Bivalent targeted conjugates

Abstract

To provide conjugates useful for targeting therapeutic nucleic acids.SOLUTION: The invention provides: conjugates that comprise a bivalent targeting moiety, a nucleic acid, and optional linking groups; synthetic intermediates and synthetic methods useful for preparing the conjugates; compositions comprising the bidentate targeting ligands and the conjugates; and methods for targeting therapeutic nucleic acids with the bidentate conjugates.SELECTED DRAWING: None

Description

Cross - reference to related applications

[0001] This patent application claims the benefit of priority based on U.S. Application No. 62 / 755,179, filed on November 2, 2018, and this application is incorporated herein by reference.

Background Art

[0002] Since the seminal paper by Ashwell and Morell elucidating the role of the asialoglycoprotein receptor (ASGPr) in the recognition and transport of circulating glycoproteins, this receptor has been the focus of intensive research (Non - Patent Document 1). This receptor is an attractive target for liver - specific delivery agents due to its high level of expression on the surface of hepatocytes. This receptor has a trivalent carbohydrate - binding domain that selectively binds to N - acetylgalactosamine. The generally accepted rule is that the binding affinity to a target - directed ligand increases with the 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 (Non - Patent Documents 2 - 6).

[0003] In most cases, the chemical synthesis of multi - valent target - directed ligands may be required, and as a result, multiple (sometimes 20 - 30) synthetic steps may be needed. This affects the manufacturing requirements and the cost of the product. Moreover, the synthesis of GalNac / siRNA conjugates is typically carried out on an immobilized controlled - pore glass (CPG) support. The access to the reactive sites on the support is related to the pore size, and thus, it is adversely affected by the size of the molecules approaching the site. The increase in the size (number of monosaccharide units, molecular weight, molecular radius, etc.) of the target - directed ligand has an adverse effect on the loading efficiency in the support. Therefore, there is a current need for target - directed ligands that have useful delivery properties and are easier to prepare, less expensive to prepare, have a smaller molecular weight, and / or have a higher loading efficiency.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Summary of the Invention

Means for Solving the Problems

[0005] Target-directed bivalent GalNac ligands containing two sugar groups (e.g., N-acetylgalactosamine moieties) have been identified that have comparable or better target-directed activity compared to known trifunctional and tetrafunctional ligands. These target-directed bivalent ligands generally have shorter synthetic routes and result in higher overall synthetic efficiency. Additionally, their smaller molecular size allows for greater penetration into CPGs, resulting in loading levels that are 30-50% higher than some trifunctional and tetrafunctional ligands. In addition, target-directed bivalent ligands are simpler to analyze compared to trifunctional and tetrafunctional ligands, whereby ADME toxicity investigations and related research activities can be facilitated. The present invention provides target-directed bivalent ligands, nucleic acid conjugates of these target-directed bivalent ligands, compositions comprising the target-directed bivalent ligands and conjugates, and methods of targeting therapeutic nucleic acids with the bivalent conjugates.

[0006] In one embodiment, the present invention provides a conjugate of formula (I): A conjugate of formula (I): [Chemical formula] Wherein, R 1 is a sugar, L 1 is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having from 0 to 20 carbon atoms, and one or more of the carbon atoms in the hydrocarbon chain are optionally replaced by -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X -, or -S-, and R X is hydrogen or (C1-C6) alkyl, and the hydrocarbon chain is optionally substituted with one or more substituents selected from oxo (=O) and halo, B is a 5- to 10-membered aryl or 5- to 10-membered heteroaryl, and the 5- to 10-membered aryl or 5- to 10-membered heteroaryl 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, L 2 is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 0 to 20 carbon atoms, and one or more of the carbon atoms in the hydrocarbon chain are optionally replaced by -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X -, or -S-, where R X is hydrogen or (C1-C6)alkyl, and the hydrocarbon chain is optionally substituted with one or more substituents selected from oxo(=O) and halo, R 2 is a sugar, L 3 is absent or a linking group, A is absent, a 3- to 20-membered cycloalkyl, a 5- to 20-membered aryl, a 5- to 20-membered heteroaryl, or a 3- to 20-membered heterocycloalkyl, Each R A is independently selected from the group consisting of hydrogen, hydroxy, CN, F, Cl, Br, I, -C 1-2 alkyl-OR a , C 1-10 alkyl C 2-10 alkenyl, and C 2-10 alkynyl, and C 1-10 alkyl C 2-10 alkenyl, and C 2-10 alkynyl are optionally substituted with one or more groups independently selected from halo, hydroxy, and C 1-3 alkoxy, n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, L4 is absent or is a linking group, R 3 is a nucleic acid, R a is hydrogen, a protecting group, a covalent bond to a solid support, or a linking group L attached to a solid support 5 is a bond to, L 5 is a linking group), or a salt thereof is provided.

[0007] 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.

[0008] The present invention also provides synthetic intermediates and methods disclosed herein that are useful for preparing the conjugate of formula I.

[0009] Other objects, features and advantages of the present invention will become apparent to those skilled in the art from the following detailed description and figures.

Mode for Carrying Out the Invention

[0010] As used herein, the following terms have the meanings ascribed to them unless otherwise specified.

[0011] The terms "alkoxy" and "alkylthio" are used in their conventional meanings and refer to an alkyl group attached to the remainder of the molecule through an oxygen atom ("oxy") or a thio group, and further include their monohalogenated and polyhalogenated variants.

[0012] The term "alkyl", unless otherwise specified, by itself or as part of another substituent, refers to the specified number of carbon atoms (i.e., C 1-8refers to a straight-chain or branched-chain hydrocarbon radical having 1 to 8 carbons (meaning 1 to 8 carbons). Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. The term "alkenyl" refers to an unsaturated alkyl radical having one or more double bonds. Similarly, the term "alkynyl" refers to an unsaturated alkyl radical having one or more triple bonds. Examples of such unsaturated alkyl groups include vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and higher homologs and isomers.

[0013] The term "animal" includes mammalian species such as humans, mice, rats, dogs, cats, hamsters, guinea pigs, rabbits, domestic animals, and the like.

[0014] As used herein, the term "aryl" refers to a monocyclic all-carbon aromatic ring or a polycyclic fused all-carbon ring system in which at least one of the rings is an aromatic ring. For example, in certain embodiments, an aryl group has 6 to 20 carbon atoms, 6 to 14 carbon atoms, 6 to 12 carbon atoms, or 6 to 10 carbon atoms. Aryl includes phenyl radicals. Aryl also includes polycyclic fused carbon ring systems having about 9 to 20 carbon atoms and at least one ring being an aromatic ring and the other rings being aromatic or non-aromatic (e.g., cycloalkyl) rings (e.g., ring systems containing 2, 3, or 4 rings). The rings of the polycyclic fused ring system can be linked to each other by fused bonds, spiro bonds, and bridging bonds when permitted by valence requirements. It should be understood that the point of attachment of the polycyclic fused ring system defined above can be at any position of the ring system, e.g., the aromatic ring portion or the carbon ring portion of the ring. Non-limiting examples of aryl groups include phenyl, indenyl, indanyl, naphthyl, 1,2,3,4-tetrahydronaphthyl, anthracenyl, and the like, but are not limited thereto.

[0015] The term "cycloalkyl" refers to a saturated or partially unsaturated (non-aromatic) all-carbon ring having 3 to 8 carbon atoms (i.e., a (C3-C8) carbon ring). The term includes polycyclic fused saturated all-carbon ring systems (e.g., ring systems containing 2, 3, or 4 carbon rings). Thus, the carbon rings include polycyclic carbon rings such as bicyclic carbon rings (e.g., bicyclic carbon rings having about 3 to 15 carbon atoms, about 6 to 15 carbon atoms, or about 6 to 12 carbon atoms, such as bicyclo[3.1.0]hexane and bicyclo[2.1.1]hexane), and polycyclic carbon rings (e.g., tricyclic and tetracyclic carbon rings having up to about 20 carbon atoms). The rings of a polycyclic fused ring system can be linked to each other by fused bonds, spiro bonds, and bridging bonds when permitted by valence requirements. For example, polycyclic carbon rings may be linked to each other by a single carbon atom to form a spiro-type linkage (e.g., spiropentane, spiro[4,5]decane, etc.), or by two adjacent carbon atoms to form a fused-type linkage (e.g., carbon rings such as decahydronaphthalene, norsabinane, norcarane, etc.), or by two non-adjacent carbon atoms to form a bridging-type linkage (e.g., norbornane, bicyclo[2.2.2]octane, etc.). Non-limiting examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo[2.2.1]heptane, pinane, and adamantane.

[0016] The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence that includes a partial-length or full-length coding sequence necessary for the production of a polypeptide or precursor polypeptide.

[0017] "Gene product", as used herein, refers to the product of a gene, e.g., an RNA transcript or a polypeptide.

[0018] The term "halo" or "halogen" means a fluorine, chlorine, bromine, or iodine atom, unless otherwise specified.

[0019] As used herein, the term "heteroaryl" refers to a monocyclic aromatic ring having at least one atom other than carbon selected from the group consisting of oxygen, nitrogen, and sulfur within the ring, and "heteroaryl" includes polycyclic fused ring systems having at least one such aromatic ring, which are further described below. Thus, "heteroaryl" includes monocyclic aromatic rings having about 1 to 6 carbon atoms and about 1 to 4 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur. As long as the ring is an aromatic ring, sulfur and nitrogen atoms may be present in an oxidized form. Exemplary heteroaryl ring systems include, but are not limited to, pyridyl, pyrimidinyl, oxazolyl, and furyl. "Heteroaryl" also includes polycyclic fused ring systems (e.g., ring systems containing 2, 3, or 4 rings) in which one or more rings selected from cycloalkyl, aryl, heterocycle, and heteroaryl are fused to the heteroaryl group defined above. It should be understood that the point of attachment to the heteroaryl or heteroaryl polycyclic fused ring system can be any suitable atom including carbon and heteroatoms (e.g., nitrogen) of the heteroaryl or heteroaryl polycyclic fused ring system. Exemplary heteroaryls include, but are not limited to, pyridyl, pyrrolyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrazolyl, thienyl, indolyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, furyl, oxadiazolyl, thiadiazolyl, quinolyl, isoquinolyl, benzothiazolyl, benzoxazolyl, indazolyl, quinoxalyl, and quinazolinyl.

[0020] The term "heterocyclic ring" refers to a saturated or partially unsaturated monocyclic ring having at least one atom other than carbon selected from the group consisting of oxygen, nitrogen, and sulfur, and the term includes polycyclic condensed ring systems having at least one such saturated or partially unsaturated ring, which are further described below. Thus, the term includes saturated or partially unsaturated monocyclic rings (e.g., 3, 4, 5, 6, or 7-membered rings) in which there are about 1 to 6 carbon atoms in the ring and about 1 to 3 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur. The sulfur and nitrogen atoms may be present in their oxidized forms. Exemplary heterocyclic rings include, but are not limited to, azetidinyl, tetrahydrofuranyl, and piperidinyl. The term "heterocyclic ring" can also include polycyclic condensed ring systems (e.g., ring systems containing 2, 3, or 4 rings) that can be formed by the condensation of a single (as defined above) heterocyclic ring with one or more groups selected from cycloalkyl, aryl, and heterocyclic rings. The rings of the polycyclic condensed ring system can be linked to each other by fused bonds, spiro bonds, and bridging bonds when permitted by valence requirements. It should be understood that the individual rings of the polycyclic condensed ring system can be linked to each other in any order. It should also be understood that the point of attachment of the polycyclic condensed ring system (as defined above for heterocyclic rings) can be at any position of the polycyclic condensed ring system, including the heterocyclic, aryl, and carbocyclic moieties of the ring. In one embodiment, the term "heterocyclic ring" includes 3- to 15-membered heterocyclic rings. In one embodiment, the term "heterocyclic ring" includes 3- to 10-membered heterocyclic rings. In one embodiment, the term "heterocyclic ring" includes 3- to 8-membered heterocyclic rings. In one embodiment, the term "heterocyclic ring" includes 3- to 7-membered heterocyclic rings. In one embodiment, the term "heterocyclic ring" includes 3- to 6-membered heterocyclic rings. In one embodiment, the term "heterocyclic ring" includes 4- to 6-membered heterocyclic rings. In one embodiment, the term "heterocyclic ring" includes monocyclic or bicyclic 3- to 10-membered heterocyclic rings containing 1 to 4 heteroatoms. In one embodiment, the term "heterocyclic ring" includes monocyclic or bicyclic 3- to 8-membered heterocyclic rings containing 1 to 3 heteroatoms. In one embodiment, the term "heterocyclic ring" includes monocyclic or bicyclic 3- to 6-membered heterocyclic rings containing 1 to 2 heteroatoms.In one embodiment, the term "heterocyclic ring" includes monocyclic 4- to 6-membered heterocyclic rings containing 1 to 2 heteroatoms. Exemplary heterocyclic rings include aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, homopiperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, tetrahydrofuranyl, dihydrooxazolyl, tetrahydropyranyl, tetrahydrothiopyranyl, 1,2,3,4-tetrahydroquinolyl, benzoxazinyl, dihydrooxazolyl, chromanyl, 1,2-dihydropyridinyl, 2,3-dihydrobenzofuranyl, 1,3-benzodioxolyl, 1,4-benzodioxanyl, spiro[cyclopropane-1,1'-isoindolinyl]-3'-one, isoindolinyl-1-one, 2-oxa-6-azaspiro[3.3]heptanyl, imidazolidin-2-one imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin, dioxolane, phthalimide and 1,4-dioxane, but are not limited thereto.

[0021] The term "sugar" includes monosaccharides, disaccharides and trisaccharides, all of which may optionally be substituted. The term includes glucose, sucrose fructose, galactose and ribose, as well as deoxysugars such as deoxyribose, and amino sugars such as galactosamine. Sugar derivatives can be conveniently prepared as described in International Patent Application Publications WO96 / 34005 and 97 / 03995. The sugar can be conveniently linked to the remainder of the compound of formula I by an ether bond, a thioether bond (e.g., S-glycoside), an amine nitrogen (e.g., N-glycoside) or a carbon-carbon bond (e.g., C-glycoside). In one embodiment, the sugar can be conveniently linked to the remainder of the compound of formula I by an ether bond.

[0022] As used herein, the term "small interfering RNA" or "siRNA" refers to a double-stranded RNA (i.e., a duplex RNA) that can reduce or inhibit the expression of a target gene or sequence when the siRNA is in the same cell as the target gene or sequence (e.g., by mediating the degradation of mRNA that is complementary to the siRNA sequence or by inhibiting translation). The siRNA may have substantial or complete identity with the target gene or sequence, or may contain regions of mismatch (i.e., mismatch motifs). In certain embodiments, the siRNA can be about 19-25 (duplex) nucleotides in length, preferably about 20-24, 21-22, or 21-23 (duplex) nucleotides in length. The siRNA duplex can include a 3' overhang of about 1 to about 4 nucleotides, or about 2 to about 3 nucleotides, and a 5' phosphate terminus. Examples of siRNAs include, but are not limited to, double-stranded polynucleotide molecules assembled from two separate molecular strands, one with a sense strand and the other with a complementary antisense strand.

[0023] In certain embodiments, the 5' and / or 3' overhangs on one or both strands of the siRNA include 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 having complementarity to the target sequence (e.g., a 3' overhang in the antisensor strand) or its complementary strand (e.g., a 3' overhang in the sense strand).

[0024] Preferably, the siRNA is chemically synthesized. The siRNA can also be generated by cleavage of longer dsRNA (e.g., dsRNA longer than about 25 nucleotides) using E. coli RNase III or Dicer. These enzymes process the dsRNA into biologically active siRNA (see, for example, Yang et al., Proc. Natl. Acad. Sci. USA, 99:9942-9947 (2002), Calegari et al., Proc. Natl. Acad. Sci. USA, 99:14236 (2002), Byrom et al., Ambion TechNotes, 10(1):4-6 (2003), Kawasaki et al., Nucleic Acids Res., 31:981-987 (2003), Knight et al., Science, 293:2269-2271 (2001), and Robertson et al., J. Biol. Chem., 243:82 (1968)). Preferably, the dsRNA is at least 50 nucleotides to about 100, 200, 300, 400 or 500 nucleotides in length. The dsRNA can be 1000, 1500, 2000, 5000 nucleotides in length or longer. The dsRNA can encode the entire gene transcript or a partial gene transcript. In some cases, the siRNA can be encoded in a plasmid (e.g., transcribed as a sequence that automatically folds into a duplex with a hairpin loop).

[0025] The term "inhibiting 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. To examine the degree of gene silencing, a test sample (e.g., a biological sample from an organism of interest in which the target gene is expressed, or a sample of cells in a culture in which the target gene is expressed) is contacted with an siRNA that silences, reduces or inhibits the expression of the target gene. The expression of the target gene in the test sample is compared with the expression of the target gene in a control sample (e.g., a biological sample from an organism of interest in which the target gene is expressed, or a sample of cells in a culture in which the target gene is expressed) that has not been contacted with the siRNA. A value of 100% can be assigned to the control sample (e.g., a sample in which the target gene is expressed). In a specific embodiment, 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% compared to a control sample (e.g., buffer only, an siRNA sequence targeting a different gene, a scrambled siRNA sequence, etc.), and silencing, inhibition or reduction of the expression of the target gene is achieved. Suitable assays include, but are not limited to, dot blot, Northern blot, in situ hybridization, ELISA, immunoprecipitation, techniques known to those skilled in the art such as enzyme function, and tests at the protein or mRNA level using phenotypic assays known to those skilled in the art.

[0026] The "effective amount" or "therapeutically effective amount" of a therapeutic nucleic acid such as siRNA is an amount sufficient to produce the desired effect, for example, inhibition of the expression of a target sequence when compared to the normal expression level detected in the absence of siRNA. In a particular embodiment, the value obtained using the siRNA is about 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or 0% compared to a control (e.g., buffer only, siRNA sequences targeting different genes, scrambled siRNA sequences, etc.), inhibition of the expression of the target gene or target sequence is achieved. Assays suitable for measuring the expression of the target gene or target sequence include, for example, dot blot, Northern blot, in situ hybridization, ELISA, immunoprecipitation, techniques known to those skilled in the art such as enzyme function, and tests at the protein or mRNA level using phenotypic assays known to those skilled in the art, but are not limited thereto.

[0027] As used herein, the term "nucleic acid" refers to a polymer containing at least two nucleotides (i.e., deoxyribonucleotides or ribonucleotides) in either single-stranded or double-stranded form, including DNA and RNA. A "nucleotide" contains a sugar, deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together by phosphate groups. "Bases" include purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, as well as synthetic derivatives of purines and pyrimidines, including, but not limited to, modified forms having new reactive groups, such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages that have binding properties comparable to the reference nucleic acid, whether synthetic, naturally occurring, or non-naturally occurring. Examples of such analogs and / or modified residues include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2'-O-methyl ribonucleotides, and peptide nucleic acids (PNA). In addition, a nucleic acid may contain one or more UNA moieties.

[0028] The term "protecting group" refers to a substituent commonly employed to block or protect a specific functional group on a compound. For example, an "amino protecting group" is a substituent attached to an amino group that blocks or protects the amino functionality of the compound. Suitable amino protecting groups include acetyl, trifluoroacetyl, t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ), and 9-fluorenylmethyloxycarbonyl (Fmoc). Similarly, a "hydroxy protecting group" refers to a substituent of a hydroxy group that blocks or protects the hydroxy functionality. Suitable protecting groups include acetyl, silyl, and 2,2-dimethoxypropene. A "carboxy protecting group" refers to a substituent of a carboxy group that blocks or protects the carboxy functionality. Common carboxy protecting groups include phenylsulfonylethyl, cyanoethyl, 2-(trimethylsilyl)ethyl, 2-(trimethylsilyl)ethoxymethyl, 2-(p-toluenesulfonyl)ethyl, 2-(p-nitrophenylsulfenyl)ethyl, 2-(diphenylphosphino)-ethyl, nitroethyl, and the like. For a general description of protecting groups and their use, see P.G.M. Wuts and T.W.G. Greene, Greene’s Protective Groups in Organic Synthesis 4 th edition, Wiley-Interscience, New York, 2006.

[0029] The term "synthetic activating group" refers to a group that can bind to an atom and activate that atom to form a covalent bond with another reactive group. It is understood that the nature of the synthetic activating group can be determined by the atom it activates. For example, when a synthetic activating group is attached to an oxygen atom, the synthetic activating group is a group that activates that oxygen atom to form a bond with another reactive group (e.g., an ester, carbamate, or ether bond). Such synthetic activating groups are known. Examples of synthetic activating groups that can be attached to an oxygen atom include, but are not limited to, acetate, succinate, triflate, and mesylate. When a synthetic activating group is attached to the oxygen atom of a carboxylic acid, the synthetic activating group can be a group derived from a known coupling reagent (e.g., a known amide coupling reagent). Such coupling reagents are known. Examples of such coupling reagents include, but are not limited to, N,N'-dicyclohexylcarbodiimide (DCC), hydroxybenzotriazole (HOBt), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC), (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), propylphosphonic anhydride solution (T3P), or O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU).

[0030] Nucleic acid The term "nucleic acid" includes any oligonucleotide or polynucleotide, with fragments of 60 nucleotides or less generally referred to as oligonucleotides and longer fragments as polynucleotides. Deoxyribooligonucleotides consist of a pentose sugar called deoxyribose covalently bonded to phosphate at the 5' and 3' carbons of this sugar to form an alternating unbranched polymer. DNA can be in the form of, for example, antisense molecules, plasmid DNA, pre-aggregated DNA, PCR products, vectors, expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups. Ribooligonucleotides consist of a similar repeating structure where the pentose sugar is ribose. RNA can be in the form of, for example, small interfering RNA (siRNA), Dicer substrate dsRNA, short hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, tRNA, viral RNA (vRNA), self-amplifying RNA (saRNA), and combinations thereof. Thus, in the context of the present invention, the terms "polynucleotide" and "oligonucleotide" refer to polymers or oligomers of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars, and sugar-sugar (backbone) linkages. The terms "polynucleotide" and "oligonucleotide" also include polymers or oligomers containing non-naturally occurring monomers or portions thereof that function similarly. Such modified or substituted oligonucleotides are often preferred over their native forms due to properties such as enhanced cellular uptake, reduced immunogenicity, and improved stability in the presence of nucleases.

[0031] Unless otherwise indicated, a particular nucleic acid sequence encompasses not only the explicitly recited sequence, but also its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixture of 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)).

[0032] In certain embodiments, the target-directed bivalent ligands described herein can be conjugated to a nucleic acid. In certain embodiments, the nucleic acid is a nucleic acid described herein. For example, the nucleic acids used herein can be single-stranded DNA or RNA, or double-stranded DNA or RNA, or DNA-RNA hybrid strands. Examples of double-stranded RNA are described herein and include, for example, siRNA and other RNAi agents, such as aiRNA and precursor miRNA. Examples of single-stranded nucleic acids include, for example, antisense oligonucleotides, ribozymes, mature miRNA, and triplex-forming oligonucleotides.

[0033] In certain embodiments, the nucleic acid is an oligonucleotide. In a more specific embodiment, the oligonucleotide ranges in length from about 10 to about 100 nucleotides. In various related embodiments, single-stranded, double-stranded, and triple-stranded oligonucleotides can each range in length from about 10 to about 60 nucleotides, from about 15 to about 60 nucleotides, from about 20 to about 50 nucleotides, from about 15 to about 30 nucleotides, or from about 20 to about 30 nucleotides.

[0034] In certain embodiments, the nucleic acid is selected from the group consisting of small interfering RNA (siRNA), Dicer-substrate dsRNA, short hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), tRNA, rRNA, tRNA, viral RNA (vRNA), self-amplifying RNA (sa-RNA), and combinations thereof.

[0035] 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 siRNA. Suitable siRNAs, as well as methods and intermediates useful for preparing the same, are reported in International Patent Application Publication No. WO2016 / 054421.

[0036] Target gene In certain embodiments, the nucleic acid (e.g., siRNA) can be used to downregulate or silence the translation (i.e., expression) of a gene of interest. Genes of interest include, but are not limited to, genes associated with viral infection and survival, genes associated with metabolic diseases and disorders (e.g., liver diseases and disorders), genes associated with tumorigenesis and cell transformation (e.g., cancer), angiogenic genes, immunomodulatory factor genes, e.g., those associated with inflammation and immune response, ligand receptor genes, and genes associated with neurodegenerative disorders. In certain embodiments, the gene of interest is expressed in hepatocytes.

[0037] Genes related to viral infection and survival include those expressed by the virus for binding, entry, and replication in cells. Of particular interest are viral sequences associated with chronic viral diseases. Particularly interesting viral sequences include filoviruses, such as Ebola virus and Marburg virus (see, for example, Geisbert et al., J. Infect. Dis., 193:1650-1657 (2006)), arenaviruses, such as Lassa virus, Funin virus, Machupo virus, Guanarito virus, and Sabia virus (Buchmeier et al., Arenaviridae: the viruses and their replication, In: FIELDS VIROLOGY, Knipe et al. (eds.), 4th ed., Lippincott-Raven, Philadelphia, (2001)), influenza viruses, such as influenza A, B, and C viruses (see, for example, Steinhauer et al., Annu Rev Genet., 36:305-332 (2002), and Neumann et al., J Gen Virol., 83:2635-2662 (2002)), hepatitis viruses (see, for example, Hamasaki et al., FEBS Lett., 543:51 (2003), Yokota et al., EMBO Rep., 4:602 (2003), Schlomai et al., Hepatology, 37:764 (2003), Wilson et al., Proc. Natl. Acad. Sci. USA, 100:2783 (2003), Kapadia et al., Proc. Natl. Acad. Sci. USA, 100:2014 (2003), and FIELDS VIROLOGY, Knipe et al. (eds.), 4th ed., Lippincott-Raven, Philadelphia (2001)), human immunodeficiency virus (HIV) (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)) sequences are included.

[0038] 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-bound proteins (e.g., VP40, glycoprotein (GP), VP24). The complete genomic sequences of Ebola virus are shown, for example, in Genbank accession numbers NC_002549, AY769362, NC_006432, NC_004161, AY729654, AY354458, AY142960, AB050936, AF522874, AF499101, AF272001, and AF086833. The Ebola virus VP24 sequence is shown, for example, in Genbank accession numbers U77385 and AY058897. The Ebola virus L-pol sequence is shown, for example, in Genbank accession number X67110. The Ebola virus VP40 sequence is shown, for example, in Genbank accession number AY058896. The Ebola virus NP sequence is shown, for example, in Genbank accession number AY058895. The Ebola virus GP sequence is shown, for example, in Genbank accession numbers 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 shown, for example, in Genbank accession numbers L11365 and X61274. The complete genomic sequences of Marburg virus are shown, for example, in Genbank accession numbers NC_001608, AY430365, AY430366, and AY358025. The Marburg virus GP sequence is shown, for example, in Genbank accession numbers AF005734, AF005733, and AF005732. The Marburg virus VP35 sequence is shown, for example, in Genbank accession numbers AF005731 and AF005730. Additional Marburg virus sequences are shown, for example, in Genbank accession numbers 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 entire disclosure of which is hereby incorporated by reference for all purposes.

[0039] 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), non-structural proteins (NS1 and NS2), RNA polymerase (PA, PB1, PB2), neuraminidase (NA), and hemagglutinin (HA). The type A influenza NP sequences are shown, for example, in Genbank accession numbers NC_004522, AY818138, AB166863, AB188817, AB189046, AB189054, AB189062, AY646169, AY646177, AY651486, AY651493, AY651494, AY651495, AY651496, AY651497, AY651498, AY651499, AY651500, AY651501, AY651502, AY651503, AY651504, AY651505, AY651506, AY651507, AY651509, AY651528, AY770996, AY790308, AY818138, and AY818140. The type A influenza PA sequences are shown, for example, in Genbank accession numbers AY818132, AY790280, AY646171, AY818132, AY818133, AY646179, AY818134, AY551934, AY651613, AY651610, AY651620, AY651617, AY651600, AY651611, AY651606, AY651618, AY651608, AY651607, AY651605, AY651609, AY651615, AY651616, AY651640, AY651614, AY651612, AY651621, AY651619, AY770995, and AY724786. 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 hereby incorporated by reference in its entirety for all purposes.

[0040] Exemplary hepatitis virus nucleic acid sequences that can be silenced include nucleic acid sequences involved in transcription and translation (e.g., En1, En2, X, P), as well as nucleic acid sequences encoding structural proteins (e.g., core proteins including C and C-related proteins, capsid and envelope proteins including S, M, and / or L proteins, or fragments thereof), but are not limited thereto (see, e.g., the above FIELDS VIROLOGY). Exemplary hepatitis C virus (HCV) nucleic acid sequences that can be silenced include the 5' untranslated region (5'-UTR), 3'-untranslated region (3'-UTR), polyprotein translation initiation codon region, internal ribosome entry site (IRES) sequence within the sequence, and / or nucleic acid sequences encoding core protein, E1 protein, E2 protein, p7 protein, NS2 protein, NS3 protease / helicase, NS4A protein, NS4B protein, NS5A protein, and / or NS5B RNA-dependent RNA polymerase, but are not limited thereto. The HCV genomic sequences are shown, for example, in Genbank accession numbers NC_004102 (HCV genotype 1a), AJ238799 (HCV genotype 1b), NC_009823 (HCV genotype 2), NC_009824 (HCV genotype 3), NC_009825 (HCV genotype 4), NC_009826 (HCV genotype 5), and NC_009827 (HCV genotype 6). The hepatitis A virus nucleic acid sequence is shown, for example, in Genbank accession number NC_001489. The hepatitis B virus nucleic acid sequence is shown, for example, in Genbank accession number NC_003977, the hepatitis D virus nucleic acid sequence is shown, for example, in Genbank accession number NC_001653, the hepatitis E virus nucleic acid sequence is shown, for example, in Genbank accession number NC_001434, and the hepatitis G virus nucleic acid sequence is shown, for example, in Genbank accession number NC_001710. Silencing of sequences encoding genes related to viral infection and survival can be advantageously used in combination with the administration of conventional drugs used to treat viral symptoms.Non-limiting examples of siRNA molecules targeting hepatitis virus 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 on March 20, 2009, the entire disclosures of which are hereby incorporated by reference for all purposes.

[0041] Genes associated with metabolic diseases and disorders (e.g., disorders targeting the liver, as well as liver diseases and liver disorders) include, for example, genes involved in dyslipidemia (e.g., liver X receptor, e.g., LXRα and LXRβ (Genbank accession number NM_007121), farnesoid X receptor (FXR) (Genbank accession number NM_005123), sterol regulatory element-binding protein (SREBP), site-1 protease (SIP), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG coenzyme A reductase), apolipoprotein B (ApoB) (Genbank accession number NM_000384), apolipoprotein CIII (ApoC3) (Genbank accession numbers NM_000040 and NG_008949 REGION:5001.8164), and apolipoprotein E (ApoE) (Genbank accession numbers NM_000041 and NG_007084 REGION:5001.8612)), and genes expressed in diabetes (e.g., glucose 6-phosphatase) (see, for example, 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)). One of ordinary skill in the art will recognize that genes associated with metabolic diseases and disorders (e.g., diseases and disorders targeting the liver, as well as liver diseases and liver 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 advantageously used in combination with the administration of conventional agents used to treat such diseases or disorders. 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 hereby incorporated 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 hereby incorporated by reference in its entirety for all purposes.

[0042] Examples of gene sequences associated with tumor formation and cell transformation (e.g., cancer or other neoplasms) include mitotic kinesins such as Eg5 (KSP, KIF11, Genbank accession number NM_004523), serine / threonine kinases such as polo-like kinase 1 (PLK-1) (Genbank accession number NM_005030, Barr et al., Nat. Rev. Mol. Cell Biol., 5:429-440 (2004)), tyrosine kinases such as WEE1 (Genbank accession numbers NM_003390 and NM_001143976), apoptosis inhibitors such as XIAP (Genbank accession number NM_001167), COP9 signalosome 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 and the like. Non-limiting examples of siRNA molecules targeting the 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 hereby incorporated by reference in its entirety for all purposes. Non-limiting examples of siRNA molecules targeting the PLK-1 gene include those described in U.S. Patent Publications Nos. 20050107316 and 20070265438, and U.S. Patent Application No. 12 / 343,342, filed December 23, 2008, the disclosures of which are hereby incorporated by reference in their entirety for all purposes. Non-limiting examples of siRNA molecules targeting the CSN5 gene include those described in U.S. Provisional Application No. 61 / 045,251, filed April 15, 2008, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.

[0043] Further examples of gene sequences associated with tumor formation and cell transformation include translocation sequences, such as the MLL fusion gene, BCR-ABL (Wilda et al., Oncogene, 21:5716 (2002), Scherr et al., Blood, 101:1566 (2003)), TEL-AML1, EWS-FLI1, TLS-FUS, PAX3-FKHR, BCL-2, AML1-ETO, and AML1-MTG8 (Heidenreich et al., Blood, 101:3157 (2003)), overexpression sequences, such as the multidrug resistance gene (Nieth et al., FEBS Lett., 545:144 (2003), Wu et al, Cancer Res. 63:1515 (2003)), cyclin (Li et al., Cancer Res., 63:3593 (2003), Zou et al., Genes Dev., 16:2923 (2002)), beta-catenin (Verma et al., Clin Cancer Res., 9:1291 (2003)), the telomerase gene (Kosciolek et al., Mol Cancer Ther., 2:209 (2003)), c-MYC, N-MYC, BCL-2, growth factor receptors (e.g., EGFR / ErbB1 (Genbank accession numbers NM_005228, NM_201282, NM_201283 and NM_201284, see also Nagy et al. Exp. Cell Res., 285:39-49 (2003), ErbB2 / HER-2 (Genbank accession numbers NM_004448 and NM_001005862), ErbB3 (Genbank accession numbers NM_001982 and NM_001005915), and ErbB4 (Genbank accession numbers 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 the EGFR gene include those described in U.S. Patent Application No. 11 / 807,872, filed May 29, 2007, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.

[0044] Silencing of the sequence encoding the DNA repair enzyme is utilized in combination with the administration of chemotherapeutic agents (Collis et al., Cancer Res., 63:1550 (2003)). Genes encoding proteins involved in tumor migration are also target sequences of interest, and these include, for example, integrins, selectins, and metalloproteins. The above examples are not exclusive. One of ordinary skill in the art will understand that any entire or partial gene sequence that facilitates or promotes tumor formation or cell transformation, tumor growth, or tumor migration can be included as a template sequence.

[0045] Angiogenesis genes can promote the formation of new blood vessels. Of particular interest are vascular endothelial growth factor (VEGF) (Reich et al., Mol. Vis., 9:210 (2003)) or VEGFR. siRNA sequences targeting VEGFR are shown, for example, in GB2396864, US Patent Publication No. 20040142895, and CA2456444, the disclosures of which are hereby incorporated by reference in their entirety for all purposes.

[0046] Angiogenesis-inhibiting genes can inhibit angiogenesis. These genes are particularly useful in treating cancers in which angiogenesis plays a role in the pathological progression of the disease. Examples of angiogenesis-inhibiting 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 disclosures of which are hereby incorporated by reference in their entirety for all purposes. Immunomodulatory factor genes are genes that regulate one or more immune responses. Examples of immunomodulatory factor 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 factor target sequences of interest (Song et al., Nat. Med., 9:347 (2003)). Genes encoding accessory signaling molecules in hematopoietic and lymphoid cells are also included in the present invention, including, for example, Tec family kinases such as Bruton's tyrosine kinase (Btk) (Heinonen et al., FEBS Lett., 527:274 (2002)).

[0047] Cell receptor ligands include ligands that can bind to cell surface receptors (e.g., insulin receptor, EPO receptor, G protein-coupled receptor, receptor with tyrosine kinase activity, cytokine receptor, growth factor receptor, etc.) and regulate (e.g., inhibit, activate, etc.) the physiological pathways (e.g., blood glucose regulation, blood cell development, induction of mitosis, etc.) in which the receptors are involved. 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 the extension of trinucleotide repeats (e.g., CAG repeats) are used to silence the pathogenic sequences of neurodegenerative disorders caused by the extension of trinucleotide repeats, such as spinal bulbar muscular atrophy and Huntington's disease (Caplen et al., Hum. Mol. Genet., 11:175 (2002)).

[0048] Other certain target genes that can be targeted by nucleic acids (e.g., siRNA) that downregulate or silence gene expression include aortic smooth muscle alpha-2 actin (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), aldose-ketose reductase family 1 member C4 (AKR1C4), serum albumin (ALB), alpha-1-microglobulin / bikunin precursor (AMBP), angiopoietin-related protein 3 (ANGPTL3), serum amyloid P component (APCS), apolipoprotein A-II (APOA2), apolipoprotein B-100 (APOB), apolipoprotein C3 (APOC3), apolipoprotein C-IV (APOC4), apolipoprotein F (APOF), beta-2-glycoprotein 1 (APOH), aquaporin-9 (AQP9), bile acid-CoA:amino acid N-acyltransferase (BAAT), C4b-binding protein beta chain (C4BPB), putative protein of unknown properties encoded by LINC01554 (C5orf27), complement factor 3 (C3), complement factor 5 (C5), complement component C6 (C6), complement component C8 alpha chain (C8A), complement component C8 beta chain (C8B), complement component C8 gamma chain (C8G), complement component C9 (C9), calmodulin-binding transcription activator 1 (CAMTA1), CD38 (CD38), complement factor B (CFB), complement factor H-related protein 1 (CFHR1), complement factor H-related protein 2 (CFHR2), complement factor H-related protein 3 (CFHR3), cannabinoid receptor 1 (CNR1), ceruloplasmin (CP), carboxypeptidase B2 (CPB2), connective tissue growth factor (CTGF), C-X-C motif chemokine 2 (CXCL2), cytochrome P450 1A2 (CYP1A2), cytochrome P450 2A6 (CYP2A6), cytochrome P450 2C8 (CYP2C8), cytochrome P450 2C9 (CYP2C9), cytochrome P450 family 2 subfamily D member 6 (CYP2D6), cytochrome P450 2E1 (CYP2E1),Phylloquinone omega hydroxylase CYP4F2 (CYP4F2), 7-alpha-hydroxy cholesterol-4-en-3-one 12-alpha-hydroxylase (CYP8B1), dipeptidyl peptidase 4 (DPP4), coagulation factor 12 (F12), coagulation factor II (thrombin) (F2), coagulation factor IX (F9), fibrinogen alpha chain (FGA), fibrinogen beta chain (FGB), fibrinogen gamma chain (FGG), fibrinogen-like 1 (FGL1), flavin-containing monooxygenase 3 (FMO3), flavin-containing monooxygenase 5 (FMO5), group-specific component (vitamin D binding protein) (GC), growth hormone receptor (GHR), glycine N-methyltransferase (GNMT), hyaluronan binding protein 2 (HABP2), hepcidin antimicrobial peptide (HAMP), hydroxyacid oxidase (glycolic acid oxidase) 1 (HAO1), HGF activator (HGFAC), haptoglobin-related protein, haptoglobin (HPR), hemopexin (HPX), histidine-rich glycoprotein (HRG), hydroxysteroid (11-beta) dehydrogenase 1 (HSD11B1), hydroxysteroid (17-beta) dehydrogenase 13 (HSD17B13), inter-alpha-trypsin inhibitor heavy chain H1 (ITIH1), inter-alpha-trypsin inhibitor heavy chain H2 (ITIH2), inter-alpha-trypsin inhibitor heavy chain H3 (ITIH3), inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), prekallikrein (KLKB1), lactate dehydrogenase A (LDHA), liver-expressed antimicrobial peptide 2 (LEAP2), leukocyte cell-derived chemotaxin 2 (LECT2), lipoprotein(a) (LPA), mannose-binding lectin serine peptidase 2 (MASP2), S-adenosylmethionine synthase isoform 1 type (MAT1A), NADPH oxidase 4 (NOX4), poly[ADP-ribose] polymerase 1 (PARP1), paraoxonase 1 (PON1), paraoxonase 3 (PON3), vitamin K-dependent protein C (PROC), retinol dehydrogenase 16 (RDH16), constitutive serum amyloid A4 (SAA4), serine dehydratase (SDS), serpin family A member 1 (serpin A1),Serpin A11 (SERPINA11), calistatin (SERPINA4), corticosteroid-binding globulin (SERPINA6), antithrombin III (SERPINC1), heparin cofactor 2 (SERPIND1), serpin family H member 1 (SERPINH1), solute carrier family 5 member 2 (SLC5A2), sodium / bile acid cotransporter (SLC10A1), solute carrier family 13 member 5 (SLC13A5), solute carrier family 22 member 1 (SLC22A1), solute carrier family 25 member 47 (SLC25A47), solute carrier family 2, facilitated 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 acid 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) are included, but not limited to these.

[0049] In addition to its usefulness in silencing the expression of any of the above genes for therapeutic purposes, certain nucleic acids (e.g., siRNA) described herein are also useful in research and development applications, as well as in diagnostic, preventive, prognostic, clinical, and other medical applications. By way of non-limiting example, certain nucleic acids (e.g., siRNA) can be used in target validation studies aimed at testing whether a gene of interest may be a potential therapeutic target. Certain nucleic acids (e.g., siRNA) can also be used in target identification studies aimed at discovering genes as potential therapeutic targets.

[0050] Generation of siRNA molecules siRNA can be provided in various forms, 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 generated enzymatically or by partial / total organic synthesis, and modified ribonucleotides can be introduced by enzymatic or organic synthesis in vitro. In some cases, each strand is chemically prepared. Methods for synthesizing RNA molecules are known in the art, for example, those described in Verma and Eckstein (1998) or the chemical synthesis methods described herein.

[0051] Methods for isolating RNA, synthesizing RNA, hybridizing nucleic acids, creating and screening cDNA libraries, and performing PCR are well known in the art (see, for example, Gubler and Hoffman, Gene, 25:263-269 (1983), Sambrook et al. supra, Ausubel et al. supra), and the same is true for 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., eds, 1990)). Expression libraries are also well known to those skilled in the art. Additional basic documents disclosing general methods of 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., eds., 1994). The disclosures of these references are hereby incorporated by reference in their entirety for all purposes.

[0052] siRNAs are typically chemically synthesized. Oligonucleotides containing the siRNA molecules of the invention can be synthesized using any of a variety of techniques known in the art, such as those described in Usman et al., J. Am. Chem. Soc., 109:7845 (1987), Scaringe et al., Nucl. Acids Res., 18:5433 (1990), Wincott et al., Nucl. Acids Res., 23:2677-2684 (1995), and Wincott et al., Methods Mol. Bio., 74:59 (1997). The synthesis of oligonucleotides utilizes common protecting and coupling groups for nucleic acids, such as dimethoxytrityl at the 5' end and phosphoramidite at the 3' end. By way of non-limiting example, small scale synthesis can be carried out using a 0.2 μmol scale protocol on a synthesis apparatus from Applied Biosystems. Alternatively, synthesis on a 0.2 μmol scale can be carried out on a 96 well plate synthesis apparatus provided by Protogene (Palo Alto, CA). However, larger or smaller scale syntheses are also within the scope of the invention. Reagents suitable for oligonucleotide synthesis, methods for RNA deprotection, and methods for RNA purification are known to those of skill in the art.

[0053] siRNA molecules can be assembled from two separate oligonucleotides, in which case one oligonucleotide contains the sense strand of the siRNA and the other contains the antisense strand. For example, each strand can be synthesized separately and joined by hybridization or ligation following synthesis and / or deprotection.

[0054] linker group The compounds and conjugates of the invention may have one or more linker groups (e.g., L 3 or L 4) may be included. The structure of each linking group can be various as long as 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 may regulate the solubility, stability, or aggregation characteristics of the conjugate.

[0055] In one embodiment, each linking group contains about 3 to 1000 atoms. In one embodiment, each linking group contains about 3 to 500 atoms. In one embodiment, each linking group contains about 3 to 200 atoms. In one embodiment, each linking group contains about 3 to 50 atoms. In one embodiment, each linking group contains about 10 to 1000 atoms. In one embodiment, each linking group contains about 10 to 500 atoms. In one embodiment, each linking group contains about 10 to 200 atoms. In one embodiment, each linking group contains about 10 to 50 atoms.

[0056] In one embodiment, each linking group contains atoms selected from H, C, N, S, and O.

[0057] In one embodiment, each linking group contains atoms selected from H, C, N, S, P, and O.

[0058] In one embodiment, each linking group contains a branched or unbranched saturated or unsaturated hydrocarbon chain having about 1 to 1000 (or 1 to 750, 1 to 500, 1 to 250, 1 to 100, 1 to 50, 1 to 25, 1 to 10, 1 to 5, 5 to 1000, 5 to 750, 5 to 500, 5 to 250, 5 to 100, 5 to 50, 5 to 25, 5 to 10, or 2 to 5 carbon atoms), and one or more of the carbon atoms are independently optionally replaced by -O-, -S, -N(R a )-, a 3- to 7-membered heterocycle, a 5- to 6-membered heteroaryl, or a carbocycle, where each chain, 3- to 7-membered heterocycle, 5- to 6-membered heteroaryl, or carbocycle is independently (C1-C6)alkyl, (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azide, cyano, nitro, halo, -N(Ra ) 2) optionally substituted with one or more (e.g., 1, 2, 3, 4, 5 or more) substituents selected from hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl and heteroaryloxy, each R a is independently H or (C1-C6) alkyl. In one embodiment, the linker comprises a branched or unbranched saturated or unsaturated hydrocarbon chain having from about 1 to 1000 (or 1 to 750, 1 to 500, 1 to 250, 1 to 100, 1 to 50, 1 to 25, 1 to 10, 1 to 5, 5 to 1000, 5 to 750, 5 to 500, 5 to 250, 5 to 100, 5 to 50, 5 to 25, 5 to 10, or 2 to 5 carbon atoms), with one or more of the carbon atoms independently optionally replaced by -O-, -S, -N(R a )-, and each R a is independently H or (C1-C6) alkyl.

[0059] In one embodiment, each linking group comprises polyethylene glycol. In one embodiment, the linking group comprises polyethylene glycol linked to the remainder of the target-directed conjugate by a carbonyl group. In one embodiment, the polyethylene glycol comprises from about 1 to about 500, or from about 5 to about 500, or from about 3 to about 100 repeating units (e.g., -CH2CH2O-) (Greenwald, R.B., et al., Poly(ethylene glycol) Prodrugs: Altered Pharmacokinetics and Pharmacodynamics, Chapter, 2.3.1., 283-338, Filpula, D., et al., Releasable PEGylation of proteins with customized linkers, Advanced Drug Delivery, 60, 2008, 29-49, Zhao, H., et al., Drug Conjugates with Poly(Ethylene Glycol), Drug Delivery in Oncology, 2012, 627-656).

[0060] Embodiments of the present invention One aspect of the present invention is a compound of formula I shown in the summary of the invention, or a salt thereof.

[0061] In one embodiment, A is absent.

[0062] In one embodiment, A is 3- to 20-membered cycloalkyl, 5- to 20-membered aryl, 5- to 20-membered heteroaryl, or 3- to 20-membered heterocycloalkyl.

[0063] In one embodiment, B is 5- to 10-membered aryl.

[0064] In one embodiment, B is naphthyl or phenyl.

[0065] In one embodiment, B is phenyl.

[0066] In one embodiment, the group:

Chemical formula

Chemical formula

[0067] In one embodiment, B is 5- to 10-membered heteroaryl.

[0068] In one embodiment, B is pyridyl, pyrimidyl, quinolyl, isoquinolyl, imidazolyl, thiazolyl, oxadiazolyl or oxazolyl.

[0069] In one embodiment, the group:

Chemical formula

Chemical formula

[0070] In one embodiment, the group:

Chem.

Chem.

[0071] In one embodiment, L 1 is a divalent non-branched saturated hydrocarbon chain having 0 to 20 carbon atoms, and one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are optionally replaced by -O-, -NR X -, -NR X -, -C(=O)-, -C(=O)-NR X -, or -S-, R X is hydrogen or (C1-C6) alkyl, and the hydrocarbon chain is optionally substituted with one or more substituents selected from oxo (=O) and halo.

[0072] In one embodiment, L 1 is a divalent non-branched saturated hydrocarbon chain having 0 to 12 carbon atoms, and one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are optionally replaced by -O-, -NR X -, -C(=O)-, or -C(=O)-NR X -, and R X is hydrogen or (C1-C6) alkyl.

[0073] In one embodiment, L 1 is -C(=O)N(H)-CH2CH2OCH2CH2OCH2CH2-, -C(=O)N(H)-CH2CH2OCH2CH2OCH2CH2OCH2CH2-, -C(=O)N(CH3)-CH2CH2OCH2CH2OCH2CH2-, or -C(=O)N(CH3)-CH2CH2OCH2CH2OCH2CH2OCH2CH2- as follows.

[0074] In one embodiment, L 2 is a divalent unbranched saturated hydrocarbon chain having 0 to 20 carbon atoms, and one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are optionally replaced by -O-, -NR X -, -NR X -, -C(=O)-, -C(=O)-NR X -, or -S-, where R X is hydrogen or (C1-C6) alkyl, and the hydrocarbon chain is optionally substituted with one or more substituents selected from oxo (=O) and halo.

[0075] In one embodiment, L 2 is a divalent unbranched saturated hydrocarbon chain having 0 to 12 carbon atoms, and one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are optionally replaced by -O-, -NR X -, -C(=O)-, or -C(=O)-NR X -, where R X is hydrogen or (C1-C6) alkyl.

[0076] In one embodiment, L 2 is -C(=O)N(H)-CH2CH2OCH2CH2OCH2CH2-, -C(=O)N(H)-CH2CH2OCH2CH2OCH2CH2OCH2CH2-, -C(=O)N(CH3)-CH2CH2OCH2CH2OCH2CH2-, or -C(=O)N(CH3)-CH2CH2OCH2CH2OCH2CH2OCH2CH2- is.

[0077] In one embodiment, R 1 is

Chemical formula

[0078] In one embodiment, R 1 is

Chemical formula

[0079] In one embodiment, R 1 is

Chemical formula

[0080] In one embodiment, R 1is [Chem.] .

[0081] In one embodiment, R 1 is [Chem.] .

[0082] In one embodiment, R 2 is [Chem.] [wherein, X is NR 20 , Y is -(C=O)R 21 , -SO2R 22 , and -(C=O)NR 23 R 24 is selected from; or X is -(C=O)- and Y is NR 25 R 26 ; or X is -NR 37 R 38 and Y is absent, R 20 is hydrogen or (C1-C4) alkyl, R 21 , R 22 , R 23 , R 24 , R 25 and R 26 are each independently selected from the group consisting of hydrogen, (C1-C8) alkyl, (C1-C8) alkoxy and (C3-C6) cycloalkyl, and any (C1-C8) alkyl, (C1-C8) alkoxy and (C3-C6) cycloalkyl are optionally substituted with one or more groups independently selected from the group consisting of halo, (C1-C4) alkyl and (C1-C4) alkoxy, R 27 is -OH, -NR 25 R 26 , or -F, R 28 is -OH, -NR25 R 26 or -F, and R 29 is -OH, -NR 25 R 26 , -F, -N3, -NR 35 R 36 or is a 5-membered heterocyclic ring optionally substituted with one or more groups independently selected from the group consisting of halo, hydroxyl, carboxyl, amino, (C1-C4)alkyl, aryl, and (C1-C4)alkoxy, wherein any (C1-C4)alkyl and (C1-C4)alkoxy are optionally substituted with one or more groups independently selected from the group consisting of halo, any aryl is optionally substituted with one or more groups independently selected from the group consisting of halo, hydroxyl, nitro, cyano, amino, (C1-C8)alkyl, (C1-C8)alkoxy, (C1-C8)alkanoyl, (C1-C8)alkoxycarbonyl, (C1-C8)alkanoyloxy, and (C3-C6)cycloalkyl, and any (C1-C8)alkyl, (C1-C8)alkoxy, (C1-C8)alkanoyl, (C1-C8)alkoxycarbonyl, (C1-C8)alkanoyloxy, and (C3-C6)cycloalkyl are optionally substituted with one or more groups independently selected from the group consisting of halo, (C1-C4)alkyl, and (C1-C4)alkoxy; each R 35 and R 36 are independently selected from the group consisting of hydrogen, (C1-C8)alkyl, (C1-C8)alkoxy, and (C3-C6)cycloalkyl, wherein any (C1-C8)alkyl, (C1-C8)alkoxy, and (C3-C6)cycloalkyl are optionally substituted with one or more groups independently selected from the group consisting of halo and (C1-C4)alkoxy; or R 35 and R 36together with the nitrogen to which they are attached form a 5- to 6-membered heteroaryl ring, and the heteroaryl ring is optionally substituted with one or more groups independently selected from the group consisting of (C1-C8) alkyl, (C1-C8) alkoxy, aryl, and (C3-C6) cycloalkyl, and any aryl and (C3-C6) cycloalkyl are optionally substituted with one or more groups R 39 optionally substituted with; each R 37 and R 38 are 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, and any (C1-C8) alkyl, (C1-C8) alkoxy, (C1-C8) alkanoyl, (C1-C8) alkoxycarbonyl, (C1-C8) alkanoyloxy, and (C3-C6) cycloalkyl are optionally substituted with one or more groups independently selected from the group consisting of halo, (C1-C4) alkyl, and (C1-C4) alkoxy; or R 37 and R 38 together with the nitrogen to which they are attached form a 5- to 8-membered heterocyclic ring optionally substituted with one or more groups independently selected from the group consisting of halo, hydroxyl, carboxyl, amino, oxo (=O), (C1-C4) alkyl, and (C1-C4) alkoxy, and any (C1-C4) alkyl and (C1-C4) alkoxy are optionally substituted with one or more groups independently selected from halo, each R 39 is independently selected from the group consisting of (C1-C8) alkyl, (C1-C8) alkoxy, and (C3-C6) cycloalkyl, and any (C1-C8) alkyl, (C1-C8) alkoxy, and (C3-C6) cycloalkyl are optionally substituted with one or more groups independently selected from halo) is.

[0083] In one embodiment, R 2 is

Chem.

[0084] In one embodiment, R 2 is

Chem.

[0085] In one embodiment, R 2 is

Chem.

[0086] In one embodiment, R 2 is

Chem.

[0087] In one embodiment, L 3 is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 0 to 50 carbon atoms, and one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are optionally replaced by -O-, -NR X -, -NR X -, -C(=O)-, -C(=O)-NR X -, or -S-, and R X is hydrogen or (C1-C6) alkyl, and the hydrocarbon chain is optionally substituted with one or more (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, azide, cyano, nitro, halo, hydroxy, oxo(=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0088] In one embodiment, L 3is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 1 to 20 carbon atoms, and one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are optionally replaced by -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X -, or -S-, and R X is hydrogen or (C1-C6) alkyl, and the hydrocarbon chain is optionally substituted with one or more (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, azide, cyano, nitro, halo, hydroxy, oxo(=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0089] In one embodiment, L 3 is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 1 to 30 carbon atoms, and one or more of the carbon atoms are optionally replaced by -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X -, or -S-, and R X is hydrogen or (C1-C6) alkyl, and the hydrocarbon chain is optionally substituted with one or more halo or oxo(=O).

[0090] In one embodiment, L 3 is

Chemical formula

[0091] In one embodiment, L 3 is linked to B by -NH-, -O-, -S-, -(C=O)-, -(C=O)-NH-, -NH-(C=O)-, -(C=O)-O-, -NH-(C=O)-NH-, or -NH-(SO2)-.

[0092] In one embodiment, L 4 is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 0 to 50 carbon atoms, and one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are optionally replaced by -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X -, or -S-, and R X is hydrogen or (C1-C6) alkyl, and the hydrocarbon chain is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from (C1-C6) alkoxy, (C3-C6) cycloalkyl, (C1-C6) alkanoyl, (C1-C6) alkanoyloxy, (C1-C6) alkoxycarbonyl, (C1-C6) alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0093] In one embodiment, L 4 is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 1 to 20 carbon atoms, and one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are optionally replaced by -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X -, or -S-, and R X is hydrogen or (C1-C6) alkyl, and the hydrocarbon chain is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from (C1-C6) alkoxy, (C3-C6) cycloalkyl, (C1-C6) alkanoyl, (C1-C6) alkanoyloxy, (C1-C6) alkoxycarbonyl, (C1-C6) alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0094] In one embodiment, L 4 is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 1 to 30 carbon atoms, and one or more of the carbon atoms are optionally replaced by -O-, -NR X -, -NR X -, -C(=O)-, -C(=O)-NR X -, or -S-, R X is hydrogen or (C1-C6) alkyl, and the hydrocarbon chain is optionally substituted with one or more halo or oxo (=O).

[0095] In one embodiment, L 4 is linked to R 3 by -O-.

[0096] In one embodiment, the nucleic acid molecule R 3 (e.g., siRNA) is bound to the remainder of the conjugate by the oxygen of the phosphate of the nucleic acid molecule.

[0097] In one embodiment, the nucleic acid molecule R 3 (e.g., siRNA) is bound to the remainder of the conjugate by the oxygen of the phosphate at the 5' end of the sense or antisense strand.

[0098] In one embodiment, the nucleic acid molecule R 3 (e.g., siRNA) is bound to the remainder of the conjugate by the oxygen of the phosphate at the 3' end of the sense or antisense strand.

[0099] In one embodiment, the nucleic acid molecule R 3 (e.g., siRNA) is bound to the remainder of the conjugate by the oxygen of the phosphate at the 3' end of the sense strand.

[0100] In one embodiment, the group:

Chemical formula

Chemical formula

[0101] In one embodiment, the group:

Chemical formula

Chemical formula

[0102] In one embodiment, the group:

Chemical formula

Chemical formula

[0103] The present invention further provides synthetic intermediates and methods described herein that are useful for preparing the conjugates of formula (I). For example, the present invention includes compounds of formula (Ia): [Chemical formula] [wherein, R 1 is a sugar, L 1 is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 0 to 20 carbon atoms, and one or more of the carbon atoms in the hydrocarbon chain are optionally -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X -, or -S-, and R X is hydrogen or (C1-C6) alkyl, and the hydrocarbon chain is optionally substituted with one or more substituents selected from oxo (=O) and halo, B is a 5- to 10-membered aryl or 5- to 10-membered heteroaryl, and the 5- to 10-membered aryl or 5- to 10-membered heteroaryl is 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 Optionally substituted with one or more groups selected, L 2 is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 0 to 20 carbon atoms, and one or more of the carbon atoms in the hydrocarbon chain are optionally -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X -, or -S-, and R X is hydrogen or (C1-C6) alkyl, and the hydrocarbon chain is optionally substituted with one or more substituents selected from oxo (=O) and halo, R 2 is a sugar, L 3 is absent or a linking group, A is a 3- to 20-membered cycloalkyl, 5- to 20-membered aryl, 5- to 20-membered heteroaryl, or 3- to 20-membered heterocycloalkyl, each R A is independently selected from the group consisting of hydrogen, hydroxy, 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, and C 1-10 alkyl C 2-10 alkenyl, and C 2-10 alkynyl are optionally substituted with one or more groups independently selected from halo, hydroxy, and C 1-3 alkoxy, n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, L 4 is absent or is a linking group, R 3a is H, a protecting group, a synthetic activating group, a covalent bond to a solid support, or a bond to a linking group attached to a solid support, R a is hydrogen, a protecting group, a covalent bond to a solid support, or a bond to a linking group L 5 attached to a solid support, L 5 is a linking group), or a salt thereof is included.

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

[0105] In one embodiment, R 3a is a protecting group. In one embodiment, the protecting group is acetate, triflate, mesylate, or succinate.

[0106] In one embodiment, R 3a is a synthetic activating group. In one embodiment, the synthetic activating group can be derived from DCC, HOBt, EDC, BOP, PyBOP, or HBTU.

[0107] In one embodiment, R 3a is a covalent bond to a solid support.

[0108] In one embodiment, R 3a is a bond to a linking group attached to the solid support. In one embodiment, the linking group attached to the solid support is -C(=O)CH2CH2C(=O)N(H)-.

Examples

[0109] The present invention will be described in more detail by way of 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 readily recognize various non-essential parameters that can be changed or modified to yield essentially the same results.

[0110] The following Schemes 1-22 illustrate the preparation of intermediate compounds that can be used to prepare the conjugates of formula I. The intermediate compounds and synthetic processes illustrated in Schemes 1-22 are embodiments of the present invention.

[0111] Scheme 1 Preparation of Compound 6

Chemical formula

[0112] Step 2. Preparation of ((3R,4S)-1-benzyl-3,4-dimethylpyrrolidine-3,4-diyl)dimethanol 2 (3aR,6aS)-5-benzyl-3a,6a-dimethyltetrahydro-1H-furo[3,4-c]pyrrole-1,3(3aH)-dione (53.7 g, 205.7 mmol) in a cooled solution (0 °C) in anhydrous diethyl ether (750 ml) was slowly added lithium aluminum hydride pellets (17.6 g, 463 mmol) in portions over the afternoon. The solution was stirred overnight and warmed to room temperature as the ice bath melted. Immediately after completion, the reaction was cooled to 0 °C and quenched very slowly using 25 ml of 5 M NaOH followed by 12 ml of water. Stirred 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 as a colorless oil (33.6 g, 65%). Rf 0.25 10% CH3OH - CH2Cl2.

[0113] Step 3. Preparation of ((3R,4S)-3,4-dimethylpyrrolidine-3,4-diyl)dimethanol 3 To a solution of ((3R,4S)-1-benzyl-3,4-dimethylpyrrolidine-3,4-diyl)dimethanol (40.1 g, 161 mmol) in methanol (300 ml) was added 10% palladium on carbon wet (4 g). The solution was stirred vigorously for 16 h under a hydrogen atmosphere. Immediately after completion, the solution was filtered through Celite and concentrated to dryness to give ((3R,4S)-3,4-dimethylpyrrolidine-3,4-diyl)dimethanol as a colorless solid (24 g, 94%). Rf 0.05 in 10% CH3OH-CH2Cl2.

[0114] Step 4. Preparation of methyl 10-((3R,4S)-3,4-bis(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoate 4 A solution containing 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 subjected to chromatography (gradient: from 0% CH3OH-CH2Cl2 to 20%) to give 4 (41.5 g, 77%). Rf 0.55 in 10% CH3OH-CH2Cl2.

[0115] Step 5. Preparation of methyl 10-(3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoate 5 A solution containing 4 (41.5 g, 116 mmol) and 4,4'-dimethoxytrityl chloride (38.8 g, 116 mmol) in pyridine (400 ml) was stirred overnight. Then, pyridine was removed under reduced pressure and the crude material was subjected to chromatography (gradient: from 0% CH3OH-CH2Cl2 to 10%) to give 5 (29.5 g, 39%) as a yellow oil. Rf 0.5 in 5% CH3OH-CH2Cl2.

[0116] Preparation of lithium 6-(3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoate 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 h, then concentrated to remove THF. The remaining aqueous solution was lyophilized overnight to give 6 as a pale purple solid (28.5 g, 98%). Rf 0.56 in 10% CH3OH - CH2Cl2.

[0117] Scheme 2 Preparation of compound 10 [Chemical formula] Preparation of methyl 12-aminododecanoate 8 12-Aminoundecanoic acid 7 (10 g, 4.64 mmol) was stirred in MeOH at room temperature. Acetyl chloride (856 μL, 12 mmol) was added dropwise and the reaction was stirred for 1.5 h. The solvent was removed under vacuum and the residue was taken up in MTBE and cooled in the refrigerator overnight. The resulting precipitate was filtered off, washed with ice-cold MTBE and dried under high vacuum to give methyl 12-aminododecanoate 8.

[0118] Preparation of methyl 12-(10-(3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanamido)dodecanoate 9 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 under vacuum and the residue was purified by column chromatography (5% MeOH, DCM). TLC showed two spots running close together with the same mass, which were assigned to geometric isomers and were quantitatively collected together as methyl 12-(10-(3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoamide)dodecanoate (9).

[0119] Step 3. Preparation of lithium 12-(10-(3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoamide)dodecanoate 10 Methyl 12-(10-(3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoamide)dodecanoate 9 (3.1 mmol) was stirred with LiOH (88 mg, 3.7 mmol) in THF:H2O (50:50) overnight at room temperature. The reaction was confirmed by TLC and THF was removed under vacuum. The aqueous solution was frozen in liquid N2 and lyophilized over 48 hours to quantitatively obtain lithium 12-(10-(3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoamide)dodecanoate 10.

[0120] Scheme 3 Preparation of compound 13

Chemical formula

[0121] Step 2. Preparation of 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethan-1-ol 13 A solution of 12 (140 g, 403 mmol) in DMF (880 ml) was treated with sodium azide (131 g, 2.02 mol) and heated overnight (45 °C). Most of the DMF was removed under reduced pressure, and the residue was dissolved in CH2Cl2 (500 ml), washed with brine (3 × 500 ml), then dried (MgSO4), filtered, and concentrated. The residue was passed through a short silica bed (5% MeOH-CH2Cl2) and concentrated to give 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethan-1-ol 13 (65 g, 74%) as a yellow oil. R f (0.56, 10% MeOH-CH2Cl2).

[0122] Scheme 4. Preparation of Compounds 19a - 19c [Chemical formula] Step 1. Preparation of (3R,4R,5R,6R)-6-(hydroxymethyl)-3-(((E)-4-methoxybenzylidene)amino)tetrahydro-2H-pyran-2,4,5-triol 16 D-Galactosamine HCl (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 mixture was stirred vigorously until solidification. The solid reaction mixture was kept at 4 °C for 16 h. Ice-cold water (200 ml) was added and the resulting solid was collected by filtration and washed with ice-cold EtOH / 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%).

[0123] Step 2. Preparation of (3R,4R,5R,6R)-6-(acetoxymethyl)-3-(((E)-4-methoxybenzylidene)amino)tetrahydro-2H-pyran-2,4,5-triyl triacetate 17 (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) and then DMAP (100 mg, catalyst) were added and the reaction mixture was stirred for 16 h and allowed to warm slowly to room temperature. The resulting solution was poured onto crushed ice and kept at 4 °C for 16 h. The reaction mixture was extracted with EtOAc (×3), the combined organics were 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%).

[0124] Step 3. Preparation of (3R,4R,5R,6R)-6-(acetoxymethyl)-3-aminotetrahydro-2H-pyran-2,4,5-triyl triacetate hydrochloride 18 (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 to reflux in acetone (300 ml). HCl (aqueous solution) (5 N, 3.0 ml) was added and the reaction mixture was stirred for 15 minutes. After cooling, Et2O (400 ml) was added and the reaction mixture was kept at 4 °C for 16 h. 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%).

[0125] Step 4a. Preparation of (3R,4R,5R,6R)-6-(acetoxymethyl)-3-(2,2,2-trifluoroacetamido)tetrahydro-2H-pyran-2,4,5-triyl triacetate 19a (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. DCM containing TFAA (8.13 g, 38.7 mmol) was added dropwise and the reaction mixture was stirred for 1 h. The reaction mixture was diluted with DCM and washed successively with 1 M HCl, saturated NaHCO3, water and brine, dried (Na2SO4) and concentrated under vacuum. The residue was purified by automated flash chromatography (5% MeOH / DCM) to give (3R,4R,5R,6R)-6-(acetoxymethyl)-3-(2,2,2-trifluoroacetamido)tetrahydro-2H-pyran-2,4,5-triyl triacetate (19a) (9.64 g, 61.8%). The product was confirmed by MS (ESI+ve).

[0126] Step 4b. Preparation of (3R,4R,5R,6R)-6-(acetoxymethyl)-3-propionamidotetrahydro-2H-pyran-2,4,5-triyl triacetate 19b This compound was prepared in the same manner as (3R,4R,5R,6R)-6-(acetoxymethyl)-3-(2,2,2-trifluoroacetamido)tetrahydro-2H-pyran-2,4,5-triyl triacetate (19a) using propionic anhydride instead of TFAA to give (3R,4R,5R,6R)-6-(acetoxymethyl)-3-propionamidotetrahydro-2H-pyran-2,4,5-triyl triacetate (19b) (1.2 g, 85.3%). The product was confirmed by MS (ESI+ve).

[0127] Step 4c. Preparation of (3R,4R,5R,6R)-6-(acetoxymethyl)-3-(2,2-difluoropropanamido)tetrahydro-2H-pyran-2,4,5-triyl triacetate 19c (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 h. The reaction mixture was partitioned between EtOAc and water. The organic layer was separated, washed successively with 1 M 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-difluoropropanamido)tetrahydro-2H-pyran-2,4,5-triyl triacetate (19c) (15.8 g, 90%). The product was confirmed by MS (ESI+ve).

[0128] Scheme 5 Preparation of Compound 24 [Chemical formula] Step 1. Preparation of Benzyl (2-(2-(2-hydroxyethoxy)ethoxy)ethyl)carbamate 22 A solution of amino alcohol (20) (313.6 g, 2.1 mol) in THF (3.5 L) was treated portionwise with N-(benzyloxycarbonyloxy)succinimide (21) (550 g, 2.21 mol). At the end of the reaction (18 h), THF was removed under reduced pressure and the residue was dissolved in CH2Cl2 (2.5 L), which was then washed with an equal volume of HCl (1 M), NaHCO3 (saturated aqueous solution), H2O, and brine. The organic extract was dried (MgSO4), filtered, and concentrated. The crude material (600 g) was subjected to chromatography (4 kg of silica, 1 - 12% CH3OH - CH2Cl2) to give HO-Trig-NHZ (22) (468 g, 78%) as a clear, viscous yellow oil.

[0129] Step 2. Preparation of (2R,3R,4R,5R)-5-Acetamido-2-(acetoxymethyl)-6-((3-oxo-1-phenyl-2,7,10-trioxa-4-azadodecan-12-yl)oxy)tetrahydro-2H-pyran-3,4-diyl Diacetate 23 A heterogeneous mixture containing 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% of Sc(OTf)3 (34.6 g, 70.5 mmol) and heated (85 °C). After stirring (5.5 h), the solution became clear and homogeneous. The reaction mixture was cooled and washed with NaHCO3 (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 an emulsified heterogeneous mixture, which was filtered through a coarse frit, thus removing the residual pentaacetate. The filtrate was concentrated and the crude material was subjected to chromatography (5 kg of silica, 0 - 10% CH3OH - EtOAc) to give the glycosylated product (23) (751 g, 87%) as a light brown foam.

[0130] Step 3. Preparation of (2R,3R,4R,5R)-5-acetamido-2-(acetoxymethyl)-6-(2-(2-(2-((2,2,2-trifluoroacetyl)-l4-azanyl)ethoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-3,4-diyl diacetate 24 A solution containing Gal-trig-NHZ (23) (750 g, 1.22 mol), TFA (103.8 ml, 1.35 mol) and Pd / C (10% wet supported product, 75 g) was purged with H2. After vigorous stirring (4.5 h), the reaction mixture was purged with N2 (30 min), then filtered through celite and concentrated. The resulting brown foam (712 g, 99%) was used in the next step without further treatment.

[0131] Scheme 6. Preparation of Compound 34

Chemical formula

[0132] Step 2. Preparation of benzyl (2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl)-carbamate 27 2-(2-(2-(2-Aminoethoxy)ethoxy)ethoxy)ethanol (25) (61.4 g, 318 mmol) was stirred at 5 °C in H2O (500 ml) containing Na2CO3 (50.51 g, 476 mmol). THF (480 ml) containing benzyl chloroformate (26) (65.0 g, 381 mmol) was added dropwise, and the reaction mixture was stirred for 16 h and warmed to room temperature. THF was removed under vacuum, and the aqueous layer was extracted with EtOAc (×3). The combined organics were dried (Na2SO4) and concentrated under vacuum, 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).

[0133] Step 3. Preparation of benzyl (1,1-bis(4-methoxyphenyl)-1-phenyl-2,5,8,11-tetraoxatridecan-13-yl)carbamate 28 (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 mixture was stirred at room temperature for 2 h. The reaction mixture was washed successively with saturated NaHCO3, water, and brine, dried (Na2SO4), and concentrated under vacuum. The residue was purified by automated flash chromatography (50% EtOAc / hexane) 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).

[0134] Step 4. Preparation of benzyl (1,1-bis(4-methoxyphenyl)-1-phenyl-2,5,8,11-tetraoxatridecan-13-yl)(methyl)carbamate 29 (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 dry THF at 0 °C. NaH (60% dispersion in mineral oil) (2.92 g, 121.5 mmol) was added and the reaction mixture was stirred at 0 °C and then at room temperature for 1 h. The reaction mixture was partitioned between EtOAc and H2O. The organic layer was separated, dried (Na2SO4), and concentrated under vacuum. The residue was purified by automated flash chromatography (50% EtOAc / hexane) to give (1,1-bis(4-methoxyphenyl)-1-phenyl-2,5,8,11-tetraoxatridecan-13-yl)(methyl) carbamic acid benzyl ester (29) (26.06 g, 100%). The product was confirmed by MS (ESI+ve).

[0135] Step 5. Preparation of benzyl (2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl)(methyl)carbamate 30 (1,1-Bis(4-methoxyphenyl)-1-phenyl-2,5,8,11-tetraoxatridecan-13-yl)(methyl) carbamic acid benzyl ester (29) (26.06 g, 40.5 mmol) was stirred in DCM at room temperature. TFA (5.1 g, 44.5 mmol) was added and the mixture was stirred for 1 h. Another 2 equivalents of TFA were added and the reaction mixture was stirred for 16 h. The reaction mixture was concentrated under vacuum 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).

[0136] 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 (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 to reflux in DCE for 2 h. After cooling, the reaction was quenched with TEA and washed successively with 1 M HCl, saturated NaHCO3, water and brine, dried (Na2SO4) 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-azapentadecane-15-yl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (31) (9.37 g, 70.6%). The product was confirmed by MS (ESI+ve).

[0137] Step 7. Preparation of (2R,3R,4R,5R)-5-Acetamido-2-(acetoxymethyl)-6-((1,1,1-trifluoro-3-methyl-2-oxo-6,9,12-trioxa-3l4-azatetradecane-14-yl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate 32 (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 with 10% Pd-C (1 g) for approximately 2 h. The reaction was filtered through celite and concentrated under vacuum to give (2R,3R,4R,5R)-5-acetamido-2-(acetoxymethyl)-6-((1,1,1-trifluoro-3-methyl-2-oxo-6,9,12-trioxa-3l4-azatetradecan-14-yl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (32) (9.0 g, 98.9%). The product was used without purification. The product was confirmed by MS (ESI+ve).

[0138] 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 (2R,3R,4R,5R)-5-Acetamido-2-(acetoxymethyl)-6-((1,1,1-trifluoro-3-methyl-2-oxo-6,9,12-trioxa-3l4-azatetradecan-14-yl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (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 for 16 h at room temperature. The reaction was diluted with DCM and washed successively with 1 M HCl, saturated NaHCO3, water and brine, dried (Na2SO4) 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).

[0139] Scheme 7 Preparation of Compound 43

Chem.

[0140] Step 2. Preparation of benzyl (2-(2-(2-(2-(((3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamate 36 (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 in MeOH solution at room temperature for 16 h. The reaction was evaporated at 50 °C to remove ammonia and the residue was concentrated under vacuum to give benzyl (2-(2-(2-(2-(((3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamate (36) (20.3 g, 100%), which was used in the next reaction without further purification. Product confirmation was done by MS (ESI+ve).

[0141] Step 3. Preparation of benzyl (2-(2-(2-(2-(((3aR,4R,7R,7aR)-7-acetamido-4-(hydroxymethyl)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-6-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamate 37 (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)carbamic acid benzyl (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 h. The reaction was cooled to room temperature, TEA (20 ml) was added and stirred for 30 min. The solvent was removed under vacuum and the residue was taken up in MeOH / H2O (10:1) and the reaction was refluxed for 1 h. The reaction was concentrated under vacuum (azeotroped with toluene (×2)) and the residue was purified by automated flash chromatography (10% MeOH / DCM) to give (2-(2-(2-(2-(((3aR,4R,7R,7aR)-7-acetamido-4-(hydroxyl-methyl)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-6-yl)oxy)ethoxy)ethoxy)-ethoxy)ethyl)carbamic acid benzyl (37) (24.9 g, 100%). The product was confirmed by MS (ESI+ve).

[0142] Step 4. Preparation of 4-methylbenzenesulfonic acid ((3aR,4R,7R,7aR)-7-acetamido-2,2-dimethyl-6-((3-oxo-1-phenyl-2,7,10,13-tetraoxa-4-azapentadecane-15-yl)oxy)tetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-4-yl)methyl 38 (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)carbamic acid benzyl (37) (25.5 g, 44.8 mmol) and TEA (9.97 g, 98.5 mmol) were stirred in DCM at 0 °C. DCM containing p-toluenesulfonyl chloride (18.8 g, 98.5 mmol) was added and the reaction was stirred for 16 h and warmed to room temperature. The reaction was diluted with DCM and washed successively with 1 M 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 4-methylbenzenesulfonic acid ((3aR,4R,7R,7aR)-7-acetamido-2,2-dimethyl-6-((3-oxo-1-phenyl-2,7,10,13-tetraoxa-4-azapentadecane-15-yl)oxy)tetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-4-yl)methyl (38) (25.5 g, 78.8%). The product was confirmed by MS (ESI+ve).

[0143] Step 5. Preparation of benzyl (2-(2-(2-(2-(((3aS,4R,7R,7aR)-7-acetamido-4-(azidomethyl)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-6-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamate 39 4-Methylbenzenesulfonic acid ((3aR,4R,7R,7aR)-7-acetamido-2,2-dimethyl-6-((3-oxo-1-phenyl-2,7,10,13-tetraoxa-4-azapentadecan-15-yl)oxy)tetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-4-yl)methyl (38) (25.0 g, 34.5 mmol) and NaN3 (28.7 g, 434.6 mmol) were heated at 100 °C for 12 h in DMSO / H2O (200 ml / 20 ml). The reaction was cooled and partitioned between EtOAc and saturated NaHCO3. The aqueous layer was extracted two more times, and the combined organics were washed with saturated NaHCO3, water, and brine, dried (Na2SO4), 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]dioxolo[4,5-c]pyran-6-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamate (39) (16.1 g, 78.2%). Product confirmation was performed by MS (ESI+ve).

[0144] Step 6. Preparation of benzyl (2-(2-(2-(2-(((3aS,4R,7R,7aR)-7-acetamido-4-((4-(3-methoxyphenyl)-1H-1,2,3-triazol-1-yl)methyl)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-6-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamate 40 (2-(2-(2-(2-(((3aS,4R,7R,7aR)-7-Acetamido-4-(azidomethyl)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-6-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamic acid benzyl (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 of H2O), and sodium ascorbate (0.53 g, 2.7 mmol in 5 ml of H2O) were added sequentially, and the reaction was stirred at room temperature for 16 h. The solvent was removed under vacuum, and the residue was taken up in DCM (200 ml) and washed with water. The aqueous layer was re-extracted with DCM, and the combined organics were washed with brine and dried (Na2SO4). The reaction was concentrated under vacuum, and the residue was purified by automated flash chromatography (10% MeOH / EtOAc) to give (2-(2-(2-(2-(((3aS,4R,7R,7aR)-7-acetamido-4-((4-(3-methoxyphenyl)-1H-1,2,3-triazol-1-yl)methyl)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-6-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamic acid benzyl (40) (15.0 g, 76.4%). The product was confirmed by MS (ESI+ve).

[0145] Step 7. Preparation of (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)carbamic acid benzyl 41 (2-(2-(2-(2-(((3aS,4R,7R,7aR)-7-acetamido-4-((4-(3-methoxyphenyl)-1H-1,2,3-triazol-1-yl)methyl)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyran-6-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamic acid benzyl (40) (15.0 g, 20.6 mmol) was stirred in MeCN (200 ml) and 1.84% H2SO4 (180 ml) for 96 h at room temperature. The reaction mixture was extracted with EtOAc (3 × 250 ml), washed with saturated NaHCO3, water and brine, dried (Na2SO4) and concentrated in vacuo to give (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)-carbamic acid benzyl (41) (11.0 g, 16.0 mmol). The product was used in the next reaction in the crude state. The product was confirmed by MS (ESI+ve).

[0146] 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-azapentadecane-15-yl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate 42 (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)carbamic acid benzyl (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 h and then at 50 °C for 3 h. The reaction was poured into water and extracted three times with DCM (250 ml). The combined organics were washed with saturated NaHCO3 (×2), 1 N HCl (×2), 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-azapentadecane-15-yl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (42) (10.7 g, 86.7%). The product was confirmed by MS (ESI+ve).

[0147] Step 9. Preparation of (2R,3S,4R,5R)-5-acetamido-2-((4-(3-methoxyphenyl)-1H-1,2,3-triazol-1-yl)methyl)-6-((1,1,1-trifluoro-2-oxo-6,9,12-trioxa-3l4-azatetradecane-14-yl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate 43 (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-azapentadecane-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 with 10% Pd-C for 1 h. The reaction was filtered through Celite and concentrated under vacuum 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-3l4-azatetradecane-14-yl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (43) (8.8 g, 99.7%), which was used in the next reaction without purification. The product was confirmed by MS (ESI+ve).

[0148] Scheme 8 Preparation of Compound 54

Chemical formula

[0149] 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 Peracetylated galactosamine (44) (25 g, 64.21 mmol) was heated with scandium triflate (1.58 g, 3.21 mmol) in anhydrous DCE at 90 °C for 3 h. The reaction was cooled to room temperature, quenched with 5 ml of TEA, and concentrated under vacuum. The residue was purified by automated column chromatography (2 - 10% MeOH / DCM) to give (2R,3R,4R,5R)-5-acetamido-2-(acetoxymethyl)-6-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-3,4-diyl diacetate (45) (27 g, 76.5%). The product was confirmed by MS.

[0150] 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)ethane-1-aminium 2,2,2-trifluoroacetate 46 A solution containing azide 45 (7.12 g, 13 mmol) in EtOAc (150 ml) and trifluoroacetic acid (2 ml) was treated with palladium on carbon (1.5 g, 10% w / w on a wet basis). The reaction mixture was then purged with hydrogen and stirred vigorously overnight. After purging with nitrogen, the mixture was filtered through celite and washed with MeOH. The filtrate was concentrated and purified by chromatography (5% → 10% → 20% MeOH-CH2Cl2) to give 46 (5.8 g, 72%) as a brown oil. Rf (0.34, 15% MeOH-CH2Cl2).

[0151] 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. (2R,3R,4R,5R)-5-Acetamido-2-(acetoxymethyl)-6-((1,1,1-trifluoro-2-oxo-6,9,12-trioxa-3l4-azatetradecan-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 for 16 h at room temperature. The reaction mixture was diluted with DCM and washed successively with 1 M HCl, saturated NaHCO3, water and brine, dried over Na2SO4 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).

[0152] 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 (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 at room temperature for 2 h with 10% Pd-C (2.6 g). The reaction was filtered through Celite and concentrated under vacuum 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 the next reaction without further purification. The product was confirmed by MS (ESI+ve).

[0153] Step 6. Preparation of (2R,3R,4R,5R)-5-acetamido-6-((1-(3-((2-(2-(2-(2-(((3R,4R,5R,6R)-3-acetamido-5-acetoxy-6-(acetoxymethyl)-4-hydroxy-tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-5-(2-(((benzyloxy)carbonyl)amino)acetamido)phenyl)-1-oxo-5,8,11-trioxa-2-azatridecan-13-yl)oxy)-2-(acetoxymethyl)tetrahydro-2H-pyran-3,4-diyl diacetate 51 (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 at room temperature overnight. Additional T3P (0.3 eq) was added and the reaction was stirred for an additional 1 h. The reaction was washed with saturated NaHCO3 and brine, dried (Na2SO4), concentrated under vacuum, and the residue was purified by automated flash chromatography (10% MeOH / DCM) to give (2R,3R,4R,5R)-5-acetamido-6-((1-(3-((2-(2-(2-(2-(((3R,4R,5R,6R)-3-acetamido-5-acetoxy-6-(acetoxymethyl)-4-hydroxy-tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-5-(2-(((benzyloxy)carbonyl)amino)acetamido)phenyl)-1-oxo-5,8,11-trioxa-2-azatridecan-13-yl)oxy)-2-(acetoxymethyl)tetrahydro-2H-pyran-3,4-diyl diacetate (51) (0.33 g, 56.8%). The product was confirmed by MS (ESI+ve).

[0154] Step 7. Preparation of (2R,2’R,3R,3’R,4R,4’R,5R,5’R)-(((5-(2-((2,2,2-trifluoroacetyl)-l4-azanil)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 52 (2R,3R,4R,5R)-5-Acetamido-6-((1-(3-((2-(2-(2-(2-(((3R,4R,5R,6R)-3-Acetamido-5-acetoxy-6-(acetoxymethyl)-4-hydroxytetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-5-(2-(((benzyloxy)carbonyl)amino)acetamido)phenyl)-1-oxo-5,8,11-trioxa-2-azatridecan-13-yl)oxy)-2-(acetoxymethyl)tetrahydro-2H-pyran-3,4-diyl diacetate (51) (3.3 g, 2.39 mmol) and TFA (0.29 g, 2.51 mmol) were stirred in MeOH at room temperature. The reaction mixture was hydrogenated over 10% Pd-C (400 mg) for 2 h, filtered through celite and concentrated under vacuum to give (2R,2’R,3R,3’R,4R,4’R,5R,5’R)-(((5-(2-((2,2,2-trifluoroacetyl)-14-azanil)-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 (52) (3.21 g, 98.7%), which was used in the next reaction without further purification. The product was confirmed by MS (ESI+ve).

[0155] Step 8. Preparation of (2R,2’R,3R,3’R,4R,4’R,5R,5’R)-(((5-(2-(10-(3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanamido)acetamido)-1,3-phenylene)bis(1-oxo-5,8,11-trioxa-2-azatridecane-1,13-diyl))bis(oxy))bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl) triacetate 53 (2R,2’R,3R,3’R,4R,4’R,5R,5’R)-(((5-(2-((2,2,2-Trifluoroacetyl)-14-azanil)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 (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 at room temperature. The reaction mixture was diluted with DCM and washed successively with 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,2’R,3R,3’R,4R,4’R,5R,5’R)-(((5-(2-(10-(3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanamido)acetamido)-1,3-phenylene)bis(1-oxo-5,8,11-trioxa-2-azatridecane-1,13-diyl))bis(oxy))bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl) tetraacetate (53) (1.02 g, 75.2%). The product was confirmed by MS (ESI+ve).

[0156] Preparation of 54 ((1 - (10 - ((2 - ((3,5 - bis((2 - (2 - (2 - (2 - ((((3R,4R,5R,6R)-3 - acetamido - 4,5 - diacetoxy - 6 - (acetoxymethyl)tetrahydro - 2H - pyran - 2 - yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamoyl)phenyl)amino)-2 - oxoethyl)amino)-10 - oxodecanoyl)-4 - ((bis(4 - methoxyphenyl)(phenyl)methoxy)methyl)-3,4 - dimethylpyrrolidin - 3 - yl)methoxy)-4 - oxobutanoic acid (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-oxodecanamide)acetamide)-1,3-phenylene)bis(1-oxo-5,8,11-trioxa-2-azatridecane-1,13-diyl))bis(oxy))bis(5-acetamide-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (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 dehydrated DCE at 60 °C for 2 h. MeOH (5 ml) was added and the reaction mixture was stirred for a further 30 min, then cooled and concentrated in vacuo. The residue was taken up in DCM and washed successively with saturated NaHCO3 (×4), water and brine. The organic layer was dried (Na2SO4) and concentrated in vacuo to give 4-((1-(10-((2-((3,5-bis((2-(2-(2-(2-(((3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamoyl)phenyl)amino)-2-oxoethyl)amino)-10-oxodecanoyl)-4-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3,4-dimethylpyrrolidin-3-yl)methoxy)-4-oxobutanoic acid (54) (1.1 g, 99.4%), which was used as the crude product in the next reaction. The product was confirmed by MS (ESI+ve).

[0157] Scheme 9 Preparation of Compound 56 [Chemical Structure]< Step 1. Preparation of (2R,2’R,3R,3’R,4R,4’R,5R,5’R)-(((5-(10-(3-((Bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanamide)-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 (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) (4 g, 3.36 mmol), 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 at room temperature for 16 h. The reaction was washed successively 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-(hydroxyl-methyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanamide)-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).

[0158] Step 2.4 - Preparation of 56: (1-(10-((3,5-bis((2-(2-(2-(2-(((3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamoyl)phenyl)amino)-10-oxodecanoyl)-4-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3,4-dimethylpyrrolidin-3-yl)methoxy)-4-oxobutanoic acid This compound was prepared in the same manner as 4-((1-(10-((2-((3,5-bis((2-(2-(2-(2-(((3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamoyl)phenyl)amino)-2-oxoethyl)amino)-10-oxodecanoyl)-4-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3,4-dimethylpyrrolidin-3-yl)methoxy)-4-oxobutanoic acid (54).

[0159] Scheme 10 - Preparation of Compound 57

Chemical Structure

[0160] Scheme 11 Preparation of Compound 66 [Chemical formula] Step 1. Preparation of Dimethyl 5-(hydroxymethyl)isophthalate 59 Trimethyl benzene-1,3,5-tricarboxylate (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 mixture was refluxed for 30 minutes. After cooling, the reaction was quenched with 1 M HCl and extracted with EtOAc. The organic layer was washed successively with 1 M HCl, NaHCO3, water, and brine, dried (Na2SO4), and concentrated under vacuum. The residue was purified by automated flash chromatography (50 / 50 EtOAc / hexane) to afford dimethyl 5-(hydroxymethyl)isophthalate (59) (20.5 g, 53.2%). 1 1H 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).

[0161] Step 2. Preparation of Dimethyl 5-(chloromethyl)isophthalate 60 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 mixture was cooled, diluted with DCM, washed successively with 0.1 M NaOH (×2), water and brine, dried (Na2SO4), and concentrated under vacuum. The residue was purified by automated flash chromatography (20% EtOAc / hexane) to give dimethyl 5-(chloromethyl)isophthalate (60) (10.84 g, 53%). 1 1H 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).

[0162] Step 3. Preparation of dimethyl 5-(azidomethyl)isophthalate 61 Dimethyl 5-(chloromethyl)isophthalate (60) (10.84 g, 45 mmol) and NaN3 (18 g, 270 mmol) were refluxed in acetone / water (3 / 1) for 16 h. The reaction mixture was cooled, concentrated under vacuum, and the residue was taken up in DCM. The organic layer was washed with water and brine, dried (Na2SO4), and concentrated under vacuum. The residue was purified by flash chromatography (15% EtOAc / hexane) to give dimethyl 5-(azidomethyl)isophthalate (61) (9.84 g, 88%). 1 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).

[0163] Step 4. Preparation of 5-(azidomethyl)isophthalic acid 62 Dimethyl 5-(azidomethyl)isophthalate (61) (9.84 g, 39.5 mmol) and LiOH (2.1 g, 87 mmol) were stirred in THF / H2O / MeOH at room temperature for 48 h. The organic solvents were removed under vacuum, and the residue was acidified with 1 M HCl. The aqueous solution was extracted with EtOAc (×3), and the combined organics were dried (Na2SO4) and concentrated under vacuum to give 5-(azidomethyl)isophthalic acid (62) (8.0 g, 91.6%), which was used in the next reaction without further purification.

[0164] 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 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)ethane-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 successively with 1 M HCl, saturated NaHCO3, water and brine, dried (Na2SO4) and concentrated under vacuum. 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).

[0165] Step 6. Preparation of (2R,2’R,3R,3’R,4R,4’R)-(((5-(((2,2,2-Trifluoroacetyl)-l4-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 (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 with 10% Pd-C for 1 h. The reaction was filtered through Celite and concentrated under vacuum, 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).

[0166] 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-oxodecanamide)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 (2R,2’R,3R,3’R,4R,4’R)-(((5-(((2,2,2-trifluoroacetyl)-14-azanil)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), lithium 10-(3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoate (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 reaction was diluted with DCM, washed with 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,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-oxodecanamide)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) (1.8 g, 43.0%). The product was confirmed by MS (ESI+ve).

[0167] Step 8.4 - Preparation of 66: (1 - (10 - ((3,5 - bis((2 - (2 - (2 - (2 - ((((4R,5R,6R) - 3 - acetamido - 4,5 - diacetoxy - 6 - (acetoxymethyl)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 This compound was prepared in the same manner as 4 - ((1 - (10 - ((2 - ((3,5 - bis((2 - (2 - (2 - (2 - ((((3R,4R,5R,6R) - 3 - acetamido - 4,5 - diacetoxy - 6 - (acetoxymethyl)tetrahydro - 2H - pyran - 2 - yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamoyl)phenyl)amino) - 2 - oxoethyl)amino) - 10 - oxodecanoyl) - 4 - ((bis(4 - methoxyphenyl)(phenyl)methoxy)methyl) - 3,4 - dimethylpyrrolidin - 3 - yl)methoxy) - 4 - oxobutanoic acid (54). The product was confirmed by MS (ESI+ve).

[0168] Scheme 12 Preparation of Compound 67 [Chemical formula] Synthesis of 4 - ((1 - (10 - ((2 - ((3,5 - bis((2 - (2 - (2 - (2 - ((((3R,4R,5R,6R) - 4,5 - diacetoxy - 6 - (acetoxymethyl) - 3 - (2,2,2 - trifluoroacetamido)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 This compound was prepared in the same manner as 54 (Scheme 8) using (3R,4R,5R,6R)-6-(acetoxymethyl)-3-(2,2,2-trifluoroacetamido)tetrahydro-2H-pyran-2,4,5-triyl triacetate in place of peracetylated galactosamine (6). The product was confirmed by MS (ESI+ve).

[0169] Scheme 13 Preparation of Compound 68

Chem.

[0170] Scheme 14 Preparation of Compound 69

Chem.

[0171] Scheme 15 Preparation of compound 70 [Chemical formula] Synthesis of 4-((1-(10-((2-((3,4-bis((2-(2-(2-(2-(((3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)(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 This compound was prepared in the same manner as compound 54 (Scheme 8) using (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) in place 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).

[0172] Scheme 16 Preparation of Compound 71 [Chemical formula] Synthesis of 4-((1-(10-((2-((3,5-bis((2-(2-(2-(2-(((3R,4R,5S,6R)-3-acetamido-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 This compound was prepared in the same manner as compound 54 (Scheme 8) by 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-3l4-azatetradecan-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)ethane-1-aminium 2,2,2-trifluoroacetate (46).

[0173] Scheme 17 Preparation of Compound 72 [Chemical Structure] Synthesis of 4-((4-(10-((2-((3,5-bis((2-(2-(2-(((3R,4R,5R,6R)-3-acetamido-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 This compound was prepared in the same manner as compound 54 (Scheme 8) by using (2R,3R,4R,5R)-5-acetamido-2-(acetoxymethyl)-6-(2-(2-(2-((2,2,2-trifluoroacetyl)-14-azanil)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)ethane-1-aminium 2,2,2-trifluoroacetate (46).

[0174] Scheme 18 Preparation of Compound 81

Chemical Structure

[0175] 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-oxododecanamide)benzamide)ethoxy)ethoxy)ethoxy)-2-(acetoxymethyl)tetrahydro-2H-pyran-3,4-diyl Diacetate 78 (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-aminobenzamide)ethoxy)ethoxy)ethoxy)-2-(acetoxymethyl)-tetrahydro-2H-pyran-3,4-diyl diacetate (77) (4.0 g, 3.6 mmol), 12-(Benzyloxy)-12-oxododecanoic acid (76) (1.3 g, 4.1 mmol) and triethylamine (1.5 ml, 10.8 mmol) were dissolved in dichloromethane (75 ml). T3P (4.5 g, ca. 9 ml, 50% solution in ethyl acetate) was added dropwise to the solution. The solution was stirred at room temperature overnight. Immediately after completion, the reaction mixture was diluted with dichloromethane and carefully quenched with a saturated solution of sodium bicarbonate (200 ml). The biphasic solution was stirred vigorously for 30 minutes. The DCM layer was separated and the aqueous phase was extracted with dichloromethane (1 x 100 ml). The combined extracts were dried over magnesium sulfate, filtered and concentrated to dryness under vacuum. The residue was purified by column chromatography on silica gel 60 (gradient: 0 to 10% MeOH in DCM) to give the title compound as a colorless solid (1.5 g, 30%).

[0176] Step 3. Preparation of 12-((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-(2-(2-(((3S,4S,5S,6S)-3-Acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)carbamoyl)phenyl)amino)-12-oxododecanoic acid 79 (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-oxododecanamide)benzamide)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 gas was bubbled through the solution for 1 hour. Immediately after completion, nitrogen was bubbled through the solution, filtered through celite, and concentrated to dryness under vacuum to give a colorless solid (1.1 g, 79%).

[0177] 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)dodecanamide)benzamide)ethoxy)ethoxy)ethoxy)-2-(acetoxymethyl)tetrahydro-2H-pyran-3,4-diyl diacetate 81 12 - ((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 - (2 - (2 - (((3S,4S,5S,6S)-3 - acetamido - 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) were added to a solution in dichloromethane (50 ml), and pentafluorophenyl trifluoroacetate (80) (150 mg, 1.1 mmol) was added. The solution was stirred at room temperature for 30 minutes and then concentrated to dryness under vacuum. The residue was purified by column chromatography on silica gel 60 (gradient: 0 to 10% methanol in dichloromethane) to give the title compound as a colorless solid (475 mg, 70%). Mass (ESI+) m / z 741.0 (M + 2H). 1H NMR (400 MHz, DMSO - d6) δ 10.12 (s, 1H), 8.52 (t, J = 5.6 Hz, 2H), 8.14 (d, J = 1.4 Hz, 2H), 7.91 (t, J = 1.6 Hz, 1H), 7.80 (d, J = 9.2 Hz, 2H), 5.21 (d, J = 3.4 Hz, 2H), 4.97 (dd, J = 11.2, 3.4 Hz, 2H), 4.54 (d, J = 8.5 Hz, 2H), 4.06 - 3.99 (m, 7H), 3.88 (dt, J = 11.2, 8.8 Hz, 2H), 3.77 (ddd, J = 11.1, 5.6, 3.9 Hz, 2H), 3.62 - 3.46 (m, 22H), 3.46 - 3.38 (m, 5H), 2.77 (t, J = 7.2 Hz, 2H), 2.31 (t, J = 7.4 Hz, 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).

[0178] Scheme 19 Preparation of Compound 90 [Chemical Structure] TIFF2025105782000047.tif180167 Step 1. Preparation of 12-((tert-butoxycarbonyl)amino)dodecanoic acid 84 A solution containing 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 at 60 °C for 3 hours and then allowed to stand overnight at room temperature. Immediately after completion, the solution was concentrated to dryness under vacuum and used in the next step without further purification.

[0179] Step 2. Preparation of benzyl 12-((tert-butoxycarbonyl)amino)dodecanoate 85 A solution containing 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 at room temperature. Immediately 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. Purification by column chromatography on silica gel 60 (Gradient: 0 to 50% ethyl acetate in hexane) gave the title compound as a colorless solid (2.0 g, 21% over 2 steps).

[0180] Step 3. Preparation of 12-(benzyloxy)-12-oxododecan-1-aminium trifluoroacetate 87 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).

[0181] 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-oxododecanamide)benzamide)ethoxy)ethoxy)ethoxy)-2-(acetoxymethyl)tetrahydro-2H-pyran-3,4-diyl diacetate 88 A solution containing 12-((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-(2-(2-(((3S,4S,5S,6S)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)-ethyl)carbamoyl)phenyl)amino)-12-oxododecanoic acid (88) (750 mg, 0.54 mmol), 12-(benzyloxy)-12-oxododecane-1-aminium trifluoroacetate (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 at room temperature 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 under vacuum. The residue was purified by column chromatography on silica gel 60 (gradient: 0 - 10% methanol in dichloromethane) to give the title compound (88) as a colorless solid (605 mg, 70%).

[0182] Preparation of 89: 5.12-(12-((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-(2-(2-(((3S,4S,5S,6S)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)carbamoyl)phenyl)amino)-12-oxododecanamide)dodecanoic acid Hydrogenation was carried out as described above to give (89) (350 mg, 55%).

[0183] Preparation of 90: (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)dodecanamide)benzamide)ethoxy)ethoxy)ethoxy)-2-(acetoxymethyl)tetrahydro-2H-pyran-3,4-diyl diacetate PFP ester formation was carried out as described above to give the required product (90) (112 mg, 23%). 11H NMR (400 MHz, DMSO-d6) δ 10.12 (s, 1H), 8.91 (s, 1H), 8.65 (t, J = 5.5 Hz, 1H), 8.52 (t, J = 5.6 Hz, 1H), 8.23 (d, J = 1.5 Hz, 1H), 8.14 (t, J = 1.4 Hz, 2H), 7.91 (d, J = 1.6 Hz, 1H), 7.80 (d, J = 9.2 Hz, 2H), 7.68 (t, J = 5.6 Hz, 1H), 5.21 (d, J = 3.4 Hz, 2H), 4.97 (dd, J = 11.2, 3.4 Hz, 2H), 4.54 (d, J = 8.5 Hz, 2H), 4.07 - 3.96 (m, 6H), 3.88 (dt, J = 11.2, 8.9 Hz, 2H), 3.81 - 3.74 (m, 2H), 3.64 - 3.36 (m, 24H), 3.15 - 3.03 (m, 6H), 2.99 (q, J = 6.5 Hz, 2H), 2.76 (t, J = 7.2 Hz, 1H), 2.31 (t, J = 7.4 Hz, 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 = 7.1 Hz, 2H), 1.40 - 1.10 (m, 29H). Mass (ESI+) m / z 839.7 (M + 2H).

[0184] Scheme 20 Preparation of Compound 94 [Chemical formula] TIFF2025105782000049.tif55144 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-oxododecanamido)acetamido)benzamido)ethoxy)ethoxy)ethoxy)-2-(acetoxymethyl)tetrahydro-2H-pyran-3,4-diyldiacetate 92 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-(2-(2-(((3S,4S,5S,6S)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)carbamoyl)phenyl)amino)-2-oxoethan-1-aminium trifluoroacetate (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 at room temperature overnight. Immediately 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 under vacuum. The residue was purified by column chromatography on silica gel 60 (gradient: 0 to 10% methanol in dichloromethane) to give the title compound as a colorless solid (0.8 g, 68%).

[0185] Step 2. Preparation of 12-((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-(2-(2-(((3S,4S,5S,6S)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)carbamoyl)phenyl)amino)-2-oxoethyl)amino)-12-oxododecanoic acid 93 Compound 93 was prepared using conditions similar to those described herein for similar transformations (450 mg, 60%).

[0186] 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)dodecanamide)acetamido)benzamide)ethoxy)ethoxy)ethoxy)-2-(acetoxymethyl)tetrahydro-2H-pyran-3,4-diyl diacetate 94 Compound 94 was prepared using conditions similar to those described herein for similar conversions (460 mg, 91%). Mass (ESI+) m / z 1537.8 (M+H).

[0187] Scheme 21 Preparation of Compound 95 [Chemical formula] (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)phosphanyl)-oxy)methyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanamide)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 synthesis (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-(10-(3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanamide)acetamido)benzamide)-ethoxy)ethoxy)ethoxy)-2-(acetoxymethyl)tetrahydro-2H-pyran-3,4-diyl diacetate (72) (1.6 g, 0.9 mmol) and diisopropylethylamine (0.4 ml, 1.8 mmol) were added to a solution in anhydrous dichloromethane (25 ml), and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.3 ml, 1.35 mmol) was added. The solution was stirred at room temperature for 75 minutes and then concentrated to dryness. The residue was purified by column chromatography (gradient: 0 to 10% MeOH in DCM (0.1% TEA)) to give the product as a colorless solid (1.1 g, 62%). 31P NMR (400 MHz, DMSO-d6): δ 146.76 (s), 146.42 (s, two overlapping signals), 146.34 (s).1H NMR (400 MHz, DMSO-d6) δ 10.20 (s, 1H), 8.54 (t, J = 5.6 Hz, 2H), 8.17 - 8.09 (m, 3H), 7.94 (s, 1H), 7.80 (d, J = 9.2 Hz, 2H), 7.39 - 7.26 (m, 4H), 7.26 - 7.17 (m, 6H), 6.91 - 6.83 (m, 4H), 5.21 (d, J = 3.4 Hz, 2H), 4.97 (dd, J = 11.2, 3.4 Hz, 2H), 4.54 (d, J = 8.5 Hz, 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, 5.8 Hz, 2H), 2.10 (m, 12H), 1.99 (s, 6H), 1.89 (s, 6H), 1.77 (s, 6H), 1.47 (d, J = 7.2 Hz, 4H), 1.23 (dq, J = 13.9, 6.4 Hz, 18H), 1.17 - 1.04 (m, 10H), 0.98 (dt, J = 13.4, 5.9 Hz, 10H).

[0188] Scheme 22 Preparation of Compound 96

Chemical Structure

[0189] Step 2: Preparation of 96 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-((((2-cyanoethoxy)(diisopropylamino)phosphanyl)oxy)methyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecyl)amino)dodecanamide)acetamido)benzamide)-ethoxy)ethoxy)ethoxy)-2-(acetoxymethyl)tetrahydro-2H-pyran-3,4-diyl diacetate Compound 96 was prepared using conditions similar to those described herein for similar conversions (1.35 g, 65%). 31 P NMR (400 MHz, DMSO-d6): δ 146.79 (s), 146.76 (s), 146.42 (s), 146.36 (s). 11H NMR (400 MHz, DMSO-d6) δ 10.19 (s, 1H), 8.54 (t, J = 5.6 Hz, 2H), 8.13 (dd, J = 6.1, 3.5 Hz, 3H), 7.94 (s, 1H), 7.80 (d, J = 9.2 Hz, 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 = 3.4 Hz, 2H), 4.97 (dd, J = 11.2, 3.4 Hz, 2H), 4.54 (d, J = 8.5 Hz, 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 = 8.0 Hz, 24H), 1.17 (t, J = 7.3 Hz, 11H), 1.09 (dt, J = 6.7, 3.3 Hz, 9H), 1.03 - 0.92 (m, 9H).

[0190] Scheme 23 General synthesis of a conjugate of Formula I having an oligonucleotide linked by the 3'-end of the oligonucleotide (Compound 73)

Chemical formula

[0191] Scheme 24 and Scheme 25 General synthesis of conjugate of formula I (compound 98) having an oligonucleotide linked by the 5’ of the oligonucleotide The pentafluorophenyl ester was linked to a C6 5’-amino modifier having a phosphate / phosphorothioate linkage on the sense strand oligonucleotide using standard coupling conditions. The desired sense strand conjugate was obtained by standard cleavage and deprotection. For example, using pentafluorophenyl ester 81, the following conjugate 98 was obtained.

Chemical formula

[0192] Phosphoramidite was linked to the 5'-hydroxyl of the sense strand terminal nucleotide using standard phosphoramidite coupling chemistry. The desired sense strand conjugate was obtained by standard cleavage and deprotection. For example, using phosphoramidite 95, the following conjugate 99 was obtained. [Chemical formula]

[0193] Examples 1-9 The following conjugates of the present invention were prepared using the general procedure shown in Scheme 23, where R 3b is the modified TTR siRNA described in Table A below. The mass is that of the single-stranded product, not the annealed double-stranded one.

[0194] Example 1 [Chemical formula] MS(+VE) Calculated: 8184.7, Measured: 8184.2.

[0195] Example 2 [Chemical formula] MS(+VE) Calculated: 8212.7, Measured: 8211.9.

[0196] Example 3 [Chemical formula] MS(+VE) Calculated: 8212.7, Measured: 8212.8.

[0197] Example 4 [Chemical formula] MS(+VE) Calculated: 8096.6, Measured: 8097.0.

[0198] Example 5 [Chemical formula] MS(+VE) calculated value: 8499.0, measured value: 8498.7.

[0199] Example 6

Chemical formula

[0200] Example 7

Chemical formula

[0201] Example 8

Chemical formula

[0202] Example 9

Chemical formula

[0203] Examples 10 - 11 The following conjugates of the present invention were prepared using the general procedure shown in Scheme 24, where R 3b is the modified TTR siRNA described in Table A below. The mass is that of the single-stranded product, not the annealed double-stranded one.

[0204] Example 10

Chemical formula

[0205] Example 11 [Chemical] MS(+VE) calculated value: 8254.0, measured value: 8253.5.

[0206] Example 12: In Vivo Testing of a TTR siRNA Bivalent Conjugate Bivalent conjugates (Examples 1 - 8) with the oligonucleotide as the modified TTR siRNA described in Table A were tested in a wild - type mouse model of TTR knockdown for in - vivo activity. The TTR conjugate is expected to be a therapeutic agent for the rare disease of TTR (transthyretin) amyloidosis. In persons suffering from this disease, misfolding and aggregation of the transthyretin protein are known to be associated with disease progression. By using this siRNA conjugate, the amount of misfolded / aggregated protein in patients can be reduced, and as a result, the disease progression may be halted.

[0207] [Table 1]

[0208] Both the TTR siRNA sequence and the animal model are described by Nair et al. J. Am. Chem. Soc., 2014, 136(49), 16958 - 16961. All procedures involving animals were conducted in accordance with written procedures following the guidelines of the Canadian Council on Animal Care (CCAC) for appropriate animal - related operations and were approved by the local Institutional Animal Care and Use Committee (IACUC).

[0209] siRNA treatment: Female C57BL / 6 mice (n = 4) were administered a single dose of 2 mg / kg of the TTR siRNA conjugate by subcutaneous injection into the scapular region on day 0 (one dose per animal). One group of animals was administered only the vehicle (PBS) which served as a control.

[0210] Blood sampling: Test blood sampling of all animals was performed at defined time points (days 2, 4, 5, 7, 8, 9, 14, and 21) after administration of the test article to determine the maximum reduction in plasma TTR levels and the duration of pharmacological activity.

[0211] Analysis: The TTR protein levels in plasma samples were determined using an Abnova prealbumin (mouse) ELISA kit (Cedar Lane, catalog number KA2070) according to the manufacturer's instructions. TTR protein values in plasma were calculated for each individual plasma sample, and the mean value for each group was determined. From these mean values, the TTR protein level (%) relative to the control (relative to PBS-treated animals) was determined.

[0212] Results: The results from the test are shown in Table B. The values represent the % of TTR protein levels (relative to the PBS control) on days 2, 4, 5, 7, 8, 9, 14, and 21 after treatment.

[0213] Conclusion: Animals treated with the TTR dimer conjugate exhibited significant knockdown of the target mRNA and protein, and the maximum knockdown of the TTR protein occurred on days 4 - 9 after subcutaneous injection.

[0214]

Table 2

[0215] Example 13: In Vivo Testing of TTR siRNA Dimer Conjugates Mono-, di-, tri-, and tetravalent conjugates (Compounds A - D) [wherein R 3b is the modified TTR siRNA described in Table A above] were tested in a wild-type mouse model of TTR knockdown for in vivo activity. Compound A (monovalent)

Chemical Structure

Chemical Structure

[0216] Both the TTR siRNA sequence and the animal model are described by Nair et al. J. Am. Chem. Soc., 2014, 136(49), 16958 - 16961. All procedures involving animals were conducted according to written operating procedures in accordance with the guidelines of the Canadian Council on Animal Care (CCAC) for appropriate animal-related operations and were approved by the local Institutional Animal Care and Use Committee (IACUC).

[0217] siRNA treatment: Female C57BL / 6 mice (n = 4) were administered a single dose of 2 mg / kg of the TTR siRNA conjugate by subcutaneous injection into the scapular region on day 0 (1 dose per animal). One animal group was administered only the vehicle (PBS) which served as a control.

[0218] Blood sampling: Test blood samples were taken from all animals at defined time points (days 2, 5, 7, 14, and 21) after administration of the test article to determine the maximum reduction in plasma TTR levels and the duration of pharmacological activity.

[0219] Analysis: The 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. The TTR protein values in plasma were calculated for each individual plasma sample, and the mean value for each group was determined. From these mean values, the TTR protein levels (%) relative to the control (relative to PBS-treated animals) were determined.

[0220] Results: The results from the test are shown in Table C. The values represent the % of TTR protein levels (relative to the PBS control) on days 2, 5, 7, 14, and 21 after treatment.

[0221] Conclusion: Animals treated with TTR bivalent, trivalent, and tetravalent conjugates exhibited knockdown of the target mRNA and protein at comparable levels, and the maximum knockdown of the TTR protein occurred between days 2 and 7 after subcutaneous injection. The TTR monovalent conjugate showed only a slight, if any, knockdown of the target mRNA and protein.

[0222] [Table 3] Finally, the preferred embodiments of the present invention are described item by item.

[0223] [Embodiment 1] Conjugate of formula (I): [Chemical formula] [Wherein, R 1 is a sugar, L 1 is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having from 0 to 20 carbon atoms, and one or more of the carbon atoms in the hydrocarbon chain are optionally replaced by -O-, -NR X -, -NR X -, -C(=O)-, -C(=O)-NR X -, or -S-, and R Xis hydrogen or (C1-C6) alkyl, and the hydrocarbon chain is optionally substituted with one or more substituents selected from oxo (=O) and halo, B is 5- to 10-membered aryl or 5- to 10-membered heteroaryl, and the 5- to 10-membered aryl or 5- to 10-membered heteroaryl 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, L 2 is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 0 to 20 carbon atoms, and one or more of the carbon atoms in the hydrocarbon chain are optionally replaced by -O-, -NR X -, -NR X -, -C(=O)-, -C(=O)-NR X -, or -S-, and R X is hydrogen or (C1-C6) alkyl, and the hydrocarbon chain is optionally substituted with one or more substituents selected from oxo (=O) and halo, R 2 is a sugar, L 3 is absent or a linking group, A is absent, 3- to 20-membered cycloalkyl, 5- to 20-membered aryl, 5- to 20-membered heteroaryl, or 3- to 20-membered heterocycloalkyl, Each R A is independently selected from the group consisting of hydrogen, hydroxy, CN, F, Cl, Br, I, -C 1-2 alkyl-OR a 、C 1-10 alkyl C 2-10 alkenyl, and C 2-10 alkynyl, and the C 1-10 alkyl C 2-10 alkenyl, and C 2-10 alkynyl are optionally substituted with halo, hydroxy and C 1-3Optionally substituted with one or more groups independently selected from alkoxy, n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, L 4 is absent or a linking group, R 3 is a nucleic acid, R a is hydrogen, a protecting group, a covalent bond with a solid support, or a linking group L bonded to the solid support 5 and the bond with L, L 5 is a linking group], or a salt thereof.

[0224] [Embodiment 2] The conjugate or salt according to Embodiment 1, wherein A is absent.

[0225] [Embodiment 3] The conjugate or salt according to Embodiment 1, wherein A is 3- to 20-membered cycloalkyl, 5- to 20-membered aryl, 5- to 20-membered heteroaryl, or 3- to 20-membered heterocycloalkyl.

[0226] [Embodiment 4] The conjugate or salt according to any one of Embodiments 1 to 3, wherein B is 5- to 10-membered aryl.

[0227] [Embodiment 5] The conjugate or salt according to any one of Embodiments 1 to 3, wherein B is naphthyl or phenyl.

[0228] [Embodiment 6] The conjugate or salt according to any one of Embodiments 1 to 3, wherein B is phenyl.

[0229] [Embodiment 7] The group:

Chemical formula

Chemical formula

[0230] [Embodiment 8] The conjugate or salt according to any one of Embodiments 1 to 3, wherein B is a 5- to 10-membered heteroaryl.

[0231] [Embodiment 9] The conjugate or salt according to any one of Embodiments 1 to 3, wherein B is pyridyl, pyrimidyl, quinolyl, isoquinolyl, imidazolyl, thiazolyl, dioxazolyl or oxazolyl.

[0232] [Embodiment 10] The group: [Chemical formula] is [Chemical formula] The conjugate or salt according to any one of Embodiments 1 to 3, which is as defined above.

[0233] [Embodiment 11] The group: [Chemical formula] is [Chemical formula] The conjugate or salt according to any one of Embodiments 1 to 3, which is as defined above.

[0234] [Embodiment 12] L 1 is a divalent unbranched saturated hydrocarbon chain having 0 to 20 carbon atoms, and one or more (for example, 1, 2, 3 or 4) of the carbon atoms in the hydrocarbon chain are optionally replaced by -O-, -NR X -, -NR X -, -C(=O)-, -C(=O)-NR X -, or -S-, and R Xis hydrogen or (C1-C6) alkyl, and the hydrocarbon chain is optionally substituted with one or more substituents selected from oxo (=O) and halo, the conjugate or salt according to any one of embodiments 1 to 11.

[0235] [Embodiment 13] L 1 is a divalent unbranched saturated hydrocarbon chain having 0 to 12 carbon atoms, and one or more (for example, 1, 2, 3 or 4) of the carbon atoms in the hydrocarbon chain are optionally -O-, -NR X -, -C(=O)-, or -C(=O)-NR X -, and R X is hydrogen or (C1-C6) alkyl, the conjugate or salt according to any one of embodiments 1 to 11.

[0236] [Embodiment 14] L 1 is -C(=O)N(H)-CH2CH2OCH2CH2OCH2CH2-, -C(=O)N(H)-CH2CH2OCH2CH2OCH2CH2OCH2CH2-, -C(=O)N(CH3)-CH2CH2OCH2CH2OCH2CH2-, or -C(=O)N(CH3)-CH2CH2OCH2CH2OCH2CH2OCH2CH2 is the conjugate or salt according to any one of embodiments 1 to 11.

[0237] [Embodiment 15] L 2 is a divalent unbranched saturated hydrocarbon chain having 0 to 20 carbon atoms, and one or more (for example, 1, 2, 3 or 4) of the carbon atoms in the hydrocarbon chain are optionally -O-, -NR X -, -NR X -, -C(=O)-, -C(=O)-NR X -, or -S-, and R Xis hydrogen or (C1-C6) alkyl, and the hydrocarbon chain is optionally substituted with one or more substituents selected from oxo (=O) and halo, the conjugate or salt according to any one of Embodiments 1 to 14.

[0238] [Embodiment 16] L 2 is a divalent unbranched saturated hydrocarbon chain having 0 to 12 carbon atoms, and one or more (for example, 1, 2, 3 or 4) of the carbon atoms in the hydrocarbon chain are optionally -O-, -NR X -C(=O)-, or -C(=O)-NR X -, and R X is hydrogen or (C1-C6) alkyl, the conjugate or salt according to any one of Embodiments 1 to 14.

[0239] [Embodiment 17] L 2 is -C(=O)N(H)-CH2CH2OCH2CH2OCH2CH2-, -C(=O)N(H)-CH2CH2OCH2CH2OCH2CH2OCH2CH2-, -C(=O)N(CH3)-CH2CH2OCH2CH2OCH2CH2-, or -C(=O)N(CH3)-CH2CH2OCH2CH2OCH2CH2OCH2CH2- is the conjugate or salt according to any one of Embodiments 1 to 14.

[0240] [Embodiment 18] R 1 is [Chemical formula] [wherein, X is NR 20 and Y is -(C=O)R 21 , -SO2R 22 , and -(C=O)NR 23 R 24 is selected from; or X is -(C=O)- and Y is NR 25 R26 or X is -NR 37 R 38 and Y is absent, R 20 is hydrogen or (C1-C4) alkyl, R 21 R 22 R 23 R 24 R 25 and R 26 are each independently selected from the group consisting of hydrogen, (C1-C8) alkyl, (C1-C8) alkoxy, and (C3-C6) cycloalkyl, and any (C1-C8) alkyl, (C1-C8) alkoxy, and (C3-C6) cycloalkyl are optionally substituted with one or more groups independently selected from the group consisting of halo, (C1-C4) alkyl, and (C1-C4) alkoxy, R 27 is -OH, -NR 25 R 26 or -F, R 28 is -OH, -NR 25 R 26 or -F, R 29 is -OH, -NR 25 R 26 -F, -N3, -NR 35 R 36or a 5-membered heterocyclic ring optionally substituted with one or more groups independently selected from the group consisting of halo, hydroxyl, carboxyl, amino, (C1-C4)alkyl, aryl and (C1-C4)alkoxy, wherein any (C1-C4)alkyl and (C1-C4)alkoxy are optionally substituted with one or more groups independently selected from the group consisting of halo, and any aryl is optionally substituted with one or more groups independently selected from the group consisting of halo, hydroxyl, nitro, cyano, amino, (C1-C8)alkyl, (C1-C8)alkoxy, (C1-C8)alkanoyl, (C1-C8)alkoxycarbonyl, (C1-C8)alkanoyloxy and (C3-C6)cycloalkyl, and any (C1-C8)alkyl, (C1-C8)alkoxy, (C1-C8)alkanoyl, (C1-C8)alkoxycarbonyl, (C1-C8)alkanoyloxy and (C3-C6)cycloalkyl are optionally substituted with one or more groups independently selected from the group consisting of halo, (C1-C4)alkyl and (C1-C4)alkoxy, each R 35 and R 36 are independently selected from the group consisting of hydrogen, (C1-C8)alkyl, (C1-C8)alkoxy and (C3-C6)cycloalkyl, wherein any (C1-C8)alkyl, (C1-C8)alkoxy and (C3-C6)cycloalkyl are optionally substituted with one or more groups independently selected from the group consisting of halo and (C1-C4)alkoxy; or R 35 and R 36 together with the nitrogen to which they are attached form a 5- to 6-membered heteroaryl ring, said heteroaryl ring being optionally substituted with one or more groups independently selected from the group consisting of (C1-C8)alkyl, (C1-C8)alkoxy, aryl and (C3-C6)cycloalkyl, and any aryl and (C3-C6)cycloalkyl being optionally substituted with one or more groups R 39 ; each R 37 and R 38is 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, and any (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 R 37 and R 38 together with the nitrogen to which they are attached, optionally form a 5- to 8-membered heterocyclic ring optionally substituted with one or more groups independently selected from the group consisting of halo, hydroxyl, carboxyl, amino, oxo (=O), (C1-C4) alkyl and (C1-C4) alkoxy, and any (C1-C4) alkyl and (C1-C4) alkoxy are optionally substituted with one or more groups independently selected from halo, each R 39 is independently selected from the group consisting of (C1-C8) alkyl, (C1-C8) alkoxy and (C3-C6) cycloalkyl, and any (C1-C8) alkyl, (C1-C8) alkoxy and (C3-C6) cycloalkyl are optionally substituted with one or more groups independently selected from halo] is a conjugate or salt according to any of embodiments 1 to 17.

[0241] [Embodiment 19] R 1 is [Chemical formula] is a conjugate or salt according to any of embodiments 1 to 17.

[0242] [Embodiment 20] R 1 is [Chemical formula] The conjugate or salt according to any one of Embodiments 1 to 17.

[0243] [Embodiment 21] R 1 is [Chemical formula] The conjugate or salt according to any one of Embodiments 1 to 17.

[0244] [Embodiment 22] R 1 is [Chemical formula] The conjugate or salt according to any one of Embodiments 1 to 15.

[0245] [Embodiment 23] R 2 is [Chemical formula] [In the formula, X is NR 20 and Y is -(C=O)R 21 , -SO2R 22 , and -(C=O)NR 23 R 24 selected from; or X is -(C=O)- and Y is NR 25 R 26 ; or X is -NR 37 R 38 and Y is absent, R 20 is hydrogen or (C1-C4) alkyl, R 21 R 22 R 23 R 24 R 25 and R 26Each is independently selected from the group consisting of hydrogen, (C1-C8) alkyl, (C1-C8) alkoxy, and (C3-C6) cycloalkyl, and any (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, (C1-C4) alkyl, and (C1-C4) alkoxy, R 27 is -OH, -NR 25 R 26 or -F, R 28 is -OH, -NR 25 R 26 or -F, R 29 is -OH, -NR 25 R 26 -F, -N3, -NR 35 R 36 or is a 5-membered heterocyclic ring optionally substituted with one or more groups independently selected from the group consisting of halo, hydroxyl, carboxyl, amino, (C1-C4) alkyl, aryl, and (C1-C4) alkoxy, where any (C1-C4) alkyl and (C1-C4) alkoxy are optionally substituted with one or more groups independently selected from the group consisting of halo, any aryl is optionally substituted with one or more groups independently selected from the group consisting of halo, hydroxyl, nitro, cyano, amino, (C1-C8) alkyl, (C1-C8) alkoxy, (C1-C8) alkanoyl, (C1-C8) alkoxycarbonyl, (C1-C8) alkanoyloxy, and (C3-C6) cycloalkyl, and any (C1-C8) alkyl, (C1-C8) alkoxy, (C1-C8) alkanoyl, (C1-C8) alkoxycarbonyl, (C1-C8) alkanoyloxy, and (C3-C6) cycloalkyl are optionally substituted with one or more groups independently selected from the group consisting of halo, (C1-C4) alkyl, and (C1-C4) alkoxy, Each R 35 and R 36is independently selected from the group consisting of hydrogen, (C1-C8) alkyl, (C1-C8) alkoxy, and (C3-C6) cycloalkyl, and any (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 R 35 and R 36 together with the nitrogen to which they are attached form a 5- to 6-membered heteroaryl ring, and the heteroaryl ring is optionally substituted with one or more groups independently selected from the group consisting of (C1-C8) alkyl, (C1-C8) alkoxy, aryl, and (C3-C6) cycloalkyl, and any aryl and (C3-C6) cycloalkyl are optionally substituted with one or more groups R 39 ; and each R 37 and R 38 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, and any (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 R 37 and R 38 together with the nitrogen to which they are attached form a 5- to 8-membered heterocyclic ring optionally substituted with one or more groups independently selected from the group consisting of halo, hydroxyl, carboxyl, amino, oxo (=O), (C1-C4) alkyl, and (C1-C4) alkoxy, and any (C1-C4) alkyl and (C1-C4) alkoxy are optionally substituted with one or more groups independently selected from halo, each R 39is independently selected from the group consisting of (C1-C8) alkyl, (C1-C8) alkoxy and (C3-C6) cycloalkyl, and any (C1-C8) alkyl, (C1-C8) alkoxy and (C3-C6) cycloalkyl is optionally substituted with one or more groups independently selected from halo] The conjugate or salt according to any one of Embodiments 1 to 22, which is as defined above.

[0246] [Embodiment 24] R 2 is [Chemical formula] The conjugate or salt according to any one of Embodiments 1 to 22, which is as defined above.

[0247] [Embodiment 25] R 2 is [Chemical formula] The conjugate or salt according to any one of Embodiments 1 to 22, which is as defined above.

[0248] [Embodiment 26] R 2 is [Chemical formula] The conjugate or salt according to any one of Embodiments 1 to 22, which is as defined above.

[0249] [Embodiment 27] R 2 is [Chemical formula] The conjugate or salt according to any one of Embodiments 1 to 22, which is as defined above.

[0250] [Embodiment 28] L 3is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 0 to 50 carbon atoms, and one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are optionally, -O-, -NR X -, -NR X -, -C(=O)-, -C(=O)-NR X -, or -S-, and R X is hydrogen or (C1-C6) alkyl, and the hydrocarbon chain is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from (C1-C6) alkoxy, (C3-C6) cycloalkyl, (C1-C6) alkanoyl, (C1-C6) alkanoyloxy, (C1-C6) alkoxycarbonyl, (C1-C6) alkylthio, azido, cyano, nitro, halo, hydroxy, oxo(=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy, the conjugate or salt according to any one of embodiments 1 to 27.

[0251] [Embodiment 29] L 3 is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 1 to 20 carbon atoms, and one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are optionally, -O-, -NR X -, -NR X -, -C(=O)-, -C(=O)-NR X -, or -S-, and R X is hydrogen or (C1-C6) alkyl, and the hydrocarbon chain is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from (C1-C6) alkoxy, (C3-C6) cycloalkyl, (C1-C6) alkanoyl, (C1-C6) alkanoyloxy, (C1-C6) alkoxycarbonyl, (C1-C6) alkylthio, azido, cyano, nitro, halo, hydroxy, oxo(=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy, the conjugate or salt according to any one of embodiments 1 to 27.

[0252] [Embodiment 30] L 3 is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 1 to 30 carbon atoms, and one or more of the carbon atoms are optionally replaced by -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X -, or -S-, and R X is hydrogen or (C1-C6) alkyl, and the hydrocarbon chain is optionally substituted with one or more halo or oxo(=O), the conjugate or salt according to any one of Embodiments 1 to 27.

[0253] [Embodiment 31] L 3 is [Chemical formula] and is the conjugate or salt according to any one of Embodiments 1 to 27.

[0254] [Embodiment 32] L 3 is linked to B by -NH-, -O-, -S-, -(C=O)-, -(C=O)-NH-, -NH-(C=O)-, -(C=O)-O-, -NH-(C=O)-NH-, or -NH-(SO2)-, the compound or salt according to any one of Embodiments 1 to 27.

[0255] [Embodiment 33] L 4 is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 0 to 50 carbon atoms, and one or more (for example, 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are optionally replaced by -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X -, or -S-, and R Xis hydrogen or (C1-C6) alkyl, and the hydrocarbon chain is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from (C1-C6) alkoxy, (C3-C6) cycloalkyl, (C1-C6) alkanoyl, (C1-C6) alkanoyloxy, (C1-C6) alkoxycarbonyl, (C1-C6) alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy, the compound or salt according to any one of Embodiments 1 to 32.

[0256] [Embodiment 34] L 4 is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 1 to 20 carbon atoms, and one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are optionally replaced by -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X -, or -S-, and R X is hydrogen or (C1-C6) alkyl, and the hydrocarbon chain is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from (C1-C6) alkoxy, (C3-C6) cycloalkyl, (C1-C6) alkanoyl, (C1-C6) alkanoyloxy, (C1-C6) alkoxycarbonyl, (C1-C6) alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy, the conjugate or salt according to any one of Embodiments 1 to 32.

[0257] [Embodiment 35] L 4 is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 1 to 30 carbon atoms, and one or more of the carbon atoms are optionally replaced by -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X- or is replaced by -S-, R X is hydrogen or (C1-C6) alkyl, and the hydrocarbon chain is optionally substituted with one or more halo or oxo (=O), the conjugate or salt according to any one of Embodiments 1 to 32.

[0258] [Embodiment 36] L 4 is linked to R by -O- 3 the conjugate or salt according to any one of Embodiments 1 to 32.

[0259] [Embodiment 37] the group: [Chemical formula] is [Chemical formula] [wherein each R' is independently C 1-9 alkyl, C 2-9 alkenyl or C 2-9 alkynyl, and the C 1-9 alkyl, C 2-9 alkenyl or C 2-9 alkynyl is optionally substituted with halo or hydroxyl] selected from the group consisting of, the conjugate or salt according to Embodiment 1 and any one of Embodiments 3 to 36.

[0260] [Embodiment 38] the group: [Chemical formula] is [Chemical formula] [wherein each R' is independently C 1-9 alkyl, C 2-9 alkenyl or C 2-9 alkynyl, and the C 1-9 alkyl, C 2-9An alkenyl or C 2-9 alkynyl is optionally substituted with halo or hydroxyl, * the valence indicated by is L 3 is attached to, ** the valence indicated by is R 3 is attached to] A conjugate or salt according to any one of Embodiment 1 and Embodiments 3 to 36, selected from the group consisting of.

[0261] [Embodiment 39] Said group:

Chemical formula

Chemical formula

[0262] [Embodiment 40]

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0263] [Embodiment 41] A pharmaceutical composition comprising a conjugate according to any one of Embodiments 1 to 40 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0264] [Embodiment 42] The compound of formula (Ia): [Chemical formula] [wherein, R 1 is a sugar, L 1 is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 0 to 20 carbon atoms, and one or more of the carbon atoms in the hydrocarbon chain are optionally -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X -, or -S-, and R X is hydrogen or (C1-C6) alkyl, and the hydrocarbon chain is optionally substituted with one or more substituents selected from oxo (=O) and halo, B is a 5- to 10-membered aryl or 5- to 10-membered heteroaryl, and the 5- to 10-membered aryl or 5- to 10-membered heteroaryl is independently selected from 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. Optionally substituted with one or more groups selected from the group consisting of, L 2 is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 0 to 20 carbon atoms, and one or more of the carbon atoms in the hydrocarbon chain are optionally -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X -, or -S-, and R X is hydrogen or (C1-C6) alkyl, and the hydrocarbon chain is optionally substituted with one or more substituents selected from oxo (=O) and halo, R 2 is a sugar, L3 is absent or is a linking group, A is absent, C3-20 cycloalkyl, C5-20 aryl, C5-20 heteroaryl, or C3-20 heterocycloalkyl, each R A is independently selected from the group consisting of hydrogen, hydroxy, CN, F, Cl, Br, I, -C 1-2 alkyl-OR a , C 1-10 alkyl C 2-10 alkenyl, and C 2-10 alkynyl, and said C 1-10 alkyl C 2-10 alkenyl, and C 2-10 alkynyl are optionally substituted with one or more groups independently selected from halo, hydroxy and C 1-3 alkoxy, n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, L 4 is absent or is a linking group, R 3a is H, a protecting group, a synthetic activating group, a covalent bond with a solid support, or a bond with a linking group attached to a solid support, R a is hydrogen, a protecting group, a covalent bond with a solid support, or a bond with a linking group L 5 attached to a solid support, L 5 is a linking group], or a salt thereof.

[0265] [Embodiment 43] The compound according to Embodiment 42, wherein A is absent.

[0266] [Embodiment 44] The compound according to Embodiment 42, wherein A is C3-20 cycloalkyl, C5-20 aryl, C5-20 heteroaryl, or C3-20 heterocycloalkyl.

[0267] [Embodiment 45] R 3aThe compound according to any one of Embodiments 42 to 44, wherein it is H.

[0268] [Embodiment 46] R 3a The compound according to any one of Embodiments 42 to 44, wherein it is a protecting group.

[0269] [Embodiment 47] The compound according to Embodiment 46, wherein the protecting group is acetate, triflate, mesylate or succinate.

[0270] [Embodiment 48] R 3a The compound according to any one of Embodiments 42 to 44, wherein it is a synthetic activating group.

[0271] [Embodiment 49] The compound according to Embodiment 48, wherein the synthetic activating group can be derived from DCC, HOBt, EDC, BOP, PyBOP or HBTU.

[0272] [Embodiment 50] R 3a The compound according to any one of Embodiments 42 to 44, wherein it is a covalent bond with a solid support.

[0273] [Embodiment 51] R 3a The compound according to any one of Embodiments 42 to 44, wherein it is a bond with a linking group attached to the solid support.

[0274] [Embodiment 52] The compound according to Embodiment 51, wherein the linking group attached to the solid support is -C(=O)CH2CH2C(=O)N(H)-.

[0275] [Embodiment 53] [Chemical formula] [Chemical formula] [Chem.] [Chem.] [wherein, Pg is a protecting group] a compound selected from the group consisting of, or a salt thereof.

Claims

1. The conjugate of formula (I): 【Chemical 1】 〔wherein, R １ is a sugar, L １ is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having from 0 to 20 carbon atoms, one or more of said carbon atoms in said hydrocarbon chain being optionally replaced by —O—, —NR Ｘ —, —NR Ｘ —C(═O)—, —C(═O)—NR Ｘ —, or —S—, R Ｘ is hydrogen or (C １ —C ６ )alkyl, and said hydrocarbon chain is optionally substituted with one or more substituents selected from oxo(═O) and halo, B is a 5- to 10-membered aryl or 5- to 10-membered heteroaryl, and the 5- to 10-membered aryl or 5- to 10-membered heteroaryl is halo, hydroxy, cyano, trifluoromethyl, trifluoromethoxy, (C １ -C ６ )alkyl, (C １ -C ６ )alkoxy, (C １ -C ６ )alkoxycarbonyl, (C １ -C ６ )alkanoyloxy, (C ３ -C ６ )cycloalkyl, and (C ３ -C ６ )cycloalkyl(C １ -C ６ )alkyl, optionally substituted with one or more groups independently selected from the group consisting of: L ２ is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 0 to 20 carbon atoms, and one or more of the carbon atoms in the hydrocarbon chain are optionally replaced by -O-, -NR Ｘ -, -NR Ｘ -, -C(=O)-, -C(=O)-NR Ｘ -, or -S-, and R Ｘ is hydrogen or (C １ -C ６ )alkyl, and the hydrocarbon chain is optionally substituted with one or more substituents selected from oxo(=O) and halo. R ２ is a sugar, L ３ is non-existent or a linking group, A is absent, C3-20 cycloalkyl, C5-20 aryl, C5-20 heteroaryl, or C3-20 heterocycloalkyl, Each R Ａ is independently selected from the group consisting of hydrogen, hydroxy, CN, F, Cl, Br, I, -C １－２ alkyl - OR ａ , C １－１０ alkyl C ２－１０ alkenyl, and C ２－１０ alkynyl, and said C １－１０ alkyl C ２－１０ alkenyl, and C ２－１０ alkynyl are optionally substituted with one or more groups independently selected from halo, hydroxy and C １－３ alkoxy, n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, L ４ is non-existent or a linking group, and R ３ is a nucleic acid, R ａ is a covalent bond with hydrogen, a protecting group, a solid support, or a linking group L attached to the solid support ５ and the bond therewith L ５ is a linking group), or a salt thereof.

2. The conjugate or salt according to Claim 1, wherein A is absent.

3. The conjugate or salt according to Claim 1, wherein A is C3-20 cycloalkyl, C5-20 aryl, C5-20 heteroaryl, or C3-20 heterocycloalkyl.

4. The conjugate or salt according to any one of Claims 1 to 3, wherein B is C5-10 aryl.

5. The conjugate or salt according to any one of Claims 1 to 3, wherein B is naphthyl or phenyl.

6. The conjugate or salt according to any one of Claims 1 to 3, wherein B is phenyl.

7. The group: 【Chemical 2】 is 【Chemical Formula 3】 The conjugate or salt according to any one of Claims 1 to 3.

8. The conjugate or salt according to any one of Claims 1 to 3, wherein B is C5-10 heteroaryl.

9. The conjugate or salt according to any one of Claims 1 to 3, wherein B is pyridyl, pyrimidyl, quinolyl, isoquinolyl, imidazolyl, thiazolyl, dioxazolyl or oxazolyl.

10. The group: [Chemical Formula 4] is [Chemical Formula 5] The conjugate or salt according to any one of Claims 1 to 3.

11. The group: ​ is 【Chemical Formula 7】 The conjugate or salt according to any one of Claims 1 to 3.

12. L １ is a divalent unbranched saturated hydrocarbon chain having 0 to 20 carbon atoms, and one or more (for example, 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are optionally, -O-, -NR Ｘ -, -NR Ｘ -, -C(=O)-, -C(=O)-NR Ｘ -, or -S-, and R Ｘ is hydrogen or (C １ -C ６ )alkyl, and the hydrocarbon chain is optionally substituted with one or more substituents selected from oxo(=O) and halo, the conjugate or salt according to any one of claims 1 to 11.

13. L １ is a divalent unbranched saturated hydrocarbon chain having 0 to 12 carbon atoms, and one or more (for example, 1, 2, 3 or 4) of the carbon atoms in the hydrocarbon chain are optionally, -O-, -NR Ｘ -, -C(=O)-, or -C(=O)-NR Ｘ -, and R Ｘ is hydrogen or (C １ -C ６ )alkyl, the conjugate or salt according to any one of claims 1 to 11.

14. L １ is -C(=O)N(H)-CH ２ CH ２ OCH ２ CH ２ OCH ２ CH ２ -, -C(=O)N(H)-CH ２ CH ２ OCH ２ CH ２ OCH ２ CH ２ OCH ２ CH ２ -, -C(=O)N(CH ３ )-CH ２ CH ２ OCH ２ CH ２ OCH ２ CH ２ -, or -C(=O)N(CH ３ )-CH ２ CH ２ OCH ２ CH ２ OCH ２ CH ２ OCH ２ CH ２ -[[]]END]] is the conjugate or salt according to any one of Claims 1 to 11.

15. L ２ is a divalent unbranched saturated hydrocarbon chain having 0 to 20 carbon atoms, and one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are optionally, -O-, -NR Ｘ -, -NR Ｘ -, -C(=O)-, -C(=O)-NR Ｘ -, or -S-, and R Ｘ is hydrogen or (C １ -C ６ )alkyl, and the hydrocarbon chain is optionally substituted with one or more substituents selected from oxo(=O) and halo, the conjugate or salt according to any one of claims 1 to 14.

16. L ２ is a divalent unbranched saturated hydrocarbon chain having 0 to 12 carbon atoms, and one or more (for example, 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are optionally replaced by -O-, -NR Ｘ -, -C(=O)-, or -C(=O)-NR Ｘ -, and R Ｘ is hydrogen or (C １ -C ６ )alkyl, the conjugate or salt according to any one of claims 1 to 14.

17. L ２ is -C(=O)N(H)-CH ２ CH ２ OCH ２ CH ２ OCH ２ CH ２ -, -C(=O)N(H)-CH ２ CH ２ OCH ２ CH ２ OCH ２ CH ２ OCH ２ CH ２ -, -C(=O)N(CH ３ )-CH ２ CH ２ OCH ２ CH ２ OCH ２ CH ２ -, or -C(=O)N(CH ３ )-CH ２ CH ２ OCH ２ CH ２ OCH ２ CH ２ OCH ２ CH ２ -[[]]END]] is the conjugate or salt according to any one of Claims 1 to 14.

18. R １ is [Chemical Formula 8] 〔wherein, X is NR ２０ and Y is -(C=O)R ２１ , -SO ２ R ２２ , and -(C=O)NR ２３ R ２４ selected from; or X is -(C=O)- and Y is NR ２５ R ２６ ; or X is -NR ３７ R ３８ and Y is absent R ２０ is hydrogen or (C １ -C ４ ) alkyl, and R ２１ 、R ２２ 、R ２３ 、R ２４ 、R ２５ and R ２６ each independently is selected from the group consisting of hydrogen, (C １ -C ８ alkyl, (C １ -C ８ alkoxy and (C ３ -C ６ cycloalkyl, and any (C １ -C ８ alkyl, (C １ -C ８ alkoxy and (C ３ -C ６ cycloalkyl is optionally substituted with one or more groups independently selected from the group consisting of halo, (C １ -C ４ alkyl and (C １ -C ４ alkoxy). R ２７ is —OH, —NR ２５ R ２６ , or —F, and R ２８ is -OH, -NR ２５ R ２６ , or -F, and R ２９ is a 5-membered heterocyclic ring optionally substituted with one or more groups independently selected from the group consisting of -OH, -NR ２５ R ２６ , -F, -N ３ , -NR ３５ R ３６ , or halo, hydroxyl, carboxyl, amino, (C １ -C ４ )alkyl, aryl and (C １ -C ４ )alkoxy, and any (C １ -C ４ )alkyl and (C １ -C ４ )alkoxy are optionally substituted with one or more groups independently selected from the group consisting of halo, and any aryl is optionally substituted with one or more groups independently selected from the group consisting of halo, hydroxyl, nitro, cyano, amino, (C １ -C ８ )alkyl, (C １ -C ８ )alkoxy, (C １ -C ８ )alkanoyl, (C １ -C ８ )alkoxycarbonyl, (C １ -C ８ )alkanoyloxy and (C ３ -C ６ )cycloalkyl, and any (C １ -C ８ )alkyl, (C １ -C ８ )alkoxy, (C １ -C ８ )alkanoyl, (C １ -C ８ )alkoxycarbonyl, (C １ -C ８ )alkanoyloxy and (C ３ -C ６ )cycloalkyl are optionally substituted with halo, (C １ -C ４ )alkyl and (C １ -C ４ optionally substituted with one or more groups independently selected from the group consisting of alkoxy; Each R ３５ and R ３６ is independently selected from the group consisting of hydrogen, (C １ -C ８ ), alkyl, (C １ -C ８ ), alkoxy and (C ３ -C ６ ), cycloalkyl, and any (C １ -C ８ ), alkyl, (C １ -C ８ ), alkoxy and (C ３ -C ６ ), cycloalkyl is optionally substituted with one or more groups independently selected from the group consisting of halo and (C １ -C ４ ), alkoxy; or R ３５ and R ３６ together with the nitrogen to which they are attached form a 5- or 6-membered heteroaryl ring, said heteroaryl ring being optionally substituted with one or more groups independently selected from the group consisting of (C １ -C ８ ), alkyl, (C １ -C ８ ), alkoxy, aryl and (C ３ -C ６ ), cycloalkyl, and any aryl and (C ３ -C ６ ), cycloalkyl is optionally substituted with one or more groups R ３９ ; Each R ３７ and R ３８ are independently selected from the group consisting of hydrogen, (C １ -C ８ ), alkyl, (C １ -C ８ ), alkoxy, (C １ -C ８ ), alkanoyl, (C １ -C ８ ), alkoxycarbonyl, (C １ -C ８ ), alkanoyloxy and (C ３ -C ６ ), cycloalkyl, and any (C １ -C ８ ), alkyl, (C １ -C ８ ), alkoxy, (C １ -C ８ ), alkanoyl, (C １ -C ８ ), alkoxycarbonyl, (C １ -C ８ ), alkanoyloxy and (C ３ -C ６ ), cycloalkyl is optionally substituted with one or more groups independently selected from the group consisting of halo, (C １ -C ４ ), alkyl and (C １ -C ４ ), alkoxy; or R ３７ and R ３８ together with the nitrogen to which they are attached form a 5- to 8-membered heterocyclic ring optionally substituted with one or more groups independently selected from the group consisting of halo, hydroxyl, carboxyl, amino, oxo (=O), (C １ -C ４ ), alkyl and (C １ -C ４ ), alkoxy, and any (C １ -C ４ ), alkyl and (C １ -C ４ ), alkoxy is optionally substituted with one or more groups independently selected from halo, Each R ３９ are independently １ -C ８ ) alkyl, (C １ -C ８ ) alkoxy and (C ３ -C ６ ) cycloalkyl; any (C １ -C ８ ) alkyl, (C １ -C ８ ) alkoxy and (C ３ -C ６ ) cycloalkyl is optionally substituted with one or more groups independently selected from halo. is the conjugate or salt according to any one of Claims 1 to 17.

19. R １ is 【Chemical Formula 9】 is the conjugate or salt according to any one of Claims 1 to 17.

20. R １ is 【Chemical Formula 10】 is the conjugate or salt according to any one of Claims 1 to 17.

21. R １ is 【Chemical Formula 11】 is the conjugate or salt according to any one of Claims 1 to 17.

22. R １ is 【Chemical 12】 is the conjugate or salt according to any one of Claims 1 to 15.

23. R ２ is 【Chemical 13】 〔wherein, X is NR ２０ and Y is -(C=O)R ２１ , -SO ２ R ２２ , and -(C=O)NR ２３ R ２４ selected from; or X is -(C=O)- and Y is NR ２５ R ２６ ; or X is -NR ３７ R ３８ and Y is absent R ２０ is hydrogen or (C １ -C ４ ) alkyl, and R ２１ 、 R ２２ 、 R ２３ 、 R ２４ 、 R ２５ and R ２６ are each independently selected from the group consisting of hydrogen, (C １ -C ８ ), alkyl, (C １ -C ８ ), alkoxy and (C ３ -C ６ ), cycloalkyl, and any (C １ -C ８ ), alkyl, (C １ -C ８ ), alkoxy and (C ３ -C ６ ), cycloalkyl is optionally substituted with one or more groups independently selected from the group consisting of halo, (C １ -C ４ ), alkyl and (C １ -C ４ ), alkoxy, R ２７ is -OH, -NR ２５ R ２６ , or -F, and R ２８ is -OH, -NR ２５ R ２６ , or -F, and R ２９ is a 5-membered heterocyclic ring optionally substituted with one or more groups independently selected from the group consisting of -OH, -NR ２５ R ２６ , -F, -N ３ , -NR ３５ R ３６ , or halo, hydroxyl, carboxyl, amino, (C １ -C ４ )alkyl, aryl and (C １ -C ４ )alkoxy, and any (C １ -C ４ )alkyl and (C １ -C ４ )alkoxy are optionally substituted with one or more groups independently selected from the group consisting of halo, and any aryl is optionally substituted with one or more groups independently selected from the group consisting of halo, hydroxyl, nitro, cyano, amino, (C １ -C ８ )alkyl, (C １ -C ８ )alkoxy, (C １ -C ８ )alkanoyl, (C １ -C ８ )alkoxycarbonyl, (C １ -C ８ )alkanoyloxy, and (C ３ -C ６ )cycloalkyl, and any (C １ -C ８ )alkyl, (C １ -C ８ )alkoxy, (C １ -C ８ )alkanoyl, (C １ -C ８ )alkoxycarbonyl, (C １ -C ８ )alkanoyloxy, and (C ３ -C ６ )cycloalkyl are optionally substituted with halo, (C １ -C ４ )alkyl and (C １ -C ４ optionally substituted with one or more groups independently selected from the group consisting of alkoxy; Each R ３５ and R ３６ are independently selected from the group consisting of hydrogen, (C １ -C ８ ), alkyl, (C １ -C ８ ), alkoxy and (C ３ -C ６ ), cycloalkyl, and any (C １ -C ８ ), alkyl, (C １ -C ８ ), alkoxy and (C ３ -C ６ ), cycloalkyl is optionally substituted with one or more groups independently selected from the group consisting of halo and (C １ -C ４ ), alkoxy; or R ３５ and R ３６ together with the nitrogen to which they are attached form a 5- to 6-membered heteroaryl ring, and the heteroaryl ring is (C １ -C ８ ), alkyl, (C １ -C ８ ), alkoxy, aryl and (C ３ -C ６ ), cycloalkyl, and any aryl and (C ３ -C ６ ), cycloalkyl is optionally substituted with one or more groups R ３９ ; Each R ３７ and R ３８ is independently selected from the group consisting of hydrogen, (C １ -C ８ ), alkyl, (C １ -C ８ ), alkoxy, (C １ -C ８ ), alkanoyl, (C １ -C ８ ), alkoxycarbonyl, (C １ -C ８ ), alkanoyloxy and (C ３ -C ６ ), cycloalkyl, and any (C １ -C ８ ), alkyl, (C １ -C ８ ), alkoxy, (C １ -C ８ ), alkanoyl, (C １ -C ８ ), alkoxycarbonyl, (C １ -C ８ ), alkanoyloxy, and (C ３ -C ６ ), cycloalkyl is optionally substituted with one or more groups independently selected from the group consisting of halo, (C １ -C ４ ), alkyl and (C １ -C ４ ), alkoxy; or R ３７ and R ３８ together with the nitrogen to which they are attached form a 5- to 8-membered heterocyclic ring optionally substituted with one or more groups independently selected from the group consisting of halo, hydroxyl, carboxyl, amino, oxo (=O), (C １ -C ４ ), alkyl and (C １ -C ４ ), alkoxy, and any (C １ -C ４ ), alkyl and (C １ -C ４ ), alkoxy is optionally substituted with one or more groups independently selected from halo, Each R ３９ is independently selected from the group consisting of (C １ -C ８ )alkyl, (C １ )alkoxy and (C ８ )cycloalkyl, and any (C ３ )alkyl, (C ６ )alkoxy and (C １ )alkyl, (C ８ )alkoxy and (C １ )alkyl, (C ８ )alkoxy and (C ３ )alkyl, (C ６ )cycloalkyl is optionally substituted with one or more groups independently selected from halo) is the conjugate or salt according to any one of Claims 1 to 22.

24. R ２ is 【Chemical 14】 The conjugate or salt according to any one of claims 1 to 22.

25. R ２ is 【Chemical Formula 15】 The conjugate or salt according to any one of claims 1 to 22.

26. R ２ is 【Chemical 16】 The conjugate or salt according to any one of claims 1 to 22.

27. R ２ is 【Chemical 17】 The conjugate or salt according to any one of claims 1 to 22.

28. L ３ is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 0 to 50 carbon atoms, and one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are optionally replaced by -O-, -NR Ｘ -, -NR Ｘ -, -C(=O)-, -C(=O)-NR Ｘ -, or -S-, and R Ｘ is hydrogen or (C １ -C ６ )alkyl, and the hydrocarbon chain is optionally substituted by one or more (e.g., 1, 2, 3, or 4) substituents selected from (C １ -C ６ )alkoxy, (C ３ -C ６ )cycloalkyl, (C １ -C ６ )alkanoyl, (C １ -C ６ )alkanoyloxy, (C １ -C ６ )alkoxycarbonyl, (C １ -C ６ )alkylthio, azide, cyano, nitro, halo, hydroxy, oxo(=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy. The conjugate or salt according to any one of claims 1 to 27, which is optionally substituted.​

29. L ３ is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 1 to 20 carbon atoms, and one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are optionally, -O-, -NR Ｘ -, -NR Ｘ -, -C(=O)-, -C(=O)-NR Ｘ -, or -S-, and R Ｘ is hydrogen or (C １ -C ６ )alkyl, and the hydrocarbon chain is (C １ -C ６ )alkoxy, (C ３ -C ６ )cycloalkyl, (C １ -C ６ )alkanoyl, (C １ -C ６ )alkanoyloxy, (C １ -C ６ )alkoxycarbonyl, (C １ -C ６ )alkylthio, azide, cyano, nitro, halo, hydroxy, oxo(=O), carboxy, aryl, aryloxy, heteroaryl, and one or more (e.g., 1, 2, 3, or 4) substituents selected from heteroaryloxy, optionally substituted, the conjugate or salt according to any one of claims 1 to 27.

30. L ３ is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 1 to 30 carbon atoms, one or more of said carbon atoms being optionally replaced by -O-, -NR Ｘ -, -NR Ｘ -, -C(=O)-, -C(=O)-NR Ｘ -, or -S-, and R Ｘ is hydrogen or (C １ -C ６ )alkyl, and said hydrocarbon chain is optionally substituted with one or more halo or oxo(=O), the conjugate or salt according to any one of claims 1 to 27.

31. L ３ is 【Chemical Formula 18】 The conjugate or salt according to any one of claims 1 to 27.

32. L ３ is linked to B by -NH-, -O-, -S-, -(C=O)-, -(C=O)-NH-, -NH-(C=O)-, -(C=O)-O-, -NH-(C=O)-NH-, or -NH-(SO ２ )-, the compound or salt according to any one of claims 1 to 27.

33. L ４ is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 0 to 50 carbon atoms, and one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are optionally, -O-, -NR Ｘ -, -NR Ｘ -, -C(=O)-, -C(=O)-NR Ｘ -, or -S-, and R Ｘ is hydrogen or (C １ -C ６ )alkyl, and the hydrocarbon chain is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from (C １ -C ６ )alkoxy, (C ３ -C ６ )cycloalkyl, (C １ -C ６ )alkanoyl, (C １ -C ６ )alkanoyloxy, (C １ -C ６ )alkoxycarbonyl, (C １ -C ６ )alkylthio, azide, cyano, nitro, halo, hydroxy, oxo(=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy. The compound or salt according to any one of claims 1 to 32.

34. L ４ is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 1 to 20 carbon atoms, and one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are optionally replaced by -O-, -NR Ｘ -, -NR Ｘ -, -C(=O)-, -C(=O)-NR Ｘ -, or -S-, and R Ｘ is hydrogen or (C １ -C ６ )alkyl, and the hydrocarbon chain is optionally substituted by one or more (e.g., 1, 2, 3, or 4) substituents selected from (C １ -C ６ )alkoxy, (C ３ -C ６ )cycloalkyl, (C １ -C ６ )alkanoyl, (C １ -C ６ )alkanoyloxy, (C １ -C ６ )alkoxycarbonyl, (C １ -C ６ )alkylthio, azido, cyano, nitro, halo, hydroxy, oxo(=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy. The conjugate or salt according to any one of claims 1 to 32.

35. L ４ is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 1 to 30 carbon atoms, one or more of said carbon atoms being optionally replaced by -O-, -NR Ｘ -, -NR Ｘ -, -C(=O)-, -C(=O)-NR Ｘ -, or -S-, and R Ｘ is hydrogen or (C １ -C ６ )alkyl, and said hydrocarbon chain is optionally substituted with one or more halo or oxo(=O); the conjugate or salt according to any one of claims 1 to 32.

36. L ４ is linked to R by -O- ３ The conjugate or salt according to any one of claims 1 to 32, wherein the L is linked to R by -O-.

37. The group: 【Chemical Formula 19】 is 【Chemical 20】 [wherein each R' is independently C １－９ alkyl, C ２－９ alkenyl or C ２－９ alkynyl, and the C １－９ alkyl, C ２－９ alkenyl or C ２－９ alkynyl is optionally substituted with halo or hydroxyl)] The conjugate or salt according to any one of claims 1 and 3 to 36, which is selected from the group consisting of.

38. The group: 【Chemical 21】 is 【Chemical 22】 〔In the formula, Each R' is independently C １－９ alkyl, C ２－９ alkenyl or C ２－９ alkynyl, and said C １－９ alkyl, C ２－９ alkenyl or C ２－９ alkynyl is optionally substituted with halo or hydroxyl, ＊ The valence indicated by is L ３ is attached to , ＊＊ The valence indicated by is R ３ attached to . The conjugate or salt according to any one of claims 1 and 3 to 36, which is selected from the group consisting of.

39. The group: 【Chemical 23】 is 【Chemical Formula 24】 The conjugate or salt according to any one of claims 1 and 3 to 36, which is.

40. 【Fig. 25】 【Chemical 26】 【Chemical 27】 【Chemical Formula 28】 ​ 〔wherein, R ３ is a nucleic acid〕 The conjugate selected from the group consisting of, or a salt thereof.

41. A pharmaceutical composition comprising the conjugate according to any one of claims 1 to 40 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

42. The compound of formula (Ia): 【Chemical Formula 30】 〔In the formula, R １ is a sugar, L １ is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 0 to 20 carbon atoms, and one or more of the carbon atoms in the hydrocarbon chain are optionally replaced by -O-, -NR Ｘ -, -NR Ｘ -, -C(=O)-, -C(=O)-NR Ｘ -, or -S-, and R Ｘ is hydrogen or (C １ -C ６ ) alkyl, and the hydrocarbon chain is optionally substituted with one or more substituents selected from oxo (=O) and halo. B is a 5- to 10-membered aryl or 5- to 10-membered heteroaryl, and the 5- to 10-membered aryl or 5- to 10-membered heteroaryl is halo, hydroxy, cyano, trifluoromethyl, trifluoromethoxy, (C １ -C ６ )alkyl, (C １ -C ６ )alkoxy, (C １ -C ６ )alkoxycarbonyl, (C １ -C ６ )alkanoyloxy, (C ３ -C ６ )cycloalkyl, and (C ３ -C ６ )cycloalkyl(C １ -C ６ )alkyl, optionally substituted with one or more groups independently selected from the group consisting of: L ２ is a divalent branched or unbranched saturated or unsaturated hydrocarbon chain having 0 to 20 carbon atoms, and one or more of the carbon atoms in the hydrocarbon chain are optionally replaced by -O-, -NR Ｘ -, -NR Ｘ -, -C(=O)-, -C(=O)-NR Ｘ -, or -S-, and R Ｘ is hydrogen or (C １ -C ６ )alkyl, and the hydrocarbon chain is optionally substituted with one or more substituents selected from oxo(=O) and halo. R ２ is a sugar, L ３ is non-existent or a linking group, and A is absent, 3- to 20-membered cycloalkyl, 5- to 20-membered aryl, 5- to 20-membered heteroaryl, or 3- to 20-membered heterocycloalkyl, Each R Ａ is independently selected from the group consisting of hydrogen, hydroxy, CN, F, Cl, Br, I, -C １－２ alkyl - OR ａ , C １－１０ alkyl C ２－１０ alkenyl, and C ２－１０ alkynyl, and said C １－１０ alkyl C ２－１０ alkenyl, and C ２－１０ alkynyl are optionally substituted with one or more groups independently selected from halo, hydroxy and C １－３ alkoxy, n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, L ４ is non-existent or a linking group, R ３ａ is a bond with H, a protecting group, a synthetic activating group, a covalent bond with a solid support, or a bond with a linking group attached to the solid support, R ａ is a covalent bond with hydrogen, a protecting group, a solid support, or a linking group L attached to the solid support ５ and the bond L ５ is a linking group〕 or a salt thereof.

43. The compound according to claim 42, wherein A is absent.

44. The compound according to claim 42, wherein A is 3- to 20-membered cycloalkyl, 5- to 20-membered aryl, 5- to 20-membered heteroaryl, or 3- to 20-membered heterocycloalkyl.

45. R ３ａ The compound according to any one of claims 42 to 44, wherein R is H.

46. R ３ａ The compound according to any one of claims 42 to 44, wherein R is a protecting group.

47. The compound according to claim 46, wherein the protecting group is acetate, triflate, mesylate or succinate.

48. R ３ａ The compound according to any one of claims 42 to 44, wherein R is a synthetic activating group.

49. The compound according to claim 48, wherein the synthetic activating group can be derived from DCC, HOBt, EDC, BOP, PyBOP or HBTU.

50. R ３ａ The compound according to any one of claims 42 to 44, wherein R is a covalent bond with a solid support.

51. R ３ａ The compound according to any one of claims 42 to 44, wherein the bond is a bond with a linking group bonded to a solid support.

52. The linking group bonded to the solid support is -C(=O)CH ２ CH ２ C(=O)N(H)-, and the compound according to claim 51.

53. 【Figure 31】 【Chemical 32】 【Chemical 33】 【Chemical Formula 34】 〔In the formula, Pg is a protecting group〕 The compound selected from the group consisting of, or a salt thereof.

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