New 2' and / or 5'-amino acid ester phosphoramidate 3'-deoxyadenosine derivatives as anticancer compounds

Abstract

The present invention relates to compounds, compounds for use in a method of treatment, in particular in a method for the prevention or treatment of cancer, methods for preparing the compounds and pharmaceutical compositions comprising the compounds. The compounds are particularly useful for treating leukemia, lymphoma and / or solid tumors in Homo sapiens. The compounds are derivatives of cordycepin (3'-deoxyadenosine) with 2' and / or 5'-amino acid ester phosphoramidate moieties.

Description

[0001] This application is a divisional application of the patent application with application number 201580063936.1, filing date 27 November 2015, and with the title "Novel 2' and / or 5'-amino acid ester phosphoramidate 3'-deoxyadenosine derivatives as anticancer compounds". TECHNICAL FIELD

[0002] The present invention relates to compounds, compounds for use in a method of treatment, in particular in a method for the prophylaxis or treatment of cancer, to methods of preparing the compounds and to pharmaceutical compositions comprising the compounds.

[0003] In particular, but not exclusively, the present invention relates to compounds for use in the treatment of leukemia, lymphoma and / or solid tumors in Homo sapiens. BACKGROUND

[0004] Cordycepin is 3'-deoxyadenosine (3'dA). It is a nucleoside analogue of adenosine lacking the 3'-hydroxyl group on the ribose moiety.

[0005] Cordycepin is one of the main bioactive substances produced by Cordyceps militaris, a parasitic fungus used in traditional Chinese medicine for its immunostimulant, anti-aging and anti-tumor effects. Reference can be made to Tuli, H.S. et al., 3 Biotech (2014) 4: 1-12.

[0006] Cordycepin can be produced from adenosine synthesis. Reference can be made to Robins, J.R. et al., J. Org. Chem. 1995, 60, 7902-7908 and Aman, S. et al., Organic Process Research & Development 2000, 4, 601-605 for this synthesis procedure.

[0007] Cordycepin is most extensively studied as an anticancer agent.

[0008] Due to its structure, 3'dA and its triphosphate form can potentially interfere with any process requiring adenosine or adenosine triphosphate (ATP), respectively.

[0009] However, after administration, 3'dA is rapidly deaminated by adenosine deaminase (ADA) and rapidly metabolized into the inactive metabolite 3'-deoxyinosine in vivo. Reference can be made to Tsai, Y-J et al., J. Agri. Food Chem. 584638-43 (2010).

[0010] Cancer Research 38, 2233-2238 (1978), when used in combination with an adenosine deaminase inhibitor, demonstrated anti-cancer efficacy as pentostatine (2-deoxycoformycin, dCF). Other ADA inhibitors were also proposed as alternative co-drugs to be administered with cordycepin, but it was the combination of 3'dA-dCF that has been used in clinical trials. However, as recognized in Wehbe-Janek, H. et al., Anticancer Research 27:3143-3146 (2007), 2-deoxycoformycin is known to be a relatively toxic drug.

[0011] 2-fluorocordycepin (3'deoxy-2-fluoroadenosine) is also known to be cytotoxic (see, e.g., Montgomery et al., J. Med. Chem., 1969, 12(3), 498-504 and Dickinson et al., J. Med. Chem., 1967, 10(6), 1165-1166).

[0012] Antiviral activity of 2-chlorocordycepin (3'deoxy-2-fluoroadenosine) has been evaluated (Rosowsky et al., J. Med. Chem., 1989, 32, 1135-40). SUMMARY

[0013] It is an object of the present invention to provide a solution to the problem of improving the efficacy of purine-based 3'-deoxynucleosides, such as cordycepin (3'-deoxyadenosine), in a prophylactic or therapeutic method, in particular but not limited to, in anticancer chemotherapy including chemotherapy for treating leukemia, lymphoma and / or solid tumors.

[0014] It is another object of the present invention to provide a solution to the problem of purine-based 3'-deoxynucleosides, such as cordycepin (3'-deoxyadenosine), being deaminated by ADA upon administration and then rapidly metabolized into inactive metabolites.

[0015] It is another object of the present invention to provide a solution to the problem of purine-based 3'-deoxynucleosides, such as cordycepin (3'-deoxyadenosine), being deaminated by ADA upon administration and then rapidly metabolized into inactive metabolites, to completely rule out or at least to reduce to some extent the need for co-administering an ADA inhibitor, in the use of purine-based 3'-deoxynucleosides in a prophylactic or therapeutic method, in particular but not limited to, in anticancer chemotherapy including chemotherapy for treating leukemia, lymphoma and / or solid tumors.

[0016] According to a first aspect of the application, there is provided a compound which is a compound of formula (la):

[0017]

[0018] wherein:

[0019] W1and W2are each independently selected from -P(=O)(U)(V) and H, provided that at least one of W1and W2is -P(=O)(U)(V),

[0020] wherein for each of W1and W2, U and V are independently selected from:

[0021] (a) U is -OAr, in combination with V is -NR4-CR1R2-C(=O)OR3,

[0022] wherein Ar is selected from C 6-30 aryl and 5-30 heteroaryl, each of which is optionally substituted;

[0023] R1and R2are each independently selected from H and the group consisting of C 1-20 alkyl, C 6-30 aryl C 1-6 alkyl, C 2-20 alkenyl, C 1-20 alkoxy, C 1-20 alkoxy C 1-20 alkyl, C 1-20 alkoxy C 6-30 aryl, C 2-20 alkynyl, C 3-20 cycloalkyl C 6-30 aryl, C 6-30 aryloxy and 5-20 heterocyclyl, any of which is optionally substituted;

[0024] R3is selected from H and the group consisting of C 1-20 alkyl, C 6-30 aryl C 1-20 alkyl, C 2-20 alkenyl, C 1-20 alkoxy C 1-20 alkyl, C 1-20 alkoxy C 6-30 aryl, C 2-20 alkynyl, C 3-20 cycloalkyl C 6-30 aryl and 5-20 heterocyclyl, any of which is optionally substituted;

[0025] R4is selected from H and the group consisting of C 1-20 alkyl, C 6-30 aryl C 1-20alkyl, C 2-20 alkenyl, C 1-20 alkoxy, C 1-20 alkoxy C 1-20 alkyl, C 1-20 alkoxy C 6-30 aryl, C 2-20 alkynyl, C 3-20 cycloalkyl C 6-30 aryl, C 6-30 aryloxy and 5-20 heterocyclyl, any of which is optionally substituted;

[0026] and

[0027] (b) U and V are each independently selected from -NR5R6,

[0028] wherein R5is selected from H and C 1-6 alkyl and R6is -CR7R8CO2R9, wherein R7and R8are independently selected from the side chains of naturally occurring alpha amino acids, including H, and R9is selected from H and C 1-20 alkyl, C 6-30 aryl C 1-20 alkyl-, C 2-20 alkenyl, C 1-20 alkoxy C 1-20 alkyl, C 1-20 alkoxy C 6-30 aryl, C 2-20 alkynyl, C 3-20 cycloalkyl C 6-30 aryl and 5-20 heterocyclyl, any of which is optionally substituted; or

[0029] R5and R6together with the N atom to which they are attached form a ring moiety comprising 5 to 8 ring atoms;

[0030] Q is selected from O, S and CR 10 R 11 , wherein R 10 and R 11 are independently selected from H, F and C 1-6 alkyl;

[0031] X and Z are each independently selected from H, OH, F, Cl, Br, I, C 1-6 alkyl, -NR 12 R 13 , wherein R 12 and R 13 are each independently selected from H and C 1-6 alkyl, and -SR 14 , wherein R 14 is selected from H and C 1-6 alkyl; and

[0032] Y is selected from H, OH, F, CI, Br, I, -OC 1-6 alkyl, C 1-6 alkyl, C 2-8 alkynyl, -NR 15 R 16 wherein R 15 and R 16 are each independently selected from H and C 1-6 alkyl, and -SR 17 wherein R 17 is selected from H and C 1-6 alkyl,

[0033] or a pharmaceutically acceptable salt, ester, salt of an ester, solvate or prodrug of a compound of Formula (Ia).

[0034] The compounds of the present application are purine-based 3'-deoxynucleosides wherein each 3' substituent position on the sugar moiety of the nucleoside is occupied by H.

[0035] In another embodiment, the compounds of the present application can be a compound of Formula (Ib):

[0036]

[0037] wherein:

[0038] W1and W2are each independently selected from -P(=O)(U)(V) and H, provided that at least one of W1and W2is -P(=O)(U)(V),

[0039] wherein U and V are independently selected for each of W1and W2from:

[0040] (a) U is -OAr and V is -NR4-CR1R2-C(=O)OR3,

[0041] wherein Ar is selected from C 6-30 aryl and 5-30 heteroaryl, each of which is optionally substituted;

[0042] R1and R2are each independently selected from H and C 1-20 alkyl, C 6-30 aryl C 1-6 alkyl, C 2-20 alkenyl, C 1-20 alkoxy, C 1-20 alkoxy C 1-20 alkyl, C 1-20 alkoxy C 6-30 aryl, C 2-20 alkynyl, C 3-20 cycloalkyl, C 6-30aryl, C 6-30 aryloxy and 5-20 heterocyclyl groups, any of which is optionally substituted;

[0043] R3is selected from H and a group consisting of C 1-20 alkyl, C 6-30 aryl C 1-20 alkyl, C 2-20 alkenyl, C 1-20 alkoxy C 1-20 alkyl, C 1-20 alkoxy C 6-30 aryl, C 2-20 alkynyl, C 3-20 cycloalkyl, C 6-30 aryl and 5-20 heterocyclyl groups, any of which is optionally substituted;

[0044] R4is selected from H and a group consisting of C 1-20 alkyl, C 6-30 aryl C 1-20 alkyl, C 2-20 alkenyl, C 1-20 alkoxy, C 1-20 alkoxy C 1-20 alkyl, C 1-20 alkoxy C 6-30 aryl, C 2-20 alkynyl, C 3-20 cycloalkyl, C 6-30 aryl, C 6-30 aryloxy and 5-20 heterocyclyl groups, any of which is optionally substituted;

[0045] and

[0046] (b) U and V are each independently selected from -NR5R6,

[0047] wherein R5is selected from H and C 1-6 alkyl and R6is -CR7R8CO2R9, wherein R7and R8are independently selected from the side chains of naturally occurring alpha amino acids, including H, and R9is selected from H and a group consisting of C 1-20 alkyl, C 6-30 aryl C 1-20 alkyl-, C 2-20 alkenyl, C 1-20 alkoxy C 1-20 alkyl, C 1-20 alkoxy C 6-30 aryl, C 2-20 alkynyl, C 3-20 cycloalkyl, C 6-30 aryl and 5-20a group consisting of heterocyclyl, any of which is optionally substituted; or

[0048] R5and R6together with the N atom to which they are attached form a ring moiety comprising 5 to 8 ring atoms;

[0049] X is selected from NR 12 R 13 wherein R 12 and R 13 are each independently selected from H and C 1-6 alkyl; and -SR 14 wherein R 14 is selected from H and C 1-6 alkyl;

[0050] Z is independently selected from H, OH, F, Cl, Br, I, C 1-6 alkyl, -NR 12 R 13 , and -SR 14 wherein R 14 is selected from H and C 1-6 alkyl; and

[0051] Y is selected from H, OH, F, Cl, Br, I, -OC 1-6 alkyl, C 1-6 alkyl, C 2-8 alkynyl, -NR 15 R 16 wherein R 15 and R 16 are each independently selected from H and C 1-6 alkyl, and -SR 17 wherein R 17 is selected from H and C 1-6 alkyl,

[0052] or a pharmaceutically acceptable salt, ester, salt of an ester, solvate, or prodrug of a compound of Formula (Ib).

[0053] A compound of Formula (Ib) can be a compound of Formula (II):

[0054]

[0055] wherein Ar, Y, Z, R 2 and R 3 are as described above for Formula (Ib) and wherein X is -NR 12 R 13 .

[0056] A compound of Formula (Ib) can be a compound of Formula (III):

[0057]

[0058] wherein Ar, R 2 and R 3 As described above for formula (lb) and wherein Y is selected from H, F, Cl and OMe.

[0059] The following statements apply to any compound of formula (la), (lb), (II) and (III). The statements are independent and interchangeable. In other words, any feature described in any one of the following statements can be combined with features described in one or more of the other statements, where chemically permissible. In particular, where a compound is exemplified or described in the specification, any two or more of the following statements expressing features of that compound at any level of generality can be combined to express a subject matter considered to form part of the disclosure of the invention in the specification.

[0060] In the present specification, the term "naturally occurring alpha amino acid" means an amino acid which can have L or D stereochemistry selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serine, threonine, lysine, arginine, histidine, aspartic acid, glutamic acid, asparagine, glutamine, cysteine and methionine. Thus, in the present specification, the side chain of a naturally occurring alpha amino acid is a member selected from the group consisting of H, CH3, -CH(CH3)2, -CH2CH(CH3)2, -CH(CH3)(CH2CH3), -CH2Ph, -CH2Ph-OH, -CH2SH, -CH2CH2SCH3, -CH2OH, -(CH3)(OH), -CH2CH2CH2CH2NH3 + , -CH2CH2CH2NHC(=NH2 + )NH2, -CH2C(O)O-, -CH2CH2C(O)O-, -CH2C(O)NH2, -CH2CH2C(O)NH2.

[0061]

[0062] It is possible that W1 is -P(=O)(U)(V) and W2 is H, and the compound of the invention is a 5'-phosphoramidate of the parent 3'-deoxynucleoside. In certain preferred embodiments, W1 is -P(=O)(U)(V) wherein U is -OAr and V is -NR4-CR1R2-C(=O)OR3, and W2 is H.

[0063] It is possible that W1 is H and W2 is -P(=O)(U)(V), and the compound of the application is a 2'-amino phosphate of the parent 3'-deoxynucleoside. In certain preferred embodiments, W1 is H and W2 is -P(=O)(U)(V), wherein U is -OAr and V is -NR4-CR1R2-C(=O)OR3.

[0064] It is possible that W1 and W2 are each -P(=O)(U)(V), and the compound of the application is a 2',5'-amino phosphate of the parent 3'-deoxynucleoside. In certain preferred embodiments, wherein W1 and W2 are each -P(=O)(U)(V), U is -OAr and V is -NR4-CR1R2-C(=O)OR3. In certain preferred embodiments, W1 is the same as W2.

[0065] Ar can be unsubstituted. Ar can be substituted. Where Ar is substituted, it can be substituted with one, two, three, four or five substituents. The substituents can be selected from the group consisting of: halo, C1-C4-alkyl, C1-C4-alkoxy, nitro and cyano.

[0066] Ar, whether substituted or unsubstituted, can be selected from the group consisting of phenyl, pyridyl, naphthyl and quinolyl. In certain preferred embodiments, Ar is selected from the group consisting of phenyl and naphthyl. In further preferred embodiments, wherein Ar is naphthyl, the bond to -O-P is at the 1 -position on the naphthyl group. In still further preferred embodiments, Ar is unsubstituted phenyl or unsubstituted naphthyl, the bond to -O-P being at the 1 -position on the naphthyl group.

[0067] R1 and R2 can be selected such that the moiety -CR1R2COO- corresponds to the corresponding moiety of a naturally occurring alpha amino acid.

[0068] It is possible that R1 and R2 are each independently selected from the group consisting of Me and H. In certain preferred embodiments, one of R1 and R2 is Me and one of R1 and R2 is H such that the C atom bearing R1 and R2 has the same absolute configuration as L-alanine.

[0069] It is possible that R 1 is H. It is possible that R 2 is C1-C4 alkyl. It is possible that R 2 is methyl. It is possible that the C atom bearing R1 and R2 has the same absolute configuration as L-alanine.

[0070] It is possible that R1 and R2 are each Me. It is possible that R1 and R2 are each H.

[0071] It is possible that R3 is selected from the group consisting of C 6-30 aryl C 1-6 alkyl and unsubstituted C 1-20alkyl. In certain preferred embodiments, R3is selected from the group consisting of benzyl (-CH2-Ph), unsubstituted methyl (-CH3), and unsubstituted n-pentyl (-n-C5H 11 ) In further preferred embodiments, R3is benzyl.

[0072] R4may be H.

[0073] U and V can be independently selected from -NR5R6. Preferably, U and V are each the same. In further preferred embodiments, R8is H and R7is selected from the group consisting of H, methyl, isopropyl, -CH2Ph, -CH2CH(CH3)2, and -CH(CH3)(CH2H5). In further preferred embodiments, R7is methyl. In further preferred embodiments, the stereochemistry of the C atom having R7and R8has the same absolute configuration as L-alanine. Alternatively, the stereochemistry of the C atom having R7and R8may have the same absolute configuration as D-alanine. In certain preferred embodiments, R9is selected from the group consisting of branched and unbranched C1-C 13 unsubstituted C1-C 18 alkyl, and C 6-30 aryl C 1-6 alkyl, any of which is optionally substituted. In certain preferred embodiments, R9is benzyl.

[0074] In certain embodiments, the compounds of the present application comprise U and V, wherein U and V are each independently selected from -NR5R6, wherein R5and R6together with the N atom to which they are attached form a ring moiety comprising 5 to 8 ring atoms. U and V can be the same.

[0075] Q can be O.

[0076] It is possible that W1is -P(=O)(U)(V), wherein U is -O-1-naphthyl and V is -NH-(L)CH(CH3)-C(=O)-O-CH2-Ph, W2is H, and Q is O.

[0077] It is possible that X and Z are each independently selected from H, OH, F, Cl, NH2, SH, and -SC 1-6 alkyl, and Y is selected from H, OH, F, Cl, -OC 1-6 alkyl, NH2, C 2-8 alkynyl, SH, and -SC 1-6 alkyl. It is possible that X is NR 12 R 13, for example, NH2. In certain preferred embodiments, Z is H. In further preferred embodiments, X is NH2and Z is H. In preferred embodiments, X is NH2, Y is H and Z is H; X is NH2, Y is F and Z is H; X is NH2, Y is Cl and Z is H; or X is NH2, Y is -OCH3and Z is H. In certain preferred embodiments, X is NH2, Y is H and Z is H and thus provide compounds of the application that are derivatives of cordycepin (3'dA).

[0078] In certain particularly preferred embodiments, Ar is phenyl, R 3 is benzyl and R 2 is methyl.

[0079] Compounds of the application wherein when P is not symmetrical, the compound can consist of a mixture of diastereomers R p , diastereomers S p or diastereomers R p and S p .

[0080] Preferred compounds of the application include:

[0081] (2S)-Benzyl 2-(((((2S,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2- yl)methoxy)(naphthalen- 1 -yloxy)phosphoryl)amino)propanoate;

[0082] Benzyl 2-(((((2S,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2- yl)methoxy)(phenoxy)phosphoryl)amino)acetate;

[0083] (2S)-Pentyl 2-(((((2S,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2- yl)methoxy)(naphthalen- 1 -yloxy)phosphoryl)amino)-4-methylpentanoate;

[0084] Methyl 2-(((((2S,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2- yl)methoxy)(naphthalen- 1 -yloxy)phosphoryl)amino)-2-methylpropanoate;

[0085] (2S)-Benzyl 2-(((((2S,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2- yl)methoxy)(2-(3-ethoxy-3-oxopropyl)phenoxy)phosphoryl)amino)propanoate;

[0086] (2S)-Benzyl 2-(((((2R,3R,5S)-2-(6-amino-9H-purin-9-yl)-5- (hydroxymethyl)tetrahydrofuran-3-yl)oxy)(phenoxy)phosphoryl)amino)propanoate;

[0087] Benzyl 2-(((((2S,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-((((1- (benzyloxy)-1-oxopropan-2-yl)amino)(phenoxy)phosphoryl)oxy)tetrahydrofuran-2- yl)methoxy)(phenoxy)phosphoryl)amino)propanoate;

[0088] (2S)-Benzyl 2-(((((2R,3R,5S)-2-(6-amino-9H-purin-9-yl)-5- (hydroxymethyl)tetrahydrofuran-3-yl)oxy)(naphthalen-1-yloxy)phosphoryl)amino)propanoate;

[0089] Benzyl 2-[({[5-(6-amino-9H-purin-9-yl)-4-hydroxyhydantoin-2-yl]methoxy}({[1- (benzyloxy)-1-oxopropan-2-yl]amino})phosphoryl)amino]propanoate;

[0090] (2S)-Benzyl 2-((((2S,4R,5R)-5-(6-amino-2-methoxy-9H-purin-9-yl)-4- hydroxytetrahydrofuran-2-yl)methoxy)(naphthalen-1-yloxy)phosphorylamino)propanoate;

[0091] (2S)-Benzyl 2-((((2S,4R,5R)-5-(6-amino-2-methoxy-9H-purin-9-yl)-4- hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphorylamino)propanoate;

[0092] (2S)-Benzyl 2-(((((2S,4R,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-4- hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate;

[0093] (2S)-Hexyl 2-(((((2S,4R,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-4- hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate;

[0094] (2R)-Benzyl 2-((((2S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-4- hydroxytetrahydrofuran-2-yl)methoxy)(naphthalen-1-yloxy)phosphorylamino)propanoate;

[0095] 3'-Deoxyadenosine-5'-O-[phenyl(benzyloxy-L-alaninyl)]phosphonate;

[0096] 2-O-methyl-3'-deoxyadenosine-5'-O-[1 -naphthyl(1 -pentyloxy-L-leucinyl)] phosphonate;

[0097] 2-O-methyl-3'-deoxyadenosine-5'-O-[phenyl(1 -hexyloxy-L-alaninyl)] phosphonate;

[0098] 2-Fluoro-3'-deoxyadenosine-5'-O-[1 -naphthyl(benzyloxy-L-alaninyl)] phosphonate;

[0099] 2-Fluoro-3'-deoxyadenosine-5'-O-[1 -naphthyl(1 -pentyloxy-L-leucinyl)] phosphonate;

[0100] 2-Chloro-3'deoxyadenosine 5'-O-[1 -phenyl(2,2-dimethylpropoxy-L-alaninyl)] phosphonate;

[0101] 2-Chloro-3'deoxyadenosine 5'-O-[1 -naphthyl(2,2-dimethylpropoxy-L-alaninyl)] phosphonate;

[0102] 2-Chloro-3'deoxyadenosine 5'-O-[1 -phenyl(ethoxy-L-alaninyl)] phosphonate; and

[0103] (2S)-isopropyl-2-(((((2S,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2- yl)methoxy)(phenoxy)phosphoryl)amino)propanoate

[0104] and pharmaceutically acceptable salts, esters, salts of esters, solvates, or prodrugs thereof.

[0105] In certain embodiments, the compounds of the present application are not:

[0106] (2S)-isopropyl-2-(((((2S,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2- yl)methoxy)(phenoxy)phosphoryl)amino)propanoate.

[0107] According to a second aspect of the present application, there is provided a compound of the present application for use in a method of treatment. The compound can be used in the prevention or treatment of cancer.

[0108] According to a third aspect of the present application, there is provided the use of a compound of the present application for the manufacture of a medicament for the prevention or treatment of, in particular but not exclusively, cancer.

[0109] According to a fourth aspect of the application, there is provided a method of preventing or treating, particularly but not exclusively, cancer, comprising administering to a patient in need of such treatment an effective amount of a compound of the application.

[0110] With respect to each of the second, third and fourth aspects of the application, embodiments of the application include cancers selected from hematological and solid tumors. In particular, the cancer can be selected from leukemia, multiple myeloma, liver cancer, breast cancer, head and neck cancer, neuroblastoma, thyroid cancer, skin cancer (including melanoma), oral squamous cell carcinoma, bladder cancer, mesenchymal tumor, colon cancer, colorectal cancer, lung cancer (non-small cell and small cell), biliary cancer, pancreatic cancer, sarcoma, prostate cancer, central nervous system cancer, Ewing's sarcoma, Cholangiocarcinoma and gynecological cancers including ovarian cancer, uterine cancer and cervical cancer (including epithelial cervical cancer). In preferred embodiments, the cancer is leukemia or lymphoma, for example a cancer selected from acute lymphoblastic leukemia, acute myelogenous leukemia, acute promyelocytic leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, monocytic leukemia, hairy cell leukemia, Hodgkin's lymphoma and non-Hodgkin's lymphoma. In further preferred embodiments, the cancer is acute lymphoblastic leukemia.

[0111] Each of the second, third and fourth aspects of the application can include embodiments of treating cancer in combination with other cancer therapies. Examples of other cancer therapies include radiation therapy and / or other chemotherapy. Without being bound by theory or mechanism, it is reported (e.g., Robertson, J. B., et al., Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 1978 34(5):417-29, Hiraoka, W., et al., Radiat. Res. (1988) 114(2):231-9 and Hiraoka, W., et al., J. Radiat. Res. (Tokyo) (1990) 31(2):156-61) that 3'-deoxyadenosine inhibits repair of X-ray induced DNA damage. In certain preferred embodiments of each of the second, third and fourth aspects of the application, the compound of the application is for use in or used in a method of treating cancer comprising administering to a patient in need of such treatment a compound of the application in combination with radiation therapy.

[0112] With respect to each of the second, third and fourth aspects of the application, other embodiments of the application include a compound of the application for use in or used in a method of preventing or treating myelodysplastic syndrome.

[0113] Without being bound by theory or mechanism: Tuli et al. (supra) report that cordycepin, not only has anti-tumour and apoptotic activity, but also shows antioxidant, anti-inflammatory, anti-malarial, anti-fungal, immunomodulatory, anti-diabetic / hypoglycaemic, steroid synthesis and anti-aging activity; Vodnala, S. K. et al. J. Med. Chem. 2013, 56, 9861-9873 report that cordycepin and 2-fluorocordycepin each show anti-parasitic activity; Ahn, Y. J. et al. J. Agric. Food Chem. 2000 48(7) 2744-8 report that cordycepin shows anti-bacterial activity and de Julian-Ortiz J. V. et al. J. Med. Chem. 1999 42(17) 3308-14 report that cordycepin shows anti-viral activity; Sugar et al., Antimicrob. Agents. Chemother. With respect to each of the second, third and fourth aspects of the application, embodiments of the application include the compounds of the application for use in, or in methods of, preventing or treating a patient suffering from a disease or condition in need of at least one treatment selected from antioxidant, anti-inflammatory, anti-malarial, anti-fungal, immunomodulatory, anti-diabetic / hypoglycaemic, steroid synthesis, anti-aging, anti-parasitic, anti-bacterial and anti-viral activity.

[0114] With respect to each of the second, third and fourth aspects of the application, embodiments of the application include the compounds of the application for use in, or in methods of, preventing or treating a patient suffering from a disease or condition in need of at least one treatment selected from antioxidant, anti-inflammatory, anti-malarial, anti-fungal, immunomodulatory, anti-diabetic / hypoglycaemic, steroid synthesis, anti-aging, anti-parasitic, anti-bacterial and anti-viral activity.

[0115] However, with respect to each of the second, third and fourth aspects of the application, if desired, an ADA inhibitor can be used as a co-agent. A suitable ADA inhibitor for co-administration with the compounds of the application is hydroxyurea or pentostatin.

[0116] According to a further aspect of the application there is provided a pharmaceutical composition comprising a compound of the application in combination with a pharmaceutically acceptable carrier, diluent or excipient.

[0117] According to a further aspect of the application there is provided a method of preparing a pharmaceutical composition comprising the step of combining a compound of the application with a pharmaceutically acceptable carrier, diluent or excipient.

[0118] According to a further aspect of the application there is provided a method of preparing a compound of formula (la):

[0119] I

[0120]

[0121] by reacting a compound of formula IV:

[0122]

[0123] with

[0124] (a) a compound of formula V:

[0125]

[0126] or

[0127] (b) POCl3, followed by N + R5R6H2,

[0128] wherein W1, W2, Q, X, Y, Z, Ar, R1, R2, R3, R4, R5 and R6 have the meanings set out herein in relation to formula (la).

[0129] Surprisingly it has been found that the compounds of the present application exhibit enhanced pharmaceutical activity, in particular enhanced anti-cancer activity, compared to their parent purine-based 3'-deoxynucleosides (i.e. where W1 and W2 are H), especially when used in the treatment of leukaemias, lymphomas and / or solid tumours.

[0130] It has been found that the activity is enhanced without the administration of a complexing agent to inhibit adenosine deaminase compared to the parent purine-based nucleoside administered without the presence of a complexing agent to inhibit ADA.

[0131] Thus, the present application unexpectedly provides a method of using a derivative of 3'-deoxyadenosine or a derivative of an analogue of 3'-deoxyadenosine as a pharmaceutical agent, in particular as an anti-cancer agent, which alleviates the problem of deamination caused by adenosine deaminase, while completely avoiding the use of a complexing agent as an inhibitor of adenosine deaminase, including the relatively toxic 2-deoxycoformycin, if desired.

[0132] Without being bound by any theory, the efficacy, in particular the anti-cancer efficacy, exhibited by the compounds of the present application is believed to be due to the intracellular phosphorylation of the 3'-deoxynucleoside compounds of the present application to 3'-deoxyadenosine triphosphate, or triphosphate of a 3'-deoxyadenosine analogue. In the case of compounds of the present application having W1 in the form of -P-(=0)(U)(V), it is believed that the enzymatic cleavage of U and V within the cell converts the compound directly to 3'-deoxyadenosine monophosphate or monophosphate of a 3'-deoxyadenosine analogue, prior to phosphorylation to triphosphate.

[0133] The intracellular activity of the above-mentioned compounds of the present application cannot be predicted in advance.

[0134] The above benefits additionally enhance the cell membrane permeability of the phosphoramidate nucleosides of the present application as compared to the 3'-deoxyadenosine parent or 3'-deoxyadenosine analog parent, where the enhanced cell membrane permeability is attributed to the phosphoramidate structure of the compounds of the present application. Moreover, the benefit of enhanced cell membrane permeability cannot be assumed a priori for any phosphoramidate of a nucleoside. The compounds of the present application are believed to be the first example of a phosphoramidate of a 3'-deoxynucleoside that exhibits enhanced anticancer efficacy relative to their 3'-deoxynucleoside parent. Thus, the benefit of enhanced cell membrane permeability by the compounds of the present application is surprising.

[0135] Preferred embodiments of the compounds of the present application have the features set forth above in connection with the embodiments of the compounds of the present application.

[0136] Each of Ar, R1, R2, R3, and R4may be substituted with one, two, three, four, or five substituents.

[0137] The substituents on Ar can be located at the ortho, meta, para, or other position on the aromatic group. The substituents on Ar are independently selected from the group consisting of hydroxyl, C 1-6 acyl, C 1-6 acyloxy, nitro, amino, carboxyl, C 2-6 ester, C 1-6 aldehyde, cyano, C 1-6 alkylamino, diC 1-6 alkylamino, thiol, chloro, bromo, fluoro, iodo, C 1-6 alkyl, C 2-6 alkenyl, C 1-6 alkoxy C 1-6 alkyl, C 1-6 alkoxy C 6-10 aryl, C 5-7 cycloalkyl, C 5-11 cycloalkyl C 1-6 alkyl, C 5-7 cycloalkenyl, C 8-12 cycloalkynyl, C 6-11 aryl C 1-6 alkyl, C 1-6 alkyl C 6-11 aryl, C 6-11 aryl, C 1-6 fluoroalkyl, C 2-6 fluoroalkenyl, SO3H, SH, and SR', where R' is independently selected from the same groups as R1set forth above in connection with Formula la. Each substituent can be substituted with any other substituent.

[0138] The substituents on R1, R2, R3, and R4are independently selected from the group consisting of hydroxyl, C 1-6 acyl, C 1-6acyloxy, nitro, amino, amido, carboxy, C 2-6 ester, C 1-6 aldehyde, cyano, C 1-6 alkylamino, diC 1-6 alkylamino, thiol, chloro, bromo, fluoro, iodo, C 5-7 cycloalkyl, C 5-7 cycloalkenyl, C 8-12 cycloalkynyl, C 6-11 aryl, C 6-11 arylC 1-6 alkyl, 5-20 heterocyclyl, SO3H, SH, and SR', where R' is independently selected from the same groups as R1 set forth above with respect to Formula Ib.

[0139] In certain preferred embodiments, R1and R2are independently selected from H, C 1-10 alkyl, C 6-30 arylC 1-6 alkyl, C 2-10 alkenyl, C 2-10 alkoxyC 1-10 alkyl, C 1-10 alkoxyC 6-10 aryl, C 2-10 alkynyl, C 3-20 cycloalkyl, C 3-20 cycloalkenyl, C 8-20 cycloalkynyl, and 5-10 heterocyclyl.

[0140] In certain embodiments, R1and / or R2correspond to the side chain of a naturally occurring alpha amino acid (including H) which can have L or D stereochemistry. Thus, it is possible that R1and / or R2(e.g., either one alone or both R 1 and R 2 ) are selected from H, CH3, -CH(CH3)2, -CH2CH(CH3)2, -CH(CH3)(CH2CH3), -CH2Ph, -CH2Ph-OH, -CH2SH, -CH2CH2SCH3, -CH2OH, CH(CH3)(OH), -CH2CH2CH2CH2NH3 + , -CH2CH2CH2NHC(=NH2 + )NH2, -CH2C(O)O-, -CH2CH2C(O)O-, -CH2C(O)NH2, -CH2CH2C(O)NH2,

[0141]

[0142] In certain preferred embodiments, R1and R2are independently selected from H, -CH3, and -CH2CH(CH3)2. In further preferred embodiments, R1and R2together correspond to the side chain of L-alanine.

[0143] R3may be selected from H, C 1-20 alkyl, C 6-30 aryl C 1-6 alkyl, C 2-10 alkenyl, C 1-10 alkoxy C 1-10 alkyl, C 1-10 alkoxy C 6-10 aryl, C 2-10 alkynyl, C 3-20 cycloalkyl, C 3-20 cycloalkenyl, C 8-20 cycloalkynyl, and 5-20 heterocyclyl.

[0144] R3may be selected from H, C 1-20 alkyl, C 6-30 aryl C 1-6 alkyl, and C 3-20 cycloalkyl. R3may be selected from C 6-30 aryl C 1-6 alkyl, and unsubstituted C 1-20 alkyl. In certain preferred embodiments, R3is selected from benzyl (-CH2Ph), unsubstituted methyl (-CH3), and unsubstituted n-pentyl (-n-C5H 11 ). R3may be benzyl.

[0145] R4may be selected from H, C 1-20 alkyl, C 6-30 aryl C 1-6 alkyl, C 2-10 alkenyl, C 1-10 alkoxy, C 1-10 alkoxy C 1-10 alkyl, C 1-10 alkoxy C 6-10 aryl, C 2-10 alkynyl, C 3-20 cycloalkyl, C 3-20 cycloalkenyl, C 8-20 cycloalkynyl, and 5-20 heterocyclyl.

[0146] R4may be selected from H, C 1-18 alkyl, C 6-30 aryl C 1-6 alkyl, C 3-20 cycloalkyl, and 5-20 heterocyclyl. R4may be selected from H, methyl, ethyl, propyl, butyl, pentyl, hexyl, and cyclohexyl. R4may be H.

[0147] The present application provides a compound of the present application for use in targeting cancer stem cells.

[0148] The present application provides use of a compound of the present application in the manufacture of a medicament for targeting cancer stem cells.

[0149] The present application provides a method of targeting cancer stem cells, the method comprising providing a population of cancer stem cells and an amount of a compound of the present application sufficient to target such cancer stem cells.

[0150] Targeting of cancer stem cells referred to in the present application can be used to prevent or treat cancer. In such embodiments, the population of cancer stem cells can be in a cancer or precancerous state in a patient in need of such targeting, and the method can comprise administering to the patient a therapeutically effective amount of a compound of the present application.

[0151] The present application provides a compound of the present application for use as an anticancer stem cell medicament. Such use of a compound of the present application can also be used to prevent or treat cancer.

[0152] The present application provides a method of determining whether a patient having a cancer or precancerous state will benefit from prevention or treatment of cancer with a compound of the present application, the method comprising:

[0153] determining the presence of cancer stem cells in a biological sample representative of the cancer or precancerous state of the patient; wherein the presence of cancer stem cells in the biological sample indicates that the patient will benefit from treatment with a compound of the present application.

[0154] The present application provides a method of determining an appropriate treatment regimen for a patient having a cancer or precancerous state, the method comprising:

[0155] determining the presence of cancer stem cells in a biological sample representative of the cancer or precancerous state of the patient; wherein the presence of cancer stem cells in the biological sample indicates that an appropriate treatment regimen will comprise treating the patient with a compound of the present application.

[0156] The present application provides a compound of the present application for use in preventing or treating cancer in a patient selected for such treatment by a method comprising:

[0157] determining the presence of cancer stem cells in a biological sample representative of the cancer or precancerous state of the patient; wherein the presence of cancer stem cells in the biological sample indicates that the patient is suitable for treatment with a compound of the present application.

[0158] The above method can further comprise the step of preventing or treating cancer or a precancerous state using a compound of the present application.

[0159] In a suitable embodiment of the method of the application, the cancer is a relapsed or refractory cancer. The compounds of the application can be used to treat such a relapsed or refractory cancer.

[0160] The present application provides a compound of the application for use in treating a refractory cancer in a subject. The subject can be a human patient. The subject can be a domestic animal, for example a mammal.

[0161] The present application provides the use of a compound of the application in the manufacture of a medicament for treating a relapsed or refractory cancer in a subject. The subject can be a domestic animal, for example a mammal.

[0162] The present application provides a method of treating a relapsed or refractory cancer in a subject, the method comprising providing to a subject in need of such treatment a therapeutically effective amount of a compound of the application. The subject can be a domestic animal, for example a mammal.

[0163] The present application provides a compound of the application for use in treating a cancer, wherein the compound of the application is used in at least one initial treatment cycle at a dose of between about 25 mg / m 2 and 4000 mg / m 2 per week, and then at a lower weekly dose in at least one further treatment cycle. The cancer can be a relapsed or refractory cancer.

[0164] Various aspects of the present application are based on the discovery that the compounds of the application are able to reduce the number of cancer stem cells, and can do so preferentially compared to other cell types. This discovery is surprising because cancer stem cells are known to be resistant to many chemotherapeutic agents, and it has not previously been suggested that the compounds of the application, or cordycepin or 2-fluorocordycepin, the parent prodrug compound from which the compounds of the application are derived, are able to target cancer stem cells. The discovery that the compounds of the application are able to target cancer stem cells and thereby reduce their numbers has therefore been recognised by the present inventors as representing a surprising breakthrough which is applicable to a wide range of cancers and represents a new series of therapeutic applications for the compounds of the application.

[0165] The biological activity exerted by the compounds of the application, which has not previously been reported, indicates that these compounds are able to provide a treatment which can be effective in patients with relapsed or refractory cancers. This kind of treatment using the compounds of the application can result in a reduction in tumour size and / or a reduction in clinically relevant biomarkers, both of which can be associated with a more favourable prognosis. Furthermore, treatment with the compounds of the application can help to maintain a reduction in tumour size in patients with relapsed or refractory cancers. Thus, treatment using the compounds of the application can achieve a high degree of durable disease control (DCR) in patients with relapsed or refractory cancers.

[0166] Without wishing to be bound by any hypothesis, the inventors believe that the ability of the compounds of the application to target cancer stem cells contributes to the therapeutic utility of these compounds in the treatment of relapsed or refractory cancers.

[0167] Except where the context requires otherwise, reference in this disclosure to the "use" of a compound of the application according to the application can be taken to mean any medical use of a compound of the application as described herein. Similarly, reference to a "method" of the application using a compound of the application should be taken to mean any method of the application as described herein.

[0168] The ability of the compounds of the application to target cancer stem cells provides a new therapy against those cancer cells which are considered to be the most difficult to treat and are believed to play a major role in limiting the effectiveness of many existing cancer therapies. This ability also provides a means of targeting cells which are believed to be involved in cancer development, progression, relapse and dissemination. Thus, it will be appreciated that this anti-cancer stem cell activity of the compounds of the application gives rise to benefits in the context of the long-standing search for new and effective therapies. BRIEF DESCRIPTION OF DRAWINGS

[0169] Embodiments of the application are further described hereinafter with reference to the accompanying drawings in which:

[0170] Figure 1 . LD of cordycepin, Compound A, 2-F-cordycepin, Compounds O, P, Q and R 50 Values are compared. All assays were performed using KG1a cells and data are presented as the mean (±SD) of five independent experiments.

[0171] Figure 2 . Analysis of the leukemia stem cell (LSC) targeting ability of cordycepin and Compound A. Previously generated data (ii) is shown for comparison. All data are the mean (±SD) of three independent experiments.

[0172] Figure 3 . Analysis of the LSC targeting ability of 2-F-cordycepin and Compounds O, P, Q. All data are the mean (±SD) of three independent experiments.

[0173] Figure 4 . Comparison of the LSC targeting ability of 2-F-cordycepin and individual proTides. All data are the mean (±SD) of three independent experiments. DETAILED DESCRIPTION

[0174] As used herein, the term "alkyl" refers to a straight-chain or branched-chain saturated monovalent (except where the context requires otherwise) acyclic or cyclic hydrocarbon radical having the number of carbon atoms indicated (or in the absence of an indication, acyclic alkyl groups can have 1-20, 1-18, 1-10, 1-6, or 1-4 carbon atoms, and cyclic alkyl groups can have 3-20, 3-10, or 3-7 carbon atoms), optionally substituted with one, two, or three substituents independently selected from the groups set forth above with respect to substituents that can be present on R1, R2, R3, and R4. By way of non-limiting example, alkyl groups can include methyl, ethyl, propyl, butyl, pentyl, hexyl, octyl, nonyl, and dodecyl.

[0175] As used herein, the term "alkenyl" refers to a straight-chain or branched-chain unsaturated monovalent (except where the context requires otherwise) acyclic or cyclic hydrocarbon radical having one or more C=C double bonds and having the number of carbon atoms indicated (or in the absence of an indication, acyclic alkenyl groups can have 2-20, 2-10, 2-6, or 2-4 carbon atoms, and cyclic alkenyl groups can have 3-20 or 5-7 carbon atoms), optionally substituted with one, two, or three substituents independently selected from the groups set forth above with respect to substituents that can be present on R1, R2, R3, and R4. By way of non-limiting example, alkenyl groups can include ethenyl, propenyl, butenyl, pentenyl, and hexenyl.

[0176] As used herein, the term "alkynyl" refers to a straight-chain or branched-chain unsaturated monovalent (except where the context requires otherwise) acyclic or cyclic hydrocarbon radical having one or more C≡C triple bonds and having the number of carbon atoms indicated (or in the absence of an indication, acyclic alkynyl groups can have 2-20, 2-10, 2-6, or 2-4 carbon atoms, and cyclic alkynyl groups can have 8-20 carbon atoms), optionally substituted with one, two, or three substituents independently selected from the groups set forth above with respect to substituents that can be present on R1, R2, R3, and R4.

[0177] As used herein, the term "alkoxy" refers to the group alkyl-O-, where alkyl is as defined above, and where the alkyl portion can be optionally substituted with one, two, or three substituents set forth above with respect to alkyl. Bonded through the -O- group. By way of non-limiting example, alkoxy groups can include methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, t-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, and 1,2-dimethylbutoxy.

[0178] As used herein, the term "aryloxy" refers to the group aryl-O-, where aryl is as defined below, and where the aryl portion can be optionally substituted with one, two, or three substituents set forth above with respect to the group Ar. Bonded through the -O- group.

[0179] As used herein, the term "alkoxyalkyl" refers to an alkyl group having an alkoxy substituent. Bonding is through the alkyl group. The alkyl portion and the alkoxy portion are each as defined herein with respect to alkyl and alkoxy, respectively. The alkoxy and alkyl portions can each be substituted with one, two, or three substituents as set forth above with respect to the definition of alkyl. In preferred embodiments, the alkoxyalkyl group is methoxymethyl, which is -CH2-O-CH3.

[0180] As used herein, the term "arylalkyl" refers to an alkyl group having an aryl substituent. Bonding is through the alkyl group. The aryl portion and the alkyl portion are each as defined herein with respect to aryl and alkyl, respectively. The aryl and alkyl portions can each be substituted with one, two, or three substituents as defined herein with respect to those substituents that can be present for aryl and alkyl, respectively. In preferred embodiments, the arylalkyl group is benzyl, which is Ph-CH2-.

[0181] As used herein, the term "alkoxyaryl" refers to an aryl group having an alkoxy substituent. Bonding is through the aryl group. The alkoxy portion and the aryl portion are each as defined herein with respect to alkoxy and aryl, respectively. The alkoxy and aryl portions can each be substituted with one, two, or three substituents as defined herein with respect to those substituents that can be present for alkoxy and aryl, respectively.

[0182] As used herein, the term "cycloalkylaryl" refers to an aryl group having a cycloalkyl substituent. Bonding is through the aryl group. The cycloalkyl portion and the aryl portion are each as defined herein with respect to cycloalkyl and aryl, respectively. The cycloalkyl portion and the aryl portion can each be optionally substituted with one, two, or three substituents as set forth herein with respect to the definition of alkyl and aryl, respectively.

[0183] As used herein, the term "aryl" refers to a monovalent (except where context requires otherwise) aromatic carbocyclic radical having one, two, three, four, five, or six rings and having the indicated number of carbon atoms (or, where not indicated, 6 to 30, 6 to 12, or 6 to 11 carbon atoms). Preferred embodiments have one, two, or three rings. The aryl group can be optionally substituted with one, two, three, four, or five substituents as set forth above with respect to the optional substituents that can be present on the group Ar. In preferred embodiments, the aryl group includes: an aromatic monocyclic ring comprising 6 carbon atoms; an aromatic fused bicyclic ring system comprising 7, 8, 9, or 10 carbon atoms; or an aromatic fused tricyclic ring system comprising 10, 11, 12, 13, or 14 carbon atoms. Non-limiting examples of aryl groups include phenyl and naphthyl. In preferred embodiments, the optional substituents on the aryl group can be independently selected from the group consisting of: hydroxy, C 1-6 acyl, C 1-6 acyloxy, nitro, amino, carboxy, cyano, C 1-6 alkylamino, diC 1-6alkylamino, thiol, chlorine, bromine, fluorine, iodine, SO3H, SH, and SR', wherein R' is independently selected from the same groups as R1 with respect to Formula Ia.

[0184] As used herein, the term “ 5-30 "Heteroaryl" refers to a monovalent (unless the context requires otherwise) unsaturated aromatic heterocyclic group having from 5 to 30 ring members in the form of one, two, three, four, five or six fused rings and containing within at least one ring at least one heteroatom selected from N, O and S. Preferred embodiments have one, two or three fused rings. The available carbon atoms and / or heteroatoms in the ring system may be substituted on the ring with one, two, three, four or five substituents as described above for the substituents that may be present on the group Ar. Heteroaryl may include groups containing six ring members. An aromatic monocyclic ring system wherein at least one ring member is a N, O, or S atom, and which optionally contains one, two, or three additional ring N atoms; an aromatic monocyclic ring having six members, wherein one, two, or three ring members are N atoms; an aromatic bicyclic fused ring system having nine members, wherein at least one ring member is a N, O, or S atom, and which optionally contains one, two, or three additional ring N atoms; or an aromatic bicyclic fused ring system having ten ring members, wherein one, two, or three ring members are N atoms. Examples include, but are not limited to, pyridyl and quinolinyl.

[0185] As used herein, the term “ 5-20 "Heterocyclyl" refers to a monovalent (unless the context requires otherwise) saturated or partially unsaturated heterocyclic group having 5 to 20 ring members, at least one of which is selected from N, O and S, and in the form of one, two, three, four, five or six fused rings. In preferred embodiments, the group has one, two or three rings. In preferred embodiments, the group has 5 to 10 ring members. Heterocyclic groups may include: monocyclic ring systems having five ring members, at least one of which is a N, O or S atom, and which optionally contain one additional ring O atom or one, two or three an additional ring N atom; a monocyclic ring system having six ring members, one, two, or three of which are N atoms and optionally include an O atom; a bicyclic fused ring system having nine ring members, at least one of which is a N, O, or S atom, and which optionally contains one, two, or three additional ring N atoms; or a bicyclic fused ring system having ten ring members, one, two, or three of which are N atoms. Examples include, but are not limited to, pyrrolinyl, pyrrolidinyl, 1,3-dioxolanyl, imidazolinyl, imidazolidinyl, pyrazolinyl, pyrazolidinyl, piperidinyl, morpholinyl, or piperazinyl.

[0186] The available ring carbon atoms and / or ring heteroatoms of the above "heterocyclyl" ring systems can be substituted with one, two, three, four, or five substituents. In the event that the ring(s) is / are substituted with one or more heteroatoms, the heteroatom substituents are selected from the group consisting of halogen (F, Cl, Br, and I) and from the group consisting of oxygen, nitrogen, and sulfur, wherein the oxygen, nitrogen, or sulfur forms part of the substituent moiety. In the event that the ring(s) is / are substituted with one or more heteroatoms, preferably there are 1, 2, 3, or 4 heteroatom substituents selected from the group consisting of oxygen, nitrogen, sulfur, and halogen. Examples of substituents that can be present on the heterocyclyl ring system can be independently selected from the group consisting of hydroxyl, C 1-6 acyl, C 1-6 acyloxy, nitro, amino, carboxyl, cyano, C 1-6 alkylamino, diC 1-6 alkylamino, thiol, chloro, bromo, fluoro, iodo, SO3H, SH, and SR', wherein R' is independently selected from the same groups as R1 with respect to Formula la.

[0187] As used herein, the term "acyl" refers to a straight-chained or branched, saturated or unsaturated, substituted or unsubstituted, monovalent (except where context requires otherwise) radical including the moiety -C(=0)-, wherein bonding is through the -C- atom of the -C(=0)- moiety, and having the number of carbon atoms indicated (or in the absence of an indication, the acyl group has 1-6, or 1-4, 1-2 carbon atoms, including the C atom of the -C(=0)- moiety), optionally substituted with one, two, or three substituents independently selected from the groups set forth above with respect to substituents that can be present on R1, R2, R3, and R4. By way of non-limiting example, acyl includes HC(=0)-, CH3C(=0)-, C2H5C(=0)-, C3H7C(=0)-, C4H9C(=0)-, and C5H 11 C(=0)-.

[0188] As used herein, the term "acyloxy" refers to a straight-chained or branched, saturated or unsaturated, substituted or unsubstituted, monovalent (except where context requires otherwise) radical including the moiety -C(=0)-0-, wherein bonding is through the -0- atom, and having the number of carbon atoms indicated, including the C atoms of the -C(=0)-0- moiety (or in the absence of an indication, the acyloxy group has 1-6, 1-4, or 1-2 carbon atoms, including the carbon atoms of the -C(=0)-0- moiety), optionally substituted with one, two, or three substituents that can be present on R1, R2, R3, and R4. By way of non-limiting example, acyloxy includes HC(=0)-0-, CH3C(=0)-0-, C2H5C(=0)-0-, C3H7C(=0)-0-, C4H9C(=0)-0-, and C5H 11 C(=0)=0-.

[0189] As used herein, the term "C2-6 "Ester" means a linear or branched, saturated or unsaturated, substituted or unsubstituted, divalent (except where context requires otherwise) radical comprising R 18 C(=0)-0-R 19 , wherein R 18 is selected from H and C 1-4 alkyl, and R 19 is selected from C 1-5 alkyl, subject to the proviso that the maximum total number of C atoms, including the C atoms of the -C(=0)-0- moiety, of R 18 C(=0)-0-R 19 is 6. Bonding is through R 18 or R 19 , where H of the respective radical is not present such that the alkyl through which it is bonded is divalent, or through the C of the -C(=0)-0- moiety when R 18 is H. In preferred embodiments, the C 2-6 atoms, including the C atoms of the -C(=0)-0- moiety, of R 2-6 ester have 2-5 carbon atoms. The C 2-6 ester can be optionally substituted with one, two, or three substituents independently selected from the groups set forth above with respect to substituents that can be present on R1, R2, R3, and R4. By way of non-limiting example, the C 20 ester can be -C2H4-C(=0)-0-C2H5, wherein the -C2H4- moiety is -CH2-CH2- and bonding is through the -C2H4- moiety.

[0190] As used herein, the term "aldehyde" means a linear or branched, saturated or unsaturated, substituted or unsubstituted, monovalent (except where context requires otherwise) radical comprising HC(=0)-R 20 , wherein bonding is through -R 10 , which has the indicated number of carbon atoms, including the C atom of the -C(=0)- moiety (or, where not indicated, the aldehyde group has 1-6, 1-4, or 1-2 carbon atoms, including the C atom of the -C(=0)- moiety), optionally substituted with one, two, or three substituents that can be present on R1, R2, R3, or R4. By way of non-limiting example, aldehyde groups include HC(=0)-CH2-, HC(=0)-C2H4-, HC(=0)-C3H6-, HC(=0)-C4H8-, and HC(=0)-C5H + -.

[0191] As used herein, the term "fluoroalkyl" means an alkyl group that is a linear or branched saturated monovalent (except where context requires otherwise) cyclic or acyclic hydrocarbon radical substituted with 1 to 6 F atoms, which has the indicated number of carbon atoms (or, where not indicated, acyclic alkyl groups have 1-6 or 1-4 carbon atoms and cyclic alkyl groups have 3-6 carbon atoms).

[0192] As used herein, the term "fluoroalkenyl" refers to an alkenyl group which is a straight chain or branched chain unsaturated monovalent (except where context requires otherwise) acyclic or cyclic hydrocarbon group, having one or more C=C double bonds and having the number of carbon atoms indicated (or, where not indicated, acyclic alkenyl groups having from 2 to 6 or 2 to 4 carbon atoms and cyclic alkenyl groups having from 4 to 6 carbon atoms), substituted with from 1 to 6 F atoms.

[0193] Preferably, the process for preparing a compound of formula la or lb is carried out in the presence of a suitable solvent.

[0194] Suitable solvents include hydrocarbon solvents such as benzene and toluene; ether type solvents such as diethyl ether, tetrahydrofuran, diphenyl ether, anisole and dimethoxybenzene; halogenated hydrocarbon solvents such as methylene chloride, chloroform and chlorobenzene; ketone type solvents such as acetone, methyl ethyl ketone and methyl isobutyl ketone; alcohol type solvents such as methanol, ethanol, propanol, isopropanol, n-butanol and t-butyl alcohol; nitrile type solvents such as acetonitrile, propionitrile and benzonitrile; ester type solvents such as ethyl acetate and butyl acetate; carbonate type solvents such as ethylene carbonate and propylene carbonate and the like. These can be used individually or two or more of them can be used in admixture.

[0195] Preferably, an inert solvent is used in the process of the application. The term "inert solvent" refers to a solvent which is inert under the reaction conditions described in connection therewith, including, for example, benzene, toluene, acetonitrile, tetrahydrofuran, dimethylformamide, chloroform, methylene chloride (or dichloromethane), diethyl ether, ethyl acetate, acetone, methyl ethyl ketone, methanol, ethanol, propanol, isopropanol, t-butyl alcohol, dioxane, pyridine and the like. Tetrahydrofuran is particularly preferred.

[0196] Preferably, the process of the application is carried out under substantially dry conditions.

[0197] The chlorophosphates can be prepared from aryloxydichlorophosphates and suitably protected amino acid derivatives. Alternatively, phosphonate chemistry can be used with a suitable condensing agent.

[0198] Preferably, the process for preparing a compound of formula lb can include a step of protecting the free OH group on the nucleoside other than the one to which the phosphoramidate is attached. For example, the reaction of a 3'-deoxynucleoside with the desired chlorophosphonate in the presence of t-BuMgCl allows the preparation of a 2'-phosphoramidate.

[0199] The 3'-deoxynucleoside is reacted with POCl3followed by N +The salt reaction of R5R6H2 allows the preparation of compounds wherein U and V are each -NR5R6. Suitable salts include chlorides, toluenesulfonates, sulfonates and ester salts such as 4-methylbenzenesulfonate. The subsequent addition of a base such as diisopropylethylamine can assist the process.

[0200] As used herein, the term "stereoisomers" defines all possible compounds composed of the same atoms bonded by the same sequence of bonding, but having different three-dimensional structures that the compounds of the present invention may have.

[0201] In the case where the compound according to the present invention has at least one chiral center, they can therefore exist in the form of enantiomers. In the case where the compound has two or more chiral centers, they can additionally exist in the form of diastereomers. In the case where the preparation method of the compound according to the present invention produces a mixture of stereoisomers, these isomers can be separated by conventional techniques such as preparative chromatography. These compounds can be prepared in stereochemically mixed forms, or single enantiomers can be prepared by standard techniques known to those skilled in the art, such as by enantiospecific synthesis or splitting, by forming a diastereoisomer pair with an optically active acid salt, followed by fractional crystallization and regeneration of the free base. These compounds can also be split by forming diastereomeric esters or amides, then chromatographic separation and removal of chiral auxiliary agents. Alternatively, a chiral HPLC column can be used to split the compound. It should be understood that all such isomers and mixtures thereof are included within the scope of the present invention.

[0202] Furthermore, it is understood that the phosphate center is chiral in the compounds of the present invention and that the compounds may be R P and S P The compound can be a mixture of R P and S P or a pure diastereomer. In a preferred embodiment, the compound is R P or S P A substantially pure single diastereomer of P or S P In another embodiment, there may be a 1:1 ratio of R P With S P Alternatively, the compound may comprise R P and S P A mixture of diastereomers, R P With S PThe ratio of diastereoisomers is 1 :90 to 90:1, 1 :50 to 50:1, 1 :20 to 20:1, 1 :15 to 15:1, 1 :10 to 10:1, 1 :9 to 9:1, 1 :8 to 8:1, 1 :7 to 7:1, 1 :6 to 6:1, 1 :5 to 5:1, 1 :4 to 4:1, 1 :3 to 3:1 or 1 :2 to 2:1. In preferred embodiments, the compounds of the application can comprise more than 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, 1 :10, 1 :15, 1 :20, 1 :50, 1 :90, 1 :95 or 1 :99 of R P with S P the ratio of diastereoisomers, or vice versa.

[0203] The term "solvate" refers to a compound of Formula la or Formula lb as defined herein wherein the molecules of a suitable solvent are incorporated in the crystal lattice. Suitable solvents are physiologically tolerable at the administered dose. Examples of suitable solvents are ethanol, water and the like. When water is the solvent, the molecules are referred to as hydrates.

[0204] The compounds of the present application can also exist as pharmaceutically acceptable salts. For use in medicine, the salts of the compounds of this application refer to "pharmaceutically acceptable salts". The FDA approved pharmaceutically acceptable salt forms (reference: International J. Pharm. 1986, 33, 201-217; J. Pharm. Sci., 1977, Jan, 66(1)) include pharmaceutically acceptable acidic / anionic or basic / cationic salts.

[0205] Pharmaceutically acceptable acidic / anionic salts include, but are not limited to, acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, glycollylarsanilate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, pamoate, pantothenate, phosphate, diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, teoclate, tosylate, and triethiodide.

[0206] Pharmaceutically acceptable basic / cationic salts include, but are not limited to, aluminum, benzathine, calcium, chloroprocaine, choline, diethanolamine, ethylenediamine, lithium, magnesium, potassium, procaine, sodium, and zinc.

[0207] The present application includes within its scope prodrugs of the compounds of this application. In general, such prodrugs will be functional derivatives of the compounds which are readily convertible in vivo into the desired compound. Thus, in the methods of treatment of the present application, the term "administering" shall encompass the treatment of the various conditions described with the compound specifically disclosed or with a compound which might not be specifically disclosed, but which converts to the specified compound in vivo after administration to the subject. Conventional procedures for the selection and

[0208] Pharmaceutically acceptable ester derivatives wherein one or more of the free hydroxyl groups are esterified with pharmaceutically acceptable esters are specific examples of prodrug esters which are convertible by solvolysis under physiological conditions to compounds of this application having free hydroxyl groups.

[0209] The pharmaceutical compositions for use according to the present application can be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. These pharmaceutical compositions can be manufactured in a manner known per se, for example, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Proper formulation is dependent upon the route of administration chosen.

[0210] The compounds or pharmaceutical compositions according to the present application can be administered to a patient, which can be Homo sapiens or an animal, by any suitable means.

[0211] The pharmaceuticals used in the present application can be administered orally or parenterally, including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal, vaginal and topical (including buccal and sublingual) administration.

[0212] For oral administration, the compounds of the present application are typically provided in tablet or capsule form, in powder or granule form, or in aqueous solution or suspension.

[0213] Tablets for oral use can include the active ingredient in admixture with a pharmaceutical acceptable excipient such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose or starch. Starches, and alginic acid are suitable disintegrating agents. Binding agents can include starch and gelatin, while the lubricating agent, if desired, is magnesium stearate, stearic acid or talc. If desired, tablets can be coated by known techniques to delay gastrointestinal in vivo dissolution and absorption and thereby provide a sustained action over an extended period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate can be employed.

[0214] Capsules for oral use include hard gelatin capsules in which the active ingredient is mixed with a solid diluent, and soft gelatin capsules wherein the active ingredient is mixed with water or an oil such as peanut oil, liquid paraffin or olive oil.

[0215] Formulations for rectal administration can be provided as suppositories by mixing the active ingredient with a suitable non-irritating excipient which is solid at room temperature but liquid at body temperature to thereby form a gelatinous mass which is therapeutically effective and lubricious.

[0216] Formulations suitable for vaginal administration can be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

[0217] For intramuscular, intraperitoneal, subcutaneous and intravenous use, the compounds of the application are generally provided in sterile aqueous solutions or suspensions, buffered to an appropriate pH and isotonicity with substances such as buffers, antioxidants or bentonite. Suitable buffers include phosphate, citrate and other organic acids. Suitable antioxidants include sodium bisulfate, ascorbic acid and methionine. Suitable water soluble preservatives include parabens (methyl or propyl), benzalkonium chloride and sorbic acid. Suitable water miscible solvents include propylene glycol and polyethylene glycol.

[0218] The compounds of the application can also be provided in the form of liposome formulations.

[0219] Generally, a suitable dose will be in the range of 0.1 mg to 300 mg per kilogram body weight of recipient per day. More suitably, the dose can be in the range of 0.5 mg to 150 mg per kilogram body weight of recipient per day, 0.5 mg to 100 mg per kilogram body weight of recipient per day, 1 mg to 50 mg per kilogram body weight of recipient per day, or 1 mg to 10 mg per kilogram body weight of recipient per day. A suitable lower dose can be 0.5 mg per kilogram body weight of recipient per day or 1 mg per kilogram body weight of recipient per day. Alternatively, a suitable dose can be in the range of 1 mg to 100 mg per square meter of body surface area of recipient per day or 5 mg to 50 mg per square meter of body surface area of recipient per day. A suitable dose can be 6 mg, 12 mg, 24 mg or 48 mg per square meter of body surface area of recipient per day. The desired dose can be provided and administered in a single daily dose, or administered in two, three, four, five or six or more sub-doses at appropriate intervals throughout the day. The dose can be administered in unit dosage form, e.g., containing 10 mg to 1500 mg, preferably 20 mg to 1000 mg, and most preferably 50 mg to 700 mg of active ingredient per unit dosage form. Suitably, the total daily dose is 1000 mg to 3000 mg, whether administered in a single dose, or as two, three, four, five or six sub-doses, at appropriate intervals throughout the day.

[0220] "Cancer stem cell"

[0221] Cancer stem cells, sometimes also referred to in other ways as "tumour initiating cells", are well known to those skilled in the art. As used herein, the term "cancer stem cell" is to be interpreted in accordance with its widely accepted meaning, as a cell which has the capacity to self-renew through asymmetric division to induce tumour formation and to generate more mature non-stem cell cancer progeny through differentiation.

[0222] Cancer stem cells play a major role in the development, progression, recurrence and dissemination of cancer. Thus, the finding that the compounds of the application are able to target cancer stem cells so as to reduce their number provides a therapeutic possibility to prevent or treat these activities.

[0223] As discussed in more detail elsewhere in the specification, cancer stem cells are found in pre-cancerous states, where their presence is thought to contribute to the development of such states into cancer. Thus, the methods of treatment and medical uses of the application, in which the compounds of the application are used to target cancer stem cells, can be used to reduce the number of cancer stem cells in a pre-cancerous state (e.g. myelodysplastic syndrome or other state considered elsewhere in the specification), so as to prevent the development of such pre-cancerous state into cancer.

[0224] As described above, asymmetric cell division of cancer stem cells produces differentiated non-stem cancer cells. Thus, cancer stem cells are responsible for the formation and maintenance of bulk tumours.

[0225] Accumulation of such non-stem cancer cells plays a major role in the progression of cancer. Targeting cancer stem cells by the compounds of the application is able to reduce the number of cancer stem cells, which in turn reduces the number of non-stem cancer cell progeny. Thus, the methods of treatment and medical uses of the compounds of the application according to the application are beneficial in treating cancer by preventing cancer progression. Such embodiments are described in more detail elsewhere in the specification.

[0226] Cancer stem cells are also able to act as a reservoir of cancer cells, which can lead to cancer recurrence after remission. Even in cases where cancer cells have been removed (for example, by surgery, radiotherapy or chemotherapy, alone or in combination) such that there are no observable signs of cancer, the continued presence of cancer stem cells can, over time, nucleate the recurrence of cancer. Targeting cancer stem cells by the compounds of the application provides a new modality that can reduce the number of cancer stem cells and kill cancer stem cells. Thus, as discussed in more detail elsewhere in the specification, in suitable embodiments the application provides methods and medical uses in which the compounds of the application prevent or delay cancer recurrence.

[0227] Furthermore, movement of cancer stem cells from a cancer site to another location in the body can contribute to the spread of cancer, for example by metastasis. Thus, the ability of the compounds of the application to target cancer stem cells therefore provides new therapeutic methods and medical uses for preventing or treating cancer spread.

[0228] In addition to the biological activity of cancer stem cells, they can be identified by their expression of certain characteristic cell surface markers. In haematological malignancies cancer stem cells are often CD34 + whereas in solid tumours, CD44 + , CD133 + and CD90 + have been identified as cancer stem cell markers. The table below summarises examples of known cancer stem cell surface phenotypes. It is expected that each of these forms of cancer stem cell can be targeted using the compounds of the application according to the application, and thus methods or uses using the compounds of the application can be used to prevent or treat cancers associated with cancer stem cells expressing any of these groups of markers.

[0229]

[0230] The data in the examples demonstrate that the compounds of the application are able to target cancer stem cells of the leukemic stem cell line, in particular cancer stem cells present in the acute myeloid leukemia cell line KG1a. This cell line shows a small stem cell-like compartment with different immunophenotypes (Lin - / CD34 + / CD38 - / CD123 + ) that are targeted by the compounds of the application. Thus, the methods of treatment or medical uses of the compounds of the application according to the application can be used to prevent or treat leukemias or other cancers associated with cancer stem cells expressing these characteristic markers.

[0231] The application also provides methods and medical uses of using the compounds of the application to select patients for the prevention or treatment of cancer on the basis of identifying the presence of cancer stem cells in a biological sample representative of the cancer or precancerous state of the patient. The above markers provide suitable examples that can be used to identify the presence of cancer stem cells according to these embodiments of the application. Suitable techniques to investigate the expression of these markers in a biological sample are further considered elsewhere in this specification.

[0232] "targeting cancer stem cells"

[0233] The application provides a first indication that the compounds of the application can be used to target cancer stem cells. The ability of the compounds of the application to target cancer stem cells is demonstrated in the examples disclosed in this specification.

[0234] It can be seen that when a compound of the application is provided to a population of cancer cells containing cancer stem cells, it targets the cancer stem cells present, resulting in a reduction in the total number of cancer cells. As discussed elsewhere in this specification, certain compounds of the application preferentially target cancer stem cells relative to the bulk of tumor cells, and the activity of such compounds is not only able to reduce the total number of cancer cells present, but also to reduce the proportion of total cancer cells that display the phenotypic markers of cancer stem cells.

[0235] It is believed that the compounds of the application enter cancer cells and bind to nucleic acids (RNA and / or DNA) within the cell. Without being bound by any theory, it is believed that the efficacy, in particular the anti-cancer efficacy, exhibited by the compounds of the application demonstrates that the compounds of the application are phosphorylated to the triphosphate of cordycepin or a cordycepin derivative (e.g., 2-fluorocordycepin or 2-Cl-cordycepin), and it is believed that prior to phosphorylation to the triphosphate, enzymes within the cell cleave the compounds of the application to directly convert to 8-chloroadenosine monophosphate.

[0236] It is also believed that the compounds of the application have enhanced cell membrane permeability (as compared to cordycepin), and that this contributes to the enhanced anticancer potency of the compounds of the application as compared to the parent from which they are derived.

[0237] Without wishing to be bound by any hypothesis, the inventors believe that the reduction in the number of cancer stem cells is a result of the targeted killing of cancer stem cells in the cancer cell population. Thus, the compounds of the application are capable of causing the death of cancer stem cells. Furthermore, the results set out elsewhere in this specification indicate that certain compounds of the application appear to preferentially kill cancer stem cells as compared to killing non-stem cancer cells, thereby not only causing the death of cancer stem cells, but also reducing the proportion of cancer stem cells in the total cancer cell population.

[0238] Although the inventors believe that the compounds of the application that preferentially target cancer stem cells preferentially kill cancer stem cells as compared to non-stem cancer cells, other mechanisms can also contribute to the reduction in the proportion of cancer stem cells caused by the compounds of the application that target these cells.

[0239] By way of example only, treatment with the compounds of the application can result in an increase in the differentiation of cancer stem cells, thereby reducing the number of cancer stem cells and the proportion of total cancer cells represented by cancer stem cells. Alternatively, the compounds of the application can cause cancer stem cells to lose their stem cell phenotype, for example to lose their ability to self-renew, thereby reducing the number of cancer stem cells.

[0240] References to targeting of cancer stem cells in the present disclosure should be interpreted accordingly. For the purposes of the present disclosure, "targeting" of cancer stem cells can be taken to include any mechanism by which the compounds of the application reduce the number of cancer stem cells present in a cell population, whether in vitro or in vivo. In particular, targeting of cancer stem cells can be taken to include preferential reduction in the number of cancer stem cells as compared to other cell types, in particular as compared to non-stem cancer cells. References to targeting in the present specification can be taken to include killing of, and optionally preferential killing of, cancer stem cells as compared to non-stem cancer cells.

[0241] "Prevention or treatment of cancer"

[0242] The present application provides medical uses and methods of treatment in which a compound of the present application is used to prevent or treat cancer. In the context of the present application, the "prevention" of cancer is considered to relate to the prophylactic application of a compound of the present application used prior to the development of cancer and is intended to stop the development of cancer. On the other hand, the "treatment" of cancer is considered to relate to the use of a compound of the present application after the occurrence of cancer with the aim of ameliorating cancer by slowing down or stopping the proliferation of cancer cells and the growth of tumours. Advantageously, the treatment of cancer can result in a partial or complete reduction in the number of cancer cells and the size of tumours. An effective treatment of cancer can result in a disease that is "stable" or "responsive" according to the RECIST (Response Evaluation Criteria in Solid Tumours) guidelines.

[0243] As described in more detail below, the prevention of cancer according to the present application can be particularly beneficial for patients having a precancerous state with an increased likelihood of developing cancer.

[0244] "Prevention of cancer"

[0245] The prevention of cancer according to the present application can be achieved by treating a precancerous state with a compound of the present application according to the various aspects or embodiments of the present application described herein.

[0246] In particular, in the context of the present application, the prevention of cancer can be achieved by the methods or medical uses of the present application in which a compound of the present application is provided to a patient having a precancerous state. The method of treatment or medical use according to this embodiment can prevent the precancerous state being treated from developing into cancer, thereby providing an effective prevention of cancer.

[0247] The reference to the prevention of cancer in the context of the present application can also include other prophylactic applications of a compound of the present application. For example, the ability of a compound of the present application to target cancer stem cells thereby preventing the development of cancer, and / or preventing the progression of cancer and / or preventing the recurrence of cancer and / or preventing the spread of cancer.

[0248] "Pre-cancerous state"

[0249] Cancer often occurs after the development of a precancerous state, which is not cancerous in itself but is associated with an increased risk of cancer. The accumulation of genetic or epigenetic changes can lead to a previously normal cell developing into a cancer stem cell phenotype. Thus, cancer stem cells can also be present in such precancerous states as well as in cancerous states.

[0250] It is believed that the presence of cancer stem cells in pre-cancerous states contributes to the development of these states into cancer. The methods and medical uses of the present application can be used to target cancer stem cells present in pre-cancerous states, thereby treating such states. It will be appreciated that the novel and unexpected finding that the compounds of the present application target cancer stem cells means that the use of such compounds to treat pre-cancerous states can be used to prevent the development of the state into cancer. This represents a way in which the compounds of the present application can be used in medicine to prevent cancer, as considered elsewhere in this specification.

[0251] Examples of pre-cancerous states that can be treated in accordance with the present application include, but are not limited to, those selected from the group consisting of actinic keratosis, Barrett's oesophagus, atrophic gastritis, dyskeratosis congenita, sideropenic dysphagia, lichen planus, oral submucous fibrosis, solar elastosis, cervical dysplasia, leukoplakia, erythema, monoclonal gammopathy of unknown significance (MGUS), monoclonal B-cell lymphocytosis (MBL), myelodysplastic syndrome and pre-cancerous states of the stomach such as atrophic gastritis, gastric ulcer, pernicious anaemia, residual stomach, gastric polyps and Ménétrier's disease. Of the listed pre-cancerous states of the stomach, atrophic gastritis, pernicious anaemia, residual stomach and certain types of gastric polyps can be particularly at risk of developing into cancer.

[0252] Pre-cancerous states often take the form of a lesion comprising dysplastic or hyperplastic cells. Thus, in addition or as an alternative to the presence of cells having an expression marker or phenotypic characteristic of a cancer stem cell, the presence of dysplasia or hyperplasia can be used to identify a pre-cancerous state.

[0253] The severity of dysplasia can vary between different pre-cancerous states or over time with the development of a single pre-cancerous state. Generally, the more advanced the dysplasia associated with a pre-cancerous state, the more likely it is to be a pre-cancerous state that develops into cancer. Dysplasia is generally classified as mild, moderate or severe. If left untreated, severe dysplasia generally develops into cancer. Accordingly, appropriately, the methods of treatment or medical uses using the compounds of the present application can be used to treat patients having a pre-cancerous state associated with severe dysplasia.

[0254] In a suitable embodiment of the present application, the compounds of the present application are used to treat a patient having severe cervical dysplasia. Severe cervical dysplasia can be diagnosed by a Pap smear. In another embodiment of the present application, the compounds of the present application are used to treat severe oesophageal dysplasia ("Barrett's oesophagus"). Severe oesophageal dysplasia can be diagnosed following a biopsy of tissue.

[0255] It has recently been reported that pre-malignancies can also be identified by detecting somatic mutations in cells of individuals who are not known to have cancer. In particular, age-related clonal hematopoiesis has been reported to be a common pre-cancerous state that is associated with increased overall mortality and increased risk of cardiovascular metabolic disease. Most mutations detected in blood cells occur in three genes: DNMT3A, TET2 and ASXL1. Thus, patients who would benefit from targeting cancer stem cells and thereby treating a pre-cancerous state using a compound of the application can be identified by assaying a sample comprising blood cells for the presence of a genetic mutation in at least one of DNMT3A and / or TET2 and / or ASXL1 that is indicative of a pre-cancerous state.

[0256] A pre-cancerous state that can benefit from treatment targeting cancer stem cells using a compound of the application according to the application can also be identified by determining the presence of cancer stem cells by any technique based on the expression of a marker characteristic of cancer stem cells or cancer stem cell phenotype, discussed elsewhere in this specification.

[0257] "Treatment of cancer"

[0258] The skilled person understands that there are many methods of evaluation by which the "treatment of cancer" can be assessed. By way of example only, any reduction or prevention of cancer development, cancer progression, cancer recurrence or cancer dissemination can be considered to indicate effective treatment of cancer.

[0259] In certain embodiments, the compounds of the application can be used to: reduce the proportion of cancer stem cells in a population of cancer cells; and / or inhibit tumour growth; and / or reduce tumourigenicity; and / or prevent or treat a primary cancer; and / or prevent or treat a recurrent cancer; and / or prevent or treat a metastatic or secondary cancer; and / or treat, prevent or inhibit metastasis or recurrence; and / or treat or prevent a refractory cancer.

[0260] The ability to treat cancer using a compound of the application to cause a reduction in tumour size and to maintain the reduction in tumour size during / after the time of administration of the treatment represents a particularly relevant indication of effective treatment of cancer. As set out in the Examples, surprisingly, the therapeutic or medical uses of the application have been demonstrated to be effective in this respect even in models using cells representing a recurrent or refractory cancer that had previously been resistant to treatment using other therapies.

[0261] The data provided in the Examples indicate that treatment with the compounds of the application reduces the proportion of cancer stem cells in a population of cancer cells. Characteristic biological activities or cell surface markers that can identify cancer stem cells are described elsewhere in this specification. In suitable embodiments, treatment of a cancer according to the application can result in a reduction of at least 10%, at least 20%, at least 30%, or at least 40% in the proportion of cancer stem cells present in a patient's cancer. In suitable embodiments, treatment of a cancer according to the application can result in a reduction of at least 50%, at least 60%, at least 70%, or at least 80% in the proportion of cancer stem cells present in a patient's cancer. Treatment of a cancer according to the application can result in a reduction of at least 85%, at least 90%, or at least 95% in the proportion of cancer stem cells present in a patient's cancer. Indeed, treatment of a cancer according to the application can result in a reduction of at least 96%, at least 97%, at least 98%, at least 99%, or even 100% in the proportion of cancer stem cells present in a patient's cancer (such that substantially no cancer stem cells remain).

[0262] Asymmetric division of cancer stem cells contributes to the growth of a tumour. Treatment of a cancer with a compound of the application according to the application can result in at least 10%, at least 20%, at least 30% or at least 40% inhibition of tumour growth. Suitable treatment of a cancer according to the application can result in at least 50%, at least 60%, at least 70% or at least 80% inhibition of tumour growth. Treatment of a cancer according to the application can result in at least 85%, at least 90% or at least 95% inhibition of tumour growth in a patient so treated. Indeed, treatment of a cancer according to the application can result in at least 96%, at least 97%, at least 98%, at least 99%, or even 100% inhibition of tumour growth in a treated cancer.

[0263] Tumour growth can be assessed by any suitable method, in which changes in tumour size are assessed over time. Suitably, the size of a tumour prior to cancer treatment is compared to the size of the same tumour during or after cancer treatment. Many methods in which tumour size can be assessed are known. For example, the size of a tumour can be assessed by imaging the tumour in situ in a patient. Suitable techniques such as imaging techniques can allow determination of the volume of a tumour and assessment of changes in tumour volume.

[0264] As shown by the results set out in the Examples of the present specification, the methods of treatment and medical uses of the compounds of the present application are not only capable of arresting tumour growth, but in fact are capable of achieving a reduction in tumour volume in cancer patients, including patients with relapsed or refractory cancer. Suitable treatment of cancer according to the present application can result in a reduction in tumour volume of at least 10%, at least 20%, at least 30% or at least 40%. In suitable embodiments, treatment of cancer according to the present application can result in a reduction in tumour volume of at least 50%, at least 60%, at least 70% or at least 80%. Treatment of cancer according to the present application can result in a reduction in tumour volume of at least 85%, at least 90% or at least 95%. Indeed, treatment of cancer according to the present application can result in a reduction in tumour volume of at least 96%, at least 97%, at least 98%, at least 99% or even 100%.

[0265] Reduction in tumour volume of the type described above can be calculated with reference to a suitable control. For example, in studies carried out in vitro or in vivo in suitable animal models, reduction in tumour volume can be determined by direct comparison between the volume of a tumour treated with a compound of the present application and the volume of a control tumour, which can be untreated or can have received treatment other than with a compound of the present application. It will be appreciated that in the context of a clinical trial or the management of treatment of a patient, such a model lacking tumour treatment can not be ethically acceptable, and in such a case reduction in tumour volume can be assessed by comparison of the volume of a treated tumour with the volume of the same tumour prior to treatment, or with a predicted volume that would have been reached by the tumour had no treatment been applied.

[0266] The methods of treatment and medical uses of the compounds of the present application can result in a reduction in a biomarker indicative of cancer. Such a reduction in a biomarker provides a further assessment by which effective treatment of cancer can be demonstrated. Suitable examples of such biomarkers can be selected based on the type of cancer to be treated: in the case of gynaecological cancer, CA125 represents a suitable example of a biomarker, while in the case of pancreatic or biliary cancer, CA19.9 represents a suitable example of a biomarker, and in the case of colorectal cancer, CEA can be a suitable biomarker.

[0267] Suitable treatment of cancer according to the present application can result in a reduction in a cancer biomarker of at least 10%, at least 20%, at least 30% or at least 40%. In suitable embodiments, treatment of cancer according to the present application can result in a reduction in a cancer biomarker of at least 50%, at least 60%, at least 70% or at least 80%. Treatment of cancer according to the present application can result in a reduction in a cancer biomarker of at least 85%, at least 90% or at least 95%. Indeed, treatment of cancer according to the present application can result in a reduction in a cancer biomarker of at least 96%, at least 97%, at least 98%, at least 99% or even 100%.

[0268] The beneficial effects observed in the treatment of cancer according to the present application, such as a reduction in the proportion of cancer stem cells present, a reduction in tumor growth, or a reduction in tumor volume or cancer biomarkers, can be maintained for at least one month. Suitably, such beneficial effects can be maintained for at least two months, at least three months, at least four months, at least five months, or at least six months. Indeed, such beneficial effects can be maintained for at least 12 months, at least 18 months, or at least 24 months. Suitably, beneficial effects can be maintained for at least three years, at least four years, at least five years, at least six years, at least seven years, at least eight years, at least nine years, or ten years or more.

[0269] In suitable embodiments of the application, by targeting cancer stem cells, the compounds of the application can be used in methods of preventing or treating cancer or a precancerous condition. In a suitable embodiment, the present application provides the use of a compound of the application in a method of preventing or treating cancer or a precancerous condition, wherein the method reduces the tumorigenicity of one or more cancer stem cells. Suitably, such a method can prevent the progression of cancer or inhibit tumor growth.

[0270] When a compound of the application is used in a method or medical use of the application to prevent or treat the progression of cancer, such prevention or treatment can result in a slowing, delay or complete cessation of the progression of cancer.

[0271] Progression of cancer is typically determined by assigning a stage to the cancer. Staging is typically performed by assigning a number from I to IV to the cancer, where I is a cancer that is isolated and IV is a cancer that has spread to the limits of the assessment measured. Details of staging vary between cancers, but the stage typically takes into account the size of the tumor, whether it has invaded adjacent organs, how many regions (nearby) lymph nodes it has spread to, and whether it appears in more distant sites (metastatic).

[0272] Generally, Stage I is localized to a portion of the body and can be treated by surgical resection (for solid tumors that are small enough). Stage II is locally advanced and can be treated by chemotherapy, radiation therapy, surgery, or a combination thereof. Stage III is also locally advanced and the designation of Stage II or Stage III depends on the particular cancer type, although Stage III is generally accepted as "advanced" locally advanced. Stage IV cancers often metastasize to a second organ. Treatment of cancer using the compounds of the invention in the methods or medical uses of the invention can be used to treat Stage I, II, III, or IV cancers by targeting cancer stem cells. Treatment using the compounds of the invention can be used to prevent progression of cancer from one stage to the next. In one embodiment, treatment using the compounds of the invention is used to prevent progression from Stage I to Stage II. In another embodiment, treatment using the compounds of the invention is used to prevent progression from Stage II to Stage III. In another embodiment, treatment using the compounds of the invention is used to prevent progression from Stage III to Stage IV.

[0273] Preventing or inhibiting progression of cancer is particularly important in preventing spread of cancer, for example, progression from Stage I to Stage II in the case of local spread of cancer, or progression from Stage III to Stage IV in the case of metastasis of cancer to other organs. Cancer stem cells are tumorigenic and are therefore believed to play a key role in local and metastatic spread of cancer. Thus, the methods of treatment or medical uses of the invention using the compounds of the invention can be used to prevent spread of cancer by targeting tumorigenic cancer stem cells and thus reducing their numbers.

[0274] "Cancer"

[0275] The compounds of the invention exhibit increased anti-cancer activity compared to the parent nucleosides from which the compounds of the invention are derived. This increased anti-cancer activity appears to be provided due to the increased activity against cancer stem cells and non-stem cancer cells.

[0276] Cancer stem cells play a role in the biological activity of a wide range of cancers. Thus, there is a wide range of cancers that can be prevented or treated in accordance with the invention.

[0277] As discussed elsewhere herein, cancer stem cells are known to exist in many tumor types, including liquid tumors (including blood tumors such as leukemia and lymphoma) and solid tumors (e.g., breast, lung, colon, prostate, ovarian, skin, bladder, biliary duct, and pancreatic tumors). Thus, the methods of treatment and medical uses of the invention in which the compounds of the invention target cancer stem cells are expected to be useful in preventing or treating such cancers.

[0278] Suitably, the compounds of the application can be used to prevent or treat a cancer selected from the group consisting of: leukemia, lymphoma, multiple myeloma, lung cancer, liver cancer, breast cancer, head and neck cancer, neuroblastoma, thyroid cancer, skin cancer (including melanoma), oral squamous cell carcinoma, bladder cancer, interstitial cell tumor, biliary cancer (e.g. cholangiocarcinoma or bile duct cancer), pancreatic cancer, colon cancer, colorectal cancer and gynecological cancers including ovarian cancer, endometrial cancer, fallopian tube cancer, uterine cancer and cervical cancer (including epithelial cervical cancer). In suitable embodiments, the cancer is leukemia, and can be selected from the group consisting of: acute lymphoblastic leukemia, acute myelocytic leukemia (also known as acute myelogenous leukemia or acute nonlymphocytic leukemia), acute promyelocytic leukemia, acute lymphocytic leukemia, chronic myelocytic leukemia (also known as chronic myelogenous leukemia, chronic myeloid leukemia or chronic granulocytic leukemia), chronic lymphocytic leukemia, monocytic leukemia and hairy cell leukemia. In further preferred embodiments, the cancer is acute lymphoblastic leukemia. In one particular embodiment, the leukemia is refractory TdT positive leukemia. In suitable embodiments, the cancer is lymphoma, which can be selected from the group consisting of: Hodgkin's lymphoma; non-Hodgkin's lymphoma; Burkitt's lymphoma; and small lymphocytic lymphoma.

[0279] Suitably targeting cancer stem cells in such cancers can achieve effective treatment of the cancer by preventing or treating development of the cancer, by preventing or treating progression of the cancer, by preventing or treating recurrence of the cancer, or by preventing or treating metastasis of the cancer.

[0280] In suitable embodiments, the present application provides a compound of the application for use in targeting cancer stem cells to prevent or treat metastatic cancer.

[0281] In suitable embodiments, the present application provides a compound of the application for use in targeting cancer stem cells to treat recurrent or refractory cancer.

[0282] In suitable embodiments, the present application provides a compound of the application for use in targeting cancer stem cells to treat primary cancer. Suitably, the primary cancer treated can be a second primary cancer.

[0283] The present application provides a compound of the application for use in targeting cancer stem cells to treat secondary cancer. In suitable embodiments, the secondary cancer is metastatic cancer.

[0284] In suitable embodiments, the present application provides compounds of the present application for use in targeting cancer stem cells, wherein targeting cancer stem cells prevents or inhibits: (i) recurrence of a cancer; (ii) occurrence of a second primary cancer; or (iii) metastasis of a cancer.

[0285] The methods of treatment or medical uses using compounds of the present application based on the ability of the compounds of the present application to target cancer stem cells can be used to treat recurrent or refractory cancers. The considerations in this embodiment with respect to recurrent or refractory cancers (except where context requires otherwise) are the same as those with respect to the treatment of recurrent or refractory cancers in respect of the various aspects of the present application.

[0286] "recurrent or refractory cancer"

[0287] As mentioned above, certain aspects and embodiments of the present application are particularly directed to the use of compounds of the present application in the treatment of recurrent or refractory cancers.

[0288] For the purposes of the present application, a refractory cancer can be considered to be a cancer which shows resistance to treatment by anti-cancer therapies other than therapy using a compound of the present application. For example, a compound of the present application can be used to treat a refractory cancer which is resistant to radiotherapy. Alternatively or additionally, a compound of the present application can be used to treat a refractory cancer which is resistant to a biological agent used in the treatment of cancer. In one suitable embodiment, a compound of the present application can be used to treat a refractory cancer which is resistant to treatment using a chemotherapeutic agent other than a compound of the present application.

[0289] In particular, refractory cancers which can benefit from the methods of treatment or medical uses of the present application using compounds of the present application include those which are resistant to cordycepin or 2-fluorocordycepin.

[0290] Recurrent cancers (or recurrent cancers) are those which return after a period of remission in which the cancer cannot be detected. Cancer recurrence can occur at the original cancer site (local cancer recurrence), at a site proximal to the original cancer site (regional cancer recurrence), or at a site distal from the original cancer site (distant cancer recurrence). Cancer stem cells are believed to play a role in the recurrence of cancer, providing a source of recurrent cancer cells. Thus, the methods of treatment and medical uses of the present application according to the present application of compounds of the present application which are able to target cancer stem cells can be highly beneficial in the context of recurrent cancers. The ability of the compounds of the present application to target cancer stem cells can be used to deplete the population of such cells which are able to cause recurrence, thereby preventing the occurrence of recurrent cancers. The anti-cancer stem cell activity of the compounds of the present application can also be used to target cancer stem cells in cancers which have already recurred, as well as potentially exerting a cytotoxic effect on non-stem cancer cells, thereby providing a treatment for recurrent cancers.

[0291] In view of the foregoing, it will be appreciated that the compounds of the present application can be used in the methods or uses of the present application for preventing or treating recurrent cancer. The compounds of the present application can be used in the methods or uses of the present application for preventing or treating locally, regionally, or distantly recurrent cancer.

[0292] The compounds of the present application can be used in the methods or uses of the present application to prevent recurrence of cancer by providing a remission period of at least 2 months, at least 6 months, at least 12 months, at least 18 months, at least 24 months, or at least 30 months. Indeed, the compounds of the present application can be used to prevent recurrence of cancer by providing a remission period of at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, or at least 10 years.

[0293] The compounds of the present application can be used in the methods or uses of the present application to treat recurrent cancer that recurs after a remission period of at least 2 months, at least 6 months, at least 12 months, at least 18 months, at least 24 months, or at least 30 months. Indeed, the compounds of the present application can be used to treat recurrent cancer that recurs after a remission period of at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, or at least 10 years.

[0294] The ability of the compounds of the present application to target cancer stem cells, according to the medical uses or treatment methods of the present application, results in the ability of these compounds to prevent or treat cancer. However, it should be noted that the compounds of the present application also have a direct cytotoxic effect on non-stem cancer cells that make up the majority of tumors. While the activity of cancer stem cells can be the basis of much of the resistance that makes recurrent or refractory cancer so difficult to treat, non-stem cancer cells are also a major component of such recurrent or refractory cancer.

[0295] The compounds of the present application have a greater cytotoxic effect on non-stem cancer cells than the chemotherapeutic molecule from which the compounds of the present application are derived, cordycepin or 2-fluorocordycepin. Thus, the mechanism by which the compounds of the present application act to treat recurrent or refractory cancer can not be limited to the anti-cancer stem cell activity of the compound, but can also take advantage of the effect of the compounds of the present application on non-stem cancer cells. In such uses, treatment with the compounds of the present application will reduce the total number of both cancer stem cells and non-stem cancer cells. When certain compounds of the present application are used, such treatment will preferentially reduce the proportion of cancer stem cells that remain after treatment.

[0296] Therapeutically effective doses of the compounds of the present application

[0297] A therapeutically effective amount of a compound of the application can be an amount sufficient to induce death of cancer cells. A therapeutically effective amount of a compound of the application can be an amount sufficient to induce death of cancer stem cells. In some embodiments, particularly those involving treatment of relapsed or refractory cancer, a therapeutically effective amount of a compound of the application can be an amount sufficient to induce death of cancer stem cells and also to induce death of non-stem cancer cells.

[0298] There are a variety of different ways in which the amount of a therapeutically effective compound (e.g. a compound of the application) to be administered to a patient can be calculated and expressed. One such way, which is considered to be particularly relevant to the dosage of an agent for use in the prevention or treatment of cancer, is the amount of agent administered per unit of patient body surface area. Such dosages are typically expressed in terms of the amount of agent (which can be determined by mass) per square meter (m2) of surface area. 2 ) surface area.

[0299] The use of a compound of the application for the prevention or treatment of cancer can use a weekly dosage of 10 mg / m 2 to 1000 mg / m 2 . Such treatment can for example use a weekly dosage of 375 mg / m 2 to 900 mg / m 2 . For example, when a patient is provided with a weekly dosage of a compound of the application in the range of about 500 mg / m 2 to 825 mg / m 2 , effective treatment of relapsed or refractory cancer can be provided.

[0300] Without wishing to be bound by any hypothesis, the inventors believe that the ability of a compound of the application to target cancer stem cells allows therapeutic effectiveness to be achieved using a lower dosage of that compound than would otherwise be expected. By way of example only, a weekly dosage of a compound of the application as low as 825 mg / m 2 , 750 mg / m 2 , 600 mg / m 2 or 500 mg / m 2 may prove to be therapeutically effective in the uses and methods of the application.

[0301] A selected weekly dosage of a compound of the application can be provided in a single administration or in multiple administrations over a week. For example, a weekly dosage of a compound of the application can be provided in two administrations, in three administrations or in multiples. Thus, where the weekly dosage is 750 mg / m 2 , this can be achieved by three administrations of 250 mg / m 2 over the course of a week or by two administrations of 375 mg / m 2 over a week. Similarly, where the weekly dosage is 600 mg / m 2In the case of a weekly dose, this can be achieved by three doses of 200 mg / m 2 administration or 300 mg / m2 twice a week 2 The application is realized.

[0302] In order to provide the desired dose of the compound within a week, a suitable amount of the compound of the invention administered at a single therapeutic rate may be about 100 mg / m 2 Up to 300 mg / m 2 .

[0303] The weekly dose of the compound of the present invention may be reduced over the course of treatment. For example, treatment may be at about 1000 mg / m 2 , 900mg / m 2 , 825mg / m 2 , 750mg / m 2 or 725 mg / m 2 Weekly doses of 10 mg / m2 are initiated, and during treatment, the required dose may be reduced to approximately 750 mg / m2 2 (in case the initial dose is higher than this amount), about 650 mg / m 2 , about 625mg / m 2 , even about 500mg / m 2 or about 375 mg / m 2 .

[0304] Of course, dosages of the compounds of the invention may be presented in other ways. The most common of these is the amount of active agent provided per unit body weight. It has been calculated that for an average human patient, 1 mg / m 2 The dose is equivalent to about 0.025 mg / kg of body weight. Therefore, the data show that the compounds of the present invention are effective for treating recurrent or refractory cancers at a dose ranging from about 6.25 mg / kg to about 25 mg / kg. Suitable dosages may, for example, be from about 9.5 mg / kg to 22.5 mg / kg. In suitable embodiments, when a weekly dose ranging from about 12.5 mg / kg to 20.5 mg / kg is provided to the patient, the compounds of the present invention achieve effective treatment of recurrent or refractory cancers.

[0305] Notes on formulations of the compounds of the invention suitable for use in the methods of prevention or treatment of the invention and for medical use are described elsewhere in this disclosure. In the case of injectable formulations of the compounds of the invention, these may be administered intravenously. Intravenous administration may be achieved within any suitable timeframe, such as within a ten-minute injection, etc.

[0306] Types of treatment

[0307] In suitable embodiments, the compounds of the application are useful for targeting cancer stem cells as a first line treatment for cancer.

[0308] However, the finding that the compounds of the application are able to target cancer stem cells, thereby treating relapsed or refractory cancer, indicates that the compounds of the application are able to provide effective treatment of cancer where other treatments have proven ineffective. Thus, in suitable embodiments, the application provides the compounds of the application for targeting cancer stem cells as a second line treatment for cancer. Indeed, in suitable embodiments, the application provides the compounds of the application for targeting cancer stem cells as a third line or further treatment for cancer.

[0309] In suitable embodiments, the compounds of the application are provided for use as a neoadjuvant for the treatment of cancer. Neoadjuvants are agents provided to a patient to reduce the size of a tumour prior to a "primary" anti-cancer treatment, for example surgical removal of the cancer. The compounds of the application can be used as a new adjuvant treatment for a patient who is subsequently subjected to surgical treatment of the cancer and / or radiation treatment of the cancer.

[0310] Alternatively or additionally, the application provides the compounds of the application for use as an adjuvant in the treatment of cancer. Adjuvants are agents provided to a patient after a "primary" anti-cancer treatment, for example surgical removal of the cancer, to prevent recurrence of the cancer after the primary treatment. The compounds of the application can be used as an adjuvant for a patient who has been subjected to surgical treatment of the cancer and / or radiation treatment of the cancer.

[0311] The compounds of the application can be used in the methods or uses of the application in monotherapy, i.e. in prophylaxis or treatment where the compound of the application provides substantially all of the therapeutic activity used in prophylaxis or treatment.

[0312] Alternatively, the methods or uses of the application can use the compounds of the application in combination therapy. In such embodiments, the compounds of the application are used in conjunction with at least one other cancer therapy. The other cancer therapy can comprise surgery and / or radiation therapy. Additionally or alternatively, the other cancer therapy can comprise the use of at least one other therapeutic agent which contributes to the prophylaxis or treatment of cancer to be achieved. Suitably, such agent can be a chemotherapeutic or biological agent for the prophylaxis or treatment of cancer.

[0313] In suitable embodiments of combination therapy, the compound of the application and the other therapeutic agent can be provided to the patient simultaneously. In suitable examples, the compound of the application and the other therapeutic agent can be formulated as part of the same pharmaceutical composition. Alternatively, the compound of the application and the other therapeutic agent can be formulated separately for provision to the patient at substantially the same time.

[0314] In another suitable embodiment of a combination therapy, the compound of the application and the other therapeutic agent can be provided to the patient at different times. The compound of the application and the other therapeutic agent can be provided to the patient sequentially. For example, the compound of the application can be provided to the patient prior to the provision of the other therapeutic agent. Alternatively, the compound of the application can be provided to the patient after the provision of the other therapeutic agent.

[0315] "other therapeutic agent"

[0316] The compound of the application can be used in combination with a wide range of other therapeutic agents for the prevention or treatment of cancer. These include biological agents, immunotherapeutic agents and chemotherapeutic agents that can be used for the prevention or treatment of cancer.

[0317] While specific examples of suitable other agents are considered in the following paragraphs, these should not be taken as limiting the scope of other therapeutic agents suitable for use with the compound of the application. Indeed, the ability of the compound of the application to target cancer stem cells suggests that it can be beneficially used in combination with any other therapeutic agent for the prevention or treatment of cancer, whether such other agent targets cancer stem cells, non-stem cancer cells or other cells or components involved in the development, maintenance, recurrence, spread or otherwise of cancer.

[0318] Examples of other therapeutic agents that can be used in combination with the compound of the application include:

[0319] (a) an anti-angiogenic agent, optionally wherein the anti-angiogenic agent is: (i) an inhibitor of the VEGF pathway, optionally bevacizumab; (ii) a tyrosine kinase inhibitor, optionally sorafenib, sunitinib or pazopanib; or (iii) an mTOR inhibitor, optionally everolimus;

[0320] (b) an alkylating agent;

[0321] (c) an anti-metabolite;

[0322] (d) an anti-tumour antibiotic;

[0323] (e) a topoisomerase;

[0324] (f) a mitotic inhibitor;

[0325] (g) a monoclonal antibody;

[0326] (h) a metal agent; or

[0327] (i) active or passive immunotherapy.

[0328] Except where the context requires otherwise, the other therapeutic agents listed in the above list are to be considered as applicable in any of the embodiments of combination therapy with the compound of the application considered above.

[0329] Selection of patients

[0330] The inventors have found that the compounds of the application are able to target cancer stem cells, enabling a number of approaches by which it is possible to determine whether a particular patient is likely to benefit from receiving a compound of the application in the prevention or treatment of a cancer, such as a relapsed or refractory cancer.

[0331] The application therefore provides a method of determining whether a patient having a cancer or precancerous condition will benefit from the prevention or treatment of the cancer using a compound of the application, the method comprising: determining the presence of cancer stem cells in a biological sample representative of the cancer or precancerous condition from the patient; wherein the presence of cancer stem cells in the biological sample indicates that the patient will benefit from treatment using a compound of the application.

[0332] The application further provides a method of determining a suitable treatment regimen for a patient having a cancer or precancerous condition, the method comprising: determining the presence of cancer stem cells in a biological sample representative of the cancer or precancerous condition from the patient; wherein the presence of cancer stem cells in the biological sample indicates that a suitable treatment regimen will include treatment using a compound of the application.

[0333] The application also provides a compound of the application for use in the prevention or treatment of a cancer in a patient selected for such treatment by a method comprising: determining the presence of cancer stem cells in a biological sample representative of the cancer or precancerous condition from the patient; wherein the presence of cancer stem cells in the biological sample indicates that the patient is suitable for treatment using a compound of the application.

[0334] In suitable embodiments, cancer stem cells in a biological sample can be identified by the expression of a profile of markers thereof discussed previously in this application.

[0335] The skilled person will appreciate that there are many suitable examples of biological samples that can be used in embodiments of the application, such as those set out above. Suitably, such a sample can comprise cells from a cancer or precancerous condition. A suitable biological sample can be a tissue sample, for example a sample for histology. The expression of cancer stem cell markers, for example those set out above, in cells in such a sample can be assessed directly.

[0336] Alternatively or additionally, a suitable biological sample can comprise target molecules representative of the gene expression of cells from a cancer or precancerous condition. Examples of such target molecules include proteins encoded by expressed genes or nucleic acids, for example mRNA, representative of gene expression.

[0337] Suitable examples of techniques by which expression of cancer stem cell markers can be assessed can be selected with reference to the type of sample. Techniques for studying markers of expression are often used in the context of clinical assessment (e.g. for diagnostic or prognostic purposes) and their use will be familiar to those required to implement them in the context of the present application. By way of example only, in a sample comprising protein, the presence of a cancer stem cell marker can be assessed by suitable techniques using antibodies reactive with the cancer stem cell marker in question. Examples of such samples comprising protein cancer stem cell markers include histological samples (in which the presence of the marker can be visualised by suitable immuno-cytochemical techniques) or samples derived from circulation. Here, the presence of circulating cancer stem cells (which are thought to contribute to the spread of cancer by metastasis) can be assessed using techniques such as flow cytometry.

[0338] In a sample comprising nucleic acids representative of expression of a cancer stem cell marker, such expression can be assessed by suitable molecular biology techniques (e.g. by polymerase chain reaction (PCR) amplification using suitable primers).

[0339] Example 1 - Synthetic Methods

[0340] The compounds of the application can be prepared according to or analogously to the following general and exemplary synthetic procedures.

[0341] General Procedure 1 (for compounds A-F and L-U)

[0342] A solution of N-methylimidazole (1.0 mmol) and the appropriate chlorophosphonate (0.6 mmol) in dry THF (2 mL) was added dropwise to a suspension of 3'-deoxyadenosine (0.20 mmol) or substituted 3'-deoxyadenosine in dry THF (10 mL) and the reaction mixture was stirred at room temperature over a period of 16 hours. Purification by column chromatography and preparative TLC gave the target compound as a white solid. The amount of components used can vary and actual amounts are given in the following examples.

[0343] General Procedure 2 (for compound J)

[0344] (0.80 mmol) was added dropwise at -5°C. The reaction mixture was allowed to reach room temperature and stirring was maintained for 4 hours. At -78°C, a solution of the appropriate amino acid ester salt (4.0 mmol) dissolved in dry CH2Cl2(5 mL) was added, followed by diisopropylethylamine (8.0 mmol). After stirring at room temperature for 20 hours, water was added and the layers were separated. The aqueous phase was extracted with dichloromethane and the organic phase was washed with brine. The combined organic layers were dried over Na2SO4and concentrated. The residue was purified by column chromatography (elution gradient CH2Cl2 / MeOH = 100 / 0 to 93 / 7) to give the target product as a white foam. The amounts of the components used can vary and are given in the following examples.

[0345] General Procedure 3 (for compounds G-I)

[0346] (0.20 mmol) was suspended in dry THF (5 mL) and a solution of the appropriate amino acid ester salt (0.20 mmol) dissolved in dry THF (5 mL) was added dropwise at room temperature. The reaction mixture was stirred at room temperature for 16 hours. The residue was purified by column chromatography (elution gradient CH2Cl2 / MeOH = 100 / 0 to 93 / 7) to give the target product as a white foam. The amounts of the components used can vary and are given in the following examples. t BuMgCl (1.0 M in THF, 0.22 mmol). A solution of the appropriate chlorophosphonate (0.6 mmol) in dry THF (2 mL) was added dropwise and the reaction mixture was stirred at room temperature during 16 hours. Purification by column chromatography and preparative TLC gave the target compound as a white solid. The amounts of the components used can vary and are given in the following examples.

[0347] General Procedure 4 (for compound V)

[0348] To a solution of the appropriate 3'-deoxyadenosine derivative (1 mol / eq) in dry DMF was added tert-butyldimethylsilyl chloride (3.3 mol / eq) and imidazole 6.6 (mol / eq) and the reaction mixture was stirred at room temperature overnight (16-20 hours). NH4Cl was then added to the mixture and washed twice with ethyl acetate. The organic layers were combined, dried over Na2SO4and the solvent was removed under vacuum. The mixture was purified by column chromatography to give intermediate Cl. Intermediate Cl was then dissolved in a water solution of THF / H2O / TFA 4 / 1 / 1 (6 ml / eq) and stirred at 0°C for 4 hours. The solution was then carefully neutralized with a saturated aqueous NaHCO3solution and the mixture was washed twice with ethyl acetate. The organic layers were combined, dried over Na2SO4and the solvent was removed under vacuum. The mixture was purified by column chromatography to give intermediate C2. General procedure B was then applied and intermediate C3 was obtained. Intermediate C3 was dissolved in a water solution of THF / H2O / TFA 1 / 1 / 1 (6 ml / eq) at 0°C and stirred at room temperature for 24 hours. Purification by chromatography gave the target compound as a white solid.

[0349] General Procedure 5 (for preparation of 3'-deoxyadenosine and 3'-deoxy-2- chloroadenosine used in the examples):

[0350] A solution of H2O / CH3CN 1:9 and α-AIBBr (4.0 mol / eq) was added successively to a suspension of dry adenosine or 2-chloroadenosine in dry CH3CN and stirring was continued at room temperature (20°C). After 1 hour, a saturated NaHCO3solution was carefully added and the solution was extracted with EtOAc. The combined organic phases were washed with brine. The aqueous phase was extracted with EtOAc and the combined organic phases were dried over Na2SO4, filtered and evaporated to give a white gum. The crude mixture was dissolved in dry MeOH and stirred with Amberlite (2x OH - ) resin previously washed thoroughly with dry MeOH for 1 hour. The solution was then filtered and the resin was carefully washed with dry methanol. The combined filtrates were evaporated to give 2',3'-didehydroadenosine or 2',3'-didehydro-2-chloroadenosine as a white solid.

[0351] A solution of LiEt3BH (1 M in THF 4-4.3 mol / eq) was added dropwise to a cold (4°C) solution of 2',3'-dehydrothymidine or 2',3'-dehydro-2-chlorothymidine (1 mol / eq) in anhydrous DMSO / THF (1 / 10). Stirring was continued at 4°C for 1 h and at room temperature overnight (16 h). The reaction mixture was carefully acidified (5% AcOH / H2O), purged with N2for 1 h (under a fume hood) to remove pyrophoric triethylborane, and evaporated. The residue was chromatographed to give 3'-deoxythymidine or 3'-deoxy-2-chlorothymidine as a white powder.

[0352] 2,3'-Dehydrothymidine was prepared using General Procedure 5: from 10.0 g (37.4 mmol) of thymidine, 7.5 mL of H2O / CH3CN (1 / 9), 22 mL (149.7 mmol) of a- AIBBr in 500 mL of anhydrous CH3CN, and 300 mL of Amberlite (2x OH - ) resin in 400 mL of anhydrous methanol. 2',3'-Dehydrothymidine was obtained as a white solid (9.12 g, 98%). 3'-Deoxythymidine was prepared from 9.12 g (36.6 mmol) of 2',3'-dehydrothymidine and 159 mL (159 mmol) of LiEt3BH / THF 1 M in anhydrous DMSO / THF (1 / 10, 50 mL). Purification by column chromatography on silica gel (eluent system 3-18% MeOH in DCM) gave 3'-deoxythymidine as a white powder (7.12 g, 77%).

[0353] 1 H NMR (500 MHz, DMSO-d6) δ 8.37 (s, 1H, H8), 8.17 (s, 1H, H2), 7.29 (br s, 2H, NH2), 5.89 (d, J = 2.5 Hz, 1H, H1'), 5.68 (d, J = 4.5 Hz, 1H, OH-2'), 5.19 (t, J = 6.0 Hz, 1H, OH-5'), 4.63-4.58 (m, 1H, H2'), 4.40-4.34 (m, 1H, H4'), 3.71 (ddd, J = 12.0, 6.0, 3.0 Hz, 1H, H5'), 3.53-3.49 (ddd, J = 12.0, 6.0, 4.0 Hz, 1H, H5'), 2.30-2.23 (m, 1H, H3'), 1.98-1.90 (m, 1H, H3'). 13C NMR (125 MHz, DMSO-d6) δ 156.00 (C6), 152.41 (C2), 148.82 (C4), 139.09 (C8), 119.06 (C5), 90.79 (C1'), 80.66 (C4'), 74.56 (C2'), 62.61 (C5'), 34.02 (C3').

[0354] 2,3'-Dideoxy-2-fluoroadenosine was prepared using General Procedure 5: from 5.0 g (16.6 mmol) of 2-chloroadenosine, 3.0 mL of H2O / CH3CN (1 / 9), 9.7 mL (66.2 mmol) of a-AIBBr in 38 mL of anhydrous CH3CN and 150 mL of Amberlite (2x OH - ) resin in 200 mL of anhydrous methanol. 2,3'-Dideoxy-2-fluoroadenosine was obtained as a white solid (3.03 g, 60%). 3'-Deoxy-2-fluoroadenosine was prepared from 2.18 g (7.68 mmol) of 2',3'-dideoxy-2-fluoroadenosine and 30.7 mL (30.7 mmol) of LiEt3BH / THF 1 M in anhydrous DMSO / THF (1 / 10, 30 mL). Purification by column chromatography on silica gel (eluent system 2-20% MeOH in DCM) gave 3'-deoxy-2-fluoroadenosine as a white powder (1.20 g, 55%).

[0355] 1 H NMR (500 MHz, CD3OD): δH 8.41 (s, 1H, H8), 5.93 (d, J = 2.5 Hz, 1H, H1'), 4.68-4.66 (m, 1H, H2'), 4.56-4.52 (m, 1H, H4'), 3.95 (dd, J = 3, 12.5 Hz, 1H, H5'), 3.70 (dd, J = 3, 12.5 Hz, 1H, H5'), 2.39-2.33 (m, 1H, H3'), 2.08-2.03 (m, 1H, H3') 13 C NMR (125 MHz, CD3OD): δC 158.14 (C6), 155.19 (C2), 151.15 (C4), 141.30 (C8), 119.56 (C5), 93.58 (C1'), 82.80 (C4'), 76.81 (C2'), 64.01 (C5'), 34.33 (C3').

[0356] Preparation of 3'-deoxy-2-fluoroadenosine:

[0357] A solution of H20 / CH3CN (1 :9; 1.4 mL) then a-AIBBr (4.10 mL, 28.05 mmol) was added sequentially to a suspension of dry 2-fluoroadenosine (2.0 g, 7.01 mmol) in anhydrous CH3CN (50 mL) and stirring was continued at room temperature (20 °C). After 1 h, a saturated NaHC03solution was carefully added and the solution was extracted with EtOAc (2 x 100 mL). The combined organic phases were washed with brine (1 x 50 mL). The aqueous phase was extracted with EtOAc (2 x 50 mL) and the combined organic phases were dried over Na2S04, filtered and evaporated to give a white gum. The crude mixture was dissolved in a mixture of THF / H20 (4 / 1, 50 mL) and stirred for 1 h with 60 mL of Amberlite (2 x OH - ) resin (previously well washed with THF). The solution was then filtered and the resin was carefully washed with THF. The combined filtrates were evaporated and the residue was crystallized from EtOH to give 2',3'-didehydro-2-fluoroadenosine as a white solid (1.13 g, 60%).

[0358] A solution of LiEt3BH / THF (1 M; 18.01 mL, 18.01 mmol) was added dropwise to a cold (4 °C, ice bath) solution of 2',3'-didehydro-2-fluoroadenosine (1.13 g, 4.18 mmol) in anhydrous DMSO / THF (1 / 10, 15 mL). Stirring was continued at 4 °C for 1 h and at room temperature overnight (16 h). The reaction mixture was carefully acidified (5% AcOH / H20), purged with N2for 1 h (under a fume hood) to remove pyrophoric triethylborane and evaporated. The residue was chromatographed on silica gel (3-18% MeOH in DCM) to give 3'-deoxy-2-fluoroadenosine as a white powder (7.12 g, 77%).

[0359] 19 F NMR (470 MHz, DMSO-d6): d F -52.19. 1 H NMR (500 MHz, DMSO-d6) δH8.34 (s, 1H, H8), 7.80 (br s, 2H, NH2), 5.78 (d, J = 2.25 Hz, 1H, H1’), 5.68 (br s, 1H, OH-2’), 5.01 (br s, 1H, OH-5’), 4.55-4.51 (m, 1H, H2’), 4.39-4.32 (m, 1H, H4’), 3.73-3.76 (m, 1H, H5’), 3.56-3.50 (m, 1H, H5’), 2.26-2.18 (m, 1H, H3’), 1.94-1.85 (m, 1H, H3’).13 C NMR(125MHz,DMSO-d6)δC158.51(d, 1 J C-F =202.7Hz,C2),157.55(d, 3 J C-F =21.2Hz,C6),150.11(d, 3 J C-F =20.3Hz,C4),139.22(d, 6 J C-F =2.2Hz,C8),117.37(d, 4 J C-F =4.1Hz,C5),90.67(C1'),80.90(C4'),74.73(C2'),62.35(C5'),33.89(C3').

[0360] Preparation of 3'-deoxy-2-methoxyadenosine:

[0361] A solution of H2O / CH3CN (1:9; 1.4mL) and then α-AIBBr (4.10mL, 28.05mmol) was added to a suspension of dry 2-fluoroadenosine (2.0g, 7.01mmol) in anhydrous CH3CN (50mL) and continued stirring at room temperature (20°C). After 1 hour, saturated NaHCO3 solution was carefully added and the solution was extracted with EtOAc (2x100mL). The combined organic phase was washed with brine (1x50mL). The aqueous phase was extracted with EtOAc (2x50mL) and the combined organic phase was dried over Na2SO4, filtered and evaporated to give a white gum. The crude mixture was dissolved in anhydrous MeOH (50mL) and washed with 60mL of Amberlite (2x OH - ) resin (previously washed thoroughly with anhydrous MeOH) was stirred for 1 hour. The solution was then filtered, and the resin was carefully washed with THF. The combined filtrates were evaporated, and the residue was crystallized from EtOH to yield 2',3'-dehydro-2-methoxyadenosine (1.57 g, 84%) as a white solid.

[0362] A solution of LiEt3BH (1 M in THF; 8.53 mL, 8.53 mmol) was added dropwise to a cold (4 °C) solution of 2',3'-dehydro-2-methoxyadenosine (762 mg, 2.84 mmol) in dry DMSO / THF (1 / 10, 15 mL) under argon. Stirring was continued at 4 °C for 1 h and at room temperature overnight (16 h). The reaction mixture was carefully acidified (5% AcOH / H2O), purged with N2for 1 h (under a hood) to remove pyrophoric triethylborane, and evaporated. The residue was chromatographed on silica gel (3-17% MeOH in DCM) to give 3'-deoxy-2-methoxyadenosine as a white powder (650 mg, 81%).

[0363] 1 H NMR (500 MHz, CD3OD) δH8.20 (s, 1H, H8), 5.90 (d, J = 2.4 Hz, 1H, H1'), 4.75-4.71 (m, 1H, H2'), 4.54-4.48 (m, 1H, H4'), 3.91 (dd, J = 12.3, 2.5 Hz, 1H, H5'), 3.69 (dd, J = 12.30, 4.0 Hz, 1H, H5'), 3.37 (s, 3H, OCH3), 2.43-2.35 (m, 1H, H3'), 2.08-2.02 (m, 1H, H3'). 13 C NMR (125 MHz, CD3OD) δC163.68 (C2), 158.12 (C6), 151.94 (C4), 139.71 (C8), 116.64 (C5), 93.36 (C1'), 82.53 (C4'), 76.59 (C2'), 64.24 (C5'), 55.29 (OCH3), 34.81 (C3').

[0364] The chlorophosphates are prepared by the published method from aryl phosphorodichloridates and amino acid ester hydrochlorides.

[0365] 3'-Deoxyadenosine-5'-O-[phenyl(benzyloxy-L-alaninyl)]phosphonate A

[0366]

[0367] Compound A was prepared according to General Procedure 1 using 3'-deoxyadenosine (50 mg, 0.20 mmol), N-methylimidazole (80 μL, 1.0 mmol) and phenyl(benzyloxy-L-alanyl)phosphochloroate (212 mg, 0.6 mmol). Purification by column chromatography (eluent system CH3OH / CH2Cl2 0 / 100 to 7 / 93) with a gradient of CH2Cl2 / MeOH (100% to 95:5%) and preparative TLC (1000 μm, eluent system CH3OH / CH2Cl2 5 / 95) gave the title compound (31 mg, 28%) as a white solid.

[0368] 1 H NMR (500MHz, CD3OD): δH 8.26(s,0.5H,H8),8.24(s,0.5H,H8),8.22(s,0.5H,H2),8.21(s,0.5H,H2),7.34-7.25(m,7H,Ar),7.21 -7.13(m,3H,Ar),6.01(d,J=2.9Hz,1H,H1'),6.00(d,J=2.9Hz,1H,H1'),5.15-5.04(m,2H,OCH2Ph),4.73 -4.63(m,2H,H2',H4'),4.43-4.35(m,1H,H5'),4.27-4.20(m,1H,H5'),4.03-3.91(m,1H,CHCH3),2.35-2 .28(m,1H,H3'),2.09-2.02(m,1H,H3'),1.32(d,J=7.4Hz,1.5H,CHCH3),1.28(d,J=7.4Hz,1.5H,CHCH3).

[0369] 13 C NMR (125MHz, CD3OD): δC 174.84(d, 3 J C-P =4.5Hz,C=O),174.63(d, 3 J C-P= 4.5 Hz, C=0), 157.32 (C6), 157.31 (C6), 153.86 (C2), 153.84 (C2), 152.13 (C4), 152.07 (C4), 150.20 (C-Ar), 150.18 (C-Ar), 140.47 (C8), 137.26 (C-Ar), 137.19 (C-Ar), 130.76 (CH-Ar), 130.74 (CH-Ar), 129.57 (CH-Ar), 129.32 (CH-Ar), 129.31 (CH-Ar), 129.29 (CH-Ar), 129.26 (CH-Ar), 126.16 (CH-Ar), 126.14 (CH-Ar), 121.46 (d, 3 J C-P = 4.7 Hz, CH-Ar) 121.38 (d, 3 J C-P = 4.7 Hz, CH-Ar) 120.54 (C5), 120.53 (C5), 93.24 (C1'), 93.18 (C1'), 80.43 (d, 3 J C-P = 3.6 Hz, C4'), 80.36 (d, 3 J C-P = 3.6 Hz, C4'), 76.62 (C2'), 68.62 (d, 2 J C-P = 5.3 Hz, C5'), 68.30 (d, 2 J C-P = 5.3 Hz, C5'), 67.95 (OCH2Ph), 67.92 (OCH2Ph), 51.74 (CHCH3), 51.60 (CHCH3), 34.91 (C3'), 34.70 (C3'), 20.45 (d, 3 J C-P = 7.0 Hz, CHCH3), 20.28 (d, 3 J C-P = 7.0 Hz, CHCH3).

[0370] 31 P NMR (202 MHz, CD3OD): δΡ 3.9, 3.7.

[0371] MS (ES+) m / z: Found: 569.2 (M+H + ), 591.2 (M+Na + ), 1159.4 (2M+Na + ) C 26 H 29N6O7P required value: (M) 568.2.

[0372] HPLC using reverse phase HPLC, eluting with H2O / CH3CN from 100 / 10 to 0 / 100 over 30 minutes, 1 ml / min, l = 254 nm, showed two peaks of diastereomers with tR 14.02 min and tR 14.26 min.

[0373] (2S)-Benzyl 2-(((((2S,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2- yl)methoxy)(naphthalen-1-yloxy)phosphoryl)amino)propanoate B Benzyl 2-(((((2S,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2- yl)methoxy)(phenoxy)phosphoryl)amino)acetate C

[0374]

[0375] Using the general procedure 1 above, a solution of (2S)-benzyl 2-((chloro(naphthalen-1-yloxy)phosphoryl)amino)propanoate (727 mg, 1.8 mmol) in anhydrous THF (10 mL) and N-methylimidazole (240 μL, 3.0 mmol) were added dropwise to a suspension of 3'-deoxyadenosine (150 mg, 0.6 mmol) in anhydrous THF, and the reaction mixture was stirred at room temperature over a period of 16 hours. Purification by column chromatography (eluent system CH3OH / CH2Cl2 0 / 100 to 6 / 94) and preparative TLC (2000 μM, eluent system CH3OH / CH2Cl2 5 / 95) gave the title compound (45 mg, 12%) as a white solid.

[0376] MS (ES+) m / z: Found: 619.2 (M+H + ),641.2(M+Na + ),1259.4(2M+Na + )C 30 H 31 N6O7P required value: (M) 618.58.

[0377] 31 P NMR (202MHz, CH3OD): δP 4.3(s), 4.1(s).

[0378] 1H NMR (500 MHz, CH3OD): δH 8.24 (s, 0.5H, H8), 8.22 (s, 0.5H, H8), 8.20 (s, 0.5H, H2), 8.19 (s, 0.5H, H2), 8.14-8.09 (m, 1H, Ar), 7.89-7.85 (m, 1H, Ar), 7.70-7.67 (m, 1H, Ar), 7.53-7.42 (m, 3H, Ar), 7.39-7.34 (m, 1H, Ar), 7.31-7.25 (m, 5H, Ar), 5.99 (d, J = 2.0 Hz, 0.5H, H1’), 5.98 (d, J = 2.0 Hz, 0.5H, H1’), 5.10-5.01 (m, 2H, CH2Ph), 4.72-4.61 (m, 2H, H2’, H4’), 4.47-4.40 (m, 1H, H5’), 4.33-4.24 (m, 1H, H5’), 4.09-3.98 (m, 1H, CH ala) 2.35-2.26 (m, 1H, H3’), 2.07-1.98 (m, 1H, H3’), 1.30-1.24 (m, 3H, CH3).

[0379] 13 C NMR (125 MHz, CH3OD): δC 174.85 (d, 3 J C-P = 3.7 Hz, C=0), 174.56 (d, 3 J C-P = 3.7 Hz, C=0), 157.33 (C6), 157.31 (C6), 153.87 (C2), 153.85 (C2), 150.24 (C4), 150.23 (C4), 147.91 (d, 3 J C-P = 7.5 Hz, ‘ipso’ Nap), 147.95, (d, 3 J C-P = 7.5 Hz, ‘ipso’ Nap), 140.56 (C8), 140.50 (C8), 137.22 (C-Ar), 137.17 (C-Ar), 136.28 (C-Ar), 129.55 (CH-Ar), 129.53 (CH-Ar), 129.30 (CH-Ar), 129.25 (CH-Ar), 128.88 (CH-Ar), 128.82 (CH-Ar), 127.91 (d, 2 J C-P = 6.25 Hz, C-Ar), 127.83 (d, 2 J C-P= 6.25 Hz, C-Ar), 127.77 (CH-Ar), 127.75 (CH-Ar), 127.49 (CH-Ar), 127.45 (CH-Ar), 126.48 (CH-Ar), 126.47 (CH-Ar), 126.02 (CH-Ar), 125.97 (CH-Ar), 122.77 (CH-Ar), 122.63 (CH-Ar), 120.58 (C5), 120.53 (C5), 116.35 (d, 3 J C-P = 3.75 Hz, CH-Ar), 116.15 (d, 3 J C-P = 3.75 Hz, CH-Ar), 93.22 (C1'), 93.20 (C1'), 80.30 (d, 3 J C-P = 2.75 Hz, C4'), 80.24 (d, 3 J C-P = 2.75 Hz, C4'), 76.51 (C2'), 76.44 (C2'), 68.87 (d, 2 J C-P = 5.2 Hz, C5'), 68.64 (d, 2 J C-P = 5.2 Hz, C5'), 67.93 (OCH2Ph), 51.82 (CH ala), 51.73 (CH ala), 35.01 (C-3'), 34.76 (C3'), 20.41 (d, 3 J C-P = 6.7 Hz, CH3 ala), 20.22 (d, 3 J C-P = 6.7, CH3 ala).

[0380] HPLC using H2O / CH3CN from 100 / 10 to 0 / 100 elution in 30 min in reverse phase HPLC, 1 ml / min, l = 200 nm, showed two peaks of diastereoisomers with tR16.36 min and tR16.60 min.

[0381] (2S)-Pentyl 2-(((((2S,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2- yl)methoxy)(naphthalen-1-yloxy)phosphoryl)amino)-4-methylpentanoate D Methyl 2-(((((2S,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2- yl)methoxy)(naphthalen-1-yloxy)phosphoryl)amino)-2-methylpropanoate E

[0382]

[0383] Using the above general procedure 1, a solution of benzyl 2-((chloro(phenoxy)phosphoryl)amino)acetate (204 mg, 0.6 mmol) and N-methylimidazole (80 μί, 1.0 mmol) in dry THF (2 mL) was added dropwise to a suspension of 3'-deoxyadenosine (50 mg, 0.20 mmol) in dry THF and the reaction mixture was stirred at room temperature for 16 h. Purification by column chromatography (eluent system CH3OH / CH2Cl20 / 100 to 6 / 94) and preparative TLC (500 μΜ, eluent system CH3OH / CH2Cl25 / 95) afforded the target compound as a white solid (21 mg, 19%).

[0384] (ES+) m / z, found: 555.2 (M+H + ), 577.2 (M+Na + ), 1131.4 (2M+Na + ). C 25 H 27 N6O7P Required: (M) 554.2.

[0385] 31 P NMR (202 MHz, CH3OD) δ 5.1, 4.9.

[0386] 1 H NMR (500 MHz, CH3OD) δ 8.27 (s, 0.5H, H8), 8.24 (s, 0.5H, H8), 8.22 (s, 0.5H, H2), 8.21 (s, 0.5H, H2), 7.37 - 7.26 (m, 7H, Ph), 7.22 - 7.13 (m, 3H, Ph), 6.02 (d, J = 1.8 Hz, 0.5H, H1'), 6.00 (d, J = 1.8 Hz, 0.5H, H1'), 5.14 - 5.11 (m, 2H, OCH2Ph), 4.73 - 4.64 (m, 2H, H2', H4'), 4.50 - 4.39 (m, 1H, H5'), 4.36 - 4.24 (m, 1H, H5'), 3.53 - 3.71 (m, 2H, CH2gly), 2.39 - 2.25 (m, 1H, H3'), 2.13 - 2.02 (m, 1H, H3').

[0387] 13 C NMR (125 MHz, CH3OD) δ 172.30 (d, 3 J C-P = 5.0 Hz, C=0), 172.27 (d, 3 J C-P= 5.0 Hz, C=0), 157.34 (C6), 157.32 (C6), 153.88 (C2), 153.87 (C2), 152.08 (d, 3 J C-P = 7.5 Hz, C-Ar), 152.05 (d, 3 J C-P = 7.5 Hz, C-Ar), 150.20 (C4), 150.19 (C4), 140.52 (C8), 140.42 (C8), 137.15 (C-Ar), 130.79 (CH-Ar), 129.57 (CH-Ar), 129.55 (CH-Ar), 129.35 (CH-Ar), 129.34 (CH-Ar), 129.33 (CH-Ar), 126.22 (CH-Ar), 121.44 (d, C-P = 3.7 Hz, CH-Ar), 121.40 (d, C-P = 3.7 Hz, CH-Ar), 120.51 (C5), 120.49 (C5), 93.19, 93.14 (C1’), 80.46 (d, 3 J C-P = 4.60 Hz, C4’), 80.39 (d, 3 J C-P = 4.60, C4’), 76.66 (C2’), 68.68 (d, 2 J C-P = 5.42 Hz, C5’), 68.24 (d, 2 J C-P = 5.42 Hz, C5’), 67.95 (OCH2Ph), 67.93 (OCH2Ph), 43.90 (CH2gly), 43.83 (CH2gly), 34.83 (C3’), 34.54 (C3’).

[0388] HPLC using H2O / CH3CN from 100 / 10 to 0 / 100 elution in 30 min in reverse phase HPLC, 1 ml / min, l = 200 nm, showed two peaks of diastereoisomers with tR13.63 min and tR13.41 min.

[0389] (2S)-Benzyl 2-(((((2S,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2- yl)methoxy)(2-(3-ethoxy-3-oxopropyl)phenoxy)phosphoryl)amino)propanoate F (2S)-Benzyl 2-(((((2R,3R,5S)-2-(6-amino-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran- 3-yl)oxy)(phenoxy)phosphoryl)amino)propanoate G

[0390]

[0391] Using the above General Procedure 1, a solution of (2S)-pentyl 2-((chloro(naphthalen- 1-yloxy)phosphoryl)amino)-4-methylpentanoate (250 mg, 0.6 mmol) in dry THF (1 mL) and N-methylimidazole (76 μL, 0.95 mmol) was added dropwise to a suspension of 3'-deoxyadenosine (48 mg, 19 mmol) in dry THF (5 mL) and the reaction mixture was stirred at room temperature during 16 hours. Purification by column chromatography (eluent system CH3OH / CH2Cl20 / 100 to 5 / 95) and preparative TLC (1000 μM, eluent system CH3OH / CH2Cl24 / 96) afforded the target compound as a white solid (27 mg, 22%).

[0392] MS (ES+) m / z: found: 641.3 (M+H + ), 663.3 (M+Na + ), 1303.6 (2M+Na + )C 31 H 41 N6O7P requires: (M) 640.3.

[0393] 31 P NMR (202 MHz, CH3OD) δ 4.64, 4.37.

[0394] 1H NMR (500 MHz, CH3OD) δ 8.28 (s, 0.5H, H-8), 8.25 (s, 0.5H, H-8), 8.21 (s, 0.5H, H-2), 8.20 (s, 0.5H, H-2), 8.17-8.12 (m, 1H, Nap), 7.88-7.83 (m, 1H, Nap), 7.69-7.66 (m, 1H, Nap), 7.54-7.42 (m, 3H, Nap), 7.40-7.35 (m, 1H, Nap), 7.31-7.26 (m, 5H, Ar), 6.01 (d, J = 2.1 Hz, 0.5H, H1’), 6.00 (d, J = 2.1 Hz, 0.5H, H1’), 4.47-4.67 (m, 2H, H2’, H4’), 4.55-4.44 (m, 1H, H5’), 4.43-4.31 (m, 1H, H5’), 4.00-3.87 (m, 3H, CH leu, CH2 Pen), 2.44-2.30 (m, 1H, H3’), 2.14-2.04 (m, 1H, H3’), 1.66-1.39 (m, 5H, CH2CH leu, CH2 Pen), 1.1.28-1.21 (m, 4H, CH2CH2 Pen), 0.86-0.81 (m, 3H, CH3 Pen), 0.81-0.68 (m, 6H, (CH3)2leu).

[0395] 13 C NMR (125 MHz, CH3OD) δ 175.42 (d, 3 J C-P = 2.5 Hz, C=0), 175.04 (d, 3 J C-P = 2.5 Hz, C=0), 157.32 (C6), 153.87 (C2), 153.86 (C2), 150.23 (C4), 147.97 (d, 3 J C-P= 6.2 Hz, 'ipso' Nap), 140.55 (C8), 136.30 (C-Ar), 136.29 (C-Ar), 128.89 (CH-Ar), 128.84 (CH-Ar), 127.95 (C-Ar), 127.91 (C-Ar), 127.84 (C-Ar), 127.78 (CH-Ar), 127.76 (CH-Ar), 127.46 (CH-Ar), 126.50 (C-Ar), 126.48 (C-Ar), 126.46 (C-Ar), 126.01 (CH-Ar), 125.91 (CH-Ar), 122.80 (CH-Ar), 122.70 (CH-Ar), 120.58 (C5), 120.56 (C5), 116.40 (d, 3 J C-P = 3.7 Hz, CH-Ar), 116.01 (d, 3 J C-P = 3.7 Hz, CH-Ar), 93.31 (C1'), 93.27 (C1'), 80.35 (d, 3 J C-P = 3.5 Hz, C4'), 80.29 (d, 3 J C-P = 3.5 Hz, C4'), 76.54 (C2'), 76.50 (C2'), 69.07 (d, 2 J C-P = 5.5 Hz, C5'), 68.85 (d, 2 J C-P = 5.5 Hz, C5'), 66.33 (CH2 Pent), 66.32 (CH2 Pent), 54.81 (CH leu), 54.71 (CH leu), 44.22 (d, 3 J C-P = 7.6 Hz, CH2 leu), 43.93 (d, 3 J C-P = 7.6 Hz, CH2 leu), 35.15 (C3'), 34.86 (C3'), 29.32 (CH2 pent), 29.30 (CH2 Pent), 29.11 (CH2 pent), 25.67 (CH leu), 25.45 (CH leu), 23.30 (CH2 pent), 23.12 (CH3 leu), 23.02 (CH3 leu), 22.04 (CH3 leu), 21.78 (CH3 leu), 14.28 (CH3 pent).

[0396] HPLC using reverse phase HPLC eluting with H20 / CH3CN from 100 / 10 to 0 / 100 for 30 min, 1 ml / min, l = 200 nm showed one peak for two overlapping diastereoisomers with tR20.84 min.

[0397] Benzyl 2-(((((2S,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-((((1-(benzyloxy)-1-oxopropan-2- yl)amino)(phenoxy)phosphoryl)oxy)tetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)- amino)propanoate H (2S)-Benzyl 2-(((((2R,3R,5S)-2-(6-amino-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran- 3-yl)oxy)(naphthalen-1-yloxy)phosphoryl)amino)propanoate I

[0398]

[0399] Using the above General Procedure 1, a solution of methyl 2-((chloro(naphthalen-1-yloxy)phosphoryl)amino)-2-methylpropanoate (612 mg, 1.8 mmol) in dry THF (1 mL) and N-methylimidazole (24 μL, 3.0 mmol) was added dropwise to a suspension of 3'-deoxyadenosine (150 mg, 0.6 mmol) in dry THF (15 mL) and the reaction mixture was stirred at room temperature during 16 hours. Purification by column chromatography (eluent system CH3OH / CH2Cl20 / 100 to 7 / 93) and preparative TLC (1000 μM, eluent system CH3OH / CH2Cl24 / 96) afforded the target compound as a white solid (20 mg, 6%).

[0400] MS (ES+) m / z: found: 557.2 (M+H + ), 579.2 (M+Na + ), 1135.4 (2M+Na + ) C 25 H 29 N6O7P requires: (M) 556.51.

[0401] 31 P NMR (202 MHz, CH3OD) δ 2.73.

[0402] 1H NMR (500 MHz, CH3OD) δ 8.28 (s, 0.5H, H8), 8.25 (s, 0.5H, H8), 8.21 (s, 0.5H, H2), 8.19 (s, 0.5H, H2), 8.18-8.14 (m, 1H, Nap), 7.90-7.84 (m, 1H, Nap), 7.71-7.66 (m, 1H, Nap), 7.53-7.47 (m, 3H, Nap), 7.41-7.35 (m, 1H, Nap), 6.03 (d, J = 2.1 Hz, 0.5H, H1’), 5.99 (d, J = 2.1 Hz, 0.5H, H1’), 4.76-4.67 (m, 2H, H2’, H4’), 4.52-4.44 (m, 1H, H5’), 4.42-4.33 (m, 1H, H5’), 3.65 (s, 1.5H, OCH3), 3.64 (s, 1.5H, OCH3), 2.48-2.41 (m, 0.5H, H3’), 2.37-2.30 (m, 0.5H, H3’), 2.15-2.09 (m, 0.5H, H3’), 2.08-2.02 (m, 0.5H, H3’), 1.47-1.44 (m, 6H, CH3).

[0403] 13 C NMR (125 MHz, CH3OD) δ 177.25 (d, 3 J C-P = 3.7 Hz, C=0), 157.53 (C6), 157.51 (C6), 153.86 (C2), 150.28 (C4), 150.25 (C4), 148.06 (d, 3 J C-P = 7.5 Hz, ‘ipso’ Nap), 148.04 (d, 3 J C-P = 7.5, ‘ipso’ Nap), 140.67 (C8), 140.60 (C8), 136.28 (C-Ar), 136.27 (C-Ar), 128.82 (CH-Ar), 128.80 (CH-Ar), 127.93 (d, 2 J C-P = 6.25 Hz, C-Ar), 127.92 (d, 2 J C-P = 6.25 Hz, C-Ar), 127.71 (CH-Ar), 127.69 (CH-Ar), 127.32 (CH-Ar), 126.44 (CH-Ar), 125.84 (CH-Ar), 122.93 (CH-Ar), 120.56 (C5), 120.50 (C5), 116.38 (d,3 J C-P = 3.75 Hz, CH-Ar), 116.36 (d, 3 J C-P = 3.75 Hz, CH-Ar), 93.25 (C1'), 80.40 (d, 3 J C-P = 8.0 Hz, C4'), 80.33 (d, 3 J C-P = 8.0 Hz, C4'), 76.57 (C2'), 76.43 (C2'), 68.99 (d, 2 J C-P = 5.5 Hz, C5'), 68.84 (d, 2 J C-P = 5.5 Hz, C5'), 53.01 (OCH3), 35.22 (C-3'), 34.90 (C3'), 27.85 (d, 3 J C-P = 6.0 Hz, CH3), 27.80 (d, 3 J C-P = 6.0, CH3), 27.60 (d, 3 J C-P = 6.0, CH3), 27.56 (d, 3 J C-P = 6.0, CH3).

[0404] HPLC using reverse phase HPLC with H2O / CH3CN elution from 100 / 10 to 0 / 100 in 30 min, 1 ml / min, l = 254 nm showed two peaks with tR16.51 min and tR16.75 min.

[0405] Benzyl 2-[({[5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl]methoxy}({[1-(benzyloxy)- 1-oxopropan-2-yl]amino} phosphoryl)amino]propanoate J (2S)-Benzyl 2-((((2S,4R,5R)-5-(6-amino-2-methoxy-9H-purin-9-yl)-4-hydroxytetrahydrofuran- 2-yl)methoxy)(naphthalen-1-yloxy)phosphoryl)amino)propanoate K

[0406]

[0407] Using the above General Procedure 1, a solution of (2S)-benzyl 2-((chloro(2-(3- ethoxy-3-oxopropyl)phenoxy)phosphoryl)amino)propanoate (1.14 g, 2.5 mmol) in anhydrous THF (2 mL) and N-methylimidazole (32 μL, 4.2 mmol) was added dropwise to a suspension of 3'-deoxyadenosine (210 mg, 0.84 mmol) in anhydrous THF (10 mL) and the reaction mixture was stirred at room temperature during 16 hours. Purification by column chromatography (eluent system CH3OH / CHCl30 / 100 to 8 / 92) and preparative TLC (1000 μM, eluent system CH3OH / CH2Cl25 / 95) afforded the target compound as a white solid (123 mg, 22%).

[0408] MS (ES+) m / z: found: 669.3 (M+H + ), 691.3 (M+Na + ), C 31 H 37 N6O9P requires: (M) 668.63.

[0409] 31 P NMR (202 MHz, CH3OD): δP 3.95, 3.65.

[0410] 1H NMR (500 MHz, CH3OD): δH 8.25 (s, 0.5H, H8), 8.21 (s, 1H, H8, H2), 8.20 (s, 0.5H, H2), 7.35-7.29 (m, 6H, Ph), 7.25-7.21 (m, 1H, Ph), 7.16-7.07 (m, 2H, Ar), 6.00 (d, J = 1.9 Hz, 0.5H, H1'), 5.98 (d, J = 1.9 Hz, 0.5H, H1'), 5.17-5.05 (m, 2H, OCH2Ph), 4.76-4.73 (m, 0.5H, H2'), 4.70-4.59 (m, 1.5H, H2', H4'), 4.45-4.34 (m, 1H, H5'), 4.30-4.22 (m, 1H, H5'), 4.08-3.96 (m, 3H, CH2CH3, CH ala), 2.98-2.92 (m, 2H, CH2CH2), 2.62-2.56 (m, 2H, CH2CH2), 2.40-2.29 (m, 1H, H3'), 2.11-2.03 (m, 1H, H3'), 1.36 (d, J = 6.9 Hz, 1.5H, CH3 ala), 1.33 (d, J = 6.9 Hz, 1.5H, CH3 ala), 1.17 (t, J = 7.0 Hz, 1.5H, CH2CH3), 1.16 (t, J = 7.0 Hz, 1.5H, CH2CH3).

[0411] 13 C NMR (125 MHz, CH3OD): δC 174.82 (d, 3 J C-P = 3.7 Hz, C=0), 174.62 (C=0), 174.58 (C=0), 174.55 (d, 3 J C-P = 3.7 Hz, C=0), 157.34 (C6), 157.32 (C6), 153.86 (C2), 153.84 (C2), 150.48 (d, J C-P = 2.5 Hz, C-Ar), 150.44 (C4), 150.22 (d, J C-P=2.5Hz,C-Ar),140.49(C8),137.29(C-Ar),137.21(C-Ar),133.09(d,J=7.5Hz,C-Ar),132.94(d, J=7.5Hz,C-Ar),131.62(CH-Ar),131.59(CH-Ar),129.58(CH-Ar),129.34(CH-Ar),129.31(CH-Ar) ,129.28(CH-Ar),128.70(d,J=5.0Hz,CH-Ar),128.69(d,J=5.0Hz,CH-Ar),126.18(CH-Ar),121.02 (d,J=2.5Hz,CH-Ar),120.49(d,J=2.5Hz,CH-Ar),120.58(C5),93.28(C1'),93.24(C1'),80.32(d, 3 J C-P =8.7Hz,C4'),76.57(C2'),68.86(d, 2 J C-P =5.0Hz,C5'),68.53(d, 2 J C-P =5.0Hz,C5'),67.98(OCH2Ph),67.95(OCH2Ph),61.57(CH2CH3),51.76(CH ala),51.65(CH ala),35.37(CH2CH2),35.30(CH2CH2),35.08(C3'),34.85(C3'),26.77(CH2CH2),26.72(CH2CH2),20.55(d, 3 J C-P =6.2Hz,CH3 ala),20.33(d, 3 J C-P =6.2Hz,CH3ala),14.53(CH2CH3).

[0412] HPLC using reverse phase HPLC, eluting with H2O / CH3CN from 100 / 10 to 0 / 100 over 30 minutes, 1 ml / min, l=245 nm, showed one peak with tR 15.99 min.

[0413] ​ ​

[0414]

[0415] Using the general procedure 3 above, 3'-deoxyadenosine (50 mg, 0.20 mmol) was suspended in dry THF (5 mL) and a solution of (2S)-benzyl 2-((chloro(phenoxy)phosphoryl)amino)propanoate (212 mg, 0.6 mmol) in dry THF (2 mL) was added dropwise and the reaction mixture was stirred at room temperature during 16 hours. Purification by column chromatography (eluent system CH3OH / CH2Cl20 / 100 to 8 / 92) and preparative TLC (500 μM, eluent system CH3OH / CH2Cl2= 5 / 95) afforded the target compound as a white solid (6 mg, 5%). t BuMgCl (1.0 M in THF, 0.22 mL, 0.22 mmol). A solution of (2S)-benzyl 2-((chloro(phenoxy)phosphoryl)amino)propanoate (212 mg, 0.6 mmol) in dry THF (2 mL) was added dropwise and the reaction mixture was stirred at room temperature during 16 hours. Purification by column chromatography (eluent system CH3OH / CH2Cl20 / 100 to 8 / 92) and preparative TLC (500 μM, eluent system CH3OH / CH2Cl2= 5 / 95) afforded the target compound as a white solid (6 mg, 5%).

[0416] MS (ES+) m / z: found: 569.2 (M+H + ), 591.2 (M+Na + ), 1159.4 (2M+Na + )C 26 H 29 N6O7P requires: (M) 568.2.

[0417] 31 P NMR (202 MHz, CH3OD): δP 2.44 (s), 2.92 (s).

[0418] 1 H NMR (500 MHz, CH3OD): δH 8.41 (s, 0.5H, H8), 8.28 (s, 0.5H, H8), 8.19 (s, 0.5H, H2), 8.18 (s, 0.5H, H2), 7.39 - 7.30 (m, 4H, Ar), 7.28 - 7.18 (m, 4H, Ar), 7.17 - 7.11 (m, 1H, Ar), 7.08 - 7.03 (m, 1H, Ar), 6.23 (d, J = 2.0 Hz, 0.5H, H1'), 6.08 (d, J = 3.4 Hz, 0.5H, H1'), 5.52 - 5.43 (m, 1H, C2'), 5.19 - 5.12 (m, 1H, CH2Ph), 5.07 - 4.95 (m, 1H, CH2Ph), 4.48 - 4.42 (m, 1H, H4'), 4.05 - 3.97 (m, 1H, CH ala), 3.95 - 3.87 (m, 1H, H5'), 3.69 - 3.61 (m, 1H, H5'), 2.59 - 2.45 (m, 1H, H3'), 2.31 - 2.23 (m, 1H, H3'), 1.36 - 1.27 (m, 3H, CH3 ala).

[0419] 13 C NMR (125 MHz, CH3OH): δc 174.76 (d, 3 J C-P = 5.0 Hz, C=0), 174.52 (d, 3 J C-P = 5.0 Hz, C=0), 157.44 (C6), 153.76 (C2), 151.93 (C4), 150.06 (C-Ar), 149.93 (C-Ar), 141.38 (C8), 141.18 (C8), 137.33 (C-Ar), 137.10 (C-Ar), 130.69 (CH-Ar), 130.79 (CH-Ar), 129.61 (CH-Ar), 129.51 (CH-Ar), 129.40 (CH-Ar), 129.30 (CH-Ar), 129.23 (CH-Ar), 126.33 (CH-Ar), 126.16 (CH-Ar), 121.53 (d, 3 J C-P = 4.5 Hz, CH-Ar), 121.20 (d, 3 J C-P = 4.5 Hz, CH-Ar), 120.76 (C5), 91.56 (d, 3 J C-P = 7.7 Hz, C1'), 91.45 (d, 3 J C-P = 7.7 Hz, C1'), 82.78 (C4'), 82.28 (C4'), 81.83 (d, 2 J C-P = 4.7 Hz, C2'), 80.96 (2 x d, 2 J C-P = 4.7 Hz, C2'), 67.95 (OCH2Ph), 67.92 (OCH2Ph), 64.13 (C5'), 63.59 (C5'), 51.88 (CH ala), 51.75 (CH ala), 33.75 (d, 3 J C-P = 3.0 Hz, C3'), 33.59 (d, 3 J C-P = 3.0 Hz, C3'), 20.33 (d, 3 J C-P = 7.1 CH3 ala), 20.18 (d, 3 J C-P = 7.1 CH3 ala).

[0420] HPLC using reverse phase HPLC with H20 / CH30H elution from 90 / 10 to 0 / 100 for 30 min, 1 ml / min, l=254 nm, showed two peaks of diastereoisomers with tR 22.16 min and tR 22.43 min.

[0421] ​ ​ ​

[0422]

[0423] Using the above general procedure 3, 3'-deoxyadenosine (50 mg, 0.20 mmol) was suspended in dry THF (5 mL) and added dropwise t BuMgCl (1.0 M in THF, 0.22 mL, 0.22 mmol). A solution of (2S)-benzyl 2-((chloro(phenoxy)phosphoryl)amino)propanoate (212 mg, 0.6 mmol) in dry THF (2 mL) was added dropwise and the reaction mixture was stirred at room temperature during 16 hours. Purification by column chromatography (eluent system CH3OH / CH2Cl20 / 100 to 8 / 92) and preparative TLC (500 μM, eluent system CH3OH / CH2Cl25 / 95) afforded the target compound as a white solid (19 mg, yield 11%).

[0424] MS (ES+) m / z, found: 886.3 (M+H + ), 1771.6 (2M+H + ), 751.2 (molecule without nucleobase M). C 42 H 45 N7O 11 P2 required value: (M+) 885.3.

[0425] 31 P NMR (202 MHz, CH30D): δΡ 3.98, 3.88, 3.59, 3.12, 3.05, 2.45, 2.32.

[0426] 1H NMR (500 MHz, CH3OD): δH 8.24-8.13 (m, 2H, H8, H2), 7.39-7.08 (m, 20H, Ph), 6.27-6.23 (m, 0.5H, H1'), 6.16-6.13 (m, 0.5H, H1'), 5.61-5.48 (m, 1H, H2'), 5.17-4.91 (m, 4H, CH2Ph), 4.57-4.49 (m, 1H, H4'), 4.41-4.29 (m, 1H, H5'), 4.25-4.15 (m, 1H, H5'), 4.10-4.01 (m, 1H, CH ala), 3.99-3.89 (m, 1H, CH ala), 2.57-2.41 (m, 1H, H3'), 2.28-2.17 (m, 1H, H3'), 1.38-1.23 (m, 6H, CH3 ala).

[0427] 13C NMR (125 MHz, CH3OD): δc174.88 (C=0), 174.83 (C=0), 174.79 (C=0), 174.73 (C=0), 174.61 (C=0), 174.57 (C=0), 174.53 (C=0), 157.36 (C6), 157.34 (C6), 157.32 (C6), 157.29 (C6), 154.04 (C2), 154.01 (C2), 153.97 (C2), 153.94 (C2), 152.09 (C4), 152.04 (C4), 152.02 (C4), 151.97 (C4), 150.31 (C-Ar), 150.29 (C-Ar), 150.16 (C-Ar), 140.98 (C8), 140.91 (C8), 140.81 (C8), 137.31 (C-Ar), 137.28 (C-Ar), 137.22 (C-Ar), 137.09 (C-Ar), 130.86 (CH-Ar), 130.78 (CH-Ar), 130.77 (CH-Ar), 129.65 (CH-Ar), 129.61 (CH-Ar), 129.58 (CH-Ar), 129.55 (CH-Ar), 129.44 (CH-Ar), 129.42 (CH-Ar), 129.38 (CH-Ar), 129.34 (CH-Ar), 129.32 (CH-Ar), 129.30 (CH-Ar), 129.28 (CH-Ar), 129.23 (CH-Ar), 129.21 (CH-Ar), 12.42 (CH-Ar), 126.23 (CH-Ar), 126.20 (CH-Ar), 126.17 (CH-Ar), 121.65 (CH-Ar), 121.63 (CH-Ar), 121.61 (CH-Ar), 121.59 (CH-Ar), 121.52 (CH-Ar), 121.50 (CH-Ar), 121.47 (CH-Ar), 121.46 (CH-Ar), 121.40 (CH-Ar), 121.39 (CH-Ar), 121.36 (CH-Ar), 121.35 (CH-Ar), 121.30 (CH-Ar), 121.28 (CH-Ar), 121.26 (CH-Ar), 121.24 (CH-Ar), 120.61 (C5), 120.57 (C5), 120.56 (C5), 120.54 (C5), 91.56 (C1’), 91.51 (C1’), 91.45 (C1’), 91.25 (C1’), 91.20 (C1’), 81.84 (C2’), 81.82 (C2’), 81.79 (C2’), 81.27 (C2'), 81.22 (C2'), 81.18 (C2'), 80.49 (C4'), 80.43 (C4'), 80.06 (C4'), 79.99 (C4'), 68.29 (C5', OCH2Ph), 68.25 (C5', OCH2Ph), 68.00 (C5', OCH2Ph), 67.96 (C5', OCH2Ph), 67.94 (C5', OCH2Ph), 67.90 (C5', OCH2Ph), 67.71 (C5', OCH2Ph), 67.67 (C5', OCH2Ph), 51.91 (CH ala), 51.74 (CH ala), 51.70 (CH ala), 51.59 (CH ala), 34.22 (C3'), 34.20 (C3'), 34.16 (C3'), 33.97 (C3'), 33.94 (C3'), 33.91 (C3'), 20.44 (CH3 ala), 20.43 (CH3 ala), 20.39 (CH3 ala), 20.29 (CH3 ala), 20.27 (CH3 ala), 20.24 (CH3 ala), 20.21 (CH3 ala), 20.19 (CH3 ala).

[0428] HPLC using reverse phase HPLC eluting with H2O / CH3CN from 100 / 10 to 0 / 100 for 30 min, 1 ml / min, l = 254 nm showed one broad peak with tR15.97 min.

[0429] ​ ​

[0430]

[0431] Using the above general procedure 3, 3'-deoxyadenosine (50 mg, 0.20 mmol) was suspended in dry THF (5 mL) and added dropwise t BuMgCl (1.0 M in THF, 0.3 mL, 0.3 mmol). A solution of (2S)-benzyl 2-((chloro(naphthalen-1-yloxy)phosphoryl)amino)propanoate (323 mg, 0.8 mmol) in dry THF (2 mL) was added dropwise and the reaction mixture was stirred at room temperature during 16 hours. Purification by column chromatography (eluent system CH3OH / CH2Cl20 / 100 to 6 / 94) and preparative TLC (500 μM, eluent system CH3OH / CH2Cl25 / 95) afforded the target compound as a white solid (14 mg, 11%).

[0432] (ES+) m / z, found: 619.2 (M+H + ), 641.2 (M+Na + ), 1259.4 (2M+Na + ) C 30 H 31 N6O7P requires: (M) 618.20.

[0433] 31 P NMR (202 MHz, CH3OD): δΡ 3.27 (s), 2.75 (s).

[0434] 1 H NMR (500 MHz, CH3OD): δΗ 8.37 (s, 1H, H8), 8.18 (s, 1H, H8), 8.14 (s, 1H, H2), 8.13-8.11 (m, 0.5H, Nap) 8.11 (s, 1H, H2), 7.94-7.90 (m, 0.5H, Ar), 7.90-7.87 (m, 0.5H, Ar), 7.86-7.82 (m, 0.5H, Ar), 7.74-7.70 (m, 0.5H, Ar), 7.66-7.61 (m, 0.5H, Ar), 7.57-7.47 (m, 1.5H, Ar), 7.46-7.37 (m, 2.5H, Ar), 7.34-7.27 (m, 4H, Ar), 7.25-7.17 (m, 1H, Ar), 6.19 (d, J = 2.4 Hz, 0.5H, H1’), 6.04 (d, J = 2.4 Hz, 0.5H, H1’), 5.60-5.54 (m, 0.5H, H2’), 5.50-5.42 (m, 0.5H, H2’), 5.16-4.99 (m, 2H, OCH2Ph), 4.46-4.40 (m, 0.5H, H4’), 4.36-4.30 (m, 0.5H, H4’), 4.13-4.04 (m, 1H, CH ala), 3.90-3.83 (m, 1H, H5’), 3.64-3.56 (m, 1H, H5’), 2.61-2.54 (m, 0.5H, H3’), 2.49-2.41 (m, 0.5H, H3’), 2.35-2.27 (m, 0.5H, H3’), 2.22-2.16 (m, 0.5H, H3’), 1.35-1.24 (m, 3H, CH3 ala).

[0435] 13C NMR (125 MHz, CH3OH): δc 174.52 (C=0), 174.49 (C=0), 157.27 (C6), 153.58 (C2), 149.97 (C4), 149.93 (C-4), 147.70 (d, 3 J C-P = 7.5, 'ipso' Nap), 147.48 (d, 3 J C-P = 7.5, 'ipso' Nap), 141.36 (C8), 141.19 (C8), 137.25 (C-Ar), 137.05 (C-Ar), 136.31 (C-Ar), 136.20 (C-Ar), 129.58 (CH-Ar), 129.48 (CH-Ar), 129.37 (CH-Ar), 129.26 (CH-Ar), 129.22 (CH-Ar), 128.88 (CH-Ar), 127.84 (CH-Ar), 127.75 (CH-Ar), 127.49 (CH-Ar), 127.44 (CH-Ar), 126.48 (CH-Ar), 126.39 (CH-Ar), 126.26 (CH-Ar), 126.05 (CH-Ar), 122.76 (CH-Ar), 122.38 (CH-Ar), 120.68 (C5), 120.61 (C5), 116.64 (d, 3 J C-P = 3.75 Hz, CH-Ar), 116.13 (d, 3 J C-P = 3.75, CH-Ar), 91.60 (d, 3 J C-P = 7.5 Hz, C1'), 91.43 (d, 3 J C-P = 7.5 Hz, C1'), 82.74 (C4'), 82.27 (C4'), 81.99 (d, 2 J C-P = 5.5 Hz, C2'), 81.12 (d, 2 J C-P = 5.5 Hz, C2'), 67.97 (OCH2Ph), 67.94 (OCH2Ph), 64.16 (C5'), 63.51 (C5'), 51.96 (CH ala), 51.89 (CH ala), 33.89 (d, 3 J C-P = 7.5 Hz, CH3 ala), 33.63 (d, 3 J C-P= 7.5 Hz, CH3 ala).

[0436] HPLC using H2O / CH3ON from 100 / 10 to 0 / 100 elution for 30 min in reverse phase HPLC, 1 ml / min, l = 200 nm, showed two peaks of diastereoisomers with tR24.84 min and tR25.43 min.

[0437] ​ ​

[0438]

[0439] Using the above general procedure 2, 3'-deoxyadenosine (200 mg, 0.80 mmol) was suspended in (CH3O)3PO (5 mL) and POCl3(75 μL, 0.80 mmol) was added dropwise at -5 °C. The reaction mixture was allowed to reach room temperature and stirring was maintained for 4 h. At -78 °C, a solution of (S)-1-(benzyloxy)-1-oxopropan-2-aminium 4-methylbenzenesulfonate (1.4 g, 4.0 mmol) in dry CH2Cl2(5 mL) was added, followed by diisopropylethylamine (1.4 mL, 8.0 mmol). After stirring at room temperature for 20 h, water was added and the layers were separated. The aqueous phase was extracted with dichloromethane and the organic phase was washed with brine. The combined organic layers were dried over Na2SO4and concentrated. The residue was purified by column chromatography (elution gradient CH2Cl2 / MeOH = 100 / 0 to 93 / 7) to give a white foam (256 mg, 49%).

[0440] MS (ES+) m / z: found: 654.2 (M+H + ), 676.2 (M+Na + ), 1329.5 (2M+Na + ) C 30 H 36 N7O8P requires: (M) 653.62.

[0441] 31 P NMR (202 MHz, CH3OD) δ 13.9.

[0442] 1H NMR (500 MHz, CH3OD) δ 8.28 (s, 1H, H8), 8.22 (s, 1H, H2), 7.37-7.26 (m, 10H, Ph), 6.00 (d, J = 1.9 Hz, 1H, H1’), 5.15-5.05 (m, 4H, OCH2Ph), 4.74-4.70 (m, 1H, H2’), 4.63-4.56 (m, 1H, H4’), 4.24-4.18 (m, 1H, H5’), 4.11-4.05 (m, 1H, H5’), 3.97-3.87 (m, 1H, CH ala), 2.35-2.27 (m, 1H, H3’), 2.07-2.01 (m, 1H, H3’), 1.34-1.27 (m, 3H, CH3 ala).

[0443] 13 C NMR (125 MHz, CH3OD) δ 175.40 (d, 3 J C-P = 5.0 Hz, C=0), 175.36 (d, 3 J C-P = 5.0 Hz, C=0), 157.36 (C6), 153.91 (C2), 150.25 (C4), 140.64 (C8), 137.33 (C-Ar), 137.29 (C-Ar), 129.58 (CH-Ar), 129.57 (CH-Ar), 129.33 (CH-Ar), 129.31 (CH-Ar), 129.29 (CH-Ar), 120.55 (C5), 93.18 (C1’), 80.67 (d, 3 J C-P = 8.4 Hz, C4’), 76.59 (C2’), 67.90 (OCH2Ph), 67.47 (d, 2 J C-P = 5.2 Hz, C5’), 51.14 (d, 2 J C-P = 1.7 Hz, CH ala), 51.11 (d, 2 J C-P = 1.7 Hz, CH ala), 35.08 (C3’), 20.77 (d, 3 J C-P = 6.5 Hz, CH3 ala), 20.59 (d, 3 J C-P = 6.5 Hz, CH3 ala).

[0444] HPLC using reverse phase HPLC with H20 / CH3CN elution from 90 / 10 to 0 / 100 for 30 min, 1 ml / min, l=254 nm showed one peak with tR 13.87 min.

[0445] ​ Furan-2-yl)methoxy)(naphthalen-1-yloxy)phosphorylaminol)propionic acid ester K

[0446]

[0447] Using the above General Procedure 1, a solution of (2S)-benzyl 2-((chloro(naphthalen- 1-yloxy)phosphoryl)amino)propanoate (303 mg, 0.75 mmol) in anhydrous THF (5 mL) and N-methylimidazole (99 μί, 1.24 mmol) was added dropwise to a suspension of 2-0-methyl-3'-deoxyadenosine (70 mg, 0.25 mmol) in anhydrous THF (10 mL) and the reaction mixture was stirred at room temperature during 16 hours. Purification by column chromatography (eluent system CH3OH / CH2Cl20 / 100 to 6 / 94) and preparative TLC (eluent system CH3OH / CH2Cl25 / 95) afforded the target compound as a white solid (96 mg, 60%).

[0448] MS (ES+) m / z: found: 649.2 (M+H + )C 31 H 33 N6O8P requires: 648.21 (M). 31 P NMR (202 MHz, CD3OD): δP 4.38 (s), 4.08 (s). 1H NMR (500 MHz, CD3OD): δH 8.14-8.11 (d, J = 8.0 Hz, 0.5H, Ar), 8.07 (d, J = 8.0 Hz, 0.5H, Ar), 8.05 (s, 0.5H, H8), 8.02 (s, 0.5H, H8), 7.82-7.80 (m, 1H, Ar), 7.61 (d, J = 7.0 Hz, Ar), 7.47-7.44 (m, 4H, Ar), 7.35-7.29 (m, 2H, Ar), 7.24-7.22 (m, 3H, Ar), 5.88 (s, 1H, H1’), 4.71-4.68 (m, 1H, H4’), 4.65-6.60 (m, 1H, H2’), 4.42-4.40 (m, 1H, H5’), 4.30-4.27 (m, 1H, H5’), 4.08-3.98 (m, 1H, CH ala) 3.88 (s, 1.5H, OCH3), 3.86 (s, 1.5H, OCH3), 2.37-2.33 (m, 1H, H3’), 2.04-2.01 (m, 1H, H3’), 1.27 (d J = 7.0 Hz, 1.5H, CH3), 1.24 (d J = 7.0 Hz, 1.5H, CH3). 13 C NMR (125 MHz, CH3OD): δC 174.83 (d, 3 J C-P = 3.7 Hz, C=0), 174.60 (d, 3 J C-P = 3.7 Hz, C=0), 163.70 (C-2), 158.10 (C6), 151.95 (C4), 147.95 (d, 3 J C-P = 7.5 Hz, ‘ipso’ Nap), 147.91, (d, 3 J C-P = 7.5 Hz, ‘ipso’ Nap), 139.39 (C8), 139.37 (C8), 137.12, 137.17 (C-ipso CH2Ph), 136.22 (C-Ar), 129.57, 129.54, 129.48, 129.32, 129.27, 129.12, 129.24 128.89, 128.83, (CH-Ar), 127.85 (d, 2 J C-P= 6.25 Hz, C-Ar), 127.86, 127.76, 127.51, 127.48, 126.49, 126.00, 125.97, 122.73, 122.63 (CH-Ar), 116.86 (C5), 116.72 (C5), 116.29 (d, 3 J C-P = 3.75 Hz, CH-Ar), 116.22 (d, 3 J C-P = 3.75 Hz, CH-Ar), 93.33 (C1'), 93.31 (C1'), 80.24 (d, 3 J C-P = 2.75 Hz, C4'), 76.29 (C2'), 76.26 (C2'), 69.09 (d, 2 J C-P = 5.0 Hz, C5'), 68.16 (d, 2 J C-P = 8.2 Hz, C5'), 67.95 (OCH2Ph), 55.28, 55.32 (OCH3), 51.79 (CH ala), 51.71 (CH ala), 35.40 (C-3'), 35.12 (C3'), 20.49 (d, 3 J C-P = 6.7 Hz, CH3 ala), 20.35 (d, 3 J C-P = 6.7, CH3 ala). HPLC shows two peaks of diastereoisomers with t R 16.22 min and t R 16.48 min using reverse phase HPLC with H20 / CH3CN elution from 100 / 10 to 0 / 100 in 30 min, F = 1 ml / min, λ = 280 nm.

[0449] (2S)-Benzyl 2-((((2S,4R,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-4- hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate P Furan-2-yl)methoxy)(naphthalen-1-yloxy)phosphorylaminol)propionic acid ester K

[0450]

[0451] Using the above general procedure 1, a solution of (2S)-benzyl 2-((chloro(phenoxy)phosphoryl)amino)propanoate (264 mg, 0.75 mmol) in dry THF (2 mL) and N-methylimidazole (99 μί, 1.24 mmol) was added dropwise to a suspension of 2-0-methyl-3'-deoxyadenosine (70 mg, 0.25 mmol) in dry THF and the reaction mixture was stirred at room temperature for 16 h. Purification by column chromatography (eluent system CH3OH / CH2Cl20 / 100 to 6 / 94) and preparative TLC (eluent system CH3OH / CH2Cl25 / 95) afforded the target compound as a white solid (13 mg, 10%).

[0452] (ES+) m / z, found: 599.2 (M+H + ), C 27 H 31 N6O8P requires: 598.19 (M).

[0453] 31 P NMR (202 MHz, CD3OD) δ 3.97, 3.64. 1 H NMR (500 MHz, CD3OD) δ 8.06 (s, 0.5H, H8), 8.04 (s, 0.5H, H8), 7.33-7.28 (m, 7H, Ph), 7.20-7.14 (m, 3H, Ph), 5.92 (d, J = 1.5 Hz, 0.5H, H1'), 5.90 (d, J = 1.5 Hz, 0.5H, H1'), 5.14-5.04 (m, 2H, OCH2Ph), 4.78-4.76 (m, 0.5H, H4'), 4.74-4.72 (m, 0.5H, H4'), 4.63-4.59 (m, 1H, H2'), 4.10-4.34 (m, 1H, H5'a), 4.25-4.20 (m, 1H, H5'b), 3.94, 3.95 (OCH3), 3.99-3.90 (m, 1H, CHaia), 2.40-2.37 (m, 1H, H3'), 2.07-2.04 (m, 1H, H3'), 1.31 (d J = 7.0 Hz, CH3), 1.26 (d, J = 7.0 Hz, CH3). 13 C NMR (125 MHz, CD3OD) δ 174.82 (d, 3 J C-P = 3.7 Hz, C=0), 174.62 (d, 3 J C-P= 4.8 Hz, C-ipso Ph), 152.00 (d, 3 J C-P = 4.8 Hz, C-ipso Ph), 152.00 (d, 3 J C-P = 4.8 Hz, C-ipso Ph), 139.39 (C8), 137.30, 137.21 (C-ipso CH2Ph), 130.72, 129.57, 129.31, 129.27, 126.122 (CH-Ar), 121.42 (d, J C-P = 4.5 Hz, CH-Ar), 121.37 (d, J C-P = 4.5 Hz, CH-Ar), 116.72 (C5), 116.69 (C5), 93.33, 93.24 (C1'), 80.26 (d, 3 J C-P = 8.87, C4'), 80.19 (d, 3 J C-P = 8.87, C4'), 76.35 (C2'), 68.78 (d, 2 J C-P = 5.0 Hz, C5'), 68.35 (d, 2 J C-P = 5.0 Hz, C5'), 67.94 (OCH2Ph), 67.92 (OCH2Ph), 55.25, 55.28 (OCH3), 51.69, 51.57 (CH ala), 35.23 (C3'), 34.96 (C3'), 20.38 (d, 3 J C-P = 6.7, CH3 ala), 20.26 (d, 3 J C-P = 6.7, CH3 ala). HPLC shows two peaks of diastereoisomers with t R 14.22 min and t R 14.51 min using reverse phase HPLC with H2O / CH3CN elution from 100 / 10 to 0 / 100 in 30 min, F = 1 ml / min, λ = 280 nm.

[0454] 2-O-methyl-3'-deoxyadenosine-5'-O-[1-naphthyl(1-pentyloxy-L-leucinyl)] phosphate M

[0455]

[0456] Compound M was prepared according to general procedure 1 using 2-0-methyl-3'- deoxyadenosine (70 mg, 0.25 mmol), N-methylimidazole (99 μL, 1.24 mmol) and naphthyl(pentyloxy-L-leucinyl) chlorophosphate (330 mg, 0.75 mmol). Purification by column chromatography (eluent system gradient CH3OH / CH2Cl20 / 100 to 6 / 94) and preparative TLC (2000 μM, eluent system CH3OH / CH2Cl27 / 93) afforded the target compound as a white solid (50 mg, 30%).

[0457] 31 P NMR (202 MHz, CD3OD) δP 4.53, 4.28.

[0458] 1 H NMR (500 MHz, CD3OD) δH 8.04 - 7.96 (m, 1H, H8), 7.77 - 7.71 (m, 1H, Nap), 7.58 - 7.53 (m, 1H, Nap), 7.45 - 7.17 (m, 5H, Nap), 5.83 - 5.75 (m, 1H, H1'), 4.64 - 4.51 (m, 2H, H2', H4'), 4.40 - 4.16 (m, 2H, H5'), 3.88 - 3.75 (m, 6H, OCH3, O(CH2)4CH3, CHCH2CH(CH3)2), 2.38 - 2.24 (m, 1H, H3'), 2.00 - 1.91 (m, 1H, H3'), 1.53 - 1.05 (m, 11H, O(CH2)4CH3, CHCH2CH(CH3)2), 0.77 - 0.55 (m, 9H, O(CH2)4CH3, CHCH2CH(CH3)2).

[0459] 13 C NMR (125 MHz, CD3OD) δC 175.02 (d, 3 J C-P = 2.5 Hz, C=0), 174.78 (d, 3 J C-P = 2.5 Hz, C=0), 163.76 (C2), 158.14 (C6), 151.03 (C4), 147.96 (d, 3 J C-P= 7.2, 'ipso' Nap), 138.96 (C8), 136.30 (C-Ar), 136.28 (C-Ar), 136.22 (C-Ar), 128.93 (CH-Ar), 128.88 (CH-Ar), 128.81 (CH-Ar), 128.48 (CH-Ar), 127.77 (CH-Ar), 127.73 (CH-Ar), 127.44 (CH-Ar), 127.42 (CH-Ar), 127.06 (CH-Ar), 126.86 (CH-Ar), 126.45 (CH-Ar), 126.44 (CH-Ar), 126.31 (CH-Ar), 125.98 (CH-Ar), 125.88 (CH-Ar), 123.83 (CH-Ar), 123.43 (CH-Ar), 123.24 (CH-Ar), 122.81 (CH-Ar), 122.77 (CH-Ar), 122.69 (CH-Ar), 116.34 (d, 3 J C-P = 3.7 Hz, CH-Ar), 116.02 (d, 3 J C-P = 3.7 Hz, CH-Ar), 115.71 (C5), 93.42 (C1'), 93.32 (C1'), 80.22 (d, 3 J C-P = 5.3 Hz, C4'), 80.15 (d, 3 J C-P = 5.3 Hz, C4'), 76.29 (C2'), 76.27 (C2'), 69.22 (d, 2 J C-P = 5.2 Hz, C5'), 69.028 (d, 2 J C-P = 5.2 Hz, C5'), 66.31 (O(CH2)4CH3), 66.30 (O(CH2)4CH3), 55.29 (OCH3), 55.24 (OCH3), 54.79 (CHCH2CH(CH3)2), 54.68 (CHCH2CH(CH3)2), 44.20 (d, 3 J C-P = 7.25 Hz, CHCH2CH(CH3)2), 43.93 (d, 3 J C-P= 7.25 Hz, CHCH2CH(CH3)2), 35.49 (C3'), 35.17 (C3'), 29.31 (0(CH2)4CH3), 29.11 (0(CH2)4CH3), 25.67 (CHCH2CH(CH3)2), 25.44 (CHCH2CH(CH3)2), 23.30 (0(CH2)4CH3), 23.10 (CHCH2CH(CH3)2), 23.00 (CHCH2CH(CH3)2), 22.94 (CHCH2CH(CH3)2), 22.81 (CHCH2CH(CH3)2), 14.27 (0(CH2)4CH3).

[0460] (ES+) m / z, found: 671.3 (M+H + ), C 32 H 43 N6O8P requires: 670.69 (M).

[0461] HPLC using H20 / CH3CN from 100 / 10 to 0 / 100 elution in 30 min, reverse phase HPLC, 1 ml / min, l = 254 nm, showed two peaks of diastereoisomers with tR20.83 min and tR20.93 min.

[0462] 2-O-methyl-3'-deoxyadenosine-5'-O-[phenyl(1-hexyloxy-L-alaninyl)] phosphate N

[0463]

[0464] Compound N was prepared according to general procedure 1 using 2-0-methyl-3'- deoxyadenosine (70 mg, 0.25 mmol), N-methylimidazole (99 μL, 1.24 mmol) and phenyl(hexyloxy-L-alaninyl) chlorophosphate (261 mg, 0.75 mmol). Purification by column chromatography (eluent system gradient CH3OH / CH2Cl20 / 100 to 6 / 94) and preparative TLC (1000 μM, eluent system CH3OH / CH2Cl27 / 93) afforded the target compound as a white solid (26 mg, 18%).

[0465] 31 P NMR (202 MHz, CD3OD) δΡ 3.87, 3.65.

[0466] 1H NMR (500 MHz, CD3OD) δH 8.08 (s, 0.5H, H8), 8.07 (s, 0.5H, H8), 7.36-7.29 (m, 2H, Ph), 7.24-7.14 (m, 3H, Ph), 5.94 (d, J = 2.0 Hz, 0.5H, H1’), 5.92 (d, J = 2.0 Hz, 0.5H, H1’), 4.81-4.76 (m, 1H, H2’), 4.71-4.62 (m, 1H, H4’), 4.48-4.43 (m, 0.5H, H5’), 4.42-4.36 (m, 0.5H, H5’), 4.33-4.25 (m, 1H, H5’), 4.10-3.83 (m, 6H, OCH3, O(CH2)5CH3, CHCH3), 2.48-2.40 (m, 1H, H3’), 2.13-2.07 (m, 1H, H3’), 1.61-1.51 (m, 2H, O(CH2)5CH3), 1.33-1.24 (m, 9H, O(CH2)5CH3, CHCH3), 0.89 (m, 3H, O(CH2)5CH3).

[0467] 13 C NMR (125 MHz, CD3OD) δC 175.13 (d, 3 J C-P = 4.3 Hz, C=0), 174.94 (d, 3 J C-P = 4.3 Hz, C=0), 163.80 (C2), 163.78 (C2), 158.17 (C6), 158.15 (C6), 152.17 (d, 2 J C-P = 6.3 Hz, C-Ar), 152.15 (d, 2 J C-P = 6.3 Hz, C-Ar), 152.03 (C4), 151.99 (C4), 139.42 (C8), 139.39 (C8), 130.75 (CH-Ar), 130.74 (CH-Ar), 126.13 (CH-Ar), 121.43 (CH-Ar), 121.41 (CH-Ar), 121.39 (CH-Ar), 121.37 (CH-Ar), 116.74 (C5), 116.69 (C5), 93.40 (C1’), 93.27 (C1’), 80.30 (C4’), 80.23 (C4’), 76.40 (C2’), 68.85 (d, 2 J C-P = 5.2 Hz, C5’), 68.42 (d, 2 JC-P = 5.2 Hz, C5'), 66.43 (O(CH2)5CH3), 55.30 (OCH3), 55.26 (OCH3), 51.64 (CHCH3), 51.54 (CHCH3), 35.30 (C3'), 35.04 (C3'), 32.58 (O(CH2)5CH3), 29.67 (O(CH2)5CH3), 29.64 (O(CH2)5CH3), 26.61 (O(CH2)5CH3), 23.59 (O(CH2)5CH3), 20.56 (d, 3 J C-P = 6.4 Hz, CHCH3), 20.41 (d, 3 J C-P = 6.4 Hz, CHCH3), 14.36 (O(CH2)5CH3).

[0468] (ES+) m / z, found: 593.3 (M+H + ), C 32 H 43 N6O8P Required: 592.58 (M).

[0469] HPLC using H2O / CH3CN from 100 / 10 to 0 / 100 elution in 30 min, reverse phase HPLC, 1 ml / min, l = 254 nm, showed two peaks of diastereoisomers with tR17.02 min and tR17.23 min.

[0470] 2-Fluoro-3'-deoxyadenosine-5'-O-[1-naphthyl(benzyloxy-L-alaninyl)] phosphate O

[0471]

[0472] Compound O was prepared according to general procedure 1 using 2-fluoro-3'deoxyadenosine (50 mg, 0.18 mmol), N-methylimidazole (74 μL, 0.93 mmol) and phenyl(benzyloxy-L-alaninyl) phosphorochloridate (196 mg, 0.56 mmol). Purification by column chromatography (eluent system gradient CH3OH / CH2Cl20 / 100 to 6 / 94) and preparative TLC (500 μM, eluent system CH3OH / CH2Cl25 / 95) afforded the target compound as a white solid (5 mg, 4%).

[0473] 31 P NMR (202 MHz, CD3OD) δΡ 4.33, 4.08.

[0474] 1H NMR (500 MHz, CD3OD) δH 8.17 (s, 0.5H, H8), 8.14 (s, 0.5H, H8), 8.14-8.09 (m, 1H, Ar), 7.89-7.85 (m, 1H, Ar), 7.70-7.66 (m, 1H, Ar), 7.54-7.42 (m, 4H, Ar), 7.40-7.24 (m, 5H, Ar), 5.89 (d, J = 2.3 Hz, 0.5H, H1’), 5.88 (d, J = 2.3 Hz, 0.5H, H1’), 5.08-5.01 (m, 2H, OCH2Ph), 4.70-4.60 (m, 2H, H2’, C4’), 4.46-4.39 (m, 1H, C5’), 4.32-4.24 (m, 1H, C5’), 4.09-3.97 (m, 1H, CHCH3), 2.36-2.25 (m, 1H, H3’), 2.06-1.98 (m, 1H, H3’), 1.32-1.25 (m, 3H, CHCH3).

[0475] 13 C NMR (125 MHz, CD3OD) δC 175.54 (CO), 175.22 (CO), 161.02 (d, 1 J C-F = 207.3 Hz, C2), 160.89 (d, 1 J C-F = 207.3 Hz, C2), 158.45 (d, 3 J C-F = 18.2 Hz, C6), 158.23 (d, 3 J C-F = 18.2 Hz, C6), 150.63 (d, 3 J C-F= 18.4 Hz, C4), 140.67 (C8), 136.26 (C-Ar), 131.62, 131.54, 129.56 (CH-Ar), 129.52 (CH-Ar), 129.37 (CH-Ar), 129.31 (CH-Ar), 129.26 (CH-Ar), 128.87 (CH-Ar), 128.81 (CH-Ar), 128.29 (CH-Ar), 128.02 (CH-Ar), 127.79 (CH-Ar), 127.76 (CH-Ar), 127.51 (CH-Ar), 127.49 (CH-Ar), 127.47 (CH-Ar), 126.47 (CH-Ar), 126.33 (C-Ar), 126.27 (C-Ar), 125.97 (CH-Ar), 122.78 (CH-Ar), 122.74 (CH-Ar), 122.64 (CH-Ar), 122.62 (CH-Ar), 116.35 (d, 4 J C-F = 3.0 Hz, C5), 116.15 (d, 4 J C-F = 3.0 Hz, C5), 93.25 (C1'), 93.20 (C1'), 80.41 (d, 3 J C-P = 7.5 Hz, C4'), 80.33 (d, 3 J C-P = 7.5 Hz, C4'), 76.43 (C2'), 76.35 (C2'), 68.84 (d, 2 J C-P = 5.5 Hz, C5'), 68.45 (d, 2 J C-P = 5.5 Hz, C5'), 67.92 (OCH2Ph), 67.92 (OCH2Ph), 51.75 (CHCH3), 51.52 (CHCH3), 34.97 (C3'), 34.74 (C3'), 20.42 (d, 3 J C-P = 6.7 Hz, CHCH3), 20.20 (d, 3 J C-P = 6.7 Hz, CHCH3).

[0476] 19 F NMR (470 MHz, CD3OD) δF -53.14, -53.22.

[0477] (ES+) m / z, found: 637.2 (M+H + ), C30 H 30 FN6O7P requires value: 636.57 (M).

[0478] HPLC using reverse phase HPLC eluting with H20 / CH3CN from 100 / 10 to 0 / 100 for 30 min, 1 ml / min, l = 254 nm showed two peaks for diastereoisomers with tR17.09 min and tR17.34 min.

[0479] (2S)-Benzyl 2-((((2S,4R,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-4- hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate P Furan-2-yl)methoxy)(naphthalen-1-yloxy)phosphorylaminol)propionic acid ester K

[0480]

[0481] Using the above General Procedure 1, a solution of (2S)-benzyl 2-((chloro(phenoxy)phosphoryl)amino)propanoate (196 mg, 0.56 mmol) in anhydrous THF (2 mL) and N-methylimidazole (74 μL, 0.93 mmol) was added dropwise to a suspension of 2-fluoro-3'-deoxyadenosine (50 mg, 0.18 mmol) in anhydrous THF (5 mL) and the reaction mixture was stirred at room temperature for 16 h. Purification by column chromatography (eluent system CH3OH / CH2Cl20 / 100 to 6 / 94) and preparative TLC (eluent system CH3OH / CH2Cl25 / 95) afforded the target compound as a white solid (5 mg, 7%).

[0482] (ES+) m / z, found: 587.1 (M+H + ), C 26 H 28 FN6O7P requires value: 586.17 (M). 19 F NMR (470 MHz, CD3OD): d F -53.17, -53.23. 31 P NMR (202 MHz, CD3OD): d P 3.95 (s), 3.67 (s). 1H NMR (500 MHz, CDC13): δH 8.19 (s, 0.5H, H8), 8.16 (s, 0.5H, H8), 7.36-7.27 (m, 7H, Ar), 7.22-7.13 (m, 3H, Ar), 5.91 (d, J = 1.5 Hz, 0.5H, H1'), 5.89 (d, J = 1.7 Hz, 0.5H, H1'), 5.15-5.06 (m, 2H, OCH2Ph), 4.73-4.58 (m, 2H, H2', H4'), 4.42-4.34 (m, 1H, H5'), 4.02-3.90 (m, 1H, H5'), 3.27-3.24 (m, 1H, H3'), 2.08-2.00 (m, 1H, H3'), 1.33 (d, J = 7.1 Hz, 1.5H, CH3 ala), 1.29 (d, J = 7.1 Hz, 1.5H, CH3 ala). 13 C NMR (125 MHz, CDC13): δC 175.85 (d, 3 J C-P = 3.7 Hz, C=0), 174.63 (d, 3 J C-P = 5.0 Hz, C=0), 160.58 (d, 1 J C-F = 207.5 Hz, C2), 160.53 (d, 1 J C-F = 207.5 Hz, C2), 159.06 (d, 3 J C-F = 18.7 Hz, C6), 159.05 (d, 3 J C-F = 17.5 Hz, C6), 152.11 (d, 2 J C-P = 8.75 Hz, C-Ar), 152.08 (d, 2 J C-P = 8.7 Hz, C-Ar), 151.58 (d, 3 J C-F = 19.7 Hz, C4), 151.56 (d, 3 J C-F= 19.5 Hz, C4), 140.63 (C8), 137.28 (C-Ar), 137.21 (C-Ar), 130.78 (CH-Ar), 130.75 (CH-Ar), 129.58 (CH-Ar), 129.38 (CH-Ar), 129.34 (CH-Ar), 129.32 (CH-Ar), 129.28 (CH-Ar), 128.3 (CH-Ar), 128.02 (CH-Ar), 121.16 (CH-Ar), 121.18 (CH-Ar), 121.47 (CH-Ar), 121.51 (CH-Ar), 121.42 (CH-Ar), 121.39 (CH-Ar), 121.36 (CH-Ar), 118.75 (d, 4 J C-F = 3.7 Hz, C5), 118.72 (d, 4 J C-F = 3.7 Hz, C5), 93.25 (C1'), 93.18 (C1'), 80.48 (d, 3 J C-P = 8.3 Hz, C4'), 80.46 (d, 3 J C-P = 8.1 Hz, C4'), 76.51 (C2'), 76.49 (C2'), 68.54 (d, 2 J C-P = 5.2 Hz, C5'), 68.18 (d, 2 J C-P = 5.6 Hz, C5'), 67.94 (CH2 Bn), 67.91 (CH2 Bn), 51.71 (CH ala), 51.56 (CH ala), 34.85 (C3'), 34.64 (C3'), 20.42 (d, 3 J C-P = 7.1 Hz, CH3 ala), 20.25 (d, 3 J C-P = 7.5 Hz, CH3 ala). HPLC on a reverse phase HPLC eluting with H2O / CH3CN from 100 / 10 to 0 / 100 in 30 min, 1 ml / min, l = 280 nm, showed two peaks for the diastereoisomers with t R 14.98 min and t R 15.12 min.

[0483] 2-Fluoro-3'-deoxyadenosine-5'-O-[1-naphthyl(1-pentyloxy-L-leucinyl)] phosphate Q

[0484]

[0485] Compound Q was prepared according to general procedure 1 using 2-fluoro-3’ deoxyadenosine (50 mg, 0.18 mmol), N-methylimidazole (74 μL, 0.93 mmol) and naphthyl(pentyloxy-L-leucinyl) chlorophosphate (246 mg, 0.56 mmol). Purification by column chromatography (eluent system CH3OH / CHCl30 / 100 to 6 / 94) and preparative TLC (1000 μM, eluent system CH3OH / CH2Cl25 / 95) afforded the target compound as a white solid (65 mg, 53%).

[0486] 31 P NMR (202 MHz, CD3OD): 4.60, 4.35.

[0487] 1 H NMR (500 MHz, CD3OD): δH 8.23 (s, 0.5H, H8), 8.20 (s, 0.5H, H8), 8.18-8.12 (m, 1H, Ar), 7.92-7.86 (m, 1H, Ar), 7.73-7.68 (m, 1H, Ar), 7.57-7.46 (m, 3H, Ar), 7.42-7.36 (m, 1H, Ar), 5.93-5.91 (m, 1H, H1’), 4.74-4.62 (m, 2H, H2’, H4’), 4.55-4.50 (m, 0.5H, H5’), 4.49-4.44 (m, 0.5H, H5’), 4.43-4.37 (m, 0.5H, H5’), 4.36-4.31 (m, 0.5H, H5’), 4.02-3.86 (m, 3H, CHCH2CH(CH3)2, O(CH2)4CH3), 2.43-2.29 (m, 1H, H3’), 2.12-2.04 (m, 1H, H3’), 1.67-1.20 (m, 11H, O(CH2)4CH3, CHCH2CH(CH3)2), 0.89-0.67 (m, 9H, O(CH2)4CH3, CHCH2CH(CH3)2)

[0488] 13 C NMR (125 MHz, CD3OD): δC 175.03 (d, 3 J C-P = 2.5 Hz, C=0), 174.93 (d, 3 J C-P = 2.5 Hz, C=0), 161.45 (d, 1 J C-F = 205.5 Hz, C2), 160.39 (d, 1 J C-F=205.5Hz,C2),158.33(C6),151.60(C4),147.92(C-Ar),140.69(C8),136.30(C-Ar),128.88(CH-Ar),128.83(CH-Ar),127.80(CH-Ar.7),127.7(7),140.69(CH-Ar). .49(CH-Ar),127.46(CH-Ar),126.48(CH-Ar),126.45(CH-Ar),126.02(CH-Ar),125.91(CH-Ar),123.03(C-Ar),122.81(CH-Ar),123.03(C-Ar),122.81(CH-Ar),122.69(CH-Ar),11,69(d) 3 J C-P =2.9Hz,CH-Ar),116.28(C5),116.26(C5),115.97(d, 3 J C-P =2.9Hz,CH-Ar),93.29(C1'),93.23(C1'),80.45(d, 3 J C-P =6.0Hz,C4'),80.38(d, 3 J C-P =6.0Hz,C4'),76.45(C2'),76.41(C2'),68.99(d, 2 J C-P =5.4Hz,C5'),68.78(d, 2 J C-P =5.4Hz,C5'),66.31(O(CH2)4CH3),66.29(O(CH2)4CH3),54.78(CHCH2CH(CH3)2),54.66(CHCH2CH(CH3)2),44.16(d, 3 J C-P =7.25Hz,CHCH2CH(CH3)2),43.84(d, 3 J C-P= 7.3 Hz, CHCH2CH(CH3)2), 35.09 (C3'), 34.79 (C3'), 29.31 (O(CH2)4CH3), 29.12 (O(CH2)4CH3), 25.65 (CHCH2CH(CH3)2), 25.41 (CHCH2CH(CH3)2), 23.33 (O(CH2)4CH3), 23.11 (CHCH2CH(CH3)2), 23.00 (CHCH2CH(CH3)2), 21.95 (CHCH2CH(CH3)2), 21.68 (CHCH2CH(CH3)2), 14.29 (O(CH2)4CH3).

[0489] 19 F NMR (470 MHz, CD3OD): δF-53.15, -53.20.

[0490] (ES+) m / z, found: 659.3 (M+H + ), C 31 H 40 FN6O7P requires: 658.66 (M).

[0491] HPLC using reverse phase HPLC eluting with H2O / CH3CN from 100 / 10 to 0 / 100 in 30 min, 1 ml / min, l = 254 nm showed one peak with overlapping diastereoisomers with tR21.95 min.

[0492] (2S)-Benzyl 2-((((2S,4R,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-4- hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate P Furan-2-yl)methoxy)(naphthalen-1-yloxy)phosphorylaminol)propionic acid ester K

[0493]

[0494] Using the above general procedure 1, a solution of (2S)-hexyl 2-((chloro(phenoxy)phosphoryl)amino)propanoate (196 mg, 0.56 mmol) in dry THF (2 mL) and N-methylimidazole (74 μL, 0.93 mmol) was added dropwise to a suspension of 2-fluoro-3'-deoxyadenosine (50 mg, 0.18 mmol) in dry THF (5 mL) and the reaction mixture was stirred at room temperature for 16 h. Purification by column chromatography (eluent system CH3OH / CH2Cl20 / 100 to 6 / 94) and preparative TLC (eluent system CH3OH / CH2Cl25 / 95) afforded the target compound as a white solid (5 mg, 7%).

[0495] (ES+) m / z, found: 587.1 (M+H + ), C 26 H 28FN607P requires value: 586.17 (M). 19 F NMR (470 MHz, CD3OD): δF -53.15, -53.20. 31 P NMR (202 MHz, CD3OD): 3.91 (s), 3.73 (s). 1 H NMR (500 MHz, CDC13): δH 8.21 (s, 0.5H, H8), 8.20 (s, 0.5H, H8), 7.37-7.29 (m, 7H, Ar), 7.26-7.13 (m, 3H, Ar), 5.94-5.91 (m, 1H, H1’), 4.76-4.64 (m, 2H, H2’, H4’), 4.49-4.44 (m, 0.5H, H5’), 4.43-4.37 (m, 0.5H, H5’), 4.33-4.26 (m, 1H, H5’), 4.11-3.99 (m, 2H, CH2 Hex), 3.97-3.83 (m, 1H, CH ala), 2.41-2.32 (m, 1H, H3’), 2.13-2.06 (m, 1H, H3’), 1.62-1.52 (m, 2H, CH2 Hex), 1.37-1.23 (m, 9H, CH3 ala, CH2 Hex), 0.92-0.85 (m, 3H, CH3 Hex).

[0496] 13 C NMR (125 MHz, CD3OD): δC 175.15 (d, 3 J C-P = 3.7 Hz, C=0), 174.96 (d, 3 J C-P = 5.0 Hz, C=0), 160.59 (d, 1 J C-F = 207.5 Hz, C2), 160.56 (d, 1 J C-F = 207.5 Hz, C2), 159.09 (d, 3 J C-F = 21.2 Hz, C6), 159.08 (d, 3 J C-F = 20.0 Hz, C6), 152.16 (d, 2 J C-P = 7.5 Hz, C-Ar), 152.14 (d, 2 J C-P = 6.3 Hz, C-Ar), 151.71 (d, 3 J C-F=20.0Hz,C4),151.67(d, 3 J C-F =20.0Hz,C4),140.70(d, 5 J C-F =2.5Hz,C8),140.68(d, 5 J C-F =2.5Hz,C8),130.77(CH-Ar),130.74(CH-Ar),126.16(CH-Ar),126.24(CH-Ar),121.48(CH-Ar),121.44(CH-Ar),121.41(CH-Ar),121.37(CH-Ar),118.80(d, 4 J C-F =3.7Hz,C5),118.77(d, 4 J C-F =3.7Hz,C5),93.37(C1'),93.25(C1'),80.52(d, 3 J C-P =3.7Hz,C4'),80.45(d, 3 J C-P =4.1Hz,C4'),76.52(C2'),76.49(C2'),68.69(d, 2 J C-P =5.4Hz,C5'),68.30(d, 2 J C-P =4.9Hz,C5'),66.46(CH2 Hex),51.68(CHala),51.57(CH ala),35.02(C3'),34.80(C3'),32.58(CH2 Hex),29.65(CH2 Hex),26.61(CH2 Hex),23.59(CH2 Hex),20.60(d, 3 J C-P =7.1Hz,CH3 ala),20.43(d, 3 J C-P =7.5 Hz, CH3 ala), 14.35 (CH3 Hex). HPLC using reverse phase HPLC with H2O / CH3CN eluting from 100 / 10 to 0 / 100 over 30 minutes, 1 ml / min, l = 280 nm, showed two peaks of diastereomers with tR 17.83 min and tR 18.02 min.

[0497] (2R)-Benzyl 2-((((2S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-4- hydroxytetrahydrofuran-2-yl)methoxy)(naphthalen-1-yloxy)phosphoryl)amino)propanoate S 2-Chloro-3'-deoxyadenosine 5'-O-[1-phenyl(2,2-dimethylpropoxy-L-alaninyl)] phosphate T

[0498]

[0499] To a stirred solution of 2-chloro-3'-deoxyadenosine (100 mg, 1.0 mol / eq.) in 10 mL of anhydrous THF was added dropwise 424 mg of (2S)-benzyl 2-(chloro(naphthalen-1-yloxy)phosphorylamino)propanoate (3.0 eq / mol) dissolved in 10 mL of anhydrous THF. To this reaction mixture was added dropwise 0.14 mL of NMI (5 mol / eq.) under argon atmosphere at room temperature. The reaction mixture was stirred for 88 h. The solvent was removed under reduced pressure and the residue was purified by column chromatography with eluent gradient (CH3OH / CH2Cl20 / 100 to 5 / 95) to give the target product as a yellow solid. (7 mg, yield = 3%). MS (ES+) m / z: found: 653 (M+H + ), 675 (M+Na + )C 30 H 30 ClN6O7P required: 652.16 (M); 31 P NMR (202 MHz, CD3OD): δP 4.39 (s), 4.12 (s); 1 H NMR (500 MHz, CD3OD): δH 8.10 (s, 0.5H, H8), 8.07 (s, 0.5H, H8), 8.02-7.97 (m, 3H, CH2Ph and Naph), 7.43-7.14 (m, 9H, CH2Ph and Naph), 5.80-5.81 (m, 1H, H1’), 4.89-4.97 (m, 2H, CH2Ph) 4.49-4.53 (m, 2H, H4’ and H2’), 4.30-4.35 (m, 1H, H5’), 4.15-4.21 (m, 1H, H5’), 3.87-3.95 (m, 1H, CHCH3), 2.12-2.23 (m, 1H, H3’), 1.86-1.93 (m, 1H H3’), 1.14-1.17 (m, 3H, CHCH3); 13 C NMR (125 MHz, CD3OD): δC 174.85 (d J CP = 4.0 Hz, C=0), 174.55 (d J CP = 4.3 Hz, C=0), 158.07, 158.04 (C6), 155.31, 155.28 (C2), 151.34, 151.31 (C4), 149.69 (C-Ar), 147.96 (d 3 J CP= 7.25 Hz, C-ipso Naph), 147.90 (d 3 J CP = 7.0 Hz, C-ipso Naph), 140.70 (C8), 137.21, 137.16 (C-ipso CH2Ph), 136.26 (C-Ar), 130.92, 130.80, 129.56, 129.53, 129.31, 129.27, 129.25, 128.88, 128.81 (CH-Ar), 127.78 (d J CP = 4.7 Hz, CH-Ar), 127.50 (d J CP = 6.2 Hz, CH-Ar), 126.48, 126.02, 125.97 (CH-Ar), 119.46, 119.42 (C5), 116.33 (d, J CP = 3.0, CH-Ar), 116.16 (d, J CP = 3.4, CH-Ar), 93.30, 93.27 (C1'), 80.56 (d J = 8.3 Hz, C4'), 80.51 (d J = 8.4 Hz, C4'), 76.61, 76.54 (C2'), 68.74 (d J CP = 5.3 Hz, C5'), 68.54 (d J CP = 5.1 Hz, C5'), 67.93, 67.90 (CH2Ph), 51.81, 51.70 (CHCH3), 34.79, 34.53 (C3'), 20.42 (d J CP = 6.5 Hz, CHCH3), 20.23 (d J CP = 7.7 Hz, CHCH3); HPLC reverse phase HPLC with H20 / CH3CN elution from 90 / 10 to 0 / 100 in 30 min, F = 1 ml / min, l = 254 nm, t R 18.03 min.

[0500] 2-Chloro-3'-deoxyadenosine 5'-O-[1-naphthyl(2,2-dimethylpropoxy-L-alaninyl)] phosphate U

[0501]

[0502] Compound T was prepared according to general procedure 1 using 2-chloro-3'- deoxyadenosine (350 mg, 1.25 mmol), N-methylimidazole (490 μL, 6.15 mmol) and phenyl(2,2-dimethylpropoxy-L-alaninyl) chlorophosphonate (1231 mg, 3.69 mmol). Purification by column chromatography (eluent system CH3OH / CH2Cl20 / 100 to 5 / 95) and preparative TLC (1000 μM, eluent system CH3OH / CH2Cl24 / 96) afforded the target compound as a white solid (181 mg, 25%).

[0503] 31 P NMR (202 MHz, CD3OD): δP 3.93, 3.72.

[0504] 1 H NMR (500 MHz, CD3OD): δH 8.12 (s, 0.5H, H8), 8.10 (s, 0.5H, H8), 7.19-7.23 (m, 2H, Ph), 7.03-7.12 (m, 3H, Ph), 5.84 (d J= 2, 0.5H, H1'), 5.83 (d J= 2, 0.5H, H1'), 4.54-4.60 (m, 2H, H4' and H2'), 4.34-4.38 (m, 0.5H, H5'), 4.27-4.31 (m, 0.5H, H5'), 4.16-4.23 (m, 1H, H5'), 3.80-3.90 (m, 1H, CHCH3), 3.57-3.73 (m, 2H OCH2C(CH3)3), 2.18-2.28 (m, 1H, H3'), 1.94-1.99 (m, 1H, H3'), 1.20-1.24 (m, 3H, CHCH3), 0.81 (s, 4.5H OCH2(CH3)3), 0.79 (s, 4.5H OCH2C(CH3)3).

[0505] 13 C NMR (125 MHz, CD3OD): δC 175.09 (d 3 J CP = 4.75 Hz, C=0), 174.90 (d 3 J CP = 5.37 Hz, C=0), 158.10, (C6), 155.31, 155.28 (C2), 152.14 (d 2 J CP = 6.37 Hz, C-ipso Ph), 152.13 (d 2 J CP= 6.25 Hz, CHCH3), 20.61 (d J = 7.12 Hz, CHCH3). 3 J CP = 11.75 Hz, CH-Ar), 121.41 (d 3 J CP = 11.75 Hz, CH-Ar), 119.52, 119.48 (C5), 93.49, 93.35 (C1'), 80.67 (d 3 J = 8.62 Hz, C4'), 80.65 (d 3 J = 8.25 Hz, C4'), 76.70, 76.67 (C2'), 75.43, (OCH2C(CH3)3), 68.68 (d 2 J CP = 5.12 Hz, C5'), 68.42 (d 2 J CP = 5.12 Hz, C5'), 51.77, 51.60 (CHCH3), 34.94, 34.67 (C3'), 32.36, 32.32 (OCH2C(CH3)3), 26.78, 26.76 (OCH2C(CH3)3), 20.83 (d J CP = 6.25 Hz, CHCH3), 20.61 (d J CP = 7.12 Hz, CHCH3).

[0506] MS (ES+) m / z: found: 583 (M+H + ), 605 (M+Na + ) C 24 H 32 ClN6O7P requires: 582.18 (M).

[0507] HPLC using H2O / CH3CN from 90 / 10 to 0 / 100 elution in 30 min, F = 1 ml / min, l = 254 nm, t R 16.37, 16.55 min.

[0508] 2-Chloro-3'-deoxyadenosine 5'-O-[1-phenyl(ethoxy-L-alaninyl)] phosphate V

[0509]

[0510] Compound U was prepared according to general procedure 1 using 2-chloro-3'deoxyadenosine (350 mg, 1.25 mmol), N-methylimidazole (490 μL, 6.15 mmol) and naphthyl(2,2-dimethylpropoxy-L-alaninyl) chlorophosphonate (1416 mg, 3.69 mmol). Purification by column chromatography (eluent system CH3OH / CH2Cl20 / 100 to 5 / 95) and preparative TLC (1000 μM, eluent system CH3OH / CH2Cl24 / 96) afforded the target compound as a white solid (264 mg, 34%).

[0511] 31 P NMR (202 MHz, CD3OD): δP 4.35, 4.20.

[0512] 1 H NMR (500 MHz, CD3OD): δH 8.23 (s, 0.5H, H8), 8.21 (s, 0.5H, H8), 8.11-8.16 (m, 1H, Naph), 7.86-7.89 (m, 1H, Naph), 7.69-7.70 (m, 1H, Naph), 7.54-7.46 (m, 3H, Naph), 7.37-7.41 (m, 1H, Naph), 5.95 (d J=2, 0.5H, H1'), 5.94 (d J=1.5, 0.5H, H1'), 4.67-4.73 (m, 2H, H4' and H2'), 4.34-4.55 (m, 2H, H5'), 4.00-4.08 (m, 1H, CHCH3), 3.66-3.81 (m, 2H OCH2C(CH3)3), 2.28-2.41 (m, 1H, H3'), 2.03-2.10 (m, 1H, H3'), 1.31-1.34 (m, 3H, CHCH3), 0.90 (s, 4.5H OCH2C(CH3)3), 0.89 (s, 4.5H CH2(CH3)3).

[0513] 13 C NMR (125 MHz, CD3OD): δC 175.11 (d J CP = 4.1 Hz, C=0), 174.85 (d J CP = 5.0 Hz, C=0), 158.10, 158.04 (C6), 155.32, 155.30 (C2), 151.33 (C4), 147.96 (d 2 J CP = 7.25 Hz, C-ipso Naph), 147.93 (d 2 JCP = 7.25 Hz, C-ipso Naph), 140.84, 140.76 (C8), 136.29 (C-Ar), 128.87, 128.82 (CH-Ar), 127.85 (C-Ar), 127.77, 127.74, 127.48, 127.45, 126.47, 125.99, 125.96, 122.74, 122.66 (CH-Ar), 119.47 (C5), 116.29 (d 3 J CP = 3.4 Hz, CH-Ar), 116.17 (d 3 J CP = 2.9 Hz, CH-Ar), 93.42, 93.34 (C1'), 80.57 (d 3 J CP = 8.1 Hz, C4'), 80.53 (d 3 J CP = 5.1 Hz, C4'), 76.61, 76.53 (C2'), 75.41, 75.38 (OCH2C(CH3)3), 68.95 (d 2 J CP = 5.3 Hz, C5'), 68.82 (d 2 J CP = 5.2 Hz, C5'), 51.84, 51.73 (CHCH3), 35.04, 34.75 (C3'), 32.29 (OCH2C(CH3)3), 26.70 (OCH2C(CH3)3), 20.76 (d 3 J CP = 6.4 Hz, CHCH3), 20.55 (d 3 J CP = 7.2 Hz, CHCH3).

[0514] MS (ES+) m / z: Found: 633 (M+H + ), 655 (M+Na + ) C 28 H 34 ClN6O7P Required: 652.16 (M).

[0515] HPLC Reverse phase HPLC using H2O / CH3CN elution from 90 / 10 to 0 / 100 in 30 min, F = 1 ml / min, l = 254 nm, t R 19.16 min.

[0516] (2S)-Benzyl 2-((((2S,4R,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-4- hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate P

[0517]

[0518] Compound V was prepared according to general procedure 4, using 2-chloro-3'- deoxyadenosine (343 mg, 0.66 mmol), tert-butyldimethylsilyl chloride (328 mg 2.18 mmol), imidazole (297 mg, 4.36 mmol). Purification by column chromatography (eluent system CH3OH / CH2Cl20 / 100 to 12 / 88) gave intermediate 1 in quantitative yield. Next, intermediate 1 (970 mg, 1.89 mmol) was reacted with a solution of 12 mL of THF / H2O / TFA 4 / 1 / 1. Purification by column chromatography (eluent system CH3OH / CH2Cl20 / 100 to 12 / 88) gave intermediate 2 (544 mg, 72%). Then, intermediate 2 (204 mg, 0.51 mmol) was reacted with phenyl(ethoxy-L-alaninyl) phosphorochloridate (348.56 mg, 1.02 mmol) in dry THF (5 mL) and tert- butylmagnesium chloride. Purification by column chromatography (eluent system CH3OH / CH2Cl20 / 100 to 8 / 92) gave intermediate 3 (93 mg, 28%). Finally, intermediate 3 (93 mg, 0.14 mmol) was reacted with a solution of THF / TFA / H2O 1 / 1 / 1 (3 mL). Purification by preparative TLC (2000 μm, eluent system CH3OH / CH2Cl24 / 96) gave the title compound (50 mg, 66%) as a white solid.

[0519] (overall yield 13%)

[0520] 31 P NMR (202 MHz, CD3OD): dP 3.93, 3.72.

[0521] 1H NMR (500 MHz, CD3OD): δH 8.12 (s, 0.5H, H8), 8.11 (s, 0.5H, H8), 7.18-7.23 (m, 2H, Ph), 7.03-7.12 (m, 3H, Ph), 5.85 (d J = 1.5, 0.5H, H1’), 5.84 (d J = 2, 0.5H, H1’), 4.55-4.62 (m, 2H, H4’ and H2’), 4.34-4.38 (m, 0.5H, H5’), 4.28-4.32 (m, 0.5H, H5’), 4.16-4.22 (m, 1H, H5’), 3.93-4.03 (m, 2H, OCH2CH3), 3.70-3.84 (m, 1H, CHCH3), 2.20-2.28 (m, 1H, H3’), 1.95-1.99 (m, 1H, H3’), 1.15-1.21 (m, 3H, CHCH3), 1.06-1.11 (m, 3H, OCH2CH3).

[0522] 13 C NMR (125 MHz, CD3OD): δC 173.66 (d 3 J CP = 4.5 Hz, C=0), 173.65 (d 3 J CP = 5.3 Hz, C=0), 156.68, 156.70 (C6), 153.93, 153.88 (C2), 150.72 (d 2 J CP = 6.7 Hz, C-ipso Ph), 150.71 (d 2 J CP = 6.5 Hz, C-ipso Ph), 149.89, 149.94 (C4), 139.41, 139.35 (C8), 129.33 (CH-Ar), 124.74, 124.73 (CH-Ar), 120.03 (d 3 J CP = 4.75 Hz, CH-Ar), 119.97 (d 3 J CP = 4.87 Hz, CH-Ar), 118.07, 118.03 (C5), 92.02, 91.88 (C1’), 79.26, 79.19 (C4’), 75.26, 75.24 (C2’), 67.18 (d 2 J CP = 5.25 Hz, C5’), 66.81 (d 2 J CP= 5.12 Hz, C5'), 60.96 (OCH2CH3), 50.23, 50.12 (CHCH3), 33.46, 33.21 (C3'), 19.16 (d 3 J CP = 6.3 Hz, CHCH3), 18.97 (d 3 J CP = 7.2 Hz, CHCH3), 13.10, 13.07 (OCH2CH3).

[0523] MS (ES+) m / z: found: 541 (M+H + ), 563 (M+Na + ) C 21 H 26 Cl N6O7P requires: 540 (M).

[0524] HPLC using reverse phase HPLC eluting with H2O / CH3CN from 90 / 10 to 0 / 100 in 30 min, F = 1 ml / min, l = 254 nm, t R 12.41, 12.83 min.

[0525] Table III Example 3 - Evaluation of cytotoxicity and cancer stem cell activity

[0526]

[0527] A solution of (2S)-isopropyl 2-((chloro(phenoxy)phosphoryl)amino)propanoate (546 mg, 3 mmol) in anhydrous THF (5 mL) and N-methylimidazole (240 μL, 5 mmol) was added dropwise to a suspension of (2R,3R,5S)-2-(6-amino-9H-purin-9-yl)-5- (hydroxymethyl)tetrahydrofuran-3-ol (150 mg, 0.6 mmol) in anhydrous THF (3 mL) and the reaction mixture was stirred at room temperature during 16 hours. Purification by column chromatography (eluent system CH3OH / CH2Cl20 / 100 to 6 / 94) and preparative TLC (2000 μM, eluent system CH3OH / CH2Cl25 / 95) afforded the target compound as a white solid (40 mg, 13%).

[0528] MS (ES+) m / z: found: 521.2 (M+H + ), 543.3 (M+Na + ), 1063.4 (2M+Na + ) C 31 H 33 N6O8P requires: 520.18 (M). MS (ES+) m / z: found: 521.2 (M+H + ), 543.3 (M+Na + ), 1063.4 (2M+Na + ) C 31 H 33 N6O8P requires: 520.18 (M).

[0529] 31 P NMR (202 MHz, CD3OD): dP 3.99 (s), 3.82 (s).

[0530] 1 H NMR (500 MHz, CD3OD): dH 8.16 (s, 0.5H, H8), 8.15 (s, 0.5H, H8), 8.11 (s, 1H, H-2) 7.23-7.20 (m, 2H, Ph), 7.11-7.03 (m, 3H, Ph), 5.91 (d J = 2.0 Hz, 0.5H, H1’), 5.90 (d J = 2.0 Hz, 0.5H, H1’), 4.85-4.79 (m, 1H, CH(CH3)2, 4.64-4.63 (m, 1H, H4’), 4.60-6.57 (m, 1H, H2’), 4.37-4.33 (m, 1H, H5’), 4.31-4.28 (m, 1H, H5’), 3.74-4.22-4.17 (m, 1H, H5’), 3.70 (m, 1H, CH ala), 2.02-1.97 (m, 1H, H3’), 2.04-2.01 (m, 1H, H3’), 1.18-1.14 (m, 3H, CH3), 1.24 (m, 6H, CH(CH3)2)

[0531] HPLC using reverse phase HPLC eluting with H2O / CH3CN from 100 / 10 to 0 / 100 for 30 min, F = 1 ml / min, λ = 200 nm, showed two peaks of diastereoisomers with t R 11.58 min and t R 11.92 min.

[0532] Solvents and reagents. The following anhydrous solvents were purchased from Sigma-Aldrich: dichloromethane (CH2Cl2), trimethyl phosphate ((CH3O)3PO). Commercially available amino acid esters were purchased from Sigma-Aldrich. All commercially available reagents were used without further purification.

[0533] Thin layer chromatography (TLC).

[0534] Pre-coated aluminium backing plates (60F254, 0.2 mm thickness, Merck) were visualized under short and long wave UV light (254 and 366 nm) or by combustion visualization using the following TLC indicators: (i) ammonium molybdate cerium sulfate; (ii) potassium permanganate solution. Preparative TLC plates (20 cm x 20 cm, 500-2000 pm) were purchased from Merck.

[0535] Flash column chromatography. Use silica gel (60A, 35-70 μm) supplied by Fisher to carry out flash column chromatography. Use appropriate eluent to fill the glass column homogenate, and the sample is pre-adsorbed on the silica gel or loaded onto the silica gel in the form of a concentrated solution in the same eluent. Identify the fractions containing the product by TLC, and merge and remove the solvent in vacuo.

[0536] High performance liquid chromatography (HPLC). The purity of the final compound was verified to be >95% by HPLC analysis using I) ThermoSCIENTIFIC, SPECTRA SYSTEM P4000, detector SPECTRA SYSTEM UV2000, Varian Pursuit XRs 5 C18, 150x4.6mm (as analytical column) or II) Varian Prostar (LC workstation - Varian Prostar 335LC detector), Thermo SCIENTIFIC Hypersil Gold C18, 5μ, 150x4.6mm (as analytical column). See the experimental section for elution methods.

[0537] Nuclear magnetic resonance (NMR) was recorded at 25°C on a Bruker Avance 500 MHz spectrometer. 1 H NMR (500 MHz), 13 C NMR (125 MHz), 31 P NMR (202 MHz) and 19 F NMR (470 MHz). Relative to internal standard MeOH-d4 (δ 3.34 1 H-NMR, δ49.86 13 C-NMR) and CHCl3-d4(δ7.26 1 H NMR, δ77.36 13 C NMR) or external standard 85% H3PO4 (δ0.00 31 Chemical shifts (δ) are quoted in parts per million (ppm) for P NMR. Coupling constants (J) are measured in Hertz. The following abbreviations are used in the attribution of NMR signals: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), bs (broad singlet), dd (double of doublets), dt (double of triplets), app (apparent). Analysis based on coupling constants and additional two-dimensional experiments (COZY, HSQC, HMBC, PENDANT) was performed. 1 H NMR and 13 Assignments of signals in C NMR.

[0538] Mass Spectrometry (MS). Low resolution mass spectra were performed on a Bruker Daltonics microTof-LC (atmospheric pressure ionization, electron spray mass spectrometry) in positive or negative mode.

[0539] Purity of final compounds. The purity of all final compounds was confirmed to be > 95% using HPLC analysis.

[0540] Example 2 - Cytotoxicity

[0541] The anti-cancer potency of exemplary compounds embodying the present application was evaluated in the following procedure.

[0542] In vitro viability assays were performed to assess the effect of compounds on cell viability in 7 selected cell lines using the CellTiterGlo (CTG, Promega-G7573) assay method over 72 hours. The tests were performed in duplicate, 3.16-fold titration in 96-well plates, treating compounds at 9 points over approximately 72 hours. The starting concentration of compounds was 198 mM. Cell viability assays using CellTiterGlo in 96-well plates were performed. Compound treatment was for 72 hours in duplicate under standard growth conditions. Compounds were dissolved to 40 mM, 100% thawed. Compounds were serially diluted in thawed DMSO at 3.16-fold and heated to 37°C before dissolution in culture medium (2 μl + 200 μl). After the compounds were dissolved in culture medium (culture medium was also heated to 37°C). The culture medium containing the compounds was heated to 37°C in an incubator in duplicate before the compounds in the medium were added to the cell plates (50 μl + 50 μl). The final concentration of compounds was 198 M to 19.9 nM. All compound solubility was checked again and recorded before the plates were immediately transferred to a CO2 tissue incubator and incubated for 3 days. The final concentration of DMSO was 0.5%.

[0543] The results of the primary screen are provided in Table II. A indicates a relative IC 50 from 0.1 to 5 μM, B indicates a relative IC 50 greater than 5 μM and up to 15 μM, C indicates a relative IC 50 greater than 15 μM and up to 100 μM; and D indicates a relative IC 50 greater than 100 μM.

[0544] Table II

[0545]

[0546] a MOLT-4: acute lymphoblastic leukemia; b KG-1: acute myelogenous leukemia; cHL-60: Acute promyelocytic leukemia; d CCRF-CEM: Acute lymphoblastic leukemia; e K562: Chronic myelogenous leukemia. f IC50 μM: Relative IC 50 ; g M.I. %: Maximum percent inhibition of cell viability.

[0547] Table II (continued)

[0548]

[0549]

[0550] h MCF-7: Breast carcinoma; i HepG2: Hepatocellular carcinoma

[0551] A subset of the compounds of the application was then assayed for cytotoxic activity in a more extensive array of different solid and hematological malignancies using the following assay.

[0552] Solid and hematological malignancy assays

[0553] In vitro viability assays to assess the effect of compounds on cell viability in selected cell lines were performed over 72 hours using CellTiterGlo (CTG, Promega-G7573). Tests were performed in duplicate, compounds were treated in 3.16-fold titration in 9 points in 96-well plates over approximately 72 hours. The starting concentration of compounds was 198 mM. Cell viability assays using CellTiterGlo in 96-well plates were performed. Standard growth conditions compound treatment in duplicate was 72 hours. Compounds were dissolved to 40 mM, thawed 100%. Compounds were serially diluted in thawed DMSO at 3.16-fold and heated to 37°C before solubilization in culture medium (2 μl + 200 μl). After solubilization of compounds in culture medium, the culture medium containing compounds was heated to 37°C in an incubator before adding the compounds in medium to cell plates (50 μl + 50 μl) in duplicate. The final concentration of compounds was 198 M to 19.9 nM. All compound solubility was checked again and recorded before the plates were immediately transferred to a CO2 tissue incubator and incubated for 3 days. The final concentration of DMSO was 0.5%.

[0554] The following cell lines were tested and are mentioned in Table IV below:

[0555] Figure 1

[0556]

[0557] The results of the preliminary screening are provided in Tables IV-VII. For Tables IV to VI: A represents the absolute IC from 0.1 μM to 5 μM 50 , B indicates an absolute IC greater than 5 μM and up to 15 μM 50 , C represents an absolute IC greater than 15 μM and up to 100 μM 50 ; and D indicates an absolute IC greater than 100 μM 50 For Table VII: A represents the absolute EC from 0.1 μM to 5 μM 50 , B indicates an absolute EC greater than 5 μM and up to 15 μM 50 , C represents an absolute EC greater than 15 μM and up to 100 μM 50 ; and D indicates an absolute EC greater than 100 μM 50 .

[0558] Table IV

[0559]

[0560]

[0561] Table IV (continued)

[0562]

[0563] Table IV (continued)

[0564]

[0565] Table IV (continued)

[0566]

[0567] Table IV (continued)

[0568]

[0569] Table V

[0570]

[0571]

[0572] Table V (continued)

[0573]

[0574] Table VI

[0575]

[0576] Table VI (continued)

[0577]

[0578]

[0579] Table VI (continued)

[0580]

[0581] Table VII

[0582]

[0583] Table VII (continued)

[0584]

[0585]

[0586] All compounds tested showed cytotoxic activity against the cell lines tested. In most cases, the compounds of the application were more potent than the parent nucleoside against all cell lines.

[0587] Figure 2

[0588] Further comparative analysis of toxicity of compounds in the acute myeloid leukaemia (AML) cell line KG1a was performed over an extended dose range and the relative impact of the compounds on the leukaemic stem cell (LSC) compartment within the KG1a cell line was assessed over the entire dose range.

[0589] Materials and methods

[0590] KG1a cell culture conditions

[0591] The KG1a cell line was maintained in RPMI medium (Invitrogen, Paisley, UK) supplemented with 100 units / ml penicillin, 100 μg / ml streptomycin and 20% foetal calf serum. Subsequently, cells were aliquoted (10 5 cells / 100 μl) into 96-well plates and incubated in the presence of nucleoside analogues and their respective proTides at concentrations determined experimentally for each series of compounds at 37°C in a humidified 5% carbon dioxide atmosphere for 72 hours. In addition, control cultures in which no drug was added were performed. Cells were then harvested by centrifugation and analysed by flow cytometry using the Annexin V assay.

[0592] Determination of apoptosis in vitro

[0593] Caltag Medsystems, Botolph Claydon, UK) was added to the cell suspension and the cells were incubated in the dark for 10 minutes prior to washing. Finally, the cells were resuspended in 190 μl of calcium-rich buffer together with 10 μl of propidium iodide. Apoptosis was assessed by dual colour immunofluorescence flow cytometry as previously described. The LD 50 values (dose required to kill 50% of cells in culture) were then calculated for each nucleoside analogue and ProTide.

[0594] Immunophenotypic identification of the leukemia stem cell compartment

[0595] KG1a cells were cultured for 72 hours in the presence of each compound assayed at a range of concentrations. Cells were then harvested and labelled with a cocktail of anti-lineage antibodies (PE-cy7), anti-CD34 (FITC), anti-CD38 (PE) and anti-CD123 (PERCP cy5). Subsets expressing the LSC phenotype were then identified and expressed as a percentage of all viable cells remaining in culture. The percentage of remaining stem cells was then plotted on a dose response graph and the effect of the compounds compared to each other and to the parent nucleoside.

[0596] Statistical analysis

[0597] The data obtained in these experiments were assessed using one-way ANOVA. All data were confirmed to be Gaussian or Gaussian approximations using the Bartlett's test for homogeneity of variances. LD 50 values were calculated from the analysis of the line from the non-linear regression and best fit of the sigmoidal dose-response curve. All statistical analyses were performed using Graphpad Prism 6.0 software (Graphpad Software Inc., San Diego, CA).

[0598] Results

[0599] In vitro drug sensitivity was measured using the Annexin V / propidium iodide assay. Compound A showed an increase in in vitro potency when compared to cordycepin (P < 0.0001). 2-F-cordycepin was significantly more potent than cordycepin (P < 0.0001) and all of the ProTides tested showed greater potency compared to the parent nucleoside Figure 3 ).

[0600] These experiments demonstrate that Compound A shows evidence of enhanced potency in the stem cell compartment at concentrations above 1 mM. From Figure 4As can be seen, Compound A showed the ability to not only reduce the total number of cancer stem cells, but also reduce the number of such cells as a proportion of the total number of cancer cells present in the culture medium. This demonstrates the ability of Compound A to preferentially target cancer stem cells. At the higher concentrations tested (1 mM and above), Compound A's ability to preferentially target LSCs was significantly greater than that of the parent compound.

[0601] When compared to the parent nucleoside, 2-F-cordycepin proTides compounds P, Q, and R also showed significantly enhanced preferential targeting of LSCs. In contrast, while compound O was able to reduce the proportion of LSCs present in the treated cell population (indicating the ability to target LSCs), its activity was not significantly different from that of 2-F-cordycepin at any concentration tested. Example 4 - Further cytotoxicity evaluation and inhibition studies Comparisons between 2-F-cordycepin and all tested proTides are shown, while individual comparisons are shown in ​ .

[0602] ​

[0603] Further studies were conducted to test the cytotoxic activity of certain compounds of the present invention and also to measure their activity against four hematological cancer cell lines.

[0604] TdT-positive CEM (human ALL)

[0605] TdT negative K562 (human CML)

[0606] TdT negative H1-60 (human ANLL)

[0607] RL (CRL-2261) non-HD lymphoma

[0608] The concentration of the active metabolite dATP (cordycepin triphosphate) was also measured in these cell lines.

[0609] Cytotoxic activity and intracellular 3'-dATP concentrations were also studied in CEM and RL cancer cell lines in the presence of pharmacological inhibitors of hENT1, adenosine kinase (AK), and adenosine deaminase that mimic known anticancer mechanisms.

[0610] method

[0611] Cell culture

[0612] HL-60( CCL-240 TM )、K562( CCL-243 TM )、CCRF-CEM( CRM-CCL-119TM ) and RL( CRL-2261 TM ) leukemia cell lines from the American Type Culture Collection (ATCC), Middlesex. The HL-60 and K562 cell lines are deoxynucleotidyl transferase negative (TdT-ve) and the CCRF-CEM cell line is TdT+ve.

[0613] The HL-60 cell line is an acute promyelocytic leukemia; K562 is a CML cell line, CCRF-CEM is an acute lymphoblastic leukemia (ALL) and RL is a non-Hodgkin lymphoma cell line.

[0614] Cell line maintenance

[0615] The HL-60, K562, CCRF-CEM and RL cell lines were cultured in RPMI-1640 medium (Sigma Aldrich, UK) supplemented with 10% foetal bovine serum (FBS) (PAA Laboratories), 1% amphotericin B (5.5 ml) and 1% penicillin / streptomycin (5.5 ml) (PAA Laboratories) and grown in flasks in an incubator at 37°C containing 5% C02.

[0616] Adenosine 5’- triphosphate (ATP) assay

[0617] The amount of ATP was used as a measure of cell number and cell viability. The ATP ViaLight™ plus assay kit (Lonza, USA: product number LT07-121) was used to detect ATP in cells treated in luminescence compatible 96-well plates (initial cell concentration of 1 x 105cells / well) with concentrations of cordycepin and ProTides: 0, 0.1, 0.5, 1, 5 and 10 mM, followed by incubation in an incubator at 37°C containing 5% C02for 72 hours. For inhibitor studies, 10 mM of NBTI or 1 mM of MEHNA or A-134974 were added and left for 5 minutes before addition of the drugs (see inhibitor details in section 5). 4

[0618] Following incubation, 50 mΐ of cell lysis reagent was added to the 96-well plate to release intracellular ATP, followed by 100 mΐ of ATP monitoring reagent (AMR). The luminescence value for each well was determined using a FLUOstar OPTIMA microplate reader (BMG Labtech) which converts ATP to light using luciferase. Thus, the amount of light produced is directly proportional to the amount of ATP.

[0619] Treatment of cells and extraction of samples for intracellular triphosphate analysis

[0620] ​Use a 5x10 6 Cell lines with 50 μM cordycepin and compounds A, B, D, E and F were prepared. Cells were treated with 1 μl of 50 μM of each cordycepin and compound A, B, D, E and F and incubated for 2 hours at 37°C in the presence of 5% CO2. After incubation, the cells were centrifuged (environment, 1200 rpm, 5 minutes), the culture supernatant was removed, and the cell aggregates were washed with 1 ml of PBS and centrifuged (environment, 1200 rpm, 5 minutes). The supernatant was removed; the aggregates were reconstituted in 100 μl of PBS and 100 μl of 0.8 M perchloric acid and vortexed and kept on ice for 30 minutes. The supernatant was then transferred to a new tube (environment, 1200 rpm, 5 minutes) of 180 μl and stored at -80°C until analysis time.

[0621] During analysis, 90 μl of the extract was transferred to a fresh tube. 25 μl of 1 M ammonium acetate was added to the extract, which was then neutralized by adding 10 μl of 10% ammonia and 5 μl of deionized water. The extract was then transferred to an LC-MS vial, and 10 μl was injected into the UPLC-MS / MS system.

[0622] Inhibitor research

[0623] Cell lines were treated in the same manner as above, but prior to drug treatment, a number of inhibitors were added:

[0624] Nitrobenzylthioinosine (NBTI) (Sigma-Aldrich, St. Louis, MO, Product No. N2255) blocks nucleoside transporters

[0625] EHNA hydrochloride (Sigma-Aldrich, St. Louis, MO, product number E114) blocks adenosine deaminase

[0626] Adenosine kinase inhibitor A-134974 dihydrochloride hydrate (Sigma-Aldrich, St. Louis, MO, Product No. A2846): blocks adenosine kinase

[0627] Cells were treated with 10 μM NBTI or 1 μM EHNA or A-134974 for 5 minutes before drug addition. Cells were then incubated at 37°C with 5% CO2 for 2 hours.

[0628] LC-MS / MS analysis

[0629] Analytes were separated using an ultra-high performance liquid chromatography system (Accela UPLC, Thermo Scientific, UK) equipped with a Biobasic Ax5 μm, 50×2.1 mm column (Thermo Electron Corporation, Murrieta, CA, USA) and a mobile phase consisting of a mixture of 10 mM NH Ac in ACN / H O (30:70 v / v) (pH 6.0) (A) and 1 mM NH Ac in ACN / H O (30:70 v / v) (pH 10.5) (B). A mobile phase gradient consisting of buffer A = 95% held at 0-0.5 min, 95% to 0% in 1.25 min, held at 0% for 1.75 min, 0-95% in 0.1 min, and ending at 95% for 2.9 min was used, all at a flow rate of 500 μl / min.

[0630] Eluted compounds of interest were detected using a triple quadrupole Vantage mass spectrometer system (Thermo Scientific, UK) equipped with an electrospray ion source. Samples were analyzed in multiple reaction monitoring, negative ion mode, at a spray voltage of 3000 V. Nitrogen was used as sheath gas and auxiliary gas at flow rates of 50 and 20 arbitrary units, respectively. Argon was used as collision gas at a pressure of 1.5 mTorr. The optimal transition product ion mass and collision energy for each analyst were as follows: 3'ATP 490.1→392.1 (collision energy 19 V) and internal standard ChloroATP 539.9→442.2 (collision energy 24 V).

[0631] Statistical analysis

[0632] Use nonlinear regression analysis of percent cell viability to determine the dose-response curve of drug cytotoxicity against concentration and obtain the EC 50 Each condition was repeated 5 times for intracellular determination. Intracellular determination was determined using a paired t-test (two-tailed) of 3'ATP / ATP concentration and p values ​​were obtained. For all analyses, Prism Software program (GraphPad software) was used and Microsoft Plot the results.

[0633] result

[0634] Summary IC 50 Table (μM)

[0635]

[0636] (Fd) = fold difference compared to cordycepin = cordycepin IC 50 / ProTide IC50

[0637] Summary of average intracellular 3'-dATP levels (pg / ml)

[0638]

[0639] (FD) = fold difference compared to cordycepin

[0640] Compounds A and B were the best performers, with IC 50 values superior to cordycepin by 3 to 150-fold. Compounds A and B produced intracellular 3'-dATP concentrations superior to cordycepin by 3 to 56-fold.

[0641] Summary of IC 50 Table (all in pM)

[0642]

[0643] (FD) = fold difference compared to control

[0644] Summary of average intracellular 3'-dATP levels (pg / ml)

[0645]

[0646]

[0647] (FD) = fold difference compared to control

[0648] NBTI, AK and EHNA did not affect intracellular 3'-dATP produced by the three compounds of the invention tested, indicating that in the blood cancer cell lines used in this study, these inhibitors did not interfere with the metabolism by which the compounds of the invention produce the active agent 3'-dATP. Since these inhibitors mimic known anticancer mechanisms, these results suggest that the compounds of the invention are not susceptible to the anticancer mechanisms of cordycepin.

Claims

1. A compound of formula (Ib): in: W1 is -P(=O)(U)(V), wherein U is -OAr, and V is -NR4-CR1R2-C(=O)OR3; W2 is H; X is NH2; Z is H; Y is selected from the group consisting of H, F, Cl, and OCH3; R1 and R2 are independently selected from the group consisting of H, -CH3 and -CH2CH(CH3)2; R3 is selected from the group consisting of benzyl and unsubstituted methyl, ethyl, propyl, butyl, pentyl, and hexyl; R4 is H; and Ar is selected from the group consisting of phenyl and naphthyl, each of which is optionally replaced by C 2-6 Ester substitution, or a pharmaceutically acceptable salt of the compound of formula (Ib).

2. The compound of claim 1, wherein the compound of formula (Ib) is a compound of formula (II):

3. The compound of claim 1 or 2, wherein Ar is unsubstituted.

4. The compound of claim 1, wherein R 2 It is a methyl group.

5. The compound of claim 4, wherein the C atom with R1 and R2 has the same absolute configuration as L-alanine.

6. The compound of claim 1, wherein R3 is selected from the group consisting of benzyl, unsubstituted methyl, and unsubstituted n-pentyl.

7. The compound of claim 6, wherein R3 is benzyl.

8. The compound of claim 1, wherein Y is H.

9. The compound of claim 1, wherein Y is F.

10. The compound of claim 1, wherein Y is Cl.

11. The compound of claim 1, wherein Y is -OCH3.

12. The compound of claim 1, wherein the compound of formula (Ib) is selected from: (2S)-Benzyl 2-(((((2S,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)(naphthalen-1-yloxy)phosphoryl)amino)propanoate; Benzyl 2-(((((2S,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)acetate; (2S)-Pentyl 2-(((((2S,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)(naphthalen-1-yloxy)phosphoryl)amino)-4-methylpentanoate; Methyl 2-(((((2S,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)(naphthalen-1-yloxy)phosphoryl)amino)-2-methylpropanoate; (2S)-Benzyl 2-(((((2S,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)(2-(3-ethoxy-3-oxopropyl)phenoxy)phosphoryl)amino)propanoate; (2S)-Benzyl 2-((((2S,4R,5R)-5-(6-amino-2-methoxy-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)(naphthalen-1-yloxy)phosphorylamino)propanoate; (2S)-Benzyl 2-((((2S,4R,5R)-5-(6-amino-2-methoxy-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphorylamino)propanoate; (2S)-Benzyl 2-(((((2S,4R,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate; (2S)-Hexyl 2-(((((2S,4R,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate; (2R)-benzyl 2-((((2S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)(naphthalen-1-yloxy)phosphorylamino)propanoate; 2-O-methyl-3'-deoxyadenosyl-5'-O-[1-naphthyl(1-pentyloxy-L-leucinyl)]phosphate; 2-O-methyl-3'-deoxyadenosine-5'-O-[phenyl(1-hexyloxy-L-alanyl)]phosphate; 2-Fluoro-3'-deoxyadenosine-5'-O-[1-naphthyl(benzyloxy-L-alanyl)]phosphate; and 2-Fluoro-3'-deoxyadenosine-5'-O-[1-naphthyl(1-pentyloxy-L-leucinyl)]phosphate.

13. The compound according to claim 1, wherein the compound of formula (Ib) is (S p )-3'-deoxyadenosine-5'-O-[phenyl(benzyloxy-L-alanyl)]phosphate.

14. The compound according to claim 1, wherein the compound of formula (Ib) is (R p )-3'-deoxyadenosine-5'-O-[phenyl(benzyloxy-L-alanyl)]phosphate.

15. Use of a compound according to any one of the preceding claims in the preparation of a medicament for preventing or treating cancer, wherein the cancer is selected from the group consisting of leukemia, lymphoma, multiple myeloma, liver cancer, breast cancer, pancreatic cancer, colon cancer and colorectal cancer.

16. The use according to claim 15, wherein the cancer is leukemia or lymphoma.

17. The method of claim 16, wherein the leukemia is selected from the group consisting of acute lymphoblastic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, monocytic leukemia, hairy cell leukemia, Hodgkin's lymphoma, and non-Hodgkin's lymphoma.

18. The use according to claim 17, wherein the leukemia is acute lymphoblastic leukemia.

19. A pharmaceutical composition comprising a compound according to any one of claims 1 to 14 in combination with a pharmaceutically acceptable carrier or excipient.

20. The pharmaceutical composition of claim 19, wherein the excipient is a diluent.

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