Oligonucleotides containing nucleotide analogs

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 provides targeted gene silencing with single- and double-stranded oligonucleotides, more particularly short interfering RNAs (siRNAs), comprising the modified nucleotide analogues described herein. related to the field. [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 using short synthetic oligonucleotides was first demonstrated (Non-Patent Document 1). Reference 1). Following Stephenson's discovery, the elucidation of the RNA interference pathway for modulation of gene expression and the role of siRNAs in that process greatly expanded scientists' understanding of post-transcriptional gene expression regulation 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 siRNA. Both ASOs and siRNAs act by binding target RNAs through Watson-Crick base pairing, but their mechanisms of action are different. In antisense technology, ASOs form DNA-RNA duplexes with target RNAs, inhibiting mRNA translation by blocking mechanisms or causing RNase H-dependent degradation of targeted RNAs. In RNA interference technology, siRNAs bind to an RNA-induced silencing complex ("RISC") in which one strand (the "passenger strand" or "sense strand") is displaced and the remaining strand (the "guide strand") or "antisense strand") cooperates with RISC to bind complementary RNA (target RNA); once bound, the target RNA is cleaved by Argonaute (AGO), an RNA endonuclease in RISC. and then further degraded by RNA exonucleases.

[0004] Among the most significant obstacles to the development of oligonucleotide therapeutics, including siRNA therapeutics, are (i) poor compound stability, (ii) low efficiency of in vivo delivery to target cells, and (iii) "off-target". Side effects include gene silencing and unanticipated immune enhancement. Various oligonucleotide chemical modifications have been explored to address some of these obstacles. These modifications can be grouped 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 the 2'-hydroxy group of the ribose ring. 2'-OMe (methoxy) nucleotide analogues 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 modifications are concentrated at 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 a nucleoside, creating a so-called locked nucleic acid ("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 in the resulting double-stranded oligonucleotide, Causes a significant increase in temperature. However, fully LNA-modified oligomers longer than 8 nucleotides tend to assemble. In contrast to the rigid nature of LNA modifications, highly flexible unlocked nucleic acid (“UNA”) modifications have also been developed for application in oligonucleotide therapeutics. UNA nucleosides do not have a ribose sugar C2'-C3' linkage. UNA is not conformationally restricted due to its open chain structure and has been used to modulate the flexibility of oligonucleotides (Non-Patent Document 5). Insertions of UNA can lower the melting temperature (Tm) of the duplex in some cases by 5-10°C per insertion. 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 techniques. Such systems include the six-membered morpholino ring system in which the ribose portion of the nucleoside is replaced with a morpholine ring. A morpholino-based nucleoside forms an internucleotide linkage within the oligonucleotide containing it 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 its low binding affinity to complementary RNA, antisense PMOs need to be relatively long, eg, 25 bases in length (Non-Patent Document 7, supra). Examples of morpholino subunits are also disclosed in US Pat.

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

[0009] However, most of the aforementioned phosphodiester modifications, such as phosphorothioates, give rise to chiral phosphorus during internucleotide linkages, resulting in diastereomeric mixtures of the resulting oligonucleotides. Since the number of diastereomeric oligonucleotides with each modified phosphodiester linkage can be doubled, the number of diastereomers obtained grows exponentially with increasing number of modified phosphodiester linkages. To increase. Individual diastereomers may exhibit different degrees of nuclease resistance and different hybridizing properties to target mRNAs. In addition, purification and chemical analytics of diastereomeric mixtures are complicated. Therefore, in some cases it may be desirable to avoid using phosphodiester modifications such as phosphorothioates and the resulting diastereomeric oligonucleotide mixtures.

[0010] Another important issue in RNA interference technology is the targeted delivery and cellular uptake of siRNA. The cell membrane is a bilayer of negatively charged phospholipids and is also a barrier to entry for negatively charged siRNAs. Some groups have used N-acetylgalactosamine (GalNAc) to target siRNA attached to it to hepatocytes, which express the GalNAc-associated asialoglycoprotein receptor (ASGPR) and endogenously. ASGPR-binding siRNA-GalNAc conjugates can be internalized via cytosis (see, eg, Non-Patent Document 9). [Prior art documents] [Patent document]

[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 [Outline of the invention] [Problems 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 profile and their delivery to target cells. [Means for solving the problem]

[0014] The present disclosure is a double-stranded oligonucleotide comprising a sense strand oligonucleotide and an antisense strand oligonucleotide, wherein the antisense strand oligonucleotide comprises either a 5'-overhang or a 3'-overhang of said antisense strand oligonucleotide. A double-stranded oligonucleotide comprising one or more nucleotide analogues of formula (I-A) that is neither nor nor, wherein the nucleotide analogues of formula (I-A) are as described throughout the specification.

[0015] In some embodiments of said double-stranded oligonucleotide, the antisense strand oligonucleotide comprises 1 to 10 nucleotide analogues of said Formula (I-A), preferably 1 to 10 nucleotide analogues of said Formula (I-A). Including 5 pcs.

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

[0017] In some embodiments of said double-stranded oligonucleotide, in the antisense strand oligonucleotide, the one or more nucleotide analogues of Formula (I-A) contained therein are contiguous and / or non-contiguous. is placed anywhere on the antisense strand oligonucleotide.

[0018] In some embodiments of said double-stranded oligonucleotide, the antisense strand oligonucleotide comprises a hybridizing region that hybridizes with the sense strand oligonucleotide, and in the antisense strand oligonucleotide, the formula (I-A ) in the nucleotide region of said antisense oligonucleotide starting from the nucleotide at the 5′ end of the antisense strand to the nucleotide at position 2 of said hybridizing region at position 15 It is arranged to span nucleotides.

[0019] In some embodiments of said double-stranded oligonucleotide, the antisense strand oligonucleotide comprises a hybridizing region that hybridizes with the sense strand oligonucleotide, and in the antisense strand oligonucleotide, the formula (I-A ) in the nucleotide region of said antisense oligonucleotide starting from the nucleotide at the 5′ end of said antisense strand and starting from the nucleotide at position 2 of said hybridizing region to position 8 is positioned ranging from the nucleotide at position 2 to the nucleotide at position 10 of said hybridizing region, such as to the nucleotide at position 1.

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

[0021] In some embodiments of the double-stranded oligonucleotide, in the nucleotide analogs of formulas (I-A) and (I-B) contained in the antisense strand oligonucleotide, B is pyrimidine, substituted pyrimidine, purine and substituted or any pharmaceutically acceptable salt thereof.

[0022] In some embodiments of said double-stranded oligonucleotide, said one or more nucleotide analogues of Formula (I-A) consists of its (2R,6R)-stereoisomer.

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

[0024] In some embodiments of said double-stranded oligonucleotide, the sense strand oligonucleotide comprises one or more nucleotide analogues of Formula (I-B) described herein.

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

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

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

[0028] The present disclosure further relates to single-stranded antisense oligonucleotides complementary to target mRNA or 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 drawing]

[0031]

Figure 1

Figure 2

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

[0033] According to a most preferred embodiment, said specific nucleotide analogue has a ribose sugar ring replaced by a 6-membered heterocyclic ring. As described in more detail in this disclosure, the 6-membered heterocyclic group of the specific nucleotide analogue can be a dioxane or morpholino ring. When the heterocyclic group is a morpholino ring, the nitrogen atom is either substituted or unsubstituted. In some embodiments, the 6-membered heterocyclic group is substituted with linear or cyclic groups and / or targeting moieties.

[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 presently disclosed subject matter.

[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" expressly do not Includes plural referents unless otherwise indicated.

[0037] The term "about," as used herein, refers to the normal margin of error for the respective value readily recognized by those skilled in the art. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. In some embodiments, the term "about" refers to ±10% of the given value. However, whenever the value in question refers to something that is indivisible, such as a nucleotide, or something else that would lose its identity once divided, "about" refers to ±1 of the indivisibility.

[0038] Aspects and embodiments of the disclosure described herein include "having," "including," "consisting of," and "consisting essentially of" aspects and embodiments. is understood. The words "have" and "comprise," or variations such as "has," "having," "comprises," or "comprises," refer to the stated element. It will be understood that although inclusion of (eg, a composition of matter or method steps) is indicated, no other element is excluded. The term "consisting of" is meant to include the stated elements and not including any additional elements. The term “consisting essentially of” indicates the inclusion of the stated element and, optionally, other elements where the other elements do not materially affect the novel characteristics underlying the invention.

[0039] "Alkyl" is a straight or branched chain having 1-20 (eg, 1-5, 1-10, or 1-15) carbon atoms in the chain, unless otherwise specified. means a good aliphatic hydrocarbon group. "Branched" means that a linear alkyl chain has one or more alkyl groups attached thereto, such as methyl, ethyl or propyl groups. Exemplary linear or branched alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, n-pentyl, 3-pentyl, octyl, nonyl, and decyl. are mentioned.

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

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

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

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

[0044] A "heterocycle" or "heterocyclic" is a saturated, partially unsaturated or unsaturated ring containing at least one heteroatom selected from the group of oxygen, nitrogen, selenium, phosphorus and sulfur. Refers to a saturated carbocyclic group. Nitrogen, selenium, phosphorus or sulfur may optionally be oxidized and nitrogen may optionally be quaternized. For example, a heterocycle can be a stable ring in which at least one ring member is a heteroatom. In some embodiments, the heterocyclic ring may have 3-14, such as 5-7, or 5-10 ring members, and may include one, two, or multiple rings ( ie, monocyclic, bicyclic or polycyclic rings). In certain embodiments, heteroatoms are oxygen, nitrogen and sulfur. The number of heteroatoms may vary, eg, from 1 to 3. Suitable heterocycles are also disclosed in The Handbook of Chemistry and Physics, 76th Edition, CRC Press, Inc., 1995-1996, pppp.2-25-2-26, the disclosure of which is incorporated herein by reference. incorporated herein. In some embodiments, the heterocycle is a non-aromatic heterocycle, including, but 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 phenyl groups.

[0045] "Heteroaryl" refers to an aromatic heterocyclic ring having 5-14 (eg, 5-7, or 5-10) ring members, which may be monocyclic, bicyclic or polycyclic. The number of heteroatoms may typically vary from 1 to 3 heteroatoms, eg selected from 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, oxazinyl. Azoles, isothiazolyls, triazolyls, tetrazolyls, isoquinolyls, benzothienyls, isobenzofuryls, pyrazolyls, carbazolyls, benzimidazolyls, isoxazolyls, and pyridyl-N-oxides, as well as fused systems resulting from condensation with phenyl groups. . In certain embodiments, the heteroaryl is a 5- or 6-membered heteroaryl containing one or more heteroatoms, eg, 1 to 3 heteroatoms selected from N and O. "Alkyl", "cycloalkyl", "alkenyl", "alkynyl", "aryl", "heteroaryl" and "heterocyclyl" are also the corresponding "alkylene" formed by removal of two hydrogen atoms, Also referred to as "cycloalkylene," "alkenylene," "alkynylene," "arylene," "heteroarylene," and "heterocyclene."

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

[0047] Unless otherwise specified, the term "heterocyclic nucleobase" as used herein, according to the present disclosure, can be attached to an optionally substituted dioxane ring or an optionally substituted morpholino ring, where refers to a nitrogen-containing heterocyclic group substituted by In some embodiments, heterocyclic nucleobases can be selected from optionally substituted purine bases or optionally substituted pyrimidine bases. The term "purine base" is used herein in its ordinary sense as understood by those of ordinary skill in the art and includes tautomers thereof. Similarly, the term "pyrimidine base" is used herein in its ordinary sense as understood by those skilled in the art, and includes tautomers thereof. A non-limiting list of optionally substituted purine bases include purine, adenine, guanine, hypoxanthine, xanthine, alloxanthine, 7-alkylguanine (e.g., 7-methylguanine), theobromine, caffeine, uric acid and Isoguanine can be mentioned. Examples of pyrimidine bases include, but are not limited to, cytosine, thymine, uracil, 5,6-dihydrouracil and 5-alkylcytosine (eg, 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-deazaxanthin, 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 by reference Other heterocyclic nucleobases are described in US Pat. Nos. 5,432,272 and 7,125,855, which disclose additional heterocyclic bases incorporated herein. In some embodiments, heterocyclic nucleobases are optionally substituted with amine or enol protecting groups.

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

[0049] Examples of protecting group moieties are described in T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, Third Edition, John Wiley & Sons, 1999, and J.F.W. McOmie, Protective Groups in Organic Chemistry Plenum Press, 1973, both of which are are also incorporated herein by reference for the limited purpose of disclosing suitable protecting groups. Protecting group moieties can be chosen in such a manner that they are stable to the particular reaction conditions and readily 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-butyl dimethylsilyl, triisopropylsilyloxymethyl, [2-(trimethylsilyl)ethoxy]methyl or t-butyldiphenylsilyl); esters (e.g. benzoate, 2-cyanoethyl phosphate); carbonates (e.g. methoxymethyl carbonate); sulfonates (e.g. tosylate or mesylate); acyclic ketals (e.g. dimethylacetal); 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); such as those described herein) and triarylmethyl groups (eg, trityl; monomethoxytrityl (MMT); 4,4′-dimethoxytrityl (DMT); 4,4′,4″-trimethoxytrityl). (TMT); and those described herein).

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

[0052] Unless otherwise specified, any protecting groups, abbreviations for amino acids and other compounds are their common usage, accepted abbreviations, or 1972)).

[0053] The term “solid support” (also called resin), as used herein, means an insoluble particle, typically 50-200 μm in diameter, to which oligonucleotides are attached during synthesis. Although many types of solid supports have been used, controlled pore glass (CPG) and polystyrene (highly crosslinked polystyrene beads) have proven particularly useful. Controlled pore glass is rigid, non-expanding, and has deep pores (500-1000 Å pore size) in which oligonucleotide synthesis occurs. Solid supports for conventional oligonucleotide synthesis are commercially available and are typically produced with loadings of 20-40 μmol nucleosides 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 non-commercial as shown for the building blocks described later herein. It is used for the synthesis of various building blocks. As an alternative to a solid support with the 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 appreciate that guanine, cytosine, adenine, uracil or thymine in nucleotides may be replaced by other It is expected that you understand that replacement is possible in the part of For example, a nucleotide containing inosine as its base can base pair with nucleotides containing, but not limited to, adenine, cytosine, or uracil. Thus, nucleotides containing uracil, guanine, or adenine can be replaced in the nucleotide sequences of the disclosure by nucleotides containing, for example, inosine. Sequences containing such replacement moieties are included as embodiments of the present disclosure.

[0055] By "cell targeting moiety" as used herein is meant a molecular group that ensures increased delivery of siRNA, such increase being associated with (i) a selected target receptor (e.g., target (ii) increased uptake of the siRNA by target cells; and / or ( iii) increasing the ability of the siRNA to be properly processed once it enters the target cell, including increasing intracellular release of the siRNA, e.g. by facilitating translocation of the siRNA from transport vesicles to the cytoplasm; do. Thus, cell-targeting moieties are used to direct and / or deliver oligonucleotides to specific cells, tissues, organs, and the like. A cell-targeting moiety comprised in a nucleotide, nucleotide analogue or oligonucleotide is preferentially recognized by a targeted cell type over a non-targeted cell type. imparting the characteristics of a nucleotide analog or an oligonucleotide to said nucleotide such that it is bound, bound, internalized, processed, activated, etc. For example, endothelial cells have a high affinity for the peptide cell-targeting moiety Arg-Gly-Asp (RGD); cancer and kidney cells preferentially interact with compounds with folic acid moieties; , has an affinity for mannose; and cardiomyocytes have an affinity for the peptide WLSEAGPVVTVRALRGTGSW (SEQ ID NO: 1) (see, eg, Biomaterials ZV-8081-8087, 2010). Other cell targeting / delivery moieties are known in the art. Thus, a compound containing a cell-targeting moiety will preferentially interact with and be taken up by the targeted cell type.

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

[0057] A "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, 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, the amino acid and nucleotide sequences of which can be found, for example, in the EMBL database under accession number CR456908; includes mouse TTR, the amino acid and nucleotide sequences of which can be found, for example, in the GenBank database under accession number AAH24702. Further examples of TTR mRNA sequences are readily available, eg, at GenBank.

[0059] A "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 product of RNA processing of the primary transcript.

[0060] As used herein, and unless otherwise specified, the term "complementary" refers to a first nucleotide sequence (e.g., an oligonucleotide) relative to a second nucleotide sequence (e.g., an oligonucleotide). As used to describe, as understood by those of skill in the art, refers to the ability of a first nucleotide sequence to hybridize to a second nucleotide sequence under specified conditions and form a duplex structure. Examples of this include base pairing of a first nucleotide sequence to a second nucleotide sequence over the entire length of the first or second nucleotide sequence. Such sequences can be referred to as "fully complementary" to each other herein. When a first sequence is referred to herein as being "substantially complementary" to a second sequence, the two sequences may be fully complementary or their final It may have 70% or more nucleotide identity while retaining the ability to hybridize under optimal conditions to the target. However, when two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs are not considered mismatches for determining complementarity. shall be For example, a double-stranded RNA (dsRNA) comprising a first oligonucleotide 21 nucleotides in length and a second oligonucleotide 23 nucleotides in length, wherein the second oligonucleotide is Those comprising a sequence of 21 nucleotides that are perfectly complementary to nucleotides are also referred to as "fully complementary" for the purposes of this disclosure. A "complementary" sequence may also comprise non-Watson-Crick base pairs and / or base pairs formed from non-naturally occurring modified nucleotides, as long as the above requirements regarding its ability to hybridize are met. or formed entirely therefrom. The terms "complementary," "fully complementary," and "substantially complementary," as understood from the context of their use, between the sense and antisense strands of dsRNA, or It can be used for base matching between the antisense strand of dsRNA and the target sequence. 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 the mRNA of interest. The term "double-stranded RNA" or "dsRNA", as used herein, is a ribonucleic acid having a double-stranded structure comprising two antiparallel, substantially complementary nucleic acid strands as defined above. Refers to a complex of molecules. 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 RNAs (siRNAs). The two strands are part of one larger molecule, thus connecting an uninterrupted strand of nucleotides between the 3' end of the first strand and the 5' end of the second strand When forming a heavy chain structure, the connecting RNA strands are referred to as a "hairpin loop," a "short hairpin RNA," or a "shRNA." The 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 strand of nucleotides to form a duplex structure If so, the connecting structure is referred to as a "linker." The RNA strands may have the same or different numbers of nucleotides. The maximum number of base pairs is the number of oligonucleotides in the shortest strand of the dsRNA minus any 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 includes substantial modifications at multiple nucleotides, as well as all types of modifications disclosed herein or known in the art. It may contain chemical modifications to the ribonucleotides. Any such modifications are encompassed by "dsRNA" for the purposes of this disclosure when used in siRNA-type molecules. In some embodiments, internucleotide linkages in the dsRNA may be modified, eg, as described herein.

[0061] Within the scope of this disclosure, "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, where this percentage is purely statistical, the differences between two sequences are randomly distributed along their length. A comparison of two nucleic acid sequences is conventionally performed by comparing the sequences after aligning them optimally, said comparison can be performed by segment or by using an "alignment window." Optimal alignment of sequences for comparison is performed using the local homology algorithm of Smith and Waterman (1981), the local homology algorithm of Neddleman and Wunsch (1970), Pearson and Lipman, in addition to manual comparison. (1988)) or using these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, or BLAST, a comparison software). NR or BLAST P).

[0062] The percentage identity between two nucleic acid sequences is determined by comparing two optimally aligned sequences, where for optimal alignment between the two sequences the compared nucleic acid sequences are referenced It may have additions or deletions compared to the sequence. Percentage identity is determined by determining the number of positions with identical nucleotide residues between the two sequences, preferably between the two complete sequences, dividing the number of identical positions by the total number of positions in the alignment window, and Calculated by 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 with a reference sequence has at least 71%, 72%, 73%, 74%, 75%, 76%, 77% identity with said reference sequence. %, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, Those with 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity are included.

[0064] In some embodiments, the dsRNA comprises modified ribonucleosides comprising deoxyribonucleosides, such as deoxyribonucleoside overhangs, those comprising one or more deoxyribonucleosides within the double-stranded portion of the dsRNA, and the like. mentioned. However, in no case is it self-evident that double-stranded DNA molecules are not covered by the term "dsRNA".

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

[0066] The term "contiguous nucleotides" as used herein refers to nucleotides, either natural or modified nucleotides, that are linked together via an internucleotide linkage. Two consecutive nucleotides are thus linked together via an internucleotide linkage. Also, three consecutive nucleotides means a stretch of three nucleotides, where the first nucleotide is linked to the second nucleotide via an internucleotide linkage, and the second nucleotide is similarly internucleotide Linked to the third nucleotide via ligation. Within the stretch of three nucleotides that are arranged consecutively in the oligonucleotide, the three nucleotides are called "contiguous nucleotides". By way of illustration, an oligonucleotide containing 10 consecutive nucleotide analogues consists of oligonucleotides containing in their sequence uninterrupted stretches of 10 nucleotide analogues linked together via internucleotide linkages. .

[0067] The terms "antisense strand" or "antisense strand oligonucleotide" in dsRNA, as used herein, refer to a strand of dsRNA containing a sequence substantially complementary to a target nucleotide sequence. The other strand of the dsRNA is the "sense strand," which is sometimes called the "sense strand oligonucleotide." In double-stranded oligonucleotides contained in dsRNA, the antisense strand contains a region of nucleotides, referred to herein as the "hybridizing region", which is the region of the sense strand (which hybridizes to the sense strand). , which is the ringing region). The antisense strand may further contain regions located at the 5' and / or 3' ends that do not pair with the sense strand. In dsRNA embodiments in which the dsRNA consists of an siRNA, this non-paired region is conventionally defined as a "nucleotide overhang" or "overhang", i.e., a "5'-nucleotide overhang" or a "3'-nucleotide called "Overhang". Nucleotide overhangs usually have a length of 1-8 nucleotides, including lengths of 1, 2, 3, 4, 5, 6, 7 and 8 nucleotides. In most embodiments, the nucleotide overhangs have a length of 2-3 nucleotides. In contrast, the sense strand of a double-stranded oligonucleotide, such as the sense strand of dsRNA, contains a hybridizing region that hybridizes with the hybridizing region of the antisense strand. The sense strand may also include 5'-nucleotide overhangs and / or 3'-nucleotide overhangs. The hybridizing region of the antisense strand of the double-stranded oligonucleotide, even if it is matched with a region of the sense strand, even if in some embodiments it is not completely complementary to said region of the sense strand. Often, this means that when hybridized, there will be some mismatch (i.e. some mismatch) between the hybridizing region of the antisense strand oligonucleotide and said pairing region of the sense strand oligonucleotide. non-nucleotides) may be present. Typically, in some of these embodiments, 1-3 mismatches exist between the hybridizing region of the antisense strand oligonucleotide and the pairing region of the sense strand oligonucleotide. may be

[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 unaided 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; the dsRNA may also be "introduced into a cell", in which case the cell is part of an organism. In such instances, introduction into a cell would include delivery into an organism. For example, for in vivo delivery, dsRNA can be injected at a tissue site or administered systemically. In vivo delivery is also mediated by the beta-glucan delivery system (see, eg, Tesz, G.J. 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. Additional approaches are described herein below or are known in the art.

[0069] The terms "inhibiting the expression of" or "inhibiting the expression of" as used herein, so far as they refer to the target gene, It refers to at least partial suppression of target gene expression as evidenced by a reduction in amount. The term "inhibiting," as used herein, "reduces," "silencing," "down-regulating," "inhibiting," "knocking down," and other similar terms is used interchangeably with 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))×100%. Alternatively, the degree of inhibition indicates a reduction in a parameter functionally related to transcription of the target gene, such as the amount of protein encoded by the target gene that is secreted by the cell, or a particular phenotype, such as apoptosis. It can be indicated by a reduction in the number of cells. In principle, targeted gene silencing can be determined in any cell that expresses the target, either constitutively or by genomic engineering and by any suitable assay. However, where reference is needed to determine whether a given dsRNA inhibits target gene expression to a particular degree and is therefore encompassed by the present disclosure, the assays provided in the examples below It is expected to serve as such a reference.

[0070] As used herein, the terms "treat," "treatment," etc., in the context of target gene expression, refer to the alleviation or alleviation of a pathological process mediated by target gene expression. In the context of the present disclosure, the terms "treat," "treatment," etc. relate to any of the other conditions (other than the pathological processes mediated by target expression) for which it is listed herein below. To the extent that it refers to alleviating or relieving one or more of the symptoms associated with such conditions.

[0071] As used herein, the terms “prevent” or “slow the progression of” (and grammatical variations thereof) in reference to a disease or disorder include, for example, those suspected of having the disease or developing the disease. It relates to prophylactic treatment of disease in individuals at risk of developing. Prevention includes, but is not limited to, preventing or slowing the onset or progression of a disease and / or maintaining one or more symptoms of a disease at levels below desired or pathological levels. can be mentioned.

[0072] The terms "therapeutically effective amount" and "prophylactically effective amount", as used herein, refer to a pathological process mediated by target gene expression or a pathological process mediated by target gene expression. It refers to an amount that provides therapeutic benefit in the treatment, prevention, or management of symptoms. The specific therapeutically effective amount can be readily determined by a person of ordinary skill in the art, and will depend on the type and stage of the pathological process mediated by target gene expression, the patient's medical history and age, and the target gene. This may vary depending on factors such as the administration of other therapeutic agents that inhibit the biological processes mediated.

[0073] The term "individual" or "subject" as used herein is a mammal. Mammals include, but are not limited to, farm animals (eg, cows, sheep, cats, dogs, and horses), primates (eg, human and non-human primates, such as monkeys), rabbits, and rodents ( For example, mice and rats). In some embodiments, the individual or subject is human.

[0074] The terms "internucleoside linkage," "internucleoside linking group," "internucleotide linkage," or "internucleotide linking group" are used interchangeably herein to refer to two nucleosides (i.e., the base portion of a heterocycle) and sugar moieties) units, examples of which include, but are not limited to, phosphate esters, analogs of phosphate esters (i.e., phosphodiester or phosphotriester moieties), phosphorothioates, phosphate esters, guanidinium, hydroxylamine, hydroxylhydrazinyl, amide, carbamate, alkyl, and substituted alkyl linkages, and the like. An "internucleoside linking group" can participate in a link between two nucleosides, two nucleoside analogues, or between a nucleoside and a nucleoside analogue.

[0075] Internucleoside linkages make up the backbone of nucleic acid molecules. An internucleoside linking group refers to a chemical group that links two adjacent nucleoside residues in a nucleic acid molecule by (i) linking two adjacent nucleoside residues, (ii) flanking the nucleoside residues. and (iii) a chemical group linking the first nucleoside analogue residue with the second nucleoside analogue residue, wherein the nucleoside analogue residues are identical. It includes chemical groups that may be different or distinct. Nucleoside analog residues include compounds of formula (I) disclosed herein. In one aspect, a nucleotide of a siNA molecule of the invention is the linkage of the 3'-carbon of the sugar moiety of the first nucleotide and the 5'-carbon of the sugar moiety of the second nucleotide (herein, the 3' adjacent nucleotides via internucleoside linkages). A 3′-5′ internucleoside linkage, as used herein, refers to an internucleoside linkage linking two adjacent nucleoside units, where the linkage is 3′ of the sugar moiety of the first nucleoside. - carbon and the 5'-carbon of the sugar moiety of the second nucleoside. In another embodiment, the nucleotides (including nucleotide analogues) of the siNA molecules of the invention are linked from the 2'-carbon of the sugar moiety of the first nucleotide to the 5'-carbon of the sugar moiety of the second nucleotide. It is linked to adjacent nucleotides (including nucleotide analogs) via a 2' internucleoside linkage (herein referred to as a 2' internucleoside linkage). A 2′-5′ internucleoside linkage, as used herein, refers to an internucleoside linkage linking two adjacent nucleoside units, where the linkage is 2′ of the sugar moiety of the first nucleoside. - carbon 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, the phosphorus-containing internucleoside linking groups are phosphodiesters, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates, Phosphoramidates such as 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinate esters, 3′-aminophosphoramidates and aminoalkyl phosphoramidates, thio Nophosphoramidates, thionoalkyl phosphates, thionoalkyl phosphotriesters, selenophosphates and boranophosphates with normal 3'-5' linkages, and their 2'-5' linked analogues encompasses

[0078] Representative U.S. Patents teaching the preparation of the above phosphorus-containing internucleoside linkages include U.S. Patent Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243;号;5,286,717号;5,321,131号;5,399,676号;5,405,939号;5,453,496号;5,455,233号;5,466,677号;5,476,925号;5,519,126号;5,536,821号;5,541,306号;5,550,111号;5,563,253号;5,571,799号;5,587,361号;5,194,599号; 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 phosphorothioates, phosphorodithioates, alkyl-phosphonates and phosphoramidate backbone linkages.

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

[0081] In some embodiments, oligonucleotides of the invention comprise one or more internucleoside linking groups that do not contain phosphorus atoms. Such oligonucleotides include, but are not limited to, short alkyl or cycloalkyl internucleoside linking groups, mixed heteroatoms and alkyl or cycloalkyl internucleoside linking groups, or one or more short heteroatoms. or formed by a heterocyclic internucleoside linking group. Examples of these include siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; riboacetyl backbones; methyleneimino and methylenehydrazino backbones; sulfonic acid and sulfonamide backbones; those with amide backbones; and others with mixed N, O, S and CH2 component moieties. Representative United States Patents teaching the preparation of the above non-phosphorus containing internucleoside linking groups include, but are not limited to, United States Patent Nos. 5,034,506; 5,166,315; 5,185,444; ;5,264,562号;5,264,564号;5,405,938号;5,434,257号;5,466,677号;5,470,967号;5,489,677号;5,541,307号;5,561,225号;5,596,086号;5,602,240号;5,610,289号;5,602,240号;5,608,046号;5,610,289号;5,618,704号;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, oligonucleotides of the invention comprise one or more neutral internucleoside linking groups that are non-ionic. Neutral internucleoside linking groups include nonionic linking groups including siloxanes (dialkylsiloxanes), carboxylate esters, carboxamides, sulfides, sulfonate esters and amides (e.g.: Carbohydrate Modifications in Antisense Research; Y. S. Sanghvi and P.D. Cook, ed., ACS Symposium Series 580; see chapters 3 and 4, (pp. 40-65). Additional neutral internucleoside linking groups include nonionic linkages containing mixed N, O, S and CH2 constituent moieties.

[0083] double-stranded oligonucleotides, such as double-stranded RNA (dsRNA) As previously specified herein, double-stranded oligonucleotides according to the present disclosure encompassing dsRNA include sense strand oligonucleotides and antisense strand oligonucleotides, with antisense strand oligonucleotides included therein. It hybridizes with the sense strand oligonucleotide through the hybridizing region. In contrast, the sense strand oligonucleotide also contains a hybridizing region that hybridizes with the hybridizing region contained in the antisense strand oligonucleotide. In some embodiments, the antisense strand oligonucleotide has at its 5′ end, at its 3′ end, or both its 5′ end and its 3′ end a nucleotide region that does not hybridize to the sense strand oligonucleotide. and this non-hybridizing region is referred to herein as a "nucleotide overhang" or alternatively an "overhang". The sense strand oligonucleotide may also contain, at its 5′ end, at its 3′ end, or at both its 5′ end and its 3′ end, a nucleotide region that does not hybridize to the antisense strand oligonucleotide. This non-hybridizing region is often referred to herein as a "nucleotide overhang" or alternatively as an "overhang".

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

[0085] The antisense strand oligonucleotide may further comprise a region that does not pair with the sense strand, located at its 5'end, its 3'end, or both its 5'end and its 3'end. In antisense strand oligonucleotide embodiments of dsRNA, wherein the dsRNA consists of an siRNA, the 3′ or 5′ terminal regions that do not pair with the sense strand oligonucleotide are conventionally referred to as “nucleotide overhangs” or “overhangs”. , called "5'-nucleotide overhangs" or "3'-nucleotide overhangs". In preferred embodiments of double-stranded oligonucleotides according to the present disclosure, the nucleotide overhangs, when present in the sense or antisense strand oligonucleotide, have a length of 1-8 nucleotides, which is 1 , 2, 3, 4, 5, 6, 7 and 8 nucleotides in length. In most embodiments, the nucleotide overhangs have a length of 2-3 nucleotides.

[0086] The present inventors have found that hybridizing regions contained in antisense strand oligonucleotides of double-stranded oligonucleotides, including sense strand oligonucleotides and antisense strand oligonucleotides, particularly in double-stranded oligonucleotides useful as siRNAs. , found that the presence of certain nucleotide analogues of formula (I-A) described in this disclosure allows high stability of said double-stranded oligonucleotide resulting in a long duration of action in vivo. Further, in embodiments in which the double-stranded oligonucleotide is an siRNA, the 1 described herein in the hybridizing region of the antisense strand oligonucleotide in a configuration other than a 5'-overhang or a 3'-overhang. The presence of one or more nucleotide analogues of formula (I-A) imparts to said siRNA improved properties of inhibiting expression of a target gene, improved duration of its gene inhibition properties and, in particular, antisense It is shown herein to confer improved specificity to the targeted sequence of strand oligonucleotides. As demonstrated by the examples provided herein, improved specificity includes improved properties of the antisense strand oligonucleotide that hybridizes to the target oligonucleotide while reducing the number of off-target events. .

[0087] Examples described herein include antisense strand oligonucleotides and sense strand oligonucleotides, wherein the hybridizing region of the antisense strand oligonucleotide differs from its 3'-overhang or 5'-overhang. Double-stranded oligonucleotides incorporating one or more nucleotide analogues of formula (I-A) in their configuration are siRNA duplexes with the requisite stability and specificity to ensure efficient inhibition of target genes. It is shown to allow heavy chain structures to be generated. It is also shown herein that efficient inhibition of target genes is accompanied by a reduction in the number of off-target events.

[0088] Surprisingly, the inventors have found that the high metabolic stability of siRNA duplexes comprising one or more nucleotide analogues of formula (I-A) or (I-B) of the present disclosure is associated with formula (I-A) or (I-B) obtained when linked one to the other or one to a ribose-containing nucleotide via a conventional phosphodiester bond. Surprisingly, when the nucleotide analogues of formula (I-A) or (I-B) are linked via conventional phosphodiester bonds within the oligonucleotide to form strands of siRNA, the resulting siRNA duplex is , has higher stability to nuclease degradation and thus a longer duration of action in vivo than the same siRNA having phosphorothioate-linked deoxyribonucleotides instead of nucleotide analogues of formula (I-A) or (I-B). was shown to result in

[0089] This high increase in the duration of action in vivo of the nucleotide analogues of formula (I-A) or (I-B), when linked via phosphodiester bonds, is due to the internucleotide modifications linking the phosphorus groups, such as phosphorothioates. This is a distinct technical advantage as it avoids stabilization via In this case, such unconventional phosphorothioates are reminiscent of those that introduce chiral centers, resulting in undesirable diastereomeric mixtures of the resulting siRNA. The latter may alter siRNA binding properties and possibly cause increased off-target events. As used herein, siRNAs having one or more compounds of formula (I-A) or (I-B) are treated as , have also been shown to have excellent target gene silencing activity in vitro and in vivo.

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

[0091] Therefore, more surprisingly, at a specific position in the antisense strand oligonucleotide, i.e. in the hybridizing region of the antisense strand oligonucleotide, furthermore the 5'-overhang of said antisense strand oligonucleotide or Incorporation of the nucleotide analogues of formula (I-A) described herein in a configuration other than the 3'-overhang is highly duplex destabilizing in a series of (2S,6R)-diastereomers and, in the case of the analogue (2R,6R)-diastereomer, showed less pronounced duplex destabilization. In contrast to expectations, siRNAs containing (2R,6R)-diastereoisomeric analogues of formula (I-A) in the hybridizing region of the antisense strand oligonucleotide exhibited significant off-target binding in the in vitro system. while maintaining its highly potent target-inhibiting properties in vivo.

[0092] Importantly, siRNAs that incorporate one or more nucleotide analogues of formula (I-A) in the hybridizing region of the antisense strand oligonucleotides demonstrate that these siRNAs exert target gene silencing effects in vivo. No side effects in the dose range shown to exert.

[0093] While not wishing to be bound by any particular theory, the nucleotide analogues of formula (I-A) described herein are one or more nucleotide analogues of formula (I-A), shown in the examples The inventors believe that given the specific physico-chemical properties, improved stability, binding properties and off-target profiles imparted to oligonucleotides containing These properties are particularly improved in double-stranded oligonucleotides in which the hybridizing region of the antisense strand oligonucleotide contains its (2R,6R) diastereoisomer, such as the nucleotide analogues of formula (I-A). .

[0094] Nucleotide analogues of Formula (I-A) described in this disclosure may also be referred to herein as "non-targeted" nucleotide analogues of Formula (I-A).

[0095] Antisense Strand Embodiments of Double-Stranded Oligonucleotides An important aspect of this disclosure is the provision of double-stranded oligonucleotides comprising a sense strand oligonucleotide and an antisense strand oligonucleotide, wherein the antisense strand oligonucleotide comprises one or more of the Including nucleotide analogues of formula (I-A), which are neither 5'-overhanging nucleotides nor 3'-overhanging nucleotides of said antisense strand oligonucleotide. In some embodiments, the double-stranded oligonucleotide consists of an siRNA that hybridizes to a selected target mRNA.

[0096] As used herein, included in the antisense strand oligonucleotide of a double-stranded oligonucleotide described herein, not included in the 5'-overhang, if present, and 3', if present. -Nucleotides that are not also included in overhangs are sometimes referred to herein as "hybridizing nucleotides" or alternatively as "internal nucleotides".

[0097] Thus, it is included in the antisense strand oligonucleotide of the double-stranded oligonucleotides described herein and is not included in the 5'-overhang, if present, nor is it included in the 3'-overhang, if present. Nucleotide analogues of Formula (I-A) without There is also

[0098] Likewise included in the sense strand oligonucleotide of the double-stranded oligonucleotides described herein and not included in the 5'-overhang, if present, nor included in the 3'-overhang, if present. The missing nucleotides may also be referred to as "hybridizing nucleotides" as described herein, or alternatively as "internal nucleotides."

[0099] Furthermore, if present, nucleotides located within the 5'-overhang or within the 3'-overhang of said antisense strand oligonucleotide of a double-stranded oligonucleotide according to the present disclosure are herein referred to as "overhang Also called a nucleotide.

[0100] In some embodiments of double-stranded oligonucleotides in which the antisense strand oligonucleotide comprises a 5'-overhang and / or a 3'-overhang, the antisense strand oligonucleotide comprises a 5'-overhang, or It does not contain any nucleotide analogues of formula (I-A) in its 3'-overhang or in both its 5'-overhang and its 3'-overhang. Nucleotide analogues of formula (I-A) contained in said antisense strand oligonucleotide are then all of formula (I-A), with reference to the previously specified definition of "hybridizing nucleotide" herein. It is a hybridizing nucleotide analogue.

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

[0102] The term "hybridizing" nucleotides or "overhanging" nucleotides, as defined in this disclosure, is used herein for reasons of ease of language and improved clarity of construction of the various disclosed embodiments. can do.

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

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

[0105] In some embodiments, the antisense strand oligonucleotide comprises 1-5 hybridizing nucleotide analogs of Formula (I-A).

[0106] In some embodiments, said antisense strand oligonucleotide comprising one or more nucleotide analogues of Formula (I-A) ranges in length from 9-36 nucleotides, which is from 15-30 nucleotides. contains the length of In some embodiments, the antisense strand oligonucleotides range in length from 20-25 nucleotides, including 17-25 nucleotides in length.

[0107] In some embodiments, the (2R,6R) stereoisomer of the hybridizing nucleotide analog of Formula (I-A) described herein is preferred.

[0108] The placement of the nucleotide analogues of formula (I-A) is within the antisense strand oligonucleotide.

[0109] One or more hybridizing nucleotide analogs of formula (I-A) are placed at various nucleotide positions within the hybridizing sequence of the antisense strand oligonucleotide.

[0110] Further, in embodiments where the antisense strand oligonucleotide comprises two or more hybridizing nucleotide analogues of Formula (I-A), said two or more hybridizing nucleotide analogues of Formula (I-A) are , anywhere in the hybridizing region of said antisense strand oligonucleotide, excluding its 5'-overhang or 3'-overhang, if one or both overhangs are present, and contiguously; and / or arranged non-contiguously.

[0111] In some embodiments of said antisense strand oligonucleotide, each hybridizing nucleotide analog of Formula (I-A) contained therein is linked to a nucleotide that is different from the modified nucleotide of Formula (I-A). According to these embodiments, the two or more hybridizing nucleotide analogues of formula (I-A) are separate, ie separately distributed 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 (I-A) contained therein are arranged sequentially within the antisense strand oligonucleotide. so that they are linked together. In these embodiments, the hybridizing nucleotide analogue of formula (I-A) located at the 5' end of this series of said hybridizing nucleotide analogues is at a different nucleotide than the nucleotide analogue of formula (I-A). The hybridizing nucleotide analogue of formula (I-A) that is linked and positioned at the 3' end of this series of said hybridizing nucleotide analogues is linked to a nucleotide that is different from the modified nucleotide of formula (I-A). be. According to these embodiments, two or more of the hybridizing nucleotide analogues of Formula (I-A) in succession are referred to as being in succession within the antisense strand oligonucleotide.

[0113] In yet another embodiment of said antisense strand oligonucleotide, the hybridizing nucleotide analogues of formula (I-A) are separated single nucleotide analogues of formula (I-A) and contiguous hybrid nucleotide analogues of formula (I-A). Arranged both as one or more stretches of dysing nucleotide analogues. As will be readily understood, the contiguous hybridizing nucleotide analogues of formula (I-A) that are ligated together are the contiguous nucleotide analogues of formula (I-A) comprised in said antisense strand oligonucleotide.

[0114] Illustratively, in a double-stranded oligonucleotide embodiment in which the antisense strand oligonucleotide has a length of 23 nucleotides and every nucleotide is matched to the sense strand oligonucleotide, the antisense strand oligonucleotide Nucleotide positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, with nucleotide position numbering starting from the 5' end of the strand oligonucleotide , 17, 18, 19, 20, 21, 22 or 23.

[0115] As a further illustration, the antisense strand oligonucleotide has a length of 23 nucleotides and has (i) a 21 nucleotide long hybridizing region and (ii) a 2 nucleotide long 3'-overhang. In an embodiment of a double-stranded oligonucleotide comprising: said antisense strand oligonucleotide has nucleotide positions 1, 2, 3, 4, 5, with nucleotide position numbering starting from the 5' end of the antisense strand oligonucleotide. Hybridizing Formula (I-A) at a nucleotide position selected from the group consisting of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 It may contain nucleotide analogues. In some of these embodiments, the antisense strand oligonucleotide further comprises two overhanging nucleotide analogues of formula (I-A) in its 3'-overhang.

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

[0117] Embodiments of Antisense Strands Comprising Targeted Nucleotides In some embodiments of the antisense strand oligonucleotide of a double-stranded oligonucleotide according to the present disclosure, said 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 analogs contained therein is a targeting compound of formula (I-B) as described elsewhere herein. consisting of nucleotide analogues that have been defined.

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

[0120] The targeted nucleotide analogues of Formula (I-B) may be present in various positions within the antisense strand oligonucleotide of a double-stranded oligonucleotide according to the present disclosure.

[0121] In some embodiments, the targeted nucleotide analogs of Formula (I-B) are attached to the 3'-overhang or 5'-overhang of the antisense oligonucleotide strand, e.g. Arranged in 3'-overhangs or 5'-overhangs.

[0122] In some preferred embodiments, the targeted nucleotide analogues of Formula (I-B) are placed in overhangs of the dsRNA, eg of the siRNA. For example, the targeted nucleotide analogues of formula (I-B) are placed in overhangs, eg, 3'-overhangs, of the antisense strand oligonucleotides of the siRNA.

[0123] In some embodiments, 2-10 (eg, 2-5) targeted nucleotide analogues of Formula (I-B) are present in the antisense strand oligonucleotide of a double-stranded oligonucleotide according to the present disclosure. do. 2-10 targeted nucleotide analogues of formula (I-B), as used herein, are 2, 3, 4, 5, 6, 7, 8, 9 and 10 of formula (I-B ).

[0124] Sense Strand Embodiments of Double-Stranded Oligonucleotides In some embodiments, the sense strand oligonucleotide of a double-stranded oligonucleotide according to this disclosure does not contain any modified nucleotides of formula (I-A). In some of these embodiments, the sense strand oligonucleotide of the double-stranded oligonucleotides described herein has one or more modified nucleotides that differ from the nucleotide analogues of formula (I-A); For example, it may contain methoxy- or fluoro-modified nucleotides.

[0125] In other embodiments, the sense strand oligonucleotide of a double-stranded oligonucleotide according to this disclosure comprises one or more nucleotide analogues. In some embodiments, the sense strand oligonucleotide of a double-stranded oligonucleotide according to the present disclosure comprises one or more nucleotide analogues of Formula (I-A), wherein the nucleotide analogues of Formula (I-A) are: It may be located on its 5'-overhang, on its 3'-overhang, on its hybridizing region, or on two or more of these locations within the sense strand oligonucleotide.

[0126] In certain embodiments of the sense strand oligonucleotide of the double-stranded oligonucleotides described herein, the nucleotide analogue of formula (I-A) is attached to the 3'-overhang of said sense strand oligonucleotide and the 5'- Located in overhang or in both 3'-overhang and 5'-overhang. In some of these particular embodiments, the nucleotide analogues of formula (I-A) are located exclusively in the 3'-overhangs of the sense strand oligonucleotides of the dsRNA, e.g. Placed exclusively in the hang.

[0127] In some of these specific embodiments, the nucleotide analogues of formula (I-A) are placed on both the 3'-overhang and the 5'-overhang of the (i) sense strand oligonucleotide of the siRNA.

[0128] In some embodiments, 2-10 (eg, 2-5) nucleotide analogues of Formula (I-A) are present in the sense strand oligonucleotide of a double-stranded oligonucleotide according to the present disclosure. 2-10 nucleotide analogues of formula (I-A) as used herein are 2, 3, 4, 5, 6, 7, 8, 9 and 10 nucleotide analogues of formula (I-A) This nucleotide analogue of formula (I-A) may be present at its 5'-overhang, at its 3'-overhang, at its hybridizing region, or at these locations within the sense strand oligonucleotide. May be arranged in two or more.

[0129] Sense Strand Embodiments Containing Targeted Nucleotides In some embodiments of the sense strand oligonucleotide of a double-stranded oligonucleotide according to the present disclosure, said 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 analogs contained therein is a targeting compound of formula (I-B) as described elsewhere herein. consisting of nucleotide analogues that have been defined.

[0131] Thus, in some embodiments of the sense strand oligonucleotide of a double-stranded oligonucleotide according to the present disclosure, said sense strand oligonucleotide comprises one or more of the formulas (I-B ) containing targeted nucleotide analogues.

[0132] As illustrated in the examples provided herein, the targeted nucleotide analogues of Formula (I-B) are present in various positions within the sense strand oligonucleotide of a double-stranded oligonucleotide according to the present disclosure. may

[0133] In some embodiments, the targeted nucleotide analog of Formula (I-B) is attached to the 3'-overhang or 5'-overhang of the sense oligonucleotide strand, e.g. Placed in an overhang or 5'-overhang.

[0134] In some preferred embodiments, the targeted nucleotide analogues of Formula (I-B) are placed in overhangs of the dsRNA, eg of the siRNA. For example, the targeted nucleotide analogues of formula (I-B) are placed in an overhang, such as a 5'-overhang, of the sense strand oligonucleotide of the siRNA.

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

[0136] In some embodiments, the sense strand oligonucleotide comprises (i) one or more nucleotide analogues of Formula (I-A) and (ii) one or more nucleotide analogues of Formula (I-B). include. In some of these embodiments, the one or more nucleotide analogues of Formula (I-A) comprised in the sense strand oligonucleotide consists of a hybridizing nucleotide analogue of Formula (I-A), i.e., the 5' - overhang, 3'-overhang, or in embodiments where both 5'-overhang and 3'-overhang are present, also the 5'-overhang of the sense strand oligonucleotide, or the 3'-overhang consisting of nucleotide analogues of formula (I-A) that are not also arranged in In some of these embodiments, the sense strand oligonucleotide also has one or more Includes nucleotide analogues of Formula (I-A) above.

[0137] Other Configurations of Double-Stranded Oligonucleotides A double-stranded oligonucleotide comprising one or more nucleotide analogues of formula (I-A) has (i) the ribose sugar moieties contained in most of the nucleotide monomer units contained therein, and (ii) It may also be referred to as "ribonucleic acid" or "RNA", considering the type of nucleobases it contains. Accordingly, certain aspects of the present disclosure relate to double-stranded ribonucleic acid (dsRNA) molecules that target mRNAs of interest. The dsRNA of the invention may comprise modified oligonucleotides comprising one or more compounds of Formula (I-A).

[0138] In some embodiments, the dsRNA comprises two strands, a sense strand comprising a first sequence and an antisense strand comprising a second sequence, wherein the first and second strands are double stranded Sufficiently complementary to form a structure. In some embodiments, the sense strand comprises a first sequence that is substantially complementary or fully complementary to a second sequence in the antisense strand. In some embodiments, the second sequence in the antisense strand is substantially complementary to or fully complementary to the target sequence, e.g., the sequence of the mRNA transcribed from the target gene. .

[0139] In some embodiments, each of the sense and antisense strands can range in length from 9 to 36 nucleotides. For example, each strand may be 12-30 nucleotides long, 14-30 nucleotides long, 15-30 nucleotides long, 25-30 nucleotides long, 27-30 nucleotides long, 15-26 nucleotides long. length, 15-23 nucleotides long, 15-22 nucleotides long, 15-21 nucleotides long, 15-20 nucleotides long, 15-19 nucleotides long, 15-18 nucleotides long length, length of 15-17 nucleotides, length of 17-30 nucleotides, length of 17-23 nucleotides, length of 17-21 nucleotides, length of 17-19 nucleotides, length of 18-30 nucleotides, 18-26 nucleotides long 18-25 nucleotides long 18-23 nucleotides long 18-22 nucleotides long 18-21 nucleotides long 18-20 nucleotides long 19-26 nucleotides long 30 nucleotides long 19-25 nucleotides long 19-24 nucleotides long 19-23 nucleotides long 19-22 nucleotides long 19-21 nucleotides long 19-20 nucleotides length, 20-30 nucleotides long, 20-26 nucleotides long, 20-25 nucleotides long, 20-24 nucleotides long, 20-23 nucleotides long, 20-22 nucleotides long length, length of 20-21 nucleotides, length of 21-30 nucleotides, length of 21-26 nucleotides, length of 21-25 nucleotides, length of 21-24 nucleotides, length of 21-23 nucleotides, or 21-22 nucleotides in length. In some embodiments, each strand has a length of Greater than or equal to 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides. In some embodiments, each strand has a length of Less than or equal to 28, 29, 30, 31, 32, 33, 34, 35, or 36 nucleotides. That is, each strand has 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, an independently selected upper limit of 32, 33, 34, 35, or 36 and an independently selected A range of nucleotide lengths with a lower limit of 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 has , 29, 30, 31, 32, 33, 34, 35, or 36 nucleotides in length. In some embodiments, the sense and antisense strands are the same number of nucleotides in length. In some embodiments, the sense and antisense strands are a different number of nucleotides in length.

[0140] Overhang In some embodiments, the dsRNAs of the disclosure comprise 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, one or more overhangs improve stability with a lower number of phosphorothioate groups in the overhangs.

[0141] In some embodiments, the overhangs are 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, or 6 or more contains more nucleotides. For example, overhangs are 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 overhangs of this disclosure comprise one or more ribonucleotides. In some embodiments, the overhangs of this disclosure comprise one or more deoxyribonucleotides. In some embodiments, the overhangs contain one or more thymines. In some embodiments, the dsRNA includes an overhang located at the 3' end of the antisense strand. In some embodiments, the dsRNA contains 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 includes an overhang located at the 3' end of the sense strand. In some embodiments, the dsRNA contains 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 overhangs located at both the 3' ends of the sense and antisense strands of the dsRNA.

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

[0144] In some embodiments, overhangs are 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, overhangs are the result of alternating sense and antisense strands of the same length. In some embodiments, the overhangs form mismatches with the target mRNA. In some embodiments the overhang is complementary to the target mRNA.

[0145] In some embodiments, a dsRNA of the disclosure comprises a sense strand comprising a first sequence and an antisense strand comprising a second sequence, wherein the first and second sequences are substantially complementary or complementary. In some embodiments, the first and second sequences are substantially complementary or complementary and form a double-stranded region of the dsRNA. In some embodiments, the double-stranded 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. length, length of 15-22 nucleotide pairs, length of 15-21 nucleotide pairs, length of 15-20 nucleotide pairs, length of 15-19 nucleotide pairs, length of 15-18 nucleotide pairs, length of 15-22 nucleotide pairs 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 length of nucleotide pairs, length of 18-21 nucleotide pairs, length of 18-20 nucleotide pairs, length of 19-30 nucleotide pairs, length of 19-25 nucleotide pairs, length of 19-24 nucleotide pairs , 19-23 nucleotide pairs long, 19-22 nucleotide pairs long, 19-21 nucleotide pairs long, 19-20 nucleotide pairs long, 20-30 nucleotide pairs long, 20-26 nucleotide pairs long pair length, 20-25 nucleotide pair length, 20-24 nucleotide pair length, 20-23 nucleotide pair length, 20-22 nucleotide pair length, 20-21 nucleotide pair length, 21-30 nucleotide pairs long, 21-26 nucleotide pairs long, 21-25 nucleotide pairs long, 21-24 nucleotide pairs long, 21-23 nucleotide pairs long, or 21-22 nucleotide pairs long It can be the length of a pair. In some embodiments, the double-stranded region of the dsRNA is , 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotide pairs in length. In some embodiments, the double-stranded region of the dsRNA is , 28, 29, 30, 31, 32, 33, 34, 35, or 36 nucleotide pairs in length. That is, the double-stranded region of the dsRNA is independently selected 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. is less than the upper limit. In some embodiments, the duplex region is , 28, 29, 30, 31, 32, 33, 34, 35, or 36 nucleotide pairs in length. If more than one dsRNA is used, the duplex region of each dsRNA may be of the same or different length as the one or more additional dsRNAs.

[0146] Embodiments of Targeted Double-Stranded Oligonucleotides As described above in this disclosure, in double-stranded oligonucleotides, the antisense strand oligonucleotide, the sense strand oligonucleotide, or both the antisense and sense strand oligonucleotides have one or more targeted It may contain nucleotides. In some embodiments, the antisense strand oligonucleotide, the sense strand oligonucleotide or both the antisense strand oligonucleotide and the sense strand oligonucleotide comprise one or more targeted nucleotide analogues of Formula (I-B) may contain

[0147] Accordingly, in some embodiments according to the present disclosure, the targeted double-stranded oligonucleotide comprises one or more targeted nucleotide analogues of Formula (I-B).

[0148] In some other embodiments, targeted nucleotides included in targeted double-stranded oligonucleotides according to the present disclosure are 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 analogues of formula (I-A) and different from formula (I-B) Contains one or more targeted nucleotides having a structure.

[0150] According to some of these embodiments, the targeted double-stranded oligonucleotide comprises (i) one or more targeted nucleotide analogues of formula (I-B), one or sense strand oligonucleotides comprising more targeted nucleotides, and (ii) non-targeted antisense strand oligonucleotides, i.e. antisense strand oligonucleotides containing no targeted nucleotides. . According to some other of these embodiments, the targeted double-stranded oligonucleotide comprises (i) a non-targeted sense strand oligonucleotide that does not contain targeted nucleotides, and (ii) 1 A targeted antisense strand oligonucleotide comprising one or more targeted nucleotides comprising one or more targeted nucleotide analogues of formula (I-B) may be included. Yet according to other embodiments, the targeted double-stranded oligonucleotide comprises (i) one or more targeted nucleotide analogues of formula (I-B). a sense strand oligonucleotide comprising at least one targeted nucleotide, and one or more targeted nucleotide analogs comprising one or more targeted nucleotide analogs of formula (I-B) A targeted antisense strand oligonucleotide may be included.

[0151] In some embodiments, a targeted double-stranded oligonucleotide according to the present disclosure is: - a sense strand oligonucleotide comprising (i) one or more nucleotide analogues of formula (I-A) and (ii) one or more nucleotide analogues of formula (I-B), and - if one or both overhangs are present in the antisense strand oligonucleotide, one or more hybrids of formula (I-A) that are located in neither the 5'-overhang nor the 3'-overhang; Includes dicing nucleotide analogues, ie, antisense strand oligonucleotides containing one or more nucleotide analogues of formula (I-A). In some embodiments, the antisense strand oligonucleotide has one or more formulas on its 5'-overhang or 3'-overhang in embodiments where one or both of these overhangs are present. Further includes nucleotide analogues of (I-A).

[0152] As disclosed elsewhere in this disclosure, the targeted nucleotide analogues of Formula (I-B) have group R3 as a cell-targeting moiety. Cell targeting moieties are disclosed elsewhere in this disclosure.

[0153] In a particular embodiment, said one or more targeted nucleotide analogues of formula (I-B) is a continuous strand of these targeted nucleotide analogues at selected ends of the oligonucleotide chain. i.e. the one or more nucleotide analogues of formula (I-B) above are contiguous in the selected antisense strand oligonucleotide or the selected sense strand oligonucleotide .

[0154] Further Embodiments of Double-Stranded Oligonucleotides For illustrative purposes and not limiting the disclosure, the double-stranded oligonucleotide may also include one or more additional nucleotides in the sense and / or antisense strands that have been modified. .

[0155] Modifications can be selected from substitutions or insertions with analogues of nucleic acids or bases, and chemical modifications of bases, sugars or phosphate moieties. Modifications selected were 3' terminal deoxy-thymine, 2'-O-methyl, 2'-deoxy modification, 2'-desoxy-fluoro, 2'-amino modification, 2'-alkyl modification, phosphorothioate modification, phosphoro may be individually selected from amidate modifications, 5′-phosphorothioate group modifications, 5′-phosphate or 5′-phosphate mimetic modifications, and cholesteryl derivatives or dodecanoic acid bisdecylamide group modifications and / or Alternatively, the modified nucleotides may be any one of locked nucleotides, abasic nucleotides or nucleotides containing non-natural bases. A preferred embodiment may be one in which 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, include the following: modifications, such as unlinked phosphates and / or oxygens and / or linked phosphates and oxygens one or more replacements; replacements of phosphate moieties; modifications or replacements of naturally occurring bases; replacements or modifications of the ribose-phosphate backbone; It may involve one or more of removing, modifying or replacing a phosphate group, or conjugating a moiety, such as a fluorescently labeled moiety, to either the 3' or 5' end of the RNA.

[0157] Further Embodiments of Targeted Oligonucleotide-Cell Targeting Moieties The targeted nucleotides herein can be linked to one or more ligands that target specific cells or tissues. Such ligands are also referred to as "cell-targeting moieties." Ligands can improve the efficiency of delivery of the resulting oligonucleotides, such as siRNA, into cells, for example, by improving specific cellular targeting, improving cellular internalization of oligonucleotides, and / or improving intracellular mRNA targeting. includes any molecular group that increases the Ligands are selected from the group comprising 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). be able to.

[0158] Ligands may be naturally occurring, or may be recombinant or synthetic. For example, ligands can be proteins, carbohydrates, lipopolysaccharides, lipids, synthetic polymers, polyamines, alpha helical peptides, lectins, vitamins, or cofactors. In some embodiments, the ligand is one or more dyes, crosslinkers, polycyclic aromatic hydrocarbons, peptide conjugates (eg, RGD peptide, antennapedia peptide, Tat peptide), polyethylene glycol (PEG ), enzymes, haptens, transport / absorption enhancers, synthetic ribonucleases (eg, imidazole, bisimidazole, histamine, or the imidazole cluster), human serum albumin (HSA), or LDL.

[0159] As an example, a ligand can be one or more proteins, glycoproteins, peptides, or molecules that have a specific affinity for a co-ligand. Such ligands include thyrotropin, melanotropin, glycoproteins, surfactant protein A, mucin carbohydrates, lactose (e.g. polyvalent lactose), galactose (e.g. polyvalent galactose), N-acetyl-galactosamine (e.g. polyvalent N-acetyl-galactosamine), N-acetyl-glucosamine (e.g. polyvalent N-acetyl-glucosamine), mannose (e.g. polyvalent mannose), fucose (e.g. polyvalent fucose), glycosylated polyamino acids, transferrin, bisphosphonates , polyglutamate, polyaspartate, lipids, cholesterol, steroids, bile acids, folate, 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 (eg, imidazole, bisimidazole, histamine, or the imidazole cluster), human serum albumin (HSA), or LDL.

[0161] In some embodiments, the ligand can be one or more cholesterol derivatives or lipophilic moieties. Any lipophilic compound includes, but is not limited to, cholesterol or a cholesterol derivative; cholic acid; vitamins such as folate, vitamin A, vitamin E (tocopherol), biotin, pyridoxal; bile, including both saturated and unsaturated or fatty acid conjugates (e.g. lauroyl (C12), myristoyl (C14) and palmitoyl (C16), stearoyl (C18) and docosanyl (C22), lithocholic acid and / or oliylamine lithocholic acid 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-co-glycolide) (PLG), steroids (eg, dihydrotestosterone); terpenes (eg, triterpenes); cationic lipids or peptides; , can bind to serum proteins such as human serum albumin (HSA) Lipid-based ligands can be used to modulate (eg, control) binding of the conjugate to target tissues. For example, a lipid or lipid-based ligand that binds HSA more strongly would be expected to be less likely to be targeted to the kidney and therefore less likely to be cleared from the body.The target tissue is the liver, e.g. parenchymal cells, and the like.

[0162] Polyamino acids, transferrin, cell-targeting ligands or moieties can also be antibodies that bind to receptors on specific cell types, such as hepatocytes. Exemplary cell receptor-specific monoclonal antibodies are described in Xia et al. (2009, Mol Pharm, 63(3):747-751); Cuellar et al. (2015, Nucleic Acids Research, 43(2):1189-1203); by 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) It is disclosed.

[0163] Cell-targeting ligands or moieties may also include monovalent or multivalent (eg, trivalent) GalNAc groups, such as Prakash et al. (2015, Bioorg Med Chem Lett, 25(19):4127-4130); Zu et al. (2016). (2017, Mol Ther, 25(1):71-78); Shemesh et al. (2016, Ther Nucleic Acids, 5:e319-doi); (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 A dsRNA of the 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, for example, a multivalent branched linker. Conjugating a ligand to dsRNA may alter its distribution, its cellular uptake and / or targeting to specific tissues and / or specific cell types of one or more (e.g. liver cells). can enhance uptake by and / or enhance the longevity of the dsRNA agent. In some embodiments, hydrophobic ligands are conjugated to dsRNA to facilitate direct penetration across cell membranes and / or uptake by cells (eg, liver cells).

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

[0166] The ligand-conjugated dsRNA and sequence-specific linked nucleosides and nucleotides to which the ligand molecule is attached of the disclosure can be assembled by any method known in the art, e.g., standard Nucleotide precursors, or nucleotide or nucleoside conjugate precursors already having a linking moiety, ligand-nucleotides, or precursors conjugated to nucleosides already having a ligand molecule, or nucleoside and ligand free building blocks are utilized. It can be assembled, such as by assembly on a suitable DNA synthesizer.

[0167] The ligand-conjugated dsRNA of the present disclosure can be synthesized by any method known in the art, such as those obtained by attaching a linking molecule on the dsRNA to which the pendant reactive functionalities are attached, such as the dsRNA. Can be synthesized by using. In some embodiments, the reactive oligonucleotides are directly linked to commercially available ligands, synthetic ligands attached to any of a variety of protecting groups, or ligands to which linking moieties are attached. can react to In some embodiments, the method facilitates the synthesis of ligand-conjugated dsRNA through the use of nucleoside monomers that are appropriately conjugated with ligands and optionally attached to a solid support material. do. In some embodiments, the dsRNA bound by an aralkyl ligand attached to the 3' end of the dsRNA is first covalently attached to the controlled pore glass support through the aminoalkyl group. the nucleotides are then attached via standard solid-phase synthesis techniques to the solid-support-bound monomeric building blocks. Monomeric building blocks can be nucleosides or other organic compounds amenable to solid phase synthesis.

[0168] Precursors of Nucleotide Analogues of Formulas (I-A) and (I-B) Nucleotide analogs of formulas (I-A) and (I-B) can be synthesized by starting from precursor compounds, such compounds are sometimes referred to herein as nucleotide analog precursors.

[0169] Both nucleotide analogues of formulas (I-A) and (I-B) can be synthesized from their corresponding precursor compounds of formula (II) below.

[0170] As expected to be readily understood, the nucleotide analogues of formula (I-A) can be converted to the nucleotide analogue precursors of formula (II-A) below, wherein the group R comprises a cell-targeting moiety. not available). Additionally, the nucleotide analogues of formula (I-B) can be prepared by starting with a nucleotide analogue precursor of formula (II-B) below, wherein group R3 comprises a cell-targeting moiety. can.

[0171] The present disclosure also describes nucleotide analogue precursors of general formula (II). [formation] In the formula: - 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 N-C(=O)-R1 and R1 is: optionally unsubstituted or halogen atom, (C1-C6) alkyl group, (C3-C8) cycloalkyl group, (C3-C14) heterocyclic ring, (C6-C14) aryl group, (C5-C14) hetero Aryl group, -O-Z1, -N(Z1)(Z2), -S-Z1, -CN, -C(=J)-O-Z1, -O-C(=J)-Z1, -C(=J )-N(Z1)(Z2), and (C 1 ~C 20 ) alkyl group (wherein 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); 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 meaning 0 or 1, p is an integer ranging from 0 to 10, R2 is optionally 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), -N(Z3)-C(=K)-Z4-substituted (C1-C20) alkylene group, K is O or S, each of Z3 and Z4 is independently H or (C1-C6)alkyl optionally substituted with one or more groups selected from halogen atoms and (C1-C6)alkyl groups is the basis, R3 is a hydrogen atom, (C1-C6) alkyl group, (C1-C6) alkoxy group, (C3-C8) cycloalkyl group, (C3-C14) heterocyclic ring, (C6-C14) aryl group or (C5-C14) C14) selected from the group consisting of heteroaryl groups; or R3 is a cell-targeting moiety); - X1 and X2 are each independently a hydrogen atom, -(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 encompassed by the term "nucleotide precursors." For the purposes of this disclosure, compounds of formula (II) in which the group R3 is present and which represent cell-targeting moieties are encompassed by the term "targeted nucleotide precursors."

[0173] Compounds of formula (I), such as (I-A) and (I-B), and (II), disclosed herein include isomers thereof, such as stereoisomers, examples of which include the formula ( Their (2S,6R) stereoisomers and their (2R,6R) stereoisomers, as illustrated in the formulas below, which identify the position numbering and chiral centers of the compounds of I) and (II). Stereoisomers are included and exemplified below for compounds of formulas (I) and (II): [formation]

[0174] The (2S,6R) stereoisomer of compounds of formula (I) and the (2R,6R) ) stereoisomers are endowed with the ability to generate siRNAs that allow superior inhibition of target mRNAs.

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

[0176] As shown in some examples herein, the use of a nucleotide analogue of formula (I-A), consisting of its (2R,6R) stereoisomer, in a double-stranded oligonucleotide, comprises nucleotide analogue (I-A) is incorporated as a hybridizing nucleotide analogue identified herein, e.g., when incorporated into the hybridizing region of the antisense strand oligonucleotide of the siRNA, in some embodiments the sense When incorporated into the hybridizing region of a stranded oligonucleotide, it results in superior potency of said double-stranded oligonucleotide, eg, siRNA, in vivo compared to the corresponding (2S,6R) stereoisomer.

[0177] In some embodiments of compounds of formula (II), which may also be referred to herein as "dioxane analogues," Y is O. Such nucleotide precursor embodiments in the present disclosure are referred to as prelB1, where B is as defined in formula (II), e.g., when B consists of a thymidinyl group , called pre-lT1.

[0178] In other embodiments of compounds of formula (II), which may also be referred to herein as "morpholino analogs," Y is NH, NR1 or N-C(=O)R1.

[0179] In the morpholino analogues of formula (II), the nitrogen atom is preferably functionalized so as to improve the properties of the resulting oligonucleotides containing the morpholino analogues, and in particular the resulting siRNA containing the morpholino analogues. ing.

[0180] In some embodiments of compounds of Formula (II), the compounds are morpholino analogs of the disclosure that do not contain a cell-targeting moiety. According to these embodiments, group R3, if present, does not represent a cell-targeting moiety.

[0181] Thus, in some preferred embodiments of morpholino analogues of formula (II), Y is NH, NR or N-C(=O)-R, wherein R is as defined for general formula (II). be.

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

[0183] As intended herein, a (C1-C20)alkyl group can 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 can be either an unsubstituted alkyl group or a substituted alkyl group, examples of which include C1, C2, C3, C4, C5 and C6 alkyl groups are included.

[0185] As intended herein, a (C3-C8) cycloalkyl group can be either an unsubstituted cycloalkyl group or a substituted cycloalkyl group, examples of which include C3, C4, C5 , C6, C7 and C8 cycloalkyl groups.

[0186] As intended herein, (C3-C14)heterocycle may be either 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 can be either an unsubstituted aryl group or a substituted aryl group, examples of which include C6, C7, C8, C9, Included are C10, C11, C12, C13 and C14 aryl groups.

[0188] As intended herein, a (C5-C14)heteroaryl group can be either an unsubstituted heteroaryl group or a 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, 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 are alkyl selected from the group including methyl, isopropyl, butyl, octyl, hexadecyl. groups, P1, P2, Ra, Rb, Rc, Rd, X1, X2 and B have the same meaning 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, P1 and P2 are defined with respect to general formula (II) It is as it was done. Embodiments of such nucleotide precursors in the present disclosure are referred to as prelB2, where B has the same meaning as in general formula (II); An embodiment is called pre-lT2 when B consists of 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, P1 and P2 are defined with respect to general formula (II) It is as it was done. Embodiments of such nucleotide precursors in the present disclosure are referred to as prelB3, where B has the same meaning as in general formula (II); Embodiments include pre-lT3 (wherein B consists of a thymidinyl group), pre-lU3 (wherein B consists of a uracil group), pre-lG3 (wherein B consists of a guanyl group), pre-lG3 (wherein B consists of a guanyl group), called lC3 (where B consists of cytosyl groups) and pre-lA3 (where B consists of adenyl groups).

[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 defined with respect to general formula (II). It is as it was done. An embodiment of such nucleotide precursors in the present disclosure is referred to as prelB6, where B has the same meaning as in general formula (II); An embodiment is called pre-lT6, where B consists of 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, P1 and P2 are defined with respect to general formula (II) It is as it was done. An embodiment of such nucleotide precursors in the present disclosure is referred to as prelB7, where B has the same meaning as in general formula (II); An embodiment is called pre-lT7, where B consists of 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 of the general formula As defined for (II). Embodiments of such nucleotide precursors in the present disclosure are referred to as prelB8, where B has the same meaning as in general formula (II); An embodiment is called pre-lT8, where B consists of a thymidinyl group.

[0197] In further embodiments of compounds of formula (II), wherein Y is NR1, R1 is a (C1-C20)alkyl group substituted as defined in general formula (II), examples of which include: Included are C1, C2 or C3 alkyl groups substituted as defined in general formula (II).

[0198] In some of these further embodiments, R1 is a halogen atom, (C1-C6) alkyl group, (C3-C8) cycloalkyl group, (C3-C14) heterocyclic ring, (C6-C14) aryl group and a (C1-C20) alkyl group substituted with one or more groups selected from (C5-C14) heteroaryl groups, wherein P1, P2, Ra, Rb, Rc, Rd, X1, X2 and B has the same meaning 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 meaning 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 an aryl-substituted methylene group. These embodiments also include compounds of formula (II) where Y is NR1, where R1 is a (C1-C20)alkyl group substituted with a phenyl group.

[0201] In some embodiments of compounds of Formula (II), where Y is NR1, R1 is a methyl group substituted with an unsubstituted phenyl group, and Ra, Rb, Rc, Rd, X1 and X2 are each is a hydrogen atom, and P1 and P2 are as defined in general formula (II). Embodiments of such nucleotide precursors in the present disclosure are referred to as prelB5, where B has the same meaning as in general formula (II); An embodiment is called pre-lT5, where B consists of a thymidinyl group.

[0202] In further embodiments of compounds of formula (II) wherein Y is NR1, R1 is optionally substituted with one or more groups selected from halogen atoms and (C1-C6)alkyl groups ( C3-C8) cycloalkyl group, wherein P1, P2, Ra, Rb, Rc, Rd, X1, X2 and B have the same meaning 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), wherein Y is NR1, R1 is unsubstituted cyclohexyl and Ra, Rb, Rc, Rd, X1, X2 are each hydrogen atoms. and P1 and P2 are as defined for general formula (II).

[0205] An embodiment of such nucleotide precursors in the present disclosure is referred to as prelB4, where B has the same meaning as in general formula (II); An embodiment is called pre-lT4, where B consists of a thymidinyl group.

[0206] In some other embodiments of morpholino analogs of Formula (II), Y is N-C(=O)-R1, where R1 is optionally a halogen atom, (C1-C6) alkyl group, (C3 ~C8) cycloalkyl group, (C3~C14) heterocycle, (C6~C14) aryl group, (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)- a (C1-C20) alkyl group substituted with one or more groups selected from Z2, J is O or S, each of Z1 and Z2 is independently H or (C1-C6)alkyl optionally substituted with one or more groups selected from halogen atoms and (C1-C6)alkyl groups is the basis, R1 is a (C3-C8) cycloalkyl group optionally substituted with one or more groups selected from a halogen atom or a (C1-C6) alkyl group; 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 N-C(=O)-R1, R1 is an optionally substituted (C1-C20) alkyl group, examples of which include optionally substituted (C1-C15 ) alkyl groups, wherein P1, P2, Ra, Rb, Rc, Rd, X1, X2 and B have the same meaning as defined for general formula (II).

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

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

[0210] Embodiments of such nucleotide precursors in the present disclosure are referred to as prelB9, where B has the same meaning as in general formula (II); An embodiment is called pre-lT9, where B consists of a thymidinyl group. These embodiments also provide that Y is N-C(=O)-R1, R1 is a pentadecyl group, Ra, Rb, Rc, Rd, X1, X2 each represent a hydrogen atom, and B, P1 and P2 are , also include compounds of formula (II) as defined in general formula (II). An embodiment of such nucleotide precursors in the present disclosure is referred to as prelB10, where B has the same meaning as in general formula (II); An embodiment is called pre-lT10, where B consists of a thymidinyl group.

[0211] In compounds 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, heterocyclic nucleobases can be selected from optionally substituted purine bases and optionally substituted pyrimidine bases. The term "purine base" is used herein in its ordinary sense as understood by those of ordinary skill in the art and includes tautomers thereof. Similarly, the term "pyrimidine base" is used herein in its ordinary sense as understood by those skilled in the art, and includes tautomers thereof. 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. are mentioned. Examples of pyrimidine bases include, but are not limited to, cytosine, thymine, uracil, 5,6-dihydrouracil and 5-alkylcytosine (eg, 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-deazaxanthin, 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 additional Other heterocyclic bases are described in US Pat. Nos. 5,432,272 and 7,125,855, which are incorporated herein by reference for the limited purpose of disclosing 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 comprising pyrimidines, substituted pyrimidines, purines and substituted purines, the amino groups of which, if present, are optionally protected by protecting groups.

[0212] In preferred embodiments, B is selected from the group comprising adenine, thymine, uracil, guanine and cytosine (ie 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 compounds of formula (II) the groups P1 and P2 are each independently a hydrogen atom, a reactive phosphorus group or a protecting group.

[0214] A "reactive phosphorus group," as used herein, is a phosphorus-containing group contained in a nucleotide unit or nucleotide analogue unit that, via a nucleophilic attack reaction, can be incorporated into another molecule. group capable of reacting with a hydroxyl group or an amine group contained in, in particular, another nucleotide unit or another nucleotide analogue. Generally, such a reaction involves an ester-type internucleoside linkage linking said first nucleotide unit or said first nucleotide analogue unit to said second nucleotide unit or said second nucleotide analogue unit. to generate

[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, including are, 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 are included. Protecting groups include hydroxyl, amine and phosphoramidite protecting groups, which are acetyl (Ac), benzoyl (Bzl), benzyl (Bn), isobutyryl (iBu), phenylacetyl, benzyloxymethylacetal (BOM). , beta-methoxyethoxymethyl ether (MEM), methoxymethyl ether (MOM), p-methoxybenzyl ether (PMB), methylthiomethyl ether, pivaloyl (Piv), tetrahydropyranyl (THP), triphenylmethyl (Trt), Methoxytrityl[(4-methoxyphenyl)diphenylmethyl] (MMT), dimethoxytrityl, [bis-(4-methoxyphenyl)phenylmethyl (DMT), trimethylsilyl ether (TMS), tert-butyldimethylsilyl ether (TBDMS), from the group comprising triisopropylsilyloxymethyl ether (TOM), tri-isopropylsilyl ether (TIPS), methyl ether, ethoxyethyl ether (EE), N,N-dimethylformamidine and 2-cynaonethyl (CE) can be selected.

[0216] In some embodiments of compounds of formula (II), wherein Y, B, Ra, Rb, Rc, Rd, X1 and X2 are as defined for general formula (II), P1 or One of 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 compounds of formula (II), wherein Y, B, Ra, Rb, Rc, Rd, X1 and X2 are as defined for general formula (II), P1 and One of P2 is a 2-cyanoethyl-N,N-diisopropyl phosphoramidite group and the other of P1 and P2 is a protecting group.

[0218] In some embodiments of compounds of formula (II), wherein Y, B, Ra, Rb, Rc, Rd, X1 and X2 are as defined for general formula (II), P1 and One of P2 is a 2-cyanoethyl-N,N-diisopropyl phosphoramidite group and the other of P1 and P2 is an O-4,4'-dimethoxytrityl group.

[0219] Furthermore, 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 ) is an alkyl group.

[0220] (C1-C6) alkyl groups, as used herein, include alkyl groups selected from the group comprising 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 Analogs As previously identified herein, the present disclosure provides compounds of formula (II) [wherein: - B is a heterocyclic nucleobase; - P1 and P2 are each independently H, a reactive phosphorus group or a protecting group; - Y is NR1 and R1 is the group -[C(=O)]m-R2-(O-CH 2 -CH 2 ) p-R3 (wherein m is an integer meaning 0 or 1, p is an integer ranging from 0 to 10, R2 is optionally 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), -N(Z3)-C(=K)-Z4-substituted (C1-C20) alkylene group, K is O or S, each of Z3 and Z4 is independently H or (C1-C6)alkyl optionally substituted with one or more groups selected from halogen atoms and (C1-C6)alkyl groups is the basis, 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] encompasses

[0224] These compounds of formula (II) are included in the more general family of compounds sometimes referred to in this disclosure as "targeted nucleotide precursors." In the present disclosure, compounds of formula (II) in which group R3 is present and which represent a cell-targeting moiety are referred to as "targeted nucleotide precursors of formula (II)" or "targeted nucleotide precursors (II) ” is sometimes called.

[0225] Compounds of formula (II) that do not contain the group R representing a cell-targeting moiety are not targeted nucleotide precursors and are referred to in this disclosure as "non-targeted nucleotide precursors of formula (II)" or " called "Non-targeted Nucleotide Precursor (II)".

[0226] In some embodiments of the targeted nucleotide precursors of Formula (II), R1 is the group -[C(=O)]m-R2-(O-CH 2 -CH 2 ) p-R3, m is 0, p is 0, R3 is a cell targeting moiety, B, P1, P2, Ra, Rb, Rc, Rd, X1, X2, and R2 is as described in the general definition of compounds of formula (II). In some embodiments, R2 is an ethylene group and X1 and X2 are both hydrogen atoms. In some other of these embodiments, R2 is a pentylene group and X1 and X2 are both hydrogen atoms. In some embodiments, R2 is (C12)alkylene and X1 and X2 are both hydrogen atoms.

[0227] In some embodiments of compounds of Formula (II), R1 is the group -[C(=O)]m-R2-(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, B, P1, P2 , Ra, Rb, Rc, Rd, X1, X2 and R2 are as described in the general definition of compounds 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 compounds of Formula (II), R1 is the group -[C(=O)]m-R2-(O-CH 2 -CH 2 )p-R3, m is 1, p is 0, R3 is a cell targeting moiety, R2, B, P1, P2, Ra, Rb, Rc, Rd, X1, X2 are as described in the general definition of compounds 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 defined with respect to general formula (II). Street. In further some of these embodiments, R2 is (C11)alkylene, both X1 and X2 represent hydrogen atoms, and B, P1, P2, Ra, Rb, Rc and Rd are of the general formula (as defined for (II)). In some further these embodiments, R2 is methylene, both X1 and X2 represent hydrogen atoms, and B, P1, P2, Ra, Rb, Rc and Rd are represented by the general formula ( As defined for II).

[0229] R1 is a group -[C(=O)]m-R2-(O-CH 2 -CH 2)p-R3, m is 1, p is selected from the group of integers consisting of 1 and 2, R3 is a cell-targeting moiety; R2, B, P1, P2, Ra, Rb, Rc, Rd, X1, X2 are as described in the general definition of compounds of formula (II). In some of these embodiments, R2 is a methylene group, p is 2, R3 is a cell targeting moiety, B, P1, P2, Ra, Rb, Rc, Rd, X1, X2 is as defined for general formula (II). In some other of these embodiments, R2 is a methylene group, p is 1, R3 is a cell targeting moiety, B, P1, P2, Ra, Rb, Rc, Rd, X1 and X2 are as defined for general formula (II).

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

[0231] Generally, the group R3 encompasses any cell-targeting moiety known in the art, including any of the cell-targeting moieties identified in this disclosure, examples of which include the oligo Cell-targeting moieties identified for nucleotide descriptions are included.

[0232] In some embodiments of compounds of Formula (II), R3 is Formula (III) [formation] (In the formula, A1, A2 and A3 are OH or O-C(=O)-R4 and R4 is (C1-C6)-alkyl or (C6-C10)-aryl group) have

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

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

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

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

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

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

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

[0240] The present disclosure also provides oligonucleotides containing one or more nucleotide analogues introduced into the oligonucleotide by using nucleotide analogue precursors that are compounds of formula (II) identified herein. also related to

[0241] Elsewhere in this disclosure, the hybridizing nucleotide analogue of formula (I-A), if present, is neither 5'-overhang nor 3'-overhang of the antisense strand oligonucleotide. Single- and double-stranded oligonucleotides comprising one or more hybridizing nucleotide analogues of formula (I-A), as specifically described with formula It also relates to one or more nucleotide analogues of (I-B).

[0242] The present disclosure includes one or more hybridizing nucleotide analogs of Formula (I-A) in the hybridizing region of the antisense strand oligonucleotide, optionally one or more in the sense strand oligonucleotide. single- and double-stranded oligonucleotides, particularly siRNAs, comprising the hybridizing nucleotide analogue (I-A) of In addition, double-stranded oligonucleotides, particularly siRNAs, if present, may be added to the 5'-overhang, 3'-overhang, or 5'-overhang and 3'-overhang of the sense and antisense strand oligonucleotides. may contain one or more non-targeting nucleotide analogues in both and optionally in the 5'-overhangs of the sense and antisense strand oligonucleotides, in the 3'-overhangs, or Both the 5'-overhang and the 3'-overhang may contain one or more targeted nucleotide analogues (I-B).

[0243] Nucleotide analogues of formulas (I-A) and (I-B) are further detailed below.

[0244] Nucleotide analogues of formulas (I-A) and (I-B) The compounds of formula (II) disclosed herein can be used as monomeric units of oligomeric compounds, in particular as monomeric units of oligonucleotides, e.g. A nucleotide analogue building block designed to be included as a unit, particularly as a monomeric unit of siRNA, etc., which is also referred to as a "nucleotide analogue precursor". Incorporation of the nucleotide analogue precursors described herein by compounds of formula (II) into oligonucleotides comprises the corresponding monomers described herein as compounds of formula (I) in said oligonucleotides. Resulting in the presence of body units, the compound of formula (I) consists of either (i) a nucleotide analogue of formula (I-A) or (ii) a nucleotide analogue of formula (I-B).

[0245] Nucleotide analogues of formula (I-A) comprise compounds of formula (I), wherein group R3 does not consist of a cell-targeting moiety, as further described below. Nucleotide analogues of formula (I-B) also consist of compounds of formula (I), wherein group R3 consists of a cell-targeting moiety.

[0246] The disclosure also provides formula (I): [formation] [In the formula, - B is a heterocyclic nucleobase; - one of L1 and L2 is an internucleoside linking group that links 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 a compound of formula (I); an internucleoside linking group that links to a nucleotide; - Y is O, NH, NR1 or N-C(=O)-R1 and R1 is: Optionally, a halogen atom, (C1-C6) alkyl group, (C3-C8) cycloalkyl group, (C3-C14) heterocyclic ring, (C6-C14) aryl group, (C5-C14) heteroaryl group, - O-Z1, -N(Z1)(Z2), -S-Z1, -CN, -C(=J)-O-Z1, -O-C(=J)-Z1, -C(=J)-N( (C1-C20) alkyl group substituted with one or more groups selected from Z1) (Z2), -N(Z1)-C(=J)-Z2 (wherein 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); 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 meaning 0 or 1, p is an integer ranging from 0 to 10, R2 is optionally 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 (C1-C20) alkylene group substituted with -N(Z3)-C(=K)-Z4, K is O or S, each of Z3 and Z4 is independently H or (C1-C6)alkyl optionally substituted with one or more groups selected from halogen atoms and (C1-C6)alkyl groups is the basis, R3 is a hydrogen atom, (C1-C6) alkyl group, (C1-C6) alkoxy group, (C3-C8) cycloalkyl group, (C3-C14) heterocyclic ring, (C6-C14) aryl group or (C5-C14) C14) selected from the group consisting of heteroaryl groups; or 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] and any isomer thereof, such as a stereoisomer, or a pharmaceutically acceptable salt thereof.

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

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

[0249] In some embodiments where Y is NR1, R1 is optionally a halogen atom, (C1-C6) alkyl group, (C3-C8) cycloalkyl group, (C3-C14) heterocycle, (C6-C14) ) aryl group, (C5-C14) heteroaryl group, -O-Z1, -N(Z1)(Z2), -S-Z1, -CN, -C(=J)-O-Z1, -O-C(= substituted with one or more groups selected from J)-Z1, -C(=J)-N(Z1)(Z2), -N(Z1)-C(=J)-Z2 ( C1-C20) an alkyl group, J is O or S, each of Z1 and Z2 is independently H or (C1-C6)alkyl optionally substituted with one or more groups selected from halogen atoms and (C1-C6)alkyl groups groups, in the form of bases or in the form of addition salts with acids.

[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 has the same meaning as defined for general formula (I) or is a pharmaceutically acceptable salt thereof.

[0251] In some embodiments where Y is NR1, R1 is an unsubstituted (C1-C16)alkyl group, examples of which are alkyl selected from the group including methyl, isopropyl, butyl, octyl, hexadecyl. groups, 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 defined with respect to general formula (I) It is as it was done.

[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 defined with respect to general formula (I) It is as it was done.

[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 are generally It has the same meaning as defined for formula (I) or is a pharmaceutically 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 defined with respect to general formula (I) It is as it was done.

[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 defined with respect to general formula (I) It is as it was done.

[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 of the general formula ( As defined for I).

[0258] In further embodiments of compounds of formula (I), wherein Y is NR1, R1 is a (C1-C20)alkyl group substituted as defined in general formula (I), examples of which include the general formula Included are C1, C2 or C3 alkyl groups substituted as defined in (I).

[0259] In some of these further embodiments, R1 is a halogen atom, (C1-C6) alkyl group, (C3-C8) cycloalkyl group, (C3-C14) heterocyclic ring, (C6-C14) aryl group and It is a (C1-C20) alkyl group substituted with one or more groups selected from (C5-C14) heteroaryl groups.

[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 meaning 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) wherein Y is NR1 and R1 is a methylene group substituted with an aryl group. These embodiments also include compounds of formula (I) wherein Y is NR1 and R1 is a (C1-C20)alkyl group substituted with a phenyl group.

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

[0263] In further embodiments of compounds of formula (I) wherein Y is NR1, R1 is optionally substituted with one or more groups selected from halogen atoms and (C1-C6)alkyl groups ( C3-C8) a cycloalkyl group, wherein 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) wherein Y is NR1, R1 is a cyclohexyl group, L1, L2, Ra, Rb, Rc, Rd, X1, X2, R2, R3 and B have the same meaning as defined for general formula (I) or are pharmaceutically acceptable salts thereof.

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

[0266] In some other embodiments of the oligonucleotides of formula (I), Y is N-C(=O)-R1, R1 is a (C1-C20)alkyl group, and R1 is methyl and pentadecyl. L1, L2, Ra, Rb, Rc, Rd, X1, X2, R2, R3 and B have the same meanings as defined for general formula (I) or their pharmaceutical is an acceptable salt for

[0267] In some other embodiments of the oligonucleotides of formula (I), Y is N-C(=O)-R1, where R1 is optionally a halogen atom, (C1-C6) alkyl group, (C3- C8) cycloalkyl group, (C3-C14) heterocycle, (C6-C14) aryl group, (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), and -N(Z1)-C(=J) a (C1-C20) alkyl group substituted with one or more groups selected from -Z2, J is O or S, each of Z1 and Z2 is independently H or (C1-C6)alkyl optionally substituted with one or more groups selected from halogen atoms and (C1-C6)alkyl groups is the basis, L1, L2, Ra, Rb, Rc, Rd, X1, X2 and B have the same meanings as defined for general formula (II).

[0268] In some of the embodiments where Y is N-C(=O)-R1, R1 is an optionally substituted (C1-C20)alkyl group, which is optionally substituted (C1-C15)alkyl containing groups, 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 N-C(=O)-R1, R1 is an unsubstituted (C1-C20)alkyl group, including unsubstituted (C1-C15)alkyl groups; 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 N-C(=O)-R1, R1 is selected from the group comprising methyl and pentadecyl, and L1 and L2 and B are defined with respect to general formula (I) has the same meaning as These embodiments are those wherein Y is N-C(=O)-R1, R1 is a methyl group, Ra, Rb, Rc, Rd, X1, X2 each represent a hydrogen atom, B, L1 and L2 includes compounds of formula (II) as defined in general formula (I). These embodiments also provide that Y is N-C(=O)-R1, R1 is a pentadecyl group, Ra, Rb, Rc, Rd, X1, X2 each represent a hydrogen atom, B, L1 and L2 also includes compounds of formula (I), as defined under general formula (I).

[0271] The nucleotide analogues of formula (I) consist of either nucleotide analogues of formula (I-A) or nucleotide analogues of formula (I-B) and are present in free base form or in the form of addition salts with acids. be able to. The nucleotide analogues of formula (I) consist of either nucleotide analogues of formula (I-A) or nucleotide analogues of formula (I-B) and may also exist in the form of their pharmaceutically acceptable salts. , which are also within the scope of this disclosure.

[0272] Embodiments of Nucleotide Analogs of Formula (I-B), Also Called Targeted Nucleotide Analogs As previously identified herein, the disclosure also provides nucleotide analogues of formula (I-B) in the form of bases or in the form of addition salts with acids, wherein - B is a heterocyclic nucleobase; - one of L1 and L2 is an internucleoside linking group that links 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 a compound of formula (I); is an internucleoside linking group that links nucleotide residues; - Y is NR1 and R1 is the group -[C(=O)]m-R2-(O-CH 2 -CH 2 ) p-R3 (wherein m is an integer meaning 0 or 1, p is an integer ranging from 0 to 10, R2 is optionally 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), -N(Z3)-C(=K)-Z4-substituted (C1-C20) alkylene group, K is O or S, each of Z3 and Z4 is independently H or (C1-C6)alkyl optionally substituted with one or more groups selected from halogen atoms and (C1-C6)alkyl groups is the basis, 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] , as well as any isomers thereof, such as stereoisomers, or pharmaceutically acceptable salts thereof.

[0273] These compounds of formula (I-B) are included in the more general family of compounds sometimes referred to in this disclosure as "targeted nucleotide analogs."

[0274] R1 is a group -[C(=O)]m-R2-(O-CH 2 -CH 2 )p-R3, m is 0, p is 0, R3 is a cell-targeting moiety; P1, P2, Ra, Rb, Rc, Rd, X1, X2, and R2 are as described in General Definitions for Compounds of Formula (I-B).

[0275] In some embodiments, R2 is an ethylene group, X1 and X2 are both hydrogen atoms, and B, P1, P2, Ra, Rb, Rc, Rd are compounds of formula (I-BI) as described in the general definition of

[0276] In some other of these embodiments, R2 is a pentylene group, X1 and X2 are both hydrogen atoms, and B, P1, P2, Ra, Rb, Rc, Rd are represented by formula (I-B) as described in the general definition of compounds of

[0277] In some other of these embodiments, R2 is a (C12)alkylene group, X1 and X2 are both hydrogen atoms, and B, P1, P2, Ra, Rb, Rc, Rd are of the formula As described in the general definitions of compounds of (I-B).

[0278] R1 is a group -[C(=O)]m-R2-(O-CH 2 -CH 2 )p-R3, m is 0, p is selected from the group of integers consisting of 1, 2, 3 and 4, and R3 is a cell-targeting The moieties B, L1, L2, Ra, Rb, Rc, Rd, X1, X2 and R2 are as described in the general definition of compounds of formula (II). In some of these embodiments, R2 is an ethylene group, p is 1, and X1 and X2 are both hydrogen atoms. In further these embodiments, R2 is an ethylene group, p is 2, and X1 and X2 are both hydrogen atoms. In still further these embodiments, R2 is an ethylene group, p is 3, and X1 and X2 are both hydrogen atoms. Still in other of these embodiments, R2 is an ethylene group, p is 4, and X1 and X2 are both hydrogen atoms.

[0279] R1 is a group -[C(=O)]m-R2-(O-CH 2 -CH 2 ) p-R3, m is 1, p is 0, R3 is a cell-targeting moiety, R2, B, L1, L2, Ra, Rb, Rc, Rd, X1, X2 are as described in the general definition of compounds of formula (I-B).

[0280] In some of these embodiments, R2 is butylene, both X1 and X2 represent hydrogen atoms, and B, L1, L2, Ra, Rb, Rc and Rd are defined with respect to general formula (I-B). It is as it was done.

[0281] In further some of these embodiments, R2 is (C11)alkylene, both X1 and X2 represent hydrogen atoms, and B, L1, L2, Ra, Rb, Rc and Rd are of the general formula As defined for (I-B).

[0282] Still in some further these embodiments, R2 is methylene, both X1 and X2 represent hydrogen atoms, and B, L1, L2, Ra, Rb, Rc and Rd are represented by the general formula ( as defined for I-B).

[0283] R1 is a group -[C(=O)]m-R2-(O-CH 2 -CH 2 )p-R3, m is 1, p is selected from the group of integers consisting of 1 and 2, R3 is a cell-targeting moiety; R2, B, L1, L2, Ra, Rb, Rc, Rd, X1, X2 are as described in the general definition of compounds of formula (I-B).

[0284] In some of these embodiments, R2 is a methylene group, p is 2, R3 is a cell targeting moiety, B, L1, L2, Ra, Rb, Rc, Rd, X1, X2, is as defined for general formula (I-B).

[0285] In some other of these embodiments, R2 is a methylene group, p is 1, R3 is a cell targeting moiety, B, L1, L2, Ra, Rb, Rc, Rd, X1 and X2 are as defined for general formula (I-B).

[0286] Generally, the group R3 encompasses any cell-targeting moiety known in the art, including any of the cell-targeting moieties identified in this disclosure, examples of which include the oligo Cell-targeting moieties identified for nucleotide descriptions are included.

[0287] In some embodiments of compounds of Formula (I-B), R3 is Formula (III): [formation] (wherein A1, A2 and A3 are OH, A4 is OH or NHC(=O)-R5, and R5 is a (C1-C6) alkyl group optionally substituted with a halogen atom) have

[0288] In some embodiments, R3 is N-acetyl-galactosamine of Formula (III-B): [formation]

[0289] According to the present disclosure, references to "GalNAc" or "N-acetylgalactosamine" refer to the β form: 2-(acetylamino)-2-deoxy-β-D-galactopyranose and the α form: 2-(acetylamino )-2-deoxy-α-D-galactopyranose. In certain embodiments, the β-form: 2-(acetylamino)-2-deoxy-β-D-galactopyranose and the α form; 2-(acetylamino)-2-deoxy-α-D-galactopyranose are both can also be used synonymously.

[0290] In oligonucleotides of formula (I-B), 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, heterocyclic nucleobases can be selected from optionally substituted purine bases, optionally substituted pyrimidine bases. The term "purine base" is used herein in its ordinary sense as understood by those of ordinary skill in the art and includes tautomers thereof. Similarly, the term "pyrimidine base" is used herein in its ordinary sense as understood by those skilled in the art, and includes tautomers thereof. A non-limiting list of optionally substituted purine bases include purine, adenine, guanine, hypoxanthine, xanthine, alloxanthine, 7-alkylguanine (e.g. 7-methylguanine), theobromine, caffeine, uric acid and isoguanine. is mentioned. Examples of pyrimidine bases include, but are not limited to, cytosine, thymine, uracil, 5,6-dihydrouracil and 5-alkylcytosine (eg, 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-deazaxanthin, 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 additional Other heterocyclic bases are described in US Pat. Nos. 5,432,272 and 7,125,855, which are incorporated herein by reference to disclose heterocyclic bases.

[0291] In some embodiments B is selected from the group comprising 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 containing nucleotide analogues of formula (I-B), one of the groups L1 and L2 binds the compound of formula (I) to a nucleotide residue, i.e. to the adjacent nucleotide residue. and the other of the L1 and L2 groups is H, a protecting group, or a nucleoside that links the compound of formula (I-B) to the nucleotide residue, i.e. to the adjacent nucleotide residue. It is an interlinking group.

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

[0295] Surprisingly, combining a targeted compound of formula (I) and a non-targeted compound of formula (I) within one double-stranded oligonucleotide results in a change in the behavior of the double-stranded oligonucleotide in vivo. (especially the duration of action in vivo) have been demonstrated to be significantly improved; this can also be demonstrated when the sense strand does not contain any phosphorothioate groups.

[0296] siRNAs incorporating one or more compounds of formula (I) in the sense strand, the antisense strand, or both the sense and antisense strands, particularly the sense strand, are also effective for targeted gene silencing in vivo. Active. Targeted gene silencing activity is determined by (i) the embodiments of compounds of formula (I) described herein, (ii) the number of compounds of formula (I) described herein, and (iii) It can be controlled by the placement of the compound of formula (I) within the sense or antisense strand of the siRNA.

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

[0298] For illustrative purposes and not limiting the disclosure, a double-stranded oligonucleotide may also include one or more nucleotides in the sense and / or antisense strands that have been modified.

[0299] Modifications can be selected from substitutions or insertions with analogues of nucleic acids or bases, and chemical modifications of bases, sugars or phosphate moieties. Modifications selected were 3' terminal deoxy-thymine, 2'-O-methyl, 2'-deoxy modification, 2'-desoxy-fluoro, 2'-amino modification, 2'-alkyl modification, phosphorothioate modification, phosphoro may be individually selected from amidate modifications, 5′-phosphorothioate group modifications, 5′-phosphate or 5′-phosphate mimetic modifications, and cholesteryl derivatives or dodecanoic acid bisdecylamide group modifications, and / Alternatively, the modified nucleotides may be any one of locked nucleotides, abasic nucleotides or nucleotides containing non-natural bases. A preferred embodiment may be one in which 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, include the following: modifications, such as unlinked phosphates and / or oxygens and / or linked phosphates and oxygens one or more replacements; replacements of phosphate moieties; modifications or replacements of naturally occurring bases; replacements or modifications of the ribose-phosphate backbone; It may involve one or more of removing, modifying or replacing a phosphate group, or conjugating a moiety, such as a fluorescently labeled moiety, to either the 3' or 5' end of the RNA.

[0301] Methods for Synthesis of Nucleotide Analog Precursors of Formula (II) Nucleotide analog precursors of formula (II) may be prepared according to the detailed methods illustrated in the disclosure provided herein.

[0302] The present disclosure provides a method for preparing compounds of formula (II-1), comprising: a) Formula (X) [formation] (wherein B is a heterocyclic nucleobase and P1 and P2 each independently represent a protecting group as defined in general formula (II) described herein) compound of formula (X) with an oxidizing reagent such as sodium periodate (NaIO 4 ) to give the following formula (XI): [formation] obtaining a compound of and

[0303] b) the compound of formula (XI) to the compound of formula (XII) R1-NH 2 (XII) (wherein R is as defined in general formula (I) described herein) in the presence of a reductive amination step of formula (II-1): [formation] (Wherein, B is a heterocyclic nucleobase, and P1 and P2 each independently represent a protecting group defined in general formula (II)) obtaining the compound of relating to the method, including

[0304] The disclosure also provides a method for preparing compounds of formula (II-2), comprising: a) Formula (X) [formation] (wherein B is a heterocyclic nucleobase and P1 and P2 each independently represent a protecting group as defined in general formula (II) described herein) compound of formula (X) with an oxidizing reagent such as sodium periodate (NaIO 4 ) to give the following formula (XI): [formation] obtaining a compound of and

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

[0306] c) a compound of formula (XIII) with formula (XIV) R1-C(=O)-OH (XIV) (wherein R is as defined in general formula (II) described herein) in the presence of a compound of formula (II-2), subjected to an amide coupling step to give a compound of formula (II-2) [formation] (Wherein, B is a heterocyclic nucleobase, P1 and P2 each independently represent a protecting group defined in general formula (II), R1 is defined in general formula (II) as I did) a step of obtaining

[0307] d) subjecting a compound of formula (XIII) to a step of reductive amination in the presence of an aldehyde or ketone to give formula (II-1) [formation] obtaining the compound of It also relates to a method comprising

[0308] The present disclosure also provides formula (II-3) [formation] A method for preparing a compound of formula (XV) [formation] (wherein A1, A2, A3 and A4 are as defined in Formula (III) or (III-A) herein, and -Y-CHO is converted to X by a reductive amination reaction) and X is a group of formula -(CH2-CH2-O)p-R2-, where p and R2 are as defined in general formula (I) be) The compound of formula (XIII) [formation] (Wherein, B is a heterocyclic nucleobase, and P1 and P2 each independently represent a protecting group defined in general formula (II)) by reductive amination with a compound of formula (II-3) [formation] obtaining the compound of It also relates to a method comprising

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

[0310] The present disclosure also provides formula (II-4) [formation] A method for obtaining a compound of

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

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

[0313] The disclosure further provides a method for preparing a compound of formula (II-5), comprising: a) Formula (XI) [formation] (Wherein, B is a heterocyclic nucleobase, and P1 and P2 each independently represent a protecting group defined in general formula (II)) by reducing a compound of formula (XVII) [formation] obtaining a compound of

[0314] b) conversion of compounds of formula (XVII) in the presence of a sulfonylating agent (e.g. p-toluenesulfonyl chloride Ts-Cl, methanesulfonyl chloride Ms-Cl) to give compounds of formula (XVIII) [formation] (Wherein, Ts represents a tosyl group) obtaining a compound of

[0315] c) subjecting a compound of formula (XVIII) to basic conditions to give a compound of formula (II-5) [formation] obtaining the compound of relating to the method, including

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

[0317] The disclosure also provides an alternative method for preparing compounds of formula (II-5), comprising: a) Formula (XVII) [formation] is converted 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) [formation] (Wherein, Ts represents a tosyl group) obtaining a compound of

[0318] b) the compound of formula (XIX) is deprotected by removal of group P1 to give a compound of formula (XX) [formation] obtaining a compound of

[0319] c) subjecting a compound of formula (XX) to basic conditions to give a compound of formula (XXI) [formation] obtaining a compound of and

[0320] d) replacing the tosyl group with a protecting group P1, formula (II-5) [formation] obtaining the compound of It also relates to a method comprising

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

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

[0323] Starting from the commercially available ribose derivative G1, the two primary OH-groups can be distinguished by selective benzylation according to standard literature protocols. Standard protecting group modification of the resulting benzyl ether G2 yields the fully protected ribose analogue G3, which can be converted to a nucleobase 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] [formation]

[0325] Scheme 1: Synthesis of compounds of formula (II) where Y is NH, NR1 and NC(=O)R1 Starting from the nucleoside analogue G4, by modifying the protecting group pattern by standard procedures, the two primary alcohols and the unprotected OH group at C2' and C3' of the ribose scaffold were converted to general formula (II). yields intermediate G5 with orthogonal protecting groups P1 and P2 as defined in . The cis orientation of the dihydroxy functional group in the G5 compound is NaIO as an oxidizing agent. 4 allows oxidative cleavage of the C-C bond between C2' and C3'. The resulting dialdehyde can be isolated as the monohydrate G6, which can be treated with a reducing agent such as NaCNBH 3 is converted to the desired morpholine scaffold by a reductive amination reaction with Using an amine substrate such as ammonia or ammonium diborate results in the morpholine intermediate G7 having a free NH group in the morpholine scaffold. e.g. NaCNBH 3 A 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 converted to the appropriate amine R1-NH 2 (wherein R1 is as defined in general formula (II)), which directly gives the alkylated morpholine G8. Analog acylated morpholines are obtained by standard peptide coupling reactions between the free morpholine building blocks G7 and the corresponding carboxylic acid R1-COOH, resulting in the amide intermediate G9.

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

[0327] Analogously 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 using intermediate G7 as an amine reagent It can be prepared by reductive amination or peptide coupling reactions used.

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

[0329] [formation]

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

[0331] In the following, X is the group -R2(OCH2- Defined as CH2)p-.

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

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

[0334] [formation]

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

[0336] Compound G18 consists of compounds of general formula (II) described in this disclosure.

[0337] Alternatively, the diol intermediate G16 can be bissulfonylated with, for example, excess 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 to G17 under nucleophilic substitution and formation of the desired dioxane scaffold G21 (see Scheme 3). The remaining tosylate can be replaced with sodium benzoate to again obtain the fully protected dioxane scaffold G18 with protecting groups P1 and P2 orthogonal to the primary hydroxyl groups.

[0338] [formation]

[0339] Scheme 4: Alternative Synthesis of Compounds of Formula (II) Where Y is O The stated building blocks of general formula (II) (G8, G9, G14, G15 and G18) with orthogonal protecting groups P1 and P2 are finally converted into the corresponding DMT-protected phosphoramidites to give autooligonucleotides Allowing their use as nucleotide precursors in 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 on the G22-building block by standard protocols yields the final DMT-protected phosphoramidite G23 as a nucleotide precursor for automated oligonucleotide synthesis.

[0341] [formation]

[0342] Scheme 5: Synthesis of 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 consisting of a phosphoramidite group.

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

[0344] [formation]

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

[0346] Preparation of Oligonucleotides Comprising Nucleotide Analogues of Formula (I-A) and Optionally (I-B) Oligonucleotides for use in accordance with the present disclosure may also be referred to herein as "modified oligonucleotides", which may be finalized according to any useful technique, including the methods described herein. by using one or more nucleotide analogue precursors of formula (II) as part of the starting building blocks for incorporation into selected positions of a growing strand of a growing oligonucleotide. Often, oligonucleotides containing one or more nucleotide analogues of formula (I) are produced in such a way that one or more compounds of formula (I) are selected for the final oligonucleotide. position.

[0347] Nucleotide analog precursors of formula (II) can be synthesized as described in this disclosure.

[0348] Modified oligonucleotides of the present disclosure may be double-stranded with or without overhangs, or may include at least a double-stranded portion. A double-stranded modified oligonucleotide comprises a first nucleotide sequence (e.g., sense nucleotide sequence) and a second nucleotide sequence (e.g., antisense nucleotide sequence) that is complementary to and hybridizes to the first nucleotide sequence. the second nucleotide sequence is also complementary to the target RNA sequence whose inhibition is sought. According to these embodiments, the first nucleotide sequence and the second nucleotide sequence may be on separate strands within the modified oligonucleotide; or on the same strand but with the spacer, or the second One nucleotide sequence may be present separated by additional nucleotide sequences of appropriate length that form a hairpin loop when hybridized to the second nucleotide sequence.

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

[0350] Oligonucleotides of the invention, eg, those comprising one or more nucleotide analogues of formula (I), can be chemically synthesized using protocols known in the art. For example, Caruthers et al., 1992, Methods in Enzymology, 211:3-19; Thompson et al., International PCT Publication No. WO 99 / 54459; Wincott et al., 1995, Nucleic Acids Res., 23:2677-2684; Wincott et al., 1997, Methods Mol. Bio., 74:59; Brennan et al., 1998, Biotechnol Bioeng., 61:33-45; and Brennan, US Patent No. 6,001,311. Oligonucleotide synthesis utilizes common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5' terminus and phosphoramidite at the 3' terminus. In certain embodiments, oligonucleotides comprising nucleotide analogs of formula (I) are synthesized, synthesized, and treated according to the methods described in U.S. Pat. Deprotected and analyzed. In a non-limiting synthetic example, small-scale synthesis is performed on a 394 Applied Biosystems, Inc. / Thermo Fischer Scientific Inc. synthesizer.

[0351] Alternatively, oligonucleotides containing one or more nucleotide analogues of formula (I) are synthesized separately and post-synthetically, e.g., 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 synthetic and / or Alternatively, they may be integrated by hybridization after deprotection. Various modified oligonucleotides according to the present disclosure can also be synthesized using the teachings of Scaringe et al., US Pat. Nos. 5,889,136; 6,008,400; and 6,111,086.

[0352] Composition Certain aspects of the disclosure relate to compositions (eg, pharmaceutical compositions) comprising the dsRNAs described herein. In some embodiments, the composition (eg, pharmaceutical composition) further comprises a pharmaceutically acceptable carrier. In some embodiments, the compositions (eg, pharmaceutical compositions) are useful for treating diseases or disorders associated with targeted gene expression or activity.

[0353] Compositions (eg, 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] Compositions (eg, pharmaceutical compositions) of this disclosure can be administered in dosages sufficient to inhibit expression of the targeted gene. In some embodiments, a suitable dose of dsRNA is in the range of 0.01 mg / kg to 400 mg / kg body weight of the recipient.

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

[0356] The dsRNA molecules of this disclosure can be formulated in a pharmaceutically acceptable carrier or diluent. Pharmaceutically acceptable carriers can be liquid or solid and are selected with a planned mode of administration to provide the desired bulk, viscosity, and other related transport and chemical properties. can. Any known pharmaceutically acceptable carrier or diluent can be used, examples of which include, for example, water, saline, binders such as polyvinylpyrrolidone or hydroxypropylmethylcellulose, fillers ( lactose and other sugars, gelatin, or calcium sulfate), lubricants (e.g. starch, polyethylene glycol, or sodium acetate), disintegrants (e.g. starch or sodium starch glycolate), calcium salts (e.g. calcium sulfate). , calcium chloride, calcium phosphate, etc.) and wetting agents (eg, sodium lauryl sulfate).

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

[0358] Vector and dsRNA delivery The dsRNA of this disclosure can be delivered directly or indirectly. In some embodiments, the dsRNA is delivered directly by administering a composition (eg, pharmaceutical composition) comprising the dsRNA to the subject. In some embodiments, dsRNA is delivered indirectly by administering one or more vectors described herein.

[0359] delivery The dsRNA of the present disclosure can be obtained by any method known in the art, such as by adapting methods for delivering nucleic acid molecules for use with dsRNA (eg, Akhtar, S. et al. (1992) Trends Cell. Biol. 2(5):139-144; see WO94 / 02595) or via additional methods known in the art (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. 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, V.A. and Barciszewski, J. (eds.) (2010) RNA Technologies and Their Applications, Springer-Verlag Berlin Heidelberg, DOI 10.1007. / 978-3-642-12168-5; see Xu, C. and Wang, J. (2015) Asian Journal of Pharmaceutical Sciences 10(1):1-12).

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

[0361] Liposomal formulation Liposomes are unilamellar or multilamellar vesicles having a membrane formed from a lipophilic substance and an aqueous interior. In some embodiments, liposomes are vesicles composed of amphipathic 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 be fused 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- and fat-soluble drugs; drugs encapsulated in the internal compartment can be protected from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (eds.), 1988, Marcel Dekker, Inc., New York, N.Y., vol. 1, 245 pages). Important considerations in the preparation of liposomal formulations are lipid surface charge, vesicle size and aqueous volume of the liposomes. For example, engineered cationic liposomes and spatially stabilized liposomes can be used to deliver dsRNA. See, eg, Podesta et al. (2009) Methods Enzymol. 464, 343-54; US Pat. No. 5,665,710.

[0362] nucleic acid-lipid particles In some embodiments, the dsRNAs of this disclosure are fully encapsulated in lipid formulations, eg, to form nucleic acid-lipid particles, eg, SPLP, pSPLP, or SNALP. The term "SNALP" as used herein refers to a stable nucleic acid-lipid particle comprising SPLP. The term "SPLP" as used herein refers to nucleic acid-lipid particles containing plasmid DNA encapsulated within lipid vesicles. Nucleic acid-lipid particles, such as SNALPs, typically contain cationic lipids, non-cationic lipids, cholesterol, and lipids (eg, PEG-lipid conjugates) that prevent particle aggregation and increase circulation time. SNALP and SPLP are useful for systemic application because they exhibit long circulation lifetimes after intravenous (i.v.) injection and accumulate at distal sites (eg, sites physically remote from the site of administration). SPLPs include "pSPLPs", examples of which include encapsulated condensing agent-nucleic acid complexes as described in PCT Publication No. WO00 / 03683.

[0363] In some embodiments, the dsRNA is resistant to nuclease degradation in aqueous solution when present in nucleic acid-lipid particles. Nucleic acid-lipid particles and methods for their preparation are disclosed, for example, in US Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410;

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

[0365] additional formulations Important factors to consider for successful delivery of dsRNA molecules in vivo include (1) biological stability of the 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, eg, by direct injection or implantation into tissue, or by administering preparations locally. For systemic administration of dsRNA for the treatment of disease, the dsRNA may be modified or alternatively delivered using drug delivery systems; Acts to prevent rapid degradation of dsRNA by nucleases. Modifications of RNA or pharmaceutical carriers can also permit targeting of dsRNA compositions to target tissues and avoid unwanted off-target effects. As noted above, dsRNA molecules can be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. In some embodiments, the dsRNA is delivered using a drug delivery system such as nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems. Positively charged cationic delivery systems facilitate the binding of dsRNA molecules (having a negative charge) and also enhance interactions with the negatively charged cell membrane to allow efficient uptake of the dsRNA by the cell. Cationic lipids, dendrimers, or polymers may either bind dsRNA or be induced to form vesicles or micelles that encase dsRNA (see, e.g., Kim S.H. et al. (2008) Journal of Controlled Release 129(2):107-116). Formation of vesicles or micelles also prevents degradation of dsRNA upon systemic administration. Methods for making and administering cationic dsRNA complexes are known in the art. In some embodiments, the dsRNA is complexed with cyclodextrin for systemic administration.

[0366] How to use dsRNA A particular embodiment of the disclosure is a method for inhibiting expression of a targeted gene in a mammal, comprising an effective amount of one or more dsRNA of the disclosure, one or more A method comprising administering a composition (eg, a pharmaceutical composition) of the disclosure comprising a vector of the disclosure or one or more dsRNA of the disclosure. Certain embodiments of the disclosure are methods of treating and / or preventing one or more target gene-mediated diseases or disorders, wherein one or more dsRNAs of the disclosure, and / or one or more vectors of the disclosure, and / or compositions (eg, pharmaceutical compositions) comprising one or more dsRNAs of the disclosure. In some embodiments, downregulating target gene expression in a subject reduces one or more symptoms of a disease or disorder mediated by the targeted gene in the subject.

[0367] In some embodiments, target gene expression in the subject is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 10%, at least about 15%, at least about 20%, at least about 25% after treatment compared to pretreatment levels. %, 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% inhibited. In some embodiments, target gene expression is at least about 1.1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, after treatment compared to pretreatment levels, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, At least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 75-fold, or at least about 100-fold inhibition. In some embodiments the target gene is inhibited in the subject's liver.

[0368] In some embodiments, the subject is 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 will be understood from the context of the present disclosure, the dsRNAs described herein are such that the nucleotide analogues of formula (II) have specific structural functions of their "sugar-like" groups, wherein It has its main characteristic based on the presence of one or more nucleotide analogues of formula (II) included. The dsRNAs described herein are generally designed to target selected nucleic acid sequences contained within a target nucleic acid of interest. In particular, dsRNA embodiments comprising siRNAs described herein include an antisense strand that specifically hybridizes to a nucleic acid sequence contained in a target nucleic acid of interest. The dsRNAs or compositions (eg, pharmaceutical compositions) described herein may be for use in the treatment of target gene-mediated disorders or diseases. In particular, the dsRNAs or compositions (e.g., pharmaceutical compositions) described herein, particularly dsRNAs comprising one or more targeted nucleotide analogues, and particularly one or more The targeted nucleotide analogues of formula (II) may be for use in the treatment of target gene-mediated disorders or diseases for which hepatic targeting is sought.

[0370] Certain aspects of the present disclosure also relate to methods of delivering nucleic acids to hepatocytes comprising contacting the hepatocytes with a dsRNA described herein.

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

[0372] The treatment or preventive action of dsRNA will result in a statistically significant improvement in one or more parameters of disease status, or worsen symptoms if they would otherwise be expected to worsen or develop. Or it is clear by not causing it to develop. For example, a favorable change in a measurable parameter of disease of at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more is effective. Treatment may be indicated. 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 a marker or symptom is observed.

[0373] Kits and products Certain aspects of the present disclosure are dsRNAs, vectors, or compositions useful for the treatment and / or prevention of target gene-mediated disorders or diseases as described above (e.g., the pharmaceutical compositions described herein). It relates to a product or kit containing one or more.The product or kit may further comprise a container and a label or package insert on or associated with the container.Suitable containers include, for example, bottles, Vials, syringes, IV fluid bags, etc. The container can be molded from a variety of materials, such as glass or plastic, etc. The container can be used alone or in separate compositions effective to treat or prevent disease. It may hold a composition in combination with an agent and have a sterile access port (for example the container can be an intravenous fluid bag or a vial having a stopper pierceable by a hypodermic injection needle). The at least one active agent is a dsRNA as described herein, and the label or package insert indicates that the composition is used to treat a target-mediated disorder or disease. The kit comprises (a) a first container containing a composition comprising a dsRNA described herein; and (b) a second container containing a composition comprising a second therapeutic agent. The product or kit of the disclosure in this embodiment may further comprise 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 comprise a second (or third) container containing a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution and dextrose solution. It may also contain other materials desirable from a commercial and user standpoint, such as other buffers, diluents, filters, needles, and syringes.

[0374] Nucleic acid sequences are disclosed herein, particularly in the Examples provided herein, which serve as a reference. The same sequences are also presented in a Sequence Listing formatted according to standard requirements for the purposes of patenting matter. In the event of any sequence discrepancies with the standard sequence listing, reference is made to the sequences set forth herein.

[0375] Several embodiments of the present disclosure are set forth below for purposes of illustration, without limiting the present disclosure.

Example

[0376] Nomenclature is defined according to IUPAC rules. Unless otherwise indicated, the following LC-MS methods are used: A: Column: Waters ACQUITY UPLC BEH C18, 1.7μm, 2.1x50mm Eluate: (H 2 O+0.05% FA) / (ACN+0.035% FA) 95:5 (0 minutes) to 5:95 (2 minutes) to 5:95 (2.6 minutes) to 95:5 (2.7 minutes) to 95:5 ( 3.0 min), 0.9 ml / min 55°C. B-1: Column: Phenomenex Luna C18, 3.0μm, 2.0x10mm Eluate: (H 2 O+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, 3.0μm, 2.0x10mm Eluate: (H 2 O+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, 3.0μm, 2.0x10mm Eluate: (H 2 O+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, 2.1x50mm Eluate: (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 Eluate: (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 Eluate: (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: D-N-acetylgalactosamine -FR: flow velocity -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 a vacuum -n.d.: not determined -TLC: thin layer chromatography -h: hour -min: minutes -Tm: melting temperature -r: ribonucleotide -d: desoxy-ribonucleotide -m:2'-OMe-nucleotide -f: 2'-desoxy-fluoro-nucleotide -ss: sense strand -as: antisense strand -ds: double stranded -chol: cholesterol -PO: Phosphodiester bond - * or PS:phosphorothioate linkage -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 analogue -lgB: targeted nucleotide analogue

[0378] A: Synthesis of Nucleotide Analogs of Formula (I) Where X is NH, NR1, NC(=O)R1

[0379] [formation]

[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 22.3 g (174.7 mmol) of thymine were dissolved in dry ACN (500 ml) under Ar atmosphere. After adding BSA (106.6 g, 128.2 ml, 524.1 mmol), the solution was stirred at 80° C. for 1 hour. 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 hour. The solvent was evaporated in vacuo and the residue was dissolved in EtOAc and saturated NaHCO 3 solution, H 2 Washed with O and saturated NaCl solution. MgSO 4 After drying at rt, 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 - : [M-H] - = 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, J=12.17 Hz, 1 H), 3.79 (d, J=10.16 Hz, 1 H), 3.53 (d, J=10.16 Hz, 1 H) 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) uracil were dissolved in dry ACN (200 ml). After adding BSA (20.09 g, 30.15 ml, 123.3 mmol), the solution was stirred at 81° C. for 1 hour. After cooling the solution to 0°C, TMSOTf (7.48g, 6.10ml, 33.3mmol) was added and the mixture was heated to 65°C. Stirring was continued at this temperature for 1 hour at which point complete conversion was achieved. At room temperature, saturated NaHCO 3 500ml of solution was added and the mixture was extracted with EtOAc (500ml). Separate the organic layer and add H 2 Washed with 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. rice field. 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 To a solution of compound G3 (148.5 g, 0.30 mol) in DCE (6.68 l) was added N 2 N-isobutyryl-guanine (135 g, 0.61 mol) and BSA (311.85 ml, 1.2 mol) were added at 15° C. under atmosphere. The mixture was stirred at 85° C. for 3 hours. TMSOTf (183.4 g, 0.90 mol) was then added at 85° C. and stirring was continued for 3 hours at which time TLC indicated complete conversion. The mixture was cooled to room temperature and saturated NaHCO 3 Poured into 6.5 l of solution. The organic layer was separated and the aqueous phase was extracted twice with DCM (5l). Combine the organic layers and wash with anhydrous Na 2 SO 4 , filtered and concentrated. The crude product obtained 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=6.4Hz, 1H), 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 hypoxanthine 3.32 g (23.66 mmol) as nucleobase, the title compound was synthesized following 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 (9.53g, 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-benzoyl adenine (121 g, 0.506 mol) in DCE (6.56 l) at room temperature, BSA (274 g, 1.348 mol) was added dropwise and the mixture was heated at 90°C for 1.5 hours. Stirred. After dropwise addition of TMSOTf (225 g, 1.011 mol) at 40-50° C., the mixture was stirred at 90° C. for 1 hour. Saturate the mixture with NaHCO 3 Quench with 15 L of solution and separate the layers. The aqueous layer was extracted twice with EtOAc (3 l) and the combined organic layers were washed with anhydrous Na 2 SO 4 Dried at rt, filtered and concentrated in vacuo. The crude product was purified by flash chromatography (PE / EtOAc 1:2) to give compound 1e as a mixture of anomers (224.4 g) as a yellow oil. The mixture was purified by two rounds of reverse flash chromatography (neutral) 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, 1H), 4.88 (d, J=5.77 Hz, 1H), 4.63 (s, 2H), 4.56-4.45 (m, 2H), 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 THF / EtOH (4:1) at 0° C. was added 221.9 ml (443.8 mmol) aqueous NaOH (2 M). After removing the ice bath, the mixture was stirred for 2 hours for 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 (250ml). The combined organic layer was 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, 1H), 1.63-1.51 (m, 3H).

[0386] 2b: 1-[(2R,3R,4S,5R)-4-benzyloxy-5-(benzyloxymethyl)-3-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl]pyrimidine-2,4 - Zeon 1b (9.10 g, 16.9 mmol) was dissolved in 240 ml THF / EtOH (3:1). At 0° C., 8.49 ml (84.5 mmol) of 1 M NaOH solution were added and the solution was stirred and brought to room temperature. After 1 hour, citric acid was added until pH reached 7 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 rt, the solvent was removed to give 7.66 g (99.7%) of 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, 1 H), 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) at 0° C. was added dropwise 1M NaOH solution (443 mL). The solution was stirred at 0° C. for 1 hour to allow complete conversion. The pH was adjusted to 7 by adding 1N HCl and the solvent was removed. The residue is H 2 Dissolved in O (500 mL) and extracted with DCM (3 x 500 ml). Combine the organic layers and wash with anhydrous Na 2 SO 4 and concentrated to give 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, 1H), 4.99 (s, 1H), 4.87-4.84 (m, 2H), 4.63 (d, J=15.6Hz, 1H), 4.56 (s, 2H) ), 4.24 (d, J=4.8 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-purine- 6-on Deprotection of 1d (9.34 g, 16.65 mmol) following the procedure described for 2b gave 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, 1H), 3.55 - 3.70 (m, 4H).

[0389] 2e: N-[9-[(2R,3R,4S,5R)-4-benzyloxy-5-(benzyloxymethyl)-3-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl]purine- 6-yl]benzamide Deprotection of 1e (144 g, 216 mmol) following the procedure described for 2b gave the title compound 2e (126 g, quantitative, crude) as a colorless foam, which was used without further purification. did. 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 (34.7g, 74.2mmol) and imidazole 16.8g (244.7mmol) were dissolved in dry DCM (300ml). At room temperature a solution of 16.2 g (18.0 ml, 81.6 mmol) of TIPS-chloride in DCM (200 ml) was added and the reaction was stirred for 19 hours. After quenching with EtOH, the solvent was evaporated and the residue dissolved in EtOAc. The organic solution is H 2 O, 1M HCl, H 2 O and saturated NaHCO 3 solution followed by saturated NaCl solution. MgSO 4 After drying at rt, 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. Obtained (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, 1H), 4.46 (s, 2H), 4.34-4.26 (m, 1H), 4.22 (d, J=6.27Hz, 1H), 3.90 (d, J=10.92Hz, 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, 3 H), 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 with 2b (7.66 g, 16.8 mmol), the title compound was prepared as described in the protocol for 3a, followed by silica gel chromatography (20 to 80% EtOAc in n-heptane) to yield the desired silylated product. Product 3b (8.69 g, 84.5%) was obtained. 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=4.0Hz, 1H), 5.37 (d, J=8.0Hz, 1H), 4.77 (d, J=12.0Hz, 1H), 4.62 (d, J=12.0Hz, 1H) ), 4.49 (s, 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) was added N 2 Imidazole (38 g, 559 mmol) and TIPSCl (35.9 g, 186 mmol) were added at 0° C. under atmosphere. After stirring for 12 hours 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 treated with anhydrous Na 2 SO 4 , filtered and concentrated. 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=6.8Hz, 1H), 5.72 (d, J=5.6Hz, 1H), 4.94-4.93 (m, 2H), 4.90-4.53 (m, 3H), 4.19 (d, J. =4.4 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, 21H).

[0393] 3d: 9-[(2R,3R,4S,5S)-4-benzyloxy-5-(benzyloxymethyl)-3-hydroxy-5-(triisopropylsilyloxymethyl)tetrahydrofuran-2-yl]-1H- purine-6-one Starting with 2d (7.75 g, 16.2 mmol), the title compound was prepared as described in the protocol for 3a and purified by silica gel chromatography (0 to 100% EtOAc / MeOH (9:1) in n-heptane). Afterwards the desired silylated product 3d (8.60 g, 83.6%) was obtained. 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) at room temperature was added a solution of TIPSCl (80.6 g, 0.418 mol) in DCM (1.35 l). The mixture was stirred for 24 hours. After quenching with EtOH (400 ml), the mixture was washed with 1.5 l of water. The aqueous layer was extracted twice with EtOAc (1 L) and the combined organic layers were washed with anhydrous Na 2 SO 4 Dried at rt, filtered and concentrated in vacuo. The crude product was purified by flash chromatography (PE / EtOAc 2:1) to give 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=6.40Hz, 1H), 5.21-5.10 (m, 1H), 4.94 (d, J=11.92Hz, 1H), 4.66-4.50 (m, 3H), 4.30 (d, J=4.77Hz , 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 bis-benzyl ether 3a (13.4 g, 21.4 mmol) in EtOH (100 ml) was degassed and purged with argon. catalytic amount of Pd(OH) 2 (20% on carbon) was added, then the solution was 2 Set for 1 hour under atmosphere. The Pd catalyst was filtered off and the filtrate 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, 1H), 4.02-3.95 (m, 1H), 3.94-3.89 (m, 1H), 3.88-3.77 (m, 3H), 3.76-3.67 (m, 1H) 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- Zeon Starting with 3b (8.69 g, 14.2 mmol), the title compound was prepared 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=8.0Hz, 1H), 5.28 (d, J=8.0Hz, 1H), 5.12 (t, J=4.0Hz, 1H), 5.00 (d, J=4.0Hz, 1H ), 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, 2 H), 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) was added N 2 BCl at -70°C under atmosphere 3 (921 mL) was added. The reaction solution was stirred between −75 and −60° C. for 2 hours at which point complete conversion was detected by TLC. NH in the mixture 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, 2H), 4.64 (s, 1H), 4.17 (d, J =4.0Hz, 1H), 3.89 (d, J =10.8Hz, 1H), 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-purine-6 -on Starting compound 3d (8.59g, 13.53mmol) was dissolved in DCM (200ml). After cooling from -70 to -50 °C, BCl 3 was added over 15 minutes. After stirring at -70°C for an additional 15 minutes, the cooling bath was removed and stirring was continued at room temperature for an additional 30 minutes. The reaction solution is then NH 3 was added dropwise to 180 ml of a 7M solution of in MeOH. After stirring for 20 min the mixture was evaporated and the residue dissolved in 100 ml DCM / iPrOH (4:1). saturated NaHCO 3 After washing with solution, the layers were separated and the aqueous layer was extracted three times with DCM / iPrOH (4:1). The combined organic layer was washed with MgSO 4 , 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 - : [M-H] - = 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, 1H), 5.01 - 5.14 (m, 2H), 4.64 - 4.76 (m, 1H), 4.15 - 4.20 (m, 1H), 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]purine-6 -yl]benzamide To a solution of compound 3e (31 g, 42.0 mmol) in DCM (620 ml) was added BCl 3 (252 mL, 0.252 mol) was added dropwise. N the mixture 2 Stirred at -70°C for 30 minutes and warmed to 15°C. After stirring for an additional 30 minutes at this temperature, the reaction mixture was treated with 7M NH 4 in MeOH while maintaining the temperature below -20°C. 3 (600ml) was carefully poured into. After evaporation, the residue was dissolved in 3 l DCM / iPrOH (4:1), 1 l water and saturated NaHCO 3 solution. The combined aqueous layers were extracted with 2 x 1 l of DCM / iPrOH (4:1) and the combined organic layers were washed with anhydrous Na 2 SO 4 Dried at rt, 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=10.67 Hz, 2 H), 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 Dissolved in 120 ml of O(4:1). NaIO 4 (1.69 g, 7.9 mmol) H 2 A solution in O (50 ml) was added and the mixture was stirred at room temperature for 1 hour. The precipitate was filtered off and the filtrate was concentrated in vacuo. The remaining aqueous solution was extracted with EtOAc (200ml). The organic layer is H 2 After washing with O and saturated NaCl solution, this was treated with MgSO 4 and the solvent was removed under reduced pressure to give 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 [minutes]=1.04 Ionization method: ES + :[M+H-H 2 O] + =443.2

[0401] 5b: 1-[(2R,6S)-3,5-dihydroxy-6-(hydroxymethyl)-6-(triisopropylsilyloxymethyl)-1,4-dioxan-2-yl]pyrimidine-2,4- Zeon Starting with 4b (6.21 g, 14.4 mmol), the title compound was prepared following the protocol described for 4a to give the desired product 5b (6.38 g, 99%) as a crude product, which was further purified. Used without purification. LCMS-Method B-1: UV-wavelength[nm]=220:R t [minutes]=1.00 Ionization method: ES + :[M+H-H 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 NaIO 4 A solution of (13.9g, 65mmol) in water (150ml) was added and the mixture was stirred for 12 hours. After evaporation of the solvent in vacuo, the residue was dissolved in EtOAc (500 ml) and saturated NaHCO 3 Washed with solution (500ml) and brine (500ml). The organic layer is Na 2 SO 4 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-purine-6 -on Starting material 4d (4.14 g, 9.1 mmol) was dissolved in acetone / H 2 Dissolved in 120 ml of O(4:1). NaIO 4 (2.63 g, 12.3 mmol) H 2 A solution in O (50ml) was added and the mixture was stirred at room temperature for 18 hours. The precipitate was filtered off and the filtrate was concentrated in vacuo. Add 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 washed with MgSO 4 and the solvent was removed under reduced pressure to give 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 [minutes]=1.82 Ionization method: ES + :[M+H-H 2 O] + =453.3

[0404] 6a: 1-[(2R,6S)-6-(hydroxymethyl)-6-(triisopropylsilyloxymethyl)morpholin-2-yl]-5-methyl-pyrimidine-2,4-dione To a solution of compound 5a (31 g, 67 mmol) in anhydrous MeOH (527 ml) was added N 2 (NH 4 ) 2 B. 2 O 7 4H 2 O (19.5 g, 74 mmol) was added. The mixture was stirred for 2 hours followed by AcOH (8.08 g, 134 mmol), 4 Å molecular sieves (62 g) and NaBH 3 CN (8.44g, 134mmol) was added. The mixture was quenched with N until TLC indicated complete consumption of the starting material. 2 The mixture was stirred at room temperature under atmosphere for 12 hours. The solvent was removed in vacuo and the residue dissolved in water (200ml) and extracted with DCM (3 x 200ml). Na 2 SO 4 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 Starting with compound 5c (25.7g, 46.4mmol), the title compound was synthesized following the protocol described for 6a to afford the desired morpholine 6c as a colorless solid (7.5g, 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) was added N 2 4 Å molecular sieves (39 g), acetone (13.25 g, 228 mmol) and NaBH at room temperature under atmosphere 3 CN (14.4 g, 228 mmol) was added. After stirring for 2 hours, the mixture was adjusted to pH=5-6 with AcOH (approximately 2.0 ml) and stirring continued for 12 hours, at which point complete consumption of starting material was detected by TLC. The solvent was removed in vacuo and the residue dissolved in water (500ml). After extraction with DCM (3 x 500 ml), the organic layers were combined and Na 2 SO 4 and concentrated in vacuo. After purification by silica gel chromatography (DCM / MeOH 50:1 to 10:1) the title compound 7a was obtained as a colorless solid (17g, 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 isopropylamine (1.34 ml, 15.5 mmol) were dissolved in dry MeOH (60 ml). At room temperature, 1.64 g (24.8 mmol) of sodium cyanoborohydride were added and the solution was stirred at 60° C. for 2 hours. After cooling to room temperature, the reaction mixture was treated 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 treated with saturated NaHCO 3 solution and saturated NaCl solution. MgSO 4 After drying at rt, 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%). Obtained 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, 3 H), 1.14-1.00 (m, 21 H), 0.97 ( d, J=6.53Hz, 6H).

[0408] 7b: 1-[(2R,6S)-6-(hydroxymethyl)-4-isopropyl-6-(triisopropylsilyloxymethyl)morpholin-2-yl]-pyrimidine-2,4-dione (from 5b) To a solution of 5b (6.38 g, 14.3 mmol) in MeOH (150 ml) at room temperature was added AcOH (8.62 g, 8.23 ​​ml, 142.8 mmol), 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 hour, 3.78 g (57.1 mmol) of sodium cyanoborohydride were added and stirring was continued at room temperature for 1 hour, followed by 65° C. for an additional hour. The mixture was cooled to room temperature and saturated NaHCO 3 150 ml of solution was added and the 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 rt, 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 (b s, 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, 1H), 4.62 (t, J=5.93 Hz, 1H), 3.99 (d, J=9.29 Hz, 1H), 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-purine-2 -yl]-2-methyl-propanamide (from 6c) The title compound was synthesized starting with 6c (7.5 g, 14.0 mmol) and following the protocol described for 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). NEt 3 (1.84 g, 2.53 ml, 18.1 mmol), AcOH (5.44 g, 5.19 ml, 90.6 mmol) and isopropylamine 808 mg (1.17 ml, 13.6 mmol) were added and the solution was stirred at room temperature for 1 hour. 1.80 g (27.2 mmol) of sodium cyanoborohydride 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. saturated NaHCO to the reaction mixture 3 100 ml of solution was added and the MeOH was evaporated. The remaining aqueous phase was extracted with EtOAc (250ml). NaHCO to saturate the organic layer 3 solution and NaCl solution, MgSO 4dried 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 (2.68g, 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, 1H), 4.57 (t, J=6.05Hz, 1H), 4.11 (d, J=9.05Hz, 1H), 3.77 (d, J=9.05Hz, 1H), 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, 27H).

[0411] [formation]

[0412] 1g: [(2S,3S,4R,5R)-4-acetoxy-3-benzyloxy-2-(benzyloxymethyl)-5-(6-chloropurin-9-yl)tetrahydrofuran-2-yl]methyl acetate Carbohydrate building blocks 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 (12.07g, 11.84ml, 77.7mmol) and TMSOTf (23.26g, 18.99ml, 103.6mmol) were added at 0°C. The ice bath was removed and the reaction was stirred at room temperature for 1 hour and at 80° C. for an additional hour at which point complete conversion was detected. Cool the solution to room temperature and add saturated NaHCO 3 Poured into 500 ml of solution. After stirring vigorously for 1 hour, the aqueous solution was extracted with EtOAc (300ml). Separate the organic layer and add saturated NaHCO 3 Solution / H 2 O (1:1) and saturated NaCl solution, washed with MgSO 4 dried and evaporated. The crude product (15.78 g) was purified on silica gel (10 to 100% EtOAc in n-heptane) to give 1 g (12.31 g, 81.8%) of the title compound as 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=12.0Hz, 1H), 3.69 (d, J=10.1Hz, 1H), 3.60 (d, J=10.1Hz, 1H), 2.05 (s, 3H), 1.99 (s, 3H).

[0413] 2h: (2R,3R,4S,5R)-2-(6-aminopurin-9-yl)-4-benzyloxy-5-(benzyloxymethyl)-5-(hydroxymethyl)tetrahydrofuran-3-ol 1 g 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 hours. The reaction mixture was evaporated and the aqueous residue was extracted three times with 150 ml of DCM / isopropanol (4:1). Combine the organic layers and wash with MgSO 4 Drying and evaporation gave 9.37g of crude product which was recrystallized from ACN (400ml) to give adenosine analogue 2h (6.92g, 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]purine- 6-yl]benzamide 2h (6.91 g, 14.5 mmol) was dissolved in 100 ml of pyridine under 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 hour. The solvent was removed in vacuo and the residue was 2 O and extracted with EtOAc. Separate the organic layer, 1N HCl (2×250 ml), H 2 O (1 x 200 ml), saturated NaHCO 3 solution (2×200 ml) and saturated NaCl solution (1×200 ml). MgSO 4 After drying at rt, the solvent was removed and the crude product (14.66 g, yellow foam) was dissolved in 140 ml 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 minutes. After adding AcOH (11 ml), the reaction solution was concentrated in vacuo. The residue is H 2 Treated with O (200ml) and extracted with EtOAc (200ml). The organic layer was washed with 1N HCl (2×200 ml), H 2 O (1 x 200 ml) and saturated NaHCO 3 solution (1 x 200 ml) and MgSO 4 dried and evaporated. 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. rice field. 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] [formation]

[0416] 8: [(2S,6R)-6-(2,4-dioxopyrimidin-1-yl)-4-isopropyl-2-(triisopropylsilyloxymethyl)-morpholin-2-yl]methylbenzoate Starting material 7b (6.00 g, 13.2 mmol) was dissolved in 80 ml dry pyridine. At room temperature 2.80 g (2.32 ml, 19.75 mmol) of benzoyl chloride was added and the reaction was stirred for 20 hours to give complete conversion. After evaporation of the solvent, the residue was dissolved in EtOAc and treated with 2×100 ml of 10% citric acid solution, H 2 O, saturated NaHCO 3 solution and NaCl solution. 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]methylbenzoate Starting compound 8 (7.99 g crude, 13.2 mmol) was dissolved in dry ACN (200 ml). NEt 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, then the solution was cooled to 0° C. and POCl 3 A solution of (6.06g, 3.68ml, 39.5mmol) in dry ACN (50ml) was added with vigorous stirring. The ice bath was removed and the solution was stirred for 2 hours. About 200 ml of solvent was evaporated under reduced pressure and the remaining solution was treated with saturated NaHCO 3 Solution / H 2 Poured into 500 ml of O(1:1). The aqueous mixture was extracted three times with DCM (150ml) and the combined organic extracts were washed with MgSO. 4 dried with After evaporation of the solvent, the crude intermediate (9.1 g, yellow foam) was dissolved in ACN (200 ml) and 100 ml aqueous ammonia solution (32%) was added. The reaction solution was stirred at room temperature for 18 hours, at which point complete conversion was achieved. The solvent is removed under reduced pressure and H 2 O (100ml) was added to the residue. Extract with DCM (2 x 200 ml) and extract the organic phase 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 - :[M-H] - =557.4

[0418] 10: [(2S,6R)-6-(4-benzamido-2-oxo-pyrimidin-1-yl)-4-isopropyl-2-(triisopropylsilyloxymethyl)morpholin-2-yl]methylbenzoate Starting compound 9 (7.64 g, 13.2 mmol) was dissolved in 130 ml 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 hours, at which point complete conversion was achieved. After evaporation of the solvent, the residue was dissolved in EtOAc and treated with 2×100 ml of 10% citric acid solution, H 2 O, saturated NaHOC 3 solution and NaCl solution. The organic layer was washed with MgSO 4 dried and evaporated to give 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 Obtained as a foam. LCMS-Method A: UV-wavelength[nm]=220:R t [minutes]=2.29 Ionization method: ES - :[M-H] - =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 pyridine / EtOH (2:1). At 0° C., 27.4 ml (27.4 mmol) of 1 M NaOH solution were added. The solution was stirred at 0° C. for 30 minutes and at room temperature for an additional 3 hours to achieve 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. 150 ml of 10% citric acid solution, H 2 O, saturated NaHCO 3 solution and NaCl solution three times. MgSO 4 After drying with , the solvent was evaporated. The crude product obtained (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 pale yellow foam. obtained as an object. 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=7.5Hz, 1H), 7.52 (m, 2H), 7.37 (d, J=7.5Hz, 1H), 5.86 (dd, J=9.5, 2.6Hz, 1H), 4.67 (t, J=5.9Hz, 1H), 4.06 (d, J=9.4Hz, 1H), 3.76 (d, J=9.3Hz, 1H), 3.51 (m, 2H), 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] [formation]

[0421] 11: [(2S,6R)-4-isopropyl-6-(6-oxo-1H-purin-9-yl)-2-(triisopropylsilyloxymethyl)morpholin-2-yl]methylbenzoate Starting material 7d (2.68g, 5.59mmol), DIPEA (2.53g, 3.42ml, 19.55mmol) and DMAP (0.34g, 2.79mmol) were dissolved in dry DCM (50ml). At room temperature, 1.18 g (0.97 ml, 8.38 mmol) of benzoyl chloride was added and the reaction was stirred for 18 hours resulting in complete conversion. After adding MeOH (20 ml), the solvent was evaporated, the residue was dissolved in EtOAc, 2×100 ml 10% citric acid solution, saturated NaHCO 3 solution and NaCl solution. The organic layer was washed with MgSO 4 Dried at rt and evaporated to give the title compound 11 (3.47 g, quantitative, 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-chloropurin-9-yl)-4-isopropyl-2-(triisopropylsilyloxymethyl)morpholin-2-yl]methylbenzoate Starting compound 11 (2.86 g crude, 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). While stirring, POCl 3 (1.70 g, 1.03 ml, 11.07 mmol) was added at room temperature and the solution was refluxed for 3 hours. After cooling to room temperature, the mixture was saturated NaHCO 3 Poured into 150 ml of solution, solid NaHCO 3 (3.0 g) was additionally added. The reaction was stirred for 30 minutes and extracted with DCM (100ml). The layers were separated and the aqueous phase was extracted twice with DCM (50ml). The combined organic layer was washed with MgSO 4 dried and evaporated. After purification 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]methylbenzoate 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 dioxane. Pd(OAc) 2 (43 mg, 191.8 μmol) and xantphos (111 mg, 191.8 μmol) were added and the reaction solution was stirred at 100° C. under argon for 1 hour to achieve complete conversion. The reaction was cooled to room temperature and diluted with EtOAc (50ml). The solution was filtered and the filtrate 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. Without chromatographic purification, the title compound 7e (731 mg, 86.2%, crude) could be isolated as a pale yellow foam. 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=7.3Hz, 1H), 7.55 (m, 2H), 6.11 (dd, J=9.9, 2.7Hz, 1H), 4.61 (t, J=5.9Hz, 1H) ), 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] [formation]

[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 (873mg, 1.20ml, 8.6mmol) was dissolved in DCM (30ml). At room temperature, DMT-Cl (716 mg, 2.1 mmol) was added and the reaction was stirred at room temperature for 2 hours. An additional 0.6 equivalents of DMT-Cl was added and after stirring overnight the reaction showed complete conversion. The crude product solution was directly transferred to 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 pale 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). 948mg (2.7mmol) of DMT-chloride was added and the reaction was stirred overnight at room temperature resulting in complete conversion. After adding the isolute-sorbent, the organic solvent was removed and the solid material was separated from silica (n-heptane + 0.5% NEt). 3 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)morpholine-2- yl]-6-oxo-1H-purin-2-yl]-2-methyl-propanamide Starting compound 7c (1.00 g, 1.77 mmol) was protected following the protocol described for 14a. After purification on silica gel (0 to 5% MeOH in DCM), the desired product 14c was isolated as a yellow foam (1.46g, 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)morpholine-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 adding a solution of DMT-Cl (733mg, 2.12mmol) in DCM (20ml), the reaction was stirred for 1.5h resulting in complete conversion. After evaporation of the solvent, the residue was dissolved in EtOAc and treated with H 2 O, twice with 10% citric acid solution and saturated NaHCO 3 solution. MgSO 4 After drying at room temperature, the solvent was removed and the crude product was purified on silica (n-heptane + 0.5% NEt 3 0 to 50% EtOAc in n-heptane) to give 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 - :[M-H] - =883.5

[0430] 14f: N-[1-[(2R,6S)-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-isopropyl-6-(triisopropylsilyloxymethyl)morpholine-2- yl]-2-oxo-pyrimidin-4-yl]benzamide Starting material 7f (2.25 g, 4.02 mmol) was protected in dry pyridine following the protocol for 14e. Silica (n-heptane + 0.5% NEt 3 0 to 80% EtOAc in n-heptane), the title compound 14f (3.35 g, 96.9%) was isolated as a yellow foam after a final chromatography. LCMS-Method A: UV-wavelength[nm]=220:R t [minutes]=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 TIPS-protected morpholine 14a (1.18 g, 1.5 mmol) and 6.74 g (9.26 ml, 66.0 mmol) of triethylamine were dissolved in THF (30 ml). At room temperature, NEt 3 ·3HF (12.23 g, 12.4 ml, 73.6 mmol) was added and the reaction was stirred at 65° C. for 1 hour. After standing overnight at room temperature, NMP (10ml) was added and the reaction was stirred at 65°C for an additional 14 hours resulting in complete conversion. The solvent was concentrated in vacuo and the remaining NMP solution was treated with H 2 O / saturated NaHCO 3 added to the solution mixture (1:2). The aqueous solution was extracted with EtOAc and the organic layer was 2 Washed 3 times with O and saturated NaCl solution. MgSO 4 After drying at room temperature, the crude product was chromatographed on silica gel (1% NEt in n-heptane 3 10 to 100% ethyl acetate in n-heptane) to give 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, 7H), 6.87 (d, J=8.80Hz, 4H), 5.82 (dd, J=9.66, 2.93Hz, 1H), 4.61 (br s, 1H), 3.67 - 3.79 (m, 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). NEt 3 (4.73g, 6.50ml, 46.3mmol) and NEt 3 After adding 3HF (8.41 g, 8.50 ml, 50.6 mmol), the mixture was stirred at room temperature for 15 hours, then at 65° C. for 5 hours. The mixture was cooled to room temperature followed by saturated NaHCO 3 250 ml of solution and H 2 O (250ml) was added. After extraction with EtOAc (250 ml), the organic layer was 2 Washed with O(2x) and saturated NaCl solution. MgSO for the organic phase 4 dried with silica (n-heptane + 0.5% NEt 3 0 to 100% EtOAc in n-heptane) to give 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 =9.05Hz, 1H), 2.82 (br d, J=10.27Hz, 1H), 2.60 - 2.76 (m, 2H), 2.10 - 2.22 (m, 2H), 0.94 (d, J=6.36Hz , 6H).

[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 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 65% pyridine.HF solution, the reaction mixture was stirred at room temperature for 1 hour. With vigorous stirring, NaHCO 3 (0.9g) and H 2 O (10ml) was added and stirring continued for 2 hours. THF was removed and the aqueous solution was extracted ten times with DCM / iPrOH (4:1). The combined organic layer was washed with MgSO 4 and evaporated to give 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 - : [M-H] - = 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, 7H), 6.85 (d, J=8.80Hz, 4H), 5.92 (dd, J=8.99, 3.00Hz, 1H), 4.64 (t, J=5.14Hz, 1H), 3.71 - 3.84 (m, 8H), 2.90 - 3.08 (m, 3H), 2.55 - 2.83 (m, 4H), 2.31 - 2.47 (m, 1H), 1.12 (dd, J= 6.79, 3.36Hz, 6H), 0.96 (d, J=6.48Hz, 6H).

[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). NEt 3 After adding 3HF (3.59 g, 3.63 ml, 21.60 mmol), the solution was heated at 65 °C followed by further NEt 3 (0.95g, 1.32ml, 9.45mmol) 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 hours). After cooling to room temperature, the mixture was 2 O / saturated NaHCO 3 Poured into 150 ml solution (1:1) and stirred for 30 minutes. The aqueous solution was extracted 3 times with 50DCM and MgSO 4 dried and evaporated. The deprotected product 15e (835 mg, 84.9%) was isolated as a colorless foam after silica gel chromatography (10 to 100% EtOAc in n-heptane). 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, 1H), 3.69 - 3.73 (m, 6H), 3.01 - 3.15 (m, 2H), 2.90 - 2.99 (m, 2H), 2.68 - 2.84 (m, 2H) 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 treated with NEt as described for 15e. 3 · Treated with 3HF. After a reaction time of 20 hours at 70° C., the conversion was complete. Workup similar to 15e and final purification gave 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, 2.69Hz, 1H), 4.66 (t, J=5.26Hz, 1H), 3.91 (dd, J=10.94, 4.22Hz, 1H), 3.71 - 3.77 (m, 7H) , 3.19 (d, J=9.05 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-pyrimidine- 1-yl)morpholin-2-yl]methoxy-(diisopropylamino)phosphanyl]oxypropanenitrile To a solution of 15a (624 mg, 1.0 mmol) and 401.0 mg (513 μl, 3.0 mmol) of N,N-diisopropylethylamine in dry DCM (15 ml) was added 370.9 mg (349.6 μl) of 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite. , 1.5 mmol) was added at room temperature under Ar atmosphere. After stirring for 1 hour, the reaction was quenched by adding 200 μl of n-butanol and stirring was continued for 10 minutes. H 2 Washed with O and the aqueous phase was extracted once with DCM. The organic layer was washed with MgSO 4 After drying at rt, the solvent was removed and the crude product was chromatographed on silica gel (0.5% NEt in n-heptane). 3 10 to 100% methyl-tert-butyl ether in n-heptane, preconditioned at rt) to give the desired phosphoramidite (mixture of diastereomers) 16a (668 mg, 80.8%) as a colorless foam. rice field. 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 x s, 1 H), 7.60, 7.56 (2 x s, 1 H), 7.41 (m, 2 H), 7.20 - 7.32 ( m, 7H), 6.83 - 6.90 (m, 4H), 5.88 (m, 1H), 3.84 - 4.05 (m, 2H), 3.74 (s, 6H), 3.54 - 3.73 (m, 2H) ), 3.40 - 3.54 (m, 2H), 3.14 (m, 1H), 2.95 - 3.05 (m, 1H), 2.57 - 2.84 (m, 5H), 2.15 - 2.37 (m, 2H), 1.76, 1.73 (2 x s, 3H), 0.93 - 1.14 (m, 18H). 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 according to the protocol described in 16a. Starting with primary alcohol 15b (1.95 g, 3.2 mmol), silica gel (n-heptane + 0.5% NEt 3 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 x d, 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 x d, 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, 1H), 2.84 (br d, J=10.15 Hz, 1H), 2.55 - 2.76 (m, 4H), 2.10 - 2.33 (m, 2H), 0.89 - 1.12 (m , 18H). 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 molecular sieves (4 Å) and 12.7 mg (70 μmol) 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 hours to achieve complete conversion. saturated NaHCO 3 After adding 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 4 dried and evaporated. Silica (DCM + 0.5% NEt 3 Purification on 0 to 10% MeOH in DCM) afforded 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 x s, 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, 2H), 3.39 - 3.50 (m, 2H), 3.11 (m, 1H), 2.92 - 3.07 (m, 2H), 2.54 - 2.81 (m, 6H), 2.26 - 2.38 (m, 1H), 0.94 - 1.16 (m, 18H), 0.83 - 0.91 (m, 6H). 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 (50ml) was added and the layers were separated. The aqueous layer was extracted once with DCM (30ml) and the combined organic phases were washed with MgSO. 4 dried with After evaporation of the solvent, the crude product was chromatographed on silica gel (n-heptane + 0.5% NEt 3Purification by 0 to 100% MTB-ether / DCM (1:1) in n-heptane, preconditioned at , gave 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 x s, 1 H), 8.61, 8.59 (2 x s, 1 H), 8.04 (d, J=8.09Hz, 2H), 7.64 (t, J=7.37Hz, 1H), 7.51 - 7.60 (m, 2H), 7.37 (m, 2H), 7.16 - 7.28 (m, 7H), 6.77 - 6.84 (m, 4H), 6.19 (m, 1H), 3.98 - 4.15 (m, 2H), 3.71 (2 x s, 6H), 3.56 - 3.69 (m, 2H), 3.41 - 3.56 (m, 2H), 3.03 - 3.17 (m, 2H), 2.89 - 3.01 (m, 2H), 2.74 - 2.88 (m, 2H), 2.70 (t, J=6.15Hz, 1H), 2.59 (m, 1H), 2.36 (m, 1H), 0.92 - 1.18 (m, 18H). 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 Primary alcohol 15f (550 mg, 0.78 mmol) was phosphitylated following the protocol described for 16e. Silica gel (n-heptane + 0.5% NEt 3 16f (560 mg, 79.3%) was isolated as a colorless foam after chromatographic purification on 0 to 100% MTB-ether / DCM (1:1) in n-heptane, preconditioned at . 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.29 (br s, 1 H), 8.12 (2 x d, 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 x d, J=8.89 , 4 H), 5.95, 6.01 (2 x m, 1 H), 3.89 - 4.14 (m, 2 H), 3.74, 3.75 (2 x s, 6 H), 3.55 - 3.72 (m, 2 H), 3.39 - 3.55 (m, 2H), 3.24 (d, J=8.85Hz, 1H), 2.93 - 3.05 (m, 2H), 2.68 - 2.87 (m, 3H), 2.55 - 2.64 (m, 1H), 2.10 - 2.23 (m, 2H), 0.90 - 1.22 (m, 18H). 31P-NMR (DMSO-d6, 162 MHz) δ[ppm]: 147,4, 146,7.

[0441] [formation]

[0442] 17a: [(2S,6R)-4-isopropyl-6-(5-methyl-2,4-dioxo-pyrimidin-1-yl)-2-(triisopropylsilyloxymethyl)morpholin-2-yl]methylbenzoate Free alcohol 7a (810 mg, 1.7 mmol) was dissolved in DCM (20 ml). After adding 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 hours. For complete conversion, another 0.2 eq of benzoyl chloride was added and stirring was continued for 18 hours. The reaction solution was directly transferred to a silica gel column eluting with 0-65% EtOAc in n-heptane. Benzoylated product 17a (726 mg, 73.3%) was obtained as a colorless foam. LCMS-Method A: UV-wavelength[nm]=220:R t [minutes]=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]methylbenzoate Primary alcohol 7b (1.45 g, 3.18 mmol) was treated with benzoyl chloride as described for 17a to give a mixture of mono- and dibenzylated products. Therefore the reaction mixture was evaporated and the crude product dissolved in 30 ml 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 hours. After evaporation of the solvent, the residue was dissolved in EtOAc and washed with 10% citric acid solution and saturated NaCl solution. The organic layer was washed with MgSO 4 and the solvent was removed. A final chromatography on silica (0 to 35% EtOAc in n-heptane) gave 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)morpholine -2-yl]methyl benzoate Starting compound 7c (0.89 g, 1.58 mmol) was dissolved in 30 ml pyridine. After adding 246 mg (203 μl, 1.73 mmol) of benzoyl chloride, the reaction solution was stirred overnight at room temperature, at which point complete conversion was detected. The solvent was removed in vacuo and the residue dissolved in EtOAc. H. 2 After washing with O, the organic phase is separated and MgSO 4 dried and evaporated. Final purification on silica (0 to 5% MeOH in DCM) gave 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]methylbenzoate A solution of 17a (720 mg, 1.25 mmol) in THF (15 ml) was treated with pyridine-hydrogen fluoride 1.34 g (1.22 ml, 8.8 mmol). After stirring for 1 hour at room temperature, the reaction mixture was treated with saturated NaHCO 3 solution to bring the pH to 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 4 and the solvent was removed. The crude product was codistilled 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 colorless. Obtained as a foam. LCMS-Method A: UV-wavelength[nm]=220:R t [minutes]=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]methylbenzoate The silyl ether 17b (1.64 g, 2.47 mmol) was desilylated according to the protocol described for 18a to give the desired product 18b (1.33 g) as a crude product, which was used without chromatographic purification as follows. used for steps. LCMS-Method C: UV-wavelength[nm]=220:R t [minutes]=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]morpholine-2- yl]methyl benzoate 17c (1.0 g, 1.49 mmol) was dissolved in DMF (10 ml). NEt 3 (1.53g, 2.10ml, 15.0mmol) 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 hours. After cooling the mixture to room temperature, NaHCO 3 (2.5g) and H 2 O (10 ml) was added and the reaction mixture was stirred for 2 hours. The solvent was evaporated in vacuo and the residue dissolved in DCM / iPrOH (25ml) and 2 Washed with O. Separate the organic phase and add MgSO 4 dried and evaporated. The crude product was purified on silica (0-10% 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]-methylbenzoate Silyl ether 13 (1.23 g, 1.79 mmol) was deprotected according to the protocol described for 18a to give the title compound 18e (939 mg, 98.8%) after silica gel chromatography (0 to 100% EtOAc in n-heptane). rice field. LCMS-Method C: UV-wavelength[nm]=220:R t [minutes]=1.60 Ionization method: ES - :[M-H] - =529.3

[0449] 18f: [(2R,6R)-6-(4-benzamido-2-oxo-pyrimidin-1-yl)-2-(hydroxymethyl)-4-isopropyl-morpholin-2-yl]methylbenzoate Silyl ether 10 (3.63 g, 5.48 mmol) was deprotected following the protocol described for 18a 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 [minutes]=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]methylbenzoate Starting compound 18a (497mg, 1.2mmol) was dissolved in DCM (15ml). 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 hours. The reaction mixture was evaporated in vacuo and the crude product was purified on silica gel (0-100% EtOAc in n-heptane). The DMT protected product 19a was isolated as a colorless foam (779mg, 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]methylbenzoate Primary alcohol 18b (1.30 g, 2.41 mmol) and NEt 3 (492mg, 676[mu]l, 4.82mmol) was dissolved in DCM (20ml). After adding DMT-Cl (925 mg, 2.65 mmol), the reaction was stirred at room temperature for 6 hours followed by addition of more DMT-Cl (925 mg, 2.65 mmol). The reaction was stirred for 72 hours. After adding 2.0 ml of n-propanol, the solution was stirred for 10 minutes. H the mixture 2 Wash with O and separate the organic layer. MgSO 4 After drying with , 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]methylbenzoate Starting alcohol 18c (240 mg, 468 μmol) was dissolved in DCM (5.0 ml). NEt 3 (95.7 mg, 131.5 μl, 936 μmol) and DMT-Cl (180 mg, 515 μmol) were added and the reaction was stirred at room temperature for 3 days to achieve complete conversion. After adding 2.0 ml of n-propanol, the solution was stirred for 10 minutes. H the mixture 2 Wash with O and separate the organic layer. MgSO 4 After drying with , 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 - :[M-H] - =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 hours. The solvent was removed in vacuo and the residue was dissolved in EtOAc and treated with H 2 O, 2 x 10% citric acid solution, saturated NaHCO 3 solution and NaCl solution. MgSO for the organic phase 4 dried and evaporated. The crude product obtained (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 afford the desired DMT-ether 19e as a colorless solid. LCMS-Method C: UV-wavelength[nm]=220:R t [minutes]=3.07 Ionization method: ES - :[M-H] - =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]methylbenzoate 18f (2.45 g, 4.84 mmol) was dissolved in 40 ml 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 treated with H 2 O, 2 x 10% citric acid solution, saturated NaHCO 3 solution and NaCl solution. MgSO for the organic phase 4 dried and evaporated. The crude product was filtered through silica gel (n-heptane + 0.5% NEt 3 0 to 100% EtOAc in n-heptane) to give 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 - :[M-H] - =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 NaOH aqueous solution was added and the reaction was stirred for 90 minutes. The pH was adjusted between 7 and 8 by adding citric acid monohydrate, then the solvent was removed under reduced pressure. DCM and H 2 Dissolved in O. The organic layer was separated and the aqueous phase was extracted with DCM. The combined organic phase was washed with MgSO 4dried 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, 7H), 6.88 (d, J=8.68Hz, 4H), 5.60 (dd, J=9.96, 2.87Hz, 1H), 4.64 (t, J=5.93Hz, 1H) , 3.74 (m, 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, 3H), 0.84 - 0.96 (m, 6H).

[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 0.5M solution of NaOMe in MeOH was added and the reaction solution was stirred for 2 hours to give complete conversion. After adding 0.92 g of citric acid, the solvent was removed. The residue is H 2 O and the aqueous mixture was extracted three times with DCM / iPrOH (4:1). The combined organic layer was washed with MgSO 4 and evaporated to give the desired compound 20b (0.85g, 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, 8H), 6.88 (d, J=8.31Hz, 4H), 5.55 - 5.64 (m, 2H), 4.66 (t, J=5.93Hz, 1H), 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, 3H), 2.32 - 2.39 (m, 1H), 1.95 - 2.13 (m, 1H), 0.86 - 0.98 (m, 6H).

[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 1 M NaOH solution, the solution was stirred at room temperature for 4 hours to achieve complete conversion. The solution was neutralized with 1M HCl and the solvent was removed in vacuo. The residue is H 2 Dissolved in O and extracted with DCM. Separate the organic phase and add MgSO 4 dried and evaporated. Final purification on silica (0-10% MeOH in DCM) gave 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 - : [M-H] - = 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, 7H), 6.83 - 6.92 (m, 4H), 5.56 (dd, J=9.60, 2.75Hz, 1H), 4.59 - 4.66 (m, 1H), 3.73 (2 x s, 6H), 3.51 - 3.62 (m, 2H), 3.43 - 3.50 (m, 1H), 2.88 - 3.02 (m, 3H), 2.70 - 2.82 (m, 2H), 2.30 - 2.39 (m, 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 were added and the reaction mixture was stirred at room temperature for 1.5 hours. After adjusting the pH to 7 by adding citric acid monohydrate (958 mg), H 2 O (70ml) and EtOAc were added. Separate the organic layer, add 10% citric acid solution (3×), HO 2 O, saturated NaHCO 3 solution and NaCl solution. MgSO 4 After drying at rt, 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 afford 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 - : [M-H] - = 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, 1H), 7.56 (t, J=7.58Hz, 2H), 7.45 (d, J=7.46Hz, 2H), 7.28 - 7.36 (m, 6H), 7.20 - 7.28 (m, 1H), 6.90 (dd, J=8.99, 2.26Hz, 4H), 5.89 (dd, J=9.84, 2.63Hz, 1H), 4.69 (t, J=5.99Hz, 1H) ), 3.75 (s, 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, 1H), 2.60 - 2.78 (m, 2H), 2.45 - 2.55 (m, 1H), 0.98 (br d, J=6.48 Hz, 3H), 0.95 (br d, J=6.48 Hz, 3H).

[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 following the procedure described for 20e. The crude product was purified by silica gel chromatography (20 to 100% EtOAc in n-heptane) to afford 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 - : [M-H] - = 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=7.40Hz, 1H), 7.51 (t, J=7.70Hz, 2H), 7.20 - 7.41 (m, 10H), 6.88 (dd, J=8.99, 2.38Hz, 4H) , 5.68 (dd, J=9.35, 2.38 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, 1H), 3.35 - 3.43 (m, 1H), 3.11 (d, J=8.56Hz, 1H), 2.79 - 2.95 (m, 2H), 2.66 - 2.76 (m, 1H), 2.39 - 2.47 (m, 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-pyrimidine- 1-yl)morpholin-2-yl]methoxy-(diisopropylamino)phosphanyl]-oxypropanenitrile Starting material 20a (764 mg, 1.2 mmol) was converted to the desired phosphoramidite 21a following the protocol for 16a (Scheme 5). 842 mg (83.2%, mixture of diastereomers) were obtained as a colorless foam. 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.34, 11.32 (2 x s, 1 H), 7.59, 7.54 (2 x d, J=1.10 Hz, 1 H), 7.36 - 7.43 (m, 2 H), 7.20 - 7.34 (m, 7H), 6.89 (m, 4H), 5.59 - 5.75 (m, 1H), 3.44 - 3.95 (m, 6H), 3.74 (s, 6H), 3.36 (m, 1H), 3.22 (m, 1H), 2.60 - 2.85 (m, 5H), 2.36 (m, 1H), 2.18 (m, 1H), 1.78 (2 x d, J=8.69Hz , 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 following the protocol for 16b (Scheme 5) starting with the corresponding alcohol 20b (1.36 g, 2.26 mmol). 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 + : [M-N 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 x s, J= 8.13 Hz, 1 H), 7.35 - 7.42 (m, 2 H) , 7.20 - 7.33 (m, 7H), 6.83 - 6.92 (m, 4H), 5.60 - 5.70 (m, 2H), 3.73 (s, 6H), 3.43 - 3.95 (m, 6H), 3.29 - 3.39 (m, 1H), 3.22 (m, 1H), 2.59 - 2.83 (m, 5H), 2.33 (m, 1H), 2.13 (m, 1H), 0.84 - 1.21 (m, 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 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 ), 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 + : [M-N i PR 2 +OH+H] + = 828,2 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.70 - 12.30 (b s, 1 H), 11.53 (b s, 1 H), 8.05 (s, 1 H), 7.35 - 7.45 (m, 2 H ), 7.20 - 7.33 (m, 7H), 6.82 - 6.91 (m, 4H), 5.54 - 5.68 (m, 1H), 3.80 - 3.93 (m, 0.5H), 3.73, 3.72 (2 x s, 6 H), 3.68 - 3.76 (m, 1.5H), 3.60 - 3.67 (m, 2H), 3.35 - 3.60 (m, 4H), 3.12 (m, 1H), 2.57 - 2.98 (m, 7H) , 0.92 - 1.13 (m, 24H). 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 Starting with 20e (950 mg, 1.30 mmol), the title compound was prepared following the protocol described for 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 x s, 1 H), 8.63, 8.59 (2 x s, 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, 7H), 6.86 - 6.93 (m, 4H), 5.89 - 5.99 (m, 1H), 3.80 - 4.15 (m, 1H), 3.74 (s, 6H), 3.63 - 3.72 (m, 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 Starting with 20f (620 mg, 0.88 mmol), the title compound was prepared following the protocol described for 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 x d, J= 7.47 Hz, 1 H), 8.01 (d, J=7.65 Hz, 2H), 7.58 - 7.67 (m, 1H), 7.51 (t, J=7.58Hz, 2H), 7.35 - 7.42 (m, 3H), 7.20 - 7.35 (m, 7H), 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, 6 H), 3.74 (s, 6 H), 3.38 (m, 1 H ), 3.24 - 3.33 (m, 1H), 2.60 - 2.94 (m, 5H), 2.33 - 2.48 (m, 1H), 2.01 - 2.09 (m, 1H), 1.02 - 1.23 (m, 12H) ), 0.83 - 1.00 (m, 6H). 31P-NMR (DMSO-d6, 162 MHz) δ[ppm]: 147,5, 147,4.

[0465] [formation]

[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 To a solution of starting compound 4a (23.7 g, 53.4 mmol) in 240 ml of anhydrous pyridine was added N 2 DMT-Cl (23.13 g, 80 mmol) was added at room temperature under atmosphere. After stirring for 12 hours the solvent was removed in vacuo and the residue was purified by silica gel column chromatography (PE / EtOAc 10:1 to 1:1) to give the desired DMT protected product 22a (16.3 g, 41%). was obtained 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 To a solution of compound 4b (75 g, 174 mmol) in 750 ml of anhydrous pyridine, N 2 DMT-Cl (70.8 g, 209 mmol) was added at room temperature under atmosphere. The mixture was stirred for 12 hours to achieve complete conversion. The solvent was removed in vacuo and the residue was diluted with 500ml water and extracted with EtOAc (3 x 500ml). The organic layer was washed with brine and dried over anhydrous Na 2 SO 4 Dried at rt, filtered and concentrated in vacuo. The resulting crude product was purified by flash chromatography on silica gel (PE / EtOAc 1:1 to EtOAc / MeOH 20:1) to afford 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, 1H), 5.27 (d, J=8.03 Hz, 1H), 5.21 (d, J=5.27 Hz, 1H), 4.33-4.26 (m, 1H), 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-(triisopropylsilyloxy Methyl)tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]-2-methyl-propanamide 4c (55.8 g, 103 mmol) was protected with DMT-Cl in pyridine following the protocol described for 22b 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-(triisopropylsilyloxy Methyl)tetrahydrofuran-2-yl]purin-6-yl]benzamide 4e (57.0 g, 102 mmol) was protected with DMT-Cl in pyridine following the protocol described for 22b to afford 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, 1.38Hz, 4H), 6.00 (d, J=7.28Hz, 1H), 5.53 (d, J=6.78Hz, 1H), 5.32 (d, J=4.89Hz, 1H ), 5.00-4.87 (m, 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 Acetone / H of starting material 22a (12.6 g, 16.9 mmol) 2 O(3:1) solution in 250 ml NaIO 4 (5.05 g, 23.6 mmol) H 2 An aqueous solution in O (60ml) was added dropwise. The mixture was stirred for 12 hours for complete conversion and poured into 500 ml of ice water. The mixture was extracted with DCM (500ml) three times. The organic layers were combined, washed with brine and dried over anhydrous Na 2 SO 4 , filtered and concentrated in vacuo to give 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 The title compound 23b (80 g, quantitative, crude) was obtained as a yellow solid from diol 22b (77.0 g, 105 mmol) following the protocol described for 23a. 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, 2H), 7.39-7.20 (m, 8H), 6.94-6.81 (m, 4H), 5.94-5.87 (m, 1H), 5.78-5.63 (m, 1H), 5.59-5.51 ( m, 1 H), 5.25-5.08 (m, 1 H), 5.07-4.99 (m, 1 H), 4.32-4.20 (m, 1 H), 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 The title compound 23c (47 g, 96.1%, crude) was obtained as a yellow solid from diol 22c (50.0 g, 59 mmol) following the protocol described for 23a. 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, 1H), 1.22-0.76 (m, 29H).

[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 The title compound 23e (75.2 g, 94.6%, crude) was obtained as a white solid from diol 22e (78.0 g, 90.6 mmol) following the protocol described for 23a. 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 To a solution of starting compound 23a (2.6 g, 3.4 mmol) in anhydrous MeOH (44 ml) was added N 2 (NH 4 ) 2 B. 4 O 7 4H 2 O (0.98 g, 3.7 mmol) was added. The mixture was stirred for 2 h followed by AcOH (0.45 g, 6.8 mmol), 5.0 g 4 Å molecular sieves and NaBH. 3 CN (0.47 g, 6.8 mmol) was added. After stirring for 12 hours at room temperature, TLC indicated complete consumption of the starting material. 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=9.54Hz, 1H), 3.96 (br d, J=9.54Hz, 1H), 3.72 (d, J=0.75Hz, 6H), 3.06 (br d, J=9.29Hz, 1H), 2.97 (br d, J=9.03Hz, 1H), 2.93-2.78 (m, 2H), 2.76-2.57 (m, 3H), 1.76 (s, 3H), 0.98-0.87 (m, 22H) ).

[0475] 24b: 1-[(2R,6S)-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-(triisopropylsilyloxymethyl)morpholin-2-yl]pyrimidine-2,4 - Zeon To a solution of compound 23b (5.0 g, 6.7 mmol) in anhydrous MeOH (85 ml) was added N 2 (NH 4 ) 2 B. 4 O 7 4H 2 O (1.93 g, 7.6 mmol) was added. The mixture was stirred for 2 h followed by 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 hours, the mixture was filtered and the filtrate was concentrated in vacuo. The residue was diluted with ice water and extracted with EtOAc (50 ml) three times. The organic layers were combined, washed with brine and dried over anhydrous Na 2 SO 4 Dried at rt, 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, 7H), 6.89 (d, J=8.80Hz, 4H), 5.88 (br dd, J=10.03, 2.45Hz, 1H), 5.73 (d, J=8.07Hz, 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 for 24b, starting material 23c (7.0 g, 8.2 mmol) was converted to morpholine compound 24c, which was isolated as a colorless foam after silica gel chromatography (PE / EtOAc 5:1 to 1:1). away (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, 1H), 3.92 (br d, J=9.03 Hz, 1H), 3.72 (d, J=2.76 Hz, 6H), 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, 22H).

[0477] 24e: N-[9-[(2R,6S)-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-(triisopropylsilyloxymethyl)morpholin-2-yl]purine- 6-yl]benzamide Following the protocol described for 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=8.93Hz, 4H), 6.16 (dd, J=10.03, 2.81Hz, 1H), 4.26 (d, J=9.66Hz, 1H), 4.10-3.98 (m, 1H), 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] [formation]

[0479] 25: tert-butyl(2S,6R)-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-(2,4-dioxo-pyrimidin-1-yl)-2-(tri Isopropylsilyloxymethyl)morpholine-4-carboxylate Boc 2 O (22.9 g, 104.8 mol) was added dropwise. After stirring for 24 hours, 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-triazole-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 with 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 minutes. At the same temperature, starting material 25 (32 g, 39.21 mmol) was added to the mixture in one portion, the ice bath was removed and the reaction was stirred at 30° C. for 3 hours. The mixture was poured into 3 l of 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, then add anhydrous Na 2 SO 4 Dried at rt, filtered and concentrated in vacuo to give crude compound 26 (40 g) as a yellow oil, which was used in the next step without further purification. MS(m / z)=867.5[M+H] +

[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 dioxane at 0° C. was added portionwise NaH (12.8 g, 0.31 mol). After stirring for 30 min at 30° C., triazole 26 (40 g, 39.21 mmol, crude) was added in one portion and the mixture was stirred at 30° C. for 1 h. Saturated NH 4 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, dried over anhydrous Na 2 SO 4 Dried at rt, filtered and concentrated in vacuo to give 54 g of crude compound as a yellow oil. A final silica gel chromatography (PE / EtOAc 2:1) gave 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, 1H), 7.58-7.48 (m, 2H), 7.47-7.36 (m, 3H), 7.34-7.16 (m, 7H), 6.87 (br d, J= 7.70 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 (13.5g, 14.70mmol) in DCM (93ml) at 0°C was added TFA (31ml) dropwise. The reaction was stirred at 30° C. for 16 hours. saturated NaHCO 3 After neutralizing 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 layer was washed with anhydrous Na 2 SO 4 Dried at rt, 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% ) was obtained as a by-product, which was dissolved in a mixed solvent of THF (186 ml) and EtOH (62 ml). At 0°C, 75.8 ml of 1M NaOH aqueous solution was added. After stirring for 2 minutes, the solution was neutralized with saturated citric acid solution and treated with H 2 Diluted with O (300ml) and extracted with EtOAc (300ml). The organic layer was washed with brine and Na 2 SO 4 dried 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) at 0° C. was added portionwise 5.5 g (16.26 mmol) of Fmoc-N-hydroxy-succinimide ester. After stirring for 16 hours, the solution was poured into 100 ml water and extracted three times with EtOAc (30 ml). The combined organic layers were washed with brine and dried over anhydrous Na 2 SO 4 Dried at rt, filtered and concentrated in vacuo. Final purification on silica (PE / EtOAc 1:2) gave 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, 2H), 7.64 (br t, J=7.40 Hz, 3H), 7.57-7.49 (m, 2H), 7.48-7.40 (m, 3H), 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 pyridine at 25°C was added DMT-Cl (12.75 g, 37.62 mmol). The mixture was stirred at 25° C. for 16 hours to achieve complete conversion. After evaporation of the solvent, the residue was dissolved in EtOAc (200ml) and washed with 100ml water and brine. Na 2 SO 4 , 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 (8.4g, 8.07mmol) in DCM (85ml) at room temperature was added 17ml of piperidine. After complete conversion (approximately 20 minutes), the reaction solution was quenched with saturated NH 4 Washed with Cl solution. The aqueous phase was extracted with DCM and the combined organic layers were washed with Na 2 SO 4 , 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] [formation]

[0487] General Procedure A for Reductive Amination Reactions to Prepare Compounds 31a-31f Morpholine 24a (1.0 g, 1.4 mmol) was dissolved in MeOH (25 ml) followed by molecular sieves (4 Å) 2.0 g, NEt 3 (560 mg, 769 μl, 5.5 mmol), AcOH (827 mg, 789 μl, 13.7 mmol) and 1.0 to 3.0 equivalents of the corresponding aldehyde or ketone were added. After stirring for 15 minutes, 344 mg (5.5 mmol) of sodium cyanoborohydride were added in 4 portions every 30 minutes and the reaction was stirred at room temperature for 6 hours. After standing overnight, the mixture was filtered and the filtrate was washed with saturated NaHCO 3 Diluted with 25 ml of solution. The MeOH was evaporated and the aqueous phase was extracted with 50 ml DCM / iPrOH (5:1). After further filtration, the organic phase is separated and washed with MgSO. 4 dried and evaporated. The crude product obtained was used for 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 The title compound 31a (1.0 g) was isolated as a crude product following General Procedure A using 3.0 equivalents of formaldehyde (37% in water) and used without further purification. LCMS-Method A: UV-wavelength[nm]=220:R t [minutes]=2.29 Ionization method: ES + :[M+H] + =744.3

[0489] 31b: 1-[(2R,6S)-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-butyl-6-(triisopropylsilyloxymethyl)morpholin-2-yl]- 5-methyl-pyrimidine-2,4-dione The title compound 31b (1.19 g) was isolated as a crude product following General Procedure A using 2.0 equivalents of butyraldehyde and used without further purification. LCMS-Method A: UV-wavelength[nm]=220:R t [minutes]=2.41 Ionization method: ES + :[M+H] + =786.7

[0490] 31c: 1-[(2R,6S)-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-octyl-6-(triisopropylsilyloxymethyl)morpholin-2-yl]- 5-methyl-pyrimidine-2,4-dione Following General Procedure A, using 2.0 equivalents of octanal, the title compound 31c (1.35 g) was isolated as a crude product and used without further purification. LCMS-Method A: UV-wavelength[nm]=220:R t [minutes]=2.66 Ionization method: ES + :[M+H] + =842.7

[0491] 31d: 1-[(2R,6S)-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-hexadecyl-6-(triisopropylsilyloxymethyl)morpholin-2-yl]- 5-methyl-pyrimidine-2,4-dione The title compound 31d (1.58 g) was isolated as a crude product following General Procedure A using 1.0 equivalent of hexadecanal and used without further purification.

[0492] 31e: 1-[(2R,6S)-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-cyclohexyl-6-(triisopropylsilyloxymethyl)morpholin-2-yl]- 5-methyl-pyrimidine-2,4-dione Following General Procedure A using 1.25 equivalents of cyclohexanone, the title compound 31e (1.14 g) was isolated as a crude product and used without further purification. LCMS-Method A: UV-wavelength[nm]=220:R t [minutes]=2.45 Ionization method: ES + :[M+H] + =812.5

[0493] 31f: 1-[(2R,6S)-4-benzyl-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-(triisopropylsilyloxymethyl)morpholin-2-yl]- 5-methyl-pyrimidine-2,4-dione Following General Procedure A, using 1.25 equivalents of benzaldehyde, the title compound 31f (1.12 g) was isolated as a crude product and used without further purification. LCMS-Method A: UV-wavelength[nm]=220:R t [minutes]=2.37 Ionization method: ES + :[M+H] + =821.4

[0494] 31g: 1-[(2R,6S)-4-acetyl-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-(triisopropylsilyloxymethyl)morpholin-2-yl]- 5-methyl-pyrimidine-2,4-dione Starting compound 24a (1.0 g, 1.4 mmol) was dissolved in DCM (25 ml). After addition of AcOH (103 mg, 99 μl, 1.7 mmol), HBTU (779 mg, 2.1 mmol) and 903 mg (1.19 ml, 6.9 mmol) of diisopropyl-ethylamine, the reaction mixture was stirred at room temperature for 4 hours to achieve complete conversion. . H 2 Washed with O (25ml) and separated the organic layer. MgSO 4 After drying at rt, the solvent was evaporated and the crude product was purified by silica gel chromatography (0 to 5% MeOH in DCM) to give 31g (1.06g, quantitative) of the title compound. LCMS-Method A: UV-wavelength[nm]=220:R t [minutes]=2.20 Ionization method: ES - :[M-H] - =770.2

[0495] 31h: 1-[(2R,6S)-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-hexadecanoyl-6-(triisopropylsilyloxymethyl)morpholin-2-yl ]-5-methyl-pyrimidine-2,4-dione Following the protocol for 31g, starting compound 24a (1.0 g, 1.4 mmol) and palmitic acid were converted to title compound 31h (1.14 g, 86%) after silica gel chromatography (0 to 20% MeOH in DCM).

[0496] General Procedure B for the Desilylation Reaction to Prepare Compounds 32a-32h Crude products 31a-31h were dissolved in NMP (12 ml). NEt 3 (2.1g, 2.9ml, 20.6mmol) and NEt 3 · After adding 3HF (1.14 g, 1.15 ml, 6.9 mmol), the reaction mixture was stirred at 90° C. for 2 hours. After cooling to room temperature, the solution was treated with saturated NaHCO 3 Poured into solution and extracted twice with EtOAc. The organic layer was washed with MgSO 4 dried and evaporated. Acetonitrile / H 2 It was dissolved in O and lyophilized. The resulting crude products were purified by silica gel chromatography to give the desired compounds 32a-32h.

[0497] 32a: 1-[(2R,6R)-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-(hydroxymethyl)-4-methyl-morpholin-2-yl]-5- methyl-pyrimidine-2,4-dione Starting with crude product 31a and following general procedure B, final silica gel purification (0 to 5% MeOH in DCM) gave the title compound 32a (543 mg, 66.0%, 2 steps). LCMS-Method A: UV-wavelength [nm] = 220: R t [minutes] = 1,59 Ionization method: ES + : [M+H] + = 588,3 1H-NMR (DMSO-d6, 400 MHz) δ[ppm]: 11.37 (s, 1 H), 7.52 (s, 1 H), 7.40 (d, J=7.46 Hz, 2 H), 7.19 - 7.32 ...

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: [Formula 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): [Case 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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