target chemical composition

Abstract

The present invention provides 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 present invention provides a specific nucleic acid (e.g., a double-stranded siRNA molecule), as well as a conjugate comprising a targeting moiety, a double-stranded siRNA, and an optional binding group. Certain embodiments also provide a synthetic method useful for preparing the conjugate. The conjugate is useful for targeting therapeutic double-stranded siRNA to the liver and treating liver diseases, including hepatitis (e.g., hepatitis B and hepatitis D). The present invention also provides synthetic intermediates and methods disclosed herein that are useful for preparing compounds of formula I.

Description

Cross-reference of related applications

[0001] This patent application claims priority under 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. These applications are incorporated herein by reference. [Background technology]

[0002] For example, several diseases, such as hepatitis B and non-alcoholic steatohepatitis (NASH), are specific to the liver. Therefore, it would be beneficial to have therapeutic compositions that can primarily target the liver, kidneys, heart, pancreas, or other organs of living subjects.

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

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

[0005] The present invention provides 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).

[0006] Accordingly, in one embodiment, the present invention relates to 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), 2 The present invention provides double-stranded siRNA molecules 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 relates to a compound of formula I. [ka] (I) or provide a salt thereof, in the formula, R 1 It is a targeted ligand, L 1 It does not exist, or it is a bonding group. L 2 It does not exist, or it is a bonding group. R 2 This is a double-stranded siRNA molecule selected from the double-stranded siRNAs in Table 1. Ring A is absent, or is 3- to 20-membered cycloalkyl, 5- to 20-membered aryl, 5- to 20-membered heteroaryl, or 3- to 20-membered heterocycloalkyl, each R A is independently selected from 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 the C 1~10 alkyl C 2~10 alkenyl, and C 2~10 alkynyl are optionally substituted with one or more groups independently selected from halo, hydroxy, and C 1~3 alkoxy, R B is hydrogen, a protecting group, a covalent bond to a solid support, or a bond to a linking group bound 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, and the conjugate is not limited to conjugates comprising the ligand-linker disclosed herein. For example, one aspect of the present invention provides a GalNAc conjugate of formula X: A-B-C (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 for treating hepatitis B virus and hepatitis B virus / hepatitis D virus.

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

[0011] Those skilled in the art will see from the following detailed description and drawings that other objects, features, and advantages of the present invention will become apparent. [Brief explanation of the drawing]

[0012] [Figure 1] This figure illustrates an intermediate compound of formula Ie, in which a targeted ligand / linker is bound to a solid support and Pg1 is a protecting group DMTr. [Figure 2] This figure illustrates a typical compound of formula Id, in which a targeted ligand is bound to a solid support and a nucleic acid is covalently bonded to it. [Figure 3] This figure illustrates a representative compound of formula Id, in which the targeted ligand-nucleic acid conjugate is cleaved from the solid support and deprotected to yield the compound of formula I. [Modes for carrying out the invention]

[0013] Please understand, including the figures, examples, and schemes, that in this application, the oligonucleotides may be the double-stranded siRNA molecules listed in Table 1.

[0014] As used herein, the following terms shall have the meanings they represent unless otherwise specified.

[0015] As used herein, the term “conjugate” includes compounds of formula (I) that contain an oligonucleotide (e.g., an siRNA molecule) bound to a targeted ligand. Therefore, the terms “compound” and “conjugate” may be used interchangeably herein.

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

[0017] In a particular embodiment, the 5' and / or 3' overhangs of one or both strands of the siRNA include 1 to 4 (e.g., 1, 2, 3, or 4) modified and / or unmodified deoxythymidine (t or dT) nucleotides, 1 to 4 (e.g., 1, 2, 3, or 4) modified (e.g., 2'OMe) and / or unmodified uridine (U) ribonucleotides, and / or 1 to 4 (e.g., 1, 2, 3, or 4) modified (e.g., 2'OMe) and / or unmodified ribonucleotides or deoxyribonucleotides that are complementary to a target sequence (e.g., the 3' overhang of the antisense strand) or its complementary strand (e.g., the 3' overhang of the sense strand).

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

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

[0020] The term "synthetic activating group" refers to a group that can bond to an atom and activate that atom to form a covalent bond with another reactive group. It is understood that the properties of a synthetic activating group may depend on the atom it activates. For example, if a synthetic activating group is bonded to an oxygen atom, it is a group that activates that oxygen atom to form a bond with another reactive group (e.g., an ester bond, a carbamate bond, or an ether bond). Such synthetic activating groups are known. Examples of synthetic activating groups that can bond to an oxygen atom include, but are not limited to, acetates, succinates, triflates, and mesylates. If a synthetic activating group is bonded to the oxygen atom of a carboxylic acid, it may 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), (benzotriazole-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazole-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), or O-benzotriazole-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU).

[0021] An "effective dose" or "therapeutic effective dose" of therapeutic nucleic acids such as siRNA is the amount sufficient to produce the desired effect, for example, inhibition of the expression of a target sequence compared to the normal expression level detected in the absence of siRNA. In certain embodiments, inhibition of the expression of a target gene or target sequence is achieved when the value obtained with siRNA is approximately 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% compared to a control (e.g., buffer only, siRNA sequences targeting different genes, scrambled siRNA sequences, etc.). Suitable assays for measuring the expression of a target gene or target sequence include, but are not limited to, protein or mRNA level tests using techniques known to those skilled in the art, such as dot blotting, Northern blotting, Insights hybridization, ELISA, immunoprecipitation, and enzyme function assays, as well as phenotypic assays known to those skilled 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-stranded or double-stranded form, and includes DNA and RNA. A “nucleotide” contains the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together via a phosphate group. “Bases” include purines and pyrimidines, which further include natural compounds of purines and pyrimidines such as adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, as well as synthetic derivatives, which include, but are not limited to, modifications that introduce novel reactive groups such as amines, alcohols, thiols, carboxylates, and alkyl halides. Nucleic acids include synthetic, naturally occurring, and naturally occurring nucleic acids containing known nucleotide analogs or modified skeletal residues or bonds that have similar binding properties to the reference nucleic acid. Examples of such analogues and / or modified residues include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2'-O-methylribonucleotides, and peptide nucleic acids (PNAs). Furthermore, nucleic acids may contain one or more UNA moieties.

[0023] The term "nucleic acid" includes any oligonucleotide or polynucleotide. Fragments containing up to 60 nucleotides are generally called oligonucleotides, while longer fragments are called polynucleotides. Deoxyribooligonucleotides consist of alternating unbranched polymers formed by the covalent bonding of a five-carbon sugar called deoxyribose to phosphate at its 5' and 3' carbon atoms. DNA may take the form of, for example, antisense molecules, plasmid DNA, pre-condensed DNA, PCR products, vectors, expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations thereof. Ribooligonucleotides consist of similar repeating structures where the five-carbon sugar is ribose. RNA may take the form of, for example, small interfering RNA (siRNA), dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, tRNA, viral RNA (vRNA), and combinations thereof. Accordingly, in relation to the present invention, the terms “polynucleotide” and “oligonucleotide” refer to polymers or oligomers of nucleotides or nucleoside monomers consisting of naturally occurring bases, sugars, and intersugar (skeletal) bonds. The terms “polynucleotide” and “oligonucleotide” also include polymers or oligomers containing similarly functional monomers or portions thereof that do not exist naturally. Such modified or substituted oligonucleotides are often preferred over the natural forms for properties such as enhanced cellular uptake, reduced immunogenicity, and increased stability in the presence of nucleases.

[0024] Unless otherwise stated, a given nucleic acid sequence implicitly includes not only the explicitly stated sequence but also its conservedly modified variants (e.g., degenerate codon substitutions), alleles, orthologues, 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 a mixed base and / or deoxyinosine residue (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 (e.g., DNA or RNA) sequence that contains a coding sequence of partial or full length required 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 polypeptide.

[0027] As used herein, the term "alkyl" means, unless otherwise specified, a molecule having a specified number of carbon atoms, either alone or as part of another substituent (i.e., C 1~8 The term "alkyl group" refers to a linear or branched hydrocarbon group (meaning 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, and n-octyl. The term "alkenyl" refers to an unsaturated alkyl group having one or more double bonds. Similarly, the term "alkynyl" refers to an unsaturated alkyl group having one or more triple bonds. Examples of such unsaturated alkyl groups include vinyl, 2-propenyl, clotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1-propynyl and 3-propynyl, 3-butynyl, and their higher homologues and isomers.

[0028] The term "alkylene" refers to a divalent group derived from an alkane (including linear and branched alkanes), either alone or as part of another substituent, as exemplified by -CH2CH2CH2CH2- and -CH(CH3)CH2CH2-.

[0029] The terms "cycloalkyl," "carbocyclic," or "carbocyclic" refer to hydrocarbon ring systems with 3 to 20 total ring atoms (for example, a 3-20 membered cycloalkyl is a cycloalkyl or C20 ring atom system). 3~20 A cycloalkyl is a cycloalkyl group having 3 to 20 carbon atoms. In the case of a 3 to 5-membered cycloalkyl group, it is completely saturated or has no more than one double bond between the ring vertices. In the case of a cycloalkyl group with 6 or more members, it is completely saturated or has no more than two double bonds between the ring vertices. As used herein, "cycloalkyl," "carbocyclic formula," or "carbocyclic ring" refers to, for example, bicyclo[2.2.1]heptane, pinan, bicyclo[2.2.2]octane, adamantane, norbornene, and spirocyclic C 5~12 The terms “alkenyl,” “alkynyl,” “cycloalkyl,” “carbocyclic,” and “carbocyclic” are intended to include their monohalogenated and polyhalogenated variants.

[0030] The terms "heterocycloalkyl," "heterocyclic," or "heterocyclic" refer to saturated or partially unsaturated cyclic groups with a total of 3 to 20 ring atoms, containing 1 to 10 heteroatoms selected from N, O, and S as ring atoms (for example, a 3-20 membered heterocycloalkyl is a heterocycloalkyl group with 3 to 20 ring atoms and C 2~19A heterocycloalkyl is a heterocycloalkyl having 3 to 10 ring atoms, with 2 to 19 of them being carbon atoms, where the nitrogen and sulfur atoms are sometimes oxidized, and the nitrogen atom(s) are sometimes quaternized. Unless otherwise specified, the rings in "heterocycloalkyl," "heterocyclic," or "heterocyclic" can be monocyclic, bicyclic, spirocyclic, or polycyclic ring systems. Non-limiting examples of "heterocycloalkyl," "heterocyclic," or "heterocyclic" 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 include rufoline-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, and (1R,4R)-2-oxa-5-azabicyclo[2.2.2]octane. The "heterocycloalkyl," "heterocyclic," or "heterocyclic" group can be attached to the remainder of the molecule via one or more ring carbons or heteroatoms. The "heterocycloalkyl," "heterocyclic," or "heterocyclic" groups may include their monohalogenated and polyhalogenated variants.

[0031] The terms "alkoxy" and "alkylthio" are used in their conventional sense, referring to alkyl groups bonded to the remainder of a molecule via an oxygen atom ("oxy") or a thio group, and further including their monohalogenated and polyhalogenated variants.

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

[0033] The term "aryl" refers to a carbocyclic aromatic group having 6 to 14 carbon atoms, whether or not it is condensed with one or more other groups. Examples of aryl groups, unless otherwise specified, include phenyl, naphthyl, and biphenyl.

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

[0035] The term sugars include monosaccharides, disaccharides, and trisaccharides. This term includes glucose, sucrose, fructose, galactose, and ribose, as well as deoxy sugars such as deoxyribose and amino sugars such as galactosamine. Conveniently, sugar derivatives can be prepared as described in International Patent Application Publications WO96 / 34005 and WO97 / 03995. Sugars can conveniently be bonded to the remainder of the compound of formula I via ether bonds, thioether bonds (e.g., S-glycosides), amine nitrogen (e.g., N-glycosides), or carbon-carbon bonds (e.g., C-glycosides). In one embodiment, sugars can conveniently be bonded to the remainder of the compound of formula I via ether bonds. In one embodiment, the term sugars is defined as: [ka] The formula includes the base, X is NR 3 And Y is -(C=O)R 4 , -SO2R 5 , and -(C=O)NR 6 R 7 Either X is selected from 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 Each is independently selected from the group consisting of (C3-C6) cycloalkyl groups that are optionally substituted with one or more groups independently selected from the group consisting of hydrogen, (C1-C8) alkyl, (C1-C8) haloalkyl, (C1-C8) alkoxy, and 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 It is a five-membered heterocycle optionally substituted with one or more groups independently selected from the group consisting of -F, or halo, hydroxyl, carboxyl, amino, (C1-C4)alkyl, (C1-C4)haloalkyl, (C1-C4)alkoxy, and (C1-C4)haloalkoxy. In another embodiment, the sugar is [ka] You can choose from the group consisting of these.

[0036] In another embodiment, sugars are [ka] That's fine.

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

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

[0039] The term "lipid particles" includes lipid formulations that can be used to deliver therapeutic nucleic acids (e.g., siRNA) to target sites of interest (e.g., cells, tissues, organs, etc.). In preferred embodiments, the lipid particles of the present invention are nucleic acid-lipid particles, which are typically formed from a cationic lipid, a non-cationic lipid (e.g., phospholipid), a conjugate lipid (e.g., PEG-lipid) to prevent particle aggregation, and optionally cholesterol. Typically, therapeutic nucleic acids (e.g., siRNA) can be encapsulated in the lipid portion of the particles to protect them from enzymatic degradation.

[0040] When used to describe the lipid particles of the present invention, the term “high electron density core” refers to the interior of the lipid particles that appears dark when visualized using a cryo-transmission electron microscope ("cryo-TEM"). Some of the lipid particles of the present invention have a high electron density core and do not have a lipid bilayer structure. Some of the lipid particles of the present invention have a high electron density core and do not have a lipid bilayer structure, and have an inverse hexagonal or cubic phase structure. While we do not wish to be bound by theory, it is thought that the filling of non-bilayer lipids results in a three-dimensional network of lipid cylinders with water and nuclei inside, i.e., aqueous channels into which lipid droplets containing nucleic acids have permeated.

[0041] As used herein, the term “SNALP” refers to stable nucleic acid-lipid particles. SNALPs are particles made from lipids (e.g., cationic lipids, non-cationic lipids, and conjugate lipids to prevent particle aggregation) in which nucleic acids (e.g., siRNA) are completely encapsulated. In certain cases, SNALPs can exhibit a long circulating lifetime after intravenous (iv) injection, can accumulate at distal sites (e.g., sites physically separated from the administration site), and can mediate siRNA expression at these distal sites, making them extremely useful for systemic application. The nucleic acids may be encapsulated within the SNALPs in a complex with a condensing agent, as described in PCT Publication WO00 / 03683 (the contents of which are incorporated herein by reference in their entirety for all purposes).

[0042] The lipid particles of the present invention (e.g., SNALP) typically have wavelengths of approximately 30 nm to 150 nm, 40 nm to 150 nm, 50 nm to 150 nm, 60 nm to 130 nm, 70 nm to 110 nm, 70 nm to 100 nm, 80 nm to 100 nm, 90 nm to 100 nm, 70 nm to 90 nm, 80 nm to 90 nm, and 70 nm to 80 nm. The particles have an average diameter of m, or approximately 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 are 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 methods for preparing them are disclosed, for example, in U.S. Patent Publications 20040142025 and 20070042031, the contents 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, partial, or both complete encapsulation of therapeutic nucleic acids, such as siRNA. In preferred embodiments, the nucleic acid (e.g., siRNA) is completely encapsulated within the lipid particles (e.g., to form SNALP or other nucleic acid-lipid particles).

[0044] The term “lipid conjugate” refers to conjugated lipids that inhibit the aggregation of lipid particles. Such lipid conjugates include, but are not limited to, PEG-lipid conjugates, e.g., PEG coupled to dialkyloxypropyl (e.g., PEG-DAA conjugate), PEG coupled to diacylglycerol (e.g., PEG-DAG conjugate), PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamine, and PEG coupled to ceramide (see, e.g., U.S. Patent No. 5,885,613), cationic PEG lipids, polyoxazoline (POZ)-lipid conjugates, polyamide oligomers (e.g., ATTA-lipid conjugate), and mixtures thereof. Further examples of POZ-lipid conjugates are described in PCT Publication WO2010 / 006282. PEG or POZ may be directly conjugated to lipids or may be bound to lipids via a linker moiety. For example, any linker portion suitable for coupling PEG or POZ to lipids can be used, including ester-free and ester-containing linker portions. In certain preferred embodiments, ester-free linker portions, such as amides or carbamates, are used. The contents of each of the above-mentioned patent documents are incorporated herein by reference in their entirety for all purposes.

[0045] The term "amphiphilic lipid" refers, in part, to any suitable substance in which the hydrophobic portion of the lipid material faces the hydrophobic phase and the hydrophilic portion faces the aqueous phase. The hydrophilic characteristic derives from the presence of polar or charged groups, such as carbohydrate groups, phosphate groups, carboxylic acid groups, sulfat groups, amino groups, sulfhydryl groups, nitro groups, hydroxyl groups, and other similar groups. Hydrophobicity can be brought about by the inclusion of nonpolar groups, including but not limited to long-chain saturated and unsaturated aliphatic hydrocarbon groups, and such groups substituted with one or more aromatic, alicyclic, or heterocyclic groups. Examples of amphiphilic 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, the sphingoglycolipid family, diacylglycerols, and β-acyloxy acids, are also included in the group designated as amphiphilic lipids. Furthermore, the above-mentioned amphiphilic lipids can be mixed with other lipids, including triglycerides and sterols.

[0047] The term "neutral lipids" 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, ceramides, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerols.

[0048] The term "noncationic lipids" refers to any amphiphilic lipids and any other neutral or anionic lipids.

[0049] The term "anionic lipid" refers to any lipid that is negatively charged at physiological pH. These lipids include, but are 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 compounds having nonpolar groups, including but not limited to long-chain saturated and unsaturated aliphatic hydrocarbon groups, and such groups optionally substituted with one or more aromatic, alicyclic, or heterocyclic groups. Preferred examples include, but are not limited to, diacylglycerol, dialkylglycerol, NN-dialkylamino, 1,2-diacyloxy-3-aminopropane, and 1,2-dialkyl-3-aminopropane.

[0051] In this specification, the terms “cationic lipid” and “amino lipid” are used interchangeably to include lipids having one, two, three, or more fatty acid or fatty alkyl chains and pH titrable amino head groups (e.g., alkylamino or dialkylamino head groups) and salts thereof. Cationic lipids are typically characterized by their pK a Below a pH below 0, the substance is protonated (i.e., positively charged), and pK aAt pH levels above this, it is substantially neutral. The cationic lipids of the present invention are sometimes referred to as titrable cationic lipids. In some embodiments, the cationic lipids are protonable tertiary amine (e.g., pH titrable) head groups, and each alkyl chain independently has 0 to 3 (e.g., 0, 1, 2, or 3) double bonds. 18 The cationic lipids include alkyl chains and ether, ester, or ketal bonds between the head group and the alkyl chain. Examples of such 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 a group of the formula -N(H)R, where R is alkyl as defined herein.

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

[0054] The term "salt" includes any complex of anion and cation, such as a complex formed between a cationic lipid and one or more anions. Non-limiting examples of anions include inorganic and organic anions, e.g., hydrides, fluorides, chlorides, bromides, iodides, oxalic acid (e.g., hemioxalates), phosphoric acid, phosphonic acid, hydrogen phosphate, dihydrogen phosphate, oxides, carbonic acid, bicarbonate, nitric acid, nitrite, nitride, bisulfite, sulfurous acid, bisulfite, sulfuric acid, thiosulfate, bisulfate, boric acid, formic acid, acetic acid, benzoic acid, citric acid, tartaric acid, lactic acid, acrylic acid, polyacrylic acid. Examples include fumaric acid, maleic acid, itaconic acid, glycolic acid, gluconic acid, malic acid, mandelic acid, tigric acid, ascorbic acid, salicylic acid, polymethacrylic acid, perchloric acid, chloric acid, chlorous acid, hypochlorous acid, bromic acid, hypobromous acid, iodic acid, alkyl sulfonates, aryl sulfonates, arsenic acid, arsenous acid, chromic acid, dichromate, cyanide, cyanic acid, thiocyanic acid, hydroxides, peroxides, permanganic acid, and mixtures thereof. In certain embodiments, the cationic lipid salts disclosed herein are crystalline salts.

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

[0056] The term "membrane fusion" refers to the ability of lipid particles, such as SNALPs, to fuse with a cell membrane. This membrane may be the cell membrane or, for example, the membrane surrounding organelles such as endosomes or the nucleus.

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

[0058] As used herein, the term "organolipid solution" refers to a composition that contains, in whole or in part, an organic solvent having lipids.

[0059] As used herein, "distal region" refers to a physically separated region and includes regions widely distributed throughout the organism, not limited to adjacent capillary beds.

[0060] "Serum stability" for nucleic acid-lipid particles such as SNALPs 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 result in broad distribution of an active agent, such as siRNA, within a living organism. Some administration techniques can result in systemic delivery of a particular drug, while others cannot. Systemic delivery means that a useful (preferably therapeutic) amount of a drug is exposed to most parts of the body. To achieve broad distribution, the drug generally needs a blood lifetime such that it is not rapidly degraded or eliminated (e.g., by first-pass organs (liver, lungs, etc.) or by rapid nonspecific cell binding) before reaching disease sites distal to the administration site. Systemic delivery of lipid particles may be by any means known in the art, including, for example, intravenous, subcutaneous, and intraperitoneal delivery. 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 an active agent, such as siRNA, to a target site in a living organism. For example, a drug may be delivered locally by direct injection to a disease site, another target site, or a target organ such as the liver, heart, pancreas, or kidneys.

[0063] In this specification, when used to describe the lipid:siRNA ratio, the term "lipid" refers to the total lipids in the particles.

[0064] Those skilled in the art will notice that the compounds of the present invention having a chiral center exist in optically active and racemic forms and can be isolated in these forms. Some compounds may exhibit polymorphism. The present invention should be understood to encompass any racemic, optically active, polymorphic, or stereoisomeric forms, or mixtures thereof, of the compounds of the present invention having the useful properties described herein. Methods for preparing the optically active form (e.g., by recrystallization of the racemic form, by synthesis from optically active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase) are well known in the art.

[0065] Where a bond in a compound formula in this specification is depicted in a non-stereochemical manner (e.g., planar), the atom to which the bond is attached includes all stereochemical possibilities. Unless otherwise specified, where a bond in a compound formula in this specification is depicted in a predetermined stereochemical manner (e.g., thick line, thick wedge, dashed line, or dashed wedge), it is understood that the atom to which the stereochemical bond is attached is concentrated in the indicated absolute stereoisomer. In one embodiment, at least 51% of the compound may be the indicated absolute stereoisomer. In another embodiment, at least 60% of the compound may be the indicated absolute stereoisomer. In another embodiment, at least 80% of the compound may be the indicated absolute stereoisomer. In another embodiment, at least 90% of the compound may be the indicated absolute stereoisomer. In another embodiment, at least 95% of the compound may be the indicated absolute stereoisomer. In another embodiment, at least 99% of the compound may be the indicated absolute stereoisomer.

[0066] Unless otherwise stated herein, the term “about” when used in relation to a value or range of values ​​means plus or minus 5% of the given 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) double helix, as longer double-stranded RNA (dsRNA), or as siRNA or dsRNA transcribed from a transcription cassette within a DNA plasmid. In some embodiments, siRNA may be produced enzymatically or by partial / complete organic synthesis, and modified ribonucleotides may be introduced by in vitro enzymatic or organic synthesis. In certain cases, each strand is chemically prepared. Methods for synthesizing RNA molecules are known in the art, such as the chemical synthesis methods described, for example, Verma and Eckstein (1998), or are described herein.

[0068] Methods for isolating RNA, synthesizing RNA, hybridizing nucleic acids, preparing and screening cDNA libraries, and performing PCR are well known in the art (see, for example, Gubler and Hoffman, Gene, 25:263-269 (1983), Sambrook et al. (above), and Ausubel et al. (above)). PCR is also well known in the art (see U.S. Patent Nos. 4,683,195 and 4,683,202, and 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 of use in the present invention include Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989), Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990), and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994). The disclosures of these references are incorporated herein by reference in their entirety for all purposes.

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

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

[0072] Therefore, one aspect of the present invention is a nucleic acid molecule selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, and 73.

[0073] Another aspect of the present invention is a nucleic acid molecule selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, and 74.

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

[0075] One aspect of the present invention relates to 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), 2 The present invention provides double-stranded siRNA molecules 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 the 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 present invention is a compound of formula I as described in the summary of the invention, or a salt thereof.

[0079] In one embodiment of the compound of formula I, R 1 It is a targeted ligand, L 1 It does not exist, or it is a bonding group. L 2 It does not exist, or it is a bonding group. R 2 This is a double-stranded siRNA molecule selected from the double-stranded siRNAs in Table 1. Ring A is either absent, or is a 3-20 member cycloalkyl, 5-20 member aryl, 5-20 member heteroaryl, or 3-20 member heterocycloalkyl. Each R A These are independently hydrogen, hydroxyl, CN, F, Cl, Br, I, and -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 the alkoxy 1~8 Selected from the group consisting of alkyl groups, R B This refers to a bond to hydrogen, a protecting group, a covalent bond to a solid support, or a bond to a bonding 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 And in the formula, each L 3 The group is independently a bonding group, p is 1, 2, or 3, and sugars are monosaccharides or disaccharides.

[0081] In one embodiment, sugars are, [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 Either X is selected from 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 Each is independently selected from the group consisting of (C3-C6) cycloalkyl groups that are optionally substituted with one or more groups independently selected from the group consisting of hydrogen, (C1-C8) alkyl, (C1-C8) haloalkyl, (C1-C8) alkoxy, and 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 It is a five-membered heterocycle that is optionally substituted with one or more groups independently selected from the group consisting of -F, or halo, hydroxyl, carboxyl, amino, (C1-C4)alkyl, (C1-C4)haloalkyl, (C1-C4)alkoxy, and (C1-C4)haloalkoxy.

[0082] In one embodiment, sugars are, [ka] Selected from the group consisting of salts thereof.

[0083] In one embodiment, sugars are, [ka] That is the case.

[0084] In one embodiment, each L 3 A hydrocarbon chain is independently a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 0 to 50 carbon atoms, wherein one or more carbon atoms in the hydrocarbon chain (e.g., 1, 2, 3, or 4) are -O-, -NR X -, -NR X-C(=O)-, -C(=O)-NR X -, or -S- may be replaced depending on the case, R X The substituent is hydrogen or (C1-C6)alkyl, and the hydrocarbon chain is optionally substituted with one or more substituents (e.g., one, two, three, or four) selected from (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azide, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0085] In one embodiment, each L 3 A hydrocarbon chain is independently a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 20 carbon atoms, wherein one or more carbon atoms in the hydrocarbon chain (e.g., 1, 2, 3, or 4) are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X -, or -S- may be replaced depending on the case, R X The substituent is hydrogen or (C1-C6)alkyl, and the hydrocarbon chain is optionally substituted with one or more substituents (e.g., one, two, three, or four) selected from (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azide, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

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

[0087] In one embodiment, R1 ,

Chem.

[0088] In one embodiment, R 1 is

Chem.

[0089] In one embodiment, R C is

Chem.

[0090] In one embodiment, R 1 is

Chem.

[0091] In one embodiment, R C is

Chem.

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

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

[0094] In one embodiment, R 1 teeth, [ka] And, In the formula, each R D These are independently hydrogen, (C1~C6) alkyl, (C9~C 20 ) Alkylsilyl, (R W Selected from the group consisting of )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 These are independently selected from the group consisting of (C1-C4) alkyl and aryl groups.

[0095] In one embodiment, the binding group L 1 and L 2 A hydrocarbon chain is independently a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 50 carbon atoms, wherein one or more carbon atoms in the hydrocarbon chain (e.g., 1, 2, 3, or 4) are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X -, or -S- may be replaced depending on the case, R XThe substituent is hydrogen or (C1-C6)alkyl, and the hydrocarbon chain is optionally substituted with one or more substituents (e.g., one, two, three, or four) selected from (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azide, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

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

[0097] In one embodiment, L 1 and L 2 A is independently a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 14 carbon atoms, wherein one or more carbon atoms in the hydrocarbon chain (e.g., 1, 2, 3, or 4) are optionally replaced by -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NRX -, or -S-, R X The substituent is hydrogen or (C1-C6)alkyl, and the hydrocarbon chain is optionally substituted with one or more substituents (e.g., one, two, three, or four) selected from (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azide, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

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

[0099] In one embodiment, L 2 R is via -O- 2 It is connected.

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

[0101] In one embodiment, L 1 teeth, [ka] Selected from the group consisting of salts thereof.

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

[0103] In one embodiment, the compound of formula I is given by the following formula Ia: [ka] (Ia) Having or a salt thereof, During the ceremony, Each D is independent, [ka] The group is selected from the group consisting of and -N=.

[0104] In one embodiment, the compound of formula Ia is [ka] And selected from the group consisting of those salts, During the ceremony, Q 1 is hydrogen, Q 2 is R 2 is or Q 1 is R 2 Q 2 It is hydrogen, Z is -L 1 -R 1 That is the case.

[0105] In one embodiment, the compound of formula I is given by the following formula Ib: [ka] (Ib) It has or is a salt thereof, During the ceremony, Each D is independent, [ka] Selected from the group consisting of and -N=, Each m is independently either 1 or 2.

[0106] In one embodiment, the compound of formula Ib is [ka] And selected from the group consisting of those salts, During the ceremony, Q 1 is hydrogen, Q 2 is R 2 is or Q 1 is R 2 Q 2 It is hydrogen, Z is -L 1 -R 1 That is the case.

[0107] In one embodiment, the compound of formula I is given by the following formula (Ic): [ka] (I C) It has or is a salt thereof, In the formula, E is -O- or -CH2-, 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 a particular embodiment, the compound of formula (Ic) is [ka] And selected from the group consisting of those salts, In the formula, Z is -L 1 -R 1 That is the case.

[0109] In one embodiment, -AL 2 -R 2 The part is, [ka] or a salt thereof, During the ceremony, Q 1 is hydrogen, Q 2 is R 2 is or Q 1 is R 2 Q 2It is 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] Selected from the group consisting of salts thereof.

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

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

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

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

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

[0116] In one embodiment, L 2 These are -CH2O-, -CH2CH2O-, or -CH(OH)CH2O-.

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

[0118] In one embodiment, each R A These are independently selected from the group consisting of hydroxy, methyl, and -CH2OH.

[0119] In one embodiment, the compound of formula I is given by the following formula (Ig): [ka] (Ig) It has or is a salt thereof, In the formula, B is -N- or -CH-, L 1 It either does not exist or is -NH-, L 2 C is optionally substituted with hydroxyl or halo. 1~4 It is alkylene-O-, n is 0, 1, or 2.

[0120] In one embodiment, the compound of formula I is given by the following formula (Ig): [ka] (Ig) It has or is a salt thereof, In the formula, B is -N- or -CH-, L1 It either does not exist or is -NH-, L 2 C is optionally substituted with hydroxyl or halo. 1~4 It is alkylene-O-, n is 0, 1, 2, 3, 4, 5, 6, or 7.

[0121] In one embodiment, the compound of formula I is given by the following formula (Ig): [ka] (Ig) It has or is a salt thereof, In the formula, B is -N- or -CH-, L 1 It either does not exist or is -NH-, L 2 C is optionally substituted with hydroxyl or halo. 1~4 It is alkylene-O-, n is 0, 1, 2, 3, or 4.

[0122] In one embodiment, the compound of formula Ig is [ka] And selected from the group consisting of those salts, In the formula, R' is C 1~9 Alkyl, C 2~9 Alkenyl, or C 2~9 It is alkinyl, and this C 1~9 Alkyl, C 2~9 Alkenyl, or C 2~9 Alkynyl is sometimes substituted with a halo or hydroxyl.

[0123] In one embodiment, the compound of formula I is [ka] Selected from the group consisting of 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] It is selected from the group consisting of the following.

[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 selected from the group consisting of those pharmaceutically acceptable salts, In the formula, R 2 This refers to a double-stranded siRNA molecule selected from the double-stranded siRNA molecules listed in Table 1.

[0126] In one embodiment, the compound of formula I is [ka] or a pharmaceutically acceptable salt thereof, where R 2 This refers to a double-stranded siRNA molecule (for example, a double-stranded siRNA molecule selected from the double-stranded siRNA molecules listed in Table 1).

[0127] In one embodiment, the compound of formula I is [ka] or a pharmaceutically acceptable salt thereof, where R 2 This refers to a double-stranded siRNA molecule (for example, a double-stranded siRNA molecule selected from the double-stranded siRNA molecules listed in Table 1).

[0128] In one embodiment, the compound of formula I is [ka] or a pharmaceutically acceptable salt thereof, where R 2 This refers to a double-stranded siRNA molecule (for example, a double-stranded siRNA molecule selected from the double-stranded siRNA molecules listed in Table 1).

[0129] In one embodiment, the compound of formula I is [ka] or a pharmaceutically acceptable salt thereof, where R 2 This refers to a double-stranded siRNA molecule (for example, a double-stranded siRNA molecule selected from the double-stranded siRNA molecules listed in Table 1).

[0130] In one embodiment, the compound of formula I is [ka] or a pharmaceutically acceptable salt thereof, where R 2 This refers to a double-stranded siRNA molecule (for example, a double-stranded siRNA molecule selected from the double-stranded siRNA molecules listed in Table 1).

[0131] In one embodiment, the compound of formula I is [ka] or a pharmaceutically acceptable salt thereof, where R 2 This refers to a double-stranded siRNA molecule (for example, a double-stranded siRNA molecule selected from the double-stranded siRNA molecules listed in Table 1).

[0132] In one embodiment, the compound of formula I is [ka] or a pharmaceutically acceptable salt thereof, where R 2 This refers to a double-stranded siRNA molecule (for example, a double-stranded siRNA molecule selected from the double-stranded siRNA molecules listed in Table 1).

[0133] In one embodiment, the compound of formula I is [ka] or a pharmaceutically acceptable salt thereof, where R 2 This refers to a double-stranded siRNA molecule (for example, a double-stranded siRNA molecule selected from the double-stranded siRNA molecules listed in Table 1).

[0134] In one embodiment, the compound of formula I is [ka] or a pharmaceutically acceptable salt thereof, where R 2 This refers to a double-stranded siRNA molecule (for example, a double-stranded siRNA molecule selected from the double-stranded siRNA molecules listed in Table 1).

[0135] In one embodiment, the compound of formula I is [ka] or a pharmaceutically acceptable salt thereof, where R 2This refers to a double-stranded siRNA molecule (for example, a double-stranded siRNA molecule selected from the double-stranded siRNA molecules listed in Table 1).

[0136] In one embodiment, the compound of formula I is [ka] or a pharmaceutically acceptable salt thereof, where R 2 This refers to a double-stranded siRNA molecule (for example, a double-stranded siRNA molecule selected from the double-stranded siRNA molecules listed in Table 1).

[0137] In one embodiment, the compound of formula I is [ka] or a pharmaceutically acceptable salt thereof, where R 2 This refers to a double-stranded siRNA molecule (for example, a double-stranded siRNA molecule selected from the double-stranded siRNA molecules listed in Table 1).

[0138] In one embodiment, the compound of formula I is [ka] or a pharmaceutically acceptable salt thereof, where R 2 This refers to a double-stranded siRNA molecule (for example, a double-stranded siRNA molecule selected from the double-stranded siRNA molecules listed in 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 relates to a compound of formula (I): [ka] or provide the salt thereof. During the ceremony, L 1 It does not exist, or it is a bonding group. L 2 It does not exist, or it is a bonding group. R 2 It is a nucleic acid, Ring A is either absent, or is a 3-20 member cycloalkyl, 5-20 member aryl, 5-20 member heteroaryl, or 3-20 member heterocycloalkyl. Each R A These are independently hydrogen, hydroxyl, CN, F, Cl, Br, I, and -C. 1~2 Alkyl-OR B , C 1~10 Alkyl C 2~10 Alkenyl and C 2~10 Selected from the group consisting of alkynnyl, this C 1~10 Alkyl C 2~10 Alkenyl and C 2~10 Alkinyl is a compound of halo, hydroxy, and C. 1~3 Optionally substituted with one or more groups independently selected from the alkoxy, R B This refers to a bond to hydrogen, a protecting group, a covalent bond to a solid support, or a bond to a bonding group attached to a solid support. n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0148] In one embodiment, the present invention relates to a compound of the following formula: [ka] or provide the salt thereof. During the ceremony, L 2 It does not exist, or it is a bonding group. R 2 It is a nucleic acid, Ring A is either absent, or is a 3-20 member cycloalkyl, 5-20 member aryl, 5-20 member heteroaryl, or 3-20 member heterocycloalkyl. Each R AThese are independently hydrogen, hydroxyl, CN, F, Cl, Br, I, and -C. 1~2 Alkyl-OR B , C 1~10 Alkyl C 2~10 Alkenyl and C 2~10 Selected from the group consisting of alkynnyl, this C 1~10 Alkyl C 2~10 Alkenyl and C 2~10 Alkinyl is a compound of halo, hydroxy, and C. 1~3 Optionally substituted with one or more groups independently selected from the alkoxy, R B This refers to a bond to hydrogen, a protecting group, a covalent bond to a solid support, or a bond to a bonding 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 relates to a compound of the following formula: [ka] or provide the salt thereof. During the ceremony, L 1 It does not exist, or it is a bonding group. L 2 It does not exist, or it is a bonding group. R 2 It is a nucleic acid, B is divalent, and [ka] A group consisting of the following is selected, where, Each R' is independent of C 1~9 Alkyl, C 2~9 Alkenyl, or C 2~9 It is alkinyl, and this C 1~9 Alkyl, C 2~9 Alkenyl, or C 2~9 Alkynyl is sometimes substituted with a halo or hydroxyl. * The valence with a mark is L 1It is bound to L 1 If it does not exist, R 1 It is connected, ** The valence with a mark is L 2 It is bound to L 2 If it does not exist, R 2 It is connected.

[0150] In one embodiment, L 1 and L 2 A hydrocarbon chain is independently a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 50 carbon atoms, wherein one or more carbon atoms in the hydrocarbon chain (e.g., 1, 2, 3, or 4) are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X -, or -S- may be replaced depending on the case, R X The substituent 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, azide, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0151] In one embodiment, L 1 teeth, [ka] Or selected from the group consisting of those salts.

[0152] In one embodiment, L 1 B is formed via 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 It is connected.

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

[0154] In one embodiment, L 2 R is via -O- 2 It is connected.

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

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

[0157] In one embodiment, the present invention relates to a compound [ka] or provides a salt thereof, in the formula R 2 It is a nucleic acid.

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

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

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

[0161] A particular embodiment of the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof for use in medical therapy.

[0162] Certain embodiments of the present invention provide compounds of formula (I) or pharmaceutically acceptable salts thereof for prophylactic or therapeutic treatment of animal diseases or disorders (e.g., liver disease or viral infections such as hepatitis B virus infection).

[0163] A particular embodiment of the present invention provides the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof for preparing a drug for treating an animal disease or disorder (e.g., liver disease or viral infection such as hepatitis B virus infection).

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

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

[0166] In one embodiment, R 3d It contains a binding group that attaches the remainder of the compound of formula Id to a solid support. 2d The properties of the binding group are not important 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 in Table 1.

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

[0168] In one embodiment, R 3d The linker has a molecular weight of approximately 20 daltons to approximately 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 approximately 5 angstroms to approximately 40 angstroms (including boundary values).

[0170] In one embodiment, R 3d The linker is a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 2 to 15 carbon atoms, wherein one or more carbon atoms (e.g., 1, 2, 3, or 4) are optionally replaced with (-O-) or (-N(H)-), and the carbon atoms are optionally substituted with one or more substituents (e.g., 1, 2, 3, or 4) selected from (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azide, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0171] In one embodiment, R 3dThe linker is a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 2 to 10 carbon atoms, wherein one or more carbon atoms (e.g., 1, 2, 3, or 4) are optionally replaced with (-O-) or (-N(H)-), and the carbon atoms are optionally substituted with one or more substituents (e.g., 1, 2, 3, or 4) selected from (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azide, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

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

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

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

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

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

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

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

[0179] In another embodiment, the compound (Id) or a salt thereof is [ka] Selected from the group consisting of 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 a compound of formula (Id) or a pharmaceutically acceptable salt thereof to the animal.

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

[0183] A particular embodiment of the present invention provides a compound of formula (Id) or a pharmaceutically acceptable salt thereof for use in medical therapy.

[0184] Certain embodiments of the present invention provide compounds of formula (Id) or pharmaceutically acceptable salts thereof for prophylactic or therapeutic treatment of animal diseases or disorders (e.g., viral infections such as hepatitis B virus infection).

[0185] A particular embodiment of the present invention provides the use of a compound of formula (Id) or a pharmaceutically acceptable salt thereof for preparing a drug for treating a disease or disorder in animals (e.g., a viral infection such as hepatitis B virus infection).

[0186] In certain embodiments, the animal is a mammal such as a human (e.g., 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 an intermediate compound of formula Ie: [ka] (Ie) or a salt thereof, in the formula, R 1d teeth, [ka] Selected from, X d These are C2-8 alkylenes, n d is 0 or 1, Pg 1 is H or a suitable protecting group, R 3d This can be H, a protecting group, covalent bonding to a solid support, or bonding to a binding group attached to a solid support. Figure 1 shows a targeted ligand / linker attached to a solid support, and Pg 1 The following are examples of typical intermediate compounds of formula (Ie) in which the protecting group DMTr is used.

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

[0189] The present invention also provides a method for preparing a compound of formula (Id) described herein, wherein the compound of the corresponding formula (Ie): [ka] (Ie) (In the formula, X d These are C2-8 alkylenes, n d is 0 or 1, Pg 1 H is, R 3d(wherein R is a covalent bond to a solid support or a bond to a binding group attached to a solid support) is subjected to solid-phase nucleic acid synthesis conditions, and the corresponding compound of formula Id (wherein R is a covalent bond to a solid support or a bond to a binding group attached to a solid support) is subjected to solid-phase nucleic acid synthesis conditions, and the compound of formula Id is formed. 2d The present invention provides a method that involves obtaining a double-stranded siRNA molecule selected from the double-stranded siRNA molecules listed in Table 1.

[0190] In one embodiment, the method removes the compound from the solid support, R 3d This further includes yielding a compound of the corresponding formula Id where H is present.

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

[0192] In one embodiment, the compound is a compound of formula Ie: [ka] (Ie) or its salt (In the formula, R 1d teeth, [ka] Selected from, X d These are C2-8 alkylenes, n d is 0 or 1, Pg 1 is H or a suitable protecting group, R 3d (This is not a bond to H, a protecting group, a covalent bond to a solid support, or a bond to a bonding group attached to a solid support.)

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

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

[0195] In one embodiment, R 3d A is a bond to a bonded group attached to a solid support, the bonded group being a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 2 to 15 carbon atoms, wherein one or more carbon atoms (e.g., 1, 2, 3, or 4) are optionally replaced with (-O-) or (-N(H)-), and the carbon atoms are optionally substituted with one or more substituents (e.g., 1, 2, 3, or 4) selected from (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azide, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0196] In one embodiment, R 3dA is a bond to a bonding group attached to a solid support, the bonding group being a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 2 to 10 carbon atoms, wherein one or more carbon atoms (e.g., 1, 2, 3, or 4) are optionally replaced with (-O-) or (-N(H)-), and the carbon atoms are optionally substituted with one or more substituents (e.g., 1, 2, 3, or 4) selected from (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azide, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

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

[0198] In one embodiment, the present invention relates to a compound of formula (I): [ka] (I) or provide the salt thereof. During the ceremony, R 1 is H or a synthetic activating group, L 1 It does not exist, or it is a bonding group. L 2 It does not exist, or it is a bonding group. R 2 This is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules in Table 1. Ring A is either absent, or is a 3-20 member cycloalkyl, 5-20 member aryl, 5-20 member heteroaryl, or 3-20 member heterocycloalkyl. Each R A These are independently hydrogen, hydroxyl, CN, F, Cl, Br, I, and -C. 1~2Alkyl-OR B , C 1~10 Alkyl C 2~10 Alkenyl and C 2~10 Selected from the group consisting of alkynnyl, this C 1~10 Alkyl C 2~10 Alkenyl and C 2~10 Alkinyl is a compound of halo, hydroxy, and C. 1~3 Optionally substituted with one or more groups independently selected from the alkoxy, R B This refers to a bond to hydrogen, a protecting group, a covalent bond to a solid support, or a bond to a bonding 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 relates to a compound of formula (I): [ka] (I) or provide the salt thereof. During the ceremony, R 1 It is a targeted ligand, L 1 It does not exist, or it is a bonding group. L 2 It does not exist, or it is a bonding group. R 2 is H or a synthetic activating group, Ring A is either absent, or is a 3-20 member cycloalkyl, 5-20 member aryl, 5-20 member heteroaryl, or 3-20 member heterocycloalkyl. Each R A These are independently hydrogen, hydroxyl, CN, F, Cl, Br, I, and -C. 1~2 Alkyl-OR B , C 1~10 Alkyl C 2~10 Alkenyl and C 2~10 Selected from the group consisting of alkynnyl, this C 1~10 Alkyl C 2~10 Alkenyl and C 2~10Alkinyl is a compound of halo, hydroxy, and C. 1~3 Optionally substituted with one or more groups independently selected from the alkoxy, R B This refers to a bond to hydrogen, a protecting group, a covalent bond to a solid support, or a bond to a bonding 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 relates to a compound of formula (Ig): [ka] (Ig) or provide the salt thereof. During the ceremony, B is -N- or -CH-, L 2 C is optionally substituted with hydroxyl or halo. 1~4 It is alkylene-O-, n is 0, 1, 2, 3, 4, 5, 6, or 7.

[0201] In one embodiment, the present invention is [ka] The present invention provides compounds selected from the group consisting of 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 It is alkinyl, and this C 1~9 Alkyl, C 2~9 Alkenyl, or C 2~9 Alkynyl is sometimes substituted with a halo or hydroxyl.

[0202] In one embodiment, the present invention is [ka] The present invention provides compounds selected from the group consisting of salts thereof, In the formula, Q is -L 1 -R 1 That is the case.

[0203] In one embodiment, the present invention relates to a compound of formula (Ig): [ka] (Ig) or provide the salt thereof. During the ceremony, B is -N- or -CH-, L 1 It does not exist, or it is a bonding group. L 2 C is optionally substituted with hydroxyl or halo. 1~4 It is 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 either H or a synthetic activating group.

[0204] In one embodiment, the present invention is [ka] We provide compounds selected from the group consisting of the above, or salts thereof. In the formula, Q is -L 1 -R 1 And, L 1 It does not exist, or it is a bonding group. R' is C 1~9 Alkyl, C 2~9 Alkenyl, or C 2~9 It is alkinyl, and this C 1~9 Alkyl, C 2~9 Alkenyl, or C 2~9 Alkynyl is sometimes substituted with a halo or hydroxyl. R1 is H or a synthetic activating group, R 2 is either H or a synthetic activating group.

[0205] In one embodiment, the present invention is [ka] We provide compounds selected from the group consisting of the above, or salts thereof. During the ceremony, Q is -L 1 -R 1 And, L 1 It does not exist, or it is a bonding group. R 1 is H or a synthetic activating group, R 2 is either H or a synthetic activating group.

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

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

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

[0209] In one embodiment, R 2 These are acetates, triflates, mesylates, or succinates.

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

[0211] In one embodiment, the present invention relates to a compound of formula (XX): [ka] (XX) or provide the salt thereof. During the ceremony, R 1 It is a targeted ligand, L 1 It does not exist, or it is a bonding group. L 2 It does not exist, or it is a bonding group. R 2 This is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules in Table 1. B is divalent, and [ka] [ka] A group consisting of the following is selected, where, Each R' is independent of C 1~9 Alkyl, C 2~9 Alkenyl, or C 2~9 It is alkinyl, and this C 1~9 Alkyl, C 2~9 Alkenyl, or C 2~9 Alkynyl is sometimes substituted with a halo or hydroxyl. * The valence with a mark is L 1 It is bound to L 1 If it does not exist, R 1 It is connected, ** The valence with a mark is L2 It is bound to L 2 If it does not exist, R 2 It is connected.

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

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

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

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

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

[0217] In one embodiment, R 1 It contains 3-4 types of sugars.

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

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

[0220] In one embodiment, R 1 The following equation applies: [ka] It has, During the ceremony, B 1 It is a trivalent group containing approximately 1 to 20 atoms, L 1 , T 1 , and T 2 It is covalently bonded to it, B 2 It is a trivalent group containing approximately 1 to 20 atoms, and T 1 , T3 , and T 4 It is covalently bonded to it, B 3 It is a trivalent group containing approximately 1 to 20 atoms, and T 2 , T 5 , and T 6 It is covalently bonded to it, T 1 It does not exist, or it is a bonding group. T 2 It does not exist, or it is a bonding group. T 3 It does not exist, or it is a bonding group. T 4 It does not exist, or it is a bonding group. T 5 It does not exist, or it is a bonding group. T 6 It either does not exist or is a bonding group.

[0221] In one embodiment, each sugar is independently, [ka] 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 Either X is selected from 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 9Each is independently selected from the group consisting of (C3-C6) cycloalkyl groups that are optionally substituted with one or more groups independently selected from the group consisting of hydrogen, (C1-C8) alkyl, (C1-C8) haloalkyl, (C1-C8) alkoxy, and 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 It is a five-membered heterocycle that is optionally substituted with one or more groups independently selected from the group consisting of -F, or halo, hydroxyl, carboxyl, amino, (C1-C4)alkyl, (C1-C4)haloalkyl, (C1-C4)alkoxy, and (C1-C4)haloalkoxy.

[0222] In one embodiment, each sugar is independently, [ka] It is selected from the group consisting of the following.

[0223] In one embodiment, each sugar is independently, [ka] That is the case.

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

[0225] In one embodiment, T 1 and T 2 None of them exist.

[0226] In one embodiment, T 1 , T 2 , T 3 , T 4, T 5 , and T 6 Each of these is independently a branched or unbranched, saturated or unsaturated hydrocarbon chain having no carbon atoms or 1 to 50 carbon atoms, wherein one or more carbon atoms in the hydrocarbon chain (e.g., 1, 2, 3, or 4) are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X -, or -S- may be replaced depending on the case, R X The substituent is hydrogen or (C1-C6)alkyl, and the hydrocarbon chain is optionally substituted with one or more substituents (e.g., one, two, three, or four) selected from (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azide, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0227] In one embodiment, T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 Each of these is independently a branched or unbranched, saturated or unsaturated hydrocarbon chain having no carbon atoms or 1 to 20 carbon atoms, wherein one or more carbon atoms in the hydrocarbon chain (e.g., 1, 2, 3, or 4) are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X -, or -S- may be replaced depending on the case, R XThe substituent is hydrogen or (C1-C6)alkyl, and the hydrocarbon chain is optionally substituted with one or more substituents (e.g., one, two, three, or four) selected from (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azide, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0228] In one embodiment, T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 Each of these is independently a branched or unbranched, saturated or unsaturated hydrocarbon chain, or a salt thereof, having no carbon atoms or having 1 to 50 carbon atoms, wherein one or more carbon atoms in the hydrocarbon chain (e.g., 1, 2, 3, or 4) are -O- or -NR X - is sometimes replaced by R X The substituent is hydrogen or (C1-C6) alkyl, and the hydrocarbon chain is optionally substituted with one or more substituents (e.g., one, two, three, or four) selected from halo, hydroxy, and oxo (=O).

[0229] In one embodiment, T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 Each of these is independently a branched or unbranched, saturated or unsaturated hydrocarbon chain having no carbon atoms or 1 to 20 carbon atoms, wherein one or more carbon atoms in the hydrocarbon chain (e.g., 1, 2, 3, or 4) are optionally replaced by -O-, and the hydrocarbon chain is optionally substituted by one or more substituents (e.g., 1, 2, 3, or 4) selected from halo, hydroxy, and oxo (=O).

[0230] In one embodiment, T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 Each of these is independently a branched or unbranched, saturated or unsaturated hydrocarbon chain having no carbon atoms or 1 to 20 carbon atoms, wherein one or more carbon atoms in the hydrocarbon chain (e.g., 1, 2, 3, or 4) are optionally replaced by -O-, and the hydrocarbon chain is optionally substituted by one or more substituents (e.g., 1, 2, 3, or 4) selected from halo, hydroxy, and oxo (=O).

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

[0232] In one embodiment, T 3 , T 4 , T 5 , and T 6 Each of them is independent, [ka] 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 them is glycine.

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

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

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

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

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

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

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

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

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

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

[0244] In one embodiment, B 1 This includes Ariel.

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

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

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

[0248] In one embodiment, B 1 It contains heteroaryl compounds.

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

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

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

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

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

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

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

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

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

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

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

[0260] In one embodiment, B 2 This includes Ariel.

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

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

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

[0264] In one embodiment, B 2 It contains heteroaryl compounds.

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

[0266] In one embodiment, B 2 teeth, [ka] Or selected from the group consisting of those salts.

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

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

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

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

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

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

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

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

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

[0276] In one embodiment, B 3 This includes Ariel.

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

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

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

[0280] In one embodiment, B 3 It contains heteroaryl compounds.

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

[0282] In one embodiment, B 3 teeth, [ka] Or selected from the group consisting of those salts.

[0283] In one embodiment, L 1 and L 2 A hydrocarbon chain is independently a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 50 carbon atoms, wherein one or more carbon atoms in the hydrocarbon chain (e.g., 1, 2, 3, or 4) are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X -, or -S- may be replaced depending on the case, R X The substituent is hydrogen or (C1-C6)alkyl, and the hydrocarbon chain is optionally substituted with one or more substituents (e.g., one, two, three, or four) selected from (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azide, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0284] In one embodiment, L 1 teeth, [ka] Or selected from the group consisting of those salts.

[0285] In one embodiment, L 1 B is formed via 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 It is connected.

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

[0287] In one embodiment, L 2 R is via -O- 2 It is connected.

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

[0289] In one embodiment, L 2 R is via -O- 2 It is connected.

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

[0291] In one embodiment, the present invention is [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] The present invention provides compounds or salts selected from the group consisting of and pharmaceutically acceptable salts thereof, wherein R 2 This refers to a double-stranded siRNA molecule selected from the double-stranded siRNA molecules listed in Table 1.

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

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

[0294] In one embodiment, a nucleic acid molecule (e.g., siRNA) is bound to the remainder of the compound via the oxygen of the phosphate group at the 3' end of the sense strand.

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

[0296] If a compound contains the following group, [ka] Four stereoisomers (two cis and two trans) are possible on the ring. Unless otherwise stated, the compounds of the present invention include all four stereoisomers with respect to such a ring. In one embodiment, the two R' groups assume the cis conformation. In one embodiment, the two R' groups assume 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 in Table 1, (b) Cationic lipids and (c) Noncationic lipids and These are nucleic acid-lipid particles containing [the specified substance]. [Examples]

[0298] The present invention will be further described in detail by specific embodiments. The following embodiments are presented for illustrative purposes only and are not intended to limit the invention in any way. Those skilled in the art will readily recognize various non-essential parameters that can be changed or modified to produce essentially the same results. In one embodiment, the oligonucleotide is understood to be a double-stranded siRNA molecule as listed 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-methylbenzene sulfonate 3 [ka] A solution containing tetraethylene glycol (934 g, 4.8 mol) in THF (175 mL) and NaOH aqueous solution (5 M, 145 mL) was cooled (0°C), treated with p-toluenesulfonyl chloride (91.4 g, 480 mmol) dissolved in THF (605 mL), and then stirred for 2 hours (0°C). This mixture was diluted with water (3 L) and extracted with CH2Cl2 (3 × 500 mL). The combined extract was washed with water and brine, then dried (MgSO4), filtered, and concentrated to obtain 2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl 4-methylbenzene sulfonate (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)ethane-1-ol (4) [ka] A solution of 3 (140 g, 403 mmol) of DMF (880 mL) was treated with sodium azide (131 g, 2.02 mol) and heated overnight (45°C). Most of the DMF was removed under reduced pressure, the residue was dissolved in CH2Cl2 (500 mL), washed with brine (3 × 500 mL), then dried (MgSO4), filtered, and concentrated. The residue was passed through a short silica bed (5% MeOH-CH2Cl2) and concentrated to obtain 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethane-1-ol (4) (65 g, 74%) as a yellow oil. f (0.56, 10% MeOH-CH2Cl2).

[0302] Step 3. Preparation of hyperacetylated galactosamine (6) [ka] Pyridine (1.5 L) containing D-galactosamine hydrochloride (5) (250 g, 1.16 mol) 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 solid was filtered off, and the mixture was frozen over liquid nitrogen and then freeze-dried for 5 days to obtain peracetylated galactosamine (6) (369.4 g, 82%) as a white solid. Rf (0.58, 10% MeOH-CH2Cl2).

[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-diyldiacetate (7) [ka] A 320 mL solution of peracetylated galactosamine (6) (8.45 g, 21.7 mmol) in CHCl3 was treated by the dropwise addition of TMSOTf (4.32 mL, 23.9 mmol). After stirring (1.5 hours, 40°C), the reaction was quenched by the addition of triethylamine (5 mL), concentrated to dryness, and compound 7 was obtained as a pale yellow glassy substance (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-diyldiacetate (8) [ka] Compound 7 (7.2 g, 21.7 mmol) and 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethane-1-ol 4 (2.65 g, 15.2 mmol) were azeotropically mixed in toluene (150 mL) three times to remove trace amounts of water. The dehydrated substances were dissolved in 1,2-dichloroethane (150 mL), cooled (approximately 5°C), and treated with TMSOTf (784 μL, 4.34 mmol). After stirring overnight, the reaction mixture was quenched and concentrated by adding triethylamine (5 mL). The residue was purified by chromatography (1% → 5% MeOH-CH2Cl2) to obtain compound 8 (7.12 g, 85%) as a brown oily substance. Rf (0.3, 10% MeOH-CH2Cl2).

[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)ethane-1-aminium 2,2,2-trifluoroacetate (9) [ka] A solution containing azede 8 (7.12 g, 13 mmol) in toluene (150 mL) and trifluoroacetic acid (2 mL) was treated with palladium carbon (1.5 g, 10% w / w wet basis). The reaction mixture was then purged with hydrogen and vigorously stirred overnight. After purging with nitrogen, the mixture was filtered through Celite and rinsed with MeOH. The filtrate was concentrated and purified by chromatography (5% → 10% → 20% MeOH-CH2Cl2) to obtain 9 (5.8 g, 72%) as a brown oily substance. Rf (0.34, 15% MeOH-CH2Cl2).

[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) (13.5g, 33 mmol), 25% Na2CO2 3(水溶液) Benzyl chloroformate (14 mL, 98 mmol) was slowly added to a solution of (150 mL) and dichloromethane (300 mL). This solution was vigorously stirred overnight (16 hours) at room temperature. Upon completion, an additional 100 mL of dichloromethane was added to separate the dichloromethane layer. The aqueous layer was extracted with dichloromethane (2 × 100 mL). The combined dichloromethane extract was dried over magnesium sulfate, filtered, and concentrated to dryness. Product 11 was isolated as a colorless oil and no further purification was required (15.8 g, 88%). Rf (0.7, 1:1 toluene-hexane).

[0307] Step 8. Preparation of 4-(((benzyloxy)carbonyl)amino)-4-(2-carboxyethyl)heptanediic acid (12) [ka] 12 A 50 mL solution of 11 (15.6 g, 28.8 mmol) of formic acid was stirred at room temperature for 2 hours. This 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 obtain 12 as a colorless solid (10.2 g, 93%). Rf (0.1, 10% MeOH-CH2Cl2).

[0308] Step 9. Preparation of Compound 13 [ka] 13 A 50 mL solution of DMF containing 12 (793 mg, 2.08 mmol) and 9 (5.8 g, 9.36 mmol) 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 subjected to chromatography (1% → 2% → 5% → 10% → 15% MeOH-CH2Cl2) to obtain 13 (5.71 g [unpurified], >100% - containing coupling byproducts that did not affect the next step). Rf (0.45, 10% MeOH-CH2Cl2).

[0309] Step 10. Preparation of Compound 14 [ka] 14 Compound 13 (5.7g) was dissolved in MeOH (150mL) and TFA (1.5mL) and treated with palladium carbon (1g, 10% w / w wet basis). The reaction mixture was then purged with hydrogen and vigorously stirred overnight. After purging with nitrogen, the mixture was filtered through Celite and rinsed with MeOH. The filtrate was concentrated and purified by chromatography (5% → 10% → 20% MeOH-CH2Cl2) to obtain compound 14 as a brown oily substance (2.15g, 56% in 2 steps). Rf (0.32, 10% MeOH-CH2Cl2).

[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 hour to a reflux mixture of 3.75 equivalents of LiAlH4 (13.6 g, 358 mmol) in THF (440 mL). The mixture was stirred under reflux for a further 2 hours, then cooled to room temperature and quenched by carefully adding MeOH (27 mL) followed by water (40 mL). After stirring the quenched mixture for 2 hours, it was filtered and concentrated to dryness. The residue was recrystallized from siRNA (twice) to obtain 15 as brownish-yellow crystals (10.2 g, 70%).

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

[0312] Step 13. Preparation of methyl 10-((3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(hydroxymethyl)phenyl)amino)-10-oxodecanoate (17) [ka] To a 50 mL solution of 16 (4.3 g, 12 mmol) of pyridine, 4,4'-(chloro(phenyl)methylene)bis(methoxybenzene) (4.1 g, 12 mmol) was added. This solution was stirred under nitrogen at room temperature overnight. Upon completion, the solution was concentrated to dryness, and the residue was purified by column chromatography (0.5% → 0.75% → 1% → 1.5% MeOH-CH2Cl2) to obtain 17 as a yellow solid (2.9 g, 35%). Rf (0.6, 10% MeOH-CH2Cl2).

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

[0314] Step 15. Preparation of Compound 19. [ka] N,N-diisopropylethylamine (0.35 mL, 2.0 mmol) was added to a 25 mL anhydrous DMF solution of 14 (454 mg, 0.67 mmol), 18 (1.25 g, 0.67 mmol), and HBTU (381 mg, 1.0 mmol). This solution was stirred overnight at room temperature. 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 (CH2Cl2 containing 5% → 7.5% → 10% → 15% MeOH) yielded 19 as a pale orange foam (1.5 g, 94%). Rf (0.25, 10% MeOH-CH2Cl2).

[0315] Step 16. Preparation of Compound 20 [ka] A 50 mL solution of anhydrous CH2Cl2 containing 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) was stirred overnight at room temperature. Upon completion, the solution was concentrated to dryness and filtered through a short silica plug (100% CH2Cl2 → CH2Cl2 containing 15% MeOH) to obtain 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)acetamide)-4-(3-(tert-butoxy)-3-oxopropyl)heptanedioate (21) [ka] A CH2Cl2 solution (300 mL) 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) was sequentially treated with EDC (23 g, 120 mmol), diisopropylethylamine (32 mL, 180 mmol), and DMAP (catalyst, 17 mg). After stirring (16 hours), the reaction mixture was poured into NaHCO3 (saturated aqueous solution), extracted with CH2Cl2, washed with brine, dried (MgSO4), filtered, and concentrated to obtain di-tert-butyl 4-(2-(((benzyloxy)carbonyl)amino)acetamide)-4-(3-(tert-butoxy)-3-oxopropyl)heptanedioate 21 as an amorphous solid, which was used without further processing (36 g, quantitative). Rf (0.85, 10% MeOH-CH2Cl2).

[0319] Step 2. Preparation of 4-(2-(((benzyloxy)carbonyl)amino)acetamide)-4-(2-carboxyethyl)heptanediic acid (22) [ka] A solution of di-tert-butyl 4-(2-(((benzyloxy)carbonyl)amino)acetamide)-4-(3-(tert-butoxy)-3-oxopropyl)heptanedioate (21) (59.3 mmol, 36 g) was stirred in undiluted formic acid (150 mL) for 72 hours. Upon completion, the formic acid was removed under reduced pressure, and the crude solid was dried overnight under high vacuum to obtain 22 as a colorless solid (15.9 g, 61%). Rf (0.15, 10% MeOH-CH2Cl2).

[0320] Step 3. Preparation of Compound 23 [ka] A 250 mL DMF 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)ethane-1-aminium 2,2,2-trifluoroacetate (35 g, 56.5 mmol) 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 subjected to chromatography (100% CH2Cl2 to 15% MeOH-CH2Cl2) to obtain compound 23 (24.6 g, 89%). Rf(0.55, 15% MeOH-CH2Cl2).

[0321] Step 4. Preparation of Compound 24 [ka] Compound 23 (24.6g) was dissolved in MeOH (200mL) and TFA (1.5mL) and purged with nitrogen. Palladium carbon (1g, 10% w / w wet basis) was added, and the reaction mixture was then purged with hydrogen and vigorously stirred overnight. Upon completion, the reaction mixture was purged with nitrogen, filtered through Celite, and rinsed with MeOH. This filtrate was concentrated and purified by column chromatography on silica gel 60 (gradient: 5% → 10% → 20% MeOH-CH2Cl2) to obtain compound 24 as a light brown viscous oil (23g). Rf (0.32, 10% MeOH-CH2Cl2).

[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 under reflux for a further 2 hours. Once completed, the solution was cooled to room temperature and quenched by slowly adding MeOH (27 mL) followed by water (40 mL). After stirring for 2 hours, the mixture was filtered, concentrated, and recrystallized from siRNA to obtain (5-amino-1,3-phenylene)dimethanol (26) as off-white crystals (10.2 g, 70%). Rf 0.5 (15% MeOH-CH2Cl2).

[0323] Step 6. Preparation of 3,5-bis(hydroxymethyl)benzonitrile (27) [ka] A 100 mL solution of 26 (5 g, 33 mmol) in 2N hydrochloric acid was cooled to 0°C and treated with a 50 mL cold aqueous solution of sodium nitrite (3.53 g, 36 mmol). The reaction mixture was maintained at a temperature below 5°C for 30 minutes, and then treated with a single dose of copper(I) cyanide (3.19 g, 35.6 mmol) and a 50 mL aqueous solution of sodium cyanide (3.53 g, 72 mmol). After stirring overnight at room temperature, the mixture was filtered, extracted with dichloromethane (3 × 100 mL), concentrated, and used without further purification. The diol 3,5-bis(hydroxymethyl)benzonitrile (27) was obtained as a yellow solid (2.19 g, 41%). Rf 0.75 (15% MeOH-CH2Cl2).

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

[0325] Step 8. Preparation of (3-(aminomethyl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)phenyl)methanol (29) [ka] A 5 mL solution of 28 (100 mg, 0.22 mmol) of methyltetrahydrofuran 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 hour, the reaction was quenched by adding methanol (1 mL) followed by water (0.3 mL), and stirred for 30 minutes. The mixture was filtered and concentrated to obtain (3-(aminomethyl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)phenyl)methanol (29) (78 mg, 77%). Rf 0.15 (10% MeOH-CH2Cl2).

[0326] Step 9. Preparation of methyl 10-((3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(hydroxymethyl)benzyl)amino)-10-oxodecanoate (30) [ka] A 5 mL 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 was successively treated with EDC (48 mg, 0.25 mmol), DMAP (catalyst, 5 mg), and diisopropylethylamine (57 μL, 0.33 mmol). After stirring (3.5 hours), the reaction mixture was poured into a 50 mL solution of saturated sodium bicarbonate. 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 substance was purified by column chromatography in silica gel 60 (gradient: 2% to 5% MeOH-CH2Cl2) to obtain 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 10-((3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(hydroxymethyl)benzyl)amino)-10-oxodecanoate lithium (31). [ka] Compound 30 (188 mg, 0.28 mmol) was dissolved in tetrahydrofuran (5 mL) and treated with an aqueous solution of LiOH (7 mg, 0.30 mmol) (1 mL). Upon completion, the tetrahydrofuran was removed under vacuum, and the remaining aqueous mixture was freeze-dried to obtain lithium 10-((3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(hydroxymethyl)benzyl)amino)-10-oxodecanoate (31) as a colorless solid (180 mg, 99%). Rf 0.45 (10% MeOH-CH2Cl2).

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

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

[0330] Step 1. Preparation of Conjugate 36 Conjugate 36 was prepared using the same procedure used to synthesize compound 34 and all its corresponding intermediates. The only exception was the synthesis of compound 6, which used propanoic anhydride 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] Diisopropylethylamine (2.75 mL, 15.9 mmol) was added to a 20 mL N,N-dimethylformamide 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). This solution was stirred overnight at room temperature. Upon completion, the solution was concentrated to dryness under vacuum. The residue was purified by column chromatography on silica gel 60 (gradient: 2% to 5% MeOH / CH2Cl2) to obtain the product as a colorless solid (2.1 g, 63%).

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

[0334] Step 3. Preparation of Compound 39 [ka] To a 25 mL solution of 38 (1.5 g, 2.3 mmol) anhydrous dioxane, 25 mL of dioxane containing 2 M hydrogen chloride was added. This solution was stirred overnight at room temperature, and then concentrated to dryness under vacuum to obtain 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 using the same procedure as that 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 (100 mL) of 2,6-bis(hydroxymethyl)-p-cresol (2.7 g, 16.3 mmol), methyl 11-bromo undecanoate (5.0 g, 17.9 mmol), and potassium carbonate (4.5 g, 32.6 mmol) in acetone was refluxed for 16 hours. Upon completion, the solution was concentrated to dryness under vacuum, 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 under vacuum. The residue was purified by column chromatography on silica gel 60 (gradient 100% Hex → 50% HCl / Hex) to obtain 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] Methyl 11-(2,6-bis(hydroxymethyl)-4-methylphenoxy)undecanoate (44) (1.5 g, 4.1 mmol) was dissolved in anhydrous pyridine solution (20 mL) with 4,4'-dimethoxytrityl chloride (1.4 g, 4.1 mmol). This solution was stirred overnight at room temperature. Upon completion, the solution was concentrated to dryness under vacuum and purified by column chromatography (CH2Cl2 containing 0.5 to 1% MeOH) on silica gel 60 to obtain 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 11-(2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-6-(hydroxymethyl)-4-methylphenoxy) lithium undecanoate (46) [ka] A solution containing methyl 11-(2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-6-(hydroxymethyl)-4-methylphenoxy)undecanoate (45) (1.1 g, 1.7 mmol) was prepared by adding lithium hydroxide (44 mg, 1.8 mmol) to anhydrous tetrahydrofuran (40 mL) and water (10 mL). This solution was concentrated under vacuum to remove all of the tetrahydrofuran. The remaining aqueous solution was rapidly frozen with liquid nitrogen and then freeze-dried overnight to obtain 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 Diisopropylethylamine (0.35 mL, 2 mmol) was added to a 25 mL solution of N,N-dimethylformamide containing 10 (1.33 g, 0.66 mmol), 46 (0.5 g, 0.73 mmol), and HBTU (400 mg, 1 mmol). This solution was stirred overnight (18 hours) at room temperature. Upon completion, the solvent was removed under vacuum, and the residue was purified by silica gel column chromatography (gradient: CH2Cl2 containing 100% CH2Cl2 - 5% - 10% - 15% MeOH) to obtain 47 as a colorless solid (710 mg, 41%).

[0341] Step 5. Preparation of Compound 48 To a 15 mL dichloromethane 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), succinic anhydride (60 mg, 0.6 mmol) was added. This solution was stirred overnight at room temperature, and upon completion, it was filtered and concentrated to dryness under vacuum. The residue was purified by column chromatography on silica gel 60 (CH2Cl2 containing MeOH with a gradient of 5% to 20%) to obtain 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 containing dichloromethane (2 mL) and acetonitrile (3 mL) with 48 (100 mg, 40 μmol), N-hydroxysuccinimide (30 mg / mL acetonitrile solution, 50 μL, 13 μmol), N,N'-diisopropylcarbodiimide (40 μL, 264 μmol), and pyridine (50 μL), 1000 Å lcaa CPG (Prime Synthesis, 920 mg) was added. This solution was stirred overnight at room temperature in an orbital shaker. TLC analysis of the reaction solution showed only partial consumption of the activated N-hydroxysuccinate, so an additional CPG (500 mg) was added. This solution was stirred again 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 acetonitrile (3 mL) containing acetic anhydride and tetrahydrofuran (3 mL) containing 10% N-methylimidazole / 10% pyridine. This 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. A standard DMT loading assay (3% trichloroacetic acid in CH2Cl2, UV-VIS, A) was performed. 504 Using this method, 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 a standard procedure. After nucleotide deprotection, removal from the solid support (simultaneously with galactosamine acetate deprotection) yielded 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)ethane-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 hours. Upon completion, the reaction mixture was cooled to room temperature, diluted with water (400 mL), and extracted with ethyl acetate (3 × 150 mL). The combined ethyl acetate extract was dried over magnesium sulfate, filtered, and concentrated to dryness under vacuum. The residue was purified by filtration through a silica pad using first 50% ethyl acetate / hexane and then 50% MeOH / siRNA to obtain 51 as a pale yellow oily substance (14 g, 100%).

[0346] Step 2. Preparation of 2-(bis(4-methoxyphenyl)(phenyl)methoxy)-N-(2-((tert-butyldimethylsilyl)oxy)ethyl)ethane-1-amine (52) [ka] 2-((2-((tert-butyldimethylsilyl)oxy)ethyl)amino)ethane-1-ol (51) (14 g, 64 mmol) and triethylamine (17.5 mL, 128 mmol) were dissolved in anhydrous dichloromethane (250 mL), to which 4,4'-dimethoxytrityl chloride (24 g, 70 mmol) was added. This solution was stirred overnight at room temperature and then concentrated to dryness under vacuum. The residue was dissolved in ethyl acetate (300 mL) and washed with water (250 mL) and brine (250 mL). The ethyl acetate was dried over magnesium sulfate, filtered, and concentrated to dryness under vacuum. Purification by column chromatography on silica gel 60 (CH2Cl2 containing 1% to 5% MeOH) yielded 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 100 mL solution of 2-(bis(4-methoxyphenyl)(phenyl)methoxy)-N-(2-((tert-butyldimethylsilyl)oxy)ethyl)ethane-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 was stirred at room temperature for 3 hours. 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 under vacuum. Purification by column chromatography on silica gel 60 (hexane containing 10% to 25% ethyl acetate) yielded 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] 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) were dissolved in anhydrous tetrahydrofuran solution (20 mL), to which TBAF (1 M in THF, 3.4 mL, 3.3 mmol) was added. The solution was stirred for 6 hours, but only partial conversion was observed by TLC (CH2Cl2 containing 5% MeOH). A further 1.7 mL of TBAF was added, and the solution was stirred overnight at room temperature. Upon completion, the solution was concentrated under vacuum and purified by column chromatography on silica gel 60 (hexane containing 10% to 50% siRNA, then 100% siRNA) to obtain 54 as a viscous, colorless oil (0.5 g, 29%).

[0349] Step 5. Preparation of 10-((2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)(2-hydroxyethyl)amino)-10-oxodecanoate lithium (55) [ka] To a 40 mL THF solution of methyl 10-((2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)(2-hydroxyethyl)amino)-10-oxodecanoate (54) (0.5 g, 0.83 mmol), water (10 mL) and lithium hydroxide (24 mg, 1.0 mmol) were added. This solution was stirred overnight at room temperature, and then concentrated under vacuum to remove the THF. The remaining aqueous solution was rapidly frozen with liquid nitrogen and freeze-dried to obtain 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 as that 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 passed through this solution for 2 minutes, and the reaction mixture was stirred under reflux for 1.5 hours. Concentration of the reaction mixture under vacuum yielded methyl (2R,5R)-5-hydroxypiperidine-2-carboxylate (61) in quantitative yield, 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 amounts, and the reaction mixture was stirred for 2 hours. The reaction mixture was diluted in DCM (100 mL) and washed sequentially with 1 M HCl (2 × 75 mL), saturated NaHCO3 (2 × 75 mL), H2O (2 × 75 mL), and saturated NaCl solution (2 × 75 mL). The organic matter was separated, dried (Na2SO4), and concentrated in a vacuum to obtain 1-(tert-butyl)2-methyl(2R,5R)-5-hydroxypiperidine-1,2-dicarboxylate 62 (5.53g, 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. LiBH4 (3.0 M THF solution) (8.9 mL, 27.7 mmol) was added dropwise over 1 hour. The reaction mixture was allowed to return to room temperature and stirred for 16 hours. The reaction mixture was quenched with 1 M NaOH, the THF was removed under vacuum, and the aqueous solution was thoroughly extracted with SiO2 (10 × 100 mL). The combined organic compounds were washed with H2O (50 mL) and saturated NaCl solution (2 × 50 mL), dried to (Na2SO4), and concentrated under vacuum to yield 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)piperidine-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 Et2O at room temperature. HCl (gas) was passed through for 45 seconds, and the reaction mixture was stirred at room temperature for 45 minutes. The reaction mixture was concentrated under vacuum and dried under high vacuum to obtain (3R,6R)-6-(hydroxymethyl)piperidine-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)piperidine-1-yl)ethane-1-one (65) [ka] The crude (3R,6R)-6-(hydroxymethyl)piperidine-3-ol (64) obtained from the previous reaction was stirred at room temperature in MeCN (50 mL) containing TEA (3.5 mL, 25.2 mmol). Trifluoroethyl acetate (3 mL, 25.2 mmol) was added, and the reaction mixture was stirred at room temperature for 16 hours, followed by concentration under vacuum to yield 2,2,2-trifluoro-1-((2R,5R)-5-hydroxy-2-(hydroxymethyl)piperidine-1-yl)ethane-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-hydroxypiperidine-1-yl)-2,2,2-trifluoroethane-1-one (66) [ka] The crude 2,2,2-trifluoro-1-((2R,5R)-5-hydroxy-2-(hydroxymethyl)piperidine-1-yl)ethane-1-one (65 mL) obtained from the previous reaction was stirred at RT in DCM containing TEA (50 mL). 4,4'-dimethoxytrityl chloride (DMTrCl) (3.87 g, 11.44 mmol) was added in one addition, and the reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was diluted with DCM (50 mL) and sequentially washed with saturated NaHCO3 (2 × 75 mL), H2O (2 × 75 mL), and saturated NaCl solution (2 × 75 mL). The organic matter was separated, dried (Na2SO4), concentrated in vacuum, and purified by column chromatography (100% hexane-60% SiO / hexane) (0.1% TEA) to obtain 1-((2R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-hydroxypiperidine-1-yl)-2,2,2-trifluoroethane-1-one (66) (3.14 g, 57%).

[0358] Step 7. Preparation of (3R,6R)-6-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-piperidine-3-ol(67) [ka] 1-((2R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-hydroxypiperidine-1-yl)-2,2,2-trifluoroethane-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 mixture was stirred at room temperature for 16 hours. Further KOH (300 mg, 6 mmol) was added, and stirring was continued for another 24 hours. The reaction mixture was concentrated under vacuum, transferred to DCM (150 mL), washed with H2O (4 × 50 mL), dried to (Na2SO4), and concentrated under vacuum to yield (3R,6R)-6-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)piperidine-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-hydroxypiperidine-1-yl)-12-oxododecanoate (68) [ka] (3R,6R)-6-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)piperidine-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 mixture was stirred at room temperature for 3 hours. The resulting solid precipitate was removed by filtration, and the filtrate was concentrated under vacuum. The residue was then purified by column chromatography (2.5% MeOH / DCM, 0.1% TEA) to obtain methyl 12-((2R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-hydroxypiperidine-1-yl)-12-oxododecanoate (68) in quantitative yield.

[0360] Step 9. Preparation of 12-((2R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)-methyl)-5-hydroxypiperidine-1-yl)-12-oxododecanoate lithium (69). [ka] Methyl 12-((2R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-hydroxypiperidine-1-yl)-12-oxododecanoate (68) (5.4 mmol) and LiOH (140 mg, 5.94 mmol) were stirred in THF:H2O (1:1, 100 mL) at room temperature for 48 hours. After removing the THF under vacuum, the aqueous solution was freeze-dried and lyophilized to yield lithium 12-((2R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-hydroxypiperidine-1-yl)-12-oxododecanoate (69) (3.2 g, 91%). This was used in subsequent reactions 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 as that 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 containing gallic acid (20 g) was prepared by mixing pyridine (50 mL) and acetic anhydride (50 mL). This solution was stirred overnight at room temperature, and then poured into ice water (1 L). When this solution was acidified with concentrated hydrochloric acid, a colorless solid precipitated immediately. This solid was collected by filtration and washed with water (5 × 100 mL). This wet solid was frozen with liquid nitrogen and freeze-dried to obtain 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-triyltriacetate (74) 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) were dissolved in DMF (200 mL), to which DIPEA (17.5 mL, 101 mmol) was added. This solution was stirred for 16 hours, then diluted with ethyl acetate (250 mL), washed with brine (3 × 200 mL), dried over magnesium sulfate, filtered, and concentrated to dryness under vacuum. The crude product was purified by silica gel column chromatography (DCM containing MeOH with a gradient of 1% to 5%) to obtain 5-((2-((2-oxo-2-phenyl-1λ 2 Ethyl)amino)ethyl)carbamoyl)benzene-1,2,3-triyltriacetate 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 1:1 MeOH / CH2Cl2 solution (100 mL) of ethyl)amino)ethyl)carbamoyl)benzene-1,2,3-triyltriacetate (5 g, 1.1 mmol) was stirred at room temperature for 3 days. Upon completion, the solvent was removed, and 3,4,5-trihydroxy-N-(2-((2-oxo-2-phenyl-1λ 2 -ethyl)amino)ethyl)benzamide was obtained as a colorless solid (4 g, quantitative).

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

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

[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 that 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 that 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 that 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 that 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 in automated oligonucleotide synthesis using a standard procedure. After nucleotide deprotection, removal from the solid support (simultaneously with galactosamine acetate deprotection) yielded 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-flu[3,4-c]pyrrole-1,3(3aH)-dione(123). Trifluoroacetic acid (75 μL) was slowly added to a chilled solution (0°C) containing 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). The mixture was stirred overnight, allowing the solution to slowly return 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 using silica gel (gradient: hexane containing 20% ​​ethyl acetate to 100% ethyl acetate) yielded racemic(cis)5-benzyl-3a,6a-dimethyltetrahydro-1H-flu[3,4-c]pyrrole-1,3(3aH)-dione as a yellow oily substance (3.5 g, 56%).

[0375] Step 2. Preparation of racemic (cis) 1-benzyl-3,4-dimethylpyrrolidine-3,4-diyl)dimethanol (124) To a chilled solution (0°C) containing (3.5 g, 13.4 mmol) of (3aR,6aS)-5-benzyl-3a,6a-dimethyltetrahydro-1H-flu[3,4-c]pyrrole-1,3(3aH)-dione (50 mL) anhydrous diethyl ether, lithium aluminum hydride pellets (1.5 g, 40 mmol) were slowly added in three portions. The solution was stirred overnight, allowing it to return to room temperature as the ice bath melted. Once complete, the reaction mixture was cooled to 0°C and quenched very slowly with 1.5 mL of 5 M NaOH followed by 1.5 mL of water. The mixture was stirred for 30 minutes, then magnesium sulfate was added, and the mixture was filtered. The filtrate was concentrated to obtain 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) A methanol solution (10 mL) of ((3R,4S)-1-benzyl-3,4-dimethylpyrrolidine-3,4-diyl)dimethanol (10 g, 40 mmol) was mixed with 1 g of moistened 10% palladium activated carbon. This solution was vigorously stirred under a hydrogen atmosphere for 16 hours. Upon completion, the solution was filtered through Celite and concentrated to dryness to obtain racemic (cis) 3,4-dimethylpyrrolidine-3,4-diyl)dimethanol as a colorless solid (5.5 g, 86%).

[0377] Step 4. Preparation of racemi(cis)methyl 10-(3,4-bis(hydroxymethyl)-3,4-dimethylpyrrolidine-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 that used for compound 17. Yield: 1.8 g, 61%.

[0378] Step 5. Preparation of racemi(cis)methyl 10-(3-((bis(4-methoxyphenyl-)(phenyl)methoxy)-methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidine-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 that used for compound 18. Yield: 1.4 g, 42%.

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

[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 that 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 that 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 in automated oligonucleotide synthesis using a standard procedure. After nucleotide deprotection, removal from the solid support (simultaneously with galactosamine acetate deprotection) yielded GalNAc-oligonucleotide conjugate 145.

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

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

[0385] Step 2. Preparation of cyclopenta-3-en-1-ylmethanol (146-2) To a 1 L suspension of lithium aluminum hydride (15.2 g, 0.40 mol) in anhydrous ether at 0°C under nitrogen, a 300 mL solution of methylcyclopenta-3-enecarboxylate (50 g, 0.40 mol) in ether was added dropwise over 5 hours. The suspension was stirred overnight at room temperature. TLC indicated completion of the reaction. The reaction mixture was recooled to 0°C. The reaction mixture was quenched by adding a saturated solution of Na₂SO₄ (32 mL) dropwise. After the addition was complete, the mixture was stirred for a further 3 hours and filtered through a Celite pad. By evaporation of the solvent, cyclopenta-3-enylmethanol 146-2 (37.3 g, 95%) was obtained as a colorless liquid.

[0386] Step 3. Preparation of (6-oxabicyclo[3.1.0]hexane-3-yl)methanol 146-3 To a 150 mL solution of cyclopenta-3-enylmethanol (146-2) (4.0 g, 41 mmol) in dichloromethane at 0°C, 3-chloroperbenzoic acid (10 g, 45 mmol, 77% purity) was added in small amounts. The reaction mixture was stirred overnight. Dichloromethane (150 mL) was added. The organic matter was 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 organic matter was dried over MgSO4. By evaporation of the solvent, a mixture of cis and trans 6-oxabicyclo[3.1.0]hexane-3-ylmethanol (146-3) (2.6 g, 57%) was obtained as a yellow oily substance. 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). A methanol solution (20 mL) of 6-oxabicyclo[3.1.0]hexane-3-ylmethanol 146-3 (2.0 g, 17.6 mmol) at 0°C was purged with ammonia gas for 10 minutes. The reaction mixture was stirred overnight at room temperature. TLC showed that the reaction was incomplete. Methanol was removed, and NH3·H2O (50 mL) was added. This mixture was stirred at room temperature for one week. TLC confirmed the completion of the reaction. Water was removed by azeotrope with ethanol to obtain 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-dioxopyrrolidine-1-yl)12-methyldodecanediol (146-1) using the same procedure as described in the synthesis of 12-(2-(tert-butoxycarbonylamino)ethylamino)-12-oxododecanoate (3-2). Methyl 12-(2-hydroxy-4-(hydroxymethyl)cyclopentylamino)-12-oxododecanoate (146-5) was obtained as an off-white solid in 87.4% yield.

[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 that 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 that 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 that 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 a standard procedure. After nucleotide deprotection, removal from the solid support (simultaneously with galactosamine acetate deprotection) yielded 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 anhydrous (65.9 g, 302.5 mmol), and TEA (42 ml, 302.5 mmol) were stirred in DCM at room temperature for 16 hours. The organic matter was sequentially washed with 1 M HCl (twice), saturated NaHCO3 (twice), H2O, and brine, dried, and concentrated under vacuum to yield 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 THF solution) (37.5 mL, 75 mmol) was added dropwise. The reaction mixture was allowed to return to room temperature, stirred for 4 hours, and then quenched with saturated NH4Cl. The reaction mixture was extracted with ethyl acetate, washed with H2O and brine, dried in (Na2SO4), and concentrated under vacuum. Column chromatography 50:50 ethyl acetate / hexane purified the residue, yielding 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 N2. LiBH4 solution (48 ml, 96 mmol) was added dropwise, and the reaction mixture was stirred at room temperature for 48 hours. The reaction mixture was quenched with 1 M NaOH, THF was removed under vacuum, and the residue was extracted with SiO2 (4 × 100 ml). The organic matter was washed with H2O and brine, dried to (Na2SO4), and concentrated under vacuum. Purification of the residue by column chromatography (5% MeOH / DCM) yielded tert-butyl(2S,4R)-4-hydroxy-2-(hydroxymethyl)-2-methylpyrrolidine-1-carboxylate (135a) as the main product (8 g, 47%). The structure was assigned according to the references.

[0397] Step 4. Preparation of (3R,5S)-5-(hydroxymethyl)-5-methylpyrrolidine-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 HCl at room temperature, and HCl gas was applied for approximately 2 minutes. The reaction mixture was stirred for 1 hour, then concentrated in vacuum, and dried under high vacuum to quantitatively obtain (3R,5S)-5-(hydroxymethyl)-5-methylpyrrolidine-3-ol hydrochloride (136).

[0398] Step 5. Preparation of methyl 12-((2S,4R)-4-hydroxy-2-(hydroxymethyl)-2-methylpyrrolidine-1-yl)-12-oxododecanoate (137) (3R,5S)-5-(hydroxymethyl)-5-methylpyrrolidine-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 hours. The precipitate was removed by filtration, and the organic matter was washed with 1 M HCl (twice), saturated NaHCO3 (twice), H2O, and brine. After drying, the organic matter was concentrated in a vacuum and purified by column chromatography (5% MeOH / DCM) to obtain methyl 12-((2S,4R)-4-hydroxy-2-(hydroxymethyl)-2-methylpyrrolidine-1-yl)-12-oxododecanoate (137) (3.1g, 18.3%).

[0399] Step 6. Preparation of methyl 12-((2S,4R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)-methyl)-4-hydroxy-2-methylpyrrolidine-1-yl)-12-oxododecanoate (138) Methyl 12-((2S,4R)-4-hydroxy-2-(hydroxymethyl)-2-methylpyrrolidine-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 DC< at room temperature for 16 hours. The reaction mixture was concentrated under vacuum, and the residue was purified by column chromatography (5% MeOH / DCM, 0.1% TEA) to obtain methyl 12-((2S,4R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxy-2-methylpyrrolidine-1-yl)-12-oxododecanoate (138) (2.7 g, 45.5 mmol).

[0400] Scheme 23 [ka]

[0401] Step 7. Preparation of Compound 154-1 Triethylamine (10.4 mL, 74.6 mmol) was added dropwise to a 200 mL dichloromethane solution of N-(2-hydroxyethyl)phthalimide (4.80 g, 25.0 mmol) and 4,4'-dimethoxytrityl chloride (8.8 g, 26.0 mmol) at 0°C under nitrogen. The reaction mixture was stirred at room temperature for 3 hours. TLC indicated completion of the reaction. 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 ethanol (100 mL) containing hydrazine monohydrate (3.6 mL, 74 mmol) were stirred overnight at room temperature. TLC indicated completion of the reaction. The precipitate was removed by filtration. The filtrate was evaporated. The residue was taken off with ethyl acetate (100 mL). This organic solution was washed with 10% NaOH, water, and brine, and dried over MgSO4. By evaporation of the solvent, 2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethaneamine (154-2) was obtained as a yellow liquid (8.11 g, 89.3% yield in 2 steps). This was used in the next reaction without further purification.

[0403] Step 9. Preparation of Compound 154-3 To a solution containing L-threonine (1.19 g, 10.0 mmol) and NaHCO3 (2.3 g, 27 mmol) in water (20 mL) and dioxane (10 mL), dioxane (10 mL) containing 1-(2,5-dioxopyrrolidine-1-yl)12-methyldodecanedioate (146-1) (3.1 g, 9.1 mmol) was added dropwise. The reaction mixture was stirred overnight at room temperature. 4N HCl (10 mL) was added. The precipitate was collected by filtration and washed with water (3 × 10 mL). The solid was dried over P2O5 in a desiccator to obtain (2S,3R)-3-hydroxy-2-(12-methoxy-12-oxododecanamide)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-oxododecanamide)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 hours. Water (30 mL) was added. The organic layer was separated and washed with brine (2 × 30 mL). Following evaporation of the solvent, column chromatography (30% ethyl acetate / hexane - 50% ethyl acetate / hexane) was performed to obtain methyl 12-((2S,3R)-1-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethylamino)-3-hydroxy-1-oxobutan-2-ylamino)-12-oxododecanoate (154) as a waxy yellow semi-solid (2.60 g, 52.6%). 1 HNMR (400MHz, acetone-d6, ppm):δ 7.51 (t, J = 5.5 Hz, 1H), 7.45-7.49 (m, 2H), 7.28-7.36 (m, 6H), 7.21 (tt, J = 7.2, 1.2 Hz, 1H), 7.08 (d, J = 8.1 Hz, 1H), 6.88 (dt, J = 8.9, 2.5 Hz, 4H), 4.39 (dd, J = 8.2, 3.0 Hz, 1H), 4.20-4.27 (m, 1H), 3.78 (s,6H), 3.60 (s, 1H), 3.35-3.52 (m, 2H), 3.07-3.16 (m, 2H), 2.23-2.37 (m, 4H), 1.53-1.65(m, 4H), 1.23-1.36 (m, 12H), 1.10 (d, J = 6.4 Hz, 3H).

[0405] Scheme 24 [ka]

[0406] Step 11. Preparation of Compound 164-1 A suspension containing potassium t-butoxide (14.6 g, 130 mol) in THF (120 mL) / ether (360 mL) was prepared, to which methyltriphenylphosphonium bromide (46.6 g, 130 mmol) was added. This mixture was refluxed for 2 hours and then cooled to 0°C. Ether (50 mL) containing tert-butyl 2-formylpyrrolidine-1-carboxylate (13.0 g, 65.2 mmol) was added dropwise. The reaction mixture was stirred at 0°C and then quenched by adding water (250 mL). The organic layer was separated, and the aqueous component was extracted with ether (250 mL). The combined extracts were dried over MgSO4. Following evaporation of the solvent, column chromatography purification (5% ethyl acetate / hexane) yielded 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), AD-mix-β (47.4 g) and methanesulfonamide (2.89 g, 30.4 mmol) were added. This 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. This reaction mixture was stirred at room temperature overnight. This reaction mixture was cooled to 0°C. Sodium thiosulfate pentahydrate (96 g, 387 mmol) was added and the temperature was returned to room temperature. Water (700 mL) was added and the mixture was extracted with ethyl acetate (500 mL). The extract was then rinsed with water (2 × 5 The samples were washed with 0 mL of methanol and brine (50 mL) and dried over MgSO4. Following evaporation of the solvent, column chromatography (2% methanol / dichloromethane - 7% methanol / dichloromethane) was performed, yielding tert-butyl 3-(1,2-dihydroxyethyl)pyrrolidine-1-carboxylate (164-2) (5.4 g, 77%) as a light brown oily substance.

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

[0409] Step 14 Preparation of Compound 164-4 To an aqueous solution (30 mL) of 1-(pyrrolidine-3-yl)ethane-1,2-diol hydrochloride (164-2) (2.0 g, 12 mmol), NaHCO3 (3.7 g, 44 mmol) was added in small amounts. Then dioxane (20 mL) was added. To the above solution, dioxane (30 mL) containing 1-(2,5-dioxopyrrolidine-1-yl)12-methyldodecanedioate (146-1) (3.7 g, 11 mmol) was added. This reaction mixture was stirred overnight. This was extracted with ethyl acetate (3 × 100 mL). The combined extract was washed with 0.5 N HCl (50 mL) and brine (50 mL) and dried over MgSO4.

[0410] Step 15. Preparation of Compound 164 This substance was prepared from methyl 12-(3-(1,2-dihydroxyethyl)pyrrolidine-1-yl)-12-oxododecanoate (164-4) and 4,4-dimethoxytrityl chloride (1 equivalent) using the same procedure as described in the synthesis of 2-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)isoindoline-1,3-dione (138). The product was purified by column chromatography (1.5% methanol / dichloromethane). Methyl 12-(3-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)-1-hydroxyethyl)pyrrolidine-1-yl)-12-oxododecanoate (164) was obtained as a yellow oil in 51% yield. 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 100 mL solution of tert-butyl 2-aminoethyl carbamate (2.88 g, 18.0 mmol) and triethylamine (2.98 g, 29.4 mmol) in dichloromethane, 50 mL of dichloromethane containing 1-(2,5-dioxopyrrolidine-1-yl)12-methyldodecanedioate (146-1) (5.09 g, 14.9 mmol) was added dropwise at room temperature. The reaction mixture was stirred overnight, and TLC indicated completion of the reaction. 100 mL of brine was added, and the organic layer was separated. The organic layer was washed with 0.5 N HCl (150 mL) and brine (2 × 100 mL) and dried over MgSO4. Upon evaporation of the solvent, pure methyl 12-(2-(tert-butoxycarbonylamino)ethylamino)-12-oxododecanoate (170-1) (5.85 g, 100%) was obtained as a white solid.

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

[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·H2O (2.55 g, 60.8 mmol) were mixed in THF (50 mL) and water (50 mL) and stirred overnight. TLC indicated completion of the reaction. The THF was evaporated, and the aqueous solution was acidified to pH=1 with 1N HCl. This was extracted with ethyl acetate (5 × 50 mL). The combined extracts were dried over MgSO4. By evaporation of the solvent, (S)-2,2-dimethyl-1,3-dioxolane-4-carboxylic acid (170-3) (2.93 g, 64.3%) was obtained 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-dioxolan-4-carboxylic acid (170-3) and N-hydroxysuccinimid using the same procedure as described in the synthesis of 1-(2,5-dioxopyrrolidine-1-yl)12-methyldodecanedioate (146-1). (S)-2,5-dioxopyrrolidine-1-yl2,2-dimethyl-1,3-dioxolan-4-carboxylate (170-4) was obtained as a white solid in 86% yield.

[0416] Step 20. Preparation of Compound 170-5 Dichloromethane (25 mL) containing triethylamine (6 mL, 43.0 mmol) was added to a dichloromethane suspension (100 mL) of methyl 12-(2-aminoethylamino)-12-oxododecanoate hydrochloride (170-2) (14.4 mmol) and (S)-2,5-dioxopyrrolidine-1-yl 2,2-dimethyl-1,3-dioxolane-4-carboxylate (170-4) (3.80 g, 15.6 mmol) over 4 hours at 0°C. The reaction mixture was then stirred overnight at room temperature. LC-MS showed that the starting material 170-2 was completely converted. The organic layer was washed with brine (50 mL), 1N HCl (50 mL), and brine (50 mL), dried over MgSO4, and concentrated to dryness to obtain (S)-methyl-12-(2-(2,2-dimethyl-1,3-dioxolane-4-carboxamide)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-carboxamide)ethylamino)-12-oxododecanoate (170-5) (5.93 g, 14.3 mmol), one drop of concentrated sulfuric acid was added. This was refluxed for 6 hours and then cooled to room temperature. The solid was collected by filtration and washed twice with cold methanol. This solid was dried in air (3.32 g). A second collectible (0.42 g) was obtained from the mother liquor to obtain (S)-methyl-12-(2-(2,3-dihydroxypropanamide)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 Under nitrogen, 4,4'-dimethoxytrityl chloride (2.84 g, 8.38 mmol) was added in a single addition to a 57.5 mL solution of anhydrous pyridine containing (S)-methyl-12-(2-(2,3-dihydroxypropanamide)ethylamino)-12-oxododecanoate (170-6) (2.99 g, 7.99 mmol). The reaction mixture was stirred at room temperature for 2 days. The reaction mixture was quenched by adding methanol (5 mL). The pyridine was evaporated. Toluene was added and then evaporated. This was repeated three times. Water (100 mL) was added and extracted with ethyl acetate (5 × 250 mL). The extracts were combined and dried over MgSO4. Following evaporation of the solvent, column chromatography (1% methanol / dichloromethane - 3% methanol / dichloromethane) yielded (S)-methyl 12-(2-(3-(bis(4-methoxyphenyl)(phenyl)methoxy)-2-hydroxypropanamide)-ethylamino)-12-oxododecanoate (170) (1.70 g, 31.4%) as a viscous oily substance. 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 that used for compound 18.

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

[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 mixture was stirred for 1.5 hours. The solvent was removed under vacuum, and the residue was placed on MTBE and refrigerated overnight. The resulting precipitate was collected by filtration, washed with ice-cold MTBE, and dried under high vacuum to obtain 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-dimethylpyrrolidine-1-yl)-10-oxodecanamide)dodecanoate (129) Methyl 12-aminododecanoate (132) (778 mg, 3.1 mmol) was racemic (cis) 10-(3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidine-1-yl)-10-oxodecanoate lithium (128) (2 g, 3.1 mmol), HBTU (1.2 g, 3.1 mmol), and TEA (1.4 mL, 10 mmol) were stirred overnight (O / N) at room temperature in DCM. The precipitate was removed by filtration, the filtrate was concentrated under vacuum, and the residue was purified by column chromatography (5% MeOH, DCM). TLC showed two spots of the same mass moving in close proximity. When these were assigned as geometric isomers and pooled together, methyl 12-(12-(10-((3R,4S)-3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidine-1-yl)-10-oxodecanamide)dodecanoamide)dodecanoate (129) was obtained in a quantitative manner.

[0425] Step 3. Preparation of racemic (cis) 12-(12-(10-(-3-((bis(4-methoxyphenyl)(phenyl)-methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidine-1-yl)-10-oxodecanamide)-dodecanoate lithium (130) Racemi(cis)methyl 12-(12-(10-(3-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidine-1-yl)-10-oxodecanamide)dodecanoate (129) (3.1 mmol) was stirred overnight at room temperature in THF:H2O (50:50) containing LiOH (88 mg, 3.7 mmol). The reaction was confirmed by TLC, and the THF was removed under vacuum. When this aqueous solution was frozen in liquid N2 and freeze-dried for 48 hours, racemi(cis)12-(12-(10-(3-((bis(4-methoxyphenyl)(phenyl)-methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidine-1-yl)-10-oxodecanamide)-dodecanoate lithium (130) was quantitatively obtained.

[0426] Step 4. Preparation of Conjugate 176 Conjugate 176 was prepared from compounds 24 and 130 using the same procedure as that 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 overnight at room temperature in a DCM. This mixture was concentrated under vacuum and purified by column chromatography to obtain 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 that used for compound 14. This compound was used crudely in the subsequent reaction, and the yield was quantitatively determined.

[0430] Step 3. Preparation of Conjugate 179 Conjugate 179 was prepared from compounds 18 and 81 using the same procedure as that 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 that 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 that 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 CH2Cl2 solution (600 mL) of pentaethylene glycol (35 g, 147 mmol), TEA (41 mL, 294 mmol), and trimethylamine-HCl (1.4 g, 14.7 mmol) was treated with tosyl chloride (29.4 g, 154 mmol). After stirring (18 hours), the reaction mixture was washed with H2O-brine (1:1), dried (MgSO4), filtered, concentrated, and subjected to chromatography to obtain 82 (24.6 g, 43%) as a pale yellow oil. Rf 0.8 (10% CH3OH-CH2Cl2).

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

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

[0439] Step 4. Preparation of Compound 85 A 120 mL solution of HCl containing 84 (11.16 g, 18.8 mmol) and Pd / C (1.1 g, 10% - wet support) was treated with TFA (4.32 mL, 56.5 mmol) and purged with H2. After vigorous stirring (4.5 hours), the reaction was purged with N2, filtered through Celite, and concentrated. This crude product was subjected to chromatography to obtain 85 (5.77 g, 45%) as a colorless foam. Rf 0.5 (10% CH3OH-CH2Cl2).

[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 that 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 that 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 that used for compound 92.

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

[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)ethane-1-ol (86) Sodium azide (10g, 154mmol) was added to an aqueous solution (200mL) of 2-(2-(2-chloroethoxy)ethoxy)ethane-1-ol (13g, 77mmol). The reaction mixture was heated at 100°C for 18 hours. The reaction mixture was cooled to room temperature, poured into a 1L separatory funnel, and extracted with dichloromethane (3×200mL). The combined dichloromethane extract was dried over magnesium sulfate, filtered, and concentrated to dryness to obtain 2-(2-(2-azidoethoxy)ethoxy)ethane-1-ol as a colorless oil (11.7g).

[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 that 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 that used for compound 85. Yield: 7.63 g, 65%.

[0451] Step 4. Preparation of Compound 89 A CH2Cl2 solution (50 mL) of 88 (2 g, 3.38 mmol) and Z-glutamic acid (427 mg, 1.52 mmol) was treated with HBTU (1.41 g, 3.7 mmol) and Hünig base (1.77 mL, 10.1 mmol). After stirring (18 hours), the mixture was concentrated and subjected to chromatography to obtain 89 (871 mg, 48%) as a colorless foam. Rf 0.5 (10% CH3OH-CH2Cl2).

[0452] Step 5. Preparation of Compound 90 A 10 mL siRNA solution of 89 (870 mg, 0.72 mmol) and Pd / C (90 mg, 10% - moistened support) was treated with TFA (84 μL, 1.1 mmol) and purged with H2. After vigorous stirring (2 hours), the reaction was purged with N2, filtered through Celite, and concentrated. This crude material was used without further processing to obtain 90 (850 mg, quantitative) as a colorless foam. Rf 0.25 (10% CH3OH-CH2Cl2) .

[0453] Step 6. Preparation of Compound 91 A 10 mL solution of 90 (850 mg, 0.72 mmol) and Z-glutamic acid (91 mg, 0.32 mmol) in CH2Cl2 was treated with HBTU (300 mg, 0.79 mmol) and Hünig base (502 μL, 2.9 mmol). After stirring (1.5 hours), the mixture was diluted with CH2Cl2, washed with NaHCO3 (saturated aqueous solution), dried (MgSO4), filtered, and concentrated. This crude substance was subjected to chromatography to obtain 91 (590 mg, 76%) as a colorless foam. Rf 0.5 (10% CH3OH-CH2Cl2).

[0454] Step 7. Preparation of Compound 92 A CH3OH solution (30 mL) of 91 (590 mg, 0.25 mmol) and Pd / C (100 mg, 10% - moistened support) was treated with TFA (29 μL, 0.37 mmol) and purged with H2. After stirring (3 hours), the mixture was purged with N2, filtered through Celite, and concentrated. This crude material was used without further processing to obtain 92 (600 mg, quantitative) as a colorless foam. Rf 0.1 (10% CH3OH-CH2Cl2).

[0455] Step 8. Preparation of Compound 101 Compound 101 was prepared from (R)-2-((2-oxo-2-phenyl-112-ethyl)amino)hexanediol (2.51 g, 8.6 mmol) and 9 (11 g, 17.2 mmol) using the same procedure as that 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 that 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)hexanediol (265 mg, 0.9 mmol) and compound 102 (2.1 g, 1.8 mmol) using the same procedure as that used for compound 91. Yield: (560 mg, 24%).

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

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

[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)ethane-1-ol (86a). Sodium azide (10g, 154mmol) was added to an aqueous solution (200mL) of 2-(2-(2-chloroethoxy)ethoxy)ethane-1-ol (13g, 77mmol). The reaction mixture was heated at 100°C for 18 hours. The reaction mixture was cooled to room temperature, poured into a 1L separatory funnel, and extracted with dichloromethane (3×200mL). The combined dichloromethane extract was dried over magnesium sulfate, filtered, and concentrated to dryness to obtain 2-(2-(2-azidoethoxy)ethoxy)ethane-1-ol as a colorless oil (11.7g).

[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 that 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 that used for compound 85. Yield: 7.63 g, 65%.

[0464] Step 4. Preparation of compound 89a A CH2Cl2 solution (50 mL) of 88a (2 g, 3.38 mmol) and ZL-glutamic acid (427 mg, 1.52 mmol) was treated with HBTU (1.41 g, 3.7 mmol) and Hünig base (1.77 mL, 10.1 mmol). After stirring (18 hours), the mixture was concentrated and subjected to chromatography to obtain 89a (871 mg, 48%) as a colorless foam. Rf 0.5 (10% CH3OH-CH2Cl2).

[0465] Step 5. Preparation of compound 90a A 10 mL siRNA solution of 89a (870 mg, 0.72 mmol) and Pd / C (90 mg, 10% - moistened support) was treated with TFA (84 μL, 1.1 mmol) and purged with H2. After vigorous stirring (2 hours), the reaction was purged with N2, filtered through Celite, and concentrated. This crude material was used without further processing to obtain 90a (850 mg, quantitative) as a colorless foam. Rf 0.25 (10% CH3OH-CH2Cl2).

[0466] Step 6. Preparation of compound 91a A 10 mL solution of 90a (850 mg, 0.72 mmol) and Z-glutamic acid (91 mg, 0.32 mmol) in CH2Cl2 was treated with HBTU (300 mg, 0.79 mmol) and Hünig base (502 μL, 2.9 mmol). After stirring (1.5 hours), the mixture was diluted with CH2Cl2, washed with NaHCO3 (saturated aqueous solution), dried (MgSO4), filtered, and concentrated. This crude substance was subjected to chromatography to obtain 91a (590 mg, 76%) as a colorless foam. Rf 0.5 (10% CH3OH-CH2Cl2).

[0467] Step 7. Preparation of compound 92a A CH3OH solution (30 mL) of 91a (590 mg, 0.25 mmol) and Pd / C (100 mg, 10% - moistened support) was treated with TFA (29 μL, 0.37 mmol) and purged with H2. After stirring (3 hours), the mixture was purged with N2, filtered through Celite, and concentrated. This crude material was used without further processing to obtain 92a (600 mg, quantitative) as a colorless foam. Rf 0.1 (10% CH3OH-CH2Cl2).

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

[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)ethane-1-ol (86b). Sodium azide (10g, 154mmol) was added to an aqueous solution (200mL) of 2-(2-(2-chloroethoxy)ethoxy)ethane-1-ol (13g, 77mmol). The reaction mixture was heated at 100°C for 18 hours. The reaction mixture was cooled to room temperature, poured into a 1L separatory funnel, and extracted with dichloromethane (3×200mL). The combined dichloromethane extract was dried over magnesium sulfate, filtered, and concentrated to dryness to obtain 2-(2-(2-azidoethoxy)ethoxy)ethane-1-ol as a colorless oil (11.7g).

[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 that 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 that used for compound 85. Yield: 7.63 g, 65%.

[0473] Step 4. Preparation of compound 89b A CH2Cl2 solution (50 mL) of 88b (2 g, 3.38 mmol) and racemic Z-glutamic acid (427 mg, 1.52 mmol) was treated with HBTU (1.41 g, 3.7 mmol) and Hünig base (1.77 mL, 10.1 mmol). After stirring (18 hours), the mixture was concentrated and subjected to chromatography to obtain 89b (871 mg, 48%) as a colorless foam. Rf 0.5 (10% CH3OH-CH2Cl2).

[0474] Step 5. Preparation of compound 90b A 10 mL siRNA solution of 89b (870 mg, 0.72 mmol) and Pd / C (90 mg, 10% - moistened support) was treated with TFA (84 μL, 1.1 mmol) and purged with H2. After vigorous stirring (2 hours), the reaction was purged with N2, filtered through Celite, and concentrated. This crude material was used without further processing to obtain 90b (850 mg, quantitative) as a colorless foam. Rf 0.25 (10% CH3OH-CH2Cl2).

[0475] Step 6. Preparation of compound 91b A 10 mL solution of 90b (850 mg, 0.72 mmol) and Z-glutamic acid (91 mg, 0.32 mmol) in CH2Cl2 was treated with HBTU (300 mg, 0.79 mmol) and Hünig base (502 μL, 2.9 mmol). After stirring (1.5 hours), the mixture was diluted with CH2Cl2, washed with NaHCO3 (saturated aqueous solution), dried (MgSO4), filtered, and concentrated. This crude substance was subjected to chromatography to obtain 91b (590 mg, 76%) as a colorless foam. Rf 0.5 (10% CH3OH-CH2Cl2).

[0476] Step 7. Preparation of compound 92b A CH3OH solution (30 mL) of 91b (590 mg, 0.25 mmol) and Pd / C (100 mg, 10% - moistened support) was treated with TFA (29 μL, 0.37 mmol) and purged with H2. After stirring (3 hours), the mixture was purged with N2, filtered through Celite, and concentrated. This crude material was used without further processing to obtain 92b (600 mg, quantitative) as a colorless foam. Rf 0.1 (10% CH3OH-CH2Cl2).

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

[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)ethane-1-ol (86c). Sodium azide (10g, 154mmol) was added to an aqueous solution (200mL) of 2-(2-(2-chloroethoxy)ethoxy)ethane-1-ol (13g, 77mmol). The reaction mixture was heated at 100°C for 18 hours. The reaction mixture was cooled to room temperature, poured into a 1L separatory funnel, and extracted with dichloromethane (3×200mL). The combined dichloromethane extract was dried over magnesium sulfate, filtered, and concentrated to dryness to obtain 2-(2-(2-azidoethoxy)ethoxy)ethane-1-ol as a colorless oil (11.7g).

[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 that used for compound 84. Yield: 10 g, 77%.

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

[0482] Step 4. Preparation of compound 89c A 50 mL solution of 88c (2 g, 3.38 mmol) and racemic Z-glutamic acid (427 mg, 1.52 mmol) in CH2Cl2 was treated with HBTU (1.41 g, 3.7 mmol) and Hünig base (1.77 mL, 10.1 mmol). After stirring for 18 hours, the mixture was concentrated and subjected to chromatography to obtain 89c (871 mg, 48%) as a colorless foam. Rf 0.5 (10% CH3OH-CH2Cl2).

[0483] Step 5. Preparation of compound 90c. A 10 mL siRNA solution of 89c (870 mg, 0.72 mmol) and Pd / C (90 mg, 10% - moistened support) was treated with TFA (84 μL, 1.1 mmol) and purged with H2. After vigorous stirring (2 hours), the reaction was purged with N2, filtered through Celite, and concentrated. This crude material was used without further processing to obtain 90c (850 mg, quantitative) as a colorless foam. Rf 0.25 (10% CH3OH-CH2Cl2).

[0484] Step 6. Preparation of compound 91c A 10 mL solution of 90c (850 mg, 0.72 mmol) and Z-glutamic acid (91 mg, 0.32 mmol) in CH2Cl2 was treated with HBTU (300 mg, 0.79 mmol) and Hünig base (502 μL, 2.9 mmol). After stirring (1.5 hours), the mixture was diluted with CH2Cl2, washed with NaHCO3 (saturated aqueous solution), dried (MgSO4), filtered, and concentrated. This crude substance was subjected to chromatography to obtain 91c (590 mg, 76%) as a colorless foam. Rf 0.5 (10% CH3OH-CH2Cl2).

[0485] Step 7. Preparation of compound 92c A CH3OH solution (30 mL) of 91c (590 mg, 0.25 mmol) and Pd / C (100 mg, 10% - moistened support) was treated with TFA (29 μL, 0.37 mmol) and purged with H2. After stirring (3 hours), the mixture was purged with N2, filtered through Celite, and concentrated. This crude material was used without further processing to obtain 92c (600 mg, quantitative) as a colorless foam. Rf 0.1 (10% CH3OH-CH2Cl2).

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

[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 that used for compound 69.

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

[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 that used for compound 1.

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

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

[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)acetamide)isophthalate(105) A 50 mL solution of DMF containing 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) was stirred overnight at room temperature. Upon completion, the reaction mixture was diluted with ethyl acetate (250 mL) and washed with 1 M HCl (2 × 100 mL), saturated sodium bicarbonate (1 × 100 mL), and brine (2 × 100 mL), respectively. The mixture was dried over magnesium sulfate, filtered, concentrated to dryness, and then dimethyl 5-(2-((2-oxo-2-phenyl-1λ 2 -ethyl)amino)acetamide)isophthalate was obtained as a colorless solid (7.2 g, 79%).

[0496] Step 2. 5-(2-((2-oxo-2-phenyl-1λ 2 Preparation of ethyl)amino)acetamide)isophthalic acid (106) Methanol (25 mL) and THF (25 mL) are mixed with methyl 5-(2-((2-oxo-2-phenyl-1λ 2 A solution containing ethyl)amino)acetamido)isophthalate (7.2 g) was mixed with 1 M NaOH (25 mL). This solution was stirred at room temperature for 2 hours, then concentrated to remove THF and MeOH. The remaining aqueous solution was diluted with water (75 mL), cooled in an ice bath, and acidified to pH=1 with 6 M HCl. This solid was filtered and washed with water (3 × 100 mL). This solid was freeze-dried to obtain 5-(2-((2-oxo-2-phenyl-1λ 2 -ethyl)amino)acetamide)isophthalic acid was obtained (6.9 g, quantitative).

[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 that 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 that used for compound 100. Yield: 920 mg, quantitative.

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

[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)acetamide)isophthalate (105a) A 50 mL solution of DMF containing 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) was stirred overnight at room temperature. Upon completion, the reaction mixture was diluted with ethyl acetate (250 mL) and washed with 1 M HCl (2 × 100 mL), saturated sodium bicarbonate (1 × 100 mL), and brine (2 × 100 mL), respectively. The mixture was dried over magnesium sulfate, filtered, concentrated to dryness, and then dimethyl 5-(2-((2-oxo-2-phenyl-1λ 2 -ethyl)amino)acetamide)isophthalate was obtained as a colorless solid (7.2 g, 79%).

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

[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 that 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 that used for compound 100. Yield: 920 mg, quantitative.

[0509] Step 7. Preparation of conjugates 212a and 215a. Using the same procedure as that 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 that used for compound 89. Yield: 2.21 g, 49%.

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

[0513] Step 3. Preparation of Compound 113 Using the same procedure as that used for compound 91, compound 112 (1.08 g, 0.88 mmol) and (2-oxo-2-phenyl-1λ) were prepared. 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 that 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 that 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 that used for compound 112. Yield: 3.23 g, 39%.

[0517] Step 7. Preparation of Compound 117 Using the same procedure as that used for compound 95, compound 116 (3.23 g, 2.45 mmol) and (2-oxo-2-phenyl-1λ) were prepared. 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 that used for compound 96. Yield: 2.02 g, 91%.

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

[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 that used for compound 89. Yield: 2.21 g, 49%.

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

[0523] Step 3. Preparation of compound 113a Using the same procedure as that used for compound 91, compound 112a (1.08 g, 0.88 mmol) and (2-oxo-2-phenyl-1λ) were prepared. 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 that 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 that 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 that used for compound 11a. Yield: 3.23 g, 39%.

[0527] Step 7. Preparation of compound 117a Using the same procedure as that used for compound 95, compound 116a (3.23 g, 2.45 mmol) and (2-oxo-2-phenyl-1λ) were prepared. 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 that used for compound 96. Yield: 2.02 g, 91%.

[0529] Step 9. Preparation of conjugates 218a and 221a. Using the same procedure as that 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 that 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 that 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-aminoacetamide)isophthalic acid (106) (560 mg, 1.5 mmol) and 9 (2.24 g, 3.6 mmol) using the same procedure as used for compound 89. Yield: 1.6 g, 80%.

[0536] Step 2: Preparation of Compound 226 Compound 226 was prepared in the same manner as compound 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 compound 89. Yield: 471 mg, 45%.

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

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

[0540] Step 6 Preparation of Compound 231 Conjugate 231 was prepared using the same procedure as that 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-aminoacetamide)isophthalic acid (106) (560 mg, 1.5 mmol) and 9 (2.24 g, 3.6 mmol) using the same procedure as used for compound 89. Yield: 1.6 g, 80%.

[0543] Step 2: Preparation of Compound 226a Compound 226a was prepared using the same method as in 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 in compound 89. Yield: 471 mg, 45%.

[0545] Step 4 Preparation of Compound 228a Compound 228a was prepared using the same method as in 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) using the same method as for compound 89. The yield was 365 mg, 66%.

[0547] Step 6 Preparation of Compound 231a Conjugate 231a was prepared using the same procedure as that used for compound 1.

[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-aminoacetamide)isophthalic acid (106) (560 mg, 1.5 mmol) and 9 (2.24 g, 3.6 mmol) using the same procedure as used for compound 89. Yield: 1.6 g, 80%.

[0550] Step 2: Preparation of compound 226b Compound 226b was prepared using the same method as in 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 in compound 89. Yield: 471 mg, 45%.

[0552] Step 4 Preparation of compound 228b Compound 228b was prepared using the same method as in 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) using the same method as for compound 89. The yield was 365 mg, 66%.

[0554] Step 6 Preparation of Compound 231b Conjugate 231b was prepared using the same procedure as that used for compound 1.

[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 that 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 that 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 that 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 that used for Compound 1.

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

[0562] Chemically modified HBV siRNAs conjugated to GalNAc ligands, as listed in Table 1, were tested for in vivo activity in established HBV infection mouse models. In the AAV-HBV1.2 C57BL / 6 mouse model, injection of an adeno-associated virus (AAV) vector encoding a sequence exceeding the HBV genome length resulted in stable and sustained HBV expression, leading to hepatic expression of HBV RNA and protein, as well as secretion of viral and subviral particles into the bloodstream.

[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 the Canadian Council on Animal Care (CCAC) Guidelines on Good Animal Practices and were authorized by the local Institutional Animal Care and Use Committee (IACUC).

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

[0565] siRNA treatment: A single dose of 3 mg / kg of HBV siRNA conjugate was administered to a mouse group (typically n=5) by subcutaneous injection into the scapula on day 0 (one dose per animal). One group of animals that received only vehicle (saline) was used as a control.

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

[0567] Analysis: HBsAg levels in serum samples were determined using the BioRad EIA GS HBsAg 3.0 kit (BioRad, catalog number 32591) according to the manufacturer's instructions. Group-mean HBsAg levels at individual time points were determined using pooled serum from each treatment group. The data were analyzed and expressed as HBsAg levels relative to pre-treatment baseline (percentage of day 0).

[0568] Results: The results of testing each of the chemically modified HBV siRNAs listed in Table 1 are shown in Table 2. The values ​​represent the percentage (%) of HBsAg levels (relative to baseline on day 0) at 7, 14, 21, 28, 42, 49, 56, and 70 days post-treatment. [Table 1-1] [Table 1-2] 2'-O-methylnucleotide = lowercase; 2'-fluoronucleotide = 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 linker [ka]

[0570] Step 1. Preparation of racemic(cis)5-benzyl-3a,6a-dimethyltetrahydro-1H-flu[3,4-c]pyrrole-1,3(3aH)-dione(301). Trifluoroacetic acid (75 μL) was slowly added to a chilled solution (0°C) containing 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). The mixture was stirred overnight, allowing the solution to slowly return 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 silica gel column chromatography (gradient: hexane containing 20% ​​ethyl acetate to 100% ethyl acetate) yielded (3aR,6aS)-5-benzyl-3a,6a-dimethyltetrahydro-1H-flu[3,4-c]pyrrole-1,3(3aH)-dione as a yellow oily substance (3.5 g, 56%).

[0571] Step 2. Preparation of racemic (cis)(1-benzyl-3,4-dimethylpyrrolidine-3,4-diyl)dimethanol (302) To a chilled solution (0°C) containing (3.5 g, 13.4 mmol) of (3aR,6aS)-5-benzyl-3a,6a-dimethyltetrahydro-1H-flu[3,4-c]pyrrole-1,3(3aH)-dione (50 mL) anhydrous diethyl ether, lithium aluminum hydride pellets (1.5 g, 40 mmol) were slowly added in three portions. The solution was stirred overnight, allowing it to return to room temperature as the ice bath melted. Once complete, the reaction mixture was cooled to 0°C and quenched very slowly with 1.5 mL of 5 M NaOH followed by 1.5 mL of water. The mixture was stirred for 30 minutes, then magnesium sulfate was added, and the mixture was filtered. The filtrate was concentrated to obtain ((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). A methanol solution (10 mL) of ((3R,4S)-1-benzyl-3,4-dimethylpyrrolidine-3,4-diyl)dimethanol (10 g, 40 mmol) was mixed with 1 g of moistened 10% palladium activated carbon. This solution was vigorously stirred under a hydrogen atmosphere for 16 hours. Upon completion, the solution was filtered through Celite and concentrated to dryness to obtain ((3R,4S)-3,4-dimethylpyrrolidine-3,4-diyl)dimethanol as a colorless solid (5.5 g, 86%).

[0573] Step 4. Preparation of racemi(cis)methyl 10-(3,4-bis(hydroxymethyl)-3,4-dimethylpyrrolidine-1-yl)-10-oxodecanoate (304) A CH2Cl2 solution (100 mL) of 3 (1.3 g, 8.2 mmol) and monomethyl sebacate (1.8 g, 8.2 mmol) was treated with HBTU (3.41 g, 9.02 mmol) and Hünig base (5.71 mL, 32.8 mmol). After stirring overnight, the mixture was washed with NaHCO3 (saturated aqueous solution), water, and brine, then dried (MgSO4), filtered, and concentrated. This crude substance was subjected to chromatography (gradient: 0% CH3OH-CH2Cl2 to 20%) to obtain 4 (1.8 g, 61%).

[0574] Step 5. Preparation of racemi(cis)methyl 10-(3-((bis(4-methoxyphenyl)(phenyl)-methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidine-1-yl)-10-oxodecanoate (305) A pyridine solution (180 mL) of 304 (1.8 g, 5.0 mmol) and 4,4'-dimethoxytrityl chloride (1.7 g, 5.0 mmol) was stirred overnight. The pyridine was then removed under reduced pressure, and the crude substance was subjected to chromatography (gradient: 0% CH3OH-CH2Cl2 to 10%) to obtain 5 (1.4 g, 42%) as a yellow oily substance.

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

[0576] Scheme 51 Synthesis of hyperacetylated galactosamine (307) [ka] Pyridine (1.5 L) containing D-galactosamine hydrochloride (250 g, 1.16 mol) 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 solid was filtered off, and the mixture was frozen over liquid nitrogen and then freeze-dried for 5 days to obtain peracetylated galactosamine (307) (369.4 g, 82%) as a white solid. Rf (0.58, 10% MeOH-CH2Cl2).

[0577] Scheme 52 Synthesis of GalNAc monomer [ka]

[0578] Step 1: Preparation of Compound 309 An aqueous solution (1 L) of 2-[2-(2-chloroethoxy)]ethanol (308) (100 g, 593 mmol) was treated with NaN3 (77 g, 1.19 mol) and heated (90°C). After stirring (72 hours), the solution was cooled (room temperature) and extracted with CH2Cl2 (4 times). The combined organic matter was washed with brine, dried (MgSO4), filtered, concentrated, and used without further treatment. Compound 9 (88.9 g, 86%) was obtained as a pale yellow oily substance.

[0579] Step 2 Preparation of Compound 310 A 1,2-dichloroethane solution (40 mL) of 7 (2.76 g, 7.1 mmol) and 309 (1.37 g, 7.8 mmol) was treated with Sc(OTf)3 (174 mg, 0.36 mmol) and heated (85°C). After stirring (2 hours), the mixture was cooled (room temperature), quenched by adding TEA (4 mL), and concentrated. This crude substance was subjected to chromatography to obtain 310 (3.03 g, 85%) as a pale yellow foam.

[0580] Step 3 Preparation of Compound 311 A 30 mL ethyl phosphate solution of 310 (3.02 g, 5.99 mmol) and Pd / C (300 mg, 10% Pd-loaded - moistened support) was treated with TFA (576 μL, 7.5 mmol). The reaction mixture was purged with hydrogen gas (45 minutes), then purged with nitrogen gas (10 minutes), and then filtered through Celite. The filtrate was concentrated and then subjected to chromatography to obtain 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)acetamide)isophthalate (312) A 50 mL solution of DMF containing 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) was stirred overnight at room temperature. Upon completion, the reaction mixture was diluted with ethyl acetate (250 mL) and washed with 1 M HCl (2 × 100 mL), saturated sodium bicarbonate (1 × 100 mL), and brine (2 × 100 mL), respectively. The mixture was dried over magnesium sulfate, filtered, concentrated to dryness, and then dimethyl 5-(2-((2-oxo-2-phenyl-1λ 2 Ethyl)amino)acetamide)isophthalate was obtained as a colorless solid (7.2 g, 79%).

[0583] Step 2. 5-(2-((2-oxo-2-phenyl-1λ 2 Preparation of ethyl(amino)acetamide)isophthalic acid (313) Methanol (25 mL) and THF (25 mL) are mixed with methyl 5-(2-((2-oxo-2-phenyl-1λ 2A solution containing ethyl)amino)acetamido)isophthalate (7.2 g) was mixed with 1 M NaOH (25 mL). This solution was stirred at room temperature for 2 hours, then concentrated to remove THF and MeOH. The remaining aqueous solution was diluted with water (75 mL), cooled in an ice bath, and acidified to pH=1 with 6 M HCl. This solid was filtered and washed with water (3 × 100 mL). This solid was freeze-dried to obtain 5-(2-((2-oxo-2-phenyl-1λ 2 -ethyl)amino)acetamide)isophthalic acid was obtained (6.9 g, quantitative).

[0584] Scheme 54: Preparation of tetramers [ka] [ka]

[0585] Step 1: Preparation of Compound 314 CH2Cl2 solutions (150 mL) of 313 (2.09 g, 5.6 mmol) and 311 (8.34 g, 14.07 mmol) were treated with HBTU (6.4 g, 16.9 mmol) and Hünig base (7.35 mL, 42.2 mmol). After stirring (overnight), the reaction mixture was poured into NaHCO3 (saturated aqueous solution), then washed with water and brine, dried (MgSO4), filtered, and concentrated. This crude substance was subjected to chromatography (CH3OH-CH2Cl2 gradient of 1-12%) to obtain 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% loaded - wet support), and trifluoroacetic acid (308 μL, 4 mmol) were purged with H2. After stirring under H2 (overnight), the mixture was purged with N2 (15-20 minutes), then filtered through Celite and concentrated. This crude substance was subjected to chromatography to obtain compound 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 compound 314. Yield: 1.66 g, 60%.

[0588] Step 4 Preparation of Compound 317 Compound 317 was prepared using the same method as compound 315. Yield: 1.65 g, quantitative analysis.

[0589] Scheme 55 Preparation of a complete conjugate [ka]

[0590] Step 1: Preparation of Compound 318 A 100 mL solution of 317 (1.91 g, 0.75 mmol) in CH2Cl2 was first treated with Hünig 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 NaHCO3 (saturated aqueous solution), then washed with water and brine, dried (MgSO4), filtered, and concentrated. This crude substance was subjected to chromatography to obtain 318 (1.19 g, 52%) as a white foam.

[0591] Step 2: Preparation of Compound 319 A 100 mL solution of 318 (1.19 g, 0.39 mmol) 1,2-dichloroethane 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 hours), the solution was removed from the heat source, treated with CH3OH (10 mL), and stirred (1 hour). After stirring, the mixture was poured into NaHCO3 (saturated aqueous solution), then washed with brine, dried (MgSO4), filtered, and concentrated. The resulting residue was used without further processing. Yield = 1.4 g, quantitative.

[0592] Step 3: Preparation of Conjugate 320 Using standard amide coupling chemistry, succinate 319 was loaded onto a 1000 Å LCAA (long-chain aminoalkyl) 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 anhydrous dichloromethane (0.2 mL) containing 319 (20.6 mg, 8 μmol). This mixture was added to LCAA CPG (183 mg). The suspension was gently mixed overnight at room temperature. After 319 disappeared (HPLC), the reaction mixture was filtered, and the CPG was washed with dichloromethane, acetonitrile, a THF solution of 5% anhydrous acetic acid / 5% N-methylimidazole / 5% pyridine, and then with 1 mL each of THF, acetonitrile, and dichloromethane. The CPG was then dried overnight under high vacuum. The loading was determined to be 19 μmol / g by a standard DMTr assay using UV / Vis (504 nm). The resulting GalNAc-loaded CPG solid support was used for automated oligonucleotide synthesis using a standard procedure. After nucleotide deprotection, removal from the solid support (simultaneously with galactosamine acetate deprotection) yielded GalNAc-oligonucleotide conjugate 320.

[0593] Example 27 Synthesis of Conjugate 520 Scheme 56 Preparation of Activated Linker [ka]

[0594] Step 1. Preparation of racemic(cis)5-benzyl-3a,6a-dimethyltetrahydro-1H-flu[3,4-c]pyrrole-1,3(3aH)-dione(301). Trifluoroacetic acid (75 μL) was slowly added to a chilled solution (0°C) containing 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). The mixture was stirred overnight, allowing the solution to slowly return 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 silica gel column chromatography (gradient: hexane containing 20% ​​ethyl acetate to 100% ethyl acetate) yielded (3aR,6aS)-5-benzyl-3a,6a-dimethyltetrahydro-1H-flu[3,4-c]pyrrole-1,3(3aH)-dione as a yellow oily substance (3.5 g, 56%).

[0595] Step 2. Preparation of racemic (cis)(1-benzyl-3,4-dimethylpyrrolidine-3,4-diyl)dimethanol (302) To a chilled solution (0°C) containing (3.5 g, 13.4 mmol) of (3aR,6aS)-5-benzyl-3a,6a-dimethyltetrahydro-1H-flu[3,4-c]pyrrole-1,3(3aH)-dione (50 mL) anhydrous diethyl ether, lithium aluminum hydride pellets (1.5 g, 40 mmol) were slowly added in three portions. The solution was stirred overnight, allowing it to return to room temperature as the ice bath melted. Once complete, the reaction mixture was cooled to 0°C and quenched very slowly with 1.5 mL of 5 M NaOH followed by 1.5 mL of water. The mixture was stirred for 30 minutes, then magnesium sulfate was added, and the mixture was filtered. The filtrate was concentrated to obtain ((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). A methanol solution (10 mL) of ((3R,4S)-1-benzyl-3,4-dimethylpyrrolidine-3,4-diyl)dimethanol (10 g, 40 mmol) was mixed with 1 g of moistened 10% palladium activated carbon. This solution was vigorously stirred under a hydrogen atmosphere for 16 hours. Upon completion, the solution was filtered through Celite and concentrated to dryness to obtain ((3R,4S)-3,4-dimethylpyrrolidine-3,4-diyl)dimethanol as a colorless solid (5.5 g, 86%).

[0597] Step 4. Preparation of racemi(cis)methyl 10-(3,4-bis(hydroxymethyl)-3,4-dimethylpyrrolidine-1-yl)-10-oxodecanoate (304) A CH2Cl2 solution (100 mL) of 3 (1.3 g, 8.2 mmol) and monomethyl sebacate (1.8 g, 8.2 mmol) was treated with HBTU (3.41 g, 9.02 mmol) and Hünig base (5.71 mL, 32.8 mmol). After stirring overnight, the mixture was washed with NaHCO3 (saturated aqueous solution), water, and brine, then dried (MgSO4), filtered, and concentrated. This crude substance was subjected to chromatography (gradient: 0% CH3OH-CH2Cl2 to 20%) to obtain 4 (1.8 g, 61%).

[0598] Step 5. Preparation of racemi(cis)methyl 10-(3-((bis(4-methoxyphenyl)(phenyl)-methoxy)methyl)-4-(hydroxymethyl)-3,4-dimethylpyrrolidine-1-yl)-10-oxodecanoate (305) A pyridine solution (180 mL) of 304 (1.8 g, 5.0 mmol) and 4,4'-dimethoxytrityl chloride (1.7 g, 5.0 mmol) was stirred overnight. The pyridine was then removed under reduced pressure, and the crude substance was subjected to chromatography (gradient: 0% CH3OH-CH2Cl2 to 10%) to obtain 5 (1.4 g, 42%) as a yellow oily substance.

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

[0600] Scheme 57 Synthesis of hyperacetylated galactosamine (507) [ka] Pyridine (1.5 L) containing galactosamine hydrochloride (250 g, 1.16 mol) 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 solid was filtered off, and the mixture was frozen over liquid nitrogen and then freeze-dried for 5 days to obtain peracetylated galactosamine (507) (369.4 g, 82%) as a white solid. Rf (0.58, 10% MeOH-CH2Cl2).

[0601] Scheme 58 Synthesis of GalNAc monomer [ka]

[0602] Step 1: Preparation of Compound 509 An aqueous solution (1 L) of 2-[2-(2-chloroethoxy)]ethanol (508) (100 g, 593 mmol) was treated with NaN3 (77 g, 1.19 mol) and heated (90°C). After stirring (72 hours), the solution was cooled (room temperature) and extracted with CH2Cl2 (4 times). The combined organic matter was washed with brine, dried (MgSO4), filtered, concentrated, and used without further treatment. Compound 509 (88.9 g, 86%) was obtained as a pale yellow oily substance.

[0603] Step 2 Preparation of Compound 510 A 1,2-dichloroethane solution (40 mL) of 507 (2.76 g, 7.1 mmol) and 509 (1.37 g, 7.8 mmol) was treated with Sc(OTf)3 (174 mg, 0.36 mmol) and heated (85°C). After stirring (2 hours), the mixture was cooled (RT), quenched by adding TEA (4 mL), and concentrated. This crude substance was subjected to chromatography to obtain 510 (3.03 g, 85%) as a pale yellow foam.

[0604] Step 3 Preparation of Compound 511 A 30 mL ethyl phosphate solution of 510 (3.02 g, 5.99 mmol) and Pd / C (300 mg, 10% Pd-loaded - moistened support) was treated with TFA (576 μL, 7.5 mmol). The reaction mixture was purged with hydrogen gas (45 minutes), then purged with nitrogen gas (10 minutes), and then filtered through Celite. The filtrate was concentrated and then subjected to chromatography to obtain 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)acetamide)isophthalate (312) A 50 mL solution of DMF containing 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) was stirred overnight at room temperature. Upon completion, the reaction mixture was diluted with ethyl acetate (250 mL) and washed with 1 M HCl (2 × 100 mL), saturated sodium bicarbonate (1 × 100 mL), and brine (2 × 100 mL), respectively. The mixture was dried over magnesium sulfate, filtered, concentrated to dryness, and then dimethyl 5-(2-((2-oxo-2-phenyl-1λ 2 Ethyl)amino)acetamide)isophthalate was obtained as a colorless solid (7.2 g, 79%).

[0607] Step 2. 5-(2-((2-oxo-2-phenyl-1λ 2 Preparation of ethyl(amino)acetamide)isophthalic acid (313) Methanol (25 mL) and THF (25 mL) are mixed with methyl 5-(2-((2-oxo-2-phenyl-1λ 2A solution containing ethyl)amino)acetamido)isophthalate (7.2 g) was mixed with 1 M NaOH (25 mL). This solution was stirred at room temperature for 2 hours, then concentrated to remove THF and MeOH. The remaining aqueous solution was diluted with water (75 mL), cooled in an ice bath, and acidified to pH=1 with 6 M HCl. This solid was filtered and washed with water (3 × 100 mL). This solid was freeze-dried to obtain 5-(2-((2-oxo-2-phenyl-1λ 2 -ethyl)amino)acetamide)isophthalic acid was obtained (6.9 g, quantitative).

[0608] Scheme 60: Preparation of tetramers [ka] [ka]

[0609] Step 1: Preparation of Compound 514 CH2Cl2 solutions (150 mL) of 313 (2.09 g, 5.6 mmol) and 511 (8.34 g, 14.07 mmol) were treated with HBTU (6.4 g, 16.9 mmol) and Hünig base (7.35 mL, 42.2 mmol). After stirring (overnight), the reaction mixture was poured into NaHCO3 (saturated aqueous solution), then washed with water and brine, dried (MgSO4), filtered, and concentrated. This crude substance was subjected to chromatography (CH3OH-CH2Cl2 gradient of 1-12%) to obtain 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% loaded - wet support), and trifluoroacetic acid (308 μL, 4 mmol) were purged with H2. After stirring under H2 (overnight), the mixture was purged with N2 (15-20 minutes), then filtered through Celite and concentrated. This crude substance was subjected to chromatography to obtain compound 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 compound 514. Yield: 1.66 g, 60%.

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

[0613] Scheme 61 Preparation of a complete conjugate [ka] [ka]

[0614] Step 1: Preparation of Compound 518 A 100 mL solution of 517 (1.91 g, 0.75 mmol) in CH2Cl2 was first treated with Hünig 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 NaHCO3 (saturated aqueous solution), then washed with water and brine, dried (MgSO4), filtered, and concentrated. This crude substance was subjected to chromatography to obtain 518 (1.19 g, 52%) as a white foam.

[0615] Step 2 Preparation of Compound 519 A 100 mL solution of 518 (1.19 g, 0.39 mmol) 1,2-dichloroethane 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 hours), the solution was removed from the heat source, treated with CH3OH (10 mL), and stirred (1 hour). After stirring, the mixture was poured into NaHCO3 (saturated aqueous solution), then washed with brine, dried (MgSO4), filtered, and concentrated. The resulting residue was used without further processing. Yield = 1.4 g, quantitative.

[0616] Step 3: Preparation of Conjugate 520 Using standard amide coupling chemistry, succinate 519 was loaded onto a 1000 Å LCAA (long-chain aminoalkyl) 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 anhydrous dichloromethane (0.2 mL) containing 519 (20.6 mg, 8 μmol). This mixture was added to LCAA CPG (183 mg). The suspension was gently mixed overnight at room temperature. After 519 disappeared (HPLC), the reaction mixture was filtered, and the CPG was washed with dichloromethane, acetonitrile, a THF solution of 5% anhydrous acetic acid / 5% N-methylimidazole / 5% pyridine, and then with 1 mL each of THF, acetonitrile, and dichloromethane. The CPG was then dried overnight under high vacuum. The loading was determined to be 19 μmol / g by a standard DMTr assay using UV / Vis (504 nm). The resulting GalNAc-loaded CPG solid support was used for automated oligonucleotide synthesis using a standard procedure. After nucleotide deprotection, removal from the solid support (simultaneously with galactosamine acetate deprotection) yielded a GalNAc-oligonucleotide conjugate (520).

[0617] Example 28. In vivo testing of TTR siRNA conjugates. Regarding the in vivo activity of TTR knockdown in a wild-type mouse model, compound 320 (wherein R is used in the formula) was investigated. 2 The compounds tested included the modified TTR siRNAs listed in Table 3. In this example, compound 320 (wherein R is the compound of the form R) was used. 2 The compound 320 (containing modified TTR siRNA) is presented as a potential treatment for the rare disease TTR (transthyretin) amyloidosis. In patients with this disease, misfolding and aggregation of the transthyretin protein are associated with disease progression. By using this siRNA-GalNAc conjugate, the amount of protein misfolding / aggregation in patients can be reduced, potentially resulting in halting disease progression. Therefore, certain embodiments of compound 320 (wherein R) are shown. 2 This includes modified TTR siRNA and provides its use for treating transthyretin amyloidosis. [Table 3] 2'-O-methylnucleotide = lowercase; 2'-fluoronucleotide = uppercase underlined; phosphorothioate linker = s; unmodified = uppercase

[0618] Both the TTR siRNA sequences and animal models were those 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 approved by the local Institutional Animal Care and Use Committee (IACUC).

[0619] siRNA treatment: Female C57BL / 6 mice (n=4) were given a single dose of compound 320(R) at a dose of 2 mg / kg. 2The animals received a modified TTR siRNA (containing siRNA) once on day 0 (one dose per animal) by subcutaneous injection into the scapula. One group of animals, administered only vehicle (PBS), served as a control.

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

[0621] Analysis: The Abnova Prealbumin (Mouse) ELISA kit (Cedar Lane, catalog number KA2070) was used according to the manufacturer's instructions to determine TTR protein levels in plasma samples. TTR plasma protein values ​​were calculated for each individual plasma sample, and the mean was determined for each group. From these means, the TTR protein level relative to the control (percentage relative to PBS-treated animals) was determined.

[0622] Results: The test results are shown in Table 4. The values ​​represent the percentage (%) of TTR protein levels (relative to PBS control) on days 2, 4, 7, 9, 14, and 21 post-treatment. [Table 4]

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

[0624] Example 29. In vivo testing of HBV siRNA conjugates. Chemically modified HBV siRNAs, conjugated to GalNAc ligands as described in Table 1 of Example 25, were tested for in vivo activity in established HBV infection mouse models. In the AAV-HBV1.2 C57BL / 6 mouse model, injection of an adeno-associated virus (AAV) vector encoding a sequence exceeding the HBV genome length resulted in stable and sustained HBV expression, leading to hepatic expression of HBV RNA and protein, as well as secretion of viral and subviral particles into the bloodstream.

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

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

[0627] siRNA treatment: A single dose of 3 mg / kg of HBV siRNA conjugate was administered to a mouse group (typically n=5) by subcutaneous injection into the scapula on day 0 (one dose per animal). One group of animals that received only vehicle (saline) was used as a control.

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

[0629] Analysis: HBsAg levels in serum samples were determined using the BioRad EIA GS HBsAg 3.0 kit (BioRad, catalog number 32591) according to the manufacturer's instructions. Group-mean HBsAg levels at individual time points were determined using pooled serum from each treatment group. The data were analyzed and expressed as HBsAg levels relative to pre-treatment baseline (percentage of day 0).

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

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

[0632] [Embodiment 1] A nucleic acid molecule selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, and 73.

[0633] [Embodiment 2] Nucleic acid molecules selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, and 74.

[0634] [Embodiment 3] A composition comprising the nucleic acid molecule described in Embodiment 1, the nucleic acid molecule described in 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) A double-stranded siRNA molecule selected from the group consisting of ), 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 described in Embodiment 4.

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

[0638] [Embodiment 7] Compound of formula (I): [ka] or a salt thereof During the ceremony, R 1 It is a targeted ligand, L 1 It does not exist, or it is a bonding group. L 2 It does not exist, or it is a bonding group. R 2 This is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules described in Embodiment 4, Ring A is either absent, or is a 3-20 member cycloalkyl, 5-20 member aryl, 5-20 member heteroaryl, or 3-20 member heterocycloalkyl. Each R A These are independently hydrogen, hydroxyl, CN, F, Cl, Br, I, and -C. 1~2 Alkyl-OR B , C 1~10 Alkyl C 2~10 Alkenyl and C 2~10 Selected from the group consisting of alkynyl, the C 1~10 Alkyl C 2~10 Alkenyl and C 2~10 Alkinyl is a compound of halo, hydroxy, and C. 1~3 Optionally substituted with one or more groups independently selected from the alkoxy, R B This refers to a bond to hydrogen, a protecting group, a covalent bond to a solid support, or a bond to a bonding 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 It is a targeted ligand, L 1 It does not exist, or it is a bonding group. L 2 It does not exist, or it is a bonding group. R 2 This is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules described in Embodiment 4, Ring A is either absent, or is a 3-20 member cycloalkyl, 5-20 member aryl, 5-20 member heteroaryl, or 3-20 member heterocycloalkyl. Each R AThese are independently hydrogen, hydroxyl, CN, F, Cl, Br, I, and -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 the alkoxy 1~8 Selected from the group consisting of alkyl groups, R B This refers to a bond to hydrogen, a protecting group, a covalent bond to a solid support, or a bond to a bonding group attached to a solid support. n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The compound described in 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 These are independent bonding groups, p is 1, 2, or 3. Sugars are monosaccharides or disaccharides. The compound described in Embodiment 8, or a salt thereof.

[0641] [Embodiment 10] The aforementioned 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 Either X is selected from 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 Each is independently selected from the group consisting of (C3-C6) cycloalkyl groups that are optionally substituted with one or more groups independently selected from the group consisting of hydrogen, (C1-C8) alkyl, (C1-C8) haloalkyl, (C1-C8) alkoxy, and 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 A five-membered heterocycle optionally substituted with one or more groups independently selected from the group consisting of -F, or halo, hydroxyl, carboxyl, amino, (C1-C4)alkyl, (C1-C4)haloalkyl, (C1-C4)alkoxy, and (C1-C4)haloalkoxy. The compound or a salt thereof as described in Embodiment 9.

[0642] [Embodiment 11] The aforementioned sugars are, [ka] A compound according to embodiment 9 or 10, selected from the group consisting of salts thereof.

[0643] [Embodiment 12] The aforementioned sugars are, [ka] The compound or salt thereof according to any one of embodiments 9 to 10.

[0644] [Embodiment 13] Each L 3A is independently a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 0 to 50 carbon atoms, wherein one or more carbon atoms in the hydrocarbon chain (e.g., 1, 2, 3, or 4) are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X -, or -S- may be replaced depending on the case, R X The compound or salt thereof according to any of Embodiments 9 to 12, wherein the hydrocarbon chain is hydrogen or (C1-C6) alkyl, and the hydrocarbon chain is optionally substituted with one or more substituents (e.g., one, two, three, or four) selected from (C1-C6) alkoxy, (C3-C6) cycloalkyl, (C1-C6) alkanoyl, (C1-C6) alkanoyloxy, (C1-C6) alkoxycarbonyl, (C1-C6) alkylthio, azide, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0645] [Embodiment 14] Each L 3 Each is independently a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 20 carbon atoms, wherein one or more carbon atoms in the hydrocarbon chain (e.g., 1, 2, 3, or 4) are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X -, or -S- may be replaced depending on the case, R XThe compound or salt thereof according to any of Embodiments 9 to 13, wherein the hydrocarbon chain is hydrogen or (C1-C6) alkyl, and the hydrocarbon chain is optionally substituted with one or more substituents (e.g., one, two, three, or four) selected from (C1-C6) alkoxy, (C3-C6) cycloalkyl, (C1-C6) alkanoyl, (C1-C6) alkanoyloxy, (C1-C6) alkoxycarbonyl, (C1-C6) alkylthio, azide, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0646] [Embodiment 15] L 3 teeth, [ka] The compound according to any one of embodiments 9 to 14, or a salt thereof.

[0647] [Embodiment 16] R 1 teeth, [ka] The compound according to any one of embodiments 8 to 15, or a salt thereof.

[0648] [Embodiment 17] R 1 teeth, [ka] And, During the ceremony, G is -NH- or -O-, R C These are hydrogen, (C1-C8) alkyl, (C1-C8) haloalkyl, (C1-C8) alkoxy, (C1-C6) alkanoyl, (C3-C 20 )Cycloalkyl, (C3~C 20) a heterocyclic, aryl, heteroaryl, monosaccharide, disaccharide, or trisaccharide, wherein the cycloalkyl, heterocyclic, aryl, heteroaryl, and sugars 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. The compound described in Embodiment 8, or a salt thereof.

[0649] [Embodiment 18] R C teeth, [ka] The compound described in Embodiment 17, or a salt thereof.

[0650] [Embodiment 19] R 1 teeth, [ka] The compound or salt thereof according to any one of embodiments 8, 17, and 18.

[0651] [Embodiment 20] R C teeth, [ka] The compound described in Embodiment 17, or a salt thereof.

[0652] [Embodiment 21] A compound or salt thereof according to any one of embodiments 17 to 20, wherein G is -NH-.

[0653] [Embodiment 22] R 1 teeth, [ka] The compound or salt thereof according to any one of embodiments 8, 17, 20, and 21.

[0654] [Embodiment 23] R 1 teeth, [ka] And, In the formula, each R D These are independently hydrogen, (C1~C6) alkyl, (C9~C 20 ) Alkylsilyl, (R W Selected from the group consisting of )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 (These are independently selected from the group consisting of (C1-C4) alkyl and aryl elements.) The compound described in Embodiment 8, or a salt thereof.

[0655] [Embodiment 24] L 1 and L 2 Each is independently a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 50 carbon atoms, wherein one or more carbon atoms in the hydrocarbon chain (e.g., 1, 2, 3, or 4) are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X -, or -S- may be replaced depending on the case, R XThe compound or salt thereof according to any of Embodiments 8 to 23, wherein the hydrocarbon chain is hydrogen or (C1-C6) alkyl, and the hydrocarbon chain is optionally substituted with one or more substituents (e.g., one, two, three, or four) selected from (C1-C6) alkoxy, (C3-C6) cycloalkyl, (C1-C6) alkanoyl, (C1-C6) alkanoyloxy, (C1-C6) alkoxycarbonyl, (C1-C6) alkylthio, azide, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0656] [Embodiment 25] L 1 and L 2 Each is independently a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 20 carbon atoms, wherein one or more carbon atoms in the hydrocarbon chain (e.g., 1, 2, 3, or 4) are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X -, or -S- may be replaced depending on the case, R X The compound or salt thereof according to any of Embodiments 8 to 24, wherein is hydrogen or (C1-C6) alkyl, and the hydrocarbon chain is optionally substituted with one or more substituents (e.g., one, two, three, or four) selected from (C1-C6) alkoxy, (C3-C6) cycloalkyl, (C1-C6) alkanoyl, (C1-C6) alkanoyloxy, (C1-C6) alkoxycarbonyl, (C1-C6) alkylthio, azide, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0657] [Embodiment 26] L 1 and L 2Each is independently a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 14 carbon atoms, wherein one or more carbon atoms (e.g., 1, 2, 3, or 4) in the hydrocarbon chain are optionally replaced by -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X -, or -S-, R X The compound or salt thereof according to any of Embodiments 8 to 25, wherein the hydrocarbon chain is hydrogen or (C1-C6) alkyl, and the hydrocarbon chain is optionally substituted with one or more substituents (e.g., one, two, three, or four) selected from (C1-C6) alkoxy, (C3-C6) cycloalkyl, (C1-C6) alkanoyl, (C1-C6) alkanoyloxy, (C1-C6) alkoxycarbonyl, (C1-C6) alkylthio, azide, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

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

[0659] [Embodiment 28] L 2 R is via -O- 2 A compound according to any of embodiments 8 to 27, or a salt thereof, bonded thereto.

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

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

[0662] [Embodiment 31] Compound of formula (Ia): [ka] And, During the ceremony, Each D is independent, [ka] Selected from the group consisting of and -N= The compound described in Embodiment 8, or a salt thereof.

[0663] [Embodiment 32] [ka] And selected from the group consisting of those salts, During the ceremony, Q 1 is hydrogen, Q 2 is R 2 is or Q 1 is R 2 Q 2 It is hydrogen, Z is -L 1 -R 1 That is, The compound described in Embodiment 8 or Embodiment 31, or a salt thereof.

[0664] [Embodiment 33] Compound of formula (Ib): [ka] And, During the ceremony, Each D is independent, [ka] Selected from the group consisting of and -N=, Each m is independently either 1 or 2. The compound described in Embodiment 8, or a salt thereof.

[0665] [Embodiment 34] [ka] And selected from the group consisting of those salts, During the ceremony, Q 1 is hydrogen, Q 2 is R 2 is or Q 1 is R 2 Q 2 It is hydrogen, Z is -L 1 -R 1 That is, The compound or salt thereof according to Embodiment 8 or Embodiment 33.

[0666] [Embodiment 35] Compounds of formula (Ic): [ka] or a salt thereof, During the ceremony, E is either -O- or -CH2-, 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. The compound described in Embodiment 8, or a salt thereof.

[0667] [Embodiment 36] [ka] A selection from the group consisting of salts thereof, where Z is -L 1 -R 1 (is), The compound described in Embodiment 8 or Embodiment 35, or a salt thereof.

[0668] [Embodiment 37] The aforementioned -AL 2 -R 2 The part is, [ka] And, During the ceremony, Q 1 is hydrogen, Q 2 is R 2 is or Q 1 is R 2 Q 2 It is hydrogen, Each q is independently 0, 1, 2, 3, 4, or 5. The compound described in Embodiment 8, or a salt thereof.

[0669] [Embodiment 38] [ka] A compound or salt thereof according to Embodiment 8, selected from the group consisting of 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 either 1 or 2. The compound described in Embodiment 7, or a salt thereof.

[0671] [Embodiment 40] L 1teeth, [ka] A compound or salt thereof according to embodiment 7 or 39, selected from the group consisting of the above.

[0672] [Embodiment 41] A is either absent or is phenyl, pyrrolidinyl, or cyclopentyl, as described in any of embodiments 7, 39, and 40, or a salt thereof.

[0673] [Embodiment 42] L 2 C is sometimes substituted with hydroxyl. 1~4 A compound or salt thereof according to any one of Embodiments 7 and 39-41, which is alkylene-O-.

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

[0675] [Embodiment 44] Each R A C is independently and optionally substituted with hydroxyl or hydroxyl. 1~8 A compound or salt thereof according to any of Embodiments 7 and 39-43, which is alkyl.

[0676] [Embodiment 45] Each R A This is a compound or salt thereof, independently selected from the group consisting of hydroxy, methyl, and -CH2OH, as described in any of Embodiments 7 and 39-44.

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

[0678] [Embodiment 47] [ka] And selected from the group consisting of those salts, In the formula, Q is -L 1 -R 1 And, R' is C 1~9 Alkyl, C 2~9 Alkenyl, or C 2~9 It is an alkynyl, and the C 1~9 Alkyl, C 2~9 Alkenyl, or C 2~9 Alkynyl is sometimes substituted with a halo or hydroxyl. The compound or salt thereof according to Embodiment 7 or 46.

[0679] [Embodiment 48] [ka] And selected from the group consisting of those salts, In the formula, Q is -L 1 -R 1 That is, The compound described in Embodiment 7, or a salt thereof.

[0680] [Embodiment 49] [ka] [ka] [ka] [ka] [ka] Furthermore, selected from the group consisting of pharmaceutically acceptable salts thereof, in the formula, R 2 This is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules described in Embodiment 4. The compound described in Embodiment 7, or a salt thereof.

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

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

[0683] [Embodiment 52] Compound of formula (Id): [ka] or a salt thereof, During the ceremony, R 1d teeth, [ka] Selected from, X d C2~ 10 It is alkylene, n d is 0 or 1, R 2d This is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules described in Embodiment 4, R 3dThis refers to H, a protecting group, a covalent bond to a solid support, or a bond to a bonding group attached to a solid support. The compound described in Embodiment 8, or a salt thereof.

[0684] [Embodiment 53] R 1d teeth, [ka] The compound or salt thereof as described in Embodiment 52.

[0685] [Embodiment 54] R 1d teeth, [ka] The compound or salt thereof as described in Embodiment 52.

[0686] [Embodiment 55] X d The compound or salt according to any of embodiments 52 to 54, wherein the compound is a C8 alkylene.

[0687] [Embodiment 56] n d The compound or salt according to any of embodiments 52 to 54, wherein is 0.

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

[0689] [Embodiment 58] R 3d The compound or salt described in any of embodiments 52 to 56 is covalently bonded to a solid support.

[0690] [Embodiment 59] R 3dThe compound or salt thereof according to any of embodiments 52 to 56, wherein the bond is to a bonding group bonded to a solid support, the bonding group being a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 2 to 15 carbon atoms, one or more of the carbon atoms (e.g., one, two, three, or four) are optionally replaced with (-O-) or (-N(H)-), and the chain is optionally substituted with one or more substituents (e.g., one, two, three, or four) selected from (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azide, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0691] [Embodiment 60] R 3d The compound or salt thereof according to any of embodiments 52 to 56, wherein the bond is to a bonding group bonded to a solid support, the bonding group being a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 2 to 10 carbon atoms, one or more of the carbon atoms (e.g., one, two, three, or four) are optionally replaced with (-O-) or (-N(H)-), and the chain is optionally substituted with one or more substituents (e.g., one, two, three, or four) selected from (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azide, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0692] [Embodiment 61] R 3dThe compound or salt thereof according to any one of embodiments 52 to 56, wherein the bond is to a binding group bonded to a solid support, and the binding group is -C(=O)CH2CH2C(=O)N(H)-.

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

[0694] [Embodiment 63] A method for delivering siRNA to the liver of an animal, comprising administering to the animal a compound of formula Id as described in any 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 as described in Embodiment 48, wherein the compound of the corresponding formula (Ie): [ka] (In the formula, X d These are C2-8 alkylenes, n d is 0 or 1, Pg 1 H is, R 3d (wherein R is a covalent bond to a solid support or a bond to a binding group attached to a solid support) is subjected to solid-phase nucleic acid synthesis conditions, and the corresponding compound of formula Id (wherein R is a covalent bond to a solid support or a bond to a binding group attached to a solid support) is subjected to solid-phase nucleic acid synthesis conditions, and the compound of formula Id is formed. 2d The method comprising providing a double-stranded siRNA molecule (which is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules described in Embodiment 4).

[0696] [Embodiment 65] The compound is removed from the solid support, R 3d The method according to embodiment 64, further comprising yielding a compound of the corresponding formula Id in which is H.

[0697] [Embodiment 66] [ka] A compound or salt thereof according to Embodiment 52, selected from the group consisting of salts thereof.

[0698] [Embodiment 67] Compound of formula (I): [ka] or a salt thereof During the ceremony, R 1 is H or a synthetic activating group, L 1 It does not exist, or it is a bonding group. L 2 It does not exist, or it is a bonding group. R 2 This is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules described in Embodiment 4, Ring A is either absent, or is a 3-20 member cycloalkyl, 5-20 member aryl, 5-20 member heteroaryl, or 3-20 member heterocycloalkyl. Each R A These are independently hydrogen, hydroxyl, CN, F, Cl, Br, I, and -C. 1~2 Alkyl-OR B , C 1~10 Alkyl C 2~10 Alkenyl and C 2~10 Selected from the group consisting of alkynyl, the C 1~10 Alkyl C 2~10 Alkenyl and C 2~10 Alkinyl is a compound of halo, hydroxy, and C. 1~3 Optionally substituted with one or more groups independently selected from the alkoxy, R B This refers to a bond to hydrogen, a protecting group, a covalent bond to a solid support, or a bond to a bonding 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 C is optionally substituted with hydroxyl or halo. 1~4 It is alkylene-O-, n is 0, 1, 2, 3, 4, 5, 6, or 7. The compound described in Embodiment 67, or a salt thereof.

[0700] [Embodiment 69] [ka] And selected from the group consisting of those salts, During the ceremony, Q is -L 1 -R 1 And, R' is C 1~9 Alkyl, C 2~9 Alkenyl, or C 2~9 It is an alkynyl, and the C 1~9 Alkyl, C 2~9 Alkenyl, or C 2~9 Alkynyl is sometimes substituted with a halo or hydroxyl. The compound described in Embodiment 67, or a salt thereof.

[0701] [Embodiment 70] [ka] And selected from the group consisting of those salts, In the formula, Q is -L 1 -R 1 The compound or salt thereof according to Embodiment 67.

[0702] [Embodiment 71] R 1 The compound or salt thereof according to any of Embodiments 66 to 73, wherein H is a synthetic activating group that can be derived from DCC, HOBt, EDC, BOP, PyBOP, or HBTU.

[0703] [Embodiment 72] R 1 The compound according to any of Embodiments 66 to 73, or a salt thereof, is a synthetic activating group that can be derived from DCC, HOBt, EDC, BOP, PyBOP, or HBTU.

[0704] [Embodiment 73] L 1 The compound or salt thereof according to any of Embodiments 66 to 77, wherein the compound is a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 5 to 20 carbon atoms, wherein one or more carbon atoms (e.g., one, two, three, or four) in the hydrocarbon chain are optionally replaced by -O-, -NH-, -NH-C(=O)-, -C(=O)-NH-, or -S-.

[0705] [Embodiment 74] Compound of formula (XX): [ka] or a salt thereof During the ceremony, R 1 It is a targeted ligand, L 1 It does not exist, or it is a bonding group. L 2 It does not exist, or it is a bonding group. R 2 This is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules described in Embodiment 4, B is divalent, and [ka] A group consisting of the following is selected, where, Each R' is independent of C 1~9 Alkyl, C 2~9 Alkenyl, or C 2~9 It is an alkynyl, and the C 1~9 Alkyl, C 2~9 Alkenyl, or C 2~9 Alkynyl is sometimes substituted with a halo or hydroxyl. * The valence with a mark is L 1 It is bound to L 1 If it does not exist, R 1 It is connected, ** The valence with a mark is L 2 It is bound to L 2 If it does not exist, R 2 It is connected, The compound, or a salt thereof.

[0706] [Embodiment 75] The aforementioned targeted ligand R 1 This is the compound according to Embodiment 74, or a salt thereof, comprising 2 to 8 sugars.

[0707] [Embodiment 76] The aforementioned targeted ligand R 1 This is the compound according to Embodiment 74, or a salt thereof, comprising 2 to 4 sugars.

[0708] [Embodiment 77] The aforementioned targeted ligand R 1 This is the compound according to Embodiment 74, or a salt thereof, comprising 3 to 8 sugars.

[0709] [Embodiment 78] The aforementioned targeted ligand R 1 This is the compound according to Embodiment 74, or a salt thereof, comprising 3 to 6 sugars.

[0710] [Embodiment 79] The aforementioned targeted ligand R 1 This is the compound according to Embodiment 74, or a salt thereof, comprising 3 to 4 sugars.

[0711] [Embodiment 80] The aforementioned targeted ligand R 1 This is the compound according to Embodiment 74, or a salt thereof, comprising three sugars.

[0712] [Embodiment 81] The aforementioned targeted ligand R 1 This is the compound according to Embodiment 74, or a salt thereof, comprising four sugars.

[0713] [Embodiment 82] The targeted portion R 1 The following equation applies: [ka] It has, During the ceremony, B 1 It is a trivalent group containing approximately 1 to 20 atoms, L 1 , T 1 , and T 2 It is covalently bonded to it, B 2 It is a trivalent group containing approximately 1 to 20 atoms, and T 1 , T 3 , and T 4 It is covalently bonded to it, B 3 It is a trivalent group containing approximately 1 to 20 atoms, and T 2 , T 5 , and T 6 It is covalently bonded to it, T 1 It does not exist, or it is a bonding group. T 2 It does not exist, or it is a bonding group. T 3 It does not exist, or it is a bonding group. T 4 It does not exist, or it is a bonding group. T 5 It does not exist, or it is a bonding group. T 6 It is either absent or is a bonding group. The 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 saccharide is independently [Chemical formula] selected from the group consisting of, in the formula, X is NR 3 , Y is -(C=O)R 4 , -SO2R 5 , and -(C=O)NR 6 R 7 is selected from the group consisting of, or X is -(C=O)- and Y is NR 8 R 9 , 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 is -OH, -NR 8 R 9 , or -F, R 11 is -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, The compound described in Embodiment 82, or a salt thereof.

[0715] [Embodiment 84] Each of the aforementioned sugars is independent, [ka] A compound or salt thereof, selected from the group consisting of the compounds described in Embodiment 82.

[0716] [Embodiment 85] Each sugar is independent of the others. [ka] The compound or salt thereof as described in Embodiment 82.

[0717] [Embodiment 86] T 1 and T 2 A compound or salt thereof according to any one of embodiments 82 to 85, wherein one of the compounds is absent.

[0718] [Embodiment 87] T 1 and T 2 None of the compounds described in any of embodiments 82 to 85, or their salts, exist.

[0719] [Embodiment 88] T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 Each of these is independently a branched or unbranched, saturated or unsaturated hydrocarbon chain having no carbon atoms or 1 to 50 carbon atoms, wherein one or more carbon atoms (e.g., 1, 2, 3, or 4) in the hydrocarbon chain are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X -, or -S- may be replaced depending on the case, R XThe compound or salt thereof according to any of Embodiments 82 to 85, wherein is hydrogen or (C1-C6) alkyl, and the hydrocarbon chain is optionally substituted with one or more substituents (e.g., one, two, three, or four) selected from (C1-C6) alkoxy, (C3-C6) cycloalkyl, (C1-C6) alkanoyl, (C1-C6) alkanoyloxy, (C1-C6) alkoxycarbonyl, (C1-C6) alkylthio, azide, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0720] [Embodiment 89] T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 Each of these is independently a branched or unbranched, saturated or unsaturated hydrocarbon chain having no carbon atoms or 1 to 20 carbon atoms, wherein one or more carbon atoms (e.g., 1, 2, 3, or 4) in the hydrocarbon chain are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X -, or -S- may be replaced depending on the case, R X The compound or salt thereof according to any of Embodiments 82 to 85, wherein is hydrogen or (C1-C6) alkyl, and the hydrocarbon chain is optionally substituted with one or more substituents (e.g., one, two, three, or four) selected from (C1-C6) alkoxy, (C3-C6) cycloalkyl, (C1-C6) alkanoyl, (C1-C6) alkanoyloxy, (C1-C6) alkoxycarbonyl, (C1-C6) alkylthio, azide, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0721] [Embodiment 90] T 1 , T 2 , T 3, T 4 , T 5 , and T 6 Each of these is independently a branched or unbranched, saturated or unsaturated hydrocarbon chain, or a salt thereof, having no carbon atoms or having 1 to 50 carbon atoms, wherein one or more carbon atoms (e.g., 1, 2, 3, or 4) in the hydrocarbon chain are -O- or -NR X - is sometimes replaced by R X The compound according to any of embodiments 82 to 85, or a salt thereof, wherein is hydrogen or (C1-C6) alkyl, and the hydrocarbon chain is optionally substituted with one or more substituents (e.g., one, two, three, or four) selected from halo, hydroxy, and oxo (=O).

[0722] [Embodiment 91] T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 Each of these is independently a branched or unbranched, saturated or unsaturated hydrocarbon chain having no carbon atoms or 1 to 20 carbon atoms, wherein one or more carbon atoms in the hydrocarbon chain (e.g., 1, 2, 3, or 4) are optionally replaced by -O-, and the hydrocarbon chain is optionally substituted with one or more substituents (e.g., 1, 2, 3, or 4) selected from halo, hydroxy, and oxo (=O), the compound or salt thereof according to any of embodiments 82 to 85.

[0723] [Embodiment 92] T 1 , T 2 , T 3 , T 4 , T 5 , and T 6Each of these is independently a branched or unbranched, saturated or unsaturated hydrocarbon chain having no carbon atoms or 1 to 20 carbon atoms, wherein one or more carbon atoms in the hydrocarbon chain (e.g., 1, 2, 3, or 4) are optionally replaced by -O-, and the hydrocarbon chain is optionally substituted with one or more substituents (e.g., 1, 2, 3, or 4) selected from halo, hydroxy, and oxo (=O), the compound or salt thereof according to any of embodiments 82 to 85.

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

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

[0727] [Embodiment 96] T 1 and T 2 are each glycine, the compound according to any one of embodiments 82 to 85, or a salt thereof.

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

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

[0730] [Embodiment 99] B 1 contains (C1~C6) alkyl, the compound according to any one of embodiments 82 to 96, or a salt thereof.

[0731] [Embodiment 100] B 1 contains C 3~8 cycloalkyl, the compound according to any one of embodiments 82 to 96, or a salt thereof.

[0732] [Embodiment 101] B 1 contains a silyl group, the compound according to any one of embodiments 82 to 96, or a salt thereof.

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

[0734] [Embodiment 103] B 1The compound is a compound according to any one of embodiments 82 to 96, which contains sugars, or a salt thereof.

[0735] [Embodiment 104] B 1 The compound comprising a phosphate group, as described in any of embodiments 82 to 96, or a salt thereof.

[0736] [Embodiment 105] B 1 The compound comprising a phosphonic acid group, as described in any of embodiments 82 to 96, or a salt thereof.

[0737] [Embodiment 106] B 1 The compound comprising an aryl compound as described in any of embodiments 82 to 96, or a salt thereof.

[0738] [Embodiment 107] B 1 The compound comprising a phenyl ring, as described in any of embodiments 82 to 96, or a salt thereof.

[0739] [Embodiment 108] B 1 The compound or salt thereof according to any of embodiments 82 to 96, wherein is a phenyl ring.

[0740] [Embodiment 109] B 1 A compound or salt thereof according to any of embodiments 82 to 96, wherein is CH.

[0741] [Embodiment 110] B 1 The compound comprising a heteroaryl compound as described in any of embodiments 82 to 96, or a salt thereof.

[0742] [Embodiment 111] B 1 teeth, [ka] The compound or salt thereof according to any of embodiments 82 to 96.

[0743] [Embodiment 112] B 2 It is a trivalent group containing 1 to 15 atoms, L 1 , T 1 , and T 2 A compound according to any of embodiments 82 to 111, or a salt thereof, which is covalently bonded to a compound.

[0744] [Embodiment 113] B 2 It is a trivalent group containing 1 to 10 atoms, L 1 , T 1 , and T 2 A compound according to any of embodiments 82 to 111, or a salt thereof, which is covalently bonded to a compound.

[0745] [Embodiment 114] B 2 The compound is one of the compounds described in any of embodiments 82 to 111, which contains a (C1-C6) alkyl group, or a salt thereof.

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

[0747] [Embodiment 116] B 2 The compound comprising a silyl group, as described in any of embodiments 82 to 111, or a salt thereof.

[0748] [Embodiment 117] B 2 The compound is one of the embodiments described in any of embodiments 82 to 111, comprising a D-amino acid or an L-amino acid, or a salt thereof.

[0749] [Embodiment 118] B 2 The compound is a compound according to any of embodiments 82 to 111, which contains sugars, or a salt thereof.

[0750] [Embodiment 119] B 2 The compound comprising a phosphate group, as described in any of embodiments 82 to 111, or a salt thereof.

[0751] [Embodiment 120] B 2 The compound comprising a phosphonic acid group, as described in any of embodiments 82 to 111, or a salt thereof.

[0752] [Embodiment 121] B 2 The compound comprising an aryl compound, or a salt thereof, according to any of embodiments 82 to 111.

[0753] [Embodiment 122] B 2 The compound comprising a phenyl ring, as described in any of embodiments 82 to 111, or a salt thereof.

[0754] [Embodiment 123] B 2 A compound or salt thereof according to any of embodiments 82 to 111, wherein is a phenyl ring.

[0755] [Embodiment 124] B 2 A compound or salt thereof according to any of embodiments 82 to 111, wherein is CH.

[0756] [Embodiment 125] B 2 The compound is a heteroaryl compound as described in any of embodiments 82 to 111, or a salt thereof.

[0757] [Embodiment 126] B 2 teeth, [ka] A compound or salt thereof, selected from the group consisting of any of embodiments 82 to 111.

[0758] [Embodiment 127] B 3 It is a trivalent group containing 1 to 15 atoms, L 1 , T 1 , and T 2 A compound according to any of embodiments 82 to 126, or a salt thereof, which is covalently bonded to a compound.

[0759] [Embodiment 128] B 3 It is a trivalent group containing 1 to 10 atoms, L 1 , T 1 , and T 2 A compound according to any of embodiments 82 to 126, or a salt thereof, which is covalently bonded to a compound.

[0760] [Embodiment 129] B 3 The compound is one of the compounds described in any of embodiments 82 to 126, or a salt thereof, comprising a (C1-C6) alkyl group.

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

[0762] [Embodiment 131] B 3 The compound comprising a silyl group, as described in any of embodiments 82 to 126, or a salt thereof.

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

[0764] [Embodiment 133] B 3 The compound is a compound according to any of embodiments 82 to 126, which contains sugars, or a salt thereof.

[0765] [Embodiment 134] B 3 The compound comprising a phosphate group, as described in any of embodiments 82 to 126, or a salt thereof.

[0766] [Embodiment 135] B 3 The compound comprising a phosphonic acid group, as described in any of embodiments 82 to 126, or a salt thereof.

[0767] [Embodiment 136] B 3 The compound comprising an aryl compound as described in any of embodiments 82 to 126, or a salt thereof.

[0768] [Embodiment 137] B 3 The compound comprising a phenyl ring, as described in any of embodiments 82 to 126, or a salt thereof.

[0769] [Embodiment 138] B 3 The compound or salt thereof according to any of embodiments 82 to 126, wherein is a phenyl ring.

[0770] [Embodiment 139] B 3 A compound or salt thereof according to any of embodiments 82 to 126, wherein is CH.

[0771] [Embodiment 140] B 3 The compound comprising a heteroaryl compound, or a salt thereof, according to any of embodiments 82 to 126.

[0772] [Embodiment 141] B 3 teeth, [ka] A compound or salt thereof as described in any of embodiments 82 to 126, selected from the group consisting of salts thereof.

[0773] [Embodiment 142] L 1 and L 2 Each is independently a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 50 carbon atoms, wherein one or more carbon atoms in the hydrocarbon chain (e.g., 1, 2, 3, or 4) are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X -, or -S- may be replaced depending on the case, R X The compound or salt thereof according to any of Embodiments 87 to 146, wherein the hydrocarbon chain 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, azide, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0774] [Embodiment 143] L 1 teeth, [ka] A compound or salt thereof as described in any of embodiments 82 to 141, selected from the group consisting of salts thereof.

[0775] [Embodiment 144] L 1 B is formed via 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 A compound according to any of embodiments 82 to 141, or a salt thereof, bonded thereto.

[0776] [Embodiment 145] L 1 teeth, [ka] A compound or salt thereof, selected from the group consisting of any of embodiments 87 to 141.

[0777] [Embodiment 146] L 2 R is via -O- 2 A compound according to any of embodiments 82 to 145, or a salt thereof, bonded thereto.

[0778] [Embodiment 147] L 2 C is sometimes substituted with hydroxyl. 1~4 A compound or salt thereof according to any of embodiments 82 to 145, which is alkylene-O-.

[0779] [Embodiment 148] L 2 R is via -O- 2 A compound according to any of embodiments 82 to 145, or a salt thereof, bonded thereto.

[0780] [Embodiment 149] L 2 The compound described in any of embodiments 82 to 145, or a salt thereof, does not exist.

[0781] [Embodiment 150] [ka] [ka] [ka] Furthermore, selected from the group consisting of pharmaceutically acceptable salts thereof, in the formula, R 2 This is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules described in Embodiment 4. The compound described in Embodiment 87, or a salt thereof.

[0782] [Embodiment 151] compound [ka] or a salt thereof, in the formula R 2 The compound or a salt thereof is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules described in Embodiment 4.

[0783] [Embodiment 152] GalNAc conjugate of formula X: ABC (X) And, In the formula, A is the targeted ligand, B is an optional linker, C is the siRNA molecule described in Embodiment 4. The aforementioned GalNAc conjugate.

[0784] [Embodiment 153] The following compound: [ka] or a pharmaceutically acceptable salt thereof, in the formula R 2 This is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules described in Embodiment 4. The aforementioned compound, or a pharmaceutically acceptable salt thereof.

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

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

[0787] [Embodiment 156] The following compound: [ka] or a pharmaceutically acceptable salt thereof, in the formula R 2 This is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules described in Embodiment 4. The aforementioned compound, or a pharmaceutically acceptable salt thereof.

[0788] [Embodiment 157] The following compound: [ka] or a pharmaceutically acceptable salt thereof, in the formula R 2 This is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules described in Embodiment 4. The aforementioned compound, or a pharmaceutically acceptable salt thereof.

[0789] [Embodiment 158] The following compound: [ka] or a pharmaceutically acceptable salt thereof, in the formula R 2 This is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules described in Embodiment 4. The aforementioned compound, or a pharmaceutically acceptable salt thereof.

[0790] [Embodiment 159] The following compound: [ka] or a pharmaceutically acceptable salt thereof, in the formula R 2 This is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules described in Embodiment 4. The aforementioned compound, or a pharmaceutically acceptable salt thereof.

[0791] [Embodiment 160] The following compound: [ka] or a pharmaceutically acceptable salt thereof, in the formula R 2 This is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules described in Embodiment 4. The aforementioned compound, or a pharmaceutically acceptable salt thereof.

[0792] [Embodiment 161] The following compound: [ka] or a pharmaceutically acceptable salt thereof, in the formula R 2 This is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules described in Embodiment 4. The aforementioned compound, or a pharmaceutically acceptable salt thereof.

[0793] [Embodiment 162] The following compound: [ka] or a pharmaceutically acceptable salt thereof, in the formula R 2 This is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules described in Embodiment 4. The aforementioned compound, or a pharmaceutically acceptable salt thereof.

[0794] [Embodiment 163] The following compound: [ka] or a pharmaceutically acceptable salt thereof, in the formula R 2 This is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules described in Embodiment 4. The aforementioned compound, or a pharmaceutically acceptable salt thereof.

[0795] [Embodiment 164] The following compound: [ka] or a pharmaceutically acceptable salt thereof, in the formula R 2 This is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules described in Embodiment 4. The aforementioned compound, or a pharmaceutically acceptable salt thereof.

[0796] [Embodiment 165] The following compound: [ka] or a pharmaceutically acceptable salt thereof, in the formula R 2 This is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules described in Embodiment 4. The aforementioned compound, or a pharmaceutically acceptable salt thereof.

[0797] [Embodiment 166] The following compound: [ka] or a pharmaceutically acceptable salt thereof.

[0798] [Embodiment 167] The following compound: [ka] or a pharmaceutically acceptable salt thereof.

[0799] [Embodiment 168] The following compound: [ka] or a pharmaceutically acceptable salt thereof.

[0800] [Embodiment 169] The following compound: [ka] or a pharmaceutically acceptable salt thereof.

[0801] [Embodiment 170] The following compound: [ka] or a pharmaceutically acceptable salt thereof.

[0802] [Embodiment 171] The following compound: [ka] or a pharmaceutically acceptable salt thereof.

[0803] [Embodiment 172] The following compound: [ka] or a pharmaceutically acceptable salt thereof.

[0804] [Embodiment 173] The following compound: [ka] or a pharmaceutically acceptable salt thereof.

[0805] [Embodiment 174] Compound of formula (I): [ka] or a salt thereof During the ceremony, L 1 It does not exist, or it is a bonding group. L 2 It does not exist, or it is a bonding group. R 2 It is a nucleic acid, Ring A is either absent, or is a 3-20 member cycloalkyl, 5-20 member aryl, 5-20 member heteroaryl, or 3-20 member heterocycloalkyl. Each R A These are independently hydrogen, hydroxyl, CN, F, Cl, Br, I, and -C. 1~2 Alkyl-OR B , C 1~10 Alkyl C 2~10 Alkenyl and C 2~10 Selected from the group consisting of alkynyl, the C 1~10 Alkyl C 2~10 Alkenyl and C 2~10 Alkinyl is a compound of halo, hydroxy, and C. 1~3 Optionally substituted with one or more groups independently selected from the alkoxy, R B This refers to a bond to hydrogen, a protecting group, a covalent bond to a solid support, or a bond to a bonding 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] Compound of formula (Ia): [ka] or a salt thereof During the ceremony, L 2 It does not exist, or it is a bonding group. R 2 It is a nucleic acid, Ring A is either absent, or is a 3-20 member cycloalkyl, 5-20 member aryl, 5-20 member heteroaryl, or 3-20 member heterocycloalkyl. Each R A These are independently hydrogen, hydroxyl, CN, F, Cl, Br, I, and -C. 1~2 Alkyl-OR B , C 1~10 Alkyl C 2~10 Alkenyl and C 2~10 Selected from the group consisting of alkynyl, the C 1~10 Alkyl C 2~10 Alkenyl and C 2~10 Alkinyl is a compound of halo, hydroxy, and C. 1~3 Optionally substituted with one or more groups independently selected from the alkoxy, R B This refers to a bond to hydrogen, a protecting group, a covalent bond to a solid support, or a bond to a bonding 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] Compound of formula (XXa): [ka] or a salt thereof During the ceremony, L 1 It does not exist, or it is a bonding group. L 2 It does not exist, or it is a bonding group. R 2 It is a nucleic acid, B is divalent, and [ka] A group consisting of the following is selected, where, Each R' is independent of C 1~9 Alkyl, C 2~9 Alkenyl, or C 2~9 It is an alkynyl, and the C1~9 Alkyl, C 2~9 Alkenyl, or C 2~9 Alkynyl is sometimes substituted with a halo or hydroxyl. * The valence with a mark is L 1 It is bound to L 1 If it does not exist, R 1 It is connected, ** The valence with a mark is L 2 It is bound to L 2 If it does not exist, R 2 It is connected, The compound, or a salt thereof.

[0808] [Embodiment 177] L 1 and L 2 Each is independently a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 1 to 50 carbon atoms, wherein one or more carbon atoms in the hydrocarbon chain (e.g., 1, 2, 3, or 4) are -O-, -NR X -, -NR X -C(=O)-, -C(=O)-NR X -, or -S- may be replaced depending on the case, R X The compound according to Embodiment 175, or a salt thereof, wherein 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, azide, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0809] [Embodiment 178] L 1 teeth, [ka] A compound or salt thereof as described in Embodiment 176, selected from the group consisting of salts thereof.

[0810] [Embodiment 179] L 1 B is formed via 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 A compound according to Embodiment 176, or a salt thereof, bonded thereto.

[0811] [Embodiment 180] L 1 teeth, [ka] A compound or salt thereof according to Embodiment 176, selected from the group consisting of the following.

[0812] [Embodiment 181] L 2 R is via -O- 2 A compound according to Embodiment 176, or a salt thereof, bonded thereto.

[0813] [Embodiment 182] L 2 C is sometimes substituted with hydroxyl. 1~4 The compound according to Embodiment 176, or a salt thereof, which is alkylene-O-.

[0814] [Embodiment 183] L 2 The compound described in Embodiment 176, or a salt thereof, does not exist.

[0815] [Embodiment 184] compound [ka] or a salt thereof, in the formula R 2 It is a nucleic acid. The compound, or a salt thereof.

[0816] [Embodiment 185] compound [ka] or a salt thereof, in the formula R 2 It is a nucleic acid. The compound, or a salt thereof.

[0817] [Embodiment 186] compound [ka] or a salt thereof, in the formula R 2 It is a nucleic acid. The compound, or a salt thereof.

[0818] [Embodiment 187] R 2 The compound is one of the compounds described in any of Embodiments 174 to 186, which is a double-stranded siRNA molecule selected from the double-stranded siRNA molecules described in Embodiment 4.

[0819] [Embodiment 188] A pharmaceutical composition comprising a compound described in any 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 as described in any of embodiments 174 to 186 or a pharmaceutically acceptable salt thereof.

[0821] [Embodiment 190] A method for treating hepatitis B virus infection in an animal, comprising administering to the animal in an effective amount a compound of formula I or Id or a pharmaceutically acceptable salt thereof as described in any of embodiments 1 to 49, 52 to 61, or 174 to 186.

[0822] [Embodiment 191] The method according to Embodiment 190, wherein the compound of formula I or Id or a pharmaceutically acceptable salt thereof is administered subcutaneously.

[0823] [Embodiment 192] Compounds of formula I or Id as described in any of embodiments 1-49, 52-61, or 174-186, or pharmaceutically acceptable salts thereof, for use in medical therapy.

[0824] [Embodiment 193] Compounds of formula I or Id as described in any of embodiments 1-49, 52-61, or 174-186, or pharmaceutically acceptable salts thereof, for prophylactic or therapeutic treatment of hepatitis B virus infection in animals.

[0825] [Embodiment 194] Use of a compound of formula I or Id, or a pharmaceutically acceptable salt thereof, as described in any of embodiments 1-49, 52-61, or 174-186, for preparing a drug for treating hepatitis B virus infection in animals.

[0826] [Embodiment 195] The animal is a human, according to any of embodiments 190 to 194, the method, compound, or use.

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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