A novel method for preparing nucleotide P(V) monomers.

Abstract

The present invention relates to an optimized process for preparing P(V) monomers of formula (IIIa) or (IIIb) from nucleosides using DBU as a base in an amount of about 0.8 equivalents relative to the nucleoside. TIFF2022529342000052.tif59128

Description

[Technical field]

[0001] The present invention relates to a novel method for preparing nucleotide P(V) monomers. [Background technology]

[0002] Nucleotide monomers are building blocks in the synthesis of oligonucleotides. Locked nucleic acid (LNA) oligonucleotides are synthetic oligonucleotides that are particularly useful as therapeutic synthetic antisense oligonucleotides. The use of LNA oligonucleotides as therapeutic agents has seen significant progress over the last few decades, leading to the development of molecules that act by diverse mechanisms, including gapmers, splice-switching oligonucleotides, microRNA inhibitors, siRNAs or aptamers that activate RNase H (ST Crooke, Antisense drug technology: principles, strategies, and applications, 2nd ed. Boca Raton, FL: CRC Press, 2008). In this regard, phosphorothioate oligonucleotides, meaning that at least some internucleotide linkages are phosphorothioates instead of natural phosphodiesters, have shown to be of particular pharmacological interest.

[0003] One consequence of using nucleotide monomers that are not chirally defined around phosphorus is that each phosphorothioate linkage can be found in both the R and S configurations (i.e., stereorandom phosphorothioate linkages). This results in a particular phosphorothioate antisense oligonucleotide containing many different diastereoisomers within the same sequence. For example, in the case of a phosphorothioate oligonucleotide of a given set sequence of 16 nucleotides in length, up to 2 15There are different diastereoisomers.This results in more than 32,000 potentially pharmacologically distinct compounds for the same set sequence of 16 oligonucleotides.Therefore, it is highly desirable to identify, synthesize and / or isolate pharmacologically optimal compound or set of pharmacologically optimal compounds from such mixtures.In fact, it can be reasonably estimated that such optimal compound will have even higher therapeutic potential than standard stereorandom phosphorothioate oligonucleotides.

[0004] One of the key steps in synthesizing such phosphorothioate oligonucleotides is the preparation of their building blocks, i.e., their monomers, which can advantageously be performed stereoselectively to direct the synthesis of antisense oligonucleotides toward a preferred set of diastereoisomers or even a single diastereoisomer.

[0005] Traditionally, the synthesis of monomers for phosphorothioate oligonucleotides is carried out with a phosphorus / [P(III)]-based reagent system. Recently, Baran et al., Science, 2018, 361, pp. 1234-1238 (Non-Patent Document 2) presented a different approach using a phosphorus / [P(V)]-based reagent system coined "platform for programmable, traceless, diastereoselective phosphorus-sulfur incorporation". Baran et al. further propose that "the power of this reagent system is demonstrated by the robust and stereocontrolled synthesis of various nucleotide structures, including ASOs and CDNs, through an efficient, inexpensive, and easy-to-operate protocol."

[0006] Unfortunately, Baran et al. are unable to guide us in the preparation of the desired LNA nucleotide P(V) monomer, but only in the preparation of DNA nucleotide P(V) monomer. After an extensive review of many parameters, reagents and method conditions, among many other recommendations, Baran et al. teach the use of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as a base in an amount of 1.3 equivalents relative to the nucleoside as the optimal condition.

[0007] The present inventors have discovered a new method for preparing LNA nucleotide P(V) monomers by a P(V) reagent-based system with surprisingly optimized conditions, resulting in the cheapest method with relatively higher yields than described in the art. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] S.T. Crooke, Antisense drug technology: principles, strategies, and applications, 2nd ed. Boca Raton, FL: CRC Press, 2008 [Non-Patent Document 2] Baran et al., Science, 2018, 361, 1234-1238 Summary of the Invention

[0009] In a first aspect, the present invention provides a P(V) monomer of formula (IIIa) or (IIIb): TIFF0007625536000001.tif59128 relates to a method for the preparation of TIFF0007625536000001.tif59128 starting from a nucleoside and using 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as a base (DBU is used in an amount of about 0.8 equivalents relative to the nucleoside).

[0010] In a second aspect, the present invention provides a compound of formula (IIIa) or (IIIb): TIFF0007625536000002.tif59128[where, R 1 , R 2 , R 3 , and R 4 is defined herein] Regarding.

[0011] In a third aspect, the present invention relates to a compound of formula (IIIa) or (IIIb) prepared by the method of the present invention.

[0012] In a fourth aspect, the present invention relates to an LNA oligonucleotide produced by the method of the present invention.

[0013] [The present invention 1001] A process for preparing a compound of formula (IIIa) or (IIIb), comprising the steps of: Modified nucleosides of formula (I): TIFF0007625536000003.tif27128 wherein PG is a hydroxyl protecting group; R 1 is H and R 2 -O(CH 2 ) 2 OCH 3 or R 1 and R 2 together, -CH 2 -O- or -CH(CH 3 )O-, and the oxygen forms a 2'-4' bridge selected from the group consisting of position R 2 are connected via Compounds of formula (IIa) or (IIb): TIFF0007625536000004.tif38128 [In the formula, R 3 is C replaced by halo 6 ~C 10 aryl, halo is selected from F, Cl and Br; R 4 is a linear or branched C 1 ~C 6 -alkyl], Reacting with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as a base (DBU is used in an amount of about 0.8 equivalents relative to the nucleoside of formula (I)), Monomers of formula (IIIa) or (IIIb): TIFF0007625536000005.tif59128 The method includes the step of generating [The present invention 1002] The nucleoside of formula I has the following formula (Ia): TIFF0007625536000006.tif29128 The method of the present invention 1001, comprising: [The present invention 1003] The nucleoside of formula I may be represented by the following formula (Ib): TIFF0007625536000007.tif29128 The method of the present invention 1001, comprising: [The present invention 1004] The nucleoside of formula I may be represented by the following formula (Ic): TIFF0007625536000008.tif46128 The method of the present invention 1001, comprising: [The present invention 1005] The method of any of claims 1001 to 1004, wherein the nucleobase is selected from the group consisting of A, T, G, C, and 5-methylcytosine. [The present invention 1006] The method of any of claims 1001 to 1004, wherein the nucleobase is selected from the group consisting of A, T and G. [The present invention 1007] The method of any one of claims 1005 to 1006, wherein the exocyclic amino moiety in A, G, C or 5-methylcytosine is protected by an amino-protecting group. [The present invention 1008] The process of claim 1007, wherein the amino protecting group is selected from the group consisting of DMF or iBu. [The present invention 1009] The method of any of claims 1001 to 1008, further comprising coupling a compound of formula (IIIa) or (IIIb) as a P(V) monomer to prepare an antisense oligonucleotide. [The present invention 1010] R 1 is H and R 2 -O(CH 2 ) 2 OCH 3 Any of the methods of 1001 to 1009 of the present invention. [The present invention 1011] R 1 and R 2 together, -CH 2 -O-, and the oxygen forms a 2'-4' bridge selected from the group consisting of position R 2 Any of the methods of 1001 to 1009, wherein the compound is bonded via [The present invention 1012] R1 and R 2 Together, -CH(CH 3 )O-, and the oxygen forms a 2'-4' bridge selected from the group consisting of position R 2 Any of the methods of 1001 to 1009, wherein the compound is bonded via [The present invention 1013] R 3 is phenyl substituted with 1 to 5 F. [The present invention 1014] R 4 is methyl or ethyl. [The present invention 1015] Use of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as a base in the production of a compound of formula (IIIa) or (IIIb) starting from a nucleoside of formula (I) of any of the present inventions 1001 to 1014, wherein DBU is used in an amount of about 0.8 equivalents relative to the nucleoside. [The present invention 1016] Compounds of formula (IIIa) or (IIIb): TIFF0007625536000009.tif59128 [In the formula, R 1 、R 2 、R 3 and R 4 is as defined in invention 1001 to 1014]. [The present invention 1017] A compound of formula (IIIa) or (IIIb) of invention 1016 prepared by any of the methods of inventions 1001 to 1014. [The present invention 1018] An oligonucleotide produced by any one of the methods of 1001 to 1014. [The present invention 1019] The invention described herein. Other aspects of the invention are described in more detail below. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] definition The term "alkyl", alone or in combination, denotes a straight or branched chain alkyl group having 1 to 8 carbon atoms, particularly a straight or branched chain alkyl group having 1 to 6 carbon atoms, more particularly a straight or branched chain alkyl group having 1 to 4 carbon atoms. Examples of straight and branched chain C1-C8 alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, the isomeric pentyls, isomeric hexyls, isomeric heptyls and isomeric octyls, particularly methyl, ethyl, propyl, butyl and pentyl. Particular examples of alkyl are methyl, ethyl and propyl.

[0015] The term "amino," alone or in combination, refers to a primary amino group (-NH2), a secondary amino group (-NH-), or a tertiary amino group (-N-).

[0016] The term "amino protecting group" refers to a protecting group for the amino group. Amines are functional groups that often require a protecting group during organic reactions. Carbamates such as t-butoxycarbonyl (Boc, e.g., removed by concentrated strong acid (such as HCl or CF3COOH) or by heating above 80°C), benzyloxycarbonyl (Cbz, e.g., removed by hydrogenolysis), or 9-fluorenylmethoxycarbonyl (Fmoc, e.g., removed by base, e.g. piperidine) are commonly used amine protecting groups. Additional options for protecting groups with different deprotection conditions can be used as described by Peter Wipf, University of Pittsburgh, PA, USA, and associates, who developed a useful protecting group for primary, secondary, and heterocyclic amines: 2,2,6,6-tetramethylpiperidin-1-yloxycarbonyl (Tempoc). Acetyl (Ac) groups are common in oligonucleotide synthesis for the protection of N4 in cytosine nucleobases and N6 in adenine nucleobases, and can be removed by treatment with base, most frequently aqueous or gaseous ammonia or methylamine. Ac is too stable to be easily removed from aliphatic amides. Benzoyl (Bz) groups are also common in oligonucleotide synthesis for the protection of N4 in cytosine nucleobases and N6 in adenine nucleobases, and can be removed by treatment with base, most frequently aqueous or gaseous ammonia or methylamine. Bz is too stable to be easily removed from aliphatic amides. Other suitable amino protecting groups commonly used, such as DMF or iBu, can also be considered by those skilled in the art.

[0017] The term "aryl", alone or in combination, denotes a monovalent aromatic carbocyclic mono- or bicyclic ring system containing 6 to 10 carbon ring atoms, optionally substituted with 1 to 3 substituents independently selected from halogen, hydroxyl, alkyl, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkenyloxy, carboxyl, alkoxycarbonyl, alkylcarbonyl, and formyl. Examples of aryl include phenyl and naphthyl, especially phenyl.

[0018] When referring to DBU, the expression "in an amount of about 0.8 equivalents relative to the nucleoside" refers to a range of 0.7-0.9 equivalents relative to the nucleoside. In this context, equivalents refers to molar equivalents. In other words, this indicates a molar ratio of about 0.7-0.9 DBU:1 nucleoside.

[0019] The terms "halogen" or "halo", alone or in combination, refer to fluorine, chlorine, bromine or iodine, particularly fluorine, chlorine or bromine, more particularly fluorine. The term "halo", in combination with another group, refers to the substitution of said group with at least one halogen, particularly 1 to 5 halogens, particularly 1 to 4 halogens, i.e., substitution with 1, 2, 3 or 4 halogens.

[0020] The terms "hydroxyl" and "hydroxy", alone or in combination, refer to an --OH group.

[0021] A "hydroxyl protecting group" is a protecting group for a hydroxyl group, and is also used to protect a thiol group. Examples of hydroxyl protecting groups are acetyl (Ac), benzoyl (Bz), benzyl (Bn), β-methoxyethoxymethyl ether (MEM), dimethoxytrityl (or bis-(4-methoxyphenyl)phenylmethyl) (DMT), trimethoxytrityl (or tris-(4-methoxyphenyl)phenylmethyl) (TMT), methoxymethyl ether (MOM), methoxytrityl[(4-methoxyphenyl)diphenylmethyl (MMT), p-methoxybenzyl ether (PMB), methylthiomethyl ether, pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl or triphenylmethyl (Tr), silyl ethers (e.g. trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), tri-iso-propylsilyloxymethyl (TOM) and triisopropylsilyl (TIPS) ethers), methyl ether and ethoxyethyl ether (EE). Particular examples of hydroxyl protecting groups are DMT and TMT, especially DMT.

[0022] The term "LNA monomer" refers to an LNA nucleotide, meaning a nucleotide wherein the nucleoside is an LNA nucleoside as defined herein.

[0023] The term "pharmaceutical acceptable salts" refers to those salts that retain the biological effectiveness and properties of the free base or free acid, which are not biologically or otherwise undesirable. Salts are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, particularly hydrochloric acid, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, N-acetylcysteine, and the like. Furthermore, these salts can be prepared from the addition of inorganic or organic bases to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, polyamine resins, etc. The oligonucleotides of the present invention can also exist in zwitterionic form.The particularly preferred pharmaceutically acceptable salts of the present invention are sodium, lithium, potassium and trialkylammonium salts.

[0024] The term "protecting group", alone or in combination, refers to groups that selectively block a reactive site in a multifunctional compound so that a chemical reaction can be selectively carried out at an otherwise unprotected reactive site. The protecting group may be removed. Exemplary protecting groups are amino-protecting groups, carboxy-protecting groups, or hydroxy-protecting groups.

[0025] A "phosphate protecting group" is a protecting group for a phosphate group. Examples of phosphate protecting groups are 2-cyanoethyl and methyl. A particular example of a phosphate protecting group is 2-cyanoethyl.

[0026] If one of the starting materials or compounds of the present invention contains one or more functional groups that are not stable or are reactive under the reaction conditions of one or more reaction steps, suitable protecting groups (for example, as described in "Protective Groups in Organic Chemistry" by TW Greene and PG M Huts, 3rd Edition, 1999, Wiley, New York) can be introduced before the critical step applying methods well known in the art. Such protecting groups can be removed at a later stage of the synthesis using standard methods described in this literature. Examples of protecting groups are tert-butoxycarbonyl (Boc), 9-fluorenylmethylcarbamate (Fmoc), 2-trimethylsilylethylcarbamate (Teoc), carbobenzyloxy (Cbz) and p-methoxybenzyloxycarbonyl (Moz).

[0027] The compounds described herein may contain several asymmetric centers and may exist in the form of optically pure enantiomers, mixtures of enantiomers, e.g. racemates, mixtures of diastereomers, diastereomeric racemates or mixtures of diastereomeric racemates.

[0028] Oligonucleotides The term "oligonucleotide" as used herein is defined as a molecule that comprises two or more covalently linked nucleosides, as it is generally understood by those skilled in the art. Such covalently linked nucleosides may also be referred to as nucleic acid molecules or oligomers. Oligonucleotides are usually produced in a laboratory by solid-phase chemical synthesis followed by purification. When referring to the sequence of an oligonucleotide, it refers to the sequence or order of the nucleobase moieties of the covalently linked nucleotides or nucleosides, or modifications thereof. The oligonucleotides of the present invention are artificial, chemically synthesized, and typically purified or isolated. The oligonucleotides of the present invention may comprise one or more modified nucleosides or nucleotides.

[0029] Antisense oligonucleotides The term "antisense oligonucleotide" as used herein is defined as an oligonucleotide that can modulate the expression of a target gene by hybridizing with a target nucleic acid, particularly a continuous sequence in the target nucleic acid.Antisense oligonucleotides are not double-stranded in nature, and therefore are not siRNA or shRNA.Preferably, the antisense oligonucleotides of the present invention are single-stranded.The single-stranded oligonucleotides of the present invention can form hairpin or intermolecular duplex structures (duplexes between two molecules of the same oligonucleotide), as long as the degree of self-complementarity within or between them is less than 50% of the total length of the oligonucleotide.

[0030] Contiguous nucleotide sequence The term "contiguous nucleotide sequence" refers to a region of an oligonucleotide that is complementary to a target nucleic acid. This term is used interchangeably herein with the terms "contiguous nucleobase sequence" and "oligonucleotide motif sequence". In some embodiments, all nucleotides of an oligonucleotide constitute a contiguous nucleotide sequence. In some embodiments, an oligonucleotide comprises a contiguous nucleotide sequence, such as an FG-F' gapmer region, and may optionally comprise a nucleotide linker region that can be used to attach additional nucleotides, such as functional groups, to the contiguous nucleotide sequence. The nucleotide linker region may or may not be complementary to the target nucleic acid.

[0031] nucleotide Nucleotides are the building blocks of oligonucleotides and polynucleotides, and for the purposes of the present invention, include both naturally occurring and non-naturally occurring nucleotides. Essentially, nucleotides, such as DNA and RNA nucleotides, contain a ribose sugar moiety, a nucleobase moiety, and one or more phosphate groups (not present in nucleosides). Nucleosides and nucleotides may be referred to interchangeably as "units" or "monomers."

[0032] Modified Nucleosides The term "modified nucleoside" or "nucleoside modification" as used herein refers to a nucleoside that is modified compared to a corresponding DNA or RNA nucleoside by the introduction of one or more modifications in the sugar or (nucleic acid) base moiety. In a preferred embodiment, the modified nucleoside comprises a modified sugar moiety. The term modified nucleoside may be used interchangeably with the terms "nucleoside analog" or modified "unit" or modified "monomer". Nucleosides that contain unmodified DNA or RNA sugar moieties are referred to herein as DNA or RNA nucleosides. Nucleosides that contain modifications in the base region of DNA or RNA nucleosides are generally still referred to as DNA or RNA if they allow Watson-Crick base pairing.

[0033] Nucleic acid bases The term nucleobase includes the purine (e.g., adenine and guanine) and pyrimidine (e.g., uracil, thymine and cytosine) moieties present in nucleosides and nucleotides that form hydrogen bonds in nucleic acid hybridization. In the context of the present invention, the term nucleobase also encompasses modified nucleobases that may differ from naturally occurring nucleobases but are functional during nucleic acid hybridization. In this context, "nucleobase" refers to both naturally occurring nucleobases such as adenine, guanine, cytosine, thymidine, uracil, xanthine and hypoxanthine, and non-naturally occurring mutants. Such mutants are described, for example, in Hirao et al. (2012) Accounts of Chemical Research Vol. 45, p. 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl.37 1.4.1.

[0034] In some embodiments, the nucleobase moiety is modified by changing the purine or pyrimidine to a modified purine or pyrimidine, e.g., a substituted purine or substituted pyrimidine, e.g., a nucleobase selected from isocytosine, pseudoisocytosine, 5-methyl-cytosine, 5-thiozolo-cytosine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-bromouracil 5-thiazolo-uracil, 2-thio-uracil, 2'thio-thymine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine, and 2-chloro-6-aminopurine.

[0035] Nucleobase moiety may be represented by the letter code for each corresponding nucleobase, for example, A, T, G, C or U, where each letter may optionally include a modified nucleobase of equivalent function.For example, in the exemplified oligonucleotide, the nucleobase moiety is selected from A, T, G, C and 5-methylcytosine.Optionally, in the LNA gapmer, 5-methylcytosine LNA nucleoside may be used.

[0036] Modified Oligonucleotides The term modified oligonucleotide describes an oligonucleotide containing one or more sugar-modified nucleosides and / or modified internucleoside linkages. The term chimeric oligonucleotide is a term used in this document to describe oligonucleotides containing modified nucleosides.

[0037] Stereodefined oligonucleotides A stereodefined oligonucleotide is an oligonucleotide in which at least one of the internucleoside linkages is a stereodefined internucleoside linkage.

[0038] A stereodefined phosphorothioate oligonucleotide is an oligonucleotide in which at least one of the internucleoside linkages is a stereodefined phosphorothioate internucleoside linkage.

[0039] Complementarity The term "complementarity" describes the ability of nucleosides / nucleotides to Watson-Crick base pairing. Watson-Crick base pairs are guanine (G)-cytosine (C) and adenine (A)-thymine (T) / uracil (U). Oligonucleotides may contain nucleosides with modified nucleobases, for example 5-methylcytosine is often used instead of cytosine, and thus the term complementarity is understood to include Watson-Crick base pairing between unmodified and modified nucleobases (see, for example, Hirao et al. (2012) Accounts of Chemical Research Vol. 45, p. 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl.37 1.4.1).

[0040] The term "% complementary" as used herein refers to the percentage of nucleotides in a contiguous nucleotide sequence in a nucleic acid molecule (e.g., an oligonucleotide) that is complementary (i.e., forms Watson-Crick base pairs) to a contiguous nucleotide sequence in another nucleic acid molecule (e.g., a target nucleic acid) at a given position. This percentage is calculated by counting the number of aligned (when aligned with the oligonucleotide sequence from the target sequence 5'-3' and 3'-5') bases that form pairs between the two sequences, dividing by the total number of nucleotides in the oligonucleotide, and multiplying by 100. In such a comparison, non-aligned (non-base-paired) nucleic acid bases / nucleotides are called mismatches. Preferably, insertions and deletions are not allowed in the calculation of the % complementarity of contiguous nucleotide sequences.

[0041] The term "fully complementary" refers to 100% complementarity.

[0042] identity The term "identity" as used herein refers to the percentage of the number of nucleotides of a contiguous nucleotide sequence in a nucleic acid molecule (e.g., an oligonucleotide) that is identical (i.e., capable of forming Watson-Crick base pairs with complementary nucleosides) at a given position to a contiguous nucleotide sequence in another nucleic acid molecule (e.g., a target nucleic acid) at a given position. This percentage is calculated by counting the number of aligned bases that are identical between the two sequences, dividing by the total number of nucleotides in the oligonucleotide, and multiplying by 100. Percent identity = (number of matches x 100) / length of aligned region. Preferably, insertions and deletions are not allowed in the calculation of the % complementarity of contiguous nucleotide sequences.

[0043] Hybridization The term "hybridizing" or "hybridizing" as used herein should be understood as two nucleic acid strands (e.g., an oligonucleotide and a target nucleic acid) forming a duplex by forming hydrogen bonds between base pairs on opposing strands. The affinity of the binding between the two nucleic acid strands is the strength of the hybridization. This strength is measured by the melting temperature (T), which is defined as the temperature at which half of the oligonucleotides form a duplex with the target nucleic acid. m ) is often described as T m is not strictly proportional to affinity (Mergny and Lacroix, 2003, Oligonucleotides 13:515-537). The standard Gibbs free energy, ΔG°, is a more accurate representation of binding affinity, ΔG°=-RTln(K d ) where R is the gas constant and T is the absolute temperature, giving the dissociation constant (K d) is related to the energy of the reaction between the oligonucleotide and the target nucleic acid. Thus, a very small ΔG° of the reaction between the oligonucleotide and the target nucleic acid reflects strong hybridization between the oligonucleotide and the target nucleic acid. ΔG° is the energy associated with a reaction in which the aqueous solution has a concentration of 1M, a pH of 7, and a temperature of 37°C. Hybridization of an oligonucleotide to a target nucleic acid is a spontaneous reaction, and in spontaneous reactions, ΔG° is less than zero. ΔG° can be experimentally measured, for example, by using the isothermal titration calorimetry (ITC) method described in Hansen et al., 1965, Chem.Comm. 36-38 and Holdgate et al., 2005, Drug Discov Today. Those skilled in the art will know that commercially available equipment is available for measuring ΔG°. ΔG° can also be evaluated numerically using the nearest neighbor model described in SantaLucia, 1998, Proc Natl Acad Sci USA. 95:1460-1465, using the roughly derived thermodynamic parameters described in Sugimoto et al., 1995, Biochemistry 34:11211-11216 and McTigue et al., 2004, Biochemistry 43:5388-5405. To be able to modulate their intended nucleic acid targets by hybridization, the oligonucleotides of the invention hybridize to target nucleic acids with estimated ΔG° values ​​of less than -10 kcal for oligonucleotides that are 10-30 nucleotides in length. In some embodiments, the degree of hybridization strength is measured by the standard Gibbs free energy ΔG°. The oligonucleotides may hybridize to the target nucleic acid with an estimated ΔG° value in the range of less than -10 kcal, such as less than -15 kcal, such as less than -20 kcal and less than -25 kcal for oligonucleotides that are 8 to 30 nucleotides in length. In some embodiments, the oligonucleotides hybridize to the target nucleic acid with an estimated ΔG° value of -10 to -60 kcal, such as -12 to -40 kcal, such as -15 to -30 kcal or -16 to -27 kcal, such as -18 to -25 kcal.

[0044] sugar modification Oligomers of the invention may contain one or more nucleosides having modified sugar moieties, ie, modifications in the sugar moiety as compared to the ribose sugar moiety found in DNA and RNA.

[0045] Many nucleosides with modifications of the ribose sugar moiety have been engineered primarily for the purpose of improving certain properties of oligonucleotides, such as affinity and / or nuclease resistance.

[0046] Such modifications include, for example, those in which the ribose ring structure is modified by replacement with a hexose ring (HNA), or a bicyclic ring (LNA) typically having a biradical bridge between the C2 and C4 carbons on the ribose ring, or an unlinked ribose ring (e.g., UNA) typically lacking a bond between the C2 and C3 carbons. Other sugar-modified nucleosides include, for example, bicyclohexose nucleic acids (WO2011 / 017521) or tricyclic nucleic acids (WO2013 / 154798). Modified nucleosides also include nucleosides in which the sugar moiety is replaced with a non-sugar moiety, for example, in the case of peptide nucleic acids (PNAs) or morpholino nucleic acids.

[0047] Sugar modifications also include modifications made by changing the substituents on the ribose ring to groups other than hydrogen or the 2'-OH group found naturally in DNA and RNA nucleosides. Substituents may be introduced, for example, at the 2', 3', 4', or 5' positions.

[0048] 2' sugar modified nucleosides A 2' sugar modified nucleoside is a nucleoside that either has a substituent other than H or -OH at the 2' position (2' substituted nucleoside) or contains a 2' linked biradical capable of forming a bridge between the 2' carbon and the second carbon in the ribose ring, such as an LNA (2'-4' biradical bridged) nucleoside.

[0049] In fact, much attention has been paid to developing 2' modified nucleosides, and many 2' modified nucleosides have been found to have beneficial properties when incorporated into oligonucleotides.For example, 2' modified sugars can provide oligonucleotides with enhanced binding affinity and / or increased nuclease resistance.Examples of 2' substituted modified nucleosides are 2'-O-alkyl-RNA nucleosides, 2'-O-methyl-RNA nucleosides, 2'-alkoxy-RNA nucleosides, 2'-O-methoxyethyl-RNA (MOE) nucleosides, 2'-amino-DNA nucleosides, 2'-fluoro-RNA nucleosides and 2'-F-ANA nucleosides. Further examples can be found, for example, in Freier and Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213, and Deleavey and Damha, Chemistry and Biology 2012, 19, 937. Below are examples of some 2'-substituted modified nucleosides. TIFF0007625536000010.tif71128

[0050] In the context of the present invention, 2' modifications do not include 2' bridging molecules such as LNA.

[0051] Locked Nucleoside (LNA) Nucleoside "LNA nucleosides" are 2' modified nucleosides that contain a biradical (also referred to as a "2'-4' bridge") linking C2' and C4' of the ribose sugar ring of said nucleoside, which restricts or fixes the conformation of the ribose ring. These nucleosides are also referred to in the literature as bridged nucleic acids or bicyclic nucleic acids (BNAs). Fixation of the ribose conformation is associated with enhanced hybridization affinity to complementary RNA or DNA molecules (duplex stabilization) when LNA is incorporated into an oligonucleotide. This can be routinely determined by measuring the melting temperature of the oligonucleotide / complement duplex.

[0052] Non-limiting exemplary LNA nucleosides are described in WO99 / 014226, WO00 / 66604, WO98 / 039352, WO2004 / 046160, WO00 / 047599, WO2007 / 134181, WO2010 / 077578, WO2010 / 036698, WO2007 / 090071, WO2009 / 006478, WO2011 / 156202, WO2008 / 154401, WO2009 / 067647, WO2008 / 150729, Morita et al., Bioorganic & Med. Chem. Lett. 12, pp. 73-76, Seth et al. J. Org. Chem. 2010, 75(5), 1569-81, and Mitsuoka et al., Nucleic Acids Research 2009, 37(4), 1225-1238.

[0053] The 2'-4' bridge includes atoms bridging positions 2 through 4, and in particular has the formula -XY- (Y is linked to C4' and X is linked to C2'): [In the formula, X is oxygen, sulfur, -CR a R b -, -C(R a )=C(R b )-, -C(=CR a R b )-, -C(R a )=N-, -Si(R a )2-, -SO2-, -NR a-; -O-NR a -, -NR a -O-, -C(=J)-, Se, -O-NR a -, -NR a -CR a R b -, -N(R a )-O- or -O-CR a R b - and; Y is oxygen, sulfur, -(CR a R b ) n -, -CR a R b -O-CR a R b -, -C(R a )=C(R b )-, -C(R a )=N-, -Si(R a )2-, -SO2-, -NR a -, -C(=J)-, Se, -O-NR a -, -NR a -CR a R b -, -N(R a )-O- or -O-CR a R b - and; However, -XY- is -OO-, Si(R a )2-Si(R a )2-, -SO2-SO2-, -C(R a )=C(R b )-C(R a )=C(R b ), -C(R a )=NC(R a )=N-, -C(R a )=NC(R a )=C(R b ), -C(R a )=C(R b )-C(R a )=N- or -Se-Se-; J is oxygen, sulfur, =CH2 or =N(R a ) and; R a and R bis hydrogen, halogen, hydroxyl, cyano, thiohydroxyl, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkoxyalkyl, alkenyloxy, carboxyl, alkoxycarbonyl, alkylcarbonyl, formyl, aryl, heterocyclyl, amino, alkylamino, carbamoyl, alkylaminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, alkylcarbonylamino, carbamido, alkanoyloxy, sulfonyl, alkylsulfonyloxy, nitro, azido, thiohydroxyl sulfidoalkylsulfanyl, aryloxycarbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxycarbonyl, heteroaryloxy, heteroarylcarbonyl, -OC(=X a )R c , -OC(=X a )NR c R d and -NR e C(=X a )NR c R d Are independently selected from; or 2 geminal R's a and R b together form an optionally substituted methylene; or 2 geminal R's a and R b together with the carbon atom to which they are attached form a cycloalkyl or halocycloalkyl having only one carbon atom, -XY-; wherein substituted alkyl, substituted alkenyl, substituted alkynyl, substituted alkoxy and substituted methylene are alkyl, alkenyl, alkynyl and methylene substituted with 1 to 3 substituents independently selected from halogen, hydroxyl, alkyl, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkenyloxy, carboxyl, alkoxycarbonyl, alkylcarbonyl, formyl, heterosilyl, aryl and heteroaryl; Xa is oxygen, sulfur or -NR c and; R c , R d and R e is independently selected from hydrogen and alkyl; n is 1, 2 or 3. It is of the following.

[0054] In further particular embodiments of the invention, X is oxygen, sulfur, -NR a -, -CR a R b -or-C(=CR a R b )—, particularly oxygen, sulfur, —NH—, —CH2— or —C(═CH2)—, more particularly oxygen.

[0055] In another particular embodiment of the invention, Y is -CR a R b -, -CR a R b -CR a R b -OR-CR a R b- CR a R b- CR a R b -, in particular -CH2-CHCH3-, -CHCH3-CH2-, -CH2-CH2- or -CH2-CH2-CH2-.

[0056] In certain embodiments of the present invention, -XY- is -O-(CR a R b ) n -,-S-CR a R b -, -N(R a )CR a R b -, -CR a R b -CR a R b -, -O-CR a R b -O-CR a R b -, -CRa R b -O-CR a R b -, -C(=CR a R b )-CR a R b -, -N(R a )CR a R b -,-ON(R a )-CR a R b -or-N(R a )-O-CR a R b -It is.

[0057] In certain embodiments of the present invention, R a and R b are independently selected from the group consisting of hydrogen, halogen, hydroxyl, alkyl and alkoxyalkyl, particularly hydrogen, halogen, alkyl and alkoxyalkyl.

[0058] In another embodiment of the present invention, R a and R b are independently selected from the group consisting of hydrogen, fluoro, hydroxyl, methyl and -CH2-O-CH3, in particular hydrogen, fluoro, methyl and -CH2-O-CH3.

[0059] Advantageously, R of -XY- a and R b is as defined above and all the others are simultaneously hydrogen.

[0060] In a further particular embodiment of the present invention, R a is hydrogen or alkyl, in particular hydrogen or methyl.

[0061] In another embodiment of the present invention, R b is hydrogen or alkyl, in particular hydrogen or methyl.

[0062] In certain embodiments of the present invention, R a and R bOne or both of are hydrogen.

[0063] In certain embodiments of the present invention, R a and R b Only one of the groups is hydrogen.

[0064] In one particular embodiment of the present invention, R a and R b One of them is methyl and the other is hydrogen.

[0065] In certain embodiments of the present invention, R a and R b are both simultaneously methyl.

[0066] In certain embodiments of the invention, -XY- is -O-CH2-, -S-CH2-, -S-CH(CH3)-, -NH-CH2-, -O-CH2CH2-, -O-CH(CH2-O-CH3)-, -O-CH(CH2CH3)-, -O-CH(CH3)-, -O-CH 2- O-CH2-, -O-CH2-O-CH2-, -CH2-O-CH2-, -C(=CH2)CH2-, -C(=CH2)CH(CH3)-, -N(OCH3)CH2- or -N(CH3)CH2-;

[0067] In certain embodiments of the present invention, -XY- is -O-CR a R b - where R a and R b are independently selected from the group consisting of hydrogen, alkyl and alkoxyalkyl, in particular hydrogen, methyl and -CH2-O-CH3.

[0068] In certain embodiments, -XY- is -O-CH2- or -O-CH(CH3)-, in particular -O-CH2-.

[0069] The 2'-4' bridge may be positioned either below the plane of the ribose ring (beta-D-configuration) or above the plane of the ring (alpha-L-configuration), as illustrated in Formula (A) and Formula (B), respectively.

[0070] Unless specified, it is understood that LNA nucleosides may exist in either beta-D or alpha-L stereoisoforms.

[0071] Particular examples of LNA nucleosides of the present invention are depicted in Scheme 1, where B is as defined above. Scheme 1 TIFF0007625536000011.tif202123TIFF0007625536000012.tif216117TIFF0007625536000013.tif134128

[0072] Particular LNA nucleosides are beta-D-oxy-LNA, 6'-methyl-beta-D-oxy LNA, such as (S)-6'-methyl-beta-D-oxy-LNA ((S)-cET), and ENA.

[0073] RNase H activity and recruitment RNase H activity of an antisense oligonucleotide refers to its ability to recruit RNase H when in a duplex with a complementary RNA molecule. WO01 / 23613 provides an in vitro method for determining RNase H activity, which can be used to determine the ability to recruit RNase H. Typically, an oligonucleotide is considered capable of recruiting RNase H if, when provided with a complementary target nucleic acid sequence, it has an initial rate, measured in pmol / l / min, of at least 5%, e.g., at least 10% or 20% higher than the initial rate determined when using an oligonucleotide having the same base sequence as the modified oligonucleotide being tested, but containing only DNA monomers with phosphorothioate linkages between all monomers in the oligonucleotide, and using the method provided by Examples 91-95 of WO01 / 23613 (incorporated herein by reference). For use in determining RNase H activity, recombinant human RNase H1 is available from Lubio Science GmbH, Lucerne, Switzerland.

[0074] Detailed Description of the Invention In one aspect, the present invention provides a method for preparing a P(V) monomer of formula (IIIa) or (IIIb), comprising the step of: TIFF0007625536000014.tif27128 [wherein PG is a hydroxyl protecting group; R 1 is H and R 2 is -O(CH2)2OCH3, or R 1 and R 2 together form a 2'-4' bridge selected from -CH2-O- or -CH(CH3)O-, and the oxygen is at position R 2 are connected via Compounds of formula (IIa) or (IIb): TIFF0007625536000015.tif38128[where, R 3 is C6-C substituted by halo10 aryl, halo is selected from F, Cl and Br; R 4 is a linear or branched C1-C6 alkyl; Reacting with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as a base (DBU is used in an amount of about 0.8 equivalents relative to the nucleoside of formula (I)), Monomers of formula (IIIa) or (IIIb): The present invention relates to a method for generating TIFF0007625536000016.tif59128.

[0075] In one or all embodiments of the invention, the compound of formula (IIa) or (IIb) is: The file is TIFF0007625536000017.tif99128.

[0076] In one or all embodiments of the present invention, the nucleoside of formula I has the following formula (Ia): I have TIFF0007625536000018.tif27128.

[0077] In one or all embodiments of the present invention, the nucleoside of formula I has the following formula (Ib): has TIFF0007625536000019.tif27128.

[0078] In one or all embodiments of the present invention, the nucleoside of formula I has the following formula (Ic): I have TIFF0007625536000020.tif46128.

[0079] In one or all embodiments of the invention, the nucleobase is selected from the group consisting of A, T, G, C, and 5-methylcytosine.

[0080] In one or all embodiments of the invention, the nucleobase is selected from the group consisting of A, T and G.

[0081] In one or all embodiments of the invention, the nucleobase is A, G, C or 5-methylcytosine and the exocyclic amino site is protected by an amino protecting group. The amino protecting group may be selected from the group consisting of DMF and iBu.

[0082] In one or all embodiments, the method of the present invention further comprises coupling a compound of formula (IIIa) or (IIIb) as a P(V) monomer to prepare an antisense oligonucleotide, which can be done by methods known in the art.

[0083] In one or all embodiments of the present invention, R 1 is H and R 2 is -O(CH2)2OCH3.

[0084] In one or all embodiments of the present invention, R 1 and R 2 together form a 2'-4' bridge selected from -CH2-O-, and the oxygen is at position R 2 are connected via

[0085] In one or all embodiments of the present invention, R 1 and R 2 together form a 2'-4' bridge selected from -CH(CH3)O-, and the oxygen is at position R 2 are connected via

[0086] In one or all embodiments of the present invention, R 3 is phenyl substituted by 1 to 5 F, for example, phenyl substituted by 1, 2, 3, 4 or 5 F, for example, phenyl substituted by 4 or 5 F, for example, phenyl substituted by 5 F.

[0087] In one or all embodiments of the present invention, R 4 is methyl or ethyl, for example methyl.

[0088] In one embodiment of the present invention, the method of the present invention is a method for preparing a compound of formula (IIIa1) or (IIIb1), comprising reacting a modified nucleoside of formula (I): TIFF0007625536000021.tif26128, where PG is a hydroxyl protecting group, Compounds of formula (IIa1) or (IIb1): TIFF0007625536000022.tif99128 and Reacting with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as a base in MeCN (DBU is used in an amount of about 0.8 equivalents relative to the nucleoside of formula (Ia)), Monomers of formula (IIIa1) or (IIIb1): generating TIFF0007625536000023.tif59128.

[0089] In another aspect, the present invention relates to the use of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as a base in the production of compounds of formula (IIIa) or (IIIb) starting from nucleosides of formula (I) as described herein, wherein DBU is used in an amount of about 0.8 equivalents relative to the nucleoside.

[0090] In another aspect, the present invention provides a compound of formula (IIIa) or (IIIb): TIFF0007625536000024.tif59128[where, R 1 , R 2 , R 3 and R 4 is as defined herein. Regarding.

[0091] In an embodiment, the compound of formula (IIIa) or (IIIb) according to the invention has the formula (IIIa1) or (IIIb1): The file is TIFF0007625536000025.tif59128.

[0092] In an embodiment, the compound of formula (IIIa) or (IIIb) according to the invention is the following compound: TIFF0007625536000026.tif120128TIFF0007625536000027.tif169128.

[0093] In an embodiment, the compound of formula (IIIa) or (IIIb) according to the invention is the following compound: TIFF0007625536000028.tif56128.

[0094] In an embodiment, the compound of formula (IIIa) or (IIIb) according to the invention is the following compound: The file is TIFF0007625536000029.tif60128.

[0095] In an embodiment, the compound of formula (IIIa) or (IIIb) according to the invention is the following compound: The file is TIFF0007625536000030.tif56128.

[0096] In an embodiment, the compound of formula (IIIa) or (IIIb) according to the invention is the following compound: The file is TIFF0007625536000031.tif109128.

[0097] Another aspect of the present invention is a compound of formula (IIIa) or (IIIb) prepared by the method of the present invention. In one embodiment, this is a compound of formula (IIIa) or (IIIb) obtained directly by the method of the present invention.

[0098] In another aspect, the invention relates to an oligonucleotide produced by the method of the invention.

[0099] In one or all aspects or embodiments of the present invention, DBU can be reacted in a solution of MeCN. Other suitable solvents may be contemplated by one of skill in the art.

[0100] In one or all aspects or embodiments of the invention, the reaction may be carried out in a solution of MeCN. Other suitable solvents may be contemplated by one of skill in the art.

[0101] In an embodiment, the compound of formula (I) is TIFF0007625536000032.tif96137.

[0102] As can be demonstrated from Table 6 below, the present inventors have found a new method for preparing LNA monomers by a P(V) reagent-based system with surprisingly optimized conditions, resulting in a method that is the cheapest and has a higher yield than described in the art.For example, in the preparation of LNA-T, LNA-A and LNA-G, when 0.8 equivalents of DBU are used, the yield of isolated compound III is significantly higher than when 1.3 equivalents of DBU are used as recommended in the prior art.In the preparation of LNA-C, when 0.8 equivalents of DBU are used, the same or better yield is obtained compared to when 1.3 equivalents of DBU are used.This means that the method can be significantly cheaper because fewer equivalents of DBU are required. EXAMPLES

[0103] (S p -General synthesis of LNA-A TIFF0007625536000033.tif45141(R p General synthesis of -LNA TIFF0007625536000034.tif47144

[0104] General synthesis for P(V)LNA A monomer using either 0.8 eq DBU or 1.3 eq DBU To a suspension of 5'-ODMTr-LNA-A (500 mg, 0.73 mmol) and (+)-1 (423 mg, 0.95 mmol) in MeCN (9 mL) was added a solution of DBU (87 μL, 0.58 mmol, 0.8 eq) in MeCN (2 ml) at room temperature, then stirred for 40 min at room temperature (TLC-full conv.). The reaction mixture was filtered through a plug of silica gel and rinsed with EtOAc (35 mL). The filtrate was washed with water (15 mL), 10% Na2HPO4 (20 mL), water (20 mL), saturated NaHCO3 (20 mL), dried over Na2SO4 and evaporated. The residue was purified by column chromatography (eluted with 50%-70% EtOAc in hexane).

[0105] (Table 1) TIFF0007625536000035.tif38128

[0106] General synthesis for P(V)LNA T monomer using either 0.8 eq DBU or 1.3 eq DBU To a suspension of 5'-ODMTr-LNA-T (500 mg, 0.87 mmol) and (-)-1 (507 mg, 1.13 mmol) in MeCN (9 mL) was added a solution of DBU (104 μL, 0.58 mmol, 0.8 eq) in MeCN (2 ml) at room temperature, then stirred for 40 min at room temperature (TLC-full conv.). The reaction mixture was filtered through a silica gel plug and rinsed with EtOAc (35 mL). The filtrate was washed with 10% Na2HPO4 (20 mL), water (20 mL), saturated NaHCO3 (20 mL), dried over Na2SO4 and evaporated. The residue was purified by column chromatography (eluted with EtOAc / Hexane + 1 / 1).

[0107] (Table 2) TIFF0007625536000036.tif38128

[0108] General synthesis for P(V)LNA C monomer using either 0.8 eq DBU or 1.3 eq DBU To a solution of 5'-ODMTr-LNA-C (588 mg, 0.87 mmol) and (+)-1 (505 mg, 1.13 mmol) in MeCN (11 mL) was added a solution of DBU (DBU 106 μL, 0.58 mmol, 0.8 eq) in MeCN (2 ml) at room temperature, then stirred at room temperature for 40 min (TLC-full conv.). The reaction mixture was filtered through a silica gel plug and rinsed with EtOAc (35 mL). The filtrate was washed with 10% Na2HPO4 (20 mL), water (20 mL), saturated NaHCO3 (20 mL), dried over Na2SO4 and evaporated. The residue was purified by column chromatography (eluted with EtOAc / Hexane + 1 / 2, then 1 / 1).

[0109] (Table 3) TIFF0007625536000037.tif38128

[0110] General synthesis for P(V)LNA G-iBu monomers using either 0.8 eq DBU or 1.3 eq DBU 5'-ODMTr-LNA-G in MeCN (11 mL) iBu To a solution of (500 mg, 0.75 mmol) and (+)-1 (434 mg, 0.97 mmol) was added a solution of DBU (91.2 μL, 0.60 mmol, 0.8 eq) in MeCN (1 ml) at room temperature, then stirred for 40 min at room temperature (TLC-full conv.). The reaction mixture was filtered through a plug of silica gel and rinsed with EtOAc (35 mL). The filtrate was washed with 10% Na2HPO4 (20 mL), water (20 mL), saturated NaHCO3 (20 mL), dried over Na2SO4 and evaporated. The residue was purified by column chromatography (eluted with 50%-75% EtOAc in hexanes).

[0111] (Table 4) TIFF0007625536000038.tif44128

[0112] General synthesis for P(V)LNA G-DMF monomer using either 0.8 eq DBU or 1.3 eq DBU 5'-ODMTr-LNA-G in MeCN (6 mL) DMF To a suspension of (500 mg, 0.77 mmol) and THF (13 mL) was added a solution of (+)-1 (444 mg, 1.00 mmol) in MeCN (5 mL), followed by a solution of DBU (91.5 μL DBU, 0.61 mmol, 0.8 eq) in MeCN (1 ml). The reaction mixture was stirred at room temperature for 1 h (gradually increasing LNA-G DMF The reaction mixture was filtered through a plug of silica gel and the product was washed through the plug with EtOAc (100 mL), EtOAc / THF (1 / 2 80 mL), and THF (50 mL). The filtrate was washed with 10% Na2HPO4 (100 mL), water (20 mL), saturated NaHCO3 (100 mL), brine (100 mL), dried over Na2SO4, and evaporated. The residue was purified by column chromatography (eluting with 10%-30% EtOAc in THF).

[0113] (Table 5) TIFF0007625536000039.tif44128

[0114] Table 6. Yield Summary TIFF0007625536000040.tif134128

[0115] 1.1 Analysis data TIFF0007625536000041.tif56128 1H NMR (300 MHz, CDCl3, ppm): 8.71 (1H, s), 7.64 (1H, d, J= 1.2 Hz), 7.46-7.41 (2H, m), 7.36-7.26 (6H, m), 7.25-7.20 (1H, m) 6.87-7.60 (4H, m), 5.70 (1H, s), 5.16 (1H, d, J= 7.3 Hz), 4.95 (1H, s), 4.85 (1H, s), 4.66 (1H, s), 4.39 (1H, dt, J= 12.8 3.5 Hz), 3.81 (2H, s), 3.78 (6H, s), 3.47 (1H, d, J= 11.1 Hz), 3.42 (1H, d, J= 11.1 Hz), 2.60-2.52 (1H, m), 2.35-2.25 (1H, m), 2.09-1.62 (5H, m), 1.76 (3H, s), 1.67-1.64 (6H, m) 13 C NMR (100 MHz, CDCl3, ppm) 163.7, 158.8, 149.9, 145.0, 144.2, 135.3, 135.2, 134.2, 130.3, 128.3, 128.2, 127.2, 113.5, 111.8, 111.0, 87.7, 87.6, 87.2, 86.9, 85.6, 78.4, 74.3, 74.2, 72.4, 66.6, 57.7, 55.4, 39.0, 33.8, 27.7, 27.6, 23.6, 22.6, 22.0, 12.7. 31 P NMR (160 MHz, CDCl3, ppm) 101.4. [α] D 20 = -37.0 (c .1, DCM). LCMS ESI (m / z): 817.3 [M-H] - TIFF0007625536000042.tif52128 1H NMR (300 MHz, CDCl3, ppm): 8.8-8.6 (1H, br. s), 7.64 (1H, d, J= 1.0 Hz), 7.52-7.46 (2H, m), 7.40-7.33 (4H, m), 7.33-7.26 (2H. m), 7.26-7.19 (1H, m) 6.87-7.79 (4H, m), 5.69 (1H, s), 5.25 (1H, d, J= 11.0 Hz), 4.90-4.88 (1H, m), 4.57 (1H, s), 4.60 (1H, s), 4.29 (1H, dt, J= 12.6 3.1 Hz), 3.87 (1H, d, J= 8.3 Hz), 3.79-3.76 (1H, m), 3.78 (3H, s), 3.78 (3H, s), 3.59 (1H, d, J= 11.0 Hz), 3.42 (1H, d, J= 11.0 Hz), 2.57-2.48 (1H, m), 2.08-1.65 (6H, m), 1.65 (6H, s), 1.58 (3H, d, J= 1.0 Hz) 13 C NMR (100 MHz, CDCl3, ppm):163.7, 158.8, 149.9, 144.9, 144.2, 135.3, 135.2, 134.2, 130.5, 130.4, 128.5, 128.1, 127.2, 113.4, 113.4, 111.9, 111.1, 87.4, 87.1, 86.1, 78.3, 78.3, 74.6, 74.6, 72.2, 65.9, 57.9, 55.3, 39.0, 33.8, 33.7, 27.8, 27.6, 23.5, 22.7, 21.8, 12.6. 31 P NMR (160 MHz, CDCl3, ppm): 100.9. [α] D 20 = +94.9 (c .1, DCM). LCMS ESI (m / z): 817.25 [M-H] - TIFF0007625536000043.tif601281 H NMR (300 MHz, CDCl3, ppm): 13.5-13.2 (1H, br. s), 8.35-8.28 (2H, m), 7.82 (1H, J= 1.0 Hz), 7.55-7.40 (5H, m), 7.39-7.28 (6H, m), 7.27-7.22 (1H, m), 6.86-6.83 (4H, m), 5.75 (1H, s), 5.19 (1H, d, J= 7.5 Hz), 4.95 (1H, s), 4.86 (1H, s), 4.71 (1H, s), 4.40 (1H, dt, J= 12.7, 3.5 Hz), 3.84-3.79 (7H, m), 3.50 (1H, d, J= 11.1 Hz), 3.44 (1H, d, J= 11.1 Hz), 2.62-2.52 (1H, m), 2.36-2.25 (1H, m), 2.09-1.66 (6H, m), 1.85 (3H, d, J= 1.0 Hz), 1.76 (3H, s), 1.65 (3H, s). 13 C NMR (100 MHz, CDCl3, ppm): 159.8, 158.8, 147.7, 145.0, 144.2, 137.3, 135.4, 135.3, 135.2, 132.6, 130.3, 130.3, 130.1, 128.3, 128.4, 127.2, 113.5, 112.2, 111.8, 87.9, 87.8, 87.5, 87.6, 85.6, 78.3, 74.1, 74.0, 72.4, 66.7, 57.7, 55.4, 39.0, 33.8, 33.7, 27.7, 27.6, 23.6, 22.7, 22.0, 13.8. 31 P NMR (160 MHz, CDCl3, ppm) 101.4 [α] D 20 = +15.9 (c .1, DCM). LCMS ESI (m / z): ): 922.81 [M+H] + TIFF0007625536000044.tif51128 1 H NMR (300 MHz, CDCl3, ppm): 13.5-13.3 (1H, br. s), 8.33-8.28 (2H, m), 7.82 (1H, d, J= 1.0 Hz), 7.55-7.48 (3H, m), 7.46-7.37 (6H, m), 7.35-7.29 (2H, m), 7.27-7.22 (1H, m), 6.89-6.81 (4H, m), 5.74 (1H, s), 5.28 (1H, d, J= 11.2 Hz), 4.92-4.88 (1H, m), 4.86 (1H, s), 4.65 (1H, s), 4.30 (1H, dt, J= 12.7, 3.2 Hz), 3.89 (1H, d, J= 8.2 Hz), 3.81-3.77 (1H, m), 3.79 (3H, s), 3.79 (3H, s), 3.62 (1H,. d, J= 11.0 Hz), 3.44 (1H, d, J= 11.0 Hz), 2.57-2.48 (1H, m), 2.08-1.62 (6H, m), 1.78 (3H, d, J= 1.0 Hz), 1.66 (3H, s), 1.65 (3H, s) 13 C NMR (100 MHz, CDCl3, ppm):159.8, 158.9, 158.8, 147.7, 144.9, 144.2, 137.2, 135.4, 135.3, 135.2, 132.6, 130.5, 130.1, 128.5, 128.3, 128.2, 127.2, 113.4, 113.4, 112.2, 111.9, 87.6, 87.5, 87.4, 87.1, 86.1, 78.2, 78.1, 74.5, 74.4, 72.3, 65.9, 57.9, 55.4, 38.9, 33.8, 33.7, 27.8, 27.6, 23.5, 22.7, 21.8, 13.7. 31 P NMR (160 MHz, CDCl3, ppm): 100.9. [α] D 20= +152.3 (c .1, DCM). LCMS ESI (m / z): 922.79 [M+H] + TIFF0007625536000045.tif56128 1 H NMR (300 MHz, CDCl3, ppm): 9.04 (1H, s), 8.82 (1H, s), 8.38 (1H, s), 8.06-7.97 (2H, m), 7.63-7.55 (1H, m), 7.55-7.47 (2H, m), 7.46-7.39 (2H, m), 7.36-7.25 (1H, m), 7.24-7.17 (1H, m) 6.88-6.78 (4H, m), 6.22 (1H, s), 5.32 (1H, d, J= 6.75 Hz), 4.98 (1H, s,), 4.96 (1H, s), 4.88 (1H, s), 4.42 (1H, dt, J= 12.7 3.5 Hz), 4.05 (1H, d, J= 8.4 Hz), 3.96 (1H, d, J= 8.4 Hz), 3.78 (6H, s), 3.49 (2H, s), 2.62-2.53 (1H, m), 2.35-1.2.25 (1H, m), 2.11-1.64 (5H, m), 1.78 (3H, s), 1.63 (3H, s) 13 C NMR (100 MHz, CDCl3, ppm): 164.6, 158.7, 153.2, 151.2, 149.7, 145.0, 144.3, 140.2, 135.4, 135.3, 133.7, 132.9, 130.2, 129.0, 128.2, 128.1, 128.0, 127.1, 123.4, 113.4, 111.8, 87.5, 87.4, 86.7, 85.9, 85.6, 78.7, 75.7, 75.6, 73.0, 66.6, 58.5, 55.4, 39.0, 33.8, 33.7, 27.7, 27.6, 23.5, 22.6, 22.0. 31 P NMR (160 MHz, CDCl3, ppm) 101.6. [α] D 20 = -71.7 (c .1, DCM). LCMS ESI (m / z): 932.7 [M+H] + TIFF0007625536000046.tif45128 1 H NMR (300 MHz, CDCl3, ppm): 9.1-9.0 (1H, br, s), 8.80 (1H, s), 8.33 (1H, s), 8.04-7.98 (2H, m), 7.62-7.56 (1H, m) 7.54-7.43 (4H, m), 7.37-7.26 (6H, m), 7.24-7.18 (1H, m) 6.85-6.80 (4H, m), 6.20 (1H, s), 5.42 (1H, d, J= 10.4 Hz), 4.95 (1H, s,), 4.84 (1H, s), 4.66 (1H, s), 4.31 (1H, dt, J= 12.6 3.3 Hz), 4.14 (1H, d, J= 8.4 Hz), 3.95 (1H, d, J= 8.4 Hz), 3.77 (6H, s), 3.59 (1H, d, J= 11.0 Hz), 3.53 (1H, d, J= 11.0 Hz), 2.56-2.47 (1H, m), 2.10-1.60 (6H, m), 1.64-1.60 (6H, m). 13 C NMR (100 MHz, CDCl3, ppm): 164.6, 158.7, 153.0, 151.0, 149.6, 144.8, 144.2, 140.3, 135.4, 135.3, 133.6, 132.9, 130.2, 129.9, 128.2, 128.0, 127.9, 127.0, 123.4, 113.3, 111.8, 87.2, 87.1, 86.7, 86.1, 85.9, 78.6, 78.5, 76.0, 75.9, 72.8, 65.8, 58.9, 55.2, 38.0, 33.6, 33.6, 27.6, 27.5, 23.3, 22.5, 21.6. 31P NMR (160 MHz, CDCl3, ppm): 101.1. [α] D 20 = +48.5 (c.1, DCM). LCMS ESI (m / z): 932.7 [M+H] + TIFF0007625536000047.tif50128 1 H NMR (400 MHz, CDCl3, ppm): 12.02 (1H, s), 8.97 (1H, s), 7.79 (1H, s), 7.44 - 7.38 (2H, m), 7.34 - 7.23 (6H, m), 7.22 - 7.17 (1H, m), 6.84 - 6.77 (4H, m), 5.99 (1H, d, J = 10.3 Hz), 5.83 (1H, s), 4.97 (1H, s), 4.91 - 4.88 (1H, m), 4.73 (1H, s), 4.40 (1H, dt, J = 12.6, 3.2 Hz), 4.15 (1H, d, J = 8.7 Hz), 3.95 (1H, d, J = 8.7 Hz), 3.78 (3H, s), 3.78 (3H, s), 3.55 - 3.49 (2H, m), 2.64 (1H, septet, J = 6.8 Hz), 2.60 - 2.53 (1H, m), 2.20 - 2.11 (1H, m), 2.10 - 1.65 (5H, m), 1.70 (3H, s), 1.69 (3H, s), 1.27 (3H, d, J = 6.8 Hz), 1.25 (3H, d, J = 6.8 Hz). 1313C NMR (100 MHz, CDCl3, ppm): 178.7, 158.7, 155.5, 147.5, 147.2, 144.8, 144.4, 138.6, 135.4, 130.3, 128.3, 128.0, 127.0, 122.3, 113.30, 111.9, 87.1, 87.0, 86.6, 86.5, 78.4, 73.0, 66.5, 59.3, 55.3, 38.9, 36.7, 33.9, 33.8, 27.8, 27.7, 23.5, 22.7, 21.8, 19.2, 19.0. 31 31P NMR (160 MHz, CDCl3, ppm): 100.3. [α] D 20 = +85.3 (c.1, DCM). LCMS ESI (m / z): 914.73 [M+H] + TIFF0007625536000048.tif55128 1 1H NMR (400 MHz, CDCl3, ppm): 11.99 (1H, s), 8.89 (1H, s), 7.81 (1H, s), 7.43 - 7.37 (2H, m), 7.33 - 7.23 (6H, m), 7.22 - 7.17 (1H, m), 6.84 - 6.78 (4H, m), 5.97 (1H, J = 7.7 Hz), 5.84 (1H, s), 5.01 - 4.87 (1H, m), 4.93 - 4.87 (2H, m), 4.51 (1H, dt, J = 12.7 3.5 Hz), 4.07 (1H, J = 8.5 Hz), 4.00 (1H, J = 68.5 Hz), 3.78 (6H, s), 3.48 - 3.41 (2H, m), 2.63 - 2.56 (1H, m), 2.57 (1H, septet, J = 6.9 Hz), 2.36 - 2.25 (1H, m), 2.14 - 1.64 (5H, m), 1.78 (3H, s), 1.68 (3H, s), 1.24 (3H, d, J = 6.9 Hz), 1.22 (3H, d, J = 6.9 Hz). 13 C NMR (100 MHz, CDCl3, ppm): 178.5, 158.7, 155.5, 147.5, 147.3, 144.8, 144.4, 138.5, 135.4, 130.2, 128.2, 128.0, 127.0, 122.2, 113.3, 111.9, 87.2, 87.1, 86.6, 86.5, 86.1, 79.2, 73.0, 66.6, 58.9, 55.3, 39.0, 36.6, 33.7, 33.6, 27.9, 27.8, 23.5, 22.7, 22.0, 19.1, 18.9. 31 P NMR (160 MHz, CDCl3, ppm): 103.3. [α] D 20 = -14.6 (c .1, DCM). LCMS ESI (m / z): 914.77 [M+H] + TIFF0007625536000049.tif57128 1H NMR (400 MHz, CDCl3, ppm): 9.2-9.0 (1H, br. s), 8.59 (1H, s), 8.0-7.8 (1H, br. s), 7.44-7.39 (2H, m), 7.34-7.23 (6H, m), 7.22-7.16 (1H, m), 6.75-6.77 (4H, m), 5.97 (1H, s), 5.39 (1H, d, J= 6.5 Hz), 4.98-4.92 (2H, m), 4.88-4.85 (1H, m), 4.41 (1H, dt, J= 12.5 3.5 Hz), 4.03 (1H, d, J= 8.2 Hz), 3.93 (1H, d, J= 8.2 Hz), 3.77 (6H, s), 3.51 (1H, d, J= 10.9 Hz), 3.43 (1H, d, J= 10.9 Hz), 3.10 (3H, s), 3.05 (3H, s), 2.59-2.52 (1H, m), 2.32-2.22 (1H, m), 2.07-1.65 (5H, m), 1.76 (3H, s), 1.62 (3H, s). 13 C NMR (100 MHz, CDCl3, ppm): 158.7, 158.5, 157.1, 149.6, 145.1, 144.3, 135.5, 135.4, 130.2, 128.3, 128.1, 127.0, 113.4, 111.8, 87.1, 87.1, 86.6, 86.1, 85.7, 79.1, 75.9, 75.8, 73.0, 66.3, 58.8, 55.4, 41.4, 39.0, 35.3, 33.8, 33.7, 27.8, 27.6, 23.5, 22.7, 21.9, 21.3. 31 P NMR (160 MHz, CDCl3, ppm): 100.6. [α] D 20 = -37.4 (c .1, THF). LCMS ESI (m / z): 899.71 [M+H] + TIFF0007625536000050.tif49128 1 H NMR (400 MHz, CDCl3, ppm): 9.2-9.0 (1H, br. s), 8.55 (1H, s), 8.0-7.8 (1H, br. s), 7.48-7.41 (2H, m), 7.37-7.34 (6H, m), 7.22-7.16 (1H, m), 6.84-6.74 (4H, m), 5.96 (1H, s), 5.39 (1H, d, J= 9.5 Hz), 4.96-4.75 (2H, m), 4.85-4.81 (1H, m), 4.27 (1H, dt, J= 12.7 3.1 Hz), 4.14 (1H, d, J= 8.3 Hz), 3.92 (1H, d, J= 8.3 Hz), 3.76 (6H, s), 3.64-3.50 (2H, m), 3.12 (3H, s), 3.06 (3H, s), 2.55-2.46 (1H, m), 2.09-1.58 (6H, m), 1.53 (3H, s), 1.61 (3H, s). 13 C NMR (100 MHz, CDCl3, ppm):158.7, 158.3, 157.0, 144.9, 144.4, 135.5, 135.4, 130.3, 130.2, 128.3, 128.1, 127.5, 127.1, 113.4, 113.4, 111.8, 87.0, 86.7, 86.2, 79.0, 76.3, 76.2, 73.0, 65.8, 59.4, 55.3, 41.5, 38.9, 35.3, 33.7, 33.7, 27.7, 27.5, 23.4, 22.6, 21.7. 31 P NMR (160 MHz, CDCl3, ppm) :100.0. [α] D 20 = +70.8 (c .1, THF). LCMS ESI (m / z): 899.74 [M+H] +

Claims

1. A process for preparing a compound of formula (IIIa) or (IIIb), comprising the steps of: Modified nucleosides of formula (I): wherein PG is a hydroxyl protecting group; R １ is H, and R ２ Ha -O(CH ２ ) ２ OCH ３ or R １ and R ２ together, -CH ２ -O- or -CH(CH ３ )O-, and the oxygen forms a 2'-4' bridge selected from position R ２ is bonded via Compounds of formula (IIa) or (IIb): [In the formula, R ３ is a C6-C10 aryl substituted by halo, the halo being selected from F, Cl and Br; R ４ is linear or branched C1-C6-alkyl; Reacting with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as a base (DBU is used in an amount of 0.8 equivalents relative to the nucleoside of formula (I)), Monomers of formula (IIIa) or (IIIb): The method includes the step of generating

2. The nucleoside of formula I has the following formula (Ia):

2. The method of claim 1, comprising:

3. The nucleoside of formula I has the following formula (Ib):

2. The method of claim 1, comprising:

4. The nucleoside of formula I has the following formula (Ic):

2. The method of claim 1, comprising:

5. 5. The method of any one of claims 1 to 4, wherein the nucleobase is selected from the group consisting of A, T, G, C, and 5-methylcytosine.

6. 5. The method of claim 1, wherein the nucleobase is selected from the group consisting of A, T and G.

7. The method of claim 5 or 6, wherein the exocyclic amino moiety in A, G, C or 5-methylcytosine is protected by an amino protecting group.

8. 8. The method of claim 7, wherein the amino protecting group is selected from the group consisting of DMF and iBu.

9. 9. The method of claim 1, further comprising coupling a compound of formula (IIIa) or (IIIb) as a P(V) monomer to prepare an antisense oligonucleotide.

10. R １ is H, and R ２ -O(CH ２ ) ２ OCH ３ 10. The method according to claim 1, wherein

11. R １ and R ２ Together, -CH ２ -O-, the oxygen forms a 2'-4' bridge selected from position R ２ The method according to any one of claims 1 to 9, wherein the bond is via

12. R １ and R ２ Together, -CH(CH ３ )O-, and the oxygen forms a 2'-4' bridge selected from position R ２ The method according to any one of claims 1 to 9, wherein the bond is via

13. R ３ The method of any one of claims 1 to 12, wherein is phenyl substituted by 1 to 5 F.

14. R ４ The method of any one of claims 1 to 12, wherein is methyl or ethyl.

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