Nucleotide precursors, nucleotide analogs and oligomeric compounds containing the same

Abstract

The present disclosure relates to novel nucleotide precursors and nucleotide analogs that can be incorporated into oligonucleotides, including double-stranded oligonucleotides, such as siRNA. Oligonucleotides containing these analogs have excellent biological activity, for example, increased in vitro stability and improved in vivo efficacy, particularly duration of action. Improved oligonucleotides can be used to silence (e.g., reduce or eliminate) the expression of target genes. In a specific embodiment, the present invention encompasses specific nucleotide analogs to be included in double-stranded RNA (dsRNA) and especially siRNA, which can hybridize with the messenger RNA (mRNA) of interest, thereby reducing or blocking the expression of the target gene of interest. The compounds of the present invention have the general formula (I), wherein Ra, Rb, Rc, and Rd are each independently H or (C1-C6) alkyl, and B is a heterocyclic nucleobase. #imgabs0#

Description

Technical Field

[0001] The present disclosure relates to the field of targeted gene silencing using small interfering RNA (siRNA), and more particularly to nucleotide precursors and analogs for siRNA. Background Art

[0002] The concept of using synthetic oligonucleotides to control the expression of specific genes dates back to the late 1970s, when targeted gene silencing using short synthetic oligonucleotides was demonstrated for the first time (Stephenson et al., 1978, Proc Natl Acad Sci USA, 75:285-288). Stephenson's discovery subsequently clarified the RNA interference pathway for regulating gene expression and the role of siRNA in this process, greatly expanding scientists' understanding of post-transcriptional gene expression control in eukaryotic cells.

[0003] Synthetic oligonucleotides include single-stranded oligonucleotides, such as antisense oligonucleotides ("ASOs"), antimiRs and antagomiRs, and double-stranded oligonucleotides, such as siRNAs. Both ASOs and siRNAs bind to target RNAs through Watson-Crick base pairing, but their mechanisms of action are different. In antisense technology, ASOs form DNA-RNA duplexes with target RNAs and inhibit mRNA translation through a blocking mechanism, or cause RNase H-dependent degradation of the targeted RNA. In RNA interference technology, siRNAs bind to RNA-induced silencing complexes ("RISCs"), in which one strand (the "passenger strand" or "sense strand") is displaced, and the remaining strand (the "guide strand" or "antisense strand") collaborates with RISC to bind to complementary RNA (target RNA); once bound, the target RNA is cleaved by the RNA endonuclease Argonaute (AGO) in RISC and then further degraded by RNA exonucleases.

[0004] The biggest obstacles to developing oligonucleotide therapeutics, including siRNA, include (i) poor compound stability, (ii) low in vivo delivery efficiency to target cells, and (iii) side effects such as "off-target" gene silencing and unintended immune stimulation. To address some of these obstacles, various oligonucleotide chemical modifications have been attempted. These modifications can be divided into three categories, namely (i) sugar modifications, (ii) internucleotide linkage modifications, and (iii) nucleobase modifications.

[0005] The chemical modification of sugar group is included in the modification of 2 '-carbon atom or 2 '-hydroxyl place of ribose ring.2 '-OMe (methoxy) nucleotide analog is one of the most widely used modification.2 '-F (fluorine) nucleotide and 2 '-O-methoxyethyl nucleotide have also been used.Although most of sugar changes are positioned at 2 '-position, modification (people such as Leydler, 1995, Antisense Res Dev, 5:161-174) such as 4 '-position in other positions has also been reported.

[0006] Other chemical modifications of the sugar group include 2'-oxygen and 4'-carbon connecting the ribose scaffold in the nucleoside, thereby forming so-called locked nucleic acids ("LNA"). LNA is also known as bicyclic nucleic acid, and has been shown to have increased RNA binding affinity (Koshin et al., 1998, Tetrahedron, 54:3607-3630; Prakash et al., 2011, Chem.Biodivers, 8:1616-1641), resulting in a significant increase in the melting temperature of the resulting double-stranded oligonucleotide. However, oligomers longer than 8 nucleotides that are fully modified through LNA tend to aggregate. In contrast to the rigid properties of LNA modifications, highly flexible unlocked nucleic acids ("UNA") modifications have also been developed for application in oligonucleotide therapeutics. UNA nucleosides do not have the C2'-C3' bond of ribose. Due to its open chain structure, UNA is not constrained in conformation and has been used to regulate the flexibility of oligonucleotides (Mangos et al., 2003, J Am Chem Soc, 125: 654-661). In some cases, each insertion of UNA inserts can reduce the double-stranded melting temperature (Tm) by 5°C-10°C. In addition, UNA inserts can promote RISC to select the antisense strand, and UNA modification of the seed region of the siRNA guide strand can reduce off-target events (Vaish et al., 2011, Nucleic Acids Res, 39: 1823-1832). Bramsen et al. have reported siRNA containing UNA and LNA (2010, Nucleic Acids Research, 38 (17): 5761-5773).

[0007] In addition, an expanded sugar ring system has also been developed and applied to gene silencing technology. Such a system includes a six-membered morpholino ring system, in which the ribose moiety of the nucleoside is replaced by a morpholine ring. Through the nitrogen atom of the morpholino subunit, the nucleoside based on morpholino forms an internucleotide bond in the oligonucleotide containing them. Oligonucleotides based on phosphorodiamidate morpholino (" PMO ") have been used in antisense technology (Corey et al., 2001, Genome Biology, 2 (5): reviews 1015.1-1015; Partridge et al., 1996, Antisense Nucleic Acid Drug Dev, 6: 169-175). However, due to the low binding affinity with complementary RNA, antisense PMO needs to be relatively long, for example, 25 base lengths (Corey et al., the same). 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 No. US2016US / 0186174.

[0008] Internucleotide linkages can also be chemically modified to reduce susceptibility to nuclease degradation by replacing the 3'-5' phosphodiester bond with a more stable moiety. A widely used modification is the partial or complete replacement of the phosphodiester backbone with a phosphorothioate bond, where a sulfur atom replaces the oxygen atom. Another backbone modification that imparts increased biological stability to nucleic acids is the boranophosphate bond. In boranophosphate oligonucleotides, the non-bridging phosphodiester oxygen is partially replaced by an isoelectronic borane (-BH3).

[0009] However, most of the aforementioned phosphodiester modifications such as phosphorothioate produce chiral phosphorus in the internucleotide bond, resulting in a diastereomeric mixture of the oligonucleotides obtained. Since the number of diastereomeric oligonucleotides of each modified phosphodiester bond can be doubled, the number of diastereoisomers obtained increases exponentially with the increase in the number of modified phosphodiester bonds. Each diastereoisomer can exhibit varying degrees of nuclease resistance and different hybridization properties with the target mRNA. In addition, the purification and chemical analysis of the diastereomeric mixture are complicated. Therefore, in some cases, it is desirable to avoid the use of phosphodiester modifications such as phosphorothioate and the resulting diastereomeric oligonucleotide mixture.

[0010] Other major challenges in RNA interference technology are targeted delivery and cellular uptake of siRNA. Cell membrane is a double-layer structure of negatively charged phospholipids and is an entry barrier for negatively charged siRNA. Some groups have used N-acetylgalactosamine (GalNAc) to target siRNA attached to hepatocytes, which express asialoglycoprotein receptors (ASGPRs) that bind GalNAc, and the siRNA-GalNAc conjugates bound by ASGPR can be internalized by endocytosis (see, e.g., Nair et al., 2014, J Am Chem Soc, 136: 16958-16961).

[0011] Despite advances in RNA interference technology, there remains a need in the art for siRNA oligonucleotides with improved stability and delivery to their target cells. Summary of the Invention

[0012] The present application discloses a compound of formula (I)

[0013]

[0014] in:

[0015] -B is a heterocyclic nucleobase;

[0016] - P1 and P2 are each independently H, a reactive phosphorus group or a protecting group;

[0017] -Y is O, NH, NR1 or NC(=O)-R1, wherein R1 is:

[0018] a (C1-C20)alkyl group, which is optionally substituted by one or more groups selected from the group consisting of a halogen atom, a (C1-C6)alkyl group, a (C3-C8)cycloalkyl group, a (C3-C14)heterocycle, a (C6-C14)aryl group, a (C5-C14)heteroaryl group, -O-Z1, -N(Z1)(Z2), -S-Z1, -CN, -C(=J)-O-Z1, -OC(=J)-Z1, -C(=J)-N(Z1)(Z2) and -N(Z1)-C(=J)-Z2, wherein

[0019] J is O or S,

[0020] Z1 and Z2 are each independently H, a (C1-C6)alkyl group, which is optionally substituted with one or more groups selected from the group consisting of halogen atoms and (C1-C6)alkyl groups,

[0021] a (C3-C8)cycloalkyl group, which is optionally substituted by one or more groups selected from the group consisting of halogen atoms and (C1-C6)alkyl groups, or

[0022] -[C(=O)]m-R2-(O-CH2-CH2)p-R3, wherein:

[0023] m is an integer, with a value of 0 or 1.

[0024] p is an integer ranging from 0 to 10,

[0025] R2 is a (C1-C20)alkylene group, which is optionally substituted by a (C1-C6)alkyl group, -O-Z3, -N(Z3)(Z4), -S-Z3, -CN, -C(=K)-O-Z3, -OC(=K)-Z3, C(=K)-N(Z3)(Z4) and N(Z3)-C(=K)-Z4, wherein

[0026] K is O or S,

[0027] Z3 and Z4 are each independently H, a (C1-C6)alkyl group, which is optionally substituted with one or more groups selected from the group consisting of halogen atoms and (C1-C6)alkyl groups,

[0028] and

[0029] R3 is selected from a hydrogen atom, a (C1-C6)alkyl group, a (C1-C6)alkoxy group, a (C3-C8)cycloalkyl group, a (C3-C14)heterocycle, a (C6-C14)aryl group or a (C5-C14)heteroaryl group, or

[0030] R3 is the cell targeting moiety,

[0031] -X1 and X2 are each independently a hydrogen atom, a -(C1-C6)alkyl group, and

[0032] -Ra, Rb, Rc and Rd are each independently H or a (C1-C6)alkyl group.

[0033] In some embodiments of the compound of Formula (I), Y is NR1, wherein R1 is an optionally substituted (C1-C20)alkyl group, and P1, P2, Ra, Rb, Rc, Rd, X1, X2 and B are as defined for Formula (I).

[0034] In some embodiments of the compound of formula (I), Y is NR1, wherein R1 is an unsubstituted (C1-C16)alkyl group, including an alkyl group selected from methyl, isopropyl, butyl, octyl, hexadecyl, and P1, P2, Ra, Rb, Rc, Rd, X1, X2 and B have the same meanings as defined for the general formula (I).

[0035] In some embodiments of the compound of formula (I), Y is NR1, wherein R1 is a (C3-C8)cycloalkyl group, which is optionally substituted with one or more groups selected from the group consisting of halogen atoms and (C1-C6)alkyl groups, and P1, P2, Ra, Rb, Rc, Rd, X1, X2 and B have the same meanings as defined for the general formula (I).

[0036] In some embodiments of the compound of Formula (I), Y is NR1, wherein R1 is a cyclohexyl group, and P1, P2, Ra, Rb, Rc, Rd, X1, X2 and B have the same meanings as defined for the general formula (I).

[0037] In some embodiments of the compound of Formula (I), Y is NR1, wherein R1 is a (C1-C20)alkyl group substituted by a (C6-C14)aryl group, and P1, P2, Ra, Rb, Rc, Rd, X1, X2 and B have the same meanings as defined for the general formula (I).

[0038] In some embodiments of the compound of Formula (I), Y is NR1, wherein R1 is a (C1-C20)alkyl group substituted with a phenyl group, and P1, P2, Ra, Rb, Rc, Rd, X1, X2 and B have the same meanings as defined for the general formula (I).

[0039] In some embodiments of the compound of Formula (I), Y is NR1, wherein R1 is a methyl group substituted with a phenyl group, and P1, P2, Ra, Rb, Rc, Rd, X1, X2 and B have the same meanings as defined for the general formula (I).

[0040] In some embodiments of the compound of Formula (I), Y is NC(═O)—R1, wherein R1 is an optionally substituted (C1-C20)alkyl group, and P1, P2, Ra, Rb, Rc, Rd, X1, X2, and B have the same meanings as defined for the general formula (I).

[0041] In some embodiments of the compound of Formula (I), Y is NC(═O)—R1, wherein R1 is selected from methyl and pentadecyl, and P1, P2, Ra, Rb, Rc, Rd, X1, X2 and B have the same meanings as defined for the general formula (I).

[0042] In some further embodiments of the compound of formula (I), Y is NR1 and R1 is a group -[C(=O)]m-R2-(O-CH2-CH2)p-R3, wherein

[0043] m is an integer, with a value of 0 or 1.

[0044] p is an integer ranging from 0 to 10,

[0045] R2 is a (C1-C20)alkylene group, which is optionally substituted by a (C1-C6)alkyl group, -O-Z3, -N(Z3)(Z4), -S-Z3, -CN, -C(=K)-O-Z3, -OC(=K)-Z3, -C(=K)-N(Z3)(Z4), -N(Z3)-C(=K)-Z4, wherein

[0046] K is O or S,

[0047] Z3 and Z4 are each independently H, a (C1-C6)alkyl group, which is optionally substituted with one or more groups selected from the group consisting of halogen atoms and (C1-C6)alkyl groups,

[0048] and

[0049] R3 is the cell targeting moiety,

[0050] - X1 and X2 are each independently a hydrogen atom, a (C1-C6) alkyl group, and

[0051] -Ra, Rb, Rc and Rd are each independently H or a (C1-C6)alkyl group.

[0052] In some of these further embodiments of compounds of formula (I), R1 is a group -[C(=O)]m-R2-(O-CH2-CH2)p-R3, m is 0, p is 0, R3 is a cell targeting moiety, and B, P1, P2, Ra, Rb, Rc, Rd, X1, X2 and R2 are as in the general definition of compounds of formula (I).

[0053] In some of these further embodiments of the compound of formula (I), R2 is an ethylene group, p is 0, and X1 and X2 are both hydrogen atoms.

[0054] In some of these further embodiments of compounds of formula (I), R2 is a pentylene group, and X1 and X2 are both hydrogen atoms.

[0055] In some of these further embodiments of the compound of formula (I), R2 is a (C12)alkylene group, and X3 and X4 are both hydrogen atoms.

[0056] In some of these further embodiments of compounds of formula (I), R1 is a group -[C(=O)]m-R2-(O-CH2-CH2)p-R3, m is 0, p is selected from the integers 1, 2, 3 and 4, R3 is a cell targeting moiety, and B, P1, P2, Ra, Rb, Rc, Rd, X1, X2 and R2 are as in the general definition of compounds of formula (I).

[0057] In some of these further embodiments of compounds of formula (I), R2 is an ethylene group, p is 1, and X1 and X2 are both hydrogen atoms.

[0058] In some of these further embodiments of compounds of formula (I), R2 is an ethylene group, p is 2, and X1 and X2 are both hydrogen atoms.

[0059] In some of these further embodiments of the compound of formula (I), R2 is an ethylene group, p is 3, and X1 and X2 are both hydrogen atoms.

[0060] In some of these further embodiments of compounds of formula (I), R2 is an ethylene group, p is 4, and X1 and X2 are both hydrogen atoms.

[0061] In some of these further embodiments of compounds of formula (I), m is 1, p is 0, R3 is a cell targeting moiety, and R2, B, P1, P2, Ra, Rb, Rc, Rd, X1, X2 are as in the general definition of compounds of formula (I).

[0062] In some of these further embodiments of compounds of formula (I), R2 is butylene, X1 and X2 both represent hydrogen atoms, and B, P1, P2, Ra, Rb, Rc and Rd are as defined for general formula (I).

[0063] In some of these further embodiments of compounds of formula (I), R2 is (C11)alkylene, X1 and X2 both represent hydrogen atoms, and B, P1, P2, Ra, Rb, Rc and Rd are as defined for general formula (I).

[0064] In some of these further embodiments of compounds of formula (I), R2 is methylene, X1 and X2 both represent hydrogen atoms, and B, P1, P2, Ra, Rb, Rc and Rd are as defined for general formula (I).

[0065] In some of these further embodiments of the compounds of formula (I), m is 1, p is selected from the integers 1 and 2, R3 is a cell targeting moiety, and R2, B, P1, P2, Ra, Rb, Rc, Rd, X1, X2 are as in the general definition of compounds of formula (I).

[0066] In some of these further embodiments of compounds of formula (I), R2 is a methylene group, p is 2, R3 is a cell targeting moiety, and B, P1, P2, Ra, Rb, Rc, Rd, X1, and X2 are as defined for general formula (I).

[0067] In some of these further embodiments of compounds of formula (I), R2 is a methylene group, p is 1, R3 is a cell targeting moiety, and B, P1, P2, Ra, Rb, Rc, Rd, X1, and X2 are as defined for general formula (I).

[0068] In some of these further embodiments of the compound of formula (I), R3 is a compound of formula (III):

[0069]

[0070] wherein A1, A2 and A3 are OC(=O)-R4,

[0071] wherein R4 is a (C1-C6)-alkyl or (C6-C10)-aryl group;

[0072] A4 is OC(=O)-R4 or NHC(=O)-R5, R4 is as defined above, and R5 is a (C1-C6)-alkyl group optionally substituted by a halogen atom.

[0073] In some of these further embodiments of the compound of formula (I), R3 is a compound of formula (III), wherein A1, A2 and A3 are OC(=O)-R4, wherein R4 is a (C1-C6)-alkyl or (C6-C10)-aryl group;

[0074] A4 is OC(=O)-R4 or NHC(=O)-R5, wherein R4 is as defined above and R5 is a (C1-C6)-alkyl group optionally substituted by a halogen atom.

[0075] In some of these further embodiments of compounds of formula (I), R3 is a compound of formula (III) wherein A1, A2 and A3 are OC(=O)-R4, wherein R4 is a methyl group, A4 is OC(=O)-R4 or NHC(=O)-R5, wherein R4 and R5 are each a methyl group.

[0076] In some embodiments of the compound of Formula (I), B is optionally protected and is selected from the group consisting of pyrimidine, substituted pyrimidine, purine, and substituted purine.

[0077] In some embodiments of the compound of formula (I), one of P1 or P2 is an O-4,4'-dimethoxytrityl group and the other of P1 and P2 is H, a reactive phosphorus group, or a protecting group, and Y, B, Ra, Rb, Rc, and Rd are as defined in formula (I).

[0078] In some embodiments of the compound of Formula (I), one of P1 and P2 is a 2-cyanoethyl-N,N-diisopropylphosphoramidite group, and the other of P1 and P2 is a protecting group, and Y, B, Ra, Rb, Rc and Rd are as defined in Formula (I).

[0079] In some embodiments of the compound of formula (I), one of P1 and P2 is a 2-cyanoethyl-N,N-diisopropylphosphoramidite group, and the other of P1 and P2 is an O-4,4'-dimethoxytrityl group, and Y, B, Ra, Rb, Rc, and Rd are as defined in formula (I).

[0080] The present invention also relates to oligonucleotides comprising one or more nucleotides of formula (II):

[0081]

[0082] in:

[0083] -B is a heterocyclic nucleobase;

[0084] - one of L1 and L2 is an internucleoside linking group that links the compound of formula (II) to the oligomeric compound, and the other of L1 and L2 is H, a protecting group, a phosphorus moiety, or an internucleoside linking group that links the compound of formula (II) to the oligomeric compound,

[0085] -Y is O, NH, NR1 or NC(=O)-R1, wherein R1 is:

[0086] a (C1-C20)alkyl group, which is optionally substituted by one or more groups selected from the group consisting of a halogen atom, a (C1-C6)alkyl group, a (C3-C8)cycloalkyl group, a (C3-C14)heterocycle, a (C6-C14)aryl group, a (C5-C14)heteroaryl group, -O-Z1, -N(Z1)(Z2), -S-Z1, -CN, -C(=J)-O-Z1, -OC(=J)-Z1, -C(=J)-N(Z1)(Z2) and -N(Z1)-C(=J)-Z2, wherein

[0087] J is O or S,

[0088] Z1 and Z2 are each independently H, a (C1-C6)alkyl group, which is optionally substituted with one or more groups selected from the group consisting of halogen atoms and (C1-C6)alkyl groups,

[0089] a (C3-C8)cycloalkyl group, which is optionally substituted by one or more groups selected from the group consisting of halogen atoms and (C1-C6)alkyl groups, or

[0090] -[C(=O)]m-R2-(O-CH2-CH2)p-R3, wherein

[0091] m is an integer, with a value of 0 or 1.

[0092] p is an integer ranging from 0 to 10,

[0093] R2 is a (C1-C20)alkylene group, which is optionally substituted by a (C1-C6)alkyl group, -O-Z3, -N(Z3)(Z4), -S-Z3, -CN, -C(=K)-O-Z3, -OC(=K)-Z3, -C(=K)-N(Z3)(Z4) and -N(Z3)-C(=K)-Z4, wherein

[0094] K is O or S,

[0095] Z3 and Z4 are each independently H, a (C1-C6)alkyl group, which is optionally substituted with one or more groups selected from the group consisting of halogen atoms and (C1-C6)alkyl groups,

[0096] and

[0097] R3 is selected from a hydrogen atom, a (C1-C6)alkyl group, a (C1-C6)alkoxy group, a (C3-C8)cycloalkyl group, a (C3-C14)heterocycle, a (C6-C14)aryl group or a (C5-C14)heteroaryl group, or R3 is a cell targeting moiety,

[0098] - X1 and X2 are each independently a hydrogen atom, a (C1-C6) alkyl group, and

[0099] - Ra, Rb, Rc and Rd are each independently H or a (C1-C6)alkyl group,

[0100] or a pharmaceutically acceptable salt thereof.

[0101] In some embodiments of the oligonucleotide, in one or more compounds of formula (II), Y is NR1, R1 is an unsubstituted (C1-C20) alkyl group, and L1, L2, Ra, Rb, Rc, Rd, X1, X2 R2, R3 and B have the same meanings as defined for the general formula (II), or a pharmaceutically acceptable salt thereof.

[0102] In some embodiments of the oligonucleotides, in one or more compounds of formula (II), Y is NR1, R1 is unsubstituted (C1-C 16) alkyl group, which includes an alkyl group selected from methyl, isopropyl, butyl, octyl, hexadecyl, and L1, L2, Ra, Rb, Rc, Rd, X1, X2, R2, R3 and B have the same meanings as defined in formula (II), or a pharmaceutically acceptable salt thereof.

[0103] In some embodiments of the oligonucleotide, in one or more compounds of formula (II), Y is NR1, R1 is a (C3-C8)cycloalkyl group, which is optionally substituted by one or more groups selected from the following: halogen atoms and (C1-C6)alkyl groups, and L1, L2, Ra, Rb, Rc, Rd, X1, X2, R2, R3 and B have the same meanings as defined in formula (II), or a pharmaceutically acceptable salt thereof.

[0104] In some embodiments of the oligonucleotide, in one or more compounds of formula (II), Y is NR1, R1 is a cyclohexyl group, and L1, L2, Ra, Rb, Rc, Rd, X1, X2, R2, R3 and B have the same meanings as defined in formula (II), or a pharmaceutically acceptable salt thereof.

[0105] In some embodiments of the oligonucleotide, in one or more compounds of formula (II), Y is NR1, R1 is a (C1-C20)alkyl group substituted by a (C6-C14)aryl group, and L1, L2, Ra, Rb, Rc, Rd, X1, X2, R2, R3 and B have the same meanings as defined in formula (II), or a pharmaceutically acceptable salt thereof.

[0106] In some embodiments of the oligonucleotide, in one or more compounds of formula (II), Y is NR1, R1 is a methyl group substituted by a phenyl group, and L1, L2, Ra, Rb, Rc, Rd, X1, X2, R2, R3 and B have the same meanings as defined in formula (II), or a pharmaceutically acceptable salt thereof.

[0107] In some embodiments of the oligonucleotide, in one or more compounds of formula (II), Y is NC(=O)-R1, R1 is an optionally substituted (C1-C20)alkyl group, and L1, L2, Ra, Rb, Rc, Rd, X1, X2, R2, R3 and B have the same meanings as defined in formula (II), or a pharmaceutically acceptable salt thereof.

[0108] In some embodiments of the oligonucleotide, in one or more compounds of formula (II), Y is NC(=O)-R1, R1 is selected from methyl and pentadecyl, and L1, L2, Ra, Rb, Rc, Rd, X1, X2, R2, R3 and B have the same meanings as defined in formula (II), or a pharmaceutically acceptable salt thereof.

[0109] In some embodiments of the oligonucleotide, in one or more compounds of Formula (II), B is selected from the group consisting of pyrimidine, substituted pyrimidine, purine, and substituted purine, or a pharmaceutically acceptable salt thereof.

[0110] In some embodiments of the oligonucleotides, in one or more compounds of formula (II), the internucleoside linking group is independently selected from phosphodiester, phosphotriester, phosphorothioate, phosphorodithioate, alkyl-phosphonate, and phosphoramidate backbone linking groups.

[0111] In some embodiments, the oligonucleotide comprises 2 to 10 compounds of Formula (II).

[0112] In some embodiments, the oligonucleotide comprises one or more targeting nucleotides.

[0113] In some embodiments of the oligonucleotide, in one or more compounds of formula (II), R3 has formula (III):

[0114]

[0115] wherein A1, A2 and A3 are O, A4 is OH or NHC(=O)-R5, and R5 is a (C1-C6)-alkyl group optionally substituted by a halogen atom.

[0116] In some embodiments of the oligonucleotide, in one or more compounds of Formula (II), R3 is N-acetyl-galactosamine.

[0117] The present invention also relates to a double-stranded oligonucleotide comprising one or more compounds of formula (II) as described herein, or a pharmaceutically acceptable salt thereof.

[0118] The present invention also relates to siRNA comprising one or more compounds of formula (II) as described herein, or a pharmaceutically acceptable salt thereof.

[0119] The present disclosure also relates to methods for obtaining the nucleotide precursors, nucleotide analogs, and single-stranded or double-stranded oligonucleotides specified herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0120] Figure 1: In vitro knockdown of siRNAs 58 to 79 with 1T3, 1T3b, 1T1, and 1T1b overhangs

[0121] Vertical axis: Percentage of AHA-1 expression in HepG2 cells transfected with control siRNA

[0122] Horizontal axis: siRNA number, concentration

[0123] Figure 2 In vitro knockdown of siRNAs 29 to 55 with 1T3 and 1T3b overhangs with or without additional cholesterol substitutions

[0124] Vertical axis: Percentage of AHA-1 expression in transfected HepG2 cells relative to the control

[0125] Horizontal axis: siRNA number

[0126] Figure 3a and 3b : Relative TTR protein serum levels at blood sampling time points before / after subcutaneous administration of the substance as indicated on the X-axis labels

[0127] Vertical axis: relative to TTR serum level before administration + / - SEM

[0128] Horizontal axis: days after subcutaneous administration

[0129] Figure 4 : Relative TTR mRNA expression levels in the liver were recorded 48 h after subcutaneous administration of the substances as indicated on the X-axis labels.

[0130] Vertical axis: relative expression of TTR mRNA in liver + / -SD

[0131] Horizontal axis: siRNA number, dosage

[0132] Figure 5 : As shown on the X-axis labels, the study recorded TTR protein serum levels 48 h after subcutaneous administration of the substance

[0133] Vertical axis: serum TTR concentration expressed as ng / ml + / - SD

[0134] Horizontal axis: siRNA number, dosage

[0135] Figure 6a and 6b : Relative TTR protein serum levels at blood sampling time points after subcutaneous administration of the substance as indicated on the X-axis labels

[0136] Vertical axis: relative to TTR serum level before administration + / - SEM

[0137] Horizontal axis: days after subcutaneous administration

[0138] Figure 7 : Relative TTR mRNA expression levels in the liver were recorded 48 h after subcutaneous administration of the substances as indicated on the X-axis labels.

[0139] Vertical axis: relative expression of TTR mRNA in liver + / -SD

[0140] Horizontal axis: siRNA number, dosage

[0141] Figure 8 : As shown on the X-axis labels, the study recorded TTR protein serum levels 48 h after subcutaneous administration of the substance

[0142] Vertical axis: serum TTR concentration expressed as ng / ml + / - SD

[0143] Horizontal axis: siRNA number, dosage

[0144] Figure 9a and 9b : Relative TTR protein serum levels at blood sampling time points after subcutaneous administration of the substance as shown in the graph

[0145] Vertical axis: relative to TTR serum level before administration + / - SEM

[0146] Horizontal axis: days after subcutaneous administration

[0147] Figure 10 : Relative TTR mRNA expression levels in the liver were recorded 48 h after subcutaneous administration of the substances as indicated on the X-axis labels.

[0148] Vertical axis: relative expression of TTR mRNA in liver + / -SD

[0149] Horizontal axis: siRNA number, dosage

[0150] Figure 11 : As shown on the X-axis labels, the study recorded TTR protein serum levels 48 h after subcutaneous administration of the substance

[0151] Vertical axis: serum TTR concentration expressed as ng / ml + / - SD

[0152] Horizontal axis: siRNA number, dosage

[0153] Figure 12a and 12b : Relative TTR protein serum levels at blood sampling time points after subcutaneous administration of the substance as shown in the graph

[0154] Vertical axis: relative to TTR serum level before administration + / - SEM

[0155] Horizontal axis: days after subcutaneous administration

[0156] Figure 13 : Relative TTR mRNA expression levels in the liver were recorded 48 h after subcutaneous administration of the substances as indicated on the X-axis labels.

[0157] Vertical axis: relative expression of TTR mRNA in liver + / -SD

[0158] Horizontal axis: siRNA number, dosage

[0159] Figure 14 : As shown on the X-axis labels, the study recorded TTR protein serum levels 48 h after subcutaneous administration of the substance

[0160] Vertical axis: serum TTR concentration expressed as ng / ml + / - SD

[0161] Horizontal axis: siRNA number, dosage DETAILED DESCRIPTION

[0162] The present disclosure provides novel nucleotide analogs that can be incorporated into oligonucleotides, including double-stranded oligonucleotides, such as siRNA. Oligonucleotides containing these analogs have excellent biological activity, for example, improved in vitro stability and in vivo action duration. Improved oligonucleotides can be used to silence (e.g., reduce or eliminate) the expression of a target gene. In a specific embodiment, the present invention encompasses specific nucleotide analogs to be included in double-stranded RNA (dsRNA) and especially siRNA, which can hybridize with the messenger RNA (mRNA) of interest, thereby reducing or blocking the expression of the target gene of interest.

[0163] In some embodiments, the present invention provides nucleotide analogs in which the ribose ring is substituted with a six-membered heterocyclic ring. As described in further detail below, the six-membered heterocyclic group can be a dioxane or a morpholino ring. When the heterocyclic group is a morpholino ring, the nitrogen atom is substituted or unsubstituted. In some embodiments, the six-membered heterocyclic group can be substituted with a linear or cyclic group and / or a targeting moiety.

[0164] definition

[0165] The terms used in this specification generally have their ordinary meanings in the art. Certain terms are discussed below or elsewhere in this disclosure to provide additional guidance in describing the products and methods of the presently disclosed subject matter.

[0166] The following definitions apply in the context of this disclosure:

[0167] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0168] As used herein, the term "about" refers to the usual error range for each value readily known to those skilled in the art. References herein to "about" a value or parameter include (and describe) embodiments for that value or parameter itself. In some embodiments, the term "about" refers to ±10% of a given value. However, as long as the value in question refers to an indivisible object, such as a nucleotide or other object, which loses its identity once subdivided, "about" refers to ±1 of the indivisible object.

[0169] It should be understood that aspects and embodiments of the present disclosure described herein include "having," "comprising," "consisting of," and "consisting essentially of." The words "have" and "comprise," or variations such as "has or having" or "comprises or comprising" should be understood to mean including the stated elements (e.g., compositions of matter or method steps) but not excluding any other elements. The term "consisting of" means including the stated elements but not any other elements. The term "consisting essentially of" means including the stated elements and may include additional elements, wherein the additional elements do not materially affect the basic and novel characteristics of the invention.

[0170] Unless otherwise indicated, "alkyl" refers to an aliphatic hydrocarbon group that can be straight or branched and has from 1 to 20 (e.g., 1-5, 1-10, or 1-15) carbon atoms in the chain. "Branched" means that one or more alkyl groups, such as methyl, ethyl, or propyl, are attached to a linear alkyl chain. Exemplary straight or branched alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, 3-pentyl, octyl, nonyl, and decyl.

[0171] "Cycloalkyl" refers to a cyclic saturated alkyl group as defined above. Examples are, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

[0172] "Alkoxy" is defined as a -OR group where R is an alkyl group as defined above, including cycloalkyl groups. Examples are, but are not limited to, methoxy, ethoxy, 1-propoxy, 2-propoxy, butoxy, and pentoxy.

[0173] "Halogen atom" refers to a fluorine, chlorine, bromine or iodine atom. In some embodiments, a fluorine or chlorine atom may be preferred.

[0174] Unless otherwise indicated, "aryl" refers to an aromatic monocyclic or polycyclic hydrocarbon ring system having 6 to 14 carbon atoms, for example 6 to 10 carbon atoms. Exemplary aryl groups include phenyl and naphthyl groups.

[0175] "Heterocycle" or "heterocyclic" refers to a saturated, partially unsaturated or unsaturated carbocyclic group containing at least one heteroatom selected from oxygen, nitrogen, selenium, phosphorus and sulfur. The nitrogen, selenium, phosphorus or sulfur may optionally be oxidized, and the nitrogen may optionally be quaternized. For example, the heterocycle may be a stable ring in which at least one ring member is a heteroatom. In some embodiments, the heterocycle may have 3 to 14 members, such as 5 to 7 or 5 to 10 members, and may have 1, 2 or more rings (i.e., monocyclic, bicyclic or polycyclic). In specific embodiments, the heteroatoms are oxygen, nitrogen and sulfur. The number of heteroatoms may vary, for example, from 1 to 3. In The Handbook of Chemistry and Physics, 76 th Suitable heterocycles are also disclosed in Serial No. 25 to 26 of the American Society of Hematology, 1995-1996, pppp. 2-25 to 2-26, the disclosure of which is incorporated herein by reference. In some embodiments, the heterocycle is a non-aromatic heterocycle including, but not limited to, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, oxiranyl, tetrahydrofuranyl, dioxolanyl, tetrahydropyranyl, dioxanyl, dioxolanyl, piperidinyl, piperazinyl, morpholinyl, pyranyl, imidazolinyl, pyrrolinyl, pyrazolinyl, thiazolidinyl, tetrahydrothiopyranyl, dithianyl, thiomorpholinyl, dihydro-pyranyl, tetrahydropyranyl, dihydropyranyl, tetrahydropyridinyl, dihydropyridinyl, tetrahydropyrinidinyl, dihydrothiopyranyl, and azepanyl, as well as fused systems resulting from condensation with a phenyl group.

[0176] "Heteroaryl" refers to an aromatic heterocycle having 5 to 14 (e.g., 5 to 7 or 5 to 10) members, and may be monocyclic, bicyclic, or polycyclic. The number of heteroatoms may typically vary from 1 to 3 heteroatoms, for example, selected from N and O. Examples of heteroaryl groups include pyrrolyl, pyridyl, pyrazolyl, thienyl, pyrimidinyl, pyrazinyl, tetrazolyl, indolyl, quinolyl, purinyl, imidazolyl, thienyl, thiazolyl, benzothiazolyl, furyl, benzofuranyl, 1,2,4-thiadiazolyl, oxadiazole, isothiazolyl, triazolyl, tetrazolyl, isoquinolyl, benzothienyl, isobenzofuranyl, pyrazolyl, carbazolyl, benzimidazolyl, isoxazolyl, and pyridyl-N-oxide, as well as fused systems resulting from condensation with phenyl groups. In certain embodiments, heteroaryl is a 5- or 6-membered heteroaryl group containing one or more heteroatoms, for example, 1-3 heteroatoms selected from N and O. "Alkyl," "cycloalkyl," "alkenyl," "alkynyl," "aryl," "heteroaryl," and "heterocyclyl" also refer to the corresponding "alkylene," "cycloalkylene," "alkenylene," "alkynylene," "arylene," "heteroarylene," and "heterocyclyl" groups formed by removing two hydrogen atoms.

[0177] The term "heterocyclic nucleobase" refers to any nitrogen-containing heterocyclic moiety that is capable of pairing with a complementary nucleobase or nucleobase analog (ie, a derivative of a nucleobase) to form Watson-Crick type hydrogen bonding and stacking interactions when the nucleobase is incorporated into a polymer structure.

[0178] Unless otherwise stated, according to the present disclosure, the term "heterocyclic nucleobase" refers to an optionally substituted nitrogen-containing heterocyclic group that can be connected to an optionally substituted dioxane ring or an optionally substituted morpholino ring in this article. In some embodiments, a heterocyclic nucleobase can be selected from an optionally substituted purine-base or an optionally substituted pyrimidine-base. The term "purine-base" is used herein with its common meaning as understood by those skilled in the art, and includes its tautomers. Similarly, the term "pyrimidine-base" is used herein with its common meaning as understood by those skilled in the art, and includes its tautomers. The non-limiting list of the optionally substituted purine-base includes purine, adenine, guanine, hypoxanthine, xanthine, alloxanthine, 7-alkylguanines (for example, 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-alkylcytosines (eg, 5-methylcytosine). Other non-limiting examples of heterocyclic nucleobases include diaminopurine, 8-oxo-N6 alkyladenine (e.g., 8-oxo-N6 methyladenine), 7-deazaxanthine, 7-deazaguanine, 7-deazaadenine, N4, N4-ethanocytosine (ethanocytosin), N6, N6-ethano-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 are incorporated herein by reference for the disclosure of additional heterocyclic bases. In some embodiments, the heterocyclic nucleobase can be substituted with an amine or enol protecting group.

[0179] As used herein, the term "protecting group" refers to any atom or group of atoms added to a molecule to prevent an existing group in the molecule from undergoing an undesirable chemical reaction. A "protecting group" may be an unstable chemical moiety known in the art that is used to protect reactive groups, such as hydroxyl, amino, and thiol groups, to prevent undesirable or inappropriate reactions during chemical synthesis. Protecting groups are typically used selectively and / or orthogonally to protect sites during reactions at other reactive sites and can then be removed to leave the unprotected group intact or available for further reactions.

[0180] Examples of protecting group moieties are described in TW Greene and PGM Wuts, Protective Groups in Organic Synthesis, 3. 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 group moieties may be selected in such a way that they are stable to certain reaction conditions and are readily removable at a convenient stage using methods known in the art.

[0181] A non-limiting list of protecting groups includes benzyl; substituted benzyl; alkylcarbonyl and alkoxycarbonyl (e.g., tert-butyloxycarbonyl (BOC), acetyl, or isobutyryl); arylalkylcarbonyl and arylalkoxycarbonyl (e.g., benzyloxycarbonyl); substituted methyl ethers (e.g., methoxymethyl ether); substituted ethyl ethers; substituted benzyl ethers; tetrahydropyranyl ethers; silyl (e.g., trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tri-isopropylsilyloxymethyl, [2-(trimethylsilyl)ethoxy]methyl, or tert-butyldiphenylsilyl); esters (e.g., benzoate); carbonates (e.g., methoxymethyl carbonate); esters); sulfonates (e.g., tosylate or mesylate); acyclic ketals (e.g., dimethyl acetal); cyclic ketals (e.g., 1,3-dioxane, 1,3-dioxolane, and those described herein); acyclic acetals; cyclic acetals (e.g., those described herein); acyclic hemiacetals; cyclic hemiacetals; cyclic dithioketals (e.g., 1,3-dithiane or 1,3-dithiolane); orthoesters (e.g., those described herein) and triarylmethyl groups (e.g., trityl; monomethoxytrityl (MMTr); 4,4′-dimethoxytrityl (DMTr); 4,4′,4″-trimethoxytrityl (TMTr); and those described herein).

[0182] Preferred protecting groups are selected from acetyl (Ac), benzoyl (Bzl), isobutyryl (iBu), phenylacetyl, dimethoxytrityl (DMT), methoxytrityl (MMT), triphenylmethyl (Trt), N,N-dimethylformamidine and 2-cyanoethyl (CE).

[0183] Unless otherwise indicated, any abbreviations for protecting groups, amino acids, and other compounds are in accordance with their common usage, recognized abbreviations, or the IUPAC-IUB Biochemical Nomenclature Commission (see, Biochem. 11:942-944 (1972)).

[0184] As used herein, the term "solid support" (also called resin) refers to insoluble particles, typically 50-200 μm in diameter, to which the oligonucleotides are bound during synthesis. Various types of solid supports have been used, but controlled pore glass (CPG) and polystyrene (highly cross-linked polystyrene microbeads) have proven particularly useful. Controlled pore glass is rigid, does not swell, and has deep pores (pore diameters between 500 and 100 μm). In some embodiments, oligonucleotide synthesis can be carried out by a plurality of methods.For example ... For example, oligonucleotide synthesis can be carried out by a plurality of methods. For example, oligonucleotide synthesis can be carried out by a plurality of methods. For example, oligonucleotide synthesis can be carried out by a plurality of methods. For example, oligonucleotide synthesis can be carried out by a plurality of methods. For example, oligonucleotide synthesis can be carried out by a plurality of methods. For example, oligonucleotide synthesis can be carried out by a plurality of methods. For example, oligonucleotide synthesis can be carried out by a plurality of methods. For example, oligonucleotide synthesis can be carried out by a plurality of methods.

[0185] As defined below, the term "ribonucleotide" or "nucleotide" includes naturally occurring or modified nucleotides, or alternative replacement moieties, as further described below. Modified nucleotides are non-naturally occurring nucleotides and are also referred to herein as "nucleotide analogs." It will be understood by those of ordinary skill in the art that guanine, cytosine, adenine, uracil, or thymine in a nucleotide can be replaced by other moieties without substantially changing the base pairing properties of the oligonucleotide comprising the nucleotide with such replacement moiety. For example, but not limited to, a nucleotide comprising inosine as a base can be base paired with a nucleotide comprising adenine, cytosine, or uracil. Therefore, in the nucleotide sequences of the present disclosure, a nucleotide containing uracil, guanine, or adenine can be replaced by a nucleotide containing, for example, inosine. Sequences comprising such replacement moieties are included as embodiments of the present disclosure.

[0186] As used herein, "cell targeting moiety" refers to a molecular group that ensures increased delivery of siRNA, which encompasses (i) increased specificity of siRNA binding to a selected target receptor (e.g., a target protein), including increased specificity of siRNA binding to cells expressing the selected target receptor; (ii) increased uptake of siRNA by target cells; and / or (iii) enhanced ability to properly process siRNA once it enters the target cell, such as by increasing the release of siRNA within the cell, for example, by promoting transport of siRNA from transport vesicles to the cytoplasm. Thus, cell targeting moieties are used to direct and / or deliver oligonucleotides to specific cells, tissues, organs, and the like. The cell targeting moiety contained in a nucleotide, nucleotide analog, or oligonucleotide imparts characteristics to the nucleotide, nucleotide analog, or oligonucleotide such that the nucleotide, nucleotide analog, or oligonucleotide is preferentially recognized, bound, internalized, processed, activated, and the like by a targeted cell type relative to a non-targeted cell type. For example, endothelial cells have a high affinity for the peptide cell targeting moiety Arg-Gly-Asp (RGD); cancer cells and kidney cells preferentially interact with compounds having a folic acid moiety; immune cells have an affinity for mannose; and cardiomyocytes have an affinity for the peptide WLSEAGPVVTVRALRGTGSW (SEQ ID NO: 118) (see, e.g., Biomaterials ZV-8081-8087, 2010). Other cell targeting / delivery moieties are known in the art. Thus, compounds comprising a cell targeting moiety preferentially interact with and are taken up by the targeted cell type.

[0187] Cell targeting moieties encompass cell targeting peptide groups and cell targeting non-peptide groups.

[0188] As used herein, "target cell" or "targeted cell" refers to a cell of interest. These cells can be found in vitro or in vivo in a tissue or organ of an organism. The organism can be an animal, preferably a mammal, more preferably a human, and most preferably a human patient.

[0189] As used herein, the term "TTR" refers to the transthyretin gene or protein. As used herein, the term "TTR" includes human TTR, whose amino acid and nucleotide sequences can be found, for example, in the EMBL database under accession number CR456908, and mouse TTR, whose amino acid and nucleotide sequences can be found, for example, in the GenBank database under accession number AAH24702. Other examples of TTR mRNA sequences are readily available, for example, in GenBank.

[0190] As used herein, the term "AHA-1" refers to the AHSA1 gene or protein. As used herein, the term "AHA-1" includes human AHSA-1, whose amino acid and nucleotide sequences can be found, for example, in the EMBL database under accession number AK300766.

[0191] As used herein, "target sequence" refers to a contiguous nucleotide sequence found in the RNA transcript of a target gene or portion thereof, including mRNA, which is the product of RNA processing of a primary transcript.

[0192] As used herein, and unless otherwise indicated, the term "complementary" when used to describe a first nucleotide sequence (e.g., oligonucleotide) related to a second nucleotide sequence (e.g., oligonucleotide), as will be understood by those of ordinary skill in the art, refers to the ability of a first nucleotide sequence to hybridize with a second nucleotide sequence and form a duplex structure under certain conditions. This includes base pairing of the first nucleotide sequence with the second nucleotide sequence over the entire length of the first or second nucleotide sequence. Such sequences may be referred to herein as "fully complementary" relative to each other. In this article, where a first sequence is referred to as "substantially complementary" relative to a second sequence, the two sequences may be fully complementary, or they may have 70% or higher nucleotide identity while retaining the ability to hybridize under conditions most relevant to their ultimate target. However, when two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs should not be considered mismatches with respect to determining complementarity. For example, a double-stranded RNA (dsRNA) comprising a first oligonucleotide of 21 nucleotides in length and a second oligonucleotide of 23 nucleotides in length, wherein the second oligonucleotide comprises a 21-nucleotide sequence that is completely complementary to the first oligonucleotide, may still be referred to as "completely complementary" for the purposes of this disclosure. As long as the above-mentioned requirements regarding its hybridization ability are met, the "complementary" sequence may also include non-Watson-Crick base pairs and / or base pairs formed by non-natural and modified nucleotides, or may be formed entirely of non-Watson-Crick base pairs and / or base pairs formed by non-natural and modified nucleotides. With respect to base pairing between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, the terms "complementary," "completely complementary," and "substantially complementary" may be used, as will be understood from the context in which they are used. As used herein, a polynucleotide that is "substantially complementary to at least a portion of an mRNA" refers to a polynucleotide that is substantially complementary to a contiguous portion of the target mRNA. As used herein, the term "double-stranded RNA" or "dsRNA" refers to a complex of ribonucleic acid molecules having a duplex structure comprising two antiparallel and substantially complementary (as defined above) nucleic acid chains. The two chains forming the duplex structure can be different parts of a larger RNA molecule, or they can be separate RNA molecules. In the case of separate RNA molecules, such dsRNAs are referred to as short interfering RNAs (siRNAs) in the literature. In the case where the two chains are part of a larger molecule, and therefore are connected by an uninterrupted nucleotide chain between the 3'-end of the first chain and the 5'-end of the second chain forming the duplex structure, the connecting RNA chains are referred to as "hairpin loops," "short hairpin RNAs," or "shRNAs."In the case where the two chains are covalently linked by a method different from the uninterrupted nucleotide chain between the 3'-end of the first chain and the 5'-end of the second chain forming a duplex structure, the connection structure is called a "joint". The RNA chain may have the same or different numbers of nucleotides. The maximum number of base pairs is the number of oligonucleotides in the shortest chain of the dsRNA minus any overhangs present in the duplex. In addition to the duplex structure, the dsRNA may also include one or more nucleotide overhangs. In addition, as used herein, the term "dsRNA" may include chemical modifications to ribonucleotides, including substantial modifications on multiple nucleotides, and includes modifications disclosed herein or known in the art of all types. For the purposes of this disclosure, any such modifications used in siRNA type molecules are encompassed within the "dsRNA". In some embodiments, the internucleotide bond in the dsRNA may be modified, for example, modified as described herein.

[0193] In the scope of the present disclosure, " identity percentage " between two nucleotide sequences means the percentage of identical nucleotide residues between two sequences to be compared obtained after the best comparison, and this percentage is purely statistical, and the difference between the two sequences is randomly distributed along its length. Traditionally, the comparison of two nucleotide sequences is carried out by comparing after the sequence is carried out the best comparison, and the comparison can be carried out by fragment or by using " comparison window ". Except manual comparison, it is also possible to use the local homology algorithm of Smith and Waterman (1981), the local homology algorithm of Neddleman and Wunsch (1970), the similarity search method of Pearson and Lipman (1988), or by using the computer software (Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, WI) of these algorithms, GAP, BESTFIT, FASTA and TFASTA, or by comparing software BLAST NR or BLAST P) for the best comparison of the sequence compared.

[0194] The percent identity between two nucleic acid sequences is determined by comparing two optimally aligned sequences, wherein the nucleic acid sequence to be compared may have additions or deletions compared to the reference sequence to provide for optimal alignment between the two sequences. The percent identity is calculated by determining the number of positions at which the nucleotide residues are identical between the two sequences, preferably between the two complete sequences, dividing the number of identical positions by the total number of positions in the comparison window, and then multiplying the result by 100 to obtain the percent identity between the two sequences.

[0195] As contemplated herein, a nucleotide sequence having at least 70% nucleotide identity to a reference sequence encompasses 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 to the reference sequence.

[0196] In some embodiments, the dsRNA comprises modified ribonucleosides, including deoxyribonucleosides, including, for example, deoxyribonucleoside overhangs, one or more deoxyribonucleosides within the double-stranded portion of the dsRNA, etc. However, it is understood that in no case does the term "dsRNA" encompass double-stranded DNA molecules.

[0197] As used herein, the term "nucleotide overhang" refers to unpaired nucleotides or nucleotides that protrude from the duplex structure of a dsRNA when the 3'-end of the first strand of a dsRNA extends beyond the 5'-end of the second strand, or vice versa. "Blunt" or "blunt-end" means that there are no unpaired nucleotides at that end of the dsRNA, i.e., there are no nucleotide overhangs. A "blunt-ended" dsRNA is a double-stranded dsRNA over its entire length, i.e., there are no nucleotide overhangs at either end of the molecule. For the sake of clarity, chemical caps or non-nucleotide chemical moieties conjugated to the 3'-end and / or 5'-end of the dsRNA chain are not taken into account when determining whether a dsRNA has an overhang or is blunt-ended.

[0198] As used herein, the term "antisense strand" in dsRNA refers to the dsRNA strand that contains a sequence that is substantially complementary to the target sequence. The other strand in the dsRNA is the "sense strand."

[0199] As used herein, the term "introduced into a cell" means promoting uptake or absorption into the cell as understood by one of ordinary skill in the art. The absorption or uptake of dsRNA can occur by unassisted diffusion or active cellular processes or by adjuvants or devices. The meaning of this term is not limited to cells in vitro; dsRNA can also be "introduced into a cell" where the cell is part of a living organism. In this case, introduction into a cell will include delivery to the organism. For example, for in vivo delivery, the dsRNA can be injected into a tissue site or administered systemically. In vivo delivery can also be mediated by a β-glucan delivery system (see, for example, Tesz, GJ et al., 2011, Biochem J. 436 (2): 351-62). In vitro introduction into cells includes methods known in the art, such as electroporation and lipofection. Other methods are described below or are known in the art.

[0200] As defined herein, the term "inhibit expression" or "inhibit expression of ... " refers to at least partially inhibiting the expression of a target gene when it relates to a target gene, which is manifested by a reduction in the number of mRNA transcribed from the target gene. As used herein, the term "inhibit" is used interchangeably with "reduce," "silence," "downregulate," "suppress," "knock-down," 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 can be given according to the reduction of a parameter functionally connected to target gene transcription, for example, the amount of protein encoded by the target gene secreted by the cell, or the number of cells showing a certain phenotype such as apoptosis. In principle, the silencing of a target gene can be determined in any cell expressing the target by constitutive or genomic engineering and by any appropriate assay method. However, when a reference is needed to determine whether a given dsRNA inhibits the expression of a target gene to some extent and is therefore encompassed by the present disclosure, the assay provided in the following examples should serve as such a reference.

[0201] As used herein, in the context of target gene expression, the terms "treat," "treatment," and the like refer to alleviating or relieving a pathological process mediated by target gene expression. In the context of the present disclosure, in relation to any other condition described below (except a pathological process mediated by target expression), the terms "treat," and the like refer to alleviating or relieving one or more symptoms associated with such a condition.

[0202] As used herein, the terms "preventing" or "delaying the progression of a disease" (and grammatical variations thereof) with respect to a disease or condition refer to prophylactic treatment of a disease, for example, in an individual suspected of having the disease or at risk of developing the disease. Prevention can include, but is not limited to, preventing or delaying the onset or progression of a disease and / or maintaining one or more symptoms of a disease at a desired or subpathological level.

[0203] As used herein, the terms "therapeutically effective amount" and "prophylactically effective amount" refer to an amount that provides a therapeutic benefit in the treatment, prevention, or management of a pathological process mediated by target gene expression or a significant symptom of a pathological process mediated by target gene expression. The specific amount that is therapeutically effective can be readily determined by an ordinary physician and may depend on factors such as the type and stage of the pathological process mediated by target gene expression, the patient's medical history and age, and the administration of other therapeutic agents that inhibit biological processes mediated by the target gene.

[0204] As used herein, the term "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans 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.

[0205] The terms "internucleoside linkage," "internucleoside linking group," "internucleotide linkage," or "internucleotide linking group" are used interchangeably herein and refer to any linker or bond between two nucleoside (i.e., heterocyclic base moiety and sugar moiety) units, as known in the art, including but not limited to, phosphate, analogs of phosphate, phosphorothioate, phosphonate, guanidinium, hydroxylamine, hydroxyhydrazine, amide, carbamate, alkyl, and substituted alkyl linkages. A bond between two nucleosides, between two nucleoside analogs, or between a nucleoside and a nucleoside analog can be referred to as an "internucleoside linking group."

[0206] Internucleoside bonds constitute the backbone of nucleic acid molecules. An internucleoside linking group refers to a chemical group that connects two adjacent nucleoside residues contained in a nucleic acid molecule, including (i) a chemical group that connects two adjacent nucleoside residues, (ii) a chemical group that connects a nucleoside residue to an adjacent nucleoside analog residue, and (iii) a chemical group that connects a first nucleoside analog residue to a second nucleoside analog residue, which nucleoside analog residues may be the same or different. Nucleoside analog residues encompass compounds of formula (II) disclosed herein. On the one hand, the nucleotides of the siNA molecule of the present invention can be connected to adjacent nucleotides through a 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 (referred to herein as a 3' internucleoside bond). As used herein, a 3'-5' internucleoside bond refers to an internucleoside bond connecting two adjacent nucleoside units, wherein the bond is located between the 3'-carbon of the sugar moiety of the first nucleoside and the 5'-carbon of the sugar moiety of the second nucleoside. In another aspect, the nucleotides (including nucleotide analogs) of the siNA molecules of the invention can be linked to adjacent nucleotides (including nucleotide analogs) via a 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 (referred to herein as a 2' internucleoside bond). As used herein, a 2'-5' internucleoside bond refers to an internucleoside bond connecting two adjacent nucleoside units, wherein the bond is located between the 2'-carbon of the sugar moiety of the first nucleoside and the 5'-carbon of the sugar moiety of the second nucleoside.

[0207] As used herein, the term "internucleoside linking group" encompasses both phosphorus-containing and non-phosphorus-containing internucleoside linking groups.

[0208] In some embodiments, phosphorus-containing internucleoside linking groups encompass phosphodiesters, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates (including 3'-alkylene phosphonates, 5'-alkylene phosphonates, and chiral phosphonates), phosphinates, phosphoramidates (including 3'-aminophosphoramidate and aminoalkylphosphoramidates), thionylphosphoramidates, thionylalkylphosphonates, thionylalkylphosphotriesters, selenophosphates, and boranophosphates with normal 3'-5' linkages and their 2'-5' linked analogs.

[0209] Representative U.S. patents that teach the preparation of the above-mentioned phosphorus-containing internucleoside linkages 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, and 5,625,050, each of which is incorporated herein by reference.

[0210] In one embodiment, the non-phosphodiester backbone linkage is selected from phosphorothioate, phosphorodithioate, alkyl-phosphonate, and phosphoramidate backbone linking groups.

[0211] In one embodiment, the phosphorus-containing internucleoside linking groups encompass phosphodiesters, phosphotriesters, and phosphorothioates.

[0212] In some embodiments, the oligonucleotides of the present invention include one or more internucleoside linking groups that do not include a phosphorus atom. Such oligonucleotides include but are not limited to those formed by short-chain alkyl or cycloalkyl internucleoside linking groups, mixed heteroatoms and alkyl or cycloalkyl internucleoside linking groups, or one or more short-chain heteroatoms or heterocyclic internucleoside linking groups. These include those with the following: siloxane backbone; sulfide, sulfoxide and sulfone backbone; formacetyl and thioformacetyl backbone; methylene formacetyl and thioformacetyl backbone; ribose acetyl backbone; olefin-containing backbone; sulfamic acid ester backbone; methylene imino and methylene hydrazine backbone; sulfonic acid ester and sulfonamide backbone; amide backbone; and other N, O, S and CH2 components with mixing. Representative U.S. patents that teach the preparation of the above-mentioned phosphorus-free internucleoside 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; 5,5 5,677,439, each of which is incorporated herein by reference.

[0213] In one embodiment, the oligonucleotide of the present invention comprises one or more nonionic neutral internucleoside linking groups. Neutral internucleoside linking groups encompass nonionic linking groups, which include siloxanes (dialkylsiloxanes), carboxylates, formamides, sulfides, sulfonates, and amides (e.g., referring to: Carbohydrate Modifications in Antisense Research; YS Tanghvi and PDCook Eds.ACS Symposium Series 580; Chapters 3 and 4, (pp.40-65)). Other neutral internucleoside linking groups encompass nonionic bonds comprising mixed N, O, S, and CH2 components.

[0214] Compounds of formula (I)

[0215] The present invention relates to compounds of general formula (I):

[0216]

[0217] in:

[0218] -B is a heterocyclic nucleobase;

[0219] - P1 and P2 are each independently H, a reactive phosphorus group or a protecting group;

[0220] -Y is O, NH, NR1 or NC(=O)-R1, wherein R1 is:

[0221] a (C1-C20)alkyl group, which is optionally unsubstituted or substituted by one or more groups selected from the group consisting of a halogen atom, a (C1-C6)alkyl group, a (C3-C8)cycloalkyl group, a (C3-C14)heterocycle, a (C6-C14)aryl group, a (C5-C14)heteroaryl group, -O-Z1, -N(Z1)(Z2), -S-Z1, -CN, -C(=J)-O-Z1, -OC(=J)-Z1, -C(=J)-N(Z1)(Z2) and -N(Z1)-C(=J)-Z2, wherein

[0222] J is O or S,

[0223] Z1 and Z2 are each independently H, a (C1-C6)alkyl group, which is optionally substituted with one or more groups selected from the group consisting of halogen atoms and (C1-C6)alkyl groups,

[0224] a (C3-C8)cycloalkyl group, which is optionally substituted by one or more groups selected from the group consisting of halogen atoms and (C1-C6)alkyl groups, or

[0225] -[C(=O)]m-R2-(O-CH2-CH2)p-R3, wherein

[0226] m is an integer, with a value of 0 or 1.

[0227] p is an integer ranging from 0 to 10,

[0228] R2 is a (C1-C20)alkylene group, which is optionally substituted by a (C1-C6)alkyl group, -O-Z3, -N(Z3)(Z4), -S-Z3, -CN, -C(=K)-O-Z3, -OC(=K)-Z3, -C(=K)-N(Z3)(Z4), -N(Z3)-C(=K)-Z4, wherein

[0229] K is O or S,

[0230] Z3 and Z4 are each independently H, a (C1-C6)alkyl group, which is optionally substituted with one or more groups selected from the group consisting of halogen atoms and (C1-C6)alkyl groups,

[0231] and

[0232] R3 is selected from a hydrogen atom, a (C1-C6)alkyl group, a (C1-C6)alkoxy group, a (C3-C8)cycloalkyl group, a (C3-C14)heterocycle, a (C6-C14)aryl group or a (C5-C14)heteroaryl group, or

[0233] R3 is the cell targeting moiety,

[0234] -X1 and X2 are each independently a hydrogen atom, a -(C1-C6)alkyl group, and

[0235] -Ra, Rb, Rc and Rd are each independently H or a (C1-C6)alkyl group.

[0236] For the purposes of this disclosure, compounds of formula (I) are encompassed by the term "nucleotide precursors." For the purposes of this disclosure, compounds of formula (I) wherein the group R3 is present and contemplates a cell targeting moiety are encompassed by the term "targeted nucleotide precursors."

[0237] The compounds of Formula (I) and (II) as disclosed herein encompass stereoisomers thereof, including the (2S,6R) stereoisomer thereof and the (2R,6R) stereoisomer thereof, as specifically depicted in the following formula, which designates the position numbers and chiral centers of the compounds of Formula (I) and (II):

[0238]

[0239] As shown in the Examples herein, when incorporated into oligonucleotides, the (2S,6R) stereoisomer of the compound of formula (I) and the (2R,6R) stereoisomer of the compound of formula (I) have the same ability to generate siRNA, thereby effectively inhibiting target mRNA.

[0240] As mentioned above, the inventors have conceived that specific nucleotide precursors of formula (I) containing a dioxane ring or a morpholino ring can be used as building blocks for the synthesis of gene silencing oligonucleotides, in particular for the synthesis of siRNA.

[0241] Thus, in some embodiments of compounds of formula (I), which may be referred to herein as "dioxane analogs," Y is O. Embodiments of such nucleotide precursors are referred to in the present disclosure as pre-1B1, wherein B is as defined in formula (I), such as pre-1T1 when B consists of a thymidine group.

[0242] In other embodiments of compounds of Formula (I), which may be referred to herein as "morpholino analogs," Y is NH, NR1, or NC(=O)R1.

[0243] In the morpholino analogs of formula (I), the nitrogen atom is preferably functionalized to improve the properties of the resulting morpholino analog-containing oligonucleotides and in particular the resulting morpholino analog-containing siRNAs.

[0244] In some embodiments of the compounds of formula (I), these compounds are morpholino analogs of the present disclosure that do not comprise a cell targeting moiety. According to these embodiments, the group R3, when present, does not represent a cell targeting moiety.

[0245] Thus, in some preferred embodiments of the morpholino analogs of formula (I), Y is NH, NR1 or NC(=O)-R1, and R1 is as defined for general formula (I).

[0246] In some embodiments, wherein Y is NR1, R1 is:

[0247] a (C1-C20)alkyl group, which is optionally substituted by one or more groups selected from the group consisting of a halogen atom, a (C1-C6)alkyl group, a (C3-C8)cycloalkyl group, a (C3-C14)heterocycle, a (C6-C14)aryl group, a (C5-C14)heteroaryl group, -O-Z1, -N(Z1)(Z2), -S-Z1, -CN, -C(=J)-O-Z1, -OC(=J)-Z1, -C(=J)-N(Z1)(Z2) and -N(Z1)-C(=J)-Z2, wherein

[0248] J is O or S, and

[0249] Z1 and Z2 are each independently H, a (C1-C6)alkyl group, which is optionally substituted with one or more groups selected from the group consisting of halogen atoms and (C1-C6)alkyl groups, or

[0250] a (C3-C8)cycloalkyl group, which is optionally substituted by one or more groups selected from the group consisting of halogen atoms and (C1-C6)alkyl groups,

[0251] -[C(=O)]m-R2-(O-CH2-CH2)p-R3, wherein

[0252] m is an integer, and its value is 0 or 1

[0253] p is an integer ranging from 0 to 10

[0254] R2 is a (C1-C20)alkylene group, which is optionally substituted by a (C1-C6)alkyl group, -O-Z3, -N(Z3)(Z4), -S-Z3, -CN, -C(=K)-O-Z3, -OC(=K)-Z3, -C(=K)-N(Z3)(Z4), -N(Z3)-C(=K)-Z4, wherein

[0255] K is O or S,

[0256] Z3 and Z4 are each independently H, a (C1-C6)alkyl group, which is optionally substituted with one or more groups selected from the group consisting of halogen atoms and (C1-C6)alkyl groups,

[0257] and

[0258] R3 is selected from a hydrogen atom, a (C1-C6)-alkyl group, a (C1-C6)-alkoxy group, a (C3-C8)-cycloalkyl group, a (C3-C14)-heterocycle, a (C6-C14)-aryl group or a (C5-C14)-heteroaryl group,

[0259] X1 and X2 are each independently a hydrogen atom, a (C1-C6) alkyl group

[0260] Ra, Rb, Rc and Rd are each independently H or a (C1-C6)alkyl group.

[0261] As contemplated herein, a (C1-C20)alkyl group, which can be an unsubstituted alkyl group or a substituted alkyl group, includes C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, and C20 alkyl groups.

[0262] As contemplated herein, a (C1-C6)alkyl group, which may be an unsubstituted alkyl group or a substituted alkyl group, includes C1, C2, C3, C4, C5, and C6 alkyl groups.

[0263] As contemplated herein, a (C3-C8)cycloalkyl group, which may be an unsubstituted cycloalkyl group or a substituted cycloalkyl group, includes C3, C4, C5, C6, C7, and C8 cycloalkyl groups.

[0264] As contemplated herein, (C3-C14)heterocycles, which may be unsubstituted or substituted, include C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, and C14 heterocycles.

[0265] As contemplated herein, a (C6-C14)aryl group, which can be an unsubstituted aryl group or a substituted aryl group, includes C6, C7, C8, C9, C10, C11, C12, C13, and C14 aryl groups.

[0266] As contemplated herein, a (C5-C14)heteroaryl group, which can be an unsubstituted heteroaryl group or a substituted heteroaryl group, includes C5, C6, C7, C8, C9, C10, C11, C12, C13, and C14 heteroaryl groups.

[0267] In some embodiments of the compound of Formula (I), wherein Y is NR1, R1 is an optionally substituted (C1-C20)alkyl group, and P1, P2, Ra, Rb, Rc, Rd, X1, X2 and B are as defined for Formula (I).

[0268] In some of these embodiments, where Y is NR1, R1 is an unsubstituted (C1-C20)alkyl group.

[0269] In some of the embodiments, wherein Y is NR1, R1 is an unsubstituted (C1-C16)alkyl group, including an alkyl group selected from methyl, isopropyl, butyl, octyl, hexadecyl, and P1, P2, Ra, Rb, Rc, Rd, X1, X2 and B have the same meanings as defined for formula (I).

[0270] In some embodiments, wherein Y is NR1, R1 is a methyl group, Ra, Rb, Rc, Rd, X1 and X2 are hydrogen atoms, and P1 and P2 are as defined for formula (I). Embodiments of such nucleotide precursors in the present disclosure are referred to as pre-1B2, wherein B has the same definition as in formula (I); for example, when B consists of a thymidine group, embodiments of such nucleotide precursors in the present disclosure are referred to as pre-1T2.

[0271] In some embodiments, wherein Y is NR1, R1 is an isopropyl group, Ra, Rb, Rc, Rd, X1 and X2 are hydrogen atoms, and P1 and P2 are as defined for formula (I). Embodiments of such nucleotide precursors in the present disclosure are referred to as pre-1B3, wherein B has the same definition as in formula (I); for example, embodiments of such nucleotide precursors in the present disclosure are referred to as pre-1T3, wherein B consists of a thymidine group; as pre-1U3, wherein B consists of a uracil group; as pre-1G3 when B consists of a guanyl group; as pre-1C3, wherein B consists of a cytosyl group; as pre-1A3, wherein B consists of an adenyl group.

[0272] In some embodiments, wherein Y is NR1, R1 is a butyl group, Ra, Rb, Rc, Rd, X1 and X2 are hydrogen atoms, and P1 and P2 are as defined for formula (I). Embodiments of such nucleotide precursors in the present disclosure are referred to as pre-1B6, wherein B has the same definition as in formula (I); for example, embodiments of such nucleotide precursors in the present disclosure are referred to as pre-1T6, wherein B consists of a thymidine group.

[0273] In some embodiments, wherein Y is NR1, R1 is an octyl group, Ra, Rb, Rc, Rd, X1 and X2 are hydrogen atoms, and P1 and P2 are as defined for formula (I). Embodiments of such nucleotide precursors in the present disclosure are referred to as pre-1B7, wherein B has the same definition as in formula (I); for example, embodiments of such nucleotide precursors in the present disclosure are referred to as pre-1T7, wherein B consists of a thymidine group.

[0274] In some embodiments, wherein Y is NR1, R1 is a linear C16-alkyl group, Ra, Rb, Rc, Rd, X1 and X2 are hydrogen atoms, and P1 and P2 are as defined for Formula (I). Embodiments of such nucleotide precursors in the present disclosure are referred to as pre-1B8, wherein B has the same definition as in Formula (I); for example, embodiments of such nucleotide precursors in the present disclosure are referred to as pre-1T8, wherein B consists of a thymidine group.

[0275] In further embodiments of the compounds of formula (I), wherein Y is NR1, R1 is a (C1-C20)alkyl group, which is substituted as defined in formula (I), including a C1, C2 or C3 alkyl group substituted as defined in formula (I).

[0276] In some of these further embodiments, R1 is a (C1-C20)alkyl group substituted by one or more groups selected from the group consisting of a halogen atom, a (C1-C6)alkyl group, a (C3-C8)cycloalkyl group, a (C3-C14)heterocycle, a (C6-C14)aryl group and a (C5-C14)heteroaryl group, and P1, P2, Ra, Rb, Rc, Rd, X1, X2 and B have the same meanings as defined for formula (I).

[0277] In some of these further embodiments, R1 is a (C1-C20)alkyl group substituted with a (C6-C14)aryl group, and P1, P2, Ra, Rb, Rc, Rd, X1, X2 and B have the same meanings as defined for formula (I).

[0278] In some embodiments of the compound of formula (I), wherein Y is NR1, R1 is a (C1-C20)alkyl group substituted with a (C6-C14)aryl group. These embodiments encompass compounds of formula (I) wherein Y is NR1, R1 is a methylene group substituted with an aryl group. These embodiments also encompass compounds of formula (I) wherein Y is NR1, R1 is a (C1-C20)alkyl group substituted with a phenyl group.

[0279] In some embodiments of the compound of formula (I), wherein Y is NR1, R1 is a methyl group substituted with an unsubstituted phenyl group, Ra, Rb, Rc, Rd, X1 and X2 are each a hydrogen atom, and P1 and P2 are as defined in formula (I). Embodiments of such nucleotide precursors in the present disclosure are referred to as pre-1B5, wherein B has the same definition as in formula (I); for example, embodiments of such nucleotide precursors in the present disclosure are referred to as pre-1T5, wherein B consists of a thymidine group.

[0280] In a further embodiment of the compound of formula (I), wherein Y is NR1, R1 is a (C3-C8)cycloalkyl group, which is optionally substituted by one or more groups selected from the group consisting of halogen atoms and (C1-C6)alkyl groups, and P1, P2, Ra, Rb, Rc, Rd, X1, X2 and B have the same meanings as defined for the general formula (I).

[0281] In some of these further embodiments of compounds of Formula (I), wherein Y is NR1, R1 is cyclohexyl.

[0282] In some of these further embodiments of compounds of formula (I), wherein Y is NR1, R1 is unsubstituted cyclohexyl, Ra, Rb, Rc, Rd, X1, X2 are each a hydrogen atom, and P1 and P2 are as defined for formula (I).

[0283] An embodiment of such a nucleotide precursor is referred to in the present disclosure as pre-1B4, wherein B has the same definition as in formula (I); for example, an embodiment of such a nucleotide precursor is referred to in the present disclosure as pre-1T4, wherein B consists of a thymidine group.

[0284] In some other embodiments of the morpholino analog of formula (I), Y is NC(═O)-R1, wherein R1 is a (C1-C20)alkyl group, which is optionally substituted with one or more groups selected from the group consisting of a halogen atom, a (C1-C6)alkyl group, a (C3-C8)cycloalkyl group, a (C3-C14)heterocycle, a (C6-C14)aryl group, a (C5-C14)heteroaryl group, -O-Z1, -N(Z1)(Z2), -S-Z1, -CN, -C(═J)-O-Z1, -OC(═J)-Z1, -C(═J)-N(Z1)(Z2), -N(Z1)-C(═J)-Z2, wherein

[0285] J is O or S,

[0286] Z1 and Z2 are each independently H, a (C1-C6)alkyl group, which is optionally substituted with one or more groups selected from the group consisting of halogen atoms and (C1-C6)alkyl groups, and

[0287] R1 is a (C3-C8)cycloalkyl group, which is optionally substituted by one or more groups selected from the group consisting of a halogen atom or a (C1-C6)alkyl group, and

[0288] P1, P2, Ra, Rb, Rc, Rd, X1, X2 and B have the same meanings as defined for the general formula (I).

[0289] In some of the embodiments, wherein Y is NC(═O)—R1, R1 is an optionally substituted (C1-C20)alkyl group, including an optionally substituted (C1-C15)alkyl group, and P1, P2, Ra, Rb, Rc, Rd, X1, X2 and B have the same meanings as defined for formula (I).

[0290] According to some of these embodiments, wherein Y is NC(═O)—R1, R1 is selected from a methyl group and a pentadecyl group, and P1, P2, Ra, Rb, Rc, Rd, X1, X2 and B have the same meanings as defined for formula (I).

[0291] These embodiments encompass compounds of formula (I) wherein Y is NC(═O)—R1, R1 is a methyl group, Ra, Rb, Rc, Rd, X1, X2 each represent a hydrogen atom, and B, P1 and P2 are as defined in formula (I).

[0292] Embodiments of such nucleotide precursors in the present disclosure are referred to as pre-1B9, wherein B has the same definition as in formula (I); for example, embodiments of such nucleotide analogs in the present disclosure are referred to as pre-1T9, wherein B consists of a thymidine group. These embodiments also encompass compounds of formula (I), wherein Y is NC(=O)-R1, R1 is a pentadecyl group, Ra, Rb, Rc, Rd, X1, X2 each represents a hydrogen atom, and B, P1, and P2 are as defined in formula (I). Embodiments of such nucleotide precursors in the present disclosure are referred to as pre-1B10, wherein B has the same definition as in formula (I); for example, embodiments of such nucleotide precursors in the present disclosure are referred to as pre-1T10, wherein B consists of a thymidine group.

[0293] In the compound of formula (I), B is a heterocyclic core base moiety. As used herein, the term "heterocyclic core base" refers to an optionally substituted nitrogen-containing heterocycle, which is covalently linked to a dioxane ring or a morpholino ring. In some embodiments, the heterocyclic core base can be selected from optionally substituted purine-bases and optionally substituted pyrimidine-bases. The term "purine-base" is used as the common meaning understood by those skilled in the art herein, and includes its tautomers. Similarly, the term "pyrimidine-base" is used as the common meaning understood by those skilled in the art herein, and includes its tautomers. The non-limiting list of optionally substituted purine-bases includes purine, adenine, guanine, hypoxanthine, xanthine, alloxanthine, 7-alkylguanines (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). Non-limiting examples of other heterocyclic bases include diaminopurine, 8-oxo-N 6 Alkyl adenine (e.g., 8-oxo-N 6 methyladenine), 7-deazaxanthine, 7-deazaguanine, 7-deazaadenine, N4,N4-ethanoylcytosine, N <6> ,N <6> -ethoxy-2,6-diaminopurine, 5-halouracil (e.g., 5-fluorouracil and 5-bromouracil), pseudoisocytosine, isocytosine, isoguanine, 1,2,4-triazole-3-carboxamide, and other heterocyclic bases described in U.S. Patent Nos. 5,432,272 and 7,125,855, which are incorporated herein by reference for the limited purpose of disclosing additional heterocyclic bases. In some embodiments, the heterocyclic base may be optionally substituted with an amine or enol protecting group. In some embodiments, B is selected from pyrimidine, substituted pyrimidine, purine, and substituted purine, and its amino group (when present) is optionally protected by a protecting group.

[0294] In a preferred embodiment, B is selected from adenine, thymine, uracil, guanine and cytosine (i.e., adenyl, thymine, uracil, guaninyl and cytosine). Adenine, guanine and cytosine are optionally protected by an amine protecting group. Amine protecting groups encompass acyl groups, such as benzoyl, phenylacetyl and isobutyryl protecting groups, or formamidine protecting groups, such as N,N-dimethyl-formamidine.

[0295] As already mentioned, in the compounds of formula (I), the groups P1 and P2 are each independently a hydrogen atom, a reactive phosphorus group or a protecting group.

[0296] As used herein, a "reactive phosphorus group" refers to a phosphorus-containing group contained in a nucleotide unit or a nucleotide analog unit that can react with a hydroxyl or amine group contained in another molecule, particularly another nucleotide unit or another nucleotide analog, via a nucleophilic attack reaction. Typically, such a reaction produces an ester-type internucleoside bond connecting the first nucleotide unit or the first nucleotide analog unit to the second nucleotide unit or the second nucleotide analog unit.

[0297] In some embodiments, the reactive phosphorus group can be selected from phosphoramidites, H-phosphonates, alkyl-phosphonates, phosphates or phosphate mimetics, including but not limited to: natural phosphates, phosphorothioates, phosphorodithioates, boranophosphates, boranophosphorothioates, phosphonates, halogen-substituted phosphonates and phosphates, phosphoramidates, phosphodiesters, phosphotriesters, phosphorothioate diesters, phosphorothioate triesters, diphosphates and triphosphates. The protecting groups encompass hydroxyl, amine and phosphoramidite protecting groups which may be selected from 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, pivaloyl (Piv), tetrahydropyranyl (THP), triphenylmethyl (Trt), methoxytrityl [(4-methoxyphenyl)diphenylmethyl] (MMT), dimethoxytrityl, [bis-(4-methoxyphenyl)phenylmethyl (DMT), trimethylsilyl ether (TMS), tert-butyldimethylsilyl ether (TBDMS), tri-iso-propylsilyloxymethyl ether (TOM), tri-isopropylsilyl ether (TIPS), methyl ether, ethoxyethyl ether (EE) N,N-dimethylformamidine and 2-cyanoethyl (CE).

[0298] In some embodiments of the compound of formula (I), wherein Y, B, Ra, Rb, Rc, Rd, X1 and X2 are as defined for general formula (I), one of P1 or P2 is an O-4,4'-dimethoxytrityl group (DMT) and the other of P1 and P2 is H, a reactive phosphorus group or a protecting group.

[0299] In some embodiments of the compound of Formula (I), wherein Y, B, Ra, Rb, Rc, Rd, X1 and X2 are as defined for general formula (I), one of P1 and P2 is a 2-cyanoethyl-N,N-diisopropylphosphoramidite group, and the other of P1 and P2 is a protecting group.

[0300] In some embodiments of the compound of formula (I), wherein Y, B, Ra, Rb, Rc, Rd, X1 and X2 are as defined for general formula (I), one of P1 and P2 is a 2-cyanoethyl-N,N-diisopropylphosphoramidite group and the other of P1 and P2 is an O-4,4'-dimethoxytrityl group.

[0301] Further, Ra, Rb, Rc and Rd are each independently H or a (C1-C6)alkyl group, and preferably H or an unsubstituted (C1-C6)alkyl group.

[0302] As used herein, a (C1-C6)alkyl group encompasses an alkyl group selected from C1, C2, C3, C4, C5, and C6 alkyl groups.

[0303] In the most preferred embodiment, X1 and X2 both represent hydrogen atoms.

[0304] In the most preferred embodiment, Ra, Rb, Rc and Rd all represent hydrogen atoms.

[0305] Embodiments of compounds of formula (I) comprising targeted nucleotide analogues

[0306] As previously specified herein, the present disclosure encompasses compounds of formula (I), wherein:

[0307] -B is a heterocyclic nucleobase;

[0308] - P1 and P2 are each independently H, a reactive phosphorus group or a protecting group;

[0309] -Y is NR1 and R1 is a group -[C(=O)]m-R2-(O-CH2-CH2)p-R3, wherein

[0310] m is an integer, with a value of 0 or 1.

[0311] p is an integer ranging from 0 to 10,

[0312] R2 is a (C1-C20)alkylene group, which is optionally substituted by a (C1-C6)alkyl group, -O-Z3, -N(Z3)(Z4), -S-Z3, -CN, -C(=K)-O-Z3, -OC(=K)-Z3, -C(=K)-N(Z3)(Z4), -N(Z3)-C(=K)-Z4, wherein

[0313] K is O or S,

[0314] Z3 and Z4 are each independently H, a (C1-C6)alkyl group, which is optionally substituted with one or more groups selected from the group consisting of halogen atoms and (C1-C6)alkyl groups,

[0315] and

[0316] R3 is the cell targeting moiety,

[0317] - X1 and X2 are each independently a hydrogen atom, a (C1-C6) alkyl group, and

[0318] -Ra, Rb, Rc and Rd are each independently H or a (C1-C6)alkyl group.

[0319] These compounds of formula (I) are encompassed within a more general family of compounds that may be referred to in the present disclosure as "targeted nucleotide precursors." Such compounds of formula (I), wherein the group R3 is present and represents a cell targeting moiety, may be referred to in the present disclosure as "targeted nucleotide precursors of formula (I)" or "targeted nucleotide precursor (I)."

[0320] Compounds of formula (I) that do not comprise a group R3 representing a cell targeting moiety are not targeted nucleotide precursors and are referred to in this disclosure as "non-targeted nucleotide precursors of formula (I)" or "non-targeted nucleotide precursor (I)".

[0321] In some embodiments of the targeted nucleotide precursors of formula (I), R1 is a group -[C(=O)]m-R2-(O-CH2-CH2)p-R3, m is 0, p is 0, R3 is a cell targeting moiety, and B, P1, P2, Ra, Rb, Rc, Rd, X1, X2 and R2 are as in the general definition of compounds of formula (I). In some embodiments, R2 is an ethylene group, and X1 and X2 are both hydrogen atoms. In some other embodiments of these embodiments, R2 is a pentylene group, and X1 and X2 are both hydrogen atoms. In some embodiments, R2 is a (C12)alkylene group, and X1 and X2 are both hydrogen atoms.

[0322] In some embodiments of the compound of formula (I), R1 is a group -[C(=O)]m-R2-(O-CH2-CH2)p-R3, m is 0, p is an integer selected from 1, 2, 3 and 4, R3 is a cell targeting moiety, and B, P1, P2, Ra, Rb, Rc, Rd, X1, X2 and R2 are as in the general definition of compounds of formula (I). In some embodiments, R2 is an ethylene group, p is 1, and X1 and X2 are both hydrogen atoms. In some embodiments, R2 is an ethylene group, p is 2, and X1 and X2 are both hydrogen atoms. In some embodiments, R2 is an ethylene group, p is 3, and X1 and X2 are both hydrogen atoms. In some embodiments, R2 is an ethylene group, p is 4, and X1 and X2 are both hydrogen atoms.

[0323] In some embodiments of the compound of formula (I), R1 is a group -[C(=O)]m-R2-(O-CH2-CH2)p-R3, m is 1, p is 0, R3 is a cell targeting moiety, and R2, B, P1, P2, Ra, Rb, Rc, Rd, X1, X2 are as in the general definition of compounds of formula (I). In some of these embodiments, R2 is a butylene group, X1 and X2 each represent a hydrogen atom, and B, P1, P2, Ra, Rb, Rc and Rd are as defined for general formula (I). In some further embodiments of these embodiments, R2 is a (C11)alkylene group, X1 and X2 both represent a hydrogen atom, and B, P1, P2, Ra, Rb, Rc and Rd are as defined for general formula (I)). In some further embodiments of these embodiments, R2 is methylene, X1 and X2 both represent hydrogen atoms, and B, P1, P2, Ra, Rb, Rc and Rd are as defined for formula (I).

[0324] In some embodiments of compounds of formula (I), wherein R1 is a group -[C(=O)]m-R2-(O-CH2-CH2)p-R3, m is 1, p is selected from the integers 1 and 2, R3 is a cell targeting moiety, and R2, B, Pl, P2, Ra, Rb, Rc, Rd, Xi, X2 are as in the general definition of compounds of formula (I). In some of these embodiments, R2 is a methylene group, p is 2, R3 is a cell targeting moiety, and B, Pl, P2, Ra, Rb, Rc, Rd, Xi, X2 are as defined for formula (I). In some other embodiments of these embodiments, R2 is a methylene group, p is 1, R3 is a cell targeting moiety, and B, Pl, P2, Ra, Rb, Rc, Rd, Xi, X2 are as defined for formula (I).

[0325] In some embodiments of the compound of Formula (I), Ra, Rb, Rc, and Rd are hydrogen atoms.

[0326] Generally, the group R3 encompasses any cell targeting moiety known in the art, including any cell targeting moiety specified in this disclosure, including those specified in this disclosure with respect to the description of targeted oligonucleotides.

[0327] In some embodiments of the compound of formula (I), R3 has the formula (III)

[0328]

[0329] in

[0330] A1, A2 and A3 are OH or OC(=O)-R4, where R4 is a (C1-C6)-alkyl or (C6-C10)-aryl group.

[0331] A4 is OH, OC(=O)-R4, NHC(=O)-R5, R4 is as defined above, and R5 is a (C1-C6)-alkyl group optionally substituted by a halogen atom.

[0332] In some preferred embodiments, A1, A2 and A3 are OC(=O)-R4, wherein R4 is a (C1-C6)-alkyl or (C6-C10)-aryl group.

[0333] In some preferred embodiments, A1, A2 and A3 are OC(=O)-R4, and R4 is a methyl or phenyl group.

[0334] In some preferred embodiments, A1, A2, and A3 are OC(=O)-R4, and R4 is methyl.

[0335] In some preferred embodiments, A4 is OC(=O)-R4 or NHC(=O)-R5, wherein R4 is a (C1-C6)alkyl or (C6-C10)-aryl group, and R5 is a (C1-C6)-alkyl group optionally substituted by a halogen atom.

[0336] In some preferred embodiments, A1, A2 and A3 are OC(=O)-R4, wherein R4 is methyl, and A4 is OC(=O)-R4 or NHC(=O)-R5, wherein R4 and R5 are each methyl.

[0337] In some preferred embodiments, R3 is 3,4,6-tri-O-acetyl-DN-acetylgalactosamine of formula (III-A):

[0338]

[0339] The present disclosure also relates to oligonucleotides comprising one or more nucleotide analogues introduced into the oligonucleotide by using nucleotide analogue precursors which are compounds of formula (I) as specified herein.

[0340] As will be described in detail elsewhere in this disclosure, the present invention further relates to single-stranded and double-stranded oligonucleotides, and in particular siRNA, comprising one or more compounds of formula (II).

[0341] Modified oligonucleotides

[0342] The compounds of formula (I) disclosed herein are nucleotide analog building blocks, also referred to as "nucleotide precursors," which have been envisioned as monomeric units of oligomeric compounds, particularly as monomeric units of oligonucleotides, including as monomeric units of double-stranded RNA ("dsRNA") oligomers, and especially as monomeric units of siRNAs. The nucleotide precursors described herein under the compounds of formula (I) are incorporated into oligonucleotides to produce the corresponding monomeric units of oligonucleotides, which are referred to herein as compounds of formula (II).

[0343] The terms "oligomeric compound" and "oligonucleotide" are used interchangeably herein.

[0344] Therefore, the present invention also relates to oligonucleotides into which non-targeted, targeted, or non-targeted and targeted one or more compounds of formula (I) are incorporated, thereby resulting in compounds of formula (II).

[0345] As will be described in further detail in this disclosure, oligonucleotides according to the present invention may be in single-stranded form or in double-stranded form.

[0346] The present invention relates to oligonucleotides comprising one or more compounds of formula (II):

[0347]

[0348] wherein, for each compound of formula (II), independently:

[0349] -B is a heterocyclic nucleobase;

[0350] - one of L1 and L2 is an internucleoside linking group that links the compound of formula (II) to the oligomeric compound, and the other of L1 and L2 is H, a protecting group, a phosphorus moiety, or an internucleoside linking group that links the compound of formula (II) to the oligomeric compound

[0351] -Y is O, NH, NR1 or NC(=O)-R1, wherein R1 is:

[0352] a (C1-C20)alkyl group, which is optionally substituted by one or more groups selected from the group consisting of a halogen atom, a (C1-C6)alkyl group, a (C3-C8)cycloalkyl group, a (C3-C14)heterocycle, a (C6-C14)aryl group, a (C5-C14)heteroaryl group, -O-Z1, -N(Z1)(Z2), -S-Z1, -CN, -C(=J)-O-Z1, -OC(=J)-Z1, -C(=J)-N(Z1)(Z2), -N(Z1)-C(=J)-Z2, wherein

[0353] J is O or S,

[0354] Z1 and Z2 are each independently H, a (C1-C6)alkyl group, which is optionally substituted with one or more groups selected from the group consisting of halogen atoms and (C1-C6)alkyl groups,

[0355] a (C3-C8)cycloalkyl group, which is optionally substituted by one or more groups selected from the group consisting of halogen atoms and (C1-C6)alkyl groups,

[0356] -[C(=O)]m-R2-(O-CH2-CH2)p-R3, wherein

[0357] m is an integer, with a value of 0 or 1.

[0358] p is an integer ranging from 0 to 10,

[0359] R2 is a (C1-C20)alkylene group, which is optionally substituted by a (C1-C6)alkyl group, -O-Z3, -N(Z3)(Z4), -S-Z3, -CN, -C(=K)-O-Z3, -OC(=K)-Z3, -C(=K)-N(Z3)(Z4) and -N(Z3)-C(=K)-Z4, wherein

[0360] K is O or S,

[0361] Z3 and Z4 are each independently H, a (C1-C6)alkyl group, which is optionally substituted with one or more groups selected from the group consisting of halogen atoms and (C1-C6)alkyl groups,

[0362] and

[0363] R3 is selected from a hydrogen atom, a (C1-C6)alkyl group, a (C1-C6)alkoxy group, a (C3-C8)cycloalkyl group, a (C3-C14)heterocycle, a (C6-C14)aryl group or a (C5-C14)heteroaryl group, or

[0364] R3 is the cell targeting moiety,

[0365] - X1 and X2 are each independently a hydrogen atom, a (C1-C6) alkyl group, and

[0366] - Ra, Rb, Rc and Rd are each independently H or a (C1-C6)alkyl group,

[0367] or a pharmaceutically acceptable salt thereof.

[0368] In some preferred embodiments of the oligonucleotides as described herein, in the compound of formula (II), Y is O.

[0369] In some other preferred embodiments of the oligonucleotides as described herein, in the compound of formula (II), Y is NR1 or NC(=O)-R1, and R1 is as defined for general formula (I).

[0370] In some embodiments, wherein Y is NR1, R1 is a (C1-C20)alkyl group, which is optionally substituted by one or more groups selected from the group consisting of a halogen atom, a (C1-C6)alkyl group, a (C3-C8)cycloalkyl group, a (C3-C14)heterocycle, a (C6-C14)aryl group, a (C5-C14)heteroaryl group, -O-Z1, -N(Z1)(Z2), -S-Z1, -CN, -C(=J)-O-Z1, -OC(=J)-Z1, -C(=J)-N(Z1)(Z2), -N(Z1)-C(=J)-Z2, wherein

[0371] J is O or S,

[0372] Z1 and Z2 are each independently H, a (C1-C6)alkyl group, which is optionally substituted by one or more groups selected from the following: halogen atoms and (C1-C6)alkyl groups, in the form of a base or an addition salt with an acid.

[0373] In some of these embodiments, wherein Y is NR1, R1 is an unsubstituted (C1-C20) alkyl group, and L1, L2, Ra, Rb, Rc, Rd, X1, X2, R2, R3 and B have the same meanings as defined for the general formula (II), or a pharmaceutically acceptable salt thereof.

[0374] In some of the embodiments, wherein Y is NR1, R1 is an unsubstituted (C1-C16)alkyl group, including an alkyl group selected from methyl, isopropyl, butyl, octyl, hexadecyl, and L1, L2, Ra, Rb, Rc, Rd, X1, X2 and B have the same meanings as defined for formula (II).

[0375] In some embodiments, wherein Y is NR1, R1 is a methyl group, Ra, Rb, Rc, Rd, X1 and X2 are hydrogen atoms, and L1 and L2 are as defined for formula (II).

[0376] In some embodiments, wherein Y is NR1, R1 is an isopropyl group, Ra, Rb, Rc, Rd, X1 and X2 are hydrogen atoms, and L1 and L2 are as defined for formula (II).

[0377] In some embodiments, wherein Y is NR1, R1 is a methyl group substituted by a phenyl group, and L1, L2, Ra, Rb, Rc, Rd, X1, X2, R2, R3 and B have the same meanings as defined for the general formula (II), or a pharmaceutically acceptable salt thereof.

[0378] In some embodiments, wherein Y is NR1, R1 is a butyl group, Ra, Rb, Rc, Rd, X1 and X2 are hydrogen atoms, and L1 and L2 are as defined for formula (II).

[0379] In some embodiments, wherein Y is NR1, R1 is an octyl group, Ra, Rb, Rc, Rd, X1 and X2 are hydrogen atoms, and L1 and L2 are as defined for formula (II).

[0380] In some embodiments, wherein Y is NR1, R1 is a linear C16 alkyl group, Ra, Rb, Rc, Rd, X1 and X2 are hydrogen atoms, and L1 and L2 are as defined for formula (II).

[0381] In further embodiments of the compound of formula (II), wherein Y is NR1, R1 is a (C1-C20)alkyl group, which is substituted as defined in formula (II), including a C1, C2 or C3 alkyl group substituted as defined in formula (II).

[0382] In some of these further embodiments, R1 is a (C1-C20)alkyl group substituted with one or more groups selected from the group consisting of: a halogen atom, a (C1-C6)alkyl group, a (C3-C8)cycloalkyl group, a (C3-C14)heterocycle, a (C6-C14)aryl group, and a (C5-C14)heteroaryl group.

[0383] In some of these further embodiments, R1 is a (C1-C20)alkyl group substituted by a (C6-C14)aryl group, and L1, L2, Ra, Rb, Rc, Rd, X1, X2 and B have the same meanings as defined for the general formula (II).

[0384] In some embodiments of the compound of formula (II), wherein Y is NR1, R1 is a (C1-C20)alkyl group substituted with a (C6-C14)aryl group. These embodiments encompass compounds of formula (II) wherein Y is NR1, R1 is a methylene group substituted with an aryl group. These embodiments also encompass compounds of formula (II) wherein Y is NR1, R1 is a (C1-C20)alkyl group substituted with a phenyl group.

[0385] In some embodiments of the compound of formula (II), wherein Y is NR1, R1 is a methyl group substituted with an unsubstituted phenyl group, Ra, Rb, Rc, Rd are each a hydrogen atom, and L1 and L2 are as defined in formula (II).

[0386] In a further embodiment of the compound of formula (II), wherein Y is NR1, R1 is a (C3-C8)cycloalkyl group, which is optionally substituted by one or more groups selected from the group consisting of halogen atoms and (C1-C6)alkyl groups, and L1, L2, Ra, Rb, Rc, Rd, X1, X2 and B have the same meanings as defined for the general formula (II).

[0387] In some of these further embodiments of compounds of formula (II), wherein Y is NR1, R1 is a cyclohexyl group, and L1, L2, Ra, Rb, Rc, Rd, X1, X2, R2, R3 and B have the same meanings as defined for said general formula (II), or a pharmaceutically acceptable salt thereof.

[0388] In some of these further embodiments of compounds of formula (II), wherein Y is NR1, R1 is unsubstituted cyclohexyl, Ra, Rb, Rc, Rd, X1, X2 are each a hydrogen atom, and L1 and L2 are as defined for formula (II).

[0389] In some other embodiments of the oligonucleotide of formula (II), Y is NC(=O)-R1, wherein R1 is a (C1-C20)alkyl group, R1 is selected from methyl and pentadecyl, and L1, L2, Ra, Rb, Rc, Rd, X1, X2, R2, R3 and B have the same meanings as defined for the general formula (II), or a pharmaceutically acceptable salt thereof.

[0390] In some other embodiments of the oligonucleotide of formula (II), Y is NC(═O)-R1, wherein R1 is a (C1-C20)alkyl group, which is optionally substituted with one or more groups selected from the group consisting of a halogen atom, a (C1-C6)alkyl group, a (C3-C8)cycloalkyl group, a (C3-C14)heterocycle, a (C6-C14)aryl group, a (C5-C14)heteroaryl group, -O-Z1, -N(Z1)(Z2), -S-Z1, -CN, -C(═J)-O-Z1, -OC(═J)-Z1, -C(═J)-N(Z1)(Z2) and -N(Z1)-C(═J)-Z2, wherein

[0391] J is O or S, and

[0392] Z1 and Z2 are each independently H, a (C1-C6)alkyl group, which is optionally substituted with one or more groups selected from the group consisting of halogen atoms and (C1-C6)alkyl groups, and

[0393] L1, L2, Ra, Rb, Rc, Rd, X1, X2 and B have the same meanings as defined for the general formula (II).

[0394] In some of the embodiments, wherein Y is NC(=O)-R1, R1 is an optionally substituted (C1-C20)alkyl group, including an optionally substituted (C1-C15)alkyl group, and L1, L2, Rb, Rc, Rd, X1, X2 and B have the same meanings as defined for formula (II).

[0395] In some of the embodiments, wherein Y is NC(═O)—R1, R1 is an unsubstituted (C1-C20)alkyl group, including an unsubstituted (C1-C15)alkyl group, and L1, L2, Ra, Rb, Rc, Rd, X1, X2 and B have the same meanings as defined for formula (II).

[0396] According to some of these embodiments, wherein Y is NC(=O)-R1, R1 is selected from methyl and pentadecyl, and L1 and L2 and B have the same meanings as defined for formula (II). These embodiments encompass compounds of formula (II) wherein Y is NC(=O)-R1, R1 is a methyl group, Ra, Rb, Rc, Rd, X1, X2 each represents a hydrogen atom, and B, L1 and L2 are as defined in formula (II). These embodiments also encompass compounds of formula (II) wherein Y is NC(=O)-R1, R1 is a pentadecyl group, Ra, Rb, Rc, Rd, X1, X2 each represents a hydrogen atom, and B, L1 and L2 are as defined in formula (II).

[0397] The compound of formula (II) can exist in the form of a free base or an addition salt formed with an acid. The compound of formula (II) can also exist in the form of its pharmaceutically acceptable salt, which also falls within the scope of the present disclosure.

[0398] Embodiments of oligonucleotides comprising targeted nucleotide analogs of formula (II)

[0399] As previously specified herein, the present disclosure also encompasses compounds of formula (II), wherein:

[0400] -B is a heterocyclic nucleobase;

[0401] - one of L1 and L2 is an internucleoside linking group that links the compound of formula (II) to the oligomeric compound, and the other of L1 and L2 is H, a protecting group, a phosphorus moiety, or an internucleoside linking group that links the compound of formula (II) to the oligomeric compound;

[0402] -Y is NR1, and R1 is a group -[C(=O)]m-R2-(O-CH2-CH2)p-R3, wherein

[0403] m is an integer, with a value of 0 or 1.

[0404] p is an integer ranging from 0 to 10,

[0405] R2 is a (C1-C20)alkylene group, which is optionally substituted by a (C1-C6)alkyl group, -O-Z3, -N(Z3)(Z4), -S-Z3, -CN, -C(=K)-O-Z3, -OC(=K)-Z3, -C(=K)-N(Z3)(Z4), -N(Z3)-C(=K)-Z4, wherein

[0406] K is O or S,

[0407] Z3 and Z4 are each independently H, a (C1-C6)alkyl group, which is optionally substituted with one or more groups selected from the group consisting of halogen atoms and (C1-C6)alkyl groups,

[0408] and

[0409] R3 is the cell targeting moiety,

[0410] - X1 and X2 are each independently a hydrogen atom, a (C1-C6) alkyl group, and

[0411] - Ra, Rb, Rc and Rd are each independently H or a (C1-C6)alkyl group,

[0412] It is in the form of a base or an addition salt with an acid.

[0413] These compounds of formula (II) are encompassed within a more general family of compounds that may be referred to in this disclosure as "targeted nucleotide analogs."

[0414] In some embodiments of the targeted nucleotide analog of formula (II), wherein R1 is a group -[C(=O)]m-R2-(O-CH2-CH2)p-R3, m is 0, p is 0, R3 is a cell targeting portion, and B, P1, P2, Ra, Rb, Rc, Rd, X1, X2 and R2 are as described in the general definition of compounds of formula (II).

[0415] In some embodiments, R2 is an ethylene group, and X1 and X2 are both hydrogen atoms, and B, P1, P2, Ra, Rb, Rc, and Rd are as defined in the general definition of the compound of formula (II).

[0416] In some other embodiments of these embodiments, R2 is a pentylene group, and X1 and X2 are both hydrogen atoms, and B, P1, P2, Ra, Rb, Rc, and Rd are as described in the general definition of the compound of formula (II).

[0417] In some other embodiments of these embodiments, R2 is a (C12)alkylene group, and X1 and X2 are both hydrogen atoms, and B, P1, P2, Ra, Rb, Rc, and Rd are as described in the general definition of compounds of formula (II).

[0418] In some embodiments of compounds of formula (II), wherein R1 is a group -[C(=O)]m-R2-(O-CH2-CH2)p-R3, m is 0, p is selected from the integers 1, 2, 3 and 4, R3 is a cell targeting moiety, and B, L1, L2, Ra, Rb, Rc, Rd, X1, X2 and R2 are as described in the general definition of compounds of formula (II). In some of these embodiments, R2 is an ethylene group, p is 1, and X1 and X2 are both hydrogen atoms. In further embodiments of these embodiments, R2 is an ethylene group, p is 2, and X1 and X2 are both hydrogen atoms. In further embodiments of these embodiments, R2 is an ethylene group, p is 3, and X1 and X2 are both hydrogen atoms. In still other embodiments of these embodiments, R2 is an ethylene group, p is 4, and X1 and X2 are both hydrogen atoms.

[0419] In some embodiments of the compound of formula (II), wherein R1 is a group -[C(=O)]m-R2-(O-CH2-CH2)p-R3, m is 1, p is 0, R3 is a cell targeting portion, and R2, B, L1, L2, Ra, Rb, Rc, Rd, X1, X2 are as described in the general definition of the compound of formula (II).

[0420] In some of these embodiments, R2 is a butylene group, X1 and X2 both represent hydrogen atoms, and B, L1, L2, Ra, Rb, Rc and Rd are as defined for formula (II).

[0421] In some further embodiments of these embodiments, R2 is (C11) alkylene, X1 and X2 both represent hydrogen atoms, and B, L1, L2, Ra, Rb, Rc and Rd are as defined for formula (II).

[0422] In still further embodiments of these embodiments, R2 is methylene, X1 and X2 both represent hydrogen atoms, and B, L1, L2, Ra, Rb, Rc and Rd are as defined for formula (II).

[0423] In some embodiments of the compound of formula (II), wherein R1 is a group -[C(=O)]m-R2-(O-CH2-CH2)p-R3, m is 1, p is selected from the integers 1 and 2, R3 is a cell targeting portion, and R2, B, L1, L2, Ra, Rb, Rc, Rd, X1, and X2 are as described in the general definition of the compound of formula (II).

[0424] In some of these embodiments, R2 is a methylene group, p is 2, R3 is a cell targeting moiety, and B, L1, L2, Ra, Rb, Rc, Rd, X1, and X2 are as defined for Formula (II).

[0425] In some other of these embodiments, R2 is a methylene group, p is 1, R3 is a cell targeting moiety, and B, L1, L2, Ra, Rb, Rc, Rd, X1, and X2 are as defined for formula (II).

[0426] Generally, the group R3 encompasses any cell targeting moiety known in the art, including any cell targeting moiety specified in this disclosure, including those specified in this disclosure with respect to the description of targeted oligonucleotides.

[0427] In some embodiments of the compound of formula (II), R3 has formula (III):

[0428]

[0429] Wherein A1, A2 and A3 are OH,

[0430] A4 is OH or NHC(=O)-R5, wherein R5 is a (C1-C6)alkyl group which is optionally substituted by a halogen atom.

[0431] In some embodiments, R3 is N-acetyl-galactosamine of formula (III-B):

[0432]

[0433] According to the present disclosure, references to "GalNAc" or "N-acetylgalactosamine" include both the β-form: 2-(acetylamino)-2-deoxy-β-D-galactopyranose and the α-form: 2-(acetylamino)-2-deoxy-α-D-galactopyranose. In certain embodiments, the β-form: 2-(acetylamino)-2-deoxy-β-D-galactopyranose and the α-form: 2-(acetylamino)-2-deoxy-α-D-galactopyranose are used interchangeably.

[0434] In the oligonucleotide of formula (II), B is a heterocyclic core base moiety. As used herein, the term "heterocyclic core base" refers to an optionally substituted nitrogenous heterocycle, which is covalently linked to a dioxane ring or a morpholino ring. In some embodiments, the heterocyclic core base can be selected from optionally substituted purine-bases, optionally substituted pyrimidine-bases. The term "purine-bases" is used as the common meaning understood by those skilled in the art herein, and includes its tautomers. Similarly, the term "pyrimidine-bases" is used as the common meaning understood by those skilled in the art herein, and includes its tautomers. The non-limiting list of optionally substituted purine-bases includes purine, adenine, guanine, hypoxanthine, xanthine, alloxanthine, 7-alkylguanines (such as 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). Non-limiting examples of other heterocyclic bases include diaminopurine, 8-oxo-N 6 Alkyl adenine (e.g., 8-oxo-N 6 methyladenine), 7-deazaxanthine, 7-deazaguanine, 7-deazaadenine, N4,N4-ethanoylcytosine, N <6> ,N <6> -ethano--2,6-diaminopurine, 5-halouracil (e.g., 5-fluorouracil and 5-bromouracil), pseudoisocytosine, isocytosine, isoguanine, 1,2,4-triazole-3-carboxamide, and other heterocyclic bases described in U.S. Pat. Nos. 5,432,272 and 7,125,855, which are incorporated herein by reference for disclosure of additional heterocyclic bases.

[0435] In some embodiments, B is selected from the group consisting of a pyrimidine, a substituted pyrimidine, a purine, and a substituted purine.

[0436] In a preferred embodiment, B is selected from the group consisting of adenine, thymine, uracil, guanine, and cytosine (ie, adenyl, thymine, uracil, guanine, and cytosine).

[0437] As mentioned, in the oligonucleotide of formula (II), one of L1 and L2 is an internucleoside linking group that connects the compound of formula (II) to the oligomeric compound, and the other of L1 and L2 is H, a protecting group or an internucleoside linking group that connects the compound of formula (II) to the oligomeric compound.

[0438] As will be shown in the examples, the compound of formula (I), when incorporated into a first oligonucleotide forming a duplex with a second oligonucleotide having a complementary sequence, shows that the Tm value of the resulting oligonucleotide containing nucleotide analogs is significantly reduced. The Tm value of the resulting duplex decreases as the number of compounds of formula (II) increases. Illustratively, for an initial 21-mer oligonucleotide with a Tm value of about 74°C, for a modified oligonucleotide comprising five compounds of formula (II) at various positions within the resulting modified oligonucleotide, the Tm value can be reduced to as low as about 50°C. Therefore, when one or more compounds of formula (I) comprising a dioxane ring or one or more compounds of formula (I) comprising a morpholino ring are incorporated, a reduction in the Tm value occurs. However, in most embodiments, before the antisense strand of the resulting siRNA hybridizes with the desired target sequence, this reduced Tm value still ensures suitable duplex formation in the RISC complex and then suitable duplex uptake.

[0439] The Examples herein demonstrate that oligonucleotides into which one or more compounds of Formula (I) have been incorporated allow for the generation of siRNA duplex structures having the stability required to ensure effective inhibition of target genes.

[0440] Extremely unexpectedly, these examples also demonstrate that when compounds of formula (II) are linked to each other or to ribose-containing nucleotides via conventional phosphodiester bonds, high metabolic stability of siRNA duplexes comprising one or more compounds of formula (II) is achieved. Unexpectedly, it was shown that when compounds of formula (II) are linked via conventional phosphodiester bonds within an oligonucleotide to form a single siRNA strand, the resulting siRNA duplex has greater stability against nuclease degradation compared to the same siRNA having phosphorothioate-linked deoxyribonucleotides rather than compounds of formula (II). This unexpected increase in stability is particularly evident for embodiments of siRNAs in which compounds of formula (II) are present at the overhangs of both their sense and antisense strands, including siRNAs in which compounds of formula (II) are present at the 3'-end or 5'-end or both, 3'-end and 5'-end overhangs of their sense strand.

[0441] When connected by phosphodiester bonds, the high increase in metabolic stability of compounds of formula (II) is a significant technical advantage because stabilization of phosphorus groups (such as thiophosphates) by modified internucleotide linkages can be avoided. It is reminded that this unconventional thiophosphate introduces chiral centers, resulting in undesirable diastereomeric mixtures of the generated siRNA. The latter may reduce the siRNA specificity of the targeting sequence, resulting in increased off-target events. It is also shown herein that siRNAs with one or more compounds of formula (II) have good target gene silencing activity in vitro, even when the siRNA is internalized by the target cell in the absence of any transfection agent. As disclosed in the Examples, embodiments of compounds of formula (II) are shown to have IC values ​​in the picomolar range. 50 The value exerts target gene silencing activity.

[0442] Additionally, examples described herein of double-stranded oligonucleotides incorporating one or more compounds of Formula (II) having a targeting moiety attached to a morpholine-nitrogen (GalNAc residue) demonstrated robust delivery into the liver, resulting in in vivo knockdown of target mRNA and corresponding protein levels.

[0443] Surprisingly, when the targeted compound of formula (II) and the non-targeted compound of formula (II) are combined in a double-stranded oligonucleotide, a significant improvement in the in vivo behavior of the double-stranded oligonucleotide (particularly the duration of action in vivo) has been demonstrated; this can also be shown when the sense strand does not contain any phosphorothioate groups. Even the additional attachment of the non-targeted compound of formula (II) as an overhang in the antisense strand without phosphorothioate stabilization has shown robust in vivo efficacy of the resulting siRNA.

[0444] SiRNAs that have incorporated one or more compounds of formula (I) in the sense strand, antisense strand, or both the sense strand and the antisense strand, particularly in the sense strand, also exert effective in vivo target gene silencing activity. The target gene silencing activity can be controlled based on (i) the embodiment of the compound of formula (I) present therein, (ii) the amount of the compound of formula (I) present therein, and (iii) the position of the compound of formula (I) in the sense strand or antisense strand of the siRNA.

[0445] Importantly, siRNAs that have incorporated one or more compounds of Formula (II) have no adverse effects in vivo within the dosage range in which those siRNAs have been shown to exert target gene silencing effects.

[0446] For purposes of illustration, and not to limit the present disclosure, a double-stranded oligonucleotide may further comprise one or more nucleotides on the modified sense and / or antisense strand.

[0447] The modification can be selected from the replacement or insertion of the analog of nucleic acid or base, and the chemical modification of base, sugar or phosphate moiety.Selected modification can be each independently selected from 3 '-terminal deoxidation-thymidine, 2 '-O-methyl, 2 '-deoxidation modification, 2 '-deoxidation-fluorine, 2 '-amino modification, 2 '-alkyl modification, phosphorothioate modification, phosphoramidate modification, 5 '-phosphorothioate base modification, 5 '-phosphate or 5 '-phosphate simulation modification and cholesteryl derivative or dodecanoic acid didecyl amide group modification and / or modified nucleotide can be any one in locked nucleotide, abasic nucleotide or the nucleotide comprising non-natural base.One of preferred embodiments can be at least one modification to 2 '-O-methyl and / or at least one modification to 2 '-deoxidation-fluorine.

[0448] As used herein, other examples of modified oligonucleotides can include one or more of the following: modification, e.g., replacement of one or two non-linked phosphate oxygens and / or one or more linked phosphate oxygens; replacement of a phosphate moiety; modification or replacement of a natural base; replacement or modification of the ribose-phosphate backbone; modification of the 3'-end or 5'-end of the RNA, e.g., removal, modification or replacement of a terminal phosphate group, or conjugation of a moiety (e.g., a fluorescently labeled moiety) to or to the 3'- or 5'-end of the RNA.

[0449] Synthesis method of compound of formula (I)

[0450] Compounds of formula (I) can be prepared according to the detailed methods shown in the disclosure herein.

[0451] The present disclosure relates to a process for preparing a compound of formula (IA), comprising the following steps:

[0452] a) Oxidation of the compound of formula (X)

[0453]

[0454] wherein B is a heterocyclic nucleobase, and P1 and P2 each independently represent a protecting group as defined in formula (I) herein

[0455] By reacting the compound of formula (X) with an oxidizing agent such as sodium periodate (NaIO4), a compound of formula (XI) is obtained:

[0456] and

[0457] b) subjecting the compound of formula (XI) to reductive amination in the presence of a compound of formula (XII) R1-NH2(XII)

[0458] wherein R1 is as defined in formula (I) herein,

[0459] To obtain a compound of formula (IA):

[0460]

[0461] wherein B is a heterocyclic nucleobase, and P1 and P2 each independently represent a protecting group as defined in general formula (I).

[0462] The present disclosure also relates to a process for preparing a compound of formula (IB), comprising the following steps:

[0463] a) Oxidation of the compound of formula (X)

[0464]

[0465] wherein B is a heterocyclic nucleobase, and P1 and P2 each independently represent a protecting group as defined in formula (I) herein

[0466] By reacting the compound of formula (X) with an oxidizing agent such as sodium periodate (NaIO4), a compound of formula (XI) is obtained:

[0467] and

[0468] b) subjecting the compound of formula (XI) to a reductive amination step in the presence of an amine, such as ammonia or ammonium diborate, to obtain a compound of formula (XIII)

[0469]

[0470] c) amide coupling of the compound of formula (XIII) in the presence of the compound of formula (XIV)

[0471] R1-C(=O)-OH(XIV)

[0472] wherein R1 is as defined in formula (I) herein,

[0473] To obtain a compound of formula (IB)

[0474]

[0475] wherein B is a heterocyclic nucleobase, and P1 and P2 each independently represent a protecting group as defined in formula (I), and R1 is as defined in formula (I).

[0476] d) subjecting the compound of formula (XIII) to reductive amination in the presence of an aldehyde or ketone to obtain a compound of formula (IA)

[0477]

[0478] The present disclosure also relates to methods for preparing compounds of formula (IC)

[0479]

[0480] It comprises the following steps: reductive amination of a compound of formula (XV)

[0481]

[0482] wherein A1, A2, A3 and A4 are as defined in formula (III) or (III-A) herein, -Y-CHO is transferred to -Y-CH2- by the reductive amination reaction, which is equivalent to X, and X is a group of the formula -(CH2-CH2-O)p-R2-, wherein p and R2 are as defined in general formula (I),

[0483] Reaction with a compound of formula (XIII)

[0484]

[0485] wherein P1, P2 and B are as defined in formula (I) herein,

[0486] , to obtain a compound of formula (IC)

[0487]

[0488] The above method is described in Scheme 2 of the present disclosure.

[0489] The present disclosure also relates to a process for obtaining compounds of formula (ID)

[0490]

[0491] It includes the following steps:

[0492] a) Under peptide coupling conditions, the compound of formula (XVI)

[0493]

[0494] wherein A1, A2, A3 and A4 are as defined herein in formula (III) or (III-A), and X is a group of formula -(CH2-CH2-O)p-R2-, wherein p and R2 are as defined in general formula (I),

[0495] Reaction with a compound of formula (XIII)

[0496]

[0497] wherein P1, P2 and B are as defined in formula (I) herein,

[0498] To obtain the compound of formula (ID)

[0499] The above method is described in Scheme 2 of the present disclosure.

[0500] The present disclosure further relates to a process for preparing a compound of formula (IE), comprising the following steps:

[0501] a) Reduction of the compound of formula (XI)

[0502]

[0503] wherein P1, P2 and B are as defined in general formula (I), thereby obtaining a compound of formula (XVII)

[0504]

[0505] b) transferring the compound of formula (XVII) in the presence of a sulfonylating agent (e.g. p-toluenesulfonyl chloride Ts-Cl, methanesulfonyl chloride Ms-Cl) to obtain a compound of formula (XVIII)

[0506]

[0507] wherein Ts represents a tosyl group,

[0508] c) subjecting said compound of formula (XVIII) to basic conditions, thereby obtaining a compound of formula (IE)

[0509]

[0510] The above method is described in Scheme 3 of this disclosure.

[0511] The present disclosure also relates to an alternative process for preparing a compound of formula (IE) comprising the following steps:

[0512] a) Transferring the compound of formula (XVII)

[0513]

[0514] In the presence of an excess of a sulfonylating agent (e.g., p-toluenesulfonyl chloride Ts-Cl, methanesulfonyl chloride Ms-Cl), a compound of formula (XIX) is obtained.

[0515]

[0516] Wherein Ts represents a tosyl group,

[0517] b) deprotecting the compound of formula (XIX) by removing group P1 to obtain a compound of formula (XX)

[0518]

[0519] c) subjecting the compound of formula (XX) to basic conditions, thereby obtaining a compound of formula (XXI)

[0520] and

[0521] d) replacing the tosyl group with a protecting group P1 to obtain a compound of formula (IE)

[0522]

[0523] The above method is described in Scheme 3 of this disclosure.

[0524] Compounds of Formula (I), (IA), (IB), (IC), (ID) and (IE) can be prepared according to the detailed methods shown in the following Schemes 1 to 8 disclosed herein.

[0525] Starting from a commercially available ribose derivative G1, the two primary OH-groups can be distinguished by selective benzylation according to standard literature protocols. The resulting benzyl ether G2 is subjected to standard protecting group modification to give the fully protected ribose analog G3, which can be used at the nucleobase B (e.g., T, U, C Bzl , I, G iBu 、A Bzl ) was used as a glycosyl donor to obtain the nucleoside derivative G4 (Tetrahedron, 1998, 54, 3607-3630).

[0526]

[0527] Scheme 1: Synthesis of compounds of formula (I) wherein Y is N

[0528] Starting from nucleoside analog G4, the protecting group pattern is modified by standard procedures to obtain intermediate G5, wherein as defined in general formula (I), orthogonal protecting groups P1 and P2 are on two primary alcohols at C2 ' and C3 ' of the ribose scaffold and unprotected OH-group. The cis orientation of the dihydroxy functional group in the G5-compound allows the use of NaIO4 as an oxidant to oxidative cleavage of the C-C bond between C2 ' and C3 '. The resulting dialdehyde can be separated into a monohydrate G6, which is converted into the desired morpholine scaffold by a reductive amination reaction with a reducing agent (such as NaCNBH3). Using an amine substrate, such as ammonia or ammonium diborate, a morpholine intermediate G7 with a free NH-group is produced in the morpholine scaffold. In the presence of, for example, NaCNBH3, a second reductive amination reaction is carried out with the corresponding aldehyde or ketone to obtain alkylated morpholine G8, wherein R1 is as defined in general formula (I). Alternatively, intermediate G6 can be subjected to reductive amination in the presence of an appropriate amine R1-NH2, wherein R1 is as defined in formula (I), to directly afford the alkylated morpholine G8. The analogous acylated morpholine can be obtained by standard peptide coupling between the free morpholine building block G7 and the corresponding carboxylic acid R1-COOH, thereby affording the amide intermediate G9.

[0529] Compounds G7, G8, and G9 consist of embodiments of compounds of formula (I) described in the present disclosure.

[0530] Similar to the synthesis described in Scheme 1 for forming compounds of formula (I), the targeted compounds of formula (I) described herein can be prepared by reductive amination or peptide coupling reactions using intermediate G7 as an amine reducing agent.

[0531] The synthesis of compounds of general formula (I) is described in Scheme 2 below using peracetylated N-acetylgalactosamine G11 as the protected cell targeting moiety.

[0532]

[0533] Scheme 2: Synthesis of compounds of general formula (I) with amide (G14) and amine (G15) attachments, protected GalNAc as cell targeting moiety and different X linker groups, where X is generated from the linker fragment -Y-CHO in (G13) and converted to -Y-CH2- after reductive amination.

[0534] X is defined below as a group -R2(OCH2-CH2)p-, which is contained in a group -[C(=O)]m-R2-(O-CH2-CH2)p-R3, as defined in the compounds of formula (I).

[0535] Under standard glycosylation conditions, the peracetylated GalNAc derivative G11 is treated with an ω-hydroxycarboxylate (HO-X-COOBn) to generate the carboxylic acid G12 after ester cleavage, which forms the desired amide G14 under peptide coupling conditions in the presence of a morpholine derivative G7. Alternatively, G11 is glycosylated with an ω-hydroxybenzyl ether (HO-X-OBn) after benzyl ether cleavage and oxidation of the corresponding alcohol to generate the aldehyde intermediate G13. Reductive amination using morpholine G7 as the amine component forms the alkylated morpholine G15. Compounds G14 and G15 are compounds of formula (I) described in the present disclosure.

[0536] Scheme 3 shows the synthetic route of the compound of formula (I) (Y is O) in the dioxane series

[0537]

[0538] Scheme 3: Synthesis of compounds of formula (I) (wherein Y is O)

[0539] Starting from the described cis-diol intermediate G5 and its oxidative cleavage to the building block G6 (see Scheme 1), treatment with a reducing agent such as sodium borohydride provides the diol intermediate G16, which carries two primary OH-groups at the 2'- and 3'-C positions. Under basic conditions, in the presence of a stoichiometric amount of a sulfonylating agent such as p-toluenesulfonyl chloride, the 2'-OH-function can be selectively tosylated to form the mono-tosylate G17. Under basic conditions, using, for example, NaOH in MeOH or aqueous NaOMe, G17 undergoes nucleophilic substitution of the primary tosylate at the 3'-C position with the free OH-group, thereby forming the desired dioxane scaffold G18.

[0540] Compound G18 consists of the compound of general formula (I) as described in this disclosure.

[0541] Alternatively, the diol intermediate G16 can be bis-sulfonylated with, for example, an excess of p-toluenesulfonyl chloride and increased reaction time to afford the bis-toluenesulfonate G19. After deprotection of one orthogonal protecting group P1 or P2, the resulting primary alcohol G20 reacts analogously to G17 (see Scheme 3) under nucleophilic substitution to form the desired dioxane scaffold G21. The remaining tosylate can be replaced with sodium benzoate to again afford the fully protected dioxane scaffold G18, which bears orthogonal protecting groups P1 and P2 on the primary hydroxyl groups.

[0542]

[0543] Scheme 4: Alternative Synthesis of Compounds of Formula (I) Wherein Y is O

[0544] The building blocks of general formula (I) (G8, G9, G14, G15 and G18) with orthogonal protecting groups P1 and P2 are finally converted into the corresponding DMT-protected phosphoramidites for use as nucleotide precursors in automated oligonucleotide synthesis.

[0545] For this purpose, fully protected compounds of general formula (I) (G8, G9, G14, G15 and G18) were converted to the mono-DMT protected intermediate G22 by standard protecting group modification. The free OH-group in the G22 building block was phosphorylated by standard protocols to give the final DMT protected phosphoramidite G23, which served as a nucleotide precursor for automated oligonucleotide synthesis.

[0546]

[0547] Scheme 5: Synthesis of final phosphoramidite building blocks of compounds of formula (I) and (V) for automated oligonucleotide synthesis

[0548] Compound G23 is a compound of formula (I) wherein group P1 is a DMT protecting group and group P2 is a reactive phosphorus group consisting of a phosphoramidite group.

[0549] Depending on the protecting group modifications available to obtain intermediate G22, the (2S,6R) and (2R,6R) diastereomers of compounds having the general structure (I) can be synthesized.

[0550]

[0551] Scheme 6: Diastereomeric structures of compound G23 of general formula (I)

[0552] The starting nucleotide at the 3'-terminus of the single-stranded oligonucleotide can be prepared by reacting with the corresponding phosphoramidite G23 as the first nucleotide scaffold in automated synthesis using a universal solid support material (see Experimental Section, Synthesis of Oligonucleotides) by standard procedures.

[0553] Alternatively, solid support materials for compounds of general formula (I) described herein can be synthesized as shown in General Scheme 7.

[0554]

[0555] Scheme 7: Final CPG (controlled pore glass) solid support material for synthesis of compounds of general formula (I) for automated oligonucleotide synthesis

[0556] Alcohol G22 is acylated with an acylating agent such as succinic anhydride to provide the corresponding succinate G24. The free carboxylic acid moiety is coupled to a free amino group of a solid support material (CPG: controlled pore glass) under peptide coupling conditions to generate the desired solid support G25, which can be used as a starting material for automated oligonucleotide synthesis.

[0557] Analogously to the diastereoselective route used for the synthesis of phosphoramidite G23 (see Scheme 6), two diastereomeric series of solid supports G25 can be synthesized (see Scheme 8).

[0558]

[0559] Scheme 8: Diastereomeric structures of compound G25

[0560] Solid support materials for succinate derivatives and compounds of formula (I), as defined in formula (I'):

[0561]

[0562] wherein T1 and T2 are each independently a protecting group, -C(=O)(CH2)r-COOH or -C(=O)(CH2)rC(=O)NH-R7, wherein R7 represents a solid support material, r is an integer selected from 2, 3 and 4, and wherein Y, B, X1, X2, Ra, Rb, Rc and Rd are as defined in general formula (I).

[0563] In some embodiments, one of T1 and T2 is -C(=O)(CH2)r-COOH, and the other is a protecting group.

[0564] In some embodiments, one of T1 and T2 is -C(=O)(CH2)rC(=O)NH-R7, wherein R7 is a CPG solid support or a polystyrene solid support, and the other of T1 and T2 is a protecting group.

[0565] In some embodiments, r is 2.

[0566] Preparation of oligonucleotides comprising compounds of formula (II)

[0567] Oligonucleotides for use according to the present invention, which may also be referred to herein as "modified oligonucleotides", may be prepared according to any useful technique, including the methods described herein, by using one or more compounds of formula (I) as certain starting building blocks to be incorporated into selected positions of the growing chain of the final oligonucleotide, thereby generating an oligonucleotide comprising one or more compounds of formula (II) at selected positions of the final oligonucleotide.

[0568] Compounds of formula (I) can be synthesized as described in this disclosure.

[0569] The modified oligonucleotides of the present invention may be double-stranded with or without overhangs, or may comprise at least one double-stranded portion. Double-stranded modified oligonucleotides may be formed by a single oligonucleotide chain comprising a first nucleotide sequence (e.g., a sense nucleotide sequence) and a second nucleotide sequence (e.g., an antisense nucleotide sequence) that is complementary to and hybridizes with the first nucleotide sequence, and wherein the second nucleotide sequence is also complementary to the target RNA sequence, seeking inhibition of the target RNA sequence. According to these embodiments, the first nucleotide sequence and the second nucleotide sequence may be on separate chains within the modified oligonucleotide; or on the same chain, but separated by a spacer or other nucleotide sequence of appropriate length so that a hairpin loop is formed once the first nucleotide sequence hybridizes to the second nucleotide sequence.

[0570] In some embodiments, the modified oligonucleotides of the invention are single-stranded and may comprise the sense or antisense strand of a double-stranded RNA, such as an siRNA.

[0571] Oligonucleotides of the present invention, such as those comprising one or more compounds of formula (II), can be chemically synthesized using protocols known in the art. See, for example, Caruthers et al., 1992, Methods in Enzymology, 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 No. 6,001,311. Oligonucleotides are synthesized using common nucleic acid protecting groups and coupling groups, such as a dimethoxytrityl group at the 5'-terminus and a phosphoramidite at the 3'-terminus. In certain embodiments, oligonucleotides comprising compounds of formula (II) are 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 a non-limiting synthetic example, small-scale synthesis is performed on a 394 Applied Biosystems, Inc. / Thermo Fischer Scientific Inc. synthesizer.

[0572] Alternatively, oligonucleotides comprising one or more compounds of formula (II) can be synthesized separately and joined together after synthesis, for example by ligation (Moore et al., 1992, Science 256:9923; Draper et al., International PCT Publication No. WO93 / 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. Oligonucleotides of various modifications according to the present 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.

[0573] Double-stranded RNA (dsRNA)

[0574] An important aspect of the present invention is the provision of compounds of formula (I) which allow the preparation of modified oligonucleotides comprising one or more compounds of formula (II). The modified oligonucleotides can be used to generate double-stranded oligonucleotides, such as siRNAs that specifically hybridize to selected target mRNAs.

[0575] Taking into account (i) the ribose moiety in the majority of the nucleotide monomer units contained in the oligomeric compound of formula (II), and (ii) the type of nucleobase contained therein, the oligomeric compound of formula (II) can be referred to as "ribonucleic acid" or "RNA". Therefore, certain aspects of the present disclosure relate to double-stranded ribonucleic acid (dsRNA) molecules that target target mRNA. The dsRNA of the present invention may comprise a modified oligonucleotide comprising one or more compounds of formula (II).

[0576] In some embodiments, the dsRNA comprises two strands, a sense strand comprising a first sequence and an antisense strand comprising a second sequence, wherein the first strand and the second strand are sufficiently complementary to form a duplex structure. In some embodiments, the sense strand comprises a first sequence that is substantially complementary or fully complementary to the second sequence in the antisense strand. In some embodiments, the second sequence in the antisense strand is substantially complementary or fully complementary to the target sequence (e.g., the sequence of an mRNA transcribed from a target gene).

[0577] In some embodiments, the sense strand and antisense strand of dsRNA are in two separated molecules. In some embodiments, the duplex region is formed between the first sequence in the sense strand of the two separated molecules and the second sequence in the antisense strand. In some embodiments, dsRNA is siRNA. In some embodiments, the two separated molecules are not covalently linked to each other. In some embodiments, the two separated molecules are covalently linked to each other by means other than hairpin loops. In some embodiments, the two separated molecules are covalently linked to each other via a connecting structure (referred to herein as "covalent linker").

[0578] In some embodiments, the sense and antisense strands can each range from 9 to 36 nucleotides in length. For example, each strand can be 12 to 30 nucleotides in length, 14 to 30 nucleotides in length, 15 to 30 nucleotides in length, 25 to 30 nucleotides in length, 27 to 30 nucleotides in length, 15 to 26 nucleotides in length, 15 to 23 nucleotides in length, 15 to 22 nucleotides in length, 15 to 21 nucleotides in length, 15 to 20 nucleotides in length, 15 to 19 nucleotides in length, 15 to 18 nucleotides in length, 15 to 17 nucleotides in length, 17 to 30 nucleotides in length, 17 to 23 nucleotides in length, 17 to 21 nucleotides in length, 17 to 19 nucleotides in length, 18 to 30 nucleotides in length, 18 to 26 nucleotides in length, 18 to 25 nucleotides in length, 18 to 23 nucleotides in length, 18 to 22 nucleotides in length, nucleotides in length, a length of 18-21 nucleotides, a length of 18-20 nucleotides, a length of 19-30 nucleotides, a length of 19-25 nucleotides, a length of 19-24 nucleotides, a length of 19-23 nucleotides, a length of 19-22 nucleotides, a length of 19-21 nucleotides, a length of 19-20 nucleotides, a length of 20-30 nucleotides, a length of 20-26 nucleotides, a length of 20-25 nucleotides, 20-24 nucleotides, a length of 20-23 nucleotides, 20-22 nucleotides, a length of 20-21 nucleotides, a length of 21-30 nucleotides, a length of 21-26 nucleotides, a length of 21-25 nucleotides, 21-24 nucleotides, a length of 21-23 nucleotides or a length of 21-22 nucleotides. In some embodiments, each strand is greater than or equal to 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, or 35 nucleotides in length. In some embodiments, each strand is less than or equal to 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 in length.That is, each strand can be any range of nucleotides in length with an upper limit of 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, and an independently selected lower limit of 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 35, wherein the lower limit is less than the upper limit. In some embodiments, 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 in length. In some embodiments, the sense strand and the antisense strand are the same number of nucleotides in length. In some embodiments, the sense strand and the antisense strand are different numbers of nucleotides in length.

[0579] protrusion

[0580] In some embodiments, the dsRNA of the present disclosure comprises one or more overhangs at the 3'-end, 5'-end, or both ends of the sense strand and the antisense strand. In some embodiments, the one or more overhangs improve stability by a smaller number of PS-groups in the overhangs.

[0581] In some embodiments, the overhang comprises one or more, two or more, three or more, four or more, five or more, or six or more nucleotides. For example, the overhang may comprise 1-8 nucleotides, 2-8 nucleotides, 3-8 nucleotides, 4-8 nucleotides, 5-8 nucleotides, 1-5 nucleotides, 2-5 nucleotides, 3-5 nucleotides, 4-5 nucleotides, 1-4 nucleotides, 2-4 nucleotides, 3-4 nucleotides, 1-3 nucleotides, 2-3 nucleotides, or 1-2 nucleotides. In some embodiments, the overhang is 1, 2, 3, 4, 5, or 6 nucleotides in length.

[0582] In some embodiments, the overhang of the present disclosure comprises one or more ribonucleotides. In some embodiments, the overhang of the present disclosure comprises one or more deoxyribonucleotides. In some embodiments, the overhang comprises one or more thymines. In some embodiments, the dsRNA comprises an overhang at the 3'-end of the antisense strand. In some embodiments, the dsRNA comprises a blunt end at the 5'-end of the antisense strand. In some embodiments, the dsRNA comprises an overhang 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 comprises an overhang at the 3'-end of the sense strand. In some embodiments, the dsRNA comprises a blunt end at the 5'-end of the sense strand. In some embodiments, the dsRNA comprises an overhang 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 comprises an overhang 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 comprises an overhang 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 comprises an overhang at the 3'-end of the sense strand and an overhang at the 3'-end of the sense strand.

[0583] In some embodiments, the dsRNA comprises an overhang at the 5'-end of the antisense strand. In some embodiments, the dsRNA comprises a blunt end at the 3'-end of the antisense strand. In some embodiments, the dsRNA comprises an overhang 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 comprises an overhang at the 5'-end of the sense strand. In some embodiments, the dsRNA comprises a blunt end at the 3'-end of the sense strand. In some embodiments, the dsRNA comprises an overhang 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 comprises an overhang 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 comprises overhangs at both strands of the dsRNA.

[0584] In some embodiments, the overhang is the result of the sense strand being longer than the antisense strand. In some embodiments, the overhang is the result of the antisense strand being longer than the sense strand. In some embodiments, the overhang is the result of the sense and antisense strands being of the same length interleaved. In some embodiments, the overhang forms a mismatch with the target mRNA. In some embodiments, the overhang is complementary to the target mRNA.

[0585] In some embodiments, the dsRNA of the present disclosure comprises a sense strand comprising a first sequence and an antisense strand comprising a second sequence, wherein the first and second sequences are substantially complementary or complementary. In some embodiments, the first and second sequences are substantially complementary or complementary and form a duplex region of the dsRNA. In some embodiments, the length of the duplex region of the dsRNA is 9-36 nucleotide pairs. For example, the duplex region can be 12-30 nucleotide pairs in length, 14-30 nucleotide pairs in length, 15-30 nucleotide pairs in length, 15-26 nucleotide pairs in length, 15-23 nucleotide pairs in length, 15-22 nucleotide pairs in length, 15-21 nucleotide pairs in length, 15-20 nucleotide pairs in length, 15-19 nucleotide pairs in length, 15-18 nucleotide pairs in length, 15-17 nucleotide pairs in length, 17-30 nucleotide pairs in length, 27-30 nucleotide pairs in length, 17-23 nucleotide pairs in length, 17-21 nucleotide pairs in length, 17-19 nucleotide pairs in length, 18-30 nucleotide pairs in length, 18-26 nucleotide pairs in length, 18-25 nucleotide pairs in length, 18-24 nucleotide pairs in length, 18-23 nucleotide pairs in length, 18-22 nucleotide pairs in length, nucleotide pairs in length, 18-21 nucleotide pairs in length, 18-20 nucleotide pairs in length, 19-30 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-24 nucleotide pairs in length, 19-23 nucleotide pairs in length, 19-22 nucleotide pairs in length, 19-21 nucleotide pairs in length, 19-20 nucleotide pairs in length, 20-30 nucleotide pairs in length, 20-26 nucleotide pairs in length, 20-25 nucleotide pairs in length, 20-24 nucleotide pairs in length, 20-23 nucleotide pairs in length, 20-22 nucleotide pairs in length, 20-21 nucleotide pairs in length, 21-30 nucleotide pairs in length, 21-26 nucleotide pairs in length, 21-25 nucleotide pairs in length, 21-24 nucleotide pairs in length, 21-23 nucleotide pairs in length, or 21-22 nucleotide pairs in length. In some embodiments, the duplex region of the dsRNA is greater than or equal to 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, or 35 nucleotide pairs in length. In some embodiments, the duplex region of the dsRNA is less than or equal to 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 nucleotide pairs in length.That is, the duplex region of the dsRNA can be any one of a range of nucleotide pairs in length having an upper limit of 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, and an independently selected lower limit of 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 35, wherein the lower limit is less than the upper limit. In some embodiments, the duplex region 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 nucleotide pairs in length. If more than one dsRNA is used, the duplex region of each dsRNA can be the same or different in length from one or more of the other dsRNAs.

[0586] The position of the compound of formula (II) in double-stranded RNA (dsRNA)

[0587] As shown in the Examples herein, the nucleotide analogues of formula (II) may be present at each position on each strand of a double-stranded oligonucleotide, ie, at each position on each strand of a double-stranded ribonucleotide.

[0588] Compounds of formula (II), wherein the group R3 is present and represents a cell targeting moiety, may be referred to in the present disclosure as "targeted nucleotide analogs of formula (II)" or "targeted nucleotide analogs (II)".

[0589] Compounds of formula (II) that do not comprise a group R3 representing a cell targeting moiety are non-targeted nucleotide analogs and are referred to in this disclosure as "non-targeted nucleotide analogs of formula (II)" or "non-targeted nucleotide analogs (II)".

[0590] Position of the non-targeted nucleotide analog of formula (II)

[0591] In certain embodiments, the nucleotide analog of formula (II) is located at the 3'-end, the 5'-end, or both the 3'-end and the 5'-end of the nucleic acid chain of the dsRNA, such as at the 3'-end or the 5'-end of the nucleic acid chain of the siRNA. In some of these certain embodiments, the nucleotide analog of formula (II) is located only at the 3'-end of the nucleic acid chain of the dsRNA, such as at the 3'-end of the nucleic acid chain of the siRNA.

[0592] In some of these specific embodiments, the nucleotide analog of formula (II) is located at the 3'-end of the sense strand of the siRNA. In some other embodiments, the nucleotide analog of formula (II) is located at the 3'-end of the antisense strand of the siRNA. In still other embodiments, the nucleotide analog of formula (II) is located at the 3'-end of the sense strand of the siRNA and the 3'-end of the antisense strand of the siRNA.

[0593] In some of these specific embodiments, the nucleotide analog of formula (II) is located (i) at the 3'-end and the 5'-end of the sense strand of the siRNA, and (ii) at the 3'-end of the antisense strand of the siRNA.

[0594] In some embodiments, 2 to 10 (e.g., 2 to 5) nucleotide analogs of formula (II) are present in the oligonucleotide. As used herein, 2 to 10 nucleotide analogs of formula (II) encompass 2, 3, 4, 5, 6, 7, 8, 9, and 10 nucleotide analogs of formula (II).

[0595] Position of the targeted nucleotide analog of formula (II)

[0596] As shown in the Examples herein, the targeted nucleotide analogs of formula (II) may be present at various positions of each chain of a double-stranded oligonucleotide. For example, the targeted nucleotide analogs of formula (II) are located at the 3'-end or 5'-end of a nucleic acid chain, such as the 3'-end or 5'-end of a nucleic acid chain of an siRNA. In some of these embodiments, the targeted nucleotide analogs of formula (II) are located at the 3'-end or 5'-end of the sense strand of the siRNA.

[0597] In some preferred embodiments, the targeted nucleotide analog of formula (II) is located at the overhang of the dsRNA, such as the overhang of the siRNA. For example, the targeted nucleotide analog of formula (II) is located at the overhang of the sense strand of the siRNA, such as the 5'-overhang.

[0598] In some embodiments, 2 to 10 (e.g., 2 to 5) targeted nucleotide analogs of formula (II) are present in an oligonucleotide. As used herein, 2 to 10 targeted nucleotide analogs of formula (II) encompass 2, 3, 4, 5, 6, 7, 8, 9, and 10 targeted nucleotide analogs of formula (II).

[0599] Targeted oligonucleotides

[0600] A targeted oligonucleotide according to the present disclosure is an oligonucleotide comprising at least a nucleotide analogue of formula (II) and further comprising a cell targeting moiety comprised within the targeted nucleotide in said oligonucleotide.

[0601] In some embodiments, the targeted nucleotide contained in the targeted oligonucleotide according to the present disclosure has the structure of formula (II). Therefore, in some embodiments, the targeted oligonucleotide according to the present disclosure comprises one or more targeted nucleotide analogs of formula (II).

[0602] In some other embodiments, the targeted nucleotides contained in the targeted oligonucleotides according to the present disclosure are selected from targeted nucleotides known in the art. Therefore, in some embodiments, the targeted oligonucleotides according to the present disclosure comprise one or more non-targeted nucleotide analogs of formula (II) and one or more targeted nucleotides having a structure different from that of formula (II).

[0603] In still other embodiments, targeted oligonucleotides encompass those comprising (i) one or more non-targeted nucleotide analogs of Formula (II), and (ii) one or more targeted nucleotide analogs of Formula (II).

[0604] As disclosed elsewhere in this disclosure, non-targeted nucleotide analogs of formula (II) encompass embodiments wherein Y is O, embodiments wherein Y is NR1, embodiments wherein Y is NH, and embodiments wherein Y is NC(=O)-R1. Non-targeted nucleotides particularly include nucleotide analogs of formula (II) wherein Y is selected from NR1 and NC(=O)-R1, and wherein R1 is a group -[C(=O)]m-R2-(O-CH2-CH2)p-R3, wherein m, p, and R2 are as defined as disclosed for general formula (II), and wherein the group R3 is selected from a hydrogen atom, a (C1-C6)alkyl group, a (C1-C6)alkoxy group, a (C3-C8)cycloalkyl group, a (C3-C14)heterocycle, a (C6-C14)aryl group, and a (C5-C14)heteroaryl group.

[0605] As disclosed elsewhere in this disclosure, the targeted nucleotide analogues of formula (II) have the group R3 as a cell targeting moiety.Cell targeting moieties are disclosed elsewhere in this disclosure.

[0606] In some embodiments, the targeted oligonucleotides according to the present disclosure are single-stranded oligonucleotides.

[0607] In some other embodiments, the targeted oligonucleotides according to the present disclosure are double-stranded oligonucleotides.

[0608] In some embodiments of the targeted oligonucleotides (single-stranded or double-stranded oligonucleotides) according to the present disclosure, the oligonucleotide chain comprises one or more targeted nucleotide analogs of formula (II), which may be located at various positions within the oligonucleotide chain, for example, within the oligonucleotide chain and / or at its 3'-end or 5'-end.

[0609] As used herein, a nucleotide analogue is defined as being located at the 3'-terminus or 5'-terminus of an oligonucleotide chain when (i) the nucleotide analogue is a nucleotide located at the 3'-terminus or 5'-terminus, or when (ii) the nucleotide analogue is a nucleotide contained in a continuous chain of continuous nucleotide analogues having a terminal nucleotide located at the 3'-terminus or 5'-terminus of the oligonucleotide chain.

[0610] In some embodiments of the targeted oligonucleotide (single-stranded or double-stranded oligonucleotide) according to the present disclosure, its oligonucleotide chain comprises one or more targeted nucleotide analogs of formula (II), which are located at the 3'-end or 5'-end or at both ends of the oligonucleotide chain.

[0611] In some embodiments of the targeted oligonucleotides according to the present disclosure (which are single-stranded or double-stranded oligonucleotides), the oligonucleotide chain comprises 1 to 10 targeted nucleotide analogs of formula (II), which are located at the 3'-end or 5'-end of the chain, or at one or more other positions within the chain.

[0612] In some embodiments, the targeted oligonucleotide further comprises 1 to 10 non-targeted nucleotide analogs of formula (II), which can be located at various positions within the oligonucleotide chain, such as within it and / or at its 3'-end or 5'-end.

[0613] In some embodiments of the targeted oligonucleotide (single-stranded or double-stranded oligonucleotide) according to the present disclosure, its oligonucleotide chain comprises (A) one or more targeted nucleotide analogs of formula (II), which are located at the 3'-end or 5'-end or at both ends of the oligonucleotide chain, and (B) one or more non-targeted nucleotide analogs of formula (II), which are located at the 3'-end or 5'-end or at both ends of the oligonucleotide chain, wherein the targeted nucleotide analogs of formula (II) and the non-targeted nucleotide analogs of formula (II) are located at different positions within the oligonucleotide chain.

[0614] In some embodiments of the targeted oligonucleotides according to the present disclosure (being single-stranded or double-stranded oligonucleotides), the oligonucleotide chain thereof comprises 1 to 10 targeted nucleotide analogs of formula (II), which are located at the 3'-end or 5'-end of the chain. In some embodiments, the targeted oligonucleotide further comprises 1 to 10 non-targeted nucleotide analogs of formula (II), which are located at the opposite ends of the oligonucleotide chain. Therefore, according to these embodiments, the number of targeted nucleotide analogs of formula (II) at the selected end of the oligonucleotide chain may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. According to some of these embodiments, if present, the number of non-targeted nucleotide analogs of formula (II) at the selected end of the oligonucleotide chain may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0615] In certain embodiments, the one or more targeted nucleotide analogs of formula (II) are linked to each other, thereby forming a continuous chain of these targeted nucleotide analogs located at the selected termini of the oligonucleotide chain.

[0616] In certain embodiments, the one or more targeted nucleotide analogs of formula (II) are located at the 5'-end of the targeted oligonucleotide chain, the oligonucleotide being a single-stranded or double-stranded oligonucleotide. In some of these embodiments, the 5'-terminal nucleotide is a targeted nucleotide analog of formula (II).

[0617] In some embodiments of the targeted oligonucleotides according to the present disclosure (being single-stranded or double-stranded oligonucleotides), the oligonucleotide chain thereof comprises one or more non-targeting nucleotide analogs of formula (II) at its 3'-end or 5'-end, and in particular at the end opposite to the end comprising one or more targeted nucleotide analogs of formula (II).

[0618] In some embodiments of the targeted oligonucleotides according to the present disclosure (being single-stranded or double-stranded oligonucleotides), the oligonucleotide chain thereof comprises 1 to 10 non-targeted nucleotide analogs of formula (II) at the 3'-end or at the 5'-end, and preferably comprises 1 to 10 non-targeted nucleotide analogs of formula (II) at its opposite end. Thus, in these embodiments, the number of non-targeted nucleotide analogs of formula (II) at the selected end of the oligonucleotide chain may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. According to some of these embodiments, if present, the number of targeted nucleotide analogs of formula (II) at the selected end of the oligonucleotide chain may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0619] In certain embodiments, the one or more non-targeted nucleotide analogs of formula (II) are linked to each other to form a continuous chain of these non-targeted nucleotide analogs located at the selected termini of the oligonucleotide chain.

[0620] In a specific embodiment, the one or more non-targeted nucleotide analogues of formula (II) are located at the 3'-terminus of the oligonucleotide chain of the targeted oligonucleotide. In a further embodiment, the 3'-terminal nucleotide is a targeted nucleotide analogue of formula (II).

[0621] Thus, the present disclosure encompasses single-stranded targeted oligonucleotides comprising (i) one or more targeted nucleotide analogs of formula (II), preferably 1 to 10 targeted nucleotide analogs of formula (II), which may be continuous in the oligonucleotide chain and located at the 5'-end of the single-stranded targeted oligonucleotide. In some of these embodiments, the single-stranded targeted oligonucleotide further comprises (ii) one or more non-targeted nucleotide analogs of formula (II), such as 1 to 10 non-targeted nucleotide analogs of formula (II), which are continuous in the oligonucleotide chain and located at the 3'-end of the single-stranded targeted oligonucleotide.

[0622] Disclosed in the Examples herein are descriptions of single-stranded targeted oligonucleotides comprising (i) three targeted nucleotide analogs of formula (II) at its 5'-terminus, and (ii) two non-targeting nucleotide analogs of formula (II) at its 3'-terminus.

[0623] The present disclosure also encompasses double-stranded oligonucleotides, wherein (i) the first strand is a targeted oligonucleotide comprising one or more targeted nucleotide analogs of Formula (II) and one or more non-targeted nucleotide analogs of Formula (II), as described above, and wherein (ii) the second strand is another targeted oligonucleotide comprising one or more targeted nucleotide analogs of Formula (II) and one or more non-targeted nucleotide analogs of Formula (II).

[0624] The present disclosure further encompasses double-stranded oligonucleotides wherein at least one strand thereof is a targeted oligonucleotide as described above, such as a targeted oligonucleotide comprising one or more targeted nucleotide analogs of formula (II) and one or more non-targeted nucleotide analogs of formula (II), as described above, and (ii) the second strand is a non-targeted oligonucleotide or a targeted oligonucleotide.

[0625] The present disclosure further encompasses double-stranded oligonucleotides, wherein (i) the first strand is a targeted oligonucleotide comprising one or more targeted nucleotide analogs of formula (II) and one or more non-targeted nucleotide analogs of formula (II), as described above, and (ii) the second strand is a non-targeted oligonucleotide comprising one or more non-targeted nucleotide analogs of formula (II).

[0626] The present disclosure also describes siRNA comprising:

[0627] a sense strand comprising (i) one or more targeted nucleotide analogs of formula (II), in particular 1 to 10 targeted nucleotide analogs of formula (II) located at its 5' terminus, and (ii) one or more non-targeted nucleotide analogs of formula (II), in particular 1 to 10 non-targeted nucleotide analogs of formula (II) located at its 3' terminus, and

[0628] - the antisense strand, which is a non-targeting oligonucleotide or a targeted oligonucleotide.

[0629] The present disclosure also describes siRNA comprising:

[0630] a sense strand comprising (i) one or more targeted nucleotide analogues of formula (II), in particular 1 to 10 targeted nucleotide analogues of formula (II) located at its 3' end, and

[0631] - the antisense strand, which does not comprise any nucleotide analogues of formula (II).

[0632] The present disclosure further describes siRNA comprising:

[0633] a sense strand comprising (i) one or more targeted nucleotide analogs of formula (II), in particular 1 to 10 targeted nucleotide analogs of formula (II) located at its 5' terminus, and (ii) one or more non-targeted nucleotide analogs of formula (II), in particular 1 to 10 non-targeted nucleotide analogs of formula (II) located at its 3' terminus, and

[0634] - an antisense strand comprising one or more non-targeted nucleotide analogues of formula (II), in particular 1 to 10 non-targeted nucleotide analogues of formula (II) located at its 3' end.

[0635] The present disclosure further describes siRNA comprising:

[0636] a sense strand comprising (i) one or more non-targeted nucleotide analogs of formula (II), in particular 1 to 10 non-targeted nucleotide analogs of formula (II) located at its 3' end, and

[0637] - an antisense strand, wherein said antisense strand most preferably does not comprise any non-targeted or targeted analogs of formula (II).

[0638] The present disclosure further describes siRNA comprising:

[0639] a sense strand comprising (i) one or more non-targeted nucleotide analogs of formula (II), in particular 1 to 10 non-targeted nucleotide analogs of formula (II) located at its 3' end, and

[0640] - an antisense strand comprising (i) one or more non-targeted nucleotide analogues of formula (II), in particular 1 to 10 non-targeted nucleotide analogues of formula (II) located at its 3' end.

[0641] The present disclosure further describes siRNA comprising:

[0642] - a sense strand comprising (i) one or more non-targeted nucleotide analogs of formula (II), in particular 1 to 10 non-targeted nucleotide analogs of formula (II) located at its 3' end, and (ii) one or more non-targeted nucleotide analogs of formula (II), in particular 1 to 10 non-targeted nucleotide analogs of formula (II) located at its 5' end

[0643] - an antisense strand, wherein said antisense strand most preferably does not comprise any non-targeted or targeted analogs of formula (II).

[0644] The present disclosure further describes siRNA comprising:

[0645] - a sense strand comprising (i) one or more non-targeted nucleotide analogs of formula (II), in particular 1 to 10 non-targeted nucleotide analogs of formula (II) located at its 3' end, and (ii) one or more non-targeted nucleotide analogs of formula (II), in particular 1 to 10 non-targeted nucleotide analogs of formula (II) located at its 5' end

[0646] - an antisense strand comprising (i) one or more non-targeted nucleotide analogues of formula (II), in particular 1 to 10 non-targeted nucleotide analogues of formula (II) located at its 3' end.

[0647] The present disclosure further describes siRNA comprising:

[0648] - a sense strand, wherein the sense strand most preferably does not comprise any non-targeting or targeted analogs of formula (II)

[0649] - an antisense strand comprising (i) one or more targeted nucleotide analogues of formula (II), in particular 1 to 10 non-targeted nucleotide analogues of formula (II).

[0650] Further embodiments of targeted oligonucleotides

[0651] Oligonucleotides comprising one or more compounds of formula (II), in particular oligonucleotides comprising one or more non-targeted nucleotide analogs of formula (II), may further comprise one or more modified nucleotides ("targeted nucleotides") that allow the oligonucleotide to be targeted to a target cell or cell receptor protein. For example, the oligonucleotide is double-stranded, wherein one or both oligonucleotide chains comprise one or more compounds of formula (II), and wherein one or both oligonucleotide chains further comprise one or more targeted nucleotides. For the purposes of this disclosure, such double-stranded oligonucleotides may also be referred to as "dsRNA conjugates."

[0652] Targeted nucleotides

[0653] In some embodiments of the targeted oligonucleotides based on formula (II), the targeting ligand (e.g., cell targeting moiety) is directly and covalently bound to the nitrogen atom of the morpholino group. In some other embodiments, the ligand is covalently bound to the nitrogen atom of the morpholino group via a linker group. For example, the targeted nucleotide analogs of formula (II) may include, for example, compounds of formula (II), wherein Y is NR1, and R1 is a group -[C(=O)]m-R2-(O-CH2-CH2)p-R3, and R3 is a cell targeting moiety, and wherein B, L1, L2, m, p, R2, X1, X2, Ra, Rb, Rc, and Rd are as defined for general formula (II).

[0654] Cell targeting moiety

[0655] The nucleotide of the targeting herein can be connected with one or more ligands of target specific cells or tissues.This ligand is also referred to as " cell targeting moiety ". The ligand encompasses any molecular group that improves the efficiency of the resulting oligonucleotide (such as siRNA) delivered into the cell, for example, by improving specific cell targeting, improving the cellular internalization of the oligonucleotide and / or improving intracellular mRNA targeting. The ligand may be selected from receptor-specific peptides, receptor-specific proteins (for example, monoclonal antibodies or fusion proteins) and receptor-specific small molecule ligands (for example, carbohydrates, such as GalNAc groups).

[0656] The ligand may be naturally occurring, recombinant or synthetic. For example, the ligand may be a protein, a carbohydrate, a lipopolysaccharide, a lipid, a synthetic polymer, a polyamine, an alpha helical peptide, a lectin, a vitamin or a cofactor. In some embodiments, the ligand is one or more dyes, cross-linking agents, polycyclic aromatic hydrocarbons, peptide conjugates (e.g., RGD peptide, antennapedia mutant peptide, Tat peptide), polyethylene glycol (PEG), enzymes, haptens, transport / absorption facilitators, synthetic ribonucleases (e.g., imidazole, biimidazole, histamine or imidazole clusters), human serum albumin (HSA) or LDL.

[0657] For example, the ligand can be one or more proteins, glycoproteins, peptides, or molecules that have a specific affinity for the co-ligand. Such ligands can include thyrotropin, melanocyte-stimulating hormone, glycoproteins, surfactant protein A, mucin carbohydrates, lactose (e.g., multivalent lactose), galactose (e.g., multivalent galactose), N-acetyl-galactosamine (e.g., multivalent N-acetyl-galactosamine), N-acetyl-glucosamine (e.g., multivalent N-acetyl-glucosamine), mannose (e.g., multivalent mannose), fucose (e.g., multivalent fucose), glycosylated polyamino acids, transferrin, bisphosphonates, polyglutamate, polyaspartate, lipids, cholesterol, steroids, bile acids, folic acid, vitamin B12, and biotin.

[0658] In some embodiments, the cell targeting moiety is one or more dyes, cross-linkers, polycyclic aromatic hydrocarbons, peptide conjugates (e.g., Antennapedia mutant peptides, Tat peptides), polyethylene glycol (PEG), enzymes, haptens, transport / absorption enhancers, synthetic ribonucleases (e.g., imidazole, biimidazole, histamine, or imidazole clusters), human serum albumin (HSA), or LDL.

[0659] In some embodiments, the ligand may be one or more cholesterol derivatives or lipophilic moieties. Any lipophilic compound may include, but is not limited to, cholesterol or cholesterol derivatives; bile acid; vitamins (e.g., folic acid, vitamin A, vitamin E (tocopherol), biotin, pyridoxal); bile or fatty acid conjugates, including saturated and unsaturated (e.g., lauroyl (C 12 ), myristoyl (C 14 ) and palmitoyl (C 16 ), stearoyl (C 18 ) and behenyl (C 22 ), lithocholic acid and / or lithocholic acid oleylamine conjugate (lithocholic acid oleyl, C 43); a polymer backbone or scaffold (e.g., PEG, triethylene glycol (TEG), hexaethylene glycol (HEG), poly(lactic-co-glycolic acid) (PLGA), poly(lactide-co-glycolide) (PLG), a hydrodynamic polymer; a steroid (e.g., dihydrotestosterone); a terpene (e.g., a triterpene); a cationic lipid or peptide; and / or a lipid or lipid-based molecule. Such a lipid or lipid-based molecule can bind to a serum protein, such as human serum albumin (HSA). The lipid-based ligand can be used to modulate (e.g., control) the binding of the conjugate to the target tissue. For example, a lipid or lipid-based ligand that binds more strongly to HSA is less likely to be targeted to the kidney and, therefore, less likely to be cleared from the body. The target tissue can be the liver, including parenchymal cells of the liver.

[0660] Polyamino acids, transferrin, cell targeting ligands or moieties can also be antibodies that bind to receptors on specific cell types such as hepatocytes. Exemplary cell receptor-specific monoclonal antibodies are by Xia et al. (2009, Mol Pharm, 63(3):747-751); Cuellar et al. (2015, Nucleic Acids Research, 43(2):1189-1203); et al. (2016, Nat Protocol, 11(1):22-36); Ibtejah et al. (2017, Clin Immunol, 176:122-130); and Sugo et al. (2016, J Control Release, 237:1-13).

[0661] Cell targeting ligands or moieties also encompass monovalent or multivalent (e.g., trivalent) GalNAc groups, e.g., Prakash et al. (2015, Bioorg Med Chem Lett, 25(19):4127-4130); Zu et al. (2016, Mol Ther-Nucleic Acids, e317, doi:10.1038 / mnta.2016.26); Zimmermann et al. (2017, Mol Ther, 25(1):71-78); Shemesh et al. (2016, Mol Ther Nucleic Acids, 5:e319-doi:10.1038 / mnta.2016.31); Huang et al. (2016, Mol Ther-Nucleic Acids, 6:116-132); Rozema et al. (2007, Proc Natl Acad Sci USA, 104(32):12982-12987); Rajeev et al. (2015, Chembiochem, 16(6):903-908); and Nair et al. (2014, J Am Chem Soc, 136(49):16958-16961).

[0662] Preparation of modified dsRNA

[0663] The dsRNA of the present disclosure can be chemically / physically connected to one or more ligands, moieties or conjugates. In some embodiments, dsRNA is conjugated / attached to one or more ligands by a joint. Any joint known in the art can be used, for example, a multivalent branched joint. Conjugating a ligand to a dsRNA can change its distribution, enhance its cellular absorption and / or target specific tissues and / or be taken up by one or more specific cell types (for example, hepatocytes), and / or extend the lifespan of a dsRNA agent. In some embodiments, a hydrophobic ligand is conjugated to a dsRNA to facilitate direct penetration through the cell membrane and / or be taken up by cells (for example, hepatocytes).

[0664] In some embodiments of the dsRNA conjugate, one or more nucleotides may comprise a group bearing a targeting moiety, e.g., one or more nucleotides comprise a group bearing a targeting moiety, wherein the targeting moiety may be covalently attached to the nucleotide backbone via a linker group. According to these embodiments, one or more nucleotides of the dsRNA are conjugated to a group bearing a targeting moiety comprising a targeting moiety, and wherein the targeting moiety may be a ligand (e.g., a cell penetrating moiety or agent) that enhances intracellular delivery of the composition.

[0665] The ligand-conjugated dsRNAs of the present disclosure and sequence-specifically linked nucleosides and nucleotides with ligand molecules can be assembled by any method known in the art, including, for example, by utilizing standard nucleotide precursors, or nucleotides or nucleoside conjugated precursors already bearing a linking moiety, ligand-nucleotide or nucleoside conjugated precursors already bearing a ligand molecule, or building blocks with non-nucleoside ligands, on a suitable DNA synthesizer.

[0666] The ligand-conjugated dsRNA disclosed herein can be synthesized by any method known in the art, including, for example, by using a dsRNA with a side chain reactive functional group, such as one derived from a linker attached to the dsRNA. In some embodiments, the reactive oligonucleotide can react directly with a commercially available ligand, a synthetic ligand with any of a variety of protective groups, or a ligand with a linking portion attached thereto. In some embodiments, the method promotes the synthesis of ligand-conjugated dsRNA by using nucleoside monomers that have been appropriately conjugated to the ligand and can be further attached to a solid support material. In some embodiments, a dsRNA with an arylalkyl ligand attached to the 3'-end of the dsRNA is prepared by first covalently attaching the monomer building block to a controlled pore glass support via an aminoalkyl group; then, the nucleotide is bonded to the monomer building block bound to the solid support by standard solid phase synthesis techniques. The monomer building block can be a nucleoside or other organic compound compatible with solid phase synthesis.

[0667] Composition

[0668] Certain aspects of the present disclosure relate to compositions (e.g., pharmaceutical compositions) comprising the dsRNA described herein. In some embodiments, the compositions (e.g., pharmaceutical compositions) further comprise a pharmaceutically acceptable carrier. In some embodiments, the compositions (e.g., pharmaceutical compositions) can be used to treat diseases or conditions associated with the expression or activity of a targeted gene.

[0669] Compositions (eg, pharmaceutical compositions) of the present disclosure are formulated based on the mode of delivery, including, for example, compositions formulated for parenteral delivery to the liver.

[0670] The compositions (eg, pharmaceutical compositions) of the present disclosure can be administered at a dosage sufficient to inhibit expression of the targeted gene. In some embodiments, a suitable dosage of dsRNA is in the range of 0.01 mg to 400 mg per kg of recipient body weight.

[0671] Those of ordinary skill in the art will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including, but not limited to, the severity of the disease or condition, previous treatment, the subject's overall health and / or age, and the presence of one or more other diseases. In addition, treating a subject with a therapeutically effective amount of a pharmaceutical composition may include a single treatment or a series of treatments. As disclosed herein, estimates of the effective dosage and in vivo half-life of a dsRNA can be made using conventional methods or based on in vivo testing using appropriate animal models.

[0672] The dsRNA molecules of the present disclosure can be formulated in a pharmaceutically acceptable carrier or diluent. Pharmaceutically acceptable carriers can be liquid or solid and can be selected with the planned mode of administration in mind to provide the required volume, consistency, and other relevant transportation and chemical properties. Any known pharmaceutically acceptable carrier or diluent can be used, including, for example, water, saline solutions, adhesives (such as polyvinyl pyrrolidone or hydroxypropyl methylcellulose), fillers (such as lactose and other sugars, gelatin, or calcium sulfate), lubricants (such as starch, polyethylene glycol, or sodium acetate), disintegrants (such as starch or sodium starch glycolate), calcium salts (such as calcium sulfate, calcium chloride, calcium phosphate, etc.), and wetting agents (such as sodium lauryl sulfate).

[0673] The dsRNA molecules of the present disclosure can be formulated into compositions (e.g., pharmaceutical compositions) containing dsRNA mixed, encapsulated, conjugated, or otherwise associated with other molecules, molecular structures, or nucleic acid mixtures. For example, a composition comprising one or more dsRNAs as described herein can include 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 vehicle (as described herein).

[0674] Vector and dsRNA delivery

[0675] The dsRNA of the present disclosure can be delivered directly or indirectly. In some embodiments, the dsRNA is delivered directly by administering a composition (e.g., a pharmaceutical composition) comprising the dsRNA to the subject. In some embodiments, the dsRNA is delivered indirectly by administering one or more carriers described herein.

[0676] deliver

[0677] The dsRNAs of the present disclosure can be delivered by any method known in the art, including, for example, by adapting a method for delivering nucleic acid molecules for use with dsRNA (see, e.g., Akhtar, S. et al. (1992) Trends Cell. Biol. 2(5):139-144; WO 94 / 02595) or by other methods known in the art (see, e.g., Kanasty, R. et al. (2013) Nature Materials 12:967-977; Wittrup, A. and Lieberman, J. (2015) Nature Reviews Genetics 16:543-552; Whitehead, K. et al. (2009) Nature Reviews Drug Discovery 8:129-138; Gary, D. et al. (2007) 121(1-2):64-73; Wang, J. et al. (2010) AAPS J.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 BerlinHeidelberg, DOI 10.1007 / 978-3-642-12168-5; Xu, C. and Wang, J. (2015) Asian Journal of Pharmaceutical Sciences 10(1):1-12).

[0678] In some embodiments, the dsRNA of the present disclosure is delivered by a delivery vehicle comprising the dsRNA. In some embodiments, the delivery vehicle is a liposome, a lipid complex, a complex, or a nanoparticle.

[0679] Liposomal preparations

[0680] Liposomes are unilamellar or multilamellar vesicles having a membrane formed of a lipophilic material and an aqueous interior. In some embodiments, liposomes are vesicles composed of amphiphilic lipids arranged into a spherical double layer or double layer. The aqueous portion comprises the composition to be delivered. Cationic liposomes have the advantage of being able to fuse to the cell wall. The advantages of liposomes include, for example, that liposomes obtained from natural phospholipids have biocompatibility and biodegradability; liposomes can be incorporated with a variety of water-soluble and fat-soluble drugs; liposomes can protect the encapsulated drugs in the internal compartment from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.245). The important considerations for preparing liposome formulations are lipid surface charge, vesicle size, and the aqueous volume of the liposomes. For example, engineered cationic liposomes and sterically stabilized liposomes can be used to deliver dsRNA. See, e.g., Podesta et al. (2009) Methods Enzymol. 464, 343-54; U.S. Patent 5,665,710.

[0681] Nucleic acid-lipid particles

[0682] In some embodiments, dsRNA of the present disclosure is fully encapsulated in lipid formulations, for example, to form nucleic acid-lipid particles, such as SPLP, pSPLP or SNALP. As used herein, term " SNALP " refers to stable nucleic acid-lipid particles, including SPLP. As used herein, term " SPLP " refers to nucleic acid-lipid particles comprising the plasmid DNA encapsulated in lipid vesicles. Nucleic acid-lipid particles, such as SNALP, generally contain cationic lipids, non-cationic lipids, cholesterol and prevent particle aggregation and increase the lipid (such as, PEG-lipid conjugate) of circulation time. SNALP and SPLP are very useful for systemic application because they show the circulation life of extension after intravenous (iv) injection, and accumulate in distal position (such as, the position physically separated from the administration site). SPLP includes " pSPLP ", which includes the condensing agent-nucleic acid complex of the package as described in PCT Publication No. WO 00 / 03683.

[0683] In some embodiments, when present in nucleic acid-lipid particles, the dsRNA is resistant to nuclease degradation in aqueous solution. Nucleic acid-lipid particles and methods for preparing them are disclosed, for example, in U.S. Patent Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; and 6,815,432; and PCT Publication No. WO 96 / 40964.

[0684] In some embodiments, the nucleic acid-lipid particle comprises a cationic lipid. Any cationic lipid known in the art or its mixture can be used. In some embodiments, the nucleic acid-lipid particle comprises a non-cationic lipid. Any non-cationic lipid known in the art or its mixture can be used. In some embodiments, the nucleic acid-lipid particle comprises a conjugated lipid (e.g., to prevent accumulation). Any conjugated lipid known in the art can be used.

[0685] Other preparations

[0686] For the successful in vivo delivery of dsRNA molecules, important considerations include: (1) the biological stability 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 by local administration, for example by direct injection or implantation into tissue or by local administration of the formulation. For systemic administration of dsRNA for the treatment of disease, the dsRNA can be modified or delivered using a drug delivery system; both of these approaches serve to prevent the rapid degradation of the dsRNA in vivo by endonucleases and exonucleases. 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 described above, dsRNA molecules can be modified by chemical conjugation to lipophilic groups, such as cholesterol, to enhance cellular uptake and prevent degradation. In some embodiments, the dsRNA is delivered using a drug delivery system, such as a nanoparticle, a dendrimer, a polymer, a liposome, or a cationic delivery system. Positively charged cationic delivery systems promote the binding of dsRNA molecules (negatively charged) and also enhance the interaction on the negatively charged cell membrane, thereby allowing cells to effectively absorb dsRNA. Cationic lipids, dendrimers or polymers can be combined with dsRNA, or induce the formation of vesicles or micelles that encapsulate dsRNA (see, for example, Kim S.H. et al. (2008) Journal of Controlled Release 129 (2): 107-116). When systemically administered, the formation of vesicles or micelles further prevents the degradation of dsRNA. The preparation and administration methods of cation-dsRNA complexes are known in the art. In some embodiments, dsRNA forms a complex with cyclodextrin for systemic administration.

[0687] Methods using dsRNA

[0688] Certain aspects of the present disclosure relate to methods for inhibiting targeted gene expression in mammals, comprising administering an effective amount of one or more dsRNAs of the present disclosure, one or more vectors of the present disclosure, or a composition of the present disclosure (e.g., a pharmaceutical composition), comprising one or more dsRNAs of the present disclosure. Certain aspects of the present disclosure relate to methods for treating and / or preventing one or more target gene-mediated diseases or conditions, comprising administering one or more dsRNAs of the present disclosure and / or one or more vectors of the present disclosure and / or a composition (e.g., a pharmaceutical composition) comprising one or more dsRNAs of the present disclosure. In some embodiments, down-regulating the expression of a target gene in a subject alleviates one or more symptoms of a target gene-mediated disease or condition in the subject.

[0689] In some embodiments, after treatment, expression of the target gene in the subject is inhibited 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%, compared to pre-treatment levels. In some embodiments, after treatment, target gene expression is inhibited 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 compared to pre-treatment levels. In some embodiments, the target gene is inhibited in the liver of the subject.

[0690] In some embodiments, the subject is a human. In some embodiments, the subject suffers from or has been diagnosed with a target gene-mediated condition or disease. In some embodiments, the subject is suspected of suffering from a target gene-mediated condition or disease. In some embodiments, the subject is at risk of developing a target gene-mediated condition or disease.

[0691] It can be understood from the content of the present disclosure that the main feature of dsRNA as described herein is to be present in the nucleotide analogs of one or more formula (II) contained therein, and the nucleotide analogs of the formula (II) have the specific structural characteristics of their "sugar-like" group. It is generally envisioned that dsRNA as described herein is used to target the selected nucleic acid sequence contained in the target nucleic acid. In particular, the embodiment of the dsRNA composed of siRNA described herein comprises an antisense strand that specifically hybridizes with the nucleic acid sequence contained in the target nucleic acid. DsRNA as described herein or compositions (for example, pharmaceutical compositions) can be used to treat target gene-mediated illness or disease. In particular, dsRNA as described herein or compositions (for example, pharmaceutical compositions), especially dsRNA of the nucleotide analogs of the targeting comprising one or more targeted nucleotide analogs, especially one or more formula (II), can be used to treat target gene-mediated illness or disease where liver targeting is required.

[0692] Certain aspects of the present disclosure also relate to methods of delivering a nucleic acid to a hepatocyte comprising contacting the hepatocyte with a dsRNA described herein.

[0693] DsRNA as herein described or compositions (such as pharmaceutical compositions) can be administered by any means 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 a mammal suffering from hyperlipidemia, the dsRNA molecules are systemically administered by parenteral means. In some embodiments, dsRNA and / or compositions are administered by subcutaneous administration. In some embodiments, dsRNA and / or compositions are administered by intravenous administration. In some embodiments, dsRNA and / or compositions are administered by pulmonary administration.

[0694] The therapeutic or preventive effect of the dsRNA is evident when there is a statistically significant improvement in one or more parameters of the disease state, or when there is no expected worsening or progression of symptoms. For example, a favorable change of at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more in a measurable disease parameter can indicate an effective treatment. The efficacy of a given dsRNA or composition comprising a dsRNA can also be judged using experimental animal models of a given disease or condition known in the art. When using an experimental animal model, the efficacy of the treatment is confirmed when a statistically significant reduction in a marker or symptom is observed.

[0695] Kits and Products

[0696] Certain aspects of the present disclosure relate to an article of manufacture or kit comprising one or more dsRNAs, vectors, or compositions (e.g., pharmaceutical compositions described herein that can be used to treat and / or prevent target gene-mediated conditions or diseases as described above). The article of manufacture or kit may further comprise a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be formed from a variety of materials, such as glass or plastic. The container may hold a composition that is effective for treating or preventing a disease by itself or in combination with another composition and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is a dsRNA described herein. The label or package insert indicates that the composition is used to treat a target-mediated condition or disease. In addition, the article of manufacture or kit may comprise (a) a first container containing a composition, wherein the composition contains a dsRNA described herein; and (b) a second container containing a composition, wherein the composition contains a second therapeutic agent. The article or kit in this embodiment of the present disclosure may further include a package insert indicating that the composition can be used to treat a specific disease. Alternatively or additionally, the article or kit may also include a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. From a commercial and user perspective, it may also include other materials, including other buffers, diluents, filters, needles, and syringes.

[0697] In this specification, particularly in the examples herein, nucleotide sequences are disclosed for reference. For the purpose of patent matters, identical sequences are also provided in the form of sequence listings formatted according to standard requirements. If there are any differences between the sequence and the standard sequence listing, the sequence described in this specification is used as a reference.

[0698] Without limiting the present disclosure, several embodiments of the present disclosure are described below for illustrative purposes.

[0699] Example

[0700] Abbreviations used:

[0701] -AcOH: acetic acid

[0702] -FA: Formic acid

[0703] -ACN: acetonitrile

[0704] -DCM: dichloromethane

[0705] -DMA: dimethylacetamide

[0706] -DCE: dichloroethane

[0707] -DMF: dimethylformamide

[0708] -DMSO: dimethyl sulfoxide

[0709] -EtOAc: ethyl acetate

[0710] -EtOH: ethanol

[0711] -Et2O: ether

[0712] -iPrOH: isopropyl alcohol

[0713] -THF: Tetrahydrofuran

[0714] -MeOH: methanol

[0715] -NMP: N-methyl-2-pyrrolidone

[0716] -PE: Petroleum ether

[0717] -Pyr: pyridine

[0718] -iPr: isopropyl

[0719] -cHex: cyclohexyl

[0720] -MTB: methyl-tert-butyl

[0721] -DIPEA: diisopropylethylamine

[0722] -DMAP: 4-(dimethylamino)-pyridine

[0723] -HBTU: (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate)

[0724] -TBTU: O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate

[0725] -DDTT: 3-((N,N-dimethyl-aminomethylene)amino)-3H-1,2,4-dithiazole-5-thione

[0726] -NEt3: triethylamine

[0727] -NEM: N-ethyl-morpholine

[0728] -BSA: N,O-bis-trimethylsilylacetamide

[0729] -TMSOTf: trimethylsilyl trifluoromethanesulfonate

[0730] -Ts: p-toluenesulfonyl

[0731] -Tf: trifluoromethanesulfonyl

[0732] -Trifluoromethanesulfonate

[0733] -TFA: trifluoroacetic acid

[0734] -DCA: dichloroacetic acid

[0735] -TEA: triethylammonium

[0736] -TIPS: triisopropylsilyl

[0737] -TBDMS: tert-butyldimethylsilyl

[0738] -DMT: 4,4'-dimethoxytrityl

[0739] -Bzl: benzoyl

[0740] -Bn: benzyl

[0741] -BOM: benzyloxymethyl

[0742] -Ac: acetyl

[0743] - i Bu: isobutyryl

[0744] -Boc: tert-butyloxycarbonyl

[0745] -Fmoc: fluorenylmethyloxycarbonyl

[0746] -Fmoc-OSu: N-(9-fluorenylmethoxycarbonyloxy)succinimide

[0747] -CE: Cyanoethyl

[0748] -CPG: Controlled Pore Glass

[0749] -T: thymine

[0750] -U: uracil

[0751] -C: cytosine

[0752] -A: adenine

[0753] -G: Guanine

[0754] -I: Hypoxanthine Hypoxanthine

[0755] -T BOM : N-benzyloxymethyl-thymine

[0756] -U BOM :N-Benzyloxymethyl-uracil

[0757] -U Bzl :N-benzoyl-uracil

[0758] -C Bzl :N-benzoyl-cytosine

[0759] -A Bzl :N-benzoyl-adenine

[0760] -G iBu :N-isobutyryl-guanine

[0761] -GalNAc: DN-acetylgalactosamine

[0762] -FR: flow rate

[0763] -HPLC: High-pressure liquid chromatography

[0764] -MS-TOF: Mass Spectrometry - Time of Flight

[0765] -LC-MS: High-pressure liquid chromatography-mass spectrometry

[0766] -R t : Retention time

[0767] -RT: Room temperature

[0768] -Hal: Halogen

[0769] -ELSD: Evaporative Light Scattering Detector

[0770] -quant.: quantitative

[0771] -sat.: saturation

[0772] -i.vac.: vacuum

[0773] -nd: not determined

[0774] -TLC: Thin layer chromatography

[0775] -h: hours

[0776] -min: minutes

[0777] -Tm: melting temperature

[0778] -r: ribonucleotide

[0779] -d: deoxy-ribonucleotide

[0780] -m: 2'-OMe-nucleotide

[0781] -f: 2'-deoxy-fluoro-nucleotide

[0782] - - ss: sense strand

[0783] -as: antisense strand

[0784] --ds: double chain

[0785] -chol: cholesterol

[0786] -PO: phosphodiester bond

[0787] -* or PS: phosphorothioate bond

[0788] -mpk: mg / kg

[0789] -M: Moore

[0790] -#: number, n°

[0791] -FBS: Fetal Bovine Serum

[0792] -ATP: adenosine triphosphate

[0793] -pre-lB: precursor nucleotide

[0794] -pre-lgB: targeted precursor nucleotide

[0795] -lB: nucleotide analogs

[0796] -lgB: targeted nucleotide analog

[0797] Example

[0798] Nomenclature has been established according to IUPAC rules.

[0799] A. Synthesis of Nucleotide Analogs of Formula (I) Wherein X is N

[0800] Unless otherwise stated, the following LC-MS methods have been used:

[0801] A:

[0802] Column: Waters ACQUITY UPLC BEH C18, 1.7μm, 2.1x50mm

[0803] Eluent: (H2O+0.05% FA) / (ACN+0.035% FA) 95:5 (0 min) to 5:95 (2 min) to 5:95 (2.6 min) to 95:5 (2.7 min) to 95:5 (3.0 min), 0.9 ml / min 55°C.

[0804] B-1:

[0805] Column: Phenomenex Luna C18, 3.0 μm, 2.0 x 10 mm

[0806] Eluent: (H2O+0.05% TFA) / ACN 93:7 (0 min) to 5:95 (1.20 min) to 5:95 ACN (1.40 min) to 93:7 (1.50 min); 1.1 ml / min, room temperature.

[0807] B-2:

[0808] Column: Phenomenex Luna C18, 3.0 μm, 2.0 x 10 mm

[0809] Eluent: (H2O+0.05% TFA) / ACN 93:7 (0 min) to 5:95 (1.00 min) to 5:95 ACN (1.45 min) to 93:7 (1.50 min); 1.1 ml / min, room temperature.

[0810] B-3:

[0811] Column: Phenomenex Luna C18, 3.0 μm, 2.0 x 10 mm

[0812] Eluent: (H2O+0.05% TFA) / ACN 20:80 (0 min) to 5:95 (0.60 min) to 5:95 ACN (1.45 min) to 80:20 (1.50 min); 1.1 ml / min, room temperature.

[0813] C:

[0814] Column: Waters ACQUITY UPLC BEH C18, 1.7μm, 2.1x50mm

[0815] Eluent: (H2O+0.05% FA) / (ACN+0.035% FA) 98:2 (0 min) to 98:2 (0.2 min) to 2:98 (3.8 min) to 2:98 (4.3 min) to 98:2 (4.5 min), 1.0 ml / min, 55°C.

[0816] D:

[0817] Column: Chromolith@Flash RP-18E 2x25mm

[0818] Eluent: (H2O + 0.0375% TFA) / (ACN + 0.01875% TFA) 95:5 (0 min) to 5:95 (0.80 min) to 5:95 (1.20 min) to 95:5 (1.21 min) to 95:5 (1.55 min), 1.5 ml / min, 50°C.

[0819] E:

[0820] Column: Kinetex EVO C18E 2.1x30mm, 5μm

[0821] Eluent: (H2O + 0.0375% TFA) / (ACN + 0.01875% TFA) 95:5 (0 min) to 5:95 (0.80 min) to 5:95 (1.20 min) to 95:5 (1.21 min) to 95:5 (1.55 min), 1.5 ml / min, 50°C.

[0822] Example A.1:

[0823] Synthesis Scheme 1

[0824]

[0825] 1a: [(2S,3S,4R,5R)-4-acetoxy-3-benzyloxy-2-(benzyloxymethyl)-5-(5-methyl-2,4-dioxo-pyrimidin-1-yl)tetrahydrofuran-2-yl]methyl acetate

[0826] In an argon atmosphere, 42.5 g (87.4 mmol) of starting material G3 and 22.3 g (174.7 mmol) of thymine were dissolved in 500 ml of dry ACN. After adding 106.6 g (128.2 ml, 524.1 mmol) of BSA, the solution was stirred at 80° C. for 1 hour. After cooling the solution to 50° C., 31.8 g (25.9 ml, 141.5 mmol) of TMSOTf were added and heating was continued at 80° C. for 1 hour. The solvent was evaporated in vacuo and the residue was dissolved in EtOAc and washed with saturated NaHCO solution, H O and saturated NaCl solution. After drying with MgSO , the organic layer was evaporated and the crude product was purified by silica gel chromatography (10 to 100% ethyl acetate in n-heptane) to obtain the desired thymidine derivative 1a as a colorless foam (40.9 g, 84.7%).

[0827] LCMS-Method A:

[0828] UV-wavelength [nm] = 220:R t [min]=1.82

[0829] Ionization method: ES - :[MH] - =551.3

[0830] 1H-NMR (CDCl3, 400MHz) δ [ppm]: 8.55 (s, 1H) 7.51-7.17 (m, 12H), 6.23 (d, J = 5.27Hz, 1H), 5.44 (t, J = 5.65Hz, 1H), 4.72-4.35 ( m, 6H), 4.19 (d, J=12.17Hz, 1H), 3.79 (d, J=10.16Hz, 1H), 3.53 (d, J=10.16Hz, 1H) 2.17-2.06 (m, 6H), 1.55 (d, J=0.75Hz, 3H).

[0831] 1b: [(2S,3S,4R,5R)-4-acetoxy-3-benzyloxy-2-(benzyloxymethyl)-5-(2,4-dioxopyrimidin-1-yl)-tetrahydrofuran-2-yl]methyl acetate

[0832] 10.0g (20.55mmol) G3 and 3.49g (30.8mmol) uracil are dissolved in 200ml dry ACN. After adding 20.09g (30.15ml, 123.3mmol) BSA, solution was stirred at 81 ℃ for 1 hour. After cooling solution to 0 ℃, 7.48g (6.10ml, 33.3mmol) TMSOTf was added, and the mixture was heated to 65 ℃. When reaching complete conversion, stirring was continued at this temperature for 1 hour. At room temperature, 500ml saturated NaHCO solution was added, and the mixture was extracted with 500ml EtOAc. The organic layer was separated and washed with H O and saturated NaCl solution. After drying with MgSO , the crude product was purified on silica gel (20 to 100% EtOAc in the normal heptane) to obtain protected nucleoside analog 1b, 82.3% productive rate (9.11g), which is colorless foam.

[0833] LCMS-Method A:

[0834] UV-wavelength [nm] = 220:R t [min]=1.82

[0835] Ionization method: ES + :[M+H] + =539.1

[0836] 1H-NMR (CDCl3, 400MHz) δ [ppm]: 8.97 (s, 1H), 7.66 (d, J=8.0Hz, 1H), 7.38-7.35 (m, 10H), 6.19 (d, J=4.0Hz, 1H), 5.38-5.30 (m, 2H), 4.63 (d, J=12.0Hz, 1H), 4.46-4.41 (m, 5H), 4.19 (d, J=12.0Hz, 1H), 3.79 (d, J=12.0Hz, 1H), 3.51 (d, J=12.0Hz, 1H), 2.12 (s, 3H), 2.07 (s, 3H).

[0837] 1c: [(2S,3S,4R,5R)-4-acetoxy-3-benzyloxy-2-(benzyloxymethyl)-5-[2-(2-methylpropionylamino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl]methyl acetate

[0838] At 15 ° C, in an N2 atmosphere, N-isobutyryl-guanine (135 g, 0.61 mol) and BSA (311.85 mL, 1.2 mol) were added to a 6.68 l DCE solution of compound G3 (148.5 g, 0.30 mol). The mixture was stirred at 85 ° C for 3 hours. TMSOTf (183.4 g, 0.90 mol) was then added at 85 ° C, and stirring was continued for 3 hours when TLC showed complete conversion. The mixture was cooled to room temperature and poured into 6.5 l saturated NaHCO3 solution. The organic layer was separated, and the aqueous phase was extracted twice with 5 l DCM. The organic layers were combined and dried over anhydrous Na2SO4, filtered and concentrated. The crude product obtained was purified by preparative HPLC (0.1% TFA / ACN) to obtain compound 1c (128 g, 64%) as a white solid.

[0839] 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).

[0840] 1d: [(2S,3S,4R,5R)-4-acetoxy-3-benzyloxy-2-(benzyloxymethyl)-5-(6-oxo-1H-purin-9-yl)-tetrahydrofuran-2-yl]methyl acetate

[0841] The title compound was synthesized according to the protocol described for 1c starting from 10.0 g (20.55 mmol) of G3 and 3.32 g (23.66 mmol) of hypoxanthine as the nucleobase. After silica gel purification (10 to 100% EtOAc / MeOH (9:1) in n-heptane), the title compound 1d was isolated as a colorless foam (9.53 g, 81.1%).

[0842] LCMS-Method A:

[0843] UV-wavelength [nm] = 220:R t [min]=1.73

[0844] Ionization method: ES + :[M+H] + =563.3

[0845] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 12.42 (s, 1H), 8.22 (s, 1H), 8.01 (s, 1H), 7.25-7.39 (m, 10H), 6.21 (d, J=4.89Hz, 1H), 5.90-5.97 (m , 1H), 4.78 (d, J = 5.75Hz, 1H), 4.45-4.61 (m, 4H), 4.32-4.41 (m, 1H), 4.17-4.25 (m, 1H), 3.59-3.74 (m, 2H), 2.04 (m, 3H), 1.98 (m, 3H).

[0846] 1e: [(2S,3S,4R,5R)-4-acetoxy-5-(6-benzoylaminopurin-9-yl)-3-benzyloxy-2-(benzyloxymethyl)-tetrahydrofuran-2-yl]methyl acetate

[0847] In room temperature, to compound G3 (164g, 0.337mol) and N-benzoyl adenine (121g, 0.506mol) in the mixture of 6.56l DCE, dropwise add BSA (274g, 1.348mol) and the mixture is stirred at 90 DEG C for 1.5 hours.After TMSOTf (225g, 1.011mol) is added dropwise at 40-50 DEG C, the mixture is stirred at 90 DEG C for 1 hour.The mixture is saturated with 15l NaHCO solution is quenched, and each layer is separated.Water layer is extracted 2x with 3l EtOAc, and the organic layer merged is through anhydrous Na sO dried, filtered and concentrated in vacuo.The crude product is purified by flash chromatography (PE / EtOAc 1:2), obtain compound 1e as anomer mixture (224.4g), it is yellow oily thing. The mixture was purified by two reverse phase flash chromatography (neutral) to give pure compound 1e (75.5 g, 33.7%) as a colorless foam.

[0848] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 11.25 (s, 1H), 8.72 (s, 1H), 8.62 (s, 1H), 8.05 (d, J=7. 40Hz, 2H), 7.71-7.62 (m, 1H), 7.60-7.50 (m, 2H), 7.43-7.21 (m, 10H), 6.38 (d, J=4.77Hz, 1 H), 6.12 (t, J=5.33Hz, 1H), 4.88 (d, J=5.77Hz, 1H), 4.63 (s, 2H), 4.56-4.45 (m, 2H), 4.41 ( d, J=11.92Hz, 1H), 4.25 (d, J=11.80Hz, 1H), 3.75-3.59 (m, 2H), 2.06 (s, 3H), 2.00 (s, 3H).

[0849] 2a: 1-[(2R,3R,4S,5R)-4-benzyloxy-5-(benzyloxymethyl)-3-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl]-5-methyl-pyrimidine-2,4-dione

[0850] At 0 ℃, to the solution of 40.9g (74.0mmol) 1a in 500ml THF / EtOH (4: 1), add 221.9ml (443.8mmol) of NaOH aqueous solution (2N). After removing ice bath, the mixture was stirred for 2 hours to reach complete conversion. At 0 ℃, the solution was made to neutral pH by adding 2N HCl solution. The solvent was concentrated in vacuo and the remaining aqueous phase was extracted twice with 250ml of DCM. The organic layer was dried and evaporated with MgSO4 to obtain 34.8g (quantitative) of the deprotected product 2a, which was used in the following steps without further purification.

[0851] LCMS-Method A:

[0852] UV-wavelength [nm] = 220:R t [min]=1.64

[0853] Ionization method: ES + :[M+H] + =469.2

[0854] 1H-NMR (CDCl3, 400MHz) δ [ppm]: 9.24-8.77 (m, 1H), 7.45 (br s, 1H), 7.41-7.30 (m, 8H), 7.27 (br d, J=7.78Hz, 2H), 6.04 (br d, J=4.02Hz, 1H), 4.86 (br d, J=11.67Hz, 1H), 4.63-4.51(m, 3H), 4.45-4.31(m, 2H), 4.31-3.96(m, 1H), 3.84(br d, J=11.29Hz, 1H), 3.76-3.64 (m, 2H), 3.58 (d, J=10.42Hz, 1H), 2.96-2.62 (m, 1H), 1.63-1.51 (m, 3H).

[0855] 2b: 1-[(2R,3R,4S,5R)-4-benzyloxy-5-(benzyloxymethyl)-3-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl]pyrimidine-2,4-dione

[0856] 9.10 g (16.9 mmol) of 1b was dissolved in 240 ml of THF / EtOH (3:1). At 0°C, 8.49 ml (84.5 mmol) of 1M NaOH solution was added and the solution was stirred and allowed to reach room temperature. After 1 hour, citric acid was added until pH 7 was reached and the solvent was evaporated. The aqueous residue was extracted with EtOAc. The organic layer was separated and washed with saturated NaCl solution. After drying over MgSO4, the solvent was removed to obtain 7.66 g (99.7%) of crude product as a colorless foam, which was used without further purification.

[0857] LCMS-Method A:

[0858] UV-wavelength [nm] = 220:R t [min]=1.63

[0859] Ionization method: ES + :[M+H] + =455.1

[0860] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 11.29 (s, 1H), 7.69 (d, J = 8.2Hz, 1H), 7.27-7.41 (m, 10H), 5.88 (d, J = 5.7Hz, 1H), 5.57 (d, J = 7.2Hz, 1H), 5.3 7(d, J=8.1Hz, 1H), 4.98 (t, J=5.4Hz, 1H), 4.80 (d, J=11.7Hz, 1H), 4.46 -4.58(m, 3H), 4.27-4.37(m, 1H), 4.00-4.14(m, 1H), 3.50-3.69(m, 4H).

[0861] 2c: N-[9-[(2R,3R,4S,5R)-4-benzyloxy-5-(benzyloxymethyl)-3-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]-2-methyl-propionamide

[0862] At 0 ℃, 1M NaOH solution (443mL) was added dropwise to a 1.7l THF / EtOH (4:1) solution of compound 1c (72g, 0.11mol). The solution was stirred at 0 ℃ for 1 hour to achieve complete conversion. The pH was adjusted to 7 and the solvent was removed by adding 1N HCl. The residue was dissolved in H2O (500mL) and extracted with 3x500ml DCM. The organic layers were merged, dried over anhydrous Na2SO4 and concentrated to obtain compound 2c (113g, quantitative) as a colorless solid, which was used in the next step without further purification.

[0863] 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).

[0864] 2d: 9-[(2R,3R,4S,5R)-4-Benzyloxy-5-(benzyloxymethyl)-3-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl]-1H-purin-6-one

[0865] 9.34 g (16.65 mmol) of 1d were deprotected according to the procedure described for 2b to afford 7.76 g (97.4%) of the title compound 2d as a crude product which was used without additional purification.

[0866] LCMS-Method A:

[0867] UV-wavelength [nm] = 220:R t [min]=1.53

[0868] Ionization method: ES + :[M+H] + =479.3

[0869] 1H-NMR (DMSO-d6, 400MHz) d[ppm]: 8.16 (s, 1H), 7.97-8.04 (m, 1H), 7.27-7.41 (m, 10H), 5.97 (d, J=5.75Hz, 1H), 5.75 (s, 1H), 5.03 (br s, 1H), 4.79-4.92 (m, 2H), 4.47-4.62 (m, 3H), 4.24-4.34 (m, 1H), 3.55-3.70 (m, 4H).

[0870] 2e: N-[9-[(2R,3R,4S,5R)-4-benzyloxy-5-(benzyloxymethyl)-3-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl]purin-6-yl]benzamide

[0871] 144 g (216 mmol) of 1e were deprotected according to the procedure described for 2b to give 126 g (quantitative, crude) of the title compound 2e as a colorless foam which was used without additional purification.

[0872] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 11.23 (s, 1H), 8.73 (s, 1H), 8.59 (s, 1H), 8.05 (br d, J=7.46Hz, 2H), 7.71-7.63 (m, 1H), 7.61-7.52 (m, 2H), 7.49-7.23 (m, 10H), 6.16 (d, J=5.75Hz, 1H), 5.85 (d, J=7.46Hz, 1H), 5.14-4.99 (m, 2H), 4.86 (d, J=11.86Hz, 1H), 4.63 (d, J=11.74Hz, 1H), 4.53 (s, 2H), 4.38 (d, J=5.14Hz, 1H), 3.79-3.57 (m, 4H).

[0873] 3a: 1-[(2R,3R,4S,5S)-4-benzyloxy-5-(benzyloxymethyl)-3-hydroxy-5-(triisopropylsilyloxy-methyl)tetrahydrofuran-2-yl]-5-methyl-pyrimidine-2,4-dione

[0874] The starting compound 2a of 34.7g (74.2mmol) and 16.8g (244.7mmol) imidazoles are dissolved among the dry DCM of 300ml.At room temperature, add 200ml DCM solution of 16.2g (18.0ml, 81.6mmol) TIPS-chloride, and with reaction stirring 19 hours.After with EtOH cancellation, evaporating solvent and resistates are dissolved among the EtOAc.With organic solution H O, 1N HCl, H O and saturated NaHCO Solution, saturated NaCl solution washing then.Use MgSO After drying, remove desolvation and with crude product (47.1g) by silica gel chromatography purification (10 to 100%EtOAc in the normal heptane), the product 3a that obtains required TIPS protection is colorless foam (37.5g, 80.9%).

[0875] LCMS-Method A:

[0876] UV-wavelength [nm] = 220:R t [min]=2.22

[0877] Ionization method: ES + :[M+H] + =625.3

[0878] 1H-NMR (CDCl3, 400MHz) δ [ppm]: 8.52 (s, 1H), 7.35-7.13 (m, 11H), 5.88 (d, J=3.89Hz, 1H), 4.72-4.63 (m, 1H), 4.60-4.51 (m, 1H), 4.46 (s, 2H), 4.34-4 .26 (m, 1H), 4.22 (d, J = 6.27Hz, 1H), 3.90 (d, J = 10.92Hz, 1H), 3.77 (d, J = 1. 00Hz, 2H), 3.63-3.51 (m, 2H), 1.52 (d, J=0.88Hz, 3H), 1.12-0.90 (m, 21H).

[0879] 3b: 1-[(2R,3R,4S,5S)-4-benzyloxy-5-(benzyloxymethyl)-3-hydroxy-5-(triisopropylsilyloxymethyl)tetrahydrofuran-2-yl]pyrimidine-2,4-dione

[0880] Starting from 7.66 g (16.8 mmol) of 2b, the title compound was prepared according to the protocol described for 3a to give 8.69 g (84.5%) of the desired silylated product 3b after silica gel chromatography (20 to 80% EtOAc in n-heptane).

[0881] LCMS-Method A:

[0882] UV-wavelength [nm] = 220:R t [min]=2.23

[0883] Ionization method: ES + :[M+H] + =611.2

[0884] 1H-NMR (CDCl3, 400MHz) δ [ppm]: 9.04 (s, 1H), 7.63 (d, J=8.0Hz, 1H), 7.37-7.34 (m, 8H), 7.26-7.24 (m, 2H), 5.96 (d, J = 4.0Hz, 1H), 5.37 (d, J = 8.0Hz, 1H), 4.77 ( d, J=12.0Hz, 1H), 4.62 (d, J=12.0Hz, 1H), 4.49 (s, 2H), 4.26 (d, J=8.0Hz, 1H), 3 .83 (dd, J=8.0, 10.0Hz, 2H), 3.63 (dd, J=8.0, 10.0Hz, 2H), 1.15-1.04 (m, 21H).

[0885] 3c: N-[9-[(2R,3R,4S,5S)-4-benzyloxy-5-(benzyloxymethyl)-3-hydroxy-5-(triisopropylsilyloxy-methyl)tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]-2-methyl-propionamide

[0886] At 0 ℃, under N2 atmosphere, to anhydrous DCM (1568mL) solution of compound 2c (75g, 133mmol), add imidazoles (38g, 559mmol) and TIPSCl (35.9g, 186mmol). After stirring for 12 hours at 10 to 15 ℃, solution was poured into ice water (2L) and extracted with DCM (3x1.5l). The organic layers were merged and washed with salt solution (1l), through anhydrous Na2SO4 drying, filtered and concentrated. Residue was purified by column chromatography (PE / EtOAc 2:1 to EtOAc) on silica gel to obtain 65g (68%) of title compound 3c as white foam.

[0887] 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).

[0888] 3d: 9-[(2R,3R,4S,5S)-4-Benzyloxy-5-(benzyloxymethyl)-3-hydroxy-5-(triisopropylsilyloxymethyl)tetrahydrofuran-2-yl]-1H-purin-6-one

[0889] Starting from 7.75 g (16.2 mmol) of 2d, the title compound was prepared according to the protocol described for 3a to give 8.60 g (83.6%) of the desired silylated product 3d after silica gel chromatography (0 to 100% EtOAc / MeOH (9:1) in n-heptane).

[0890] LCMS-Method A:

[0891] UV-wavelength [nm] = 220:R t [min]=2.19

[0892] Ionization method: ES + :[M+H] + =635.5

[0893] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 12.38 (br s, 1H), 8.22 (s, 1H), 8.03 (s, 1H), 7.26-7.39 (m, 10H), 5.94 (d, J=6.85Hz, 1H), 5.67 (d, J=6.48Hz, 1H), 4.98- 4.74 (m, 2H), 4.61-4.48 (m, 3H), 4.24 (d, J=4.89Hz, 1H), 3.97-3.82 (m, 2H), 3.69 (s, 2H), 0.80-1.18 (m, 21H).

[0894] 3e: N-[9-[(2R,3R,4S,5S)-4-benzyloxy-5-(benzyloxymethyl)-3-hydroxy-5-(triisopropylsilyloxymethyl)tetrahydrofuran-2-yl]purin-6-yl]benzamide

[0895] At room temperature, to the mixture of compound 2e (135g, 0.232mol) and imidazoles (47.4g, 0.696mol) in 1.35l DCM solution of TIPSCl (80.6g, 0.418mol) was added. The mixture was stirred for 24 hours. After quenching with 400ml of EtOH, the mixture was washed with 1.5l water. The water layer was extracted twice with 1l EtOAc, and the organic layer combined was dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The crude product was purified by flash chromatography (PE / EtOAc 2:1) to obtain silyl ether 3e (145g, 84.8%) as a white foam.

[0896] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 11.23 (s, 1H), 8.74 (s, 1H), 8.63 (s, 1H), 8.06 (d, J= 7.28Hz, 2H), 7.71-7.62(m, 1H), 7.60-7.51(m, 2H), 7.44-7.23(m, 10H), 6.13(d, J=6.7 8Hz, 1H), 5.77 (d, J=6.40Hz, 1H), 5.21-5.10 (m, 1H), 4.94 (d, J=11.92Hz, 1H), 4.66-4. 50 (m, 3H), 4.30 (d, J=4.77Hz, 1H), 4.00-3.86 (m, 2H), 3.74 (s, 2H), 1.11-0.89 (m, 21H).

[0897] 4a: 1-[(2R,3R,4S,5S)-3,4-dihydroxy-5-(hydroxymethyl)-5-(triisopropylsilyloxymethyl)-tetrahydrofuran-2-yl]-5-methyl-pyrimidine-2,4-dione

[0898] In an autoclave, a solution of 13.4 g (21.4 mmol) of bis-benzyl ether 3a in 100 ml of EtOH was degassed and purged with argon. After adding a catalytic amount of Pd(OH)2 (20% on carbon), the solution was placed under a 4 bar H2 atmosphere for 1 hour. The Pd catalyst was filtered off and the filtrate was evaporated to give 9.74 g (quantitative) of the desired debenzylated product as a colorless foam.

[0899] LCMS-Method A:

[0900] UV-wavelength [nm] = 220:R t [min]=1.84

[0901] Ionization method: ES + :[M+H] + =445.3

[0902] 1H-NMR (CDCl3, 400MHz) δ [ppm]: 8.85 (s, 1H), 7.25 (d, J = 1.00Hz, 1H), 5.58 (d, J = 6.15Hz, 1H), 4.71-4.61 (m, 1H), 4.51-4.44(m, 1H), 4.02-3.95(m, 1H), 3.94-3.89(m, 1H), 3.88-3.77(m, 3H), 3.76-3.67(m, 1H), 3.22(br dd, J=6.71, 3.83Hz, 1H), 1.93 (d, J=1.00Hz, 3H), 1.00-1.24 (m, 21H).

[0903] 4b: 1-[(2R,3R,4S,5S)-3,4-dihydroxy-5-(hydroxymethyl)-5-(triisopropylsilyloxymethyl)-tetrahydrofuran-2-yl]pyrimidine-2,4-dione

[0904] Starting from 8.69 g (14.2 mmol) of 3b, the title compound was prepared according to the protocol described for 4a to give 6.21 g (quantitative, crude) of the desired product 4b which was used without purification.

[0905] LCMS-Method A:

[0906] UV-wavelength [nm] = 220:R t [min]=1.78

[0907] Ionization method: ES + :[M+H] + =431.1

[0908] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 11.29 (s, 1H), 7.91 (d, J=8.0Hz, 1H), 5.87 ( d, J=8.0Hz, 1H), 5.69 (d, J=8.0Hz, 1H), 5.28 (d, J=8.0Hz, 1H), 5.12 (t, J=4.0H z, 1H), 5.00 (d, J=4.0Hz, 1H), 4.19 (dd, J=8.0, 12.0Hz, 1H), 4.05 (t, J=4.0Hz, 2H), 3.81 (dd, J=8.0, 12.0Hz, 2H), 3.62 (d, J=4.0Hz, 2H), 1.05-1.00 (m, 21H).

[0909] 4c: N-[9-[(2R,3R,4S,5S)-3,4-dihydroxy-5-(hydroxymethyl)-5-(triisopropylsilyloxymethyl)-tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]-2-methyl-propionamide

[0910] At -70 ° C, under N2 atmosphere, BCl3 (921 mL) was added to a solution of compound 3c (95 g, 0.132 mol) in anhydrous DCM (300 mL). When TLC detected complete conversion, the reaction solution was stirred at -75 ° C to -60 ° C for 2 hours. About 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 column chromatography on silica gel (PE / EtOAc 20:1 to 4:1) to obtain the debenzylated product 4c (51 g, 71.6% yield) as a yellow solid.

[0911] 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).

[0912] 4d: 9-[(2R,3R,4S,5S)-3,4-dihydroxy-5-(hydroxymethyl)-5-(triisopropylsilyloxymethyl)-tetrahydrofuran-2-yl]-1H-purin-6-one

[0913] 8.59g (13.53mmol) of starting compound 3d was dissolved in 200ml DCM. After being cooled to -70 ℃ to -50 ℃, 74.4ml (74.4mmol) of 1M BCl were added in 15 minutes toluene solution. After stirring at -70 ℃ for an additional 15 minutes, the cooling bath was removed and further stirring was continued at room temperature for 30 minutes. The reaction solution was subsequently dropwise added to 180ml of 7M NH MeOH solution. After stirring for 20min, the mixture was evaporated and residue was dissolved in 100ml of DCM / iPrOH (4:1). After washing with saturated NaHCO solution, each layer was separated, and water layer was extracted 3 times with DCM / iPrOH (4:1). The organic layer MgSO was dried, filtered and evaporated. The crude product was purified by silica gel chromatography (0 to 10% MeOH in DCM) to afford 4.14 g (67.3%) of the title compound 4d as a colorless foam.

[0914] LCMS-Method A:

[0915] UV-wavelength [nm] = 220:R t [min]=1.74

[0916] Ionization method: ES - :[MH] - =453.4

[0917] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 12.38 (m, 1H), 8.33 (s, 1H), 8.07 (s, 1H), 5.87 (d, J = 7.34Hz, 1H), 5.37 (d, J = 6.97Hz, 1H) , 5.01-5.14 (m, 2H), 4.64-4.76 (m, 1H), 4.15-4.20 (m, 1H), 3.81-3.93 (m, 2H), 3.65 (d, J=5.14Hz, 2H), 0.94-1.14 (m, 21H).

[0918] 4e: N-[9-[(2R,3R,4S,5S)-3,4-dihydroxy-5-(hydroxymethyl)-5-(triisopropylsilyloxymethyl)-tetrahydrofuran-2-yl]purin-6-yl]benzamide

[0919] At -70 ℃, BCl is added dropwise in the solution of 620ml DCM of compound 3e (31g, 42.0mmol) (252mL, 0.252mol).The mixture is stirred at -70 ℃, in N2, 30 minutes, and warmed to 15 ℃.After stirring another 30 minutes at this temperature, the reaction mixture is carefully poured into the NH in 600ml 7M MeOH, while keeping the temperature below -20 ℃.After evaporation, residue is dissolved in 3l DCM / iPrOH (4: 1), and with 1l water and saturated NaHCO3 solution washing.The water layer merged is extracted with 2x1l DCM / iPrOH (4: 1), and the organic layer merged is through anhydrous Na2SO4 drying, filter and vacuum concentration.Residue is purified by flash chromatography (PE / EtOAc 1: 4), obtains compound 4e (16.9g, productive rate 72.2%) as white solid.

[0920] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 11.23 (s, 1H), 8.77 (s, 1H), 8.72 (s, 1H), 8. 06(d, J=7.28Hz, 2H), 7.70-7.61(m, 1H), 7.60-7.51(m, 2H), 6.06(d, J=7.53Hz , 1H), 5.48 (d, J=6.90Hz, 1H), 5.22-5.11 (m, 2H), 4.93-4.83 (m, 1H), 4.24 (t, J =4.77Hz, 1H), 3.92 (q, J = 10.67Hz, 2H), 3.78-3.62 (m, 2H), 1.14-1.01 (m, 21H).

[0921] 5a: 1-[(2R,6S)-3,5-dihydroxy-6-(hydroxymethyl)-6-(triisopropylsilyloxymethyl)-1,4-dioxan-2-yl]-5-methyl-pyrimidine-2,4-dione

[0922] 2.93g (6.6mmol) of starting material 4a was dissolved in 120ml acetone / H2O (4:1). 1.69g (7.9mmol) of NaIO4 in 50ml H2O solution was added, and the mixture was stirred at room temperature for 1 hour. The precipitate was filtered off, and the filtrate was concentrated in vacuo. The remaining aqueous solution was extracted with 200ml EtOAc. After washing the organic layer with H2O and saturated NaCl solution, it was dried and the solvent was removed under reduced pressure to obtain 2.86g (94.1%) of the required product as a colorless solid, which was used without further purification.

[0923] LCMS-Method B-1:

[0924] UV-wavelength [nm] = 220:R t [min]=1.04

[0925] Ionization method: ES + :[M+H-H2O] + =443.2

[0926] 5b: 1-[(2R,6S)-3,5-dihydroxy-6-(hydroxymethyl)-6-(triisopropylsilyloxymethyl)-1,4-dioxan-2-yl]pyrimidine-2,4-dione

[0927] Starting from 6.21 g (14.4 mmol) of 4b, the title compound was prepared according to the protocol described in 4a to give 6.38 g (99%) of the desired product 5b as a crude product which was used without additional purification.

[0928] LCMS-Method B-1:

[0929] UV-wavelength [nm] = 220:R t [min]=1.00

[0930] Ionization method: ES + :[M+H-H2O] + =429.5

[0931] 5c: N-[9-[(2R,6S)-3,5-dihydroxy-6-(hydroxymethyl)-6-(triisopropylsilyloxymethyl)-1,4-dioxan-2-yl]-6-oxo-1H-purin-2-yl]-2-methyl-propionamide

[0932] At 25 ℃, to the solution of compound 4c (25g, 46mmol) in the mixed solvent of acetone (485ml) and water (145ml), add NaIO4(13.9g, 65mmol) water (150ml) solution, and the mixture was stirred for 12 hours. After the solvent was evaporated in vacuo, the residue was dissolved in EtOAc (500ml) and washed with saturated NaHCO3 solution (500ml) and salt solution (500ml). With organic layer Na2SO4 drying, filter and vacuum concentration, obtain rough 5c (25.7g, quantitative) as white solid, without the need to purify it and use it.

[0933] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 12.05 (br s, 1H), 11.79 (s, 1H), 8.17 (s, 1H), 7.52 (d, J=5.62Hz, 1H), 6.23 (s, 1H), 5.34(d, J=5.62Hz, 1H), 5.23(s, 1H), 4.46(d, J=9.54Hz, 1H), 4.15-4.04( m, 1H), 3.93 (d, J=9.78Hz, 1H), 3.85 (d, J=10.03Hz, 1H), 3.71 (d, J=10.0 3Hz, 1H), 2.85-2.66 (m, 1H), 1.12 (d, J=6.85Hz, 6H), 1.07-0.95 (m, 21H).

[0934] 5d: 9-[(2R,6S)-3,5-dihydroxy-6-(hydroxymethyl)-6-(triisopropylsilyloxymethyl)-1,4-dioxan-2-yl]-1H-purin-6-one

[0935] 4.14g (9.1mmol) of starting material 4d was dissolved in 120ml acetone / H2O (4:1). 2.63g (12.3mmol) of NaIO4 in 50ml H2O solution was added, and the mixture was stirred at room temperature for 18 hours. The precipitate was filtered off, and the filtrate was concentrated in vacuo. 100ml of saturated NaHCO3 solution was added to the remaining aqueous solution, and the water layer was extracted twice with 100ml DCM / iPrOH (4:1). The organic layer was dried with MgSO4 and the solvent was removed under reduced pressure to obtain 4.27g (99.7%) of the desired product 5d as a colorless foam, which was used without further purification.

[0936] LCMS-Method A:

[0937] UV-wavelength [nm] = 220:R t [min]=1.82

[0938] Ionization method: ES + :[M+H-H2O] + =453.3

[0939] 6a:1-[(2R,6S)-6-(hydroxymethyl)-6-(triisopropylsilyloxymethyl)morpholin-2-yl]-5-methyl-pyrimidine-2,4-dione

[0940] To a solution of compound 5a (31 g, 67 mmol) in anhydrous MeOH (527 ml) was added (NH4)2B2O7.4H2O (19.5 g, 74 mmol) at room temperature under N2 atmosphere. The mixture was stirred for 2 hours, and then AcOH (8.08 g, 134 mmol) and To the 4-nitro-2-oxo-2-nitro-4-oxo-5-nitro-2-oxo-5-nitro-2-oxo-3 ...

[0941] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 11.27 (br s, 1H), 7.71-7.52 (m, 1H), 5.90-5.68 (m, 1H), 4.68-4.46 (m, 1H), 4.05-3.83 (m, 2H), 3.40 (br d, J=5.38Hz, 2H), 2.83 (br d, J=9.78Hz, 1H), 2.79-2.72 (m, 1H), 2.66 (br d, J=12.96Hz, 2H), 1.79 (s, 3H), 1.22-0.83 (m, 23H).

[0942] 6c: N-[9-[(2R,6S)-6-(hydroxymethyl)-6-(triisopropylsilyloxymethyl)morpholin-2-yl]-6-oxo-1H-purin-2-yl]-2-methyl-propionamide

[0943] Starting from 25.7 g (46.4 mmol) of compound 5c, the title compound was synthesized according to the protocol described for 6a to give the desired morpholine 6c as a colorless solid (7.5 g, 30.6%).

[0944] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 11.56 (br s, 1H), 8.13-8.25 (m, 1H), 5.67-5.81 (m, 2H), 4.56 (br s, 1H), 4.08 (br d, J=9.54Hz, 1H), 3.83 (br d, J=9.29Hz, 1H), 3.39-3.48 (m, 2H), 3.10 (br d, J=9.54Hz, 1H), 2.65-2.93 (m, 4H), 1.12 (br d, J=6.60Hz, 7H), 0.96-1.07 (m, 21H).

[0945] 7a: 1-[(2R,6S)-6-(hydroxymethyl)-4-isopropyl-6-(triisopropylsilyloxymethyl)morpholin-2-yl]-5-methyl-pyrimidine-2,4-dione (starting from 6a)

[0946] To a solution of compound 6a (19.5 g, 45.7 mmol) in anhydrous MeOH (390 ml) was added at room temperature under N2 atmosphere. To the 4-nitro-benzene of 1-nitro-2-oxo-1-oxo-2-oxo-4-oxo-5-nitro-1-oxo-2-oxo-3-oxo-4-oxo-3 ...

[0947] 7a: 1-[(2R,6S)-6-(hydroxymethyl)-4-isopropyl-6-(triisopropylsilyloxymethyl)morpholin-2-yl]-5-methyl-pyrimidine-2,4-dione (starting from 5a)

[0948] 2.85g (6.2mmol) of starting compound 5a, 1.26g (1.74ml, 12.4mmol) of NEt , 3.73g (3.56ml, 61.9mmol) of AcOH and 924mg (1.34ml, 15.5mmol) of isopropylamine were dissolved in 60ml of dry MeOH. At room temperature, 1.64g (24.8mmol) of sodium cyanoborohydride were added and the solution was stirred at 60°C for 2 hours. After cooling to room temperature, the reaction mixture was poured into 100ml of saturated NaHCO solution and the solvent was concentrated under reduced pressure. The remaining aqueous phase was extracted with EtOAc and washed with saturated NaHCO solution and saturated NaCl solution. After drying with MgSO , the solvent was evaporated and the crude product (3.0g) was purified by silica gel chromatography (10 to 80% ethyl acetate in n-heptane) to obtain 1.76g (60.7%) of required morpholine 7a as a colorless solid.

[0949] LCMS-Method A:

[0950] UV-wavelength [nm] = 220:R t [min]=1.80

[0951] Ionization method: ES + :[M+H] + =470.2

[0952] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 11.30 (s, 1H), 7.63 (s, 1H), 5.77 (dd, J=10.42, 2.89Hz, 1H), 4.61 (t, J=5 .90Hz, 1H), 4.00 (d, J=9.29Hz, 1H), 3.74 (d, J=9.03Hz, 1H), 3.53-3.41 (m, 2H), 2.83-2.63 (m, 3H), 2.26 (br d, J=11.29Hz, 1H), 2.21-2.07 (m, 1H), 1.80 (s, 3H), 1.14-1.00 (m, 21H), 0.97 (d, J=6.53Hz, 6H).

[0953] 7b: 1-[(2R,6S)-6-(hydroxymethyl)-4-isopropyl-6-(triisopropylsilyloxymethyl)morpholin-2-yl]pyrimidine-2,4-dione (starting from 5b)

[0954] To a solution of 5b (6.38 g, 14.3 mmol) in 150 ml of MeOH at room temperature were added 8.62 g (8.23 ml, 142.8 mmol) of AcOH, 2.90 g (3.98 ml, 28.6 mmol) of NEt and 2.13 g (3.09 ml, 35.7 mmol) of isopropylamine. After 1 hour, 3.78 g (57.1 mmol) of sodium cyanoborohydride were added and stirring was continued at room temperature for 1 hour, then at 65° C. for another hour. The mixture was cooled to room temperature, 150 ml of saturated NaHCO solution was added, and the MeOH was evaporated in vacuo. The aqueous solution was extracted with 350 ml of EtOAc, and the organic layer was washed with saturated NaHCO solution and saturated NaCl solution. After drying over MgSO4, the solvent was removed and the crude product was purified by silica gel chromatography (0 to 5% MeOH in DCM) to afford 3.18 g (48.9%) of the title compound 7b as a colorless solid.

[0955] LCMS-Method A:

[0956] UV-wavelength [nm] = 220:R t [min]=1.76

[0957] Ionization method: ES + :[M+H] + =456.4

[0958] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 11.32 (bs, 1H), 7.77 (d, J=8.07Hz, 1H), 5.75 (dd, J=9.90, 2.81Hz, 1H), 5.61 (d, J=8.07 Hz, 1H), 4.62 (t, J=5.93Hz, 1H), 3.99 (d, J=9.29Hz, 1H), 3.74 (d, J=9.17Hz, 1H), 3.45 (dd, J=10.33, 6.05Hz, 2H), 2.82 (br d, J=10.27Hz, 1H), 2.64-2.77 (m, 2H), 2.24 (d, J=11.37Hz, 1H), 2.03-2.13 (m, 1H), 1.00-1.10 (m, 21H), 0.96 (m, 6H).

[0959] 7c: N-[9-[(2R,6S)-6-(hydroxymethyl)-4-isopropyl-6-(triisopropylsilyloxymethyl)morpholin-2-yl]-6-oxo-1H-purin-2-yl]-2-methyl-propionamide (starting from 6c)

[0960] Starting from 7.5 g (14.0 mmol) of 6c, the title compound was synthesized according to the protocol described for 7a (starting from 6a).After final purification on silica gel (PE / EtOAc 4:1 to 1:1), 7c was isolated as a colorless solid (5.13 g, 63%).

[0961] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 12.09 (s, 1H), 11.56 (s, 1H), 8.22 (s, 1H), 5.80 (dd, J=9.79, 2.76Hz, 1H ), 4.60 (t, J=6.27Hz, 1H), 4.09 (d, J=8.78Hz, 1H), 3.71 (d, J=8.78Hz, 1H), 3.48 (d, J=6.27Hz, 2H), 2.98 (br d, J=10.04Hz, 1H), 2.89-2.65 (m, 3H), 2.43 (t, J=10.42Hz, 1H), 2.34 (d, J=11. 29Hz, 1H), 1.13 (d, J=6.78Hz, 6H), 1.08-1.01 (m, 21H), 0.99 (d, J=6.27Hz, 6H).

[0962] 7d: 9-[(2R,6S)-6-(hydroxymethyl)-4-isopropyl-6-(triisopropylsilyloxymethyl)morpholin-2-yl]-1H-purin-6-one (from 5d)

[0963] 4.27g (9.1mmol) of starting compound 5d is dissolved in 100ml MeOH. After adding 1.84g (2.53ml, 18.1mmol) NEt , 5.44g (5.19ml, 90.6mmol) AcOH and 808mg (1.17ml, 13.6mmol) isopropylamine, the solution is stirred at room temperature for 1 hour. Add 1.80g (27.2mmol) sodium cyanoborohydride, and the reaction is stirred at room temperature for 30 minutes, and when detecting complete conversion, stir another 30 minutes at 60 ℃. In the reaction mixture, add 100ml of saturated NaHCO solution and evaporate MeOH. Remaining aqueous phase is extracted with 250ml EtOAc. With saturated NaHCO solution and saturated NaCl solution washing of the organic layer, use MgSO dry and evaporate. The crude product was purified by silica gel chromatography (0 to 5% MeOH in DCM) to afford the title compound 7d as a colorless solid (2.68 g, 61.7%).

[0964] LCMS-Method A:

[0965] UV-wavelength [nm] = 220:R t [min]=1.80

[0966] Ionization method: ES + :[M+H] + =480.4

[0967] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 12.35 (br s, 1H), 8.31 (s, 1H), 8.06 (s, 1H), 5.91 (dd, J=9.90, 2.81Hz, 1H), 4.57 (t, J=6.05Hz, 1H), 4.11 (d, J=9.05Hz, 1H), 3.77 (d, J=9.05H z, 1H), 3.40-3.50 (m, 2H), 2.94-3.02 (m, 1H), 2.67-2.87 (m, 2H), 2.54-2.63 (m, 1H), 2.33 (d, J=11.37Hz, 1H), 0.97-1.11 (m, 27H).

[0968] Example A.2:

[0969] Synthesis Scheme 2

[0970]

[0971] 8: Benzoic acid [(2S,6R)-6-(2,4-dioxopyrimidin-1-yl)-4-isopropyl-2-(triisopropylsilyloxymethyl)-morpholin-2-yl]methyl ester

[0972] The starting material 7b of 6.00g (13.2mmol) is dissolved in the dry pyridine of 80ml.In room temperature, add the Benzoyl chloride of 2.80g (2.32ml, 19.75mmol), and with reaction stirring 20 hours to reach complete conversion.Behind the evaporating solvent, resistates is dissolved in EtOAc, with 2x100ml 10% citric acid solution, H O, saturated NaHCO Solution and saturated NaCl solution washing.With organic layer MgSO Drying and evaporation, the title compound that obtains 7.99g (quantitative) is colorless foam, and it is used without further purification.

[0973] LCMS-Method A:

[0974] UV-wavelength [nm] = 220:R t [min]=2.23

[0975] Ionization method: ES + :[M+H] + =560.5

[0976] 9: Benzoic acid [(2S,6R)-6-(4-amino-2-oxo-pyrimidin-1-yl)-4-isopropyl-2-(triisopropylsilyloxymethyl)-morpholin-2-yl]methyl ester

[0977] Starting compound 8 (7.99g rough, 13.2mmol) is dissolved in 200ml dry ACN. After adding 22.88g (31.4ml, 223.8mmol) NEt and 11.13g (158.0mmol) 1H-1,2,4-triazole, the solution is cooled to 0 ℃, and under vigorous stirring, 6.06g (3.68ml 39.5mmol) POCl is added 50ml dry ACN solution. The ice bath is removed and the solution is stirred for 2 hours. Under reduced pressure, about 200ml of solvent is evaporated, and the remaining solution is poured into 500ml saturated NaCHO solution / H O (1:1). The aqueous mixture is extracted 3 times with 150ml DCM, and the organic extracts combined are dried with MgSO. After evaporating solvent, crude intermediate (9.1g, yellow foam) is dissolved among the 200ml ACN, and add the ammonia solution (32%) of 100ml.When reaching complete conversion, reaction solution is stirred at room temperature 18 hours.Remove solvent under reduced pressure, and with 100ml H O is added in the resistates.Extract with 2x200ml DCM, use MgSO Dry organic phase, and evaporate solvent, obtain 7.65g (quantitative) crude compound 9, it is used without the need for additional purification.

[0978] LCMS-Method A:

[0979] UV-wavelength [nm] = 220:R t [min]=2.02

[0980] Ionization method: ES - :[MH] - =557.4

[0981] 10: Benzoic acid [(2S,6R)-6-(4-benzamido-2-oxo-pyrimidin-1-yl)-4-isopropyl-2-(triisopropylsilyloxy-methyl)morpholin-2-yl]methyl ester

[0982] The starting compound 9 of 7.64g (13.2mmol) is dissolved in 130ml dry pyridine.In room temperature, add 2.90g (2.40ml, 20.5mmol) Benzoyl chloride and solution was stirred 20 hours, when reaching complete conversion.After evaporating solvent, resistates is dissolved in EtOAc, with 2x100ml 10% citric acid solution, H O, saturated NaHOC Solution and saturated NaCl solution washing.With organic layer MgSO Drying and evaporation obtain the crude product of 8.88g, by silica gel chromatography with its purification (0 to 100%EtOAc in the normal heptane), the title compound (10) that obtains 6.69g (76.7%) is faint yellow foam.

[0983] LCMS-Method A:

[0984] UV-wavelength [nm] = 220:R t [min]=2.29

[0985] Ionization method: ES - :[MH] - =661.5

[0986] 7f: N-[1-[(2R,6S)-6-(hydroxymethyl)-4-isopropyl-6-(triisopropylsilyloxymethyl)morpholin-2-yl]-2-oxo-pyrimidin-4-yl]benzamide

[0987] 3.63g (5.48mmol) of compound 10 is dissolved in 45ml pyridine / EtOH (2: 1). At 0 ℃, add the 1M NaOH solution of 27.4ml (27.4mmol). At 0 ℃, the solution is stirred 30 minutes, and stirred at room temperature for another 3 hours, to reach complete conversion. By adding citric acid monohydrate (about 2.0g), pH is adjusted to about 6. At 35 ℃, organic solvent is removed and the aqueous solution is extracted with EtOAc. With 150ml 10% citric acid solution, H o, saturated NaHCO and saturated NaCl solution washing three times. After using MgSO dried, solvent is evaporated. The crude product (2.73g, yellow foam) of gained is purified by silica gel chromatography (10 to 70% EtOAc in normal heptane), and the title compound (7f) obtaining 2.25g (73.4%) is light yellow foam.

[0988] LCMS-Method A:

[0989] UV-wavelength [nm] = 220:R t [min]=1.89

[0990] Ionization method: ES + :[M+H] +=559.5

[0991] Example A.3:

[0992] Synthesis Scheme 3

[0993]

[0994] 11: Benzoic acid [(2S,6R)-4-isopropyl-6-(6-oxo-1H-purin-9-yl)-2-(triisopropylsilyloxymethyl)morpholin-2-yl]methyl ester

[0995] 2.68g (5.59mmol) starting material 7d, 2.53g (3.42ml, 19.55mmol) DIPEA and 0.34g (2.79mmol) DMAP are dissolved among the dry DCM of 50ml.At room temperature, add 1.18g (0.97ml, 8.38mmol) Benzoyl chloride, and reaction is stirred 18 hours to reach complete conversion.After adding 20ml MeOH, evaporating solvent and resistates are dissolved in EtOAc, use 2x100ml 10% citric acid solution, saturated NaHCO Solution and saturated NaCl solution washing.With organic layer mutually MgSO Drying and evaporation, the title compound (11) that obtains 3.47g (quantitative, crude product) is colorless foam, and it is used without further purification.

[0996] LCMS-Method A:

[0997] UV-wavelength [nm] = 220:R t [min]=2.18

[0998] Ionization method: ES + :[M+H] + =584.4

[0999] 12: Benzoic acid [(2S,6R)-6-(6-chloropurin-9-yl)-4-isopropyl-2-(triisopropylsilyloxymethyl)morpholin-2-yl]methyl ester

[1000] Starting compound 11 (2.86g crude product, 4.90mmol), N, N-dimethylaniline (1.28g, 1.34ml, 10.53mmol) and tetraethylammonium chloride (1.24g, 7.35mmol) are dissolved in 30ml dry ACN. Under stirring, 1.70g (1.03ml, 11.07mmol) POCl is added at room temperature, and the solution is refluxed for 3 hours. After cooling to room temperature, the mixture is poured into 150ml saturated NaHCO solution, and 3.0g of solid NaHCO is additionally added. The reaction is stirred for 30 minutes and extracted with 100ml DCM. Separate each layer, and the aqueous phase is extracted twice with 50ml DCM. The organic layer merged is dried and evaporated with MgSO. After purifying (0 to 50% EtOAc in n-heptane) crude product on silica gel, 1.52g (51.4%) of title compound 12 can be separated as light yellow foam.

[1001] LCMS-Method A:

[1002] UV-wavelength [nm] = 220:R t [min]=2.33

[1003] Ionization method: ES + :[M+H] + =602.4

[1004] 13: Benzoic acid [(2S,6R)-6-(6-benzoylaminopurin-9-yl)-4-isopropyl-2-(triisopropylsilyloxymethyl)morpholin-2-yl]methyl ester

[1005] Chloropurine 12 (1.54g, 2.56mmol), benzamide (646mg, 3.84mmol) and Cs CO (1.25g, 3.84mmol) are dissolved in 30ml dioxane. After adding Pd (OAc) (43mg, 191.8 μmol) and xantphos (111mg, 191.8 μmol), at 100 ℃, the reaction solution is stirred for 1 hour in argon, to reach complete conversion. The reaction is cooled to room temperature and diluted with 50ml EtOAc. The solution is filtered and the filtrate is evaporated in vacuo. The crude product (2.48g) is purified on silica gel (10 to 100% EtOAc in n-heptane) to obtain 1.29g (73.4%) of the required compound 13 as a colorless foam.

[1006] LCMS-Method A:

[1007] UV-wavelength [nm] = 220:R t [min]=2.26

[1008] Ionization method: ES + :[M+H] + =687.6

[1009] 7e: N-[9-[(2R,6S)-6-(hydroxymethyl)-4-isopropyl-6-(triisopropylsilyloxymethyl)morpholin-2-yl]purin-6-yl]benzamide

[1010] 1.00 g (1.46 mmol) of benzoate 13 were saponified according to the protocol described for 7f. Without chromatographic purification, 731 mg (86.2%, crude) of the title compound 7e could be isolated as a light yellow foam.

[1011] LCMS-Method A:

[1012] UV-wavelength [nm] = 220:R t [min]=1.97

[1013] Ionization method: ES + :[M+H] + =583.5

[1014] Example A.4:

[1015] Synthesis Scheme 4

[1016]

[1017] 14a: 1-[(2R,6S)-6-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-isopropyl-6-(triisopropylsilyloxymethyl)morpholin-2-yl]-5-methyl-pyrimidine-2,4-dione

[1018] With the primary alcohol 7a of 810mg (1.7mmol) and 873mg (1.20ml, 8.6mmol) NEt3 be dissolved in 30ml DCM.In room temperature, add 716mg (2.1mmol) DMT-Cl, and will be reacted at stirring at room 2 hours.After adding the DMT-Cl of another 0.6 equivalent and stirring and spending the night, reaction display is converted fully.The solution of crude product is directly transferred to silica column and purifies with 0 to 65%EtOAc in the normal heptane, and obtaining the required product (14a) of 1.19g (89.0%) is faint yellow foam.

[1019] LCMS-Method A:

[1020] UV-wavelength [nm] = 220:R t [min]=2.38

[1021] Ionization method: ES + :[M+H]+ =772.5

[1022] 14b: 1-[(2R,6S)-6-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-isopropyl-6-(triisopropylsilyloxymethyl)morpholin-2-yl]pyrimidine-2,4-dione

[1023] 1.00g (2.2mmol) 7b and 1.42g (1.92ml, 11.0mmol) DIPEA are dissolved in 30ml DCM.Add 948mg (2.7mmol) DMT-chloride, and reaction is spent the night at stirring at room temperature to reach complete conversion.After adding the Isolute sorbent material, remove organic solvent and with solid material purified on silicon-dioxide (with normal heptane+0.5%NEt , 0 to 75%EtOAc carries out pre-treatment in the normal heptane), the product (14b) that obtains 1.61g (96.8%) DMT protection is faint yellow foam.

[1024] LCMS-Method A:

[1025] UV-wavelength [nm] = 220:R t [min]=2.34

[1026] Ionization method: ES + :[M+H] + =758.3

[1027] 14c: N-[9-[(2R,6S)-6-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-isopropyl-6-(triisopropylsilyloxymethyl)morpholin-2-yl]-6-oxo-1H-purin-2-yl]-2-methyl-propionamide

[1028] 1.00 g (1.77 mmol) of the starting compound 7c was protected following the protocol described for 14a.After purification on silica gel (0 to 5% MeOH in DCM), the desired product (14c) was isolated as a yellow foam (1.46 g, 94.8%).

[1029] LCMS-Method C:

[1030] UV-wavelength [nm] = 220:R t [min]=3.66

[1031] Ionization method: ES + :[M+H] + =867.6

[1032] 14e: N-[9-[(2R,6S)-6-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-isopropyl-6-(triisopropylsilyloxymethyl)morpholin-2-yl]purin-6-yl]benzamide

[1033] 950mg (1.63mmol) starting material 7e is dissolved among the 10ml DCM / pyridine (1:1).After adding the 20ml DCM solution of 733mg (2.12mmol) DMT-Cl, with reaction stirring 1.5 hours, to reach complete conversion.After evaporating solvent, resistates is dissolved among the EtOAc, and uses H o, 2x10% citric acid solution and saturated NaHCO solution washing.Use MgSO after drying, remove desolvation and also with crude product purify (with normal heptane+0.5%NEt on silica ; 0 to 50%EtOAc carries out pre-treatment in the normal heptane), the DMT-ether 14e that obtains 1.20g (83.2%) is colorless foam.

[1034] LCMS-Method A:

[1035] UV-wavelength [nm] = 220:R t [min]=2.38

[1036] Ionization method: ES - :[MH] - =883.5

[1037] 14f: N-[1-[(2R,6S)-6-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-isopropyl-6-(triisopropylsilyloxymethyl)morpholin-2-yl]-2-oxo-pyrimidin-4-yl]benzamide

[1038] Following the protocol described for 14e, 2.25 g (4.02 mmol) of the starting material 7f were protected in dry pyridine. After final chromatography on silica (pre-treated with n-heptane + 0.5% NEt; 0 to 80% EtOAc in n-heptane), 3.35 g (96.9%) of the title compound (14f) were isolated as a yellow foam.

[1039] LCMS-Method A:

[1040] UV-wavelength [nm] = 220:R t [min]=2.40

[1041] Ionization method: ES + :[M+H] + =861.6

[1042] 15a:1-[(2R,6R)-6-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-(hydroxymethyl)-4-isopropyl-morpholin-2-yl]-5-methyl-pyrimidine-2,4-dione

[1043] 1.18 g (1.5 mmol) of TIPS-protected morpholine 14a and 6.74 g (9.26 ml, 66.0 mmol) of triethylamine were dissolved in 30 ml of THF. At room temperature, 12.23 g (12.4 ml, 73.6 mmol) of NEt 3 · 3 HF were added and the reaction was stirred at 65 ° C for 1 hour. After standing at room temperature overnight, 10 ml of NMP was added and the reaction was stirred at 65 ° C for an additional 14 hours to achieve complete conversion. The solvent was concentrated in vacuo, and the remaining NMP solution was added to a mixture of H2O / saturated NaHCO3 solution (1:2). The aqueous solution was extracted with EtOAc, and the organic layer was washed three times with H2O and saturated NaCl solution. After drying over MgSO4, the crude product was purified by silica gel chromatography (pretreated with 1% NEt3 in n-heptane, 10 to 100% ethyl acetate in n-heptane) to give 645 mg (68.8%) of the desilylated product 15a as a colorless foam.

[1044] LCMS-Method A:

[1045] UV-wavelength [nm] = 220:R t [min]=1.67

[1046] Ionization method: ES + :[M+H] + =616.4

[1047] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 11.36 (s, 1H), 7.53-7.58 (m, 1H), 7.41 (d, J=7.34Hz, 2H), 7.20-7.31 (m, 7H), 6.87 (d, J=8.80Hz, 4H), 5.82 (dd, J=9.66, 2.93Hz, 1H), 4.61 (br s, 1H), 3.67-3.79 (m, 8H), 3.07 (s, 1H), 2.97-3.10 (m, 1H), 2.80 (br d, J=9.78Hz, 1H), 2.63-2.76 (m, 2H), 2.17-2.31 (m, 2H), 1.69 (s, 3H), 0.95 (d, J=6.60Hz, 6H).

[1048] 15b: 1-[(2R,6R)-6-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-(hydroxymethyl)-4-isopropyl-morpholin-2-yl]pyrimidine-2,4-dione

[1049] 1.60 g (2.1 mmol) of 14b was dissolved in 20 ml of NMP. After adding 4.73 g (6.50 ml, 46.3 mmol) of NEt and 8.41 g (8.50 ml, 50.6 mmol) of NEt.3 HF, the mixture was stirred at room temperature for 15 hours and then at 65° C. for 5 hours. The mixture was cooled to room temperature, then 250 ml of saturated NaHCO solution and 250 ml of H O were added. After extraction with 250 ml of EtOAc, the organic layer was washed with H O (2×) and saturated NaCl solution. The organic phase was dried over MgSO and purified on silica (pre-treated with n-heptane+0.5% NEt, 0 to 100% EtOAc in n-heptane) to give the desired alcohol 15b as a colorless foam (1.16 g, 91.1%).

[1050] LCMS-Method A:

[1051] UV-wavelength [nm] = 220:R t [min]=1.63

[1052] Ionization method: ES + :[M+H] + =602.3

[1053] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 11.36 (br s, 1H), 7.63 (d, J=8.07Hz, 1H), 7.40 (br d, J=7.82Hz, 2H), 7.20-7.33 (m, 7H), 6.88 (d, J=8.56Hz, 4H), 5.81 (br d, J=7.34Hz, 1H), 5.62 (d, J=8.07Hz, 1H), 4.62 (br s, 1H), 3.78-3.95 (m, 1H), 3.68-3.76 (m, 7H), 3.11 (d, J=9.05Hz, 1H), 2.96 (br d, J=9.05Hz, 1H), 2.82 (br d, J=10.27Hz, 1H), 2.60-2.76 (m, 2H), 2.10-2.22 (m, 2H), 0.94 (d, J=6.36Hz, 6H).

[1054] 15c: N-[9-[(2R,6R)-6-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-(hydroxymethyl)-4-isopropyl-morpholin-2-yl]-6-oxo-1H-purin-2-yl]-2-methyl-propionamide

[1055] The alcohol 14c of TIPS protection of 1.44g (1.66mmol) is dissolved in 17ml THF. After adding 65% pyridine .HF solution of 1.27g (1.15ml, 8.30mmol), the reaction mixture was stirred at room temperature for 1 hour. Under vigorous stirring, 0.9g NaHCO and 10ml H O and continued to stir 2 hours. Remove THF and the aqueous solution is extracted 10 times with DCM / iPrOH (4: 1). The organic layer MgSO that merges is dried and evaporated to obtain 0.83g (70.1%, crude product) 15c, which is used for the next step without further purification.

[1056] LCMS-Method C:

[1057] UV-wavelength [nm] = 220:R t [min]=2.11

[1058] Ionization method: ES - :[MH] - =709.5

[1059] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 12.09 (s, 1H), 11.67 (s, 1H), 8.05 (s, 1H), 7.38 ( d, J=7.34Hz, 2H), 7.18-7.30 (m, 7H), 6.85 (d, J=8.80Hz, 4H), 5.92 (dd, J=8.99, 3.0 0Hz, 1H), 4.64 (t, J=5.14Hz, 1H), 3.71-3.84 (m, 8H), 2.90-3.08 (m, 3H), 2.55-2.83 (m, 4H), 2.31-2.47 (m, 1H), 1.12 (dd, J=6.79, 3.36Hz, 6H), 0.96 (d, J=6.48Hz, 6H).

[1060] 15e: N-[9-[(2R,6R)-6-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-(hydroxymethyl)-4-isopropyl-morpholin-2-yl]purin-6-yl]benzamide

[1061] 1.20g (1.35mmol) of starting compound 14e and 1.91g (2.63ml, 18.90mmol) NEt were dissolved in 20ml THF. After adding 3.59g (3.63ml, 21.60mmol) NEt 3HF, the solution was heated at 65°C and then another 0.95g (1.32ml, 9.45mmol) NEt and 1.80g (1.82ml, 10.8mmol) NEt 3HF were added. The reaction mixture was stirred at 65°C until complete conversion (approximately 10h) was reached. After cooling to room temperature, the mixture was poured into 150ml H O / saturated NaHCO solution (1:1) and stirred for 30 minutes. The aqueous solution was extracted 3 times with 50DCM, dried and evaporated with MgSO. After silica gel chromatography (10 to 100% EtOAc in n-heptane), 835 mg (84.9%) of the deprotected product 15e was isolated as a colorless foam.

[1062] LCMS-Method A:

[1063] UV-wavelength [nm] = 220:R t [min]=1.74

[1064] Ionization method: ES + :[M+H] + =729.5

[1065] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 11.21 (br s, 1H), 8.74 (s, 1H), 8.54 (s, 1H), 8.04 (d, J=7.62Hz, 2H), 7.62-7.67 (m, 1H), 7.52-7.58 (m, 2H), 7.37 (d, J=7.34Hz, 2H), 7.15-7.29 (m, 7H), 6.82 (d, J=7.95Hz, 4H), 6.16 (dd, J=9.17, 2.93Hz, 1H ), 4.67 (t, J = 5.20Hz, 1H), 3.86-3.96 (m, 1H), 3.79 (dd, J = 11.00, 5.87Hz, 1H), 3.69-3.73 (m, 6H) , 3.01-3.15 (m, 2H), 2.90-2.99 (m, 2H), 2.68-2.84 (m, 2H), 2.40 (m, 1H), 1.00 (d, J=6.60Hz, 6H).

[1066] 15f: N-[1-[(2R,6R)-6-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-(hydroxymethyl)-4-isopropyl-morpholin-2-yl]-2-oxo-pyrimidin-4-yl]benzamide

[1067] 3.35 g (3.88 mmol) of the starting compound 14f were treated with NEt3·3HF as described for 15e. After 20 hours at 70°C, complete conversion was achieved. Workup and final purification as for 15e gave 2.57 g (93.7%) of the title compound 15f as a colorless foam.

[1068] LCMS-Method A:

[1069] UV-wavelength [nm] = 220:R t [min]=1.79

[1070] Ionization method: ES + :[M+H] + =705.4

[1071] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 11.28 (br s, 1H), 8.10 (br d, J=7.58Hz, 1H), 8.00 (d, J=7.69Hz, 2H), 7.58-7.67 (m, 1H), 7.51 (t, J=7.64Hz, 2H), 7.21-7 .43 (m, 10H), 6.89 (d, J=8.80Hz, 4H), 5.93 (dd, J=9.41, 2.69Hz, 1H), 4.66 (t, J=5.26Hz, 1H), 3 .91 (dd, J=10.94, 4.22Hz, 1H), 3.71-3.77 (m, 7H), 3.19 (d, J=9.05Hz, 1H), 2.92-3.06 (m, 2H), 2.65-2.79 (m, 2H), 2.18 (d, J=11.49Hz, 1H), 2.07 (t, J=10.21Hz, 1H), 0.95 (d, J=6.36Hz, 6H).

[1072] 16a: 3-[[(2S,6R)-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-isopropyl-6-(5-methyl-2,4-dioxo-pyrimidin-1-yl)morpholin-2-yl]methoxy-(diisopropylamino)phosphinoyl]oxy-propionitrile

[1073] To a solution of 624 mg (1.0 mmol) of 15a and 401.0 mg (513 μl, 3.0 mmol) of N,N-diisopropylethylamine in 15 ml of dry DCM was added 370.9 mg (349.6 μl, 1.5 mmol) of 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite at room temperature under argon. After stirring for 1 hour, the reaction was quenched by adding 200 μl of n-butanol and stirring was continued for 10 minutes. The reaction solution was washed with H2O, and the aqueous phase was extracted once with DCM. After drying the organic layer over MgSO4, the solvent was removed and the crude product was purified by silica gel chromatography (pre-treated with 0.5% NEt3 in n-heptane, 10 to 100% methyl-tert-butyl ether in n-heptane) to obtain 668 mg (80.8%) of the desired phosphoramidite (mixture of diastereomers) 16a as a colorless foam.

[1074] LCMS-Method B-2:

[1075] UV-wavelength [nm] = 220:R t [min]=0.82

[1076] Ionization method: ES + :[M] + =733.1(M- i Pr2N+OH+H + )

[1077] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 11.37.11.36 (2x s, 1H), 7.60, 7.56 (2x s, 1H), 7.41 (m, 2H), 7.20-7.32 (m, 7H), 6.83-6.90 (m, 4H), 5.88 (m, 1H), 3.84-4.05 (m, 2H), 3.74 (s, 6H), 3.54-3.7 3(m, 2H), 3.40-3.54(m, 2H), 3.14(m, 1H), 2.95-3.05(m, 1H), 2.57-2.84(m, 5H), 2.15-2.37(m, 2H), 1.76, 1.73(2x s, 3H), 0.93-1.14 (m, 18H). 31P-NMR (DMSO-d6, 162MHz) δ [ppm]: 148.1, 147.6.

[1078] 16b: 3-[[(2S,6R)-2-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-(2,4-dioxopyrimidin-1-yl)-4-isopropyl-morpholin-2-yl]methoxy-(diisopropylamino)phosphinoyl]oxypropionitrile

[1079] The title compound was prepared according to the protocol described for 16a. Starting from 1.95 g (3.2 mmol) of primary alcohol 15b, 1.70 g (65.7%) of the desired phosphoramidite 16b was isolated as a colorless foam after purification on silica gel (pretreated with n-heptane + 0.5% NEt3, 0 to 80% EtOAc in n-heptane).

[1080] LCMS-Method A:

[1081] UV-wavelength [nm] = 220:R t [min]=1.85

[1082] Ionization method: ES + :[M] + =719.2(M- i Pr2N+OH+H + )

[1083] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 11.36 (br s, 1H), 7.67, 7.63 (2x d, J=8.19Hz, 1H), 7.35-7.44(m, 2H), 7.20-7.32(m, 7H), 6.84-6.89(m, 4H), 5.81-5.89(m, 1H), 5.67, 5.63(2x d, J=8.13Hz, 1H), 3.89-4.08 (m, 2H), 3.73 (s, 6H), 3.54-3.72 (m, 2H), 3.41-3.54 (m, 2H), 3.16 (d, J=9.05Hz, 1H), 2.96 (t, J=9.05Hz, 1H), 2.84 (br d, J=10.15Hz, 1H), 2.55-2.76 (m, 4H), 2.10-2.33 (m, 2H), 0.89-1.12 (m, 18H).

[1084] 31P-NMR (DMSO-d6, 162MHz) δ [ppm]: 148.3, 147.6.

[1085] 16c: N-[9-[(2R,6S)-6-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-[[2-cyanoethoxy-(diisopropylamino)phosphinoyl]oxymethyl]-4-isopropyl-morpholin-2-yl]-6-oxo-1H-purin-2-yl]-2-methyl-propionamide

[1086] 100 mg (141 μmol) of alcohol 15c, 0.2 g of molecular sieve To the 16c of 40ml (50ml) 4-nitropropane-2-nitropropane, 1 ...

[1087] LCMS-Method C:

[1088] UV-wavelength [nm] = 220:R t [min]=2.50,2.52

[1089] Ionization method: ES + :[M- i Pr2N+OH+H] + =828.3

[1090] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 12.08 (br s, 1H), 11.62 (br s, 1H), 8.09, 8.04 (2x s, 1H), 7.33-7.40 (m, 2H), 7.18-7.29 (m, 7H), 6.81-6.87 (m, 4H), 5.91 (m, 1H), 3.93-4.07 (m, 2H), 3.73 (s, 6H), 3.54-3.72 (m, 2H), 3.39-3.50 (m, 2H), 3.11 (m, 1H), 2.92-3.07 (m, 2H), 2.54-2.81 (m, 6H), 2.26-2.38 (m, 1H), 0.94-1.16 (m, 18H), 0.83-0.91 (m, 6H).

[1091] 31P-NMR (DMSO-d6, 162MHz) δ [ppm]: 147.5, 146.7.

[1092] 16e: N-[9-[(2R,6S)-6-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-[[2-cyanoethoxy-(diisopropylamino)phosphinoyl]oxymethyl]-4-isopropyl-morpholin-2-yl]purin-6-yl]benzamide

[1093] 820mg (1.13mmol) of 15e and 584mg (3.38mmol) of diisopropylammonium salt tetrazolium are dissolved in 20ml of dry DCM. In an atmosphere of argon, 524mg (1.69mmol) of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite are added, and the solution is stirred at room temperature. After stirring overnight, 50ml of H2O is added, and the layers are separated. The aqueous layer is extracted once with 30mlDCM, and the organic phase combined is dried with MgSO4. After evaporating the solvent, the crude product is purified by silica gel chromatography (using n-heptane+0.5%NEt3, 0 to 100% MTB-ether / DCM (1:1) in n-heptane is pre-treated), obtaining 913mg (87.3%) of the title compound (16e) as a colorless solid.

[1094] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 11.19 (s, 1H), 8.76, 8.74 (2x s, 1H), 8.61, 8.59 (2x s, 1H), 8.04 (d, J=8.09Hz, 2H), 7.64 (t, J=7.37Hz, 1H), 7.51-7.60 (m, 2H), 7.37 (m, 2H), 7.16-7.28(m, 7H), 6.77-6.84(m, 4H), 6.19(m, 1H), 3.98-4.15(m, 2H), 3.71(2x s, 6H), 3.56-3.69 (m, 2H), 3.41-3.56 (m, 2H), 3.03-3.17 (m, 2H), 2.89-3.01 (m, 2H), 2. 74-2.88 (m, 2H), 2.70 (t, J=6.15Hz, 1H), 2.59 (m, 1H), 2.36 (m, 1H), 0.92-1.18 (m, 18H).

[1095] 31P-NMR (DMSO-d6, 162MHz) δ [ppm]: 147.0, 146.7.

[1096] 16f: N-[1-[(2R,6S)-6-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-[[2-cyanoethoxy-(diisopropylamino)phosphinoyl]oxymethyl]-4-isopropyl-morpholin-2-yl]-2-oxo-pyrimidin-4-yl]benzamide

[1097] Following the protocol described for 16e, 550 mg (0.78 mmol) of the primary alcohol 15f was phosphitylated. After chromatography on silica gel (pre-treated with n-heptane + 0.5% NEt, 0 to 100% MTB-ether / DCM (1:1) in n-heptane), 560 mg (79.3%) of 16f was isolated as a colorless foam.

[1098] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 11.29 (br s, 1H), 8.12 (2x d, J=7.50Hz, 1H), 7.98-8.04 (m, 2H), 7.63 (t, J=7.40Hz, 1H), 7.48-7.55 (m, 2H), 7.36-7.45 (m, 3H), 7.21-7.35 (m, 7H), 6.88 (2x d, J=8.89, 4H), 5.95, 6.01 (2x m, 1H), 3.89-4.14 (m, 2H), 3.74, 3.75 (2x s, 6H), 3.55-3.72(m, 2H), 3.39-3.55(m, 2H), 3.24(d, J=8.85Hz, 1H), 2.93-3.05(m , 2H), 2.68-2.87(m, 3H), 2.55-2.64(m, 1H), 2.10-2.23(m, 2H), 0.90-1.22(m, 18H).

[1099] 31P-NMR (DMSO-d6, 162MHz) δ [ppm]: 147.4, 146.7.

[1100] Example A.5:

[1101] Synthesis Scheme 5

[1102]

[1103] 17a: Benzoic acid [(2S,6R)-4-isopropyl-6-(5-methyl-2,4-dioxo-pyrimidin-1-yl)-2-(triisopropylsilyloxy-methyl)morpholin-2-yl]methyl ester

[1104] 810mg (1.7mmol) of free alcohol 7a is dissolved among the 20ml DCM.After adding 1.14g (1.45ml, 8.6mmol) DIPEA and 294mg (243 μ l, 2.1mmol) Benzoyl chloride, solution is stirred at room temperature for 2 hours.In order to reach complete conversion, add another 0.2 equivalent of Benzoyl chloride, and continue to stir 18 hours.The reaction soln is directly transferred on the silica gel column, with 0 to 65%EtOAc wash-out in the normal heptane.The benzoylated product 17a that obtains 726mg (73.3%) is colorless foam.

[1105] LCMS-Method A:

[1106] UV-wavelength [nm] = 220:R t [min]=2.29

[1107] Ionization method: ES + :[M+H] + =574.3

[1108] 17b: Benzoic acid [(2S,6R)-6-(3-benzoyl-2,4-dioxo-pyrimidin-1-yl)-4-isopropyl-2-(triisopropylsilyloxy-methyl)morpholin-2-yl]methyl ester

[1109] As described in 17a, 1.45g (3.18mmol) of primary alcohol 7b was treated with benzoyl chloride to obtain a mixture of monobenzylated and dibenzylated products. Therefore, the reaction mixture was evaporated and the crude product was dissolved in 30ml of pyridine. After adding 565mg (3.98mmol) of benzoyl chloride and a catalytic amount of DMAP, the reaction solution was stirred at room temperature for 4 hours. After evaporating the solvent, the residue was dissolved in EtOAc and washed with 10% citric acid solution and saturated NaCl solution. The organic layer was dried and desolvated with MgSO4. Finally, chromatographic separation on silica (0 to 35% EtOAc in n-heptane) gave 1.64g (77.8%) of dibenzoylated product 17b.

[1110] LCMS-Method C:

[1111] UV-wavelength [nm] = 220:R t [min]=3.67

[1112] Ionization method: ES + :[M+H] + =664.4

[1113] 17c: Benzoic acid [(2S,6R)-4-isopropyl-6-[2-(2-methylpropionylamino)-6-oxo-1H-purin-9-yl]-2-(triisopropylsilyloxymethyl)morpholin-2-yl]methyl ester

[1114] The starting compound 7c of 0.89g (1.58mmol) is dissolved in 30ml pyridine.After adding the Benzoyl chloride of 246mg (203 μ l, 1.73mmol), when detecting complete conversion, the reaction solution is stirred at room temperature overnight. Remove the solvent in vacuo and the residue is dissolved in EtOAc. After washing with H o, the organic phase is separated and MgSO dried and evaporated. Finally purify on silicon dioxide (0 to 5% MeOH in DCM), obtaining title compound 17c is colorless foam (1.01g, 95.4%).

[1115] LCMS-Method C:

[1116] UV-wavelength [nm] = 220:R t [min]=3.28

[1117] Ionization method: ES + :[M+H] + =669.2

[1118] 18a: Benzoic acid [(2R,6R)-2-(hydroxymethyl)-4-isopropyl-6-(5-methyl-2,4-dioxo-pyrimidin-1-yl)morpholin-2-yl]methyl ester

[1119] The 15ml THF solution of 720mg (1.25mmol) 17a is handled with 1.34g (1.22ml, 8.8mmol) pyridine-hydrogen fluoride. After stirring at room temperature for 1 hour, the reaction mixture is used saturated NaHCO Solution is handled to reach pH 7.5. The solvent is concentrated under reduced pressure, and the aqueous solution is extracted twice with DCM. The organic layer is used MgSO Drying and desolvation. The crude product is co-distilled 2 times with the toluene of 50ml, and with remaining resistates by silica gel chromatography purification (0 to 100% EtOAc in the normal heptane), obtaining 502mg (95.8%) of required product (18a) is colorless foam.

[1120] LCMS-Method A:

[1121] UV-wavelength [nm] = 220:R t [min]=1.30

[1122] Ionization method: ES + :[M+H] + =418.2

[1123] 18b: Benzoic acid [(2R,6R)-6-(3-benzoyl-2,4-dioxo-pyrimidin-1-yl)-2-(hydroxymethyl)-4-isopropyl-morpholin-2-yl]methyl ester

[1124] 1.64 g (2.47 mmol) of silyl ether 17b were desilylated according to the protocol described for 18a to afford 1.33 g of the desired product 18b as a crude product which was used in the following step without chromatographic purification.

[1125] LCMS-Method C:

[1126] UV-wavelength [nm] = 220:R t [min]=1.81

[1127] Ionization method: ES + :[M+H] + =508.2

[1128] 18c: Benzoic acid [(2R,6R)-2-(hydroxymethyl)-4-isopropyl-6-[2-(2-methylpropionylamino)-6-oxo-1H-purin-9-yl]morpholin-2-yl]methyl ester

[1129] 1.0g (1.49mmol) of 17c was dissolved in 10ml DMF. After adding 1.53g (2.10ml, 15.0mmol) NEt and 1.48g (1.49ml, 9.0mmol) NEt 3 HF, the reaction solution was stirred at 90°C for 2 hours. After the mixture was cooled to room temperature, 2.5g NaHCO was added and 10ml H O was added, and the reaction mixture was stirred for 2 hours. The solvent was evaporated in vacuo, and the residue was dissolved in 25ml DCM / iPrOH and washed with H O. The organic phase was separated and dried and evaporated with MgSO. The crude product was purified on silica (0 to 10% MeOH in DCM) to obtain 245mg (32.0%) of the desilylated product 18c as a colorless foam.

[1130] LCMS-Method C:

[1131] UV-wavelength [nm] = 220:R t [min]=1.45

[1132] Ionization method: ES + :[M+H] + =513.1

[1133] 18e: Benzoic acid [(2R,6R)-6-(6-benzoylaminopurin-9-yl)-2-(hydroxymethyl)-4-isopropyl-morpholin-2-yl]methyl ester

[1134] 1.23 g (1.79 mmol) of silyl ether 13 were deprotected according to the protocol described for 18a to afford 939 mg (98.8%) of the title compound (18e) after silica gel chromatography (0 to 100% EtOAc in n-heptane).

[1135] LCMS-Method C:

[1136] UV-wavelength [nm] = 220:R t [min]=1.60

[1137] Ionization method: ES - :[MH] - =529.3

[1138] 18f: Benzoic acid [(2R,6R)-6-(4-benzamido-2-oxo-pyrimidin-1-yl)-2-(hydroxymethyl)-4-isopropyl-morpholin-2-yl]methyl ester

[1139] 3.63 g (5.48 mmol) of the silyl ether 10 were deprotected according to the protocol described for 18a to give 2.45 g (88.4%) of the title compound (18f) after silica gel chromatography (5% MeOH in DCM).

[1140] LCMS-Method A:

[1141] UV-wavelength [nm] = 220:R t [min]=1.53

[1142] Ionization method: ES + :[M+H] + =507.3

[1143] 19a: Benzoic acid [(2R,6R)-2-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-isopropyl-6-(5-methyl-2,4-dioxo-pyrimidin-1-yl)morpholin-2-yl]methyl ester

[1144] The starting compound 18a of 497mg (1.2mmol) is dissolved among the 15ml DCM.After adding 502mg (643 μ l, 3.8mmol) DIPEA and 659mg (1.90mmol) DMT-Cl, the reaction solution is stirred at room temperature for 2 hours.The reaction mixture is evaporated in vacuo and the crude product is purified on silica gel (0 to 100% EtOAc in the normal heptane).The product 19a of DMT protection is separated as colorless foam (779mg, 90.9%).LCMS-method A:

[1145] UV-wavelength [nm] = 220:R t[min]=2.14

[1146] Ionization method: ES + :[M+H] + =720.4

[1147] 19b: Benzoic acid [(2R,6R)-6-(3-benzoyl-2,4-dioxo-pyrimidin-1-yl)-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-isopropyl-morpholin-2-yl]methyl ester

[1148] The primary alcohol 18b of 1.30g (2.41mmol) and 492mg (676 μ l, 4.82mmol) NEt are dissolved among the 20ml DCM.After adding 925mg (2.65mmol) DMT-Cl, the reaction is stirred at room temperature for 6 hours, and then other 925mg (2.65mmol) DMT-Cl is added. The reaction is stirred for 72 hours.After adding 2.0ml n-propyl alcohol, the solution is stirred for 10 minutes. With the mixture H O washing, and the organic layer is separated. After using MgSO Drying, remove the solvent in vacuo. The crude product is purified by silica gel chromatography (0 to 25% EtOAc in the normal heptane), obtains 1.74g (89.2%) of required DMT-ether 19b.

[1149] LCMS-Method C:

[1150] UV-wavelength [nm] = 220:R t [min]=3.25

[1151] Ionization method: ES + :[M+H] + =810.3

[1152] 19c: Benzoic acid [(2R,6R)-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-isopropyl-6-[2-(2-methylpropionylamino)-6-oxo-1H-purin-9-yl]morpholin-2-yl]methyl ester

[1153] 240mg (468 μ mol) of starting alcohol 18c is dissolved among the 5.0ml DCM.After adding 95.7mg (131.5 μ l, 936 μ mol) NEt 3 and 180mg (515 μ mol) DMT-Cl, will react at stirring at room temperature 3 days, to reach complete conversion.After adding 2.0ml n-propyl alcohol, solution was stirred 10 minutes.With mixture H 2 O washing, and organic layer is separated.After using MgSO 4 drying, remove solvent in vacuo.Crude product is passed through silica gel chromatography purification (0 to 25% EtOAc in the normal heptane), obtains 303mg (79.4%) of required DMT-ether 19c.

[1154] LCMS-Method C:

[1155] UV-wavelength [nm] = 220:R t [min]=2.93

[1156] Ionization method: ES - :[MH] - =813.2

[1157] 19e: Benzoic acid [(2R,6R)-6-(6-benzoylaminopurin-9-yl)-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-isopropyl-morpholin-2-yl]methyl ester

[1158] 934mg (1.76mmol) of starting material 18e is dissolved in 20ml DMC / pyridine (1: 1). After adding 974mg (2.82mmol) DMT-Cl, the reaction solution is stirred at room temperature for 17 hours. The solvent is removed in vacuo and the residue is dissolved in EtOAc, and washed with H o, 2x10% citric acid solution, saturated NaHCO and NaCl solution. The organic phase is dried and evaporated with MgSO. The crude product (1.77g) gained is dissolved in 20ml EtOAc / ether (1: 2). After adding 40ml of n-pentane, centrifuge the precipitate and discard the supernatant. Repeat the precipitation procedure twice to obtain the required DMT-ether 19e as a colorless solid. LCMS-method C:

[1159] UV-wavelength [nm] = 220:R t [min]=3.07

[1160] Ionization method: ES - :[MH] - =831.5

[1161] 19f: Benzoic acid [(2R,6R)-6-(4-benzamido-2-oxo-pyrimidin-1-yl)-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-isopropyl-morpholin-2-yl]methyl ester

[1162] 18f of 2.45g (4.84mmol) is dissolved in 40ml pyridine.After adding 2.51g (7.26mmol) DMT-Cl, with reaction mixture stirring at room temperature 4 days.Desolventizing in vacuo and resistates is dissolved among the EtOAc, and uses H o, 2x10% citric acid solution, saturated NaHCO solution and NaCl solution washing.With organic phase MgSO dry and evaporation.Crude product is purified on silica gel (with normal heptane+0.5%NEt , 0 to 100%EtOAc carries out pre-treatment in the normal heptane), the title compound 19f that obtains 3.69g (94.3%) is colorless foam.

[1163] LCMS-Method A:

[1164] UV-wavelength [nm] = 220:R t [min]=2.14

[1165] Ionization method: ES - :[MH] - =807.4

[1166] 20a: 1-[(2R,6S)-6-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-(hydroxymethyl)-4-isopropyl-morpholin-2-yl]-5-methyl-pyrimidine-2,4-dione

[1167] Benzoyl ester 19a (775 mg, 1.1 mmol) is dissolved in 20 ml of THF-methanol (4: 1). At room temperature, 4.31 ml (8.6 mmol) of 2N NaOH aqueous solution are added, and the reaction is stirred for 90 minutes. By adding citric acid monohydrate, the pH is adjusted to 7 to 8, and then the solvent is removed under reduced pressure. Residue is dissolved in DCM and H O. The organic layer is separated, and the aqueous phase is extracted with DCM. The organic phase merged is dried and evaporated with MgSO. The crude product is purified by silica gel chromatography (5 to 100% EtOAc in n-heptane) to obtain 663 mg (quantitative) of the desired product (20a) as colorless foam.

[1168] LCMS-Method A:

[1169] UV-wavelength [nm] = 220:R t [min]=1.73

[1170] Ionization method: ES +:[M+H] + =616.3

[1171] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 11.29 (s, 1H), 7.60 (d, J = 1.10Hz, 1H), 7.39 (d, J = 6.93Hz, 2H), 7.19-7.33 (m, 7H), 6.88 (d, J = 8.68Hz, 4H), 5.60 (dd , J=9.96, 2.87Hz, 1H), 4.64 (t, J=5.93Hz, 1H), 3.74 (m, 6H), 3.53-3.60 (m, 1H), 3.44-3.52(m, 1H), 3.32-3.37(m, 1H), 3.09(d, J=8.56Hz, 1H), 2.76(br d, J=11.62Hz, 1H), 2.61-2.72 (m, 2H), 2.37 (d, J=11.49Hz, 1H), 2.10 (t, J=10.45Hz, 1H), 1.78 (s, 3H), 0.84-0.96 (m, 6H).

[1172] 20b: 1-[(2R,6S)-6-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-(hydroxymethyl)-4-isopropyl-morpholin-2-yl]pyrimidine-2,4-dione

[1173] 1.40g (1.73mmol) of starting compound 19b is dissolved among the 80ml MeOH.At room temperature, add the MeOH solution of 0.5M NaOMe of 13.83ml (6.91mmol), and the reaction solution is stirred 2 hours, to reach complete conversion.After adding 0.92g citric acid, desolvate.Resistates is dissolved in H Among the O, and aqueous mixture is extracted 3 times with DCM / iPrOH (4: 1).With the organic layer MgSO that merges Dry and evaporate, obtain 0.85g (81.2%, crude product) required compound 20b, it is used for following step, without further purification.

[1174] LCMS-Method C:

[1175] UV-wavelength [nm] = 220:R t [min]=2.04

[1176] Ionization method: ES + :[M+H] + =602.2

[1177] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 11.31 (s, 1H), 7.75 (d, J=8.07Hz, 1H), 7.39 (d, J=7.34Hz, 2H), 7.10-7.33 (m, 8H), 6. 88 (d, J=8.31Hz, 4H), 5.55-5.64 (m, 2H), 4.66 (t, J=5.93Hz, 1H), 3.74 (s, 6H), 3.57 (dd, J=11.31, 6.79Hz, 1H), 3.46 (br dd, J=11.25, 5.14Hz, 1H), 3.08 (d, J=8.56Hz, 1H), 2.54-2.79 (m, 3H), 2.32-2.39 (m, 1H), 1.95-2.13 (m, 1H), 0.86-0.98 (m, 6H).

[1178] 20c: N-[9-[(2R,6S)-6-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-(hydroxymethyl)-4-isopropyl-morpholin-2-yl]-6-oxo-1H-purin-2-yl]-2-methyl-propionamide

[1179] 260mg (319 μmol) of starting material 19c is dissolved among the 10ml EtOH.After adding the 1MNaOH solution of 1.0ml (1.0mmol), the solution is stirred at room temperature for 4 hours to reach complete conversion. The solution is neutralized with 1M HCl, and the solvent is removed in vacuo. Residue is dissolved in H Among the O, and extracted with DCM. The organic phase is separated, used MgSO Drying and evaporation. Finally, purification on silicon dioxide (0 to 10% MeOH among the DCM) obtains 114mg (50.3%) of required alcohol 20c as a yellow solid.

[1180] LCMS-Method C:

[1181] UV-wavelength [nm] = 220:R t [min]=2.36

[1182] Ionization method: ES - :[MH] - =709.3

[1183] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 12.08 (s, 1H), 11.52 (s, 1H), 8.20 (s, 1H), 7.35-7.43 (m, 2H), 7.20-7.33 (m, 7H), 6.83-6.92 (m, 4H), 5.56 (dd, J=9.60, 2.75Hz, 1H), 4.59-4.66 (m, 1H), 3.73 (2x s, 6H), 3.51-3.62 (m, 2H), 3.43-3.50 (m, 1H), 2.88-3.02 (m, 3H), 2.70-2.82 (m, 2H ), 2.30-2.39 (m, 1H), 1.11 (m, 6H), 1.01 (d, J = 6.48Hz, 3H), 0.97 (d, J = 6.48Hz, 3H).

[1184] 20e: N-[9-[(2R,6S)-6-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-(hydroxymethyl)-4-isopropyl-morpholin-2-yl]purin-6-yl]benzamide

[1185] Starting compound 19e (1.27g, 1.52mmol) is dissolved in 14ml EtOH / pyridine (1:1). At 0 ℃, 7.59ml (15.19mmol) of 1M NaOH solution is added, and the reaction mixture is stirred at room temperature for 1.5 hours. After pH is adjusted to 7 by adding citric acid monohydrate (958mg), 70ml H2O and EtOAc are added. The organic layer is separated and washed with 10% citric acid solution (3 times), H2O, saturated NaHCO3 and NaCl solution. After drying with MgSO4, the organic phase is evaporated to obtain 1.08g of crude product as colorless foam, which is dissolved in 20ml EtOAc / ether (1:1). After adding 40ml of n-pentane, the precipitate is centrifuged and the supernatant is discarded. The precipitation procedure is repeated twice to obtain 964mg (87.1%) of title compound (20e) as colorless foam.

[1186] LCMS-Method C:

[1187] UV-wavelength [nm] = 220:R t [min]=2.42

[1188] Ionization method: ES - :[MH] - =727.5

[1189] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 11.18 (s, 1H), 8.71 (s, 1H), 8.69 (s, 1H), 8.05 (d, J=7 .34Hz, 2H), 7.61-7.68 (m, 1H), 7.56 (t, J=7.58Hz, 2H), 7.45 (d, J=7.46Hz, 2H), 7.28-7. 36 (m, 6H), 7.20-7.28 (m, 1H), 6.90 (dd, J=8.99, 2.26Hz, 4H), 5.89 (dd, J=9.84, 2.63Hz, 1H), 4.69 (t, J=5.99Hz, 1H), 3.75 (s, 6H), 3.45-3.61 (m, 3H), 3.24-3.30 (m, 1H), 3.00 (br d, J=10.15Hz, 1H), 2.85 (br d, J=11.62Hz, 1H), 2.60-2.78 (m, 2H), 2.45-2.55 (m, 1H), 0.98 (br d, J=6.48Hz, 3H), 0.95 (br d, J=6.48Hz, 3H).

[1190] 20f: N-[1-[(2R,6S)-6-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-(hydroxymethyl)-4-isopropyl-morpholin-2-yl]-2-oxo-pyrimidin-4-yl]benzamide

[1191] 3.68 g (4.55 mmol) of 19f were saponified according to the procedure described for 20e.The crude product was purified by silica gel chromatography (20 to 100% EtOAc in n-heptane) to afford 1.72 g (53.5%) of 20f as a light yellow foam.

[1192] LCMS-Method A:

[1193] UV-wavelength [nm] = 220:R t [min]=1.81

[1194] Ionization method: ES - :[MH] - =703.3

[1195] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 11.24 (s, 1H), 8.29 (d, J = 7.46Hz, 1H), 8.01 (d, J = 7.46Hz, 2H), 7.62 (t, J = 7.40Hz, 1 H), 7.51 (t, J=7.70Hz, 2H), 7.20-7.41 (m, 10H), 6.88 (dd, J=8.99, 2.38Hz, 4H), 5.68 (dd, J=9.35, 2.38Hz, 1H), 4.71 (t , J=6.11Hz, 1H), 3.74 (s, 6H), 3.58-3.69 (m, 1H), 3.48-3.58 (m, 1H), 3.35-3.43 (m, 1H), 3.11 (d, J=8.56Hz, 1H), 2.79- 2.95 (m, 2H), 2.66-2.76 (m, 1H), 2.39-2.47 (m, 1H), 1.91-2.00 (m, 1H), 0.96 (d, J=6.54Hz, 3H), 0.94 (d, J=6.54Hz, 3H).

[1196] 21a: 3-[[(2R,6R)-2-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-isopropyl-6-(5-methyl-2,4-dioxo-pyrimidin-1-yl)morpholin-2-yl]methoxy-(diisopropylamino)phosphinoyl]-oxypropionitrile

[1197] Following the protocol for 16a (Scheme 4), 764 mg (1.2 mmol) of starting material 20a were converted to the desired phosphoramidite 21a. 842 mg (83.2%, mixture of diastereomers) were obtained as a colorless foam.

[1198] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 11.34, 11.32 (2x s, 1H), 7.59, 7.54 (2x d, J=1.10Hz, 1H), 7.36-7.43(m, 2H), 7.20-7.34(m, 7H), 6.89(m, 4H), 5.59-5.75(m, 1H), 3.44-3.95( m, 6H), 3.74 (s, 6H), 3.36 (m, 1H), 3.22 (m, 1H), 2.60-2.85 (m, 5H), 2.36 (m, 1H), 2.18 (m, 1H), 1.78 (2x d, J=8.69Hz, 3H), 0.83-1.17 (m, 18H).

[1199] 31P-NMR (DMSO-d6, 162MHz) δ [ppm]: 148.6, 148.3.

[1200] 21b: 3-[[(2R,6R)-2-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-(2,4-dioxopyrimidin-1-yl)-4-isopropyl-morpholin-2-yl]methoxy-(diisopropylamino)phosphinoyl]oxypropionitrile

[1201] Following the protocol for 16b, starting from 1.36 g (2.26 mmol) of the corresponding alcohol 20b, the title compound 21b was synthesized. 1.09 g (60.1%) of the title compound was isolated as a colorless foam.

[1202] LCMS-Method B-1:

[1203] UV-wavelength [nm] = 220:R t [min]=0.80

[1204] Ionization method: ES + :[MN i Pr2+OH+H] + =718.3

[1205] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 11.32 (br s, 1H), 7.71, 7.66 (2x s, J=8.13Hz, 1H), 7.35-7.42 (m, 2H), 7.20-7.33 (m, 7H), 6.83-6.92 (m, 4H), 5.60-5.70 (m, 2H), 3.73 (s, 6H), 3.43 -3.95 (m, 6H), 3.29-3.39 (m, 1H), 3.22 (m, 1H), 2.59-2.83 (m, 5H), 2.33 (m, 1H), 2.13 (m, 1H), 0.84-1.21 (m, 18H).

[1206] 31P-NMR (DMSO-d6, 162MHz) δ [ppm]: 148.5, 148.3.

[1207] 21c: N-[9-[(2R,6R)-6-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-[[2-cyanoethoxy-(diisopropylamino)phosphinoyl]oxymethyl]-4-isopropyl-morpholin-2-yl]-6-oxo-1H-purin-2-yl]-2-methyl-propionamide

[1208] Following the protocol described for the synthesis of 16c, 40 mg (56 μmol) of starting compound 20c gave 23 mg (44.9%) of the desired phosphoramidite 21c after silica gel chromatography (0 to 100% methyl-tert-butyl ether in n-heptane, pre-conditioning the column with n-heptane+1% NEt3).

[1209] LCMS-Method C:

[1210] UV-wavelength [nm] = 220:R t [min]=2.57

[1211] Ionization method: ES + :[MN i Pr2+OH+H] + =828.2

[1212] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 11.70-12.30 (bs, 1H), 11.53 (bs, 1H), 8.05 (s, 1H), 7.35-7.45 (m , 2H), 7.20-7.33(m, 7H), 6.82-6.91(m, 4H), 5.54-5.68(m, 1H), 3.80-3.93(m, 0.5H), 3.73, 3.72(2x s, 6H), 3.68-3.76 (m, 1.5H), 3.60-3.67 (m, 2H), 3.35-3.60 (m, 4H), 3.12 (m, 1H), 2.57-2.98 (m, 7H), 0.92-1.13 (m, 24H).

[1213] 31P-NMR (DMSO-d6, 162MHz) δ [ppm]: 147.6, 147.4.

[1214] 21e: N-[9-[(2R,6R)-6-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-[[2-cyanoethoxy-(diisopropylamino)phosphinoyl]oxymethyl]-4-isopropyl-morpholin-2-yl]purin-6-yl]benzamide

[1215] Starting from 950 mg (1.30 mmol) of 20e, the title compound was prepared according to the protocol described for 16e (Scheme 4) to give 1.10 g (91.0%) of 21e as a colorless foam.

[1216] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 11.19 (br s, 1H), 8.73, 8.71 (2x s, 1H), 8.63, 8.59 (2x s, 1H), 8.04 (d, J=7.65Hz, 2H), 7.64 (t, J=7.34Hz, 1H), 7.55 (t, J=7.43Hz, 2H), 7.44 (d, J=7.78Hz, 2H), 7.21-7.35 (m, 7H ), 6.86-6.93(m, 4H), 5.89-5.99(m, 1H), 3.80-4.15(m, 1H), 3.74(s, 6H), 3.63-3.72(m, 2H), 3.31-3.60(m, 5H), 3.01(br d, J=10.79Hz, 1H), 2.66-2.90 (m, 4H), 2.59-2.64 (m, 1H), 2.42-2.47 (m, 1H), 0.91-1.12 (m, 18H).

[1217] 31P-NMR (DMSO-d6, 162MHz) δ [ppm]: 147.6, 147.5.

[1218] 21f: N-[1-[(2R,6R)-6-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-[[2-cyanoethoxy-(diisopropylamino)phosphinoyl]oxymethyl]-4-isopropyl-morpholin-2-yl]-2-oxo-pyrimidin-4-yl]benzamide

[1219] Starting from 620 mg (0.88 mmol) of 20f, the title compound was prepared according to the protocol described for 16e (Scheme 4) to give 681 mg (85.5%) of 21f as a colorless foam.

[1220] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 11.28 (br s, 1H), 8.12-8.23 (2x d, J=7.47Hz, 1H), 8.01 (d, J=7.65Hz, 2H), 7.58-7.67 (m, 1H), 7.51 (t, J=7.58H z, 2H), 7.35-7.42 (m, 3H), 7.20-7.35 (m, 7H), 6.83-6.93 (m, 4H), 5.71-5.80 (2x dd, J=9.49, 2.54Hz, 1H), 3.44-4.00 (m, 6H), 3.74 (s, 6H), 3.38 (m, 1H), 3.24-3.33 (m, 1H), 2.6 0-2.94 (m, 5H), 2.33-2.48 (m, 1H), 2.01-2.09 (m, 1H), 1.02-1.23 (m, 12H), 0.83-1.00 (m, 6H).

[1221] 31P-NMR (DMSO-d6, 162MHz) δ [ppm]: 147.5, 147.4.

[1222] Example A.6:

[1223] Synthesis Scheme 6

[1224]

[1225] 22a: 1-[(2R,3R,4S,5S)-5-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3,4-dihydroxy-5-(triisopropylsilyloxymethyl)tetrahydrofuran-2-yl]-5-methyl-pyrimidine-2,4-dione

[1226] To a solution of 23.7 g (53.4 mmol) of the starting compound 4a in 240 ml of anhydrous pyridine was added 23.13 g (80 mmol) of DMT-Cl at room temperature under a N2 atmosphere. After stirring for 12 hours, the solvent was removed in vacuo and the residue was purified by silica gel column chromatography (PE / EtOAc 10:1 to 1:1) to give 16.3 g (41%) of the desired DMT-protected product (22a) as a colorless solid.

[1227] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 11.34 (s, 1H), 7.50 (d, J = 0.75Hz, 1H), 7.42-7.37 (m , 2H), 7.32 (t, J=7.53Hz, 2H), 7.29-7.22 (m, 6H), 6.90 (dd, J=8.91, 2.13Hz, 4H), 5.89 (d , J=7.28Hz, 1H), 5.45 (d, J=6.53Hz, 1H), 5.15 (d, J=5.02Hz, 1H), 4.48-4.39 (m, 1H), 4. 31(t, J=5.14Hz, 1H), 3.86-3.80(m, 1H), 3.74(s, 6H), 1.28(s, 3H), 0.98-0.83(m, 22H).

[1228] 22b:1-[(2R,3R,4S,5S)-5-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3,4-dihydroxy-5-(triisopropylsilyloxymethyl)tetrahydrofuran-2-yl]pyrimidine-2,4-dione

[1229] At room temperature, in N2 atmosphere, DMT-Cl (70.8g, 209mmol) was added to a 750ml anhydrous pyridine solution of compound 4b (75g, 174mmol). The mixture was stirred for 12 hours to achieve complete conversion. The solvent was removed in vacuo, and the residue was diluted with 500ml water and extracted with 3x500ml EtOAc. The organic layer was washed with salt water, dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The crude product of the gained was purified on silica gel by flash chromatography (PE / EtOAc 1:1 to EtOAc / MeOH 20:1), obtaining 80g (62.6%) of 22b as a colorless foam.

[1230] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 11.35 (s, 1H), 7.61 (d, J=8.28Hz, 1H), 7.42-7.36 (m, 2H), 7 .32 (t, J=7.65Hz, 2H), 7.28-7.19 (m, 5H), 6.90 (d, J=8.03Hz, 4H), 5.83 (d, J=6.53Hz, 1H), 5.48 (d, J=6.02Hz, 1H), 5.27 (d, J=8.03Hz, 1H), 5.21 (d, J=5.27Hz, 1H), 4.33-4.26 (m, 1H), 4.22 (q , J=6.02Hz, 1H), 3.89-3.79 (m, 2H), 3.74 (s, 6H), 3.24 (d, J=10.04Hz, 1H), 1.01-0.84 (m, 21H).

[1231] 22c: N-[9-[(2R,3R,4S,5S)-5-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3,4-dihydroxy-5-(triisopropylsilyloxymethyl)tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]-2-methyl-propionamide

[1232] 55.8 g (103 mmol) of 4c were protected with DMT-Cl in pyridine according to the protocol described for 22b to afford 50.0 g (57.4%) of 22c as a colorless solid.

[1233] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 12.12 (br s, 1H), 11.67 (br s, 1H), 7.99-7.83 (m, 1H), 7.38 (br d, J=7.28Hz, 2H), 7.32-7.13 (m, 8H), 6.84 (dd, J=8.78, 2.26Hz, 4H), 5.86 (d, J=7.03Hz, 1H), 5.70-5.45 (m, 1H), 5.18 (br s, 1H), 4.73-4.62 (m, 1H), 4.25 (br d, J=4.77Hz, 1H), 3.97 (br d, J=10.29Hz, 1H), 3.84 (br d, J=10.29Hz, 1H), 3.73 (s, 6H), 3.20 (br d, J=10.04Hz, 1H), 2.76 (dt, J=13.61, 6.87Hz, 1H), 1.12 (d, J=6.78Hz, 6H), 1.01-0.87 (m, 21H).

[1234] 22e: N-[9-[(2R,3R,4S,5S)-5-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3,4-dihydroxy-5-(triisopropylsilyloxymethyl)tetrahydrofuran-2-yl]purin-6-yl]benzamide

[1235] 57.0 g (102 mmol) of 4e were protected with DMT-Cl in pyridine according to the protocol described for 22b to afford 22e as a colorless solid (70.0%).

[1236] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 11.22 (s, 1H), 8.68-8.58 (m, 1H), 8.49 (s, 1H), 8.05 (d, J=7.28Hz, 2H), 7.70-7.60 (m, 1H), 7.59-7.50 (m, 2H), 7.43 (d, J=7.28Hz, 2H), 7.33-7.17 (m, 7H), 6.86 (dd, J=8.91, 1.38H z, 4H), 6.00 (d, J = 7.28Hz, 1H), 5.53 (d, J = 6.78Hz, 1H), 5.32 (d, J = 4.89Hz, 1H), 5.00-4.87 (m, 1H), 4.34 (t , J=4.89Hz, 1H), 4.08-4.04 (m, 1H), 3.98 (d, J=10.79Hz, 1H), 3.73 (s, 6H), 3.40 (d, J=9.54Hz, 1H), 3.30 (br d, J=9.54Hz, 1H), 1.12-0.93 (m, 21H).

[1237] 23a: 1-[(2R,6S)-6-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3,5-dihydroxy-6-(triisopropylsilyloxymethyl)-1,4-dioxan-2-yl]-5-methyl-pyrimidine-2,4-dione

[1238] At room temperature, 5.05 g (23.6 mmol) of NaIO in a 250 ml acetone / H O (3:1) solution of 12.6 g (16.9 mmol) starting material 22a was dropwise added in an H O (60 ml) aqueous solution. The mixture was stirred for 12 hours to achieve complete conversion and poured into 500 ml of ice water. The mixture was extracted 3 times with 500 ml of DCM. The organic layers were combined and washed with salt water, dried over anhydrous Na SO , filtered and concentrated in vacuo to afford 11.20 g (87%) of the desired product 23a as a white foam, which was used in the next step without further purification.

[1239] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 11.62-11.33 (m, 1H), 7.80-7.65 (m, 1H), 7.57-7. 38(m, 3H), 7.33-7.13(m, 9H), 6.88-6.73(m, 5H), 6.08-5.86(m, 1H), 5.71-5.51(m, 1H ), 5.24-5.06(m, 1H), 5.01-4.94(m, 1H), 4.29-4.16(m, 1H), 3.75-3.70(m, 7H), 3.34- 3.24 (m, 1H), 3.06-2.81 (m, 1H), 2.02-1.59 (m, 2H), 1.03 (m, 1H), 1.09-0.74 (m, 21H).

[1240] 23b: 1-[(2R,6S)-6-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3,5-dihydroxy-6-(triisopropylsilyloxymethyl)-1,4-dioxan-2-yl]pyrimidine-2,4-dione

[1241] Following the protocol described for 23a, 77.0 g (105 mmol) of diol 22b gave 80 g (quantitative, crude) of the title compound 23b as a yellow solid.

[1242] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 11.67-11.36 (m, 1H), 7.87-7.77 (m, 1H), 7.76-7.67 (m, 1 H), 7.55-7.41 (m, 2H), 7.39-7.20 (m, 8H), 6.94-6.81 (m, 4H), 5.94-5.87 (m, 1H), 5.78-5.63 (m, 1H), 5.59-5.51 (m, 1H), 5.25-5.08 (m, 1H), 5.07-4.99 (m, 1H), 4.32-4.20 (m, 1H), 3.82- 3.70 (m, 6H), 3.34-3.26 (m, 1H), 3.15-3.04 (m, 1H), 2.95-2.86 (m, 1H), 1.14-0.81 (m, 22H).

[1243] 23c: N-[9-[(2R,6S)-6-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3,5-dihydroxy-6-(triisopropylsilyloxymethyl)-1,4-dioxan-2-yl]-6-oxo-1H-purin-2-yl]-2-methyl-propionamide

[1244] Following the protocol described for 23a, 50.0 g (59 mmol) of diol 22c gave 47 g (96.1%, crude) of the title compound 23c as a yellow solid.

[1245] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 12.25-12.06 (m, 1H), 11.78-11.55 (m, 1H), 8.19 (d, J=8.78Hz, 1H), 7.44 (br d, J=7.28Hz, 1H), 7.38 (br d, J=6.53Hz, 1H), 7.34-7.14 (m, 10H), 6.99 (d, J=6.78Hz, 1H), 6.90-6.71 (m, 5H), 6.05-5.98 (m, 1H), 5.74 (d, J=7.78Hz , 1H), 5.61-5.53 (m, 1H), 5.48 (q, J=6.78Hz, 1H), 5.24-5.17 (m, 1H), 5.06 (d, J=6.53Hz, 1H), 4.35-4.22 (m, 1H), 3.83 (br d, J=11.54Hz, 1H), 3.72 (d, J=3.26Hz, 7H), 3.27(br d, J=9.29Hz, 1H), 3.29-3.23 (m, 1H), 3.12-3.04 (m, 1H), 2.91-2.75 (m, 1H), 1.22-0.76 (m, 29H).

[1246] 23e: N-[9-[(2R,6S)-6-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3,5-dihydroxy-6-(triisopropylsilyloxymethyl)-1,4-dioxan-2-yl]purin-6-yl]benzamide

[1247] Following the protocol described for 23a, 78.0 g (90.6 mmol) of diol 22e gave 75.2 g (94.6%, crude) of the title compound 23e as a white solid.

[1248] MS (m / z) = 876.2 [M+H] +

[1249] 24a: 1-[(2R,6S)-6-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-(triisopropylsilyloxy-methyl)morpholin-2-yl]-5-methyl-pyrimidine-2,4-dione

[1250] To a solution of 2.6 g (3.4 mmol) of the starting compound 23a in 44 ml of anhydrous MeOH was added 0.98 g (3.7 mmol) of (NH4)2B4O7.4H2O at room temperature under N2 atmosphere. The mixture was stirred for 2 hours, and then 0.45 g (6.8 mmol) of AcOH, 5.0 g of To the mixture of 4-nitropropene methyl esters and 0.47 g (6.8 mmol) of NaBH 3 CN. After stirring at room temperature for 12 hours, TLC showed that the starting material was completely consumed. The solvent was removed in vacuo, and the residue was purified by silica gel chromatography (PE / EtOAc 4:1 to 1:1) to give 1.59 g (64%) of the desired morpholine 24a as a white foam.

[1251] MS (m / z) = 752.5 [M+Na] +

[1252] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 11.39 (s, 1H), 7.62 (s, 1H), 7.43 (br d, J=7.28Hz, 2H), 7.33-7.23 (m, 8H), 6.84 (d, J=8.78Hz, 5H), 5.86 (br dd, J=9.91, 2.64Hz, 1H), 5.89-5.81 (m, 1H), 4.13 (br d, J=9.54Hz, 1H), 3.96 (br d, J=9.54Hz, 1H), 3.72 (d, J=0.75Hz, 6H), 3.06 (br d, J=9.29Hz, 1H), 2.97(br d, J=9.03Hz, 1H), 2.93-2.78 (m, 2H), 2.76-2.57 (m, 3H), 1.76 (s, 3H), 0.98-0.87 (m, 22H).

[1253] 24b: 1-[(2R,6S)-6-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-(triisopropylsilyloxy-methyl)morpholin-2-yl]pyrimidine-2,4-dione

[1254] To a solution of compound 23b (5.0 g, 6.7 mmol) in 85 ml of anhydrous MeOH was added (NH4)2B4O7.4H2O (1.93 g, 7.6 mmol) at 25°C under N2 atmosphere. The mixture was stirred for 2 hours, and then 0.80 g (13.4 mmol) of AcOH, 10 g of To the product was added 4-nitropropene (2-nitropropene)-2 ...

[1255] MS (m / z) = 738.5 [M+H] +

[1256] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 11.44 (br s, 1H), 7.75 (d, J=8.07Hz, 1H), 7.45 (br d, J=7.34Hz, 2H), 7.37-7.20 (m, 7H), 6.89 (d, J=8.80Hz, 4H), 5.88 (br dd, J=10.03, 2.45Hz, 1H), 5.73 (d, J=8.07Hz, 1H), 4.17 (br d, J=9.78Hz, 1H), 4.11-4.02(m, 2H), 3.77(d, J=0.73Hz, 6H), 3.12(br d, J=9.05Hz, 1H), 3.02-2.83 (m, 3H), 2.70-2.58 (m, 2H), 1.06-0.90 (m, 21H).

[1257] 24c: N-[9-[(2R,6S)-6-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-(triisopropylsilyl-oxymethyl)morpholin-2-yl]-6-oxo-1H-purin-2-yl]-2-methyl-propionamide

[1258] Following the protocol described for 24b, 7.0 g (8.2 mmol) of starting material 23c was converted to the morpholine compound 24c, which was isolated as a colorless foam (73.2%) after silica gel chromatography (PE / EtOAc 5:1 to 1:1). MS (m / z) = 825.2 [M+H] +

[1259] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 12.12 (br s, 1H), 11.59 (s, 1H), 8.15 (s, 1H), 7.40 (br d, J=7.03Hz, 2H), 7.30-7.12 (m, 7H), 6.81 (br d, J=7.78Hz, 4H), 5.83 (dd, J=9.66, 3.14Hz, 1H), 4.23 (br d, J=9.03Hz, 1H), 3.92 (br d, J=9.03Hz, 1H), 3.72 (d, J=2.76Hz, 6H), 2.97-3.18 (m, 3H), 2.96-2.86 (m, 2H), 2.80 (dt, J=13.55, 6.78Hz, 1H), 2.67 (br d, J=12.05Hz, 1H), 1.13 (t, J=6.15Hz, 6H), 1.03-0.82 (m, 22H).

[1260] 24e: N-[9-[(2R,6S)-6-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-(triisopropylsilyl-oxymethyl)morpholin-2-yl]purin-6-yl]benzamide

[1261] Following the protocol described for 24b, 5.0 g (5.7 mmol) of starting material 23e were converted to the morpholine compound 24e, which was isolated as a colorless foam (73.4%) after silica gel chromatography (PE / EtOAc 1:4). MS (m / z) = 843.1 [M+H] +

[1262] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 11.22 (br s, 1H), 8.75 (s, 1H), 8.66 (s, 1H), 8.05 (d, J = 7.34Hz, 2H), 7.70-7.62 (m, 1H), 7.60-7.51 (m, 2H), 7.38 (br d, J=6.97Hz, 2H), 7.26-7.16 (m, 7H), 6.78 (d, J=8.93Hz, 4H), 6.16 (dd, J=10.03, 2.8 1Hz, 1H), 4.26 (d, J=9.66Hz, 1H), 4.10-3.98 (m, 1H), 3.71 (d, J=0.73Hz, 6H), 3.28 (br d, J=10.64Hz, 1H), 3.16(br d, J=9.17Hz, 1H), 3.06(br d, J=9.17Hz, 1H), 2.98-2.90(m, 2H), 2.79(br d, J=12.47Hz, 1H), 1.10-0.82 (m, 21H).

[1263] Example A.7:

[1264] Synthesis Scheme 7

[1265]

[1266] 25: (2S,6R)-2-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-(2,4-dioxo-pyrimidin-1-yl)-2-(triisopropylsilyloxymethyl)morpholine-4-carboxylic acid tert-butyl ester

[1267] To a mixture of morpholine 24b (50 g, 69.8 mmol) and DIPEA (18 g, 139.7 mol) in 500 ml of DCM was added dropwise Boc O (22.9 g, 104.8 mol) at room temperature. After stirring for 24 hours, the solvent was removed in vacuo and residue was purified by column chromatography (PE / EtOAc 1:1) to give compound 25 (60 g, 93% yield) as a white foam.

[1268] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 7.58 (d, J=8.0Hz, 1H), 7.45 (d, J=7.2Hz, 2H), 7.33- 7.26 (m, 7H), 6.85-6.81 (m, 4H), 6.02-5.98 (dd, J=10.03.6Hz, 1H), 5.77 (d, J=8.0Hz, 1 H), 4.24-4.11(m, 2H), 3.99(d, J=9.6Hz, 1H), 3.80(s, 1H), 3.75(m, 1H), 3.28-3.20(m, 2H), 3.08 (d, J=12.8Hz, 1H), 2.69 (t, J=11.4Hz, 1H), 1.48 (m, 9H), 1.03-0.95 (m, 21H).

[1269] 26: (2S,6R)-2-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-[2-oxo-4-(1,2,4-triazol-1-yl)pyrimidin-1-yl]-2-(triisopropylsilyloxymethyl)morpholine-4-carboxylic acid tert-butyl ester

[1270] To a mixture of 1H-1,2,4-triazole (65 g, 0.94 mol) in 650 ml of ACN was added POCl3 (49.79 g, 0.31 mol) dropwise at 30°C. After cooling to 0°C, DIPEA (198 g, 1.53 mol) was added dropwise and stirring was continued at 0°C for 30 minutes. At the same temperature, starting material 25 (32 g, 39.21 mmol) was added to the mixture in one portion, the ice bath was removed, and the reaction was stirred at 30°C for 3 hours. The mixture was poured into 3 liters of a mixed solvent of EtOAc / water (1:2). After separation, the organic layer was washed with 500 ml of saturated NaHCO3 solution and brine, dried over anhydrous Na2SO4, filtered and concentrated in vacuo to give crude compound 26 (40 g) as a yellow oil, which was used in the next step without further purification. MS (m / z) = 867.5 [M+H] +

[1271] 27: (2S,6R)-6-(4-Benzamido-2-oxo-pyrimidin-1-yl)-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-2-(triisopropylsilyloxymethyl)morpholine-4-carboxylic acid tert-butyl ester

[1272] To a mixture of benzamide (38 g, 0.31 mol) in 380 ml of dioxane was added NaH (12.8 g, 0.31 mol) at 0°C. After stirring at 30°C for 30 minutes, triazole 26 (40 g, 39.21 mmol, crude product) was added in one portion and the mixture was stirred at 30°C for 1 hour. The reaction was quenched with 24 liters of saturated NH4Cl solution and extracted with 3 liters of EtOAc. The organic layer was washed with 2 liters of water and brine, dried over anhydrous Na2SO4, filtered and concentrated in vacuo to give 54 g of the crude compound as a yellow oil. Finally, silica gel chromatography (PE / EtOAc 2:1) gave 20.4 g (56.6%) of 27 as a yellow foam.

[1273] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 11.31 (br s, 1H), 8.20 (br d, J=7.21Hz, 1H), 8.01 (br d, J=7.46Hz, 2H), 7.70-7.59 (m, 1H), 7.58-7.48 (m, 2H), 7.47-7.36 (m, 3H), 7.34-7.16 (m, 7H), 6.87 (br d, J=7.70Hz, 4H), 5.96 (br dd, J=10.09, 2.63Hz, 1H), 4.12(br d, J=11.25Hz, 1H), 4.00-3.83 (m, 3H), 3.74 (s, 6H), 3.28-3.16 (m, 2H), 3.06 (br d, J=9.17Hz, 2H), 1.55-1.32 (m, 9H), 1.11-0.80 (m, 21H).

[1274] 28: N-[1-[(2R,6S)-6-(hydroxymethyl)-6-(triisopropylsilyloxymethyl)morpholin-2-yl]-2-oxo-pyrimidin-4-yl]benzamide

[1275] At 0 ℃, 31ml of TFA was added dropwise in 93ml DCM solution of 27 (13.5g, 14.70mmol). The reaction was stirred at 30 ℃ for 16 hours. After being neutralized to pH=7-8 with saturated NaHCO solution, the organic layer was separated, and aqueous phase was extracted twice with 100ml DCM. The organic layer merged was dried over anhydrous Na SO , filtered and concentrated in vacuo. Residue was purified by column chromatography (EtOAc / EtOH=30:1), and 28 (2.7g, 35.6% yield) was obtained as yellow foam, and corresponding trifluoro-acetic acid ester was a by product (4.78g, 53.1%), which was dissolved in a mixed solvent of 186ml THF and 62ml EtOH. At 0 ℃, 75.8ml of 1M NaOH-water solution was added. After stirring for 2 minutes, the solution was neutralized with saturated citric acid solution, diluted with 300 ml of H2O, and extracted with 300 ml of EtOAc. The organic layer was washed with brine, dried over Na2SO4, and evaporated. Purification by silica gel chromatography (EtOAc / EtOH 30:1) gave 5.2 g (68.5%) of 28 as a white solid.

[1276] MS (m / z) = 517.4 [M+H + ] +

[1277] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 11.26 (br s, 1H), 8.28 (d, J = 7.53Hz, 1H), 8.01 (d, J = 7.28Hz, 2H), 7.67-7.58 (m, 1H), 7.5 7-7.47 (m, 2H), 7.34 (d, J=7.40Hz, 1H), 5.86 (dd, J=9.66, 2.64Hz, 1H), 4.64 (br s, 1H), 4.01-3.90 (m, 2H), 3.46 (br s, 2H), 3.05 (dd, J=11.98, 2.57Hz, 1H), 3.11-3.00 (m, 1H), 2.87-2.75 (m, 1H), 2.74-2.63 (m, 1H), 2.40-2.27 (m, 1H), 1.15-0.99 (m, 21H).

[1278] 29: (2S,6R)-6-(4-Benzoylamino-2-oxo-pyrimidin-1-yl)-2-(hydroxymethyl)-2-(triisopropylsilyloxymethyl)morpholine-4-carboxylic acid 9H-fluoren-9-ylmethyl ester

[1279] To a solution of 7.0 g (13.55 mmol) of morpholine 28 (13.55 mmol) in 70 ml of DMF was added 5.5 g (16.26 mmol) of Fmoc-N-hydroxysuccinimide ester in portions at 0°C. After stirring for 16 hours, the solution was poured into 100 ml of water and extracted three times with 30 ml of EtOAc. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. Finally, purification on silica (PE / EtOAc 1:2) afforded 10.0 g (quantitative) of compound 29 as a yellow solid.

[1280] MS (m / z) = 739.4 [M+H] +

[1281] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 11.26 (br s, 1H), 8.33 (br s, 1H), 8.02 (br d, J=7.46Hz, 2H), 7.90 (d, J=7.58Hz, 2H), 7.64 (br t, J=7.40Hz, 3H), 7.57-7.49 (m, 2H), 7.48-7.40 (m, 3H), 7.39-7.30 (m, 2H), 6.11-5 .79(m, 1H), 4.98(t, J=6.05Hz, 1H), 4.52-4.12(m, 4H), 3.89-3.69(m, 2H), 3.54(br s, 3H), 3.21-3.07 (m, 1H), 3.04-2.77 (m, 1H), 1.11-0.89 (m, 21H).

[1282] 30: (2S,6R)-6-(4-Benzamido-2-oxo-pyrimidin-1-yl)-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-2-(triisopropylsilyloxymethyl)morpholine-4-carboxylic acid 9H-fluoren-9-ylmethyl ester

[1283] At 25 ° C, 12.75 g (37.62 mmol) of DMT-Cl was added to a 140 ml pyridine solution of compound 29 (13.9 g, 18.83 mmol). The mixture was stirred at 25 ° C for 16 hours to achieve complete conversion. After evaporation of the solvent, the residue was dissolved in 200 ml of EtOAc and washed with 100 ml of water and brine. The organic phase was dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography (PE / EtOAc 1:1) to obtain 17.3 g (88.1%) of compound 30 as a yellow foam.

[1284] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 11.32 (br s, 1H), 8.21 (br s, 1H), 8.02 (br d, J=7.58Hz, 2H), 7.88 (br s, 2H), 7.76-7.60 (m, 3H), 7.59-7.50 (m, 2H), 7.42 (br d, J=7.21Hz, 5H), 7.38-7.19 (m, 9H), 6.89 (br d, J=8.44Hz, 4H), 6.19-5.85(m, 1H), 4.28(br s, 4H), 3.91(br d, J=8.80Hz, 2H), 3.75(s, 6H), 3.23(br s, 1H), 3.10 (br d, J=9.05Hz, 3H), 1.06-0.78 (m, 21H).

[1285] 24f: N-[1-[(2R,6S)-6-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-(triisopropylsilyloxymethyl)morpholin-2-yl]-2-oxo-pyrimidin-4-yl]benzamide

[1286] At room temperature, 17ml piperidines is added to a solution of 85ml DCM of 30 of 8.4g (8.07mmol). After complete conversion (about 20min), the reaction solution is washed with saturated NH4Cl solution. Aqueous phase is extracted with DCM, and the organic layer combined is dried over Na2SO4, filtered and concentrated in vacuo. The crude product is purified by column chromatography (PE / EtOAc 1:4), and the title compound 24f obtaining 4.7g (71.2%) is white foam.

[1287] MS (m / z) = 819.4 [M+H] +

[1288] 1H-NMR (DMSO-d6, 400MHz) δ [ppm]: 11.31 (br s, 1H), 8.18 (d, J = 7.58Hz, 1H), 8.01 (br d, J = 7.34Hz, 2H), 7.67-7.58 (m, 1H), 7.57-7.47 (m, 2H), 7.42 (br d, J=7.46Hz, 3H), 7.34-7.15 (m, 7H), 6.86 (br d, J=8.56Hz, 4H), 6.04-5.89 (m, 1H), 4.18 (br d, J=9.54Hz, 1H), 4.07 (br d, J=9.54Hz, 1H), 3.73 (d, J=1.34Hz, 6H), 3.15 (br d, J=9.17Hz, 1H), 3.05 (br d, J=10.15Hz, 1H), 2.97 (br d, J=9.17Hz, 1H), 2.88 (br d, J=12.47Hz, 1H), 2.62 (brd, J=12.47Hz, 1H), 2.46 (br s, 1H), 1.08-0.83 (m, 21H).

[1289] Example A.8:

[1290] Synthesis Scheme 8

[1291]

[1292] General Procedure A for Reductive Amination Reactions to Prepare Compounds 31a-31f

[1293] 1.0 g (1.4 mmol) of morpholine 24a was dissolved in 25 ml of MeOH, and then 2.0 g of molecular sieves were added. 560mg (769 μ l, 5.5mmol) NEt , 827mg (789 μ l, 13.7mmol) AcOH and 1.0 to 3.0 equivalents of the corresponding aldehyde or ketone. After stirring for 15 minutes, the sodium cyanoborohydride of 344mg (5.5mmol) was added in four parts every 30 minutes, and the reaction was stirred at room temperature for 6 hours. After standing overnight, the mixture was filtered, and the filtrate was diluted with 25ml saturated NaHCO solution. Evaporation MeOH, and aqueous phase was extracted with 50ml DCM / iPrOH (5: 1). After filtering once more, the organic phase was separated, and MgSO was used Drying and evaporation. The crude product gained is used for the next step without the need to further purification.

[1294] 31a: 1-[(2R,6S)-6-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-methyl-6-(triisopropylsilyloxymethyl)morpholin-2-yl]-5-methyl-pyrimidine-2,4-dione

[1295] 1.0 g of the title compound 31a was isolated as a crude product according to general procedure A using 3.0 equivalents of formaldehyde (37% in water) and used without further purification.

[1296] LCMS-Method A:

[1297] UV-wavelength [nm] = 220:R t [min]=2.29

[1298] Ionization method: ES + :[M+H] + =744.3

[1299] 31b: 1-[(2R,6S)-6-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-butyl-6-(triisopropylsilyloxymethyl)morpholin-2-yl]-5-methyl-pyrimidine-2,4-dione

[1300] Following general procedure A using 2.0 equivalents of butyraldehyde, 1.19 g of the title compound 31b was isolated as a crude product and used without further purification.

[1301] LCMS-Method A:

[1302] UV-wavelength [nm] = 220:R t [min]=2.41

[1303] Ionization method: ES +:[M+H] + =786.7

[1304] 31c: 1-[(2R,6S)-6-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-octyl-6-(triisopropylsilyloxymethyl)morpholin-2-yl]-5-methyl-pyrimidine-2,4-dione

[1305] Following general procedure A using 2.0 equivalents of octanal, 1.35 g of the title compound 31c was isolated as a crude product and used without further purification.

[1306] LCMS-Method A:

[1307] UV-wavelength [nm] = 220:R t [min]=2.66

[1308] Ionization method: ES + :[M+H] + =842.7

[1309] 31d: 1-[(2R,6S)-6-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-hexadecyl-6-(triisopropylsilyloxymethyl)morpholin-2-yl]-5-methyl-pyrimidine-2,4-dione

[1310] Following general procedure A using 1.0 equivalent of hexadecanal, 1.58 g of the title compound 31d was isolated as a crude product and used without further purification.

[1311] 31 e :1-[(2R,6S)-6-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-cyclohexyl-6-(triisopropylsilyloxymethyl)morpholin-2-yl]-5-methyl-pyrimidine-2,4-dione

[1312] According to general procedure A, 1.25 equivalents of cyclohexanone were used to isolate 1.14 g of the title compound 31e as a crude product and used without further purification.

[1313] LCMS-Method A:

[1314] UV-wavelength [nm] = 220:R t [min]=2.45

[1315] Ionization method: ES + :[M+H] + =812.5

[1316] 31f: 1-[(2R,6S)-4-Benzyl-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-(triisopropylsilyloxymethyl)morpholin-2-yl]-5-methyl-pyrimidine-2,4-dione

[1317] According to general procedure A, 1.25 equivalents of benzaldehyde were used to isolate 1.12 g of the title compound 31f as a crude product and used without further purification.

[1318] LCMS-Method A:

[1319] UV-wavelength [nm] = 220:R t [min]=2.37

[1320] Ionization method: ES + :[M+H] + =821.4

[1321] 31g: 1-[(2R,6S)-4-acetyl-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-(triisopropylsilyloxymethyl)morpholin-2-yl]-5-methyl-pyrimidine-2,4-dione

[1322] The starting compound 24a of 1.0g (1.4mmol) is dissolved among the 25ml DCM.After adding 103mg (99 μ l, 1.7mmol) AcOH, 779mg (2.1mmol) HBTU and 903mg (1.19ml, 6.9mmol) diisopropyl-ethylamine, with reaction mixture stirring at room temperature 4 hours, to reach complete conversion.With reaction soln 25ml H O washing, and with the organic layer separation.Use MgSO After drying, evaporating solvent also obtains the title compound 31g of 1.06g (quantitatively) by silica gel chromatography purification (0 to 5%MeOH among the DCM) of crude product.

[1323] LCMS-Method A:

[1324] UV-wavelength [nm] = 220:R t [min]=2.20

[1325] Ionization method: ES - :[MH] - =770.2

[1326] 31h: 1-[(2R,6S)-6-[[Bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-hexadecanoyl-6-(triisopropylsilyloxymethyl)morpholin-2-yl]-5-methyl-pyrimidine-2,4-dione

[1327] Following the protocol for 31 g, 1.0 g (1.4 mmol) of starting compound 24a and palmitic acid were converted to 1.14 g (86%) of the title compound 31 h after silica gel chromatography (0 to 20% MeOH in DCM).

[1328] General Procedure B for Desilylation Reactions to Prepare Compounds 32a-32h

[1329] The crude products 31a-31h were dissolved in 12 ml of NMP. After adding ...

Claims

1. Compounds of formula (I): in: -B is a heterocyclic nucleobase; - P1 and P2 are each independently a protecting group selected from O-4,4'-dimethoxytrityl and 2-cyanoethyl-N,N-diisopropylphosphoramidite; -Y is O, NH or NR1, wherein R1 is: (C1-C20)alkyl group, a (C3-C8)cycloalkyl group, which is optionally substituted by one or more groups selected from the group consisting of halogen atoms and (C1-C6)alkyl groups, or The group -[C(=O)]m-R2-(O-CH2-CH2)p-R3, wherein: m is an integer, with a value of 0 or 1. p is an integer ranging from 0 to 10, R2 is a (C1-C20)alkylene group, which is optionally substituted by a (C1-C6)alkyl group, -O-Z3, -N(Z3)(Z4), -S-Z3, -CN, -C(=K)-O-Z3, -OC(=K)-Z3, -C(=K)-N(Z3)(Z4) or -N(Z3)-C(=K)-Z4, wherein K is O or S, Z3 and Z4 are each independently H or a (C1-C6)alkyl group, which is optionally substituted by one or more groups selected from the group consisting of a halogen atom and a (C1-C6)alkyl group, and R3 is H or a compound of formula (III-A) -X1 and X2 are both hydrogen atoms, and -Ra, Rb, Rc and Rd are each independently H or a (C1-C6)alkyl group.

2. A compound of formula (I) according to claim 1, wherein Y is NR1, and wherein R1 is an unsubstituted (C1-C16)alkyl group selected from methyl, isopropyl, butyl, octyl and hexadecyl groups, and P1, P2, Ra, Rb, Rc, Rd, X1, X2 and B are as defined for general formula (I).

3. A compound of formula (I) according to claim 1, wherein Y is NR1, and wherein R1 is a (C3-C8)cycloalkyl group, which is optionally substituted by one or more groups selected from the group consisting of halogen atoms and (C1-C6)alkyl groups, and P1, P2, Ra, Rb, Rc, Rd, X1, X2 and B are as defined for the general formula (I).

4. A compound of formula (I) according to claim 1, wherein Y is NR1, and wherein R1 is a cyclohexyl group, and P1, P2, Ra, Rb, Rc, Rd, X1, X2 and B are as defined for general formula (I).

5. A compound of formula (I) according to claim 1, wherein Y is NR1, and wherein R1 is a methyl group substituted by a phenyl group, and P1, P2, Ra, Rb, Rc, Rd, X1, X2 and B are as defined for the general formula (I).

6. A compound of formula (I) according to claim 1, wherein Y is NR1 and R1 is -[C(=O)]m-R2-(O-CH2-CH2)p-R3, wherein m is an integer, the value is 0 or 1, p is an integer ranging from 0 to 10, R2 is a (C1-C20)alkylene group, which is optionally substituted by a (C1-C6)alkyl group, -O-Z3, -N(Z3)(Z4), -S-Z3, -CN, -C(=K)-O-Z3, -OC(=K)-Z3, -C(=K)-N(Z3)(Z4) or -N(Z3)-C(=K)-Z4, wherein K is O or S, Z3 and Z4 are each independently H or a (C1-C6)alkyl group, which is optionally substituted by one or more groups selected from the group consisting of a halogen atom and a (C1-C6)alkyl group, -X1 and X2 are both hydrogen atoms, and -Ra, Rb, Rc and Rd are each independently H or a (C1-C6)alkyl group.

7. A compound of formula (I) according to claim 6, wherein R1 is the group -[C(=O)]m-R2-(O-CH2-CH2)p-R3, m is 0, p is 0, 1, 2, 3 or 4, and B, P1, P2, Ra, Rb, Rc, Rd, X1, X2 and R2 are as in the general definition of compounds of formula (I).

8. A compound of formula (I) according to claim 6, wherein -R2 is an ethylene group, p is 0, or -R2 is a pentylene group, or -R2 is a (C12) alkylene group.

9. The compound of formula (I) according to claim 7, wherein R2 is an ethylene group and p is 1, 2, 3 or 4.

10. A compound of formula (I) according to claim 8, wherein m is 1, p is 0, 1 or 2, and R2, B, P1, P2, Ra, Rb, Rc, Rd, X1, X2 are as in the general definition of compounds of formula (I).

11. A compound of formula (I) according to claim 10, wherein R2 is -butylene, or -(C11)alkylene, or -methylene, and B, P1, P2, Ra, Rb, Rc, Rd, X1 and X2 are as defined for the general formula (I).

12. A compound of formula (I) according to claim 6, wherein R2 is a methylene group, p is 1 or 2, and B, P1, P2, Ra, Rb, Rc, Rd, X1, and X2 are as defined for general formula (I).

13. A compound of formula (I) according to claim 1, wherein B is optionally protected and is a pyrimidine or a purine.

14. An oligonucleotide comprising one or more compounds of formula (II): in: -B is a heterocyclic nucleobase; - one of L1 and L2 is O-4,4'-dimethoxytrityl, and the other of L1 and L2 is 2-cyanoethyl-N,N-diisopropylphosphoramidite group, wherein -Y is O, NH or NR1, wherein R1 is: (C1-C20)alkyl group, (C3-C8) cycloalkyl groups, which are optionally substituted by one or more groups selected from Substituted with: halogen atoms and (C1-C6) alkyl groups, or The group -[C(=O)]m-R2-(O-CH2-CH2)p-R3, wherein m is an integer, the value is 0 or 1, p is an integer ranging from 0 to 10, R2 is a (C1-C20)alkylene group, which is optionally substituted by a (C1-C6)alkyl group, -O-Z3, -N(Z3)(Z4), -S-Z3, -CN, -C(=K)-O-Z3, -OC(=K)-Z3, -C(=K)-N(Z3)(Z4) or -N(Z3)-C(=K)-Z4, wherein K is O or S, Z3 and Z4 are each independently H or a (C1-C6)alkyl group, which is optionally substituted by one or more groups selected from the group consisting of a halogen atom and a (C1-C6)alkyl group, and R3 is H or a compound of formula (III-A) -X1 and X2 are both hydrogen atoms, and -Ra, Rb, Rc and Rd are each independently H or a (C1-C6)alkyl group.

15. The oligonucleotide according to claim 14, comprising one or more compounds of formula (II), wherein Y is NR1, wherein R1 is an unsubstituted (C1-C20) alkyl group, and L1, L2, Ra, Rb, Rc, Rd, X1, X2, R2, R3 and B have the same meanings as defined for the general formula (II).

16. An oligonucleotide according to claim 14, comprising one or more compounds of formula (II), wherein Y is NR1, wherein R1 is an unsubstituted (C1-C16) alkyl group, which is an alkyl group selected from methyl, isopropyl, butyl, octyl, hexadecyl, and L1, L2, Ra, Rb, Rc, Rd, X1, X2, R2, R3 and B have the same meanings as defined for the general formula (II).

17. An oligonucleotide according to claim 14, comprising one or more compounds of formula (II), wherein Y is NR1, wherein R1 is a (C3-C8)cycloalkyl group, which is optionally substituted by one or more groups selected from the following: halogen atoms and (C1-C6)alkyl groups, and L1, L2, Ra, Rb, Rc, Rd, X1, X2, R2, R3 and B have the same meanings as defined for the general formula (II).

18. An oligonucleotide according to claim 14, comprising one or more compounds of formula (II), wherein Y is NR1, wherein R1 is a cyclohexyl group, and L1, L2, Ra, Rb, Rc, Rd, X1, X2, R2, R3 and B have the same meanings as defined for the general formula (II).

19. The oligonucleotide according to claim 14, comprising one or more compounds of formula (II), wherein B is pyrimidine or purine.

20. The oligonucleotide according to claim 14, comprising 2 to 10 compounds of formula (II), and / or one or more targeted nucleotides.

21. A double-stranded oligonucleotide comprising one or more compounds of formula (II) as defined in any one of claims 14 to 20.

22. Small interfering RNA (siRNA) comprising one or more compounds of formula (II) as defined in any one of claims 14 to 20.

23. A process for preparing a compound of formula (IA), It includes the following steps: a) Oxidation of the compound of formula (X) wherein B is a heterocyclic nucleobase, and P1 and P2 each independently represent a protecting group as defined in the general formula (I) according to claim 1, The compound of formula (X) is reacted with an oxidizing agent to obtain a compound of formula (XI): and b) subjecting the compound of formula (XI) to reductive amination in the presence of a compound of formula (XII) R1-NH2(XII) wherein R1 is as defined in the general formula (I) according to claim 1, To obtain the compound of formula (IA): wherein B is a heterocyclic nucleobase, and P1 and P2 each independently represent a protecting group as defined in the general formula (I) according to claim 1.

24. The method according to claim 23, wherein the oxidizing agent is sodium periodate (NalO4).

25. A process for preparing a compound of formula (IC): It includes the following steps: a) Reductive amination of the compound of formula (XV) in, -A1, A2 and A3 are OH or OC(=O)-R4, wherein R4 is a (C1-C6)alkyl or (C6-C10)aryl group; -A4 is OH, OC(=O)-R4 or NHC(=O)-R5, R4 is as defined above, and R5 is a (C1-C6)alkyl group which is optionally substituted by a halogen atom; -Y-CHO is transferred to -Y-CH2- through the reductive amination reaction, which is equivalent to X, and X is a group of the formula -(CH2-CH2-O)p-R2-, wherein p and R2 are as defined in the general formula (I) according to claim 1, Reaction with a compound of formula (XIII) Wherein P1, P2 and B are as defined in the general formula (I) according to claim 1, To obtain the compound of formula (IC) 26. A method for preparing a compound of formula (IE), comprising the following steps: a) Reduction of the compound of formula (XI) Wherein P1, P2 and B are as defined in the general formula (I) according to claim 1, thereby obtaining a compound of formula (XVII) b) transferring the compound of formula (XVII) in the presence of a sulfonylating agent to obtain a compound of formula (XVIII) wherein Ts represents a tosyl group, and c) subjecting the compound of formula (XVIII) to basic conditions to obtain the compound of formula (IE) 27. The method according to claim 26, wherein the sulfonylating agent is selected from p-toluene-sulfonyl chloride Ts-Cl or methanesulfonyl chloride Ms-Cl.

28. A process for preparing a compound of formula (IE), comprising the following steps: a) in the presence of an excess of a sulfonylating agent, transferring the compound of formula (XVII) Wherein P1, P2 and B are as defined in the general formula (I) according to claim 1, to obtain a compound of formula (XIX) wherein Ts represents a tosyl group, b) deprotecting the compound of formula (XIX) by removing the group P1 to obtain a compound of formula (XX) c) subjecting the compound of formula (XX) to basic conditions, thereby obtaining a compound of formula (XXI) and d) replacing the tosyl group with a protecting group P1 to obtain the compound of formula (IE)

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