4 apos is synthesized; methods of-phosphate analogue nucleotide phosphoramidite

Abstract

The present invention discloses a process for the preparation of a 4 '-phosphate analogue phosphoramidite, referred to as MeMOP, which can be used in the synthesis of oligonucleotides, such as therapeutic oligonucleotides.

Description

Technical Field

[0001] The present invention generally relates to an improved method for preparing nucleotide phosphoramidites, which nucleotide phosphoramidites include 4'-phosphate analogs, such as 2-cyanoethyl ((2R,3S,4R,5R)-2-((dimethoxyphosphoryl)methoxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-3-yl)diisopropylphosphoramidite (methoxy, phosphonate-4'-oxy-2'-O-methyluridine, MePhosphonate-4O-mU or MeMOP) and can be used for preparing therapeutic oligonucleotides. Background Art

[0002] Oligonucleotides are short polymeric sequences of nucleotides and have a wide range of applications, including being used as primers, probes, and therapeutic agents. Oligonucleotides can be chemically synthesized using various known methods, such as therapeutic oligonucleotides. Various chemical modifications can be made to one or more nucleotides in therapeutic oligonucleotides to introduce improved properties for in vivo administration (e.g., to stabilize the oligonucleotide against nucleases, increase cellular uptake of the oligonucleotide, and / or enhance other pharmacodynamic and / or pharmacokinetic properties of the oligonucleotide).

[0003] International Patent Application Publication No. WO 2018 / 045317 describes a method for preparing a 4'-phosphate analog called MeMOP to improve therapeutic oligonucleotides for in vivo administration. The method described therein uses a lead (Pb)-based reagent, which cannot be obtained on a large scale, is highly toxic, and is harmful to the environment.

[0004] In view of the above, there is a need for an improved method for preparing MeMOP. Summary of the Invention

[0005] The present invention describes a method for preparing MeMOP. In one example, a method for preparing MeMOP is provided, which method comprises the following steps:

[0006] (1) Oxidizing 1-((2R,3R,4R,5R)-4-hydroxy-5-(hydroxymethyl)-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione to obtain (2S,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-methoxytetrahydrofuran-2-carboxylic acid;

[0007] (2) Amidation and silylation of (2S,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-methoxytetrahydrofuran-2-carboxylic acid to obtain (2S,3S,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-N,4-dimethoxy-N-methyltetrahydrofuran-2-carboxamide;

[0008] (3) Addition of an organometallic moiety to (2S,3S,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-N,4-dimethoxy-N-methyltetrahydrofuran-2-carboxamide to obtain 1-((2R,3R,4S,5S)-5-acetyl-4-((tert-butyldimethylsilyl)oxy)-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione;

[0009] (4) Oxidation of 1-((2R,3R,4S,5S)-5-acetyl-4-((tert-butyldimethylsilyl)oxy)-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione to obtain (2R,3S,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-2-yl acetate;

[0010] (5) Desilylation and benzoylation of (2R,3S,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-2-yl acetate to obtain (2R,3S,4R,5R)-2-acetoxy-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-3-yl benzoate;

[0011] (6) Hydrolysis and alkylation of (2R,3S,4R,5R)-2-acetoxy-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-3-yl benzoate to obtain [(2R,3S,4R,5R)-2-(dimethoxyphosphorylmethoxy)-5-(2,4-dioxopyrimidin-1-yl)-4-methoxy-tetrahydrofuran-3-yl] benzoate;

[0012] (7) Deprotect benzoyl benzoic acid [(2R,3S,4R,5R)-2-(dimethoxyphosphorylmethoxy)-5-(2,4-dioxopyrimidin-1-yl)-4-methoxytetrahydrofuran-3-yl] ester to obtain dimethyl ((((2R,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-methoxytetrahydrofuran-2-yl)oxy)methyl)phosphonate; and

[0013] (8) Phosphorylate dimethyl ((((2R,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-methoxytetrahydrofuran-2-yl)oxy)methyl)phosphonate to obtain 2-cyanoethyl ((2R,3S,4R,5R)-2-((dimethoxyphosphoryl)methoxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-3-yl)diisopropylphosphoramidite (MeMOP).

[0014] In some instances, a compound represented by the following structure:

[0015]

[0016] can be prepared by a method comprising the following steps:

[0017] (1) Oxidize 1-((2R,3R,4R,5R)-4-hydroxy-5-(hydroxymethyl)-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione to obtain (2S,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-methoxytetrahydrofuran-2-carboxylic acid;

[0018] (2) Amidate and silylate (2S,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-methoxytetrahydrofuran-2-carboxylic acid to obtain (2S,3S,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-N,4-dimethoxy-N-methyltetrahydrofuran-2-carboxamide;

[0019] (3) An organometallic moiety is added to (2S,3S,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-N,4-dimethoxy-N-methyltetrahydrofuran-2-carboxamide to obtain 1-((2R,3R,4S,5S)-5-acetyl-4-((tert-butyldimethylsilyl)oxy)-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione;

[0020] (4) 1-((2R,3R,4S,5S)-5-acetyl-4-((tert-butyldimethylsilyl)oxy)-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione is oxidized to obtain (2R,3S,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-2-yl acetate;

[0021] (5) The (2R,3S,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-2-yl acetate is desilylated and benzoylated to obtain (2R,3S,4R,5R)-2-acetoxy-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-3-yl benzoate; and

[0022] (6) The (2R,3S,4R,5R)-2-acetoxy-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-3-yl benzoate is hydrolyzed and alkylated to obtain [(2R,3S,4R,5R)-2-(dimethoxyphosphorylmethoxy)-5-(2,4-dioxopyrimidin-1-yl)-4-methoxy-tetrahydrofuran-3-yl] benzoate.

[0023] In some instances, the step of oxidizing 1-((2R,3R,4S,5S)-5-acetyl-4-((tert-butyldimethylsilyl)oxy)-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione may include a Baeyer-Villiger reaction. In some instances, the Baeyer-Villiger reaction includes using meta-chloroperbenzoic acid (mCPBA) or urea hydrogen peroxide (UHP).

[0024] An advantage of the methods herein is that the materials used in the methods are available on a commercial scale.

[0025] An advantage of the method herein is that the materials used therein are non-toxic.

[0026] An advantage of the method herein is that the materials used therein are environmentally friendly.

[0027] An advantage of the method herein is that, compared to known methods for preparing MeMOP, it is more cost-effective and provides MeMOP with higher yield and purity.

[0028] An advantage of the method herein is that it uses a Baeyer-Villiger reaction to prepare MeMOP, which is a stereospecific process that produces only the desired β-anomer (relative to the α-anomer) at the 4'-OH position of ribose. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Advantages, effects, features, and objects other than those set forth above will become more apparent when considering the following detailed description. This detailed description refers to one or more of the following drawings, in which:

[0030] Figure 1A and Figure 1B depicts an exemplary process for preparing MeMOP. DETAILED DESCRIPTION

[0031] OVERVIEW

[0032] Chemical modifications can be introduced into therapeutic oligonucleotides to confer properties that may be required under specific conditions, such as those experienced after in vivo administration. These modifications can be introduced into the bases, sugars, and / or phosphate groups of one or more nucleotides of the oligonucleotide. These modifications include those that are designed, for example: (i) to stabilize the oligonucleotide against nucleases or other enzymes that degrade or interfere with the structure or activity of the oligonucleotide; (ii) to increase cellular uptake of the oligonucleotide; and / or (iii) to improve the pharmacokinetic properties of the oligonucleotide.

[0033] For example, a therapeutic oligonucleotide may include a hydroxyl group at the 5' end or 3' end. It is possible to replace the hydroxyl group with a phosphate group, for example, to attach a linker, an adapter, a label, and / or a targeting ligand, or to directly conjugate the oligonucleotide to another nucleic acid. Additionally, the phosphate group can enhance the interaction between the oligonucleotide and a protein such as Argonaute 2 (Ago2). However, the phosphate group at the 5' end can be readily degraded via phosphatases or other enzymes, which may limit its in vivo bioavailability. Therefore, phosphate analogs that can be incorporated into therapeutic oligonucleotides have been developed, which not only provide the functional effects of the phosphate group but are also more stable in vivo. One such phosphate analog is MeMOP, which is a 4'-phosphate analog nucleotide phosphoramidite. See International Patent Application Publication No. WO 2018 / 045317.

[0034] Abbreviations and Definitions

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the methods, exemplary methods and materials are described herein.

[0036] In addition, unless the context clearly requires that there be one and only one element, the use of the indefinite article "a" or "an" in reference to "an element" does not exclude the possibility of there being more than one element. Thus, the indefinite article "a" or "an" generally refers to "at least one".

[0037] Furthermore, the use of "including" and other forms such as "include", "includes", and "included" is not restrictive.

[0038] Certain abbreviations used herein are as follows:

[0039] "ACN" refers to acetonitrile (C2H3N); "DCM" refers to dichloromethane (CH2Cl2); "DMAP" refers to 4-dimethylaminopyridine (C7H 10 N2); "DMSO" refers to dimethyl sulfoxide (C2H6OS);

[0040] "DMHMP" refers to dimethyl (hydroxymethyl)phosphonate (C3H9O4P); "DNA" refers to deoxyribonucleic acid; "EDCI" refers to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (C8H 17N3); "ES-MS" refers to electrospray mass spectrometry; "EtOAc" refers to ethyl acetate (C4H8O2); "eq" refers to equivalent; "hr" refers to hour; "mCPBA" refers to meta-chloroperoxybenzoic acid (C7H5ClO3);

[0041] "Me" refers to methyl (-CH3); "MeOH" refers to methanol (CH4O); "min" refers to minute;

[0042] "MTBE" refers to methyl tert-butyl ether (C5H 12 O); "m / z" refers to mass-to-charge ratio; "NMI" refers to N-methylimidazole (C4H6N2); "NODMHA×HCl" refers to N,O-dimethylhydroxylamine hydrochloride (C2H8ClNO or C2H7NO*HCl); "RNA" refers to ribonucleic acid; "TBSCl" refers to tert-butyldimethylsilyl chloride (C6H 15 ClSi); "TBSO" refers to tert-butyldimethylsilyl ether; "TEA" refers to triethylamine (C6H 15 N); "TEMPO" refers to 2,2,6,6-tetramethyl-1-piperidinyloxy (C9H 18 NO); "THF" refers to tetrahydrofuran (C4H8O); "TMSOTf" refers to trimethylsilyl trifluoromethanesulfonate (C4H9F3O3SSi); "UHP" refers to urea peroxide; "V" refers to volume.

[0043] Certain definitions used herein are as follows:

[0044] As used herein, "about" refers to a range or value within a statistically significant value, such as a stated concentration, length, molecular weight, pH, sequence similarity, time range, temperature, volume, etc. Such values or ranges can be within 20%, within 15%, within 10%, or more typically within 5% of the given value or range. Alternatively, in the context of biological systems or processes, "about" can refer to within an order of magnitude, such as within five-fold or more typically within two-fold of the given value. The allowable differences encompassed by "about" will depend on the particular system being studied and will be readily understood by those skilled in the art.

[0045] As used herein, "modified nucleobase" refers to a nucleobase that includes a modified purine or pyrimidine base (e.g., adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U)). Examples of modified nucleobases include, but are not limited to, 2,6-diaminopurine and its derivatives, alkylated purines or pyrimidines, acylated purines or pyrimidines, thiolated purines or pyrimidines, and analogs thereof. Other modified nucleobases include analogs of purines and pyrimidines, including but not limited to 1-methyladenine, 2-methyladenine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentyladenine, N,N-dimethyladenine, 8-bromoadenine, 2-thiocytosine, 3-methylcytosine, 5-methylcytosine, 5-ethylcytosine, 4-acetylcytosine, 1-methylguanine, 2-methylguanine, 7-methylguanine, 2,2-dimethylguanine, 8-bromoguanine, 8-chloroguanine, 8-aminoguanine, 8-methylguanine, 8-thioguanine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, 5-ethyluracil, 5-propyluracil, 5-methoxyuracil, 5-hydroxymethyluracil, 5-(carboxyhydroxymethyl)uracil, 5-(methylaminomethyl)uracil, 5-(carboxymethylaminomethyl)-uracil, 2-thiouracil, 5-methyl-2-thiouracil, 5-(2-bromovinyl)uracil, uracil-5-oxyacetic acid, methyl uracil-5-oxyacetate, pseudouracil, 1-methylpseudouracil, queosine, hypoxanthine, xanthine, 2-aminopurine, 6-hydroxyaminopurine, nitropyrrolyl, nitroindolyl, and difluorotolyl, 6-thiopurine, and 2,6-diaminopurine nitropyrrolyl, nitroindolyl, and difluorotolyl. Alternatively, a modified nucleobase may not contain a nitrogen atom (i.e., a universal base). See also International Patent Application Publication No. WO 2003 / 040395. Alternatively, a modified nucleobase is abasic (i.e., does not include a nucleobase).

[0046] As used herein, "modified nucleoside" refers to a nucleoside that includes a modified or universal nucleobase and / or a modified sugar. The modified or universal nucleobase (also referred to herein as a base analog) may be located at the 1' position of the sugar moiety and refers to a nucleobase other than adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U) at the 1' position. In some instances, the modified nucleotide does not contain a nucleobase (is abasic). Modified sugars (also referred to herein as sugar analogs) include modified deoxyribose or ribose moieties (e.g., where the modification occurs at the 2', 3', 4', or 5' carbon positions of the sugar). Modified sugars may also include non-natural alternative carbon structures, such as those present in bridged nucleic acids ("BNAs"), locked nucleic acids ("LNAs"), and / or unlocked nucleic acids ("UNAs").

[0047] As used herein, "modified nucleotide" refers to a nucleotide that includes a modified or universal nucleobase, a modified sugar, and / or a modified phosphate or phosphate group as described above. A modified phosphate can be a modification of a phosphate or phosphate group not present in natural nucleotides and includes non-naturally occurring phosphate mimics known in the art. Modified phosphates or phosphate groups also include non-naturally occurring internucleotide linking groups, which include both phosphorus-containing linking groups and non-phosphorus-containing linking groups known in the art. Suitable modified or universal nucleobases, modified sugars, and modified phosphates or phosphate groups are described herein.

[0048] As used herein, "nucleobase" refers to a heterocyclic nitrogenous base that, when incorporated into a polymeric structure, is capable of forming Watson-Crick type hydrogen bonds and pairing with a complementary nucleobase or nucleobase analog (i.e., a derivative of a nucleobase) for stacking interactions. Natural heterocyclic nitrogenous bases include purines and pyrimidines, such as adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U).

[0049] As used herein, "nucleoside" refers to a heterocyclic nitrogenous base linked to a sugar moiety (e.g., deoxyribose, ribose, or an analog thereof) by an N-glycosidic bond.

[0050] As used herein, "nucleoside phosphoramidite" refers to a derivative of a natural or synthetic nucleoside in which the reactive hydroxyl groups and exocyclic amino groups present in the natural or synthetic nucleoside are appropriately protected to prevent unwanted side reactions during nucleic acid synthesis.

[0051] As used herein, "nucleotide" refers to a heterocyclic nitrogenous base linked to a sugar moiety (e.g., deoxyribose, ribose, or an analog thereof) by an N-glycosidic bond, where the sugar moiety is linked to a phosphate or phosphate group (i.e., a nucleoside plus a phosphate or phosphate group). As above, natural heterocyclic nitrogenous bases include adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U).

[0052] As used herein, "nucleotide phosphoramidite" refers to a derivative of a natural or synthetic nucleotide in which the reactive hydroxyl groups and exocyclic amino groups present in the natural or synthetic nucleotide are appropriately protected to prevent unwanted side reactions during nucleic acid synthesis.

[0053] As used herein, "oligonucleotide" refers to a short nucleic acid having ribonucleotides, deoxyribonucleotides, or a combination thereof (e.g., less than about 100 nucleotides in length). Oligonucleotides can be single-stranded (ss) or double-stranded (ds). Oligonucleotides may or may not have double-helical regions. As a non-limiting set of examples, oligonucleotides can be (but are not limited to) small interfering RNAs (siRNAs), microRNAs (miRNAs), short hairpin RNAs (shRNAs), dicer substrate interfering RNAs (dsiRNAs), antisense oligonucleotides (ASOs), short siRNAs, or ss siRNAs.

[0054] As used herein, "phosphate analog" refers to a chemical moiety that mimics the electrostatic and / or steric properties of a phosphate group. A phosphate analog can be located at the 5'-terminal nucleotide of an oligonucleotide in place of a 5'-phosphate and can include a phosphatase-resistant bond. Examples of phosphate analogs include, but are not limited to, 5'-phosphonates such as 5'-methylenephosphonate (5'-MP) and 5'-(E)-vinylphosphonate (5'-VP). Similarly, a phosphate analog can be located at the 4'-carbon site of the 5'-terminal nucleotide of the sugar (referred to as a "4'-phosphate analog"). An example of a 4'-phosphate analog is oxy-methylenephosphonate, where the oxygen atom of the oxy-methyl group is bonded to the sugar moiety (e.g., at its 4'-carbon) or an analog thereof. See, for example, International Patent Application Publication No. WO 2018 / 045317. Other modifications have been developed for the 5'-terminus of oligonucleotides (see, for example, International Patent Application No. WO 2011 / 133871; U.S. Patent No. 8,927,513; and Prakash et al. (2015) Nuc. Acids Res. 43:2993-3011).

[0055] As used herein, "phosphoramidite" refers to a nitrogen-containing, trivalent phosphorus derivative that can have the formula (RO)2PNR2.

[0056] As used herein, "protecting group" refers to a group that reversibly renders a functional group non-reactive under certain conditions of a desired reaction. After the desired reaction, the protecting group can be removed to deprotect the protected functional group. The protecting group should be removable under conditions that do not degrade a substantial proportion of the synthesized molecules (i.e., oligonucleotides).

[0057] As used herein, "ribonucleotide" refers to a natural or modified nucleotide having a hydroxyl group at the 2'-site of the sugar moiety.

[0058] As used herein, "targeting ligand" refers to a chemical moiety that facilitates entry of an oligonucleotide (such as an RNAi agent) into a cell. It can be a compound (such as an amino sugar, carbohydrate, cholesterol, lipid, or polypeptide) that selectively binds to a cognate compound (e.g., a receptor) of a tissue or cell of interest and can be conjugated to another substance to target the other substance to the tissue or cell of interest. For example, for the purpose of targeting an oligonucleotide to a specific tissue or cell of interest, a targeting ligand can be conjugated to the oligonucleotide. A targeting ligand can selectively bind to a cell surface receptor. Thus, when conjugated to an oligonucleotide, the targeting ligand facilitates delivery of the oligonucleotide into a specific cell by selective binding to a receptor presented on the cell surface and intracellular endocytosis of the complex comprising the oligonucleotide, targeting ligand, and receptor by the cell. In addition, the targeting ligand can conjugate the oligonucleotide via a linker that cleaves after or during cell internalization to release the oligonucleotide from the targeting ligand in the cell.

[0059] Composition

[0060] MeMOP:

[0061] The structure of MeMOP is as follows:

[0062] See International Patent Application Publication No. WO 2018 / 045317.

[0063] MeMOP-modified oligonucleotides:

[0064] MeMOP can be incorporated into oligonucleotides, such as therapeutic oligonucleotides. In some instances, MeMOP can bind to the 4'-carbon of the sugar moiety of a nucleotide within the oligonucleotide (e.g., ribose, deoxyribose, or an analogue thereof). In some instances, MeMOP can be incorporated at the 3'-end of the oligonucleotide. In other instances, MeMOP can be incorporated at the 5'-end of the oligonucleotide. In other instances, MeMOP can be incorporated at both the 5'-end and 3'-end of the oligonucleotide. In other instances, MeMOP can be incorporated at one or more internal sites of the oligonucleotide. See, for example, International Patent Application Publication Nos. WO 2018 / 045317, WO 2021 / 188795, WO 2022 / 032288, and WO 2022 / 221430.

[0065] Oligonucleotides (e.g., ds oligonucleotides, such as MeMOP-modified oligonucleotides) can be prepared using methods and / or techniques known to those of ordinary skill in the art (such as conventional solid-phase nucleic acid synthesis). Oligonucleotide nucleotides can be assembled using standard nucleotide or nucleoside precursors (e.g., phosphoramidites) on a suitable nucleic acid synthesizer. Automated nucleic acid synthesizers, including DNA / RNA synthesizers, are commercially available from, for example, Applied Biosystems (Foster City, CA), BioAutomation (Irving, TX), and GE Healthcare Life Sciences (Pittsburgh, PA).

[0066] In some instances, oligonucleotide synthesis steps can be carried out in an alternating order to obtain the desired compound. Other synthetic chemical transformations, protecting groups (e.g., for hydroxyl, amino, etc. present on the bases), and protecting group methods (protection and deprotection) suitable for oligonucleotide synthesis are known in the art and are described, for example, in Larock, “Comprehensive Organic Transformations”, VCH Publishers (1989); Greene and Wuts, “Protective Groups in Organic Synthesis”, 2nd ed., John Wiley & Sons (1991); Fieser and Fieser, “Fieser & Fieser's Reagents for Organic Synthesis”, John Wiley & Sons (1994); and Paquette, ed., “Encyclopedia of Reagents for Organic Synthesis”, John Wiley & Sons (1995).

[0067] Pharmaceutical composition:

[0068] MeMOP-modified oligonucleotides (or pharmaceutically acceptable salts thereof, such as trifluoroacetate, acetate, or hydrochloride salts) can be incorporated into a pharmaceutical composition that contains an effective amount of the MeMOP-containing oligonucleotide and a pharmaceutically acceptable carrier, delivery agent, or excipient. See, for example, International Patent Application Publication Nos. WO 2018 / 045317, WO 2021 / 188795, WO 2022 / 032288, and WO 2022 / 221430.

[0069] A variety of formulations have been developed to facilitate the use of oligonucleotides. In some instances, formulations can be used to deliver oligonucleotides to an individual or cellular environment that minimize degradation, facilitate delivery and / or uptake, or provide another beneficial property to the oligonucleotides in the formulation. In some instances, oligonucleotides can be formulated in buffer solutions (such as phosphate buffered saline solutions), liposomes, micellar structures, and capsids.

[0070] In some instances, oligonucleotides can react with inorganic and organic acids / bases to form pharmaceutically acceptable acid / base addition salts. In some instances, forming pharmaceutically acceptable acid / base addition salts can improve the in vivo compatibility and / or performance of the oligonucleotides. Pharmaceutically acceptable salts and general methods for preparing them are well known in the art (see, for example, Stahl et al., “Handbook of Pharmaceutical Salts: Properties, Selection and Use,” 2nd Revised Edition (Wiley-VCH, 2011)). Pharmaceutically acceptable salts used herein include sodium salts, trifluoroacetates, hydrochlorides, and acetates.

[0071] In some instances, pharmaceutical compositions can be formulated to be compatible with the intended route of administration. Routes of administration include but are not limited to parenteral (e.g., intravenous, intramuscular, intraperitoneal, intradermal, and subcutaneous), oral (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration.

[0072] In addition, those skilled in the art of preparing such pharmaceutical formulations can consider factors such as solubility, bioavailability, biological half-life, route of administration, product shelf-life, and other pharmacological considerations, and thus, various dosages and treatment regimens may be required.

[0073] In some instances, a pharmaceutical composition can comprise one or more additional therapeutic agents.

[0074] Methods

[0075] Method for preparing MeMOP:

[0076] The method for preparing MeMOP or its salt can include the steps described herein, which can be, but need not be, in the order described. However, other orders are also conceivable. In addition, individual or multiple steps can be parallel and / or overlapping in time, and / or carried out individually or in multiple repeated steps. The products of the following steps can be recovered by conventional methods, including chromatography, crystallization, evaporation, extraction, filtration, precipitation, and grinding.

[0077] In one instance, MeMOP can be prepared according to the following method, which can include the following steps:

[0078] (1) Oxidize 1-((2R,3R,4R,5R)-4-hydroxy-5-(hydroxymethyl)-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione,

[0079]

[0080] to obtain (2S,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-methoxytetrahydrofuran-2-carboxylic acid,

[0081]

[0082] (2) Amidate and silylate (2S,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-methoxytetrahydrofuran-2-carboxylic acid to obtain (2S,3S,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-N,4-dimethoxy-N-methyltetrahydrofuran-2-carboxamide,

[0083]

[0084] (3) Add an organometallic moiety (e.g., an organomagnesium compound) to (2S,3S,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-N,4-dimethoxy-N-methyltetrahydrofuran-2-carboxamide to obtain 1-((2R,3R,4S,5S)-5-acetyl-4-((tert-butyldimethylsilyl)oxy)-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione,

[0085]

[0086] (4) Oxidize (e.g., via the Baeyer-Villiger reaction) 1-((2R,3R,4S,5S)-5-acetyl-4-((tert-butyldimethylsilyl)oxy)-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione to obtain (2R,3S,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-2-yl acetate,

[0087]

[0088] (5) Desilylation and benzoylation of (2R,3S,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-2-yl acetate to obtain (2R,3S,4R,5R)-2-acetoxy-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-3-yl benzoate,

[0089]

[0090] (6) Hydrolysis and alkylation of (2R,3S,4R,5R)-2-acetoxy-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-3-yl benzoate to obtain [(2R,3S,4R,5R)-2-(dimethoxyphosphorylmethoxy)-5-(2,4-dioxopyrimidin-1-yl)-4-methoxy-tetrahydrofuran-3-yl] benzoate,

[0091]

[0092] (7) Debenzoylation of [(2R,3S,4R,5R)-2-(dimethoxyphosphorylmethoxy)-5-(2,4-dioxopyrimidin-1-yl)-4-methoxy-tetrahydrofuran-3-yl] benzoate to obtain dimethyl (((2R,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-methoxytetrahydrofuran-2-yl)oxy)methyl)phosphonate,

[0093]

[0094] (8) Phosphorylation of (2R,3S,4R,5R)-2-acetoxy-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-3-yl benzoate to obtain 2-cyanoethyl ((2R,3S,4R,5R)-2-((dimethoxyphosphoryl)methoxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-3-yl) diisopropylphosphoramidite (MeMOP),

[0095]

[0096] In some instances, [(2R,3S,4R,5R)-2-(dimethoxyphosphorylmethoxy)-5-(2,4-dioxopyrimidin-1-yl)-4-methoxy-tetrahydrofuran-3-yl] benzoate,

[0097]

[0098] It can be prepared according to the following method, which may include the following steps:

[0099] (1) Oxidize 1-((2R,3R,4R,5R)-4-hydroxy-5-(hydroxymethyl)-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione to obtain (2S,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-methoxytetrahydrofuran-2-carboxylic acid;

[0100] (2) Amidate and silylate (2S,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-methoxytetrahydrofuran-2-carboxylic acid to obtain (2S,3S,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-N,4-dimethoxy-N-methyltetrahydrofuran-2-carboxamide;

[0101] (3) Add an organometallic moiety to (2S,3S,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-N,4-dimethoxy-N-methyltetrahydrofuran-2-carboxamide to obtain 1-((2R,3R,4S,5S)-5-acetyl-4-((tert-butyldimethylsilyl)oxy)-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione;

[0102] (4) Oxidize 1-((2R,3R,4S,5S)-5-acetyl-4-((tert-butyldimethylsilyl)oxy)-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione to obtain (2R,3S,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-2-yl acetate;

[0103] (5) Desilylate and benzoylate (2R,3S,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-2-yl acetate to obtain (2R,3S,4R,5R)-2-acetoxy-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-3-yl benzoate; and

[0104] (6) Hydrolyze and alkylate benzoic acid (2R,3S,4R,5R)-2-acetoxy-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-3-yl ester to obtain benzoic acid [(2R,3S,4R,5R)-2-(dimethoxyphosphorylmethoxy)-5-(2,4-dioxopyrimidin-1-yl)-4-methoxy-tetrahydrofuran-3-yl] ester.

[0105] In some instances, the step of oxidizing 1-((2R,3R,4S,5S)-5-acetyl-4-((tert-butyldimethylsilyl)oxy)-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione can be a Baeyer-Villiger reaction, which is a stereospecific process that produces the desired β-anomer only at the 4'-OH position of the ribose. In some instances, the Baeyer-Villiger reaction can be carried out using meta-chloroperoxybenzoic acid (mCPBA) or urea hydrogen peroxide (UHP).

[0106] Examples

[0107] The following non-limiting examples are provided for illustrative purposes only and do not limit the scope of the invention.

[0108] Example 1: Synthesis of (2S,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-methoxytetrahydrofuran-2-carboxylic acid

[0109]

[0110] Method: At 15 °C to 25 °C, 1-((2R,3R,4R,5R)-4-hydroxy-5-(hydroxymethyl)-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (1.0 eq) was slowly added in portions to a solution of ACN (5V), H2O (5V), TEMPO (0.5 eq), NaHCO3 (4.0 eq), and mCPBA (4.0 eq). The resulting mixture was warmed to 30 °C to 40 °C and stirred at room temperature for 4 hours. The mixture was cooled, and the solution was quenched by adding 20% aqueous NaHSO3. Subsequently, the mixture was diluted with EtOAc (10V), and the pH was adjusted to 1 to 2 with 36% aqueous HCl. The mixture was filtered to obtain a first wet filter cake. The first filtrate was collected, and the organic phase was removed. The aqueous phase was concentrated to 3V to form a suspension. The suspension was filtered to obtain a second wet filter cake. The first and second wet filter cakes were combined and washed successively with EtOAc (4V) and water (1V). The solid was dried in vacuo to obtain the title compound as a solid (70%).

[0111] Results: 11H-NMR (DMSO-d6) δ 3.32 (s, 3H, OCH3), 3.83 (q, 1H, C4’H), 4.35 (s, 2H, C2’H, C3’H), 5.76 (q, 1H, C5-H), 5.84 (br, 1H, C3’OH), 6.05 (d, 1H, C1’H), 8.14 (d, 1H, C6-H), 11.40 (s, 1H, NH). ES-MS m / z 271.00 (M-H).

[0112] Example 2: Synthesis of (2S,3S,4R,5R)-3-((tert-Butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-N,4-dimethoxy-N-methyltetrahydrofuran-2-carboxamide

[0113]

[0114] Method: Dissolve (2S,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-methoxytetrahydrofuran-2-carboxylic acid (1.0 eq) from Example 1 in a mixture of ACN (10V), DMAP (0.13 eq), and pyridine (3.3 eq). Slowly add EDCI (1.6 eq) in portions while maintaining the temperature at -5°C to 5°C. Slowly add N,O-NODMHA*HCl (1.5 eq) in portions while maintaining the temperature at -5°C to 5°C. Warm the solution to 10°C to 20°C and stir for 2 hours. Add imidazole (3.0 eq) and TBSCl (2.5 eq) and stir at 10°C to 20°C for 16 hours. Quench the mixture by adding H2O (2V) and concentrate (3V). Extract the mixture with EtOAc (10V) and then wash successively with 1M aqueous HCl (5V × 2), saturated aqueous NaHCO3 (5V), and 8% aqueous Na2SO4 (5V). Concentrate the mixture to 4V and add n-heptane (10V). Subsequently, concentrate the mixture to 10V to crystallize the mixture. Filter the mixture and then dry to obtain the title compound as a solid (80%).

[0115] Results: 11H NMR (DMSO-d6) δ 0.11 (d, 6H, SiCH3), 0.988 (s, 9H, SiCCH3,), 3.17 (s, 3H, NCH3), 3.29 (s, 3H, C2’OCH3), 3.71 (s, 3H, NOCH3), 3.85 (t, 1H, C4’H), 4.47 (dd, 1H, C3’H), 4.76 (s, 1H, C2’H), 5.76 (d, 1H, C5-H), 6.06 (d, 1H, C1’H), 8.50 (d, 1H, C6-H), 11.39 (s, 1H, CONH). ES-MS m / z 430.30 (M+H).

[0116] Example 3: Synthesis of 1 - ((2R,3R,4S,5S)-5-acetyl-4-((tert-butyldimethylsilyl)oxy)-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione

[0117]

[0118] Method: Dissolve (2S,3S,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-N,4-dimethoxy-N-methyltetrahydrofuran-2-carboxamide (1.0 eq) from Example 2 in 2-MeTHF (15V). Dropwise add a solution of 3N MeMgCl in THF (2.3 eq) while maintaining the temperature at -20 °C to 10 °C. Stir the resulting mixture at -20 °C to 10 °C for 16 h. Add H2O (1V) and adjust the pH to 4 to 5 with 1M aqueous HCl. Separate the two phases and wash the organic phase successively with saturated aqueous NaHCO3 (5V×2) and 8% aqueous Na2SO4 (5V). Concentrate the mixture to 3V and add n-heptane (10V) to crystallize the mixture. Filter the mixture and dry the cake to obtain the title compound as a solid (85%).

[0119] Result: Weight: 1 1H NMR (CDCl3) δ 0.14 (s, 6H, SiCH3), 0.93 (s, 9H, SiCCH3,), 2.28 (s, 3H, COCH3), 3.49 (s, 3H, C2’OCH3), 3.72 (t, 1H, C4’H), 4.14 (dd, 1H, C3’H), 4.64 (d, 1H, C2’H), 5.80 (d, 1H, C5-H), 5.94 (d, 1H, C1’H), 8.11 (d, 1H, C6-H), 8.60 (s, 1H, CONH). ES-MS m / z 385.20 (M+H).

[0120] Example 4: Synthesis of (2R,3S,4R,5R)-3-((tert-Butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-2-yl acetate

[0121]

[0122] Method: Dissolve 1-((2R,3R,4S,5S)-5-acetyl-4-((tert-butyldimethylsilyl)oxy)-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (1.0 eq) from Example 3 in ACN (10 V). Add NaHCO3 solution (3.0 eq), and then slowly add mCPBA (1.5 eq) at 5 °C to 15 °C. Stir the mixture at room temperature for 3 hours, then dilute with water (6 V) and quench with 20% aqueous NaHSO3 solution. Extract the solution with EtOAc (10 V) and separate the layers. Wash the organic phase successively with saturated aqueous NaHCO3 solution (5 V × 2) and 8% aqueous Na2SO4 solution (5 V). Concentrate the mixture to 3 V and add n-heptane (14 V). Filter the mixture and dry the cake to obtain the title compound as a solid (85%).

[0123] Results: 1 1H NMR (CDCl3) δ 0.15 (s, 6H, SiCH3), 0.93 (s, 9H, SiCCH3,), 2.17 (s, 3H, COCH3), 3.41 (s, 3H, C2’OCH3), 3.90 (dd, 1H, C3’H), 4.26 (d, 1H, C2’H), 5.80 (d, 1H, C5-H), 6.03 (s, 1H, C4’H), 6.26 (d, 1H, C1’H), 7.39 (d, 1H, C6-H), 8.28 (br, 1H, CONH). ES-MS m / z 341.20 (M + H).

[0124] Example 5: Synthesis of (2R,3S,4R,5R)-2-acetoxy-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-3-yl benzoate

[0125]

[0126] Method: Dissolve acetic acid (2R,3S,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-2-yl ester (1.0 eq) of Example 4 in ACN (10 V), TEA (6.0 eq) and 3HF·TEA (3.0 eq). Warm the mixture to 25 °C to 35 °C and then stir for 6 h. Add DMAP (0.1 eq) and Bz2O (1.5 eq), and stir the mixture at 20 °C to 30 °C for 2 h. Add H2O (2 V), and concentrate the mixture to 4 V in vacuo. Extract the mixture with EtOAc (8 V) and separate the layers. Wash the organic phase successively with saturated aqueous NaHCO3 solution (5 V), 1 M aqueous HCl solution (5 V) and 8% aqueous Na2SO4 solution (5 V). Concentrate the mixture to 2 V and add EtOAc (5 V). Concentrate the mixture to 2 V and add n-heptane (10 V). Stir the suspension at 0 °C to 10 °C and then filter. Dry the cake to obtain the title compound as a solid (82%).

[0127] Results: 1 1H NMR (CDCl3) δ 2.21 (s, 3H, COCH3), 3.44 (s, 3H, C2’OCH3), 4.26 (d, 1H, C2’H), 5.60 (d, 1H, C3’H), 5.85 (dd, 1H, C5-H), 6.36 (d, 1H, C1’H), 6.38 (s, 1H, C4’H), 7.37 - 7.65 (m, 6H, C6-H, Ph-H), 8.17 (s, 1H, CONH). ES-MS m / z 389.10 (M-H).

[0128] Example 6: Synthesis of [(2R,3S,4R,5R)-2-(dimethoxyphosphorylmethoxy)-5-(2,4-dioxopyrimidin-1-yl)-4-methoxy-tetrahydrofuran-3-yl] benzoate,

[0129]

[0130] Method: Dissolve benzoic acid (2R,3S,4R,5R)-2-acetoxy-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-3-yl ester (1.0 eq) from Example 5 in ACN (5V). Add TMSOTf (2.4 eq) dropwise while maintaining the temperature at -15 °C to -5 °C. Add DMHMP (5.0 eq) dropwise to the mixture while maintaining the temperature at -15 °C to -5 °C. Warm the solution to 15 °C to 25 °C and then stir for 16 h. Add saturated aqueous NaHCO3 (6V), and extract the mixture with DCM (4V×2). Wash the organic phase with H2O (10V×5) and then concentrate to give the title compound as an oil (85%).

[0131] Results: 1 1H NMR (CDCl3) δ 3.41 (s, 3H, C2’OCH3), 3.84 - 4.09 (m, 8H, PCH2, POCH3), 4.26 (dd, 1H, C2’H), 5.23 (s, 1H, C4’H), 5.59 (d, 1H, C3’H), 5.90 (dd, 1H, C5-H), 6.50 (d, 1H, C1’H), 7.48 - 8.09 (m, 6H, C6-H, Ph-H), 8.11 (s, 1H, CONH). ES-MS m / z 369.10 (M-H).

[0132] Example 7: Synthesis of dimethyl ((((2R,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-methoxytetrahydrofuran-2-yl)oxy)methyl)phosphonate,

[0133]

[0134] Method: Dissolve benzoic acid [(2R,3S,4R,5R)-2-(dimethoxyphosphorylmethoxy)-5-(2,4-dioxopyrimidin-1-yl)-4-methoxy-tetrahydrofuran-3-yl] ester (1.0 eq) from Example 6 in MeOH (10V), then add K2CO3 (1.5 eq). Stir the mixture at 20 °C to 30 °C for 6 h and filter. Subsequently, adjust the pH to about 4 to 5 by adding formic acid. Concentrate the mixture to a gummy solid and purify by silica gel chromatography using a gradient of CH2Cl2:MeOH from 60:1 to 30:1 to give the title compound as a solid (60%).

[0135] Results: 11H NMR (CD3OD) δ 3.43 (s, 3H, C2’OCH3), 3.82 - 3.86 (m, 6H, POCH3), 4.00 - 4.10 (m, 3H, C3’H, PCH2), 4.26 (dd, 1H, C2’H), 5.05 (s, 1H, C4’H), 5.81 (dd, 1H, C5-H), 6.32 (d, 1H, C1’H), 7.70 (d, 1H, C6-H). ES-MS m / z 367.20 (M+1).

[0136] Example 8: Synthesis of 2-cyanoethyl ((2R,3S,4R,5R)-2-((dimethoxyphosphoryl)methoxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-3-yl)diisopropylphosphoramidite (MeMOP)

[0137]

[0138] Method: Dissolve dimethyl (((2R,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-methoxytetrahydrofuran-2-yl)oxy)methyl)phosphonate (1.0 eq) from Example 7 in DCM (3V). Add a solution of DCM (10V) and NMI (0.3 eq) to the mixture, and then add tetrazole (0.7 eq) and 3-((bis(diisopropylamino)phosphoryl)oxy)propanenitrile (1.3 eq) dropwise while maintaining the temperature at 0 °C to 10 °C. Warm the solution to 15 °C to 25 °C, stir for 3 hours, and then add 8% aqueous NaHCO3 solution (8V). Separate the layers and wash the organic phase successively with 8% aqueous NaHCO3 solution (5V) and H2O (5V × 4). Concentrate the mixture to approximately 1.5V and add MTBE (15V). Filter the mixture and dry the cake to obtain the title compound as a solid (64%).

[0139] Results: 11H NMR (CD3CN) δ 1.22 (m, 12H, NCCH3), 2.73 (m, 2H, 2H, NCH), 3.39 (d, 3H, C2’OCH3), 3.68 - 3.76 (m, 2H, CNCH2), 3.76 - 3.80 (m, 6H, POCH3), 3.80 - 3.95 (m, 2H, OCH2), 2.94 - 4.07 (m, 2H, PCH2), 4.24 (dd, 1H, C2’H), 4.45 (dd, 1H, C3’H), 5.15 (d, 1H, C4’H), 5.74 (d, 1H, C5-H), 6.22 (d, 1H, C1’H), 7.60 (dd, 1H, C6-H), 9.09 (s, 1H, CONH). ES-MS (+ve mode) = 567.20 (M+1).

Claims

1. A method for preparing a compound represented by the following structure: The method comprises the following steps: Oxidizing 1-((2R,3R,4R,5R)-4-hydroxy-5-(hydroxymethyl)-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione to obtain (2S,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-methoxytetrahydrofuran-2-carboxylic acid; Amidating and silylating (2S,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-methoxytetrahydrofuran-2-carboxylic acid to obtain (2S,3S,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-N,4-dimethoxy-N-methyltetrahydrofuran-2-carboxamide; Adding an organometallic moiety to (2S,3S,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-N,4-dimethoxy-N-methyltetrahydrofuran-2-carboxamide to obtain 1-((2R,3R,4S,5S)-5-acetyl-4-((tert-butyldimethylsilyl)oxy)-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione; Oxidizing 1-((2R,3R,4S,5S)-5-acetyl-4-((tert-butyldimethylsilyl)oxy)-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione to obtain (2R,3S,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-2-yl acetate; Desilylating and benzoylating (2R,3S,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-2-yl acetate to obtain (2R,3S,4R,5R)-2-acetoxy-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-3-yl benzoate; Hydrolyzing and alkylating (2R,3S,4R,5R)-2-acetoxy-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-3-yl benzoate to obtain [(2R,3S,4R,5R)-2-(dimethoxyphosphorylmethoxy)-5-(2,4-dioxopyrimidin-1-yl)-4-methoxy-tetrahydrofuran-3-yl] benzoate; Deprotect benzoyl benzoic acid [(2R,3S,4R,5R)-2-(dimethoxyphosphorylmethoxy)-5-(2,4-dioxopyrimidin-1-yl)-4-methoxytetrahydrofuran-3-yl] ester to obtain dimethyl ((((2R,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-methoxytetrahydrofuran-2-yl)oxy)methyl)phosphonate; and Phosphorylate dimethyl ((((2R,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-methoxytetrahydrofuran-2-yl)oxy)methyl)phosphonate to obtain 2-cyanoethyl ((2R,3S,4R,5R)-2-((dimethoxyphosphoryl)methoxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-3-yl)diisopropylphosphoramidite (MeMOP).

2. A method for preparing a compound represented by the following structure: The method comprises the following steps: Oxidize 1-((2R,3R,4R,5R)-4-hydroxy-5-(hydroxymethyl)-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione to obtain (2S,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-methoxytetrahydrofuran-2-carboxylic acid; Amidate and silylate (2S,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-methoxytetrahydrofuran-2-carboxylic acid to obtain (2S,3S,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-N,4-dimethoxy-N-methyltetrahydrofuran-2-carboxamide; Add an organometallic moiety to (2S,3S,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-N,4-dimethoxy-N-methyltetrahydrofuran-2-carboxamide to obtain 1-((2R,3R,4S,5S)-5-acetyl-4-((tert-butyldimethylsilyl)oxy)-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione; Oxidize 1-((2R,3R,4S,5S)-5-acetyl-4-((tert-butyldimethylsilyl)oxy)-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione to obtain (2R,3S,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-2-yl acetate; Desilylation and benzoylation of (2R,3S,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-2-yl acetate to obtain (2R,3S,4R,5R)-2-acetoxy-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-3-yl benzoate; and Hydrolysis and alkylation of (2R,3S,4R,5R)-2-acetoxy-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-3-yl benzoate to obtain [(2R,3S,4R,5R)-2-(dimethoxyphosphorylmethoxy)-5-(2,4-dioxopyrimidin-1-yl)-4-methoxy-tetrahydrofuran-3-yl] benzoate.

3. The method according to claim 1 or claim 2, wherein the step of oxidizing 1-((2R,3R,4S,5S)-5-acetyl-4-((tert-butyldimethylsilyl)oxy)-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione comprises a Baeyer-Villiger reaction.

4. The method according to claim 3, wherein the Baeyer-Villiger reaction comprises using meta-chloroperoxybenzoic acid (mCPBA) or urea peroxide (UHP).

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