Method for synthesizing 4'-phosphate analog nucleotide phosphoramidites

Abstract

Disclosed is a method for making the 4'-phosphate analog phosphoramidite known as MeMOP, which can be used in the synthesis of oligonucleotides, such as therapeutic oligonucleotides.

Description

[Technical Field]

[0001] The present disclosure relates generally to improved methods for making nucleotide phosphoramidites containing 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), which can be used to make therapeutic oligonucleotides. [Background technology]

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

[0003] WO 2018 / 045317 describes a method for making 4'-phosphate analogs known as MeMOP to improve therapeutic oligonucleotides for in vivo administration. The method described therein uses lead (Pb)-based reagents that are not available on a large scale, are highly toxic, and are harmful to the environment.

[0004] In view of the above, there is a need for improved methods of producing MeMOPs. Summary of the Invention

[0005] The present disclosure describes a method for making MeMOP. In one example, the method comprises the following steps: (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; (2) 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; (3) 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; (4) 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; (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; (6) 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; (7) debenzoylating [(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; (8) phosphorylating 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).

[0006] In some examples, the compound represented by the structure:

[0007] [ka] The following steps: (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; (2) 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; (3) 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; (4) 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; (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; (6) 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.

[0008] In some examples, 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 include a Baeyer-Villiger reaction. In some examples, the Baeyer-Villiger reaction includes the use of meta-chloroperoxybenzoic acid (mCPBA) or urea hydrogen peroxide (UHP).

[0009] An advantage of the methods herein is that the materials used in the methods are available on a commercial scale.

[0010] An advantage of the methods herein is that the materials used therein are non-toxic.

[0011] An advantage of the method herein is that the materials used therein are not harmful to the environment.

[0012] An advantage of the methods herein is that they are more cost-effective and provide MeMOP in higher yield and purity compared to known methods of making MeMOP.

[0013] An advantage of the methods herein is that they use the Baeyer-Villiger reaction to make MeMOP, a stereospecific process that gives exclusively the desired β-anomer (versus the α-anomer) at the 4'OH position of ribose. [Brief explanation of the drawings]

[0014] Further advantages, benefits, features and objects will become more readily apparent from a consideration of the following detailed description, which refers to the following drawings. [Figure 1A] 1 shows an exemplary scheme for making MeMOP. [Figure 1B] 1 shows an exemplary scheme for making MeMOP. DETAILED DESCRIPTION OF THE INVENTION

[0015] overview Chemical modifications can be introduced into therapeutic oligonucleotides to confer desirable properties under certain conditions, such as those experienced after their in vivo administration. These modifications can be introduced into the base, sugar, and / or phosphate groups of one or more nucleotides of the oligonucleotide. Such modifications include, for example, those designed to (i) stabilize the oligonucleotide against nucleases or other enzymes that degrade or interfere with the structure or activity of the oligonucleotide, (ii) increase cellular uptake of the oligonucleotide, and / or (iii) improve the pharmacokinetic properties of the oligonucleotide.

[0016] For example, therapeutic oligonucleotides may contain a hydroxyl group at the 5' or 3' end. The hydroxyl group can be replaced with a phosphate group to attach, for example, a linker, an adapter, a label, and / or a targeting ligand, or to directly ligate the oligonucleotide to another nucleic acid. Furthermore, the phosphate group can enhance the interaction between the oligonucleotide and proteins such as Argonaute 2 (Ago2). However, phosphate groups at the 5' end are susceptible to degradation by phosphatases or other enzymes, which can limit their in vivo bioavailability. Therefore, phosphate analogs have been developed that not only provide the functional effects of a phosphate group but can also be incorporated into more stable therapeutic oligonucleotides in vivo. One such phosphate analog is MeMOP, a 4'-phosphate analog nucleotide phosphoramidite. See International Publication No. WO 2018 / 045317.

[0017] Abbreviations and Definitions 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.

[0018] In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one element is present, unless the context clearly requires that there is one and only one element. Thus, the indefinite article "a" or "an" normally means "at least one."

[0019] Furthermore, the use of "including" and other forms such as "include," "includes," and "included" is not limiting.

[0020] Certain abbreviations used herein are as follows:

[0021] "ACN" refers to acetonitrile (C2H3N), "DCM" refers to dichloromethane (CH2Cl2), and "DMAP" refers to 4-dimethylaminopyridine (C7H 10 N2), "DMSO" refers to dimethyl sulfoxide (C2H6OS), "DMHMP" refers to dimethyl P-(hydroxymethyl)phosphonate (C3H9O4P), "DNA" refers to deoxyribonucleic acid, and "EDCI" refers to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (C8H 17 N3), "ES / MS" refers to electrospray mass spectrometry, "EtOAc" refers to ethyl acetate (C4H8O2), "eq" refers to equivalents, "hr" refers to hours, "mCPBA" refers to meta-chloroperoxybenzoic acid (C7H5ClO3), "Me" refers to methyl (-CH3), "MeOH" refers to methanol (C4O), "min" refers to minutes, and "MTBE" refers to methyl tert-butyl ether (C5H 12 "m / z" refers to mass-to-charge ratio, "NMI" refers to N-methylimidazole (C4H6N2), and "NODMHA" refers to methylimidazole (C4H6N2). * HCl" is N,O-dimethylhydroxylamine hydrochloride (C2H8ClNO or C2H7NO * HCl), "RNA" refers to ribonucleic acid, and "TBSCl" refers to tert-butyldimethylsilyl chloride (CH 15 ClSi), "TBSO" refers to tert-butyldimethylsilyl ether, and "TEA" refers to triethylamine (CH 15 N), and "TEMPO" refers to 2,2,6,6-tetramethyl-1-piperidinyloxy (CH 18 NO), "THF" refers to tetrahydrofuran (C4H8O), "TMSOTf" refers to trimethylsilyl trifluoromethanesulfonate (C4H9F3O3SSi), "UHP" refers to urea hydrogen peroxide, and "V" refers to volume.

[0022] Certain definitions used herein are as follows:

[0023] As used herein, "about" means within a statistically significant range of values, e.g., a specified concentration, length, molecular weight, pH, sequence similarity, time frame, temperature, volume, etc. Such values ​​or ranges can be within 20%, within 15%, within 10%, or more typically within 5% of a given value or range. Alternatively, with respect to biological systems or processes, "about" can mean within an order of magnitude, e.g., within 5-fold, or more typically within 2-fold, of a given value. The acceptable variation encompassed by "about" will depend on the system under study and can be readily appreciated by one of ordinary skill in the art.

[0024] As used herein, "modified nucleobase" means a nucleobase comprising 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, diaminopurine and derivatives thereof, alkylated purines or pyrimidines, acylated purines or pyrimidines, thiolated purines or pyrimidines, etc. Other modified nucleobases include 1-methyladenine, 2-methyladenine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentyladenine, N,N-dimethyladenine, 8-bromoadenine, 2-thiocytosine, 3-methylcytosine, 5-methylcytosine, 5-ethycytosine, 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- Purine and pyrimidine analogs include, but are not limited to, (carboxyhydroxymethyl)uracil, 5-(methylaminomethyl)uracil, 5-(carboxymethylaminomethyl)-uracil, 2-thiouracil, 5-methyl-2-thiouracil, 5-(2-bromovinyl)uracil, uracil-5-oxyacetic acid, uracil-5-oxyacetic acid methyl ester, pseudouracil, 1-methylpseudouracil, queosine, hypoxanthine, xanthine, 2-aminopurine, 6-hydroxyarninopurine, nitropyrrolyl, nitroindolyl, and difluorotolyl, 6-thiopurine, and 2,6-diaminopurine. Alternatively, the modified nucleobase may not contain a nitrogen atom (i.e., a universal base. See also WO 2003 / 040395. Alternatively, the modified nucleobase is abasic (i.e., does not include a nucleobase).

[0025] As used herein, "modified nucleoside" refers to a nucleoside comprising a modified or universal nucleobase and / or a modified sugar. A modified or universal nucleobase (also referred to herein as a base analog) can 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, a modified nucleotide does not contain a nucleobase (abasic). A modified sugar (also referred to herein as a sugar analog) comprises a modified deoxyribose or ribose moiety (e.g., the modification occurs at the 2'-, 3'-, 4'-, or 5'-carbon position of the sugar). Modified sugars can also contain non-natural alternative carbon structures, such as those present in bridged nucleic acids ("BNA"), locked nucleic acids ("LNA"), and / or unlocked nucleic acids ("UNA").

[0026] As used herein, "modified nucleotide" refers to a nucleotide comprising a modified or universal nucleobase, as described above, a modified sugar, as described above, and / or a modified phosphate or phosphate group. The modified phosphate can be a modification of a phosphate or phosphate group that does not occur in naturally occurring nucleotides, including non-naturally occurring phosphate mimetics as known in the art. Modified phosphate or phosphate groups also include non-naturally occurring internucleotide linkage groups, including both phosphorus-containing and non-phosphorus-containing linkage groups as known in the art. Suitable modified or universal nucleobases, modified sugars, and modified phosphates or phosphate groups are described herein.

[0027] As used herein, "nucleobase" refers to a heterocyclic nitrogenous base that can pair with a complementary nucleobase or nucleobase analog (i.e., a nucleobase derivative) to form Watson-Crick hydrogen bonding and stacking interactions when the nucleobase is incorporated into a polymer structure. Naturally occurring heterocyclic nitrogenous bases include purines and pyrimidines, such as adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U).

[0028] As used herein, "nucleoside" means a heterocyclic nitrogenous base in N-glycosidic linkage with a sugar moiety (e.g., deoxyribose, ribose, or an analog thereof).

[0029] As used herein, "nucleoside phosphoramidite" means a derivative of a natural or synthetic nucleoside in which the reactive hydroxy and exocyclic amino groups present in the natural or synthetic nucleoside are suitably protected to prevent undesired side reactions during nucleic acid synthesis.

[0030] As used herein, "nucleotide" means a heterocyclic nitrogenous base in N-glycosidic linkage with a sugar moiety (e.g., deoxyribose, ribose, or an analog thereof) linked to a phosphate or phosphate group (i.e., nucleoside plus phosphate or phosphate group). As noted above, naturally occurring heterocyclic nitrogenous bases include adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U).

[0031] As used herein, "nucleotide phosphoramidite" means a derivative of a natural or synthetic nucleotide in which the reactive hydroxy and exocyclic amino groups present in the natural or synthetic nucleotide are appropriately protected to prevent undesired side reactions during nucleic acid synthesis.

[0032] As used herein, "oligonucleotide" refers to a short nucleic acid (e.g., less than about 100 nucleotides in length) of ribonucleotides, deoxyribonucleotides, or a combination thereof. An oligonucleotide may be single-stranded (ss) or double-stranded (ds). An oligonucleotide may or may not have a duplex region. As a non-limiting set of examples, an oligonucleotide may be, but is not limited to, a small interfering RNA (siRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), a dicer substrate interfering RNA (dsiRNA), an antisense oligonucleotide (ASO), a short siRNA, or a ss siRNA.

[0033] As used herein, "phosphate analog" refers to a chemical moiety that mimics the electrostatic and / or steric properties of a phosphate group. Phosphate analogs can be located at the 5'-terminal nucleotide of an oligonucleotide in place of the 5'-phosphate and can include a phosphatase-resistant linkage. Examples of phosphate analogs include, but are not limited to, 5' phosphonates, such as 5' methylene phosphonate (5'-MP) and 5'-(E)-vinyl phosphonate (5'-VP). Similarly, a phosphate analog can have a phosphate analog at the 4'-carbon position of the sugar of a 5'-terminal nucleotide (referred to as a "4'-phosphate analog"). One example of a 4'-phosphate analog is an oxymethyl phosphonate, in which the oxygen atom of an oxymethyl group is attached to the sugar moiety (e.g., at the 4'-carbon) or an analog thereof. See, e.g., WO 2018 / 045317. Other modifications of the 5' end of oligonucleotides have been developed (see, e.g., WO 2011 / 133871, U.S. Pat. No. 8,927,513, and Prakash et al. (2015) Nuc. Acids Res. 43:2993-3011).

[0034] As used herein, "phosphoramidite" means a nitrogen-containing trivalent phosphorus derivative that may have the formula: (RO)2PNR2.

[0035] As used herein, a "protecting group" refers to a group that reversibly renders a functional group unreactive 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 substantially reduce the amount of the molecule (i.e., oligonucleotide) being synthesized.

[0036] As used herein, "ribonucleotide" means a natural or modified nucleotide that has a hydroxyl group at the 2' position of the sugar moiety.

[0037] As used herein, a "targeting ligand" refers to a chemical moiety that facilitates the entry of an oligonucleotide, such as an RNAi agent, into a cell. It can be a compound (e.g., an amino sugar, carbohydrate, cholesterol, lipid, or polypeptide) that selectively binds to a cognate compound (e.g., a receptor) in 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, a targeting ligand can be conjugated to an oligonucleotide for the purpose of targeting the oligonucleotide to a specific tissue or cell of interest. The targeting ligand can selectively bind to a cell surface receptor. Thus, when conjugated to an oligonucleotide, the targeting ligand facilitates delivery of the oligonucleotide to a specific cell through selective binding to a receptor expressed on the surface of the cell and endosomal internalization by the cell of a complex comprising the oligonucleotide, targeting ligand, and receptor. Furthermore, the targeting ligand can be conjugated to the oligonucleotide via a linker that is cleaved after or during cellular internalization, thereby releasing the oligonucleotide from the targeting ligand within the cell.

[0038] composition MeMOP: The structure of MeMOP is as follows:

[0039] [ka] See, for example, WO 2018 / 045317.

[0040] MeMOP-modified oligonucleotides: MeMOP can be incorporated into oligonucleotides, such as therapeutic oligonucleotides. In some examples, MeMOP can be attached to the 4'-carbon of a sugar moiety (e.g., ribose, deoxyribose, or an analog thereof) of a nucleotide in an oligonucleotide. In some examples, MeMOP can be incorporated at the 3' end of an oligonucleotide. In other examples, MeMOP can be incorporated at the 5' end of an oligonucleotide. In still other examples, MeMOP can be incorporated at both the 5' and 3' ends of an oligonucleotide. In still other examples, MeMOP can be incorporated at one or more internal positions of an oligonucleotide. See, for example, International Publication Nos. 2018 / 045317, 2021 / 188795, 2022 / 032288, and 2022 / 221430.

[0041] Oligonucleotides (e.g., ds oligonucleotides such as MeMOP-modified oligonucleotides) can be prepared using methods and / or techniques known to those skilled in the art, such as, for example, conventional solid-phase nucleic acid synthesis. The nucleotides of the oligonucleotide can be assembled on a suitable nucleic acid synthesizer using standard nucleotide or nucleoside precursors (e.g., phosphoramidites). 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).

[0042] In some cases, oligonucleotide synthesis steps may be performed in alternating order to obtain the desired compound. Other synthetic chemical transformations, protecting groups (e.g., present on bases for hydroxyl, amino, etc.), and protecting group methodologies (protection and deprotection) useful in synthesizing oligonucleotides are known in the art, see, for example, Larock, "Comprehensive Organic Transformations," VCH Publishers (1989); Greene & Wuts, "Protective Groups in Organic Synthesis," 2000; nd Ed., John Wiley & Sons (1991), Fieser & 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).

[0043] Pharmaceutical Composition: MeMOP-modified oligonucleotides (or pharmaceutically acceptable salts thereof, such as, for example, trifluoroacetate, acetate, or hydrochloride) can be incorporated into pharmaceutical compositions comprising an effective amount of a MeMOP-containing oligonucleotide and a pharmaceutically acceptable carrier, delivery agent, or excipient. See, e.g., WO 2018 / 045317, WO 2021 / 188795, WO 2022 / 032288, and WO 2022 / 221430.

[0044] Various formulations have been developed to facilitate the use of oligonucleotides. In some instances, oligonucleotides can be delivered to an individual or cellular environment using formulations that minimize degradation, facilitate delivery and / or uptake, or provide other beneficial properties to the oligonucleotides in the formulation. In some instances, oligonucleotides can be formulated in buffers such as phosphate-buffered saline, liposomes, micellar structures, and capsids.

[0045] 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 improves the in vivo compatibility and / or efficacy of the oligonucleotide. Pharmaceutically acceptable salts and general methodologies for preparing them are well known in the art (e.g., Stahl et al., "Handbook of Pharmaceutical Salts: Properties, Selection and Use," 2004). nd Revised Edition (Wiley-VCH, 2011). Pharmaceutically acceptable salts for use herein include sodium, trifluoroacetate, hydrochloride, and acetate salts.

[0046] In some instances, a pharmaceutical composition can be formulated to be compatible with its intended route of administration, including, but not limited to, parenteral (e.g., intravenous, intramuscular, intraperitoneal, intradermal, and subcutaneous), oral (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration.

[0047] Additionally, factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations can be contemplated by those skilled in the art of preparing such pharmaceutical formulations, and therefore, various dosages and treatment regimens may be desirable.

[0048] In some examples, the pharmaceutical composition may include one or more additional therapeutic agents.

[0049] method How to make MeMOP: The method for making MeMOP or a salt thereof can include the steps described herein, which may, but need not necessarily, be performed in the order described. However, other orders are contemplated. Furthermore, individual or multiple steps may be performed in parallel and / or overlapping time, and / or individually or in multiple repeated steps. The products of each of the following steps can be recovered by conventional methods, including chromatography, crystallization, evaporation, extraction, filtration, precipitation, and trituration.

[0050] In one example, MeMOP can be prepared according to the following method, which comprises the following steps: (1) To obtain (2S,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-methoxytetrahydrofuran-2-carboxylic acid,

[0051] [ka] oxidizing 1-((2R,3R,4R,5R)-4-hydroxy-5-(hydroxymethyl)-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione;

[0052] [ka] (2) 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;

[0053] [ka] (3) adding 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;

[0054] [ka] (4) oxidizing 1-((2R,3R,4S,5S)-5-acetyl-4-((tert-butyldimethylsilyl)oxy)-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (e.g., via a Baeyer-Villiger reaction) 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;

[0055] [ka] (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;

[0056] [ka] (6) 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;

[0057] [ka] (7) debenzoylating [(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;

[0058] [ka] (8) phosphorylating dimethyl (2R,3S,4R,5R)-2-acetoxy-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-3-ylbenzoate 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).

[0059] [ka]

[0060] In some instances, [(2R,3S,4R,5R)-2-(dimethoxyphosphorylmethoxy)-5-(2,4-dioxopyrimidin-1-yl)-4-methoxy-tetrahydrofuran-3-yl]benzoate

[0061] [ka] It can be prepared according to the following method, which comprises the following steps: (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; (2) 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; (3) 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; (4) 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; (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; (6) 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.

[0062] 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, a stereospecific process that gives exclusively the desired β-anomer at the 4'OH position of ribose. In some instances, the Baeyer-Villiger reaction can be carried out using meta-chloroperoxybenzoic acid (mCPBA) or urea hydrogen peroxide (UHP). [Example]

[0063] The following non-limiting examples are provided for illustrative purposes and are not intended to limit the scope of the present disclosure.

[0064] Example 1: (2S,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-methoxytetrahydrofuran-2-carboxylic acid,

[0065] [ka] Synthesis of.

[0066] Procedure: 1-((2R,3R,4R,5R)-4-hydroxy-5-(hydroxymethyl)-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (1.0 equiv.) was added slowly in portions to a solution of ACN (5 V), HO (5 V), TEMPO (0.5 equiv.), and NaHCO (4.0 equiv.) and mCPBA (4.0 equiv.) at 15°C-25°C. The resulting mixture was warmed to 30°C-40°C and stirred at room temperature for 4 hours. The mixture was cooled, and the solution was quenched by adding 20% ​​aqueous NaHSO. The mixture was then diluted with EtOAc (10 V), and the pH was adjusted to 1-2 with 36% aqueous HCl. The mixture was filtered to obtain a first wet cake. The first filtrate was collected, and the organic phase was removed. The aqueous phase was concentrated to 3 V to form a suspension. The suspension was filtered to obtain a second wet cake. The first and second wet cakes were combined and washed successively with EtOAc (4V) and water (1V). The solid was dried under vacuum to obtain the title compound (70%) as a solid.

[0067] result: 1 H-NMR(DMSO-d6)δ3.32(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(MH).

[0068] Example 2: (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,

[0069] [ka] Synthesis of.

[0070] Procedure: (2S,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-methoxytetrahydrofuran-2-carboxylic acid (1.0 equiv.) from Example 1 was dissolved in a mixture of ACN (10 V), DMAP (0.13 equiv.), and pyridine (3.3 equiv.). EDCI (1.6 equiv.) was added slowly in portions while maintaining the temperature at -5°C to 5°C. N,O-NODMHA was added while maintaining the temperature at -5°C to 5°C. * HCl (1.5 equiv.) was added slowly in portions. The solution was warmed to 10-20°C and stirred for 2 h. Imidazole (3.0 equiv.) and TBSCl (2.5 equiv.) were added and stirred at 10-20°C for 16 h. The mixture was quenched by adding HO (2 V) and concentrated (3 V). The mixture was extracted with EtOAc (10 V) and then washed successively with 1 M aqueous HCl (5 V × 2), saturated aqueous NaHCO (5 V), and 8% aqueous NaSO (5 V). The mixture was concentrated to approximately 4 V, and n-heptane (10 V) was added. The mixture was then concentrated to 10 V, allowing the mixture to crystallize. The mixture was filtered and then dried to give the title compound (80%) as a solid.

[0071] result: 1H NMR(DMSO-d6)δ0.11(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).

[0072] Example 3: 1-((2R,3R,4S,5S)-5-acetyl-4-((tert-butyldimethylsilyl)oxy)-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione,

[0073] [ka] Synthesis of.

[0074] Procedure: (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 equiv.) from Example 2 was dissolved in 2-MeTHF (15 V). A solution of 3 N MeMgCl in THF (2.3 equiv.) was added dropwise while maintaining the temperature between -20 and 10 °C. The resulting mixture was stirred at -20 to 10 °C for 16 h. HO (1 V) was added, and the pH was adjusted to 4-5 with 1 M aqueous HCl. The two phases were separated, and the organic phase was washed successively with saturated aqueous NaHCO (5 V × 2) and 8% aqueous NaSO (5 V). The mixture was concentrated to 3 V and n-heptane (10 V) was added to crystallize the mixture. The mixture was filtered and the filter cake was dried to give the title compound (85%) as a solid.

[0075] Result: Weight: 1H NMR(CDCl3)δ0.14(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).

[0076] Example 4: (2R,3S,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-2-yl acetate,

[0077] [ka] Synthesis of.

[0078] Procedure: 1-((2R,3R,4S,5S)-5-acetyl-4-((tert-butyldimethylsilyl)oxy)-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (1.0 equiv.) from Example 3 was dissolved in ACN (10 V). A solution of NaHCO (3.0 equiv.) was added, followed by the slow addition of mCPBA (1.5 equiv.) at 5°C to 15°C. The mixture was stirred at room temperature for 3 h, then diluted with water (6 V) and quenched with 20% aqueous NaHSO. The solution was extracted with EtOAc (10 V) and the layers were separated. The organic phase was washed successively with saturated aqueous NaHCO (5 V × 2) and 8% aqueous NaSO (5 V). The mixture was concentrated to approximately 3 V, and n-heptane (14 V) was added. The mixture was filtered and the filter cake was dried to give the title compound (85%) as a solid.

[0079] result: 1H NMR(CDCl3)δ0.15(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).

[0080] Example 5: (2R,3S,4R,5R)-2-acetoxy-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-3-yl benzoate,

[0081] [ka] Synthesis of.

[0082] Procedure: (2R,3S,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-2-yl acetate (1.0 equiv.) from Example 4 was dissolved in ACN (10 V), TEA (6.0 equiv.), and 3HF·TEA (3.0 equiv.). The mixture was warmed to 25°C-35°C and then stirred for 6 h. DMAP (0.1 equiv.) and BzO (1.5 equiv.) were added, and the mixture was stirred at 20°C-30°C for 2 h. HO (2 V) was added, and the mixture was concentrated in vacuo to 4 V. The mixture was extracted with EtOAc (8 V), and the layers were separated. The organic phase was washed successively with saturated aqueous NaHCO3 (5 V), 1 M aqueous HCl (5 V), and 8% aqueous Na2SO4 (5 V). The mixture was concentrated to 2 V, and EtOAc (5 V) was added. The mixture was concentrated to approximately 2 V, and n-heptane (10 V) was added. The suspension was stirred at 0°C to 10°C and then filtered. The filter cake was dried to give the title compound (82%) as a solid.

[0083] result: 1H NMR(CDCl3)δ2.21(s,3H,COCH3),3.44(s,3H,C2'OCH3),4.26(d,1H,C2'H),5.60(d,1H,C3'H),5.85(d d,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(MH).

[0084] Example 6: (2R,3S,4R,5R)-2-(dimethoxyphosphorylmethoxy)-5-(2,4-dioxopyrimidin-1-yl)-4-methoxy-tetrahydrofuran-3-yl]benzoate,

[0085] [ka] Synthesis of.

[0086] Procedure: (2R,3S,4R,5R)-2-acetoxy-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-3-yl benzoate (1.0 equiv.) from Example 5 was dissolved in ACN (5 V). TMSOTf (2.4 equiv.) was added dropwise while maintaining the temperature at -15 to -5 °C. DMHMP (5.0 equiv.) was added dropwise to the mixture while maintaining the temperature at -15 to -5 °C. The solution was warmed to 15 to 25 °C and then stirred for 16 h. Saturated aqueous NaHCO3 (6 V) was added, and the mixture was extracted with DCM (4 V × 2). The organic phase was washed with HO (10 V × 5) and then concentrated to give the title compound (85%) as an oil.

[0087] result: 1H NMR(CDCl3)δ3.41(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(MH).

[0088] Example 7: dimethyl((((2R,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-methoxytetrahydrofuran-2-yl)oxy)methyl)phosphonate,

[0089] [ka] Synthesis of.

[0090] Method: [(2R,3S,4R,5R)-2-(dimethoxyphosphorylmethoxy)-5-(2,4-dioxopyrimidin-1-yl)-4-methoxy-tetrahydrofuran-3-yl]benzoate (1.0 equiv.) from Example 6 was dissolved in MeOH (10 V) and then KCO (1.5 equiv.) was added. The mixture was stirred at 20-30°C for 6 h and then filtered. The pH was then adjusted to approximately 4-5 by adding formic acid. The mixture was concentrated to give a gummy solid, which was purified by silica gel chromatography using a gradient of 60:1 to 30:1 CHCl:MeOH to give the title compound (60%) as a solid.

[0091] result: 1H NMR(CD3OD)δ3.43(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).

[0092] Example 8: 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),

[0093] [ka] Synthesis of.

[0094] Procedure: 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 equiv.) from Example 7 was dissolved in DCM (3 V). A solution of DCM (10 V) and NMI (0.3 equiv.) was added to the mixture, followed by the dropwise addition of tetrazole (0.7 equiv.) and 3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile (1.3 equiv.) while maintaining the temperature between 0 and 10 °C. The solution was warmed to 15 and 25 °C and stirred for 3 h, after which 8% aqueous NaHCO (8 V) was added. The layers were separated and the organic phase was washed with 8% aqueous NaHCO (5 V) followed by HO (5 V × 4). The mixture was concentrated to about 1.5 V and MTBE (15 V) was added. The mixture was filtered and the filter cake was dried to give the title compound (64%) as a solid.

[0095] result: 1H NMR(CD3CN)δ1.22(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モード)=567.20(M+1)。

Claims

1. 1. A method of making a compound represented by the structure: 【Chemistry 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; 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 provide 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; 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-ylbenzoate; 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; debenzoylating [(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; and phosphorylating 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. 1. A method of making a compound represented by the structure: 【Chemistry 2】 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 provide 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; 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-ylbenzoate; and 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.

3. 3. The method of claim 1, 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. 4. The method of claim 3, wherein the Baeyer-Villiger reaction involves the use of meta-chloroperoxybenzoic acid (mCPBA) or urea hydrogen peroxide (UHP).

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