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Title:
ADENOSINE DERIVATIVES FOR USE IN THE TREATMENT OF CANCER
Document Type and Number:
WIPO Patent Application WO/2017/207989
Kind Code:
A1
Abstract:
The present invention relates to chemical compounds of formula (I) as defined in the amended claims, their preparation and their use in the treatment of cancer.

Inventors:
GRIFFITH HUGH (GB)
SERPI MICHAELA (GB)
SLUSARCZYK MAGDALENA (GB)
MCGUIGAN CHRISTOPHER
Application Number:
GB2017/051554
Publication Date:
December 07, 2017
Filing Date:
May 31, 2017
Export Citation:
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Assignee:
NUCANA BIOMED LTD (GB)
International Classes:
C07H19/167; A61K31/7076; A61P35/00; A61P35/02; C07H19/20
Domestic Patent References:
WO2006100439A12006-09-28
WO2015081133A22015-06-04
WO2014076490A12014-05-22
Foreign References:
Other References:
TADEUSZ ROBAK: "New nucleoside analogs for patients with hematological malignancies", EXPERT OPINION ON INVESTIGATIONAL D, INFORMA HEALTHCARE, UK, vol. 20, no. 3, 1 January 2011 (2011-01-01), pages 343 - 359, XP009186868, ISSN: 1354-3784, DOI: 10.1517/13543784.2011.554822
JONG HYUN CHO ET AL: "Efficient synthesis of nucleoside aryloxy phosphoramidate prodrugs utilizing benzyloxycarbonyl protection", TETRAHEDRON, vol. 67, no. 30, 1 July 2011 (2011-07-01), pages 5487 - 5493, XP055092851, ISSN: 0040-4020, DOI: 10.1016/j.tet.2011.05.046
Attorney, Agent or Firm:
HGF LIMITED (1 City Walk, Leeds West Yorkshire LS11 9DX, GB)
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Claims:
CLAIMS

1. A compound of formula I:

wherein: each of R1 and R2 is independently selected from H, and the group consisting of Ci-8alkyl, benzyl, C2-8alkenyl, Ci-4alkoxyCi-4alkyl, C2-8alkynyl, C3-8cycloalkyl, and 4-8heterocycloalkyl, any of which is optionally substituted;

R3 is selected from H, and the group consisting of Ci-8alkyl, benzyl, C2-8alkenyl, Ci- 4alkoxyCi-4alkyl, C2-8alkynyl, C3-8cycloalkyl, 4-8heterocycloalkyl, phenyl, napthyl, 5- or 6- membered monocyclic heteroaryl and 9- or 10 membered bicyclic heteroaryl, any of which is optionally substituted;

R4 is selected fromH, and the group consisting of Ci-8alkyl, benzyl, C2-8alkenyl, Ci-4alkoxyCi- 4alkyl, C2-8alkynyl, C3-8cycloalkyl, and 4-8heterocycloalkyl, any of which is optionally substituted

R5 is independently selected from OAr and ;

Ar is selected from the group consisting of phenyl, napthyl, 5- or 6- membered monocyclic heteroaryl and 9- or 10 membered bicyclic heteroaryl, each of which is optionally substituted; or a pharmaceutically acceptable salt, ester, salt of an ester, solvate, prodrug or salt of a prodrug thereof.

2. A compound of claim 1, wherein the compound of formula (I) is a compound of formula (II)

3. A compound of claim 1 or claim 2, wherein Ar is phenyl.

4. A compound of claim 3, wherein Ar is naphthyl.

5. A compound of claim 4, wherein Ar is 1 -naphthyl.

6. A compound of claim 1, wherein the compound of formula (I) is a compound of formula (III)

A compound of any one of claims 1 to 6, wherein R1 and R2 are each independently at occurrence selected from H, Ci-4-alkyl and -CH2-phenyl.

8. A compound of claim 7, wherein R1 is H and R2 is Me.

9. A compound of any one of claims 1 to 8, wherein R3 is independently at each occurrence selected from benzyl, C3-7cycloalkyl and Ci-6alkyl.

10. A compound of claim 9, wherein R3 is benzyl.

11. A compound of any one of claims 1 to 10, wherein R4 is H.

12. A compound of any one of claims 1 to 11, wherein Ar, R1, R2, R3 and R4 are unsubstituted.

13. A compound of claim 1, wherein the compound of formula (I) is a compound selected from:

Cyclohexyl 2-(((((2R,3^,4R,5R)-5-(6-amino-8-chloro-9H-purin-9-yl)-3,4-dihydroxy- tetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoiyl)amino)acetate;

Benzyl 2-(((((2R,3^,4R,5R)-5-(6-amino-8-chloro-9H-purin-9-yl)-3,4-dihydroxytetra- hydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)acetate;

(25)-Benzyl 2-(((((2R,3^,4R,5R)-5-(6-amino-8-chloro-9H-purin-9-yl)-3,4-dihydroxy- tetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate;

(25)-Benzyl 2-(((((2R,3^,4R,5R)-5-(6-amino-8-chloro-9H-purin-9-yl)-3,4-dihydroxy- tetrahydrofuran-2-yl)methoxy)(naphthalen-l-yloxy)phosphoryl)amino)propanoate;

(2S)-Pentyl 2-(((((2R,3^,4R,5R)-5-(6-amino-8-chloro-9H-purin-9-yl)-3,4-dihydroxy- tetrahydrofuran-2-yl)methoxy)(naphthalen-l-yloxy)phosphoryl)amino)propanoate; 5'- ([(Ethyloxy-L-alanin-N-yl)-naphth- 1 -yl]-phosphatyl) 8-chloroadenosine;

5'-([(Cyclohexyloxy-L-alanin-N-yl)-naphth-l-yl]-phosphatyl) 8-chloroadenosine;

5'-([(Benzyloxy-L-leucin-N-yl)-naphth-l-yl]-phosphatyl) 8-chloroadenosine;

5 ' -([(Pent- 1 -yloxy-L-leucin-N-yl)-naphth- 1 -yl]-phosphatyl) 8-chloroadenosine;

5'-([(Cyclohexyloxy-L-leucin-N-yl)-naphth-l-yl]-phosphatyl) 8-chloroadenosine;

5'-([(ethyloxy-L-leucin-N-yl)-naphth-l-yl]-phosphatyl) 8-chloroadenosine;

5 ' -([(benzyloxy -glycin-N-yl)-naphth- 1 -yl]-phosphatyl) 8-chloroadenosine;

5'-([(Cyclohexyloxy-glycin-N-yl)-naphth-l-yl]-phosphatyl) 8-chloroadenosine;

5 ' -([(ethyloxy-glycin-N-yl)-naphth- 1 -yl]-phosphatyl) 8-chloroadenosine;

5 ' -([(Pent- 1 -yloxy-glycin-N-yl)-naphth- 1 -yl]-phosphatyl) 8-chloroadenosine;

5'-([(Benzyloxy-L-phenylalanin-N-yl)-naphth-l-yl]-phosphatyl) 8-chloroadenosine;

5'-([(Pent-l-yloxy-L-phenylalanin-N-yl)-naphth-l-yl]-phosphatyl) 8-chloroadenosine;

5'-([(Cyclohexyloxy-L-phenylalanin-N-yl)-naphth-l-yl]-phosphatyl) 8-chloroadenosine; 5'-([(Ethyloxy-L-phenylalanin-N-yl)-naphth-l-yl]-phosphatyl) 8-chloroadenosine;

5'-([(Benzyloxy-D-alanin-N-yl)-phenyl]-phosphatyl) 8-chloroadenosine;

5'-([(Benzyloxy-L-phenylalanin-N-yl)-phenyl]-phosphatyl) 8-chloroadenosine;

5'-([(Benzyloxy-L-leucin-N-yl)-phenyl]-phosphatyl) 8-chloroadenosine;

5'-([(Isopropyloxy-L-alanin-N-yl)-phenyl]-phosphatyl) 8-chloroadenosine;

5'-([(Cyclohexyloxy-L-alanin-N-yl)-phenyl]-phosphatyl) 8-chloroadenosine;

5'-([(Neopentyloxy-2,2-dimethylglycin-N-yl)-phenyl]-phosphatyl) 8-chloroadenosine;

5'-([(Methyloxy-L-methionin-N-yl) phenylj-phosphatyl) 8-chloroadenosine;

5'-([(Benzyloxy-L-valin-N-yl)-phenyl]-phosphatyl) 8-chloroadenosine;

8-Chloroadenosine 5'-O-bis(benzyloxy-L-alanin-N-yl) phosphate; and

8-Chloroadenosine 5'-O-bis(benzyloxy-glycin-N-yl) phosphate.

14. A compound of claim 13, wherein the compound of formula (I) is

(25)-Benzyl 2-(((((2R,3^,4R,5R)-5-(6-amino-8-chloro-9H-purin-9-yl)-3,4-dihydroxy- tetrahydrofuran-2-yl)methoxy)(naphthalen-l-yloxy)phosphoryl)amino)propanoate.

15. A compound of claim 14, wherein the compound of formula (I) is

(25)-Benzyl 2-(((((2R,3^,4R,5R)-5-(6-amino-8-chloro-9H-purin-9-yl)-3,4-dihydroxy- tetrahydrofuran-2-yl)methoxy)(naphthalen- 1 -yloxy)-(,Sp)-phosphoryl)amino)propanoate in substantially diastereoisomerically pure form.

16. A compound of claim 14, wherein the compound of formula (I) is

(25)-Benzyl 2-(((((2R,3^,4R,5R)-5-(6-amino-8-chloro-9H-purin-9-yl)-3,4-dihydroxy- tetrahydrofuran-2-yl)methoxy)(naphthalen- 1 -yloxy)-(Rp)-phosphoryl)amino)propanoate in substantially diastereoisomerically pure form.

17. A compound of claim 13, wherein the compound of formula (I) is benzyl 2- (((((2R,3^,4R,5R)-5-(6-amino-8-chloro-9H-purin-9-yl)-3,4-dihydroxytetra-hydrofuran-2- yl)methoxy)(phenoxy)phosphoryl) amino)acetate.

18. A compound of claim 17, wherein the compound of formula (I) is benzyl 2- (((((2R,3^,4R,5R)-5-(6-amino-8-chloro-9H-purin-9-yl)-3,4-dihydroxytetra-hydrofuran-2- yl)methoxy)(phenoxy)-(Sp)-phosphoryl)amino)acetate in substantially diastereoisomerically pure form.

19. A compound of claim 17, wherein the compound of formula (I) is benzyl 2- (((((2R,3^,4R,5R)-5-(6-amino-8-chloro-9H-purin-9-yl)-3,4-dihydroxytetra-hydrofuran-2- yl)methoxy)(phenoxy)-(Rp)-phosphoryl)amino)acetate in substantially diastereoisomerically pure form.

20. A compound of any one of claims 1 to 19 for medical use.

21. A compound of any one of claims 1 to 19 for use in treating cancer.

22. A compound for use of claim 21, wherein the cancer is selected from the group consisting of leukaemia, multiple myeloma, lung cancer, liver cancer, breast cancer, head and neck cancer, neuroblastoma, thyroid carcinoma, skin cancer, oral squamous cell carcinoma, urinary bladder cancer, Leydig cell tumour, colon cancer, colorectal cancer and gynaecological cancers.

23. A compound for use of claim 22, wherein the cancer is leukaemia.

24. The compound for use of claim 23, wherein the leukaemia is selected from the group comprising acute lymphoblastic leukaemia, acute myelogenous leukaemia, acute premyelocytic leukaemia, acute lymphocytic leukaemia, chronic myelogenous leukaemia, chronic lymphocytic leukaemia, monoblastic leukaemia and hairy cell leukaemia.

25. A pharmaceutical formulation comprising a compound of any one of claims 1 to 19 and a pharmaceutically acceptable excipient.

26. A pharmaceutical formulation of claim 25, which further comprises a polar aprotic organic solvent. 27. A pharmaceutical formulation of claim 26, wherein the polar aprotic organic solvent is dimethylacetamide.

Description:
ADENOSINE DERIVATIVES FOR USE IN THE TREATMENT OF CANCER

The present invention relates to chemical compounds, their preparation and their use in the treatment of cancer. Particularly, although not exclusively, the present invention relates to chemical compounds useful in the treatment of leukaemia in homo sapiens. WO 2006/100439 A discloses phosphoramidates of cladribine, isocladribine, fludarabine and clofarabine and their use in treating cancer.

8-Chloroadenosine (8-Cl-A) is a nucleoside analogue that has shown some cytotoxic activity in many cancers, including leukaemia. 8-Cl-A is currently under phase I clinical study for chronic lymphocytic leukaemia. 8-Cl-A is composed of an adenine base which has a chlorine group attached at position 8. 8-Cl-A is believed to exert cytotoxicity by causing cell cycle arrest at G2/M phase, mediating catastrophic mitotic division, thereby inducing apoptosis and autophagy.

8-Cl-A uptake into the cells is mediated by nucleoside transporters such as equilibrative nucleoside transporters (ENT) type and concentrative nucleoside transporters (CNT) type. ENTs, including human ENT 1 (hENTl), are crucial for the drugs to be therapeutically effective. Once inside the cancer cells 8-Cl-A goes through a series of phosphorylation steps by adenosine kinases (AK) to form the active anti-cancer metabolite 8-Chloro-adenosine triphosphate, 8-Cl-ATP. Adenosine kinases (AK) phosphorylate 8-Cl-A by transferring γ- phosphate from ATP to 5'-hydroxyl of adenosine of 8-Cl-A, to produce 8-Chloro-adenosine monophosphate (8-Cl-AMP) and as a result 8-Cl-A becomes activated, ultimately generating 8-Cl-ATP. Therefore, as the 8-Cl-ATP accumulates, the intracellular ATP pool decreases. As a result the ratio of 8-Cl-ATP to ATP in the cell increases and favours the incorporation of 8-Cl-ATP into DNA as a false nucleotide, causing double-strand breaks in the DNA and preventing repair, ultimately leading to cell death through apoptosis. The development of resistance mechanisms of cancer cells to 8-Cl-A can limit its effectiveness. There are several mechanisms proposed through which resistance occurs: limited cellular uptake (hENTl); poor activation (AK); extensive degradation by Adenosine Deaminase (ADA). hENTl. Cancer cells may downregulate hENTl or express a mutated form of the protein which may be less functional or non- functional. For example, single missense mutations can lead to the substitution of glycine to arginine at position 24 (G24) in the transmembrane 1 domain of hENTl, generating an aberrant protein, which cannot bind to nucleoside analogues. Therefore, the mutated hENTl protein cannot mediate the uptake of 8-Cl-A across the cell membrane into the cancer cell. AK. Cancer cells can develop mutations in the catalytic core of AK, preventing efficient 8- Cl-A binding and phosphorylation. Therefore 8-Cl-A remains largely in an inactive non- phosphorylated form in the cells and can no longer exert its cytotoxic effects. In vitro studies have shown that when AK is inhibited, cells become resistant to 8-Cl-A and no detectable 8- Cl-ATP is produced. Therefore, adenosine kinase is required to phosphorylate and activate 8- CI- A.

ADA. Adenosine deaminase degrades 8-Cl-A in the plasma and intracellularly, thereby decreasing the drug levels and limiting the anti-cancer effects.

The putative existence of a cancer stem cell has been suggested in many human cancers including leukaemias and solid tumours. The cancer stem cell hypothesis considers that only a small sub-population of tumour cells is responsible for the formation and maintenance of the bulk of the tumour.

It is an object of certain embodiments of the present invention to provide a therapeutic agent that, in use, has therapeutic efficacy, particularly enhanced therapeutic efficacy in the prophylaxis or treatment of cancer. It is an object of certain embodiments of the present invention to provide a means for delivering 8-Cl-ATP to cancer cells. It is an object of certain embodiments to provide a means of generating 8-Cl-ATP which overcomes, at least in part, the abovementioned resistance mechanisms to 8-Cl-A.

A further object of certain embodiments of the present invention is to provide a therapeutic agent having therapeutic efficacy against cancer stem cells.

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

Summary of the Invention

According to a first aspect of the present invention there is provided a compound of formula I:

each of R 1 and R 2 is independently selected from H, and the group consisting of Ci-2oalkyl, C6-3oarylCi-6alkyl, C 2-20 alkenyl, C 1-20 alkoxyC 1-20 alkyl, C 1 -20 alkoxyC6-3 0 aryl, C 2-20 alkynyl, C 3- 2ocycloalkyl, C3-2ocycloalkylC6-3oaryl, and 4-2oheterocycloalkyl, any of which is optionally substituted;

R 3 is selected from H, and the group consisting of Ci-2oalkyl, C6-3oarylCi-2oalkyl, C2-2oalkenyl, Ci-2oalkoxyCi-2oalkyl, Ci-2oalkoxyC6-3oaryl, C2-2oalkynyl, C3-2ocycloalkyl, C3-2ocycloalkylC6- 3oaryl, 5-2oheterocycloalkyl, C6-3oaryl and 5-3oheteroaryl, any of which is optionally substituted;

R 4 is selected from H, and the group consisting of Ci-2oalkyl, C6-3oarylCi-2oalkyl, C2-2oalkenyl, Ci-2oalkoxy, Ci-2oalkoxyCi-2oalkyl, C3-2ocycloalkyl, Ci-2oalkoxyC6-3oaryl, C2-2oalkynyl, C3- 2ocycloalkylC6-3oaryl, C6-3oaryloxy, 5-2oheterocycloaklyl, C6-3oaryl and 5-3oheteroaryl, any of which is optionally substituted;

R 5 is independently selected from OAr and ;

Ar is selected from the group consisting of C6-3oaryl and 5-3oheteroaryl, each of which is optionally substituted; or a pharmaceutically acceptable salt, ester, salt of an ester, solvate, prodrug or salt of a prodrug thereof. Surprisingly, the phosphoramidate compounds of the present invention show, compared to the parent nucleoside 8-Cl-A, an enhanced anti-proliferative effect, as measured by IC50 or EC50 assays. Surprisingly, at least some of the phosphoramidate compounds of the present invention show, compared to the parent nucleoside 8-Cl-A, an enhanced anti-cancer stem cell activity. Thus, at least some of the phosphoramidate compounds of the present invention show, compared to the parent nucleoside 8-chloroadenosine, an enhanced anti-proliferative effect, as measured by IC50 or EC50 assays, in combination with an ability to target cancer stem cells.

Compounds embodying the present invention are particularly suitable for treating cancer in homo sapiens.

In certain preferred embodiments, Ar is selected from the group consisting of phenyl, napthyl, 5- or 6- membered monocyclic heteroaryl and 9- or 10 membered bicyclic heteroaryl, each of which is optionally substituted. each of R 1 and R 2 is independently selected from H, and the group consisting of Ci -8 alkyl, benzyl, C 2-8 alkenyl, Ci-4alkoxyCi-4alkyl, C 2-8 alkynyl, C3- 8 cycloalkyl, and 4- 8 heterocycloalkyl, any of which is optionally substituted;

R 3 is selected from H, and the group consisting of Ci -8 alkyl, benzyl, C 2 - 8 alkenyl, Ci- 4alkoxyCi-4alkyl, C 2 - 8 alkynyl, C3- 8 cycloalkyl, 4- 8 heterocycloalkyl, phenyl, napthyl, 5- or 6- membered monocyclic heteroaryl and 9- or 10 membered bicyclic heteroaryl, any of which is optionally substituted;

R 4 is selected fromH, and the group consisting of Ci -8 alkyl, benzyl, C 2-8 alkenyl, Ci-4alkoxyCi- 4alkyl, C 2-8 alkynyl, C3- 8 cycloalkyl, and 4- 8 heterocycloalkyl, any of which is optionally substituted.

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

embodiment, the compound of formula (I) is a compound of formula (III)

The following embodiments apply to compounds of any of formulae (I) to (III). These embodiments are independent and interchangeable. Any one embodiment may be combined with any other embodiment, where chemically allowed. In other words, any of the features described in the following embodiments may (where chemically allowable) be combined with the features described in one or more other embodiments. In particular, where a compound is exemplified or illustrated in this specification, any two or more of the embodiments listed below, expressed at any level of generality, which encompass that compound may be combined to provide a further embodiment which forms part of the present disclosure.

Ar may be selected from the group consisting of phenyl, napthyl, 5- or 6- membered monocyclic heteroaryl and 9- or 10 membered bicyclic heteroaryl, each of which is optionally substituted.

Ar may be unsubstituted. Ar may be substituted. Where Ar is substituted, it can be substituted with one, two, three, four or five substituents.

Ar, whether substituted or unsubstituted, may be selected from the group consisting of naphthyl, phenyl, quinolyl and pyridyl. Ar may be selected from the group consisting of naphthyl and phenyl. Where Ar is naphthyl it may be bound to -O-P at the 1 -position on naphthyl. Ar may be selected from unsubstituted naphthyl or unsubstituted phenyl. In certain preferred embodiments, Ar is unsubstituted naphthyl and is bound to -O-P at the 1 -position on naphthyl. R 1 and R 2 may each be unsubstituted.

It may be that R 1 and R 2 are selected such that -CR 1 R 2 COO- represents the corresponding part of a proteinogenic alpha amino acid or (where the proteinogenic alpha amino acid is not glycine) its D-enantiomer.

Thus, it may be that one of R 1 and R 2 is H and the other is independently at each occurrence selected from H and Ci-C4alkyl, said Ci-C4-alkyl being optionally substituted with a group selected from phenyl (optionally substituted with a single hydroxyl substituent), imidazole, indole, SR a , OR a , C0 2 R a , C0 2 R a R a , R a R b and H(= H) H 2 ; wherein R a is independently at each occurrence selected from: H and Ci-C4-alkyl; and R b is independently at each occurrence selected from: H, and Ci-C4-alkyl and C(0)-Ci-C4-alkyl . Thus, it may be that R 1 is H and R 2 is independently at each occurrence selected from H, CH 2 - phenyl (optionally substituted with a single hydroxyl substituent) and Ci-C4-alkyl, said C1-C4- alkyl being optionally substituted with a group selected from imidazole, indole, SR a , OR a , C0 2 R a , C0 2 R a R a , R a R b and NH(= H) H 2

It may be that R 1 and R 2 are each independently at each occurrence selected from H, C1-C4 alkyl and CH 2 -phenyl.

In certain preferred embodiments, R 1 and R 2 are independently at each occurrence selected from the group consisting of H, -CH 3 , -CH 2 -Ph and -CH 2 CH(CH 3 ) 2 .

R 1 and R 2 may at each occurrence independently be selected from CH 3 and H. In certain preferred embodiments, one of R 1 and R 2 is CH 3 and one of R 1 and R 2 is H. Thus, in further preferred embodiments, R 2 is CH 3 and R 1 is H (i.e. such that the C atom bearing R 1 and R 2 has the same absolute configuration as L-alanine).

It may be that one of R 1 and R 2 is H and one of R 1 and R 2 is -CH 2 -CH(CH 3 ) 2 or -CH 2 -Ph. It may be that each of R 1 and R 2 is H. In the present specification, the term "proteinogenic alpha amino acid" may mean one of the 21 amino acids that are directly encoded for protein synthesis by the genetic code of eukaryotes. Thus a proteinogenic amino acid may mean an amino acid 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. In the present specification, a side chain of a naturally occurring alpha amino acid is thus a member selected from the group consisting of

H, CH 3 , -CH(CH 3 ) 2 , -CH 2 CH(CH 3 ) 2 , -CH(CH 3 )(CH 2 CH 3 ), -CH 2 Ph, -CH 2 Ph-OH, -CH 2 SH, CH 2 CH 2 SCH 3 , -CH 2 OH, -CH(CH 3 )(OH), -CH 2 CH 2 CH 2 CH 2 NH 3 + , -CH 2 CH 2 CH 2 NHC(=NH 2 )NH 2 , CH 2 C(0)0-, -CH 2 CH 2 C(0)0-, -CH 2 C(0)NH 2 , -CH 2 CH 2 C(0)NH 2 ,

R 3 may be unsubstituted.

R 3 may be independently at each occurrence selected from C6-3oarylCi-6alkyl and unsubstituted Ci-2oalkyl. R 3 may be independently at each occurrence selected from benzyl, C3-7cycloalkyl and Ci-6alkyl. Thus, R 3 may be independently at each occurrence selected from unsubstituted benzyl, unsubstituted C3-7cycloalkyl and unsubstituted Ci-6alkyl, any of which is optionally substituted. In certain preferred embodiments, R 3 is independently at each occurrence selected from the group consisting of benzyl (-CH2-PI1), unsubstituted methyl (- CH3) unsubstituted n-pentyl (-n-CsHn), unsubstituted cyclohexyl (-CeHn) and unsubstituted ethyl (-CH2CH3). In certain preferred embodiments, R 3 is benzyl. R 3 may be selected from H, and the group consisting of Ci -8 alkyl, benzyl, C 2-8 alkenyl, Ci- 4alkoxyCi-4alkyl, C 2 -8alkynyl, C3- 8 cycloalkyl, 4-sheterocycloalkyl, phenyl, napthyl, 5- or 6- membered monocyclic heteroaryl and 9- or 10 membered bicyclic heteroaryl, any of which is optionally substituted. R 3 may be selected from the group consisting of Ci -8 alkyl, benzyl, C 2 - 8 alkenyl, Ci-4alkoxyCi-4alkyl, C 2-8 alkynyl, C3- 8 cycloalkyl, 4- 8 heterocycloalkyl, phenyl, napthyl, 5- or 6- membered monocyclic heteroaryl and 9- or 10 membered bicyclic heteroaryl, any of which is optionally substituted. R 3 may be selected from the group consisting of H, Ci- 2oalkyl, C6-3oarylCi-6alkyl, C2-ioalkenyl, Ci-ioalkoxyCi-ioalkyl, Ci-ioalkoxyC6-ioaryl, C2- loalkynyl, C3- 2 ocycloalkyl, C3-2ocycloalkenyl, Cs-2ocycloalkynyl, and 4-2oheterocycloalkyl.

In certain preferred embodiments, R 3 is selected from the group consisting of H, Ci-2oalkyl, C6-3oarylCi-6alkyl and C3-2ocycloalkyl. Thus, R 3 may be selected from the group consisting of H, Ci-6alkyl, benzyl and C3-6cycloalkyl. Thus, R 3 may be selected from the group consisting of Ci-6alkyl, benzyl and C3-6cycloalkyl. R 3 may be selected from the group consisting of C 6 - 3oarylCi-6alkyl and unsubstituted Ci-2oalkyl. Thus, R 3 may be selected from the group consisting of Ci-6alkyl, and benzyl. In further preferred embodiments, R 3 is selected from the group consisting of benzyl (-CH2PI1), unsubstituted methyl (-CH3) and unsubstituted n-pentyl (-n-C 5 Hii). In yet further preferred embodiments, R 3 is benzyl.

In certain preferred embodiments, R 4 is H.

It may be that R 4 is not optionally substituted, i.e. R 4 may be unsubstituted.

R 4 may be independently at each occurrence selected from the group consisting of H, Ci- 2oalkyl, C6-3oarylCi-6alkyl, C2-ioalkenyl, Ci-ioalkoxy, Ci-ioalkoxyCi-ioalkyl, Ci-ioalkoxyC6- loaryl, C2-ioalkynyl, C3-2ocycloalkyl, C3-2ocycloalkenyl, Cs-2ocycloalkynyl, and 4- 2oheterocy cloalkyl .

In certain preferred embodiments, R 4 is independently at each occurrence selected from the group consisting of H, Ci-isalkyl, C6-3oarylCi-6alkyl, C3-2ocy cloalkyl and 4-2oheterocyclyl. Thus, R 4 may be independently at each occurrence selected from the group consisting of H, Ci-salkyl, benzyl, C3-scycloalkyl and 4-8heterocyclyl. In a further preferred embodiment, R 4 is independently at each occurrence selected from the group consisting of H, methyl, ethyl, propyl, butyl, pentyl, hexyl and cyclohexyl.

In certain specific embodiments: Ar is selected from phenyl (C6H5-) and naphthyl (C10H7-), each of which is optionally substituted; each of R 1 and R 2 is independently selected from H and methyl (CH3-), wherein methyl is optionally substituted; R 3 is selected from the group consisting of benzyl (PI1-CH2-) and n-pentyl (n-CsHn-), each of which is optionally substituted; and R 4 is H.

In certain specific embodiments Ar is selected from unsubstituted naphthyl (C10H7-); each of R 1 and R 2 is independently selected from H and methyl (CH3-), wherein methyl is optionally substituted; R 3 is selected from the group consisting of benzyl (Ph-CH 2 -) and n-pentyl (n- C5H11-), each of which is optionally substituted; and R 4 is H.

In certain specific embodiments Ar is 1-naphthyl, which is optionally substituted; each of R 1 and R 2 is independently selected from H and methyl (CH3-), wherein methyl is optionally substituted; R 3 is selected from the group consisting of benzyl (Ph-CH 2 -) and n-pentyl (n- C5H11-), each of which is optionally substituted; and R 4 is H.

In certain specific embodiments Ar is unsubstituted phenyl (C6H5-); each of R 1 and R 2 is independently selected from H and methyl (CH3-), wherein methyl is optionally substituted; R 3 is selected from the group consisting of benzyl (Ph-CH 2 -) and n-pentyl (n-CsHn-), each of which is optionally substituted; and R 4 is H.

In certain specific embodiments Ar is selected from phenyl (C6H5-) and naphthyl (C10H7-), each of which is optionally substituted; one of R 1 and R 2 is Me and one of R 1 and R 2 is H such that the C atom bearing R 1 and R 2 has chirality as in natural alanine; R 3 is selected from the group consisting of benzyl (Ph-CH 2 -) and n-pentyl (n-CsHn-), each of which is optionally substituted; and R 4 is H.

In certain specific embodiments Ar is selected from phenyl (C6H5-), which is optionally substituted; each of R 1 and R 2 is H; R 3 is selected from the group consisting of benzyl (Ph- CH 2 -) and n-pentyl (n-CsHn-), each of which is optionally substituted; and R 4 is H.

In certain specific embodiments: Ar is selected from phenyl (C6H5-) and naphthyl (C10H7-), each of which is optionally substituted; each of R 1 and R 2 is independently selected from H and methyl (CH3-), wherein methyl is optionally substituted; R 3 is unsubstituted benzyl (Ph- CH 2 -); and R 4 is H.

Each of Ar, R 1 , R 2 , R 3 and R 4 can be substituted with one, two, three, four or five substituents. Alternatively, Ar, R 1 R 2 , R 3 and R 4 may each be unsubstituted. The compound may be or may be selected from a group of more than one of the compounds A- AC described in the Examples.

In certain preferred embodiments, the compound is (2,S)-benzyl 2-(((((2R,3,S',4R,5R)-5-(6- amino-8-chloro-9H-purin-9-yl)-3,4-dihydroxy-tetrahydrofuran- 2-yl)methoxy)(naphthalen-l- yloxy)phosphoryl)amino)propanoate (compound D from the examples below). The compound may be (2S)-benzyl 2-(((((2R,3^,4R,5R)-5-(6-amino-8-chloro-9H-purin-9-yl)- 3 ,4-dihydroxy-tetrahydrofuran-2-yl)methoxy)(naphthalen- 1 -yloxy)-(Sp)- phosphoryl)amino)propanoate in substantially diastereoisomerically pure form.

The compound may be (2S)-benzyl 2-(((((2R,3^,4R,5R)-5-(6-amino-8-chloro-9H-purin-9-yl)- 3 ,4-dihydroxy-tetrahydrofuran-2-yl)methoxy)(naphthalen- 1 -yloxy)-(Rp)- phosphoryl)amino)propanoate in substantially diastereoisomerically pure form.

The compound may be isomer A of (2S)-benzyl 2-(((((2R,3^,4R,5R)-5-(6-amino-8-chloro-9H- purin-9-yl)-3,4-dihydroxy-tetrahydrofuran-2-yl)methoxy)(naph thalen-l- yloxy)phosphoryl)amino)propanoate in substantially diastereoisomerically pure form. Thus, the compound may be the isomer of (2,S)-benzyl 2-(((((2R,3,S',4R,5R)-5-(6-amino-8-chloro- 9H-purin-9-yl)-3,4-dihydroxy-tetrahydrofuran-2-yl)methoxy)(n aphthalen-l- yloxy)phosphoryl)amino)propanoate that has a 31 P NMR peak at 3.93±0.04 when the NMR spectrum has been obtained on a 202 MHz NMR machine in CD3OD, said isomer being in substantially diastereoisomerically pure form. The compound may be the isomer of (25)- benzyl 2-(((((2R,35,4R,5R)-5-(6-amino-8-chloro-9H-purin-9-yl)-3,4-d ihydroxy- tetrahydrofuran-2-yl)methoxy)(naphthalen-l-yloxy)phosphoryl) amino)propanoate that has a retention time of 16.43 ± 0.10 minutes when analytical HPLC is performed on a Varian Pursuit XRs 5 C18, 150 x 4.6 mm eluting with H 2 0/ CH3CN from 100/10 to 0/100 in 30 min at 1 mL/min, , said isomer being in substantially diastereoisomerically pure form.

The compound may be isomer B of (25)-Benzyl 2-(((((2R,3,S',4R,5R)-5-(6-amino-8-chloro- 9H-purin-9-yl)-3,4-dihydroxy-tetrahydrofuran-2-yl)methoxy)(n aphthalen-l-yloxy)

phosphoryl)amino)propanoate in substantially diastereoisomerically pure form. Thus, the compound may be the isomer of (25)-Benzyl 2-(((((2R,35,4R,5R)-5-(6-amino-8-chloro-9H- purin-9-yl)-3,4-dihydroxy-tetrahydrofuran-2-yl)methoxy)(naph thalen-l-yloxy)phosphoryl) amino)propanoate that has a 31 P NMR peak at 3.83±0.04 when the NMR spectrum has been obtained on a 202 MHz NMR machine in CD3OD, said isomer being in substantially diastereoisomerically pure form. The compound may be the isomer of (25)-benzyl 2- (((((2R,35,4R,5R)-5-(6-amino-8-chloro-9H-purin-9-yl)-3,4-dih ydroxy-tetrahydrofuran-2- yl)methoxy)(naphthalen-l-yloxy)phosphoryl)amino)propanoate that has a retention time of 16.59 ± 0.10 minutes when analytical HPLC is performed on a Varian Pursuit XRs 5 C18, 150 x 4.6 mm eluting with H 2 0/ CH 3 CN from 100/10 to 0/100 in 30 min at 1 mL/min, said isomer being in substantially diastereoisomerically pure form.

In certain preferred embodiments, the compound is benzyl 2-(((((2R,3,S',4R,5R)-5-(6-amino-8- chloro-9H-purin-9-yl)-3,4-dihydroxytetra-hydrofuran-2-yl)met hoxy)(phenoxy)phosphoryl) amino)acetate (compound B from the examples below).

The compound may be benzyl 2-(((((2R,3^,4R,5R)-5-(6-amino-8-chloro-9H-purin-9-yl)-3,4- dihydroxytetra-hydrofuran-2-yl)methoxy)(phenoxy)-(Sp)-phosph oryl)amino)acetate in substantially diastereoisomerically pure form.

The compound may be benzyl 2-(((((2R,3^,4R,5R)-5-(6-amino-8-chloro-9H-purin-9-yl)-3,4- dihydroxytetra-hydrofuran-2-yl)methoxy)(phenoxy)-(Rp)-phosph oryl)amino)acetate in substantially diastereoisomerically pure form.

The compound may be isomer A of benzyl 2-(((((2R,3,S',4R,5R)-5-(6-amino-8-chloro-9H- purin-9-yl)-3,4-dihydroxytetra-hydrofuran-2-yl)methoxy)(phen oxy)phosphoryl)amino) acetate in substantially diastereoisomerically pure form. Thus, the compound may be the isomer of benzyl 2-(((((2R,35',4R,5R)-5-(6-amino-8-chloro-9H-purin-9-yl)-3,4- dihydroxytetra-hydrofuran-2-yl)methoxy)(phenoxy)phosphoryl) amino)acetate that has a 31 P NMR peak at a chemical shift of 4.64±0.04 when the NMR spectrum has been obtained on a 202 MHz NMR machine in CD 3 OD, said isomer being in substantially diastereoisomerically pure form. The compound may be the isomer of benzyl 2-(((((2R,3S,4R,5R)-5-(6-amino-8- chloro-9H-purin-9-yl)-3,4-dihydroxytetra-hydrofuran-2-yl)met hoxy)(phenoxy)

phosphoryl)amino)acetate that has a retention time of 12.58 ± 0.10 minutes when analytical HPLC is performed on a Varian Pursuit XRs 5 CI 8, 150 x 4.6 mm eluting with H 2 0/ CH 3 CN from 100/10 to 0/100 in 30 min at 1 mL/min, said isomer being in substantially diastereoisomerically pure form. The compound may be isomer B of benzyl 2-(((((2R,3,S',4R,5R)-5-(6-amino-8-chloro-9H- purin-9-yl)-3,4-dihydroxytetra-hydrofuran-2-yl)methoxy)(phen oxy)phosphoryl)amino) acetate. Thus, the compound may be the isomer of benzyl 2-(((((2R,3,S',4R,5R)-5-(6-amino-8- chloro-9H-purin-9-yl)-3,4-dihydroxytetra-hydrofuran-2-yl)met hoxy)(phenoxy)phosphoryl) amino)acetate that has a 31 P NMR peak at a chemical shift of 3.57±0.04 when the NMR spectrum has been obtained on a 202 MHz NMR machine in CD 3 OD, said isomer being in substantially diastereoisomerically pure form. The compound may be the isomer of benzyl 2- (((((2R,3^,4R,5R)-5-(6-amino-8-chloro-9H-purin-9-yl)-3,4-dih ydroxytetra-hydrofuran-2- yl)methoxy)(phenoxy)phosphoryl) amino)acetate that has a retention time of 12.88 ± 0.10 minutes when analytical HPLC is performed on a Varian Pursuit XRs 5 C18, 150 x 4.6 mm eluting with Η 2 0/ CH3CN from 100/10 to 0/100 in 30 min at 1 mL/min, said isomer being in substantially diastereoisomerically pure form.

Compounds in accordance with the said specific embodiments of the first aspect of present invention have surprisingly been shown to provide enhanced targeting of cancer stem cells, compared to 8-chloroadenosine.

According to a second aspect of the present invention, there is provided a compound of the present invention for use in a method of treatment.

In certain embodiments, there is provided a compound of the present invention for use in the prophylaxis or treatment of cancer.

According to a third aspect of the present invention there is provided use of a compound of the present invention in the manufacture of a medicament for the prophylaxis or treatment of cancer.

According to a fourth aspect of the present invention, there is provided a method of prophylaxis or treatment of cancer comprising administration to a patient in need of such treatment an effective dose of a compound of the present invention.

Each of the second, third and fourth aspects of the invention can comprise embodiments for treating cancer employed in combination with other cancer therapy. Examples of other cancer therapy include radiotherapy and/or other chemotherapy.

With respect to each of the second, third and fourth aspects of the present invention, embodiments of the invention comprise a cancer selected from among haematological and solid tumours. In particular, the cancer can be selected from the group consisting of leukaemia, multiple myeloma, lung cancer, liver cancer, breast cancer, head and neck cancer, neuroblastoma, thyroid carcinoma, skin cancer (including melanoma), oral squamous cell carcinoma, urinary bladder cancer, Leydig cell tumour, colon cancer, colorectal cancer and gynaecological cancers, including ovarian cancer, uterine cancer and cervical cancer, including epithelia cervix carcinoma. In preferred embodiments, the cancer is leukaemia and can be selected from the group consisting of acute lymphoblastic leukaemia, acute myelogenous leukaemia (also known as acute myeloid leukaemia or acute nonlymphocytic leukaemia), acute promyelocytic leukaemia, acute lymphocytic leukaemia, chronic myelogenous leukaemia (also known as chronic myeloid leukaemia, chronic myelocytic leukaemia or chronic granulocytic leukaemia), chronic lymphocytic leukaemia, monoblastic leukaemia and hairy cell leukaemia. In further preferred embodiments, the cancer is acute lymphoblastic leukaemia.

Compounds embodying the present invention have been found to have enhanced anti-cancer activity, compared to the 8-chloroadenosine, in treating solid tumours, as well as leukaemia. Examples of solid tumours that are suitable for treatment by compounds of the present invention include breast cancer, prostate cancer, lung cancer, colon cancer, cervical cancer and lymphomas. Examples of lymphomas suitable for treatment by compounds of the invention include Hodgkin Lymphoma and non-Hodgkin Lymphoma. Examples of leukaemia which are suitable for treatment by compounds of the present invention include myeloid leukaemia, multiple myeloma, chronic myelogenous leukaemia, acute myelogenous leukaemia and acute lymphocytic leukaemia.

It is believed that the compounds of the present invention enter into cancer cells and are incorporated into the cell's RNA and/or DNA. Without being bound by any theory, it is believed that the efficacy, particularly the anticancer efficacy, exhibited by compounds of the present invention demonstrates that compounds of the present invention are phosphorylated to 8-chloroadenosine triphosphate and it is believed that enzymatic cleavage within the cell converts a compound of the invention directly into 8-chloroadenosine monophosphate prior to phosphorylation to the triphosphate. The anti-cancer potency of the compounds of the present invention is believed additionally to be due to their cellular membrane permeability.

The invention provides a compound of the invention for use in targeting cancer stem cells.

The invention provides the use of a compound of the invention in the manufacture of a medicament for targeting cancer stem cells. The invention provides a method of targeting cancer stem cells, the method comprising providing a population of cancer stem cells with an amount of a compound of the invention sufficient to target such cancer stem cells.

The targeting of cancer stem cells referred to in the present invention may be employed in the prevention or treatment of cancer. In such embodiments the population of cancer stem cells may be in a cancer or pre-cancerous condition in a patient in need of such targeting, and the method may comprise administering a therapeutically effective amount of a compound of the invention to the patient.

The invention provides a compound of the invention for use as an anti-cancer stem cell medicament. This use of a compound of the invention may also be employed in the prevention or treatment of cancer.

The invention provides a method of determining whether a patient with cancer or a precancerous condition will benefit from prevention or treatment of cancer with a compound of the invention, the method comprising:

assaying a biological sample representative of cancer or a pre-cancerous condition in the patient for the presence of cancer stem cells; wherein the presence of cancer stem cells in the biological sample indicates that the patient will benefit from treatment with a compound of the invention.

The invention provides a method of determining a suitable treatment regimen for a patient with cancer or a pre-cancerous condition, the method comprising:

assaying a biological sample representative of cancer or a pre-cancerous condition in the patient for the presence of cancer stem cells; wherein the presence of cancer stem cells in the biological sample indicates that a suitable treatment regimen will comprise treatment of the patient with a compound of the invention. The invention provides a compound of the invention for use in the prevention or treatment of cancer in a patient selected for such treatment by a method comprising:

assaying a biological sample representative of cancer or a pre-cancerous condition in the patient for the presence of cancer stem cells; wherein the presence of cancer stem cells in the biological sample indicates that the patient is suitable for treatment with a compound of the invention. The methods set out above may further comprise a step of preventing or treating the cancer or pre-cancerous condition using a compound of the invention.

In suitable embodiments of the methods of the invention the cancer is relapsed or refractory cancer. A compound of the invention may be used for the treatment of such relapsed or refractory cancer.

The invention provides a compound of the invention for use in treatment of refractory cancer in a subject. The subject may be a human patient.

The invention provides the use of a compound of the invention in the manufacture of a medicament for the treatment of relapsed or refractory cancer in a human patient.

The invention provides a method of treating relapsed or refractory cancer in a subject, the method comprising providing a therapeutically effective amount of a compound of the invention to a subject in need of such treatment.

According to further aspects of the present invention, there are provided a compound of the present invention for use in a method of prophylaxis or treatment, a use of a compound of the present invention in the manufacture of medicament for use in a method of prophylaxis or treatment and a method of prophylaxis or treatment of a patient comprising administration to a patient in need thereof a compound of the present invention, wherein, in each instance, the method of prophylaxis or treatment comprises a method of prophylaxis or treatment of myelodysplastic syndrome.

According to a fifth aspect of the invention, there is provided a pharmaceutical composition comprising a compound of the present invention, in combination with a pharmaceutically acceptable carrier, diluent or excipient.

In certain embodiments of the fifth aspect of the present invention, the pharmaceutical composition comprises a compound of the invention and a second anti-cancer compound, in combination with a pharmaceutically acceptable carrier, diluent or excipient.

According to a sixth aspect of the present invention, there is provided a method of preparing a pharmaceutical composition comprising the step of combining a compound of the present invention with a pharmaceutically acceptable excipient, carrier or diluent. According to a seventh aspect of the present invention there is provided a method of preparing a compound of the present invention comprising reacting a compound of formula II:

II

with a compound of formula III:

III

where Ar, R 1 , R 2 , R 3 and R 4 have the meanings set out above and wherein LG is a leaving group.

Suitable leaving groups will be familiar to those skilled in the art but would include, for example, a halogen atom (e.g. CI), a sulfonate, an aryl group (e.g. phenyl) that is substituted with one or more electron withdrawing substituents or a heteroaryl group (e.g. monocyclic C5 or C 6 membered heteroaryl group) that is substituted with one or more electron withdrawing substituents. Suitable electron withdrawing substituents include fluoride, chloride, nitro, cyano, C02-Ci-C4alkyl, S(0)2-Ci-C4alkyl. A preferred leaving group is CI.

The process for preparing a compound of formula I is preferably carried out in the presence of a suitable solvent. Suitable solvents include hydrocarbon solvents such as hexane, pentane, heptane, cyclohexane, benzene and toluene; ether type solvents such as diethyl ether, tetrahydrofuran, diphenyl ether, anisole and dimethoxybenzene; halogenated hydrocarbon solvents such as methylene chloride, chloroform, 1,2, dichloroethane 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-butyl alcohol and tert-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 may be used singly or two or more of them may be used in admixture.

Preferably an inert solvent is used in the process of the present invention. The term "inert solvent" means a solvent inert under the conditions of the reaction being described in conjunction therewith including, for example, benzene, toluene, acetonitrile, tetrahydrofuran, dimethylformamide, chloroform, methylene chloride (or dichloromethane), diethyl ether, ethyl acetate, acetone, methylethyl ketone, methanol, ethanol, propanol, isopropanol, tert- butanol, dioxane, pyridine, and the like. Tetrahydrofuran is particularly preferred.

Preferably the process of the present invention is carried out under substantially dry conditions.

The phosphorochloridate may be prepared from an aryloxy phosphorodichloridate and a suitably protected amino acid derivative. Alternatively, phosphate chemistry may be used with suitable condensing agents.

Preferably the process for preparing the compound of formula I can include the step of protecting free OH groups, on the nucleoside other than that to which the phosphoramidate is to be attached. Various aspects of the invention are based upon the finding that a compound of the invention is able to preferentially reduce cancer stem cell numbers. This finding is surprising in that cancer stem cells are known to be resistant to many chemotherapeutic agents, and there has previously been no suggestion that either a compound of the invention or 8-chloroadenosine, the parent prodrug compound from which a compound of the invention is derived, were able to target cancer stem cells. Thus the finding that a compound of the invention is able to target cancer stem cells and thus reduce their numbers, a finding which the inventors have confirmed is applicable across a broad range of cancers, represents a surprising breakthrough that enables a range of new therapeutic applications of a compound of the invention.

The biological activities exerted by the compounds of the invention indicate that they will be useful in the treatment of cancer. These activities may be useful in inhibiting the development or progression of cancers. In particular, the biological properties disclosed herein, which have not previously been reported, indicate that these compounds are able to provide treatment that is likely to be effective in patients with relapsed or refractory cancers. Treatment of this sort, using the compounds of the invention, may bring about a reduction in tumour size and/or a reduction in clinically relevant biomarkers, either of which may be associated with more favourable prognosis. Furthermore, treatment with a compound of the invention may help to maintain a reduction in the size of tumours in patients with relapsed or refractory cancer. Accordingly, treatment using a compound of the invention may achieve a high, durable Disease Control Rate (DCR) in patients with relapsed or refractory cancers. Without wishing to be bound by any hypothesis, the inventors believe that the ability of the compounds of the invention to target cancer stem cells contributes to the therapeutic utility of these compounds in the treatment of relapsed or refractory cancer.

Except for where the context requires otherwise, references within this disclosure to a "use" of a compound of the invention in accordance with the invention may be taken as applying to any of the medical uses of compounds of the invention described herein. Similarly, references to "methods" of the invention using a compound of the invention should be taken as applying to any of the methods of the invention herein described.

The ability of a compound of the invention to target cancer stem cells provides new therapies directed against those cancer cells that are considered most difficult to treat, and that are considered to play a major role in the resistance that limits effectiveness of many existing cancer therapies. This ability also provides a way of targeting cells that are believed to be associated with the development, progression, recurrence, and propagation of cancers. Accordingly, it will be recognised that this anti-cancer stem cell activity of a compound of the invention yields benefits in contexts in which new and effective therapies have long been sought. Brief Description of the Drawings

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

Figure 1 compares the LD50 values for 8-chloroadenosine and compounds D, B and E in cells of the acute myeloid leukaemia cell line KGla. All data are the mean ± Standard Deviation (± SD) of three independent experiments.

Figure 2 shows, in Figure 2(i), the individual sigmoidal dose response curve for 8- chloroadenosine and phosphoramidate compounds D, B and E. In Figure 2(ii) the dose response curves for compounds B and E have been removed, to highlight the significant differences (p<0.05) between the responses to 8-chloroadenosine and compound D.

Figure 3 shows the intracellular concentration of 8-Cl-A (A), compound A (B), compound B (C), and compound D (D) in HL-60 leukaemic cells in the absence or presence of the nucleoside transporter inhibitor NBTI and the adenosine kinase inhibitor A-134974, mimicking drug resistance mechanisms.

Figure 4 shows the intracellular concentration of 8-Cl-A (A), compound A (B), compound B (C), and compound D (D) in CTS leukaemic cells in the absence or presence of inhibitors (NBTI, A- 134974).

Figure 5 shows the intracellular concentration of 8-Cl-A (A), compound A (B), compound B (C), and compound D (D) in K-562 leukaemic cells in the absence or presence of inhibitors (NBTI, A-134974).

Figure 6 shows the stability of 8-Cl-A and compounds A, B and C in human plasma ^g/ml) in the absence or presence of the adenosine deaminase inhibitor EHNA.

Figure 7 shows individual sigmoidal dose response curves for Compound E, and Isomers (A and B) of Compounds B and D and Compound AC.

Figure 8 shows the results of a study investigating the ability of Compound E, and Isomers (A and B) of Compounds B and D, and Compound AC, to selectively target cancer stem cells. Figure 9 shows HPLC traces for (25)-Benzyl 2-(((((2R,3^,4R,5R)-5-(6-amino-8-chloro-9H- purin-9-yl)-3,4-dihydroxy-tetrahydrofuran-2-yl)methoxy)(naph thalen-l- yloxy)phosphoryl)amino)propanoate: A) both isomers; B) isomer A; and C) isomer B. Figure 10 shows HPLC traces for benzyl 2-(((((2R,3S,4R,5R)-5-(6-amino-8-chloro-9H-purin- 9-yl)-3,4-dihydroxytetra-hydrofuran-2-yl)methoxy)(phenoxy)ph osphoryl)amino)acetate: A) both isomers; B) isomer A; and C) isomer B.

Detailed description Substituents on Ar can be located anywhere on the aromatic group, where Ar is a 6 membered ring, substituents can be located ortho-, meta- or para-. Substituents on Ar are independently selected from the group consisting of hydroxyl, Ci-6acyl, Ci-6acyloxy, nitro, amino, carboxyl, Chester, Ci-6aldehyde, cyano, Ci-6alkylamino, diCi-6alkylamino, chloro, bromo, fluoro, iodo, Ci-6alkyl, C 2 -6alkenyl, Ci-6alkoxyCi-6alkyl, Ci-6alkoxyC6-ioaryl, C 5 -7cycloalkyl, C 5 - ncycloalkylCi-6alkyl, C5-7cycloalkenyl, Cs-i 2 cycloalkynyl, C6-iiarylCi-6alkyl, Ci-6alkylC6- liaryl, C6-iiaryl, Ci-6fluoroalkyl, C 2- 6fluoroalkenyl, SO3H, SH and SR', wherein R' is independently selected from the same group set out above as R 1 with respect to formula I. Typically each substituent is itself unsubstituted. However, each substituent may optionally be substituted by any other substituents, e.g. 1, 2, 3, 4 or 5 substituents taken from the above list.

Substituents on R 1 , R 2 , R 3 and R 4 are independently selected from the group consisting of hydroxyl, Ci-6acyl, Ci-6acyloxy, nitro, amino, amido, carboxy, Chester, Ci-6aldehyde, cyano, Ci-6alkylamino, diCi-6alkylamino, chloro, bromo, fluoro, iodo, C 5 -7cycloalkyl, C5- 7cycloalkenyl, Cs-i 2 cycloalkynyl, C6-iiaryl, C6-iiarylCi-6alkyl, 5- 2 oheterocyclyl, SO3H, SH and SR', wherein R' is independently selected from the same group set out above as R 1 with respect to formula I. Typically each substituent is itself unsubstituted. However, each substituent may optionally be substituted with 1, 2, 3, 4 or 5 other substituents from the above list.

As used herein, the term "alkyl" refers to a straight or branched saturated monovalent (except where the context requires otherwise) acyclic hydrocarbon radical, having the number of carbon atoms as indicated (an acyclic alkyl group can have 1-20, 1-18, 1-10, 1-6 or 1-4 carbon atoms), optionally substituted with one, two, three, four or five substituents (e.g. one, two or three substituents) independently selected from the group set out above with respect to substituents that may be present on R 1 , R 2 , R 3 and R 4 . It may be that any given alkyl group is unsubstituted. By way of non-limiting examples, alkyl groups can include methyl, ethyl, propyl, butyl, pentyl, hexyl, octyl, nonyl and dodecyl. As used herein, the term "cycloalkyl" refers to a monovalent (except where the context requires otherwise) cyclic hydrocarbon radical, having the number of carbon atoms as indicated (a cycloalkyl group can have 3-20, 3-10 or 3-7 carbon atoms; a cyclic alkenyl group can have 3-20 or 5-7 carbon atoms; a cyclic alkynyl group can have 8-20 carbon atoms), optionally substituted with one, two, three, four or five substituents (e.g. one, two or three substituents) independently selected from the group set out above with respect to substituents that may be present on R 1 , R 2 , R 3 and R 4 . A cycloalkyl group may be saturated or partially saturated, thus the term cycloalkyl encompasses cycloalkenyl and cycloalkynyl groups. It may be that any given cycloalkyl group is un substituted. By way of non-limiting examples, alkyl groups can include cyclopentyl, cyclohexyl, cyclohexenyl and cyclooctenyl.

As used herein, the term "alkenyl" refers to a straight or branched unsaturated monovalent (except where the context requires otherwise) acyclic hydrocarbon radical having one or more C=C double bonds and having the number of carbon atoms as indicated (or where not indicated, an acyclic alkenyl group can have 2-20, 2-10, 2-6 or 2-4 carbon atoms), optionally substituted with one, two, three, four or five substituents (e.g. one, two or three substituents) independently selected from the group set out above with respect to substituents that may be present on R 1 , R 2 , R 3 and R 4 . Any given alkenyl group may be unsubstituted. By way of non-limiting examples, alkenyl groups can include vinyl, propenyl, butenyl, pentenyl and hexenyl. As used herein, the term "alkynyl" refers to a straight or branched unsaturated monovalent (except where the context requires otherwise) acyclic hydrocarbon radical having one or more C≡C triple bonds and having the number of carbon atoms as indicated (or where not indicated, an acyclic alkynyl group can have 2-20, 2-10, 2-6 or 2-4 carbon atoms), optionally substituted with one, two, three, four or five substituents (e.g. one, two or three substituents) independently selected from the group set out above with respect to substituents that may be present on R 1 , R 2 , R 3 and R 4 . Any given alkynyl group may be unsubstituted. By way of non-limiting examples, alkynyl groups can include -C≡CH, -CH2-C≡CH, -C≡C-CH2, -CH 2 - CH 2 -C≡CH, -CH 2 -C≡C-CH 3 and -CH(CH 3 )-C≡CH.

As used herein, the term "alkoxy" refers to the group alkyl-O-, where alkyl is as defined above and where the alkyl moiety may optionally be substituted by one, two, three, four or five substituents (e.g. one, two or three substituents) as set out above for alkyl. Any given alkoxy group may be unsubstituted. Binding is through -0-. By way of non-limiting examples, alkoxy groups can include methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy and 1,2-dimethylbutoxy.

As used herein, the term "aryloxy" refers to the group aiyl-O-, where aryl is as defined below and where the aryl moiety may optionally be substituted by one, two, three, four or five substituents (e.g. one, two or three substituents) as set out above with respect to the group Ar. Any given aryloxy group may be unsubstituted. Binding is through -0-. By way of non- limiting examples, aryloxy groups can include phenyloxy and naphthyloxy.

As used herein, the term "alkoxyalkyl" refers to an alkyl group having an alkoxy substituent. Binding is through the alkyl group. The alkyl moiety and the alkoxy moiety are as defined herein with respect to the definitions of alkyl and alkoxy, respectively. The alkoxy and alkyl moieties may each be substituted by one, two, three, four or five substituents (e.g. one, two or three substituents) as set out above with regard to the definition of alkyl. Any given alkoxyalkyl may be unsubstituted. By way of non-limiting examples, alkoxyalkyl groups can include -CH2-O-CH3, -CH2CH2-O-CH3 and -CH2-O-CH2CH3.

As used herein, the term "arylalkyl" refers to an alkyl group having an aryl substituent. Binding is through the alkyl group. The aryl moiety and the alkyl group are as defined herein with respect to the definitions of aryl and alkyl, respectively. The aryl and alkyl moieties may each be substituted by one, two, three, four or five substituents (e.g. one, two or three substituents), the substituents being as defined herein with respect to the definitions of those substituents that may be present with respect to aryl and alkyl, respectively. Any given arylalkyl groupmay be unsubstituted. In a preferred embodiment, arylalkyl is benzyl, which is Ph-CH 2 -.

As used herein, the term "alkoxy aryl" refers to an aryl group having an alkoxy substituent. Binding is through the aryl group. The alkoxy moiety and the aryl moiety are as defined herein with respect to the definitions of alkoxy and aryl, respectively. The alkoxy and aryl moieties may each be substituted by one, two, three, four or five substituents (e.g. one, two or three substituents), the substituents being as defined herein with respect to the definitions of those substituents that may be present with respect to alkoxy and aryl, respectively. In an embodiment alkoxyaryl is unsubstituted. As used herein, the term "cycloalkylaryl" refers to an aryl group having a cyclic alkyl substituent. Binding is through the aryl group. The cycloalkyl moiety and the aryl moiety are as defined herein with respect to the definitions of cycloalkyl and aryl, respectively. The cycloalkyl moiety and the aryl moiety may each be optionally substituted by one, two, three, four or five substituents (e.g. one, two or three substituents) as set out herein with regard to the definitions of alkyl and aryl, respectively. Any given cycloalkylaryl group may be unsubstituted.

As used herein, the term "aryl" refers to a monovalent (except where the context requires otherwise) aromatic (i.e. the ring system has 4n +2 electrons in a conjugated π system within a ring where all atoms contributing to the conjugated π system are in the same plane) carbocyclic radical having one, two, three, four, five or six rings and having the number of carbon atoms indicated (or where not indicated 6 to 30, 6 to 12 or 6 to 11 carbon atoms). A preferred embodiment has one, two or three rings. An aryl group may optionally be substituted by one, two, three, four or five substituents (e.g. one, two or three substituents), as set out above with respect to optional substituents that may be present on the group Ar. In preferred embodiments, an aryl group comprises: an aromatic monocyclic ring containing 6 carbon atoms; an aromatic fused bicyclic ring system containing 7, 8, 9 or 10 carbon atoms; or an aromatic fused tricyclic ring system containing 10, 11, 12, 13 or 14 carbon atoms. Non- limiting examples of aryl include phenyl and naphthyl. In a preferred embodiment, optional substituent groups on an aryl group can be independently selected from hydroxyl, Ci-6acyl, Ci-6acyloxy, nitro, amino, carboxyl, cyano, Ci-6alkylamino, diCi-6alkylamino, chloro, bromo, fluoro, iodo, SO3H, SH and SR', wherein R' is independently selected from the same groups as R 1 with respect to formula I. Any given aryl group may be unsubstituted.

As used herein, the term "m-nheteroaryl" refers to a monovalent (except where the context requires otherwise) unsaturated aromatic (i.e. a ring system containing (4n + 2) π- or n- electrons in a conjugated π system within a ring where all atoms contributing to the conjugated π system are in the same plane) heterocyclic radical having m to n ring members in the form of one, two, three, four, five or six rings and contained within at least one ring at least one heteroatom (e.g. one, two or three heteroatoms) selected from the group consisting of N, O and S. A preferred embodiment has one, two or three rings. Available carbon atoms and/or heteroatoms in the ring system may be substituted on the ring with one, two, three, four or five substituents (e.g. one, two or three substituents), as set out above with respect to the substituents that may be present on the group Ar. In an embodiment heteroaryl is unsubstituted. Heteroaryl groups can include an aromatic monocyclic ring system containing five ring members of which at least one ring member is a N, O or S atom and which optionally contains one, two or three additional ring N atoms (also referred to in this specification as a 5-membered monocyclic heteroaryl group); an aromatic monocyclic ring having six members of which one, two or three ring members are a N atom (also referred to in this specification as a 6-membered monocyclic heteroaryl group); an aromatic bicyclic fused ring system having nine members of which at least one ring member is a N, O or S atom and which optionally contains one, two or three additional ring N atoms (also referred to in this specification as a 9-membered bicyclic heteroaryl group); or an aromatic bicyclic fused ring system having ten ring members of which one, two or three ring members are a N atom (also referred to in this specification as a 10-membered bicyclic heteroaryl group). Examples include, and are not limited to, pyrrole, furan, thiophene, pyrazole, imidazole, oxazole, isoxazole, triazole, oxadiazole, thiadiazole, tetrazole; pyridine, pyridazine, pyrimidine, pyrazine, triazine, indole, isoindole, benzofuran, isobenzofuran, benzothiophene, indazole, benzimidazole, benzoxazole, benzthiazole, benzisoxazole, purine, quinoline, isoquinoline, cinnoline, quinazoline, quinoxaline, pteridine, phthalazine, naphthyridine. Preferred examples include pyridyl and quinolyl.

As used herein, the term " m -nheterocycloalkyl" refers to a monovalent (except where the context requires otherwise) saturated or partially unsaturated heterocyclic radical having m to n ring members, with at least one ring member (e.g. one, two or three substituents) selected from the group consisting of N, O and S, and being in the form of one, two, three, four, five or six rings. In a preferred embodiment, the radical has one, two or three rings. In a preferred embodiment, the radical has 5 to 10 ring members. Heterocyclyl radicals can include: a monocyclic ring system having five ring members of which at least one ring member is a N, O or S atom and which optionally contains one additional ring O atom or one, two or three additional ring N atoms; a monocyclic ring system having six ring members of which one, two or three ring members are a N atom and which optionally includes an O atom; a bicyclic fused ring system having nine ring members of which 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 a bicyclic fused ring system having ten ring members of which one, two or three ring members are a N atom. Examples include, and are not limited to, azetidinyl, pyrrolinyl, pyrrolidinyl, 1,3- dioxolanyl, imidazolinyl, imidazolidinyl, pyrazolinyl, pyrazolidinyl, piperidinyl, morpholinyl or piperazinyl.

Available ring carbon atoms and/or ring heteroatoms of the "heterocycloalkyl" ring systems described above may be substituted with one, two, three, four or five substituents. Where the ring(s) is substituted with one or more heteroatoms, the heteroatom substituents are selected from halogen (F, CI, Br and I) and from oxygen, nitrogen and sulphur, where the oxygen, nitrogen or sulphur form part of a substituent moiety. Where the ring(s) is substituted with one or more heteroatoms, preferably there are 1, 2, 3 or 4 heteroatom substituents selected from the group consisting of oxygen, nitrogen, sulphur and halogen. Examples of substituent groups that can be present on the heterocyclic ring system can be independently selected from hydroxyl, Ci-6acyl, Ci-6acyloxy, nitro, amino, carboxyl, cyano, Ci-6alkylamino, diCi- 6alkylamino, chloro, bromo, fluoro, iodo, SO3H, SH and SR', wherein R' is independently selected from the same groups as R 1 with respect to formula I. In an embodiment heterocyclyl is unsubstituted. As used herein, the term "acyl" refers to a straight or branched, saturated or unsaturated, substituted or unsubstituted, monovalent (except where the context requires otherwise) radical that includes the moiety -C(=0)-, where binding is through the -C- atom of -C(=0)- moiety, and has the number of carbon atoms indicated (or where not indicated, an acyl group has 1-6, or 1-4 or 1-2 carbon atoms, including the C atom of the -C(=0)- moiety). Acyl may be optionally substituted with one, two, three, four or five substituents (e.g. one, two or three substituents) independently selected from the group set out above with respect to the substituents that may be present on R 1 , R 2 , R 3 and R 4 . Any given acyl group may be unsubstituted. By way of non-limiting examples, acyl groups include HC(=0)-, CH3C(=0)-, C 2 H 5 C(=0)-, C 3 H 7 C(=0)-, C 4 H 9 C(=0)- and C 5 HnC(=0)-. As used herein, the term "acyloxy" refers to a straight or branched, saturated or unsaturated, substituted or unsubstituted monovalent (except where the context requires otherwise) radical that includes the moiety -C(=0)-0-, where binding is through the -O- atom, and has the number of carbon atoms indicated, including the C atom of the -C(=0)-0- moiety (or where not indicated, an acyloxy group has 1-6, 1-4 or 1-2 carbon atoms, including the carbon atom of the -C(=0)-0)- moiety). Acycloxy may be optionally substituted with one, two, three, four or five substituents (e.g. one, two or three of the substituents) that may be present on R 1 , R 2 , R 3 and R 4 . Any given acyloxy group may be unsubstituted. By way of non-limiting examples, acyloxy groups include HC(=0)-0-, CH 3 C(=0)-0-, C 2 H 5 C(=0)-0-, C 3 H 7 C(=0)- 0-, C 4 H 9 C(=0)-0- and C 5 HnC(=0)-0-.

As used herein, the term "C 2 -6ester" refers to a substituted or unsubstituted monovalent (except where the context requires otherwise) radical that comprises R 18 C(=0)-0-R 19 , where R 18 is selected from the group consisting of H and Ci-4alkyl and R 19 is selected from the group consisting of Ci-salkyl, subject to the maximum total number of C atoms, including the C atom of the -C(=0)-0- moiety, of R 18 C(=0)-0-R 19 being six. Binding is through R 18 or R 19 , with an H of the respective group absent such that the alkyl group through which binding occurs is divalent, or, when R 18 is H, through the C of the -C(=0)-0- moiety. In a preferred embodiment, the Chester, including the C atom of the -C(=0)-0 moiety, has 2-5 carbon atoms. The Chester can optionally be substituted with one, two, three, four or five substituents (e.g. one, two or three substituents) independently selected from the group set out above with respect to the substituents that may be present on R 1 , R 2 , R 3 and R 4 . In certain embodiments Chester is unsubstituted. By way of a non-limiting example, Chester can be - C 2 H4-C(=0)-0-C 2 H5, where the -C 2 H4- moiety is -CH 2 -CH 2 - and binding is through the - C 2 H4- moiety.

As used herein, the term "aldehyde" refers to a straight or branched, saturated or unsaturated, substituted or unsubstituted monovalent (except where the context requires otherwise) radical that comprises HC(=0)-R 20 -, where binding is through -R 20 -. "Aldehyde" has the number of carbon atoms indicated, including the C atom of the -C(=0)- moiety (or where not indicated, an aldehyde group has 1-6, 1-4 or 1-2 carbon atoms, including the C atom of the -C(=0)- moiety) and may be optionally substituted with one, two, three, four or five substituents (e.g. one, two or three of the substituents) that may present on R 1 , R 2 , R 3 or R 4 . Any given aldehyde group may be unsubstituted. By way of non-limiting examples, aldehyde groups include HC(=0)-CH 2 -, HC(=0)-C 2 H 4 -, HC(=0)-C 3 H 6 -, HC(=0)-C 4 H 8 - and

As used herein, the term "fluoroalkyl" refers to an alkyl group, where the alkyl group is a straight or branched saturated monovalent (except where the context requires otherwise) cyclic or acyclic hydrocarbon radical, having the number of carbon atoms as indicated (or where not indicated, an acyclic alkyl group has 1-6 or 1-4 carbon atoms and a cyclic alkyl group has 3-6 carbon atoms) substituted with 1 to 6 F atoms. By way of non-limiting examples, fluoroalkyl groups include -CF 3 , -CFIF2 and -CF2-CF3.

As used herein, the term "fluoroalkenyl" refers to an alkenyl group, where the alkenyl group is a straight or branched 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 as indicated (or where not indicated, an acyclic alkenyl group has 2-6 or 2-4 carbon atoms and a cyclic alkenyl group has 4-6 carbon atoms) substituted with 1 to 6 F atoms.

As used herein, the term "stereoisomer" defines all possible compounds made up of the same atoms bonded by the same sequence of bonds but having different three-dimensional structures which the compounds of the present invention may possess.

Compounds according to this invention have at least one chiral centre and accordingly exist as enantiomers. Where the compounds possess two or more chiral centres, they may additionally exist as diastereoisomers. Where the processes for the preparation of the compounds according to the invention give rise to mixture of stereoisomers, these isomers may be separated by conventional techniques such as preparative chromatography. The compounds may be prepared in stereochemically mixed form or individual enantiomers may be prepared by standard techniques known to those skilled in the art, for example, by enantiospecific synthesis or resolution, formation of diastereoisomeric pairs by salt formation with an optically active acid, followed by fractional crystallization and regeneration of the free base. The compounds may also be resolved by formation of diastereoisomeric esters or amides, followed by chromatographic separation and removal of the chiral auxiliary. Alternatively, the compounds may be resolved using a chiral UPLC column. It is to be understood that all such isomers and mixtures thereof are encompassed within the scope of the present invention.

The phosphate centre is chiral in the compounds of the present invention and the compounds may exist as Rp and Sp diastereoisomers. The composition of the compound may be mixed Rp and Sp or one pure diastereoisomer. In a preferred embodiment, the compound is a either Rp or Sp in substantially diastereoisomerically pure form Throughout this specification, by "substantially diastereoisomerically pure form" is meant that the compound consists of 90% (e.g. 95% or more or 98% or more) or more of either the Rp or the Sp diastereoisomer. In another embodiment, there may be a mixture of 1 : 1 Rp to Sp diastereoisomers. Alternatively, the compound may comprise a mixture of Rp and Sp diastereoisomers in a ratio of Rp to Sp diastereoisomers of 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 compound of the invention may comprise a ratio of Rp to Sp diastereoisomers of greater 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 or vice versa.

The compound of formula (I) may be the Rp diastereoisomer. The compound of formula (I) may consist of at least 98wt% of the Rp diastereoisomer. The compound of formula (I) may be the Sp diastereoisomer. The compound of formula (I) may consist of at least 98wt% of the Sp diastereoisomer.

The term "solvate" means a compound of formula I as defined herein, wherein molecules of a suitable solvent are incorporated in the crystal lattice. A suitable solvent is physiologically tolerable at the dosage administered. Examples of suitable solvents are ethanol, water and the like. When water is the solvent, the molecule is referred to as a hydrate.

The compounds of the present invention may also be present in the form of pharmaceutical acceptable salts. For use in medicine, the salts of the compounds of this invention refer to "pharmaceutically acceptable salts." FDA approved pharmaceutical acceptable salt forms (Ref. International J. Pharm. 1986, 33, 201-217; J. Pharm. Sci., 1977, Jan, 66 (1)) include pharmaceutically acceptable acidic/anionic or basic/cationic salts.

Pharmaceutically acceptable acidic/anionic salts include, and are not limited to acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, glyceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methyl sulfate, mucate, napsylate, nitrate, pamoate, pantothenate, phosphate, diphospate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulphate, tannate, tartrate, teoclate, tosylate and triethiodide. Pharmaceutically acceptable basic/cationic salts include, and are not limited to aluminium, benzathine, calcium, chloroprocaine, choline, diethanolamine, ethylenediamine, lithium, magnesium, potassium, procaine, sodium and zinc.

The present invention includes within its scope prodrugs of the compounds of this invention. In general, such prodrugs will be functional derivatives of the compounds which are readily convertible in vivo into the required compound. Thus, in the methods of treatment of the present invention, the term "administering" shall encompass the treatment of the various disorders described with the compound specifically disclosed or with a compound which may not be specifically disclosed, but which converts to the specified compound in vivo after administration to the subject. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in "Design of Prodrugs", ed. H. Bundgaard, Elsevier, 1985.

Examples of prodrugs of compounds of formula I are esters and amides of the compounds of formula I. For example, where the compound of formula I contains a carboxyl group (-COOH), the hydrogen atom of the carboxyl group may be replaced in order to form an ester {e.g. the hydrogen atom may be replaced by Ci-6alkyl). Where the compound of formula I contains an alcohol group (-OH), the hydrogen atom of the alcohol group may be replaced in order to form an ester {e.g. the hydrogen atom may be replaced by -C(0)Ci-6alkyl. Where the compound of formula I contains a primary or secondary amino group, one or more hydrogen atoms of the amino group may be replaced in order to form an amide {e.g. one or more hydrogen atoms may be replaced by -C(0)Ci-6alkyl). Pharmaceutical compositions for use in accordance with the present invention may 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 may be manufactured in a manner that is itself known, e.g. 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.

The compound or pharmaceutical composition according to the present invention can be administered to a patient, which may be homo sapiens or other animal, by any suitable means. The medicaments employed in the present invention can be administered by oral or parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal, vaginal and topical (including buccal and sublingual) administration.

For oral administration, the compounds of the invention will generally be provided in the form of tablets or capsules, as a powder or granules, or as an aqueous solution or suspension.

Tablets for oral use may include the active ingredient mixed with pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavouring agents, colouring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while cornstarch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.

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.

Formulations for rectal administration may be presented as a suppository with a suitable base comprising for example cocoa butter or a salicylate.

Formulations suitable for vaginal administration may 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.

For intramuscular, intraperitoneal, subcutaneous and intravenous use, the compounds of the invention will generally be provided in sterile aqueous solutions or suspensions, buffered to an appropriate pH and isotonicity. Suitable aqueous vehicles include Ringer's solution and isotonic sodium chloride. Aqueous suspensions according to the invention may include suspending agents such as cellulose derivatives, sodium alginate, polyvinyl-pyrrolidone and gum tragacanth, and a wetting agent such as lecithin. Suitable preservatives for aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate. The formulation (and in particular the formulation for intravenous administration) may comprise a polar aprotic organic solvent, e.g. dimethylsulfoxide (DMSO), N- methylpyrrolidinone ( MP) or N, N-dimethylacetamide (DMA). It may be that the formulation (e.g. the formulation for intravenous administration) comprises DMA. The compounds of the invention may also be presented as liposome formulations.

In general a suitable dose will be in the range of 0.1 to 350 mg per kilogram body weight of recipient per day. A more suitable dose may be in the range of 0.5 mg to 150 mg per kilogram body weight of recipient per day, in the range of 0.5 to 100 mg per kilogram body weight of recipient per day, in the range of 1 to 50 mg per kilogram body weight of recipient per day, or in the range of 1 to 10 mg per kilogram body weight of recipient per day. A suitable a lower dose may 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 may be in the range of 1 to 100 mg per m 2 of body surface area of recipient per day or 5 to 50 mg per m 2 of body surface area of recipient per day. Suitable doses may be 6, 12, 24 or 48 mg per m 2 of body surface area of recipient per day. The desired dose may be presented and administered as a single daily dose or as two, three, four, five or six or more sub-doses administered at appropriate intervals throughout the day. Doses may be administered in unit dosage forms, for example, containing 10 to 1500 mg, preferably 20 to 1000 mg, and most preferably 50 to 700 mg of active ingredient per unit dosage form. The total daily dose is suitably 1000 to 3000 mg, whether taken as a single dose or as sub-doses at intervals throughout the day.

Detailed Description of the Invention "Cancer stem cells"

Cancer stem cells, which are sometimes otherwise referred to 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, which is a cell that possesses the capacity to self-renew through asymmetric division, to initiate tumour formation, and to give rise to more mature non-stem cell cancer progeny by differentiation.

Cancer stem cells play a major role in the development, progression, recurrence and propagation of cancers. Accordingly, the finding that compounds of the invention are able to target cancer stem cells, and thereby reduce their numbers, offers therapeutic possibilities in preventing or treating these activities.

As discussed in more detail elsewhere in the specification, cancer stem cells are found in precancerous conditions, where their presence is believed to contribute to the development of such conditions into cancers. Accordingly the methods of treatment and medical uses of the invention, in which a compound of the invention is used to target cancer stem cells, may be used to reduce cancer stem cell numbers in pre-cancerous conditions (such as myelodyplastic syndrome, or other conditions considered elsewhere in the specification), and thus to prevent progression of such pre-cancerous conditions into cancer. As referred to above, asymmetric cell division of cancer stem cells gives rise to differentiated non-stem cancer cells. Thus cancer stem cells are responsible for the formation and maintenance of the bulk of the tumour.

The accumulation of such non-stem cancer cells plays a major role in the progression of cancers. Targeting of cancer stem cells by a compound of the invention is able to reduce cancer stem cell numbers, which in turn reduces the number of non-stem cancer cell progeny. Thus methods of treatment and medical uses of a compound of the invention in accordance with the present invention are of benefit in treating cancer by preventing cancer progression. Such embodiments are described in more details elsewhere in the present specification.

Cancer stem cells are also able to act as a reservoir of cancer cells that may cause the recurrence of cancer after remission. Even in the event that the majority of a patient's cancer cells have been removed (for example by surgery, radiotherapy, or chemotherapy, either alone or in combination), so that no observable signs of a cancer remain, the continued presence of cancer stem cells may nucleate the recurrence of the cancer over time. Targeting of cancer stem cells by a compound of the invention provides a new mode by which cancer stem cell numbers may be reduced and cancer stem cells killed. Accordingly, and as discussed in more detail elsewhere in the specification, in suitable embodiments the present invention provides methods and medical uses in which a compound of the invention prevents or delays recurrence of cancer.

Furthermore, movement of cancer stem cells from the site of a cancer to another location within the body can contribute to propagation of cancer, for example by giving rise to metastases. Consequently, the ability of a compound of the invention to target cancer stem cells therefore provides new methods of treatment and medical uses in preventing or treating cancer propagation.

In addition to their biological activities, cancer stem cells may be identified by their expression of certain characteristic cell surface markers. Cancer stem cells identified in haematological malignancies are typically CD34 + , while in solid tumours, CD44 + , CD133 + and CD90 + have been identified as cancer stem cell markers. The following table 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 a compound of the invention in accordance with the invention, and so methods or uses employing a compound of the invention may be used in the prevention or treatment of cancers associated with cancer stem cells expressing any of these sets of markers.

Tumour type Reported cell surface markers for cancer stem cells

Solid Tumours

Breast CD447CD247 low /Lineage " /ESA +

CNS CD133 +

Colon CD133 +

Colon ES A gh /CD44 + /Lineage " /(CD 166 + )

Ewing's CD133 +

Head and Neck CD44 + /Lineage "

Melanoma ABCB5 +

Liver CD907CD457(CD44 + )

Cholangiocarinoma CD447GLir (Glioma-associated oncogene homolog-1)

Ovarian CD447CD117 +

Pancreas CD447CD247ESA +

Pancreas CD133 +

Non-small-cell lung cancer CD447Ber-EP4 +

Bladder cancer CD447ALDH1A1 +

Haematological tumours Acute myeloid leukaemia Lin7CD347CD387CD123 +

B-Acute lymphoblastic leukaemia CD347CD10 " or CD347CD19

B-Acute lymphoblastic leukaemia CD347CD387CD19 +

Multiple myeloma CD34 /CD 138

T-Acute lymphoblastic leukaemia CD34 /CD4 or CD347CD7

The data presented in the Examples demonstrate that a compound of the invention is able to target cancer stem cells of leukaemic stem cell lines, specifically cancer stem cells present in the acute myeloid leukaemia cell line KGla. This cell line manifests a minor stem cell-like compartment with a distinct immunophenotype (Lin7CD347CD387CD123 + ) which is targeted by a compound of the invention. Accordingly, methods of treatment or medical uses of a compound of the invention in accordance with the present invention may be used to prevent or treat leukaemia or other cancers associated with cancer stem cells expressing these characteristic markers.

The present invention also provides methods and medical uses in which patients are selected for prevention or treatment of cancer, utilising a compound of the invention, on the basis of the identification of the presence of cancer stem cells in a biological sample representative of the patient's cancer or pre-cancerous condition. The markers set out above provide suitable examples that can be used to identify the presence of cancer stem cells in accordance with such embodiments of the invention. Suitable techniques by which expression of these markers may be investigated in a biological sample are considered further elsewhere in this specification.

"Targeting of cancer stem cells"

The present invention provides the first indication that compounds of the invention can be used for targeting cancer stem cells. The ability of compounds of the invention to target cancer stem cells is illustrated in the Examples disclosed in this specification.

It can be seen from the Examples that when a compound of the invention is provided to populations of cancer cells containing cancer stem cells it targets the cancer stem cells present, leading to a reduction in the total number of cancer cells and in the proportion of total cancer cells exhibiting phenotypic markers of cancer stem cells. While the parent prodrug compound 8-chloroadenosine is able to target cancer stem cells at certain, higher, concentrations, the compounds of the invention demonstrate the ability to achieve such targeting across a broader range of concentrations. Notably, in vitro studies, the results of which are reported in the present application, demonstrate that the compounds of the invention are able to target cancer stem cells at low concentrations more effectively than 8- chloroadenosine. At concentrations in the region of 1 x 10 "6 to 5 x 10 "6 , the improvement in cancer stem cell targeting is such that the proportion of cancer stem cells remaining in a population of cells treated with a compound of the invention is significantly lower than for a population of cells treated with 8-chloroadenosine. It will be appreciated that the ability to achieve effective targeting of cancer stem cells at low concentrations of cytotoxic agents will generally be desirable since this reduces the likelihood of unwanted side effects.

It is believed that the compounds of the present invention enter into cancer cells and are incorporated into the nucleic acids (RNA and/or DNA). Without being bound by any theory, it is believed that the efficacy, particularly the anti-cancer efficacy, exhibited by compounds of the present invention demonstrates that compounds of the present invention are phosphorylated to 8-chloroadenosine triphosphate and it is believed that enzymatic cleavage within the cell converts a compound of the invention directly into 8-chloroadenosine monophosphate prior to phosphorylation to the triphosphate.

It is also believed that the compounds of the invention possess enhanced cellular membrane permeability (as compared to 8-chloroadenosine), and that this contributes to the enhanced anti-cancer potency of the compounds of the invention compared to the parent from which they are derived.

Without wishing to be bound by any hypothesis, the inventors believe that the reduction in cancer stem cell numbers arises as a result of targeted killing of the cancer stem cells among the cancer cell population. That is to say, that compounds of the invention appear to kill cancer stem cells preferentially as compared to killing of non-stem cancer cells, thereby causing the death of cancer stem cells, and a reduction of the proportion of cancer stem cells among the total cancer cell population.

While the inventors believe that compounds of the invention preferentially kills cancer stem cells as compared to non-stem cancer cells, other mechanisms may also contributed to the reduction in the proportion of cancer stem cells caused by a compound of the invention's targeting of these cells.

Merely by way of example, treatment with a compound of the invention may cause an increase in cancer stem cell differentiation, thereby reducing cancer stem cell numbers and also the proportion of total cancer cells represented by cancer stem cells. Alternatively, a compound of the invention may cause cancer stem cells to lose their stem cell phenotype, for example losing their ability to self-renew, thereby reducing cancer stem cell numbers.

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 may be taken as encompassing any mechanism by which a compound of the invention reduces the proportion of cancer stem cells present in a population of cells, whether in vitro or in vivo. In particular targeting of cancer stem cells may be taken as encompassing preferential killing of cancer stem cells as compared to other cell types, particularly as compared to non-stem cancer cells. "Prevention or treatment of cancer"

The invention provides medical uses and methods of treatment in which a compound of the invention is used for the prevention or treatment of cancer. In the context of the present invention, "prevention" of cancer is to be considered as relating to prophylactic applications of a compound of the invention used before the development of cancer, and with an aim of stopping cancer from developing. On the other hand "treatment" of cancer is taken as concerning the use of a compound of the invention after cancer has occurred, with a view to ameliorating cancer by slowing or stopping cancer cell proliferation and tumour growth. Advantageously treatment of cancer may cause partial or total reduction in cancer cell numbers and tumour size. Effective treatment of cancer may bring about disease that either "stabilizes" or "responds" in accordance with the RECIST (Response Evaluation Criteria In Solid Tumours) rules.

As described in more detail below, prevention of cancer in accordance with the present invention may be of particular benefit in patients who have a pre-cancerous condition that increases their likelihood of developing cancer. "Prevention of cancer"

Prevention of cancer in accordance with the present invention may be effected by treatment of a pre-cancerous condition using a compound of the invention in accordance with the various aspects or embodiments of the invention described herein. In particular, prevention of cancer, in the context of the present invention, may be achieved by the methods or medical uses of the invention in which a compound of the invention is provided to a patient with a pre-cancerous condition. Methods of treatment or medical uses in accordance with this embodiment may prevent development of the treated pre-cancerous condition into cancer, thereby providing effective prevention of cancer. References to prevention of cancer in the context of the present invention may also encompass other prophylactic applications of a compound of the invention. For example, the ability of a compound of the invention to target cancer stem cells and thereby prevent the development of cancer, and/or prevent the progression of cancer, and/or prevent the recurrence of cancer, and/or prevent the propagation of cancer. "Pre-cancerous conditions "

Cancer is frequently preceded by the development of a pre-cancerous condition, which is not itself cancerous, but is associated with an increased risk of cancer. Accumulation of genetic or epigenetic changes may cause previously normal cells to develop a cancer stem cell phenotype. Accordingly, cancer stem cells may also be present in such pre-cancerous conditions, as well as in cancerous conditions.

It is believed that the presence of cancer stem cells in pre-cancerous conditions contributes to the development of these conditions into cancer. The methods and medical uses of the invention may be employed to target cancer stem cells present in pre-cancerous conditions, and thereby treat such conditions. It will be appreciated that the new and unexpected finding that compounds of the invention target cancer stem cells means that treatment of precancerous conditions with such compounds may be used to prevent the treated conditions developing into cancer. This represents a way in which a compound of the invention can be used medically in the prevention of cancer, as considered elsewhere in this specification. Examples of pre-cancerous conditions that may be treated in accordance with the present invention include, but are not limited to, those selected from the group consisting of: actinic keratosis, Barrett's oesophagus, atrophic gastritis, dyskeratosis congenital, Sideropenic dysphagia, Lichen planus, oral submucous fibrosis, solar elastosis, cervical dysplasia, leukoplakia, erythroplakia, monoclonal gammopathy of unknown significance (MGUS), monoclonal B-cell lymphocytosis (MBL), myelodysplastic syndromes, as well as precancerous conditions of the stomach such as atrophic gastritis, gastric ulcer, pernicious anaemia, gastric stumps, gastric polyps, and Menetrier' s disease. Among the listed precancerous conditions of the stomach, atrophic gastritis, pernicious anaemia, gastric stumps, and certain types of gastric polyp may have particularly heightened risk of developing into cancers.

Pre-cancerous conditions often take the form of lesions comprising dysplastic or hyperplastic cells. Accordingly, the presence of dysplasia or hyperplasia, as an alternative or addition to the presence of cells with expressed markers or phenotypes characteristic of cancer stem cells, may be used in the identification of pre-cancerous conditions.

The severity of dysplasia can vary between different pre-cancerous conditions, or with the development of a single pre-cancerous condition over time. Generally, the more advanced dysplasia associated with a pre-cancerous condition is, the more likely it is that the precancerous condition will to develop into cancer. Dysplasia is typically classified as mild, moderate or severe. Severe dysplasia usually develops into cancer if left untreated. Suitably, methods of treatment or medical uses employing a compound of the invention may therefore be used to treat a patient with a pre-cancerous condition associated with severe dysplasia.

In a suitable embodiment of the invention a compound of the invention is used to treat a patient with severe cervical dysplasia. Severe cervical dysplasia may be diagnosed by means of a smear test. In another embodiment of the invention a compound of the invention is used to treat severe oesophageal dysplasia ("Barrett's oesophagus"). Severe oesophageal dysplasia may be diagnosed following a tissue biopsy.

It has recently been reported that pre-malignancies can also be identified by detecting somatic mutations in cells in individuals not known to have cancer. In particular, it has been reported that age-related clonal haematopoiesis is a common pre-malignant condition that is associated with increased overall mortality and increased risk of cardiometabolic disease. The majority of mutations detected in blood cells occurred in three genes: D MT3A, TET2, and ASXL1. Accordingly, patients that will benefit from the use of a compound of the invention to target cancer stem cells, and thereby treat a pre-cancerous condition, may be identified by assaying a sample comprising blood cells for the presence of genetic mutations indicative of a pre- cancerous condition in at least one of the genes: DNMT3 A and/or TET2 and/or ASXL1.

Pre-cancerous conditions that may benefit from treatment with a compound of the invention in accordance with the invention to target cancer stem cells may also be identified by determination of the presence of cancer stem cells with reference to any of the techniques based upon expression of markers characteristic of cancer stem cells, or cancer stem cell phenotypes, discussed elsewhere in the specification.

"Treatment of cancer"

The skilled person will appreciate that there are many measurements by which "treatment" of cancer may be assessed. Merely by way of example, any reduction or prevention of cancer development, cancer progression, cancer recurrence, or cancer propagation may be considered to indicate effective treatment of cancer.

In certain embodiments, a compound of the invention may be used: to reduce the proportion of cancer stem cells in a population of cancer cells; and/or to inhibit tumour growth; and/or to reduce tumourigenicity; and/or to prevent or treat a primary cancer; and/or to prevent or treat a relapsed cancer; and/or to prevent or treat a metastatic or secondary cancer; and/or to treat, prevent or inhibit metastasis or recurrence; and/or to treat or prevent refractory cancer.

The ability of cancer treatment using a compound of the invention to bring about a reduction in tumour size, and also to maintain the reduction in tumour size during/after the period in which the treatment is administered represents a particularly relevant indication of effective cancer treatment. As set out in the Examples, the treatments or medical uses of the invention have proven surprisingly effective in this respect, even in models using cells representative of relapsed or refractory cancers that have previously been resistant to treatment with other therapies.

The data presented in the Examples illustrate that treatment with a compound of the invention reduces the proportion of cancer stem cells in a population of cancer cells. Characteristic biological activities or cell surface markers by which cancer stem cells may be identified are described elsewhere in the specification. In a suitable embodiment, treatment of cancer in accordance with the present invention may give rise to a reduction in the proportion of cancer stem cells present in a patient's cancer of at least 10%, at least 20%, at least 30%, or at least 40%). In suitable embodiments, treatment of cancer in accordance with the present invention may give rise to a reduction in the proportion of cancer stem cells present in a patient's cancer of at least 50%, at least 60%, at least 70%, or at least 80%. Treatment of cancer in accordance with the invention may give rise to a reduction in the proportion of cancer stem cells present in a patient's cancer of at least 85%, at least 90%, or at least 95%. Indeed, treatment of cancer in accordance with the present invention may give rise to a reduction in the proportion of cancer stem cells present in a patient's cancer of at least 96%, at least 97%, at least 98%, at least 99%), or even 100% (such that substantially no detectable cancer stem cells remain).

Asymmetric division of cancer stem cells contributes to the growth of tumours. Treatment of cancer with a compound of the invention in accordance with the present invention may bring about an inhibition of tumour growth of at least 10%, at least 20%, at least 30%, or at least 40%). Suitably treatment of cancer in accordance with the invention may give rise to an inhibition of tumour growth of at least 50%, at least 60%, at least 70%, or at least 80%. Treatment of cancer in accordance with the invention may give rise to an inhibition of tumour growth of at least 85%, at least 90%, or at least 95% in a patient so treated. Indeed, treatment of cancer in accordance with the invention may give rise to an inhibition of tumour growth of at least 96%, at least 97%, at least 98%, at least 99%, or even 100% in a treated cancer.

Tumour growth may be assessed by any suitable method in which the change in size of a tumour is assessed over time. Suitably, the size of a tumour prior to cancer treatment may be compared with the size of the same tumour during or after cancer treatment. A number of ways in which the size of a tumour may be assessed are known. For example, the size of a tumour may be assessed by imaging of the tumour in situ within a patient. Suitable techniques, such as imaging techniques, may allow the size of a tumour to be determined, and changes in tumour size to be assessed.

As shown in the results set out in the Examples of this specification, the methods of treatment and medical uses of a compound of the invention are able not only to arrest tumour growth, but are actually able to bring about a reduction in tumour size in patients with cancers, including patients with relapsed or refractory cancers. Suitably, treatment of cancer in accordance with the present invention may give rise to a reduction in tumour size of at least 10%, at least 20%, at least 30%, or at least 40%. In suitable embodiments, treatment of cancer in accordance with the present invention may give rise to a reduction in tumour size of at least 50%), at least 60%>, at least 70%, or at least 80%. Treatment of cancer in accordance with the invention may give rise to a reduction in tumour size of at least 85%, at least 90%, or at least 95%. Indeed, treatment of cancer in accordance with the present invention may give rise to a reduction in tumour size of at least 96%, at least 97%, at least 98%, at least 99%, or even 100%.

A reduction in tumour size of the sort 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, the reduction in tumour size may be determined by direct comparison between the size of a tumour treated with a compound of the invention and the size of a control tumour (which may be untreated, or may have received treatment other than with a compound of the invention). It will be appreciated that such models requiring lack of treatment of a tumour may not be ethically acceptable in the context of clinical studies or therapeutic management of patients, and in this case a reduction in tumour size may be assessed by comparing the size of a treated tumour with the size of the same tumour prior to treatment, or with a predicted size that would have been attained by the tumour had no treatment been administered.

The methods of treatment and medical uses of a compound of the invention may bring about a reduction in biomarkers indicative of cancer. The reduction of such biomarkers provides a further assessment by which effective treatment of cancer may be demonstrated. Suitable examples of such biomarkers may be selected on the basis of the type of cancer to be treated. For example, in the case of gynaecological cancers CA125 represents a suitable biomarker, while in the case of pancreatic or biliary cancers CA19.9 represents a suitable biomarker, and in the case of colorectal cancers CEA may be a suitable biomarker.

Suitably, treatment of cancer in accordance with the present invention may give rise to a reduction in the expression levels of cancer biomarkers by at least 10%, at least 20%, at least 30%), or at least 40%. In suitable embodiments, treatment of cancer in accordance with the invention may give rise to a reduction in the expression levels of cancer biomarkers by at least 50%), at least 60%, at least 70%, or at least 80%. Treatment of cancer in accordance with the invention may give rise to a reduction in the expression levels of cancer biomarkers by at least 85%, at least 90%, or at least 95%. Indeed, treatment of cancer in accordance with the invention may give rise to a reduction in the expression levels of cancer biomarkers by at least 96%, at least 97%, at least 98%, at least 99%, or even 100%.

Beneficial effects, such as a reduction in the proportion of cancer stem cells present, reduction in tumour growth, or reduction in tumour size or the expression levels of cancer biomarkers, observed on treatment of cancer in accordance with the present invention may be maintained for at least one month. Suitably, such beneficial effects may 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 may be maintained for at least 12 months, at least 18 months, or at least 24 months. Suitably, the beneficial effects may 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 for ten years or more.

In a suitable embodiment of the invention a compound of the invention is used in a method of preventing or treating cancer or a pre-malignant condition, by targeting cancer stem cells. In a suitable embodiment the invention provides the use of a compound of the invention in a method of preventing or treating cancer or a pre-malignant condition, wherein the method reduces the tumourigenicity of one or more cancer stem cells. Suitably, such methods may prevent the progression of cancer, or inhibit tumour growth.

When a compound of the invention is used in methods or medical uses of the present invention to prevent or treat the progression of a cancer, such prevention or treatment may cause the cancer progression to be slowed, delayed or stopped entirely.

The progress of a cancer is typically determined by assigning a stage to the cancer. Staging is usually carried out by assigning a number from I to IV to the cancer, with I being an isolated cancer and IV being a cancer that has spread to the limit of what the assessment measures. Specifics of staging vary between cancers, but the stage generally takes into account the size of a tumour, whether it has invaded adjacent organs, how many regional (nearby) lymph nodes it has spread to (if any), and whether it has appeared in more distant locations (metastasised).

Generally, Stage I is localised to one part of the body and may be treated by surgical resection (for solid tumours that are small enough). Stage II is locally advanced, and is treatable 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 specific type of cancer, although Stage III is generally accepted to be "late" locally advanced. Stage IV cancers have often metastasised to a second organ. Treatment of cancer using a compound of the invention in the methods or medical uses of the present invention may be used to treat a stage I, II, III or IV cancer by targeting cancer stem cells. Treatment with a compound of the invention may be used to prevent the progression of a cancer from one stage to the next. In one embodiment, treatment with a compound of the invention is used to prevent progression from Stage I to Stage II. In another embodiment, treatment with a compound of the invention is used to prevent progression from Stage II to Stage III. In still another embodiment, treatment with a compound of the invention is used to prevent progression from Stage III to Stage IV.

Preventing or inhibiting progression of the cancer is particularly important for preventing the spread of the cancer, for example the progression from Stage I to Stage II where the cancer spreads locally, or the progression from Stage III to Stage IV where the cancer metastasises to other organs. Cancer stem cells are tumourigenic and so are believed to play a critical role in the spread of cancer, both locally and metastatically. Methods of treatment or medical uses of the invention employing a compound of the invention can therefore be used to prevent the spread of cancer, by targeting tumourigenic cancer stem cells and thus reducing their numbers.

"Cancers"

Compounds of the invention demonstrate increased anti-cancer activity as compared to 8- chloroadenosine from which they are derived. This increased anti-cancer activity appears to be provided as a result of increased activity against both cancer stem cells and non-stem cancer cells.

Cancer stem cells play a role in the biological activity of a wide range of cancers. Accordingly, there are a wide range of cancers that may be prevented or treated in accordance with the present invention.

As discussed elsewhere herein, cancer stem cells are known to be present in many tumour types including liquid tumours (including haematological tumours such as leukaemias and lymphomas) and solid tumours (such as breast, lung, colon, prostate, ovarian, skin, bladder, biliary and pancreas tumours). Methods of treatment and medical uses of a compound of the invention to target cancer stem cells are therefore expected to be useful in the prevention or treatment of such cancers.

Suitably, a compound of the invention may be used in the prevention or treatment of a cancer selected from the group consisting of: leukaemia, lymphoma, multiple myeloma, lung cancer, liver cancer, breast cancer, head and neck cancer, neuroblastoma, thyroid carcinoma, skin cancer (including melanoma), oral squamous cell carcinoma, urinary bladder cancer, Leydig cell tumour, biliary cancer, such as cholangiocarcinoma or bile duct cancer, pancreatic cancer, colon cancer, colorectal cancer and gynaecological cancers, including ovarian cancer, endometrial cancer, fallopian tube cancer, uterine cancer and cervical cancer, including epithelia cervix carcinoma. In suitable embodiments, the cancer is leukaemia and can be selected from the group consisting of acute lymphoblastic leukaemia, acute myelogenous leukaemia (also known as acute myeloid leukaemia or acute non-lymphocytic leukaemia), acute promyelocytic leukaemia, acute lymphocytic leukaemia, chronic myelogenous leukaemia (also known as chronic myeloid leukaemia, chronic myelocytic leukaemia or chronic granulocytic leukaemia), chronic lymphocytic leukaemia, monoblastic leukaemia and hairy cell leukaemia. In further preferred embodiments, the cancer is acute lymphoblastic leukaemia. In a suitable embodiment the cancer is lymphoma, which may be selected from the group consisting of: Hodgkin's lymphoma; non-Hodgkin lymphoma; Burkitt's lymphoma; and small lymphocytic lymphoma. Suitably, targeting cancer stem cells in such cancers may achieve effective treatment of the cancer by preventing or treating the development of the cancer, by preventing or treating the progression of the cancer, by preventing or treating the recurrence of the cancer, or by preventing or treating the propagation of the cancer.

In a suitable embodiment the present invention provides a compound of the invention for use in targeting cancer stem cells in the prevention or treatment of metastatic cancer.

In a suitable embodiment the present invention provides a compound of the invention for use in targeting cancer stem cells in the treatment of relapsed or refractory cancer.

In a suitable embodiment the present invention provides a compound of the invention for use in targeting cancer stem cells in the treatment of a primary cancer. Suitably, the primary cancer treated may be a second primary cancer. The invention provides a compound of the invention for use in targeting cancer stem cells in the treatment of secondary cancer. In a suitable embodiment the secondary cancer is a metastatic cancer.

In a suitable embodiment the present invention provides a compound of the invention for use in targeting cancer stem cells, wherein the targeting of cancer stem cells prevents or inhibits: (i) recurrence of a cancer; (ii) occurrence of second primary cancer; or (iii) metastasis of a cancer.

Methods of treatment or medical uses in which a compound of the invention is employed on the basis of its ability to target cancer stem cells may be used in the treatment of relapsed or refractory cancer. The considerations regarding relapsed or refractory cancer in such embodiments are, except for where the context requires otherwise, the same as for the treatment of relapsed or refractory cancer in connection with the other aspects of the invention.

"Relapsed or refractory cancer" As noted above, certain aspects and embodiments of the invention particularly relate to the use of a compound of the invention in the treatment of relapsed or refractory cancers.

For the purposes of the present invention, refractory cancers may be taken as cancers that demonstrate resistance to treatment by anti-cancer therapies other than those utilising a compound of the invention. For example, a compound of the invention may be used in the treatment of refractory cancers that are resistant to treatment with radiotherapy. Alternatively, or additionally, a compound of the invention may be used in the treatment of refractory cancers that are resistant to biological agents used in the treatment of cancer. In a suitable embodiment a compound of the invention may be used in the treatment of refractory cancers that are resistant to treatment with chemotherapeutic agents other than a compound of the invention.

In particular, refractory cancers that may benefit from the methods of treatment of medical uses of the invention employing a compound of the invention include those cancers that are resistant to 8-chloroadenosine. Relapsed cancers (or recurrent cancers) are those that return after a period of remission during which the cancer cannot be detected. Cancer recurrence may occur at the site of the original cancer (local cancer recurrence), at a site close to that of the original cancer (regional cancer recurrence), or at a site distant from that of the original cancer (distal cancer recurrence). Cancer stem cells are believed to play a role in the recurrence of cancer, providing a source from which cells of the relapsed cancer are generated. Accordingly, the methods of treatment and medical uses of a compound of the invention in accordance with the invention, which enable targeting of cancer stem cells, may be of great benefit in the context of relapsed cancers. The ability of a compound of the invention to target cancer stem cells may be used to reduce, or even remove, the populations of such cells that are able to give rise to recurrence, thus preventing incidences of relapsed cancer. The anti-cancer stem cell activity of a compound of the invention may also be used to target cancer stem cells in cancers that have recurred, as well as potentially exerting cytotoxic effects on non-stem cancer cells, thereby providing treatment of relapsed cancers. In view of the above, it will be appreciated that a compound of the invention may be used in the methods or uses of the invention for the prevention or treatment of a relapsed cancer. A compound of the invention may be used in the methods or uses of the invention for the prevention or treatment of a local, regional or distant relapsed cancer.

A compound of the invention may be used in the methods or uses of the invention to prevent the recurrence of cancer by providing 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 of remission. Indeed, a compound of the invention may be used to prevent recurrence of cancer by providing 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 of remission. A compound of the invention may be used in the methods or uses of the invention to treat a relapsed cancer which has recurred after 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 of remission. Indeed, a compound of the invention may be used to treat a relapsed cancer which has recurred after 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 of remission. The ability of the compounds of the invention to target cancer stem cells gives rise to the ability of these compounds to prevent or treat cancers in accordance with the medical uses or methods of treatment of the invention. However, it should be noted that compounds of the invention also exert a direct cytotoxic effect upon non-stem cancer cells that make up the bulk of tumours. While cancer stem cells may underlie much of the difficulty to treat relapsed or refractory cancers, non-stem cancer cells are also a major constituent of such relapsed or refractory cancers.

Compounds of the invention exert greater cytotoxic effects on non-stem cancer cells than does 8-chloroadenosine, the chemotherapeutic molecule from which the compounds of the invention are derived. Accordingly, the mechanism by which a compound of the invention acts in the treatment of relapsed or refractory cancer may not be limited solely to the anticancer stem cell activity of this compound, but may also make use of the action of a compound of the invention on non-stem cancer cells. In such uses treatment with a compound of the invention will reduce the total number of both cancer stem cells and non-stem cancer cells, but will preferentially reduce the proportion of cancer stem cells that remain after treatment.

Therapeutically effective doses of a compound of the invention

A therapeutically effective amount of a compound of the invention may be an amount sufficient to induce death of cancer cells. A therapeutically effective amount of a compound of the invention may be an amount sufficient to induce death of cancer stem cells. In some embodiments, particularly those relating to the treatment of relapsed or refractory cancer, a therapeutically effective amount of a compound of the invention may be an amount sufficient to induce death of cancer stem cells and also to induce death of non-stem cancer cells.

There are various different ways in which the amount of a therapeutically effective compound, such as a compound of the invention, to be administered to a patient may be calculated and expressed. One such way which is considered particularly relevant in doses of agents for the prevention or treatment of cancer, is in the amount of the agent to be administered per unit of body surface area of the patient. Such doses are typically expressed in terms of the amount of the agent (which may be determined by mass) per square meter (m 2 ) of surface area. Uses of a compound of the invention for the prevention or treatment of cancer may utilise a weekly dose of between 250 mg/m 2 and 1000 mg/m 2 . Such treatments may, for example utilise a weekly dose of between 375 mg/m 2 and 900 mg/m 2 . For example, effective treatment of relapsed or refractory cancers may be provided when patients are provided with weekly doses of a compound of the invention that range between approximately 500 mg/m 2 and 825 mg/m 2 .

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

A chosen weekly dose of a compound of the invention may be provided in a single incidence of administration, or in multiple incidences of administration during a week. For example, a weekly dose of a compound of the invention may be provided in two incidences of administration, in three incidences of administration, or more. Thus, in the case of a weekly dose of 750 mg/m 2 , this may be achieved by three administrations of 250 mg/m 2 over the course of a week, or two administrations of 375 mg/m 2 during a week. Similarly, in the case of a weekly dose of 600 mg/m 2 , this may be achieved by three administrations of 200 mg/m 2 over the course of a week, or two administrations of 300 mg/m 2 during a week.

A suitable amount of a compound of the invention to be administered in a single incidence of treatment in order to provide a required dose of this compound over the course of week may be between approximately 100 mg/m 2 and 300 mg/m 2 .

The weekly dose of a compound of the invention provided may decrease over the course of treatment. For example, treatment may be started at a weekly dose of around 1000 mg/m 2 , 900 mg/m 2 , 825 mg/m 2 , 750 mg/m 2 , or 725 mg/m 2 , and over the course of treatment the dose needed may decrease to around 750 mg/m 2 (in cases where the initial dose is above this amount), around 650 mg/m 2 , around 625 mg/m 2 , or even around 500 mg/m 2 or around 375 mg/m 2 . Doses of a compound of the invention can, of course, be presented in other manners. The most common of these is the amount of the active agent to be provided per unit body mass. It has been calculated that for an average human patient a dose of 1 mg/m 2 is equivalent to approximately 0.025 mg/kg body mass. Accordingly, the data indicate that a compound of the invention is effective for the treatment of relapsed or refractory cancer at doses ranging from approximately 6.25 mg/kg to approximately 25 mg/kg. A suitable dose may, for example, be of between about 9.5 mg/kg and 22.5 mg/kg. In a suitable embodiment a compound of the invention achieves effective treatment of relapsed or refractory cancers when patients are provided with weekly doses ranging between approximately 12.5 mg/kg and 20.5 mg/kg. Considerations regarding formulations of a compound of the invention suitable for use in the methods of prevention or treatment and medical uses of the present invention are described elsewhere in this disclosure. In the case of injectable formulations of a compound of the invention, these may be administered intravenously. Intravenous administration may be achieved over any suitable time frame, for example in a ten minute injection, or the like. Types of treatment

In a suitable embodiment a compound of the invention may be used for targeting cancer stem cells as a first line treatment of cancer.

However, the finding that compounds of the invention are able to target cancer stem cells and thereby treat relapsed or refractory cancer illustrates that a compound of the invention is able to provide effective treatment of cancer in contexts in which other treatments have proved ineffective. Accordingly, in a suitable embodiment the present invention provides a compound of the invention for targeting cancer stem cells as a second line treatment of cancer. Indeed, in a suitable embodiment the present invention provides a compound of the invention for targeting cancer stem cells as a third, or further, line treatment of cancer. In a suitable embodiment there is provided a compound of the invention for use as a neoadjuvant in the treatment of cancer. A neoadjuvant is an agent provided to a patient in order to reduce the size of a tumour prior to a "main" anti-cancer therapy, such as surgical removal of cancer. A compound of the invention may be used as a neoadjuvant therapy for a patient who will subsequently undergo surgical treatment of cancer and/or radiotherapy for cancer. Alternatively, or additionally, the invention provides a compound of the invention for use as an adjuvant in the treatment of cancer. An adjuvant is an agent provided to a patient after a "main" anti-cancer therapy, such as surgical removal of cancer, in order to prevent the return of cancer after the main therapy. A compound of the invention may be used as an adjuvant for a patient who has undergone surgical treatment of cancer and/or radiotherapy for cancer.

A compound of the invention may be employed in the methods or uses of the invention in a monotherapy, which is to say in preventions or treatments in which a compound of the invention provides substantially all of the therapeutic activity that is made use of in the prevention or treatment. Alternatively, the methods or uses of the invention may employ a compound of the invention in a combination therapy. In such embodiments a compound of the invention is used in conjunction with at least one further cancer therapy. The further cancer therapy may comprise surgery and/or radiotherapy. Additionally, or alternatively, the further cancer therapy may comprise use of at least one further therapeutic agent that contributes to the prevention or treatment of cancer to be achieved. Suitably, such an agent may be a chemotherapeutic agent or a biological agent used in the prevention or treatment of cancer.

In a suitable embodiment of a combination therapy a compound of the invention and a further therapeutic agent may be provided to a patient at the same time. In a suitable example, the compound of the invention and a further therapeutic agent may be formulated as part of the same pharmaceutical composition. Alternatively the compound of the invention and a further therapeutic agent may be formulated separately for provision to the patient at substantially the same time. The amount of the compound of the invention required may be substantially reduced when the compounds are used in such embodiments.

In another suitable embodiment of a combination therapy, a compound of the invention and a further therapeutic agent may be provided to a patient at different times. The compound of the invention and a further therapeutic agent may be provided to a patient sequentially. For example, the compound of the invention may be provided to the patient prior to provision of the further therapeutic agent. Alternatively a compound of the invention may be provided to the patient after provision of the further therapeutic agent. "Further therapeutic agents"

A compound of the invention may be used in combination with a wide range of further therapeutic agents for the prevention or treatment of cancer. These include biological agents, immunotherapeutic agents, and chemotherapeutic agents that may be used for the prevention or treatment of cancer.

While specific examples of suitable further agents are considered in the following paragraphs, these should not be taken as limiting the range of further therapeutic agents suitable for use with a compound of the invention. Indeed, the ability of a compound of the invention to target cancer stem cells indicates that it may be beneficially used in combination with any further therapeutic agent used in the prevention or treatment of cancer, whether such further agent targets cancer stem cells, non-stem cancer cells, or other cells or constituents involved in the development, maintenance, recurrence and propagation of cancer.

Examples of further therapeutic agents that may be used in combination with a compound of the invention include: (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;

(b) an alkylating agent;

(c) an anti-metabolite;

(d) an anti-tumour antibiotic;

(e) a topoisomerase inhibitor;

(f) a mitotic inhibitor;

(g) a monoclonal antibody;

(h) a metallic agent; or

(i) an active or passive immunotherapy.

Except for where the context requires otherwise, the further therapeutic agents set out in the preceding list should all be considered suitable for use in any of the embodiments of combination therapies with a compound of the invention considered above. Selection of patients

The inventors' finding that a compound of the invention is able to target cancer stem cells makes possible a number of methods by which it is possible to determine whether a particular patient is likely to benefit from receiving a compound of the invention in the prevention or treatment of cancer, such as relapsed or refractory cancer.

Accordingly, the invention provides a method of determining whether a patient with cancer or a pre-cancerous condition will benefit from prevention or treatment of cancer with a compound of the invention, the method comprising: assaying a biological sample representative of cancer or a pre-cancerous condition in the patient for the presence of cancer stem cells; wherein the presence of cancer stem cells in the biological sample indicates that the patient will benefit from treatment with a compound of the invention.

The invention further provides a method of determining a suitable treatment regimen for a patient with cancer or a pre-cancerous condition, the method comprising: assaying a biological sample representative of cancer or a pre-cancerous condition in the patient for the presence of cancer stem cells; wherein the presence of cancer stem cells in the biological sample indicates that a suitable treatment regimen will comprise treatment of the patient with a compound of the invention.

The invention also provides a compound of the invention for use in the prevention or treatment of cancer in a patient selected for such treatment by a method comprising: assaying a biological sample representative of cancer or a pre-cancerous condition in the patient for the presence of cancer stem cells; wherein the presence of cancer stem cells in the biological sample indicates that the patient is suitable for treatment with a compound of the invention.

In suitable embodiments cancer stem cells in a biological sample may be identified by their expression of characteristic patterns of markers discussed previously in the application. The skilled person will appreciate that there are many suitable examples of biological samples that may be used in embodiments of the invention such as those set out above. Suitably, such a sample may include cells from the cancer or pre-cancerous condition. A suitable biological sample may be a tissue sample, such as a sample for use in histology. Cells in such samples may be directly assessed for their expression of cancer stem cell markers, such as those set out above. Alternatively or additionally, a suitable biological sample may comprise target molecules representative of gene expression by cells of the cancer or pre-cancerous condition. Examples of such target molecules include proteins or nucleic acids, such as mRNA, representative of gene expression. Suitable examples of techniques by which expression of cancer stem cell markers may be assessed may be selected with reference to the sample type. Techniques for the investigation of expressed markers are frequently used in the context of clinical assessments (such as for diagnostic or prognostic purposes) and their use will be familiar to those required to practice them in the context of the present invention. Merely by way of example, in samples containing proteins the presence of cancer stem cell markers may be assessed by suitable techniques using antibodies that react with the cancer stem cell markers in question. Examples of such samples containing protein cancer stem cell markers include histology samples (where the presence of the markers may be visualised by suitable immunocytochemistry techniques), or samples derived from the circulation. Here the presence of circulating cancer stem cells (which are believed to contribute to the propagation of cancer through metastasis) may be assessed using techniques such as flow cytometry.

In samples containing nucleic acids representative of expression of cancer stem cell markers, such expression may be assessed by suitable molecule biology techniques, such as by polymerase chain reaction (PCR) amplification using suitable primers or RNAscope in situ hybridisation technique.

Synthetic Examples

Examples of the present invention will now be described with respect to the accompanying drawings. The compounds of the invention can be made according to or analogously to the following examples. General Experimental

Solvents and Reagents. The following anhydrous solvents were purchased from Sigma- Aldrich: dichloromethane (CH2CI2), trimethylphosphate ((CH 3 0) 3 PO). Amino acid esters commercially available were purchased from Sigma-Aldrich. All reagents commercially available were used without further purification. Thin Layer Chromatography (TLC): Precoated aluminium backed plates (60 F254, 0.2 mm thickness, Merck) were visualized under both short and long wave ultraviolet light (254 and 366 nm) or by burning using the following TLC indicators: (i) molybdate ammonium cerium sulphate; (ii) potassium permanganate solution. Preparative TLC plates (20 cm χ 20 cm, 500- 2000 μιη) were purchased from Merck.

Flash Column Chromatography: Flash column chromatography was carried out using silica gel supplied by Fisher (60A, 35-70 μιη). Glass columns were slurry packed using the appropriate eluent with the sample being loaded as a concentrated solution in the same eluent or preadsorbed onto silica gel. Fractions containing the product were identified by TLC, and pooled and the solvent was removed in vacuo. In an alternative method, flash chromatography was performed with a Biotage Isolera automated purification system using prepacked SNAP KP or SNAP ultra silica gel columns for direct phase chromatography; SNAP Ultra C-18 for reverse phase chromatography.

High Performance Liquid Chromatography (FIPLC): The purity of the final compounds was verified to be >95% by HPLC analysis using either I) ThermoSCIENTIFIC, SPECTRA SYSTEM P4000, detector SPECTRA SYSTEM UV2000, Varian Pursuit XRs 5 CI 8, 150 x 4.6 mm (as an analytic column) or II) Varian Prostar (LC Workstation- Varian Prostar 335 LC detector), Thermo SCIENTIFIC Hypersil Gold C18, 5μ, 150 x 4.6 mm (as an analytic column). For the method of elution see the experimental part. Nuclear Magnetic Resonance (NMR): ¾ NMR (500 MHz), 13 C NMR (125 MHz), 31 P NMR (202 MHz) and 19 F NMR (470 MHz) were recorded on a Bruker Avance 500 MHz spectrometer at 25 °C. Chemical shifts (δ) are quoted in parts per million (ppm) relative to internal MeOH-d 4 (δ 3.34 1 H-NMR, δ 49.86 13 C-NMR) and CHCl 3 -d 4 (δ 7.26 ¾ NMR, δ 77.36 13 C NMR) or external 85 % Η 3 Ρ0 4 (δ 0.00 31 P NMR). Coupling constants (J) are measured in Hertz. The following abbreviations are used in the assignment of NMR signals: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), bs (broad singlet), dd (doublet of doublet), dt (doublet of triplet), app (apparent). The assignment of the signals in ¾ NMR and 13 C NMR was done based on the analysis of coupling constants and additional two- dimensional experiments (COSY, HSQC, HMBC, PENDANT). Mass spectrometry (MS): Low resolution mass spectra were performed on Bruker Daltonics microToF-LC, (atmospheric pressure ionization, electron spray mass spectroscopy) in either positive or negative mode.

Purity of final compounds: The >95% purity of all the final compounds was confirmed using HPLC analysis.

Example 1

Cyclohexyl 2-(((((2R,3S,4R,5R)-5-(6-amino-8-chloro-9H-purin-9-yl)-3,4-d ihydroxy- tetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)aceta te A

A

8- hloroadenosine

Procedure 1 : Adenosine (1.97g, 7.37 mmol) was suspended in anh. DMF (20 mL) and BzCl (0.9 mL, 7.7 mmol) was added. wCPBA (70-75 %, 2.03 g, 8.6 mmol) was dissolved in anh. DMF (10 mL) and added dropwise. The reaction mixture was stirred at rt for 20 min before it was poured into ice-cold water (100 mL) and filtered. The filtrate was extracted with Et 2 0 (3 x 100 mL). The combined aqueous phases were evaporated in vacuo to give an oil. The oil was absorbed on silica and purified by silica gel CC (packed in 5% MeOH/CHCb, eluted with 7% MeOH/CHCh) to yield a white solid (0.53 g, 24 %).

Procedure 2: Adenosine (1.09g, 4.08 mmol) was suspended in DMF (50 mL) and glacial acetic acid (10 mL) was added. A solution of N-chlorosuccinimide (2 g, 15 mmol) in DMF (15 mL) was added dropwise. The reaction mixture was stirred at rt for 48 hours and the volatiles were evaporated in vacuo to give yellow gum. The crude was absorbed on silica and purified by silica gel CC (packed in 5% MeOH/CHCh, eluted with 7% MeOH/CHCb) to yield a white powder (0.65 g, 52 %). H MR (500 MHz, OMSO-d6) δ 8.16 (s, 1H, H-2), 7.55 (br s, 2H, H 2 ), 5.86 (d, J = 6.8 Hz, 1H, HI '), 5.48 (d, J = 6.2 Hz, 1H, 2ΌΗ), 5.45 (d, J = 4.0 Hz, 1H, 5'-OH), 5.23 (d, J = 4.6 Hz, 1H, 3ΌΗ), 5.08-5.06 (m, 1H, H-2'), 4.23-4.18 (m, 1H, H3'), 4.01-3.96 (m, 1H, H4'), 3.72-3.65 (m, 1H, H5'), 3.57-3.50 (m, 1H, H5').

5-([Cyclohexyloxyglycin-N-yl)-phenyl]-phosphatyl) 8-chloroadenosine, A, was prepared according to the following procedure: 8-chloroadenosine (0.21 g, 0.70 mmol) was dissolved in anh. THF (30 mL), and t-BuMgCl (1.0 M in THF, 0.70 mmol, 0.70 mL) was added dropwise followed by a solution of phenyl- 1-yl-L-gly cine cyclohexyl ester phosphorochloridate (0.88 g, 2.65 mmol) dissolved in anh. THF (5 mL). The mixture was stirred at room temperature overnight. Purification first by silica gel CC (0-5% MeOH/CH 2 Cl 2 ) and secondly by prep. TLC (10% MeOH/DCM) yielded 55 mg of the desired compound as a white solid (yield = 14%).

3 1 P MR (202 MHz, CD 3 OD) δ 4.7 (s), 4.6 (s).

'H NMR (500 MHz, CD3OD) δ 8.19 (s, 0.5H, H2), 8.17 (s, 0.5H, H2), 7.38-7.27 (m, 3H, Ph), 7.24-7.12 (m, 2H, Ph), 6.05 (d, J = 4.9 Hz, 1H, HI '), 6.03 (d, J = 4.9 Hz, 1H, HI '), 5.34 (t, J = 5.2 Hz, 0.5H, H-2'), 5.31 (t, J = 5.2 Hz, 0.5H, H-2'), 4.71 (m, 1H, CH cyclohexyl), 4.66 (t, J = 5.2 Hz, 0.5H, H-3 '), 4.60 (t, J = 5.2 Hz, 0.5H, H-3 ') , 4.53-4.33 (m, 2H, H5'), 4.24-4.17 (m, 1H, H4'), 3.71-3.57 (m, 2H, CH 2 gly), 1.93-1.21 (m, 10H, CH 2 gly).

HPLC Reverse-phase HPLC eluting with H 2 0/CH 3 OH from 90/10 to 0/100 in 35 minutes, 1 ml/min, λ = 254 nm, two peaks with tR 17.50 min., tR 18.12 min.

Example 2

Benzyl 2-(((((2R,3S,4R,5R)-5-(6-amino-8-chloro-9H-purin-9-yl)-3,4-d ihydroxytetra- hydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)acetate B

5-([Benzyloxyglycin-N-yl)-phenyl]-phosphatyl) 8-chloroadenosine, B, was prepared according to the following procedure: 8-chloroadenosine (0.15 g, 0.50 mmol) was dissolved in anh. THF (20 mL), and t-BuMgCl (1.0 M in THF, 0.50 mmol, 0.50 mL) was added dropwise followed by a solution of phenyl- 1-yl-L-glycine benzyl ester phosphorochloridate (1.08 g, 3.05 mmol) dissolved in anh. THF (5 mL). The mixture was stirred at room temperature overnight. Purification first by silica gel CC (0-7% MeOH/CHiCh) and secondly by prep. TLC (10% MeOH/DCM) yielded 82 mg of the desired compound as a white solid (yield = 28%).

31 P NMR (202 MHz, CD 3 OD) δ 4.6 (br s).

¾ NMR (500 MHz, CD3OD) δ 8.18 (s, 0.5H, H2), 8.16 (s, 0.5H, H2), 7.36-7.14 (m, 10H, Ph), 6.04 (d, J= 5.2 Hz, 1H, HI '), 6.02 (d, J= 5.2 Hz, 1H, HI '), 5.34 (t, J= 5.3 Hz, 0.5H, H- 2'), 5.31 (t, J= 5.3 Hz, 0.5H, H-2'), 5.16-5.09 (m, 2H, OC%Ph), 4.64 (t, J= 5.1 Hz, 0.5H, H- 3 '), 4.61 (t, J = 5.1 Hz, 0.5H, H-3 '), 4.49-4.30 (m, 2H, H5'), 4.23-4.17 (m, 1H, H4'), 3.74-

HPLC Reverse-phase HPLC eluting with H2O/CH3CN from 90/10 to 0/100 in 35 minutes, 1 ml/min, λ = 254 nm, two peaks with tR 11.30 min., tR 11.01 min.

Example 3

(2S)-Benzyl 2-(((((2R,3S,4R,5R)-5-(6-amino-8-chloro-9H-purin-9-yl)-3,4-d ihydroxy- tetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propa noate C

5-([Benzyloxy-L-alanin-/V-yl)-phenyl]-phosphatyl) 8-chloroadenosine, C, was prepared according to the following procedure: 8-chloroadenosine (0.23 g, 0.76 mmol) was dissolved in anh. THF (30 mL), and t-BuMgCl (1.0 M in THF, 0.76 mmol, 0.76 mL) was added dropwise followed by a solution of phenyl- 1-yl-L-alanine benzyl ester phosphorochloridate (1.08 g, 3.05 mmol) dissolved in anh. THF (5 mL). The mixture was stirred at room temperature overnight. Purification fist by silica gel CC (0-7% MeOH/CHiCh) and secondly by prep. TLC (7% MeOH/DCM) yielded 9 mg of the desired compound as a white solid (yield = 2%).

31 P NMR (202 MHz, CD 3 OD) δ 3.5 (s), 3.4 (s).

¾ NMR (500 MHz, CD3OD) δ 8.18 (s, 0.5H, H2), 8.15 (s, 0.5H, H2), 7.44-7.10 (m, 10H, Ph), 6.05-6.00 (m, 1H, HI '), 5.34 (t, J= 5.2 Hz, 0.5H, H-2'), 5.28 (t, J= 5.2 Hz, 0.5H, H-2'), 5.12-5.05 (m, 2H, OC%Ph), 4.66 (t, J= 5.4 Hz, 0.5H, H-3 '), 4.63 (t, J= 5.4 Hz, 0.5H, H-3 '), 4.45-4.25 (m, 1H, H5'), 4.23-4.15 (m, 2H, H4', H5'), 3.95-3.86 (m, 1H, CH L-Ala), 1.30-1.21 (m, 3H, CH 3 L-Ala).

HPLC Reverse-phase HPLC eluting with H2O/CH3CN from 90/10 to 0/100 in 35 minutes, 1 ml/min, λ = 254 nm, two peaks with tR 11.30 min., tR 11.01 min.

Example 4

(2S)-Benzyl 2-(((((2R,3S,4R,5R)-5-(6-amino-8-chloro-9H-purin-9-yl)-3,4-d ihydroxy- t rahydrofuran-2-yl)methoxynaphthalene-l-yloxy)phosphoryl)amin o)propanoate D

D 5-([Benzyloxy-L-alanin-/V-yl)-naphth-l-yl]-phosphatyl) 8-chloroadenosine, D, was prepared according to the following procedure: 8-Chloroadenosine (0.40 g, 1.33 mmol) was suspended in acetone (30 mL) and HCIO4 (70%, 0.2 mL) was added dropwise. The mixture was stirred at rt for 30 min before neutralization by NaHC0 3 . The solvent was evaporated and the crude purified by silicagel CC (3% MeOH/CHCl 3 ) to yield 2',3 '-0-Isopropylidene-8- chloroadenosine as a white solid (0.36 g, 79%).

2',3 '-0-Isopropylidene-8-chloroadenosine (157 mg, 0.52 mmol) was then dissolved in anh. THF (24 mL), and t-BuMgCl (1.0 M in THF, 3eq, 1.56 mL, 1.56 eq) was added slowly followed by naphtha- 1-yl-L-alanine benzyl ester phosphorochloridate (540 mg, 1.04 mmol) dissolved in anh. THF (1.5 mL). The mixture was stirred at room temperature overnight. Purification by silica gel CC (0-5% MeOH/DCM) gave a residue that was stirred at room temperature in HCOOH/H2O (3/2, 10 mL) overnight. The solvents were evaporated in vacuo and the crude purified first by silica gel CC (0-7% MeOH/DCM) and secondly by prep. TLC (7% MeOH/DCM) to yield 67 as a white foam (53 mg, yield 17 % over two steps).

3 1 P MR (202 MHz, CD 3 OD) 5 3.8 (s), 3.9 (s).

¾ NMR (500 MHz, CD 3 OD) δ 8.12-8.03 (m, 3H, H2, Nap), 7.81-7.83 (m, 1H, Nap), 7.66- 7.61 (m, 1H, Nap, Ph), 7.52-7.18 (m, 9H, Nap, Ph) 6.05-6.01 (m, 1H, HI '), 5.35-5.31 (m, 0.5H, H-2'X 5.30-5.26 (m, 0.5 H, H-2'), 5.05-4.91 (m, 2H, OCH 2 Ph), 4.69-4.63 (m, 1H, H3 '), 4.53-4.45 (m, 1H, H5') 4.43-4.34 (m, 1H, H5'), 4.26-4.20 (m, 1H, H4'), 4.08-4.00 (m, 0.5H, CH ala), 3.97-3.89 (m, 0.5H, CH Ala), 1.28-1.14 (m, 3H, CH 3 ala).

HPLC Reverse-phase HPLC eluting with H 2 0/CH 3 CN from 90/10 to 0/100 in 35 minutes, 1 ml/min, λ = 254 nm, two peaks with tR 16.48 min., tR 17.07 min.

Example 5

(2S)-Pentyl 2-(((((2R,3S,4R,5R)-5-(6-amino-8-chloro-9H-purin-9-yl)-3,4-d ihydroxy- tetrahydrofuran-2-yl)methoxnaphthalene-l-yloxy)phosphoryl)am ino)propanoate E

E 5'-([(Pentyl-l-oxy-L-alanin-/V-yl)-naphth-l-yl]-phosphatyl) 8-chloroadenosine, E, was prepared according to the following procedure: 8-Chloroadenosine (0.40 g, 1.33 mmol) was suspended in acetone (30 mL) and HCIO4 (70%, 0.2 mL) was added dropwise. The mixture was stirred at rt for 30 min before neutralization by NaHC0 3 . The solvent was evaporated and the crude purified by silica gel CC (3% MeOH/CHCb) to yield 2',3 '-0-Isopropylidene-8- chloroadenosine as a white solid (0.36 g, 79%).

2',3 '-O-Isopropylidene-8-chloroadenosine (200 mg, 0.59 mmol) was then dissolved in anh. THF (30 mL), and t-BuMgCl (1.0 M in THF, 2.93 mmol, 2.93 mL) was added slowly followed by naphthalene- 1-yl-L-alanine pent-l-yl ester phosphorochloridate (674.7 mg, 1.76 mmol) dissolved in anh. THF (5mL). The mixture was stirred at rt overnight. Purification first by silica gel CC (0-3% MeOH/DCM) and secondly by prep. TLC (3% MeOH/DCM) gave a residue that was stirred at rt in HCOOH/H2O (3/2, 10 mL) overnight. The solvents were evaporated in vacuo and the crude purified by prep. TLC (10% MeOH/DCM) to yield a white solid (15 mg, yield = 4% over two steps).

3 1 P MR (202 MHz, CD 3 OD) 5 3. 8 (s), 3.9 (s).

¾ NMR (500 MHz, CD 3 OD) δ 8.13 (s, 0.5H, H2) 8.12-8.09 (m, 1H, Nap), 8.06 (s, 0.5 H, H2), 7.88-7.85 (m, 1H, Nap), 7.68 (d, J = 8.18 Hz, 1H, Nap), 7.55-7.48 (m, 2H, Nap), 7.45- 7.33 (m, 2H, Nap), 6.04 (d, J= 4.7 Hz, 0.5H, HI '), 6.03 (d, J= 4.7 Hz, 0.5H, HI '), 5.36-5.32 (m, 0.5H, H2'), 5.30-5.27 (m, 0.5 H, H2'), 4.69-4.64 (m, 1H, H3 '), 4.55-4.49 (m, 1H, H5'), 4.45-4.36 (m, 1H, H5'), 4.27- 4.22 (m, 1H, H4'), 4.02-3.83 (m, 3H, CH ala, C%CH 2 CH 2 CH 2 CH 3 Pen), 1.55-1.46 (m, 2H, CH 3 ala), 1.32-1.16 (m, 7H, CH 3 ala, CH2CH2CH2CH2CH3 Pen), 0.86 (t, J = 6.9 Hz, 1.5H, CH 2 CH 2 CH 2 CH 2 C% Pen), 0.82 (t, J = 6.9 Hz, 1.5H, CH 2 CH 2 CH 2 CH 2 C% Pen).

HPLC Reverse-phase HPLC eluting with H 2 0/CH 3 CN from 90/10 to 0/100 in 35 minutes, 1 ml/min, λ = 254 nm, two peaks with tR 18.44 min., tR 17.79 min.

Example 6 - 5'-([(Ethyloxy-L-alanin-N-yl)-naphth-l-yl]-phosphatyl) 8-chloroadenosine

F -0-Isopropylidene-8-chloroadenosine

Procedure: 8-Chloroadenosine (0.40 g, 1.33 mmol) was suspended in acetone (30 mL) and HCIO4 (70 %, 0.2 mL) was added dropwise. The mixture was stirred at rt for 30 min before neutralization by NaHC0 3 . The solvent was evaporated and the crude purified by silicagel CC (3% MeOH/CHCl 3 ) to yield a white solid.

Yield: (0.36 g, 79 %).

H NMR (500 MHz, MeOD) δ 8.18 (s, 1H, H2), 7.58 (br s, 2H, H 2 ), 6.05 (d, J= 2.5 Hz, 1H, HI '), 5.68 (dd, J = 6.3, 2.5 Hz, 1H, H2'), 5.07 (t, J = 5.9 Hz, OH-5'), 5.04 (dd, J = 6.3, 3.1 Hz, 1H, H3'), 4.23-4.18 (m, 1H, H-3'), 4.18-4.14 (m, 1H, H-4'), 3.54-3.48 (m, 1H, H5'), 3.46-3.41 (m, 1H, H5'), 1.55 (s, 3H, CH 3 ), 1.34 (s, 3H, CH 3 ).

2',3'-O-Isopropylidene-8-chloroadenosine (234 mg, 0.69 mmol) was dissolved in anh. THF (31 mL), and t-BuMgCl (1.0 M in THF, 3.41 mL, 3.41 mmol) was added slowly followed by 52 (510 mg, 1.49 mmol) dissolved in anh. THF (2 mL). The mixture was stirred at rt overnight. Purification by silica gel CC (0-5% MeOH/DCM) gave a residue that was stirred at rt in HCOOH/H2O (3/2, 5 mL) overnight. The solvents were evaporated in vacuo and the crude purified twice by preparative TLC (10% MeOH/DCM) to yield 5'-([(Ethyloxy-L- alanin-N-yl)-naphth-l-yl]-phosphatyl) 8-chloroadenosine F as a white solid.

Yield: (26 mg, 7.4 % over two steps).

¾ MR (500 MHz, CD3OD) δ 8.12 (s, 0.5H, H2) 8.11-8.07 (m, 1H, Naph) 8.05 (s, 0.5 H, H2), 7.87-7.84 (m, 1H, Nap), 7.67 (d, J = 8.1 1H, Nap), 7.54-7.31 (m, 6H, Nap), 6.05-6.02 (m, 1H, HI '), 5.36-5.32 (m, 0.5H, H2'), 5.31-5.28 (m, 0.5H, H2'), 4.72-4.66 (m, 1H, H-3'), 4.55-4.49 (m, 1H, H5') 4.46-4.37 (m, 1H, H5'), 4.28-4.22 (m, 1H, H4'), 4.06-3.91 (m, 2.5H, CH ala, C%CH 3 Et), 3.87-3.79 (m, 0.5H, CH ala), 1.28-1.25 (m, 1H, CH 3 ala), 1.19-1.08 (m, 5H, CH 3 ala, CH 2 C% Et).

31 P NMR (202 MHz, CD3OD) δ 3. 98 (s), 3.80 (s). MS (ES+) m/z: Found: 629.14 (M + Na + ), 645.11 (M + K + ), CisHisClNeOsP required: (M + ) 606.95.

HPLC Reverse-phase HPLC eluting with H2O/CH3CN from 90/10 to 0/100 in 35 minutes, 1 ml/min, λ = 254 nm, two peaks with tR 14.43 min., tR 13.73 min.

Example 7 - 5'-([(Cyclohexyloxy-L-alanin-N-yl)-naphth-l-yl]-phosphatyl) 8- chloroadenosine G

Procedure: Prepared according to the general procedure D.2: 2',3'-O-Isopropylidene-8- chloroadenosine (220 mg, 0.64 mmol) was dissolved in anh. THF (30 mL), and ¾uMgCl (1.0 M in THF, 1.93 mL, 1.93 mmol) was added slowly followed by napht-l-yl(cyclohexyloxy-L- alaninyl) phosphorochloridate (510 mg, 1.29 mmol) dissolved in anh. THF (1.5mL). The mixture was stirred at rt overnight. Purification by silica gel CC (0-2.5% MeOH/DCM) gave a residue that was stirred at rt in HCOOH/H2O (3/2, 20 mL) overnight. The solvents were evaporated in vacuo and the crude purified by silica gel CC (0-4% MeOH/DCM) to yield 69 as a white solid.

Yield: (293 mg, 75 % over two steps).

¾ MR (500 MHz, CD3OD) δ 8.13 (s, 0.5H, H2) 8.11-8.07 (m, 1H, Nap), 8.06 (s, 0.5 H, H2), 7.83 (d, J = 8.3 Hz, 1H, Nap), 7.64 (d, J = 8.3 Hz, 1H, Nap), 7.52-7.39 (m, 3H, Nap), 7.37-7.30 (m, 1H, Nap), 6.06-6.02 (m, 1H, HI '), 5.35-5.31 (m, 0.5H, H2'), 5.30-5.27 (m, 0.5 H, H2'), 4.70-4.65 (m, 1H, H3 '), 4.65-4.38 (m, 3H, H5', Ch CHex), 4.29-4.23 (m, 1H, H4'), 4.00-3.92 (m, 0.5H, CH ala), 3.91-3.83 (m, 0.5H, CH ala), 1.79-1.13 (m, 13H, CH 3 ala, CH 2 cHex).

31 P NMR (202 MHz, CD3OD) δ 3. 93 (s), 3.90 (s).

MS (ES+) m/z: Found: 683.19 (M + Na + ), 699.17 (M + K + ), C29H34CIN6O8P

required: (M + ) 660.19.

HPLC Reverse-phase HPLC eluting with H2O/CH3CN from 90/10 to 0/100 in 35 minutes, 1 ml/min, λ = 254 nm, two peaks with tR 17.36 min., tR 17.93 min. Example 8 - 5'-([(Benzyloxy-L-leucin-N-yl)-naphth-l-yl]-phosphatyl) 8-chloroadenosine

Procedure: Prepared according to the general procedure D.2: 2',3'-O-Isopropylidene-8- chloroadenosine (200 mg, 0.59 mmol) was dissolved in anh. THF (30 mL), and t-BuMgCl (1.0 M in THF, 3eq, 1.76 mL, 1.76 eq) was added slowly, followed by Naphth-l-yl-L-leucine benzyl ester phosphorochloridate (657.67 mg, 1.47 mmol) dissolved in anh. TFIF (3mL). The mixture was stirred at rt overnight. Purification by silica gel CC (0-4% MeOH/DCM) gave a residue that was stirred at rt in HCOOH/H2O (3/2, 10 mL) overnight. The solvents were evaporated in vacuo and the crude purified first by silica gel CC (3-6% MeOH/DCM) and secondly by prep. TLC (10% MeOH/DCM) to yield the product as a white foam.

Yield: (110 mg, 26 % over two steps).

¾ NMR (500 MHz, MeOD) δ 8.10 (s, 0.5H, H2) 8.09-8.05 (m, 1H, H8 Nap), 8.04 (s, 0.5 H, H2), 7.83-7.79 (m, 1H, H5 Nap), 7.62 (d, J = 8.37 Hz, 1H, H4 Nap), 7.49- 7.38 (m, 3H, H2, H6, H7 Nap), 7.32-7.19 (m, 6H, H3 Nap, Ph), 6.07-6.02 (m, 1H, HI '), 5.36-5.32 (m, 0.5H, H2'), 5.29-5.25 (m, 0.5H, H2'), 5.02-4.91 (m, 2H, CH 2 Ph), 4.67-4.60 (m, 1H, H3'), 4.55-4.41 (m, 2.5H, H5'), 4.40-4.33 (m, 0.5H, H5'), 4.29-4.22 (m, 1H, H4'), 3.99-3.86 (m, 1H, CH leu), 1.65-1.54 (m, 0.5H, CH leu), 1.51-1.31 (m, 2.5 H, CH leu, CH 2 leu, CH 3 Pen), 0.80-0.62 (m, 6H, CH 3 leu).

3 1 P NMR (202 MHz, CD 3 OD) δ 4.13 (s), 3.95 (s).

MS (ES+) m/z: Found: 733.20 (M + Na + ), 749.19 (M + K + ), C 33 H 36 C1N 6 0 8 P required: (M + ) 711.10.

HPLC Reverse-phase HPLC eluting with H 2 0/CH 3 CN from 90/10 to 0/100 in 35 minutes, 1 ml/min, 1 = 254 nm, two peaks with tR 19.33 min., tR 18.93 min. Example 9 - 5'-([(Pent-l-yloxy-L-leucin-N-yl)-naphth-l-yl]-phosphatyl) 8- chloroadenosine I

Procedure: Prepared according to the general procedure D.2: 2',3'-O-Isopropylidene-8- chloroadenosine (180 mg, 0.53 mmol) was dissolved in anh. THF (30 mL), and TJuMgCl (1.0 M in THF, 1.59 mL, 1.59 mmol) was added slowly, followed by Naphth-l-yl-L-leucine pent- 1-yl ester phosphorochloridate (677.16 mg, 1.59 mmol) dissolved in anh. THF (3mL). The mixture was stirred at rt overnight. Purification by silica gel CC (0-3% MeOH/DCM) gave a residue that was stirred at rt in HCOOH/H2O (3/2, 20 mL) overnight. The solvents were evaporated in vacuo and the crude purified by silica gel CC (0-6% MeOH/DCM) to yield a white foam. Yield: (132 mg, 36% over two steps).

¾ NMR (500 MHz, MeOD) δ 8.12 (s, 0.5H, H2) 8.11-8.06 (m, 1H, H8 Nap), 8.06 (s, 0.5H, H2), 7.84-7.79 (m, 1H, H5 Nap), 7.65-7.60 (m, 1H, H4 Nap), 7.51- .40 (m, 3H, H2, H6, H7 Nap), 7.32 (t, J = 8.0 Hz, 1H, H3 Nap), 6.07-6.03 (m, 1H, HI '), 5.37-5.33 (m, 0.5H, H2'), 5.30-5.26 (m, 0.5H, H2'), 4.69-4.62 (m, 1H, H-3'), 4.57-4.46 (m, 1.5H, H5'), 4.42-4.36 (m, 0.5H, H5'), 4.32-4.24 (m, 1H, H4'), 3.95-3.89 (m, 2H, CH2CH2CH2CH2CH3 Pen), 3.89-3.83 (m, 1H, CH leu), 1.70-1.57 (m, 0.5H, CH leu), 1.52-1.35 (m, 4.5H, CH 2 leu, CH2CH2CH2CH2CH3 Pen, CH leu), 1.26-1.15 (m, 4H, CH2CH2CH2CH2CH3 Pen), 0.85-0.80 (m, 3H, CH2CH 2 CH2CH 2 C% Pen), 0.80-0.68 (m, 6H, CH 3 leu).

3 1 P NMR (202 MHz, CD3OD) δ 4.06 (s), 4.02 (s).

MS (ES+) m/z: Found: 713.24 (M + Na + ), 729.20 (M + K + ), C3iH 4 oClN 6 0 8 P

required: (M + ) 691.11.

HPLC Reverse-phase HPLC eluting with H2O/CH3CN from 90/10 to 0/100 in 35 minutes, 1 ml/min, λ = 254 nm, two peaks with tR 21.33 min., tR 20.87 min.

Example 10 - 5'-([(Cyclohexyloxy-L-leucin-N-yl)-naphth-l-yl]-phosphatyl) 8- chloroadenosine J

Procedure: Prepared according to the general procedure D.2: 2',3'-O-Isopropylidene-8- chloroadenosine (64 mg, 0.19 mmol) was dissolved in anh. THF (15 mL), and TJuMgCl (1.0 M in TFIF, 0.57 mL, 0.57 mmol) was added slowly, followed by Naphth-l-yl-L-leucine cyclohexyl ester phosphorochloridate (250 mg, 0.57 mmol) dissolved in anh. TFIF (1.5 mL). The mixture was stirred at rt overnight. Purification by silica gel CC (0-3% MeOH/DCM) gave a residue that was stirred at rt in HCOOH/H2O (3/2, 10 mL) overnight. The solvents were evaporated in vacuo and the crude purified twice by prep. TLC (0-5% MeOH/DCM) to yield a white solid.

Yield: (10 mg, 8 % over two steps).

¾ NMR (500 MHz, CD3OD) δ 8.13 (s, 0.5H, H2) 8.12-8.07 (m, 1H, H8 Nap), 8.07 (s, 0.5 H, H2), 7.89-7.84 (m, 1H, H5 Nap), 7.69-7.64 (m, 1H, H4 Nap), 7.54-7.40 (m, 3H, H2, H6, H7 Nap), 7.37-7.32 (m, 1H, H3 Nap), 6.06-6.02 (m, 1H, HI '), 5.36-5.32 (m, 0.5H, H2'), 5.29- 5.25 (m, 0.5H, H2'), 4.67-4.45 (m, 4.5H, H3', H5', CH cHex), 4.41-4.34 (m, 0.5H, H5'), 4.30-4.21 (m, 1H, H4'), 3.91-3.79 (m, 1H, CH leu), 1.74-1.22 (m, 13H, CH 2 CH leu, CH 2 cHex), 0.85-0.70 (m, 6H, CH 3 leu).

3 1 P NMR (202 MHz, CD3OD) δ 4.12 (s), 4.02 (s).

MS (ES-VE) m/z: Found: 701.21 (M - H + ), 737.19 (M + CI " ), C32H40CIN6O8P required: (M + ) 702.2.

HPLC Reverse-phase HPLC eluting with H 2 0/CH 3 CN from 90/10 to 0/100 in 35 minutes, 1 ml/min, λ = 254 nm, two peaks with tR 20.53 min., tR 20.00 min.

Example 11 - 5'-([(ethyloxy-L-leucin-N-yl)-naphth-l-yl]-phosphatyl) 8-chloroadenosine K

Procedure: 2',3'-O-Isopropylidene-8-chloroadenosine (80 mg, 0.23 mmol) was dissolved in anh. THF (15 mL), and ¾uMgCl (1.0 M in THF, 0.69 mL, 0.69 mmol) was added slowly, followed by Naphth-l-yl-L-leucine rthyl ester phosphorochloridate (254.5 mg, 0.69 mmol) dissolved in anh. THF (1.5 mL). The mixture was stirred at rt overnight. Purification by silica gel CC (0-3% MeOH/DCM) gave a residue that was stirred at rt in HCOOH/H 2 0 (3/2, 10 mL) overnight. The solvents were evaporated in vacuo and the crude purified twice by prep. TLC (10% MeOH/DCM) to yield a white solid.

Yield: (30 mg, 35 % over two steps).

'H NMR (500 MHz, CD 3 OD) δ 8.12 (s, 0.5H, H2) 8.10-8.06 (m, 1H, H8 Nap), 8.05 (s, 0.5H, H2), 7.86-7.82 (m, 1H, H5 Nap), 7.67-7.63 (m, 1H, H4 Nap), 7.53-7.39 (m, 3H, H2, H6, H7 Nap), 7.36-7.31 (m, 1H, H3 Nap), 6.06-6.03 (m, 1H, HI '), 5.36-5.33 (m, 0.5H, H2'), 5.30- 5.27 (m, 0.5H, H2'), 4.68-4.62 (m, 1H, H3'), 4.57-4.46 (m, 1.5H, H5'), 4.41-4.35 (m, 0.5H, H5'), 4.30-4.23 (m, 1H, H4'), 4.02-3.92 (m, 2H, CH2CH3 Et), 3.91-3.81 (m, 1H, CH leu), 1.70-1.61 (m, 0.5H, CH leu), 1.50-1.35 (m, 2.5H, CH 2 CH leu), 1.16-1.08 (m, 3H, CH 2 C% Et), 0.84-0.68 (m, 6H, CH 3 leu).

3 1 P NMR (202 MHz, CD3OD) δ 4.11 (s), 4.04 (s).

MS (-S -VE) m/z: Found: 647.22 -M - H + ), 683.14 (M + CI " ), C^ft^lNeOsP

required: (M + ) 649.03.

HPLC Reverse-phase HPLC eluting with H 2 0/CH 3 CN from 90/10 to 0/100 in 35 minutes, 1 ml/min, λ = 254 nm, two peaks with 1R 17.88 min., tR 17.43 min.

Example 12 - 5'-([(benzylo-y -glycin-N-yl)-naphth-l-yl]-phosphatyl) 8-chloroadenosine

L Procedure: 8-Chloroadenosine (150 mg, 0.50 mmol) was dissolved in anh. THF (40 mL), and t-BuMgCl (1.0 M in THF, leq, 0.50 mL, 0.50 eq) was added slowly, followed by Naphth-1- yl-glycine benzyl ester phosphorochloridate (779.54 mg, 2 mmol) dissolved in anh. TFIF (5 mL). The mixture was stirred at rt overnight. The crude was purified first by silica gel CC (0- 6% MeOH/DCM) and secondly by prep. TLC (5% MeOH/DCM) to yield 76 as a white powder.

Yield: (13 mg, 4%).

¾ MR (500 MHz, CD 3 OD) δ 8.13-8.06 (m, 1.5H, H2, H8 Nap), 8.05 (s, 0.5H, H2), 7.87- 7.84 (m, 1H, H5 Nap), 7.68-7.64 (m, 1H, H4 Nap), 7.53-7.26 (m, 9H, H2, H6, H7, H3 Nap, Ph), 6.04 (d, J = 5.1 Hz, 1H, HI '), 6.02 (d, J = 5.1 Hz, 1H, HI '), 5.33-5.28 (m, 1H, H2'), 5.10-5.01 (m, 2H, CH 2 Ph), 4.67-4.63 (m, 0.5H, H3'), 4.63-4.59 (m, 0.5H, H3'), 4.55-4.48 (m, 1H, H5'), 4.48-4.40 (m, 1H, H5'), 4.26-4.20 (m, 1H, H4'), 3.79-3.71 (m, 2H, CH 2 gly).

3 1 P NMR (202 MHz, CD3OD) δ 4.93 (s).

MS (ES-VE) m/z: Found: 653.19 -M - H + ), 688.64 (M + CI " ), C^H^ClNeOsP required: (M + ) 654.99.

HPLC Reverse-phase HPLC eluting with H 2 0/CH 3 CN from 90/10 to 0/100 in 35 minutes, 1 ml/min, λ = 254 nm, two peaks with tR 15.84 min., tR 15.35 min.

Example 13 - 5'-([(Cyclohexyloxy-glycin-N-yl)-naphth-l-yl]-phosphatyl) 8- chloroadenosine M

Procedure: 2',3'-O-Isopropylidene-8-chloroadenosine (100 mg, 0.29 mmol) was dissolved in anhydrous THF (20 mL), and *BuMgCl (1.0 M in THF, 0.88 mL, 0.88 mmol) was added slowly, followed by naphthalene- 1-yl glycine cyclohexyl ester phosphorochloridate (335 mg, 0.88 mmol) dissolved in anhydrous THF (1.5 mL). The mixture was stirred at room temperature overnight. Purification by silica gel CC (0-3% MeOH/CH 2 Cl 2 ) gave a residue that was stirred at room temperature in HCOOH/H 2 0 (3/2, 10 mL) overnight. The solvents were evaporated in vacuo and the product purified twice by preparative TLC (10% MeOH/ CH2CI2) to afford the product as a white solid.

Yield: (7 mg, 4 % over two steps).

1 H MR (500 MHz, CD3OD) δ 8.15-8.04 (m, 2 H, H2, H8 Nap), 7.89-7.85 (m, 1H, H5 Nap), 7.70-7.65 (m, 1H, H4 Nap), 7.55-7.44 (m, 3H, H2, H6, H7 Nap), 7.42-7.33 (m, 1H, H3 Nap), 6.04 (d, J = 6.0 Hz, 0.5 H, HI '), 6.02 (d, J = 6.0 Hz, 0.5H, HI '), 5.32-5.28 (m, 1H, H2'), 4.70-4.63 (m, 1.5H, H3', CH cHex), 4.61-4.58 (m, 1H, H3'), 4.57-4.50 (m, 1H, H5'), 4.49- 4.42 (m, 1H, H5'), 4.27-4.23 (m, 1H, H4'), 3.72-3.62 (m, 2H, CH 2 gly), 1.81-1.64 (m, 4H, CH 2 cHex), 1.56-1.49 (m, 1H, CH 2 gly), 1.39-1.22 (m, 5H, CH 2 gly).

3 1 P NMR (202 MHz, CD3OD) δ 4.96 (s), 4.94 (s).

MS (ES+) m/z: Found: 669.01 (M + Na + ), 685.12 (M + K + ), C 2 8H 32 C1N 6 0 8 P required: (M + ) 647.02.

HPLC Reverse-phase HPLC eluting with H 2 0/CH 3 CN from 90/10 to 0/100 in 35 minutes, 1 ml/min, λ = 254 nm, two peaks with tR 17.52 min., tR 17.00 min.

Example 14 - 5'-([(ethyloxy-glycin-N-yl)-naphth-l-yl]-phosphatyl) 8-chloroadenosine N

N

Procedure: 8-Chloroadenosine (100 mg, 0.33 mmol) was dissolved in anh. THF (30 mL), and ¾uMgCl (1.0 M in THF, 0.33 mL, 0.33 mmol) was added slowly, followed by Naphth-l-yl- glycine ethyl ester phosphorochloridate (434.5 mg, 1.33 mmol) dissolved in anh. THF (3 mL). The mixture was stirred at rt overnight. The crude was purified first by silica gel CC (0-6% MeOH/DCM) and secondly by prep. TLC (5% MeOH/DCM) to yield a white solid.

Yield: (7 mg, 4 %).

¾ NMR (500 MHz, CD3OD) δ 8.15-8.07 (m, 1.5H, H2, H8 Nap) 8.05 (s, 0.5 H, H2), 7.89- 7.85 (m, 1H, H5 Nap), 7.69-7.66 (m, 1H, H4 Nap), 7.55-7.33 (m, 4H, H2, H6, H7, H3 Nap), 6.04 (d, J= 5.1 Hz, 0.5H, HI '), 6.02 (d, J= 5.1 Hz, 0.5H, HI '), 5.35-5.29 (m, 1H, H2'), 4.69- 4.66 (m, 0.5H, H3'), 4.64-4.61 (m, 0.5H, H3'), 4.56-4.50 (m, 1H, H5'), 4.49-4.43 (m, 1H, H5'), 4.27-4.22 (m, 1H, H4'), 4.10-4.00 (m, 2H, CH 2 Et), 3.73-3.62 (m, 2H, CH 2 gly), 1.21- 1.13 (m, 3H, CH 3 Et). 31 P NMR (202 MHz, CD 3 OD) δ 4.96 (s), 4.94 (s).

MS (ES -VE) m/z: Found: 591.10 -M - H + ), 627.08 (M + CI " ), Ci+KzeClNeOgP

required: (M + ) 592.93.

HPLC Reverse-phase HPLC eluting with H2O/CH3CN from 90/10 to 0/100 in 35 minutes, 1 ml/min, λ = 254 nm, two peaks with tR 13.88

Example 15 - 5'-([(Pent-l-yloxy-glycin-N-yl)-naphth-l-yl]-phosphatyl) 8- chloroadenosine O

O

Procedure: 8-Chloroadenosine (130 mg, 0.43 mmol) was dissolved in anh. THF (40 mL), and t-BuMgCl (1.0 M in THF, leq, 0.43 mL, 0.43 eq) was added slowly, followed by Naphth-1- yl-glycine pent-l-yl ester phosphorochloridate (637.36 mg, 1.72 mmol) dissolved in anh. THF (5 mL). The mixture was stirred at rt overnight. The crude was purified first by silica gel CC (0-6% MeOH/DCM) and secondly by prep. TLC (7% MeOH/DCM), to yield the product as a white solid.

Yield: (39 mg, 15 %.)

¾ NMR (500 MHz, CD3OD) δ 8.15-8.07 (m, 1.5H, H2, H8 Nap), 8.05 (s, 0.5H, H2), 7.87- 7.82 (m, 1H, H5 Nap), 7.68-7.64 (m, 1H, H4 Nap), 7.54- .42 (m, 3H, H2, H6, H7 Nap), 7.35 (t, J = 8.0 Hz, 1H, H3 Nap), 6.04 (d, J = 4.7 Hz, 1H, HI '), 6.02 (d, J = 4.7 Hz, 1H, HI '), 5.37-5.25 (m, 1H, H2'), 4.69-4.65 (m, 0.5H, H3 '), 4.64-4.60 (m, 0.5H, H3'), 4.58-4.50 (m, 1H, H5'), 4.50-4.41 (m, 1H, H5'), 4.29-4.22 (m, 1H, H4'), 4.03-9.94 (m, 2H, CH2CH2CH2CH2CH3 Pen), 3.75-3.64 (m, 2H, CH 2 gly), 1.57-1.47 (m, 2H, CH2CH2CH2CH2CH3 Pen), 1.31-1.22 (m, 4H, CH2CH2CH2CH2CH3 Pen), 0.86 (t, J = 6.9 Hz, 3H, CH2CH2CH2CH2CH5 Pen).

3 1 P NMR (202 MHz, CD3OD) δ 4.93 (s).

MS (ES+) m/z: Found: 657.19 (M + Na + ), C27H32CIN6O8P required: (M + ) 635.01.

HPLC Reverse-phase HPLC eluting with H2O/CH3CN from 90/10 to 0/100 in 35 minutes, 1 ml/min, λ = 254 nm, two peaks with tR 17.91min., tR 17.39 min. ( [(Benzyloxy-L-phenylalanin-N-yl)-naphth- 1-yl] -phosphatyl)

Procedure: 2',3'-O-Isopropylidene-8-chloroadenosine (100 mg, 0.29 mmol) was dissolved in anhydrous THF (20 mL), and ¾uMgCl (1.0 M in THF, 0.88 mL, 0.88 mmol) was added slowly, followed by naphthalene- 1-yl-L-phenylalanine cyclohexyl ester phosphorochloridate (422 mg, 0.88 mmol) dissolved in anhydrous THF (2.5 mL). The mixture was stirred at room temperature overnight. Purification by silica gel CC (0-2% MeOH/CH 2 Cl 2 ) gave a residue that was stirred at room temperature in HCOOH/H2O (3/2, 10 mL) overnight. The solvents were evaporated in vacuo and the product purified twice by preparative TLC (7% MeOH/ CH2CI2) to obtain a white foam.

Yield: (12 mg, 6 % over two steps).

¾ NMR (500 MHz, CD3OD) δ 8.08 (s, 0.5H, H2), 8.03 (s, 0.5H, H2), 8.00-7.97 (m, 1H, H8 Naph), 7.85-7.82 (m, 1H, H5 Nap), 7.64-7.61 (m, 1H, H4 Nap), 7.52-7.47 (m, 1H, H2 Nap), 7.44-7.38 (m, 1H, H6 Nap), 7.28-7.20 (m, 5H, Ph), 7.19-7.11 (m, 5H, Ph), 7.05-7.02 (m, 1H, H7 Nap), 7.00-6.96 (m, 1H, H3 Nap), 6.02-6.00 (m, 1 H, HI '), 5.32-5.25 (m, 1H, H2'), 4.98- 4.88 (m, 2H, CH 2 Ph), 4.58-4.54 (m, 0.5H, H3 '), 4.52-4.49 (m, 0.5H, H3'), 4.34-4.10 (m, 4H, H4', H5', CH phe), 3.01-2.90 (m, 1H, CH 2 phe), 2.83-2.76 (m, 1H, CH 2 phe).

3 1 P NMR (202 MHz, CD3OD) δ 3.66 (s), 3.46 (s).

MS (ES+) m/z: Found: 767.21 (M + Na + ), C36H34C1N 6 0 8 P required: (M + ) 745.12.

HPLC Reverse-phase HPLC eluting with H2O/CH3CN from 90/10 to 0/100 in 35 minutes, 1 ml/min, λ = 254 nm, two peaks with tR 20.48 min., tR 20.03 min.

Example 16 - 5'-([(Pent-l-yloxy-L-phenylalanin-N-yl)-naphth-l-yl]-phospha tyl) 8- chloroadenosine Q

Procedure: 2',3'-O-Isopropylidene-8-chloroadenosine (100 mg, 0.29 mmol) was dissolved in anhydrous THF (20 mL), and ¾uMgCl (1.0 M in THF, 0.88 mL, 0.88 mmol) was added slowly, followed by naphthalene- 1-yl-L-phenylalanine-l-pentyl ester phosphorochloridate (404 mg, 0.88 mmol) dissolved in anhydrous THF (2 mL). The mixture was stirred at room temperature overnight. Purification by silica gel CC (0-2% MeOH/CH 2 Cl 2 ) gave a residue that was stirred at room temperature in HCOOH/H2O (3/2, 10 mL) overnight. The solvents were evaporated in vacuo and the product purified twice by preparative TLC (7% MeOH/ CH2CI2) to obtain a white foam.

Yield: (20 mg, 10 % over two steps).

¾ NMR (500 MHz, CD3OD) δ 8.11 (s, 0.5H, H2),8.05 (s, 0.5H, H2), 8.04-8.00 (m, 1H, H8 Nap), 7.87-7.83 (m, 1H, H5 Nap), 7.67-7.61 (m, 1H, H4 Nap), 7.54-7.43 (m, 2H, H2, H6 Nap), 7.32-7.26 (m, 2H, Ph), 7.22-7.12 (m, 3H, Ph), 7.11-7.08 (m, 1H, H7 Nap), 7.07-7.04 (m, 1H, H3 Nap), 6.04-6.00 (m, 1H, HI '), 5.32-5.26 (m, 1H, H2'), 4.58-4.54 (m, 0.5H, H3 '), 4.53-4.50 (m, 0.5H, H3 '), 4.37-4.31 (m, 0.5H, H5'), 4.29-4.24 (m, 0.5H, H5'), 4.18-4.05 (m, 3H, H5', H4', CH phe), 3.90-3.79 (m, 2H, CH2CH2CH2CH2CH3 Pen), 3.00-2.90 (m, 1H, CH 2 phe), 2.85-2.78 (m, 1H, CH 2 phe), 1.45-1.35 (m, 2H, CH2CH2CH2CH2CH3 Pen), 1.25-1.09 (m, 4H, CH2CH2CH2CH2CH3 Pen), 0.83 (t, J= 7.2 Hz, 3H, CH2CH2CH2CH2CH5 Pen), 0.80 (t, J= 7.2 Hz, 3H, CH2CH2CH2CH2CH5 Pen).

31 P NMR (202 MHz, CD3OD) δ 3.72 (s), 3.39 (s).

MS (ES+) m/z: Found: 747.23 (M + Na + ), C34H38C1N 6 0 8 P required: (M + ) 725.13

HPLC Reverse-phase HPLC eluting with H2O/CH3CN from 90/10 to 0/100 in 35 minutes, 1 ml/min, λ = 254 nm, two peaks with tR 22.04 min., tR 21.48 min. Example 17 - 5'-([(Cyclohexyloxy-L-phenylalanin-N-yl)-naphth-l-yl]-phosph atyl) 8- chloroadenosine R

Procedure: 2',3'-O-Isopropylidene-8-chloroadenosine (123 mg, 0.36 mmol) was dissolved in anhydrous THF (20 mL), and ¾uMgCl (1.0 M in THF, 1.1 mL, 1.1 mmol) was added slowly, followed by naphthalene- 1-yl-L-phenylalaninecyclohexyl ester phosphorochloridate (498 mg, 1.1 mmol) dissolved in anhydrous THF (2 mL). The mixture was stirred at room temperature overnight. Purification by silica gel CC (0-2.5 % MeOH/CH 2 Cl 2 ) gave a residue that was stirred at room temperature in HCOOH/H2O (3/2, 10 mL) overnight. The solvents were evaporated in vacuo and the product purified twice by preparative TLC (10 % MeOH/ CH2CI2) to obtain a white foam.

Yield: (53 mg, 21 % over two steps).

¾ MR (500 MHz, CD3OD) δ 8.10 (s, 0.5H, H2), 8.05 (s, 0.5H, H2), 8.04-8.00 (m, 1H, H8 Nap), 7.86-7.82 (m, 1H, H5 Nap), 7.65-7.61 (m, 1H, H4 Nap), 7.53-7.48 (m, 1H, H2), 7.47- 7.42 (m, 1H, H6), 7.32-7.22 (m, 2H, Ph), 7.21-7.12 (m, 3H, Ph), 7.1 1-7.08 (m, 1H, H7 Nap), 7.07-7.04 (m, 1H, H3 Nap), 6.04-6.01 (m, 1H, HI '), 5.32-5.27 (m, 1H, H2'), 4.59-4.50 (m, 2H, H3 ', CH cHex), 4.39-4.26 (m, 1H, H5'), 4.20-4.04 (m, 3H, H5', H4', CH phe), 2.99-2.90 (m, 1H, CH 2 phe), 2.86-2.78 (m, 1H, CH 2 phe), 1.69-1.52 (m, 4H, CH 2 cHex), 1.50-1.41 (m, 1H, CH 2 cHex), 1.32-1.12 (m, 5H, CH 2 cHex).

3 1 P NMR (202 MHz, CD3OD) δ 3.73 (s), 3.74 (s).

MS (ES+) m/z: Found: 759.19 (M + Na + ), CssHssClNeOsP required: (M + ) 737.14.

HPLC Reverse-phase HPLC eluting with H2O/CH3CN from 90/10 to 0/100 in 35 minutes, 1 ml/min, λ = 254 nm, two peaks with tR 21.81 min., tR 21.27 min.

Example 18 - 5'-([(Ethyloxy-L-phenylalanin-N-yl)-naphth-l-yl]-phosphatyl) 8- chloroadenosine S

Procedure: 2',3'-O-Isopropylidene-8-chloroadenosine (170 mg, 0.50 mmol) was dissolved in anhydrous THF (30 mL), and ¾uMgCl (1.0 M in THF, 1.5 mL, 1.5 mmol) was added slowly, followed by naphthalene- 1-yl-L-phenylalanine-l -ethyl ester phosphorochloridate (1.05 g, 2.5 mmol) dissolved in anhydrous THF (2 mL). The mixture was stirred at room temperature overnight. Purification by silica gel CC (0-2% MeOH/QHbCk) gave a residue that was stirred at room temperature in HCOOH/H2O (3/2, 10 mL) overnight. The solvents were evaporated in vacuo and the product purified twice by preparative TLC (7% MeOH/ CH2CI2) to obtain a white foam.

Yield: 90 mg, 26 % over two steps.

¾ MR (500 MHz, CD3OD) δ 8.09 (s, 0.5H, H2), 8.04 (s, 0.5H, H2), 8.00 (d, J = 8.4 Hz, 1H, H8 Nap), 7.80 (d, J = 8.2 Hz, 1H, H5 Nap), 7.62-7.58 (m, 1H, H4 Nap), 7.50-7.40 (m, 2H, H2, H6), 7.32-7.23 (m, 1H, Ph), 7.20-7.07 (m, 4H, H7, Ph), 7.05-7.01 (m, 1H, H3 Nap), 6.05-6.01 (m, 1H, HI '), 5.33-5.28 (m, 1H, H2'), 4.60-4.53 (m, 1H, H3'), 4.38-4.31 (m, 0.5H, H5'), 4.30-4.24 (m, 0.5H, H5'), 4.20-4.10 (m, 4.5H, H5', H4', CH phe, CH 2 Et), 4.09-4.03 (m, 0.5H, H5'), 3.97-3.85 (m, 2H, CH 2 phe), 1.07-0.98 (m, 3H, CH 3 Et).

3 1 P NMR (202 MHz, CD3OD) δ 3.73 (s), 3. 42 (s).

MS (ES+) m/z: Found: 705.18 (M + Na + ), C 3 iH 32 ClN 6 0 8 P required: (M + ) 683.05.

HPLC Reverse-phase HPLC eluting with H 2 0/CH 3 CN from 90/10 to 0/100 in 35 minutes, 1 ml/min, λ = 254 nm, two peaks with tR 17.00 min., tR 16.53 min.

Example 19 - 5'-([(Benzyloxy-D-alanin-N-yl)-phenyl]-phosphatyl) 8-chloroadenosine T

8-Chloroadenosine (0.31 g; 1.03 mmol) was dissolved in anh. THF under Ar and tBuMgCl (1.0 M in THF, 1 eq, 1.2 mL, 1.2 mmol) was added dropwise at rt. The mixture was stirred for 10 min before addition of the appropriate phosphorochloridate (0.91 g, 2.57 mmol) in anh. THF. The mixture was stirred overnight and the solvent evaporated in vacuo. The residue was purified by silica gel CC (3-5% MeOH/DCM) followed by another CC (4% MeOH/CHCh) and prep. TLC (mobile phase: 5% MeOH/CHCb) and EtOAc/H 2 0 wash. Yield: 62 mg, 11%.

¾ NMR (500 MHz, CD 3 OD) δ 8.18, 8.16 (2 x s, 1H, H-2), 7.36 - 7.07 (m, 10H, Ar), 6.05, 6.02 (2 x d, J= 4.6, 5.0, 1H, H-l '), 5.32 (m, 1H, H-2'), 5.14 - 4.46 (m, 2H, CH 2 (Bn)), 4.67, 4.61 (2 x t, J= 5.3, 5.2, 1H, H-3'), 4.46 - 4.26 (m, 2H, H-5'), 4.21 (m, 1H, H-4'), 3.89 (m, 1H, CHCH3), 1.24, 1.21 (2 x d, J= 7.3, 7.0, 3H, CH 3 ).

3 1 P NMR (202 MHz, CD3OD) δ 3.6, 3.4 (2 x s, diastereoisomers).

HR-MS (ES+) m/z: Found: (M + H + ) Found 619.1490. C 2 6H 28 C1N60 8 P required: (M + ) 619.1473.

HPLC Reverse-phase HPLC eluting with H 2 0/CH 3 OH from 90/10 to 0/100 in 30 minutes, 1 ml/min, λ = 254 nm, one peak with tR 17.00 min.

HPLC Reverse-phase HPLC eluting with H 2 0/CH 3 CN from 90/10 to 0/100 in 30 minutes, 1 ml/min, λ = 254 nm, one peak with tR 11.6 min.

Example 20 - 5'-([(Benzyloxy-L-phenylalanin-N-yl)-phenyl]-phosphatyl) 8- chloroadenosine U

2',3'-O-isopropylidene-8-chloroadenosine (0.25 g, 0.73 mmol) was dissolved in anh. THF (30 mL), and tBuMgCl (1.0 M in THF, 6 eq, 4.4 mL, 4.4 mmol) was added. The appropriate phosphorochloridate (1.61 g, 3.75 mmol) in anh. THF(3 mL) was added and the mixture was stirred overnight. The solvent was evaporated in vacuo. Purification by silica gel CC (3% MeOH/DCM) gave a residue that was stirred at rt in HCOOH/H2O (30 + 20 mL) overnight. Silica gel CC (3-5.5% MeOH/DCM) followed by prep. TLC (10% MeOH/DCM) gave the desired product. Yield: 76 mg, 15% over 2 steps.

'H NMR (500 MHz, CD3OD) δ 8.14, 8.13 (2 x s, 1H, H-2, diastereoisomers), 7.29 - 6.96 (m, 15H, Ar), 6.03 (m, 1H, H-l '), 5.32, 5.28 (2 x t, J = 5.4, 5.1, 1H, H-2'), 5.03 - 4.98 (m, 2H, CH 2 (Bn)), 4.56, 4.51 (2 x t, J = 5.3, 5.1, 1H, H-3'), 4.26 - 4.04 (m, 4H, H-4', H-5', CHCH 2 (Phe)), 2.97, 2.83 (2 x m, 2H, CH 2 CH (Phe)).

3 1 P MR (202 MHz, CD3OD) δ 3.3, 3.1 (2 x s, diastereoisomers).

HR-MS (ES+) m/z: Found: (M + Na + ) Found 717.1632. C3 2 H 32 C1N 6 0 8 P required: (M+Na+) 717.1605.

Example 21 - 5'-([(Benzyloxy-L-leucin-N-yl)-phenyl]-phosphatyl) 8-chloroadenosine V

V 2',3'-O-isopropylidene-8-chloroadenosine (80 mg, 0.23 mmol) was dissolved in anh. THF (30 mL), and tBuMgCl (1.0 M in THF, 6 eq, 1.4 mL, 1.4 mmol) was added. The appropriate phosphorochloridate (0.49 g, 1.24 mmol) in anh. THF (3 mL) was added and the mixture was stirred overnight. Purification by silica gel CC (2% MeOH/DCM) gave a residue that was stirred at rt in HCOOH/H2O (18 + 12 mL) overnight. The product was purified by prep. TLC (3-3.55% MeOH/DCM). Yield: 83 mg, 55% over 2 steps.

¾ MR (500, MHz, MeOD) δ 8.17, 8.15 (2 x s, 1H, H-2), 7.38 - 7.25, 7.20 - 7.09 (2 x m, 10H, Ar), 6.03 (m, 1H, H-l '), 5.35, 5.27 (2 x t, J = 5.4, 5.1, 1H, H-2'), 5.11 - 5.04 (m, 2H, CH 2 (Bn)), 4.61, 4.57 (2 x t, J = 5.3, 5.1, 1H, H-3'), 4.45 - 4.24 (m, 2H, H-5'), 4.19 (m, 1H, H-4'), 3.88 (m, 1H, CHNH), 1.71 - 1.38 (m, 3H, CH2_CH(CH 3 ) 2 ), 0.86, 0.83, 0.79, 0.76 (4 x d, J= 6.7, 6.6, 6.3, 6.2, 6H, CH(CH 3 ) 2 , diastereoisomers).

3 1 P NMR (202 MHz, CD 3 OD) δ 3.8, 3.6 (2 x s, diastereoisomers).

HR-MS (ES+) m/z: Found: (M + H + ) Found 661.1965. C29H 3 4C1N 6 0 8 P required: (M + ) 661.1943.

HPLC Reverse-phase HPLC eluting with H 2 0/CH 3 CN from 90/10 to 0/100 in 30 minutes, 1 ml/min, λ = 254 nm, two peaks with tR 13.7 min, tR 13.9 min.

Example 22 - 5'-([(Isopropyloxy-L-alanin-N-yl)-phenyl]-phosphatyl) 8-chloroadenosine

8-Chloroadenosine (0.30 g, 0.99 mmol) was dissolved in a mixture of anh. pyridine and THF (8 + 12 mL). The solution was cooled to 0°C and NMI (0.4 mL, 5.0 mmol) was added dropwise. The mixture was stirred for 10 min before phosphorochloridate (1.26 g, 4.12 mmol) dissolved in anh. THF (2 mL) was added slowly. After overnight stirring, additional NMI (0.4 mL, 5.0 mmol) was added and the mixture stirred for 48 h. tBuMgCl (0.1 M in THF, 1.0 mL, 1.0 mmol) and phosphorochloridate (1.33 g, 4.35 mmol) was added and the mixture was stirred for 24 h. The solvents were evaporated. The residue was purified twice by silica gel CC (5-10% MeOH/DCM and 4-5% MeOH/DCM) followed by preparative TLC (4 x 5% MeOH/DCM). The desired product was finally dissolved in EtOAc and washed with water. Yield: 15 mg, 2%

31 P MR (202 MHz, CD 3 OD) δ 3.60, 3.55 (2 x s, diastereoisomers). ¾ NMR (500 MHz, CD3OD) δ 7.35 - 7.27, 7.19 - 7.13 (2 x m, 6H, Ph, H-2), 6.04 (m, 1H, H-l '), 5.34, 5.29 (2 x t, J= 5.0, 4.9, 1H, H-2', diastereoisomers), 4.91 (m, 1H, CH (iPr)), 4.66 (m, 1H, H-3'), 4.45, 4.33 (2 x m, 2H, H-5'), 4.22 (m, 1H, H-4'), 3.85, 3.77 (2 x m, 1H, CH (Ala), diastereoisomers), 1.34 - 1.27, 1.23 - 1.17 (2 x m, 9H, 3 x CH 3 ).

HPLC Reverse-phase HPLC eluting with H2O/CH3OH from 90/10 to 0/100 in 30 minutes, 1 ml/min, λ = 254 nm, two peaks with tR 15.1 min, tR 15.9 min.

Example 23 - 5'-([(Cyclohexyloxy-L-alanin-N-yl)-phenyl]-phosphatyl) 8-chloroadenosine

8-Chloroadenosine (0.21 g, 0.70 mmol) was dissolved in a mixture of anh. pyridine and anh. THF (8 + 12 mL), tBuMgCl (1.0 M in THF, 0.7 mL, 0.7 mmol) was added dropwise at 0°C and the mixture was stirred for 10 min. The appropriate phosphorochloridate (0.5 g, 1.4 mmol) was dissolved in anh. THF (2 mL) and added slowly to the mixture. The mixture was stirred for 24 h before the solvents were evaporated. The residue was purified by silica gel CC (5% MeOH/DCM) followed by preparative TLC (5% MeOH/DCM). This was followed by another preparative TLC (10% MeOH/DCM) and subsequent EtOAc/H 2 0 wash. The product was obtained by evaporation of the organic phase. Yield: 8 mg, 2%.

¾ NMR (500 MHz, CD 3 OD) δ 8.19, 8.17 (2 x s, H-2, diastereoisomers), 7.34 - 7.27 (m, 2H, Ph), 7.19 - 7.13 (m, 3H, Ph.), 6.04 (d, J = 4.5, 1H, H-l '), 5.35, 5.29 (2 x m, 1H, H-2', diastereoisomers), 4.73 - 4.62 (m, 2H, H-3', CH (cHx)), 4.45, 4.34 (2 x m, 2H, H-5'), 4.22 (m, 1H, H-4'), 3.88, 3.80 (2 x m, 1H, CH (Ala)), 1.82 - 1.67, 1.54, 1.46 - 1.31 (3 x m, 10H, cHx), 1.29, 1.22 (2 x d, J= 7.0, 7.1, 3H, CH 3 ).

31 P NMR (202 MHz, CD 3 OD) δ 3.6, 3.5 (2 x s, diastereoisomers).

HPLC Reverse-phase HPLC eluting with H2O/CH3OH from 90/10 to 0/100 in 30 minutes, 1 ml/min, λ = 254 nm, two peaks with tR 18.0 min, tR 18.6 min

Example 24 - 5'-([(Neopentyloxy-2,2-dimethylglycin-N-yl)-phenyl]-phosphat yl) 8- chloroadenosine Y

Y 8-Chloroadenosine (0.30 g, 0.99 mmol) was dissolved in a mixture of anh. pyridine and anh. THF (2 + 3), tBuMgCl (1.0 M in THF, 1.0 mL, 1.0 mmol) was added dropwise at 0°C and the mixture was stirred for 10 min. The appropriate phosphorochloridate (1.6 g, 4.5 mmol) was dissolved in anh. THF and added slowly to the mixture. The mixture was stirred at rt for 48 h. The solvent was evaporated and the residue purified by silica gel CC (3% MeOH/DCM), prep. TLC (mobile phase: 10% MeOH/DCM) and EtOAc/H 2 0 wash. This was followed by prep. TLC (mobile phase: 10% MeOH/CHCl 3 ) to give the desired product. Yield: 13 mg, 2%.

¾ NMR (500 MHz, CD 3 OD) δ 8.2, 8.1 (2 x s, 1H, H-2, diastereoisomers), 7.33 - 7.27 (m, 2H, Ph), 7.19 - 7.12 (m, 3H, Ph), 6.04 (m, ΙΗ, Η-Ι '), 5.33, 5.28 ( 2 x t, J= 5.2, 4.9, 1H, H-2') 4.67, 4.63 (2 x t, J = 5.4, 5.2, 1H, H-3'), 4.46, 4.37 (2 x m, 2H, H-5'), 4.22 (m, 1H, H-4'), 3.79 - 3.76 (m, 2H, CH 2 tBu), 1.45, 1.44, 1.43, 1.41 (4 x s, CH 3 dimethylglycine, diastereoisomers), 0.94 (s, 9H, tBu).

31 P NMR (202 MHz, CD3OD) δ 2.0, 1.9 (2 x s, diastereoisomers).

HPLC Reverse-phase HPLC eluting with H 2 0/CH 3 OH from 90/10 to 0/100 in 30 minutes, 1 ml/min, λ = 254 nm, two peaks with tR 18.5 min, tR 18.8 min. Example 25 - 5'-([(Methyloxy-L-methionin-N-yl) phenylj-phosphatyl) 8-chloroadenosine

z

2',3'-O-Isopropylidene-8-chloroadenosine (0.18 g, 0.49 mmol) was dissolved in anh. THF (20 mL) and tBuMgCl (1.0 M in THF, 10 eq, 5.0 mL, 5.0 mmol) was added. Phosphorochloridate (1.26 g, 3.73 mmol) in anh. TFIF (3 mL) was added and the mixture was stirred overnight. The solvent was evaporated in vacuo. The residue obtained after silica gel CC (3% MeOH/DCM) was stirred overnight in a HCOOH/H 2 0 mixture (6 + 4 mL). Evaporation of the solvents gave a residue that was purified twice by prep. TLC (5% MeOH/DCM, 10% MeOH/DCM), dissolved in EtOAc and washed with H 2 0. Yield: 15 mg, 5% over 2 steps.

31 P MR (202 MHz, CD 3 OD) δ 3.63, 3.61 (2 x s, diastereoisomers).

'H NMR (500 MHz, CD3OD) δ 8.20, 8.17 (2 x s, 1H, H-2, diastereoisomers), 7.36 - 7.28 (m, 2H, Ph), 7.21 - 7.14 (m, 3H, Ph), 6.05, 6.03 (2 x d, J= 5.0, 4.7, 1H, H-l '), 5.37, 5.30 (2 x t, J

= 5.3, 5.1, 1H, H-2'), 4.65 (t, J = 5.2, 1H, H-3'), 4.51 - 4.29 (m, 2H, H-5'), 4.24, 4.20 (2 x q,

J = 4.6, 4.9, 1H, H-4'), 4.03, 3.93 (2 x m, 1H, CH (Met)), 3.653, 3.646 (2 x s, 3H, CH3O),

2.51 - 2.44, 2.40 - 2.28 (2 x m, 2H, CH 2 S), 2.01, 1.98 (2 x s, 3H, CH 3 S), 1.95 - 1.81, 1.79 -

1.70 (2 x m, 2H, CH 2 CH (Met)).

HR-MS (ES+) m/z: Found: (M + Na + ) Found 625.1006. C 22 H 28 C1N 6 0 8 P required: (M+Na + )

625.1013.

Example 26 - 5'-([(Benzyloxy-L-valin-N-yl)-phenyl]-phosphatyl) 8-chloroadenosine AA

2',3'-O-Cyclopentylidene-8-chloroadenosine (0.15 g, 0.41 mmol) was dissolved in anh. THF (15) and tBuMgCl (1.0 M in THF, 3.eq, 1.2 mL. 1.2 mmol) was added slowly followed by phosphorochloridate (0.89 g, 2.33 mmol) dissolved in anh. THF (3 mL). The mixture was stirred at rt overnight. Purification by silica gel CC (0-2.5% MeOH/DCM) gave a residue that was stirred at rt in HCOOH/H2O (6 + 4 mL) overnight. The solvents were evaporated in vacuo and the product purified twice by silica gel CC (4%MeOH/DCM, 3.5%-4% MeOH/DCM). Yield: 0.11 g, 41% over 2 steps.

MS: m/z = [M( 35 C1) + H] + calc./exp.: 647.1786/647.1786 31 P MR (202 MHz, CD3OD) δ 4.3, 4.2 (2 x s, diastereoisomers). 'H NMR (500 MHz, CD3OD) δ 8.17, 8.15 (2 x s, 1H, H-2, diastereoisomers), 7.45 - 7.08 (m, 10H, Ar), 6.04 (m 1H, H-l '), 5.34, 5.28 (2 x m, 1H, H-2'), 5.11 - 5.03 (m, 2H, CH 2 (Bn)), 4.65, 4.61 (2 x t, J = 5.3, 5.2, 1H, H-3'), 4.42, 4.31 (2 x m, 2H, H-5'), 4.20 (m, 1H, H-4'), 3.64 (m, 1H, CHNH (Val)), 1.94 (m, 1H, CH(CH 3 ) 2 ), 0.84, 0.81, 0.77, 0.76 (4 x d, J = 6.8, 6.8, 5.4, 6H, 2 x CH3, diastereoisomers.

HR-MS (ES+) m/z: Found: (M + H + ) Found 647.1786. C28H3 2 C1N 6 0 8 P required: (M + ) 647.1786.

HPLC Reverse-phase HPLC eluting with H 2 0/CH 3 OH from 90/10 to 0/100 in 30 minutes, 1 ml/min, λ = 254 nm, one peak with tR 19.0 min.

HPLC Reverse-phase HPLC eluting with H 2 0/CH 3 CN from 90/10 to 0/100 in 30 minutes, 1 ml/min, λ = 254 nm, two peaks with tR 13.1 min.

Example 27 - 8-Chloroadenosine 5'-0-bis(benzyloxy-L-alanin-N-yl) phosphate AB

Procedure: 8-chloroadenosine (250 mg, 0.83 mmol) was suspended in (CH 3 ) 3 P0 3 (5 mL), and POCh (77.36 \L, 0.83 mmol) was added dropwise at -5 °C. The reaction mixture was left stirring for 4 hours at room temperature. A solution of L-alanine-O-benzyl ester /?-TSA salt (1.46 g, 4.15 mmol) dissolved in anhydrous CH2CI2 (5 mL) was added followed by diisopropyl ethyl amine (1.45 mL, 8.3 mmol) at -78 °C. 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 washed with brine. The combined organic layers were dried over Na 2 S04 and concentrated. The residue was purified by column chromatography (gradient elution of CH 2 Cl2/MeOH=96/4 to 93/7) to give a white foam.

Yield: (245 mg, 42 % over two steps).

1 H MR (500 MHz, CD 3 OD) δ 8.21 (s, 1H, H2), 7.36-7.26 (m, 10H, Ph), 6.02 (d, J= 4.8 Hz, 1H, HI '), 5.30 (t, J = 5.5 Hz, 1H, H2'), 5.14-5.02 (m, 4H, CH 2 Ph), 4.62 (dd, J = 7.5, 5.0 Hz, 1H, H3'), 4.31-4.24 (m, 1H, H5'), 4.18-4.11 (m, 2H, H5', H4'), 3.95-3.82 (m, 2H, CH ala), 1.28 (d, J= 7.2 Hz, 3H, CH 3 ala), 1.23 (d, J= 7.2 Hz, 3H, CH 3 ala).

3 1 P MR (202 MHz, CD 3 OD) δ 13.54 (s).

MS (ES+) m/z: Found: 704.1 (M + H + ), 726.1 (M + Na + ), C 30 H 35 ClN 7 O9P required: (M + ) 704.07.

HPLC Reverse-phase HPLC eluting with H 2 0/CH 3 CN from 90/10 to 0/100 in 35 minutes, 1 ml/min, 1 = 254.

Example 28 - 8-Chloroadenosine 5'-0-bis(benzyloxy-glycin-N-yl) phosphate AC

AC Procedure: 8-chloroadenosine (100 mg, 0.33 mmol) was suspended in (CH 3 ) 3 P0 3 (5 mL), and POCh (30.89 \L, 0.33 mmol) was added dropwise at -5 °C. The reaction mixture was left stirring for 4 hours at room temperature. A solution of glycine-O-benzyl ester hydrochloride salt (333 mg, 1.65 mmol) dissolved in anhydrous CH2CI2 (5 mL) was added followed by diisopropyl ethyl amine (0.57 mL, 3.3 mmol) at -78 °C. 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 washed with brine. The combined organic layers were dried over Na 2 S04 and concentrated. The residue was purified by column chromatography (gradient elution of CH 2 Cl2/MeOH=96/4 to 93/7) to give a white foam.

Yield: (60 mg, 27 %).

1 H MR (500 MHz, CD 3 OD) δ 8.23 (s, 1H, H2), 7.35-7.26 (m, 10H, Ph), 6.03 (d, J= 5.1 Hz, 1H, HI '), 5.31 (dd, J = 5.5, 5.1 Hz, 1H, H2'), 5.11-5.08 (m, 4H, CH 2 Ph), 4.59 (t, 5.5, 5.1 Hz, 1H, H3'), 4.34-4.28 (m, 1H, H5'), 4.27-4.16 (m, 2H, H5', H4'), 3.70-3.64 (m, 4H, CH 2 gly). 31 P MR (202 MHz, CD 3 OD) δ 15.96 (s).

MS (ES+) m/z: Found: 704.1 (M + H + ), 726.1 (M + Na + ), C28H 3 iClN 7 0 9 P required: (M + ) 676.01.

HPLC Reverse-phase HPLC eluting with H 2 0/CH 3 OH from 100/0 to 0/100 in 35 minutes, 1 ml/min, λ = 254 nm, one peak with tR 23.23 min.

Stereochemistry and Method of Determination Isomer separation and characterization

Both benzyl 2-(((((2R,3^,4R,5R)-5-(6-amino-8-chloro-9H-purin-9-yl)-3,4-d ihydroxytetra- hydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)acetate (compound B) and (2S)- Benzyl 2-(((((2R,3^,4R,5R)-5-(6-amino-8-chloro-9H-purin-9-yl)-3,4-d ihydroxy- tetrahydrofuran-2-yl)methoxy)(naphthalen- 1 -yloxy)phosphoryl)amino)propanoate (compound D) may exist as diastereomeric forms in the same way as other proTides such as NUC-1031 (acelarin) may do due to the presence of a chiral phosphorus atom. During synthesis, the coupling reaction typically involves the formation of a new chiral centre and the above 2 proTides are each formed as a mixture of two diastereoisomers. The same is true for other compounds described in this application. Since the phosphate centre is chiral in the compounds of the present invention and the compounds may independently exist as Rp and Sp diastereoisomers it is possible for the compounds to be present as mixed Rp and Sp or one pure diastereoisomer.

The diastereomers can be detected individually by 31 P- MR and separated by chromatography, each isolated peak providing one of the two 31 P- MR signals present in the mixture. The separation technique may be chromatography, e.g. column chromatography, preparative thin layer chromatography, preparative HPLC or flash purification by Biotage Isolera™. When the separation technique is preparative HPLC a reverse phase column C-18 maybe used. An example of reverse phase column is Varian Pursuit XRs 5 C-18. When the separation technique is Biotage Isolera™ a Reverse phase cartridge may be used. An example of reverse phase cartridge is SNAPUltra C-18.

On the basis of comparison of 31 P chemical shift, 1H MR spectra, and HPLC retention times with those of other ProTides known in the literature, isomer A of compound B was tentatively assigned as the Rp diastereomer and isomer B of compound B was tentatively assigned as the Sp diastereomer. Similarly, isomer A of compound D was tentatively assigned as the Sp diastereomer and isomer B of compound D was tentatively assigned as the Rp diastereomer. The absolute structures may also be determined by conventional X-ray crystallographic analysis.

The (Rp) and Sp) isomers of the two compounds were separated by prep-HPLC under the following conditions: Equipment: Varian Prostar (LC Workstti on- Varian Prostar 335 LC detector) Flow rate: 20 mL/min

Column: Varian Pursuit XRs 5 C-18, 150x21 Temperature: ambient Feed: dissolved in MeOH Loading: 20 mg

Solvent: Isocratic 55% MeOH in water

The (Rp) and Sp) isomer of each compound was separated by isolera Biotage under the following condition: Equipment: Isolera Biotage™

Flow rate: 12 mL/min

Column: SNAP Ultra C-18 12g

Temperature: ambient Feed: dissolved in MeOH

Loading: 50 mg

Solvent: Isocratic 55% MeOH in water

The supporting experimental evidence is shown below

HPLC traces for (25)-Benzyl 2-(((((2R,35',4R,5R)-5-(6-amino-8-chloro-9H-purin-9-yl)-3,4- dihydroxy-tetrahydrofuran-2-yl)methoxy)(naphthalen- 1 -yloxy)phosphoryl)amino)propanoate (compound D) are shown in Figure 9.

Compound D isomer A

¾-NMR (500 MHz; MeOO-d4): δ Η 8.07 (IH, d J= 8.5Hz, H-Napht), 8.05 (IH, s, 2-H), 7.87 (IH, d J= 8.5Hz, H-Napht), 7.67 (IH, d J= 8.5Hz, H-Napht) 7.54-7.48 (2H, m, H-Napht), 7.41-7.30 (IH, m, H-Napht), 7.36-7.33 (IH, m, H-Napht), 7.26-7.22 (5H, m, - R T PH), 6.03 (IH, d J= 5.0 Hz, H-l '), 5.33 (IH, t J= 5.0Hz, H-2'), 5.01, 4.98 (AB, JAB = 12.3 Hz, C¾Ph), 4.65 (IH t J= 5.5Hz, H-3'), 4.49-4.45 (IH, m, H a -5'), 4.41-4.36 (IH, m, H b -5'), 4.22-4.20 (IH, m, H-4'), 3.94-3.90 (IH, m, -CHCH 3 ), 1.17 (IH, d J = 7.0 Hz, CH 3 ). 31 P NMR (202 MHz, MeOD-i¾): δ Ρ 3.93 (IP, s). Reverse-phase HPLC, eluting with H 2 0/ CH 3 CN from 100/10 to 0/100 in 30 min; 1 mL/min, λ = 254 nm, showed a peak with t R = 16.43 min

31 P NMR (202 MHz, MeOD-i¾): δ Ρ 3.93 Compound D isomer B

1 H-NMR (500 MHz; MeOD-i¾): δ Η 8.10 (IH, s, H-2), 8.08 (IH, d J= 8.5Hz, H-Napht), 7.87 (IH, d J= 8.5Hz, H-Napht),7.67 (IH, d J= 8.5Hz, H-Napht), 7.53-7.50 (IH, m, H-Napht), 7.48-7.44 (IH, m, H-Napht), 7.40-7.38 (IH, m, H-Napht), 7.33-7.27 (6H, m, H-Napht and - CH2 /2), 6.02 (IH, d J = 5.0 Hz, H-l '), 5.28 (IH, t J= 5.0Hz, H-2'), 5.04, 5.02 (AB, JAB = 12.2 Hz, C¾Ph), 4.63 (lH t J= 5.5Hz, H-3'), 4.48-4.46 (IH, m, H a -5'), 4.38-4.35 (IH, m, H b - 5'), 4.23-4.20 (IH, m, H-4'),4.05-4.01 (IH, m, -CHCH 3 ), 1.17 (IH, d J = 7.0 Hz, CH 3 ). 31 P NMR (202 MHz, MeOO-d4): δ Ρ 3.83 (IP, s). Reverse-phase HPLC, eluting with H2O/CH3CN from 100/10 to 0/100 in 30 min; 1 mL/min, λ = 254 nm, showed a peak with tR = 16.59 min

31 P NMR (202 MHz, MeOO-d4): δ Ρ 3.83

HPLC traces for benzyl 2-(((((2R,3^,4R,5R)-5-(6-amino-8-chloro-9H-purin-9-yl)-3,4- dihydroxytetra-hydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)a mino)acetate (compound B) are shown in Figure 10.

Compound B isomer A

1 H-NMR (500 MHz; MeOO-d4): δ Η 8.03 (1H, s, H-2), 8.23-7.13 (7H, m, C hPh and Ph), 7.05-6.99 (3H, m, Ph), 5.90 (1H, d J= 5.0 Hz, H-l '), 5.22 (1H, t J= 5.0Hz, H-2'), 5.01, 4.97 (AB, JAB = 12.2 Hz, C¾Ph), 4.48 (lH t J= 5.5Hz, H-3'), 4.53-4.47 (1H, m, H a -5'), 4.23-4.18 (1H, m, Hb-5'), 4.08-4.050 (1H, m, H-4'),3.56 (1H, d J= 8.5Hz, -C% a NH 2 ), 3.54 (1H, d J= 8.5Hz, -CH2aNH 2 ). 31 P NMR (202 MHz, MeOD-i¾): δ Ρ 4.63 (IP, s). 31 P NMR (202 MHz,

Reverse-phase HPLC, eluting with H 2 0/ CH3CN from 100/10 to 0/100 in 30 min; 1 mL/min, λ = 254 nm, showed a peak with tR = 12.58 min

Compound B isomer B

1 H-NMR (500 MHz; MeOD-i¾): δ Η 8.06 (1H, s, 2-H), 7.23-7.16 (7H, m, -C hPh and Ph), 7.05-6.99 (3H, m, Ph), 5.92 (1H, d J = 5.0 Hz, H-l '), 5.20 (1H, t J= 5.0Hz, H-2'), 5.01, 4.99 (AB, JAB = 12.2 Hz, C¾Ph), 4.53 (1H t J= 5.5Hz, H-3'), 4.33-4.23 (2H, m, H-5'), 4.12-4.07 (1H, m, H-4'), 361-3.53 (2H, m, -C¾NH 2 ). 31 P NMR (202 MHz, MeOD-i¾): δ Ρ 4.57 (IP, s).

Reverse-phase HPLC, eluting with H 2 0/CH 3 CN from 100/10 to 0/100 in 30 min; 1 mL/min, λ = 254 nm, showed a peak with tR = 12.88 min

2. Biological Experimental Examples

Exemplified compounds embodying the present invention were assessed for their ability to target cancer stem cells and for their anti-cancer potency.

Example 29 - In vitro cytotoxicity analyses

The following cell lines are referred to in the experimental data set out in Table 2 below: Lovo: cell line derived from a metastatic site in a patient with colon cancer

MCF7: human oestrogen sensitive mammary epithelial cell line

PC3 : human prostate cancer cell line

A549: cancerous lung tissue derived cells

L1210: lymphoblast cell line derived from a mouse with lymphocytic leukaemia

FM3 A: mammary carcinoma murine cells

CEM: human T-lymphoblasts, derived from the blood of a patient with acute lymphoblastic leukaemia

HeLa: epithelial cells from a patient with cervical adenocarcinoma

Certain compounds of the invention were assayed for their cytotoxic activity in cancer cell lines, according to the assays below.

L1210, CEM and HeLa cell line Cytotoxicity Assay

Murine leukemia L1210, human T-lymphocyte CEM, and human cervix carcinoma HeLa cells were obtained from the American Type Culture Collection (ATCC) (Rockville, MD). All cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Carlsbad, CA) with 10% fetal bovine serum (FBS) (Biochrom AG, Berlin, Germany), 10 mM Hepes, and lmM sodiumpyruvate (Invitrogen). Cells were grown at 37 °C in a humidified incubator with a gas phase of 5% C0 2 . Monolayer cells (HeLa and HeLa) were seeded in 96- well microtiter plates (Nunc, Roskilde, Denmark) at 10 000 cells/well. After 24 h, an equal volume of fresh medium containing the test compounds was added. On day 5, cells were trypsinized and counted in a Coulter counter (Analis, Suarlee, Belgium). Suspension cells (L1210, CEM) were seeded in 96-well microtiter plates (Nunc) at 60 000 cells/well in the presence of a given amount of the test compounds. The cells were allowed to proliferate for 48 h (LI 210) or 72 h (CEM) and were then counted in a Coulter counter. The 50% inhibitory concentration (IC50) was defined as the compound concentration required to reduce cell proliferation by 50%. The cytostatic activity of the compounds was determined after 3 days, as outlined above.

MCF7, L0V0, A549 and PC3 cell lines Cytotoxicity Assay

Anti-tumour evaluation in MCF7, LoVo, A549 and PC3 cell lines was performed by MTT assay. Compounds were prepared as 0.1-100 mM stock solutions dissolved in DMSO and stored at -20°C. Cells were seeded into 96-well microtitre plates at a density of 5xl0 3 cells per well and allowed 24 hours to adhere. Decimal compound dilutions were prepared in medium immediately prior to each assay (final concentration 0.1-100 μΜ). Experimental medium was DMEM +10% FCS (PC3 and Lovo) or RPMI +10% heat inactivated FCS (A549 and MCF7). Following 96h compound exposure at 37°C, 5% CO2, MTT reagent (Sigma Aldrich) was added to each well (final concentration 0.5 mg/ml). Incubation at 37°C for 4h allowed reduction of MTT by viable cells to an insoluble formazan product. MTT was removed and formazan solubilised by addition of 10% Triton X-100 in PBS. Absorbance was read on a Tecan Sunrise spectrophotometer at 540 nm as a measure of cell viability; thus inhibition relative to control was determined (IC50).

The results of the initial screening are presented in Table 2. A represents an absolute IC50 of from 0.1 to 5, B represents an absolute IC50 greater than 5 and up to 15, C represents an absolute IC50 of greater than 15 and up to 100; and D represents an absolute IC50 of greater than 100. Table 2

D B A A

F C B A

G C B A

E B A A

H B A A

I B B A J B B B

K C B A

L B A A

M C C C

N C C A

0 A A A

P B A A

Q B A A

B B A

S C B A

A C C C

B B B A

T C C B

U C B A

c C C B

V C C A

AB B A A

AC A A A

All of the compounds tested showed inhibitory activity against certain cell lines. Example 30 - Further in vitro cytotoxicity analyses

A subset of compounds of the invention were then assayed for their cytotoxic activity in a broader array of different solid tumours and haematological malignancies using the following assay.

Solid tumour and haematological malignancy assay

In vitro viability assay were performed to assess the effects of compounds on cell viability in selected cell lines over 72 hrs using the CellTiterGlo (CTG, Promega-G7573) assay. The tests were performed in duplicates with treatment of compounds at 9 points, 3.16 folds titration in 96 well plates over -72 hrs. The compound starting concentrations were 198 μΜ. Cell viability assay using CellTiterGlo in 96-well plate were performed. Compound treatment 72 hrs, standard growth conditions, duplicates. Compounds were dissolved to 40mM with thawed 100%. Compounds were serially diluted at 3.16 fold in thawed DMSO, and warmed to 37°C before being dissolved in media (2μ1+200μ1). After compounds were dissolved in media, media containing compounds were warmed to 37°C in incubator and then compounds in media were added to cell plates (50μ1+50μ1) in duplicates. The compounds' final concentrations were from 198μΜ to 19.9nM. All compound solubilities were checked and recorded again, then the plates were transferred to CO2 tissue culture incubator immediately and incubated for 3 days. DMSO final concentration is 0.5%.

The following cell lines were tested and are referred to in the Table 3 below:

Table 3

Cell line Malignancy Cell line Malignancy

MOLT-4 Acute lymphoblastic leukaemia HEL92.1.7 Erythroleukaemia

CCRFCEM Acute lymphoblastic leukaemia HL-60 Promyelocytic leukaemia

RL Non-Hodgkin' s lymphoma MV4-1 1 Biphenotypic B myelomonocytic leukemia

HS445 Hodgkin lymphoma HepG2 Hepatocellular carcinoma

RPMI8226 Human multiple myeloma HT29 Colon adenocarcinoma

K562 Chronic myelogenous leukaemia BxPC-3 Pancreatic cancer

KG-1 Acute myelogenous leukaemia MCF-7 Breast adenocarcinoma

THP-1 Acute monocytic leukaemia MiaPaCa2 Breast adenocarcinoma

Z- 138 Mantle cell lymphoma MDAMB231 Human breast adenocarcinoma

NCI-H929 Plasmacytoma SW620 Colon adenocarcinoma

Jurkat acute T cell leukaemia

The results of the further screening are presented in Table 4. "A" represents an absolute IC50 of from 0.1 to 5; "B" represents an absolute IC50 greater than 5 and up to 15; "C" represents an absolute IC50 of greater than 15 and up to 100; and "D" represents an absolute IC50 of greater than 100.

Table 4 (Cont.)

MiaPaCa-2 SW620 HepG2 HT29 MCF7 BxPC-3 MDAMB231

Cpnd

ICso MI% ICso MI% ICso MI% ICso MI% ICso MI% ICso MI% ICso MI%

8C1A A 93 A 88 A 82 A 79 A 75 B 67 A 74

C B 98 C 97 C 84 B 98 C 100 C 80

A B 106 c 85 B 85 C 86 c 94 c 66

B A 96 A 87 A 86 A 86 A 92 B 68 B 76

V A 99 A 100 A 100 A 92 A 97 B 99 C 99

D A 99 A 99 A 102 A 92 A 99 B 99

E A 100 A 99 A 101 A 91 A 98 B 92

L

O

H A 99 B 99

P

Q

AC A 94 A 94 A 87 A 87 A 87 B 68

Table 4 (Cont.)

NCI-H929 MV4-11 RL RPMI-8226 MOLT-4 HL-60 Z138

CpnD

IC 50 MI% ICso MI% ICso Ml IC 50 MI% IC 50 MI% IC 50 MI% IC50 M I

8C1A A 98 A 100 B 85 A 98 A 99 A 99 A 100

C B 101 A 92 C 94 B 91 A 95 B 84 A 86

A C 103 A 100 B 98 B 96 C 110 B 94 A 99

B B 98 A 99 B 73 A 99 A 99 A 99 A 100

V A 99 A 99 C 51 A 100 A 100 B 100 A 102

D A 98 A 100 B 74 A 100 A 99 B 100 A 100

E A 98 A 100 B 77 A 100 A 99 A 100 A 100

L C 98 A 97 B 74 A 98 B 94

O C 99 A 96 C 27 B 100 C 100

H A 98 A 100 C 28 A 100 B 100

P A 99 A 100 B 66 A 100 A 100

Q B 99 A 100 B 79 A 100 B 100

AC A 103 A 100 A 94 A 98 A 100 A 100 A 98

Isomers A and B of compound D were tested separately and the results are presented in Table 5

Example 31 - Assessment of cytotoxicity and cancer stem cell activity

A further comparative analysis of the toxicity of compounds in the KGla cell line over an extended dose range was carried out, and the relative effect assessed of the compounds on the leukaemic stem cell compartment within the KGla cell line, across the entire dose range. Thus, experimental tests were performed on certain compounds of the present invention to assess their ability to target cancer stem cells in a leukaemic cell line. The acute myeloid leukaemia (AML) cell line, KGla, was employed to assess the relative effect of compounds on the stem cell compartment. The KGla cell line was selected because it manifests a minor stem cell-like compartment with a distinct immunophenotype (Lin7CD34 + /CD387CD123 + ). Materials and Methods

KGla cell culture conditions

Cells of the KGla cell line were maintained in RPMI medium (Invitrogen, Paisley, UK) supplemented with lOOunits/ml penicillin, 10C^g/ml streptomycin and 20% foetal calf serum. Cells were subsequently aliquoted (10 5 cells/ΙΟΟμΙ) into 96-well plates and were incubated at 37 ° C in a humidified 5% carbon dioxide atmosphere for 72 hrs in the presence of nucleoside analogues and their respective nucleotide analogues (known as ProTides) at concentrations that were experimentally determined for each series of compounds. In addition, control cultures were carried out to which no drug was added. Cells were subsequently harvested by centrifugation and were analyzed by flow cytometry using the Annexin V assay. Measurement of in vitro apoptosis

Cultured cells were harvested by centrifugation and then resuspended in 195μ1 of calcium- rich buffer. Subsequently, 5μ1 of Annexin V (Caltag Medsystems, Botolph Claydon, UK) was added to the cell suspension and cells were incubated in the dark for 10 mins prior to washing. Cells were finally resuspended in 190μ1 of calcium-rich buffer together with ΙΟμΙ of propidium iodide. Apoptosis was assessed by dual-colour immunofluorescent flow cytometry as described previously. Subsequently LD50 values (the dose required to kill 50% of the cells in a culture) were calculated for each nucleoside analogue and ProTide.

Immunophenotypic identification of the leukaemic stem cell compartment

KGla cells were cultured for 72 hrs in the presence of a wide range of concentrations of each compound assayed. 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). The sub-population expressing a leukaemic stem cell (LSC) phenotype were subsequently identified and were expressed as a percentage of all viable cells left in the culture. The percentages of stem cells remaining were then plotted on a dose-response graph and the effects of the compounds were compared with 8-chloroadenosine. Statistical analysis

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

Results

In vitro cytotoxicity

The in vitro drug sensitivity was measured using the Annexin V/propidium iodide assay.

Figure 1 compares the LD50 values for 8-chloroadenosine and compounds D, B and E in unsorted populations of KGla cells. As can be seen from this Figure, Compound D is significantly (P<0.0001) more cytotoxic in respect of cancer cell populations as a whole than is the parent prodrug compound 8-chloroadenosine. The LD50 values calculated are also shown in Table 6.

Table 6

Anti-Cancer Stem Cell Activity

The leukaemic stem cell (LSC) targeting capacity of 8-chloroadenosine and phosphoramidate compounds B, C, D and E are also illustrated in Table 6, as well as in Figure 2. All data presented in the Table and Figure are the mean (± SD) of three independent experiments. Figure 2 shows the individual sigmoidal dose response curve for 8-chloroadenosine phosphoramidate compounds D, B and E.

All of the compounds tested, including 8-chloroadenosine, at certain doses, reduced the proportion of leukaemic stem cells (LSCs) present among the KGla population. Of these compounds, B and D demonstrated particularly effective preferential targeting of LSCs when compared to 8-chloroadenosine. These effects were observed at concentrations up to Ι μΜ. There was no significant difference with respect to each other in the ability of B and D to deplete LSCs at the concentrations shown in Figure 2(i). The significant differences between the anti-cancer stem cell selectivity of Compound D and 8-chloroadenosine is clearly represented in Figure 2(ii) where the results generated in respect of Compounds B and E have been removed from the graph. Targeting of cancer stem cells by Compound D leads to a statistically significantly reduction in the proportion of LSCs present after treatment with Compound D or 8-chloroadenosine at concentrations of 1 x 10 "6 and 5 x 10 "6 M.

The results of further investigation of the anti-cancer stem cell activity of Compounds AC and E, and Isomers A and B of both Compounds B and D are shown in Figures 7 and 8.

From the results set out in Figure 7, it can be seen that Isomer A of Compound B was significantly more potent than either 8-chloroadenosine, or Isomer B of Compound B. Similarly, Isomer A of Compound D was significantly more potent than either 8- chloroadenosine, or Isomer B of Compound D. Compound E was less potent than 8- chloroadenosine, whilst Compound AC was not as cytotoxic as Isomer A of Compound B, or Isomer A of Compound D.

The results of cancer stem cell targeting studies, set out in Figure 8, illustrate that both Isomer A of Compound B and Isomer A of Compound D have a statistically significantly increased ability to target cancer stem cells as compared to 8-chloroadenosine. There was no statistically significant difference in the cancer stem cell targeting activity of Isomer A of Compound B as compared to Isomer A of Compound D. In contrast, Compound AC showed no evidence of a capacity to target cancer stem cells. Table 7 compares the control of stem cells demonstrated by compound D of this invention with that of equivalent experiments conducted on ProTides of clofarabine and cladnbine (comparative compounds 1 and 2 respectively; WO2006/100439).

comparative compound 1

comparative compound 2

As can be seen, compound D provides better control of cancer stem cells than the clofarabine or cladribine equivalent.

KG-la unsorted KG- la Change relative to

(CD347CD387CD123 + ) control (%) LDso (μΜ)

% compartment

Control - 8-Cl-A series - 3.3 -

8-chloroadenosine 9.7 3.7 +0.4

Compound D 3.8 2.5 -0.8

Control - clofarabine series - 3.5 -

Clofarabine 0.02 3.3 -0.2

Comparative compound 1 0.14 3.3 -0.2

Control - cladribine series - 4.0 -

Cladribine 0.18 6.0 +2.0

Comparative compound 2 0.82 3.5 -0.5 Example 32 - Cytotoxicity assessment under both normal conditions and under induced resistance.

The cytotoxicity and intracellular accumulation of 8-Cl-ATP for three compounds of the invention were compared with 8-Cl-A under both normal conditions and under induced resistance (i.e. with inhibitors).

Cell Culture

HL-60, CTS, and K-562 are leukaemia cell lines, which were obtained from the American Type Culture Collection (ATCC), Middlesex. HL-60 cell line is of acute promyelocytic leukaemia; CTS cell line is of acute myeloid leukaemia; and K-562 cell line is of chronic myeloid leukaemia.

HL-60, CTS, and K-562 cell lines were cultured in RPMI-1640 medium (Sigma Aldrich, UK), which was supplemented with 10% Fetal Bovine Serum (FBS) (PAA Laboratories), 1% amphotericin B (5.5 ml) and 1% penicillin/streptomycin (5.5 ml) (PAA Laboratories) and grown in flasks at 37°C incubator with 5% C0 2 . Treating Cells and Extracting Samples

Cell lines with 3xl0 6 cells/ml were used. Cells were treated with Ι μΐ of 20μΜ of each 8-Cl-A, compound A, compound B and compound D and incubated for 2 hours at 37°C with 5% CO2. After incubation, the cells were centrifuged (ambient, 1200 rpm, 5 minutes), the supernatant was removed, and the pellet was washed with 1ml of PBS and centrifuged (ambient, 1200 rpm, 5 minutes). The supernatant was removed, the pellet was reconstituted in ΙΟΟμΙ of PBS and ΙΟΟμΙ of 0.8M perchloric acid. This was then centrifuged (ambient, 1200 rpm, 5 minutes) and 180μ1 of the supernatant was transferred to new tubes and stored at -80°C until time of analysis.

For inhibitor studies the cell lines were treated in the same way as described above but before treatment with drugs, a number of inhibitors were added:

1) Nitrobenzylthioinosine ( BTI) (Sigma- Aldrich, St. Louis, MO) inhibits hENTl .

2) A- 134974 dihydrochloride hydrate (Sigma- Aldrich, St. Louis, MO) inhibits adenosine kinase.

3) EHNA hydrochloride (Sigma-Aldrich, St. Louis, MO) inhibits adenosine deaminase. Cells were treated with 1 μΐ of 10μΜ of each inhibitor and left for 5 minutes before adding the drug. The cells were then incubated for 2 hours at 37°C with 5% CO2. LC-MS/MS Analysis

The analytes were resolved using an ultra-performance liquid chromatography system (Accela UPLC, Thermo Scientific, UK) equipped with a Biobasic Αχ5μπι, 50x2. lmm column (Thermo Electron Corporation, Murrieta, CA, USA) and mobile phase consisting of a mixture of lOmM H4Ac in ACN/H 2 0 (30:70v/v), pH6.0 (A), and ImM H4Ac in ACN/H 2 0 (30:70v/v), pH10.5 (B). The mobile phase gradient was employed, comprising: buffer A=95% at 0-0.5 minutes, from 95 to 0% over 1.25 minutes, held at 0% for 1.75 minutes, from 0-95% over 0.1 minutes, ending with 95% for 2.9 minutes, all at a flow rate of 500μ1/πήη. For compounds detection a triple stage quadrupole Vantage mass spectrometry system (Thermo Scientific, UK) equipped with an electrospray ion source was used. Samples were analysed in the Multiple Reaction Monitoring, negative ion modes at spray voltage 3000V.

Adenosine 5 '-triphosphate (ATP) Assay

ATP ViaLightTM plus assay kit (Lonza, USA: Product No. LT07-121) was used for treating the cells in luminescence compatible 96 well plates (initial concentration of cells was lxlO 4 cells/well) with 8-Cl-A and ProTides, concentrations: 0, 0.1, 0.5, 1, 5 and 10μΜ were added to cells, respectively, followed by incubation for 72 hours at 37°C incubator with 5% C0 2 . For inhibitor studies, 10μΜ concentration of each inhibitor was added before drugs and incubated for 72 hours at 37°C with 5% C0 2 .

After incubation, 50μ1 of cell lysis reagent was added to the 96 well plates to release the intracellular ATP, followed by ΙΟΟμΙ of ATP monitoring reagent (AMR). The luminescent values of each well were determined using FLUOstar OPTFMA microplate reader (BMG Labtech) which converted ATP into light by using luciferase enzyme.

Drug Stability

10ml of human plasma sample (Sera laboratories, UK) was treated with 1 μΜ of EFINA. 1ml from this was taken along with Ι μΐ of 20μΜ concentration of each drug (8-Cl-A, compound A, compound B and compound D) which was added to new tubes and incubated for 2 hours at 37°C with 5% C0 2 . After incubation, ΙΟΟμΙ of each sample was taken and 300μ1 of 100% methanol was added and left on ice for 30 minutes. After 30 minutes, the mixture was centrifuged (14000 rpm, 4 °C, 10 minutes), the supernatant was transferred to new tubes and evaporated under speed vac. Then the dried pellet was reconstituted in ΙΟΟμΙ of 10% Acetonitrile and transferred to LC-MS vials for injection in to the UPLC-MS/MS system. Statistical A nalysis

The dose-response curves of cytotoxicity of the drugs was determined using non-linear regression analysis of percentage cell viability versus concentration and EC50 values were obtained. The intracellular assay was conducted in five repeats for each condition. Intracellular assay was determined using paired T test (two-tailed) analysis of 8- ChloroATP/ATP concentration and p values were obtained. Drug stability in human plasma was determined using two-way ANOVA analysis. For all the analysis, Prism Software program (GraphPad Software) was used.

Cytotoxicity assay in haematological cancer cell lines

The cytotoxicity assay results in the presence or absence of inhibitors are summarised in Table 8.

Table 8: EC50 values of 8-Cl-A and ProTides when HL-60, CTS and K-562 cancer cell lines are treated under various conditions.

Intracellular levels of 8-ChloroATP/ATP in haematological cancer cell lines

Figure 3 A shows the intracellular accumulation of the active metabolite, 8-Cl-ATP after treating HL-60 cell line with 8-Cl-A alone or 8-Cl-A with the inhibitors NBTI and A- 134974. 8-Cl-A alone produced 8-ChloroATP in equal concentrations to the endogenous metabolite ATP. Thus, the mean 8-Cl-ATP/ ATP ratio produced was found to be nearly 1. After inhibiting hENTl, this ratio was reduced by 50% (p value=0.0156), indicating that hENTl inhibition partially blocked the uptake of 8-Cl-A inside the cells. However, after inhibiting adenosine kinase, ratio was almost zero (p value=0.0004), indicating that adenosine kinase is required to activate 8-Cl-A into 8-Cl-ATP.

Figure 3B shows the intracellular accumulation 8-Cl-ATP after treating HL-60 cells with compound A alone or compound A with the inhibitors BTI and A- 134974. Compound A alone produced 8-Cl-ATP/ ATP ratio of 0.02. Inhibiting hENTl did not produce significant difference in intracellular accumulation of 8-Cl-ATP (p value=0.9568), showing that compound A uptake is independent of hENTl . Blocking adenosine kinase did not produce significant difference in intracellular accumulation of 8-Cl-ATP (p value=0.1016), indicating that compound A activation is independent of adenosine kinase.

Figure 3C shows the intracellular accumulation of 8-Cl-ATP after treating HL-60 cells with compound B alone or compound B with the inhibitors NBTI and A- 134974. Compound B alone produced 8-Cl-ATP/ ATP ratio of 0.13. Blocking hENTl did not produce significant difference in intracellular accumulation of 8-Cl-ATP (p value=0.9064), indicating that compound B uptake is independent of hENTl . However, after inhibiting adenosine kinase, this ratio decreased by 50% (p value=0.0386), showing that compound B activation is partially dependent on adenosine kinase. Figure 3D shows the intracellular accumulation of 8-Cl-ATP after treating HL-60 cells with compound D alone or compound D with the inhibitors NBTI and A- 134974. Compound D alone produced 8-Cl-ATP/ATP ratio of 0.6. Inhibiting hENTl did not produce significant difference in intracellular accumulation of 8-Cl-ATP (p value=0.4483), demonstrating that compound D uptake is independent of hENTl . However, after inhibiting adenosine kinase, the 8-Cl-ATP/ ATP ratio was reduced by 50% (p value=0.0082), indicating that compound D activation is partially dependent on adenosine kinase.

Figure 4A shows the intracellular accumulation of 8-Cl-ATP after treating CTS cell line with 8-Cl-A alone or 8-Cl-A with the inhibitors NBTI and A- 134974. 8-Cl-A alone produced 8- Cl-ATP/ATP ratio of 0.7. However, after inhibiting hENTl, the 8-Cl-ATP/ATP ratio decreased by 50% (p value=0.0156), indicating that 8-Cl-A partially depends on hENTl for entering the cell. However, after inhibiting adenosine kinase with A- 134974, this ratio was almost zero (p value=0.0004), indicating that 8-Cl-A requires adenosine kinase for activation into 8-Cl-ATP.

Figure 4B shows the intracellular accumulation of 8-Cl-ATP after treating CTS cells with compound A alone or compound A with the inhibitors BTI and A-134974. Compound A alone produced 8-Cl-ATP/ATP ratio of 0.04. Blocking hENTl did not produce significant difference in intracellular accumulation of 8-Cl-ATP (p value=0.2627), demonstrating that compound A did not require hENTl transporter for entering the cell. Likewise, A-134974 did not produce significant difference in intracellular accumulation of 8-Cl-ATP (p value=0.1588), indicating that compound A activation is independent of adenosine kinase. Figure 4C shows the intracellular accumulation of 8-Cl-ATP after treating CTS with compound B alone or compound B with inhibitors NBTI and A-134974. Compound B alone produced 8-Cl-ATP/ ATP ratio of 0.10. Inhibiting hENTl did not produce significant difference in intracellular accumulation of 8-Cl-ATP (p value=0.2725), showing that compound B enters the cell without using hENT. Likewise, inhibiting adenosine kinase did not produce significant difference in intracellular accumulation of 8-Cl-ATP (p value=0.9138), indicating compound B is activated independent of adenosine kinase.

Figure 4D shows the intracellular accumulation of 8-Cl-ATP after treating CTS cells with compound D alone or compound D with inhibitors NBTI and A-134974. Compound D alone produced 8-Cl-ATP/ATP ratio of 0.8. Inhibiting hENTl did not produce significant difference in intracellular accumulation of 8-Cl-ATP (p value=0.1252), demonstrating that compound D crosses the cell membrane without using hENTl . However, blocking adenosine kinase decreased this ratio by 60% (p value=0.0009), showing that compound D activation partially requires adenosine kinase.

Figure 5A shows the intracellular accumulation of the active metabolite, 8-Cl-ATP after treating K-562 cell line with 8-Cl-A alone or 8-Cl-A with the inhibitors NBTI and A-134974. 8-Cl-A alone produced 8-Cl-ATP in equal concentrations to the endogenous metabolite ATP, the ratio was nearly 1. However, after inhibiting hENTl, the mean 8-Cl-ATP/ATP ratio was reduced by 30% (p value=0.0149), demonstrating that 8-Cl-A is partially dependent on hENTl . However, after inhibiting adenosine kinase, the mean 8-Cl-ATP/ATP ratio was almost zero (p value=0.0002), showing that 8-Cl-A activation requires adenosine kinase. Figure 5B shows the intracellular accumulation of 8-Cl-ATP after treating K-562 cells with compound A alone or compound A with the inhibitors BTI and A- 134974. Compound A alone produced 8-Cl-ATP/ATP ratio of 0.04. Blocking hENTl did not produce significant difference in intracellular accumulation of 8-ChloroATP (p value=0.1960), indicating that compound A enters the cells independently of hENTl . Similarly, inhibiting adenosine kinase did not produce significant difference in 8-Cl-ATP concentration (p value=0.1061), showing that compound A activation is independent of adenosine kinase.

Figure 5C shows that the intracellular accumulation of 8-Cl-ATP after treating K-562 with compound B alone or compound B with the inhibitors NBTI and A- 134974. Compound B alone produced 8-Cl-ATP/ ATP ratio of 0.14. Inhibiting hENTl did not produce significant difference in 8-Cl-ATP concentration (p value=0.4797), indicating that compound B enters the cells independently of hENTl . However, inhibiting adenosine kinase decreased the 8-C1- ATP/ATP ratio by 50% (p value=0.0092). This signifies that compound B activation is partially dependent on adenosine kinase. Figure 5D shows the intracellular accumulation of 8-Cl-ATP after treating K-562 cells with compound D alone or compound D with the inhibitors NBTI and A- 134974. Compound D alone produced 8-Cl-ATP/ATP ratio of 0.6. Blocking hENTl did not produce significant difference in intracellular accumulation of 8-Cl-ATP (p value=0.5517), showing that compound D enters the cells independently of hENTl . However, blocking adenosine kinase decreased the 8-Cl-ATP/ATP ratio by 50% (p value O.0001). This indicates that compound D activation is partially dependent on adenosine kinase.

Stability of the drugs in human plasma

Figure 6 shows that plasma concentration of 8-Cl-A was increased by ~3 -folds when adenosine deaminase was blocked by EHNA inhibitor compared to control when adenosine deaminase was not inhibited. The plasma concentration of compound D was the highest when compared with all the other compounds, whether adenosine deaminase was inhibited or not. This indicates that compound D is the most stable compound in human plasma, resisting degradation by adenosine deaminase. Compound B had a low plasma concentration in the presence of EHNA and control compared to other compounds, indicating that compound A resists adenosine deaminase degradation more effectively than compound B. Discussion

In HL-60 cell line, compound D was found to be potent, with EC50 0.33 μΜ. However, compound D generated less intracellular 8-Cl-ATP/ATP than 8-Cl-A (compare Figure 3 A and 3D, control). When the hENTl inhibitor NBTI was added, cytotoxicity assay showed compound D to be most potent in CTS and K-562 cell lines. Similarly, intracellular accumulation assay showed that compound D generated higher intracellular 8-Cl-ATP/ATP in CTS and K-562 cell lines. Furthermore, when comparing the potency of 8-Cl-A and compound D in HL-60 cancer cell line, 8-Cl-A was found to be more potent as it had lower EC50 value (0.96μΜ) than compound D (1.23μΜ). When comparing the intracellular accumulation of 8-Cl-ATP generated by 8-C1- A and compound D, 8-Cl-A was found to have slightly higher 8-Cl-ATP/ATP ratio than compound D. The data shows that compound D was most effective in CTS AND K-562 cell lines but less effective in HL-60. The data shows that compound D cellular uptake is independent of the membrane transporter hENTl . Therefore, compound D could be effective in treating patients with cancer who have a non- functional hENTl transporter.

When the adenosine kinase A- 134974 was added, cytotoxicity assay showed compound B to be most potent in HL-60 cell line. Likewise, intracellular assay showed that compound B generated a higher ratio of 8-Cl-ATP/ATP than the other compounds in HL-60 cells in the presence of the inhibitor. This indicates that compound B is potent and activated independent of adenosine kinase. However, cytotoxicity assay and intracellular assay showed compound D to be the most potent drug, which generated a high 8-Cl-ATP/ATP ratio in CTS and K-562 cell lines. This shows that compound D activation is independent of adenosine kinase. Hypoxia leads to ATP depletion, and the activity of adenosine kinase becomes low in hypoxia. Therefore, compound D could be useful in treating patients with hypoxic tumours. The drug stability assay showed that there was a ~3-fold increase in human plasma concentration of 8-Cl-A when adenosine deaminase was inhibited by EHNA. This shows that 8-Cl-A is a much stronger substrate for adenosine deaminase in plasma (e.g. the plasma concentration of 8-Cl-A without EHNA was 3μg/ml which increased to 10μg/ml in the presence of EHNA). This suggests that following administration in patients 8-Cl-A could be extensively degraded in the plasma before reaching the tissues to exert its effects. Therefore, 8-Cl-A would not be potent in patients with high plasma activity of adenosine deaminase. Cytotoxicity assay showed that after 8-Cl-A, compound B was most potent in all the cell lines. However, intracellular accumulation showed compound D generated more than 70% 8- Cl-ATP/ATP than compound B in all the cell lines. Plasma concentrations also showed that compound D had the highest plasma concentration when compared to other compounds. Out of all the agents tested, compound A showed the least potency with the least intracellular accumulation of 8-Cl-ATP/ATP. On the other hand, compound D showed the strongest potency as well as the highest intracellular accumulation of 8-Cl-ATP.

Conclusion

Compound B and compound D cellular uptake is independent of hENTl nucleoside transporter proteins. The activation of both compounds is independent of adenosine kinase. This means that compounds of the invention are able to overcome the resistance mechanisms associated with 8-Cl-A.




 
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