Treatment method

Abstract

To provide a method and a composition for delivering a nucleic acid to a cell or a cytosol of a target cell.SOLUTION: A method includes bringing a cell in contact with 1) a membrane-destabilizing polymer and 2) a nucleic acid complex. The nucleic acid complex includes an optionally selected linker and a target-directional ligand bonded to a nucleic acid.SELECTED DRAWING: None

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims the benefit of priority to U.S. Application No. 62 / 755,196, filed November 2, 2018, which is incorporated herein by reference. [Background technology]

[0002] Targeted nucleic acid complexes are effective drug delivery systems for biologically active nucleic acids (see WO2017 / 177326). Nucleic acid-based drugs, including large nucleic acid molecules (e.g., in vitro transcribed messenger RNA (mRNA)) as well as small polynucleotides that interact with messenger RNA or genes, must be delivered to the appropriate intracellular compartment to be effective.

[0003] For example, double-stranded nucleic acids (e.g., double-stranded RNA molecules (dsRNA) (e.g., including siRNA)) suffer from the problem of cell impermeability due to their physicochemical properties. Once delivered to the appropriate compartment, siRNA blocks gene expression through a highly conserved regulatory mechanism known as RNA interference (RNAi). Typically, siRNAs are large, with molecular weights ranging from 12 to 17 kDa, and are highly anionic due to their phosphate backbone, carrying up to 50 negative charges. Furthermore, the two complementary RNA strands form a rigid helix. These characteristics contribute to the poor "drug-like" properties of siRNA. Upon intravenous administration, siRNAs are rapidly excreted from the body, with a half-life typically of only approximately 10 minutes. Furthermore, siRNAs are rapidly degraded by nucleases present in blood and other body fluids or tissues, and have been shown to stimulate potent immune responses in vitro and in vivo. For example, see Non-Patent Document 1. mRNA molecules also suffer from similar problems of impermeability, fragility, and immunogenicity.

[0004] By introducing appropriate chemical modifications, stability against nucleases can be improved, and at the same time, immune stimulation can be suppressed. Binding a certain ligand to siRNA can improve the pharmacokinetic characteristics of the double-stranded RNA molecule. A certain small molecule siRNA complex has been shown to be effective in specifically downregulating genes expressed in rodent hepatocytes. However, large doses are required to induce the desired biological effect. See Non-Patent Document 2.

[0005] Despite the efforts made to date, improved methods for delivering nucleic acids to cells are needed. For example, methods are needed that improve efficacy, reduce the required dose, and / or reduce the frequency of dosing. There is also a need for methods and formulations that can be used for subcutaneous delivery of nucleic acids. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Robbins et al.,Oligonucleotides 19:89-102,2009 [Non-patent document 2] Soutschek et al,Nature 432:173-178,2004 Summary of the Invention [Means for solving the problem]

[0007] In one embodiment, the present invention provides a method for delivering a nucleic acid to a cell, comprising treating the cell with 1) a membrane destabilizing polymer, and 2) a nucleic acid molecule of formula (X): [ka] wherein A is a targeting ligand, B is an optional linker, and C is a nucleic acid.

[0008] In one embodiment, the present invention provides a method for delivering a nucleic acid to the cytosol of a target cell in a subject, the method comprising: (a) a membrane-destabilizing polymer; and (b) a nucleic acid molecule of formula (X): [ka] (wherein A is a targeting ligand, B is an optional linker, and C is a nucleic acid) to a subject, wherein the nucleic acid is delivered to the cytosol of the target cell.

[0009] In one embodiment, the present invention provides a compound comprising 1) a membrane destabilizing polymer and 2) a compound of formula (X): [ka] wherein A is a targeting ligand, B is an optional linker, and C is a nucleic acid.

[0010] In one embodiment, the present invention provides a composition comprising a) a pharmaceutically acceptable carrier; b) a membrane destabilizing polymer; and c) a compound of formula (X): [ka] wherein A is a targeting ligand, B is an optional linker, and C is a nucleic acid. In one embodiment, the composition is formulated for administration by injection. In one embodiment, the composition is formulated for administration by subcutaneous injection.

[0011] In one embodiment, the present invention provides a method for treating a disease characterized by overexpression of a polypeptide, the method comprising: (a) a membrane destabilizing polymer; and b) a polypeptide of formula (X): [ka] (wherein A is a targeting ligand, B is an optional linker, and C is an siRNA that targets expression of the overexpressed polypeptide) in a therapeutically effective amount to an animal having the disease.

[0012] In one embodiment, the present invention provides a method for delivering siRNA to the liver of an animal, the method comprising: (a) administering to the animal a targeting moiety (T) selected to facilitate hepatocyte-specific delivery of the siRNA; 5 ), and b) a membrane destabilizing polymer comprising formula (X): [ka] wherein A is a targeting ligand, B is an optional linker, and C is an siRNA, and administering the nucleic acid complex of the formula:

[0013] In one embodiment, the present invention provides a method for treating a hepatitis B virus infection in an animal, the method comprising administering to an animal a targeting moiety (T 5 ), and (b) a membrane destabilizing polymer comprising formula (X): [ka] wherein A is a targeting ligand selected to facilitate hepatocyte-specific delivery of the complex, B is an optional linker, and C is an siRNA effective in treating hepatitis B virus infection, to an animal.

[0014] In one embodiment, the present invention provides a compound comprising 1) a membrane destabilizing polymer and 2) a compound of formula (X): [ka] wherein A is a targeting ligand, B is an optional linker, and C is a nucleic acid; and 3) instructions for delivering the nucleic acid to a cell, comprising contacting the cell with the nucleic acid complex and a membrane-destabilizing polymer.

[0015] In one embodiment, the present invention provides a compound comprising 1) a membrane destabilizing polymer and 2) a compound of formula (X): [ka] wherein A is a targeting ligand, B is an optional linker, and C is a nucleic acid; and 3) instructions for delivering the nucleic acid to the cytosol of a target cell in a subject by administering the nucleic acid complex and a membrane-destabilizing polymer to the subject.

[0016] In one embodiment, the present invention provides a compound comprising 1) a membrane destabilizing polymer and 2) a compound of formula (X): [ka] wherein A is a targeting ligand, B is an optional linker, and C is a nucleic acid; and 3) instructions for administering the nucleic acid complex and membrane-destabilizing polymer to an animal.

[0017] In one embodiment, the present invention provides a membrane destabilizing polymer and a compound of formula (X): [ka] A nucleic acid complex is provided, where A is a targeting ligand, B is an optional linker, and C is a nucleic acid.

[0018] In one embodiment, the present invention provides a nucleic acid-treatable compound of formula (X): [ka] wherein A is a targeting ligand, B is an optional linker, and C is a nucleic acid.

[0019] In one embodiment, the present invention provides a nucleic acid-treatable compound of formula (X): [ka] wherein A is a targeting ligand, B is an optional linker, and C is a nucleic acid.

[0020] In one embodiment, the present invention provides a compound of formula (X): [ka] wherein A is a targeting ligand, B is an optional linker, and C is a nucleic acid, wherein the nucleic acid complex is non-covalently associated with a membrane-destabilizing polymer.

[0021] In one embodiment, the present invention provides a compound of formula (X): [ka] wherein A is a targeting ligand, B is an optional linker, and C is a nucleic acid, wherein the nucleic acid complex is partially or completely encapsulated by a micelle comprising a plurality of membrane-destabilizing polymers.

[0022] In one embodiment, the present invention provides a compound of formula (X): [ka] wherein A is a targeting ligand, B is an optional linker, and C is a nucleic acid, wherein the nucleic acid complex is partially encapsulated by a micelle comprising a plurality of membrane-destabilizing polymers.

[0023] In one embodiment, the present invention provides a compound of formula (X): [ka] wherein A is a targeting ligand, B is an optional linker, and C is a nucleic acid, wherein the nucleic acid complex is completely encapsulated by a micelle comprising a plurality of membrane-destabilizing polymers.

[0024] In one embodiment, the present invention provides a compound comprising a pharmaceutically acceptable carrier and a compound of formula (X): [ka] wherein A is a targeting ligand, B is an optional linker, and C is a nucleic acid; and wherein the nucleic acid complex is partially or completely encapsulated by a micelle comprising a plurality of membrane-destabilizing polymers.

[0025] In one embodiment, the present invention provides compounds, compositions, and methods that can be used to deliver therapeutic nucleic acids to a target (e.g., the liver). Specifically, the present invention involves the use of polymeric micelles as an efficacy enhancer for subcutaneously administered complex platforms for targeted delivery of nucleic acid therapeutics to the liver. The polymeric micelles typically remain intact during delivery to hepatocytes and exert their functionality, e.g., upon subcutaneous administration. Gene silencing is examined by measuring the suppression or reduction of target gene expression compared to a vehicle control. Results in mice were favorable, with coadministration of membrane-destabilizing polymers and nucleic acid complexes resulting in an approximately five-fold increase in efficacy. Rapid onset of action and prolonged duration of effect were also observed.

[0026] 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. DETAILED DESCRIPTION OF THE INVENTION

[0027] Membrane-destabilizing polymers Membrane destabilizing polymers have been reported in U.S. Patent Application Publication Nos. US2010 / 0160216, US2010 / 0210504, US2011 / 0143434, US2011 / 0123636, US2016 / 0250338, US2017 / 0239360, and US2016 / 0206750, and International Patent Application Publication Nos. WO2009 / 140427, WO2009 / 140429, WO2015 / 017519, and WO2016 / 118697. Furthermore, in the supplemental section of Prieve et al., Mol. Ther., 2018, 26, 3, you can find a description of the synthesis of certain specific membrane-destabilizing polymers.

[0028] In one embodiment, the membrane destabilizing polymer comprises three distinct regions: The first region can contain a targeting moiety to achieve targeting to hepatocytes. This targeting moiety can be a single N-acetylgalactosamine monosaccharide unit that interacts with one of the three domains (trivalent) of the ASGPr receptor, which is highly expressed on the surface of hepatocytes. This monosaccharide unit forms the "head" of the polymer chain. N-acetylgalactosamine (GalNAc or NAG) can be attached to the second functional domain of the polymer via a PEG12 amino acid spacer coupled to ethyl carbonotrithioate (ECT). This serves as the starting "chain transfer agent" (CTA). Subsequent polymerization reactions can be performed with the monosaccharide fully deprotected.

[0029] The second, "solubilizing" or hydrophilic region is composed of polyethylene glycol methacrylate 4-5 (PEGMA4-5) (the numbers 4 and 5 refer to the number of ethylene glycol repeats in the monomer) and hydroxyethyl methacrylate (HMA). Typically, the PEGMA / HMA ratio is approximately 75 / 25. Polymerization can be carried out using reversible addition-fragmentation chain transfer (RAFT), which allows for control of the molecular weight and polydispersity of the resulting polymer during free-radical polymerization initiated with azobisisobutyronitrile (AIBN). Proceeding the reaction at a specific concentration and temperature for a fixed time yields a hydrophilic polymer of approximately 4 kDa end-capped with trithiocarbonate functional groups, which allow further polymerization.

[0030] The third region of the polymer provides the functionality for endosomal escape. This region can also be synthesized using RAFT polymerization, but in this case the monomer units in the reaction are dimethylaminoethyl acrylate (DMAEA), butyl methacrylate (BMA), and propylacrylic acid (PAA) (typically in a ratio of about 33% / 55% / 12%). This second polymerization step expands the polymer by an additional 5 kDa. After polymerization, the polymer end groups (trithiocarbonate) can be removed by radical-induced reduction, and the final polymer is characterized by 1H NMR, HPLC, and GPC (which determines MW and polydispersity).

[0031] Combining these two polymer regions helps maximize efficacy. At physiological or neutral pH, the polymer is typically neutral. Furthermore, at neutral pH, the second region, which provides endosomal escape functionality, exhibits hydrophobic characteristics. When combined with the hydrophilic domain, small micellar structures spontaneously form when the polymer exceeds the critical micelle concentration (CMC) in aqueous media. These have been shown to have pH-responsive membrane-destabilizing activity in red blood cell hemolysis assays: hemolysis rapidly decreases below the CMC. During endocytosis and the subsequent pH drop, the polymer becomes positively charged, which may facilitate endosomal escape.

[0032] In one embodiment, the membrane destabilizing polymer has the formula (XX): [ka] is a polymer of During the ceremony: PEGMA is a polyethylene glycol methacrylate residue having 2 to 20 ethylene glycol units; M 2 teeth, (C4-C 18 ) alkyl-methacrylate residue, (C4-C 18 ) branched alkyl-methacrylate residues, cholesteryl methacrylate residue, (C4-C 18 ) alkyl-methacrylate residues, and (C4-C 18 ) Branched alkyl methacrylate residue is a methacrylate residue selected from the group consisting of: BMA is the residue of butyl methacrylate, PAA is the residue of propyl acrylic acid, DMAEMA is the residue of dimethylaminoethyl methacrylate, m and n are each a molar fraction greater than 0, m is greater than n, and m+n=1; q is a molar ratio between 0.2 and 0.75; r is a molar ratio between 0.05 and 0.6; s is a molar ratio between 0.2 and 0.75; q+r+s=1, v is 1 to 25 kDa; w is 1 to 25 kDa; T 5 is a targeting moiety (e.g., a peptide, polymer, or sugar), L is absent or a linking moiety.

[0033] In one embodiment, M 2 teeth, 2,2,3,3,4,4,4-heptafluorobutyl methacrylate residue, 3,3,4,4,5,6,6,6-octafluoro-5(trifluoromethyl)hexyl methacrylate residue, 2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-pentadecafluorooctyl 2-methylacrylate residue, 3,3,4,4,5,5,6,6,6-nonafluorohexyl methacrylate residue, 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl methacrylate residue, 1,1,1-trifluoro-2-(trifluoromethyl)-2-hydroxy-4-methyl-5-pentyl methacrylate residue, 2-[(1',1',1'-trifluoro-2'-(trifluoromethyl)-2'-hydroxy)propyl]-3-norbornyl methacrylate residue, 2-ethylhexyl methacrylate residue, butyl methacrylate residue, hexyl methacrylate residue, octyl methacrylate residue, n-decyl methacrylate residue, lauryl methacrylate residue, myristyl methacrylate residue, stearyl methacrylate residue, cholesteryl methacrylate residue, ethylene glycol phenyl ether methacrylate residue, 2-propenoic acid, 2-methyl-, 2-phenylethyl ester residue, 2-Propenoic acid, 2-methyl-, 2-[[(1,1-dimethylethoxy)carbonyl]amino]ethyl ester residue, 2-Propenoic acid, 2-methyl-, 2-(1H-imidazol-1-yl)ethyl ester residue, 2-propenoic acid, 2-methyl-, cyclohexyl ester residue, 2-Propenoic acid, 2-methyl-, 2-[bis(1-methylethyl)amino]ethyl ester residue, 2-propenoic acid, 2-methyl-, 3-methylbutyl ester residue, Neopentyl methacrylate residue, tert-butyl methacrylate residue, 3,3,5-trimethylcyclohexyl methacrylate residue, 2-hydroxypropyl methacrylate residue, 5-nonyl methacrylate residue, 2-butyl-1-octyl methacrylate residue, 2-hexyl-1-decyl methacrylate residue, and 2-(tert-butylamino)ethyl methacrylate residue is selected from the group consisting of:

[0034] Targeting part T 5 is a moiety that can be, for example, a peptide, a polymer, or a sugar. 5 In certain embodiments, T is targeted to a location within the body, for example, to a particular organ or cell type. 5 is a peptide. In certain embodiments, T 5 is a polymer. In certain embodiments, T 5 is a sugar.

[0035] In one embodiment, the membrane destabilizing polymer has formula (XXI): [ka] In some embodiments, px is an integer from about 2 to about 50 (e.g., from about 2 to about 20, e.g., from 4 to 12) (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50). In some embodiments, px is an integer from about 8 to about 16 (e.g., 8, 9, 10, 11, 12, 13, 14, 15, or 16). In some embodiments, px is about 12. In some embodiments, py is an integer from about 2 to about 20 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). In some embodiments, py is an integer from about 2 to about 10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, py is an integer from about 4 to about 5 (e.g., 4 or 5).

[0036] In the polymer of formula (X), the polymer block: [ka] The notation indicates that the polymer block has a and b groups distributed throughout the block: [ka] where it is understood that about 75.5 weight percent of the block is monomer group a and about 24.5 weight percent of the block is monomer group b. [ka] does not indicate a polymer block comprising one homopolymer block of monomer group a and one homopolymer block of monomer group b. The same applies to the polymer block: [ka] This also applies to the notation, where the three monomer units are distributed throughout the block in approximately the total weight proportion indicated.

[0037] Targeted nucleic acid complex The terms "alkoxy" and "alkylthio" are used in their ordinary sense to refer to an alkyl group attached to the remainder of the molecule through an oxygen atom ("oxy") or a thio group, and further include mono- and poly-halogenated variants thereof.

[0038] The term “alkyl,” by itself or as part of another substituent, means, unless otherwise stated, alkyl having the specified number of carbon atoms (i.e., C 1-8 means a straight-chain or branched-chain hydrocarbon radical having 1 to 8 carbon atoms. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. The term "alkenyl" refers to an unsaturated alkyl radical having one or more double bonds. Similarly, the term "alkynyl" refers to an unsaturated alkyl radical 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 and 3-propynyl, 3-butynyl, and higher homologs and isomers.

[0039] The term "animal" includes mammalian species (such as humans, mice, rats, dogs, cats, hamsters, guinea pigs, rabbits, farm animals, and the like).

[0040] As used herein, the term "aryl" refers to a single all-carbon aromatic ring or to an all-carbon multiple fused ring system in which at least one of the rings is aromatic. For example, in certain embodiments, an aryl group has 6 to 20 carbon atoms, 6 to 14 carbon atoms, 6 to 12 carbon atoms, or 6 to 10 carbon atoms. Aryl includes phenyl radicals. Aryl also includes multiple fused carbocyclic ring systems (e.g., ring systems containing two, three, or four rings) having approximately 9 to 20 carbon atoms, where at least one ring is aromatic and the remaining rings can be aromatic or non-aromatic (e.g., cycloalkyl). The rings of a multiple fused ring system can be connected to each other via fused bonds, spiro bonds, and bridged bonds, where valency requirements permit. It is understood that the point of attachment of the multiple fused ring systems defined above can be at any position on the ring system, including the aromatic or carbocyclic portions of the ring. Non-limiting examples of aryl groups include, but are not limited to, phenyl, indenyl, indanyl, naphthyl, 1,2,3,4-tetrahydronaphthyl, anthracenyl, and the like.

[0041] The term "cycloalkyl" refers to an all-carbon (non-aromatic) saturated or (non-aromatic) partially unsaturated ring (i.e., (C3-C8 carbocycle)) having 3 to 8 carbon atoms. The term also includes all-carbon multiple fused saturated ring systems (e.g., ring systems containing two, three, or four carbocyclic rings). Thus, carbocycle includes multiple carbocycles, such as bicyclic carbocycles (e.g., bicyclic carbocycles having about 3 to 15 carbon atoms, about 6 to 15 carbon atoms, or 6 to 12 carbon atoms, such as bicyclo[3.1.0]hexane and bicyclo[2.1.1]hexane) and polycyclic carbocycles (e.g., tricyclic and tetracyclic carbocycles having up to about 20 carbon atoms). Rings of multiple fused ring systems can be connected to each other through fused, spiro, and bridged bonds, where valency requirements permit. For example, multicyclic carbocycles can be linked together through a single carbon atom to form a spiro bond (e.g., spiropentane, spiro[4,5]decane, etc.), can form a fused bond through two adjacent carbon atoms (e.g., carbocycles such as decahydronaphthalene, norsabinane, norcarane, etc.), or can form a bridged bond through two non-adjacent carbon atoms (e.g., norbornane, bicyclo[2.2.2]octane, etc.). Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo[2.2.1]heptane, pinane, and adamantane.

[0042] The term "gene" refers to a nucleic acid (eg, DNA or RNA) sequence that comprises partial or full-length coding sequences necessary for the production of a polypeptide or precursor polypeptide.

[0043] As used herein, "gene product" refers to the product of a gene (such as an RNA transcript or a polypeptide).

[0044] The term "halo" or "halogen" means, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.

[0045] As used herein, the term "heteroaryl" refers to a single aromatic ring having at least one atom other than carbon (selected from the group consisting of oxygen, nitrogen, and sulfur) in the ring. "Heteroaryl" also includes multiple condensed ring systems having at least one such aromatic ring, which are further described below. Thus, "heteroaryl" includes a single aromatic ring having about 1 to 6 carbon atoms and about 1 to 4 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur. When the ring is aromatic, the sulfur and nitrogen atoms may also be present in oxidized form. Examples of heteroaryl ring systems include, but are not limited to, pyridyl, pyrimidinyl, oxazolyl, and furyl. "Heteroaryl" also includes multiple condensed ring systems (e.g., ring systems containing 2, 3, or 4 rings) in which a heteroaryl group as defined above is fused to one or more rings selected from cycloalkyl, aryl, heterocycle, and heteroaryl. It will be understood that the point of attachment of a heteroaryl or heteroaryl multiple fused ring system can be at any suitable atom position of the heteroaryl or heteroaryl multiple fused ring system, including carbon atoms and heteroatoms (e.g., nitrogen). Examples of heteroaryls include, but are not limited to, pyridyl, pyrrolyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrazolyl, thienyl, indolyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, furyl, oxadiazolyl, thiadiazolyl, quinolyl, isoquinolyl, benzothiazolyl, benzoxazolyl, indazolyl, quinoxalyl, and quinazolyl.

[0046] The term "heterocycle" refers to a single saturated or partially unsaturated ring containing at least one atom other than carbon (selected from the group consisting of oxygen, nitrogen, and sulfur) in the ring. The term also includes multiple condensed ring systems containing at least one such saturated or partially unsaturated ring, which are further described below. Thus, the term includes a single saturated or partially unsaturated ring (e.g., a 3-, 4-, 5-, 6-, or 7-membered ring) containing about 1 to 6 carbon atoms in the ring and about 1 to 3 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur in the ring. The sulfur and nitrogen atoms may also be present in their oxidized forms. Examples of heterocycles include, but are not limited to, azetidinyl, tetrahydrofuranyl, and piperidinyl. The term "heterocycle" also includes multiple fused ring systems (e.g., ring systems containing two, three, or four rings) that can be formed by fusing a single heterocycle (as defined above) to one or more groups selected from cycloalkyl, aryl, and heterocycle. The rings of a multiple fused ring system can be connected to each other via fused bonds, spiro bonds, and bridged bonds, where valency requirements permit. It is understood that the individual rings of a multiple fused ring system can be connected to each other in any order. It is also understood that the point of attachment of a multiple fused ring system (as defined above for heterocycle) can be at any position in the multiple fused ring system, including the heterocyclic, aryl, and carbocyclic portions of the ring. In one embodiment, the term heterocycle includes a 3- to 15-membered heterocycle. In one embodiment, the term heterocycle includes a 3- to 10-membered heterocycle. In one embodiment, the term heterocycle includes a 3- to 8-membered heterocycle. In one embodiment, the term heterocycle includes a 3- to 7-membered heterocycle. In one embodiment, the term heterocycle includes a 3- to 6-membered heterocycle. In one embodiment, the term heterocycle includes 4- to 6-membered heterocycles. In one embodiment, the term heterocycle includes 3- to 10-membered monocyclic or bicyclic heterocycles containing 1 to 4 heteroatoms. In one embodiment, the term heterocycle includes 3- to 8-membered monocyclic or bicyclic heterocycles containing 1 to 3 heteroatoms. In one embodiment, the term heterocycle includes 3- to 6-membered monocyclic heterocycles containing 1 to 2 heteroatoms.In one embodiment, the term heterocycle includes 4-6 membered monocyclic heterocycles containing 1-2 heteroatoms. Examples of heterocycles include, but are not limited to, aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, homopiperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, tetrahydrofuranyl, dihydrooxazolyl, tetrahydropyranyl, tetrahydrothiopyranyl, 1,2,3,4-tetrahydroquinolyl, benzoxazinyl, dihydrooxazolyl, chromanyl, 1,2-dihydropyridinyl, 2,3-dihydrobenzoyl, and the like. furanyl, 1,3-benzodioxolyl, 1,4-benzodioxanyl, spiro[cyclopropane-1,1'-isoindolinyl]-3'-one, isoindolinyl-1-one, 2-oxa-6-azaspiro[3.3]heptanyl, imidazolidin-2-one, imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin, dioxolane, phthalimide, and 1,4-dioxane.

[0047] The term "sugar" includes monosaccharides, disaccharides, and trisaccharides, all of which may be optionally substituted. The term includes glucose, sucrose, fructose, galactose, and ribose, as well as deoxysugars (such as deoxyribose) and aminosugars (such as galactosamine). Sugar derivatives may be conveniently prepared as described in International Patent Applications WO 96 / 34005 and WO 97 / 03995. The sugar may be conveniently linked to the remainder of the compound of Formula I via an ether bond, a thioether bond (e.g., S-glycoside), an amine nitrogen (e.g., N-glycoside), or a carbon-carbon bond (e.g., C-glycoside). In one embodiment, the sugar may be conveniently linked to the remainder of the compound of Formula I via an ether bond.

[0048] As used herein, the term "small interfering RNA" or "siRNA" refers to a double-stranded RNA (i.e., duplex RNA) capable of reducing or suppressing expression of a target gene or sequence (e.g., by mediating degradation or translational inhibition of mRNA complementary to the siRNA sequence) when the siRNA is present 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 mismatched regions (i.e., mismatch motifs). In certain embodiments, the siRNA may have a length of about 19-25 (double-stranded) nucleotides, preferably about 20-24, about 21-22, or about 21-23 (double-stranded) nucleotides. The siRNA duplex may include a 3' overhang of about 1-4 nucleotides or about 2-3 nucleotides and a 5' phosphate terminus. Examples of siRNA include, but are not limited to, double-stranded polynucleotide molecules constructed from two separate strands, one of which is the sense strand and the other the complementary antisense strand.

[0049] In certain embodiments, the 5' overhang and / or 3' overhang present on one or both strands of the siRNA comprises 1 to 4 (e.g., 1, 2, 3, or 4) modified deoxythymidine (t or dT) nucleotides and / or unmodified deoxythymidine (t or dT) nucleotides, 1 to 4 (e.g., 1, 2, 3, or 4) modified (e.g., 2'OMe) uridine (U) ribonucleotides and / or unmodified uridine (U) ribonucleotides, and / or 1 to 4 (e.g., 1, 2, 3, or 4) modified (e.g., 2'OMe) ribonucleotides or deoxyribonucleotides and / or unmodified ribonucleotides or deoxyribonucleotides that are complementary to a target sequence (e.g., a 3' overhang in the antisense strand) or its complementary strand (e.g., a 3' overhang in the sense strand).

[0050] Preferably, siRNAs are chemically synthesized. siRNAs can also be generated by E. coli RNase III or Dicer cleavage of longer dsRNAs (e.g., dsRNAs greater than about 25 nucleotides in length). These enzymes process dsRNA into biologically active siRNA (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, the dsRNA has a length of at least 50 to about 100, 200, 300, 400, or 500 nucleotides. The dsRNA can be 1000, 1500, 2000, or 5000 nucleotides in length, or longer. The dsRNA can encode the entire gene transcript or a portion of the gene transcript. In certain cases, the siRNA can be encoded by a plasmid (e.g., transcribed as a sequence that automatically folds into a double-stranded sequence with a hairpin loop).

[0051] The phrase " target gene expression suppression " refers to the ability of the siRNA of the present invention to silence, reduce or suppress the expression of target gene.To examine the degree of gene silencing, a test sample (for example, a biological sample obtained from a target organism that expresses a target gene, or a sample of cultured cells that express a target gene) is contacted with the siRNA that silences, reduces or suppresses 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 (for example, a biological sample obtained from a target organism that expresses a target gene, or a sample of cultured cells that express a target gene) that is not contacted with siRNA.The control sample (for example, the sample that expresses a target gene) can be assigned a value of 100%. In certain embodiments, silencing, suppression, or reduction of expression of a target gene is achieved by a value of about 100%, about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 8%, or more in the test sample compared to a control sample (e.g., buffer only, an siRNA sequence targeting a different gene, a scrambled siRNA sequence, etc.). This is achieved when the expression level is 9%, about 88%, about 87%, about 86%, about 85%, about 84%, about 83%, about 82%, about 81%, about 80%, about 79%, about 78%, about 77%, about 76%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, or about 0%. Suitable assays include, but are not limited to, those examining protein or mRNA levels using techniques known to those of skill in the art, such as, for example, dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays known to those of skill in the art.

[0052] An "effective amount" or "therapeutically effective amount" of a therapeutic nucleic acid (e.g., siRNA) is an amount sufficient to achieve the desired effect (e.g., suppression of expression of a target sequence compared to the normal expression level detected in the absence of the siRNA). In certain embodiments, suppression of expression of a target gene or target sequence is achieved with an siRNA by about 100%, about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 8%, or more, as compared to a control (e.g., buffer only, an siRNA sequence targeting a different gene, a scrambled siRNA sequence, etc.). This is achieved when the expression of a target gene or sequence is 9%, about 88%, about 87%, about 86%, about 85%, about 84%, about 83%, about 82%, about 81%, about 80%, about 79%, about 78%, about 77%, about 76%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, or about 0%. Suitable assays for measuring expression of a target gene or target sequence include, but are not limited to, those that examine protein or mRNA levels using techniques known to those of skill in the art, such as, for example, dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays known to those of skill in the art.

[0053] As used herein, the term "nucleic acid" 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" comprises a sugar (deoxyribose (DNA) or ribose (RNA)), a base, and a phosphate group. Nucleotides are linked together via the phosphate group. A "base" includes purines and pyrimidines, which further include naturally occurring compounds (adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs) as well as synthetic derivatives of purines and pyrimidines, including modifications that introduce new reactive groups (such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides). Nucleic acids include known nucleotide analogs or nucleic acids containing modified backbone residues or modified backbone linkages, which are synthetic, naturally occurring, and non-naturally occurring, and have similar binding properties to the 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). Additionally, nucleic acids can contain one or more UNA moieties.

[0054] The term "protecting group" refers to a substituent that is commonly employed to block or protect a particular functional group on a compound. For example, an "amino-protecting group" is a substituent that is added to an amino group to block or protect the amino functionality in the compound. Suitable amino-protecting groups include acetyl, trifluoroacetyl, t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ), and 9-fluorenylmethyleneoxycarbonyl (Fmoc). Similarly, a "hydroxy-protecting group" refers to a substituent of a hydroxy group that blocks or protects the hydroxy functionality. Suitable protecting groups include acetyl, silyl, and 2,2-dimethoxypropene. A "carboxy-protecting group" refers to a substituent of a carboxy group that blocks or protects the carboxy functionality. Common carboxy protecting groups include phenylsulfonylethyl, cyanoethyl, 2-(trimethylsilyl)ethyl, 2-(trimethylsilyl)ethoxymethyl, 2-(p-toluenesulfonyl)ethyl, 2-(p-nitrophenylsulfenyl)ethyl, 2-(diphenylphosphino)-ethyl, nitroethyl, and the like. For a general description of protecting groups and their use, see P.G.M.Wuts and T.W.Greene, Greene's Protective Groups in Organic Synthesis 4 th edition, Wiley-Interscience, New York, 2006.

[0055] 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 will be understood that the nature of the synthetic activating group can depend on the atom being activated. For example, when a 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 a synthetic activating group is attached to the oxygen atom of a carboxylic acid, the synthetic activating group can be a group 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), (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), propylphosphonic anhydride solution (T3P), or O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU).

[0056] nucleic acid The term "nucleic acid" includes any oligonucleotide or polynucleotide; fragments containing up to 60 nucleotides are generally referred to as oligonucleotides, while longer fragments are referred to as polynucleotides. Deoxyribooligonucleotides consist of a five-carbon sugar called deoxyribose covalently linked to phosphate at its 5' and 3' carbons to form an alternating, unbranched polymer. DNA forms can be, for example, antisense molecules, plasmid DNA, pre-condensed DNA, PCR products, vectors, expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups. Ribooligonucleotides consist of a similar repeating structure in which the five-carbon sugar is ribose. RNA forms can be, for example, small interfering RNA (siRNA), Dicer substrate dsRNA, short hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, tRNA, viral RNA (vRNA), self-amplifying RNA (sa-RNA), 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 composed of naturally occurring bases, sugars, and intersugar (backbone) linkages. The terms "polynucleotide" and "oligonucleotide" also include polymers or oligomers or portions thereof that contain non-naturally occurring monomers, which function similarly. Such modified or substituted oligonucleotides are often preferable to naturally occurring forms because of their properties, such as enhanced cellular uptake, reduced immunogenicity, and increased stability in the presence of nucleases.

[0057] Unless otherwise specified, a particular nucleic acid sequence implicitly includes not only the sequence explicitly set forth, but also conservatively modified variants thereof (e.g., those with degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected codons (or all codons) is substituted 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)).

[0058] As described herein, certain embodiments of the present invention provide methods and compositions for delivering nucleic acids to cells. In certain embodiments, the nucleic acid is a nucleic acid described herein. For example, the nucleic acid used herein can be single-stranded DNA or RNA, or double-stranded DNA or RNA, or a DNA-RNA hybrid. Examples of double-stranded RNA are described herein, and examples of double-stranded RNA include, for example, siRNA and other RNAi agents (such as aiRNA and pre-miRNA). Single-stranded nucleic acids include, for example, antisense oligonucleotides, ribozymes, mature miRNAs, and triplex-forming oligonucleotides.

[0059] In certain embodiments, the nucleic acid is an oligonucleotide. In certain embodiments, the length range of the oligonucleotide is from about 10 to about 100 nucleotides. In various related embodiments, the length range of the oligonucleotide (both single-stranded, double-stranded, and triple-stranded) can be from about 10 to about 60, from about 15 to about 60, from about 20 to about 50, from about 15 to about 30, or from about 20 to about 30 nucleotides.

[0060] In certain embodiments, the nucleic acid is selected from the group consisting of small interfering RNA (siRNA), Dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), tRNA, rRNA, tRNA, viral RNA (vRNA), self-amplifying RNA (sa-RNA), and combinations thereof.

[0061] In certain embodiments, the nucleic acid is an antisense molecule. In certain embodiments, the nucleic acid is an miRNA molecule. In certain embodiments, the nucleic acid is an siRNA. Suitable siRNA, as well as methods and intermediates useful for its preparation, are reported in International Patent Application Publication No. WO2016 / 054421.

[0062] Target gene In certain embodiments, nucleic acids (e.g., siRNAs) can be used to downregulate or silence the translation (i.e., expression) of a gene of interest. Genes of interest include, but are not limited to, genes associated with viral infection and survival, genes associated with metabolic diseases and disorders (e.g., liver diseases and disorders), genes associated with tumorigenesis and cell transformation (e.g., cancer), angiogenesis genes, immunoregulatory genes (such as those associated with inflammatory and autoimmune responses), ligand receptor genes, and genes associated with neurodegenerative disorders. In certain embodiments, the gene of interest is expressed in hepatocytes.

[0063] Genes associated with viral infection and survival include those expressed by viruses in order to bind to, enter, and replicate in cells. Viral sequences associated with chronic viral diseases are of particular interest. Viral sequences of particular interest include filovirus (such as Ebola virus and Marburg virus) sequences (see, e.g., Geisbert et al., J. Infect. Dis., 193:1650-1657 (2006)), arenavirus (such as Lassa virus, Junin virus, Machupo virus, Guanarito virus, and Sabia virus) sequences (see, e.g., Buchmeier et al., Arenaviridae: the viruses and their replication, In: FIELDS VIROLOGY, Knipe et al. (eds.), 4th ed., Lippincott-Raven, Philadelphia, (2001)), influenza virus (such as influenza A virus, influenza B virus, and influenza C virus) sequences (see, e.g., Steinhauer et al., Annu Rev Genet., 36:305-332 (2002), and Neumann et al., J Gen Virol., 83:2635-2662 (2002)), hepatitis virus sequences (see, e.g., Hamasaki et al., FEBS Lett., 543:51 (2003), Yokota et al., EMBO Rep., 4:602 (2003), Schlomai et al., Hepatology, 37:764 (2003), Wilson et al., Proc. Natl. Acad. Sci. USA, 100:2783 (2003), Kapadia et al., Proc. Natl. Acad. Sci. USA, 100:2014 (2003), and FIELDS VIROLOGY, Knipe et al. (eds.), 4th ed., Lippincott-Raven, Philadelphia (2001)), human immunodeficiency virus (HIV) sequences (Banerjea et al., Mol. Ther., 8:62 (2003); Song et al., J. Virol., 77:7174 (2003), Stephenson, JAMA, 289:1494 (2003), Qin et al., Proc. Natl. Acad. Sci. USA, 100:183 (2003)), herpesvirus sequences (Jia et al., J. Virol., 77:3301 (2003)), and human papillomavirus (HPV) sequences (Hall et al., J. Virol., 77:6066 (2003), Jiang et al., Oncogene, 21:6041 (2002)).

[0064] Examples of filovirus nucleic acid sequences that can be silenced include, but are not limited to, nucleic acid sequences encoding structural proteins (e.g., VP30, VP35, nucleoprotein (NP), polymerase protein (L-pol)) and membrane-associated proteins (e.g., VP40, glycoprotein (GP), VP24). The complete genome sequence of Ebola virus is shown, for example, in GenBank accession numbers NC_002549, AY769362, NC_006432, NC_004161, AY729654, AY354458, AY142960, AB050936, AF522874, AF499101, AF272001, and AF086833. The sequence of Ebola virus VP24 is shown, for example, in GenBank accession numbers U77385 and AY058897. The sequence of Ebola virus L-pol is shown, for example, in Genbank accession number X67110. The sequence of Ebola virus VP40 is shown, for example, in Genbank accession number AY058896. The sequence of Ebola virus NP is shown, for example, in Genbank accession number AY058895. The sequence of Ebola virus GP is shown, for example, in Genbank accession number AY058898, Sanchez et al., Virus Res., 29:215-240 (1993), Will et al., J. Virol., 67:1203-1210 (1993), Volchkov et al., FEBS Lett., 305:181-184 (1992), and U.S. Patent No. 6,713,069. Additional Ebola virus sequences are set forth, for example, in Genbank accession numbers L11365 and X61274. The complete genome sequence of Marburg virus is set forth, for example, in Genbank accession numbers NC_001608, AY430365, AY430366, and AY358025. The sequence of Marburg virus GP is set forth, for example, in Genbank accession numbers AF005734, AF005733, and AF005732. The sequence of Marburg virus VP35 is set forth, for example, in Genbank accession numbers AF005731 and AF005730.Additional Marburg virus sequences are set forth, for example, in Genbank Accession Nos. X64406, Z29337, AF005735, and Z12132. Examples of siRNA molecules targeting Ebola virus nucleic acid sequences and siRNA molecules targeting Marburg virus nucleic acid sequences include, but are not limited to, those described in U.S. Patent Publication No. 20070135370, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0065] Examples of influenza virus nucleic acid sequences that can be silenced include, but are not limited to, nucleic acid sequences encoding nucleoprotein (NP), matrix proteins (M1 and M2), nonstructural proteins (NS1 and NS2), RNA polymerases (PA, PB1, PB2), neuraminidase (NA), and hemagglutinin (HA). The sequences of influenza A NP are listed, for example, under GenBank accession numbers NC_004522, AY818138, AB166863, AB188817, AB189046, AB189054, AB189062, AY646169, AY646177, AY651486, AY651493, AY651494, AY651495, AY651496, and AY651497. These are shown in Y651497, AY651498, AY651499, AY651500, AY651501, AY651502, AY651503, AY651504, AY651505, AY651506, AY651507, AY651509, AY651528, AY770996, AY790308, AY818138, and AY818140. Influenza A PA sequences are listed, for example, under GenBank accession numbers AY818132, AY790280, AY646171, AY818132, AY818133, AY646179, AY818134, AY551934, AY651613, AY651610, AY651620, AY651617, AY651600, and AY AY651611, AY651606, AY651618, AY651608, AY651607, AY651605, AY651609, AY651615, AY651616, AY651640, AY651614, AY651612, AY651621, AY651619, AY770995, and AY724786.Examples of siRNA molecules that target influenza virus nucleic acid sequences include, but are not limited to, those described in U.S. Patent Publication No. 20070218122, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0066] Examples of nucleic acid sequences of hepatitis viruses that can be silenced include, but are not limited to, nucleic acid sequences involved in transcription and translation (e.g., En1, En2, X, P), and nucleic acid sequences encoding structural proteins (e.g., core protein (including C protein and C-related protein), capsid, and envelope proteins (including S protein, M protein, and / or L protein), or fragments thereof) (see, e.g., FIELDS VIROLOGY, supra). Examples of nucleic acid sequences of hepatitis C virus (HCV) that can be silenced include, but are not limited to, the 5'-untranslated region (5'-UTR), the 3'-untranslated region (3'-UTR), the polyprotein translation initiation codon region, the internal ribosome entry site (IRES) sequence, and / or a nucleic acid sequence encoding a core protein, a nucleic acid sequence encoding an E1 protein, a nucleic acid sequence encoding an E2 protein, a nucleic acid sequence encoding a p7 protein, a nucleic acid sequence encoding an NS2 protein, a nucleic acid sequence encoding an NS3 protease / helicase, a nucleic acid sequence encoding an NS4A protein, a nucleic acid sequence encoding an NS4B protein, a nucleic acid sequence encoding an NS5A protein, and / or a nucleic acid sequence encoding an NS5B RNA-dependent RNA polymerase. The genome sequence of HCV is shown, for example, in GenBank accession numbers NC_004102 (HCV genotype 1a), AJ238799 (HCV genotype 1b), NC_009823 (HCV genotype 2), NC_009824 (HCV genotype 3), NC_009825 (HCV genotype 4), NC_009826 (HCV genotype 5), and NC_009827 (HCV genotype 6). The nucleic acid sequence of hepatitis A virus is shown, for example, in GenBank accession number NC_001489. The nucleic acid sequence of hepatitis B virus is shown, for example, in GenBank accession number NC_003977. The nucleic acid sequence of hepatitis D virus is shown, for example, in GenBank accession number NC_001653. The nucleic acid sequence of Hepatitis E virus is shown, for example, in Genbank Accession No. NC_001434. The nucleic acid sequence of Hepatitis G virus is shown, for example, in Genbank Accession No. NC_001710.Silencing the sequence that encodes the gene that is related to viral infection and survival can be advantageously combined with the administration of common drugs that are used to treat viral disease.Examples of siRNA molecules that target the nucleic acid sequence of hepatitis virus include but are not limited to those described in U.S. Patent Publication No. 20060281175, U.S. Patent Publication No. 20050058982 and U.S. Patent Publication No. 20070149470, U.S. Patent No. 7,348,314 and U.S. Provisional Application No. 61 / 162,127 filed on March 20, 2009, the disclosures of which are incorporated herein by reference in their entirety for any purpose.

[0067] Genes associated with metabolic diseases and disorders (e.g., disorders in which the liver is a target, and diseases and disorders of the liver) include, for example, genes expressed in dyslipidemia (e.g., liver X receptors (LXRα and LXRβ (Genbank accession number NM_007121)), farnesoid X receptor (FXR) (Genbank accession number NM_005123), sterol regulatory element binding protein (SREBP), site-1 protease (SIP), 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMG coenzyme-A reductase), apolipoprotein B (ApoB) (Genbank accession number NM_000384), apolipoprotein CIII (ApoC3) (Genbank accession numbers NM_000040 and NM_008949)). REGION:5001.8164), and apolipoprotein E (ApoE) (Genbank accession numbers NM_000041 and NG_007084 REGION:5001.8612), as well as genes expressed in diabetes (e.g., glucose 6-phosphatase) (e.g., Forman et al., Cell, 81:687 (1995); Seol et al., Mol. Endocrinol., 9:72 (1995); Zavacki et al., Proc. Natl. Acad. Sci. USA, 94:7909 (1997); Sakai et al., Cell, 85:1037-1046 (1996); Duncan et al., J. Biol. Chem., 272:12778-12785 (1997); Willy et al., Genes Dev., 9:1033-1045 (1995); Lehmann et al., J. Biol. Chem., 272:3137-3140 (1997); Janowski et al., Nature, 383:728-731 (1996); and Peet et al., Cell, 93:693-704 (1998). Those skilled in the art will understand that genes associated with metabolic diseases and disorders (e.g., diseases and disorders in which the liver is a target, and diseases and disorders of the liver) include not only genes expressed in the liver itself, but also genes expressed in other organs and tissues.Silencing of sequences encoding genes associated with metabolic diseases and disorders can be advantageously combined with the administration of common drugs used to treat diseases or disorders. Examples of siRNA molecules targeting the ApoB gene include, but are not limited to, those described in U.S. Patent Publication No. 20060134189, the disclosure of which is incorporated herein by reference in its entirety for all purposes. Examples of siRNA molecules targeting the ApoC3 gene include, but are not limited to, those described in U.S. Provisional Application No. 61 / 147,235, filed January 26, 2009, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0068] Examples of gene sequences associated with tumorigenesis and cell transformation (e.g., cancer or other neoplasms) include mitotic kinesins (such as Eg5 (KSP, KIF11; Genbank accession number NM_004523)), serine / threonine kinases (such as polo-like kinase 1 (PLK-1) (Genbank accession number NM_005030, Barr et al., Nat. Rev. Mol. Cell. Biol., 5:429-440 (2004))), tyrosine kinases (such as WEE1 (Genbank accession numbers NM_003390 and NM_001143976)), inhibitors of apoptosis (such as XIAP (Genbank accession number NM_001167)), COP9 signalosome subunits (CSN1, CSN2, CSN3, CSN4, CSN5 (JAB1; Genbank accession number NM_001167)), and the like. Examples of siRNA molecules targeting the Eg5 gene and XIAP gene include, but are not limited to, those described in U.S. Patent Application No. 11 / 807,872, filed May 29, 2007, the disclosure of which is incorporated herein by reference in its entirety for all purposes. Examples of siRNA molecules targeting the PLK-1 gene include, but are not limited to, those described in U.S. Patent Publication Nos. 20050107316 and 20070265438, and U.S. Patent Application No. 12 / 343,342, filed December 23, 2008, the disclosures of which are incorporated herein by reference in their entirety for all purposes. Examples of siRNA molecules targeting the CSN5 gene include, but are not limited to, those described in U.S. Provisional Application No. 61 / 045,251, filed April 15, 2008, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0069] Additional examples of gene sequences associated with tumorigenesis and cell transformation include translocation sequences (such as MLL fusion genes (BCR-ABL (Wilda et al., Oncogene, 21:5716 (2002); Scherr et al., Blood, 101:1566 (2003)), TEL-AML1, EWS-FLI1, TLS-FUS, PAX3-FKHR, BCL-2, AML1-ETO, and AML1-MTG8 (Heidenreich et al., Blood, 101:3157 (2003))), overexpression sequences (multidrug resistance genes (Nieth et al., FEBS Lett., 545:144 (2003); Wu et al., Cancer Res., 63:1515 (2003)), cyclins (Li et al., Cancer Res., 63:3593 (2003); Zou et al. al., Genes Dev., 16:2923 (2002)), beta-catenin (Verma et al., Clin Cancer Res., 9:1291 (2003)), telomerase gene (Kosciolek et al., Mol Cancer Ther., 2:209 (2003)), c-MYC, N-MYC, BCL-2, growth factor receptors (e.g., EGFR / ErbB1 (Genbank accession numbers NM_005228, NM_201282, NM_201283, and NM_201284 (Nagy et al. Exp. Cell Res., 285:39-49 (2003)), ErbB2 / HER-2 (Genbank Accession Nos. NM_004448 and NM_001005862), ErbB3 (Genbank Accession Nos. NM_001982 and NM_001005915), and ErbB4 (Genbank Accession Nos. NM_005235 and NM_001042599)), and mutant sequences such as RAS (reviewed in Tuschl and Borkhardt, Mol. Interventions, 2:158 (2002)).Examples of siRNA molecules targeting the EGFR gene include, but are not limited to, those described in U.S. Patent Application No. 11 / 807,872, filed May 29, 2007, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0070] Silencing sequences encoding DNA repair enzymes is useful in combination with the administration of chemotherapeutic agents (Collis et al., Cancer Res., 63:1550 (2003)). Genes encoding proteins associated with tumor cell migration are also target sequences of interest (e.g., integrins, selectins, and metalloproteinases). The foregoing examples are not exhaustive. Those skilled in the art will understand that any whole or partial gene sequence that facilitates or promotes tumorigenesis or cell transformation, tumor growth, or tumor cell migration can be included as a template sequence.

[0071] Angiogenic genes can promote the formation of new blood vessels. Vascular endothelial growth factor (VEGF) (Reich et al., Mol. Vis., 9:210 (2003)) or VEGFR is of particular interest. siRNA sequences targeting VEGFR are shown, for example, in GB2396864, U.S. Patent Publication No. 20040142895, and CA2456444, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0072] Anti-angiogenic genes can inhibit angiogenesis. Such genes are particularly useful for treating cancers in which angiogenesis plays a role in the pathogenesis of the disease. Examples of anti-angiogenic genes include, but are not limited to, endostatin (see, e.g., U.S. Pat. No. 6,174,861), angiostatin (see, e.g., U.S. Pat. No. 5,639,725), and VEGFR2 (see, e.g., Decaussin et al., J. Pathol., 188:369-377 (1999)), the disclosures of which are incorporated herein by reference in their entirety for all purposes. Immunomodulatory genes are genes that regulate one or more immune responses. Examples of immunomodulatory genes include, but are not limited to, cytokines, such as growth factors (e.g., TGF-α, TGF-β, EGF, FGF, IGF, NGF, PDGF, CGF, GM-CSF, SCF, etc.), interleukins (e.g., IL-2, IL-4, IL-12 (Hill et al., J. Immunol., 171:691 (2003)), IL-15, IL-18, IL-20, etc.), interferons (e.g., IFN-α, IFN-β, IFN-γ, etc.), and TNF. The Fas gene and Fas ligand gene are also immunomodulatory target sequences of interest (Song et al., Nat. Med., 9:347 (2003)). The present invention also encompasses genes encoding secondary signaling molecules in hematopoietic and lymphoid cells, such as Tec family kinases (Bruton's tyrosine kinase (Btk) (Heinonen et al., FEBS Lett., 527:274 (2002))).

[0073] Cell receptor ligands include ligands that can bind to cell surface receptors (e.g., insulin receptors, EPO receptors, G protein-coupled receptors, receptors with tyrosine kinase activity, cytokine receptors, growth factor receptors, etc.) and thereby modulate (e.g., inhibit, activate, etc.) physiological pathways in which the receptor participates (e.g., regulation of glucose levels, blood cell development, mitogenesis, etc.). Examples of cell receptor ligands include, but are not limited to, cytokines, growth factors, interleukins, interferons, erythropoietin (EPO), insulin, glucagon, G protein-coupled receptor ligands, etc. Templates encoding expanded trinucleotide repeats (e.g., CAG repeats) are useful for silencing pathogenic sequences in neurodegenerative disorders caused by trinucleotide repeat expansions, such as spinal-bulbar muscular atrophy and Huntington's disease (Caplen et al., Hum. Mol. Genet., 11:175 (2002)).

[0074] Certain other target genes whose expression can be targeted by nucleic acids (e.g., siRNA) to downregulate or silence include, but are not limited to, actin, alpha 2, smooth muscle, aortic (ACTA2), alcohol dehydrogenase 1A (ADH1A), alcohol dehydrogenase 4 (ADH4), alcohol dehydrogenase 6 (ADH6), afamin (AFM), angiotensinogen (AGT), serine-pyruvate aminotransferase (AGXT), alpha -2-HS-glycoprotein (AHSG), aldo-keto reductase family 1 member C4 (AKR1C4), serum albumin (ALB), alpha-1-microglobulin / bikunin precursor (AMBP), angiopoietin-related protein 3 (ANGPTL3), serum amyloid P component (APCS), apolipoprotein A-II (APOA2), apolipoprotein B-100 (APOB), apolipoprotein C3 (APOC3), apolipoprotein C-IV (APOC4), apolipoprotein B-II (APOA2), apolipoprotein B ... Polyprotein F (APOF), beta-2-glycoprotein 1 (APOH), aquaporin-9 (AQP9), bile acid-CoA:amino acid N-acyltransferase (BAAT), C4b-binding protein beta chain (C4BPB), putative protein of unknown nature encoded by LINC01554 (C5orf27), complement factor 3 (C3), complement factor 5 (C5), complement component C6 (C6), complement component C8 alpha chain (C8A), complement component C8 beta chain (C8B), complement component C8 gamma chain (C8G), complement component C8 alpha chain (C8G), complement component C8 beta chain (C8G ... 8G), complement component C9 (C9), calmodulin-binding transcription activator 1 (CAMTA1), CD38 (CD38), complement factor B (CFB), complement factor H-related protein 1 (CFHR1), complement factor H-related protein 2 (CFHR2), complement factor H-related protein 3 (CFHR3), cannabinoid receptor 1 (CNR1), ceruloplasmin (CP), carboxypeptidase B2 (CPB2), connective tissue growth factor (CTGF), C-X-C motif chemokine 2 (CXCL2), cytochrome P450 1A2 (CYP1A2), cytochrome P450 2A6 (CYP2A6), cytochrome P450 2C8 (CYP2C8), cytochrome P450 2C9 (CYP2C9),Cytochrome P450 family 2 subfamily D member 6 (CYP2D6), cytochrome P450 2E1 (CYP2E1), phylloquinone omega-hydroxylase CYP4F2 (CYP4F2), 7-alpha-hydroxycholest-4-en-3-one 12-alpha-hydroxylase (CYP8B1), dipeptidyl peptidase 4 (DPP4), coagulation factor 12 (F12), coagulation factor II (thrombin) (F2), coagulation factor IX (F9), fibrinogen alpha chain (FGA), fibrinogen beta chain (FGB), fibrinogen gamma chain (FGG), fibrinogen-like 1 (FGL1), flavin-containing monoiodophosphate (FMP) Oxygenase 3 (FMO3), flavin-containing monooxygenase 5 (FMO5), group-specific component (vitamin D binding protein) (GC), growth hormone receptor (GHR), glycine N-methyltransferase (GNMT), hyaluronic acid binding protein 2 (HABP2), hepcidin antimicrobial peptide (HAMP), hydroxyacid oxidase (glycolate oxidase) 1 (HAO1), HGF activator (HGFAC), haptoglobin-related protein, haptoglobin (HPR), hemopexin (HPX), high histamine cysteine ​​glycoprotein (HRG), hydroxysteroid (11-beta) dehydrogenase 1 (HSD11B1), hydroxysteroid (17-beta) dehydrogenase 13 (HSD17B13), inter-alpha-trypsin inhibitor heavy chain H1 (ITIH1), inter-alpha-trypsin inhibitor heavy chain H2 (ITIH2), inter-alpha-trypsin inhibitor heavy chain H3 (ITIH3), inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), prekallikrein (KLKB1) ), lactate dehydrogenase (LDHA), liver-expressed antimicrobial peptide 2 (LEAP2), leukocyte-derived chemotaxin 2 (LECT2), lipoprotein(a) (LPA), mannan-binding lectin serine peptidase 2 (MASP2), S-adenosylmethionine synthase isoform type 1 (MAT1A), NADPH oxidase 4 (NOX4), poly[ADP-ribose] polymerase 1 (PARP1), paraoxonase 1 (PON1), paraoxonase 3 (PON3), vitamin K-dependent protein C (PROC),Retinol dehydrogenase 16 (RDH16), constitutive serum amyloid A4 (SAA4), serine dehydratase (SDS), serpin family member 1 (SERPINA1), serpin A11 (SERPINA11), kallistatin (SERPINA4), corticosteroid-binding globulin (SERPINA6), antithrombin-III (SERPINC1), heparin cofactor 2 (SERPIND1), serpin family H member 1 (SERPINH1), solute carrier family 5 member 2 (SLC5A2), sodium / bile acid cotransporter (SLC10A1), solute carrier family 13 member 5 (SLC13A5), solute carrier family 22 member 1 (SLC22A1), solute carrier family 25 member 47 (SLC25A47), solute carrier family These include glucose transporter 2 (SLC2A2), facilitated diffusion type glucose transporter member 2 (SLC38A4), sodium-dependent neutral amino acid transporter 4 (SLC38A4), solute carrier organic anion transporter family member 1B1 (SLCO1B1), sphingomyelin phosphodiesterase 1 (SMPD1), bile salt sulfotransferase (SULT2A1), tyrosine aminotransferase (TAT), tryptophan 2,3-dioxygenase (TDO2), UDP-glucuronosyltransferase 2 family, polypeptide B10 (UGT2B10), UDP-glucuronosyltransferase 2 family, polypeptide B15 (UGT2B15), UDP-glucuronosyltransferase 2 family, polypeptide B4 (UGT2B4), and vitronectin (VTN).

[0075] In addition to their usefulness in silencing the expression of any of the above genes for therapeutic purposes, certain nucleic acids (e.g., siRNAs) described herein are also useful in research and development applications, as well as diagnostic, preventive, predictive, clinical, and other medical applications. As a non-limiting example, certain nucleic acids (e.g., siRNAs) can be used in target validation studies to test whether a gene of interest may be a potential therapeutic target. Certain nucleic acids (e.g., siRNAs) can also be used in target identification studies to discover genes as potential therapeutic targets.

[0076] Generation of siRNA molecules siRNA can be provided in several forms, such as one or more isolated short interfering RNA (siRNA) duplexes, longer double-stranded RNA (dsRNA), or siRNA or dsRNA transcribed from transcription cassette in DNA plasmid.In some embodiments, siRNA can be produced enzymatically or by partial / total organic synthesis, and modified ribonucleotides can be introduced by in vitro enzymatic synthesis or organic synthesis.In certain cases, each strand is chemically prepared.Methods for synthesizing RNA molecules are known in the art, such as the chemical synthesis method described in Verma and Eckstein (1998) or herein.

[0077] Methods for isolating RNA, synthesizing RNA, hybridizing nucleic acids, preparing 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 also well known in the art (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 skilled in the art. Additional basic textbooks that disclose 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 such references are incorporated herein by reference in their entireties for all purposes.

[0078] Typically, siRNA is chemically synthesized.The oligonucleotide that constitutes the siRNA molecule of the present invention can be synthesized by any of various methods 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).In the synthesis of oligonucleotide, common nucleic acid protecting group and nucleic acid coupling group are used, such as dimethoxytrityl for 5'-end and phosphoramidite for 3'-end. As a non-limiting example, small-scale synthesis can be performed on an Applied Biosystems synthesizer using a 0.2 μmol scale protocol. Alternatively, 0.2 μmol scale synthesis can be performed on a 96-well plate synthesizer supplied by Protogene (Palo Alto, CA). However, larger or smaller scale syntheses are also within the scope of the present invention. Reagents suitable for oligonucleotide synthesis, methods for RNA deprotection, and methods for RNA purification are known to those skilled in the art.

[0079] An siRNA molecule can be constructed from two different oligonucleotides, one constituting the sense strand of the siRNA and the other constituting the antisense strand of the siRNA. For example, each strand can be synthesized separately and, after synthesis and / or deprotection, linked together by hybridization or ligation.

[0080] linking group The conjugates of the invention may comprise one or more linking groups (e.g., L 3 or L 4) The structure of each linking group can vary depending on the complex function described herein. For example, the structure of each linking group can vary in length and atomic composition, and each linking group can be branched, unbranched, cyclic, or a combination thereof. The linking group can also modulate the solubility, stability, or aggregation properties of the complex.

[0081] In one embodiment, each linking group contains about 3 to 1000 atoms. In one embodiment, each linking group contains about 3 to 500 atoms. In one embodiment, each linking group contains about 3 to 200 atoms. In one embodiment, each linking group contains about 3 to 50 atoms. In one embodiment, each linking group contains about 10 to 1000 atoms. In one embodiment, each linking group contains about 10 to 500 atoms. In one embodiment, each linking group contains about 10 to 200 atoms. In one embodiment, each linking group contains about 10 to 50 atoms.

[0082] In one embodiment, each linking group comprises an atom selected from H, C, N, S, and O.

[0083] In one embodiment, each linking group comprises an atom selected from H, C, N, S, P, and O.

[0084] In one embodiment, each linking group comprises a branched or unbranched saturated or unsaturated hydrocarbon chain having about 1 to 1000 carbon atoms (or 1 to 750, 1 to 500, 1 to 250, 1 to 100, 1 to 50, 1 to 25, 1 to 10, 1 to 5, 5 to 1000, 5 to 750, 5 to 500, 5 to 250, 5 to 100, 5 to 50, 5 to 25, 5 to 10, or 2 to 5 carbon atoms), wherein one or more of the carbon atoms are selected from the group consisting of -O-, -S, -N(R a)-, a 3- to 7-membered heterocycle, a 5- to 6-membered heteroaryl, or a carbocycle, each of which is optionally replaced by (C-C)alkyl, (C-C)alkoxy, (C-C)cycloalkyl, (C-C)alkanoyl, (C-C)alkanoyloxy, (C-C)alkoxycarbonyl, (C-C)alkylthio, azido, cyano, nitro, halo, -N(R a )2, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy; and each R a are independently H or (C1-C6) alkyl. In one embodiment, the linker comprises a branched or unbranched saturated or unsaturated hydrocarbon chain having about 1 to 1000 carbon atoms (or 1 to 750, 1 to 500, 1 to 250, 1 to 100, 1 to 50, 1 to 25, 1 to 10, 1 to 5, 5 to 1000, 5 to 750, 5 to 500, 5 to 250, 5 to 100, 5 to 50, 5 to 25, 5 to 10, or 2 to 5 carbon atoms), wherein one or more of the carbon atoms are selected from the group consisting of -O-, -S, -N(R a )-, each R a is independently H or (C1-C6) alkyl.

[0085] In one embodiment, each linking group comprises polyethylene glycol. In one embodiment, the linking group comprises polyethylene glycol linked to the remainder of the targeting conjugate by a carbonyl group. In one embodiment, the polyethylene glycol comprises about 1 to about 500, or about 5 to about 500, or about 3 to about 100 repeat (e.g., —CHCHO—) units (Greenwald, R.B., et al., Poly(ethylene glycol) Prodrugs: Altered Pharmacokinetics and Pharmacodynamics, Chapter, 2.3.1., 283-338; Filpula, D., et al., Releasable PEGylation of proteins with customized linkers, Advanced Drug Delivery, 60, 2008, 29-49; Zhao, H., et al., Drug Conjugates with Poly(Ethylene Glycol), Drug Delivery in Oncology, 2012, 627-656).

[0086] Embodiment In one embodiment, A is a targeting ligand that specifically binds to a molecule on the surface of a target cell.

[0087] In one embodiment, the nucleic acid complex and the membrane destabilizing polymer are administered separately.

[0088] In one embodiment, the membrane destabilizing polymer is administered after administration of the nucleic acid complex.

[0089] In one embodiment, the nucleic acid complex and the membrane destabilizing polymer are administered together in a single composition.

[0090] In one embodiment, the targeting ligand and T 5 are different and either (i) specifically bind to the same cell surface molecule or (ii) specifically bind to different cell surface molecules on the target cell.

[0091] In one embodiment, the targeting ligand and T 5 are identical and each specifically binds to the same cell surface molecule.

[0092] In one embodiment, the cell is a secretory cell, a chondrocyte, an epithelial cell, a neuronal cell, a muscle cell, a blood cell, an endothelial cell, a pericyte, a fibroblast, a glial cell, or a dendritic cell.

[0093] In one embodiment, the cell is a cancer cell, an immune cell, a bacterially infected cell, a virally infected cell, or a cell with abnormal metabolic activity.

[0094] In one embodiment, the targeting ligand specifically binds to a cell surface molecule selected from the group consisting of transferrin receptor type 1, transferrin receptor type 2, EGF receptor, HER2 / Neu, VEGF receptor, PDGF receptor, integrin, NGF receptor, CD2, CD3, CD4, CD8, CD19, CD20, CD22, CD33, CD43, CD38, CD56, CD69, asialoglycoprotein receptor (ASGPR), prostate-specific membrane antigen (PSMA), folate receptor, and sigma receptor.

[0095] In one embodiment, the targeting ligand comprises a small molecule targeting moiety.

[0096] In one embodiment, the small molecule targeting moiety is a sugar, vitamin, bisphosphonate, or analog thereof.

[0097] In one embodiment, the sugar is selected from lactose, galactose, N-acetylgalactosamine (NAG), mannose, and mannose-6-phosphate (M6P).

[0098] In one embodiment, the vitamin is folic acid.

[0099] In one embodiment, the targeting ligand comprises a protein.

[0100] In one embodiment, the protein is an antibody, a peptide aptamer, or a protein derived from the natural ligand of a cell surface molecule.

[0101] In one embodiment, the targeting ligand comprises a peptide.

[0102] In one embodiment, the peptide is an integrin-binding peptide, a LOX-1-binding peptide, and an epidermal growth factor (EGF) peptide, a neurotensin peptide, an NL4 peptide, or a YIGSR laminin peptide.

[0103] In one embodiment, the cells are hepatocytes.

[0104] In one embodiment, the targeting ligand specifically binds to the asialoglycoprotein receptor (ASGPR).

[0105] In one embodiment, the targeting ligand comprises an N-acetylgalactosamine (NAG) residue.

[0106] In one embodiment, the membrane destabilizing polymer is monosaccharide, a hydrophilic region comprising polyethylene glycol methacrylate 4-5 (PEGMA 4-5) and hydroxyethyl methacrylate (HMA); and Regions that mediate endosomal escape It includes three areas:

[0107] In one embodiment, the membrane destabilizing polymer has the formula (XX): [ka] is a polymer of During the ceremony: PEGMA is a polyethylene glycol methacrylate residue having 2 to 20 ethylene glycol units; M 2 teeth, (C4-C 18 ) alkyl-methacrylate residue, (C4-C 18 ) branched alkyl-methacrylate residues, cholesteryl methacrylate residue, (C4-C 18 ) alkyl-methacrylate residues, and (C4-C 18 ) Branched alkyl methacrylate residue is a methacrylate residue selected from the group consisting of: BMA is the residue of butyl methacrylate, PAA is the residue of propyl acrylic acid, DMAEMA is the residue of dimethylaminoethyl methacrylate, m and n are each a molar fraction greater than 0, m is greater than n, and m+n=1; q is a molar ratio between 0.2 and 0.75; r is a molar ratio between 0.05 and 0.6; s is a molar ratio between 0.2 and 0.75; q+r+s=1, v is 1 to 25 kDa; w is 1 to 25 kDa; T 5 is a targeting moiety (e.g., a peptide, polymer, or sugar), L is absent or a linking moiety.

[0108] In one embodiment, M 2 teeth, 2,2,3,3,4,4,4-heptafluorobutyl methacrylate residue, 3,3,4,4,5,6,6,6-octafluoro-5(trifluoromethyl)hexyl methacrylate residue, 2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-pentadecafluorooctyl 2-methylacrylate residue, 3,3,4,4,5,5,6,6,6-nonafluorohexyl methacrylate residue, 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl methacrylate residue, 1,1,1-trifluoro-2-(trifluoromethyl)-2-hydroxy-4-methyl-5-pentyl methacrylate residue, 2-[(1',1',1'-trifluoro-2'-(trifluoromethyl)-2'-hydroxy)propyl]-3-norbornyl methacrylate residue, 2-ethylhexyl methacrylate residue, butyl methacrylate residue, hexyl methacrylate residue, octyl methacrylate residue, n-decyl methacrylate residue, lauryl methacrylate residue, myristyl methacrylate residue, stearyl methacrylate residue, cholesteryl methacrylate residue, ethylene glycol phenyl ether methacrylate residue, 2-propenoic acid, 2-methyl-, 2-phenylethyl ester residue, 2-Propenoic acid, 2-methyl-, 2-[[(1,1-dimethylethoxy)carbonyl]amino]ethyl ester residue, 2-Propenoic acid, 2-methyl-, 2-(1H-imidazol-1-yl)ethyl ester residue, 2-propenoic acid, 2-methyl-, cyclohexyl ester residue, 2-Propenoic acid, 2-methyl-, 2-[bis(1-methylethyl)amino]ethyl ester residue, 2-propenoic acid, 2-methyl-, 3-methylbutyl ester residue, Neopentyl methacrylate residue, tert-butyl methacrylate residue, 3,3,5-trimethylcyclohexyl methacrylate residue, 2-hydroxypropyl methacrylate residue, 5-nonyl methacrylate residue, 2-butyl-1-octyl methacrylate residue, 2-hexyl-1-decyl methacrylate residue, and 2-(tert-butylamino)ethyl methacrylate residue is selected from the group consisting of: In one embodiment, the PEGMA has 4 to 5 ethylene glycol units or 7 to 8 ethylene glycol units. In one embodiment, T l and L exist, and T l contains an N-acetylgalactosamine (NAG) residue.

[0109] In one embodiment, L comprises a polyethylene glycol (PEG) moiety having 2 to 20 ethylene glycol units.

[0110] In one embodiment, the membrane destabilizing polymer has formula (XXI): [ka] is a polymer of wherein px is an integer from about 2 to about 50 (e.g., from about 2 to about 20, e.g., from 4 to 12) (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50). In some embodiments, px is an integer from about 8 to about 16 (e.g., 8, 9, 10, 11, 12, 13, 14, 15, or 16). In some embodiments, px is about 12. In some embodiments, py is an integer from about 2 to about 20 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). In some embodiments, py is an integer from about 2 to about 10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, py is an integer from about 4 to about 5 (e.g., 4 or 5).

[0111] In one embodiment, the compound of formula (X) has formula (I): [ka] or a salt thereof, During the ceremony: R 1 is a targeting ligand, L 1 is 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 is 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, independently selected from the group consisting of C 1-10 Alkyl C 2-10 Alkenyl, and C 2-10 Alkynyl includes halo, hydroxy, and C 1-3 optionally 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; n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0112] In one embodiment, R 1 is a targeting ligand, L 1 is 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 is hydrogen, hydroxy, CN, F, Cl, Br, I, -C 1-2 Alkyl-OR B , and C 1-8 alkyl, independently selected from the group consisting of C 1-8 Alkyl is substituted with halo, hydroxy, and C 1-3 optionally 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; n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0113] In one embodiment, R 1 is -C(H) (3-p) (L 3 -sugar) p and In the formula, each L 3 are independently a linking group, p is 1, 2, or 3; The sugar may be a monosaccharide or a disaccharide.

[0114] 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 selected from 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, (C-C)alkyl, (C-C)haloalkyl, (C-C)alkoxy, and (C-C)cycloalkyl, wherein the (C-C)cycloalkyl is optionally substituted with one or more groups independently selected from the group consisting of halo, (C-C)alkyl, (C-C)haloalkyl, (C-C)alkoxy, and (C-C)haloalkoxy; R 10 -OH, -NR 8 R 9 , or -F, R 11 -OH, -NR 8 R 9 , -F, or a 5-membered heterocycle, wherein the 5-membered heterocycle is 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.

[0115] In one embodiment, the sugar is [ka] is selected from the group consisting of:

[0116] In one embodiment, the sugar is [ka] is.

[0117] In one embodiment, each L 3 is independently a branched or unbranched saturated or unsaturated divalent hydrocarbon chain having 0 to 50 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are -O-, -NRX -, -NR X -C(=O)-, -C(=O)-NR X - or -S-, optionally replaced by R X is hydrogen or (C-C) alkyl, wherein the hydrocarbon chain is optionally substituted with one or more substituents selected from (C-C) alkoxy, (C-C) cycloalkyl, (C-C) alkanoyl, (C-C) alkanoyloxy, (C-C) alkoxycarbonyl, (C-C) alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0118] In one embodiment, each L 3 are independently branched or unbranched saturated or unsaturated divalent hydrocarbon chains having 1 to 20 carbon atoms, and one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X - or -S-, optionally replaced by R X is hydrogen or (C-C) alkyl, and the hydrocarbon chain is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from (C-C) alkoxy, (C-C) cycloalkyl, (C-C) alkanoyl, (C-C) alkanoyloxy, (C-C) alkoxycarbonyl, (C-C) alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0119] In one embodiment, L 3 teeth, [ka] is.

[0120] In one embodiment, R 1 teeth, [ka] is.

[0121] In one embodiment, R 1 teeth, [ka] and During the ceremony: 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 ) heterocycle, aryl, heteroaryl, monosaccharide, disaccharide, or trisaccharide, wherein the cycloalkyl, heterocycle, aryl, heteroaryl, and 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.

[0122] In one embodiment, R C teeth, [ka] is.

[0123] In one embodiment, R 1 teeth, [ka] is.

[0124] In one embodiment, R C teeth, [ka] is.

[0125] In one embodiment, G is -NH-.

[0126] In one embodiment, R 1 teeth, [ka] is.

[0127] 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 )Si—, (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.

[0128] In one embodiment, L 1 and L 2 is independently a branched or unbranched saturated or unsaturated divalent hydrocarbon chain having 1 to 50 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X - or -S-, optionally replaced by R Xis hydrogen or (C-C) alkyl, and the hydrocarbon chain is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from (C-C) alkoxy, (C-C) cycloalkyl, (C-C) alkanoyl, (C-C) alkanoyloxy, (C-C) alkoxycarbonyl, (C-C) alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0129] In one embodiment, L 1 and L 2 are independently branched or unbranched saturated or unsaturated divalent hydrocarbon chains having 1 to 20 carbon atoms, and one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X - or -S-, optionally replaced by R X is hydrogen or (C-C) alkyl, and the hydrocarbon chain is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from (C-C) alkoxy, (C-C) cycloalkyl, (C-C) alkanoyl, (C-C) alkanoyloxy, (C-C) alkoxycarbonyl, (C-C) alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0130] In one embodiment, L 1 and L 2 are independently branched or unbranched saturated or unsaturated divalent hydrocarbon chains having 1 to 14 carbon atoms, and one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X- or -S-, optionally replaced by R X is hydrogen or (C-C) alkyl, and the hydrocarbon chain is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from (C-C) alkoxy, (C-C) cycloalkyl, (C-C) alkanoyl, (C-C) alkanoyloxy, (C-C) alkoxycarbonyl, (C-C) alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0131] In one embodiment, L 1 is connected to R via -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.

[0132] In one embodiment, L 2 -O- through R 2 is linked to.

[0133] In one embodiment, L 1 teeth, [ka] is selected from the group consisting of:

[0134] In one embodiment, L 2 is —CH2—O— or —CH2—CH2—O—.

[0135] In one embodiment, the compound of formula (I) has the formula (Ia): [ka] is a compound of During the ceremony: Each D is [ka] and -N=, or a salt thereof.

[0136] In one embodiment, the compound of formula (I) is [ka] is selected from the group consisting of During the ceremony: Q 1 is hydrogen and Q 2 is R 2 or Q 1 is R 2 and Q 2 is hydrogen, Z is -L 1 -R 1 is.

[0137] In one embodiment, the compound of formula (I) has formula (Ib): [ka] is a compound of During the ceremony: Each D is [ka] and -N=, Each m is independently 1 or 2.

[0138] In one embodiment, the compound of formula (I) is [ka] is selected from the group consisting of During the ceremony: Q 1 is hydrogen and Q 2 is R 2 or Q 1 is R 2 and Q 2 is hydrogen, Z is -L 1 -R1 is.

[0139] In one embodiment, the compound of formula (I) has the formula (Ic): [ka] is a compound of During the ceremony: E is —O— or —CH—; n is selected from the group consisting of 0, 1, 2, 3, and 4; n1 and n2 are each independently selected from the group consisting of 0, 1, 2, and 3.

[0140] In one embodiment, the compound of formula (I) is [ka] is selected from the group consisting of Wherein: Z is -L 1 -R 1 is.

[0141] In one embodiment, -AL 2 -R 2 teeth, [ka] and During the ceremony: Q 1 is hydrogen and Q 2 is R 2 or Q 1 is R 2 and Q 2 is hydrogen, Each q is independently 0, 1, 2, 3, 4, or 5.

[0142] In one embodiment, the compound of formula (I) is [ka] is selected from the group consisting of:

[0143] In one embodiment, R 1 teeth, [ka] is selected from the group consisting of During the ceremony: R S teeth, [ka] and n is 2, 3, or 4; x is 1 or 2.

[0144] In one embodiment, L 1 teeth, [ka] is selected from the group consisting of:

[0145] In one embodiment, A is absent, phenyl, pyrrolidinyl, or cyclopentyl.

[0146] In one embodiment, L 2 is C optionally substituted by hydroxy 1-4 It is alkylene-O-.

[0147] In one embodiment, L 2 is —CH2O—, —CH2CH2O—, or —CH(OH)CH2O—.

[0148] In one embodiment, each R A are independently hydroxy or C 1-8 alkyl, C 1-8 The alkyl is optionally substituted with a hydroxyl.

[0149] In one embodiment, each R A is independently selected from the group consisting of hydroxy, methyl, and —CHOH.

[0150] In one embodiment, the compound of formula (I) is a compound of formula (Ig): [ka] is a compound of During the ceremony: B is -N- or -CH-; L 2 is C optionally substituted by hydroxyl or halo 1-4 alkylene-O-; n is 0, 1, 2, 3, 4, 5, 6, or 7.

[0151] In one embodiment, the compound of formula (I) is [ka] is selected from the group consisting of 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, C 1-9 Alkyl, C 2-9 Alkenyl, or C 2-9 The alkynyl is optionally substituted by halo or hydroxyl.

[0152] In one embodiment, the compound of formula (I) is [ka] is selected from the group consisting of In the formula, Q is -L 1 -R 1 is.

[0153] In one embodiment, the compound of formula (I) is [ka] [ka] [ka] [ka] [ka] is selected from the group consisting of:

[0154] In one embodiment, the compound of formula (I) has the formula (Id): [ka] is a compound of During the ceremony: R 1d teeth, [ka] is selected from X d is C2- 10 is alkylene, n d is either 0 or 1, R 2d is a nucleic acid, R 3d is H.

[0155] In one embodiment, R 1d teeth, [ka] is.

[0156] In one embodiment, R 1d teeth, [ka] is.

[0157] In one embodiment, X d is a C8 alkylene.

[0158] In one embodiment, n dis 0.

[0159] In one embodiment, R 3d is H.

[0160] In one embodiment, the compound of formula (I) is [ka] is selected from the group consisting of:

[0161] In one embodiment, the compound of formula (I) has the formula (Ig): [ka] is a compound of During the ceremony: B is -N- or -CH-; L 2 is C optionally substituted by hydroxyl or halo 1-4 alkylene-O-; n is 0, 1, 2, 3, 4, 5, 6, or 7.

[0162] In one embodiment, the compound of formula (I) is [ka] is 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, C 1-9 Alkyl, C 2-9 Alkenyl, or C 2-9 The alkynyl is optionally substituted by halo or hydroxy.

[0163] In one embodiment, the compound of formula (I) is [ka] [ka] is selected from the group consisting of Wherein: Q is -L 1 -R 1 is.

[0164] In one embodiment, the compound of formula (X) has the formula (XX): [ka] is a compound of During the ceremony: R 1 is a targeting ligand, L 1 is absent or a linking group, L 2 is absent or a linking group, R 2 is a nucleic acid, B is divalent, [ka] [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, C 1-9 Alkyl, C 2-9 Alkenyl, or C 2-9 The alkynyl is optionally substituted by halo or hydroxyl; * Valences marked with L 1 or L 1 If does not exist, R 1 is added to ** Valences marked with L 2 or L 2 If does not exist, R2 is added to.

[0165] In one embodiment, R 1 contains 2 to 8 sugars.

[0166] In one embodiment, R 1 contains two to four sugars.

[0167] In one embodiment, R 1 contains 3 to 8 sugars.

[0168] In one embodiment, R 1 contains 3 to 6 sugars.

[0169] In one embodiment, R 1 contains 3 to 4 sugars.

[0170] In one embodiment, R 1 contains two sugars.

[0171] In one embodiment, R 1 contains three sugars.

[0172] In one embodiment, R 1 contains four sugars.

[0173] In one embodiment, R 1 is the following formula: [ka] and During the ceremony: B 1 is a trivalent group containing from about 1 to about 20 atoms, and L 1 , T 1 , and T 2 is covalently bound to B 2 is a trivalent group containing about 1 to about 20 atoms, and T 1 , T 3 , and T 4 is covalently bound to B 3is a trivalent group containing about 1 to about 20 atoms, and T 2 , T 5 , and T 6 is covalently bound to T 1 is absent or a linking group, T 2 is absent or a linking group, T 3 is absent or a linking group, T 4 is absent or a linking group, T 5 is absent or a linking group, T 6 is absent or a linking group.

[0174] In one embodiment, each sugar is [ka] are independently 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 selected from 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, (C-C)alkyl, (C-C)haloalkyl, (C-C)alkoxy, and (C-C)cycloalkyl, wherein the (C-C)cycloalkyl is optionally substituted with one or more groups independently selected from the group consisting of halo, (C-C)alkyl, (C-C)haloalkyl, (C-C)alkoxy, and (C-C)haloalkoxy; R 10 -OH, -NR 8 R 9 , or -F, R 11 -OH, -NR 8 R 9 , -F, or a 5-membered heterocycle, wherein the 5-membered heterocycle is 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.

[0175] In one embodiment, each sugar is [ka] are independently selected from the group consisting of:

[0176] In one embodiment, each sugar is independently selected from the group consisting of: [ka] is.

[0177] In one embodiment, T 1 and T 2 One of them does not exist.

[0178] In one embodiment, T 1 and T 2 Both do not exist.

[0179] 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 50 carbon atoms, and one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X - or -S-, optionally replaced by R X is hydrogen or (C-C) alkyl, and the hydrocarbon chain is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from (C-C) alkoxy, (C-C) cycloalkyl, (C-C) alkanoyl, (C-C) alkanoyloxy, (C-C) alkoxycarbonyl, (C-C) alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0180] 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, and one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X - or -S-, optionally replaced by R Xis hydrogen or (C-C) alkyl, and the hydrocarbon chain is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from (C-C) alkoxy, (C-C) cycloalkyl, (C-C) alkanoyl, (C-C) alkanoyloxy, (C-C) alkoxycarbonyl, (C-C) alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0181] 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 50 carbon atoms, or a salt thereof, wherein one or more of the carbon atoms in the hydrocarbon chain are -O- or -NR X - is optionally replaced by 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 halo, hydroxy, and oxo (=O).

[0182] 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 in the hydrocarbon chain are optionally replaced 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).

[0183] 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 in the hydrocarbon chain are optionally replaced 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).

[0184] 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.

[0185] In one embodiment, T 3 , T 4 , T 5 , and T 6 are respectively, [ka] are independently selected from the group consisting of During the ceremony: n=1, 2, 3.

[0186] In one embodiment, T 1 and T 2 At least one of the amino acids is glycine.

[0187] In one embodiment, T 1 and T 2 are glycines.

[0188] In one embodiment, B 1 is a trivalent group containing 1 to 15 atoms, and L 1 , T 1 , and T 2 is covalently bonded to

[0189] In one embodiment, B 1 is a trivalent group containing 1 to 10 atoms, and L 1 , T 1 , and T 2 is covalently bonded to

[0190] In one embodiment, B 1 includes (C1-C6) alkyl.

[0191] In one embodiment, B 1 is C 3-8 Includes cycloalkyl.

[0192] In one embodiment, B 1 contains a silyl group.

[0193] In one embodiment, B 1 includes D-amino acids or L-amino acids.

[0194] In one embodiment, B 1 contains sugar.

[0195] In one embodiment, B 1 contains a phosphate group.

[0196] In one embodiment, B 1 contains a phosphonic acid group.

[0197] In one embodiment, B 1 includes aryl.

[0198] In one embodiment, B 1 contains a phenyl ring.

[0199] In one embodiment, B 1 is a phenyl ring.

[0200] In one embodiment, B 1 is CH.

[0201] In one embodiment, B 1 includes heteroaryl.

[0202] In one embodiment, B 1 teeth, [ka] is selected from.

[0203] In one embodiment, B 2 is a trivalent group containing 1 to 15 atoms, and T 2 , T 5 , and T 6 is covalently bonded to

[0204] In one embodiment, B 2 is a trivalent group containing 1 to 10 atoms, and T 2 , T 5 , and T 6 is covalently bonded to

[0205] In one embodiment, B 2 includes (C1-C6) alkyl.

[0206] In one embodiment, B 2 is C 3-8 Includes cycloalkyl.

[0207] In one embodiment, B 2 contains a silyl group.

[0208] In one embodiment, B 2 includes D-amino acids or L-amino acids.

[0209] In one embodiment, B 2 contains sugar.

[0210] In one embodiment, B 2contains a phosphate group.

[0211] In one embodiment, B 2 contains a phosphonic acid group.

[0212] In one embodiment, B 2 includes aryl.

[0213] In one embodiment, B 2 contains a phenyl ring.

[0214] In one embodiment, B 2 is a phenyl ring.

[0215] In one embodiment, B 2 is CH.

[0216] In one embodiment, B 2 includes heteroaryl.

[0217] In one embodiment, B 2 teeth, [ka] is selected from the group consisting of:

[0218] In one embodiment, B 3 is a trivalent group containing 1 to 15 atoms, and L 1 , T 1 , and T 2 is covalently bonded to

[0219] In one embodiment, B 3 is a trivalent group containing 1 to 10 atoms, and L 1 , T 1 , and T 2 is covalently bonded to

[0220] In one embodiment, B 3 includes (C1-C6) alkyl.

[0221] In one embodiment, B3 is C 3-8 Includes cycloalkyl.

[0222] In one embodiment, B 3 contains a silyl group.

[0223] In one embodiment, B 3 includes D-amino acids or L-amino acids.

[0224] In one embodiment, B 3 contains sugar.

[0225] In one embodiment, B 3 contains a phosphate group.

[0226] In one embodiment, B 3 contains a phosphonic acid group.

[0227] In one embodiment, B 3 includes aryl.

[0228] In one embodiment, B 3 contains a phenyl ring.

[0229] In one embodiment, B 3 is a phenyl ring.

[0230] In one embodiment, B 3 is CH.

[0231] In one embodiment, B 3 includes heteroaryl.

[0232] In one embodiment, B 3 teeth, [ka] is selected from the group consisting of:

[0233] In one embodiment, B 3 teeth, [ka] or a salt thereof.

[0234] In one embodiment, L 1 and L 2 is independently a branched or unbranched saturated or unsaturated divalent hydrocarbon chain having 1 to 50 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X - or -S-, optionally replaced by R X is hydrogen or (C-C) alkyl, and the hydrocarbon chain is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from (C-C) alkoxy, (C-C) cycloalkyl, (C-C) alkanoyl, (C-C) alkanoyloxy, (C-C) alkoxycarbonyl, (C-C) alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0235] In one embodiment, L 1 teeth, [ka] or a salt thereof.

[0236] In one embodiment, L 1 is connected to B through a bond 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.

[0237] In one embodiment, L 1 teeth, [ka] is selected from the group consisting of:

[0238] In one embodiment, L 2 -O- through R 2 is linked to.

[0239] In one embodiment, L 2 is C optionally substituted by hydroxy 1-4 It is alkylene-O-.

[0240] In one embodiment, L 2 -O- through R 2 is linked to.

[0241] In one embodiment, L 2 does not exist.

[0242] In one embodiment, the compound of formula (I) is [ka] [ka] [ka] is selected from the group consisting of:

[0243] In one embodiment, the compound of formula (I) is: [ka] [ka]

[0244] In one embodiment, the compound of formula (I) is: [ka]

[0245] In one embodiment, the compound of formula (I) is: [ka]

[0246] In one embodiment, the compound of formula (I) is: [ka]

[0247] In one embodiment, the compound of formula (I) is: [ka]

[0248] In one embodiment, the compound of formula (I) is: [ka]

[0249] In one embodiment, the compound of formula (I) is: [ka]

[0250] In one embodiment, the compound of formula (I) is: [ka]

[0251] In one embodiment, the compound of formula (I) is: [ka]

[0252] In one embodiment, the compound of formula (I) is: [ka]

[0253] In one embodiment, the compound of formula (I) is: [ka]

[0254] In one embodiment, the compound of formula (I) is: [ka]

[0255] In one embodiment, the compound of formula (I) is: [ka]

[0256] In one embodiment, the compound of formula (I) is: [ka]

[0257] In one embodiment, the compound of formula (I) is [ka] During the ceremony: L 1 is 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 Ais 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, independently selected from the group consisting of C 1-10 Alkyl C 2-10 Alkenyl, and C 2-10 Alkynyl includes halo, hydroxy, and C 1-3 optionally 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; n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0258] In one embodiment, the compound of formula (I) is the following compound or a salt thereof: [ka] During the ceremony: 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 is 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, independently selected from the group consisting of C 1-10 Alkyl C 2-10 Alkenyl, and C 2-10 Alkynyl includes halo, hydroxy, and C 1-3 optionally substituted with one or more groups independently selected from alkoxy; R Bis 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.

[0259] In one embodiment, the compound of formula (I) is the following compound or a salt thereof: [ka] R 2 is a nucleic acid.

[0260] In one embodiment, the compound of formula (I) is: [ka]

[0261] In one embodiment, the compound of formula (I) is: [ka]

[0262] In one embodiment, the compound of formula (I) is the following compound or a salt thereof: [ka] During the ceremony: R 1c is a sugar, L 1c is a branched or unbranched saturated or unsaturated divalent hydrocarbon chain having 0 to 20 carbon atoms, wherein one or more of the carbon atoms in the hydrocarbon chain are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X - or -S-, optionally replaced by R X is hydrogen or (C1-C6) alkyl, wherein the hydrocarbon chain is optionally substituted with one or more substituents selected from oxo (=O) and halo; B cis a 5- to 10-membered aryl or a 5- to 10-membered heteroaryl, wherein the 5- to 10-membered aryl or the 5- to 10-membered heteroaryl is optionally substituted by one or more groups independently selected from the group consisting of halo, hydroxy, cyano, trifluoromethyl, trifluoromethoxy, (C-C)alkyl, (C-C)alkoxy, (C-C)alkoxycarbonyl, (C-C)alkanoyloxy, (C-C)cycloalkyl, and (C-C)cycloalkyl(C-C)alkyl; L 2c is a branched or unbranched saturated or unsaturated divalent hydrocarbon chain having 0 to 20 carbon atoms, wherein one or more of the carbon atoms in the hydrocarbon chain are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X - or -S-, optionally replaced by R X is hydrogen or (C1-C6) alkyl, wherein the hydrocarbon chain is optionally substituted with one or more substituents selected from oxo (=O) and halo; R 2c is a sugar, L 3c is absent or a linking group, A c is 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 Ac is hydrogen, hydroxy, CN, F, Cl, Br, I, -C 1-2 Alkyl-OR a , C 1-10 Alkyl C 2-10 Alkenyl, and C 2-10 alkynyl, independently selected from the group consisting of C 1-10 Alkyl C 2-10 Alkenyl, and C 2-10 Alkynyl includes halo, hydroxy, and C 1-3 optionally substituted with one or more groups independently selected from alkoxy; nc is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, L 4c is absent or a linking group, R 3c is a nucleic acid, R ac is hydrogen, L 5c is a linking group.

[0263] In one embodiment, B c is a 5- to 10-membered aryl.

[0264] In one embodiment, B c is naphthyl or phenyl.

[0265] In one embodiment, B c is phenyl.

[0266] In one embodiment, the group: [ka] teeth, [ka] is.

[0267] In one embodiment, B c is a 5-10 membered heteroaryl.

[0268] In one embodiment, B c is pyridyl, pyrimidyl, quinolyl, isoquinolyl, imidazolyl, thiazolyl, oxadiazolyl, or oxazolyl.

[0269] In one embodiment, the group: [ka] teeth, [ka] is.

[0270] In one embodiment, the group: [ka] teeth, [ka] is.

[0271] In one embodiment, L 1c is an unbranched saturated divalent hydrocarbon chain having 0 to 20 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X - or -S-, optionally replaced by R X is hydrogen or (C1-C6) alkyl, and the hydrocarbon chain is optionally substituted with one or more substituents selected from oxo (=O) and halo.

[0272] In one embodiment, L 1c is an unbranched saturated divalent hydrocarbon chain having 0 to 12 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are -O-, -NR X -C(=O)- or -C(=O)-NR X - is optionally replaced by R X is hydrogen or (C1-C6) alkyl.

[0273] In one embodiment, L 1c teeth, -C(=O)N(H)-CH2CH2OCH2CH2OCH2CH2-, -C(=O)N(H)-CH2CH2OCH2CH2OCH2CH2OCH2CH2-, -C(=O)N(CH3)-CH2CH2OCH2CH2OCH2CH2-, or -C(=O)N(CH3)-CH2CH2OCH2CH2OCH2CH2OCH2CH2- is.

[0274] In one embodiment, L 2c is an unbranched saturated divalent hydrocarbon chain having 0 to 20 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X - or -S-, optionally replaced by R X is hydrogen or (C1-C6) alkyl, and the hydrocarbon chain is optionally substituted with one or more substituents selected from oxo (=O) and halo.

[0275] In one embodiment, L 2c is an unbranched saturated divalent hydrocarbon chain having 0 to 12 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are -O-, -NR X -C(=O)- or -C(=O)-NR X - is optionally replaced by R X is hydrogen or (C1-C6) alkyl.

[0276] In one embodiment, L 2c teeth, -C(=O)N(H)-CH2CH2OCH2CH2OCH2CH2-, -C(=O)N(H)-CH2CH2OCH2CH2OCH2CH2OCH2CH2-, -C(=O)N(CH3)-CH2CH2OCH2CH2OCH2CH2-, or -C(=O)N(CH3)-CH2CH2OCH2CH2OCH2CH2OCH2CH2- is.

[0277] In one embodiment, R 1c teeth, [ka] and During the ceremony: X is NR 20 and Y is —(C═O)R 21 , -SO2R 22 , and —(C═O)NR 23 R 24 or X is —(C═O)— and Y is selected from NR 25 R 26 or X is -NR 37 R 38 and Y is absent, R 20 is hydrogen or (C1-C4) alkyl, R 21 , R 22 , R 23 , R 24 , R 25 , and R 26 are each independently selected from the group consisting of hydrogen, (C-C) alkyl, (C-C) alkoxy, and (C-C) cycloalkyl, wherein any of the (C-C) alkyl, (C-C) alkoxy, and (C-C) cycloalkyl are optionally substituted with one or more groups independently selected from the group consisting of halo, (C-C) alkyl, and (C-C) alkoxy; R 27 -OH, -NR 25 R 26 , or -F, R 28 -OH, -NR 25 R 26 , or -F, R 29 -OH, -NR 25 R 26 , -F, -N3, -NR 35 R 36or a 5-membered heterocycle, wherein the 5-membered heterocycle is optionally substituted with one or more groups independently selected from the group consisting of halo, hydroxyl, carboxyl, amino, (C1-C4) alkyl, aryl, and (C1-C4) alkoxy, wherein each of the (C1-C4) alkyl and (C1-C4) alkoxy is optionally substituted with one or more groups independently selected from the group consisting of halo, and each of the aryl is optionally substituted with one or more groups independently selected from the group consisting of halo, hydroxyl, nitro, cyano, amino, (C1-C8) alkyl, (C1-C8) alkoxy, (C1-C8) alkanoyl. (C-C)alkyl, (C-C)alkoxy, (C-C)alkanoyl, (C-C)alkoxycarbonyl, (C-C)alkanoyloxy, and (C-C)cycloalkyl, each of which is optionally substituted by one or more groups independently selected from the group consisting of halo, (C-C)alkyl, and (C-C)alkoxy; Each R 35 and R 36 are independently selected from the group consisting of hydrogen, (C-C) alkyl, (C-C) alkoxy, and (C-C) cycloalkyl, each of which is optionally substituted with one or more groups independently selected from the group consisting of halo and (C-C) alkoxy; or R 35 and R 36 together with the nitrogen to which they are attached form a 5-6 membered heteroaryl ring, the heteroaryl ring being optionally substituted with one or more groups independently selected from the group consisting of (C-C) alkyl, (C-C) alkoxy, aryl, and (C-C) cycloalkyl, each of which may be substituted with one or more R 39 optionally substituted by a group, Each R 37 and R 38is independently selected from the group consisting of hydrogen, (C-C)alkyl, (C-C)alkoxy, (C-C)alkanoyl, (C-C)alkoxycarbonyl, (C-C)alkanoyloxy, and (C-C)cycloalkyl, wherein any of the (C-C)alkyl, (C-C)alkoxy, (C-C)alkanoyl, (C-C)alkoxycarbonyl, (C-C)alkanoyloxy, and (C-C)cycloalkyl is optionally substituted with one or more groups independently selected from the group consisting of halo, (C-C)alkyl, and (C-C)alkoxy; or R 37 and R 38 together with the nitrogen to which they are attached form a 5-8 membered heterocycle, which is optionally substituted with one or more groups independently selected from the group consisting of halo, hydroxyl, carboxyl, amino, oxo (=O), (C1-C4) alkyl, and (C1-C4) alkoxy, wherein both the (C1-C4) alkyl and (C1-C4) alkoxy are optionally substituted with one or more groups independently selected from halo; Each R 39 is independently selected from the group consisting of (C-C) alkyl, (C-C) alkoxy, and (C-C) cycloalkyl, wherein any of the (C-C) alkyl, (C-C) alkoxy, and (C-C) cycloalkyl are optionally substituted with one or more groups independently selected from halo.

[0278] In one embodiment, R 1c teeth, [ka] [ka] is.

[0279] In one embodiment, R 1c teeth, [ka] is.

[0280] In one embodiment, R 1c teeth, [ka] is.

[0281] In one embodiment, R 1c teeth, [ka] is.

[0282] In one embodiment, R 2c teeth, [ka] and During the ceremony: X is NR 20 and Y is —(C═O)R 21 , -SO2R 22 , and —(C═O)NR 23 R 24 or X is —(C═O)— and Y is selected from NR 25 R 26 or X is -NR 37 R 38 and Y is absent, R 20 is hydrogen or (C1-C4) alkyl, R 21 , R 22 , R 23 , R 24 , R 25 , and R 26 are each independently selected from the group consisting of hydrogen, (C-C) alkyl, (C-C) alkoxy, and (C-C) cycloalkyl, wherein any of the (C-C) alkyl, (C-C) alkoxy, and (C-C) cycloalkyl are optionally substituted with one or more groups independently selected from the group consisting of halo, (C-C) alkyl, and (C-C) alkoxy; R27 -OH, -NR 25 R 26 , or -F, R 28 -OH, -NR 25 R 26 , or -F, R 29 -OH, -NR 25 R 26 , -F, -N3, -NR 35 R 36 or a 5-membered heterocycle, wherein the 5-membered heterocycle is optionally substituted with one or more groups independently selected from the group consisting of halo, hydroxyl, carboxyl, amino, (C1-C4) alkyl, aryl, and (C1-C4) alkoxy, wherein each of the (C1-C4) alkyl and (C1-C4) alkoxy is optionally substituted with one or more groups independently selected from the group consisting of halo, and each of the aryl is optionally substituted with one or more groups independently selected from the group consisting of halo, hydroxyl, nitro, cyano, amino, (C1-C8) alkyl, (C1-C8) alkoxy, (C1-C8) alkanoyl. (C-C)alkyl, (C-C)alkoxy, (C-C)alkanoyl, (C-C)alkoxycarbonyl, (C-C)alkanoyloxy, and (C-C)cycloalkyl, each of which is optionally substituted by one or more groups independently selected from the group consisting of halo, (C-C)alkyl, and (C-C)alkoxy; Each R 35 and R 36 are independently selected from the group consisting of hydrogen, (C-C) alkyl, (C-C) alkoxy, and (C-C) cycloalkyl, each of which is optionally substituted with one or more groups independently selected from the group consisting of halo and (C-C) alkoxy; or R 35 and R 36together with the nitrogen to which they are attached form a 5-6 membered heteroaryl ring, the heteroaryl ring being optionally substituted with one or more groups independently selected from the group consisting of (C-C) alkyl, (C-C) alkoxy, aryl, and (C-C) cycloalkyl, each of which may be substituted with one or more R 39 optionally substituted by a group, Each R 37 and R 38 is independently selected from the group consisting of hydrogen, (C-C)alkyl, (C-C)alkoxy, (C-C)alkanoyl, (C-C)alkoxycarbonyl, (C-C)alkanoyloxy, and (C-C)cycloalkyl, wherein any of the (C-C)alkyl, (C-C)alkoxy, (C-C)alkanoyl, (C-C)alkoxycarbonyl, (C-C)alkanoyloxy, and (C-C)cycloalkyl is optionally substituted with one or more groups independently selected from the group consisting of halo, (C-C)alkyl, and (C-C)alkoxy; or R 37 and R 38 together with the nitrogen to which they are attached form a 5-8 membered heterocycle, which is optionally substituted with one or more groups independently selected from the group consisting of halo, hydroxyl, carboxyl, amino, oxo (=O), (C1-C4) alkyl, and (C1-C4) alkoxy, wherein both the (C1-C4) alkyl and (C1-C4) alkoxy are optionally substituted with one or more groups independently selected from halo; Each R 39 is independently selected from the group consisting of (C-C) alkyl, (C-C) alkoxy, and (C-C) cycloalkyl, wherein any of the (C-C) alkyl, (C-C) alkoxy, and (C-C) cycloalkyl are optionally substituted with one or more groups independently selected from halo.

[0283] In one embodiment, R 2c teeth, [ka] is.

[0284] In one embodiment, R 2c teeth, [ka] is.

[0285] In one embodiment, R 2c teeth, [ka] is.

[0286] In one embodiment, R 2c teeth, [ka] is.

[0287] In one embodiment, L 3c is a branched or unbranched saturated or unsaturated divalent hydrocarbon chain having 0 to 50 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X - or -S-, optionally replaced by R X is hydrogen or (C-C) alkyl, and the hydrocarbon chain is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from (C-C) alkoxy, (C-C) cycloalkyl, (C-C) alkanoyl, (C-C) alkanoyloxy, (C-C) alkoxycarbonyl, (C-C) alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0288] In one embodiment, L 3c is a branched or unbranched saturated or unsaturated divalent hydrocarbon chain having 1 to 20 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X - or -S-, optionally replaced by R X is hydrogen or (C-C) alkyl, and the hydrocarbon chain is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from (C-C) alkoxy, (C-C) cycloalkyl, (C-C) alkanoyl, (C-C) alkanoyloxy, (C-C) alkoxycarbonyl, (C-C) alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0289] In one embodiment, L 3c is a branched or unbranched saturated or unsaturated divalent hydrocarbon chain having 1 to 30 carbon atoms, one or more of which may be -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X - or -S-, optionally replaced by R X is hydrogen or (C1-C6) alkyl, wherein the hydrocarbon chain is optionally substituted with one or more halo or oxo (=O).

[0290] In one embodiment, L 3c teeth, [ka] is.

[0291] In one embodiment, L 3cis linked to B via -NH-, -O-, -S-, -(C=O)-, -(C=O)-NH-, -NH-(C=O)-, -(C=O)-O-, -NH-(C=O)-NH-, or -NH-(SO2)-.

[0292] In one embodiment, L 4c is a branched or unbranched saturated or unsaturated divalent hydrocarbon chain having 0 to 50 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X - or -S-, optionally replaced by R X is hydrogen or (C-C) alkyl, and the hydrocarbon chain is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from (C-C) alkoxy, (C-C) cycloalkyl, (C-C) alkanoyl, (C-C) alkanoyloxy, (C-C) alkoxycarbonyl, (C-C) alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0293] In one embodiment, L 4c is a branched or unbranched saturated or unsaturated divalent hydrocarbon chain having 1 to 20 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X - or -S-, optionally replaced by R Xis hydrogen or (C-C) alkyl, and the hydrocarbon chain is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from (C-C) alkoxy, (C-C) cycloalkyl, (C-C) alkanoyl, (C-C) alkanoyloxy, (C-C) alkoxycarbonyl, (C-C) alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0294] In one embodiment, L 4c is a branched or unbranched saturated or unsaturated divalent hydrocarbon chain having 1 to 30 carbon atoms, one or more of which may be -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X - or -S-, optionally replaced by R X is hydrogen or (C1-C6) alkyl, wherein the hydrocarbon chain is optionally substituted with one or more halo or oxo (=O).

[0295] In one embodiment, the group: [ka] teeth, [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, C 1-9 Alkyl, C 2-9 Alkenyl, or C 2-9 The alkynyl is optionally substituted by halo or hydroxyl.

[0296] In one embodiment, the group: [ka] teeth, [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, C 1-9 Alkyl, C 2-9 Alkenyl, or C 2-9 The alkynyl is optionally substituted by halo or hydroxyl; * Valences marked with L 3c is added to ** Valences marked with R 3c is added to.

[0297] In one embodiment, the group: [ka] teeth, [ka] is.

[0298] In one embodiment, L 4c -O- through R 3c is linked to.

[0299] In one embodiment, R 3c is attached to the rest of the complex via a phosphate oxygen of the nucleic acid molecule.

[0300] In one embodiment, R 3c is attached to the rest of the complex via the phosphate oxygen at the 5' end of either the sense or antisense strand.

[0301] In one embodiment, R 3cis attached to the rest of the complex via the oxygen of the phosphate at the 3' end of either the sense or antisense strand.

[0302] In one embodiment, R 3c is added to the rest of the complex via the oxygen of the phosphate at the 3' end of the sense strand.

[0303] In one embodiment, the compound of formula (I) is [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] is selected from the group consisting of:

[0304] In one embodiment, the targeted nucleic acid complex is selected from the group consisting of WO2015 / 006740, WO2016 / 028649, US8,106,022B2, US8,450,467B2, US8,828,956B2, WO2016 / 149020, WO2017 / 156012, WO2018 / 044350, WO2016 / 100401, WO2018 / 039364, WO2018 / 044350, WO2017 / 174657, WO2018 / 185210, WO2018 / 185252, WO2018 / 185253, US9,943,604B2, or US9,714,421B2.

[0305] 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 invention in any manner. Those skilled in the art will recognize a variety of non-critical parameters that can be changed or modified to achieve essentially the same results. [Example]

[0306] The membrane destabilizing polymers can be prepared using starting materials and synthetic methods similar to those described in International Patent Application Publication Nos. WO2015 / 017519 and WO2016 / 118697.

[0307] The targeted nucleic acid complexes can be prepared as described in International Patent Application Publication No. WO2017 / 177326 or as described below.

[0308] Example 1. Synthesis of Complex 1 [ka] [ka] [ka] [ka] [ka]

[0309] Step 1. Preparation of 2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate 3 [ka] A cooled (0 °C) solution of tetraethylene glycol (934 g, 4.8 mol) in THF (175 mL) and aqueous NaOH (5 M, 145 mL) was treated with p-toluenesulfonyl chloride (91.4 g, 480 mmol) dissolved 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 x 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. f (0.57, 10% MeOH-CH2Cl2).

[0310] 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), washed with brine (3 x 500 mL), 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. f (0.56, 10% MeOH-CH2Cl2).

[0311] 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) over 45 min. After stirring overnight, the reaction mixture was divided into three 1 L portions. Each 1 L portion was poured into 3 L of ice water and mixed for 1 h. After mixing, the solid was isolated by filtration, combined, frozen in liquid nitrogen, and lyophilized for 5 days to give peracetylated galactosamine 6 (369.4 g, 82%) as a white solid. Rf (0.58, 10% MeOH-CH2Cl2).

[0312] 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 CHCl (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 the solution was concentrated to dryness to give compound 7 as a pale yellow glass (7.2 g, quantitative). This product was used without further purification. Rf (0.59, 10% MeOH-CH2Cl2).

[0313] 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 (three times) with toluene (150 mL) to remove traces of water. The dehydrated 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 the solution was concentrated. The residue was purified by chromatography (1% → 5% MeOH-CHCl) to give 8 (7.12 g, 85%) as a brown oil. Rf (0.3, 10% MeOH-CHCl).

[0314] 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 activated carbon (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).

[0315] Step 7. Preparation of di-tert-butyl 4-(((benzyloxy)carbonyl)amino)-4-(3-(tert-butoxy)-3-oxopropyl)heptanedioate 11 [ka] To a solution of di-tert-butyl 4-amino-4-(3-(tert-butoxy)-3-oxopropyl)heptanedioate 10 (13.5 g, 33 mmol) in 25% aqueous NaCO (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, an additional 100 mL of dichloromethane was added, and the dichloromethane layer was separated. The aqueous layer was extracted with dichloromethane (2 x 100 mL). The dichloromethane extracts were combined, 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).

[0316] 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 (approximately 25 mL). The product crystallized as a colorless solid upon standing. The solid was filtered, washed with ethyl acetate, and air-dried to give 12 as a colorless solid (10.2 g, 93%). Rf (0.1, 10% MeOH-CH2Cl2).

[0317] 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-CHCl) to give 13 (5.71 g [crude] (>100%), which contained a coupling by-product but did not affect the next step). Rf (0.45, 10% MeOH-CHCl).

[0318] 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 activated carbon (1 g, 10 w / w% wet basis). The reaction mixture was then purged with hydrogen and vigorously stirred 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% yield over two steps). Rf (0.32, 10% MeOH-CH2Cl2).

[0319] Step 11. Preparation of (5-amino-1,3-phenylene)dimethanol 15 [ka] To a refluxing mixture of 3.75 equiv. LiAlH (13.6 g, 358 mmol) in THF (440 mL) was added dropwise over 1 h a solution of dimethyl 5-aminoisophthalate (20.0 g, 96 mmol) in THF (350 mL). The mixture was stirred at reflux for an additional 2 h, then cooled to room temperature and quenched by the careful addition of MeOH (27 mL) followed by water (40 mL). The quenched mixture was stirred for 2 h, filtered, and concentrated to dryness. The residue was recrystallized (twice) from EtOAc to give 15 as brownish-yellow crystals (10.2 g, 70%).

[0320] 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).

[0321] 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 a nitrogen atmosphere. Upon completion, the solution was concentrated to dryness. The residue was purified by column chromatography (0.5% → 0.75% → 1% → 1.5% MeOH-CHCl) to give 17 as a yellow solid (2.9 g, 35%). Rf (0.6, 10% MeOH-CHCl).

[0322] 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).

[0323] 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 poured into ethyl acetate (250 mL) and washed with brine (3 x 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 CHCl) afforded 19 as a pale orange foam (1.5 g, 94%). Rf (0.25, 10% MeOH-CHCl).

[0324] 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 silica plug (100% CHCl → 15% MeOH in CHCl) to give 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).

[0325] ステップ17. Complex 1 modulation

change

[0326] Example 2: Synthesis of Conjugate 34 [ka] [ka] [ka]

[0327] 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, 17 mg). After stirring (16 h), the reaction mixture was poured into NaHCO3 (sat. aq.), extracted with CHCl2, 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 (36 g, quantitative). This amorphous solid was used without further treatment. Rf (0.85, 10% MeOH-CHCl2).

[0328] 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) in neat formic acid (150 mL) was stirred 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-CHCl).

[0329] Step 3. Preparation of compound 23 [ka] A solution of 22 (6.2 g, 14.1 mmol) and 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 (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 → 15% MeOH—CHCl) to give compound 23 (24.6 g, 89%). Rf (0.55, 15% MeOH-CH2Cl2).

[0330] 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 activated carbon (1 g, 10 w / w% wet basis) was added, and the reaction mixture was purged with hydrogen and vigorously stirred overnight. Upon completion, the reaction was purged with nitrogen, filtered through Celite, and rinsed with MeOH. The filtrate was concentrated and purified by column chromatography on silica gel 60 (gradient: 5% → 10% → 20% MeOH-CHCl) to give 24 as a light brown viscous oil (23 g). Rf (0.32, 10% MeOH-CHCl).

[0331] 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 a nitrogen atmosphere and treated dropwise with a solution of dimethyl-5-aminoisophthalate 25 (20 g, 96 mmol) in anhydrous tetrahydrofuran (350 mL). After the addition was complete, the mixture was heated to 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-CHCl).

[0332] Step 6. Preparation of 3,5-bis(hydroxymethyl)benzonitrile 27 [ka] A solution of 26 (5 g, 33 mmol) in 2 N 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 maintained at a temperature below 5 °C for 30 min and then treated in one portion 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 x 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-CHCl).

[0333] 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 at room temperature overnight. Upon completion, the mixture was concentrated, dispersed in diethyl ether (25 mL), filtered, and concentrated. The crude product was purified by column chromatography on silica gel 60 (gradient: 10% to 50% EtOAc-hexanes) to give 28 as a yellow solid (725 mg, 47%). Rf 0.5 (1:1 EtOAc-hexanes).

[0334] 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 adding methanol (1 mL) followed by water (0.3 mL), and the solution was 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-CHCl).

[0335] 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 treated sequentially with EDC (48 mg, 0.25 mmol), DMAP (catalytic, 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 column chromatography on silica gel 60 (gradient: 2% to 5% MeOH-CHCl) 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-CHCl).

[0336] 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 under reduced pressure, 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-CHCl).

[0337] Step 11. Preparation of Compounds 32, 33, and 34 Compounds 32, 33, and 34 were prepared according to the same procedures used to synthesize compounds 19, 20, and 1, respectively.

[0338] Example 3. Synthesis of Conjugate 36 [ka]

[0339] Step 1. Preparation of Complex 36 Conjugate 36 was prepared using the same procedure used to synthesize compound 34 and all corresponding intermediates, with the only exception being the synthesis of compound 6, in which propanoic anhydride was used instead of acetic anhydride.

[0340] Example 4. Synthesis of Conjugate 42 [ka] [ka]

[0341] Step 1. Preparation of compound 37 [ka] 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) was added diisopropylethylamine (2.75 mL, 15.9 mmol). The solution was stirred overnight at room temperature. Upon completion, the solution was concentrated to dryness under reduced pressure. The residue was purified by column chromatography on silica gel 60 (gradient: 2% to 5% MeOH / CHCl) to give the product as a colorless solid (2.1 g, 63%).

[0342] 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 at room temperature overnight. 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%).

[0343] 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 at room temperature overnight and then concentrated to dryness under reduced pressure to give a light brown solid (1.3 g, 96%).

[0344] Step 4. Preparation of Compound 40, Compound 41, and Compound 42 Compounds 40, 41, and 42 were prepared according to the same procedures used to synthesize Compounds 19, 20, and 1, respectively.

[0345] Example 5. Synthesis of Conjugate 43 [ka] [ka]

[0346] 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 under reduced pressure, suspended in ethyl acetate (150 mL), washed with water (2 x 100 mL), and brine (100 mL). The ethyl acetate layer was dried over magnesium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was purified by column chromatography on silica gel 60 (gradient 100% hexanes to 50% EtOAc / hexanes) to give methyl 11-(2,6-bis(hydroxymethyl)-4-methylphenoxy)undecanoate 44 as a colorless oil (1.6 g, 27%).

[0347] 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 at room temperature overnight. Upon completion, the solution was concentrated to dryness under reduced pressure and purified by column chromatography on silica gel 60 (0.5 to 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%).

[0348] 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 under reduced pressure to remove all tetrahydrofuran. The remaining aqueous solution was flash-frozen in liquid nitrogen and 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%).

[0349] 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 at room temperature overnight (18 h). Upon completion, the solvent was removed under reduced pressure. The residue was purified by column chromatography on silica gel (gradient: 100% CHCl→5%→10%→15% MeOH in CHCl) to give 47 as a colorless solid (710 mg, 41%).

[0350] 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 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 under reduced pressure. The residue was purified by column chromatography on silica gel 60 (5% to 20% MeOH in CHCl gradient) to give 48 as a pale yellow solid (570 mg, 70%). 1 H 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).

[0351] Step 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 Icaa CPG (prime synthesis, 920 mg). The solution was stirred overnight at room temperature on an orbital shaker. Analysis of the reaction by TLC revealed that the activated N-hydroxysuccinate ester was only partially consumed, so an additional 500 mg of CPG 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 hours, 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 (CHCl containing 3% trichloroacetic acid, UV-VIS, A) was used. 504 ) the ligand loading efficiency was 22 μmol / g.

[0352] Step 7. Preparation of Complex 43 The resulting GalNAc-loaded CPG solid support 49 was used in automated oligonucleotide synthesis using standard procedures. Deprotection of the nucleotide followed by cleavage from the solid support (with simultaneous deprotection of the galactosamine acetate) gave GalNAc-oligonucleotide conjugate 43.

[0353] Example 6. Synthesis of Conjugate 50 [ka] [ka]

[0354] 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 x 150 mL). The ethyl acetate extracts were combined, dried over magnesium sulfate, filtered, and concentrated to dryness under reduced pressure. Purification by filtering the residue through a pad of silica first with 50% ethyl acetate / hexanes, then 50% MeOH / EtOAc gave 51 as a pale yellow oil (14 g, 100%).

[0355] 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 at room temperature overnight and then concentrated to dryness under reduced pressure. 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 under reduced pressure. Purification by column chromatography on silica gel 60 (1% → 5% MeOH in CHCl) gave 52 as a pale yellow viscous oil (13 g, 39%).

[0356] 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 at room temperature for 3 h. Upon completion, the solution was poured into water (400 mL) and extracted with ethyl acetate (1 x 500 mL). The ethyl acetate extract was washed with brine (2 x 250 mL), dried over magnesium sulfate, filtered, and concentrated to dryness under reduced pressure. Purification by column chromatography on silica gel 60 (10% to 25% ethyl acetate in hexanes) gave 53 as a yellow viscous oil (6.5 g, 87%).

[0357] 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, with only partial conversion as monitored by TLC (5% MeOH in CHCl). An additional 1.7 mL of TBAF was added, and the solution was stirred at room temperature overnight. Upon completion, the solution was concentrated under reduced pressure and purified by column chromatography on silica gel 60 (10% → 50% EtOAc in hexanes, then 100% EtOAc) to give 54 as a colorless viscous oil (0.5 g, 29%).

[0358] 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 under reduced pressure 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%).

[0359] Step 6. Preparation of Compound 56, Compound 57, Compound 58, and Compound 50 Compounds 56, 57, 58, and 50 were prepared using the same procedures used to synthesize compounds 47, 48, 49, and 43, respectively.

[0360] Example 7. Synthesis of Conjugate 59 [ka] [ka]

[0361] 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 (gas) was bubbled through the solution for 2 min, and the reaction was stirred at reflux for 1.5 h. The reaction was concentrated under reduced pressure to give methyl (2R,5R)-5-hydroxypiperidine-2-carboxylate 61 in quantitative yield, which was used without further purification.

[0362] 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 in portions, and the reaction was stirred for 2 h. The reaction was diluted with DCM (100 mL) and washed successively with 1 M HCl (2 x 75 mL), saturated NaHCO (2 x 75 mL), HO (2 x 75 mL), and saturated NaCl solution (2 x 75 mL). The organic solution was separated, dried (NaSO), and concentrated under reduced pressure to give 1-(tert-butyl) 2-methyl(2R,5R)-5-hydroxypiperidine-1,2-dicarboxylate 62 (5.53 g, 88%). This was used without further purification.

[0363] 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. LiBH (3.0 M solution 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 under reduced pressure, and the aqueous solution was exhaustively extracted with EtOAc (10 x 100 mL). The combined organic solutions were washed with HO (50 mL), saturated NaCl solution (2 x 50 mL), dried (NaSO), and concentrated under reduced pressure to give tert-butyl (2R,5R)-5-hydroxy-2-(hydroxymethyl)piperidine-1-carboxylate 63 (2.4 g, 49.0%). This was used without further purification.

[0364] 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 (gas) was bubbled through for 45 seconds, and the reaction was stirred at room temperature for 45 minutes. The reaction was concentrated under reduced pressure and dried under high vacuum to give (3R,6R)-6-(hydroxymethyl)piperidin-3-ol 64. This product was used without further purification.

[0365] 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 with TEA (3.5 mL, 25.2 mmol) in MeCN (50 mL) 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 under reduced pressure 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.

[0366] 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 with TEA (50 mL) in DCM 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 organic solution was separated, dried (NaSO), concentrated under reduced pressure, and purified by column chromatography (100% hexanes → 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%).

[0367] 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. An additional aliquot of KOH (300 mg, 6 mmol) was added and stirring was continued for an additional 24 h. The reaction was concentrated under reduced pressure, taken up in DCM (150 mL), washed with HO (4 x 50 mL), dried (NaSO), and concentrated under reduced pressure 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.

[0368] 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 under reduced pressure, 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.

[0369] 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 under reduced pressure, and the aqueous solution 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 subsequent reaction without further purification.

[0370] Step 10. Preparation of Compound 70, Compound 71, Compound 72, and Compound 59 Compounds 70, 71, 72, and 59 were prepared using the same procedures used to synthesize compounds 47, 48, 49, and 43, respectively.

[0371] Example 8. Synthesis of Conjugate 142 [ka] [ka]

[0372] Step 1. Preparation of 3,4,5-triacetoxybenzoic acid 73 To a solution of gallic acid (20 g) in pyridine (50 mL) was added 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 the precipitation of a colorless solid. 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%).

[0373] Step 2.5-((2-((2-oxo-2-phenyl-1λ 2Preparation 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 x 200 mL), dried over magnesium sulfate, filtered, and concentrated to dryness under reduced pressure. The crude product was purified by column chromatography on silica gel (1% to 5% MeOH in DCM gradient) to give 5-((2-((2-oxo-2-phenyl-1λ 2 The resulting product was 1,2,3-triyl-2-(2-ethylamino)ethyl)carbamoyl)benzene-1,2,3-triyl triacetate as an off-white solid (5.5 g).

[0374] 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 2 A 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 (-ethyl)amino)ethyl)benzamide was obtained as a colorless solid (4 g, quantitative).

[0375] 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 (-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 at 60 °C overnight. Upon completion, the solution was cooled to room temperature, diluted with ethyl acetate (200 mL), washed with water (200 mL), washed with brine (3 x 100 mL), dried over magnesium sulfate, filtered, and concentrated to dryness under reduced pressure. The crude product was purified by column chromatography on silica gel (2% to 10% MeOH gradient in DCM) to give trimethyl 2,2',2''-((5-((2-((2-oxo-2-phenyl-1λ 2 -ethyl)amino)ethyl)carbamoyl)benzene-1,2,3-triyl)tris(oxy))-triacetate as a beige solid (5 g, 79%).

[0376] Step 5. 2,2',2''-((5-((2-((2-oxo-2-phenyl-1λ 2 Preparation of (ethyl)amino)ethyl)carbamoyl)benzene-1,2,3-triyl)tris(oxy))triacetic acid 77 Trimethyl 2,2',2''-((5-((2-((2-oxo-2-phenyl-1λ 2 A solution of 2,2',2''-((5-((2-((2-oxo-2-phenyl-1λ-phenyl)-2,2',2''-trimethylamino)ethyl)-carbamoyl)benzene-1,2,3-triyl)tris(oxy))triacetate (5 g, 9.2 mmol) and 1 M NaOH (30 mL) in methanol (100 mL) was stirred at room temperature for 2 hours. Upon completion, the reaction was concentrated to remove the methanol and diluted with water (75 mL). The mixture was cooled to 0°C, acidified with 2 M HCl, and extracted with ethyl acetate (5 x 150 mL). The ethyl acetate extracts were combined, dried over magnesium sulfate, filtered, and evaporated to dryness under reduced pressure to give 2,2',2''-((5-((2-((2-oxo-2-phenyl-1λ-phenyl)-2,2',2''-trimethylamino)ethyl)-carbamoyl)benzene-1,2,3-triyl)tris(oxy)triacetate. 2(-ethyl)amino)ethyl)carbamoyl)benzene-1,2,3-triyl)tris(oxy))triacetic acid was obtained as a colorless solid (2.3 g, 50%).

[0377] Step 6. Preparation of Compound 78 Compound 78 was prepared from compound 9 (2.75 g, 4.3 mmol) and compound 77 (0.5 g, 0.96 mmol) using the same procedure as used for compound 13. Yield: 600 mg.

[0378] 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.

[0379] 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%.

[0380] 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.

[0381] Step 10. Preparation of Complex 142 Conjugate 142 was prepared from compound 141 (200 mg) and 1000A lcaa CPG (1.8 g) using the same procedure as that used for compound 1. Yield: 1.9 g, loading: 22 μmol / g CPG. The resulting GalNAc-loaded CPG solid support was used in automated oligonucleotide synthesis using standard procedures. Deprotection of the nucleotide followed by cleavage from the solid support (with simultaneous deprotection of the galactosamine acetate) afforded GalNAc-oligonucleotide conjugate 142.

[0382] Example 9. Synthesis of Conjugate 145 [ka] [ka]

[0383] Step 1. Preparation of racemic (cis) 5-benzyl-3a,6a-dimethyltetrahydro-1H-furo[3,4-c]pyrrole-1,3(3aH)-dione 123 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) was added trifluoroacetic acid (75 μL) slowly. The solution was stirred overnight, allowing the ice bath to melt and gradually warm to room temperature. 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 column chromatography on silica gel (gradient: 20% ethyl acetate in hexane → 100% ethyl acetate) gave racemic (cis) 5-benzyl-3a,6a-dimethyltetrahydro-1H-furo[3,4-c]pyrrole-1,3(3aH)-dione as a yellow oil (3.5 g, 56%).

[0384] Step 2. Preparation of racemic (cis)(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 added lithium aluminum hydride pellets (1.5 g, 40 mmol) slowly in three portions. The solution was stirred overnight, allowing the solution 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 5 M 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 racemic (cis)1-benzyl-3,4-dimethylpyrrolidine-3,4-diyl)dimethanol as a colorless oil (2.7 g).

[0385] Step 3. Preparation of racemic (cis)(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 on wet activated carbon (1 g). The solution was vigorously stirred under a hydrogen atmosphere for 16 hours. Upon completion, the solution was filtered through Celite and concentrated to dryness to give racemic (cis)(3,4-dimethylpyrrolidine-3,4-diyl)dimethanol as a colorless solid (5.5 g, 86%).

[0386] Step 4. Preparation of racemic (cis)methyl 10-(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%.

[0387] Step 5. Preparation of racemic (cis)methyl 10-(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%.

[0388] Step 6. Preparation of racemic (cis) lithium 10-(3-((bis(4-methoxyphenyl)-(phenyl)methoxy)-methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoate 128 To a solution of compound 127 (3.0 g, 4.6 mmol) in THF (50 mL) and water (50 mL) was added lithium hydroxide (121 mg, 5.0 mmol). The solution was stirred at room temperature for 4 hours and then concentrated to remove the THF. The remaining aqueous solution was lyophilized overnight to give a pale pink solid (2.9 g, quantitative yield).

[0389] 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%.

[0390] 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.

[0391] Step 9. Preparation of Complex 145 Conjugate 145 was prepared from compound 144 (200 mg) and 1000A lcaa CPG (1.8 g) using the same procedure as that used for compound 1. Yield: 1.9 g, loading: 20 μmol / g CPG. The resulting GalNAc-loaded CPG solid support was used in automated oligonucleotide synthesis using standard procedures. Deprotection of the nucleotide followed by cleavage from the solid support (with simultaneous deprotection of the galactosamine acetate) afforded GalNAc-oligonucleotide conjugate 145.

[0392] Example 10. Synthesis of Conjugate 150 [ka]

[0393] 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 at room temperature overnight, and the reaction became a clear solution. Analysis by TLC confirmed the reaction was complete. The organic solution was 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%).

[0394] 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 a nitrogen atmosphere, 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. Analysis by TLC confirmed the reaction was complete. The reaction was cooled back to 0 °C. The reaction was quenched by the dropwise addition of saturated Na2SO4 solution (32 mL). After the addition was complete, the mixture was stirred for an additional 3 h and filtered through a pad of celite. Evaporation of the solvent gave cyclopent-3-enylmethanol 146-2 (37.3 g, 95%) as a colorless liquid.

[0395] 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, 3-chloroperbenzoic acid (10 g, 45 mmol, 77% purity) was added slowly. The reaction was stirred overnight. Dichloromethane (150 mL) was added. The organic solution was washed with sodium thiosulfate (12 g in 10 mL of water) followed by saturated NaHCO3 (40 mL). This procedure was repeated until all remaining 3-chloroperbenzoic acid was washed away. The organic solution was dried over MgSO4. Evaporation of the solvent gave a mixture of cis-6-oxabicyclo[3.1.0]hexan-3-ylmethanol and trans-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).

[0396] 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. Analysis by TLC showed that the reaction was incomplete. The methanol was removed, and NH3·H2O (50 mL) was added, and this was stirred at room temperature for 1 week. Analysis by TLC showed that the reaction was complete. Water was removed by azeotropy with ethanol to give 2-amino-4-(hydroxymethyl)cyclopentanol 146-4 (2.1 g, 91%) as a yellow oil.

[0397] 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 in 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 as an off-white solid in 87.4% yield.

[0398] Step 6. Preparation of Compound 147 Compound 147 was prepared quantitatively from compound 146 (1.4 g, 2.33 mmol) using the same procedure as used for compound 18.

[0399] 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%.

[0400] 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%.

[0401] Step 9. Preparation of Complex 150 Compound 150 was prepared from compound 149 (436 mg) and 1000A lcaa CPG (2.62 g) using the same procedure as that used for compound 1. Yield: 2.7 g, loading: 21.3 μmol / g CPG. The resulting GalNAc-loaded CPG solid support was used in automated oligonucleotide synthesis using standard procedures. Deprotection of the nucleotide followed by cleavage from the solid support (with simultaneous deprotection of the galactosamine acetate) gave GalNAc-oligonucleotide conjugate 150.

[0402] Example 11. Synthesis of Conjugate 153, Conjugate 158, Conjugate 163, Conjugate 168, and Conjugate 173 [ka]

[0403] 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 organic solution was washed successively with 1 M HCl (twice), saturated NaHCO (twice), HO, and brine, dried, and concentrated under reduced pressure to give 1-(tert-butyl) 2-methyl(2S,4R)-4-hydroxypyrrolidine-1,2-dicarboxylate (133) (58.1 g, 85%).

[0404] 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 solution in THF) (37.5 mL, 75 mmol) was added dropwise. The reaction was warmed to room temperature and stirred for 4 h. After that, the reaction was quenched with saturated NH₄Cl. The reaction was extracted with EtOAc, washed with HO and brine, dried (Na₂SO₄), and concentrated under reduced pressure. 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%).

[0405] 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 a N atmosphere. LiBH solution (48 mL, 96 mmol) was added dropwise, and the reaction was stirred at room temperature for 48 h. The reaction was quenched with 1 M NaOH, the THF was removed under reduced pressure, and the residue was extracted with EtOAc (4 x 100 mL). The organic solution was washed with H2O and brine, dried (Na2SO4), and concentrated under reduced pressure. 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%). Structural assignments were performed according to references.

[0406] 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 HCl gas was applied for approximately 2 min. After stirring for 1 h, the reaction was concentrated under reduced pressure and dried under high vacuum to give (3R,5S)-5-(hydroxymethyl)-5-methylpyrrolidin-3-ol hydrochloride (136) in quantitative yield.

[0407] 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) were stirred in DCM at room temperature for 16 h. The precipitate was removed by filtration, and the organic solution was washed with 1 M HCl (twice), saturated NaHCO (twice), HO, and brine. After dehydration, the organic solution was concentrated under reduced pressure 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%).

[0408] 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 hours. The reaction mixture was concentrated under reduced pressure, 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).

[0409] [ka]

[0410] Step 7. Preparation of compound 154-1 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 a nitrogen atmosphere, triethylamine (10.4 mL, 74.6 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 3 h. Analysis by TLC confirmed the reaction was complete. The organic layer was washed with brine (100 mL), dried over MgSO4, and concentrated to dryness. This was used directly in the next reaction without purification.

[0411] Step 8. Preparation of compound 154-2 A mixture of 2-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)isoindoline-1,3-dione (154-1) obtained as above and hydrazine monohydrate (3.6 mL, 74 mmol) in ethanol (100 mL) was stirred at room temperature overnight. Analysis by TLC confirmed the reaction was complete. The precipitate was removed by filtration. The filtrate was evaporated. The residue was taken up in ethyl acetate (100 mL). The organic solution was washed with 10% NaOH, water, and brine and dried over MgSO. Evaporation of the solvent afforded 2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethanamine (154-2) as a yellow liquid (8.11 g, 89.3% yield over two steps), which was used in the next reaction without further purification.

[0412] 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 dropwise 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 overnight at room temperature. 4 N HCl (10 mL) was added. The precipitate was collected by filtration and washed with water (3 x 10 mL). The solid was dried in a desiccator over PO5 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 + ).

[0413] 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 → 50% ethyl acetate / hexane) gave 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%). 1 HNMR (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).

[0414] [ka] Step 11. Preparation of compound 164-1

[0415] 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 quenched by the addition of water (250 mL). The organic layer was separated, and the aqueous solution was extracted with ether (250 mL). The extracts were combined and dried over MgSO4. Evaporation of the solvent followed by column chromatography (5% ethyl acetate / hexane) afforded 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).

[0416] Step 12. Preparation of compound 164-2 To a mixture of t-BuOH (140 mL) and water (70 mL) were added AD-mix-β (47.4 g) and methanesulfonamide (2.89 g, 30.4 mmol). The mixture was stirred at room temperature for 30 minutes and then cooled to 0°C. tert-Butyl 3-vinylpyrrolidine-1-carboxylate 164-1 (6.00 g, 30.4 mmol) was added. The reaction mixture 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 warm 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), brine (50 mL), and dried over MgSO4. Evaporation of the solvent followed by column chromatography (2% methanol / dichloromethane → 7% methanol / dichloromethane) gave tert-butyl 3-(1,2-dihydroxyethyl)pyrrolidine-1-carboxylate 164-2 (5.4 g, 77%) as a light brown oil.

[0417] 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. Analysis by TLC confirmed the reaction was complete. The ethanol was evaporated. Toluene was added and evaporated. This procedure 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).

[0418] 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 slowly added NaHCO3 (3.7 g, 44 mmol). Dioxane (20 mL) was then added. To this 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.

[0419] 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 equivalent) using the same procedure described in 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. 1 HNMR (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).

[0420] [ka]

[0421] 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) dropwise at room temperature. The reaction mixture was stirred overnight and analyzed by TLC, which confirmed 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. Evaporation of the solvent gave pure methyl 12-(2-(tert-butoxycarbonylamino)ethylamino)-12-oxododecanoate 170-1 (5.85 g 100%) as a white solid.

[0422] 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, thionyl chloride (3.3 mL, 45.5 mmol) was added dropwise. The reaction was then stirred overnight at room temperature. Analysis by TLC confirmed the reaction was complete. The solvent and volatile organics were evaporated. The residue was then co-evaporated twice with heptane to quantitatively afford methyl 12-(2-aminoethylamino)-12-oxododecanoate hydrochloride 170-2 as a white solid. LC-MS (ESI): m / z: 287 (100), (M + H + , free amine).

[0423] Step 18. Preparation of compound 170-3 A mixture of (-)-methyl (S)-2,2-dimethyl-1,3-dioxolane-4-carboxylate (5.01 g, 31.2 mmol) and LiOH·HO (2.55 g, 60.8 mmol) in THF (50 mL) and water (50 mL) was stirred overnight. Analysis by TLC confirmed the reaction was complete. The THF was evaporated, and the aqueous solution was acidified with 1 N HCl to pH = 1. This was extracted with ethyl acetate (5 x 50 mL). The extracts were combined and dried over MgSO. Evaporation of the solvent gave (S)-2,2-dimethyl-1,3-dioxolane-4-carboxylic acid 170-3 (2.93 g, 64.3%) as a pale yellow liquid.

[0424] 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 in 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.

[0425] 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) at 0 °C over 4 h. The reaction mixture was then stirred at room temperature overnight. Analysis by LC-MS confirmed complete conversion of starting material 170-2. The organic layer was washed with brine (50 mL), 1 N 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.

[0426] Step 21. Preparation of compound 170-6 To a solution containing (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-dihydroxypropanamido)ethylamino)-12-oxododecanoate 170-6 (3.74 g, 69.4% total) 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).

[0427] 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 anhydrous pyridine (57.5 mL) under a nitrogen atmosphere 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 evaporated. This procedure was repeated three times. Water (100 mL) was added, and the mixture 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-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).

[0428] [ka]

[0429] Step 23. Preparation of Compound 139, Compound 155, Compound 160, Compound 165, and Compound 170 Compounds 139, 155, 160, 165, and 170 were prepared from compounds 138, 154, 159, 164, and 169 using the same procedure as that used for compound 18.

[0430] Step 24. Preparation of Conjugate 153, Conjugate 158, Conjugate 163, Conjugate 168, and Conjugate 173 Conjugate 153, conjugate 158, conjugate 163, conjugate 168, and conjugate 173 were prepared from compounds 139, 154, 159, 164, and 169 using the same procedure as that used for compound 1.

[0431] Example 12. Synthesis of Conjugate 176 [ka] [ka]

[0432] 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 under reduced pressure, and the residue was taken up in MTBE and cooled 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.

[0433] Step 2. Preparation of racemic (cis)methyl 12-(12-(10-(3-((bis(4-methoxyphenyl)-(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanamido)dodecanamido) dodecanoate 129 Lithium racemic (cis) 10-(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) were stirred in DCM at room temperature overnight. The precipitate was removed by filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography (5% MeOH, DCM). TLC analysis revealed the presence of two closely spaced spots of identical mass, which were assigned as geometric isomers. These were 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 fashion.

[0434] Step 3. Preparation of racemic (cis) lithium 12-(12-(10-(-3-((bis(4-methoxyphenyl)(phenyl)-methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanamido)-dodecanamido) dodecanoate 130 Racemic (cis)methyl 12-(12-(10-(3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanamido)dodecanamido) dodecanoate (129) (3.1 mmol) was stirred with LiOH (88 mg, 3.7 mmol) in THF:HO (50:50) at room temperature overnight. The reaction was followed by TLC and the THF was removed under reduced pressure. The aqueous solution was frozen in liquid N2 and lyophilized for 48 hours to quantitatively obtain racemic (cis) lithium 12-(12-(10-(3-((bis(4-methoxyphenyl)(phenyl)-methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanamido)-dodecanamido) dodecanoate 130.

[0435] Step 4. Preparation of Complex 176 Conjugate 176 was prepared from compound 24 and compound 130 using the same procedure as used for compound 1.

[0436] Example 13. Synthesis of Conjugate 179 [ka] [ka]

[0437] 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 under reduced pressure and purified by column chromatography to give compound 80 (2.88 g, 83%).

[0438] 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. This compound was used crude in subsequent reactions, and the yield obtained was quantitative.

[0439] Step 3. Preparation of Complex 179 Conjugate 179 was prepared from compound 18 and compound 81 using the same procedure as used for compound 1.

[0440] Example 14. Synthesis of Conjugate 182 [ka] [ka]

[0441] Step 1. Preparation of Compound 93 Compound 93 was prepared by the same procedure as 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.

[0442] 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.

[0443] Step 3. Preparation of Complex 182 Conjugate 182 was prepared from compound 18 and compound 94 using the same procedure as used for compound 1.

[0444] Example 15. Synthesis of Conjugate 185 and Conjugate 188 [ka] [ka] [ka]

[0445] 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 HO-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).

[0446] 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%.

[0447] 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 (to room temperature) and quenched by the addition of TEA (40 mL), and the solution was concentrated. Chromatography of the crude material afforded 84 (11.16 g, 60%) as a pale yellow foam. Rf 0.7 (10% CH3OH-CH2Cl2).

[0448] 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 H. After vigorous stirring (4.5 h), the reaction was purged with N, filtered through Celite, and concentrated. Chromatography of the crude material gave 85 (5.77 g, 45%) as a colorless foam. Rf 0.5 (10% CHOH-CHCl).

[0449] Step 5. Preparation of Compound 95 Compound 95 was prepared by the same procedure as that used for compound 91 (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.

[0450] 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.

[0451] Step 7. Preparation of Compound 97 Compound 97 was prepared using the same procedure as that used for compound 89 (2-oxo-2-phenyl-1λ). 2-ethyl)-D-glutamic acid (2 g, 7.1 mmol) and 85 (12.1 g, 17.8 mmol). Yield: 10 g, quantitative.

[0452] 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%.

[0453] 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).

[0454] 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.

[0455] Step 11. Preparation of Conjugate 185 and Conjugate 188 Conjugates 185 and 188 were prepared from compounds 18 and 96, or compounds 18 and 100, using the same procedure as that used for compound 1.

[0456] Example 16. Synthesis of Conjugate 191, Conjugate 194, Conjugate 197, and Conjugate 200 [ka] [ka] [ka]

[0457] 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 at 100 °C for 18 h. The reaction was cooled to room temperature, poured into a 1 L separatory funnel, and extracted with dichloromethane (3 x 200 mL). The dichloromethane extracts were combined, 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).

[0458] Step 2. Preparation of Compound 87 Compound 87 is 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%.

[0459] Step 3. Preparation of Compound 88 Compound 88 is prepared from 87 (10 g, 19.8 mmol) using the same procedure as used for compound 85. Yield: 7.63 g, 65%.

[0460] 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).

[0461] 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 H. After vigorous stirring (2 h), the reaction was purged with N, 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% CHOH-CHCl).

[0462] 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. Chromatography of the crude material gave 91 (590 mg, 76%) as a colorless foam. Rf 0.5 (10% CHOH-CHCl).

[0463] Step 7. Preparation of Compound 92 A solution of 91 (590 mg, 0.25 mmol) and Pd / C (100 mg, 10% wet support) in CHOH (30 mL) was treated with TFA (29 μL, 0.37 mmol) and purged with H. After stirring (3 h), the mixture was purged with N, 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% CHOH-CHCl).

[0464] Step 8. Preparation of Compound 101 Compound 101 is 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%.

[0465] Step 9. Preparation of Compound 102 Compound 102 is prepared from compound 101 (4.2 g, 3.2 mmol) using the same procedure as used for compound 90. Yield: 2.1 g, 47%.

[0466] 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 that used for compound 91. Yield: (560 mg, 24%).

[0467] Step 11. Preparation of Compound 104 Compound 104 is prepared quantitatively from compound 103 (560 mg) using the same procedure as used for compound 92. The compound is used without purification.

[0468] Step 12. Preparation of Conjugate 191, Conjugate 194, and Conjugate 197 Conjugates 191, 194, and 197 are prepared from compound 128 and compounds 92, 96, and 100 using the same procedure as that used for compound 1.

[0469] Example 16a. Synthesis of Conjugate 191a [ka] [ka] [ka]

[0470] Step 1. Preparation of 2-(2-(2-azidoethoxy)ethoxy)ethan-1-ol 86a 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 at 100 °C for 18 h. The reaction was cooled to room temperature, poured into a 1 L separatory funnel, and extracted with dichloromethane (3 x 200 mL). The dichloromethane extracts were combined, 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).

[0471] Step 2. Preparation of compound 87a Compound 87a was prepared from 86a (4.95 g, 28.3 mmol) and 6a (10 g, 25.7 mmol) using the same procedure as used for compound 84. Yield: 10 g, 77%.

[0472] Step 3. Preparation of compound 88a Compound 88a was prepared from 87a (10 g, 19.8 mmol) using the same procedure as used for compound 85. Yield: 7.63 g, 65%.

[0473] Step 4. Preparation of compound 89a A solution of 88a (2 g, 3.38 mmol) and ZL-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 89a (871 mg, 48%) as a colorless foam. Rf 0.5 (10% CHOH-CHCl).

[0474] Step 5. Preparation of compound 90a A solution of 89a (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 H. After vigorous stirring (2 h), the reaction was purged with N, filtered through Celite, and concentrated. The crude material was used without further manipulation to give 90a (850 mg, quantitative) as a colorless foam. Rf 0.25 (10% CHOH-CHCl).

[0475] Step 6. Preparation of compound 91a A solution of 90a (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. Chromatography of the crude material afforded 91a (590 mg, 76%) as a colorless foam. Rf 0.5 (10% CHOH-CHCl).

[0476] Step 7. Preparation of compound 92a A solution of 91a (590 mg, 0.25 mmol) and Pd / C (100 mg, 10% wet support) in CHOH (30 mL) was treated with TFA (29 μL, 0.37 mmol) and purged with H. After stirring (3 h), the mixture was purged with N, filtered through Celite, and concentrated. The crude material was used without further treatment to give 92a (600 mg, quantitative) as a colorless foam. Rf 0.1 (10% CHOH-CHCl).

[0477] Step 8. Preparation of Complex 191a Conjugate 191a was prepared from compound 128 and compound 92a using the same procedure as used for compound 1.

[0478] Example 16b. Synthesis of Conjugate 191b [ka] [ka] [ka]

[0479] Step 1. Preparation of 2-(2-(2-azidoethoxy)ethoxy)ethan-1-ol 86b 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 at 100 °C for 18 h. The reaction was cooled to room temperature, poured into a 1 L separatory funnel, and extracted with dichloromethane (3 x 200 mL). The dichloromethane extracts were combined, 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).

[0480] Step 2. Preparation of compound 87b Compound 87a is prepared from 86b (4.95 g, 28.3 mmol) and 6b (10 g, 25.7 mmol) using the same procedure as used for compound 84. Yield: 10 g, 77%.

[0481] Step 3. Preparation of compound 88b Compound 88a is prepared from 87b (10 g, 19.8 mmol) using the same procedure as used for compound 85. Yield: 7.63 g, 65%.

[0482] Step 4. Preparation of compound 89b A solution of 88b (2 g, 3.38 mmol) and racemic 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 89b (871 mg, 48%) as a colorless foam. Rf 0.5 (10% CHOH-CHCl).

[0483] Step 5. Preparation of compound 90b A solution of 89b (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 H. After vigorous stirring (2 h), the reaction was purged with N, filtered through Celite, and concentrated. The crude material was used without further treatment to give 90b (850 mg, quantitative) as a colorless foam. Rf 0.25 (10% CHOH-CHCl).

[0484] Step 6. Preparation of compound 91b A solution of 90b (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. Chromatography of the crude material gave 91b (590 mg, 76%) as a colorless foam. Rf 0.5 (10% CHOH-CHCl).

[0485] Step 7. Preparation of compound 92b A solution of 91b (590 mg, 0.25 mmol) and Pd / C (100 mg, 10% wet support) in CHOH (30 mL) was treated with TFA (29 μL, 0.37 mmol) and purged with H. After stirring (3 h), the mixture was purged with N, filtered through Celite, and concentrated. The crude material was used without further treatment to give 92b (600 mg, quantitative) as a colorless foam. Rf 0.1 (10% CHOH-CHCl).

[0486] Step 8. Preparation of Complex 191b Conjugate 191b is prepared from compound 128 and compound 92b using the same procedure as used for compound 1.

[0487] Example 16c. Synthesis of Conjugate 191c [ka] [ka] [ka]

[0488] Step 1. Preparation of 2-(2-(2-azidoethoxy)ethoxy)ethan-1-ol 86c 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 at 100 °C for 18 h. The reaction was cooled to room temperature, poured into a 1 L separatory funnel, and extracted with dichloromethane (3 x 200 mL). The dichloromethane extracts were combined, 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).

[0489] Step 2. Preparation of compound 87c Compound 87c is prepared from 86c (4.95 g, 28.3 mmol) and 6c (10 g, 25.7 mmol) using the same procedure as used for compound 84. Yield: 10 g, 77%.

[0490] Step 3. Preparation of compound 88c Compound 88c is prepared from 87c (10 g, 19.8 mmol) using the same procedure as used for compound 85. Yield: 7.63 g, 65%.

[0491] Step 4. Preparation of compound 89c A solution of 88c (2 g, 3.38 mmol) and racemic 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 89c (871 mg, 48%) as a colorless foam. Rf 0.5 (10% CHOH-CHCl).

[0492] Step 5. Preparation of compound 90c A solution of 89c (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 H. After vigorous stirring (2 h), the reaction was purged with N, filtered through Celite, and concentrated. The crude material was used without further treatment to give 90c (850 mg, quantitative) as a colorless foam. Rf 0.25 (10% CHOH-CHCl).

[0493] Step 6. Preparation of compound 91c A solution of 90c (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. Chromatography of the crude material gave 91c (590 mg, 76%) as a colorless foam. Rf 0.5 (10% CHOH-CHCl).

[0494] Step 7. Preparation of compound 92c A solution of 91c (590 mg, 0.25 mmol) and Pd / C (100 mg, 10% wet support) in CHOH (30 mL) was treated with TFA (29 μL, 0.37 mmol) and purged with H. After stirring (3 h), the mixture was purged with N, filtered through Celite, and concentrated. The crude material was used without further treatment to give 92c (600 mg, quantitative) as a colorless foam. Rf 0.1 (10% CHOH-CHCl).

[0495] Step 8. Preparation of Complex 191c Conjugate 191c is prepared from compound 128 and compound 92c using the same procedure as used for compound 1.

[0496] Example 17. Synthesis of Conjugate 203 and Conjugate 206 [ka]

[0497] 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.

[0498] Step 2. Preparation of Complexes 203 and 206 Conjugates 203 and 206 were prepared from compounds 96 and 100 using the same procedure as that used for compound 1.

[0499] Example 18. Synthesis of Conjugate 209 [ka]

[0500] Step 1. Preparation of Complex 209 Conjugate 209 was prepared from compound 96 and compound 160 using the same procedure as used for compound 1.

[0501] Example 18a. Synthesis of Conjugate 209a [ka]

[0502] Step 1. Preparation of complex 209a Conjugate 209a is prepared from compound 96a and compound 160 using the same procedure as used for compound 1.

[0503] Example 19. Synthesis of Conjugate 212 and Conjugate 215 [ka] [ka]

[0504] Step 1. Dimethyl 5-(2-((2-oxo-2-phenyl-1λ) 2 Preparation of (ethyl)amino)acetamido)-isophthalate 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), and 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 x 100 mL), saturated sodium bicarbonate (1 x 100 mL), and brine (2 x 100 mL), respectively. Dimethyl 5-(2-((2-oxo-2-phenyl-1λ) was obtained by drying over magnesium sulfate, filtering, and concentrating to dryness. 2 -ethyl)amino)acetamido)isophthalate as a colorless solid (7.2 g, 79%).

[0505] Step 2.5-(2-((2-oxo-2-phenyl-1λ) 2 Preparation of (-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 1 M NaOH (25 mL). The solution was stirred at room temperature for 2 hours and then concentrated to remove the THF and MeOH. The remaining aqueous solution was diluted with water (75 mL), cooled on an ice-water bath, and acidified to pH = 1 with 6 M 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). The solution was stirred at room temperature for 2 hours and then concentrated to remove the THF and MeOH. The remaining aqueous solution was diluted with water (75 mL), cooled on an ice-water bath, and acidified to pH = 1 with 6 M HCl. The solid was filtered and washed with water (3 x 100 mL). 2 (-ethyl)amino)acetamido)-isophthalic acid (6.9 g, quantitative yield) was obtained.

[0506] Step 3. Preparation of Compound 107 Compound 107 was prepared by cleaving 5-(2-((2-oxo-2-phenyl-1λ))-2-(2-oxo-2-phenyl-1λ)-isopropyl ether using the same procedure as that used for compound 95. 2 Prepared from 106 (200 mg, 0.54 mmol) and 94 (1.7 g, 1.3 mmol). Yield: 600 mg.

[0507] 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.

[0508] Step 5. Preparation of Compound 109 Compound 109 was prepared by the same procedure 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.

[0509] 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.

[0510] Step 7. Preparation of Conjugate 212 and Conjugate 215 Conjugates 212 and 215 were prepared from compound 128 and either compound 108 or compound 110 using the same procedure as that used for compound 1.

[0511] Example 19a. Synthesis of Conjugate 212a and Conjugate 215a [ka] [ka]

[0512] Step 1. Dimethyl 5-(2-((2-oxo-2-phenyl-1λ) 2 Preparation of (ethyl)amino)acetamido)-isophthalate 105a 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), and 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 x 100 mL), saturated sodium bicarbonate (1 x 100 mL), and brine (2 x 100 mL), respectively. Dimethyl 5-(2-((2-oxo-2-phenyl-1λ) was obtained by drying over magnesium sulfate, filtering, and concentrating to dryness. 2 -ethyl)amino)acetamido)isophthalate as a colorless solid (7.2 g, 79%).

[0513] Step 2.5-(2-((2-oxo-2-phenyl-1λ) 2 Preparation of (-ethyl)amino)acetamido)isophthalic acid 106a 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) is added 1 M NaOH (25 mL). The solution is stirred at room temperature for 2 hours and then concentrated to remove the THF and MeOH. The remaining aqueous solution is diluted with water (75 mL), cooled on an ice-water bath, and acidified to pH = 1 with 6 M HCl. The solid is filtered and washed with water (3 x 100 mL). The solid is lyophilized to give 5-(2-((2-oxo-2-phenyl-1λ-ethyl)amino)acetamido)isophthalate. 2 -ethyl)amino)acetamido)-isophthalic acid (6.9 g, quantitative yield) was obtained.

[0514] Step 3. Preparation of compound 107a Compound 107a was prepared by the same procedure used for compound 95: 5-(2-((2-oxo-2-phenyl-1λ) 2-ethyl)amino)acetamido)isophthalic acid 106a (200 mg, 0.54 mmol) and 94a (1.7 g, 1.3 mmol). Yield: 600 mg.

[0515] Step 4. Preparation of compound 108a Compound 108a is prepared from compound 107a (600 mg) using the same procedure as used for compound 96a. Yield: 650 mg, quantitative.

[0516] Step 5. Preparation of compound 109a Compound 109a was prepared by the same procedure used for compound 99: 5-(2-((2-oxo-2-phenyl-1λ) 2 Prepared from 106a (180 mg, 0.48 mmol) and 9a8 (1.5 g, 1.1 mmol). Yield: 900 mg.

[0517] Step 6. Preparation of compound 110a Compound 110a is prepared from compound 109 (900 mg) using the same procedure as used for compound 100. Yield: 920 mg, quantitative.

[0518] Step 7. Preparation of Conjugate 212a and Conjugate 215a Conjugates 212a and 21a5 are prepared from compound 128 and compound 108a or compound 110a using the same procedure as that used for compound 1.

[0519] Example 20. Synthesis of Conjugate 218 and Conjugate 221 [ka] [ka] Example 19. Synthesis of Conjugate 212 and Conjugate 215

[0520] 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%.

[0521] Step 2. Preparation of Compound 112 A solution of 111 (2.21 g, 1.87 mmol) in CHCl (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% CHOH-CHCl).

[0522] 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%.

[0523] Step 4. Preparation of Compound 114 Compound 114 was prepared from compound 113 using the same procedure as used for compound 92.

[0524] 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%.

[0525] 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%.

[0526] 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%.

[0527] 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%.

[0528] Step 9. Preparation of Conjugate 218 and Conjugate 221 Conjugates 218 and 221 were prepared from compound 128 and compound 114 or compound 118 using the same procedure as that used for compound 1.

[0529] Example 20a. Synthesis of Conjugate 218a and Conjugate 221a [ka] [ka]

[0530] Step 1. Preparation of compound 111a Compound 111a is 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%.

[0531] Step 2. Preparation of compound 112a A solution of 111a (2.21 g, 1.87 mmol) in CHCl (40 mL) was slowly treated with TFA (5 mL). After stirring (2 h), the mixture was concentrated and chromatographed to give 112a (1.08 g, 47%) as a colorless foam, Rf 0.1 (10% CHOH-CHCl).

[0532] Step 3. Preparation of compound 113a Compound 113a was prepared from compound 112a (1.08 g, 0.88 mmol) and (2-oxo-2-phenyl-1λ) using the same procedure as used for compound 91. 2 -ethyl)-D-glutamic acid (112 mg, 0.39 mmol). Yield: 600 mg, 62%.

[0533] Step 4. Preparation of compound 114a Compound 114a is prepared from compound 113a using the same procedure as used for compound 92.

[0534] Step 5. Preparation of compound 115a Compound 115a is 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%.

[0535] Step 6. Preparation of compound 116a Compound 116a is prepared from compound 115a (8 g, 6.3 mmol) using the same procedure as used for compound 11a. Yield: 3.23 g, 39%.

[0536] Step 7. Preparation of compound 117a Compound 117a was prepared by the same procedure used for compound 95 from compound 116a (3.23 g, 2.45 mmol) and (2-oxo-2-phenyl-1λ 2 -ethyl)glutamic acid (192 mg, 1.1 mmol). Yield: 2.22 g, 34%.

[0537] Step 8. Preparation of compound 118a Compound 118a is prepared from compound 117a (2.22 g, 0.84 mmol) using the same procedure as used for compound 96. Yield: 2.02 g, 91%.

[0538] Step 9. Preparation of Conjugate 21a8 and Conjugate 221a Conjugates 218a and 22a1 are prepared from compound 128 and compound 114a or compound 118a using the same procedure as that used for compound 1.

[0539] Example 21. Synthesis of Conjugate 224 [ka]

[0540] Step 1. Preparation of Compound 224 Conjugate 224 was prepared from compound 96 and compound 130 using the same procedure as used for compound 1.

[0541] Example 21a. Synthesis of Conjugate 224b [ka]

[0542] Step 1. Preparation of compound 224b Conjugate 224b is prepared from compound 96b and compound 130 using the same procedure as used for compound 1.

[0543] Example 22 Synthesis of Conjugate 231 [ka] [ka]

[0544] 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%.

[0545] Step 2 Preparation of compound 226 Compound 226 was prepared in the same manner as 14. Yield 1.22 g, 78%.

[0546] 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%.

[0547] Step 4 Preparation of compound 228 Compound 228 was prepared in the same manner as 14. Yield 460 mg, quantitative.

[0548] 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%.

[0549] Step 6 Preparation of compound 231 Conjugate 231 was prepared using the same procedure as that used for compound 1.

[0550] Example 22a Synthesis of Conjugate 231a [ka] [ka]

[0551] Step 1 Preparation of compound 225a Compound 225a 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%.

[0552] Step 2 Preparation of compound 226a Compound 226a is prepared in the same manner as 14. Yield 1.22 g, 78%.

[0553] Step 3 Preparation of compound 227a Compound 227a was prepared from Z-glutamic acid (108 mg, 0.38 mmol) and 226a (1.22 g, 0.92 mmol) in the same manner as 89. Yield 471 mg, 45%.

[0554] Step 4 Preparation of compound 228a Compound 228a is prepared in the same manner as 14. Yield 460 mg, quantitative.

[0555] Step 5 Preparation of compound 229a Compound 229a was prepared from 228a (460 mg, 0.17 mmol) and 128 (125 mg, 0.19 mmol) in the same manner as 89. Yield 365 mg, 66%.

[0556] Step 6 Preparation of compound 231a Conjugate 231a is prepared using the same procedure as that used for compound 1.

[0557] Example 22b Synthesis of Conjugate 231b [ka] [ka]

[0558] Step 1 Preparation of compound 225b Compound 225b 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%.

[0559] Step 2 Preparation of compound 226b Compound 226b is prepared in the same manner as 14. Yield 1.22 g, 78%.

[0560] Step 3 Preparation of compound 227b Compound 227b was prepared from Z-glutamic acid (108 mg, 0.38 mmol) and 226b (1.22 g, 0.92 mmol) in the same manner as 89. Yield 471 mg, 45%.

[0561] Step 4 Preparation of compound 228b Compound 228b is prepared in the same manner as 14. Yield 460 mg, quantitative.

[0562] Step 5 Preparation of compound 229b Compound 229b was prepared from 228b (460 mg, 0.17 mmol) and 128 (125 mg, 0.19 mmol) in the same manner as 89. Yield 365 mg, 66%.

[0563] Step 6 Preparation of compound 231b Conjugate 231b is prepared using the same procedure as that used for compound 1.

[0564] Example 23. Synthesis of Conjugate 233 [ka]

[0565] 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%.

[0566] Step 2. Preparation of Compound 233 Compound 233 was prepared from compound 232 using the same procedure as used for compound 1.

[0567] Example 24. Synthesis of Conjugate 235 [ka]

[0568] 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%.

[0569] Step 2. Preparation of Compound 235 Compound 235 was prepared from compound 234 using the same procedure as used for compound 1.

[0570] Example 25 Synthesis of Conjugate 320 [ka]

[0571] Step 1. Preparation of racemic (cis) 5-benzyl-3a,6a-dimethyltetrahydro-1H-furo[3,4-c]pyrrole-1,3(3aH)-dione 301 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) was added trifluoroacetic acid (75 μL) slowly. The solution was stirred overnight, allowing the ice bath to melt and gradually warm to room temperature. 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 column chromatography on silica gel (gradient: 20% ethyl acetate in hexane → 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%).

[0572] Step 2. Preparation of racemic (cis)(1-benzyl-3,4-dimethylpyrrolidine-3,4-diyl)dimethanol 302 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 added lithium aluminum hydride pellets (1.5 g, 40 mmol) slowly in three portions. The solution was stirred overnight, allowing the solution 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 5 M 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).

[0573] Step 3. Preparation of racemic (cis)(3,4-dimethylpyrrolidine-3,4-diyl)dimethanol 303 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 on wet activated carbon (1 g). The solution was vigorously stirred under a hydrogen atmosphere for 16 hours. 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%).

[0574] Step 4. Preparation of racemic (cis) methyl 10-(3,4-bis(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoate 304 A solution of 3 (1.3 g, 8.2 mmol) and monomethyl sebacate (1.8 g, 8.2 mmol) in CHCl (100 mL) was treated with HBTU (3.41 g, 9.02 mmol) and Hunig's base (5.71 mL, 32.8 mmol). After stirring overnight, the mixture was washed with NaHCO (saturated aqueous solution), water, and brine, then dried (MgSO), filtered, and concentrated. The crude material was chromatographed (gradient: 0% CHOH-CHCl → 20%) to give 4 (1.8 g, 61%).

[0575] Step 5. Preparation of racemic (cis)methyl 10-(3-((bis(4-methoxyphenyl)(phenyl)-methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoate 305 A solution of 304 (1.8 g, 5.0 mmol) and 4,4'-dimethoxytrityl chloride (1.7 g, 5.0 mmol) in pyridine (180 mL) was stirred overnight. The pyridine was then removed under reduced pressure, and the crude material was chromatographed (gradient: 0% CHOH-CHCl to 10%) to give 5 (1.4 g, 42%) as a yellow oil.

[0576] Step 6. Preparation of racemic (cis) lithium 10-(3-((bis(4-methoxyphenyl)-(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoate 306 To a solution of compound 305 (3.0 g, 4.6 mmol) in THF (50 mL) and water (50 mL) was added lithium hydroxide (121 mg, 5.0 mmol). The solution was stirred at room temperature for 4 hours and then concentrated to remove the THF. The remaining aqueous solution was lyophilized overnight to give a pale pink solid (2.9 g, quantitative yield). Compound 306 was prepared as a mixture of two cis-diastereomers.

[0577] [ka] D-Galactosamine hydrochloride (250 g, 1.16 mol) in pyridine (1.5 L) was treated with acetic anhydride (1.25 L, 13.2 mol) over 45 min. After stirring overnight, the reaction mixture was divided into three 1 L portions. Each 1 L portion was poured into 3 L of ice water and mixed for 1 h. After mixing, the solid was isolated by filtration, combined, frozen in liquid nitrogen, and lyophilized for 5 days to give peracetylated galactosamine 7 (369.4 g, 82%) as a white solid. Rf (0.58, 10% MeOH-CH2Cl2).

[0578] [ka]

[0579] Step 1 Preparation of compound 309 A solution of 2-[2-(2-chloroethoxy)]ethanol 308 (100 g, 593 mmol) in water (1 L) was treated with NaN (77 g, 1.19 mol) and heated (90 °C). After stirring (72 h), the solution was cooled (room temperature) and extracted (4 times) with CHCl. ​​The organic solutions were combined, washed with brine, dried (MgSO), filtered, concentrated, and used without further treatment. Compound 9 (88.9 g, 86%) was obtained as a pale yellow oil.

[0580] Step 2 Preparation of compound 310 A solution of 7 (2.76 g, 7.1 mmol) and 309 (1.37 g, 7.8 mmol) in 1,2-dichloroethane (40 mL) was treated with Sc(OTf) (174 mg, 0.36 mmol) and heated (85 °C). After stirring (2 h), the mixture was cooled (room temperature) and quenched by the addition of TEA (4 mL), and the mixture was concentrated. Chromatography of the crude material afforded 310 (3.03 g, 85%) as a pale yellow foam.

[0581] Step 3 Preparation of compound 311 A solution of 310 (3.02 g, 5.99 mmol) and Pd / C (300 mg, 10% Pd loading - wet support) in EtOAc (30 mL) was treated with TFA (576 μL, 7.5 mmol). The reaction mixture was purged with hydrogen gas (45 min) followed by nitrogen gas (10 min) and then filtered through celite. The filtrate was concentrated and chromatographed to give 311 (2.67 g, 75%) as a brown foam.

[0582] [ka]

[0583] Step 1. Dimethyl 5-(2-((2-oxo-2-phenyl-1λ) 2 Preparation of (ethyl)amino)acetamido)-isophthalate 312 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), and 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 x 100 mL), saturated sodium bicarbonate (1 x 100 mL), and brine (2 x 100 mL), respectively. Dimethyl 5-(2-((2-oxo-2-phenyl-1λ) was obtained by drying over magnesium sulfate, filtering, and concentrating to dryness. 2 (-ethyl)amino)-acetamido)isophthalate as a colorless solid (7.2 g, 79%).

[0584] Step 2.5-(2-((2-oxo-2-phenyl-1λ) 2 Preparation of (-ethyl)amino)acetamido)isophthalic acid 313 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 1 M NaOH (25 mL). The solution was stirred at room temperature for 2 hours and then concentrated to remove the THF and MeOH. The remaining aqueous solution was diluted with water (75 mL), cooled on an ice-water bath, and acidified to pH = 1 with 6 M 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). The solution was stirred at room temperature for 2 hours and then concentrated to remove the THF and MeOH. The remaining aqueous solution was diluted with water (75 mL), cooled on an ice-water bath, and acidified to pH = 1 with 6 M HCl. The solid was filtered and washed with water (3 x 100 mL). 2 (-ethyl)amino)acetamido)-isophthalic acid (6.9 g, quantitative yield) was obtained.

[0585] [ka]

[0586] Step 1 Preparation of compound 314 A solution of 313 (2.09 g, 5.6 mmol) and 311 (8.34 g, 14.07 mmol) in CHCl (150 mL) was treated with HBTU (6.4 g, 16.9 mmol) and Hunig's base (7.35 mL, 42.2 mmol). After stirring (overnight), the reaction mixture was poured into NaHCO (saturated aqueous solution), washed with water and brine, dried (MgSO), filtered, and concentrated. The crude material was chromatographed (gradient 1 to 12% CHOH-CHCl) to give 6 (3.97 g, 55%) as a pale yellow foam.

[0587] Step 2 Preparation of compound 315 Compound 314 (3.92 g, 3.07 mmol), Pd / C (400 mg, 10% loading—wet support), and trifluoroacetic acid (308 μL, 4 mmol) were purged with H. After stirring under H overnight, the mixture was purged with N (15–20 min), filtered through Celite, and concentrated. Chromatography of the crude material afforded 7 (3.36 g, 86%) as a white-cream foam.

[0588] Step 3 Preparation of compound 316 Compound 316 was prepared from Z-glutamic acid (306 mg, 1.09 mmol) and 315 (3.3 g, 2.6 mmol) in the same manner as 314. Yield 1.66 g, 60%.

[0589] Step 4 Preparation of compound 317 Compound 317 was prepared in the same manner as 315. Yield 1.65 g, quantitative.

[0590] [ka]

[0591] Step 1 Preparation of compound 318 A solution of 317 (1.91 g, 0.75 mmol) in CHCl (100 mL) was first treated with Hunig's base (392 μL, 2.25 mmol), then with 6 (a mixture of two cis-diastereomers, 509 mg, 0.79 mmol), followed by HBTU (356 mg, 0.94 mmol). After stirring (overnight), the solution was poured into NaHCO (saturated aqueous solution), washed with water and brine, dried (MgSO), filtered, and concentrated. Chromatography of the crude material afforded 318 (1.19 g, 52%) as a white foam.

[0592] Step 2 Preparation of compound 319 A solution of 318 (1.19 g, 0.39 mmol) in 1,2-dichloroethane (100 mL) was treated with TEA (542 μL, 3.9 mmol), DMAP (238 mg, 1.95 mmol), and succinic anhydride (195 mg, 1.95 mmol) and heated (85 °C). After stirring (2.5 h), the solution was removed from the heat, treated with CHOH (10 mL), and stirred (1 h). After stirring, the mixture was poured into NaHCO (saturated aqueous solution), washed with brine, dried (MgSO), filtered, and concentrated. The resulting residue was used without further treatment. Yield = 1.4 g, quantitative.

[0593] Step 3 Preparation of Complex 320 Succinate 319 was loaded onto 1000 Å LCAA (long-chain amino alkyl) CPG (controlled pore glass) using standard amide coupling chemistry. A solution of diisopropylcarbodiimide (52.6 μmol), N-hydroxysuccinimide (0.3 mg, 2.6 μmol), and pyridine (10 μL) in anhydrous acetonitrile (0.3 mL) was added to 319 (20.6 mg, 8 μmol) in anhydrous dichloromethane (0.2 mL). This mixture was added to LCAA CPG (183 mg). The suspension was gently mixed overnight at room temperature. Upon disappearance of 319 (HPLC), the reaction mixture was filtered, and the CPG was washed with 1 mL each of dichloromethane, acetonitrile, 5% acetic anhydride / 5% N-methylimidazole / 5% pyridine in THF, followed by THF, acetonitrile, and dichloromethane. The CPG was then dried overnight under high vacuum. The loading was 19 μmol / g, as determined by a standard DMTr assay by UV / Vis (504 nm). The resulting GalNAc-loaded CPG solid support was used in automated oligonucleotide synthesis using standard procedures. Deprotection of the nucleotide followed by cleavage from the solid support (with simultaneous deprotection of the galactosamine acetate) gave GalNAc-oligonucleotide conjugate 320.

[0594] Example 26 Synthesis of Conjugate 520 [ka]

[0595] Step 1. Preparation of racemic (cis) 5-benzyl-3a,6a-dimethyltetrahydro-1H-furo[3,4-c]pyrrole-1,3(3aH)-dione 301 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) was added trifluoroacetic acid (75 μL) slowly. The solution was stirred overnight, allowing the ice bath to melt and gradually warm to room temperature. 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 column chromatography on silica gel (gradient: 20% ethyl acetate in hexane → 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%).

[0596] Step 2. Preparation of racemic (cis)(1-benzyl-3,4-dimethylpyrrolidine-3,4-diyl)dimethanol 302 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 added lithium aluminum hydride pellets (1.5 g, 40 mmol) slowly in three portions. The solution was stirred overnight, allowing the solution 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 5 M 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).

[0597] Step 3. Preparation of racemic (cis)(3,4-dimethylpyrrolidine-3,4-diyl)dimethanol 303 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 on wet activated carbon (1 g). The solution was vigorously stirred under a hydrogen atmosphere for 16 hours. 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%).

[0598] Step 4. Preparation of racemic (cis)methyl 10-(3,4-bis(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoate 304 A solution of 3 (1.3 g, 8.2 mmol) and monomethyl sebacate (1.8 g, 8.2 mmol) in CHCl (100 mL) was treated with HBTU (3.41 g, 9.02 mmol) and Hunig's base (5.71 mL, 32.8 mmol). After stirring overnight, the mixture was washed with NaHCO (saturated aqueous solution), water, and brine, then dried (MgSO), filtered, and concentrated. The crude material was chromatographed (gradient: 0% CHOH-CHCl → 20%) to give 4 (1.8 g, 61%).

[0599] Step 5. Preparation of racemic (cis)methyl 10-(3-((bis(4-methoxyphenyl)(phenyl)-methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoate 305 A solution of 304 (1.8 g, 5.0 mmol) and 4,4'-dimethoxytrityl chloride (1.7 g, 5.0 mmol) in pyridine (180 mL) was stirred overnight. The pyridine was then removed under reduced pressure, and the crude material was chromatographed (gradient: 0% CHOH-CHCl to 10%) to give 5 (1.4 g, 42%) as a yellow oil.

[0600] Step 6. Preparation of racemic (cis) lithium 10-(3-((bis(4-methoxyphenyl)-(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoate 306 To a solution of compound 305 (3.0 g, 4.6 mmol) in THF (50 mL) and water (50 mL) was added lithium hydroxide (121 mg, 5.0 mmol). The solution was stirred at room temperature for 4 hours and then concentrated to remove the THF. The remaining aqueous solution was lyophilized overnight to give a pale pink solid (2.9 g, quantitative yield). Compound 306 was prepared as a mixture of two cis-diastereomers.

[0601] [ka] Galactosamine hydrochloride (250 g, 1.16 mol) in pyridine (1.5 L) was treated with acetic anhydride (1.25 L, 13.2 mol) over 45 min. After stirring overnight, the reaction mixture was divided into three 1 L portions. Each 1 L portion was poured into 3 L of ice water and mixed for 1 h. After mixing, the solid was filtered, isolated, combined, frozen in liquid nitrogen, and lyophilized for 5 days to give peracetylated galactosamine 507 (369.4 g, 82%) as a white solid. Rf (0.58, 10% MeOH-CH2Cl2).

[0602] [ka]

[0603] Step 1 Preparation of compound 509 A solution of 2-[2-(2-chloroethoxy)]ethanol 508 (100 g, 593 mmol) in water (1 L) was treated with NaN (77 g, 1.19 mol) and heated (90 °C). After stirring (72 h), the solution was cooled (room temperature) and extracted (4 times) with CHCl. ​​The organic solutions were combined, washed with brine, dried (MgSO), filtered, concentrated, and used without further treatment. Compound 509 (88.9 g, 86%) was obtained as a pale yellow oil.

[0604] Step 2 Preparation of compound 510 A solution of 507 (2.76 g, 7.1 mmol) and 509 (1.37 g, 7.8 mmol) in 1,2-dichloroethane (40 mL) was treated with Sc(OTf) (174 mg, 0.36 mmol) and heated (85 °C). After stirring (2 h), the mixture was cooled (room temperature) and quenched by the addition of TEA (4 mL) and concentrated. Chromatography of the crude material gave 510 (3.03 g, 85%) as a pale yellow foam.

[0605] Step 3 Preparation of compound 511 A solution of 510 (3.02 g, 5.99 mmol) and Pd / C (300 mg, 10% Pd loading - wet support) in EtOAc (30 mL) was treated with TFA (576 μL, 7.5 mmol). The reaction mixture was purged with hydrogen gas (45 min) followed by nitrogen gas (10 min) and then filtered through celite. The filtrate was concentrated and chromatographed to give 511 (2.67 g, 75%) as a brown foam.

[0606] [ka]

[0607] Step 1. Dimethyl 5-(2-((2-oxo-2-phenyl-1λ) 2 Preparation of (ethyl)amino)acetamido)-isophthalate 312 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), and 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 x 100 mL), saturated sodium bicarbonate (1 x 100 mL), and brine (2 x 100 mL), respectively. Dimethyl 5-(2-((2-oxo-2-phenyl-1λ) was obtained by drying over magnesium sulfate, filtering, and concentrating to dryness. 2 (-ethyl)amino)-acetamido)isophthalate as a colorless solid (7.2 g, 79%).

[0608] Step 2.5-(2-((2-oxo-2-phenyl-1λ) 2 Preparation of (-ethyl)amino)acetamido)isophthalic acid 313 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 1 M NaOH (25 mL). The solution was stirred at room temperature for 2 hours and then concentrated to remove the THF and MeOH. The remaining aqueous solution was diluted with water (75 mL), cooled on an ice-water bath, and acidified to pH = 1 with 6 M 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). The solution was stirred at room temperature for 2 hours and then concentrated to remove the THF and MeOH. The remaining aqueous solution was diluted with water (75 mL), cooled on an ice-water bath, and acidified to pH = 1 with 6 M HCl. The solid was filtered and washed with water (3 x 100 mL). 2 (-ethyl)amino)acetamido)-isophthalic acid (6.9 g, quantitative yield) was obtained.

[0609] [ka]

[0610] Step 1 Preparation of compound 514 A solution of 313 (2.09 g, 5.6 mmol) and 511 (8.34 g, 14.07 mmol) in CHCl (150 mL) was treated with HBTU (6.4 g, 16.9 mmol) and Hunig's base (7.35 mL, 42.2 mmol). After stirring (overnight), the reaction mixture was poured into NaHCO (saturated aqueous solution), washed with water and brine, dried (MgSO), filtered, and concentrated. The crude material was chromatographed (gradient 1 → 12% CHOH-CHCl) to give 6 (3.97 g, 55%) as a pale yellow foam.

[0611] Step 2 Preparation of compound 515 Compound 514 (3.92 g, 3.07 mmol), Pd / C (400 mg, 10% loading - wet support), and trifluoroacetic acid (308 μL, 4 mmol) were purged with H. After stirring under H overnight, the mixture was purged with N for 15–20 min, filtered through Celite, and concentrated. Chromatography of the crude material gave 7 (3.36 g, 86%) as a white-to-cream foam.

[0612] Step 3 Preparation of compound 516 Compound 516 is prepared from Z-glutamic acid (306 mg, 1.09 mmol) and 515 (3.3 g, 2.6 mmol) in the same manner as 514. Yield 1.66 g, 60%.

[0613] Step 4 Preparation of compound 517 Compound 517 is prepared in the same manner as 515. Yield 1.65 g, quantitative.

[0614] [ka]

[0615] Step 1 Preparation of compound 518 A solution of 517 (1.91 g, 0.75 mmol) in CHCl (100 mL) was first treated with Hunig's base (392 μL, 2.25 mmol), then with 306 (a mixture of two cis-diastereomers, 509 mg, 0.79 mmol), followed by HBTU (356 mg, 0.94 mmol). After stirring (overnight), the solution was poured into NaHCO (saturated aqueous solution), washed with water and brine, dried (MgSO), filtered, and concentrated. Chromatography of the crude material afforded 518 (1.19 g, 52%) as a white foam.

[0616] Step 2 Preparation of compound 519 A solution of 518 (1.19 g, 0.39 mmol) in 1,2-dichloroethane (100 mL) was treated with TEA (542 μL, 3.9 mmol), DMAP (238 mg, 1.95 mmol), and succinic anhydride (195 mg, 1.95 mmol) and heated (85 °C). After stirring (2.5 h), the solution was removed from the heat, treated with CHOH (10 mL), and stirred (1 h). After stirring, the mixture was poured into NaHCO (saturated aqueous solution), washed with brine, dried (MgSO), filtered, and concentrated. The resulting residue was used without further treatment. Yield = 1.4 g, quantitative.

[0617] Step 3 Preparation of Complex 520 Succinate 519 was loaded onto 1000 Å LCAA (long-chain amino alkyl) CPG (controlled pore glass) using standard amide coupling chemistry. A solution of diisopropylcarbodiimide (52.6 μmol), N-hydroxysuccinimide (0.3 mg, 2.6 μmol), and pyridine (10 μL) in anhydrous acetonitrile (0.3 mL) was added to 519 (20.6 mg, 8 μmol) in anhydrous dichloromethane (0.2 mL). This mixture was added to LCAA CPG (183 mg). The suspension was gently mixed overnight at room temperature. Upon disappearance of 519 (HPLC), the reaction mixture was filtered, and the CPG was washed with 1 mL each of dichloromethane, acetonitrile, 5% acetic anhydride / 5% N-methylimidazole / 5% pyridine in THF, followed by THF, acetonitrile, and dichloromethane. The CPG is then dried overnight under high vacuum. The loading was 19 μmol / g, as determined by a standard DMTr assay by UV / Vis (504 nm). The resulting GalNAc-loaded CPG solid support is used in automated oligonucleotide synthesis using standard procedures. Deprotection of the nucleotide followed by cleavage from the solid support (with simultaneous deprotection of the galactosamine acetate) yields GalNAc-oligonucleotide conjugate 520.

[0618] Example 27 Synthesis of target-directed nucleic acid complexes Schemes 101-122 below illustrate the preparation of intermediate compounds that can be used in the preparation of conjugates of Formula I. The intermediate compounds and synthetic processes illustrated in Schemes 1-22 are embodiments of the present invention. [ka]

[0619] Step 1. Preparation of (3aR,6aS)-5-benzyl-3a,6a-dimethyltetrahydro-1H-furo[3,4-c]pyrrole-1,3(3aH)-dione 601 To a cold (0 °C) solution of 3,4-dimethylfuran-2,5-dione (40 g, 317 mmol) and N-benzyl-1-methoxy-N-((trimethylsilyl)methyl)methanamine (94.1 g, 396.5 mmol) in DCM (600 mL) was added trifluoroacetic acid (732 μL) slowly. The solution was allowed to warm to room temperature slowly by stirring overnight. The reaction mixture was concentrated to dryness, dissolved in EtOAc (500 mL), washed with saturated sodium bicarbonate (2 x 500 mL), dried over magnesium sulfate, filtered, and concentrated to dryness. Purification by column chromatography on silica gel (gradient: 20% ethyl acetate in hexanes 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 (53.7 g, 65%). Rf 0.85 40% EtOAc-hexanes

[0620] Step 2. Preparation of ((3R,4S)-1-benzyl-3,4-dimethylpyrrolidine-3,4-diyl)dimethanol 602 To a cold (0 °C) solution of (3aR,6aS)-5-benzyl-3a,6a-dimethyltetrahydro-1H-furo[3,4-c]pyrrole-1,3(3aH)-dione (53.7 g, 205.7 mmol) in anhydrous diethyl ether (750 mL) was added lithium aluminum hydride pellets (17.6 g, 463 mmol) gradually in portions over the afternoon. The solution was stirred overnight, allowing the solution to warm to room temperature as the ice-water bath melted. Upon completion, the reaction was cooled to 0 °C and very slowly quenched with 25 mL of 5 M NaOH followed by 12 mL of water. After stirring for 30 minutes, magnesium sulfate was added and the mixture was filtered. The filtrate was concentrated to give ((3R,4S)-1-benzyl-3,4-dimethylpyrrolidine-3,4-diyl)dimethanol as a colorless oil (33.6 g, 65%). Rf 0.25 10% CH3OH-CH2Cl2

[0621] Step 3. Preparation of ((3R,4S)-3,4-dimethylpyrrolidine-3,4-diyl)dimethanol 603 To a solution of ((3R,4S)-1-benzyl-3,4-dimethylpyrrolidine-3,4-diyl)dimethanol (40.1 g, 161 mmol) in methanol (300 ml) was added 10% palladium on wet activated carbon (4 g). The solution was stirred vigorously under a hydrogen atmosphere for 16 hours. 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 (24 g, 94%). Rf 0.05 10% CH3OH-CH2Cl2

[0622] Step 4. Preparation of methyl 10-((3R,4S)-3,4-bis(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoate 604 A solution of 3 (24 g, 151 mmol) and monomethyl sebacate (34.2 g, 159 mmol) in CHCl (1 L) was treated with HBTU (62.9 g, 166 mmol) and Hunig's base (105 mL, 604 mmol). After stirring overnight, the mixture was washed with NaHCO (saturated aqueous solution), water, and brine, then dried (MgSO), filtered, and concentrated. The crude material was chromatographed (gradient: 0% CHOH-CHCl → 20%) to give 604 (41.5 g, 77%). Rf 0.55 10% CHOH-CHCl

[0623] Step 5. Preparation of methyl 10-(3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoate 605 A solution of 604 (41.5 g, 116 mmol) and 4,4'-dimethoxytrityl chloride (38.8 g, 116 mmol) in pyridine (400 mL) was stirred overnight. The pyridine was then removed under reduced pressure, and the crude material was chromatographed (gradient: 0% CHOH-CHCl to 10%) to give 605 (29.5 g, 39%) as a yellow oil. Rf 0.5 5% CHOH-CHCl

[0624] Step 6. Preparation of lithium 10-(3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoate 606 To a solution of compound 605 (29.5 g, 45 mmol) in THF (250 mL) and water (250 mL) was added lithium hydroxide (1.19 g, 50 mmol). The solution was stirred at room temperature for 18 hours and then concentrated to remove the THF. The remaining aqueous solution was lyophilized overnight to give 606 as a pale purple solid (28.5 g, 98%). Rf 0.56 10% CHOH-CHCl

[0625] [ka]

[0626] Step 1. Preparation of methyl 12-aminododecanoate 608 12-Aminoundecanoic acid 607 (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 under reduced pressure, and the residue was taken up in MTBE and cooled 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 608.

[0627] Step 2. Preparation of methyl 12-(10-(3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanamido) dodecanoate 609 Lithium 10-(3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoate (606) (2 g, 3.1 mmol), methyl 12-aminododecanoate (608) (778 mg, 3.1 mmol), HBTU (1.2 g, 3.1 mmol), and TEA (1.4 mL, 10 mmol) were stirred in DCM at room temperature overnight. The precipitate was removed by filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography (5% MeOH, DCM). TLC analysis revealed the presence of two closely spaced spots of identical mass, which were assigned as geometric isomers. These were pooled together to give methyl 12-(10-(3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanamido)dodecanoate (609) in quantitative fashion.

[0628] Step 3. Preparation of lithium 12-(10-(3-((bis(4-methoxyphenyl)(phenyl)methoxy)-methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanamido)-dodecanoate 610 Methyl 12-(10-(3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanamido) dodecanoate 609 (3.1 mmol) was stirred with LiOH (88 mg, 3.7 mmol) in THF:HO (50:50) at room temperature overnight. The reaction was monitored by TLC, and the THF was removed under reduced pressure. The aqueous solution was frozen in liquid N and lyophilized for 48 hours to quantitatively afford lithium 12-(10-(3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanamido) dodecanoate 610.

[0629] [ka]

[0630] Step 1. Preparation of 2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate 612 A cooled (0 °C) solution of tetraethylene glycol (611) (934 g, 4.8 mol) in THF (175 mL) and aqueous NaOH (5 M, 145 mL) was 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 x 500 mL). The combined extracts were washed with water and brine, dried (MgSO), filtered, and concentrated to give 2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (612) (140 g, 84%) as a pale yellow oil. f (0.57, 10% MeOH-CH2Cl2).

[0631] Step 2. Preparation of 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethan-1-ol 613 A solution of 612 (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), washed with brine (3 x 500 mL), 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 613 (65 g, 74%) as a yellow oil. f (0.56, 10% MeOH-CH2Cl2).

[0632] [ka]

[0633] Step 1. Preparation of (3R,4R,5R,6R)-6-(hydroxymethyl)-3-(((E)-4-methoxybenzylidene)amino)tetrahydro-2H-pyran-2,4,5-triol 616 D-Galactosamine HCl (614) (9 g, 41.7 mmol) was stirred in 1 M NaOH solution at room temperature. Anisaldehyde (51 mL, 420 mmol) was added, and the reaction mixture was stirred vigorously until solidification occurred. The solid mixture was kept at 4 °C for 16 h. Ice-cold water (200 mL) was added, and the resulting solid was collected by filtration and washed with ice-cold EtOH / EtO (1:1). The solid was dried to constant weight to give (3R,4R,5R,6R)-6-(hydroxymethyl)-3-(((E)-4-methoxybenzylidene)amino)tetrahydro-2H-pyran-2,4,5-triol (616) (9.81 g, 78%).

[0634] Step 2. Preparation of (3R,4R,5R,6R)-6-(acetoxymethyl)-3-(((E)-4-methoxybenzylidene)amino)tetrahydro-2H-pyran-2,4,5-triyl triacetate 617 (3R,4R,5R,6R)-6-(hydroxymethyl)-3-(((E)-4-methoxybenzylidene)amino)tetrahydro-2H-pyran-2,4,5-triol (616) (9.81 g, 30 mmol) was stirred in pyridine at 0 °C. Acetic anhydride (34 mL) was added followed by DMAP (100 mg, catalytic) and the reaction was allowed to warm gradually to room temperature by stirring for 16 h. The resulting solution was poured onto crushed ice and kept at 4 °C for 16 h. The reaction was extracted with EtOAc (3x), and the organic solutions were combined, washed with HO and brine, dried (NaSO), and concentrated under reduced pressure to give (3R,4R,5R,6R)-6-(acetoxymethyl)-3-(((E)-4-methoxybenzylidene)amino)tetrahydro-2H-pyran-2,4,5-triyl triacetate (617) (6.0 g, 43%).

[0635] Step 3. Preparation of (3R,4R,5R,6R)-6-(acetoxymethyl)-3-aminotetrahydro-2H-pyran-2,4,5-triyl triacetate hydrochloride 618 (3R,4R,5R,6R)-6-(acetoxymethyl)-3-(((E)-4-methoxybenzylidene)amino)tetrahydro-2H-pyran-2,4,5-triyl triacetate (617) (6.0 g, 43%) was heated at reflux in acetone (300 mL). HCl (aq) (5N, 3.0 mL) was added and the reaction was stirred for 15 minutes. After cooling, EtO (400 mL) was added and the reaction was kept at 4 °C for 16 hours. The resulting solid was collected by filtration and washed twice with ice-cold EtO. The solid was dried to constant weight to give (3R,4R,5R,6R)-6-(acetoxymethyl)-3-aminotetrahydro-2H-pyran-2,4,5-triyl triacetate hydrochloride (618) (4.17 g, 84.4%).

[0636] Step 4a. Preparation of (3R,4R,5R,6R)-6-(acetoxymethyl)-3-(2,2,2-trifluoroacetamido)tetrahydro-2H-pyran-2,4,5-triyl triacetate 619a (3R,4R,5R,6R)-6-(acetoxymethyl)-3-aminotetrahydro-2H-pyran-2,4,5-triyl triacetate hydrochloride (618) (13.5 g, 35.2 mmol) and TEA (7.83 g, 77.4 mmol) were stirred in DCM at room temperature. TFAA (8.13 g, 38.7 mmol) in DCM was added dropwise, and the reaction was stirred for 1 h. The reaction was diluted with DCM and washed successively with 1 M HCl, saturated NaHCO, water, and brine, dried (NaSO), and concentrated under reduced pressure. The residue was purified by automated flash chromatography (5% MeOH / DCM) to give (3R,4R,5R,6R)-6-(acetoxymethyl)-3-(2,2,2-trifluoroacetamido)tetrahydro-2H-pyran-2,4,5-triyl triacetate (619a) (9.64 g, 61.8%), confirmed by MS (ESI+ve).

[0637] Step 4b. Preparation of (3R,4R,5R,6R)-6-(acetoxymethyl)-3-propionamidotetrahydro-2H-pyran-2,4,5-triyl triacetate 619b This compound was prepared in a manner similar to (3R,4R,5R,6R)-6-(acetoxymethyl)-3-(2,2,2-trifluoroacetamido)tetrahydro-2H-pyran-2,4,5-triyl triacetate (619a). In this preparation, propionic anhydride was used instead of TFAA to give (3R,4R,5R,6R)-6-(acetoxymethyl)-3-propionamidotetrahydro-2H-pyran-2,4,5-triyl triacetate (619b) (1.2 g, 85.3%). Product confirmation was confirmed by MS (ESI+ve).

[0638] Step 4c. Preparation of (3R,4R,5R,6R)-6-(acetoxymethyl)-3-(2,2-difluoropropanamido)tetrahydro-2H-pyran-2,4,5-triyl triacetate 619c (3R,4R,5R,6R)-6-(acetoxymethyl)-3-aminotetrahydro-2H-pyran-2,4,5-triyl triacetate hydrochloride (618) (15.34 g, 39.98 mmol), 2,2-difluoropropionic acid (4.4 g, 39.98 mmol), HATU (24.37 g, 64 mmol), and TEA (12.14 g, 120 mmol) were stirred in DMF at room temperature for 16 h. The reaction was partitioned between EtOAc and water. The organic solution was separated, washed successively with 1 M HCl, saturated NaHCO, water, and brine, dried (NaSO), and concentrated under reduced pressure. The residue was purified by automated flash chromatography (3% MeOH / DCM) to give (3R,4R,5R,6R)-6-(acetoxymethyl)-3-(2,2-difluoropropanamido)-tetrahydro-2H-pyran-2,4,5-triyl triacetate (619c) (15.8 g, 90%), confirmed by MS (ESI+ve).

[0639] [ka]

[0640] Step 1. Preparation of benzyl (2-(2-(2-hydroxyethoxy)ethoxy)ethyl)carbamate 622 A solution of amino alcohol 620 (313.6 g, 2.1 mol) in THF (3.5 L) was slowly treated with N-(benzyloxycarbonyloxy)succinimide 621 (550 g, 2.21 mol). Upon reaction completion (18 h), the THF was removed under reduced pressure, and the residue was dissolved in CHCl (2.5 L) and washed with equal volumes of 1 M HCl, saturated aqueous NaHCO, HO, and brine. The organic extract was dried (MgSO), filtered, and concentrated. Chromatography of the crude material (600 g) (4 kg silica; 1 → 12% CHOH-CHCl) afforded HO-Trig-NHZ 622 (468 g, 78%) as a clear, yellow, viscous oil.

[0641] Step 2. Preparation of (2R,3R,4R,5R)-5-acetamido-2-(acetoxymethyl)-6-((3-oxo-1-phenyl-2,7,10-trioxa-4-azadodecan-12-yl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate 623 A heterogeneous mixture of galactosamine pentaacetate (715.2 g, 1.84 mol) and HO-Trig-NHZ (622) (400 g, 1.41 mol) in 1,2-dichloroethane (10 L) was treated with 5 mol% Sc(OTf) (34.6 g, 70.5 mmol) and heated (85 °C). After stirring (5.5 h), the solution became clear and homogeneous. The reaction was cooled and washed with NaHCO (sat. aq.), HCl (1 M), HO, and brine. The organic extract was dried (MgSO), filtered, and concentrated. The crude material (900 g) was treated with EtOAc (900 mL) to give a milky heterogeneous mixture, which was filtered through a coarse frit to remove residual pentaacetate. The filtrate was concentrated and the crude material was chromatographed (5 kg silica; 0→10% CH 3 OH-EtOAc) to give the glycosylated product (623) (751 g, 87%) as a light brown foam.

[0642] Step 3. Preparation of (2R,3R,4R,5R)-5-acetamido-2-(acetoxymethyl)-6-(2-(2-(2-((2,2,2-trifluoroacetyl)-l4-azaneyl)ethoxy)ethoxy)tetrahydro-2H-pyran-3,4-diyl diacetate 624 A solution containing Gal-trig-NHZ (623) (750 g, 1.22 mol), TFA (103.8 mL, 1.35 mol), and Pd / C (10%-wet support, 75 g) was purged with H. After vigorous stirring (4.5 h), the reaction mixture was purged with N (30 min), filtered through Celite, and concentrated. The resulting brown foam (712 g, 99%) was used in the next step without further treatment.

[0643] [ka] [ka]

[0644] Step 1. Preparation of 2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethan-1-ol 625 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethan-1-ol (613) (70.0 g, 318 mmol) was stirred in MeOH at room temperature. The reaction was hydrogenated with 10% PD-C (7 g) for 16 h. The reaction was filtered through celite and concentrated under reduced pressure to give 2-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethan-1-ol (625) (61.4 g, 100%), which was used without further purification. The product was characterized by MS (ESI+ve).

[0645] Step 2. Preparation of benzyl (2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl)-carbamate 627 2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethan-1-ol (625) (61.4 g, 318 mmol) was stirred in HO (500 mL) containing NaCO (50.51 g, 476 mmol) at 5 °C. Benzyl chloroformate (626) (65.0 g, 381 mmol) in THF (480 mL) was added dropwise and the reaction was stirred for 16 h, warming to room temperature. The THF was removed under reduced pressure and the aqueous layer was extracted (3 times) with EtOAc. The organic solutions were combined, dried (NaSO), concentrated under reduced pressure, and the residue was purified by automated flash chromatography (5% MeOH / DCM) to give benzyl (2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl)carbamate (627) (23.6 g, 22.7%). Product confirmation was confirmed by MS (ESI+ve).

[0646] Step 3. Preparation of benzyl (1,1-bis(4-methoxyphenyl)-1-phenyl-2,5,8,11-tetraoxatridecan-13-yl)carbamate 628 (2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl)carbamate (627) (23.6 g, 72.1 mmol) and TEA (7.7 g, 75.7 mmol) were stirred in DCM at room temperature. DMTr-Cl (25.65 g, 75.7 mmol) was added, and the reaction was stirred at room temperature for 2 h. The reaction was washed successively with saturated NaHCO, water, and brine, dried (NaSO), and concentrated under reduced pressure. The residue was purified by automated flash chromatography (50% EtOAc / hexanes) to give (1,1-bis(4-methoxyphenyl)-1-phenyl-2,5,8,11-tetraoxatridecan-13-yl)carbamate (v28) (25.5 g, 56.2%). Product confirmation was confirmed by MS (ESI+ve).

[0647] Step 4. Preparation of benzyl (1,1-bis(4-methoxyphenyl)-1-phenyl-2,5,8,11-tetraoxatridecan-13-yl)(methyl)carbamate 629 (1,1-Bis(4-methoxyphenyl)-1-phenyl-2,5,8,11-tetraoxatridecan-13-yl)carbamate (628) (25.5 g, 40.5 mmol) and MeI (46.0 g, 324 mmol) were stirred in anhydrous THF at 0 °C. NaH (60% dispersion in mineral oil) (2.92 g, 121.5 mmol) was added and the reaction was stirred at 0 °C and then at room temperature for 1 h. The reaction was partitioned between EtOAc and HO. The organic solution was separated, dried (NaSO), and concentrated under reduced pressure. The residue was purified by automated flash chromatography (50% EtOAc / hexanes) to give benzyl (1,1-bis(4-methoxyphenyl)-1-phenyl-2,5,8,11-tetraoxatridecan-13-yl)(methyl)carbamate (629) (26.06 g, 100%). Product characterization was confirmed by MS (ESI+ve).

[0648] Step 5. Preparation of benzyl (2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl)(methyl)carbamate 630 Benzyl (1,1-bis(4-methoxyphenyl)-1-phenyl-2,5,8,11-tetraoxatridecan-13-yl)(methyl)carbamate (629) (26.06 g, 40.5 mmol) was stirred in DCM at room temperature. TFA (5.1 g, 44.5 mmol) was added and stirred for 1 hour. Two additional equivalents of TFA were added and the reaction was stirred for 16 hours. The reaction was concentrated under reduced pressure, and the residue was purified by automated flash chromatography (5% MeOH / DCM) to give benzyl (2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl)(methyl)carbamate (630) (6.76 g, 48.9%). Product confirmation was confirmed by MS (ESI+ve).

[0649] Step 6. Preparation of (2R,3R,4R,5R)-5-acetamido-2-(acetoxymethyl)-6-((4-methyl-3-oxo-1-phenyl-2,7,10,13-tetraoxa-4-azapentadecan-15-yl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate 631 (2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl)(methyl)carbamate (630) (6.76 g, 19.8 mmol), (3R,4R,5R,6R)-3-acetamido-6-(acetoxymethyl)tetrahydro-2H-pyran-2,4,5-triyl triacetate (7.71 g, 19.8 mmol), and Sc(III)OTf (0.49 g, 1.0 mmol) were heated at reflux in DCE for 2 h. After cooling, the reaction was quenched with TEA, and the solution was washed successively with 1 M HCl, saturated NaHCO, water, and brine, dried (NaSO), and concentrated under reduced pressure. The residue was purified by automated flash chromatography to give (2R,3R,4R,5R)-5-acetamido-2-(acetoxymethyl)-6-((4-methyl-3-oxo-1-phenyl-2,7,10,13-tetraoxa-4-azapentadecan-15-yl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (631) (9.37 g, 70.6%). Product characterization was confirmed by MS (ESI+ve).

[0650] Step 7. Preparation of (2R,3R,4R,5R)-5-acetamido-2-(acetoxymethyl)-6-((1,1,1-trifluoro-3-methyl-2-oxo-6,9,12-trioxa-3l4-azatetradecan-14-yl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate 632 (2R,3R,4R,5R)-5-acetamido-2-(acetoxymethyl)-6-((4-methyl-3-oxo-1-phenyl-2,7,10,13-tetraoxa-4-azapentadecan-15-yl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (631) (9.37 g, 14.0 mmol) and TFA (1.76 g, 15.4 mmol) were stirred in MeOH at room temperature. The reaction mixture was hydrogenated over 10% Pd-C (1 g) for approximately 2 hours. The reaction mixture was filtered through Celite and concentrated under reduced pressure to give (2R,3R,4R,5R)-5-acetamido-2-(acetoxymethyl)-6-((1,1,1-trifluoro-3-methyl-2-oxo-6,9,12-trioxa-314-azatetradecan-14-yl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (632) (9.0 g, 98.9%). This product was used without further purification. Product characterization was confirmed by MS (ESI+ve).

[0651] Step 8. Preparation of (2R,2'R,3R,3'R,4R,4'R,5R,5'R)-(((4-nitro-1,2-phenylene)bis(2-methyl-1-oxo-5',8',11'-trioxa-2'-azatridecane-1,13-diyl))bis(oxy))bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate 634 (2R,3R,4R,5R)-5-acetamido-2-(acetoxymethyl)-6-((1,1,1-trifluoro-3-methyl-2-oxo-6,9,12-trioxa-314-azatetradecan-14-yl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (32) (4.5 g, 6.93 mmol), 4-nitrophthalic acid (33) (0.73 g, 3.46 mmol), HATU (8.45 g, 22.18 mmol), and TEA (4.21 g, 41.6 mmol) in DCM were stirred at room temperature for 16 h. The reaction mixture was diluted with DCM and washed successively with 1 M HCl, saturated NaHCO, water, and brine, dried (NaSO), and concentrated under reduced pressure. The residue was purified by automated flash column chromatography (10% MeOH / DCM) to give (2R,2'R,3R,3'R,4R,4'R,5R,5'R)-(((4-nitro-1,2-phenylene)bis(2-methyl-1-oxo-5',8',11'-trioxa-2'-azatridecan-1,13-diyl))bis(oxy))bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (634) (5.0 g, 57.4%). Product characterization was confirmed by MS (ESI+ve).

[0652] [ka] [ka] [ka]

[0653] Step 1. Preparation of (2R,3R,4R,5R)-5-acetamido-2-(acetoxymethyl)-6-((3-oxo-1-phenyl-2,7,10,13-tetraoxa-4-azapentadecan-15-yl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (2R,3R,4R,5R)-5-acetamido-2-(acetoxymethyl)-6-((1,1,1-trifluoro-2-oxo-6,9,12-trioxa-314-azatetradecan-14-yl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (45.0 g, 70.8 mmol) and Na2CO3 (11.3 g, 106 mmol) were stirred in THF / HO (50:50) at room temperature. Benzyl chloroformate (626) (14.5 g, 85 mmol) was added dropwise, and the reaction was stirred for 16 h. The THF was removed under reduced pressure, and the aqueous solution was extracted (3x) with EtOAc. The organic solution was washed successively with 1 M HCl, saturated NaHCO3, water, and brine, dried (Na2SO4), and concentrated under reduced pressure. The residue was purified by automated flash chromatography (5% MeOH / DCM) to give (2R,3R,4R,5R)-5-acetamido-2-(acetoxymethyl)-6-((3-oxo-1-phenyl-2,7,10,13-tetraoxa-4-azapentadecan-15-yl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (635) (25.12 g, 54%). Product characterization was confirmed by MS (ESI+ve).

[0654] Step 2. Preparation of benzyl (2-(2-(2-(2-(((3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamate 636 (2R,3R,4R,5R)-5-acetamido-2-(acetoxymethyl)-6-((3-oxo-1-phenyl-2,7,10,13-tetraoxa-4-azapentadecan-15-yl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (635) (25.12 g, 38.3 mmol) was stirred in a sealed reaction vessel containing a solution of 7 N ammonia in MeOH at room temperature for 16 hours. The reaction mixture was evaporated at 50° C. to remove ammonia, and the residue was concentrated under reduced pressure to give benzyl (2-(2-(2-(2-(((3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamate (636) (20.3 g, 100%), which was used in the subsequent reaction without further purification. The product was confirmed by MS (ESI+ve).

[0655] Step 3. Preparation of benzyl (2-(2-(2-(2-(((3aR,4R,7R,7aR)-7-acetamido-4-(hydroxymethyl)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-6-yl)oxy)-ethoxy)ethoxy)ethoxy)ethyl)carbamate 637 Benzyl (2-(2-(2-(2-(((3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamate (636) (20.3 g, 38.3 mmol) was stirred in DMF (200 mL) at room temperature. 2,2-Dimethoxypropane (274 g, 1.6 mol) and pTsOH (catalytic) were added, and the reaction was heated at 65 °C for 16 h. The reaction was cooled to room temperature, and TEA (20 mL) was added and stirred for 30 min. The solvent was removed under reduced pressure, the residue was taken up in MeOH / HO (10:1), and the reaction was refluxed for 1 h. The reaction was concentrated under reduced pressure (azeotroped twice with toluene), and the residue was purified by automated flash chromatography (10% MeOH / DCM) to give benzyl (2-(2-(2-(2-(((3aR,4R,7R,7aR)-7-acetamido-4-(hydroxyl-methyl)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-6-yl)oxy)ethoxy)ethoxy)-ethoxy)ethyl)carbamate (637) (24.9 g, 100%). Product characterization was confirmed by MS (ESI+ve).

[0656] Step 4. Preparation of ((3aR,4R,7R,7aR)-7-acetamido-2,2-dimethyl-6-((3-oxo-1-phenyl-2,7,10,13-tetraoxa-4-azapentadecan-15-yl)oxy)tetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-4-yl)methyl 4-methylbenzenesulfonate 638 Benzyl (2-(2-(2-(2-(((3aR,4R,7R,7aR)-7-acetamido-4-(hydroxymethyl)-2,2-dimethyl-tetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-6-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamate (637) (25.5 g, 44.8 mmol) and TEA (9.97 g, 98.5 mmol) in DCM were stirred at 0 °C. p-Toluene-sulfonyl chloride (18.8 g, 98.5 mmol) in DCM was added and the reaction was stirred for 16 h, warming to room temperature. The reaction was diluted with DCM and washed successively with 1 M HCl, saturated NaHCO, water, and brine, dried (NaSO), and concentrated under reduced pressure. The residue was purified by automated flash chromatography (5% MeOH / DCM) to give ((3aR,4R,7R,7aR)-7-acetamido-2,2-dimethyl-6-((3-oxo-1-phenyl-2,7,10,13-tetraoxa-4-azapentadecan-15-yl)oxy)tetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-4-yl)methyl 4-methylbenzenesulfonate (638) (25.5 g, 78.8%). Product characterization was confirmed by MS (ESI+ve).

[0657] Step 5. Preparation of benzyl (2-(2-(2-(2-(((3aS,4R,7R,7aR)-7-acetamido-4-(azidomethyl)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-6-yl)oxy)ethoxy)ethoxy)ethoxy)-ethyl)carbamate 639 ((3aR,4R,7R,7aR)-7-Acetamido-2,2-dimethyl-6-((3-oxo-1-phenyl-2,7,10,13-tetraoxa-4-azapentadecan-15-yl)oxy)tetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-4-yl)methyl 4-methylbenzenesulfonate (638) (25.0 g, 34.5 mmol) and NaN (28.7 g, 434.6 mmol) in DMSO / HO (200 mL / 20 mL) were heated at 100 °C for 12 h. The reaction was cooled and partitioned between EtOAc and saturated NaHCO. The aqueous solution was extracted twice more, and the organic solutions were combined, washed with saturated NaHCO, water, and brine, dried (NaSO), and concentrated under reduced pressure. The residue was purified by automated flash chromatography (5% MeOH / DCM) to give benzyl (2-(2-(2-(2-(((3aS,4R,7R,7aR)-7-acetamido-4-(azidomethyl)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-6-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamate (639) (16.1 g, 78.2%). Product characterization was confirmed by MS (ESI+ve).

[0658] Step 6. Preparation of benzyl (2-(2-(2-(2-(((3aS,4R,7R,7aR)-7-acetamido-4-((4-(3-methoxyphenyl)-1H-1,2,3-triazol-1-yl)methyl)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-6-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamate 640 Benzyl (2-(2-(2-(2-(((3aS,4R,7R,7aR)-7-acetamido-4-(azidomethyl)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-6-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamate (39) (16.1 g, 27.0 mmol) was stirred in MeOH (200 ml) at room temperature. 1-Ethynyl-3-methoxybenzene (4.28 g, 32.4 mmol), tris(benzyltriazolylmethyl)amine (0.72 g, 1.35 mmol), CuSO (0.07 g, 0.27 mmol in 1 mL of HO), and sodium ascorbate (0.53 g, 2.7 mmol in 5 mL of HO) were added sequentially, and the reaction was stirred at room temperature for 16 h. The solvent was removed under reduced pressure, and the residue was taken up in DCM (200 mL) and washed with water. The aqueous layer was back-extracted with DCM, and the organic solutions were combined, washed with brine, and dried (NaSO). The reaction was concentrated under reduced pressure, and the residue was purified by automated flash chromatography (10% MeOH / EtOAc) to give benzyl (2-(2-(2-(2-(((3aS,4R,7R,7aR)-7-acetamido-4-((4-(3-methoxyphenyl)-1H-1,2,3-triazol-1-yl)methyl)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-6-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamate (640) (15.0 g, 76.4%). Product characterization was confirmed by MS (ESI+ve).

[0659] Step 7. Preparation of benzyl (2-(2-(2-(2-(((3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-((4-(3-methoxyphenyl)-1H-1,2,3-triazol-1-yl)methyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamate 641 Benzyl (2-(2-(2-(2-(((3aS,4R,7R,7aR)-7-acetamido-4-((4-(3-methoxyphenyl)-1H-1,2,3-triazol-1-yl)methyl)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-6-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamate (640) (15.0 g, 20.6 mmol) was stirred in MeCN (200 mL) and 1.84% HSO (180 mL) at room temperature for 96 hours. The reaction was extracted with EtOAc (3 x 250 mL), washed with saturated NaHCO, water, and brine, dried (NaSO), and concentrated under reduced pressure to give benzyl (2-(2-(2-(2-(((3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-((4-(3-methoxyphenyl)-1H-1,2,3-triazol-1-yl)methyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamate (641) (11.0 g, 16.0 mmol). This product was used crude in subsequent reactions. Product characterization was confirmed by MS (ESI+ve).

[0660] Step 8. Preparation of (2R,3S,4R,5R)-5-acetamido-2-((4-(3-methoxyphenyl)-1H-1,2,3-triazol-1-yl)methyl)-6-((3-oxo-1-phenyl-2,7,10,13-tetraoxa-4-azapentadecan-15-yl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate 642 Benzyl (2-(2-(2-(2-(((3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-((4-(3-methoxyphenyl)-1H-1,2,3-triazol-1-yl)methyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)-ethyl)carbamate (641) (11.0 g, 16.0 mmol) was stirred in pyridine (200 mL) at room temperature. Acetic anhydride (16.3 g, 160 mmol) was added, and the reaction was stirred at room temperature for 16 hours, then at 50 °C for 3 hours. The reaction was poured into water and extracted three times with DCM (250 mL). The combined organic solution was washed with saturated NaHCO (twice), 1 N HCl (twice), water, and brine, dried (NaSO), and concentrated under reduced pressure. The residue was purified by automated flash chromatography (5% MeOH / DCM) to give (2R,3S,4R,5R)-5-acetamido-2-((4-(3-methoxyphenyl)-1H-1,2,3-triazol-1-yl)methyl)-6-((3-oxo-1-phenyl-2,7,10,13-tetraoxa-4-azapentadecan-15-yl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (642) (10.7 g, 86.7%). Product characterization was confirmed by MS (ESI+ve).

[0661] Step 9. Preparation of (2R,3S,4R,5R)-5-acetamido-2-((4-(3-methoxyphenyl)-1H-1,2,3-triazol-1-yl)methyl)-6-((1,1,1-trifluoro-2-oxo-6,9,12-trioxa-3l4-azatetradecan-14-yl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate 643 (2R,3S,4R,5R)-5-acetamido-2-((4-(3-methoxyphenyl)-1H-1,2,3-triazol-1-yl)methyl)-6-((3-oxo-1-phenyl-2,7,10,13-tetraoxa-4-azapentadecan-15-yl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (642) (9.06 g, 11.74 mmol) and TFA (1.47 g, 12.91 mmol) were stirred in MeOH at room temperature. The reaction mixture was hydrogenated over 10% Pd—C for 1 h. The reaction mixture was filtered through Celite and concentrated under reduced pressure to give (2R,3S,4R,5R)-5-acetamido-2-((4-(3-methoxyphenyl)-1H-1,2,3-triazol-1-yl)methyl)-6-((1,1,1-trifluoro-2-oxo-6,9,12-trioxa-314-azatetradecan-14-yl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (643) (8.8 g, 99.7%), which was used in the subsequent reaction without further purification. The product was confirmed by MS (ESI+ve).

[0662] [ka] [ka]

[0663] Step 1. Preparation of peracetylated galactosamine 644 D-Galactosamine hydrochloride (614) (250 g, 1.16 mol) in pyridine (1.5 L) was treated with acetic anhydride (1.25 L, 13.2 mol) over 45 min. After stirring overnight, the reaction mixture was divided into three 1 L portions. Each 1 L portion was poured into 3 L of ice water and mixed for 1 h. After mixing, the solid was isolated by filtration, combined, frozen in liquid nitrogen, and lyophilized for 5 days to give peracetylated galactosamine (644) (369.4 g, 82%) as a white solid. Rf (0.58, 10% MeOH-CH2Cl2).

[0664] Step 2. 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 645 Peracetylated galactosamine (644) (25 g, 64.21 mmol) was heated with scandium trifluoromethanesulfonate (1.58 g, 3.21 mmol) in dry DCE at 90 °C for 3 h. The reaction was cooled to room temperature, quenched with 5 mL of TEA, and concentrated under reduced pressure. The residue was purified by automated column chromatography (2 → 10% MeOH / DCM) to give (2R,3R,4R,5R)-5-acetamido-2-(acetoxymethyl)-6-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-3,4-diyl diacetate (645) (27 g, 76.5%). Product confirmation was confirmed by MS.

[0665] Step 3. 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 646 A solution of azide 645 (7.12 g, 13 mmol) in EtOAc (150 mL) and trifluoroacetic acid (2 mL) was treated with palladium on activated carbon (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. 6Rf (0.34, 15% MeOH-CH2Cl2).

[0666] Step 4. Preparation of (2R,2'R,3R,3'R,4R,4'R,5R,5'R)-(((5-nitro-1,3-phenylene)bis(1-oxo-5,8,11-trioxa-2-azatridecane-1,13-diyl))bis(oxy))bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate 648 (2R,3R,4R,5R)-5-acetamido-2-(acetoxymethyl)-6-((1,1,1-trifluoro-2-oxo-6,9,12-trioxa-314-azatetradecan-14-yl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (646) (13.25 g, 20.84 mmol), 5-nitroisophthalic acid (647) (2.0 g, 9.5 mmol), HATU (12.3 g, 32.21 mmol), and TEA (5.75 g, 59.0 mmol) in DCM were stirred at room temperature for 16 h. The reaction mixture was diluted with DCM, washed successively with 1 M HCl, saturated NaHCO, water, and brine, dried over NaSO, and concentrated under reduced pressure. The residue was purified by automated flash chromatography (5% MeOH / DCM) to give (2R,2'R,3R,3'R,4R,4'R,5R,5'R)-(((5-nitro-1,3-phenylene)bis(1-oxo-5,8,11-trioxa-2-azatridecane-1,13-diyl))bis(oxy))bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (648) (4.43 g, 38.3%). Product characterization was confirmed by MS (ESI+ve).

[0667] Step 5. Preparation of (2R,2'R,3R,3'R,4R,4'R,5R,5'R)-(((5-amino-1,3-phenylene)bis(1-oxo-5,8,11-trioxa-2-azatridecane-1,13-diyl))bis(oxy))bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate 649 (2R,2'R,3R,3'R,4R,4'R,5R,5'R)-(((5-nitro-1,3-phenylene)bis(1-oxo-5,8,11-trioxa-2-azatridecane-1,13-diyl))bis(oxy))bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (648) (26.1 g, 23.05 mmol) was stirred in MeOH at room temperature. The reaction mixture was hydrogenated over 10% Pd-C (2.6 g) at room temperature for 2 hours. The reaction mixture was filtered through celite and concentrated under reduced pressure to give (2R,2'R,3R,3'R,4R,4'R,5R,5'R)-(((5-amino-1,3-phenylene)bis(1-oxo-5,8,11-trioxa-2-azatridecane-1,13-diyl))bis(oxy))bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (649) (28.0 g, 99.9%), which was used in the subsequent reaction without further purification. The product was confirmed by MS (ESI+ve).

[0668] Step 6. Preparation of (2R,3R,4R,5R)-5-acetamido-6-((1-(3-((2-(2-(2-(2-(((3R,4R,5R,6R)-3-acetamido-5-acetoxy-6-(acetoxymethyl)-4-hydroxytetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-5-(2-(((benzyloxy)carbonyl)amino)acetamido)phenyl)-1-oxo-5,8,11-trioxa-2-azatridecan-13-yl)oxy)-2-(acetoxymethyl)tetrahydro-2H-pyran-3,4-diyl diacetate 651 (2R,2'R,3R,3'R,4R,4'R,5R,5'R)-(((5-amino-1,3-phenylene)bis(1-oxo-5,8,11-trioxa-2-azatridecane-1,13-diyl))bis(oxy))bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (649) (0.5 g, 0.45 mmol) and CBZ-gly (650) (0.09 g, 0.45 mmol) were stirred in EtOAc at room temperature. T3P (50% solution in EtOAc) (0.29 g, 0.91 mmol) was added and the reaction was stirred overnight at room temperature. An additional 0.3 equiv. of T3P was added and the reaction was stirred for an additional 1 h. The reaction was washed with saturated NaHCO3 and brine, dried (Na2SO4), concentrated under reduced pressure and the residue purified by automated flash chromatography (10% MeOH / DCM) to give (2R,3R,4R,5R)-5-acetamido-6-((1-(3-((2-(2-(2-(2-((3R,4R,5R,6R)-3-acetamido-5-acetoxy-6-(acetoxymethyl)-4-hydroxytetrahydrofuran). Tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)-ethoxy)ethyl)carbamoyl)-5-(2-(((benzyloxy)carbonyl)amino)acetamido)phenyl)-1-oxo-5,8,11-trioxa-2-azatridecan-13-yl)oxy)-2-(acetoxymethyl)tetrahydro-2H-pyran-3,4-diyl diacetate (651) (0.33 g, 56.8%) was obtained. The product was confirmed by MS (ESI+ve).

[0669] Step 7. Preparation of (2R,2'R,3R,3'R,4R,4'R,5R,5'R)-(((5-(2-((2,2,2-trifluoroacetyl)-l4-azaneyl)acetamido)-1,3-phenylene)bis(1-oxo-5,8,11-trioxa-2-azatridecane-1,13-diyl))bis(oxy))bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate 652 (2R,3R,4R,5R)-5-acetamido-6-((1-(3-((2-(2-(2-(2-(((3R,4R,5R,6R)-3-acetamido-5-acetoxy-6-(acetoxymethyl)-4-hydroxytetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-5-(2-(((benzyloxy)carbonyl)amino)acetamido)phenyl)-1-oxo-5,8,11-trioxa-2-azatridecan-13-yl)oxy)-2-(acetoxymethyl)tetrahydro-2H-pyran-3,4-diyl diacetate (651) (3.3 g, 2.39 mmol) and TFA (0.29 g, 2.51 mmol) were stirred in MeOH at room temperature. The reaction was hydrogenated over 10% Pd-C (400 mg) for 2 hours, filtered through Celite, and concentrated under reduced pressure to give (2R,2'R,3R,3'R,4R,4'R,5R,5'R)-(((5-(2-((2,2,2-trifluoroacetyl)-14-azaneyl)-acetamido)-1,3-phenylene)bis(1-oxo-5,8,11-trioxa-2-azatridecan-1,13-diyl))bis(oxy))bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (652) (3.21 g, 98.7%), which was used in subsequent reactions without further purification. The product was characterized by MS (ESI+ve).

[0670] Step 8. Preparation of (2R,2'R,3R,3'R,4R,4'R,5R,5'R)-(((5-(2-(10-(3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanamido)acetamido)-1,3-phenylene)bis(1-oxo-5,8,11-trioxa-2-azatridecane-1,13-diyl))bis(oxy))bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate 653 (2R,2'R,3R,3'R,4R,4'R,5R,5'R)-(((5-(2-((2,2,2-trifluoroacetyl)-14-azaneyl)acetamido)-1,3-phenylene)bis(1-oxo-5,8,11-trioxa-2-azatridecan-1,13-diyl))bis(oxy))bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate A mixture of 652 (1.0 g, 0.73 mmol), lithium 10-(3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethyl-pyrrolidin-1-yl)-10-oxodecanoate (606) (0.45 g, 0.73 mmol), HATU (0.47 g, 1.25 mmol), and TEA (0.22 g, 2.2 mmol) in DCM was stirred at room temperature for 4 h. The reaction was diluted with DCM, washed successively with saturated NaHCO, water, and brine, dried (NaSO), and concentrated under reduced pressure. The residue was purified by automated flash chromatography (5% MeOH / DCM) to give (2R,2'R,3R,3'R,4R,4'R,5R,5'R)-(((5-(2-(10-(3-((bis(4-methoxy-phenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanamido)acetamido)-1,3-phenylene)bis(1-oxo-5,8,11-trioxa-2-azatridecane-1,13-diyl))bis(oxy))bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (653) (1.02 g, 75.2%). Product characterization was confirmed by MS (ESI+ve).

[0671] Step 9. Preparation of 4-((1-(10-((2-((3,5-bis((2-(2-(2-(2-(((3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamoyl)phenyl)amino)-2-oxoethyl)amino)-10-oxodecanoyl)-4-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3,4-dimethylpyrrolidin-3-yl)methoxy)-4-oxobutanoic acid 654 (2R,2'R,3R,3'R,4R,4'R,5R,5'R)-(((5-(2-(10-(3-((bis(4-methoxyphenyl)(phenyl)methoxy)-methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanamido)acetamido)-1,3-phenylene)bis(1-oxo-5,8,11-trioxa-2-azatridecane-1,13 (5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (653) (1.05 g, 0.57 mmol), succinic anhydride (0.28 g, 2.84 mmol), DMAP (0.35 g, 2.84 mmol), and TEA (0.58 g, 5.68 mmol) were heated in dry DCE at 60 °C for 2 h. MeOH (5 mL) was added, and the reaction was stirred for an additional 30 min, then cooled and concentrated under reduced pressure. The residue was taken up in DCM and washed successively with saturated NaHCO (4 times), water, and brine. The organic solution was dried (NaSO) and concentrated under reduced pressure to give 4-((1-(10-((2-((3,5-bis((2-(2-(2-(2-(((3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamoyl)phenyl)amino)-2-oxoethyl)amino)-10-oxodecanoyl)-4-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3,4-dimethylpyrrolidin-3-yl)methoxy)-4-oxobutanoic acid (654) (1.1 g, 99.4%), which was used crude in the subsequent reaction. The product was characterized by MS (ESI+ve).

[0672] [ka]

[0673] Step 1. Preparation of (2R,2'R,3R,3'R,4R,4'R,5R,5'R)-(((5-(10-(3-((bis(4-methoxy-phenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanamido)-1,3-phenylene)bis(1-oxo-5,8,11-trioxa-2-azatridecane-1,13-diyl))bis(oxy))bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate 655 (2R,2'R,3R,3'R,4R,4'R,5R,5'R)-(((5-amino-1,3-phenylene)bis(1-oxo-5,8,11-trioxa-2-azatridecane-1,13-diyl))bis(oxy))bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (649) (4 g, 3.36 mmol), lithium Umium 10-(3-((bis(4-methoxyphenyl)-(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoate (6) (2.13 g, 3.36 mmol), TEA (1 mL, 6.7 mmol), and T3P (50 w / w% solution in EtOAc) (4.3 g, 6.72 mmol) in DCM were stirred at room temperature for 16 h. The reaction was washed successively with saturated NaHCO3, water, and brine, dried (Na2SO4), and concentrated under reduced pressure. The residue was purified by automated flash chromatography (10% MeOH / DCM) to give 2R,2'R,3R,3'R,4R,4'R,5R,5'R)-(((5-(10-(3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxyl-methyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanamido)-1,3-phenylene)bis(1-oxo-5,8,11-trioxa-2-azatridecane-1,13-diyl))bis(oxy))bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (655) (1.37 g, 22.5%). Product characterization was confirmed by MS (ESI+ve).

[0674] Step 2. Preparation of 4-((1-(10-((3,5-bis((2-(2-(2-(2-(((3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamoyl)phenyl)amino)-10-oxodecanoyl)-4-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3,4-dimethylpyrrolidin-3-yl)methoxy)-4-oxobutanoic acid 656 This compound was prepared in a similar manner to 4-((1-(10-((2-((3,5-bis((2-(2-(2-(2-(((3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamoyl)phenyl)amino)-2-oxoethyl)amino)-10-oxodecanoyl)-4-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3,4-dimethylpyrrolidin-3-yl)methoxy)-4-oxobutanoic acid (654).

[0675] [ka]

[0676] Synthesis of 3-((((1-(10-((3,5-bis((2-(2-(2-(((3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)carbamoyl)phenyl)amino)-10-oxodecanoyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-3-yl)methoxy)carbonyl)oxy)propanoic acid 657 This compound was prepared in a similar manner to 4-((1-(10-((3,5-bis((2-(2-(2-(2-(((3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamoyl)phenyl)amino)-10-oxodecanoyl)-4-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3,4-dimethylpyrrolidin-3-yl)methoxy)-4-oxobutanoic acid (654).

[0677] [ka] [ka]

[0678] Step 1. Preparation of dimethyl 5-(hydroxymethyl)isophthalate 659 Trimethylbenzene-1,3,5-tricarboxylate (658) (40 g, 159 mmol) and NaBH4 were stirred in THF at room temperature. MeOH (30 mL) in THF (120 mL) was slowly added dropwise. After the addition was complete, the reaction was refluxed for 30 minutes. After cooling, the reaction was quenched with 1 M HCl, and the solution was extracted with EtOAc. The organic solution was washed successively with 1 M HCl, NaHCO3, water, and brine, dried (Na2SO4), and concentrated under reduced pressure. The residue was purified by automated flash chromatography (50 / 50 EtOAc / hexanes) to give dimethyl 5-(hydroxymethyl)isophthalate (659) (20.5 g, 53.2%). 1 H NMR (400 MHz, CDCl) δ 8.59 (s, 1H), 8.23 ​​(s, 2H), 4.81 (s, 2H), 3.95 (s, 6H). The product was confirmed by MS (ESI+ve).

[0679] Step 2. Preparation of dimethyl 5-(chloromethyl)isophthalate 660 Dimethyl 5-(hydroxymethyl)isophthalate (659) (20.5 g, 80.5%) in SOCl (11.1 g, 94 mmol) was refluxed for 1.5 h. The reaction was cooled, diluted with DCM, washed successively with 0.1 M NaOH (twice), water, and brine, dried (NaSO), and concentrated under reduced pressure. The residue was purified by automated flash chromatography (20% EtOAc / hexanes) to give dimethyl 5-(chloromethyl)isophthalate (660) (10.84 g, 53%). 1 H NMR (400 MHz, CDCl) δ 8.65 (s, 1H), 8.27 (s, 2H), 4.66 (s, 2H), 3.97 (s, 6H). The product was confirmed by MS (ESI+ve).

[0680] Step 3. Preparation of dimethyl 5-(azidomethyl)isophthalate 661 Dimethyl 5-(chloromethyl)isophthalate (660) (10.84 g, 45 mmol) and NaN (18 g, 270 mmol) were refluxed in acetone / water (3 / 1) for 16 h. The reaction was cooled, concentrated under reduced pressure, and the residue was taken up in DCM. The organic solution was washed with water and brine, dried (NaSO), and concentrated under reduced pressure. The residue was purified by flash chromatography (15% EtOAc / hexanes) to give dimethyl 5-(azidomethyl)isophthalate (661) (9.84 g, 88%). 1 H NMR (400 MHz, CDCl) δ 8.66 (s, 2H), 8.2 (s, 2H), 4.49 (s, 2H), 3.97 (s, 2H). The product was confirmed by MS (ESI+ve).

[0681] Step 4. Preparation of 5-(azidomethyl)isophthalic acid 662 Dimethyl 5-(azidomethyl)isophthalate (661) (9.84 g, 39.5 mmol) and LiOH (2.1 g, 87 mmol) were stirred in THF / HO / MeOH at room temperature for 48 h. The organic solvent was removed under reduced pressure, and the residue was acidified with 1 M HCl. The aqueous solution was extracted with EtOAc (3 times), and the organic solutions were combined, dried (NaSO), and concentrated under reduced pressure to give 5-(azidomethyl)isophthalic acid (662) (8.0 g, 91.6%), which was used in the subsequent reaction without further purification.

[0682] Step 5. Preparation of (2R,2'R,3R,3'R,4R,4'R)-(((5-(azidomethyl)-1,3-phenylene)bis(1-oxo-5,8,11-trioxa-2-azatridecane-1,13-diyl))bis(oxy))bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate 663 5-(Azidomethyl)isophthalic acid (662) (4.42 g, 20 mmol), 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)ethane-1-aminium 2,2,2-trifluoroacetate (646) (25 g, 40 mmol), HATU (24.4 g, 64 mmol), and TEA (17 mL, 120 mmol) were stirred in DCM at room temperature for 16 h. The reaction was washed successively with 1 M HCl, saturated NaHCO, water, and brine, dried (NaSO), and concentrated under reduced pressure. The residue was purified by automated flash chromatography (7% MeOH / DCM) to give (2R,2'R,3R,3'R,4R,4'R)-(((5-(azidomethyl)-1,3-phenylene)bis(1-oxo-5,8,11-trioxa-2-azatridecane-1,13-diyl))bis(oxy))bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (663) (10.9 g, 44.5%). Product characterization was confirmed by MS (ESI+ve).

[0683] ...

Claims

1. A pharmaceutical combination for delivering a nucleic acid to a cell, comprising: 1) a membrane destabilizing polymer; and 2) a nucleic acid complex, The nucleic acid complex has the formula (I): 【Chemistry 1】 [In the formula, R 1 is a targeting ligand having the formula: 【Chemistry 2】 (In the formula, B 1 is 【Chemistry 3】 and is covalently bonded to L 1 , T 1 , and T 2 ; B2 is 【Chemistry 4】 and is covalently attached to T 1 , T 3 , and T 4 ; B3 is 【Chemistry 5】 and is covalently attached to T 2 , T 5 , and T 6 ; T 1 and T 2 are each absent or a glycine linking group; T 3 , T 4 , T 5 , and T 6 are each 【Chemistry 6】 (Wherein, n=1, 2, 3) are independently selected from the group consisting of: each sugar is a monosaccharide); L 1 is a linking group that is independently an unbranched saturated divalent hydrocarbon chain having 1 to 20 carbon atoms, one or more of the carbon atoms in said hydrocarbon chain may be optionally replaced by -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X -, or -S-, where R X is hydrogen or (C 1 -C 6 )alkyl, and said hydrocarbon chain may be optionally substituted by one or more oxo (=O); Ring A is a 3-6 membered monocyclic heterocycloalkyl containing zero or one or two heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur; each R A is independently selected from the group consisting of hydrogen, hydroxy, -C 1-2 alkyl-OR B , and C 1-4 alkyl; R B is hydrogen; n is 0, 1, 2, 3, or 4; L 2 is a linking group that is independently a branched or unbranched saturated divalent hydrocarbon chain having 1 to 14 carbon atoms, one or more of the carbon atoms in said hydrocarbon chain may be optionally replaced by -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X -, or -S-, where R X is hydrogen or (C 1 -C 6 )alkyl, and said hydrocarbon chain may be optionally substituted by one or more oxo (=O); R 2 is the nucleic acid, and the nucleic acid is an oligonucleotide having a length of about 10 to about 100 nucleotides. or a salt thereof, The membrane destabilizing polymer has the formula (XX): 【Chemistry 7】 [In the formula, PEGMA is a polyethylene glycol methacrylate residue having 2-20 ethylene glycol units; M ２ (C ４ -C １８ ) alkyl-methacrylate residue; BMA is the residue of butyl methacrylate; PAA is the residue of propylacrylic acid; DMAEMA is the residue of dimethylaminoethyl methacrylate; m and n are each a molar fraction greater than 0, m is greater than n, and m+n=1; q is a molar ratio from 0.2 to 0.75; r is a molar ratio between 0.05 and 0.6; s is a molar ratio between 0.2 and 0.75; q+r+s=1; v is 1-25 kDa; w is 1-25 kDa; T ５ is a targeting moiety that includes an N-acetylgalactosamine (NAG) residue; L is a linking moiety comprising a polyethylene glycol (PEG) moiety having 2 to 20 ethylene glycol units. The combination drug,

2. The combination pharmaceutical of claim 1, wherein the nucleic acid complex and the membrane destabilizing polymer are administered separately.

3. The combination pharmaceutical of claim 1, wherein the membrane destabilizing polymer is administered after administration of the nucleic acid complex.

4. The combination pharmaceutical of claim 1, wherein the nucleic acid complex and the membrane destabilizing polymer are administered together in a single composition.

5. A pharmaceutical combination according to any one of claims 1 to 4 formulated for administration by injection.

6. The pharmaceutical combination of any one of claims 1 to 5 formulated for administration by subcutaneous injection.

7. The combination pharmaceutical of claim 1, wherein the cell is a secretory cell, a chondrocyte, an epithelial cell, a neuronal cell, a muscle cell, a blood cell, an endothelial cell, a pericyte, a fibroblast, a glial cell, or a dendritic cell.

8. The combination pharmaceutical of any one of claims 1 to 6, wherein the cell is a cancer cell, an immune cell, a bacterially infected cell, a virally infected cell, or a cell with abnormal metabolic activity.

9. The combination pharmaceutical of claim 1, wherein the cells are hepatocytes.

10. The combination pharmaceutical of any one of claims 1 to 6, wherein the targeting ligand specifically binds to a cell surface molecule selected from the group consisting of transferrin receptor type 1, transferrin receptor type 2, EGF receptor, HER2 / Neu, VEGF receptor, PDGF receptor, integrin, NGF receptor, CD2, CD3, CD4, CD8, CD19, CD20, CD22, CD33, CD43, CD38, CD56, CD69, asialoglycoprotein receptor (ASGPR), prostate-specific membrane antigen (PSMA), folate receptor, and sigma receptor.

11. The combination pharmaceutical of any one of claims 1 to 10, wherein the nucleic acid is selected from the group consisting of small interfering RNA (siRNA), Dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), tRNA, rRNA, tRNA, viral RNA (vRNA), self-amplifying RNA (sa-RNA), and combinations thereof.

12. The combination pharmaceutical described in any one of claims 1 to 11, wherein the nucleic acid is siRNA.

13. The membrane destabilizing polymer of claim 1, wherein the membrane destabilizing polymer has formula (XXI): 【Chemistry 8】 (Wherein, px is 11 or 12, and py is 4 or 5.) The pharmaceutical combination according to any one of claims 1 to 12, which is a polymer of the formula:

14. The combination pharmaceutical of any one of claims 1 to 13, wherein ring A is absent or pyrrolidinyl.

15. The pharmaceutical combination according to any one of claims 1 to 14, wherein each R A is independently hydroxy or C 1-8 alkyl, said C 1-8 alkyl being optionally substituted by hydroxyl.

16. The compound of formula (I), 【Chemistry 9】 wherein Q is -L 1 -R 1 and R' is C 1-9 alkyl. The pharmaceutical combination according to any one of claims 1 to 14, selected from the group consisting of:

17. The compound of formula (I), 【Chemistry 10】 wherein Q is -L 1 -R 1 and R' is C 1-9 alkyl. The pharmaceutical combination according to any one of claims 1 to 14 and 16, 18. The combination pharmaceutical according to any one of claims 1 to 17, wherein L 1 is linked to R 1 via -NH-, -O-, -S-, -(C═O)-, -(C═O)-NH-, -NH-(C═O)-, -(C═O)-O-, -NH-(C═O)-NH-, or -NH-(SO 2 )-.

19. L 1 is 【Chemistry 11】 The pharmaceutical combination according to any one of claims 1 to 17, selected from the group consisting of:

20. L 1 is 【Chemistry 12】 The pharmaceutical combination according to any one of claims 1 to 17, selected from the group consisting of:

21. The pharmaceutical combination according to any one of claims 1 to 15, wherein L 2 is linked to R 2 via -O-.

22. The combination pharmaceutical according to any one of claims 1 to 15, wherein L 2 is -CH 2 O-, -CH 2 CH 2 O-, or -CH(OH)CH 2 O-.

23. The combination pharmaceutical of any one of claims 1 to 22, wherein T 1 and T 2 are both absent.

24. The combination pharmaceutical of any one of claims 1 to 22, wherein at least one of T 1 and T 2 is glycine.

25. The pharmaceutical combination of claim 24, wherein T 1 and T 2 are each glycine.

26. B 1 is 【Chemistry 13】 The combination pharmaceutical according to any one of claims 1 to 25,

27. ​​B2 is 【Chemistry 14】 The pharmaceutical combination according to any one of claims 1 to 26, selected from the group consisting of:

28. B3 is 【Chemistry 15】 The pharmaceutical combination according to any one of claims 1 to 27, selected from the group consisting of:

29. Each monosaccharide is 【Chemistry 16】 The pharmaceutical combination according to any one of claims 1 to 28, independently selected from the group consisting of:

30. Each monosaccharide is independently: 【Chemistry 17】 The pharmaceutical combination according to any one of claims 1 to 29,

31. The compound of formula (I) has the following formula: 【Chemistry 18】 (wherein L 1 , L 2 , R 2 , ring A, R A , R B , and n are as defined in claim 1 ). The pharmaceutical combination according to any one of claims 1 to 13, which is a compound of the formula:

32. A composition comprising: (a) a nucleic acid complex which is a compound of formula (I) or a salt thereof: 【Chemistry 19】 [In the formula, R 1 is a targeting ligand having the formula: 【Chemistry 20】 (In the formula, B 1 is 【Chemistry 21】 and is covalently bonded to L 1 , T 1 , and T 2 ; B2 is 【Chemical 22】 and is covalently attached to T 1 , T 3 , and T 4 ; B3 is 【Chemistry 23】 and is covalently attached to T 2 , T 5 , and T 6 ; T 1 and T 2 are each absent or a glycine linking group; T 3 , T 4 , T 5 , and T 6 are each 【Chemistry 24】 (Wherein, n=1, 2, 3) are independently selected from the group consisting of: each sugar is a monosaccharide); L 1 is a linking group that is independently an unbranched saturated divalent hydrocarbon chain having 1 to 20 carbon atoms, one or more of the carbon atoms in said hydrocarbon chain may be optionally replaced by -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X -, or -S-, where R X is hydrogen or (C 1 -C 6 )alkyl, and said hydrocarbon chain may be optionally substituted by one or more oxo (=O); Ring A is a 3-6 membered monocyclic heterocycloalkyl containing zero or one or two heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur; each R A is independently selected from the group consisting of hydrogen, hydroxy, -C 1-2 alkyl-OR B , and C 1-4 alkyl; R B is hydrogen; n is 0, 1, 2, 3, or 4; L 2 is a linking group that is independently a branched or unbranched saturated divalent hydrocarbon chain having 1 to 14 carbon atoms, one or more of the carbon atoms in said hydrocarbon chain may be optionally replaced by -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X -, or -S-, where R X is hydrogen or (C 1 -C 6 )alkyl, and said hydrocarbon chain may be optionally substituted by one or more oxo (=O); R 2 is a nucleic acid, which is an oligonucleotide having a length of about 10 to about 100 nucleotides; (b) a membrane destabilizing polymer of formula (XX): 【Chemistry 25】 [In the formula, PEGMA is a polyethylene glycol methacrylate residue having 2-20 ethylene glycol units; M 2 is a (C 4 -C 18 )alkyl-methacrylate residue; BMA is the residue of butyl methacrylate; PAA is the residue of propylacrylic acid; DMAEMA is the residue of dimethylaminoethyl methacrylate; m and n are each a molar fraction greater than 0, m is greater than n, and m+n=1; q is a molar ratio from 0.2 to 0.75; r is a molar ratio between 0.05 and 0.6; s is a molar ratio between 0.2 and 0.75; q+r+s=1; v is 1-25 kDa; w is 1-25 kDa; T 5 is a targeting moiety that contains an N-acetylgalactosamine (NAG) residue; L is a linking moiety that includes a polyethylene glycol (PEG) moiety having 2 to 20 ethylene glycol units; and (c) a pharma- ceutically acceptable carrier The composition comprising:

33. The composition of claim 32 formulated for administration by injection.

34. The composition of claim 32 or 33, formulated for administration by subcutaneous injection.

35. A method for delivering a nucleic acid to a cell in vitro, the method comprising contacting the cell with 1) a membrane destabilizing polymer and 2) a nucleic acid complex; The nucleic acid complex has the formula (I): 【Chemistry 26】 [In the formula, R 1 is a targeting ligand having the formula: 【Chemical 27】 (In the formula, B 1 is 【Chemistry 28】 and is covalently bonded to L 1 , T 1 , and T 2 ; B2 is 【Chemical 29】 and is covalently attached to T 1 , T 3 , and T 4 ; B3 is 【Chemistry 30】 and is covalently attached to T 2 , T 5 , and T 6 ; T 1 and T 2 are each absent or a glycine linking group; T 3 , T 4 , T 5 , and T 6 are each 【Chemistry 31】 (Wherein, n=1, 2, 3) are independently selected from the group consisting of: each sugar is a monosaccharide); L 1 is a linking group that is independently an unbranched saturated divalent hydrocarbon chain having 1 to 20 carbon atoms, one or more of the carbon atoms in said hydrocarbon chain may be optionally replaced by -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X -, or -S-, where R X is hydrogen or (C 1 -C 6 )alkyl, and said hydrocarbon chain may be optionally substituted by one or more oxo (=O); Ring A is a 3-6 membered monocyclic heterocycloalkyl containing zero or one or two heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur; each R A is independently selected from the group consisting of hydrogen, hydroxy, -C 1-2 alkyl-OR B , and C 1-4 alkyl; R B is hydrogen; n is 0, 1, 2, 3, or 4; L 2 is a linking group that is independently a branched or unbranched saturated divalent hydrocarbon chain having 1 to 14 carbon atoms, one or more of the carbon atoms in said hydrocarbon chain may be optionally replaced by -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X -, or -S-, where R X is hydrogen or (C 1 -C 6 )alkyl, and said hydrocarbon chain may be optionally substituted by one or more oxo (=O); R 2 is the nucleic acid, and the nucleic acid is an oligonucleotide having a length of about 10 to about 100 nucleotides. or a salt thereof, The membrane destabilizing polymer has the formula (XX): 【Chemistry 32】 [In the formula, PEGMA is a polyethylene glycol methacrylate residue having 2-20 ethylene glycol units; M 2 is a (C 4 -C 18 )alkyl-methacrylate residue; BMA is the residue of butyl methacrylate; PAA is the residue of propylacrylic acid; DMAEMA is the residue of dimethylaminoethyl methacrylate; m and n are each a molar fraction greater than 0, m is greater than n, and m+n=1; q is a molar ratio from 0.2 to 0.75; r is a molar ratio between 0.05 and 0.6; s is a molar ratio between 0.2 and 0.75; q+r+s=1; v is 1-25 kDa; w is 1-25 kDa; T 5 is a targeting moiety that contains an N-acetylgalactosamine (NAG) residue; L is a linking moiety comprising a polyethylene glycol (PEG) moiety having 2 to 20 ethylene glycol units. The polymer of the above formula (1) is a polymer of the above formula (1).

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