EA042712B1 - NUCLEOSIDE DIPHOSPHATE-PHOSPHORAMIDATE SALTS AS ANTI-CANCER COMPOUNDS - Google Patents

Description

The present invention relates to compounds of formula (I) or their pharmaceutically acceptable salts useful in the treatment of cancer, such as leukemia. The compounds include the diphosphate phosphoramidate salt.

State of the art invention

Nucleoside drugs have become a powerful tool in the treatment of human disease. In the treatment of cancer, in particular, the use of nucleoside drugs such as gemcitabine, clofarabine, cytarabine, fludarabine is widely used.

However, the efficacy of all nucleoside drugs may be limited by both innate and acquired resistance mechanisms.

One way in which the efficacy of nucleoside drugs can be increased is by administering as a drug of the ProTide class. The ProTide class of drugs are prodrugs of monophosphorylated nucleosides and have been shown to be particularly effective therapeutic agents in both the antiviral and oncology fields. These compounds appear to avoid many of the 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; 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; WO2005/012327; WO2006/100439; WO2012/117246 and WO2016/083830). An example of ProTide is NUC-1031, the ProTide of gemcitabine

While the ProTide strategy has proven to be very effective for certain nucleosides, it is not as effective with other nucleoside drugs. Therefore, there is a need to find additional ways to potentiate the molecules of nucleoside drugs.

Drugs of the ProTide class may also be poorly soluble in aqueous vehicles and this may make administration problematic.

The purpose of certain embodiments of the present invention is to provide a therapeutic agent that, when used, has therapeutic efficacy in the prevention or treatment of cancer.

The purpose of certain embodiments of the present invention is to provide a therapeutic agent that, when used, has a greater therapeutic efficacy for the prevention or treatment of cancer than the original nucleotide or the corresponding ProTide.

The purpose of certain embodiments of the present invention is to provide a therapeutic agent that is more convenient to enter than the corresponding ProTide.

Certain embodiments of the present invention solve some or all of the above problems.

Statement of invention

In a first aspect of the present invention, a compound of formula (I) or a pharmaceutically acceptable salt thereof is provided

Z ¹ o II II ^R k _l O-X ⁺ o l ^R

R ² V ⁵ (I) in which R ¹ independently at each occurrence is selected from C ₁ -C ₈ -alkyl, C ₀ -C ₄ -alkylene-C ₃ -C ₈ cycloalkyl and C ₀ -C ₄ -alkylenaryl;

each R ² and R ³ independently at each occurrence is selected from H, C ₁ -C ₆ -alkyl and C ₁ -C ₃ -alkylene-R ⁷ ;

R ⁴ independently at each occurrence is H or R ⁴ , a group selected from R ² and R ³ , and the atoms to which they are attached may form a 3-6 membered heterocycloalkyl group containing 1 or 2 heteroatoms independently selected from O, S and N;

R ⁵ is independently selected at each occurrence from aryl and C ₁ -C ₈ alkyl, with aryl optionally fused to C ₆ -C ₈ cycloalkyl;

- 1 042712

R ⁶ independently selected from

and he is OH;

R ⁷ is independently selected from aryl and SR ^a at each occurrence;

R ⁸ is independently selected from H and

each Z ¹ and Z ² is independently selected from O and S; Y is independently selected from H, F, Cl and OMe;

X ⁺ independently at each occurrence is a pharmaceutically acceptable metal cation or ammonium cation;

where any aryl group is either phenyl or naphthyl;

where when any of R ¹ , R ² , R ³ , R ⁴ , R ⁵ or R ⁷ is alkyl, cycloalkyl, heterocycloalkyl or aryl or heteroaryl, then the alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl group is optionally substituted with 1-4 substituents , selected from halogen, nitro, cyano, NR ^a R ^a , NR ^a S(O)2R ^a , NR ^a C(O)R ^a , NR ^a CONR ^a R ^a , NR ^a CO2R ^a , OR ^a , SR ^a , SOR ^a , SO3R ^a , SO2R ^a , SO2NR ^a R ^a , CO2R ^a , C(O)R ^a , CONR ^a R ^a , CR ^a R ^a NR ^a R ^a , ^^-alkyl, C2-C4-alkenyl, C2 -C4 alkynyl and C1-C4 haloalkyl;

where R ^a independently at each occurrence is selected from H and C ₁ -C ₄ -alkyl and

R ^b independently at each occurrence is selected from H, and C 1 -C ₄ -alkyl and C (O) -C ₁ -C ₄ -alkyl.

The inventors have found that the phosphoramidate diphosphates of formula (I) have good activities against a number of cancer cell lines, including solid tumor and hemoblastosis lines. In many cases, the diphosphates of formula (I) are more active than the corresponding ProTide.

Diphosphate prodrugs reported in the literature, such as, for example, nucleoside pyrophosphate diester, are known to be based on glycerides (K.Y. Hostetler et al., J. Biol. Chem. 1990, 265, 6112-6117; G.M.T. can Wijk, K.Y. Hostetler et al., Biochim. Biophys. Acta Lipids Lipid Metab. 1991, 1084, 307-310) are unstable due to cleavage of the P-O-P bond of the group, thus releasing the corresponding nucleoside monophosphate instead of the required diphosphate. The inventors have found, however, that deprotonation of the α-phosphate group of diphosphate phosphoramidates provides high stability. The ionic nature of the compounds of the present invention also means that they are more water soluble than the corresponding ProTide.

A number of enzymatic processes are required to convert ProTide to the corresponding monophosphate nucleotide, including cleavage of the ester by carboxylesterases and cleavage of the resulting amino acid from the monophosphate nucleoside by phosphoramidase-type enzymes. The diphosphate phosphoramidates of the present invention are larger molecules having different shapes and are ionic, which makes it doubtful that they can interact with the corresponding enzymes in the same way to be converted into a diphosphate nucleotide. Indeed, preliminary data have shown that the diphosphate phosphoridates of formula (I) are stable with respect to carboxypeptidases, eg carboxypeptidase Y, enzymes which carry out the ester cleavage step in converting a ProTide class drug to monophosphates. However, the diphosphate phosphoramidates of formula (I) are still active in cells, suggesting an alternative mechanism of action compared to the ProTide class of drugs. The inventors also showed that where

- 2 042712 an alkyloxy group is present at the phosphorus of the phosphoramidate (OR ⁵ in formula (I)), the compounds of the present invention are active. In drugs of the ProTide class, this is usually not the case, and only compounds having aryloxy groups at this position provide good activity. It is clear that this is due to the fact that the aryloxy group acts as a leaving group in the activation of drugs of the ProTide class. Again, this indicates that the diphosphate phosphoramidates of the present invention are activated by a mechanism different from that of the ProTide class of drugs.

In embodiments, the compound of formula (I) is a compound of formula (II)

Clofarabine diphosphate phosphoramidates, such as compounds of formula (II), were more active than the corresponding ProTide against a number of cancer cell lines, including the hematological cell line.

In embodiments, the compound of formula (I) is a compound of formula (III)

In embodiments, the compound of formula (I) is a compound of formula (IV)

In embodiments, the compound of formula (I) is a compound of formula (V)

HoN

In embodiments, the compound of formula (I) is a compound of formula (VI)

In embodiments, the compound of formula (I) is a compound of formula (VII) h ₂ n k O o N b \

II II / 1 /-γ o R ⁵ oy IC /- ^NH OX ^{+ 0} X \ / o o

R ² R ³ ij ΰ _h

O^j^/^OR ⁵ O °- ^χ+

R ² R ³ (Vii)

In these embodiments, it may be that R ¹ , R ² , R ³ , R ⁵ and X are the same in both occurrences.

The following statements apply to compounds of any formulas (I)-(VII). These statements are independent and interchangeable. In other words, any of the features described in any of the following statements may (where chemically acceptable) be combined with the features described in one or more of the other statements below. In particular, when a compound is exemplary or illustrated herein, any two or more of the following statements that describe a feature of that compound, expressed at any level of generalization, may be combined to represent the subject matter that is considered to be 042712 which is part of the description of the present invention in this description.

R ¹ can be independently selected at each occurrence from C ₁ -C ₈ alkyl, C ₀ -C ₄ alkylene-C ₃ -C ₈ cycloalkyl, and C ₀ -C ₄ alkylenaryl.

R ¹ can be independently selected at each occurrence from C1-C8 alkyl, C0-C4 alkylene-C3-C8 cycloalkyl and CH2 aryl. R ¹ can be independently selected at each occurrence from C 1 -C ₈ alkyl, C ₄ -C ₆ cycloalkyl and benzyl. R ¹ can be independently selected at each occurrence from C ₁ -C ₈ alkyl, C ₆ cycloalkyl and benzyl.

R ¹ may be C ₁ -C ₈ alkyl.

R ¹ can be chosen to contain three or more carbon atoms. R ¹ can be chosen to contain five or more carbon atoms. R ¹ can therefore be chosen to contain six or more carbon atoms. R ¹ is preferably chosen to contain only carbon and hydrogen atoms. R ¹ may be selected from C5- _C7 cycloalkyl, _C5 - _C8 alkyl and benzyl, optionally when these groups are unsubstituted. R ¹ may be unsubstituted benzyl. R ¹ may be neopentyl. R ¹ may be ethyl.

It may be that R ³ is H. It may be that R ³ is C1-C4 alkyl. It may be that R ³ is methyl. It may be that R ² is selected from C1-C ₆ alkyl and C ₁ -C ₃ alkylene-R ⁷ . It may be that R ² is C ₁ -C ₄ alkyl. It may be that R ² is selected from methyl and isopropyl. R ² may be methyl. R ² may be H.

R ⁴ is preferably H.

It may be that R ⁴ , a group selected from R ² and R ³ , and the atoms to which it is attached form a 3-6 membered heterocycloalkyl group. It may be that R ⁴ , a group selected from R ² and R ³ , and the atoms to which it is attached do not form a 3-6 membered heterocycloalkyl group containing 1 or 2 heteroatoms independently selected from O, S and N It may be that each R ² and R ³ is independently selected at each occurrence from H, C ₁ -C ₆ alkyl and C ₁ -C ₃ alkylene-R ⁷ ; and R ⁴ is independently H at each occurrence.

It may be that R ⁵ is unsubstituted phenyl, which may optionally be fused to a C ₆ -C ₈ cycloalkyl ring, eg a cyclohexane ring. It may be that R ⁵ is unsubstituted phenyl. It may be that R ⁵ is an unsubstituted naphthyl (eg 1-naphthyl). Preferably R ⁵ is selected from unsubstituted phenyl or unsubstituted naphthyl (eg 1-naphthyl). Thus R ⁵ may be unsubstituted phenyl. Alternatively, R ⁵ may be an unsubstituted naphthyl (eg, 1 naphthyl).

R ⁶ may be

R ⁶ may be

R ⁶ may be

R ⁶ may be

Y may be H. Y may be F.

- 4 042712

R ⁸ may be H. R ⁸ may be

O Z ² II II

O-X ^{+ R 0}

R ² R ³

Z ² may be S. Z ² may be O.

where R ⁸ is ^s X \ it may be that R ¹ , R ² , R ³ , R ⁵ , X and Z ² are the same in both occurrences.

R ⁶ may be

He

R ⁶ may be

HoN

HE

R ⁶ may be

-5 042712

- 6 042712

- 7 042712

- 8 042712

- 9 042712

X ⁺ may be a metal cation, or it may be an ammonium cation. X ⁺ may be a metal cation, for example an alkali or alkaline earth metal cation. X ⁺ can be an ammonium cation, for example a trialkylammonium cation or a nitrogen heterocycle ammonium cation. Exemplary cations include those derived from aluminum, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine, triethylamine, and zinc cations. X ⁺ may be a triethylamine cation.

The compounds of the present invention contain a chiral center at the phosphorus atom (β-phosphorus), which is connected to OR ⁵ . The compound may be present as a mixture of phosphate diastereoisomers, as the (S)-epimerα at the phosphorus atom in substantially diastereomerically pure form, or as the (R)-epimer at the phosphorus atom in essentially diastereomerically pure form. Substantially diastereomeric purity for the purposes of the present invention is defined as a diastereomeric purity greater than about 90%. When present in a substantially diastereomeric pure form, the compound may have a diastereomeric purity greater than 95%, 98%, 99%, or even 99.5%. Alternatively, the compound may be present as a mixture of phosphate diastereoisomers.

The (R)- and/or (8)-epimers of a compound can be obtained in substantially diastereomeric pure form by chromatography, eg HPLC, optionally using a chiral column. Alternatively, the (R) and/or (S) epimers of a compound can be obtained in substantially diastereomerically pure form by crystallization from a suitable solvent or solvent system.

According to a fourth aspect of the present invention, the use of a compound of formula (I), or a pharmaceutically acceptable salt thereof, is provided in the preparation of a medicament for the prevention or treatment of cancer.

In certain embodiments, the cancer is leukemia or lymphoma.

The cancer may be leukemia.

There are four main types of leukemia depending on whether it is chronic or acute, and myeloid or lymphoid in origin. These are: acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML) and chronic lymphocytic leukemia (CLL).

Mixtures of these leukemias are also known; for example, acute biphenotypic leukemia (BAL; which is a mixture of AML and ALL).

In specific embodiments, the leukemia is selected from the group consisting of acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), and acute biphenotypic leukemia (BAL; which is a mixture of AML and ALL).

Chronic lymphocytic leukemia (CLL) includes the subtypes B-cell CLL (B-CLL), B-cell prolymphatic leukemia (PLL), and T-cell chronic prolymphatic leukemia (T-PLL) and several subtypes that differ at the genetic level.

Acute lymphoblastic leukemia (ALL) includes the subtypes: progenitor B cell ALL (ALL), progenitor T cell ALL (T-ALL), Burkitt-type ALL, and Philadelphia chromosome-positive (BCR-ABL fusion) ALL.

Acute myeloid leukemia (AML) includes subtypes: myeloid leukemia, monocytic leukemia, and acute promyelocytic leukemia (APL).

Chronic myelogenous leukemia (CML) includes subtypes: chronic granulocytic leukemia (CGL) (CGL), juvenile CML (JCML), chronic neutrophilic leukemia (CNL), chronic myelocytic leukemia (CML) and atypical CML (aCML).

In specific embodiments, any of the subtypes above CLL, ALL, AML and CML are suitable for treatment with the compounds of the present invention or pharmaceutical compositions containing them.

Other forms of leukemia that may be considered outside of these four major groups include erythroleukemia, which arises from erythrocyte precursors; and lymphoma that has entered the bloodstream.

The cancer may be a lymphoma, eg solid lymphoma.

There are two main types of lymphoma: Hodgkin's lymphoma and non-Hodgkin's lymphoma. Each of them has different subtypes.

In specific embodiments, the lymphoma is selected from the group consisting of lymphoma

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Hodgkin's and non-Hodgkin's lymphoma.

In specific embodiments, the lymphoma is selected from the group consisting of Burkitt's lymphoma (BL), mantle cell lymphoma (MCL), follicular lymphoma (FL), small lymphocytic lymphoma (SLL), indolent B-cell non-Hodgkin's lymphoma, histiocytic lymphoma (also known as as immunoblastic lymphoma; IBL) and diffuse large B-cell lymphoma (DLBCL), including diffuse activated-cell large B-cell lymphoma (DLBCL-ABC) and diffuse terminal center B-cell lymphoma (DLBCL-GCB).

Cancer can be recurrent. The cancer may be metastatic. Cancer may be previously untreatable.

The cancer may be refractory cancer that has been previously treated but has been refractory to prior treatment. Alternatively, a patient with cancer may not tolerate previous therapy, for example, they may develop side effects that make the patient intolerant of further treatment with an administered agent.

The compound of formula (I) may be as described in the following numbered paragraphs.

1. In a first aspect of the present invention, a compound of formula (I) or a pharmaceutically acceptable salt thereof is provided

in which R ¹ independently at each occurrence is selected from C ₁ -C ₈ -alkyl, C ₀ -C ₄ -alkylene-C ₃ -C ₈ cycloalkyl and C ₀ -C ₄ -alkylene;

each R ² and R ³ is independently selected at each occurrence from H, C1-C6 alkyl and C ₁ -C ₃ alkylene-R ⁷ ;

R ⁴ independently at each occurrence is H or R ⁴ , a group selected from R ² and R ³ , and the atoms to which they are attached may form a 3-6 membered heterocycloalkyl group containing 1 or 2 heteroatoms independently selected from O, S and N;

R ⁵ independently at each occurrence is selected from aryl and C 1 -C ₈ -alkyl, and aryl is optionally fused with C ₆ -C ₈ -cycloalkyl;

R ⁶ independently selected from

R ⁷ is independently selected from aryl and SR ^a at each occurrence; R ⁸ is independently selected from H and

each Z ¹ and Z ² is independently selected from O and S;

Y is independently selected from H, F, Cl and OMe;

X ⁺ independently at each occurrence is a pharmaceutically acceptable metal cation or ammonium cation;

where any aryl group is either phenyl or naphthyl;

where when any of R ¹ , R ² , R ³ , R ⁴ , R ⁵ or R ⁷ is alkyl, cycloalkyl, heterocycloalkyl or aryl or heteroaryl, then the alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl group is optionally substituted with 1-4 substituents , selected from halogen, nitro, cyano, NR ^a R ^a , NR ^a S(O)2R ^a , NR ^a C(O)R ^a , NR ^a CONR ^a R ^a , NR ^a CO2R ^a , OR ^a ; SR ^a , SOR ^a , SO3R ^a , SO2R ^a , SO2NR ^a R ^a , CO2R ^a , C(O)R ^a , CONR ^a R ^a , CR ^a R ^a NR ^a R ^a , C ₁ -C ₄ -alkyl, C ₂ -C ₄ alkenyl, C ₂ -C ₄ alkynyl and C ₁ -C ₄ haloalkyl;

- 11 042712 where R ^a is independently selected at each occurrence from H and C ₁ -C ₄ -alkyl and R ^b independently at each occurrence is selected from H and C ₁ -C ₄ -alkyl and C (O) -C ₁ -C ₄ -alkyl.

2. The compound according to claim 1, in which R ⁶ represents h ₂ n

3. The compound according to claim 1, in which R ⁶ represents

4. Connection

5. The compound according to claim 1, in which R ⁶ represents

1, wherein R ⁶ is

where Y and R ⁸ have the meanings specified in paragraph 1.

6. A compound according to claim 5, wherein Y is H.

7. A compound according to claim 5, wherein Y is F.

8. A compound according to any one of claims 5 to 7, wherein R ⁸ is H.

9. A compound according to any one of claims 5 to 7, wherein R ⁸ is

where R ¹ , R ² , R ³ , R ⁴ , R ⁵ , Z ² and X ⁺ have the meanings specified in paragraph 1.

10. A compound according to claim 9, wherein Z ² is O.

11. A compound according to claim 9, wherein Z ² is S.

12. The compound according to claim 1, in which R ⁶ represents

13. The compound according to claim 1, in which R ⁶ represents

14. The compound according to claim 1, in which R ⁶ represents

15. A compound according to any one of claims 1 to 14, wherein R ¹ is selected from C ₅ -C ₇ cycloalkyl, C 1 -C ₈ alkyl and benzyl.

16. A compound according to claim 15, wherein R ¹ is benzyl.

17. The compound according to claim 15, in which R ¹ represents C 1 -C ₈ -alkyl, for example ethyl.

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18. A compound according to any one of claims 1-17, wherein R ³ is H.

19. A compound according to any one of claims 1 to 18, wherein R ² is C ₁ -C ₄ alkyl.

20. A compound according to any one of claims 1-18, wherein R ² is H.

21. A compound according to any one of claims 1 to 20, wherein R ⁵ is phenyl.

22. A compound according to any one of claims 1-20, wherein R ⁵ is naphthyl.

23. The compound according to claim 1, in which the compound of formula (I) is selected from

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- 14 042712

- 15 042712

where X ⁺ has the meaning specified in paragraph 1.

24. The use of a compound according to any one of claims 1 to 23 for the treatment of cancer.

25. Use according to claim 24, wherein the cancer is leukemia or lymphoma.

26. Use according to claim 25, wherein the cancer is a leukemia selected from the group consisting of acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphoid leukemia (CLL), and acute biphenotypic leukemia (BAL).

27. Use according to claim 25, wherein the cancer is a lymphoma selected from the group consisting of Hodgkin's lymphoma and non-Hodgkin's lymphoma.

Brief description of the figures

Embodiments of the present invention are further described herein with reference to the accompanying drawings, in which:

fig. 1 shows sigmoidal dose-response curves for (A) clofarabine, (B) example 1 and (C) example 2. Clofarabine and example 1 showed the highest activity. All analyzes were performed using KG1a cells and data are presented as the mean (±SD) of three independent experiments;

fig. 2 shows LSC targeting analysis of clofarabine, Example 1 and Example 2. Under normoxic conditions, all compounds showed stem cell targeting at concentrations above 1 x 10 ^-7 M. All data are mean (±SD) of three independent experiments. ;

fig. 3 shows a comparison of the proportion of KG1a cells expressing the LSC phenotype under normoxic and hypoxic conditions. (A) KG1a cells show a time-dependent increase in cells expressing the LSC phenotype under hypoxic conditions, which (B) was reversed when the cells were transferred to normoxic conditions. All data are mean (±SD) of four independent experiments;

fig. 4 shows a comparison of the average LD ₅₀ values for (A) clofarabine, (B) Example 1, and (C) Example 2 under normoxic and hypoxic conditions. Clofarabine showed a significant decrease in activity under hypoxic conditions. On the contrary, the compounds of the present invention retained their activity under hypoxic conditions. All data are mean (±SD) of three independent experiments;

fig. 5 shows a comparison of DNA damage as measured by the YH2A.X phosphorylation assay stimulated in purified LSC and bulk tumor cells after 2 hours of exposure to (A) clofarabine, (B) Example 1 and (C) Example 2 under hypoxic conditions (5% _O2 ). All data are the mean (±SD) of three independent experiments.

Detailed description

The term brine is intended to refer to an aqueous solution of sodium chloride. Saline solutions of the present invention will generally be sterile and will generally be at a concentration suitable for parenteral use. Suitable concentrations are up to 2 or up to 1 wt./about. To optimize osmolarity in the compositions of the present invention, different concentrations of saline can be used, for example, 0.9 or 0.45%.

The compositions of the present invention can be used in the treatment of the human body. They can be used in the treatment of the animal body.

The compounds in the compositions of the present invention may be prepared, stored and/or administered as a pharmaceutically acceptable salt. Suitable pharmaceutically acceptable salts include, but are not limited to, salts of pharmaceutically acceptable inorganic acids such as hydrochloric, sulfuric, phosphoric, nitric, carbonic, boric, sulfamic and hydrobromic acids, or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymalic, fumaric, malic, citric, lactic, mucinic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulfonic, toluenesulfonic, benzenesulfonic, salicylic, sulfanilic, aspartic, glutamic, ethylenediaminetetraacetic, stearic, palmitic, lauric, oleic pantothenic, tannic, ascorbic and valeric acids. Examples include salts of aluminum, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc. Hemisalts of acids and bases can also be prepared, for example, hemisulphate, hemioxalate and hemicalcium salts.

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For the above formulations of the present invention, the dose administered will, of course, vary depending on the compound used, the precise route of administration, the treatment desired, and the disease in question. It is expected that dosage levels, frequency of administration and duration of treatment with the compounds of the present invention will vary depending on the composition and clinical indications, age and comorbidities of the patient. The dosage size for therapeutic purposes of the compounds of the present invention will naturally vary with the nature and severity of the disease, the age and sex of the animal or patient, and the route of administration, in accordance with well known principles of medicine.

The pharmaceutical composition can usually be in the form of a composition in which the active compounds or their pharmaceutically acceptable salts are in combination with a pharmaceutically acceptable adjuvant, diluent or carrier. One such pharmaceutically acceptable adjuvant, diluent or carrier in the composition of the present invention is a polar aprotic solvent. Conventional methods for selecting and preparing suitable pharmaceutical formulations are described, for example, in Pharmaceuticals-The Science of Dosage Form Designs, M.E. Aulton, Churchill Livingstone, 1988.

The formulations may be suitable for topical application (eg, to the skin or bladder), for oral administration, or for parenteral administration (eg, for intravenous administration).

Any solvents used in the pharmaceutical compositions of the present invention must be of pharmaceutical grade, which means that they have an impurity profile that makes them suitable for administration (eg, intravenous administration) to humans.

For oral administration, the compositions of the present invention may contain the active compound admixed with an adjuvant or carrier, eg lactose, sucrose, sorbitol, mannitol; starch, for example potato starch, corn starch or amylopectin; a cellulose derivative; a binder, such as gelatin or polyvinylpyrrolidone; and/or a lubricant such as magnesium stearate, calcium stearate, polyethylene glycol, wax, paraffin and the like, and then compressed into tablets. If coated tablets are required, the cores prepared as described above may be coated with a concentrated sugar solution which may contain, for example, gum arabic, gelatin, talc and titanium dioxide. Alternatively, the tablet may be coated with a suitable polymer dissolved in a highly volatile organic solvent.

To obtain soft gelatin capsules, the active compounds can be mixed with, for example, vegetable oil or polyethylene glycol. Hard gelatin capsules may contain granules of the compound using any of the tablet excipients mentioned above. Also, liquid or semi-solid formulations of the active compounds can be filled into hard gelatin capsules.

Liquid oral preparations may be in the form of syrups or suspensions, for example solutions, containing a compound of the present invention, the balance of which is sugar and a mixture of ethanol, water, glycerol and propylene glycol. Optionally, these liquid preparations may contain colors, flavors, sweeteners (such as saccharin), preservatives and/or carboxymethyl cellulose as a thickening agent, or other excipients known to those skilled in the art.

The formulations may be for parenteral (eg, intravenous) administration. For parenteral (eg, intravenous) administration, the active compounds may be administered as a sterile aqueous or oily solution. Preferably, the active compounds are administered as a sterile aqueous solution. The aqueous solution may further contain at least one surfactant and/or an organic solvent. Exemplary organic solvents include dimethylacetamide, ethanol, ethylene glycol, N-methylpyrrolidone, dimethylsulfoxide, dimethylformamide, and isopropanol. Exemplary surfactants include polyethoxylated fatty acids and fatty acid esters and mixtures thereof. Suitable surfactants include polyethoxylated castor oil (eg sold under the trade name Kolliphor® ELP); or polyethoxylated stearic acid (eg sold under the names Solutol® or Kolliphor® HS15); or polyethoxylated (eg polyoxyethylene (20)) sorbitan monooleate (eg sold under the tradenames Polysorbate 80 or Tween® 80).

The pharmaceutical composition of the present invention will preferably contain 0.05-99 wt% of the compound of the present invention, more preferably 0.05-80 wt% of the compound of the present invention, even more preferably 0.10-70 wt% of the compound of the present invention, and even more preferably 0.10-50% by weight of a compound of the present invention, all percentages being by weight relative to the total composition.

Cyclodextrins have been shown to be widely used for drug delivery (Rasheed et al., Sci. Pharm., 2008, 76, 567-598). Cyclodextrins are a family of cyclic oligosaccharides. They act as a molecular cage that encapsulates drug molecules and alters the properties of those drug molecules, such as solubility. Cyclodextrins contain (a-1,4)-linked a-D-glucopyranose units. Cyclodextry

- 17 042712 we can contain 6, 7 or 8 glucopyranose units (designated as α-, β- and γ-cyclodextrins, respectively). Cyclodextrins used in pharmaceutical formulations are often β-cyclodextrins. The pendent hydroxyl groups can be alkylated with C1-C6 substituted or unsubstituted alkyl groups. Examples of cyclodextrins are α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin (HP-P-CD), sulfobutyl ether sodium salt β-cyclodextrin, partially methylated β-cyclodextrin. The compositions of the present invention may also contain at least one cyclodextrin.

The term Cm-C _n refers to a group with mn carbon atoms.

The term alkyl refers to a straight or branched hydrocarbon group. The alkyl group is monovalent. For example, C1- _C6 alkyl may refer to methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, t-butyl, n-pentyl, and n-hexyl. The alkyl groups are preferably unsubstituted.

The term alkylene refers to a linear hydrocarbon chain. The alkylene group is divalent. For example, C1-alkylene may refer to the CH ₂ group. C ₂ -alkylene may refer to the -CH2CH2- group. The alkylene groups are preferably unsubstituted.

The term haloalkyl refers to a hydrocarbon chain substituted with at least one halogen atom independently selected at each occurrence from fluorine, chlorine, bromine and iodine. The halogen atom may be present at any position on the hydrocarbon chain. For example, C1-C4 haloalkyl may refer to chloromethyl, fluoromethyl, trifluoromethyl, chloroethyl, for example 1-chloromethyl and 2-chloroethyl, trichloroethyl, for example 1,2,2-trichloroethyl, 2,2,2-trichloroethyl, fluoroethyl, for example 1 -fluoromethyl and 2-fluoroethyl, trifluoroethyl, for example, 1,2,2-trifluoroethyl and 2,2,2trifluoroethyl, chloropropyl, trichloropropyl, fluoropropyl, trifluoropropyl. The haloalkyl group may be a fluoroalkyl group, i. e. a hydrocarbon chain substituted with at least one fluorine atom.

The term alkenyl refers to a branched or linear hydrocarbon chain containing at least one carbon-carbon double bond. The double bond(s) may be present as the E or Z isomer. The double bond can be in any possible position on the hydrocarbon chain. For example, C ₂ -C ₄ -alkenyl may refer to ethenyl, allyl and butenyl. Alkenyl groups are preferably unsubstituted.

The term alkynyl refers to a branched or linear hydrocarbon chain containing at least one carbon-carbon triple bond. The triple bond can be at any possible position on the hydrocarbon chain. For example, C ₂ -C ₆ alkynyl may refer to ethynyl, propynyl, butynyl. Alkynyl groups are preferably unsubstituted.

The term cycloalkyl refers to a saturated hydrocarbon ring system containing 3, 4, 5 or 6 carbon atoms. For example, 3-6 membered cycloalkyl may refer to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl. Cycloalkyl groups are preferably unsubstituted.

The term heterocycloalkyl may refer to a saturated or partially saturated monocyclic group containing 1 or 2 heteroatoms independently selected from O, S and N in the ring system (in other words, 1 or 2 atoms forming the ring system are selected from O, S and N). Examples of heterocycloalkyl groups include piperidine, piperazine, morpholine, thiomorpholine, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, tetrahydropyran, dihydropyran, dioxane, azepine. Heterocycloalkyl groups are preferably unsubstituted or substituted.

The present invention also includes formulations of all pharmaceutically acceptable isotopically labeled forms of the compound in which one or more atoms have been replaced by atoms having the same atomic number but an atomic mass or mass number different from that of the predominant isotope normally found in nature.

Examples of isotopes suitable for inclusion in the compounds of the present invention include isotopes of hydrogen such as ² H and ³ H, carbon such as 11 C, ¹³ C and ¹⁴ C, chlorine such as ³⁶ Cl, fluorine such as ¹⁸ F, iodine , such as ¹²³ I and ¹²⁵ I, nitrogen, such as ¹³ N and ¹⁵ N, oxygen, such as ¹⁵ O, ¹⁷ O, and ¹⁸ O, phosphorus, such as ³² P, and sulfur, such as ³⁵ S.

Certain isotopically labeled compounds, for example compounds that contain a radioactive isotope, are useful in drug and/or substrate tissue distribution studies. Radioactive isotopes of tritium, i.e. ³ H, and carbon-14, i.e. ¹⁴ C, and ¹⁸ F are particularly suitable for this purpose due to their ease of incorporation and ready means of detection.

Replacement with heavier isotopes such as deuterium, i.e. ² H may confer certain therapeutic benefits due to greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements, and therefore may be preferred in some circumstances.

Isotopically labeled compounds can usually be obtained by conventional methods known to the person skilled in the art, or by methods similar to the methods described using

- 18 042712 a suitable isotopically labeled reagent instead of the previously used unlabeled reagent.

A method of treatment or formulation for use in the treatment of cancer, including lymphoma or leukemia, may include, in addition to the compositions of the present invention, conventional surgery or radiation therapy or chemotherapy. This chemotherapy may include the administration of one or more other active agents.

If an additional active agent is administered as part of the method of treatment of the present invention, this combined treatment can be achieved by the simultaneous, sequential or separate administration of the individual components of the treatment. These combination products use the compounds of the present invention in the therapeutically effective dosage range described above and one or more other pharmaceutically active agents within their approved dosage range.

Thus, the pharmaceutical compositions of the present invention may contain another active agent.

One or more other active ingredients may be one or more of the following categories of anticancer agents:

(i) anti-proliferative/anti-blastoma drugs and combinations thereof, such as alkylating agents (eg, cyclophosphamide, nitrogen mustard, bendamustine, melphalan, chlorambucil, busulfan, temozolomide, and nitrosoureas); antimetabolites (eg, gemcitabine and antifolates such as fluoropyrimidines like 5-fluorouracil and tegafur, reltitrexed, methotrexate, pemetrexed, cytosine arabinoside, and hydroxyurea); antibiotics (eg anthracyclines such as adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin and mithramycin); antimitotic agents (eg vinca alkaloids such as vincristine, vinblastine, vindesine and vinorelbine and taxoids such as taxol, taxotere and polokinase inhibitors); proteasome inhibitors such as carfilzomib and bortezomib; interferon therapy; and topoisomerase inhibitors (eg epipodophyllotoxins such as etoposide and teniposide, amsacrine, topotecan, mitoxantrone and camptothecin);

(ii) cytostatic agents such as antiestrogen (eg, tamoxifen, fulvestrant, toremifene, raloxifene, droloxifene, and iodoxifene), antiandrogens (eg, bicalutamide, flutamide, nilutamide, and cyproterone acetate), LHRH antagonists, or LHRH agonists (eg, goserelin, leuprorelin and buserelin) progestogens (eg megestrol acetate), aromatase inhibitors (eg anastrozole, letrozole, vorazole and exemestane) and 5a-reductase inhibitors such as finasteride;

(iii) anti-invasive agents, eg dasatinib and bosutinib (SKI-606) and metalloproteinase inhibitors, inhibitors of urokinase plasminogen activator receptor function, or anti-heparanase antibodies;

(iv) inhibitors of growth factor function: for example, these inhibitors include anti-growth factor antibodies and anti-growth factor receptor antibodies, e.g. anti-erbB2 antibody trastuzumab [Herceptin™], anti-EGFR antibody panitimumab, anti-erbB1 antibody cetuximab, tyrosine kinase inhibitors, for example, epidermal growth factor family inhibitors (e.g. EGFR family tyrosine kinase inhibitors such as gefitinib, erlotinib, and 6-acrylamido-M-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)quinazoline-4-amine (CI 1033 ), erbB2 tyrosine kinase inhibitors such as lapatinib); hepatocyte growth factor family inhibitors; inhibitors of the insulin growth factor family; modulators of protein regulators of cell apoptosis (eg Bcl-2 inhibitors); platelet growth factor family inhibitors such as imatinib and/or nilotinib (AMN107); serine/threonine kinase inhibitors (eg, inhibitors of Ras/Raf signaling, such as farnesyl transferase inhibitors, eg, sorafenib, tipifarnib, and lonafarnib), inhibitors of cellular MEK and/or AKT kinase signaling, c-kit inhibitors, αN-kinase inhibitors , PI3 kinase inhibitors, Plt3 kinase inhibitors, CSF-1R kinase inhibitors, IGF receptor, kinase inhibitors; aurorakinase inhibitors and cyclin-dependent kinase inhibitors such as CDK2 and/or CDK4 inhibitors;

(v) anti-angiogenic agents, such as agents that inhibit the effects of vascular endothelial growth factor [eg, anti-vascular endothelial cell growth factor antibody, bevacizumab (Avastin™); thalidomide; lenalidomide; and, for example, a VEGF receptor tyrosine kinase inhibitor such as vandetanib, vatalanib, sunitinib, axitinib, and pazopanib;

(vi) gene therapy approaches, including, for example, approaches to replace aberrant genes such as aberrant p53 or aberrant BRCA1 or BRCA2;

(vii) immunotherapy approaches, including, for example, adaptive cell transfer such as CAR T cell therapy; antibody therapy such as alemtuzumab, rituximab, ibritumomab tiuxetan (Zevalin®)) and ofatumumab; interferons such as interferon α; interleukins such as IL-2 (aldesleukin); interleukin inhibitors, eg IRAK4 inhibitors; cancer vaccines, including prophylactic and therapeutic vaccines such as HPV vaccines such as Gardazil, Cervarix, Oncophage and Sipuleucel-T (Provenge); and toll-like receptor modulators, such as TLR-7 agonists

- 19 042712 or TLR-9;

(viii) cytotoxic agents, eg fludarabine (fludara), cladribine, pentostatin (Nipent™);

(ix) steroids such as corticosteroids, including glucocorticosteroids and mineralocorticosteroids, eg aclomethasone, aclomethasone dipropionate, aldosterone, amcinonides, beclomethasone, beclomethasone dipropionate, betamethasone, betamethasone dipropionate, betamethasone sodium phosphate, betamethasone valerate, budesonide, clobetasone, clobetasone, propionate cloprednol, cortisone, cortisone acetate, cortivazole, deoxycortone, desonide, deoxymethasone, dexamethasone, dexamethasone sodium phosphate, dexamethasone isonicotinate, difluorocortolone, fluclorolone, flumethasone, flunisolide, fluocinolone, fluocinolone acetonide, fluocinonide, beer fluocortine butyl, fluocortolone, fluocortolonide, fluorocortolone, fluorocortolone , fluorometholone, fluprednidene, fluprednidene acetate, flurandrenolone, fluticasone, fluticasone propionate, chalcinonide, hydrocortisone, hydrocortisone acetate, hydrocortisone butyrate, hydrocortisone aceponate, hydrocortisone buteprate, hydrocortisone valerate, icomethasone, icomethasone enbutate, meprednisone, prednisone methylprednisomethasone, mometrazone monohydrate parana , prednisolone, prednisone, thixocortol, thixocortol pivalate, triamcinolone, triamcinolone acetonide, triamcinolone alcohol and their respective pharmaceutically acceptable derivatives. You can use a combination of steroids, for example, a combination of two or more steroids listed in this paragraph;

(x) targeted therapies, eg PDKd inhibitors, eg idelalisib and perifosine; or compounds that are checkpoint inhibitors, including anti-PD-1, anti-PD-L1 and anti-CTLA4 molecules such as nivolumab, pembrolizumab, pidilizumab, atezolizumab, durvalumab and avelumab.

One or more other active ingredients may also be an antibiotic.

Throughout the description and claims of this specification, the word contain and its variations means including, but not limited to, and it is not intended to (and does not exclude) other groups, additives, components, integers, or steps. Throughout the specification and claims of this specification, the singular encompasses the plural unless the context requires otherwise. In particular, when the indefinite article is used, the description is to be understood as referring to plurality as well as singularity, unless the context otherwise requires.

Features, integers, characteristics, compounds, chemical moieties, or groups described in connection with a particular aspect, embodiment, or example of the present invention are to be understood as applicable to any other aspect, embodiment, or example described herein, unless they are incompatible with it. All of the features described in the present invention (including any appended claims, abstract and drawings) and/or all of the steps of any method so described may be combined in any combination, except combinations where at least some of these features/ or stages are mutually exclusive. The present invention is not limited to the details of any of the above embodiments. The present invention extends to any new or any new combination of features described in the present invention (including any accompanying claims, abstract and drawings), or to any new or any new combination of steps of any method so described.

The attention of the reader is directed to all articles and documents that are filed simultaneously with or precede this specification in connection with this application and which are open to public review of this specification, and the contents of all these articles and documents are incorporated into the present invention by reference.

Examples

Throughout the description, these abbreviations have the following meanings.__________

AcCN acetonitrile MS Mass spectrometry aq Water NMR Nuclear magnetic resonance DCM dichloromethane Np 1 - naphthyl DMSO dimethyl sulfoxide rt Room temperature EC so Half maximum effective concentration TEA triethylamine eq equivalents TEAB Bicarbonate buffer triethylammonium HPLC Liquid chromatography TER triethyl phosphate

- 20 042712

high pressure h Hours retention time 1C ₅₀ Half maximum inhibitory concentration

General method for the preparation of diphosphate phosphoramidates.

Dry nucleoside A (~1.0 mmol, 1.0 eq.) was dissolved in 5 ml of triethyl phosphate (TEP) and stirred for 5 min at room temperature. The reaction mixture was cooled to 4°C and dry 1,8-bis-(dimethylamino)naphthalene (Proton Sponge®, 1.5 eq.) was added, followed by dropwise addition of POCl ₃ (1.3 eq.). The resulting suspension was stirred at 4° C. for 2-3 hours. After this time, cold 0.5 M aq. triethylammonium bicarbonate buffer (TEAB) (pH 7.5), and the resulting mixture was stirred for 30 minutes, and then warmed to room temperature and stirred for an additional 1 h. TEP was extracted with tert-butyl methyl ether (4x50 ml), and aqueous the solution was evaporated and dried under high vacuum overnight to give the corresponding triethylammonium salt of nucleoside 5'-monophosphate B as a glassy colorless oil or white powder, which was used in the next step without further purification. The formation of nucleoside 5'-monophosphate was confirmed by mass spectrometry in the negative ion detection mode.

A 0.5 M TEAB solution was prepared by bubbling CO ₂ through a 0.5 M solution of triethylamine (TEA) in water at 0-4°C for 30-45 min (pH approximately 7.4-7.6 unless otherwise noted) .

Preparation of the triethylammonium salt of nucleoside 5'-monophosphate in the first step was carried out following the modified reference: El-Tayeb A. et al., J. Med. Chem. 2006, 49(24), 7076-7087.

A mixture of triethylammonium salt of nucleoside 5'-monophosphate B (1 mmol) in 1:1 1,4dioxane/acetonitrile (20 ml) was stirred at room temperature (rt) under argon and the appropriate phosphorochloridate C (1.0 eq. ) dissolved in anhydrous acetonitrile. The resulting reaction mixture was stirred for 18 hours at room temperature. The crude mixture was evaporated under reduced pressure and the residue was dissolved in DCM and loaded onto silica gel deactivated with TEA (3%) in DCM/MeOH (92/5%). Purification of crude material (obtained for dry loading) of an automated Biotage Isolera One on a SNAP Ultra 50 g cartridge pretreated with 3CV TEA (3%) in DCM/MeOH (92/5%) with MeOH gradient in DCM (2-20%, flow 50 ml/min) gave the desired compounds, which were further purified on Biotage Isolera One (C18 SNAP Ultra 30 g cartridge with a gradient of 0.1 M TEAB in AcCN (acetonitrile) from 100 to 0% as eluent over 60 min, flow 15 ml/min) or HPLC on a semi-preparative column (Varian Pursuit XRs 5C18, 150x21.22 mm) applying a gradient of 0.1 M TEAB in AcCN 90/10-0/100 over 30 min.

Example 1 Triethylammonium salt of 2-chloro-9-(2'-deoxy-2'-fluoro-β-D-α-rabinofuranosyl)adenine-5'O-[phenyl(benzoxy-1-alaninyl)]diphosphate.

Prepared according to the general method from TEA salt of 2-chloro-9-(2'-deoxy-2'-fluoro-β-Darabinofuranosyl)adenine-5'-monophosphate (0.58 g, 0.98 mmol), phenyl(benzo- L-alaninyl)phosphorochloridate (0.35 g, 0.98 mmol) as a white foam (0.04 g, 6%).

³¹ R NMR (202 MHz, MeOD): δ _P -7.73 (d, J=21.0 Hz), -7.93 (d, J=21.2 Hz), -11.96 (d, J =18.4 Hz), -12.06 (d, J=20.8 Hz).

1 H NMR (500 MHz, MeOD): δ _H 8.29, 8.28 (2x0.5H, H-8), 7.33-7.24 (9H, m, H-Ar), 7.18-7 .14 (1H, m, HAr), 6.44, 6.41 (2x0.5H, 2xd, J=3.5 Hz, H-1'), 5.20-5.19 (0.5H, m , H-2'), 5.11 (2H, conditional s, OCH ₂ Ph), 5.10-5.08 (0.5H, m, H-2'), 4.58-4.54 (1H , m, H-3'), 4.26-4.24 (2H, m, 2xH-5'), 4.16-4.13 (2H, m, H-4', NHCHCH3), 3.08 (6H, q, J=7.0 Hz, 3xCH ₂ CH ₃ ), 1.39, 1.35 (3H, 2xd, J=7.0 Hz, NHCHCH3), 1.26 (9H, t, J= 7.0 Hz, 3xCH ₂ CH ₃ ). MS (ES) negative m/z mode. 699 ([M-H] ^- ), exact mass: C ₂₅ H ₂₈ ClFN ₆ O ₁₀ P ₂ required 700.93, found 699.20 ([M-H] ^- ). Reverse phase HPLC eluting with 0.1 M TEAB/AcCN 90/100/100 for 30 min, F=1 ml/min, λ=254 nm, fR=14.05 min.

Example 2 Triethylammonium salt of 2-chloro-9-(2'-deoxy-2'-fluoro-β-D-arabinofuranosyl)adenine-5'O-[phenyl(ethoxy-L-alaninyl)]diphosphate.

Prepared according to the general method from TEA salt of 2-chloro-9-(2'-deoxy-2'-fluoro-β-Darabinofuranosyl)adenine-5'-monophosphate (0.58 g, 0.98 mmol), phenyl(ethoxy- L-alaninyl)phosphorochloridate (0.28 g, 0.98 mmol) as a white solid residue (0.07 g, 10%).

³¹ R NMR (202 MHz, MeOD): δρ -7.72 (d, J=20.2 Hz), -7.89 (d, J=20.2 Hz), -11.94 (d, J= 18.2 Hz), -12.10 (d, J=18.2 Hz).

1H NMR (500 MHz, MeOD): δ _H 8.29 (1H, s, H-8), 7.34-7.27 (4H, m, H-Ar), 7.17-7.15 (1H , m, H-Ar), 6.45, 6.42 (2x0.5H, 2x conditional t, J=1.5 Hz, H-1'), 5.24, 5.10 (2x0.5H, 2x conventional t, J=1.5 Hz, H-2'), 4.59, 4.55 (2x0.5H, 2x conventional t, J=2.0 Hz, H-3'), 4.26-4, 24 (2H, m, OCH2CH3), 4.17-4.07 (4H, m, H-5', NHCHCH3, H-4'), 3.05 (6H, q, J=7.0 Hz, 3XCH2CH3 ), 1.39, 1.34 (2x1.5H, 2xd, J=7.0 NHCHCH3), 1.26-1.20 (12H, m, OCH2CH3, 3xCI2CI3). MS (ES) negative mode m/z: 637 ([M-H] ^- ), exact mass: C21H26CIFN6O10P2 required 638.86, found 637.12 ([M-H] ^- ). Reverse phase HPLC, eluting with H ₂ O/AcCN 90/10-0/100 for 30 min, F=1 ml/min, X=254 nm, two peaks for two diastereoisomers with tR=14.20 min.

Example 3 Triethylammonium salt of 2-chloro-9-(2'-deoxy-2'-fluoro-β-D-arabinofuranosyl)adenine-5'O-[phenyl(benzoxy-L-glycinyl)]diphosphate.

Received according to the general method from TEA salt of 2-chloro-9-(2'-deoxy-2'-fluoro-β-Darabinofuranosyl)adenine-5'-monophosphate (0.58 g, 0.98 mmol), phenyl (benzo- L-glycinyl)phosphorochloridate (0.335 g, 0.98 mmol) as a white solid (0.10 g, 13%).

³¹ P NMR (202 MHz, MeOD): δρ -6.70 (d, J=20.2 Hz), -11.86 (d, J=22.2 Hz).

1H NMR (500 MHz, MeOD): δ _H 8.30, 8.29 (2x0.5H, 2xd, J=2.0 Hz, H-8), 7.36-7.28 (9H, m, H -Ar), 7.187.14 (1H, m, H-Ar), 6.44, 6.41 (2x0.5H, 2xd, J=4.5 Hz, H-1'), 5.20-5, 18 (0.5H, m, H-2'), 5.16 (2H, conditional s, OCH2Ph), 5.09-5.07 (0.5H, m, H-2'), 4.61- 4.55 (1H, m, H-3'), 4.29-4.25 (2H, m, 2x H-5'), 4.17-4.13 (1H, m, H4'), 3 .96-3.84 (2H, m, NHCH2), 2.89 (6H, q, J=7.0 Hz, 3xCH ₂ CH ₃ ), 1.19 (9H, t, J=7.0 Hz, 3xCH ₂ CH ₃ ). MS (ES) negative mode m/z: 685 ([M-H] ^- ), exact mass: C25H26ClFN6O10P2 required 686, found 685.119 ([M-H] ^- ). Reverse phase HPLC, eluting with 0.1 M TEAB/AcCN 90/10-0/100 for 30 min, F=1 ml/min, λ=254 nm, tR=13.6 min.

Example 4 Triethylammonium salt of 5-fluoro-2'-deoxyuridine-5'-O-[1-naphthyl(benzoxy-1alaninyl)]diphosphate.

CW ^F V%

9 shAe ^ο \u003d Ρ-°-Ρ-° ι

NH O y" MM M (4)

Received according to the general method from TEA salt of 5-fluoro-2'-deoxyuridine-5'-monophosphate (1.07 g, 2.03 mmol), 1-naphthyl (benzoxy-L-alaninyl) phosphorochloridate (0.82 g, 2 .03 mmol) as a white solid residue (0.16 g, 10%).

³¹ R NMR (202 MHz, MeOD): δρ-7.43 (d, J=22.2 Hz), -7.79 (d, J=22.2 Hz), -11.89 (d, J= 20.2 Hz), -12.02 (d, J=20.2 Hz).

1H NMR (500 MHz, MeOD) δH 8.39-8.35 (1H, m, H-Ar), 7.99 (1H, r.m., J=6.0 Hz, H-Ar), 7.877.86 (1H, m, H-Ar), 7.69 (1H, t, J=7.0 Hz, H-Ar), 7.58 (1H, ref. d, J=7.0 Hz, H-Ar) , 7.53-7.51 (2H, m,

- 22 042712

H-Ar, H-6), 7.43-7.36 (1H, m, H-Ar), 7.29-7.26 (5H, m, H-Ar), 6.26 (1H, apparent q, J=6.5 Hz, H-1'), 5.02-4.94 (2H, 2xd, J=13.5, J=12.5 Hz, OCH2Ph), 4.46-4, 43 (1H, m, H-3'), 4.24-4.16 (3H, m, 3H, 2xH-5', NHCHCH3), 4.03 (1H, conditional s, H-4'), 3 .09 (6H, q, J=7.0 Hz, 3XCH2CH3), 2.22-2.19 (1H, m, H-2'), 2.162.4 (1H, m, H-2'), 1 .37 (1.5H, d, J=7.0 Hz, NHCHCH3), 1.29 (1.5H, d, J=7.0 Hz, NHCHCH3), 1.26 (9H, t, J=7 .0 Hz, 3xCH ₂ CH ₃ ). MS (ES) negative mode m/z: 692 ([M-N] ^- ), exact mass: C ₂₉ H ₃₀ FN ₃ O ₁₂ P ₂ required 693, found 692.128 ([M-N] ^- ). Reverse-phase HPLC, eluting with 0.1 M TEAB/AcCN 90/10-0/100 for 30 min, F=1 ml/min, λ=254 nm, tR=4.24 min, 14.34 min.

Example 5 Triethylammonium salt of 8-chloroadenosine-5'-O-[1-naphthyl(benzoxy-1-αlaninyl)]diphosphate.

Prepared according to the general method from TEA 8-chloroadenosine-5'-monophosphate salt (0.46 g, 0.795 mmol), 1-naphthyl(benzoxy-L-alaninyl)phosphorochlorideα (0.32 g, 0.795 mmol) after two Biotage purifications Isolera One (C18 30 g cartridge) and HPLC (C18 semi-preparative column) as a white solid (0.02 g, 4%).

³¹ P NMR (202 MHz, MeOD): 6p -7.58 (d, J=22.2 Hz), -8.0 (d, J=20.2 Hz), -11.85 (d, J= 22.2 Hz), -12.05 (d, J=20.2 Hz).

1H NMR (500 MHz, CD3OD): d 8.36-8.33 (1H, m, H-Ar), 8.14, 8.10 (2x0.5H, 2xs, H-2), 7.86- 7.84 (1H, m, H-Ar), 7.66 (1H, d, J=8.0 Hz, H-Ar), 7.56-7.45 (3H, m, H-Ar), 7.38-7.33 (1H, m, H-Ar), 7.29-7.24 (5H, m, H-Ar), 6.02 (1H, t, J=6.0 Hz, H -1'), 5.35-5.32 (1H, m, H-2'), 5.00, 4.98, 4.97, 4.94 (2H, AB, Jab=12.3 Hz, OCH2Ph), 4.54-4.50(1H, m, H-3'), 4.40-4.33(1H, m, H-5'), 4.26-4.13(3H, m , H-5', H-4', NHCHCH3), 3.06 (6H, q, J=7.0 Hz, 3XCH2CH3), 1.36 (1.5H, d, J=7.0 Hz, NHCHCH3 ), 1.26 (1.5H, d, J=7.0 Hz, NHCHCH3), 1.25 (9H, t, J=7.0 Hz, 3xCH ₂ CH ₃ ). MS (ES) negative mode m/z: 747 ([M-H] ^- ), exact mass: C ₃₀ H ₃₁ ClN ₆ O11P ₂ required 749, found 747.1102 ([M-H] ^- ). Reverse-phase HPLC, eluting with 0.1 M TEAB/AcCN 90/100/100 for 30 min, F=1 ml/min, λ=254 nm, tR=14.32, 14.48 min.

Examples 6 and 7 Triethylammonium salt of 3'-deoxyadenosine-5'-O-[phenyl(benzoxy-Lalaninyl)]diphosphate (6) and triethylammonium salt of 3'-deoxyadenosine-5',2'-bis-O-[phenyl( benzoxy-Lalaninyl)]diphosphate (7).

Received according to the General method from TEA salt of 3'-deoxyadenosine-5'-monophosphate (0.63 g, 1.186 mmol), phenyl (benzoxy-L-alaninyl) phosphorchloridate (0.42 g, 1.186 mmol). Two products were isolated: triethylammonium salt of 3'-deoxyadenosine-5',2'-O-[phenyl(benzoxy-E-alaninyl)]diphosphate (0.05 g, 5%) and 3'-deoxyadenosine-5'-O- [phenyl(benzoxy-E-alaninyl)]diphosphate as a colorless film (0.01 g, 1.3%).

³¹ R NMR (202 MHz, MeOD): δρ -7.76 (d, J=21.0 Hz), -8.03 (d, J=21.8 Hz), -11.70 (d, J= 20.4 Hz), -11.81 (d, J=16.76 Hz).

1H NMR (500 MHz, CD3OD): b _H 8.45 (1H, s, H-8), 8.21 (1H, s, H-2), 7.34-7.23 (9H, m, H -Ar), 7.14-7.11 (1H, m, H-Ar), 6.02 (1H, ref. c, H-1'), 5.10 (2H, AB system, ref. t, JAB=16 .5 Hz, OCH ₂ Ph), 4.664.60 (2H, m, H-2', H-4'), 4.29-4.27 (1H, m, H-5'), 4.19- 4.13 (2H, m, H-5', NHCHCH3), 3.06 (6H, q, J=7.0 Hz, 3xCH _{2 CH3} ), 2.42-2.33 (1H, m, H -3'), 2.11-2.06 (1H, m, H-3'), 1.40 (1.5H, d, J=7.0 Hz, NHCHCH3), 1.35 (1.5H , d, J=7.0 Hz, NHCHCH3), 1.25 (9H, t, J=7.0 Hz, 3XCH2CH3). MS (ES) negative m/z mode. 647 ([M-N] ^- ), exact mass: C26H30N6O10P2 required 648, found 647.154 ([M-N] ^- ). Reverse phase HPLC eluting with 0.1 M TEAB/AcCN 90/10-0/100 for 20 min, F=1 ml/min, λ=254 nm, tR=8.20 min.

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³¹ R NMR (202 MHz, MeOD): δρ -7.78 (d, J=20.4 Hz), -7.84 (d, J=22.08 Hz), -8.01 (d, J= 21.0 Hz), -12.08 (d, J=21.6 Hz), -12.18 (d, J=22.0 Hz), -13.50 (d, J=20.6 Hz) , -13.56 (d, J=20.2 Hz).

1H NMR (500 MHz, CD3OD): _δH 8.20 (0.5H, s, H-8), 8.19 (0.5H, s, H-8), 8.04 (1H, s, H -2), 7.21-7.19 (12H, m, H-Ar), 7.14-6.97 (8H, m, H-Ar), 6.16 (0.5H, s, H- 1'), 6.15 (0.5H, s, H-1'), 5.27 (0.5H, ref. t, J=7.0 Hz, H-2'), 5.15 (0. 5H, conventional t, J=5.0 Hz, H-2'), 5.0-4.94 (4H, m, 2xOC^Ph), 4.48-4.60 (1H, m, H-4 '), 4.13-4.08 (1H, m, H-5'), 4.05-3.95 (3H, m, H-5', 2x NHCHCH3), 3.06 (12H, q, J=7.0 Hz, 3xCH ₂ CH ₃ ), 2.50-2.42 (1H, m, H-3'), 2.27-2.20 (1H, m, H-3'), 1 .26-1.17 (6H, m, 2XNHCHCH3), 1.25 (18H, t, J=7.0 Hz, 3XCH2CH3). MS (ES) negative m/z mode. 1044 ([M-N] ^- ), exact mass: C42H47N7Oi7P4 required 1045, found 1044.232 ([M-N] ^- ). Reverse phase HPLC eluting with 0.1 M TEAB/AcCN 90/10-0/100 for 30 min, F=1 ml/min, λ=254 nm, tR=9.75 min, 10.37 min.

Example 8 Triethylammonium salt of 2'-deoxy-2',2'-difluoro-O-cytidine-5'-O-[phenyl(benzoxy-Lalaninyl)]diphosphate.

Obtained according to the general method from

TEA salts of 2'-deoxy-2',2'-difluoro-O-cytidine 5' monophosphate (0.62 g, 1.13 mmol), phenyl (benzoxy-L-alaninyl) phosphorochloridate (0.40 g, 1.13 mmol) 13 mmol) as a white solid residue (0.05 g, 6%).

³¹ R NMR (202 MHz, MeOD): δ _Ρ -7.65 (d, J=21.0 Hz), -7.87 (d, J=21.0 Hz), -12.12 (d, J =16.9 Hz), -12.21 (d, J=21.4 Hz).

1H NMR (500 MHz, MeOD): δ _H 7.83 (1H, d, J=7.5 Hz, H-6), 7.35-7.25 (9H, m, H-Ar), 7, 16 (1H, ref., J=7.0 Hz, H-Ar), 6.27-6.24 (1H, m, H-1'), 5.96, 5.95 (1H, 2xd, J =5.0 Hz, H-5), 5.13 (2H, AB system, ref. s, OCH ₂ Ph), 4.37-4.25 (3H, m, 2xH-5', H-3') , 4.18-4.12 (1H, m, H-4'), 4.00-3.97 (1H, m, NHCHCH3), 3.20 (6H, q, J=7.0 Hz, 3xCH ₂ CH ₃ ), 1.40, 1.35 (3H, 2xd, J=7.5 Hz, NHCHCH3), 1.31 (9H, t, J=7.5 Hz, 3xCH ₂ CH3). MS (ES) negative mode m/z: 659 ([M-H] ^- ), exact mass: C ₂₅ H ₂₈ F ₂ N ₄ OnP ₄ required 660, found 659 ([M-H] ^- ). Reverse phase HPLC, eluting with 0.1 M TEAB/AcCN 90/10-0/100 for 30 min, F=1 ml/min, λ=254 nm, tR=2.55 min, 12.85 min.

Example 9 Triethylammonium salt of 2-chloro-9-(2'-deoxy-2'-fluoro-β-D-arabinofuranosyl)adenine-5'O-[phenyl(neopentoxy-E-alaninyl)]diphosphate.

Received according to the General method from TEA salt 2-chloro-9-(2'-deoxy-2'-fluoro-β-Darabinofuranosyl)adenine-5'-monophosphate (0.77 g, 1.32 mmol), phenyl(neopentoxy- L-alaninyl)phosphorchloridate (0.43 g, 1.32 mmol) and TEA (0.53 ml, 3.81 mmol) as a white solid (0.065 g, 6%).

³¹ R NMR (202 MHz, CD3OD): δp -7.67 (d, J=21.0 Hz), -7.93 (d, J=21.0 Hz), -11.93 (d, J= 21.21 Hz), -12.04 (d, J=21.21 Hz).

1H NMR (500 MHz, CD3OD): δH 8.18 (0.5H, s, H-8), 8.17 (0.5H, s, H-8), 7.22-7.15 (4H, m, H-Ar), 7.127.09 (1H, m, H-Ar), 6.33 (0.5H, t, J=1.5 Hz, H-1'), 6.30 (0.5H , t, J=1.5 Hz, H-1'), 5.08 (0.5N, conditional t, J=1.5 Hz, H-2'), 4.96 (0.5N, conditional t , J=1.5 Hz, H-2'), 4.46 (0.5N, ref. t, J=2.0 Hz, H-3'), 4.43 (0.5N, ref. t, J =2.0 Hz, H-3'), 4.15-4.11 (2H, m, H-5'), 4.05-3.99 (2H, m, NHCHCH3, H-4'), 3.72, 3.69, 3.63, 3.61 (2H, AB system, J=10.50 Hz, OCH ₂ C(CH ₃ ) ₃ ), 3.05 (6H, q, J=7, 0 Hz, N(CH ₂ CH ₃ ) ₃ ), 1.31, 1.25 (2x1.5H, 2xd,

- 24 042712

J=7.0 NHCHCH3), 1.31 (9H, t, J=7.5 Hz, N(CH ₂ CH ₃ ) ₃ ), 0.80 (9H, s, OCH ₂ C(CH ₃ ) ₃ ) .

¹³ C NMR (125 MHz, CD3OD): δc 175.10 (d, ³ Jc-p=6.4 Hz, C=O, ether), 173.56 (d, ³ Jc-p=6.4 Hz, C=O, ether), 156.72 (C-6), 154.16 (C-2), 153.42 (d, ² Jc-p=3.8 Hz, C-Ar), 150.90 ( e, ² Jc-p=5.8 Hz, С-Ar), 150.26 (С-4), 142.68 (С-8), 129.26, 128.73, 124.65, 124.60 , 122.40 (CH-Ar), 120.38, 120.15 (2xd, ³ Jc-p=4.6 Hz, CH-Ar), 117.15 (C-5), 94.99 (d, ¹ Jc-f=191.4 Hz, C-2'), 82.92 (d, ³ Jc-f=8.25 Hz, C-4'), 82.78 (d, ² Jc-f=8 .5 Hz, C-1'), 73.96, 73.66 (OCH2C(CH3)3), 73.61 (d, ² Jc-f=24.5 Hz, C-3'), 65.05 (C-5'), 50.59, 50.29 (NHCHCH3), 46.26 (N(CH2CH3)3), 30.91 (OCH2C(CH3)3), 25.36 (OCH2C(CH3)3) , 19.75, 19.63 (2xd, ³ Jc-p=5.4 Hz, NHCHCH3), 7.84 (N(CH ₂ CH ₃ ) ₃ ). C ₂₄ H ₃₂ ClFN ₆ O ₁₀ P ₂ required mass m/z 680.94; found MS (ES ^- ) m/z: 679.19 ([MH] ^- ).

Reverse-phase HPLC, eluting with 0.1 M TEAB/AcCN 90/10-0/100 for 30 min, F=1 ml/min, λ=254 nm, one peak for two diastereoisomers with tR=15.83 min.

Example 10 2-Chloro-9-(2'-deoxy-2'-fluoro-β-D-arabinofuranosyl)adenine 5'-O-[phenyl(benzoxydimethylglycinyl)]diphosphate triethylammonium salt.

Prepared according to the general method from TEA salt of 2-chloro-9-(2'-deoxy-2'-fluoro-β-Darabinofuranosyl)adenine-5'-monophosphate (0.96 g, 1.64 mmol), phenyl(benzoxydimethylglycinyl) phosphorochloridate (0.60 g, 1.64 mmol) and TEA (0.66 ml, 4.75 mmol) as a white solid (0.085 g, 6%).

³¹ R NMR (202 MHz, CD3OD): δρ -9.79 (d, J=22.6 Hz), -9.82 (d, J=22.6 Hz), -12.17 (d, J= 21.20 Hz), -12.20 (d, J=21.0 Hz).

1H NMR (500 MHz, CD3OD): _δH 8.29 (0.5H, s, H-8), 8.27 (0.5H, s, H-8), 7.36-7.26 (9H , m, H-Ar), 7.167.13 (1H, m, H-Ar), 6.43 (0.5H, d, J=1.5 Hz, H-1'), 6.39 (0. 5H, d, 4=1.5 Hz, H-1'), 5.20-5.17 (0.5H, m, H-2'), 5.15-5.13 (2H, m, OCH2Ph ), 5.11-5.07(0.5H, m, H-2'), 4.60-4.52(1H, m, H-3'), 4.26-4.23(2H, m, H-5'), 4.154.11 (1H, m, H-4'), 3.05 (6H, q, J=7.0 Hz, N(CH ₂ CH ₃ ) ₃ ), 1.54 (6H, s, NHC(CH ₃ ) ₂ ), 1.31 (9H, t, J=7.5 Hz, N(CH2CH3)3).

¹³ C NMR (125 MHz, CD3OD): δ. 174.96 (d, ³ Jc-p=6.6 Hz, C=O, ether), 156.69 (C-6), 154.13 (C-2), 151.06 (d, ² Jc- p=7.3 Hz, C-Ar), 150.22 (C-4), 140.21 (C-8), 136.04 (C-Ar), 129.15. 128.12. 127.76, 127.68, 124.48, 120.47, 120.43 (CH-Ar), 117.12 (C-5), 95.49 (d, 14c-F=192.5 Hz, C -2'), 82.84 (d, ³ Jc-f=3.6 Hz, C-4'), 82.68 (d, ² Jc-f=11.5 Hz, C-1'), 73 .59 (d, ² Jc-f=11.5 Hz, C-3'), 73.41 (d, ² J _cf =10.5 Hz, C-3'), 66.74 (OCH ₂ Ph) , 64.90 (C-5'), 48.47 (NHC(CH3) ₂ ), 46.35 (N(CH ₂ CH3)3), 26.14, 26.12 (NHC(CH3) ₂ , 9 _.85 C ₂₂ H30ClFN6O10P ₂ required mass m/z 714.96; found MS (ES) m/z: 713.13 ([MH] ^- ).

Reverse-phase HPLC, eluting with 0.1 M TEAB/AcCN 90/10-0/100 for 30 min, F=1 ml/min, λ=254 nm, one peak for two diastereoisomers with tR=14.51 min.

Example 11 Triethylammonium salt of 2-chloro-9-(2'-deoxy-2'-fluoro-β-D-arabinofuranosyl)adenine 5'-O-[1-naphthyl(benzoxydimethylglycinyl)]diphosphate.

Oh (11)

Prepared according to the general method from TEA salt of 2-chloro-9-(2'-deoxy-2'-fluoro-β-Darabinofuranosyl)adenine-5'-monophosphate (0.96 g, 1.64 mmol), 1-naphthyl( benzoxydimethylglycinyl)phosphorochloridate (0.68 g, 1.64 mmol) and TEA (0.66 ml, 4.75 mmol) as a white solid, which was further purified by preparative HPLC (0.14 g, 10%).

³¹ R NMR (202 MHz, CD3OD): δ _Ρ -9.79 (d, J=22.6 Hz), -9.82 (d, J=22.6 Hz), -12.17 (d, J =21.20 Hz), -12.20 (d, J=21.0 Hz).

1H NMR (500 MHz, CD3OD): δn 8.38-8.34 (2H, m, H-Ar), 8.24 (0.5H, d, ⁵ Jh-f=2.0 Hz, H-8 ), 8.22 (0.5H, d, ⁵ J _h _ _f =2.0 Hz, H-8), 7.85-7.83 (2H, m, H-Ar), 7.65-7 .60 (3H, m, H-Ar), 7.48-7.44 (3H, m, H-Ar), 7.38-7.29 (2H, m, H-Ar), 6.39 ( 0.5H, t, J=1.5Hz, H-1'), 6.36(0.5H, t, J=1.5Hz, H-1'), 5.17-5.15( 0.5H, m, H-2'), 5.14-5.07 (2H, m, OCH ₂ Ph), 5.05-5.02 (0.5H, m, H-2'), 4 .57-4.50(1H, m, H-3'), 4.27-4.24(2H, m, H-5'), 4.15-4.10(1H, m, H-4 '), 3.05 (6H, q, J=7.0 Hz, N(CH ₂ CH ₃ ) ₃ ), 1.90 (6H, s, NHC(CH ₃ ) ₂ ), 1.31 (9H, t, J=7.5 Hz, N(CH ₂ CH ₃ ) ₃ ).

¹³ C NMR (125 MHz, CD3OD): δc 176.53, 174.95 (2xd, ³ Jc-p=6.7 Hz, C=O, ether), 156.64 (C-6), 154.08 (C-2), 150.16, 149.36 (2xd, ³ Jc-p=7.0 Hz, C-Ar), 146.91, 146.85 (C-4), 142.89 (C- 8), 136.23, 135.99, 134.79 (СAr), 128.07, 127.70, 127.65, 127.61, 127.16, 126.99, 126.18, 125.83, 125.59, 125.43, 125.06, 124.76, 124.20,

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122.57, 122.25, 122.20, 121.44 (CH-Ar), 117.07 (C-5), 115.9 (d, ⁴ JC-P=3.2 Hz, CH-Ar) , 113.15 (d, ⁴ JC-P=3.2 Hz,

CH-Ar), 94.98, 94.95 (2xd, ¹ JC-F=191.5 Hz, C-2'), 82.80 (d, ³ JC-F=3.8 Hz C-4' ), 82.68 (d, ² J _CF =24.5 Hz C-1'),

73.57, 73.45 (2xd, ² Jc-f=24.5 Hz, C-3'), 66.69, 66.37 (OCH ₂ Ph), 64.94 (C-5'), 56.95, 55.87 (NHC(CH3)2), 46.28 (N(CH ₂ CH ₃ ) ₃ ), 26.10 (d, 3J _CP =4.1 Hz, NHC(CH ₃ )2) , 7.86 (N(CH ₂ CH ₃ ) ₃ ).

C ₃₁ H ₃₂ ClFN ₆ O ₁₀ P ₂ required mass m/z 764.13; found MS (ES ^- ) m/z: 763.15 ([M-H] ^- ).

Reverse phase HPLC, eluting with 0.1 M TEAB/AcCN 90/10-0/100 for 30 min, F=1 ml/min, λ=254 nm, one peak for two diastereoisomers with tR=14.85 min.

Example 12 Triethylammonium salt of 2-chloro-9-(2'-deoxy-2'-fluoro-β-D-arabinofuranosyl)adenine 5'-O-[phenyl(cycohexoxy-L-valinyl)]diphosphate.

Prepared according to the general method from TEA salt of 2-chloro-9-(2'-deoxy-2'-fluoro-β-Darabinofuranosyl)adenine-5'-monophosphate (0.96 g, 1.64 mmol), phenyl(cyclohexoxy- L-valinyl)phosphorochloridate (0.61 g, 1.64 mmol) and TEA (0.66 ml, 4.75 mmol) as a white solid (0.19 g, 14%).

³¹ P NMR (202 MHz, CD3OD): δ _Ρ -6.74 (d, J=21.2 Hz), -6.78 (d, J=21.0 Hz), -12.0 (d, J =21.4 Hz).

1H NMR (500 MHz, CD3OD): 5n 8.18 (0.5H, d, ⁵ Jh-f=1.0 Hz, H-8), 8.17 (0.5H, d, ⁵ Jh-f= 1.0 Hz, H-8), 7.227.15 (4H, m, H-Ar), 7.01-6.98 (1H, m, H-Ar), 6.34 (0.5H, t, J=1.5 Hz, H-1'), 6.30(0.5H, t, J=1.5 Hz, H-1'), 5.085.06(0.5H, m, H-2' ), 4.97-4.96 (0.5H, m, H-2'), 4.62-4.54 (1H, m, OCH), 4.46 (0.5H, t, J= 2.0 Hz, H-3'), 4.43 (0.5H, conventional t, J=2.0 Hz, H-3'), 4.15-4.12 (2H, m, H-5 '), 4.06-4.02 (1H, m, H-4'), 3.68 (0.5H, conditional dd, J=10.0 Hz, J=5.5 Hz, NHCHCH(CH ₃ ) ₂ ), 3.64 (0.5H, conditional dd, J=10.0 Hz, J=5.5 Hz, NHCHCH(CH ₃ ) ₂ ), 3.05 (6H, q, J=7.0 Hz, N(CH ₂ CH ₃ ) ₃ ), 1.91-1.80 (1H, m, NHCHCH(CH ₃ ) ₂ ), 1.67-1.58 (5H, m, CH2), 1.39 -1.37 (1H, m, CH2), 1.36-1.31 (3H, m, CH2), 1.31 (9H, t, J=7.5 Hz, N(CH ₂ CH ₃ ) ₃ ), 0.86-0.78 (6H, m, NHCHCH(CH3)2).

¹³ C NMR (125 MHz, CD3OD): OC 171.98, 171.95 (2xd, ³ Jc-p=4.5 Hz, C=O, ether), 156.69 (C-6), 154.14 (C-2), 151.02 (d, ² Jc-p=3.8 Hz, C-Ar), 150.23 (C-4), 142.25 (C-8), 129.16, 128 .60, 124.56, 124.47, 120.44 (CHAr), 117.13 (C-5), 94.96 (d, ^=191.4 Hz, C-2'), 82.90 ( e, ³ Jc-f=3.8 Hz C-4'), 82.81 (e, ² Jc-f=32.5 Hz C-1'), 73.59, (e, ² Jc-f= 24.5 Hz, С-3*), 73.42 (d, ² Jc-f=16.8 Hz, С-3*), 62.88 (d, ² Jc-p=7.5 Hz, С -C-5'), 60.45, 60.20 (NHCHCH(CH3)2), 46.42 (N(CH ₂ CH ₃ ) ₃ ), 32.56, 32.09 (NHCHCH(CH3)2) , 31.14 (CH ₂ ), 25.00 (CH ₂ ), 23.25 (CH 2 ), 17.79, 16.76 (NHCHCHCH 3 ), 7.82 (N(CH 2 CH 3 ) 3 ).

C ₂₇ H ₃₆ ClFN ₆ O ₁₀ P ₂ required mass m/z 720.16; found MS (ES ^- ) m/z: 719.17 ([MH] ^- ).

Reverse-phase HPLC, eluting with 0.1 M TEAB/AcCN 90/10-0/100 for 30 min, F=1 ml/min, λ=254 nm, one peak for two diastereoisomers with t _R =15.73 min .

Example 13 Triethylammonium salt of 2-chloro-9-(2'-deoxy-2'-fluoro-β-D-arabinofuranosyl)adenine 5'-O-[1-naphthyl(neopentoxy-L-alaninyl)]diphosphate.

Prepared according to the general method from TEA salt 2-chloro-9-(2'-deoxy-2'-fluoro-β-Darabinofuranosyl)adenine-5'-monophosphate (0.57 g, 0.97 mmol), 1-naphthyl( neopentoxy-Lalaninyl)phosphorochloridate (0.37 g, 0.97 mmol) and TEA (0.39 ml, 2.86 mmol) as a white solid (0.13 g, 16%).

³¹ R NMR (202 MHz, CD3OD): δρ -7.41 (d, J=23.5 Hz), -7.82 (d, J=22.5 Hz), -11.82 (d, J= 22.69 Hz), -11.95 (d, J=21.80 Hz).

¹ H NMR (500 MHz, CD3OD): δ _H 8.36-8.25 (2H, m, H-Ar), 7.85-7.79 (2H, m, H-Ar), 7.68- 7.65 (2H, m, H-8), 7.58-7.36 (2H, m, H-Ar), (6H, m, H-Ar), 6.4 (1H, dd, J= 16.68 Hz, 4.1 Hz, H-1'), 5.19-5.05 (1H, m, H-2'), 4.58-4.53 (1H, m, H-3' ), 4.29-4.25 (2H, m, H-5'), 4.17-4.14 (1.5H, m, H-4' and NHCHCH3), 4.06-4.01 ( 0.5H, m, NHCHCH3), 3.75-3.66 (2H, m, OCH ₂ C(CH ₃ ) ₃ ), 3.14 (6H, q, J=7.0 Hz, N(CH ₂ CH ₃ ) ₃ ), 1.31-1.26 (12H, m, NHCHCH3, M(CH2CH3)3).

C ₂₈ H ₃ 4ClFN ₆ O ₁₀ P ₂ required mass m/z 730.15; found MS (ES ^- ) m/z: 729.09 ([M-H] ^- ).

Reverse-phase HPLC, eluting with 0.1 M TEAB (pH 7.2)/AcCN 90/10-0/100 for 30 min, F=1 ml/min, λ=254 nm, one peak for two diastereoisomers with t _R = 14.63 min.

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Example 14 Triethylammonium salt of 2-chloro-9-(2'-deoxy-2'-fluoro-β-D-arabinofuranosyl)adenine 5'-O-[phenyl(neopentoxy-D-alaninyl)]diphosphate.

Prepared according to the general method from TEA salt of 2-chloro-9-(2'-deoxy-2'-fluoro-β-Darabinofuranosyl)adenine-5'-monophosphate (0.86 g, 1.48 mmol), phenyl(neopentoxy- Dalaninyl)phosphorochloridate (0.49 g, 1.48 mmol) and TEA (0.59 ml, 4.25 mmol) as a white solid (0.16 g, 14%).

³¹ R NMR (202 MHz, CD3OD): δp -7.68 (d, J=21.0 Hz), -7.91 (d, J=21.0 Hz), -12.04 (d, J= 20.08 Hz), -12.14 (d, J=20.08 Hz).

¹ H NMR (500 MHz, CD3OD): δ _H 8.30 (0.5H, d, J _H- F=2.0 Hz, H-8), 8.29 (0.5 H, d, J _H- F=2.0 Hz, H-8), 7.357.27 (4H, m, H-Ar), 7.18-7.14 (1H, m, H-Ar), 6.45 (0.5H, t, J=3.5 Hz, H-1'), 6.42 (0.5N, t, J=3.5 Hz, H-1'), 5.20 (0.5N, conditional t, J =3.5 Hz, H-2'), 5.09 (0.5H, conventional t, J=3.5 Hz, H-2'), 4.61-4.54 (1H, m, H- 3'), 4.27-4.23 (2H, m, H-5'), 4.17-4.10 (2H, m, NHCHCH3, H-4'), 3.80-3.72 ( 2H, m, OCH ₂ C(CH ₃ ) ₃ ), 3.33 (6H, q, J=7.0 Hz, N(CH2CH3)3), 1.42 (1.5H, dd, J=7, 0 Hz, J=0.5 Hz, NHCHCH3), 1.36 (1.5H, dd, J=7.0 Hz, J=0.5 Hz, NHCHCH3), 1.31 (9H, t, J= 7.5 Hz, N(CH ₂ CH ₃ ) ₃ ), 0.80 (9H, s, OCH ₂ C(CH ₃ ) ₃ ).

¹³ C NMR (125 MHz, CD3OD): δC 173.56, 173.48 (r.m., ³ JC-P=7.6 Hz, C=O, ether), 156.70 (C-6), 154, 13 (C-2), 150.89 (conventional t, ² JC-P=6.6 Hz, C-Ar), 150.24 (C-4), 142.68 (C-8), 129.22 , 128.67, 124.65, 124.60, 122.34 (CH-Ar), 120.38, 120.36 (2xd, ³ Jc-p=4.6 Hz, CH-Ar), 117.14 (C-5), 95.0, 94.99 (2xd, 9^=191.6 Hz, C-2'), 82.76 (d, ^3Jc -f=8.25 Hz, C-4' ), 82.68 (d, ² Jc-f=8.5 Hz, C-1'), 73.95, 73.92 (OCH2C(CH3b), 73.58, 73.50 (2xd, ² Jc- f=24.5 Hz, C-3'), 64.98, 64.86 (2xd, ² JC-P=4.6 Hz, C-5'), 50.36, 50.27 (2xd, ³ JC-P=2.4Hz, NHCHCH3), 46.36(N(CH2CH3)3), 30.92, 30.89(OCH2C(CH3)3), 25.35, 25.33(OCH2C(CH3) 3), 19.75, 19.61 (2xd, ³ JC-P=5.6 Hz, NHCHCH3), 7.82 (N(CH2CH3)3).

C ₂₄ H ₃₂ ClFN ₆ O ₁₀ P ₂ required mass m/z 680.94; found MS (ES-) m/z: 679.19 ([MH] ^- ).

Reverse-phase HPLC, eluting with 0.1 M TEAB/AcCN 90/10-0/100 for 30 min, F=1 ml/min, λ=254 nm, one peak for two diastereoisomers with tR=14.23 min.

Example 15 Triethylammonium salt of 2-chloro-9-(2'-deoxy-2'-fluoro-β-D-arabinofuranosyl)adenine 5'-O-[1-phenyl(isopropoxy-L-alaninyl)]diphosphate.

Prepared according to the general method from TEA salt of 2-chloro-9-(2'-deoxy-2'-fluoro-β-Darabinofuranosyl)adenine-5'-monophosphate (0.86 g, 1.48 mmol), phenyl(isopropoxy- Lalaninyl)phosphorochloridate (0.45 g, 1.48 mmol) and TEA (0.59 ml, 4.25 mmol) as a white solid (0.126 g, 11%).

³¹ Р NMR (202 MHz, CD3OD): δρ -7.62 (d, J=21.0 Hz), -7.85 (d, J=21.0 Hz), -12.14 (r.m., J =20.2 Hz).

¹ H NMR (500 MHz, CD3OD): 8H 8.30 (1H, d, ⁵ JH-F=2.0 Hz, H-8), 7.35-7.27 (4H, m, H-Ar) , 7.18-7.14 (1H, m, H-Ar), 6.45 (0.5H, t, J=3.5 Hz, H-1'), 6.42 (0.5H, t , J=3.5Hz, H-1'), 5.22(0.5H, q, J=3.5Hz, H-2'), 5.13(0.5H, q, J=3 .5 Hz, H-2 ¹ ), 4.96-4.94 (1H, m, OCH(CH ₃ ) ₂ ), 4.61-4.58 (0.5H, m, H-3'), 4.57-4.55(0.5H, m, H-3'), 4.29-4.25(2H, m, H-5'), 4.19-4.15(1H, m, NHCHCH3), 4.07-4.00 (1H, m, H-4'), 3.33 (6H, q, J=7.0 Hz, N(CH2CH3)3), 1.38 (1H, d , J=7.5 Hz, NHCHCH3), 1.33 (2H, dd, J=7.5 Hz, NHCHCH ₃ ), 1.31 (9H, t, J=7.5 Hz, N(CH ₂ CH ₃ ) ₃ ), 1.20 (6H, conventional t, J=6.0 Hz, OCH(CH ₃ ) ₂ ).

¹³ C NMR (125 MHz, CD3OD): δ, 173.10 (d, ³ Jc-p=7.1 Hz, C=O, ether), 173.02 (C=O, ether), 156.73 ( C-6), 154.16 (C-2), 150.92, 150.86 (2xd, ² Jc-p=5.2 Hz, C-Ar), 150.24 (C-4), 142, 55 (C-8), 129.26, 124.64, 124.62 (CH-Ar), 120.34 (d, ³ JC-P=4.5 Hz, CH-Ar), 117.16 (C -5), 95.00 (d, ¹ JC-F=191.5 Hz, C-2'), 82.90 (d, ³ JC-F=6.5 Hz, C-4'), 82, 80, 82.76 (2xd, ² Jc-f=10.5 Hz, C-1'), 73.60, 73.59 (2xd, ² Jc-f=24.5 Hz, C-3'), 68.71 (OCH(CH3)2), 65.08 (d, ² Jc-p=4.2 Hz, C-5'), 50.39, 50.34 (2xd, ² Jc-p=1, 8 Hz, NHCHCH3), 46.23 (N(CH2CH3)3), 20.61, 20.54 (OCH(CH3)2), 19.60, 19.47 (2xd, ³ Jc-p=6.0 Hz, NHCHCH3), 7.86 (N(CH2CH3)3).

C ₂₂ H ₂ 8ClFN ₆ O ₁₀ P ₂ required mass m/z 652.10; found MS (ES-) m/z: 651.13 ([M-H]-).

Reverse phase HPLC, eluting with 0.1 M TEAB/AcCN 90/10-0/100 for 30 min, F=1 ml/min, λ=254 nm, one peak for two diastereoisomers with tR=12.32 min.

- 27 042712

Example 16 Triethylammonium salt of 2-chloro-9-(2'-deoxy-2'-fluoro-β-D-arabinofuranosyl)adenine 5'-O-[ 1 -phenyl(methoxy-L-alaninyl)]diphosphate.

Prepared according to the general method from TEA salt of 2-chloro-9-(2'-deoxy-2'-fluoro-β-Darabinofuranosyl)adenine-5'-monophosphate (0.86 g, 1.48 mmol), phenyl(methoxy- L-alaninyl)phosphorochloridate (0.41 g, 1.48 mmol) and TEA (0.59 ml, 4.25 mmol) as a white solid (0.182 g, 17%).

³¹ R NMR (202 MHz, CD3OD): δρ -7.73 (d, J=20.7 Hz), -7.84 (d, J=20.8 Hz), -12.04 (d, J= 21.0 Hz), -12.11 (d, J=20.8 Hz).

1H NMR (500 MHz, CD3OD): δ _H 8.31 (0.5H, d, J _H- F=2.5 Hz, H-8), 8.30 (0.5H, d, J _H- F =2.0 Hz, H-8), 7.357.27 (4H, m, H-Ar), 7.18-7.14 (1H, m, H-Ar), 6.44 (0.5H, dd , Jh-f=16.4 Hz, J=3.9 Hz, H-G), 6.42 (0.5H, dd, Jh-f=16.4 Hz, J=3.7 Hz, H- 1'), 5.20-5.09(1H, m, H-2'), 4.68-4.52(1H, m, H-3'), 4.28-4.24(2H, m, H-5'), 4.184.15 (1H, m, H-4'), 4.12-4.07 (1H, m, NHCHCH3), 3.66 (1.5H, s, OCH3), 3.65 (1.5H, s, OCH3), 3.78 (6H, q, J=7.0 Hz, N(CH ₂ CH ₃ ) ₃ ), 1.37 (1.5H, dd, J= 7.0 Hz, J=0.5 Hz, NHCHCH3), 1.32 (1.5H, dd, J=7.0 Hz, J=0.5 Hz, NHCHCH3), 1.14 (9H, t, J=7.5 Hz, N(CH ₂ CH ₃ ) ₃ ).

C ₂₀ H ₂₄ ClFN6O ₁₀ P ₂ required mass m/z 724.07; found MS (ES ^- ) m/z: 723.01 ([M-H] ^- ).

Reverse-phase HPLC, eluting with 0.1 M TEAB/AcCN 90/10-0/100 for 30 min, F=1 ml/min, λ=254 nm, one peak for two diastereoisomers with tR=12.24 min.

Example 17 Triethylammonium salt of 2-chloro-9-(2'-deoxy-2'-fluoro-β-D-arabinofuranosyl)adenine 5'-O-[1-1-naphthyl(benzoxy-L-alaninyl)]diphosphate.

Prepared according to the general method from TEA salt of 2-chloro-9-(2'-deoxy-2'-fluoro-β-Darabinofuranosyl)adenine-5'-monophosphate (0.86 g, 1.48 mmol), 1-naphthyl( benzoxy-Lalaninyl) phosphorochloridate (0.59 g, 1.48 mmol) and TEA (0.59 ml, 4.25 mmol) as a white solid (0.226 g, 18%).

³¹ R NMR (202 MHz, CD3OD): δρ -7.26 (d, J=20.2 Hz), -7.61 (d, J=20.2 Hz), -12.07 (d, J= 20.40 Hz), -12.27 (d, J=20.40 Hz).

1H NMR (500 MHz, CD3OD): d 8.19-8.17 (2H, m, H-Ar), 8.13 (0.5H, d, Jh-f=2.0 Hz, H-8) , 8.12 (0.5H, d, Jh-f=2.0 Hz, H-8), 7.72-7.68 (2H, m, H-Ar), 7.54-7.52 ( 1H, conditional d, J=9.5 Hz, H-Ar), 7.44-7.40 (4H, m, H-Ar), 7.37-7.35 (1H, m, H-Ar) , 6.28 (0.5H, t, J=4.0Hz, H-1'), 6.25 (0.5H, t, J=4.0Hz, H-1'), 5.06 -5.02 (0.5H, m, H-2'), 4.95-4.93 (0.5H, m, H-2'), 4.89-4. 80 (2H, OCH2Ph), 4.43-4.43 (0.5H, m, H-3'), 4.41-4.40 (0.5H, m, H-3'), 4.15 -4.12 (2H, m, H-5'), 4.09-4.06 (1H, m, NHCHCH3), 4.03-4.01 (1H, m, H-4'), 3, 33 (6H, q, J=7.0 Hz, N(CH2CH3)3), 1.23 (1.5H, dd, J=7.0 Hz, J=0.5 Hz, NHCHCH3), 1.20 (1.5H, dd, J=7.0Hz, J=0.5Hz, NHCHCH3), 1.31 (9H, t, J=7.5Hz, N(CH2CH3)3).

C ₃₀ H ₃₀ ClFN6O ₁₀ P ₂ required mass m/z 750.99; found MS (ES ^- ) m/z: 749.19 ([M-H] ^- ).

Reverse phase HPLC eluting with 0.1 M TEAB/AcCN 90/10-0/100 for 30 min, F=1 ml/min, λ=254 nm, tR=14.69, 14.81 min.

Example 18 2-Chloro-9-(2'-deoxy-2'-fluoro-β-D-arabinofuranosyl)adenine 5'-O-[ 1 -phenyl(hexoxy-L-alaninyl)]diphosphate triethylammonium salt.

Prepared according to the general method from arabinofuranosyl)adenine-5'-monophosphate (0.86

TEA salts of 2-chloro-9-(2'-deoxy-2'-fluoro-β-Dg, 1.48 mmol), 1-phenyl(hexoxy-L- 28 042712 alaninyl) phosphorochloridate (0.51 g, 1, 48 mmol) and TEA (0.59 ml, 4.25 mmol) as a white solid residue (0.235 g, 20%).

³¹ R NMR (202 MHz, CD3OD): δp -7.55 (d, J=23.00 Hz), -7.77 (d, J=19.40 Hz), -12.09 (d, J= 19.40 Hz), 12.19 (d, J=20.10 Hz).

1H NMR (500 MHz, CD3OD): δH 8.18 (0.5H, d, Jh-f=2.0 Hz, H-8), 8.17 (0.5H, d, Jh-f=2, 0 Hz, H-8), 7.237.11 (4H, m, H-Ar), 7.08-7.03 (1H, m, H-Ar), 6.33 (0.5H, dd, J= 4.0, 1.5 Hz, H-1'), 6.29 (0.5H, dd, J=4.0 Hz, J=1.5 Hz, H-1'), 5.08-5 .06 (0.5H, m, H-2'), 4.98-4.96 (0.5H, m, H-2'), 4.47-4.45 (0.5H, m, H -3'), 4.44-4.42 (0.5H, m, H-3'), 4.15-4.11 (2H, m, H-5'), 4.06-4.03 (1H, m, NHCHCH3), 3.97-3.90 (5H, m, H-4', 2xCH2, OCH2(CH2)4CH3), 3.84-3.78 (1H, m, OCH2(CH2) 4CH ₃ ), 3.33 (6H, q, J=7.0 Hz, N(CH2CH3)3, 1.50-1.43 (4H, m, 2xCH ₂ , OCH ₂ (CH ₂ ) ₄ CH ₃ ) , 1.31-1.22 (6H, m, CH ₂ , OCH ₂ (CH ₂ ) ₄ CH ₃ , NHCHCH3), 1.31 (9H, t, J=7.5 Hz, N(CH ₂ CH3) ₃ ), 0.81-0.76 (3H, m, OCH ₂ (CH ₂ ) ₄ CH ₃ ).

C ₂₅ H ₃₄ ClFN ₆ O ₁₀ P ₂ required mass m/z 694.97; found MS (ES ^- ) m/z: 693.14 ([M-H] ^- ).

Reverse phase HPLC eluting with 0.1 M TEAB/AcCN 90/10-0/100 for 30 min, F=1 ml/min, λ=254 nm, tR=15.13 and 15.21 min.

Example 19 2-Chloro-9-(2'-deoxy-2'-fluoro-β-D-arabinofuranosyl)adenine 5'-O-[1-5,6,7,8-tetrahydro-1-naphthyl( benzoxy-L-alaninyl)]diphosphate.

Prepared according to the general method from TEA salt of 2-chloro-9-(2'-deoxy-2'-fluoro-β-Darabinofuranosyl)adenine-5'-monophosphate (0.86 g, 1.48 mmol), 5.6, 7,8-tetrahydro-1-naphthyl(benzoxy-Lalaninyl)phosphorochloridate (0.603 g, 1.48 mmol) and TEA (0.59 ml, 4.25 mmol) as a white solid (0.19 g, 15% ).

³¹ R NMR (202 MHz, CD3OD): δ _Ρ -7.06 (d, J=24.0 Hz), -7.55 (d, J=21.2 Hz), -11.24 (d, J =21.2 Hz), -11.38 (d, J=24.0 Hz).

1H NMR (500 MHz, CD3OD): δ _Η 8.27 (0.5H, d, J _HF = 2.0 Hz, H-8), 8.25 (0.5 H, d, J _HF = 2.0 Hz, H-8), 7.337.30 (3H, m, H-Ar), 7.22-7.18 (2H, m, H-Ar), 7.00-6.93 (2H, m, H -Ar), 6.84 (1H, ref. t, J=6.5 Hz, H-Ar), 6.72 (1H, ref. d, J=7.5 Hz, H-Ar), 6.43 ( 0.5N, dd, J=4.0Hz, J=2.0Hz, H-1'), 6.39 (0.5N, dd, J=4.0Hz, J=2.0Hz, H-1'), 5.19-5.16 (0.5H, m, H-2'), 5.09-5.06 (0.5H, m, H-2'), 5.06- 5.03 (2H, m, OC^Ph), 4.56-4.49 (1H, m, H-3'), 4.27-4.10 (3H, m, H-5', H- 4'), 4.02-3.97 (1H, m, NHCHCH3), 3.33 (6H, q, J=7.0 Hz, N(CH ₂ CH ₃ ) ₃ ), 2.73 (4H, bt, 2xCH2), 1.75-1.72 (4, m, 2xCH2), 1.42 (1.5H, d, J=7.5 Hz, NHCHCH3), 1.38 (1.5H, dd, J=7.0 Hz, NHCHCH3), 1.31 (9H, t, J=7.5 Hz, N(CH2CH3)3).

C ₃₀ H ₃₄ ClFN ₆ O _i0 P ₂ required mass m/z 754.15; found MS (ES ^- ) m/z: 753.23 ([MH] ^- ).

Reverse phase HPLC, eluting with 0.1 M TEAB/AcCN 90/10-0/100 for 30 min, F=1 ml/min, λ=254 nm, tR=15.32 and 15.44 min.

Example 20 Triethylammonium salt of 2-chloro-9-(2'-deoxy-2'-fluoro-β-D-arabinofuranosyl)adenine 5'-O-[1-ethyl(benzoxy-G-alaninyl)]diphosphate.

Received according to the General method from TEA salt of 2-chloro-9-(2'-deoxy-2'-fluoro-β-Darabinofuranosyl)adenine-5'-monophosphate (0.86 g, 1.48 mmol), ethyl (benzo- L-alaninyl)phosphorochloridate (0.45 g, 1.48 mmol) and TEA (0.59 ml, 4.25 mmol) as a white solid (0.218 g, 20%).

³¹ P NMR (202 MHz, CD3OD): δ _P -2.73 (d, J=20.2 Hz), -3.04 (d, J=20.4 Hz), -11.96 (d, J =20.2 Hz), -12.07 (d, J=20.04 Hz).

1H NMR (500 MHz, CD3OD): δ _Η 8.32 (0.5H, d, J _HF = 2.0 Hz, H-8), 8.31 (0.5 H, d, J _HF = 2.0 Hz, H-8), 7.377.27 (5H, m, H-Ar), 6.46 (0.5 H, dd, J=4.0 Hz, J=2.0 Hz, H-1') , 6.43(0.5H, dd, J=4.0Hz, J=2.0Hz, H-1'), 5.245.22(0.5H, m, H-2'), 5.20 -5.14 (2H, m, OC^Ph), 5.13-5. 11 (0.5H, m, H-2'), 4.62-4.60 (0.5H, m, H-3'), 4.594.57 (0.5H, m, H-3'), 4.29-4.26 (2H, m, H-5'), 4.19-4.17 (2H, m, NHCHCH3), 4.12-4.04 (3H, m, H-4', OCH2CH3), 2.86 (6H, q, J=7.0 Hz, N(CH2CH3)3), 1.43 (1.5H, dd, J=7.0 Hz, J=0.5 Hz, NHCHCH3 ), 1.42 (1.5H, dd, J=7.0 Hz, J=0.5 Hz, NHCHCH3), 1.27-1.23 (3H, m, OCH2CH3), 1.19 (9H, t, J=7.5 Hz, N(CH2CH ₃ ) ₃ ).

C ₂₂ H ₂₈ ClFN ₆ O ₁₀ P ₂ required mass m/z 652.89; found MS (ES ^- ) m/z: 651.15 ([M-H] ^- ).

- 29 042712

Reverse-phase HPLC, eluting with 0.1 M TEAB/AcCN 90/10-0/100 for 30 min, F=1 ml/min, λ=254 nm, one peak for two diastereoisomers with tR=14.12 min.

Example 21 Triethylammonium salt of 2-chloro-9-(2'-deoxy-2'-fluoro-β-D-arabinofuranosyl)adenine 5'-O-[1-phenyl(benzoxy-L-leucinyl)]diphosphate.

Prepared according to the general method from TEA salt of 2-chloro-9-(2'-deoxy-2'-fluoro-β-Darabinofuranosyl)adenine-5'-monophosphate (0.86 g, 1.48 mmol), phenyl(benzo- L-leucinyl)phosphorochloridate (0.58 g, 1.48 mmol) and TEA (0.59 ml, 4.25 mmol) as a white solid (0.30 g, 24%).

³¹ R NMR (202 MHz, CD3OD): δp-7.28 (d, J=21.4 Hz), -7.57 (d, J=21.6 Hz), -12.18 (d, J= 21.6 Hz).

1H NMR (500 MHz, CD3OD): 3 _H 8.18 (1H, s, H-8), 7.19-7.10 (8H, m, H-Ar), 7.06-6.99 (2H , m, H-Ar), 6.32 (0.5H, t, J=3.5 Hz, H-1'), 6.29 (0.5H, t, J=3.5 Hz, H- 1'), 5.10-5.08 (0.5H, m, H-2'), 4.97-4.88 (2.5H, m, H-2', OCH ₂ Ph), 4, 46 (0.5H, conventional t, J=3.5 Hz, H-3'), 4.43 (0.5H, conventional t, J=3.5 Hz, H-3'), 4.17- 4.13 (2H, m, H-5'), 4.06-4.04 (1H, m, H-4'), 3.94-3.88 (1H, m, NHCHCH ₂ CH(CH ₃ ) ₂ ), 3.33 (6H, q, J=7.0 Hz, N(CH ₂ CH ₃ ) ₃ ), 1.13 (9H, t, J=7.5 Hz, N(CH ₂ CH ₃ ) ₃ ), 1.43-1.37 (3H, m, NHCHCH ₂ CH(CH ₃ ) ₂ ), 0.75-0.72 (3H, m, NHCHCH2CH(CH3) ₂ ), 0.65-0 .64 (3H, conditional dd, J=14.0 Hz, J=6.0 Hz, NHCHCH ₂ CH(CH ₃ ) ₂ ).

¹³ C NMR (125 MHz, CD3OD): 0C 173.43, 173.22 (2xd, ³ JC-P=3.5 Hz, C=O, ether), 156.78, 156.72 (C-6) , 154.16 (C-2), 150.92 (d, ² Jc-p=7.0 Hz, C-Ar), 150.24 (C-4), 135.84, 84, 135.81 ( C-8), 129.26, 128.74, 128.26, 128.13, 127.95, 127.90, 127.88, 127.83, 124.68, 124.57, 122.30 (CH -Ar), 120.44 (d, ³ JC-P=4.6 Hz, CH-Ar), 120.27 (d, ³ JC-P=5.2 Hz, CH-Ar), 119.98, 119.94 (CH-Ar), 117.17 (C-5), 95.00 (d, 1J _CF =191.6 Hz, C-2'), 82.90 (d, ³ Jc-f=4 ,1 Hz C-4'), 82.74 (d, ² JC-F=11.0 Hz C-1'), 73.63, 73.58 (2xd, ² JC-F=24.5 Hz, C-3'), 66.43, 66.05 (OCH2Ph), 65.10 (d, ² Jc-p=5.5 Hz, C-5'), 53.70 (NHCH), 53.31 ( e, ² JC-P=5.5 Hz, NHCH), 46.21 (N(CH2CH3)3), 43.20, 43.00 (2xd, ³ JC-P=7.0 Hz, NHCHCH2), 24 .13, 24.08 (NHCHCH ₂ CH(CH ₃ ) ₂ ), 21.66, 21.45, 21.07, 20.99 (NHCHCH2CH(CH3)2), 7.83 (N(CH2CH3)3) .

C ₂₉ H3 ₄ ClFN ₆ O ₁₀ P ₂ required mass m/z 742.15; found MS (ES-) m/z: 741.18 ([MH]-).

Reverse-phase HPLC, eluting with 0.1 M TEAB/AcCN 90/10-0/100 for 30 min, F=1 ml/min, λ=254 nm, one peak for two diastereoisomers with tR=14.91 min.

Example 22 Triethylammonium salt of 2-chloro-9-(2'-deoxy-2'-fluoro-β-D-arabinofuranosyl)adenine 5'-O-[1-phenyl(benzoxy-L-phenylalaninyl)]diphosphate.

Received according to the General method arabinofuranosyl)adenine-5'-monophosphate (0.86 from TEA g, 1.48 salts of 2-chloro-9-(2'-deoxy-2'-fluoro-in -D mmol), phenyl (benzo- L-phenylalaninyl)phosphorochloridate (0.63 g, 1.48 mmol) and TEA (0.59 ml, 4.25 mmol) as a white solid (0.185 g, 14%).

³¹ P NMR (202 MHz, CD3OD): δ _P -7.88 (d, J=20.6 Hz), -7.98 (d, J=20.0 Hz), -12.00 (d, J =21.21 Hz).

1H NMR (500 MHz, CD3OD): 8 _H 8.17 (0.5H, d, J _HF = 2.5 Hz, H-8), 8.14 (0.5 H, d, J _HF = 2.5 Hz, H-8), 7.187.08 (10, m, H-Ar), 6.97-6.87 (5H, m, H-Ar), 6.30 (0.5 H, dd, J= 4.0 Hz, J=1.5 Hz, H-1'), 6.27 (0.5H, dd, J=4.0 Hz, J=1.5 Hz, H-1'), 5, 08-5.05 (0.5H, m, H-T), 4.97-4.95 (0.5H, m, H-2'), 4.86-4.77 (4H, m, OCH ₂ Ph, NHCHCH ₂ Ph), 4.46-4.44 (0.5H, m, H-3'), 4.43-4.41 (0.5H, m, H-3'), 4, 25-4.17 (1H, m, NHCHCH ₂ Ph), 4.16-4.12 (2H, m, H-5'), 4.10-4.05 (1H, m, H-4') , 3.33 (6H, q, J=7.0 Hz, N(CH ₂ CH ₃ ) ₃ ), 1.31 (9H, t, J=7.5 Hz, N(CH2CH3)3).

¹³ C NMR (125 MHz, CD3OD): δ( 173.46 (d, ³ JC-P=4.6 Hz, C=O, ether), 172.05 (d, ³ JC-P=4.5 Hz , C=O, ether), 156.68 (C-6), 154.13 (C-2), 153.36 (d, ² JC-P=7.1 Hz, C-Ar), 150.85 , 150.83 (С-4), 150.23 (С-Ar), 136.73, 136.11 (d, ² Jc-p=4.7 Hz, С-Ar), 135.69 (С- 8), 135.52, 135.50 (С-Ar), 129.20 (d, ³ JC-P=3.1 Hz, CH-Ar), 128.74, 128.09, 128.05 (d , ³ Jc-p=2.8 Hz, CH-Ar), 127.90, 127.84, 127.78, 126.44, 126.25, 124.59, 122.40, 120.38, 120, 34, 120.30, 120.26, 120.15, 120.11 (CH-Ar), 117.15 (C-5), 95.01 (d, 1JC-P=191.0 Hz, C-2 '), 95.00 (d, 1JC. F=191.0 Hz, C-2'), 82.84 (d, ³ JC-F=3.8 Hz C-4'), 82.68 (d , ² JC-F=32.5 Hz C-1'), 73.58 (d, ² JC-F=24.5 Hz C-3'), 66.45, 66.44 (OCH ₂ Ph), 66.11 (OCH ₂ Ph), 65.04 (d, ² Jc-p=4.8 Hz, C-5'), 56.44 (NHCHCH ₂ Ph), 56.19

- 30 042712 (NHCHCH2Ph), 46.21 (MCIH'I hh), 40.79, 40.74 (CH2Ph), 9.85 (K(CH2CH3)3).

C ₃₂ H ₃₂ ClFN ₆ O ₁₀ P ₂ required mass m/z 776.13; found MS (ES ^- ) m/z: 775.17 ([MH] ^- ).

Reverse-phase HPLC, eluting with 0.1 M TEAB/AcCN 90/10-0/100 for 30 min, F=1 ml/min, λ=254 nm, one peak for two diastereoisomers with tR=24.28 min.

Example 23 Triethylammonium salt of 2-chloro-9-(2'-deoxy-2'-fluoro-β-D-arabinofuranosyl)adenine 5'-O-[1-phenyl(benzoxy-L-prolinyl)]diphosphate.

Prepared according to the general method from TEA salt of 2-chloro-9-(2'-deoxy-2'-fluoro-β-Oarabinofuranosyl)adenine-5'-monophosphate (0.86 g, 1.48 mmol), phenyl(benzo- Eprolinyl)phosphorchloridate (0.55 g, 1.48 mmol) and TEA (0.59 ml, 4.25 mmol) as a white solid (0.17 g, 14%).

³¹ R NMR (202 MHz, CD3OD): δρ-9.05 (d, J=17.6 Hz), -9.72 (d, J=21.6 Hz), -11.75 (d, J= 17.6 Hz), -12.12 (d, J=21.6 Hz).

1H NMR (500 MHz, CD3OD): δ _Η 8.17 (0.5H, d, J _HF = 2.5 Hz, H-8), 8.16 (0.5 H, d, J _HF = 2.5 Hz, H-8), 7.217.15 (8H, m, H-Ar), 7.11-7.09 (1H, m, H-Ar), 7.04-7.00 (1H, m, H -Ar), 6.32 (0.5H, d, J=4.0 Hz, H-G), 6.31 (0.5H, d, J=4.0 Hz, Y-G), 5, 08-5.06 (0.5H, m, H-2'), 5.01-4.99 (0.5H, m, H-2'), 4.98-4.91 (2H, m, OCH ₂ Ph), 4.484.45 (1H, m, NCH), 4.43-4.41 (0.5H, m, H-3'), 4.34-4.30 (0.5H, m, H-3'), 4.16-4.09 (2H, m, H-5'), 4.05-4.00 (1H, m, H-4'), 3.46-3.38 ( 2H, m, NCH ₂ ), 2.21-2.05 (1H, m, 1xH, NCH2CH2), 1.85-1.76 (3H, m, 1xH, NCH ₂ CH ₂ , 2xH, NCH ₂ CH ₂ CH ₂ ), 3.33 (6H, q, J=7.0 Hz, N(CH ₂ CH ₃ ) ₃ , 1.31 (9H, t, J=7.5 Hz, N(CH ₂ CH ₃ ) ₃ ).

¹³ C NMR (125 MHz, CD3OD): δ( 173.60, 173.31 (C=O, ether), 156.69 (C-6), 154.14 (C-2), 150.78 (d , 2JC-P=3.8 Hz, C-Ar), 150.22 (C-4), 136.37 (C-8), 136.03, 135.96 (C-Ar), 129.29, 129.23, 128.13, 127.80, 127.76, 127.67, 124.57 (CH-Ar), 120.15 (d, ³ Jc-p=5.2 Hz, CH-Ar), 119.96 (d, ³ Jc-p=5.2 Hz, CH-Ar), 117.14 (C-5), 95.02 (d, 1JC-F=191.7 Hz, C-2') , 94.99 (d, 1J _CF =191.7 Hz, C-2'), 82.85 (d, ³ JC-F=5.6 Hz, C-4'), 82.71 (d, 2JC -F=32.5 Hz, C-1'), 73.59 (d, ² Jc-f=24.5 Hz, C-3'), 73.55 (d, ² Jc-f=24.5 Hz, C-3'), 66.32, 66.25 (OCH2Ph), 65.02 (d, ^2Jc -p=4.0 Hz, C-5'), 64.90 (d, 2JC-P =4.0 Hz, C-5'), 60.72 (2J _CP = 7.75 Hz, NCH), 60.35 (2J _CP = 7.75 Hz, NCH), 46.31 (N(CH ₂ CH ₃ ) ₃ ), 30.99 ( ² Jc-p=11.0 Hz, NCH2), 30.89 ( ² Jc-p=11.0 Hz, NCH2), 24.84 ( ³ Jc-p=9 .0 Hz, NCH2CH ₂ ), 24.40 ( ³ JC-P=9.0 Hz, NCHCH2), 7.85 (N(CH2CH3)3).

C ₂₈ H ₃₀ C1FN ₆ Oi ₀ P ₂ required mass m/z 726.97; found MS (ES ^- ) m/z: 725.67 ([MH] ^- ).

Reverse-phase HPLC, eluting with 0.1 M TEAB/AcCN 90/10-0/100 for 30 min, F=1 ml/min, λ=254 nm, one peak for two diastereoisomers with tR=15.39 min.

Example 24 Triethylammonium salt of 2-chloro-9-(2'-deoxy-2'-fluoro-β-D-arabinofuranosyl)adenine 5'-O-[1-phenyl(methoxy-E-methioninyl)]diphosphate.

Prepared according to the general method from TEA salt of 2-chloro-9-(2'-deoxy-2'-fluoro-β-Darabinofuranosyl)adenine-5'-monophosphate (0.86 g, 1.48 mmol), phenyl(methoxy- E-methioninyl)phosphorochloridate (0.49 g, 1.48 mmol) and TEA (0.59 ml, 4.25 mmol) as a white solid (0.25 g, 22%).

³¹ R NMR (202 MHz, CD3OD): δ _ρ -7.41 (d, J=21.33 Hz), -7.69 (d, J=21.6 Hz), -12.29 (d, J =20.6 Hz).

1 H NMR (500 MHz, CD3OD): 5 _H 8.19 (0.5 H, d, J _HF = 2.1 Hz, H-8), 8.18 (0.5 H, d, J _H . _F = 2 .0 Hz, H-8), 7.217.16 (4H, m, H-Ar), 7.09-7.02 (1H, m, H-Ar), 6.32 (0.5H, dd, J _{H. F} =16.6 Hz, J=4.3 Hz, H-1'), 6.31 (0.5H, dd, Jh-f=16.6 Hz, J=4.2 Hz, H- 1'), 5.02 (0.5H, dd Jh-f=51.9 Hz, Jh-f=2.2 Hz, H-2') 5.00 (0.5H, dd, Jh-f= 51.9 Hz, J _{H .H} = 2.2 Hz, H-2'), 4.48-4.43 (1H, m, H-3'), 4.17-4.13 (2H, m , H-5'), 4.08-4.00 (m, 2H, NHCHCH3, H-4'), 3.56, 3.43 (3H, 2s, OCH3), 2.80 (6H, q, J=7.0 Hz, N(CH2CH3)3, 2.42-2.39 (1H, m, CH3SCH2CH2a), 2.28-2.24 (1H, m, CH3SCH2CH26), 1.90, 1.86 (3H, 2xs, SCH3), 1.90-1.71 (2H, m, CH3SCH2CH2a) C22H28CIFN6O10P2S mass required 684.96, found MS (ES ^- ) m/z: 683 ([M-H] ^- ).

Reverse-phase HPLC, eluting with 0.1 M TEAB/AcCN 90/10-0/100 for 30 min, F=1 ml/min, λ=254 nm, one peak for two diastereoisomers with tR=12.28 min.

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Example 25 Triethylammonium salt of 2-chloro-9-(2'-deoxy-2'-fluoro-v-E-arabinofuranosyl)adenine 5'-O-[1-phenyl(ethoxy-L-isoleucinyl)]diphosphate.

Prepared according to the general method from TEA salt of 2-chloro-9-(2'-deoxy-2'-fluoro-β-Darabinofuranosyl)adenine-5'-monophosphate (0.86 g, 1.48 mmol), phenyl(ethoxy- L-isoleucinyl)phosphorochloridate (0.49 g, 1.48 mmol) and TEA (0.59 ml, 4.25 mmol) as a white solid (0.26 g, 23%).

³¹ R NMR (202 MHz, CD3OD): δp-6.84 (d, J=21.5 Hz), -6.99 (d, J=22.3 Hz), -12.00 (d, J= 21.6 Hz).

1 H NMR (500 MHz, CD3OD): 5 _H 8.30, (0.5H, d, J _hf = 2.4 Hz, H-8), 8.29 (0.5 H, d, J _hf = 2, 3 Hz, H-8), 7.35-7.23 (4H, m, H-Ar), 7.19-7.13 (1H, m, H-Ar), 6.44 (0.5H, dd, J _hf =16.7 Hz, J=3.9 Hz, H-1'), 6.43 (0.5H, dd, Jh-f=16.7 Hz, Jh-h=4.2 Hz , H-1'), 5.21-5.08 (1H, m, H-2'), 4.59-4.54 (1H, m, H-3'), 4.28-4.25 (2H, m, OCH2CH3), 4.18-4.07 (3H, m, H-5'a, H-4', NHCHCH3), 3.83 (0.5H, dd, J=9.8 Hz , J=5.7 Hz, H-5'b) 3.82 (0.5H, dd, J=9.8 Hz, J=5.7 Hz, H _b -5'), 3.33 (6H , q, J=7.0 Hz, N(CH ₂ CH ₃ ) ₃ , 1.77-1.68 (1H, m, CHCH3), 1.55-1.42 (1H, m, CHCH _2a ) 1 .15-1.09 (1H, m, CHCH _2b ), 1.27-1.20 (12H, m, N(CH ₂ CH ₃ ) ₃ , OCH ₂ CH ₃ ), 0.91-0.82 ( 6H, CHCH ₃ , CH ₂ CH ₃ ).

C ₂₄ H ₃₂ ClFN ₆ O ₁₀ P ₂ required weight 680.14; found MS (ES ^- ) m/z: 680.18 ([M-H] ^- ).

Reverse-phase HPLC, eluting with 0.1 M TEAB/AcCN 90/10-0/100 for 30 min, F=1 ml/min, λ=254 nm, one peak for two diastereoisomers with t _R =13.57 min .

Example 26 Triethylammonium salt of 2-chloro-9-(2'-deoxy-2'-fluoro-β-D-arabinofuranosyl)adenine 5'-O-[1-naphthyl(benzoxy-E-alaninyl)]phosphorthiol phosphate.

Prepared according to the general method from TEA salt of 2-chloro-9-(2'-deoxy-2'-fluoro-β-Darabinofuranosyl)adenine-5'-monophosphate (0.86 g, 1.48 mmol), 1-naphthyl( benzoxy-Lalaninyl)phosphorthiol chloridate (0.62 g, 1.48 mmol) and TEA (0.59 ml, 4.25 mmol) as a white solid (0.054 g, 4%).

³¹ R NMR (202 MHz, CD3OD): δp 55.05 (d, J=28.5 Hz), 53.71 (d, J=28.5 Hz), -12.24 (br d, J=28 .7 Hz).

1 H NMR (500 MHz, CD3OD): δ _H 8.37-8. 34 (1H, m, H-Ar), 8.28 (0.5H, d, J _HF =2.0 Hz, H-8), 8.26 (0.5H, d, J _hf = 2.0 Hz, H-8), 7.85-7.82 (1H, m, H-Ar), 7.66-7.64 (2H, m, H-Ar), 7.50-7.43 (2H , m, H-Ar), 7.38-7.19 (5H, m, H-Ar), 6.42 (0.5H, t, J=4.0 Hz, H-1'), 6, 39 (0.5H, t, J=4.0 Hz, H-1'), 5.19-5.16 (1H, m, H-2'), 5.11-5.02 (2H, m , OCH2Ph), 4.60-4.57 (0.5H, m, H-3'), 4.56-4.54 (0.5H, m, H-3'), 4.44-4, 28 (3H, m, H-5', NHCHCH3), 4.20-4.17 (1H, m, H-4'), 3.33 (6H, q, J=7.0 Hz, N(CH ₂ CH ₃ ) ₃ ), 1.47-1.41 (3H, m, NHCHCH3), 1.31 (9H, t, J=7.5 Hz, N(CH2CH3)3).

C ₃₀ H ₃₀ ClFN ₆ O ₉ P ₂ S required mass m/z 767.06; found MS (ES ^- ) m/z 766.12 ([M-H] ^- ).

Reverse-phase HPLC, eluting with 0.1 M TEAB/AcCN 90/10-0/100 for 30 min, F=1 ml/min, λ=254 nm, one peak for two diastereoisomers with t _R =16.39 min .

Example 27 In Vitro Cytotoxicity Assays

A subset of the compounds of the present invention was tested for their cytotoxic activity in a variety of solid tumors and hematological malignancies using the following assay.

Analysis of solid tumors and hematological malignancies.

An in vitro viability assay was performed to evaluate the effects of compounds on cell survival in selected cell lines for 72 hours using the CellTiterGlo (CTG, Promega-G7573) assay. Tests were run in duplicate with compound treatment at 9 points, 3.16 fold titration in 96 well plates for ~72 hours. Initial compound concentrations were 198 μM. Cell viability assay was performed using CellTiterGlo in a 96-well plate. Treatment with compounds for 72 h, standard growth conditions, in duplicate. Compounds were dissolved to 40 mM with 100% thawing. Compounds were serially diluted 3.16-fold in thawed DMSO and heated to 37° C. before being dissolved in medium (2+200 µl). After the compounds were dissolved in the medium, the media containing the compounds were heated to 37° C. in an incubator, and then the compounds in the medium were added to the cell plates (50+50 μl) in duplicate. The final concentrations of compounds ranged from 198 μM to 19.8 nM. The solubility of all compounds was checked and recorded again, then the plates were immediately transferred to a CO2 tissue culture incubator and incubated for 3 days. The final concentration of DMSO was 0.5%. The parent nucleoside was tested as a reference in each case as it was an exemplary ProTide. The ProTide class drugs tested were as follows:

HoN

O

HoN

H _? N

The following cell lines were analyzed and shown in table. 1 below.

- 33 042712

Table 1

Cellular Malignant Cellular

malignant tumor line tumor line

Acute T lymphoblastic Adenocarcinoma dairy MOLT-4 MCF-7 leukemia glands

CCRF- Acute lymphoblastic

HL-60 Promyelocytic leukemia

CEM leukemia

non-Hodgkin

RL lymphoma biphenotypic B

MV4-11 myelomoncytic leukemia multiple myeloma

RPMI-8226 Human HepG2

Hepatocellular carcinoma

Chronic

K562 HT29 myelogenous leukemia colon adenocarcinoma

Adenocarcinoma

OVCAR-3 ovarian pancreatic carcinoma

Mia-Pa-Ca-2 glands

Acute myelocytic

KG-1 leukemia

The results of screening are presented in table. 2-4.

In table. 3A is an EC50 greater than 0.2 µm; B is an EC50 greater than 0.2 µm but less than 2 µm; C is EC ₅₀ greater than 2 μm but less than 5 μm; D is EC ₅₀ greater than 5 μm but less than 10 μm; and E is an EC ₅₀ greater than 10 µm. The absence of a record means that the specified compound was not screened against the specified cell line.

In table. 4A is an EC ₅₀ greater than 0.2 µm; B is an EC ₅₀ greater than 0.2 μm but less than 1 μm; C is EC ₅₀ greater than 1 μm but less than 5 μm; D is EC ₅₀ greater than 5 μm but less than 10 μm; and E is an EC ₅₀ greater than 10 µm. Bold letters represent EC ₅₀ less than the EC ₅₀ of the exemplary ProTide A.

In table. 2-4, Ab EC ₅₀ refers to absolute EC ₅₀ and maximum inhibition refers to the percentage inhibition achieved at the highest concentration tested.

table 2

Mia-Pa-Ca-2 HT29 HepG2 OVCAR.3 MCF-7 About S bubbling bubbling bubbling bubbling bubbling To th 1- - b I s 1 O AB (m Maximum C AB (m Max. C AB (m Maximum C AB (m Maximum C AB (m Maximum C Gemcite 0.03 79 0.22 65 28.0 54 >198 44 0.011 53 abin 8 0.07 79 0.48 62 83.13 52 >198 48.5 0.07 52 IN 0.53 82 8.55 62 21.33 57 >198 33.5 2.71 54 FUDR 0.35 90 0.22 82 84.02 61 2.32 66 7.23 73 4 0.11 67 0.25 77 >198 48 2.81 67.5 15.80 66 WITH 1.01 89 1.91 81 70.60 65 27.25 89 8-ST- Aadenosis 0.42 85 1.96 73 - - 1.83 85.7 in 5 0.43 87.6 1.41 76.5 1.69 88 D 1.97 99.7 3.82 94 - - 4.30 99.6 cordyce 84.73 88 32.74 87 - - - - pin 7 3.32 98.90 7.79 79 - - 6 40.89 95.7 24.34 82 - - - - E 7.92 98 10.75 97 - -

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Table 3

ΗΤ29 HepG2 OVCAR3 MCF-7 (D s X X X eu ₅₀ κΜ) small evolution (? eu ₅₀ km) small 1 coating %) eu ₅₀ km) small o/l eu ₅₀ km) small shaping %) -Q S Sa -About SX ⁴ ' M S X l S s >g ω Ο ο 'os th Yu S £ Xs < g S her h to < g S < g S her h s § Clofarabine At 64 C 64 E 48 At 55 1 At 66 E - At 59 2 At 62 E 48 At 54 3 At 61 C 53 E 49 C 59 Α C 94 - - D 99.6

- 35 042712

ABOUT ABOUT ω ABOUT O th S I X sch E To I S Sat Sat Sat Sat oZ Sat co CQ CQ CQ d CQ ABOUT O ABOUT ABOUT ABOUT about S ? i km) Ibir ΐ I ? i Oh' I 2nd and I km) Ibir I 2 and 2 i 2 b 2 b 2 fc 2 b O I I Oh me ⁴ - ^zt I X o I x° o S'o ° ^s o I ° I th i G? υ L And I d) U V w 3 o g w i th i I g -Ω l <! St. < § § 5 ί 5 2 2 2 2 2 2 I I S I O O O And Sat 03 Sat Sat Sat Sat 2 2 2 2 2 Klofa A 98.7 A 84.1 E 23 A 97 At 77 D89 rabin 1 A 98.7 A 82.7 E 24 A 97 At 70 D91 2 A 98.7 A 98 E 29 A 98 At 65 D92 3 A 99.6 A 96 E 24 A 97 At 63 D 91.6 A A 99.4 At 93 E 32 At 97 From 99.8 C 97

Table 4

compound

HT29 HepG2 MCF-7 M0LT-4 Ma KSI Max Max Maxi small imal imal maln but th th Ab EU so o AB EC ₅₀ AB EC ₅₀ AB EC ₅₀ e INGI INGI (μM) inhibition (µM) (µM) (µM) ING biro biro irov ibi vani vani % moat e% e% ani e% At 68 C 57 At 54 A 94 C 86 D99 D99 IN 100 At 70 At 53 - 45 A 92 At 70 C 55 E 51 A 93 At 69 C 58 E 57 A 91 0.37 71 0.29 54 - 44 0.04 91 At 68 - 49 - 49 A 89 At 71 At 52 - 48 A 95 0.33 69 0.37 51 - 47 0.03 90 At 71 At 53 - 47 A 97 At 69 At 52 - 46 A 88 At 66 At 53 - 44 A 88 At 68 At 51 - 47 A 90 At 69 At 54 - 48 A 89 At 69 At 53 - 49 A 90 At 69 - 47 - 50 A 89 At 68 C 51 - 48 A 89 At 70 C 54 - 49 A 88 At 67 - 49 - 43 A 86 At 64 - 49 - 44 A 84 0VCAR3 CCRF-CEM K 562 HL-60 Max Ma Max Max imal KSI imal imal AB EC ₅ AB EC ₅ o small AB EC ₅ AB EC ₅₀ th (µM) INGI (µM) but (µM) INGI (µM) INGI biro e biro biro vani ING vani vani

- 36 042712 e % ibi e % e % equal e%

Clofarabine E 42 A 99 E 32 A 97 A E 94 A 99 E 85 IN 97 9 E 43 A 99 E 40 A 96 10 E 42 A 99 E 37 A 96 eleven E 70 A 99 E 50 A 96 12 >198 41 <0.02 99 >198 40 0.07 96 13 E 80 A 99 E 44 A 96 14 E 43 A 99 E 36 A 96 15 >198 41 <0.02 99 >198 36 0.06 96 16 E 44 A 99 E 33 A 96 17 E 47 A 99 E 37 A 96 18 E 47 A 99 E 40 A 96 19 E 51 A 99 E 37 A 95 20 E 49 A 99 E 35 A 96 21 E 47 A 99 E 32 A 96 22 E 73 A 99 E 62 A 95 23 E 63 A 99 E 35 A 96 24 E 67 A 99 E 49 A 95 25 E 24 A 99 E 33 A 95 26 E 62 A 99 E 69 A 95 KG-1 I <L RPMI-8226 Maximum —— maxima maxima AB EC 5o noe AB EC ₅₀ flax AB EC ₅₀ flax compound (µM) inhibitors (µM) inhibitiro (μM) inhibition % % % Clofarabine A 93 IN 61 Έ 80 A IN 93 WITH 90 WITH 94 9 A 92 V 53 Έ 81 10 A 92 48 I 80 eleven A 92 47 I 81 12 0.14 9 1 47 39.55 79 13 A 93 48 Έ 88 14 A 93 49 Έ 78 15 0.09 92 49 30.28 80 16 A 93 49 I 84 17 A 92 48 Έ 82 18 A 9 1 48 Έ 84 19 A 92 V 57 Έ 79 20 A 92 V 69 Έ 80 21 A 92 With 70 I 81 22 A 92 42 ( 86 23 A 93 39 Έ 85 24 A 9 1 39 Έ 86 25 A 91 44 ( 83 26 A 9 1 E 56 ( 93

As can be seen from Table. 3 and 4, certain compounds of the present invention were more active than ProTide A on a number of different cancer cell lines.

Example 28 Metabolic Stability

The analysis was carried out according to the published method (Kuhnz, W.; Gieschen, H. DrugMetab. Dispos. 1998, 26, 1120-1127).

Pooled cryopreserved hepatocytes were thawed, washed and suspended in Krebs-Henseleit buffer (pH 7.3). The reaction was initiated by adding the test compound (1

- 37 042712 μm final concentration) into the cell suspension and incubated in a final volume of 100 μl in a flat-bottomed 96-well plate for 0 and 120 min, respectively, at 37°C/5% CO ₂ . The reaction was stopped by adding 100 μl of acetonitrile to the incubation mixture. Then, the samples were gently and briefly mixed on a plate shaker, completely transferred to a 0.8 ml 96-well V-bottom plate, and centrifuged at 2500 g for 15 min at room temperature. Each supernatant (150 μl) was transferred to a clean cassette tube for subsequent HPLC/MS analysis on a Thermo Electron triproquadrupole system.

For comparison, the parent nucleoside and the corresponding ProTide were tested in the same assay. The results are shown in table. 5 and 6.

compound Human hepatocytes mean half-life (min) A 1 2 3 Clofarabine 24 In the range of 20-100 In the range of 20-100 In the range of 20-100 >120

Example 29 Evaluation of Clofarabine and Selected Clofarabine Diphosphate Phosphoramidates in the KG1a Acute Myeloid Leukemia (AML) Cell Line Model.

Summary.

Clofarabine and Examples 1 and 2 were selected for analysis by determining:

(1) whether the compounds of the present invention have increased activity compared to the parent compound; and (2) whether the compounds of the present invention preferentially target leukemic stem cells (LSCs).

In order to establish this, the acute myeloid leukemia cell line, KG1a, was used because they have a small stem cell-like compartment with a distinct immunophenotype (Lin7CD34 ⁺ /CD387CD123 ⁺ ). Compounds were evaluated over an extended dose range. In addition, the effects of the compounds on the stem cell compartment were evaluated over the entire dose range. The mean LD50 for clofarabine (drug concentration required to kill 50% of cells) was 1.69 x 10 ^-8 M. Example 1 showed a similar mean LD50 (1.37 x 10 ^-8 M) while Example 2 showed a significantly increased mean LD ₅₀ (4.38x10 ^-8 M and 7.10x10 ^-8 M, respectively).

Under normoxic conditions, the LSC (Lm7CD34 ⁺ /CD387CD123+) compartment accounted for ~3.5% of the total cell line. However, under hypoxic conditions (1 and 5% oxygen), the proportion of LSC showed a time-dependent increase (up to 23.7%). Moreover, this was completely reversed when the cell line was brought back to normoxic conditions. There was no significant difference in the proportion of LSC in cultures between 1 and 5% oxygen. When considering the relative activity of the compounds under normoxic and hypoxic conditions (5% oxygen), clofarabine showed a significant increase in the mean LD50 value under hypoxic conditions (1.69 x 10 ^-8 M-5.31 x 10 ^-8 M; P=0.01). In contrast, Example 1 showed no significant difference in mean LD50 values.

Goals.

1. Obtain a complete cytotoxic dose response curve for a series of selected compounds and their respective parent nucleoside in KG1a cells.

- 38 042712

2. Establish the effects of test compounds on the LSC compartment over the entire range of applied concentrations.

3. Compare the effects of test compounds under hypoxic conditions (1 and 5% oxygen).

4. To study the persistence of the γΗ2Α.Χ focus in LSC cells and whole tumor cells after treatment with clofarabine and test compounds under hypoxic conditions.

Materials and methods.

Conditions for KG1a cell culture.

The acute myeloid leukemia (AML) cell line KG1a was maintained in RPMI medium (Invitrogen, Paisley, UK) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin and 20% fetal bovine serum. Cells were then aliquoted (105 cells/100 µl) into 96-well plates and incubated at 37°C in a humidified normoxic (20% oxygen) or hypoxic (1 or 5% oxygen) atmosphere for 48 h in the presence of clofarabine or clofarabine diphosphate phosphoramidates. of the present invention (1x10'10 M- ^1x10'6 M). In addition, control cultures were obtained to which no drug was added. Then, the cells were collected by centrifugation and analyzed by flow cytometry using Annexin V analysis.

Measurement of in vitro apoptosis.

The cultured cells were collected by centrifugation, and then suspended in 195 μl of calcium-rich buffer. Subsequently, 5 μl of Annexin V (Caltag Medsystems, Botolph Claydon, UK) was added to the cell suspension and the cells were incubated in the dark for 10 min before washing. Finally, the cells were suspended in 190 µl of calcium rich buffer along with 10 µl of propidium iodide. Apoptosis was assessed by two color immunofluorescent flow cytometry as described previously. Then, LD50 values (the dose required to kill 50% of the cells in culture) were calculated for each nucleoside analog and diphosphate phosphoramidate.

Immunophenotypic identification of the leukemia stem cell compartment.

KG1a cells were grown for 48 h in the presence of a wide range of concentrations of each nucleoside analog and their respective phosphoramidate diphosphate. Then, the cells were harvested and labeled with a cocktail of anti-lineage antibodies (PE-cy7), anti-CD34 (FITC), anti-VD38 (PE) and anti-CD123 (PERCP cy5). Then, a subpopulation expressing the leukemic stem cell (LSC) phenotype was identified and expressed as the percentage of all viable cells remaining in culture. Then, the percentage of stem cells remaining was plotted on a dose-response plot and the effects of phosphoramidate diphosphate were compared to the parent nucleosides.

cell sorting.

KG1a cells were grown under hypoxic conditions for 7 days to obtain 10 ⁷ cells and the percentage of cells expressing the LSC phenotype was increased. Then, Lin'/CD34'/CD38'/CD123 ⁺ LSC and Lin'/CD34'/CD38-/CD123+ bulk tumor cells were purified by high speed cell sorting using a FACS Melody cell sorter (Becton Dickenson) and placed back under hypoxic conditions for cell culture. cultures before adding clofarabine or clofarabine diphosphate phosphoramidates.

γΗ2Α.Χ phosphorylation assay.

Phosphorylation of the histone variant γΗ2Α.Χ occurs as a rapid response to double-stranded DNA breaks. The γH2Α.Χ Phosphorylation Assay Kit (Flow Cytometry) is a cell-based assay formatted for flow cytometric detection of γH2Α.Χ phosphorylated histone levels (Merck, UK). LSC cells and bulk tumors were cultured in 96-well plates in the presence of clofarabine or clofarabine diphosphate phosphoramidates. After 2 hours of drug exposure, cells were harvested by centrifugation and then fixed and permeable in preparation for staining and detection. The histone γH2A.X phosphorylated at serine 139 was detected by adding the anti-phospho histone γH2A.X conjugated to ΑPC. Then, the cells were run through a flow cytometer to quantify the number of cells stained positively for the phosphorylated histone γH2A.X.

Statistical analysis.

The data obtained in these experiments were evaluated using one-way analysis of variance. All data were validated as Gaussian or Gaussian fit using the K2 universal test. LD50 values were calculated from non-linear regression and line of best fit to sigmoidal dose-response curves. All statistical analyzes were performed using Graphpad Prism 6.0 software (Graphpad Software Inc., San Diego, CA).

Results.

Αin vitro cytotoxicity assay.

In vitro drug susceptibility of KG1a cells was assessed using the Annexin V/propidium iodide assay. Sigmoid dose-response curves for each test compound are shown in FIG. 1Α-S. The results are summarized in table. 7.

- 39 042712

Clofarabine and the compounds of the present invention showed activity in the nanomolar range; clofarabine and example 1 had similar LD50 values, with example 1 being slightly more active.

The fraction of KG1a cells expressing the LSC phenotype is modulated by hypoxia.

KG1a cells were grown under normoxic and hypoxic conditions and LSC phenotype was monitored over time. Under normoxic conditions, the LSC phenotype was stably maintained in approximately 3.5% of the cells in culture. In contrast, under hypoxic conditions, the proportion of cells in culture expressing the LSC phenotype increased in a time-dependent manner (FIG. 3A). When the cells were then transferred back to normoxic culture conditions, the proportion of cells expressing the LSC phenotype reverted back to ~3.5% in a time dependent manner (FIG. 3B).

Comparison of clofarabine and compounds of the present invention under normoxic and hypoxic conditions.

Given the observed increase in the LSC phenotype under hypoxic conditions, the relative activity of clofarabine and compounds was evaluated in KG1a cells cultured under normoxic and hypoxic (5% O ₂ ) conditions. Fig. 4 shows superimposed sigmoid dose-response curves for clofarabine and the compounds of the present invention. The results are summarized in table. 8.

compound Normoxia average LD ₅₀ (nM) Hypoxia average LD ₅₀ (nM) ratio normoxia/hypoxia Clofarabine 16.9 53.1 31.8 Example 1 13.7 16.3 84.0 Example 2 71.0 95.0 74.7

As can be seen, clofarabine showed a significant decrease in activity when applied to cells cultured under hypoxic conditions. This decrease in activity was not observed when Examples 1 and 2 were used under the same hypoxic conditions.

Clofarabine causes significantly less DNA damage in LSCs than in bulk tumor cells under hypoxic conditions.

In an attempt to understand why clofarabine-treated cells were less drug-responsive under hypoxic conditions, the cells were grown under hypoxic conditions and then LSC fractions and bulk tumors were purified using high speed cell sorting. Then, the purified cells were exposed to the drug for 2 hours, and the amount of DNA damage was quantified using the γH2A.X phosphorylation assay. Fig. 5 shows that the level of DNA damage in LSC was significantly lower than in bulk tumor cells after clofarabine treatment. In contrast, Examples 1 and 2 caused the same levels of DNA damage in both LSC and bulk tumor cells.

1. With regard to stem cell targeting, all of the compounds tested showed stem cell targeting at concentrations above 10 ^-8 M. Example 1 showed a trend towards increased selectivity for Lin7CD347CD387CD123 ⁺ LSC.

2. The LSC phenotype was shown to be induced under hypoxic conditions and this was reversed when cells were introduced into normoxic culture conditions. The dynamics of change in the LSC fraction suggests plasticity in the phenotype rather than selective expansion/contraction in a fixed LSC subpopulation.

3. Clofarabine showed a significant decrease in activity when applied to cells cultured under hypoxic conditions. This decrease in activity was not observed when Examples 1 and 2 were used under the same hypoxic conditions. Many types of cancer exist in a hypoxic state in the human body.

One explanation for the reduced activity of clofarabine under hypoxic conditions is that under these conditions there is a significant expansion of the LSC fraction, and LSC showed significantly less DNA damage, as measured by γH2A.Χ phosphorylation after short-term exposure to clofarabine. In contrast, Examples 1 and 2 induced the same levels of DNA damage in bulk tumor cells and LSC under the same conditions.

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Claims (27)

CLAIM 1. A compound of formula (I) or a pharmaceutically acceptable salt thereof in which R ¹ independently at each occurrence is selected from C ₁ -C ₈ -alkyl, C ₀ -C ₄ -alkylene-C3-C ₈ cycloalkyl and C ₀ -C ₄ -alkylene; each R ² and R 3 is independently selected at each occurrence from H, C 1 -C ₆ alkyl and C ₁ -C ₃ alkylene-R ⁷ ; R ⁴ independently at each occurrence is H; or R ⁴ , a group selected from R ² and R ³ , and the atoms to which they are connected may form a 3-6 membered heterocycloalkyl group containing 1 or 2 heteroatoms independently selected from O, S and N; R ⁵ independently at each occurrence is selected from aryl and C 1 -C ₈ -alkyl, and aryl is optionally fused with C ₆ -C ₈ -cycloalkyl; R ⁶ independently selected from R ⁷ is independently selected from aryl and SR ^a at each occurrence; R ⁸ is independently selected from H and each Z ¹ and Z ² is independently selected from O and S; Y is independently selected from H, F, Cl and OMe; X ⁺ independently at each occurrence is a pharmaceutically acceptable metal cation or ammonium cation; where any aryl group is either phenyl or naphthyl; where when any of R ¹ , R ² , R ³ , R ⁴ , R ⁵ or R ⁷ is alkyl, cycloalkyl, heterocycloalkyl or aryl or heteroaryl, then the alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl groups are optionally substituted with 1-4 substituents , selected from halogen, nitro, cyano, NR ^a R ^a , NR ^a S(O)2R ^a , NR ^a C(O)R ^a , NR ^a CONR ^a R ^a , NR ^a CO2R ^a , OR ^a ; SR ^a , SOR ^a , SO3R ^a , SO2R ^a , SO2NR ^a R ^a , CO2R ^a , C(O)R ^a , CONR ^a R ^a , CR ^a R ^a NR ^a R ^a , C1-C ₄ -alkyl, C ₂ -C ₄ -alkenyl, C ₂ -C ₄ alkynyl and C ₁ -C ₄ haloalkyl; where R ^a independently at each occurrence is selected from H and C ₁ -C ₄ -alkyl and R ^b independently at each occurrence is selected from H and C ₁ -C ₄ -alkyl and C(O) -C ₁ -C ₄ -alkyl. 2. The compound according to claim 1, in which R ⁶ represents He - 41 042712 3. The compound according to claim 1, in which R ⁶ represents NH? 4. The compound according to claim 1, in which R ⁶ represents o He . 5. The compound according to claim 1, in which R ⁶ represents H _? N where Y and R ⁸ have the meanings specified in paragraph 1. 6. A compound according to claim 5, wherein Y is H. 7. A compound according to claim 5, wherein Y is F. 8. A compound according to any one of claims 5 to 7, wherein R ⁸ is H. 9. A compound according to any one of claims 5 to 7, wherein R ⁸ is where R ¹ , R ² , R ³ , R ⁴ , R ⁵ , Z ² and X ⁺ have the meanings specified in paragraph 1. 10. A compound according to claim 9, wherein Z ² is O. 11. A compound according to claim 9, wherein Z ² is S. 12. The compound according to claim 1, in which R ⁶ represents H?N HE 13. The compound according to claim 1, in which R ⁶ represents 14. The compound according to claim 1, in which R ⁶ represents h ₉ n he he . 15. A compound according to any one of claims 1 to 14, wherein R ¹ is selected from C ₅ -C ₇ cycloalkyl, C 1 -C ₈ alkyl and benzyl. 16. A compound according to claim 15, wherein R ¹ is benzyl. 17. The compound according to claim 15, in which R ¹ represents C 1 -C ₈ -alkyl, for example ethyl. 18. A compound according to any one of claims 1-17, wherein R ³ is H. 19. A compound according to any one of claims 1 to 18, wherein R ² is C ₁ -C ₄ alkyl. 20. A compound according to any one of claims 1-18, wherein R ² is H. - 42 042712 21. A compound according to any one of claims 1 to 20, wherein R ⁵ is phenyl. 22. A compound according to any one of claims 1-20, wherein R ⁵ is naphthyl. 23. The compound according to claim 1, in which the compound of formula (I) is selected from - 43 042712 - 44 042712 - 45 042712 - 46 042712 he where X ⁺ has the meaning specified in paragraph 1. 24. The use of a compound according to any one of claims 1 to 23 for the treatment of cancer. 25. Use according to claim 24, wherein the cancer is leukemia or lymphoma. 26. Use according to claim 25, wherein the cancer is a leukemia selected from the group consisting of acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphoid leukemia (CLL), and acute biphenotypic leukemia (BAL). 27. Use according to claim 25, wherein the cancer is a lymphoma selected from the group consisting of Hodgkin's lymphoma and non-Hodgkin's lymphoma. -