Synthesis of nucleoside derivative NUC-3373

Abstract

The present invention relates to a novel method for preparing 5-fluoro-2'-deoxyuridine-5'-O-[1-naphthyl(benzoxy-L-alaninyl)]phosphate (NUC-3373) and its derivatives. NUC-3373 produced according to this method is more stable than NUC-3373 produced according to other methods.

Description

[Technical Field]

[0001] The present invention relates to a novel process for preparing 5-fluoro-2'-deoxyuridine-5'-O-[1-naphthyl(benzoxy-L-alaninyl)]phosphate (NUC-3373) and its derivatives. [Background technology]

[0002] NUC-3373 The ProTide class of drugs is a masked phosphate derivative of a nucleoside. They have been shown to be particularly potent therapeutic agents in both antiviral and oncology fields. More specifically, the ProTide class of drugs is a prodrug of a monophosphorylated nucleoside. These compounds appear to circumvent many of the innate and acquired resistance mechanisms that limit the availability of the parent nucleoside.

[0003] 5-Fluoro-2'-deoxyuridine-5'-O-[1-naphthyl(benzoxy-L-alaninyl)]phosphate (NUC-3373) and a range of related compounds have demonstrated in vitro activity against a range of cancer models; in many cases, and particularly for NUC-3373, the activity is outstanding and significantly superior to results obtained with 5-fluorouracil. The addition of a phosphoramidate moiety to 5-fluorouracil / FUDR offers the unique advantage of delivering the key active form of the agent (FUDR monophosphate) to tumor cells. In nonclinical studies, NUC-3373 has been shown to overcome key cancer cell resistance mechanisms associated with 5-FU and its oral prodrug, capecitabine, providing high intracellular levels of the active FUDR monophosphate metabolite, resulting in significant inhibition of tumor cell growth. Furthermore, in formal canine toxicology studies, NUC-3373 was significantly better tolerated than 5-FU (see WO 2012 / 117246; McGuigan et al., "Phosphoramidate ProTides of the Anticancer Drug FUDR Deliver Preformed Bioactive Monophosphates in Cells, Offering Advantages Over the Parent Nucleoside," J. Med. Chem., 2011, 54, 7247-7258; and Vande Voorde et al., "The Cytostatic Activity of NUC-3073, a Phosphoramidate Prodrug of 5-Fluoro-2'-Deoxyuridine, Is Independent of Activation by Thymidine Kinase and Insensitive to Phosphatases," Biochem. Pharmacol.; 2011, 82, 441-452). [ka] (NUC-3373)

[0004] NUC-3373 is generally prepared as a mixture of two diastereomers (S- and R-epimers) epimeric at the phosphate center. Summary of the Invention [Problem to be solved by the invention]

[0005] It is an object of the present invention to provide an alternative method for preparing NUC-3373, e.g., a method for preparing NUC-3373 that is stable upon prolonged storage in solution, whereby NUC-3373 is provided in a substantially diastereomerically pure form.

[0006] Particular embodiments of the present invention may satisfy some or all of the above objectives. [Means for solving the problem]

[0007] According to a first aspect of the present invention, there is provided NUC-3373(I): [ka] (I) A method for preparing a compound of formula (I) is provided, the method comprising the steps of: (a) converting one or more solvents into formula (Ic): [ka] (I C) (where P 1 is a protecting group; (b) Removal of the protecting group P from the compound of formula (Ic) 1 to provide NUC-3373(I). The compound comprises:

[0008] The method further comprises reacting a compound of formula (Ia): [ka] (Ia) with a compound of formula (Ib): [ka] (Ib) to provide a compound of formula (Ic) may include:

[0009] Crystallization of protected compound (Ic), followed by deprotection, results in the formation of NUC-3373 with increased stability relative to a similar method that does not involve crystallization of the compound of formula (Ic) prior to deprotection. For example, this can provide NUC-3373 with increased stability relative to NUC-3373 obtained from a method that does not involve crystallization of protected compound (Ic), but in which NUC-3373 itself is subjected to a purification process that includes crystallization.

[0010] According to some embodiments, the compound of formula (Ib) [ka] is.

[0011] According to some embodiments, the compound of formula (Ic) [ka] is.

[0012] According to some embodiments, step (a) comprises: obtaining a solution of a compound of formula (Ic) in one or more solvents; crystallizing the compound of formula (Ic); and recovering the crystallized compound of formula (Ic). The compound comprises:

[0013] According to some embodiments, the step of obtaining a solution of the compound of Formula (Ic) in one or more solvents comprises dissolving the compound of Formula (Ic) in one or more solvents.

[0014] According to some embodiments, the one or more solvents comprise a solvent selected from ethanol, acetonitrile, N,N-dimethylformamide (DMF), methanol, dichloromethane (DCM), acetone, diethyl ether, toluene, n-hexane, tetrahydrofuran (THF), isopropyl alcohol (IPA), ethyl acetate, dimethyl sulfoxide (DMSO), n-heptane, cyclohexane, and methyl tertiary-butyl ether (MTBE).

[0015] According to some embodiments, the one or more solvents comprise an ether, such as diethyl ether, THF, MTBE. According to some embodiments, the ether comprises MTBE.

[0016] In some embodiments, the one or more solvents comprise an alcohol, for example, selected from ethanol, methanol, and IPA. In some embodiments, the alcohol is IPA.

[0017] According to some embodiments, the step of crystallizing the compound of formula (Ic) is carried out in the presence of an anti-solvent.

[0018] According to some embodiments, step (a) can further comprise combining an anti-solvent with the one or more solvent solutions of the compound of Formula (Ic). Step (a) can comprise adding an anti-solvent to the one or more solvent solutions of the compound of Formula (Ic). Alternatively, step (a) can comprise adding the one or more solvent solutions of the compound of Formula (Ic) to an anti-solvent.

[0019] According to some embodiments, the anti-solvent is water. This is particularly the case when the solvent is an alcohol, such as IPA.

[0020] According to another embodiment, the anti-solvent is an alkane, such as hexane, cyclohexane, pentane, petroleum ether, heptane. The anti-solvent is a hexane, such as n-hexane. This is particularly the case when the solvent is an ether, such as MTBE.

[0021] Combining an alkane (e.g., hexane or heptane) with a solution of a compound of Formula (Ic) in one or more solvents (e.g., MTBE) will produce a mixture of an ether (e.g., MTBE) and an alkane (e.g., hexane) containing the compound of Formula (Ic). According to some embodiments, the mixture comprises a 3:1 to 1:3 mixture of ether (e.g., MTBE):alkane (e.g., hexane or heptane). According to some embodiments, the mixture comprises a 1:1 to 1:2 mixture of ether (e.g., MTBE):alkane (e.g., hexane or heptane).

[0022] Combining water with a solution of a compound of Formula (Ic) in one or more solvents (e.g., IPA) will produce a mixture of alcohol (e.g., IPA) and water containing the compound of Formula (Ic). According to some embodiments, the mixture comprises a 1:1 to 1:4 mixture of alcohol (e.g., IPA):water. According to some embodiments, the mixture comprises a 1:1 to 1:3 mixture of alcohol (e.g., IPA):water.

[0023] According to some embodiments, the step of crystallizing the compound of formula (Ic) is carried out at a temperature ranging from 10 to 45° C. According to some embodiments, the crystallization step is carried out at a temperature ranging from 25 to 35° C. According to some embodiments, the crystallization step is carried out at a temperature ranging from 10 to 20° C.

[0024] According to some embodiments, the step of crystallizing the compound of Formula (Ic) can include cooling a solution of the compound of Formula (Ic). According to some embodiments, the step of crystallizing the compound of Formula (Ic) can include introducing a seed material into the solution.

[0025] According to some embodiments, the step of recovering the crystallized compound of Formula (Ic) comprises filtration, vacuum filtration, centrifugation, solvent evaporation, or crystal fishing. According to some embodiments, the step of recovering the crystallized compound of Formula (Ic) comprises filtration.

[0026] According to some embodiments, step (a) may further comprise the step of drying the recovered crystalline compound of Formula (Ic).

[0027] According to some embodiments, step (a) comprises: obtaining a solution of formula (Ic) in alcohol (e.g., IPA); adding water to the alcoholic solution of formula (Ic); crystallizing the compound of formula (Ic); and filtering the mixture to obtain a crystalline compound of formula (Ic). The compound comprises:

[0028] According to some embodiments, step (a) comprises: obtaining a solution of formula (Ic) in an ether (e.g., MTBE); adding the ethereal solution of Formula (Ic) to an alkane (e.g., hexane or heptane); crystallizing the compound of formula (Ic); and filtering the mixture to obtain a crystalline compound of formula (Ic). The compound comprises:

[0029] According to some embodiments, step (a) comprises: obtaining a solution of formula (Ic) in an ether (e.g., MTBE); adding an alkane (e.g., hexane or heptane) to the ethereal solution of Formula (Ic); crystallizing the compound of formula (Ic); and filtering the mixture to obtain a crystalline compound of formula (Ic). The compound comprises:

[0030] According to some embodiments, the alkane in step (a) is a heptane, such as n-heptane.

[0031] According to some embodiments, the step of crystallizing the compound of Formula (Ic) is carried out for 4 hours or less. According to some embodiments, the step of crystallizing the compound of Formula (Ic) is carried out for 1 hour or less.

[0032] The crystallization may be carried out once, or may be carried out more than once, for example, 2 to 8 times. One or more different crystallization conditions may be used.

[0033] Thus, the continuous process: obtaining a solution of a compound of formula (Ic) in an alcohol (e.g., IPA); adding water to an alcoholic solution of a compound of formula (Ic); crystallizing the compound of formula (Ic); and filtering the mixture to obtain a crystalline compound of formula (Ic). This step may be repeated 2 to 4 times. In this case, the crystallized compound of formula (Ic) obtained in the fourth step is dissolved in alcohol (e.g., IPA) to obtain a solution of the compound of formula (Ic) to be used in the first step of the next repeated series of steps.

[0034] Similarly, the continuous process: obtaining a solution of the compound of formula (Ic) in an ether (e.g., MTBE); adding an alkane (e.g., hexane or heptane) to an ethereal solution of a compound of formula (Ic); crystallizing the compound of formula (Ic); and filtering the mixture to obtain a crystalline compound of formula (Ic). This step may be repeated 2 to 4 times. In this case, the crystalline compound of formula (Ic) obtained in the fourth step is dissolved in ether (e.g., MTBE) to obtain a solution of the compound of formula (Ic) to be used in the first step of the next repeated sequence.

[0035] Continuous process: obtaining a solution of the compound of formula (Ic) in an ether (e.g., MTBE); adding an alkane (e.g., hexane or heptane) to an ethereal solution of a compound of formula (Ic); crystallizing the compound of formula (Ic); and filtering the mixture to obtain a crystalline compound of formula (Ic). This step may be repeated 2 to 4 times. In this case, the crystalline compound of formula (Ic) obtained in the fourth step is dissolved in ether (e.g., MTBE) to obtain a solution of the compound of formula (Ic) to be used in the first step of the next repeated sequence.

[0036] Alternatively, at least two different crystallization processes can be performed, at least one of which is one of the processes described above in which an alcohol (e.g., IPA) is the solvent (e.g., with water as an anti-solvent) and at least one of which is one of the processes described above in which an ether (e.g., MTBE) is the solvent (e.g., with an alkane (e.g., hexane or heptane) as an anti-solvent).

[0037] Step (a) is a crystallization process: Obtaining a solution of a compound of formula (Ic) in an ether (e.g., MTBE): adding an ethereal solution of a compound of formula (Ic) to an alkane (e.g., hexane or heptane); crystallizing the compound of formula (Ic); filtering the mixture to obtain a crystalline compound of formula (Ic); and Crystallization Process: obtaining a solution of the compound of formula (Ic) in an ether (e.g., MTBE); adding an alkane (e.g., hexane or heptane) to an ethereal solution of a compound of formula (Ic); crystallizing the compound of formula (Ic); filtering the mixture to obtain a crystalline compound of formula (Ic). It may also comprise both of the above.

[0038] The two crystallization processes can be carried out in any order.

[0039] According to some embodiments, the step of reacting compound (Ia) with a compound of formula (Ib) to provide a compound of formula (Ic) is carried out in the presence of a base. The base is a nitrogen base. Nitrogen 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, diisopropylethylamine). Alternatively, the base may be an organometallic base or a metal hydride base (e.g., NaH). Thus, the base may be a Grignard reagent (i.e., an alkylmagnesium halide). Exemplary Grignard reagents include t-butylmagnesium halides, such as tBuMgCl and tBuMgBr. Preferably, the base is tBuMgCl.

[0040] In some embodiments, the step of reacting compound (Ia) with a compound of formula (Ib) to provide a compound of formula (Ic) is carried out in the presence of a solvent. The solvent is an organic solvent. Examples of organic solvents include, but are not limited to, ethers (e.g., tetrahydrofuran, diethyl ether, methyl t-butyl ether); ketones (e.g., acetone, methyl isobutyl ketone); halogenated solvents (e.g., dichloromethane, chloroform, 1,2-dichloroethane); and amides (e.g., DMF, NMP); or mixtures thereof. When the step of reacting compound (Ia) with a compound of formula (Ib) to provide a compound of formula (Ic) is carried out in the presence of a Grignard reagent, the organic solvent is preferably an ether. More preferably, the solvent is tetrahydrofuran.

[0041] When the step of reacting compound (Ia) with a compound of formula (Ib) to provide a compound of formula (Ic) is carried out in the presence of a nitrogen base, the organic solvent is most preferably a halogenated solvent or an amide.

[0042] The step of reacting compound (Ia) with a compound of formula (Ib) to provide a compound of formula (Ic) is generally carried out at a suitable temperature, for example, in the range of about -5 to about 40°C. Preferably, the reaction temperature is in the range of about 25 to about 30°C. The step of reacting compound (Ia) with a compound of formula (Ib) to provide a compound of formula (Ic) may be carried out at a temperature in the range of about -5 to about 10°C. The reaction is stirred for about 15 minutes to about 16 hours, preferably about 30 to about 60 minutes. The reaction is stirred for about 15 minutes to about 16 hours, preferably about 1 to about 6 hours.

[0043] Protecting groups for hydroxyl groups (e.g., P 1 ) 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-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.

[0044] If the protecting group is acid-sensitive (e.g., trityl, C(O)OtBu, MOM, MEM, 2,4-dimethoxybenzyl, 2,3-dimethoxybenzyl, -C(Me)-), the deprotection step (step (b)) can be carried out using a suitable acid. The acid can be a Bronsted acid (e.g., TFA, phosphoric acid, HCl, or formic acid) or a Lewis acid (e.g., ZnBr, CeCl). Lewis acids (e.g., ZnBr) are less preferred. Likewise, HCl is less preferred. Preferably, the acid is TFA.

[0045] Alternatively, when the protecting group is C(O)OtBu, step (b) is achieved using a C1-C4-alcohol and / or water (eg, a mixture of isopropyl alcohol (IPA) and water).

[0046] If the protecting group is base-sensitive (e.g., acetyl and benzoyl), the deprotection step is carried out using a suitable base, e.g., aqueous NH3 or aqueous NaOH. However, base-sensitive groups are less preferred.

[0047] 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, or HF).

[0048] 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), however, such protecting groups are less preferred.

[0049] When the protecting group is 4-methoxybenzyl, 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).

[0050] When the protecting group is -C(O)-O-allyl, the deprotection step is carried out using (PPh3)4Pd.

[0051] When the protecting group is -C(O)-O-CH2-fluorenyl, the deprotection step is carried out using piperidine.

[0052] The deprotection step is carried out in an organic solvent or mixture thereof. Exemplary organic solvents include, but are not limited to, halogenated solvents (e.g., dichloromethane, chloroform, dichloroethane); alcohols (e.g., methanol, ethanol, isopropanol); and ethers (e.g., tetrahydrofuran, diethyl ether).

[0053] 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.

[0054] The deprotection reaction is carried out, for example, at a temperature ranging from -10 to about 30°C, for example, about 10°C. The temperature is in the range of -5 to 5°C. The reaction is 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.

[0055] If the deprotection step is carried out using a C1-C4-alcohol and / or water (e.g., a mixture of isopropyl alcohol (IPA) and water), the reaction mixture is heated, for example, to a temperature in the range of 30 to 100°C or to a temperature in the range of 70 to 90°C.

[0056] When the deprotection step is carried out in the presence of an acid (e.g., TFA), the product obtained after deprotection is generally recovered by quenching the excess acid used in the deprotection step, extracting the product with a water-immiscible organic solvent, and evaporating the organic solvent.

[0057] Examples of water-immiscible organic solvents useful for extraction include esters such as ethyl acetate, methyl acetate, isopropyl acetate, etc.; halogenated solvents such as dichloromethane, chloroform, etc.; aromatic hydrocarbons such as toluene, xylene, etc., preferably ethyl acetate.

[0058] In certain embodiments, it may be desirable to further purify the compound of Formula (I) obtained from the process of the first aspect of the present invention. Purification methods are known to those skilled in the art and include chromatography and recrystallization. In other embodiments, no purification is necessary.

[0059] As used herein, the term "stable" refers to the chemical stability of a compound of Formula (I), for example, in solution or as a dry solid. It can also refer to the physical stability of a solution of a compound of Formula (I). In certain embodiments, a compound of Formula (I) prepared according to the method of the first aspect of the present invention is stable in solution at a temperature ranging from 15 to 30°C for up to 2 weeks, up to 6 months, up to 12 months, or more. In certain embodiments, a compound of Formula (I) is stable in solution at a temperature ranging from 2 to 8°C for up to 2 weeks, up to 6 months, up to 12 months, or more. The solution comprises DMA. In certain embodiments, a compound of Formula (I) prepared according to the method of the first aspect of the present invention is stable (as a dry solid) at a temperature ranging from 15 to 30°C for up to 1 month, up to 6 months, up to 12 months, or more. In certain embodiments, a compound of Formula (I) is stable (as a dry solid) at a temperature ranging from 2 to 8°C for up to 1 month, up to 6 months, up to 12 months, or more. In certain embodiments, the compound of formula (I) is stable (as a dry solid) for 1 month, 6 months, up to 12 months, or longer at temperatures ranging from 15 to 60° C. The compound of formula (I) is stable (as a dry solid) at 40° C. for at least 6 months.

[0060] In a second aspect of the invention, there is provided NUC-3373 obtainable (obtained) by the method of the first aspect of the invention.

[0061] In a third aspect of the present invention, there is provided a pharmaceutical composition comprising NUC-3373 obtained by the method of the first aspect of the present invention. The formulation may include dimethylacetamide (DMA). The formulation may include water, such as saline.

[0062] NUC-3373 produced according to the methods of the present invention is a mixture of phosphate diastereomers, or is the S-phosphate diastereomer with a diastereomeric purity of 85% or more (e.g., 95% or more, or 99% or more), or is the R-phosphate diastereomer with a diastereomeric purity of 85% or more (e.g., 95% or more, or 99% or more).

[0063] The compound of formula (Ic) is a mixture of phosphate diastereomers, or is an S-phosphate diastereomer with a diastereomeric purity of 85% or more (e.g., 95% or more, or 99% or more), or is an R-phosphate diastereomer with a diastereomeric purity of 85% or more (e.g., 95% or more, or 99% or more).

[0064] The compound of formula (Ia) is a mixture of phosphate diastereomers, or is an S-phosphate diastereomer with a diastereomeric purity of 85% or more (e.g., 95% or more, or 99% or more), or is an R-phosphate diastereomer with a diastereomeric purity of 85% or more (e.g., 95% or more, or 99% or more).

[0065] When the compound of formula (Ia) is a mixture of phosphate diastereomers, a compound of formula (Ic) (and thus NUC-3373) is provided, which is also a mixture of phosphate diastereomers. When the compound of formula (Ia) is a single diastereomer, the reaction proceeds stereoselectively via inversion of the phosphate stereocenter, providing a compound of formula (Ic) (and thus NUC-3373) that is also a single diastereomer.

[0066] In a fourth aspect of the present invention, there is provided a method for preparing a pharmaceutical formulation of NUC-3373(I), said method comprising: performing the method of the first aspect to provide NUC-3373; and Preparing a Pharmaceutical Formulation Comprising NUC-3373 It comprises:

[0067] The formulation may include a polar aprotic solvent. The formulation may include dimethylacetamide (DMA). The formulation may also include water, e.g., saline. The process for preparing the pharmaceutical formulation comprises dissolving NUC-3373 in a solvent. The solvent may include a polar aprotic solvent. The solvent may include DMA. The solvent may include a mixture of DMA and water, e.g., saline. If NUC-3373 is initially dissolved in DMA, water (e.g., saline) may be added to form the formulation.

[0068] In a fifth aspect, the present invention provides a pharmaceutical composition obtainable (eg obtained) by the method of the fourth aspect of the invention. DETAILED DESCRIPTION OF THE INVENTION

[0069] Throughout this specification and the claims, the terms "comprise" and "include" and variations thereof mean "including, but not limited to," and they are not intended to (and do not) exclude other moieties, additives, components, integers, or steps. Throughout this specification and the claims, the singular includes the plural unless the context requires otherwise. In particular, where the indefinite article is used, the specification should be understood to contemplate the plural as well as the singular, unless the context requires otherwise.

[0070] It should be understood that any feature, integer, property, compound, chemical moiety, or group described in connection with a particular aspect, embodiment, or example of the invention may be compatible with any other aspect, embodiment, or example described herein, unless incompatible therewith. All features and / or all method or process steps disclosed in this specification (including the claims, abstract, and drawings) may be combined in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive. The present invention is not specifically limited to the above-described examples. The present invention extends to any novel one or any novel combination of features disclosed in this specification (including the claims, abstract, and drawings), or to any novel one or any novel combination of steps of any method or process disclosed.

[0071] The reader's attention is directed to all documents filed contemporaneously with or prior to this application as a supplemental specification and open to public inspection herewith, and all such documents are hereby incorporated by reference. [Definition]

[0072] The term "alkyl" refers to a monovalent, straight or branched, saturated hydrocarbon chain. The term "C1-C6-alkyl" refers to an alkyl group containing 1 to 6 carbon atoms in the hydrocarbon chain. For example, C1-C6-alkyl includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, 3-methylpentyl, 2,3-dimethylbutyl, and neohexyl.

[0073] The term "aryl" refers to any monovalent aromatic carbocyclic ring system (i.e., a ring system containing 2(2n+1) pi electrons). Aryl can be monocyclic or polycyclic. Aryl groups have 6 to 12 carbon atoms in the ring system. Aryl groups are typically phenyl groups. However, aryl groups can also be naphthyl or biphenyl groups.

[0074] The optionally substituted -Si(C1-C6-alkyl)3 group may also be a -Si(C1-C4-alkyl)3 group. The group (i.e., the alkyl group) is preferably unsubstituted. Illustrative examples include triethylsilyl and t-butyl-dimethylsilyl.

[0075] The optionally substituted -C(O)-C1-C6-alkyl group may also be a -C(O)-C1-C4-alkyl group. The group (i.e., the alkyl group) is preferably unsubstituted. Illustrative examples include acetyl and propionyl.

[0076] The optionally substituted -C(O)-aryl group may be a -C(O)-phenyl group. The group (i.e., the phenyl group) is preferably unsubstituted. An illustrative example is benzoyl.

[0077] The optionally substituted -C(O)-O-C-alkyl group may also be a -C(O)-O-C-alkyl group. The group (i.e., the alkyl group) is preferably unsubstituted. Illustrative examples include -C(O)-O-methyl and -C(O)-O-ethyl. A particularly preferred example is C(O)OtBu.

[0078] The optionally substituted -(C1-C3-alkylene)-aryl group is preferably an optionally substituted benzyl group. Illustrative examples include benzyl, phenethyl, 4-methoxybenzyl, 4-nitrobenzyl, 4-bromobenzyl, 2,3-dimethoxybenzyl, and 2,4-dimethoxybenzyl.

[0079] The optionally substituted -C(O)OCH-aryl group may be an optionally substituted -C(O)Obenzyl group. Illustrative examples include -C(O)Obenzyl and -C(O)O-(4-methylbenzyl).

[0080] The optionally substituted -C1-C4-alkyl-O-C1-C4-alkyl group may also be -C1-C2-alkyl-O-C1-C2-alkyl. The group (i.e., the alkyl group) is preferably unsubstituted. Illustrative examples include methoxy-methyl (MOM) and 2-methoxy-ethoxy-methyl (MEM).

[0081] The optionally substituted -S(O)2-C1-C6-alkyl group may also be -S(O)2-C1-C4-alkyl. The group (i.e., the alkyl group) is preferably unsubstituted. Illustrative examples include methanesulfonate.

[0082] The optionally substituted -S(O)2-aryl group may be a -S(O)2-phenyl group. Illustrative examples include phenylsulfonate, 4-methylphenylsulfonate, and 4-nitrophenylsulfonate.

[0083] An optionally substituted -C(aryl)3 may be a -C(phenyl)3 group. Illustrative examples include trityl.

[0084] Throughout this specification, "diastereomerically enriched" and "substantially diastereomerically pure" refer to a diastereomeric purity of 95% or greater. "Diastereomerically enriched" and "substantially diastereomerically pure" refer to a diastereomeric purity of 98% or greater, 99% or greater, or 99.5% or greater.

[0085] Any of the above alkyl groups may independently in each occurrence be selected from the group consisting of oxo, ═NR, ═H ... a , =NOR a , 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 , S.R.a , SOR a , SO3R a , SO2R a , SO2NR a R a , CO2R a C(O)R a , and CONR a R a (where R a is optionally substituted with 1 to 3 substituents selected from the group consisting of: H, C1-C4 alkyl, and C1-C4 haloalkyl).

[0086] Any of the above alkyl groups may, where chemically possible, independently be selected from halo, nitro, cyano, NR a R a , OR a , and S.R. a (where R a is optionally substituted with 1 to 3 substituents selected from the group consisting of: H, C1-C4 alkyl, and C1-C4 haloalkyl).

[0087] Any of the above alkyl groups may be unsubstituted.

[0088] Any of the above aryl groups (e.g., phenyl group (including the phenyl group in a benzyl group)) may independently in each occurrence, if chemically possible, be selected from oxo, ═NR a , =NOR a , 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 , S.R. 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-alkynyl, and C1-C4 haloalkyl (wherein R a is optionally substituted with 1 to 3 substituents selected from the group consisting of: H, C1-C4 alkyl, and C1-C4 haloalkyl).

[0089] Any of the above aryl groups (e.g., phenyl groups (including the phenyl group in a benzyl group)) may, where chemically possible, be independently selected from halo, nitro, cyano, NR a R a , OR a , S.R. 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-alkynyl, and C1-C4 haloalkyl (wherein R a is optionally substituted with 1 to 3 substituents selected from the group consisting of: H, C1-C4 alkyl, and C1-C4 haloalkyl).

[0090] Any of the above aryl groups (e.g., phenyl groups (including the phenyl group in a benzyl group)) may, where chemically possible, be independently selected from halo, nitro, OR, a , C1-C4-alkyl, C1-C4 haloalkyl (wherein R a is optionally substituted with 1 to 3 substituents selected from the group consisting of: H, C1-C4 alkyl, and C1-C4 haloalkyl).

[0091] It will be understood that various modifications can be made to the embodiments disclosed herein. Therefore, the above description should not be construed as limiting, but merely as exemplifications of preferred embodiments. For example, the functions set forth above as the best mode for operating the invention are for illustrative purposes. Other arrangements and methods will be implemented by those skilled in the art without departing from the scope and spirit of the invention. Moreover, those skilled in the art will be able to make other modifications within the scope and spirit of the specification set forth herein.

[0092] The following abbreviations are used throughout this specification: CAN: acetonitrile; Boc: t-butyl carbonate; DCM: dichloromethane; DMSO: dimethyl sulfoxide; DM: demineralized (water); DMA: dimethylacetamide; DMF: N,N-dimethylformamide; FUDR: 5-fluoro-2'-deoxyuridine; HDPE: high-density polyethylene; IPA: isopropyl alcohol; LDPE: low-density polyethylene; MEM: 2-methoxyethoxymethyl; MOM: methoxymethyl; MTBE: methyl t-butyl ether; NMI: N-methylimidazole; NMP: N-methyl-2-pyrrolidone; RRT: relative retention time; RT: room temperature; TBAF: tetrabutylammonium fluoride; TBDMS: tert-butyldimethylsilyl; TFA: trifluoroacetic acid; THF: tetrahydrofuran; TsOH: para-toluenesulfonic acid (tosylate); HPLC: high-performance liquid chromatography; UPLC: ultra-high performance liquid chromatography. [Example]

[0093] The present invention is further described by the following examples, which are intended for illustrative purposes only and are not intended to limit the scope of the invention. [Example]

[0094] Preparation of Compounds of Formula (Ic) [ka] To a mixture of 3'-Boc-floxuridine 1 (90 g; WO2019 / 053476; WO2018 / 229493) in THF (720 mL) was added PFP ligand (mixture of phosphate diastereomers; 150.48 g; WO2018 / 229493) at 30 °C. 135 mL of THF was added at 30 °C, and the reaction mixture was cooled to 0 °C. 299 mL of a 2.0 M T-butylmagnesium chloride solution in THF was added to the reaction mixture at 0 °C over approximately 60 minutes. 45 mL of THF was added at 0 °C. The reaction was maintained at 0 °C for 3-4 hours.

[0095] A second reactor was charged with 846 mL of water and 54 mL of hydrochloric acid and cooled to 5°C. The contents of the first reactor were added to the second reactor at 5°C over approximately 60 minutes. 180 mL of THF was added to the second reactor, and the temperature was maintained at 5°C for 2-3 hours. 1350 mL of ethyl acetate was added to the second reactor at 5°C, and the temperature of the second reactor was then increased to 30°C. The reaction mass was allowed to settle over 30 minutes into an aqueous (bottom) and organic (top) phase. The phases were then separated by extraction with water. The organic phase was added to a sodium chloride solution at the same temperature. The solvent was removed under vacuum at below 45°C. 1530 mL of MTBE was added to the resulting brown syrup at 30°C and maintained for 20-30 minutes before filtering through Celite and concentrating under vacuum. [Example]

[0096] Crystallization of the compound of formula (Ic) At 25°C, 1800 mL of MTBE was added to the product from Example 1. The resulting solution was slowly added to 2700 mL of n-heptane. The reaction mass was maintained at 25°C for 3-5 hours. The mixture was filtered to obtain a solid and dried under vacuum. At 25°C, 1800 mL of MTBE was added to the solid, and 2700 mL of n-heptane was slowly added to the resulting solution. The reaction mass was maintained at 25°C for 3-5 hours. The mixture was filtered to obtain a solid and dried under vacuum. The solid was dissolved in 900 mL of isopropyl alcohol, and the solution was maintained at 40°C for 20-30 minutes, then cooled to 25°C. 1800 mL of water was added, and the mixture was maintained at 25°C for 2-3 hours, then filtered. The solid was again dissolved in 900 mL of isopropyl alcohol, and the mixture was maintained at 40°C for 20-30 minutes, then cooled to 25°C. 1800 mL of water was added, and the reaction mixture was maintained at 25°C for 2-3 hours, then filtered. The solid was dried under vacuum.

[0097] Yield: 143.3 g; Purity by HPLC (% area): 99.45% [Example]

[0098] Preparation of NUC-3373(I) 130 g of 3'-Boc NUC-3373 (product of Example 2) was dissolved in 975 mL of isopropyl alcohol and 1950 mL of water, and the mixture was heated to 80°C and held at 80°C for 7-9 hours. The mixture was cooled to 30°C, and 1300 mL of isopropyl alcohol and 1300 mL of cyclohexane were added. The aqueous and organic phases were separated. The aqueous phase was mixed with 1300 mL of cyclohexane, and the aqueous and organic phases were separated. To the aqueous phase was added sodium bicarbonate solution (prepared by dissolving 65.0 g of sodium bicarbonate in 950 mL of demineralized water at 30°C) and 1300 mL of ethyl acetate. The aqueous phase was again mixed with 1300 mL of ethyl acetate, and the phases were separated. The combined organic phases from the two ethyl acetate separations were washed with NaCl and concentrated under vacuum to give NUC-3373. got

[0099] Yield: 78.0 g [Example]

[0100] [Comparative Example] Part 1: Preparation of Compounds of Formula (Ic) [ka] To a mixture of 3'-Boc-floxuridine 1 (10 g) in THF (80 mL) was added PFP ligand (a mixture of phosphate diastereomers: 19.11 g) at 30 °C. 20 mL of THF was added at 30 °C, and the reaction mixture was cooled to 5 °C. 36.10 mL of 2.0 M t-butylmagnesium chloride in THF was added to the reaction mixture at a temperature below 15 °C. The reaction mixture was stirred at 20 °C for 3 to 4 h.

[0101] 100 mL of 10% ammonium chloride solution was added dropwise, and the reaction mixture was stirred at 30°C for 15 minutes. 100 mL of ethyl acetate was added to the reactor. The reaction mass was allowed to settle for 30 minutes into an aqueous phase (bottom) and an organic phase (top). The phases were then separated by extraction with water. The organic phase was added to a sodium chloride solution at the same temperature. The reaction mass was allowed to settle for 30 minutes into an aqueous phase (bottom) and an organic phase (top). The phases were then separated, and the solvent was removed from the organic phase under vacuum at 45°C or below. Part 2: Preparation of NUC-3373(I)

[0102] The product of Comparative Example Part 1 (3'-Boc NUC-3373) was dissolved in 150 mL of DCM. 50 mL of trifluoroacetic acid was added dropwise, and the mixture was heated to 30°C and maintained at 30°C for 3-4 hours. The mixture was cooled to 5°C, and 150 mL of DM water was added. The aqueous and organic phases were separated. The aqueous phase was mixed with 100 mL of DCM, and the organic phase was separated. To the combined organic phases was added sodium bicarbonate solution (prepared by dissolving 7 g of sodium bicarbonate in 100 mL of DM water at 30°C). The aqueous and organic phases were separated. The organic phase was washed with NaCl, followed by sodium sulfate, and then concentrated under vacuum to give NUC-3373.

[0103] The product was purified by column chromatography using silica as the stationary phase and ethyl acetate and DCM as the mobile phase. Fractions were monitored by TLC. Pure fractions were combined and the solvent was removed under vacuum below 45°C.

[0104] To the product was added 15 mL of ethyl acetate at 30°C. The resulting solution was added dropwise to 180 mL of cyclohexane at 7°C. The reaction mass was stirred at 0°C for 3-4 hours. The mixture was filtered to obtain a solid, which was dried under vacuum. To the product was added 100 mL of DCM at 30°C. To the resulting solution was added 1 g of activated carbon, stirred for 30-45 minutes, and then filtered. The solvent was then removed under vacuum at 45°C or lower. To the solid was added 50 mL of DCM at 30°C. The solvent was then removed under vacuum at 45°C or lower. To the solid was added 50 mL of cyclohexane at 30°C. The solvent was then removed under vacuum at 45°C or lower.

[0105] Yield: 9.7g [Example]

[0106] Stability Data 5.1: 12-month dry powder NUC-3373 prepared according to Example 3 above was packaged in a clear LDPE bag, which was then flushed with nitrogen and closed with a seal strip. This bag was then placed in another clear LDPE bag, which was similarly flushed with nitrogen. A silica gel packet was placed between the two bags, and the outer bag was then closed with a seal strip. These bags were then placed in a triple-layered light-tight barrier bag and sealed with a heat sealer. The bags were maintained in an HDPE container. NUC-3373 was analyzed by HPLC initially after manufacturing, and then after 3, 6, 9, and 12 months of storage. Stability data are shown in Table 1. [Table 1] TIFF2025539267000011.tif47169

[0107] The data in Table 1 demonstrate that NUC-3373 prepared according to the methods of the present invention is chemically stable at 15-30°C for at least 12 months. 5.2: Dry powder for 6 months

[0108] NUC-3373 prepared according to Example 3 and Example 4 (Comparative Example) above was packaged in a clear LDPE bag, which was then filled with nitrogen and sealed with a seal strip. This bag was then placed in another clear LDPE bag, which was also filled with nitrogen. A silica gel packet was placed between the two bags, and the outer bag was then sealed with a seal strip. These bags were then placed in a triple-layered light-tight barrier bag and sealed with a heat sealer. The bags were then kept in an HDPE container.

[0109] NUC-3373 was analyzed by HPLC first at 0 months after manufacture, and then at 1 and 2 months and / or 3 and 6 months after storage at 2-8°C, 25°C, or 40°C. Stability data are shown in Table 2. [Table 2] TIFF2025539267000012.tif89158

[0110] The alpha-naphthol content of the same batches was also determined by HPLC. The level of alpha-naphthol impurity is an important indicator of the stability of the test article. The data are shown in Table 3. [Table 3] TIFF2025539267000013.tif95160

[0111] The data in Tables 2 and 3 show that NUC-3373 prepared according to the method of the present invention (i.e., protected NUC-3373 is purified by crystallization prior to deprotection) is more thermally stable than that prepared by other methods (including methods in which NUC-3373 is itself subjected to extensive purification once deprotected, rather than beforehand). 5.3: Solution stability

[0112] Stability data are shown in Table 4, which compares the levels of alpha naphthol (0.60 RRT) present in 400 mg / mL NUC-3373 solutions in DMA:saline (80:20 v / v) prepared according to Example 3 or Example 4 (comparative), measured at different temperatures over a set period of time. The solutions tested are suitable for infusion and injection, and the temperatures and times measured represent typical storage environments for solutions suitable for infusion and injection.

[0113] In Table 4, the values ​​provided for each temperature at each time (separated by " / ") represent different formulations (samples) of each synthesis method. [Table 4] TIFF2025539267000014.tif87126

[0114] The data in Table 4 demonstrate that NUC-3373 prepared according to the methods of the present invention (i.e., purifying protected NUC-3373 by crystallization) is more stable than NUC-3373 prepared by other methods, including methods in which NUC-3373 is itself subjected to extensive purification after deprotection, rather than before, particularly when samples are stored at room temperature.

[0115] In further studies, the inventors found that a DMA:saline (80:20 v / v) solution of a sample prepared according to the method of the present invention retained its purity or assay and alpha naphthol impurity intact for up to 12 months at 2-8° C. The stability data (obtained by UPLC) are shown in Table 5. [Table 5] TIFF2025539267000015.tif36141

[0116] The data in Table 5 demonstrate that solutions comprising NUC-3373 prepared according to the methods of the present invention are both chemically and physically stable for at least 12 months.

Claims

1. NUC-3373(I): 【Chemistry 1】 （I） 1. A method for preparing a compound comprising: (a) reacting one or more solvents with a compound of formula (Ic): 【Chemistry 2】 (I C) (where P 1 is a protecting group; (b) removing the protecting group P from the compound of formula (Ic) 1 to provide NUC-3373 (I). The method comprising:

2. Furthermore, the formula (Ia): 【Transformation 3】 (Ia) with a compound of formula (Ib): 【Chemistry 4】 (Ib) to provide a compound of formula (Ic) 2. The method of claim 1, comprising:

3. P 1 3. The method of claim 1 or 2, wherein is C(O)OtBu.

4. 10. The method of claim 1, wherein the one or more solvents in step a) comprise a solvent selected from water, ethanol, acetonitrile, DMF, methanol, DCM, acetone, diethyl ether, toluene, n-hexane, THF, IPA, ethyl acetate, DMSO, n-heptane, cyclohexane, and MTBE or a mixture thereof.

5. Step a) obtaining a solution of a compound of formula (Ic); Crystallizing the compound of formula (Ic); and recovering the crystallized compound of formula (Ic).

2. The method of claim 1, comprising:

6. Step a) obtaining an alcoholic solution of the compound of formula (Ic); adding water to an alcoholic solution of a compound of formula (Ic); Crystallizing the compound of formula (Ic); and filtering the mixture to obtain the crystallized compound of formula (Ic).

6. The method of claim 5, comprising:

7. 7. The method of claim 6, wherein the alcohol is IPA.

8. Step a) obtaining an ether solution of the compound of formula (Ic); adding an ethereal solution of a compound of formula (Ic) to an alkane; Crystallizing the compound of formula (Ic); and filtering the mixture to obtain the crystallized compound of formula (Ic).

6. The method of claim 5, comprising:

9. Step a) obtaining an ether solution of the compound of formula (Ic); adding an alkane to an ethereal solution of a compound of formula (Ic); Crystallizing the compound of formula (Ic); and filtering the mixture to obtain the crystallized compound of formula (Ic).

6. The method of claim 5, comprising:

10. 10. The method of claim 8 or 9, wherein the ether is MTBE.

11. 10. The method according to claim 8 or 9, wherein the alkane is hexane.

12. 10. The method according to claim 8 or 9, wherein the alkane is n-heptane.

13. 10. The method of claim 1, comprising at least one crystallization according to claim 6 and at least one crystallization according to claim 8 or 9.

14. NUC-3373 (I) obtained by the method according to claim 1 or 2.

15. A pharmaceutical preparation comprising NUC-3373 (I) obtained by the method of claim 1 or 2.

16. 16. The pharmaceutical formulation of claim 15, further comprising dimethylacetamide (DMA).

17. 1. A method for preparing a pharmaceutical formulation comprising NUC-3373 (I), comprising: performing the method of claim 1 or 2 to provide NUC-3373; and Preparing a pharmaceutical formulation comprising NUC-3373 (I) The method comprising:

18. 18. The method of claim 17, wherein the formulation comprises DMA.

19. A pharmaceutical formulation of NUC-3373 (I) obtained by the method of claim 17.

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