EA043691B1 - OBTAINING PHOSPHATE DERIVATIVES - Google Patents

Description

Field of technology

The present invention generally relates to a new process for preparing certain ProTides as specific phosphate diastereoisomers. Specific ProTides include those useful in the treatment of cancer, such as NUC-3373 (5-fluoro-2'-deoxyuridine-5'-O-[1-nαphthyl(benzyloxy-Lalaninyl)]phosphate).

State of the art

ProTides are protected phosphate nucleoside derivatives. They have been shown to be particularly potent therapeutic agents in both the antiviral and oncology fields. ProTides, more specifically, are prodrugs of monophosphorylated nucleosides. These compounds appear to avoid many of the intrinsic and acquired resistance mechanisms that limit the usefulness of the parent nucleosides (see, for example, 'Application of ProTide Technology to Gemcitabine: A Successful Approach to Overcome the Key Cancer Resistance Mechanisms Leads to a New Agent (NUC) -1031) in Clinical Development'; Slusarczyk et al; J. Med. Chem.; 2014, 57, 1531-1542).

NUC-3 373 (5-fluoro-2'-deoxyuridine-5'-O-[1-naphthyl(benzoxy-L-alaninyl)]phosphate) is a ProTide adaptation of 5FU/FUDR, the current mainstream treatment for colorectal cancer. NUC-3373 and a number of related compounds have shown in vitro activity in a number of cancer models, in many cases and in particular for NUC-3373, this activity was outstanding and far superior to the results obtained with 5-fluorouracil. The addition of ProTide's phosphoramidate moiety to the 5fluorouracil/FUDR molecule offers the particular benefit of delivering the key activated form of the agent (FdUMP) to tumor cells. Preclinical studies have shown that NUC-3373 overcomes key cancer cell resistance mechanisms associated with 5-FU and its oral prodrug, capecitabine, by generating high intracellular levels of the active metabolite FdUMP, resulting in much greater inhibition of tumor cell growth. Additionally, in formal toxicology studies in dogs, NUC-3373 was significantly better tolerated than 5-FU (see WO2012/117246; McGuigan et al.; Phosphoramidate ProTides of the anticancer agent FUDR successfully deliver the preformed bioactive monophosphate in cells and confer advantage 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 degradation by phosphorolytic enzymes; Biochem. Pharmacol; 2011, 82, 441-452).

ProTides are typically prepared as a mixture of two diastereoisomers, epimeric at the phosphate center. Diastereoisomers of NUC-3373, for example, have the following structures (in which Np is 1-naphthyl):

- 1 043691

WO 2014/076490 describes a method for the preparation of nucleoside prodrugs such as gemcitabine[phenyl(benzoxy-L-alaninyl)]phosphate by reacting gemcitabine or structural variants thereof with a diastereomeric mixture of phosphorus chloridates in the presence of a catalyst including a metal salt such as Cu(OTf) ₂ . CuCl, CuBr, CuI, Cu(OAc) ₂ , CuSO ₄ , Cu(OC(O)CF ₃ ) ₂ , Cu(OTf) ₂ , Yb(OTf) ₃ , Fe(OTf) ₃ , La(OTf) ₃ , with a yield of ~45%.

A method for obtaining NUC-1031 in diastereoisomerically pure form is described in WO 2017/098252 (PCT/GB 2016/053875).

The purpose of some embodiments of the present invention is to provide a method for producing NUC-3373 in substantially diastereoisomerically pure form.

The purpose of some embodiments of the present invention is to provide the (Sp) and/or (R _p )-epimer(s) of NUC-3373 in substantially diastereoisomerically pure form(s) that is scalable, economical and/or effective, e.g., more scalable, economical and/or efficient than methods using HPLC. Thus, the object of some embodiments of the present invention is to provide a process for producing (Sp) and/or (R _p )-epimer(s) in substantially diastereoisomerically pure form(s) that is suitable for large-scale production.

The purpose of some embodiments of the present invention is to provide a simple method, i.e. a process that includes a minimum number of process steps and or reagents to produce (Sp) and/or (R _p )-epimer(s) in substantially diastereomeric pure form(s).

Another object of some embodiments of the present invention is to provide a process that provides the separated (Sp)- or (R _p )-epimer in substantially diastereoisomerically pure form and, at the same time, meets or exceeds the necessary criteria established by organizations such as US FDA regarding the amount and nature of any trace impurities resulting from the synthesis and separation.

Some embodiments of the present invention satisfy some or all of the objectives above.

Summary of the present invention

According to a first aspect of the present invention there is provided a method for preparing the Xdiastereoisomer of a compound of formula IIa

(R% about

Formula Pa, where R ¹ represents a halogen, and a represents an integer from 1 to 5, including:

a) suspending or dissolving the Y-diastereoisomer of a compound of formula IIa or a mixture of Y and X-diastereoisomers of a compound of formula IIa in a solvent (S2),

b) treating the solution or suspension with a base (B2) to obtain the X-diastereoisomer in substantially diastereomerically enriched form, and

c) isolating the X-diastereoisomer of formula IIa, where B2 is a tertiary amine;

where S2 is a mixture of heptane or hexane and a polar organic solvent, where the mixture contains more than 50% by volume of hexane or heptane; and where the X-diastereoisomer of compound IIa is an (S _p )-diastereoisomer, and the Y diastereoisomer of compound IIa is an (R _p )-diastereoisomer, except when OPh(R1) _a leaving group has a lower distribution priority according to the Cana rule IngoldPrelog than the naphthyloxy group, wherein the X-diastereoisomer of compound IIa is an (R _p ) diastereoisomer and the Y-diastereoisomer of compound IIa is an (S _p ) diastereoisomer.

According to a second aspect of the present invention there is provided a method for preparing a slow eluting diastereoisomer of a compound of formula IIa

- 2 043691

where R ¹ represents a halogen, and a represents an integer from 1 to 5, including:

a) suspending or dissolving a fast eluting compound of formula IIa or a mixture of fast eluting and slow eluting diastereoisomers of a compound of formula IIa in a solvent (S2),

b) treating the solution or suspension with a base (B2), obtaining a slowly eluting diastereoisomer in substantially diastereomerically enriched form, and

c) isolating the slowly eluting diastereoisomer of formula IIa, where B2 is a tertiary amine;

where S2 is a mixture of heptane or hexane and a polar organic solvent, where the mixture contains more than 50% by volume of hexane or heptane;

wherein the slow eluting diastereoisomer of compound IIa is the isomer that elutes more slowly on reverse phase HPLC, and the fast eluting diastereoisomer of compound IIa is the isomer that elutes faster on reverse phase HPLC.

According to a third aspect of the present invention, there is provided a method for preparing the (S _p )diastereoisomer of NUC-3373 (Formula Ia) in substantially diastereoisomerically pure form:

comprising a method according to any of the first and second aspects, step d) and step e):

d) reacting the X-diastereoisomer of the compound of formula IIa isolated in step c) with a compound of formula IIIa in the presence of a base (B1) to obtain a compound of formula IVa in substantially diastereomerically pure form; where P ¹ is independently selected from hydrogen and a protecting group:

where B1 is a Grignard reagent;

And

e) wherein P ¹ represents a protecting group, removing the protecting group P ¹ from the compound of formula IVa, yielding the (S _p )-diastereoisomer NUC-3373 in substantially diastereomerically pure form.

Substitution of the substituted phenoxy group occurs selectively with inversion of the phosphate stereocenter.

Typically, the (S _p )-diastereoisomer of the precursor (compound of formula IIa) gives the (S _p )-diastereoisomer ProTide, and the (R _p )-diastereoisomer of the precursor gives the (R _p )-diastereoisomer ProTide. An exception is when OPh(R1) _a leaving group has a lower Cahn-Ingold-Prelog distribution priority than the naphthyl group (for example, when OPh(R1) _a is paranitrophenoxy). In these cases, the SH diastereoisomer of the precursor (compound of formula IIa) gives the (S _p )-diastereoisomer ProTide, and the (S _p )-diastereoisomer of the precursor gives the (R _p )diastereoisomer ProTide. Throughout this specification, the isomer of the compound of formula IIa that gives the (Sp)isomer of ProTide is called the X-diastereoisomer, and the isomer of the compound of formula IIa that gives the (R _p )isomer of ProTide is called the Y-diastereoisomer.

Thus, it may be that the method of the third aspect is a method for producing the (S _p )-diastereoisomer of ProTide in a diastereomerically enriched form, and the compound of formula IIa is in a diastereomerically enriched form.

The base (B1) may be a Grignard reagent (ie, an alkyl magnesium halide). Examples of Grignard reagents include tert-butylmagnesium halides such as tert-BuMgCl, mpem-BuMgBr. Preferably the base is mpem-BuMgCl.

Step a) can be carried out in solvent S1.

The inventors have surprisingly discovered that upon treatment of compounds of formula IIa with a base, they isomerize, preferentially forming the X-diastereoisomer over the Y-diastereoisomer. Thus, a Y-diastereoisomer can be converted to an X-diastereoisomer, or an epimer mixture of a Y-diastereoisomer and an X-diastereoisomer can be converted to an X-diastereoisomer. This increases the overall efficiency of any synthetic sequence to produce the Xdiastereoisomer of NUC-3373, which includes the process of the third aspect, since it means that all compounds of formula IIa can be used, even a portion of what was originally formed as the Ydiastereoisomer.

The compound of formula IIa used in the method of the third aspect may be an Xdiastereoisomer. Typically, the X-diastereoisomer is an (S _p )-diastereoisomer, and the Y diastereoisomer is an (R _p )-diastereoisomer.

The base (B2) is a tertiary amine. Thus, B2 may be a trialkylamine. Most preferably, B2 is triethylamine.

Solvent S2 may be a mixture that contains more than 50% (eg, more than 70%) hydrocarbon. The hydrocarbon may be hexane. The hydrocarbon may be heptane. S2 may be a mixture of hexane or heptane and a polar organic solvent (eg, ether, ester, alcohol or halogenated solvent). S2 may be a mixture of hexane or heptane and a polar organic solvent, the mixture containing more than 50% (eg, more than 70%) by volume of hexane or heptane. S2 may be a mixture of hexane or heptane and ethyl acetate. S2 may be a mixture of heptane and ethyl acetate. S2 may be a mixture of hexane or heptane and ethyl acetate, the mixture containing more than 50% (eg, more than 70%) by volume of hexane or heptane. S2 may be a mixture of heptane and ethyl acetate, the mixture containing more than 50% (eg, more than 70%) by volume of heptane. S2 may be a mixture of hexane or heptane and methyl tert-butyl ether. S2 may be a mixture of hexane and methyl tert-butyl ether. S2 may be a mixture of hexane or heptane and methyl tert-butyl ether, the mixture containing more than 50% (eg, more than 70%) by volume of hexane or heptane. S2 may be a mixture of hexane and methyl tert-butyl ether, the mixture containing more than 50% (eg, more than 70%) by volume of hexane.

Step b) may involve stirring a mixture of the compound of formula IIa and base B2 for 24 hours or more. Step b) may involve stirring a mixture of the compound of formula IIa and base B2 for 48 hours or more. Step b) may involve stirring a mixture of the compound of formula IIa and base B2 for 60 hours or more. Step b) may involve stirring a mixture of the compound of formula IIa and base B2 for 72 hours or more. Step b) may involve stirring a mixture of the compound of formula IIa and base B2 for up to 100 hours.

Step b) may involve stirring a mixture of the compound of formula IIa and base B2 at a temperature of from 0 to 60°C. Step b) may involve stirring a mixture of the compound of formula IIa and base B2 at a temperature of from 20 to 40°C.

The compound of formula IIa may be compound 12:

connection 12

The compound of formula IIa may be (R _p ) compound 12 in substantially diastereomerically pure form. The compound may be a rapidly eluting isomer of compound 12 in substantially diastereomeric pure form. Thus, the compound may be an isomer of compound 12, which has ^{a 31} P NMR peak at -1.41 ± 0.02, where the NMR spectrum was obtained on a 202 MHz NMR instrument in CDCl ₃ , and the said isomer is essentially in the diastereoisomer number 4 043691 that form. The compound may be an isomer of compound 12, which has a retention time of 12.96 ± 0.20 min by analytical HPLC on a Varian Pursuit XRs 5 C18, 1504.6 mm, eluting with H ₂ O/MeOH at 20/80 for 35 min at 1 ml/min, and the specified isomer is essentially in diastereoisomerically pure form.

The compound of formula IIa may be (S _p )-compound 12 in substantially diastereomerically pure form. The compound may be a slowly eluting isomer of compound 12 in substantially diastereomeric pure form. Thus, the compound may be an isomer of compound 12, which has ^{a 31} P NMR peak at -1.36±0.02 when the NMR spectrum was obtained on a 202 MHz NMR instrument in CDCl ₃ , said isomer being in substantially diastereoisomerically pure form . The compound may be an isomer of compound 12, which has a retention time of 14.48 ± 0.20 minutes by analytical HPLC on a Varian Pursuit XRs 5 C18, 150 x 4.6 mm, eluting with H ₂ O/MeOH at 20/80 for 35 minutes at 1 ml/min, and the specified isomer is essentially in diastereoisomerically pure form.

NUC-3373 may be a rapidly eluting isomer of NUC-3373 in substantially diastereomeric pure form. Thus, NUC-3373 may be an isomer of NUC-3373 which has ^{a 31} P NMR peak at 4.27 ± 0.10 when the NMR spectrum was obtained on a 202 MHz NMR instrument at CD3OD, said isomer being substantially diastereoisomerically pure form. NUC3373 may be an isomer of NUC-3373, which has a retention time of 16.03 ± 0.20 minutes by analytical HPLC on a Varian Pursuit XRs 5 C18, 150 x 4.6 mm, eluting H2O/CH3CN from 100/10 to 0/100 v for 35 minutes at 1 ml/min, said isomer being in substantially diastereoisomerically pure form.

NUC-3373 may be a slowly eluting isomer of NUC-3373 in substantially diastereomeric pure form. Thus, NUC-3373 may be an isomer of NUC3373 which has ^{a 31} P NMR peak at 4.62 ± 0.10 when the NMR spectrum was obtained on a 202 MHz NMR instrument in CD ₃ OD, said isomer being substantially diastereoisomerically pure form. NUC3373 may be an isomer of NUC-3373, which has a retention time of 16.61 ± 0.20 min by analytical HPLC on a Varian Pursuit XRs 5 C18, 150x4.6 mm, eluting H ₂ O/CH ₃ CN from 90/10 to 0/100 for 35 min at 1 ml/min, said isomer being in substantially diastereomeric pure form.

In the fourth aspect of the present invention, (S _p )-NUC-3373 is prepared:

ABOUT

OH is essentially in diastereoisomerically pure form. The preferred isomerization to form the X-diastereoisomer of the compound of formula IIa means that the S _p isomer of NUC-3373 is easier to obtain than the R _p isomer.

The compound of the fourth aspect of the present invention may be for medical use. The compound of the fourth aspect of the present invention may be for the treatment of cancer (eg, solid tumor or leukemia).

The product of the fourth aspect of the present invention can be (or is) obtained by the first or second aspect of the present invention.

The protecting group for the hydroxyl group (eg, P ¹ ) can be independently selected from optionally substituted -Si(C ₁ -C ₆ -alkyl) ₃ , optionally substituted -C(O)-C ₁ -C ₆ -alkyl, optionally substituted -C (O)-aryl, optionally substituted -C(O)-OC1-C ₆ -alkyl, -C(O)-O-allyl, -C(O)-O-CH ₂ -fluorenyl, optionally substituted -C(aryl ) _3; optionally substituted -(C1-C ₃ -alkylene)aryl, optionally substituted -C(O)OCH ₂ -aryl and -C ₁ -C ₄ -alkyl-O-C ₁ -C ₄ -alkyl.

Many of the protected starting compounds of formula IIIa are known in the art and/or can be prepared by known methods. For example, the parent compounds of formula IIIa can be prepared from the parent nucleoside by protecting the hydroxy and/or amino groups with suitable protecting groups. Protecting groups can generally be introduced and removed using standard protecting group methodology, for example, as described in Protective Groups in Organic Chemistry, edited by JWF McOmie (1973); Protective Groups in Organic Synthesis, ^2nd edition, T W Greene (1991); and Protecting Groups, ^3rd addition P. J. Koscienski (1995).

It is usually necessary to prepare compounds of formula IIIa by first protecting the 5'-hydroxy group of the parent nucleoside with a protecting group that is orthogonal to the protecting groups that will be

- 5 043691 used to protect the 3' and/or 2'-hydroxy and/or amino group (i.e. a group that can be removed from the 5'hydroxyl group also without removing the required 3'-hydroxyl, 2'-hydroxyl and/or amino protecting groups). Simultaneously or subsequently, the 3', 2'-hydroxyl and/or amino groups are protected with the desired protecting group(s), and the 5'-hydroxyl protecting group can be removed, yielding a compound of formula IIIa. Certain protecting groups can be simultaneously introduced at the 3' and/or 2'hydroxyl and the 5'-hydroxyl and optionally the amino group, and then selectively removed from the 5' hydroxyl group without removing the 3' and/or 2'-hydroxyl and optionally the amino group.

In some embodiments, P ¹ is independently selected from optionally substituted -Si(C ₁ -C ₆ -alkyl) ₃ , optionally substituted -C(O)-C ₁ -C6 -alkyl, optionally substituted C(O)-aryl, optionally substituted -C(O)-OC ₁ -Cb-alkyl, -C(O)-O-allyl, -C(O)-O-CH ₂ fluorenyl, optionally substituted -C(aryl) _3; optionally substituted -(C ₁ -C ₃ -alkylene)aryl, optionally substituted -C(O)OCH ₂ -aryl and -C ₁ -C ₄ -alkyl-O-C ₁ -C4-alkyl.

P ¹ can 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-allyla. Preferably, P ¹ is selected from -C(O)O-tBu, -C(O)O-benzyl and -C(O)OCH2-allyl. Thus, P ¹ may be -C(O)OCH ₂ -aryl. P ¹ may be -C(O)O-tBu.

Alternatively, P ¹ can be independently selected from optionally substituted -C(O)-C ₁ -C ₆ -alkyl and optionally substituted -C(O)-aryl, for example, P ¹ can be independently selected from benzoyl and acetyl.

As a further alternative, P ¹ may be an optionally substituted Si(C1-C6 alkyl)3. P ¹ may be -Si(C1-C ₄ -alkyl) ₃ . Alkyl groups may be unsubstituted. P ¹ may be tert-butyldimethylsilyl.

The optionally substituted -Si(C ₁ -C ₆ -alkyl) ₃ group may be a -Si(C ₁ -C ₄ -alkyl) ₃ group. The group is (ie alkyl groups are) preferably unsubstituted. Illustrative examples include triethylsilyl and tert-butyldimethylsilyl.

The optionally substituted -C(O)-C ₁ -Cb-alkyl group may be a -C(O)-C ₁ -Cbalkyl group. The group (ie, alkyl group) is preferably unsubstituted. Illustrative examples include acetyl and propionyl.

The optionally substituted -C(O)-aryl group may be a -C(O)-phenyl group. The group (ie phenyl group) is preferably unsubstituted. Illustrative examples include benzoyl.

The optionally substituted -C(O)-OC ₁ -C ₆ -alkyl group may be a -C(O)-OC ₁ -C ₄ alkyl group. The group (ie, 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.

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

The optionally substituted -C(O)OCH ₂ -aryl group is preferably an optionally substituted -C(O)obenzyl group. Illustrative examples include -C(O)benzyl and C(O)O-(4-methoxybenzyl).

An optionally substituted -C ₁ -C ₄ -alkyl-O-C ₁ -C ₄ -alkyl group may be a -C ₁ -C ₂ alkyl-O-C ₁ -C ₂ -alkyl group. The group (ie alkyl groups are) is preferably unsubstituted. Illustrative examples include methoxymethyl (MOM) and 2-methoxyethoxymethyl (MEM).

An optionally substituted -S(O) ₂ -C ₁ -C6 alkyl group may be an -S(O) ₂ -C ₁ -C ₄ alkyl group. The group (ie, alkyl group) is preferably unsubstituted. Illustrative examples include methanesulfonate.

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

The optionally substituted -C(aryl) ₃ group may be a -C(phenyl) ₃ group. Illustrative examples include trityl.

Throughout this specification, a diastereomeric enriched form and a substantially diastereomeric pure form denote a diastereomeric purity of greater than 95%. A diastereomerically enriched form and a substantially diastereomerically pure form can mean a diastereoisomeric purity greater than 98%, greater than 99%, or greater than 99.5%.

The methods of the present invention may also include deprotection of hydroxy protecting groups.

It may happen that the deblocking step (step d) is carried out without purifying the product of step c).

When the protecting group is acid sensitive (eg trityl, C(O)OtBu, MOM, MEM, 2,4-dimethoxybenzyl, 2,3-dimethoxybenzyl, -C(Me) ₂ -), the deprotection step can be carried out

- 6 043691 using a suitable acid. The acid may be a Bronsted acid (eg TFA, phosphoric acid, HCl or formic acid) or a Lewis acid (eg ZnBr2, CeCl ₃ ). Lewis acids (eg ZnBr2) are less preferred. HCl likewise is less preferred. Preferably, the acid is TFA.

When the protecting group is a silyl group (eg triethylsilyl or tert-butyldimethylsilyl), the deprotection step can be carried out using a suitable acid (eg TFA) or using a suitable fluorine source (eg tetrabutylammonium fluoride, fluorosilicic acid, HF).

When the protecting group is a benzyl group or a C(O)benzyl group, the deprotection step can be carried out using H2 and a suitable catalyst (eg Pd/C). These protecting groups may be less preferred.

When the protecting group is 4-methoxybenzyl, 2,3-dimethoxybenzyl, 2,4-dimethoxybenzyl or C(O)O-(4-methoxybenzyl), the deprotection step can be carried out using a suitable oxidizing reagent (eg meta-chloroperbenzoic acid).

When the protecting group is -C(O)-O-allyl, the deprotection step can be carried out using (PPh ₃ ) ₄ Pd.

When the protecting group is -C(O)-O-CH ₂ -fluorenyl, the deprotection step can be carried out using piperidine.

The deblocking step can be carried out in an organic solvent or a mixture thereof. Examples of organic solvents include, but are not limited to, halogenated solvents (eg, dichloromethane, chloroform, dichloroethane); alcohols (eg methanol, ethanol, isopropanol) and ethers (eg tetrahydrofuran, diethyl ether).

When the deprotection step is carried out in the presence of an acid (for example, TFA, the organic solvent is preferably a halogenated solvent, for example, dichloromethane.

The deblocking reaction can be carried out at a temperature ranging from, for example, -10°C to about 30°C, for example to about 10°C. A suitable temperature for the reaction is -5°C to 5°C. The reaction can be stirred for a period of time from about 15 minutes to about 16 hours, and preferably from about 1 hour to about 4 hours, and more preferably from about 2 hours to about 3 hours.

When step e) is carried out using a C1- _C4 alcohol and/or water (eg a mixture of isopropyl alcohol (IPA) and water), the reaction mixture is heated, for example, to a temperature of from 30°C to 90°C or to a temperature of 60°C to 85°C.

When deblocking is carried out in the presence of an acid (eg, TFA), recovery of the deblocking product is typically accomplished by quenching the excess acid used in the deblocking step and extracting the product with a water-immiscible organic solvent and recovering the product by evaporation of the organic solvent.

Examples of water-immiscible organic solvents suitable for extraction include ethers such as ethyl acetate, methyl acetate, isopropyl acetate and the like; chlorinated solvents such as dichloromethane, chloroform and the like; aromatic hydrocarbon solvents such as toluene, xylene and the like; preferably ethyl acetate.

In certain embodiments, it may still be desirable to purify ProTide obtained in the method of the first aspect of the present invention. Likewise, it may still be desirable to purify the compound of formula IIa obtained in the method of the fourth aspect of the present invention. Purification methods are well known to one skilled in the art and include chromatography (eg, column chromatography), recrystallization and distillation. In other embodiments, purification is not necessary.

It is clear that various modifications can be made to the embodiments described in the present invention. Therefore, the above description should not be considered as limiting, but merely as examples of preferred embodiments. For example, the functions described above and implemented as the best mode for operating the present invention are provided for illustration purposes only. Other schemes and methods may be implemented by those skilled in the art without departing from the scope and spirit of the present invention. Moreover, those skilled in the art will appreciate other modifications within the scope and spirit of the description appended to the present invention.

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The following abbreviations apply throughout the description:

ACN-acetonitrile

AIBBr-acetoxyisobutyryl bromide

B QC-mpem-butyl carbonate DCM-dichloromethane

DMAP-Λζ A-dimethyl-4-aminopyridine

DMF-Λζ A-dimethylformamide eq.-molar equivalent

R1LV-5-fluoro-2’-deoxyuridine

IPA-isopropyl alcohol

MEM-2-methoxyethoxymethyl

MOM-methoxymethyl

MTBE-methyl-/?ulm butyl ether

MMP-A-methyl-2-pyrrolidone

Np-1-naphthyl

PTSA-p-toluenesulfonic acid

RT-room temperature TVAG-tetrabutylammonium fluoride TBDMS-tert-butyl dimethyl silyl TEA-triethylamine

Tf-trifluoromethylsulfonate (triflate) TFA-trifluoroacetic acid

THF-tetrahydrofuran

V is used to denote the volume (in ml) per weight (in g) of the parent compound. So, if there is 1 g of the parent compound, 10 V will represent 10 ml of said liquid.

Examples

The present invention is further illustrated by the following examples, which are provided by way of illustration only and should not be construed as limiting the scope of the present invention.

Illustrative Example 1: Preparation of a diastereoisomeric mixture of 2-[(2,3,4,5,6pentafluorophenoxy)phenoxyphosphorylamino]propionic acid benzyl ester 5 (illustrative example of a compound of formula IIb).

To a stirred mixture of L-alanine benzyl ester hydrochloride 1 (100 g) in methylene chloride (1 L) was added phenyl dichlorophosphate 2 (77 ml) at 25-35°C, and the resulting mixture was cooled to -70°C-78°C , triethylamine (130.5 ml) was added and the mixture was stirred for 1 hour at the same temperature. The temperature of the reaction mass was increased to 25-35°C and stirred for 2 hours. After completion of the reaction, the reaction mass was concentrated in vacuum below 35°C, obtaining 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 mixture was filtered and washed with diisopropyl ether.

- 8 043691 (500 ml), followed by concentrating the filtrate in vacuo below 35°C, obtaining phenyl(benzoxyL-alaninyl)phosphorochloridate 3. The resulting compound was dissolved in methylene chloride (1 l) at 2535°C and cooled to -5°C -10°C. Pentafluorophenol 4 (85.5 g) and triethylamine (65.2 ml) were added to the reaction mass at the same temperature and stirred for 2 hours. After completion of the reaction, the reaction mass was concentrated in a vacuum below 35°C and ethyl acetate (1 l) was added ) at 25-35°C and stirred for 30 minutes at the same temperature. The solid residue was filtered and washed with ethyl acetate (1 L). To the filtrate was added water (1 L), 10% sodium carbonate (2x1 L), brine (1 L) and dried the organic layer with anhydrous sodium sulfate, concentrated in vacuo at 35-45°C to obtain a diastereomeric mixture of the title compound 5 in the form of a white semi-solid residue.

Yield: 210 g.

HPLC chiral purity (% area): 33.74:66.26% (R _P : S _P ).

Illustrative Example 2: Separation of the Sp-diastereoisomer of 2-[(2,3,4,5,6pentafluorophenoxy)phenoxyphosphorylamino]propionic acid benzyl ester 5 (illustrative example of a compound of formula IIb).

F

(Rp-diastereomer)

To the diastereoisomeric mixture of compound 5 (210 g; R _P :Sp-33.74:66.26%) was added 20% ethyl acetate in hexane (1.2 L) at 25-35°C and stirred for 1 hour. Filter the solid and wash with 20% ethyl acetate in hexane (300 ml) to obtain a diastereomeric mixture of compound 5.

Yield: 112 g.

HPLC chiral purity (% 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; RP:SP-65.43:34.57%).

To a diastereomeric mixture of the compound of formula IIb (112 g; R _P : S _P -22.13:77.87%) was added 20% ethyl acetate in hexane (1.2 L) at 25-35° C. and stirred for 1 hour. Filter the solid and wash with 20% ethyl acetate in hexane (300 ml) to give an essentially pure Sp diastereoisomer of compound 5.

Yield: 80 g.

Chiral purity by HPLC (% area): 0.20:99.80% (R _P : S _P ).

Ή NMR (300 MHz, DMSO- _d6 ): 7.18-7.41(m, 10H), 6.91-6.99(d, 1H), 5.10(s, 2H), 4.01 -4.11(m, 1H), 1.301.32(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%).

Illustrative Example 3: S-isomer enrichment of 2-[(2,3,4,5,6pentafluorophenoxy)phenoxyphosphorylamino]propionic acid benzyl ester 5 (illustrative example of a compound of formula IIb).

(Sp and Rp mixture) 5(5p-diastereomer)

To a stirred solution of 2-[(2,3,4,5,6- 9 043691 pentafluorophenoxy)phenoxyphosphorylamino]propionic acid benzyl ester 5 (75 g; R _P : S _P -65.43:34.57%) in 20% ethyl acetate in hexane (1.1 L), triethylamine (7.5 ml) was added at 25-35°C and stirred for 6 hours at the same temperature. After completion of the reaction, the reaction mass was quenched with water (750 ml) and extracted with ethyl acetate (750 ml). The organic layer was dried with anhydrous sodium sulfate and concentrated in vacuo to obtain the title compound as a solid residue.

Yield: 45 g.

HPLC chiral purity (% area): 91.29: 8.71% (S _P : R _P ).

The above R _p and Sp diastereoisomeric mixture of 2-[(2,3,4,5,6pentafluorophenoxy)phenoxyphosphorylamino]propionic acid benzyl ester 5 (45 g; R _P :S _P -8.71:91.29%) was suspended in 20% ethyl acetate in hexane (1.1 l) at 25-30°C and stirred for 1 hour at the same temperature. Filter the solid and wash with 20% ethyl acetate in hexane (225 ml) to obtain the Spdiastereoisomer of the title compound as a solid.

Yield: 19 g.

Chiral purity by HPLC (% area): 99.92: 0.08% (S _P : R _p ).

Illustrative Example 4: Preparation of a diastereoisomeric mixture of 2-[(4nitrophenoxy)phenoxyphosphorylamino]propionic acid benzyl ester 7 (illustrative example of a compound of formula IIb.

To a stirred mixture of L-alanine benzyl ester hydrochloride 1 (50 g) in methylene chloride (500 ml) was added phenyl dichlorophosphate 2 (54 g) at 25-35°C and the resulting mixture was cooled to -70°C-78°C , triethylamine (65.2 ml) was added and stirred for 1 hour at the same temperature. The temperature of the reaction mass was increased to 25-35°C and stirred for 2 hours. After completion of the reaction, the reaction mass was concentrated in a vacuum below 35°C, obtaining 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 mass was filtered and washed with diisopropyl ether (250 ml), followed by concentrating the filtrate in vacuum below 35°C, obtaining phenyl(benzoxyL-alaninyl)phosphorochloridate 3. The resulting compound was dissolved in methylene chloride (500 ml) at 25-35°C and cooled to -5°C-10°C. 4-nitrophenol 6 (27.5 g) and triethylamine (65.2 ml) were added to the reaction mass at the same temperature and stirred for 2 hours. After completion of the reaction, the reaction mass was concentrated in a vacuum below 35°C and ethyl acetate ( 500 ml) at 25-35°C and stirred for 30 minutes at the same temperature. The solid residue was filtered and washed with ethyl acetate (500 ml). Water (500 ml), 10% sodium carbonate (2x500 ml), brine (500 ml) was added to the filtrate and the organic layer was dried with anhydrous sodium sulfate, concentrated in vacuo at 35-40°C to obtain a diastereomeric mixture of the title compound 7 in the form of a thick oily liquid.

Yield: 90 g.

HPLC chiral purity (% area): 45.6: 54.94% (R _P : S _P ).

The above diastereoisomeric mixture of benzyl ester 2-[(4nitrophenoxy)phenoxyphosphorylamino]propionic acid 7 (40 g; R _p : S _p -45.6: 54.94%) was separated into pure S _p and R _p diastereoisomers by preparative HPLC and concentrated pure fractions in vacuum, obtaining S _p and K _p diastereoisomers separately.

Yield: Sp-diastereoisomer: 8 g, ^1H NMR (300 MHz, CDCl ₃ ): 8.15-8.19 (d, 2H), 7.15-7.37 (m, 12H), 5.12 ( s, 2H), 4.02-4.24 (m, 2H), 1.39-

- 10 043691

1.42 (d, 3H).

ESI-MS (m/z): 479 (M+Na).

Rp-diastereoisomer: 6 g, ^1H NMR (300 MHz, CDCl ₃ ): 8.08-8.13 (d, 2H), 7.15-7.34 (m, 12H), 5.10 (s, 2H), 4.48-4.56 (m, 1H), 4.114.20 (m, 1H), 1.39-1.41 (d, 3H).

ESI-MS (m/z): 457 (M+1) +

Sp and Rp diastereomeric mixture: 20 g.

Illustrative Example 5: Preparation of benzyl ester (Sp)-2-[(2,3,4,5,6pentafluorophenoxy)phenoxyphosphorylamino]propionic acid 5 (illustrative example of a compound of formula IIb).

About ci—P-CI

To a stirred mixture of benzyl ester L-alanine 1 hydrochloride (100 g) in 1000 ml of methylene chloride, phenyl dichlorophosphate 2 (97.8 g) was added to the reaction mass 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 was stirred for 1 hour at -20°C, then heated to 10°C (10±5) and stirred for an additional 1.5 hours.

A solution of pentafluorophenol 4 (85.3 g) in 100 ml methylene chloride was added slowly at 10°C, followed by trimethylamine (46.8 g), which was added slowly while maintaining the temperature at 10°C. 46.9 g of triethylamine was slowly added to the reaction mass at 10°C (10 ± 5) under a nitrogen atmosphere. The mixture was stirred for 2 hours at 10°C before stopping the reaction by slowly adding 0.5 NHCl solution, maintaining the temperature at 10°C. After warming to room temperature, the mixture was separated and the organic layer was washed with saturated bicarbonate solution, distilled water and brine before concentration in vacuo.

The crude mixture was suspended in 1500 ml of 20% 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 residue was dissolved in 2500 ml of ethyl acetate, which was washed with water and brine and concentrated in vacuo. The solid residue was suspended in 1200 ml of 20% ethyl acetate in n-heptane, stirred for 45-60 minutes and filtered. The material was dried under vacuum to obtain the desired product 5-(Sp). Yields were in the range of 40-80%, and diastereoisomeric purities were above 99%.

Example 6: Preparation of a diastereoisomeric mixture of 2-[(2,3,4,5,6pentafluorophenoxy)naphth-1-oxyphosphorylamino]propionic acid benzyl ester 12 (illustrative example of a compound of formula IIa).

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ABOUT

Alpha-naphthol 8 (100 g) was dissolved in DCM (1 L) at 25°C and POCl ₃ 9 (1.1 eq.) was added at 25°C and stirred for 10 minutes before the mixture was cooled to -70 °C and stirred for 10 minutes. Triethylamine (1.1 eq.) was added slowly while maintaining the temperature below -70°C and the mixture was stirred for 1 hour at -70°C. The mixture was heated to 25°C and stirred for 1 hour before cooling to -50°C. Added to the mixture of L-alanine benzyl ester 1 (HCl salt; 1 eq.), which was stirred for 10 min before triethylamine (2.2 eq.) in DCM (200 ml) was added at -50°C for 30 min. The mixture was stirred for 1 hour at -50°C before warming to 25°C and stirring for an additional 1 hour. The mixture was cooled to -10°C and stirred for 10 minutes before pentafluorophenol 4 in DCM (200 ml) to the reaction mass below -10°C. The mixture was stirred at -10°C for 10 minutes before triethylamine (1.1 eq.) was added over 30 minutes at -10°C. The mixture was stirred at -10°C for 1 hour before the mixture was heated to 0°C. Water (1 L) was added and the mixture was stirred for 30 minutes at 0°C. The mixture was heated to 25°C and stirred for 5-10 minutes before the organic layer was separated. The aqueous layer was extracted with DCM (500 ml). The combined organic layers were washed with 7% sodium bicarbonate solution (2X1 L) and the organic layer was dried over anhydrous sodium sulfate before concentration in vacuo.

50% IPA/water (2.4 L) was added to the crude compound and stirred for 1 hour at 25°C. The solid was filtered and the wet filter cake was washed with 50% IPA/water (500 ml) before vacuum drying. 50% IPA/water (2.4 L) was again added to the crude compound and stirred for 1 hour at 25° C. before filtering, and the wet filter cake was again washed with 50% IPA/water (500 ml) before vacuum drying. The half-dried compound was washed with cyclohexane (10 v/w) at 25-30°C for 1 hour before the semi-solid compound was washed with cyclohexane (2 L) and the wet compound 12 was dried in vacuum at 55-60°C for 12 h.

Results.

Connection weight: 252 g.

Overall yield: 66%.

HPLC purity: 98.31% (diastereoisomeric ratio is 1:1).

³¹ P NMR (202 MHz, CDCl3): δp-1.35,-1.41; ^1H NMR (500 MHz, CDCl3): δН 8.13-8.10 (1H, m, H-Ar), 7.907.88 (1H, m, H-Ar), 7.73 (1H, visible d, J=8.5 Hz, 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, visible with, CH ₂ Ph), 5.12 (1H, sq, J=12.5 Hz, CH ₂ Ph), 4.38 -4.31 (1H, m, NHCHCH3), 4.17-4.08 (1H, m, NHCHCH3), 1.49, 1.47 (3H, 2 x d, J = 3.5 NHCHCH3); MS (ES+) m/z: 574 (M+Na+, 100%), Exact mass: C ₂₆ Hi ₉ F ₅ NO ₅ P required 551.40 found 574.05 (M+Na+); Reversed phase HPLC, eluting H ₂ O/MeOH in 20/80 for 35 min, F = 1 ml/min, λ = 254, two peaks for two diastereoisomers with tR = 12.96, 14.48 min.

Diastereoisomers of compound 12 were separated by Biotage Isolera HPLC using a C18 SNAP Ultra (30 g) cartridge with a mixture of MeOH/H ₂ O (70%/30%) as eluent, obtaining: a fast eluting isomer (considered the Rp diastereoisomer) and a slow eluting isomer ( Sp is considered to be a diastereoisomer)

Note: The isomers were named fast eluting (FE) and slow eluting (SE) based on retention time on the C18 (reversed phase) cartridge and HPLC analytical column.

Rapidly eluting isomer (considered to be the Rp diastereoisomer): ³¹ P NMR (202 MHz, CDCl ₃ ): δ _P 1.41; 1H NMR (500 MHz, CDCl ₃ ): δн 8.02 (1H, dd, J=7.0, 2.0 Hz, H-Ar), 7.79 (1H, dd, J=6.5, 3 ,0 Hz, H-Ar), 7.64 (1H, d, J=8.5 Hz, H-Ar), 7.53-7.45 (3H, m, H-Ar), 7.33 ( 1H, t, J=8.0 Hz, H-Ar), 7.28-7.23 (5H, m, H-Ar), 5.09 (s, 2H, CH ₂ Ph), 4.29- 4.21 (1H, m, NHCHCH3), 4.02-3.97 (1H, m, NHCHCH3), 1.38 (3H, d, J=7.0 Hz, NHCHCH3); MS (ES+) m/z: MS (ES+) m/z: 574 (M+Na+, 100%), Exact mass: C ₂₆ H _i9 F ₅ NO ₅ P required 551.40 found 574.05 (M+Na+ ); Reverse phase HPLC, eluting H ₂ O/MeOH in 20/80 for 35 min, F=1 ml/min, λ=254, t _R =2.96.

- 12 043691

Slowly eluting isomer (considered the Sp diastereoisomer): ³¹ P NMR (202 MHz, CDCl ₃ ): δ _P 1.36; 1H NMR (500 MHz, CDCl ₃ ): 5 _H 8.14-8.11 (1H, m, H-Ar), 7.90-7.87 (1H, m, H-Ar), 7.74 ( 1H, d, J=8.0 Hz, H-Ar), 7.60 (1H, d, J=8.0 Hz, H-Ar), 7.58-7.55 (2H, m, H- Ar), 7.44 (1H, t, J=8.0 Hz, H-Ar), 7.34-7.30 (5H, m, H-Ar), 5.12 (2H, kv, J= 12.5 Hz, CH ₂ Ph), 4.35-4.29 (1H, m, NHCHCH3), 4.04-4.00 (1H, m, NHCHCH3), 1.48 (3H, d, J= 7.0 Hz, NHCHCH3); MS (ES+) m/z: MS (ES+) m/z: 574 (M+Na+, 100%), Exact mass: C ₂₆ H ₁₉ F ₅ NO ₅ P required 551.40 found 574.05 (M+Na+ ); Reversed phase HPLC, eluting

H ₂ O/MeOH in 20/80 for 35 min, F = 1 ml/min, λ = 254, tR = 14.48.

Illustrative Example 7: Sp-diastereoisomer enrichment of 2-[(2,3,4,5,6pentafluorophenoxy)naph-1-oxyphosphorylamino]propionic acid benzyl ester 12 (illustrative example of a compound of formula IIb).

(Sp and Rp mixture) ¹² (Sp-diastereomer)

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 for 80 hours at 30°C. The mixture was filtered and the wet filter cake was washed with 10% MTBE/n-hexane (75 ml) before vacuum drying for 30 min. Add 50% IPA/water (200 ml) to the crude compound above and stir for 1 hour at 25-35°C before filtering. The wet filter residue was washed with 50% IPA/water (100 ml) before vacuum drying at 55-60°C for 12 hours.

Result:

compound weight: 17 g, yield: 68%,

HPLC purity: 97.66%.

Slowly eluting isomer (considered to be an Sp-diastereoisomer): ³¹ P NMR (202 MHz, CDCl ₃ ): δ _P 1.36; 1H NMR (500 MHz, CDCl ₃ ): 5 _H 8.14-8.11 (1H, m, H-Ar), 7.90-7.87 (1H, m, H-Ar), 7.74 ( 1H, d, J=8.0 Hz, H-Ar), 7.60 (1H, d, J=8.0 Hz, H-Ar), 7.58-7.55 (2H, m, H- Ar), 7.44 (1H, t, J=8.0 Hz, H-Ar), 7.34-7.30 (5H, m, H-Ar), 5.12 (2H, kv, J= 12.5 Hz, CN ₂ Pb), 4.35-4.29 (1H, m, NHCHCH3), 4.04-4.00 (1H, m, NHCHCH3), 1.48 (3H, d, J= 7.0 Hz, NHCHCH3); MS (ES+) m/z: MS (ES+) m/z: 574 (M+Na+, 100%), Exact mass: C ₂₆ Hi ₉ F ₅ NO ₅ P required 551.40 found 574.05 (M+Na+ ); Reverse phase HPLC, eluting with H2O/MeOH at 20/80 for 35 min, F=1 ml/min, λ=254, tR= 14.48.

The stereochemistry (Rp relative to Sp) of the two isomers of compound 12 described above was tentatively assigned based on comparison of the ^31P chemical shifts, 1H NMR spectra and HPLC retention times of NUC-3373 isomers obtained using the isomers of compound 12 with those of other ProTides known in literature. As mentioned above, the stereochemistry of the phosphate stereocenter is reversed in the method of the present invention, therefore the (Sp)-diastereoisomer of the compound of formula 12 will form the (Sp)-diastereoisomer of NUC-3373, and similarly, the (R)-diastereoisomer of the compound of formula 12 will form (R) -diastereoisomer of NUC-3373. The assignment of stereochemistry is supported by powder X-ray diffraction and differential scanning calorimetry, which were performed on the two isomers of compound 12, but is not itself conclusive.

Example 8: Preparation of Sp and Rp Isomers NUC-3373 3'-BOC protected FUDR 16 can be prepared according to the following scheme.

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imidazole, DMAP,

TBDMSCI, DMF, RT, 4 h

(BOC) ₂ O, TEA, DCM, RT

Compound 16 is then condensed with a compound of formula IIa.

Compound 16 (1 g) and the Sp isomer of compound 12 (1.2 eq) were dissolved in THF (10 ml) and the mixture was cooled to 0°C. Tert-butyl magnesium chloride (2.5 eq., 2.0 M in THF) was added to the mixture over 15 minutes. The mixture was heated 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 layers were washed with deionized water (5 ml), followed by 20% sodium chloride solution (5 ml). The organic layers were dried over anhydrous sodium sulfate before concentration in vacuo to give 2.16 g of compound 17 (100% crude yield).

Crude 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 heated to 30°C and stirred

- 14 043691 for 6 hours. The mixture was cooled to 10°C and deionized water (5 ml) was added slowly, 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 (2X5 ml), 7% sodium bicarbonate (2X5 ml) and 20% sodium chloride (5 ml) before drying with anhydrous sodium sulfate (1 w/w) and concentrated in vacuo. The crude compound was purified by ethyl acetate/DCM column chromatography using silica gel (100-200 mesh). The pure compound was eluted in 50% ethyl acetate/DCM-100% ethyl acetate. The combined pure fractions were concentrated in vacuo before the compound suspension was washed with cyclohexane (5 ml).

Results:

weight NUC-3373 (Sp isomer): 9.3 g,

Total yield: 70%,

HPLC purity: 96.86%.

¹ H-NMR (500 MHz, MeOD): δ _H 8.16-8.14 (m, 1H, H-Ar), 7.90-7.80 (m, 1H, H-Ar), 7.72 -7.70 (m, 2H, HAr), 7.54-7.49 (m, 3H, H-Ar, H-6), 7.43 (visible t, 1H, J=8.0 Hz, H -Ar), 7.35-7.27 (m, 5H, H-Ar), 6.16-6.13 (m, 1H, H-1'), 5.11 (AB system, J=12, 0 Hz, 2H, OCH ₂ Ph), 4.35-4.33 (m, 2H, 2 x H-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=7.0 Hz, 3H, CHCH3);

¹³ C-NMR (125 MHz, MeOD): δC 174.92 (d, ³ JC-P=3.75 Hz, C=O, ether), 159.37 (d, ² JC-F=25.9 Hz , C=O, base), 150.54 (d, ⁴ JC-F=4.0 Hz, C=O, base), 147.99 (d, ² JC-P=7.1 Hz, C-Ar , Naph), 141.75 (d, Jc-f=232.1 Hz, CF-base), 137.18, 136.29 (C-Ar), 129.59, 129.36, 128.90, 127 .91 (CH-Ar), 127.83 (d, ³ JC-P=5.4 Hz, C-Ar, Naph), 127.59, 126.52, 126.50, 126.18 (CH-Ar ), 125.54 (d, ² J _CF =34.1 Hz, CH-base), 122.64 (CH-Ar), 116.29 (d, ³ Jc-p = 2.75 Hz, CH-Ar , Naph), 86.95 (C-1'), 86.67 (d, ³ Jc-p=8.1 Hz, C-4'), 72.12 (C3'), 68.05 (OCH2Ph) , 67.85 (d, ² Jc-p=5.3 Hz, C-5'), 51.96 (CHCH3), 40.84 (C-2'), 20.52 (d, ³ Jc-p =7.5 Hz, CHSN3).

³¹ P-NMR (202 MHz, MeOD): _δ 4.62;

¹⁹ F NMR (470 MHz, MeOD): δF -167.19;

(ES+) m/z: found: (M+Na+) 636.1520. C ₂₉ H ₂₉ N ₃ O ₉ FNaP required: (M+), 613.15.

Reverse HPLC (Varian Pursuit XRs 5 C18, 150x4.6 mm) eluting (H ₂ O/AcCN 90/10 to 0/100) for 35 min, t _R 16.61 min.

The Rp isomer of NUC-3373 can be prepared by following the method above, but starting from the Rp diasteremer of compound 12:

¹ H-NMR (500 MHz, MeOD): _δΜ 8.17-8.15 (m, 1H, H-Ar), 7.91-7.88 (m, 1H, H-Ar), 7.72 -7.69 (m, 2H, HAr), 7.56-7.52 (m, 2H, H-Ar, H-6), 7.50-7.48 (m, 1H, H-Ar), 7.39 (visible t, J=8.0 Hz, 1H, H-Ar), 7.35-7.28 (m, 5H, H-Ar), 6.16-6.09 (m, 1H, H-1'), 5.13 (s, 2H, OCH ₂ Ph), 4.35-4.25 (m, 3H, 2 x H-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=7.0 Hz, 3H, CHCH3);

¹³ C-NMR (125 MHz, MeOD): δα 174.58 (d, ³ JC-P=5.0 Hz, C=O, ester), 159.38 (d, ² JC-F=26.3 Hz , C=O), 150.48 (C=O base), 147.80 (d, ² JC-P=6.5 Hz, C-Ar, Naph), 141.67 (d, 1J _CF =232, 5 Hz, CF-base), 137.15, 136.26 (C-Ar), 129.62, 129.40, 129.36, 128.96, 127.89 (CH-Ar), 127.84 ( d, ³ Jc-p=5.5 Hz, C-Ar, Naph), 127.59, 126.57, 126.55, 126.21 (CH-Ar), 125.61 (d, ² Jc-f =34.0 Hz, CH-base), 122.62 (CH-Ar), 116.55 (d, ³ JC-P=3.75 Hz, CH-Ar, Naph), 86.97 (C-1 '), 86.66 (d, ³ JC-P=7.5 Hz, C-4'), 72.01 (C-3'), 68.07 (OCH ₂ Ph), 67.84 (d, ² Jc-p=5.0 Hz, С-5'), 51.83 (СНСН3), 40.89 (С-2'), 20.42 (d, ³ Jc-p=7.5 Hz, СНСН3 ).

³¹ P-NMR (202 MHz, MeOD): _δ 4.27;

¹⁹ F NMR (470 MHz, MeOD): δF -167.27;

(ES+) m/z: found: (M+Na+) 636.1520. C ₂₉ H ₂₉ N ₃ O ₉ FNaP required: (M+), 613.15.

Reverse HPLC (Varian Pursuit XRs 5 C18, 150x4.6 mm) eluting (H ₂ O/MeOH 90/10 to 0/100) for 35 min, t _R 16.03 min.

The stereochemistry (Rp relative to Sp) of the two NCU-3373 isomers described above was tentatively assigned based on comparison of ³¹ P chemical shift, 1 H NMR spectra and HPLC retention time with those of ProTides known in the literature. The stereochemistry of compound 12 was assigned tentatively based on which isomer of NUC-3373 forms a given isomer of compound 12.

Illustrative Example 9: Preparation of Sp and Rp Isomers of NUC-7738.

2'-TBDMS protected 3'-deoxyadenosine 21 can be prepared according to the following scheme.

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Compound 21 is then condensed with the compound of Formula IIb using the conditions of the condensation method described in Example 8. To prepare NUC-7738, the TBDMS group can be removed using TFA in THF.

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Sp isomer OH

Adenosine (18) to epoxide 19.

One equivalent of adenosine (18) was dissolved in 10 V 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 warmed to room temperature and stirred for 8 hours. The reaction was quenched with sodium bicarbonate solution and extracted with ethyl acetate. The combined organic layers were washed with 5% sodium chloride solution and the organic layers were concentrated in vacuo.

The product was dissolved in 15 V methanol and 1 equivalent by weight of potassium carbonate was added before stirring for 2 hours. The mixture was concentrated in vacuo and the product was washed with water before drying the product in vacuo at 60°C to give 2',3'-anhydradenosine in 70-85% yield.

an equivalent of 2',3'-anhydradenosine and 1.6 equivalents of imidazole were dissolved in 5 V DMF. The mixture was cooled to 15°C and 0.8 equivalents of TBDMSCl was added. The mixture was stirred for 1-2 hours at 30°C before adding an additional 0.4 equivalents of imidazole and 0.4 equivalents of TBDMSCl. The mixture was stirred for the next 1-2 hours at 30°C before adding water (5 V). The mixture was extracted with ethyl acetate. The combined organic layers were washed successively with 7% sodium bicarbonate, water and 5% sodium chloride before concentration in vacuo. The product was washed with heptane before drying in vacuum at 50°C, obtaining epoxide 19 in 75-90% yield.

Epoxide 19 in 5'-silylcordycepin 21.

An equivalent of epoxide 19 was dissolved in a mixture of DMSO (5V) and THF (5V). The mixture was cooled to 0°C and the mixture was purged with nitrogen. Add 1M lithium triethylborohydride (1 equiv) in THF at 0(±5)°C for 1-2 hours. The mixture was stirred at 0°C for 30 minutes, heated to 30°C and stirred for 2

- 17043691 hours before methanol (10 V) was slowly added at 5°C. 10 V 10% sodium hydroxide, and then 10 V 10% hydrogen peroxide solution were added dropwise at 5°C. The mixture was extracted with ethyl acetate and the combined organic layers were washed successively with 10% sodium metabisulfite, water, 7% sodium bicarbonate and 10% sodium chloride before concentration in vacuo. The product was washed with heptane before drying in vacuum at 50°C, obtaining 2'silylcordycepin in 70-100% yield.

2'-silylcordycepin, 2.5 equivalents of imidazole and 0.15 equivalents of DMAP were dissolved in 5 V DMF. The mixture was cooled to 15°C before adding 2.5 equivalents of TBDMSCl in portions. The reaction was stirred for 4 hours at 30°C before cooling to 15°C. 10 V water was added and the mixture was extracted with ethyl acetate. The organic layers were washed with 7% sodium bicarbonate solution, water and 5% sodium chloride before concentration in vacuo.

The mixture was dissolved in 8V and 2V water was added before the mixture was cooled to 0°C. 2.5 eq. trifluoroacetic acid was added to the reaction mixture at 0°C for 30-60 minutes. The mixture was heated to 10°C and stirred for 4-6 hours at 10°C. Water was added and the mixture was extracted with ethyl acetate. The combined organic layers were washed with 7% sodium bicarbonate, water (twice) and 5% sodium chloride before concentration in vacuo. The product was washed with heptane and dried in vacuum, obtaining 5'-silylcordycepin 21 in 40-70% yield.

5'-Silylcordycepin 21 in Sp-NUC-7738.

5'-silylcordycepin 21 was dissolved in 10 vol THF and cooled to 0°C. 2.0 M tert-BuMgCl (2.5 equivalents) was added and the mixture was stirred for 15 minutes. The Sp isomer of compound 5 (2.5 eq.) was dissolved in 5 V THF and added to the reaction at 0°C. The mixture was stirred at 0°C for 15 minutes before heating to 25°C and stirred for a further 2 hours. The reaction was quenched with 10% ammonium chloride solution (10 V) and extracted with ethyl acetate. The combined organic layers were washed with water and 10% brine before concentration in vacuo.

The product was dissolved in THF (10V) before cooling to 0°C. A mixture of 10 V TFA and water (1:1) was added to the reaction over 30 minutes before the mixture was stirred for 45 minutes, heated to 30°C and stirred for a further 16 hours. The reaction was quenched with 7% NaHCO ₃ solution ( 90 volumes) at 0°C before extraction with ethyl acetate. The combined organic layers were washed with water, 7% sodium bicarbonate and 10% brine before concentration 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 Sp-NUC-7738 in 40% yield. HPLC purity of the product was 99.50%, and chiral HPLC showed that the Sp isomer was 99.90% and the Rp isomer was 0.10%.

The same method was carried out to obtain Rp-NUC-7738.

Rp-NUC-7738:

1H NMR (500 MHz, CDCl ₃ ) 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=1.5 Hz, 1H, H1'), 5.12 (AB kv, J _AB =12.0 Hz, Δδ _AB = 0.04, 2H, CH ₂ Ph), 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, CHCH3 L-Ala), 2.352.27 (m, 1H , H3'), 2.09-2.02 (m, 1H, H3'), 1.29 (d, J=7.0 Hz, 3H, CHCH3 L-Ala).

³¹ P NMR (202 MHz, CD3OD) δp 3.91.

MS (ES+) m/z found 569.2 [M+H+], 591.2 [M+Na ⁺ ], 1159.4 [2M+Na+] C26H ₂₉ N ₆ O ₇ P required m/z 568.2 [M].

Reverse phase HPLC (Varian Pursuit XRs 5 C18, 150x4.6 mm) eluting H2O/CH3CN from 90/10 to 0/100 over 30 min, F: 1 ml/min, λ=200 nm, showed one peak with t _R 14.02 min.

Sp-NUC-7738:

1H NMR (500 MHz, CDQ3) δ _B 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=1.5 Hz, 1H, H1'), 5.08 (AB kv, Jab=12.0 Hz, A6ab=O.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, CHCH3 L-Ala), 2.362.27 (m, 1H, H3'), 2.08-2.01 (m, 1H, H3'), 1.34-1.30 (m, 3H, CHCH3 L-Ala).

³¹ P NMR (202 MHz, CD3OD) δp 3.73.

MS (ES+) m/z found 569.2 [M+H+], 591.2 [M+Na ⁺ ], 1159.4 [2M+Na+] C ₂₆ H ₂₉ N ₆ O ₇ P required m/z 568 ,2 [M].

Reversed phase HPLC (Varian Pursuit XRs 5 C18, 150x4.6 mm) eluting H2O/CH3CN from 90/10 to 0/100 in 30 minutes, F: 1 ml/min, λ=200 nm, showed one peak with tR 14.26 min.

The stereochemistry (R _p relative to S _p ) of the two NUC-7738 isomers described above was confirmed by standard x-ray crystallographic analysis.

Illustrative Example 10: Receipt of NUC-9701.

8-Chloroadenosine dimethyl acetal 25 24 can be prepared according to the following scheme (also described in WO 2017/207989).

- 18 043691

Compound 25 can then be condensed with the compound of Formula IIa using the condensation process conditions described in Example 8. To prepare NUC-9701, dimethyl acetal can be removed using 1:1 TPA:water at 0°C for 5 hours.

HoN

Compound 25 (30 g; 1 equivalent) and the desired isomer of compound 12 (58.08 g; 1.2 equivalent) were dissolved in 300 ml (10 volumes) THF. The mixture was cooled to 0° C. before tert-butyl magnesium chloride (76.8 ml 2.0 M in THF; 1.75 equivalents) was slowly added, maintaining the temperature at 0° C., and the mixture was stirred for 4 hours. 300 ml (10 volumes) of 10% ammonium chloride solution was added to the reaction mixture, maintaining the temperature below 15°C. The mixture was extracted with ethyl acetate and the combined organic layers were washed with 7% sodium bicarbonate (twice), water (twice) and 20% sodium chloride before drying with anhydrous sodium sulfate and filtering. Ethyl acetate was removed in vacuo.

To the resulting product was added 600 ml (20 volumes) of 60% formic acid in water and the reaction was stirred for 65-70 hours at 25° C. before slowly adding ethyl acetate (600 ml; 20 volumes). 600 ml (20 volumes) of 20% sodium chloride solution were added and the layers were separated. The aqueous layer was extracted with ethyl acetate before 600 ml (20 volumes) of 10% ammonia solution was added dropwise to the combined organic layers and the layers were separated. The organic layer was washed with water (three times) and 20% sodium chloride solution, dried with 30 g (1 w/w) anhydrous sodium sulfate and filtered before concentration in vacuo. The crude product was purified by column chromatography to obtain 20-45 g of NUC-9701.

Sp-NUC-9701:

Ή-NMR (500 MHz; MeOD-d4): δ _H 8.07 (1H, d J= 8.5 Hz, H-Napht), 8.05 (1H, s, 2-H), 7.87 ( 1H, d J=8.5 Hz, H-Napht), 7.67 (1H, d J= 8.5 Hz, H-Napht) 7.54-7.48 (2H, m, H-Napht), 7.41-7.30 (1H, m, H-Napht), 7.367.33 (1H, m, H-Napht), 7.26-7.22 (5H, m, -CH ₂ Ph), 6, 03 (1H, d J=5.0 Hz, Н-1'), 5.33 (1H, TJ= 5.0 Hz, Н-2'), 5.01, 4.98 (АВ, JAB=12 ,3 Hz, CH ₂ Ph), 4.65 (1H t J = 5.5 Hz, 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, d J=7.0 Hz, CH ₃ ).

³¹ P NMR (202 MHz, MeOD-d4): δ _P 3.93 (1P, s).

Reverse phase HPLC, eluting _H2O /CH3CN from 90/10 to 0/100 over 30 min; 1 ml/min, λ=254 np, showed a peak with t _R = 16.43 min.

Rp-NUC-9701:

^1H -NMR (500 MHz; MeOD-d4): _δH 8.10 (1H, s, H-2), 8.08 (1H, d J= 8.5 Hz, H-Napht), 7.87 (1H, d J= 8.5

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Claims (12)

Hz, H-Napht),7.67 (1H, d J= 8.5 Hz, H-Napht), 7.53-7.50 (1H, m, H-Napht), 7.48-7.44 (1H, m, H-Napht), 7.407.38 (1H, m, H-Napht), 7.33-7.27 (6H, m, H-Napht and -CH ₂ Ph), 6.02 (1H , d J=5.0 Hz, H-1'), 5.28 (1H, TJ= 5.0 Hz, H-2'), 5.04, 5.02 (AB, Jab=12.2 Hz, CH2Ph), 4.63 (1H t J= 5.5 Hz, H-3'), 4.48 -4.46 (1H, m, H _a -5'), 4.38-4.35 (1H, m, Hb-5'), 4.23-4.20 (1H, m, H-4' ), 4.05-4.01 (1H, m, -CHCH3), 1.17 (1H, d J=7.0 Hz, CH ₃ ). ³¹ P NMR (202 MHz, MeOD-d ₄ : δ _P 3.83 (1P, s). Reversed phase HPLC, eluting H ₂ O/CH ₃ CN from 90/10 to 0/100 over 30 min; 1 ml/min, λ=254 nm, showed a peak with tR=16.59 min. The stereochemistry (R _p relative to S _p ) of the two NUC-9701 isomers described above was assigned tentatively based on comparison of ³¹ P chemical shift, 1 H NMR spectra and HPLC retention time with those of other ProTides known in the literature. CLAIM 1. Method for obtaining the X-diastereoisomer of a compound of formula IIa (R ¹ ^ O Formula Pa, where R ¹ represents a halogen and a represents an integer from 1 to 5, including: a) suspending or dissolving the Y-diastereoisomer of a compound of formula IIa or a mixture of Y and X-diastereoisomers of a compound of formula IIa in a solvent (S2), b) treating the solution or suspension with a base (B2), obtaining the X-diastereoisomer in substantially diastereomerically enriched form, c) isolating the X-diastereoisomer of formula IIa, where B2 is a tertiary amine; where S2 is a mixture of heptane or hexane and a polar organic solvent, where the mixture contains more than 50% by volume of hexane or heptane; and wherein the X-diastereoisomer of compound IIa is an (S _p )-diastereoisomer, and the Y diastereoisomer of compound IIa is an (R _p )-diastereoisomer, unless OPh(R1) _a leaving group has a lower distribution priority according to the Cana rule IngoldPrelog than the naphthyloxy group, wherein the X-diastereoisomer of compound IIa is an (R _p ) diastereoisomer and the Y-diastereoisomer of compound IIa is an (S _p ) diastereoisomer. 2. Method for preparing a slowly eluting diastereoisomer of a compound of formula IIa where R ¹ represents a halogen and a represents an integer from 1 to 5, including: a) suspending or dissolving a fast-eluting compound of formula IIa or a mixture of fast-eluting and slow-eluting diastereoisomers of a compound of formula IIa in a solvent (S2), b) treating the solution or suspension with a base (B2), obtaining a slowly eluting diastereoisomer in substantially diastereomerically enriched form, and c) isolating the slowly eluting diastereoisomer of formula IIa, where B2 is a tertiary amine; where S2 is a mixture of heptane or hexane and a polar organic solvent, where the mixture contains more than 50% by volume of hexane or heptane; wherein the slow eluting diastereoisomer of compound IIa is the isomer that elutes more slowly on reverse phase HPLC, and the fast eluting diastereoisomer of compound IIa is the isomer that elutes faster on reverse phase HPLC. - 20 043691 3. Method according to claim 1 or 2, where B2 represents triethylamine. 4. A method according to any one of the preceding claims, wherein S2 is a mixture of heptane or hexane and tert-butyl methyl ether, the mixture containing more than 50% by volume of heptane or hexane. 5. The method according to any of the preceding claims, wherein step b) comprises stirring a mixture of a compound of formula Pa and a base B2 for 24 hours or more. 6. The method according to any of the preceding paragraphs, where step b) includes stirring a mixture of a compound of formula Pa and a base B2 at a temperature of 0-60°C. 7. The method according to any of the preceding paragraphs, where the compound of formula Pa is: 8. Process for preparing the ( _8p )-diastereoisomer NUC-3373 (Formula 1a) in substantially diastereoisomerically pure form Formula 1a including the method according to any one of claims 1 to 9, stage d) and stage e): d) reacting the X-diastereoisomer of the compound of formula Pa isolated in step c) with a compound of formula IIIa in the presence of a base (B1) to obtain a compound of formula IVa in substantially diastereomerically pure form; where P ¹ is independently selected from hydrogen and a protecting group: Formula Sha Formula IVa where B1 is a Grignard reagent; And e) wherein P ¹ represents a protecting group, removing the protecting group P ¹ from the compound of formula IVa, yielding the (8 _p )-diastereoisomer NUC-3373 in substantially diastereomerically pure form. 9. The method according to claim 8, where P ¹ represents -C(O)O ^t Bu. 10. Method according to claim 8 or claim 9, where B1 represents ^uMgCl. 11. Method according to any one of paragraphs 8-10, where stage d) is carried out in an ethereal solvent. 12. Method according to claim 11, where step d) is carried out in THF. Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky lane, 2