Bivalent Targeted Conjugates

Abstract

The present invention provides a conjugate comprising a bivalent targeting moiety, a nucleic acid and an optional linking group, as well as a synthetic intermediate and a synthetic method for preparing the conjugate, a composition comprising a bidentate targeting ligand and the conjugate, and a method for targeting therapeutic nucleic acids with the bidentate conjugate. The conjugate can be used to target therapeutic nucleic acids.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This patent claims priority to U.S. Application No. 62 / 755,179, filed on November 2, 2018, which is incorporated herein by reference. Background Art

[0003] Since Ashwell and Morell's seminal paper elucidating the role of the asialoglycoprotein receptor (ASGPr) in the recognition and transport of circulating glycoproteins, this receptor has become the focus of intensive research (D'Souza et al., 2015, J. Control Release, 203, 126-139). High levels of expression on the surface of hepatocytes make this receptor an attractive target for liver-specific delivery agents. The receptor has a trivalent carbohydrate binding domain that selectively binds to N-acetylgalactosamine. It is a generally accepted rule that the binding affinity for a targeting ligand increases with increasing number of GalNac units in the following order: six GalNac units are greater than four GalNac units, four GalNac units are greater than three GalNac units, three GalNac units are greater than two GalNac units, and two GalNac units are greater than one GalNAc unit (Meier et al., 2000, J Mol Biol, 300, 857-865; Spiess M, 1990, Biochemistry, 29, 43, 10009-10018; Grewal P., 2010, Methods in Enzymology, 479, 223-241; Lee et al., 1983, J Biol Chem, 258, 1, 199-202; and Valentijn et al., 1997, Tetrahedron, 53, 2, 759-770).

[0004] In most cases, the chemical synthesis of multi-dentate targeting ligands may be involved, which requires multiple synthesis steps (sometimes between 20 and 30). This will affect manufacturing requirements and commodity costs. In addition, the synthesis of GalNAc / siRNA conjugates is usually carried out on a fixed controlled pore glass (CPG) support. The reactive sites close to the support are related to the pore size, and are therefore adversely affected by the molecular size close to the site. The increase in the size of the targeting ligand (number of monosaccharide units, molecular weight, molecular radius, etc.) will have an adverse effect on the loading efficiency on the support. Therefore, it is currently necessary to have useful delivery characteristics, but it is easier to prepare, cheaper to prepare, the molecular weight is lower, and / or the loading efficiency is higher. Summary of the invention

[0005] Bidentate GalNac targeting ligands containing two sugar groups (e.g., N-acetylgalactosamine moieties) have been identified, and the targeting activity of the targeting ligands is as good as or better than the known triantennary and tetraantennary ligands. These bidentate targeting ligands generally have a shorter synthetic pathway, so that the overall synthesis efficiency is higher. In addition, the smaller molecular size of the bidentate targeting ligands allows more penetration onto the CPG, so that the loading level is about 30-50% higher than some triantennary and tetraantennary ligands. In addition, compared with triantennary and tetraantennary ligands, the bidentate targeting ligands have a simplified analysis, which can speed up ADME toxicity studies and related research activities. The present invention provides bidentate targeting ligands, nucleic acid conjugates of these bidentate targeting ligands, compositions comprising bidentate targeting ligands and conjugates, and methods for targeting therapeutic nucleic acids with bidentate conjugates.

[0006] In one embodiment, the present invention provides a conjugate of formula (I):

[0007]

[0008] in:

[0009] R 1 It’s sugar;

[0010] L 1 is a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 0 to 20 carbon atoms, wherein one or more carbon atoms in the hydrocarbon chain are optionally replaced by -O-, -NR X -、-NR X -C(=O)-, -C(=O)-NR X - or -S- substitution, and wherein R X is hydrogen or (C1-C6)alkyl, and wherein the hydrocarbon chain is optionally substituted with one or more substituents selected from oxo (=O) and halo;

[0011] B is a 5-10 membered aryl or 5-10 membered heteroaryl, which is optionally substituted with one or more groups independently selected from the group consisting of halo, hydroxy, cyano, trifluoromethyl, trifluoromethoxy, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkanoyloxy, (C3-C6)cycloalkyl, and (C3-C6)cycloalkyl(C1-C6)alkyl;

[0012] L 2 is a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 0 to 20 carbon atoms, wherein one or more carbon atoms in the hydrocarbon chain are optionally replaced by -O-, -NR X -、-NR X-C(=O)-, -C(=O)-NR X - or -S- substitution, and wherein R X is hydrogen or (C1-C6)alkyl, and wherein the hydrocarbon chain is optionally substituted with one or more substituents selected from oxo (=O) and halo;

[0013] R 2 It’s sugar;

[0014] L 3 There is no or a linking group;

[0015] A is absent and is 3-20 membered cycloalkyl, 5-20 membered aryl, 5-20 membered heteroaryl or 3-20 membered heterocycloalkyl;

[0016] Each R A independently selected from hydrogen, hydroxyl, CN, F, Cl, Br, I, -C 1-2 Alkyl-OR a , C 1-10 Alkyl C 2-10 Alkenyl and C 2-10 Alkynyl group; wherein the C 1-10 Alkyl C 2-10 Alkenyl and C 2-10 Alkynyl is optionally substituted by one or more independently selected from halo, hydroxy and C 1-3 Alkoxy group substitution;

[0017] n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0018] L 4 There is no or a linking group;

[0019] R 3 It is nucleic acid;

[0020] R a is hydrogen, a protecting group, a covalent bond to a solid support, or a linker group L bound to a solid support 5 and

[0021] L 5 is a linking group;

[0022] or a salt of the conjugate.

[0023] The present invention also provides a pharmaceutical composition comprising a conjugate of formula I as described herein or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0024] The present invention also provides synthetic intermediates and methods disclosed herein that can be used to prepare the conjugates of Formula I.

[0025] Other objects, features and advantages of the present invention will be apparent to those skilled in the art from the following detailed description and drawings. DETAILED DESCRIPTION

[0026] As used herein, the following terms have the meanings ascribed to them unless otherwise indicated.

[0027] The terms "alkoxy" and "alkylthio" are used in their conventional sense and refer to those alkyl groups attached to the remainder of the molecule via an oxygen atom ("oxy") or a thiol group, and further include mono- and poly-halogenated versions thereof.

[0028] The term "alkyl" by itself or as part of another substituent means, unless otherwise specified, a straight or branched chain hydrocarbon radical (i.e., C 1-8 The term "alkynyl" refers to an unsaturated alkyl group having one or more double bonds. Similarly, the term "alkynyl" refers to an unsaturated alkyl group 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 homologues and isomers.

[0029] The term "animal" includes mammalian species such as humans, mice, rats, dogs, cats, hamsters, guinea pigs, rabbits, livestock, and the like.

[0030] The term "aryl" as used herein refers to a single all-carbon aromatic ring or a plurality of fused all-carbon ring systems, wherein at least one ring is aromatic. For example, in certain embodiments, the aryl group has 6 to 20 carbon atoms, 6 to 14 carbon atoms, 6 to 12 carbon atoms, or 6 to 10 carbon atoms. The aryl group includes a phenyl group. The aryl group also includes a plurality of fused carbocyclic ring systems (e.g., a ring system comprising 2, 3, or 4 rings) having about 9 to 20 carbon atoms, wherein at least one ring is aromatic and wherein the other rings may be aromatic or non-aromatic (e.g., cycloalkyl). When the valence requirement allows, the rings of the plurality of fused ring systems may be interconnected via fused bonds, spiro bonds, and bridge bonds. It should be understood that the connection points of the plurality of fused ring systems as defined above may be at any position in the ring system, including the aromatic or carbocyclic portion 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, etc.

[0031] The term "cycloalkyl" refers to a saturated or partially unsaturated (non-aromatic) all-carbon ring (i.e., (C3-C8) carbocycle) having 3 to 8 carbon atoms. This term also includes multiple fused, saturated all-carbon ring systems (e.g., ring systems containing 2, 3 or 4 carbocycles). Thus, carbocycles include polycyclic 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). When valence requirements permit, the rings of multiple fused ring systems may be interconnected via fused bonds, spiro bonds, and bridge bonds. For example, polycyclic carbocyclic rings can be connected to each other via a single carbon atom to form a spiro connection (e.g., spiropentane, spiro[4,5]decane, etc.), connected to each other via two adjacent carbon atoms to form a fused connection (e.g., decahydronaphthalene, norsabinane, norcarane, etc. carbocyclic rings), or connected to each other via two non-adjacent carbon atoms to form a bridged connection (e.g., norbornane, bicyclo[2.2.2]octane, etc.). Non-limiting examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo[2.2.1]heptane, pinane, and adamantane.

[0032] The term "gene" refers to a nucleic acid (eg, DNA or RNA) sequence that contains a partial or full-length coding sequence necessary to produce a polypeptide or precursor polypeptide.

[0033] As used herein, "gene product" refers to the product of a gene, such as an RNA transcript or a polypeptide.

[0034]

[0043] Unless otherwise specified, the term "halo" or "halogen" means a fluorine, chlorine, bromine, or iodine atom.

[0035] As used herein, the term "heteroaryl" refers to a single aromatic ring having at least one atom other than carbon in the ring, wherein the atom is selected from the group consisting of oxygen, nitrogen and sulfur; "heteroaryl" also includes multiple fused ring systems having at least one such aromatic ring, the multiple fused ring systems being further described below. Thus, "heteroaryl" includes a single aromatic ring of about 1 to 6 carbon atoms and about 1-4 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur. Sulfur and nitrogen atoms may also exist in oxidized form, provided that the ring is aromatic. Exemplary heteroaryl ring systems include, but are not limited to, pyridyl, pyrimidyl, oxazolyl and furanyl. "Heteroaryl" also includes multiple fused ring systems (e.g., ring systems comprising 2, 3 or 4 rings), wherein the heteroaryl as defined above is fused to one or more rings selected from cycloalkyl, aryl, heterocycle and heteroaryl. It should be understood that the point of attachment of the heteroaryl or heteroaryl multiple fused ring systems can be on any suitable atom of the heteroaryl or heteroaryl multiple fused ring systems, including carbon atoms and heteroatoms (e.g., nitrogen). Exemplary heteroaryl groups include, but are not limited to, pyridinyl, pyrrolyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrazolyl, thienyl, indolyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, furanyl, oxadiazolyl, thiadiazolyl, quinolyl, isoquinolyl, benzothiazolyl, benzoxazolyl, indazolyl, quinoxalinyl, and quinazolinyl.

[0036] The term "heterocycle" refers to a single saturated or partially unsaturated ring having at least one atom other than carbon in the ring, wherein the atom is selected from the group consisting of oxygen, nitrogen and sulfur; the term also includes multiple condensed ring systems having at least one such saturated or partially unsaturated ring, and the multiple condensed ring systems are further described below. Therefore, the term includes a single saturated or partially unsaturated ring (e.g., 3, 4, 5, 6 or 7-membered ring) having about 1 to 6 carbon atoms and about 1 to 3 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur in the ring. Sulfur and nitrogen atoms can also exist in their oxidized form. Exemplary heterocycles include but are not limited to azetidinyl, tetrahydrofuranyl and piperidinyl. The term "heterocycle" also includes multiple condensed ring systems (e.g., a ring system comprising 2, 3 or 4 rings), wherein a single heterocycle (as defined above) can be fused with one or more groups selected from cycloalkyl, aryl and heterocycle to form multiple condensed ring systems. When the valence requirement allows, the rings of multiple condensed ring systems can be interconnected via condensed bonds, spiro bonds and bridge bonds. It should be understood that each ring of a plurality of condensed ring systems can be connected in any order relative to each other. It should also be understood that the connection point of a plurality of condensed ring systems (as defined above for heterocycle) can be at any position of a plurality of condensed ring systems, including heterocycles, aryls and carbocyclic moieties of the ring. In one embodiment, the term heterocycle includes 3-15 heterocycles. In one embodiment, the term heterocycle includes 3-10 heterocycles. In one embodiment, the term heterocycle includes 3-8 heterocycles. In one embodiment, the term heterocycle includes 3-7 heterocycles. In one embodiment, the term heterocycle includes 3-6 heterocycles. In one embodiment, the term heterocycle includes 4-6 heterocycles. In one embodiment, the term heterocycle includes 3-10 monocyclic or bicyclic heterocycles containing 1 to 4 heteroatoms. In one embodiment, the term heterocycle includes 3-8 monocyclic or bicyclic heterocycles containing 1 to 3 heteroatoms. In one embodiment, the term heterocycle includes 3-6 monocyclic heterocycles containing 1 to 2 heteroatoms. In one embodiment, the term heterocycle includes 4-6 membered monocyclic heterocycles containing 1 to 2 heteroatoms. Exemplary heterocycles include, but are not limited to, aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, homopiperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, tetrahydrofuranyl, dihydrooxazolyl, tetrahydropyranyl, tetrahydrothiopyranyl, 1,2,3,4-tetrahydroquinolinyl, benzoxazinyl, dihydrooxazolyl, chromanyl, 1,2-dihydropyridinyl, 2,3-dihydrobenzofuranyl , 1,3-benzodioxolyl, 1,4-benzodioxanyl, spiro[cyclopropane-1,1'-isoindolyl]-3'-one, isoindolyl-1-one, 2-oxa-6-azaspiro[3.3]heptyl, imidazolidin-2-one imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin, dioxolane, phthalimide and 1,4-dioxane.

[0037] The term "sugar" includes monosaccharides, disaccharides and trisaccharides, all of which can be optionally substituted. The term includes glucose, sucrose, galactose and ribose, and deoxy sugars, such as deoxyribose and amino sugars, such as galactosamine. Sugar derivatives can be easily prepared as described in International Patent Application Publication Nos. WO 96 / 34005 and 97 / 03995. Sugar can be easily connected to the remainder of the compound of formula I by ether bond, thioether bond (for example, S-glycosides), amine nitrogen (for example, N-glycosides) or carbon-carbon bond (for example, C-glycosides). In one embodiment, sugar can be easily connected to the remainder of the compound of formula I by ether bond.

[0038] As used herein, the term "small interfering RNA" or "siRNA" refers to a double-stranded RNA (i.e., duplex RNA) that can reduce or inhibit the expression of a target gene or sequence (e.g., by mediating the degradation of an mRNA complementary to the siRNA sequence or inhibiting the translation of an mRNA complementary to the siRNA sequence) when the siRNA is in the same cell as the target gene or sequence. The siRNA may have substantial or complete identity to the target gene or sequence, or may contain a mismatch region (i.e., a mismatch motif). In certain embodiments, the siRNA may be about 19-25 (duplex) nucleotides in length, and preferably about 20-24, 21-22, or 21-23 (duplex) nucleotides in length. The siRNA duplex may contain a 3' overhang and a 5' phosphate end of about 1 to about 4 nucleotides or about 2 to about 3 nucleotides. Examples of siRNA include, but are not limited to, a double-stranded polynucleotide molecule assembled from two separate strands of molecules, one of which is a sense strand and the other is a complementary antisense strand.

[0039] In certain embodiments, the 5' and / or 3' overhangs on one or both strands of the siRNA comprise 1-4 (e.g., 1, 2, 3, or 4) modified and / or unmodified deoxythymidine (t or dT) nucleotides, 1-4 (e.g., 1, 2, 3, or 4) modified (e.g., 2'OMe) and / or unmodified uridine (U) ribonucleotides, and / or 1-4 (e.g., 1, 2, 3, or 4) modified (e.g., 2'OMe) and / or unmodified ribonucleotides or deoxyribonucleotides that are complementary to the target sequence (e.g., the 3' overhang in the antisense strand) or its complementary strand (e.g., the 3' overhang in the sense strand).

[0040] Preferably, the siRNA is chemically synthesized. siRNA can also be produced by cleavage of longer dsRNAs (eg, dsRNAs greater than about 25 nucleotides in length) using E. coli RNase III or Dicer. 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 Tech Notes, 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 is at least 50 nucleotides to about 100, 200, 300, 400 or 500 nucleotides in length. The length of dsRNA can be up to 1000, 1500, 2000, 5000 nucleotides or longer. dsRNA can encode the entire gene transcript or a portion of the gene transcript. In some cases, siRNA can be encoded by a plasmid (e.g., transcribed as a sequence that automatically folds into a duplex with a hairpin loop).

[0041] The phrase "inhibits the expression of a target gene" refers to the ability of the siRNA of the present invention to silence, reduce or inhibit the expression of a target gene. In order to examine the extent of gene silencing, a test sample (e.g., a biological sample from an organism of interest expressing a target gene, or a cultured cell sample expressing a target gene) is contacted with an siRNA that silences, reduces or inhibits the expression of a target gene. The expression of the target gene in the test sample is compared with the expression of the target gene in a control sample (e.g., a biological sample from an organism of interest expressing a target gene, or a cultured cell sample expressing a target gene) that is not contacted with siRNA. A value of 100% can be assigned to a control sample (e.g., a sample expressing a target gene). In certain embodiments, silencing, inhibition or reduction of target gene expression is achieved when the value of the test sample is about 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or 0% relative to a control sample (e.g., buffer alone, a siRNA sequence targeting a different gene, a scrambled siRNA sequence, etc.). Suitable assays include, but are not limited to, examining protein or mRNA levels using techniques known to those of skill in the art, e.g., dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays known to those of skill in the art.

[0042] An "effective amount" or "therapeutically effective amount" of a therapeutic nucleic acid (eg, siRNA) is an amount sufficient to produce the desired effect, such as inhibition of expression of the target sequence as compared to normal expression levels detected in the absence of the siRNA. In specific embodiments, inhibition of target gene or target sequence expression is achieved when the value obtained with the siRNA is about 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or 0% relative to a control (e.g., buffer alone, siRNA sequence targeting a different gene, scrambled siRNA sequence, etc.). Suitable assays for measuring expression of a target gene or target sequence include, but are not limited to, examining protein or mRNA levels using techniques known to those of skill in the art, e.g., dot blots, Northrop blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays known to those of skill in the art.

[0043] As used herein, the term "nucleic acid" refers to a polymer containing at least two nucleotides (i.e., deoxyribonucleotides or ribonucleotides) in a single-strand or double-stranded form, and includes DNA and RNA." Nucleotide" contains sugar deoxyribose (DNA) or ribose (RNA), bases and phosphate groups. Nucleotides are linked together by phosphate groups. "Bases" include purines and pyrimidines, which further include natural compounds adenine, thymine, guanine, cytosine, uracil, inosine and natural analogs, and synthetic derivatives of purines and pyrimidines, including but not limited to placing new reactive groups, such as but not limited to the modification of amines, alcohols, thiols, carboxylates and alkyl halides. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or connections, which are synthetic, naturally occurring and non-naturally occurring, and have binding properties similar to reference nucleic acids. 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 (PNA). Additionally, the nucleic acid may comprise one or more UNA moieties.

[0044] The term "protecting group" refers to a substituent that is usually used to block or protect a specific functional group on a compound. For example, an "amino protecting group" is a substituent connected to an amino group that blocks or protects an amino functional group in a compound. Suitable amino protecting groups include acetyl, trifluoroacetyl, tert-butyloxycarbonyl (BOC), benzyloxycarbonyl (CBZ) and 9-fluorenylmethyleneoxycarbonyl (Fmoc). Similarly, a "hydroxy protecting group" refers to a substituent of a hydroxyl group that blocks or protects a hydroxyl functional group. Suitable protecting groups include acetyl, silyl and 2,2-dimethoxypropylene. A "carboxyl protecting group" refers to a substituent of a carboxyl group that blocks or protects a carboxyl functional group. Common carboxyl protecting groups include phenylsulfonylethyl, cyanoethyl, 2-(trimethylsilyl)ethyl, 2-(trimethylsilyl)ethoxymethyl, 2-(p-toluenesulfonyl)ethyl, 2-(p-nitrophenylsulfinyl)ethyl, 2-(diphenylphosphino)-ethyl, nitroethyl, etc. For a general description of protecting groups and their use, see PGM Wuts and TW Greene, Greene's Protective Groups in Organic Synthesis 4th edition, Wiley-Interscience, New York, 2006.

[0045] The term "synthetic activation group" refers to a group that can be connected to an atom to activate that atom so that it forms a covalent bond with another reactive group. It should be understood that the properties of the synthetic activation group can depend on the atom it activates. For example, when the synthetic activation group is connected to an oxygen atom, the synthetic activation group is a group that will activate that oxygen atom to form a bond (e.g., ester, carbamate or ether bond) with another reactive group. Such synthetic activation groups are known. Examples of synthetic activation groups that can be connected to oxygen atoms include, but are not limited to, acetate, succinate, triflate and mesylate. When the synthetic activation group is connected to the oxygen atom of a carboxylic acid, the synthetic activation group can be a group that can be derived from a known coupling reagent (e.g., a known amide coupling reagent). Such coupling reagents are known. Examples of such coupling agents include, but are not limited to, N,N'-dicyclohexylcarbodiimide (DCC), hydroxybenzotriazole (HOBt), N-(3-dimethylaminopropyl)-N'-ethyl carbonate (EDC), (benzotriazol-1-yloxy)tris(dimethylamino)phosphine hexafluorophosphate (BOP), benzotriazol-1-yl-oxytripyrrolylphosphine hexafluorophosphate (PyBOP), (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridine 3-oxide hexafluorophosphate (HATU), propylphosphonic anhydride solution (T3P), or O-benzotriazol-1-yl-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU).

[0046] Nucleic Acids

[0047] The term "nucleic acid" includes any oligonucleotide or polynucleotide, wherein fragments containing up to 60 nucleotides are generally referred to as oligonucleotides, and longer fragments are referred to as polynucleotides. Deoxyribonucleotides are composed of a 5-carbon sugar called deoxyribose, which is covalently linked to phosphate on the 5' and 3' carbons of this sugar to form an alternating unbranched polymer. DNA can be, for example, in the form of antisense molecules, plasmid DNA, precondensed DNA, PCR products, vectors, expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups. Ribonucleotides are composed of similar repeating structures, in which 5-carbon sugars are ribose. RNA can be, for example, small interfering RNA (siRNA), Dicer-substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, tRNA, viral RNA (vRNA), self-amplification RNA (saRNA) and the form of their combination. Therefore, in the context of the present invention, the terms "polynucleotide" and "oligonucleotide" refer to polymers or oligomers of nucleotide or nucleoside monomers composed of naturally occurring bases, sugars, and intersugar (backbone) connections. The terms "polynucleotide" and "oligonucleotide" also include polymers or oligomers comprising non-naturally occurring monomers or portions thereof with similar functions. Such modified or substituted oligonucleotides are generally superior to the native form due to their properties, such as enhanced cellular uptake, reduced immunogenicity, and increased stability in the presence of nucleases.

[0048] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as sequences explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced by mixed bases 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)).

[0049] In certain embodiments, the bidentate targeting ligands described herein can be conjugated to nucleic acids. In certain embodiments, nucleic acids are nucleic acids described herein. For example, nucleic acids used herein can be single-stranded DNA or RNA, or double-stranded DNA or RNA, or DNA-RNA hybrids. Examples of double-stranded RNA are described herein, and include, for example, siRNA and other RNAi agents, such as aiRNA and precursor miRNA. Single-stranded nucleic acids include, for example, antisense oligonucleotides, ribozymes, mature miRNAs, and triplex-forming oligonucleotides.

[0050] In certain embodiments, nucleic acid is an oligonucleotide. In a specific embodiment, the length of the oligonucleotide is in the range of about 10 to about 100 nucleotides. In various related embodiments, the length of single strand, double strand and three strand oligonucleotides can be in the range of about 10 to about 60 nucleotides, about 15 to about 60 nucleotides, about 20 to about 50 nucleotides, about 15 to about 30 nucleotides or about 20 to about 30 nucleotides.

[0051] 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), micro RNA (miRNA), tRNA, rRNA, tRNA, viral RNA (vRNA), self-amplifying RNA (sa-RNA), and combinations thereof.

[0052] In certain embodiments, the nucleic acid is an antisense molecule. In certain embodiments, the nucleic acid is a miRNA molecule. In certain embodiments, the nucleic acid is a siRNA. Suitable siRNA and methods and intermediates that can be used for its preparation are reported in International Patent Application Publication No. WO2016 / 054421.

[0053] Target Gene

[0054] In certain embodiments, nucleic acid (for example, siRNA) can be used to make the translation (that is, expression) of the gene being paid attention to be down-regulated or silent.The gene being paid attention to includes but is not limited to the gene relevant to viral infection and survival, the gene relevant to metabolic disease and illness (for example, liver disease and illness), the gene relevant to tumor generation and cell transformation (for example, cancer), angiogenesis gene, immunomodulatory gene (for example, those genes relevant to inflammatory and autoimmune response), ligand receptor gene and the gene relevant to neurodegenerative disease.In certain embodiments, the gene being paid attention to is expressed in hepatocyte.

[0055] Genes associated with viral infection and survival include those genes expressed by the virus in order to bind to, enter and replicate in cells. Of particular interest are viral sequences associated with chronic viral diseases.Viral sequences of particular interest include sequences of the following viruses: filoviruses, such as Ebola virus and Marburg virus (see, e.g., Geisbert et al., J. Infect. Dis., 193: 1650-1657 (2006)); arenaviruses, such as Lassa virus, Junin virus, Machupo virus, Guanarito virus, and Sabia virus (Buchmeier et al., Arenaviridae: the viruses and their replication, FIELDS VIROLOGY, Knipe et al. (eds.), 4th edition, Lippincott-Raven, Philadelphia, (2001)); influenza viruses, such as influenza A, B, and C viruses (see, e.g., Steinhauer et al., Annu Rev Genet., 36: 305-332 (2002); and Neumann et al., J Gen Genet., 37: 317-331 (2003); Virol., 83:2635-2662 (2002)); hepatitis viruses (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 edition, Lippincott-Raven, Philadelphia (2001)); human immunodeficiency virus (HIV) (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)); herpes virus (Jia et al., J. Virol., 77:3301 (2003)); and human papillomavirus (HPV) (Hall et al., J. Virol., 77:6066 (2003); Jiang et al., Oncogene, 21:6041 (2002)).

[0056] Exemplary 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 listed in, for example, Genbank Accession Nos. NC_002549; AY769362; NC_006432; NC_004161; AY729654; AY354458; AY142960; AB050936; AF522874; AF499101; AF272001; and AF086833. The Ebola virus VP24 sequence is listed in, for example, Genbank Accession Nos. U77385 and AY058897. The Ebola virus L-pol sequence is listed in, for example, Genbank Accession No. X67110. The Ebola virus VP40 sequence is listed, for example, in Genbank Accession No. AY058896. The Ebola virus NP sequence is listed, for example, in Genbank Accession No. AY058895. The Ebola virus GP sequence is listed, for example, in Genbank Accession No. 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 listed, for example, in Genbank Accession Nos. L11365 and X61274. The complete genome sequence of Marburg virus is listed, for example, in Genbank Accession Nos. NC_001608; AY430365; AY430366; and AY358025. The Marburg virus GP sequence is listed, for example, in Genbank Accession Nos. AF005734; AF005733; and AF005732. The Marburg virus VP35 sequence is listed, for example, in Genbank Accession Nos. AF005731 and AF005730. Additional Marburg virus sequences are listed, for example, in Genbank Accession Nos. X64406; Z29337; AF005735; and Z12132. Non-limiting examples of siRNA molecules targeting Ebola virus and Marburg virus nucleic acid sequences include those described in U.S. Patent Publication No. 20070135370, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0057] Exemplary 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). Influenza A NP sequences are, for example, in Genbank accession numbers NC_004522; AY818138; AB166863; AB188817; AB189046; AB189054; AB189062; AY646169; AY646177; AY651486; AY651493; AY651494; AY651495; AY651496; AY651497; AY651498; AY651499; AY651491; AY651493; AY651494; AY651495; AY651496; AY651497; AY651498; AY651499; AY651498; AY651499; AY651498; AY651499; AY651499; AY651491; AY651493; AY651494; AY651495; AY666863; AB188817; AB189046; AB189054; AB189062; AY646169; AY646177; AY651486; AY651493; AY651494; AY651495 listed in 1497; AY651498; AY651499; AY651500; AY651501; AY651502; AY651503; AY651504; AY651505; AY651506; AY651507; AY651509; AY651528; AY770996; AY790308; AY818138; and AY818140. The influenza A PA sequences are in Genbank accession numbers AY818132; AY790280; AY646171; AY818132; AY818133; AY646179; AY818134; AY551934; AY651613; AY651610; AY651620; AY651617; AY651600; AY651 Non-limiting examples of siRNA molecules targeting influenza virus nucleic acid sequences include those described in U.S. Patent Publication No. 20070218122, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0058] Exemplary hepatitis virus nucleic acid sequences 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 proteins including C proteins and C-related proteins, capsid and envelope proteins (including S, M and / or L proteins) or fragments thereof) (see, e.g., FIELDS VIROLOGY, supra). Exemplary hepatitis C virus (HCV) nucleic acid sequences that can be silenced include, but are not limited to, 5'-untranslated regions (5'-UTRs), 3'-untranslated regions (3'-UTRs), polyprotein translation start codon regions, internal ribosome entry site (IRES) sequences, and / or nucleic acid sequences encoding core proteins, E1 proteins, E2 proteins, p7 proteins, NS2 proteins, NS3 protease / helicases, NS4A proteins, NS4B proteins, NS5A proteins, and / or NS5B RNA-dependent RNA polymerases. HCV genome sequences are listed, 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). Hepatitis A virus nucleic acid sequences are listed, for example, in Genbank Accession No. NC_001489; hepatitis B virus nucleic acid sequences are listed, for example, in Genbank Accession No. NC_003977; hepatitis D virus nucleic acid sequences are listed, for example, in Genbank Accession No. NC_001653; hepatitis E virus nucleic acid sequences are listed, for example, in Genbank Accession No. NC_001434; and hepatitis G virus nucleic acid sequences are listed, for example, in Genbank Accession No. NC_001710. Silencing of sequences encoding genes associated with viral infection and survival can be conveniently used in combination with the administration of conventional agents for treating viral disorders. Non-limiting examples of siRNA molecules targeting hepadnavirus nucleic acid sequences include those described in U.S. Patent Publication Nos. 20060281175, 20050058982, and 20070149470; U.S. Patent No. 7,348,314; and U.S. Provisional Application No. 61 / 162,127, filed March 20, 2009, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0059] Genes associated with metabolic diseases and disorders (e.g., liver-targeted disorders and liver diseases and disorders) include, for example, genes expressed in dyslipidemia (e.g., liver X receptors, such as LXRα and LXRβ (Genbank Accession No. NM_007121), farnesoid X receptor (FXR) (Genbank Accession No. NM_005123), sterol regulatory element binding protein (SREBP), site-1 protease (SIP), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase), apolipoprotein B (ApoB) (Genbank Accession No. NM_000384), apolipoprotein CIII (ApoC3) (Genbank Accession Nos. NM_000040 and NG_0004999), region: 5001.81. 64) and apolipoprotein E (ApoE) (Genbank accession numbers NM_000041 and NG_007084 region: 5001.8612)); and diabetes (e.g., glucose 6-phosphatase) (see, 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)). It will be appreciated by those skilled in the art that genes associated with metabolic diseases and disorders (e.g., diseases and disorders targeting the liver and liver diseases and disorders) include genes expressed in the liver itself as well as genes expressed in other organs and tissues. The silencing of sequences encoding genes associated with metabolic diseases and disorders can be conveniently used in combination with the administration of conventional agents for treating the disease or disorder. Non-limiting examples of siRNA molecules targeting the ApoB gene include those described in U.S. Patent Publication No. 20060134189, the disclosure of which is incorporated herein by reference in its entirety for all purposes. Non-limiting examples of siRNA molecules targeting the ApoC3 gene include 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.

[0060] Examples of gene sequences associated with tumorigenesis and cellular transformation (e.g., cancer or other neoplasia) include mitotic kinesins, such as Eg5 (KSP, KIF11; Genbank Accession No. NM_004523); serine / threonine kinases, such as polo-like kinase 1 (PLK-1) (Genbank Accession No. NM_005030; Barr et al., Nat. Rev. Mol. Cell. Biol., 5:429-440 (2004)); tyrosine kinases, such as WEE1 (Genbank Accession Nos. NM_003390 and NM_001143976); apoptosis inhibitors, such as XIAP (Genbank Accession No. NM_004523); k accession number NM_001167); COP9 signal transducer subunits, such as CSN1, CSN2, CSN3, CSN4, CSN5 (JAB1; Genbank accession number NM_006837); CSN6, CSN7A, CSN7B and CSN8; ubiquitin ligases, such as COP1 (RFWD2; Genbank accession numbers NM_022457 and NM_001001740); and histone deacetylases, such as HDAC1, HDAC2 (Genbank accession number NM_001527), HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, etc. Non-limiting examples of siRNA molecules targeting Eg5 and XIAP genes include 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. Non-limiting examples of siRNA molecules targeting PLK-1 genes include 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 disclosure of which is incorporated herein by reference in its entirety for all purposes. Non-limiting examples of siRNA molecules targeting CSN5 genes include those described in U.S. Provisional Application No. 61 / 045,251, filed April 15, 2008, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0061] 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)); overexpressed sequences, such as multidrug resistance genes (Nieth et al., FEBS Lett., 545:144 (2003); Wu et al., Cancer Res. 63:1515 (2003)), cell cycle proteins (Li et al., Cancer Res., 63:3593 (2003); Zou et al., Genes Dev., 16:2923 (2002)), β-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 / ErbBl (Genbank Accession Nos. NM_005228, NM_201282, NM_201283, and NM_201284; see also 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)). Non-limiting examples of siRNA molecules targeting EGFR genes include 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.

[0062] Silencing of sequences encoding DNA repair enzymes can be used in combination with the administration of chemotherapeutic agents (Collis et al., Cancer Res., 63:1550 (2003)). Genes encoding proteins associated with tumor migration are also target sequences of interest, for example, integrins, selectins, and metalloproteinases. The foregoing examples are not exclusive. Those skilled in the art will appreciate that any complete or partial gene sequence that contributes to or promotes tumorigenesis or cell transformation, tumor growth, or tumor migration can be included as a template sequence.

[0063] Angiogenesis genes can promote the formation of new blood vessels. Of particular interest is vascular endothelial growth factor (VEGF) (Reich et al., Mol. Vis., 9:210 (2003)) or VEGFR. siRNA sequences targeting VEGFR are listed in, for example, GB 2396864; U.S. Patent Publication No. 20040142895; and CA 2456444, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0064] Anti-angiogenic genes can inhibit new blood vessel formation. These genes are particularly useful for treating those cancers in which angiogenesis plays a role in the pathological development of the disease. Examples of anti-angiogenic genes include, but are not limited to, endostatin (see, e.g., U.S. Patent No. 6,174,861), angiostatin (see, e.g., U.S. Patent No. 5,639,725), and VEGFR2 (see, e.g., Decaussin et al., J. Pathol., 188: 369-377 (1999)), the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0065] 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. Fas and Fas ligand genes are also immunomodulatory target sequences of interest (Song et al., Nat. Med., 9: 347 (2003)). Genes encoding secondary signaling molecules in hematopoietic and lymphoid cells are also included in the present invention, for example, Tec family kinases, such as Bruton's tyrosine kinase (Btk) (Heinonen et al., FEBS Lett., 527:274 (2002)).

[0066] Cell receptor ligands include those 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.) to regulate (e.g., inhibit, activate, etc.) physiological pathways involved in the receptors (e.g., glucose level regulation, blood cell development, mitosis, 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 trinucleotide repeat sequences (e.g., CAG repeat sequences) expansion can be used to silence pathogenic sequences of neurodegenerative disorders caused by trinucleotide repeat sequence expansion, such as spinobulbar muscular atrophy and Huntington's Disease (Caplen et al., Hum. Mol. Genet., 11: 175 (2002)).

[0067] Certain other target genes that can be targeted by nucleic acids (e.g., by siRNA) to downregulate or silence the expression of the gene include, but are not limited to, smooth muscle aortic actin alpha 2 (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), aldehyde-keto reductase family 1 member (ACET), alpha-2-HS-glycoprotein (AHSG ... 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 F (APOF), beta-2-glycoprotein 1 (APOH) , Aquaporin-9 (AQP9), Bile acid-CoA:amino acid N-acyltransferase (BAAT), C4b-binding protein β chain (C4BPB), Hypothetical uncharacterized protein encoded by LINC01554 (C5orf27), Complement factor 3 (C3), Complement factor 5 (C5), Complement factor C6 (C6), Complement factor C8 α chain (C8A), Complement factor C8 β chain (C8B), Complement factor C8 γ chain (C8G), Complement factor 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), CXC 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 ω-hydroxylase CYP4F2 (CYP4F2), 7-α-hydroxycholester-4-en-3-one 12-α-hydroxylase (CYP8B1), dipeptidyl peptidase 4 (DPP4), coagulation factor 12 (F12), coagulation factor II (thrombin) (F2), coagulation factor IX (F9), fibrinogen α chain (FGA), fibrinogen β chain (FGB), fibrinogen γ chain (FGG),Fibrinogen-like 1 (FGL1), flavin-containing monooxygenase 3 (FMO3), flavin-containing monooxygenase 5 (FMO5), population-specific component (vitamin D binding protein) (GC), growth hormone receptor (GHR), glycine N-methyltransferase (GNMT), hyaluronan binding protein 2 (HABP2), hepcidin antimicrobial peptide (HAMP), hydroxy acid oxidase (glycolate oxidase) 1 (HAO1), HGF activating factor (HGFAC), haptoglobin-related protein; haptoglobin (HPR), heme-binding protein (HPX), histidine-rich glycoprotein (HRG), hydroxysteroid (11-β) dehydrogenase 1 (HSD11B1), hydroxysteroid (17-β) dehydrogenase 13 (HSD17B13), inter-α-trypsin inhibitor heavy chain H1 (ITIH1), inter-α-trypsin inhibitor heavy chain H2 (ITIH2), inter-α-trypsin inhibitor heavy chain H3 (ITIH3), inter-α-trypsin inhibitor heavy chain H4 (ITIH4), kallikrein (KLKB1), lactate dehydrogenase A (LDHA), liver expressed antimicrobial peptide 2 (LEAP2), leukocyte-derived chemokine 2 (LECT2), lipoprotein (a) (LPA), mannan-binding lectin serine peptidase 2 (MASP2), S-adenosylmethionine synthase isoform 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), serine protease inhibitor (Serpin) family A member 1 (SERPINA1), serine protease inhibitor A11 (SERPINA11), human kallistatin binding protein (Kallistatin, SERPINA4), corticosteroid binding globulin (SERPINA6), antithrombin-III (SERPINC1), heparin cofactor 2 (SERPIND1), serine protease inhibitor family H member 1 (SERPINH1), solute carrier family 5 member 2 (SLC5A2), sodium / bile acid co-transporter (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 2 glucose transporter member 2 (SLC2A2), sodium-coupled 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).

[0068] In addition to silencing the expression of any one of the above-mentioned genes to achieve the effectiveness of therapeutic purposes, some nucleic acids described herein (e.g., siRNA) can also be used for research and development applications and diagnosis, prevention, prognosis, clinical and other health care applications. As non-limiting examples, some nucleic acids (e.g., siRNA) can be used in target validation studies, and the target validation studies are intended to test whether the gene of interest has the potential as a therapeutic target. Some nucleic acids (e.g., siRNA) can also be used in target identification studies, and the target identification studies are intended to find genes as potential therapeutic targets.

[0069] Generation of siRNA molecules

[0070] Several forms of siRNA can be provided, including, for example, as one or more isolated small interfering RNA (siRNA) duplexes, as longer double-stranded RNA (dsRNA), or as siRNA or dsRNA transcribed from a transcription cassette of a DNA plasmid. In some embodiments, siRNA can be produced enzymatically or by partial / complete organic synthesis, and modified ribonucleotides can be introduced by in vitro enzymatic or organic synthesis. In some cases, each strand is chemically prepared. Methods for synthesizing RNA molecules are known in the art, for example, chemical synthesis methods described in Verma and Eckstein (1998) or described herein.

[0071] Methods for isolating RNA, synthesizing RNA, hybridizing nucleic acids, making and screening cDNA libraries, and performing PCR are well known in the art (see, e.g., Gubler and Hoffman, Gene, 25:263-269 (1983); Sambrook et al., supra; Ausubel et al., supra), as are PCR methods (see U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., ed., 1990)). Expression libraries are also well known to those skilled in the art. Additional basic texts disclosing general methods for use 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., ed., 1994). The disclosures of these references are incorporated herein by reference in their entirety for all purposes.

[0072] Typically, siRNA is chemically synthesized. Oligonucleotides comprising siRNA molecules of the present invention can be synthesized using any of a variety of techniques known in the art, such as those described in the following literature: 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). The synthesis of oligonucleotides utilizes common nucleic acid protection and coupling groups, such as dimethoxytrityl at the 5' end and phosphoramidite at the 3' end. As a non-limiting example, small-scale synthesis can be performed on an Applied Biosystems synthesizer using a 0.2 μmol scale protocol. Alternatively, synthesis at a 0.2 μmol scale can be performed on a 96-well plate synthesizer from Protogene (Palo Alto, CA). However, larger or smaller scale synthesis is also within the scope of the present invention. Suitable reagents for oligonucleotide synthesis, methods for RNA deprotection, and methods for RNA purification are known to those skilled in the art.

[0073] The siRNA molecule can be assembled from two different oligonucleotides, one of which contains the sense strand of the siRNA and the other contains the antisense strand of the siRNA. For example, each strand can be synthesized separately and linked together by hybridization or ligation after synthesis and / or deprotection.

[0074] Linking group

[0075] The compounds and conjugates of the invention may include one or more linking groups (e.g., L 3 or L 4 ). The structure of each linking group can vary, provided that the conjugate functions as described herein. For example, the structure of each linking group varies in length and atomic composition, and each linking group can be branched, unbranched, cyclic, or a combination thereof. The linking group can also adjust the solubility, stability, or aggregation characteristics of the conjugate.

[0076] In one embodiment, each linking group comprises about 3-1000 atoms. In one embodiment, each linking group comprises about 3-500 atoms. In one embodiment, each linking group comprises about 3-200 atoms. In one embodiment, each linking group comprises about 3-50 atoms. In one embodiment, each linking group comprises about 10-1000 atoms. In one embodiment, each linking group comprises about 10-500 atoms. In one embodiment, each linking group comprises about 10-200 atoms. In one embodiment, each linking group comprises about 10-50 atoms.

[0077] In one embodiment, each linking group comprises atoms selected from H, C, N, S and O.

[0078] In one embodiment, each linking group comprises atoms selected from H, C, N, S, P and O.

[0079] In one embodiment, each linking group comprises a branched or unbranched, saturated or unsaturated hydrocarbon chain having about 1 to 1000 (or 1-750, 1-500, 1-250, 1-100, 1-50, 1-25, 1-10, 1-5, 5-1000, 5-750, 5-500, 5-250, 5-100, 5-50, 5-25, 5-10 or 2-5 carbon atoms), wherein one or more carbon atoms are optionally independently replaced by -O-, -S, -N (R a)-, 3-7 membered heterocyclic, 5-6 membered heteroaryl or carbocyclic ring, and each chain, 3-7 membered heterocyclic, 5-6 membered heteroaryl or carbocyclic ring is optionally and independently replaced by one or more (e.g., 1, 2, 3, 4, 5 or more) selected from (C1-C6) alkyl, (C1-C6) alkoxy, (C3-C6) cycloalkyl, (C1-C6) alkanoyl, (C1-C6) alkanoyloxy, (C1-C6) alkoxycarbonyl, (C1-C6) alkylthio, azido, cyano, nitro, halogen, -N(R a )2, hydroxy, oxo (=O), carboxyl, aryl, aryloxy, heteroaryl and heteroaryloxy substituents, wherein each R a In one embodiment, the linker comprises a branched or unbranched, saturated or unsaturated hydrocarbon chain having about 1 to 1000 (or 1-750, 1-500, 1-250, 1-100, 1-50, 1-25, 1-10, 1-5, 5-1000, 5-750, 5-500, 5-250, 5-100, 5-50, 5-25, 5-10 or 2-5 carbon atoms), wherein one or more carbon atoms are optionally independently replaced by -O-, -S, -N (R a )-permutation, where each R a are independently H or (C1-C6)alkyl.

[0080] In one embodiment, each linking group comprises polyethylene glycol. In one embodiment, the linking group comprises polyethylene glycol connected to the rest 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 repeating (e.g., -CH2CH2O-) units (Greenwald, RB, etc., Poly (ethylene glycol) Prodrugs: Altered Pharmacokinetics and Pharmacodynamics, Chapter 2.3.1., 283-338; Filpula, D., etc., Releasable PEGylation of proteins with customized linkers, Advanced Drug Delivery, 60, 2008, 29-49; Zhao, H., etc., Drug Conjugates with Poly (Ethylene Glycol), Drug Delivery in Oncology, 2012, 627-656).

[0081] Embodiments of the present invention

[0082] One aspect of the present invention is a compound of formula I or a salt thereof as described in the Summary of the Invention.

[0083] In one embodiment, A is absent.

[0084] In one embodiment, A is 3-20 membered cycloalkyl, 5-20 membered aryl, 5-20 membered heteroaryl, or 3-20 membered heterocycloalkyl.

[0085] In one embodiment, B is a 5-10 membered aryl group.

[0086] In one embodiment, B is naphthyl or phenyl.

[0087] In one embodiment, B is phenyl.

[0088] In one embodiment, the group:

[0089]

[0090] yes:

[0091]

[0092] In one embodiment, B is a 5-10 membered heteroaryl.

[0093] In one embodiment, B is pyridinyl, pyrimidinyl, quinolyl, isoquinolyl, imidazolyl, thiazolyl, oxadiazolyl or oxazolyl.

[0094] In one embodiment, the group:

[0095]

[0096] yes:

[0097]

[0098] In one embodiment, the group:

[0099]

[0100] yes:

[0101]

[0102] In one embodiment, L 1 is a divalent, unbranched, saturated hydrocarbon chain having 0 to 20 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) carbon atoms in the hydrocarbon chain are optionally replaced by -O-, -NR X -、-NR X -C(=O)-, -C(=O)-NR X- or -S- substitution, and wherein R X is hydrogen or (C1-C6)alkyl, and wherein the hydrocarbon chain is optionally substituted with one or more substituents selected from oxo (=O) and halo.

[0103] In one embodiment, L 1 is a divalent, unbranched, saturated hydrocarbon chain having 0 to 12 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) carbon atoms in the hydrocarbon chain are optionally replaced by -O-, -NR X -C(=O)- or -C(=O)-NR X - permutation, and where R X It is hydrogen or (C1-C6) alkyl.

[0104] In one embodiment, L 1 yes:

[0105] -C(=O)N(H)-CH2CH2OCH2CH2OCH2CH2-,

[0106] -C(=O)N(H)-CH2CH2OCH2CH2OCH2CH2OCH2CH2-,

[0107] -C(=O)N(CH3)-CH2CH2OCH2CH2OCH2CH2-, or

[0108] -C(=O)N(CH3)-CH2CH2OCH2CH2OCH2CH2OCH2CH2-.

[0109] In one embodiment, L 2 is a divalent, unbranched, saturated hydrocarbon chain having 0 to 20 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) carbon atoms in the hydrocarbon chain are optionally replaced by -O-, -NR X -、-NR X -C(=O)-, -C(=O)-NR X - or -S- substitution, and wherein R X is hydrogen or (C1-C6)alkyl, and wherein the hydrocarbon chain is optionally substituted with one or more substituents selected from oxo (=O) and halo.

[0110] In one embodiment, L 2 is a divalent, unbranched, saturated hydrocarbon chain having 0 to 12 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) carbon atoms in the hydrocarbon chain are optionally replaced by -O-, -NR X -C(=O)- or -C(=O)-NR X - permutation, and where R XIt is hydrogen or (C1-C6) alkyl.

[0111] In one embodiment, L 2 yes:

[0112] -C(=O)N(H)-CH2CH2OCH2CH2OCH2CH2-,

[0113] -C(=O)N(H)-CH2CH2OCH2CH2OCH2CH2OCH2CH2-,

[0114] -C(=O)N(CH3)-CH2CH2OCH2CH2OCH2CH2-, or

[0115] -C(=O)N(CH3)-CH2CH2OCH2CH2OCH2CH2OCH2CH2-.

[0116] In one embodiment, R 1 yes:

[0117]

[0118] in:

[0119] X is NR 20 and Y is selected from -(C=O)R 21 、-SO2R 22 and -(C=O)NR 23 R 24 ; or X is -(C=O)- and Y is NR 25 R 26 ; or X is -NR 37 R 38 And Y does not exist

[0120] R 20 is hydrogen or (C1-C4)alkyl;

[0121] R 21 , R 22 , R 23 , R 24 , R 25 and R 26 Each independently selected from hydrogen, (C1-C8) alkyl, (C1-C8) alkoxy and (C 3- any of (C1-C8)alkyl, (C1-C8)alkoxy and (C3-C6)cycloalkyl groups optionally substituted with one or more groups independently selected from the group consisting of halo, (C1-C4)alkyl and (C1-C4)alkoxy;

[0122] R 27 Yes -OH, -NR25 R 26 or -F;

[0123] R 28 Yes -OH, -NR 25 R 26 or -F;

[0124] R 29 Yes -OH, -NR 25 R 26 、-F、-N3、-NR 35 R 36 or a 5-membered heterocyclic ring, the 5-membered heterocyclic ring is optionally substituted by one or more groups independently selected from the group consisting of halogen, hydroxy, carboxyl, amino, (C1-C4) alkyl, aryl and (C1-C4) alkoxy, wherein any (C1-C4) alkyl and (C1-C4) alkoxy are optionally substituted by one or more groups independently selected from the group consisting of halogen, and wherein any aryl is optionally substituted by one or more groups independently selected from halogen, hydroxy, nitro, cyano, amino, (C1-C8) alkyl, C1-C8) alkoxy (C1-C8)alkyl, (C1-C8)alkoxy, (C1-C8)alkanoyl, (C1-C8)alkoxycarbonyl, (C1-C8)alkanoyloxy and (C3-C6)cycloalkyl, wherein any of the (C1-C8)alkyl, (C1-C8)alkoxy, (C1-C8)alkanoyl, (C1-C8)alkoxycarbonyl, (C1-C8)alkanoyloxy and (C3-C6)cycloalkyl groups are optionally substituted with one or more groups independently selected from the group consisting of halo, (C1-C4)alkyl and (C1-C4)alkoxy;

[0125] Each R 35 and R 36 R is independently selected from the group consisting of hydrogen, (C1-C8)alkyl, (C1-C8)alkoxy and (C3-C6)cycloalkyl, wherein any of (C1-C8)alkyl, (C1-C8)alkoxy and (C3-C6)cycloalkyl is optionally substituted with one or more groups independently selected from the group consisting of halo and (C1-C4)alkoxy; or 35 and R 36 Together with the nitrogen to which they are attached, they form a 5-6 membered heteroaryl ring, which is optionally substituted by one or more groups independently selected from the group consisting of (C1-C8)alkyl, (C1-C8)alkoxy, aryl and (C3-C6)cycloalkyl, wherein any aryl and (C3-C6)cycloalkyl are optionally substituted by one or more groups R 39 replace;

[0126] Each R 37 and R 38R is independently selected from the group consisting of hydrogen, (C1-C8) alkyl, (C1-C8) alkoxy, (C1-C8) alkanoyl, (C1-C8) alkoxycarbonyl, (C1-C8) alkanoyloxy and (C3-C6) cycloalkyl, wherein any of (C1-C8) alkyl, (C1-C8) alkoxy, (C1-C8) alkanoyl, (C1-C8) alkoxycarbonyl, (C1-C8) alkanoyloxy and (C3-C6) cycloalkyl is optionally substituted with one or more groups independently selected from the group consisting of halo, (C1-C4) alkyl and (C1-C4) alkoxy; or 37 and R 38 Together with the nitrogen to which they are attached, they form a 5-8 membered heterocyclic ring, which is optionally substituted by one or more groups independently selected from the group consisting of halo, hydroxy, carboxyl, amino, oxo (═O), (C1-C4) alkyl and (C1-C4) alkoxy, wherein any (C1-C4) alkyl and (C1-C4) alkoxy is optionally substituted by one or more groups independently selected from halo; and

[0127] Each R 39 Independently selected from the group consisting of (C1-C8)alkyl, (C1-C8)alkoxy and (C3-C6)cycloalkyl, wherein any of the (C1-C8)alkyl, (C1-C8)alkoxy and (C3-C6)cycloalkyl groups are optionally substituted by one or more groups independently selected from halogen.

[0128] In one embodiment, R 1 yes:

[0129]

[0130] In one embodiment, R 1 yes:

[0131]

[0132] In one embodiment, R 1 yes:

[0133]

[0134] In one embodiment, R 1 yes:

[0135]

[0136] In one embodiment, R 2 yes:

[0137]

[0138] in:

[0139] X is NR 20 and Y is selected from -(C=O)R 21 、-SO2R 22 and -(C=O)NR 23 R 24 ; or X is -(C=O)- and Y is NR 25 R 26 ; or X is -NR 37 R 38 And Y does not exist

[0140] R 20 is hydrogen or (C1-C4)alkyl;

[0141] R 21 , R 22 , R 23 , R 24 , R 25 and R 26 Each independently selected from hydrogen, (C1-C8) alkyl, (C1-C8) alkoxy and (C 3- any of (C1-C8)alkyl, (C1-C8)alkoxy and (C3-C6)cycloalkyl groups optionally substituted with one or more groups independently selected from the group consisting of halo, (C1-C4)alkyl and (C1-C4)alkoxy;

[0142] R 27 Yes -OH, -NR 25 R 26 or -F;

[0143] R 28 Yes -OH, -NR 25 R 26 or -F;

[0144] R 29 Yes -OH, -NR 25 R 26 、-F、-N3、-NR 35 R 36or a 5-membered heterocyclic ring, the 5-membered heterocyclic ring is optionally substituted by one or more groups independently selected from the group consisting of halogen, hydroxy, carboxyl, amino, (C1-C4) alkyl, aryl and (C1-C4) alkoxy, wherein any (C1-C4) alkyl and (C1-C4) alkoxy are optionally substituted by one or more groups independently selected from the group consisting of halogen, and wherein any aryl is optionally substituted by one or more groups independently selected from halogen, hydroxy, nitro, cyano, amino, (C1-C8) alkyl, C1-C8) alkoxy (C1-C8)alkyl, (C1-C8)alkoxy, (C1-C8)alkanoyl, (C1-C8)alkoxycarbonyl, (C1-C8)alkanoyloxy and (C3-C6)cycloalkyl, wherein any of the (C1-C8)alkyl, (C1-C8)alkoxy, (C1-C8)alkanoyl, (C1-C8)alkoxycarbonyl, (C1-C8)alkanoyloxy and (C3-C6)cycloalkyl groups are optionally substituted with one or more groups independently selected from the group consisting of halo, (C1-C4)alkyl and (C1-C4)alkoxy;

[0145] Each R 35 and R 36 R is independently selected from the group consisting of hydrogen, (C1-C8)alkyl, (C1-C8)alkoxy and (C3-C6)cycloalkyl, wherein any of (C1-C8)alkyl, (C1-C8)alkoxy and (C3-C6)cycloalkyl is optionally substituted with one or more groups independently selected from the group consisting of halo and (C1-C4)alkoxy; or 35 and R 36 Together with the nitrogen to which they are attached, they form a 5-6 membered heteroaryl ring, which is optionally substituted by one or more groups independently selected from the group consisting of (C1-C8)alkyl, (C1-C8)alkoxy, aryl and (C3-C6)cycloalkyl, wherein any aryl and (C3-C6)cycloalkyl are optionally substituted by one or more groups R 39 replace;

[0146] Each R 37 and R 38 R is independently selected from the group consisting of hydrogen, (C1-C8) alkyl, (C1-C8) alkoxy, (C1-C8) alkanoyl, (C1-C8) alkoxycarbonyl, (C1-C8) alkanoyloxy and (C3-C6) cycloalkyl, wherein any of (C1-C8) alkyl, (C1-C8) alkoxy, (C1-C8) alkanoyl, (C1-C8) alkoxycarbonyl, (C1-C8) alkanoyloxy and (C3-C6) cycloalkyl is optionally substituted with one or more groups independently selected from the group consisting of halo, (C1-C4) alkyl and (C1-C4) alkoxy; or 37 and R 38Together with the nitrogen to which they are attached, they form a 5-8 membered heterocyclic ring, which is optionally substituted by one or more groups independently selected from the group consisting of halo, hydroxy, carboxyl, amino, oxo (═O), (C1-C4) alkyl and (C1-C4) alkoxy, wherein any (C1-C4) alkyl and (C1-C4) alkoxy is optionally substituted by one or more groups independently selected from halo; and

[0147] Each R 39 Independently selected from the group consisting of (C1-C8)alkyl, (C1-C8)alkoxy and (C3-C6)cycloalkyl, wherein any of the (C1-C8)alkyl, (C1-C8)alkoxy and (C3-C6)cycloalkyl groups are optionally substituted by one or more groups independently selected from halogen.

[0148] In one embodiment, R 2 yes:

[0149]

[0150] In one embodiment, R 2 yes:

[0151]

[0152] In one embodiment, R 2 yes:

[0153]

[0154] In one embodiment, R 2 yes

[0155]

[0156] In one embodiment, L 3 is a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 0 to 50 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) carbon atoms in the hydrocarbon chain are optionally replaced by -O-, -NR X -、-NR X -C(=O)-, -C(=O)-NR X - or -S- substitution, and wherein R Xis hydrogen or (C1-C6)alkyl, and wherein the hydrocarbon chain is optionally substituted by one or more (e.g., 1, 2, 3 or 4) substituents selected from (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (═O), carboxyl, aryl, aryloxy, heteroaryl and heteroaryloxy.

[0157] In one embodiment, L 3 is a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 20 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) carbon atoms in the hydrocarbon chain are optionally replaced by -O-, -NR X -、-NR X -C(=O)-, -C(=O)-NR X - or -S- substitution, and wherein R X is hydrogen or (C1-C6)alkyl, and wherein the hydrocarbon chain is optionally substituted by one or more (e.g., 1, 2, 3 or 4) substituents selected from (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (═O), carboxyl, aryl, aryloxy, heteroaryl and heteroaryloxy.

[0158] In one embodiment, L 3 is a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 30 carbon atoms, wherein one or more carbon atoms are optionally replaced by -O-, -NR X -、-NR X -C(=O)-, -C(=O)-NR X - or -S- substitution, and wherein R X is hydrogen or (C1-C6)alkyl, and wherein the hydrocarbon chain is optionally substituted by one or more halo or oxo (=O).

[0159] In one embodiment, L 3 yes:

[0160]

[0161] In one embodiment, L 3 B is connected through -NH-, -O-, -S-, -(C=O)-, -(C=O)-NH-, -NH-(C=O)-, -(C=O)-O-, -NH-(C=O)-NH- or -NH-(SO2)-.

[0162] In one embodiment, L 4 is a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 0 to 50 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) carbon atoms in the hydrocarbon chain are optionally replaced by -O-, -NR X -、-NR X -C(=O)-, -C(=O)-NR X - or -S- substitution, and wherein R X is hydrogen or (C1-C6)alkyl, and wherein the hydrocarbon chain is optionally substituted by one or more (e.g., 1, 2, 3 or 4) substituents selected from (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (═O), carboxyl, aryl, aryloxy, heteroaryl and heteroaryloxy.

[0163] In one embodiment, L 4 is a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 20 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) carbon atoms in the hydrocarbon chain are optionally replaced by -O-, -NR X -、-NR X -C(=O)-, -C(=O)-NR X - or -S- substitution, and wherein R X is hydrogen or (C1-C6)alkyl, and wherein the hydrocarbon chain is optionally substituted by one or more (e.g., 1, 2, 3 or 4) substituents selected from (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (═O), carboxyl, aryl, aryloxy, heteroaryl and heteroaryloxy.

[0164] In one embodiment, L 4 is a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 30 carbon atoms, wherein one or more carbon atoms are optionally replaced by -O-, -NR X -、-NR X -C(=O)-, -C(=O)-NR X - or -S- substitution, and wherein R X is hydrogen or (C1-C6)alkyl, and wherein the hydrocarbon chain is optionally substituted by one or more halo or oxo (=O).

[0165] In one embodiment, L4 By-O- and R 3 connect.

[0166] In one embodiment, the nucleic acid molecule R 3 (e.g., siRNA) is linked to the rest of the conjugate through the oxygen of the phosphate of the nucleic acid molecule.

[0167] In one embodiment, the nucleic acid molecule R 3 (eg, siRNA) is linked to the remainder of the conjugate via the oxygen of the phosphate at the 5' end of the sense or antisense strand.

[0168] In one embodiment, the nucleic acid molecule R 3 (eg, siRNA) is linked to the remainder of the conjugate via the oxygen of the phosphate at the 3' end of the sense or antisense strand.

[0169] In one embodiment, the nucleic acid molecule R 3 (eg, siRNA) is linked to the remainder of the conjugate via the oxygen of the phosphate at the 3' end of the sense strand.

[0170] In one embodiment, the group:

[0171]

[0172] Selected from the group consisting of:

[0173]

[0174] in

[0175] Each R' is independently C 1-9 Alkyl, C 2-9 Alkenyl or C 2-9 Alkynyl; wherein the C 1-9 Alkyl, C 2-9 Alkenyl or C 2-9 The alkynyl group is optionally substituted with halo or hydroxy.

[0176] In one embodiment, the group:

[0177]

[0178] Selected from the group consisting of:

[0179]

[0180] in:

[0181] Each R' is independently C 1-9 Alkyl, C 2-9 Alkenyl or C 2-9 Alkynyl; wherein the C1-9 Alkyl, C 2-9 Alkenyl or C 2-9 Alkynyl is optionally substituted with halo or hydroxy;

[0182] The bonds marked with * are 3 Connect; and

[0183] The bond marked with ** is 3 connect.

[0184] In one embodiment, the group:

[0185]

[0186] yes:

[0187]

[0188] The present invention also provides synthetic intermediates and methods disclosed herein that can be used to prepare conjugates of formula (I). For example, the present invention includes compounds of formula (Ia):

[0189]

[0190] in:

[0191] R 1 It’s sugar;

[0192] L 1 is a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 0 to 20 carbon atoms, wherein one or more carbon atoms in the hydrocarbon chain are optionally replaced by -O-, -NR X -、-NR X -C(=O)-, -C(=O)-NR X - or -S- substitution, and wherein R X is hydrogen or (C1-C6)alkyl, and wherein the hydrocarbon chain is optionally substituted with one or more substituents selected from oxo (=O) and halo;

[0193] B is a 5-10 membered aryl or 5-10 membered heteroaryl, which is optionally substituted with one or more groups independently selected from the group consisting of halo, hydroxy, cyano, trifluoromethyl, trifluoromethoxy, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkanoyloxy, (C3-C6)cycloalkyl and (C3-C6)cycloalkyl(C1-C6)alkyl

[0194] L 2is a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 0 to 20 carbon atoms, wherein one or more carbon atoms in the hydrocarbon chain are optionally replaced by -O-, -NR X -、-NR X -C(=O)-, -C(=O)-NR X - or -S- substitution, and wherein R X is hydrogen or (C1-C6)alkyl, and wherein the hydrocarbon chain is optionally substituted with one or more substituents selected from oxo (=O) and halo;

[0195] R 2 It’s sugar;

[0196] L 3 There is no or a linking group;

[0197] A is 3-20 membered cycloalkyl, 5-20 membered aryl, 5-20 membered heteroaryl or 3-20 membered heterocycloalkyl;

[0198] Each R A independently selected from hydrogen, hydroxyl, CN, F, Cl, Br, I, -OR a , -C 1-2 Alkyl-OR a , C 1-10 Alkyl C 2-10 Alkenyl and C 2-10 Alkynyl group; wherein the C 1-10 Alkyl C 2-10 Alkenyl and C 2-10 Alkynyl is optionally substituted by one or more independently selected from halo, hydroxy and C 1-3 Alkoxy group substitution;

[0199] n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0200] L 4 There is no or a linking group;

[0201] R 3a is H, a protecting group, a synthesis activating group, a covalent bond to a solid support, or a bond to a linker group bound to a solid support;

[0202] R a is hydrogen, a protecting group, a covalent bond to a solid support, or a linker group L bound to a solid support 5 and

[0203] L 5 is a linking group;

[0204] or a salt thereof.

[0205] In one embodiment, R 3a It's H.

[0206] In one embodiment, R 3a In one embodiment, the protecting group is acetate, triflate, mesylate or succinate.

[0207] In one embodiment, R 3a is a synthetic activating group. In one embodiment, the synthetic activating group may be derived from DCC, HOBt, EDC, BOP, PyBOP or HBTU.

[0208] In one embodiment, R 3a It is a covalent bond to the solid support.

[0209] In one embodiment, R 3a is a bond to a linker group bound to a solid support. In one embodiment, the linker group bound to a solid support is -C(=O)CH2CH2C(=O)N(H)-.

[0210] Example

[0211] The present invention will be described in more detail by specific examples. The following examples are provided for illustrative purposes and are not intended to limit the present invention in any way. Those skilled in the art will easily recognize a variety of noncritical parameters that can be changed or modified to obtain substantially the same result.

[0212] The following Schemes 1-22 illustrate the preparation of intermediate compounds that can be used to prepare conjugates of Formula I. The intermediate compounds and synthetic processes illustrated in Schemes 1-22 are embodiments of the present invention.

[0213] Scheme 1 Preparation of Compound 6

[0214]

[0215] Step 1. Preparation of (3aR,6aS)-5-benzyl-3a,6a-dimethyltetrahydro-1H-furo[3,4-c]pyrrole-1,3(3aH)-dione 1

[0216] To a cooled solution (0°C) 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 slowly added trifluoroacetic acid (732 μl). Stir overnight to allow the solution to slowly warm to room temperature. 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 silica gel column chromatography (gradient: 20% ethyl acetate to 100% ethyl acetate in hexanes) gave (3aR,6aS)-5-benzyl-3a,6a-dimethyltetrahydro-1H-furo[3,4-c]pyrrole-1,3(3aH)-dione (53.7 g, 65%) as a yellow oil. Rf 0.85 40% EtOAc-hexanes

[0217] Step 2. Preparation of ((3R,4S)-1-benzyl-3,4-dimethylpyrrolidine-3,4-diyl) dimethanol 2

[0218] Over the afternoon, to a cooled (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 ether (750 ml) was slowly added lithium aluminum hydride pellets (17.6 g, 463 mmol) in portions. The solution was stirred overnight 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 25 ml of 5M NaOH followed by 12 ml of water. Stir for 30 minutes, then magnesium sulfate was added and filtered. The filtrate was concentrated to give ((3R,4S)-1-benzyl-3,4-dimethylpyrrolidine-3,4-diyl)dimethanol (33.6 g, 65%) as a colorless oil. Rf 0.25 10% CH3OH-CH2Cl2

[0219] Step 3. Preparation of ((3R,4S)-3,4-dimethylpyrrolidine-3,4-diyl)dimethanol 3

[0220] 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 wet 10% palladium on 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 (24 g, 94%) as a colorless solid. Rf 0.05 10% CH3OH-CH2Cl2

[0221] Step 4. Preparation of 10-((3R,4S)-3,4-bis(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoic acid methyl ester

[0222] A solution of 3 (24 g, 151 mmol) and monomethyl sebacate (34.2 g, 159 mmol) in CH2Cl2 (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 NaHCO3 (saturated aqueous solution), water and brine, then dried (MgSO4), filtered and concentrated. The crude material was chromatographed (gradient: 0% CH3OH-CH2Cl2 to 20%) to give 4 (41.5 g, 77%). Rf 0.55 10% CH3OH-CH2Cl2

[0223] Step 5. Preparation of methyl 10-(3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoate 5

[0224] A solution of 4 (41.5 g, 116 mmol) and 4,4′-dimethoxytrityl chloride (38.8 g, 116 mmol) in pyridine (400 ml) was stirred overnight. Pyridine was then removed under reduced pressure and the crude material was chromatographed (gradient: 0% CH3OH-CH2Cl2 to 10%) to give 5 (29.5 g, 39%) as a yellow oil. Rf 0.5 5% CH3OH-CH2Cl2

[0225] Step 6. Preparation of lithium 10-(3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoate 6

[0226] To a solution of compound 5 (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 THF. The remaining aqueous solution was freeze-dried overnight to give 6 (28.5 g, 98%) as a light purple solid. Rf 0.56 10% CH3OH-CH2Cl2

[0227] Scheme 2 Preparation of Compound 10

[0228]

[0229] Step 1. Preparation of 12-aminododecanoic acid methyl ester 8

[0230] At room temperature, stir 12-aminoundecanoic acid 7 (10 g, 4.64 mmol) in MeOH. Add acetyl chloride (856 μ l, 12 mmol) dropwise, and stir the reaction for 1.5 hours. Remove solvent in a vacuum, and the residue is dissolved in MTBE and cooled in a refrigerator overnight. Collect the resulting precipitate by filtering, wash with ice-cold MTBE and dry under high vacuum to obtain 12-aminododecanoic acid methyl ester 8.

[0231] Step 2. Preparation of methyl 12-(10-(3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoylamino)dodecanoate 9

[0232] Lithium 10-(3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoate (6) (2 g, 3.1 mmol), methyl 12-aminododecanoate (8) (778 mg, 3.1 mmol), HBTU (1.2 g, 3.1 mmol) and TEA (1.4 ml, 10 mmol) were stirred in DCM overnight at room temperature. The precipitate was removed by filtration, the filtrate was concentrated in vacuo and the residue was purified by column chromatography (5% MeOH, DCM). TLC showed two close spots with the same mass, which were assigned as geometric isomers and pooled together in a quantitative manner as methyl 12-(10-(3-((bis(4-(methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoylamino)dodecanoate (9).

[0233] Step 3. Preparation of 12-(10-(3-((bis(4-methoxyphenyl)(phenyl)methoxy)-methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoylamino)-dodecanoic acid lithium 10

[0234] Methyl 12-(10-(3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoylamino)dodecanoate 9 (3.1 mmol) was stirred overnight in THF:HO (50:50) with LiOH (88 mg, 3.7 mmol) at room temperature. The reaction was confirmed by TLC and THF was removed in vacuo. The aqueous solution was frozen in liquid N2 and lyophilized for 48 hours to quantitatively obtain lithium 12-(10-(3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoylamino)dodecanoate 10.

[0235] Scheme 3 Preparation of Compound 13

[0236]

[0237] Step 1. Preparation of 2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate 12

[0238] A solution of tetraethylene glycol (11) (934 g, 4.8 mol) in THF (175 ml) and aqueous NaOH (5 M, 145 ml) was cooled (0 ° C) and treated with p-toluenesulfonyl chloride (91.4 g, 480 mmol) dissolved in THF (605 ml) and then stirred for two hours (0 ° C). The reaction mixture was diluted with water (3 L) and extracted with CH2Cl2 (3 x 500 ml). The combined extracts were washed with water and brine, then dried (MgSO4), filtered and concentrated to give 4-methylbenzenesulfonic acid 2-(2-(2-hydroxyethoxy)ethoxy)ethyl ester (12) (140 g, 84%) as a light yellow oil. f (0.57, 10% MeOH-CH2Cl2).

[0239] Step 2. Preparation of 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethan-1-ol 13

[0240] A solution of 12 (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 CH2Cl2 (500 ml) and washed with brine (3 x 500 ml), then dried (MgSO4), filtered and concentrated. The residue was passed through a short bed of silica (5% MeOH-CH2Cl2) and concentrated to give 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)ethane-1-ol 13 (65 g, 74%) as a yellow oil. f (0.56, 10% MeOH-CH2Cl2).

[0241] Scheme 4 Preparation of Compounds 19a-19c

[0242]

[0243] Step 1. Preparation of (3R,4R,5R,6R)-6-(hydroxymethyl)-3-(((E)-4-methoxybenzylidene)amino)tetrahydro-2H-pyran-2,4,5-triol 16

[0244] D-galactosamine hydrochloride (14) (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 was stirred vigorously until solidified. The solid reaction was kept at 4 ° C for 16 hours. Ice-cold water (200 ml) was added and the resulting solid was collected by filtration and washed with ice-cold EtOH / Et2O (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 (16) (9.81 g, 78%).

[0245] Step 2. Preparation of (3R,4R,5R,6R)-6-(acetoxymethyl)-3-(((E)-4-methoxybenzylidene)amino)tetrahydro-2H-pyran-2,4,5-triyl triacetate 17

[0246] (3R,4R,5R,6R)-6-(Hydroxymethyl)-3-(((E)-4-methoxybenzylidene)amino)tetrahydro-2H-pyran-2,4,5-triol (16) (9.81 g, 30 mmol) was stirred in pyridine at 0°C. Acetic anhydride (34 ml) was added followed by DMAP (100 mg, catalyst) and the reaction was stirred for 16 hours, allowing it to slowly warm to room temperature. The resulting solution was poured onto crushed ice and kept at 4°C for 16 hours. The reaction was extracted with EtOAc (x 3) and the combined organics washed with H2O and brine, dried (Na2SO4) and concentrated in vacuo to give (3R,4R,5R,6R)-6-(acetoxymethyl)-3-(((E)-4-methoxybenzylidene)amino)tetrahydro-2H-pyran-2,4,5-triyl triacetate (17) (6.0 g, 43%).

[0247] Step 3. Preparation of (3R,4R,5R,6R)-6-(acetoxymethyl)-3-aminotetrahydro-2H-pyran-2,4,5-triyl triacetate hydrochloride 18

[0248] (3R,4R,5R,6R)-6-(acetoxymethyl)-3-(((E)-4-methoxybenzylidene)amino)tetrahydro-2H-pyran-2,4,5-triyl triacetate (17) (6.0 g, 43%) was heated in acetone (300 ml) at reflux. HCl (aq) (5N, 3.0 ml) was added and the reaction was stirred for 15 minutes. After cooling, Et2O (400 ml) was added and the reaction was maintained at 4°C for 16 hours. The resulting solid was collected by filtration and washed twice with ice-cold Et2O. 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 (18) (4.17 g, 84.4%).

[0249] Step 4a. Preparation of (3R,4R,5R,6R)-6-(acetoxymethyl)-3-(2,2,2-trifluoroacetylamino)tetrahydro-2H-pyran-2,4,5-triyl triacetate 19a

[0250] (3R,4R,5R,6R)-6-(acetoxymethyl)-3-aminotetrahydro-2H-pyran-2,4,5-triyl triacetate hydrochloride (18) (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 hour. The reaction was diluted with DCM, washed sequentially with 1M HCl, saturated NaHCO, water and brine, dried (NaSO) and concentrated in vacuo. The residue was purified by automated flash chromatography (5% MeOH / DCM) to give (3R,4R,5R,6R)-6-(acetoxymethyl)-3-(2,2,2-trifluoroacetylamino)tetrahydro-2H-pyran-2,4,5-triyl triacetate (19a) (9.64 g, 61.8%). The product was confirmed by MS (ESI+ve).

[0251] Step 4b. Preparation of (3R,4R,5R,6R)-6-(acetoxymethyl)-3-propionylaminotetrahydro-2H-pyran-2,4,5-triyl triacetate 19b

[0252] This compound was prepared in a similar manner to (3R,4R,5R,6R)-6-(acetoxymethyl)-3-(2,2,2-trifluoroacetylamino)tetrahydro-2H-pyran-2,4,5-triyl triacetate (19a) using propionic anhydride instead of TFAA to give (3R,4R,5R,6R)-6-(acetoxymethyl)-3-propionylaminotetrahydro-2H-pyran-2,4,5-triyl triacetate (19b) (1.2 g, 85.3%). The product was confirmed by MS (ESI+ve).

[0253] Step 4c. Preparation of (3R,4R,5R,6R)-6-(acetoxymethyl)-3-(2,2-difluoropropionylamino)tetrahydro-2H-pyran-2,4,5-triyl triacetate 19c

[0254] (3R,4R,5R,6R)-6-(acetoxymethyl)-3-aminotetrahydro-2H-pyran-2,4,5-triyl triacetate hydrochloride (18) (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 hours. The reaction was partitioned between EtOAc and water. The organics were separated, washed sequentially with 1M HCl, saturated NaHCO3, water and brine, dried (Na2SO4) and concentrated in vacuo. The residue was purified by automated flash chromatography (3% MeOH / DCM) to give (3R,4R,5R,6R)-6-(acetoxymethyl)-3-(2,2-difluoropropionylamino)tetrahydro-2H-pyran-2,4,5-triyl triacetate (19c) (15.8 g, 90%). The product was confirmed by MS (ESI+ve).

[0255] Scheme 5 Preparation of Compound 24

[0256]

[0257] Step 1. Preparation of benzyl (2-(2-(2-hydroxyethoxy)ethoxy)ethyl)carbamate 22

[0258] A solution of the amino alcohol (20) (313.6 g, 2.1 mol) in THF (3.5 L) was treated in portions with N-(benzyloxycarbonyloxy)succinimide (21) (550 g, 2.21 mol). Once the reaction was complete (18 h), the THF was removed under reduced pressure and the residue was dissolved in CH2Cl2 (2.5 L) and washed with equal volumes of HCl (1 M), NaHCO3 (saturated aqueous solution), H2O and brine. The organic extracts were dried (MgSO4), filtered and concentrated. The crude material (600 g) was chromatographed (4 kg silica; 1-12% CH3OH-CH2Cl2) to give HO-Trig-NHZ (22) (468 g, 78%) as a clear-yellow viscous oil.

[0259] Step 2. Preparation of (2R,3R,4R,5R)-5-acetamido-2-(acetoxymethyl)-6-((3-oxo-1-phenyl-2,7,10-trioxa-4-azadodec-12-yl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate

[0260] A heterogeneous mixture of galactosamine pentaacetate (715.2 g, 1.84 mol) and HO-Trig-NHZ (22) (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 hours), the solution became clear and homogeneous, and the reaction was cooled and washed with NaHCO (saturated aqueous solution), HCl (1 M), H2O, and brine. The organic extract was dried (MgSO4), 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 filter to remove residual pentaacetate. The filtrate was concentrated and the crude material was chromatographed (5 kg silica; 0-10% CH3OH-EtOAc) to afford the glycosylated product (23) (751 g, 87%) as a light brown foam.

[0261] Step 3. Preparation of (2R,3R,4R,5R)-5-acetamido-2-(acetoxymethyl)-6-(2-(2-(2-((2,2,2-trifluoroacetyl)-14-azanyl)ethoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-3,4-diyl diacetate 24

[0262] A solution of Gal-trig-NHZ (23) (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), then filtered through celite and concentrated. The resulting brown foam (712 g, 99%) was used in the next step without further treatment.

[0263] Scheme 6 Preparation of Compound 34

[0264]

[0265] Step 1. Preparation of 2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethan-1-ol 25

[0266] 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)ethane-1-ol (13) (70.0 g, 318 mmol) was stirred in MeOH at room temperature. The reaction was hydrogenated over 10% PD-C (7 g) for 16 hours. The reaction was filtered through celite and concentrated in vacuo to give 2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethane-1-ol (25) (61.4 g, 100%) which was used without further purification. The product was confirmed by MS (ESI+ve).

[0267] Step 2. Preparation of (2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl)-carbamic acid benzyl ester 27

[0268] 2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethan-1-ol (25) (61.4 g, 318 mmol) was stirred in H2O (500 ml) with Na2CO3 (50.51 g, 476 mmol) at 5°C. THF (480 ml) containing benzyl chloroformate (26) (65.0 g, 381 mmol) was added dropwise and the reaction was stirred for 16 hours and allowed to warm to room temperature. THF was removed in vacuo and the aqueous layer was extracted with EtOAc (×3). The combined organics were dried (Na2SO4), concentrated in vacuo, and the residue was purified by automated flash chromatography (5% MeOH / DCM) to give benzyl (2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl)carbamate (27) (23.6 g, 22.7%). The product was confirmed by MS (ESI+ve).

[0269] Step 3. Preparation of benzyl (1,1-bis(4-methoxyphenyl)-1-phenyl-2,5,8,11-tetraoxatridecan-13-yl)carbamate 28

[0270] (2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl)carbamate (27) (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 for 2 hours at room temperature. The reaction was washed sequentially with saturated NaHCO, water and brine, dried (NaSO) and concentrated in vacuo. 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 (28) (25.5 g, 56.2%). The product was confirmed by MS (ESI+ve).

[0271] Step 4. Preparation of benzyl (1,1-bis(4-methoxyphenyl)-1-phenyl-2,5,8,11-tetraoxatridecan-13-yl)(methyl)carbamate 29

[0272] (1,1-bis(4-methoxyphenyl)-1-phenyl-2,5,8,11-tetraoxatridecan-13-yl)carbamate (28) (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 hour. The reaction was partitioned between EtOAc and H2O. The organics were separated, dried (Na2SO4) and concentrated in vacuo. 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 (29) (26.06 g, 100%). The product was confirmed by MS (ESI+ve).

[0273] Step 5. Preparation of benzyl (2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl)(methyl)carbamate 30

[0274] Benzyl (1,1-bis(4-methoxyphenyl)-1-phenyl-2,5,8,11-tetraoxatridecan-13-yl)(methyl)carbamate (29) (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. Another 2 equivalents of TFA were added and the reaction was stirred for 16 hours. The reaction was concentrated in vacuo 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 (30) (6.76 g, 48.9%). The product was confirmed by MS (ESI+ve).

[0275] 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 31

[0276] (2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl)(methyl)carbamate (30) (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 in DCE at reflux for 2 h. After cooling, the reaction was quenched with TEA and washed sequentially with 1 M HCl, saturated NaHCO, water and brine, dried (NaSO) and concentrated in vacuo. 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 (31) (9.37 g, 70.6%). The product was confirmed by MS (ESI+ve).

[0277] 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-314-azatetradec-14-yl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate 32

[0278] (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 (31) (9.37 g, 14.0 mmol) and TFA (1.76 g, 15.4 mmol) were stirred in MeOH at room temperature. The reaction was hydrogenated over 10% Pd-C (1 g) for about 2 hours. The reaction was filtered through celite and concentrated in vacuo to give (2R,3R,4R,5R)-5-acetamido-2-(acetoxymethyl)-6-((1,1,1-trifluoro-3-methyl-2-oxo-6,9,12-trioxa-314-azatetradec-14-yl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (32) (9.0 g, 98.9%). The product was used without purification. The product was confirmed by MS (ESI+ve).

[0279] 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 34

[0280] (2R,3R,4R,5R)-5-acetamido-2-(acetoxymethyl)-6-((1,1,1-trifluoro-3-methyl-2-oxo-6,9,12-trioxa-314-azatetradec-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) were stirred in DCM at room temperature for 16 h. The reaction was diluted with DCM and washed sequentially with 1M HCl, saturated NaHCO, water and brine, dried (NaSO) and concentrated in vacuo. 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'-azatridecane-1,13-diyl))bis(oxy))bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (34) (5.0 g, 57.4%). The product was confirmed by MS (ESI+ve).

[0281] Scheme 7 Preparation of Compound 43

[0282]

[0283] 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

[0284] (2R,3R,4R,5R)-5-acetamido-2-(acetoxymethyl)-6-((1,1,1-trifluoro-2-oxo-6,9,12-trioxa-314-azatetradec-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 / H2O (50:50) at room temperature. Benzyl chloroformate (26) (14.5 g, 85 mmol) was added dropwise and the reaction was stirred for 16 hours. THF was removed in vacuo and the aqueous layer was extracted with EtOAc (×3). The organics were washed sequentially with 1M HCl, saturated NaHCO3, water and brine, dried (Na2SO4) and concentrated in vacuo. 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 (35) (25.12 g, 54%). The product was confirmed by MS (ESI+ve).

[0285] Step 2. Preparation of benzyl (2-(2-(2-(((3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)carbamate 36

[0286] (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 (35) (25.12 g, 38.3 mmol) was stirred in a 7N ammonia solution in MeOH at room temperature for 16 hours in a gas-tight sealed reaction vessel. The reaction was evaporated at 50°C to remove ammonia and the residue was concentrated in vacuo to give benzyl (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 (36) (20.3 g, 100%) which was used in subsequent reactions without further purification. The product was confirmed by MS (ESI+ve).

[0287] Step 3. Preparation of (2-(2-(2-(2-(((3aR,4R,7R,7aR)-7-acetamido-4-(hydroxymethyl)-2,2-dimethyltetrahydro-4H-[1,3]dioxol[4,5-c]pyran-6-yl)oxy)-ethoxy)ethoxy)ethyl)carbamic acid benzyl ester 37

[0288] 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 (36) (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 (catalyst) were added and the reaction was heated at 65°C for 16 hours. The reaction was cooled to room temperature, TEA (20 ml) was added and stirred for 30 minutes. The solvent was removed in vacuo, the residue was dissolved in MeOH / H2O (10:1), and the reaction was refluxed for 1 hour. The reaction was concentrated in vacuo (azeotroped with toluene (×2)) 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-(hydroxy-methyl)-2,2-dimethyltetrahydro-4H-[1,3]dioxol[4,5-c]pyran-6-yl)oxy)ethoxy)ethoxy)-ethoxy)ethyl)carbamate (37) (24.9 g, 100%). The product was confirmed by MS (ESI+ve).

[0289] 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]dioxol[4,5-c]pyran-4-yl)methyl 4-methylbenzenesulfonate 38

[0290]

[0136] 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 (37) (25.5 g, 44.8 mmol) and TEA (9.97 g, 98.5 mmol) were stirred in DCM 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, allowing to warm to rt. The reaction was diluted with DCM, washed sequentially with 1M HCl, saturated NaHCO3, water and brine, dried (Na2SO4) and concentrated in vacuo. 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]dioxol[4,5-c]pyran-4-yl)methyl 4-methylbenzenesulfonate (38) (25.5 g, 78.8%). The product was confirmed by MS (ESI+ve).

[0291] Step 5. Preparation of (2-(2-(2-(2-(((3aS,4R,7R,7aR)-7-acetamido-4-(azidomethyl)-2,2-dimethyltetrahydro-4H-[1,3]dioxol[4,5-c]pyran-6-yl)oxy)ethoxy)ethoxy)ethyl)carbamic acid benzyl ester 39

[0292] ((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]dioxol[4,5-c]pyran-4-yl)methyl 4-methylbenzenesulfonate (38) (25.0 g, 34.5 mmol) and NaN (28.7 g, 434.6 mmol) were heated in DMSO / HO (200 ml / 20 ml) at 100 °C for 12 h. The reaction was cooled and partitioned between EtOAc and saturated NaHCO. The aqueous layer was further extracted twice and the combined organics were washed with saturated NaHCO, water and brine, dried (NaSO) and concentrated in vacuo. 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]dioxol[4,5-c]pyran-6-yl)oxy)ethoxy)ethoxy)ethyl)carbamate (39) (16.1 g, 78.2%). The product was confirmed by MS (ESI+ve).

[0293] 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]dioxol[4,5-c]pyran-6-yl)oxy)ethoxy)ethoxy)ethyl)carbamate 40

[0294] Benzyl (2-(2-(2-(2-(((3aS,4R,7R,7aR)-7-acetamido-4-(azidomethyl)-2,2-dimethyltetrahydro-4H-[1,3]dioxol[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), CuSO4 (0.07 g, 0.27 mmol in 1 ml H2O) and sodium ascorbate (0.53 g, 2.7 mmol in 5 ml H2O) were added sequentially and the reaction was stirred at room temperature for 16 hours. In vacuum, remove solvent, residue is dissolved in DCM (200ml) and washed with water. With DCM back extraction water layer, and with salt water washing merged organic matter and dry (Na SO ). Concentrate reactant in vacuum, and by automatic flash chromatography (10%MeOH / EtOAc) purification of residue, obtain (2-(2-(2-(2-(((3aS, 4R, 7R, 7aR)-7-acetylamino-4-((4-(3-methoxyphenyl)-1H-1,2,3-triazol-1 base) methyl)-2,2-dimethyltetrahydro-4H-[1,3] dioxol [4,5-c] pyrans-6-yl) oxygen base) ethoxy) ethoxy) ethoxy) ethyl) benzyl carbamate (40) (15.0g, 76.4%). Confirm product by MS (ESI+ve).

[0295] 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 41

[0296] 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]dioxol[4,5-c]pyran-6-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamate (40) (15.0 g, 20.6 mmol) was stirred in MeCN (200 ml) and 1.84% H2SO4 (180 ml) at room temperature for 96 h. The reaction was extracted with EtOAc (3 x 250 ml), washed with saturated NaHCO, water and brine, dried (NaSO) and concentrated in vacuo to give (2-(2-(2-(2-(((3R,4R,5R,6R)-3-acetylamino-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)-carbamic acid benzyl ester (41) (11.0 g, 16.0 mmol). This product was used crude in subsequent reactions. The product was confirmed by MS (ESI+ve).

[0297] 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

[0298]

[0136] 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 (41) (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 followed by 3 hours at 50°C. The reaction was poured onto water and extracted three times with DCM (250 ml). The combined organics were washed with saturated NaHCO3 (x2), 1N HCl (x2), water and brine, dried (Na2SO4) and concentrated in vacuo. 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 (42) (10.7 g, 86.7%). The product was confirmed by MS (ESI+ve).

[0299] 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-314-azatetradec-14-yl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate

[0300] (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 (42) (9.06 g, 11.74 mmol) and TFA (1.47 g, 12.91 mmol) were stirred in MeOH at room temperature. The reaction was hydrogenated over 10% Pd-C for 1 hour. The reaction was filtered through celite and concentrated in vacuo 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-azatetradec-14-yl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (43) (8.8 g, 99.7%) which was used in subsequent reactions without purification. The product was confirmed by MS (ESI+ve).

[0301] Scheme 8 Preparation of Compound 54

[0302]

[0303]

[0304] Step 1. Preparation of peracetylated galactosamine 44

[0305] After 45 minutes, pyridine (1.5 L) containing D-galactosamine hydrochloride (14) (250 g, 1.16 mol) was treated with acetic anhydride (1.25 L, 13.2 mol). 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 one hour. After mixing, the solid was filtered off, combined, frozen on liquid nitrogen, and then lyophilized for five days to obtain peracetylated galactosamine (44) (369.4 g, 82%) as a white solid. Rf (0.58, 10% MeOH-CH2Cl2).

[0306] 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 45

[0307] Peracetylated galactosamine (44) (25 g, 64.21 mmol) and scandium trifluoromethanesulfonate (1.58 g, 3.21 mmol) were heated in anhydrous DCE at 90°C for 3 h. The reaction was cooled to room temperature, quenched with 5 ml of TEA and concentrated in vacuo. 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-azidoethoxy)ethoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-3,4-diyl diacetate (45) (27 g, 76.5%). The product was confirmed by MS.

[0308] 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)eth-1-ammonium 2,2,2-trifluoroacetate 46

[0309] A solution of azide 45 (7.12 g, 13 mmol) in EtOAc (150 ml) and trifluoroacetic acid (2 ml) was treated with palladium / charcoal (1.5 g, 10% w / w, wet basis). The reaction mixture was then purged with hydrogen and stirred vigorously overnight. After purging with nitrogen, the mixture was filtered through diatomaceous earth while rinsing with MeOH. The filtrate was concentrated and purified via chromatography (5% → 10% → 20% MeOH-CH2Cl2) to give 46 (5.8 g, 72%) as a brown oil. Rf (0.34, 15% MeOH-CH2Cl2).

[0310] 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 48

[0311] (2R,3R,4R,5R)-5-acetamido-2-(acetoxymethyl)-6-((1,1,1-trifluoro-2-oxo-6,9,12-trioxa-314-azatetradec-14-yl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (46) (13.25 g, 20.84 mmol), 5-nitroisophthalic acid (47) (2.0 g, 9.5 mmol), HATU (12.3 g, 32.21 mmol) and TEA (5.75 g, 59.0 mmol) were stirred in DCM at room temperature for 16 h. The reaction was diluted with DCM, washed sequentially with 1M HCl, saturated NaHCO, water and brine, dried over NaSO and concentrated in vacuo. 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 (48) (4.43 g, 38.3%). The product was confirmed by MS (ESI+ve).

[0312] 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 49

[0313] (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 (48) (26.1 g, 23.05 mmol) was stirred in MeOH at room temperature. The reaction was hydrogenated over 10% Pd-C (2.6 g) at room temperature for 2 hours. The reaction was filtered through celite and concentrated in vacuo 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 (49) (28.0 g, 99.9%) which was used in subsequent reactions without further purification. The product was confirmed by MS (ESI+ve).

[0314] Step 6. Preparation of (2R,3R,4R,5R)-5-acetylamino-6-((1-(3-((2-(2-(2-(((3R,4R,5R,6R)-3-acetylamino-5-acetyloxy-6-(acetyloxymethyl)-4-hydroxytetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-5-(2-(((benzyloxy)carbonyl)amino)acetylamino)phenyl)-1-oxo-5,8,11-trioxa-2-azatridec-13-yl)oxy)-2-(acetyloxymethyl)tetrahydro-2H-pyran-3,4-diyl diacetate 51

[0315] (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 (49) (0.5 g, 0.45 mmol) and CBZ-gly (50) (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. Additional T3P (0.3 eq) was added and the reaction was stirred for an additional hour. The reaction was washed with saturated NaHCO and brine, dried (NaSO), concentrated in vacuo, and the residue purified by automated flash chromatography (10% MeOH / DCM) to afford (2R,3R,4R,5R)-5-acetylamino-6-((1-(3-((2-(2-(2-(2-(((3R,4R,5R,6R)-3-acetylamino-5-acetoxy-6-(acetoxy)-4-nitropropanediol)-1,2-dihydro ... 4-diyl)-5-(((benzyloxy)carbonyl)amino)acetylamino)phenyl)-1-oxo-5,8,11-trioxa-2-azatridec-13-yl)oxy)-2-(acetoxymethyl)tetrahydro-2H-pyran-3,4-diyl ester (51) (0.33 g, 56.8%). The product was confirmed by MS (ESI+ve).

[0316] Step 7. Preparation of (2R,2'R,3R,3'R,4R,4'R,5R,5'R)-(((5-(2-((2,2,2-trifluoroacetyl)-14-azanyl)acetylamino)-1,3-phenylene)bis(1-oxo-5,8,11-trioxa-2-azatridecane-1,13-diyl))bis(oxy))bis(5-acetylamino-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl) tetraacetate 52

[0317] (2R,3R,4R,5R)-5-acetamido-6-((1-(3-((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 (51) (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 two hours, filtered through celite and concentrated in vacuo to give (2R, 2'R, 3R, 3'R, 4R, 4'R, 5R, 5'R)-(((5-(2-((2,2,2-trifluoroacetyl)-14-azanyl)-acetylamino)-1,3-phenylene)bis(1-oxo-5,8,11-trioxa-2-azatridecane-1,13-diyl))bis(oxy))bis(5-acetylamino-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (52) (3.21 g, 98.7%), which was used in subsequent reactions without further purification. The product was confirmed by MS (ESI+ve).

[0318] 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-oxodecanoylamino)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 53

[0319] Tetraacetic acid (2R,2'R,3R,3'R,4R,4'R,5R,5'R)-(((5-(2-((2,2,2-trifluoroacetyl)-14-azanyl)acetylamino)-1,3-phenylene)bis(1-oxo-5,8,11-trioxa-2-azatridecane-1,13-diyl))bis(oxy))bis(5-acetylamino-2-(acetyloxymethyl)tetrahydro-2H-pyran-6,3,4- =Toluene tris(trimethyl) ester (52) (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 (6) (0.45 g, 0.73 mmol), HATU (0.47 g, 1.25 mmol) and TEA (0.22 g, 2.2 mmol) were stirred in DCM for 4 h. The reaction was diluted with DCM and washed sequentially with saturated NaHCO, water and brine, dried (NaSO) and concentrated in vacuo. 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-oxodecanoylamino)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 (53) (1.02 g, 75.2%). The product was confirmed by MS (ESI+ve).

[0320] Step 9. Preparation of 4-((1-(10-((2-((3,5-bis((2-(2-(2-(2-(((3R,4R,5R,6R)-3-acetylamino-4,5-diacetyloxy-6-(acetyloxymethyl)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 54

[0321] (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-oxodecanoylamino)acetamido)-1,3-phenylene)bis(1-oxo-5,8,11-trioxa)-2-azadecanoyl)tetraacetic acid was added at 60°C. bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)-1-(trioxane-1,13-diyl)bis(oxy)bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)ester (54) (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 anhydrous DCE for 2 hours. MeOH (5 ml) was added and the reaction was stirred for an additional 30 minutes, then cooled and concentrated in vacuo. The residue was dissolved in DCM and washed sequentially with saturated NaHCO (x4), water and brine. The organics were dried (Na2SO4) and concentrated in vacuo to give 4-((1-(10-((2-((3,5-bis((2-(2-(2-(((3R,4R,5R,6R)-3-acetylamino-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 (54) (1.1 g, 99.4%) which was used crude in subsequent reactions. The product was confirmed by MS (ESI+ve).

[0322] Scheme 9 Preparation of Compound 56

[0323]

[0324] 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-oxodecanoylamino)-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 55

[0325] (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-acetylamino-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (49) (4 g, 3.36 mmol) was stirred at room temperature. ), lithium 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) were stirred in DCM for 16 hours. The reaction was washed sequentially with saturated NaHCO3, water and brine, dried (Na2SO4) and concentrated in vacuo. 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-(hydroxy-methyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoylamino)-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 (55) (1.37 g, 22.5%). The product was confirmed by MS (ESI+ve).

[0326] Step 2. Preparation of 4-((1-(10-((3,5-bis((2-(2-(2-(((3R,4R,5R,6R)-3-acetylamino-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 56

[0327] This compound was prepared in a manner similar to 4-((1-(10-((2-((3,5-bis((2-(2-(2-(((3R,4R,5R,6R)-3-acetylamino-4,5-diacetyloxy-6-(acetyloxymethyl)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 (54)

[0328] Scheme 10 Preparation of Compound 57

[0329]

[0330] Synthesis of 3-((((1-(10-((3,5-bis((2-(2-(2-(((3R,4R,5R,6R)-3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)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 57

[0331] This compound was prepared in a manner similar to 4-((1-(10-((3,5-bis((2-(2-(2-(((3R,4R,5R,6R)-3-acetylamino-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 (54)

[0332] Scheme 11 Preparation of Compound 66

[0333]

[0334] Step 1. Preparation of dimethyl 5-(hydroxymethyl)isophthalate 59

[0335] Benzene-1,3,5-tricarboxylic acid trimethyl ester (58) (40 g, 159 mmol) and NaBH4 were stirred in THF at room temperature. THF (120 ml) containing MeOH (30 ml) was slowly added dropwise. After the addition was complete, the reaction was refluxed for 30 minutes. After cooling, the reaction was quenched with 1M HCl and extracted into EtOAc. The organics were washed sequentially with 1M HCl, NaHCO3, water and brine, dried (Na2SO4) and concentrated in vacuo. The residue was purified by automated flash chromatography (50 / 50 EtOAc / hexanes) to give 5-(hydroxymethyl) dimethyl isophthalate (59) (20.5 g, 53.2%). 1 H NMR (400 MHz, CDCl3) 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).

[0336] Step 2. Preparation of dimethyl 5-(chloromethyl)isophthalate 60

[0337] Dimethyl 5-(hydroxymethyl)isophthalate (59) (20.5 g, 80.5%) was refluxed in SOCl2 (11.1 g, 94 mmol) for 1.5 h. The reaction was cooled, diluted with DCM, and washed sequentially with 0.1 M NaOH (×2), water, and brine, dried (Na2SO4), and concentrated in vacuo. The residue was purified by automated flash chromatography (20% EtOAc / hexanes) to afford dimethyl 5-(chloromethyl)isophthalate (60) (10.84 g, 53%). 1 H NMR (400 MHz, CDCl3) 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).

[0338] Step 3. Preparation of dimethyl 5-(azidomethyl)isophthalate 61

[0339] Dimethyl 5-(chloromethyl)isophthalate (60) (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 in vacuo, and the residue was dissolved in DCM. The organics were washed with water and brine, dried (Na2SO4) and concentrated in vacuo. The residue was purified by flash chromatography (15% EtOAc / hexanes) to give dimethyl 5-(azidomethyl)isophthalate (61) (9.84 g, 88%). 1H NMR (400 MHz, CDCl3) 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).

[0340] Step 4. Preparation of 5-(azidomethyl)isophthalic acid 62

[0341] Dimethyl 5-(azidomethyl)isophthalate (61) (9.84 g, 39.5 mmol) and LiOH (2.1 g, 87 mmol) were stirred in THF / H2O / MeOH for 48 h at room temperature. The organic solvent was removed in vacuo and the residue was acidified with 1M HCl. The aqueous layer was extracted with EtOAc (×3) and the combined organics were dried (Na2SO4) and concentrated in vacuo to give 5-(azidomethyl)isophthalic acid (62) (8.0 g, 91.6%) which was used in subsequent reactions without further purification.

[0342] 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 63

[0343] 5-(Azidomethyl)isophthalic acid (62) (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)ethoxy)eth-1-aminium 2,2,2-trifluoroacetate (46) (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 sequentially with 1 M HCl, saturated NaHCO, water and brine, dried (NaSO) and concentrated in vacuo. 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 (63) (10.9 g, 44.5%). The product was confirmed by MS (ESI+ve).

[0344] Step 6. Preparation of (2R,2'R,3R,3'R,4R,4'R)-(((5-(((2,2,2-trifluoroacetyl)-14-azanyl)methyl)-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 64

[0345] (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 (63) (10.9 g, 8.9 mmol) and TFA (0.68 ml, 8.9 mmol) were stirred in MeOH at room temperature. The reaction was hydrogenated over 10% Pd-C for 1 hour. The reaction was filtered through celite, concentrated in vacuo, and the residue was purified by automated flash chromatography (15% MeOH / DCM) to give (2R, 2'R, 3R, 3'R, 4R, 4'R)-(((5-(((2,2,2-trifluoroacetyl)-14-azanyl)methyl)-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 (64) (6.41 g, 54.7%). The product was confirmed by MS (ESI+ve).

[0346] Step 7. Preparation of (2R,2'R,3R,3'R,4R,4'R)-(((5-((10-(3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoylamino)methyl)-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 65

[0347] (2R,2'R,3R,3'R,4R,4'R)-(((5-(((2,2,2-trifluoroacetyl)-14-azanyl)methyl)-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 (3.0 g, 2.3 mmol), 10-(3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoic acid lithium (65) (1.5 g, 2.3 mmol), HATU (1.4 g, 3.7 mmol) and TEA (1 ml, 7.0 mmol) were stirred at room temperature overnight. The reactant was diluted with DCM, washed with saturated NaHCO , water and brine, dried (Na SO ) and concentrated in vacuo. The residue was purified by automatic flash chromatography (5% MeOH / DCM) to give tetraacetic acid (2R, 2'R, 3R, 3'R, 4R, 4'R)-(((5-((10-(3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoylamino)methyl)-1,3-phenylene)bis(1-oxo-5,8,11-trioxa-2-azatridecane-1,13-diyl))bis(oxygen))bis(5-acetylamino-2-(acetoxymethyl)tetrahydro-2H-pyrans-6,3,4-triyl)ester (65) (1.8 g, 43.0%). The product was confirmed by MS (ESI+ve).

[0348] Step 8. Preparation of 4-((1-(10-((3,5-bis((2-(2-(2-(((4R,5R,6R)-3-acetylamino-4,5-diacetyloxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamoyl)benzyl)amino)-10-oxodecanoyl)-4-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3,4-dimethylpyrrolidin-3-yl)methoxy)-4-oxobutanoic acid 66

[0349] This compound was prepared in a manner similar to 4-((1-(10-((2-((3,5-bis((2-(2-(2-(2-(((3R,4R,5R,6R)-3-acetylamino-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 (54). The product was confirmed by MS (ESI+ve).

[0350] Scheme 12 Preparation of Compound 67

[0351]

[0352] Synthesis of 4-((1-(10-((2-((3,5-bis((2-(2-(2-(((3R,4R,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-3-(2,2,2-trifluoroacetylamino)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 67

[0353] This compound was prepared in a manner similar to 54 (Scheme 8) using (3R,4R,5R,6R)-6-(acetoxymethyl)-3-(2,2,2-trifluoroacetylamino)tetrahydro-2H-pyran-2,4,5-triyl triacetate instead of peracetylated galactosamine (6). The product was confirmed by MS (ESI+ve).

[0354] Scheme 13 Preparation of Compound 68

[0355]

[0356] Synthesis of 4-((1-(10-((2-((3,5-bis((2-(2-(2-(((3R,4R,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-3-propionamidotetrahydro-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 68

[0357] This compound was prepared in a manner similar to 54 (Scheme 8) using (3R,4R,5R,6R)-6-(acetoxymethyl)-3-propionamidotetrahydro-2H-pyran-2,4,5-triyl triacetate (19b) instead of peracetylated galactosamine (44). The product was confirmed by MS (ESI+ve).

[0358] Scheme 14 Preparation of Compound 69

[0359]

[0360] Synthesis of 4-((1-(10-((2-((3,5-bis((2-(2-(2-(((3R,4R,5R,6R)-4,5-diacetoxy-6-(acetoxymethyl)-3-(2,2-difluoropropionylamino)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 69

[0361] This compound was prepared in a manner similar to 54 (Scheme 8) using (3R,4R,5R,6R)-6-(acetoxymethyl)-3-(2,2-difluoropropionylamino)tetrahydro-2H-pyran-2,4,5-triyl triacetate (19c) instead of peracetylated galactosamine (44). The product was confirmed by MS (ESI+ve).

[0362] Scheme 15 Preparation of Compound 70

[0363]

[0364] Synthesis of 4-((1-(10-((2-((3,4-bis((2-(2-(2-(((3R,4R,5R,6R)-3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)(methyl)carbamoyl)phenyl)amino)-2-oxoethyl)amino)-10-oxodecanoyl)-4-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3,4-dimethylpyrrolidin-3-yl)methoxy)-4-oxobutanoic acid 70

[0365] In a manner similar to compound 54 (Scheme 8), (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-diyl)-tetrahydro- This compound was prepared by replacing (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 (48) with (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 (48).

[0366] Scheme 16 Preparation of Compound 71

[0367]

[0368] Synthesis of 4-((1-(10-((2-((3,5-bis((2-(2-(2-(2-(((3R,4R,5S,6R)-3-acetylamino-4,5-diacetoxy-6-((4-(3-methoxyphenyl)-1H-1,2,3-triazol-1-yl)methyl)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 71

[0369] This compound was prepared in a manner analogous to compound 54 (Scheme 8) using (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-azatetradec-14-yl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (43) instead 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)ethoxy)ethan-1-aminium 2,2,2-trifluoroacetate (46).

[0370] Scheme 17 Preparation of Compound 72

[0371]

[0372] Synthesis of 4-((4-(10-((2-((3,5-bis((2-(2-(2-(((3R,4R,5R,6R)-3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)carbamoyl)phenyl)amino)-2-oxoethyl)amino)-10-oxodecanoyl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-1,2-dimethylcyclopentyl)methoxy)-4-oxobutanoic acid 72

[0373] This compound was prepared in a manner analogous to compound 54 (Scheme 8) using (2R,3R,4R,5R)-5-acetamido-2-(acetoxymethyl)-6-(2-(2-(2-((2,2,2-trifluoroacetyl)-14-azanyl)ethoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-3,4-diyl diacetate (24) instead 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)ethoxy)eth-1-ammonium 2,2,2-trifluoroacetate (46).

[0374] Scheme 18 Preparation of Compound 81

[0375]

[0376]

[0377] Step 1. Preparation of 12-(Benzyloxy)-12-oxododecanoic acid 76

[0378] Potassium carbonate (10 g, 72.4 mmol) and benzyl bromide (75) (10 ml, 84.2 mmol) were added to a solution of dodecanedioic acid (74) (21.0 g, 91.3 mmol) in DMF (200 ml). The solution was stirred at 80 ° C for 4 hours, cooled to 0 ° C, and then carefully acidified with 6M HCl. Diluted with water (250 ml) and extracted with ethyl acetate (500 ml). The ethyl acetate extract was washed with brine (3 x 250 ml), dried over magnesium sulfate, filtered and concentrated to dryness. The solid was suspended in dichloromethane (200 ml) and filtered. The filtrate, now enriched with the product, was concentrated and then purified by column chromatography on silica gel 60 (gradient: DCM containing 0 to 10% methanol) to give 12-(benzyloxy)-12-oxododecanoic acid (76) (13 g, 45%) as a colorless solid. The structure was confirmed by mass spectrometry

[0379] Step 2. Preparation of (2S,3S,4S,5S)-5-acetamido-6-(2-(2-(2-(3-((2-(2-(2-(((3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)carbamoyl)-5-(12-(benzyloxy)-12-oxododecanoylamino)benzamido)ethoxy)ethoxy)ethoxy)-2-(acetoxymethyl)tetrahydro-2H-pyran-3,4-diyl diacetate 78

[0380] To (2S,3S,4S,5S)-5-acetylamino-6-(2-(2-(2-(2-(3-((2-(2-(2-(((3R,4R,5R,6R)-3-acetylamino-4,5-diacetyloxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)-ethoxy)ethyl)carbamoyl)-5-aminobenzamido)ethoxy)ethoxy)ethoxy)-2-acetyl -(acetoxymethyl)-tetrahydro-2H-pyran-3,4-diyl ester (77) (4.0g, 3.6mmol), 12-(benzyloxy)-12-oxododecanoic acid (76) (1.3g, 4.1mmol) and triethylamine (1.5ml, 10.8mmol) in dichloromethane (75ml) were added dropwise T3P (4.5g, about 9ml, 50% solution in ethyl acetate). The solution was stirred at room temperature overnight. After completion, the reaction mixture was diluted with dichloromethane and carefully quenched with a saturated solution of sodium bicarbonate (200ml). The two-phase solution was stirred vigorously for 30 minutes. The DCM layer was separated and the aqueous phase was extracted with dichloromethane (1x 100ml). The combined extracts were dried over magnesium sulfate, filtered and concentrated to dryness in vacuo. The residue was purified by column chromatography on silica gel 60 (gradient: 0-10% MeOH in DCM) to give the title compound (1.5 g, 30%) as a colorless solid.

[0381] Step 3. Preparation of 12-((3-((2-(2-(((3R,4R,5R,6R)-3-acetylamino-4,5-diacetyloxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)carbamoyl)-5-((2-(2-(2-(((3S,4S,5S,6S)-3-acetylamino-4,5-diacetyloxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)carbamoyl)phenyl)amino)-12-oxododecanoic acid 79

[0382] To a solution of (2S,3S,4S,5S)-5-acetamido-6-(2-(2-(2-(3-((2-(2-(2-(((3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)-ethyl)carbamoyl)-5-(12-(benzyloxy)-12-oxododecanoylamino)benzamido)ethoxy)ethoxy)-ethoxy)-2-(acetoxymethyl)tetrahydro-2H-pyran-3,4-diyl diacetate (78) (1.5 g, 1.1 mmol) in methanol (25 ml) was added 10% palladium on carbon (wet basis, 150 mg, 10% wt / wt). Hydrogen was slowly bubbled into the solution over 1 hour. After completion, the solution was bubbled with nitrogen, filtered through celite, and concentrated to dryness in vacuo to afford a colorless solid (1.1 g, 79%).

[0383] Step 4. Preparation of (2S,3S,4S,5S)-5-acetamido-6-(2-(2-(2-(3-((2-(2-(2-(((3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)carbamoyl)-5-(12-oxo-12-(perfluorophenoxy)dodecanoylamino)benzamido)ethoxy)ethoxy)ethoxy)-2-(acetoxymethyl)tetrahydro-2H-pyran-3,4-diyl diacetate 81

[0384] To a solution of 12-((3-((2-(2-(2-(((3R,4R,5R,6R)-3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)carbamoyl)-5-((2-(2-(2-(((3S,4S,5S,6S)-3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)carbamoyl)phenyl)amino)-12-oxododecanoic acid (79) (0.6 g, 0.46 mmol) and triethylamine (125 μL, 0.92 mmol) in dichloromethane (50 ml) was added pentafluorophenyl trifluoroacetate (80) (150 mg, 1.1 mmol). The solution was stirred at room temperature for 30 minutes and then concentrated to dryness in vacuo. The residue was purified by column chromatography on silica gel 60 (gradient: 0 to 10% methanol in dichloromethane) to give the title compound (475 mg, 70%) as a colorless solid. Mass (ESI+) m / z 741.0 (M+2H). 1H NMR (400MHz, DMSO-d6) δ10.12(s,1H),8.52(t,J=5.6Hz,2H),8.14(d,J=1.4Hz,2H),7.91(t,J=1.6Hz,1H),7.80(d,J =9.2Hz,2H),5.21(d,J=3.4Hz,2H),4.97(dd,J=11.2,3.4Hz,2H),4.54(d,J=8.5Hz,2H),4.06-3.99(m,7H),3.88(dt, J=11.2,8.8Hz,2H),3.77(ddd,J=11.1,5.6,3.9Hz,2H),3.62-3.46(m,22H),3.46-3.38(m,5H),2.77(t,J=7.2Hz,2H ), 2.31 (t, J = 7.4Hz, 2H), 2.10 (s, 7H), 1.99 (s, 7H), 1.89 (s, 7H), 1.77 (s, 7H), 1.69-1.54 (m, 4H), 1.40-1.20 (m, 14H). Mass (ESI+)m / z 741.0(M+2H).

[0385] Scheme 19 Preparation of Compound 90

[0386]

[0387]

[0388] Step 1. Preparation of 12-((tert-butoxycarbonyl)amino)dodecanoic acid 84

[0389] A solution of 12-aminododecanoic acid (82) (5.0 g, 23.3 mmol), di-tert-butyl dicarbonate (83) (6.1 g, 27.9 mmol) and triethylamine (6.3 ml, 46.6 mmol) in methanol (75 ml) was heated to 60°C for 3 hours and then at room temperature overnight. Upon completion, the solution was concentrated to dryness in vacuo and used in the next step without further purification.

[0390] Step 2. Preparation of 12-((tert-butoxycarbonyl)amino)dodecanoic acid benzyl ester 85

[0391] At room temperature, a solution of crude 12-((tert-butoxycarbonyl)amino)dodecanoic acid (84) (9.0 g, 30.0 mmol), benzyl alcohol (85) (3.1 g, 30.0 mmol), EDC hydrochloride (6.9 g, 36.0 mmol) and triethylamine (12 ml, 90.0 mmol) in dichloromethane (100 ml) was stirred overnight. After completion, the solution was washed with saturated sodium bicarbonate solution (100 ml) and brine (100 ml). The dichloromethane solution was dried over magnesium sulfate, filtered and concentrated to dryness. Purified by column chromatography (gradient: hexane containing 0 to 50% ethyl acetate) on silica gel 60 to obtain the title compound (2.0 g, 21% over two steps) as a colorless solid.

[0392] Step 3. Preparation of 12-(Benzyloxy)-12-oxododecan-1-ammonium trifluoroacetate 87

[0393] A solution of benzyl 12-((tert-butoxycarbonyl)amino)dodecanoate (86) (2.0 g, 4.9 mmol), dichloromethane (15 ml) and TFA (5 ml) was stirred overnight at room temperature. The reaction mixture was concentrated to dryness to give the product as a viscous oil (2.1 g, quantitative).

[0394] Step 4. Preparation of (2S,3S,4S,5S)-5-acetamido-6-(2-(2-(2-(3-((2-(2-(2-(((3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)carbamoyl)-5-(12-((12-(benzyloxy)-12-oxododecyl)amino)-12-oxododecanoylamino)benzamido)ethoxy)ethoxy)ethoxy)-2-(acetoxymethyl)tetrahydro-2H-pyran-3,4-diyl diacetate 88

[0395] 12-((3-((2-(2-(((3R,4R,5R,6R)-3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)carbamoyl)-5-((2-(2-(((3S,4S,5S,6S)-3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl) A solution of 12-(benzyloxy)-12-oxododecanoic acid (88) (750 mg, 0.54 mmol), 12-(benzyloxy)-12-oxododecanoic acid (87) (225 mg, 0.54 mmol), HBTU (210 mg, 0.54 mmol) and diisopropylethylamine (0.3 ml, 1.62 mmol) in dichloromethane (30 ml) was stirred overnight. The solution was diluted with dichloromethane (50 ml) and washed with saturated bicarbonate solution (100 ml). The dichloromethane was dried over magnesium sulfate, filtered and concentrated to dryness in vacuo. The residue was purified by column chromatography on silica gel 60 (gradient: dichloromethane containing 0 to 10% methanol) to give the title compound (88) (605 mg, 70%) as a colorless solid.

[0396] Step 5. Preparation of 12-(12-((3-((2-(2-(((3R,4R,5R,6R)-3-acetylamino-4,5-diacetyloxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)carbamoyl)-5-((2-(2-(2-(((3S,4S,5S,6S)-3-acetylamino-4,5-diacetyloxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)carbamoyl)phenyl)amino)-12-oxododecanoylamino)dodecanoic acid 89

[0397] Hydrogenation was performed as previously described to give (89) (350 mg, 55%)

[0398] Step 6. Preparation of (2S,3S,4S,5S)-5-acetamido-6-(2-(2-(2-(3-((2-(2-(2-(((3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)carbamoyl)-5-(12-oxo-12-((12-oxo-12-(perfluorophenoxy)-dodecyl)amino)dodecanoylamino)benzamido)ethoxy)ethoxy)ethoxy)-2-(acetoxymethyl)-tetrahydro-2H-pyran-3,4-diyl diacetate 90

[0399] Formation of the PFP ester was performed as previously described to afford the desired product (90) (112 mg, 23%). 1 H NMR (400MHz, DMSO-d6) δ10.12(s,1H),8.91(s,1H),8.65(t,J=5.5Hz,1H),8.52(t,J=5.6Hz,1H),8.23(d,J=1.5Hz,1H),8.14(t,J=1.4Hz,2H),7 .91(d,J=1.6Hz,1H),7.80(d,J=9.2Hz,2H),7.68(t,J=5.6Hz,1H),5.21(d,J=3.4Hz,2H),4.97(dd,J=11.2,3.4Hz,2H),4.54(d,J=8.5Hz,2H),4. 07-3.96(m,6H),3.88(dt,J=11.2,8.9Hz,2H),3.81-3.74(m,2H),3.64- 3.36(m,24H),3.15-3.03(m,6H),2.99(q,J=6.5Hz,2H),2.76(t,J=7.2H z,1H),2.31(t,J=7.4Hz,1H),2.10(s,6H),1.99(s,7H),1.89(s,7H),1. 76(s,6H),1.70-1.53(m,3H),1.47(q,J=7.1Hz,2H),1.40-1.10(m,29H). Mass (ESI+)m / z 839.7(M+2H).

[0400] Scheme 20 Preparation of Compound 94

[0401]

[0402]

[0403] Step 1. Preparation of (2S,3S,4S,5S)-5-acetamido-6-(2-(2-(2-(3-((2-(2-(2-(((3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)carbamoyl)-5-(2-(12-(benzyloxy)-12-oxododecanoylamino)acetamido)benzamido)ethoxy)ethoxy)ethoxy)-2-(acetoxymethyl)tetrahydro-2H-pyran-3,4-diyl diacetate 92

[0404] 2-((3-((2-(2-(((3R,4R,5R,6R)-3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)carbamoyl)-5-((2-(2-(2-(((3S,4S,5S,6S)-3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)carbamoyl)- A solution of (1,2-(pyran-2-yl)oxy)ethoxy)-ethoxy)ethyl)carbamoyl)phenyl)amino)-2-oxoethyl-1-ammonium (91) (1.0 g, 0.8 mmol), 12-(benzyloxy)-12-oxododecanoic acid (76) (256 mg, 0.8 mmol), HBTU (341 mg, 0.9 mmol) and diisopropylethylamine (0.4 ml, 2.4 mmol) in dichloromethane (20 ml) was stirred overnight. After completion, the reaction mixture was diluted with dichloromethane (80 ml) and washed with saturated sodium bicarbonate (100 ml). The solution was dried over magnesium sulfate, filtered and concentrated to dryness in a vacuum. The residue was purified by column chromatography on silica gel 60 (gradient: dichloromethane containing 0 to 10% methanol) to give the title compound (0.8 g, 68%) as a colorless solid.

[0405] Step 2. Preparation of 12-((2-((3-((2-(2-(2-(((3R,4R,5R,6R)-3-acetylamino-4,5-diacetyloxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)carbamoyl)-5-((2-(2-(2-(((3S,4S,5S,6S)-3-acetylamino-4,5-diacetyloxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)carbamoyl)phenyl)amino)-2-oxoethyl)amino)-12-oxododecanoic acid 93

[0406] Compound 93 (450 mg, 60%) was prepared using conditions analogous to those described herein for similar transformations.

[0407] Step 3. Preparation of (2S,3S,4S,5S)-5-acetamido-6-(2-(2-(2-(3-((2-(2-(2-(((3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)-ethoxy)ethyl)carbamoyl)-5-(2-(12-oxo-12-(perfluorophenoxy)dodecanoylamino)acetamido)benzamido)ethoxy)ethoxy)ethoxy)-2-(acetoxymethyl)tetrahydro-2H-pyran-3,4-diyl diacetate 94

[0408] Compound 94 (460 mg, 91%) was prepared using conditions analogous to those described herein for similar transformations. Mass (ESI+) m / z 1537.8 (M+H).

[0409] Scheme 21 Preparation of Compound 95

[0410]

[0411] Synthesis of (2R,2'R,3R,3'R,4R,4'R,5R,5'R)-(((((((5-(2-(10-(3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((((2-cyanoethoxy)(diisopropylamino)phosphoalkyl)-oxy)methyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoylamino)acetamido)-isophthaloyl)bis(azanediyl))bis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl))bis(oxy))-bis(ethane-2,1-diyl))bis(oxy))bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl) tetraacetate 95

[0412] To (2S,3S,4S,5S)-5-acetylamino-6-(2-(2-(2-(3-((2-(2-(2-(2-(((3R,4R,5R,6R)-3-acetylamino-4,5-diacetyloxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)carbamoyl)-5-(2-(10-(3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3-(4-methoxyphenyl)-1-yl)-1-yl)-2-thiazolyl-4-thiazolyl-2 ... To a solution of (4-dimethylpyrrolidin-1-yl)-10-oxodecanoylamino)acetamido)benzamido)-ethoxy)ethoxy)ethoxy)-2-(acetoxymethyl)tetrahydro-2H-pyran-3,4-diyl ester (72) (1.6 g, 0.9 mmol) and diisopropylethylamine (0.4 ml, 1.8 mmol) in anhydrous dichloromethane (25 ml) was added 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.3 ml, 1.35 mmol). The solution was stirred at room temperature for 75 minutes and then concentrated to dryness. The residue was purified by column chromatography (gradient: DCM (0.1% TEA) containing 0 to 10% MeOH) to give the product (1.1 g, 62%) as a colorless solid. 31P NMR (400 MHz, DMSO-d6): δ 146.76 (s), 146.42 (s, 2 overlapping signals), 146.34 (s). 1HNMR(400MHz,DMSO-d6)δ10.20(s,1H),8.54(t,J=5.6Hz,2H),8.17-8.09(m,3H),7.94(s,1H),7.80(d,J=9.2Hz,2H),7.39-7.26(m,4H), 7.26-7.17(m,6H),6.91-6.83(m,4H),5.21(d,J=3.4Hz,2H),4.97(dd,J=11.2,3.4Hz,2H),4.54(d,J=8.5Hz,2H),4.02(s,6H),3.93-3.82 (m,4H),3.73(s,10H),3.66-3.36(m,35H),3.28-3.06(m,6H),3.06-2.87(m,3H),2.72-2.63(m,J=11.5,5.8Hz,2H),2.10(m,12H),1.99(s ,6H),1.89(s,6H),1.77(s,6H),1.47(d,J=7.2Hz,4H),1.23(dq,J=13.9,6.4Hz,18H),1.17-1.04(m,10H),0.98(dt,J=13.4,5.9Hz,10H).

[0413] Scheme 22 Preparation of Compound 96

[0414]

[0415] Step 1. Preparation of (2S,3S,4S,5S)-5-acetamido-6-(2-(2-(2-(3-((2-(2-(2-(((3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)carbamoyl)-5-(2-(12-((10-(3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanyl)amino)dodecanoylamino)acetamido)benzamido)ethoxy)ethoxy)ethoxy)-2-(acetoxymethyl)tetrahydro-2H-pyran-3,4-diyl diacetate 95

[0416] Compound 95 (1.9 g, 61%) was prepared using conditions analogous to those described herein for similar transformations.

[0417] Step 2: Preparation of diacetyl (2S,3S,4S,5S)-5-acetylamino-6-(2-(2-(2-(3-((2-(2-(2-(((3R,4R,5R,6R)-3-acetylamino-4,5-diacetyloxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)-ethoxy)ethyl)carbamoyl)-5-(2-(12-((10-( 3-((bis(4-methoxyphenyl)(phenyl)methoxy)-methyl)-4-((((2-cyanoethoxy)(diisopropylamino)phosphoalkyl)oxy)methyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanyl)amino)dodecanoylamino)acetylamino)benzamido)ethoxy)ethoxy)ethoxy)-2-(acetoxymethyl)tetrahydro-2H-pyran-3,4-diyl ester 96

[0418] Compound 96 (1.35 g, 65%) was prepared using conditions similar to those described herein for similar transformations. 31 P NMR (400MHz, DMSO-d6): δ146.79(s), 146.76(s), 146.42(s), 146.36(s). 1H NMR (400MHz, DMSO-d6) δ10.19(s,1H),8.54(t,J=5.6Hz,2H),8.13(dd,J=6.1,3.5Hz,3H),7.94(s,1H),7.80(d,J=9.2Hz,2H),7.71-7.65(m,1H),7. 39-7.25(m,4H),7.25-7.17(m,4H),6.92-6.83(m,4H),5.21(d,J=3.4Hz, 2H),4.97(dd,J=11.2,3.4Hz,2H),4.54(d,J=8.5Hz,2H),4.07-3.97(m,6H ),3.94-3.82(m,4H),3.82-3.74(m,2H),3.73(s,6H),3.62-3.45(m,23H) ,3.42(m,6H),3.27-2.92(m,14H),2.73-2.62(m,2H),2.10(s,8H),1.99(s ,9H),1.89(s,6H),1.77(s,6H),1.52-1.42(m,6H),1.22(d,J=8.0Hz,24H ),1.17(t,J=7.3Hz,11H),1.09(dt,J=6.7,3.3Hz,9H),1.03-0.92(m,9H).

[0419] Scheme 23 General synthesis of conjugates of formula I with oligonucleotides coupled via the 3' end of the oligonucleotide (Compound 73)

[0420]

[0421] General method for synthesizing bidentate ASGPr targeting ligands from succinate ligands exemplified for 4-((1-(10-((2-((3,5-bis((2-(2-(2-(((3R,4R,5R,6R)-3-acetylamino-4,5-diacetyloxy-6-(acetyloxymethyl)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 97

[0422] The succinate was loaded onto the LCAA (long chain aminoalkyl) CPG (controlled pore glass). LCAA CPG (2.0 g) was suspended in DCM (5 ml) and MeCN (7.6 ml). Diisopropylcarbodiimide (100 μl), N-hydroxysuccinimide (110 μl, 30 μM / g), pyridine (110 μL) and 56 (200 mg, 0.1 mmol) were added, and the suspension was slowly mixed at room temperature for 16 hours. CPG was recovered by filtration, washed with DCM (×3) and MeCN (×3) and dried under high vacuum. A solution of 5% acetic anhydride / 5% N-methylimidazole / 5% pyridine in THF was added, and the suspension was stirred at room temperature for 2 hours. CPG was recovered by filtration, washed with DCM (×3) and MeCN (×3) and dried under high vacuum. The load was determined to be 31.3 μmol / g (DMTr determination, by UV / Vis 504nm). The resulting GalNAc-loaded CPG solid support was used in automated oligonucleotide synthesis using standard procedures. Nucleotide deprotection followed by removal from the solid support (with simultaneous deprotection of galactosamine acetate) afforded GalNAc-oligonucleotide conjugate 97.

[0423] Schemes 24 and 25 General synthesis of conjugates of Formula I with oligonucleotides coupled via the 5' end of the oligonucleotide (Compound 98)

[0424] Pentafluorophenyl ester was coupled to C6 5'-amino modifier with phosphate / phosphorothioate linkage on sense strand oligonucleotide using standard coupling conditions. Standard cleavage and deprotection gave the desired sense strand conjugate. For example, pentafluorophenyl ester 81 was used to give the following conjugate 98.

[0425]

[0426] The phosphoramidite was coupled to the 5' hydroxyl group of the terminal nucleotide of the sense strand using standard phosphoramidite coupling chemistry. Standard cleavage and deprotection afforded the desired sense strand conjugate. For example, phosphoramidite 95 was used to obtain the following conjugate 99.

[0427]

[0428] Examples 1-9

[0429] Using the general procedure illustrated in Scheme 23, the following conjugates of the invention were prepared, wherein R 3b are the modified TTR siRNAs described below in Table A. Masses apply to single stranded products - not to annealed duplexes.

[0430] Example 1

[0431]

[0432] MS (+VE) calculated value: 8184.7; measured value: 8184.2

[0433] Example 2

[0434]

[0435] MS (+VE) calculated value: 8212.7; measured value 8211.9

[0436] Example 3

[0437]

[0438] MS (+VE) calculated value: 8212.7; measured value: 8212.8

[0439] Example 4

[0440]

[0441] MS (+VE) calculated value: 8096.6; measured value: 8097.0

[0442] Example 5

[0443]

[0444] MS (+VE) calculated: 8499.0; measured: 8498.7

[0445] Example 6

[0446]

[0447] MS (+VE) calculated value: 8284.7; measured value: 8283.8

[0448] Example 7

[0449]

[0450] MS (+VE) calculated: 7596.0; measured: 7596.8

[0451] Example 8

[0452]

[0453] MS (+VE) calculated value: 8140.6; measured value: 8139.6

[0454] Example 9

[0455]

[0456] MS (+VE) calculated value: 8038.5; measured value: 8037.5

[0457] Embodiment 10-11

[0458] Using the general procedure illustrated in Scheme 24, the following conjugates of the invention were prepared, wherein R 3b are the modified TTR siRNAs described below in Table A. Masses apply to single stranded products - not to annealed duplexes.

[0459] Example 10

[0460]

[0461] MS (+VE) calculated value: 8056.7; measured value: 8056.1

[0462] Embodiment 11

[0463]

[0464] MS (+VE) calculated value: 8254.0; measured value: 8253.5

[0465] Example 12: In vivo testing of TTR siRNA bidentate conjugates

[0466] The in vivo activity of bidentate conjugates (Examples 1-8) in which the oligonucleotide is a modified TTR siRNA described in Table A was tested in a wild-type mouse model of TTR knockdown. The TTR conjugate is a possible treatment for the rare disease of TTR (transthyretin) amyloidosis. In those suffering from this disease, misfolding and aggregation of transthyretin are known to be associated with disease progression. By using this siRNA conjugate, the amount of misfolded / aggregated protein in the patient can be reduced, thereby potentially halting the progression of the disease.

[0467] Table A. Chemically modified TTR siRNA duplexes

[0468]

[0469] 2'-O-methyl nucleotides = lowercase; 2'-fluoro nucleotides = capital; Phosphorothioate linker = s; unmodified = uppercase

[0470] TTR siRNA sequences and animal models are described by Nair et al. J. Am. Chem. Soc., 2014, 136(49), 16958-16961. All animal-related procedures were performed according to written procedures, in accordance with the Good Animal Practice Guidelines of the Canadian Council on Animal Care (CCAC), and approved by the local Institutional Animal Care and Use Committee (IACUC).

[0471] siRNA Treatment: Female C57BL / 6 mice (n=4) were administered a single dose of 2 mg / kg of TTR siRNA conjugate via subcutaneous injection in the scapular region on day 0 (1 dose per animal). A group of animals administered vehicle only (PBS) served as control.

[0472] Collection: All animals were test bled at defined time points after test article administration (Days 2, 4, 5, 7, 8, 9, 14, and 21) to determine the maximal reduction in plasma TTR levels and the duration of pharmacological activity.

[0473] Analysis: TTR protein levels in plasma samples were determined using the Abnova Prealbumin (mouse) ELISA kit (Cedar Lane, catalog number KA2070) according to the manufacturer's instructions. TTR plasma protein values ​​were calculated for each plasma sample and the mean for each group was determined. From these mean values, TTR protein levels relative to controls were determined (% relative to PBS-treated animals).

[0474] Results: The test results are presented in Table B. Values ​​represent % TTR protein levels (relative to PBS control) on days 2, 4, 5, 7, 8, 9, 14 and 21 after treatment.

[0475] Conclusions: Animals treated with the TTR bidentate conjugates exhibited significant target mRNA and protein knockdown, with maximal TTR protein knockdown occurring between days 4 and 9 following subcutaneous injection.

[0476] Table B. Plasma TTR protein levels in mice after a single intravenous administration (2 mg / kg) of GalNAc bidentate-conjugated siRNAs from Table A.

[0477] TTR protein data are expressed as percentage of the values ​​for PBS-treated mice

[0478]

[0479] Example 13: In vivo testing of TTR siRNA bidentate conjugates

[0480] The R 3b

[00136] The in vivo activities of the monovalent, bidentate, trivalent, and tetravalent conjugates of the modified TTR siRNA described in Table A above (Compounds AD).

[0481] Compound A (monovalent)

[0482]

[0483] Compound B (bidate)

[0484]

[0485] Compound C (trivalent)

[0486]

[0487] Compound D (quadrivalent)

[0488]

[0489] TTR conjugate is a possible treatment for the rare disease of TTR (transthyretin) amyloidosis. In those suffering from this disease, misfolding and aggregation of transthyretin is known to be associated with disease progression. By using this siRNA conjugate, the amount of misfolded / aggregated protein in the patient's body can be reduced, thereby potentially halting the progression of the disease.

[0490] TTR siRNA sequences and animal models are described by Nair et al. J. Am. Chem. Soc., 2014, 136(49), 16958-16961. All animal-related procedures were performed according to written procedures, in accordance with the Good Animal Practice Guidelines of the Canadian Council on Animal Care (CCAC), and approved by the local Institutional Animal Care and Use Committee (IACUC).

[0491] siRNA Treatment: Female C57BL / 6 mice (n=4) were administered a single dose of 2 mg / kg of TTR siRNA conjugate via subcutaneous injection in the scapular region on day 0 (1 dose per animal). A group of animals administered vehicle only (PBS) served as control.

[0492] Collection: All animals were test bled at defined time points (Day 2, Day 5, Day 7, Day 14, and Day 21) after administration of the test article to determine the maximal reduction in plasma TTR levels and the duration of pharmacological activity.

[0493] Analysis: TTR protein levels in plasma samples were determined using the Abnova Prealbumin (mouse) ELISA kit (Cedar Lane, catalog number KA2070) according to the manufacturer's instructions. TTR plasma protein values ​​were calculated for each plasma sample and the mean for each group was determined. From these mean values, TTR protein levels relative to controls were determined (% relative to PBS-treated animals).

[0494] Results: The test results are presented in Table C. Values ​​represent % TTR protein levels (relative to PBS control) on days 2, 5, 7, 14 and 21 after treatment.

[0495] Conclusions: Animals treated with TTR bidentate, trivalent, and tetravalent conjugates exhibited similar levels of target mRNA and protein knockdown, with maximal TTR protein knockdown occurring between days 2 and 7 following subcutaneous injection. TTR monovalent conjugates showed little, if any, target mRNA and protein knockdown.

[0496] Table C. Plasma TTR protein levels in mice after a single intravenous administration (2 mg / kg) of GalNAc-conjugated siRNA Compounds AD.

[0497] TTR protein data are expressed as percentage of the values ​​for PBS-treated mice

[0498]

Claims

1. A conjugate selected from the group consisting of: in: R 3 is a nucleic acid; or a salt thereof.

2. A compound selected from the group consisting of: wherein Pg is a protecting group; or a salt thereof.

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