Novel ligands for desialyl glycoprotein receptors

Abstract

This disclosure provides novel piperidine and guanosine-derived ligands that specifically bind to the desialyl glycoprotein receptor (ASGPR) and nucleotide analogs conjugated thereto, said nucleotide analogs being incorporated into oligonucleotides, including double-stranded oligonucleotides such as siRNA.

Description

Technical Field

[0001] sequence list

[0002] The nucleic acid sequences disclosed in this specification are for reference only. For patent purposes, identical sequences are also presented in a sequence listing formatted according to standard requirements. In the event of any discrepancy between the sequence and the standard sequence listing, the sequence described in this specification shall prevail. Background Technology

[0003] The concept of using synthetic oligonucleotides to control gene expression dates back to the late 1970s, when the use of short synthetic oligonucleotides for targeted gene silencing was first demonstrated (Stephenson et al., Proc Natl Acad Sci. USA (1978) 75: 285-88). Following Stephenson's discovery, the elucidation of the RNA interference pathway regulating gene expression and the role of siRNA in this process greatly expanded scientists' understanding of posttranscriptional gene expression control in eukaryotic cells.

[0004] Synthetic oligonucleotides include single-stranded oligonucleotides, such as antisense oligonucleotides (“ASO”), antimiR, or antagomiR; and double-stranded oligonucleotides, such as small interfering RNA (siRNA). Both ASO and siRNA function by binding to target RNA through Watson-Crick base pairing, but their mechanisms of action differ. In antisense techniques, ASO forms a DNA-RNA duplex with the target RNA and inhibits mRNA translation or induces RNase H-dependent degradation of the target RNA through blocking mechanisms. In RNA interference techniques, siRNA binds to an RNA-induced silencing complex (“RISC”), in which one strand (“lagging strand” or “sense strand”) is substituted, and the remaining strand (“guide strand” or “antisense strand”) cooperates with the RISC to bind to complementary RNA (target RNA). Once bound, the target RNA is cleaved by the RNA endonuclease Argonaute (AGO) in the RISC and subsequently further degraded by RNA exonucleases.

[0005] The main challenges in developing oligonucleotide therapeutics, such as siRNA therapeutics, include (i) poor compound stability, (ii) low efficiency of in vivo delivery to target cells, and (iii) side effects such as “off-target” gene silencing and unintended immune stimulation. Among these, the most significant obstacles are the targeted delivery and subsequent cellular uptake of siRNA. To overcome some of these obstacles, researchers have explored various chemical modifications of oligonucleotides, including (i) sugar modifications, (ii) nucleotide inter-bond modifications, and (iii) nucleobase modifications. While these chemical modifications have led to increased stability and reduced immunogenicity of siRNA, they are still insufficient to deliver these large, negatively charged macromolecules across the negatively charged phospholipid bilayer of the cell membrane and into the cytoplasm.

[0006] To this end, some research groups have used N-acetylgalactosamine (GalNAc) to target siRNAs attached to it to hepatocytes that express GalNAc-binding asialic acid glycoprotein receptor (ASGPR) and internalize ASGPR-bound siRNA-GalNAc conjugates via endocytosis (see, for example, Nair et al., J Am Chem Soc. (2014) 136: 16958-61). ASGPR is a calcium-dependent, carbohydrate-specific, transmembrane C-type lectin that is mainly expressed on the sinusoidal surface of hepatocytes. It plays a key role in serum glycoprotein turnover by mediating the endocytosis and lysosomal degradation of glycoproteins with exposed terminal galactose or GalNAc residues (Roggenbuck et al., Auto Immun Highlights. (2012) 3(3): 119-25; D'Souza et al., J Controlled Release (2015) 203: 126-39).

[0007] Given the importance of delivering therapeutic siRNAs to target cells in a tissue-specific manner, there is still a need to develop stable molecular moieties that can be readily conjugated to therapeutic agents (e.g., siRNA and ASO) and bind specifically to molecules expressed in the target tissue. Summary of the Invention

[0008] This disclosure provides for compounds of formula (I).

[0009] Or its pharmaceutically acceptable salt.

[0010] in:

[0011] B is a heterocyclic nucleobase;

[0012] P1 and P2 are each independently H, reactive phosphorus group, or protecting group;

[0013] Y is NR1 or NC (=0)-R1, where R1 is -L-R3.

[0014] L is a C1-C25 hydrocarbon chain optionally interrupted or terminated by one or more -O-, -C(O)-, -N(Re)-, -N(Re)-C(O)-O-, -OC(O)-N(Re)-, -N(Re)-C(O)-N(Rf)-, -C(O)-N(Re)-, -N(Re)-C(O)-, -OC(O)-, -C(O)-O-, or -OC(O)-O-; Re and Rf are each independently hydrogen, alkyl, alkenyl, alkynyl, alkoxy, aryloxy, Hydroxyl, hydroxy, or haloalkyl, wherein the C1-C25 hydrocarbon chain is optionally substituted with one or more -L'-R3, wherein L' is a C1-C25 hydrocarbon chain optionally interrupted by one or more -O-, -C(O)-, -N(Re)-, -N(Re)-C(O)-O-, -OC(O)-N(Re)-, -N(Re)-C(O)-N(Rf)-, -C(O)-N(Re)-, -N(Re)-C(O)-, -OC(O)-, -C(O)-O- or -OC(O)-O-;

[0015] R3 is the cell-targeting portion of formula (II) or its protected derivative:

[0016]

[0017] in:

[0018] R3 targets the mammalian (optionally human) desialylate glycoprotein receptor (ASGPR).

[0019] A1, A2, and A3 are independently H, hydroxyl, alkoxy, acyloxy, aryloxy, aryloxy, alkoxycarbonyl, aryloxycarbonyl, oxo (=O), or unsubstituted or optionally substituted by one or more groups selected from the following (C1-C20) alkyl groups: OH, (C3-C8) cycloalkyl, (C3-C14) heterocyclic, (C6-C14) aryl, (C5-C14) heteroaryl, -O-Z5, -N(Z5)(Z6), -S-Z5, -CN, -C(=M)-O-Z5, -OC(=M)-Z5, -C(=M)-N(Z5)(Z6), and -N(Z5)-C(=M)-Z6, wherein:

[0020] M is O or S.

[0021] Z5 and Z6 are each independently H, (C1-C6)alkyl or (C6-C14)aryl, wherein the alkyl and aryl groups are unsubstituted or optionally substituted by one or more groups selected from the following: halogen, amino, hydroxyl, thiol, cyano, alkyl, alkoxy, aryloxy, acyloxy, aryloxy, carboxyl, alkoxycarbonyl, aryloxycarbonyl and arylalkoxycarbonyl.

[0022] A4 is -N(R4)2, -NH-C(=O)-R4 or in:

[0023] D2 and D3 are N, O or S;

[0024] R4 is H or an unsubstituted or optionally substituted (C1-C20) alkyl group selected from one or more of the following groups: halogen, amino, hydroxyl, thiol, cyano, alkyl, alkoxycarbonyl, aryloxycarbonyl, alkoxy, aryloxy, acyloxy, aryloxy, and carboxyl; and

[0025] X1, X2, Ra, Rb, Rc, and Rd are each independently H or -(C1-C6) alkyl.

[0026] On the other hand, this disclosure provides compounds of formula (I).

[0027] Or its pharmaceutically acceptable salt.

[0028] in:

[0029] B is a heterocyclic nucleobase;

[0030] P1 and P2 are each independently H, reactive phosphorus group, or protecting group;

[0031] Y is NR1 or NC (=0)-R1, where R1 is -L-R3.

[0032] L is a C1-C25 hydrocarbon chain optionally interrupted or terminated by one or more -O-, -C(O)-, -N(Re)-, -N(Re)-C(O)-O-, -OC(O)-N(Re)-, -N(Re)-C(O)-N(Rf)-, -C(O)-N(Re)-, -N(Re)-C(O)-, -OC(O)-, -C(O)-O-, or -OC(O)-O-; Re and Rf are each independently hydrogen, alkyl, alkenyl, alkynyl, alkoxy, aryloxy, Hydroxyl, hydroxy, or haloalkyl, wherein the C1-C25 hydrocarbon chain is optionally substituted with one or more -L'-R3, wherein L' is a C1-C25 hydrocarbon chain optionally discontinuously substituted with one or more -O-, -C(O)-, -N(Re)-, -N(Re)-C(O)-O-, -OC(O)-N(Re)-, -N(Re)-C(O)-N(R()-, -C(O)-N(Re)-, -N(Re)-C(O)-, -OC(O)-, -C(O)-O- or -OC(O)-O-;

[0033] R3 is the cell-targeting moiety of formula (IVA) or (IVB) or its protected derivative:

[0034]

[0035] in:

[0036] R3 targets the mammalian (optionally human) desialylate glycoprotein receptor (ASGPR).

[0037] R6 is H or an unsubstituted or optionally substituted (C1-C6) alkyl group selected from one or more of the following groups: halogen, amino, hydroxyl, thiol, alkyl, alkoxy, aryloxy, carboxyl, alkoxycarbonyl and aryloxycarbonyl.

[0038] A5, A6, A7, and A'7 are independently H, hydroxyl, alkoxy, acyloxy, aryloxy, aryloxy, alkoxycarbonyl, aryloxycarbonyl, amino, or unsubstituted or optionally substituted with one or more groups selected from the following (C1-C20) alkyl groups: OH, (C3-C8) cycloalkyl, (C3-C14) heterocyclic, (C6-C14) aryl, (C5-C14) heteroaryl, -O-Z7, -N(Z7)(Z8), -S-Z7, -CN, -C(=Q)-O-Z7, -OC(=Q)-Z7, -C(=Q)-N(Z7)(Z8), and -N(Z7)-C(=Q)-Z8, wherein:

[0039] Q is either O or S.

[0040] Z7 and Z8 are each independently H, (C1-C6)alkyl or (C6-C14)aryl, and neither group is substituted or optionally substituted by one or more groups selected from halogen atoms and (C1-C6)alkyl.

[0041] A8 and A9 are each independently H, halogen, OH (or its tautomeric oxo group (=O)), -N(R7)2, -NHR7, or -NH-C(=O)-R7, wherein R7 is hydrogen or an unsubstituted or optionally substituted (C1-C20) alkyl group selected from: halogen atom, alkoxy, aryloxy, (C1-C6) alkyl, (C3-C8) cycloalkyl, (C3-C14) heterocyclic, (C6-C14) aryl, (C5-C14) heteroaryl; and

[0042] X1, X2, Ra, Rb, Rc, and Rd are each independently H or -(C1-C6) alkyl.

[0043] On the other hand, this disclosure provides compounds of formula (III).

[0044] Or its pharmaceutically acceptable salt.

[0045] in:

[0046] A1, A2, and A3 are independently H, hydroxyl, alkoxy, acyloxy, aryloxy, aryloxy, alkoxycarbonyl, aryloxycarbonyl, oxo (=O), or unsubstituted or optionally substituted by one or more groups selected from the following (C1-C20) alkyl groups: halogen, hydroxyl, (C3-C8) cycloalkyl, (C3-C14) heterocyclic, (C6-C14) aryl, (C5-C14) heteroaryl, -O-Z5, -N(Z5)(Z6), -S-Z5, -CN, -C(=M)-O-Z5, -OC(=M)-Z5, -C(=M)-N(Z5)(Z6), and -N(Z5)-C(=M)-Z6, wherein:

[0047] M is O or S.

[0048] Z5 and Z6 are each independently H, an unsubstituted or optionally substituted (C1-C6) alkyl group selected from one or more of the following groups: halogen, amino, hydroxyl, alkoxy, aryloxy, carboxyl, alkoxycarbonyl, aryloxycarbonyl and carbonyloxy.

[0049] A4 is -N(R4)2, -NC(=O)-R4 or in:

[0050] D2 and D3 are N, O or S;

[0051] R4 is H or an unsubstituted or optionally substituted (C1-C20) alkyl group selected from one or more of the following groups: halogen, amino, hydroxyl, thiol, cyano, alkyl, alkoxycarbonyl, aryloxycarbonyl, alkoxy, aryloxy, acyloxy, aryloxy, and carboxyl.

[0052] B1 can be H, benzyl ester, -L-R5, or -(CO)-L-R5, where:

[0053] L is a C1-C25 hydrocarbon chain optionally interrupted or terminated by one or more -O-, -C(O)-, -N(Re)-, -N(Re)-C(O)-O-, -OC(O)-N(Re)-, -N(Re)-C(O)-N(Rf)-, -C(O)-N(Re)-, -N(Re)-C(O)-, -OC(O)-, -C(O)-O-, or -OC(O)-O-; Re and Rf are each independently hydrogen, alkyl, alkenyl, alkynyl, alkoxy, aryloxy, hydroxyalkyl, hydroxyl The C2-C25 hydrocarbon chain is optionally substituted with one or more -L'-R5 groups, wherein L' is a C2-C25 hydrocarbon chain optionally interrupted by one or more -O-, -C(O)-, -N(Re)-, -N(Re)-C(O)-O-, -OC(O)-N(Re)-, -N(Re)-C(O)-N(Rf)-, -C(O)-N(Re)-, -N(Re)-C(O)-, -OC(O)-, -C(O)-O- or -OC(O)-O-; and

[0054] R5 is H, OH, benzyl, benzyloxy, or a nucleoside, nucleoside analog, nucleotide, or nucleotide analog, such as a nucleoside analog of formula (I).

[0055] On the other hand, this disclosure provides compounds of formula (V).

[0056] Or its pharmaceutically acceptable salt.

[0057] in:

[0058] R6 is H or an unsubstituted or optionally substituted (C1-C6) alkyl group selected from one or more of the following groups: halogen, amino, hydroxyl, thiol, alkoxy, aryloxy, carboxyl, alkoxycarbonyl and aryloxycarbonyl.

[0059] A5, A6, A7, and A'7 are each independently a (C1-C20) alkyl group consisting of H, hydroxyl, alkoxy, acyloxy, aryloxy, aryloxy, amino, or unsubstituted or optionally substituted with one or more groups selected from the following: halogen, OH, (C3-C8)cycloalkyl, (C3-C14) heterocyclic, (C6-C14) aryl, (C5-C14) heteroaryl, -O-Z7, -N(Z7)(Z8), -S-Z7, -CN, -C(=Q)-O-Z7, -OC(=Q)-Z7, -C(=Q)-N(Z7)(Z8), and -N(Z7)-C(=Q)-Z8, wherein:

[0060] Q is either O or S.

[0061] Z7 and Z8 are each independently H or (C1-C6) alkyl groups that are unsubstituted or optionally substituted by one or more groups selected from halogen atoms and (C1-C6) alkyl groups;

[0062] A8 and A9 are each independently H, halogen, OH (or its tautomeric oxo group (=O)), -N(R7)2, -NHR7 or -NC(=O)-R7, wherein R7 is hydrogen or an unsubstituted or optionally substituted (C1-C20) alkyl group selected from one or more of the following groups: halogen atom, alkoxy, aryloxy, (C1-C6) alkyl, (C3-C8) cycloalkyl, (C3-C14) heterocyclic, (C6-C14) aryl, (C5-C14) heteroaryl;

[0063] B2 and B'2 are each independently -H, -OH, -OR8, -COOH, -C(O)-NR8R'8, -NH2, -NHR8, -NH-C(O)-R8, -OP(O)(OH)2, -OP(O)(OR8)(OR'8) or optionally substituted with -OH (C1-C6) alkyl groups, wherein R8 and R'8 are independently H or -L-R9, wherein

[0064] L is a C1-C25 hydrocarbon chain optionally interrupted or terminated by one or more -O-, -C(O)-, -N(Re)-, -N(Re)-C(O)-O-, -OC(O)-N(Re)-, -N(Re)-C(O)-N(Rf)-, -C(O)-N(Re)-, -N(Re)-C(O)-, -OC(O)-, -C(O)-O- or -OC(O)-O-; Re and Rf are each independently hydrogen, alkyl, alkenyl, alkynyl, alkoxy, aryloxy, hydroxyl Alkyl, hydroxyl, or haloalkyl, wherein the C1-C25 hydrocarbon chain is optionally substituted with one or more -L'-R9, wherein L' is a C1-C25 hydrocarbon chain optionally interrupted by one or more -O-, -C(O)-, -N(Re)-, -N(Re)-C(O)-O-, -OC(O)-N(Re)-, -N(Re)-C(O)-N(Rf)-, -C(O)-N(Re), -OC(O)-(Re), -C(O)-O-(Re) or -OC(O)-O-;

[0065] R9 is H, OH, benzyl, benzyloxy, or a nucleoside, nucleoside analog, or nucleotide or nucleotide analog, such as a nucleoside analog of formula (I); and

[0066] When B2 is CH2OH, B2' is OH, A5 is H, A6 is OH, A7 is H, A7' is OH, A9 is H, R6 is H, and A8 is not NH2.

[0067] On the other hand, this disclosure provides oligonucleotides comprising one or more compounds of formula (VI):

[0068] Or its pharmaceutically acceptable salt.

[0069] in:

[0070] B is a heterocyclic nucleobase;

[0071] One of T1 and T2 is a compound of formula (VI) attached to an internucleotide linking group of an oligomer, and the other of T1 and T2 is H, a protecting group, a phosphorus moiety, or a compound of formula (VI) attached to an internucleotide linking group of an oligomer.

[0072] Y is NR1 or NC (=0)-R1, where R1 is -L-R3.

[0073] L is a C1-C25 hydrocarbon chain optionally interrupted or terminated by one or more -O-, -C(O)-, -N(Re)-, -N(Re)-C(O)-O-, -OC(O)-N(Re)-, -N(Re)-C(O)-N(Rf)-, -C(O)-N(Re)-, -N(Re)-C(O)-, -OC(O)-, -C(O)-O-, or -OC(O)-O-; Re and Rf are each independently hydrogen, alkyl, alkenyl, alkynyl, alkoxy, aryloxy, hydroxyalkyl, Hydroxyl or haloalkyl, wherein the C1-C25 hydrocarbon chain is optionally substituted with one or more -L'-R3, wherein L' is a C1-C25 hydrocarbon chain optionally interrupted by one or more -O-, -C(O)-, -N(Re)-, -N(Re)-C(O)-O-, -OC(O)-N(Re)-, -N(Re)-C(O)-N(Rf)-, -C(O)-N(Re)-, -N(Re)-C(O)-, -OC(O)-, -C(O)-O- or -OC(O)-O-;

[0074] R3 is the ASGPR-binding cell-targeting fraction of formula (II), (IVA), or (IVB); and

[0075] X1, X2, Ra, Rb, Rc, and Rd are each independently H or -(C1-C6) alkyl.

[0076] This disclosure also provides a method for delivering oligonucleotides to liver (hepatic) cells of a human subject in need, the method comprising administering (e.g., via subcutaneous or intravenous injection or via portal vein injection) the oligonucleotides of this disclosure to the subject.

[0077] This disclosure also relates to the use of the oligonucleotides of this specification in the manufacture of pharmaceutical preparations for the treatment of human subjects in need.

[0078] This disclosure also provides oligonucleotides, as described herein, for the treatment of human subjects in need.

[0079] This disclosure also provides a method for preparing a liver-targeted therapeutic agent (e.g., a protein, peptide, peptide mimic, small molecule, or oligonucleotide), the method comprising reacting a therapeutic portion with a compound of this specification to allow the compound to conjugate with the therapeutic portion, thereby producing a liver-targeted therapeutic agent.

[0080] This disclosure also provides a method for delivering a therapeutic agent (e.g., a protein, peptide, peptide mimic, small molecule, or polynucleotide) to liver (hepatic) cells of a human subject in need, the method comprising administering to the subject a therapeutic portion of a compound conjugated to this specification.

[0081] Other features, objects, and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that while the detailed description indicates embodiments and aspects of the invention, it is given by way of illustration only and not by way of limitation. Various changes and modifications within the scope of the invention will be apparent to those skilled in the art based on the detailed description. Attached Figure Description

[0082] Figure 1 Describe the schemes for synthesizing compounds 2, 3, and 23.

[0083] Figure 2 Describe a scheme for the synthesis of compound 30.

[0084] Figure 3 Describe a scheme for the synthesis of compound 37.

[0085] Figure 4 Describe the scheme for synthesizing compound 47.

[0086] Figure 5 Describe the scheme for synthesizing compound 58.

[0087] Figure 6 Describe the schemes for synthesizing compounds 71, 72 and 73.

[0088] Figure 7 A scheme is described for synthesizing a simplified precursor for piperidine-derived ASGPR-binding molecules.

[0089] Figure 8 Describe a scheme for synthesizing the precursor of the connector.

[0090] Figure 9 Schemes for synthesizing compounds 112, 117, 119, 120 and 121 are described.

[0091] Figure 10 Schemes for synthesizing compounds 128, 129, 131, and 132 are described.

[0092] Figure 11 Describe the schemes for synthesizing compounds 138 and 140.

[0093] Figure 12 Schemes for synthesizing compounds 146, 147, 148, 153, and 154 are described.

[0094] Figure 13 Describe the schemes for synthesizing compounds 160, 161 and 162.

[0095] Figure 14 Describe the schemes for synthesizing compounds 180 and 181.

[0096] Figure 15 Describe a scheme for the synthesis of trimerized piperidine precursors.

[0097] Figure 16 Describe the protocol for synthesizing the targeted nucleotide precursor 218 (pre-lsT1).

[0098] Figure 17 Describe a protocol for synthesizing the targeted nucleotide precursor 230 (pre-lsT2).

[0099] Figure 18 Describe a protocol for synthesizing the targeted nucleotide precursor 246 (pre-lsT3).

[0100] Figure 19 A scheme for synthesizing the targeted nucleotide precursor 249 (pre-lpT1) is described.

[0101] Figure 20A Describe a scheme for the synthesis of targeted nucleosides 254 and 258.

[0102] Figure 20B Describe a scheme for the synthesis of targeted nucleoside 260.

[0103] Figure 21A Describe the scheme for synthesizing trimeric targeted nucleotides 261 and 262.

[0104] Figure 21B Describe the schemes for synthesizing trimeric targeted nucleotides 263, 264, and 265.

[0105] Figure 22 A scheme for synthesizing trimeric ASGPR binder 267 is described.

[0106] Figure 23 A scheme for synthesizing trimeric ASGPR binder 268 is described.

[0107] Figure 24 A scheme for synthesizing trimeric ASGPR binder 269 is described.

[0108] Figure 25A This is a graph showing the relative serum TTR protein levels at blood collection time points before and after subcutaneous (sc) administration of siRNA 1-0 (negative control), siRNA 1-1 (positive control), and siRNA 1-3 as indicated. The vertical axis represents the TTR serum level relative to pre-administration serum SEM. The horizontal axis represents the number of days after subcutaneous administration.

[0109] Figure 25BThis is a graph showing the relative serum TTR protein levels at blood collection time points before and after subcutaneous administration of siRNA1-0 (negative control), siRNA1-2 (positive control), siRNA1-4, siRNA1-5, and siRNA1-6 as indicated. The vertical axis represents the TTR serum level relative to pre-administration serum SEM. The horizontal axis represents the number of days after subcutaneous administration. Detailed Implementation

[0110] This disclosure provides novel ligands for asialic acid glycoprotein receptors (ASGPRs), such as human ASGPRs. These ASGPR-binding ligands or their chemically protected analogs are piperidine or guanosine derivatives listed in Tables C, D, E, F, G, H, J, K, L, and M or described in Examples 1-25, and can be conjugated with therapeutic nucleic acid molecules to target them to tissues expressing ASGPRs, such as the liver. For example, the ASGPR ligands of the present invention can be conjugated with nucleotides or nucleotide analogs incorporated into therapeutic oligonucleotides, including double-stranded oligonucleotides, such as dsRNA (e.g., siRNA), and single-stranded oligonucleotides, such as antisense oligonucleotides. Oligonucleotides containing these ASGPR-targeting nucleotide analogs exhibit superior biological activity, including efficient delivery and uptake in specific cells or tissues (e.g., hepatocytes), superior in vivo potency, and significant in vitro stability. These ASGPR-targeting oligonucleotides can be used to silence (e.g., reduce or eradicate) the expression of target genes. In specific embodiments, the present invention includes specific piperidine and guanosine-derived ASGPR-binding ligands and nucleotide analogues conjugated thereto, for incorporation into double-stranded RNA (dsRNA), such as siRNA, which can hybridize with messenger RNA (mRNA) of interest in order to reduce or block the expression of target genes of interest.

[0111] Unless otherwise specified, all technical and scientific terms used herein are the same as those commonly used by one of ordinary skill in the art to which this invention pertains.

[0112] "alkyl" or "hydrocarbon chain" refers to 1-20, 1-18, 1-16, 1-12, 1-10, preferably 1-8, more preferably 1-6 unsubstituted or substituted hydrogen-saturated carbon groups, said hydrogen-saturated carbons being linked in a linear, branched, or cyclic manner, including combinations of linear, branched, and cyclic linkages. Non-limiting examples include methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, and pentyl.

[0113] "Cycloalkyl" refers to a monocyclic or polycyclic non-aromatic group containing carbon and hydrogen and which may be saturated or partially unsaturated. Cycloalkyl groups include groups having 3 to 10 ring atoms (e.g., C3-C4). 10(Cycloalkyl). Whenever appearing herein, numerical ranges such as “3 to 10” refer to each integer within the given range; for example, “3 to 10 carbon atoms” means that a cycloalkyl group can consist of 3, 4, 5, etc., carbon ring atoms, up to and including 10 carbon ring atoms. In some embodiments, it is a C3-C8 cycloalkyl group. In some embodiments, it is a C3-C5 cycloalkyl group. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, and norbornyl. The term “cycloalkyl” also refers to a spirocyclic linked ring system in which the cycloalkyl rings share a single carbon atom.

[0114] "Heterocyclic alkyl" refers to a 3- to 18-membered nonaromatic ring (e.g., C3-C1) containing two to twelve ring carbon atoms and one to six heteroatoms selected from nitrogen, oxygen, and sulfur. 18 Heterocyclic alkyl groups. Whenever appearing herein, numerical ranges such as “3 to 18” refer to each integer within the given range; for example, “3 to 18 ring atoms” means that a heterocyclic alkyl group can consist of 3 ring atoms, 4 ring atoms, etc., up to and including 18 ring atoms. In some embodiments, it is C5-C 10 Heterocyclic alkyl groups. In some embodiments, they are C4-C6. 10 Heterocyclic alkyl groups. In some embodiments, they are C3-C 10 Heterocyclic alkyl groups. Heterocyclic alkyl groups can be monocyclic, bicyclic, tricyclic, or tetracyclic ring systems, and may include fused or bridged ring systems. The heteroatoms in the heterocyclic alkyl group may optionally be oxidized. If present, one or more nitrogen atoms may optionally be quaternized. Heterocyclic alkyl groups may be partially or fully saturated. Heterocyclic alkyl groups may be attached to the remainder of the molecule by any atom of the ring. Examples of such heterocyclic alkyl groups include, but are not limited to, 6,7-dihydro-5H-cyclopentyl[b]pyridine, dioxopentyl, thienyl[1,3]disulfidecyclopentyl, decahydroisoquinolinyl, imidazolinyl, imidazoalkyl, isothiazolyl, isoxazolyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopiperidinyl, 2-oxopiperylalkyl, oxazolyl, piperidinyl, piperazinyl, 4-piperidinyl, pyrrolylalkyl, pyrazolylalkyl, quininecycloyl, thiazoalkyl, tetrahydrofuranyl, trisulfidecyclopentyl, tetrahydropyranyl, thiomorpholinyl, thiomorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. In some embodiments, the heterocyclic alkyl group is aziridinyl, aziridine, pyrrolyl, piperidinyl, homopiperidinyl, morpholinyl, thiomorpholinyl, piperazine, tetrahydrofuranyl, tetrahydrothiopheneyl, dihydrooxazolyl, tetrahydropyranyl, tetrahydrothiopheneyl, dihydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and benzoxazine, preferably dihydrooxazolyl and tetrahydrofuranyl.

[0115] "Halogen" refers to any one of the halogen atoms: fluorine (F), chlorine (Cl), bromine (Br), or iodine (I). A specific example of such a halogen group is fluorine.

[0116] "Amino" refers to both unsubstituted and substituted amino groups, such as primary, secondary, tertiary, and quaternary amines. Specifically, "amino" refers to -NR. a R b , where R a and R b All are directly bonded to nitrogen (N) and can be independently selected from hydrogen, deuterium, hydroxyl, cyano, formyl, alkyl, haloalkyl, alkenyl, haloalkenyl, alkynyl, haloalkynyl, alkoxycarbonyl, alkoxy, haloalkoxy, thioalkoxy, halothioalkoxy, alkanoyl (acyl), haloalkanoyl, thioalkanoyl, halothioalkanoyl, carboxyl, carbonyloxy, halocarbonyloxy, carbonylthio, halocarbonylthio, thiocarbonyloxy, halothiocarbonylthio, halothiocarbonylthio, nitrogen-protecting group, -(CO)-alkyl, -(CO)-O-alkyl, or -S(O). n R c (n = 0 to 2, R) c Directly connected to S), where R c It is independently selected from hydrogen, deuterium, amino, hydroxyl, thiol, alkyl, haloalkyl, aryl, heteroaryl, alkenyl, haloalkenyl, alkynyl, haloalkynyl, alkoxy, haloalkoxy, thioalkoxy and halothioalkoxy.

[0117] "Aryl" refers to unsubstituted or substituted C6-C 14 Aromatic hydrocarbons. For example, the aryl group can be phenyl, naphthyl, or fluorenyl.

[0118] "Heteroaryl" refers to a C6-C group having one or more heteroatoms, such as N, O, or S. 14Aromatic hydrocarbons. Heteroaryl groups can be substituted or unsubstituted. Examples of heteroaryl groups include, but are not limited to, acrylonitrile, acrylidine, benzimidazolyl, benzoindolyl, 1,3-benzodioxazolyl, benzoxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dihydrodibenzoxepinyl, benzo[b][1,4]oxazinyl, 1,4-benzodioxane, benzonaphthuronyl, benzoxazolyl, benzodioxacyclopentenyl, benzodioxinyl, benzopyranyl, benzopyranoneyl, benzofuranyl, benzofuranoneyl, benzofurazanyl, benzothiazolyl, benzothiophene, benzothiophene[3,2-d]pyrimidinyl, benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridyl, and carbazoleyl. , cyclopentanyl, cyclopentanyl[d]pyrimidinyl, 6,7-dihydro-5H-cyclopentanyl[4,5]thieno[2,3-d]pyrimidinyl, 5,6-dihydrobenzo[h]quinazolinyl, 5,6-dihydrobenzo[h]cynolinyl, 6,7-dihydro-5H-benzo[6,7]cycloheptane[1,2-c]pyridazinyl, dibenzofuranyl, dibenzothienoyl, furanyl, furazanyl, furanoneyl, furano[3,2-c]pyridinyl, 5,6,7,8,9,10-hexahydrocyclooctyl[d]pyrimidinyl, 5,6,7,8,9,10-hexahydrocyclooctyl[d]pyridazinyl, 5,6,7,8,9,10-hexahydrocyclooctyl[d]pyridazinyl, isothiazolyl, imidazolyl, indazoleyl, Indole, indazole, isoindole, indololin, isoindololin, isoquinolin, indene, isoxazolyl, 5,8-methyl-5,6,7,8-tetrahydroquinazolinyl, naphridinyl, 1,6-naphthidone, oxadiazolyl, 2-oxo-achenginyl, oxazolyl, ethylene oxide, 5,6,6a,7,8,9,10,10a-octahydrobenzo[h]quinazolinyl, 1-phenyl-1H-pyrroleyl, phenazinyl, phenothiazinyl, phenotoxazinyl, phthalazinyl, pteridinyl, purine, pyranyl, pyrroleyl, pyrazolyl, pyrazolo[3,4-d]pyrimidinyl, pyridinyl, pyrido[3,2-d]pyrimidinyl, pyrido[3,4-d]pyrimidinyl, pyrazinyl, pyrimidinyl The following are listed: pyridyl, pyrroleyl, quinazolinyl, quinoxalyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, 5,6,7,8-tetrahydroquinazolinyl, 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl, 6,7,8,9-tetrahydro-5H-cycloheptane[4,5]thieno[2,3-d]pyrimidinyl, 5,6,7,8-tetrahydropyrido[4,5-c]pyridyl, thiazolyl, thiadiazolyl, thiaranyl, triazolyl, tetrazolyl, triazineyl, thieno[2,3-d]pyrimidinyl, thieno[3,2-d]pyrimidinyl, thieno[2,3-c]pyrimidinyl, and thiophenyl / thienyl.In some embodiments, the heteroaryl group may be dithiazinyl, furanyl, imidazolyl, indolyl, isoquinolinyl, isoxazolyl, oxadiazolyl (e.g., (1,3,4)-oxadiazolyl or (1,2,4)-oxadiazolyl), oxazolyl, pyrazinyl, pyrazolyl, pyridinyl, pyrimidinyl, m-diazophenyl, pyrroloyl, quinolinyl, tetrazolyl, thiazolyl, thiophene, triazinyl, (1,2,3)-triazolyl, (1,2,4)-triazolyl, 1,3,4-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,4-triazolyl, 1,3,4-thiadiazolyl, 5-amino-1,2,4-oxadiazolyl, 5-amino-1,3,4-oxadiazolyl, 5-amino-1,3,4-oxadiazolyl, 3-methyl-1,2,4-oxadiazolyl, 5-methyl 5-(trifluoromethyl)-1,2,4-oxadiazolyl, 5-(methylamino)-1,2,4-oxadiazolyl, 5-(aminomethyl)-1,2,4-oxadiazolyl, 5-(aminomethyl)-1,3,4-oxadiazolyl, 5-amino-4-cyanoxazolyl, 5,6-dichloro-1H-indolyl, 5,6-difluoro-1H-indolyl 5-Chloro-1H-indolyl, 5,6-dibromo-1H-indolyl, 5-fluoro-1H-indolyl, 5-methoxy-1H-indolyl, 7-fluoro-1H-indolyl, 6-cyano-1H-indolyl, 5-cyano-1H-indolyl, 4-fluoro-1H-indolyl, 5,6-difluoro-1H-indolyl, 6-fluoro-1H-indolyl, or 5,7-difluoro-1h-indolyl.

[0119] The substituents on the aryl or heteroaryl group can be alkyl (e.g., C1-C6 alkyl), alkoxy (e.g., C1-C6 alkoxy), amino, cyano, halo (e.g., fluorine, bromine, and chlorine), alkylamino (e.g., C1-C6 alkylamino), methyleneamino, nitro, or hydroxyl. The heteroaryl group can have two, three, or four substituents.

[0120] "Heterocycle" refers to an unsubstituted or substituted C6-C ring containing one or more heteroatoms, such as N, O, or S. 14 Cyclic hydrocarbons.

[0121] "Alkoxy" refers to an alkyl group (-O-alkyl) bonded to an oxygen atom.

[0122] "Aryl group" refers to an aryl group (-O-aryl) bonded to an oxygen atom.

[0123] "Carbonyl" refers to -(CO)-, where (CO) represents oxygen bonded to carbon via a double bond.

[0124] "Alkyl" or "acyl" refers to an alkyl group [-(CO)-alkyl] that is attached to a carbonyl group.

[0125] "Aromatic acyl group" refers to an aryl group [-(CO)-aryl] that is attached to a carbonyl group.

[0126] "Carboxyl group" refers to the carboxylic acid group [-(CO)-OH].

[0127] "Alkoxycarbonyl" refers to a carboxylic acid ester group [-(CO)-O-alkyl], wherein the alkyl group may be further substituted with, for example, an aryl group.

[0128] "Aryloxycarbonyl" refers to a carboxylic acid ester group [-(CO)-O-aryl], wherein the aryl group may be further substituted by, for example, alkyl or aryl groups.

[0129] "Arylalkoxycarbonyl" refers to a carboxylic acid ester group [-(CO)-O-alkylaryl], wherein the aryl group may be further substituted with, for example, alkyl or aryl groups.

[0130] "Carbonyloxy group" refers to an alkyl acyl group (or acyl group) [-O-(CO)-alkyl] bonded to an oxygen atom.

[0131] "Aryloxy group" refers to an aryl group [-O-(CO)-aryl] bonded to an oxygen atom.

[0132] The terms “alkyl,” “cycloalkyl,” “alkenyl,” “alkynyl,” “aryl,” “heteroaryl,” and “heterocyclic” can also refer to the corresponding “alkylene,” “cycloalkylene,” “alkenyl,” “alkynyl,” “arylene,” “heteroaryl,” and “heterocyclic” formed by removing two hydrogen atoms, respectively.

[0133] The term "heterocyclic nucleobase" refers to any nitrogen-containing heterocyclic moiety that, when incorporated into a polymeric structure, can pair with complementary nucleobases or nucleobase analogs (i.e., derivatives of nucleobases) to form Watson-Crick type hydrogen bonds and stacking interactions.

[0134] Unless otherwise stated, the term "heterocyclic nucleobase" herein refers to a nitrogen-containing heterocyclic group that may be linked to a optionally substituted ribose ring, optionally substituted deoxyribose ring, optionally substituted dioxane ring, or optionally substituted morpholinyl ring according to this disclosure. In some embodiments, the heterocyclic nucleobase may be selected from optionally substituted purine bases or optionally substituted pyrimidine bases. The term "purine base" is used herein in its ordinary sense as understood by those skilled in the art and includes its tautomers. Similarly, the term "pyrimidine base" is used herein in its ordinary sense as understood by those skilled in the art and includes its tautomers. A non-limiting list of optionally substituted purine bases includes purine, adenine, guanine, hypoxanthine, xanthine, alloxanthine, 7-alkylguanine (e.g., 7-methylguanine), theobromine, caffeine, uric acid, and isoguanine. Examples of pyrimidine bases include, but are not limited to, cytosine, thymine, uracil, 5,6-dihydrouracil, and 5-alkylcytosine (e.g., 5-methylcytosine). Other non-limiting examples of heterocyclic nucleobases include diaminopurine, 8-oxo-N6 alkyladenine (e.g., 8-oxo-Nemethyladenine), 7-denitroxanthine, 7-denitroguanine, 7-denitroadenine, N4,N4-bridged ethylidene cytosine, N6,N6-bridged ethylidene-2,6-diaminopurine, 5-halouracil (e.g., 5-fluorouracil and 5-bromouracil), pseudoisocytosine, isocytosine, isoguanine, 1,2,4-triazole-3-carboxamide, and other heterocyclic nucleobases described in U.S. Patent Nos. 5,432,272 and 7,125,855, which disclose additional heterocyclic bases, which are incorporated herein by reference. In some embodiments, the heterocyclic nucleobase may optionally be substituted with an amine or enol protecting group.

[0135] As used herein, the term "protecting group" refers to any atom or group of atoms added to a molecule to prevent unwanted chemical reactions of existing groups in the molecule. A "protecting group" can be an unstable chemical motif known in the art for protecting reactive groups such as hydroxyl, amino, and thiol groups from unwanted or untimely reactions during chemical synthesis. Protecting groups are typically used selectively and / or orthogonally to protect sites at other reaction sites during a reaction, and can then be removed to leave the unprotected group as is, or can be used for further reactions.

[0136] Examples of protecting groups are described in TW Greene and P GMWuts, Protective Groups in Organic Synthesis, 3rd ed., John Wiley & Sons, 1999, and JFW McOmie, Protective Groups in Organic Chemistry, Plenum Press, 1973, both of which are incorporated herein by reference for the limited purpose of disclosing suitable protecting groups. Protecting groups can be selected in a manner that makes them stable to certain reaction conditions and readily removable at convenient stages using methods known in the art.

[0137] A non-limiting list of protecting groups includes benzyl; substituted benzyl; alkyl carbonyl (acetyl or isobutyryl), aryl carbonyl, alkoxy carbonyl, and aryloxy carbonyl (e.g., tert-butoxy carbonyl (BOC)); arylalkyl carbonyl and arylalkoxy carbonyl (e.g., benzyloxy carbonyl); substituted methyl ether (e.g., methoxymethyl ether); substituted diethyl ether; substituted benzyl ether; tetrahydropyran ether; silyl ether (e.g., trimethylsilyl-, triethylsilyl-, triisopropylsilyl-, tert-butyl-). Dimethylsilyl-, triisopropylsiloxymethyl-, [2-(trimethylsilyl)ethoxy]methyl- or tert-butyldiphenylsilyl-); esters (e.g., benzoates); carbonates (e.g., methoxymethyl carbonates); sulfonates (e.g., toluenesulfonate or methanesulfonate); acyclic ketals (e.g., dimethyl acetals); cyclic ketals (e.g., 1,3-dioxane, 1,3-dioxolane, and those described herein); acyclic acetals; cyclic acetals (e.g., those described herein). Some); acyclic hemiacetals; cyclic hemiacetals; cyclic dithioacetals (e.g., 1,3-dithiane or 1,3-dithiopentane); orthoesters (e.g., those described herein) and triarylmethyl groups (e.g., triphenylmethyl; monomethoxytriphenylmethyl (MMTr); 4,4′-dimethoxytriphenylmethyl (DMTr); 4,4′,4″-trimethoxytriphenylmethyl (TMTr); and those described herein). Preferred protecting groups are selected from the group consisting of acetyl (Ac), benzene Formyl (Bzl), isobutyryl (iBu), phenylacetyl, dimethoxytribenzyl (DMT), methoxytribenzyl (MMT), triphenylmethyl (Trt), N,N-dimethylformamidinium, and 2-cyanoethyl (CE). Unless otherwise stated, abbreviations for any protecting groups, amino acids, and other compounds are as commonly used, recognized abbreviations, or those of the IUPAC-IUB Committee on Biochemical Nomenclature (see Biochem. 11: 942-944 (1972)).

[0138] As used herein, a “reactive phosphorus group” refers to a phosphorus-containing group contained in a nucleotide unit or nucleotide analog unit that can react with a hydroxyl or amino group contained in another molecule, and particularly in another nucleotide unit or another nucleotide analog unit, via a nucleophilic attack reaction. Typically, such reactions followed by an oxidation step produce a phosphate ester-type nucleoside internucleotide bond that links a first nucleotide unit or first nucleotide analog unit to a second nucleotide unit or second nucleotide analog unit.

[0139] In some embodiments, the reactive phosphorus group may be selected from the group consisting of: phosphoramide, H-phosphonate, alkyl-phosphonate, phosphate ester or phosphate ester analog, including but not limited to: native phosphate ester, thiophosphate ester, dithiophosphate ester, boroalkyl phosphate ester, boroalkyl thiophosphate ester, phosphonate ester, halogen-substituted phosphonate ester and phosphate ester, aminophosphate ester, phosphate diester, phosphate triester, thiophosphate diester, thiophosphate triester, diphosphate ester and triphosphate ester. Protecting groups at the nucleotide or nucleotide analog include hydroxyl, amine and phosphoramide protecting groups, which may be selected from the group consisting of: acetyl (Ac), benzoyl (Bzl), benzyl (Bn), isobutyryl (iBu), phenylacetyl, benzyloxymethyl acetal (BOM), β-methoxyethoxymethyl ether (MEM), methoxymethyl ether (MOM), p-methoxybenzyl ether (PMB), methylthiomethyl ether, neopentanoyl (Piv), tetrahydropyranyl (THP), triphenylmethyl (Trt), methoxytriphenylmethyl[(4-methoxyphenyl))diphenyl-1-methyl](MMT), dimethoxytriphenylmethyl, [bis-(4-methoxyphenyl)phenylmethyl (DMT), trimethylsilyl ether (TMS), tert-butyldimethylsilyl ether (TBDMS), triisopropylsiloxymethyl ether (TOM), triisopropylsilyl ether (TIPS), methyl ether, ethoxyethyl ether (EE), N,N-dimethylformamidinium and 2-cyanoethyl (CE).

[0140] I. Nucleotide modification

[0141] As used herein, the term "nucleotide" includes naturally occurring or modified nucleotides, or substituted portions. Modified nucleotides, also referred to herein as "nucleotide analogs," are non-naturally occurring nucleotides. Those skilled in the art will understand that guanine, cytosine, adenine, uracil, or thymine in a nucleotide can be substituted with other portions without significantly altering the base-pairing properties of the modified nucleotide. For example, a nucleotide containing inosine as its base can base-pair with a nucleotide containing adenine, cytosine, or uracil. Therefore, a nucleotide containing uracil, guanine, or adenine can be substituted in the nucleotide sequences of this disclosure with a nucleotide containing, for example, inosine. Sequences containing such substitution portions are included as embodiments of this disclosure. A modified nucleotide can also be a nucleotide in which its ribose portion is substituted with a non-ribose portion. As used herein, the terms "nucleoside" and "nucleoside analog" refer to nucleotides and nucleotide analogs, respectively, without their phosphate groups.

[0142] The nucleotide analogues disclosed herein may contain any modifications known in the art, including, for example, terminal modifications, base modifications, sugar modifications / substitutions, and backbone modifications.

[0143] End modifications may include, for example, 5' end modifications (e.g., phosphorylation, conjugation, and reverse linkage) and 3' end modifications (e.g., conjugation, DNA nucleotides, and reverse linkage).

[0144] Base modifications may include, for example, substitution with a stable base, a destabilized base, or a base that pairs with an expanded library of coordinates; base removal (base-free modification of nucleotides); or base conjugation.

[0145] Modification of the glycosyl group can include chemical modifications at the 2'-carbon atom or 2'-hydroxyl group of the ribosome, such as 2'-deoxy-2'-F (fluorine), 2'-OMe (methoxy), and 2'-O-methoxyethyl modifications. Although most sugar changes are located at the 2'-position, modifications at other positions (e.g., the 4'-position) are also permitted (Leydler et al., Antisense Res Dev. (1995) 5: 161-74).

[0146] Other chemical modifications to the sugar group may include linking the 2'-oxygen and 4'-carbon of the ribose scaffold in the nucleoside, producing so-called locked nucleic acids (“LNAs”). LNAs, also known as bicyclic nucleic acids, have been shown to have increased RNA-binding affinity (Koshin et al., Tetrahedron (1998) 54: 3607-30; Prakash et al., Chem Biodivers. (2011) 8: 1616-41), which leads to a significant increase in the melting temperature of the resulting double-stranded oligonucleotides. However, oligomers of fully LNA-modified oligonucleotides longer than eight nucleotides tend to aggregate. In contrast to the rigid nature of LNA modifications, highly flexible unlocked nucleic acids (“UNA”) modifications can also be incorporated into the nucleotide analogs described herein. UNA nucleosides do not have a C2'-C3' bond in the ribose. Due to their open-chain structure, UNA are conformationally unrestricted and have been used to modulate oligonucleotide flexibility (Mangos et al., J Am ChemSoc. (2003) 125: 654-61). In some cases, UNA inserts can reduce the double-strand melting temperature (Tm) by 5-10 °C / insertion. Furthermore, UNA inserts can promote antisense strand selection in RISC, and UNA modification of the siRNA guide strand seed region can reduce off-target events (Vaish et al., Nucleic Acids Res. (2011) 39: 1823-32). Bramsen et al. (Nucleic Acids Research (2010) 38(17): 5761-73) have reported siRNAs containing both UNA and LNA.

[0147] Furthermore, extended sugar ring systems, including six-membered morpholino ring systems (in which the ribose portion of the nucleoside is replaced by a morpholino ring), can also be incorporated into the nucleotide analogs described herein. Morpholino-based nucleosides form internucleotide bonds within oligonucleotides containing said nucleosides via the nitrogen atom of the morpholino subunit. Phosphodiamidomorpholino-based oligonucleotides (“PMO”) have been used in antisense technology (Corey et al., Genome Biology (2001) 2(5): Review 1015.1-1015; Partridge et al., Antisense Nucleic Acid Drug Dev. (1996) 6: 169-75). Examples of morpholino subunits are also disclosed in U.S. Patents 5,034,506; 5,166,315; 5,185,444; 5,698,685; and U.S. Patent Publication US2016US / 0186174.

[0148] The nucleotides or nucleotide analogs disclosed herein can be conjugated to a cell-targeting portion. Such nucleotides or nucleotide analogs are referred to as "targeting nucleotides". Nucleotides or nucleotide analogs that are not conjugated to a cell-targeting portion are referred to as "non-targeting nucleotides".

[0149] The nucleotide or nucleotide analog building blocks (targeted and untargeted) disclosed herein are used to synthesize oligonucleotides and to incorporate such nucleotides into oligonucleotides, and are referred to as nucleotide precursors. These targeted or untargeted nucleotide precursors exhibit specific chemical modifications necessary for automated oligonucleotide synthesis. Common functional groups are reactive phosphorus groups, such as phosphoramide, and specific protecting groups, such as DMT protecting groups.

[0150] Nucleotide bonds form the backbone of nucleic acid molecules. A nucleotide linker group is a chemical group that connects two adjacent nucleoside residues in a nucleic acid molecule. This includes (i) chemical groups connecting two adjacent nucleoside residues, (ii) chemical groups connecting a nucleoside residue to an adjacent nucleoside analog residue, and (iii) chemical groups connecting a first nucleoside analog residue to a second nucleoside analog residue, wherein the nucleoside analog residues may be the same or different. The terms “nucleoside bond,” “nucleoside linker group,” “nucleotide bond,” or “nucleotide linker group” are used interchangeably herein and refer to any linker or bond between two nucleoside units (i.e., a heterocyclic base moiety and a sugar moiety) as known in the art, including but not limited to phosphate esters, phosphate ester analogs, thiophosphate esters, phosphonates, guanidines, hydroxylamines, hydroxyhydrazines, amides, carbamates, alkyl groups, and substituted alkyl bonds.

[0151] Backbone modifications can include chemically modifying the internucleotide bonds by partially replacing the 3'-5' phosphodiester bonds with more stable ones to reduce susceptibility to nuclease degradation. A widely used modification is the partial or complete replacement of the phosphodiester backbone with thiophosphate bonds, where sulfur atoms replace non-bridging oxygen atoms. Backbone modifications can also include modifying or replacing phosphodiester bonds with one or more dithiophosphates, triphosphates, methyl and other alkylphosphonates, hypophosphonates, or aminophosphates. Another backbone modification that imparts greater stability to nucleic acids is the borane phosphate bond. In borane phosphate oligonucleotides, the non-bridging phosphodiester oxygen is partially replaced by an isoelectronic borane (-BH3).

[0152] The non-targeted nucleotide precursors discovered in this disclosure are described by Hofmeister et al. in WO 2019 / 170731. Examples are listed in Table A. Morpholine-type nucleotide precursors in the (2S,6R)-diastere series are abbreviated as “pre-l”, followed by a nucleobase (T, U, C, A, or G) and a number that specifies the substituent at the morpholine nitrogen. Analogs (2R,6R)-diastereomers are abbreviated with the additional “b”. The abbreviations of the corresponding nucleotides in oligonucleotide sequences are constructed according to the same rules, but without “pre”, and are also shown in Table A.

[0153] Table A

[0154]

[0155]

[0156]

[0157]

[0158] Based on the same scaffold, the targeted nucleotide precursor is described in the same application (Hofmeister et al., WO 2019 / 170731). ASGPR targeting is performed using GalNAc residues; the precursor molecule is abbreviated as "pre-lg," followed by a nucleobase and a number, specifying the linker between the morpholine nitrogen and the GalNAc residue. Examples are shown in Table B.

[0159] Table B:

[0160]

[0161]

[0162]

[0163] In some embodiments, the nucleotide analog precursors disclosed herein are compounds of general formula (I):

[0164]

[0165] in:

[0166] B is a heterocyclic nucleobase;

[0167] P1 and P2 are each independently H, reactive phosphorus group, or protecting group;

[0168] Y is NR1 or NC (=0)-R1, where R1 is -L-R3.

[0169] L is a C1-C25 hydrocarbon chain optionally interrupted or terminated by one or more -O-, -C(O)-, -N(Re)-, -N(Re)-C(O)-O-, -OC(O)-N(Re)-, -N(Re)-C(O)-N(Rf)-, -C(O)-N(Re)-, -N(Re)-C(O)-, -OC(O)-, -C(O)-O-, or -OC(O)-O-; Re and Rf are each independently hydrogen, alkyl, alkenyl, alkynyl, alkoxy, aryloxy, Hydroxyl, hydroxy, or haloalkyl, wherein the C1-C25 hydrocarbon chain is optionally substituted with one or more -L'-R3, wherein L' is a C1-C25 hydrocarbon chain optionally interrupted by one or more -O-, -C(O)-, -N(Re)-, -N(Re)-C(O)-O-, -OC(O)-N(Re)-, -N(Re)-C(O)-N(Rf)-, -C(O)-N(Re)-, -N(Re)-C(O)-, -OC(O)-, -C(O)-O- or -OC(O)-O-;

[0170] R3 is the cell-targeting moiety of ASGPR or its protected form, wherein the cell-targeting moiety may be a piperidine, a piperidine-derived ligand, a guanosine, or a guanosine-derived ligand that specifically binds to ASGPR; and

[0171] X1, X2, Ra, Rb, Rc, and Rd are each independently H or (C1-C6) alkyl.

[0172] As described in this disclosure, L can be a branched or unbranched linker group. The branched linker group may have 2, 3, 4, or 5 cell-targeting moieties or their protected forms.

[0173] In some embodiments of the compound of formula (I), Y is NR1, wherein R1 is -L-R3, wherein L is a C2-C25 hydrocarbon chain, and R3 is the ASGPR-binding cell-targeting moiety or its protected form.

[0174] In some embodiments of the compound of formula (I), Y is NR1, wherein R1 is -L-R3, wherein L is a C1-C10 hydrocarbon chain, and R3 is the ASGPR-binding cell-targeting moiety or its protected form.

[0175] In some embodiments of the compound of formula (I), Y is NC(=O)-R1, wherein R1 is -L-R3, wherein L is a C2-C25 hydrocarbon chain, and R3 is the ASGPR-binding cell-targeting moiety or its protected form.

[0176] In some embodiments of the compound of formula (I), Y is NC(=O)-R1, wherein R1 is -L-R3, wherein L is a C1-C10 hydrocarbon chain, and R3 is the ASGPR-binding cell-targeting moiety or its protected form.

[0177] In some embodiments of the compound of formula (I), Y is NR1, wherein R1 is -L-R3, wherein L is a C2-C25 hydrocarbon chain optionally capped with -C(O)-, and R3 is the ASGPR-binding cell-targeting moiety or its protected form.

[0178] In some embodiments of the compound of formula (I), Y is NR1, wherein R1 is -L-R3, wherein L is a C1-C10 hydrocarbon chain optionally capped with -C(O)-, and R3 is the ASGPR-binding cell-targeting moiety or its protected form.

[0179] In some embodiments of the compound of formula (I), Y is NC(=O)-R1, wherein R1 is -L-R3, wherein L is a C2-C25 hydrocarbon chain optionally capped with -C(O)-, and R3 is the ASGPR-binding cell-targeting moiety or its protected form.

[0180] In some embodiments of the compound of formula (I), Y is NC(=O)-R1, wherein R1 is -L-R3, wherein L is a C1-C10 hydrocarbon chain optionally capped with -C(O)-, and R3 is the ASGPR-binding cell-targeting moiety or its protected form.

[0181] In some embodiments of the compound of formula (I), Y is NR1, wherein R1 is -L-R3, wherein L is a C2-C25 hydrocarbon chain optionally interrupted by one or more -O-, and R3 is the ASGPR-binding cell-targeting moiety or its protected form.

[0182] In some embodiments of the compound of formula (I), Y is NR1, wherein R1 is -L-R3, wherein L is a C2-C10 hydrocarbon chain optionally interrupted by one or more -O-, and R3 is the ASGPR-binding cell-targeting moiety or its protected form.

[0183] II. ASGPR ligands

[0184] The nucleotides or nucleotide analogs disclosed herein can be conjugated to one or more ligands that target specific cells or tissues. Such ligands are also referred to as “cell-targeting moieties.” As used herein, a “cell-targeting ligand or moiety” refers to a molecular group that ensures the efficient delivery of an oligonucleotide, such as dsRNA, to a target cell or tissue by adding: (i) the affinity of the dsRNA for a target receptor (e.g., a target protein) or a cell expressing the target receptor; (ii) the uptake of the dsRNA by the target cell; and / or (iii) the ability of the dsRNA to be appropriately processed once it enters the target cell, including efficient intracellular release of the dsRNA, for example, by facilitating the translocation of the dsRNA from transport vesicles into the cytoplasm. Thus, cell-targeting moieties are used to guide and / or deliver oligonucleotides to specific cells, tissues, organs, etc. Cell-targeting moieties conjugated to nucleotides, nucleotide analogs, or oligonucleotides endow the nucleotides, nucleotide analogs, or oligonucleotides with characteristics such that the nucleotides, nucleotide analogs, or oligonucleotides are preferentially recognized, bound, internalized, processed, activated, etc., by the target cell type relative to non-target cell types. Therefore, compounds containing a cell-targeting moiety preferentially interact with and are absorbed by the target cell type. In some embodiments, the cell-targeting moiety may be chemically protected using protecting groups well known in the art.

[0185] As used herein, "target cell" or "targeting cell" refers to a cell of interest. The cell may be found in vitro, in vivo, in vitro, or in the tissues or organs of an organism. The organism may be an animal, preferably a mammal, more preferably a human, and most preferably a human patient. In a specific embodiment, the target cell is a hepatocyte.

[0186] II.1 Piperidine-derived cell-targeting ligands

[0187] The cell-targeting portion of this disclosure may be a piperidine-derived ligand that specifically binds to ASGPR. In some embodiments, the piperidine-derived ASGPR-binding ligand is a portion of formula (II) or a compound of formula (III).

[0188]

[0189] in:

[0190] A1, A2, and A3 are independently H, hydroxyl, alkoxy, acyloxy, aryloxy, aryloxy, alkoxycarbonyl, aryloxycarbonyl, oxo (=O), or unsubstituted or optionally substituted by one or more groups selected from the following (C1-C20) alkyl or alkenyl groups: OH, (C3-C8) cycloalkyl, (C3-C14) heterocyclic, (C6-C14) aryl, (C5-C14) heteroaryl, -O-Z5, -N(Z5)(Z6), -S-Z5, -CN, -C(=M)-O-Z5, -OC(=M)-Z5, -C(=M)-N(Z5)(Z6), and -N(Z5)-C(=M)-Z6, wherein:

[0191] M is O or S.

[0192] Z5 and Z6 are each independently H, (C1-C6)alkyl or (C6-C14)aryl, wherein the alkyl and aryl groups may be unsubstituted or optionally substituted by one or more groups selected from the following: halogen, amino, hydroxyl, thiol, cyano, alkyl, alkoxy, aryloxy, acyloxy, aryloxy, carboxyl, alkoxycarbonyl, aryloxycarbonyl and arylalkoxycarbonyl.

[0193] A4 is -N(R4)2, -NH-C(=O)-R4 or in:

[0194] D2 and D3 are N, O or S;

[0195] R4 is H or an unsubstituted or optionally substituted (C1-C20) alkyl group selected from one or more of the following groups: halogen, amino, hydroxyl, thiol, cyano, alkyl, alkoxy, aryloxy, acyloxy, aryloxy, carboxyl, alkoxycarbonyl, aryloxycarbonyl and arylalkoxycarbonyl.

[0196] B1 can be H, benzyl ester, -L-R5, or (CO)-L-R5, wherein:

[0197] L is a C1-C25 hydrocarbon chain optionally interrupted or terminated by one or more -O-, -C(O)-, -N(Re)-, -N(Re)-C(O)-O-, -OC(O)-N(Re)-, -N(Re)-C(O)-N(Rf)-, -C(O)-N(Re)-, -N(Re)-C(O)-, -OC(O)-, -C(O)-O-, or -OC(O)-O-; Re and Rf are each independently hydrogen, alkyl, alkenyl, alkoxy, aryloxy, hydroxyalkyl, hydroxyl, or haloalkyl, and the C1-C25 hydrocarbon chain is optionally... The base is replaced by one or more -L'-R5, wherein L' is a C1-C25 hydrocarbon chain optionally interrupted by one or more -O-, -C(O)-, -N(Re)-, -N(Re)-C(O)-O-, -OC(O)-N(Re)-, -N(Re)-C(O)-N(R()-, -C(O)-N(Re)-, -N(Re)-C(O)-, -OC(O)-, -C(O)-O- or -OC(O)-O-; and R5 is H, OH, benzyl, benzyloxy, nucleoside, nucleoside analog, nucleotide or nucleotide analog.

[0198] The cell-targeting portion of formula (II) or formula (III) also consists of the specific targets of this disclosure.

[0199] As described in this disclosure, L can be a branched or unbranched linker. The branched linker may have 2, 3, 4, or 5 cell-targeting moieties.

[0200] In some embodiments of the formula (II) portion or the formula (III) compound, A1 is H, an oxo group (=O) or an (C1-C6) alkyl or alkenyl group optionally substituted with a hydroxyl or alkoxy group.

[0201] In some embodiments of the (II) portion or the (III) compound, A1 is an (C1-C6) alkyl group optionally substituted with -OC (=M)-Z5, wherein M is O and Z5 is an (C1-C6) alkyl group optionally substituted with alkoxycarbonyl or arylalkoxycarbonyl.

[0202] In some embodiments of the (II) portion or the (III) compound, A1 is an (C1-C6) alkyl group optionally substituted with -OC (=M)-Z5, wherein M is O and Z5 is an (C1-C6) alkyl group optionally substituted with benzyl ester.

[0203] In some embodiments of the formula (II) portion or the formula (III) compound, A2 and A3 are hydroxyl or acyloxy groups.

[0204] In some embodiments of the formula (II) portion or the formula (III) compound, A2 and A3 are acetoxy groups.

[0205] In some embodiments of the formula (II) portion or the formula (III) compound, A4 is -NH-C(=O)-R4, wherein R4 is an (C1-C6) alkyl group optionally substituted with a carboxyl or alkoxycarbonyl group.

[0206] In some embodiments of the formula (II) portion or the formula (III) compound, A4 is -NH-C(=O)-R4, wherein R4 is an (C1-C6) alkyl group optionally substituted with a methyl ester group.

[0207] In some embodiments of the formula (II) portion or the compound of formula (III), A4 is Wherein D2 and D3 are N, and R4 is an (C1-C6) alkyl group optionally substituted with alkoxy or aryloxy groups.

[0208] In some embodiments of the formula (II) portion or the compound of formula (III), A4 is Wherein D2 and D3 are N, and R4 is an (C1-C6) alkyl group optionally substituted with a phenoxy group.

[0209] In some embodiments of the compound of formula (III), B1 is H.

[0210] In some embodiments of the compound of formula (III), B1 is benzyloxycarbonyl.

[0211] In some embodiments of compounds of formula (III), L is a C1-C6 hydrocarbon chain.

[0212] In some embodiments of compounds of formula (III), L is a C1-C6 hydrocarbon chain optionally terminated with C(O).

[0213] In some embodiments of compounds of formula (III), R5 is H, OH, benzyl or benzyloxy.

[0214] In some embodiments of the compound of formula (III), L is a C1-C6 hydrocarbon chain optionally terminated with -C(O)-, and R5 is H, OH, benzyl or benzyloxy.

[0215] In some embodiments of compounds of formula (III), A1 is H, (=O), or optionally substituted with a hydroxyl, alkoxy, or aryl group (C1-C6) alkyl or alkenyl group.

[0216] In some embodiments of the compound of formula (III), A1 is a (C1-C6) alkyl group optionally substituted with -OC (=M)-Z5, wherein M is O and Z5 is a (C1-C6) alkyl group optionally substituted with alkoxycarbonyl or arylalkoxycarbonyl.

[0217] In some embodiments of compounds of formula (III), A1 is a (C1-C6) alkyl group optionally substituted with -OC (=M)-Z5, wherein M is O and Z5 is a (C1-C6) alkyl group optionally substituted with benzyl ester.

[0218] In some embodiments of the compound of formula (III), A2 and A3 are hydroxyl groups.

[0219] In some embodiments of compounds of formula (III), A4 is -NH-C(=O)-R4, wherein R4 is a (C1-C6) alkyl group optionally substituted with a carboxyl group, an alkoxycarbonyl group, or an aryloxycarbonyl group.

[0220] In some embodiments of compounds of formula (III), A4 is -NH-C(=O)-R4, wherein R4 is a (C1-C6) alkyl group substituted with a methyl ester group.

[0221] In some embodiments of the compound of formula (III), A4 is... Wherein D2 and D3 are N, and R4 is an (C1-C6) alkyl group optionally substituted with alkoxy or aryloxy groups.

[0222] In some embodiments of the compound of formula (III), A4 is... Where D2 and D3 are N, and R4 is a (C1-C6) alkyl group substituted with phenoxy group.

[0223] In some embodiments of compounds of formula (III), B1 is H or benzyl ester group.

[0224] Exemplary piperidine-derived ASGPR-binding ligands of formula (III) are shown in Table C below:

[0225] Table C:

[0226]

[0227]

[0228] II.2 Guanosine-derived cell-targeting ligands

[0229] The cell-targeting portion of this disclosure may be a guanosine-derived ligand that specifically binds to ASGPR. In some embodiments, the guanosine-derived ASGPR-binding ligand is a fraction of formula (IVA) or (IVB) or a compound of formula (V).

[0230]

[0231] in:

[0232] Each R6 is an H or an unsubstituted or optionally substituted (C1-C6) alkyl group selected from one or more of the following groups: halogen, amino, hydroxyl, thiol, alkyl, alkoxy, aryloxy, carboxyl, alkoxycarbonyl and aryloxycarbonyl.

[0233] A5, A6, A7, and A'7 are independently H, hydroxyl, alkoxy, acyloxy, aryloxy, aryloxy, alkoxycarbonyl, aryloxycarbonyl, amino, or unsubstituted or optionally substituted with one or more groups selected from the following (C1-C20) alkyl groups: OH, (C3-C8) cycloalkyl, (C3-C14) heterocyclic, (C6-C14) aryl, (C5-C14) heteroaryl, -O-Z7, -N(Z7)(Z8), -S-Z7, -CN, -C(=Q)-o-Z7, -OC(=Q)-Z7, -C(=Q)-N(Z7)(Z8), and -N(Z7)-C(=Q)-Z8, wherein:

[0234] Q is either O or S.

[0235] Z7 and Z8 are each independently H, (C1-C6)alkyl or (C6-C14)aryl, wherein the alkyl and aryl groups may be unsubstituted or optionally substituted by one or more groups selected from halogen atoms and (C1-C6)alkyl groups;

[0236] A8 and A9 are each independently H, halogen, OH (or its tautomeric oxo group (=O)), -N(R7)2, -NHR7 or -NH-C(=O)-R7, wherein R7 is hydrogen or an unsubstituted or optionally substituted (C1-C20) alkyl group: halogen atom, alkoxy, aryloxy, (C1-C6) alkyl, (C3-C8) cycloalkyl, (C3-C14) heterocyclic, (C6-C14) aryl, (C5-C14) heteroaryl;

[0237] B2 and B'2 are each independently -H, -R8, -OH, -OR8, -COOH, -C(O)-NR8R'8, -NH2, -NHR8, -NH-C(O)-R8, -OP(O)(OH)2, -OP(O)(OR8)(OR'8) or optionally substituted with -OH (C1-C6) alkyl groups, wherein R8 and R'8 are independently H or -L-R9, wherein

[0238] L is a C1-C25 hydrocarbon chain optionally interrupted or terminated by one or more -O-, -C(O)-, -N(Re)-, -N(Re)-C(O)-O-, -OC(O)-N(Re)-, -N(Re)-C(O)-N(Rf)-, -C(O)-N(Re)-, -N(Re)-C(O)-, -OC(O)-, -C(O)-O-, or -OC(O)-O-; Re and Rf are each independently hydrogen, alkyl, alkenyl, alkynyl, alkoxy, aryloxy, hydroxyalkyl, Hydroxyl or haloalkyl, wherein the C1-C25 hydrocarbon chain is optionally substituted with one or more -L'-R9, wherein L' is a C1-C25 hydrocarbon chain optionally interrupted by one or more -O-, -C(O)-, -N(Re)-, -N(Re)-C(O)-O-, -OC(O)-N(Re)-, -N(Re)-C(O)-N(Rf)-, -C(O)-N(Re)-, -N(Re)-C(O)-, -OC(O)-, -C(O)-O- or -OC(O)-O-;

[0239] R9 can be H, OH, benzyl, benzyloxy, nucleoside, nucleoside analog, nucleotide, or nucleotide analog.

[0240] As described in this disclosure, L can be a branched or unbranched linker. The branched linker may have 2, 3, 4, or 5 cell-targeting moieties.

[0241] The cell-targeting portions of formulas (IVA), (IVB), and (V) consist of specific targets of this disclosure.

[0242] In some embodiments of the (IVA) or (IVB) portion or the (V) compound, A5 is H or an (C1-C6) alkyl group optionally substituted with one or more hydroxyl or acyloxy groups.

[0243] In some embodiments of the (IVA) or (IVB) portion or the (V) compound, A6 and A7 are hydroxyl groups.

[0244] In some embodiments of the formula (IVA) or (IVB) portion or the compound of formula (V), A6 and A7 are acyloxy groups, such as acetoxy groups.

[0245] In some embodiments of the (IVA) or (IVB) fraction or the (V) compound, A'7 is H or a (C1-C6) alkyl group, such as methyl.

[0246] In some embodiments of the formula (IVA) or (IVB) portion or the formula (V) compound, A5 is H or optionally a (C1-C6) alkyl group substituted with one or more hydroxyl or acyloxy groups, such as acetoxy groups.

[0247] In some embodiments of the formula (IVA) or (IVB) portion or the compound of formula (V), A8 is H, a halogen (e.g., Cl), OH, or an oxo group (=O).

[0248] In some embodiments of the (IVA) or (IVB) portion or the (V) compound, A8 is -N(R7)2, wherein R7 is H or (C1-C6) alkyl.

[0249] In some embodiments of the (IVA) or (IVB) portion or the (V) compound, A8 is -N(R7)2 or -NHR7, wherein R7 is a (C1-C6) alkyl group, such as methyl.

[0250] In some embodiments of the (IVA) or (IVB) portion or the (V) compound, A8 is -NH-C(=O)-R7, wherein R7 is a (C1-C6) alkyl group, such as methyl, ethyl or isopropyl.

[0251] In some embodiments of the formula (IVA) or (IVB) portion or the compound of formula (V), A9 is H, OH, oxo group (=O) or NH2.

[0252] In some embodiments of the (IVA) or (IVB) fraction or the (V) compound, R6 is H or a (C1-C6) alkyl group, such as methyl.

[0253] In some embodiments of the compound of formula (V), B2 is CH2OH, B2' is OH, A5 is H, A6 is OH, A7 is H, A7' is OH, A9 is H, R6 is H and A8 is not NH2.

[0254] In some embodiments of the compound of formula (V), each of B2 and B'2 is independently H, OH, -NH2 or -COOH.

[0255] In some embodiments of the compound of formula (V), B2 is -NH-C(O)-R8, -C(O)-NR8R'8 or -C(O)-NHR8, wherein R8 and R'8 are independently H or -L-R9, wherein L is a C1-C6 hydrocarbon chain optionally terminated with -C(O).

[0256] In some embodiments of the compound of formula (V), B2 is -NH-C(O)-R8, -C(O)-NR8R'8 or -C(O)-NHR8, wherein R8 and R'8 are independently H or -L-R9, wherein L is a C1-C6 hydrocarbon chain optionally terminated with -C(O), and R9 is H, OH or a nucleoside analogue.

[0257] In some embodiments of the compound of formula (V), B'2 is H and B2 is OH.

[0258] In some embodiments of the compound of formula (V), B'2 is H and B2 is -OP(O)(OH)2 or -OP(O)(OR8)(OR'8), wherein R8 and R'8 are H or -L-R9 and R9 is H or a nucleoside analog.

[0259] In some embodiments of the compound of formula (V), B'2 is H and B2 is -NH2.

[0260] In some embodiments of the compound of formula (V), B'2 is H and B2 is -NH-C(O)-R8, wherein R8 is -L-R9, wherein L is a C1-C6 hydrocarbon chain and R9 is H.

[0261] In some embodiments of the compound of formula (V), B'2 is H and B2 is -NH-C(O)-R8, wherein R8 is -L-R9, wherein L is a C1-C6 hydrocarbon chain and R9 is OH.

[0262] In some embodiments of the compound of formula (V), B'2 is -OH and B2 is a (C1-C6)-alkyl group substituted with OH.

[0263] In some embodiments of the compound of formula (V), B'2 is H and B2 is COOH.

[0264] In some embodiments of the compound of formula (V), B'2 is H and B2 is -C(O)-NHR8, where R8 is -L-R9, and L is a C1-C6 hydrocarbon chain, such as methyl or butyl.

[0265] In some embodiments of the compound of formula (V), B'2 is H and B2 is -C(O)-NR8R'8, where R8 and R'8 are -L-R9, where L is a C1-C6 hydrocarbon chain, such as methyl.

[0266] In some embodiments of the compound of formula (V), B'2 is H and B2 is OR8, wherein R8 is L-R9 and R9 is a nucleoside analog.

[0267] In some embodiments of the compound of formula (V), A5 is H or an (C1-C6) alkyl group optionally substituted with one or more hydroxyl groups.

[0268] In some embodiments of the compound of formula (V), A6 and A7 are hydroxyl groups.

[0269] In some embodiments of compounds of formula (V), A'7 is H or (C1-C6) alkyl.

[0270] In some embodiments of the compound of formula (V), A8 is H, a halogen or a hydroxyl group or its corresponding oxo (=O) tautomer.

[0271] In some embodiments of the compound of formula (V), A8 is -N(R7)2 or -NHR7, wherein R7 is H or (C1-C6) alkyl.

[0272] In some embodiments of the compound of formula (V), A8 is -NH-C(=O)-R7, wherein R7 is a (C1-C6) alkyl group.

[0273] In some embodiments of the compound of formula (V), A9 is H, OH or their corresponding oxo (=O) tautomer or NH2.

[0274] In some embodiments of the compound of formula (V), R6 is H or (C1-C6) alkyl.

[0275] Exemplary formula (V) guanosine-derived ASGPR binding ligands (where R9 may be a nucleoside analog) are shown in Table D below:

[0276] Table D

[0277]

[0278]

[0279]

[0280] II.3 Trimeric ASGPR binder

[0281] Exemplary trimeric ASGPR-binding molecules comprising the three cell-targeting moieties of formula (II) are shown in Table E below:

[0282] Table E

[0283]

[0284]

[0285] In some embodiments, the nucleotide analog precursor of formula (I) described herein may be conjugated directly or via a linker to one or more ASGPR binding moieties of formula (II), (IVA), or (IVB). In some embodiments, the nucleotide analog precursor of formula (I) described herein may be conjugated directly or via a linker to one, two, three, or four ASGPR binding moieties of formula (II), (IVA), or (IVB). In a particular embodiment, the nucleotide analog precursor of formula (I) described herein may be conjugated directly or via a linker to three ASGPR binding moieties of formula (II), (IVA), or (IVB). In a particular embodiment, the nucleotide analog precursor of formula (I) described herein may be a nucleotide analog of a compound of formula (III) or a compound of formula (V).

[0286] As described herein, in the ASGPR-targeted nucleotide analog precursor of formula (I), an ASGPR-binding ligand, such as that of formula (II), (IVA), or (IVB), is partially and directly and covalently bound to the nitrogen atom of the morpholino group. In certain embodiments, the ASGPR-binding ligand is covalently bound to the nitrogen atom of the morpholino group via a linker group.

[0287] Exemplary nucleotide precursors of formula (I) that are conjugated directly or via a linker to the ASGPR-binding portion of formula (II), (IVA), or (IVB) are shown in Table F below. The abbreviations for ASGPR-targeted nucleotide analog precursors are as described above, but “1p” or “1s” are used instead of “1g”.

[0288] Table F

[0289]

[0290] Another aspect of the invention relates to ASGPR-targeting oligonucleotides comprising one or more targeting nucleotide analogs derived from precursor compounds having the structure of formula (I), optimized for delivery to specific cells or tissues, such as hepatocytes. The compound of formula (I) disclosed herein is a nucleotide analog precursor that is converted into a monomeric unit of an oligomeric compound during oligonucleotide synthesis, particularly as a monomeric unit of an oligonucleotide, including monomeric units as double-stranded RNA (“dsRNA”) oligomers, and especially as monomeric units as siRNA. Incorporating the ASGPR-targeting nucleotide analog precursor of formula (I) described herein into an oligonucleotide results in the corresponding monomeric unit of the oligonucleotide described herein being a compound of formula (VI).

[0291] The ASGPR-targeting oligonucleotides disclosed herein comprise one or more compounds of formula (VI):

[0292]

[0293] in:

[0294] B is a heterocyclic nucleobase;

[0295] One of T1 and T2 is a compound of formula (VI) attached to an internucleotide linking group of an oligomer, and the other of T1 and T2 is H, a protecting group, a phosphorus moiety, or a compound of formula (VI) attached to an internucleotide linking group of an oligomer.

[0296] Y is NR1 or NC (=0)-R1, where R1 is -L-R3.

[0297] L is a C1-C25 hydrocarbon chain optionally interrupted or terminated by one or more -O-, -C(O)-, -N(Re)-, -N(Re)-C(O)-O-, -OC(O)-N(Re)-, -N(Re)-C(O)-N(Rf)-, -C(O)-N(Re)-, -N(Re)-C(O)-, -OC(O)-, -C(O)-O-, or -OC(O)-O-; Re and Rf are each independently hydrogen, alkyl, alkenyl, alkynyl, alkoxy, aryloxy, hydroxyalkyl, Hydroxyl or haloalkyl, wherein the C1-C25 hydrocarbon chain is optionally substituted with one or more -L'-R3, wherein L' is a C1-C25 hydrocarbon chain optionally interrupted by one or more -O-, -C(O)-, -N(Re)-, -N(Re)-C(O)-O-, -OC(O)-N(Re)-, -N(Re)-C(O)-N(Rf)-, -C(O)-N(Re)-, -N(Re)-C(O)-, -OC(O)-, -C(O)-O- or -OC(O)-O-;

[0298] R3 is the ASGPR-binding cell-targeting fraction of formula (II), (IVA), or (IVB); and

[0299] X1, X2, Ra, Rb, Rc, and Rd are each independently H or -(C1-C6) alkyl.

[0300] As described in this disclosure, L can be a branched or unbranched linker. The branched linker may have 2, 3, 4, or 5 cell-targeting moieties.

[0301] In some embodiments of the compound of formula (VI), Y is NR1, wherein R1 is -L-R3, wherein L is a C2-C25 hydrocarbon chain, and R3 is the ASGPR-binding cell-targeting portion of formula (II), (IVA), or (IVB).

[0302] In some embodiments of compounds of formula (VI), Y is NR1 and L is a C1-C10 hydrocarbon chain.

[0303] In some embodiments of the compound of formula (VI), Y is NR1 and L is a C1-C10 hydrocarbon chain optionally end-capped with -C(O).

[0304] In some embodiments of the compound of formula (VI), Y is NR1, wherein R1 is -L-R3, wherein L is a C1-C10 hydrocarbon chain, and R3 is the ASGPR-binding cell-targeting portion of formula (II), (IVA), or (IVB).

[0305] In some embodiments of the compound of formula (VI), Y is NC(=O)-R1 and L is a C1-C10 hydrocarbon chain optionally terminated with -C(O)-.

[0306] In some embodiments of the compound of formula (VI), Y is NC(=O)-R1, wherein R1 is -L-R3, wherein L is a C2-C25 hydrocarbon chain, and R3 is the ASGPR-binding cell-targeting portion of formula (II), (IVA), or (IVB).

[0307] In some embodiments of the compound of formula (VI), Y is NC(=O)-R1, wherein R1 is -L-R3, wherein L is a C1-C10 hydrocarbon chain, and R3 is the ASGPR-binding cell-targeting portion of formula (II), (IVA), or (IVB).

[0308] In some embodiments of the compound of formula (VI), Y is NR1, wherein R1 is -L-R3, wherein L is a C2-C25 hydrocarbon chain optionally capped with -C(O)-, and R3 is the ASGPR-binding cell-targeting moiety of formula (II), (IVA), or (IVB).

[0309] In some embodiments of the compound of formula (VI), Y is NR1, wherein R1 is -L-R3, wherein L is a C1-C10 hydrocarbon chain optionally capped with -C(O)-, and R3 is the ASGPR-binding cell-targeting moiety of formula (II), (IVA), or (IVB).

[0310] In some embodiments of the compound of formula (VI), Y is NC(=O)-R1, wherein R1 is -L-R3, wherein L is a C2-C25 hydrocarbon chain optionally capped with -C(O)-, and R3 is the ASGPR-binding cell-targeting moiety of formula (II), (IVA), or (IVB).

[0311] In some embodiments of the compound of formula (VI), Y is NC(=O)-R1, wherein R1 is -L-R3, wherein L is a C1-C10 hydrocarbon chain optionally capped with -C(O)-, and R3 is the ASGPR-binding cell-targeting moiety of formula (II), (IVA) or (IVB).

[0312] In some embodiments of the compound of formula (VI), Y is NR1, wherein R1 is -L-R3, wherein L is a C2-C25 hydrocarbon chain optionally interrupted by one or more -O-, and R3 is the ASGPR-binding cell-targeting portion of formula (II), (IVA), or (IVB).

[0313] In some embodiments of the compound of formula (VI), Y is NR1, wherein R1 is -L-R3, wherein L is a C2-C10 hydrocarbon chain optionally interrupted by one or more -O-, and R3 is the ASGPR-binding cell-targeting portion of formula (II), (IVA), or (IVB).

[0314] The trimeric oligonucleotide of exemplary formula (VI), where each nucleotide is an ASGPR-targeting nucleotide analog, can be understood as a trivalent ASGPR binder and is therefore a functional analog of the structure shown in Table E. Examples are shown in Table G below.

[0315] Table G

[0316]

[0317]

[0318] In some embodiments, the ASGPR-targeting oligonucleotide according to this disclosure is a single-stranded oligonucleotide, such as ASO.

[0319] In some embodiments, the ASGPR-targeting oligonucleotide according to this disclosure is an antisense oligonucleotide targeting human mRNA.

[0320] In some other embodiments, the ASGPR-targeting oligonucleotide according to this disclosure is a double-stranded oligonucleotide, such as siRNA.

[0321] In some other embodiments, the ASGPR-targeting oligonucleotide according to this disclosure is a double-stranded interfering RNA that targets human mRNA and comprises a sense strand and an antisense strand.

[0322] In some embodiments, the ASGPR-targeting oligonucleotide according to this disclosure comprises one or more non-targeting nucleotides or nucleotide analogs and one or more ASGPR-targeting nucleotide analogs of formula (VI).

[0323] In some embodiments of the ASGPR-targeting oligonucleotides as single-stranded or double-stranded oligonucleotides according to this disclosure, the oligonucleotide chain comprises one or more ASGPR-targeting nucleotide analogs of formula (VI), which may be located at different positions within the oligonucleotide chain, such as inside and / or at its 3' or 5' end.

[0324] As used herein, nucleotide analogues are compounds that function as nucleotides in relation to the phosphate backbone of a nucleic acid molecule and / or the ability to form base pairs with another nucleotide.

[0325] In some embodiments of the ASGPR-targeting oligonucleotides as single-stranded or double-stranded oligonucleotides according to this disclosure, the oligonucleotide chain comprises one or more ASGPR-targeting nucleotide analogs of formula (VI), said analogs being located at the 5' end or 3' end, or both ends, of the oligonucleotide chain.

[0326] In some embodiments of the ASGPR-targeting oligonucleotides as single-stranded or double-stranded oligonucleotides according to this disclosure, the oligonucleotide chain comprises 1 to 10 ASGPR-targeting nucleotide analogs of formula (VI), said analogs being located at the 5' or 3' end of the chain, or at one or more other positions within the chain.

[0327] In some embodiments, the ASGPR-targeted oligonucleotide also includes 1 to 10 non-targeted nucleotide analogs, which may be located at different positions within the oligonucleotide chain, such as inside and / or at its 3' or 5' end.

[0328] In some embodiments of the ASGPR-targeting oligonucleotides according to this disclosure as single-stranded or double-stranded oligonucleotides, the oligonucleotide chain comprises (A) one or more ASGPR-targeting nucleotide analogs of formula (VI) located at the 3' or 5' end, or both ends, of the oligonucleotide chain, and (B) one or more non-targeting nucleotide analogs located at the 3' or 5' end, or both ends, of the oligonucleotide chain, wherein the ASGPR-targeting nucleotide analog of formula (VI) and the non-targeting nucleotide analog are located at different positions within the oligonucleotide chain.

[0329] In some embodiments of the ASGPR-targeting oligonucleotides according to this disclosure, which are single-stranded or double-stranded oligonucleotides, the oligonucleotide chain comprises 1 to 10 ASGPR-targeting nucleotide analogs of formula (VI) located at the 3' or 5' end of the chain. In some embodiments, the ASGPR-targeting oligonucleotide further comprises 1 to 10 non-targeting nucleotide analogs located at opposite ends of the oligonucleotide chain. Thus, according to these embodiments, the number of ASGPR-targeting nucleotide analogs of formula (VI) at selected ends of the oligonucleotide chain can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. According to some of these embodiments, the number of non-targeting nucleotide analogs (if present) at selected ends of the oligonucleotide chain can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0330] In a specific embodiment, one or more ASGPR-targeting nucleotide analogues of formula (VI) are interconnected to form a continuous chain of these targeting nucleotide analogues at selected ends of the oligonucleotide chain.

[0331] In specific embodiments, one or more ASGPR-targeting nucleotide analogs of formula (VI) are located at the 5' end of an ASGPR-targeting oligonucleotide chain, said oligonucleotide being a single-stranded or double-stranded oligonucleotide. In some of these embodiments, the 5' nucleotide is an ASGPR-targeting nucleotide analog of formula (VI).

[0332] In some embodiments of the ASGPR-targeting oligonucleotides as single-stranded or double-stranded oligonucleotides according to this disclosure, the oligonucleotide chain comprises one or more non-targeting nucleotide analogs at its 3' or 5' end, and particularly at the end opposite to the end containing one or more targeting nucleotide analogs of formula (VI).

[0333] In a specific embodiment, one or more non-targeted nucleotide analogs are interconnected to form a continuous chain of these non-targeted nucleotide analogs at selected ends of the oligonucleotide chain.

[0334] In a specific embodiment, one or more non-targeted nucleotide analogs are located at the 3' end of the oligonucleotide chain of the ASGPR-targeted oligonucleotide.

[0335] Therefore, this disclosure covers single-stranded ASGPR-targeting oligonucleotides comprising (i) one or more ASGPR-targeting nucleotide analogs of formula (VI), preferably 1 to 10 ASGPR-targeting nucleotide analogs of formula (VI), which may be continuous in the oligonucleotide chain and located at the 5' end of the single-stranded targeting oligonucleotide. In some of these embodiments, the single-stranded targeting oligonucleotide also comprises (ii) one or more non-targeting nucleotide analogs, for example, 1 to 10 non-targeting nucleotide analogs, which may be continuous in the oligonucleotide chain and located at the 3' end of the single-stranded targeting oligonucleotide.

[0336] Examples of single-stranded targeted oligonucleotides disclosed in the embodiments herein comprise (i) three targeted nucleotide analogs of formula (VI) at their 5' end and (ii) two non-targeted nucleotide analogs at their 3' end.

[0337] This disclosure also includes double-stranded oligonucleotides, wherein (i) the first strand is a targeting oligonucleotide comprising one or more targeting nucleotide analogs of formula (VI) and one or more non-targeting nucleotides or nucleotide analogs, as described above, and wherein (ii) the second strand is another targeting oligonucleotide comprising one or more targeting nucleotide analogs of formula (VI) and one or more non-targeting nucleotides or nucleotide analogs.

[0338] This disclosure also includes double-stranded oligonucleotides, wherein (i) the first strand is a targeted oligonucleotide comprising one or more targeted nucleotide analogs of formula (VI) and one or more non-targeted nucleotides or nucleotide analogs, as described above, and (ii) the second strand is a non-targeted oligonucleotide not comprising any targeted nucleotides or nucleotide analogs.

[0339] IV. Double-stranded RNA

[0340] An important aspect of this disclosure relates to double-stranded ribonucleic acid (dsRNA) molecules, particularly siRNA, which contain nucleotide analogs conjugated to the ASGPR targeting portion, wherein said nucleotide analogs have the structure shown in Formula (VI). As used herein, the term "double-stranded RNA" or "dsRNA" refers to an oligonucleotide molecule containing a double-stranded structure having two antiparallel and substantially complementary nucleic acid strands. The two strands forming the double-stranded structure may be different parts of a larger RNA molecule, or they may be located on different RNA molecules. When the two strands are located on different RNA molecules, the dsRNA structure can act as small interfering RNA (siRNA). If the two strands are part of a larger molecule and are linked by an uninterrupted nucleotide chain between the 3' end of the first strand and the 5' end of the second strand, the linking RNA strands are called a "hairpin loop," and the RNA molecule may be called a "short hairpin RNA" or "shRNA." The RNA strands may have the same or different numbers of nucleotides. In addition to the double-stranded structure, dsRNA may also contain one or more (e.g., 1, 2, or 3) nucleotide overhangs.

[0341] As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides with a length of at least 10 bases: ribonucleotides or deoxyribonucleotides or modified forms of any type of nucleotide, or combinations thereof. The term includes both single-stranded and double-stranded forms.

[0342] As used herein, the term "oligonucleotide" refers to a polymeric form of nucleotides with a length not exceeding 50 bases: ribonucleotides or deoxyribonucleotides or modified forms of any type of nucleotide, or combinations thereof. The term includes both single-stranded and double-stranded forms.

[0343] “dsRNA” may include naturally occurring ribonucleotides and / or their chemically modified analogs. The dsRNA disclosed herein may contain one or more modifications that enhance its cellular uptake, affinity for target sequences, inhibitory activity, and / or stability. Furthermore, the dsRNA disclosed herein may include one or more modified nucleotides known in the art, including but not limited to 2'-O-methyl modified nucleotides, 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, 2'-O-methoxyethyl modified nucleotides, modified nucleotides containing alternating nucleotide internucleotide bonds such as thiophosphate and phosphorothioate, phosphotriester modified nucleotides, modified nucleotides with terminals linked to cholesterol derivatives or lipophilic moieties, and peptide nucleic acids (PNAs; see examples). Examples include Nielsen et al., Science (1991) 254: 1497-500), restricted ethyl (cEt) modified nucleotides, reverse deoxy modified nucleotides, reverse dideoxy modified nucleotides, locked nucleic acid modified nucleotides (LNA), unlocked nucleic acid modified nucleotides (UNA), base-free modified nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino modified nucleotides, aminophosphate modified nucleotides, modified nucleotides including those modified at other sites of the sugar or base of oligonucleotides, and modified nucleotides containing non-natural bases.

[0344] In some embodiments, at least one of the one or more modified nucleotides is a 2'-O-methyl nucleotide, a 5'-phosphothioester nucleotide, or a terminal nucleotide moiety linked to a cholesterol derivative, a lipophilic group, or any other cell-targeting group. The incorporation of a 2'-O-methyl, 2'-O-ethyl, 2'-O-propyl, 2'-O-alkyl, 2'-O-aminoalkyl, or 2'-deoxy-2'-fluoro (i.e., 2'-fluoro) group into the nucleoside or nucleotide of the oligonucleotide can confer enhanced hybridization properties and / or enhanced nuclease stability. Furthermore, oligonucleotides containing a phosphothioester backbone (e.g., a phosphothioester bond between two consecutive nucleotides at one or more positions on the dsRNA) may have enhanced nuclease stability. In some embodiments, the dsRNA may contain a nucleotide having a modified ribose, such as a locked nucleic acid (LNA) unit.

[0345] In some embodiments, the dsRNA of this disclosure comprises one or more 2'-O-methylnucleotides and one or more 2'-fluoronucleotides. In some embodiments, the dsRNA comprises two or more 2'-O-methylnucleotides and two or more 2'-fluoronucleotides. In some embodiments, the dsRNA comprises two or more 2'-O-methylnucleotides (OMe) and two or more 2'-fluoronucleotides (F) in an alternating pattern, such as the pattern OMe-F-OMe-F or the pattern F-OMe-F-OMe. In some embodiments, the dsRNA comprises up to 10 consecutive nucleotides, each being a 2'-O-methylnucleotide. In some embodiments, the dsRNA comprises up to 10 consecutive nucleotides, each being a 2'-fluoronucleotide. In some embodiments, the dsRNA comprises two or more 2'-fluoronucleotides at the 5' or 3' end of the antisense strand.

[0346] As used herein, “dsRNA” is not limited to those containing ribonucleotides. The dsRNAs described herein include double-stranded polynucleotide (e.g., oligonucleotide) molecules in which the ribose portion of some or all of the nucleotides has been replaced by another portion, provided that the resulting double-stranded molecule can repress the expression of a target gene by RNA interference. dsRNAs may also include one or more, but not more than 60% (e.g., not more than 50%, 40%, 30%, 20%, or 10%) of deoxyribonucleotides or their chemically modified analogues.

[0347] In some embodiments, the nucleotides or nucleotide analogs of this disclosure can be linked to adjacent nucleotides or nucleotide analogs via a bond (referred to herein as a 3'-5' internucleotide bond) between the 3'-carbon of the sugar moiety of the first nucleotide and the 5'-carbon of the sugar moiety of the second nucleotide. In another embodiment, the nucleotides or nucleotide analogs of this disclosure can be linked to adjacent nucleotides or nucleotide analogs via a bond (referred to herein as a 2'-5' internucleotide bond) between the 2'-carbon of the sugar moiety of the first nucleotide and the 5'-carbon of the sugar moiety of the second nucleotide.

[0348] As used herein, the term "nucleotide linker" includes both phosphorus-containing and phosphorus-free nucleotide linkers.

[0349] In some embodiments of the dsRNA disclosed herein, the internucleotide backbone is a phosphorus-containing internucleotide linker group, such as phosphodiester, thiophosphate, dithiophosphate, triphosphate, aminoalkyl phosphate, methyl and other alkylphosphonates (including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates), hypophosphonates, aminophosphates (including 3'-aminoaminophosphate and aminoalkylaminophosphate), thioaminophosphate, thioalkylphosphonates, thioalkyl phosphate, selenophosphate and boron phosphate having normal 3'-5' bonds, and analogs thereof linked at 2'-5'.

[0350] In some embodiments, the dsRNA of this disclosure comprises one or more phosphate thioester groups. In some embodiments, the dsRNA of this disclosure comprises two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more phosphate thioester groups. In some embodiments, the dsRNA does not contain any phosphate thioester groups.

[0351] In some embodiments, the dsRNA disclosed herein comprises one or more phosphotriester groups. In some embodiments, the dsRNA comprises two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more phosphotriester groups. In some embodiments, the dsRNA does not contain any phosphotriester groups.

[0352] In some embodiments of the dsRNA disclosed herein, the internucleotide backbone bonds are non-phosphodiester bonds, such as thiophosphate, dithiophosphate, alkylphosphonate, and aminophosphate backbone linkers.

[0353] In some embodiments, the dsRNA disclosed herein comprises one or more phosphorus-free internucleotide linking groups. Such oligonucleotides include, but are not limited to, oligonucleotides formed from short-chain alkyl or cycloalkyl internucleotide linking groups, mixed heteroatom and alkyl or cycloalkyl internucleotide linking groups, or one or more short-chain heteroatom or heterocyclic internucleotide linking groups. These oligonucleotides include those having the following: siloxane backbone; sulfide, sulfoxide, and sulfone backbone; formyl and thioformyl backbone; methyleneformyl and thioformyl backbone; riboacetyl backbone; olefin-containing backbone; aminosulfonate backbone; methyleneimino and methylenehydrazine backbone; sulfonate and sulfonamide backbone; amide backbone; and other backbones having a mixed N, O, S, and CH2 composition.

[0354] Representative U.S. patents teaching the preparation of the aforementioned phosphorus-containing nucleotide interbonds include U.S. patents 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,4 55,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, each of which is incorporated herein by reference.

[0355] Representative U.S. patents teaching the preparation of the above-mentioned non-phosphoronucleotide linking groups include, but are not limited to, U.S. patents 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; and 5,541. ,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, each of which is incorporated herein by reference.

[0356] In some embodiments, the dsRNA disclosed herein comprises one or more nonionic neutral nucleoside linker groups. Neutral nucleoside linker groups include nonionic linker groups comprising siloxanes (dialkylsiloxanes), carboxylic esters, carboxamides, sulfides, sulfonates, and amides (see, for example: Carbohydrate Modifications in Antisense Research; YSSanghvi and PDCook, eds., ACS Symposium Series 580; Chapters 3 and 4, (pp. 40–65)). Additional neutral nucleoside linker groups comprise nonionic bonds comprising a mixture of N, O, S, and CH2 components.

[0357] The dsRNA disclosed herein contains a sense strand comprising a sense sequence and an antisense strand comprising an antisense sequence, wherein the sense and antisense sequences are substantially or completely complementary to each other. Unless otherwise stated, the term “complementary” herein refers to the ability of a polynucleotide comprising a first consecutive nucleotide sequence to hybridize with another polynucleotide comprising a second consecutive nucleotide sequence under certain conditions, such as physiological conditions, and to form a duplex structure. This may include base pairing of two polynucleotides (e.g., two oligonucleotides) along the full length of the first or second consecutive nucleotide sequence; in this case, the two nucleotide sequences are considered “completely complementary” to each other. For example, when the dsRNA comprises a first oligonucleotide of 21 nucleotides in length and a second oligonucleotide of 23 nucleotides in length, and the two oligonucleotides form 21 consecutive base pairs, the two oligonucleotides may be described as “completely complementary” to each other. When a first polynucleotide (e.g., oligonucleotide) sequence is described as “substantially complementary” to a second polynucleotide sequence, the two sequences may be base-paired with each other for 80% or more (e.g., 90% or more) of their length, with no more than 20% (e.g., no more than 10%) of mismatched base pairs (e.g., no more than 4 or 2 mismatched base pairs for a 20-nucleotide duplex). When the two oligonucleotides are designed as a duplex with one or more single-stranded overhangs, such overhangs should not be considered as mismatches for determining complementarity. The complementarity of the two sequences may be based on Watson-Crick base pairs and / or non-Watson-Crick base pairs. As used herein, a polynucleotide “substantially complementary to at least a portion of the mRNA” means a polynucleotide substantially complementary to a continuous portion of the mRNA of interest.

[0358] In some embodiments, the dsRNA is siRNA, wherein the sense and antisense strands are not covalently linked to each other. In some embodiments, the sense and antisense strands of the dsRNA are covalently linked to each other, for example, through a hairpin loop (e.g., in the case of shRNA), or through a means other than a hairpin loop (e.g., through a linker structure called a "covalent linker").

[0359] IV.1 Length

[0360] In some embodiments, each sense sequence (in the sense strand) and antisense sequence (in the antisense strand) is 9-30 nucleotides in length. For example, each sequence may be within any nucleotide length range with an upper limit of 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 and an independently chosen lower limit of 9, 10, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, the number of nucleotides in each sequence may be 15-25 (i.e., 15 to 25 nucleotides in each sequence), 15-30, 16-29, 17-28, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, or 19-21.

[0361] In some embodiments, the length of each sequence is greater than 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the length of each sequence is less than 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 nucleotides. In some embodiments, the length of each sequence is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

[0362] In some embodiments, the length of each of the sense and antisense sequences is at least 15 and no more than 25 nucleotides. In some embodiments, the length of each of the sense and antisense sequences is at least 19 and no more than 23 nucleotides. For example, the sequence length is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides.

[0363] In some embodiments, the dsRNA has a sense strand and an antisense strand of the same or different lengths. For example, the sense strand may be 1, 2, 3, 4, 5, 6, or 7 nucleotides longer than the antisense strand. Alternatively, the sense strand may be 1, 2, 3, 4, 5, 6, or 7 nucleotides shorter than the antisense strand.

[0364] In some embodiments, the length of each sense strand and antisense strand is 9-36 nucleotides. For example, each strand can be any nucleotide length with an upper limit of 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 and an independently chosen lower limit of 9, 10, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, the number of nucleotides in each chain may be 15-25, 15-30, 16-29, 17-28, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, or 19-21.

[0365] In some embodiments, the length of each strand is greater than 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the length of each strand is 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37 nucleotides. In some embodiments, the length of each strand is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 nucleotides.

[0366] In some embodiments, the length of each of the sense and antisense strands is at least 15 and no more than 25 nucleotides. In some embodiments, the length of each of the sense and antisense strands is at least 19 and no more than 23 nucleotides. For example, the strand length is 19, 20, 21, 22, or 23 nucleotides.

[0367] In some embodiments, the sense strand may have 21, 22, 23, or 24 nucleotides, including any modified nucleotides, while the antisense strand may have 21 nucleotides, including any modified nucleotides; in some embodiments, the sense strand may have a sense sequence of 17, 18, or 19 nucleotides, while the antisense strand may have an antisense sequence of 19 nucleotides.

[0368] IV.2 Protruding End

[0369] In some embodiments, the dsRNA of this disclosure includes one or more overhangs at the 5' end, 3' end, or both ends of one or both of the sense and antisense strands. In some embodiments, one or more overhangs enhance the delivery capability, repressive activity, and / or stability of the dsRNA.

[0370] In this paper, "protruding end" refers to an unpaired nucleotide that protrudes from the double-stranded structure of dsRNA when the 3' end of the first strand extends beyond the 5' end of the second strand (or vice versa). "Blunt end" means that one end of the dsRNA has no unpaired nucleotide, i.e., no nucleotide protrusion. A "blunt end" dsRNA is a double-stranded dsRNA that is double-stranded throughout its entire length, meaning that neither end of the double-stranded molecule has nucleotide protrusions. In determining whether dsRNA has a protrusion, this paper does not consider chemical caps or non-nucleotide chemical portions conjugated to the 3' and / or 5' ends of the dsRNA.

[0371] In some embodiments, the overhang contains one or more, two or more, three or more, or four or more nucleotides. For example, the overhang may contain 1, 2, 3, or 4 nucleotides.

[0372] In some embodiments, the overhang of this disclosure comprises one or more nucleotides (e.g., ribonucleotides or deoxyribonucleotides, their naturally occurring or chemically modified analogs). In some embodiments, the overhang comprises one or more thymines or their chemically modified analogs. In certain embodiments, the overhang comprises one or more thymines.

[0373] In some embodiments, the dsRNA includes a protruding end at the 3' end of the antisense strand. In some embodiments, the dsRNA includes a blunt end at the 5' end of the antisense strand. In some embodiments, the dsRNA includes a protruding end at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand. In some embodiments, the dsRNA includes a protruding end at the 3' end of the sense strand. In some embodiments, the dsRNA includes a blunt end at the 5' end of the sense strand. In some embodiments, the dsRNA includes a protruding end at the 3' end of the sense strand and a blunt end at the 5' end of the sense strand. In some embodiments, the dsRNA includes protruding ends at the 3' ends of both the sense and antisense strands of the dsRNA.

[0374] In some embodiments, the dsRNA includes a protruding end at the 5' end of the antisense strand. In some embodiments, the dsRNA includes a blunt end at the 3' end of the antisense strand. In some embodiments, the dsRNA includes a protruding end at the 5' end of the antisense strand and a blunt end at the 3' end of the antisense strand. In some embodiments, the dsRNA includes a protruding end at the 5' end of the sense strand. In some embodiments, the dsRNA includes a blunt end at the 3' end of the sense strand. In some embodiments, the dsRNA includes a protruding end at the 5' end of the sense strand and a blunt end at the 3' end of the sense strand. In some embodiments, the dsRNA includes protruding ends at the 5' ends of both the sense and antisense strands of the dsRNA.

[0375] In some embodiments, the dsRNA includes a 3' end protrusion on the antisense strand and a 5' end protrusion on the antisense strand. In some embodiments, the dsRNA includes a 3' end protrusion on the sense strand and a 5' end protrusion on the sense strand.

[0376] In some embodiments, dsRNA has two blunt ends.

[0377] In some embodiments, the bulge is the result of the sense strand being longer than the antisense strand. In some embodiments, the bulge is the result of the antisense strand being longer than the sense strand. In some embodiments, the bulge is the result of sense and antisense strands of equal length interleaved. In some embodiments, the bulge forms a mismatch with the target mRNA. In some embodiments, the bulge is complementary to the target mRNA.

[0378] In some embodiments, dsRNA comprises a modified ribonucleoside, such as a deoxyribonucleoside, including, for example, a deoxyribonucleoside overhang, and one or more deoxyribonucleosides within the double-stranded portion of the dsRNA. However, it is self-evident that double-stranded DNA molecules are not included in the term "dsRNA" under any circumstances.

[0379] In some embodiments, the dsRNA comprises two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more different modified nucleotides described herein. In some embodiments, the dsRNA comprises at most two consecutive modified nucleotides, at most three consecutive modified nucleotides, at most four consecutive modified nucleotides, at most five consecutive modified nucleotides, at most six consecutive modified nucleotides, at most seven consecutive modified nucleotides, at most eight consecutive modified nucleotides, at most nine consecutive modified nucleotides, or at most ten consecutive modified nucleotides. In some embodiments, the consecutive modified nucleotides are the same modified nucleotide. In some embodiments, the consecutive modified nucleotides are two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more different modified nucleotides.

[0380] As used herein, the “antisense strand” in the term dsRNA refers to the strand of dsRNA containing a sequence that is substantially complementary to the target sequence. The other strand in dsRNA is the “sense strand.”

[0381] In some embodiments, the targeting nucleotide analog of formula (VI) is located at the 5' end, 3' end, or both of the 5' and 3' ends of the nucleic acid strand of dsRNA, such as the 5' end or 3' end of the nucleic acid strand of siRNA. In a specific embodiment, the targeting nucleotide analog of formula (VI) is located at the 5' end, 3' end, or both of the 5' and 3' ends of the sense strand of siRNA. In some other embodiments, the targeting nucleotide analog of formula (VI) is located at the 5' end, 3' end, or both of the 5' and 3' ends of the antisense strand of siRNA.

[0382] In some embodiments, the targeting nucleotide analog of formula (VI) is exclusively located at the 5' end of the nucleic acid strand of dsRNA, for example, exclusively located at the 5' end of the nucleic acid strand of siRNA. In a specific embodiment, the targeting nucleotide analog of formula (VI) is located at the 5' end of the positive strand of siRNA.

[0383] In some embodiments, the targeting nucleotide analog of formula (VI) is located at the 5' end of the sense strand and the 5' end of the antisense strand of the siRNA. In some other embodiments, the targeting nucleotide analog of formula (VI) is located at the 3' end of the sense strand and the 3' end of the antisense strand of the siRNA. In other embodiments, the targeting nucleotide analog of formula (VI) is located at the 5' end of the sense strand and the 3' end of the antisense strand of the siRNA, or at the 3' end of the sense strand and the 5' end of the antisense strand of the siRNA.

[0384] In some embodiments, the targeting nucleotide analog of formula (VI) is located at (i) the 5' and 3' ends of the sense strand of the siRNA, and (ii) at the 5' end of the antisense strand of the siRNA. In some other embodiments, the targeting nucleotide analog of formula (VI) is located at (i) the 5' and 3' ends of the sense strand of the siRNA, and (ii) at the 3' end of the antisense strand of the siRNA.

[0385] In some embodiments, the oligonucleotide contains 2 to 10 (e.g., 2 to 5) nucleotide analogs of formula (VI). As used herein, nucleotide analogs of formula (VI) of 2 to 10 include nucleotide analogs of formula (VI) of 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0386] In a specific embodiment, the targeting nucleotide analog of formula (VI) is located in the overhang of dsRNA, such as siRNA. For example, the targeting nucleotide analog of formula (VI) is located in the overhang, such as the 5'-overhang of the positive strand of siRNA.

[0387] This disclosure also describes an siRNA comprising:

[0388] A positive chain comprising (i) one or more ASGPR-targeting nucleotide analogs of formula (VI) at its 5' end, particularly 1 to 10 ASGPR-targeting nucleotide analogs of formula (VI), and (ii) one or more non-targeting nucleotide analogs at its 3' end, particularly 1 to 10 non-targeting nucleotide analogs, and

[0389] The antisense strand is either a non-targeting oligonucleotide or an ASGPR-targeting oligonucleotide.

[0390] This disclosure also describes an siRNA comprising:

[0391] A positive chain comprising (i) one or more ASGPR-targeting nucleotide analogs of formula (VI) at its 3' end, particularly 1 to 10 ASGPR-targeting nucleotide analogs of formula (VI), and (ii) one or more non-targeting nucleotide analogs at its 5' end, particularly 1 to 10 non-targeting nucleotide analogs, and

[0392] The antisense strand is either a non-targeting oligonucleotide or an ASGPR-targeting oligonucleotide.

[0393] This disclosure further describes an siRNA comprising:

[0394] A positive chain comprising (i) one or more ASGPR-targeting nucleotide analogs of formula (VI) at its 5' end, particularly 1 to 10 ASGPR-targeting nucleotide analogs of formula (VI), and (ii) one or more non-targeting nucleotide analogs at its 3' end, particularly 1 to 10 non-targeting nucleotide analogs, and

[0395] The antisense strand contains one or more non-targeted nucleotides or nucleotide analogs, especially 1 to 10 non-targeted nucleotides or nucleotide analogs.

[0396] This disclosure further describes an siRNA comprising:

[0397] A positive chain comprising (i) one or more ASGPR-targeting nucleotide analogs of formula (VI) at its 5' end, particularly 1 to 10 ASGPR-targeting nucleotide analogs of formula (VI), and (ii) one or more non-targeting nucleotide analogs at its 3' end, particularly 1 to 10 non-targeting nucleotide analogs, and

[0398] The antisense strand may or may not include such nucleotide analogues.

[0399] Within the scope of this disclosure, "percentage identity" between two nucleic acid sequences refers to the percentage of identical nucleotide residues between the two sequences to be compared after optimal alignment. This percentage is purely statistical, and the differences between the two sequences are randomly distributed along their length. Comparison of two nucleic acid sequences is conventionally performed by comparing sequences after optimal alignment, which can be done segmentally or by using an "alignment window." In addition to manual comparison, optimal alignment of the sequences for comparison can be performed using Smith and Waterman's (1981) local homology algorithm, Neddleman and Wunsch's (1970) local homology algorithm, Pearson and Lipman's (1988) similarity search method, or by using computer software containing these algorithms (GAP, BESTFIT, FASTA, and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, or by comparison software BEAST NR or BEAST P). The percentage identity between two nucleic acid sequences is determined by comparing two optimally aligned sequences, where the nucleic acid sequence to be compared may have additions or deletions compared to the reference sequence to achieve optimal alignment between the two sequences. Percentage identity is calculated as follows: by determining the number of identical nucleotide residues between the two sequences, preferably between two complete sequences, the number of identical positions is divided by the total number of positions in the alignment window, and the result is multiplied by 100 to obtain the percentage identity between the two sequences.

[0400] As anticipated herein, nucleotide sequences having at least 70% nucleotide identity with a reference sequence include nucleotide sequences having at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% nucleotide identity with a reference sequence.

[0401] As used herein, the term “introduced into cells” means to facilitate uptake or absorption into cells, as understood by one of ordinary skill in the art. Absorption or uptake of dsRNA can occur through unassisted diffusion or active cellular processes, or through an adjuvant or device. The meaning of this term is not limited to in vitro cells; dsRNA can also be “introduced into cells” where the cells are part of a living organism. In this case, introducing into cells will include delivery to the organism. For example, for in vivo delivery, dsRNA can be injected into a tissue site or administered systemically. In vivo delivery can also be mediated by β-glucan delivery systems (see, for example, Tesz et al., Biochem J. (2011) 436(2): 351-62). In vitro introduction into cells includes methods known in the art, such as electroporation and lipid transfection. Other methods are described below or are known in the art.

[0402] As used herein, the terms “inhibit the expression of” or “inhibiting expression of” refer, with respect to the target gene, to at least a partial inhibition of the expression of the target gene, as indicated by a reduction in the amount of mRNA transcribed from the target gene. As used herein, the term “inhibit” is used interchangeably with “reduction,” “silence,” “downregulation,” “suppression,” “knockdown,” and other similar terms, and includes any level of inhibition. The degree of inhibition is typically expressed as (((mRNA in control cells) - (mRNA in treated cells)) / (mRNA in control cells)) * 100%. Alternatively, the degree of inhibition may be given based on a reduction in parameters associated with the transcriptional function of the target gene, such as the amount of protein encoded by the target gene secreted by the cell, or the number of cells exhibiting a certain phenotype (e.g., apoptosis). In principle, target gene silencing can be determined in any cell expressing the target, whether constitutively or through genome engineering, and by any appropriate assay. However, when reference is needed to determine whether a given dsRNA inhibits the expression of a target gene to some extent and is therefore included in this disclosure, the assays provided in the following examples should be used as such reference.

[0403] As used herein, in the context of target gene expression, the terms “treat,” “treatment,” etc., refer to the reduction or relief of a pathological process mediated by target gene expression. In the context of this disclosure, with respect to any other conditions listed below (other than pathological processes mediated by target expression), the terms “treat,” “treatment,” etc., refer to the reduction or relief of one or more symptoms associated with such conditions.

[0404] As used herein, the terms “prevention” or “delay of progression of…” (and their grammatical variations) relating to a disease or condition refer to preventive treatment of the disease, for example, in an individual suspected of having or at risk of having the disease. Prevention may include, but is not limited to, preventing or delaying the onset or progression of the disease and / or maintaining one or more symptoms of the disease at a desired or subpathological level. As used herein, the terms “therapeutic effective amount” and “preventive effective amount” refer to the amount that provides therapeutic benefit in treating, preventing, or controlling a pathological process mediated by the expression of a target gene or a significant symptom of a pathological process mediated by the expression of a target gene. The specific amount of therapeutic effectiveness can be readily determined by a general practitioner and can vary depending on factors such as the type and stage of the pathological process mediated by the expression of the target gene, the patient’s medical history and age, and the administration of other therapeutic agents that inhibit the biological process mediated by the target gene.

[0405] As used herein, the terms “individual” or “subject” refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0406] v. Methods of synthesis

[0407] Preparation of compounds of formula (I) V.1

[0408] In some embodiments, the compound of formula (I) may be prepared according to the method described in WO 2019 / 170731, which is incorporated herein by reference in its entirety. In some other embodiments, the compound of formula (I) may be prepared according to the detailed method illustrated in Examples 1-25 of this disclosure.

[0409] V.2 Preparation of oligonucleotides containing formula (VI)

[0410] The oligonucleotides of the present invention, such as oligonucleotides comprising one or more compounds of formula (VI), can be chemically synthesized using methods known in the art. See, for example, Caruthers et al., Methods in Enzymology (1992) 211:3-19; Thompson et al., International PCT Publication No. WO 99 / 54459; Wincott et al., 1995, Nucleic Acids Res., 23:2677-2684; Wincott et al., 1997, Methods Mol. Bio., 74:59; Brennan et al., 1998, Biotechnol Bioeng., 61:33-45; and Brennan, U.S. Patent 6,001,311. The synthesis of oligonucleotides utilizes common nucleic acid protecting and coupling groups, such as a 5'-terminal dimethoxytriphenylmethyl and a 3'-terminal phosphoramide. In some embodiments, the oligonucleotide comprising formula (II) was synthesized, deprotected, and analyzed according to the methods described in U.S. Patents 6,995,259; 6,686,463; 6,673,918; 6,649,751; 6,989,442; and 7,205,399. In non-limiting synthetic examples, small-scale synthesis was performed on a 394 Applied Biosystems, Inc. / Thermo Fischer Scientific Inc. synthesizer.

[0411] Alternatively, oligonucleotides comprising one or more compounds of formula (VI) can be synthesized individually and then linked together post-synthetically, for example by conjugation (Moore et al., 1992, Science 256:9923; Draper et al., International PCT Publication WO 93 / 23569; Shabarova et al., 1991, Nucleic Acids Research 19:4247; Bellon et al., 1997, Nucleosides & Nucleotides, 16:951; Bellon et al., 1997, Bioconjugate Chem., 8:204), or by hybridization after synthesis and / or deprotection. Various modified oligonucleotides according to this disclosure can also be synthesized using the teachings of Scaringe et al., U.S. Patents 5,889,136; 6,008,400; and 6,111,086.

[0412] V.3 Preparation of Modified dsRNA

[0413] The dsRNA disclosed herein can be chemically / physically linked to one or more ligands, portions, or conjugates. In some embodiments, the dsRNA is conjugated / linked to one or more ligands via a adapter. Any adapter known in the art can be used, including, for example, a multivalent branched adapter. Conjugating a ligand to the dsRNA can alter its distribution, enhance its cellular uptake and / or target specific tissues and / or be taken up by one or more specific cell types (e.g., hepatocytes), and / or prolong the lifetime of the dsRNA reagent. In some embodiments, a hydrophobic ligand is conjugated to the dsRNA to facilitate direct permeation across the cell membrane and / or uptake by cells (e.g., hepatocytes).

[0414] In some embodiments of the dsRNA conjugate, one or more nucleotides may contain a group with a targeting moiety, such as one or more nucleotides containing a group with a targeting moiety, wherein the targeting moiety is covalently linked to the nucleotide backbone, possibly via a linker group. According to these embodiments, one or more nucleotides of the dsRNA are conjugated to a group with a targeting moiety, and wherein the targeting moiety may be a ligand (e.g., a cell-penetrating portion or reagent) that enhances the intracellular delivery of the composition.

[0415] The ligand-conjugated dsRNA and sequence-specific linked nucleosides and nucleotides with ligand molecules disclosed herein can be assembled by any method known in the art, including, for example, by using standard nucleotide precursors, or nucleotide or nucleoside conjugate precursors already with linking portions, ligand-nucleotides, or nucleoside conjugate precursors already with ligand molecules, or building blocks with non-nucleoside ligands, on a suitable DNA synthesizer.

[0416] The ligand-conjugated dsRNAs of this disclosure can be synthesized by any method known in the art, including, for example, by using dsRNAs with a pendant reactive functional group, such as those derived from linker molecules attached to double-stranded RNA. In some embodiments, this reactive oligonucleotide can react directly with a commercially available ligand, a synthetic ligand with any of a variety of protecting groups, or a ligand having a linker portion to which it is attached. In some embodiments, the method facilitates the synthesis of ligand-conjugated dsRNAs by using nucleoside monomers that have been suitably conjugated to a ligand and can be further attached to a solid support material. In some embodiments, dsRNAs with an aralkyl ligand attached to the 3' end of the dsRNA are prepared by first covalently attaching a monomer building block to a glass support with controlled porosity via an aminoalkyl group; then, the nucleotide is bound to the monomer building block bound to the solid support using standard solid-phase synthesis techniques. The monomer building block can be a nucleoside or other organic compound compatible with solid-phase synthesis.

[0417] This disclosure also relates to a method for preparing a liver-targeted therapeutic agent, the method comprising mixing a therapeutic portion with a compound of any one of claims 1-44 to allow the compound to conjugate with the therapeutic portion, thereby producing a liver-targeted therapeutic agent.

[0418] VI. Composition

[0419] Certain aspects of this disclosure relate to compositions (e.g., pharmaceutical compositions) comprising dsRNA as described herein. In some embodiments, the composition (e.g., pharmaceutical composition) further comprises a pharmaceutically acceptable carrier. In some embodiments, the composition (e.g., pharmaceutical composition) may be used to treat diseases or conditions associated with the expression or activity of a target gene.

[0420] The compositions disclosed herein (e.g., pharmaceutical compositions) are formulated based on delivery modes, including, for example, compositions formulated for delivery to the liver via parenteral delivery.

[0421] The compositions disclosed herein (e.g., pharmaceutical compositions) can be administered at doses sufficient to inhibit the expression of targeted genes. In some embodiments, a suitable dose of dsRNA is in the range of 0.01 mg / kg to 400 mg / kg of the recipient's body weight.

[0422] Those skilled in the art will understand that certain factors can influence the dosage and duration required for effective treatment of a subject, including but not limited to the severity of the disease or condition, prior treatment, the subject's general health and / or age, and the presence of one or more other diseases. Furthermore, treatment of a subject with a therapeutically effective amount of the pharmaceutical composition may include a single treatment or a series of treatments. The effective dosage and in vivo half-life of the dsRNA disclosed herein can be estimated using conventional methods or based on in vivo testing using appropriate animal models.

[0423] The dsRNA molecules disclosed herein can be formulated in pharmaceutically acceptable carriers or diluents. Pharmaceutically acceptable carriers can be liquids or solids and can be selected based on the planned administration method to provide the desired volume, consistency, and other relevant transport and chemical properties. Any known pharmaceutically acceptable carrier or diluent can be used, including, for example, water, saline solutions, binders (e.g., polyvinylpyrrolidone or hydroxypropyl methylcellulose), fillers (e.g., lactose and other sugars, gelatin or calcium sulfate), lubricants (e.g., starch, polyethylene glycol, or sodium acetate), disintegrants (e.g., starch or sodium glycolate), calcium salts (e.g., calcium sulfate, calcium chloride, calcium phosphate, etc.), and wetting agents (e.g., sodium dodecyl sulfate).

[0424] The dsRNA molecules disclosed herein can be formulated into compositions (e.g., pharmaceutical compositions) containing dsRNA that is mixed, encapsulated, conjugated, or otherwise bound to other molecules, molecular structures, or nucleic acid mixtures. For example, compositions containing one or more dsRNAs as described herein may contain other therapeutic agents, such as other lipid-lowering agents (e.g., statins). In some embodiments, the composition (e.g., pharmaceutical composition) further comprises a delivery medium (as described herein).

[0425] VII. Vector and dsRNA Delivery

[0426] The dsRNA disclosed herein can be delivered directly or indirectly. In some embodiments, dsRNA is delivered directly by administering a composition containing dsRNA (e.g., a pharmaceutical composition) to a subject. In some embodiments, dsRNA is delivered indirectly by administering one or more of the vectors described herein.

[0427] The dsRNA disclosed herein can be delivered by any method known in the art, including, for example, by employing a method of delivering nucleic acid molecules for use with dsRNA (see, for example, Akhtar et al., Trends Cell Biol. (1992) 2(5): 139-44; WO 94 / 02595), or by other methods known in the art (see, for example, Kanasty et al., Nature Materials (2013) 12: 967-77; Wittrup, A. and Lieberman, J. (2015) Nature Reviews Genetics 16: 543-552; Whitehead et al., Nature Reviews Drug Discovery (2009) 8: 129-38; Gary et al., (2007) 121(1-2): 64-73; Wang, J. et al. (2010) AAPSJ. 12(4): 492-503; Draz, M. et al. (2014) Theranostics 4(9): 872-892; Wan, C. et al. (2013) Drug Deliv. And Transl. Res. 4(1): 74-83; Erdmann, VA and Barciszewski, J. (eds.) (2010) "RNA Technologies and Their Applications", Springer-Verlag Berlin Heidelberg, DOI 10.1007 / 978-3-642-12168-5; Xu, C. and Wang, J. (2Q\5) Asian Journal of Pharmaceutical Sciences 10(1): 1-12).

[0428] In some embodiments, the dsRNA disclosed herein is delivered by a delivery medium containing dsRNA. In some embodiments, the delivery medium is a liposome, a lipid complex, a complex, or nanoparticles.

[0429] VII.1 Liposome Formulation

[0430] Liposomes are monolayered or multilayered vesicles having a membrane formed of a lipophilic material and an aqueous interior. In some embodiments, liposomes are vesicles composed of amphiphilic lipids arranged in one or more spherical bilayers. The aqueous portion contains the composition to be delivered. Cationic liposomes have the advantage of being able to fuse with cell walls. Advantages of liposomes include, for example, the biocompatibility and biodegradability of liposomes derived from natural phospholipids; the ability to incorporate a variety of water-soluble and lipid-soluble drugs; and the ability of liposomes to protect the drug encapsulated in their internal compartments from metabolism and degradation (Rosoff, Pharmaceutical Dosage Forrms, Lieberman, Rieger, and Banker (eds.), 1988, Marcel Dekker, Inc., New York, NY, Vol. 1, p. 245). Important considerations in preparing liposome formulations are lipid surface charge, vesicle size, and the water volume of the liposome. For example, engineered cationic liposomes and spatially stable liposomes can be used for the delivery of dsRNA. See, for example, Podesta et al. (2009) Methods Enzymol. 464, 343-54; U.S. Patent 5,665,710.

[0431] VII.2 Nucleic Acid-Lipid Particles

[0432] In some embodiments, the dsRNA of this disclosure is completely encapsulated in a lipid formulation, for example, to form nucleic acid-lipid particles, such as SPLP, pSPLP, or SNALP. As used herein, the term “SNALP” refers to a stable nucleic acid-lipid particle, including SPLP. As used herein, the term “SPLP” refers to a nucleic acid-lipid particle containing plasmid DNA encapsulated within lipid vesicles. Nucleic acid-lipid particles, such as SNALP, typically contain cationic lipids, non-cationic lipids, cholesterol, and lipids (e.g., PEG-lipid conjugates) that prevent particle aggregation and increase circulation time. SNALP and SPLP can be used for systemic application because they have a prolonged circulation life after intravenous (iv.) injection and accumulate at distal sites (e.g., sites physically separated from the application site). SPLP includes “pSPLP”, which comprises an encapsulated agglutinant-nucleic acid complex as described in PCT Publication WO 00 / 03683.

[0433] In some embodiments, dsRNAs, when present in nucleic acid-lipid particles, resist degradation by nucleases in aqueous solutions. Nucleic acid-lipid particles and methods for their preparation are disclosed, for example, in U.S. Patents 5,976,567; 5,981,501; 6,534,484; 6,586,410; and 6,815,432; and PCT Publication WO 96 / 40964.

[0434] In some embodiments, the nucleic acid-lipid particles comprise cationic lipids. Any cationic lipid or mixture thereof known in the art may be used. In some embodiments, the nucleic acid-lipid particles comprise non-cationic lipids. Any non-cationic lipid or mixture thereof known in the art may be used. In some embodiments, the nucleic acid-lipid particles comprise conjugated lipids (e.g., to prevent aggregation). Any conjugated lipid known in the art may be used.

[0435] VII.3 Additional delivery of preparations

[0436] Important factors to consider for successful in vivo delivery of dsRNA molecules include: (1) the biostability of the delivered molecule, (2) prevention of nonspecific effects, and (3) accumulation of the delivered molecule in the target tissue. Nonspecific effects of dsRNA can be minimized through local administration, such as by direct injection or implantation into tissues or by topical application of the formulation. For systemic administration of dsRNA to treat disease, dsRNA can be modified or delivered using a drug delivery system; both methods prevent rapid degradation of dsRNA by endonucleases and exonucleases in vivo. Modification of the RNA or drug carrier can also allow the dsRNA composition to be targeted to the target tissue and avoid undesirable off-target effects. As mentioned above, dsRNA molecules can be modified by chemical conjugation with lipophilic groups (e.g., cholesterol) to enhance cellular uptake and prevent degradation. In some embodiments, dsRNA is delivered using a drug delivery system, such as nanoparticles, dendritic polymers, polymers, liposomes, or cationic delivery systems. Positively charged cationic delivery systems promote the binding of dsRNA molecules (which are negatively charged) and also enhance interactions at negatively charged cell membranes to allow for efficient cellular uptake of dsRNA. Cationic lipids, dendritic polymers, or polymers can bind to dsRNA or induce the formation of vesicles or micelles (see, for example, Kim SH et al. (2008) Journal of Controlled Release 129(2): 107-116), which enhance dsRNA. When administered systemically, the formation of vesicles or micelles further prevents the degradation of dsRNA. Methods for preparing and administering cationic-dsRNA complexes are known in the art. In some embodiments, dsRNA forms a complex with cyclodextrin for systemic administration.

[0437] VIII. Methods using dsRNA

[0438] Some aspects of this disclosure relate to methods for inhibiting the expression of a target gene in mammals, the methods comprising administering an effective amount of one or more of the disclosed dsRNAs, one or more of the disclosed vectors, or compositions of the disclosed comprising one or more of the disclosed dsRNAs (e.g., pharmaceutical compositions). Some aspects of this disclosure relate to methods for treating and / or preventing diseases or conditions mediated by one or more target genes, the methods comprising administering one or more of the disclosed dsRNAs and / or one or more of the disclosed vectors and / or compositions comprising one or more of the disclosed dsRNAs (e.g., pharmaceutical compositions). In some embodiments, downregulating the expression of a target gene in a subject alleviates one or more symptoms of a disease or condition mediated by the target gene in the subject.

[0439] This disclosure also relates to a method of delivering oligonucleotides to liver (hepatic) cells of a human subject in need, the method comprising administering the oligonucleotides described herein to the subject.

[0440] In some embodiments of the method, administration is performed via intravenous or subcutaneous injection or via the portal vein.

[0441] This disclosure also relates to the use of oligonucleotides as described herein in the manufacture of pharmaceutical agents for the treatment of human subjects in need. In some of these embodiments, the oligonucleotides as described herein are used to treat human subjects in need. This disclosure also relates to a method of delivering a therapeutic agent to hepatocytes of a human subject in need, the method comprising administering to the subject a therapeutic portion of a compound described herein, particularly compounds of formula (I) or (II) described herein, and even more specifically, a therapeutic portion comprising an oligonucleotide conjugate of one or more of said compounds.

[0442] This disclosure also relates to the use of compounds described herein, particularly compounds of formula (I) or formula (II) described herein, and even more specifically, the use of oligonucleotides comprising one or more of said compounds for the manufacture of agents that target therapeutic agents to liver (hepatic) cells of human subjects in need.

[0443] This disclosure also relates to compounds described herein, particularly compounds of formula (I) or formula (II) described herein, and even more specifically to oligonucleotides comprising one or more of said compounds for delivering therapeutic agents to liver (hepatic) cells of human subjects in need.

[0444] In some embodiments of the above uses or methods, the therapeutic agent is a protein, peptide, peptide mimic, small molecule, or polynucleotide.

[0445] In some embodiments, the expression of the target gene in the subject is suppressed by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or about 100% after treatment, compared to pre-treatment levels. In some embodiments, the expression of the target gene is suppressed by at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 75-fold, or at least about 100-fold after treatment compared to pre-treatment levels. In some embodiments, the target gene is suppressed in the liver of the subject.

[0446] In some embodiments, the subject is a human being. In some embodiments, the subject has or has been diagnosed with a target gene-mediated condition or disease. In some embodiments, the subject is suspected of having a target gene-mediated condition or disease. In some embodiments, the subject is at risk of developing a target gene-mediated condition or disease.

[0447] As will be understood from this disclosure, the key feature of the dsRNAs described herein is that they contain one or more nucleotide analogs of formula (II), wherein the nucleotide analogs of formula (IV) have specific structural features of their “sugar-like” groups. The dsRNAs described herein are generally envisioned for targeting selected nucleic acid sequences contained in target nucleic acids of interest. In particular, embodiments of dsRNAs composed of siRNAs described herein include an antisense strand that specifically hybridizes to a nucleic acid sequence contained in a target nucleic acid of interest. The dsRNAs or compositions (e.g., pharmaceutical compositions) described herein can be used to treat target gene-mediated conditions or diseases. In particular, the dsRNAs or compositions (e.g., pharmaceutical compositions) described herein, and especially dsRNAs containing one or more targeting nucleotide analogs, and especially one or more ASGPR targeting nucleotide analogs of formula (IV), can be used to treat target gene-mediated conditions or diseases where targeting the liver is required.

[0448] Some aspects of this disclosure also relate to a method for delivering nucleic acids to hepatocytes, the method comprising contacting the hepatocytes with the dsRNA described herein.

[0449] The dsRNA or composition (e.g., pharmaceutical composition) described herein may be administered in any manner known in the art, including but not limited to oral or parenteral routes, including intravenous, intramuscular, subcutaneous, pulmonary, transdermal, and airway (aerosol) administration. Typically, when treating mammals with hyperlipidemia, dsRNA molecules are administered systemically via a parenteral route. In some embodiments, dsRNA and / or the composition are administered subcutaneously. In some embodiments, dsRNA and / or the composition are administered intravenously. In some embodiments, dsRNA and / or the composition are administered pulmonaryly.

[0450] The therapeutic or preventative effect of dsRNA is evident when one or more parameters of a disease state show statistically significant improvement, or when the condition does not worsen or when other expected symptoms appear. For example, a favorable change of at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more in a measurable parameter of the disease indicates effective treatment. The efficacy of a given dsRNA or a composition containing said dsRNA can also be determined using experimental animal models of a given disease or symptom known in the art. When using experimental animal models, therapeutic efficacy is demonstrated when a statistically significant reduction in biomarkers or symptoms is observed.

[0451] IX. Reagent Kits and Products

[0452] Certain aspects of this disclosure relate to articles or kits containing one or more dsRNAs, vectors, or compositions (e.g., pharmaceutical compositions) as described herein for the treatment and / or prevention of diseases. The articles or kits may also include containers and labels or instructions for use on or associated with the containers. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. Containers may be formed from a variety of materials, such as glass or plastic. The container contains a composition that, on its own or in combination with another composition, is effective in treating or preventing diseases and may have a sterile inlet (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a hypodermic needle). At least one active agent in the composition is dsRNA as described herein. The label or instructions for use indicate that the composition is for the treatment of a disease. Furthermore, the articles or kits may contain (a) a first container in which the composition contains dsRNA as described herein; and (b) a second container in which the composition contains a second therapeutic agent (e.g., an additional pharmaceutical agent as described herein). Articles or kits of this aspect of the disclosure may also include instructions for use indicating that the composition is for the treatment of a specific disease. Alternatively, the product or kit may also include a second (or third) container containing pharmaceutically acceptable buffers, such as water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and glucose solution. It may also include other materials required from a commercial and / or user perspective, including additional buffers, diluents, filters, needles, and syringes.

[0453] Unless otherwise defined herein, scientific and technical terms relating to this disclosure shall have the meanings commonly understood by one of ordinary skill in the art. Exemplary methods and materials are described below, although similar or equivalent methods and materials to those described herein may also be used in practice or testing of this disclosure. In case of conflict, this specification (including definitions) shall prevail.

[0454] Generally, the terms and techniques used herein related to cell and tissue culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal chemistry, and protein and nucleic acid chemistry and hybridization are those well-known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as is commonly done in the art or as described herein.

[0455] Furthermore, unless the context otherwise requires, singular terms shall include plural terms, and plural terms shall include singular terms. Throughout this specification and embodiments, the words “have” and “comprise” or variations such as “has,” “having,” “comprises,” or “comprising” shall be understood to imply inclusion of the specified integers or groups of integers but not exclude any other integers or groups of integers.

[0456] All publications and other references mentioned herein are incorporated herein by reference in their entirety. While this document cites numerous sources, such citations do not constitute an acknowledgment that any of these documents constitutes part of general knowledge in the field.

[0457] Example

[0458] The following examples are illustrated to better understand this disclosure. These examples are for illustrative purposes only and should not be construed as limiting the scope of this disclosure in any way.

[0459] Abbreviations used:

[0460] -AcOH: Acetic acid

[0461] -FA: Formic acid

[0462] -ACN: Acetonitrile

[0463] -DCM: Dichloromethane

[0464] -DMA: Dimethylacetamide

[0465] -DCE: Dichloroethane

[0466] -DMF: Dimethylformamide

[0467] -DMSO: Dimethyl sulfoxide

[0468] -EtOAc: Ethyl acetate

[0469] -EtOH: Ethanol

[0470] -Et2O: Diethyl ether

[0471] -iPrOH: Isopropanol

[0472] -THF: Tetrahydrofuran

[0473] -MeOH: Methanol

[0474] -NMP: N-methyl-2-pyrrolidone

[0475] -PE: Petroleum ether

[0476] -Pyr: Pyridine

[0477] -iPr: Isopropyl

[0478] -iBu: Isobutyryl

[0479] -cHex: Cyclohexyl

[0480] -MTB: Methyl-tert-butyl

[0481] -DIPEA: Diisopropylethylamine

[0482] -DMAP: 4-(dimethylamino)-pyridine

[0483] -DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene

[0484] -HBTU: (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea-hexafluorophosphate)

[0485] -TBTU: O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethylurea tetrafluoroborate

[0486] -DDTT: 3-((N,N-dimethyl-aminomethylene)amino)-3H-1,2,4-dithiazolyl-5-thione

[0487] -NEt3: Triethylamine

[0488] -NEM: N-ethylmorpholine

[0489] -BSA: N,O-bis-trimethylsilylacetamide

[0490] -TMSOTf: Trimethylsilyl trifluoromethanesulfonate

[0491] -Ts: p-Toluenesulfonyl group

[0492] -Tf: Trifluoromethanesulfonyl group

[0493] -trifluoromethanesulfonate

[0494] -TFA: Trifluoroacetic acid

[0495] -DCAA: Dichloroacetic acid

[0496] -TEA: Triethylammonium

[0497] -TIPS: Triisopropylsilyl

[0498] -TBDMS: tert-butyldimethylsilyl

[0499] -DMT: 4,4'-Dimethoxytriphenylmethyl

[0500] -Bzl: Benzoyl group

[0501] -Bn: Benzyl

[0502] -BOM: Benzyloxymethyl

[0503] -Ac: Acetyl group

[0504] - 1 Bu: Isobutyryl

[0505] -Boc: tert-Butyloxycarbonyl

[0506] -Fmoc: fluorenylmethoxycarbonyl

[0507] -Fmoc-OSu:N-(9-fluorenylmethoxycarbonyloxy)succinimide

[0508] -CE: Cyanoethyl

[0509] -CPG: Controlled Porosity Glass

[0510] -T: Thymine

[0511] -U: Uracil

[0512] -C: Cytosine

[0513] -A: Adenine

[0514] -G: Guanine

[0515] -I: Hypoxanthine

[0516] -T BOM N-Benzyloxymethyl-thymine

[0517] -U BOM N-Benzyloxymethyl-uracil

[0518] -U Bzl N-benzoyl-uracil

[0519] -C Bzl N-benzoyl-cytosine

[0520] -A Bzl N-benzoyl-adenine

[0521] -G iBu N-Isobutyryl-Guanine

[0522] -GalNAc: DN-acetylgalactosamine

[0523] -FR: Flow rate

[0524] HPLC: High-performance liquid chromatography

[0525] MS-TOF: Mass Spectrometry - Time of Flight

[0526] LC-MS: High-performance liquid chromatography-mass spectrometry

[0527] -R t Retention time

[0528] -RT: Room temperature

[0529] -Hal: Halogen

[0530] -ELSD: Evaporative Light Scattering Detector

[0531] -quant.: quantitative

[0532] -sat.: saturation

[0533] -i.vac.: in a vacuum

[0534] -nd: Not determined

[0535] -TLC: Thin-layer chromatography

[0536] _h: hours

[0537] -min: minutes

[0538] -Tm: Melting temperature

[0539] -r: ribonucleotides

[0540] -d: deoxyribonucleotide

[0541] -m: 2'-OMe-nucleotide

[0542] -f: 2'-deoxy-fluorine-nucleotide

[0543] -ss: Chain of Justice

[0544] -as: antichain

[0545] -ds: doubly linked

[0546] -chol: cholesterol

[0547] -PO: Phosphodiester bond

[0548] -* or PS: Thiophosphate bond

[0549] -mpk: mg / kg

[0550] -M: Moore

[0551] -#: Number, n°

[0552] -FBS: Fetal bovine serum

[0553] -ATP: Adenosine triphosphate

[0554] -pre-lB: Pre-nucleotide

[0555] -pre-lgB: Targeting precursor nucleotides

[0556] -1B: Nucleotide analogues

[0557] -lgB: Targeted nucleotide analogue example 1: Synthetic examples of compounds 2, 3, and 23

[0558] Example 1.1: Synthesis of N-[[(2R,3S,4R,5R)-5-(2-amino-6-oxo-1H-purin-9-yl)-3,4-dihydroxy-tetrahydrofuran-2-yl]methyl]acetamide (2)

[0559] 5'-Deoxy-5'-amino-guanosine (1,100 mg, 0.34 mmol) was dissolved in 1.4 mL of pyridine, and 89 mg (0.67 mmol) of NEt3 was added at room temperature. After adding 139 mg (1.35 mmol) of acetic anhydride, the reaction solution was stirred overnight at room temperature. The solvent was removed under vacuum, and the residue was dissolved in 5 mL of MeOH / H2O (1:1). After adding 1 mL (1.0 mmol) of 1M NaOH solution, the reaction mixture was stirred at room temperature for 2 hours. The solution was diluted with 2.5 mL of H2O and neutralized with 2N HCl. After adding 130 mg of Amberlite IRN 150 ion exchanger, the mixture was stirred for 15 minutes. The mixture was filtered and the MeOH was evaporated. The aqueous solution was lyophilized to give 64 mg (58.6%) of the title compound N-[[(2R,3S,4R,5R)-5-(2-amino-6-oxo-1H-purine-9-yl)-3,4-dihydroxy-tetrahydrofuran-2-yl]methyl]acetamide (2) as a colorless foam.

[0560] LC-MS (Method A):

[0561] R t [min](ELSD-signal): 0.30

[0562] MS (calculated value: 324.1)(m / z) = 325.3[M+H + ]

[0563] 1H-NMR (600MHz, DMSO-d6) [ppm]: 10.68, (s, 1H), 8.00 (t, J=5.9Hz, 1H), 7.91 (s, 1H), 6.52 (br s, 2H), 5.66 (d, J = 5.9Hz, 1H), 5.42 (d, J = 6.1Hz, 1H), 5.15 (br d, J=4.6Hz, 1H), 4.42 (dd, J=11.2, 5.5Hz, 1H), 4.02 (m, 1H), 3.83 (m, 1H), 3.44 (dt, J=13.9, 5.7Hz1H), 3.21 (dt, J=13.8, 6.2Hz 1H), 1.81 (s, 3H).

[0564] Example 1.2: Synthesis of N-[[(2R,3S,4R,5R)-5-(2-amino-6-oxo-1H-purin-9-yl)-3,4-dihydroxy-tetrahydrofuran-2-yl]methyl]propionamide (3)

[0565] 5'-Deoxy-5'-amino-guanosine (1,100 mg, 0.34 mmol) was dissolved in 1.4 mL of pyridine, and 89 mg (0.67 mmol) of NEt3 was added at room temperature. After adding 177 mg (1.35 mmol) of propionic anhydride, the reaction solution was stirred overnight at room temperature. The solvent was removed under vacuum, and the residue was dissolved in 5 mL of MeOH / H2O (1:1). After adding 1 mL (1.0 mmol) of 1 M NaOH solution, the reaction mixture was stirred at room temperature for 2 hours. The MeOH was removed under vacuum, and the precipitate was collected by filtration. After drying the precipitate under vacuum, 62 mg (54.4%) of the title compound N-[[(2R,3S,4R,5R)-5-(2-amino-6-oxo-1H-purine-9-yl)-3,4-dihydroxy-tetrahydrofuran-2-yl]-methyl]propionamide (3) was obtained as a colorless solid.

[0566] LC-MS (Method A):

[0567] R t [min](ELSD-signal): 0.51

[0568] MS (calculated value: 338.1)(m / z) = 339.3[M+H + ]

[0569] 1H-NMR (600MHz, DMSO-d6) [ppm]: 10.87 (br s, 1H), 7.92 (t, J=5.9Hz, 1H), 7.88 (s, 1H), 6.54 (br s, 2H), 5.66 (d, J=6.1Hz, 1H), 5.42 (br s, 1H), 5.14 (br s, 1H), 4.41 (t, J=5.6Hz, 1H), 4.02 (t, J=4.4Hz, 1H), 3.83 (m, 1H), 3.44 (dt, J=13.9, 5 .6Hz, 1H), 3.23 (dt, J=13.9, 6.2Hz, 1H), 2.09 (q, J=7.5Hz, 2H), 0.98 (t, J=7.6Hz, 3H).

[0570] Example 1.3: Synthesis of methyl 4-methylbenzenesulfonic acid [(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]dioxacyclopenten-6-yl]methyl ester (5)

[0571] TsCl (68 g, 0.356 mol, 1.5 equivalent) was added fractionally to a mixture of methyl-2,3-di-O-isopropylidene-D-riboside (4, 48.5 g, 0.237 mol, 1.0 equivalent) in pyridine (97 mL) at 0 °C. The mixture was stirred at 25 °C for 5 hours to achieve complete conversion. After adding 100 mL of cold water, the mixture was stirred at 25 °C for 1 hour. The precipitate was filtered, washed with 2 × 100 mL of cold water, and dried under vacuum to give 79.6 g (93.5%, crude matter) of toluenesulfonate 4-methylbenzenesulfonic acid [(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofuran[3,4-d][1,3]dioxane-6-yl]methyl ester (5), which was separated as a white solid.

[0572] 1 H-NMR (400MHz, CDCl3) δ [ppm]: 7.70-7.78 (m, 2H), 7.29 (d, J=8.0Hz, 2H), 4.86 (s, 1H), 4.51-4.56 (m, 1H), 4.4 3-4.49 (m, 1H), 4.24 (t, J=7.2Hz, 1H), 3.90-3.99 (m, 2H), 3.17 (s, 3H), 2.39 (s, 3H), 1.38 (s, 3H), 1.22 (s, 3H).

[0573] Example 1.4: Synthesis of (3aR,6R,6aR)-6-(azidomethyl)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano-[3,4-d][1,3]dioxacyclopentene; 4-methylbenzenesulfonic acid [(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano-[3,4-d][1,3]dioxacyclopenten-6-yl]methyl ester (6)

[0574] A mixture of toluenesulfonate 4-methylbenzenesulfonic acid [(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]dioxacyclopenten-6-yl]methyl ester (5.36 g, 0.100 mol, 1.0 equivalent) and NaN3 (13 g, 0.201 mol, 2.0 equivalent) in DMF (360 mL) was heated to 120 °C and maintained for 4 hours. The heating bath was removed and the mixture was allowed to reach room temperature. 200 mL of acetone was added and stirring was continued for 30 minutes. The acetone was removed under vacuum, and the remaining solution was poured into 1000 mL of water. After extraction with 3 × 1000 mL, the organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The residue was purified by column chromatography (PE / EtOAc 30:1) to give 21.0 g (91.2%) of the desired azide (3aR,6R,6aR)-6-(azidomethyl)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano-[3,4-d][1,3]dioxacyclopentene; 4-methylbenzenesulfonic acid [(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofuran[3,4-d][1,3]dioxacyclopentene-6-yl]methyl ester (6).

[0575] 1 H-NMR (400MHz, CDCl3) δ [ppm]: 4.93 (s, 1H), 4.49-4.58 (m, 2H), 4.22 (t, J==7.3Hz, 1H), 3.38 (d d, J=12.5, 7.64Hz, 1H), 3.31 (s, 3H), 3.20 (dd, J=12.53, 6.8Hz, 1H), 1.42 (s, 3H), 1.25 (s, 3H).

[0576] Example 1.5: Synthesis of [(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]dioxacyclopenten-6-yl]methylamine (7)

[0577] PPh3 (20.6 g, 78.524 mmol, 1.2 equivalents) was added fractionally to a mixture of (3aR,6R,6aR)-6-(azidomethyl)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano-[3,4-d][1,3]dioxacyclopentene;4-methylbenzenesulfonic acid [(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano-[3,4-d][1,3]dioxacyclopenten-6-yl]methyl ester (6,15 g, 65.4 mmol, 1.0 equivalent) in THF (75 ml). The solution was stirred for 16 hours to achieve complete conversion. After adding 75 ml of H2O, stirring was continued for 5 hours. The mixture was extracted with 2 × 200 ml DCM, and the combined organic layers were dried over anhydrous Na2SO4. After filtration, the solvent was evaporated under vacuum to give 30 g of the title compound [(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofuran[3,4-d][1,3]dioxane-6-yl]methylamine (7) as a white solid (crude substance, purity approximately 44%), which can be used without further purification.

[0578] Example 1.6: Synthesis of N-[[(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofuran[3,4-d][1,3]-dioxacyclopenten-6-yl]methyl]-2-methylpropionamide (8)

[0579] Amine [(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]-dioxacyclopenten-6-yl]methylamine (7,4-d][1,3]-dioxacyclopenten-6-yl]methylamine (44% crude purity) was dissolved in 43 mL of NEt3 and 173 mL of DCM. After adding dropwise 2-methylpropionyl chloride (8.4 g, 78.5 mmol) dissolved in 108 mL of DCM at 0 °C, the ice bath was removed and the solution was stirred at room temperature for 3 hours. The solution was diluted with 300 mL of DCM and washed with water (2 × 150 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The residue was purified by rapid chromatography (PE / EtOAc 1∶1) to give 17.86 g (65.3%, two steps) of the title compound N-[[(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]-dioxacyclopenten-6-yl]methyl]-2-methylpropionamide (8) as a colorless oil.

[0580] 1H-NMR (400MHz, CDCl3) δ [ppm]: 6.17 (br s, 1H), 4.91 (s, 1H), 4.45-4.57 (m, 2H), 4.30 (t, J=5.1Hz, 1H), 3.51 (dt, J==14.1, 6.4Hz, 1H), 3.34 (s, 3H) , 3.26 (dt, J=14.2, 4.5Hz, 1H), 2.30 (spt, J=6.9Hz, 1H), 1.40 (s, 3H), 1.23 (s, 3H), 1.09 (d, J==7.0Hz, 6H).

[0581] Example 1.7: Synthesis of 2-methyl-N-[[(2R,3S,4R)-3,4,5-trihydroxytetrahydrofuran-2-yl]methyl]acrylamide (11)

[0582] The starting material N-[[(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano-[3,4-d][1,3]-dioxacyclopenten-6-yl]methyl]-2-methylpropionamide (8, 12.86 g, 47.0 mmol, 1.0 equivalent) was dissolved in 0.1 N H₂SO₄ (155 mL, 15.5 mmol, 0.33 equivalent) and dioxane (77 mL). The reaction mixture was refluxed for 2 hours to achieve complete conversion. The reaction mixture was cooled to room temperature and neutralized with Ba(OH)₂8H₂O. After vacuum evaporation of the solvent, the residue was co-evaporated three times with 100 ml of dioxane to obtain 10.3 g (crude substance) of the deprotected product 2-methyl-N-[[(2R,3S,4R)-3,4,5-trihydroxytetrahydrofuran-2-yl]methyl]acrylamide (11), which is a white solid and can be used without further purification.

[0583] Example 1.8: Synthesis of acetate [(2R,3R,4R)-4,5-diacetoxy-2-[(2-methylpropionamido)-methyl]tetrahydrofuran-3-yl] ester (14)

[0584] The ribose derivative 2-methylN-[[(2R,3S,4R)-3,4,5-trihydroxytetrahydrofuran-2-yl]methyl]acrylamide (11, 10.3 g, 47.0 mmol, 1.0 equivalent) was co-evaporated three times with 100 mL of pyridine and dissolved in pyridine (155 mL). After adding 52 mL of acetic anhydride at room temperature, the solution was stirred for 16 hours. The reaction solution was concentrated under vacuum, and the residue was purified by column chromatography (PE / EtOAc 1:1) to give 14.4 g (88.5%, two steps) of a yellow oily acetoriboside acetate [(2R,3R,4R)-4,5-diacetoxy-2-[(2-methylpropionylamino)methyl]tetrahydrofuran-3-yl] ester (14).

[0585] 1 H-NMR (400MHz, CDCl3) δ [ppm]: 6.14 (s, 1H), 5.80-5.91 (m, 1H), 5.33 (d, J==4.8Hz, 1H), 5.13-5.20 (m, 1H), 4.23-4.32 (m , 1H), 3.67 (ddd, J=14.3, 6.02, 3.9Hz, 1H), 3.35-3.46 (m, 1H), 2.30-2.44 (m, 1H), 2.05-2.17 (m, 9H), 1.11-1.22 (m, 6H).

[0586] Example 1.9: Synthesis of acetate [(2R,3R,4R,5R)-4-acetoxy-2-[(2-methylpropionylamino)-methyl]-5-[2-(2-methyl-propionyl-amino)-6-oxo-1H-purine-9-yl]tetrahydrofuran-3-yl] ester (17)

[0587] The starting material, acetate [(2R,3R,4R)-4,5-diacetoxy-2-[(2-methylpropionylamino)methyl]-tetrahydrofuran-3-yl] ester (14, 12 g, 34.7 mmol, 1.0 equivalent), and the compound isobutyryl-guanine (11.5 g, 52.1 mmol, 1.5 equivalent), were dissolved in DCE (480 mL). BSA (28.3 g, 0.139 mol, 4.0 equivalent) was added dropwise at room temperature, and the solution was stirred at 95 °C for 2 h. TMSOTf (23 g, 0.104 mol, 3.0 equivalent) was added at 90 °C, and stirring continued at this temperature for 7 h. The heating bath was removed, and the solution was cooled to room temperature. After adding 200 mL of water, the mixture was extracted with DCM (3 × 200 mL), and the combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum. The residue was purified by rapid chromatography (4% MeOH / EtOAc) to give 13.3 g of yellow foaming guanosine analogue (17) (75.7%, purity 66%). 5 g was purified by reverse rapid chromatography (TFA) to give 3.4 g of white foaming pure acetate [(2R,3R,4R,5R)-4-acetoxy-2-[(2-methylpropionylamino)methyl]-5-[2-(2-methyl-propionyl-amino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-3-yl] ester ((17)).

[0588] 1 H-NMR (400MHz, DMSO-d6) δ [ppm]: 12.12 (s, 1H), 11.37-11.74 (m, 1H), 8.31 (s, 1H), 8.01-8.13 (m, 1H), 6.05 (d, J=6.5Hz, 1H ), 5.71 (t, J=6.2Hz, 1H), 5.45 (dd, J=5.7, 3.6Hz, 1H), 4.18 (td, J=5.9, 3.8Hz, 1H), 3.63 (dt, J==13.9, 6.1Hz, 1H), 3.39 (br d, J=2.0Hz, 1H), 2.79 (spt, J=6.8Hz, 1H), 2.32-2.45 (m, 1H), 2.08-2.16 (m, 3H), 1.97-2.05 (m, 3H), 1.14 (dd, J=6.9, 2.1Hz, 6H), 1.00 (t, J=6.48Hz, 6H).

[0589] MS (calculated value: 506.2) (m / z) = 507.5 [M+H + ].

[0590] Example 1.10: Synthesis of N-[[(2R,3S,4R,5R)-5-(2-amino-6-oxo-1H-purin-9-yl)-3,4-dihydroxy-tetrahydrofuran-2-yl]methyl]-2-methylpropionamide (19)

[0591] The starting material, acetate [(2R,3R,4R,5R)-4-acetoxy-2-[(2-methylpropionylamino)methyl]-5-[2-(2-methyl-propionyl-amino)-6-oxo-1H-purine-9-yl]tetrahydrofuran-3-yl] ester (17.2 g, 3.95 mmol, 1.0 equivalent), was dissolved in a MeOH solution containing 0.1 N NaOMe (13 mL, 1.303 mmol, 0.33 equivalent) and heated at 60 °C for 5 hours. After the solution cooled to room temperature, the pH was adjusted to 6 by adding 1 N HCl aqueous solution. The precipitate was filtered and wet-milled with water (20 ml) and acetone (5 ml) to give 1.12 g (80.6%) of a colorless solid of the guanosine analog N-[[(2R,3S,4R,5R)-5-(2-amino-6-oxo-1H-purine-9-yl)-3,4-dihydroxy-tetrahydrofuran-2-yl]methyl]-2-methyl-propionamide (19).

[0592] MS (calculated value: 352.1)(m / z) = 353.1[M+H + ].

[0593] 1 H-NMR (400MHz, DMSO-d6) δ [ppm]: 10.66 (s, 1H), 7.83-8.00 (m, 2H), 6.48 (br s, 2H), 5.67 (d, J=6.1Hz, 1H), 5.45 (br d, J==5.8Hz, 1H), 5.16 (br d, J==3.9Hz, 1H), 4.40 (q, J=5.4Hz, 1H), 4.02(br d, J=3.4Hz, 1H), 3.78-3.90 (m, 1H), 3.41-3.51 (m, 1H), 3.15-3.31 (m, 1H), 2.39 (spt, J==6.9Hz, 1H), 0.98 (dd, J=6.9, 3.1Hz, 6H).

[0594] Example 1.11: Synthesis of N-[[(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]-dioxacyclopenten-6-yl]methyl]butyramide (9)

[0595] The starting material [(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]dioxacyclopenten-6-yl]methylamine (7, 22.5 g, purity 39%, impurity Ph3PO, 43.6 mmol, 1.0 equivalent) was synthesized according to the synthesis of N-[[(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]dioxacyclopenten-6-yl]methyl]-2-methyl-propionamide (8) with propionyl chloride (5.6 g, 52.3 mmol, 1.2 equivalent) and subjected to column chromatography (PE / EtOAc). After 2:1), 17.2 g (69% purity, 100% impurity Ph3PO) of the desired amide N-[[(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d]-[1,3]-dioxacyclopenten-6-yl]methyl]butyramide (9) was obtained as a colorless oil.

[0596] Example 1.12: Synthesis of N-[[(2R,3S,4R)-3,4,5-trihydroxytetrahydrofuran-2-yl]methyl]-butyramide (12)

[0597] The ribose derivative N-[[(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano-[3,4-d][1,3]-dioxacyclopenten-6-yl]methyl]butyramide (9, 17.2 g, 69% purity, 43.6 mmol, 1.0 equivalent) was dissolved in 0.1 N H₂SO₄ (140 mL, 14.4 mmol, 0.33 equivalent) and dioxane (70 mL). After heating under reflux for 2 hours, the reactants were cooled to room temperature and the mixture was neutralized with Ba(OH)₂·8H₂O (solid). The solution was evaporated under vacuum, and the residue was diluted with water (100 mL) and washed with EtOAc (3 × 50 mL). The aqueous layer was concentrated under vacuum, and the residue was co-evaporated with dioxane (3 × 100 ml) to obtain the title compound N-[[(2R,3S,4R)-3,4,5-trihydroxytetrahydrofuran-2-yl]methyl]butyramide (12) (10.2 g, purity 94%, 100%) as a white solid, which can be used without further purification.

[0598] Example 1.13: Synthesis of acetate [(2R,3R,4R)-4,5-diacetoxy-2-[(butyrylamino)methyl]tetrahydrofuran-3-yl] ester (15)

[0599] Following the method described for synthesizing [(2R,3R,4R)-4,5-diacetoxy-2-[(2-methylpropionylamino)methyl]tetrahydrofuran-3-yl] ester (14), the starting material N-[[(2R,3S,4R)-3,4,5-trihydroxytetrahydrofuran-2-yl]methyl]butyramide (12, 10.2 g, 43.6 mmol, 1.0 equivalent) was acetylated, and 10.1 g (67.3%) of yellow oily triacetate [(2R,3R,4R)-4,5-diacetoxy-2-[(butyroxyamino)methyl]tetrahydrofuran-3-yl] ester (15) was produced after silica gel chromatography (PE / EtOAc 1∶1).

[0600] Example 1.14: Synthesis of acetate [(2R,3R,4R,5R)-4-acetoxy-2-[(butyrylamino)methyl]-5-[2-(2-methylpropionylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-3-yl] ester (18)

[0601] The starting compound, acetate [(2R,3R,4R)-4,5-diacetoxy-2-[(butyrylamino)methyl]tetrahydro-furan-3-yl] ester (15.5 g, 14.5 mmol, 1.0 equivalent), was synthesized with isobutyryl-guanine glycosylation according to the scheme described for the synthesis of acetate [(2R,3R,4R,5R)-4-acetoxy-2-[(2-methylpropionylamino)methyl]-5-[2-(2-methyl-propionyl-amino)-6-oxo-1H-purine-9-yl]tetrahydrofuran-3-yl] ester (17). Following silica gel chromatography (EtOAc / MeOH 20:1), 4.5 g (61.6%) of the title compound, acetate [(2R,3R,4R,5R)-4-acetoxy-2-[(butyrylamino)methyl]-5-[2-(2-methylpropionyl-amino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-3-yl] ester (18), was separated as a white foam. MS (calculated value: 506.2) (m / z) = 507.3 [M+H + ].

[0602] Example 1.15: Synthesis of N-[[(2R,3S,4R,5R)-5-(2-amino-6-oxo-1H-purin-9-yl)-3,4-dihydroxy-tetrahydrofuran-2-yl]methyl]butyramide (20)

[0603] Following the synthesis of N-[[(2R,3S,4R,5R)-5-(2-amino-6-oxo-1H-purin-9-yl)-3,4-dihydroxy-tetrahydrofuran-2-yl]methyl]-2-methyl-propionamide (19), the starting material acetate [(2R,3R,4R,5R)-4-acetoxy-2-[(butyrylamino)methyl]-5-[2-(2-methylpropyl-amino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-3-yl] ester (18, 3.0 g, 5.9 mmol, 1.0 equivalent) was treated with NaOMe / MeOH at 60 °C for 8 hours. After treatment as described for (19), 1.46 g (70.2%) of the title compound N-[[(2R,3S,4R,5R)-5-(2-amino-6-oxo-1H-purine-9-yl)-3,4-dihydroxy-tetrahydrofuran-2-yl]methyl]butyramide (20) was isolated as a white solid.

[0604] MS (calculated value: 352.1)(m / z) = 353.0 [M+H + ].

[0605] 1 H-NMR (400MHz, DMSO-d6) δ [ppm]: 10.71 (s, 1H), 8.01-7.92 (m, 2H), 6.52 (br s, 2H), 5.68 (d, J=6.0Hz, 1H), 5.57-4.89 (m, 2H), 4.42 (t, J=5.6Hz, 1H), 4.07-3.98 (m, 1H), 3.88-3.78 (m, 1H), 3 .45 (td, J=5.7, 13.7Hz, 1H), 3.30-3.17 (m, 1H), 2.12-2.02 (m, 2H), 1.50 (sxt, J=7.4Hz, 2H), 0.93-0.74 (m, 3H).

[0606] Example 1.16: Synthesis of methyl 4-[[(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]-dioxacyclopenten-6-yl]methylamino]-4-oxo-butyrate (10)

[0607] At 0 °C, NEt3 (36.4 mL) was added dropwise to a solution of the starting compound [(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]dioxane-6-methylamine (7,21.3 g, crude material, 1.0 equivalent) in DCM (150 mL), followed by the addition of monomethyl succinate (7.55 g, 50.3 mmol, 1.2 equivalent) in DCM (92 mL). After stirring at room temperature for 3 hours, the solution was diluted with DCM (200 mL), washed with water (100 mL), dried over anhydrous a₂SO₄, filtered, and concentrated under vacuum. The residue was purified by column chromatography (PE / EtOAc 1∶1) to yield 20.0 g (66%, 46% purity) of the title compound methyl 4-[[(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]-dioxacyclopenten-6-yl]methylamino]-4-oxo-butyrate (10).

[0608] 1 H-NMR (400MHz, CDCl3) δ [ppm]: 4.99 (s, 1H), 4.67-4.59 (m, 2H), 4.37 (t, J=5.3Hz, 1H), 3.73-3.68 (m, 3H), 3.63-3. 54(m, 1H), 3.45-3.40(m, 3H), 3.39-3.29(m, 1H), 2.79-2.59(m, 2H), 2.51-2.44(m, 2H), 1.48(s, 3H),, 1.32(s, 3H).

[0609] Example 1.17: Synthesis of methyl 4-oxo-4-[[(2R,3S,4R)-3,4,5-trihydroxytetrahydrofuran-2-yl]methylamino]-butyrate (13)

[0610] At room temperature, a single addition of 0.1 N H₂SO₄ solution (24 mL, 2.4 mmol, 0.33 equivalences) was made to a solution of methyl 4-[[(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofuran-[3,4-d][1,3]-dioxane-6-yl]methylamino]-4-oxoyl-butyrate (10.5 g, 7.25 mmol, 1.0 equivalence) in dioxane (30 mL). The solution was stirred at 120 °C for 2 h to achieve complete conversion. The reaction mixture was adjusted to pH 7 with Ba(OH)₂₈H₂O (solid) and filtered. The filtrate was washed with EtOAc (2 × 30 mL). The aqueous layer was separated and concentrated under vacuum to obtain 2.0 g (crude substance, quantitative) of a yellow oily deprotected ribose derivative, methyl 4-oxo-4-[[(2R,3S,4R)-3,4,5-trihydroxytetrahydrofuran-2-yl]methylamino]-butyrate (13), which can be used without further purification.

[0611] Example 1.18: Synthesis of methyl 4-oxo-4-[[(2R,3R,4R)-3,4,5-triacetoxytetrahydrofuran-2-yl]methylamino]-butyrate (16)

[0612] Methyl 4-oxo-4-[[(2R,3R,4R)-3,4,5-triacetoxy-tetrahydrofuran-2-yl]methylamino]-butyrate (13, 2.0 g, 7.6 mmol, 1.0 equivalent) was added dropwise to a solution of starting material in pyridine (20 mL) at room temperature with 10 mL of Ac2O. The solution was stirred for 12 h and evaporated under vacuum. The residue was purified by column chromatography (PE / EtOAc 2:1) to give 1.2 g (41%) of the title compound methyl 4-oxo-4-[[(2R,3R,4R)-3,4,5-triacetoxy-tetrahydrofuran-2-yl]methylamino]-butyrate (16) as a yellow oil.

[0613] MS (calculated value: 389.1) (m / z) = 412.2 [M + Na] + ].

[0614] 1 H-NMR (400MHz, CDCl3) δ [ppm]: 6.15-6.11 (m, 1H), 5.41-5.36 (m, 1H), 5.28 (d, J=5.4Hz, 1H), 5.26-5.23 (m, 1H) , 5.23-5.07(m, 1H), 4.51-4.44(m, 1H), 3.88-3.67(m, 4H), 2.73(s, 3H), 2.18(s, 3H), 2.13(s, 3H), 2.08(s, 3H).

[0615] Example 1.19: Synthesis of acetate [(2R,3R,4R,5R)-4-acetoxy-2-[(2,5-dioxopyrrolidone-1-yl)methyl]-5-[2-(2-methyl-propionyl-amino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-3-yl] ester (21)

[0616] BSA (5.84 g, 28.8 mmol, 4.0 equivalent) was added dropwise to a solution of starting materials methyl 4-oxo-4-[[(2R,3R,4R)-3,4,5-triacetoxy-tetrahydrofuran-2-yl]methylamino]-butyrate (16 g, 2.8 g, 7.2 mmol, 1.0 equivalent) and isobutyrylguanine (2.38 g, 10.8 mmol, 1.5 equivalent) in DCE (110 mL). After stirring at 95 °C for 2 hours, TMSOTf (4.8 g, 21.6 mmol, 3.0 equivalent) was added dropwise at 90 °C, and stirring was continued at 90 °C for 12 hours. The solution was cooled to room temperature and 150 mL of H₂O was added. The layers were separated and the aqueous layer was extracted with DCM (3 × 100 mL). The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum. The residue was purified by rapid chromatography (10% MeOH / EtOAc) and reverse rapid chromatography (FA) to give 1.5 g (40%) of the title compound acetate [(2R,3R,4R,5R)-4-acetoxy-2-[(2,5-dioxopyrrolidone-1-yl)methyl]-5-[2-(2-methyl-propionyl-amino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-3-yl] ester (21) as a white foam.

[0617] MS (calculated value: 518.2)(m / z) = 519.5[M+H + ].

[0618] 1 H-NMR (400MHz, CDCl3) δ [ppm]: 12.06 (br s, 1H), 9.70 (s, 1H), 7.61 (s, 1H), 5.89-5.79 (m, 2H), 5.69 (t, J=5.4Hz, 1H), 4.64-4.53 (m, 1H), 4.36 (dd, J==9.4, 13.3Hz, 1H) , 3.68 (dd, J==5.7, 13.3Hz, 1H), 2.74-2.63 (m, 5H), 2.04 (s, 3H), 1.99 (s, 3H), 1.24 (d, J==6.8Hz, 3H), 1.19 (d, J==6.8Hz, 3H).

[0619] Example 1.20: Synthesis of 1-[[(2R,3S,4R,5R)-5-(2-amino-6-oxo-1H-purin-9-yl)-3,4-dihydroxy-tetrahydrofuran-2-yl]methyl]pyrrolidine-2,5-dione (22)

[0620] At room temperature, a freshly prepared 1M methanol-NaOMe solution (0.63 mL, 0.63 mmol, 0.33 equivalents) was added dropwise to a solution of acetate [(2R,3R,4R,5R)-4-acetoxy-2-[(2,5-dioxopyrrolidone-1-yl)methyl]-5-[2-(2-methyl-propionyl-amino)-6-oxo-1H-purine-9-yl]tetrahydrofuran-3-yl] ester (21, 1.2 g, 2.1 mmol, 1.0 equivalent) in MeOH (6.3 mL). The solution was heated at 60 °C for 9 hours, and then allowed to reach room temperature. The mixture was filtered and the residue was vacuum dried to give 720 g (85.7%) of a white solid guanosine analog 1-[[(2R,3S,4R,5R)-5-(2-amino-6-oxo-1H-purine-9-yl)-3,4-dihydroxy-tetrahydrofuran-2-yl]methyl]pyrrolidine-2,5-dione (22).

[0621] MS (calculated value: 364.1)(m / z) = 365.0 [M+H + ].

[0622] Example 1.21: Synthesis of 4-[[(2R,3S,4R,5R)-5-(2-amino-6-oxo-1H-purin-9-yl)-3,4-dihydroxy-tetrahydrofuran-2-yl]methylamino]-4-oxo-butyric acid (23)

[0623] At room temperature, LiOH H2O (80 mg, 1.9 mmol, 1.0 equivalent) was added to a mixture of 1-[[(2R,3S,4R,5R)-5-(2-amino-6-oxo-1H-purine-9-yl)-3,4-dihydroxy-tetrahydrofuran-2-yl]methyl]pyrrolidine-2,5-dione (22,720 mg, 2.0 mmol, 1.0 equivalent) in a mixed solvent of THF (7 ml) and water (7 ml). The solution was stirred at 30 °C for 2 hours. The solvent was evaporated under vacuum to give 770 mg of the title compound 4-[[(2R,3S,4R,5R)-5-(2-amino-6-oxo-1H-purine-9-yl)-3,4-dihydroxy-tetrahydrofuran-2-yl]methylamino]-4-oxo-butyric acid (23) (Li-salt) as a yellow solid.

[0624] MS (calculated value: 382.1)(m / z) = 383.1[M+H + ].

[0625] 1 H-NMR (400MHz, D2O) δ [ppm]: 7.78 (s, 1H), 5.79 (d, J==5.4Hz, 1H), 4.63 (br t, J==5.3Hz, 1H), 4.23 (brt, J==4.5Hz, 1H), 4.12 (br d, J==4.6Hz, 1H), 3.56-3.42 (m, 2H), 2.37 (br s, 4H).

[0626] Example 2: Synthetic scheme for example compound 30

[0627] Example 2.1: Synthesis of methyl acetate [(2S,3S,4R,5R)-4-acetoxy-3-benzyloxy-2-(benzyloxymethyl)-5-[2-(2-methylpropionamido)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl] (25)

[0628] N-Isobutyryl-guanine (135 g, 0.61 mol) and BSA (311.85 ml, 1.2 mol) were added to a solution of the starting material [(2S,3S,4R)-4,5-diacetoxy-3-benzyloxy-2-(benzyloxy-methyl)tetrahydrofuran-2-yl]methyl acetate (24,148.5 g, 0.30 mol) in 6,681 DCE at 15 °C under a nitrogen atmosphere. The mixture was stirred at 85 °C for 3 hours. TMSOTf (183.4 g, 0.90 mol) was added at 85 °C and stirring was continued for 3 hours to achieve complete conversion. The mixture was cooled to room temperature and poured into a 6,51 saturated NaHCO3 solution. The organic layer was separated, and the aqueous phase was extracted twice with 51 DCM. The organic layers were combined, dried over anhydrous Na2SO4, filtered, and concentrated. The crude product obtained by preparative HPLC (0.1% TFA / ACN) yielded a white solid compound, methyl acetate [(2S,3S,4R,5R)-4-acetoxy-3-benzyloxy-2-(benzyloxymethyl)-5-[2-(2-methylpropionamido)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl] (25) (128 g, 64%).

[0629] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 12.09 (s, 1H), 11.62 (s, 1H), 8.14 (s, 1H), 7.41-7. 30 (m, 10H), 6.12 (d, J=6.4Hz, 1H), 5.90 (t, J1=J2=5.6Hz, 1H), 4.71 (d, J=5.2Hz, 1H), 4.63-4.55(m, 4H), 4.34(d, J=5.6Hz, 1H), 4.23(d, J=5.6Hz, 1H), 3.71-3.66(m, 2H), 3 .18(d, J=4.8Hz, 1H), 2.76-2.51(m, 1H), 2.05(s, 3H), 1.99(s, 3H), 1.20-1.12(s, 6H).

[0630] Example 2.2: Synthesis of N-[9-[(2R,3R,4S,5R)-4-benzyloxy-5-(benzyloxymethyl)-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]-2-methylpropionamide (26)

[0631] At 0 °C, 1 M NaOH solution (443 mL) was added dropwise to a solution of methyl acetate [(2S,3S,4R,5R)-4-acetoxy-3-benzyloxy-2-(benzyloxymethyl)-5-[2-(2-methylpropionamido)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl] (25.72 g, 0.11 mol) in 1,71 THF / EtOH (4:1). The solution was stirred at this temperature for 1 hour to achieve complete conversion. The pH was adjusted to 7 by adding 1 N HCl aqueous solution and the solvent was removed. The residue was dissolved in H2O (500 mL) and extracted with 3 × 500 mL DCM. The organic layers were combined, dried over anhydrous Na2SO4, and concentrated to obtain a colorless solid compound N-[9-[(2R,3R,4S,5R)-4-benzyloxy-5-(benzyloxymethyl)-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-6-oxo-1H-purine-2-yl]-2-methylpropionamide (26) (113 g, quantitative), which can be used in the next step without further purification.

[0632] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 12.07 (s, 1H), 11.66 (s, 1H), 8.10 (s, 1H), 7.42-7.30 (m, 10H), 5.92 (d, J=6.8Hz, 1H), 4.99 (s, 1H), 4 .87-4.84 (m, 2H), 4.63 (d, J=15.6Hz, 1H), 4.56 (s, 2H), 4.24 (d, J=4.8Hz, 1H), 3.69-3.62 (m, 4H), 2.76-2.73 (m, 1H), 1.13-1.04 (m, 7H).

[0633] Example 2.3: Synthesis of N-[9-[(2R,3R,4S,5S)-4-benzyloxy-5-(benzyloxymethyl)-3-hydroxy-5-(triisopropyl-silyloxymethyl)tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]-2-methylpropionamide (27)

[0634] Imidazole (38 g, 559 mmol) and TIPSCl (35.9 g, 186 mmol) were added to a solution of N-[9-[(2R,3R,4S,5R)-4-benzyloxy-5-(benzyloxymethyl)-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-6-oxo-1H-purine-2-yl]-2-methylpropionamide (26, 75 g, 133 mmol) in anhydrous DCM (1568 ml) at 0 °C under a N2 atmosphere. After stirring between 10 °C and 15 °C for 12 hours, the solution was poured into ice water (21) and extracted with DCM (3 × 1.5 L). The organic layers were combined and washed with brine (11), dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by silica gel column chromatography (PE / EtOAc 2:1 to EtOAc) to give 65 g (68%) of white foamy silyl ether N-[9-[(2R,3R,4S,5S)-4-benzyloxy-5-(benzyloxymethyl)-3-hydroxy-5-(triisopropyl-silyloxymethyl)tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]-2-methyl-propionamide (27).

[0635] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 12.07 (s, 1H), 11.61 (s, 1H), 8.14 (s, 1H) ,7.37-7.22(m,10H),5.89(d,J==6.8Hz,1H),5.72(d,J==5.6Hz,1H),4.94- 4.93 (m, 2H), 4.90-4.53 (m, 3H), 4.19 (d, J=4.4Hz, 1H), 3.92-3.88 (m, 2H), 3 .85-3.71(m, 2H), 2.78-2.71(m, 1H), 1.13-1.05(m, 6H), 1.00-0.94(m, 21H).

[0636] Example 2.4: Synthesis of N-[9-[(2R,3R,4S,5S)-3,4-dihydroxy-5-(hydroxymethyl)-5-(triisopropylsilyloxy-methyl)tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]-2-methylpropionamide (28)

[0637] BCl3 (921 ml) was added to a solution of N-[9-[(2R,3R,4S,5S)-4-benzyloxy-5-(benzyloxymethyl)-3-hydroxy-5-(triisopropylsilyloxymethyl)tetrahydrofuran-2-yl]-6-oxo-1H-purine-2-yl]-2-methylpropionamide (27, 95 g, 0.132 mol) in anhydrous DCM (300 ml) at -70 °C under a nitrogen atmosphere. When complete conversion was detected, the reaction solution was stirred between -75 °C and -60 °C for 2 hours. Approximately 200 ml of a saturated solution of NH3 in MeOH was added to the mixture. The pH was adjusted to 10-11 and the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography (PE / EtOAc 20:1 to 4:1) to produce a yellow solid debenzylated product N-[9-[(2R,3R,4S,5S)-3,4-dihydroxy-5-(hydroxymethyl)-5-(triisopropylsilyloxy-methyl)tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]-2-methylpropionamide (28) (51 g, 71.6%).

[0638] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 11.86 (s, 2H), 8.27 (s, 1H), 5.83 (d, J=7.2Hz, 1H), 5.42 (s, 1H), 5.06 (s, 2H), 4.64 (s, 1H), 4.17 (d, J=4.0Hz, 1H), 3.89 (d, J=10.8Hz, 1H), 3.79 (d, J=10.4Hz, 1H), 3.67 (s, 2H), 2.80-2.73 (m, 1H), 1.17-1.08 (m, 6H), 1.02-0.92 (m, 21H).

[0639] Example 2.5: Synthesis of N-[9-[(2R,3R,4S)-3,4-dihydroxy-5,5-bis(hydroxymethyl)-tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]-2-methylpropionamide (29)

[0640] TBAF (15 mL, 15.0 mmol, 2.5 equivalences, 1 mol / L in THF) was added dropwise to a solution of N-[9-[(2R,3R,4S,5S)-3,4-dihydroxy-5-(hydroxymethyl)-5-(triisopropyl-silyloxymethyl)tetrahydrofuran-2-yl]-6-oxo-1H-purine-2-yl]-2-methylpropionamide (28 g, 3.2 g, 6.10 mmol, 1.0 equivalence) in 15 mL of THF at 15 °C. The mixture was stirred at this temperature for 12 hours to achieve complete deprotection. The reaction solution was concentrated under vacuum to obtain a yellow oily compound N-[9-[(2R,3R,4S)-3,4-dihydroxy-5,5-bis(hydroxymethyl)tetrahydrofuran-2-yl]-6-oxo-1H-purine-2-yl]-2-methyl-propionamide (29) (2.5 g, crude substance), which was used in the next step without further purification.

[0641] Example 2.6: Synthesis of 2-amino-9-[(2R,3R,4S)-3,4-dihydroxy-5,5-bis(hydroxymethyl)-tetrahydrofuran-2-yl]-1H-purine-6-one (30)

[0642] A solution of NaOMe (2.8 ml, 2.8 mmol, 0.5 equivalence, 1 mol / L in MeOH) was added dropwise to a solution of N-[9-[(2R,3R,4S)-3,4-dihydroxy-5,5-bis(hydroxymethyl)tetrahydrofuran-2-yl]-6-oxo-1H-purine-2-yl]-2-methyl-propionamide (29 g, 2.2 g, 5.74 mmol, 1.0 equivalence) in MeOH (22 ml). The mixture was stirred at 60 °C for 4 hours to achieve complete conversion. After cooling to room temperature, the precipitate was filtered and the filter cake was wet-milled with MeOH (5 ml). After drying, 1.4 g (78%) of 2-amino-9-[(2R,3R,4S)-3,4-dihydroxy-5,5-bis(hydroxymethyl)tetrahydrofuran-2-yl]-1H-purine-6-one (30) was separated into a white solid.

[0643] MS (calculated value: 313.1)(m / z) = 314.1 [M+H] + ].

[0644] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 10.76 (br s, 1H), 7.92 (s, 1H), 6.48 (br s, 2H), 5.73 (d, J=7.4Hz, 1H), 5.33 (br s, 1H), 5.09 (br s, 2H), 4.80-4.41 (m, 2H), 4.15 (d, J=5.1Hz, 1H), 3.65-3.47 (m, 4H).

[0645] Example 3: Synthetic scheme for example compound 37

[0646]

[0647] Example 3.1: Synthesis of acetate [(3aR,5R,6R,6aR)-5-[(4R)-2,2-dimethyl-1,3-dioxolane-4-yl]-2,2-dimethyl-3a,5,6,6a-tetrahydrofuran[2,3-d][1,3]dioxacyclopenten-6-yl] ester (32)

[0648] Acetic anhydride (25 ml) was added dropwise to a solution of allosugar derivative (3aR,5S,6R,6aR)-5-[(4R)-2,2-dimethyl-1,3-dioxolane-4-yl]-2,2-dimethyl-3a,5,6,6a-tetrahydrofuran[2,3-d][1,3]dioxacyclopenten-6-ol (31, 10.0 g, 38.4 mmol, 1.0 equivalent) in pyridine (25 ml). The solution was stirred for 12 hours to achieve complete conversion. The reaction mixture was concentrated under vacuum and the residue was poured into a mixture of EtOAc (100 ml) and water (100 ml). After separation of the layers, the aqueous layer was extracted with EtOAc (2 × 100 ml). The combined organic phases were washed with saturated citric acid solution (100 ml) and brine (100 ml), dried over anhydrous Na2SO4, filtered, and concentrated under vacuum to produce 13.3 g (crude) of a yellow solid acetyl-protected product, [(3aR,5R,6R,6aR)-5-[(4R)-2,2-dimethyl-1,3-dioxolane-4-yl]-2,2-dimethyl-3a,5,6,6a-tetrahydrofurano[2,3-d][1,3]dioxacyclopenten-6-yl] ester (32), which could be used without further purification.

[0649] Example 3.2: Synthesis of acetate [(3aR,5R,6R,6aR)-5-[(1R)-1,2-dihydroxyethyl]-2,2-dimethyl-3a,5,6,6a-tetrahydrofuran[2,3-d][1,3]dioxacyclopenten-6-yl] ester (33)

[0650] A solution of diisopropylidene-protected starting material acetate [(3aR,5R,6R,6aR)-5-[(4R)-2,2-dimethyl-1,3-dioxolane-4-yl]-2,2-dimethyl-3a,5,6,6a-tetrahydrofuran[2,3-d][1,3]dioxacyclopenten-6-yl] ester (32, 13.3 g, 38.4 mmol, 1.0 equivalent) was prepared by stirring at 40 °C in 90% AcOH (140 ml). After 12 hours, the reaction mixture was concentrated under vacuum to obtain 13.8 g (crude matter) of the desired diol acetic acid [(3aR,5R,6R,6aR)-5-[(1R)-1,2-dihydroxyethyl]-2,2-dimethyl-3a,5,6,6a-tetrahydrofurano[2,3-d][1,3]dioxane-6-yl] ester (33), which was ready for use without further purification.

[0651] Example 3.3: Synthesis of acetate [(2R)-2-[(3aR,5R,6R,6aR)-6-acetoxy-2,2-dimethyl-3a,5,6,6a-tetrahydrofuran[2,3-d]-[1,3]dioxacyclopenten-5-yl]-2-acetoxy-ethyl] ester (34)

[0652] Acetic anhydride (30 ml) was added dropwise to a solution of the starting material, acetate [(3aR,5R,6R,6aR)-5-[(1R)-1,2-dihydroxyethyl]-2,2-dimethyl-3a,5,6,6a-tetrahydrofuran[2,3-d][1,3]dioxane-6-yl] ester (33 g, 13.8 g, 38.4 mmol, 1.00 equivalent), in pyridine (30 ml). After 12 hours, the solvent was removed under vacuum, and the residue was dissolved in EtOAc (100 ml). The organic layer was washed with water (50 ml) and brine (50 ml), dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. After purification on silica (PE / EtOAc 2:1), 7.5 g (56.4%, three steps) of the title compound acetate [(2R)-2-[(3aR,5R,6R,6aR)-6-acetoxy-2,2-dimethyl-3a,5,6,6a-tetrahydrofuran[2,3-d]-[1,3]dioxane-5-yl]-2-acetoxy-ethyl] ester (34) was separated into a colorless solid.

[0653] 1H-NMR (CDCl3, 400MHz) δ [ppm]: 5.79 (d, J=3.2Hz, 1H), 5.30 (ddd, J=3.7, 4.8, 6.7Hz, 1H), 4.88-4.78 (m, 2H), 4.39 (dd, J=3.7, 12.1Hz, 1H), 4.27 (ddd, J=2.6, 5.2, 8.1Hz, 1H), 4.12 (dd, J=6.8, 12.0Hz, 1H), 2.14 (s, 3H), 2.09 (s, 3H), 2.06 (s, 3H), 1.56 (s, 3H), 1.34 (s, 3H).

[0654] Example 3.4: Synthesis of acetate [(2R)-2-acetoxy-2-[(2R,3R,4R)-3,4,5-triacetoxytetrahydrofuran-2-yl]ethyl] ester (35)

[0655] The starting material, acetic acid [(2R)-2-[(3aR,5R,6R,6aR)-6-acetoxy-2,2-dimethyl-3a,5,6,6a-tetrahydrofurano[2,3-d]-[1,3]dioxacyclopenten-5-yl]-2-acetoxy-ethyl] ester (34 g, 6.5 g, 18.8 mmol, 1.00 equivalent), was dissolved in AcOH (35 mL). Acetic anhydride (7 mL) and H₂SO₄ (150 mg, catalytic amount) were added dropwise at 0 °C, and the mixture was stirred at room temperature for 12 hours to achieve complete conversion. The reaction mixture was diluted with EtOAc (200 mL) and washed with water (100 mL) and brine (100 mL). The organic layer was separated, dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum. Silica gel chromatography (PE / EtOAc 2:1) yielded 6.5 g (89.0%) of a yellow oily, fully acetylated product, [(2R)-2-acetoxy-2-[(2R,3R,4R)-3,4,5-triacetoxy-tetrahydrofuran-2-yl]ethyl] ester (35).

[0656] 1H-NMR (CDCl3, 400MHz) δ [ppm]: 6.47-6.14 (m, 1H), 5.55-5.47 (m, 1H), 5.3 6-5.17 (m, 2H), 4.47-4.29 (m, 2H), 4.17-4.02 (m, 1H), 2.14-2.05 (m, 15H).

[0657] Example 3.5: Synthesis of acetate [(2R)-2-acetoxy-2-[(2R,3R,4R,5R)-3,4-diacetoxy-5-[2-(2-methylpropionamido)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl]ethyl] ester (36)

[0658] Glycosyl donor acetate [(2R)-2-acetoxy-2-[(2R,3R,4R)-3,4,5-triacetoxytetrahydrofuran-2-yl]ethyl] ester (35, 5.5 g, 14.1 mmol, 1.0 equivalent) and isobutyrylguanosine (4.7 g, 21.1 mmol, 1.5 equivalent) were dissolved in DCE (220 ml). BSA (11.5 g, 56.4 mmol, 4.0 equivalent) was added dropwise at room temperature, and the mixture was stirred at 95 °C for 2 hours. TMSOTf (9.4 g, 242.3 mmol, 3.0 equivalent) was added dropwise at 90 °C, and the solution was stirred at this temperature for 12 hours to achieve complete conversion. After cooling the reaction solution to room temperature, the mixture was filtered, and the filtrate was poured into water (100 ml). After extraction with DCM (3 × 100 ml), the combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The residue was purified by reversed-phase rapid chromatography (FA) to give 4.4 g (57.1%) of a white, foamy guanosine analogue acetate [(2R)-2-acetoxy-2-[(2R,3R,4R,5R)-3,4-diacetoxy-5-[2-(2-methylpropionamido)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl]ethyl] ester (36).

[0659] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 12.12 (br s, 1H), 11.54 (s, 1H), 8.33 (s, 1H), 6.07 (d, J=7.0Hz, 1H), 5.81 (t, J=6.6Hz, 1H), 5. 58 (dd, J=3.6, 6.1Hz, 1H), 5.41 (dt, J=3.1, 5.9Hz, 1H), 4.40-4.26 (m, 2H), 4.09 (br dd, J=5.7, 12.3Hz, 1H), 2.78 (td, J=6.8, 13.6Hz, 1H), 2.14 (s, 3H), 2.08 (s, 3H), 2.03 (s, 3H), 2.00 (s, 3H), 1.14 (d, J=6.7Hz, 6H).

[0660] Example 3.6: Synthesis of 2-amino-9-[(2R,3R,4S,5R)-5-[(1R)-1,2-dihydroxyethyl]-3,4-dihydroxy-tetrahydrofuran-2-yl]-1H-purine-6-one (37)

[0661] NaOMe (1.8 ml, 1.8 mmol, 0.33 equivalents, 1 mol / L in MeOH) was added dropwise to a solution of the starting material [(2R)-2-acetoxy-2-[(2R,3R,4R,5R)-3,4-diacetoxy-5-[2-(2-methylpropionylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl]ethyl] ester (36 g, 3.0 g, 5.4 mmol, 1.0 equivalent) in MeOH (18 ml) at 15 °C. The mixture was stirred at 60 °C for 8 hours to achieve complete conversion. The mixture was filtered and the filter cake was vacuum dried to produce 1.68 g (98.8%) of guanosine analogue 2-amino-9-[(2R,3R,4S,5R)-5-[(1R)-1,2-dihydroxyethyl]-3,4-dihydroxy-tetrahydrofuran-2-yl]-1H-purine-6-one (37) as a colorless solid.

[0662] MS (calculated value: 313.1)(m / z) = 313.9[M+H + ].

[0663] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 7.88 (s, 1H), 6.77 (br s, 2H), 5.67 (d, J=7.1Hz, 1H), 5.59-5.29 (m, 2H), 5.28-4.95 (m, 1H), 4.83-4.55 (m, 1H), 4.51-4.42 (m, 1H), 4.16 (br dd, J=1.5, 4.8Hz, 1H), 3.96-3.88 (m, 1H), 3.72-3.61 (m, 1H), 3.47-3.40 (m, 2H).

[0664] Example 4: Synthetic scheme for example compound 47

[0665]

[0666] Example 4.1: Synthesis of (1R)-1-[(3aR,5R,6R,6aR)-6-benzyloxy-2,2-dimethyl-3a,5,6,6a-tetrahydrofuran[2,3-d][1,3]dioxacyclopenten-5-yl]-2-[tert-butyl(dimethyl)silyl]oxy-ethanol (39)

[0667] At room temperature, imidazole (4.36 g, 64.2 mmol, 3.0 equivalence) and TBDMSCl (3.7 g, 24.6 mmol, 1.15 equivalence) were added fractionally to a solution of diol (1R)-1-[(3aR,5R,6R,6aR)-6-benzyloxy-2,2-dimethyl-3a,5,6,6a-tetrahydrofurano[2,3-d][1,3]dioxacyclopenten-5-yl]ethane-1,2-diol (38 g, 6.64 g, 21.4 mmol, 1.0 equivalence) in DCM (130 mL). After stirring for 12 hours, the solvent was evaporated under vacuum, and the residue was poured into a mixture of EtOAc (100 mL) and water (100 mL). The aqueous layer was extracted with EtOAc (2 × 100 mL), and the combined organic phases were washed with brine (100 mL). The organic solution was dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. After purification by column chromatography (PE / EA 5:1), 7.0 g (77.8%) of silyl ether (1R)-1-[(3aR,5R,6R,6aR)-6-benzyloxy-2,2-dimethyl-3a,5,6,6a-tetrahydrofurano[2,3-d][1,3]dioxacyclopenten-5-yl]-2-[tert-butyl(dimethyl)silyl]oxy-ethanol (39) was separated into a white solid.

[0668] MS (calculated value: 424.2) (m / z) = 447.3 [M + Na] + ].

[0669] 1H-NMR (CDCl3, 400MHz) δ [ppm]: 7.44-7.29 (m, 5H), 5.75 (d, J=3.7Hz, 1H), 4.79 (d , J=11.7Hz, 1H), 4.62 (d, J=11.7Hz, 1H), 4.57 (t, J=4.1Hz, 1H), 4.11-4.05 (m, 1H), 3.97 (dd, J=4.4, 8.7Hz, 1H), 3.95-3.89 (m, 1H), 3.75-3.65 (m, 2H), 2.53 (d, J=3.1 Hz, 1H), 1.64-1.57 (m, 3H), 1.37 (s, 3H), 0.95-0.89 (m, 9H), 0.08 (d, J=0.7Hz, 6H).

[0670] Example 4.2: Synthesis of 4-methylbenzenesulfonic acid [(1R)-1-[(3aR,5S,6R,6aR)-6-benzyloxy-2,2-dimethyl-3a,5,6,6a-tetrahydrofuran[2,3-d][1,3]dioxacyclopenten-5-yl]-2-[tert-butyl(dimethyl)silyl]oxy-ethyl] ester (40)

[0671] At room temperature, NET3 (4.7 g, 15.5 mmol, 3.0 equivalent) and DMAP (1.69 g, 15.5 mmol, 1.0 equivalent) were added fractionally to a solution of starting material (1R)-1-[(3aR,5R,6R,6aR)-6-benzyloxy-2,2-dimethyl-3a,5,6,6a-tetrahydrofurano[2,3-d][1,3]dioxacyclopenten-5-yl]-2-[tert-butyl(dimethyl)silyl]oxy-ethanol (39 g, 6.6 g, 15.5 mmol, 1.0 equivalent) in DCM (70 mL), followed by p-toluenesulfonyl chloride (5.93 g, 31.1 mmol, 2.0 equivalent). The solution was stirred for 12 hours and the solvent was evaporated under vacuum. The residue was dissolved in EtOAc (150 mL) and washed with water (100 mL) and brine (100 mL). After drying with Na2SO4 and evaporating the solvent under vacuum, the crude product was purified by column chromatography (PE / EA 7:1) to obtain 7.0 g (77.7%) of the title compound 4-methylbenzenesulfonic acid [(1R)-1-[(3aR,5S,6R,6aR)-6-benzyloxy-2,2-dimethyl-3a,5,6,6a-tetrahydrofuran[2,3-d][1,3]dioxacyclopenten-5-yl]-2-[tert-butyl(dimethyl)silyl]oxy-ethyl] ester (40) as a white solid.

[0672] 1H-NMR (CDCl3, 400MHz) δ [ppm]: 7.77 (d, J = 8.3Hz, 2H), 7.39-7.32 (m, 5H), 7.30-7.25 (m , 2H), 5.34 (d, J = 3.5Hz, 1H), 4.88 (dt, J = 2.0, 6.5Hz, 1H), 4.72 (d, J = 11.5Hz, 1H), 4.54 (d , J=11.5Hz, 1H), 4.47-4.43 (m, 1H), 4.24 (dd, J=2.1, 8.8Hz, 1H), 3.94-3.89 (1n, 1H), 3. 85-3.76 (m, 2H), 2.44 (s, 3H), 1.52 (s, 3H), 1.33 (s, 3H), 0.86 (s, 9H), 0.03-0.04 (m, 6H).

[0673] Example 4.3: Synthesis of (3aR,5R,6R,6aR)-6-benzyloxy-2,2-dimethyl-5-[(2S)-epoxyethylene-2-yl]-3a,5,6,6a-tetrahydrofuran[2,3-d][1,3]dioxane (41)

[0674] Furanose 4-methylbenzenesulfonic acid [(1R)-1-[(3aR,5S,6R,6aR)-6-benzyloxy-2,2-dimethyl-3a,5,6,6a-tetrahydrofuran-[2,3-d][1,3]dioxacyclopenten-5-yl]-2-[tert-butyl(dimethyl)silyl]oxy-ethyl] ester (40.7 g, 12.1 mmol, 1.00 equivalent) and TBAF (30 mL, 30 mmol, 2.50 equivalent, 1 mol / L in THF) were stirred at room temperature for 12 hours to achieve complete conversion. The solution was concentrated under vacuum and the residue was dissolved in EtOAc (150 mL). After washing with water (100 mL) and brine (100 mL), the organic phase was dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by column chromatography (PE / EA 5∶1) to obtain 4 g (88.8%) of colorless oily ethylene oxide (3aR,5R,6R,6aR)-6-benzyloxy-2,2-dimethyl-5-[(2S)-ethylene oxide-2-yl]-3a,5,6,6a-tetrahydrofurano[2,3-d][1,3]dioxane (41).

[0675] 1H-NMR (CDCl3, 400MHz) δ [ppm]: 7.34-7.22 (m, 5H), 5.63 (d, J=3.7Hz, 1H), 4.72 (d, J=12.0Hz, 1H), 4.55 (d, J=12.0Hz, 1H), 4.49 (t, J=3.9Hz, 1H), 3. 96 (dd, J=4.0, 9.0Hz, 1H), 3.72 (dd, J=4.2, 9.0Hz, 1H), 3.05-2.97 (m, 1H), 2.80 (dd, J=2.7, 5.4Hz, 1H), 2.74-2.69 (m, 1H), 1.51 (s, 3H), 1.28 (s, 3H).

[0676] Example 4.4: Synthesis of (1S)-1-[(3aR,5R,6R,6aR)-6-benzyloxy-2,2-dimethyl-3a,5,6,6a-tetrahydrofuran[2,3-d][1,3]dioxacyclopenten-5-yl]ethane-1,2-diol (42)

[0677] The starting material (3aR,5R,6R,6aR)-6-benzyloxy-2,2-dimethyl-5-[(2S)-epoxyethylene-2-yl]-3a,5,6,6a-tetrahydrofurano[2,3-d][1,3]dioxazole (41.4 g, 13.7 mmol, 1.00 equivalent) was dissolved in a mixed solvent of THF (80 mL) and H₂O (40 mL). After adding 1 M NaOH solution (30 mL) at room temperature, the mixture was stirred at 90 °C for 48 hours. After cooling the reaction solution to room temperature, THF was removed under vacuum, and the aqueous layer was adjusted to pH 2-3 by adding 2 M HCl. The aqueous mixture was extracted with EtOAc (3 × 100 mL), and the combined organic layers were dried over anhydrous Na₂SO₄. After vacuum evaporation, 4.2 g (crude matter) of diol (1S)-1-[(3aR,5R,6R,6aR)-6-benzyloxy-2,2-dimethyl-3a,5,6,6a-tetrahydrofuran[2,3-d][1,3]dioxacyclopenten-5-yl]ethane-1,2-diol (42) was separated into a yellow oily substance that could be used without further purification.

[0678] 1H-NMR (CDCl3, 400MHz) δ [ppm]: 7.34-7.23 (m, 5H), 5.70-5.64 (m, 1H), 4.73-4.67 (m, 1H), 4.55-4.46 (m, 2H), 4 .04-3.97 (m, 1H), 3.86 (dd, J=4.3, 8.9Hz, 1H), 3.74-3.60 (m, 3H), 2.42-2.18 (m, 1H), 1.52 (s, 3H), 1.29 (s, 3H).

[0679] Example 4.5: Synthesis of (1S)-1-[(3aR,5R,6R,6aR)-6-hydroxy-2,2-dimethyl-3a,5,6,6a-tetrahydrofurano[2,3-d][1,3]dioxacyclopenten-5-yl]ethane-1,2-diol (43)

[0680] Pd / C (1 g, 10%) was added fractionally to a solution of benzyl ether (1S)-1-[(3aR,5R,6R,6aR)-6-benzyloxy-2,2-dimethyl-3a,5,6,6a-tetrahydrofurano[2,3-d][1,3]dioxacyclopenten-5-yl]ethane-1,2-diol (42.4 g, 13.5 mmol, 1.00 equivalent) in MeOH (80 mL). The mixture was stirred at 45 °C under a 3.5 bar H2 atmosphere. After 12 hours, the mixture was cooled to room temperature. The catalyst was separated by filtration and the filtrate was concentrated under vacuum to obtain 2.9 g (97%) of the title compound (1S)-1-[(3aR,5R,6R,6aR)-6-hydroxy-2,2-dimethyl-3a,5,6,6a-tetrahydrofuran[2,3-d][1,3]dioxacyclopenten-5-yl]ethane-1,2-diol (43) as a colorless oil.

[0681] 1H-NMR (CDCl3, 400MHz) δ [ppm]: 5.84 (d, J=3.8Hz, 1H), 4.65-4.58 (m, 1H), 4.09 (br dd, J=5.3, 8.1Hz, 1H), 3.86-3.76 (m, 4H), 2.99-2.62 (m, 3H), 1.62-1.56 (m, 3H), 1.40 (s, 3H).

[0682] Example 4.6: Synthesis of acetate [(2S)-2-[(3aR,5R,6R,6aR)-6-acetoxy-2,2-dimethyl-3a,5,6,6a-tetrahydrofuran[2,3-d][1,3]dioxacyclopenten-5-yl]-2-acetoxy-ethyl] ester (44)

[0683] Acetic anhydride (15 ml) was added dropwise to a solution of the starting material (1S)-1-[(3aR,5R,6R,6aR)-6-hydroxy-2,2-dimethyl-3a,5,6,6a-tetrahydrofurano[2,3-d][1,3]dioxacyclopenten-5-yl]ethane-1,2-diol (43 g, 2.9 g, 13.1 mmol, 1.00 equivalent) in pyridine (30 ml). After stirring for 12 hours, the solvent was removed under vacuum, and the residue was dissolved in EtOAc (100 ml). The organic layer was washed with water (50 ml) and brine (50 ml), dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by column chromatography (PE / EA 4:1) to give 4 g (88%) of the title compound, acetate [(2S)-2-[(3aR,5R,6R,6aR)-6-acetoxy-2,2-dimethyl-3a,5,6,6a-tetrahydrofuran[2,3-d][1,3]dioxacyclopenten-5-yl]-2-acetoxy-ethyl] ester (44), which was a colorless oil.

[0684] 1H-NMR (CDCl3, 400MHz) δ [ppm]: 5.75 (d, J=3.7Hz, 1H), 5.25-5.14 (m, 1H), 4.77-4.69 (m, 1H), 4.59 (dd, J=4.8, 9.2Hz, 1 H), 4.35-4.19 (m, 2H), 4.14 (dd, J=7.5, 11.8Hz, 1H), 2.06 (s, 3H), 2.05 (s, 3H), 1.97 (s, 3H), 1.49 (s, 3H), 1.27 (s, 3H).

[0685] Example 4.7: Synthesis of acetate [(2S)-2-acetoxy-2-[(2R,3R,4R)-3,4,5-triacetoxytetrahydrofuran-2-yl]ethyl] ester (45)

[0686] Following the method described for synthesizing [(2R)-2-acetoxy-2-[(2R,3R,4R)-3,4,5-triacetoxytetrahydrofuran-2-yl]ethyl] ester (35), 4 g (11.5 mmol, 1.0 equivalent) of isopropylidene protected starting material [(2S)-2-[(3aR,5R,6R,6aR)-6-acetoxy-2,2-dimethyl-3a,5,6,6a-tetrahydrofurano[2,3-d][1,3]dioxacyclopenten-5-yl]-2-acetoxy-ethyl] ester (44) was converted into the fully acetylated furanose derivative [(2S)-2-acetoxy-2-[(2R,3R,4R)-3,4,5-triacetoxytetrahydrofuran-2-yl]ethyl] ester (45). Following column chromatography (PE / EtOAc 3:1), 3.4 g (75.5%) of the title compound was separated into a colorless oil.

[0687] Example 4.8: Synthesis of acetate [(2S)-2-acetoxy-2-[(2R,3R,4R,5R)-3,4-diacetoxy-5-[2-(2-methylpropionamido)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl]ethyl] ester (46)

[0688] Following the method described for synthesizing acetate [(2R)-2-acetoxy-2-[(2R,3R,4R,5R)-3,4-diacetoxy-5-[2-(2-methylpropionylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl]ethyl] ester (36), 3.4 g (8.7 mmol, 1.0 equivalent) of the starting material acetate [(2S)-2-acetoxy-2-[(2R,3R,4R,5R)-3,4-diacetoxy-5-[2-(2-methylpropionylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl]ethyl] ester was used. )-3,4,5-triacetoxytetrahydrofuran-2-yl]ethyl] ester (45) was glycosylated to give 2.0 g (41.6%) of the title compound [(2S)-2-acetoxy-2-[(2R,3R,4R,5R)-3,4-diacetoxy-5-[2-(2-methylpropionylamino)-6-oxo-1H-purine-9-yl]tetrahydrofuran-2-yl]ethyl] ester (46) as a colorless foam.

[0689] MS (calculated value: 551.2)(m / z) = 552.3[M+H + ].

[0690] 1H-NMR (CDCl3, 400MHz) δ [ppm]: 5.75 (d, J=3.7Hz, 1H), 5.25-5.14 (m, 1H), 4.77-4.69 (m, 1H), 4.59 (dd, J=4.8, 9.2Hz, 1 H), 4.35-4.19 (m, 2H), 4.14 (dd, J=7.5, 11.8Hz, 1H), 2.06 (s, 3H), 2.05 (s, 3H), 1.97 (s, 3H), 1.49 (s, 3H), 1.27 (s, 3H).

[0691] Example 4.9: Synthesis of 2-amino-9-[(2R,3R,4S,5R)-5-[(1S)-1,2-dihydroxyethyl]-3,4-dihydroxy-tetrahydrofuran-2-yl]-1H-purine-6-one (47)

[0692] NaOMe (0.87 ml, 0.87 mmol, 0.5 equivalent, 1 mol / L in MeOH) was added dropwise to a solution of the starting material [(2R)-2-acetoxy-2-[(2R,3R,4R,5R)-3,4-diacetoxy-5-[2-(2-methylpropionylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl]ethyl] ester (46,960 mg, 1.74 mmol, 1.0 equivalent) in MeOH (10 ml) at 60 °C. The mixture was heated to 60 °C and maintained for 4 hours to achieve complete conversion. The mixture was filtered and the filter cake was vacuum dried to produce 500 mg (91.7%) of the desired product 2-amino-9-[(2R,3R,4S,5R)-5-[(1S)-1,2-dihydroxyethyl]-3,4-dihydroxy-tetrahydrofuran-2-yl]-1H-purine-6-one (47).

[0693] MS (calculated value: 313.1)(m / z) = 314.1 [M+H] + ].

[0694] 1H-NMR (D2O, 400MHz) δ [ppm]: 7.83 (s, 1H), 5.77 (d, J=6.4Hz, 1H), 4.68-4.66 (m, 1H), 4.36 (dd, J=2. 9, 5.2Hz, 1H), 4.17-4.13 (m, 1H), 3.84 (ddd, J=2.4, 5.2, 7.5Hz, 1H), 3.63-3.51 (m, 2H), 3.26 (s, 1H).

[0695] Example 5: Synthetic scheme for example compound 58

[0696]

[0697] Example 5.1: Synthesis of (3aR,6S,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]dioxacyclopentene-6-carboxaldehyde (49)

[0698] 2-Iodobenzoic acid (19.2 g, 0.069 mol, 1.25 equivalents) was added fractionally to a solution of ribose derivative [(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]dioxacyclopenten-6-yl]methanol (48 g, 11.2 g, 0.055 mol, 1.0 equivalent) in ACN (450 mL). After stirring at 90 °C for 3 hours, the reaction mixture was cooled to room temperature, filtered, and the filtrate was concentrated under vacuum. The residue was dissolved in EtOAc (200 mL) and washed with saturated Na2S2O3 (50 mL) and brine (50 mL). The organic layer was dried with anhydrous Na2SO4, filtered, and concentrated under vacuum to obtain 9.7 g (87.3%) of a white solid aldehyde (3aR,6S,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]dioxacyclopentene-6-carboxaldehyde (49).

[0699] 1H-NMR (CDCl3, 400MHz) δ [ppm]: 9.50 (s, 1H), 5.01 (s, 1H), 4.97 (d, J=5.9Hz, 1 H), 4.42 (d, J=6.1Hz, 1H), 4.39 (s, 1H), 3.37 (s, 3H), 1.41 (s, 3H), 1.25 (s, 3H).

[0700] Example 5.2: Synthesis of (3aR,6R,6aR)-4-methoxy-6-[(E)-2-methoxyvinyl]-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]dioxane (50)

[0701] t-BuOK (96 mL, 96.4 mmol, 2.5 mmol, 1 mol / L in THF) was added dropwise to a suspension of (methoxymethyl)triphenylphosphine chloride (40 g, 0.116 mol, 3.0 equivalence) in THF (580 mL) at 0 °C. The resulting red mixture was stirred at 0 °C for 1 hour, and then a solution of (3aR,6S,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]-dioxacyclopentene-6-carboxaldehyde (49 g, 7.8 g, 38.6 mmol, 1.0 equivalence) in THF (116 mL) was added. Stirring was continued at 0 °C for 1 hour to achieve complete conversion. The mixture was washed with saturated NaHCO3 (120 mL) and extracted with methyl tert-butyl ether (3 × 300 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by rapid chromatography (PE / EtOAc 5:1) to give 5.1 g (57.3%) of a yellow oily Wittig product (3aR,6R,6aR)-4-methoxy-6-[(E)-2-methoxyvinyl]-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]dioxane (50) (a mixture of E and Z isomers).

[0702] Example 5.3: Synthesis of 2-[(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydro-furan[3,4-d][1,3]dioxacyclopenten-6-yl]acetaldehyde (51)

[0703] At 10 °C, a mixture of vinyl ether (3aR,6R,6aR)-4-methoxy-6-[(E)-2-methoxyvinyl]-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]dioxane (50, 5.2 g, 22.6 mmol, 1.0 equivalent) in acetone (113 ml) was added dropwise with 0.56 ml of 1 mol / L aqueous solution in water. After stirring for 2 hours, another 0.56 ml of 1 N HCl aqueous solution was added, and stirring was continued at 10 °C for 4 hours. The solution was neutralized by adding NEt3, and the solvent was removed under vacuum. The residue was purified by rapid chromatography (PE / EA 3:1) to give 3.9 g (80%) of aldehyde 2-[(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofuran[3,4-d][1,3]dioxacyclopenten-6-yl]acetaldehyde (51) as a colorless oil.

[0704] 1H-NMR (CDCl3, 400MHz) δ [ppm]: 9.50 (s, 1H), 5.01 (s, 1H), 4.97 (d, J=5.9Hz, 1 H), 4.42 (d, J=6.1Hz, 1H), 4.39 (s, 1H), 3.37 (s, 3H), 1.41 (s, 3H), 1.25 (s, 3H).

[0705] Example 5.4: Synthesis of 2-[(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]dioxacyclopenten-6-yl]acetic acid (52)

[0706] Aldehyde 2-[(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]-dioxacyclopenten-6-yl]acetaldehyde (51.4 g, 18.5 mmol, 1.0 equivalent) was dissolved in a mixed solvent of t-BuOH (184 ml) and 2-methylbut-2-ene (60 ml). A solution of NaClO2 (16.7 g, 0.185 mol, 10.0 equivalent) in water (18.5 ml) and a solution of NaH2PO4 (22 g, 0.185 mol, 10.0 equivalent) in water (18.5 ml) were added sequentially at 10 °C. The mixture was then stirred at 10 °C for 16 hours to achieve complete conversion. The reaction mixture was diluted with saturated NH4Cl (300 ml) and extracted with EtOAc (3 × 500 ml). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by rapid chromatography (EtOAc) to give 4.1 g (95.4%) of a yellow oily carboxylic acid 2-[(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]dioxacyclopenten-6-yl]acetic acid (52). 1H-NMR (CDCl3, 400MHz) δ [ppm]: 4.90 (s, 1H), 4.50-4.66 (m, 3H), 3.23-3.35 (m, 3H), 2.62 (m, 2H), 1.42 (s, 3H), 1.25 (s, 3H).

[0707] Example 5.5: Synthesis of methyl 2-[(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]dioxacyclopenten-6-yl]acetate (53)

[0708] K₂CO₃ (7.14 g, 51.7 mmol, 1.2 equivalent) and MeI (9.17 g, 64.6 mmol, 1.5 equivalent) were added fractionally to a solution of carboxylic acid 2-[(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]dioxacyclopenten-6-yl]acetic acid (52, 10 g, 43.1 mmol, 1.0 equivalent) in DMF (600 mL) at 0 °C. After stirring at this temperature for 2 hours, the mixture was poured into ice water (11) and extracted with methyl tert-butyl ether (3 × 11). The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum. Following rapid chromatography (PE / EtOAc 4∶1), 10.0 g (94.3%) of methyl 2-[(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofuran[3,4-d][1,3]dioxacyclopenten-6-yl]acetate (53) was separated into a colorless oil.

[0709] 1H-NMR (CDCl3, 400MHz) δ [ppm]: 4.97 (s, 1H), 4.59-4.71 (m, 3H), 3.73 (s, 3H), 3.29-3.40 (m, 3H), 2.55-2.76 (m, 2H), 1.50 (s, 3H), 1.33 (s, 3H).

[0710] Example 5.6: Synthesis of methyl 2-[(3aR,6R,6aR)-4-acetoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]dioxacyclopenten-6-yl]acetate (54)

[0711] Eight drops of concentrated H₂₈O₄ were added to a mixture of methyl glycoside 2-[(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]dioxacyclopenten-6-yl]acetate (53.5 g, 20.3 mmol, 1.0 equivalent) in a mixed solvent of acetic anhydride (17 ml), AcOH (50 ml), and DCM (50 ml). After stirring at this temperature for 16 hours, the mixture was diluted with DCM (100 ml) and neutralized with a saturated NaHCO₃ solution (approximately 200 ml). The organic layer was separated and washed with brine (50 ml), dried over anhydrous Na₂SO₄, and concentrated under vacuum. Rapid chromatography (PE / EtOAc 2:1) of the crude product yielded 4.3 g (76.9%) of the title compound methyl 2-[(3aR,6R,6aR)-4-acetoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]dioxacyclopenten-6-yl]acetate (54) as a white solid.

[0712] 1H-NMR (CDCl3, 400MHz) δ [ppm]: 6.22 (m, 1H), 4.68-4.83 (m, 3H), 3.74 (s, 3H), 2.55-2.79 (m, 2H), 2.10 (s, 3H), 1.52 (s, 3H), 1.36 (s, 3H).

[0713] Example 5.7: Synthesis of methyl 2-[(2R,3R,4R)-3,4,5-triacetoxytetrahydrofuran-2-yl]-acetate (55)

[0714] Ten drops of concentrated H₂SO₄ were added to a mixture of the starting compound methyl 2-[(3aR,6R,6aR)-4-acetoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]dioxacyclopenten-6-yl]acetate (54 g, 3.8 g, 13.9 mmol, 1.0 equivalent) in a mixed solvent of acetic anhydride (19 mL) and AcOH (9.5 mL). After stirring at 10 °C for 16 hours, the mixture was diluted with EtOAc (100 mL) and washed with saturated aqueous NaHCO₃ solution (2 × 150 mL) and brine (100 mL), dried over anhydrous Na₂SO₄, and concentrated under vacuum. The crude product was purified by rapid chromatography (PE / EtOAc 2:1) to yield 1.78 g (40.5%) of the title compound methyl 2-[(2R,3R,4R)-3,4,5-triacetoxytetrahydrofuran-2-yl]acetate (55), which was a yellow oil.

[0715] 1H-NMR (CDCl3, 400MHz) δ [ppm]: 6.08 (m, 1H), 5.17-5.31 (m, 2H), 4.44-4.58 (m, 1H), 3.64 (s, 3H), 2.58-2.81 (m, 2H), 2.00-2.07 (m, 9H).

[0716] Example 5.8: Synthesis of methyl 2-[(2R,3R,4R,5R)-3,4-diacetoxy-5-[2-(2-methylpropionamido)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl]acetate (56)

[0717] A solution of the starting compound methyl 2-[(2R,3R,4R)-3,4,5-triacetoxytetrahydrofuran-2-yl]acetate (55 g, 1.26 g, 4.0 mmol, 1.0 equivalent), isobutyrylguanine (1.05 g, 4.8 mmol, 1.2 equivalent), and BSA (3.22 g, 15.8 mmol, 4.0 equivalent) in a DCE (50 mL) was stirred at 100 °C for 2 h, followed by the addition of TMSOTf (2.64 g, 11.9 mmol, 3.0 equivalent). Stirring was continued at 100 °C for 1 h. The solution was cooled to room temperature and diluted with DCM (100 mL). The white precipitate was filtered off, and the organic solution was dried over anhydrous Na₂SO₄. After evaporating the solvent, the crude product was purified by preparative TLC (EtOAc / MeOH20∶1) to obtain 780 mg (41.1%) of methyl 2-[(2R,3R,4R,5R)-3,4-diacetoxy-5-[2-(2-methylpropionamido)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl]acetate (56) as a colorless foam.

[0718] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 12.17 (s, 1H), 11.66 (s, 1H), 8.35 (s, 1H), 6.11 (d, J=6.7Hz, 1H), 5.97 (s, 1H), 5.48 (dd, J =5.6, 3.7Hz, 1H), 4.45-4.58(m, 1H), 3.66(s, 3H), 2.95-3.18(m, 2H), 2.75-2.91(m, 1H), 2.19(s, 3H), 2.08(s, 3H), 1.20(br d, J=6.7Hz, 6H).

[0719] Example 5.9: Synthesis of 2-[(2R,3S,4R,5R)-3,4-dihydroxy-5-[2-(2-methylpropionamido)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl]acetic acid (57)

[0720] The starting material, methyl 2-[(2R,3R,4R,5R)-3,4-diacetoxy-5-[2-(2-methylpropionyl-amino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl]acetate (56 g, 1.61 g, 3.4 mmol, 1.0 equivalent), was dissolved in a mixed solvent of THF (65 mL) and water (13 mL). LiOH·H₂O (479 mg, 11.4 mmol, 3.4 equivalent) was added in portions at 0 °C, and the reaction mixture was stirred at this temperature for 3 hours. The solution was diluted with water (50 mL) and washed with DCM (100 mL). The aqueous layer was separated and neutralized with 1 N HCl aqueous solution. The precipitate was filtered, and the aqueous filtrate was washed with DCM (2 × 100 mL). The combined organic layers were dried over Na₂SO₄ and concentrated under vacuum. The crude product was dissolved in water and DMF (20 ml, v / v = 10 / 1) and purified by reversed rapid chromatography (neutral) to produce 1.15 g (62.9%) of the title compound 2-[(2R,3S,4R,5R)-3,4-dihydroxy-5-[2-(2-methylpropionamido)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl]acetic acid (57) as a yellow foam.

[0721] MS (calculated value: 381.1)(m / z) = 382.0 [M+H] + ].

[0722] 1H-NMR (D2O, 400MHz) δ [ppm]: 8.09 (s, 1H), 5.91 (br d, J=5.0Hz, 1H), 4.76-4.80 (m, 1H), 4.41 (br s, 1H), 4.20-4.31 (m, 1H), 2.54-2.76 (m, 3H), 1.16 (br d, J==6.9Hz, 6H).

[0723] Example 5.10: Synthesis of 2-[(2R,3S,4R,5R)-5-(2-amino-6-oxo-1H-purin-9-yl)-3,4-dihydroxy-tetrahydrofuran-2-yl]acetic acid (58)

[0724] A solution of NaOMe (1.7 ml, 1.7 mmol, 1.0 equivalence) was added dropwise to a solution of the starting material 2-[(2R,3S,4R,5R)-3,4-dihydroxy-5-[2-(2-methylpropionylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl]acetic acid (57,650 mg, 1.7 mmol, 1.0 equivalence) in MeOH (13 ml). After stirring at 60 °C for 10 hours, the solvent was removed under vacuum and the residue was washed with MeOH (5 ml). After vacuum drying, 444 mg (87% purity, 72.8%) of the guanosine analog 2-[(2R,3S,4R,5R)-5-(2-amino-6-oxo-1H-purine-9-yl)-3,4-dihydroxy-tetrahydrofuran-2-yl]acetic acid (58) was separated into a yellow solid.

[0725] MS (calculated value: 311.1)(m / z) = 311.9[M+H + ].

[0726] 1H-NMR (D2O, 400MHz) δ [ppm]: 7.76 (m, 1H), 5.73 (br d, J=5.5Hz, 1H), 4.57 (br t, J=5.4Hz, 1H), 4.21-4.36 (m, 1H), 4.11 (br t, J==4.6Hz, 1H), 2.39-2.64 (m, 2H).

[0727] Example 6: Synthetic schemes for example compounds 71, 72 and 73

[0728]

[0729] Example 6.1: Synthesis of 2-[(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]-dioxacyclopenten-6-yl]-N-methylacetamide (59)

[0730] HATU (7.37 g, 19.4 mmol, 1.5 equivalents) and NET3 (3.51 g, 27.1 mmol, 2.1 equivalents) were added to a solution of carboxylic acid 2-[(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]dioxacyclopenten-6-yl]acetic acid (52.3 g, 12.9 mmol, 1.0 equivalents) and MeNH2·HCl (959 mg, 14.2 mmol, 1.1 equivalents) in DMF (129 mL) at 10 °C. The solution was stirred at 10 °C for 3 hours, diluted with EtOAc (500 mL), and washed with water (2 × 200 mL). The organic layer was separated, dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by rapid chromatography (EtOAc) to obtain 3.54 g (89.5%, purity approximately 80%) of the desired amide 2-[(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofuran[3,4-d][1,3]-dioxacyclopenten-6-yl]-N-methylacetamide (59) as a yellow oil.

[0731] 1H-NMR (CDCl3, 400MHz) δ [ppm]: 6.05 (br s, 1H), 4.90 (s, 1H), 4.44-4.61 (m, 3H), 3.28 (s, 3H), 2.74 (s, 3H), 2.31-2.53 (m, 2H), 1.41 (s, 3H), 1.24 (s, 3H).

[0732] Example 6.2: Synthesis of 2-[(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]-dioxacyclopenten-6-yl]-N,N-dimethylacetamide (60)

[0733] Following the synthesis of 2-[(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]-dioxacyclopenten-6-yl]-N-methyl-acetamide (59), 6.0 g (25.8 mmol, 1.0 equivalent) carboxylic acid 2-[(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]-dioxacyclopenten-6-yl]-N-methyl-acetamide (59) -d][1,3]dioxacyclopenten-6-yl]acetic acid (52) and 2.3 g (28 mmol, 1.1 equivalent) of dimethylamine hydrochloride yielded 5.6 g (84.8%) of the desired dimethylamide 2-[(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]-dioxacyclopenten-6-yl]-N,N-dimethylacetamide (60) in a yellow oil.

[0734] 1H-NMR (CDCl3, 400MHz) δ [ppm]: 4.95 (s, 1H), 4.72-4.65 (m, 2H), 4.60 (d, J=5.9Hz, 1H), 3.34 (s, 3H), 3.00(s, 3H), 2.80(s, 3H), 2.70-2.80(m, 1H), 2.50-2.60(m, 1H), 1.49(s, 3H), 1.31(s, 3H).

[0735] Example 6.3: Synthesis of 2-[(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]-dioxacyclopenten-6-yl]N-butylacetamide (61)

[0736] Following the synthesis of 2-[(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]-dioxacyclopenten-6-yl]-N-methylacetamide (59), 6.0 g (25.8 mmol, 1.0 equivalent) carboxylic acid 2-[(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]dioxacyclopenten-6-yl]acetic acid (52) and 2.1 g (28 mmol, 1.1 equivalent) n-butylamine were subjected to silica gel chromatography (PE / Et=Ac) After 1:1), 7.0 g (93.3%) of the desired n-butylamine 2-[(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofuran[3,4-d][1,3]-dioxacyclopenten-6-yl]N-butyl-acetamide (61) was obtained.

[0737] 1H-NMR (CDCl3, 400MHz) δ [ppm]: 6.06 (br s, 1H), 4.99 (s, 1H), 4.71-4.50 (m, 3H), 3.37 (s, 3H), 3.33-3.25 (m, 2H), 2.60-2.51 (m , 1H), 2.50-2.39 (m, 1H), 1.56-1.46 (m, 5H), 1.42-1.30 (m, 5H), 0.94 (t, J=7.3Hz, 3H).

[0738] Example 6.4: Synthesis of N-methyl-2-[(2R,3S,4R)-3,4,5-trihydroxytetrahydrofuran-2-yl]acetamide (62)

[0739] The starting material 2-[(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]-dioxacyclopenten-6-yl]-N-methylacetamide (59 g, 2 g, 8.2 mmol, 1.0 equivalent) was dissolved in a mixed solvent of 0.1 NH₂SO₄ (27 mL, 2.7 mmol, 0.33 equivalent) and dioxane (13.5 mL). After stirring at 120 °C (oil bath) for 2 hours, the reaction mixture was cooled to 10 °C and neutralized with Ba(OH)₂·8H₂O (solid). The mixture was concentrated under vacuum, and the residue was co-evaporated with dioxane (3 × 50 ml) to obtain 3 g (crude substance) of deprotected furanose derivative N-methyl-2-[(2R,3S,4R)-3,4,5-trihydroxytetrahydrofuran-2-yl]acetamide (62) as a yellow solid, which could be used in the next step without further purification.

[0740] Example 6.5: Synthesis of N,N-dimethyl-2-[(2R,3S,4R)-3,4,5-trihydroxytetrahydrofuran-2-yl]acetamide (63)

[0741] Following the synthesis protocol described for N-methyl-2-[(2R,3S,4R)-3,4,5-trihydroxytetrahydrofuran-2-yl]acetamide (62), 5.6 g (21.6 mmol, 1.0 equivalent) of the starting material 2-[(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]-dioxacyclopenten-6-yl]-N,N-dimethyl-acetamide (60) was hydrolyzed with 0.1 NH2SO4. After neutralization with Ba(OH)2·8H2O, the mixture was poured into water (150 ml) and washed with EtOAc (2 × 50 ml). The aqueous layer was separated and concentrated under vacuum to obtain the title compound N,N-dimethyl-2-[(2R,3S,4R)-3,4,5-trihydroxytetrahydrofuran-2-yl]acetamide (63) (8 g, crude substance), which was a yellow oil and could be used in the next step without further purification.

[0742] Example 6.6: Synthesis of N-butyl-2-[(2R,3S,4R)-3,4,5-trihydroxytetrahydrofuran-2-yl]-acetamide (64)

[0743] Following the method described for synthesizing N-methyl-2-[(2R,3S,4R)-3,4,5-trihydroxytetrahydrofuran-2-yl]acetamide (62), 2-[(3aR,6R,6aR)-4-methoxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]-dioxacyclopenten-6-yl]-N-butyl-acetamide (61, 7.0 g, 24.0 mm) was prepared. Hydrolysis of 1.0 mol (equivalent) followed by treatment as described for N,N-dimethyl-2-[(2R,3S,4R)-3,4,5-trihydroxytetrahydrofuran-2-yl]acetamide (63) yields the title compound N-butyl-2-[(2R,3S,4R)-3,4,5-trihydroxytetrahydrofuran-2-yl]acetamide (64) (6 g, crude material), which is used in the next step without further purification.

[0744] Example 6.7: Synthesis of acetate [(2R,3R,4R)-4,5-diacetoxy-2-[2-(methylamino)-2-oxo-ethyl]-tetrahydrofuran-3-yl] ester (65)

[0745] The crude product N-methyl-2-[(2R,3S,4R)-3,4,5-trihydroxytetrahydrofuran-2-yl]acetamide (62, 3 g, 8.2 mmol, purity 52%, 1.0 equivalent) was co-evaporated with pyridine (3 × 20 ml) and dissolved in pyridine (20 ml). Acetic anhydride (20 ml) was added dropwise at 10 °C, and the solution was stirred at this temperature for 16 hours to achieve complete conversion. The reaction mixture was concentrated under vacuum, and the residue was purified by rapid chromatography (EtOAc) to give 1.46 g (56.1%, two steps) of the peracetylated product [(2R,3R,4R)-4,5-diacetoxy-2-[2-(methylamino)-2-oxo-ethyl]tetrahydrofuran-3-yl] ester (65), which was a yellow oil.

[0746] 1H-NMR (CDCl3, 400MHz) δ [ppm]: 6.02-6.39 (m, 1H), 5.60-5.94 (m, 1H), 5.11-5.33 (m, 2H), 4.37-4.56 (m, 1H), 2.74 (d, J=4.89Hz, 3H), 2.38-2.60 (m, 2H), 1.96-2.09 (m, 9H).

[0747] Example 6.8: Synthesis of acetate [(2R,3R,4R)-4,5-diacetoxy-2-[2-(dimethylamino)-2-oxo-ethyl]-tetrahydrofuran-3-yl] ester (66)

[0748] Following the method described in the synthesis of [(2R,3R,4R)-4,5-diacetoxy-2-[2-(dimethylamino)-2-oxo-ethyl]tetrahydrofuran-3-yl] ester (65), crude N,N-dimethyl-2-[(2R,3S,4R)-3,4,5-trihydroxytetrahydrofuran-2-yl]acetamide (63, 8 g) was acetylated, and 4.0 g (55.9%, two steps) of the title compound [(2R,3R,4R)-4,5-diacetoxy-2-[2-(dimethylamino)-2-oxo-ethyl]tetrahydrofuran-3-yl] ester (66) was obtained as a colorless oil after silica gel chromatography (EtOAc).

[0749] 1H-NMR (CDCl3, 400MHz) δ [ppm]: 6.01-6.34 (m, 1H), 5.19-5.31 (m, 2H), 4.52-4.70 (m, 1H), 2.91-2.96 (m, 3 H), 2.83-2.89(m, 3H), 2.66-2.73(m, 1H), 2.57-2.65(m, 1H), 2.04-2.07(m, 3H), 2.02(s, 3H), 2.00(s, 3H).

[0750] Example 6.9: Synthesis of acetate [(2R,3R,4R)-4,5-diacetoxy-2-[2-(butylamino)-2-oxo-ethyl]-tetrahydrofuran-3-yl] ester (67)

[0751] Following the method described in the synthesis of [(2R,3R,4R)-4,5-diacetoxy-2-[2-(methylamino)-2-oxo-ethyl]tetrahydrofuran-3-yl] ester (65), crude N-butyl-2-[(2R,3S,4R)-3,4,5-trihydroxytetrahydrofuran-2-yl]acetamide (64, 6 g) was acetylated and, after silica gel chromatography (PE / EtOAc 1:1), 6.5 g (75.4%, two steps) of the title compound [(2R,3R,4R)-4,5-diacetoxy-2-[2-(butylamino)-2-oxo-ethyl]tetrahydrofuran-3-yl] ester (67) was obtained as a yellow oil.

[0752] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: .84-7.98 (m, 1H), 5.92-6.33 (m, 1H), 5.16-5.36 (m, 2H), 4.40-4.49 (m, 1H), 2. 98-3.09 (m, 2H), 2.37-2.49 (m, 2H), 2.01-2.11 (m, 9H), 1.32-1.41 (m, 2H), 1.21-1.31 (m, 2H), 0.83-0.91 (m, 3H).

[0753] Example 6.10: Synthesis of acetate [(2R,3R,4R,5R)-4-acetoxy-2-[2-(methylamino)-2-oxo-ethyl]-5-[2-(2-methylpropionamido)-6-oxo-1H-purine-9-yl]tetrahydrofuran-3-yl] ester (68)

[0754] BSA (6.6 g, 32.5 mmol, 4.0 equivalent) was added dropwise to a solution of ribose derivative acetate [(2R,3R,4R)-4,5-diacetoxy-2-[2-(methylamino)-2-oxo-ethyl]tetrahydrofuran-3-yl] ester (65 g, 2.58 g, 8.1 mmol, 1.0 equivalent) and isobutyrylguanine (2.16 g, 9.8 mmol, 1.2 equivalent) in DCE (103 mL). The reaction solution was stirred at 100 °C for 2 hours, and then TMSOTf (5.4 g, 24.4 mmol, 3.0 equivalent) was added. The mixture was stirred at 100 °C for another hour, and then cooled to 10 °C and diluted with DCM (100 mL). The organic solution was washed with saturated NaHCO3 (100 mL) and brine (100 mL), dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by preparative TLC (EtOAc / MeOH 4∶1) to obtain 1.48 g (38%) of a yellow, foamy guanosine analogue acetate [(2R,3R,4R,5R)-4-acetoxy-2-[2-(methylamino)-2-oxo-ethyl]-5-[2-(2-methylpropionylamino)-6-oxo-1H-purine-9-yl]tetrahydrofuran-3-yl] ester (68).

[0755] MS (calculated value: 478.2)(m / z) = 479.1 [M+H + ].

[0756] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 12.13 (br s, 1H), 11.66 (br s, 1H), 8.26 (s, 1H), 7.92 (br d, J=4.7Hz, 1H), 6.01 (d, J=6.5Hz, 1H), 5.86 (t, J=6.1Hz, 1H), 5.47 (dd, J=5.8, 3.9Hz, 1H), 4.39-4.49 (m, 1H), 2.79 (dt, J=13.7, 6.9Hz, 1H), 2.62-2.73 (m, 2H), 2.57 (d, J=4.5Hz, 3H), 2.12 (s, 3H), 2.02 (s, 3H), 1.14 (d, J=6.9Hz, 6H).

[0757] Example 6.11: Synthesis of acetate [(2R,3R,4R,5R)-4-acetoxy-2-[2-(dimethylamino)-2-oxo-ethyl]-5-[2-(2-methylpropionyl-amino)-6-oxo-1H-purine-9-yl]tetrahydrofuran-3-yl] ester (69)

[0758] Following the synthesis of acetate [(2R,3R,4R,5R)-4-acetoxy-2-[2-(methylamino)-2-oxo-ethyl]-5-[2-(2-methylpropionylamino)-6-oxo-1H-purine-9-yl]tetrahydrofuran-3-yl] ester (68), the starting material acetate [(2R,3R,4R)-4,5-diacetoxy-2-[2-(dimethylamino)-2-oxo-ethyl]tetrahydrofuran-3-yl] ester (66, 3 g, 9.05 mmol, 1.0 equivalent) was glycosylated with isobutyrylguanine (2.4 g, 10.8 mmol, 1.2 equivalent). The reaction was completed after 3 hours at 100 °C. The product was processed as described in (68) and purified by reverse rapid chromatography (FA) and SFC (neutral-MeOH, REG2S(s,s)) to give 1.8 g (40%) of a yellow foamy guanosine analogue acetate [(2R,3R,4R,5R)-4-acetoxy-2-[2-(dimethylamino)-2-oxo-ethyl]-5-[2-(2-methylpropionylamino)-6-oxo-1H-purine-9-yl]tetrahydrofuran-3-yl] ester (69).

[0759] MS (calculated value: 492.2)(m / z) = 493.1[M+H + ].

[0760] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 12.11 (br s, 1H), 11.66 (br s, 1H), 8.33 (s, 1H), 6.08-6.14 (m, 1H), 6.02-6.07 (m, 1H), 5.43 (dd, J=2.5, 5.4Hz, 1H), 4.45-4.58 (m, 1H), 3. 01-3.10 (m, 1H), 2.89-3.00 (m, 4H), 2.73-2.85 (m, 4H), 2.15 (s, 3H), 2.00 (s, 3H), 1.13 (dd, J=1.7, 6.8Hz, 6H).

[0761] Example 6.12: Synthesis of acetate [(2R,3R,4R,5R)-4-acetoxy-2-[2-(butylamino)-2-oxo-ethyl]-5-[2-(2-methylpropylamino)-6-oxo-1H-purine-9-yl]tetrahydrofuran-3-yl] ester (70)

[0762] BSA (6.8 g, 33.4 mmol, 4.0 equivalent) was added dropwise to a solution of the starting compound acetate [(2R,3R,4R)-4,5-diacetoxy-2-[2-(methylamino)-2-oxo-ethyl]tetrahydrofuran-3-yl] ester (67.3 g, 8.35 mmol, 1.0 equivalent) and isobutyrylguanine (2.2 g, 10.0 mmol, 1.2 equivalent) in a DCE (120 mL). The mixture was stirred at 100 °C for 1 hour, and then TMSOTf (5.5 g, 25.1 mmol, 3.0 equivalent) was added. After another 3 hours at 100 °C, the reaction mixture was cooled to 10 °C and poured into water (100 mL). The layers were separated and the aqueous phase was extracted with DCM (2 × 100 mL). The combined organic phases were dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum. The crude product was purified by reversed-fast chromatography (FA) and SFC (neutral-MeOH, REG2S(s,s)) to give 1.5 g (30%) of yellow foamy guanosine derivative acetate [(2R,3R,4R,5R)-4-acetoxy-2-[2-(butylamino)-2-oxo-ethyl]-5-[2-(2-methylpropionylamino)-6-oxo-1H-purine-9-yl]tetrahydrofuran-3-yl] ester (70).

[0763] MS (calculated value: 520.2)(m / z) = 521.1[M+H + ].

[0764] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 11.30-12.37 (2x br s, 2H), 8.26 (s, 1H), 7.89 (t, J=5.5Hz, 1H), 6.02 (d, J=6.8Hz, 1H), 5.87 (t, J= 6.2Hz, 1H), 5.47 (dd, J=3.6, 5.6Hz, 1H), 4.36-4.51 (m, 1H), 3.04 (q, J=6.3Hz , 2H), 2.83-2.74(m, 1H), 2.71-2.62(m, 2H), 2.12(s, 3H), 2.02(s, 3H), 1.30- 1.39 (m, 2H), 1.18-1.28 (m, 2H), 1.14 (d, J=6.9Hz, 6H), 0.82 (t, J=7.2Hz, 3H).

[0765] Example 6.13: Synthesis of 2-[(2R,3S,4R,5R)-5-(2-amino-6-oxo-1H-purin-9-yl)-3,4-dihydroxy-tetrahydrofuran-2-yl]-N-methylacetamide (71)

[0766] The starting material, acetate [(2R,3R,4R,5R)-4-acetoxy-2-[2-(methylamino)-2-oxo-ethyl]-5-[2-(2-methylpropionylamino)-6-oxo-1H-purine-9-yl]tetrahydrofuran-3-yl] ester (68,930 mg, 1.9 mmol, 1.0 equivalent), was dissolved in MeOH (18.6 ml) and NaOMe solution (0.64 ml, 0.64 mmol, 0.33 equivalent, 1 mol / L in MeOH) was added dropwise at 10 °C. After stirring at 60°C for 3 hours, the solvent was removed under vacuum, and the residue was washed with MeOH (5 ml) to produce 501 mg (79.5%) of the title compound 2-[(2R,3S,4R,5R)-5-(2-amino-6-oxo-1H-purine-9-yl)-3,4-dihydroxy-tetrahydrofuran-2-yl]-N-methyl-acetamide (71) as a white solid.

[0767] MS (calculated value: 324.1)(m / z) = 324.8[M+H + ].

[0768] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 7.89 (br d, J=4.5Hz, 1H), 7.85 (s, 1H), 6.88 (br s, 2H), 5.70 (d, J=5.4Hz, 1H), 4.54 (t, J=5.1Hz, 1H), 4.17-4.26 (m, 1H), 4.08 (t, J=4.6Hz, 1H), 2.61 (d, J=4.4Hz, 3H), 2.43-2.54 (m, 2H).

[0769] 1H-NMR (D2O, 400MHz) δ [ppm]: 7.73 (s, 1H), 5.72 (d, J=4.9Hz, 1H), 4.62 (brt, J=5 .0Hz, 1H), 4.24-4.35(m, 1H), 4.13-4.22(m, 1H), 2.54-2.69(m, 2H), 2.52(s, 3H).

[0770] Example 6.14: Synthesis of 2-[(2R,3S,4R,5R)-5-(2-amino-6-oxo-1H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl]-N,N-dimethylacetamide (72)

[0771] The starting material, acetate [(2R,3R,4R,5R)-4-acetoxy-2-[2-(dimethylamino)-2-oxo-ethyl]-5-[2-(2-methylpropionyl-amino)-6-oxo-1H-purine-9-yl]tetrahydrofuran-3-yl] ester (69, 1.6 g, 3.25 mmol, 1.0 equivalent), was dissolved in MeOH (30 ml) and 1.62 ml (1.62 mmol, 0.5 equivalent, 1 mol / L in MeOH) of NaOMe solution was added dropwise at 10 °C. After stirring at 60°C for 4 hours, the solvent was evaporated under vacuum, and the residue was ground with 10 ml of MeOH to obtain 0.99 g (90%) of the desired guanosine analog 2-[(2R,3S,4R,5R)-5-(2-amino-6-oxo-1H-purine-9-yl)-3,4-dihydroxy-tetrahydrofuran-2-yl]-N,N-dimethylacetamide (72) in the form of a brown solid.

[0772] MS (calculated value: 338.1)(m / z) = 339.1 [M+H + ].

[0773] 1H-NMR (D2O, 400MHz) δ [ppm]: 7.70 (br s, 1H), 5.70 (m, 1H), 4.68 (br s, 1H), 4.34-4.22 (m, 2H), 2.83 (br s, 3H), 2.82-2.73 (m, 2H), 2.71 (br s, 3H).

[0774] Example 6.15: Synthesis of 2-[(2R,3S,4R,5R)-5-(2-amino-6-oxo-1H-purin-9-yl)-3,4-dihydroxy-tetrahydrofuran-2-yl]N-butyl-acetamide (73)

[0775] Following the synthesis of 2-[(2R,3S,4R,5R)-5-(2-amino-6-oxo-1H-purin-9-yl)-3,4-dihydroxy-tetrahydrofuran-2-yl]-N,N-dimethyl-acetamide (72), 1.4 g (2.7 mmol, 1.0 equivalent) of the starting material acetic acid [(2R,3R,4R,5R)-4-acetoxy-2-[2-(butylamino)-2-oxo-ethyl]-5-[ 2-(2-methyl-propionylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-3-yl] ester (70) was treated with NaOMe / MeOH to produce 800 mg (81.2%) of the title compound 2-[(2R,3S,4R,5R)-5-(2-amino-6-oxo-1H-purin-9-yl)-3,4-dihydroxy-tetrahydrofuran-2-yl]-N-butyl-acetamide (73) as a white solid. MS (calculated value: 366.2)(m / z) = 366.9 [M+H + ].

[0776] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 7.69-7.84 (m, 2H), 6.81 (br s, 2H), 5.65 (d, J=5.6Hz, 1H), 4.97-5.59 (m, 1H), 4.51 (t, J=5.3Hz, 1H), 4.11-4.23 (m, 1H), 4.02 (t, J=4.3 Hz, 1H), 3.02 (q, J=6.3Hz, 2H), 2.48 (m, 2H), 1.28-1.38 (m, 2H), 1.18-1.26 (m, 2H), 0.82 (t, J=7.3Hz, 3H).

[0777] 1H-NMR (D2O, 400MHz) δ [ppm]: 7.84 (s, 1H), 5.80 (d, J=5.9Hz, 1H), 4.93 (t, J=5.5Hz, 1H), 4.33-4.41 (m, 1H), 4.25-4.32 (m, 1H), 3.13 ( td, J=6.7, 13.4Hz, 1H), 2.93 (td, J=6.4, 13.2Hz, 1H), 2.58-2.73 (m, 2H), 1.10-1.28 (m, 2H), 0.85-1.01 (m, 2H), 0.60 (t, J=7.3Hz, 3H).

[0778] Example 7: Synthesis of a simplified precursor for piperidine-derived ASGPR binders

[0779]

[0780] Example 7.1: Synthesis of (3aR,6R,6aR)-6-(hydroxymethyl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]dioxacyclopenten-4-ol (75)

[0781] Concentrated sulfuric acid (3.50 g, 35.65 mmol, 0.11 equivalents) was added to a solution of d-ribose (74 g, 50.25 g, 334.71 mmol, 1.00 equivalents) in acetone (500 mL). The reaction mixture was stirred overnight until TLC indicated complete conversion of the starting material. A saturated aqueous solution of NaHCO3 (300 mL) and toluene (300 mL) were added, and the mixture was concentrated under vacuum to remove acetone from the mixture.

[0782] Add EtOAc (300 mL), saturated NaHCO3 aqueous solution (100 mL), and water (50 mL) to separate the layers. The aqueous layer was then extracted again with EtOAc (3 × 100 mL). The combined organic layers were washed with saturated NaCl aqueous solution (100 mL), dried (MgSO4), filtered, and concentrated under vacuum. The crude product was purified by rapid chromatography (silica, 0.100% EtOAc / n-heptane) to yield a colorless oily (3aR,6R,6aR)-6-(hydroxymethyl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofuran[3,4-d][1,3]dioxane-4-ol (75 g, 33.79 g, 177.66 mmol, 53%).

[0783] Example 7.2: Synthesis of methyl 4-methylbenzenesulfonic acid [(3aR,6R,6aR)-4-hydroxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofuran[3,4-d][1,3]dioxacyclopenten-6-yl]methyl ester (76)

[0784] A solution of (3aR,6R,6aR)-6-(hydroxymethyl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]dioxanepenten-4-ol (75 g, 8.02 g, 42.18 mmol, 1.00 equivalent) in anhydrous pyridine (20 mL) was cooled to 0 °C and toluenesulfonyl chloride (9.85 g, 51.67 mmol, 1.20 equivalent) was added. The reaction mixture was stirred at 0 °C for 1.5 h until LC / MS indicated complete conversion of the starting material. EtOAc (300 mL) and 1N HCl aqueous solution (150 mL) were added to separate the layers, and the aqueous layer was re-extracted with EtOAc (2 × 100 mL). The combined organic layers were washed with saturated NaHCO3 aqueous solution (2 × 50 mL) and saturated NaCl aqueous solution (2 × 50 mL), dried (MgSO4), filtered, and concentrated under vacuum. The crude material is used directly in the next step.

[0785] LC-MS (Method D):

[0786] R t [min](UV signal 220nm): 1.24

[0787] M [g / mol]: 327.0 [M+H-H2O] + ]

[0788] Example 7.3: Synthesis of (3aR,6R,6aR)-6-(aminomethyl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]dioxacyclopenten-4-ol (77)

[0789] From methyl 4-methylbenzenesulfonic acid [(3aR,6R,6aR)-4-hydroxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofuran[3,4-d][1,3]dioxacyclopenten-6-yl] ester (76): Crude methyl 4-methylbenzenesulfonic acid [(3aR,6R,6aR)-4-hydroxy-2,2-dimethyl-3a,4,6,6a-tetrahydrofuran[3,4-d][1,3]dioxacyclopenten-6-yl] ester (76, maximum 42.18 mmol, 1.00 equivalent) was dissolved in DMF (10 mL), LiN3 (2 M in DMF, 60.0 mL, 120 mmol, 2.80 equivalent) was added, and the mixture was stirred overnight at 75 °C. Add EtOAc (300 mL) and water (100 mL), separate the layers, and re-extract the aqueous layer with EtOAc (3 × 50 mL). The combined organic layers were washed with saturated NaCl aqueous solution (50 mL), dried (MgSO4), filtered, and concentrated under vacuum. The crude product was purified by rapid chromatography (silica, 0.70% EtOAc / n-heptane) to yield a colorless oil of (3aR,6R,6aR)-6-(aminomethyl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]dioxane-4-ol (77 g, 3.51 g, 16.32 mmol, two-step yield 39%).

[0790] From (3aR,6R,6aR)-6-(aminomethyl)-2,2-dimethyl-6,6a-dihydro-3aH-furano[3,4-d][1,3]dioxacyclopenten-4-one (87): A solution of (3aR,6R,6aR)-6-(aminomethyl)-2,2-dimethyl-6,6a-dihydro-3aH-furano[3,4-d][1,3]dioxacyclopenten-4-one (87, 3.05 g, 14.28 mmol, 1.00 equivalent) in anhydrous DCM (50 mL) was cooled to 78 °C and DiBA1-H (1 M in toluene, 24.00 mL, 24.00 mmol, 1.68 equivalent) was added. The reaction mixture was stirred at 78 °C for 30 min until LC / MS indicated complete conversion of the starting material. Add saturated Rochelle salt aqueous solution (30 mL) and EtOAc (150 mL), stir the mixture at room temperature for 1 hour, separate the layers, and re-extract the aqueous layer with EtOAc (60 mL). The combined organic layers were washed with saturated NaCl aqueous solution (50 mL), dried (MgSO4), filtered, and concentrated under vacuum. The crude product (3aR,6R,6aR)-6-(aminomethyl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]dioxanepenten-4-ol (77 g, 2.90 g, 13.48 mmol, 94%) was obtained as a colorless oil and was used in the next step without further purification.

[0791] LC-MS (Method D):

[0792] R t [min](UV signal 220nm): 0.80

[0793] M [g / mol]: 170.0 [M+H-N2-H2O] + ]

[0794] Example 7.4: Synthesis of (3aS,7R,7aR)-2,2-dimethyl-3a,4,5,6,7,7a-hexahydro-[1,3]dioxacyclopenteno[4,5-c]pyridine-7-ol (78)

[0795] (3aR,6R,6aR)-6-(aminomethyl)-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]dioxacyclopenten-4-ol (77, 3.37 g, 15.66 mmol, 1.00 equivalent) was dissolved in THF (50 mL), 10% Pd / C (0.17 g, 0.16 mmol, 0.01 equivalent) was added, and the mixture was hydrogenated in an autoclave at room temperature and 4 bar of hydrogen for 4 days. The reaction mixture was filtered because piperidine was completely formed as detected by LC / MS. The crude product (3aS,7R,7aR)-2,2-dimethyl-3a,4,5,6,7,7a-hexahydro-[1,3]dioxacyclopenteno[4,5-c]pyridine-7-ol (78) was used directly as a stock solution in THF for further reactions.

[0796] LC-MS (Method D):

[0797] R t [min](TIC signal): 0.14

[0798] M [g / mol]: 173.9 [M+H] + ]

[0799] Example 7.5: Synthesis of (3aS,7R,7aR)-7-hydroxy-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopentenano[4,5-c]pyridine-5-carboxylic acid benzyl ester (79)

[0800] To a solution of (3aS,7R,7aR)-2,2-dimethyl-3a,4,6,6a-tetrahydrofurano[3,4-d][1,3]dioxacyclopenten-4-ol (77, max 15.66 mmol, 1.00 equivalent) in THF (90 mL), a saturated aqueous solution of NaHCO3 (30 mL) and Cbz-Cl (2.67 g, 15.66 mmol, 1.00 equivalent) were added and the reaction mixture was stirred overnight. Add EtOAc (200 mL), saturated NaHCO3 aqueous solution (50 mL), and water (30 mL) to separate the layers. The aqueous layer was then extracted again with EtOAc (2 × 50 mL). The combined organic layers were washed with saturated NaCl aqueous solution (50 mL), dried (MgSO4), filtered, and concentrated under vacuum. The crude product was purified by rapid chromatography (silica, 0.100% EtOAc / n-heptane) to give a colorless oily (3aS,7R,7aR)-7-hydroxy-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxanepenteno[4,5-c]pyridine-5-carboxylic acid benzyl ester (79 g, 3.37 g, 10.96 mmol, two-step yield 70%).

[0801] LC-MS (Method D):

[0802] R t [min](UV-signal 220nm): 1.08

[0803] M [g / mol]: 308.0 [M+H] + ]

[0804] Example 7.6: Synthesis of (3aS,7R,7aS)-2,2-dimethyl-7-methylsulfonyloxy-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopentenano[4,5-c]pyridine-5-carboxylic acid benzyl ester (80)

[0805] A solution of (3aS,7R,7aR)-7-hydroxy-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridine-5-carboxylic acid benzyl ester (79 g, 5.00 g, 16.27 mmol, 1.00 equivalent) in anhydrous DCM (80 mL) was cooled to 0 °C and pyridine (5.25 mL, 65.07 mmol, 4.00 equivalent) and methanesulfonic anhydride (11.33 g, 65.07 mmol, 4.00 equivalent) were added. The reaction mixture was stirred at 0 °C for 1.5 h until LC / MS indicated complete conversion of the starting material. 1 N HCl aqueous solution (100 mL) and EtOAc (250 mL) were added, the layers were separated, the organic layer was washed with saturated NaCl aqueous solution (50 mL), dried (MgSO4), filtered, and concentrated under vacuum. The crude product was purified by rapid chromatography (silica, 0.100% EtOAc / n-heptane) to give a colorless oil of (3aS,7R,7aS)-2,2-dimethyl-7-methylsulfonyloxy-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridine-5-carboxylic acid benzyl ester (80, 3.63 g, 9.41 mmol, 58%). LC-MS (Method D):

[0806] R t [min](UV signal 220nm): 1.34

[0807] M [g / mol]: 386.0 [M+H] + ]

[0808] Example 7.7: Synthesis of (3aS,7S,7aR)-7-hydroxy-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopentenano[4,5-c]pyridine-5-carboxylic acid benzyl ester (81)

[0809] LiN3 (2M in DMF, 12 mL, 24 mmol, 2.5 equivalence) was added to a solution of (3aS,7R,7aS)-2,2-dimethyl-7-methylsulfonyloxy-4,6,7,7a-tetrahydro-3aH-[1,3]dioxanepenteno[4,5-c]pyridine-5-carboxylic acid benzyl ester (80, 3.63 g, 9.41 mmol, 1.00 equivalent) in DMF (3 mL), and the mixture was stirred at 100 °C for 2 days. The reaction was stopped due to the detection of a large amount of elimination products by LC / MS. EtOAc (100 mL) and water (50 mL) were added, the layers were separated, and the aqueous layer was re-extracted with EtOAc (3 × 20 mL). The combined organic layers were washed with saturated NaCl aqueous solution (30 mL), dried (MgSO4), filtered, and concentrated under vacuum. The crude product was purified by rapid chromatography (silica, 0.100% EtOAc / n-heptane) to give a colorless oil of (3aS,7S,7aR)-7-hydroxy-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridine-5-carboxylate (81, 410 mg, 1.23 mmol, 13%) and recovered (3aS,7R,7aS)-2,2-dimethyl-7-methylsulfonyloxy-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridine-5-carboxylate (80, 1.77 g, 4.58 mmol, 49%).

[0810] LC-MS (Method D):

[0811] R t [min](UV-signal 220nm): 1.52

[0812] M [g / mol]: 305.1 [M+H-N2+]

[0813] Example 7.8: Synthesis of (3aS,7S,7aR)-7-amino-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopentenano[4,5-c]pyridine-5-carboxylic acid benzyl ester (82)

[0814] Trimethylphosphine (1M in THF, 2.50 mL, 2.50 mmol, 1.50 equivalent) was added to a solution of (3aS,7S,7aR)-7-hydroxy-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxanepenteno[4,5-c]pyridine-5-carboxylic acid benzyl ester (81,533 mg, 1.66 mmol, 1.00 equivalent) in 10 mL of THF, and the reaction mixture was stirred for 1 hour until the starting material was completely consumed as monitored by LC / MS. Water (1 mL) was added, and the reaction mixture was concentrated under vacuum. The crude product was directly acetylated.

[0815] LC-MS (Method D):

[0816] R t [min](UV signal 220nm): 0.87

[0817] M [g / mol]: 307.1 [M+H] + ]

[0818] Example 7.9: Synthesis of (3aS,7S,7aR)-7-acetamido-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopentenano[4,5-c]pyridine-5-carboxylic acid benzyl ester (83)

[0819] Crude (3aS,7S,7aR)-7-amino-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridine-5-carboxylic acid benzyl ester (82, maximum 1.66 mmol, 1.00 equivalent) was dissolved in EtOAc (10 mL), pyridine (0.70 mL, 8.65 mmol, 5.20 equivalent) and acetic anhydride (0.80 mL, 8.34 mL, 5.01 equivalent) were added, and the reaction mixture was stirred overnight at room temperature. LC / MS indicated complete acetylation, so the crude mixture was concentrated under vacuum and purified by rapid chromatography (silica, 0.100% EtOAc / n-heptane) to give (3aS,7S,7aR)-7-acetamido-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridine-5-carboxylic acid benzyl ester (83,474 mg, 1.36 mmol, two-step yield 82%).

[0820] LC-MS (Method D):

[0821] R t [min](UV-signal 220nm): 1.08

[0822] M [g / mol]: 349.0 [M+H]+ ]

[0823] Example 7.10: Synthesis of (3aR,6R,6aR)-6-(hydroxymethyl)-2,2-dimethyl-6,6a-dihydro-3aH-furano[3,4-d][1,3]dioxacyclopenten-4-one (85)

[0824] A solution of d-ribose-1,4-lactone (84, 20.21 g, 136.45 mmol, 1.00 equivalence) in acetone (400 mL) was mixed with concentrated HCl (37%, 9.50 mL, 113.77 mmol, 0.83 equivalence). The reaction mixture was stirred overnight until TLC indicated complete conversion of the starting material. Solid NaHCO3 was added, the reaction mixture was filtered, and the filter cake was washed with acetone (100 mL). The combined filtrates were concentrated under vacuum, and (3aR,6R,6aR)-6-(hydroxymethyl)-2,2-dimethyl-6,6a-dihydro-3aH-furano[3,4-d][1,3]dioxane-4-one (85, 25.21 g, 133.97 mmol, 98%) was used directly as the crude product for the next step.

[0825] Example 7.11: Synthesis of methyl 4-methylbenzenesulfonic acid [(3aR,6R,6aR)-2,2-dimethyl-4-oxo-6,6a-dihydro-3aH-furan[3,4-d][1,3]dioxacyclopenten-6-yl]methyl ester (86)

[0826] A solution of (3aR,6R,6aR)-6-(hydroxymethyl)-2,2-dimethyl-6,6a-dihydro-3aH-furano[3,4-d][1,3]dioxacyclopenten-4-one (85, 5.22 g, 27.74 mmol, 1.00 equivalent) in anhydrous DCM (50 mL) was cooled to 0 °C and toluenesulfonic anhydride (13.38 g, 41.00 mmol, 1.48 equivalent) and pyridine (6.65 mL, 82.22 mmol, 2.96 equivalent) were added. The reaction mixture was stirred at 0 °C for 1.5 h and then at room temperature overnight until LC / MS indicated complete conversion of the starting material. Add EtOAc (300 mL) and 1N HCl aqueous solution (150 mL), separate the layers, and re-extract the aqueous layer with EtOAc (2 × 100 mL). The combined organic layers are washed with saturated NaHCO3 aqueous solution (2 × 50 mL) and saturated NaCl aqueous solution (30 mL), dried (MgSO4), filtered, and concentrated under vacuum. The crude product, 4-methylbenzenesulfonic acid [(3aR,6R,6aR)-2,2-dimethyl-4-oxo-6,6a-dihydro-3aH-furano[3,4-d][1,3]dioxacyclopenten-6-yl]methyl ester (86, 9.31 g, 27.20 mmol, 98%), is used directly in the next step.

[0827] LC-MS (Method D):

[0828] R t [min](UV signal 220nm): 1.34

[0829] M [g / mol]: 343.0 [M+H-H2O] + ]

[0830] Example 7.12: Synthesis of (3aR,6R,6aR)-6-(aminomethyl)-2,2-dimethyl-6,6a-dihydro-3aH-furano[3,4-d][1,3]dioxacyclopenten-4-one (87)

[0831] 4-Methylbenzenesulfonic acid [(3aR,6R,6aR)-2,2-dimethyl-4-oxo-6,6a-dihydro-3aH-furano[3,4-d][1,3]dioxacyclopenten-6-yl]methyl ester (86, 8.29 g, 24.23 mmol, 1.00 equivalent) was dissolved in DMF (10 mL), and NaN3 (6.47 g, 99.52 mmol, 4.11 equivalent) was added. The mixture was stirred at 70 °C for 6 days. EtOAc (300 mL) and water (100 mL) were added, the layers were separated, and the aqueous layer was re-extracted with EtOAc (3 × 50 mL). The combined organic layers were washed with saturated NaCl aqueous solution (50 mL), dried (MgSO4), filtered, and concentrated under vacuum. The crude product was purified by rapid chromatography (silica, 0.35% EtOAc / n-heptane) to produce a colorless oily (3aR,6R,6aR)-6-(aminomethyl)-2,2-dimethyl-6,6a-dihydro-3aH-furano[3,4-d][1,3]dioxane-4-one (87 g, 3.26 g, 15.29 mmol, 63%).

[0832] LC-MS (Method D):

[0833] R t [min](UV signal 220nm): 0.94

[0834] M [g / mol]: 214.0 [M+H] + ]

[0835] Example 7.13: Synthesis of (3aR,7R,7aR)-7-hydroxy-2,2-dimethyl-5,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridin-4-one (88)

[0836] (3aR,6R,6aR)-6-(aminomethyl)-2,2-dimethyl-6,6a,4,6,6a-tetrahydrofurano[3,4-d][1,3]dioxacyclopenten-4-ol (87 g, 2.83 g, 13.27 mmol, 1.00 equivalent) was dissolved in THF (50 mL), 10% Pd / C (0.14 g, 0.13 mmol, 0.01 equivalent) was added, and the mixture was hydrogenated in an autoclave at room temperature and 4 bar of hydrogen for 2 days. Since piperidine was completely formed as detected by LC / MS, the reaction mixture was filtered and concentrated under vacuum. The crude product (3aR,7R,7aR)-7-hydroxy-2,2-dimethyl-5,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridin-4-one (88, 2.65 g, quantitative) was obtained as a yellow solid and used directly for further reactions.

[0837] LC-MS (Method D):

[0838] R t [min](TIC signal): 0.11

[0839] M [g / mmol]: 188.2 [M+H] + ]

[0840] Example 7.14: Synthesis of (3aR,7R,7aS)-7-[tert-butyl(dimethyl)silyl]oxy-2,2-dimethyl-5,6,7,7a-tetrahydro-3aH-[1,3]dioxane[4,5-c]pyridin-4-one (89)

[0841] To a solution of (3aR,7R,7aR)-7-hydroxy-2,2-dimethyl-5,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridin-4 one (88, 2.88 g, 15.37 mmol, 1.00 equivalent) in DCM (100 mL), imidazole (3.30 g, 48.47 mmol, 3.15 equivalent) and TBSC1 (4.64 g, 30.79 mmol, 2.00 equivalent) were added. The reaction mixture was stirred overnight at room temperature until TLC showed complete conversion of the starting material. EtOAc (250 mL) and citric acid aqueous solution (10%, 100 mL) were added, the layers were separated, and the aqueous layer was re-extracted with EtOAc (20 mL). The combined organic layers were washed with saturated NaCl aqueous solution (30 mL), dried (MgSO4), filtered, and concentrated under vacuum. The crude product was purified by rapid chromatography (silica, 580% EtOAc / n-heptane) to give (3aR,7R,7aS)-7-[tert-butyl(dimethyl)silyl]oxy-2,2-dimethyl-5,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridin-4-one (89 g, 4.22 g, 14.00 mmol, 91%) as a colorless oil.

[0842] LC-MS (Method D):

[0843] R t [min](TIC signal): 1.38

[0844] M [g / mol]: 302.1 [M+H] + ]

[0845] Example 8: Synthetic connector precursor.

[0846]

[0847] Example 8.1: Synthesis of 6-benzyloxyhexyl-1-ol (91)

[0848] A solution of hexane-1,6-diol (90, 9.96 g, 84.28 mmol, 1.00 equivalent) and tetrabutylammonium iodide (934 mg, 2.53 mmol, 0.03 equivalent) in anhydrous THF (100 mL) was cooled to 0 °C and sodium hydride (60% in mineral oil, 3.80 g, 95.01 mmol, 1.13 equivalent) was added in small amounts. The reaction mixture was stirred at 0 °C for 10 min, then at room temperature for 30 min, and benzyl bromide (15.86 g, 92.71 mmol, 1.10 equivalent) was added. The reaction mixture was stirred at room temperature overnight and saturated aqueous NH4Cl solution (100 mL) and EtOAc (250 mL) were added. The layers were separated, the organic layer was washed with saturated aqueous NaCl solution (100 mL), dried (MgSO4), filtered, and concentrated under vacuum. The crude product was purified by rapid chromatography (silica, 1-38% EtOAc / n-heptane) to give 6-benzyloxyhexyl-1-ol (91, 9.08 g, 43.58 mmol, 52%) as a colorless oil.

[0849] LC-MS (Method D):

[0850] R t [min](UV signal 220nm): 1.27

[0851] M [g / mol]: 209.1 [M+H] + ]

[0852] Example 8.2: Synthesis of 6-benzyloxyhexyl methanesulfonate (92)

[0853] A solution of 6-benzyloxyhexyl-1-ol (91, 9.08 g, 43.58 mmol, 1.00 equivalent) in anhydrous DCM (100 mL) was cooled to 0 °C and pyridine (17.62 mL, 217.88 mmol, 5.00 equivalent) and methanesulfonyl chloride (8.47 mL, 108.94 mmol, 2.50 equivalent) were added. The reaction mixture was stirred at 0 °C for 1.5 h until LC / MS indicated complete conversion of the starting material. The layers were separated by adding 1 N HCl aqueous solution (100 mL) and EtOAc (250 mL). The organic layer was washed with saturated NaCl aqueous solution (50 mL), dried (MgSO4), filtered, and concentrated under vacuum. The crude product was purified by rapid chromatography (silica, 0.35% EtOAc / n-heptane) to give 6-benzyloxyhexyl methanesulfonate (92, 10.59 g, 36.98 mmol, 85%) as a colorless oil.

[0854] LC-MS (Method D):

[0855] R t [min](UV signal 220nm): 1.49

[0856] M [g / mol]: 287.1 [M+H] + ]

[0857] Example 8.3: Synthesis of 6-benzyloxyhexanoic acid (93) / 6-benzoyloxyhexanoic acid (94) (inseparable mixture):

[0858] To a solution of 6-benzyloxyhexyl methanesulfonate (92, 9.22 g, 44.24 mmol, 1.00 equivalent) and TEMPO (693 mg, 4.44 mmol, 0.10 equivalent) in acetonitrile (50 mL) and pH 4 buffered aqueous solution (50 mL), simultaneously add NaClO2 (technical mass, about 80%, 30.01 g, about 6.00 equivalent) in water (50 mL) and NaOCl aqueous solution (technical mass, about 10%, 14.00 mL, about 0.51 equivalent). Stir the reaction mixture overnight at room temperature until LC / MS indicates complete conversion of the starting material. Add EtOAc (200 mL) and saturated Na2SO3 aqueous solution (100 mL), separate the layers, acidify the aqueous layer by adding 1N HCl aqueous solution (50 mL) and re-extract with EtOAc (3 × 50 mL). The combined organic layers were washed with saturated NaCl aqueous solution (30 mL), dried (MgSO4), filtered, and concentrated under vacuum. The crude product (10.05 g, quantitative) was used directly for the next step. LC / MS indicated that the desired product, 6-benzyloxyhexanoic acid (93), was partially peroxidized to 6-benzoyloxyhexanoic acid (94), which appeared as an inseparable mixture.

[0859] LC-MS (Method D):

[0860] R t [min](UV-signal 220nm): 1.25

[0861] M [g / mol]: 223.2 [M+H] + ](93, major product), 237.1[M+H + (94, secondary products)

[0862] Example 8.4: Synthesis of 6-benzyloxyhexanoyl chloride (95) / 6-benzoyloxyhexanoyl chloride (96) (inseparable mixture)

[0863] Oxaloyl chloride (6.00 mL, 67.18 mmol, 1.52 equivalents) and one drop of DMF were added to a crude mixture of 6-benzyloxyhexanoic acid (93) / 6-benzoyloxyhexanoic acid (94) (maximum 44.24 mmol, 1.00 equivalents) in DCM (50 mL) to catalyze and accelerate the reaction. The reaction mixture was stirred overnight at room temperature until LC / MS indicated complete conversion of the starting material (an aliquot of the reaction mixture was added to methanol and the acyl chloride was detected as its corresponding methyl ester). The solvent was removed under vacuum, and the crude product was used for the next step without purification.

[0864] LC-MS (Method D):

[0865] R t [min](UV-signal 220nm): 1.54

[0866] M [g / mol]: 237.2 [M-Cl+OMe+H] + ](95, major product), 251.1[M-Cl+OMe+H + (96, secondary products)

[0867] Example 8.5: Synthesis of benzyl 6-bromohexanoate (98)

[0868] To a solution of 6-bromohexanoic acid (97, 20.19 g, 103.51 mmol, 1.00 equivalent) in DCM (100 mL), oxalyl chloride (14.00 mL, 159.94 mmol, 1.55 equivalent) and one drop of DMF were added to catalyze and accelerate the reaction. The reaction mixture was stirred overnight at room temperature until LC / MS indicated complete conversion of the starting material (an aliquots of the reaction mixture were added to methanol and the acyl chloride was detected as its corresponding methyl ester). The solvent was removed under vacuum, and the crude product was used for the next step without purification.

[0869] Benzyl alcohol (23.30 g, 215.46 mmol, 2.08 equivalents) and pyridine (26.00 mL, 321.46 mmol, 3.11 equivalents) were added to a solution of crude acyl chloride (maximum 103.51 mmol, 1.00 equivalents) in DCM (100 mL). The reaction mixture was stirred overnight at room temperature until LC / MS indicated complete conversion of the starting material. The layers were separated by adding 1N HCl (100 mL) and an aqueous solution of EtOAc (500 mL). The aqueous layer was re-extracted with EtOAc (3 × 50 mL), and the organic layer was washed with a saturated aqueous solution of NaCl (50 mL), dried (MgSO4), filtered, and concentrated under vacuum. The crude product was purified by rapid chromatography (silica, 0.35% EtOAc / n-heptane) to give benzyl 6-bromohexanoate (98 g, 22.05 g, 77.32 mmol, 75%) as a colorless oil.

[0870] LC-MS (Method D):

[0871] R t [min](UV-signal 220nm): 1.68

[0872] M[g / mol]: 302.1 / 304.1[M+H2O+H + ]

[0873] Example 8.6: Synthesis of 6-benzyloxy-6-oxo-hexanoic acid (99)

[0874] To a solution of benzyl 6-bromohexanoate (98 g, 5.73 g, 20.09 mmol, 1.00 equivalence) in DMSO (40 mL), NaNO₂ (5.54 g, 80.30 mmol, 4.00 equivalence) and acetic acid (12 mL) were added. The reaction mixture was stirred at 40 °C for 2 days. EtOAc (300 mL) and 1N HCl aqueous solution (100 mL) were added to separate the layers, and the aqueous layer was extracted with EtOAc (3 × 20 mL). The combined organic layers were washed with saturated NaCl aqueous solution (3 × 50 mL), dried (MgSO₄), filtered, and concentrated under vacuum. The crude product was purified by rapid chromatography (silica, 150% EtOAc / n-heptane) to give 6-benzyloxy-6-oxo-hexanoic acid (99 g, 2.12 g, 8.97 mmol, 45%) as a colorless oil.

[0875] LC-MS (Method D):

[0876] R t [min](UV signal 220nm): 1.14

[0877] M [g / mol]: 237.1 [M+H] + ]

[0878] Example 8.7: Synthesis of benzyl 6-chloro-6-oxo-hexanoate (100)

[0879] To a solution of 6-benzyloxy-6-oxo-hexanoic acid (99 g, 2.31 g, 9.77 mmol, 1.00 equivalence) in DCM (30 mL), oxalyl chloride (1.20 mL, 13.44 mmol, 1.37 equivalence) and one drop of DMF were added to catalyze and accelerate the reaction. The reaction mixture was stirred overnight at room temperature until LC / MS indicated complete conversion of the starting material (an aliquots of the reaction mixture were added to methanol and the acyl chloride was detected as its corresponding methyl ester). The solvent was removed under vacuum, and the crude product, benzyl 6-chloro-6-oxo-hexanoate (100), was used for the next step without purification.

[0880] LC-MS (Method D):

[0881] R t [min](UV-signal 220nm): 1.39

[0882] M [g / mol]: 251.1 [M-Cl+OMe+H] + ]

[0883] Example 8.8: Synthesis of 4-benzyloxy-4-oxo-butyric acid (102)

[0884] A solution of benzyl alcohol (9.73 g, 89.93 mmol, 1.00 equivalent) in anhydrous THF (100 mL) was cooled to 0 °C, and sodium hydride (60% in mineral oil, 3.74 g, 93.51 mmol, 1.04 equivalent) was added in small amounts. The reaction mixture was stirred at 0 °C for 1 hour, and succinic anhydride (101 g, 9.00 g, 89.93 mmol, 1.00 equivalent) was added. The reaction mixture was stirred overnight at room temperature, and water (300 mL), EtOAc (300 mL), and solid Na2CO3 (10.00 g, excess) were added. The layers were separated, the aqueous layer was re-extracted with EtOAc (50 mL), the combined organic layer was discarded, and the aqueous layer was acidified to pH 1 by adding 1 N HCl aqueous solution. The aqueous layer was extracted with EtOAc (3 × 50 mL), and the organic layer was washed with a saturated NaCl aqueous solution (50 mL), dried (MgSO4), filtered, and concentrated under vacuum. A crude product of 4-benzyloxy-4-oxo-butyric acid (102 g, 12.29 g, 59.02 mmol, 66%) was obtained as a colorless solid, with a purity sufficient for further transformation.

[0885] LC-MS (Method D):

[0886] R t [min](UV-signal 220nm): 1.06

[0887] M [g / mol]: 209.1 [M+H] + ]

[0888] Example 8.9: Synthesis of benzyl 4-chloro-4-oxo-butyrate (103)

[0889] To a solution of 4-benzyloxy-4-oxo-butyric acid (102, 866 mg, 4.16 mmol, 1.00 equivalent) in DCM (10 mL), oxalyl chloride (0.72 mL, 8.32 mmol, 2.00 equivalent) and one drop of DMF were added to catalyze and accelerate the reaction. The reaction mixture was stirred overnight at room temperature until LC / MS indicated complete conversion of the starting material (an aliquots of the reaction mixture were added to methanol and the acyl chloride was detected as its corresponding methyl ester). The solvent was removed under vacuum, and the crude product, benzyl 4-chloro-4-oxo-butyrate (103), was used for the next step without purification.

[0890] LC-MS (Method D):

[0891] R t [min](UV-signal 220nm): 1.35

[0892] M [g / mol]: 223.1 [M-Ci+OMe+H] + ]

[0893] Example 8.10: Synthesis of 5-benzyloxy-5-oxo-valeric acid (105)

[0894] A solution of benzyl alcohol (9.49 g, 87.76 mmol, 1.00 equivalent) in anhydrous THF (100 mL) was cooled to 0 °C, and sodium hydride (60% in mineral oil, 3.51 g, 87.76 mmol, 1.00 equivalent) was added in small amounts. The reaction mixture was stirred at 0 °C for 1 hour, and glutaric anhydride (104, 10.01 g, 87.76 mmol, 1.00 equivalent) was added. The reaction mixture was stirred overnight at room temperature, and water (300 mL), EtOAc (300 mL), and solid Na2CO3 (10.00 g, excess) were added. The layers were separated, and the aqueous layer was re-extracted with EtOAc (50 mL). The combined organic layer was discarded, and the aqueous layer was acidified to pH 1 by adding 1 N HCl aqueous solution. The aqueous layer was extracted with EtOAc (3 × 50 mL), and the organic layer was washed with a saturated NaCl aqueous solution (50 mL), dried (MgSO4), filtered, and concentrated under vacuum. A crude product, 5-benzyloxy-5-oxo-pentanoic acid (105 g, 9.86 g, 44.37 mmol, 51%), was obtained as a colorless solid, with a purity sufficient for further transformation.

[0895] LC-MS (Method D):

[0896] R t [min](UV-signal 220nm): 1.11

[0897] M [g / mol]: 223.1 [M+H] + ]

[0898] Example 8.11: Synthesis of benzyl 5-chloro-5-oxo-valerate (106)

[0899] To a solution of 5-benzyloxy-5-oxo-pentanoic acid (105, 930 mg, 4.18 mmol, 1.00 equivalent) in DCM (10 mL), oxalyl chloride (0.75 mL, 8.37 mmol, 2.00 equivalent) and one drop of DMF were added to catalyze and accelerate the reaction. The reaction mixture was stirred overnight at room temperature until LC / MS indicated complete conversion of the starting material (an aliquots of the reaction mixture were added to methanol and the acyl chloride was detected as its corresponding methyl ester). The solvent was removed under vacuum, and the crude benzyl 5-chloro-5-oxo-pentanoic acid ester (106) was used for the next step without purification.

[0900] LC-MS (Method D):

[0901] R t [min](UV signal 220nm): 1.37

[0902] M [g / mol]: 237.1 [M-Cl+OMe+H] + ]

[0903] Example 8.12: Synthesis of methyl 5-chloro-5-oxo-valerate (108)

[0904] Oxaloyl chloride (3.30 mL, 37.69 mmol, 2.00 equivalent) and one drop of DMF were added to a solution of commercially available 107 (3.02 g, 18.85 mmol, 1.00 equivalent) in DCM (20 mL) to accelerate the reaction. The reaction mixture was stirred overnight at room temperature until LC / MS indicated complete conversion of the starting material (an aliquot of the reaction mixture was added to methanol and the acyl chloride was detected as its corresponding methyl ester). The solvent was removed under vacuum, and the crude product, methyl 5-chloro-5-oxo-valerate (108), was used for the next step without purification.

[0905] LC-MS (Method D):

[0906] R t [min](UV signal 220nm): 0.98

[0907] M [g / mmol]: 175.1 [M-Cl+OMe+H] + ]

[0908] Example 9: Synthesis of compounds 112, 117, 119, 120 and 121.

[0909]

[0910] Example 9.1: Synthesis of benzyl 6-[(3aS,7R,7aR)-7-hydroxy-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopentenano[4,5-c]pyridin-5-yl]-6-oxo-hexanoate (109)

[0911] LiAlH4 (15% in toluene / THF, 3.5 M, 5.00 mL, 17.50 mmol, 2.73 equivalents) was added to a solution of (3aR,7R,7aR)-7-hydroxy-2,2-dimethyl-5,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridin-4-one (88 g, 1.20 g, 6.41 mmol, 1.00 equivalents) in anhydrous THF (100 mL) and the reaction mixture was stirred overnight at room temperature. Excess LiAlH4 was quenched by carefully adding saturated aqueous solution of NaHCO3 (50 mL) and water (20 mL). The crude product (3aS,7R,7aR)-2,2-dimethyl-3a,4,5,6,7,7a-hexahydro-[1,3]dioxacyclopenteno[4,5-c]pyridine-7-ol (78) was used directly as a solution of THF / saturated NaHCO3 aqueous solution, with the addition of acyl chloride 6-chloro-6-oxo-benzyl hexanoate (100, 2.49 g, 9.79 mmol, 1.53 equivalents) in THF (10 mL). The reaction mixture was stirred at room temperature for 6 hours. EtOAc (200 mL) and water (50 mL) were added, and the reaction mixture was filtered through diatomaceous earth to remove insoluble aluminum salts. The layers were separated, and the organic layer was washed with 2N NaOH aqueous solution (3 × 30 mL) and saturated NaCl aqueous solution (30 mL), dried (MgSO4), filtered, and concentrated under vacuum. The crude product was purified by rapid chromatography (silica, 180% EtOAc / n-heptane) to give benzyl 6-[(3aS,7R,7aR)-7-hydroxy-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridin-5-yl]-6-oxoyl-hexanoate (109 g, 1.69 g, 4.32 mmol, two-step yield 67%).

[0912] LC-MS (Method D):

[0913] R t [min](UV-signal 220nm): 1.09

[0914] M [g / mol]: 392.2 [M+H] + ]

[0915] Example 9.2: Synthesis of benzyl 6-[(3aS,7R,7aS)-2,2-dimethyl-7-methylsulfonyloxy-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopentenano[4,5-c]pyridin-5-yl]-6-oxo-hexanoate (110)

[0916] A solution of 6-[(3aS,7R,7aR)-7-hydroxy-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridin-5-yl]-6-oxoyl-hexanoate benzyl ester (109 g, 1.69 g, 4.32 mmol, 1.00 equivalent) in anhydrous DCM (30 mL) was cooled to 0 °C and pyridine (1.10 mL, 13.53 mmol, 3.13 equivalent) and methanesulfonic anhydride (1.17 g, 6.59 mmol, 1.53 equivalent) were added. The reaction mixture was stirred at 0 °C for 3 hours until LC / MS indicated complete conversion of the starting material. The layers were separated by adding 1 N HCl aqueous solution (100 mL) and EtOAc (250 mL). The organic layer was washed with saturated NaCl aqueous solution (50 mL), dried (MgSO4), filtered, and concentrated under vacuum. The crude product 6-[(3aS,7R,7aS)-2,2-dimethyl-7-methylsulfonyloxy-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopentenano[4,5-c]pyridin-5-yl]-6-oxo-hexanoic acid benzyl ester (110) was used directly in the next step.

[0917] LC-MS (Method D):

[0918] R t [min](UV signal 220nm): 1.30

[0919] M [g / mol]: 470.1 [M+H] + ]

[0920] Example 9.3: Synthesis of benzyl 6-[(3aS,7S,7aR)-7-azido-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridin-5-yl]-6-oxo-hexanoate (111)

[0921] The crude product 6-[(3aS,7R,7aS)-2,2-dimethyl-7-methylsulfonyloxy-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridin-5-yl]-6-oxoyl-hexanoate benzyl ester (110, maximum 4.32 mmol, 1.00 equivalent) was dissolved in DMF (5 mL), and NaN3 (1.14 g, 17.49 mmol, 4.04 equivalent) and 15-crown-5 ether (1.51 g, 6.86 mmol, 1.58 equivalent) were added. The mixture was stirred at 100 °C for 1 day. EtOAc (100 mL) and water (50 mL) were added, the layers were separated, and the aqueous layer was re-extracted with EtOAc (3 × 10 mL). The combined organic layers were washed with saturated NaCl aqueous solution (20 mL), dried (MgSO4), filtered, and concentrated under vacuum. The crude product was purified by rapid chromatography (silica, 180% EtOAc / n-heptane) to give benzyl 6-[(3aS,7S,7aR)-7-azido-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridin-5-yl]-6-oxo-hexanoate as a colorless oil (111,593 mg, 1.42 mmol, two-step yield 33%).

[0922] LC-MS (Method D):

[0923] R t [min](UV signal 220nm): 1.44

[0924] M [g / mol]: 417.2 [M+H] + ]

[0925] Example 9.4: Synthesis of benzyl 6-[(3S,4R,5S)-3-acetamido-4,5-dihydroxy-1-piperidinyl]-6-oxo-hexanoate (112)

[0926] PMe3 (1N in THF, 0.40 mL, 0.40 mmol, 1.75 equivalence) was added to a solution of 6-[(3aS,7S,7aR)-7-azido-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridin-5-yl]-6-oxo-hexanoate benzyl ester (111, 95 mg, 0.228 mmol, 1.00 equivalent) in THF (5 mL) and water (0.1 mL), and the reaction mixture was stirred at room temperature for 1 hour. Since LC / MS indicated complete conversion of the starting material, water (0.9 mL) and acetic acid (5 mL) were added, and the reaction mixture was stirred at 80 °C for 3 hours to allow complete hydrolysis of the formed phosphineimine. Acetic anhydride (0.25 mL) was added at room temperature, and the reaction mixture was stirred for 1 hour. Add EtOAc (30 mL) and saturated NaHCO3 aqueous solution (50 mL), separate the layers, and re-extract the aqueous layer with EtOAc (3 × 10 mL). The combined organic layers were washed with saturated NaCl aqueous solution (20 mL), dried (MgSO4), filtered, and concentrated under vacuum. The crude product was dissolved in acetic acid (4 mL) and water (1 mL) and heated to 80 °C and maintained for 1 hour until the acetone was completely deprotected by LC / MS. The solvent was removed under vacuum, and the crude mixture was purified by HPLC (15 min, 590% acetonitrile / water + 0.1% TFA) to give benzyl 6-[(3S,4R,5S)-3-acetamido-4,5-dihydroxy-1-piperidinyl]-6-oxo-hexanoate (112,83 mg, 0.211 mmol, 93%) as a colorless solid. LC-MS (Method D):

[0927] R t [min](UV signal 220nm): 0.90

[0928] M[g / mol]:393.2[M+H + ]

[0929] 1 ¹H NMR (400MHz, DMSO-d⁶) δ [ppm]: 7.88 / 7.69 (d, J = 7.5 / 8.4Hz, 1H), 7.45-7.35 (m, 5H), 5.12 (s, 2H), 4.14-4.07 (m, 1H), 4.00-3.78 (m, 7H), 3.18-3.05 (m, 1H), 2.40-2.17 (m, 4H), 1.81 / 1.80 (s, 3H), 1.58-1.43 (m, 4H) (two main conformational isomers due to amide resonance)

[0930] Example 9.5: Synthesis of (3aS,7R,7aR)-7-hydroxy-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridine-5-carboxylic acid 9H-fluorene-9-ylmethyl ester (113)

[0931] LiAlH4 (15% in toluene / THF, 3.5 M, 5.00 mL, 17.50 mmol, 2.65 equivalents) was added to a solution of (3aR,7R,7aR)-7-hydroxy-2,2-dimethyl-5,6,7,7a-tetrahydro-3aH-[1,3]dioxanepenteno[4,5-c]pyridin-4-one (88 g, 1.24 g, 6.61 mmol, 1.00 equivalents) in anhydrous THF (100 mL). The reaction mixture was stirred overnight at room temperature and saturated aqueous solution of NaHCO3 (50 mL) and water (20 mL) were added. The crude product 3aS,7R,7aR)-2,2-dimethyl-3a,4,5,6,7,7a-hexahydro-[1,3]dioxacyclopenteno[4,5-c]pyridine-7-ol (78) was used directly as a THF / NaHCO3 aqueous solution, with FmocOSu (3.38 g, 10.02 mmol, 1.51 equivalents) added to a solution of THF (10 mL). The reaction mixture was stirred overnight at room temperature. EtOAc (100 mL) and saturated NaHCO3 aqueous solution (50 mL) were added, and the reaction mixture was filtered through diatomaceous earth to remove insoluble aluminum salts. The layers were separated, the aqueous layer was re-extracted with EtOAc (3 × 30 mL), the combined organic layers were washed with saturated NaCl aqueous solution (20 mL), dried (MgSO4), filtered, and concentrated under vacuum. The crude product was purified by rapid chromatography (silica, 130% EtOAc / n-heptane) to give a colorless oily (3aS,7R,7aR)-7-hydroxy-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridine-5-carboxylic acid 9H-fluorene-9-ylmethyl ester (113, 1.99 g, 5.03 mmol, two-step yield 76%).

[0932] LC-MS (Method D):

[0933] R t [min](UV-signal 220nm): 1.35

[0934] M [g / mol]: 338.1 [M-C3H6O+H + ]

[0935] Example 9.6: Synthesis of (3aS,7R,7aS)-2,2-dimethyl-7-methylsulfonyloxy-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridine-5-carboxylic acid 9H-fluorene-9-ylmethyl ester (114)

[0936] A solution of (3aS,7R,7aR)-7-hydroxy-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridine-5-carboxylic acid 9H-fluorene-9-ylmethyl ester (113, 1.99 g, 5.03 mmol, 1.00 equivalent) in anhydrous DCM (50 mL) was cooled to 0 °C and pyridine (1.30 mL, 16.80 mmol, 3.34 equivalent) and methanesulfonic anhydride (1.49 g, 8.54 mmol, 1.70 equivalent) were added. The reaction mixture was stirred at 0 °C for 3 hours until LC / MS indicated complete conversion of the starting material. The layers were separated by adding 1N HCl aqueous solution (100 mL) and EtOAc (250 mL). The organic layer was washed with saturated NaCl aqueous solution (50 mL), dried (MgSO4), filtered, and concentrated under vacuum. The crude product (3aS,7R,7aS)-2,2-dimethyl-7-methylsulfonyloxy-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridine-5-carboxylic acid 9H-fluorene-9-ylmethyl ester (114) was used directly in the next step.

[0937] LC-MS (Method D):

[0938] R t [min](UV-signal 220nm): 1.57

[0939] M [g / mol]: 474.0 [M+H] + ]

[0940] Example 9.7: Synthesis of (3aS,7S,7aR)-7-azido-2,2-dimethyl-3a,4,5,6,7,7a-hexahydro-[1,3]dioxacyclopenteno[4,5-c]pyridine (115)

[0941] The crude product (3aS,7R,7aS)-2,2-dimethyl-7-methylsulfonyloxy-4,6,7,7a-tetrahydro-3aH-[1,3]dioxanepenteno[4,5-c]pyridine-5-carboxylic acid 9H-fluorene-9-ylmethyl ester (114, maximum 5.03 mmol, 1.00 equivalent) was dissolved in DMF (10 mL), and NaN3 (1.32 g, 20.26 mmol, 4.03 equivalent) and 15-crown-5 ether (1.67 g, 7.60 mmol, 1.51 equivalent) were added. The mixture was stirred at 100 °C for 1 day. LC / MS indicated completely unexpected Fmoc deprotection and formation of the azide (3aS,7S,7aR)-7-azido-2,2-dimethyl-3a,4,5,6,7,7a-hexahydro-[1,3]dioxacyclopenteno[4,5-c]pyridine (115). EtOAc (100 mL) and water (50 mL) were added to separate the layers, and the aqueous layer was re-extracted with EtOAc (3 × 10 mL). The combined organic layers were washed with a saturated aqueous solution of NaCl (20 mL), dried (MgSO4), filtered, and concentrated under vacuum. The crude product was purified by rapid chromatography (silica, 1100% EtOAc / n-heptane) to give (3aS,7S,7aR)-7-azido-2,2-dimethyl-3a,4,5,6,7,7a-hexahydro-[1,3]dioxacyclopenteno[4,5-c]pyridine (115) (857 mg, 4.32 mmol, 85%) as a colorless oil.

[0942] LC-MS (Method D):

[0943] R t [min](TIC signal): 0.37

[0944] M [g / mol]: 199.1 [M+H] + ]

[0945] Example 9.8: Synthesis of (3aS,7S,7aR)-7-azido-5-(6-benzyloxyhexyl)-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridine (116)

[0946] (3aS,7S,7aR)-7-azido-2,2-dimethyl-3a,4,5,6,7,7a-hexahydro-[1,3]dioxacyclopenteno[4,5-c]pyridine (115,570 mg, 2.88 mmol, 1.00 equivalent) was dissolved in DMF (5 mL), and NaI (665 mg, 4.44 mmol, 1.54 equivalent), K₂CO₃ (2.00 g, 14.45 mmol, 5.02 equivalent), and 6-benzyloxyhexyl methanesulfonate (92, 1.06 g, 3.69 mmol, 1.28 equivalent) were added. The mixture was stirred at room temperature for 3 days. EtOAc (100 mL) and water (50 mL) were added, the layers were separated, and the aqueous layer was re-extracted with EtOAc (3 × 20 mL). The combined organic layers were washed with saturated NaCl aqueous solution (30 mL), dried (MgSO4), filtered, and concentrated under vacuum. The crude product was purified by rapid chromatography (silica, 0.40% EtOAc / n-heptane) to give a colorless oily (3aS,7S,7aR)-7-azido-5-(6-benzyloxyhexyl)-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridine (116,345 mg, 0.88 mmol, 31%).

[0947] LC-MS (Method D):

[0948] R t [min](UV signal 220nm): 1.14

[0949] M [g / mol]: 389.2 [M+H] + ]

[0950] Example 9.9: Synthesis of N-[(3S,4R,5S)-1-(6-benzyloxyhexyl)-4,5-dihydroxy-3-piperidinyl]acetamide (117)

[0951] PMe3 (1N in THF, 0.40 mL, 0.40 mmol, 1.75 equivalent) was added to a solution of (3aS,7S,7aR)-7-azido-5-(6-benzyloxyhexyl)-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridine (116, 86 mg, 0.221 mmol, 1.00 equivalent) in THF (5 mL) and water (0.1 mL), and the reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under vacuum as LC / MS indicated complete conversion of the starting material. The residue was dissolved in EtOAc (5 mL), and acetic anhydride (0.20 mL) and pyridine (0.10 mL) were added at room temperature, and the reaction mixture was stirred for 1 hour. The reaction mixture was concentrated under vacuum as LC / MS indicated complete formation of acetamide. The crude product was dissolved in acetic acid (4 mL) and water (1 mL) and heated to 80 °C for 3 hours until the acetone was completely deprotected by LC / MS. The solvent was removed under vacuum and the crude mixture was purified by HPLC (15 min, 590% acetonitrile / water + 0.1% TFA) to give N-[(3S,4R,5S)-1-(6-benzyloxyhexyl)-4,5-dihydroxy-3-piperidinyl]acetamide (117, TFA-salt, 65 mg, 0.138 mmol, 62%) as a colorless solid.

[0952] LC-MS (Method D):

[0953] R t [min](UV signal 220nm): 0.80

[0954] M[g / mol]:365.2[M+H + ]

[0955] 1 H NMR (400MHz, DMSO-d6) δ [ppm]: 9.55 (s, br, 1H), 7.93 (d, J==7.9Hz, 1H), 7.407.24 (m, 5H), 5.605.35 (m, 2H), 4.44 (s, 2H), 4.123.9 8(m, 1H), 3.803.71(m, 2H), 3.513.37(m, 2H), 3.213.03(m, 4H), 2.942.70(m, 2H), 1.86(s, 3H), 1.681.49(m, 4H), 1.411.21(m, 4H).

[0956] Example 9.10: Synthesis of (3aS,7S,7aR)-7-[(6-methoxy-6-oxo-hexanoyl)amino]-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopentenano[4,5-c]pyridine-5-carboxylic acid benzyl ester (118)

[0957] PMe3 (1N in THF, 0.41 mL, 0.41 mmol, 1.50 equivalent) was added to a solution of (3aS,7S,7aR)-7-hydroxy-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridine-5-carboxylic acid benzyl ester (81,90 mg, 0.27 mmol, 1.00 equivalent) in THF (5 mL) and water (0.1 mL), and the reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under vacuum as LC / MS indicated complete conversion of the starting material. The residue was dissolved in EtOAc (20 mL) and used as a stock solution for parallel reactions. Using 8.00 mL of this stock solution (0.11 mmol), methyl 5-chloro-5-oxo-valerate (108, 39 mg, 0.22 mmol, 2.00 equivalents) and pyridine (0.05 mL, about 6 equivalents) were added at room temperature, and the reaction mixture was stirred for 1 hour. Since LC / MS indicated complete formation of acetamide, the reaction mixture was concentrated under vacuum. The crude product (3aS,7S,7aR)-7-[(6-methoxy-6-oxo-hexanoyl)amino]-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridine-5-carboxylate (118) was used directly for the next step. LC-MS (Method D):

[0958] R t [min](UV-signal 220nm): 1.21

[0959] M [g / mol]: 449.2 [M+H] + ]

[0960] Example 9.11: Synthesis of (3S,4R,5S)-3,4-dihydroxy-5-[(6-methoxy-6-oxo-hexanoyl)amino]piperidine-1-carboxylic acid benzyl ester (119)

[0961] The crude product (3aS,7S,7aR)-7-[(6-methoxy-6-oxo-hexanoyl)amino]-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridine-5-carboxylic acid benzyl ester (118, maximum 0.11 mmol, 1.00 equivalent) was dissolved in acetic acid (4 mL) and water (1 mL) and heated to 80 °C for 3 hours until the acetone was completely deprotected as monitored by LC / MS. The solvent was removed under vacuum, and the crude mixture was purified by HPLC (15 min, 1060% acetonitrile / water + 0.1% TFA) to give (3S,4R,5S)-3,4-dihydroxy-5-[(6-methoxy-6-oxo-hexanoyl)amino]piperidine-1-carboxylic acid benzyl ester (119, 32 mg, 0.08 mmol, two-step yield 72%) as a colorless solid.

[0962] LC-MS (Method D):

[0963] R t [min](UV signal 220nm): 0.99

[0964] M [g / mol]: 409.2 [M+H] + ]

[0965] 1 H NMR (400MHz, DMSO-d6) δ [ppm]: 7.69 (m, 1H), 7.417.27 (m, 5H), 5.07 (d, J = 12.8Hz, 1 H), 5.03 (d, J=12.8Hz, 1H), 4.74 (d, J==4.5Hz, 1H), 4.69 (s, br, 1H), 3.84 (m, 1H), 3 .78 3.59 (m, 3H), 3.57 (s, 3H), 3.48 3.38 / 3.24 3.15 (m, 2H), 3.12 3.00 / 2.84 2.74 (m, 1H), 2.34 2.24 (m, 2H), 2.12 2.04 (m, 2H), 1.54 1.42 (m, 4H) (two major conformational isomers resulting from amide resonance).

[0966] Example 9.12: Synthesis of methyl 6-[[(3S,4R,5S)-1-acetyl-4,5-dihydroxy-3-piperidinyl]amino]-6-oxo-hexanoate (120)

[0967] A solution of (3S,4R,5S)-3,4-dihydroxy-5-[(6-methoxy-6-oxo-hexanoyl)amino]piperidine-1-carboxylic acid benzyl ester (119, 25 mg, 0.061 mmol, 1.00 equivalent) in EtOH (10 mL) was hydrogenated in an H-Cube (10% Pd(OH)2 / C, all-hydrogen mode, 60 °C, flow rate 1 mL / min). Complete hydrogenation was detected after three cycles. The solvent was removed under vacuum, and the crude product was dissolved in EtOAc (10 mL). Acetic anhydride (0.02 mL, about 4 equivalents) and pyridine (0.02 mL, about 4 equivalents) were added at room temperature, and the reaction mixture was stirred for 1 hour. Since LC / MS indicated complete formation of acetamide, the reaction was terminated by adding water (1 mL) and the mixture was concentrated under vacuum. The crude mixture was purified by HPLC (15 min, 252% acetonitrile / water + 0.1% TFA) to give methyl 6-[[(3S,4R,5S)-1-acetyl-4,5-dihydroxy-3-piperidinyl]amino]-6-oxo-hexanoate (120, 5 mg, 0.016 mmol, 26%) as a colorless solid.

[0968] LC-MS (Method D):

[0969] R t [min](UV signal 220nm): 0.48

[0970] M [g / mol]: 317.2 [M+H] + ]

[0971] 1 ¹H NMR (400MHz, DMSO-d⁶) δ [ppm]: 7.80 / 7.59 (d, J = 7.5 / 8.2Hz, 1H), 4.93-4.47 (m, 2H), 4.09-4.02 / 3.85-3.78 (m, 1H), 3.78-3.70 (m, 1H), 3.68-3.54 (m, 1H), 3.58 (s, 3H), 3.53-3.08 (m, 4H), 2.34-2.27 (1n, 2H), 2.14-2.04 (m, 2H), 1.98 / 1.91 (s, 3H), 1.55-1.45 (m, 4H) (two major conformational isomers due to amide resonance).

[0972] Example 9.13: Synthesis of (3S,4R,5S)-3-acetamido-4,5-dihydroxy-piperidine-1-carboxylic acid benzyl ester (121)

[0973] PMe3 (1N in THF, 0.41 mL, 0.41 mmol, 1.50 equivalence) was added to a solution of (3aS,7S,7aR)-7-hydroxy-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridine-5-carboxylic acid benzyl ester (81,90 mg, 0.27 mmol, 1.00 equivalence) in THF (5 mL) and water (0.1 mL), and the reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under vacuum as LC / MS indicated complete conversion of the starting material. The residue was dissolved in EtOAc (20 mL) and used as a stock solution for parallel reactions. Using 8.00 mL of this stock solution (0.11 mmol), acetic anhydride (0.10 mL, about 10 equivalences) and pyridine (0.05 mL, about 6 equivalences) were added at room temperature, and the reaction mixture was stirred for 1 hour. Since LC / MS indicated complete formation of acetamide, the reaction mixture was concentrated under vacuum. The crude product was dissolved in acetic acid (4 mL) and water (1 mL) and heated to 80 °C and maintained for 3 h until complete deprotection of the acetone compound was detected by LC / MS. The solvent was removed under vacuum, and the crude mixture was purified by HPLC (15 min, 150% acetonitrile / water + 0.1% TFA) to give (3S,4R,5S)-3-acetamido-4,5-dihydroxy-piperidine-1-carboxylic acid benzyl ester (121, 27 mg, 0.087 mmol, 80%) as a colorless solid.

[0974] LC-MS (Method D):

[0975] R t [min](UV signal 220nm): 0.81

[0976] M [g / mol]: 309.2 [M+H] + ]

[0977] 1 ¹H NMR (400MHz, DMSO-d⁶) δ [ppm]: 7.75 (m, 1H), 7.40-7.27 (m, 5H), 5.14-4.20 (s, br, 2H), 5.08 (d, J = 12.8Hz, 1H), 5.03 (d, J = 12.8Hz, 1H), 3.83 (m, 1H), 3.79-3.57 (m, 3H), 3.52-3.15 (m, 2H), 3.10-2.99 / 2.85-2.74 (m, 1H), 1.80 (s, 3H), (two major conformational isomers due to amide resonance).

[0978] Example 10: Synthesis of compounds 128, 129, 131 and 132.

[0979]

[0980] Example 10.1: Synthesis of 1-[(3aS,7R,7aR)-7-hydroxy-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridin-5-yl]-6-benzyloxy-hexyl-1-one (122) and benzoic acid [6-[(3aS,7R,7aR)-7-hydroxy-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridin-5-yl]-6-oxo-hexyl] ester (123, an inseparable mixture)

[0981] To a solution of (3aR,7R,7aR)-7-hydroxy-2,2-dimethyl-5,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridin-4-one (88 g, 0.93 g, 4.97 mmol, 1.00 equivalent) in anhydrous THF (100 mL), LiAlH4 (15% in toluene / THF, 3.5 M, 5.00 mL, 17.50 mmol, 3.52 equivalent) was added. The reaction mixture was stirred overnight at room temperature and saturated aqueous solution of NaHCO3 (50 mL) and water (20 mL) were added. The crude product (3aS,7R,7aR)-2,2-dimethyl-3a,4,5,6,7,7a-hexahydro-[1,3]dioxacyclopenteno[4,5-c]pyridine-7-ol (78) was used directly as a solution of THF / saturated NaHCO3 aqueous solution with added acyl chloride 6-benzyloxyhexanoyl chloride (95) / 6-benzoyloxyhexanoyl chloride (96) (an inseparable mixture, 2.36 g, 9.81 mmol, 1.97 equivalents) in THF (10 mL). The reaction mixture was stirred at room temperature for 6 hours. EtOAc (200 mL) and water (50 mL) were added, and the reaction mixture was filtered through diatomaceous earth to remove insoluble aluminum salts. The layers were separated, and the organic layer was washed with 2N NaOH aqueous solution (3 × 30 mL) and saturated NaCl aqueous solution (30 mL), dried (MgSO4), filtered, and concentrated under vacuum. The crude product was purified by rapid chromatography (silica, 5100% EtOAc / n-heptane) to give a colorless oily mixture of 1-[(3aS,7R,7aR)-7-hydroxy-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridin-5-yl]-6-benzyloxy-hexyl-1-one (122) and benzoic acid [6-[(3aS,7R,7aR)-7-hydroxy-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridin-5-yl]-6-oxo-hexyl] ester (123, an inseparable mixture, 0.73 g, 1.93 mmol, two-step yield 39%).

[0982] LC-MS (Method D):

[0983] R t [min](UV-signal 220nm): 1.19

[0984] M [g / mol]: 378.2 [M+H] + ](122, major product), 392.1[M+H + (123, secondary products)

[0985] Example 10.2: Synthesis of methanesulfonic acid [(3aS,7R,7aS)-5-(6-benzyloxyhexanoyl)-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridin-7-yl] ester (124) and benzoic acid [6-[(3aS,7R,7aS)-2,2-dimethyl-7-methylsulfonyloxy-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridin-5-yl]-6-oxo-hexyl] ester (125, an inseparable mixture)

[0986] 1-[(3aS,7R,7aR)-7-hydroxy-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxanepenteno[4,5-c]pyridin-5-yl]-6-benzyloxy-hexane-1-one (122) and benzoic acid [6-[(3aS,7R,7aR)-7-hydroxy-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxanepenteno[4,5-c]pyridin-5-yl]-6-benzyloxy-hexane-1-one (122) were mixed with benzoic acid [6-[(3aS,7R,7aR)-7-hydroxy-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxanepenteno[4,5-c]pyridin-5-yl]-6-benzyloxy-1-one (122) A solution of oxacyclopenteno[4,5-c]pyridin-5-yl]-6-oxo-hexyl] ester (123, an indivisible mixture, 0.73 g, 1.93 mmol, 1.00 equivalent) in anhydrous DCM (10 mL) was cooled to 0 °C, and pyridine (0.40 mL, 4.92 mmol, 2.54 equivalent) and methanesulfonic anhydride (0.57 g, 3.27 mmol, 1.69 equivalent) were added. The reaction mixture was stirred at 0 °C for 3 hours until LC / MS indicated complete conversion of the starting material. The layers were separated by adding 1 N HCl aqueous solution (30 mL) and EtOAc (50 mL). The organic layer was washed with saturated NaCl aqueous solution (30 mL), dried (MgSO4), filtered, and concentrated under vacuum. A mixture of crude products of methanesulfonic acid [(3aS,7R,7aS)-5-(6-benzyloxyhexanoyl)-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridin-7-yl] ester (124) and benzoic acid [6-[(3aS,7R,7aS)-2,2-dimethyl-7-methylsulfonyloxy-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridin-5-yl]-6-oxo-hexyl] ester (125) was used directly for the next step.

[0987] LC-MS (Method D):

[0988] R t [min](UV signal 220nm): 1.43

[0989] M [g / mol]: 456.2 [M+H] + ](124, major product), 470.2[M+H+ (125, secondary product)

[0990] Example 10.3: Synthesis of 1-[(3aS,7S,7aR)-7-azido-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridin-5-yl]-6-benzyloxy-hexyl-1-one (126) and benzoic acid [6-[(3aS,7S,7aR)-7-azido-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridin-5-yl]-6-oxo-hexyl] ester (127, an inseparable mixture)

[0991] The methanesulfonic acid [(3aS,7R,7aS)-5-(6-benzyloxyhexanoyl)-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxanepenten[4,5-c]pyridin-7-yl] ester (124) and benzoic acid [6-[(3aS,7R,7aS)-2,2-dimethyl-7-methylsulfonyloxy-4,6,7,7a-tetrahydro-3aH-[1,3]dioxane] A crude mixture of penteno[4,5-c]pyridin-5-yl]-6-oxo-hexyl] ester (125) (maximum 1.93 mmol, 1.00 equivalent) was dissolved in DMF (5 mL), and NaN3 (0.45 g, 6.97 mmol, 4.01 equivalent) and 15-crown-5 ether (0.60 g, 2.73 mmol, 1.57 equivalent) were added, and the mixture was stirred at 100 °C for 1 day. EtOAc (50 mL) and water (50 mL) were added, the layers were separated, and the aqueous layer was re-extracted with EtOAc (3 × 10 mL). The combined organic layers were washed with a saturated aqueous solution of NaCl (20 mL), dried (MgSO4), filtered, and concentrated under vacuum. The crude product was purified by rapid chromatography (silica, 180% EtOAc / n-heptane) to give a colorless oil of 1-[(3aS,7S,7aR)-7-azido-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridin-5-yl]-6-benzyloxy-hexyl-1-one (126) and benzoic acid [6-[(3aS,7S,7aR)-7-azido-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridin-5-yl]-6-oxo-hexyl] ester (127, inseparable mixture, 189 mg, 0.47 mmol, two-step yield 27%). LC-MS (Method D):

[0992] R t [min](UV-signal 220nm): 1.48

[0993] M [g / mol]: 403.2 [M+H] + ](126, major product), 417.2[M+H + (127, secondary products)

[0994] Example 10.4: Synthesis of N-[(3S,4R,5S)-1-(6-benzyloxyhexanoyl)-4,5-dihydroxy-3-piperidinyl]acetamide (128) and N-[(3S,4R,5S)-4,5-dihydroxy-1-(6-hydroxyhexanoyl)-3-piperidinyl]acetamide (129)

[0995] To 1-[(3aS,7S,7aR)-7-azido-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxanepenteno[4,5-c]pyridin-5-yl]-6-benzyloxy-hex-1-one (126) and benzoic acid [6-[(3aS,7S,7aR)-7-azido-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxanepenteno[4,5-c]pyridin-5-yl]-6-benzyloxy-hex-1-one (126) and benzoic acid [1 A mixture of [3]dioxacyclopenteno[4,5-c]pyridin-5-yl]-6-oxo-hexyl] ester (127, 95 mg, 0.24 mmol, 1.00 equivalent) was added to a solution of THF (5 mL) and water (0.1 mL), along with PMe3 (1 N in THF, 0.41 mL, 0.41 mmol, 1.50 equivalent), and the reaction mixture was stirred at room temperature for 1 hour. Since LC / MS indicated complete conversion of the starting material, the reaction mixture was concentrated under vacuum. The residue was dissolved in EtOAc (20 mL), and acetic anhydride (0.09 mL, approximately 4 equivalents) and pyridine (0.05 mL, approximately 2.5 equivalents) were added, and the reaction mixture was stirred at room temperature for 1 hour. Since LC / MS indicated complete formation of acetamide, the reaction mixture was concentrated under vacuum. The crude product was dissolved in methanol (6 mL) and 1 N HCl aqueous solution (3 mL), and heated to 45 °C and maintained for 5 hours until complete deprotection of the acetone compound was observed by LC / MS. The solvent was removed under vacuum, and the crude mixture was dissolved in methanol (10 mL) with the addition of solid NaOH (0.04 g, 1.00 mmol, 4.17 mmol). The reaction mixture was stirred until complete transesterification of the benzoyl ester was observed by LC / MS. Acetic acid (0.10 mL) was added, the solvent was removed under vacuum, and the crude mixture was purified by HPLC (15 min, 150% acetonitrile / water + 0.1% TFA) to give N-[(3S,4R,5S)-1-(6-benzyloxyhexanoyl)-4,5-dihydroxy-3-piperidinyl]acetamide (128, 15 mg, 0.040 mmol, 17%) and N-[(3S,4R,5S)-4,5-dihydroxy-1-(6-hydroxyhexanoyl)-3-piperidinyl]acetamide (129, 15 mg, 0.052 mmol, 22%) as colorless solids.

[0996] N-[(3S,4R,5S)-1-(6-benzyloxyhexanoyl)-4,5-dihydroxy-3-piperidinyl]acetamide (128):

[0997] LC-MS (Method D):

[0998] R t [min](UV signal 220nm): 0.93

[0999] M [g / mol]: 379.2 [M+H]+ ]

[1000] 1 ¹H NMR (400MHz, DMSO-d⁶) δ [ppm]: 7.82 / 7.64 (d, J = 7.4 / 8.2Hz, 1H), 7.41-7.23 (m, 5H), 4.78 / 4.73 (d, J = 4.9 / 3.7Hz, 1H), 4.68 / 4.65 (d, J = 5.1 / 5.7Hz, 1H), 4.44 (s, 2H), 4.14-3.24 (m, 8H), 3.16-3.04 (m, 1H), 2.38-2.12 (m, 2H), 1.82 / 1.81 (s, 3H), 1.59-1.43 (m, 4H), 1.37-1.22 (m, 2H) (two main conformational isomers due to amide resonance)

[1001] N-[(3S,4R,5S)-4,5-dihydroxy-1-(6-hydroxyhexanoyl)-3-piperidinyl]acetamide (129):

[1002] R t [min](UV signal 220nm): 0.18

[1003] M [g / mol]: 289.1 [M+H] + ]

[1004] 1 ¹H NMR (400MHz, DMSO-d⁶) δ [ppm]: 7.82 / 7.64 (d, J = 7.3 / 7.9Hz, 1H), 4.69 (s, 2H), 4.41 4.35 / 4.13 4.06 (m, 1H), 3.82 3.04 (m, 9H), 2.35–2.11 (m, 2H), 1.82 / 1.81 (s, 3H), 1.59 1.18 (m, 6H) (two main conformational isomers due to amide resonance)

[1005] Example 10.5: 6-benzyloxy-1-[(3S,4R,5S)-3,4-dihydroxy-5-[4-(phenoxy-methyl)triazol-1-yl]-1-piperidinyl]hex-1-one (131) and 1-[(3S,4R,5S)-3,4-dihydroxy-5-[4-(phenoxy-methyl)triazol-1-yl]-1-piperidinyl]-6-hydroxy-hex-1-one (132)

[1006] To 1-[(3aS,7S,7aR)-7-azido-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridin-5-yl]-6-benzyloxy-hex-1-one (126) and benzoic acid [6-[(3aS,7S,7aR)-7-azido-2,2-dimethyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridin-5-yl]-6-oxo-hexyl] ester (127, 107 mg, 0.26 mmol) In a methanol (4 mL) solution, (prop-2-yn-1-yloxy)benzene (130 mg, 42 mg, 0.32 mmol, 1.20 equivalent), tris-(2-(1-benzyl-1H-1,2,3-triazol-4-yl)ethyl)amine (TBTA, 8 mg, 0.01 mmol, 0.05 equivalent), copper(II) acetate (9 mg, 0.05 mmol, 0.18 equivalent), and sodium ascorbate (529 mg, 2.67 mmol, 10.08 equivalent) were added, and the reaction mixture was stirred at room temperature for 1 hour. Since LC / MS indicated complete conversion of the starting material, EtOAc (40 mL) and water (30 mL) were added, the layers were separated, and the aqueous layer was re-extracted with EtOAc (3 × 10 mL). The combined organic layers were washed with saturated NaCl aqueous solution (20 mL), dried (MgSO4), filtered, and concentrated under vacuum.

[1007] The crude mixture was dissolved in methanol (10 mL) and solid NaOH (0.04 g, 1.00 mmol, 4.17 mmol) was added. The reaction mixture was stirred until complete transesterification of benzoyl ester was observed by LC / MS. Acetic acid (0.10 mL) was added, the solvent was removed under vacuum, and the crude mixture was purified by HPLC (15 min, 150% acetonitrile / water + 0.1% TFA) to give 6-benzyloxy-1-[(3S,4R,5S)-3,4-dihydroxy-5-[4-(phenoxy-methyl)triazol-1-yl]-1-piperidinyl]hex-1-one (131, 47 mg, 0.094 mmol, 36%) and 1-[(3S,4R,5S)-3,4-dihydroxy-5-[4-(phenoxy-methyl)triazol-1-yl]-1-piperidinyl]-6-hydroxy-hex-1-one (132, 22 mg, 0.054 mmol, 21%) as colorless solids.

[1008] 6-Benzyloxy-1-[(3S,4R,5S)-3,4-dihydroxy-5-[4-(phenoxy-methyl)triazol-1-yl]-1-piperidinyl]hex-1-one (131): LC-MS (Method D):

[1009] R t[min](UV signal 220nm): 1.28

[1010] M [g / mol]: 495.3 [M+H] + ]

[1011] 1 ¹H NMR (400MHz, DMSO-d6) δ [ppm]: 8.28 / 8.27 (s, 1H), 7.38-7.24 (m, 7H), 7.07-7.02 (m, 2H), 6.96 (m, 1H), 5.32-4.81 (m, 2H), 5.13 (s, 2H), 4.72-4.40 (m, 4H), 4.12-3.84 (m, 3H), 3.53-3.25 (m, 3H), 3.07-2.82 (m, 1H), 2.42-2.29 (m, 2H), 1.60-1.44 (m, 4H), 1.39-1.28 (m, 2H) (two major conformational isomers due to amide resonance).

[1012] 1-[(3S,4R,5S)-3,4-dihydroxy-5-[4-(phenoxymethyl)triazol-1-yl]-1-piperidinyl]-6-hydroxy-hexane-1-one (132):

[1013] LC-MS (Method D):

[1014] R t [min](UV-signal 220nm): 0.85

[1015] M [g / mol]: 405.3 [M+H] + ]

[1016] 1 ¹H NMR (400MHz, DMSO-d6) δ [ppm]: 8.28 / 8.28 (s, 1H), 7.97-7.92 / 7.08-7.02 (m, 2H), 7.66-7.59 / 6.99-6.93 (m, 1H), 7.53-7.47 / 7.34-7.27 (m, 2H), 5.20-4.91 (m, 4H), 4.73-4.27 (m, 3H), 4.14-3.81 (m, 3H), 3.54-3.25 (m, 3H), 3.08-2.81 (m, 1H), 2.43-2.29 (m, 2H), 1.57-1.36 (m, 4H), 1.36-1.20 (m, 2H) (two major conformational isomers due to amide resonance).

[1017] Example 11: Synthesis of compounds 138 and 140.

[1018]

[1019] Example 11.1: Synthesis of 6-benzyloxyhexyl 4-methylbenzenesulfonic acid (133)

[1020] Pyridine (1.94 mL, 24.00 mmol, 5.00 equivalence) and toluenesulfonic anhydride (3.92 g, 12.00 mmol, 2.50 equivalence) were added to a solution of 6-benzyloxyhexyl-1-ol (91, 1.00 g, 4.80 mmol, 1.00 equivalence) in anhydrous DCM (30 mL). The reaction mixture was stirred at room temperature for 3 hours until LC / MS indicated complete conversion of the starting material. Water (50 mL) was added, the layers were separated, and the aqueous layer was re-extracted with DCM (3 × 40 mL). The combined organic layers were dried (Na₂SO₄), filtered, and concentrated under vacuum. The crude product was purified by rapid chromatography (silica, 10% EtOAc / n-heptane) to give 6-benzyloxyhexyl 4-methylbenzenesulfonic acid ester (133, 1.10 g, 3.03 mmol, 63%) as a colorless oil.

[1021] LC-MS (Method E):

[1022] R t [min](UV-signal 214nm): 2.02

[1023] M [g / mol]: 363.5 [M+H] + ]

[1024] Example 11.2: Synthesis of (3aR,7R,7aS)-5-(6-benzyloxyhexyl)-7-[tert-butyl(dimethyl)silyl]oxy-2,2-dimethyl-3a,6,7,7a-tetrahydro-[1,3]dioxacyclopentenano[4,5-c]pyridin-4-one (134)

[1025] A solution of (3aR,7R,7aS)-7-[tert-butyl(dimethyl)silyl]oxy-2,2-dimethyl-5,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridin-4-one (89 g, 1.00 g, 3.32 mmol, 1.00 equivalent) in anhydrous THF (50 mL) was cooled to 0 °C and sodium hydride (60% in mineral oil, 528 mg, 13.20 mmol, 3.98 equivalent) was added in small amounts. The reaction mixture was stirred at 0 °C for 10 minutes and 6-benzyloxyhexyl 4-methylbenzenesulfonate (133 g, 1.40 g, 3.86 mmol, 1.16 equivalent) was added. The reaction mixture was stirred at 80 °C for 5 hours. Water (50 mL) and EtOAc (40 mL) were added to separate the layers, and the aqueous layer was extracted again with EtOAc (2 × 40 mL). The combined organic layers were washed with saturated NaCl aqueous solution (40 mL), dried (Na28O4), filtered, and concentrated under vacuum. The crude product was purified by rapid chromatography (silica, 16% EtOAc / DCM) to give a colorless oily (3aR,7R,7aS)-5-(6-benzyloxyhexyl)-7-[tert-butyl(dimethyl)silyl]oxy-2,2-dimethyl-3a,6,7,7a-tetrahydro-[1,3]dioxacyclopenteno[4,5-c]pyridin-4-one (134,723 mg, 1.47 mmol, 44%).

[1026] LC-MS (Method F):

[1027] R t [min](UV-signal 214nm): 2.39

[1028] M [g / mol]: 491.9 [M+H] + ]

[1029] 1 H NMR (400MHz, DMSO-d6) δ [ppm]: 7.377.24 (m, 5H), 4.43 (s, 2H), 4.394.33 (m, 2H), 4.15 (m, 1H), 3.453.32 (m, 4H ), 3.183.03(m, 2H), 1.581.37(m, 4H), 1.371.16(m, 5H), 1.31(s, 3H), 1.29(s, 3H), 0.86(s, 9H), 0.09(s, 6H).

[1030] Example 11.3: Synthesis of (3aR,7R,7aR)-5-(6-benzyloxyhexyl)-7-hydroxy-2,2-dimethyl-3a,6,7,7a-tetrahydro-[1,3]dioxacyclopenteno[4,5-c]pyridin-4-one (135)

[1031] Because hydrofluoric acid was used, the reaction was carried out in a plastic bottle. 3HF·NEt3 (2.34 g, 14.53 mmol, 9.88 equivalents) was added to a solution of (3aR,7R,7aS)-5-(6-benzyloxyhexyl)-7-[tert-butyl(dimethyl)silyl]oxy-2,2-dimethyl-3a,6,7,7a-tetrahydro-[1,3]dioxacyclopenteno[4,5-c]pyridin-4-one (134,723 mg, 1.47 mmol, 1.00 equivalents) in acetonitrile (10 mL), and the solution was stirred at room temperature for 16 hours until LC / MS indicated complete conversion. EtOAc (150 mL) and a saturated aqueous solution of NaHCO3 (50 mL) were added, the layers were separated, and the aqueous layer was re-extracted with EtOAc (2 × 20 mL). The combined organic layers were washed with a saturated aqueous solution of NaCl (30 mL), dried (MgSO4), filtered, and concentrated under vacuum. The crude product (3aR,7R,7aR)-5-(6-benzyloxyhexyl)-7-hydroxy-2,2-dimethyl-3a,6,7,7a-tetrahydro-[1,3]dioxacyclopenteno[4,5-c]pyridin-4-one (135,492 mg, 1.30 mmol, 89%) was obtained as a colorless oil with sufficient purity for the next step.

[1032] LC-MS (Method D):

[1033] R t [min](UV signal 220nm): 0.76

[1034] M [g / mol]: 378.2 [M+H] + ]

[1035] Example 11.4: Synthesis of methanesulfonic acid [(3aR,7R,7aS)-5-(6-benzyloxyhexyl)-2,2-dimethyl-4-oxo-3a,6,7,7a-tetrahydro-[1,3]dioxacyclopenteno[4,5-c]pyridin-7-yl] ester (136)

[1036] A solution of (3aR,7R,7aR)-5-(6-benzyloxyhexyl)-7-hydroxy-2,2-dimethyl-3a,6,7,7a-tetrahydro-[1,3]dioxacyclopenteno[4,5-c]pyridin-4-one (135,492 mg, 1.30 mmol, 1.00 equivalent) in anhydrous DCM (20 mL) was cooled to 0 °C, and pyridine (0.30 mL, 3.69 mmol, 2.83 equivalent) and methanesulfonic anhydride (0.40 g, 2.30 mmol, 1.77 equivalent) were added. The reaction mixture was stirred at 0 °C for 3 hours until LC / MS indicated complete conversion of the starting material. The layers were separated by adding 1 N HCl aqueous solution (30 mL) and EtOAc (50 mL). The organic layer was washed with saturated NaCl aqueous solution (30 mL), dried (MgSO4), filtered, and concentrated under vacuum. The crude product, methanesulfonic acid [(3aR,7R,7aS)-5-(6-benzyloxyhexyl)-2,2-dimethyl-4-oxo-3a,6,7,7a-tetrahydro-[1,3]dioxacyclopenteno[4,5-c]pyridin-7-yl] ester (136), was used directly in the next step.

[1037] LC-MS (Method D):

[1038] R t [min](UV signal 220nm): 1.42

[1039] M [g / mol]: 456.1 [M+H] + ]

[1040] Example 11.5: Synthesis of (3aR,7S,7aR)-7-azido-5-(6-benzyloxyhexyl)-2,2-dimethyl-3a,6,7,7a-tetrahydro-[1,3]dioxo[4,5-c]pyridin-4-one (137)

[1041] The crude product, methanesulfonic acid [(3aR,7R,7aS)-5-(6-benzyloxyhexyl)-2,2-dimethyl-4-oxo-3a,6,7,7a-tetrahydro-[1,3]dioxacyclopenteno[4,5-c]pyridin-7-yl] ester (136, maximum 1.30 mmol, 1.00 equivalent), was dissolved in DMF (5 mL), and NaN3 (0.34 g, 5.23 mmol, 4.00 equivalent) and 15-crown-5 ether (0.43 g, 1.96 mmol, 1.50 equivalent) were added. The mixture was stirred at 100 °C for 1 day. EtOAc (50 mL) and water (50 mL) were added, the layers were separated, and the aqueous layer was re-extracted with EtOAc (3 × 10 mL). The combined organic layers were washed with saturated NaCl aqueous solution (20 mL), dried (MgSO4), filtered, and concentrated under vacuum. The crude product was purified by rapid chromatography (silica, 1-40% EtOAc / n-heptane) to give a colorless oily (3aR,7S,7aR)-7-azido-5-(6-benzyloxyhexyl)-2,2-dimethyl-3a,6,7,7a-tetrahydro-[1,3]dioxo[4,5-c]pyridin-4-one (137, 135 mg, 0.34 mmol, two-step yield 26%).

[1042] LC-MS (Method D):

[1043] R t [min](UV signal 220nm): 1.60

[1044] M [g / mol]: 403.2 [M+H] + ]

[1045] Example 11.6: Synthesis of N-[(3S,4R,5R)-1-(6-benzyloxyhexyl)-4,5-dihydroxy-6-oxo-3-piperidinyl]acetamide (138)

[1046] PMe3 (1N in THF, 0.29 mL, 0.29 mmol, 1.50 equivalence) was added to a solution of (3aR,7S,7aR)-7-azido-5-(6-benzyloxyhexyl)-2,2-dimethyl-3a,6,7,7a-tetrahydro-[1,3]dioxo[4,5-c]pyridin-4-one (137.79 mg, 0.19 mmol, 1.00 equivalence) in THF (5 mL) and water (0.1 mL), and the reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under vacuum as LC / MS indicated complete conversion of the starting material. The residue was dissolved in EtOAc (20 mL), and acetic anhydride (0.09 mL, approximately 4 equivalences) and pyridine (0.05 mL, approximately 2.5 equivalences) were added, and the reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under vacuum as LC / MS indicated complete formation of acetamide. The crude product was dissolved in methanol (6 mL) and 1N HCl aqueous solution (3 mL), and heated to 45 °C and maintained for 5 hours until the acetone was completely deprotected as monitored by LC / MS. The solvent was removed under vacuum, and the crude mixture was purified by HPLC (15 min, 1095% acetonitrile / water + 0.1% TFA) to give N-[(3S,4R,5R)-1-(6-benzyloxyhexyl)-4,5-dihydroxy-6-oxo-3-piperidinyl]acetamide (138, 15 mg, 0.040 mmol, 17%) as a colorless solid.

[1047] LC-MS (Method D):

[1048] R t [min](UV signal 220nm): 1.04

[1049] M [g / mol]: 379.2 [M+H] + ]

[1050] 1 H NMR (400MHz, DMSO-d6) δ [ppm]: 8.09 (d, J=7.7Hz, 1H), 7.377.24 (m, 5H), 5.464.53 (m, 2H), 4.44 (s, 2H), 4.043.95 (m, 2H), 3.82 (m, 1H) , 3.56 (dd, J=12.7, 5.6Hz, 1H), 3.41 (t, J=6.6Hz, 2H), 3.393.09 (m, 1H), 2.97 (dd, J=12.8, 5.0Hz, 1H), 1.83 (s, 3H), 1.581.17 (m, 8H).

[1051] Example 11.7: Synthesis of (3aR,7S,7aR)-5-(6-benzyloxyhexyl)-2,2-dimethyl-7-[4-(phenoxymethyl)triazol-1-yl]-3a,6,7,7a-tetrahydro-[1,3]dioxacyclopentenano[4,5-c]pyridin-4-one (139)

[1052] To a solution of (3aR,7S,7aR)-7-azido-5-(6-benzyloxyhexyl)-2,2-dimethyl-3a,6,7,7a-tetrahydro-[1,3]dioxo[4,5-c]pyridin-4-one (137, 100 mg, 0.25 mmol, 1.00 equivalent) in methanol (4 mL), (prop-2-yn-1-yloxy)benzene (130, 39 mg, 0.30 mmol, 1.20 equivalent), tris-(2-(1-benzyl-1H-1,2,3-triazol-4-yl)ethyl)amine (TBTA, 8 mg, 0.01 mmol, 0.05 equivalent), copper(II) acetate (9 mg, 0.05 mmol, 0.18 equivalent), and sodium ascorbate (495 mg, 2.50 mmol, 10.00 equivalent) were added, and the reaction mixture was stirred at room temperature for 1 hour. Since LC / MS indicated complete conversion of the starting material, EtOAc (40 mL) and water (30 mL) were added to separate the layers, and the aqueous layer was re-extracted with EtOAc (3 × 10 mL). The combined organic layers were washed with saturated NaCl aqueous solution (20 mL), dried (MgSO4), filtered, and concentrated under vacuum.

[1053] The crude mixture was purified by HPLC (15 min, 1090% acetonitrile / water + 0.1% TFA) to obtain an acetonitrile-water mixture containing (3aR, 7S, 7aR)-5-(6-benzyloxyhexyl)-2,2-dimethyl-7-[4-(phenoxymethyl)triazol-1-yl]-3a,6,7,7a-tetrahydro-[1,3]dioxacyclopenteno[4,5-c]pyridin-4-one (139), which was directly used for acetone deprotection.

[1054] LC-MS (Method D):

[1055] R t [min](UV-signal 220nm): 1.65

[1056] M [g / mol]: 535.2 [M+H] + ]

[1057] Example 11.8: Synthesis of (3R,4R,5S)-1-(6-benzyloxyhexyl)-3,4-dihydroxy-5-[4-(phenoxymethyl)triazol-1-yl]piperidin-2-one (140)

[1058] The acetonitrile-water mixture containing the product (3aR,7S,7aR)-5-(6-benzyloxyhexyl)-2,2-dimethyl-7-[4-(phenoxymethyl)triazol-1-yl]-3a,6,7,7a-tetrahydro-[1,3]dioxacyclopenteno[4,5-c]pyridin-4-one (139) purified by HPLC was concentrated under vacuum to remove most of the acetonitrile. It was then redissolved in methanol (6 mL) and 1N HCl aqueous solution (3 mL) and heated to 45 °C and maintained for 16 hours until the acetone was completely deprotected by LC / MS. The solvent was removed under vacuum, and the crude mixture was purified by HPLC (15 min, 2090% acetonitrile / water + 0.1% TFA) to give (3R,4R,5S)-1-(6-benzyloxyhexyl)-3,4-dihydroxy-5-[4-(phenoxymethyl)triazol-1-yl]piperidin-2-one (140, 44 mg, 0.089 mmol, two-step yield 36%) as a colorless solid.

[1059] LC-MS (Method D):

[1060] R t [min](UV signal 220nm): 1.44

[1061] M [g / mol]: 495.2 [M+H] + ]

[1062] 1 H NMR (400MHz, DMSO-d6) δ [ppm]: 8.36 (s, 1H), 7.387.23 (m, 7H), 7.077.02 (m, 2H), 6.9 6 (m, 1H), 5.48 (s, br, 2H), 5.14 (s, 2H), 4.96 (m, 1H), 4.44 (s, 2H), 4.25 (dd, J=7.0, 3. 7Hz, 1H), 4.02 (d, J = 3.7Hz, 1H), 3.76 (dd, J = 12.8, 6.4Hz, 1H), 3.69 (dd, J = 12.8, 8.3Hz, 1H), 3.41 (t, J = 6.4Hz, 2H), 3.39 3.19 (m, 2H), 1.58 1.42 (m, 4H), 1.38 1.19 (m, 4H). Example 12: Synthesis of compounds 146, 147, 148, 153 and 154.

[1063]

[1064] Example 12.1: Synthesis of (3aR,6R,6aR)-4-allyl-6-(aminomethyl)-2,2-dimethyl-6,6a-dihydro-3aH-furano[3,4-d][1,3]dioxacyclopenten-4-ol (141)

[1065] A solution of (3aR,6R,6aR)-6-(aminomethyl)-2,2-dimethyl-6,6a-dihydro-3aH-furano[3,4-d][1,3]dioxane-4-one (87, 2.22 g, 10.43 mmol, 1.00 equivalent) in THF (50 mL) was cooled to 78 °C. Allyl magnesium chloride (1.7 M, 9.20 mL, 15.64 equivalent, 1.50 equivalent) was added, and the reaction mixture was stirred for 30 min until LC / MS indicated complete conversion of the starting material. Saturated NH4Cl aqueous solution (40 mL), EtOAc (50 mL), and water (50 mL) were added to separate the layers. The aqueous layer was re-extracted with EtOAc (3 × 50 mL). The combined organic layers were washed with saturated NaCl aqueous solution (30 mL), dried (MgSO4), filtered, and concentrated under vacuum. The crude product was purified by rapid chromatography (silica, 130% EtOAc / n-heptane) to yield a colorless oil, (3aR,6R,6aR)-4-allyl-6-(aminomethyl)-2,2-dimethyl-6,6a-dihydro-3aH-furano[3,4-d][1,3]dioxane-4-ol (141, 2.17 g, 8.49 mmol, 81%). LC-MS (Method D):

[1066] R t [min](UV-signal 220nm): 1.20

[1067] M [g / mol]: 238.1 [M-H2O+H + ], 210.1238.1[M-H2O-N2+H + ]

[1068] Example 12.2: Synthesis of (3aS,7R,7aR)-2,2-dimethyl-4-propyl-3a,4,5,6,7,7a-hexahydro-[1,3]dioxacyclopenteno[4,5-c]pyridine-7-ol (142)

[1069] (3aR,6R,6aR)-4-allyl-6-(aminomethyl)-2,2-dimethyl-6,6a-dihydro-3aH-furano[3,4-d][1,3]dioxane-4-ol (141,325 mg, 1.27 mmol, 1.00 equivalent) was dissolved in THF (20 mL), 10% Pd / C (0.07 g, 0.06 mmol, 0.05 equivalent) was added, and the mixture was hydrogenated in an autoclave at room temperature and 4 bar of hydrogen for 1 day. Since piperidine was completely formed as detected by LC / MS, the reaction mixture was filtered and concentrated under vacuum. The crude product (3aS,7R,7aR)-2,2-;methyl-4-propyl-3a,4,5,6,7,7a-hexahydro-[1,3]dioxacyclopenteno[4,5-c]pyridine-7-ol (142) was used directly in the solution of the next step.

[1070] LC-MS (Method D):

[1071] R t [min](TIC signal): 0.48

[1072] M [g / mol]: 216.1 [M+H] + ]

[1073] Example 12.3: Synthesis of benzyl 6-[(3aS,7R,7aR)-7-hydroxy-2,2-dimethyl-4-propyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopentenano[4,5-c]pyridin-5-yl]-6-oxo-hexanoate (143)

[1074] A solution of crude (3aS,7R,7aR)-2,2-dimethyl-4-propyl-3a,4,5,6,7,7a-hexahydro-[1,3]dioxacyclopenteno[4,5-c]pyridine-7-ol (142, maximum 1.27 mmol, 1.00 equivalent) in THF (30 mL) and saturated NaHCO3 aqueous solution (20 mL) was added to a solution of acyl chloride 6-chloro-6-oxo-hexanoate benzyl ester (100, 486 mg, 1.91 mmol, 1.51 equivalent) in THF (10 mL), and the reaction mixture was stirred at room temperature for 16 hours. EtOAc (100 mL) and water (20 mL) were added, the layers were separated, and the organic layer was washed with 2NNaOH aqueous solution (3 × 30 mL) and saturated NaCl aqueous solution (30 mL), dried (MgSO4), filtered, and concentrated under vacuum. The crude product was purified by rapid chromatography (silica, 570% EtOAc / n-heptane) to give an inseparable mixture of 6-[(3aS,7R,7aR)-7-hydroxy-2,2-dimethyl-4-propyl-4,6,7,7a-tetrahydro-3aH-[1,3]dioxacyclopenteno[4,5-c]pyridin-5-yl]-6-oxoyl-hexanoate benzyl ester (143,392 mg, 0.90 mmol, two-step yield 72%) and the epimer ...

Claims

1. A compound of formula (I) Or its pharmaceutically acceptable salt, in: B is a pyrimidine base; P1 and P2 are each independently a protecting group, wherein the protecting group is selected from O-4,4'-dimethoxytriphenylmethyl and phosphoramidinyl; Y is NR1 or NC (=0)-R1, where R1 is -L-R3. L represents a C1-C10 hydrocarbon chain or a C1-C10 hydrocarbon chain terminated with -(CO)-. R3 is the cell-targeting portion of formula (II) or its protected derivative: (II) in: R3 targets the mammalian desialyl glycoprotein receptor (ASGPR). A l It can be H, oxo (=O), (C1-C6)alkyl, or (C1-C6)alkenyl; A2 and A3 are independently hydroxyl or acetoxy groups; A4 is -NH-C(=O)-R4, where: R4 is a (C1-C6) alkyl group; and X1, X2, Ra, Rb, Rc, and Rd are each independently H or -(C1-C6) alkyl. The condition is that the (C1-C6) alkenyl group does not include the C1 alkenyl group.

2. The compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, wherein L is a C1-C10 hydrocarbon chain.

3. The compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, wherein L is a C1-C10 hydrocarbon chain terminated with -(CO)-.

4. The compound of formula (I) according to claim 1 or a pharmaceutically acceptable salt thereof, wherein Y is NR1, wherein R1 is -L-R3, and wherein L is a C1-C10 hydrocarbon chain or a C1-C10 hydrocarbon chain terminated with -(CO)-.

5. The compound of formula (I) according to claim 1 or a pharmaceutically acceptable salt thereof, wherein A1 is H, (C1-C6)alkyl or (C1-C6)alkenyl.

6. The compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, wherein A1 is a (C1-C6) alkyl group.

7. The compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, wherein A2 and A3 are acetoxy groups.

8. The compound of formula (I) according to claim 1 or a pharmaceutically acceptable salt thereof, wherein A4 is -NH-C(=O)-R4, and R4 is methyl.

9. A compound of formula (III) (III), or a pharmaceutically acceptable salt thereof. in: Al can be H, oxo group (=O), (C1-C6)alkyl or (C1-C6)alkenyl; A2 and A3 are hydroxyl or acetoxy groups; A4 is -NC(=O)-R4, where: R4 is a (C1-C6) alkyl group; B1 can be H, benzyl ester, -L-R5, or -(CO)-L-R5, where: L is a C1-C10 hydrocarbon chain or a C1-C10 hydrocarbon chain terminated with -(CO)-; and R5 can be H, OH, benzyl, benzyloxy, nucleoside, or nucleotide. The condition is that the (C1-C6) alkenyl group does not include the C1 alkenyl group.

10. The compound of claim 9 or a pharmaceutically acceptable salt thereof, wherein L is optionally a C1-C6 hydrocarbon chain terminated with –(CO)– and R5 is H, OH, benzyl or benzyloxy.

11. The compound of claim 9 or a pharmaceutically acceptable salt thereof, wherein A1 is H, an oxo group (=O) or a (C1-C6) alkyl group.

12. The compound of claim 9 or a pharmaceutically acceptable salt thereof, wherein A1 is a (C1-C6) alkyl group.

13. The compound of claim 9 or a pharmaceutically acceptable salt thereof, wherein A4 is -NH-C(=O)-R4, and R4 is methyl.

14. The compound of claim 9 or a pharmaceutically acceptable salt thereof, wherein B1 is H or a benzyl ester group.

15. An oligonucleotide comprising one or more compounds of formula (VI): (WE), in: B is a pyrimidine base; T l One of T2 is to attach a compound of formula (VI) to a nucleoside linker group of an oligomeric compound, and T... l The other one in T2 is selected from H, O-4,4'-dimethoxytriphenylmethyl, phosphoramidyl and nucleoside linker group that links the compound of formula (VI) to the oligomeric compound; Y is NR1 or NC (=0)-R1, where R1 is -L-R3. L represents a C1-C10 hydrocarbon chain or a C1-C10 hydrocarbon chain terminated with -(CO)-. R3 is the ASGPR-binding cell-targeting component of formula (II): (II); A l It can be H, oxo (=O), (C1-C6)alkyl, or (C1-C6)alkenyl; A2 and A3 are independently hydroxyl or acetoxy groups; A4 is -NH-C(=O)-R4, where: R4 is a (C1-C6) alkyl group; and X1, X2, Ra, Rb, Rc, and Rd are each independently H or -(C1-C6) alkyl. The condition is that the (C1-C6) alkenyl group does not include the C1 alkenyl group.

16. The oligonucleotide of claim 15, wherein Y is selected from the group consisting of NR1 and NC(=O)-R1, and L is a C1-C10 hydrocarbon chain.

17. The oligonucleotide of claim 15, wherein Y is selected from the group consisting of NR1 and NC(=O)-R1, and L is a C1-C10 hydrocarbon chain capped with -C(O)-.

18. The oligonucleotide of claim 15, wherein Y is NR1 and L is a C1-C10 hydrocarbon chain or a C1-C10 hydrocarbon chain capped with -(CO)-.

19. The oligonucleotide of claim 15, wherein the oligonucleotide is single-stranded or double-stranded.

20. The oligonucleotide of claim 19, wherein the oligonucleotide is an antisense oligonucleotide targeting human mRNA.

21. The oligonucleotide of claim 19, wherein the oligonucleotide is a double-stranded interfering RNA that targets human mRNA and comprises a sense strand and an antisense strand.

22. The oligonucleotide of claim 19, wherein the oligonucleotide has a protruding end at the 5' or 3' end of the sense strand or antisense strand.

23. The oligonucleotide according to claim 22, wherein the compound of formula (VI) is located at the overhang.

24. The oligonucleotide of claim 19, wherein the compound of formula (VI) is located at the 5' or 3' end of the positive strand.

25. The oligonucleotide of claim 24, wherein the compound of formula (VI) is located at the protruding end.

26. Use of the oligonucleotide according to any one of claims 15 to 25 in the preparation of a medicament for delivering the oligonucleotide to liver cells of a human subject in need, optionally the delivery comprising administering the oligonucleotide to the subject by intravenous or subcutaneous injection or by portal vein injection.

27. Use of the compound of any one of claims 1 to 14 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for delivering a therapeutic agent to liver cells of a human subject in need.

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