Oligonucleotides Containing Nucleotide Analogues

Abstract

The present disclosure relates to a double-stranded oligonucleotide, including a double-stranded oligonucleotide such as siRNA, comprising a sense strand oligonucleotide and an antisense strand oligonucleotide, wherein the antisense strand oligonucleotide comprises one or more nucleotide analogs of formula (IA), which are not the 5'-overhanging nucleotide or the 3'-overhanging nucleotide of the antisense strand oligonucleotide, and the nucleotide analogs of formula (IA) are as described in the present disclosure.The oligonucleotides containing these analogs have excellent biological activity, for example, increased in vitro stability and improved in vivo efficacy, particularly improved off-target profile.Improved oligonucleotides are useful for silencing (for example, reducing or eradicating) the expression of target genes.

Description

[Technical field]

[0001] The present disclosure relates to the field of targeted gene silencing with single- and double-stranded oligonucleotides, more particularly small interfering RNAs (siRNAs), that contain the modified nucleotide analogs described herein. [Background technology]

[0002] The concept of using synthetic oligonucleotides to control the expression of specific genes dates back to the late 1970s, when targeted gene silencing was first demonstrated using short synthetic oligonucleotides (Non-Patent Document 1). Following Stephenson's discovery, the elucidation of the RNA interference pathway for modulation of gene expression and the role of siRNA in the process has greatly expanded scientists' understanding of post-transcriptional gene expression control in eukaryotic cells.

[0003] Synthetic oligonucleotides include antisense oligonucleotides ("ASOs"), single-stranded oligonucleotides such as anti-miRs and antagomirs, and double-stranded oligonucleotides such as siRNAs. Both ASOs and siRNAs act by binding to target RNA through Watson-Crick base pairing, but their mechanisms of action are different. In antisense technology, ASOs form DNA-RNA duplexes with target RNA, inhibiting mRNA translation by blocking the mechanism or causing RNase H-dependent degradation of the targeted RNA. In RNA interference technology, siRNAs bind to RNA-induced silencing complexes ("RISCs"), where one strand ("passenger strand" or "sense strand") is displaced, and the remaining strand ("guide strand" or "antisense strand") works 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 most significant obstacles to the development of oligonucleotide therapeutics, including siRNA therapeutics, include (i) poor compound stability, (ii) low efficiency of in vivo delivery to target cells, and (iii) side effects such as "off-target" gene silencing and unexpected immune promotion. To address some of these obstacles, various oligonucleotide chemical modifications have been investigated. These modifications can be divided into three categories: (i) sugar modifications, (ii) internucleotide linkage modifications, and (iii) nucleobase modifications.

[0005] Chemical modifications to sugar groups include modifications at the 2'-carbon atom or 2'-hydroxyl group of the ribose ring. 2'-OMe (methoxy) nucleotide analogs are one of the most widely used modifications. 2'-F (fluoro) nucleotides and 2'-O-methoxyethyl nucleotides have also been used. Most of the sugar changes are focused on the 2' position, but modifications at other positions, such as the 4' position, have also been reported (Non-Patent Document 2).

[0006] Other chemical modifications of sugar groups include linking the 2'-oxygen and 4'-carbon of the ribose scaffold of the nucleoside to create so-called locked nucleic acids ("LNA"). LNAs, also referred to as bicyclic nucleic acids, have been shown to have increased RNA binding affinity (Non-Patent Document 3; Non-Patent Document 4), which causes a significant increase in the melting temperature of the resulting double-stranded oligonucleotide. However, fully LNA-modified oligomers longer than 8 nucleotides tend to aggregate. In contrast to the rigid nature of the LNA modification, the highly flexible unlocked nucleic acid ("UNA") modification has also been developed for application in oligonucleotide therapeutics. UNA nucleosides do not have the C2'-C3' bond of the ribose sugar. UNAs are not conformationally restricted due to their open chain structure and have been used to modulate the flexibility of oligonucleotides (Non-Patent Document 5). Insertion of UNAs can reduce the melting temperature (Tm) of a duplex by 5°C to 10°C per insertion in some cases. siRNAs containing UNA and LNA have been reported by Non-Patent Document 6.

[0007] In addition, extended sugar ring systems have also been developed and applied to gene silencing technology. Such systems include the six-membered morpholino ring system, in which the ribose moiety of the nucleoside is replaced with a morpholine ring. Morpholino-based nucleosides form internucleotide linkages in oligonucleotides containing them through the nitrogen atom of the morpholine subunit. Phosphorodiamidate morpholino-based oligonucleotides ("PMOs") have been used in antisense technology (Non-Patent Document 7; Non-Patent Document 8). However, due to low binding affinity to complementary RNA, antisense PMOs need to be relatively long, e.g., 25 bases in length (Non-Patent Document 7, supra). Examples of morpholino subunits are also disclosed in Patent Document 1; Patent Document 2; Patent Document 3; Patent Document 4; and Patent Document 5.

[0008] To reduce susceptibility to nuclease degradation, chemical modifications may be performed on the internucleotide linkages by replacing the 3'-5' phosphodiester linkages with more stable moieties. A widely used modification is the partial or complete replacement of the phosphodiester backbone with phosphorothioate linkages, in which a sulfur atom is substituted for the oxygen atom. An alternative backbone modification that confers increased biological stability to nucleic acids is the boranophosphate linkage. In boranophosphate oligonucleotides, the oxygen of the non-bridging phosphodiester is replaced by an isoelectronic borane (-BH 3 ) part has been replaced.

[0009] However, most of the aforementioned phosphodiester modifications, such as phosphorothioates, result in chiral phosphorus in the internucleotide linkage, resulting in diastereomeric mixtures of the resulting oligonucleotides. The number of diastereomeric oligonucleotides with each modified phosphodiester linkage can double, so the number of diastereomeric oligonucleotides obtained increases exponentially with the increase in the number of modified phosphodiester linkages. Individual diastereomers can exhibit different degrees of nuclease resistance and different hybridization properties to target mRNA. In addition, the purification and chemical analysis of diastereomeric mixtures are complicated. Therefore, in some cases, it may be desirable to avoid the use of phosphodiester modifications, such as phosphorothioates, and the resulting diastereomeric oligonucleotide mixtures.

[0010] Another important problem in RNA interference technology is the targeted delivery and intracellular uptake of siRNA. The cell membrane is a bilayer of negatively charged phospholipids, which is an entry barrier for siRNA that is also negatively charged. Some groups have used N-acetylgalactosamine (GalNAc) to target siRNA attached to it to hepatocytes, which express GalNAc-binding asialoglycoprotein receptor (ASGPR) and can internalize ASGPR-binding siRNA-GalNAc conjugates via endocytosis (see, for example, Non-Patent Document 9). [Prior art documents] [Patent documents]

[0011] [Patent Document 1] U.S. Patent No. 5,034,506 [Patent Document 2] U.S. Patent No. 5,166,315 [Patent Document 3] U.S. Patent No. 5,185,444 [Patent Document 4] U.S. Patent No. 5,698,685 [Patent Document 5] U.S. Patent Publication No. 2016 / 0186174 [Non-patent literature]

[0012] [Non-Patent Document 1] Stephenson et al., 1978, Proc Natl Acad Sci USA, 75:285~288 [Non-Patent Document 2] Leydler et al., 1995, Antisense Res Dev, 5:161–174 [Non-Patent Document 3] Koshin et al., 1998, Tetrahedron, 54:3607-3630 [Non-Patent Document 4] Prakash et al., 2011, Chem. Biodivers, 8:1616-1641 [Non-Patent Document 5] Mangos et al., 2003, J Am Chem Soc, 125:654~661 [Non-Patent Document 6] Bramsen et al. (2010, Nucleic Acids Research, 38(17):5761~5773) [Non-Patent Document 7] Corey et al., 2001, Genome Biology, 2(5):reviews 1015.1~1015 [Non-Patent Document 8] Partridge et al., 1996, Antisense Nucleic Acid Drug Dev, 6:169–175 [Non-Patent Document 9] Nair et al., 2014, J Am Chem Soc, 136:16958-16961 Summary of the Invention [Problem to be solved by the invention]

[0013] While RNA interference technology continues to advance, there remains a need in the field for siRNA oligonucleotides with improved stability and reduced off-target profiles, and their delivery to target cells. [Means for solving the problem]

[0014] The present disclosure relates to a double-stranded oligonucleotide comprising a sense strand oligonucleotide and an antisense strand oligonucleotide, wherein the antisense strand oligonucleotide comprises one or more nucleotide analogs of formula (IA) that are not in the 5'-overhang or the 3'-overhang of the antisense strand oligonucleotide, wherein the nucleotide analogs of formula (IA) are as described throughout this specification.

[0015] In some embodiments of the double-stranded oligonucleotide, the antisense strand oligonucleotide comprises 1 to 10 nucleotide analogues of formula (IA), preferably 1 to 5 nucleotide analogues of formula (IA).

[0016] In some embodiments of the double-stranded oligonucleotide, the antisense strand oligonucleotide has a nucleotide length in the range of 15 to 30 nucleotides, preferably 20 to 25 nucleotides, and most preferably 17 to 25 nucleotides.

[0017] In some embodiments of the double-stranded oligonucleotide, the one or more nucleotide analogues of formula (IA) contained therein in the antisense strand oligonucleotide are positioned contiguous and / or non-contiguous anywhere in the antisense strand oligonucleotide.

[0018] In some embodiments of the double-stranded oligonucleotide, the antisense strand oligonucleotide comprises a hybridizing region that hybridizes to the sense strand oligonucleotide, and in the antisense strand oligonucleotide, the one or more nucleotide analogues of formula (IA) contained therein are positioned in the nucleotide region of the antisense oligonucleotide ranging from the nucleotide at position 2 to the nucleotide at position 15 of the hybridizing region, starting from the 5'-terminal nucleotide of the antisense strand.

[0019] In some embodiments of the double-stranded oligonucleotide, the antisense strand oligonucleotide comprises a hybridizing region that hybridizes to the sense strand oligonucleotide, and in the antisense strand oligonucleotide, the one or more nucleotide analogues of formula (IA) contained therein are positioned in the nucleotide region of the antisense oligonucleotide in the range from the nucleotide at position 2 to the nucleotide at position 10 of the hybridizing region, such as from the nucleotide at the 5' end of the antisense strand to the nucleotide at position 8 of the hybridizing region.

[0020] In some embodiments of the double-stranded oligonucleotide, the antisense strand oligonucleotide further comprises one or more nucleotide analogues of formula (IB) described herein.

[0021] In some embodiments of the double-stranded oligonucleotide, in the nucleotide analogs of formula (IA) and (IB) contained in the antisense strand oligonucleotide, B is selected from the group consisting of pyrimidine, substituted pyrimidine, purine and substituted purine, or any pharma- ceutically acceptable salt thereof.

[0022] In some embodiments of the double-stranded oligonucleotide, one or more of the nucleotide analogues of formula (IA) are in their (2R,6R)-stereoisomers.

[0023] In some embodiments of the double-stranded oligonucleotide, the sense strand oligonucleotide comprises one or more nucleotide analogues of formula (IA) described herein.

[0024] In some embodiments of the double-stranded oligonucleotides, the sense strand oligonucleotide comprises one or more nucleotide analogues of formula (IB) described herein.

[0025] In some embodiments of the double-stranded oligonucleotide, the sense strand comprises one or more nucleotide analogues of formula (IB) as defined in claim 7, wherein at least one nucleotide of formula (IB) consists of a 5'-terminal or 3'-terminal nucleotide, and the one or more modified nucleotides of formula (IB) are contiguous.

[0026] In some embodiments of the double-stranded oligonucleotide, the sense strand comprises one to three nucleotide analogues of formula (IB) as defined in claim 7, wherein the modified nucleotides of formula (IB) are contiguous and form an oligonucleotide stretch that is located at the 5'-end or the 3'-end of the sense strand.

[0027] In some embodiments, the double-stranded oligonucleotide consists of a short interfering RNA (siRNA), or a pharma- ceutically acceptable salt thereof.

[0028] The present disclosure further relates to single-stranded antisense oligonucleotides complementary to a target mRNA or a target (double-stranded) DNA as defined throughout this specification.

[0029] The present disclosure also relates to compositions comprising the double-stranded oligonucleotides or single-stranded antisense oligonucleotides described herein.

[0030] The present disclosure also relates to a double-stranded oligonucleotide as described herein for its use as a medicament. [Brief description of the drawings]

[0031] [Figure 1] Figure 1 shows in vivo knockdown of siRNAs 2-0, 1-8, 3-3, 3-8 and 3-11. Ordinate: TTR serum levels compared to control siRNA. Abscissa: days after sc administration. [Diagram 2] Figure 1 shows in vivo knockdown of siRNAs 2-0, 1-8, 3-8 and 3-11. Ordinate: TTR mRNA expression levels in liver compared to control siRNA. Abscissa: dose in mg / kg of each of the administered siRNAs 2-0, 1-8, 3-8 and 3-11. [Diagram 3] Figure 1 shows in vivo knockdown of siRNAs 2-0, 1-8, 3-8 and 3-11. Ordinate: TTR serum levels in ng / ml. Abscissa: dose in mg / kg of each of the administered siRNAs 2-0, 1-8, 3-8 and 3-11. [Figure 4] Figure 1 shows in vivo knockdown of siRNAs 2-0, 5-1, 5-4, 5-5, 5-6, 5-8, 5-10 and 5-11. Ordinate: TTR serum levels compared to control siRNA. Abscissa: days after scsiRNA administration. [Diagram 5] Figure 1 shows in vivo knockdown of siRNAs 2-0, 5-1, 5-4, 5-5, 5-6, 5-8, 5-10 and 5-11. Ordinate: TTR serum levels compared to control siRNA. Abscissa: days after scsiRNA administration. [Figure 6] Figure 1 shows in vivo knockdown of siRNAs 2-0, 5-1, 5-11 and 5-13. Ordinate: TTR mRNA expression levels in liver compared to control siRNA. Abscissa: dose in mg / kg of each of the administered siRNAs 2-0, 5-1, 5-11 and 5-13. [Figure 7] Figure 1 shows in vivo knockdown of siRNAs 2-0, 5-1, 5-11 and 5-13. Ordinate: TTR serum levels in ng / ml. Abscissa: dose in mg / kg of each of the administered siRNAs 2-0, 5-1, 5-11 and 5-13. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] The present disclosure provides novel double-stranded and single-stranded oligonucleotides that contain one or more nucleotide analogues.The oligonucleotides that contain these analogues have superior biological activity, such as improved in vitro stability, off-target profile and in vivo duration of action.The improved oligonucleotides are useful for silencing (e.g., reducing or eradicating) the expression of target genes.In certain embodiments, the present disclosure encompasses double-stranded RNAs (dsRNAs) that contain specific nucleotide analogues, particularly siRNAs that contain specific nucleotide analogues, that can hybridize to the messenger RNA (mRNA) of interest to reduce or block the expression of the target gene of interest.

[0033] According to the most preferred embodiment, the specific nucleotide analogue has a ribose sugar ring replaced with a six-membered heterocyclic ring.As described in more detail in this disclosure, the six-membered heterocyclic ring of the specific nucleotide analogue can be a dioxane or morpholino ring.When the heterocyclic ring is a morpholino ring, the nitrogen atom is either substituted or unsubstituted.In some embodiments, the six-membered heterocyclic ring is replaced with a linear or cyclic group and / or a targeting moiety.

[0034] definition The terms used herein generally have their ordinary meaning 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 subject matter of this disclosure.

[0035] The following definitions apply in the context of this disclosure.

[0036] 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.

[0037] The term "about" as used herein refers to a normal error range for each value that is easily recognized by those skilled in the art. Reference herein to a value or parameter of "about" includes (and describes) embodiments that are directed to the value or parameter itself. In some embodiments, the term "about" refers to ±10% of a given value. However, whenever the value in question refers to something that cannot be divided, such as a nucleotide, or something else that is expected to lose its identity once divided, "about" refers to ±1 of the thing that cannot be divided.

[0038] It is understood that the aspects and embodiments of the disclosure described herein include "having," "including," "consisting," and "consisting essentially of" the aspects and embodiments. The words "have" and "comprise," or variations such as "has," "having," "comprises," or "comprising," will be understood to indicate the inclusion of the stated elements (e.g., a composition of matter or method step) but not the exclusion of any other elements. The term "consisting of" refers to the inclusion of the stated elements, without including any additional elements. The term "consisting essentially of" indicates the inclusion of the stated elements and, optionally, other elements, if the other elements do not significantly affect the novel characteristics underlying the invention.

[0039] "Alkyl" means, unless otherwise specified, an aliphatic hydrocarbon group which may be straight or branched having 1 to 20 (e.g., 1 to 5, 1 to 10, or 1 to 15) carbon atoms in the chain. "Branched" means that a straight alkyl chain has one or more alkyl groups, such as methyl, ethyl or propyl groups, attached to it. Exemplary straight or branched alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, n-pentyl, 3-pentyl, octyl, nonyl, and decyl.

[0040] "Cycloalkyl" means a cyclic, saturated alkyl group as defined above. Examples, but not limited to, are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

[0041] "Alkoxy" is defined as the radical -OR, where R is an alkyl group as defined above, including cycloalkyl groups. Examples include, but are not limited to, methoxy, ethoxy, 1-propoxy, 2-propoxy, butoxy, and pentoxy.

[0042] "Halogen atom" refers to a fluorine, chlorine, bromine, or iodine atom. In some embodiments, a fluorine or chlorine atom may be preferred.

[0043] "Aryl" means, unless otherwise specified, an aromatic monocyclic or polycyclic hydrocarbon ring system of 6 to 14 carbon atoms, e.g., 6 to 10 carbon atoms. Exemplary aryl groups include phenyl and naphthyl groups.

[0044] "Heterocycle" or "heterocyclic" refers to a saturated, partially unsaturated, or unsaturated carbocyclic group containing at least one heteroatom selected from the group of oxygen, nitrogen, selenium, phosphorus, and sulfur. The nitrogen, selenium, phosphorus, or sulfur may be optionally oxidized, and the nitrogen may be optionally 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, e.g., 5 to 7, or 5 to 10 ring members, and may have one, two, or multiple rings (i.e., monocyclic, bicyclic, or polycyclic rings). In certain embodiments, the heteroatoms are oxygen, nitrogen, and sulfur. The number of heteroatoms may vary, e.g., from 1 to 3. Suitable heterocycles are also disclosed in The Handbook of Chemistry and Physics, 76th Edition, CRC Press, Inc., 1995-1996, pp. 2-25 to 2-26, the disclosure of which is incorporated herein by reference. In some embodiments, the heterocycle is a non-aromatic heterocycle, examples of which include, but are not limited to, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, oxiranyl, tetrahydrofuranyl, dioxolanyl, tetrahydro-pyranyl, dioxanyl, dioxolanyl, piperidyl, piperazinyl, morpholinyl, pyranyl, imidazolinyl, pyrrolinyl, pyrazolinyl, thiazolidinyl, tetrahydrothiopyranyl, dithianyl, thiomorpholinyl, dihydropyranyl, tetrahydropyranyl, dihydropyranyl, tetrahydro-pyridyl, dihydropyridyl, tetrahydropyridinyl, dihydrothiopyranyl, and azepanyl, as well as fused systems resulting from condensation with a phenyl group.

[0045] "Heteroaryl" refers to an aromatic heterocycle having 5-14 (e.g., 5-7, or 5-10) ring members, which may be monocyclic, bicyclic, or polycyclic. The number of heteroatoms may vary from 1 to 3 heteroatoms, typically selected from, for example, N and O. Examples of heteroaryl groups include pyrrolyl, pyridyl, pyrazolyl, thienyl, pyrimidinyl, pyrazinyl, tetrazolyl, indolyl, quinolinyl, purinyl, imidazolyl, thienyl, thiazolyl, benzothiazolyl, furanyl, benzofuranyl, 1,2,4-thiadiazolyl, oxadiazole, isothiazolyl, triazolyl, tetrazolyl, isoquinolyl, benzothienyl, isobenzofuryl, pyrazolyl, carbazolyl, benzimidazolyl, isoxazolyl, and pyridyl-N-oxide, as well as fused systems resulting from condensation with a phenyl group. In certain embodiments, the heteroaryl is a 5- or 6-membered heteroaryl containing one or more heteroatoms, e.g., 1 to 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 "heterocyclene" formed by the removal of two hydrogen atoms.

[0046] The term "heterocyclic nucleobase" refers to any nitrogen-containing heterocyclic moiety capable of forming Watson-Crick type hydrogen bonding and stacking interactions upon pairing with a complementary nucleobase or nucleobase analog (i.e., a derivative of a nucleobase) when the nucleobase is incorporated into a polymeric structure.

[0047] Unless otherwise specified, the term "heterocyclic nucleobase" herein refers to an optionally substituted nitrogen-containing heterocyclic group that can be attached to an optionally substituted dioxane ring or an optionally substituted morpholino ring according to the present disclosure. In some embodiments, the heterocyclic nucleobase can be selected from an optionally substituted purine base or an optionally substituted pyrimidine base. The term "purine base" is used herein in its ordinary sense as understood by those skilled in the art, and includes its tautomers. Similarly, the term "pyrimidine base" is used herein in its ordinary sense as understood by those skilled in the art, and includes its tautomers. A non-limiting list of optionally substituted purine bases includes purine, adenine, guanine, hypoxanthine, xanthine, alloxanthine, 7-alkylguanine (e.g., 7-methylguanine), theobromine, caffeine, uric acid, and isoguanine. Examples of pyrimidine bases include, but are not limited to, cytosine, thymine, uracil, 5,6-dihydrouracil, and 5-alkylcytosine (e.g., 5-methylcytosine). Other non-limiting examples of heterocyclic nucleobases include diaminopurine, 8-oxo-N 6 Alkyl adenines (e.g., 8-oxo-N 6 methyladenine), 7-deazaxanthine, 7-deazaguanine, 7-deazaadenine, N 4 ,N 4 -Ethanocytosine, 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 nucleobases described in U.S. Patent Nos. 5,432,272 and 7,125,855, which disclose additional heterocyclic bases, which are incorporated herein by reference.In some embodiments, the heterocyclic nucleobase is optionally substituted with an amine or enol protecting group.

[0048] The terms "protecting group" and "protecting groups", as used herein, refer to any atom or group of atoms that is added to a molecule to prevent existing groups in the molecule from undergoing unwanted chemical reactions. A "protecting group" may be a labile chemical moiety known in the art to protect reactive groups from undesired or premature reactions during chemical synthesis, such as a hydroxyl, amino, thiol, carboxylic acid or phosphate group. Protecting groups are typically used selectively and / or orthogonally to protect sites at other reactive sites during a reaction, and can then be removed to leave unprotected groups intact or available for further reactions.

[0049] Examples of protecting group moieties are described in TW Greene and PGM Huts, Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, 1999, and JFW MacOmie, 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 can be selected in such a way that they are stable to the particular reaction conditions and are easily removed at a convenient stage using techniques known in the art.

[0050] A non-limiting list of protecting groups includes benzyl; substituted benzyl; alkylcarbonyl and alkoxycarbonyl (e.g., t-butoxycarbonyl (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 ethers (e.g., trimethylsilyl, triethylsilyl, triisopropylsilyl, t-butyldimethylsilyl, triisopropylsilyloxymethyl, [2-(trimethylsilyl)ethoxy]methyl, or t-butyldiphenylsilyl); esters (e.g., benzoate esters, 2-cyanoethylphosphonic acid ... acid esters); carbonates (e.g., methoxymethyl carbonate); sulfonates (e.g., tosylates or mesylates); 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 (MMT); 4,4'-dimethoxytrityl (DMT); 4,4',4"-trimethoxytrityl (TMT); and those described herein).

[0051] Preferred protecting groups are selected from the group comprising acetyl (Ac), benzoyl (Bzl), isobutyryl (iBu), phenylacetyl, dimethoxytrityl (DMT), methoxytrityl (MMT), triphenylmethyl (Trt), N,N-dimethylformamidine and 2-cyanoethyl (CE).

[0052] Unless otherwise specified, abbreviations for any protecting groups, amino acids and other compounds follow their common usage, accepted abbreviations, or those of the IUPAC-IUB Commission on Biochemical Nomenclature (see Biochem. 11:942-944 (1972)).

[0053] The term "solid support" (also called resin), as used herein, refers to insoluble particles, typically 50-200 μm in diameter, to which oligonucleotides are attached during synthesis. Many types of solid supports have been used, but controlled pore glass (CPG) and polystyrene (highly cross-linked polystyrene beads) have proven to be particularly useful. Controlled pore glass is rigid, non-swelling, and has deep pores (pore size 500-1000 Å) in which oligonucleotide synthesis occurs. Solid supports for conventional oligonucleotide synthesis are commercially available and are typically manufactured with loadings of 20-40 μmol of nucleoside per gram of resin for CPG solid supports. Polystyrene-based solid supports exhibit higher loadings of up to 300 μmol per gram of resin. Solid support materials with standard nucleotides already attached are commercially available, and amino-functionalized CPG and polystyrene materials are used for the synthesis of non-commercial building blocks as shown later with respect to the building blocks described herein. As an alternative to a solid support having a first nucleotide building block already attached, commercially available universal solid support materials as described later in this disclosure can be used.

[0054] The term "ribonucleotide" or "nucleotide" as used herein includes naturally occurring or modified nucleotides or surrogate replacement moieties, as further detailed below. Modified nucleotides are non-naturally occurring nucleotides, also referred to herein as "nucleotide analogs". Those skilled in the art will understand that guanine, cytosine, adenine, uracil, or thymine in a nucleotide can be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide containing such a replacement moiety to which the nucleotide is attached. For example, a nucleotide containing inosine as its base can base pair with nucleotides containing, but not limited to, adenine, cytosine, or uracil. Thus, a nucleotide containing uracil, guanine, or adenine can be replaced in the nucleotide sequences of the present disclosure by, for example, a nucleotide containing inosine. Sequences containing such replacement moieties are included as embodiments of the present disclosure.

[0055] "Cell targeting moiety" as used herein means a molecular group that ensures increased delivery of siRNA, including (i) increasing the specificity of siRNA binding to a selected target receptor (e.g., a target protein), such as increasing the specificity of siRNA binding to cells expressing the selected target receptor; (ii) increasing the uptake of siRNA by the target cell; and / or (iii) increasing the ability of siRNA to be properly processed once in the target cell, such as increasing the intracellular release of siRNA, for example by facilitating the transfer of siRNA from a transport vesicle to the cytoplasm. Thus, cell targeting moieties are used to direct and / or deliver oligonucleotides to specific cells, tissues, organs, etc. Cell targeting moieties included in nucleotides, nucleotide analogs, or oligonucleotides confer characteristics of nucleotide analogs or oligonucleotides to the nucleotides, such that the nucleotides, nucleotide analogs, or oligonucleotides are preferentially recognized, bound, internalized, processed, activated, etc. by targeted cell types versus non-targeted cell types. For example, endothelial cells have high affinity for the peptide cell targeting moiety Arg-Gly-Asp (RGD); cancer and kidney cells preferentially interact with compounds having a folate moiety; immune cells have affinity for mannose; and cardiomyocytes have affinity for the peptide WLSEAGPVVTVRALRGTGSW (SEQ ID NO: 1) (see, for example, Biomaterials ZV-8081-8087, 2010). Other cell targeting / delivery moieties are known in the art. Thus, compounds containing cell targeting moieties preferentially interact with and are taken up by the targeted cell type.

[0056] Cell-targeting moieties include cell-targeting peptidic and non-peptidic groups.

[0057] "Target cell" or "targeted cell" as used herein refers to a cell of interest. Such cells can be found in vitro, in vivo, in situ, or 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.

[0058] The term "TTR" as used herein refers to the transthyretin gene or protein. The term "TTR" as used herein 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. Further examples of TTR mRNA sequences are readily available, for example, in GenBank.

[0059] "Target sequence," as used herein, refers to a contiguous nucleotide sequence found in an RNA transcript or portion thereof of a target gene, including mRNA, which is the RNA processing product of the primary transcription product.

[0060] As used herein, unless otherwise specified, the term "complementary" when used to describe a first nucleotide sequence (e.g., an oligonucleotide) to a second nucleotide sequence (e.g., an oligonucleotide) refers to the ability of the first nucleotide sequence to hybridize with the second nucleotide sequence under certain conditions and form a duplex structure, as understood by those skilled in the art. An example of this includes base pairing of the first nucleotide sequence to the second nucleotide sequence over the entire length of the first or second nucleotide sequence. Such sequences can be referred to herein as "fully complementary" to each other. When a first sequence is referred to herein as "substantially complementary" to a second sequence, the two sequences may be fully complementary or may have 70% or more nucleotide identity while retaining the ability to hybridize under optimal conditions to its ultimate target. However, when two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs shall not be considered as mismatches for determining complementarity. For example, a double-stranded RNA (dsRNA) comprising a first oligonucleotide having a length of 21 nucleotides and a second oligonucleotide having a length of 23 nucleotides, the second oligonucleotide comprising a 21-nucleotide sequence that is completely complementary to the first oligonucleotide, is also referred to as "fully complementary" for the purposes of this disclosure. A "complementary" sequence may also include or be entirely formed from non-Watson-Crick base pairs and / or base pairs formed from non-natural modified nucleotides, so long as the above requirements for its ability to hybridize are met. The terms "complementary", "fully complementary" and "substantially complementary" can be used in reference to base matching between the sense strand and antisense strand of a dsRNA or between the antisense strand of a dsRNA and a target sequence, as understood from the context of their use.A polynucleotide "substantially complementary to at least a portion" of an mRNA, as used herein, refers to a polynucleotide that is substantially complementary to a contiguous portion of an mRNA of interest. The term "double-stranded RNA" or "dsRNA", as used herein, refers to a complex of ribonucleic acid molecules having a duplex structure comprising two antiparallel, substantially complementary nucleic acid strands as defined above. The two strands forming the duplex structure may be different parts of one larger RNA molecule, or they may be separate RNA molecules. In the case of separate RNA molecules, such dsRNAs are sometimes referred to in the literature as short interfering RNA (siRNA). When the two strands are part of one larger molecule, and thus connected by an uninterrupted chain of nucleotides between the 3' end of the first strand and the 5' end of the second strand to form a duplex structure, the connecting RNA strands are referred to as "hairpin loops", "short hairpin RNAs", or "shRNAs". When two strands are covalently connected between the 3' end of the first strand and the 5' end of the second strand by means other than an uninterrupted chain of nucleotides to form a duplex structure, the connecting structure is referred to as a "linker". The RNA strands may have the same or different number of nucleotides. The maximum number of base pairs is the number of oligonucleotides in the shortest strand of the dsRNA minus all overhangs present in the duplex. In addition to the duplex structure, the dsRNA may contain one or more nucleotide overhangs. In addition, the term "dsRNA" as used herein may include substantial modifications in multiple nucleotides as well as chemical modifications to ribonucleotides, such as all types of modifications disclosed herein or known in the art. Any such modifications are encompassed by "dsRNA" for the purposes of this disclosure when used in siRNA type molecules. In some embodiments, the internucleotide linkages in the dsRNA may be modified, for example as described herein.

[0061] Within the scope of the present disclosure, the "percentage identity" between two sequences of nucleic acids means the percentage of identical nucleotide residues between the two sequences being compared, obtained after optimal alignment, and this percentage is purely statistical, and the differences between the two sequences are randomly distributed along their length. Comparison of two nucleic acid sequences is conventionally carried out by comparing the sequences after optimally aligning them, and said comparison can be carried out by segments or by using "alignment windows". Optimal alignment of sequences for comparison can be performed using the local homology algorithm of Smith and Waterman (1981), using the local homology algorithm of Neddleman and Wunsch (1970), using the similarity search method of Pearson and Lipman (1988), or using computer software which employs these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, or with the comparison software BLAST NR or BLAST P).

[0062] The percentage identity between two nucleic acid sequences is determined by comparing two optimally aligned sequences, where the nucleic acid sequences being compared may have additions or deletions compared to the reference sequence for optimal alignment between the two sequences. The percentage identity is calculated by determining the number of positions where nucleotide residues are identical between the two sequences, preferably between the two complete sequences, dividing the number of identical positions in the alignment window by the total number of positions, and multiplying the result by 100 to obtain the percentage identity between the two sequences.

[0063] As intended herein, a nucleotide sequence having at least 70% nucleotide identity to a reference sequence includes those 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 said reference sequence.

[0064] In some embodiments, dsRNA comprises modified ribonucleosides, including deoxyribonucleosides, for example, deoxyribonucleoside overhangs, one or more deoxyribonucleosides in the double-stranded portion of dsRNA, etc. However, it is self-evident that in any case, double-stranded DNA molecules are not included in the term "dsRNA".

[0065] The term "nucleotide overhang" as used herein refers to one or more unpaired nucleotides that protrude from the double-stranded structure of a dsRNA when the 3'-end of the first strand of the dsRNA extends beyond the 5'-end of the second strand, or vice versa. "Blunt" or "blunt-ended" means that there are no unpaired nucleotides at the end of the dsRNA, i.e., there are no nucleotide overhangs. A "blunt-ended" dsRNA is a dsRNA that is double-stranded throughout its entire length, i.e., there are no nucleotide overhangs at either end of the molecule. For clarity, chemical caps or non-nucleotide chemical moieties conjugated to the 3'-end and / or 5'-end of the strand of the dsRNA are not considered in determining whether the dsRNA has an overhang or is blunt-ended.

[0066] The term "consecutive nucleotides" as used herein refers to nucleotides, either natural or modified, that are linked to each other via an internucleotide linkage. Thus, two consecutive nucleotides are linked to each other via an internucleotide linkage. Three consecutive nucleotides also refer to a stretch of three nucleotides, where a first nucleotide is linked to a second nucleotide via an internucleotide linkage, which is also linked to a third nucleotide via an internucleotide linkage. Within the stretch of three nucleotides that are consecutively arranged in an oligonucleotide, the three nucleotides are referred to as "consecutive nucleotides". By way of illustration, an oligonucleotide that comprises 10 consecutive nucleotide analogues consists of an oligonucleotide that comprises in its sequence an uninterrupted stretch of 10 nucleotide analogues that are linked to each other via an internucleotide linkage.

[0067] The term "antisense strand" or "antisense strand oligonucleotide" in dsRNA, as used herein, refers to a strand of dsRNA that contains a sequence that is substantially complementary to a target nucleotide sequence. The other strand of the dsRNA is the "sense strand", which may also be referred to as a "sense strand oligonucleotide". In the double-stranded oligonucleotide contained in the dsRNA, the antisense strand comprises a nucleotide region, referred to herein as the "hybridizing region", which is paired with a region of the sense strand (which is the hybridizing region of the sense strand). The antisense strand may further comprise a region located at the 5'-end and / or 3'-end that is not paired with the sense strand. In an embodiment of the dsRNA in which the dsRNA is comprised of an siRNA, this unpaired region is conventionally referred to as a "nucleotide overhang" or "overhang", i.e., a "5'-nucleotide overhang" or a "3'-nucleotide overhang". Typically, the nucleotide overhang has a length of 1 to 8 nucleotides, which includes lengths of 1, 2, 3, 4, 5, 6, 7 and 8 nucleotides. In most embodiments, the nucleotide overhang has a length of 2-3 nucleotides. Symmetrically, the sense strand of a double-stranded oligonucleotide, such as the sense strand of a dsRNA, comprises a hybridizing region that hybridizes with the hybridizing region of the antisense strand. The sense strand may also comprise a 5'-nucleotide overhang and / or a 3'-nucleotide overhang. The hybridizing region of the antisense strand of a double-stranded oligonucleotide may be paired with a region of the sense strand, but in some embodiments may not be completely complementary to said region of the sense strand, which means that when hybridized, there may be some mismatches (i.e., some unpaired nucleotides) between the hybridizing region of the antisense strand oligonucleotide and the paired region of the sense strand oligonucleotide.Typically, in some of these embodiments, one to three mismatches may exist between the hybridizing region of the antisense strand oligonucleotide and the hybridizing region of the sense strand oligonucleotide.

[0068] The term "introducing into a cell" as used herein means facilitating uptake or absorption into a cell, as understood by those skilled in the art. Absorption or uptake of dsRNA may occur through unassisted diffusive or active cellular processes, or by auxiliary agents or devices. The meaning of this term is not intended to be limited to cells in vitro; dsRNA may also be "introduced into a cell", where the cell is part of an organism. In such an instance, introduction into a cell would include delivery to an organism. For example, in the case of in vivo delivery, dsRNA may be injected into a tissue site or administered systemically. In vivo delivery is also mediated by a beta-glucan delivery system (see, for example, Tesz, GJ et al., 2011, Biochem J. 436(2):351-62). Introduction into cells in vitro includes methods known in the art, such as electroporation and lipofection. Further approaches are described herein below or known in the art.

[0069] The terms "inhibit expression of" or "inhibiting expression of" as used herein, insofar as they refer to a target gene, refer to at least partial suppression of expression of the target gene as evidenced by a reduction in the amount of mRNA transcribed from the target gene. The term "inhibiting" as used herein is used synonymously with "reducing", "silencing", "downregulating", "suppressing", "knockdown" and other similar terms, and includes any level of inhibition. The degree of inhibition is usually expressed as (((mRNA in control cells)-(mRNA in treated cells)) / (mRNA in control cells)) x 100%. Alternatively, the degree of inhibition can be shown in a reduction of a parameter functionally related to the transcription of the target gene, for example, the amount of protein encoded by the target gene secreted by the cell, or a reduction in the number of cells exhibiting a particular phenotype, for example apoptosis. In principle, target gene silencing can be determined in any cell expressing the target, either constitutively or by genomic engineering, and by any suitable assay. However, if a reference is needed to determine whether a given dsRNA inhibits expression of a target gene to a particular degree, and is therefore encompassed by this disclosure, it is expected that the assays provided in the examples below will serve as such a reference.

[0070] As used herein, in the context of target gene expression, the terms "treat", "treatment" and the like refer to the alleviation or reduction of a pathological process mediated by the expression of a target gene. In the context of the present disclosure, the terms "treat", "treatment" and the like, insofar as they relate to any of the other conditions listed herein below (other than pathological processes mediated by target expression), refer to the alleviation or reduction of one or more symptoms associated with such a condition.

[0071] As used herein, the terms "prevent" or "delay the progression of" (and grammatical variations thereof) in reference to a disease or disorder refer to prophylactic treatment of the disease, for example in an individual suspected of having or at risk of developing the disease. Prevention can include, but is not limited to, preventing or delaying the onset or progression of the disease and / or maintaining one or more symptoms of the disease at a desirable or subpathological level.

[0072] The terms "therapeutically effective amount" and "prophylactically effective amount" as used herein refer to an amount that provides a therapeutic benefit in treating, preventing, or managing a pathological process mediated by target gene expression, or an obvious symptom of a pathological process mediated by target gene expression. The specific therapeutically effective amount can be easily determined by a medical technician of ordinary skill, and can vary depending 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 the biological process mediated by the target gene.

[0073] The term "individual" or "subject" as used herein is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, 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.

[0074] 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., the base and sugar portions of a heterocycle) units, examples of which include, but are not limited to, phosphate ester, phosphate ester analogs (i.e., phosphodiester or phosphotriester moieties), phosphorothioate, phosphate ester, guanidinium, hydroxylamine, hydroxylhydrazinyl, amide, carbamate, alkyl, and substituted alkyl linkages, as known in the art. An "internucleoside linking group" may involve a linkage between two nucleosides, between two nucleoside analogs, or between a nucleoside and a nucleoside analog.

[0075] Internucleoside linkages constitute the backbone of a nucleic acid molecule. Internucleoside linkage groups refer to chemical groups that link two adjacent nucleoside residues contained in a nucleic acid molecule, and include (i) chemical groups that link two adjacent nucleoside residues, (ii) chemical groups that link a nucleoside residue to an adjacent nucleoside analog residue, and (iii) chemical groups that link a first nucleoside analog residue to a second nucleoside analog residue, where the nucleoside analog residues can be the same or different. Nucleoside analog residues include compounds of formula (I) disclosed herein. In one embodiment, the nucleotides of the siNA molecules of the present invention are linked to adjacent nucleotides via a linkage 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 3' internucleoside linkage). 3'-5' internucleoside linkage, as used herein, refers to an internucleoside linkage that connects two adjacent nucleoside units, where the linkage is 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 embodiment, a nucleotide (including a nucleotide analog) of the siNA molecule of the present invention is linked to an adjacent nucleotide (including a nucleotide analog) through a linkage 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 linkage). 2'-5' internucleoside linkage, as used herein, refers to an internucleoside linkage that connects two adjacent nucleoside units, where the linkage is between the 2'-carbon of the sugar moiety of the first nucleoside and the 5'-carbon of the sugar moiety of the second nucleoside.

[0076] The term "internucleoside linking group," as used herein, includes phosphorus and non-phosphorus containing internucleoside linking groups.

[0077] In some embodiments, phosphorus-containing internucleoside linking groups include phosphodiesters, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates, such as 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, such as 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkyl phosphates, thionoalkyl phosphotriesters, selenophosphates and boranophosphates having normal 3'-5' linkages, and 2'-5' linked analogs thereof.

[0078] Representative U.S. patents which teach the preparation of the above phosphorus-containing internucleoside linkages include, but are not limited to, U.S. Pat. Nos. 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; 5,455, Nos. 233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, each of which is incorporated herein by reference.

[0079] In one embodiment, the non-phosphodiester backbone linkages are selected from the group consisting of phosphorothioate, phosphorodithioate, alkyl-phosphonate, and phosphoroamidate backbone linkage groups.

[0080] In one embodiment, phosphorus-containing internucleoside linking groups include phosphodiesters, phosphotriesters, and phosphorothioates.

[0081] In some embodiments, the oligonucleotide of the present invention comprises one or more internucleoside linking groups that do not contain phosphorus atom.Such oligonucleotides include, but are not limited to, short chain alkyl or cycloalkyl internucleoside linking groups, mixed heteroatom and alkyl or cycloalkyl internucleoside linking groups, or those formed by one or more short chain heteroatom or heterocyclic internucleoside linking groups.These examples include siloxane backbone; sulfide, sulfoxide and sulfone backbone; formacetyl and thioformacetyl backbone; methyleneformacetyl and thioformacetyl backbone; riboacetyl backbone; backbone containing alkene; sulfamic acid backbone; methyleneimino and methylenehydrazino backbone; sulfonic acid and sulfonamide backbone; those with amide backbone; and others with mixed N, O, S and CH2 component parts. Representative United States patents which teach the preparation of the above non-phosphorus containing internucleoside linking groups include, but are not limited to, U.S. Patent Nos. 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; 5,54 Nos. 1,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, each of which is incorporated herein by reference.

[0082] In one embodiment, the oligonucleotide of the present invention comprises one or more neutral internucleoside linking groups that are non-ionic. Neutral internucleoside linking groups include non-ionic linking groups that include siloxanes (dialkylsiloxanes), carboxylate esters, carboxamides, sulfides, sulfonate esters and amides (see, for example: Carbohydrate Modifications in Antisense Research; YS Sanghvi and PD Cook, eds., ACS Symposium Series 580; Chapters 3 and 4, (pages 40-65)). Further neutral internucleoside linking groups include non-ionic linkages that include hybrid N, O, S and CH2 component moieties.

[0083] Double-stranded oligonucleotides, such as double-stranded RNA (dsRNA) As previously specified herein, the double-stranded oligonucleotide according to the present disclosure, including dsRNA, comprises a sense strand oligonucleotide and an antisense strand oligonucleotide, and the antisense strand oligonucleotide hybridizes with the sense strand oligonucleotide through the hybridizing region contained therein. Symmetrically, the sense strand oligonucleotide also comprises a hybridizing region that hybridizes with the hybridizing region contained in the antisense strand oligonucleotide. In some embodiments, the antisense strand oligonucleotide further comprises a nucleotide region at its 5' end, at its 3' end, or at both its 5' end and its 3' end that does not hybridize with the sense strand oligonucleotide, and this non-hybridizing region is referred to herein as a "nucleotide overhang" or alternatively as an "overhang". The sense strand oligonucleotide may also further comprise a nucleotide region at its 5' end, at its 3' end, or at both its 5' end and its 3' end that does not hybridize with the antisense strand oligonucleotide, and this non-hybridizing region is referred to herein as a "nucleotide overhang" or alternatively as an "overhang".

[0084] In a preferred embodiment of the double-stranded oligonucleotide according to the present disclosure, the hybridizing region contained in the antisense strand oligonucleotide is completely complementary to the hybridizing region contained in the sense strand oligonucleotide.

[0085] The antisense strand oligonucleotide may further comprise a region that is not paired with the sense strand, located at its 5' end, its 3' end, or both the 5' end and its 3' end. In an embodiment of an antisense strand oligonucleotide of a dsRNA in which the dsRNA is composed of an siRNA, the 3' end or 5' end region that is not paired with the sense strand oligonucleotide is conventionally called a "nucleotide overhang" or "overhang", i.e., a "5'-nucleotide overhang" or a "3'-nucleotide overhang". In a preferred embodiment of a double-stranded oligonucleotide according to the present disclosure, the nucleotide overhang, when present in the sense strand oligonucleotide or the antisense strand oligonucleotide, has a length of 1 to 8 nucleotides, including lengths of 1, 2, 3, 4, 5, 6, 7 and 8 nucleotides. In most embodiments, the nucleotide overhang has a length of 2 to 3 nucleotides.

[0086] The present inventors have found that the presence of a specific nucleotide analogue of formula (IA) as described herein in the hybridizing region of the antisense strand oligonucleotide of a double-stranded oligonucleotide, including a sense strand oligonucleotide and an antisense strand oligonucleotide, particularly in a double-stranded oligonucleotide useful as an siRNA, allows for high stability of the double-stranded oligonucleotide, resulting in a long duration of action in vivo.Furthermore, in an embodiment in which the double-stranded oligonucleotide is an siRNA, it is shown herein that the presence of one or more nucleotide analogues of formula (IA) as described herein in the hybridizing region of the antisense strand oligonucleotide in a configuration other than a 5'-overhang or a 3'-overhang confers on the siRNA an improved property of inhibiting the expression of a target gene, an improved duration of its gene expression inhibition property, and an improved specificity, particularly to the targeted sequence of the antisense strand oligonucleotide.As shown in the examples described herein, the improved specificity includes an improved property of the antisense strand oligonucleotide to hybridize with a target oligonucleotide, while reducing the number of off-target events.

[0087] The examples described herein show that the double-stranded oligonucleotide, including antisense strand oligonucleotide and sense strand oligonucleotide, incorporates one or more nucleotide analogues of formula (IA) in the hybridizing region of antisense strand oligonucleotide at different positions from its 3'-overhang or 5'-overhang, can generate siRNA duplex structure with the necessary stability and specificity to ensure efficient inhibition of target gene.It is also shown herein that efficient inhibition of target gene is accompanied by a reduction in the number of off-target events.

[0088] Surprisingly, the inventors have shown that the high metabolic stability of the siRNA duplex comprising one or more nucleotide analogues of formula (IA) or (IB) of the present disclosure can be obtained when the nucleotide analogues of formula (IA) or (IB) are linked one to the other or one to the nucleotide containing ribose via conventional phosphodiester bond.Surprisingly, when the nucleotide analogues of formula (IA) or (IB) are linked in oligonucleotide via conventional phosphodiester bond to form the strand of siRNA, the obtained siRNA duplex has higher stability against nuclease degradation, thereby resulting in a longer duration of action in vivo than the same siRNA that has phosphorothioate-linked deoxyribonucleotide instead of the nucleotide analogues of formula (IA) or (IB).

[0089] This high increase in the duration of action of the nucleotide analogues of formula (IA) or (IB) in vivo is a clear technical advantage, since when linked via phosphodiester bonds, it is possible to avoid stabilization via modified internucleotides linking phosphorus groups, such as phosphorothioates. In this case, such non-conventional phosphorothioates are reminiscent of introducing chiral centers, which lead to undesirable diastereomeric mixtures of the resulting siRNA. The latter may alter siRNA binding properties, possibly causing an increase in off-target events. It is also shown herein that siRNAs having one or more compounds of formula (IA) or (IB) have excellent target gene silencing activity in vitro and in vivo, even when the siRNAs are internalized by target cells in the absence of any transfection reagent.

[0090] As described in the literature, incorporation of duplex-destabilizing modified nucleotides into the antisense strand of a double-stranded oligonucleotide results in an improved off-target profile and therefore fewer side effects of the corresponding siRNA (see, e.g., Nucleic Acid Research, 2010, 38(17), 5761-5773).

[0091] Therefore, more surprisingly, the incorporation of the nucleotide analogue of formula (IA) described herein in specific positions in antisense strand oligonucleotide, i.e. in the hybridizing region of antisense strand oligonucleotide, and in positions other than the 5'-overhang or 3'-overhang of said antisense strand oligonucleotide, shows high duplex destabilizing properties in a series of (2S,6R)-diastereomers, and shows less pronounced duplex destabilizing properties in the case of analogue (2R,6R)-diastereomers.In contrast to expectations, the siRNA that comprises the (2R,6R)-diastereoisomeric analogue of formula (IA) in the hybridizing region of antisense strand oligonucleotide shows significant reduction of off-target binding in in vitro systems, while at the same time maintaining its highly potent target inhibiting properties in vivo.

[0092] Importantly, siRNAs incorporating one or more nucleotide analogues of formula (IA) in the hybridizing region of the antisense strand oligonucleotide are free of side effects in vivo in the dose range in which these siRNAs have been shown to exert their target gene silencing effects.

[0093] Without wishing to be bound by any particular theory, the inventors believe that the nucleotide analogues of formula (IA) described herein are endowed with specific physicochemical properties, improved stability, binding properties and off-target profile conferred to oligonucleotides comprising one or more nucleotide analogues of formula (IA), as shown in the examples. These properties are particularly improved in double-stranded oligonucleotides in which the hybridizing region of the antisense strand oligonucleotide comprises its (2R,6R) diastereoisomer, such as the nucleotide analogues of formula (IA).

[0094] The nucleotide analogues of formula (IA) described in the present disclosure may also be referred to herein as "non-targeted" nucleotide analogues of formula (IA).

[0095] Antisense Strand of Double-Stranded Oligonucleotides An important aspect of the present disclosure is the provision of a double-stranded oligonucleotide comprising a sense strand oligonucleotide and an antisense strand oligonucleotide, wherein the antisense strand oligonucleotide comprises one or more nucleotide analogues of formula (IA) as described herein, and the nucleotide analogues are not the 5'-overhanging nucleotides or the 3'-overhanging nucleotides of the antisense strand oligonucleotide. In some embodiments, the double-stranded oligonucleotide comprises an siRNA that hybridizes to a selected target mRNA.

[0096] As used herein, a nucleotide contained in the antisense strand oligonucleotide of a double-stranded oligonucleotide described herein that is not contained in the 5'-overhang, if present, and not contained in the 3'-overhang, if present, may be referred to herein as a "hybridizing nucleotide" or, alternatively, as an "internal nucleotide."

[0097] Thus, a nucleotide analog of formula (IA) that is included in the antisense strand oligonucleotide of a double-stranded oligonucleotide described herein and that is not included in the 5'-overhang, if present, and not included in the 3'-overhang, if present, may also be referred to as a "hybridizing nucleotide" of formula (IA) described herein, or alternatively, an "internal nucleotide" of formula (IA) described herein.

[0098] Similarly, a nucleotide contained in a sense strand oligonucleotide of a double-stranded oligonucleotide described herein that is not contained in a 5'-overhang, if present, and not contained in a 3'-overhang, if present, may also be referred to as a "hybridizing nucleotide" as described herein, or alternatively as an "internal nucleotide."

[0099] Furthermore, if present, nucleotides located within the 5'-overhang or 3'-overhang of the antisense strand oligonucleotide of the double-stranded oligonucleotide according to the present disclosure may also be referred to herein as "overhanging" nucleotides.

[0100] In some embodiments of the double-stranded oligonucleotide, in which the antisense strand oligonucleotide comprises a 5'-overhang and / or a 3'-overhang, the antisense strand oligonucleotide does not comprise any nucleotide analogue of formula (IA) in its 5'-overhang, or in its 3'-overhang, or in both its 5'-overhang and its 3'-overhang.Then, the nucleotide analogue of formula (IA) contained in the antisense strand oligonucleotide is all the hybridizing nucleotide analogue of formula (IA) with reference to the definition of "hybridizing nucleotide" specified hereinabove.

[0101] In some other embodiments of double-stranded oligonucleotides, where the antisense strand oligonucleotide comprises a 5'-overhang and / or a 3'-overhang, the antisense strand oligonucleotide may further comprise one or more nucleotide analogs of formula (IA) in its 5'-overhang, or in its 3'-overhang, or in both its 5'-overhang and its 3'-overhang.

[0102] As used herein, the terms "hybridizing" nucleotides or "overhanging" nucleotides, as defined in this disclosure, may be used for reasons of simplifying the language and improving clarity of the configurations of the various disclosed embodiments.

[0103] The number of hybridizing nucleotide analogues of formula (IA) contained in the antisense strand of the double-stranded oligonucleotide may vary.

[0104] In some embodiments, the antisense strand oligonucleotide comprises 1-10 hybridizing nucleotide analogs of formula (IA), which as used herein includes 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotide analogs of formula (IA).

[0105] In some embodiments, the antisense strand oligonucleotide comprises from 1 to 5 hybridizing nucleotide analogs of formula (IA).

[0106] In some embodiments, the antisense strand oligonucleotides comprising one or more nucleotide analogues of formula (IA) range in length from 9 to 36 nucleotides, including lengths of 15 to 30 nucleotides, hi some embodiments, the antisense strand oligonucleotides range in length from 20 to 25 nucleotides, including lengths of 17 to 25 nucleotides.

[0107] In some embodiments, the (2R,6R) stereoisomer of the hybridizing nucleotide analogues of formula (IA) described herein is preferred.

[0108] The location of the nucleotide analogue of formula (IA) is within the antisense strand oligonucleotide.

[0109] One or more hybridizing nucleotide analogues of formula (IA) are positioned at various nucleotide positions within the hybridizing sequence of the antisense strand oligonucleotide.

[0110] Furthermore, in embodiments in which the antisense strand oligonucleotide comprises two or more hybridizing nucleotide analogs of formula (IA), the two or more hybridizing nucleotide analogs of formula (IA) may be positioned contiguously and / or non-contiguously anywhere in the hybridizing region of the antisense strand oligonucleotide, thus excluding either the 5'-overhang or the 3'-overhang, if one or both overhangs are present.

[0111] In some embodiments of the antisense strand oligonucleotide, each hybridizing nucleotide analog of formula (IA) contained therein is linked to a nucleotide different from the modified nucleotide of formula (IA). According to these embodiments, two or more hybridizing nucleotide analogs of formula (IA) are separate, i.e., distributed separately within the hybridizing sequence of the antisense strand oligonucleotide.

[0112] In some other embodiments of the antisense strand oligonucleotide, all hybridizing nucleotide analogs of formula (IA) contained therein are linked together so that they are arranged consecutively in the antisense strand oligonucleotide. In these embodiments, the hybridizing nucleotide analog of formula (IA) located at the 5'-end of the series of hybridizing nucleotide analogs is linked to a different nucleotide than the nucleotide analog of formula (IA), and the hybridizing nucleotide analog of formula (IA) located at the 3'-end of the series of hybridizing nucleotide analogs is linked to a different nucleotide than the modified nucleotide of formula (IA). According to these embodiments, two or more consecutive hybridizing nucleotide analogs of formula (IA) are said to be consecutive in the antisense strand oligonucleotide.

[0113] In yet another embodiment of the antisense strand oligonucleotide, the hybridizing nucleotide analogues of formula (IA) are arranged both as a single nucleotide analogue of formula (IA) separated from each other, and as one or more stretches of consecutive hybridizing nucleotide analogues of formula (IA).As can be easily understood, consecutive hybridizing nucleotide analogues of formula (IA) linked together are consecutive nucleotide analogues of formula (IA) contained in the antisense strand oligonucleotide.

[0114] By way of illustration, in an embodiment of a double-stranded oligonucleotide in which the antisense strand oligonucleotide has a length of 23 nucleotides and all nucleotides are paired to the sense strand oligonucleotide, the antisense strand oligonucleotide may comprise a hybridizing nucleotide analog of formula (IA) at a nucleotide position selected from the group consisting of nucleotide positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23, numbering the nucleotide positions starting from the 5' end of the antisense strand oligonucleotide.

[0115] By way of further illustration, in an embodiment of a double-stranded oligonucleotide in which the antisense strand oligonucleotide has a length of 23 nucleotides and comprises (i) a hybridizing region 21 nucleotides in length, and (ii) a 3'-overhang 2 nucleotides in length, the antisense strand oligonucleotide may comprise a hybridizing nucleotide analog of formula (IA) at a nucleotide position selected from the group consisting of nucleotide positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21, numbering the nucleotide positions starting from the 5' end of the antisense strand oligonucleotide. In some such embodiments, the antisense strand oligonucleotide further comprises two overhanging nucleotide analogs of formula (IA) in its 3'-overhang.

[0116] In another exemplary embodiment of a double-stranded oligonucleotide, wherein the antisense strand oligonucleotide has a length of 23 nucleotides, said antisense strand oligonucleotide may comprise a hybridizing nucleotide analog of formula (IA) at a nucleotide position selected from the group consisting of nucleotide positions 2, 3, 4, 5, 6, 7 or 8, numbering the nucleotide positions starting from the 5' end of the antisense strand oligonucleotide.

[0117] Antisense Strand Embodiments Containing Targeted Nucleotides In some embodiments of the antisense strand oligonucleotide of a double-stranded oligonucleotide according to the present disclosure, the antisense strand oligonucleotide further comprises one or more targeted nucleotide analogues.

[0118] In some of these embodiments, at least one of the one or more targeted nucleotide analogues comprised therein consists of a targeted nucleotide analogue of formula (IB) as described elsewhere herein.

[0119] Thus, in some embodiments of the antisense strand oligonucleotide of a double-stranded oligonucleotide according to the present disclosure, the antisense strand oligonucleotide comprises one or more targeted nucleotide analogues of formula (IB) as described elsewhere herein.

[0120] The targeted nucleotide analogue of formula (IB) may be present in various locations within the antisense strand of a double-stranded oligonucleotide according to the present disclosure.

[0121] In some embodiments, the targeted nucleotide analogue of formula (IB) is positioned in the 3'- or 5'-overhang of the antisense oligonucleotide strand, e.g., in the 3'- or 5'-overhang of the antisense strand oligonucleotide of a siRNA.

[0122] In some preferred embodiments, the targeted nucleotide analog of formula (IB) is placed in the overhang of dsRNA, for example, siRNA.For example, the targeted nucleotide analog of formula (IB) is placed in the overhang of the antisense strand oligonucleotide of siRNA, for example, in the 3'-overhang.

[0123] In some embodiments, 2-10 (e.g., 2-5) targeted nucleotide analogs of formula (IB) are present in the antisense strand oligonucleotide of a double-stranded oligonucleotide according to the present disclosure. 2-10 targeted nucleotide analogs of formula (IB), as used herein, include 2, 3, 4, 5, 6, 7, 8, 9 and 10 targeted nucleotide analogs of formula (IB).

[0124] Sense Strand Embodiments of Double-Stranded Oligonucleotides In some embodiments, the sense strand oligonucleotide of the double-stranded oligonucleotide according to the present disclosure does not contain any modified nucleotide of formula (IA).In some of these embodiments, the sense strand oligonucleotide of the double-stranded oligonucleotide described herein may contain one or more modified nucleotides different from the nucleotide analog of formula (IA), such as methoxy or fluoro modified nucleotides.

[0125] In other embodiments, the sense strand oligonucleotide of the double-stranded oligonucleotide according to the present disclosure comprises one or more nucleotide analogues. In some embodiments, the sense strand oligonucleotide of the double-stranded oligonucleotide according to the present disclosure comprises one or more nucleotide analogues of formula (IA), which may be located in its 5'-overhang, in its 3'-overhang, in its hybridizing region, or in two or more of these locations within the sense strand oligonucleotide.

[0126] In certain embodiments of the sense strand oligonucleotide of double-stranded oligonucleotide described herein, the nucleotide analog of formula (IA) is located in the 3'-overhang, the 5'-overhang, or both the 3'-overhang and the 5'-overhang of said sense strand oligonucleotide.In some of these certain embodiments, the nucleotide analog of formula (IA) is located exclusively in the 3'-overhang of the sense strand oligonucleotide of dsRNA, for example, exclusively in the 3'-overhang of the nucleic acid strand of siRNA.

[0127] In some of these particular embodiments, a nucleotide analogue of formula (IA) is positioned at (i) both the 3'-overhang and the 5'-overhang of the sense strand oligonucleotide of the siRNA.

[0128] In some embodiments, 2-10 (e.g., 2-5) nucleotide analogs of formula (IA) are present in the sense strand oligonucleotide of a double-stranded oligonucleotide according to the present disclosure. 2-10 nucleotide analogs of formula (IA), as used herein, encompass 2, 3, 4, 5, 6, 7, 8, 9 and 10 nucleotide analogs of formula (IA), which may be located in the 5'-overhang, in the 3'-overhang, in the hybridizing region, or in two or more of these locations within the sense strand oligonucleotide.

[0129] Sense Strand Embodiments Containing Targeted Nucleotides In some embodiments of the sense strand oligonucleotide of the double-stranded oligonucleotide according to the present disclosure, the sense strand oligonucleotide further comprises one or more targeted nucleotide analogues.

[0130] In some of these embodiments, at least one of the one or more targeted nucleotide analogues comprised therein consists of a targeted nucleotide analogue of formula (IB) as described elsewhere herein.

[0131] Thus, in some embodiments of the sense strand oligonucleotide of a double-stranded oligonucleotide according to the present disclosure, the sense strand oligonucleotide comprises one or more targeted nucleotide analogues of formula (IB) as described elsewhere herein.

[0132] As illustrated in the examples provided herein, the targeted nucleotide analogue of formula (IB) may be present in various locations within the sense strand oligonucleotide of a double-stranded oligonucleotide according to the present disclosure.

[0133] In some embodiments, the targeted nucleotide analogue of formula (IB) is positioned in the 3'-overhang or 5'-overhang of the sense oligonucleotide strand, for example in the 3'-overhang or 5'-overhang of the sense strand oligonucleotide of a siRNA.

[0134] In some preferred embodiments, the targeted nucleotide analog of formula (IB) is placed in the overhang of dsRNA, for example, siRNA.For example, the targeted nucleotide analog of formula (IB) is placed in the overhang, for example, 5'-overhang, of the sense strand oligonucleotide of siRNA.

[0135] In some embodiments, 2-10 (e.g., 2-5) targeted nucleotide analogs of formula (IB) are present in the sense strand oligonucleotide of a double-stranded oligonucleotide according to the present disclosure. 2-10 targeted nucleotide analogs of formula (IB), as used herein, includes 2, 3, 4, 5, 6, 7, 8, 9 and 10 targeted nucleotide analogs of formula (IB).

[0136] In some embodiments, the sense strand oligonucleotide comprises (i) one or more nucleotide analogues of formula (IA) and (ii) one or more nucleotide analogues of formula (IB). In some of these embodiments, the one or more nucleotide analogues of formula (IA) contained in the sense strand oligonucleotide comprise hybridizing nucleotide analogues of formula (IA), i.e., in embodiments where the 5'-overhang, the 3'-overhang, or both the 5'-overhang and the 3'-overhang are present, the nucleotide analogues of formula (IA) are not located in the 5'-overhang or the 3'-overhang of the sense strand oligonucleotide. In some of these embodiments, the sense strand oligonucleotide also comprises one or more nucleotide analogues of formula (IA) in the 5'-overhang, the 3'-overhang, or both the 5'-overhang and the 3'-overhang.

[0137] Other configurations of double-stranded oligonucleotides A double-stranded oligonucleotide comprising one or more nucleotide analogues of formula (IA) may also be referred to as "ribonucleic acid" or "RNA", taking into account (i) the ribose sugar moiety contained in most of the nucleotide monomer units contained therein, and (ii) the type of nucleobases contained therein. Accordingly, certain aspects of the present disclosure relate to double-stranded ribonucleic acid (dsRNA) molecules that target mRNA of interest. The dsRNA of the present invention may include modified oligonucleotides comprising one or more compounds of formula (IA).

[0138] In some embodiments, dsRNA comprises two strands, that is, the sense strand comprises a first sequence and the antisense strand comprises a second sequence, and the first strand and the second strand are sufficiently complementary to form a double-stranded structure.In some embodiments, the sense strand comprises the first sequence that is substantially complementary to the second sequence in the antisense strand or is completely complementary to it.In some embodiments, the second sequence in the antisense strand is substantially complementary to the sequence of target sequence, for example, the mRNA transcribed from target gene, or is completely complementary to it.

[0139] In some embodiments, each of the sense and antisense strands can range in length from 9 to 36 nucleotides. For example, each strand can be 12 to 30 nucleotides long, 14 to 30 nucleotides long, 15 to 30 nucleotides long, 25 to 30 nucleotides long, 27 to 30 nucleotides long, 15 to 26 nucleotides long, 15 to 23 nucleotides long, 15 to 22 nucleotides long, 15 to 21 nucleotides long, 15 to 20 nucleotides long, 15 to 19 nucleotides long, 15 to 18 nucleotides long, 15 to 17 nucleotides long, 17 to 30 nucleotides long, 17 to 23 nucleotides long, 17 to 21 nucleotides long, 17 to 19 nucleotides long, 18 to 30 nucleotides long, 18 to 26 nucleotides long, 18 to 25 nucleotides long, 18 to 23 nucleotides long, 18 to 22 nucleotides long, It may be 18-21 nucleotides in length, 18-20 nucleotides in length, 19-30 nucleotides in length, 19-25 nucleotides in length, 19-24 nucleotides in length, 19-23 nucleotides in length, 19-22 nucleotides in length, 19-21 nucleotides in length, 19-20 nucleotides in length, 20-30 nucleotides in length, 20-26 nucleotides in length, 20-25 nucleotides in length, 20-24 nucleotides in length, 20-23 nucleotides in length, 20-22 nucleotides in length, 20-21 nucleotides in length, 21-30 nucleotides in length, 21-26 nucleotides in length, 21-25 nucleotides in length, 21-24 nucleotides in length, 21-23 nucleotides in length, or 21-22 nucleotides in length. 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 of a range of nucleotide lengths 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, or 35, where 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, sense strand and antisense strand are the same number of nucleotides in length.In some embodiments, sense strand and antisense strand are different number of nucleotides in length.

[0140] Overhang In some embodiments, the dsRNA of the present disclosure comprises one or more overhangs at the 3' end, 5' end, or both ends of one or both of the sense and antisense strands.In some embodiments, the one or more overhangs improve stability with fewer phosphorothioate groups in the overhang.

[0141] In some embodiments, the overhang comprises 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, or 6 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.

[0142] 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 located 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 located at the 3' end of the antisense strand and a blunt end located at the 5' end of the antisense strand. In some embodiments, the dsRNA comprises an overhang located 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 located at the 3' end of the sense strand and a blunt end located at the 5' end of the sense strand. In some embodiments, the dsRNA comprises an overhang located at both the 3' end of the sense and antisense strands of the dsRNA.

[0143] In some embodiments, the dsRNA comprises an overhang disposed 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 disposed at the 5' end of the antisense strand and a blunt end disposed at the 3' end of the antisense strand. In some embodiments, the dsRNA comprises an overhang disposed 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 disposed at the 5' end of the sense strand and a blunt end disposed at the 3' end of the sense strand. In some embodiments, the dsRNA comprises an overhang disposed on both strands of the dsRNA.

[0144] 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 of the same length being staggered. In some embodiments, the overhang forms a mismatch with the target mRNA. In some embodiments, the overhang is complementary to the target mRNA.

[0145] 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, and the first and second sequences are substantially complementary or complementary.In some embodiments, the first and second sequences are substantially complementary or complementary to each other, forming a duplex region of the dsRNA.In some embodiments, the duplex region of the dsRNA is 9-36 nucleotide pairs in length. For example, the duplex region may be 12-30 nucleotide pairs long, 14-30 nucleotide pairs long, 15-30 nucleotide pairs long, 15-26 nucleotide pairs long, 15-23 nucleotide pairs long, 15-22 nucleotide pairs long, 15-21 nucleotide pairs long, 15-20 nucleotide pairs long, 15-19 nucleotide pairs long, 15-18 nucleotide pairs long, 15-17 nucleotide pairs long, 17-30 nucleotide pairs long, 27-30 nucleotide pairs long, 17-23 nucleotide pairs long, 17-21 nucleotide pairs long, 17-19 nucleotide pairs long, 18-30 nucleotide pairs long, 18-26 nucleotide pairs long, 18-25 nucleotide pairs long, 18-24 nucleotide pairs long, 18-23 nucleotide pairs long, 18-22 nucleotide pairs long. The length may be 18-21 nucleotide pairs, 18-20 nucleotide pairs, 19-30 nucleotide pairs, 19-25 nucleotide pairs, 19-24 nucleotide pairs, 19-23 nucleotide pairs, 19-22 nucleotide pairs, 19-21 nucleotide pairs, 19-20 nucleotide pairs, 20-30 nucleotide pairs, 20-26 nucleotide pairs, 20-25 nucleotide pairs, 20-24 nucleotide pairs, 20-23 nucleotide pairs, 20-22 nucleotide pairs, 20-21 nucleotide pairs, 21-30 nucleotide pairs, 21-26 nucleotide pairs, 21-25 nucleotide pairs, 21-24 nucleotide pairs, 21-23 nucleotide pairs, or 21-22 nucleotide pairs.In some embodiments, the duplex region of 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 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 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, or 35, where 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. When more than one dsRNA is used, the duplex region of each dsRNA may be the same or different length as the one or more additional dsRNAs.

[0146] Targeted Double-Stranded Oligonucleotide Embodiments As described above in the present disclosure, in double-stranded oligonucleotide, antisense strand oligonucleotide, sense strand oligonucleotide, or both antisense strand oligonucleotide and sense strand oligonucleotide may contain one or more targeted nucleotides.In some embodiments, antisense strand oligonucleotide, sense strand oligonucleotide, or both antisense strand oligonucleotide and sense strand oligonucleotide may contain one or more targeted nucleotide analogs of formula (IB).

[0147] Thus, in some embodiments according to the present disclosure, the targeted double-stranded oligonucleotide comprises one or more targeted nucleotide analogues of formula (IB).

[0148] In some other embodiments, the targeted nucleotide included in a targeted double-stranded oligonucleotide according to the present disclosure is selected from targeted nucleotides known in the art.

[0149] Thus, in some embodiments, a targeted double-stranded oligonucleotide according to the present disclosure comprises one or more non-targeted nucleotide analogs of formula (IA) and one or more targeted nucleotides having a structure different from formula (IB).

[0150] According to some of these embodiments, the targeted double-stranded oligonucleotide may include (i) a sense strand oligonucleotide comprising one or more targeted nucleotides, including one or more targeted nucleotide analogues of formula (IB), and (ii) a non-targeted antisense strand oligonucleotide, i.e., an antisense strand oligonucleotide that does not include targeted nucleotides. According to other of these embodiments, the targeted double-stranded oligonucleotide may include (i) a non-targeted sense strand oligonucleotide comprising no targeted nucleotides, and (ii) a targeted antisense strand oligonucleotide comprising one or more targeted nucleotides, including one or more targeted nucleotide analogues of formula (IB). According to still other embodiments, the targeted double-stranded oligonucleotide may include (i) a sense strand oligonucleotide comprising one or more targeted nucleotides, including one or more targeted nucleotide analogues of formula (IB), and a targeted antisense strand oligonucleotide comprising one or more targeted nucleotides, including one or more targeted nucleotide analogues of formula (IB).

[0151] In some embodiments, the targeted double-stranded oligonucleotide according to the present disclosure is: - a sense strand oligonucleotide comprising (i) one or more nucleotide analogues of formula (IA) and (ii) one or more nucleotide analogues of formula (IB), and - when one or both overhangs are present in the antisense strand oligonucleotide, it comprises one or more hybridizing nucleotide analogues of formula (IA) that are not located in the 5'-overhang or the 3'-overhang, i.e. the antisense strand oligonucleotide comprises one or more nucleotide analogues of formula (IA). In some embodiments, the antisense strand oligonucleotide further comprises one or more nucleotide analogues of formula (IA) in its 5'-overhang or 3'-overhang in embodiments where one or both of these overhangs are present.

[0152] As disclosed elsewhere in this disclosure, the targeted nucleotide analogue of formula (IB) has the group R3 as a cell-targeting moiety. The cell-targeting moiety is disclosed elsewhere in this disclosure.

[0153] In a particular embodiment, the one or more targeted nucleotide analogues of formula (IB) are linked to each other to form a continuous strand of these targeted nucleotide analogues at a selected end of the oligonucleotide strand, i.e., the one or more nucleotide analogues of formula (IB) are contiguous in a selected antisense strand oligonucleotide or a selected sense strand oligonucleotide.

[0154] Further embodiments of the double-stranded oligonucleotide For purposes of illustration, and without limiting the disclosure, the double-stranded oligonucleotide may also contain one or more additional nucleotides in the sense and / or antisense strand that are modified.

[0155] The modification can be selected from substitution or insertion with nucleic acid or base analogs, and chemical modification of base, sugar or phosphate moiety. The selected modification can be selected individually from 3'-terminal deoxy-thymine, 2'-O-methyl, 2'-deoxy modification, 2'-desoxy-fluoro, 2'-amino modification, 2'-alkyl modification, phosphorothioate modification, phosphoroamidate modification, 5'-phosphorothioate group modification, 5'-phosphate or 5'-phosphate mimetic modification, and cholesteryl derivative or dodecanoic acid bisdecylamide group modification, and / or the modified nucleotide can be any one of locked nucleotide, abasic nucleotide, or nucleotide containing non-natural base. One of the preferred embodiments can be that at least one modification is 2'-O-methyl, and / or at least one modification is 2'-desoxy-fluoro.

[0156] Other examples of modified oligonucleotides, as used herein, may include one or more of the following: modifications, e.g., replacement of one or both of a free phosphate and oxygen and / or one or more of a linked phosphate and oxygen; replacement of a phosphate moiety; modification or replacement of a naturally occurring base; replacement or modification of the ribose-phosphate backbone; modification of the 3' 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 either the 3' or 5' end of the RNA.

[0157] Targeted Oligonucleotides - Further Embodiments of Cell Targeting Moieties The targeted nucleotide herein can be linked to one or more ligands that target specific cells or tissues. Such ligands are also called "cell targeting moieties". Ligands include any molecular group that increases the efficiency of delivery of the resulting oligonucleotide, such as siRNA, to cells, for example, by improving specific cell targeting, improving cellular internalization of the oligonucleotide, and / or improving intracellular mRNA targeting. Ligands can be selected from the group including receptor-specific peptides, receptor-specific proteins (e.g., monoclonal antibodies or fusion proteins), and receptor-specific small molecule ligands (e.g., carbohydrates such as GalNAc groups).

[0158] The ligand may be naturally occurring or recombinant or synthetic. For example, the ligand may be a protein, carbohydrate, lipopolysaccharide, lipid, synthetic polymer, polyamine, alpha helical peptide, lectin, vitamin, or cofactor. In some embodiments, the ligand is one or more dyes, crosslinkers, polycyclic aromatic hydrocarbons, peptide conjugates (e.g., RGD peptide, antennapedia peptide, Tat peptide), polyethylene glycol (PEG), enzymes, haptens, transport / absorption enhancers, synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, or imidazole clusters), human serum albumin (HSA), or LDL.

[0159] By way of example, the ligand may be one or more proteins, glycoproteins, peptides, or molecules that have a specific affinity for the co-ligand. Such ligands may include thyrotropin, melanotropin, 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, polyglutamates, polyaspartates, lipids, cholesterol, steroids, bile acids, folates, vitamin B12, and biotin.

[0160] In some embodiments, the cell targeting moiety is one or more dyes, crosslinkers, polycyclic aromatic hydrocarbons, peptide conjugates (e.g., antennapedia peptide, Tat peptide), polyethylene glycol (PEG), enzymes, haptens, transport / absorption enhancers, synthetic ribonucleases (e.g., imidazole, bis-imidazole, histamine, or imidazole clusters), human serum albumin (HSA), or LDL.

[0161] In some embodiments, the ligand may be one or more cholesterol derivatives or lipophilic moieties. Optional lipophilic compounds include, but are not limited to, cholesterol or cholesterol derivatives; cholic acid; vitamins (e.g., folate, vitamin A, vitamin E (tocopherol), biotin, pyridoxal; bile or fatty acid conjugates, including both saturated and unsaturated (e.g., lauroyl (C12), myristoyl (C14) and palmitoyl (C16), stearoyl (C18) and docosanyl (C22), lithocholic acid and / or lithocholic acid oleylamine conjugates (lithocholic acid-oleyl, C43); polymeric backbones or scaffolds (e.g., PEG, triethylene glycol (TEG), hexaethylene glycol (HEG), poly(lactic-co-glycolic acid) (PLGA), poly(lactide ... The lipid or lipid-based ligand may include tetrahydropyridine-co-glycolide (PLG), hydrodynamic polymers; steroids (e.g., dihydrotestosterone); terpenes (e.g., triterpenes); cationic lipids or peptides; and / or lipids or lipid-based molecules. Such lipids or lipid-based molecules may bind to serum proteins, such as human serum albumin (HSA). The lipid-based ligand may be used to modulate (e.g., control) the binding of the conjugate to a target tissue. For example, lipids or lipid-based ligands that bind more strongly to HSA are expected to be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. The target tissue may be the liver, such as liver parenchymal cells.

[0162] The polyamino acid, transferrin, cell targeting ligand or moiety can also be an antibody that binds to a receptor in a specific cell type, such as a hepatocyte. Exemplary cell receptor-specific monoclonal antibodies are those disclosed by Xia et al. (2009, Mol Pharm, 63(3):747-751); Cuellar et al. (2015, Nucleic Acids Research, 43(2):1189-1203); Baumer 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).

[0163] The cell targeting ligand or moiety may also be a monovalent or polyvalent (e.g., trivalent) GalNAc group, e.g., as described by 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, 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).

[0164] Preparation of modified dsRNA The dsRNA of the present disclosure may be chemically / physically linked to one or more ligands, moieties or conjugates. In some embodiments, the dsRNA is conjugated / attached to one or more ligands via a linker. Any linker known in the art can be used, such as a multivalent branched linker. Conjugating a ligand to the dsRNA can change its distribution, enhance its cellular absorption and / or targeting to a specific tissue and / or uptake by one or more specific cell types (e.g., liver cells), and / or enhance the lifespan of the dsRNA agent. In some embodiments, a hydrophobic ligand is conjugated to the dsRNA to facilitate direct penetration through cell membranes and / or uptake by cells (e.g., liver cells).

[0165] In some embodiments of dsRNA conjugate, one or more nucleotides can comprise a group to which targeting moiety is attached, for example, one or more nucleotides comprise a group to which targeting moiety is attached, where targeting moiety is optionally covalently linked to nucleotide backbone via linking group.According to these embodiments, one or more nucleotides of dsRNA are conjugated to a group to which targeting moiety is attached, which comprises a targeting moiety, where targeting moiety can be a ligand (e.g., cell-permeable moiety or drug) that enhances intracellular delivery of composition.

[0166] The ligand-conjugated dsRNAs and sequence-specific linked nucleosides and nucleotides having attached ligand molecules of the present disclosure can be assembled by any method known in the art, such as by assembly on a suitable DNA synthesizer utilizing standard nucleotide precursors, or nucleotide or nucleoside conjugate precursors that already have a linking moiety, ligand-nucleotides, or precursors conjugated to nucleosides that already have a ligand molecule, or nucleoside and non-liganded building blocks.

[0167] The ligand-conjugated dsRNA of the present disclosure can be synthesized by any method known in the art, such as by using a dsRNA with pendant reactive functionalities attached, such as those obtained by attachment of a linking molecule on the dsRNA. In some embodiments, the reactive oligonucleotide can be directly reacted with a commercially available ligand, a synthesized ligand with any of a variety of protecting groups attached, or a ligand with a linking moiety attached. In some embodiments, the method facilitates the synthesis of the ligand-conjugated dsRNA by using a nucleoside monomer that is appropriately conjugated with a ligand and may also be attached to a solid support material. In some embodiments, a dsRNA with an aralkyl ligand attached to the 3' end of the dsRNA is prepared by first covalently attaching a monomer building block to a controlled pore glass support via an aminoalkyl group; then, a nucleotide is attached to the solid support-bound monomer building block via standard solid-phase synthesis techniques. The monomer building block can be a nucleoside or other organic compound compatible with solid-phase synthesis.

[0168] Precursors of nucleotide analogues of formula (IA) and (IB) The nucleotide analogues of formula (IA) and (IB) can be synthesized by starting with precursor compounds, such compounds are sometimes referred to herein as nucleotide analogue precursors.

[0169] Both the nucleotide analogues of formula (IA) and (IB) can be synthesized from their corresponding precursor compounds of formula (II) below.

[0170] As will be readily understood, the nucleotide analogues of formula (IA) can be prepared by starting with a nucleotide analogue precursor of formula (II-A) below, where the group R3 does not comprise a cell-targeting moiety. Additionally, the nucleotide analogues of formula (IB) can be prepared by starting with a nucleotide analogue precursor of formula (II-B) below, where the group R3 comprises a cell-targeting moiety.

[0171] The present disclosure also describes nucleotide analog precursors of general formula (II). [ka] During the ceremony: - B is a heterocyclic nucleobase; - P1 and P2 are each independently H, a reactive phosphorus group or a protecting group; - Y is O, NH, NR1 or NC(=O)-R1, where R1 is: (C) optionally unsubstituted or substituted by one or more groups selected from halogen atoms, (C1-C6) alkyl groups, (C3-C8) cycloalkyl groups, (C3-C14) heterocycles, (C6-C14) aryl groups, (C5-C14) heteroaryl groups, -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; 1 ~C 20 ) an alkyl group, J is O or S; Each of Z1 and Z2 is independently H, optionally substituted with one or more groups selected from halogen atoms and (C1-C6) alkyl groups; a (C3-C8)cycloalkyl group, optionally substituted with one or more groups selected from halogen atoms and (C1-C6)alkyl groups, or ·Group-[C(=O)]m-R2-(O-CH 2 -CH 2 )p-R3 (wherein, m is an integer having a value of 0 or 1; p is an integer ranging from 0 to 10; R2 is a (C1-C20) alkylene group optionally substituted with 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; K is O or S; Each of Z3 and Z4 is independently H or a (C1-C6) alkyl group optionally substituted with one or more groups selected from halogen atoms and (C1-C6) alkyl groups; R3 is selected from the group consisting of 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; - X1 and X2 each independently represent a hydrogen atom or a -(C1-C6)alkyl group; Each of Ra, Rb, Rc and Rd is independently H or a (C1-C6) alkyl group.

[0172] For the purposes of this disclosure, compounds of formula (II) are included in the term "nucleotide precursor". For the purposes of this disclosure, compounds of formula (II) in which the group R3 is present and which represent a cell targeting moiety are included in the term "targeted nucleotide precursor".

[0173] The compounds of formula (I), e.g., (IA), and (IB), and (II) disclosed herein include their isomers, e.g., stereoisomers, including their (2S,6R) stereoisomers and their (2R,6R) stereoisomers, as specifically set forth in the following formulas, which identify the numbering of positions and chiral centers of the compounds of formula (I) and (II), and are exemplified below for the compounds of formula (I) and (II): [ka]

[0174] As will be shown in the examples provided 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) are endowed with the ability to generate siRNAs that enable superior inhibition of target mRNA.

[0175] However, in some embodiments of double-stranded oligonucleotides of the present disclosure, such as siRNA, stereoisomers of the nucleotide analogues of formula (IA) having the configuration (2R,6R) are preferred.

[0176] As shown in some examples herein, the use of a nucleotide analog of formula (IA) in its (2R,6R) stereoisomer in a double-stranded oligonucleotide results in superior in vivo efficacy of said double-stranded oligonucleotide, e.g., siRNA, when the nucleotide analog (IA) is incorporated as a hybridizing nucleotide analog as specified herein, e.g., when incorporated in the hybridizing region of an antisense strand oligonucleotide of an siRNA, and in some embodiments, when incorporated in the hybridizing region of a sense strand oligonucleotide of an siRNA, compared to the corresponding (2S,6R) stereoisomer.

[0177] In some embodiments of compounds of formula (II), sometimes referred to herein as "dioxane analogs," Y is O. Such nucleotide precursor embodiments of the present disclosure are referred to as pre-lB1, where B is as defined in formula (II), e.g., when B comprises a thymidinyl group, they are referred to as pre-lT1.

[0178] In other embodiments of compounds of formula (II), sometimes referred to herein as "morpholino analogs," Y is NH, NR1, or NC(=O)R1.

[0179] In the morpholino analogs of formula (II), the nitrogen atoms are preferably functionalized to improve the properties of oligonucleotides containing the resulting morpholino analogs, and particularly siRNAs containing the resulting morpholino analogs.

[0180] In some embodiments of the compound of formula (II), the compound is a morpholino analog of the present disclosure that does not include a cell targeting moiety. According to these embodiments, the group R3, when present, does not represent a cell targeting moiety.

[0181] Thus, in some preferred embodiments of the morpholino analog of formula (II), Y is NH, NR1, or NC(=O)-R1, where R1 is as defined with respect to general formula (II).

[0182] In some embodiments where Y is NR1, R1 is: (C1-C20) alkyl groups optionally substituted with one or more groups selected from halogen atoms, (C1-C6) alkyl groups, (C3-C8) cycloalkyl groups, (C3-C14) heterocycles, (C6-C14) aryl groups, (C5-C14) heteroaryl groups, -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, J is O or S; Each of Z1 and Z2 is independently H or optionally substituted with one or more groups selected from halogen atoms and (C1-C6) alkyl groups; or a (C3-C8)cycloalkyl group optionally substituted with one or more groups selected from halogen atoms and (C1-C6)alkyl groups, ·Group-[C(=O)]m-R2-(O-CH 2 -CH 2 )p-R3 (wherein, m is an integer having a value of 0 or 1; p is an integer ranging from 0 to 10; R2 is a (C1-C20) alkylene group optionally substituted with 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; K is O or S; Each of Z3 and Z4 is independently H or a (C1-C6) alkyl group optionally substituted with one or more groups selected from halogen atoms and (C1-C6) alkyl groups; R3 is selected from the group consisting of a hydrogen atom, a (C1-C6)-alkyl, a (C1-C6)-alkoxy, a (C3-C8)-cycloalkyl group, a (C3-C14)-heterocycle, a (C6-C14)-aryl group or a (C5-C14)-heteroaryl group; X1 and X2 are each independently a hydrogen atom or a (C1-C6) alkyl group, and Ra, Rb, Rc and Rd are each independently H or a (C1-C6) alkyl group.

[0183] As intended herein, a (C1-C20) alkyl group may be either an unsubstituted alkyl group or a substituted alkyl group, examples of which include C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19 and C20 alkyl groups.

[0184] As intended herein, a (C1-C6) alkyl group may be either an unsubstituted alkyl group or a substituted alkyl group, examples of which include C1, C2, C3, C4, C5 and C6 alkyl groups.

[0185] As intended herein, a (C-C) cycloalkyl group may be either an unsubstituted cycloalkyl group or a substituted cycloalkyl group, examples of which include C, C, C, C, C, C, and C cycloalkyl groups.

[0186] As intended herein, a (C3-C14) heterocycle may be either an unsubstituted or substituted heterocycle, examples of which include C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13 and C14 heterocycles.

[0187] As intended herein, a (C6-C14) aryl group may be either an unsubstituted aryl group or a substituted aryl group, examples of which include C6, C7, C8, C9, C10, C11, C12, C13 and C14 aryl groups.

[0188] As intended herein, a (C5-C14) heteroaryl group may be either an unsubstituted or substituted heteroaryl group, examples of which include C5, C6, C7, C8, C9, C10, C11, C12, C13 and C14 heteroaryl groups.

[0189] In some embodiments of compounds of formula (II) where 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 general formula (II).

[0190] In some of these embodiments where Y is NR1, R1 is an unsubstituted (C1-C20) alkyl group.

[0191] In some embodiments where Y is NR1, R1 is an unsubstituted (C1-C16) alkyl group, examples of which include alkyl groups selected from the group including methyl, isopropyl, butyl, octyl, and hexadecyl, and P1, P2, Ra, Rb, Rc, Rd, X1, X2, and B have the same meanings as defined for general formula (II).

[0192] In some embodiments where 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 general formula (II). Such nucleotide precursor embodiments in the present disclosure are referred to as pre-1B2, with B having the same meaning as in general formula (II); for example, such nucleotide precursor embodiments in the present disclosure are referred to as pre-1T2 when B is a thymidinyl group.

[0193] In some embodiments where 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 with respect to general formula (II). Such nucleotide precursor embodiments in the present disclosure are referred to as pre-1B3, with B having the same meaning as in general formula (II); for example, such nucleotide precursor embodiments in the present disclosure are referred to as pre-1T3 (wherein B is composed of a thymidinyl group), pre-1U3 (wherein B is composed of a uracil group), pre-1G3 (wherein B is composed of a guanyl group), pre-1C3 (wherein B is composed of a cytosyl group), and pre-1A3 (wherein B is composed of an adenyl group).

[0194] In some embodiments where 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 general formula (II). Such nucleotide precursor embodiments in the present disclosure are referred to as pre-1B6, where B has the same meaning as in general formula (II); for example, such nucleotide precursor embodiments in the present disclosure are referred to as pre-1T6, where B is a thymidinyl group.

[0195] In some embodiments where 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 general formula (II). Such nucleotide precursor embodiments in the present disclosure are referred to as pre-1B7, where B has the same meaning as in general formula (II); for example, such nucleotide precursor embodiments in the present disclosure are referred to as pre-1T7, where B is a thymidinyl group.

[0196] In some embodiments where 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 general formula (II). Such nucleotide precursor embodiments in the present disclosure are referred to as pre-1B8, where B has the same meaning as in general formula (II); for example, such nucleotide precursor embodiments in the present disclosure are referred to as pre-1T8, where B is a thymidinyl group.

[0197] In a further embodiment of the compound of formula (II) where Y is NR1, R1 is a (C1-C20) alkyl group substituted as defined in general formula (II), examples of which include a C1, C2 or C3 alkyl group substituted as defined in general formula (II).

[0198] In some of these further embodiments, R1 is a (C1-C20) alkyl group substituted with one or more groups selected from 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 general formula (II).

[0199] 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 general formula (II).

[0200] In some embodiments of compounds of formula (II) where Y is NR1, R1 is a (C1-C20) alkyl group substituted with a (C6-C14) aryl group. These embodiments include compounds of formula (II) where Y is NR1 and R1 is a methylene group substituted with an aryl group. These embodiments also include compounds of formula (II) where Y is NR1 and R1 is a (C1-C20) alkyl group substituted with a phenyl group.

[0201] In some embodiments of the compound of formula (II) where 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 general formula (II). Such an embodiment of the nucleotide precursor in the present disclosure is called pre-1B5, where B has the same meaning as in general formula (II); for example, such an embodiment of the nucleotide precursor in the present disclosure is called pre-1T5, where B is a thymidinyl group.

[0202] In a further embodiment of the compound of formula (II) where Y is NR1, R1 is a (C3-C8)cycloalkyl group optionally substituted with one or more groups selected from 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 general formula (II).

[0203] In some of these further embodiments of compounds of Formula (II) where Y is NR1, R1 is cyclohexyl.

[0204] In some of these further embodiments of compounds of formula (II) where Y is NR1, R1 is unsubstituted cyclohexyl, Ra, Rb, Rc, Rd, X1, and X2 are each a hydrogen atom, and P1 and P2 are as defined for general formula (II).

[0205] An embodiment of such a nucleotide precursor in the present disclosure is referred to as pre-lB4, where B has the same meaning as in general formula (II); for example, an embodiment of such a nucleotide precursor in the present disclosure is referred to as pre-lT4, where B consists of a thymidinyl group.

[0206] In some other embodiments of the morpholino analog of formula (II), Y is NC(=O)-R1, where R1 is a (C1-C20) alkyl group optionally substituted with one or more groups selected from 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; J is O or S; Each of Z1 and Z2 is independently H or a (C1-C6) alkyl group optionally substituted with one or more groups selected from halogen atoms and (C1-C6) alkyl groups; R1 is a (C3-C8)cycloalkyl group optionally substituted with one or more groups selected from halogen atoms or (C1-C6)alkyl groups; P1, P2, Ra, Rb, Rc, Rd, X1, X2 and B have the same meanings as defined for general formula (II).

[0207] In some embodiments where Y is NC(=O)-R1, R1 is an optionally substituted (C1-C20) alkyl group, such as 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 general formula (II).

[0208] According to some of these embodiments where Y is NC(=O)-R1, R1 is selected from the group including methyl and pentadecyl groups, and P1, P2, Ra, Rb, Rc, Rd, X1, X2 and B have the same meanings as defined for general formula (II).

[0209] These embodiments include compounds of formula (II) in which Y is NC(=O)-R1, R1 is a methyl group, Ra, Rb, Rc, Rd, X1, and X2 each represent a hydrogen atom, and B, P1, and P2 are as defined in general formula (II).

[0210] The embodiment of such a nucleotide precursor in the present disclosure is called pre-1B9, B has the same meaning as in general formula (II); for example, the embodiment of such a nucleotide analog in the present disclosure is called pre-1T9, B is composed of a thymidinyl group. These embodiments also include compounds of formula (II) where Y is NC(=O)-R1, R1 is a pentadecyl group, Ra, Rb, Rc, Rd, X1, X2 each represent a hydrogen atom, and B, P1 and P2 are as defined in general formula (II). The embodiment of such a nucleotide precursor in the present disclosure is called pre-1B10, B has the same meaning as in general formula (II); for example, the embodiment of such a nucleotide precursor in the present disclosure is called pre-1T10, B is composed of a thymidinyl group.

[0211] In the compound of formula (II), B is a heterocyclic nucleobase moiety. The term "heterocyclic nucleobase" as used herein refers to an optionally substituted nitrogen-containing heterocycle covalently linked to a dioxane or morpholino ring. In some embodiments, the heterocyclic nucleobase can be selected from an optionally substituted purine base and an optionally substituted pyrimidine base. The term "purine base" is used herein in its ordinary sense as understood by those skilled in the art, and includes its tautomers. Similarly, the term "pyrimidine base" is used herein in its ordinary sense as understood by those skilled in the art, and includes its tautomers. A non-limiting list of optionally substituted purine bases includes purine, adenine, guanine, hypoxanthine, xanthine, alloxanthine, 7-alkylguanine (e.g., 7-methylguanine), theobromine, caffeine, uric acid, and isoguanine. Examples of pyrimidine bases include, but are not limited to, cytosine, thymine, uracil, 5,6-dihydrouracil, and 5-alkylcytosine (e.g., 5-methylcytosine). Other non-limiting examples of heterocyclic bases include diaminopurine, 8-oxo-N 6 Alkyl adenines (e.g., 8-oxo-N 6 methyladenine), 7-deazaxanthine, 7-deazaguanine, 7-deazaadenine, N 4 N 4 Ethanocytosine, 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. 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 is optionally substituted with an amine or enol protecting group. In some embodiments, B is selected from the group including pyrimidine, substituted pyrimidine, purine, and substituted purine, and their amino groups, if present, are optionally protected by a protecting group.

[0212] In a preferred embodiment, B is selected from the group including adenine, thymine, uracil, guanine and cytosine (i.e. adenyl, thyminyl, uracil, guanyl and cytosyl groups). Adenine, guanine and cytosine are optionally protected by amine protecting groups. Amine protecting groups include acyl groups, such as benzoyl, phenylacetyl and isobutyryl-protecting groups, or formamidine protecting groups, such as N,N-dimethyl-formamidine.

[0213] As already mentioned, in the compound of formula (II), the groups P1 and P2 are each independently a hydrogen atom, a reactive phosphorus group or a protecting group.

[0214] "Reactive phosphorus group", as used herein, refers to a phosphorus-containing group in a nucleotide or nucleotide analog unit that can react with a hydroxyl or amine group 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 linkage that links the first nucleotide or first nucleotide analog unit to the second nucleotide or second nucleotide analog unit.

[0215] In some embodiments, the reactive phosphorus group can be selected from the group consisting of phosphoramidites, H-phosphates, alkyl phosphates, phosphates or phosphate mimetics, examples of which include, but are not limited to: natural phosphates, phosphorothioates, phosphorodithioates, boranophosphates, boranothiophosphates, phosphates, halogen substituted phosphonates and phosphates, phosphoramidates, phosphodiesters, phosphotriesters, thiophosphodiesters, thiophosphotriesters, diphosphates and triphosphates. Protecting groups include hydroxyl, amine and phosphoramidite protecting groups, including acetyl (Ac), benzoyl (Bzl), benzyl (Bn), isobutyryl (iBu), phenylacetyl, benzyloxymethyl acetal (BOM), beta-methoxyethoxymethyl ether (MEM), methoxymethyl ether (MOM), p-methoxybenzyl ether (PMB), methylthiomethyl ether, pivaloyl (Piv), tetrahydropyranyl (THP), triphenylmethyl (Trt), methoxytrityl [(4-methylphenyl)-2-phenylpropionyl], phenylacetyl, benzyloxymethyl ether ... [(4-methylphenyl)-2-phenylpropionyl], phenylacetyl, benzyloxymethyl ether (BOM), beta-methoxyethoxymethyl ether (MEM), methoxymethyl ether [(4-methylphenyl)-2-phenylpropionyl], phenylacetyl, benzyloxymethyl ether (BOM), beta-methoxyethoxymethyl ether (MEM), methoxymethyl ether [(4-methylphenyl)-2-phenylpropionyl], phenylacetyl, benzyloxymethyl ether (BOM), [bis-(4-methoxyphenyl)diphenylmethyl] (MMT), dimethoxytrityl, [bis-(4-methoxyphenyl)phenylmethyl (DMT), trimethylsilyl ether (TMS), tert-butyldimethylsilyl ether (TBDMS), triisopropylsilyloxymethyl ether (TOM), tri-isopropylsilyl ether (TIPS), methyl ether, ethoxyethyl ether (EE), N,N-dimethylformamidine and 2-cyanoethyl (CE).

[0216] In some embodiments of the compound of formula (II) (wherein Y, B, Ra, Rb, Rc, Rd, X1 and X2 are as defined for general formula (II)), 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.

[0217] In some embodiments of the compound of formula (II) (wherein Y, B, Ra, Rb, Rc, Rd, X1 and X2 are as defined for general formula (II)), one of P1 and P2 is a 2-cyanoethyl-N,N-diisopropylphosphoramidite group, and the other of P1 and P2 is a protecting group.

[0218] In some embodiments of the compound of formula (II) (wherein Y, B, Ra, Rb, Rc, Rd, X1 and X2 are as defined for general formula (II)), 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.

[0219] Further, each of Ra, Rb, Rc and Rd is independently H or a (C1-C6) alkyl group, preferably H or an unsubstituted (C 1 ~C 6 ) alkyl group.

[0220] A (C1-C6) alkyl group, as used herein, encompasses alkyl groups selected from the group including C1, C2, C3, C4, C5 and C6 alkyl groups.

[0221] In a most preferred embodiment, both X1 and X2 represent a hydrogen atom.

[0222] In a most preferred embodiment, Ra, Rb, Rc and Rd all represent a hydrogen atom.

[0223] Embodiments of compounds of formula (II) containing targeted nucleotide analogues As hereinbefore specified, the present disclosure relates to a compound of formula (II), - B is a heterocyclic nucleobase; - P1 and P2 are each independently H, a reactive phosphorus group or a protecting group; - Y is NR1, R1 is a group -[C(=O)]m-R2-(O-CH 2 -CH2 )p-R3 (wherein, m is an integer having a value of 0 or 1; p is an integer ranging from 0 to 10; R2 is a (C1-C20) alkylene group optionally substituted with 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; K is O or S; Each of Z3 and Z4 is independently H or a (C1-C6) alkyl group optionally substituted with one or more groups selected from halogen atoms and (C1-C6) alkyl groups; R3 is a cell targeting moiety; - X1 and X2 each independently represent a hydrogen atom or a (C1-C6) alkyl group; - each of Ra, Rb, Rc and Rd is independently H or a (C1-C6) alkyl group; Includes:

[0224] These compounds of formula (II) are included in a more general family of compounds that may be referred to in the present disclosure as "targeted nucleotide precursors". In the present disclosure, compounds of formula (II) in which the group R3 is present and represents a cell targeting moiety may be referred to as "targeted nucleotide precursors of formula (II)" or "targeted nucleotide precursors (II)".

[0225] Compounds of formula (II) that do not include the group R3 representing a cell targeting moiety are not targeted nucleotide precursors and are referred to in the present disclosure as "non-targeted nucleotide precursors of formula (II)" or "non-targeted nucleotide precursors (II)."

[0226] In some embodiments of the targeted nucleotide precursor of formula (II), R is a group -[C(=O)]-R-(O-CH 2 -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 described in the general definition of the compound of formula (II). In some embodiments, R2 is an ethylene group, and X1 and X2 are both hydrogen atoms. In other parts of these embodiments, R2 is a pentylene group, and X1 and X2 are both hydrogen atoms. In some embodiments, R2 is a (C12) alkylene, and X1 and X2 are both hydrogen atoms.

[0227] In some embodiments of the compound of Formula (II), R is a group -[C(=O)]-R-(O-CH 2 -CH 2 )p-R3, m is 0, p is an integer selected from the group consisting of 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 described in the general definition of the compound of formula (II). 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.

[0228] In some embodiments of the compound of Formula (II), R is a group -[C(=O)]-R-(O-CH 2 -CH 2)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 described in the general definition of the compound of formula (II). In some of these embodiments, R2 is butylene, X1 and X2 each represent a hydrogen atom, and B, P1, P2, Ra, Rb, Rc, and Rd are as defined for general formula (II). In a further part of these embodiments, R2 is (C11) alkylene, X1 and X2 both represent a hydrogen atom, and B, P1, P2, Ra, Rb, Rc, and Rd are as defined for general formula (II). In some further of these embodiments, R2 is methylene, X1 and X2 both represent a hydrogen atom, and B, P1, P2, Ra, Rb, Rc and Rd are as defined for general formula (II).

[0229] R1 is the group -[C(=O)]m-R2-(O-CH 2 -CH 2 In some embodiments of the compound of formula (II) where m is 1, p is selected from the group of integers consisting of 1 and 2, R is a cell-targeting moiety, and R, B, P, P, Ra, Rb, Rc, Rd, X, X are as described in the general definition of the compound of formula (II). In some of these embodiments, R is a methylene group, p is 2, R is a cell-targeting moiety, and B, P, P, Ra, Rb, Rc, Rd, X, X are as defined for general formula (II). In other of these embodiments, R is a methylene group, p is 1, R is a cell-targeting moiety, and B, P, P, Ra, Rb, Rc, Rd, X, X are as defined for general formula (II).

[0230] In some embodiments of the compounds of Formula (I), Ra, Rb, Rc and Rd are hydrogen atoms.

[0231] In general, the group R3 includes any cell targeting moiety known in the art, including any cell targeting moiety specified in this disclosure, including, for example, those cell targeting moieties specified for the description of targeted oligonucleotides in this disclosure.

[0232] In some embodiments of the compound of Formula (II), R3 is a group represented by Formula (III): [ka] (In the formula, A1, A2 and A3 are OH or OC(=O)-R4, R4 being a (C1-C6)-alkyl or (C6-C10)-aryl group. has.

[0233] A4 is OH, OC(=O)-R4, NHC(=O)-R5, where R4 is as defined above and R5 is a (C1-C6)-alkyl group optionally substituted with a halogen atom.

[0234] In some preferred embodiments, A1, A2 and A3 are OC(=O)-R4, where R4 is a (C1-C6)-alkyl or (C6-C10)-aryl group.

[0235] In some preferred embodiments, A1, A2, and A3 are OC(=O)-R4, where R4 is a methyl or phenyl group.

[0236] In some preferred embodiments, A1, A2, and A3 are OC(=O)-R4, where R4 is methyl.

[0237] In some preferred embodiments, A4 is OC(=O)-R4 or NHC(=O)-R5, where R4 is a (C1-C6) alkyl or (C6-C10)-aryl group and R5 is a (C1-C6)-alkyl group optionally substituted with a halogen atom.

[0238] In some preferred embodiments, A1, A2, and A3 are OC(=O)-R4, where R4 is methyl, and A4 is OC(=O)-R4 or NHC(=O)-R5, where each of R4 and R5 is methyl.

[0239] In some preferred embodiments, R3 is 3,4,6-tri-O-acetyl-DN-acetylgalactosylamine of formula (III-A): [ka]

[0240] The present disclosure also relates to oligonucleotides comprising one or more nucleotide analogues incorporated into the oligonucleotide by using a nucleotide analogue precursor which is a compound of formula (II) as specified herein.

[0241] The present disclosure relates to single-stranded and double-stranded oligonucleotides, particularly siRNAs, comprising one or more hybridizing nucleotide analogs of formula (IA), as detailed elsewhere herein, where the hybridizing nucleotide analogs of formula (IA), if present, are not in the 5'-overhang or 3'-overhang of the antisense strand oligonucleotide, and optionally also to one or more nucleotide analogs of formula (IB).

[0242] The present disclosure relates to single-stranded and double-stranded oligonucleotides, particularly siRNAs, which contain one or more hybridizing nucleotide analogs of formula (IA) in the hybridizing region of the antisense strand oligonucleotide, and optionally contain one or more hybridizing nucleotide analogs (IA) in the sense strand oligonucleotide.Furthermore, the double-stranded oligonucleotides, particularly siRNAs, may contain one or more non-targeting nucleotide analogs in the 5'-overhang, 3'-overhang, or both the 5'-overhang and 3'-overhang of the sense and antisense strand oligonucleotides, if present, and may optionally contain one or more targeted nucleotide analogs (IB) in the 5'-overhang, 3'-overhang, or both the 5'-overhang and 3'-overhang of the sense and antisense strand oligonucleotides.

[0243] Nucleotide analogues of formulae (IA) and (IB) are described in further detail below.

[0244] Nucleotide analogues of formula (IA) and (IB) The compounds of formula (II) disclosed herein are nucleotide analog building blocks, also referred to as "nucleotide analog precursors", that are designed to be included as monomeric units of oligomeric compounds, particularly oligonucleotides, such as double-stranded RNA ("dsRNA") oligomers, particularly siRNAs, etc. The incorporation of the nucleotide analog precursors described herein as compounds of formula (II) into an oligonucleotide results in the presence of the corresponding monomeric units described herein as compounds of formula (I) in said oligonucleotide, which are either (i) nucleotide analogs of formula (IA) or (ii) nucleotide analogs of formula (IB).

[0245] As further described below, the nucleotide analogue of formula (IA) comprises a compound of formula (I) where the group R3 does not comprise a cell-targeting moiety, and the nucleotide analogue of formula (IB) comprises a compound of formula (I) where the group R3 comprises a cell-targeting moiety.

[0246] The present disclosure also provides a compound of formula (I): [ka] [In the formula, - B is a heterocyclic nucleobase; - one of L1 and L2 is an internucleoside linking group linking the compound of formula (I) to a nucleotide residue, and the other of L1 and L2 is H, a protecting group, a phosphorus moiety or an internucleoside linking group linking the compound of formula (I) to a nucleotide residue, - Y is O, NH, NR1 or NC(=O)-R1, where R1 is: (C1-C20) alkyl groups optionally substituted with one or more groups selected from halogen atoms, (C1-C6) alkyl groups, (C3-C8) cycloalkyl groups, (C3-C14) heterocycles, (C6-C14) aryl groups, (C5-C14) heteroaryl groups, -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, J is O or S; Each of Z1 and Z2 is independently H, optionally substituted with one or more groups selected from halogen atoms and (C1-C6) alkyl groups; a (C3-C8)cycloalkyl group optionally substituted with one or more groups selected from halogen atoms and (C1-C6)alkyl groups, ·Group-[C(=O)]m-R2-(O-CH 2 -CH 2 )p-R3 (wherein, m is an integer having a value of 0 or 1; p is an integer ranging from 0 to 10; R2 is a (C1-C20) alkylene group optionally substituted with 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; K is O or S; Each of Z3 and Z4 is independently H or a (C1-C6) alkyl group optionally substituted with one or more groups selected from halogen atoms and (C1-C6) alkyl groups; R3 is selected from the group consisting of 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; - X1 and X2 each independently represent a hydrogen atom or a (C1-C6) alkyl group; - each of Ra, Rb, Rc and Rd is independently H or a (C1-C6) alkyl group; The present invention also relates to nucleotide analogues of the formula:

[0247] In some preferred embodiments of the oligonucleotides described herein, in the compounds of formula (I), Y is O.

[0248] In some other preferred embodiments of the oligonucleotides described herein, in the compounds of formula (I), Y is NR1 or NC(=O)-R1, where R1 is as defined for general formula (I).

[0249] In some embodiments where Y is NR1, R1 is a (C1-C20) alkyl group optionally substituted with one or more groups selected from 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; J is O or S; Each of Z1 and Z2 is independently H or a (C1-C6) alkyl group optionally substituted with one or more groups selected from halogen atoms and (C1-C6) alkyl groups, in the form of a base or in the form of an addition salt with an acid.

[0250] In some of these embodiments where 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 general formula (I), or a pharma- ceutically acceptable salt thereof.

[0251] In some embodiments where Y is NR1, R1 is an unsubstituted (C1-C16) alkyl group, examples of which include alkyl groups selected from the group including methyl, isopropyl, butyl, octyl, and hexadecyl, and L1, L2, Ra, Rb, Rc, Rd, X1, X2, and B have the same meanings as defined for general formula (I).

[0252] In some embodiments where 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 general formula (I).

[0253] In some embodiments where 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 general formula (I).

[0254] In some embodiments where Y is NR1, R1 is a methyl group substituted with a phenyl group, and L1, L2, Ra, Rb, Rc, Rd, X1, X2, R2, R3 and B have the same meanings as defined for general formula (I), or a pharma- ceutically acceptable salt thereof.

[0255] In some embodiments where 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 with respect to general formula (I).

[0256] In some embodiments where 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 with respect to general formula (I).

[0257] In some embodiments where 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 general formula (I).

[0258] In a further embodiment of the compound of formula (I) where Y is NR1, R1 is a (C1-C20) alkyl group substituted as defined in general formula (I), examples of which include a C1, C2 or C3 alkyl group substituted as defined in general formula (I).

[0259] In some of these further embodiments, R1 is a (C1-C20) alkyl group substituted with one or more groups selected from 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.

[0260] In some of these further embodiments, R1 is a (C1-C20) alkyl group substituted with a (C6-C14) aryl group, and L1, L2, Ra, Rb, Rc, Rd, X1, X2 and B have the same meanings as defined for general formula (I).

[0261] In some embodiments of compounds of formula (I) where Y is NR1, R1 is a (C1-C20) alkyl group substituted with a (C6-C14) aryl group. These embodiments include compounds of formula (I) where Y is NR1 and R1 is a methylene group substituted with an aryl group. These embodiments also include compounds of formula (I) where Y is NR1 and R1 is a (C1-C20) alkyl group substituted with a phenyl group.

[0262] In some embodiments of the compound of formula (I) where Y is NR1, R1 is a methyl group substituted with an unsubstituted phenyl group, Ra, Rb, Rc, and Rd are each a hydrogen atom, and L1 and L2 are as defined in general formula (I).

[0263] In a further embodiment of the compound of formula (I) where Y is NR1, R1 is a (C3-C8)cycloalkyl group optionally substituted with one or more groups selected from 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 general formula (I).

[0264] In some of these further embodiments of compounds of formula (I) where 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 general formula (I) or a pharma- ceutically acceptable salt thereof.

[0265] In some of these further embodiments of compounds of formula (I) where Y is NR1, R1 is unsubstituted cyclohexyl, Ra, Rb, Rc, Rd, X1, and X2 are each a hydrogen atom, and L1 and L2 are as defined for general formula (I).

[0266] In some other embodiments of the oligonucleotide of formula (I), Y is NC(=O)-R1, R1 is a (C1-C20) alkyl group, R1 is selected from the group including methyl and pentadecyl, and L1, L2, Ra, Rb, Rc, Rd, X1, X2, R2, R3 and B have the same meaning as defined for general formula (I) or a pharma- ceutically acceptable salt thereof.

[0267] In some other embodiments of the oligonucleotide of formula (I), Y is NC(=O)-R1, where R1 is a (C1-C20) alkyl group optionally substituted with one or more groups selected from 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; J is O or S; Each of Z1 and Z2 is independently H or a (C1-C6) alkyl group optionally substituted with one or more groups selected from halogen atoms and (C1-C6) alkyl groups; L1, L2, Ra, Rb, Rc, Rd, X1, X2 and B have the same meanings as defined for general formula (II).

[0268] In some embodiments where 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 general formula (I).

[0269] In some embodiments where 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 general formula (I).

[0270] According to some of these embodiments where Y is NC(=O)-R1, R1 is selected from the group including methyl and pentadecyl, and L1, L2 and B have the same meaning as defined for general formula (I). These embodiments include compounds of formula (II) where Y is NC(=O)-R1, R1 is a methyl group, Ra, Rb, Rc, Rd, X1, X2 each represent a hydrogen atom, and B, L1 and L2 are as defined in general formula (I). These embodiments also include compounds of formula (I) where Y is NC(=O)-R1, R1 is a pentadecyl group, Ra, Rb, Rc, Rd, X1, X2 each represent a hydrogen atom, and B, L1 and L2 are as defined in general formula (I).

[0271] The nucleotide analogue of formula (I) can be either a nucleotide analogue of formula (IA) or a nucleotide analogue of formula (IB) and can exist in the form of a free base or in the form of an addition salt with an acid. The nucleotide analogue of formula (I) can be either a nucleotide analogue of formula (IA) or a nucleotide analogue of formula (IB) and can also exist in the form of their pharma- ceutically acceptable salts, which are also within the scope of the present disclosure.

[0272] Embodiments of the nucleotide analogues of formula (IB), also called targeted nucleotide analogues As previously specified herein, the present disclosure also relates to a nucleotide analogue of formula (IB), in the form of a base or in the form of an addition salt with an acid, - B is a heterocyclic nucleobase; - one of L1 and L2 is an internucleoside linking group linking the compound of formula (I) to a nucleotide residue, and the other of L1 and L2 is H, a protecting group, a phosphorus moiety or an internucleoside linking group linking the compound of formula (I) to a nucleotide residue; - Y is NR1, R1 is a group -[C(=O)]m-R2-(O-CH 2 -CH 2 )p-R3 (wherein, m is an integer having a value of 0 or 1; p is an integer ranging from 0 to 10; R2 is a (C1-C20) alkylene group optionally substituted with 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; K is O or S; Each of Z3 and Z4 is independently H or a (C1-C6) alkyl group optionally substituted with one or more groups selected from halogen atoms and (C1-C6) alkyl groups; R3 is a cell targeting moiety; - X1 and X2 each independently represent a hydrogen atom or a (C1-C6) alkyl group; - each of Ra, Rb, Rc and Rd is independently H or a (C1-C6) alkyl group; and any isomers thereof, such as stereoisomers thereof, or pharma- ceutically acceptable salts thereof.

[0273] These compounds of formula (IB) fall within a more general family of compounds sometimes referred to in this disclosure as "targeted nucleotide analogs."

[0274] R1 is the group -[C(=O)]m-R2-(O-CH 2 -CH 2 In some embodiments of the targeted nucleotide analog of formula (IB), m is 0, p is 0, R is a cell-targeting moiety, and B, P, P, Ra, R, R, R, X, X, and R are as described in the general definition of the compound of formula (IB).

[0275] In some embodiments, R2 is an ethylene group, 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 (I-BI).

[0276] In other embodiments, R2 is a pentylene group, 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 (IB).

[0277] In other embodiments, R2 is a (C12) alkylene group, 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 (IB).

[0278] R1 is the group -[C(=O)]m-R2-(O-CH 2 -CH 2In some embodiments of the compound of formula (IB) where m is 0, p is selected from the group of integers consisting of 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 the compound 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 still these embodiments, R2 is an ethylene group, p is 2, and X1 and X2 are both hydrogen atoms. In even further still these embodiments, R2 is an ethylene group, p is 3, and X1 and X2 are both hydrogen atoms. In still other still these embodiments, R2 is an ethylene group, p is 4, and X1 and X2 are both hydrogen atoms.

[0279] R1 is the group -[C(=O)]m-R2-(O-CH 2 -CH 2 In some embodiments of compounds of formula (IB), m is 1, p is 0, R is a cell targeting moiety, and R, B, L, L, Ra, R, R, R, X, X are as described in the general definition of compounds of formula (IB).

[0280] In some of these embodiments, R2 is butylene, X1 and X2 both represent a hydrogen atom, and B, L1, L2, Ra, Rb, Rc, and Rd are as defined with respect to general formula (IB).

[0281] In some further of these embodiments, R2 is (C11)alkylene, X1 and X2 both represent a hydrogen atom, and B, L1, L2, Ra, Rb, Rc and Rd are as defined for general formula (IB).

[0282] In still some further of these embodiments, R2 is methylene, both X1 and X2 represent a hydrogen atom, and B, L1, L2, Ra, Rb, Rc and Rd are as defined for general formula (IB).

[0283] R1 is the group -[C(=O)]m-R2-(O-CH 2 -CH 2 In some embodiments of the compound of formula (II), m is 1, p is selected from the group of integers consisting of 1 and 2, R is a cell-targeting moiety, and R, B, L, L, Ra, R, R, X, X are as described in the general definition of the compound of formula (IB).

[0284] 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, X2, are as defined with respect to general formula (IB).

[0285] In 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 with respect to general formula (IB).

[0286] In general, the group R3 includes any cell targeting moiety known in the art, including any cell targeting moiety specified in this disclosure, including, for example, those cell targeting moieties specified for the description of targeted oligonucleotides in this disclosure.

[0287] In some embodiments of the compound of formula (IB), R3 is selected from the group consisting of formula (III): [ka] (Wherein, A1, A2 and A3 are OH, A4 is OH or NHC(=O)-R5, where R5 is a (C1-C6) alkyl group optionally substituted with a halogen atom. has.

[0288] In some embodiments, R3 is an N-acetyl-galactosamine of formula (III-B): [ka]

[0289] According to the present disclosure, reference to "GalNAc" or "N-acetylgalactosamine" includes 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, both the β-form: 2-(acetylamino)-2-deoxy-β-D-galactopyranose and the α-form: 2-(acetylamino)-2-deoxy-α-D-galactopyranose can be used interchangeably.

[0290] In the oligonucleotide of formula (IB), B is a heterocyclic nucleobase moiety. The term "heterocyclic nucleobase" as used herein refers to an optionally substituted nitrogen-containing heterocycle covalently linked to a dioxane or morpholino ring. In some embodiments, the heterocyclic nucleobase can be selected from an optionally substituted purine base, an optionally substituted pyrimidine base. The term "purine base" is used herein in its ordinary sense as understood by those skilled in the art, and includes its tautomers. Similarly, the term "pyrimidine base" is used herein in its ordinary sense as understood by those skilled in the art, and includes its tautomers. A non-limiting list of optionally substituted purine bases includes purine, adenine, guanine, hypoxanthine, xanthine, alloxanthine, 7-alkylguanine (e.g., 7-methylguanine), theobromine, caffeine, uric acid, and isoguanine. Examples of pyrimidine bases include, but are not limited to, cytosine, thymine, uracil, 5,6-dihydrouracil, and 5-alkylcytosine (e.g., 5-methylcytosine). Other non-limiting examples of heterocyclic bases include diaminopurine, 8-oxo-N 6 Alkyl adenines (e.g., 8-oxo-N 6 methyladenine), 7-deazaxanthine, 7-deazaguanine, 7-deazaadenine, N 4 N4 Ethanocytosine, N <6> ,N <6> -ethano-2,6-diaminopurine, 5-halouracils (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 by reference herein for their disclosure of additional heterocyclic bases.

[0291] In some embodiments, B is selected from the group consisting of pyrimidines, substituted pyrimidines, purines, and substituted purines.

[0292] In preferred embodiments, B is adenine, thymine, uracil, guanine and cytosine (ie, selected from the group comprising adenyl, thyminyl, uracil, guanyl and cytosyl groups).

[0293] As already mentioned, in oligonucleotides comprising a nucleotide analogue of formula (IB), one of the groups L1 and L2 is an internucleoside linking group linking the compound of formula (I) to the nucleotide residue, i.e. to the adjacent nucleotide residue, and the other of the groups L1 and L2 is H, a protecting group or an internucleoside linking group linking the compound of formula (IB) to the nucleotide residue, i.e. to the adjacent nucleotide residue.

[0294] In addition, examples described herein of double-stranded oligonucleotides incorporating one or more compounds of formula (I) having a targeting moiety attached to the morpholine-nitrogen (GalNAc residue) demonstrate robust delivery to the liver, resulting in in vivo knockdown of target mRNA and corresponding protein levels.

[0295] Surprisingly, it has been demonstrated that combining targeted and non-targeted compounds of formula (I) within one double-stranded oligonucleotide significantly improves the behavior of the double-stranded oligonucleotide in vivo, particularly its duration of action in vivo; this can also be shown when the sense strand does not contain any phosphorothioate groups.

[0296] The siRNA that incorporates one or more compounds of formula (I) in the sense strand, antisense strand, or both sense strand and antisense strand, especially in the sense strand, also exerts efficient target gene silencing activity in vivo.Target gene silencing activity can be controlled by (i) the embodiment of the compound of formula (I) described herein, (ii) the number of compounds of formula (I) described herein, and (iii) the arrangement of the compound of formula (I) in the sense strand or antisense strand of siRNA.

[0297] Importantly, siRNAs incorporating one or more compounds of formula (I) are devoid of side effects in vivo at the dose ranges in which these siRNAs have been shown to exert their targeted gene silencing effects.

[0298] For purposes of illustration, and without limiting the disclosure, the double-stranded oligonucleotide may also contain one or more nucleotides in the sense and / or antisense strand that are modified.

[0299] The modification can be selected from the substitution or insertion of nucleic acid or base analogs, and chemical modification of base, sugar or phosphate moiety. The selected modification can be selected individually from 3'-terminal deoxy-thymine, 2'-O-methyl, 2'-deoxy modification, 2'-desoxy-fluoro, 2'-amino modification, 2'-alkyl modification, phosphorothioate modification, phosphoroamidate modification, 5'-phosphorothioate group modification, 5'-phosphate or 5'-phosphate mimetic modification, and cholesteryl derivative or dodecanoic acid bisdecylamide group modification, and / or the modified nucleotide can be any one of locked nucleotide, abasic nucleotide, or nucleotide containing non-natural base. One of the preferred embodiments can be that at least one modification is 2'-O-methyl, and / or at least one modification is 2'-desoxy-fluoro.

[0300] Other examples of modified oligonucleotides, as used herein, may include one or more of the following: modifications, e.g., replacement of one or both of a free phosphate and oxygen and / or one or more of a linked phosphate and oxygen; replacement of a phosphate moiety; modification or replacement of a naturally occurring base; replacement or modification of the ribose-phosphate backbone; modification of the 3' 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 either the 3' or 5' end of the RNA.

[0301] Methods for the synthesis of nucleotide analogue precursors of formula (II) Nucleotide analogue precursors of formula (II) may be prepared according to the detailed methods exemplified in the disclosure contained herein.

[0302] The present disclosure relates to a method for preparing a compound of formula (II-1), comprising: a) Formula (X) [ka] wherein B is a heterocyclic nucleobase, and P1 and P2 each independently represent a protecting group as defined in general formula (II) herein. The compound of formula (X) is reacted with an oxidizing agent, such as sodium periodate (NaIO 4 ) to obtain a compound of formula (XI): [ka] obtaining a compound of formula (I),

[0303] b) reacting a compound of formula (XI) with a compound of formula (XII) R1-NH 2 (XII) (wherein R1 is as defined in general formula (I) described herein). to a reductive amination step in the presence of formula (II-1): [ka] (wherein B is a heterocyclic nucleobase, and P1 and P2 each independently represent a protecting group as defined in general formula (II). A process for obtaining a compound of The present invention relates to a method comprising the steps of:

[0304] The present disclosure also provides a method for preparing a compound of formula (II-2), comprising: a) Formula (X) [ka] wherein B is a heterocyclic nucleobase, and P1 and P2 each independently represent a protecting group as defined in general formula (II) herein. The compound of formula (X) is reacted with an oxidizing agent, such as sodium periodate (NaIO 4 ) to obtain a compound of formula (XI): [ka] obtaining a compound of formula (I),

[0305] b) subjecting the compound of formula (XI) to a step of reductive amination, for example in the presence of an amine such as ammonia or ammonium diborate, to give a compound of formula (XIII) [ka] obtaining a compound of formula (I),

[0306] c) reacting a compound of formula (XIII) with a compound of formula (XIV) R1-C(=O)-OH (XIV) (wherein R1 is as defined in general formula (II) described herein). to obtain a compound of formula (II-2) by subjecting the compound to an amide coupling step in the presence of a compound of formula (II-3) [ka] (wherein B is a heterocyclic nucleobase, P1 and P2 each independently represent a protecting group as defined in general formula (II), and R1 is as defined in general formula (II). obtaining

[0307] d) subjecting the compound of formula (XIII) to a reductive amination step in the presence of an aldehyde or ketone to obtain a compound of formula (II-1) [ka] A process for obtaining a compound of The present invention also relates to a method, comprising:

[0308] The present disclosure also relates to a compound represented by formula (II-3): [ka] A process for preparing a compound of formula (XV) [ka] wherein A1, A2, A3 and A4 are as defined in formula (III) or (III-A) described herein, -Y-CHO is converted by a reductive amination reaction to -Y-CH2- equal to X, X being a group of formula -(CH2-CH2-O)p-R2-, where p and R2 are as defined in general formula (I). with a compound of formula (XIII) [ka] (wherein B is a heterocyclic nucleobase, and P1 and P2 each independently represent a protecting group as defined in general formula (II). by reductive amination to obtain a compound of formula (II-3) [ka] A process for obtaining a compound of The present invention also relates to a method, comprising:

[0309] The above method is illustrated in Scheme 2 in this disclosure.

[0310] The present disclosure also relates to a compound represented by formula (II-4): [ka] A method for obtaining a compound of formula (I), comprising:

[0311] a) Formula (XVI) [ka] (wherein A1, A2, A3 and A4 are as defined in formula (III) or (III-A) described herein, X is a group of formula -(CH2-CH2-O)p-R2-, p and R2 are as defined in general formula (I). with a compound of formula (XIII) [ka] (wherein B is a heterocyclic nucleobase, and P1 and P2 each independently represent a protecting group as defined in general formula (II). under peptide coupling conditions to obtain a compound of formula (II-4) The present invention also relates to a method, comprising:

[0312] The above method is illustrated in Scheme 2 in this disclosure.

[0313] The present disclosure further provides a method for preparing a compound of formula (II-5), comprising: a) Formula (XI) [ka] (wherein B is a heterocyclic nucleobase, and P1 and P2 each independently represent a protecting group as defined in general formula (II). to reduce a compound of formula (XVII) [ka] obtaining a compound of formula (I),

[0314] b) Compounds of formula (XVII) are converted in the presence of a sulfonylating agent (e.g. p-toluenesulfonyl chloride Ts-Cl, methanesulfonyl chloride Ms-Cl) to give compounds of formula (XVIII): [ka] (In the formula, Ts represents a tosyl group.) obtaining a compound of formula (I),

[0315] c) subjecting the compound of formula (XVIII) to basic conditions to obtain a compound of formula (II-5) [ka] A process for obtaining a compound of The present invention relates to a method comprising the steps of:

[0316] The above method is illustrated in Scheme 3 in this disclosure.

[0317] The present disclosure also provides an alternative method for preparing a compound of formula (II-5), comprising the steps of: a) Formula (XVII) [ka] in the presence of an excess of a sulfonylating agent (e.g., p-toluenesulfonyl chloride Ts-Cl, methanesulfonyl chloride Ms-Cl) to give a compound of formula (XIX) [ka] (In the formula, Ts represents a tosyl group.) obtaining a compound of formula (I),

[0318] b) deprotecting the compound of formula (XIX) by removal of group P1 to give the compound of formula (XX) [ka] obtaining a compound of formula (I),

[0319] c) subjecting the compound of formula (XX) to basic conditions to obtain a compound of formula (XXI) [ka] obtaining a compound of formula (I),

[0320] d) replacing the tosyl group with a protecting group P1, to obtain a compound represented by formula (II-5) [ka] A process for obtaining a compound of The present invention also relates to a method, comprising:

[0321] The above method is illustrated in Scheme 3 in this disclosure.

[0322] Compounds of formula (II), (II-1), (II-2), (II-3), (II-4) and (II-5) may be prepared according to the detailed methods disclosed herein and illustrated in Schemes 1-8 below.

[0323] Starting from commercially available ribose derivative G1, the two primary OH-groups can be differentiated by selective benzylation according to standard literature protocols. Standard protecting group modification of the resulting benzyl ether G2 leads to the fully protected ribose analogue G3, which can be coupled to nucleobases B (e.g.: T, U, C, Bzl , I, G iBu , A Bzl ) can be used as a glycosyl donor to give the nucleoside derivative G4 (Tetrahedron, 1998, 54, 3607-3630).

[0324] [ka]

[0325] Scheme 1: Synthesis of compounds of formula (II) where Y is NH, NR1 and NC(=O)R1 Starting with nucleoside analogue G4, the protecting group pattern is modified by standard procedures to give intermediate G5, which bears orthogonal protecting groups P1 and P2 as defined by general formula (II) on the two primary alcohols and the unprotected OH group at C2' and C3' of the ribose scaffold. The cis orientation of the dihydroxy functionality in G5 compound is achieved by the addition of NaIO as the oxidizing agent. 4 The resulting dialdehyde can be isolated as monohydrate G6, which can be converted to a dialdehyde by oxidation using a reducing agent such as NaCNBH 3 The desired morpholine scaffold is converted by reductive amination with NaCNBH. The use of amine substrates such as ammonia or ammonium diborate gives rise to the morpholine intermediate G7, which has a free NH group in the morpholine scaffold. 3A second reductive amination reaction with the corresponding aldehyde or ketone in the presence of gives the alkylated morpholine G8 (R1 is as defined in general formula (II)). Alternatively, intermediate G6 can be prepared by reaction of the appropriate amine R1-NH 2 where R1 is as defined in general formula (II)), which directly gives the alkylated morpholine G8. The analogous acylated morpholines can be obtained by standard peptide coupling reaction of the free morpholine building block G7 with the corresponding carboxylic acid R1-COOH, resulting in the amide intermediate G9.

[0326] Compounds G7, G8 and G9 consist of embodiments of compounds of formula (II) described in the present disclosure.

[0327] Similar to the synthesis described in Scheme 1 for the formation of compounds of general formula (II), the targeted compounds of general formula (II) described herein can be prepared by reductive amination or peptide coupling reactions using intermediate G7 as an amine reagent.

[0328] Scheme 2 below describes the synthesis of compounds of general formula (II) using peracetylated N-acetylgalactosamine G11 as the protected cell targeting moiety.

[0329] [ka]

[0330] Scheme 2: Synthesis of compounds of general formula (II) with attachment of amide (G14) and amine (G15), protected GalNAc as cell targeting moiety and different X linker groups, where in G15 synthesis X is generated from the linker fragment -Y-CHO in (G13) and after reductive amination to -Y-CH 2 - is converted to

[0331] In the following, X is defined as the group -R2(OCH2-CH2)p- contained in the group -[C(=O)]m-R2-(O-CH2-CH2)p-R3 as defined for compounds of general formula (II).

[0332] Treatment of peracetylated GalNAc-derivative G11 with ω-hydroxycarboxylic acid ester (HO-X-COOBn) under standard glycosylation conditions gives carboxylic acid G12 after ester cleavage, which in the presence of morpholine derivative G7 under peptide coupling conditions forms the desired amide G14. Alternatively, glycosylation of G11 with ω-hydroxy-benzyl ether (HO-X-OBn) provides aldehyde intermediate G13 after benzyl ether cleavage and oxidation of the corresponding alcohol. Reductive amination with morpholine G7 as the amine component leads to the formation of alkylated morpholine G15. Compounds G14 and G15 are compounds of formula (II) as described in the present disclosure.

[0333] Scheme 3 shows a synthetic route to compounds of general formula (II) where Y is O in a series of dioxanes.

[0334] [ka]

[0335] Scheme 3: Synthetic route to compounds of general formula (II) where Y is O Starting from the already described cis-diol intermediate G5 and its cleavage by oxidation into building block G6 (see Scheme 1), treatment with a reducing agent such as sodium borohydride gives the diol intermediate G16 bearing two primary OH groups at the 2'- and 3'-C. In the presence of a stoichiometric amount of a sulfonylating agent such as p-toluenesulfonyl chloride under basic conditions, the 2'-OH function can be selectively tosylated to form the monotosylate G17. Under basic conditions, for example using aqueous NaOH or NaOMe in MeOH, G17 undergoes nucleophilic displacement of the primary tosylate with the free OH group at the 3'-C, which leads to the formation of the desired dioxane scaffold G18.

[0336] Compound G18 consists of a compound of general formula (II) as described in the present disclosure.

[0337] Alternatively, the diol intermediate G16 can be bissulfonylated, for example with an excess of p-toluenesulfonyl chloride and increased reaction time, to give the bistosylate G19. After deprotection of one of the orthogonal protecting groups P1 or P2, the resulting primary alcohol G20 reacts similarly with G17 under nucleophilic displacement and formation of the desired dioxane scaffold G21 (see Scheme 3). The remaining tosylate can be displaced with sodium benzoate to again give the fully protected dioxane scaffold G18 bearing orthogonal protecting groups P1 and P2 at the primary hydroxyl group.

[0338] [ka]

[0339] Scheme 4: Alternative synthesis of compounds of formula (II) where Y is O The described building blocks of general formula (II) (G8, G9, G14, G15 and G18) bearing orthogonal protecting groups P1 and P2 are finally converted to the corresponding DMT-protected phosphoramidites, enabling their use as nucleotide precursors in automated oligonucleotide synthesis.

[0340] For this purpose, the fully protected compounds of general formula (II) (G8, G9, G14, G15 and G18) are converted to the single DMT-protected intermediate G22 by standard protecting group modifications. Phosphitylation of the free OH group in the G22-building block by standard protocols leads to the final DMT-protected phosphoramidite G23 as a nucleotide precursor for automated oligonucleotide synthesis.

[0341] [ka]

[0342] Scheme 5: Synthesis of the final phosphoramidite building blocks of compounds of general formula (II) for automated oligonucleotide synthesis Compound G23 is a compound of formula (II) in which group P1 is a DMT protecting group and group P2 is a reactive phosphorus group comprising a phosphoramidite group.

[0343] Depending on the protecting group modification resulting in intermediate G22, the (2S,6R)- and (2R,6R)-diastereomers of compounds of general structure (II) can be synthesized.

[0344] [ka]

[0345] Scheme 6: Diastereomeric structure of compound G23 of general formula (II) The starting nucleotide at the 3' end of the oligonucleotide single strand can be prepared by standard procedures using universal solid support materials which react with the corresponding phosphoramidite G23 as the first nucleotide scaffold in an automated synthesis (see Experimental Section, Synthesis of Oligonucleotides).

[0346] Preparation of oligonucleotides containing nucleotide analogues of formula (IA) and optionally (IB) Oligonucleotides for use in accordance with the present disclosure, sometimes referred to herein as "modified oligonucleotides," may be prepared according to any useful technique, including the methods described herein, by using one or more nucleotide analog precursors of formula (II) as part of the starting building blocks to be incorporated at selected positions in a growing chain of the final oligonucleotide, thus producing an oligonucleotide containing one or more nucleotide analogs of formula (I), where one or more compounds of formula (I) are positioned at selected positions in the final oligonucleotide.

[0347] The nucleotide analog precursor of formula (II) can be synthesized as described in this disclosure.

[0348] The modified oligonucleotide of the present disclosure may be double-stranded, with or without overhang, or may at least include a double-stranded portion.The double-stranded modified oligonucleotide may be formed from a single oligonucleotide strand that contains 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 the second nucleotide sequence is also complementary to the target RNA sequence that is sought to be inhibited.According to these embodiments, the first nucleotide sequence and the second nucleotide sequence may be present on separate strands within the modified oligonucleotide; or may be present on the same strand, but separated by a spacer or an additional nucleotide sequence of suitable length such that the first nucleotide sequence forms a hairpin loop when hybridized to the second nucleotide sequence.

[0349] In some embodiments, the modified oligonucleotides of the present invention are single-stranded and may comprise either the sense or antisense strand of a double-stranded RNA, such as an siRNA.

[0350] The oligonucleotides of the present invention, such as those containing one or more nucleotide analogs of formula (I), 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. WO99 / 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. Synthesis of oligonucleotides utilizes common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-terminus and phosphoramidites at the 3'-terminus. In certain embodiments, oligonucleotides containing nucleotide analogs of formula (I) are synthesized, deprotected, and analyzed according to the methods described in U.S. Patent Nos. 6,995,259; 6,686,463; 6,673,918; 6,649,751; 6,989,442; and 7,205,399. In a non-limiting synthesis example, small-scale synthesis is carried out on a 394 Applied Biosystems, Inc. / Thermo Fischer Scientific Inc. synthesizer.

[0351] Alternatively, oligonucleotides containing one or more nucleotide analogs of formula (I) may 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. Various modified oligonucleotides according to the present disclosure can also be synthesized using the teachings of Scaringe et al., U.S. Patent Nos. 5,889,136; 6,008,400; and 6,111,086.

[0352] composition Certain aspects of the present disclosure relate to compositions (e.g., pharmaceutical compositions) that comprise dsRNA as described herein.In some embodiments, the compositions (e.g., pharmaceutical compositions) further comprise a pharma- ceutical acceptable carrier.In some embodiments, the compositions (e.g., pharmaceutical compositions) are useful for treating diseases or disorders related to the expression or activity of targeted genes.

[0353] Compositions (e.g., pharmaceutical compositions) of the present disclosure are formulated based on the mode of delivery, including, for example, compositions formulated for delivery to the liver via parenteral delivery.

[0354] The compositions (e.g., pharmaceutical compositions) of the present disclosure can be administered in a dosage sufficient to inhibit expression of the targeted gene. In some embodiments, a suitable dose of dsRNA is within the range of 0.01 mg / kg to 400 mg / kg of recipient body weight.

[0355] Those skilled in the art will understand that certain factors, such as, but not limited to, the severity of disease or disorder, previous treatment, the general health and / or age of the subject, and one or more other existing diseases, may affect the dosage and timing required to effectively treat the subject.Furthermore, the treatment of the subject with a therapeutically effective amount of the pharmaceutical composition may include a single treatment or a series of treatments.Effective dosage and in vivo half-life of the dsRNA disclosed herein can be estimated using conventional methodology or based on in vivo testing using suitable animal models.

[0356] The dsRNA molecule of the present disclosure can be formulated in pharmaceutically acceptable carrier or diluent.Pharmaceutically acceptable carrier can be liquid or solid, and can be selected with the planned administration method in consideration of providing desired bulk, viscosity and other related transport and chemical properties.Any known pharmaceutically acceptable carrier or diluent can be used, examples of which include, for example, water, saline, binder (e.g., polyvinylpyrrolidone or hydroxypropylmethylcellulose), filler (e.g., lactose and other sugars, gelatin, or calcium sulfate), lubricant (e.g., starch, polyethylene glycol, or sodium acetate), disintegrant (e.g., starch or sodium starch glycolate), calcium salt (e.g., calcium sulfate, calcium chloride, calcium phosphate, etc.) and wetting agent (e.g., sodium lauryl sulfate).

[0357] The dsRNA molecules of the present disclosure can be formulated into compositions (e.g., pharmaceutical compositions) that contain dsRNA mixed with, encapsulated with, conjugated with, or otherwise associated with a mixture of other molecules, molecular structures, or nucleic acids.For example, a composition that contains one or more dsRNAs as described herein may contain other therapeutic agents, such as other lipid-lowering agents (e.g., statins).In some embodiments, the composition (e.g., pharmaceutical composition) further comprises a delivery vehicle (as described herein).

[0358] Vectors and dsRNA delivery 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., pharmaceutical composition) that contains the dsRNA to a subject.In some embodiments, the dsRNA is delivered indirectly by administering one or more vectors as described herein.

[0359] delivery The dsRNAs of the disclosure can be delivered to or from a host cell by any method known in the art, for example, by adapting methods of 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 through additional 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 Berlin Heidelberg, DOI 10.1007 / 978-3-642-12168-5; Xu, C. and Wang, J. (2015) Asian Journal of Pharmaceutical Sciences 10(1):1-12).

[0360] In some embodiments, the dsRNA of the present disclosure is delivered by a delivery vehicle that contains the dsRNA. In some embodiments, the delivery vehicle is a liposome, a lipoplex, a complex, or a nanoparticle.

[0361] Liposomal Formulations Liposomes are unilamellar or multilamellar vesicles with a membrane formed from a lipophilic material and an aqueous interior. In some embodiments, liposomes are vesicles composed of amphiphilic lipids arranged in a spherical bilayer or bilayer. The aqueous portion contains the composition to be delivered. Cationic liposomes have the advantage that they can fuse to the cell wall. Advantages of liposomes include, for example, that liposomes derived from natural phospholipids are biocompatible and biodegradable; liposomes can entrap a wide variety of water-soluble and lipid-soluble drugs; and liposomes can protect drugs encapsulated in their internal compartment from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (eds.), 1988, Marcel Dekker, Inc., New York, NY, Vol. 1, p. 245). Important considerations in the preparation of liposomal formulations are the lipid surface charge, the size of the vesicle and the aqueous volume of the liposomes. For example, engineered cationic liposomes and spatially stabilized liposomes can be used to deliver dsRNA.See, for example, Podesta et al. (2009) Methods Enzymol. 464, 343-54; U.S. Patent No. 5,665,710.

[0362] Nucleic acid-lipid particles In some embodiments, the dsRNA of the present disclosure is fully encapsulated in lipid formulations to form, for example, nucleic acid-lipid particles, such as SPLPs, pSPLPs, or SNALPs. The term "SNALP" as used herein refers to stable nucleic acid-lipid particles, including SPLPs. The term "SPLP" as used herein refers to nucleic acid-lipid particles that include plasmid DNA encapsulated within lipid vesicles. Nucleic acid-lipid particles, such as SNALPs, typically contain cationic lipids, non-cationic lipids, cholesterol, and lipids (e.g., PEG-lipid conjugates) that prevent particle aggregation and increase circulation time. SNALPs and SPLPs exhibit long circulation lifetimes after intravenous (iv) injection and accumulate at distal sites (e.g., sites physically distant from the site of administration), making them useful for systemic application. SPLPs include "pSPLPs," examples of which include encapsulated condensing agent-nucleic acid complexes as described in PCT Publication WO00 / 03683.

[0363] In some embodiments, when dsRNA is present in nucleic acid-lipid particle, it is resistant to decomposition by nuclease in aqueous solution.Nucleic acid-lipid particle and its preparation method are disclosed in, for example, United States Patent No. 5,976,567; No. 5,981,501; No. 6,534,484; No. 6,586,410; and No. 6,815,432; and PCT publication WO96 / 40964.

[0364] In some embodiments, the nucleic acid-lipid particles comprise a cationic lipid. Any cationic lipid or mixture thereof known in the art can be used. In some embodiments, the nucleic acid-lipid particles comprise a non-cationic lipid. Any non-cationic lipid or mixture thereof known in the art can be used. In some embodiments, the nucleic acid-lipid particles comprise a conjugated lipid (e.g., to prevent aggregation). Any conjugated lipid known in the art can be used.

[0365] Additional Compounding Agents Important factors to consider for successful delivery of dsRNA molecules in vivo include (1) biological stability of delivered molecules, (2) prevention of non-specific effects, and (3) accumulation of delivered molecules in target tissues. Non-specific effects of dsRNA can be minimized by local administration, for example, by direct injection or implantation into tissues, or by administering preparations locally. To administer dsRNA systemically for disease treatment, dsRNA can be modified or alternatively delivered using a drug delivery system; both methods act to prevent rapid degradation of dsRNA by endo- and exonucleases in vivo. RNA modification or pharmaceutical carriers can also allow targeting of dsRNA compositions to target tissues and avoid undesirable off-target effects. As mentioned 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, dsRNA is delivered using drug delivery systems such as nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems. Cationic delivery systems with positive charges facilitate the binding of dsRNA molecules (which have negative charges) and also enhance the interaction with the negatively charged cell membrane to allow efficient uptake of dsRNA by cells. Cationic lipids, dendrimers, or polymers can either bind to dsRNA or be induced to form vesicles or micelles that encapsulate dsRNA (see, for example, Kim SH et al. (2008) Journal of Controlled Release 129(2):107-116). The formation of vesicles or micelles further prevents the degradation of dsRNA when administered systemically. Methods for making and administering cationic dsRNA complexes are known in the art. In some embodiments, dsRNA is complexed with cyclodextrin for systemic administration.

[0366] Methods for using dsRNA Certain aspects of the present disclosure relate to a method for inhibiting the expression of a targeted gene in a mammal, comprising administering an effective amount of one or more of the dsRNAs of the present disclosure, one or more of the vectors of the present disclosure, or a composition (e.g., pharmaceutical composition) of the present disclosure that comprises one or more of the dsRNAs of the present disclosure. Certain aspects of the present disclosure relate to a method for treating and / or preventing a disease or disorder mediated by one or more target genes, comprising administering a composition (e.g., pharmaceutical composition) that comprises one or more of the dsRNAs of the present disclosure, and / or one or more of the vectors of the present disclosure, and / or one or more of the dsRNAs of the present disclosure. In some embodiments, downregulating the target gene expression in a subject alleviates one or more symptoms of the disease or disorder mediated by the targeted gene in the subject.

[0367] In some embodiments, 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% after treatment compared to pretreatment levels. In some embodiments, the expression of the target gene is inhibited after treatment by at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 4.5 times, at least about 5 times, at least about 5 times, at least about 5.5 times, at least about 6 times, at least about 6.5 times, at least about 7 times, at least about 7.5 times, at least about 8 times, at least about 8.5 times, at least about 9 times, at least about 9.5 times, at least about 10 times, at least about 25 times, at least about 50 times, at least about 75 times, or at least about 100 times, compared to pretreatment levels. In some embodiments, the target gene is inhibited in the liver of the subject.

[0368] In some embodiments, the subject is a human. In some embodiments, the subject has or has been diagnosed with a target gene-mediated disorder or disease. In some embodiments, the subject is suspected of having a target gene-mediated disorder or disease. In some embodiments, the subject is at risk of developing a target gene-mediated disorder or disease.

[0369] As can be understood from the contents of this disclosure, the dsRNA described herein has its main feature based on the presence of one or more nucleotide analogues of formula (II) contained therein, where the nucleotide analogues of formula (II) have specific structural functions of their "sugar-like" groups. The dsRNA described herein is generally designed to target a selected nucleic acid sequence contained in a target nucleic acid of interest. In particular, the dsRNA embodiment of the siRNA described herein comprises an antisense strand that specifically hybridizes with a nucleic acid sequence contained in a target nucleic acid of interest. The dsRNA or composition (e.g., pharmaceutical composition) described herein can be for use in the treatment of a target gene-mediated disorder or disease. In particular, the dsRNA or composition (e.g., pharmaceutical composition) described herein, in particular the dsRNA comprising one or more targeted nucleotide analogues, and in particular the one or more targeted nucleotide analogues of formula (II), can be for use in the treatment of a target gene-mediated disorder or disease where liver targeting is required.

[0370] Certain embodiments of the present disclosure also relate to methods of delivering nucleic acid to a hepatocyte comprising contacting the hepatocyte with a dsRNA described herein.

[0371] The dsRNA or composition (e.g., pharmaceutical composition) described herein can be administered by any means known in the art, including, but not limited to, oral or parenteral routes, such as intravenous, intramuscular, subcutaneous, pulmonary, transdermal, and airway (aerosol) administration. Typically, when treating a mammal with hyperlipidemia, the dsRNA molecule is administered systemically via parenteral means. In some embodiments, the dsRNA and / or composition is administered by subcutaneous administration. In some embodiments, the dsRNA and / or composition is administered by intravenous administration. In some embodiments, the dsRNA and / or composition is administered by pulmonary administration.

[0372] The therapeutic or preventive effect of dsRNA is evident when there is a statistically significant improvement in one or more parameters of the disease status, or when the symptoms do not worsen or develop when they would normally be expected. For example, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more favorable changes in measurable parameters of the disease may indicate effective treatment. Efficacy for a given dsRNA or composition comprising dsRNA can also be determined using experimental animal models for a given disease or disorder known in the art. When using experimental animal models, efficacy of treatment is demonstrated when a statistically significant reduction in markers or symptoms is observed.

[0373] Kits and Products Certain embodiments of the present disclosure relate to articles of manufacture or kits that include one or more of the dsRNAs, vectors, or compositions (e.g., pharmaceutical compositions described herein) useful for treating and / or preventing a target gene-mediated disorder or disease, as described above. The articles of manufacture or kits 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 fluid bags, and the like. The containers can be molded from a variety of materials such as glass or plastic. The containers hold the composition, alone or in combination with another composition effective to treat or prevent a disease, and may have a sterile access port (e.g., the container can be an intravenous fluid bag or vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a dsRNA, vector, or composition useful for treating and / or preventing a target gene-mediated disorder or disease, as described herein. The label or package insert indicates that the composition is used to treat a target-mediated disorder or disease. In addition, the product or kit may include (a) a first container containing a composition comprising the dsRNA described herein; and (b) a second container containing a composition comprising a second therapeutic agent. The product or kit of the present disclosure in this embodiment may further include a package insert indicating that the composition can be used to treat a particular disease. Alternatively, or in addition, the product or kit may further include a second (or third) container containing a pharma- ceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, such as other buffers, diluents, filters, needles, and syringes.

[0374] The nucleic acid sequences are disclosed herein, in particular in the examples described herein, which serve as reference. The same sequences are also presented in the sequence listing, which is formatted according to standard requirements for patent purposes. In the event of any sequence discrepancy with the standard sequence listing, the sequences described herein shall be referred to.

[0375] Without intending to limit the disclosure, several embodiments of the disclosure are described below for illustrative purposes. EXAMPLES

[0376] The nomenclature is governed by IUPAC rules. Unless otherwise indicated, the following LC-MS methods were used: A: Column: Waters ACQUITY UPLC BEH C18, 1.7μm, 2.1×50mm Eluent: (H 2 O+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 at 55°C. B-1: Column: Phenomenex Luna C18, 3.0 μm, 2.0 × 10 mm Eluent: (H 2 0+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. B-2: Column: Phenomenex Luna C18, 3.0 μm, 2.0 × 10 mm Eluent: (H 2 0+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. B-3: Column: Phenomenex Luna C18, 3.0 μm, 2.0 × 10 mm Eluent: (H 2 0+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. C: Column: Waters ACQUITY UPLC BEH C18, 1.7μm, 2.1×50mm Eluent: (H 2 O+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. D: Column: Chromolith@Flash RP-18E 2×25mm Eluent: (H 2 O+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. E: Column: Kinetex EVO C18E 2.1×30mm, 5μm Eluent: (H 2 O+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.

[0377] Abbreviations used: -AcOH: acetic acid -FA: Formic acid -ACN: Acetonitrile -DCM: dichloromethane -DMA: Dimethylacetamide -DCE: dichloroethane -DMF: Dimethylformamide -DMSO: Dimethyl sulfoxide - EtOAc: ethyl acetate -EtOH: Ethanol -Et 2 O: Diethyl ether -iPrOH: isopropanol -THF: Tetrahydrofuran -MeOH: Methanol -NMP: N-methyl-2-pyrrolidone -PE: Petroleum ether -Pyr: Pyridine -iPr: Isopropyl -iBu: Isobutyryl -cHex: cyclohexyl -MTB: Methyl tert-butyl -DIPEA: Diisopropylethylamine -DMAP: 4-(dimethylamino)-pyridine -DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene -HBTU: (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) -TBTU: O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate -DDTT: 3-((N,N-dimethyl-aminomethylidene)amino)-3H-1,2,4-dithiazole-5-thione -NEt 3 : Triethylamine -NEM: N-ethyl-morpholine -BSA: N,O-bis-trimethylsilylacetamide -TMSOTf: Trimethylsilyl trifluoromethanesulfonate -Ts: p-toluenesulfonyl -Tf: Trifluoromethanesulfonyl -Trifluoromethanesulfonate -TFA: Trifluoroacetic acid -DCAA: dichloroacetic acid -TEA: Triethylammonium -TIPS: Triisopropylsilyl -TBDMS: tert-butyldimethylsilyl -DMT: 4,4'-dimethoxytrityl -Bzl: Benzoyl -Bn: Benzyl -BOM: benzyloxymethyl -Ac: Acetyl -iBu: Isobutyryl -Boc: tert-butyloxycarbonyl -Fmoc: Fluorenylmethyloxycarbonyl -Fmoc-OSu: N-(9-fluorenylmethoxycarbonyloxy)succinimide -CE: Cyanoethyl -CPG: Controlled Pore Glass -T: Thymine -U: Uracil -C: Cytosine -A: Adenine -G: Guanine -I: Hypoxanthine -T BOM :N-benzyloxymethyl-thymine -U BOM :N-benzyloxymethyl-uracil -U Bzl :N-benzoyl-uracil -C Bzl :N-benzoyl-cytosine -A Bzl :N-benzoyl-adenine -G iBu :N-isobutyryl-guanine -GalNAc:DN-acetylgalactosamine -FR:Flow rate - HPLC: High Pressure Liquid Chromatography -MS-TOF: Time-of-flight mass spectrometry -LC-MS: High pressure liquid chromatography-mass spectrometry -Rt: Retention time -RT: room temperature -Hal: Halogen -ELSD: Evaporative Light Scattering Detector -quant.:Quantitative -sat.: saturation -i.vac.: in vacuum -nd:Not decided -TLC: Thin Layer Chromatography -h: time -min:minutes -Tm: melting temperature -r: ribonucleotide -d: Desoxyribonucleotide -m: 2'-OMe-nucleotide -f: 2'-desoxy-fluoro-nucleotide -ss: sense strand -as: antisense strand -ds: double strand -chol: cholesterol -PO: phosphodiester bond - * or PS: phosphorothioate bond -mpk:mg / kg -M: Molar concentration -#: number, n° -FBS: Fetal bovine serum -ATP: Adenosine triphosphate -pre-lB: precursor nucleotide -pre-lgB: targeted precursor nucleotide -lB: nucleotide analogues -lgB: targeted nucleotide analogues

[0378] A: Synthesis of nucleotide analogues of formula (I) where X is NH, NR1, NC(=O)R1

[0379] [ka]

[0380] 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 Starting material G3 (42.5 g, 87.4 mmol) and thymine 22.3 g (174.7 mmol) were dissolved in dry ACN (500 ml) under Ar atmosphere. BSA (106.6 g, 128.2 ml, 524.1 mmol) was added and the solution was stirred at 80° C. for 1 h. After cooling the solution to 50° C., TMSOTf (31.8 g, 25.9 ml, 141.5 mmol) was added and heating was continued at 80° C. for 1 h. The solvent was evaporated in vacuum and the residue was dissolved in EtOAc and saturated NaHCO 3 solution, H 2 O and saturated NaCl solution. MgSO 4 After drying at 40° C., the organic layer was evaporated and the crude product was purified by silica gel chromatography (10 to 100% ethyl acetate in n-heptane) to give the desired thymidine derivative 1a as a colorless foam (40.9 g, 84.7%). LCMS-Method A: UV-wavelength [nm] = 220: R t [minutes] = 1,82 Ionization method: ES - : [MH] - = 551,3 1H-NMR (CDCl 3 , 400 MHz) δ[ppm]: 8.55 (s, 1 H) 7.51-7.17 (m, 12 H), 6.23 (d, J=5.27 Hz, 1 H), 5.44 (t, J=5.65 Hz, 1 H), 4.72-4.35 (m, 6 H), 4.19 (d, 2.17-2.06 (m, 6 H), 1.55 (d, J=0.75 Hz, 3 H).

[0381] 1b: [(2S,3S,4R,5R)-4-acetoxy-3-benzyloxy-2-(benzyloxymethyl)-5-(2,4-dioxopyrimidin-1-yl)-tetrahydrofuran-2-yl]methyl acetate G3 (10.0 g, 20.55 mmol) and 3.49 g (30.8 mmol) of uracil were dissolved in dry ACN (200 ml). BSA (20.09 g, 30.15 ml, 123.3 mmol) was added and the solution was stirred at 81° C. for 1 h. After cooling the solution to 0° C., TMSOTf (7.48 g, 6.10 ml, 33.3 mmol) was added and the mixture was heated to 65° C. Stirring was continued at this temperature for 1 h, at which point complete conversion had occurred. At room temperature, saturated NaHCO 3 500 mL of solution was added and the mixture was extracted with EtOAc (500 mL). The organic layer was separated and 2 O and saturated NaCl solution. MgSO 4 After drying at rt, the crude product was purified on silica gel (20 to 100% EtOAc in n-heptane) to give the protected nucleoside analog 1b in 82.3% yield (9.11 g) as a colorless foam. LCMS-Method A: UV-wavelength [nm] = 220: R t [minutes] = 1,82 Ionization method: ES + : [M+H] + = 539,1 1H-NMR (CDCl 3 , 400 MHz) δ[ppm]: 8.97 (s, 1 H), 7.66 (d, J=8.0 Hz, 1 H), 7.38-7.35 (m, 10 H), 6.19 (d, J=4.0 Hz, 1 H), 5.38-5.30 (m, 2 H), 4.63 (d, J=12.0 Hz, 1 H), 4.46-4.41 (m, 5 H), 4.19 (d, J=12.0 Hz, 1 H), 3.79 (d, J=12.0 Hz, 1 H), 3.51 (d, J=12.0 Hz, 1 H), 2.12 (s, 3 H), 2.07 (s, 3 H).

[0382] 1c: [(2S,3S,4R,5R)-4-acetoxy-3-benzyloxy-2-(benzyloxymethyl)-5-[2-(2-methylpropanoyl-amino)-6-oxo-1H-purin-9-yl]tetrahydrofuran-2-yl]methyl acetate A solution of compound G3 (148.5 g, 0.30 mol) in DCE (6.68 L) was added to 2 N-isobutyryl-guanine (135 g, 0.61 mol) and BSA (311.85 ml, 1.2 mol) were added under atmosphere at 15° C. The mixture was stirred at 85° C. for 3 h. TMSOTf (183.4 g, 0.90 mol) was then added at 85° C. and stirring was continued for 3 h, at which point TLC showed complete conversion. The mixture was cooled to room temperature and washed with saturated NaHCO 3 The solution was poured into 6.5 L. The organic layer was separated and the aqueous phase was extracted twice with DCM (5 L). The organic layers were combined and washed with anhydrous Na 2 SO 4 The resulting crude product was purified by preparative HPLC (0.1% TFA / ACN) to give compound 1c (128 g, 64%) as a white solid. 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 12.09 (s,1 H), 11.62 (s, 1 H), 8.14 (s, 1 H), 7.41-7.30 (m, 10 H), 6.12 (d, J=6.4 Hz, 1 H), 5.90 (t, J 1 =J 2 =5.6 Hz, 1 H), 4.71 (d, J=5.2 Hz, 1 H), 4.63-4.55 (m, 4 H), 4.34 (d, J=5.6 Hz, 1 H), 4.23 (d, J=5.6 Hz, 1 H), 3.71-3.66 (m, 2 H), 3.18 (d, J=4.8 Hz, 1 H), 2.76-2.51 (m, 1 H), 2.05 (s, 3 H), 1.99 (s, 3 H), 1.20-1.12 (s, 6 H).

[0383] 1d: [(2S,3S,4R,5R)-4-acetoxy-3-benzyloxy-2-(benzyloxymethyl)-5-(6-oxo-1H-purin-9-yl)-tetrahydrofuran-2-yl]methyl acetate Starting with G3 (10.0 g, 20.55 mmol) and 3.32 g (23.66 mmol) of hypoxanthine as the nucleobase, the title compound was synthesized according to the protocol described in 1c. 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%). LCMS-Method A: UV-wavelength [nm] = 220: R t [minutes] = 1,73 Ionization method: ES + : [M+H] + = 563,3 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 12.42 (s, 1 H), 8.22 (s, 1 H), 8.01 (s, 1 H), 7.25 - 7.39 (m, 10 H), 6.21 (d, J=4.89 Hz, 1 H), 5.90 - 5.97 (m, 1 H), 4.78 (d, J=5.75 Hz, 1 H), 4.45 - 4.61 (m, 4 H), 4.32 - 4.41 (m, 1 H), 4.17 - 4.25 (m, 1 H), 3.59 - 3.74 (m, 2 H), 2.04 (m, 3 H), 1.98 (m, 3 H).

[0384] 1e: [(2S,3S,4R,5R)-4-acetoxy-5-(6-benzamidopurin-9-yl)-3-benzyloxy-2-(benzyloxymethyl)-tetrahydrofuran-2-yl]methyl acetate To a mixture of compound G3 (164 g, 0.337 mol) and N-benzoyladenine (121 g, 0.506 mol) in DCE (6.56 L), BSA (274 g, 1.348 mol) was added dropwise at room temperature, and the mixture was stirred at 90 °C for 1.5 h. After TMSOTf (225 g, 1.011 mol) was added dropwise at 40-50 °C, the mixture was stirred at 90 °C for 1 h. The mixture was diluted with saturated NaHCO 3 The solution was quenched with 15 L and the layers were separated. The aqueous layer was extracted twice with EtOAc (3 L) and the combined organic layers were washed with anhydrous Na 2 SO 4 The mixture was dried at 40° C., filtered and concentrated in vacuo. The crude product was purified by flash chromatography (PE / EtOAc 1:2) to give compound 1e as an anomeric mixture (224.4 g) as a yellow oil. The mixture was purified by reverse flash chromatography (neutral) twice to give pure compound 1e (75.5 g, 33.7%) as a colorless foam. 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.25 (s, 1 H), 8.72 (s, 1 H), 8.62 (s, 1 H), 8.05 (d, J=7.40 Hz, 2 H), 7.71-7.62 (m, 1 H), 7.60-7.50 (m, 2 H), 7.43-7.21 (m, 10 H), 6.38 (d, J=4.77 Hz, 1 H), 6.12 (t, J=5.33 Hz, 1 H), 4.88 (d, J=5.77 Hz, 1 H), 4.63 (s, 2 H), 4.56-4.45 (m, 2 H), 4.41 (d, J=11.92 Hz, 1 H), 4.25 (d, J=11.80 Hz, 1 H), 3.75-3.59 (m, 2 H), 2.06 (s, 3 H), 2.00 (s, 3 H).

[0385] 2a: 1-[(2R,3R,4S,5R)-4-benzyloxy-5-(benzyloxymethyl)-3-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl]-5-methyl-pyrimidine-2,4-dione To a solution of 1a (40.9 g, 74.0 mmol) in 500 ml of THF / EtOH (4:1) was added 221.9 ml (443.8 mmol) of aqueous NaOH (2 M) at 0° C. After removing the ice bath, the mixture was stirred for 2 h to allow complete conversion. The solution was brought to neutral pH by adding 2N HCl solution at 0° C. The solvent was concentrated in vacuo and the remaining aqueous phase was extracted twice with DCM (250 ml). The combined organic layers were washed with MgSO 4 Drying at rt and evaporation gave the deprotected product 2a (34.8 g, quantitative), which was used in the subsequent step without further purification. LCMS-Method A: UV-wavelength [nm] = 220: R t [minutes] = 1,64 Ionization method: ES + : [M+H] + = 469,2 1H-NMR (CDCl 3 , 400 MHz) δ[ppm]: 9.24-8.77 (m, 1 H), 7.45 (br s, 1 H), 7.41-7.30 (m, 8 H), 7.27 (br d, J=7.78 Hz, 2 H), 6.04 (br d, J=4.02 Hz, 1 H), 4.86 (br d, J=11.67 Hz, 1 H), 4.63-4.51 (m, 3 H), 4.45-4.31 (m, 2 H), 4.31-3.96 (m, 1 H), 3.84 (br d, J=11.29 Hz, 1 H), 3.76-3.64 (m, 2 H), 3.58 (d, J=10.42 Hz, 1 H), 2.96-2.62 (m, 1 H), 1.63-1.51 (m, 3 H).

[0386] 2b: 1-[(2R,3R,4S,5R)-4-benzyloxy-5-(benzyloxymethyl)-3-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl]pyrimidine-2,4-dione 1b (9.10 g, 16.9 mmol) was dissolved in 240 ml of THF / EtOH (3:1). At 0° C., 8.49 ml (84.5 mmol) of 1 M NaOH solution was added and the solution was stirred and allowed to come to room temperature. After 1 h, citric acid was added until a pH of 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. MgSO 4 After drying at 40° C., the solvent was removed to give 7.66 g (99.7%) of the crude product as a colorless foam which was used without further purification. LCMS-Method A: UV-wavelength [nm] = 220: R t [minutes] = 1,63 Ionization method: ES + : [M+H] + = 455,1 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.29 (s, 1 H), 7.69 (d, J=8.2 Hz, 1 H), 7.27 - 7.41 (m, 10 H), 5.88 (d, J=5.7 Hz, 1 H), 5.57 (d, J=7.2 Hz, 1 H), 5.37 (d, J=8.1 Hz, 1 H), 4.98 (t, J=5.4 Hz, 1 H), 4.80 (d, J=11.7 Hz, 1 H), 4.46 - 4.58 (m, 3 H), 4.27 - 4.37 (m, 1 H), 4.00 - 4.14 (m, 1H), 3.50 - 3.69 (m, 4 H).

[0387] 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-propanamide To a solution of compound 1c (72 g, 0.11 mol) in 1.7 L of THF / EtOH (4:1) was added 1 M NaOH solution (443 mL) dropwise at 0° C. The solution was stirred at 0° C. for 1 h to ensure complete conversion. The pH was adjusted to 7 by adding 1 N HCl and the solvent was removed. The residue was purified by HCl.2 The mixture was dissolved in 2×O (500 mL) and extracted with DCM (3×500 mL). The organic layers were combined and washed with anhydrous Na 2 SO 4 Drying at rt and concentration afforded compound 2c (113 g, quantitative) as a colorless solid, which was used in the next step without further purification. 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 12.07(s,1 H), 11.66 (s, 1 H), 8.10 (s, 1 H), 7.42-7.30 (m, 10 H), 5.92 (d, J=6.8Hz, 1 H), 4.99 (s, 1 H) , 4.87-4.84 (m, 2 H), 4.63 (d, J=15.6 Hz, 1 H), 4.56 (s, 2 H), 4.24 (d, J=4.8 Hz, 1 H), 3.69-3.62 (m, 4 H), 2.76-2.73 (m, 1 H), 1.13-1.04 (m, 7 H).

[0388] 2d: 9-[(2R,3R,4S,5R)-4-benzyloxy-5-(benzyloxymethyl)-3-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl]-1H-purin-6-one Following the procedure described for 2b, 1d (9.34 g, 16.65 mmol) was deprotected to give the title compound 2d (7.76 g, 97.4%) as a crude product which was used without further purification. LCMS-Method A: UV-wavelength [nm] = 220: R t [minutes] = 1,53 Ionization method: ES + : [M+H] + = 479,3 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 8.16 (s, 1 H), 7.97 - 8.04 (m, 1 H), 7.27 - 7.41 (m, 10 H), 5.97 (d, J=5.75 Hz, 1 H), 5.75 (s, 1 H), 5.03 (br s, 1 H), 4.79 - 4.92 (m, 2 H), 4.47 - 4.62 (m, 3 H), 4.24 - 4.34 (m, 1 H), 3.55 - 3.70 (m, 4 H).

[0389] 2e: N-[9-[(2R,3R,4S,5R)-4-benzyloxy-5-(benzyloxymethyl)-3-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl]purin-6-yl]benzamide Following the procedure described for 2b, 1e (144 g, 216 mmol) was deprotected to give the title compound 2e (126 g, quantitative, crude) as a colorless foam which was used without further purification. 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.23 (s, 1 H), 8.73 (s, 1 H), 8.59 (s, 1 H), 8.05 (br d, J=7.46 Hz, 2 H), 7.71-7.63 (m, 1 H), 7.61-7.52 (m, 2 H), 7.49-7.23 (m, 10 H), 6.16 (d, J=5.75 Hz, 1 H), 5.85 (d, J=7.46 Hz, 1 H), 5.14-4.99 (m, 2 H), 4.86 (d, J=11.86 Hz, 1 H), 4.63 (d, J=11.74 Hz, 1 H), 4.53 (s, 2 H), 4.38 (d, J=5.14 Hz, 1 H), 3.79-3.57 (m, 4 H).

[0390] 3a: 1-[(2R,3R,4S,5S)-4-benzyloxy-5-(benzyloxymethyl)-3-hydroxy-5-(triisopropylsilyloxymethyl)tetrahydrofuran-2-yl]-5-methyl-pyrimidine-2,4-dione Starting compound 2a (34.7 g, 74.2 mmol) and imidazole 16.8 g (244.7 mmol) were dissolved in dry DCM (300 ml). At room temperature, a solution of TIPS-chloride 16.2 g (18.0 ml, 81.6 mmol) in DCM (200 ml) was added and the reaction was stirred for 19 h. After quenching with EtOH, the solvent was evaporated and the residue was dissolved in EtOAc. The organic solution was diluted with H 2 O, 1M HCl, H 2 O and saturated NaHCO 3 solution, followed by saturated NaCl solution. MgSO 4 After drying at 40° C., the solvent was removed and the crude product (47.1 g) was purified by silica gel chromatography (10 to 100% EtOAc in n-heptane) to give the desired TIPS-protected product 3a as a colorless foam (37.5 g, 80.9%). LCMS-Method A: UV-wavelength [nm] = 220: R t [minutes] = 2,22 Ionization method: ES + : [M+H] + = 625,3 1H-NMR (CDCl 3 , 400 MHz) δ[ppm]: 8.52 (s, 1 H), 7.35-7.13 (m, 11 H), 5.88 (d, J=3.89 Hz, 1 H), 4.72-4.63 (m, 1 H), 4.60-4.51 (m, 1 H), 4.46 (s, 2 H), 4.34-4.26 (m, 1 H), 4.22 (d, J=6.27 Hz, 1 H), 3.90 (d, J=10.92 Hz, 1 H), 3.77 (d, J=1.00 Hz, 2 H), 3.63-3.51 (m, 2 H), 1.52 (d, J=0.88 Hz, 3H), 1.12-0.90 (m, 21 H).

[0391] 3b: 1-[(2R,3R,4S,5S)-4-benzyloxy-5-(benzyloxymethyl)-3-hydroxy-5-(triisopropylsilyloxymethyl)tetrahydrofuran-2-yl]pyrimidine-2,4-dione Starting from 2b (7.66 g, 16.8 mmol), the title compound was prepared as described in the protocol for 3a to give the desired silylated product 3b (8.69 g, 84.5%) after silica gel chromatography (20 to 80% EtOAc in n-heptane). LCMS-Method A: UV-wavelength [nm] = 220: R t [minutes] = 2,23 Ionization method: ES + : [M+H] + = 611,2 1H-NMR (CDCl 3 , 400 MHz) δ[ppm]: 9.04 (s, 1 H), 7.63 (d, J=8.0 Hz, 1 H), 7.37-7.34 (m, 8 H), 7.26-7.24 (m, 2 H), 5.96 (d, J=4.0 Hz, 1 H), 5.37 (d, J=8.0 Hz, 1 H), 4.77 (d, J=12.0 Hz, 1 H), 4.62 (d, J=12.0 Hz, 1 H), 4.49 (s, 2 H), 4.26 (d, J=8.0 Hz, 1 H), 3.83 (dd, J=8.0, 10.0 Hz, 2 H), 3.63 (dd, J=8.0, 10.0 Hz, 2 H), 1.15-1.04 (m, 21 H).

[0392] 3c: N-[9-[(2R,3R,4S,5S)-4-benzyloxy-5-(benzyloxymethyl)-3-hydroxy-5-(triisopropylsilyloxymethyl)tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]-2-methyl-propanamide To a solution of compound 2c (75 g, 133 mmol) in anhydrous DCM (1568 ml), 2Imidazole (38 g, 559 mmol) and TIPSCl (35.9 g, 186 mmol) were added under ambient conditions at 0° C. After stirring for 12 h at 10-15° C., the solution was poured into ice water (2 L) and extracted with DCM (3×1.5 L). The organic layers were combined, washed with brine (1 L) and diluted with anhydrous Na 2 SO 4 The residue was purified by column chromatography on silica gel (PE / EtOAc 2:1 to EtOAc) to give the title compound 3c (65 g, 68%) as a white foam. 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 12.07 (s,1 H), 11.61 (s, 1 H), 8.14 (s, 1 H), 7.37-7.22 (m, 10 H), 5.89 (d, J=6.8 Hz, 1 H), 5.72 (d, J=5.6 Hz, 1 H), 4.94-4.93 (m, 2 H), 4.90-4.53 (m, 3 H), 4.19 (d, J =4.4 Hz, 1 H), 3.92-3.88 (m, 2 H), 3.85-3.71 (m, 2 H), 2.78-2.71 (m, 1 H), 1.13-1.05 (m, 6 H), 1.00-0.94 (m, 21 H).

[0393] 3d: 9-[(2R,3R,4S,5S)-4-benzyloxy-5-(benzyloxymethyl)-3-hydroxy-5-(triisopropylsilyloxymethyl)tetrahydrofuran-2-yl]-1H-purin-6-one Starting from 2d (7.75 g, 16.2 mmol), the title compound was prepared as described in the protocol for 3a to give the desired silylated product 3d (8.60 g, 83.6%) after silica gel chromatography (0 to 100% EtOAc / MeOH (9:1) in n-heptane). LCMS-Method A: UV-wavelength [nm] = 220: R t [minutes] = 2,19 Ionization method: ES +: [M+H] + = 635,5 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 12.38 (br s, 1 H), 8,22 (s, 1 H), 8,03 (s, 1 H), 7.26 - 7.39 (m, 10 H), 5.94 (d, J=6.85 Hz, 1 H), 5.67 (d, J=6.48 Hz, 1 H), 4.98 - 4.74 (m, 2 H), 4.61 - 4.48 (m, 3 H), 4.24 (d, J=4.89 Hz, 1 H), 3.97 - 3.82 (m, 2 H), 3.69 (s, 2 H), 0.80 - 1.18 (m, 21 H).

[0394] 3e: N-[9-[(2R,3R,4S,5S)-4-benzyloxy-5-(benzyloxymethyl)-3-hydroxy-5-(triisopropylsilyloxymethyl)tetrahydrofuran-2-yl]purin-6-yl]benzamide To a mixture of compound 2e (135 g, 0.232 mol) and imidazole (47.4 g, 0.696 mol) in DCM (1.35 L) was added a solution of TIPSCl (80.6 g, 0.418 mol) in DCM (1.35 L) at room temperature. The mixture was stirred for 24 h. After quenching with EtOH (400 ml), the mixture was washed with water (1.5 L). The aqueous layer was extracted twice with EtOAc (1 L) and the combined organic layers were washed with anhydrous Na 2 SO 4 The mixture was dried at 40° C., filtered and concentrated in vacuo. The crude product was purified by flash chromatography (PE / EtOAc 2:1) to give the silyl ether 3e (145 g, 84.8%) as a white foam. 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.23 (s, 1 H), 8.74 (s, 1 H), 8.63 (s, 1 H), 8.06 (d, J=7.28 Hz, 2 H), 7.71-7.62 (m, 1 H), 7.60-7.51 (m, 2 H), 7.44-7.23 (m, 10 H), 6.13 (d, J=6.78 Hz, 1 H), 5.77 (d, J=6.40 Hz, 1 H), 5.21-5.10 (m, 1 H), 4.94 (d, J=11.92 Hz, 1 H), 4.66-4.50 (m, 3H), 4.30 (d, J=4.77 Hz, 1 H), 4.00-3.86 (m, 2 H), 3.74 (s, 2 H), 1.11-0.89 (m, 21 H).

[0395] 4a: 1-[(2R,3R,4S,5S)-3,4-dihydroxy-5-(hydroxymethyl)-5-(triisopropylsilyloxymethyl)-tetrahydrofuran-2-yl]-5-methyl-pyrimidine-2,4-dione In an autoclave, a solution of the bis-benzyl ether 3a (13.4 g, 21.4 mmol) in EtOH (100 ml) was degassed and purged with argon. 2 (20% carbon supported) was added and the solution was pressurized under 4 bar H 2 Set under atmosphere for 1 h. 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. LCMS-Method A: UV-wavelength [nm] = 220: R t [minutes] = 1,84 Ionization method: ES + : [M+H] + = 445,3 1H-NMR (CDCl 3, 400 MHz) δ[ppm]: 8.85 (s, 1 H), 7.25 (d, J=1.00 Hz, 1 H), 5.58 (d, J=6.15 Hz, 1 H), 4.71-4.61 (m, 1 H), 4.51-4.44 (m, 1 H), 4.02-3.95 (m, 1 H), 3.94-3.89 (m, 1 H), 3.88-3.77 (m, 3 H), 3.76-3.67 (m, 1 H), 3.22 (br dd, J=6.71, 3.83 Hz, 1 H), 1.93 (d, J=1.00 Hz, 3 H), 1.00 - 1.24 (m, 21 H).

[0396] 4b: 1-[(2R,3R,4S,5S)-3,4-dihydroxy-5-(hydroxymethyl)-5-(triisopropylsilyloxymethyl)-tetrahydrofuran-2-yl]pyrimidine-2,4-dione The title compound was prepared starting from 3b (8.69 g, 14.2 mmol) as described in the protocol for 4a to give the desired product 4b (6.21 g, quantitative, crude), which was used without purification. LCMS-Method A: UV-wavelength [nm] = 220: R t [minutes] = 1,78 Ionization method: ES + : [M+H] + = 431,1 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.29 (s, 1 H), 7.91 (d, J=8.0 Hz, 1 H), 5.87 (d, J=8.0 Hz, 1 H), 5.69 (d, J=8.0 Hz, 1 H), 5.28 (d, J=8.0 Hz, 1 H), 5.12 (t, J=4.0 Hz, 1 H), 5.00 (d, J=4.0 Hz, 1 H), 4.19 (dd, J=8.0, 12.0 Hz, 1 H), 4.05 (t, J=4.0 Hz, 2 H), 3.81 (dd, J=8.0, 12.0 Hz, 2H), 3.62 (d, J=4.0 Hz, 2 H), 1.05-1.00 (m, 21 H).

[0397] 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-propanamide To a solution of compound 3c (95 g, 0.132 mol) in anhydrous DCM (300 mL), 2 BCl at -70 °C under atmospheric pressure 3 (921 mL) was added. The reaction solution was stirred between -75 and -60 °C for 2 h, at which point complete conversion was detected by TLC. The mixture was treated with NH 3 About 200 mL of a saturated solution of in MeOH was added. 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 give the debenzylated product 4c (51 g, 71.6% yield) as a yellow solid. 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.86 (s, 2 H), 8.27 (s, 1 H), 5.83 (d, J=7.2 Hz, 1 H), 5.42 (s, 1 H), 5.06 (s, 2 H), 4.64 (s, 1 H), 4.17 (d, J =4.0Hz, 1H), 3.89 (d, J =10.8 Hz, 1 H), 3.79 (d, J =10.4 Hz, 1 H), 3.67 (s, 2 H), 2.80-2.73 (m, 1 H),1.17-1.08 (m, 6 H), 1.02-0.92 (m, 21 H).

[0398] 4d: 9-[(2R,3R,4S,5S)-3,4-dihydroxy-5-(hydroxymethyl)-5-(triisopropylsilyloxymethyl)-tetrahydrofuran-2-yl]-1H-purin-6-one Starting compound 3d (8.59 g, 13.53 mmol) was dissolved in DCM (200 ml). After cooling to -70 to -50 °C, BCl 3 74.4 ml (74.4 mmol) of a 1M solution in toluene was added over 15 min. After stirring for an additional 15 min at -70°C, the cooling bath was removed and stirring was continued at room temperature for an additional 30 min. The reaction solution was then diluted with NH 3 In 180 ml of a 7M solution of in MeOH, the mixture was added dropwise. After stirring for 20 min, the mixture was evaporated and the residue was dissolved in 100 ml of DCM / iPrOH (4:1). Saturated NaHCO 3 After washing with the solution, the layers were separated and the aqueous layer was extracted three times with DCM / iPrOH (4:1). The combined organic layers were washed with MgSO 4 It was dried at 40° C., filtered and evaporated. The crude product was purified by silica gel chromatography (0 to 10% MeOH in DCM) to give the title compound 4d (4.14 g, 67.3%) as a colorless foam. LCMS-Method A: UV-wavelength [nm] = 220: R t [minutes] = 1,74 Ionization method: ES - : [MH] - = 453,4 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 12.38 (m, 1 H), 8.33 (s, 1 H), 8.07 (s, 1 H), 5.87 (d, J=7.34 Hz, 1 H), 5.37 (d, J=6.97 Hz, 1 H), 5.01 - 5.14 (m, 2 H), 4.64 - 4.76 (m, 1 H), 4.15 - 4.20 (m, 1 H), 3.81 - 3.93 (m, 2 H), 3.65 (d, J=5.14 Hz, 2 H), 0.94 - 1.14 (m, 21 H).

[0399] 4e: N-[9-[(2R,3R,4S,5S)-3,4-dihydroxy-5-(hydroxymethyl)-5-(triisopropylsilyloxymethyl)-tetrahydrofuran-2-yl]purin-6-yl]benzamide A solution of compound 3e (31 g, 42.0 mmol) in DCM (620 ml) was added with BCl 3 (252 mL, 0.252 mol) was added dropwise. The mixture was then rinsed with N 2 The mixture was stirred at −70° C. for 30 min and then warmed to 15° C. After stirring at this temperature for an additional 30 min, the reaction mixture was diluted with 7M NH 3 (600 ml). After evaporation, the residue was dissolved in 3 L of DCM / iPrOH (4:1) and diluted with 1 L of water and saturated NaHCO 3 The combined aqueous layers were extracted with DCM / iPrOH (4:1) 2×1 L, and the combined organic layers were washed with anhydrous Na 2 SO 4 The mixture was dried over 1000 ml of ethyl acetate, filtered and concentrated in vacuo. The residue was purified by flash chromatography (PE / EtOAc 1:4) to give compound 4e (16.9 g, 72.2% yield) as a white solid. 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.23 (s, 1 H), 8.77 (s, 1 H), 8.72 (s, 1 H), 8.06 (d, J=7.28 Hz, 2 H), 7.70-7.61 (m, 1 H), 7.60-7.51 (m, 2 H), 6.06 (d, J=7.53 Hz, 1 H), 5.48 (d, J=6.90 Hz, 1 H), 5.22-5.11(m, 2 H), 4.93-4.83 (m, 1 H), 4.24 (t, J=4.77 Hz, 1 H), 3.92 (q, J=10.67Hz, 2H), 3.78-3.62 (m, 2 H), 1.14-1.01 (m, 21 H).

[0400] 5a: 1-[(2R,6S)-3,5-dihydroxy-6-(hydroxymethyl)-6-(triisopropylsilyloxymethyl)-1,4-dioxan-2-yl]-5-methyl-pyrimidine-2,4-dione Starting material 4a (2.93 g, 6.6 mmol) was dissolved in acetone / H 2 The mixture was dissolved in 120 ml of NaIO (4:1). 4 (1.69g, 7.9mmol) of H 2 A solution of 1H2O (50 ml) was added and the mixture was stirred at room temperature for 1 h. The precipitate was filtered off and the filtrate was concentrated in vacuo. The remaining aqueous solution was extracted with EtOAc (200 ml). The organic layer was 2 After washing with O and saturated NaCl solution, it was diluted with MgSO 4 Drying at rt and removal of the solvent under reduced pressure gave 2.86 g (94.1%) of the desired product as a colorless solid which was used without further purification. LCMS-Method B-1: UV-wavelength [nm]=220:R t [minute]=1.04 Ionization method: ES + :[M+HH 2 O] + =443.2

[0401] 5b: 1-[(2R,6S)-3,5-dihydroxy-6-(hydroxymethyl)-6-(triisopropylsilyloxymethyl)-1,4-dioxan-2-yl]pyrimidine-2,4-dione The title compound was prepared starting from 4b (6.21 g, 14.4 mmol) and following the protocol described in 4a to give the desired product 5b (6.38 g, 99%) as a crude product which was used without further purification. LCMS-Method B-1: UV-wavelength [nm]=220:R t [minute]=1.00 Ionization method: ES + :[M+HH 2 O] + =429.5

[0402] 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-propanamide A solution of compound 4c (25 g, 46 mmol) in a mixed solvent of acetone (485 mL) and water (145 mL) was added to NaIO at 25 °C. 4 A solution of (13.9 g, 65 mmol) in water (150 ml) was added and the mixture was stirred for 12 h. After evaporation of the solvent in vacuo, the residue was dissolved in EtOAc (500 ml) and saturated NaHCO 3 The organic layer was washed with sodium hydroxide (500 ml) and brine (500 ml). 2 SO 4 The mixture was dried at rt, filtered and concentrated in vacuo to give crude 5c (25.7 g, quantitative) as a white solid which was used without further purification. 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 12.05 (br s, 1 H), 11.79 (s, 1 H), 8.17 (s, 1 H), 7.52 (d, J=5.62 Hz, 1 H), 6.23 (s, 1 H), 5.34 (d, J=5.62 Hz, 1 H), 5.23 (s, 1 H), 4.46 (d, J=9.54 Hz, 1 H), 4.15-4.04 (m, 1 H), 3.93 (d, J=9.78 Hz, 1 H), 3.85 (d, J=10.03 Hz, 1 H), 3.71 (d, J=10.03 Hz, 1 H), 2.85-2.66 (m, 1 H), 1.12 (d, J=6.85 Hz, 6 H), 1.07-0.95 (m, 21 H).

[0403] 5d: 9-[(2R,6S)-3,5-dihydroxy-6-(hydroxymethyl)-6-(triisopropylsilyloxymethyl)-1,4-dioxan-2-yl]-1H-purin-6-one Starting material 4d (4.14 g, 9.1 mmol) was dissolved in acetone / H 2 The mixture was dissolved in 120 ml of NaIO (4:1). 4 (2.63g, 12.3mmol) of H 2 A solution of 200 ml in 50 ml of HO was added and the mixture was stirred at room temperature for 18 h. The precipitate was filtered off and the filtrate was concentrated in vacuo. The remaining aqueous solution was diluted with saturated NaHCO 3 100 ml of solution was added and the aqueous layer was extracted twice with 100 ml of DCM / iPrOH (4:1). The organic layer was diluted with MgSO 4 Drying at rt and removal of the solvent under reduced pressure gave the desired product 5d (4.27 g, 99.7%) as a colorless foam which was used without further purification. LCMS-Method A: UV-wavelength [nm]=220:R t [minute]=1.82 Ionization method: ES + :[M+HH 2 O] + =453.3

[0404] 6a: 1-[(2R,6S)-6-(hydroxymethyl)-6-(triisopropylsilyloxymethyl)morpholin-2-yl]-5-methyl-pyrimidine-2,4-dione A solution of compound 5a (31 g, 67 mmol) in anhydrous MeOH (527 ml) was added with N 2 At room temperature under atmospheric (NH 4 ) 2 B 2 O 7 4H 2 2H2O (19.5 g, 74 mmol) was added. The mixture was stirred for 2 h, followed by the addition of AcOH (8.08 g, 134 mmol), 4 Å molecular sieves (62 g) and NaBH 3 CN (8.44 g, 134 mmol) was added. The mixture was irradiated with N until TLC showed complete consumption of the starting material. 2 The mixture was stirred at room temperature under atmospheric pressure for 12 hours. The solvent was removed in vacuo and the residue was dissolved in water (200 ml) and extracted with DCM (3×200 ml). The combined organic layers were washed with Na 2 SO 4 The mixture was dried at 40° C. and concentrated in vacuo. The residue was purified by column chromatography on silica gel (DCM / MeOH 50:1 to 10:1) to give compound 6a (19.5 g, 67%) as a colorless solid. 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.27 (br s, 1 H), 7.71-7.52 (m, 1 H), 5.90-5.68 (m, 1 H), 4.68-4.46 (m, 1 H), 4.05-3.83 (m, 2 H), 3.40 (br d, J=5.38 Hz, 2 H), 2.83 (br d, J=9.78 Hz, 1 H), 2.79-2.72 (m, 1 H), 2.66 (br d, J=12.96 Hz, 2 H),1.79 (s, 3 H), 1.22-0.83 (m, 23 H).

[0405] 6c: N-[9-[(2R,6S)-6-(hydroxymethyl)-6-(triisopropylsilyloxymethyl)morpholin-2-yl]-6-oxo-1H-purin-2-yl]-2-methyl-propanamide The title compound was synthesized starting from compound 5c (25.7 g, 46.4 mmol) following the protocol described in 6a to afford the desired morpholine 6c as a colorless solid (7.5 g, 30.6%). 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.56 (br s, 1 H), 8.13 - 8.25 (m, 1 H), 5.67 - 5.81 (m, 2 H), 4.56 (br s, 1 H), 4.08 (br d, J=9.54 Hz, 1 H), 3.83 (br d, J=9.29 Hz, 1 H), 3.39 - 3.48 (m, 2 H), 3.10 (br d, J=9.54 Hz, 1 H), 2.65 - 2.93 (m, 4 H), 1.12 (br d, J=6.60 Hz, 7 H), 0.96 - 1.07 (m, 21 H).

[0406] 7a: 1-[(2R,6S)-6-(hydroxymethyl)-4-isopropyl-6-(triisopropylsilyloxymethyl)morpholin-2-yl]-5-methyl-pyrimidine-2,4-dione (from 6a) To a solution of compound 6a (19.5 g, 45.7 mmol) in anhydrous MeOH (390 mL), 2 At room temperature under atmospheric pressure, 4 Å molecular sieves (39 g), acetone (13.25 g, 228 mmol) and NaBH 3 CN (14.4 g, 228 mmol) was added. After stirring for 2 h, the mixture was adjusted to pH=5-6 with AcOH (ca. 2.0 ml) and stirring was continued for 12 h, at which point complete consumption of starting material was detected by TLC. The solvent was removed in vacuo and the residue was dissolved in water (500 ml). After extraction with DCM (3×500 ml), the organic layers were combined and washed with Na 2 SO 4 After purification by silica gel chromatography (DCM / MeOH 50:1 to 10:1), the title compound 7a was obtained as a colorless solid (17 g, 81%).

[0407] 7a: 1-[(2R,6S)-6-(hydroxymethyl)-4-isopropyl-6-(triisopropylsilyloxymethyl)morpholin-2-yl]-5-methyl-pyrimidine-2,4-dione (from 5a) Starting compound 5a (2.85 g, 6.2 mmol), NEt 3 (1.26 g, 1.74 ml, 12.4 mmol), AcOH (3.73 g, 3.56 ml, 61.9 mmol) and 924 mg (1.34 ml, 15.5 mmol) of isopropylamine were dissolved in dry MeOH (60 ml). At room temperature, 1.64 g (24.8 mmol) of sodium cyanoborohydride was added and the solution was stirred at 60° C. for 2 h. After cooling to room temperature, the reaction mixture was diluted with saturated NaHCO 3 The solution was poured into 100 mL and the solvent was concentrated under reduced pressure. The remaining aqueous phase was extracted with EtOAc and this was diluted with saturated NaHCO 3 solution and saturated NaCl solution. MgSO 4 After drying at 40° C., the solvent was evaporated and the crude product (3.0 g) was purified by silica gel chromatography (10 to 80% ethyl acetate in n-heptane) to give the desired morpholine 7a (1.76 g, 60.7%) as a colorless solid. LCMS-Method A: UV-wavelength [nm] = 220: R t [minutes] = 1,80 Ionization method: ES + : [M+H] + = 470,2 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.30 (s, 1 H), 7.63 (s, 1 H), 5.77 (dd, J=10.42, 2.89 Hz, 1 H), 4.61 (t, J=5.90 Hz, 1 H), 4.00 (d, J=9.29 Hz, 1 H), 3.74 (d, J=9.03 Hz, 1 H), 3.53-3.41 (m, 2 H), 2.83-2.63 (m, 3 H), 2.26 (br d, J=11.29 Hz, 1 H), 2.21-2.07 (m, 1 H), 1.80 (s, 3H), 1.14-1.00 (m, 21 H), 0.97 (d, J=6.53 Hz, 6 H).

[0408] 7b: 1-[(2R,6S)-6-(hydroxymethyl)-4-isopropyl-6-(triisopropylsilyloxymethyl)morpholin-2-yl]-pyrimidine-2,4-dione (from 5b) A solution of 5b (6.38 g, 14.3 mmol) in MeOH (150 ml) was added with AcOH (8.62 g, 8.23 ​​ml, 142.8 mmol) and NEt 3 (2.90 g, 3.98 ml, 28.6 mmol) and 2.13 g (3.09 ml, 35.7 mmol) of isopropylamine were added. After 1 h, 3.78 g (57.1 mmol) of sodium cyanoborohydride was added and stirring was continued for 1 h at room temperature followed by an additional 1 h at 65° C. The mixture was cooled to room temperature and saturated NaHCO 3 150 ml of solution was added and MeOH was evaporated in vacuo. The aqueous solution was extracted with EtOAc (350 ml) and the organic layer was washed with saturated NaHCO 3 solution and saturated NaCl solution. MgSO 4 After drying at 40° C., the solvent was removed and the crude product was purified by silica gel chromatography (0 to 5% MeOH in DCM) to give the title compound 7b (3.18 g, 48.9%) as a colorless solid. LCMS-Method A: UV-wavelength [nm] = 220: R t [minutes] = 1,76 Ionization method: ES + : [M+H] + = 456,4 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.32 (bs, 1 H), 7.77 (d, J=8.07 Hz, 1 H), 5.75 (dd, J=9.90, 2.81 Hz, 1 H), 5.61 (d, J=8.07 Hz, 1 H), 4.62 (t, J=5.93 Hz, 1 H), 3.99 (d, J=9.29 Hz, 1 H), 3.74 (d, J=9.17 Hz, 1 H), 3.45 (dd, J=10.33, 6.05 Hz, 2 H), 2.82 (br d, J=10.27 Hz, 1 H), 2.64 - 2.77 (m, 2 H), 2.24 (d, J=11.37 Hz, 1 H), 2.03 - 2.13 (m, 1 H), 1.00 - 1.10 (m, 21 H), 0.96 (m, 6 H).

[0409] 7c: N-[9-[(2R,6S)-6-(hydroxymethyl)-4-isopropyl-6-(triisopropylsilyloxymethyl)morpholin-2-yl]-6-oxo-1H-purin-2-yl]-2-methyl-propanamide (from 6c) Starting from 6c (7.5 g, 14.0 mmol), the title compound was synthesized according to the protocol described in 7a (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%). 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 12.09 (s, 1 H), 11.56 (s, 1 H), 8.22 (s, 1 H), 5.80 (dd, J=9.79, 2.76 Hz, 1 H), 4.60 (t, J=6.27 Hz, 1 H), 4.09 (d, J=8.78 Hz, 1 H), 3.71 (d, J=8.78 Hz, 1 H), 3.48 (d, J=6.27 Hz, 2 H), 2.98 (br d, J=10.04 Hz, 1 H), 2.89-2.65 (m, 3 H), 2.43 (t, J = 10.42 Hz, 1 H), 2.34 (d, J=11.29 Hz, 1 H), 1.13 (d, J=6.78 Hz, 6 H), 1.08-1.01 (m, 21 H), 0.99 (d, J=6.27 Hz, 6 H).

[0410] 7d: 9-[(2R,6S)-6-(hydroxymethyl)-4-isopropyl-6-(triisopropylsilyloxymethyl)morpholin-2-yl]-1H-purin-6-one (from 5d) Starting compound 5d (4.27 g, 9.1 mmol) was dissolved in MeOH (100 ml). 3 (1.84 g, 2.53 ml, 18.1 mmol), AcOH (5.44 g, 5.19 ml, 90.6 mmol) and 808 mg (1.17 ml, 13.6 mmol) of isopropylamine were added and the solution was stirred at room temperature for 1 hour. Sodium cyanoborohydride (1.80 g, 27.2 mmol) was added and the reaction was stirred at room temperature for 30 minutes and at 60° C. for an additional 30 minutes, at which point complete conversion was detected. The reaction mixture was diluted with saturated NaHCO 3 100 ml of solution was added and MeOH was evaporated. The remaining aqueous phase was extracted with EtOAc (250 ml). The organic layer was washed with saturated NaHCO 3 solution and NaCl solution, and washed with MgSO 4 The mixture was dried at rt and evaporated. The crude product was purified by silica gel chromatography (0 to 5% MeOH in DCM) to give the title compound 7d as a colorless solid (2.68 g, 61.7%). LCMS-Method A: UV-wavelength [nm] = 220: R t [minutes] = 1,80 Ionization method: ES + : [M+H] + = 480,4 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 12.35 (br s, 1 H), 8.31 (s, 1 H), 8.06 (s, 1 H), 5.91 (dd, J=9.90, 2.81 Hz, 1 H), 4.57 (t, J=6.05 Hz, 1 H), 4.11 (d, J=9.05 Hz, 1 H), 3.77 (d, J=9.05 Hz, 1 H), 3.40 - 3.50 (m, 2 H), 2.94 - 3.02 (m, 1 H), 2.67 - 2.87 (m, 2 H), 2.54 - 2.63 (m, 1 H), 2.33 (d, J=11.37 Hz, 1 H), 0.97 - 1.11 (m, 27 H).

[0411] [ka]

[0412] 1g: [(2S,3S,4R,5R)-4-acetoxy-3-benzyloxy-2-(benzyloxymethyl)-5-(6-chloropurine-9-yl)tetrahydrofuran-2-yl]methyl acetate Carbohydrate building block G3 (12.60 g, 25.9 mmol) and 6.19 g (38.9 mmol) of 6-chloro-purine were dissolved in dry ACN (175 ml). Under Ar atmosphere, DBU (12.07 g, 11.84 ml, 77.7 mmol) and TMSOTf (23.26 g, 18.99 ml, 103.6 mmol) were added at 0° C. The ice bath was removed and the reaction was stirred at room temperature for 1 h and at 80° C. for an additional 1 h, at which point complete conversion was detected. The solution was cooled to room temperature and saturated NaHCO 3The solution was poured into 500 ml. After vigorous stirring for 1 h, the aqueous solution was extracted with EtOAc (300 ml). The organic layer was separated and washed with saturated NaHCO 3 solution / H 2 HO (1:1) and saturated NaCl solution, then washed with MgSO 4 It was dried at 40° C. and evaporated. The crude product (15.78 g) was purified on silica gel (10 to 100% EtOAc in n-heptane) to give the title compound 1g (12.31 g, 81.8%) as a pale yellow foam. LCMS-Method C: UV-wavelength [nm] = 220: R t [minutes] = 2,57 Ionization method: ES - : [M-H+FA] - = 625,1 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 8.80 (s, 1 H), 8.75 (s, 1 H), 7.16 - 7.39 (m, 10 H), 6.38 (d, J=4.2 Hz, 1 H), 6.10 (dd, J=5.6, 4.3 Hz, 1 H), 4.86 (d, J=5.7 Hz, 1 H), 4.54 - 4.68 (m, 2 H), 4.37 - 4.47 (m, 3 H), 4.24 (d, J=12.0 Hz, 1 H), 3.69 (d, J=10.1 Hz, 1 H), 3.60 (d, J=10.1 Hz, 1 H), 2.05 (s, 3 H), 1.99 (s, 3 H).

[0413] 2h: (2R,3R,4S,5R)-2-(6-aminopurin-9-yl)-4-benzyloxy-5-(benzyloxymethyl)-5-(hydroxymethyl)tetrahydrofuran-3-ol 1g of chloropurine riboside (12.30 g, 21.2 mmol) was dissolved in ACN (40 ml). After adding 68.0 g (77.2 ml, 1.40 mol) of aqueous ammonia (35%), the reaction solution was transferred into an autoclave and heated at 70° C. for 18 h. The reaction mixture was evaporated and the aqueous residue was extracted three times with 150 ml of DCM / isopropanol (4:1). The organic layers were combined and washed with MgSO 4 The mixture was dried at 400° C. and evaporated to give 9.37 g of crude product, which was recrystallized from ACN (400 ml) to give the adenosine analog 2h (6.92 g, 68.5%) as a colorless solid. LCMS-Method C: UV-wavelength [nm] = 220: R t [minutes] = 1,69 Ionization method: ES + : [M+H] + = 477,9 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 8.22 (s, 1 H), 8.12 (s, 1 H), 7.24 - 7.42 (m, 12 H), 6.01 (d, J=5.7 Hz, 1 H), 5.72 (d, J=7.3 Hz, 1 H), 5.01 (br t, J=5.1 Hz, 1 H), 4.91 - 4.98 (m, 1 H), 4.84 (d, J=11.9 Hz, 1 H), 4.60 (d, J=11.7 Hz, 1 H), 4.51 (s, 2 H), 4.34 (d, J=5.3 Hz, 1 H), 3.57 - 3.72 (m, 4 H).

[0414] 2e: N-[9-[(2R,3R,4S,5R)-4-benzyloxy-5-(benzyloxymethyl)-3-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl]purin-6-yl]benzamide 2h (6.91 g, 14.5 mmol) was dissolved in 100 mL of pyridine under an Ar atmosphere. At room temperature, 10.17 g (8.41 mL, 72.4 mmol) of benzoyl chloride was added and the reaction was stirred for 1 h. The solvent was removed in vacuo and the residue was purified by H 2The mixture was dissolved in 250 mL of 1N HCl (2×250 mL), and extracted with EtOAc. The organic layer was separated and washed with 1N HCl (2×250 mL), H 2 O (1 x 200 ml), saturated NaHCO 3 The mixture was washed with saturated NaCl solution (1×200 ml) and MgSO 4 After drying at 40° C., the solvent was removed and the crude product (14.66 g, yellow foam) was dissolved in 140 ml of pyridine / EtOH (1:1). At room temperature, 2N NaOH (108.5 ml, 217.1 mmol) was added and the reaction mixture was stirred for 30 min. After adding AcOH (11 ml), the reaction solution was concentrated in vacuo. The residue was diluted with H 2 The organic layer was washed with 1N HCl (2×200 mL), H2O (200 mL) and extracted with EtOAc (200 mL). 2 O (1 × 200 ml) and saturated NaHCO 3 Wash with solution (1 × 200 ml) and MgSO 4 The crude product (9.0 g) was purified on silica (20 to 100% MeOH / EtOAc (9:1) in n-heptane) to give the title compound 2e (7.25 g, 86.1%) as a colorless foam. LCMS-Method C: UV-wavelength [nm] = 220: R t [minutes] = 2,12 Ionization method: ES + : [M+H] + = 582,4 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.19 (s, 1 H), 8.72 (s, 1 H), 8.58 (s, 1 H), 8.05 (br d, J=7.3 Hz, 2 H), 7.64 (br t, J=7.3 Hz, 1 H), 7.52 - 7.60 (br t, 2 H), 7.25 - 7.44 (m, 10 H), 6.15 (d, J=5.6 Hz, 1 H), 5.82 (d, J=7.5 Hz, 1 H), 4.99 - 5.08 (m, 2 H), 4.86 (d, J=11.9 Hz, 1 H), 4.62 (d, J=11.9 Hz, 1 H), 4.52 (s, 2 H), 4.38 (d, J=5.1 Hz, 1 H), 3.59 - 3.74 (m, 4 H).

[0415] [ka]

[0416] 8: [(2S,6R)-6-(2,4-dioxopyrimidin-1-yl)-4-isopropyl-2-(triisopropylsilyloxymethyl)-morpholin-2-yl]methyl benzoate Starting material 7b (6.00 g, 13.2 mmol) was dissolved in 80 ml of dry pyridine. Benzoyl chloride (2.80 g, 2.32 ml, 19.75 mmol) was added at room temperature and the reaction was stirred for 20 h to achieve complete conversion. After evaporation of the solvent, the residue was dissolved in EtOAc and washed with 2×100 ml of 10% citric acid solution, H 2 O, saturated NaHCO 3 The organic layer was washed with MgSO 4 Drying at rt and evaporation gave 7.99 g (quantitative) of the title compound as a colorless foam which was used without further purification. LCMS-Method A: UV-wavelength [nm]=220:R t [minutes]=2.23 Ionization method: ES + :[M+H] + =560.5

[0417] 9: [(2S,6R)-6-(4-amino-2-oxo-pyrimidin-1-yl)-4-isopropyl-2-(triisopropylsilyloxymethyl)-morpholin-2-yl]methyl benzoate Starting compound 8 (crude 7.99 g, 13.2 mmol) was dissolved in dry ACN (200 ml). 3 (22.88 g, 31.4 ml, 223.8 mmol) and 11.13 g (158.0 mmol) of 1H-1,2,4-triazole were added, and the solution was cooled to 0 °C and POCl 3 A solution of (6.06 g, 3.68 ml, 39.5 mmol) in dry ACN (50 ml) was added with vigorous stirring. The ice bath was removed and the solution was stirred for 2 h. Approximately 200 ml of solvent was evaporated under reduced pressure and the remaining solution was diluted with saturated NaHCO 3 solution / H 2 The aqueous mixture was poured into 500 ml of DCM (1:1). The aqueous mixture was extracted three times with DCM (150 ml) and the combined organic extracts were washed with MgSO 4 After evaporation of the solvent, the crude intermediate (9.1 g, yellow foam) was dissolved in ACN (200 ml) and 100 ml of aqueous ammonia (32%) was added. The reaction solution was stirred at room temperature for 18 h, at which point complete conversion was achieved. The solvent was removed under reduced pressure and H 2 O (100 ml) was added to the residue. Extraction was performed with DCM (2×200 ml) and the organic phase was washed with MgSO 4 Drying at rt and evaporation of the solvent gave crude compound 9 (7.65 g, quantitative), which was used without further purification. LCMS-Method A: UV-wavelength [nm]=220:R t [minutes]=2.02 Ionization method: ES - :[MH] - =557.4

[0418] 10: [(2S,6R)-6-(4-benzamido-2-oxo-pyrimidin-1-yl)-4-isopropyl-2-(triisopropylsilyloxymethyl)morpholin-2-yl]methyl benzoate Starting compound 9 (7.64 g, 13.2 mmol) was dissolved in 130 ml of dry pyridine. At room temperature, 2.90 g (2.40 ml, 20.5 mmol) of benzoyl chloride was added and the solution was stirred for 20 h, at which point complete conversion had occurred. After evaporation of the solvent, the residue was dissolved in EtOAc and washed with 2×100 ml of 10% citric acid solution, H 2 O, saturated NaHOC 3 The organic layer was washed with MgSO 4 Drying at rt and evaporation gave 8.88 g of crude product, which was purified by silica gel chromatography (0 to 100% EtOAc in n-heptane) to give the title compound 10 (6.69 g, 76.7%) as a pale yellow foam. LCMS-Method A: UV-wavelength [nm]=220:R t [minute]=2.29 Ionization method: ES - :[MH] - =661.5

[0419] 7f: N-[1-[(2R,6S)-6-(hydroxymethyl)-4-isopropyl-6-(triisopropylsilyloxymethyl)morpholin-2-yl]-2-oxo-pyrimidin-4-yl]benzamide Compound 10 (3.63 g, 5.48 mmol) was dissolved in 45 ml of pyridine / EtOH (2:1). At 0° C., 27.4 ml (27.4 mmol) of 1 M NaOH solution was added. The solution was stirred at 0° C. for 30 min and at room temperature for another 3 h to ensure complete conversion. The pH was adjusted to about 6 by adding citric acid monohydrate (about 2.0 g). The organic solvent was removed at 35° C. and the aqueous solution was extracted with EtOAc. The organic phase was washed with 150 ml of 10% citric acid solution, H 2 O, saturated NaHCO 3 The solution was washed three times with NaCl solution and MgSO 4 After drying at 40° C., the solvent was evaporated. The resulting crude product (2.73 g, yellow foam) was purified by silica gel chromatography (10 to 70% EtOAc in n-heptane) to give the title compound 7f (2.25 g, 73.4%) as a light yellow foam. LCMS-Method A: UV-wavelength [nm] = 220: R t [minutes] = 1,89 Ionization method: ES + : [M+H] + = 559,5 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.23 (s, 1 H), 8.31 (d, J=7.5 Hz, 1 H), 8.01 (d, J=7.3 Hz, 2 H), 7.63 (t, J=7.5 Hz, 1 H), 7.52 (m, 2 H), 7.37 (d, J=7.5 Hz, 1 H), 5.86 (dd, J=9.5, 2.6 Hz, 1 H), 4.67 (t, J=5.9 Hz, 1 H), 4.06 (d, J=9.4 Hz, 1 H), 3.76 (d, J=9.3 Hz, 1 H), 3.51 (m, 2 H), 2.98 (br d, J=10.5 Hz, 1 H), 2.64 - 2.83 (m, 2 H), 2.31 (m, 1 H), 2.00 (m, 1 H), 0.93 - 1.14 (m, 27 H).

[0420] [ka]

[0421] 11: [(2S,6R)-4-isopropyl-6-(6-oxo-1H-purin-9-yl)-2-(triisopropylsilyloxymethyl)morpholin-2-yl]methyl benzoate Starting material 7d (2.68 g, 5.59 mmol), DIPEA (2.53 g, 3.42 ml, 19.55 mmol) and DMAP (0.34 g, 2.79 mmol) were dissolved in dry DCM (50 ml). At room temperature, 1.18 g (0.97 ml, 8.38 mmol) of benzoyl chloride was added and the reaction was stirred for 18 h to achieve complete conversion. After addition of MeOH (20 ml), the solvent was evaporated and the residue was dissolved in EtOAc and washed with 2×100 ml of 10% citric acid solution, saturated NaHCO 3The organic layer was washed with MgSO 4 Drying at rt and evaporation afforded the title compound 11 (3.47 g, quant., crude) as a colorless foam which was used without further purification. LCMS-Method A: UV-wavelength [nm]=220:R t [minutes]=2.18 Ionization method: ES + :[M+H] + =584.4

[0422] 12: [(2S,6R)-6-(6-chloropurine-9-yl)-4-isopropyl-2-(triisopropylsilyloxymethyl)morpholin-2-yl]methyl benzoate Starting compound 11 (crude 2.86 g, 4.90 mmol), N,N-dimethylaniline (1.28 g, 1.34 ml, 10.53 mmol) and tetraethylammonium chloride (1.24 g, 7.35 mmol) were dissolved in dry ACN (30 ml). 3 (1.70 g, 1.03 ml, 11.07 mmol) was added at room temperature and the solution was refluxed for 3 h. After cooling to room temperature, the mixture was diluted with saturated NaHCO 3 Pour into 150 ml of solution and add solid NaHCO 3 (3.0 g) was added. The reaction was stirred for 30 min and extracted with DCM (100 ml). The layers were separated and the aqueous phase was extracted twice with DCM (50 ml). The combined organic layers were washed with MgSO 4 After drying and evaporation of the crude product on silica gel (0 to 50% EtOAc in n-heptane), the title compound 12 (1.52 g, 51.4%) was isolated as a pale yellow foam. LCMS-Method A: UV-wavelength [nm]=220:R t [minutes]=2.33 Ionization method: ES + :[M+H] + =602.4

[0423] 13: [(2S,6R)-6-(6-benzamidopurin-9-yl)-4-isopropyl-2-(triisopropylsilyloxymethyl)morpholin-2-yl]methyl benzoate Chloropurine 12 (1.54 g, 2.56 mmol), benzamide (646 mg, 3.84 mmol) and Cs 2 CO 3 (1.25 g, 3.84 mmol) was dissolved in 30 ml of dioxane. 2 After addition of 1,2-dichloromethane (43 mg, 191.8 μmol) and xantphos (111 mg, 191.8 μmol), the reaction solution was stirred at 100° C. under argon for 1 h to achieve complete conversion. The reaction was cooled to room temperature and diluted with EtOAc (50 ml). The solution was filtered and the filtrate was evaporated in vacuo. The crude product (2.48 g) was purified on silica gel (10 to 100% EtOAc in n-heptane) to give the desired compound 13 (1.29 g, 73.4%) as a colorless foam. LCMS-Method A: UV-wavelength [nm]=220:R t [minutes]=2.26 Ionization method: ES + :[M+H] + =687.6

[0424] 7e: N-[9-[(2R,6S)-6-(hydroxymethyl)-4-isopropyl-6-(triisopropylsilyloxymethyl)morpholin-2-yl]purin-6-yl]benzamide Benzoate 13 (1.00 g, 1.46 mmol) was saponified according to the protocol described in 7f. The title compound 7e (731 mg, 86.2%, crude) could be isolated as a pale yellow foam without chromatographic purification. LCMS-Method A: UV-wavelength [nm] = 220: R t [minutes] = 1,97 Ionization method: ES + : [M+H] + = 583,5 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.17 (s, 1 H), 8.75 (s, 1 H), 8.71 (s, 1 H), 8.04 (d, J=7.2 Hz, 2 H), 7.65 (t, J=7.3 Hz, 1 H), 7.55 (m, 2 H), 6.11 (dd, J=9.9, 2.7 Hz, 1 H), 4.61 (t, J=5.9 Hz, 1 H), 4.15 (d, J=9.1 Hz, 1 H), 3.85 (d, J=9.2 Hz, 1 H), 3.46 (br d, J=6.7 Hz, 2 H), 3.08 (br d, J=10.4 Hz, 1 H), 2.71 - 2.89 (m, 3 H), 2.37 (d, J=11.5 Hz, 1 H), 0.95 - 1.19 (m, 27 H).

[0425] [ka]

[0426] 14a: 1-[(2R,6S)-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-isopropyl-6-(triisopropylsilyloxymethyl)morpholin-2-yl]-5-methyl-pyrimidine-2,4-dione Primary alcohol 7a (810 mg, 1.7 mmol) and NEt 3 (873 mg, 1.20 ml, 8.6 mmol) was dissolved in DCM (30 ml). At room temperature, DMT-Cl (716 mg, 2.1 mmol) was added and the reaction was stirred at room temperature for 2 h. An additional 0.6 eq. of DMT-Cl was added and after stirring overnight the reaction showed complete conversion. The crude product solution was transferred directly onto a silica column and purified using 0 to 65% EtOAc in n-heptane to give the desired product 14a (1.19 g, 89.0%) as a light yellow foam. LCMS-Method A: UV-wavelength [nm]=220:R t [minutes]=2.38 Ionization method: ES + :[M+H]+ =772.5

[0427] 14b: 1-[(2R,6S)-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-isopropyl-6-(triisopropylsilyloxymethyl)morpholin-2-yl]pyrimidine-2,4-dione 7b (1.00 g, 2.2 mmol) and DIPEA (1.42 g, 1.92 ml, 11.0 mmol) were dissolved in DCM (30 ml). 948 mg (2.7 mmol) of DMT-chloride was added and the reaction was stirred at room temperature overnight to achieve complete conversion. After addition of isolute-sorbent, the organic solvent was removed and the solid material was precipitated on silica (n-heptane + 0.5% NEt 3 Preconditioned with 0 to 75% EtOAc in n-heptane) to give the DMT-protected product 14b (1.61 g, 96.8%) as a pale yellow foam. LCMS-Method A: UV-wavelength [nm]=220:R t [minutes]=2.34 Ionization method: ES + :[M+H] + =758.3

[0428] 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-propanamide Following the protocol described in 14a, starting compound 7c (1.00 g, 1.77 mmol) was protected. 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%). LCMS-Method C: UV-wavelength [nm]=220:R t [minutes]=3.66 Ionization method: ES + :[M+H] + =867.6

[0429] 14e: N-[9-[(2R,6S)-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-isopropyl-6-(triisopropylsilyloxymethyl)morpholin-2-yl]purin-6-yl]benzamide Starting material 7e (950 mg, 1.63 mmol) was dissolved in 10 ml of DCM / pyridine (1:1). After addition of a solution of DMT-Cl (733 mg, 2.12 mmol) in DCM (20 ml), the reaction was stirred for 1.5 h to achieve complete conversion. After evaporation of the solvent, the residue was dissolved in EtOAc and H 2 2x with 10% citric acid solution and saturated NaHCO 3 The solution was washed with MgSO 4 After drying over 1000 s, the solvent was removed and the crude product was purified by column chromatography on silica (n-heptane + 0.5% NEt 3 Purification on HPLC (pretreated with 0 to 50% EtOAc in n-heptane) afforded the DMT-ether 14e (1.20 g, 83.2%) as a colorless foam. LCMS-Method A: UV-wavelength [nm]=220:R t [minutes]=2.38 Ionization method: ES - :[MH] - =883.5

[0430] 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 Following the protocol for 14e, starting material 7f (2.25 g, 4.02 mmol) was protected in dry pyridine on silica (n-heptane + 0.5% NEt 3 After final chromatography on hexane (pretreated with 0 to 80% EtOAc in n-heptane), the title compound 14f (3.35 g, 96.9%) was isolated as a yellow foam. LCMS-Method A: UV-wavelength [nm]=220:R t [minute]=2.40 Ionization method: ES + :[M+H] + =861.6

[0431] 15a: 1-[(2R,6R)-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-(hydroxymethyl)-4-isopropyl-morpholin-2-yl]-5-methyl-pyrimidine-2,4-dione The TIPS-protected morpholine 14a (1.18 g, 1.5 mmol) and triethylamine (6.74 g, 9.26 mL, 66.0 mmol) were dissolved in THF (30 mL). 3 3HF (12.23 g, 12.4 ml, 73.6 mmol) was added and the reaction was stirred at 65° C. for 1 h. After standing at room temperature overnight, NMP (10 ml) was added and the reaction was stirred at 65° C. for an additional 14 h to achieve complete conversion. The solvent was concentrated in vacuo and the remaining NMP solution was diluted with H 2 O / saturated NaHCO 3 The solution was added to the mixture (1:2). The aqueous solution was extracted with EtOAc and the organic layer was washed with H 2 2H and saturated NaCl solution three times. 4 After drying at 40°C, the crude product was purified by silica gel chromatography (1% NEt in n-heptane). 3 Purification by 10 to 100% ethyl acetate in n-heptane (preconditioned with ethyl acetate) afforded the desilylated product 15a (645 mg, 68.8%) as a colorless foam. LCMS-Method A: UV-wavelength [nm] = 220: R t [minutes] = 1,67 Ionization method: ES + : [M+H] + = 616,4 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.36 (s, 1 H), 7.53 - 7.58 (m, 1 H), 7.41 (d, J=7.34 Hz, 2 H), 7.20 - 7.31 (m, 7 H), 6.87 (d, J=8.80 Hz, 4 H), 5.82 (dd, J=9.66, 2.93 Hz, 1 H), 4.61 (br s, 1 H), 3.67 - 3.79 (m, 8 H), 3.07 (s, 1 H), 2.97 - 3.10 (m, 1 H), 2.80 (br d, J=9.78 Hz, 1 H), 2.63 - 2.76 (m, 2 H), 2.17 - 2.31 (m, 2 H), 1.69 (s, 3 H), 0.95 (d, J=6.60 Hz, 6 H).

[0432] 15b: 1-[(2R,6R)-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-(hydroxymethyl)-4-isopropyl-morpholin-2-yl]pyrimidine-2,4-dione 14b (1.60 g, 2.1 mmol) was dissolved in NMP (20 ml). 3 (4.73 g, 6.50 ml, 46.3 mmol) and NEt 3 After adding 3HF (8.41 g, 8.50 ml, 50.6 mmol), the mixture was stirred at room temperature for 15 h and then at 65 °C for 5 h. The mixture was cooled to room temperature and then saturated NaHCO 3 250 ml of solution and H 2 O (250 ml) was added. After extraction with EtOAc (250 ml), the organic layer was 2 The organic phase was washed with O (2x) and saturated NaCl solution. 4 Dry and dissolve silica (n-heptane + 0.5% NEt 3 Purification on hexane (preconditioned with 0 to 100% EtOAc in n-heptane) afforded the desired alcohol 15b as a colorless foam (1.16 g, 91.1%). LCMS-Method A: UV-wavelength [nm] = 220: R t[minutes] = 1,63 Ionization method: ES + : [M+H] + = 602,3 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.36 (br s, 1 H), 7.63 (d, J=8.07 Hz, 1 H), 7.40 (br d, J=7.82 Hz, 2 H), 7.20 - 7.33 (m, 7 H), 6.88 (d, J=8.56 Hz, 4 H), 5.81 (br d, J=7.34 Hz, 1 H), 5.62 (d, J=8.07 Hz, 1 H), 4.62 (br s, 1 H), 3.78 - 3.95 (m, 1 H), 3.68 - 3.76 (m, 7 H), 3.11 (d, J=9.05 Hz, 1 H), 2.96 (br d, J=9.05 Hz, 1 H), 2.82 (br d, J=10.27 Hz, 1 H), 2.60 - 2.76 (m, 2 H), 2.10 - 2.22 (m, 2 H), 0.94 (d, J=6.36 Hz, 6 H).

[0433] 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-propanamide The TIPS-protected alcohol 14c (1.44 g, 1.66 mmol) was dissolved in THF (17 ml). After adding 1.27 g (1.15 ml, 8.30 mmol) of a 65% pyridine-HF solution, the reaction mixture was stirred at room temperature for 1 h. With vigorous stirring, NaHCO 3 (0.9g) and H 2 O (10 ml) was added and stirring was continued for 2 h. THF was removed and the aqueous solution was extracted 10 times with DCM / iPrOH (4:1). The combined organic layers were washed with MgSO 4 Drying at rt and evaporation gave 15c (0.83 g, 70.1%, crude), which was used in the next step without further purification. LCMS-Method C: UV-wavelength [nm] = 220: R t [minutes] = 2,11 Ionization method: ES - : [MH] - = 709,5 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 12.09 (s, 1 H), 11.67 (s, 1 H), 8.05 (s, 1 H), 7.38 (d, J=7.34 Hz, 2 H), 7.18 - 7.30 (m, 7 H), 6.85 (d, J=8.80 Hz, 4 H), 5.92 (dd, J=8.99, 3.00 Hz, 1 H), 4.64 (t, J=5.14 Hz, 1 H), 3.71 - 3.84 (m, 8 H), 2.90 - 3.08 (m, 3 H), 2.55 - 2.83 (m, 4 H), 2.31 - 2.47 (m, 1 H), 1.12 (dd, J=6.79, 3.36 Hz, 6 H), 0.96 (d, J=6.48 Hz, 6 H).

[0434] 15e: N-[9-[(2R,6R)-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-(hydroxymethyl)-4-isopropyl-morpholin-2-yl]purin-6-yl]benzamide Starting compound 14e (1.20 g, 1.35 mmol) and NEt 3 (1.91 g, 2.63 ml, 18.90 mmol) was dissolved in THF (20 ml). 3 After adding 3HF (3.59 g, 3.63 ml, 21.60 mmol), the solution was heated at 65 °C, followed by additional NEt 3 (0.95 g, 1.32 ml, 9.45 mmol) and NEt 3 3HF (1.80 g, 1.82 ml, 10.8 mmol) was added. The reaction mixture was stirred at 65° C. until complete conversion (approximately 10 h). After cooling to room temperature, the mixture was diluted with H 2 O / saturated NaHCO 3The solution was poured into 150 ml of (1:1) and stirred for 30 min. The aqueous solution was extracted three times with 50 mL of DCM and MgSO 4 The mixture was dried at rt and evaporated to dryness. The deprotected product 15e (835 mg, 84.9%) was isolated after silica gel chromatography (10 to 100% EtOAc in n-heptane) as a colorless foam. LCMS-Method A: UV-wavelength [nm] = 220: R t [minutes] = 1,74 Ionization method: ES + : [M+H] + = 729.5 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.21 (br s, 1 H), 8.74 (s, 1 H), 8.54 (s, 1 H), 8.04 (d, J=7.62 Hz, 2 H), 7.62 - 7.67 (m, 1 H), 7.52 - 7.58 (m, 2 H), 7.37 (d, J=7.34 Hz, 2 H), 7.15 - 7.29 (m, 7 H), 6.82 (d, J=7.95 Hz, 4 H), 6.16 (dd, J=9.17, 2.93 Hz, 1 H), 4.67 (t, J=5.20 Hz, 1 H), 3.86 - 3.96 (m, 1 H), 3.79 (dd, J=11.00, 5.87 Hz, 1 H), 3.69 - 3.73 (m, 6 H), 3.01 - 3.15 (m, 2 H), 2.90 - 2.99 (m, 2 H), 2.68 - 2.84 (m, 2 H), 2.40 (m, 1 H), 1.00 (d, J=6.60 Hz, 6 H).

[0435] 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 Starting compound 14f (3.35 g, 3.88 mmol) was reacted with NEt 33HF. After 20 h reaction time at 70 °C, complete conversion was achieved. Workup and final purification as for 15e afforded the title compound 15f (2.57 g, 93.7%) as a colorless foam. LCMS-Method A: UV-wavelength [nm] = 220: R t [minutes] = 1,79 Ionization method: ES + : [M+H] + = 705,4 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.28 (br s, 1 H), 8.10 (br d, J=7.58 Hz, 1 H), 8.00 (d, J=7.69 Hz, 2 H), 7.58 - 7.67 (m, 1 H), 7.51 (t, J=7.64 Hz, 2 H), 7.21 - 7.43 (m, 10 H), 6.89 (d, J=8.80 Hz, 4 H), 5.93 (dd, J=9.41, 2.69 Hz, 1 H), 4.66 (t, J=5.26 Hz, 1 H), 3.91 (dd, J=10.94, 4.22 Hz, 1 H), 3.71 - 3.77 (m, 7 H), 3.19 (d, J=9.05 Hz, 1 H), 2.92 - 3.06 (m, 2 H), 2.65 - 2.79 (m, 2 H), 2.18 (d, J=11.49 Hz, 1 H), 2.07 (t, J=10.21 Hz, 1 H), 0.95 (d, J=6.36 Hz, 6 H).

[0436] 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)phosphanyl]oxypropanenitrile To a solution of 15a (624 mg, 1.0 mmol) and N,N-diisopropylethylamine (401.0 mg, 513 μl, 3.0 mmol) in dry DCM (15 ml) was added 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (370.9 mg, 349.6 μl, 1.5 mmol) at room temperature under Ar atmosphere. After stirring for 1 h, the reaction was quenched by adding n-butanol (200 μl) and stirring was continued for 10 min. The reaction solution was diluted with H 2 O and the aqueous phase was extracted once with DCM. The organic layer was washed with MgSO 4 After drying at 40°C, the solvent was removed and the crude product was purified by silica gel chromatography (0.5% NEt in n-heptane). 3 Purification by 10 to 100% methyl tert-butyl ether in n-heptane (preconditioned with 10 to 100%) afforded the desired phosphoramidite (mixture of diastereomers) 16a (668 mg, 80.8%) as a colorless foam. LCMS-Method B-2: UV-wavelength [nm] = 220: R t [minutes] = 0,82 Ionization method: ES + : [M] + = 733.1 (M- i Pr 2 N+OH+H + ) 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.37,11.36 (2 xs, 1 H), 7.60, 7.56 (2 xs, 1 H), 7.41 (m, 2 H), 7.20 - 7.32 (m, 7 H), 6.83 - 6.90 (m, 4 H), 5.88 (m, 1 H), 3.84 - 4.05 (m, 2 H), 3.74 (s, 6 H), 3.54 - 3.73 (m, 2 H), 3.40 - 3.54 (m, 2 H), 3.14 (m, 1 H), 2.95 - 3.05 (m, 1 H), 2.57 - 2.84 (m, 5 H), 2.15 - 2.37 (m, 2 H), 1.76, 1.73 (2 xs, 3 H), 0.93 - 1.14 (m, 18 H). 31P-NMR (DMSO-d6, 162 MHz) δ[ppm]: 148,1, 147,6.

[0437] 16b: 3-[[(2S,6R)-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-(2,4-dioxopyrimidin-1-yl)-4-isopropyl-morpholin-2-yl]methoxy-(diisopropylamino)phosphanyl]oxypropanenitrile The title compound was prepared following the protocol described in 16a. Starting with primary alcohol 15b (1.95 g, 3.2 mmol), 15c was purified by cyclohexane-hexane chromatography (n-heptane + 0.5% NEt 3 After purification on a hexanes preconditioned with 0 to 80% EtOAc in n-heptane), the desired phosphoramidite 16b (1.70 g, 65.7%) was isolated as a colorless foam. LCMS-Method A: UV-wavelength [nm] = 220: R t [minutes] = 1,85 Ionization method: ES + : [M] + = 719.2 (M- i Pr 2 N+OH+H + ) 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.36 (br s, 1 H), 7.67, 7.63 (2 xd, J=8.19 Hz, 1 H), 7.35 - 7.44 (m, 2 H), 7.20 - 7.32 (m, 7 H), 6.84 - 6.89 (m, 4 H), 5.81 - 5.89 (m, 1 H), 5.67, 5.63 (2 xd, J=8.13 Hz, 1 H), 3.89 - 4.08 (m, 2 H), 3.73 (s, 6 H), 3.54 - 3.72 (m, 2 H), 3.41 - 3.54 (m, 2 H), 3.16 (d, J=9.05 Hz, 1 H), 2.96 (t, J=9.05 Hz, 1 H), 2.84 (br d, J=10.15 Hz, 1 H), 2.55 - 2.76 (m, 4 H), 2.10 - 2.33 (m, 2 H), 0.89 - 1.12 (m, 18 H). 31P-NMR (DMSO-d6, 162 MHz) δ[ppm]: 148,3, 147,6.

[0438] 16c: N-[9-[(2R,6S)-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-[[2-cyanoethoxy-(diisopropylamino)phosphanyl]oxymethyl]-4-isopropyl-morpholin-2-yl]-6-oxo-1H-purin-2-yl]-2-methyl-propanamide Alcohol 15c (100 mg, 141 μmol), 0.2 g of molecular sieves (4 Å) and 12.7 mg (70 μmol) of diisopropylammonium tetrazolide were dissolved in dry DCM (2.5 ml). Under Ar atmosphere, 42.8 mg (45 μl, 138 μmol) of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite was added and the solution was stirred at room temperature for 2 h to achieve complete conversion. Saturated NaHCO 3 After the addition of the solution, the organic phase was separated and the aqueous phase was extracted with DCM. The combined organic layers were washed with saturated NaCl solution and MgSO 4The crude product was purified by precipitation onto silica (DCM+0.5% NEt 3 Preconditioned with 0 to 10% MeOH in DCM) to afford the title compound 16c (87 mg, 67.9%) as a colorless foam. LCMS-Method C: UV-wavelength [nm] = 220: R t [minutes] = 2,50, 2,52 Ionization method: ES + : [M- i Pr 2 N+OH+H] + = 828,3 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 12.08 (br s, 1 H), 11.62 (br s, 1 H), 8.09, 8.04 (2 xs, 1 H), 7.33 - 7.40 (m, 2 H), 7.18 - 7.29 (m, 7 H), 6.81 - 6.87 (m, 4 H), 5.91 (m, 1 H), 3.93 - 4.07 (m, 2 H), 3.73 (s, 6 H), 3.54 - 3.72 (m, 2 H), 3.39 - 3.50 (m, 2 H), 3.11 (m, 1 H), 2.92 - 3.07 (m, 2 H), 2.54 - 2.81 (m, 6 H), 2.26 - 2.38 (m, 1 H), 0.94 - 1.16 (m, 18 H), 0.83 - 0.91 (m, 6 H). 31P-NMR (DMSO-d6, 162 MHz) δ[ppm]: 147,5, 146,7.

[0439] 16e: N-[9-[(2R,6S)-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-[[2-cyanoethoxy-(diisopropylamino)phosphanyl]oxymethyl]-4-isopropyl-morpholin-2-yl]purin-6-yl]benzamide 15e (820 mg, 1.13 mmol) and 584 mg (3.38 mmol) of diisopropylammonium tetrazolide were dissolved in dry DCM (20 ml). Under Ar atmosphere, 524 mg (1.69 mmol) of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite was added and the solution was stirred at room temperature. After stirring overnight, H 2 O (50 ml) was added and the layers were separated. The aqueous layer was extracted once with DCM (30 ml) and the combined organic phases were washed with MgSO 4 After evaporation of the solvent, the crude product was purified by silica gel chromatography (n-heptane + 0.5% NEt 3 Purification with 0 to 100% MTB in n-heptane-ether / DCM (1:1) preconditioned with ethyl acetate afforded the title compound 16e (913 mg, 87.3%) as a colorless solid. 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.19 (s, 1 H), 8.76, 8.74 (2 xs, 1 H), 8.61, 8.59 (2 xs, 1 H), 8.04 (d, J=8.09 Hz, 2 H), 7.64 (t, J=7.37 Hz, 1 H), 7.51 - 7.60 (m, 2 H), 7.37 (m, 2 H), 7.16 - 7.28 (m, 7 H), 6.77 - 6.84 (m, 4 H), 6.19 (m, 1 H), 3.98 - 4.15 (m, 2 H), 3.71 (2 xs, 6 H), 3.56 - 3.69 (m, 2 H), 3.41 - 3.56 (m, 2 H), 3.03 - 3.17 (m, 2 H), 2.89 - 3.01 (m, 2 H), 2.74 - 2.88 (m, 2 H), 2.70 (t, J=6.15 Hz, 1 H), 2.59 (m, 1 H), 2.36 (m, 1 H), 0.92 - 1.18 (m, 18 H). 31P-NMR (DMSO-d6, 162 MHz) δ[ppm]: 147,0, 146,7.

[0440] 16f: N-[1-[(2R,6S)-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-[[2-cyanoethoxy-(diisopropylamino)phosphanyl]oxymethyl]-4-isopropyl-morpholin-2-yl]-2-oxo-pyrimidin-4-yl]benzamide Following the protocol described in 16e, primary alcohol 15f (550 mg, 0.78 mmol) was phosphitylated. 3 After chromatographic purification on 0 to 100% MTB in n-heptane-ether / DCM (1:1) preconditioned with 16f (560 mg, 79.3%) was isolated as a colorless foam. 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.29 (br s, 1 H), 8.12 (2 xd, J=7.50 Hz, 1 H), 7.98 - 8.04 (m, 2 H), 7.63 (t, J=7.40 Hz, 1 H), 7.48 - 7.55 (m, 2 H), 7.36 - 7.45 (m, 3 H), 7.21 - 7.35 (m, 7 H), 6.88 (2 xd, J=8.89, 4 H), 5.95, 6.01 (2 xm, 1 H), 3.89 - 4.14 (m, 2 H), 3.74, 3.75 (2xs, 6H), 3.55 - 3.72 (m, 2 H), 3.39 - 3.55 (m, 2 H), 3.24 (d, J=8.85 Hz, 1 H), 2.93 - 3.05 (m, 2 H), 2.68 - 2.87 (m, 3 H), 2.55 - 2.64 (m, 1 H), 2.10 - 2.23 (m, 2 H), 0.90 - 1.22 (m, 18 H). 31P-NMR (DMSO-d6, 162 MHz) δ[ppm]: 147,4, 146,7.

[0441] [ka]

[0442] 17a: [(2S,6R)-4-isopropyl-6-(5-methyl-2,4-dioxo-pyrimidin-1-yl)-2-(triisopropylsilyloxymethyl)morpholin-2-yl]methyl benzoate The free alcohol 7a (810 mg, 1.7 mmol) was dissolved in DCM (20 ml). After addition of DIPEA (1.14 g, 1.45 ml, 8.6 mmol) and 294 mg (243 μl, 2.1 mmol) of benzoyl chloride, the solution was stirred at room temperature for 2 h. To ensure complete conversion, another 0.2 equivalents of benzoyl chloride was added and stirring was continued for 18 h. The reaction solution was directly transferred to a silica gel column eluted with 0 to 65% EtOAc in n-heptane. The benzoylated product 17a (726 mg, 73.3%) was obtained as a colorless foam. LCMS-Method A: UV-wavelength [nm]=220:R t [minute]=2.29 Ionization method: ES + :[M+H] + =574.3

[0443] 17b: [(2S,6R)-6-(3-benzoyl-2,4-dioxo-pyrimidin-1-yl)-4-isopropyl-2-(triisopropylsilyloxymethyl)morpholin-2-yl]methyl benzoate As described in 17a, primary alcohol 7b (1.45 g, 3.18 mmol) was treated with benzoyl chloride to give a mixture of mono- and dibenzylated products. Therefore, the reaction mixture was evaporated and the crude product was dissolved in 30 ml of pyridine. After adding 565 mg (3.98 mmol) of benzoyl chloride and a catalytic amount of DMAP, the reaction solution was stirred at room temperature for 4 h. 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 diluted with MgSO 4 The mixture was dried at 40° C. and the solvent was removed. Final chromatography on silica (0 to 35% EtOAc in n-heptane) afforded the dibenzoylated product 17b (1.64 g, 77.8%). LCMS-Method C: UV-wavelength [nm]=220:R t [minutes]=3.67 Ionization method: ES + :[M+H] + =664.4

[0444] 17c: [(2S,6R)-4-isopropyl-6-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]-2-(triisopropylsilyloxymethyl)morpholin-2-yl]methyl benzoate Starting compound 7c (0.89 g, 1.58 mmol) was dissolved in 30 ml of pyridine. After adding 246 mg (203 μl, 1.73 mmol) of benzoyl chloride, the reaction solution was stirred at room temperature overnight, at which point complete conversion was detected. The solvent was removed in vacuo and the residue was dissolved in EtOAc. 2 The organic phase was separated and washed with O and MgSO. 4 It was dried at rt and evaporated. Final purification on silica (0 to 5% MeOH in DCM) afforded the title compound 17c as a colorless foam (1.01 g, 95.4%). LCMS-Method C: UV-wavelength [nm]=220:R t [minutes]=3.28 Ionization method: ES + :[M+H] + =669.2

[0445] 18a: [(2R,6R)-2-(hydroxymethyl)-4-isopropyl-6-(5-methyl-2,4-dioxo-pyrimidin-1-yl)morpholin-2-yl]methyl benzoate A solution of 17a (720 mg, 1.25 mmol) in THF (15 ml) was treated with 1.34 g (1.22 ml, 8.8 mmol) of pyridine-hydrogen fluoride. After stirring at room temperature for 1 h, the reaction mixture was diluted with saturated NaHCO 3 The solution was treated with a solution until the pH reached 7.5. The solvent was concentrated under reduced pressure and the aqueous solution was extracted twice with DCM. The organic layer was washed with MgSO 4The mixture was dried at 40° C. and the solvent was removed. The crude product was co-evaporated twice with 50 mL of toluene, and the remaining residue was purified by silica gel chromatography (0 to 100% EtOAc in n-heptane) to give the desired product 18a (502 mg, 95.8%) as a colorless foam. LCMS-Method A: UV-wavelength [nm]=220:R t [minute]=1.30 Ionization method: ES + :[M+H] + =418.2

[0446] 18b: [(2R,6R)-6-(3-benzoyl-2,4-dioxo-pyrimidin-1-yl)-2-(hydroxymethyl)-4-isopropyl-morpholin-2-yl]methyl benzoate Silyl ether 17b (1.64 g, 2.47 mmol) was desilylated following the protocol described for 18a to give the desired product 18b (1.33 g) as a crude product, which was used in the next step without chromatographic purification. LCMS-Method C: UV-wavelength [nm]=220:R t [minute]=1.81 Ionization method: ES + :[M+H] + =508.2

[0447] 18c: [(2R,6R)-2-(hydroxymethyl)-4-isopropyl-6-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]morpholin-2-yl]methyl benzoate 17c (1.0 g, 1.49 mmol) was dissolved in DMF (10 ml). 3 (1.53 g, 2.10 ml, 15.0 mmol) and NEt 3 After adding 3HF (1.48 g, 1.49 ml, 9.0 mmol), the reaction solution was stirred at 90 °C for 2 h. The mixture was cooled to room temperature, and then NaHCO 3 (2.5g) and H 2HO (10 ml) was added and the reaction mixture was stirred for 2 h. The solvent was evaporated in vacuo and the residue was dissolved in DCM / iPrOH (25 ml) and H 2 The organic phase was separated and washed with MgSO 4 The crude product was purified on silica (0 to 10% MeOH in DCM) to give the desilylated product 18c (245 mg, 32.0%) as a colorless foam. LCMS-Method C: UV-wavelength [nm]=220:R t [minutes]=1.45 Ionization method: ES + :[M+H] + =513.1

[0448] 18e: [(2R,6R)-6-(6-benzamidopurin-9-yl)-2-(hydroxymethyl)-4-isopropyl-morpholin-2-yl]-methyl benzoate Following the protocol described in 18a, the silyl ether 13 (1.23 g, 1.79 mmol) was deprotected to give the title compound 18e (939 mg, 98.8%) after silica gel chromatography (0 to 100% EtOAc in n-heptane). LCMS-Method C: UV-wavelength [nm]=220:R t [minute]=1.60 Ionization method: ES - :[MH] - =529.3

[0449] 18f: [(2R,6R)-6-(4-benzamido-2-oxo-pyrimidin-1-yl)-2-(hydroxymethyl)-4-isopropyl-morpholin-2-yl]methyl benzoate Following the protocol described in 18a, silyl ether 10 (3.63 g, 5.48 mmol) was deprotected to give the title compound 18f (2.45 g, 88.4%) after silica gel chromatography (5% MeOH in DCM). LCMS-Method A: UV-wavelength [nm]=220:R t [minute]=1.53 Ionization method: ES + :[M+H] + =507.3

[0450] 19a: [(2R,6R)-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-isopropyl-6-(5-methyl-2,4-dioxo-pyrimidin-1-yl)morpholin-2-yl]methyl benzoate Starting compound 18a (497 mg, 1.2 mmol) was dissolved in DCM (15 ml). After adding DIPEA (502 mg, 643 μl, 3.8 mmol) and DMT-Cl (659 mg, 1.90 mmol), the reaction solution was stirred at room temperature for 2 h. The reaction mixture was evaporated in vacuum and the crude product was purified on silica gel (0 to 100% EtOAc in n-heptane). The DMT-protected product 19a was isolated as a colorless foam (779 mg, 90.9%). LCMS-Method A: UV-wavelength [nm]=220:R t [minutes]=2.14 Ionization method: ES + :[M+H] + =720.4

[0451] 19b: [(2R,6R)-6-(3-benzoyl-2,4-dioxo-pyrimidin-1-yl)-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-isopropyl-morpholin-2-yl]methyl benzoate Primary alcohol 18b (1.30 g, 2.41 mmol) and NEt 3 (492 mg, 676 μl, 4.82 mmol) was dissolved in DCM (20 ml). After addition of DMT-Cl (925 mg, 2.65 mmol), the reaction was stirred at room temperature for 6 h, followed by the addition of additional DMT-Cl (925 mg, 2.65 mmol). The reaction was stirred for 72 h. After addition of 2.0 ml of n-propanol, the solution was stirred for 10 min. The mixture was diluted with H 2 O and separated the organic layer. 4After drying at rt, the solvent was removed in vacuo. The crude product was purified by silica gel chromatography (0 to 25% EtOAc in n-heptane) to give the desired DMT-ether 19b (1.74 g, 89.2%). LCMS-Method C: UV-wavelength [nm]=220:R t [minutes]=3.25 Ionization method: ES + :[M+H] + =810.3

[0452] 19c: [(2R,6R)-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-isopropyl-6-[2-(2-methylpropanoylamino)-6-oxo-1H-purin-9-yl]morpholin-2-yl]methyl benzoate The starting alcohol 18c (240 mg, 468 μmol) was dissolved in DCM (5.0 ml). 3 After addition of (95.7 mg, 131.5 μl, 936 μmol) and DMT-Cl (180 mg, 515 μmol), the reaction was stirred at room temperature for 3 days to achieve complete conversion. After addition of 2.0 ml of n-propanol, the solution was stirred for 10 min. The mixture was diluted with H 2 O and separated the organic layer. 4 After drying at rt, the solvent was removed in vacuo. The crude product was purified by silica gel chromatography (0 to 25% EtOAc in n-heptane) to give the desired DMT-ether 19c (303 mg, 79.4%). LCMS-Method C: UV-wavelength [nm]=220:R t [minutes]=2.93 Ionization method: ES - :[MH] - =813.2

[0453] 19e: [(2R,6R)-6-(6-benzamidopurin-9-yl)-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-isopropyl-morpholin-2-yl]methyl benzoate Starting material 18e (934 mg, 1.76 mmol) was dissolved in 20 ml of DMC / pyridine (1:1). After adding DMT-Cl (974 mg, 2.82 mmol), the reaction solution was stirred at room temperature for 17 h. The solvent was removed in vacuo and the residue was dissolved in EtOAc and H 2 O, 2 x 10% citric acid solution, saturated NaHCO 3 The organic phase was washed with MgSO 4 The resulting crude product (1.77 g) was dissolved in 20 ml of EtOAc / diethyl ether (1:2). After adding 40 ml of n-pentane, the precipitate was centrifuged and the supernatant was discarded. The precipitation procedure was repeated two more times to give the desired DMT-ether 19e as a colorless solid. LCMS-Method C: UV-wavelength [nm]=220:R t [minutes]=3.07 Ionization method: ES - :[MH] - =831.5

[0454] 19f: [(2R,6R)-6-(4-benzamido-2-oxo-pyrimidin-1-yl)-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-isopropyl-morpholin-2-yl]methyl benzoate 18f (2.45 g, 4.84 mmol) was dissolved in 40 ml of pyridine. After adding DMT-Cl (2.51 g, 7.26 mmol), the reaction mixture was stirred at room temperature for 4 days. The solvent was removed in vacuo and the residue was dissolved in EtOAc and H 2 O, 2 x 10% citric acid solution, saturated NaHCO 3 The organic phase was washed with MgSO 4 The crude product was purified by silica gel (n-heptane + 0.5% NEt 3 Preconditioned with 0 to 100% EtOAc in n-heptane) to afford the title compound 19f (3.69 g, 94.3%) as a colorless foam. LCMS-Method A: UV-wavelength [nm]=220:R t [minutes]=2.14 Ionization method: ES - :[MH] - =807.4

[0455] 20a: 1-[(2R,6S)-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-(hydroxymethyl)-4-isopropyl-morpholin-2-yl]-5-methyl-pyrimidine-2,4-dione Benzoyl ester 19a (775 mg, 1.1 mmol) was dissolved in 20 ml of THF-methanol (4:1). At room temperature, 4.31 ml (8.6 mmol) of 2N aqueous NaOH was added and the reaction was stirred for 90 min. The pH was adjusted to between 7 and 8 by adding citric acid monohydrate, and the solvent was then removed under reduced pressure. The residue was dissolved in DCM and H 2 The organic layer was separated and the aqueous phase was extracted with DCM. The combined organic phase was diluted with MgSO 4 The mixture was dried at 40° C. and evaporated. The crude product was purified by silica gel chromatography (5 to 100% EtOAc in n-heptane) to give the desired product 20a (663 mg, quantitative) as a colorless foam. LCMS-Method A: UV-wavelength [nm] = 220: R t [minutes] = 1,73 Ionization method: ES + : [M+H] + = 616,3 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.29 (s, 1 H), 7.60 (d, J=1.10 Hz, 1 H), 7.39 (d, J=6.93 Hz, 2 H), 7.19 - 7.33 (m, 7 H), 6.88 (d, J=8.68 Hz, 4 H), 5.60 (dd, J=9.96, 2.87 Hz, 1 H), 4.64 (t, J=5.93 Hz, 1 H), 3.74 (m, 6 H), 3.53 - 3.60 (m, 1 H), 3.44 - 3.52 (m, 1 H), 3.32 - 3.37 (m, 1 H), 3.09 (d, J=8.56 Hz, 1 H), 2.76 (br d, J=11.62 Hz, 1 H), 2.61 - 2.72 (m, 2 H), 2.37 (d, J=11.49 Hz, 1 H), 2.10 (t, J=10.45 Hz, 1 H), 1.78 (s, 3 H), 0.84 - 0.96 (m, 6 H).

[0456] 20b: 1-[(2R,6S)-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-(hydroxymethyl)-4-isopropyl-morpholin-2-yl]pyrimidine-2,4-dione Starting compound 19b (1.40 g, 1.73 mmol) was dissolved in MeOH (80 ml). At room temperature, 13.83 ml (6.91 mmol) of a 0.5 M solution of NaOMe in MeOH was added and the reaction solution was stirred for 2 h to achieve complete conversion. After addition of 0.92 g of citric acid, the solvent was removed. The residue was purified by H 2 The aqueous mixture was extracted three times with DCM / iPrOH (4:1). The combined organic layers were diluted with MgSO 4 Drying at 40° C. and evaporation afforded the desired compound 20b (0.85 g, 81.2%, crude), which was used in the subsequent step without further purification. LCMS-Method C: UV-wavelength [nm] = 220: R t [minutes] = 2,04 Ionization method: ES + : [M+H]+ = 602,2 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.31 (s, 1 H), 7.75 (d, J=8.07 Hz, 1 H), 7.39 (d, J=7.34 Hz, 2 H), 7.10 - 7.33 (m, 8 H), 6.88 (d, J=8.31 ​​Hz, 4 H), 5.55 - 5.64 (m, 2 H), 4.66 (t, J=5.93 Hz, 1 H), 3.74 (s, 6 H), 3.57 (dd, J=11.31, 6.79 Hz, 1 H), 3.46 (br dd, J=11.25, 5.14 Hz, 1 H), 3.08 (d, J=8.56 Hz, 1 H), 2.54 - 2.79 (m, 3 H), 2.32 - 2.39 (m, 1 H), 1.95 - 2.13 (m, 1 H), 0.86 - 0.98 (m, 6 H).

[0457] 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-propanamide Starting material 19c (260 mg, 319 μmol) was dissolved in EtOH (10 ml). After adding 1.0 ml (1.0 mmol) of 1M NaOH solution, the solution was stirred at room temperature for 4 h to achieve complete conversion. The solution was neutralized with 1M HCl and the solvent was removed in vacuo. The residue was purified by HCl. 2 O and extracted with DCM. The organic phase was separated and washed with MgSO 4 The mixture was dried at rt and evaporated. Final purification on silica (0 to 10% MeOH in DCM) afforded the desired alcohol 20c (114 mg, 50.3%) as a yellow solid. LCMS-Method C: UV-wavelength [nm] = 220: R t [minutes] = 2,36 Ionization method: ES - : [MH] - = 709,3 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 12.08 (s, 1 H), 11.52 (s, 1 H), 8.20 (s, 1 H), 7.35 - 7.43 (m, 2 H), 7.20 - 7.33 (m, 7 H), 6.83 - 6.92 (m, 4 H), 5.56 (dd, J=9.60, 2.75 Hz, 1 H), 4.59 - 4.66 (m, 1 H), 3.73 (2 xs, 6 H), 3.51 - 3.62 (m, 2 H), 3.43 - 3.50 (m, 1 H), 2.88 - 3.02 (m, 3 H), 2.70 - 2.82 (m, 2 H), 2.30 - 2.39 (m, 1 H), 1.11 (m, 6 H), 1.01 (d, J=6.48 Hz, 3 H), 0.97 (d, J=6.48 Hz, 3 H).

[0458] 20e: N-[9-[(2R,6S)-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-(hydroxymethyl)-4-isopropyl-morpholin-2-yl]purin-6-yl]benzamide Starting compound 19e (1.27 g, 1.52 mmol) was dissolved in 14 ml of EtOH / pyridine (1:1). At 0° C., 7.59 ml (15.19 mmol) of 1 M NaOH solution was added and the reaction mixture was stirred at room temperature for 1.5 h. The pH was adjusted to 7 by adding citric acid monohydrate (958 mg), and then H 2 HO (70 ml) and EtOAc were added. The organic layer was separated and washed with 10% citric acid solution (3x), H 2 O, saturated NaHCO 3 solution and NaCl solution. MgSO 4 After drying at 40° C., the organic phase was evaporated to give 1.08 g of crude product as a colorless foam, which was dissolved in 20 ml of EtOAc / diethyl ether (1:1). After adding 40 ml of n-pentane, the precipitate was centrifuged and the supernatant was discarded. The precipitation procedure was repeated two more times to give the title compound 20e (964 mg, 87.1%) as a colorless foam. LCMS-Method C: UV-wavelength [nm] = 220: R t [minutes] = 2,42 Ionization method: ES - : [MH] - = 727,5 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.18 (s, 1 H), 8.71 (s, 1 H), 8.69 (s, 1 H), 8.05 (d, J=7.34 Hz, 2 H), 7.61 - 7.68 (m, 1 H), 7.56 (t, J=7.58 Hz, 2 H), 7.45 (d, J=7.46 Hz, 2 H), 7.28 - 7.36 (m, 6 H), 7.20 - 7.28 (m, 1 H), 6.90 (dd, J=8.99, 2.26 Hz, 4 H), 5.89 (dd, J=9.84, 2.63 Hz, 1 H), 4.69 (t, J=5.99 Hz, 1 H), 3.75 (s, 6 H), 3.45 - 3.61 (m, 3 H), 3.24 - 3.30 (m, 1 H), 3.00 (br d, J=10.15 Hz, 1 H), 2.85 (br d, J=11.62 Hz, 1 H), 2.60 - 2.78 (m, 2 H), 2.45 - 2.55 (m, 1 H), 0.98 (br d, J=6.48 Hz, 3 H), 0.95 (br d, J=6.48 Hz, 3 H).

[0459] 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 19f (3.68 g, 4.55 mmol) was saponified according to the procedure described in 20e. The crude product was purified by silica gel chromatography (20 to 100% EtOAc in n-heptane) to give 20f (1.72 g, 53.5%) as a pale yellow foam. LCMS-Method A: UV-wavelength [nm] = 220: R t[minutes] = 1,81 Ionization method: ES - : [MH] - = 703,3 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.24 (s, 1 H), 8.29 (d, J=7.46 Hz, 1 H), 8.01 (d, J=7.46 Hz, 2 H), 7.62 (t, J=7.40 Hz, 1 H), 7.51 (t, J=7.70 Hz, 2 H), 7.20 - 7.41 (m, 10 H), 6.88 (dd, J=8.99, 2.38 Hz, 4 H), 5.68 (dd, J=9.35, 2.38 Hz, 1 H), 4.71 (t, J=6.11 Hz, 1 H), 3.74 (s, 6 H), 3.58 - 3.69 (m, 1 H), 3.48 - 3.58 (m, 1 H), 3.35 - 3.43 (m, 1 H), 3.11 (d, J=8.56 Hz, 1 H), 2.79 - 2.95 (m, 2 H), 2.66 - 2.76 (m, 1 H), 2.39 - 2.47 (m, 1 H), 1.91 - 2.00 (m, 1 H), 0.96 (d, J=6.54 Hz, 3 H), 0.94 (d, J=6.54 Hz, 3 H).

[0460] 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)phosphanyl]-oxypropanenitrile Following the protocol for 16a (Scheme 5), starting material 20a (764 mg, 1.2 mmol) was converted to the desired phosphoramidite 21a, yielding 842 mg (83.2%, mixture of diastereomers) as a colorless foam. 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.34, 11.32 (2 xs, 1 H), 7.59, 7.54 (2 xd, J=1.10 Hz, 1 H), 7.36 - 7.43 (m, 2 H), 7.20 - 7.34 (m, 7 H), 6.89 (m, 4 H), 5.59 - 5.75 (m, 1 H), 3.44 - 3.95 (m, 6 H), 3.74 (s, 6 H), 3.36 (m, 1 H), 3.22 (m, 1 H), 2.60 - 2.85 (m, 5 H), 2.36 (m, 1 H), 2.18 (m, 1 H), 1.78 (2 xd, J=8.69 Hz, 3 H), 0.83 - 1.17 (m, 18 H). 31P-NMR (DMSO-d6, 162 MHz) δ[ppm]: 148,6, 148,3.

[0461] 21b: 3-[[(2R,6R)-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-(2,4-dioxopyrimidin-1-yl)-4-isopropyl-morpholin-2-yl]methoxy-(diisopropylamino)phosphanyl]oxypropanenitrile The title compound 21b was synthesized starting from the corresponding alcohol 20b (1.36 g, 2.26 mmol) following the protocol for 16b (Scheme 5). 1.09 g (60.1%) of the title compound was isolated as a colorless foam. LCMS-Method B-1: UV-wavelength [nm] = 220: R t [minutes] = 0,80 Ionization method: ES + : [MN i Pr 2 +OH+H] + = 718,3 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.32 (br s, 1 H), 7.71, 7.66 (2 xs, J= 8.13 Hz, 1 H), 7.35 - 7.42 (m, 2 H), 7.20 - 7.33 (m, 7 H), 6.83 - 6.92 (m, 4 H), 5.60 - 5.70 (m, 2 H), 3.73 (s, 6 H), 3.43 - 3.95 (m, 6 H), 3.29 - 3.39 (m, 1 H), 3.22 (m, 1 H), 2.59 - 2.83 (m, 5 H), 2.33 (m, 1 H), 2.13 (m, 1 H), 0.84 - 1.21 (m, 18 H). 31P-NMR (DMSO-d6, 162 MHz) δ[ppm]: 148,5, 148,3.

[0462] 21c: N-[9-[(2R,6R)-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-[[2-cyanoethoxy-(diisopropylamino)phosphanyl]oxymethyl]-4-isopropyl-morpholin-2-yl]-6-oxo-1H-purin-2-yl]-2-methyl-propanamide Following the protocol described for the synthesis of 16c (Scheme 5), starting compound 20c (40 mg, 56 μmol) was purified by silica gel chromatography (0 to 100% methyl tert-butyl ether in n-heptane, n-heptane + 1% NEt 3 After purification by HPLC (column preconditioned with HPLC), the desired phosphoramidite 21c (23 mg, 44.9%) was obtained. LCMS-Method C: UV-wavelength [nm] = 220: R t [minutes] = 2,57 Ionization method: ES + : [MN i Pr 2 +OH+H] + = 828,2 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.70 - 12.30 (bs, 1 H), 11.53 (bs, 1 H), 8.05 (s, 1 H), 7.35 - 7.45 (m, 2 H), 7.20 - 7.33 (m, 7 H), 6.82 - 6.91 (m, 4 H), 5.54 - 5.68 (m, 1 H), 3.80 - 3.93 (m, 0.5 H), 3.73, 3.72 (2 xs, 6 H), 3.68 - 3.76 (m, 1.5 H), 3.60 - 3.67 (m, 2 H), 3.35 - 3.60 (m, 4 H), 3.12 (m, 1 H), 2.57 - 2.98 (m, 7 H), 0.92 - 1.13 (m, 24 H). 31P-NMR (DMSO-d6, 162 MHz) δ[ppm]: 147.6, 147.4.

[0463] 21e: N-[9-[(2R,6R)-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-[[2-cyanoethoxy-(diisopropylamino)phosphanyl]oxymethyl]-4-isopropyl-morpholin-2-yl]purin-6-yl]benzamide The title compound was prepared starting from 20e (950 mg, 1.30 mmol) following the protocol described in 16e (Scheme 5) to afford 21e (1.10 g, 91.0%) as a colorless foam. 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.19 (br s, 1 H), 8.73, 8.71 (2 xs, 1 H), 8.63, 8.59 (2 xs, 1 H), 8.04 (d, J=7.65 Hz, 2 H), 7.64 (t, J=7.34 Hz, 1 H), 7.55 (t, J=7.43 Hz, 2 H), 7.44 (d, J=7.78 Hz, 2 H), 7.21 - 7.35 (m, 7 H), 6.86 - 6.93 (m, 4 H), 5.89 - 5.99 (m, 1 H), 3.80 - 4.15 (m, 1 H), 3.74 (s, 6 H), 3.63 - 3.72 (m, 2 H), 3.31 - 3.60 (m, 5 H), 3.01 (br d, J=10.79 Hz, 1 H), 2.66 - 2.90 (m, 4 H), 2.59 - 2.64 (m, 1 H), 2.42 - 2.47 (m, 1 H), 0.91 - 1.12 (m, 18 H). 31P-NMR (DMSO-d6, 162 MHz) δ[ppm]: 147,6, 147,5.

[0464] 21f: N-[1-[(2R,6R)-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-[[2-cyanoethoxy-(diisopropylamino)phosphanyl]oxymethyl]-4-isopropyl-morpholin-2-yl]-2-oxo-pyrimidin-4-yl]benzamide The title compound was prepared starting from 20f (620 mg, 0.88 mmol) following the protocol described in 16e (Scheme 5) to afford 21f (681 mg, 85.5%) as a colorless foam. 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.28 (br s, 1 H), 8.12 - 8.23 ​​(2 xd, J= 7.47 Hz, 1 H), 8.01 (d, J=7.65 Hz, 2 H), 7.58 - 7.67 (m, 1 H), 7.51 (t, J=7.58 Hz, 2 H), 7.35 - 7.42 (m, 3 H), 7.20 - 7.35 (m, 7 H), 6.83 - 6.93 (m, 4 H), 5.71 - 5.80 (2 x dd, J=9.49, 2.54 Hz, 1 H), 3.44 - 4.00 (m, 6H), 3.74 (s, 6 H), 3.38 (m, 1 H), 3.24 - 3.33 (m, 1 H), 2.60 - 2.94 (m, 5 H), 2.33 - 2.48 (m, 1 H), 2.01 - 2.09 (m, 1 H), 1.02 - 1.23 (m, 12 H), 0.83 - 1.00 (m, 6 H). 31P-NMR (DMSO-d6, 162 MHz) δ[ppm]: 147,5, 147,4.

[0465] [ka]

[0466] 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 A solution of starting compound 4a (23.7 g, 53.4 mmol) in 240 mL of anhydrous pyridine was added to 2 DMT-Cl (23.13 g, 80 mmol) was added at room temperature under atmosphere. After stirring for 12 h, 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 the desired DMT-protected product 22a (16.3 g, 41%) as a colorless solid. 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.34 (s, 1 H), 7.50 (d, J=0.75 Hz, 1 H), 7.42-7.37 (m, 2 H), 7.32 (t, J=7.53 Hz, 2 H), 7.29-7.22 (m, 6 H), 6.90 (dd, J=8.91, 2.13 Hz, 4 H), 5.89 (d, J=7.28 Hz, 1 H), 5.45 (d, J=6.53 Hz, 1 H), 5.15 (d, J=5.02 Hz, 1 H), 4.48-4.39 (m, 1 H), 4.31 (t, J=5.14 Hz, 1 H), 3.86-3.80 (m, 1 H), 3.74 (s, 6 H), 1.28 (s, 3 H), 0.98-0.83 (m, 22 H).

[0467] 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 A solution of compound 4b (75 g, 174 mmol) in 750 mL of anhydrous pyridine was added to 2 DMT-Cl (70.8 g, 209 mmol) was added at room temperature under ambient atmosphere. The mixture was stirred for 12 h to achieve complete conversion. The solvent was removed in vacuo and the residue was diluted with 500 ml water and extracted with EtOAc (3×500 ml). The organic layer was washed with brine and diluted with anhydrous Na 2 SO 4 The crude product was purified by flash chromatography on silica gel (PE / EtOAc 1:1 to EtOAc / MeOH 20:1) to give 22b (80 g, 62.6%) as a colorless foam. 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.35 (s, 1 H), 7.61 (d, J=8.28 Hz, 1 H), 7.42-7.36 (m, 2 H), 7.32 (t, J=7.65 Hz, 2 H), 7.28-7.19 (m, 5 H), 6.90 (d, J=8.03 Hz, 4 H), 5.83 (d, J=6.53 Hz, 1 H), 5.48 (d, J=6.02 Hz, 1 H), 5.27 (d, J=8.03 Hz, 1 H), 5.21 (d, J=5.27 Hz, 1 H), 4.33-4.26 (m, 1 H), 4.22 (q, J=6.02 Hz, 1 H), 3.89-3.79 (m, 2 H), 3.74 (s, 6 H), 3.24 (d, J=10.04 Hz, 1 H), 1.01-0.84 (m, 21 H).

[0468] 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-propanamide Following the protocol described in 22b, 4c (55.8 g, 103 mmol) was protected with DMT-Cl in pyridine to give 22c (50.0 g, 57.4%) as a colorless solid. 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 12.12 (br s, 1 H), 11.67 (br s, 1 H), 7.99-7.83 (m, 1 H), 7.38 (br d, J=7.28 Hz, 2 H), 7.32-7.13 (m, 8 H), 6.84 (dd, J=8.78, 2.26 Hz, 4 H), 5.86 (d, J=7.03 Hz, 1 H), 5.70-5.45 (m, 1 H), 5.18 (br s, 1 H), 4.73-4.62 (m, 1 H), 4.25 (br d, J=4.77 Hz, 1 H), 3.97 (br d, J=10.29 Hz, 1 H), 3.84 (br d, J=10.29 Hz, 1 H), 3.73 (s, 6 H), 3.20 (br d, J=10.04 Hz, 1 H), 2.76 (dt, J=13.61, 6.87 Hz, 1 H), 1.12 (d, J=6.78 Hz, 6 H), 1.01-0.87 (m, 21 H).

[0469] 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 Following the protocol described for 22b, 4e (57.0 g, 102 mmol) was protected with DMT-Cl in pyridine to give 22e as a colorless solid (70.0%). 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.22 (s, 1 H), 8.68-8.58 (m, 1 H), 8.49 (s, 1 H), 8.05 (d, J=7.28 Hz, 2 H), 7.70-7.60 (m, 1 H), 7.59-7.50 (m, 2 H), 7.43 (d, J=7.28 Hz, 2 H), 7.33-7.17 (m, 7 H), 6.86 (dd, J=8.91, 1.38 Hz, 4 H), 6.00 (d, J=7.28 Hz, 1 H), 5.53 (d, J=6.78 Hz, 1H), 5.32 (d, J=4.89 Hz, 1 H), 5.00-4.87 (m, 1 H), 4.34 (t, J=4.89 Hz, 1 H), 4.08-4.04 (m, 1 H), 3.98 (d, J=10.79 Hz, 1 H), 3.73 (s, 6 H), 3.40 (d, J=9.54 Hz, 1 H), 3.30 (br d, J=9.54 Hz, 1 H), 1.12-0.93 (m, 21 H).

[0470] 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 Starting material 22a (12.6 g, 16.9 mmol) was dissolved in acetone / H 2 A solution of NaIO in 250 mL of 2:3 HO (3:1) at room temperature was 4 (5.05g, 23.6mmol) of H 2 Aqueous solution of 1,2-dichlorophenyl ether (60 ml) was added dropwise. The mixture was stirred for 12 h to allow complete conversion and poured into 500 ml of ice water. The mixture was extracted three times with DCM (500 ml). The organic layers were combined, washed with brine and diluted with anhydrous Na 2 SO 4 Drying at rt, filtration and concentration in vacuo afforded the desired product 23a (11.20 g, 87%) as a white foam which was used in the next step without further purification. 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.62-11.33 (m, 1 H), 7.80-7.65 (m, 1 H), 7.57-7.38 (m, 3 H), 7.33-7.13 (m, 9 H), 6.88-6.73 (m, 5 H), 6.08-5.86 (m, 1 H), 5.71-5.51 (m, 1 H), 5.24-5.06 (m, 1 H), 5.01-4.94 (m, 1 H), 4.29-4.16 (m, 1 H), 3.75-3.70 (m, 7 H), 3.34-3.24 (m, 1 H), 3.06-2.81 (m, 1 H), 2.02-1.59 (m, 2 H), 1.03 (m, 1 H), 1.09-0.74 (m, 21 H).

[0471] 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 Following the protocol described in 23a, diol 22b (77.0 g, 105 mmol) gave the title compound 23b (80 g, quantitative, crude) as a yellow solid. 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.67-11.36 (m, 1 H), 7.87-7.77 (m, 1 H), 7.76-7.67 (m, 1 H), 7.55-7.41 (m, 2 H), 7.39-7.20 (m, 8 H), 6.94-6.81 (m, 4 H), 5.94-5.87 (m, 1 H), 5.78-5.63 (m, 1 H), 5.59-5.51 (m, 1 H), 5.25-5.08 (m, 1 H), 5.07-4.99 (m, 1 H), 4.32-4.20 (m, 1H), 3.82-3.70 (m, 6 H), 3.34-3.26 (m, 1 H), 3.15-3.04 (m, 1 H), 2.95-2.86 (m, 1 H), 1.14-0.81 (m, 22 H).

[0472] 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-propanamide Following the protocol described in 23a, diol 22c (50.0 g, 59 mmol) gave the title compound 23c (47 g, 96.1%, crude) as a yellow solid. 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 12.25-12.06 (m, 1 H), 11.78-11.55 (m, 1 H), 8.19 (d, J=8.78 Hz, 1 H), 7.44 (br d, J=7.28 Hz, 1 H), 7.38 (br d, J=6.53 Hz, 1 H), 7.34-7.14 (m, 10 H), 6.99 (d, J=6.78 Hz, 1 H), 6.90-6.71 (m, 5 H), 6.05-5.98 (m, 1 H), 5.74 (d, J=7.78 Hz, 1 H), 5.61-5.53 (m, 1 H), 5.48 (q, J=6.78 Hz, 1 H), 5.24-5.17 (m, 1 H), 5.06 (d, J=6.53 Hz, 1 H), 4.35-4.22 (m, 1 H), 3.83 (br d, J=11.54 Hz, 1 H), 3.72 (d, J=3.26 Hz, 7 H), 3.27 (br d, J=9.29 Hz, 1 H), 3.29-3.23 (m, 1 H), 3.12-3.04 (m, 1 H), 2.91-2.75 (m, 1 H), 1.22-0.76 (m, 29 H).

[0473] 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 Following the protocol described in 23a, diol 22e (78.0 g, 90.6 mmol) gave the title compound 23e (75.2 g, 94.6%, crude) as a white solid. MS(m / z)=876.2[M+H] +

[0474] 24a: 1-[(2R,6S)-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-(triisopropylsilyloxymethyl)morpholin-2-yl]-5-methyl-pyrimidine-2,4-dione A solution of starting compound 23a (2.6 g, 3.4 mmol) in anhydrous MeOH (44 ml) was added with N 2 At room temperature under atmospheric (NH 4 ) 2 B 4 O 7 4H 2 HO (0.98 g, 3.7 mmol) was added. The mixture was stirred for 2 h, followed by addition of AcOH (0.45 g, 6.8 mmol), 5.0 g of 4 Å molecular sieves and NaBH 3 CN (0.47 g, 6.8 mmol) was added. After stirring at room temperature for 12 h, 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 the desired morpholine 24a (1.59 g, 64%) as a white foam. MS (m / z) = 752.5 [M+Na] + 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.39 (s, 1 H), 7.62 (s, 1 H), 7.43 (br d, J=7.28 Hz, 2 H), 7.33-7.23 (m, 8 H), 6.84 (d, J=8.78 Hz, 5 H), 5.86 (br dd, J=9.91, 2.64 Hz, 1 H), 5.89-5.81 (m, 1 H), 4.13 (br d, J=9.54 Hz, 1 H), 3.96 (br d, J=9.54 Hz, 1 H), 3.72 (d, J=0.75 Hz, 6 H), 3.06 (br d, J=9.29 Hz, 1 H), 2.97 (br d, J=9.03 Hz, 1 H), 2.93-2.78 (m, 2 H), 2.76-2.57 (m, 3 H), 1.76 (s, 3 H), 0.98-0.87 (m, 22 H).

[0475] 24b: 1-[(2R,6S)-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-(triisopropylsilyloxymethyl)morpholin-2-yl]pyrimidine-2,4-dione To a solution of compound 23b (5.0 g, 6.7 mmol) in anhydrous MeOH (85 mL), 2 At 25°C under atmospheric pressure (NH 4 ) 2 B 4 O 7 4H 2 HO (1.93 g, 7.6 mmol) was added. The mixture was stirred for 2 h, followed by addition of AcOH (0.80 g, 13.4 mmol), 10 g of 4 Å molecular sieves and NaBH 3 CN (0.84 g, 13.4 mmol) was added. After stirring for 12 h, the mixture was filtered and the filtrate was concentrated in vacuo. The residue was diluted with ice water and extracted three times with EtOAc (50 ml). The organic layers were combined, washed with brine and extracted with anhydrous Na 2 SO 4The mixture was dried at 40° C., filtered and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (PE / EtOAc 10:1 to 1:1) to give the title compound 24b as a white foam (94%). MS (m / z) = 738.5 [M+H] + 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.44 (br s, 1 H), 7.75 (d, J=8.07 Hz, 1 H), 7.45 (br d, J=7.34 Hz, 2 H), 7.37-7.20 (m, 7 H), 6.89 (d, J=8.80 Hz, 4 H), 5.88 (br dd, J=10.03, 2.45 Hz, 1 H), 5.73 (d, J=8.07 Hz, 1 H), 4.17 (br d, J=9.78 Hz, 1 H), 4.11-4.02 (m, 2 H), 3.77 (d, J=0.73 Hz, 6 H), 3.12 (br d, J=9.05 Hz, 1 H), 3.02-2.83 (m, 3 H), 2.70-2.58 (m, 2 H), 1.06-0.90 (m, 21 H).

[0476] 24c: N-[9-[(2R,6S)-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-(triisopropylsilyloxymethyl)morpholin-2-yl]-6-oxo-1H-purin-2-yl]-2-methyl-propanamide Following the protocol described in 24b, starting material 23c (7.0 g, 8.2 mmol) was converted to morpholine compound 24c, which was isolated after silica gel chromatography (PE / EtOAc 5:1 to 1:1) as a colorless foam (73.2%). MS (m / z) = 825,2 [M+H] + 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 12.12 (br s, 1 H), 11.59 (s, 1 H), 8.15 (s, 1 H), 7.40 (br d, J=7.03 Hz, 2 H), 7.30-7.12 (m, 7 H), 6.81 (br d, J=7.78 Hz, 4 H), 5.83 (dd, J=9.66, 3.14 Hz, 1 H), 4.23 (br d, J=9.03 Hz, 1 H), 3.92 (br d, J=9.03 Hz, 1 H), 3.72 (d, J=2.76 Hz, 6 H), 2.97 - 3.18 (m, 3H), 2.96-2.86 (m, 2 H), 2.80 (dt, J=13.55, 6.78 Hz, 1 H), 2.67 (br d, J=12.05 Hz, 1 H), 1.13 (t, J=6.15 Hz, 6 H), 1.03-0.82 (m, 22 H).

[0477] 24e: N-[9-[(2R,6S)-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-(triisopropylsilyloxymethyl)morpholin-2-yl]purin-6-yl]benzamide Following the protocol described in 24b, starting material 23e (5.0 g, 5.7 mmol) was converted to morpholine compound 24e, which was isolated after silica gel chromatography (PE / EtOAc 1:4) as a colorless foam (73.4%). MS (m / z) = 843,1 [M+H] + 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.22 (br s, 1 H), 8.75 (s, 1 H), 8.66 (s, 1 H), 8.05 (d, J=7.34 Hz, 2 H), 7.70-7.62 (m, 1 H), 7.60-7.51 (m, 2 H), 7.38 (br d, J=6.97 Hz, 2 H), 7.26-7.16 (m, 7 H), 6.78 (d, J=8.93 Hz, 4 H), 6.16 (dd, J=10.03, 2.81 Hz, 1 H), 4.26 (d, J=9.66 Hz, 1 H), 4.10-3.98 (m, 1 H), 3.71 (d, J=0.73 Hz, 6 H), 3.28 (br d, J=10.64 Hz, 1 H), 3.16 (br d, J=9.17 Hz, 1 H), 3.06 (br d, J=9.17 Hz, 1 H), 2.98-2.90 (m, 2 H), 2.79 (br d, J=12.47 Hz, 1 H), 1.10-0.82 (m, 21 H).

[0478] [ka]

[0479] 25: tert-Butyl (2S,6R)-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-(2,4-dioxo-pyrimidin-1-yl)-2-(triisopropylsilyloxymethyl)morpholine-4-carboxylate A mixture of morpholine 24b (50 g, 69.8 mmol) and DIPEA (18 g, 139.7 mol) in DCM (500 ml) was added with Boc 2 O (22.9 g, 104.8 mol) was added dropwise. After stirring for 24 h, the solvent was removed in vacuo and the residue was purified by column chromatography (PE / EtOAc 1:1) to give compound 25 (60 g, 93% yield) as a white foam. 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 7.58 (d, J=8.0 Hz, 1 H), 7.45 (d, J=7.2 Hz, 2 H), 7.33-7.26 (m, 7 H), 6.85-6.81 (m, 4 H), 6.02-5.98 (dd, J=10.0 3.6 Hz, 1 H), 5.77 (d, J=8.0 Hz, 1 H), 4.24-4.11 (m, 2 H), 3.99 (d, J=9.6 Hz, 1 H), 3.80 (s, 1 H), 3.75 (m, 1 H), 3.28-3.20 (m, 2 H), 3.08 (d, J=12.8 Hz, 1 H), 2.69 (t, J=11.4 Hz, 1 H), 1.48 (m, 9 H), 1.03-0.95 (m, 21 H).

[0480] 26: tert-Butyl (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-carboxylate A mixture of 1H-1,2,4-triazole (65 g, 0.94 mol) in ACN (650 ml) was added to POCl at 30 °C. 3 (49.79 g, 0.31 mol) was added dropwise. After cooling to 0° C., DIPEA (198 g, 1.53 mol) was added dropwise and stirring was continued at 0° C. for 30 min. 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 h. The mixture was poured into 3 L of a mixed solvent of EtOAc / water (1:2). After separation, the organic layer was washed with saturated NaHCO 3 Wash with 500 ml of solution and brine, and add anhydrous Na 2 SO 4 Drying at rt, filtration and concentration in vacuo afforded 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] +

[0481] 27: tert-Butyl-(2S,6R)-6-(4-benzamido-2-oxo-pyrimidin-1-yl)-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-2-(triisopropylsilyloxymethyl)morpholine-4-carboxylate To a mixture of benzamide (38 g, 0.31 mol) in 380 mL of dioxane at 0° C. was added NaH (12.8 g, 0.31 mol) in one portion. After stirring at 30° C. for 30 min, triazole 26 (40 g, 39.21 mmol, crude) was added in one portion and the mixture was stirred at 30° C. for 1 h. The reaction was purified with saturated NH 4 The mixture was quenched with 24 L of Cl solution and extracted with EtOAc (3 L). The organic layer was washed with 2 L of water and brine, and then washed with anhydrous Na 2 SO 4 Drying at 40° C., filtration and concentration in vacuo afforded crude compound 54 g as a yellow oil, and final silica gel chromatography (PE / EtOAc 2:1) afforded 27 (20.4 g, 56.6%) as a yellow foam. 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.31 (br s, 1 H), 8.20 (br d, J=7.21 Hz, 1 H), 8.01 (br d, J=7.46 Hz, 2 H), 7.70-7.59 (m, 1 H), 7.58-7.48 (m, 2 H), 7.47-7.36 (m, 3 H), 7.34-7.16 (m, 7 H), 6.87 (br d, J=7.70 Hz, 4 H), 5.96 (br dd, J=10.09, 2.63 Hz, 1 H), 4.12 (br d, J=11.25 Hz, 1 H), 4.00-3.83 (m, 3 H), 3.74 (s, 6 H), 3.28-3.16 (m, 2 H), 3.06 (br d, J=9.17 Hz, 2 H), 1.55-1.32 (m, 9 H), 1.11-0.80 (m, 21 H).

[0482] 28: N-[1-[(2R,6S)-6-(hydroxymethyl)-6-(triisopropylsilyloxymethyl)morpholin-2-yl]-2-oxo-pyrimidin-4-yl]benzamide To a solution of 27 (13.5 g, 14.70 mmol) in DCM (93 ml) was added TFA (31 ml) dropwise at 0° C. The reaction was stirred at 30° C. for 16 h. Saturated NaHCO 3 After neutralization to pH=7-8 with solution, the organic layer was separated and the aqueous phase was extracted twice with DCM (100 ml). The combined organic layers were washed with anhydrous Na 2 SO 4 The mixture was dried over 1000 cc, filtered and concentrated in vacuo. The residue was purified by column chromatography (EtOAc / EtOH=30:1) to give 28 (2.7 g, 35.6% yield) as a yellow foam and the corresponding trifluoroacetate (4.78 g, 53.1%) as a by-product, which was dissolved in a mixture of THF (186 ml) and EtOH (62 ml). 75.8 ml of 1M aqueous NaOH was added at 0° C. After stirring for 2 min, the solution was neutralized with saturated citric acid solution and diluted with H 2 The mixture was diluted with 200 mL of EtOAc (300 mL) and extracted with EtOAc (300 mL). The organic layer was washed with brine and 2 SO 4 It was dried at 40° C. and evaporated. Purification by silica gel chromatography (EtOAc / EtOH 30:1) gave 28 (5.2 g, 68.5%) as a white solid. MS (m / z) = 517,4 [M+H + ] + 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.26 (br s, 1 H), 8.28 (d, J=7.53 Hz, 1 H), 8.01 (d, J=7.28 Hz, 2 H), 7.67-7.58 (m, 1 H), 7.57-7.47 (m, 2 H), 7.34 (d, J=7.40 Hz, 1 H), 5.86 (dd, J=9.66, 2.64 Hz, 1 H), 4.64 (br s, 1 H), 4.01-3.90 (m, 2 H), 3.46 (br s, 2 H), 3.05 (dd, J=11.98, 2.57 Hz, 1 H), 3.11-3.00 (m, 1 H), 2.87-2.75 (m, 1 H), 2.74-2.63 (m, 1 H), 2.40-2.27 (m, 1 H), 1.15-0.99 (m, 21 H).

[0483] 29: 9H-Fluoren-9-ylmethyl (2S,6R)-6-(4-benzamido-2-oxo-pyrimidin-1-yl)-2-(hydroxymethyl)-2-(triisopropylsilyloxymethyl)morpholine-4-carboxylate To a solution of morpholine 28 (7.0 g, 13.55 mmol) in DMF (70 ml) was added Fmoc-N-hydroxy-succinimide ester (5.5 g, 16.26 mmol) in portions at 0° C. After stirring for 16 h, the solution was poured into water (100 ml) and extracted three times with EtOAc (30 ml). The combined organic layers were washed with brine and extracted with anhydrous Na 2 SO 4 The mixture was dried at 40° C., filtered and concentrated in vacuo. Final purification on silica (PE / EtOAc 1:2) afforded compound 29 (10.0 g, quantitative) as a yellow solid. MS (m / z) = 739.4 [M+H] + 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.26 (br s, 1 H), 8.33 (br s, 1 H), 8.02 (br d, J=7.46 Hz, 2 H), 7.90 (d, J=7.58 Hz, 2 H), 7.64 (br t, J=7.40 Hz, 3 H), 7.57-7.49 (m, 2 H), 7.48-7.40 (m, 3 H), 7.39-7.30 (m, 2 H), 6.11-5.79 (m, 1 H), 4.98 (t, J=6.05 Hz, 1 H), 4.52-4.12 (m, 4 H), 3.89-3.69 (m, 2 H), 3.54 (br s, 3 H), 3.21-3.07 (m, 1 H), 3.04-2.77 (m, 1 H), 1.11-0.89 (m, 21 H).

[0484] 30: 9H-Fluoren-9-ylmethyl-(2S,6R)-6-(4-benzamido-2-oxo-pyrimidin-1-yl)-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-2-(triisopropylsilyloxymethyl)morpholine-4-carboxylate To a solution of compound 29 (13.9 g, 18.83 mmol) in 140 ml of pyridine was added DMT-Cl (12.75 g, 37.62 mmol) at 25° C. The mixture was stirred at 25° C. for 16 h to achieve complete conversion. After evaporation of the solvent, the residue was dissolved in EtOAc (200 ml) and washed with 100 ml of water and brine. The organic phase was washed with Na 2 SO 4 The mixture was dried at 40° C., filtered and concentrated in vacuo. The residue was purified by column chromatography (PE / EtOAc 1:1) to give compound 30 (17.3 g, 88.1%) as a yellow foam. 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.32 (br s, 1 H), 8.21 (br s, 1 H), 8.02 (br d, J=7.58 Hz, 2 H), 7.88 (br s, 2 H), 7.76-7.60 (m, 3 H), 7.59-7.50 (m, 2 H), 7.42 (br d, J=7.21 Hz, 5 H), 7.38-7.19 (m, 9 H), 6.89 (br d, J=8.44 Hz, 4 H), 6.19-5.85 (m, 1 H), 4.28 (br s, 4 H), 3.91 (br d, J=8.80 Hz, 2 H), 3.75 (s, 6 H), 3.23 (br s, 1 H), 3.10 (br d, J=9.05 Hz, 3 H), 1.06-0.78 (m, 21 H).

[0485] 24f: N-[1-[(2R,6S)-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-(triisopropylsilyloxymethyl)morpholin-2-yl]-2-oxo-pyrimidin-4-yl]benzamide To a solution of 30 (8.4 g, 8.07 mmol) in DCM (85 ml) was added 17 ml of piperidine at room temperature. After complete conversion (approximately 20 min), the reaction solution was diluted with saturated NH 4 The aqueous phase was extracted with DCM and the combined organic layers were washed with Na 2 SO 4 The mixture was dried at 40° C., filtered and concentrated in vacuo. The crude product was purified by column chromatography (PE / EtOAc 1:4) to give the title compound 24f (4.7 g, 71.2%) as a white foam. MS (m / z) = 819,4 [M+H] + 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.31 (br s, 1 H), 8.18 (d, J=7.58 Hz, 1 H), 8.01 (br d, J=7.34 Hz, 2 H), 7.67-7.58 (m, 1 H), 7.57-7.47 (m, 2 H), 7.42 (br d, J=7.46 Hz, 3 H), 7.34-7.15 (m, 7 H), 6.86 (br d, J=8.56 Hz, 4 H), 6.04-5.89 (m, 1 H), 4.18 (br d, J=9.54 Hz, 1 H), 4.07 (br d, J=9.54 Hz, 1 H), 3.73 (d, J=1.34 Hz, 6 H), 3.15 (br d, J=9.17 Hz, 1 H), 3.05 (br d, J=10.15 Hz, 1 H), 2.97 (br d, J=9.17 Hz, 1 H), 2.88 (br d, J=12.47 Hz, 1 H), 2.62 (br d, J=12.47 Hz, 1 H), 2.46 (br s, 1 H), 1.08-0.83 (m, 21 H).

[0486] [ka]

[0487] General Procedure A for the Reductive Amination Reaction to Prepare Compounds 31a-31f Morpholine 24a (1.0 g, 1.4 mmol) was dissolved in MeOH (25 ml), followed by 2.0 g of molecular sieves (4 Å), NEt 3 (560 mg, 769 μl, 5.5 mmol), AcOH (827 mg, 789 μl, 13.7 mmol) and 1.0 to 3.0 equiv of the corresponding aldehyde or ketone were added. After stirring for 15 min, 344 mg (5.5 mmol) of sodium cyanoborohydride was added in four portions every 30 min and the reaction was stirred at room temperature for 6 h. After standing overnight, the mixture was filtered and the filtrate was dissolved in saturated NaHCO 3The solution was diluted with 25 ml. MeOH was evaporated and the aqueous phase was extracted with 50 ml of DCM / iPrOH (5:1). After further filtration, the organic phase was separated and diluted with MgSO 4 It was dried at 40° C. and evaporated, and the crude product obtained was used in the next step without further purification.

[0488] 31a: 1-[(2R,6S)-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-methyl-6-(triisopropylsilyloxymethyl)morpholin-2-yl]-5-methyl-pyrimidine-2,4-dione Following general procedure A, using 3.0 equivalents of formaldehyde (37% in water), the title compound 31a (1.0 g) was isolated a...

Claims

1. A double-stranded oligonucleotide comprising a sense strand oligonucleotide and an antisense strand oligonucleotide, wherein the antisense strand oligonucleotide comprises one or more nucleotide analogues of formula (IA) at a position other than the 5'-overhang or other than the 3'-overhang of the antisense strand oligonucleotide, and the nucleotide analogues of formula (IA) are as described below: 【Chemistry 1】 wherein, independently for each compound of formula (IA): - B is a heterocyclic nucleobase; one of L1 and L2 is an internucleoside linking group linking the compound of formula (IA) to a nucleotide residue and the other of L1 and L2 is H, a protecting group, a phosphorus moiety or an internucleoside linking group linking the compound of formula (IA) to a nucleotide residue, Y is O, NH, NR1 or N-C(=O)-R1, R1 being: a (C1-C20) alkyl group optionally substituted with one or more groups selected from 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, -O-C(=J)-Z1, -C(=J)-N(Z1)(Z2), -N(Z1)-C(=J)-Z2, J is O or S; Each of Z1 and Z2 is independently H or a (C1-C6) alkyl group, optionally substituted with one or more groups selected from halogen atoms and a (C1-C6) alkyl group; a (C3-C8)cycloalkyl group, optionally substituted with one or more groups selected from halogen atoms and (C1-C6)alkyl groups, ・Group -[C(=O)]m-R2-(O-CH 2 -CH 2 ) p-R3, m is an integer meaning 0 or 1, p is an integer ranging from 0 to 10; R2 is a (C1-C20) alkylene group optionally substituted with a (C1-C6) alkyl group, -O-Z3, -N(Z3)(Z4), -S-Z3, -CN, -C(=K)-O-Z3, -O-C(=K)-Z3, -C(=K)-N(Z3)(Z4), and -N(Z3)-C(=K)-Z4; K is O or S; Each of Z3 and Z4 is independently H or a (C1-C6) alkyl group optionally substituted with one or more groups selected from halogen atoms and a (C1-C6) alkyl group; R3 is selected from the group consisting of 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; X1 and X2 are each independently a hydrogen atom or a (C1-C6) alkyl group; each of Ra, Rb, Rc and Rd is independently H or a (C1-C6) alkyl group; and any stereoisomer thereof or any pharma- ceutically acceptable salt thereof.

2. The double-stranded oligonucleotide according to claim 1, wherein the antisense strand oligonucleotide comprises 1 to 10 nucleotide analogues of the formula (IA).

3. The double-stranded oligonucleotide according to claim 1, wherein the antisense strand oligonucleotide comprises 1 to 5 nucleotide analogues of the formula (IA).

4. The double-stranded oligonucleotide of claim 1, wherein the antisense strand oligonucleotide has a nucleotide length ranging from 15 to 30 nucleotides.

5. The double-stranded oligonucleotide of claim 4, wherein the antisense strand oligonucleotide has a nucleotide length ranging from 20 to 25 nucleotides.

6. The double-stranded oligonucleotide of claim 5, wherein the antisense strand oligonucleotide has a nucleotide length ranging from 17 to 25 nucleotides.

7. The double-stranded oligonucleotide according to claim 1, wherein in the antisense strand oligonucleotide, one or more nucleotide analogues of formula (IA) contained therein are arranged contiguously and / or non-contiguously at any position in the antisense strand oligonucleotide.

8. The antisense strand oligonucleotide comprises a hybridizing region that hybridizes with the sense strand oligonucleotide, and in the antisense strand oligonucleotide, one or more nucleotide analogues of formula (IA) contained therein are located at the 5'-terminal nucleotide of the antisense strand in the nucleotide region of the antisense strand oligonucleotide.

2. The double-stranded oligonucleotide of claim 1, wherein the nucleotide at position 2 of the hybridizing region is located in the range of nucleotides at positions 2 to 15, or nucleotides at positions 2 to 10, or nucleotides at positions 2 to 8 of the hybridizing region starting from the nucleotide at position 15 of the hybridizing region.

9. The antisense strand oligonucleotide may comprise one or more nucleotide analogs of the following formula (IB): 【Chemistry 2】 wherein, independently for each compound of formula (IB): - B is a heterocyclic nucleobase; one of L1 and L2 is an internucleoside linking group linking the compound of formula (IB) to a nucleotide residue and the other of L1 and L2 is H, a protecting group, a phosphorus moiety or an internucleoside linking group linking the compound of formula (IB) to a nucleotide residue, Y is NR1 or N-C(=O)-R1, R1 being: ・Group -[C(=O)]m-R2-(O-CH 2 -CH 2 ) p-R3, m is an integer meaning 0 or 1, p is an integer ranging from 0 to 10; R2 is a (C1-C20) alkylene group optionally substituted with a (C1-C6) alkyl group, -O-Z3, -N(Z3)(Z4), -S-Z3, -CN, -C(=K)-O-Z3, -O-C(=K)-Z3, -C(=K)-N(Z3)(Z4), and -N(Z3)-C(=K)-Z4; K is O or S; Each of Z3 and Z4 is independently H or a (C1-C6) alkyl group optionally substituted with one or more groups selected from halogen atoms and a (C1-C6) alkyl group; R3 is a cell targeting moiety, X1 and X2 are each independently a hydrogen atom or a (C1-C6) alkyl group; each of Ra, Rb, Rc and Rd is independently H or a (C1-C6) alkyl group; 2. The double-stranded oligonucleotide of claim 1, further comprising:

10. The double-stranded oligonucleotide according to claim 9, wherein the antisense strand oligonucleotide comprises one or more nucleotide analogues of formula (IB), and at least one nucleotide of formula (IB) is a 5'- or 3'-terminal nucleotide.

11. In the nucleotide analogs of formulae (IA) and (IB) contained in the antisense strand oligonucleotide, Y is NR1, - R1 is an unsubstituted (C1-C20) alkyl group, or R1 is an unsubstituted (C1-C16) alkyl group, which alkyl group comprises an alkyl group selected from the group comprising methyl, isopropyl, butyl, octyl, hexadecyl, or R1 is a (C3-C8) cycloalkyl group optionally substituted with one or more groups selected from halogen atoms and (C1-C6) alkyl groups, or R1 is a cyclohexyl group, or R1 is a (C1-C20) alkyl group substituted with a (C6-C14) aryl group, or R1 is a methyl group substituted with a phenyl group, The double-stranded oligonucleotide according to claim 1 or 9, wherein L1, L2, Ra, Rb, Rc, Rd, X1, X2, R2, R3 and B have the same meaning as defined for general formula (IA) according to claim 1, or any pharma- ceutically acceptable salt thereof.

12. In the nucleotide analogs of formulae (IA) and (IB) contained in the antisense strand oligonucleotide, Y is N-C(=O)-R1; R1 is an optionally substituted (C1-C20) alkyl group or R1 is selected from the group comprising methyl and pentadecyl; The double-stranded oligonucleotide according to claim 1 or 9, wherein L1, L2, Ra, Rb, Rc, Rd, X1, X2, R2, R3 and B have the same meaning as defined in claim 1 or a pharma- ceutically acceptable salt thereof.

13. The double-stranded oligonucleotide according to claim 1 or 9, wherein in the nucleotide analogues of formulae (IA) and (IB) contained in the antisense strand oligonucleotide, B is selected from the group consisting of pyrimidine, substituted pyrimidine, purine and substituted purine, or any pharma- ceutically acceptable salt thereof.

14. 2. The double-stranded oligonucleotide of claim 1, wherein the one or more nucleotide analogues of formula (IA) are in their (2R,6R)-stereoisomers.

15. The double-stranded oligonucleotide of claim 1, wherein the sense strand oligonucleotide comprises one or more nucleotide analogues of formula (IA) as defined in claim 1.

16. The double-stranded oligonucleotide of claim 15, wherein the sense strand oligonucleotide comprises one or more nucleotide analogues of formula (IB) as defined in claim 9.

17. The double-stranded oligonucleotide according to claim 16, wherein the sense strand comprises one or more nucleotide analogues of formula (IB) as defined in claim 9, wherein at least one nucleotide of formula (IB) consists of a 5'-terminal or 3'-terminal nucleotide, and the one or more modified nucleotides of formula (IB) are contiguous.

18. The double-stranded oligonucleotide according to claim 16, wherein the sense strand comprises from 1 to 3 nucleotide analogues of formula (IB) as defined in claim 9, and the modified nucleotides of formula (IB) are consecutive and form an oligonucleotide stretch located at the 5'-end or the 3'-end of the sense strand.

19. The double-stranded oligonucleotide of claim 1 , which consists of a short interfering RNA (siRNA), or a pharma- ceutically acceptable salt thereof.

20. A composition comprising a double-stranded oligonucleotide as defined in any one of claims 1 to 19.

21. A pharmaceutical composition comprising the double-stranded oligonucleotide according to any one of claims 1 to 19.

22. A double-stranded oligonucleotide as defined in any one of claims 1 to 19 as a medicament.

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