Targeted compositions

Abstract

To provide nucleic acid molecules (e.g., therapeutic double stranded siRNA molecules), as well as compounds, compositions and methods that can be used to target such nucleic acids (e.g., to the liver).SOLUTION: The invention provides certain nucleic acids (e.g., double stranded siRNA molecules), as well as conjugates that comprise a targeting moiety, a double stranded siRNA and optional linking groups. Certain embodiments also provide synthetic methods useful for preparing the conjugates. The conjugates are useful to target therapeutic double stranded siRNA to the liver and to treat liver diseases including hepatitis (e.g., hepatitis B and hepatitis D). The invention also provides synthetic intermediates and methods disclosed herein that are useful to prepare compounds of formula I.SELECTED DRAWING: Figure 1

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims the benefit of priority to U.S. Patent Application No. 62 / 525,071, filed June 26, 2017, and U.S. Patent Application No. 62 / 484,247, filed April 11, 2017, which applications are incorporated herein by reference. [Background technology]

[0002] Some diseases, such as hepatitis B and nonalcoholic steatohepatitis (NASH), are liver-specific, and therefore it would be beneficial to have therapeutic compositions that can primarily target the liver, kidney, heart, pancreas, or other organs of a living subject.

[0003] Nucleic acids, including siRNAs, are useful as therapeutic agents.

[0004] Currently, there is a need for compositions and methods that can be used to deliver (eg, target) therapeutic nucleic acids, such as double-stranded siRNA, to living subjects. Summary of the Invention [Means for solving the problem]

[0005] The present invention provides nucleic acid molecules (eg, therapeutic double-stranded siRNA molecules), as well as compounds, compositions, and methods that can be used to target such nucleic acids (eg, to the liver).

[0006] Thus, in one aspect, the present invention provides siRNAs 1 (SEQ ID NOs: 1 and 2), 2 (SEQ ID NOs: 3 and 4), 3 (SEQ ID NOs: 5 and 6), 4 (SEQ ID NOs: 7 and 8), 5 (SEQ ID NOs: 9 and 10), 6 (SEQ ID NOs: 11 and 12), 7 (SEQ ID NOs: 13 and 14), 8 (SEQ ID NOs: 15 and 16), 9 (SEQ ID NOs: 17 and 18), 10 (SEQ ID NOs: 19 and 20), 11 (SEQ ID NOs: 21 and 22), 12 (SEQ ID NOs: 23 and 24), 13 (SEQ ID NOs: 25 and 26), 14 (SEQ ID NOs: 27 and 28), 15 (SEQ ID NOs: 29 and 30), 16 (SEQ ID NOs: 31 and 32), 17 (SEQ ID NOs: 33 and 34), 18 (SEQ ID NOs: 35 and 36), 19 (SEQ ID NOs: 37 and 38), 20 (SEQ ID NOs: 39 and 40), 21 (SEQ ID NOs: 31 and 32), 22 (SEQ ID NOs: 32 and 33), 23 (SEQ ID NOs: 33 and 34), 24 (SEQ ID NOs: 35 and 36), 25 (SEQ ID NOs: 37 and 38), 26 (SEQ ID NOs: 39 and 40), 27 (SEQ ID NOs: 39 and 41), 28 (SEQ ID NOs: 39 and 42), 29 (SEQ ID NOs: 4 The double-stranded siRNA molecules are selected from the group consisting of: 1 (SEQ ID NOs: 41 and 42), 22 (SEQ ID NOs: 43 and 44), 23 (SEQ ID NOs: 45 and 46), 24 (SEQ ID NOs: 47 and 48), 25 (SEQ ID NOs: 49 and 50), 26 (SEQ ID NOs: 51 and 52), 27 (SEQ ID NOs: 53 and 54), 28 (SEQ ID NOs: 55 and 56), 29 (SEQ ID NOs: 57 and 58), 30 (SEQ ID NOs: 59 and 60), 31 (SEQ ID NOs: 61 and 62), 32 (SEQ ID NOs: 63 and 64), 33 (SEQ ID NOs: 65 and 66), 34 (SEQ ID NOs: 67 and 68), 35 (SEQ ID NOs: 69 and 70), 36 (SEQ ID NOs: 71 and 72), and 37 (SEQ ID NOs: 73 and 74).

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

[0008] Another aspect of the present invention provides a GalNAc conjugate comprising one of the siRNAs described herein, which is not limited to conjugates comprising the ligand-linkers disclosed herein. For example, one aspect of the present invention provides a GalNAc conjugate of formula X: ABC (X) wherein A is a targeting ligand; B is an optional linker; C is an siRNA molecule described herein.

[0009] The therapeutic double-stranded siRNAs described herein, as well as compounds and compositions comprising such siRNAs, can be used to treat hepatitis B virus and hepatitis B virus / hepatitis D virus.

[0010] The present invention also provides synthetic intermediates and processes disclosed herein that are useful for preparing compounds of Formula I.

[0011] Other objects, features, and advantages of the present invention will become apparent to those skilled in the art from the following detailed description and drawings. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 illustrates an intermediate compound of formula Ie, where the targeting ligand / linker is attached to a solid support and Pg1 is the protecting group DMTr. [Figure 2] FIG. 1 illustrates a representative compound of Formula Id in which a targeting ligand is bound to a solid support and a nucleic acid is covalently attached. [Figure 3] FIG. 1 illustrates a representative compound of Formula Id in which the targeting ligand-nucleic acid conjugate has been cleaved from the solid support and deprotected to yield the compound of Formula I. DETAILED DESCRIPTION OF THE INVENTION

[0013] In this application, including the figures, examples, and schemes, it is understood that the oligonucleotides may be double-stranded siRNA molecules as described in Table 1.

[0014] As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0015] As used herein, the term "conjugate" includes a compound of formula (I) comprising an oligonucleotide (e.g., an siRNA molecule) bound to a targeting ligand. Thus, the terms compound and conjugate may be used interchangeably herein.

[0016] As used herein, the term "small interfering RNA" or "siRNA" refers to a double-stranded RNA (i.e., duplex RNA) that can reduce or inhibit expression of a target gene or sequence (e.g., by mediating degradation of or inhibiting translation of mRNA complementary to the siRNA sequence) when the siRNA is present in the same cell as the target gene or sequence. The siRNA may have substantial or complete identity to the target gene or sequence, or may contain regions of mismatch (i.e., mismatch motifs). In certain embodiments, the siRNA may be about 19-25 (duplex) nucleotides in length, preferably about 20-24, 21-22, or 21-23 (duplex) nucleotides in length. The siRNA duplex may include a 3' overhang of about 1 to about 4 nucleotides or about 2 to about 3 nucleotides, and a 5' phosphate terminus. Examples of siRNA include, but are not limited to, double-stranded polynucleotide molecules constructed from two separate strands, one of which is the sense strand and the other the complementary antisense strand.

[0017] In certain embodiments, the 5' and / or 3' overhangs of one or both strands of the siRNA comprise one to four (e.g., one, two, three, or four) modified and / or unmodified deoxythymidine (t or dT) nucleotides, one to four (e.g., one, two, three, or four) modified (e.g., 2'OMe) and / or unmodified uridine (U) ribonucleotides, and / or one to four (e.g., one, two, three, or four) modified (e.g., 2'OMe) and / or unmodified ribonucleotides or deoxyribonucleotides that are complementary to a target sequence (e.g., the 3' overhang of the antisense strand) or its complementary strand (e.g., the 3' overhang of the sense strand).

[0018] Preferably, the siRNA is chemically synthesized. siRNA can also be generated by cleaving longer dsRNA (e.g., dsRNA greater than about 25 nucleotides in length) with E. coli RNase III or Dicer. These enzymes process dsRNA into biologically active siRNAs (see, e.g., Yang et al., Proc. Natl. Acad. Sci. USA, 99:9942-9947 (2002); Calegari et al., Proc. Natl. Acad. Sci. USA, 99:14236 (2002); Byrom et al., Ambion TechNotes, 10(1):4-6 (2003); Kawasaki et al., Nucleic Acids Res., 31:981-987 (2003); Knight et al., Science, 293:2269-2271 (2001); and Robertson et al., J. Biol. Chem., 243:82 (1968)). Preferably, dsRNA is at least 50 nucleotides to about 100, 200, 300, 400, or 500 nucleotides in length. dsRNA may be as long as 1000, 1500, 2000, 5000 nucleotides, or longer. dsRNA can encode the entire or partial gene transcript. In certain cases, siRNA may be encoded by a plasmid (for example, transcribed as a sequence that automatically folds into a double-stranded chain with a hairpin loop).

[0019] The expression " inhibiting the expression of target gene " refers to the ability of the siRNA of the present invention to silence, reduce or inhibit the expression of target gene.To examine the degree of gene silencing, contact test sample (for example, the biological sample obtained from the target organism that expresses target gene, or the sample of cultured cells that express target gene) with siRNA that silences, reduces or inhibits the expression of target gene.The expression of target gene in this test sample is compared with the expression of target gene in control sample (for example, the biological sample obtained from the target organism that expresses target gene, or the sample of cultured cells that express target gene) that is not contacted with siRNA.Control sample (for example, the sample that expresses target gene) can be assigned a value of 100%. In certain embodiments, silencing, inhibition, or reduction of expression of a target gene is achieved when the value for the test sample relative to a control sample (e.g., buffer only, an siRNA sequence targeting a different gene, a scrambled siRNA sequence, etc.) is about 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. Suitable assays include, but are not limited to, examination of protein or mRNA levels using techniques known to those of skill in the art, such as dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and the like, as well as phenotypic assays known to those of skill in the art.

[0020] The term "synthetic activating group" refers to a group that can be attached 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 depend on the atom it is activating. 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 (e.g., an ester bond, a carbamate bond, or an ether bond) with another reactive group. Such synthetic activating groups are known. Examples of synthetic activating groups that can be attached to an oxygen atom include, but are not limited to, acetate, succinate, triflate, and mesylate. When a synthetic activating group is attached to the oxygen atom of a carboxylic acid, the synthetic activating group can be a group 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'-ethyl carbonate (EDC), (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), or O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU).

[0021] An "effective amount" or "therapeutically effective amount" of a therapeutic nucleic acid, such as an siRNA, is an amount sufficient to produce a desired effect, e.g., inhibition of expression of a target sequence compared to the normal expression level detected in the absence of the siRNA. In certain embodiments, inhibition of expression of a target gene or target sequence is achieved when the siRNA yields 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% inhibition of expression of a target gene or target sequence relative to a control (e.g., buffer only, an siRNA sequence targeting a different gene, a scrambled siRNA sequence, etc.). Suitable assays for measuring expression of a target gene or target sequence include, but are not limited to, examination of protein or mRNA levels using techniques known to those of skill in the art, such as dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzymatic function, and the like, as well as phenotypic assays known to those of skill in the art.

[0022] As used herein, the term "nucleic acid" refers to a polymer containing at least two nucleotides (i.e., deoxyribonucleotides or ribonucleotides) in either single- or double-stranded form, including DNA and RNA. A "nucleotide" contains the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together via the phosphate group. "Base" includes purines and pyrimidines, including the naturally occurring compounds adenine, thymine, guanine, cytosine, uracil, inosine, and naturally occurring analogs of purines and pyrimidines, as well as synthetic derivatives, including, but not limited to, modifications that place new reactive groups, such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides. Nucleic acids include synthetic, naturally occurring, and non-naturally occurring nucleic acids containing known nucleotide analogs or modified backbone residues or linkages that have similar binding properties as the reference nucleic acid. Examples of such analogs and / or modified residues include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides, and peptide nucleic acids (PNAs). Additionally, nucleic acids can contain one or more UNA moieties.

[0023] The term "nucleic acid" includes any oligonucleotide or polynucleotide; fragments containing up to 60 nucleotides are generally referred to as oligonucleotides, while longer fragments are referred to as polynucleotides. Deoxyribonucleotides consist of a five-carbon sugar called deoxyribose covalently linked 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, precondensed DNA, PCR products, vectors, expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups. Ribonucleotides consist of a similar repeating structure in which the five-carbon 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), and combinations thereof. Thus, in the context of the present invention, the terms "polynucleotide" and "oligonucleotide" refer to a polymer or oligomer of nucleotide or nucleoside monomers composed of naturally occurring bases, sugars, and intersugar (backbone) linkages. The terms "polynucleotide" and "oligonucleotide" also include polymers or oligomers containing non-naturally occurring monomers or portions thereof which function similarly. Such modified or substituted oligonucleotides are often preferred over natural forms because of properties such as, for example, enhanced cellular uptake, reduced immunogenicity, and increased stability in the presence of nucleases.

[0024] Unless otherwise indicated, a particular nucleic acid sequence implicitly encompasses not only the sequence explicitly indicated, but also its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences. In particular, 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 mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)).

[0025] The term "gene" refers to a nucleic acid (eg, DNA or RNA) sequence that comprises partial or full-length coding sequences necessary for the production of a polypeptide or precursor polypeptide.

[0026] As used herein, "gene product" refers to the product of a gene, such as an RNA transcript or a polypeptide.

[0027] As used herein, unless otherwise stated, the term “alkyl,” by itself or as part of another substituent, refers to an alkyl group having the specified number of carbon atoms (i.e., C 1~8 means a straight-chain or branched-chain hydrocarbon group (having 1 to 8 carbon atoms). Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. The term "alkenyl" refers to unsaturated alkyl groups having one or more double bonds. Similarly, the term "alkynyl" refers to unsaturated alkyl groups having one or more triple bonds. Examples of such unsaturated alkyl groups include vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1-propynyl and 3-propynyl, 3-butynyl, and the higher homologs and isomers.

[0028] The term "alkylene," by itself or as part of another substituent, means a divalent group derived from an alkane (including straight-chain and branched alkanes), as exemplified by -CH2CH2CH2CH2- and -CH(CH3)CH2CH2-.

[0029] The terms "cycloalkyl," "carbocyclic," or "carbocycle" refer to a hydrocarbon ring system having a total of 3 to 20 ring atoms (e.g., a 3- to 20-membered cycloalkyl is a cycloalkyl having 3 to 20 ring atoms, or C 3~20 Cycloalkyl is a cycloalkyl having 3 to 20 carbon ring atoms, and in the case of 3- to 5-membered cycloalkyl, it is either fully saturated or has no more than one double bond between the ring vertices, and in the case of 6 or more-membered cycloalkyl, it is either fully saturated or has no more than two double bonds between the ring vertices. As used herein, "cycloalkyl," "carbocyclic," or "carbocycle" refers to, for example, bicyclo[2.2.1]heptane, pinane, bicyclo[2.2.2]octane, adamantane, norbornene, spirocyclic C 5~12 It is also intended to refer to bicyclic, polycyclic, and spirocyclic hydrocarbon ring systems, such as alkanes. As used herein, the terms "alkenyl," "alkynyl," "cycloalkyl," "carbocycle," and "carbocyclic" are intended to include mono- and poly-halogenated variants thereof.

[0030] The terms "heterocycloalkyl," "heterocyclic," or "heterocycle" refer to a saturated or partially unsaturated ring system containing from 1 to 10 heteroatoms selected from N, O, and S as ring atoms, with a total of 3 to 20 ring atoms (e.g., a 3- to 20-membered heterocycloalkyl is a heterocycloalkyl group having from 3 to 20 ring atoms, and C 2~19Heterocycloalkyl is a heterocycloalkyl having 3 to 10 ring atoms, wherein 2 to 19 ring atoms are carbon), where the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom(s) are optionally quaternized. Unless otherwise specified, the ring of a "heterocycloalkyl," "heterocyclic," or "heterocycle" can be a monocyclic, bicyclic, spirocyclic, or polycyclic ring system. Non-limiting examples of "heterocycloalkyl," "heterocyclic," or "heterocycle" rings include pyrrolidine, piperidine, N-methylpiperidine, imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin, dioxolane, phthalimide, piperidine, pyrimidine-2,4(1H,3H)-dione, 1,4-dioxane, morpholine, thiomorpholine, thiomorpholine-S-oxide, thiomo Examples of heterocyclic groups include methylpyrroline-S,S-oxide, piperazine, pyran, pyridone, 3-pyrroline, thiopyran, pyrone, tetrahydrofuran, tetrahydrothiophene, quinuclidine, tropane, 2-azaspiro[3.3]heptane, (1R,5S)-3-azabicyclo[3.2.1]octane, (1s,4s)-2-azabicyclo[2.2.2]octane, (1R,4R)-2-oxa-5-azabicyclo[2.2.2]octane, and the like. A "heterocycloalkyl," "heterocyclic," or "heterocycle" group can be attached to the remainder of the molecule through one or more ring carbons or heteroatoms. "Heterocycloalkyl," "heterocyclic," or "heterocycle" can include monohalogenated and polyhalogenated variants thereof.

[0031] The terms "alkoxy" and "alkylthio" are used in their conventional sense to refer to an alkyl group attached to the remainder of the molecule through an oxygen atom ("oxy") or a thio group, and further include mono- and poly-halogenated variants thereof.

[0032] The terms "halo" or "halogen," by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. The term "(halo)alkyl" is intended to include both "alkyl" and "haloalkyl" substituents. Furthermore, the term "haloalkyl" is intended to include monohaloalkyl and polyhaloalkyl. For example, "C 1~4 The term "haloalkyl" is meant to include trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, difluoromethyl and the like.

[0033] The term "aryl" means a carbocyclic aromatic group having 6 to 14 carbon atoms, whether or not fused to one or more groups. Examples of aryl groups, unless otherwise specified, include phenyl, naphthyl, biphenyl, and the like.

[0034] The term "heteroaryl" refers to an aryl ring(s) containing 1 to 5 heteroatoms selected from N, O, and S, where the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom(s) are optionally quaternized. A heteroaryl group can be attached to the remainder of the molecule through a heteroatom. Examples of heteroaryl groups include pyridyl, pyridazinyl, pyrazinyl, pyrimidinyl, triazinyl, quinolinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, benzotriazinyl, purinyl, benzimidazolyl, benzopyrazolyl, benzotriazolyl, benzisoxazolyl, isobenzofuryl, isoindolyl, indolizinyl, benzotriazinyl, thienopyridinyl, thienopyrimidinyl, pyrazolopyrimidinyl, imidazopyridine, benzothiaxolyl, benzofuranyl, benzothienyl, indolyl, quinolyl, isoquinolyl, isothiazolyl, pyrazolyl, indazolyl, pteridinyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiadiazolyl, pyrrolyl, thiazolyl, furyl, thienyl, and the like.

[0035] The term "saccharide" includes monosaccharides, disaccharides, and trisaccharides. It includes glucose, sucrose, fructose, galactose, and ribose, as well as deoxysaccharides such as deoxyribose and aminosaccharides such as galactosamine. Sugar derivatives can be conveniently prepared as described in International Patent Applications WO 96 / 34005 and WO 97 / 03995. The saccharide can be conveniently linked to the remainder of the compound of Formula I via an ether bond, a thioether bond (e.g., S-glycoside), an amine nitrogen (e.g., N-glycoside), or a carbon-carbon bond (e.g., C-glycoside). In one embodiment, the saccharide can be conveniently linked to the remainder of the compound of Formula I via an ether bond. In one embodiment, the term "saccharide" refers to a sugar having the formula: [ka] wherein X is NR 3 and Y is -(C=O)R 4 , -SO2R 5 , and —(C═O)NR 6 R 7 or X is —(C═O)— and Y is NR 8 R 9 and R 3 is hydrogen or (C1-C4) alkyl, R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 are each independently selected from the group consisting of hydrogen, (C1-C8)alkyl, (C1-C8)haloalkyl, (C1-C8)alkoxy, and (C3-C6)cycloalkyl optionally substituted with one or more groups independently selected from the group consisting of halo, (C1-C4)alkyl, (C1-C4)haloalkyl, (C1-C4)alkoxy, and (C1-C4)haloalkoxy; R 10 -OH, -NR 8 R 9, or -F, R 11 -OH, -NR 8 R 9 , -F, or a 5-membered heterocycle optionally substituted with one or more groups independently selected from the group consisting of halo, hydroxyl, carboxyl, amino, (C1-C4) alkyl, (C1-C4) haloalkyl, (C1-C4) alkoxy, and (C1-C4) haloalkoxy. [ka] It can be selected from the group consisting of:

[0036] In another embodiment, the saccharide is [ka] It may be.

[0037] The term "animal" includes mammalian species such as humans, mice, rats, dogs, cats, hamsters, guinea pigs, rabbits, farm animals, and the like.

[0038] The term "lipid" refers to a group of organic compounds, including but not limited to esters of fatty acids, that are characterized by being insoluble in water but soluble in many organic solvents. Lipids are typically divided into at least three classes: (1) "simple lipids," which include fats and oils as well as waxes; (2) "complex lipids," which include phospholipids and glycolipids; and (3) "derived lipids," such as steroids.

[0039] The term " lipid particle " includes lipid preparations that can be used to deliver therapeutic nucleic acid (e.g., siRNA) to target site of interest (e.g., cell, tissue, organ, etc.).In a preferred embodiment, the lipid particle of the present invention is nucleic acid-lipid particle, which is typically formed by cationic lipid, non-cationic lipid (e.g., phospholipid), conjugate lipid (e.g., PEG-lipid) that prevents particle aggregation, and optionally cholesterol.Typically, therapeutic nucleic acid (e.g., siRNA) can be encapsulated in the lipid portion of particle so as to be protected from enzymatic degradation.

[0040] The term "electron-dense core," when used to describe lipid particles of the present invention, refers to the interior of the lipid particle, which appears dark when visualized using a cryo-transmission electron microscope ("cryo-TEM"). Some lipid particles of the present invention have an electron-dense core and do not have a lipid bilayer structure. Some lipid particles of the present invention have an electron-dense core and do not have a lipid bilayer structure, but have an inverted hexagonal or cubic phase structure. Without wishing to be bound by theory, it is believed that the packing of non-bilayer lipids results in a three-dimensional network of lipid cylinders with water and nuclei inside, essentially aqueous channels permeated with lipid droplets containing nucleic acids.

[0041] As used herein, the term "SNALP" refers to stable nucleic acid-lipid particles. SNALPs are particles made of lipids (e.g., cationic lipids, non-cationic lipids, and conjugated lipids that prevent particle aggregation), in which nucleic acids (e.g., siRNA) are completely encapsulated. In certain cases, SNALPs are extremely useful for systemic applications because they can exhibit long-term circulation life after intravenous (iv) injection, accumulate at distant sites (e.g., sites physically separated from the administration site), and mediate siRNA expression at these distant sites. Nucleic acids can be complexed with condensing agents and encapsulated within SNALPs, as described in PCT Publication No. WO00 / 03683 (the disclosure of which is incorporated herein by reference in its entirety for all purposes).

[0042] The lipid particles of the present invention (e.g., SNALP) typically have a diameter of about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm The average diameter of the lipid particles of the present invention is about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and is substantially non-toxic.In addition, when nucleic acids are present in the lipid particles of the present invention, they are resistant to degradation by nucleases in aqueous solution.Nucleic acid-lipid particles and their preparation methods are disclosed, for example, in U.S. Patent Publication Nos. 20040142025 and 20070042031, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0043] As used herein, "lipid-encapsulated" can refer to lipid particles that provide complete encapsulation, partial encapsulation, or both, of a therapeutic nucleic acid, such as siRNA. In a preferred embodiment, the nucleic acid (e.g., siRNA) is completely encapsulated in the lipid particle (e.g., to form a SNALP or other nucleic acid-lipid particle).

[0044] The term "lipid conjugate" refers to a conjugated lipid that inhibits lipid particle aggregation.Such lipid conjugates include, but are not limited to, PEG-lipid conjugates, such as PEG coupled to dialkyloxypropyl (e.g., PEG-DAA conjugates), PEG coupled to diacylglycerol (e.g., PEG-DAG conjugates), PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamine, and PEG coupled to ceramide (see, e.g., U.S. Pat. No. 5,885,613), cationic PEG lipids, polyoxazoline (POZ)-lipid conjugates, polyamide oligomers (e.g., ATTA-lipid conjugates), and mixtures thereof.Further examples of POZ-lipid conjugates are described in PCT Publication No. WO2010 / 006282.PEG or POZ can be directly conjugated to lipid, or can be bound to lipid via a linker moiety. Any suitable linker moiety can be used for coupling PEG or POZ to lipid, including, for example, non-ester-containing linker moiety and ester-containing linker moiety.In certain preferred embodiments, non-ester-containing linker moiety, such as amide or carbamate, is used.The disclosures of each of the above patent documents are incorporated herein by reference in their entirety for all purposes.

[0045] The term "amphipathic lipid" refers, in part, to any suitable lipid material in which the hydrophobic portion of the lipid material faces the hydrophobic phase and the hydrophilic portion faces the aqueous phase. The hydrophilic character comes from the presence of polar or charged groups, such as carbohydrate groups, phosphate groups, carboxylic acid groups, sulfato groups, amino groups, sulfhydryl groups, nitro groups, hydroxyl groups, and other similar groups. Hydrophobicity can also be achieved through the inclusion of nonpolar groups, including, but not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups, and those in which such groups are substituted with one or more aromatic, alicyclic, or heterocyclic group(s). Examples of amphipathic compounds include, but are not limited to, phospholipids, aminolipids, and sphingolipids.

[0046] Representative examples of phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, and dilinoleoylphosphatidylcholine.Other compounds lacking phosphorus, such as sphingolipids, glycosphingolipid families, diacylglycerols, and β-acyloxyacids, are also included in the group designated as amphipathic lipids.Furthermore, the above-mentioned amphipathic lipids can be mixed with other lipids, including triglycerides and sterols.

[0047] The term "neutral lipid" refers to any of several lipid species that exist in either an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerol.

[0048] The term "non-cationic lipid" refers to any amphipathic lipid and any other neutral or anionic lipid.

[0049] The term "anionic lipid" refers to any lipid that is negatively charged at physiological pH, including, but not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), and other anionic modifying groups attached to neutral lipids.

[0050] The term "hydrophobic lipid" refers to a compound having a non-polar group, including but not limited to long-chain saturated and unsaturated aliphatic hydrocarbon groups, and these groups are optionally substituted with one or more aromatic groups, alicyclic groups, or heterocyclic groups.Suitable examples include but are not limited to diacylglycerol, dialkylglycerol, N-N-dialkylamino, 1,2-diacyloxy-3-aminopropane, and 1,2-dialkyl-3-aminopropane.

[0051] As used herein, the terms "cationic lipid" and "amino lipid" are used interchangeably to include lipids and their salts having one, two, three, or more fatty acid or fatty alkyl chains and a pH-titratable amino head group (e.g., an alkylamino or dialkylamino head group). Cationic lipids are typically lipids whose pK a It is protonated (i.e., positively charged) at pH below pK aThe cationic lipids of the present invention are also referred to as titratable cationic lipids. In some embodiments, the cationic lipids are C 1 -C 2 -C 3 -C 4 -C 5 -C 6 -C 7 -C 8 -C 9 -C 10 -C 11 -C 12 -C 13 -C 14 -C 15 -C 16 -C 17 -C 18 -C 19 -C 20 -C 21 -C 22 -C 23 -C 24 -C 25 -C 26 -C 27 -C 28 -C 29 -C 30 -C 31 -C 32 -C 33 -C 44 -C 29 -C 34 -C 29 -C 35 -C 45 -C 29 -C 36 -C 29 -C 37 -C 48 -C 29 -C 38 -C 49 -C 50 -C 51 -C 52 -C 53 -C 54 -C 55 -C 56 -C 57 -C 58 -C 59 -C 60 -C 61 -C 62 -C 63 -C 64 -C 65 -C 66 -C 67 -C 68 -C 70 -C 71 -C 72 -C 73 -C 74 -C 75 -C 76 -C 77 -C 78 -C 79 -C 80 -C 81 -C 82 -C 83 -C 84 -C 85 -C 86 -C 87 -C 88 -C 89 -C 90 -C 91 -C 92 -C 93 -C 94 -C 95 -C 96 -C 97 -C 98 -C 99 -C 100 -C 18 Cationic lipids include, but are not limited to, DSDMA, DODMA, DLinDMA, DLenDMA, γ-DLenDMA, DLin-K-DMA, DLin-K-C2-DMA (also known as DLin-C2K-DMA, XTC2, and C2K), DLin-K-C3-DMA, DLin-K-C4-DMA, DLen-C2K-DMA, γ-DLen-C2K-DMA, DLin-M-C2-DMA (also known as MC2), DLin-M-C3-DMA (also known as MC3), and (DLin-MP-DMA) (also known as 1-B11).

[0052] The term "alkylamino" includes groups of the formula -N(H)R, where R is alkyl as defined herein.

[0053] The term "dialkylamino" includes groups of the formula -NR2, where each R is independently alkyl as defined herein.

[0054] The term "salt" includes any complex of anion and cation, such as the complex formed between a cationic lipid and one or more anions.Non-limiting examples of anions include inorganic and organic anions, such as hydride, fluoride, chloride, bromide, iodide, oxalate (e.g., hemioxalate), phosphate, phosphonate, hydrogen phosphate, dihydrogen phosphate, oxide, carbonate, hydrogen carbonate, nitrate, nitrite, nitride, bisulfite, sulfide, sulfite, bisulfate, sulfate, thiosulfate, hydrogen sulfate, borate, formate, acetate, benzoate, citrate, tartaric acid, lactic acid, acrylic acid, polyacrylic acid, Cationic lipid salts include fumaric acid, maleic acid, itaconic acid, glycolic acid, gluconic acid, malic acid, mandelic acid, tiglic acid, ascorbic acid, salicylic acid, polymethacrylic acid, perchloric acid, chloric acid, chlorous acid, hypochlorous acid, bromic acid, hypobromous acid, iodic acid, alkylsulfonate, arylsulfonate, arsenic acid, arsenous acid, chromic acid, dichromate, cyanide, cyanic acid, thiocyanate, hydroxide, peroxide, permanganate, and mixtures thereof.In certain embodiments, the salt of cationic lipid disclosed herein is a crystalline salt.

[0055] The term "acyl" includes any alkyl, alkenyl, or alkynyl, as defined below, in which the carbon at the point of attachment is substituted with an oxo group. The following are non-limiting examples of acyl groups: -C(=O)alkyl, -C(=O)alkenyl, and -C(=O)alkynyl.

[0056] The term "fusogenic" refers to the ability of a lipid particle, such as a SNALP, to fuse with the membrane of a cell, which may be either the plasma membrane or the membrane surrounding an organelle, such as, for example, an endosome, the nucleus, etc.

[0057] As used herein, the term "aqueous solution" refers to a composition that comprises, in whole or in part, water.

[0058] As used herein, the term "organic lipid solution" refers to a composition comprising, in whole or in part, an organic solvent with a lipid.

[0059] As used herein, "distal site" refers to a physically separate site, and is not limited to adjacent capillary beds, but includes sites widely distributed throughout an organism.

[0060] "Serum stability" with respect to nucleic acid-lipid particles such as SNALP means that the particles are not significantly degraded after exposure to serum or nuclease assays that significantly degrade free DNA or RNA. Suitable assays include, for example, standard serum assays, DNAse assays, or RNAse assays.

[0061] As used herein, "systemic delivery" refers to the delivery of lipid particles that results in widespread biodistribution of an active agent, such as siRNA, in an organism. Some administration techniques can result in systemic delivery of certain drugs, while others do not. Systemic delivery means that a useful (preferably therapeutic) amount of the drug is exposed to most parts of the body. Achieving widespread biodistribution generally requires a blood lifetime such that the drug is not rapidly degraded or eliminated (e.g., by first-pass organs (liver, lung, etc.) or by rapid nonspecific cellular binding) before reaching disease sites distal to the administration site. Systemic delivery of lipid particles can be by any means known in the art, including, for example, intravenous, subcutaneous, and intraperitoneal. In a preferred embodiment, systemic delivery of lipid particles is by intravenous delivery.

[0062] As used herein, " local delivery " refers to the direct delivery of active agent such as siRNA to target site in living organism.For example, the agent can be delivered locally by direct injection into disease site, other target site or target organ such as liver, heart, pancreas, kidney.

[0063] As used herein to describe the lipid: siRNA ratio, the term "lipid" refers to the total lipid in the particle.

[0064] Those skilled in the art will appreciate that compounds of the present invention having chiral centers may exist in and be isolated in optically active and racemic forms. Some compounds may exhibit polymorphism. It is understood that the present invention encompasses any racemic, optically active, polymorphic, or stereoisomeric form of a compound of the present invention, or mixtures thereof, that possesses the useful properties described herein. Methods for preparing optically active forms are well known in the art (e.g., by resolution of racemic forms by recrystallization techniques, by synthesis from optically active starting materials, by chiral synthesis, or by chromatographic separation using chiral stationary phases).

[0065] When a bond in a compound formula herein is drawn in a non-stereochemical manner (e.g., planar), the atom to which the bond is attached includes all stereochemical possibilities. Unless specifically stated otherwise, when a bond in a compound formula herein is drawn in a given stereochemical manner (e.g., as a bold line, a bold wedge, a dashed line, or a dashed wedge), it is understood that the atom to which the stereochemical bond is attached is enriched in the absolute stereoisomer shown. In one embodiment, at least 51% of the compounds can be the absolute stereoisomer shown. In another embodiment, at least 60% of the compounds can be the absolute stereoisomer shown. In another embodiment, at least 80% of the compounds can be the absolute stereoisomer shown. In another embodiment, at least 90% of the compounds can be the absolute stereoisomer shown. In another embodiment, at least 95% of the compounds can be the absolute stereoisomer shown. In another embodiment, at least 99% of the compounds can be the absolute stereoisomer shown.

[0066] Unless otherwise stated herein, the term "about," when used in connection with a value or range of values, means plus or minus 5% of the stated value or range of values.

[0067] Generation of siRNA molecules siRNA can be prepared in several forms, including, for example, as one or more isolated small interfering RNA (siRNA) duplexes, as longer double-stranded RNA (dsRNA), or as siRNA or dsRNA transcribed from a transcription cassette in a DNA plasmid. In some embodiments, siRNA can be produced enzymatically or by partial / complete organic synthesis, and modified ribonucleotides can be introduced by in vitro enzymatic synthesis or organic synthesis. In certain cases, each strand is chemically prepared. Methods for synthesizing RNA molecules are known in the art, such as the chemical synthesis method described in Verma and Eckstein (1998), or are described herein.

[0068] Methods for isolating RNA, synthesizing RNA, hybridizing nucleic acids, creating and screening cDNA libraries, and performing PCR are well known in the art (see, e.g., Gubler and Hoffman, Gene, 25:263-269 (1983); Sambrook et al. (supra); Ausubel et al. (supra)). PCR techniques are also well known in the art (see U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., eds, 1990)). Expression libraries are also well known to those skilled in the art. Further basic texts disclosing general methods for use in the present invention include Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989), Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990), and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994), the disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0069] Typically, siRNA is chemically synthesized.Oligonucleotides, including siRNA molecules of the present invention, can be synthesized using any of a variety of techniques known in the art, such as those described in Usman et al., J.Am.Chem.Soc., 109:7845 (1987), Scaringe et al., Nucl.Acids Res., 18:5433 (1990), Wincott et al., Nucl.Acids Res., 23:2677-2684 (1995), and Wincott et al., Methods Mol.Bio., 74:59 (1997).Synthesis of oligonucleotides utilizes common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end and phosphoramidite at the 3'-end.As a non-limiting example, small-scale synthesis can be carried out using a 0.2 μmol scale protocol on an Applied Biosystems synthesizer. Alternatively, 0.2 μmol scale syntheses can be performed on a Protogene (Palo Alto, CA) 96-well plate synthesizer. However, larger or smaller scale syntheses are also within the scope of the present invention. Suitable reagents for oligonucleotide synthesis, methods for RNA deprotection, and methods for RNA purification are known to those of skill in the art.

[0070] An siRNA molecule can be constructed from two separate oligonucleotides, one containing the sense strand of the siRNA and the other the antisense strand, e.g., each strand can be synthesized separately and joined together by hybridization or ligation after synthesis and / or deprotection.

[0071] Embodiments of the present invention Table 1 in Example 25 describes a series of chemically modified siRNA duplexes (sense and antisense strands are shown) targeting hepatitis B virus (abbreviated as "HBV"). As described herein, compounds of the invention can include such siRNAs (i.e., siRNAs 1-37).

[0072] Accordingly, one aspect of the present invention is a nucleic acid molecule selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, and SEQ ID NO:73.

[0073] Another aspect of the invention is a nucleic acid molecule selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, and SEQ ID NO:74.

[0074] One aspect of the present invention is a composition comprising the nucleic acid molecules described herein or combinations thereof.

[0075] One aspect of the present invention is siRNA 1 (SEQ ID NOs: 1 and 2), 2 (SEQ ID NOs: 3 and 4), 3 (SEQ ID NOs: 5 and 6), 4 (SEQ ID NOs: 7 and 8), 5 (SEQ ID NOs: 9 and 10), 6 (SEQ ID NOs: 11 and 12), 7 (SEQ ID NOs: 13 and 14), 8 (SEQ ID NOs: 15 and 16), 9 (SEQ ID NOs: 17 and 18), 10 (SEQ ID NOs: 19 and 20), 11 (SEQ ID NOs: 21 and 22), 12 (SEQ ID NOs: 23 and 24), 13 (SEQ ID NOs: 25 and 26), 14 (SEQ ID NOs: 27 and 28), 15 (SEQ ID NOs: 29 and 30), 16 (SEQ ID NOs: 31 and 32), 17 (SEQ ID NOs: 33 and 34), 18 (SEQ ID NOs: 35 and 36), 19 (SEQ ID NOs: 37 and 38), 20 (SEQ ID NOs: 39 and 40), 21 (SEQ ID NOs: 31 and 32), 22 (SEQ ID NOs: 32 and 33), 23 (SEQ ID NOs: 33 and 34), 24 (SEQ ID NOs: 35 and 36), 25 (SEQ ID NOs: 37 and 38), 26 (SEQ ID NOs: 39 and 40), 27 (SEQ ID NOs: 39 and 41), 28 (SEQ ID NOs: 39 and 42), 29 (SEQ ID NOs: 39 and 4 The double-stranded siRNA molecules are selected from the group consisting of: 1 (SEQ ID NOs: 41 and 42), 22 (SEQ ID NOs: 43 and 44), 23 (SEQ ID NOs: 45 and 46), 24 (SEQ ID NOs: 47 and 48), 25 (SEQ ID NOs: 49 and 50), 26 (SEQ ID NOs: 51 and 52), 27 (SEQ ID NOs: 53 and 54), 28 (SEQ ID NOs: 55 and 56), 29 (SEQ ID NOs: 57 and 58), 30 (SEQ ID NOs: 59 and 60), 31 (SEQ ID NOs: 61 and 62), 32 (SEQ ID NOs: 63 and 64), 33 (SEQ ID NOs: 65 and 66), 34 (SEQ ID NOs: 67 and 68), 35 (SEQ ID NOs: 69 and 70), 36 (SEQ ID NOs: 71 and 72), and 37 (SEQ ID NOs: 73 and 74).

[0076] Another aspect of the present invention provides a composition comprising a double-stranded siRNA molecule described herein.

[0077] In one embodiment, the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

[0078] One aspect of the invention is a compound of Formula I, or a salt thereof, as described in the Summary of the Invention.

[0079] In one embodiment of the compound of formula I, R 1 is the targeting ligand, L 1 is absent or a bonding group, L 2 is absent or a bonding group, R 2 is a double-stranded siRNA molecule selected from the double-stranded siRNAs of Table 1, Ring A is absent, a 3- to 20-membered cycloalkyl, a 5- to 20-membered aryl, a 5- to 20-membered heteroaryl, or a 3- to 20-membered heterocycloalkyl; Each R A are independently hydrogen, hydroxy, CN, F, Cl, Br, I, -C 1~2 Alkyl-OR B , as well as halo, hydroxy, and C 1~3 C optionally substituted with one or more groups independently selected from alkoxy 1~8 is selected from the group consisting of alkyl, R B is hydrogen, a protecting group, a covalent bond to a solid support, or a bond to a linking group attached to a solid support; n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0080] In one embodiment, R 1 is -C(H) (3-p) (L 3 -Sugars) p where each L 3 are independently a linking group, p is 1, 2, or 3, and the saccharide is a monosaccharide or disaccharide.

[0081] In one embodiment, the sugar is [ka] or a salt thereof, During the ceremony, X is NR 3 and Y is -(C=O)R 4 , -SO2R 5 , and —(C═O)NR 6 R 7 or X is —(C═O)— and Y is NR 8 R 9 and R 3 is hydrogen or (C1-C4) alkyl, R4 , R 5 , R 6 , R 7 , R 8 , and R 9 are each independently selected from the group consisting of hydrogen, (C1-C8)alkyl, (C1-C8)haloalkyl, (C1-C8)alkoxy, and (C3-C6)cycloalkyl optionally substituted with one or more groups independently selected from the group consisting of halo, (C1-C4)alkyl, (C1-C4)haloalkyl, (C1-C4)alkoxy, and (C1-C4)haloalkoxy; R 10 -OH, -NR 8 R 9 , or -F, R 11 -OH, -NR 8 R 9 , -F, or a 5-membered heterocycle optionally substituted with one or more groups independently selected from the group consisting of halo, hydroxyl, carboxyl, amino, (C1-C4) alkyl, (C1-C4) haloalkyl, (C1-C4) alkoxy, and (C1-C4) haloalkoxy.

[0082] In one embodiment, the sugar is [ka] and salts thereof.

[0083] In one embodiment, the sugar is [ka] is.

[0084] In one embodiment, each L 3 are independently a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 0 to 50 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are -O-, -NR X -, -NR X-C(=O)-, -C(=O)-NR X - or -S-, optionally replaced by R X is hydrogen or (C1-C6) alkyl, and the hydrocarbon chain is optionally substituted with one or more (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.

[0085] In one embodiment, each L 3 are independently a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 20 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X - or -S-, optionally replaced by R X is hydrogen or (C1-C6) alkyl, and the hydrocarbon chain is optionally substituted with one or more (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.

[0086] In one embodiment, L 3 teeth, [ka] or a salt thereof.

[0087] In one embodiment, R1 teeth, [ka] or a salt thereof.

[0088] In one embodiment, R 1 teeth, [ka] or a salt thereof, wherein G is —NH— or —O—; R C is hydrogen, (C1-C8) alkyl, (C1-C8) haloalkyl, (C1-C8) alkoxy, (C1-C6) alkanoyl, (C3-C 20 ) cycloalkyl, (C3-C 20 ) heterocycle, aryl, heteroaryl, monosaccharide, disaccharide, or trisaccharide, wherein the cycloalkyl, heterocycle, aryl, heteroaryl, and saccharide are optionally substituted with one or more groups independently selected from the group consisting of halo, carboxyl, hydroxyl, amino, (C1-C4) alkyl, (C1-C4) haloalkyl, (C1-C4) alkoxy, and (C1-C4) haloalkoxy).

[0089] In one embodiment, R C teeth, [ka] is.

[0090] In one embodiment, R 1 teeth, [ka] is.

[0091] In one embodiment, R C teeth, [ka] is.

[0092] In one embodiment, G is -NH-.

[0093] In one embodiment, R 1 teeth, [ka] is.

[0094] In one embodiment, R 1 teeth, [ka] and In the formula, each R D are independently hydrogen, (C1-C6) alkyl, (C9-C 20 ) alkylsilyl, (R W )3Si—, (C2-C6)alkenyl, tetrahydropyranyl, (C1-C6)alkanoyl, benzoyl, aryl(C1-C3)alkyl, TMTr (trimethoxytrityl), DMTr (dimethoxytrityl), MMTr (monomethoxytrityl), and Tr (trityl); Each R W is independently selected from the group consisting of (C1-C4) alkyl and aryl.

[0095] In one embodiment, the linking group L 1 and L 2 are independently a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 50 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X - or -S-, optionally replaced by R Xis hydrogen or (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.

[0096] In one embodiment, L 1 and L 2 are independently a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 20 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X - or -S-, optionally replaced by R X is hydrogen or (C1-C6) alkyl, and the hydrocarbon chain is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from (C1-C6) alkoxy, (C3-C6) cycloalkyl, (C1-C6) alkanoyl, (C1-C6) alkanoyloxy, (C1-C6) alkoxycarbonyl, (C1-C6) alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0097] In one embodiment, L 1 and L 2 are independently a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 14 carbon atoms, in which one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are optionally replaced, such as -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NRX -, 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.

[0098] In one embodiment, L 1 is connected to R via -NH-, -O-, -S-, -(C=O)-, -(C=O)-NH-, -NH-(C=O)-, -(C=O)-O-, -NH-(C=O)-NH-, or -NH-(SO2)-. 1 is bonded to.

[0099] In one embodiment, L 2 -O- through R 2 is bonded to.

[0100] In one embodiment, L 1 teeth, [ka] is selected from the group consisting of:

[0101] In one embodiment, L 1 teeth, [ka] and salts thereof.

[0102] In one embodiment, L 2 is —CH2—O— or —CH2—CH2—O—.

[0103] In one embodiment, the compound of formula I has formula Ia: [ka] (Ia) or a salt thereof, During the ceremony, Each D is independently [ka] and -N=.

[0104] In one embodiment, the compound of formula Ia is [ka] and salts thereof; During the ceremony, Q 1 is hydrogen and Q 2 is R 2 or Q 1 is R 2 and Q 2 is hydrogen, Z is -L 1 -R 1 is.

[0105] In one embodiment, the compound of formula I has the following formula Ib: [ka] (Ib) or a salt thereof, During the ceremony, Each D is independently [ka] and -N=; Each m is independently 1 or 2.

[0106] In one embodiment, the compound of formula Ib is [ka] and salts thereof; During the ceremony, Q 1 is hydrogen and Q 2 is R 2 or Q 1 is R 2 and Q 2 is hydrogen, Z is -L 1 -R 1 is.

[0107] In one embodiment, the compound of formula I has the following formula (Ic): [ka] (I C) or a salt thereof, wherein E is —O— or —CH—; n is selected from the group consisting of 0, 1, 2, 3, and 4; n1 and n2 are each independently selected from the group consisting of 0, 1, 2, and 3.

[0108] In certain embodiments, the compound of formula (Ic) is [ka] and salts thereof; In the formula, Z is -L 1 -R 1 is.

[0109] In one embodiment, -AL 2 -R 2 The part is [ka] or a salt thereof, During the ceremony, Q 1 is hydrogen and Q 2 is R 2 or Q 1 is R 2 and Q 2is hydrogen, Each q is independently 0, 1, 2, 3, 4, or 5.

[0110] In one embodiment, the compound of formula (I) is [ka] [ka] and [ka] and salts thereof.

[0111] In one embodiment, R 1 teeth, [ka] is selected from the group consisting of In the formula, R S teeth, [ka] and n is 2, 3, or 4; x is 1 or 2.

[0112] In one embodiment, L 1 teeth, [ka] is selected from the group consisting of:

[0113] In one embodiment, L 1 teeth, [ka] is selected from the group consisting of:

[0114] In one embodiment, A is absent, phenyl, pyrrolidinyl, or cyclopentyl.

[0115] In one embodiment, L 2 is C optionally substituted with hydroxy 1~4 It is alkylene-O-.

[0116] In one embodiment, L 2 is —CH2O—, —CH2CH2O—, or —CH(OH)CH2O—.

[0117] In one embodiment, each R A are independently hydroxy or C optionally substituted with hydroxyl 1~8 It is alkyl.

[0118] In one embodiment, each R A is independently selected from the group consisting of hydroxy, methyl, and —CHOH.

[0119] In one embodiment, the compound of formula I has the following formula (Ig): [ka] (Ig) or a salt thereof, wherein B is —N— or —CH—; L 1 is absent or is -NH-, L 2 is a C optionally substituted with hydroxyl or halo 1~4 alkylene-O-; n is 0, 1, or 2.

[0120] In one embodiment, the compound of formula I has the following formula (Ig): [ka] (Ig) or a salt thereof, wherein B is —N— or —CH—; L1 is absent or is -NH-, L 2 is a C optionally substituted with hydroxyl or halo 1~4 alkylene-O-; n is 0, 1, 2, 3, 4, 5, 6, or 7.

[0121] In one embodiment, the compound of formula I has the following formula (Ig): [ka] (Ig) or a salt thereof, wherein B is —N— or —CH—; L 1 is absent or is -NH-, L 2 is a C optionally substituted with hydroxyl or halo 1~4 alkylene-O-; n is 0, 1, 2, 3, or 4.

[0122] In one embodiment, the compound of formula Ig is [ka] and salts thereof; In the formula, R' is C 1~9 Alkyl, C 2~9 Alkenyl, or C 2~9 Alkynyl, and this C 1~9 Alkyl, C 2~9 Alkenyl, or C 2~9 The alkynyl is optionally substituted with halo or hydroxyl.

[0123] In one embodiment, the compound of formula I is [ka] and salts thereof.

[0124] In one embodiment, the compound of formula I or a salt thereof is [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] , and [ka] is selected from the group consisting of:

[0125] In one embodiment, the compound of formula I or a salt thereof is [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] and [ka] or a pharmaceutically acceptable salt thereof; In the formula, R 2 is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules of Table 1.

[0126] In one embodiment, the compound of formula I is [ka] or a pharmaceutically acceptable salt thereof, wherein R 2 is a double-stranded siRNA molecule (eg, a double-stranded siRNA molecule selected from the double-stranded siRNA molecules of Table 1).

[0127] In one embodiment, the compound of formula I is [ka] or a pharmaceutically acceptable salt thereof, wherein R 2 is a double-stranded siRNA molecule (eg, a double-stranded siRNA molecule selected from the double-stranded siRNA molecules of Table 1).

[0128] In one embodiment, the compound of formula I is [ka] or a pharmaceutically acceptable salt thereof, wherein R 2 is a double-stranded siRNA molecule (eg, a double-stranded siRNA molecule selected from the double-stranded siRNA molecules of Table 1).

[0129] In one embodiment, the compound of formula I is [ka] or a pharmaceutically acceptable salt thereof, wherein R 2 is a double-stranded siRNA molecule (eg, a double-stranded siRNA molecule selected from the double-stranded siRNA molecules of Table 1).

[0130] In one embodiment, the compound of formula I is [ka] or a pharmaceutically acceptable salt thereof, wherein R 2 is a double-stranded siRNA molecule (eg, a double-stranded siRNA molecule selected from the double-stranded siRNA molecules of Table 1).

[0131] In one embodiment, the compound of formula I is [ka] or a pharmaceutically acceptable salt thereof, wherein R 2 is a double-stranded siRNA molecule (eg, a double-stranded siRNA molecule selected from the double-stranded siRNA molecules of Table 1).

[0132] In one embodiment, the compound of formula I is [ka] or a pharmaceutically acceptable salt thereof, wherein R 2 is a double-stranded siRNA molecule (eg, a double-stranded siRNA molecule selected from the double-stranded siRNA molecules of Table 1).

[0133] In one embodiment, the compound of formula I is [ka] or a pharmaceutically acceptable salt thereof, wherein R 2 is a double-stranded siRNA molecule (eg, a double-stranded siRNA molecule selected from the double-stranded siRNA molecules of Table 1).

[0134] In one embodiment, the compound of formula I is [ka] or a pharmaceutically acceptable salt thereof, wherein R 2 is a double-stranded siRNA molecule (eg, a double-stranded siRNA molecule selected from the double-stranded siRNA molecules of Table 1).

[0135] In one embodiment, the compound of formula I is [ka] or a pharmaceutically acceptable salt thereof, wherein R 2is a double-stranded siRNA molecule (eg, a double-stranded siRNA molecule selected from the double-stranded siRNA molecules of Table 1).

[0136] In one embodiment, the compound of formula I is [ka] or a pharmaceutically acceptable salt thereof, wherein R 2 is a double-stranded siRNA molecule (eg, a double-stranded siRNA molecule selected from the double-stranded siRNA molecules of Table 1).

[0137] In one embodiment, the compound of formula I is [ka] or a pharmaceutically acceptable salt thereof, wherein R 2 is a double-stranded siRNA molecule (eg, a double-stranded siRNA molecule selected from the double-stranded siRNA molecules of Table 1).

[0138] In one embodiment, the compound of formula I is [ka] or a pharmaceutically acceptable salt thereof, wherein R 2 is a double-stranded siRNA molecule (eg, a double-stranded siRNA molecule selected from the double-stranded siRNA molecules of Table 1).

[0139] In one embodiment, the compound of formula I is [ka] or a pharmaceutically acceptable salt thereof.

[0140] In one embodiment, the compound of formula I is [ka] or a pharmaceutically acceptable salt thereof.

[0141] In one embodiment, the compound of formula I is [ka] or a pharmaceutically acceptable salt thereof.

[0142] In one embodiment, the compound of formula I is [ka] or a pharmaceutically acceptable salt thereof.

[0143] In one embodiment, the compound of formula I is [ka] or a pharmaceutically acceptable salt thereof.

[0144] In one embodiment, the compound of formula I is [ka] or a pharmaceutically acceptable salt thereof.

[0145] In one embodiment, the compound of formula I is [ka] or a pharmaceutically acceptable salt thereof.

[0146] In one embodiment, the compound of formula I is [ka] or a pharmaceutically acceptable salt thereof.

[0147] In one embodiment, the present invention provides a compound of formula (I): [ka] or a salt thereof, During the ceremony, L 1 is absent or a bonding group, L 2 is absent or a bonding group, R 2 is a nucleic acid, Ring A is absent, a 3- to 20-membered cycloalkyl, a 5- to 20-membered aryl, a 5- to 20-membered heteroaryl, or a 3- to 20-membered heterocycloalkyl; Each R A are independently hydrogen, hydroxy, CN, F, Cl, Br, I, -C 1~2 Alkyl-OR B , C 1~10 Alkyl C 2~10 Alkenyl, and C 2~10 alkynyl, wherein C 1~10 Alkyl C 2~10 Alkenyl, and C 2~10 Alkynyl includes halo, hydroxy, and C 1~3 optionally substituted with one or more groups independently selected from alkoxy; R B is hydrogen, a protecting group, a covalent bond to a solid support, or a bond to a linking group attached to a solid support; where n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0148] In one embodiment, the present invention provides a compound of the formula: [ka] or a salt thereof, During the ceremony, L 2 is absent or a bonding group, R 2 is a nucleic acid, Ring A is absent, a 3- to 20-membered cycloalkyl, a 5- to 20-membered aryl, a 5- to 20-membered heteroaryl, or a 3- to 20-membered heterocycloalkyl; Each R Aare independently hydrogen, hydroxy, CN, F, Cl, Br, I, -C 1~2 Alkyl-OR B , C 1~10 Alkyl C 2~10 Alkenyl, and C 2~10 alkynyl, wherein C 1~10 Alkyl C 2~10 Alkenyl, and C 2~10 Alkynyl includes halo, hydroxy, and C 1~3 optionally substituted with one or more groups independently selected from alkoxy; R B is hydrogen, a protecting group, a covalent bond to a solid support, or a bond to a linking group attached to a solid support; n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0149] In one embodiment, the present invention provides a compound of the formula: [ka] or a salt thereof, During the ceremony, L 1 is absent or a bonding group, L 2 is absent or a bonding group, R 2 is a nucleic acid, B is divalent, and [ka] wherein: Each R' is independently C 1~9 Alkyl, C 2~9 Alkenyl, or C 2~9 Alkynyl, and this C 1~9 Alkyl, C 2~9 Alkenyl, or C 2~9 The alkynyl is optionally substituted with halo or hydroxyl; * The valence with a is L 1or L 1 If does not exist, R 1 is bound to ** The valence with a is L 2 or L 2 If does not exist, R 2 is bonded to.

[0150] In one embodiment, L 1 and L 2 are independently a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 50 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X - or -S-, optionally replaced by R X is hydrogen or (C1-C6) alkyl, and the hydrocarbon chain is optionally substituted with one or more substituents selected from (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.

[0151] In one embodiment, L 1 teeth, [ka] or salts thereof.

[0152] In one embodiment, L 1 is connected to B through a bond selected from the group consisting of -O-, -S-, -(C=O)-, -(C=O)-NH-, -NH-(C=O), -(C=O)-O-, -NH-(C=O)-NH-, or -NH-(SO2)- 1 is bonded to.

[0153] In one embodiment, L 1 teeth, [ka] is selected from the group consisting of:

[0154] In one embodiment, L 2 -O- through R 2 is bonded to.

[0155] In one embodiment, L 2 is C optionally substituted with hydroxy 1~4 It is alkylene-O-.

[0156] In one embodiment, L 2 does not exist.

[0157] In one embodiment, the present invention provides the compound [ka] or a salt thereof, wherein R 2 is a nucleic acid.

[0158] One aspect of the invention is a pharmaceutical composition comprising a compound of Formula I and a pharmaceutically acceptable carrier.

[0159] Another aspect of the invention is a method for delivering double-stranded siRNA to the liver of an animal, comprising administering to the animal a compound of formula I or a pharmaceutically acceptable salt thereof.

[0160] Another aspect of the invention is a method of treating a disease or disorder in an animal (e.g., liver disease or a viral infection such as hepatitis B virus infection), comprising administering to the animal a compound of formula I, or a pharmaceutically acceptable salt thereof.

[0161] Certain embodiments of the present invention provide a compound of formula (I) or a pharmaceutically acceptable salt thereof for use in medical therapy.

[0162] Certain embodiments of the present invention provide a compound of formula (I) or a pharmaceutically acceptable salt thereof for the prophylactic or therapeutic treatment of a disease or disorder in an animal (e.g., liver disease, or a viral infection such as hepatitis B virus infection).

[0163] Certain embodiments of the present invention provide for the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof to prepare a medicament for treating a disease or disorder in an animal (e.g., liver disease, or a viral infection such as hepatitis B virus infection).

[0164] In certain embodiments, the animal is a mammal, such as a human (eg, an HBV-infected patient).

[0165] In one embodiment, the compound of formula I has the following formula (Id): [ka] (Id) and During the ceremony, R 1d teeth, [ka] and [ka] is selected from X d is C2~ 10 is alkylene, n d is either 0 or 1, R 2d is a double-stranded siRNA molecule selected from the double-stranded siRNAs of Table 1, R 3d is H, a protecting group, a covalent bond to a solid support, or a bond to a linking group attached to a solid support.

[0166] In one embodiment, R 3d contains a linking group that attaches the remainder of the compound of formula Id to a solid support. 2d The nature of the linking group is not critical, as long as it is a suitable intermediate for preparing a compound of formula Id, which is a double-stranded siRNA molecule selected from the double-stranded siRNAs of Table 1.

[0167] In one embodiment, R 3d The linker has a molecular weight of about 20 daltons to about 1,000 daltons.

[0168] In one embodiment, R 3d The linker has a molecular weight of about 20 daltons to about 500 daltons.

[0169] In one embodiment, R 3d The linker separates the solid support from the remainder of the compound of Formula I by a length of about 5 angstroms to about 40 angstroms, inclusive.

[0170] In one embodiment, R 3d is a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having from 2 to 15 carbon atoms, in which one or more (e.g., 1, 2, 3, or 4) of the carbon atoms are optionally replaced with (-O-) or (-N(H)-), and in which the carbons of the chain are optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from (C-C)alkoxy, (C-C)cycloalkyl, (C-C)alkanoyl, (C-C)alkanoyloxy, (C-C)alkoxycarbonyl, (C-C)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0171] In one embodiment, R 3dis a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 2 to 10 carbon atoms, in which one or more (e.g., 1, 2, 3, or 4) of the carbon atoms are optionally replaced with (-O-) or (-N(H)-), and the chain optionally has carbons substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from (C-C)alkoxy, (C-C)cycloalkyl, (C-C)alkanoyl, (C-C)alkanoyloxy, (C-C)alkoxycarbonyl, (C-C)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0172] In one embodiment, R 3d The linker is -C(=O)CH2CH2C(=O)N(H)-.

[0173] In one embodiment, R 1d teeth, [ka] is.

[0174] In one embodiment, R 1d teeth, [ka] is.

[0175] In one embodiment, X d is a C8 alkylene.

[0176] In one embodiment, n d is 0.

[0177] In one embodiment, R 2d is an siRNA.

[0178] In one embodiment, R 3d is H.

[0179] In another embodiment, the compound of formula (Id) or a salt thereof is [ka] and salts thereof.

[0180] One aspect of the present invention is a pharmaceutical composition comprising a compound of formula (Id) and a pharmaceutically acceptable carrier.

[0181] One aspect of the present invention is a method for delivering double-stranded siRNA to the liver of an animal, comprising administering to the animal a compound of formula (Id) or a pharmaceutically acceptable salt thereof.

[0182] Another aspect of the invention is a method of treating a disease or disorder in an animal (e.g., a viral infection such as a hepatitis B virus infection), comprising administering to the animal a compound of formula (Id) or a pharmaceutically acceptable salt thereof.

[0183] Certain embodiments of the present invention provide a compound of formula (Id) or a pharmaceutically acceptable salt thereof for use in medical therapy.

[0184] Certain embodiments of the present invention provide a compound of formula (Id) or a pharmaceutically acceptable salt thereof for the prophylactic or therapeutic treatment of a disease or disorder (e.g., a viral infection, such as a hepatitis B virus infection) in an animal.

[0185] Certain embodiments of the present invention provide the use of a compound of formula (Id) or a pharmaceutically acceptable salt thereof for the preparation of a medicament for treating a disease or disorder (e.g., a viral infection, such as a hepatitis B virus infection) in an animal.

[0186] In certain embodiments, the animal is a mammal, such as a human (eg, an HBV-infected patient).

[0187] The present invention also provides synthetic intermediates and methods disclosed herein that are useful for preparing compounds of formula (Id). For example, the present invention provides intermediate compounds of formula Ie: [ka] (Ie) or a salt thereof, wherein R 1d teeth, [ka] is selected from X d is a C2-8 alkylene, n d is either 0 or 1, Pg 1 is H or a suitable protecting group, R 3d is H, a protecting group, a covalent bond to a solid support, or a bond to a linking group attached to a solid support. Figure 1 shows a targeting ligand / linker attached to a solid support and Pg 1 is a protecting group DMTr.

[0188] In one embodiment, Pg 1 is TMTr (trimethoxytrityl), DMTr (dimethoxytrityl), MMTr (monomethoxytrityl), or Tr (trityl).

[0189] The present invention also provides a method for preparing a compound of formula (Id) as described herein, comprising reacting a compound of formula (Ie) with a corresponding compound of formula (Ie): [ka] (Ie) (In the formula, X d is a C2-8 alkylene, n d is either 0 or 1, Pg 1 is H, R 3dis a covalent bond to a solid support or a bond to a linking group attached to a solid support), is subjected to solid phase nucleic acid synthesis conditions to form a corresponding compound of formula Id, where R 2d is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules of Table 1.

[0190] In one embodiment, the method comprises removing the compound from the solid support and reacting R 3d is H.

[0191] In one embodiment, the compound is a compound of formula Id: [ka] (Id) or its salts (In the formula, R 1d teeth, [ka] and [ka] is selected from X d is C2~ 10 is alkylene, N d is either 0 or 1, R 2d is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules of Table 1, R 3d is not H, a protecting group, a covalent bond to a solid support, or a bond to a linking group attached to a solid support).

[0192] In one embodiment, the compound is a compound of formula Ie: [ka] (Ie) or its salts (In the formula, R 1d teeth, [ka] is selected from X d is a C2-8 alkylene, n d is either 0 or 1, Pg 1 is H or a suitable protecting group, R 3d is not H, a protecting group, a covalent bond to a solid support, or a bond to a linking group attached to a solid support).

[0193] In one embodiment, R 3d is H.

[0194] In one embodiment, R 3d is a covalent bond to a solid support.

[0195] In one embodiment, R 3d is a bond to a linking group attached to a solid support, the linking group being a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having from 2 to 15 carbon atoms, in which one or more (e.g., 1, 2, 3, or 4) of the carbon atoms are optionally replaced with (-O-) or (-N(H)-), and in which the chain is optionally substituted on carbons with one or more (e.g., 1, 2, 3, or 4) substituents selected from (C-C)alkoxy, (C-C)cycloalkyl, (C-C)alkanoyl, (C-C)alkanoyloxy, (C-C)alkoxycarbonyl, (C-C)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0196] In one embodiment, R 3dis a bond to a linking group attached to a solid support, the linking group being a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having from 2 to 10 carbon atoms, in which one or more (e.g., 1, 2, 3, or 4) of the carbon atoms are optionally replaced with (-O-) or (-N(H)-), and in which the chain is optionally substituted on carbon with one or more (e.g., 1, 2, 3, or 4) substituents selected from (C-C)alkoxy, (C-C)cycloalkyl, (C-C)alkanoyl, (C-C)alkanoyloxy, (C-C)alkoxycarbonyl, (C-C)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0197] In one embodiment, R 3d is the bond to a linking group attached to a solid support, the linking group being -C(=O)CH2CH2C(=O)N(H)-.

[0198] In one embodiment, the present invention provides a compound of formula (I): [ka] (I) or a salt thereof, During the ceremony, R 1 is H or a synthetic activating group, L 1 is absent or a bonding group, L 2 is absent or a bonding group, R 2 is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules of Table 1, Ring A is absent, a 3- to 20-membered cycloalkyl, a 5- to 20-membered aryl, a 5- to 20-membered heteroaryl, or a 3- to 20-membered heterocycloalkyl; Each R A are independently hydrogen, hydroxy, CN, F, Cl, Br, I, -C 1~2Alkyl-OR B , C 1~10 Alkyl C 2~10 Alkenyl, and C 2~10 alkynyl, wherein C 1~10 Alkyl C 2~10 Alkenyl, and C 2~10 Alkynyl includes halo, hydroxy, and C 1~3 optionally substituted with one or more groups independently selected from alkoxy; R B is hydrogen, a protecting group, a covalent bond to a solid support, or a bond to a linking group attached to a solid support; n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0199] In one embodiment, the present invention provides a compound of formula (I): [ka] (I) or a salt thereof, During the ceremony, R 1 is the targeting ligand, L 1 is absent or a bonding group, L 2 is absent or a bonding group, R 2 is H or a synthetic activating group, Ring A is absent, a 3- to 20-membered cycloalkyl, a 5- to 20-membered aryl, a 5- to 20-membered heteroaryl, or a 3- to 20-membered heterocycloalkyl; Each R A are independently hydrogen, hydroxy, CN, F, Cl, Br, I, -C 1~2 Alkyl-OR B , C 1~10 Alkyl C 2~10 Alkenyl, and C 2~10 alkynyl, wherein C 1~10 Alkyl C 2~10 Alkenyl, and C 2~10Alkynyl includes halo, hydroxy, and C 1~3 optionally substituted with one or more groups independently selected from alkoxy; R B is hydrogen, a protecting group, a covalent bond to a solid support, or a bond to a linking group attached to a solid support; n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0200] In one embodiment, the present invention provides a compound of formula (Ig): [ka] (Ig) or a salt thereof, During the ceremony, B is -N- or -CH-; L 2 is a C optionally substituted with hydroxyl or halo 1~4 alkylene-O-; n is 0, 1, 2, 3, 4, 5, 6, or 7.

[0201] In one embodiment, the present invention provides a method for producing a pharmaceutical composition comprising: [ka] and salts thereof, During the ceremony, Q is -L 1 -R 1 and R' is C 1~9 Alkyl, C 2~9 Alkenyl, or C 2~9 Alkynyl, and this C 1~9 Alkyl, C 2~9 Alkenyl, or C 2~9 The alkynyl is optionally substituted with halo or hydroxyl.

[0202] In one embodiment, the present invention provides a method for producing a pharmaceutical composition comprising: [ka] and salts thereof, In the formula, Q is -L 1 -R 1 is.

[0203] In one embodiment, the present invention provides a compound of formula (Ig): [ka] (Ig) or a salt thereof, During the ceremony, B is -N- or -CH-; L 1 is absent or a bonding group, L 2 is a C optionally substituted with hydroxyl or halo 1~4 alkylene-O-; n is 0, 1, 2, 3, 4, 5, 6, or 7; R 1 is H or a synthetic activating group, R 2 is H or a synthetic activating group.

[0204] In one embodiment, the present invention provides a method for producing a pharmaceutical composition comprising: [ka] or a salt thereof, In the formula, Q is -L 1 -R 1 and L 1 is absent or a bonding group, R' is C 1~9 Alkyl, C 2~9 Alkenyl, or C 2~9 Alkynyl, and this C 1~9 Alkyl, C 2~9 Alkenyl, or C 2~9 The alkynyl is optionally substituted with halo or hydroxyl; R1 is H or a synthetic activating group, R 2 is H or a synthetic activating group.

[0205] In one embodiment, the present invention provides a method for producing a pharmaceutical composition comprising: [ka] or a salt thereof, During the ceremony, Q is -L 1 -R 1 and L 1 is absent or a bonding group, R 1 is H or a synthetic activating group, R 2 is H or a synthetic activating group.

[0206] In one embodiment, R 1 is H or a synthetic activating group that can be derived from DCC, HOBt, EDC, BOP, PyBOP, or HBTU.

[0207] In one embodiment, R 2 is H, acetate, triflate, mesylate, or succinate.

[0208] In one embodiment, R 1 is a synthetic activating group that can be derived from DCC, HOBt, EDC, BOP, PyBOP, or HBTU.

[0209] In one embodiment, R 2 is acetate, triflate, mesylate, or succinate.

[0210] In one embodiment, L 1is a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 5 to 20 carbon atoms, in which one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are optionally replaced and are -O-, -NH-, -NH-C(=O)-, -C(=O)-NH-, or -S-.

[0211] In one embodiment, the present invention provides a compound of formula (XX): [ka] (XX) or a salt thereof, During the ceremony, R 1 is the targeting ligand, L 1 is absent or a bonding group, L 2 is absent or a bonding group, R 2 is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules of Table 1, B is divalent, and [ka] [ka] wherein: Each R' is independently C 1~9 Alkyl, C 2~9 Alkenyl, or C 2~9 Alkynyl, and this C 1~9 Alkyl, C 2~9 Alkenyl, or C 2~9 The alkynyl is optionally substituted with halo or hydroxyl; * The valence with a is L 1 or L 1 If does not exist, R 1 is bound to ** The valence with a is L2 or L 2 If does not exist, R 2 is bonded to.

[0212] In one embodiment, R 1 contains 2 to 8 sugars.

[0213] In one embodiment, R 1 contains 2 to 6 sugars.

[0214] In one embodiment, R 1 contains 2 to 4 sugars.

[0215] In one embodiment, R 1 contains 3 to 8 sugars.

[0216] In one embodiment, R 1 contains 3 to 6 sugars.

[0217] In one embodiment, R 1 contains 3 to 4 sugars.

[0218] In one embodiment, R 1 contains three sugars.

[0219] In one embodiment, R 1 contains four sugars.

[0220] In one embodiment, R 1 is expressed as follows: [ka] and During the ceremony, B 1 is a trivalent group containing from about 1 to about 20 atoms, and L 1 , T 1 , and T 2 is covalently bonded to B 2 is a trivalent group containing about 1 to about 20 atoms, and T 1 , T3 , and T 4 is covalently bonded to B 3 is a trivalent group containing about 1 to about 20 atoms, and T 2 , T 5 , and T 6 is covalently bonded to T 1 is absent or a bonding group, T 2 is absent or a bonding group, T 3 is absent or a bonding group, T 4 is absent or a bonding group, T 5 is absent or a bonding group, T 6 is absent or a bonding group.

[0221] In one embodiment, each saccharide is independently: [ka] is selected from During the ceremony, X is NR 3 and Y is -(C=O)R 4 , -SO2R 5 , and —(C═O)NR 6 R 7 or X is —(C═O)— and Y is NR 8 R 9 and R 3 is hydrogen or (C1-C4) alkyl, R 4 , R 5 , R 6 , R 7 , R 8 , and R 9are each independently selected from the group consisting of hydrogen, (C1-C8)alkyl, (C1-C8)haloalkyl, (C1-C8)alkoxy, and (C3-C6)cycloalkyl optionally substituted with one or more groups independently selected from the group consisting of halo, (C1-C4)alkyl, (C1-C4)haloalkyl, (C1-C4)alkoxy, and (C1-C4)haloalkoxy; R 10 -OH, -NR 8 R 9 , or -F, R 11 -OH, -NR 8 R 9 , -F, or a 5-membered heterocycle optionally substituted with one or more groups independently selected from the group consisting of halo, hydroxyl, carboxyl, amino, (C1-C4) alkyl, (C1-C4) haloalkyl, (C1-C4) alkoxy, and (C1-C4) haloalkoxy.

[0222] In one embodiment, each saccharide is independently: [ka] is selected from the group consisting of:

[0223] In one embodiment, each saccharide is independently: [ka] is.

[0224] In one embodiment, T 1 and T 2 One of them does not exist.

[0225] In one embodiment, T 1 and T 2 Neither of these exist.

[0226] In one embodiment, T 1 , T 2 , T 3 , T 4, T 5 , and T 6 are each independently absent or a branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 50 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X - or -S-, optionally replaced by R X is hydrogen or (C1-C6) alkyl, and the hydrocarbon chain is optionally substituted with one or more (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.

[0227] In one embodiment, T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 are each independently absent or a branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 20 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X - or -S-, optionally replaced by 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.

[0228] In one embodiment, T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 are each independently absent or a branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 50 carbon atoms, or a salt thereof, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are -O- or -NR X - is optionally replaced by R X is hydrogen or (C1-C6) alkyl, and the hydrocarbon chain is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from halo, hydroxy, and oxo (=O).

[0229] In one embodiment, T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 are each independently absent or a branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 20 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are optionally replaced with -O-, and the hydrocarbon chain is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from halo, hydroxy, and oxo (=O).

[0230] In one embodiment, T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 are each independently absent or a branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 20 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are optionally replaced with -O-, and the hydrocarbon chain is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from halo, hydroxy, and oxo (=O).

[0231] In one embodiment, T 3 , T 4 , T 5 , and T 6 At least one of the [ka] and During the ceremony, n=1, 2, 3.

[0232] In one embodiment, T 3 , T 4 , T 5 , and T 6 Each of the [ka] is selected from the group consisting of During the ceremony, n=1, 2, 3.

[0233] In one embodiment, T 1 and T 2 At least one of these is glycine.

[0234] In one embodiment, T 1 and T 2 Each of is glycine.

[0235] In one embodiment, B 1 is a trivalent group containing 1 to 15 atoms, and L 1 , T 1 , and T 2 is covalently bonded to

[0236] In one embodiment, B 1 is a trivalent group containing 1 to 10 atoms, and L 1 , T 1 , and T 2 is covalently bonded to

[0237] In one embodiment, B 1 includes (C1-C6) alkyl.

[0238] In one embodiment, B 1 is C 3~8 Includes cycloalkyl.

[0239] In one embodiment, B 1 contains a silyl group.

[0240] In one embodiment, B 1 includes D-amino acids or L-amino acids.

[0241] In one embodiment, B 1 contains sugars.

[0242] In one embodiment, B 1 contains a phosphate group.

[0243] In one embodiment, B 1 contains a phosphonic acid group.

[0244] In one embodiment, B 1 includes aryl.

[0245] In one embodiment, B 1 contains a phenyl ring.

[0246] In one embodiment, B 1is a phenyl ring.

[0247] In one embodiment, B 1 is CH.

[0248] In one embodiment, B 1 includes heteroaryl.

[0249] In one embodiment, B 1 teeth, [ka] is selected from the group consisting of:

[0250] In one embodiment, B 1 teeth, [ka] is selected from the group consisting of:

[0251] In one embodiment, B 2 is a trivalent group containing 1 to 15 atoms, and L 1 , T 1 , and T 2 is covalently bonded to

[0252] In one embodiment, B 2 is a trivalent group containing 1 to 10 atoms, and L 1 , T 1 , and T 2 is covalently bonded to

[0253] In one embodiment, B 2 includes (C1-C6) alkyl.

[0254] In one embodiment, B 2 is C 3~8 Includes cycloalkyl.

[0255] In one embodiment, B 2 contains a silyl group.

[0256] In one embodiment, B 2 includes D-amino acids or L-amino acids.

[0257] In one embodiment, B 2 contains sugars.

[0258] In one embodiment, B 2 contains a phosphate group.

[0259] In one embodiment, B 2 contains a phosphonic acid group.

[0260] In one embodiment, B 2 includes aryl.

[0261] In one embodiment, B 2 contains a phenyl ring.

[0262] In one embodiment, B 2 is a phenyl ring.

[0263] In one embodiment, B 2 is CH.

[0264] In one embodiment, B 2 includes heteroaryl.

[0265] In one embodiment, B 2 teeth, [ka] is selected from the group consisting of:

[0266] In one embodiment, B 2 teeth, [ka] or salts thereof.

[0267] In one embodiment, B 3is a trivalent group containing 1 to 15 atoms, and L 1 , T 1 , and T 2 is covalently bonded to

[0268] In one embodiment, B 3 is a trivalent group containing 1 to 10 atoms, and L 1 , T 1 , and T 2 is covalently bonded to

[0269] In one embodiment, B 3 includes (C1-C6) alkyl.

[0270] In one embodiment, B 3 is C 3~8 Includes cycloalkyl.

[0271] In one embodiment, B 3 contains a silyl group.

[0272] In one embodiment, B 3 includes D-amino acids or L-amino acids.

[0273] In one embodiment, B 3 contains sugars.

[0274] In one embodiment, B 3 contains a phosphate group.

[0275] In one embodiment, B 3 contains a phosphonic acid group.

[0276] In one embodiment, B 3 includes aryl.

[0277] In one embodiment, B 3 contains a phenyl ring.

[0278] In one embodiment, B 3 is a phenyl ring.

[0279] In one embodiment, B 3 is CH.

[0280] In one embodiment, B 3 includes heteroaryl.

[0281] In one embodiment, B 3 teeth, [ka] is selected from the group consisting of:

[0282] In one embodiment, B 3 teeth, [ka] or salts thereof.

[0283] In one embodiment, L 1 and L 2 are independently a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 50 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X - or -S-, optionally replaced by R X is hydrogen or (C1-C6) alkyl, and the hydrocarbon chain is optionally substituted with one or more (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.

[0284] In one embodiment, L 1 teeth, [ka] or salts thereof.

[0285] In one embodiment, L 1 is connected to B through a bond selected from the group consisting of -O-, -S-, -(C=O)-, -(C=O)-NH-, -NH-(C=O), -(C=O)-O-, -NH-(C=O)-NH-, or -NH-(SO2)- 1 is bonded to.

[0286] In one embodiment, L 1 teeth, [ka] is selected from the group consisting of:

[0287] In one embodiment, L 2 -O- through R 2 is bonded to.

[0288] In one embodiment, L 2 is C optionally substituted with hydroxy 1~4 It is alkylene-O-.

[0289] In one embodiment, L 2 -O- through R 2 is bonded to.

[0290] In one embodiment, L 2 does not exist.

[0291] In one embodiment, the present invention provides a method for producing a pharmaceutical composition comprising: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] and pharmaceutically acceptable salts thereof, wherein R 2 is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules of Table 1.

[0292] In one embodiment, the present invention provides a compound of the formula: [ka] or a salt thereof, wherein R 2 is a nucleic acid.

[0293] In one embodiment, the present invention provides a compound of the formula: [ka] or a salt thereof, wherein R 2 is a nucleic acid.

[0294] In one embodiment, the nucleic acid molecule (eg, siRNA) is linked to the rest of the compound via the phosphate oxygen at the 3' end of the sense strand.

[0295] In one embodiment, the compound or salt is administered subcutaneously.

[0296] When the compound contains a group of the formula: [ka] Four stereoisomers are possible for the ring (two cis and two trans). Unless otherwise specified, the compounds of the present invention include all four stereoisomers for such ring. In one embodiment, the two R' groups are in the cis conformation. In one embodiment, the two R' groups are in the trans conformation.

[0297] One aspect of the present invention is (a) one or more double-stranded siRNA molecules selected from the double-stranded siRNA molecules of Table 1; (b) a cationic lipid; (c) non-cationic lipids and The nucleic acid-lipid particle comprises: [Example]

[0298] The present invention will be further described in detail by specific examples.The following examples are presented for illustrative purposes and are not intended to limit the present invention in any way.Those skilled in the art will easily recognize various non-critical parameters that can be changed or modified to essentially achieve the same results.It is understood that in one embodiment, oligonucleotide is the double-stranded siRNA molecule described in Table 1.

[0299] Example 1. Synthesis of Conjugate 1 Scheme 1. [ka] Scheme 2. [ka] Scheme 3. [ka] Scheme 4. [ka] Scheme 5. [ka] [ka] [ka] [ka]

[0300] Step 1. Preparation of 2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate 3 [ka] A solution of tetraethylene glycol (934 g, 4.8 mol) in THF (175 mL) and aqueous NaOH (5 M, 145 mL) was cooled (0 °C) and treated with p-toluenesulfonyl chloride (91.4 g, 480 mmol) dissolved in THF (605 mL), then stirred (0 °C) for 2 h. The mixture was diluted with water (3 L) and extracted with CHCl (3 × 500 mL). The combined extracts were washed with water and brine, then dried (MgSO), filtered, and concentrated to give 2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (3) (140 g, 84%) as a pale yellow oil. f (0.57, 10% MeOH-CH2Cl2).

[0301] Step 2. Preparation of 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethan-1-ol (4) [ka] A solution of 3 (140 g, 403 mmol) in DMF (880 mL) was treated with sodium azide (131 g, 2.02 mol) and heated (45 °C) overnight. Most of the DMF was removed under reduced pressure, and the residue was dissolved in CHCl (500 mL), washed with brine (3 × 500 mL), then dried (MgSO), filtered, and concentrated. The residue was passed through a short silica bed (5% MeOH-CHCl) and concentrated to give 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethan-1-ol (4) (65 g, 74%) as a yellow oil. f (0.56, 10% MeOH-CH2Cl2).

[0302] Step 3. Preparation of peracetylated galactosamine (6) [ka] D-Galactosamine hydrochloride (5) (250 g, 1.16 mol) in pyridine (1.5 L) was treated with acetic anhydride (1.25 L, 13.2 mol) for 45 minutes. After stirring overnight, the reaction mixture was divided into three 1 L portions. Each 1 L portion was poured into 3 L of ice water and mixed for 1 hour. After mixing, the solids were filtered off, combined, frozen in liquid nitrogen, and lyophilized for 5 days to give peracetylated galactosamine (6) (369.4 g, 82%) as a white solid. Rf (0.58, 10% MeOH-CHCl).

[0303] Step 4. Preparation of (3aR,5R,6R,7R,7aR)-5-(acetoxymethyl)-2-methyl-3a,6,7,7a-tetrahydro-5H-pyrano[3,2-d]oxazole-6,7-diyl diacetate (7) [ka] A solution of peracetylated galactosamine 6 (8.45 g, 21.7 mmol) in CHCl (320 mL) was treated dropwise with TMSOTf (4.32 mL, 23.9 mmol). After stirring (1.5 h, 40 °C), the reaction was quenched by the addition of triethylamine (5 mL) and concentrated to dryness to give compound 7 as a pale yellow glass (7.2 g, quantitative). This product was used without further purification. Rf (0.59, 10% MeOH-CH2Cl2).

[0304] Step 5. Preparation of (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-3,4-diyl diacetate (8) [ka] Compound 7 (7.2 g, 21.7 mmol) and 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethan-1-ol 4 (2.65 g, 15.2 mmol) were azeotroped (three times) from toluene (150 mL) to remove traces of water. The dried material was dissolved in 1,2-dichloroethane (150 mL), cooled (ca. 5 °C), and treated with TMSOTf (784 μL, 4.34 mmol). After stirring overnight, the reaction was quenched by the addition of triethylamine (5 mL) and concentrated. The residue was purified by chromatography (1% to 5% MeOH-CHCl) to give 8 (7.12 g, 85%) as a brown oil. Rf (0.3, 10% MeOH-CHCl).

[0305] Step 6. Preparation of 2-(2-(2-(2-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethan-1-aminium 2,2,2-trifluoroacetate (9) [ka] A solution of azide 8 (7.12 g, 13 mmol) in EtOAc (150 mL) and trifluoroacetic acid (2 mL) was treated with palladium on carbon (1.5 g, 10% w / w wet basis). The reaction mixture was then purged with hydrogen and stirred vigorously overnight. After purging with nitrogen, the mixture was filtered through Celite and rinsed with MeOH. The filtrate was concentrated and purified by chromatography (5% → 10% → 20% MeOH-CHCl) to give 9 (5.8 g, 72%) as a brown oil. Rf (0.34, 15% MeOH-CHCl).

[0306] Step 7. Preparation of di-tert-butyl 4-(((benzyloxy)carbonyl)amino)-4-(3-(tert-butoxy)-3-oxopropyl)heptanedioate (11) [ka] Di-tert-butyl 4-amino-4-(3-(tert-butoxy)-3-oxopropyl)heptanedioate (10) (13.5 g, 33 mmol), 25% NaCO 3(水溶液) To a solution of 150 mL of hexane and 300 mL of dichloromethane was slowly added benzyl chloroformate (14 mL, 98 mmol). The solution was vigorously stirred at room temperature overnight (16 h). Upon completion, additional dichloromethane (100 mL) was added and the dichloromethane layer was separated. The aqueous layer was extracted with dichloromethane (2 x 100 mL). The combined dichloromethane extracts were dried over magnesium sulfate, filtered, and concentrated to dryness. The product 11 was isolated as a colorless oil and required no further purification (15.8 g, 88%). Rf (0.7, 1:1 EtOAc-hexane).

[0307] Step 8. Preparation of 4-(((benzyloxy)carbonyl)amino)-4-(2-carboxyethyl)heptanedioic acid (12) [ka] 12 A solution of 11 (15.6 g, 28.8 mmol) in formic acid (50 mL) was stirred at room temperature for 2 h. The solution was concentrated to dryness and dissolved in ethyl acetate (approximately 25 mL). Upon standing, the product crystallized as a colorless solid. The solid was filtered, washed with ethyl acetate, and air-dried to give 12 (10.2 g, 93%) as a colorless solid. Rf (0.1, 10% MeOH-CH2Cl2).

[0308] Step 9. Preparation of Compound 13 [ka] 13 A solution of 12 (793 mg, 2.08 mmol) and 9 (5.8 g, 9.36 mmol) in DMF (50 mL) was treated with BOP (3.67 g, 8.32 mmol) followed by N,N-diisopropylethylamine (4.31 mL, 25 mmol). After stirring overnight, the mixture was concentrated to dryness and chromatographed (1% → 2% → 5% → 10% → 15% MeOH-CHCl) to give 13 (5.71 g [crude], >100%—contained a coupling by-product that did not affect the next step). Rf (0.45, 10% MeOH-CHCl).

[0309] Step 10. Preparation of Compound 14 [ka] 14 Compound 13 (5.7 g) was dissolved in MeOH (150 mL) and TFA (1.5 mL) and treated with palladium on carbon (1 g, 10% w / w wet basis). The reaction mixture was then purged with hydrogen and vigorously stirred overnight. After purging with nitrogen, the mixture was filtered through Celite and rinsed with MeOH. The filtrate was concentrated and purified by chromatography (5% → 10% → 20% MeOH-CHCl) to give 14 as a brown oil (2.15 g, 56% over two steps). Rf (0.32, 10% MeOH-CHCl).

[0310] Step 11. Preparation of (5-amino-1,3-phenylene)dimethanol (15) [ka] A solution of dimethyl 5-aminoisophthalate (20.0 g, 96 mmol) in THF (350 mL) was added dropwise over 1 h to a refluxing mixture of 3.75 equivalents of LiAlH (13.6 g, 358 mmol) in THF (440 mL). The mixture was stirred at reflux for an additional 2 h, then cooled to room temperature and quenched by the careful addition of MeOH (27 mL) followed by water (40 mL). After stirring the quenched mixture for 2 h, it was filtered and concentrated to dryness. The residue was recrystallized from EtOAc (twice) to give 15 (10.2 g, 70%) as brownish-yellow crystals.

[0311] Step 12. Preparation of methyl 10-((3,5-bis(hydroxymethyl)phenyl)amino)-10-oxodecanoate (16) [ka] A solution of methyl sebacate (3.8 g, 17 mmol), 15 (2.5 g, 17 mmol), and EEDQ (8.1 g, 33 mmol) in 2:1 dichloromethane / methanol (200 mL) was stirred at room temperature for 2 h. Upon completion, the solution was concentrated to dryness. The resulting solid was triturated with dichloromethane (50 mL) and filtered. The solid was rinsed with cold dichloromethane and air-dried to give 16 as a colorless solid (4.3 g, 72%). Rf (0.33, EtOAc).

[0312] Step 13. Preparation of methyl 10-((3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(hydroxymethyl)phenyl)amino)-10-oxodecanoate (17) [ka] To a solution of 16 (4.3 g, 12 mmol) in pyridine (50 mL) was added 4,4'-(chloro(phenyl)methylene)bis(methoxybenzene) (4.1 g, 12 mmol). The solution was stirred at room temperature overnight under nitrogen. Upon completion, the solution was concentrated to dryness, and the residue was purified by column chromatography (0.5% → 0.75% → 1% → 1.5% MeOH-CHCl) to give 17 as a yellow solid (2.9 g, 35%). Rf (0.6, 10% MeOH-CHCl).

[0313] Step 14. Preparation of lithium 10-((3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(hydroxymethyl)phenyl)amino)-10-oxodecanoate (18) [ka] To a solution of 17 (2.9 g, 4.3 mmol) in THF (60 mL) was added water (15 mL) and lithium hydroxide (112 mg, 4.7 mmol). The solution was stirred at room temperature overnight. Upon completion, the solution was concentrated to remove the THF. The remaining aqueous solution was flash frozen in liquid nitrogen and lyophilized overnight to give a colorless solid (2.9 g, quantitative). Rf (0.3, 10% MeOH-CH2Cl2).

[0314] Step 15. Preparation of Compound 19 [ka] To a solution of 14 (454 mg, 0.67 mmol), 18 (1.25 g, 0.67 mmol), and HBTU (381 mg, 1.0 mmol) in anhydrous DMF (25 mL) was added N,N-diisopropylethylamine (0.35 mL, 2.0 mmol). The solution was stirred at room temperature overnight. Upon completion, the solution was poured into ethyl acetate (250 mL) and washed with brine (3 × 200 mL). The ethyl acetate layer was dried over magnesium sulfate, filtered, and concentrated to dryness. Purification by column chromatography (5% → 7.5% → 10% → 15% MeOH in CHCl) afforded 19 (1.5 g, 94%) as a pale orange foam. Rf (0.25, 10% MeOH-CHCl).

[0315] Step 16. Preparation of Compound 20 [ka] A solution of compound 19 (1.5 g, 0.6 mmol), succinic anhydride (120 mg, 1.2 mmol), DMAP (220 mg, 1.8 mmol), and trimethylamine (250 μL, 1.8 mmol) in anhydrous CHCl (50 mL) was stirred overnight at room temperature. Upon completion, the solution was concentrated to dryness and filtered through a short silica plug (100% CHCl → 15% MeOH in CHCl) to give product 20 as a light beige foam (1.1 g, 70%). Mass m / z (ES-TOF MS) 727.7 [M + 3H - DMTr] + , 1091.1 [M + 2H - DMTr]. 1H NMR (400 MHz, CDCl3) δ 8.92 (br s, 1H), 7.78 (s, 1H), 7.49-7.47 (m, 3H), 7.41 (br s, 1H), 7.38-7.34 (m, 5H), 7.32-7.26 (m, 4H), 7.24-7.08 (br s, 3H), 7.08 (s, 1H), 6.90-6.80 (m, 7H), 5.31 (d, 3H, J = 2.7Hz), 5.12 (s, 2H), 5.06 (dd,3H, J = 11.2, 3.2 Hz), 4.78 (d, 3H, J = 8.5 Hz), 4.24-4.08 (m, 12H), 3.95-3.88 (m, 7H), 3.85-3.76 (m, 4H), 3.78 (s, 6H), 3.68-3.56(m, 34H), 3.54-3.44 (m, 8H), 3.41-3.33 (m,6H), 2.70-2.60 (m, 4H), 2.52-2.30 (m, 30H), 2.24-2.16 (m, 8H), 2.14 (s, 9H), 2.04 (s, 9H), 2.02-1.96 (m, 6H), 1.98 (s, 9H), 1.96 (s, 9H), 1.74-1.52 (m, 4H), 1.36-1.24 (m, 12H).

[0316] ステップ17.コンジュゲート1のmodulation

change

[0317] Example 2: Synthesis of conjugate 34 Scheme 6. [ka] Scheme 7. [ka] Scheme 8. [ka]

[0318] Step 1. Preparation of di-tert-butyl 4-(2-(((benzyloxy)carbonyl)amino)acetamido)-4-(3-(tert-butoxy)-3-oxopropyl)heptanedioate (21) [ka] A solution of di-tert-butyl 4-amino-4-(3-(tert-butoxy)-3-oxopropyl)heptanedioate (25 g, 60 mmol) and Z-glycine (18.9 g, 90.2 mmol) in CHCl (300 mL) was treated sequentially with EDC (23 g, 120 mmol), diisopropylethylamine (32 mL, 180 mmol), and DMAP (catalytic, 17 mg). After stirring (16 h), the reaction mixture was poured into NaHCO (saturated aqueous solution), extracted with CHCl, washed with brine, dried (MgSO), filtered, and concentrated to give di-tert-butyl 4-(2-(((benzyloxy)carbonyl)amino)acetamido)-4-(3-(tert-butoxy)-3-oxopropyl)heptanedioate 21 as an amorphous solid, which was used without further treatment (36 g, quantitative). Rf (0.85, 10% MeOH—CHCl).

[0319] Step 2. Preparation of 4-(2-(((benzyloxy)carbonyl)amino)acetamido)-4-(2-carboxyethyl)heptanedioic acid (22) [ka] A solution of di-tert-butyl 4-(2-(((benzyloxy)carbonyl)amino)acetamido)-4-(3-(tert-butoxy)-3-oxopropyl)heptanedioate (21) (59.3 mmol, 36 g) was stirred in neat formic acid (150 mL) for 72 h. Upon completion, the formic acid was removed under reduced pressure and the crude solid was dried overnight under high vacuum to give 22 as a colorless solid (15.9 g, 61%). Rf (0.15, 10% MeOH-CHCl).

[0320] Step 3. Preparation of Compound 23 [ka] A solution of 22 (6.2 g, 14.1 mmol) and 2-(2-(2-(2-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethan-1-aminium 2,2,2-trifluoroacetate (35 g, 56.5 mmol) in DMF (250 mL) was treated with BOP (25 g, 56.5 mmol) followed by N,N-diisopropylethylamine (29 mL, 170 mmol). After stirring overnight, the mixture was concentrated to dryness and chromatographed (100% CHCl to 15% MeOH—CHCl) to give compound 23 (24.6 g, 89%). Rf (0.55, 15% MeOH-CH2Cl2).

[0321] Step 4. Preparation of Compound 24 [ka] Compound 23 (24.6 g) was dissolved in MeOH (200 mL) and TFA (1.5 mL) and purged with nitrogen. Palladium on carbon (1 g, 10% w / w wet basis) was added, and the reaction mixture was then purged with hydrogen and vigorously stirred overnight. Upon completion, the reaction was purged with nitrogen, filtered through Celite, and rinsed with MeOH. The filtrate was concentrated and purified by column chromatography on silica gel 60 (gradient: 5% → 10% → 20% MeOH-CHCl) to give 24 (23 g) as a light brown viscous oil. Rf (0.32, 10% MeOH-CHCl).

[0322] Step 5. Preparation of (5-amino-1,3-phenylene)dimethanol (26) [ka] A suspension of lithium aluminum hydride (13.6 g, 358 mmol) in anhydrous tetrahydrofuran (450 mL) was refluxed under a nitrogen atmosphere and treated dropwise with a solution of dimethyl-5-aminoisophthalate (25) (20 g, 96 mmol) in anhydrous tetrahydrofuran (350 mL). After the addition was complete, the mixture was heated at reflux for an additional 2 h. Upon completion, the solution was cooled to room temperature and quenched by the slow addition of MeOH (27 mL) followed by water (40 mL). After stirring for 2 h, the mixture was filtered, concentrated, and recrystallized from EtOAc to give (5-amino-1,3-phenylene)dimethanol (26) as off-white crystals (10.2 g, 70%). Rf 0.5 (15% MeOH-CHCl).

[0323] Step 6. Preparation of 3,5-bis(hydroxymethyl)benzonitrile (27) [ka] A solution of 26 (5 g, 33 mmol) in 2 N hydrochloric acid (100 mL) was cooled to 0 °C and treated with a cold solution of sodium nitrite (3.53 g, 36 mmol) in water (50 mL). The reaction mixture was maintained at a temperature below 5 °C for 30 min and then treated with a single portion of a solution of copper(I) cyanide (3.19 g, 35.6 mmol) and sodium cyanide (3.53 g, 72 mmol) in water (50 mL). After stirring overnight at room temperature, the mixture was filtered, extracted with dichloromethane (3 × 100 mL), concentrated, and used without further purification. The diol, 3,5-bis(hydroxymethyl)benzonitrile (27), was obtained as a yellow solid (2.19 g, 41%). Rf 0.75 (15% MeOH-CHCl).

[0324] Step 7. Preparation of 3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(hydroxymethyl)benzonitrile (28) [ka] A solution of 3,5-bis(hydroxymethyl)benzonitrile (27) (538 mg, 3.3 mmol) in pyridine (14 mL) was treated with 4,4'-dimethoxytrityl chloride (1.17 g, 3.46 mmol) and stirred at room temperature overnight. Upon completion, the mixture was concentrated, dispersed in diethyl ether (25 mL), filtered, and concentrated. The crude product was purified by column chromatography on silica gel 60 (gradient: 10% to 50% EtOAc-hexanes) to give 28 as a yellow solid (725 mg, 47%). Rf 0.5 (1:1 EtOAc-hexanes).

[0325] Step 8. Preparation of (3-(aminomethyl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)phenyl)methanol (29) [ka] A solution of 28 (100 mg, 0.22 mmol) in methyltetrahydrofuran (5 mL) was cooled to 0 °C and slowly treated with lithium aluminum hydride (0.64 mmol = 0.28 mL of 2.3 M MeTHF solution). After stirring for 1 h, the reaction was quenched by adding methanol (1 mL) followed by water (0.3 mL) and stirred for 30 min. The mixture was filtered and concentrated to give (3-(aminomethyl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)phenyl)methanol (29) (78 mg, 77%). Rf 0.15 (10% MeOH-CHCl).

[0326] Step 9. Preparation of methyl 10-((3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(hydroxymethyl)benzyl)amino)-10-oxodecanoate (30) [ka] A solution of (3-(aminomethyl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)-methyl)phenyl)methanol (29) (78 mg, 0.17 mmol) and monomethyl sebacate (38 mg, 0.17 mmol) in dichloromethane (5 mL) was treated sequentially with EDC (48 mg, 0.25 mmol), DMAP (catalytic, 5 mg), and diisopropylethylamine (57 μL, 0.33 mmol). After stirring (3.5 h), the reaction mixture was poured into saturated sodium bicarbonate solution (50 mL). The sodium bicarbonate solution was extracted with dichloromethane (3 × 50 mL), washed with brine (50 mL), dried over magnesium sulfate, filtered, and concentrated to dryness. The crude material was purified by column chromatography on silica gel 60 (gradient: 2% to 5% MeOH-CH2Cl2) to give methyl 10-((3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(hydroxymethyl)benzyl)amino)-10-oxodecanoate (30) as a yellow oil (57 mg, 53%). Rf 0.45 (10% MeOH-CH2Cl2).

[0327] Step 10. Preparation of lithium 10-((3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(hydroxymethyl)benzyl)amino)-10-oxodecanoate (31) [ka] Compound 30 (188 mg, 0.28 mmol) was dissolved in tetrahydrofuran (5 mL) and treated with a solution of LiOH (7 mg, 0.30 mmol) in water (1 mL). Upon completion, the tetrahydrofuran was removed in vacuo, and the remaining aqueous mixture was frozen and lyophilized to give lithium 10-((3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(hydroxymethyl)benzyl)amino)-10-oxodecanoate (31) as a colorless solid (180 mg, 99%). Rf 0.45 (10% MeOH-CHCl).

[0328] Step 11. Preparation of Compounds 32, 33, and 34 Compounds 32, 33, and 34 were prepared according to the same procedures used to synthesize compounds 19, 20, and 1, respectively.

[0329] Example 3. Synthesis of conjugate 36 [ka]

[0330] Step 1. Preparation of conjugate 36 The same procedure used to synthesize compound 34 and all corresponding intermediates was used to prepare conjugate 36. The only exception was the synthesis of compound 6, where propanoic anhydride was used instead of acetic anhydride.

[0331] Example 4. Synthesis of conjugate 42 Scheme 9. [ka] Scheme 10. [ka] [ka]

[0332] Step 1. Preparation of compound 37 [ka] To a solution of 18β-glycyrrhetinic acid (2.5 g, 5.3 mmol), tert-butyl (3-aminopropyl)carbamate (1.1 g, 6.4 mmol), and HBTU (3.0 g, 8.0 mmol) in N,N-dimethylformamide (20 mL) was added diisopropylethylamine (2.75 mL, 15.9 mmol). The solution was stirred at room temperature overnight. Upon completion, the solution was concentrated to dryness in vacuo. The residue was purified by column chromatography on silica gel 60 (gradient: 2% to 5% MeOH / CHCl) to give the product as a colorless solid (2.1 g, 63%).

[0333] Step 2. Preparation of Compound 38 [ka] To a solution of 37 (2.1 g, 3.3 mmol) and triethylamine (3.5 mL, 10 mmol) in dichloromethane (25 mL) was added acetic anhydride (850 μL, 5.3 mmol) and DMAP (5 mg). The solution was stirred at room temperature overnight. Upon completion, the solution was concentrated to dryness, dissolved in ethyl acetate (100 mL), washed with water (100 mL), dried over magnesium sulfate, filtered, and concentrated to dryness to give a light brown foam (1.9 g, 85%).

[0334] Step 3. Preparation of Compound 39 [ka] To a solution of 38 (1.5 g, 2.3 mmol) in anhydrous dioxane (25 mL) was added 2 M hydrogen chloride in dioxane (25 mL). The solution was stirred at room temperature overnight and then concentrated to dryness in vacuo to give a light brown solid (1.3 g, 96%).

[0335] Step 4. Preparation of compounds 40, 41, and 42 Compounds 40, 41, and 42 were prepared according to the same procedures used to synthesize compounds 19, 20, and 1, respectively.

[0336] Example 5. Synthesis of conjugate 43 Scheme 11. [ka] Scheme 12. [ka]

[0337] Step 1. Preparation of methyl 11-(2,6-bis(hydroxymethyl)-4-methylphenoxy)undecanoate (44) [ka] A solution of 2,6-bis(hydroxymethyl)-p-cresol (2.7 g, 16.3 mmol), methyl 11-bromoundecanoate (5.0 g, 17.9 mmol), and potassium carbonate (4.5 g, 32.6 mmol) in acetone (100 mL) was refluxed for 16 h. Upon completion, the solution was concentrated to dryness in vacuo, suspended in ethyl acetate (150 mL), and washed with water (2 × 100 mL) and brine (100 mL). The ethyl acetate layer was dried over magnesium sulfate, filtered, and concentrated to dryness in vacuo. The residue was purified by column chromatography on silica gel 60 (gradient 100% Hex → 50% EtOAc / Hex) to give methyl 11-(2,6-bis(hydroxymethyl)-4-methylphenoxy)undecanoate (44) as a colorless oil (1.6 g, 27%).

[0338] Step 2. Preparation of methyl 11-(2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-6-(hydroxymethyl)-4-methylphenoxy)undecanoate (45) [ka] To a solution of methyl 11-(2,6-bis(hydroxymethyl)-4-methylphenoxy)undecanoate (44) (1.5 g, 4.1 mmol) in anhydrous pyridine (20 mL) was added 4,4'-dimethoxytrityl chloride (1.4 g, 4.1 mmol). The solution was stirred at room temperature overnight. Upon completion, the solution was concentrated to dryness in vacuo and purified by column chromatography on silica gel 60 (CHCl containing 0.5 to 1% MeOH) to give methyl 11-(2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-6-(hydroxymethyl)-4-methylphenoxy)undecanoate (45) as a pale yellow solid (1.1 g, 40%).

[0339] Step 3. Preparation of lithium 11-(2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-6-(hydroxymethyl)-4-methylphenoxy)undecanoate (46) [ka] To a solution of methyl 11-(2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-6-(hydroxymethyl)-4-methylphenoxy)undecanoate (45) (1.1 g, 1.7 mmol) in anhydrous tetrahydrofuran (40 mL) and water (10 mL) was added lithium hydroxide (44 mg, 1.8 mmol). The solution was concentrated in vacuo to remove all tetrahydrofuran. The remaining aqueous solution was flash frozen in liquid nitrogen and then lyophilized overnight to give lithium 11-(2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-6-(hydroxymethyl)-4-methylphenoxy)undecanoate (46) as a pale pink solid (1.1 g, 94%).

[0340] Step 4. Preparation of Compound 47 To a solution of 10 (1.33 g, 0.66 mmol), 46 (0.5 g, 0.73 mmol), and HBTU (400 mg, 1 mmol) in N,N-dimethylformamide (25 mL) was added diisopropylethylamine (0.35 mL, 2 mmol). The solution was stirred at room temperature overnight (18 h). Upon completion, the solvent was removed in vacuo, and the residue was purified by column chromatography on silica gel (gradient: 100% CHCl-5%-10%-15% MeOH in CHCl) to give 47 (710 mg, 41%) as a colorless solid.

[0341] Step 5. Preparation of Compound 48 To a solution of 47 (0.71 g, 0.3 mmol), triethylamine (0.4 mL, 3.0 mmol), and polystyrene-DMAP (3 mmol / g loading, 200 mg, 0.6 mmol) in dichloromethane (15 mL) was added succinic anhydride (60 mg, 0.6 mmol). The solution was stirred overnight at room temperature and, upon completion, filtered and concentrated to dryness in vacuo. The residue was purified by column chromatography on silica gel 60 (gradient 5% to 20% MeOH in CHCl) to give 48 as a pale yellow solid (570 mg, 70%). 1H NMR (DMSO-d6, 400 MHz) δ 7.91 (m, 1H),7.86-7.76 (m, 6H), 7.45-7.40 (m, 2H), 7.36-7.14 (m, 10H), 7.10 (s, 1H), 6.91 (d, J = 8.9Hz, 4H), 5.21 (d, J = 3.3 Hz, 3H), 5.01 (s, 2H), 4.97 (dd, J = 11.2, 3.4 Hz, 3H), 4.56 (d,J = 8.5 Hz, 3H), 4.06-3.98 (m, 11H), 3.93-3.84 (m, 3H), 3.81-3.72 (m, 3H), 3.74 (s, 6H), 3.65-3.46 (m, 38H), 3.40-3.35 (m, 6H), 3.20-3.16 (m, 6H), 2.56-2.44 (m, 4H), 2.33 (s, 3H), 2.15-2.08 (m, 2H), 2.10 (s, 9H), 2.04-1.96 (m, 6H), 1.89 (s, 9H), 1.82-1.76 (m, 4H), 1.77 (s, 9H), 1.54-1.34 (m, 4H), 1.28-1.10 (m, 12H).

[0342] ステップ6. Modulation of compound 49 To a solution of 48 (100 mg, 40 μmol), N-hydroxysuccinimide (30 mg / mL in acetonitrile, 50 μL, 13 μmol), N,N'-diisopropylcarbodiimide (40 μL, 264 μmol), and pyridine (50 μL) in dichloromethane (2 mL) and acetonitrile (3 mL) was added 1000 Å lcaa CPG (Prime Synthesis, 920 mg). The solution was stirred overnight on an orbital shaker at room temperature. TLC analysis of the reaction solution showed only partial consumption of the activated N-hydroxysuccinate ester, so additional CPG (500 mg) was added. The solution was again stirred overnight. Upon completion, the CPG was filtered and washed with dichloromethane (25 mL), acetonitrile (25 mL), and tetrahydrofuran (25 mL). Unreacted amine residues on the CPG were acetylated (capped) by adding a 1:1 solution of acetic anhydride in acetonitrile (3 mL) and 10% N-methylimidazole / 10% pyridine in tetrahydrofuran (3 mL). The suspension was allowed to stand for 2 hours, then filtered and rinsed with equal volumes of tetrahydrofuran (25 mL), acetonitrile (25 mL), and dichloromethane (25 mL). The loaded CPG 49 was dried overnight under high vacuum. The standard DMT loading assay (3% trichloroacetic acid in CHCl, UV-VIS, A) was used. 504 ) and the ligand loading efficiency was determined to be 22 μmol / g.

[0343] Step 7. Preparation of conjugate 43 The resulting GalNAc-loaded CPG solid support 49 was used in automated oligonucleotide synthesis using standard procedures. Nucleotide deprotection followed by removal from the solid support (concurrent with galactosamine acetate deprotection) afforded the GalNAc-oligonucleotide conjugate 43.

[0344] Example 6. Synthesis of conjugate 50 Scheme 13. [ka] Scheme 14. [ka] [ka]

[0345] Step 1. Preparation of 2-((2-((tert-butyldimethylsilyl)oxy)ethyl)amino)ethan-1-ol (51) [ka] A solution of ethanolamine (77 mL, 1.25 mol) and (2-bromoethoxy)-tert-butyldimethylsilane (15 g, 62.7 mmol) in anhydrous acetonitrile (200 mL) was refluxed for 3 h. Upon completion, the reaction was cooled to room temperature, diluted with water (400 mL), and extracted with ethyl acetate (3 × 150 mL). The combined ethyl acetate extracts were dried over magnesium sulfate, filtered, and concentrated to dryness in vacuo. The residue was purified by filtration through a silica pad first using 50% ethyl acetate / hexanes, then 50% MeOH / EtOAc to give 51 as a pale yellow oil (14 g, 100%).

[0346] Step 2. Preparation of 2-(bis(4-methoxyphenyl)(phenyl)methoxy)-N-(2-((tert-butyldimethylsilyl)oxy)ethyl)ethan-1-amine (52) [ka] To a solution of 2-((2-((tert-butyldimethylsilyl)oxy)ethyl)amino)ethan-1-ol (51) (14 g, 64 mmol) and triethylamine (17.5 mL, 128 mmol) in anhydrous dichloromethane (250 mL) was added 4,4′-dimethoxytrityl chloride (24 g, 70 mmol). The solution was stirred at room temperature overnight and then concentrated to dryness in vacuo. The residue was dissolved in ethyl acetate (300 mL) and washed with water (250 mL) and brine (250 mL). The ethyl acetate was dried over magnesium sulfate, filtered, and concentrated to dryness in vacuo. Purification by column chromatography on silica gel 60 (1% to 5% MeOH in CHCl) gave 52 as a pale yellow viscous oil (13 g, 39%).

[0347] Step 3. Methyl 10-((2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)(2-((tert-butyldimethylsilyl)oxy)ethyl)amino) Preparation of 10-oxodecanoate (53) [ka] A solution of 2-(bis(4-methoxyphenyl)(phenyl)methoxy)-N-(2-((tert-butyldimethylsilyl)oxy)ethyl)ethan-1-amine (52) (5.4 g, 10.3 mmol), monomethyl sebacate (2.2 g, 10.3 g), HBTU (4.9 g, 12.9 mmol), and DIPEA (5.3 mL, 30.9 mmol) in N,N-dimethylformamide (100 mL) was stirred at room temperature for 3 h. Upon completion, the solution was poured into water (400 mL) and extracted with ethyl acetate (1 × 500 mL). The ethyl acetate extract was washed with brine (2 × 250 mL), dried over magnesium sulfate, filtered, and concentrated to dryness in vacuo. Purification by column chromatography on silica gel 60 (10% to 25% ethyl acetate in hexane) afforded 53 as a viscous yellow oil (6.5 g, 87%).

[0348] Step 4. Preparation of methyl 10-((2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)(2-hydroxyethyl)amino)-10-oxodecanoate (54) [ka] To a solution of methyl 10-((2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)(2-((tert-butyldimethylsilyl)oxy)ethyl)amino)-10-oxodecanoate (53) (2.0 g, 2.8 mmol) and triethylamine (1 mL) in anhydrous tetrahydrofuran (20 mL) was added TBAF (1 M in THF, 3.4 mL, 3.3 mmol). The solution was stirred for 6 h, but only partial conversion was observed by TLC (5% MeOH in CHCl). An additional 1.7 mL of TBAF was added, and the solution was stirred at room temperature overnight. Upon completion, the solution was concentrated in vacuo and purified by column chromatography on silica gel 60 (10% to 50% EtOAc in hexane, then 100% EtOAc) to give 54 (0.5 g, 29%) as a viscous colorless oil.

[0349] Step 5. Preparation of lithium 10-((2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)(2-hydroxyethyl)amino)-10-oxodecanoate (55) [ka] To a solution of methyl 10-((2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)(2-hydroxyethyl)amino)-10-oxodecanoate (54) (0.5 g, 0.83 mmol) in THF (40 mL) was added water (10 mL) and lithium hydroxide (24 mg, 1.0 mmol). The solution was stirred at room temperature overnight and then concentrated in vacuo to remove the THF. The remaining aqueous solution was flash frozen in liquid nitrogen and lyophilized to give 55 as a colorless solid (485 mg, 95%).

[0350] Step 6. Preparation of compounds 56, 57, 58, and 50 Compounds 56, 57, 58, and 50 were prepared using the same procedure used to synthesize compounds 47, 48, 49, and 43, respectively.

[0351] Example 7. Synthesis of conjugate 59 Scheme 15. [ka] [ka] [ka] Scheme 16. [ka]

[0352] Step 1. Preparation of methyl (2R,5R)-5-hydroxypiperidine-2-carboxylate (61) [ka] (2R,5R)-5-hydroxypiperidine-2-carboxylic acid (60) (3.5 g, 24.1 mmol) was stirred in MeOH (50 mL). HCl (gas) was bubbled through the solution for 2 min, and the reaction was stirred under reflux for 1.5 h. The reaction was concentrated in vacuo to afford a quantitative yield of methyl (2R,5R)-5-hydroxypiperidine-2-carboxylate (61), which was used without further purification.

[0353] Step 2. Preparation of 1-(tert-butyl) 2-methyl(2R,5R)-5-hydroxypiperidine-1,2-dicarboxylate (62) [ka] Methyl (2R,5R)-5-hydroxypiperidine-2-carboxylate (61) (24.1 mmol) and TEA (7.2 mL, 53.02 mmol) were stirred in DCM (100 mL) at room temperature. Di-tert-butyl-dicarbonate (5.7 g, 26.5 mmol) was added in small portions, and the reaction was stirred for 2 h. The reaction was diluted with DCM (100 mL) and washed sequentially with 1 M HCl (2 × 75 mL), saturated NaHCO (2 × 75 mL), HO (2 × 75 mL), and saturated NaCl solution (2 × 75 mL). The organics were separated, dried (Na2SO4) and concentrated in vacuo to afford 1-(tert-butyl) 2-methyl(2R,5R)-5-hydroxypiperidine-1,2-dicarboxylate 62 (5.53 g, 88%), which was used without further purification.

[0354] Step 3. Preparation of tert-butyl (2R,5R)-5-hydroxy-2-(hydroxymethyl)piperidine-1-carboxylate (63) [ka] (2R,5R)-1-(tert-butoxycarbonyl)-5-hydroxypiperidine-2-carboxylic acid (62) (5.53 g, 21.4 mmol) was stirred in THF at 0 °C. LiBH (3.0 M in THF) (8.9 mL, 27.7 mmol) was added dropwise over 1 h. The reaction was allowed to warm to room temperature and stirring was continued for 16 h. The reaction was quenched with 1 M NaOH, the THF was removed in vacuo, and the aqueous was exhaustively extracted with EtOAc (10 × 100 mL). The combined organics were washed with HO (50 mL), saturated NaCl solution (2 × 50 mL), dried (NaSO), and concentrated in vacuo to afford tert-butyl (2R,5R)-5-hydroxy-2-(hydroxymethyl)piperidine-1-carboxylate (63) (2.4 g, 49.0%), which was used without further purification.

[0355] Step 4. Preparation of (3R,6R)-6-(hydroxymethyl)piperidin-3-ol (64) [ka] tert-Butyl (2R,5R)-5-hydroxy-2-(hydroxymethyl)piperidine-1-carboxylate (63) (2.4 g, 10.4 mmol) was stirred in EtO at room temperature. HCl (gas) was bubbled through for 45 seconds, and the reaction was stirred at room temperature for 45 minutes. The reaction was concentrated in vacuo and dried under high vacuum to give (3R,6R)-6-(hydroxymethyl)piperidin-3-ol (64). This product was used without further purification.

[0356] Step 5. Preparation of 2,2,2-trifluoro-1-((2R,5R)-5-hydroxy-2-(hydroxymethyl)piperidin-1-yl)ethan-1-one (65) [ka] The crude (3R,6R)-6-(hydroxymethyl)piperidin-3-ol (64) from the previous reaction was stirred in MeCN (50 mL) containing TEA (3.5 mL, 25.2 mmol) at room temperature. Ethyl trifluoroacetate (3 mL, 25.2 mmol) was added, and the reaction was stirred at room temperature for 16 h and then concentrated in vacuo to afford 2,2,2-trifluoro-1-((2R,5R)-5-hydroxy-2-(hydroxymethyl)piperidin-1-yl)ethan-1-one (65). This product was used without further purification.

[0357] Step 6. Preparation of 1-((2R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-hydroxypiperidin-1-yl)-2,2,2-trifluoroethan-1-one (66) [ka] Crude 2,2,2-trifluoro-1-((2R,5R)-5-hydroxy-2-(hydroxymethyl)piperidin-1-yl)ethan-1-one (65) from the previous reaction was stirred in DCM with TEA (50 mL) at RT. 4,4'-Dimethoxytrityl chloride (DMTrCl) (3.87 g, 11.44 mmol) was added in one portion, and the reaction was stirred at RT for 3 h. The reaction was diluted with DCM (50 mL) and washed sequentially with saturated NaHCO3 (2 × 75 mL), HO (2 × 75 mL), and saturated NaCl solution (2 × 75 mL). The organics were separated, dried (NaSO), concentrated in vacuo, and purified by column chromatography (100% hexane-60% EtOAc / hexane) (0.1% TEA) to afford 1-((2R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-hydroxypiperidin-1-yl)-2,2,2-trifluoroethan-1-one (66) (3.14 g, 57%).

[0358] Step 7. Preparation of (3R,6R)-6-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-piperidin-3-ol (67) [ka] 1-((2R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-hydroxypiperidin-1-yl)-2,2,2-trifluoroethan-1-one (66) (3.14 g, 6.0 mmol) was stirred in MeOH (50 mL) at room temperature. KOH (672 mg, 12 mmol) was added and the reaction was stirred at room temperature for 16 h. Additional KOH (300 mg, 6 mmol) was added and stirring was continued for an additional 24 h. The reaction was concentrated in vacuo, taken up in DCM (150 mL), washed with HO (4 × 50 mL), dried (NaSO), and concentrated in vacuo to afford (3R,6R)-6-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)piperidin-3-ol (67) (2.34 g, 90%), which was used without further purification.

[0359] Step 8. Preparation of methyl 12-((2R,5R)-2-((bis(4-methoxyphenyl)(phenyl)-methoxy)methyl)-5-hydroxypiperidin-1-yl)-12-oxododecanoate (68) [ka] (3R,6R)-6-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)piperidin-3-ol (67) (2.34 g, 5.34 mmol) was stirred in DCM (75 mL) at room temperature. Triethylamine (2.2 mL, 16.2 mmol), HATU (3.5 g, 9.2 mmol), and 12-methoxy-12-oxododecanoic acid (1.32 g, 5.4 mmol) were added, and the reaction was stirred at room temperature for 3 hours. The resulting solid precipitate was removed by filtration, the filtrate was concentrated in vacuo, and the residue was purified by column chromatography (2.5% MeOH / DCM, 0.1% TEA) to afford a quantitative yield of methyl 12-((2R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-hydroxypiperidin-1-yl)-12-oxododecanoate (68).

[0360] Step 9. Preparation of lithium 12-((2R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)-methyl)-5-hydroxypiperidin-1-yl)-12-oxododecanoate (69) [ka] Methyl 12-((2R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-hydroxypiperidin-1-yl)-12-oxododecanoate (68) (5.4 mmol) and LiOH (140 mg, 5.94 mmol) were stirred in THF:HO (1:1, 100 mL) at room temperature for 48 h. The THF was removed in vacuo, and the aqueous was frozen and lyophilized to afford lithium 12-((2R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-hydroxypiperidin-1-yl)-12-oxododecanoate (69) (3.2 g, 91%), which was used in the subsequent reaction without further purification.

[0361] Step 10. Preparation of Compounds 70, 71, 72, and 59 Compounds 70, 71, 72, and 59 were prepared using the same procedure used to synthesize compounds 47, 48, 49, and 43, respectively.

[0362] Example 8. Synthesis of conjugate 142 Scheme 17. [ka] [ka] Scheme 18. [ka]

[0363] Step 1. Preparation of 3,4,5-triacetoxybenzoic acid (73) A solution of gallic acid (20 g) in pyridine (50 mL) and acetic anhydride (50 mL) was stirred overnight at room temperature and then poured into ice water (1 L). The solution was acidified with concentrated hydrochloric acid, whereupon a colorless solid precipitated. The solid was collected by filtration and washed with water (5 x 100 mL). The wet solid was frozen in liquid nitrogen and freeze-dried to give 3,4,5-triacetoxybenzoic acid (26 g, 75%).

[0364] Step 2. 5-((2-((2-oxo-2-phenyl-1λ 2 Preparation of (ethyl)amino)ethyl)carbamoyl)benzene-1,2,3-triyl triacetate (74) To a solution of 3,4,5-triacetoxybenzoic acid (10 g, 33.8 mmol), N-carbobenzoxy-1,2-diaminoethane hydrochloride (5.3 g, 33.8 mmol), and HBTU (13.5 g, 35.5 mmol) in DMF (200 mL) was added DIPEA (17.5 mL, 101 mmol). The solution was stirred for 16 h, then diluted with ethyl acetate (250 mL), washed with brine (3×200 mL), dried over magnesium sulfate, filtered, and concentrated to dryness in vacuo. The crude product was purified by column chromatography on silica gel (gradient 1% to 5% MeOH in DCM) to give 5-((2-((2-oxo-2-phenyl-1λ 2 (Ethyl)amino)ethyl)carbamoyl)benzene-1,2,3-triyl triacetate was obtained as an off-white solid (5.5 g).

[0365] Step 3. 3,4,5-Trihydroxy-N-(2-((2-oxo-2-phenyl-1λ) 2 Preparation of (-ethyl)amino)ethyl)benzamide (75) 5-((2-((2-oxo-2-phenyl-1λ 2 A solution of 3,4,5-trihydroxy-N-(2-((2-oxo-2-phenyl-1λ-ethyl)amino)ethyl)carbamoyl)benzene-1,2,3-triyl triacetate (5 g, 1.1 mmol) in 1:1 MeOH / CHCl (100 mL) was stirred at room temperature for 3 days. Upon completion, the solvent was removed to give 3,4,5-trihydroxy-N-(2-((2-oxo-2-phenyl-1λ-ethyl)amino)ethyl)carbamoyl)benzene-1,2,3-triyl triacetate. 2 (4 g, quantitative yield)-ethyl)amino)ethyl)benzamide as a colorless solid.

[0366] Step 4. Trimethyl 2,2',2''-((5-((2-((2-oxo-2-phenyl-1λ 2Preparation of (1,2,3-ethyl)amino)ethyl)carbamoyl)benzene-1,2,3-triyl)tris(oxy))triacetate (76) 3,4,5-trihydroxy-N-(2-((2-oxo-2-phenyl-1λ 2 A solution of (2,2',2''-((5-((2-((2-oxo-2-phenyl-1λ-methyl)amino)ethyl)benzamide (4 g, 11.6 mmol), methyl bromoacetate (7.7 g, 46.4 mmol), and potassium carbonate (9.6 g, 69.4 mmol) in DMF (100 mL) was stirred at 60 °C overnight. Upon completion, the solution was cooled to room temperature, diluted with ethyl acetate (200 mL), washed with water (200 mL), brine (3 x 100 mL), dried over magnesium sulfate, filtered, and concentrated to dryness in vacuo. The crude product was purified by column chromatography on silica gel (gradient 2% to 10% MeOH in DCM) to give trimethyl 2,2',2''-((5-((2-((2-oxo-2-phenyl-1λ-methyl)amino)ethyl)benzamide. 2 To the residue was obtained (5 g, 79%) of 1,2,3-tri-(ethyl)amino)ethyl)carbamoyl)benzene-1,2,3-triyl)tris(oxy))-triacetate as a beige solid.

[0367] Step 5. 2,2',2''-((5-((2-((2-oxo-2-phenyl-1λ 2 Preparation of (1,2,3-ethyl)amino)ethyl)carbamoyl)benzene-1,2,3-triyl)tris(oxy))triacetic acid (77) Trimethyl 2,2',2''-((5-((2-((2-oxo-2-phenyl-1λ 2 A solution of 2,2',2''-((5-((2-((2-oxo-2-phenyl-1λ-phenyl)-2,2',2''-amino)ethyl)-carbamoyl)benzene-1,2,3-triyl)tris(oxy))triacetate (5 g, 9.2 mmol) and 1 M NaOH (30 mL) in methanol (100 mL) was stirred at room temperature for 2 hours. Upon completion, the reaction was concentrated to remove methanol and diluted with water (75 mL). The mixture was cooled to 0°C, acidified with 2 M HCl, and extracted with ethyl acetate (5 x 150 mL). The combined ethyl acetate extracts were dried over magnesium sulfate, filtered, and concentrated to dryness in vacuo to give 2,2',2''-((5-((2-((2-oxo-2-phenyl-1λ-phenyl)-2,2',2''-amino)ethyl)-carbamoyl)benzene-1,2,3-triyl)tris(oxy))triacetate. 2(2.3 g, 50%) of 1,2,3-triyl-(-ethylamino)ethyl)carbamoyl)benzene-1,2,3-triyl)tris(oxy))triacetic acid was obtained as a colorless solid.

[0368] Step 6. Preparation of Compound 78 Compound 78 was prepared from compounds 9 (2.75 g, 4.3 mmol) and 77 (0.5 g, 0.96 mmol) using the same procedure as used for compound 13. Yield: 600 mg.

[0369] Step 7. Preparation of Compound 79 Compound 79 was prepared from compound 78 (0.6 g) using the same procedure as used for compound 14. Yield: 500 mg.

[0370] Step 8. Preparation of Compound 140 Compound 140 was prepared from compound 79 (500 mg, 0.25 mmol) and compound 18 (175 mg, 0.25 mmol) using the same procedure as used for compound 19. Yield: 250 mg, 44%.

[0371] Step 9. Preparation of Compound 141 Compound 141 was prepared from compound 140 (250 mg, 0.11 mmol) using the same procedure as used for compound 20. Yield: 200 mg.

[0372] Step 10. Preparation of conjugate 142 Conjugate 142 was prepared from compound 141 (200 mg) and 1000A lcaa CPG (1.8 g) using the same procedure as that used for compound 1. Yield: 1.9 g, 22 μmol / g CPG loading. The resulting GalNAc-loaded CPG solid support was used for automated oligonucleotide synthesis using standard procedures. Nucleotide deprotection followed by removal from the solid support (concurrent with galactosamine acetate deprotection) afforded GalNAc-oligonucleotide conjugate 142.

[0373] Example 9. Synthesis of conjugate 145 Scheme 19. [ka] Scheme 20. [ka]

[0374] Step 1. Preparation of racemic (cis) 5-benzyl-3a,6a-dimethyltetrahydro-1H-furo[3,4-c]pyrrole-1,3(3aH)-dione (123) To a cooled (0 °C) solution of 3,4-dimethylfuran-2,5-dione (3 g, 24 mmol) and N-benzyl-1-methoxy-N-((trimethylsilyl)methyl)methanamine (7 g, 29.8 mmol) in dichloromethane (75 mL) was added trifluoroacetic acid (75 μL) slowly. Stirred overnight, allowing the solution to slowly warm to room temperature as the ice bath melted. The reaction mixture was concentrated to dryness, dissolved in ethyl acetate (100 mL), washed with saturated sodium bicarbonate (2 × 100 mL), dried over magnesium sulfate, filtered, and concentrated to dryness. Purification by column chromatography on silica gel (gradient: 20% ethyl acetate in hexane to 100% ethyl acetate) gave racemic (cis) 5-benzyl-3a,6a-dimethyltetrahydro-1H-furo[3,4-c]pyrrole-1,3(3aH)-dione as a yellow oil (3.5 g, 56%).

[0375] Step 2. Preparation of racemic (cis)(1-benzyl-3,4-dimethylpyrrolidine-3,4-diyl)dimethanol (124) To a cooled (0 °C) solution of (3aR,6aS)-5-benzyl-3a,6a-dimethyltetrahydro-1H-furo[3,4-c]pyrrole-1,3(3aH)-dione (3.5 g, 13.4 mmol) in anhydrous diethyl ether (50 mL) was slowly added lithium aluminum hydride pellets (1.5 g, 40 mmol) in three portions. The solution was stirred overnight and allowed to return to room temperature as the ice-water bath melted. Upon completion, the reaction was cooled to 0 °C and quenched very slowly with 1.5 mL of 5 M NaOH followed by 1.5 mL of water. Stirring was continued for 30 minutes, followed by the addition of magnesium sulfate and filtration. The filtrate was concentrated to give racemic (cis)1-benzyl-3,4-dimethylpyrrolidine-3,4-diyl)dimethanol as a colorless oil (2.7 g).

[0376] Step 3. Preparation of racemic (cis)(3,4-dimethylpyrrolidine-3,4-diyl)dimethanol (125) To a solution of ((3R,4S)-1-benzyl-3,4-dimethylpyrrolidine-3,4-diyl)dimethanol (10 g, 40 mmol) in methanol (10 mL) was added wet 10% palladium on activated carbon (1 g). The solution was vigorously stirred under a hydrogen atmosphere for 16 hours. Upon completion, the solution was filtered through Celite and concentrated to dryness to give racemic (cis)(3,4-dimethylpyrrolidine-3,4-diyl)dimethanol as a colorless solid (5.5 g, 86%).

[0377] Step 4. Preparation of racemic (cis) methyl 10-(3,4-bis(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoate (126) Compound 126 was prepared from compound 125 (1.3 g, 8.2 mmol) and monomethyl sebacate (1.8 g, 8.2 mmol) using the same procedure as used for compound 17. Yield: 1.8 g, 61%.

[0378] Step 5. Preparation of racemic (cis)methyl 10-(3-((bis(4-methoxyphenyl-)(phenyl)methoxy)-methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoate (127) Compound 127 was prepared from compound 126 (1.8 g, 5.0 mmol) and 4,4'-dimethoxytrityl chloride (1.7 g, 5.0 mmol) using the same procedure as used for compound 18. Yield: 1.4 g, 42%.

[0379] Step 6. Preparation of racemic (cis) lithium 10-(3-((bis(4-methoxyphenyl)-(phenyl)methoxy)-methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoate (128) To a solution of compound 127 (3.0 g, 4.6 mmol) in THF (50 mL) and water (50 mL) was added lithium hydroxide (121 mg, 5.0 mmol). The solution was stirred at room temperature for 4 hours and then concentrated to remove the THF. The remaining aqueous solution was freeze-dried overnight to give a pale pink solid (2.9 g, quantitative yield).

[0380] Step 7. Preparation of Compound 143 Compound 143 was prepared from compound 128 (270 mg, 0.42 mmol) and compound 14 (800 mg, 0.42 mmol) using the same procedure as used for compound 19. Yield: 900 mg, 87%.

[0381] Step 8. Preparation of Compound 144 Compound 144 was prepared from compound 143 (500 mg, 0.2 mmol) using the same procedure as used for compound 20. Yield: 200 mg.

[0382] Step 9. Preparation of conjugate 145 Conjugate 145 was prepared from compound 144 (200 mg) and 1000A lcaa CPG (1.8 g) using the same procedure as that used for compound 1. Yield: 1.9 g, 20 μmol / g CPG loading. The resulting GalNAc-loaded CPG solid support was used for automated oligonucleotide synthesis using standard procedures. Nucleotide deprotection followed by removal from the solid support (concurrent with galactosamine acetate deprotection) afforded GalNAc-oligonucleotide conjugate 145.

[0383] Example 10. Synthesis of conjugate 150 Scheme 21. [ka] [ka]

[0384] Step 1. Preparation of 146-1 To a solution of dodecanedioic acid monomethyl ester (12.2 g, 50.0 mmol) in dichloromethane (300 mL) were added N-hydroxysuccinimide (6.10 g, 53.0 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (10.52 g, 55.0 mmol). The cloudy mixture was stirred at room temperature overnight, and the reaction became a clear solution. TLC showed the reaction was complete. The organics were washed with saturated NH4Cl (300 mL) and brine (100 mL). The organic layer was separated, dried over MgSO4, and concentrated to dryness to give pure 1-(2,5-dioxopyrrolidin-1-yl) 12-methyldodecanedioate (146-1) as a white solid (16.7 g, 97.8%).

[0385] Step 2. Preparation of cyclopent-3-en-1-ylmethanol (146-2) To a suspension of lithium aluminum hydride (15.2 g, 0.40 mol) in anhydrous ether (1 L) at 0 °C under nitrogen, a solution of methyl cyclopent-3-enecarboxylate (50 g, 0.40 mol) in ether (300 mL) was added dropwise over 5 h. The suspension was stirred at room temperature overnight. TLC showed the reaction was complete. The reaction was recooled to 0 °C. A saturated solution of Na SO (32 mL) was added dropwise to quench the reaction. After the addition was complete, the mixture was stirred for an additional 3 h and filtered through a pad of Celite. Evaporation of the solvent afforded cyclopent-3-enylmethanol 146-2 (37.3 g, 95%) as a colorless liquid.

[0386] Step 3. Preparation of (6-oxabicyclo[3.1.0]hexan-3-yl)methanol 146-3 To a solution of cyclopent-3-enylmethanol (146-2) (4.0 g, 41 mmol) in dichloromethane (150 mL) at 0 °C, 3-chloroperbenzoic acid (10 g, 45 mmol, 77% purity) was added in small portions. The reaction was stirred overnight. (150 mL) was added. The organics were washed with sodium thiosulfate (12 g in 10 mL of water) followed by saturated NaHCO3 (40 mL). This was repeated until all remaining 3-chloroperbenzoic acid was washed away. The organics were dried over MgSO4. Evaporation of the solvent afforded a mixture of cis and trans 6-oxabicyclo[3.1.0]hexan-3-ylmethanol (146-3) (2.6 g, 57%) as a yellow oil. GC-MS: m / z 114 (5) (M + ), 95 (15), 88 (100), 81 (15).

[0387] Step 4. Preparation of 2-amino-4-(hydroxymethyl)cyclopentan-1-ol (146-4) Ammonia gas was purged into a methanol solution (20 mL) of 6-oxabicyclo[3.1.0]hexan-3-ylmethanol 146-3 (2.0 g, 17.6 mmol) at 0 °C for 10 min. The reaction was stirred at room temperature overnight. TLC showed the reaction was incomplete. Methanol was removed, and NH3·H2O (50 mL) was added, and the mixture was stirred at room temperature for 1 week. TLC confirmed the reaction was complete. Water was removed by azeotroping with ethanol to give 2-amino-4-(hydroxymethyl)cyclopentanol (146-4) (2.1 g, 91%) as a yellow oil.

[0388] Step 5. Preparation of methyl 12-(2-hydroxy-4-(hydroxymethyl)cyclopentylamino)-12-oxododecanoate (146-5) Compound 146-5 was prepared from 2-amino-4-(hydroxymethyl)cyclopentanol (146-4) and 1-(2,5-dioxopyrrolidin-1-yl)12-methyldodecanedioate (146-1) using the same procedure as described for the synthesis of 12-(2-(tert-butoxycarbonylamino)ethylamino)-12-oxododecanoate (3-2). Methyl 12-(2-hydroxy-4-(hydroxymethyl)cyclopentylamino)-12-oxododecanoate (146-5) was obtained as an off-white solid in 87.4% yield.

[0389] Step 6. Preparation of Compound 147 Compound 147 was quantitatively prepared from compound 146 (1.4 g, 2.33 mmol) using the same procedure as used for compound 18.

[0390] Step 7. Preparation of Compound 148 Compound 148 was prepared from compound 147 (150 mg, 0.23 mmol) and compound 14 (431 mg, 0.23 mmol) using the same procedure as used for compound 19. Yield: 460 mg, 84%.

[0391] Step 8. Preparation of Compound 149 Compound 149 was prepared from compound 148 (460 mg, 0.19 mmol) using the same procedure as used for compound 20. Yield: 436 mg, 91%.

[0392] Step 9. Preparation of Conjugate 150 Compound 150 was prepared from compound 149 (436 mg) and 1000A lcaa CPG (2.62 g) using the same procedure as that used for compound 1. Yield: 2.7 g, 21.3 μmol / g CPG loading. The resulting GalNAc-loaded CPG solid support was used in automated oligonucleotide synthesis using standard procedures. Nucleotide deprotection followed by removal from the solid support (concurrent with galactosamine acetate deprotection) afforded GalNAc-oligonucleotide conjugate 150.

[0393] Example 11. Synthesis of conjugates 153, 158, 163, 168, and 173 Scheme 22. [ka] [ka]

[0394] Step 1. Preparation of 1-(tert-butyl) 2-methyl (2S,4R)-4-hydroxypyrrolidine-1,2-dicarboxylate (133) Methyl (2S,4R)-4-hydroxypyrrolidine-2-carboxylate (25.9 g, 46 mmol), BOC anhydride (65.9 g, 302.5 mmol), and TEA (42 mL, 302.5 mmol) were stirred in DCM at room temperature for 16 h. The organics were washed sequentially with 1 M HCl (twice), saturated NaHCO (twice), HO, and brine, dried, and concentrated in vacuo to afford 1-(tert-butyl) 2-methyl(2S,4R)-4-hydroxypyrrolidine-1,2-dicarboxylate (133) (58.1 g, 85%).

[0395] Step 2. Preparation of 1-(tert-butyl) 2-methyl (4R)-4-hydroxy-2-methylpyrrolidine-1,2-dicarboxylate (134) 1-(tert-butyl) 2-methyl(2S,4R)-4-hydroxypyrrolidine-1,2-dicarboxylate (133) (5 g, 20.4 mmol) and MeI (12 g, 84.5 mmol) were stirred in anhydrous THF at −40° C. LDA (2.0 M in THF) (37.5 mL, 75 mmol) was added dropwise. The reaction was allowed to warm to room temperature and stirred for 4 hours before being quenched with saturated NH₄Cl. The reaction was extracted with EtOAc, washed with HO and brine, dried (Na₂SO₄), and concentrated in vacuo. The residue was purified by column chromatography 50:50 EtOAc / hexane to afford 1-(tert-butyl) 2-methyl(4R)-4-hydroxy-2-methylpyrrolidine-1,2-dicarboxylate (134) as a racemic mixture (3.6 g, 68%).

[0396] Step 3. Preparation of tert-butyl (2S,4R)-4-hydroxy-2-(hydroxymethyl)-2-methylpyrrolidine-1-carboxylate (135a) 1-(Tert-butyl) 2-methyl(4R)-4-hydroxy-2-methylpyrrolidine-1,2-dicarboxylate (134) (19 g, 73.5 mmol) was stirred in anhydrous THF under N. LiBH solution (48 mL, 96 mmol) was added dropwise, and the reaction was stirred at room temperature for 48 h. The reaction was quenched with 1 M NaOH, the THF was removed in vacuo, and the residue was extracted with EtOAc (4 × 100 mL). The organics were washed with HO and brine, dried (NaSO), and concentrated in vacuo. Purification of the residue by column chromatography (5% MeOH / DCM) afforded tert-butyl (2S,4R)-4-hydroxy-2-(hydroxymethyl)-2-methylpyrrolidine-1-carboxylate (135a) as the major product (8 g, 47%). The structure was assigned according to the literature.

[0397] Step 4. Preparation of (3R,5S)-5-(hydroxymethyl)-5-methylpyrrolidin-3-ol hydrochloride (136) tert-Butyl (2S,4R)-4-hydroxy-2-(hydroxymethyl)-2-methylpyrrolidine-1-carboxylate (135a) (8 g, 34.6 mmol) was stirred in EtOAc at room temperature, and HCl gas was applied for approximately 2 min. The reaction was stirred for 1 h, then concentrated in vacuo and dried under high vacuum to afford (3R,5S)-5-(hydroxymethyl)-5-methylpyrrolidin-3-ol hydrochloride (136) in quantitative yield.

[0398] Step 5. Preparation of methyl 12-((2S,4R)-4-hydroxy-2-(hydroxymethyl)-2-methylpyrrolidin-1-yl)-12-oxododecanoate (137) (3R,5S)-5-(hydroxymethyl)-5-methylpyrrolidin-3-ol hydrochloride (136) (7.9 g, 47.4 mmol), 12-methoxy-12-oxododecanoic acid (11.5 g, 47.4 mmol), HBTU (36 g, 76 mmol), and TEA (20 mL, 142.2 mmol) were stirred in DCM at room temperature for 16 h. The precipitate was removed by filtration, and the organics were washed with 1 M HCl (twice), saturated NaHCO (twice), HO, and brine. After drying, the organics were concentrated in vacuo and purified by column chromatography (5% MeOH / DCM) to afford methyl 12-((2S,4R)-4-hydroxy-2-(hydroxymethyl)-2-methylpyrrolidin-1-yl)-12-oxododecanoate (137) (3.1 g, 18.3%).

[0399] Step 6. Preparation of methyl 12-((2S,4R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)-methyl)-4-hydroxy-2-methylpyrrolidin-1-yl)-12-oxododecanoate (138) Methyl 12-((2S,4R)-4-hydroxy-2-(hydroxymethyl)-2-methylpyrrolidin-1-yl)-12-oxododecanoate (137) (3.1 g, 9.0 mmol), DMTr-Cl (2.8 g, 8.2 mmol), and TEA (1.1 mL, 8.2 mmol) were stirred in DC3 at room temperature for 16 h. The reaction was concentrated in vacuo, and the residue was purified by column chromatography (5% MeOH / DCM, 0.1% TEA) to afford methyl 12-((2S,4R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxy-2-methylpyrrolidin-1-yl)-12-oxododecanoate (138) (2.7 g, 45.5 mmol).

[0400] Scheme 23 [ka]

[0401] Step 7. Preparation of Compound 154-1 To a solution of N-(2-hydroxyethyl)phthalimide (4.80 g, 25.0 mmol) and 4,4'-dimethoxytrityl chloride (8.8 g, 26.0 mmol) in dichloromethane (200 mL) at 0°C under nitrogen, triethylamine (10.4 mL, 74.6 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 3 hours. TLC showed the reaction was complete. The organic layer was washed with brine (100 mL), dried over MgSO4, and concentrated to dryness. This was used directly in the next reaction without purification.

[0402] Step 8. Preparation of Compound 154-2 The 2-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)isoindoline-1,3-dione (154-1) obtained above and hydrazine monohydrate (3.6 mL, 74 mmol) in ethanol (100 mL) were stirred at room temperature overnight. TLC showed the reaction was complete. The precipitate was filtered off. The filtrate was evaporated. The residue was taken up in ethyl acetate (100 mL). This organic solution was washed with 10% NaOH, water, and brine and dried over MgSO. Evaporation of the solvent gave 2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethanamine (154-2) as a yellow liquid (8.11 g, 89.3% yield for two steps), which was used in the next reaction without further purification.

[0403] Step 9. Preparation of Compound 154-3 To a solution of L-threonine (1.19 g, 10.0 mmol) and NaHCO3 (2.3 g, 27 mmol) in water (20 mL) and dioxane (10 mL) was added dropwise 1-(2,5-dioxopyrrolidin-1-yl) 12-methyldodecanedioate (146-1) (3.1 g, 9.1 mmol) in dioxane (10 mL). The reaction mixture was stirred at room temperature overnight. 4 N HCl (10 mL) was added. The precipitate was collected by filtration and washed with water (3 × 10 mL). The solid was dried over PO5 in a desiccator to give (2S,3R)-3-hydroxy-2-(12-methoxy-12-oxododecanamido)butanoic acid (154-3) as an off-white solid (2.84 g, 82.2%). LC-MS (ESI): m / z: 346 (100), (M + H + ).

[0404] Step 10. Preparation of Compound 154 (2S,3R)-3-Hydroxy-2-(12-methoxy-12-oxododecanamido)butanoic acid (154-3) (2.47 g, 7.15 mmol), 2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethanamine (154-2) (2.60 g, 7.15 mmol), EDC (1.64 g, 8.58 mmol), 1-hydroxybenzotriazole (HOBt) (1.16 g, 8.58 mmol), and TEA (2.4 mL, 17.2 mmol) were stirred in dichloromethane (72 mL) at room temperature for 2 h. Water (30 mL) was added. The organic layer was separated and washed with brine (2 × 30 mL). Evaporation of the solvent followed by column chromatography (30% ethyl acetate / hexane-50% ethyl acetate / hexane) gave methyl 12-((2S,3R)-1-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethylamino)-3-hydroxy-1-oxobutan-2-ylamino)-12-oxododecanoate (154) as a waxy yellow semisolid (2.60 g, 52.6%). 1 HNMR (400MHz, acetone-d6, ppm):δ 7.51 (t, J = 5.5 Hz, 1H), 7.45-7.49 (m, 2H), 7.28-7.36 (m, 6H), 7.21 (tt, J = 7.2, 1.2 Hz, 1H), 7.08 (d, J = 8.1 Hz, 1H), 6.88 (dt, J = 8.9, 2.5 Hz, 4H), 4.39 (dd, J = 8.2, 3.0 Hz, 1H), 4.20-4.27 (m, 1H), 3.78 (s,6H), 3.60 (s, 1H), 3.35-3.52 (m, 2H), 3.07-3.16 (m, 2H), 2.23-2.37 (m, 4H), 1.53-1.65(m, 4H), 1.23-1.36 (m, 12H), 1.10 (d, J = 6.4 Hz, 3H).

[0405] Scheme 24 [ka]

[0406] Step 11. Preparation of Compound 164-1 To a suspension of potassium t-butoxide (14.6 g, 130 mol) in THF (120 mL) / ether (360 mL) was added methyltriphenylphosphonium bromide (46.6 g, 130 mmol). The mixture was refluxed for 2 h and then cooled to 0 °C. tert-Butyl 2-formylpyrrolidine-1-carboxylate (13.0 g, 65.2 mmol) in ether (50 mL) was added dropwise. The reaction mixture was stirred at 0 °C and then quenched by the addition of water (250 mL). The organic layer was separated, and the aqueous was extracted with ether (250 mL). The combined extracts were dried over MgSO. Evaporation of the solvent followed by column chromatography purification (5% ethyl acetate / hexane) afforded tert-butyl 3-vinylpyrrolidine-1-carboxylate (164-1) (11.5 g, 89.4%) as a colorless liquid. GC-MS: m / z: 197 (2) (M + ), 141 (40), 124 (30), 57 (100).

[0407] Step 12. Preparation of Compound 164-2 To a mixture of t-BuOH (140 mL) and water (70 mL) was added AD-mix-β (47.4 g) and methanesulfonamide (2.89 g, 30.4 mmol). The mixture was stirred at room temperature for 30 minutes and then cooled to 0° C. tert-Butyl 3-vinylpyrrolidine-1-carboxylate (164-1) (6.00 g, 30.4 mmol) was added. The reaction was stirred at room temperature overnight. The reaction mixture was cooled to 0° C. Sodium thiosulfate pentahydrate (96 g, 387 mmol) was added and the temperature was allowed to return to room temperature. Water (700 mL) was added and the mixture was extracted with ethyl acetate (500 mL). The extract was diluted with water (2×5 The residue was washed with HCl (0 mL) and brine (50 mL) and dried over MgSO. Evaporation of the solvent followed by column chromatography (2% methanol / dichloromethane-7% methanol / dichloromethane) afforded tert-butyl 3-(1,2-dihydroxyethyl)pyrrolidine-1-carboxylate (164-2) (5.4 g, 77%) as a light brown oil.

[0408] Step 13. Preparation of Compound 164-3 To a solution (10 mL) of tert-butyl 3-(1,2-dihydroxyethyl)pyrrolidine-1-carboxylate (164-2) (3.1 g, 13.4 mmol) in ethanol, 30 mL of 3N HCl (90 mmol) was added. The reaction mixture was stirred at room temperature overnight. TLC showed the reaction was complete. The ethanol was evaporated. Toluene was added and evaporated. This was repeated three times to give 1-(pyrrolidin-3-yl)ethane-1,2-diol hydrochloride (164-3) (2.0 g, 89%) as a brown oil. LC-MS (ESI): m / z: 132 (100), (M + H + , free amine).

[0409] Step 14 Preparation of compound 164-4 To a solution (30 mL) of 1-(pyrrolidin-3-yl)ethane-1,2-diol hydrochloride (164-2) (2.0 g, 12 mmol), NaHCO3 (3.7 g, 44 mmol) was added in small portions. Dioxane (20 mL) was then added. To the above solution, 1-(2,5-dioxopyrrolidin-1-yl)12-methyldodecanedioate (146-1) (3.7 g, 11 mmol) in dioxane (30 mL) was added. The reaction mixture was stirred overnight. It was extracted with ethyl acetate (3 × 100 mL). The combined extracts were washed with 0.5 N HCl (50 mL) and brine (50 mL) and dried over MgSO4.

[0410] Step 15. Preparation of Compound 164 This material was prepared from methyl 12-(3-(1,2-dihydroxyethyl)pyrrolidin-1-yl)-12-oxododecanoate (164-4) and 4,4-dimethoxytrityl chloride (1 equivalent) using the same procedure as described for the synthesis of 2-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)isoindoline-1,3-dione (138). The product was purified by column chromatography (1.5% methanol / dichloromethane). Methyl 12-(3-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)-1-hydroxyethyl)pyrrolidin-1-yl)-12-oxododecanoate (164) was obtained in 51% yield as a yellow oil. 1 HNMR (400MHz, acetone-d6, ppm):δ 7.49-7.54 (m, 2H), 7.35-7.40 (m, 4H), 7.28-7.34 (m, 2H),7.19-7.25 (m, 1H), 6.86-6.91 (m, 4H), 4.11-4.20 (m, 1H), 3.79 (s, 6H), 3.68-3.77 (m, 1H), 3.60 (s, 3H), 3.29-3.59 (m, 3H), 3.06-3.20 (m, 3H), 2.33-2.55 (m, 1H), 2.29 (t, J = 7.4 Hz, 2H), 2.19 (t, J = 7.6 Hz, 2H), 1.65-2.0 (m, 2H), 1.51-1.62 (m, 4H), 1.26-1.35 (m, 12H).

[0411] Scheme 25 [ka]

[0412] Step 16. Preparation of Compound 170-1 To a solution of tert-butyl 2-aminoethylcarbamate (2.88 g, 18.0 mmol) and triethylamine (2.98 g, 29.4 mmol) in dichloromethane (100 mL) was added dropwise 1-(2,5-dioxopyrrolidin-1-yl) 12-methyldodecanedioate (146-1) (5.09 g, 14.9 mmol) in dichloromethane (50 mL) at room temperature. The reaction mixture was stirred overnight, and TLC showed the reaction was complete. 100 mL of brine was added, and the organic layer was separated. The organic layer was washed with 0.5 N HCl (150 mL), brine (2 × 100 mL), and dried over MgSO4. Evaporation of the solvent afforded pure methyl 12-(2-(tert-butoxycarbonylamino)ethylamino)-12-oxododecanoate (170-1) (5.85 g, 100%) as a white solid.

[0413] Step 17. Preparation of Compound 170-2 To a solution of 12-(2-(tert-butoxycarbonylamino)ethylamino)-12-oxododecanoate (170-1) (5.55 g, 14.4 mmol) in methanol (100 mL) at 0 °C, thionyl chloride (3.3 mL, 45.5 mmol) was added dropwise. The reaction was then stirred at room temperature overnight. TLC showed the reaction was complete. The solvent and volatile organics were evaporated. The residue was then co-evaporated twice with heptane to quantitatively afford methyl 12-(2-aminoethylamino)-12-oxododecanoate hydrochloride (170-2) as a white solid. LC-MS (ESI): m / z: 287 (100), (M + H + , free amine).

[0414] Step 18. Preparation of Compound 170-3 (-)-Methyl (S)-2,2-dimethyl-1,3-dioxolane-4-carboxylate (5.01 g, 31.2 mmol) and LiOH·HO (2.55 g, 60.8 mmol) in THF (50 mL) and water (50 mL) were stirred overnight. TLC showed the reaction was complete. The THF was evaporated, and the aqueous was acidified to pH = 1 with 1 N HCl. This was extracted with ethyl acetate (5 × 50 mL). The combined extracts were dried over MgSO. Evaporation of the solvent afforded (S)-2,2-dimethyl-1,3-dioxolane-4-carboxylic acid (170-3) (2.93 g, 64.3%) as a pale yellow liquid.

[0415] Step 19. Preparation of Compound 170-4 Compound 170-4 was synthesized in 86% yield from (S)-2,2-dimethyl-1,3-dioxolane-4-carboxylic acid 170-3 and N-hydroxysuccinimide using the same procedure as described for the synthesis of 1-(2,5-dioxopyrrolidin-1-yl) 12-methyldodecanedioate 146-1. (S)-2,5-Dioxopyrrolidin-1-yl 2,2-dimethyl-1,3-dioxolane-4-carboxylate 170-4 was obtained as a white solid in 86% yield.

[0416] Step 20. Preparation of Compound 170-5 To a suspension of methyl 12-(2-aminoethylamino)-12-oxododecanoate hydrochloride (170-2) (14.4 mmol) and (S)-2,5-dioxopyrrolidin-1-yl 2,2-dimethyl-1,3-dioxolane-4-carboxylate (170-4) (3.80 g, 15.6 mmol) in dichloromethane (100 mL) was added triethylamine (6 mL, 43.0 mmol) in dichloromethane (25 mL) over 4 h at 0 °C. The reaction mixture was then stirred at room temperature overnight. LC-MS showed complete conversion of starting material 170-2. The organic layer was washed with brine (50 mL), 1 N HCl (50 mL), brine (50 mL), dried over MgSO and concentrated to dryness to give (S)-methyl 12-(2-(2,2-dimethyl-1,3-dioxolane-4-carboxamido)ethylamino)-12-oxododecanoate (170-5) (5.93 g, 99.3%) as a white solid.

[0417] Step 21. Preparation of Compound 170-6 To a solution of (S)-methyl 12-(2-(2,2-dimethyl-1,3-dioxolane-4-carboxamido)ethylamino)-12-oxododecanoate (170-5) (5.93 g, 14.3 mmol) was added one drop of concentrated sulfuric acid. This was refluxed for 6 hours and then cooled to room temperature. The solid was collected by filtration and washed twice with cold methanol. The solid was dried in air (3.32 g). A second crop (0.42 g) was obtained from the mother liquor to give (S)-methyl 12-(2-(2,3-dihydroxypropanamido)ethylamino)-12-oxododecanoate (170-6) (total 3.74 g, 69.4%) as white crystals. LC-MS (ESI): m / z: 375 (100), (M + H + ). 1HNMR (400MHz, DMSO-d6, ppm):δ 7.79 (br, 2H),5.49 (d, J = 5.3 Hz, 1H), 4.66 (t, J = 5.8 Hz,1H), 3.83-3.88 (m, 1H), 3.55-3.61 (m, 4H), 3.41-3.47 (m, 1H), 3.05-3.15 (m, 4H), 2.29(t, J = 7.4 Hz, 2H), 2.03 (t, J = 7.6 Hz, 2H),1.42-1.52 (m, 4H), 1.18-1.29 (m, 12H).

[0418] Step 22. Preparation of Compound 170 To a solution of (S)-methyl 12-(2-(2,3-dihydroxypropanamido)ethylamino)-12-oxododecanoate (170-6) (2.99 g, 7.99 mmol) in anhydrous pyridine (57.5 mL) under nitrogen, 4,4'-dimethoxytrityl chloride (2.84 g, 8.38 mmol) was added in one portion. The reaction was stirred at room temperature for 2 days. Methanol (5 mL) was added to quench the reaction. Pyridine was evaporated. Toluene was added and then evaporated. This was repeated three times. Water (100 mL) was added, and this was extracted with ethyl acetate (5 x 250 mL). The extracts were combined and dried over MgSO4. Evaporation of the solvent followed by column chromatography (1% methanol / dichloromethane-3% methanol / dichloromethane) afforded (S)-methyl 12-(2-(3-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-hydroxypropanamido)-ethylamino)-12-oxododecanoate (170) (1.70 g, 31.4%) as a viscous oil. 1HNMR (400MHz, acetone-d6, ppm):δ 7.64-7.70 (br, 1H), 7.47-7.51 (m, 2H),7.33-7.37 (m, 4H), 7.26-7.32 (m, 2H), 7.20 (dt, J = 7.3, 2.1 Hz, 1H), 7.11 (br, 1H), 6.86 (d, J = 8.7 Hz, 4H), 4.84 (br, 1H), 4.21 (dd, J = 5.1, 3.8 Hz, 1H), 3.78 (s, 6H), 3.60 (s, 1H), 3.25-3.42 (m, 6H), 2.28 (t, J = 7.4 Hz, 2H), 1.48-1.62 (m, 4H), 1.21-1.34 (m, 12H).

[0419] Scheme 26. [ka] [ka]

[0420] Step 23. Preparation of Compounds 139, 155, 160, 165, and 170 Compounds 139, 155, 160, 165, and 170 were prepared from compounds 138, 154, 159, 164, and 169 using the same procedure as used for compound 18.

[0421] Step 24. Preparation of conjugates 153, 158, 163, 168, and 173 Conjugates 153, 158, 163, 168, and 173 were prepared from compounds 139, 154, 159, 164, and 169 using the same procedure as used for compound 1.

[0422] Example 12. Synthesis of conjugate 176 Scheme 27. [ka] Scheme 28. [ka]

[0423] Step 1. Preparation of methyl 12-aminododecanoate (132) 12-Aminoundecanoic acid (131) (10 g, 4.64 mmol) was stirred in MeOH at room temperature. Acetyl chloride (856 μL, 12 mmol) was added dropwise, and the reaction was stirred for 1.5 h. The solvent was removed in vacuo, and the residue was taken up in MTBE and chilled in the refrigerator overnight. The resulting precipitate was collected by filtration, washed with ice-cold MTBE, and dried under high vacuum to give methyl 12-aminododecanoate (132).

[0424] Step 2. Preparation of racemic (cis)methyl 12-(12-(10-(3-((bis(4-methoxyphenyl)-(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanamido)dodecanamido) dodecanoate (129) Methyl 12-aminododecanoate (132) (778 mg, 3.1 mmol) of racemic (cis) lithium 10-(3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoate (128) (2 g, 3.1 mmol), HBTU (1.2 g, 3.1 mmol), and TEA (1.4 mL, 10 mmol) were stirred in DCM at room temperature overnight (O / N). The precipitate was removed by filtration, the filtrate was concentrated in vacuo, and the residue was purified by column chromatography (5% MeOH, DCM). TLC showed two closely migrating spots with identical masses. These were assigned as geometric isomers and pooled together to afford methyl 12-(12-(10-((3R,4S)-3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanamido)dodecanamido)dodecanoate (129) in quantitative fashion.

[0425] Step 3. Preparation of racemic (cis) 12-(12-(10-(-3-((bis(4-methoxyphenyl)(phenyl)-methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanamido)-dodecanamido) lithium dodecanoate (130) Racemic (cis)methyl 12-(12-(10-(3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanamido)dodecanamido) dodecanoate (129) (3.1 mmol) was stirred in THF:HO (50:50) containing LiOH (88 mg, 3.7 mmol) at room temperature overnight. The reaction was monitored by TLC, and the THF was removed in vacuo. The aqueous solution was frozen in liquid N2 and lyophilized for 48 hours to yield racemic (cis) lithium 12-(12-(10-(3-((bis(4-methoxyphenyl)(phenyl)-methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanamido)-dodecanamido) dodecanoate (130) in quantitative yield.

[0426] Step 4. Preparation of conjugate 176 Conjugate 176 was prepared from compounds 24 and 130 using the same procedure as used for compound 1.

[0427] Example 13. Synthesis of conjugate 179 Scheme 29. [ka] Scheme 30. [ka]

[0428] Step 1. Preparation of Compound 80 Compound 24 (2 g, 0.86 mmol), N-carbobenzoxy-L-glutamic acid (120 mg, 0.43 mmol), HBTU (326 mg, 0.86 mmol), and TEA (353 μL, 2.6 mmol) were stirred in DCM at room temperature overnight. The mixture was concentrated in vacuo and purified by column chromatography to give compound 80 (2.88 g, 83%).

[0429] Step 2. Preparation of Compound 81 Compound 81 was prepared from compound 80 (670 mg, 0.17 mmol) using the same procedure as used for compound 14. This compound was used crude in subsequent reactions in quantitative yield.

[0430] Step 3. Preparation of conjugate 179 Conjugate 179 was prepared from compounds 18 and 81 using the same procedure as used for compound 1.

[0431] Example 14. Synthesis of conjugate 182 Scheme 31. [ka] Scheme 32. [ka]

[0432] Step 1. Preparation of Compound 93 Using the same procedure as used for compound 89, (2-oxo-2-phenyl-1λ 2 Compound 93 was prepared from (-ethyl)-D-glutamic acid (2.25 g, 8.1 mmol) and 9 (13 g, 21 mmol). Yield: 11.2 g.

[0433] Step 2. Preparation of Compound 94 Compound 94 was prepared from compound 93 (11.1 g) using the same procedure as used for compound 90. Yield: 10.2 g.

[0434] Step 3. Preparation of conjugate 182 Conjugate 182 was prepared from compounds 18 and 94 using the same procedure as used for compound 1.

[0435] Example 15. Synthesis of conjugates 185 and 188 Scheme 33. [ka] Scheme 34. [ka] Scheme 35. [ka]

[0436] Step 1. Preparation of 14-hydroxy-3,6,9,12-tetraoxatetradecyl 4-methylbenzenesulfonate (82) A solution of pentaethylene glycol (35 g, 147 mmol), TEA (41 mL, 294 mmol), and trimethylamine-HCl (1.4 g, 14.7 mmol) in CHCl (600 mL) was treated with tosyl chloride (29.4 g, 154 mmol). After stirring (18 h), the reaction mixture was washed with HO-brine (1:1), dried (MgSO), filtered, concentrated, and chromatographed to give 82 (24.6 g, 43%) as a pale yellow oil. Rf 0.8 (10% CHOH-CHCl).

[0437] Step 2. 14-Azido-3,6,9,12-tetraoxatetradecan-1-ol (83) 14-Azido-3,6,9,12-tetraoxatetradecan-1-ol (83) was prepared from 82 (24.6 g, 62.7 mmol) and sodium azide (7.13 g, 110 mmol) using the same procedure as used for compound 4. Yield: 14.8 g, 90%.

[0438] Step 3. Preparation of Compound 84 A solution of GalNAc 6 (12.2 g, 31.4 mmol) and HO-PEG-N383 (9.2 g, 35 mmol) in 1,2-dichloroethane (150 mL) was treated with Sc(OTf)3 (771 mg, 1.6 mmol). After stirring (85 °C, 2 h), the reaction was cooled (room temperature), quenched by the addition of TEA (40 mL), and concentrated. The crude material was chromatographed to give 84 (11.16 g, 60%) as a pale yellow foam. Rf 0.7 (10% CH3OH-CH2Cl2).

[0439] Step 4. Preparation of Compound 85 A solution of 84 (11.16 g, 18.8 mmol) and Pd / C (1.1 g, 10% wet support) in 120 mL of EtOAc was treated with TFA (4.32 mL, 56.5 mmol) and purged with H. After vigorous stirring (4.5 h), the reaction was purged with N, filtered through Celite, and concentrated. The crude material was chromatographed to give 85 (5.77 g, 45%) as a colorless foam. Rf 0.5 (10% CHOH—CHCl).

[0440] Step 5. Preparation of Compound 95 Using the same procedure as used for compound 91, (2-oxo-2-phenyl-1λ 2 Compound 95 was prepared from (-ethyl)-D-glutamic acid (1.04 g, 3.7 mmol) and compound 94 (10.2 g). Yield: 7.2 g.

[0441] Step 6. Preparation of Compound 96 Compound 96 was prepared from compound 95 (11.1 g) using the same procedure as used for compound 92. Yield: 6.5 g.

[0442] Step 7. Preparation of Compound 97 Using the same procedure as used for compound 89, (2-oxo-2-phenyl-1λ 2Compound 97 was prepared from (-ethyl)-D-glutamic acid (2 g, 7.1 mmol) and 85 (12.1 g, 17.8 mmol). Yield: 10 g, quantitative.

[0443] Step 8. Preparation of Compound 98 Compound 98 was prepared from compound 97 (10 g, 7.2 mmol) using the same procedure as used for compound 90. Yield: 3.5 g, 36%.

[0444] Step 9. Preparation of Compound 99 Using the same procedure as used for compound 91, (2-oxo-2-phenyl-1λ 2 Compound 99 was quantitatively prepared from (-ethyl)-D-glutamic acid (350 mg, 1.25 mmol) and compound 98 (2.86 mg, 2.5 mmol).

[0445] Step 10. Preparation of Compound 100 Compound 100 was quantitatively prepared from compound 99 (3.2 g, 1.25 mmol) using the same procedure as used for compound 92.

[0446] Step 11. Preparation of conjugates 185 and 188 Conjugates 185 and 188 were prepared from compounds 18 and 96 or 18 and 100 using the same procedure as used for compound 1.

[0447] Example 16. Synthesis of conjugates 191, 194, 197, and 200 Scheme 36 [ka] Scheme 37. [ka] Scheme 38. [ka]

[0448] Step 1. Preparation of 2-(2-(2-azidoethoxy)ethoxy)ethan-1-ol (86) To a solution of 2-(2-(2-chloroethoxy)ethoxy)ethan-1-ol (13 g, 77 mmol) in water (200 mL) was added sodium azide (10 g, 154 mmol). The reaction was heated to 100° C. for 18 hours. The reaction was cooled to room temperature, poured into a 1 L separatory funnel, and extracted with dichloromethane (3×200 mL). The combined dichloromethane extracts were dried over magnesium sulfate, filtered, and concentrated to dryness to give 2-(2-(2-azidoethoxy)ethoxy)ethan-1-ol as a colorless oil (11.7 g).

[0449] Step 2. Preparation of Compound 87 Compound 87 was prepared from 86 (4.95 g, 28.3 mmol) and 6 (10 g, 25.7 mmol) using the same procedure as used for compound 84. Yield: 10 g, 77%.

[0450] Step 3. Preparation of Compound 88 Compound 88 was prepared from 87 (10 g, 19.8 mmol) using the same procedure as used for compound 85. Yield: 7.63 g, 65%.

[0451] Step 4. Preparation of Compound 89 A solution of 88 (2 g, 3.38 mmol) and Z-glutamic acid (427 mg, 1.52 mmol) in CHCl (50 mL) was treated with HBTU (1.41 g, 3.7 mmol) and Hunig's base (1.77 mL, 10.1 mmol). After stirring (18 h), the mixture was concentrated and chromatographed to give 89 (871 mg, 48%) as a colorless foam. Rf 0.5 (10% CHOH-CHCl).

[0452] Step 5. Preparation of Compound 90 A solution of 89 (870 mg, 0.72 mmol) and Pd / C (90 mg, 10% wet support) in 10 mL of EtOAc was treated with TFA (84 μL, 1.1 mmol) and purged with H. After vigorous stirring (2 h), the reaction was purged with N, filtered through Celite, and concentrated. This crude material was used without further treatment to give 90 (850 mg, quantitative) as a colorless foam. Rf 0.25 (10% CHOH-CHCl). .

[0453] Step 6. Preparation of Compound 91 A solution of 90 (850 mg, 0.72 mmol) and Z-glutamic acid (91 mg, 0.32 mmol) in CHCl (10 mL) was treated with HBTU (300 mg, 0.79 mmol) and Hunig's base (502 μL, 2.9 mmol). After stirring (1.5 h), the mixture was diluted with CHCl, washed with NaHCO (sat. aq.), dried (MgSO), filtered, and concentrated. The crude material was chromatographed to give 91 (590 mg, 76%) as a colorless foam. Rf 0.5 (10% CHOH—CHCl).

[0454] Step 7. Preparation of Compound 92 A solution of 91 (590 mg, 0.25 mmol) and Pd / C (100 mg, 10% wet support) in CHOH (30 mL) was treated with TFA (29 μL, 0.37 mmol) and purged with H. After stirring (3 h), the mixture was purged with N, filtered through Celite, and concentrated. This crude material was used without further treatment to give 92 (600 mg, quantitative) as a colorless foam. Rf 0.1 (10% CHOH—CHCl).

[0455] Step 8. Preparation of Compound 101 Compound 101 was prepared from (R)-2-((2-oxo-2-phenyl-112-ethyl)amino)hexanedioic acid (2.51 g, 8.6 mmol) and 9 (11 g, 17.2 mmol) using the same procedure as used for compound 89. Yield: 4.2 g, 37%.

[0456] Step 9. Preparation of Compound 102 Compound 102 was prepared from compound 101 (4.2 g, 3.2 mmol) using the same procedure as used for compound 90. Yield: 2.1 g, 47%.

[0457] Step 10. Preparation of Compound 103 Compound 103 was prepared from (R)-2-((2-oxo-2-phenyl-112-ethyl)amino)hexanedioic acid (265 mg, 0.9 mmol) and compound 102 (2.1 g, 1.8 mmol) using the same procedure as used for compound 91. Yield: (560 mg, 24%).

[0458] Step 11. Preparation of Compound 104 Compound 104 was quantitatively prepared from compound 103 (560 mg) using the same procedure as used for compound 92. This compound was used without purification.

[0459] Step 12. Preparation of conjugates 191, 194, and 197 Conjugates 191, 194, and 197 were prepared from compounds 128 and 92, 96, and 100 using the same procedure as used for compound 1.

[0460] Example 16a. Synthesis of conjugate 191a Scheme 36a [ka] Scheme 37a. [ka] Scheme 38a. [ka]

[0461] Step 1. Preparation of 2-(2-(2-azidoethoxy)ethoxy)ethan-1-ol (86a) To a solution of 2-(2-(2-chloroethoxy)ethoxy)ethan-1-ol (13 g, 77 mmol) in water (200 mL) was added sodium azide (10 g, 154 mmol). The reaction was heated to 100° C. for 18 hours. The reaction was cooled to room temperature, poured into a 1 L separatory funnel, and extracted with dichloromethane (3×200 mL). The combined dichloromethane extracts were dried over magnesium sulfate, filtered, and concentrated to dryness to give 2-(2-(2-azidoethoxy)ethoxy)ethan-1-ol as a colorless oil (11.7 g).

[0462] Step 2. Preparation of compound 87a Compound 87a was prepared from 86a (4.95 g, 28.3 mmol) and 6a (10 g, 25.7 mmol) using the same procedure as used for compound 84. Yield: 10 g, 77%.

[0463] Step 3. Preparation of compound 88a Compound 88a was prepared from 87a (10 g, 19.8 mmol) using the same procedure as used for compound 85. Yield: 7.63 g, 65%.

[0464] Step 4. Preparation of compound 89a A solution of 88a (2 g, 3.38 mmol) and ZL-glutamic acid (427 mg, 1.52 mmol) in CHCl (50 mL) was treated with HBTU (1.41 g, 3.7 mmol) and Hunig's base (1.77 mL, 10.1 mmol). After stirring (18 h), the mixture was concentrated and chromatographed to give 89a (871 mg, 48%) as a colorless foam. Rf 0.5 (10% CHOH-CHCl).

[0465] Step 5. Preparation of Compound 90a A solution of 89a (870 mg, 0.72 mmol) and Pd / C (90 mg, 10% wet support) in 10 mL of EtOAc was treated with TFA (84 μL, 1.1 mmol) and purged with H. After vigorous stirring (2 h), the reaction was purged with N, filtered through Celite, and concentrated. This crude material was used without further treatment to give 90a (850 mg, quantitative) as a colorless foam. Rf 0.25 (10% CHOH—CHCl).

[0466] Step 6. Preparation of Compound 91a A solution of 90a (850 mg, 0.72 mmol) and Z-glutamic acid (91 mg, 0.32 mmol) in CHCl (10 mL) was treated with HBTU (300 mg, 0.79 mmol) and Hunig's base (502 μL, 2.9 mmol). After stirring (1.5 h), the mixture was diluted with CHCl, washed with NaHCO (sat. aq.), dried (MgSO), filtered, and concentrated. Chromatography of the crude material afforded 91a (590 mg, 76%) as a colorless foam. Rf 0.5 (10% CHOH—CHCl).

[0467] Step 7. Preparation of Compound 92a A solution of 91a (590 mg, 0.25 mmol) and Pd / C (100 mg, 10% wet support) in CHOH (30 mL) was treated with TFA (29 μL, 0.37 mmol) and purged with H. After stirring (3 h), the mixture was purged with N, filtered through Celite, and concentrated. This crude material was used without further treatment to give 92a (600 mg, quantitative) as a colorless foam. Rf 0.1 (10% CHOH—CHCl).

[0468] Step 8. Preparation of conjugate 191a Using the same procedure as used for compound 1, conjugate 191a was prepared from compound 128 and compound 92a.

[0469] Example 16b. Synthesis of conjugate 191b Scheme 36b [ka] Scheme 37b. [ka] Scheme 38b. [ka]

[0470] Step 1. Preparation of 2-(2-(2-azidoethoxy)ethoxy)ethan-1-ol (86b) To a solution of 2-(2-(2-chloroethoxy)ethoxy)ethan-1-ol (13 g, 77 mmol) in water (200 mL) was added sodium azide (10 g, 154 mmol). The reaction was heated to 100° C. for 18 hours. The reaction was cooled to room temperature, poured into a 1 L separatory funnel, and extracted with dichloromethane (3×200 mL). The combined dichloromethane extracts were dried over magnesium sulfate, filtered, and concentrated to dryness to give 2-(2-(2-azidoethoxy)ethoxy)ethan-1-ol as a colorless oil (11.7 g).

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

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

[0473] Step 4. Preparation of compound 89b A solution of 88b (2 g, 3.38 mmol) and racemic Z-glutamic acid (427 mg, 1.52 mmol) in CHCl (50 mL) was treated with HBTU (1.41 g, 3.7 mmol) and Hunig's base (1.77 mL, 10.1 mmol). After stirring (18 h), the mixture was concentrated and chromatographed to give 89b (871 mg, 48%) as a colorless foam. Rf 0.5 (10% CHOH-CHCl).

[0474] Step 5. Preparation of Compound 90b A solution of 89b (870 mg, 0.72 mmol) and Pd / C (90 mg, 10% wet support) in 10 mL of EtOAc was treated with TFA (84 μL, 1.1 mmol) and purged with H. After vigorous stirring (2 h), the reaction was purged with N, filtered through Celite, and concentrated. This crude material was used without further treatment to give 90b (850 mg, quantitative) as a colorless foam. Rf 0.25 (10% CHOH—CHCl).

[0475] Step 6. Preparation of Compound 91b A solution of 90b (850 mg, 0.72 mmol) and Z-glutamic acid (91 mg, 0.32 mmol) in CHCl (10 mL) was treated with HBTU (300 mg, 0.79 mmol) and Hunig's base (502 μL, 2.9 mmol). After stirring (1.5 h), the mixture was diluted with CHCl, washed with NaHCO (sat. aq.), dried (MgSO), filtered, and concentrated. Chromatography of the crude material afforded 91b (590 mg, 76%) as a colorless foam. Rf 0.5 (10% CHOH—CHCl).

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

[0477] Step 8. Preparation of conjugate 191b Using the same procedure as used for compound 1, conjugate 191b was prepared from compound 128 and compound 92b.

[0478] Example 16c. Synthesis of conjugate 191c Scheme 36c [ka] Scheme 37c. [ka] Scheme 38c. [ka]

[0479] Step 1. Preparation of 2-(2-(2-azidoethoxy)ethoxy)ethan-1-ol (86c) To a solution of 2-(2-(2-chloroethoxy)ethoxy)ethan-1-ol (13 g, 77 mmol) in water (200 mL) was added sodium azide (10 g, 154 mmol). The reaction was heated to 100° C. for 18 hours. The reaction was cooled to room temperature, poured into a 1 L separatory funnel, and extracted with dichloromethane (3×200 mL). The combined dichloromethane extracts were dried over magnesium sulfate, filtered, and concentrated to dryness to give 2-(2-(2-azidoethoxy)ethoxy)ethan-1-ol as a colorless oil (11.7 g).

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

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

[0482] Step 4. Preparation of Compound 89c A solution of 88c (2 g, 3.38 mmol) and racemic Z-glutamic acid (427 mg, 1.52 mmol) in CHCl (50 mL) was treated with HBTU (1.41 g, 3.7 mmol) and Hunig's base (1.77 mL, 10.1 mmol). After stirring (18 h), the mixture was concentrated and chromatographed to give 89c (871 mg, 48%) as a colorless foam. Rf 0.5 (10% CHOH-CHCl).

[0483] Step 5. Preparation of Compound 90c A solution of 89c (870 mg, 0.72 mmol) and Pd / C (90 mg, 10% wet support) in 10 mL of EtOAc was treated with TFA (84 μL, 1.1 mmol) and purged with H. After vigorous stirring (2 h), the reaction was purged with N, filtered through Celite, and concentrated. This crude material was used without further treatment to give 90c (850 mg, quantitative) as a colorless foam. Rf 0.25 (10% CHOH—CHCl).

[0484] Step 6. Preparation of Compound 91c A solution of 90c (850 mg, 0.72 mmol) and Z-glutamic acid (91 mg, 0.32 mmol) in CHCl (10 mL) was treated with HBTU (300 mg, 0.79 mmol) and Hunig's base (502 μL, 2.9 mmol). After stirring (1.5 h), the mixture was diluted with CHCl, washed with NaHCO (sat. aq.), dried (MgSO), filtered, and concentrated. The crude material was chromatographed to give 91c (590 mg, 76%) as a colorless foam. Rf 0.5 (10% CHOH—CHCl).

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

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

[0487] Example 17. Synthesis of conjugates 203 and 206 Scheme 39. [ka]

[0488] Step 1. Preparation of compound 69b Compound 69b was prepared from (2S,4R)-4-hydroxypyrrolidine-2-carboxylic acid using the same procedure as used for compound 69.

[0489] Step 2. Preparation of conjugates 203 and 206 Conjugates 203 and 206 were prepared from compounds 96 and 100 using the same procedure as used for compound 1.

[0490] Example 18. Synthesis of conjugate 209 Scheme 40. [ka]

[0491] Step 1. Preparation of conjugate 209 Conjugate 209 was prepared from compounds 96 and 160 using the same procedure as used for compound 1.

[0492] Example 18a. Synthesis of conjugate 209a Scheme 40a. [ka]

[0493] Step 1. Preparation of conjugate 209a Conjugate 209a was prepared from compounds 96a and 160 using the same procedure as used for compound 1.

[0494] Example 19. Synthesis of conjugates 212 and 215 Scheme 41. [ka] Scheme 42. [ka]

[0495] Step 1. Dimethyl 5-(2-((2-oxo-2-phenyl-1λ) 2 Preparation of (ethyl)amino)acetamido)isophthalate (105) A solution of dimethyl 5-aminoisophthalate (5 g, 24 mmol), Z-Gly-OH (5 g, 24 mmol), EDC (5 g, 26.3 mmol), HOBt (3.6 g, 26.3 mmol), and NMM (2.9 mL, 26.3 mmol) in DMF (50 mL) was stirred at room temperature overnight. Upon completion, the reaction mixture was diluted with ethyl acetate (250 mL) and washed with 1 M HCl (2 × 100 mL), saturated sodium bicarbonate (1 × 100 mL), and brine (2 × 100 mL), respectively. The mixture was dried over magnesium sulfate, filtered, and concentrated to dryness to give dimethyl 5-(2-((2-oxo-2-phenyl-1λ)). 2 (7.2 g, 79%) of 1-ethylaminoacetamidoisophthalate was obtained as a colorless solid.

[0496] Step 2. 5-(2-((2-oxo-2-phenyl-1λ 2 Preparation of (-ethyl)amino)acetamido)isophthalic acid (106) Methyl 5-(2-(2-oxo-2-phenyl-1λ)-isopropyl methyl ester) was dissolved in methanol (25 mL) and THF (25 mL). 2 To a solution containing 5-(2-((2-ethyl)amino)acetamido)isophthalate (7.2 g) was added 1 M NaOH (25 mL). The solution was stirred at room temperature for 2 hours and then concentrated to remove THF and MeOH. The remaining aqueous solution was diluted with water (75 mL), cooled in an ice-water bath, and acidified to pH = 1 with 6 M HCl. The solid was filtered and washed with water (3 x 100 mL). The solid was freeze-dried to give 5-(2-((2-oxo-2-phenyl-1λ)). 2 (6.9 g, quantitative yield)-ethyl)amino)acetamido)-isophthalic acid was obtained.

[0497] Step 3. Preparation of Compound 107 Using the same procedure as used for compound 95, 5-(2-((2-oxo-2-phenyl-1λ) 2 Compound 107 was prepared from (-ethyl)amino)acetamido)isophthalic acid 106 (200 mg, 0.54 mmol) and 94 (1.7 g, 1.3 mmol). Yield: 600 mg.

[0498] Step 4. Preparation of Compound 108 Compound 108 was prepared from compound 107 (600 mg) using the same procedure as used for compound 96. Yield: 650 mg, quantitative.

[0499] Step 5. Preparation of Compound 109 Using the same procedure as used for compound 99, 5-(2-((2-oxo-2-phenyl-1λ) 2 Compound 109 was prepared from (-ethyl)amino)acetamido)isophthalic acid (106) (180 mg, 0.48 mmol) and 98 (1.5 g, 1.1 mmol). Yield: 900 mg.

[0500] Step 6. Preparation of Compound 110 Compound 110 was prepared from compound 109 (900 mg) using the same procedure as used for compound 100. Yield: 920 mg, quantitative.

[0501] Step 7. Preparation of conjugates 212 and 215 Conjugates 212 and 215 were prepared from compounds 128 and 108 or 110 using the same procedure as used for compound 1.

[0502] Example 19a. Synthesis of conjugates 212a and 215a Scheme 41a. [ka] Scheme 42a. [ka]

[0503] Step 1. Dimethyl 5-(2-((2-oxo-2-phenyl-1λ) 2 Preparation of (ethyl)amino)acetamido)isophthalate (105a) A solution of dimethyl 5-aminoisophthalate (5 g, 24 mmol), Z-Gly-OH (5 g, 24 mmol), EDC (5 g, 26.3 mmol), HOBt (3.6 g, 26.3 mmol), and NMM (2.9 mL, 26.3 mmol) in DMF (50 mL) was stirred at room temperature overnight. Upon completion, the reaction mixture was diluted with ethyl acetate (250 mL) and washed with 1 M HCl (2 × 100 mL), saturated sodium bicarbonate (1 × 100 mL), and brine (2 × 100 mL), respectively. The mixture was dried over magnesium sulfate, filtered, and concentrated to dryness to give dimethyl 5-(2-((2-oxo-2-phenyl-1λ)). 2 (7.2 g, 79%) of 1-ethylaminoacetamidoisophthalate was obtained as a colorless solid.

[0504] Step 2. 5-(2-((2-oxo-2-phenyl-1λ 2 Preparation of (-ethyl)amino)acetamido)isophthalic acid (106a) Methyl 5-(2-(2-oxo-2-phenyl-1λ)-isopropyl methyl ester) was dissolved in methanol (25 mL) and THF (25 mL). 2 To a solution containing 5-(2-((2-ethyl)amino)acetamido)isophthalate (7.2 g) was added 1 M NaOH (25 mL). The solution was stirred at room temperature for 2 hours and then concentrated to remove THF and MeOH. The remaining aqueous solution was diluted with water (75 mL), cooled in an ice-water bath, and acidified to pH = 1 with 6 M HCl. The solid was filtered and washed with water (3 x 100 mL). The solid was freeze-dried to give 5-(2-((2-oxo-2-phenyl-1λ)). 2 (6.9 g, quantitative yield)-ethyl)amino)acetamido)-isophthalic acid was obtained.

[0505] Step 3. Preparation of Compound 107a Using the same procedure as used for compound 95, 5-(2-((2-oxo-2-phenyl-1λ) 2 Compound 107a was prepared from (-ethyl)amino)acetamido)isophthalic acid 106a (200 mg, 0.54 mmol) and 94a (1.7 g, 1.3 mmol). Yield: 600 mg.

[0506] Step 4. Preparation of Compound 108a Compound 108a was prepared from compound 107a (600 mg) using the same procedure as used for compound 96a. Yield: 650 mg, quantitative.

[0507] Step 5. Preparation of Compound 109a Using the same procedure as used for compound 99, 5-(2-((2-oxo-2-phenyl-1λ) 2 Compound 109a was prepared from (-ethyl)amino)acetamido)isophthalic acid 106a (180 mg, 0.48 mmol) and 98a (1.5 g, 1.1 mmol). Yield: 900 mg.

[0508] Step 6. Preparation of Compound 110a Compound 110a was prepared from compound 109 (900 mg) using the same procedure as used for compound 100. Yield: 920 mg, quantitative.

[0509] Step 7. Preparation of conjugates 212a and 215a Using the same procedure as used for compound 1, conjugates 212a and 215a were prepared from compounds 128 and 108a or 110a.

[0510] Example 20. Synthesis of conjugates 218 and 221 Scheme 43. [ka] Scheme 44. [ka]

[0511] Step 1. Preparation of Compound 111 Compound 111 was prepared from 4-(((tert-butoxycarbonyl)amino)methyl)phthalic acid (1.13 g, 3.84 mmol) and 88 (5 g, 8.44 mmol) using the same procedure as used for compound 89. Yield: 2.21 g, 49%.

[0512] Step 2. Preparation of Compound 112 A solution of 111 (2.21 g, 1.87 mmol) in CH2Cl2 (40 mL) was slowly treated with TFA (5 mL). After stirring (2 h), the mixture was concentrated and chromatographed to give 112 (1.08 g, 47%) as a colorless foam. Rf 0.1 (10% CH3OH-CH2Cl2).

[0513] Step 3. Preparation of Compound 113 Compound 112 (1.08 g, 0.88 mmol) and (2-oxo-2-phenyl-1λ) were synthesized using the same procedure as used for compound 91. 2 Compound 113 was prepared from (-ethyl)-D-glutamic acid (112 mg, 0.39 mmol). Yield: 600 mg, 62%.

[0514] Step 4. Preparation of Compound 114 Compound 114 was prepared from compound 113 using the same procedure as used for compound 92.

[0515] Step 5. Preparation of Compound 115 Compound 115 was prepared from 4-(((tert-butoxycarbonyl)amino)methyl)phthalic acid (3.94 g, 13.3 mmol) and 9 (18.2 g, 29.4 mmol) using the same procedure as used for compound 93. Yield: 9.02 g, 53%.

[0516] Step 6. Preparation of Compound 116 Compound 116 was prepared from compound 115 (8 g, 6.3 mmol) using the same procedure as used for compound 112. Yield: 3.23 g, 39%.

[0517] Step 7. Preparation of Compound 117 Compound 116 (3.23 g, 2.45 mmol) and (2-oxo-2-phenyl-1λ) were prepared using the same procedure as used for compound 95. 2 Compound 117 was prepared from (-ethyl)-D-glutamic acid (192 mg, 1.1 mmol). Yield: 2.22 g, 34%.

[0518] Step 8. Preparation of Compound 118 Compound 118 was prepared from compound 117 (2.22 g, 0.84 mmol) using the same procedure as used for compound 96. Yield: 2.02 g, 91%.

[0519] Step 9. Preparation of conjugates 218 and 221 Conjugates 218 and 221 were prepared from compounds 128 and 114 or 118 using the same procedure as used for compound 1.

[0520] Example 20a. Synthesis of conjugates 218a and 221a Scheme 43a. [ka] Scheme 44a. [ka]

[0521] Step 1. Preparation of Compound 111a Compound 111a was prepared from 4-(((tert-butoxycarbonyl)amino)methyl)phthalic acid (1.13 g, 3.84 mmol) and 88 (5 g, 8.44 mmol) using the same procedure as used for compound 89. Yield: 2.21 g, 49%.

[0522] Step 2. Preparation of Compound 112a A solution of 111a (2.21 g, 1.87 mmol) in CH2Cl2 (40 mL) was slowly treated with TFA (5 mL). After stirring (2 h), the mixture was concentrated and chromatographed to give 112a (1.08 g, 47%) as a colorless foam. Rf 0.1 (10% CH3OH-CH2Cl2).

[0523] Step 3. Preparation of Compound 113a Compound 112a (1.08 g, 0.88 mmol) and (2-oxo-2-phenyl-1λ) were synthesized using the same procedure as used for compound 91. 2 Compound 113a was prepared from (-ethyl)-D-glutamic acid (112 mg, 0.39 mmol). Yield: 600 mg, 62%.

[0524] Step 4. Preparation of Compound 114a Compound 114a was prepared from compound 113a using the same procedure as used for compound 92.

[0525] Step 5. Preparation of Compound 115a Compound 115a was prepared from 4-(((tert-butoxycarbonyl)amino)methyl)phthalic acid (3.94 g, 13.3 mmol) and 9 (18.2 g, 29.4 mmol) using the same procedure as used for compound 93. Yield: 9.02 g, 53%.

[0526] Step 6. Preparation of Compound 116a Compound 116a was prepared from compound 115a (8 g, 6.3 mmol) using the same procedure as used for compound 11a. Yield: 3.23 g, 39%.

[0527] Step 7. Preparation of Compound 117a Compound 116a (3.23 g, 2.45 mmol) and (2-oxo-2-phenyl-1λ) were prepared using the same procedure as used for compound 95. 2 Compound 117a was prepared from (-ethyl)glutamic acid (192 mg, 1.1 mmol). Yield: 2.22 g, 34%.

[0528] Step 8. Preparation of Compound 118a Compound 118a was prepared from compound 117a (2.22 g, 0.84 mmol) using the same procedure as used for compound 96. Yield: 2.02 g, 91%.

[0529] Step 9. Preparation of conjugates 218a and 221a Using the same procedure as used for compound 1, conjugates 218a and 221a were prepared from compounds 128 and 114a or 118a.

[0530] Example 21. Synthesis of conjugate 224 Scheme 45. [ka]

[0531] Step 1. Preparation of Compound 224 Conjugate 224 was prepared from compounds 96 and 130 using the same procedure as used for compound 1.

[0532] Example 21a. Synthesis of conjugate 224b Scheme 45a. [ka] [ka]

[0533] Step 1. Preparation of compound 224b Conjugate 224b was prepared from compounds 96b and 130 using the same procedure as used for compound 1.

[0534] Example 22 Synthesis of Conjugate 231 Scheme 46 [ka] [ka] Scheme 47 [ka] [ka]

[0535] Step 1 Preparation of compound 225 Compound 225 was prepared from 5-(2-aminoacetamido)isophthalic acid (106) (560 mg, 1.5 mmol) and 9 (2.24 g, 3.6 mmol) using the same procedure as used for 89. Yield 1.6 g, 80%.

[0536] Step 2 Preparation of compound 226 Compound 226 was prepared in the same manner as 14. Yield 1.22 g, 78%.

[0537] Step 3 Preparation of compound 227 Compound 227 was prepared from Z-glutamic acid (108 mg, 0.38 mmol) and 226 (1.22 g, 0.92 mmol) in the same manner as 89. Yield 471 mg, 45%.

[0538] Step 4 Preparation of compound 228 Compound 228 was prepared in the same manner as 14. Yield 460 mg, quantitative.

[0539] Step 5 Preparation of compound 229 Compound 229 was prepared from 228 (460 mg, 0.17 mmol) and 128 (125 mg, 0.19 mmol) in the same manner as 89. Yield 365 mg, 66%.

[0540] Step 6 Preparation of compound 231 Conjugate 231 was prepared using the same procedure as used for compound 1.

[0541] Example 22a Synthesis of conjugate 231a Scheme 46a [ka] [ka] Scheme 47a [ka] [ka]

[0542] Step 1 Preparation of compound 225a Compound 225a was prepared from 5-(2-aminoacetamido)isophthalic acid (106) (560 mg, 1.5 mmol) and 9 (2.24 g, 3.6 mmol) using the same procedure as used for 89. Yield 1.6 g, 80%.

[0543] Step 2 Preparation of compound 226a Compound 226a was prepared in the same manner as 14. Yield 1.22 g, 78%.

[0544] Step 3 Preparation of compound 227a Compound 227a was prepared from Z-glutamic acid (108 mg, 0.38 mmol) and 226a (1.22 g, 0.92 mmol) in the same manner as 89. Yield 471 mg, 45%.

[0545] Step 4 Preparation of compound 228a Compound 228a was prepared in the same manner as 14. Yield 460 mg, quantitative.

[0546] Step 5 Preparation of compound 229a Compound 229a was prepared from 228a (460 mg, 0.17 mmol) and 128 (125 mg, 0.19 mmol) in the same manner as 89. Yield 365 mg, 66%.

[0547] Step 6 Preparation of compound 231a Using the same procedure as used for compound 1, conjugate 231a was prepared.

[0548] Example 22b Synthesis of conjugate 231b Scheme 46b [ka] [ka] Scheme 47b [ka] [ka]

[0549] Step 1 Preparation of compound 225b Compound 225b was prepared from 5-(2-aminoacetamido)isophthalic acid (106) (560 mg, 1.5 mmol) and 9 (2.24 g, 3.6 mmol) using the same procedure as used for 89. Yield 1.6 g, 80%.

[0550] Step 2 Preparation of compound 226b Compound 226b was prepared in the same manner as 14. Yield 1.22 g, 78%.

[0551] Step 3 Preparation of compound 227b Compound 227b was prepared from Z-glutamic acid (108 mg, 0.38 mmol) and 226b (1.22 g, 0.92 mmol) in the same manner as 89. Yield 471 mg, 45%.

[0552] Step 4 Preparation of compound 228b Compound 228b was prepared in the same manner as 14. Yield 460 mg, quantitative.

[0553] Step 5 Preparation of compound 229b Compound 229b was prepared from 228b (460 mg, 0.17 mmol) and 128 (125 mg, 0.19 mmol) in the same manner as 89. Yield 365 mg, 66%.

[0554] Step 6 Preparation of compound 231b Using the same procedure as used for compound 1, conjugate 231b was prepared.

[0555] Example 23. Synthesis of conjugate 233 Scheme 48 [ka] [ka]

[0556] Step 1. Preparation of Compound 232 Compound 232 was prepared from compound 24 (650 mg, 0.33 mmol) and compound 69b (175 mg, 0.33 mmol) using the same procedure as used for compound 19. Yield: 380 mg, 47%.

[0557] Step 2. Preparation of Compound 233 Compound 233 was prepared from compound 232 using the same procedure as used for compound 1.

[0558] Example 24. Synthesis of conjugate 235 Scheme 49 [ka] [ka] [ka]

[0559] Step 1. Preparation of compound 234 Compound 234 was prepared from compound 24 (1.1 g, 0.55 mmol) and compound 18 (175 mg, 0.33 mmol) using the same procedure as used for compound 19. Yield: 685 mg, 51%.

[0560] Step 2. Preparation of Compound 235 Compound 235 was prepared from compound 234 using the same procedure as used for compound 1.

[0561] Example 25. In vivo testing of HBV siRNA conjugates Chronic HBV infection is a global disease that causes progressive liver damage. Currently available treatments can reduce viral DNA, but have little effect on viral antigens, which are a major contributor to disease progression. Therefore, we designed siRNAs targeting HBV to reduce viral antigens.

[0562] Chemically modified HBV siRNAs conjugated to GalNAc ligands, as described in Table 1, were tested for in vivo activity in an established mouse model of HBV infection. In the AAV-HBV1.2 C57BL / 6 mouse model, stable and persistent HBV expression is achieved after injection of an adeno-associated virus (AAV) vector encoding a sequence exceeding the genome length of HBV, resulting in expression of HBV RNA and proteins in the liver and secretion of viral and subviral particles into the blood.

[0563] The AAV-HBV1.2 constructs used in these studies were based on the details provided in Dion, S., et al., Journal of Virology, 2013, 87(10):5554-5563. All animal-related procedures were performed in accordance with operating procedures that adhere to the Canadian Council on Animal Care (CCAC) Guidelines on Good Animal Practices and were approved by the local Institutional Animal Care and Use Committee (IACUC).

[0564] Each animal was inoculated with the AAV-HBV1.2 vector of 1E11 vector genome (VG). Prior to treatment, all animals were bled and serum HBsAg levels were determined in each animal to confirm established HBV expression.

[0565] siRNA treatment: Groups of mice (typically n=5) were administered a single 3 mg / kg dose of HBV siRNA conjugate by subcutaneous injection in the scapular region once (one dose per animal) on day 0. One group of animals receiving vehicle only (saline) served as control.

[0566] Collection: Test bleeds were taken from all mice on day 0, prior to treatment, and at predetermined time points after test article administration (e.g., study days 7, 14, 21, 28, 35, 42, 49, 56, 63, and 70) to determine the maximum reduction in serum HBsAg levels and the duration of pharmacological activity.

[0567] Analysis: HBsAg levels were determined in serum samples using a Biorad EIA GS HBsAg 3.0 kit (BioRad, catalog number 32591) according to the manufacturer's instructions. Pooled serum from each treatment group was used to determine group mean HBsAg levels at each time point. Data were analyzed and expressed as HBsAg levels relative to pretreatment baseline (% of day 0).

[0568] Results: The results of testing each of the chemically modified HBV siRNAs listed in Table 1 are presented in Table 2. Values ​​represent the percent (%) of HBsAg levels (relative to day 0 baseline) at days 7, 14, 21, 28, 42, 49, 56, and 70 after treatment. [Table 1-1] [Table 1-2] 2'-O-methyl nucleotides = lowercase; 2'-fluoro nucleotides = uppercase underlined; phosphorothioate linker = s; unmodified = uppercase [Table 2-1] [Table 2-2] Table 2 identifies the compound numbers (column 2) and corresponding oligonucleotides (column 1) of the HBV siRNA conjugates tested.

[0569] Example 26 Synthesis of Conjugate 320 Scheme 50. Preparation of activated linkers [ka]

[0570] Step 1. Preparation of racemic (cis) 5-benzyl-3a,6a-dimethyltetrahydro-1H-furo[3,4-c]pyrrole-1,3(3aH)-dione (301) To a cooled (0 °C) solution of 3,4-dimethylfuran-2,5-dione (3 g, 24 mmol) and N-benzyl-1-methoxy-N-((trimethylsilyl)methyl)methanamine (7 g, 29.8 mmol) in dichloromethane (75 mL) was added trifluoroacetic acid (75 μL) slowly. Stirred overnight, allowing the solution to slowly warm to room temperature as the ice bath melted. The reaction mixture was concentrated to dryness, dissolved in ethyl acetate (100 mL), washed with saturated sodium bicarbonate (2 × 100 mL), dried over magnesium sulfate, filtered, and concentrated to dryness. Purification by column chromatography on silica gel (gradient: 20% ethyl acetate in hexane to 100% ethyl acetate) gave (3aR,6aS)-5-benzyl-3a,6a-dimethyltetrahydro-1H-furo[3,4-c]pyrrole-1,3(3aH)-dione as a yellow oil (3.5 g, 56%).

[0571] Step 2. Preparation of racemic (cis)(1-benzyl-3,4-dimethylpyrrolidine-3,4-diyl)dimethanol (302) To a cooled (0 °C) solution of (3aR,6aS)-5-benzyl-3a,6a-dimethyltetrahydro-1H-furo[3,4-c]pyrrole-1,3(3aH)-dione (3.5 g, 13.4 mmol) in anhydrous diethyl ether (50 mL) was slowly added lithium aluminum hydride pellets (1.5 g, 40 mmol) in three portions. The solution was stirred overnight and allowed to return to room temperature as the ice-water bath melted. Upon completion, the reaction was cooled to 0 °C and quenched very slowly with 1.5 mL of 5 M NaOH followed by 1.5 mL of water. Stirring was continued for 30 minutes, followed by the addition of magnesium sulfate and filtration. The filtrate was concentrated to give ((3R,4S)-1-benzyl-3,4-dimethylpyrrolidine-3,4-diyl)dimethanol as a colorless oil (2.7 g).

[0572] Step 3. Preparation of racemic (cis)(3,4-dimethylpyrrolidine-3,4-diyl)dimethanol (303) To a solution of ((3R,4S)-1-benzyl-3,4-dimethylpyrrolidine-3,4-diyl)dimethanol (10 g, 40 mmol) in methanol (10 mL) was added wet 10% palladium on activated carbon (1 g). The solution was vigorously stirred under a hydrogen atmosphere for 16 hours. Upon completion, the solution was filtered through Celite and concentrated to dryness to give ((3R,4S)-3,4-dimethylpyrrolidine-3,4-diyl)dimethanol as a colorless solid (5.5 g, 86%).

[0573] Step 4. Preparation of racemic (cis) methyl 10-(3,4-bis(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoate (304) A solution of 3 (1.3 g, 8.2 mmol) and monomethyl sebacate (1.8 g, 8.2 mmol) in CHCl (100 mL) was treated with HBTU (3.41 g, 9.02 mmol) and Hunig's base (5.71 mL, 32.8 mmol). After stirring overnight, the mixture was washed with NaHCO (saturated aqueous solution), water, and brine, then dried (MgSO), filtered, and concentrated. The crude material was chromatographed (gradient: 0% CHOH-CHCl to 20%) to give 4 (1.8 g, 61%).

[0574] Step 5. Preparation of racemic (cis)methyl 10-(3-((bis(4-methoxyphenyl)(phenyl)-methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoate (305) A solution of 304 (1.8 g, 5.0 mmol) and 4,4'-dimethoxytrityl chloride (1.7 g, 5.0 mmol) in pyridine (180 mL) was stirred overnight. Pyridine was then removed under reduced pressure, and the crude material was chromatographed (gradient: 0% CHOH—CHCl to 10%) to give 5 (1.4 g, 42%) as a yellow oil.

[0575] Step 6. Preparation of racemic (cis) lithium 10-(3-((bis(4-methoxyphenyl)-(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoate (306) To a solution of compound 305 (3.0 g, 4.6 mmol) in THF (50 mL) and water (50 mL) was added lithium hydroxide (121 mg, 5.0 mmol). The solution was stirred at room temperature for 4 hours and then concentrated to remove the THF. The remaining aqueous solution was lyophilized overnight to give a pale pink solid (2.9 g, quantitative). Compound 306 was prepared as a mixture of two cis-diastereomers.

[0576] Scheme 51. Synthesis of peracetylated galactosamine (307) [ka] D-Galactosamine hydrochloride (250 g, 1.16 mol) in pyridine (1.5 L) was treated with acetic anhydride (1.25 L, 13.2 mol) for 45 minutes. After stirring overnight, the reaction mixture was divided into three 1 L portions. Each 1 L portion was poured into 3 L of ice water and mixed for 1 hour. After mixing, the solids were filtered off, combined, frozen in liquid nitrogen, and lyophilized for 5 days to give peracetylated galactosamine (307) (369.4 g, 82%) as a white solid. Rf (0.58, 10% MeOH-CH2Cl2).

[0577] Scheme 52. Synthesis of GalNAc monomers [ka]

[0578] Step 1 Preparation of compound 309 A solution of 2-[2-(2-chloroethoxy)]ethanol (308) (100 g, 593 mmol) in water (1 L) was treated with NaN (77 g, 1.19 mol) and heated (90 °C). After stirring (72 h), the solution was cooled (room temperature) and extracted with CHCl (4 times). The combined organics were washed with brine, dried (MgSO), filtered, concentrated, and used without further treatment. Compound 9 (88.9 g, 86%) was obtained as a pale yellow oil.

[0579] Step 2 Preparation of compound 310 A solution of 7 (2.76 g, 7.1 mmol) and 309 (1.37 g, 7.8 mmol) in 1,2-dichloroethane (40 mL) was treated with Sc(OTf) (174 mg, 0.36 mmol) and heated (85 °C). After stirring (2 h), the mixture was cooled (room temperature), quenched by the addition of TEA (4 mL), and concentrated. The crude material was chromatographed to give 310 (3.03 g, 85%) as a pale yellow foam.

[0580] Step 3 Preparation of compound 311 A solution of 310 (3.02 g, 5.99 mmol) and Pd / C (300 mg, 10% Pd loading-wet support) in 30 mL of EtOAc was treated with TFA (576 μL, 7.5 mmol). The reaction mixture was purged with hydrogen gas (45 min), then with nitrogen gas (10 min), and then filtered through Celite. The filtrate was concentrated and chromatographed to give 311 (2.67 g, 75%) as a brown foam.

[0581] Scheme 53. Synthesis of aromatic cores [ka]

[0582] Step 1. Dimethyl 5-(2-((2-oxo-2-phenyl-1λ) 2 Preparation of (ethyl)amino)acetamido)isophthalate (312) A solution of dimethyl 5-aminoisophthalate (5 g, 24 mmol), Z-Gly-OH (5 g, 24 mmol), EDC (5 g, 26.3 mmol), HOBt (3.6 g, 26.3 mmol), and NMM (2.9 mL, 26.3 mmol) in DMF (50 mL) was stirred at room temperature overnight. Upon completion, the reaction mixture was diluted with ethyl acetate (250 mL) and washed with 1 M HCl (2 × 100 mL), saturated sodium bicarbonate (1 × 100 mL), and brine (2 × 100 mL), respectively. The mixture was dried over magnesium sulfate, filtered, and concentrated to dryness to give dimethyl 5-(2-((2-oxo-2-phenyl-1λ)). 2 (7.2 g, 79%) of 1,3-dichloro-2,4-diamino-2-acetamidoisophthalate was obtained as a colorless solid.

[0583] Step 2. 5-(2-((2-oxo-2-phenyl-1λ 2 Preparation of (-ethyl)amino)acetamido)isophthalic acid (313) Methyl 5-(2-(2-oxo-2-phenyl-1λ)-isopropyl methyl ester) was dissolved in methanol (25 mL) and THF (25 mL). 2To a solution containing 5-(2-((2-ethyl)amino)acetamido)isophthalate (7.2 g) was added 1 M NaOH (25 mL). The solution was stirred at room temperature for 2 hours and then concentrated to remove THF and MeOH. The remaining aqueous solution was diluted with water (75 mL), cooled in an ice-water bath, and acidified to pH = 1 with 6 M HCl. The solid was filtered and washed with water (3 x 100 mL). The solid was freeze-dried to give 5-(2-((2-oxo-2-phenyl-1λ)). 2 (6.9 g, quantitative yield)-ethyl)amino)acetamido)-isophthalic acid was obtained.

[0584] Scheme 54: Preparation of the tetramer [ka] [ka]

[0585] Step 1 Preparation of compound 314 A solution of 313 (2.09 g, 5.6 mmol) and 311 (8.34 g, 14.07 mmol) in CHCl (150 mL) was treated with HBTU (6.4 g, 16.9 mmol) and Hunig's base (7.35 mL, 42.2 mmol). After stirring (overnight), the reaction mixture was poured into NaHCO (saturated aqueous solution), then washed with water and brine, dried (MgSO), filtered, and concentrated. The crude material was chromatographed (gradient 1-12% CHOH-CHCl) to give 6 (3.97 g, 55%) as a pale yellow foam.

[0586] Step 2 Preparation of compound 315 Compound 314 (3.92 g, 3.07 mmol), Pd / C (400 mg, 10% loading-wet support), and trifluoroacetic acid (308 μL, 4 mmol) were purged with H. After stirring under H (overnight), the mixture was purged with N (15–20 min), then filtered through Celite and concentrated. Chromatography of the crude material afforded 7 (3.36 g, 86%) as a white to cream-colored foam.

[0587] Step 3 Preparation of compound 316 Compound 316 was prepared from Z-glutamic acid (306 mg, 1.09 mmol) and 315 (3.3 g, 2.6 mmol) in the same manner as 314. Yield 1.66 g, 60%.

[0588] Step 4 Preparation of compound 317 Compound 317 was prepared in the same manner as 315. Yield 1.65 g, quantitative.

[0589] Scheme 55. Preparation of the complete conjugate [ka]

[0590] Step 1 Preparation of compound 318 A solution of 317 (1.91 g, 0.75 mmol) in CHCl (100 mL) was treated first with Hunig's base (392 μL, 2.25 mmol), then with 6 (a mixture of two cis-diastereomers, 509 mg, 0.79 mmol), followed by HBTU (356 mg, 0.94 mmol). After stirring (overnight), the solution was poured into NaHCO (saturated aqueous solution), then washed with water and brine, dried (MgSO), filtered, and concentrated. Chromatography of the crude material afforded 318 (1.19 g, 52%) as a white foam.

[0591] Step 2 Preparation of compound 319 A solution of 318 (1.19 g, 0.39 mmol) in 1,2-dichloroethane (100 mL) was treated with TEA (542 μL, 3.9 mmol), DMAP (238 mg, 1.95 mmol), and succinic anhydride (195 mg, 1.95 mmol) and heated (85 °C). After stirring (2.5 h), the solution was removed from the heat, treated with CHOH (10 mL), and allowed to stir (1 h). After stirring, the mixture was poured into NaHCO (saturated aqueous solution), then washed with brine, dried (MgSO), filtered, and concentrated. The resulting residue was used without further treatment. Yield = 1.4 g, quantitative.

[0592] Step 3 Preparation of conjugate 320 Using standard amide coupling chemistry, succinate 319 was loaded onto 1000 Å LCAA (long-chain amino alkyl) CPG (controlled pore glass). A solution of diisopropylcarbodiimide (52.6 μmol), N-hydroxysuccinimide (0.3 mg, 2.6 μmol), and pyridine (10 μL) in anhydrous acetonitrile (0.3 mL) was added to 319 (20.6 mg, 8 μmol) in anhydrous dichloromethane (0.2 mL). This mixture was added to LCAA CPG (183 mg). The suspension was gently mixed overnight at room temperature. Once 319 had disappeared (HPLC), the reaction mixture was filtered, and the CPG was washed with 1 mL each of dichloromethane, acetonitrile, and a solution of 5% acetic anhydride / 5% N-methylimidazole / 5% pyridine in THF, followed by THF, acetonitrile, and dichloromethane. The CPG was then dried overnight under high vacuum. Loading was determined to be 19 μmol / g by standard DMTr assay by UV / Vis (504 nm). The resulting GalNAc-loaded CPG solid support was used for automated oligonucleotide synthesis using standard procedures. Nucleotide deprotection followed by removal from the solid support (concurrent with galactosamine acetate deprotection) afforded GalNAc-oligonucleotide conjugate 320.

[0593] Example 27 Synthesis of Conjugate 520 Scheme 56. Preparation of activated linkers [ka]

[0594] Step 1. Preparation of racemic (cis) 5-benzyl-3a,6a-dimethyltetrahydro-1H-furo[3,4-c]pyrrole-1,3(3aH)-dione (301) To a cooled (0 °C) solution of 3,4-dimethylfuran-2,5-dione (3 g, 24 mmol) and N-benzyl-1-methoxy-N-((trimethylsilyl)methyl)methanamine (7 g, 29.8 mmol) in dichloromethane (75 mL) was added trifluoroacetic acid (75 μL) slowly. Stirred overnight, allowing the solution to slowly warm to room temperature as the ice bath melted. The reaction mixture was concentrated to dryness, dissolved in ethyl acetate (100 mL), washed with saturated sodium bicarbonate (2 × 100 mL), dried over magnesium sulfate, filtered, and concentrated to dryness. Purification by column chromatography on silica gel (gradient: 20% ethyl acetate in hexane to 100% ethyl acetate) gave (3aR,6aS)-5-benzyl-3a,6a-dimethyltetrahydro-1H-furo[3,4-c]pyrrole-1,3(3aH)-dione as a yellow oil (3.5 g, 56%).

[0595] Step 2. Preparation of racemic (cis)(1-benzyl-3,4-dimethylpyrrolidine-3,4-diyl)dimethanol (302) To a cooled (0 °C) solution of (3aR,6aS)-5-benzyl-3a,6a-dimethyltetrahydro-1H-furo[3,4-c]pyrrole-1,3(3aH)-dione (3.5 g, 13.4 mmol) in anhydrous diethyl ether (50 mL) was slowly added lithium aluminum hydride pellets (1.5 g, 40 mmol) in three portions. The solution was stirred overnight and allowed to return to room temperature as the ice-water bath melted. Upon completion, the reaction was cooled to 0 °C and quenched very slowly with 1.5 mL of 5 M NaOH followed by 1.5 mL of water. Stirring was continued for 30 minutes, followed by the addition of magnesium sulfate and filtration. The filtrate was concentrated to give ((3R,4S)-1-benzyl-3,4-dimethylpyrrolidine-3,4-diyl)dimethanol as a colorless oil (2.7 g).

[0596] Step 3. Preparation of racemic (cis)(3,4-dimethylpyrrolidine-3,4-diyl)dimethanol (303) To a solution of ((3R,4S)-1-benzyl-3,4-dimethylpyrrolidine-3,4-diyl)dimethanol (10 g, 40 mmol) in methanol (10 mL) was added wet 10% palladium on activated carbon (1 g). The solution was vigorously stirred under a hydrogen atmosphere for 16 hours. Upon completion, the solution was filtered through Celite and concentrated to dryness to give ((3R,4S)-3,4-dimethylpyrrolidine-3,4-diyl)dimethanol as a colorless solid (5.5 g, 86%).

[0597] Step 4. Preparation of racemic (cis) methyl 10-(3,4-bis(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoate (304) A solution of 3 (1.3 g, 8.2 mmol) and monomethyl sebacate (1.8 g, 8.2 mmol) in CHCl (100 mL) was treated with HBTU (3.41 g, 9.02 mmol) and Hunig's base (5.71 mL, 32.8 mmol). After stirring overnight, the mixture was washed with NaHCO (saturated aqueous solution), water, and brine, then dried (MgSO), filtered, and concentrated. The crude material was chromatographed (gradient: 0% CHOH-CHCl to 20%) to give 4 (1.8 g, 61%).

[0598] Step 5. Preparation of racemic (cis)methyl 10-(3-((bis(4-methoxyphenyl)(phenyl)-methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoate (305) A solution of 304 (1.8 g, 5.0 mmol) and 4,4'-dimethoxytrityl chloride (1.7 g, 5.0 mmol) in pyridine (180 mL) was stirred overnight. Pyridine was then removed under reduced pressure, and the crude material was chromatographed (gradient: 0% CHOH—CHCl to 10%) to give 5 (1.4 g, 42%) as a yellow oil.

[0599] Step 6. Preparation of racemic (cis) lithium 10-(3-((bis(4-methoxyphenyl)-(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-10-oxodecanoate (306) To a solution of compound 305 (3.0 g, 4.6 mmol) in THF (50 mL) and water (50 mL) was added lithium hydroxide (121 mg, 5.0 mmol). The solution was stirred at room temperature for 4 hours and then concentrated to remove the THF. The remaining aqueous solution was lyophilized overnight to give a pale pink solid (2.9 g, quantitative). Compound 306 was prepared as a mixture of two cis-diastereomers.

[0600] Scheme 57. Synthesis of peracetylated galactosamine (507) [ka] Galactosamine hydrochloride (250 g, 1.16 mol) in pyridine (1.5 L) was treated with acetic anhydride (1.25 L, 13.2 mol) for 45 minutes. After stirring overnight, the reaction mixture was divided into three 1 L portions. Each 1 L portion was poured into 3 L of ice water and mixed for 1 hour. After mixing, the solids were filtered off, combined, frozen in liquid nitrogen, and lyophilized for 5 days to give peracetylated galactosamine (507) (369.4 g, 82%) as a white solid. Rf (0.58, 10% MeOH-CH2Cl2).

[0601] Scheme 58. Synthesis of GalNAc monomers [ka]

[0602] Step 1 Preparation of compound 509 A solution of 2-[2-(2-chloroethoxy)]ethanol (508) (100 g, 593 mmol) in water (1 L) was treated with NaN (77 g, 1.19 mol) and heated (90 °C). After stirring (72 h), the solution was cooled (room temperature) and extracted with CHCl (4 times). The combined organics were washed with brine, dried (MgSO), filtered, concentrated, and used without further treatment. Compound 509 (88.9 g, 86%) was obtained as a pale yellow oil.

[0603] Step 2 Preparation of compound 510 A solution of 507 (2.76 g, 7.1 mmol) and 509 (1.37 g, 7.8 mmol) in 1,2-dichloroethane (40 mL) was treated with Sc(OTf) (174 mg, 0.36 mmol) and heated (85 °C). After stirring (2 h), the mixture was cooled (RT), quenched by the addition of TEA (4 mL), and concentrated. The crude material was chromatographed to give 510 (3.03 g, 85%) as a pale yellow foam.

[0604] Step 3 Preparation of compound 511 A solution of 510 (3.02 g, 5.99 mmol) and Pd / C (300 mg, 10% Pd loading-wet support) in 30 mL of EtOAc was treated with TFA (576 μL, 7.5 mmol). The reaction mixture was purged with hydrogen gas (45 min), then with nitrogen gas (10 min), and then filtered through Celite. The filtrate was concentrated and chromatographed to give 511 (2.67 g, 75%) as a brown foam.

[0605] Scheme 59. Synthesis of aromatic cores [ka]

[0606] Step 1. Dimethyl 5-(2-((2-oxo-2-phenyl-1λ) 2 Preparation of (ethyl)amino)acetamido)isophthalate (312) A solution of dimethyl 5-aminoisophthalate (5 g, 24 mmol), Z-Gly-OH (5 g, 24 mmol), EDC (5 g, 26.3 mmol), HOBt (3.6 g, 26.3 mmol), and NMM (2.9 mL, 26.3 mmol) in DMF (50 mL) was stirred at room temperature overnight. Upon completion, the reaction mixture was diluted with ethyl acetate (250 mL) and washed with 1 M HCl (2 × 100 mL), saturated sodium bicarbonate (1 × 100 mL), and brine (2 × 100 mL), respectively. The mixture was dried over magnesium sulfate, filtered, and concentrated to dryness to give dimethyl 5-(2-((2-oxo-2-phenyl-1λ)). 2 (7.2 g, 79%) of 1,3-dichloro-2,4-diamino-2-acetamidoisophthalate was obtained as a colorless solid.

[0607] Step 2. 5-(2-((2-oxo-2-phenyl-1λ 2 Preparation of (-ethyl)amino)acetamido)isophthalic acid (313) Methyl 5-(2-(2-oxo-2-phenyl-1λ)-isopropyl methyl ester) was dissolved in methanol (25 mL) and THF (25 mL). 2To a solution containing 5-(2-((2-ethyl)amino)acetamido)isophthalate (7.2 g) was added 1 M NaOH (25 mL). The solution was stirred at room temperature for 2 hours and then concentrated to remove THF and MeOH. The remaining aqueous solution was diluted with water (75 mL), cooled in an ice-water bath, and acidified to pH = 1 with 6 M HCl. The solid was filtered and washed with water (3 x 100 mL). The solid was lyophilized to give 5-(2-((2-oxo-2-phenyl-1λ)). 2 (6.9 g, quantitative yield)-ethyl)amino)acetamido)-isophthalic acid was obtained.

[0608] Scheme 60: Preparation of the tetramer [ka] [ka]

[0609] Step 1 Preparation of compound 514 A solution of 313 (2.09 g, 5.6 mmol) and 511 (8.34 g, 14.07 mmol) in CHCl (150 mL) was treated with HBTU (6.4 g, 16.9 mmol) and Hunig's base (7.35 mL, 42.2 mmol). After stirring (overnight), the reaction mixture was poured into NaHCO (saturated aqueous solution), then washed with water and brine, dried (MgSO), filtered, and concentrated. The crude material was chromatographed (gradient 1-12% CHOH-CHCl) to give 6 (3.97 g, 55%) as a pale yellow foam.

[0610] Step 2 Preparation of compound 515 Compound 514 (3.92 g, 3.07 mmol), Pd / C (400 mg, 10% loading-wet support), and trifluoroacetic acid (308 μL, 4 mmol) were purged with H. After stirring under H (overnight), the mixture was purged with N (15–20 min), then filtered through Celite and concentrated. Chromatography of the crude material afforded 7 (3.36 g, 86%) as a white to cream-colored foam.

[0611] Step 3 Preparation of compound 516 Compound 516 was prepared from Z-glutamic acid (306 mg, 1.09 mmol) and 515 (3.3 g, 2.6 mmol) in the same manner as 514. Yield 1.66 g, 60%.

[0612] Step 4 Preparation of compound 517 Compound 517 was prepared in the same manner as 515. Yield 1.65 g, quantitative.

[0613] Scheme 61. Preparation of the complete conjugate [ka] [ka]

[0614] Step 1 Preparation of compound 518 A solution of 517 (1.91 g, 0.75 mmol) in CHCl (100 mL) was treated first with Hunig's base (392 μL, 2.25 mmol), then with 306 (a mixture of two cis-diastereomers, 509 mg, 0.79 mmol), followed by HBTU (356 mg, 0.94 mmol). After stirring (overnight), the solution was poured into NaHCO (saturated aqueous solution), then washed with water and brine, dried (MgSO), filtered, and concentrated. Chromatography of the crude material afforded 518 (1.19 g, 52%) as a white foam.

[0615] Step 2 Preparation of compound 519 A solution of 518 (1.19 g, 0.39 mmol) in 1,2-dichloroethane (100 mL) was treated with TEA (542 μL, 3.9 mmol), DMAP (238 mg, 1.95 mmol), and succinic anhydride (195 mg, 1.95 mmol) and heated (85 °C). After stirring (2.5 h), the solution was removed from the heat, treated with CHOH (10 mL), and allowed to stir (1 h). After stirring, the mixture was poured into NaHCO (saturated aqueous solution), then washed with brine, dried (MgSO), filtered, and concentrated. The resulting residue was used without further treatment. Yield = 1.4 g, quantitative.

[0616] Step 3 Preparation of conjugate 520 Using standard amide coupling chemistry, succinate 519 was loaded onto 1000 Å LCAA (long-chain amino alkyl) CPG (controlled pore glass). A solution of diisopropylcarbodiimide (52.6 μmol), N-hydroxysuccinimide (0.3 mg, 2.6 μmol), and pyridine (10 μL) in anhydrous acetonitrile (0.3 mL) was added to 519 (20.6 mg, 8 μmol) in anhydrous dichloromethane (0.2 mL). This mixture was added to LCAA CPG (183 mg). The suspension was gently mixed overnight at room temperature. Once 519 had disappeared (HPLC), the reaction mixture was filtered, and the CPG was washed with 1 mL each of dichloromethane, acetonitrile, and a solution of 5% acetic anhydride / 5% N-methylimidazole / 5% pyridine in THF, followed by THF, acetonitrile, and dichloromethane. The CPG was then dried overnight under high vacuum. Loading was determined to be 19 μmol / g by standard DMTr assay by UV / Vis (504 nm). The resulting GalNAc-loaded CPG solid support was used for automated oligonucleotide synthesis using standard procedures. Nucleotide deprotection followed by removal from the solid support (concurrent with galactosamine acetate deprotection) afforded GalNAc-oligonucleotide conjugate (520).

[0617] Example 28. In vivo testing of TTR siRNA conjugates Compound 320 (wherein R 2 In this example, compound 320 (wherein R 2 The modified TTR siRNA is indicated as a potential treatment for the rare disease TTR (transthyretin) amyloidosis. In patients with this disease, misfolding and aggregation of the transthyretin protein is associated with disease progression. Use of this siRNA-GalNAc conjugate can reduce the amount of protein misfolding / aggregation in patients, potentially resulting in halting disease progression. Accordingly, certain embodiments utilize compound 320 (wherein R 2 provides modified TTR siRNAs and their use for treating transthyretin amyloidosis. [Table 3] 2'-O-methyl nucleotides = lowercase; 2'-fluoro nucleotides = uppercase underlined; phosphorothioate linker = s; unmodified = uppercase

[0618] Both the TTR siRNA sequence and the animal model were as described in Nair et al., J. Am. Chem. Soc., 36(49), 16958-16961 (2014). All animal-related procedures were performed in accordance with the Canadian Council on Animal Care (CCAC) Guidelines on Good Animal Practices and were approved by the local Institutional Animal Care and Use Committee (IACUC).

[0619] siRNA treatment: Female C57BL / 6 mice (n=4) were administered a single 2 mg / kg dose of Compound 320 (R 2containing modified TTR siRNA) was administered by subcutaneous injection in the scapular region once (one dose per animal) on day 0. One group of animals received vehicle only (PBS) as a control.

[0620] Collection: Test blood samples were taken from all animals at predetermined time points (days 2, 4, 7, 9, 14, and 21) after test article administration to determine the maximum reduction in plasma TTR levels and the duration of pharmacological activity.

[0621] Analysis: TTR protein levels were determined in plasma samples using the Abnova Prealbumin (Mouse) ELISA kit (Cedar Lane, Cat. No. KA2070) according to the manufacturer's instructions. TTR plasma protein values ​​were calculated for each individual plasma sample and averaged for each group. These averages were used to determine TTR protein levels relative to controls (% of PBS-treated animals).

[0622] Results: The study results are presented in Table 4. Values ​​represent the percent (%) of TTR protein levels (relative to PBS control) on days 2, 4, 7, 9, 14, and 21 after treatment. [Table 4]

[0623] Conclusion: Compound 320 (wherein R 2 Animals treated with siRNA containing the modified TTR siRNAs listed in Table 3 exhibited significant knockdown of target mRNA and protein, with maximal knockdown of TTR protein occurring between days 4 and 9 after subcutaneous injection.

[0624] Example 29. In vivo testing of HBV siRNA conjugates Chemically modified HBV siRNAs conjugated to GalNAc ligands and described in Table 1 of Example 25 were tested for in vivo activity in an established mouse model of HBV infection. In the AAV-HBV1.2 C57BL / 6 mouse model, stable and persistent HBV expression is achieved after injection of an adeno-associated virus (AAV) vector encoding a sequence exceeding the genome length of HBV, resulting in expression of HBV RNA and proteins in the liver and secretion of viral and subviral particles into the blood.

[0625] The AAV-HBV1.2 construct used in these studies was based on the details provided in Dion et al., Journal of Virology, 87(10), 5554-5563 (2013). All animal-related procedures were performed according to operating procedures that adhered to the Canadian Council on Animal Care (CCAC) Guidelines on Good Animal Practices and were approved by the local Institutional Animal Care and Use Committee (IACUC).

[0626] Each animal was inoculated with the AAV-HBV1.2 vector of 1E11 vector genome (VG). Prior to treatment, all animals were bled and serum HBsAg levels were determined in each animal to confirm established HBV expression.

[0627] siRNA treatment: Groups of mice (typically n=5) were administered a single 3 mg / kg dose of HBV siRNA conjugate by subcutaneous injection in the scapular region once (one dose per animal) on day 0. One group of animals receiving vehicle only (saline) served as control.

[0628] Collection: Test bleeds were taken from all mice on day 0, prior to treatment, and at designated time points after test article administration (e.g., study days 7, 14, 21, 28, 42, 56, and 70) to determine the maximum reduction in serum HBsAg levels and the duration of pharmacological activity.

[0629] Analysis: HBsAg levels were determined in serum samples using a Biorad EIA GS HBsAg 3.0 kit (BioRad, catalog number 32591) according to the manufacturer's instructions. Pooled serum from each treatment group was used to determine group mean HBsAg levels at each time point. Data were analyzed and expressed as HBsAg levels relative to pretreatment baseline (% of day 0).

[0630] Results: The results of testing each of the chemically modified HBV siRNAs listed in Table 1 are presented in Table 5. Values ​​represent the percent (%) of HBsAg levels (relative to day 0 baseline) at days 7, 14, 21, 28, 42, 56, and 70 after treatment. [Table 5]

[0631] Each of the 13 compounds tested resulted in a reduction of serum HBV surface antigen after a single dose of subcutaneous treatment, with the maximum effect occurring on days 14 or 21. The four compounds that showed the most potent reduction were compound 191a, in which the oligonucleotides contained siRNA 3 or 25, and R 2 The compound was compound 320, which contained siRNA 3 or 25. These four compounds were notable for a more rapid reduction (>97%) at the first time point (day 7), a better maximum reduction (>99%), and a more sustained reduction effect (still >97% at day 56, 8 weeks after treatment). Finally, preferred embodiments of the present invention are described in sections.

[0632] [Embodiment 1] A nucleic acid molecule selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, and SEQ ID NO:73.

[0633] [Embodiment 2] A nucleic acid molecule selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, and SEQ ID NO:74.

[0634] [Embodiment 3] 10. A composition comprising a nucleic acid molecule according to embodiment 1, a nucleic acid molecule according to embodiment 2, or a combination thereof.

[0635] [Embodiment 4] siRNA 1 (SEQ ID NOs: 1 and 2), 2 (SEQ ID NOs: 3 and 4), 3 (SEQ ID NOs: 5 and 6), 4 (SEQ ID NOs: 7 and 8), 5 (SEQ ID NOs: 9 and 10), 6 (SEQ ID NOs: 11 and 12), 7 (SEQ ID NOs: 13 and 14), 8 (SEQ ID NOs: 15 and 16), 9 (SEQ ID NOs: 17 and 18), 10 (SEQ ID NOs: 19 and 20), 11 (SEQ ID NOs: 21 and 22), 12 (SEQ ID NOs: 23 and 24), 13 (SEQ ID NOs: 25 and 26), 14 (SEQ ID NOs: 27 and 28), 15 (SEQ ID NOs: 29 and 30), 16 (SEQ ID NOs: 31 and 32), 17 (SEQ ID NOs: 33 and 34), 18 (SEQ ID NOs: 35 and 36), 19 (SEQ ID NOs: 37 and 38), 20 (SEQ ID NOs: 39 and 40) ), 21 (SEQ ID NOs:41 and 42), 22 (SEQ ID NOs:43 and 44), 23 (SEQ ID NOs:45 and 46), 24 (SEQ ID NOs:47 and 48), 25 (SEQ ID NOs:49 and 50), 26 (SEQ ID NOs:51 and 52), 27 (SEQ ID NOs:53 and 54), 28 (SEQ ID NOs:55 and 56), 29 (SEQ ID NOs:57 and 58), 30 (SEQ ID NOs:59 and 60), 31 (SEQ ID NOs:61 and 62), 32 (SEQ ID NOs:63 and 64), 33 (SEQ ID NOs:65 and 66), 34 (SEQ ID NOs:67 and 68), 35 (SEQ ID NOs:69 and 70), 36 (SEQ ID NOs:71 and 72), and 37 (SEQ ID NOs:73 and 74).

[0636] [Embodiment 5] A composition comprising the double-stranded siRNA molecule of embodiment 4.

[0637] [Embodiment 6] 6. The composition of embodiment 3 or 5, which is a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

[0638] [Embodiment 7] Compounds of formula (I): [ka] or a salt thereof, During the ceremony, R 1 is the targeting ligand, L 1 is absent or a bonding group, L 2 is absent or a bonding group, R 2 is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules of embodiment 4, Ring A is absent, a 3- to 20-membered cycloalkyl, a 5- to 20-membered aryl, a 5- to 20-membered heteroaryl, or a 3- to 20-membered heterocycloalkyl; Each R A are independently hydrogen, hydroxy, CN, F, Cl, Br, I, -C 1~2 Alkyl-OR B , C 1~10 Alkyl C 2~10 Alkenyl, and C 2~10 alkynyl, 1~10 Alkyl C 2~10 Alkenyl, and C 2~10 Alkynyl includes halo, hydroxy, and C 1~3 optionally substituted with one or more groups independently selected from alkoxy; R B is hydrogen, a protecting group, a covalent bond to a solid support, or a bond to a linking group attached to a solid support; n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; The compound or a salt thereof.

[0639] [Embodiment 8] R 1 is the targeting ligand, L 1 is absent or a bonding group, L 2 is absent or a bonding group, R 2 is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules of embodiment 4, Ring A is absent, a 3- to 20-membered cycloalkyl, a 5- to 20-membered aryl, a 5- to 20-membered heteroaryl, or a 3- to 20-membered heterocycloalkyl; Each R Aare independently hydrogen, hydroxy, CN, F, Cl, Br, I, -C 1~2 Alkyl-OR B , as well as halo, hydroxy, and C 1~3 C optionally substituted with one or more groups independently selected from alkoxy 1~8 is selected from the group consisting of alkyl, R B is hydrogen, a protecting group, a covalent bond to a solid support, or a bond to a linking group attached to a solid support; n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; 8. The compound of embodiment 7, or a salt thereof.

[0640] [Embodiment 9] R 1 is -C(H) (3-p) (L 3 -Sugars) p and In the formula, each L 3 are independently a linking group, p is 1, 2, or 3; The sugars are monosaccharides or disaccharides, 9. The compound of embodiment 8, or a salt thereof.

[0641] [Embodiment 10] The sugars are [ka] and During the ceremony, X is NR 3 and Y is -(C=O)R 4 , -SO2R 5 , and —(C═O)NR 6 R 7 or X is —(C═O)— and Y is NR 8 R 9 and R 3 is hydrogen or (C1-C4) alkyl, R 4 , R 5 , R6 , R 7 , R 8 , and R 9 are each independently selected from the group consisting of hydrogen, (C1-C8)alkyl, (C1-C8)haloalkyl, (C1-C8)alkoxy, and (C3-C6)cycloalkyl optionally substituted with one or more groups independently selected from the group consisting of halo, (C1-C4)alkyl, (C1-C4)haloalkyl, (C1-C4)alkoxy, and (C1-C4)haloalkoxy; R 10 -OH, -NR 8 R 9 , or -F, R 11 -OH, -NR 8 R 9 , -F, or a 5-membered heterocycle optionally substituted with one or more groups independently selected from the group consisting of halo, hydroxyl, carboxyl, amino, (C1-C4) alkyl, (C1-C4) haloalkyl, (C1-C4) alkoxy, and (C1-C4) haloalkoxy; 10. The compound according to embodiment 9, or a salt thereof.

[0642] [Embodiment 11] The sugars are [ka] or a salt thereof.

[0643] [Embodiment 12] The sugars are [ka] 11. The compound according to any one of embodiments 9 to 10, wherein:

[0644] [Embodiment 13] Each L 3are independently a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 0 to 50 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X - or -S-, optionally replaced by R X is hydrogen or (C1-C6)alkyl, and the hydrocarbon chain is optionally substituted with one or more (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; or a salt thereof.

[0645] [Embodiment 14] Each L 3 are independently a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 20 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X - or -S-, optionally replaced by R Xis hydrogen or (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; or a salt thereof.

[0646] [Embodiment 15] L 3 teeth, [ka] or a salt thereof.

[0647] [Embodiment 16] R 1 teeth, [ka] or a salt thereof.

[0648] [Embodiment 17] R 1 teeth, [ka] and During the ceremony, G is -NH- or -O-; R C is hydrogen, (C1-C8) alkyl, (C1-C8) haloalkyl, (C1-C8) alkoxy, (C1-C6) alkanoyl, (C3-C 20 ) cycloalkyl, (C3-C 20) a heterocycle, aryl, heteroaryl, monosaccharide, disaccharide, or trisaccharide, wherein said cycloalkyl, heterocycle, aryl, heteroaryl, and saccharide are optionally substituted with one or more groups independently selected from the group consisting of halo, carboxyl, hydroxyl, amino, (C1-C4) alkyl, (C1-C4) haloalkyl, (C1-C4) alkoxy, and (C1-C4) haloalkoxy; 9. The compound of embodiment 8, or a salt thereof.

[0649] [Embodiment 18] R C teeth, [ka] 18. The compound of embodiment 17, wherein:

[0650] [Embodiment 19] R 1 teeth, [ka] 20. The compound of any of embodiments 8, 17, and 18, wherein:

[0651] [Embodiment 20] R C teeth, [ka] 18. The compound of embodiment 17, wherein:

[0652] [Embodiment 21] 21. The compound of any one of embodiments 17 to 20, or a salt thereof, wherein G is —NH—.

[0653] [Embodiment 22] R 1 teeth, [ka] 22. The compound of any one of embodiments 8, 17, 20, and 21, wherein:

[0654] [Embodiment 23] R 1 teeth, [ka] and In the formula, each R D are independently hydrogen, (C1-C6) alkyl, (C9-C 20 ) alkylsilyl, (R W )3Si—, (C2-C6)alkenyl, tetrahydropyranyl, (C1-C6)alkanoyl, benzoyl, aryl(C1-C3)alkyl, TMTr (trimethoxytrityl), DMTr (dimethoxytrityl), MMTr (monomethoxytrityl), and Tr (trityl); Each R W are independently selected from the group consisting of (C1-C4) alkyl and aryl. or a salt thereof.

[0655] [Embodiment 24] L 1 and L 2 are independently a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 50 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X - or -S-, optionally replaced by R Xis hydrogen or (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; or a salt thereof.

[0656] [Embodiment 25] L 1 and L 2 are independently a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 20 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X - or -S-, optionally replaced by R X is hydrogen or (C1-C6)alkyl, and the hydrocarbon chain is optionally substituted with one or more (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; or a salt thereof.

[0657] [Embodiment 26] L 1 and L 2are independently a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 14 carbon atoms, in which one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are optionally replaced, such as -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; or a salt thereof.

[0658] [Embodiment 27] L 1 is connected to R via -NH-, -O-, -S-, -(C=O)-, -(C=O)-NH-, -NH-(C=O)-, -(C=O)-O-, -NH-(C=O)-NH-, or -NH-(SO2)-. 1 27. The compound according to any one of embodiments 8 to 26, or a salt thereof, wherein

[0659] [Embodiment 28] L 2 -O- through R 2 28. The compound according to any one of embodiments 8 to 27, or a salt thereof, wherein

[0660] [Embodiment 29] L 1 teeth, [ka] 29. The compound according to any one of embodiments 8 to 28, or a salt thereof, selected from the group consisting of:

[0661] [Embodiment 30] L 2 30. The compound according to any one of embodiments 8 to 29, or a salt thereof, wherein is —CH2—O— or —CH2—CH2—O—.

[0662] [Embodiment 31] Compounds of formula (Ia): [ka] And, During the ceremony, Each D is independently [ka] and -N=, 9. The compound of embodiment 8, or a salt thereof.

[0663] [Embodiment 32] [ka] and salts thereof; During the ceremony, Q 1 is hydrogen and Q 2 is R 2 or Q 1 is R 2 and Q 2 is hydrogen, Z is -L 1 -R 1 That is, 32. A compound according to embodiment 8 or embodiment 31, or a salt thereof.

[0664] [Embodiment 33] Compounds of formula (Ib): [ka] And, During the ceremony, Each D is independently [ka] and -N=; each m is independently 1 or 2; 9. The compound of embodiment 8, or a salt thereof.

[0665] [Embodiment 34] [ka] and salts thereof; During the ceremony, Q 1 is hydrogen and Q 2 is R 2 or Q 1 is R 2 and Q 2 is hydrogen, Z is -L 1 -R 1 That is, 34. A compound according to embodiment 8 or embodiment 33, or a salt thereof.

[0666] [Embodiment 35] Compounds of formula (Ic): [ka] or a salt thereof, During the ceremony, E is —O— or —CH—; n is selected from the group consisting of 0, 1, 2, 3, and 4; n1 and n2 are each independently selected from the group consisting of 0, 1, 2, and 3; 9. The compound of embodiment 8, or a salt thereof.

[0667] [Embodiment 36] [ka] and salts thereof, wherein Z is -L 1 -R 1 ), 36. A compound according to embodiment 8 or embodiment 35, or a salt thereof.

[0668] [Embodiment 37] Said-AL 2 -R 2 The part is [ka] and During the ceremony, Q 1 is hydrogen and Q 2 is R 2 or Q 1 is R 2 and Q 2 is hydrogen, each q is independently 0, 1, 2, 3, 4, or 5; 9. The compound of embodiment 8, or a salt thereof.

[0669] [Embodiment 38] [ka] and salts thereof.

[0670] [Embodiment 39] R 1 teeth, [ka] selected from the group consisting of During the ceremony, R S teeth, [ka] and n is 2, 3, or 4; x is 1 or 2, 8. The compound of embodiment 7, or a salt thereof.

[0671] [Embodiment 40] L 1teeth, [ka] 40. The compound of embodiment 7 or 39, selected from the group consisting of:

[0672] [Embodiment 41] 41. The compound according to any one of embodiments 7, 39, and 40, wherein A is absent, phenyl, pyrrolidinyl, or cyclopentyl; or a salt thereof.

[0673] [Embodiment 42] L 2 is C optionally substituted with hydroxy 1~4 42. The compound according to any one of embodiments 7 and 39 to 41, wherein alkylene-O—, or a salt thereof.

[0674] [Embodiment 43] L 2 43. The compound according to any one of embodiments 7 and 39 to 42, or a salt thereof, wherein is —CH2O—, —CH2CH2O—, or —CH(OH)CH2O—.

[0675] [Embodiment 44] Each R A are independently hydroxy or C optionally substituted with hydroxyl 1~8 44. The compound according to any one of embodiments 7 and 39 to 43, or a salt thereof, wherein R is alkyl.

[0676] [Embodiment 45] Each R A is independently selected from the group consisting of hydroxy, methyl, and —CH 2 OH, or a salt thereof.

[0677] [Embodiment 46] Compounds of formula (Ig): [ka] or a salt thereof, During the ceremony, B is -N- or -CH-; L 2 is a C optionally substituted with hydroxyl or halo 1~4 alkylene-O-; n is 0, 1, 2, 3, 4, 5, 6, or 7; 8. The compound of embodiment 7, or a salt thereof.

[0678] [Embodiment 47] [ka] and salts thereof; In the formula, Q is -L 1 -R 1 and R' is C 1~9 Alkyl, C 2~9 Alkenyl, or C 2~9 alkynyl, 1~9 Alkyl, C 2~9 Alkenyl, or C 2~9 Alkynyl is optionally substituted with halo or hydroxyl. 47. A compound according to embodiment 7 or 46, or a salt thereof.

[0679] [Embodiment 48] [ka] and salts thereof; In the formula, Q is -L 1 -R 1 That is, 8. The compound of embodiment 7, or a salt thereof.

[0680] [Embodiment 49] [ka] [ka] [ka] [ka] [ka] and pharmaceutically acceptable salts thereof, wherein R 2 is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules of embodiment 4, 8. The compound of embodiment 7, or a salt thereof.

[0681] [Embodiment 50] 50. A pharmaceutical composition comprising a compound according to any one of embodiments 1 to 49, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0682] [Embodiment 51] 50. A method for delivering siRNA to the liver of an animal, comprising administering to the animal a compound of formula I, or a pharmaceutically acceptable salt thereof, as defined in any one of embodiments 1 to 49.

[0683] [Embodiment 52] Compounds of formula (Id): [ka] or a salt thereof, During the ceremony, R 1d teeth, [ka] is selected from X d is C2~ 10 is alkylene, n d is either 0 or 1, R 2d is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules of embodiment 4, R 3dis H, a protecting group, a covalent bond to a solid support, or a bond to a linking group attached to a solid support; 9. The compound of embodiment 8, or a salt thereof.

[0684] [Embodiment 53] R 1d teeth, [ka] 53. The compound of embodiment 52, wherein:

[0685] [Embodiment 54] R 1d teeth, [ka] 53. The compound of embodiment 52, wherein:

[0686] [Embodiment 55] X d 55. The compound or salt of any one of embodiments 52-54, wherein is C8 alkylene.

[0687] [Embodiment 56] n d 55. The compound or salt of any one of embodiments 52 to 54, wherein

[0688] [Embodiment 57] R 3d 57. The compound or salt of any one of embodiments 52 to 56, wherein

[0689] [Embodiment 58] R 3d 57. The compound or salt of any of embodiments 52-56, wherein is a covalent bond to a solid support.

[0690] [Embodiment 59] R 3dis a bond to a linking group attached to a solid support, wherein the linking group is a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having from 2 to 15 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms are optionally replaced with (—O—) or (—N(H)—), and wherein the chain is optionally substituted on carbons 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; or a salt thereof.

[0691] [Embodiment 60] R 3d is a bond to a linking group attached to a solid support, wherein the linking group is a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having from 2 to 10 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms are optionally replaced with (—O—) or (—N(H)—), and wherein the chain is optionally substituted on carbons 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; or a salt thereof.

[0692] [Embodiment 61] R 3dis a bond to a linking group attached to a solid support, and the linking group is —C(═O)CH2CH2C(═O)N(H)—, or a salt thereof.

[0693] [Embodiment 62] A pharmaceutical composition comprising a compound according to any one of embodiments 52 to 61, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0694] [Embodiment 63] 62. A method for delivering siRNA to the liver of an animal, comprising administering to the animal a compound of formula Id according to any one of embodiments 52 to 61, or a pharmaceutically acceptable salt thereof.

[0695] [Embodiment 64] A method for preparing a compound of formula (Id), or a salt thereof, according to embodiment 48, comprising the step of: [ka] (In the formula, X d is a C2-8 alkylene, n d is either 0 or 1, Pg 1 is H, R 3d is a covalent bond to a solid support or a bond to a linking group attached to a solid support), is subjected to solid phase nucleic acid synthesis conditions to form a corresponding compound of formula Id, where R 2d is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules of embodiment 4.

[0696] [Embodiment 65] The compound is removed from the solid support to form R 3d

[00113] The method of embodiment 64, further comprising providing the corresponding compound of formula Id, wherein

[0697] [Embodiment 66] [ka] and salts thereof.

[0698] [Embodiment 67] Compounds of formula (I): [ka] or a salt thereof, During the ceremony, R 1 is H or a synthetic activating group, L 1 is absent or a bonding group, L 2 is absent or a bonding group, R 2 is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules of embodiment 4, Ring A is absent, a 3- to 20-membered cycloalkyl, a 5- to 20-membered aryl, a 5- to 20-membered heteroaryl, or a 3- to 20-membered heterocycloalkyl; Each R A are independently hydrogen, hydroxy, CN, F, Cl, Br, I, -C 1~2 Alkyl-OR B , C 1~10 Alkyl C 2~10 Alkenyl, and C 2~10 alkynyl, 1~10 Alkyl C 2~10 Alkenyl, and C 2~10 Alkynyl includes halo, hydroxy, and C 1~3 optionally substituted with one or more groups independently selected from alkoxy; R B is hydrogen, a protecting group, a covalent bond to a solid support, or a bond to a linking group attached to a solid support; n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; The compound or a salt thereof.

[0699] [Embodiment 68] Compounds of formula (Ig): [ka] or a salt thereof, During the ceremony, B is -N- or -CH-; L 2 is a C optionally substituted with hydroxyl or halo 1~4 alkylene-O-; n is 0, 1, 2, 3, 4, 5, 6, or 7; 68. A compound according to embodiment 67, or a salt thereof.

[0700] [Embodiment 69] [ka] and salts thereof; During the ceremony, Q is -L 1 -R 1 and R' is C 1~9 Alkyl, C 2~9 Alkenyl, or C 2~9 alkynyl, 1~9 Alkyl, C 2~9 Alkenyl, or C 2~9 Alkynyl is optionally substituted with halo or hydroxyl. 68. A compound according to embodiment 67, or a salt thereof.

[0701] [Embodiment 70] [ka] and salts thereof; In the formula, Q is -L 1 -R 1 68. The compound of embodiment 67, wherein:

[0702] [Embodiment 71] R 1 is H or a synthetic activating group derivable from DCC, HOBt, EDC, BOP, PyBOP, or HBTU, or a salt thereof.

[0703] [Embodiment 72] R 1 74. The compound of any one of embodiments 66-73, or a salt thereof, wherein is a synthetic activating group derivable from DCC, HOBt, EDC, BOP, PyBOP, or HBTU.

[0704] [Embodiment 73] L 1 is a divalent branched or unbranched, saturated or unsaturated hydrocarbon chain having 5 to 20 carbon atoms, in which one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are optionally replaced and are -O-, -NH-, -NH-C(=O)-, -C(=O)-NH-, or -S-; or a salt thereof.

[0705] [Embodiment 74] Compounds of formula (XX): [ka] or a salt thereof, During the ceremony, R 1 is the targeting ligand, L 1 is absent or a bonding group, L 2 is absent or a bonding group, R 2 is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules of embodiment 4, B is divalent, and [ka] wherein: Each R' is independently C 1~9 Alkyl, C 2~9 Alkenyl, or C 2~9 alkynyl, 1~9 Alkyl, C 2~9 Alkenyl, or C 2~9 The alkynyl is optionally substituted with halo or hydroxyl; * The valence with a is L 1 or L 1 If does not exist, R 1 is bound to ** The valence with a is L 2 or L 2 If does not exist, R 2 is bonded to The compound or a salt thereof.

[0706] [Embodiment 75] the targeting ligand R 1 75. The compound of embodiment 74, or a salt thereof, comprising 2 to 8 saccharides.

[0707] [Embodiment 76] the targeting ligand R 1 75. The compound of embodiment 74, or a salt thereof, comprising 2 to 4 sugars.

[0708] [Embodiment 77] the targeting ligand R 1 75. The compound of embodiment 74, or a salt thereof, comprising 3 to 8 saccharides.

[0709] [Embodiment 78] the targeting ligand R 1 75. The compound of embodiment 74, or a salt thereof, comprising 3 to 6 sugars.

[0710] [Embodiment 79] the targeting ligand R 1 75. The compound of embodiment 74, or a salt thereof, comprising 3 to 4 sugars.

[0711] [Embodiment 80] the targeting ligand R 1 75. The compound of embodiment 74, or a salt thereof, wherein:

[0712] [Embodiment 81] the targeting ligand R 1 75. The compound of embodiment 74, or a salt thereof, wherein:

[0713] [Embodiment 82] the targeting moiety R 1 is expressed as follows: [ka] and During the ceremony, B 1 is a trivalent group containing from about 1 to about 20 atoms, and L 1 , T 1 , and T 2 is covalently bonded to B 2 is a trivalent group containing about 1 to about 20 atoms, and T 1 , T 3 , and T 4 is covalently bonded to B 3 is a trivalent group containing about 1 to about 20 atoms, and T 2 , T 5 , and T 6 is covalently bonded to T 1 is absent or a bonding group, T 2 is absent or a bonding group, T 3 is absent or a bonding group, T 4 is absent or a bonding group, T 5 is absent or a bonding group, T 6 is absent or a bonding group, A compound according to any one of embodiments 7 to 8, 31 to 36, 38 to 39, 40 to 44, 46 to 48, 67 to 69, and 74, or a salt thereof.

[0714] [Embodiment 83] Each sugar may independently be [ka] is selected from During the ceremony, X is NR 3 and Y is -(C=O)R 4 , -SO2R 5 , and —(C═O)NR 6 R 7 or X is —(C═O)— and Y is NR 8 R 9 and R 3 is hydrogen or (C1-C4) alkyl, R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 are each independently selected from the group consisting of hydrogen, (C1-C8)alkyl, (C1-C8)haloalkyl, (C1-C8)alkoxy, and (C3-C6)cycloalkyl optionally substituted with one or more groups independently selected from the group consisting of halo, (C1-C4)alkyl, (C1-C4)haloalkyl, (C1-C4)alkoxy, and (C1-C4)haloalkoxy; R 10 -OH, -NR 8 R 9 , or -F, R 11 -OH, -NR 8 R 9 , -F, or a 5-membered heterocycle optionally substituted with one or more groups independently selected from the group consisting of halo, hydroxyl, carboxyl, amino, (C1-C4) alkyl, (C1-C4) haloalkyl, (C1-C4) alkoxy, and (C1-C4) haloalkoxy; 83. A compound according to embodiment 82, or a salt thereof.

[0715] [Embodiment 84] Each of the saccharides may independently be [ka] 83. The compound according to embodiment 82, selected from the group consisting of:

[0716] [Embodiment 85] Each sugar may independently be [ka] 83. The compound of embodiment 82, wherein:

[0717] [Embodiment 86] T 1 and T 2 86. The compound according to any one of embodiments 82 to 85, or a salt thereof, wherein one of:

[0718] [Embodiment 87] T 1 and T 2 86. The compound according to any one of embodiments 82 to 85, or a salt thereof, wherein none of

[0719] [Embodiment 88] T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 are each independently absent or a branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 50 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X - or -S-, optionally replaced by R Xis hydrogen or (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; or a salt thereof.

[0720] [Embodiment 89] T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 are each independently absent or a branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 20 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X - or -S-, optionally replaced by R X is hydrogen or (C1-C6)alkyl, and the hydrocarbon chain is optionally substituted with one or more (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; or a salt thereof.

[0721] [Embodiment 90] T 1 , T 2 , T 3, T 4 , T 5 , and T 6 are each independently absent or a branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 50 carbon atoms, or a salt thereof, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are -O- or -NR X - is optionally replaced by R X is hydrogen or (C1-C6) alkyl, and the hydrocarbon chain is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from halo, hydroxy, and oxo (=O), or a salt thereof.

[0722] [Embodiment 91] T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 are independently absent or a branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 20 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) carbon atoms in the hydrocarbon chain are optionally replaced with —O—, and the hydrocarbon chain is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from halo, hydroxy, and oxo (═O), or a salt thereof.

[0723] [Embodiment 92] T 1 , T 2 , T 3 , T 4 , T 5 , and T 6are independently absent or a branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 20 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) carbon atoms in the hydrocarbon chain are optionally replaced with —O—, and the hydrocarbon chain is optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents selected from halo, hydroxy, and oxo (═O), or a salt thereof.

[0724] [Embodiment 93] T 3 , T 4 , T 5 , and T 6 At least one of the [ka] and During the ceremony, n=1, 2, 3, 86. A compound according to any one of embodiments 82 to 85, or a salt thereof.

[0725] [Embodiment 94] T 3 , T 4 , T 5 , and T 6 Each of the [ka] selected from the group consisting of During the ceremony, n=1, 2, 3, 86. A compound according to any one of embodiments 82 to 85, or a salt thereof.

[0726] [Embodiment 95] T 1 and T 2 86. The compound according to any one of embodiments 82 to 85, or a salt thereof, wherein at least one of is glycine.

[0727] [Embodiment 96] T 1 and T 2 86. The compound of any one of embodiments 82-85, or a salt thereof, wherein each of is glycine.

[0728] [Embodiment 97] B 1 is a trivalent group containing 1 to 15 atoms, and L 1 , T 1 , and T 2 97. The compound of any one of embodiments 82 to 96, or a salt thereof, wherein

[0729] [Embodiment 98] B 1 is a trivalent group containing 1 to 10 atoms, and L 1 , T 1 , and T 2 97. The compound of any one of embodiments 82 to 96, or a salt thereof, wherein

[0730] [Embodiment 99] B 1

[0082] Embodiment 97. The compound of any one of embodiments 82-96, or a salt thereof, wherein comprises (C1-C6) alkyl.

[0731] [Embodiment 100] B 1 is C 3~8 97. The compound of any one of embodiments 82 to 96, or a salt thereof, comprising cycloalkyl.

[0732] [Embodiment 101] B 1 97. The compound of any one of embodiments 82 to 96, or a salt thereof, wherein

[0733] [Embodiment 102] B 1 97. The compound according to any one of embodiments 82 to 96, or a salt thereof, wherein comprises a D-amino acid or an L-amino acid.

[0734] [Embodiment 103] B 197. The compound according to any one of embodiments 82 to 96, or a salt thereof, wherein

[0735] [Embodiment 104] B 1 97. The compound of any one of embodiments 82 to 96, or a salt thereof, wherein

[0736] [Embodiment 105] B 1 97. The compound of any one of embodiments 82 to 96, or a salt thereof, wherein

[0737] [Embodiment 106] B 1 97. The compound of any one of embodiments 82-96, or a salt thereof, wherein is comprises aryl.

[0738] [Embodiment 107] B 1 97. The compound of any one of embodiments 82-96, or a salt thereof, wherein

[0739] [Embodiment 108] B 1 is a phenyl ring; or a salt thereof.

[0740] [Embodiment 109] B 1 97. The compound of any one of embodiments 82-96, or a salt thereof, wherein is CH.

[0741] [Embodiment 110] B 1 97. The compound of any one of embodiments 82-96, or a salt thereof, wherein is heteroaryl.

[0742] [Embodiment 111] B 1 teeth, [ka] 97. The compound of any one of embodiments 82 to 96, wherein:

[0743] [Embodiment 112] B 2 is a trivalent group containing 1 to 15 atoms, and L 1 , T 1 , and T 2 112. The compound of any one of embodiments 82 to 111, or a salt thereof, wherein

[0744] [Embodiment 113] B 2 is a trivalent group containing 1 to 10 atoms, and L 1 , T 1 , and T 2 112. The compound of any one of embodiments 82 to 111, or a salt thereof, wherein

[0745] [Embodiment 114] B 2 The compound of any one of embodiments 82-111, or a salt thereof, wherein comprises (C1-C6) alkyl.

[0746] [Embodiment 115] B 2 is C 3~8 112. A compound according to any one of embodiments 82 to 111, or a salt thereof, which comprises a cycloalkyl.

[0747] [Embodiment 116] B 2 112. The compound of any one of embodiments 82 to 111, or a salt thereof, wherein

[0748] [Embodiment 117] B 2 112. The compound according to any one of embodiments 82 to 111, or a salt thereof, wherein comprises a D-amino acid or an L-amino acid.

[0749] [Embodiment 118] B 2 112. The compound according to any one of embodiments 82 to 111, or a salt thereof, wherein

[0750] [Embodiment 119] B 2 112. The compound according to any one of embodiments 82 to 111, or a salt thereof, wherein

[0751] [Embodiment 120] B 2 112. The compound of any one of embodiments 82-111, or a salt thereof, wherein

[0752] [Embodiment 121] B 2 112. The compound of any one of embodiments 82-111, or a salt thereof, wherein is comprises aryl.

[0753] [Embodiment 122] B 2 112. The compound of any one of embodiments 82-111, or a salt thereof, wherein

[0754] [Embodiment 123] B 2 is a phenyl ring; or a salt thereof.

[0755] [Embodiment 124] B 2 The compound of any one of embodiments 82-111, or a salt thereof, wherein is CH.

[0756] [Embodiment 125] B 2 112. The compound of any one of embodiments 82-111, or a salt thereof, wherein is heteroaryl.

[0757] [Embodiment 126] B 2 teeth, [ka] 112. The compound according to any one of embodiments 82 to 111, selected from the group consisting of:

[0758] [Embodiment 127] B 3 is a trivalent group containing 1 to 15 atoms, and L 1 , T 1 , and T 2 127. The compound of any one of embodiments 82-126, or a salt thereof, wherein

[0759] [Embodiment 128] B 3 is a trivalent group containing 1 to 10 atoms, and L 1 , T 1 , and T 2 127. The compound of any one of embodiments 82-126, or a salt thereof, wherein

[0760] [Embodiment 129] B 3 The compound of any one of embodiments 82-126, or a salt thereof, wherein comprises (C1-C6) alkyl.

[0761] [Embodiment 130] B 3 is C 3~8 127. A compound according to any one of embodiments 82 to 126, or a salt thereof, which comprises a cycloalkyl.

[0762] [Embodiment 131] B 3 127. The compound of any one of embodiments 82 to 126, or a salt thereof, wherein

[0763] [Embodiment 132] B 3 127. The compound according to any one of embodiments 82 to 126, or a salt thereof, wherein comprises a D-amino acid or an L-amino acid.

[0764] [Embodiment 133] B 3 127. The compound according to any one of embodiments 82 to 126, or a salt thereof, wherein

[0765] [Embodiment 134] B 3 127. The compound according to any one of embodiments 82 to 126, or a salt thereof, wherein

[0766] [Embodiment 135] B 3 127. The compound of any one of embodiments 82-126, or a salt thereof, wherein

[0767] [Embodiment 136] B 3 127. The compound of any one of embodiments 82-126, or a salt thereof, wherein is comprises aryl.

[0768] [Embodiment 137] B 3 127. The compound of any one of embodiments 82-126, or a salt thereof, wherein

[0769] [Embodiment 138] B 3 is a phenyl ring; or a salt thereof.

[0770] [Embodiment 139] B 3 The compound of any one of embodiments 82-126, or a salt thereof, wherein is CH.

[0771] [Embodiment 140] B 3 127. The compound of any one of embodiments 82-126, or a salt thereof, wherein is heteroaryl.

[0772] [Embodiment 141] B 3 teeth, [ka] or a salt thereof.

[0773] [Embodiment 142] L 1 and L 2 are independently a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 50 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X - or -S-, optionally replaced by R X is hydrogen or (C1-C6)alkyl, and the hydrocarbon chain is optionally substituted with one or more substituents selected from (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy; or a salt thereof.

[0774] [Embodiment 143] L 1 teeth, [ka] or a salt thereof.

[0775] [Embodiment 144] L 1 is connected to B through a bond selected from the group consisting of -O-, -S-, -(C=O)-, -(C=O)-NH-, -NH-(C=O), -(C=O)-O-, -NH-(C=O)-NH-, or -NH-(SO2)- 1 142. The compound of any one of embodiments 82 to 141, or a salt thereof, wherein

[0776] [Embodiment 145] L 1 teeth, [ka] 142. The compound according to any one of embodiments 87 to 141, selected from the group consisting of:

[0777] [Embodiment 146] L 2 -O- through R 2 146. The compound of any one of embodiments 82 to 145, or a salt thereof, wherein

[0778] [Embodiment 147] L 2 is C optionally substituted with hydroxy 1~4 The compound of any one of embodiments 82 to 145, wherein alkylene-O—; or a salt thereof.

[0779] [Embodiment 148] L 2 -O- through R 2 146. The compound of any one of embodiments 82 to 145, or a salt thereof, wherein

[0780] [Embodiment 149] L 2 146. The compound of any one of embodiments 82 to 145, or a salt thereof, wherein is not present.

[0781] [Embodiment 150] [ka] [ka] [ka] and pharmaceutically acceptable salts thereof, wherein R 2 is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules of embodiment 4, 88. A compound according to embodiment 87, or a salt thereof.

[0782] [Embodiment 151] compound [ka] or a salt thereof, wherein R 2 is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules of embodiment 4, or a salt thereof.

[0783] [Embodiment 152] GalNAc conjugate of formula X: ABC (X) And, where A is a targeting ligand; B is an optional linker; C is an siRNA molecule according to embodiment 4. The GalNAc conjugate.

[0784] [Embodiment 153] A compound of the formula: [ka] or a pharmaceutically acceptable salt thereof, wherein R 2 is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules of embodiment 4, The compound or a pharmaceutically acceptable salt thereof.

[0785] [Embodiment 154] A compound of the formula: [ka] or a pharmaceutically acceptable salt thereof, wherein R 2 is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules described in embodiment 4), The compound or a pharmaceutically acceptable salt thereof.

[0786] [Embodiment 155] A compound of the formula: [ka] or a pharmaceutically acceptable salt thereof, wherein R 2 is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules described in embodiment 4), The compound or a pharmaceutically acceptable salt thereof.

[0787] [Embodiment 156] A compound of the formula: [ka] or a pharmaceutically acceptable salt thereof, wherein R 2 is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules of embodiment 4, The compound or a pharmaceutically acceptable salt thereof.

[0788] [Embodiment 157] A compound of the formula: [ka] or a pharmaceutically acceptable salt thereof, wherein R 2 is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules of embodiment 4, The compound or a pharmaceutically acceptable salt thereof.

[0789] [Embodiment 158] A compound of the formula: [ka] or a pharmaceutically acceptable salt thereof, wherein R 2 is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules of embodiment 4, The compound or a pharmaceutically acceptable salt thereof.

[0790] [Embodiment 159] A compound of the formula: [ka] or a pharmaceutically acceptable salt thereof, wherein R 2 is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules of embodiment 4, The compound or a pharmaceutically acceptable salt thereof.

[0791] [Embodiment 160] A compound of the formula: [ka] or a pharmaceutically acceptable salt thereof, wherein R 2 is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules of embodiment 4, The compound or a pharmaceutically acceptable salt thereof.

[0792] [Embodiment 161] A compound of the formula: [ka] or a pharmaceutically acceptable salt thereof, wherein R 2 is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules of embodiment 4, The compound or a pharmaceutically acceptable salt thereof.

[0793] [Embodiment 162] A compound of the formula: [ka] or a pharmaceutically acceptable salt thereof, wherein R 2 is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules of embodiment 4, The compound or a pharmaceutically acceptable salt thereof.

[0794] [Embodiment 163] A compound of the formula: [ka] or a pharmaceutically acceptable salt thereof, wherein R 2 is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules of embodiment 4, The compound or a pharmaceutically acceptable salt thereof.

[0795] [Embodiment 164] A compound of the formula: [ka] or a pharmaceutically acceptable salt thereof, wherein R 2 is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules of embodiment 4, The compound or a pharmaceutically acceptable salt thereof.

[0796] [Embodiment 165] A compound of the formula: [ka] or a pharmaceutically acceptable salt thereof, wherein R 2 is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules of embodiment 4, The compound or a pharmaceutically acceptable salt thereof.

[0797] [Embodiment 166] A compound of the formula: [ka] or a pharmaceutically acceptable salt thereof.

[0798] [Embodiment 167] A compound of the formula: [ka] or a pharmaceutically acceptable salt thereof.

[0799] [Embodiment 168] A compound of the formula: [ka] or a pharmaceutically acceptable salt thereof.

[0800] [Embodiment 169] A compound of the formula: [ka] or a pharmaceutically acceptable salt thereof.

[0801] [Embodiment 170] A compound of the formula: [ka] or a pharmaceutically acceptable salt thereof.

[0802] [Embodiment 171] A compound of the formula: [ka] or a pharmaceutically acceptable salt thereof.

[0803] [Embodiment 172] A compound of the formula: [ka] or a pharmaceutically acceptable salt thereof.

[0804] [Embodiment 173] A compound of the formula: [ka] or a pharmaceutically acceptable salt thereof.

[0805] [Embodiment 174] Compounds of formula (I): [ka] or a salt thereof, During the ceremony, L 1 is absent or a bonding group, L 2 is absent or a bonding group, R 2 is a nucleic acid, Ring A is absent, a 3- to 20-membered cycloalkyl, a 5- to 20-membered aryl, a 5- to 20-membered heteroaryl, or a 3- to 20-membered heterocycloalkyl; Each R A are independently hydrogen, hydroxy, CN, F, Cl, Br, I, -C 1~2 Alkyl-OR B , C 1~10 Alkyl C 2~10 Alkenyl, and C 2~10 alkynyl, 1~10 Alkyl C 2~10 Alkenyl, and C 2~10 Alkynyl includes halo, hydroxy, and C 1~3 optionally substituted with one or more groups independently selected from alkoxy; R B is hydrogen, a protecting group, a covalent bond to a solid support, or a bond to a linking group attached to a solid support; n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; The compound or a salt thereof.

[0806] [Embodiment 175] Compounds of formula (Ia): [ka] or a salt thereof, During the ceremony, L 2 is absent or a bonding group, R 2 is a nucleic acid, Ring A is absent, a 3- to 20-membered cycloalkyl, a 5- to 20-membered aryl, a 5- to 20-membered heteroaryl, or a 3- to 20-membered heterocycloalkyl; Each R A are independently hydrogen, hydroxy, CN, F, Cl, Br, I, -C 1~2 Alkyl-OR B , C 1~10 Alkyl C 2~10 Alkenyl, and C 2~10 alkynyl, 1~10 Alkyl C 2~10 Alkenyl, and C 2~10 Alkynyl includes halo, hydroxy, and C 1~3 optionally substituted with one or more groups independently selected from alkoxy; R B is hydrogen, a protecting group, a covalent bond to a solid support, or a bond to a linking group attached to a solid support; n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; The compound or a salt thereof.

[0807] [Embodiment 176] Compounds of formula (XXa): [ka] or a salt thereof, During the ceremony, L 1 is absent or a bonding group, L 2 is absent or a bonding group, R 2 is a nucleic acid, B is divalent, and [ka] wherein: Each R' is independently C 1~9 Alkyl, C 2~9 Alkenyl, or C 2~9 alkynyl,1~9 Alkyl, C 2~9 Alkenyl, or C 2~9 The alkynyl is optionally substituted with halo or hydroxyl; * The valence with a is L 1 or L 1 If does not exist, R 1 is bound to ** The valence with a is L 2 or L 2 If does not exist, R 2 is bonded to The compound or a salt thereof.

[0808] [Embodiment 177] L 1 and L 2 are independently a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 50 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) of the carbon atoms in the hydrocarbon chain are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X - or -S-, optionally replaced by R X is hydrogen or (C1-C6)alkyl, and the hydrocarbon chain is optionally substituted with one or more substituents selected from (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy; or a salt thereof.

[0809] [Embodiment 178] L 1 teeth, [ka] or a salt thereof.

[0810] [Embodiment 179] L 1 is connected to B through a bond selected from the group consisting of -O-, -S-, -(C=O)-, -(C=O)-NH-, -NH-(C=O), -(C=O)-O-, -NH-(C=O)-NH-, or -NH-(SO2)- 1 177. The compound of embodiment 176, or a salt thereof, wherein

[0811] [Embodiment 180] L 1 teeth, [ka] 177. A compound according to embodiment 176, selected from the group consisting of:

[0812] [Embodiment 181] L 2 -O- through R 2 177. The compound of embodiment 176, or a salt thereof, wherein

[0813] [Embodiment 182] L 2 is C optionally substituted with hydroxy 1~4 The compound according to embodiment 176, wherein the compound is alkylene-O—; or a salt thereof.

[0814] [Embodiment 183] L 2 is absent, or a salt thereof.

[0815] [Embodiment 184] compound [ka] or a salt thereof, wherein R 2 is a nucleic acid, The compound or a salt thereof.

[0816] [Embodiment 185] compound [ka] or a salt thereof, wherein R 2 is a nucleic acid, The compound or a salt thereof.

[0817] [Embodiment 186] compound [ka] or a salt thereof, wherein R 2 is a nucleic acid, The compound or a salt thereof.

[0818] [Embodiment 187] R 2 The compound according to any one of embodiments 174 to 186, which is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules according to embodiment 4.

[0819] [Embodiment 188] 187. A pharmaceutical composition comprising a compound according to any one of embodiments 174 to 186, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0820] [Embodiment 189] A method for delivering siRNA to the liver of an animal, comprising administering to the animal a compound of formula I according to any of embodiments 174 to 186 or a pharmaceutically acceptable salt thereof.

[0821] [Embodiment 190] 186. A method for treating a hepatitis B virus infection in an animal, comprising administering to the animal an effective amount of a compound of Formula I or Id according to any of embodiments 1 to 49, 52 to 61, or 174 to 186, or a pharmaceutically acceptable salt thereof.

[0822] [Embodiment 191] 191. The method of embodiment 190, wherein the compound of formula I or Id or a pharmaceutically acceptable salt thereof is administered subcutaneously.

[0823] [Embodiment 192] A compound of Formula I or Id, or a pharmaceutically acceptable salt thereof, according to any of embodiments 1 to 49, 52 to 61, or 174 to 186, for use in medical therapy.

[0824] [Embodiment 193] A compound of Formula I or Id, as defined in any of embodiments 1-49, 52-61, or 174-186, or a pharmaceutically acceptable salt thereof, for the prophylactic or therapeutic treatment of hepatitis B virus infection in an animal.

[0825] [Embodiment 194] Use of a compound of Formula I or Id, as defined in any of embodiments 1-49, 52-61, or 174-186, or a pharmaceutically acceptable salt thereof, for the preparation of a medicament for treating a hepatitis B virus infection in an animal.

[0826] [Embodiment 195] The method, compound or use according to any one of embodiments 190 to 194, wherein the animal is a human.

Claims

[Claim 1] A nucleic acid molecule selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, and SEQ ID NO:73.

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