Synthesis of phosphate derivatives

Abstract

To provide a method for producing 5-fluoro-2' - deoxyuridine-5' -O - [1-naphthyl (benzyloxy-L-alaninyl)] phosphate (NUC-3373) and a method for producing 3' - deoxyadenosine-5' -O - [phenyl (benzyloxy-L-alaninyl)] phosphate (NUC-7738).SOLUTION: A compound of Formula (IIa) or (IIb) is reacted with a compound of Formula (IIIa) or (IIIb), respectively, in presence of a base (B1) and the protecting groups are removed from the resulting compound of Formula (IVa) or (IVb) to provide NUC-3373 or NUC-7738.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention generally relates to novel methods for preparing specific ProTides as particular phosphate diastereomers, including those useful in the treatment of cancer, such as NUC-3373 (5-fluoro-2'-deoxyuridine-5'-O-[1-naphthyl(benzyloxy-L-alaninyl)]phosphate), NUC-7738 (3'-deoxyadenosine-5'-O-[phenyl(benzyloxy-L-alaninyl)]phosphate), and NUC-9701 (8-chloroadenosine-5'-O-[naphthyl(benzyloxy-L-alaninyl)]phosphate). [Background technology]

[0002] ProTides are masked phosphate derivatives of nucleosides. They have been shown to be particularly potent therapeutic agents in both the antiviral and oncology fields. More specifically, ProTides are prodrugs of monophosphorylated nucleosides. These compounds appear to circumvent many of the inherent or acquired resistance mechanisms that limit the utility of the parent nucleosides (see, for example, "Application of ProTide Technology to Gemcitabine: Successful Overcoming of Important Core Resistance Mechanisms Leads to a Novel Agent (NUC-1031) in Clinical Development," Slusarczyk et al.; J. Med. Chem.; 2014, 57, 1531-1542).

[0003] NUC-3373 (5-fluoro-2'-deoxyuridine-5'-O-[1-naphthyl(benzylosyl-L-alaninyl)] phosphate) is a ProTide adaptation of 5FU / FUDR, the current standard of care for colorectal cancer. NUC-3373 and a group of related compounds have demonstrated in vitro activity against a range of cancer models, in many cases, and for NUC-3373 in particular, the activity was outstanding and far superior to results obtained with 5-fluorouracil. The addition of a ProTide phosphoramidate moiety to the 5-fluorouracil / FUDR molecule provides a unique opportunity for delivery of the key active form of the drug (FdUMP) to tumor cells. Clinical studies have demonstrated that NUC-3373 overcomes important central resistance mechanisms associated with 5-FU and its oral prodrug, capecitabine, generating high intracellular levels of the active FdUMP metabolite and, as a result, providing superior inhibition of tumor cell growth. Furthermore, in formal dog toxicology studies, NUC-3373 is significantly better tolerated than 5-FU (WO 2012 / 117246; McGuigan et al., "Phosphoramidate ProTide of the Anticancer Drug FUDR Successfully Delivers Preformed Bioactive Monophosphate in Cells, Conferring Advantages Over the Parent Nucleoside"); J. Med. Chem.; 2011, 54, 7247-7258; Vande Voorde et al.; "The cytostatic activity of NUC-3073, a phosphoramidate prodrug of 5-fluoro-2'-deoxyuridine, is dependent on activation by thymidine kinase and insensitive to degradation by phosphorylase"; Biochem. Pharmacol.; 2011, 82, 441-452). NUC-3373 [ka]

[0004] ProTide derivatives of purine nucleosides, such as 8-chloroadenosine and 3'-deoxyadenosine and related compounds, also exhibit excellent in vitro activity against a range of solid tumors, leukemias, and lymphomas (see WO 2016 / 083830 and GB 1609602.6). 3'-Deoxyadenosine itself is not a particularly potent anticancer agent.

[0005] NUC-7738 [ka]

[0006] NUC-9701 [ka]

[0007] ProTides are generally prepared as mixtures of two diastereomers (epimers at the phosphate center). For example, a diastereomer of NUC-3373 has the following structure (where Np is 1-naphthyl): (S p )-NUC-3373 [ka] (R p )-NUC-3373 [ka]

[0008] WO 2014 / 076490 discloses a method for preparing a nucleoside prodrug, e.g., gemcitabine-[phenyl(benzoxy-L-alaninyl)]phosphate, in ∼45% yield by reacting gemcitabine or a structural variant thereof with a diastereomeric mixture of phosphorochloridates in the presence of a catalyst comprising a metal salt, e.g., Cu(OTf)2, CuCl, CuBr, CuI, Cu(OAc)2, CuSO4, Cu(OC(O)CF3)2, Cu(OTf)2, Yb(OTf)3, Fe(OTf)3, La(OTf)3.

[0009] WO 2017 / 098252 (PCT / GB2016 / 053875) describes a method for synthesizing NUC-1031 in diastereomerically pure form. Summary of the Invention [Problem to be solved by the invention]

[0010] It is an object of certain embodiments of the present invention to provide NUC-3373, NUC-7738 and / or NUC-9701 in substantially diastereomerically pure form.

[0011] It is an object of certain embodiments of the present invention to provide a method for the treatment of NUC-3373, NUC-7738 and / or NUC-9701 (S p )- and (R p The present invention provides a method for providing the 2-epimer of 2-(2-methyl-2-propanol)-diastereomeric ester in substantially diastereomerically pure form, which method is scalable, economical and / or effective, e.g., more scalable, economical and / or effective than methods using HPLC.

[0012] Thus, the object of certain embodiments of the present invention is to p )- and (R p The present invention aims to provide a process suitable for large-scale production that provides the diastereomeric 2-epimer in substantially diastereomerically pure form.

[0013] The object of certain embodiments of the present invention is top )- and (R p The present invention provides a simple method for providing the 2-epimer in substantially diastereomerically pure form, i.e., a method using a minimum number of steps and reagents.

[0014] Another object of certain embodiments of the present invention is to provide a method for the production of isolated (S p )- and (R p The objective of the present invention is to provide a method for providing the diastereomeric 2-epimer in substantially diastereomerically pure form while meeting or exceeding required limits set by authorities, such as the US FDA, regarding the amount and nature of various trace impurities resulting from synthesis and separation.

[0015] Particular embodiments of the present invention may satisfy some or all of the above objectives. [Means for solving the problem]

[0016] According to a first aspect of the present invention, a compound of formula (Ia) [ka] is provided a method for preparing NUC-3373 of formula (I) in substantially diastereomerically pure form, the method comprising the steps a) and optionally b): Process a) Formula (IIa) [ka] (In the formula, R 1 is an electron withdrawing group, and a is an integer of 1 to 5), in the presence of a base (B1), a compound of formula (IIIa): [ka] to form a compound of formula (IVa) [ka] In a substantially diastereomerically pure form, 1are independently selected from hydrogen and a protecting group; and the compound of formula (IIa) is in substantially diastereomerically pure form; Step b) P 1 is a protecting group, optionally removing the protecting group P from the compound of formula (IVa) 1 is removed to provide NUC-3373 in substantially diastereomerically pure form.

[0017] According to a second aspect of the present invention, a compound of formula (Ib) [ka] is provided a method for preparing NUC-7738 of formula (I) in substantially diastereomerically pure form, the method comprising steps a) and optionally step b): Process a) Formula (IIb) [ka] (In the formula, R 1 is an electron withdrawing group, and a is an integer of 1 to 5), in the presence of a base (B1), a compound of formula (IIIb): [ka] to form a compound of formula (IVb) [ka] In a substantially diastereomerically pure form, 2 , P 3 and P 4 are independently selected from hydrogen and a protecting group; and the compound of formula (IIb) is in substantially diastereomerically pure form; Step b) P 2 , P 3 and P 4 is a protecting group, optionally by removing from the compound of formula (IVb) the protecting group P 2 , P 3and P 4 is removed to provide NUC-7738 in substantially diastereomerically pure form.

[0018] According to a third aspect of the present invention, a compound of formula (Ic) [ka] is provided a method for preparing NUC-9701 of formula (I) in substantially diastereomerically pure form, the method comprising the steps a) and optionally b): Process a) Formula (IIa) [ka] (In the formula, R 1 is an electron withdrawing group, and a is an integer of 1 to 5), in the presence of a base (B1), a compound of formula (IIIc): [ka] to form a compound of formula (IVc) [ka] In a substantially diastereomerically pure form, 5 , P 6 , P 7 and P 8 are independently selected from hydrogen and a protecting group; and the compound of formula (IIa) is in substantially diastereomerically pure form; Step b) P 5 , P 6 , P 7 and P 8 is a protecting group, optionally by removing from the compound of formula (IVc) the protecting group P 5 , P 6 , P 7 and P 8 is removed to provide NUC-9701 in substantially diastereomerically pure form. DETAILED DESCRIPTION OF THE INVENTION

[0019] R 1 is selected from the group consisting of halo groups (e.g., selected from fluorine, bromine, chlorine, and iodine), trifluoromethyl, cyano, and nitro. a is an integer from 1 to 5. R 1 At each occurrence, a may be halo, e.g., fluorine. a may be 5.

[0020] Substitution of the substituted phenoxy group is selectively effected with inversion of the phosphate stereocenter.

[0021] Generally, the precursor (compound of formula (IIa) or (IIb)) (S p )-diastereomer of ProTide p )-diastereomer of the precursor (R p )-diastereomer of ProTide p )-diastereomer. The exception to this is the leaving group OPh(R 1 ) a has a lower priority than the naphthyl group in the Cahn-Ingold-Prelog sequence (e.g., OPh(R 1 ) a In such a case, (R p )-diastereomer of ProTide p )-diastereomer of the precursor, p )-diastereomer of ProTide p Throughout this specification, the (S)-diastereomer of ProTide is provided. p The isomer of the compound of formula (IIa) that provides the (R )-isomer is referred to as the X-diastereomer, and the (R )-isomer of ProTide is referred to as the X-diastereomer. p The isomer of the compound of formula (IIa) that provides the (S)-isomer is called the Y-diastereomer. For compound IIb, the (S)-isomer of the precursor (compound of formula (IIb)) is always p )-diastereomer of ProTide p)-diastereomer of the precursor (R p )-diastereomer of ProTide p )-diastereomers.

[0022] Thus, the method of the first, second, or third aspect may comprise administering to a subject a therapeutically effective amount of ProTide (S p )-diastereomer in diastereomerically enriched form, and the compound of formula (IIa) or (IIb) may be in diastereomerically enriched form.

[0023] The method of the first, second, or third aspect may comprise administering to a subject a therapeutically effective amount of ProTide (R p )-diastereomer in diastereomerically enriched form, and the compound of formula (IIa) or (IIb) may be in diastereomerically enriched form.

[0024] The base (B1) is a nitrogen base. Examples of the nitrogen base include N-alkylimidazoles (e.g., N-methylimidazole (NMI)), imidazole, optionally substituted phenyl pyridines (e.g., collidine, pyridine, 2,6-lutidine), and trialkylamines (e.g., triethylamine and diisopropylethylamine). Alternatively, the base (B1) is an organometallic base or a metal hydride base (e.g., NaH). Thus, the base may be a Grignard compound (i.e., an alkyl magnesium hydride). For example, Grignard compounds include t-butyl magnesium hydrides, such as tBuMgCl and tBuMgBr. Preferably, the base is tBuMgCl.

[0025] Step a) is carried out in solvent S1.

[0026] In a fourth aspect of the present invention, there is provided a method for diastereomerically enriching a compound of formula (IIa), the method comprising: c) suspending or dissolving the X-diacrylate monomer of the compound of formula (IIa) or a mixture of the XR- and Y-diastereomers of the compound of formula (IIa) in a solvent (S2); d) treating the solution or suspension with a base (B2) to obtain the X-diastereomer in substantially diastereomerically enriched form; and e) isolating the X-diastereomer of formula (IIa) Generally, the X-diastereomer is the (S)-diastereomer and the Y-diastereomer is the (R)-diastereomer.

[0027] The inventors have surprisingly found that when compounds of formula (IIa) are treated with base, they isomerize to form the X-diastereomer preferentially over the Y-diastereomer. The Y-diastereomer is converted to the X-diastereomer or an epimeric mixture of the Y-diastereomer, and the X-diastereomer is converted to the X-diastereomer. This increases the net efficiency of various synthetic sequences for producing the X-diastereomer of NUC-3373 or NUC-9701 that are combined with the methods of the first or third aspects, since all of the compounds of formula (IIa) (even a portion that was originally formed as the Y-diastereomer) are used. Generally, the X-diastereomer is (S p )-diastereomer, and the Y-diastereomer is (R p )-diastereomer.

[0028] The method may comprise forming a compound of formula (IIa) as a mixture of Y- and X-diastereomers, and step c) may comprise suspending or dissolving the mixture of Y- and X-diastereomers of the compound of formula (IIa) in a solvent (S2). Generally, the X-diastereomer is (S p )-diastereomer, and the Y-diastereomer is (R p )-diastereomer.

[0029] The compound of formula (IIa) used in the method of the first or third aspect may be the X-diastereomer formed according to the method of the fourth aspect. Generally, the X-diastereomer is (S p )-diastereomer, and the Y-diastereomer is (R p )-diastereomer.

[0030] The method of the second aspect of the present invention comprises: c) (R p )-diastereomer of the compound of formula (IIb), or the (R p )- and (S p )-diastereomer mixture is suspended or dissolved in a solvent (S2), d) Treating the solution or suspension with a base (B2) to form (S p )-diastereomer in substantially diastereomerically enriched form, e) (S) of the compound of formula (IIb) p )-diastereomer is isolated It may also include the following.

[0031] Thus, the method of the second aspect of the present invention comprises reacting a compound of formula (IIb) with (R p )- and (S p )-diastereomers of the compound of formula (IIb), and step c) may comprise forming a mixture of (R p )- and (S p The method may involve suspending or dissolving the mixture of 2-diastereomers in a solvent (S2).

[0032] The base (B2) is selected from the group consisting of organic amine bases (e.g., primary, secondary, tertiary amines, cyclic amines; for example, organic amine bases include N-alkylimidazoles (e.g., N-methylimidazole (NMI)), imidazole, optionally substituted pyridines (e.g., collidine, pyridine, 2,6-lutidine), and trialkylamines (e.g., triethylamine, and diisopropylethylamine)); or inorganic bases (e.g., alkali metal hydroxides, alkali metal carbonates, alkali metal alkoxides, alkali metal aryloxides). Preferably, B2 is a tertiary amine. Thus, B2 may be a trialkylamine. Most preferably, B2 is triethylamine.

[0033] The solvent S2 is selected from the group consisting of, but not limited to, amides, ethers, esters, ketones, aromatic hydrocarbons, halogenated solvents, nitriles, sulfoxides, sulfones, and mixtures thereof. S2 may also be an organic solvent. Examples of organic solvents include, but are not limited to, ethers (e.g., tetrahydrofuran, dioxane, diethyl ether, t-butyl methyl ether); ketones (e.g., acetone and methyl isobutyl ketone); halogenated solvents (e.g., dichloromethane, chloroform, and 1,2-dichloroethane); hydrocarbons (e.g., cyclohexane, pentane, hexane, heptane), aromatic solvents (e.g., benzene and toluene), esters (e.g., ethyl acetate), and amides (e.g., DMF, NMP); or mixtures thereof. Preferably, S2 is a hydrocarbon or a mixture comprising a hydrocarbon. When S2 is a mixture, it is a mixture comprising 50% or more (e.g., 70% or more) of hydrocarbons. The hydrocarbon may be hexane. The hydrocarbon may be heptane. S2 may be a mixture of hexane or heptane and a polar organic solvent (e.g., an ether, ester, alcohol, or halogenated solvent). S2 may be a mixture of hexane or heptane and a polar organic solvent (comprising 50% or more (e.g., 70% or more) of hexane or heptane by volume). S2 may be a mixture of heptane and ethyl acetate. S2 may be a mixture of hexane or heptane and ethyl acetate (comprising 50% or more (e.g., 70% or more) of hexane or heptane by volume). S2 may be a mixture of heptane and ethyl acetate (comprising 50% or more (e.g., 70% or more) of heptane by volume). S2 may be a mixture of hexane or heptane and methyl t-butyl ether. S2 may be a mixture of hexane or heptane and methyl t-butyl ether (comprising 50% or more (e.g., 70% or more) of hexane or heptane by volume). S2 may be a mixture of hexane and methyl t-butyl ether (comprising 50% or more (eg, 70% or more) by volume of hexane).

[0034] Step d) can involve stirring the mixture of the compound of Formula (IIa) and base (B2) for 24 hours or more. Step d) can involve stirring the mixture of the compound of Formula (IIa) and base (B2) for 48 hours or more. Step d) can involve stirring the mixture of the compound of Formula (IIa) and base (B2) for 60 hours or more. Step d) can involve stirring the mixture of the compound of Formula (IIa) and base (B2) for 72 hours or more. Step d) can involve stirring the mixture of the compound of Formula (IIa) and base (B2) for 100 hours or more.

[0035] Step d) can involve stirring a mixture of the compound of formula (IIa) and the base (B2) at a temperature of 0 to 60° C. Step d) can involve stirring a mixture of the compound of formula (IIa) and the base (B2) at a temperature of 20 to 40° C.

[0036] Step d) can involve stirring the mixture of the compound of Formula (IIb) and base (B2) for 2 hours or more. Step d) can involve stirring the mixture of the compound of Formula (IIb) and base (B2) for 6 hours or more. Step d) can involve stirring the mixture of the compound of Formula (IIb) and base (B2) for 10 hours or more. Step d) can involve stirring the mixture of the compound of Formula (IIb) and base (B2) for 16 hours or more. Step d) can involve stirring the mixture of the compound of Formula (IIb) and base (B2) for 36 hours or more.

[0037] Step d) can involve stirring a mixture of the compound of formula (IIb) and the base (B2) at a temperature of 0 to 50° C. Step d) can involve stirring a mixture of the compound of formula (IIb) and the base (B2) at a temperature of 10 to 35° C.

[0038] In certain embodiments of the second aspect of the present invention, the compound of formula (IIb) is a compound selected from: [ka] TIFF2026034703000019.tif192144 TIFF2026034703000020.tif171142 Note: * indicates a chiral center in the phosphorous acid.

[0039] The compound of formula (IIb) [ka] is.

[0040] The compound of formula (IIb) [ka] That's fine too.

[0041] In certain embodiments of the first, third and fourth aspects of the present invention, the compound of formula (IIa) is a compound selected from: [ka] TIFF2026034703000024.tif208144 TIFF2026034703000025.tif187144 Note: * indicates a chiral center in the phosphorous acid.

[0042] The compound of formula (IIa) is compound 12 [ka] That's fine too.

[0043] The compound of formula (IIa) may be obtained in substantially diastereomerically pure form (R p)-Compound 12. The compound is the faster eluting isomer of Compound 12 in substantially diastereomerically pure form. Thus, the compound has a chromatogram of -1.41±0.02. 31 P NMR peak (NMR spectrum obtained in CDCl3 on a 202 MHz NMR instrument), said isomer being in substantially diastereomerically pure form. Compound 12 is an isomer of compound 12 having a retention time of 12.96 ± 0.20 minutes (analytical HPLC performed on a Varian Pursuit XRs 5 C18, 150 × 4.6 mm, eluted with HO / MeOH (20 / 80) at 1 ml / min for 35 minutes), said isomer being in substantially diastereomerically pure form.

[0044] The compound of formula (IIa) may be obtained in substantially diastereomerically pure form (S p )-Compound 12. The compound is the slower eluting isomer of Compound 12 in substantially diastereomerically pure form. Thus, the compound has a pH of -1.36±0.02. 31 P NMR peak (NMR spectrum obtained in CDCl3 on a 202 MHz NMR instrument), said isomer being in substantially diastereomerically pure form. Compound 12 is an isomer of compound 12 having a retention time of 14.48 ± 0.20 minutes (analytical HPLC performed on a Varian Pursuit XRs 5 C18, 150 × 4.6 mm, eluted with HO / MeOH (20 / 80) at 1 ml / min for 35 minutes), said isomer being in substantially diastereomerically pure form.

[0045] The compound of formula (IIa) [ka] That's fine too.

[0046] NUC-3373 is the faster eluting isomer of NUC-3373 in substantially diastereomerically pure form. 31 NUC-3373 is an isomer of NUC-3373 having a P NMR peak (NMR spectrum obtained in CD3OD on a 202 MHz NMR instrument), said isomer being in substantially diastereomerically pure form. NUC-3373 is an isomer of NUC-3373 having a retention time of 16.03 ± 0.20 minutes (analytical HPLC performed on a Varian Pursuit XRs 5 C18, 150 × 4.6 mm, eluting with HO / CH3CN (100 / 10 to 0 / 100) at 1 mL / min for 35 minutes), said isomer being in substantially diastereomerically pure form.

[0047] NUC-3373 is a substantially diastereomerically pure form of the slower eluting isomer of NUC-3373. Thus, NUC-3373 has a chromatographic resolution of 4.62±0.10. 31 NUC-3373 is an isomer of NUC-3373 having a P NMR peak (NMR spectrum obtained in CD3OD on a 202 MHz NMR instrument), said isomer being in substantially diastereomerically pure form. NUC-3373 is an isomer of NUC-3373 having a retention time of 16.61 ± 0.20 minutes (analytical HPLC performed on a Varian Pursuit XRs 5 C18, 150 × 4.6 mm, eluting with HO / CH3CN (90 / 10 to 0 / 100) at 1 mL / min for 35 minutes), said isomer being in substantially diastereomerically pure form.

[0048] NUC-9701 is the fast-eluting isomer of NUC-9701 in substantially diastereomerically pure form. Thus, NUC-9701 has a chromatogram of 3.93±0.04. 31NUC-9701 is an isomer of NUC-9701 having a P NMR peak (NMR spectrum obtained in CD3OD on a 202 MHz NMR instrument), said isomer being in substantially diastereomerically pure form. NUC-9701 is an isomer of NUC-9701 having a retention time of 16.43 ± 0.10 minutes (analytical HPLC performed on a Varian Pursuit XRs 5 C18, 150 × 4.6 mm, eluting with HO / CH3CN (90 / 10 to 0 / 100) at 1 mL / min for 30 minutes), said isomer being in substantially diastereomerically pure form.

[0049] NUC-9701 is a substantially diastereomerically pure form of the slower eluting isomer of NUC-9701. Thus, NUC-9701 has a pH of 3.83±0.04. 31 NUC-9701 is an isomer of NUC-9701 having a P NMR peak (NMR spectrum obtained in CD3OD on a 202 MHz NMR instrument), said isomer being in substantially diastereomerically pure form. NUC-9701 is an isomer of NUC-9701 having a retention time of 16.59 ± 0.10 minutes (analytical HPLC performed on a Varian Pursuit XRs 5 C18, 150 × 4.6 mm, eluting with HO / CH3CN (100 / 10 to 0 / 100) at 1 mL / min for 30 minutes), said isomer being in substantially diastereomerically pure form.

[0050] The compound of formula (IIa) is prepared according to the fourth aspect of the invention.

[0051] In a fifth aspect of the present invention, there is provided a compound of formula (IIa), which is a compound of formula (IIa) p )-isomer.

[0052] In a sixth aspect of the present invention, there is provided a method for producing a compound of formula (S p )-NUC-3373 [ka] The isomerization preferentially forms the X-diastereomer of the compound of formula (IIa), which is p The isomer is R p This means that they are more easily produced than their isomers.

[0053] In a seventh aspect of the present invention, there is provided a compound (R p )-NUC-3373 [ka] is provided.

[0054] In an eighth aspect of the present invention, there is provided a compound of formula (S p )-NUC-7738 [ka] The isomerization preferentially forms the (S)-diastereomer of the compound of formula (IIb), which is the S p The isomer is R p This means that they are more easily produced than their isomers.

[0055] In a ninth aspect of the present invention, there is provided a compound (R p )-NUC-7738 [ka] is provided.

[0056] In a tenth aspect of the present invention, there is provided a compound of formula (S p )-NUC-9701 [ka] The isomerization preferentially forms the X-diastereomer of the compound of formula (IIa), which is the S p The isomer is R pThis means that they are more easily produced than their isomers.

[0057] In an eleventh aspect of the present invention, there is provided a compound (R p )-NUC-9701 [ka] is provided.

[0058] The compounds of the fifth, sixth, seventh, eighth, ninth, tenth and eleventh aspects of the invention may be diastereomers as described above in relation to the first, second and third aspects of the invention.

[0059] The present invention also provides pharmaceutical compositions comprising a compound of the sixth, seventh, eighth, ninth, tenth and eleventh aspects of the invention and a pharmaceutically acceptable excipient.

[0060] The present invention also provides a method of treating cancer (e.g., a solid tumor or leukemia), the cancer method comprising administering to a subject in need thereof a therapeutically effective amount of a compound of the sixth, seventh, eighth, ninth, tenth, and eleventh aspects of the present invention.

[0061] The compounds of the sixth, seventh, eighth, ninth, tenth, and eleventh aspects of the invention are of medical use. The compounds of the sixth, seventh, eighth, ninth, tenth, and eleventh aspects of the invention are for use in the treatment of cancer (e.g., solid tumors or leukemia).

[0062] The products of the sixth, seventh, eighth, ninth, tenth and eleventh aspects of the invention are obtainable (or obtained) by the first, second or third aspects of the invention.

[0063] Protecting groups for hydroxyl groups (e.g., P 1 , P 2 , P 5 or P 6) are independently selected from optionally substituted -Si(C1-C6-alkyl)3, optionally substituted -C(O)-C1-C6-alkyl, optionally substituted -C(O)-aryl, optionally substituted -C(O)-O-C1-C6-alkyl, -C(O)-O-aryl, -C(O)-O-CH2-fluorenyl, optionally substituted -C(aryl)3, optionally substituted -(C1-C3-alkylene)-aryl, optionally substituted -C(O)OCH2-aryl, and -C1-C4-alkyl-O-C1-C4-alkyl. When two hydroxyl groups are bonded to adjacent carbon atoms (e.g., P 5 and P 6 ), which may be jointly protected with an optionally substituted —C(C1-C4-alkyl)2 group.

[0064] Protecting groups for amino groups (e.g., P 3 , P 4 , P 7 or P 8 ) is, at each occurrence, independently selected from -C(O)OC-C-alkyl, optionally substituted -C(O)OCH-aryl, -C(O)-O-allyl, -C(O)-O-CH-fluorenyl, optionally substituted -C(aryl)3, optionally substituted -(C-C-alkylene)-aryl, optionally substituted -C(O)-C-C-alkyl, optionally substituted -C(O)-aryl, -S(O)-C-C-alkyl, optionally substituted -S(O)-aryl, and optionally substituted -Si(C-C-alkyl)3.

[0065] Many of the protected starting compounds of formula (IIIa), (IIIb), or (IIIc) are known in the art and / or can be prepared by known methods. For example, starting compounds of formula (IIIa), (IIIb), and (IIIc) can be synthesized from a parent nucleoside by protecting the hydroxy and / or amino groups with suitable protecting groups. Protecting groups are typically added and removed using conventional protecting group methodology, as described, for example, in "Protecting Groups in Organic Chemistry," edited by J.W.F. McOmie (1973); "Protecting Groups in Organic Synthesis," 2nd ed., T.W. Greene (1991); and "Protecting Groups," 3rd Revised Edition, P. J. Koscienski (1995).

[0066] Generally, compounds of formula (IIIa), (IIIb), and (IIIc) must be prepared by first protecting the 5'-hydroxyl group of the parent nucleoside with a protecting group (i.e., a group that is removed from the 5'-hydroxyl group without removing the desired 2'-hydroxyl and / or amino protecting group) ortho to that used to protect the 3'- and / or 2'-hydroxyl and / or amino groups. Concurrently or subsequently, the 3'-, 2'-hydroxyl, and / or amino groups can be protected with the desired protecting group, and the 5'-hydroxyl protecting group can be removed to produce compounds of formula (IIIa), (IIIb), and (IIIc). The specific protecting group can be simultaneously introduced at the 3'- and / or 2'-hydroxyl and 5'-hydroxyl and, optionally, amino groups, and then selectively removed from the 5'-hydroxyl group without being removed from the 3'- and / or 2'-hydroxyl and, optionally, amino groups.

[0067] According to some embodiments, P 1are independently selected from optionally substituted —Si(C1-C6-alkyl)3, optionally substituted —C(O)—C1-C6-alkyl, optionally substituted —C(O)-aryl, optionally substituted —C(O)—O—C1-C6-alkyl, —C(O)—O-allyl, —C(O)—O—CH2-fluorenyl, optionally substituted —C(aryl)3, optionally substituted —(C1-C3-alkylene)-aryl, optionally substituted —C(O)OCH2-aryl, and —C1-C4-alkyl-O—C1-C4-alkyl.

[0068] P 1 are independently selected from optionally substituted -Si(C1-C6-alkyl)3, optionally substituted -C(O)-OC1-C6-alkyl and optionally substituted -C(O)OCH2-aryl, -C(O)-O-aryl. Preferably, P 1 is selected from -C(O)O-tBu, -C(O)O-benzyl and -C(O)OCH2-allyl. 1 may be -C(O)OCH2-aryl. 1 may also be -C(O)O-tBu.

[0069] Or, P 1 may be independently selected from optionally substituted —C(O)—C1-C6-alkyl and optionally substituted —C(O)-aryl, such as P 1 may be independently selected from benzoyl and acetyl.

[0070] Furthermore, P 1 may be an optionally substituted —Si(C1-C6-alkyl)3. 1 may be -Si(C1-C4-alkyl)3. The alkyl group may be unsubstituted. P 1 may be t-butyldimethylsilyl.

[0071] According to some embodiments, P 2is selected from optionally substituted -Si(C1-C6-alkyl)3, optionally substituted -C(O)-C1-C6-alkyl, optionally substituted -C(O)-aryl, optionally substituted -C(O)-O-C1-C6-alkyl, -C(O)-O-allyl, -C(O)-O-CH2-fluorenyl, optionally substituted -C(aryl)3, optionally substituted -(C1-C3-alkylene)-aryl, optionally substituted -C(O)OCH2-aryl and -C1-C4-alkyl-O-C1-C4-alkyl.

[0072] P 2 may be independently selected from optionally substituted -Si(C1-C6-alkyl)3, optionally substituted -C(O)-OC1-C6-alkyl and optionally substituted -C(O)OCH2-aryl, -C(O)-O-aryl. Preferably, P 2 is selected from -C(O)O-tBu, -C(O)O-benzyl, and -C(O)OCH2-allyl. 2 may be -C(O)OCH2-aryl. 2 may also be -C(O)O-tBu.

[0073] Or, P 2 may be independently selected from optionally substituted —C(O)—C1-C6-alkyl and optionally substituted —C(O)-aryl, for example, P2 may be independently selected from benzoyl and acetyl.

[0074] Furthermore, P 2 may be an optionally substituted -Si(C1-C6-alkyl)3. 2 may also be -Si(C1-C4-alkyl)3. 2 may be t-butyldimethylsilyl.

[0075] P 3are independently selected from -C(O)OC-C-alkyl, optionally substituted -C(O)OCH-aryl, -C(O)-O-allyl, -C(O)-O-CH-fluorenyl, optionally substituted -C(aryl)3, optionally substituted -(C-C-alkylene)-aryl, optionally substituted -C(O)-C-C-alkyl, optionally substituted -C(O)-aryl, -S(O)-C-C-alkyl, optionally substituted -S(O)-aryl and optionally substituted -Si(C-C-alkyl)3.

[0076] P 3 may be independently selected from -C(O)OC1-C6-alkyl, optionally substituted -C(O)OCH2-aryl, -C(O)-O-allyl, optionally substituted -C(aryl)3 and optionally substituted -Si(C1-C6-alkyl)3. Preferably, P 3 is selected from -C(O)O-tBu, -C(O)O-benzyl and -C(O)OCH2-allyl. 3 may be -C(O)OCH2-aryl.

[0077] Or, P 3 is independently selected from optionally substituted —C(O)—C1-C6-alkyl and optionally substituted —C(O)-aryl, such as P 3 is independently selected from benzoyl and acetyl.

[0078] Or, P 3 is H.

[0079] P 4are independently selected from H, -C(O)OC-C-alkyl, optionally substituted -C(O)OCH-aryl, -C(O)-O-allyl, -C(O)-O-CH-fluorenyl, optionally substituted -C(aryl)3, optionally substituted -(C-C-alkylene)-aryl, optionally substituted -C(O)-C-C-alkyl, optionally substituted -C(O)-aryl, -S(O)-C-C-alkyl, optionally substituted -S(O)-aryl and optionally substituted -Si(C-C-alkyl)3.

[0080] Preferably, P 4 is H.

[0081] P 3 and P 4 may each be H. 3 and P 4 are H and P, respectively. 2 may be t-butyldimethylsilyl.

[0082] According to some embodiments, P 5 and P 6 are each independently selected from optionally substituted -Si(C1-C6-alkyl)3, optionally substituted -C(O)-C1-C6-alkyl, optionally substituted l-C(O)-aryl, optionally substituted -C(O)-O-C1-C6-alkyl, -C(O)-O-allyl, -C(O)-O-CH2-fluorenyl, optionally substituted -C(aryl)3, optionally substituted -(C1-C3-alkylene)-aryl, optionally substituted -C(O)OCH2-aryl and -C1-C4-alkyl-O-C1-C4-alkyl, or P 5 and P 6 together form an optionally substituted —C(C1-C4-alkyl)2- group. P 5 and P 6 may be identical.

[0083] P 5 and P 6may each be selected from optionally substituted -Si(C1-C6-alkyl)3, optionally substituted -C(O)-OC1-C6-alkyl, and optionally substituted -C(O)OCH2-aryl, -C(O)-O-aryl. Preferably, P 5 and P 6 may each be selected from -C(O)O-tBu, -C(O)O-benzyl, and -C(O)OCH2-allyl. 5 and P 6 may each be -C(O)OCH2-aryl. 5 and P 6 may each be —C(O)O-tBu.

[0084] Or, P 5 and P 6 may each be selected from optionally substituted -C(O)-C1-C6-alkyl and optionally substituted -C(O)-aryl, for example, P 5 and P 6 may each be selected from benzoyl and acetyl.

[0085] Furthermore, P 5 and P 6 may each be an optionally substituted —Si(C1-C6-alkyl)3. 2 may be -Si(C1-C4-alkyl). The alkyl group may be unsubstituted. 5 and P 6 may each be t-butyldimethylsilyl.

[0086] However, preferably, P 5 and P 6 can together form an optionally substituted -C(C1-C4-alkyl)2- group. P 5 and P 6 can together form a -C(Me)2- group.

[0087] P 7are independently selected from -C(O)OC-C-alkyl, optionally substituted -C(O)OCH-aryl, -C(O)-O-allyl, -C(O)-O-CH-fluorenyl, optionally substituted -C(aryl), optionally substituted -(C-C-alkylene)-aryl, optionally substituted -C(O)-C-C-alkyl, optionally substituted -C(O)-aryl, -S(O)-C-C-alkyl, optionally substituted -S(O)-aryl, and optionally substituted -Si(C-C-alkyl).

[0088] P 7 may be independently selected from -C(O)OC1-C6-alkyl, optionally substituted -C(O)OCH2-aryl, -C(O)-O-allyl, optionally substituted -C(aryl)3 and optionally substituted -Si(C1-C6-alkyl)3. Preferably, P 7 is selected from -C(O)O-tBu, -C(O)O-benzyl and -C(O)OCH2-allyl. 7 may be -C(O)OCH2-aryl.

[0089] Or, P 7 may be independently selected from optionally substituted —C(O)—C1-C6-alkyl and optionally substituted —C(O)-aryl, such as P 7 may be independently selected from benzoyl and acetyl.

[0090] Furthermore, P 7 may be H.

[0091] Similarly, P 8are independently selected from H, —C(O)OC₁-C₆-alkyl, optionally substituted —C(O)OCH₂-aryl, —C(O)—O-allyl, —C(O)—O—CH₂-fluorenyl, optionally substituted —C(aryl)₃, optionally substituted —(C₁-C₃-alkylene)-aryl, optionally substituted —C(O)—C₁-C₆-alkyl, optionally substituted —C(O)-aryl, —S(O)₂-C₁-C₆-alkyl, optionally substituted —S(O)₂-aryl, and optionally substituted —Si(C₁-C₆-alkyl)₃.

[0092] Preferably, P 8 is H.

[0093] P 7 and P 8 may each be H. 7 and P 8 is H, and P 5 and P 6 may together form a -C(Me)2- group.

[0094] The optionally substituted -Si(C1-C6-alkyl)3 group may also be -Si(C1-C4-alkyl)3. The group (i.e., the alkyl group) is preferably unsubstituted. Examples include triethylsilyl and t-butyl-dimethylsilyl.

[0095] The optionally substituted -C(O)-C1-C6-alkyl group may be a -C(O)-C1-C6-alkyl group. The group (i.e., the alkyl group) is preferably unsubstituted. Examples include acetyl and propionyl.

[0096] The optionally substituted -C(O)-aryl may be a -C(O)-phenyl group. The group (i.e., the phenyl group) is preferably unsubstituted. Examples include benzoyl.

[0097] The optionally substituted -C(O)-O-C-alkyl group may be a -C(O)-O-C-alkyl group. The group (i.e., the alkyl group) is preferably unsubstituted. Examples include -C(O)-O-methyl and -C(O)-O-ethyl. A particularly preferred example is C(O)OtBu.

[0098] The optionally substituted -(C1-C3-alkylene)-aryl group is preferably an optionally substituted benzyl group, examples of which include benzyl, phenethyl, 4-methoxybenzyl, 4-nitrobenzyl, 4-bromobenzyl, 2,3-dimethoxybenzyl, and 2,4-dimethoxybenzyl.

[0099] The optionally substituted -C(O)OCH-aryl group is preferably an optionally substituted -C(O)Obenzyl group, examples of which include -C(O)Obenzyl and -C(O)O-(4-methoxybenzyl).

[0100] The optionally substituted -C1-C4-alkyl-O-C1-C4-alkyl group may also be a -C1-C2-alkyl-O-C1-C2-alkyl group. The group (i.e., the alkyl group) is preferably unsubstituted. Examples include methoxy-methyl (MOM) and 2-methoxy-ethoxy-methyl (MEM).

[0101] The optionally substituted -S(O)2-C1-C6-alkyl group may be an -S(O)2-C1-C4-alkyl group. The group (i.e., the alkyl group) is preferably unsubstituted. Examples include methanesulfonate.

[0102] The optionally substituted -S(O)2-aryl group may be a -S(O)2-phenyl group, examples of which include phenylsulfonate, 4-methylphenylsulfonate, and 4-nitrophenylsulfonate.

[0103] An optionally substituted -C(aryl)3 group may be a -C(phenyl)3 group. Examples include trityl.

[0104] P 2 , P 3 and P 4 or P 5 , P 6 , P 7 and P 8 are protecting groups, the deprotection step can comprise two or three individual deprotection reactions. This is the case when two or three different protecting groups are used and these two or three protecting groups are not removed under the same conditions.

[0105] However, the deprotection step can also comprise a single deprotection reaction in which all protecting groups are removed. 2 and P 3 may be a protecting group that is removed under the same conditions. 2 and P 3 Similarly, P 5 and P 6 may be a protecting group that is removed under the same conditions. 5 and P 6 may be identical.

[0106] Throughout this specification, "diastereomerically enriched" and "substantially diastereomerically pure" mean a diastereomeric purity of greater than 95%. "Diastereomerically enriched" and "substantially diastereomerically pure" may also mean a diastereomeric purity of greater than 98%, greater than 99%, or greater than 99.5%.

[0107] Any of the above alkyl and aryl groups (e.g., phenyl (including the phenyl group in a benzyl group)) are optionally substituted, if chemically possible, with 1 to 3 substituents, each of which, in each occurrence, is independently oxo, ═NR a , =NOR a , Halo, Nitro, Cyano, NRa R a , N.R. a S(O)2R a , N.R. a CONR a R a , N.R. a CO2R a , OR a ;SR a , SOR a , SO3R a , SO2R a , SO2NR a R a , CO2R a C(O)R a ,CONR a R a , C1-C4-alkyl, C2-C4-alkenyl, C2-C4-alkenyl, and C1-C4 haloalkyl (where R a is, at each occurrence, independently selected from the group consisting of H, C1-C4 alkyl, and C1-C4 haloalkyl.

[0108] Any of the above alkyl groups may be unsubstituted.

[0109] Any of the above-mentioned aryl groups (e.g., phenyl (including the phenyl group in a benzyl group)) may optionally be substituted, if chemically possible, with 1 to 3 substituents, each of which, in each occurrence, is independently selected from halo, nitro, cyano, NR a R a , N.R. a S(O)2R a , N.R. a CONR a R a , N.R. a CO2R a , OR a ;SR a , SOR a , SO3R a , SO2R a , SO2NR a R a , CO2R a C(O)R a ,CONR a R a, C1-C4-alkyl, C2-C4-alkenyl, C2-C4-alkenyl, and C1-C4 haloalkyl (where R a is, at each occurrence, independently selected from the group consisting of H, C1-C4 alkyl, and C1-C4 haloalkyl.

[0110] Any of the above-described aryl groups (e.g., phenyl (including the phenyl group in a benzyl group)) may be optionally substituted with 1 to 3 substituents, each of which, at each occurrence, is independently selected from halo, nitro, OR, a , C1-C4-alkyl, C1-C4 haloalkyl (wherein R a is, at each occurrence, independently selected from the group consisting of H, C1-C4 alkyl, and C1-C4 haloalkyl.

[0111] The aryl group optionally has 6 to 20 carbon atoms to satisfy valency requirements. The aryl group is a carbocyclic group that satisfies Hückel's rule (i.e., contains a carbocyclic ring system containing 2(2n+1) π electrons). The aryl group may be an optionally substituted phenyl group, an optionally substituted biphenyl group, an optionally substituted naphthalenyl group, or an optionally substituted anthracenyl group. Similarly, the aryl group may contain a non-aromatic carbocyclic moiety. Preferably, the aryl group is an optionally substituted phenyl group.

[0112] Alkyl groups are straight-chain or branched. Thus, for example, a C4 alkyl group is n-butyl, i-butyl, or t-butyl.

[0113] Step a) of the first, second or third embodiment is carried out in an organic solvent (S1). Organic solvents include, but are not limited to, ethers (e.g., tetrahydrofuran, dioxane, diethyl ether, methyl t-butyl ether); ketones (e.g., acetone and methyl isobutyl ketone); halogenated solvents (e.g., dichloromethane, chloroform, and 1,2-dichloroethane); and amides (e.g., DMF, NMP); and / or mixtures thereof. When step a) is carried out in the presence of a Grignard compound, the organic solvent is preferably an ether. Most preferably, the solvent is tetrahydrofuran.

[0114] When step a) of the first aspect is carried out in the presence of a nitrogen base, the organic solvent is most preferably a halogenated solvent or an amide.

[0115] The reaction is generally carried out at a suitable temperature, for example, about -5 to 40° C. Preferably, the reaction temperature is about 25 to about 30° C. During the reaction, the mixture may be stirred for about 15 minutes to about 16 hours, preferably about 30 to about 60 minutes.

[0116] The methods of the present invention can also include deprotection of hydroxy and amino protecting groups.

[0117] The deprotection step (step b)) may be carried out without purification of the product of step a).

[0118] If the protecting group is acid-sensitive (e.g., trityl, C(O)OtBu, MOM, MEM, 2,4-dimethoxybenzyl, 2,3-dimethoxybenzyl, -C(Me)2-), the deprotection step is carried out using a suitable acid. The acid is a Bronsted acid (e.g., TFA, phosphoric acid, HCl, or formic acid) or a Lewis acid (e.g., ZnBr2, CeCl3). Lewis acids (e.g., ZnBr2) are less preferred. Likewise, HCl is less preferred. Preferably, the acid is TFA.

[0119] If the protecting group is base-sensitive (e.g., acetyl, benzoyl), the deprotection step is carried out using a suitable base, e.g., aqueous NH3 or aqueous NaOH. Base-sensitive groups are less preferred.

[0120] If the protecting group is a silyl group (e.g., triethylsilyl or t-butyldimethylsilyl), the deprotection step is carried out using a suitable acid (e.g., TFA) or using a suitable fluoride source (e.g., tetrabutylammonium fluoride, fluorosilicic acid, HF).

[0121] If the protecting group is a benzyl or C(O)O benzyl group, the deprotection step is carried out using H2 and a suitable catalyst (e.g., Pd / C). Such protecting groups are less preferred.

[0122] When the protecting group is 4-methoxy-benzyl, 2,3-dimethoxybenzyl, 2,4-dimethoxybenzyl, or C(O)O-(4-methoxybenzyl), the deprotection step is carried out using a suitable oxidizing agent (e.g., meta-chloroperbenzoic acid).

[0123] If the protecting group is -C(O)-O-allyl, the deprotection step is carried out using (PPh3)4Pd.

[0124] When the protecting group is -C(O)-O-CH2-fluorenyl, the deprotection step is carried out using piperidine.

[0125] The deprotection step is carried out in an organic solvent or mixture thereof, including, but not limited to, halogenated solvents (e.g., dichloromethane, chloroform, dichloroethane), alcohols (e.g., methanol, ethanol, isopropanol), and ethers (e.g., tetrahydrofuran, diethyl ether).

[0126] When the deprotection step is carried out in the presence of an acid (eg TFA), the organic solvent is preferably a halogenated solvent, such as dichloromethane.

[0127] The deprotection reaction is carried out, for example, at a temperature in the range of −10 to about 30° C. The reaction is generally carried out at a temperature of −5 to 5° C. The reaction may be stirred for about 15 minutes to about 16 hours, preferably about 1 to about 4 hours, and more preferably about 2 to about 3 hours.

[0128] If step b) is carried out using a C1-C4-alcohol and / or water (for example a mixture of isopropyl alcohol (IPA) and water), the reaction mixture is heated, for example, to a temperature of 30 to 90°C or to a temperature of 60 to 85°C.

[0129] When deprotection is carried out in the presence of an acid (e.g., TFA), isolation of the product obtained after deprotection is generally carried out by quenching the excess acid used in the deprotection step, extracting the product with a water-immiscible organic solvent, and recovering the product by evaporation of the organic solvent.

[0130] Examples of water-immiscible organic solvents useful in the extraction include esters such as ethyl acetate, methyl acetate, isopropyl acetate, etc.; chlorinated solvents such as dichloromethane, chloroform, etc.; aromatic hydrocarbon solvents such as toluene, xylene, etc., preferably ethyl acetate.

[0131] In certain embodiments, it may be desirable to further purify the ProTide obtained from the method of the first aspect of the invention. Similarly, it may be desirable to purify the compound of Formula (IIa) obtained from the method of the fourth aspect of the invention. Purification methods are well known to those skilled in the art and include chromatography (e.g., column chromatography), recrystallization, and distillation. In other embodiments, no purification is required.

[0132] It will be understood that various modifications of the embodiments disclosed herein are possible. Therefore, the above description is not intended to be limiting, but merely to exemplify preferred embodiments. For example, the functions described and illustrated as the best mode for carrying out the invention are for illustrative purposes only. Other arrangements and methods may be implemented by those skilled in the art without departing from the spirit of the invention. Moreover, those skilled in the art will envision other modifications within the scope and spirit of the present disclosure.

[0133] The following abbreviations are used throughout this specification: ACN: acetonitrile, AIBBr: acetoxyisobutyryl bromide, BOC: t-butyl carbonate, DCM: dichloromethane, DMAP: N,N-dimethyl-4-aminopyridine, DMF: N,N-dimethylformamide, eq.: molar equivalent, FUDR: 5-fluoro-2'-deoxyuridine, IPA: isopropyl alcohol, MEM: 2-methoxyethoxymethyl, MOM: methoxymethyl, MTBE: methyl t-butyl ether, NMP: N-methyl-2-pyrrolidone, Np: 1-naphthyl, PTSA: p-toluenesulfonic (tosylated) acid, RT: room temperature, TBAF: tetrabutylammonium fluoride, TBDMS: t-butyldimethylsilyl, TEA: triethylamine, Tf: trifluoromethylsulfonate (triflate), TFA: trifluoroacetic acid, THF: tetrahydrofuran.

[0134] V is used to indicate the volume (ml) per mass (g) of an ingredient, so if there is 1g of ingredient, 10V means 10ml of the indicated liquid.

[0135] [Example] The present invention is further described by the following examples, which are for illustrative purposes only and are not intended to limit the scope of the invention. [Example]

[0136] Preparation of a diastereomeric mixture of 2-[(2,3,4,5,6-pentafluorophenoxy)-phenoxy-phosphorylamino]propionic acid benzyl ester 5 (Illustrative example of a compound of formula (IIb)) [ka] To a mixture containing L-alanine benzyl ester hydrochloride 1 (100 g) in methylene chloride (1 L) was added phenyl dichlorophosphate 2 (77 ml) at 25-35°C under stirring. The resulting mixture was cooled to -70 to -78°C, triethylamine (130.5 ml) was added, and the mixture was stirred at the same temperature for 1 hour. The temperature of the reaction system was raised to 25-35°C and stirred for 2 hours. After completion of the reaction, the reaction system was concentrated under reduced pressure at 35°C or below to obtain a residue. Diisopropyl ether (2 L) was added to the resulting residue at 25-35°C and stirred for 30 minutes at the same temperature. The reaction system was filtered and washed with diisopropyl ether (500 ml). The filtrate was then concentrated under vacuum at 35°C or below to obtain phenyl-(benzoxy-L-alaninyl)-phosphorochloridate 3. The resulting compound was dissolved in methylene chloride (1 L) at 25-35°C and cooled to -5-10°C. Pentafluorophenol 4 (85.5 g) and triethylamine (65.2 ml) were added to the reaction mixture at the same temperature, followed by stirring for 2 hours. After completion of the reaction, the reaction mixture was concentrated under vacuum at 35°C or below, and ethyl acetate (1 L) was added at 25-35°C. The mixture was stirred for 30 minutes at the same temperature. The solid was filtered and washed with ethyl acetate (1 L). The filtrate was washed with water (1 L), 10% sodium carbonate (2 x 1 L), and brine (1 L). The organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum at 35-45°C to obtain the diastereomeric mixture of the title compound 5 as a white semisolid. Yield: 210g Chiral purity by HPLC (% area): 33.74:66.26% (R P :S P ) [Example]

[0137] 2-[(2,3,4,5,6-pentafluorophenoxy)-phenoxy-phosphorylamino]propionic acid benzyl ester 5S p Separation of diastereomers (example of compound of formula (IIb)) [ka] Diastereomeric mixture of compound 5 (210 g; R P :S P The diastereomeric mixture of Compound 5 (33.74:66.26%) was added to a 20% ethyl acetate in hexane solution (1.2 L) at 25-35°C and stirred for 1 hour. The solid was filtered and washed with a 20% ethyl acetate in hexane solution (300 ml) to give a diastereomeric mixture of Compound 5. Yield: 112g Chiral purity by HPLC (% area): 22.13:77.87% (R P :S P ) The filtrate was concentrated in vacuo to give a diastereomeric mixture of compound 5 (75 g; R P :S P :65.43:34.57%). A diastereomeric mixture of the compound of formula (IIb) (112 g; R P :S P The resulting mixture (22.13:77.87%) was charged with 20% ethyl acetate in hexane (1.2 L) at 25-35°C and stirred for 1 hour. The solid was filtered and washed with 20% ethyl acetate in hexane (300 ml) to obtain substantially pure S of compound 5. p -diastereomers were obtained. Yield: 80g Chiral purity by HPLC (% area): 0.20:99.80% 1 H NMR (300MHz, DMSO-d6):7.18-7.41(m, 10H), 6.91-6.99(d, 1H), 5.10(s, 2H), 4.01-4.11(m, 1H), 1.30-1.32(d, 3H) ESI-MS (m / z): 524 (M+1) The filtrate was concentrated in vacuo to give a diastereomeric mixture of compound 5 (28 g; R P :S P :80.77:19.23%). [Example]

[0138] Enrichment of the S-isomer of 2-[(2,3,4,5,6-pentafluorophenoxy)-phenoxy-phosphorylamino]propionic acid benzyl ester 5 (Illustrative example of a compound of formula (IIb)) [ka] 2-[(2,3,4,5,6-pentafluorophenoxy)-phenoxy-phosphorylamino]propionic acid benzyl ester 5 (75 g; R p :S p To a solution of 65.43%:34.57% triethylamine (7.5 ml) was added at 25-35°C with stirring, and the mixture was stirred at the same temperature for 6 hours. After completion of the reaction, the reaction mixture was quenched with water (750 ml) and extracted with ethyl acetate (750 ml). The organic phase was dried over anhydrous sodium sulfate and concentrated under vacuum to give the title compound as a solid. Yield: 45g Chiral purity by HPLC (% area): 91.29:8.71% (S P :R P ) R of the obtained 2-[(2,3,4,5,6-pentafluorophenoxy)-phenoxy-phosphorylamino]propionic acid benzyl ester 5 p and S p -diastereomeric mixture (45g; R p :S p The resulting mixture (8.71:91.29%) was slurried in 20% ethyl acetate in hexane (1.1 L) at 25-30°C and stirred at the same temperature for 1 hour. The solid was filtered and washed with 20% ethyl acetate in hexane (225 ml) to give the title compound S. p -diastereomer was obtained as a solid. Yield: 19g Chiral purity by HPLC (% area): 99.2:0.08% (S p :R p ) [Example]

[0139] Preparation of a diastereomeric mixture of 2-[(4-nitrophenoxy)-phenoxy-phosphorylamino]propionic acid benzyl ester 7 (Illustrative example of a compound of formula (IIb)) [ka] To a mixture of L-alanine benzyl ester hydrochloride 1 (50 g) in methylene chloride (500 ml) was added phenyl dichlorophosphate 2 (54 g) with stirring at 25-35°C. The resulting mixture was cooled to -70 to -78°C, and triethylamine (65.2 ml) was added and stirred at the same temperature for 1 hour. The temperature of the reaction system was raised to 25-35°C and stirred for 2 hours. After completion of the reaction, the reaction system was concentrated under reduced pressure at 35°C or below to obtain a residue. Diisopropyl ether (1 L) was added to the resulting residue at 25-35°C and stirred for 30 minutes at the same temperature. The reaction system was filtered and washed with diisopropyl ether (250 ml). The filtrate was then concentrated under vacuum at 35°C or below to obtain phenyl-(benzoxy-L-alaninyl)-chlorophosphate 3. The resulting compound was dissolved in methylene chloride (500 ml) at 25-35°C and cooled to -5 to -10°C. 4-Nitrophenol 6 (27.5 g) and triethylamine (65.2 ml) were added to the reaction mixture at the same temperature and stirred for 2 hours. After completion of the reaction, the reaction mixture was concentrated under vacuum at 35°C or below, and ethyl acetate (500 ml) was added at 25-35°C and stirred for 30 minutes at the same temperature. The solid was filtered and washed with ethyl acetate (500 ml). The filtrate was washed with water (500 ml), 10% sodium carbonate (2 x 500 ml), and brine (500 ml). The organic phase was dried over anhydrous sodium sulfate and concentrated under vacuum at 35-40°C to obtain the diastereomeric mixture of the title compound 7 as a viscous oily liquid. Yield: 90g Chiral by HPLC (% area): 45.6:54.94% (R P :S P ) The diastereomeric mixture of 2-[(4-nitrophenoxy)-phenoxy-phosphorylamino]propionic acid benzyl ester 7 obtained as described above (40 g; R p :S p :45.6:54.94%) was obtained by preparative HPLC. p and R pThe diastereomers were separated and the pure fractions were concentrated under vacuum to give S p and R p -diastereomers were obtained. Yield; S p -Diastereomer: 8g 1 H NMR (300MHz, CDCl3):8.15-8.19 (d, 2H), 7.15-7.37 (m, 12H), 5.12 (s, 2H), 4.02-4.24 (m, 2H), 1.39-1.42 (d, 3H) ESI-MS (m / z): 479 (M+Na) R p -Diastereomer: 6g 1 H NMR (300MHz, CDCl3):8.08-8.13 (d, 2H), 7.15-7.34 (m, 12H), 5.10 (s, 2H), 4.48-4.56 (m, 1H), 4.11-4.20 (m, 1H), 1.39-1.41 (d, 3H) ESI-MS (m / z): 457 (M+1) + S p and R p -Diastereomeric mixture: 20g [Example]

[0140] (S p Preparation of 2-[(2,3,4,5,6-pentafluorophenoxy)-phenoxy-phosphorylamino]propionic acid benzyl ester 5 (Illustrative example of a compound of formula (IIb)) [ka] To a mixture of L-alanine benzyl ester·HCl (100 g) in 1000 ml of methylene dichloride was added phenyl dichlorophosphate 2 (97.8 g) with stirring at 30°C. The mixture was cooled to −20°C, and triethylamine (93.8 g) was added slowly while maintaining the temperature at −20°C. The reaction mixture was stirred at −20°C for 1 h, then warmed to 10°C (10±5°C) and stirred for an additional 1.5 h. A solution of pentafluorophenol 4 (85.3 g) in methylene dichloride (1000 ml) was slowly added, followed by the slow addition of trimethylamine (46.8 g) while maintaining the temperature at 10°C. Under a nitrogen atmosphere, 46.9 g of triethylamine was slowly added to the reaction at 10°C (10±5°C). The mixture was stirred at 10°C for 2 hours and then quenched by the slow addition of 0.5 N HCl solution while maintaining the temperature at 10°C. After warming to room temperature, the mixture was separated, and the organic phase was washed with saturated bicarbonate solution, distilled water, and brine, and then concentrated in vacuo. The crude mixture was suspended in 1500 ml of a 20% solution of ethyl acetate in n-heptane at 25° C. Triethylamine (12.2 g) was added and the mixture was stirred at 25° C. The mixture was filtered, and the solid was dissolved in 2500 ml of ethyl acetate, washed with water and brine, and concentrated in vacuo. The solid was suspended in 1200 ml of a 20% solution of ethyl acetate in n-heptane, stirred for 45-60 minutes, and filtered. The material was dried under vacuum to give the desired product 5-(S p The yields ranged from 40 to 80%, and the diastereomeric purity was over 99%. [Example]

[0141] Preparation of a diastereomeric mixture of 2-[(2,3,4,5,6-pentafluorophenoxy)-naphth-1-oxy-phosphorylamino]propionic acid benzyl ester 12 (an example of a compound of formula (IIa)) [ka] α-Naphthol 8 (100 g) was dissolved in DCM (1 L) at 25°C. POCl39 (1.1 eq) was added at 25°C and stirred for 10 minutes. The mixture was cooled to −70°C and stirred for 10 minutes. Triethylamine (1.1 eq) was slowly added while maintaining the temperature below −70°C, and the mixture was stirred at −70°C for 1 hour. The mixture was warmed to 25°C, stirred for 1 hour, and then cooled to −50°C. L-alanine benzyl ester 1 (HCl salt; 1 eq) was added to the mixture and stirred for 10 minutes. Then, triethylamine (2.2 eq) in DCM (200 ml) was added over 30 minutes at −50°C. The mixture was stirred at −50°C for 1 hour, then warmed to 25°C and stirred for an additional 1 hour. The mixture was cooled to -10°C and stirred for 10 minutes. Pentafluorophenol 4 in DCM (200 ml) was then slowly added to the reaction mixture at -10°C or below. The reaction mixture was stirred at -10°C for 10 minutes, and triethylamine (1.1 eq) was added at -10°C over 30 minutes. The mixture was stirred at -10°C for 1 hour, and then warmed to 0°C. Water (1 L) was added, and the mixture was stirred at 0°C for 30 minutes. The mixture was warmed to 25°C and stirred for 5-10 minutes. The organic layer was then separated. The aqueous layer was extracted with DCM (500 ml). The combined organic phase was washed with 7% sodium bicarbonate solution (2 x 1 L), and the organic layer was dried over anhydrous sodium sulfate and concentrated in vacuo. 50% IPA / water (2.4 L) was added to the crude compound and stirred at 25°C for 1 hour. The solid compound was filtered, and the wet cake was washed with 50% IPA / water (500 ml) and then dried under vacuum. 50% IPA / water (2.4 L) was added again to the crude compound and stirred at 25°C for 1 hour, and then filtered. The cake was washed again with 50% IPA / water (500 ml) and then dried under vacuum. The semi-dried compound was washed with cyclohexane (10 v / w) at 25-30°C for 1 hour. The solid compound was then washed with cyclohexane (2 L), and the wet compound 12 was dried under vacuum at 55-60°C for 12 hours. result: Mass of compound: 252 g Overall yield: 66% HPLC purity: 98.31% (diastereomer ratio: 1:1) 31 P NMR (202MHz, CDCl):δ P 1.35, -1.41; 1 H NMR (500MHz, CDCl):δ H 8.13-8.10 (1H, m, H-Ar), 7.90-7.88 (1H, m, H-Ar), 7.73 (1H, apparent d, J= 8.5Hz, H-Ar), 7.62-7.55 (3H, m, H-Ar), 7.45-7.41 (1H, m, H-Ar), 7.36-7.28 (5H, m, H-Ar), 5.01 (1H, apparent s, CH2Ph), 5.12 (1H, q, J=12.5Hz, CH2Ph), 4.38-4.31 (1H, m, NHCHCH3), 4.17-4.08 (1H, m, NHCHCH3), 1.49, 1.47 (3H, 2xd, J=3.5Hz, NHCHCH3); MS (ES+) m / z: 574 (M+Na + , 100%), exact mass: C 26 H 19 F5NO5P theoretical value 551.40, actual value 574.05 (M+Na + ); Reverse-phase HPLC (elution with H2O / MeOH (20 / 80), F = 1 ml / min, 35 min, λ = 254): t for the two diastereomers R Two peaks with 12.96 min and 14.48 min The diastereomers of compound 12 were separated by HPLC on a Biotage Isolera: using a C18 SNAP Ultra (30 g) cartridge with a mixture of MeOH / HO (70 / 30) as eluent, the rapidly eluting isomer (R p diastereomers) and the slower eluting isomer (S p (presumably diastereomers) were obtained. Note: The isomers are designated as fast eluting (FE) and slow eluting (SE) based on their retention times on a C18 (reverse phase) cartridge and an analytical HPLC column. Rapidly eluting isomer (R p (possibly diastereomers): 31 P NMR (202MHz, CDCl):δ P 1.41; 1 H NMR (500MHz, CDCl):δ H 8.02 (1H, dd, J=7.0, 2.0Hz, H-Ar), 7.79 (1H, dd, J=6.5, 3.0Hz, H-Ar), 7.64 (1H, d, J=8.5Hz, H-Ar), 7.53-7.45 (3H, m, H-Ar), 7.33 (1H, t, J=8.0Hz, H-Ar), 7.28-7.23 (5H, m, H-Ar), 5.09 (s, 2H, CH2Ph), 4.29-4.21 (1H, m, NHCHCH3), 4.02-3.97 (1H, m, NHCHCH3), 1.38 (3H, d, J=7.0Hz, NHCHCH3); MS (ES+) m / z: 574 (M+Na + , 100%), exact mass:C 26 H 19 F5NO5P theoretical value 551.40, actual value 574.05 (M+Na + ); Reverse-phase HPLC (elution with HO / MeOH (20 / 80), F = 1 ml / min, 35 min, λ = 254): R =12.96 Slowly eluting isomer (S p (possibly diastereomers): 31 P NMR (202MHz, CDCl):δ P 1.36; 1 H NMR (500MHz, CDCl):δ H8.14-8.11 (1H, m, H-Ar), 7.90-7.87 (1H, m, H-Ar), 7.74 (1H, d, J=8.0Hz, H-Ar), 7.60 (1H, d, J=8.0Hz, H-Ar), 7.58-7.55 (2H, m, H-Ar), 7.44 (1H, t, J=8.0Hz, H-Ar), 7.34-7.30 (5H, m, H-Ar), 5.12 (2H, q, J=12.5Hz, CH2Ph), 4.35-4.29 (1H, m, NHCHCH3), 4.04-4.00 (1H, m, NHCHCH3), 1.48 (3H, d, J=7.0Hz, NHCHCH3); MS (ES+) m / z: 574 (M+Na + , 100%), exact mass:C 26 H 19 F5NO5P Theoretical value 551.40, Measured value 574.05 (M+Na + ); Reverse-phase HPLC (elution with HO / MeOH (20 / 80), F = 1 ml / min, 35 min, λ = 254): R =14.48. [Example]

[0142] 2-[(2,3,4,5,6-Pentafluorophenoxy)-naphth-1-oxy-phosphorylamino]propionic acid benzyl ester 12S p -Diastereomeric enrichment (example of compound of formula (IIb)) [ka] A 1:1 diastereomeric mixture of compound 12 (25 g) was dissolved in 10% MTBE / n-hexane (500 ml) and triethylamine (2.5 ml) was added to the reaction mixture at 25°C. The mixture was stirred at 30°C for 80 hours. The mixture was filtered, and the wet cake was washed with 10% MTBE / n-hexane (75 ml) and then vacuum dried for 30 minutes. 50% IPA / water (200 ml) was added to the crude compound, and the mixture was stirred at 25-35°C for 1 hour and then filtered. The wet cake was washed with 50% IPA / water (100 ml) and then vacuum dried at 55-60°C. result: Mass of compound: 17g Overall yield: 68% HPLC purity: 97.66% Slowly eluting isomer (S p (possibly diastereomers): 31 P NMR (202MHz, CDCl):δ P 1.36; 1 H NMR (500MHz, CDCl):δ H 8.14-8.11 (1H, m, H-Ar), 7.90-7.87 (1H, m, H-Ar), 7.74 (1H, d, J=8.0Hz, H-Ar), 7.60 (1H, d, J=8.0Hz, H-Ar), 7.58-7.55 (2H, m, H-Ar), 7.44 (1H, t, J=8.0Hz, H-Ar), 7.34-7.30 (5H, m, H-Ar), 5.12 (2H, q, J=12.5Hz, CH2Ph), 4.35-4.29 (1H, m, NHCHCH3), 4.04-4.00 (1H, m, NHCHCH3), 1.48 (3H, d, J=7.0Hz, NHCHCH3); MS (ES+) m / z: 574 (M+Na + , 100%), exact mass:C 26 H 19 F5NO5P theoretical value 551.40, actual value 574.05 (M+Na + ); Reverse-phase HPLC (elution with HO / MeOH (20 / 80), F = 1 ml / min, 35 min, λ = 254): R =14.48 The stereochemistry of the two isomers of compound 12 (R p vs. S p ) of NUC-3373 prepared using compound 12 31The tentative assignment was based on a comparison of the P chemical shifts, 1H NMR spectrum, and HPLC retention time with those of other ProTides known in the literature. As noted above, the stereochemistry of the phosphate stereocenter is inverted during the process of the present invention, with the (Sp)-diastereomer of compound 12 forming the (Sp)-diastereomer of NUC-3373, and similarly, the (R)-diastereomer of compound 12 generating the (R)-diastereomer of NUC-3373. The stereochemical assignment is supported by powder X-ray diffraction and differential scanning calorimetry performed on two isomers of compound 12, but this is not conclusive in itself. [Example]

[0143] NUC-3373 S p and R p Isomer formation 3'-BOC protected FUDR 16 is generated according to the following scheme: [ka] Compound 16 is then coupled with a compound of formula (IIa). [ka] Compound 16 (1g) and Compound 12 S p The isomer (1.2 eq) was dissolved in THF (10 ml) and the mixture was cooled to 0°C. To the mixture was added t-butylmagnesium chloride (2.5 eq, 2.0 M THF solution) over 15 minutes. The mixture was warmed and stirred at 25°C for 4 hours. The mixture was cooled to 10°C and saturated ammonium chloride solution (10 ml) was added. Ethyl acetate (10 ml) was added to the mixture and the organic layer was separated. The aqueous layer was extracted with ethyl acetate (5 ml). The combined organic layer was washed with deionized water (5 ml) followed by 20% sodium chloride solution (5 ml). The organic phase was dried over anhydrous sodium sulfate and then concentrated under vacuum to give 2.16 g of compound 17 (crude yield 100%). Compound 17 (1 g) was dissolved in DCM (5 ml) and cooled to 10°C. TFA (2 ml) was slowly added to the mixture while maintaining the temperature below 20°C. The mixture was warmed to 30°C and stirred for 6 hours. The mixture was cooled to 10°C, and deionized water (5 ml) was slowly added while maintaining the temperature below 20°C. After stirring for 10 minutes, the organic layer was separated, and the aqueous layer was extracted with DCM (5 ml). The combined organic layers were washed with deionized water (2 × 5 ml), 7% sodium bicarbonate solution (2 × 5 ml), and 20% sodium chloride solution (5 ml), then dried over anhydrous sodium sulfate (1 w / w) and concentrated under vacuum. The crude compound was purified by column chromatography using silica gel (100-200 mesh) in ethyl acetate / DCM. The pure compound was eluted in 50% ethyl acetate / DCM to 100% ethyl acetate. The combined pure fractions were concentrated in vacuo and the compound slurry was then washed with cyclohexane (5 ml). result: NUC-3373(S p Mass of isomer: 9.3g Overall yield: 70% HPLC purity: 96.86% 1 H-NMR (500MHz, MeOD):δ H8.16 - 8.14 (m, 1H, H - Ar), 7.90 - 7.80 (m, 1H, H - Ar), 7.72 - 7.70 (m, 2H, H - Ar), 7.54 - 7.49 (m, 3H, H - Ar, H - 6), 7.43 (apparent t, 1H, J = 8.0Hz, H - Ar), 7.35 - 7.27 (m, 5H, H - Ar), 6.16 - 6.13 (m, 1H, H - 1’), 5.11 (AB system, J = 12.0Hz, 2H, OCH2Ph), 4.35 - 4.33 (m, 2H, 2xH - 5’), 4.30 - 4.28 (m, 1H, H - 3’), 4.14 - 4.08 (m, H, CHCH3), 4.07 - 4.04 (m, 1H, H - 4’), 2.14 - 2.09 (m, 1H, H - 2’), 1.74 - 1.68 (m, 1H, H - 2’), 1.35 (d, J = 7.0Hz, 3H, CHCH3) 13 C - NMR (125MHz, MeOD): δ C 174.92 (d, 3 J C-P = 3.75Hz, C = O, ester), 159.37 (d, 2 J C-F = 25.9Hz, C = O, base), 150.54 (d, 4 J C-F = 4.0Hz, C = O, base), 147.99 (d, 2 J C-P = 7.1Hz, C - Ar, Naph), 141.75 (d, 1 J C-F = 232.1Hz, CF - base), 137.18, 136.29 (C - Ar), 129.59, 129.36, 128.90, 127.91 (CH - Ar), 127.83 (d, 3 J C-P = 5.4Hz, C - Ar, Naph), 127.59, 126.52, 126.50, 126.18 (CH - Ar), 125.54 (d, 2 J C-F = 34.1Hz, CH - base), 122.64 (CH - Ar), 116.29 (d, 3 JC-P =2.75Hz, CH-Ar, Naph), 86.95 (C-1'), 86.67 (d, 3 J C-P =8.1Hz, C-4'), 72.12 (C-3'), 68.05 (OCH2Ph), 67.85 (d, 2 J C-P =5.3 Hz, C-5'), 51.96 (CHCH3), 40.84 (C-2'), 20.52 (d, 3 J C-P =7. Hz, CHCH3) 31 P NMR (202MHz, MeOD):δ P 4.62 19 F NMR (470MHz, MeOD):δ F 167.19 (ES+) m / z: Measured value (M+Na + ) 636.1520, C 29 H 29 N3O9FNaP Theoretical value: (M + ), 613.15 Reverse-phase HPLC (Varian Pursuit XRs 5 C18, 150x4.6 mm; elution with H2O / AcCN (90 / 10 to 0 / 100) in 35 min): R =16.61 minutes NUC-3373 R p The isomers can be obtained by carrying out the above method, except that R p It can be obtained using diastereomers as starting materials. 1 H-NMR (500MHz, MeOD):δ H8.17 - 8.15 (m, 1H, H - Ar), 7.91 - 7.88 (m, 1H, H - Ar), 7.72 - 7.69 (m, 2H, H - Ar), 7.56 - 7.52 (m, 2H, H - Ar, H - 6), 7.50 - 7.48 (m, 1H, H - Ar), 7.39 (apparent t, J = 8.0Hz, 1H, H - Ar), 7.35 - 7.28 (m, 5H, H - Ar), 6.16 - 6.09 (m, 1H, H - 1’), 5.13 (s, 2H, OCH2Ph), 4.35 - 4.25 (m, 3H, 2xH - 5’, H - 3’), 4.14 - 4.08 (m, 1H, CHCH3), 4.05 - 4.03 (m, 1H, H - 4’), 2.15 - 2.10 (m, 1H, H - 2’), 1.74 - 1.68 (m, 1H, H - 2’), 1.36 (d, J = 7.0Hz, 3H, CHCH3); 13 C - NMR (125MHz, MeOD): δ C 174.58 (d, 3 J C-P = 5.0Hz, C = O, ester), 159.38 (d, 2 J C-F = 26.3Hz, C = O), 150.48 (C = O base), 147.80 (d, 2 J C-P = 6.5Hz, C - Ar, Naph), 141.67 (d, 1 J C-F = 232.5Hz, CF - base), 137.15, 136.26 (C - Ar), 129.62, 129.40, 129.36, 128.96, 127.89 (CH - Ar), 127.84 (d, 3 J C-P = 5.5Hz, C - Ar, Naph), 127.59, 126.57, 126.55, 126.21 (CH - Ar), 125.61 (d, 2 J C-F = 34.0Hz, CH - base), 122.62 (CH - Ar), 116.55 (d, 3 J C-P=3.75Hz, CH-Ar, Naph), 86.97 (C-1'), 86.66 (d, 3 J C-P =7.5Hz, C-4'), 72.01 (C-3'), 68.07 (OCH2Ph), 67.84 (d, 2 J C-P =5.0Hz, C-5'), 51.83 (CHCH3), 40.89 (C-2'), 20.42 (d, 3 J C-P =7.5Hz, CHCH3); 31 P-NMR (202MHz, MeOD):δ P 4.27; 19 F NMR (470MHz, MeOD):δ F 167.27; (ES+) m / z: Measured value: (M+Na + ) 636.1520, C 29 H 29 N3O9FNaP Theoretical value: (M + ), 613.15; Reverse-phase HPLC (Varian Pursuit XRs 5 C18, 150 x 4.6 mm); elution with H2O / MeOH (90 / 10 to 0 / 100 in 35 min): R =16.03 minutes The stereochemistry of the two NUC-3373 isomers (R p vs. S p )of, 31 The stereochemistry of compound 12 has been tentatively assigned based on comparison of the P chemical shift, 1H NMR spectrum, and HPLC retention time with those of other ProTides known in the literature. The stereochemistry of compound 12 has been tentatively assigned based on either isomer of NUC-3373 forming compound 12. [Example]

[0144] NUC-7738 Sp and R p Isomer formation 2'-TBDMS protected 3'-deoxyadenosine 21 is generated according to the following scheme: [ka] Compound 21 is then coupled with a compound of formula (IIb) using the conditions of the coupling method described in Example 8. The TBDMS group can be removed using TFA in THF to form NUC-7738. [ka] [ka] Adenosine (18) to Epoxide 19 One equivalent of adenosine (18) was dissolved in 10 V of acetonitrile, and the mixture was cooled to 15° C. 3.0 molar equivalents of acetoxyisobutyryl bromide were added slowly at 15° C. The mixture was allowed to warm to room temperature and stirred for 8 hours. The reaction was quenched with sodium bicarbonate solution and extracted with ethyl acetate. The combined organic phase was washed with 5% sodium chloride solution, and the organic phase was concentrated in vacuo. The product was dissolved in 15 V of methanol, and 1 equivalent of potassium carbonate was added, followed by stirring for 2 hours. The mixture was concentrated in vacuo, and the product was washed with water and then dried under vacuum at 60°C to give 2',3'-anhydroadenosine in 70-85% yield. One equivalent of 2',3'-anhydroadenosine and 1.6 equivalents of imidazole were dissolved in 5 V of DMF. The mixture was cooled to 15 °C, and 0.8 equivalents of TBDMSCl were added. The mixture was stirred at 30 °C for 1-2 h, followed by the addition of 0.4 equivalents of imidazole and 0.4 equivalents of TBDMSCl. The mixture was stirred at 30 °C for an additional 1-2 h, followed by the addition of water (5 V). The mixture was extracted with ethyl acetate. The combined organic phase was washed sequentially with 7% sodium bicarbonate solution, water, and 5% sodium chloride solution, and then concentrated in vacuo. The product was washed with heptane and then dried under vacuum at 50 °C to give epoxide 19 in 75-90% yield.

[0145] Epoxide 19 to 5′-silylcordycepin 21 191 equivalents of epoxide were dissolved in a mixture of DMSO (5V) and THF (5V). The mixture was cooled to 0°C and degassed with nitrogen gas. 1 M lithium triethylborohydride (1 eq) in THF was added over 1-2 hours at 0 (±5)°C. The mixture was stirred at 0°C for 30 minutes, warmed to 30°C, and stirred for 2 hours. Then, at 5°C, methanol (10V) was slowly added. At 5°C, 10V of 10% sodium hydroxide was added dropwise, followed by 10V of 10% hydrogen peroxide solution. The mixture was extracted with ethyl acetate, and the combined organic phases were washed successively with 10% sodium metabisulfite solution, water, 7% sodium bicarbonate solution, and 10% sodium chloride solution, and then concentrated in vacuo. The product was washed with heptane and then concentrated in vacuo at 50° C. to give 2′-silylcordycepin in 70-100% yield. 2'-Silylcordycepin, 2.5 equivalents of imidazole, and 0.15 equivalents of DMAP were dissolved in 5 V of DMF. The mixture was cooled to 15 °C, and then 2.5 equivalents of TBDMSCl were added dropwise. The reaction was stirred at 30 °C for 4 hours. 10 V of water was added, and the mixture was extracted with ethyl acetate. The organic phase was washed with 7% sodium bicarbonate solution, water, and 5% sodium chloride solution, and concentrated in vacuo. The mixture was dissolved in 8 V of water, and 2 V of water was added. The mixture was then cooled to 0°C. 2.5 eq of trifluoroacetic acid was added to the reaction mixture at 0°C over 30-60 min. The reaction mixture was warmed to 10°C and stirred at 10°C for 4-6 h. Water was added, and the mixture was extracted with ethyl acetate. The combined organic phase was washed with 7% sodium bicarbonate solution, water (twice), and 5% sodium chloride solution, and then concentrated in vacuo. The product was washed with heptane and dried in vacuo to give 5'-silylcordycepin 21 in 40-70% yield.

[0146] 5'-Silylcordycepin 21 to S p -NUC-7738 5'-Silylcordycepin 21 was dissolved in 10 V of THF at 0° C. and cooled to 0° C. 2.0 M t-BuMgCl (2.5 eq.) was added and the mixture was stirred for 15 min. p The -isomer (2.5 eq) was dissolved in 5 V of THF and added to the reaction at 0 °C. The mixture was stirred at 0 °C for 15 min, then warmed to 25 °C and stirred for a further 2 h. The reaction was quenched by pouring into 10% ammonium chloride solution (10 vol) and extracted with ethyl acetate. The combined organic phases were washed with water and 10% brine, then concentrated in vacuo. The product was dissolved in THF (10V) and then cooled to 0°C. 10V of a 1:1 mixture of TFA and water was added to the reaction over 30 minutes, and the mixture was stirred for 45 minutes, warmed to 30°C, and stirred for an additional 16 hours. The reaction was quenched by pouring into 7% NaHCO3 solution at 0°C and then extracted with ethyl acetate. The combined organic layers were washed with water, 7% sodium bicarbonate solution, and 10% brine, and then concentrated in vacuo. The product was purified by column chromatography using silica gel (100-200 mesh), and the column was eluted with 2-10% MeOH in DCM to give S p The HPLC purity of the product was 99.50%. p The isomer is present in an amount of 99.90%, and R p The isomer was shown to be present in an amount of 0.10%. By carrying out the same method, R p -NUC-7738 can be provided. R p -NUC-7738: 1 H NMR (500MHz, CDCl):δ H 8.26 (s, 1H, H8), 8.22 (s, 1H, H2), 7.37-7.25 (m, 7H, Ar), 7.22-7.12 (m, 3H, Ar), 6.01 (d, J=1.5Hz, 1H, H1'), 5.12 (AB q, J AB =12.0Hz,AB = 0.04, 2H, CH2Ph), 4.74-4.70 (m, 1H, H2'), 4.69-4.62 (m, 1H, H4'), 4.44-4.38 (m, 1H, H5'), 4.28-4.21 (m, 1H, H5'), 3.99-3.90 (m, 1H, CHCH3L-Ala), 2.35-2.27 (m, 1H, H3'), 2.09-2.02 (m, 1H, H3'), 1.29 (d, J=7.0Hz, 3H, CHCH3L-Ala) 31 P NMR (202MHz, CD3OD):δ P 3.91 MS (ES + ) m / z: Measured value 569.2 [M+H + ], 591.2 [M+Na + ], 1159.4 [2M+Na + ], C 26 H 29 N6O7P theoretical value m / z 568.2 [M] Reverse-phase HPLC (Varian Pursuit XRs 5 C18, 150x4.6 mm, elution with H2O / CH3CN (90 / 10 to 0 / 100 in 30 min), F = 1 ml / min, λ = 200 nm): R Shows one peak at 14.02 min S p -NUC-7738: 1 H NMR (500MHz, CDCl):δ H 8.24 (s, 1H, H8), 8.22 (s, 1H, H2), 7.36-7.26 (m, 7H, Ar), 7.22-7.13 (m, 3H, Ar), 6.01 (d, J=1.5Hz, 1H, H1'), 5.08 (AB q, J AB =12.0Hz, AB=0.01, 2H, CH2Ph), 4.70-4.67 (m, 1H, H2'), 4.66-4.60 (m, 1H, H4'), 4.41-4.35 (m, 1H, H5'), 4.26-4.19 (m, 1H, H5'), 4.02-3.94 (m, 1H, CHCH3L-Ala), 2.36-2.27 (m, 1H, H3'), 2.08-2.01 (m, 1H, H3'), 1.34-1.30 (m, 3H, CHCH3L-Ala) 31 P NMR (202MHz, CD3OD):δ P 3.73 MS (ES + ) m / z Measured value 569.2 [M+H + ], 591.2 [M+Na + ], 1159.4 [2M+Na + ], C 26 H 29 N6O7P theoretical value m / z 568.2 [M] Reverse-phase HPLC (Varian Pursuit XRs 5 C18, 150x4.6mm, elution with H2O / CH3CN (90 / 10 to 0 / 100 in 30 min), F = 1 ml / min, λ = 200 nm): R Shows one peak at 14.26 min The stereochemistry of the two NUC-7738 isomers (R p vs. S p ) was confirmed by conventional X-ray crystallography. [Example]

[0147] Formation of NUC-9701 The dimethyl acetal 25 of 8-chloro-adenosine 24 is prepared according to the following scheme (WO 2017 / 207989). [ka] Compound 25 can then be coupled with a compound of formula (IIa) using the coupling conditions described in Example 8. To form NUC-9701, the dimethyl acetal is removed using TFA / water (1 / 1) at 0° C. for 5 hours. [ka] Compound 25 (30 g; 1 equivalent) and the desired isomer of compound 12 (58.08 g; 1.2 equivalents) were dissolved in 300 ml of THF (10 V). The mixture was cooled to 0°C, and then t-butylmagnesium chloride (76.8 ml of a 2.0 M THF solution; 1.75 equivalents) was slowly added while maintaining the temperature at 0°C. The mixture was stirred for 4 hours. 300 ml of 10% ammonium chloride solution (10 V) was added to the reaction mixture while maintaining the temperature at 15°C. The reaction mixture was extracted with ethyl acetate, and the combined organic layers were washed with 7% sodium bicarbonate solution (twice), water (twice), and 20% sodium chloride solution, and then dried over anhydrous sodium sulfate. The ethyl acetate was removed under vacuum. To the resulting product, 600 ml (20V) of 60% aqueous formic acid was added, and the reaction mixture was stirred at 25°C for 65-70 hours. Then, ethyl acetate (600 ml; 20V) was slowly added. 600 ml (20V) of 20% sodium chloride solution was added, and the layers were separated. The aqueous layer was extracted with ethyl acetate, and then 10% ammonia solution was added dropwise to the combined organic layers, and the layers were separated. The organic phase was washed with water (three times) and 20% sodium chloride solution, dried over 30 g (1 w / w) of anhydrous sodium sulfate, filtered, and then concentrated in vacuo. The crude product was purified by column chromatography to yield 20-45 g of NUC-9701. S p -NUC-9701: 1 H-NMR (500MHz; MeOD-d4):δ H8.07 (1H, dJ=8.5Hz, H-Naphth), 8.05 (1H, s, 2-H), 7.87 (1H, dJ=8.5Hz, H-Naphth), 7.67 (1H, dJ=8.5Hz, H-Naphth) 7.54-7.48 (2H, m, H-Naphth), 7.41-7.30 (1H, m, H-Napht), 7.36-7.33 (1H, m, H-Napht), 7.26-7.22 (5H, m, -CH2Ph), 6.03 (1H, dJ=5.0Hz, H-1'), 5.33 (1H, tJ=5.0Hz, H-2'), 4.98 (AB, J AB =12.3Hz, CH2Ph), 4.65 (1H t J=5.5Hz, H-3'), 4.49-4.45 (1H, m, H a -5'), 4.41-4.36 (1H,m,H b -5'), 4.22-4.20 (1H, m, H-4'), 3.94-3.90 (1H, m, -CHCH3), 1.17 (1H, dJ=7.0Hz, CH3) 31 P NMR (202MHz, MeOD-d4):δ P 3.93 (1P, s) Liquid HPLC(H2O / CH3CN(30 at 90 / 10 and 0 / 100) at a concentration of 1ml / water and λ=254nm): t R =16.43 is the price R p -NUC-9701: 1 H-NMR (500MHz; MeOD-d4):δ H8.10 (1H, s, H-2), 8.08 (1H, d J=8.5Hz, H-Napht), 7.87 (1H, d J=8.5Hz, H-Napht), 7.67 (1H, d J=8.5Hz, H-Napht), 7.53-7.50 (1H, m, H-Napht), 7.48-7.44 (1H, m, H-Napht), 7.40-7.38 (1H, m, H-Napht), 7.33-7.27 (6H, m, H-Napht and -CH2Ph), 6.02 (1H, d J=5.0Hz, H-1'), 5.28 (1H, t J=5.0Hz, H-2'), 5.04, 5.02 (AB, J AB =12.2 Hz, CH2Ph), 4.63 (1H t J=5.5Hz, H-3'), 4.48-4.46 (1H, m, H a -5'), 4.38-4.35 (1H, m, H b -5'), 4.23-4.20 (1H, m, H-4'),4.05-4.01 (1H, m, -CHCH3), 1.17 (1H, d J=7.0Hz, CH3) 31 P NMR (202MHz, MeOD-d4):δ P 3.83 (1P, s) Reverse-phase HPLC (elution with H2O / CH3CN (90 / 10 to 0 / 100 in 30 min), 1 ml / min, λ = 254 nm): t R = 16.59 min. The stereochemistry of the two isomers of NUC-9701 (R p vs. S p )of, 31 P chemical shift, 1 The tentative assignment was made based on comparison of the 1 H NMR spectrum and HPLC retention time with those of other ProTides known in the literature.

Claims

1. Formula (Ia) 【Chemistry 1】 1. A method for preparing NUC-3373 of the formula: a) Formula (IIa) 【Chemistry 2】 (In the formula, R 1 is an electron withdrawing group, and a is an integer of 1 to 5), in the presence of a base (B1), a compound of formula (IIIa): 【Transformation 3】 (where P 1 is selected from hydrogen and a protecting group to form a compound of formula (IVa): 【Chemistry 4】 providing a compound of formula (I); and b) P 1 is a protecting group, the protecting group P 1 and removing the compound to provide NUC-3373. The method comprising:

2. P 1 2. The method of claim 1, wherein is -C(O)O-tBu.

3. Step b) is C 1 -C 4 The process according to claim 2, which is carried out using alcohol and / or water and in which the reaction mixture is heated to a temperature in the range from 60 to 90°C.

4. 4. The method of claim 3, wherein step b) is carried out using a mixture of isopropyl alcohol and water.

5. The method according to any one of claims 1 to 4, wherein B1 is a Grignard compound.

6. 6. The method of claim 5, wherein B1 is tBuMgCl.

7. The method according to any one of claims 1 to 6, wherein step a) is carried out in an ether solvent.

8. 8. The process of claim 7, wherein step a) is carried out in THF.

9. The compound of formula (IIa) 【Transformation 5】 The method according to any one of claims 1 to 8, wherein

10. Formula (Ib) 【Transformation 6】 1. A method for preparing NUC-7738 of the formula: a) Formula (IIb) 【Transformation 7】 (In the formula, R 1 is an electron withdrawing group, and a is an integer of 1 to 5), in the presence of a base (B1), a compound of formula (IIIb): 【Transformation 8】 (where P 2 , P 3 and P 4 are each independently selected from hydrogen and a protecting group, to form a compound of formula (IVb): 【Chemistry 9】 providing a compound of formula (I); and b) P 2 , P 3 and / or P 4 is a protecting group, the protecting group P 2 , P 3 and / or P 4 removing the compound to provide NUC-7738 The method comprising:

11. P 2 11. The method of claim 10, wherein is -C(O)O-tBu.

12. 12. The process according to claim 11, wherein step b) is carried out using a C1-C4-alcohol and / or water and the reaction mixture is heated to a temperature in the range from 60 to 90°C.

13. 13. The method of claim 12, wherein step b) is carried out using a mixture of isopropyl alcohol and water.

14. P 3 and P 4 The method of any one of claims 10 to 13, wherein each of

15. The method according to any one of claims 10 to 14, wherein B1 is a Grignard compound.

16. 16. The method of claim 15, wherein B1 is tBuMgCl.

17. The method according to any one of claims 10 to 16, wherein step a) is carried out in an ether solvent.

18. 18. The method of claim 17, wherein step a) is carried out in THF.

19. The compound of formula (IIb) 【Chemistry 10】 The method according to any one of claims 10 to 18, wherein

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