hydrocarbon chain is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from halo, hydroxy, and oxo (=O).

[0153] In one embodiment, T 3 , T 4 , T 5 and T 6 At least one of the [ka] and During the ceremony, n=1, 2, 3.

[0154] In one embodiment, T 3 , T 4 , T 5 and T 6 Each of the following is independent: [ka] is selected from the group consisting of During the ceremony, n=1, 2, 3.

[0155] In one embodiment, T 1 and T 2 At least one of the is glycine.

[0156] In one embodiment, T 1 and T 2 Each of is glycine.

[0157] In one embodiment, B 1 is a trivalent group containing 1 to 15 atoms, L 1 , T 1 and T 2 is covalently bonded to

[0158] In one embodiment, B 1 is a trivalent group containing 1 to 10 atoms, L 1 , T 1 and T 2 is covalently bonded to

[0159] In one embodiment, B 1 includes (C1-C6) alkyl.

[0160] In one embodiment, B 1 is C 3-8 Includes cycloalkyl.

[0161] In one embodiment, B 1 contains a silyl group.

[0162] In one embodiment, B 1 includes D- or L-amino acids.

[0163] In one embodiment, B 1 contains sugar.

[0164] In one embodiment, B 1 contains a phosphate group.

[0165] In one embodiment, B 1 contains a phosphonate group.

[0166] In one embodiment, B 1 includes aryl.

[0167] In one embodiment, B 1 contains a phenyl ring.

[0168] In one embodiment, B 1 is a phenyl ring.

[0169] In one embodiment, B 1 is CH.

[0170] In one embodiment, B 1 includes heteroaryl.

[0171] In one embodiment, B 1 teeth, [ka] is selected from the group consisting of:

[0172] In one embodiment, B 2 is a trivalent group containing 1 to 15 atoms, L 1 , T 1 and T 2 is covalently bonded to

[0173] In one embodiment, B 2 is a trivalent group containing 1 to 10 atoms, L 1 , T 1 and T 2 is covalently bonded to

[0174] In one embodiment, B 2 includes (C1-C6) alkyl.

[0175] In one embodiment, B 2 is C 3-8 Includes cycloalkyl.

[0176] In one embodiment, B 2 contains a silyl group.

[0177] In one embodiment, B 2 includes D- or L-amino acids.

[0178] In one embodiment, B 2 contains sugar.

[0179] In one embodiment, B2 contains a phosphate group.

[0180] In one embodiment, B 2 contains a phosphonate group.

[0181] In one embodiment, B 2 includes aryl.

[0182] In one embodiment, B 2 contains a phenyl ring.

[0183] In one embodiment, B 2 is a phenyl ring.

[0184] In one embodiment, B 2 is CH.

[0185] In one embodiment, B 2 includes heteroaryl.

[0186] In one embodiment, B 2 teeth, [ka] is selected from the group consisting of:

[0187] In one embodiment, B 3 is a trivalent group containing 1 to 15 atoms, L 1 , T 1 and T 2 is covalently bonded to

[0188] In one embodiment, B 3 is a trivalent group containing 1 to 10 atoms, L 1 , T 1 and T 2 is covalently bonded to

[0189] In one embodiment, B 3 includes (C1-C6) alkyl.

[0190] In one embodiment, B 3 is C 3-8 Includes cycloalkyl.

[0191] In one embodiment, B 3 contains a silyl group.

[0192] In one embodiment, B 3 includes D- or L-amino acids.

[0193] In one embodiment, B 3 contains sugar.

[0194] In one embodiment, B 3 contains a phosphate group.

[0195] In one embodiment, B 3 contains a phosphonate group.

[0196] In one embodiment, B 3 includes aryl.

[0197] In one embodiment, B 3 contains a phenyl ring.

[0198] In one embodiment, B 3 is a phenyl ring.

[0199] In one embodiment, B 3 is CH.

[0200] In one embodiment, B 3 includes heteroaryl.

[0201] In one embodiment, B 3 teeth, [ka] or its salt is selected from the group consisting of:

[0202] In one embodiment, L 1and L 2 are independently a divalent branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 50 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms of the hydrocarbon chain are optionally -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X - or -S-, where R X is hydrogen or (C1-C6)alkyl, where the hydrocarbon chain is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0203] In one embodiment, L 1 teeth, [ka] or its salt is selected from the group consisting of:

[0204] In one embodiment, L 1 is connected to B through a linkage selected from the group consisting of -O-, -S-, -(C=O)-, -(C=O)-NH-, -NH-(C=O), -(C=O)-O-, -NH-(C=O)-NH- or -NH-(SO2)- 1 is linked to.

[0205] In one embodiment, L 1 teeth, [ka] is selected from the group consisting of:

[0206] In one embodiment, L 2through -O-, R 2 is linked to.

[0207] In one embodiment, L 2 is optionally substituted with hydroxy 1-4 It is alkylene-O-.

[0208] In one embodiment, L 2 through -O-, R 2 is linked to.

[0209] In one embodiment, L 2 does not exist.

[0210] In one embodiment, the present invention provides [ka] [ka] [ka] and pharma- ceutically acceptable salts thereof. The present invention provides a compound or salt selected from the group consisting of: EXAMPLES

[0211] The present invention will be described in more detail by specific examples. The following examples are provided for illustrative purposes and are not intended to limit the present invention in any way. Those skilled in the art will easily recognize various non-critical parameters that can be changed or modified to produce essentially the same results.

[0212] Example 1. Synthesis of Conjugate 1 Scheme 1. [ka]

[0213] Scheme 2. [ka]

[0214] Scheme 3. [ka]

[0215] Scheme 4. [ka]

[0216] Scheme 5. [ka]

[0217] Step 1. Preparation of 2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate 3 [ka] A solution of tetraethylene glycol (934 g, 4.8 mol) in THF (175 mL) and aqueous NaOH (5 M, 145 mL) was cooled (0 °C) and treated with p-toluenesulfonyl chloride (91.4 g, 480 mmol) in THF (605 mL) and stirred (0 °C) for 2 h. The reaction mixture was diluted with water (3 L) and extracted with CHCl (3 × 500 mL). The combined extracts were washed with water and brine, then dried (MgSO), filtered, and concentrated to give 2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate 3 (140 g, 84%) as a pale yellow oil. R f (0.57, 10% MeOH-CH2Cl2).

[0218] Step 2. Preparation of 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethan-1-ol 4 [ka] A solution of 3 (140 g, 403 mmol) in DMF (880 mL) was treated with sodium azide (131 g, 2.02 mol) and heated (45° C.) overnight. Most of the DMF was removed under reduced pressure and the residue was dissolved in CHCl (500 mL) and washed with brine (3×500 mL), then dried (MgSO), filtered, and concentrated. The residue was passed through a short bed of silica (5% MeOH—CHCl) and concentrated to give 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethan-1-ol 4 (65 g, 74%) as a yellow oil. R f (0.56, 10% MeOH-CH2Cl2).

[0219] Step 3. Preparation of peracetylated galactosamine 6 [ka] D-Galactosamine hydrochloride 5 (250 g, 1.16 mol) in pyridine (1.5 L) was treated with acetic anhydride (1.25 L, 13.2 mol) for 45 min. After stirring overnight, the reaction mixture was divided into three 1 L portions. Each 1 L portion was placed in 3 L of ice water and mixed for 1 h. After mixing, the solids were filtered, combined, frozen in liquid nitrogen, and lyophilized for 5 days to give the peracetylated galactosamine 6 (369.4 g, 82%) as a white solid. Rf (0.58, 10% MeOH-CH2Cl2).

[0220] Step 4. Preparation of (3aR,5R,6R,7R,7aR)-5-(acetoxymethyl)-2-methyl-3a,6,7,7a-tetrahydro-5H-pyrano[3,2-d]oxazole-6,7-diyl diacetate 7 [ka] A solution of peracetylated galactosamine 6 (8.45 g, 21.7 mmol) in CHCl3 (320 mL) was treated dropwise with TMSOTf (4.32 mL, 23.9 mmol). After stirring (1.5 h, 40 °C), the reaction was quenched by the addition of triethylamine (5 mL) and concentrated to dryness to give compound 7 as a pale yellow glass (7.2 g, quantitative). The product was used without further purification. Rf (0.59, 10% MeOH-CH2Cl2).

[0221] Step 5. Preparation of (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-3,4-diyl diacetate 8 [ka] Compound 7 (7.2 g, 21.7 mmol) and 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethan-1-ol 4 (2.65 g, 15.2 mmol) were azeotroped (3x) from toluene (150 mL) to remove traces of water. The dried material was dissolved in 1,2-dichloroethane (150 mL), cooled (ca. 5° C.), and treated with TMSOTf (784 μL, 4.34 mmol). After stirring overnight, the reaction was quenched by the addition of triethylamine (5 mL) and concentrated. The residue was purified by chromatography (1%→5% MeOH-CH2Cl2) to give 8 (7.12 g, 85%) as a brown oil. Rf (0.3, 10% MeOH-CH2Cl2).

[0222] Step 6. Preparation of 2-(2-(2-(2-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethan-1-aminium 2,2,2-trifluoroacetate 9 [ka] A solution of azide 8 (7.12 g, 13 mmol) in EtOAc (150 mL) and trifluoroacetic acid (2 mL) was treated with palladium on charcoal (1.5 g, 10% w / w, wet basis). The reaction mixture was then purged with hydrogen and stirred vigorously overnight. After purging with nitrogen, the mixture was filtered through Celite and rinsed with MeOH. The filtrate was concentrated and purified by chromatography (5% → 10% → 20% MeOH-CH2Cl2) to give 9 (5.8 g, 72%) as a brown oil. Rf (0.34, 15% MeOH-CH2Cl2).

[0223] Step 7. Preparation of di-tert-butyl 4-(((benzyloxy)carbonyl)amino)-4-(3-(tert-butoxy)-3-oxopropyl)heptanedioate 11 [ka] Di-tert-butyl 4-amino-4-(3-(tert-butoxy)-3-oxopropyl)heptanedioate 10 (13.5 g, 33 mmol), 25% NaCO 3(aq) To a solution of 1,2-dichloromethane (150 mL) and dichloromethane (300 mL) was added benzyl chloroformate (14 mL, 98 mmol) slowly. The solution was stirred vigorously at room temperature overnight (16 h). Upon completion, additional dichloromethane (100 mL) was added and the dichloromethane layer was separated. The aqueous layer was extracted with dichloromethane (2×100 mL). The combined dichloromethane extracts were dried over magnesium sulfate, filtered, and concentrated to dryness. The product 11 was isolated as a colorless oil (15.8 g, 88%) without further purification. Rf (0.7, 1:1 EtOAc-hexanes).

[0224] Step 8. Preparation of 4-(((benzyloxy)carbonyl)amino)-4-(2-carboxyethyl)heptanedioic acid 12 [ka] A solution of 11 (15.6 g, 28.8 mmol) in formic acid (50 mL) was stirred at room temperature for 2 h. The solution was concentrated to dryness and dissolved in ethyl acetate (ca. 25 mL). The product crystallized on standing as a colorless solid. The solid was filtered, washed with ethyl acetate, and air-dried to give 12 (10.2 g, 93%) as a colorless solid. Rf (0.1, 10% MeOH-CH2Cl2).

[0225] Step 9. Preparation of compound 13 [ka] A solution of 12 (793 mg, 2.08 mmol) and 9 (5.8 g, 9.36 mmol) in DMF (50 mL) was treated with BOP (3.67 g, 8.32 mmol) followed by N,N-diisopropylethylamine (4.31 mL, 25 mmol). After stirring overnight, the mixture was concentrated to dryness and chromatographed (1% → 2% → 5% → 10% → 15% MeOH-CH2Cl2) to give 13 (5.71 g [crude], >100% - contained coupling by-products that did not affect the next step). Rf (0.45, 10% MeOH-CH2Cl2).

[0226] Step 10. Preparation of compound 14 [ka] Compound 13 (5.7 g) was dissolved in MeOH (150 mL) and TFA (1.5 mL) and treated with palladium on charcoal (1 g, 10% w / w, wet basis). The reaction mixture was then purged with hydrogen and stirred vigorously overnight. After purging with nitrogen, the mixture was filtered through Celite and rinsed with MeOH. The filtrate was concentrated and purified by chromatography (5% → 10% → 20% MeOH-CH2Cl2) to give 14 as a brown oil (2.15 g, 56% for two steps). Rf (0.32, 10% MeOH-CH2Cl2).

[0227] Step 11. Preparation of (5-amino-1,3-phenylene)dimethanol 15 [ka] To a mixture of 3.75 equiv. LiAlH4 (13.6 g, 358 mmol) refluxing in THF (440 mL) was added dropwise a solution of dimethyl 5-aminoisophthalate (20.0 g, 96 mmol) in THF (350 mL) over 1 h. The mixture was stirred at reflux for an additional 2 h, then cooled to room temperature and quenched by careful addition of MeOH (27 mL) followed by water (40 mL). After stirring the quenched mixture for 2 h, the mixture was filtered and concentrated to dryness. The residue was recrystallized from EtOAc (2×) to give 15 as tan crystals (10.2 g, 70%).

[0228] Step 12. Preparation of methyl 10-((3,5-bis(hydroxymethyl)phenyl)amino)-10-oxodecanoate 16 [ka] A solution of methyl sebacate (3.8 g, 17 mmol), 15 (2.5 g, 17 mmol) and EEDQ (8.1 g, 33 mmol) in 2:1 dichloromethane / methanol (200 mL) was stirred at room temperature for 2 h. Upon completion, the solution was concentrated to dryness. The resulting solid was triturated with dichloromethane (50 mL) and filtered. The solid was rinsed with cold dichloromethane and air-dried to give 16 as a colorless solid (4.3 g, 72%). Rf (0.33, EtOAc).

[0229] Step 13. Preparation of methyl 10-((3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(hydroxymethyl)phenyl)amino)-10-oxodecanoate 17 [ka] To a solution of 16 (4.3 g, 12 mmol) in pyridine (50 mL) was added 4,4'-(chloro(phenyl)methylene)bis(methoxybenzene) (4.1 g, 12 mmol). The solution was stirred overnight at room temperature under nitrogen. Upon completion, the solution was concentrated to dryness and the residue was purified by column chromatography (0.5%→0.75%→1%→1.5% MeOH-CH2Cl2) to give 17 as a yellow solid (2.9 g, 35%). Rf (0.6, 10% MeOH-CH2Cl2).

[0230] Step 14. Preparation of lithium 10-((3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(hydroxymethyl)phenyl)amino)-10-oxodecanoate 18 [ka] To a solution of 17 (2.9 g, 4.3 mmol) in THF (60 mL) was added water (15 mL) and lithium hydroxide (112 mg, 4.7 mmol). The solution was stirred overnight at room temperature. Upon completion, the solution was concentrated to remove the THF. The remaining aqueous solution was flash frozen in liquid nitrogen and lyophilized overnight to give a colorless solid (2.9 g, quantitative). Rf (0.3, 10% MeOH-CH2Cl2).

[0231] Step 15. Preparation of compound 19 [ka] To a solution of 14 (454 mg, 0.67 mmol), 18 (1.25 g, 0.67 mmol), and HBTU (381 mg, 1.0 mmol) in anhydrous DMF (25 mL) was added N,N-diisopropylethylamine (0.35 mL, 2.0 mmol). The solution was stirred overnight at room temperature. Upon completion, the solution was taken up in ethyl acetate (250 mL) and washed with brine (3×200 mL). The ethyl acetate layer was dried over magnesium sulfate, filtered, and concentrated to dryness. Purification by column chromatography (5%→7.5%→10%→15% MeOH in CH2Cl2) afforded 19 as a pale orange foam (1.5 g, 94%). Rf (0.25, 10% MeOH-CH2Cl2).

[0232] Step 16. Preparation of compound 20 [ka] A solution of compound 19 (1.5 g, 0.6 mmol), succinic anhydride (120 mg, 1.2 mmol), DMAP (220 mg, 1.8 mmol) and trimethylamine (250 μL, 1.8 mmol) in anhydrous CHCl (50 mL) was stirred overnight at room temperature. Upon completion, the solution was concentrated to dryness and filtered through a short plug of silica (100% CHCl→15% MeOH in CHCl) to give the product 20 as a pale beige foam (1.1 g, 70%). Mass m / z (ES-TOF MS) 727.7 [M + 3H - DMTr] + , 1091.1 [M + 2H - DMTr]. 1H NMR (400 MHz, CDCl3) δ 8.92 (br s, 1H), 7.78 (s, 1H), 7.49-7.47 (m, 3H), 7.41 (br s, 1H), 7.38-7.34 (m, 5H), 7.32-7.26 (m, 4H), 7.24-7.08 (br s, 3H), 7.08 (s, 1H), 6.90-6.80 (m, 7H), 5.31 (d, 3H, J=2.7Hz), 5.12 (s, 2H), 5.06 (dd, 3H, J=11.2, 3.2 Hz), 4.78 (d, 3H, J = 8.5 Hz), 4.24-4.08 (m, 12H), 3.95-3.88 (m, 7H), 3.85-3.76 (m, 4H), 3.78 (s, 6H), 3.68-3.56 (m, 34H), 3.54-3.44 (m, 8H), 3.41-3.33 (m, 6H), 2.70-2.60 (m, 4H), 2.52-2.30 (m, 30H), 2.24-2.16 (m, 8H), 2.14 (s, 9H), 2.04 (s, 9H), 2.02-1.96 (m, 6H), 1.98 (s, 9H), 1.96 (s, 9H), 1.74-1.52 (m, 4H), 1.36-1.24 (m, 12H).

[0233] Project 17.Construction of コンジュゲート1

change

[0234] Example 2: Synthesis of conjugate 34 Scheme 6. [ka]

[0235] Scheme 7. [ka]

[0236] Scheme 8. [ka]

[0237] Step 1. Preparation of di-tert-butyl 4-(2-(((benzyloxy)carbonyl)amino)acetamido)-4-(3-(tert-butoxy)-3-oxopropyl)heptanedioate 21 [ka] A solution of di-tert-butyl 4-amino-4-(3-(tert-butoxy)-3-oxopropyl)heptanedioate (25 g, 60 mmol) and Z-glycine (18.9 g, 90.2 mmol) in CHCl (300 mL) was treated sequentially with EDC (23 g, 120 mmol), diisopropylethylamine (32 mL, 180 mmol), and DMAP (catalytic amount, 17 mg). After stirring (16 h), the reaction mixture was poured into NaHCO3 (sat. aq.), extracted with CH2Cl2, washed with brine, dried (MgSO4), filtered and concentrated to give di-tert-butyl 4-(2-(((benzyloxy)carbonyl)amino)acetamido)-4-(3-(tert-butoxy)-3-oxopropyl)heptanedioate 21 as an amorphous solid, which was used without further treatment (36 g, quant.). Rf (0.85, 10% MeOH-CH2Cl2).

[0238] Step 2. Preparation of 4-(2-(((benzyloxy)carbonyl)amino)acetamido)-4-(2-carboxyethyl)heptanedioic acid 22 [ka] A solution of di-tert-butyl 4-(2-(((benzyloxy)carbonyl)amino)acetamido)-4-(3-(tert-butoxy)-3-oxopropyl)heptanedioate 21 (59.3 mmol, 36 g) was stirred in pure formic acid (150 mL) for 72 h. Upon completion, the formic acid was removed under reduced pressure and the crude solid was dried overnight under high vacuum to give 22 as a colorless solid (15.9 g, 61%). Rf (0.15, 10% MeOH-CH2Cl2).

[0239] Step 3. Preparation of compound 23 [ka] A solution of 22 (6.2 g, 14.1 mmol) and 2-(2-(2-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethan-1-aminium 2,2,2-trifluoroacetate (35 g, 56.5 mmol) in DMF (250 mL) was treated with BOP (25 g, 56.5 mmol) followed by N,N-diisopropylethylamine (29 mL, 170 mmol). After stirring overnight, the mixture was concentrated to dryness and chromatographed (100% CHCl to 15% MeOH-CHCl) to give compound 23 (24.6 g, 89%). Rf (0.55, 15% MeOH-CH2Cl2).

[0240] Step 4. Preparation of compound 24 [ka] Compound 23 (24.6 g) was dissolved in MeOH (200 mL) and TFA (1.5 mL) and purged with nitrogen. Palladium on charcoal (1 g, 10% w / w, wet basis) was added and the reaction mixture was then purged with hydrogen and stirred vigorously overnight. Upon completion, the reaction was purged with nitrogen, filtered through Celite, and rinsed with MeOH. The filtrate was concentrated and purified by silica gel 60 column chromatography (gradient: 5%→10%→20% MeOH-CH2Cl2) to give 24 (23 g) as a light brown viscous oil. Rf (0.32, 10% MeOH-CH2Cl2).

[0241] Step 5. Preparation of (5-amino-1,3-phenylene)dimethanol 26 [ka] A suspension of lithium aluminum hydride (13.6 g, 358 mmol) in anhydrous tetrahydrofuran (450 mL) was refluxed under nitrogen and treated dropwise with a solution of dimethyl-5-aminoisophthalate 25 (20 g, 96 mmol) in anhydrous tetrahydrofuran (350 mL). Upon completion of the addition, the mixture was heated at reflux for an additional 2 h. Upon completion, the solution was cooled to room temperature and quenched by the slow addition of MeOH (27 mL) followed by water (40 mL). After stirring for 2 h, the mixture was filtered, concentrated, and recrystallized from EtOAc to give (5-amino-1,3-phenylene)dimethanol 26 as off-white crystals (10.2 g, 70%). Rf 0.5 (15% MeOH-CH2Cl2).

[0242] Step 6. Preparation of 3,5-bis(hydroxymethyl)benzonitrile 27 [ka] A solution of 26 (5 g, 33 mmol) in 2N hydrochloric acid (100 mL) was cooled to 0 °C and treated with a cold solution of sodium nitrite (3.53 g, 36 mmol) in water (50 mL). The reaction mixture was kept at a temperature of ≦5 °C for 30 min and then rapidly treated with a solution of copper(I) cyanide (3.19 g, 35.6 mmol) and sodium cyanide (3.53 g, 72 mmol) in water (50 mL). After stirring overnight at room temperature, the mixture was filtered, extracted with dichloromethane (3 × 100 mL), concentrated, and used without further purification. The diol, 3,5-bis(hydroxymethyl)benzonitrile 27, was obtained as a yellow solid (2.19 g, 41%). Rf 0.75 (15% MeOH-CH2Cl2).

[0243] Step 7. Preparation of 3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(hydroxymethyl)benzonitrile 28 [ka] A solution of 3,5-bis(hydroxymethyl)benzonitrile 27 (538 mg, 3.3 mmol) in pyridine (14 mL) was treated with 4,4'-dimethoxytrityl chloride (1.17 g, 3.46 mmol) and stirred overnight at room temperature. Upon completion, the mixture was concentrated, dispersed in diethyl ether (25 mL), filtered, and concentrated. The crude product was purified by silica gel 60 column chromatography (gradient: 10% to 50% EtOAc-hexanes) to give 28 as a yellow solid (725 mg, 47%). Rf 0.5 (1:1 EtOAc-hexanes).

[0244] Step 8. Preparation of (3-(aminomethyl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)phenyl)methanol 29 [ka] A solution of 28 (100 mg, 0.22 mmol) in methyltetrahydrofuran (5 mL) was cooled to 0° C. and slowly treated with lithium aluminum hydride (0.64 mmol = 0.28 mL of a 2.3 M solution in MeTHF). After stirring for 1 h, the reaction was quenched by the addition of methanol (1 mL) followed by water (0.3 mL) and stirred for 30 min. The mixture was filtered and concentrated to give (3-(aminomethyl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)phenyl)methanol 29 (78 mg, 77%). Rf 0.15 (10% MeOH-CH2Cl2).

[0245] Step 9. Preparation of methyl 10-((3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(hydroxymethyl)benzyl)amino)-10-oxodecanoate 30 [ka] A solution of (3-(aminomethyl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)-methyl)phenyl)methanol 29 (78 mg, 0.17 mmol) and monomethyl sebacate (38 mg, 0.17 mmol) in dichloromethane (5 mL) was successively treated with EDC (48 mg, 0.25 mmol), DMAP (catalytic amount, 5 mg) and diisopropylethylamine (57 μL, 0.33 mmol). After stirring (3.5 h), the reaction mixture was poured into saturated sodium bicarbonate solution (50 mL). The sodium bicarbonate solution was extracted with dichloromethane (3×50 mL), washed with brine (50 mL), dried over magnesium sulfate, filtered and concentrated to dryness. The crude material was purified by silica gel 60 column chromatography (gradient: 2% to 5% MeOH-CH2Cl2) to give methyl 10-((3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(hydroxymethyl)benzyl)amino)-10-oxodecanoate 30 as a yellow oil (57 mg, 53%). Rf 0.45 (10% MeOH-CH2Cl2).

[0246] Step 10. Preparation of lithium 10-((3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(hydroxymethyl)benzyl)amino)-10-oxodecanoate 31 [ka] Compound 30 (188 mg, 0.28 mmol) was dissolved in tetrahydrofuran (5 mL) and treated with a solution of LiOH (7 mg, 0.30 mmol) in water (1 mL). Upon completion, the tetrahydrofuran was removed in vacuo and the remaining aqueous mixture was frozen and lyophilized to give lithium 10-((3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(hydroxymethyl)benzyl)amino)-10-oxodecanoate 31 as a colorless solid (180 mg, 99%). Rf 0.45 (10% MeOH-CH2Cl2).

[0247] Step 11. Preparation of compounds 32, 33 and 34 Compound 32 was prepared according to the same procedure used to synthesize compound 19, compound 33 was prepared according to the same procedure used to synthesize compound 20, and compound 34 was prepared according to the same procedure used to synthesize compound 1.

[0248] Example 3. Synthesis of conjugate 36 [ka]

[0249] Step 1. Preparation of conjugate 36 The same procedure used to synthesize compound 34 and all the corresponding intermediates was used to prepare conjugate 36. The only exception was the synthesis of compound 6, where propanoic anhydride was used instead of acetic anhydride.

[0250] Example 4. Synthesis of conjugate 42 Scheme 9. [ka]

[0251] Scheme 10. [ka]

[0252] Step 1. Preparation of compound 37 [ka] Diisopropylethylamine (2.75 mL, 15.9 mmol) was added to a solution of 18β-glycyrrhetinic acid (2.5 g, 5.3 mmol), tert-butyl (3-aminopropyl)carbamate (1.1 g, 6.4 mmol) and HBTU (3.0 g, 8.0 mmol) in N,N-dimethylformamide (20 mL). The solution was stirred overnight at room temperature. Upon completion, the solution was concentrated to dryness in vacuo. The residue was purified by silica gel 60 column chromatography (gradient: 2% to 5% MeOH / CH2Cl2) to give the product as a colorless solid (2.1 g, 63%).

[0253] Step 2. Preparation of compound 38 [ka] To a solution of 37 (2.1 g, 3.3 mmol) and triethylamine (3.5 mL, 10 mmol) in dichloromethane (25 mL) was added acetic anhydride (850 μL, 5.3 mmol) and DMAP (5 mg). The solution was stirred overnight at room temperature. Upon completion, the solution was concentrated to dryness, dissolved in ethyl acetate (100 mL), washed with water (100 mL), dried over magnesium sulfate, filtered, and concentrated to dryness to give a light brown foam (1.9 g, 85%).

[0254] Step 3. Preparation of compound 39 [ka] To a solution of 38 (1.5 g, 2.3 mmol) in anhydrous dioxane (25 mL) was added 2 M hydrogen chloride in dioxane (25 mL) The solution was stirred overnight at room temperature and then concentrated to dryness in vacuo to give a light brown solid (1.3 g, 96%).

[0255] Step 4. Preparation of compounds 40, 41 and 42 Compound 40 was prepared according to the same procedure used to synthesize compound 19, compound 41 was prepared according to the same procedure used to synthesize compound 20, and compound 42 was prepared according to the same procedure used to synthesize compound 1.

[0256] Example 5. Synthesis of conjugate 43 Scheme 11. [ka]

[0257] Scheme 12. [ka]

[0258] Step 1. Preparation of methyl 11-(2,6-bis(hydroxymethyl)-4-methylphenoxy)undecanoate 44 [ka] A solution of 2,6-bis(hydroxymethyl)-p-cresol (2.7 g, 16.3 mmol), methyl 11-bromoundecanoate (5.0 g, 17.9 mmol) and potassium carbonate (4.5 g, 32.6 mmol) in acetone (100 mL) was refluxed for 16 h. Upon completion, the solution was concentrated to dryness in vacuo, suspended in ethyl acetate (150 mL) and washed with water (2×100 mL) and brine (100 mL). The ethyl acetate layer was dried over magnesium sulfate, filtered and concentrated to dryness in vacuo. The residue was purified by silica gel 60 column chromatography (gradient: 100% Hex→50% EtOAc / Hex) to give methyl 11-(2,6-bis(hydroxymethyl)-4-methylphenoxy)undecanoate 44 as a colorless oil (1.6 g, 27%).

[0259] Step 2. Preparation of methyl 11-(2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-6-(hydroxymethyl)-4-methylphenoxy)undecanoate 45 [ka] To a solution of methyl 11-(2,6-bis(hydroxymethyl)-4-methylphenoxy)undecanoate 44 (1.5 g, 4.1 mmol) in anhydrous pyridine (20 mL) was added 4,4'-dimethoxytrityl chloride (1.4 g, 4.1 mmol). The solution was stirred overnight at room temperature. Upon completion, the solution was concentrated to dryness in vacuo and purified by silica gel 60 column chromatography (0.5-1% MeOH in CHCl) to give methyl 11-(2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-6-(hydroxymethyl)-4-methylphenoxy)undecanoate 45 as a pale yellow solid (1.1 g, 40%).

[0260] Step 3. Preparation of lithium 11-(2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-6-(hydroxymethyl)-4-methylphenoxy)undecanoate 46 [ka] To a solution of methyl 11-(2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-6-(hydroxymethyl)-4-methylphenoxy)undecanoate 45 (1.1 g, 1.7 mmol) in anhydrous tetrahydrofuran (40 mL) and water (10 mL) was added lithium hydroxide (44 mg, 1.8 mmol). The solution was concentrated in vacuo to remove all tetrahydrofuran. The remaining aqueous solution was flash frozen in liquid nitrogen and then lyophilized overnight to give lithium 11-(2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-6-(hydroxymethyl)-4-methylphenoxy)undecanoate 46 as a pale pink solid (1.1 g, 94%).

[0261] Step 4. Preparation of compound 47 To a solution of 10 (1.33 g, 0.66 mmol), 46 (0.5 g, 0.73 mmol), and HBTU (400 mg, 1 mmol) in N,N-dimethylformamide (25 mL) was added diisopropylethylamine (0.35 mL, 2 mmol). The solution was stirred overnight (18 h) at room temperature. Upon completion, the solvent was removed in vacuo and the residue was purified by silica gel column chromatography (gradient: 100% CH2Cl2 to 5% to 10% to 15% MeOH in CH2Cl2) to give 47 as a colorless solid (710 mg, 41%).

[0262] Step 5. Preparation of compound 48 To a solution of 47 (0.71 g, 0.3 mmol), triethylamine (0.4 mL, 3.0 mmol), and polystyrene-DMAP (loading of 3 mmol / g, 200 mg, 0.6 mmol) in dichloromethane (15 mL) was added succinic anhydride (60 mg, 0.6 mmol). The solution was stirred overnight at room temperature and upon completion, filtered and concentrated to dryness in vacuo. The residue was purified by silica gel 60 column chromatography (gradient: 5% to 20% MeOH in CHCl) to give 48 as a pale yellow solid (570 mg, 70%). 1H NMR (DMSO-d6, 400 MHz) δ 7.91 (m, 1H), 7.86-7.76 (m, 6H), 7.45-7.40 (m, 2H), 7.36-7.14 (m, 10H), 7.10 (s, 1H), 6.91 (d, J = 8.9 Hz, 4H), 5.21 (d, J = 3.3 Hz, 3H), 5.01 (s, 2H), 4.97 (dd, J = 11.2, 3.4 Hz, 3H), 4.56 (d, J = 8.5 Hz, 3H), 4.06-3.98 (m, 11H), 3.93-3.84 (m, 3H), 3.81-3.72 (m, 3H), 3.74 (s, 6H), 3.65-3.46 (m, 38H), 3.40-3.35 (m, 6H), 3.20-3.16 (m, 6H), 2.56-2.44 (m, 4H), 2.33 (s, 3H), 2.15-2.08 (m, 2H), 2.10 (s, 9H), 2.04-1.96 (m, 6H), 1.89 (s, 9H), 1.82-1.76 (m, 4H), 1.77 (s, 9H), 1.54-1.34 (m, 4H), 1.28-1.10 (m, 12H)

[0263] Project 6. Preparation of Compound 49 To a solution of 48 (100 mg, 40 μmol), N-hydroxysuccinimide (30 mg / mL in acetonitrile, 50 μL, 13 μmol), N,N'-diisopropylcarbodiimide (40 μL, 264 μmol) and pyridine (50 μL) in dichloromethane (2 mL) and acetonitrile (3 mL) was added 1000 Å lcaa CPG (prime synthesis, 920 mg). The solution was stirred overnight at room temperature on an orbital shaker. TLC analysis of the reaction solution indicated that the activated N-hydroxysuccinate ester was only partially consumed, so additional CPG (500 mg) was added. The solution was again stirred overnight. Upon completion, the CPG was filtered and washed with dichloromethane (25 mL), acetonitrile (25 mL) and tetrahydrofuran (25 mL). Unreacted amine residues on the CPG were acetylated (capped) by adding a 1:1 solution of acetic anhydride in acetonitrile (3 mL) and 10% N-methylimidazole / 10% pyridine in tetrahydrofuran (3 mL). The suspension was allowed to stand for 2 h, then filtered and rinsed with equal volumes of tetrahydrofuran (25 mL), acetonitrile (25 mL), and dichloromethane (25 mL). The loaded CPG49 was dried overnight under high vacuum. A standard DMT loading assay (3% trichloroacetic acid in CHCl, UV-VIS, A) was used. 504 ), the ligand loading efficiency was found to be 22 μmol / g.

[0264] Step 7. Preparation of conjugate 43 The resulting GalNAc-loaded CPG solid support 49 was used in automated oligonucleotide synthesis using standard procedures. Deprotection of the nucleotides was followed by cleavage from the solid support (with simultaneous deprotection of the galactosamine acetate) to give the GalNAc-oligonucleotide conjugate 43.

[0265] Example 6. Synthesis of conjugate 50 Scheme 13. [ka]

[0266] Scheme 14. [ka]

[0267] Step 1. Preparation of 2-((2-((tert-butyldimethylsilyl)oxy)ethyl)amino)ethan-1-ol 51 [ka] A solution of ethanolamine (77 mL, 1.25 mol) and (2-bromoethoxy)-tert-butyldimethylsilane (15 g, 62.7 mmol) in anhydrous acetonitrile (200 mL) was refluxed for 3 h. Upon completion, the reaction was cooled to room temperature, diluted with water (400 mL) and extracted with ethyl acetate (3×150 mL). The combined ethyl acetate extracts were dried over magnesium sulfate, filtered and concentrated to dryness in vacuo. The residue was purified by filtration through a silica pad first with 50% ethyl acetate / hexanes and then with 50% MeOH / EtOAc to give 51 as a pale yellow oil (14 g, 100%).

[0268] Step 2. Preparation of 2-(bis(4-methoxyphenyl)(phenyl)methoxy)-N-(2-((tert-butyldimethylsilyl)oxy)ethyl)ethan-1-amine 52 [ka] To a solution of 2-((2-((tert-butyldimethylsilyl)oxy)ethyl)amino)ethan-1-ol 51 (14 g, 64 mmol) and triethylamine (17.5 mL, 128 mmol) in anhydrous dichloromethane (250 mL) was added 4,4'-dimethoxytrityl chloride (24 g, 70 mmol). The solution was stirred overnight at room temperature and then concentrated to dryness in vacuo. The residue was dissolved in ethyl acetate (300 mL) and washed with water (250 mL) and brine (250 mL). The ethyl acetate was dried over magnesium sulfate, filtered, and concentrated to dryness in vacuo. Purification by silica gel 60 column chromatography (1% to 5% MeOH in CHCl) gave 52 as a pale yellow viscous oil (13 g, 39%).

[0269] Step 3. Preparation of methyl 10-((2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)(2-((tert-butyldimethylsilyl)oxy)ethyl)amino)-10-oxodecanoate 53 [ka] A solution of 2-(bis(4-methoxyphenyl)(phenyl)methoxy)-N-(2-((tert-butyldimethylsilyl)oxy)ethyl)ethan-1-amine 52 (5.4 g, 10.3 mmol), monomethyl sebacate (2.2 g, 10.3 g), HBTU (4.9 g, 12.9 mmol), and DIPEA (5.3 mL, 30.9 mmol) in N,N-dimethylformamide (100 mL) was stirred for 3 h at room temperature. Upon completion, the solution was poured into water (400 mL) and extracted with ethyl acetate (1×500 mL). The ethyl acetate extract was washed with brine (2×250 mL), dried over magnesium sulfate, filtered, and concentrated to dryness in vacuo. Purification by silica gel 60 column chromatography (10% to 25% ethyl acetate in hexanes) afforded 53 as a yellow viscous oil (6.5 g, 87%).

[0270] Step 4. Preparation of methyl 10-((2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)(2-hydroxyethyl)amino)-10-oxodecanoate 54 [ka] To a solution of methyl 10-((2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)(2-((tert-butyldimethylsilyl)oxy)ethyl)amino)-10-oxodecanoate 53 (2.0 g, 2.8 mmol) and triethylamine (1 mL) in anhydrous tetrahydrofuran (20 mL) was added TBAF (1 M in THF, 3.4 mL, 3.3 mmol). The solution was stirred for 6 h, but only partial conversion was observed by TLC (5% MeOH in CHCl). An additional 1.7 mL of TBAF was added and the solution was stirred overnight at room temperature. Upon completion, the solution was concentrated in vacuo and purified by silica gel 60 column chromatography (10% to 50% EtOAc in hexanes → 100% EtOAc) to give 54 as a colorless viscous oil (0.5 g, 29%).

[0271] Step 5. Preparation of lithium 10-((2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)(2-hydroxyethyl)amino)-10-oxodecanoate 55 [ka] To a solution of methyl 10-((2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)(2-hydroxyethyl)amino)-10-oxodecanoate 54 (0.5 g, 0.83 mmol) in THF (40 mL) was added water (10 mL) and lithium hydroxide (24 mg, 1.0 mmol). The solution was stirred overnight at room temperature and then concentrated in vacuo to remove the THF. The remaining aqueous solution was flash frozen in liquid nitrogen and lyophilized to give 55 as a colorless solid (485 mg, 95%).

[0272] Step 6. Preparation of compounds 56, 57, 58 and 50 Compound 56 was prepared using the same procedure as that used to synthesize compound 47, compound 57 was prepared using the same procedure as that used to synthesize compound 48, compound 58 was prepared using the same procedure as that used to synthesize compound 49, and compound 50 was prepared using the same procedure as that used to synthesize compound 43.

[0273] Example 7. Synthesis of conjugate 59 Scheme 15. [ka]

[0274] Scheme 16. [ka]

[0275] Step 1. Preparation of methyl (2R,5R)-5-hydroxypiperidine-2-carboxylate 61 [ka] (2R,5R)-5-hydroxypiperidine-2-carboxylic acid 60 (3.5 g, 24.1 mmol) was stirred in MeOH (50 mL). HCl (g) was bubbled through the solution for 2 min and the reaction was stirred at reflux for 1.5 h. The reaction was concentrated in vacuo to give methyl (2R,5R)-5-hydroxypiperidine-2-carboxylate 61 in quantitative yield, which was used without further purification.

[0276] Step 2. Preparation of 1-(tert-butyl) 2-methyl (2R,5R)-5-hydroxypiperidine-1,2-dicarboxylate 62 [ka] Methyl (2R,5R)-5-hydroxypiperidine-2-carboxylate 61 (24.1 mmol) and TEA (7.2 mL, 53.02 mmol) were stirred in DCM (100 mL) at room temperature. Di-tert-butyl-dicarbonate (5.7 g, 26.5 mmol) was added portionwise and the reaction was stirred for 2 h. The reaction was diluted with DCM (100 mL) and washed successively with 1 M HCl (2×75 mL), saturated NaHCO3 (2×75 mL), H2O (2×75 mL) and saturated NaCl solution (2×75 mL). The organics were separated, dried (Na2SO4) and concentrated in vacuo to give 1-(tert-butyl) 2-methyl(2R,5R)-5-hydroxypiperidine-1,2-dicarboxylate 62 (5.53 g, 88%), which was used without further purification.

[0277] Step 3. Preparation of tert-butyl (2R,5R)-5-hydroxy-2-(hydroxymethyl)piperidine-1-carboxylate 63 [ka] (2R,5R)-1-(tert-butoxycarbonyl)-5-hydroxypiperidine-2-carboxylic acid 62 (5.53 g, 21.4 mmol) was stirred in THF at 0° C. LiBH4 (3.0 M in THF) (8.9 mL, 27.7 mmol) was added dropwise over 1 h. The reaction was allowed to warm to room temperature and stirring was continued for 16 h. The reaction was quenched with 1 M NaOH, the THF was removed in vacuo, and the aqueous was exhaustively extracted with EtOAc (10×100 mL). The combined organics were washed with H2O (50 mL), saturated NaCl solution (2×50 mL), dried (Na2SO4), and concentrated in vacuo to give tert-butyl (2R,5R)-5-hydroxy-2-(hydroxymethyl)piperidine-1-carboxylate 63 (2.4 g, 49.0%), which was used without further purification.

[0278] Step 4. Preparation of (3R,6R)-6-(hydroxymethyl)piperidin-3-ol 64 [ka] tert-Butyl (2R,5R)-5-hydroxy-2-(hydroxymethyl)piperidine-1-carboxylate 63 (2.4 g, 10.4 mmol) was stirred in EtO at room temperature. HCl (g) was bubbled in for 45 s and the reaction was stirred at room temperature for 45 min. The reaction was concentrated in vacuo and dried under high vacuum to give (3R,6R)-6-(hydroxymethyl)piperidin-3-ol 64. This product was used without further purification.

[0279] Step 5. Preparation of 2,2,2-trifluoro-1-((2R,5R)-5-hydroxy-2-(hydroxymethyl)piperidin-1-yl)ethan-1-one 65 [ka] The crude (3R,6R)-6-(hydroxymethyl)piperidin-3-ol 64 from the above reaction was stirred in MeCN (50 mL) with TEA (3.5 mL, 25.2 mmol) at room temperature. Ethyl trifluoroacetate (3 mL, 25.2 mmol) was added and the reaction was stirred at room temperature for 16 h and then concentrated in vacuo to give 2,2,2-trifluoro-1-((2R,5R)-5-hydroxy-2-(hydroxymethyl)piperidin-1-yl)ethan-1-one 65. This product was used without further purification.

[0280] Step 6. Preparation of 1-((2R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-hydroxypiperidin-1-yl)-2,2,2-trifluoroethan-1-one 66 [ka] The crude 2,2,2-trifluoro-1-((2R,5R)-5-hydroxy-2-(hydroxymethyl)piperidin-1-yl)ethan-1-one 65 from the above reaction was stirred in DCM with TEA (50 mL) at room temperature. 4,4'-Dimethoxytrityl chloride (DMTrCl) (3.87 g, 11.44 mmol) was added in one portion and the reaction was stirred at room temperature for 3 h. The reaction was diluted with DCM (50 mL) and washed successively with saturated NaHCO3 (2 x 75 mL), HO (2 x 75 mL) and saturated NaCl solution (2 x 75 mL). The organics were separated, dried (Na2SO4), concentrated in vacuo and purified by column chromatography (100% hexanes to 60% EtOAc / hexanes) (0.1% TEA) to give 1-((2R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-hydroxypiperidin-1-yl)-2,2,2-trifluoroethan-1-one 66 (3.14 g, 57%).

[0281] Step 7. Preparation of (3R,6R)-6-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-piperidin-3-ol 67 [ka] 1-((2R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-hydroxypiperidin-1-yl)-2,2,2-trifluoroethan-1-one 66 (3.14 g, 6.0 mmol) was stirred in MeOH (50 mL) at room temperature. KOH (672 mg, 12 mmol) was added and the reaction was stirred at room temperature for 16 h. Additional KOH (300 mg, 6 mmol) was added and stirring was continued for an additional 24 h. The reaction was concentrated in vacuo, taken up in DCM (150 mL), washed with HO (4×50 mL), dried (NaSO) and concentrated in vacuo to give (3R,6R)-6-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)piperidin-3-ol 67 (2.34 g, 90%), which was used without further purification.

[0282] Step 8. Preparation of methyl 12-((2R,5R)-2-((bis(4-methoxyphenyl)(phenyl)-methoxy)methyl)-5-hydroxypiperidin-1-yl)-12-oxododecanoate 68 [ka] (3R,6R)-6-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)piperidin-3-ol 67 (2.34 g, 5.34 mmol) was stirred in DCM (75 mL) at room temperature. Triethylamine (2.2 mL, 16.2 mmol), HATU (3.5 g, 9.2 mmol) and 12-methoxy-12-oxododecanoic acid (1.32 g, 5.4 mmol) were added and the reaction was stirred at room temperature for 3 h. The resulting solid precipitate was removed by filtration, the filtrate was concentrated in vacuo and the residue was purified by column chromatography (2.5% MeOH / DCM, 0.1% TEA) to give methyl 12-((2R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-hydroxypiperidin-1-yl)-12-oxododecanoate 68 in quantitative yield.

[0283] Step 9. Preparation of lithium 12-((2R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)-methyl)-5-hydroxypiperidin-1-yl)-12-oxododecanoate 69 [ka] Methyl 12-((2R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-hydroxypiperidin-1-yl)-12-oxododecanoate 68 (5.4 mmol) and LiOH (140 mg, 5.94 mmol) were stirred in THF:HO (1:1, 100 mL) at room temperature for 48 h. The THF was removed in vacuo and the aqueous was frozen and lyophilized to give lithium 12-((2R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-hydroxypiperidin-1-yl)-12-oxododecanoate 69 (3.2 g, 91%), which was used in the next reaction without further purification.

[0284] Step 10. Preparation of compounds 70, 71, 72 and 59 Compound 70 was prepared using the same procedure used to synthesize compound 47, compound 71 was prepared using the same procedure used to synthesize compound 48, compound 72 was prepared using the same procedure used to synthesize compound 49, and compound 59 was prepared using the same procedure used to synthesize compound 43.

[0285] Example 8. Synthesis of conjugate 142 Scheme 17. [ka]

[0286] Scheme 18. [ka]

[0287] Step 1. Preparation of 3,4,5-triacetoxybenzoic acid 73 Gallic acid (20 g) was dissolved in pyridine (50 mL) and acetic anhydride (50 mL). The solution was stirred overnight at room temperature and then poured into ice-water (1 L). The solution was acidified with concentrated hydrochloric acid, causing a colorless solid to precipitate. The solid was collected by filtration and washed with water (5 x 100 mL). The wet solid was frozen in liquid nitrogen and lyophilized to give 3,4,5-triacetoxybenzoic acid (26 g, 75%).

[0288] Step 2.5-((2-((2-oxo-2-phenyl-1λ 2 Preparation of (ethyl)amino)ethyl)carbamoyl)benzene-1,2,3-triyl triacetate 74 To a solution of 3,4,5-triacetoxybenzoic acid (10 g, 33.8 mmol), N-carbobenzoxy-1,2-diaminoethane hydrochloride (5.3 g, 33.8 mmol) and HBTU (13.5 g, 35.5 mmol) in DMF (200 mL) was added DIPEA (17.5 mL, 101 mmol). The solution was stirred for 16 h, then diluted with ethyl acetate (250 mL), washed with brine (3×200 mL), dried over magnesium sulfate, filtered and concentrated to dryness in vacuo. The crude product was purified by silica gel column chromatography (gradient: 1% to 5% MeOH in DCM) to give 5-((2-((2-oxo-2-phenyl-1λ 2 The resulting product was 1,2,3-triyl-1,3-dihydro-2,4-triphenylphosphine triacetate (5.5 g) as an off-white solid.

[0289] Step 3. 3,4,5-Trihydroxy-N-(2-((2-oxo-2-phenyl-1λ) 2 Preparation of (ethyl)amino)ethyl)benzamide 75 5-((2-((2-oxo-2-phenyl-1λ 2A solution of 3,4,5-trihydroxy-N-(2-((2-oxo-2-phenyl-1λ-ethyl)amino)ethyl)carbamoyl)benzene-1,2,3-triyl triacetate (5 g, 1.1 mmol) in 1:1 MeOH / CHCl (100 mL) was stirred at room temperature for 3 days. Upon completion, the solvent was removed to give 3,4,5-trihydroxy-N-(2-((2-oxo-2-phenyl-1λ-ethyl)amino)ethyl)carbamoyl)benzene-1,2,3-triyl triacetate. 2 The resulting mixture was treated with 100 ml of 1-ethylaminoethylbenzamide as a colorless solid (4 g, quantitative).

[0290] Step 4. Trimethyl 2,2',2"-((5-((2-((2-oxo-2-phenyl-1λ 2 Preparation of 1,2,3-ethyl)amino)ethyl)carbamoyl)benzene-1,2,3-triyl)tris(oxy))triacetate 76 3,4,5-Trihydroxy-N-(2-((2-oxo-2-phenyl-1λ 2 A solution of 2,2',2"-((5-((2-((2-oxo-2-phenyl-1λ-ethyl)amino)ethyl)benzamide (4 g, 11.6 mmol), methyl bromoacetate (7.7 g, 46.4 mmol) and potassium carbonate (9.6 g, 69.4 mmol) in DMF (100 mL) was stirred overnight at 60 °C. Upon completion, the solution was cooled to room temperature, diluted with ethyl acetate (200 mL), washed with water (200 mL), brine (3 x 100 mL), dried over magnesium sulfate, filtered and concentrated to dryness in vacuo. The crude product was purified by silica gel column chromatography (gradient: 2% to 10% MeOH in DCM) to give trimethyl 2,2',2"-((5-((2-((2-oxo-2-phenyl-1λ-ethyl)amino)ethyl)benzamide (4 g, 11.6 mmol), methyl bromoacetate (7.7 g, 46.4 mmol) and potassium carbonate (9.6 g, 69.4 mmol) in DMF (100 mL). 2 To the reaction mixture was obtained 1,2,3-triyl-1,2,3-tris(oxy)-triacetate (5 g, 79%) as a beige solid.

[0291] Step 5. 2,2',2"-((5-((2-((2-oxo-2-phenyl-1λ 2 Preparation of (1,2,3-ethyl)amino)ethyl)carbamoyl)benzene-1,2,3-triyl)tris(oxy))triacetic acid 77 Trimethyl 2,2',2”-((5-((2-((2-oxo-2-phenyl-1λ2 A solution of 2,2',2"-((5-((2-((2-oxo-2-phenyl-1λ-phenyl)-2,3-dihydro-1,4-dihydro-2,4-dihydro-1,5-trimethyl-1,6-dihydro-1,7-trimethyl-1,8-triphenyl-2,5-dihydro-1,9-triphenyl-2,6-dihydro-1,8-triphenyl-2,5-dihydro-1,9-triphenyl-2,6-dihydro-1,9-triphenyl-2,5-dihydro-1,8-triphenyl-2,5-dihydro-1,9 ... 2 The resulting mixture was treated with 1,2,3-triyl-1,2,3-triphenylamine (1,2,3-triyl)tris(oxy)triacetic acid as a colorless solid (2.3 g, 50%).

[0292] Step 6. Preparation of compound 78 Compound 78 was prepared from compounds 9 (2.75 g, 4.3 mmol) and 77 (0.5 g, 0.96 mmol) using the same procedure as used for compound 13. Yield: 600 mg.

[0293] Step 7. Preparation of compound 79 Compound 79 was prepared from compound 78 (0.6 g) using the same procedure as used for compound 14. Yield: 500 mg.

[0294] Step 8. Preparation of compound 140 Compound 140 was prepared from compound 79 (500 mg, 0.25 mmol) and compound 18 (175 mg, 0.25 mmol) using the same procedure as used for compound 19. Yield: 250 mg, 44%.

[0295] Step 9. Preparation of compound 141 Compound 141 was prepared from compound 140 (250 mg, 0.11 mmol) using the same procedure as used for compound 20. Yield: 200 mg.

[0296] Step 10. Preparation of conjugate 142 Conjugate 142 was prepared from 141 (200 mg) and 1000A lcaa CPG (1.8 g) using the same procedure as used for compound 1. Yield: 1.9 g, 22 μmol per gram of CPG loading. The resulting GalNAc-loaded CPG solid support was used in automated oligonucleotide synthesis using standard procedures. Deprotection of the nucleotides was followed by cleavage from the solid support (with simultaneous deprotection of the galactosamine acetate) to give GalNAc-oligonucleotide conjugate 142.

[0297] Example 9. Synthesis of conjugate 145 Scheme 19. [ka]

[0298] Scheme 20. [ka]

[0299] Step 1. Preparation of (3aR,6aS)-5-benzyl-3a,6a-dimethyltetrahydro-1H-furo[3,4-c]pyrrole-1,3(3aH)-dione 123 Trifluoroacetic acid (75 μL) was added slowly to a cold (0 °C) solution of 3,4-dimethylfuran-2,5-dione (3 g, 24 mmol) and N-benzyl-1-methoxy-N-((trimethylsilyl)methyl)methanamine (7 g, 29.8 mmol) in dichloromethane (75 mL). Stir overnight and allow the solution to warm slowly to room temperature as the ice bath melts. The reaction mixture was concentrated to dryness, dissolved in ethyl acetate (100 mL), washed with saturated sodium bicarbonate (2 x 100 mL), dried over magnesium sulfate, filtered, and concentrated to dryness. Purification by silica gel column chromatography (gradient: 20% ethyl acetate in hexane to 100% ethyl acetate) gave (3aR,6aS)-5-benzyl-3a,6a-dimethyltetrahydro-1H-furo[3,4-c]pyrrole-1,3(3aH)-dione as a yellow oil (3.5 g, 56%).

[0300] Step 2. Preparation of ((3R,4S)-1-benzyl-3,4-dimethylpyrrolidine-3,4-diyl)dimethanol 124 To a cold (0° C.) solution of (3aR,6aS)-5-benzyl-3a,6a-dimethyltetrahydro-1H-furo[3,4-c]pyrrole-1,3(3aH)-dione (3.5 g, 13.4 mmol) in anhydrous diethyl ether (50 mL) was slowly added lithium aluminum hydride pellets (1.5 g, 40 mmol) in three portions. The solution was stirred overnight and allowed to warm to room temperature as the ice-water bath melted. Upon completion, the reaction was cooled to 0° C. and quenched very slowly with 1.5 mL of 5M NaOH, followed by 1.5 mL of water. After stirring for 30 min, magnesium sulfate was added and filtered. The filtrate was concentrated to give ((3R,4S)-1-benzyl-3,4-dimethylpyrrolidine-3,4-diyl)dimethanol as a colorless oil (2.7 g).

[0301] Step 3. Preparation of ((3R,4S)-3,4-dimethylpyrrolidine-3,4-diyl)dimethanol 125 To a solution of ((3R,4S)-1-benzyl-3,4-dimethylpyrrolidine-3,4-diyl)dimethanol (10 g, 40 mmol) in methanol (10 mL) was added 10% palladium-wet activated carbon (1 g). The solution was stirred vigorously under a hydrogen atmosphere for 16 h. Upon completion, the solution was filtered through Celite and concentrated to dryness to give ((3R,4S)-3,4-dimethylpyrrolidine-3,4-diyl)dimethanol as a colorless solid (5.5 g, 86%).

[0302] Step 4. Preparation of methyl 10-((3R,4S)-3,4-bis(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoate 126 Compound 126 was prepared from compound 125 (1.3 g, 8.2 mmol) and monomethyl sebacate (1.8 g, 8.2 mmol) using the same procedure as used for compound 17. Yield: 1.8 g, 61%.

[0303] Step 5. Preparation of methyl 10-((3R,4S)-3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoate 127 Compound 127 was prepared from compound 126 (1.8 g, 5.0 mmol) and 4,4'-dimethoxytrityl chloride (1.7 g, 5.0 mmol) using the same procedure as used for compound 18. Yield: 1.4 g, 42%.

[0304] Step 6. Preparation of lithium 10-((3R,4S)-3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoate 128 Lithium hydroxide (121 mg, 5.0 mmol) was added to a solution of compound 127 (3.0 g, 4.6 mmol) in THF (50 mL) and water (50 mL). The solution was stirred for 4 h at room temperature and then concentrated to remove the THF. The remaining aqueous solution was lyophilized overnight to give a pale pink solid (2.9 g, quantitative).

[0305] Step 7. Preparation of compound 143 Compound 143 was prepared from compound 128 (270 mg, 0.42 mmol) and compound 14 (800 mg, 0.42 mmol) using the same procedure as used for compound 19. Yield: 900 mg, 87%.

[0306] Step 8. Preparation of compound 144 Compound 144 was prepared from compound 143 (500 mg, 0.2 mmol) using the same procedure as used for compound 20. Yield: 200 mg.

[0307] Step 9. Preparation of conjugate 145 Conjugate 145 was prepared from 144 (200 mg) and 1000A lcaa CPG (1.8 g) using the same procedure as that used for compound 1. Yield: 1.9 g, 20 μmol per gram of CPG loading. The resulting GalNAc-loaded CPG solid support was used in automated oligonucleotide synthesis using standard procedures. Deprotection of the nucleotides was followed by cleavage from the solid support (with simultaneous deprotection of the galactosamine acetate) to give GalNAc-oligonucleotide conjugate 145.

[0308] Example 10. Synthesis of conjugate 150 Scheme 21. [ka]

[0309] Step 1. Preparation of 146-1 To a solution of the monomethyl ester of dodecanedioic acid (12.2 g, 50.0 mmol) in dichloromethane (300 mL) was added N-hydroxysuccinimide (6.10 g, 53.0 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (10.52 g, 55.0 mmol). The cloudy mixture was stirred overnight at room temperature and the reaction became a clear solution. TLC showed the reaction was complete. The organics were washed with saturated NH4Cl (300 mL) and brine (100 mL). The organic layer was separated, dried over MgSO4, and concentrated to dryness to give pure 1-(2,5-dioxopyrrolidin-1-yl) 12-methyldodecanedioate 146-1 as a white solid (16.7 g, 97.8%).

[0310] Step 2. Preparation of cyclopent-3-en-1-ylmethanol 146-2 To a suspension of lithium aluminum hydride (15.2 g, 0.40 mol) in anhydrous ether (1 L) at 0° C. under nitrogen, a solution of methyl cyclopent-3-enecarboxylate (50 g, 0.40 mol) in ether (300 mL) was added dropwise over 5 h. The suspension was stirred at room temperature overnight. TLC showed the reaction was complete. The reaction was recooled to 0° C. A saturated solution of Na2SO4 (32 mL) was added dropwise to quench the reaction. After the addition was complete, the mixture was stirred for an additional 3 h and filtered through a pad of Celite. The solvent was evaporated to give cyclopent-3-enylmethanol 146-2 (37.3 g, 95%) as a colorless liquid.

[0311] Step 3. Preparation of (6-oxabicyclo[3.1.0]hexan-3-yl)methanol 146-3 To a solution of cyclopent-3-enylmethanol 146-2 (4.0 g, 41 mmol) in dichloromethane (150 mL) at 0 °C was added 3-chloroperbenzoic acid (10 g, 45 mmol, 77% purity) in portions. The reaction was stirred overnight. Dichloromethane (150 mL) was added. The organics were washed with sodium thiosulfate (12 g in 10 mL water) and then with saturated NaHCO3 (40 mL). This was repeated until all remaining 3-chloroperbenzoic acid had been washed away. The organics were dried over MgSO4. The solvent was evaporated to give a mixture of cis-6-oxabicyclo[3.1.0]hexan-3-ylmethanol 146-3 (2.6 g, 57%) as a yellow oil. GC-MS: m / z 114 (5) (M + ), 95 (15), 88 (100), 81 (15).

[0312] Step 4. Preparation of 2-amino-4-(hydroxymethyl)cyclopentan-1-ol 146-4 A solution of 6-oxabicyclo[3.1.0]hexan-3-ylmethanol 146-3 (2.0 g, 17.6 mmol) in methanol (20 mL) at 0° C. was purged with ammonia gas for 10 min. The reaction was stirred at room temperature overnight. TLC showed the reaction was incomplete. Methanol was removed and NH3H2O ​​(50 mL) was added and this was stirred at room temperature for 1 week. TLC confirmed the reaction was complete. Water was removed by azeotroping with ethanol to give 2-amino-4-(hydroxymethyl)cyclopentanol 146-4 (2.1 g, 91%) as a yellow oil.

[0313] Step 5. Preparation of methyl 12-(2-hydroxy-4-(hydroxymethyl)cyclopentylamino)-12-oxododecanoate 146-5 Compound 146-5 was prepared from 2-amino-4-(hydroxymethyl)cyclopentanol 146-4 and 1-(2,5-dioxopyrrolidin-1-yl)12-methyldodecanedioate 146-1 using the same procedure described for the synthesis of 12-(2-(tert-butoxycarbonylamino)ethylamino)-12-oxododecanoate (3-2). Methyl 12-(2-hydroxy-4-(hydroxymethyl)cyclopentylamino)-12-oxododecanoate 146-5 was obtained in 87.4% yield as an off-white solid.

[0314] Step 6. Preparation of compound 147 Compound 147 was quantitatively prepared from compound 146 (1.4 g, 2.33 mmol) using the same procedure as used for compound 18.

[0315] Step 7. Preparation of compound 148 Compound 148 was prepared from compound 147 (150 mg, 0.23 mmol) and compound 14 (431 mg, 0.23 mmol) using the same procedure as used for compound 19. Yield: 460 mg, 84%.

[0316] Step 8. Preparation of compound 149 Compound 149 was prepared from compound 148 (460 mg, 0.19 mmol) using the same procedure as used for compound 20. Yield: 436 mg, 91%.

[0317] Step 9. Preparation of conjugate 150 Compound 150 was prepared from compound 149 (436 mg) and 1000A lcaa CPG (2.62 g) using the same procedure as used for compound 1. Yield: 2.7 g, 21.3 μmol per gram of CPG loading. The resulting GalNAc-loaded CPG solid support was used in automated oligonucleotide synthesis using standard procedures. Deprotection of the nucleotides was followed by cleavage from the solid support (with simultaneous deprotection of the galactosamine acetate) to give GalNAc-oligonucleotide conjugate 150.

[0318] Example 11. Synthesis of conjugates 153, 158, 163, 168 and 173 Scheme 22. [ka]

[0319] Step 1. Preparation of 1-(tert-butyl) 2-methyl (2S,4R)-4-hydroxypyrrolidine-1,2-dicarboxylate (133) Methyl (2S,4R)-4-hydroxypyrrolidine-2-carboxylate (25.9 g, 46 mmol), BOC anhydride (65.9 g, 302.5 mmol) and TEA (42 mL, 302.5 mmol) were stirred in DCM at room temperature for 16 h. The organics were washed successively with 1M HCl (×2), saturated NaHCO3 (×2), HO and brine, dried and concentrated in vacuo to give 1-(tert-butyl) 2-methyl(2S,4R)-4-hydroxypyrrolidine-1,2-dicarboxylate (133) (58.1 g, 85%).

[0320] Step 2. Preparation of 1-(tert-butyl) 2-methyl (4R)-4-hydroxy-2-methylpyrrolidine-1,2-dicarboxylate (134) 1-(tert-butyl) 2-methyl(2S,4R)-4-hydroxypyrrolidine-1,2-dicarboxylate (133) (5 g, 20.4 mmol) and MeI (12 g, 84.5 mmol) were stirred in anhydrous THF at -40 °C. LDA (2.0 M in THF) (37.5 mL, 75 mmol) was added dropwise. The reaction was allowed to warm to room temperature and stirred for 4 h before being quenched with saturated NH4Cl. The reaction was extracted with EtOAc, washed with H2O and brine, dried (Na2SO4), and concentrated in vacuo. The residue was purified by column chromatography (50:50 EtOAc / Hexanes) to give 1-(tert-butyl) 2-methyl(4R)-4-hydroxy-2-methylpyrrolidine-1,2-dicarboxylate (134) as a racemic mixture (3.6 g, 68%).

[0321] Step 3. Preparation of tert-butyl (2S,4R)-4-hydroxy-2-(hydroxymethyl)-2-methylpyrrolidine-1-carboxylate (135a) 1-(Tert-butyl) 2-methyl (4R)-4-hydroxy-2-methylpyrrolidine-1,2-dicarboxylate (134) (19 g, 73.5 mmol) was stirred in anhydrous THF under N2. LiBH4 solution (48 ml, 96 mmol) was added dropwise and the reaction was stirred at room temperature for 48 h. The reaction was quenched with 1M NaOH, THF was removed in vacuo, and the residue was extracted with EtOAc (4 x 100 ml). The organics were washed with H2O, brine, dried (Na2SO4), and concentrated in vacuo. The residue was purified by column chromatography (5% MeOH / DCM) to give tert-butyl (2S,4R)-4-hydroxy-2-(hydroxymethyl)-2-methylpyrrolidine-1-carboxylate (135a) as the major product (8 g, 47%). The structure was assigned according to the literature references.

[0322] Step 4. Preparation of (3R,5S)-5-(hydroxymethyl)-5-methylpyrrolidin-3-ol hydrochloride (136) tert-Butyl (2S,4R)-4-hydroxy-2-(hydroxymethyl)-2-methylpyrrolidine-1-carboxylate (135a) (8 g, 34.6 mmol) was stirred in EtOAc at room temperature and gaseous HCl was added for about 2 min. The reaction was stirred for 1 h, then concentrated in vacuo and dried under high vacuum to give (3R,5S)-5-(hydroxymethyl)-5-methylpyrrolidin-3-ol hydrochloride (136) in quantitative form.

[0323] Step 5. Preparation of methyl 12-((2S,4R)-4-hydroxy-2-(hydroxymethyl)-2-methylpyrrolidin-1-yl)-12-oxododecanoate (137) (3R,5S)-5-(hydroxymethyl)-5-methylpyrrolidin-3-ol hydrochloride (136) (7.9 g, 47.4 mmol), 12-methoxy-12-oxododecanoic acid (11.5 g, 47.4 mmol), HBTU (36 g, 76 mmol) and TEA (20 mL, 142.2 mmol) in DCM were stirred at room temperature for 16 h. The precipitate was removed by filtration and the organics were washed with 1M HCl (×2), saturated NaHCO3 (×2), HO and brine. After drying, the organics were concentrated in vacuo and purified by column chromatography (5% MeOH / DCM) to give methyl 12-((2S,4R)-4-hydroxy-2-(hydroxymethyl)-2-methylpyrrolidin-1-yl)-12-oxododecanoate (137) (3.1 g, 18.3%).

[0324] Step 6. Preparation of methyl 12-((2S,4R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxy-2-methylpyrrolidin-1-yl)-12-oxododecanoate (138) Methyl 12-((2S,4R)-4-hydroxy-2-(hydroxymethyl)-2-methylpyrrolidin-1-yl)-12-oxododecanoate (137) (3.1 g, 9.0 mmol), DMTr-Cl (2.8 g, 8.2 mmol) and TEA (1.1 ml, 8.2 mmol) were stirred in DCM at room temperature for 16 h. The reaction was concentrated in vacuo and the residue was purified by column chromatography (5% MeOH / DCM, 0.1% TEA) to give methyl 12-((2S,4R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxy-2-methylpyrrolidin-1-yl)-12-oxododecanoate (138) (2.7 g, 45.5 mmol).

[0325] Scheme 23 [ka]

[0326] Step 7. Preparation of compound 154-1 Triethylamine (10.4 mL, 74.6 mmol) was added dropwise to a solution of N-(2-hydroxyethyl)phthalimide (4.80 g, 25.0 mmol) and 4,4'-dimethoxytrityl chloride (8.8 g, 26.0 mmol) in dichloromethane (200 mL) at 0° C. under nitrogen. The reaction mixture was stirred at room temperature for 3 h. TLC showed the reaction was complete. The organic layer was washed with brine (100 mL), dried over MgSO4, and concentrated to dryness. It was used directly in the next reaction without purification.

[0327] Step 8. Preparation of compound 154-2 2-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)isoindoline-1,3-dione (154-1) obtained above and hydrazine monohydrate (3.6 mL, 74 mmol) were stirred in ethanol (100 mL) overnight at room temperature. TLC showed the reaction was complete. The precipitate was filtered off. The filtrate was evaporated. The residue was taken up with ethyl acetate (100 mL). The organic solution was washed with 10% NaOH, water and brine, and dried over MgSO4. The solvent was evaporated to give 2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethanamine (154-2) as a yellow liquid (8.11 g, 89.3% yield for two steps), which was used in the next reaction without further purification.

[0328] Step 9. Preparation of compound 154-3 To a solution of L-threonine (1.19 g, 10.0 mmol) and NaHCO3 (2.3 g, 27 mmol) in water (20 mL) and dioxane (10 mL) was added 1-(2,5-dioxopyrrolidin-1-yl) 12-methyldodecanedioate 146-1 (3.1 g, 9.1 mmol) in dioxane (10 mL). The reaction mixture was stirred at room temperature overnight. 4N HCl (10 mL) was added. The precipitate was collected by filtration and washed with water (3 x 10 mL). The solid was dried over P2O5 in a desiccator to give (2S,3R)-3-hydroxy-2-(12-methoxy-12-oxododecanamido)butanoic acid 154-3 as an off-white solid (2.84 g, 82.2%). LC-MS (ESI): m / z: 346 (100), (M + H + )

[0329] Step 10. Preparation of compound 154 (2S,3R)-3-Hydroxy-2-(12-methoxy-12-oxododecanamido)butanoic acid 154-3 (2.47 g, 7.15 mmol), 2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethanamine 154-2 (2.60 g, 7.15 mmol), EDC (1.64 g, 8.58 mmol), 1-hydroxybenzotriazole (HOBt) (1.16 g, 8.58 mmol) and TEA (2.4 mL, 17.2 mmol) were stirred in dichloromethane (72 mL) at room temperature for 2 h. Water (30 mL) was added. The organic layer was separated and washed with brine (2 x 30 mL). Evaporation of the solvent followed by column chromatography (30% ethyl acetate / hexane to 50% ethyl acetate / hexane) afforded methyl 12-((2S,3R)-1-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethylamino)-3-hydroxy-1-oxobutan-2-ylamino)-12-oxododecanoate 154 as a waxy yellow semi-solid (2.60 g, 52.6%). 1HNMR (400MHz, acetone-d6, ppm): δ 7.51 (t, J = 5.5 Hz, 1H), 7.45-7.49 (m, 2H), 7.28-7.36 (m, 6H), 7.21 (tt, J = 7.2, 1.2 Hz, 1H), 7.08 (d, J = 8.1 Hz, 1H), 6.88 (dt, J = 8.9, 2.5 Hz, 4H), 4.39 (dd, J = 8.2, 3.0 Hz, 1H), 4.20-4.27 (m, 1H), 3.78 (s, 6H), 3.60 (s, 1H), 3.35-3.52 (m, 2H), 3.07-3.16 (m, 2H), 2.23-2.37 (m, 4H), 1.53-1.65 (m, 4H), 1.23-1.36 (m, 12H), 1.10 (d, J = 6.4 Hz, 3H)

[0330] Scheme 24 [ka]

[0331] Step 11. Preparation of compound 164-1 To a suspension of potassium t-butoxide (14.6 g, 130 mol) in THF (120 mL) / ether (360 mL) was added methyltriphenylphosphonium bromide (46.6 g, 130 mmol). The mixture was refluxed for 2 h and then cooled to 0 °C. tert-Butyl 2-formylpyrrolidine-1-carboxylate (13.0 g, 65.2 mmol) in ether (50 mL) was added dropwise. The reaction mixture was stirred at 0 °C and then quenched by adding water (250 mL). The organic layer was separated and the aqueous was extracted with ether (250 mL). The combined extracts were dried over MgSO4. The solvent was evaporated and purified by column chromatography (5% ethyl acetate / hexane) to give tert-butyl 3-vinylpyrrolidine-1-carboxylate 164-1 (11.5 g, 89.4%) as a colorless liquid. GC-MS: m / z: 197 (2) (M +), 141 (40), 124 (30), 57 (100)

[0332] Step 12. Preparation of compound 164-2 To a mixture of t-BuOH (140 mL) and water (70 mL) was added AD-mix-β (47.4 g) and methanesulfonamide (2.89 g, 30.4 mmol). The mixture was stirred at room temperature for 30 min and then cooled to 0 °C. tert-Butyl 3-vinylpyrrolidine-1-carboxylate 164-1 (6.00 g, 30.4 mmol) was added. The reaction was stirred at room temperature overnight. The reaction mixture was cooled to 0 °C. Sodium thiosulfate pentahydrate (96 g, 387 mmol) was added and the temperature was allowed to rise to room temperature. Water (700 mL) was added and the mixture was extracted with ethyl acetate (500 mL). The extract was washed with water (2 x 50 mL) and brine (50 mL) and dried over MgSO4. Evaporation of the solvent followed by column chromatography (2% methanol / dichloromethane to 7% methanol / dichloromethane) afforded tert-butyl 3-(1,2-dihydroxyethyl)pyrrolidine-1-carboxylate 164-2 (5.4 g, 77%) as a light brown oil.

[0333] Step 13. Preparation of compound 164-3 To a solution of tert-butyl 3-(1,2-dihydroxyethyl)pyrrolidine-1-carboxylate 164-2 (3.1 g, 13.4 mmol) in ethanol (10 mL) was added 3N HCl (30 mL, 90 mmol). The reaction mixture was stirred at room temperature overnight. TLC showed the reaction was complete. Ethanol was evaporated. Toluene was added and evaporated. This was repeated three times to give 1-(pyrrolidin-3-yl)ethane-1,2-diol hydrochloride 164-3 (2.0 g, 89%) as a brown oil. LC-MS (ESI): m / z: 132 (100), (M + H + , free amine)

[0334] Step 14: Preparation of compound 164-4 To a solution of 1-(pyrrolidin-3-yl)ethane-1,2-diol hydrochloride 164-2 (2.0 g, 12 mmol) in water (30 mL) was added NaHCO3 (3.7 g, 44 mmol) in small portions. Then dioxane (20 mL) was added. To the above solution was added 1-(2,5-dioxopyrrolidin-1-yl)12-methyldodecanedioate 146-1 (3.7 g, 11 mmol) in dioxane (30 mL). The reaction mixture was stirred overnight. It was extracted with ethyl acetate (3 x 100 mL). The combined extracts were washed with 0.5 N HCl (50 mL) and brine (50 mL) and dried over MgSO4.

[0335] Step 15. Preparation of compound 164 This material was prepared from methyl 12-(3-(1,2-dihydroxyethyl)pyrrolidin-1-yl)-12-oxododecanoate 164-4 and 4,4-dimethoxytrityl chloride (1 equiv.) using the same procedure as described for the synthesis of 2-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)isoindoline-1,3-dione 138. The product was purified by column chromatography (1.5% methanol / dichloromethane). Methyl 12-(3-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)-1-hydroxyethyl)pyrrolidin-1-yl)-12-oxododecanoate 164 was obtained in 51% yield as a yellow oil. 1HNMR (400MHz, acetone-d6, ppm): δ 7.49-7.54 (m, 2H), 7.35-7.40 (m, 4H), 7.28-7.34 (m, 2H), 7.19-7.25 (m, 1H), 6.86-6.91 (m, 4H), 4.11-4.20 (m, 1H), 3.79 (s, 6H), 3.68-3.77 (m, 1H), 3.60 (s, 3H), 3.29-3.59 (m, 3H), 3.06-3.20 (m, 3H), 2.33-2.55 (m, 1H), 2.29 (t, J = 7.4 Hz, 2H), 2.19 (t, J = 7.6 Hz, 2H), 1.65-2.0 (m, 2H), 1.51-1.62 (m, 4H), 1.26-1.35 (m, 12H)

[0336] Scheme 25 [ka]

[0337] Step 16. Preparation of compound 170-1 To a solution of tert-butyl 2-aminoethylcarbamate (2.88 g, 18.0 mmol) and triethylamine (2.98 g, 29.4 mmol) in dichloromethane (100 mL) was added 1-(2,5-dioxopyrrolidin-1-yl) 12-methyldodecanedioate (146-1) (5.09 g, 14.9 mmol) in dichloromethane (50 mL) at room temperature. The reaction mixture was stirred overnight and TLC showed the reaction was complete. 100 mL of brine was added and the organic layer was separated. The organic layer was washed with 0.5 N HCl (150 mL), brine (2 x 100 mL) and dried over MgSO4. The solvent was evaporated to give pure methyl 12-(2-(tert-butoxycarbonylamino)ethylamino)-12-oxododecanoate 170-1 (5.85 g, 100%) as a white solid.

[0338] Step 17. Preparation of compound 170-2 To a solution of 12-(2-(tert-butoxycarbonylamino)ethylamino)-12-oxododecanoate 170-1 (5.55 g, 14.4 mmol) in methanol (100 mL) at 0 °C was added thionyl chloride (3.3 mL, 45.5 mmol) dropwise. The reaction was then stirred at room temperature overnight. TLC showed the reaction was complete. The solvent and volatile organics were evaporated. The residue was then co-evaporated twice with heptane to give methyl 12-(2-aminoethylamino)-12-oxododecanoate hydrochloride 170-2 quantitatively as a white solid. LC-MS (ESI): m / z: 287 (100), (M + H + , free amine)

[0339] Step 18. Preparation of compound 170-3 (-)-Methyl (S)-2,2-dimethyl-1,3-dioxolane-4-carboxylate (5.01 g, 31.2 mmol) and LiOHH2O (2.55 g, 60.8 mmol) in THF (50 mL) and water (50 mL) were stirred overnight. TLC showed the reaction was complete. THF was evaporated and the aqueous was acidified with 1N HCl to pH = 1. It was extracted with ethyl acetate (5 x 50 mL). The combined extracts were dried over MgSO4. The solvent was evaporated to give (S)-2,2-dimethyl-1,3-dioxolane-4-carboxylic acid 170-3 (2.93 g, 64.3%) as a pale yellow liquid.

[0340] Step 19. Preparation of compound 170-4 Compound 170-4 was synthesized in 86% yield from (S)-2,2-dimethyl-1,3-dioxolane-4-carboxylic acid 170-3 and N-hydroxysuccinimide using the same procedure described for the synthesis of 1-(2,5-dioxopyrrolidin-1-yl) 12-methyldodecanedioate 146-1. (S)-2,5-dioxopyrrolidin-1-yl 2,2-dimethyl-1,3-dioxolane-4-carboxylate 170-4 was obtained as a white solid in 86% yield.

[0341] Step 20. Preparation of compound 170-5 To a suspension of methyl 12-(2-aminoethylamino)-12-oxododecanoate hydrochloride 170-2 (14.4 mmol) and (S)-2,5-dioxopyrrolidin-1-yl 2,2-dimethyl-1,3-dioxolane-4-carboxylate 170-4 (3.80 g, 15.6 mmol) in dichloromethane (100 mL) was added triethylamine (6 mL, 43.0 mmol) in dichloromethane (25 mL) over 4 h at 0° C. The reaction mixture was then stirred at room temperature overnight. LC-MS showed complete conversion of starting material 170-2. The organic layer was washed with brine (50 mL), 1N HCl (50 mL), brine (50 mL), dried over MgSO4, and concentrated to dryness to give (S)-methyl 12-(2-(2,2-dimethyl-1,3-dioxolane-4-carboxamido)ethylamino)-12-oxododecanoate 170-5 (5.93 g, 99.3%) as a white solid.

[0342] Step 21. Preparation of compound 170-6 To a solution of (S)-methyl 12-(2-(2,2-dimethyl-1,3-dioxolane-4-carboxamido)ethylamino)-12-oxododecanoate 170-5 (5.93 g, 14.3 mmol) was added one drop of concentrated sulfuric acid. This was refluxed for 6 h and then cooled to room temperature. The solid was collected by filtration and washed twice with cold methanol. The solid was air-dried (3.32 g). A second crop (0.42 g) was obtained from the mother liquor to give (S)-methyl 12-(2-(2,3-dihydroxycypropanamido)ethylamino)-12-oxododecanoate 170-6 (total 3.74 g, 69.4%) as white crystals. LC-MS (ESI): m / z: 375 (100), (M + H + ). 1HNMR (400MHz, DMSO-d6, ppm): δ 7.79 (br, 2H), 5.49 (d, J = 5.3 Hz, 1H), 4.66 (t, J = 5.8 Hz, 1H), 3.83-3.88 (m, 1H), 3.55-3.61 (m, 4H), 3.41-3.47 (m, 1H), 3.05-3.15 (m, 4H), 2.29 (t, J = 7.4 Hz, 2H), 2.03 (t, J = 7.6 Hz, 2H), 1.42-1.52 (m, 4H), 1.18-1.29 (m, 12H)

[0343] Step 22. Preparation of compound 170 To a solution of (S)-methyl 12-(2-(2,3-dihydroxypropanamido)ethylamino)-12-oxododecanoate 170-6 (2.99 g, 7.99 mmol) in dry pyridine (57.5 mL) under nitrogen was added 4,4'-dimethoxytrityl chloride (2.84 g, 8.38 mmol) in one portion. The reaction was stirred at room temperature for 2 days. Methanol (5 mL) was added to quench the reaction. Pyridine was evaporated. Toluene was added and then evaporated. This was repeated three times. Water (100 mL) was added and this was extracted with ethyl acetate (5 x 250 mL). The extracts were combined and dried over MgSO4. Evaporation of the solvent followed by column chromatography (1% methanol / dichloromethane to 3% methanol / dichloromethane) gave (S)-methyl 12-(2-(3-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-hydroxypropanamido)ethylamino)-12-oxododecanoate 170 (1.70 g, 31.4%) as a viscous oil. 1HNMR (400MHz, acetone-d6, ppm): δ 7.64-7.70 (br, 1H), 7.47-7.51 (m, 2H), 7.33-7.37 (m, 4H), 7.26-7.32 (m, 2H), 7.20 (dt, J = 7.3, 2.1 Hz, 1H), 7.11 (br, 1H), 6.86 (d, J = 8.7 Hz, 4H), 4.84 (br, 1H), 4.21 (dd, J = 5.1, 3.8 Hz, 1H), 3.78 (s, 6H), 3.60 (s, 1H), 3.25-3.42 (m, 6H), 2.28 (t, J = 7.4 Hz, 2H), 1.48-1.62 (m, 4H), 1.21-1.34 (m, 12H)

[0344] Scheme 26. [ka]

[0345] Step 23. Preparation of compounds 139, 155, 160, 165 and 170 Compounds 139, 155, 160, 165 and 170 were prepared from compounds 138, 154, 159, 164 and 169 using the same procedure as used for compound 18.

[0346] Step 24. Preparation of conjugates 153, 158, 163, 168 and 173 Conjugates 153, 158, 163, 168 and 173 were prepared from compounds 139, 154, 159, 164 and 169 using the same procedure as used for compound 1.

[0347] Example 12. Synthesis of conjugate 176 Scheme 27. [ka]

[0348] Scheme 28. [ka]

[0349] Step 1. Preparation of methyl 12-aminododecanoate 132 12-Aminoundecanoic acid (131) (10 g, 4.64 mmol) was stirred in MeOH at room temperature. Acetyl chloride (856 μL, 12 mmol) was added dropwise and the reaction was stirred for 1.5 h. The solvent was removed in vacuo and the residue was taken up in MTBE and chilled in the refrigerator overnight. The resulting precipitate was collected by filtration, washed with ice-cold MTBE and dried under high vacuum to give methyl 12-aminododecanoate 132.

[0350] Step 2. Preparation of methyl 12-(12-(10-((3R,4S)-3-((bis(4-methoxyphenyl)-(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanamido)dodecanamido)dodecanoate 129 Lithium 10-((3R,4S)-3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoate (128) (2 g, 3.1 mmol), methyl 12-aminododecanoate (132) (778 mg, 3.1 mmol), HBTU (1.2 g, 3.1 mmol) and TEA (1.4 mL, 10 mmol) in DCM were stirred at room temperature overnight. The precipitate was removed by filtration, the filtrate was concentrated in vacuo and the residue was purified by column chromatography (5% MeOH, DCM). TLC showed two closely spaced spots of identical mass which were determined to be geometric isomers and pooled together to give methyl 12-(12-(10-((3R,4S)-3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanamido)dodecanamido)dodecanoate (129) in quantitative form.

[0351] Step 3. Preparation of lithium 12-(12-(10-((3R,4S)-3-((bis(4-methoxyphenyl)(phenyl)-methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanamido)-dodecanamido) dodecanoate 130 Methyl 12-(12-(10-((3R,4S)-3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanamido)dodecanamido)dodecanoate (129) (3.1 mmol) in THF:HO (50:50) was stirred with LiOH (88 mg, 3.7 mmol) at room temperature overnight. The reaction was monitored by TLC and the THF was removed in vacuo. The aqueous solution was frozen in liquid N2 and lyophilized for 48 h to quantitatively obtain lithium 12-(12-(10-((3R,4S)-3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanamido)dodecanamido)dodecanoate 130.

[0352] Step 4. Preparation of conjugate 176 Conjugate 176 was prepared from compounds 24 and 130 using the same procedure as used for compound 1.

[0353] Example 13. Synthesis of conjugate 179 Scheme 29. [ka]

[0354] Scheme 30. [ka]

[0355] Step 1. Preparation of compound 80 Compound 24 (2 g, 0.86 mmol), N-carbobenzoxy-L-glutamic acid (120 mg, 0.43 mmol), HBTU (326 mg, 0.86 mmol) and TEA (353 μL, 2.6 mmol) were stirred in DCM at room temperature overnight. The mixture was concentrated in vacuum and purified by column chromatography to give compound 80 (2.88 g, 83%).

[0356] Step 2. Preparation of compound 81 Compound 81 was prepared from compound 80 (670 mg, 0.17 mmol) using the same procedure as that used for compound 14. In the next reaction, this compound was used crude and the yield was quantitative.

[0357] Step 3. Preparation of conjugate 179 Conjugate 179 was prepared from compounds 18 and 81 using the same procedure as used for compound 1.

[0358] Example 14. Synthesis of conjugate 182 Scheme 31. [ka]

[0359] Scheme 32. [ka]

[0360] Step 1. Preparation of compound 93 Compound 93 was prepared by the same procedure as that used for compound 89 (2-oxo-2-phenyl-1λ 2 -ethyl)-D-glutamic acid (2.25 g, 8.1 mmol) and 9 (13 g, 21 mmol). Yield: 11.2 g.

[0361] Step 2. Preparation of compound 94 Compound 94 was prepared from compound 93 (11.1 g) using the same procedure as used for compound 90. Yield: 10.2 g.

[0362] Step 3. Preparation of conjugate 182 Conjugate 182 was prepared from compounds 18 and 94 using the same procedure as used for compound 1.

[0363] Example 15. Synthesis of conjugates 185 and 188 Scheme 33. [ka]

[0364] Scheme 34. [ka]

[0365] Scheme 35. [ka]

[0366] Step 1. Preparation of 14-hydroxy-3,6,9,12-tetraoxatetradecyl 4-methylbenzenesulfonate 82 A solution of pentaethylene glycol (35 g, 147 mmol), TEA (41 mL, 294 mmol), and trimethylamine-HCl (1.4 g, 14.7 mmol) in CHCl (600 mL) was treated with tosyl chloride (29.4 g, 154 mmol). After stirring (18 h), the reaction mixture was washed with H0-brine (1:1), dried (MgSO), filtered, concentrated, and chromatographed to give 82 (24.6 g, 43%) as a pale yellow oil. Rf 0.8 (10% CHOH-CHCl).

[0367] Step 2. 14-Azido-3,6,9,12-tetraoxatetradecan-1-ol 83 14-Azido-3,6,9,12-tetraoxatetradecan-1-ol (83) was prepared from 82 (24.6 g, 62.7 mmol) and sodium azide (7.13 g, 110 mmol) using the same procedure as used for compound 4. Yield: 14.8 g, 90%.

[0368] Step 3. Preparation of compound 84 A solution of GalNAc6 (12.2 g, 31.4 mmol) and HO-PEG-N383 (9.2 g, 35 mmol) in 1,2-dichloroethane (150 mL) was treated with Sc(OTf)3 (771 mg, 1.6 mmol). After stirring (85 °C, 2 h), the reaction was cooled (room temperature), quenched by the addition of TEA (40 mL), and concentrated. The crude material was chromatographed to give 84 (11.16 g, 60%) as a pale yellow foam. Rf 0.7 (10% CH3OH-CH2Cl2).

[0369] Step 4. Preparation of compound 85 A solution of 84 (11.16 g, 18.8 mmol) and Pd / C (1.1 g, 10%-wet support) in EtOAc (120 mL) was treated with TFA (4.32 mL, 56.5 mmol) and purged with H2. After vigorous stirring (4.5 h), the reaction was purged with N2, filtered through Celite, and concentrated. The crude material was chromatographed to give 85 (5.77 g, 45%) as a colorless foam. Rf 0.5 (10% CH3OH-CH2Cl2).

[0370] Step 5. Preparation of Compound 95 Compound 95 was prepared by the same procedure as that used for compound 91, by the synthesis of (2-oxo-2-phenyl-1λ 2 -ethyl)-D-glutamic acid (1.04 g, 3.7 mmol) and compound 94 (10.2 g). Yield: 7.2 g.

[0371] Step 6. Preparation of Compound 96 Compound 96 was prepared from compound 95 (11.1 g) using the same procedure as used for compound 92. Yield: 6.5 g.

[0372] Step 7. Preparation of compound 97 Compound 97 was prepared by the same procedure as that used for compound 89 (2-oxo-2-phenyl-1λ 2 Prepared from 85 (12.1 g, 17.8 mmol) and (-ethyl)-D-glutamic acid (2 g, 7.1 mmol). Yield: 10 g, quantitative.

[0373] Step 8. Preparation of compound 98 Compound 98 was prepared from compound 97 (10 g, 7.2 mmol) using the same procedure as used for compound 90. Yield: 3.5 g, 36%.

[0374] Step 9. Preparation of Compound 99 Compound 99 was prepared by the same procedure as that used for compound 91, (2-oxo-2-phenyl-1λ 2 -ethyl)-D-glutamic acid (350 mg, 1.25 mmol) and compound 98 (2.86 mg, 2.5 mmol).

[0375] Step 10. Preparation of Compound 100 Compound 100 was quantitatively prepared from compound 99 (3.2 g, 1.25 mmol) using the same procedure as used for compound 92.

[0376] Step 11. Preparation of conjugates 185 and 188 Conjugates 185 and 188 were prepared from compounds 18 and 96, or 18 and 100, using the same procedure as that used for compound 1.

[0377] Example 16. Synthesis of conjugates 191, 194, 197 and 200 Scheme 36 [ka]

[0378] Scheme 37. [ka]

[0379] Scheme 38. [ka]

[0380] Step 1. Preparation of 2-(2-(2-azidoethoxy)ethoxy)ethan-1-ol 86 To a solution of 2-(2-(2-chloroethoxy)ethoxy)ethan-1-ol (13 g, 77 mmol) in water (200 mL) was added sodium azide (10 g, 154 mmol). The reaction was heated to 100° C. for 18 h. The reaction was cooled to room temperature, placed in a 1 L separatory funnel, and extracted with dichloromethane (3×200 mL). The combined dichloromethane extracts were dried over magnesium sulfate, filtered, and concentrated to dryness to give 2-(2-(2-azidoethoxy)ethoxy)ethan-1-ol as a colorless oil (11.7 g).

[0381] Step 2. Preparation of compound 87 Compound 87 was prepared from 86 (4.95 g, 28.3 mmol) and 6 (10 g, 25.7 mmol) using the same procedure as used for compound 84. Yield: 10 g, 77%.

[0382] Step 3. Preparation of compound 88 Compound 88 was prepared from 87 (10 g, 19.8 mmol) using the same procedure as used for compound 85. Yield: 7.63 g, 65%.

[0383] Step 4. Preparation of compound 89 A solution of 88 (2 g, 3.38 mmol) and Z-glutamic acid (427 mg, 1.52 mmol) in CHCl (50 mL) was treated with HBTU (1.41 g, 3.7 mmol) and Hunig's base (1.77 mL, 10.1 mmol). After stirring (18 h), the mixture was concentrated and chromatographed to give 89 (871 mg, 48%) as a colorless foam. Rf 0.5 (10% CHOH-CHCl).

[0384] Step 5. Preparation of Compound 90 A solution of 89 (870 mg, 0.72 mmol) and Pd / C (90 mg, 10%-wet support) in EtOAc (10 mL) was treated with TFA (84 μL, 1.1 mmol) and purged with H2. After vigorous stirring (2 h), the reaction was purged with N2, filtered through Celite, and concentrated. The crude material was used without further treatment to give 90 (850 mg, quantitative) as a colorless foam. Rf 0.25 (10% CH3OH-CH2Cl2).

[0385] Step 6. Preparation of Compound 91 A solution of 90 (850 mg, 0.72 mmol) and Z-glutamic acid (91 mg, 0.32 mmol) in CHCl (10 mL) was treated with HBTU (300 mg, 0.79 mmol) and Hunig's base (502 μL, 2.9 mmol). After stirring (1.5 h), the mixture was diluted with CHCl, washed with NaHCO (sat. aq.), dried (MgSO), filtered, and concentrated. The crude material was chromatographed to give 91 (590 mg, 76%) as a colorless foam. Rf 0.5 (10% CHOH-CHCl).

[0386] Step 7. Preparation of Compound 92 A solution of 91 (590 mg, 0.25 mmol) and Pd / C (100 mg, 10%-wet support) in CH3OH (30 mL) was treated with TFA (29 μL, 0.37 mmol) and purged with H2. After stirring (3 h), the mixture was purged with N2, filtered through Celite, and concentrated. The crude material was used without further treatment to give 92 (600 mg, quantitative) as a colorless foam. Rf 0.1 (10% CH3OH-CH2Cl2).

[0387] Step 8. Preparation of Compound 101 Compound 101 was prepared from (R)-2-((2-oxo-2-phenyl-112-ethyl)amino)hexanedioic acid (2.51 g, 8.6 mmol) and 9 (11 g, 17.2 mmol) using the same procedure as used for compound 89. Yield: 4.2 g, 37%.

[0388] Step 9. Preparation of Compound 102 Compound 102 was prepared from compound 101 (4.2 g, 3.2 mmol) using the same procedure as used for compound 90. Yield: 2.1 g, 47%.

[0389] Step 10. Preparation of compound 103 Compound 103 was prepared from (R)-2-((2-oxo-2-phenyl-112-ethyl)amino)hexanedioic acid (265 mg, 0.9 mmol) and compound 102 (2.1 g, 1.8 mmol) using the same procedure as used for compound 91. Yield: (560 mg, 24%).

[0390] Step 11. Preparation of compound 104 Compound 104 was quantitatively prepared from compound 103 (560 mg) using the same procedure as used for compound 92. This compound was used without purification.

[0391] Step 12. Preparation of conjugates 191, 194, 197 and 200 Conjugates 191, 194, 197 and 200 were prepared from compounds 128 and 92, 96, 100 or 104 using the same procedure as that used for compound 1.

[0392] Example 17. Synthesis of conjugates 203 and 206 Scheme 39. [ka]

[0393] Step 1. Preparation of compound 69b Compound 69b was prepared from (2S,4R)-4-hydroxypyrrolidine-2-carboxylic acid using the same procedure as used for compound 69.

[0394] Step 2. Preparation of conjugates 203 and 206 Conjugates 203 and 206 were prepared from compounds 96 and 100 using the same procedure as used for compound 1.

[0395] Example 18. Synthesis of conjugate 207 Scheme 40. [ka]

[0396] Step 1. Preparation of conjugate 209 Conjugate 209 was prepared from compounds 96 and 160 using the same procedure as used for compound 1.

[0397] Example 19. Synthesis of conjugates 212 and 215 Scheme 41. [ka]

[0398] Scheme 42. [ka]

[0399] Step 1. Dimethyl 5-(2-((2-oxo-2-phenyl-1λ 2 Preparation of 1-ethylaminoacetamidoisophthalate 105 A solution of dimethyl 5-aminoisophthalate (5 g, 24 mmol), Z-Gly-OH (5 g, 24 mmol), EDC (5 g, 26.3 mmol), HOBt (3.6 g, 26.3 mmol), NMM (2.9 mL, 26.3 mmol) in DMF (50 mL) was stirred overnight at room temperature. Upon completion, the reaction mixture was diluted with ethyl acetate (250 mL) and washed with 1 M HCl (2×100 mL), saturated sodium bicarbonate (1×100 mL), and brine (2×100 mL), respectively. It was dried over magnesium sulfate, filtered, and concentrated to dryness to give dimethyl 5-(2-((2-oxo-2-phenyl-1λ)). 2 3-Ethyl)amino)acetamido)isophthalate was obtained as a colorless solid (7.2 g, 79%).

[0400] Step 2.5-(2-((2-oxo-2-phenyl-1λ 2 Preparation of 1-ethyl)amino)acetamido)isophthalic acid 106 Methyl 5-(2-((2-oxo-2-phenyl-1λ 2 To a solution of 5-(2-((2-oxo-2-phenyl-1λ-ethyl)amino)acetamido)isophthalate (7.2 g) in methanol (25 mL) and THF (25 mL) was added 1M NaOH (25 mL). The solution was stirred at room temperature for 2 h and then concentrated to remove THF and MeOH. The remaining aqueous solution was diluted with water (75 mL), cooled in an ice-water bath, and acidified to pH = 1 with 6M HCl. The solid was filtered and washed with water (3 x 100 mL). The solid was lyophilized to give 5-(2-((2-oxo-2-phenyl-1λ-ethyl)amino)acetamido)isophthalate (7.2 g) in methanol (25 mL) and THF (25 mL). 2 (Ethyl)amino)acetamido)-isophthalic acid was obtained (6.9 g, quantitative).

[0401] Step 3. Preparation of compound 107 Compound 107 was prepared by the same procedure as used for compound 95, 5-(2-((2-oxo-2-phenyl-1λ 2 Prepared from 106 (200 mg, 0.54 mmol) and 94 (1.7 g, 1.3 mmol). Yield: 600 mg.

[0402] Step 4. Preparation of compound 108 Compound 108 was prepared from compound 107 (600 mg) using the same procedure as used for compound 96. Yield: 650 mg, quantitative.

[0403] Step 5. Preparation of Compound 109 Compound 109 was prepared by the same procedure as used for compound 99, 5-(2-((2-oxo-2-phenyl-1λ 2 Prepared from 106 (180 mg, 0.48 mmol) and 98 (1.5 g, 1.1 mmol). Yield: 900 mg.

[0404] Step 6. Preparation of compound 110 Compound 110 was prepared from compound 109 (900 mg) using the same procedure as used for compound 100. Yield: 920 mg, quantitative.

[0405] Step 7. Preparation of conjugates 212 and 215 Conjugates 212 and 215 were prepared from compounds 128 and 108 or 110 using the same procedure as that used for compound 1.

[0406] Example 20. Synthesis of conjugates 218 and 221 Scheme 43. [ka]

[0407] Scheme 44. [ka]

[0408] Step 1. Preparation of compound 111 Compound 111 was prepared from 4-(((tert-butoxycarbonyl)amino)methyl)phthalic acid (1.13 g, 3.84 mmol) and 88 (5 g, 8.44 mmol) using the same procedure as used for compound 89. Yield: 2.21 g, 49%.

[0409] Step 2. Preparation of compound 112 A solution of 111 (2.21 g, 1.87 mmol) in CH2Cl2 (40 mL) was slowly treated with TFA (5 mL). After stirring (2 h), the mixture was concentrated and chromatographed to give 112 (1.08 g, 47%) as a colorless foam. Rf 0.1 (10% CH3OH-CH2Cl2).

[0410] Step 3. Preparation of compound 113 Compound 113 was prepared by the same procedure used for compound 91 from compound 112 (1.08 g, 0.88 mmol) and (2-oxo-2-phenyl-1λ 2 -ethyl)-D-glutamic acid (112 mg, 0.39 mmol). Yield: 600 mg, 62%.

[0411] Step 4. Preparation of compound 114 Compound 114 was prepared from compound 113 using the same procedure used for compound 92.

[0412] Step 5. Preparation of compound 115 Compound 115 was prepared from 4-(((tert-butoxycarbonyl)amino)methyl)phthalic acid (3.94 g, 13.3 mmol) and 9 (18.2 g, 29.4 mmol) using the same procedure as used for compound 93. Yield: 9.02 g, 53%.

[0413] Step 6. Preparation of compound 116 Compound 116 was prepared from compound 115 (8 g, 6.3 mmol) using the same procedure as used for compound 112. Yield: 3.23 g, 39%.

[0414] Step 7. Preparation of compound 117 Compound 117 was prepared by the same procedure used for compound 95 from compound 116 (3.23 g, 2.45 mmol) and (2-oxo-2-phenyl-1λ 2 -ethyl)-D-glutamic acid (192 mg, 1.1 mmol). Yield: 2.22 g, 34%.

[0415] Step 8. Preparation of compound 118 Compound 118 was prepared from compound 117 (2.22 g, 0.84 mmol) using the same procedure as used for compound 96. Yield: 2.02 g, 91%.

[0416] Step 9. Preparation of conjugates 218 and 221 Conjugates 218 and 221 were prepared from compounds 128 and 114 or 118 using the same procedure as used for compound 1.

[0417] Example 21. Synthesis of conjugate 224 Scheme 45. [ka]

[0418] Step 1. Preparation of compound 224 Conjugate 224 was prepared from compounds 96 and 130 using the same procedure as used for compound 1.

[0419] Example 22: Synthesis of conjugate 231 Scheme 46 [ka]

[0420] Scheme 47 [ka]

[0421] Step 1: Preparation of Compound 225 Compound 225 was prepared from 5-(2-aminoacetamido)isophthalic acid 106 (560 mg, 1.5 mmol) and 9 (2.24 g, 3.6 mmol) using the same procedure as used for 89. Yield: 1.6 g, 80%.

[0422] Step 2: Preparation of Compound 226 Compound 226 was prepared in the same manner as 14. Yield 1.22 g, 78%.

[0423] Step 3: Preparation of Compound 227 Compound 227 was prepared from Z-glutamic acid (108 mg, 0.38 mmol) and 226 (1.22 g, 0.92 mmol) in the same manner as 89. Yield 471 mg, 45%.

[0424] Step 4: Preparation of Compound 228 Compound 228 was prepared in the same manner as 14. Yield 460 mg, quantitative.

[0425] Step 5: Preparation of Compound 229 Compound 229 was prepared from 228 (460 mg, 0.17 mmol) and 128 (125 mg, 0.19 mmol) in the same manner as 89. Yield 365 mg, 66%.

[0426] Step 6: Preparation of Compound 231 Conjugate 231 was prepared using the same procedure as used for compound 1.

[0427] Example 23. Synthesis of conjugate 233 Scheme 48 [ka]

[0428] Step 1. Preparation of compound 232 Compound 232 was prepared from compound 24 (650 mg, 0.33 mmol) and compound 69b (175 mg, 0.33 mmol) using the same procedure as used for compound 19. Yield: 380 mg, 47%.

[0429] Step 2. Preparation of compound 233 Compound 233 was prepared from compound 232 using the same procedure as used for compound 1.

[0430] Example 24. Synthesis of conjugate 235 Scheme 49 [ka]

[0431] Step 1. Preparation of compound 234 Compound 234 was prepared from compound 24 (1.1 g, 0.55 mmol) and compound 18 (175 mg, 0.33 mmol) using the same procedure as used for compound 19. Yield: 685 mg, 51%.

[0432] Step 2. Preparation of compound 235 Compound 235 was prepared from compound 234 using the same procedure as used for compound 1.

[0433] Example 25. In vitro and in vivo evaluation of GalNAc-siTTR conjugates Hydration of GalNAc-siTTR conjugates: In the GalNAc-siTTR conjugate having the structure of Compound 1, [ka] (GalNAc) (SEQ ID NO: 31), and the sense strand of [ka] The oligonucleotides used were the antisense strand of siRNA (SEQ ID NO:32) (listed in the 5' to 3' direction). In the former case, the underlined part is a 2'-O methyl sugar modification, and in the latter case, a 2'-fluoro sugar modification. The asterisk (*) represents a phosphorothioate (PS) bond. Before use in the study, the conjugates were hydrated with PBS and quantified for A260 values ​​using a NanoDrop instrument.

[0434] In vitro comparison of GalNAc-siTTR conjugates: Primary hepatocytes were isolated from C57 Bl / 6 female mice using standard procedures. Once isolated, the hepatocytes were plated in 96-well Primaria plates at 2.75 × 10 4Cells / well were seeded in Williams Media E (WME) containing 10% FBS. After 4 hours, the medium was replaced with WME without FBS for the remainder of the experiment. The next day, 90 μL of fresh WME was added to the cells. siRNA conjugate stocks were prepared in PBS at a concentration of 100 μg / mL. Serial dilutions were made in WME (1:2) (50 μL of conjugate dilution + 50 μL of WME). 10 μL of the conjugate dilutions were then transferred in triplicate to Primaria plates to give final conjugate concentrations in hepatocytes of 5 μg / mL, 2.5 μg / mL, 1.25 μg / mL, 0.625 μg / mL, 0.312 μg / mL, 0.156 μg / mL, and 0.078 μg / mL. Serial dilutions of PBS in WME served as negative controls. Cells were incubated for 24 hours at 37°C, 5% CO2. Cells were then lysed by removing the transfection medium and adding 200 μL of 1xLWR (containing 0.5 mg / mL proteinase K). The plates were freeze-thawed (60 min cycles, -80°C / 52°C) to aid in cell lysis. The resulting lysates were then analyzed for mouse TTR and GAPD mRNA levels using QuantiGene2.0 Assay according to the manufacturer's instructions. The relative concentration of TTR mRNA normalized to GAPD mRNA was determined and plotted. Significant knockdown of target mRNA was observed in cells treated with the conjugates.

[0435] In vivo comparison of GalNAc-siTTR conjugates: Female C57BL / 6 mice (n=4) were treated subcutaneously (scapular region) with PBS or GalNAc-siTTR conjugates (mice were administered the conjugates at a dose of 1 mg / kg diluted to 0.1 mg / mL in PBS or at a dose of 2 mg / kg diluted to 0.2 mg / mL in PBS). Blood was collected on days 2, 4, 5, 7, 8, 9, 11, 14, 21 (tail nick) and 28 (peripheral) and the resulting plasma was analyzed for TTR protein by ELISA. TTR plasma protein values ​​relative to PBS control are shown in the tables (Tables 1 and 2; see Figures 7 and 8). Significant knockdown of target mRNA and protein was observed in mice treated with the conjugates.

Claims

1. A compound of formula (I) 【Chemistry 1】 During the ceremony, R １ is the targeting ligand; L １ is absent or is a linking group, L ２ is absent or is a linking group, R ２ is a nucleic acid, Ring A is absent, a 3- to 20-membered cycloalkyl, a 5- to 20-membered aryl, a 5- to 20-membered heteroaryl or a 3- to 20-membered heterocycloalkyl; Each R Ａ are independently hydrogen, hydroxy, CN, F, Cl, Br, I, -C １－２ Alkyl-OR Ｂ , C １－１０ Alkyl C ２－１０ Alkenyl and C ２－１０ alkynyl, １－１０ Alkyl C ２－１０ Alkenyl and the C ２－１０ Alkynyl is optionally selected from halo, hydroxy and C １－３ substituted with one or more groups independently selected from alkoxy; R Ｂ is hydrogen, a protecting group, a covalent bond to a solid support, or a bond to a linking group attached to a solid support; The compound wherein n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Or its salt.

2. In the above formula, R １ is the targeting ligand; L １ is absent or is a linking group, L ２ is absent or is a linking group, R ２ is a nucleic acid, Ring A is absent, a 3- to 20-membered cycloalkyl, a 5- to 20-membered aryl, a 5- to 20-membered heteroaryl or a 3- to 20-membered heterocycloalkyl; Each R Ａ are independently hydrogen, hydroxy, CN, F, Cl, Br, I, -C １－２ Alkyl-OR Ｂ , as well as halo, hydroxy and C １－３ C optionally substituted with one or more groups independently selected from alkoxy １－８ is selected from the group consisting of alkyl, R Ｂ is hydrogen, a protecting group, a covalent bond to a solid support, or a bond to a linking group attached to a solid support; 2. The compound of claim 1, wherein n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Or its salt.

3. In the above formula, R １ But -C(H) （３－ｐ） (L ３ -sugar) ｐ and Each L ３ are independently a linking group, p is 1, 2 or 3; 3. The compound according to claim 2, wherein the sugar is a monosaccharide or a disaccharide. Or its salt.

4. The sugar is 【Chemistry 2】 And, During the ceremony, X is NR ３ and Y is -(C=O)R ４ , -SO ２ R ５ and -(C=O)NR ６ R ７ or X is -(C=O)- and Y is NR ８ R ９ and R ３ is hydrogen or (C １ -C ４ ) alkyl; R ４ , R ５ , R ６ , R ７ , R ８ and R ９ are each independently hydrogen, (C １ -C ８ ) alkyl, (C １ -C ８ ) haloalkyl, (C １ -C ８ ) alkoxy, and halo, (C １ -C ４ ) alkyl, (C １ -C ４ ) haloalkyl, (C １ -C ４ ) alkoxy and (C １ -C ４ ) haloalkoxy, optionally substituted with one or more groups independently selected from the group consisting of ３ -C ６ ) cycloalkyl; R １０ is -OH, -NR ８ R ９ or -F, R １１ is -OH, -NR ８ R ９ , -F, or halo, hydroxyl, carboxyl, amino, (C １ -C ４ ) alkyl, (C １ -C ４ ) haloalkyl, (C １ -C ４ ) alkoxy and (C １ -C ４ 4. The compound of claim 3, wherein the heterocyclic ring is a 5-membered heterocycle optionally substituted with one or more groups independently selected from the group consisting of haloalkoxy; Or its salt.

5. The sugar is 【Chemistry 3】 The compound according to claim 3 or 4, selected from the group consisting of: Or its salt.

6. The sugar is 【Chemistry 4】 The compound according to any one of claims 3 to 5, Or its salt.

7. Each of the above L ３ are each independently a divalent branched or unbranched, saturated or unsaturated hydrocarbon chain having 0 to 50 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms of the hydrocarbon chain are optionally selected from the group consisting of -O-, -NR Ｘ --, --NR Ｘ -C(=O)-, -C(=O)-NR Ｘ - or -S-, and R Ｘ is hydrogen or (C １ -C ６ ) alkyl, the hydrocarbon chain optionally being １ -C ６ ) alkoxy, (C ３ -C ６ ) cycloalkyl, (C １ -C ６ ) alkanoyl, (C １ -C ６ ) alkanoyloxy, (C １ -C ６ ) alkoxycarbonyl, (C １ -C ６ ) the compound according to any one of claims 3 to 6, which is substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy, or a salt thereof.

8. Each of the above L ３ are each independently a divalent branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 20 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms of the hydrocarbon chain are optionally selected from -O-, -NR Ｘ --, --NR Ｘ -C(=O)-, -C(=O)-NR Ｘ - or -S-, and R Ｘ is hydrogen or (C １ -C ６ ) alkyl, the hydrocarbon chain optionally being １ -C ６ ) alkoxy, (C ３ -C ６ ) cycloalkyl, (C １ -C ６ ) alkanoyl, (C １ -C ６ ) alkanoyloxy, (C １ -C ６ ) alkoxycarbonyl, (C １ -C ６ 8. The compound according to any one of claims 3 to 7, or a salt thereof, which is substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

9. Said L ３ but 【Chemistry 5】 The compound according to any one of claims 3 to 8, Or its salt.

10. The R １ but, 【Chemistry 6】 The compound according to any one of claims 2 to 9, Or its salt.

11. The R １ but 【Chemistry 7】 and During the ceremony, G is -NH- or -O-; R Ｃ is hydrogen, (C １ -C ８ ) alkyl, (C １ -C ８ ) haloalkyl, (C １ -C ８ ) alkoxy, (C １ -C ６ ) alkanoyl, (C ３ -C ２０ ) cycloalkyl, (C ３ -C ２０ ) heterocycle, aryl, heteroaryl, monosaccharide, disaccharide or trisaccharide, wherein the cycloalkyl, heterocycle, aryl, heteroaryl and sugar are selected from the group consisting of halo, carboxyl, hydroxyl, amino, (C １ -C ４ ) alkyl, (C １ -C ４ ) haloalkyl, (C １ -C ４ ) alkoxy and (C １ -C ４ ) haloalkoxy, A compound according to claim 2, Or its salt.

12. The R Ｃ but, 【Chemistry 8】 The compound according to claim 11, Or its salt.

13. The R １ but, 【Chemistry 9】 13. The compound according to any one of claims 2, 11 and 12, Or its salt.

14. The R Ｃ but, 【Chemistry 10】 12. The compound according to claim 11, wherein:

15. The compound or salt thereof according to any one of claims 11 to 14, wherein G is -NH-.

16. The R １ but, 【Chemistry 11】 16. The compound according to any one of claims 2, 11, 14 and 15, wherein

17. The R １ but 【Chemistry 12】 and In the formula, R Ｄ are independently hydrogen, (C １ -C ６ ) alkyl, (C ９ -C ２０ ) alkylsilyl, (R Ｗ ) ３ Si-, (C ２ -C ６ ) alkenyl, tetrahydropyranyl, (C １ -C ６ ) alkanoyl, benzoyl, aryl (C １ -C ３ ) alkyl, TMTr (trimethoxytrityl), DMTr (dimethoxytrityl), MMTr (monomethoxytrityl) and Tr (trityl); Each R Ｗ is independent, (C １ -C ４ ) selected from the group consisting of alkyl and aryl; The compound according to claim 2 or a salt thereof.

18. Said L １ and L ２ are independently a divalent branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 50 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms of the hydrocarbon chain are optionally selected from -O-, -NR Ｘ --, --NR Ｘ -C(=O)-, -C(=O)-NR Ｘ - or -S-, and R Ｘ is hydrogen or (C １ -C ６ ) alkyl, the hydrocarbon chain optionally being １ -C ６ ) alkoxy, (C ３ -C ６ ) cycloalkyl, (C １ -C ６ ) alkanoyl, (C １ -C ６ ) alkanoyloxy, (C １ -C ６ ) alkoxycarbonyl, (C １ -C ６ 18. The compound according to any one of claims 2 to 17, or a salt thereof, which is substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

19. Said L １ and L ２ are independently a divalent branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 20 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms of the hydrocarbon chain are optionally selected from -O-, -NR Ｘ --, --NR Ｘ -C(=O)-, -C(=O)-NR Ｘ - or S-, and R Ｘ is hydrogen or (C １ -C ６ ) alkyl, the hydrocarbon chain optionally being １ -C ６ ) alkoxy, (C ３ -C ６ ) cycloalkyl, (C １ -C ６ ) alkanoyl, (C １ -C ６ ) alkanoyloxy, (C １ -C ６ ) alkoxycarbonyl, (C １ -C ６ 19. The compound according to any one of claims 2 to 18, or a salt thereof, which is substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

20. Said L １ and L ２ are independently a divalent branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 14 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms of the hydrocarbon chain are optionally selected from -O-, -NR Ｘ --, --NR Ｘ -C(=O)-, -C(=O)-NR Ｘ - or S-, and R Ｘ is hydrogen or (C １ -C ６ ) alkyl, the hydrocarbon chain optionally being １ -C ６ ) alkoxy, (C ３ -C ６ ) cycloalkyl, (C １ -C ６ ) alkanoyl, (C １ -C ６ ) alkanoyloxy, (C １ -C ６ ) alkoxycarbonyl, (C １ -C ６ 20. The compound according to any one of claims 2 to 19, or a salt thereof, which is substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

21. Said L １ is -NH-, -O-, -S-, -(C=O)-, -(C=O)-NH-, -NH-(C=O)-, -(C=O)-O-, -NH-(C=O)-NH- or -NH-(SO ２ )-through R １ The compound or salt thereof according to any one of claims 2 to 20, wherein

22. Said L ２ But through -O-, R ２ The compound according to any one of claims 2 to 21, or a salt thereof, which is linked to

23. Said L １ but, 【Chemistry 13】 The compound according to any one of claims 2 to 22, or a salt thereof, selected from the group consisting of:

24. Said L ２ But -CH ２ -O- or -CH ２ -CH ２ The compound according to any one of claims 2 to 23, or a salt thereof, which is -O-.

25. A compound of formula (Ia): 【Chemistry 14】 During the ceremony, Each D is independent, 【Chemistry 15】 and -N=; The compound according to claim 2 Or its salt.

26. 【Catalog 16】 wherein Q １ is hydrogen, and Q ２ But, R ２ or Q １ But, R ２ And Q ２ is hydrogen, Z is -L １ -R １ That which is, and its salts is selected from the group consisting of 26. A compound according to claim 2 or claim 25, or a salt thereof.

27. A compound of formula (Ib) 【Chemistry 17】 During the ceremony, Each D is independent, 【Chemistry 18】 and -N=; each m is independently 1 or 2; The compound according to claim 2, or a salt thereof.

28. 【Chemical 19】 wherein Q １ is hydrogen, and Q ２ But, R ２ or Q １ But, R ２ And Q ２ is hydrogen, Z is -L １ -R １ That which is, and its salts is selected from the group consisting of 28. A compound according to claim 2 or claim 27, or a salt thereof.

29. A compound of formula (Ic) 【Chemistry 20】 During the ceremony, E is -O- or -CH ２ - and n is selected from the group consisting of 0, 1, 2, 3 and 4; A compound in which n1 and n2 are each independently selected from the group consisting of 0, 1, 2, and 3; or a salt thereof. The compound according to claim 2, or a salt thereof.

30. 【Chemical 21】 And, In the formula, Z is -L １ -R １ That which is, and its salts is selected from the group consisting of 30. A compound according to claim 2 or claim 29, or a salt thereof.

31. Said -A-L ２ -R ２ The part is, 【Chemical 22】 and During the ceremony, Q １ is hydrogen, and Q ２ But, R ２ or Q １ But, R ２ And Q ２ is hydrogen, each q is independently 0, 1, 2, 3, 4 or 5; The compound according to claim 2, or a salt thereof.

32. The R ２ The compound according to any one of claims 2 to 31, or a salt thereof, wherein is an oligonucleotide.

33. The R ２ The compound according to any one of claims 2 to 32, or a salt thereof, wherein 【Request 34】 【Chemical 23】 and its salts 3. The compound of claim 2, or a salt thereof, selected from the group consisting of:

35. The R １ but, 【Chemistry 24】 is selected from the group consisting of During the ceremony, R Ｓ but, 【Chemistry 25】 and n is 2, 3 or 4; x is 1 or 2; 2. The compound according to claim 1, or a salt thereof.

36. Said L １ but, 【Chemistry 26】 is selected from the group consisting of 36. A compound according to claim 1 or 35, or a salt thereof.

37. 37. The compound according to any one of claims 1, 35 and 36, or a salt thereof, wherein A is absent, phenyl, pyrrolidinyl or cyclopentyl.

38. Said L ２ is optionally substituted with hydroxy １－４ 38. The compound according to any one of claims 1 and 35 to 37, which is alkylene-O-, or a salt thereof.

39. Said L ２ But -CH ２ O-, -CH ２ CH ２ O- or -CH(OH)CH ２ 39. The compound according to any one of claims 1 and 35 to 38, or a salt thereof, which is O-.

40. Each of the R Ａ is optionally substituted with hydroxy or hydroxyl; １－８ 40. The compound according to any one of claims 1 and 35 to 39, or a salt thereof, which is alkyl.

41. Each of the R Ａ are independently selected from hydroxy, methyl and -CH ２ 41. The compound according to any one of claims 1 and 35 to 40, or a salt thereof, selected from the group consisting of: OH.

42. A compound of formula (Ig): 【Chemical 27】 During the ceremony, B is -N- or -CH-; L ２ is optionally substituted with hydroxyl or halo; １－４ alkylene-O-, The compound wherein n is 0, 1, 2, 3, 4, 5, 6 or 7; or a salt thereof.

2. The compound according to claim 1 or a salt thereof.

43. 【Catalogue 28】 And, In the formula, Q is -L １ -R １ and R' is C １－９ Alkyl, C ２－９ Alkenyl or C ２－９ alkynyl, １－９ Alkyl, C ２－９ Alkenyl or C ２－９ compounds wherein alkynyl is optionally substituted with halo or hydroxyl; and their salts 43. The compound of claim 1 or 42, or a salt thereof, selected from the group consisting of:

44. 【Catalogue 29】 And, In the formula, Q is -L １ -R １ That which is, and their salts 2. The compound of claim 1, or a salt thereof, selected from the group consisting of:

45. 【Chemical 30】 【Chemistry 31】 【Chemistry 32】 【Chemical 33】 and pharma- ceutically acceptable salts thereof.

2. The compound of claim 1, or a salt thereof, selected from the group consisting of:

46. 46. ​​A pharmaceutical composition comprising a compound as claimed in any one of claims 1 to 45, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier.

47. 46. ​​A method for delivering a nucleic acid to the liver of an animal, said method comprising administering to said animal a compound of formula I, or a pharma- ceutically acceptable salt thereof, as defined in any one of claims 1 to 45.

48. A compound of formula (Id): 【Chemical 34】 During the ceremony, R １ｄ but, 【Chemistry 35】 It is selected from X ｄ But, C ２－１０ is alkylene, n ｄ is 0 or 1, R ２ｄ is a nucleic acid, R ３ｄ is H, a protecting group, a covalent bond to a solid support, or a bond to a linking group attached to a solid support; or its salt 3. The compound according to claim 2, wherein:

49. The R １ｄ but, 【Chemical 36】 49. The compound of claim 48, wherein:

50. The R １ｄ but, 【Chemical 37】 49. The compound of claim 48, wherein:

51. The X ｄ But, C ８ 51. The compound or salt of any one of claims 48 to 50, which is alkylene.

52. The n ｄ The compound or salt according to any one of claims 48 to 51, wherein is 0.

53. The R ２ｄ The compound or salt according to any one of claims 48 to 51, wherein is siRNA.

54. The R ３ｄ The compound or salt of any one of claims 48 to 53, wherein is H.

55. The R ３ｄ 54. The compound or salt of any one of claims 48 to 53, wherein is a covalent bond to a solid support.

56. The R ３ｄ is a bond to a linking group attached to a solid support, said linking group being a divalent branched or unbranched, saturated or unsaturated hydrocarbon chain having 2 to 15 carbon atoms, one or more (e.g., 1, 2, 3 or 4) of said carbon atoms optionally being substituted by (-O-) or (-N(H)-), said chain optionally having on a carbon: (C １ -C ６ ) alkoxy, (C ３ -C ６ ) cycloalkyl, (C １ -C ６ ) alkanoyl, (C １ -C ６ ) alkanoyloxy, (C １ -C ６ ) alkoxycarbonyl, (C １ -C ６ 54. The compound according to any one of claims 48 to 53, or a salt thereof, which is substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

57. The R ３ｄ is a bond to a linking group attached to a solid support, said linking group being a divalent branched or unbranched, saturated or unsaturated hydrocarbon chain having 2 to 10 carbon atoms, one or more (e.g., 1, 2, 3 or 4) of said carbon atoms optionally being substituted by (-O-) or (-N(H)-), said chain optionally having on a carbon: (C １ -C ６ ) alkoxy, (C ３ -C ６ ) cycloalkyl, (C １ -C ６ ) alkanoyl, (C １ -C ６ ) alkanoyloxy, (C １ -C ６ ) alkoxycarbonyl, (C １ -C ６ 54. The compound according to any one of claims 48 to 53, or a salt thereof, which is substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

58. The R ３ｄ is a bond to a linking group attached to a solid support, said linking group being -C(=O)CH ２ CH ２ The compound according to any one of claims 48 to 53, wherein the compound is C(=O)N(H)-, or a salt thereof.

59. 59. A pharmaceutical composition comprising a compound as claimed in any one of claims 48 to 58, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier.

60. A method for delivering a nucleic acid to the liver of an animal, said method comprising administering to said animal a compound of formula Id as defined in any one of claims 48 to 58, or a pharma- ceutically acceptable salt thereof.

61. A compound of formula (Ie): 【Chemical 38】 During the ceremony, R １ｄ but, 【Chemical Formula 39】 It is selected from X ｄ But, C ２－８ is alkylene, n ｄ is 0 or 1, Pg １ is H or a suitable protecting group; R ３ｄ is H, a protecting group, a covalent bond to a solid support, or a bond to a linking group attached to a solid support; or its salt 3. The compound according to claim 2, wherein:

62. Pg １ 62. The compound according to claim 61, wherein is TMTr (trimethoxytrityl), DMTr (dimethoxytrityl), MMTr (monomethoxytrityl) or Tr (trityl), or a salt thereof.

63. A process for the preparation of a compound of formula (Id) or a salt thereof as claimed in claim 48, comprising the steps of: The corresponding compound of formula (Ie) 【Chemistry 40】 During the ceremony, X ｄ But, C ２－８ is alkylene, n ｄ is 0 or 1, Pg １ is H, R ３ｄ is a covalent bond to a solid support or a bond to a linking group attached to a solid support, is subjected to solid phase nucleic acid synthesis conditions to produce a corresponding compound of formula Id, ２ｄ providing said compound which is a nucleic acid; The method comprising:

64. The compound is cleaved from the solid support and the corresponding compound of formula Id is formed by cleaving the compound from the solid support. ３ｄ 64. The method of claim 63, further comprising providing said compound wherein

65. 【Catalogue 41】 and their salts 49. The compound of claim 48, or a salt thereof, selected from the group consisting of:

66. A compound of formula (I) 【Chemistry 42】 During the ceremony, R １ is H or a synthetic activating group; L １ is absent or is a linking group, L ２ is absent or is a linking group, R ２ is a nucleic acid, Ring A is absent, a 3- to 20-membered cycloalkyl, a 5- to 20-membered aryl, a 5- to 20-membered heteroaryl or a 3- to 20-membered heterocycloalkyl; Each R Ａ are independently hydrogen, hydroxy, CN, F, Cl, Br, I, -C １－２ Alkyl-OR Ｂ , C １－１０ Alkyl C ２－１０ Alkenyl, and C ２－１０ alkynyl, １－１０ Alkyl C ２－１０ Alkenyl and C ２－１０ Alkynyl is optionally selected from halo, hydroxy and C １－３ substituted with one or more groups independently selected from alkoxy; R Ｂ is hydrogen, a protecting group, a covalent bond to a solid support, or a bond to a linking group attached to a solid support; The compound wherein n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Or its salt.

67. A compound of formula (I) 【Chemistry 43】 During the ceremony, R １ is the targeting ligand; L １ is absent or is a linking group, L ２ is absent or is a linking group, R ２ is H or a synthetic activating group; Ring A is absent, a 3- to 20-membered cycloalkyl, a 5- to 20-membered aryl, a 5- to 20-membered heteroaryl, or a 3- to 20-membered heterocycloalkyl; Each R Ａ are independently hydrogen, hydroxy, CN, F, Cl, Br, I, -C １－２ Alkyl-OR Ｂ , C １－１０ Alkyl C ２－１０ Alkenyl and C ２－１０ alkynyl, １－１０ Alkyl C ２－１０ Alkenyl and C ２－１０ Alkynyl is optionally selected from halo, hydroxy and C １－３ substituted with one or more groups independently selected from alkoxy; R Ｂ is hydrogen, a protecting group, a covalent bond to a solid support, or a bond to a linking group attached to a solid support; The compound wherein n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Or its salt.

68. A compound of formula (Ig): 【Chemistry 44】 During the ceremony, B is -N- or -CH-; L ２ is optionally substituted with hydroxyl or halo; １－４ alkylene-O-, The compound wherein n is 0, 1, 2, 3, 4, 5, 6 or 7; or its salt 67. The compound of claim 65 or 66, or a salt thereof,

69. 【Catalogue 45】 And, During the ceremony, Q is -L １ -R １ and R' is C １－９ Alkyl, C ２－９ Alkenyl or C ２－９ alkynyl, １－９ Alkyl, C ２－９ Alkenyl or C ２－９ Alkynyl optionally substituted with halo or hydroxyl and their salts 68. The compound of claim 66 or 67, or a salt thereof, selected from the group consisting of: 【Request 70】 【Chemical 46】 And, In the formula, Q is -L １ -R １ and salts thereof 68. The compound of claim 66 or 67, or a salt thereof, selected from the group consisting of:

71. A compound of formula (Ig): 【Chemistry 47】 During the ceremony, B is -N- or -CH-; L １ is absent or is a linking group, L ２ is optionally substituted with hydroxyl or halo; １－４ alkylene-O-, n is 0, 1, 2, 3, 4, 5, 6 or 7; R １ is H or a synthetic activating group; R ２ is H or a synthetic activating group, Or its salt.

72. 【Catalogue 48】 And, In the formula, Q is -L １ -R １ and L １ is absent or is a linking group, R' is C １－９ Alkyl, C ２－９ Alkenyl or C ２－９ alkynyl, １－９ Alkyl, C ２－９ Alkenyl or C ２－９ alkynyl is optionally substituted with halo or hydroxyl; R １ is H or a synthetic activating group; R ２ is H or a synthetic activating group A compound selected from the group consisting of: Or its salt. 【Request 73】 【Chemical 49】 And, During the ceremony, Q is -L １ -R １ and L １ is absent or is a linking group, R １ is H or a synthetic activating group; R ２ is H or a synthetic activating group; A compound selected from the group consisting of: Or its salt.

74. The R １ 74. The compound according to any one of claims 66 to 73, or a salt thereof, wherein is H or a synthetic activating group derivable from DCC, HOBt, EDC, BOP, PyBOP or HBTU.

75. The R ２ 75. The compound according to any one of claims 66 to 74, wherein is H, acetate, triflate, mesylate or succinate, or a salt thereof.

76. The R １ 74. The compound according to any one of claims 66 to 73, or a salt thereof, wherein is a synthetic activating group derivable from DCC, HOBt, EDC, BOP, PyBOP or HBTU.

77. The R ２ The compound according to any one of claims 66 to 74, or a salt thereof, wherein is acetate, triflate, mesylate or succinate.

78. Said L １ is a divalent branched or unbranched, saturated or unsaturated hydrocarbon chain having 5 to 20 carbon atoms, and one or more (e.g., 1, 2, 3, or 4) of the carbon atoms of the hydrocarbon chain are optionally substituted by -O-, -NH-, -NH-C(=O)-, -C(=O)-NH-, or -S-, or a salt thereof.

79. A compound of the following formula (XX): 【Chemistry 50】 During the ceremony, R １ is the targeting ligand; L １ is absent or is a linking group, L ２ is absent or is a linking group, R ２ is a nucleic acid, B is divalent and 【Chemistry 51】 is selected from the group consisting of During the ceremony, Each R' is independently C １－９ Alkyl, C ２－９ Alkenyl or C ２－９ alkynyl, １－９ Alkyl, C ２－９ Alkenyl or C ２－９ alkynyl is optionally substituted with halo or hydroxyl; ＊ The bond marked with is L １ or L １ If does not exist, then R １ is bound to ＊＊ The bond marked with is L ２ or L ２ If does not exist, then R ２ The compound bound to Or its salt.

80. The targeting ligand R １ The compound according to claim 79, or a salt thereof, wherein said compound contains 2 to 8 sugars.

81. The targeting ligand R １ The compound according to claim 79, or a salt thereof, wherein said compound contains 2 to 4 sugars.

82. The targeting ligand R １ The compound according to claim 79, or a salt thereof, wherein said compound contains 3 to 8 sugars.

83. The targeting ligand R １ 80. The compound according to claim 79, or a salt thereof, wherein said compound comprises 3 to 6 sugars.

84. The targeting ligand R １ The compound according to claim 79, or a salt thereof, wherein said compound comprises 3 to 4 sugars.

85. The targeting ligand R １ 80. The compound or salt thereof of claim 79, wherein:

86. The targeting ligand R １ 80. The compound or salt thereof of claim 79, wherein:

87. The targeting moiety R １ has the formula 【Chemistry 52】 During the ceremony, B １ is a trivalent group containing from about 1 to about 20 atoms; L １ , T １ and T ２ is covalently bonded to B ２ is a trivalent group containing from about 1 to about 20 atoms; T １ , T ３ and T ４ is covalently bonded to B ３ is a trivalent group containing from about 1 to about 20 atoms; T ２ , T ５ and T ６ is covalently bonded to T １ is absent or is a linking group, T ２ is absent or is a linking group, T ３ is absent or is a linking group, T ４ is absent or is a linking group, T ５ is absent or is a linking group, T ６ is absent or is a linking group; The compound according to any one of claims 1 to 2, 25 to 30, 32 to 33, 36 to 39, 42 to 44, 66 to 69 and 79, or a salt thereof. 【Request 88】 【Chemical 53】 And, During the ceremony, X is NR ３ and Y is -(C=O)R ４ , -SO ２ R ５ and -(C=O)NR ６ R ７ or X is -(C=O)- and Y is NR ８ R ９ and R ３ is hydrogen or (C １ -C ４ ) alkyl; R ４ , R ５ , R ６ , R ７ , R ８ and R ９ are each independently hydrogen, (C １ -C ８ ) alkyl, (C １ -C ８ ) haloalkyl, (C １ -C ８ ) alkoxy, and halo, (C １ -C ４ ) alkyl, (C １ -C ４ ) haloalkyl, (C １ -C ４ ) alkoxy and (C １ -C ４ ) haloalkoxy, optionally substituted with one or more groups independently selected from the group consisting of ３ -C ６ ) cycloalkyl; R １０ is -OH, -NR ８ R ９ or -F, R １１ is -OH, -NR ８ R ９ , -F, or halo, hydroxyl, carboxyl, amino, (C １ -C ４ ) alkyl, (C １ -C ４ ) haloalkyl, (C １ -C ４ ) alkoxy and (C １ -C ４ ) a 5-membered heterocycle optionally substituted with one or more groups independently selected from the group consisting of haloalkoxy; 88. The compound of claim 87, or a salt thereof, wherein each said sugar is independently selected from:

89. each said sugar independently 【Chemical 54】 88. The compound of claim 87, or a salt thereof, selected from the group consisting of:

90. each said sugar independently 【Chemistry 55】 88. The compound of claim 87, which is: or a salt thereof.

91. Said T １ and T ２ The compound or salt thereof according to any one of claims 87 to 90, wherein one of:

92. Said T １ and T ２ The compound or salt thereof according to any one of claims 87 to 90, wherein both are absent.

93. Said T １ , T ２ , T ３ , T ４ , T ５ and T ６ each independently is absent or a branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 50 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms of the hydrocarbon chain are optionally selected from -O-, -NR Ｘ --, --NR Ｘ -C(=O)-, -C(=O)-NR Ｘ - or -S-, wherein R Ｘ is hydrogen or (C1-C6)alkyl, and the hydrocarbon chain is optionally substituted with one or more (e.g. 1, 2, 3 or 4) substituents selected from (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl and heteroaryloxy. The compound of any one of claims 87 to 90, or a salt thereof.

94. Said T １ , T ２ , T ３ , T ４ , T ５ and T ６ each independently is absent or a branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 20 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms of the hydrocarbon chain are optionally selected from -O-, -NR Ｘ --, --NR Ｘ -C(=O)-, -C(=O)-NR Ｘ - or -S-, wherein R Ｘ is hydrogen or (C1-C6)alkyl, and the hydrocarbon chain is optionally substituted with one or more (e.g. 1, 2, 3 or 4) substituents selected from (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl and heteroaryloxy. The compound of any one of claims 87 to 90, or a salt thereof.

95. Said T １ , T ２ , T ３ , T ４ , T ５ and T ６ each independently is absent or a branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 50 carbon atoms, or a salt thereof, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms of the hydrocarbon chain are optionally -O- or -NR Ｘ -, and R Ｘ is hydrogen or (C １ -C ６ 91. The compound according to any one of claims 87 to 90, or a salt thereof, wherein the hydrocarbon chain is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from halo, hydroxy, and oxo (=O).

96. Said T １ , T ２ , T ３ , T ４ , T ５ and T ６ is independently absent or a branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 20 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms of the hydrocarbon chain are optionally substituted by -O-, and the hydrocarbon chain is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from halo, hydroxy, and oxo (=O), or a salt thereof.

97. Said T １ , T ２ , T ３ , T ４ , T ５ and T ６ is independently absent or a branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 20 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms of the hydrocarbon chain are optionally substituted by -O-, and the hydrocarbon chain is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from halo, hydroxy, and oxo (=O), or a salt thereof.

98. Said T ３ , T ４ , T ５ and T ６ At least one of 【Chemistry 56】 and During the ceremony, n=1, 2, 3; The compound according to any one of claims 87 to 90, or a salt thereof.

99. Said T ３ , T ４ , T ５ and T ６ Each of these is independent. 【Chemistry 57】 is selected from the group consisting of During the ceremony, n=1, 2, 3; The compound according to any one of claims 87 to 90, or a salt thereof.

100. Said T １ and T ２ The compound or salt thereof according to any one of claims 87 to 90, wherein at least one of is glycine.

101. Said T １ and T ２ or a salt thereof. The compound according to any one of claims 87 to 90, wherein each of is glycine.

102. B １ is a trivalent group containing 1 to 15 atoms, and L １ , T １ and T ２ 102. The compound according to any one of claims 87 to 101, or a salt thereof, which is covalently bound to:

103. B １ is a trivalent group containing 1 to 10 atoms, and L １ , T １ and T ２ 102. The compound according to any one of claims 87 to 101, or a salt thereof, which is covalently bound to:

104. B １ However, (C １ -C ６ 102. The compound according to any one of claims 87 to 101, or a salt thereof, comprising:

105. B １ But, C ３－８ 102. The compound or salt thereof according to any one of claims 87 to 101, which comprises a cycloalkyl.

106. B １ The compound or salt thereof according to any one of claims 87 to 101, wherein

107. B １ The compound according to any one of claims 87 to 101, or a salt thereof, wherein comprises a D-amino acid or an L-amino acid.

108. B １ The compound or salt thereof according to any one of claims 87 to 101, wherein

109. B １ The compound according to any one of claims 87 to 101, or a salt thereof, wherein

110. B １ The compound or salt thereof according to any one of claims 87 to 101, wherein

111. B １ The compound according to any one of claims 87 to 101, or a salt thereof, wherein contains aryl.

112. B １ The compound according to any one of claims 87 to 101, or a salt thereof, wherein:

113. B １ The compound according to any one of claims 87 to 101, or a salt thereof, wherein is a phenyl ring.

114. B １ The compound or salt thereof according to any one of claims 87 to 101, wherein is CH.

115. B １ The compound according to any one of claims 87 to 101, or a salt thereof, wherein comprises heteroaryl.

116. B １ but, 【Chemistry 58】 The compound according to any one of claims 87 to 101, or a salt thereof.

117. B ２ is a trivalent group containing 1 to 15 atoms, and L １ , T １ and T ２ 117. The compound according to any one of claims 87 to 116, or a salt thereof, which is covalently bound to:

118. B ２ is a trivalent group containing 1 to 10 atoms, and L １ , T １ and T ２ 117. The compound according to any one of claims 87 to 116, or a salt thereof, which is covalently bound to:

119. B ２ However, (C １ -C ６ 117. The compound according to any one of claims 87 to 116, or a salt thereof, comprising:

120. B ２ But, C ３－８ 117. The compound or salt thereof according to any one of claims 87 to 116, which comprises a cycloalkyl.

121. B ２ The compound according to any one of claims 87 to 116, or a salt thereof, wherein

122. B ２ The compound according to any one of claims 87 to 116, or a salt thereof, wherein comprises a D-amino acid or an L-amino acid.

123. B ２ The compound or salt thereof according to any one of claims 87 to 116, wherein

124. B ２ The compound according to any one of claims 87 to 116, or a salt thereof, wherein

125. B ２ The compound according to any one of claims 87 to 116, or a salt thereof, wherein

126. B ２ The compound according to any one of claims 87 to 116, or a salt thereof, wherein comprises aryl.

127. B ２ The compound according to any one of claims 87 to 116, or a salt thereof, wherein:

128. B ２ The compound according to any one of claims 87 to 116, or a salt thereof, wherein is a phenyl ring.

129. B ２ The compound according to any one of claims 87 to 116, or a salt thereof, wherein is CH.

130. B ２ The compound according to any one of claims 87 to 116, or a salt thereof, wherein comprises heteroaryl.

131. B ２ but, 【Chemistry 59】 The compound according to any one of claims 87 to 116, or a salt thereof, selected from the group consisting of:

132. B ３ is a trivalent group containing 1 to 15 atoms, and L １ , T １ and T ２ 132. The compound according to any one of claims 87 to 131, or a salt thereof, which is covalently bound to:

133. B ３ is a trivalent group containing 1 to 10 atoms, and L １ , T １ and T ２ 132. The compound according to any one of claims 87 to 131, or a salt thereof, which is covalently bound to:

134. B ３ However, (C １ -C ６ 132. The compound according to any one of claims 87 to 131, or a salt thereof, comprising:

135. B ３ But, C ３－８ 132. The compound or salt thereof according to any one of claims 87 to 131, which comprises a cycloalkyl.

136. B ３ The compound or salt thereof according to any one of claims 87 to 131, wherein

137. B ３ The compound according to any one of claims 87 to 131, or a salt thereof, wherein comprises a D-amino acid or an L-amino acid.

138. B ３ The compound or salt thereof according to any one of claims 87 to 131, wherein

139. B ３ The compound according to any one of claims 87 to 131, or a salt thereof, wherein

140. B ３ The compound according to any one of claims 87 to 131, or a salt thereof, wherein

141. B ３ The compound according to any one of claims 87 to 131, or a salt thereof, wherein comprises aryl.

142. B ３ The compound according to any one of claims 87 to 131, or a salt thereof, wherein:

143. B ３ The compound according to any one of claims 87 to 131, or a salt thereof, wherein is a phenyl ring.

144. B ３ The compound or salt thereof according to any one of claims 87 to 131, wherein is CH.

145. B ３ The compound according to any one of claims 87 to 131, or a salt thereof, wherein comprises heteroaryl.

146. B ３ but, 【Chemistry 60】 or their salts The compound according to any one of claims 87 to 131, or a salt thereof, selected from the group consisting of:

147. Said L １ and L ２ are independently a divalent branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 50 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms of the hydrocarbon chain are optionally selected from -O-, -NR Ｘ --, --NR Ｘ -C(=O)-, -C(=O)-NR Ｘ - or -S-, and R Ｘ is hydrogen or (C1-C6)alkyl, and the hydrocarbon chain is optionally substituted with one or more substituents selected from (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy; or a salt thereof.

148. Said L １ but, 【Chemistry 61】 or their salts The compound according to any one of claims 87 to 146, or a salt thereof, selected from the group consisting of:

149. Said L １ is -O-, -S-, -(C=O)-, -(C=O)-NH-, -NH-(C=O), -(C=O)-O-, -NH-(C=O)-NH- or -NH-(SO ２ )-, through a linkage selected from the group consisting of B １ The compound according to any one of claims 87 to 146, or a salt thereof, linked to

150. Said L １ but, 【Chemistry 62】 The compound according to any one of claims 87 to 146, or a salt thereof, selected from the group consisting of:

151. Said L ２ But through -O-, R ２ The compound according to any one of claims 87 to 150, or a salt thereof, which is linked to

152. Said L ２ is optionally substituted with hydroxy １－４ The compound according to any one of claims 87 to 150, which is alkylene-O-, or a salt thereof.

153. Said L ２ But through -O-, R ２ The compound according to any one of claims 87 to 150, or a salt thereof, which is linked to

154. Said L ２ The compound or salt thereof according to any one of claims 87 to 150, wherein 【Request 155】 【Chemical 63】 【Chemistry 64】 【Chemistry 65】 and pharma- ceutically acceptable salts thereof.

88. The compound of claim 87, or a salt thereof, selected from the group consisting of: 【Request 156】 【Chemical 66】 Compound Or its salt.

157. 157. A pharmaceutical composition comprising a compound as claimed in any one of claims 79 to 156, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier.

158. 157. A method for delivering a nucleic acid to the liver of an animal, said method comprising administering to said animal a compound of formula I, as defined in any one of claims 79 to 156, or a pharma- ceutically acceptable salt thereof.

Images