NZ730919B2

NZ730919B2 - New 2' and/or 5' amino-acid ester phosphoramidate 3'-deoxy adenosine derivatives as anti-cancer compounds

Info

Publication number: NZ730919B2
Application number: NZ730919A
Authority: NZ
Inventors: Valentina Ferrari; Hugh Griffith; Antunez Carmen Jimenez; Christopher Mcguigan; Michaela Serpi
Original assignee: NuCana plc
Priority date: 2014-11-28
Filing date: 2015-11-27
Publication date: 2021-05-27

Abstract

The present invention relates to chemical compounds, the compounds for use in a method of treatment, particularly in a method of prophylaxis or treatment for cancer, a process for preparation of the compounds and pharmaceutical compositions comprising the compounds. The compounds may, in particular, be useful in the treatment of leukaemia, lymphoma and/or solid tumours inhomo sapiens. The compounds are derivatives of cordycepin (3'-deoxyadenosine) having a 2' and/or 5'- amino-acid ester phosphoramidate moeity. be useful in the treatment of leukaemia, lymphoma and/or solid tumours inhomo sapiens. The compounds are derivatives of cordycepin (3'-deoxyadenosine) having a 2' and/or 5'- amino-acid ester phosphoramidate moeity.

Description

NEW 2' AND/OR 5' AMINO-ACID ESTER PHOSPHORAMIDATE XY ADENOSINE DERIVATIVES AS ANTI-CANCER COMPOUNDS The present invention s to chemical compounds, the compounds for use in a method of treatment, particularly in a method of prophylaxis or treatment for cancer, a process for preparation of the compounds and pharmaceutical compositions comprising the compounds.

Particularly, although not exclusively, the present invention relates to chemical compounds for use in the treatment of leukaemia, ma and/or solid tumours in homo sapiens.

Cordycepin is 3’-deoxyadenosine (3’dA). It is a nucleoside analogue of adenosine that lacks the 3’-hydroxyl group on the ribose .

Cordycepin is one of the major bioactive substances produced by eps ris, a parasitic fungus used for traditional Chinese medicine because of its immune activator, anti-aging and anti-tumour effects. Reference is made to Tuli, H. S. el al 3 Biotech (2014) 4: 1-12.

Cordycepin can be ed synthetically from adenosine. Reference for such synthetic procedures is made to Robins, J. R. el al J. Org. Chem. 1995, 60, 7902-7908 and Aman, S. er a] Organic Process Research & Development 2000, 4, 601-605.

Cordycepin has been d most ively as an anti-cancer agent.

Because of its structure, 3’dA and its triphosphate form could potentially interfere with any process respectively requiring adenosine or adenosine triphosphate (ATP).

After administration, 3’dA is, however, quickly deaminated by adenosine deaminase (ADA), and rapidly metabolized to an inactive metabolite, 3’-deoxyinosine, in vivo. Reference is made to Tsai, Y-J el al J. Agri. Food Chem. 58 4638-43 (2010).

As described in Glazer, R. el al Cancer Research 38, 2233-2238 (1978), cordycepin has been shown to exhibit anti-cancer potency when used in combination with an inhibitor of ine deaminase, as pentostatine (2-deoxycoformicin, dCF). Other ADA tors have also been proposed as alternative co-drugs to be administered with cordycepin, but it is the combination of 2015/053628 3’dA-dCF which has been employed in clinical trials. As acknowledged in Wehbe-Janek, H. et a] Anticancer Research 27: 3143-3146 (2007), 2-deoxycoformicin is, however, known to be a relatively toxic drug. 2-Fluorocordycepin (3’deoxyﬁuoroadenosine) is also known to be cytotoxic (see e.g.

Montgomery et al., J. Med. Chem., 1969, 12(3), 4 and Dickinson et al, J. Med. Chem., 1967, 10(6), 1165-1166). 2-Chlorocordycepin (3’deoxyﬁuoroadenosine) has been assessed for it’s antiviral activity (Rosowsky et al. J. Med. Chem., 1989, 32, 1135-40).

The present invention has as its object a solution to the problem of enhancing the potency of a purine-based 3’-deoxynucleoside, as exempliﬁed by cordycepin oxyadenosine), in a method of prophylaxis or treatment, particularly, although not exclusively, in ancer chemotherapy, including chemotherapy to treat leukaemia, lymphoma and/or solid s.

A further object of the present invention is to e a solution to the m of purine-based xynucleosides, as exempliﬁed by epin (3’-deoxyadenosine), on administration being ated by ADA and then rapidly metabolized to an inactive metabolite.

A further object of the present invention is to provide a solution to the problem of purine-based 3’-deoxynucleosides, as exempliﬁed by cordycepin (3’-deoxyadenosine), on administration being deaminated by ADA and then rapidly metabolized to an inactive metabolite, so as to obviate entirely, or to reduce to at least some extent, the need to co-administer an ADA inhibitor when a purine-based 3’-deoxynucleoside is employed in a method of prophylaxis or treatment, particularly, although not exclusively, in anti-cancer chemotherapy, including chemotherapy to treat leukaemia, lymphoma and/or solid tumours.

According to a ﬁrst aspect of the present invention there is provided a compound which is a compound of formula (Ia): 1/"\ 3/ ’ L\\ W2 (Ia) wherein: W1 and W2 are each independently selected from the group consisting of -P(=O)(U)(V) and H, with the proviso that at least one of W1 and W2 is -P(=O)(U)(V), where U and V, independently for each of W1 and W2, are selected from the group consisting of: (a) U is -OAr in combination with V is -NR4-CR1R2-C(=O)OR3, where Ar is selected from the group consisting of C6-3oaryl and 5-3oheteroaryl, each of which is optionally substituted; each of R1 and R2 is independently selected from H, and the group consisting of C1-20alkyl, C6-3oarle1-6alkyl, C2-20alkenyl, C1-20alkoxy, C1-20alkoxyC1-2oalkyl, C1- 2oalkoxyC6.3oaryl, C2-2oalkynyl, C3-2ocycloalkle6-3oaryl, C6-3oaryloxy and 5- 2oheterocyclyl, any of which is optionally tuted, R3 is selected from H, and the group consisting of C1-2oalkyl, C6-30arle1.2oalkyl, C2-20alkenyl, C1-20alkoxyC1-20alkyl, C1-20alkoxyC6-30aryl, lkynyl, C3- 20cycloalkle6-30aryl, and terocyclyl, any of which is optionally substituted, R4 is selected from H, and the group ting of C1-2oalkyl, C6-30arle1-2oalkyl, C2-20alkenyl, C1-20alkoxy, C1-20alkoxyC1-20alkyl, C1-20alkoxyC6-3oaryl, C2- 2oalkynyl, C3.2ocycloalkle6.3oaryl, ryloxy and 5-2oheterocyclyl, any of which is optionally substituted, (b) each ofU and V is ed independently from -NR5R6, where R5 is selected from the group consisting of H and C1-6alkyl and R6 is - CR7R8C02R9, where R7 and Rs are selected independently from the group consisting of the side chains, ing H, of naturally occurring alpha amino acids and R9 is selected from H, and the group ting of lkyl, C6. 3oarle1.20alkyl-, C2-20alkenyl, C1-20alkoxyC1-20alkyl, C1-20alkoxyC6-3oaryl, C2- 20alkynyl, C3.2ocycloalkle6.30aryl, and 5-2oheterocyclyl, any of which is optionally substituted, or R5 and R6 together with the N atom to which they are attached form a ring moiety comprising 5 to 8 ring atoms, Q is selected from the group consisting of O, S and 1, where R10 and R11 are independently selected from H, F and C1.6alkyl, each of X and Z is independently selected from the group consisting of H, OH, F, Cl, Br, 1, C1- 6alkyl, -NR12R13 where each of R12 and R13 is independently selected from H and C1-6alkyl, and - SR14 where R14 is selected from the group consisting ofH and kyl, and Y is selected from the group ting of H, OH, F, Cl, Br, I, -OC1.6alkyl, C1-6alkyl, C2.galkynyl, -NR15R16 where each of R15 and R16 is independently selected from H and C1-6alkyl, and -SR17 where R17 is selected from the group consisting ofH and C1-6alkyl, or a pharmaceutically acceptable salt, ester, salt of an ester, solvate or prodrug of the compound of formula (Ia).

Compounds of the present invention are purine-based xynucleosides in which each of the 3’ substituent positions on the sugar moiety of the nucleoside is occupied by H.

In a further embodiment, the compound of the invention may be a compound of formula (Ib): N \ 2% N N Y ‘W2 (1b) W1 and W2 are each independently selected from the group consisting of -P(=O)(U)(V) and H, with the proviso that at least one of W1 and W2 is -P(=O)(U)(V), where U and V, independently for each of W1 and W2, are selected from the group consisting of: (a) U is -OAr and V is R1R2-C(=O)OR3, where Ar is selected from the group consisting of C6-3oaryl and 5-3oheteroaryl, each of which is optionally tuted; each of R1 and R2 is independently selected from H, and the group consisting of C1-20alkyl, C6-3oarle1-6alkyl, C2-20alkenyl, C1-20alkoxy, C1-20alkoxyC1-2oalkyl, C1- 2oalkoxyC6.3oaryl, lkynyl, C3-2ocycloalkyl, C6-3oaryl, C6-3oaryloxy and 5- 2oheterocyclyl, any of which is optionally substituted, R3 is selected from H, and the group consisting of C1-2oalkyl, C6-30arle1.2oalkyl, C2-20alkenyl, C1-20alkoxyC1-20alkyl, C1-20alkoxyC6-30aryl, C2.2oalkynyl, C3- 2ocycloalkyl, C6-30aryl, and 5-2oheterocyclyl, any of which is optionally substituted, R4 is selected from H, and the group consisting of C1-2oalkyl, C6-30arle1-2oalkyl, C2-20alkenyl, C1-20alkoxy, lkoxyC1-20alkyl, C1-20alkoxyC6-3oaryl, C2- goalkynyl, C3-20cycloalkyl, C6-30aryl, C6-3oaryloxy and 5-2oheterocyclyl, any of which is optionally substituted; (b) each ofU and V is selected independently from -NR5R6, where R5 is selected from the group consisting of H and C1-6alkyl and R6 is - 02R9, where R7 and Rs are selected ndently from the group consisting of the side chains, including H, of naturally occurring alpha amino acids and R9 is selected from H, and the group consisting of C1-2oalkyl, C6. 3oarle1.20alkyl-, C2-20alkenyl, C1-20alkoxyC1-20alkyl, lkoxyC6-3oaryl, C2- 20alkynyl, C3-2ocycloalkyl, C6-3oaryl, and 5-2oheterocyclyl, any of which is optionally substituted, or R5 and R6 together with the N atom to which they are attached form a ring moiety comprising 5 to 8 ring atoms, X is selected from NRIZR13 where each of R12 and R13 is independently selected from H and C1- , and -SR14 where R14 is selected from the group consisting ofH and C1-6alkyl, Z is independently selected from the group ting of H, OH, F, Cl, Br, I, C1-6alkyl, -NR12R13, and -SR14 where R14 is selected from the group consisting ofH and C1-6alkyl, and Y is selected from the group consisting of H, OH, F, Cl, Br, I, -OC1.6alkyl, C1-6alkyl, C2.galkynyl, -NR15R16 where each of R15 and R16 is independently selected from H and C1-6alkyl, and -SR17 where R17 is selected from the group consisting ofH and C1-6alkyl, or a ceutically acceptable salt, ester, salt of an ester, solvate or g of the compound of formula (lb).

The compound of formula (Ib) may be a compound of formula (II): OAr N N Y N O R3O P R2 O H OH (II) wherein Ar, Y, Z, R2 and R3 are as described above for formula (Ib) and wherein X is –NR12R13.

The compound of formula (Ib) may be a compound of formula (III): OAr N N Y N O R3O P R2 O H OH (III) wherein Ar, R2 and R3 are as described above for a (Ib) and wherein Y is selected from H, F, Cl and OMe.

The following ents apply to compounds of any of formulae (Ia), (Ib), (II) and ( III).

These statements are independent and interchangeable. In other words, any of the es described in any one of the following statements may (where chemically ble) be combined with the features described in one or more other statements below. In particular, where a compound is exemplified or illustrated in this specification, any two or more of the statements below which describe a feature of that compound, expressed at any level of generality, may be combined so as to represent subject matter which is plated as forming part of the disclosure of this invention in this specification.

In a particular aspect, the present ion provides a compound of formula 3’- Deoxyadenosine-5’-O-[phenyl(benzyloxy-L-alaninyl)] phosphate [FOLLOWED BY PAGE 7a] or a pharmaceutically acceptable salt, ester, salt of an ester, or solvate of the compound thereof.

In the present ication, the term "naturally occurring alpha amino acid" means an amino acid, which can have L or D stereochemistry, selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serine, threonine, lysine, ne, ine, aspartic acid, glutamic acid, asparagine, glutamine, cysteine and methionine.

[FOLLOWED BY PAGE 8] In the present speciﬁcation, a side chain of a naturally ing alpha amino acids is thus a member selected from the group ting of H, CH3, 3)2, -CH2CH(CH3)2, - CH(CH3)(CH2CH3), -CH2Ph, -OH, -CH2SH, -CH2CH2SCH3, -CH2OH, — (CH3)(OH), -CH2CH2CH2CH2NH3+, -CH2CH2CH2NHC(=NH2+)NH2, -CH2C(O)O-, -CH2CH2C(O)O- , -CH2C(O)NH2, 2C(O)NH2, H>C CH2 + >=\ N\/NH NH It may be that W1 is -P(=O)(U)(V) and W2 is H and the compound of the invention is a 5’- phosphoramidate of the parent 3’-deoxynucleoside. In certain preferred embodiments, W1 is - P(=O)(U)(V), wherein U is -OAr and V is R1R2-C(=O)OR3, and W2 is H.

It may be that W1 is H and W2 is -P(=O)(U)(V) and the compound of the invention is a 2’- phosphoramidate of the parent 3’-deoxynucleoside. In certain preferred embodiments, W1 is H and W2 is -P(=O)(U)(V), wherein U is -OAr and V is -NR4-CR1R2-C(=O)OR3.

It may be that each of W1 and W2 is -P(=O)(U)(V) and the compound of the invention is a 2’,5’- phosphoramidate of the parent xynucleoside. In certain preferred embodiments, where each of W1 and W2 is -P(=O)(U)(V), U is -OAr and V is -NR4-CR1R2-C(=O)OR3. In certain preferred embodiments, W1 is the same as W2.

Ar may be unsubstituted. Ar may be substituted. Where Ar is tuted, it can be substituted with one, two, three, four or ﬁve substituents. The substitutents may be selected from: halo, C1- C4-alkyl, C1-C4-alkoxy, nitro and cyano.

Ar, whether substituted or unsubstituted, may be selected from the group consisting of phenyl, pyridyl, naphthyl and quinolyl. In certain preferred embodiments, Ar is selected from the group consisting of phenyl and naphthyl. In further preferred embodiments, where Ar is naphthyl, binding to -O-P is at the l-position on naphthyl. In further red embodiments, Ar is unsubstituted phenyl or unsubstituted naphthyl, with binding to -O-P at the l-position on naphthyl.

R1 and R2 may be selected such that the moiety -CR1R2COO- corresponds to the corresponding part of a naturally occurring alpha amino acid.

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

It may be that R1 is H. It may be that R2 is C1-C4 alkyl. It may be that R2 is methyl. It may be that the C atom bearing R1 and R2 has the same absolute conﬁguration as L-alanine..

It may be that each of R1 and R2 is Me. It may be that each of R1 and R2 is H.

It may be that have R3 is ed from the group consisting of C6-30arle1.6alkyl and unsubstituted lkyl. In n preferred embodiments, R3 is selected from the group consisting of benzyl (-CH2-Ph), unsubstituted methyl (-CH3) and unsubstituted n-pentyl (-n- C5H11). In further preferred ments, R3 is benzyl.

R4 may be H.

U and V may be selected independently from -NR5R6. Preferably, each of U and V is the same.

In further preferred embodiments, R8 is H and R7 is selected from the group comprising H, methyl, i-propyl, -CH2Ph, -CH2CH(CH3)2 and -CH(CH3)(CH2H5). In further preferred embodiments, R7 is . In r preferred embodiments, the stereochemistry of the C atom g R7 and Rs has the same absolute conﬁguration as L-alanine. Alternatively, the stereochemistry of the C atom bearing R7 and R8 can have the same absolute conﬁguration as D- alanine. In certain preferred embodiments, R9 is selected from the group consisting of branched and unbranched C1-C13 acyclic alkyl, C3-C1s cyclic alkyl and C6-30arle1-6alkyl, any of which is optionally substituted. In certain preferred embodiments, R9 is benzyl.

In certain embodiments, a nd of the present invention comprises U and V, wherein each of U and V is selected, independently, from -NR5R6 wherein R5 and R6 together with the N atom to which they are attached form a ring moiety comprising 5 to 8 ring atoms. U and V may be the same.

Q may be 0.

It may be that W1 is -P(=O)(U)(V), where U is -O-l-naphthyl and V is -NH-(L)CH(CH3)-C(=O)- O-CHz-Ph, W2 is H and Q is 0.

It may be that each of X and Z ndently selected from the group consisting of H, OH, F, Cl, I\H2, SH and -SC1-6alkyl and Y selected from the group consisting of H, OH, F, Cl, -OC1.6alkyl, I\H2, C2.galkynyl, SH and -SC1.6alkyl. It may be that X is NRIZRB, e.g. NHz. In certain preferred embodiments, Z is H. In further preferred embodiments, X is NHz and Z is H. In preferred embodiments, X is NHz, Y is H and Z is H, X is NHz, Y is F and Z is H, X is NHz, Y as Cl and Z is H, or X is NHz, Y is -OCH3 and Z as H. In certain preferred embodiments, X is NHz, Y is H and Z is H and so e compounds of the invention which are derivatives of cordycepin (3’dA).

In certain particularly preferred embodiments, Ar is phenyl, R3 is benzyl and R2 is methyl.

Compounds of the present invention wherein, when P is tric, the compound can consist of the reoisomer Rp, the reoisomer Sp or a mixture of the diastereoisomers Rp and Sp.

Preferred compounds of the ion include: (ZS-Benzyl 2-(((((ZS,4R,5R)(6-amino-9H—purinyl)hydroxytetrahydrofuran yl)methoxy)(naphthalen- l )phosphoryl)amino)propanoate, Benzyl 2-(((((ZS,4R,5R)(6-amino-9H—purinyl)hydroxytetrahydrofuran yl)methoxy)(phenoxy)phosphoryl)amino)acetate, (25)—Pentyl 2-(((((ZS,4R,5R)(6-amino-9H—purinyl)hydroxytetrahydrofuran yl)methoxy)(naphthalen- l -yloxy)phosphoryl)amino)methylpentanoate, Methyl 2-(((((ZS,4R,5R)(6-amino-9H—purinyl)hydroxytetrahydrofuran yl)methoxy)(naphthalen- l -yloxy)phosphoryl)amino)methylpropanoate, 2015/053628 (25)—Benzyl 2-(((((ZS;4R;5R)(6-amino-9H—puriny1)hydroxytetrahydrofuran-Z- yl)methoxy)(2-(3 -ethoxy-3 -oxopropyl)phenoxy)phosphory1)amino)propanoate; (25)—Benzyl 2-(((((2R;3R;55)(6-amino-9H—puriny1)(hydroxymethyl)tetrahydrofuran-3 - yl)oxy)(phenoxy)phosphory1)amino)propanoate; Benzyl 2-(((((ZS;4R;5R)(6-amino-9H—puriny1)—4-((((1-(benzyloxy)oxopropan yl)amino)(phenoxy)phosphoryl)oxy)tetrahydrofuran hoxy)(phenoxy)phosphory1)amino)propanoate; enzyl 2-(((((2R;3R;55)(6-amino-9H—puriny1)(hydroxymethyl)tetrahydrofuran-3 - yl)oxy)(naphthaleny1oxy)phophory1)amino)proponate; Benzyl 2-[({ [5 -(6-amino-9H-puriny1)hydroxyoxolany1]methoxy}({ [1 -(benzyloxy) oxopropan-Z-yl]amino})phosphory1)amino]propanoate; (25)—Benzyl 2-((((ZS;4R; 5R)—5-(6-aminomethoxy-9H—puriny1)hydroxytetrahydrofuran-Z- yl)methoxy)(naphtha1eny1oxy)phosphory1amino)propanoate; (25)—Benzyl 2-((((ZS;4R; 5R)—5-(6-aminomethoxy-9H—puriny1)hydroxytetrahydrofuran-Z- yl)methoxy)(phenoxy)phosphory1amino)propanoate; (25)—Benzyl (ZS;4R; 5R)(6-aminoﬂuoro-9H—puriny1)hydroxytetrahydrofuran-Z- yl)methoxy)(phenoxy)phosphory1)amino)propanoate; (25)—Hexyl 2-(((((ZS;4R;5R)—5-(6-aminoﬂuoro-9H—puriny1)hydroxytetrahydrofuran-Z- hoxy)(phenoxy)phosphory1)amino)propanoate; (2R)-Benzy1 2-((((ZS;4R;5R)(6-aminochloro-9H-puriny1)hydroxytetrahydrofuran-Z- yl)methoxy)(naphtha1en- 1 )phosphory1amino)propanoate; 3 ’ -Deoxyadenosine-5 ’ -O-[pheny1(benzyloxy-L-alaniny1)] phosphate; 2-O-Methy1-3 ’ -deoxyadenosine-5 ’ -O-[1 -naphthy1(1 -pentyloxy-L-1euciny1)] phosphate; 2-O-Methy1-3 ’ -deoxyadenosine-5 ’ -O-[pheny1(1 -hexyloxy-L-a1aniny1)] phosphate; 2-Fluoro-3 ’ -deoxyadenosine-5 ’ -O-[1-naphthy1(benzyloxy-L-alaniny1)] phosphate; 2-F1uoro-3 ’ -deoxyadenosine-5 ’ -O-[ 1 hy1(1-pentyloxy-L-1euciny1)] phosphate; 2-Chloro-3’deoxyadenosine 5’-O-[1-pheny1 (2,2-dimethy1propoxy-L-a1anine)] phosphate; 2-Chloro-3’deoxyadenosine 5’-O-[1-naphty1 (2,2-dimethy1propoxy-L-a1anine)] phosphate; 2-Chloro-3’deoxyadenosine 5’-O-[1-pheny1 (ethoxy-L-alanine)] phosphate; and (ZS-isopropy1(((((ZS;4R;5R)—5-(6-amino-9H—puriny1)hydroxytetrahydrofuran-Z- yl)methoxy)(phenoxy)phosphoryl)amino)propanoate WO 83830 2015/053628 and pharrnaceutically acceptable salts, esters, salts of an ester, solvates or prodrugs thereof.

In certain embodiments, the nd of the invention is not: (25)—isopropyl(((((ZS,4R,5R)—5-(6-amino-9H—purinyl)hydroxytetrahydrofuran yl)methoxy)(phenoxy)phosphoryl)amino)propanoate.

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. The compound may be for use in the prophylaxis or treatment of cancer.

According to a third aspect of the present ion there is provided use of a compound of the present invention in the manufacture of a medicament for the prophylaxis or treatment of particularly, although not exclusively, of cancer.

According to a fourth aspect of the t invention, there is provided a method of prophylaxis or ent of particularly, although not ively, of cancer comprising administration to a patient in need of such treatment an ive dose of a compound of the present invention.

With respect to each of the second, third and fourth aspects of the present invention, ments of the invention comprise a cancer ed from among haematological and solid tumours. In particular, the cancer can be selected from the group consisting of leukaemia, multiple myeloma, liver cancer, breast cancer, head and neck cancer, neuroblastoma, thyroid carcinoma, skin cancer (including melanoma), oral squamous cell carcinoma, urinary bladder , Leydig cell tumour, colon cancer, colorectal cancer, lung cancer (non-small cell and small cell), biliary cancer, pancreatic , sarcoma, prostate cancer, cancer of the central nervous system, s sarcoma, Cholangiocarcinoma and gynaecological cancers, including ovarian cancer, uterine cancer and cervical cancer, including epithelia cervix carcinoma. In preferred embodiments, the cancer is leukaemia or ma, e.g. a cancer selected from the group consisting of acute lymphoblastic leukaemia, acute myelogenous leukaemia, acute promyelocytic leukaemia, acute lymphocytic leukaemia, chronic myelogenous leukaemia, chronic lymphocytic leukaemia, monoblastic leukaemia, hairy cell leukaemia, Hodgkin lymphoma and non-Hodgkin lymphoma. In further preferred embodiments, the cancer is acute lymphoblastic leukaemia.

WO 83830 Each of the second, third and fourth aspects of the invention can se embodiments for treating cancer employed in combination with other cancer therapy. Examples of other cancer therapy e radiotherapy and/or other chemotherapy. Without being bound by theory or mechanism, it has been reported (e. g. Robertson, J. B. el al Int. J. Radiat. Biol. Relat. Stud. Phys.

Chem. Med. 1978 34(5): 417-29, Hiraoka, W. el al . Res. (1988) 114(2):231-9 and Hiraoka, W. el al J. Radiat. Res. (Tokyo) (1990) 31(2): 156-61) that 3’-deoxyadenosine inhibits the repair of X-ray induced DNA damage. In certainpreferred embodiments of each of the , third and fourth aspects of the present invention the compounds of the invention are for use in, or are used in, a method of treatment of cancer comprising administration to a patient in need of such treatment a compound of the present invention in conjunction with herapy.

With respect to each of the , third and fourth aspects of the present invention, further embodiments of the invention comprise compounds of the invention for use in, or are used in, a method of prophylaxis or treatment of myelodysplastic syndrome.

Without being bound by theory or mechanism: Tuli el al (supra) reported that cordycepin, as well as having anti-tumour ty and apoptotic activity, also shows anti-oxidant, anti- inﬂammatory, anti-malarial, anti-fungal, immunomodulatory, anti-diabetic/hyopglycemic, steroidogenesis and anti-aging activities, Vodnala, S. K. el al J. Med. Chem. 2013, 56, 9861- 9873 reported that each of cordycepin and 2-ﬂuorocordycepin shows arasitic activity, Ahn, Y. J. el al J. Agric. Food Chem. 2000 48 (7) 2744-8 reported that cordycepin shows anti- bacterial activity and de Julian-Ortiz J. V. el al J. Med. Chem. 1999 42(17) 3308-14 reported that cordycepin shows anti-viral activity, Sugar et al, Antimicrob. . Chemother. 1998 42(6) 1424-7, showed that cordycepin has ngal activity. With respect to each of the second, third and fourth aspects of the present invention, embodiments of the invention se compounds of the invention for use in, or are used in, a method of prophylaxis or treatment of a patient with a disease or condition in need of at least one treatment selected from the group consisting of anti- oxidant, anti-inﬂammatory, anti-malarial, anti-fungal, immunomodulatory, anti- diabetic/hypoglycemic, steroidogenesis, anti-aging, anti-parasitic, anti-bacterial and anti-viral With respect to each of the second, third and fourth aspects of the invention, embodiments of the invention comprise compounds of the present invention for use in, or are used in, a method of prophylaxis or treatment wherein the method does not employ the administration of a co-drug which is an inhibitor of adenosine deaminase. Unlike the parent compound cordycepin, which typically needs to be co-administered with an ADA inhibitor, to be effective it may be that the compounds of the invent do not require such co-administration.

An ADA inhibitor can, however, be employed as a co-drug, if desired, with respect to each of the second, third and fourth aspects of the invention. A suitable ADA inhibitor for co- administration with a compound embodying the present invention is hydroxyurea or tatin.

According to a further aspect of the present invention, there is provided a pharmaceutical ition comprising a compound of the present ion in ation with a pharmaceutically acceptable carrier, diluent or ent.

According to a further 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 carrier, t or excipient.

According to further aspect of the present invention, there is provided a method of ing a nd of formula (Ia): by reacting a compound of formula IV: with: (a) a compound of formula V: ‘9' R1 $4 ‘9' N—FI’—CI R2 ('3 (b) POC13 followed by a salt of N+R5R6H2, where W1, W2, Q, X, Y, Z, Ar, R1, R2, R3, R4, R5 and R6 have the meanings set out herein with respect to formula (Ia).

Compounds embodying the present invention have surprisingly been found to have enhanced pharmaceutical actiVity, particularly enhanced anti-cancer actiVity, compared to their parent purine-based 3’-deoxynucleoside (i.e. n W1 and W2 are H), especially when ed in the treatment of leukaemia, lymphoma and/or solid tumours.

The enhancement in actiVity has been found in the absence of administration of a co-drug to inhibit adenosine deaminase, compared to the parent purine-based nucleoside administered in the e of a co-drug to inhibit ADA.

The present invention thus unexpectedly provides a means to employ a derivative of 3’- deoxyadenosine, or a derivative of an analogue of 3’-deoxyadenosine, as a pharmaceutical agent, particularly as an anti-cancer agent, that mitigates the problem of deamination by adenosine ase, whilst avoiding completely, if desired, the use of a co-drug which is an inhibitor of adenosine deaminase, ing the relatively toxic 2-deoxycoformycin.

Without being bound by any theory, the y, particularly the anticancer efﬁcacy, exhibited by compounds of the present invention demonstrates that the 3’-deoxynucleoside compounds of the present ion are phosphorylated intracellularly to 3’-deoxyadenosine sphate, or to the triphosphate of a 3’-deoxyadenosine analogue. Where the compounds of the present invention have W1 as -P-(=O)(U)(V), it is believed that enzymic ge of U and V within the cell ts the compounds directly into 3’-deoxyadenosine monophosphate, or the monophosphate of the 3’-deoxyadenosine analogue, prior to phosphorylation to the triphosphate.

None of the above intracellular ty of compounds of the present invention could have been predicted beforehand.

The above beneﬁts are in addition to ed cellular membrane permeability of the phosphoramidate nucleosides of the present invention, compared to the 3’-deoxyadenosine parent or the 3’-deoxyadenosine analogue parent, where enhanced cell membrane permeability is attributable to the phosphoramidate structure of the present compounds. The beneﬁt of enhanced cellular ne permeability cannot, moreover, be assumed to be present a priori for the phosphoramidate of any nucleoside. The nds of the present invention are, it is believed, the ﬁrst example of a phosphoramidate of a xynucleoside to show an enhanced anti-cancer potency, relative to their parent xynucleoside. The beneﬁt of enhanced cellular membrane permeability by compounds of the present invention is thus surprising.

Preferred embodiments of the compounds of the present invention have, in ation, the features set out above with respect to embodiments of the compounds of the invention.

Each of Ar, R1, R2, R3 and R4 can be substituted with one, two, three, four or ﬁve substituents.

Substituents on Ar can be located ortho-, meta-, para- or otherwise on the aromatic groups.

Substituents on Ar are independently selected from the group ting of hydroxy, C1-6acyl, C1- 6acyloxy, nitro, amino, yl, C2.6ester, C1.6aldehyde, cyano, C1-6alkylamino, diC1. 6alkylamino, thiol, chloro, bromo, ﬂuoro, iodo, C1-6alkyl, C2.6alkenyl, C1.6alkoxyC1.6alkyl, C1. salkoxyC6.1oaryl, C5-7cycloalkyl, C5.11cycloalkle1-6alkyl, C5-7cycloalkenyl, Cs.12cycloalkynyl, C6.11arle1.6alkyl, C1-6alkle6.11aryl, C6.11aryl, oroalkyl, C2-6ﬂuoroalkenyl, SO3H, SH and SR’, wherein R’ is independently selected from the same group set out above as R1 with respect to formula Ia. Each substituent can be substituted by any other substituent.

Substituents on R1, R2, R3 and R4 are independently selected from the group consisting of y, C1.6acyl, C1-6acyloxy, nitro, amino, amido, carboxy, C2.6ester, C1.6aldehyde, cyano, C1- salkylamino, diC1.6alkylamino, thiol, chloro, bromo, ﬂuoro, iodo, C5-7cycloalkyl, C5-7 cycloalkenyl, Cs.12cycloalkynyl, C6-11aryl, C6-11arle1-6alkyl, 5-2oheterocyclyl, SO3H, SH and SR’, wherein R’ independently selected from the same group set out above as R1 with respect to a Ib.

In n preferred embodiments, R1 and R2 are independently ed from the group consisting of H, C1-1oalkyl, C6.3oarle1-6alkyl, C2.10alkenyl, C2-1oalkoxyC1.1oalkyl, C1.1oalkoxyC6- 1oaryl, C2-1oalkynyl, C3-2ocycloalkyl, C3.2ocycloalkenyl, Cs.2ocycloalkynyl, and 5-1oheterocyclyl.

In certain embodiments, R1 and/or R2 correspond to a side chain, including H, of a naturally occurring alpha amino acid, which can have L or D stereochemistry. Thus, it may be that R1 and/or R2 (e.g. a single one or R1 and R2)are selected from the group consisting of H, CH3, - CH(CH3)2, -CH2CH(CH3)2, -CH(CH3)(CH2CH3), , -CH2Ph-OH, , -CH2CH2SCH3, - CH2OH, CH(CH3)(OH), -CH2CH2CH2CH2NH3+, -CH2CH2CH2NHC(=NH2+)NH2, -CH2C(O)O- , -CH2CH2C(O)O-, -CH2C(O)NH2, -CH2CH2C(O)NH2, \ CH2 HN\/NH NH In certain preferred embodiments, R1 and R2 are ndently selected from the group consisting of H, -CH3 and -CH2CH(CH3)2. In further preferred embodiments, R1 and R2 together correspond to the side chains of L e.

R3 may be selected from the group consisting of H, C1-2oalkyl, C6-3oarle1-6alkyl, C2-10alkenyl, C1-10alkoxyC1.1oalkyl, C1.10alkoxyC6-10aryl, C2.1oalkynyl, C3-2ocycloalkyl, C3-2ocycloalkenyl, Cg- zocycloalkynyl, and 5-2oheterocyclyl.

R3 may be selected from the group consisting of H, C1-2oalkyl, C6-3oarle1-6alkyl and C3- oalkyl. R3 may be selected from the group consisting of C6-30arle1-6alkyl and unsubstituted C1-2oalkyl. In certain preferred embodiments, R3 is selected from the group consisting of benzyl (-CH2Ph), unsubstituted methyl (-CH3) and tituted n-pentyl (-n- C5H11). R3 may be benzyl.

R4 may be ed from the group consisting of H, C1-2oalkyl, C6-3oarle1-6alkyl, C2-10alkenyl, C1-10alkoxy, C1-10alkoxyC1-10alkyl, C1-10alkoxyC6-10aryl, C2.10alkynyl, C3-2ocycloalkyl, C3- 20cycloalkenyl, Cs-2ocycloalkynyl, and 5-2oheterocyclyl.

R4 may be selected from the group consisting of H, C1-1salkyl, C6-3oarle1-6alkyl, C3-2ocycloalkyl and 5-2oheterocyclyl. R4 may be selected from the group consisting of H, , ethyl, , butyl, pentyl, hexyl and cyclohexyl. R4 may be H.

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

The invention provides the use of a nd 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 2015/053628 be in a cancer or pre-cancerous condition in a patient in need of such ing, 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 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 ce of cancer stem cells; n the presence of cancer stem cells in the biological sample indicates that the t will beneﬁt from ent 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 ent 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 ncerous 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 s set out above may further comprise a step of preventing or treating the cancer or pre-cancerous condition using a nd of the invention.

WO 83830 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 tory cancer in a t. The subject may be a human patient. The subject may be a domestic animal, e.g. mammal.

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 subject. The subject may be a domestic animal, e.g. mammal.

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 inventionto a subject in need of such treatment. The t may be a domestic animal, e.g. mammal.

The invention provides a compound of the invention for use in the treatment of cancer, wherein a compound of the invention is for use at dose of between approximately 25 mg/m2 and 4000 mg/m2 per week in at least one initial cycle of treatment, and then for use at a lower weekly dose in at least one further cycle of treatment. The cancer may be a ed or refractory cancer.

Various aspects of the invention are based upon the ﬁnding that a compound of the invention is able to reduce cancer stem cell numbers, and may reduce these preferentially as compared to other cell types. 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 cordycepin or 2-ﬂuorocordycepin, the parent g 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 ors have ed is applicable across a broad range of cancers, represents a sing breakthrough that s a range of new therapeutic ations of a compound of the invention.

The biological ties exerted by the compounds of the invention, which have not previously been ed, 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 ated 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 nd of the invention may achieve a high, e 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 ise, 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 nd 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 ty of a nd of the invention yields ts in contexts in which new and effective therapies have long been sought.

Brief description of the ﬁgures Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which: Figure 1. Comparison of the LDso values for Cordycepin, Compounds A, 2-F-Cordycepin, Compounds 0, P, Q and R. All assays were carried out using KGla cells and data are presented as mean (:SD) of ﬁve independent experiments.

Figure 2. Analysis of the leukaemic stem cell (LSC) targeting capacity of Cordycepin and nd A. The previously generated data (ii) is shown for comparison. All data are the mean (:SD) of three independent experiments.

Figure 3. Analysis of the LSC targeting capacity of 2-F-Cordycepin and Compounds 0, P, Q and. All data are the mean (:SD) of three ndent experiments.

Figure 4. Comparison of LSC targeting capacity of 2-F-Cordycepin and each proTide. All data are the mean (:SD) of three independent experiments.

Detailed description As used herein, the term " refers to a ht or branched saturated monovalent (except where the context requires otherwise) cyclic or c hydrocarbon radical, having the number of carbon atoms as indicated (or where not indicated, an acyclic alkyl group can have 1-20, 1-18, 1-10, 1-6 or 1-4 carbon atoms and a cyclic alkyl group can have 3-20, 3-10 or 3-7 carbon atoms), optionally substituted with one, two or three substituents independently selected from the group set out above with respect to substituents that may be present on R1, R2, R3 and R4. By way of non-limiting examples, alkyl groups can include methyl, ethyl, propyl, butyl, pentyl, hexyl, octyl, nonyl and dodecyl.

As used , the term yl" refers to a straight or ed 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 ted, an acyclic alkenyl group can have 2-20, 2-10, 2-6 or 2-4 carbon atoms and a cyclic alkenyl group can have 3-20 or 5-7 carbon atoms), optionally substituted with one, two or three substituents independently selected from the group set out above with respect to tuents that may be present on R1, R2, R3 and R4. By way of non-limiting examples, alkenyl groups can include vinyl, propenyl, butenyl, pentenyl and hexenyl.

As used , the term "alkynyl" refers to a straight or branched unsaturated monovalent (except where the context requires otherwise) acyclic or cyclic arbon radical having one or more CEC triple bonds and having the number of carbon atoms as indicated (or where not indicated, an c alkynyl group can have 2-20, 2-10, 2-6 or 2-4 carbon atoms and a cyclic alkynyl group can have 8-20 carbon atoms), optionally substituted with one, two or three substituents independently selected from the group set out above with respect to substituents that may be present on R1, R2, R3 and R4.

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 or three substituents as set out above for alkyl. Binding is through -O-. 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 aryl-O-, where aryl is as d below and where the aryl moiety may optionally be substituted by one, two or three substituents as set out above with respect to the group Ar. Binding is through -O-.

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 es may each be substituted by one, two or three substituents as set out above with regard to the definition of alkyl.

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 d herein with respect to the definitions of aryl and alkyl, respectively. The aryl and alkyl moieties may each be substituted by one, two or three substituents, the substituents being as deﬁned herein with respect to the tions of those substituents that may be present with respect to aryl and alkyl, respectively. In a preferred ment, arylalkyl is benzyl, which is Ph-CH2-.

As used herein, the term "alkoxyaryl" refers to an aryl group having an alkoxy substituent. g 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 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.

As used herein, the term "cycloalkylaryl" refers to an aryl group having a cyclic alkyl tutent. 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, tively. The cycloalkyl moiety and the aryl moiety may each be optionally tuted by one, two or three substituents as set out herein with regard to the deﬁnitions of alkyl and aryl, respectively.

As used herein, the term "aryl" refers to a monovalent (except where the context requires ise) aromatic 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, 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 ning 7, 8, 9 or 10 carbon atoms, or an aromatic fused tricyclic ring system containing 10, ll, 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 hydroxy, C1.6acyl, C1-6acyloxy, nitro, amino, carboxyl, cyano, C1- salkylamino, alkylamino, thiol, chloro, bromo, ﬁuoro, iodo, SO3H, SH and SR’, wherein R’ is independently selected from the same groups as R1 with respect to formula la.

As used herein, the term 4 ‘5-3oheteroaryl" refers to a monovalent (except where the context requires otherwise) unsaturated ic heterocyclic radical having 5 to 30 ring members in the form of one, two, three, four, ﬁve or six fused rings and contained within at least one ring at least one heteroatom selected from the group consisting of N, O and S. A red embodiment has one, two or three fused rings. ble carbon atoms and/or heteroatoms in the ring system may be tuted on the ring with one, two, three, four or ﬁve substituents, as set out above with respect to the sub nts that may be present on the group Ar. Heteroaryl groups can include an aromatic monocyclic ring system ning six 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; an aromatic monocyclic ring having six members of which one, two or three ring members are a N atom, 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, or an aromatic 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, pyridyl and quinolyl.

As used herein, the term (4 5-2oheterocyclyl" refers to a monovalent (except where the context requires otherwise) saturated or partially unsaturated heterocyclic l having 5 to 20 ring members, with at least one ring member selected from the group consisting of N, O and S, and being in the form of one, two, three, four, five or six fused rings. In a preferred embodiment, the radical has one, two or three rings. In a preferred ment, the radical has 5 to 10 ring s. 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 0 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 es 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, pyrrolinyl, pyrrolidinyl, l,3-dioxolanyl, imidazolinyl, olidinyl, pyrazolinyl, pyrazolidinyl, piperidinyl, morpholinyl or piperazinyl.

Available ring carbon atoms and/or ring heteroatoms of the ocyclyl" ring systems described above may be substituted with one, two, three, four or ﬁve substituents. Where the ring(s) is tuted with one or more heteroatoms, the heteroatom substituents are selected from halogen (F, Cl, Br and I) and from oxygen, nitrogen and sulphur, where the oxygen, nitrogen or r form part of a substituent moiety. Where the ring(s) is tuted with one or more heteroatoms, ably there are l, 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 ndently selected from hydroxy, C1- 6acyl, C1-6acyloxy, nitro, amino, carboxyl, cyano, C1-6alkylamino, diC1-6alkylamino, thiol, chloro, bromo, ﬂuoro, iodo, SO3H, SH and SR’, n R’ is independently selected from the same groups as R1 with t to formula Ia.

As used herein, the term "acyl" refers to a straight or branched, saturated or unsaturated, substituted or unsubstituted, monovalent (except where the context es otherwise) radical that includes the moiety —, where binding is through the -C- atom of -C(=O)- moiety, and has the number of carbon atoms indicated (or where not ted, an acyl group has 1-6, or 1-4 or 1-2 carbon atoms, including the C atom of the -C(=O)— moiety), optionally substituted with one, two or three substituents independently selected from the group set out above with respect to the substituents that may be present on R1, R2, R3 and R4. By way of non-limiting examples, acyl groups include HC(=O)—, CH3C(=O)-, C2H5C(=O)-, C3H7C(=O)-, C4H9C(=O)- and C5H11C(=O)-.

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 es the moiety -C(=O)-O-, where g is through the -O- atom, and has the number of carbon atoms indicated, including the C atom of the —C(=O)—O- moiety (or where not indicated, an acyloxy group has 1-6, 1-4 or 1-2 carbon atoms, ing the carbon atom of the - C(=O)—O)— moiety), optionally substituted with one, two or three of the substituents that may be present on R1, R2, R3 and R4. By way of non-limiting examples, acyloxy groups include HC(=O)—O-, CH3C(=O)-O-, C2H5C(=O)-O-, =O)-O-, C4H9C(=O)-O- and C5H11C(=O)=O-.

As used herein, the term ster" refers to a substituted or unsubstituted monovalent (except where the context requires otherwise) l that comprises R18C(:O)-O-Rl9, where R18 is selected from the group consisting H and C1-4alkyl and R19 is selected from the group consisting of C1-5alkyl, subject to the maximum total number of C atoms, including the C atom of the - C(=O)-O- moiety, of R1gC(=O)-O-R19 being six. Binding is through R18 or R19, with an H of the respective group absent such that the alkyl group through which binding occurs is divalent, or, when R18 is H, through the C of the -C(=O)-O- moiety. In a preferred embodiment, the C2-6ester, including the C atom of the -C(=O)-O moiety, has 2-5 carbon atoms. The C2-6ester can ally be substituted with one, two or three substituents independently selected from the group set out above with respect to the substituents that may be t on R1, R2, R3 and R4. By way of a non- limiting example, C2-6ester can be -C2H4-C(=O)-O-C2H5, where the -C2H4- moiety is -CH2-CH2- and g is through the -C2H4- 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(=O)—R20-, where binding is through —R20-, has the number of carbon atoms ted, including the C atom of the -C(=O)— 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(=O)— moiety), optionally substituted with one, two or three of the substituents that may present on R1, R2, R3 or R4. By way of non-limiting examples, de groups include HC(=O)—CH2-, HC(=O)—C2H4-, HC(=O)—C3H6-, -C4Hg- and HC(=O)—C5H1o-.

As used herein, the term "ﬂuoroalkyl" refers to an alkyl group, where the alkyl group is a straight or branched ted 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 l to 6 F atoms.

As used herein, the term "ﬂuoroalkenyl" refers to an alkenyl group, where the alkenyl group is a straight or branched rated monovalent (except where the t es 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 l to 6 F atomS.

The process for preparing a nd of formula Ia or Ib is preferably carried out in the presence of a suitable solvent.

Suitable solvents include hydrocarbon solvents such as e and toluene, ether type solvents such as diethyl ether, tetrahydrofuran, diphenyl ether, anisole and dimethoxybenzene, halogenated hydrocarbon solvents such as methylene de, chloroform and chlorobenzene, ketone type solvents such as acetone, methyl ethyl ketone and methyl isobutyl ketone, alcohol type solvents such as methanol, ethanol, propanol; isopropanol; n-butyl alcohol and tert-butyl alcohol; nitrile type solvents such as itrile; propionitrile and benzonitrile; ester type solvents such as ethyl acetate and butyl acetate; carbonate type solvents such as ethylene carbonate and propylene ate; and the like. These may be used singly or two or more of them may be used in ure.

Preferably an inert t 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 ing; 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; e; 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 ed from an aryloxy phosphorodichloridate and a suitably protected amino acid derivative. Alternatively; phosphate chemistry may be used with suitable sing .

Preferably the process for preparing the compound of formula Ib can include the step of protecting free OH groups; on the nucleoside other than that to which the oramidate is to be attached. For example; carrying out the reaction of the 3’-deoxynucleoside with the desired phosphorochloridate in the ce of t-BuMgCl allows the 2’-phosphoramidate to be prepared.

Reacting the 3’-deoxynucleoside with POC13 followed by a salt of N+R5R6H2 allows compounds to be prepared where each of U and V is -NR5R6. Suitable salts include chloride; tosylate; sulphonate and ester salts such as ylbenzene sulphonate. Subsequent addition of a base such as diisopropylethyl amine can aid the process.

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 t invention may possess.

Where the compounds according to this invention have at least one chiral , they may accordingly exist as enantiomers. Where the compounds possess two or more chiral centres, they may additionally exist as reoisomers. 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 nds may be prepared in stereochemically mixed form or dual enantiomers may be prepared by standard techniques known to those skilled in the art, for example, by enantiospecif1c synthesis or tion, formation of diastereoisomeric pairs by salt formation with an lly active acid, followed by fractional crystallization and regeneration of the free base. The compounds may also be resolved by formation of reoisomeric esters or amides, followed by chromatographic separation and removal of the chiral auxiliary. Alternatively, the compounds may be resolved using a chiral HPLC column. It is to be understood that all such isomers and es thereof are encompassed within the scope of the present ion.

Furthermore, it should be appreciated that 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 red embodiment, the compound is a ntially pure single diastereoisomer of either Rp or Sp. By "substantially pure single reoisomer" is meant that the compound consists of 98% 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 e 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 :1, 1:15 to 15:1, 1:10to 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 opr to Sp diastereoisomers ofgreater 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 term "solvate" means a compound of formula Ia or formula Ib 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.

WO 83830 The compounds of the t invention may also be present in the form of ceutical acceptable salts. For use in medicine, the salts of the compounds of this invention refer to aceutically acceptable salts." FDA ed pharmaceutical able 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, ochloride, e, edisylate, estolate, esylate, fumarate, glyceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, , isethionate, lactate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, pamoate, pantothenate, phosphate, diphospate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, teoclate, tosylate and triethiodide.

Pharmaceutically acceptable basic/cationic salts include, and are not limited to aluminum, benzathine, calcium, chloroprocaine, e, 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 onal 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 nd specif1cally 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. ceutically acceptable ester derivatives in which one or more free hydroxy groups are esterif1ed in the form of a ceutically acceptable ester are particular examples of g esters that may be convertible by solvolysis under physiological conditions to the compounds of the present invention having free hydroxy groups.

Pharmaceutical compositions for use in accordance with the present invention may be formulated in a tional manner using one or more physiologically acceptable rs comprising ents and auxiliaries which tate 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 ation is dependent upon the route of administration chosen.

The compound or pharmaceutical composition ing to the present invention can be administered to a patient, which may be homo sapiens or animal, by any suitable means.

The medicaments ed in the present invention can be administered by oral or parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), , l 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 on or suspension. s 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 vatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and e, 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 es wherein the active ingredient is mixed with water or an oil such as peanut oil, liquid parafﬁn or olive oil.

WO 83830 Formulations for rectal administration may be presented as a itory 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 uscular, intraperitoneal, subcutaneous and intravenous use, the nds of the invention will generally be ed in e aqueous solutions or suspensions, buffered to an appropriate pH and icity. Suitable aqueous vehicles include Ringer’s solution and isotonic sodium chloride. Aqueous suspensions according to the invention may e 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 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 300 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 m2 of body surface area of recipient per day or 5 to 50 mg per m2 of body surface area of recipient per day. Suitable doses may be 6, 12, 24 or 48 mg per m2 of body e 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 riate intervals throughout the day. Doses may be administered in unit dosage forms, for example, ning 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, r taken as a single dose or as sub-doses at intervals throughout the day.

"Cancer stem cells" Cancer stem cells, which are sometimes ise 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 h 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. ingly, the ﬁnding that compounds of the invention are able to target cancer stem cells, and y 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 pre- ous ions, 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 nd 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 yplastic syndrome, or other conditions considered elsewhere in the speciﬁcation), and thus to prevent ssion of such ncerous 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 bed in more details elsewhere in the present speciﬁcation.

Cancer stem cells are also able to act as a reservoir of cancer cells that they may cause the recurrence of cancer after remission. Even in the event that the majority of a patient’s cancer cells have been d (for e by y, radiotherapy, or herapy, either alone or in ation), so that no observable signs of a cancer remain, the continued presence of cancer stem cells may te 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. ingly, 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 n characteristic cell surface markers. Cancer stem cells identiﬁed in haematological malignancies are lly 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 ion, 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 CD133+ CD133+ ESAhigh/CD44+ /Lineage' /(CD166+) Head and Neck CD44+/Lineage- Melanoma ABCBS+ 2015/053628 Liver CD90+/CD45-/(CD44+) Cholangiocarinoma CD44+/GL11+ (Glioma-associated oncogene homolog—1) Ovarian CD44+/CD1 17+ Pancreas CD 13 3+ Non-small-cell lung cancer CD44+/Ber-EP4+ Bladder cancer CD44+/ALDH1A1+ Haematological tumours Acute myeloid leukaemia Lin'/CD34+/CD38-/CD123+ B-Acute lymphoblastic leukaemia CD34+/CD 10— or CD34+/CD 19— B-Acute lymphoblastic leukaemia CD38—/CD19+ Multiple myeloma CD34—/CD138— T-Acute lymphoblastic leukaemia CD34+/CD4' or CD34+/CD7' 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, cally cancer stem cells present in the acute d mia cell line KGla. This cell line manifests a minor stem cell-like compartment with a distinct immunophenotype (Lin'/CD34+/CD38'/CD123+) which is targeted by a compound of the invention. ingly, methods of ent or medical uses of a compound of the invention in ance with the present ion 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 t’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 ere in this speciﬁcation.

"Targeting ofcancer 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 rated in the Examples disclosed in this speciﬁcation.

It can be seen that when a compound of the invention is ed 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. As discussed ere in the present speciﬁcation, certain compounds of the invention preferentially target cancer stem cells as d to bulk tumour cells, and the activity of such compounds is able not only to reduce the total number of cancer cells present, but also to reduce the tion of total cancer cells that exhibit phenotypic markers of cancer stem cells.

It is believed that the compounds of the present invention enter into cancer cells and are incorporated into nucleic acids (RNA and/or DNA) with the cells. Without being bound by any theory, it is believed that the efﬁcacy, particularly the anti-cancer efﬁcacy, exhibited by compounds of the present invention trates that compounds of the t invention are phosphorylated to the triphosphate of cordycepin or a cordycepin derivate (e.g. 2- ordycepin or 2-Cl-cordycepin) and it is believed that tic 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 cordycepin), 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 e 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. Thus compounds of the invention are able to cause the death of cancer stem cells. Furthermore, the results set out elsewhere in this cation illustrate that certain nds of the invention appear to kill cancer stem cells preferentially as compared to killing of non-stem cancer cells, thereby causing not only the death of cancer stem cells, but also a reduction of the proportion of cancer stem cells among the total cancer cell population.

While the ors e that compounds of the invention that preferentially target cancer stem cells preferentially kill 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 ing of these cells.

Merely by way of example, treatment with a compound of the invention may cause an increase in cancer stem cell differentiation, y reducing cancer stem cell numbers and also the proportion of total cancer cells represented by cancer stem cells. Alternatively, a compound of the ion 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 reted accordingly. For the purposes of the t sure, "targeting" of cancer stem cells may be taken as encompassing any mechanism by which a compound of the invention s the number of cancer stem cells t in a population of cells, whether in vitro or in vivo. In particular targeting of cancer stem cells may be taken as encompassing preferential reduction of cancer stem cell numbers as compared to other cell types, particularly as compared to non-stem cancer cells. References to targeting in this specification may be taken as including the killing, and optionally preferential killing, of cancer stem cells as compared to non-stem cancer cells.

"Prevention or treatment ofcancer" The invention provides medical uses and methods of treatment in which a compound of the invention is used for the prevention or treatment of . 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) guidelines.

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

"Prevention ofcancer" tion 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 ular, prevention of , 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. s of treatment or medical uses in accordance with this embodiment may prevent development of the treated pre-cancerous condition into cancer, y 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 t the recurrence of cancer, and/or prevent the propagation of cancer.

"Pre-cancerous conditions " Cancer is frequently preceded by the development of a ncerous 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. ingly, cancer stem cells may also be t 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 ions 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 pre-cancerous 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, t’s oesophagus, atrophic gastritis, dyskeratosis congenital, Sideropenic dysphagia, Lichen planus, oral submucous ﬁbrosis, solar elastosis, cervical dysplasia, lakia, erythroplakia, monoclonal gammopathy of n signiﬁcance (MGUS), monoclonal B-cell lymphocytosis (lVﬂ3L), myelodysplastic syndromes, as well as ncerous conditions of the stomach such as atrophic gastritis, c ulcer, pernicious anaemia, gastric stumps, gastric polyps, and Ménétrier's disease. Among the listed pre-cancerous conditions of the stomach, atrophic gastritis, ious 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 sing dysplastic or hyperplastic cells. Accordingly, the presence of sia or hyperplasia, as an alternative or addition to the presence of cells with expressed markers or phenotypes teristic of cancer stem cells, may be used in the ﬁcation of pre-cancerous ions.

The severity of dysplasia can vary between ent pre-cancerous conditions, or with the development of a single pre-cancerous ion over time. Generally, the more advanced dysplasia associated with a pre-cancerous condition is, the more likely it is that the ncerous 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 sia. 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 sia may be diagnosed following a tissue biopsy.

It has recently been ed 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 lignant condition that is associated with increased overall mortality and increased risk of cardiometabolic disease. The ty of mutations detected in blood cells occurred in three genes: , TET2, and ASXLl.

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 tive of a ncerous condition in at least one of: DNMT3A and/or TET2 and/or ASXLl.

Pre-cancerous conditions that may beneﬁt from ent 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, sed ere in the speciﬁcation.

"Treatment ofcancer" 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 t or treat a metastatic or secondary cancer; and/or to treat, prevent or inhibit metastasis or recurrence; and/or to treat or t refractory cancer.

The ability of cancer treatment using a compound of the invention to bring about a reduction in tumour size; and also to in the reduction in tumour size during/after the period in which the treatment is administered represents a particularly relevant indication of ive cancer treatment. As set out in the Examples; the treatments or l uses of the ion have proven surprisingly effective in this respect; even in models using cells entative 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 ties or cell surface markers by which cancer stem cells may be identified are described elsewhere in the cation. In a suitable embodiment; treatment of cancer in accordance with the present ion 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 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 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 cancer stem cells remain).

Asymmetric division of cancer stem cells contributes to the growth of tumours. Treatment of cancer with a nd 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 ion 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 d cancer.

Tumour growth may be assessed by any le 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 silu within a patient. Suitable techniques, such as g techniques, may allow the volume of a tumour to be determined, and changes in tumour volume to be assessed.

As shown in the results set out in the Examples of this cation, the methods of treatment and medical uses of a nd of the invention of the invention are able not only to arrest tumour growth, but are actually able to bring about a reduction in tumour volume in patients with cancers, including patients with relapsed or refractory s. Suitably treatment of cancer in accordance with the present invention may give rise to a reduction in tumour volume of at least %, 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 tumour volume 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 volume of 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 tumour volume of at least 96%, at least 97%, at least 98%, at least 99%, or even 100%.

A reduction in tumour volume of the sort described above can be calculated with reference to a suitable control. For e in studies carried out in vilro, or in vivo in suitable animal models, the reduction in tumour volume may be determined by direct comparison between the volume of a tumour treated with a compound of the invention and the volume of a control tumour (which may be untreated, or may have received treatment other than with a nd of the invention).

It will be appreciated that such models requiring lack of treatment of a tumour may not be ethically acceptable in the t of clinical trials or therapeutic management of patients, and in this case a reduction in tumour volume may be assessed by comparing the volume of a treated tumour with the volume of the same tumour prior to treatment, or with a predicted volume that would have been 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: in the case of gynaecological cancers CA125 represents a suitable example of a biomarker, while in the case of pancreatic or biliary cancers CA19.9 represents a suitable example of a biomarker, and in the case of colorectal s CEA may be a le biomarker. ly treatment of cancer in accordance with the present invention may give rise to a reduction in cancer kers of at least 10%, at least 20%, at least 30%, or at least 40%. In le embodiments, treatment of cancer in accordance with the invention may give rise to a reduction in cancer biomarkers 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 cancer biomarkers of 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 cancer biomarkers of at least 96%, at least 97%, at least 98%, at least 99%, or even 100%.

Beneﬁcial effects, such as a reduction in the tion of cancer stem cells present, reduction in tumour , or reduction in tumour volume or cancer kers, 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 s 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 ng cancer or a pre-malignant condition, by ing cancer stem cells. In a le 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 WO 83830 tumourigenicity of one or more cancer stem cells. Suitably such methods may prevent the progression of , or inhibit tumour growth.

When a nd 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 , with I being an isolated cancer and IV being a cancer that has spread to the limit of what the assessment es.

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 y 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. ent 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 ing 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 ion is used to prevent progression from Stage I to Stage II. In another embodiment, treatment with a compound of the invention is used to t progression from Stage II to Stage III. In still another embodiment, treatment with a compound of the ion is used to prevent ssion 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 asises 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. s of treatment or medical uses of the invention employing a compound of the invention can therefore be used to prevent the spread of , by targeting tumourigenic cancer stem cells and thus reducing their numbers.

"Cancers " Compounds of the invention demonstrate increased anti-cancer activity as compared to parent nucleosides from which they are derived. This increase 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 ty of a wide range of cancers. Accordingly, there are a wide range of cancers that may be ted or treated in accordance with the present ion.

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 tion or treatment of such cancers.

Suitably a compound of the invention may be used in the prevention or ent of a cancer selected from the group consisting of: leukaemia, lymphoma, multiple myeloma, lung cancer, liver cancer, breast , 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 giocarcinoma or bile duct cancer, pancreatic cancer, colon cancer, colorectal cancer and gynaecological cancers, including ovarian , trial cancer, fallopian tube , uterine cancer and cervical cancer, including epithelia cervix carcinoma. In suitable embodiments, the cancer is leukaemia and can be ed from the group consisting of acute lymphoblastic leukaemia, acute myelogenous leukaemia (also known as acute myeloid leukaemia or acute non-lymphocytic leukaemia), acute promyelocytic mia, acute lymphocytic leukaemia, chronic enous leukaemia (also known as chronic myeloid leukaemia, chronic myelocytic leukaemia or chronic granulocytic leukaemia), chronic WO 83830 lymphocytic mia, monoblastic leukaemia and hairy cell leukaemia. In further preferred embodiments, the cancer is acute lymphoblastic leukaemia. In a particular embodiment, the leukaemia is refractory TdT-Positive Leukemia In a le 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 cytic lymphoma.

Suitably targeting cancer stem cells in such cancers may e ive ent of the cancer by preventing or treating the development of the cancer, by preventing or ng the progression of the cancer, by preventing or ng 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 ing cancer stem cells in the prevention or treatment of metastatic cancer.

In a le ment 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 y cancer, or (iii) metastasis of a .

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 ed or refractory cancer in connection with the 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 es of the present invention, refractory cancers may be taken as s that demonstrate resistance to treatment by anti-cancer therapies other than those utilising a compound of the invention. For example, a compound of the ion may be used in the treatment of tory 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 ical agents used in the ent of cancer. In a suitable embodiment a compound of the invention may be used in the treatment of refractory s 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 l uses of the invention employing a nd of the invention include those cancers that are resistant to cordycepin or 2-ﬂuorocordycepin.

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 , 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 nd of the ion to target cancer stem cells may be used to 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 ed, 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 ion 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 , 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 ion.

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 y of these compounds to prevent or treat cancers in accordance with the medical uses or methods of treatment of the ion. 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 activity of cancer stem cells may underlie much of the resistance that makes relapsed or refractory cancers so difﬁcult to treat, non-stem cancer cells are also a major constituent of such relapsed or refractory cancers.

Compounds of the invention exert r xic effects on non-stem cancer cells than does cordycepin or 2-ﬂuorocordycepin, the chemotherapeutic molecule from which the compounds of the invention are d. Accordingly, the mechanism by which a compound of the invention acts in the ent of relapsed or refractory cancer may not be limited solely to the anti-cancer 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 em cancer cells. When certain compounds of the invention are utilised such treatments will preferentially reduce the proportion of cancer stem cells that remain after treatment.

Therapeutically eﬂective doses ofa compound ofthe invention A eutically 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 ent 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 eutically effective nd, such as a compound of the invention, to be administered to a patient may be calculated and expressed. One such way which is ered particularly relevant in doses of agents for the prevention or ent 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 (m2) of surface area.

Uses of a compound of the invention for the prevention or treatment of cancer may utilise a weekly dose of between 10 mg/m2 and 1000 mg/mz. Such treatments may, for example utilise a weekly dose of between 375 mg/m2 and 900 mg/mz. For example, effective ent 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/m2 and 825 mg/mz.

Without wishing to be bound by any esis, the inventors believe that the ability of a compound of the invention to target cancer stem cells allows therapeutic effectiveness to be achieve using lower doses of this compound than would ise be expected. Merely by way of example, weekly doses of a compound of the invention that are as low as 825 mg/m2, 750 mg/m2, 600 mg/mz, or 500 mg/m2 may prove therapeutically effective in the uses and methods of the ion.

A chosen weekly dose of a compound of the invention may be provided in a single incidence of administration, or in le incidences of administration during a week. For e, 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/mz, this may be achieved by three administrations of 250 mg/m2 over the course of a week, or two strations of 375 mg/m2 during a week Similarly, in the case of a weekly dose of 600 mg/mz, this may be achieved by three administrations of 200 mg/m2 over the course of a week, or two administrations of 300 mg/m2 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/m2 and 300 mg/mz.

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/mz, 900 mg/mz, 825 mg/mz, 750 mg/m2, or 725 mg/m2, and over the course of treatment the dose needed may decrease to around 750 mg/m2 (in cases where the initial dose is above this amount), around 650 mg/mz, around 625 mg/mz, or even around 500 mg/m2 or around 375 mg/mz.

Doses of a nd 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/m2 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 le embodiment a nd of the invention es effective treatment of relapsed or refractory s when patients are provided with weekly doses ranging between approximately 12.5 mg/kg and 20.5 mg/kg.

WO 83830 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 le time frame, for example in a ten minute injection, or the like.

Types oftreatment In a suitable ment 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 ion is able to e ive treatment of cancer in contexts in which other treatments have proved ineffective. Accordingly, in a suitable ment the present invention es a compound of the invention for targeting cancer stem cells as a second line treatment of . , 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 neooadjuvant 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 al 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 herapy 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 nt 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 ion in a monotherapy, which is to say in preventions or treatments in which a compound of the invention provides ntially 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 butes 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 y a compound of the invention and a further therapeutic agent may be provided to a t at the same time. In a le 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 ntially the same time.

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 nd of the ion 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. er therapeutic agents " A compound of the invention may be used in combination with a wide range of further therapeutic agents for the prevention or ent 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, r such r agent targets cancer stem cells, non-stem cancer cells, or other cells or constituents involved in the development, maintenance, recurrence, propagation or 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 umour antibiotic, (e) a topoisomerase, (f) a c 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. ion ofpatients The inventors’ ﬁnding that a nd of the invention is able to target cancer stem cells makes le a number of methods by which it is possible to determine whether a particular t is likely to beneﬁt from receiving a compound of the invention in the prevention or treatment of , such as relapsed or tory cancer.

WO 83830 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 ion 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 entative 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 n 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 ical sample representative of cancer or a pre-cancerous condition in the patient for the presence of cancer stem cells, wherein the ce of cancer stem cells in the ical sample indicates that the patient is suitable for treatment with a compound of the invention.

In suitable ments 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. ly 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 ly assessed for their expression of cancer stem cell markers, such as those set out above.

Alternatively or additionally, a le biological sample may se target molecules representative of gene expression by cells of the cancer or pre-cancerous condition. Examples of such target molecules include proteins encoded by the genes sed, 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 sed markers are frequently used in the context of clinical assessments (such as for diagnostic or prognostic es) 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 s in on. es of such samples containing n 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 asis) may be assessed using techniques such as ﬂow cytometry.

In samples containing nucleic acids representative of expression of cancer stem cell s, such expression may be assessed by suitable molecule biology techniques, such as by polymerase chain reaction (PCR) amplification using suitable s.

Example 1 — Synthetic Procedures Compounds of the invention can be made according to or analogously to the following General ures and Exemplary Synthetic Procedures.

General procedure 1 (For compounds A-F and L-U) N—methylimidazole (l .0 mmol) and a solution of the appropriate phosphorochloridate (0.6 mmol) in anhydrous THF (2 mL) were added dropwisely to a sion of 3’-deoxyadenosine (0.20 mmol), or of the substituted 3’-deoxyadenosine, in anhydrous THF (10 mL) and the reaction mixture was stirred at room temperature during a period of 16 hours. Purif1cation by column chromatography and preparative TLC afforded the desired compound as a white solid. Amounts of components employed may vary and actual amounts are given in the examples below. l Procedure 2 (For compound J 1 3’-Deoxyadenosine (0.80 mmol) was suspended in (CH30)3PO (5 mL), and POC13 (0.80 mmol) was added dropwise at -5 oC. The reaction mixture was allowed to reach room temperature and left stirring for 4 hours. A solution of the appropriate amino acid ester salt (4.0 mmol) dissolved in anhydrous CHzClz (5 mL) was added followed by diisopropyl ethyl amine (8.0 mmol) at -78 0C. After stirring at room ature for 20 hours, water was added and the layers were separated. The aqueous phase was extracted with romethane and the organic phase washed with brine. The combined organic layers were dried over Na2804 and concentrated. The residue was purified by column chromatography (gradient elution of CHzClz/MeOH=100/0 to 93/7) to give the desired product as a white foam. Amounts of components employed may vary and actual amounts are given in the examples below.

General ure 3 (For compounds G—I) 3’-Deoxyadenosine (0.20 mmol) was ded in anhydrous THF (5 mL) and tBuMgCl (1.0 M solution in THF, 0.22 mmol) was added dropwisely at room temperature. A solution of the appropriate phosphorochloridate (0.6 mmol) in anhydrous THF (2 mL) was added dropwisely and the reaction mixture was stirred at room temperature during a period of 16 hours.

Purif1cation by column chromatography and preparative TLC afforded the desired compound as a white solid. The amounts of the components employed may vary and actual amounts are given in the examples below. l Procedure 4 (For compound V1 TerlButyldimethylsilyl chloride (3.3 .) and imidazole 6.6 (mol/eq) were added tp a solution of the appropriate 3’-deoxyadenosine te (l mol/eq) in anhydrous DMF and the reaction mixture was stirred at room temperature overnight (16-20 h). Then NH4Cl was added to the mixture and washed twice with ethylacetate. Organic layers were combined, dried on Na2804 and solvent was removed under vacuum. Purification of the mixture by column chromatography ed ediate Cl. Intermediate Cl was then dissolved at in an aqueous solution THF/HzO/TFA 4/l/l (6 ml/eq) and was stirring at 0 °C for 4 h. The solution was then carefully neutralized with an s saturated solution of NaHC03 and the mixture was washed twice with ethylacetate. Organic layers were combined, dried on Na2804 and solvent was d under vaccuo. Purification of mixture by column chromatography afforded intermediate C2.

Then l ure B was applied, and intermediate C3 was afforded. Intermediate C3 was dissolved in an aqueous solution of THF/HzO/TFA 1/1/1 (6 ml/eq) at 0 °C and was stirred at RT for 24 h. ation by chromatography afforded the d compounds as white solids.

General Procedure 5 for re arin 3’-deox adenosine and x chloroadenosine employed in the es): A solution of HzO/CH3CN 1:9 and then d-AIBBr (4.0 mol/eq) were added sequentially to a suspension of dried adenosine or 2-chloroadenosine in anhydrous CH3CN and stirring was continued at room temperature (20 °C). After 1 h, a saturated solution of NaHCOg was added cautiously and the solution was extracted with EtOAc. The combined organic phase was washed with brine. The aqueous phase was extracted with EtOAc and the combined organic phase was dried over Na2804, ﬁltered and evaporated to give a white gum. The crude mixture was dissolved in anhydrous MeOH and stirred for 1 h with ite (2 x OH') resin previously washed well with anhydrous MeOH. The solution was then ﬁltered and the resin carefully washed with anhydrous methanol. Evaporation of the combined ﬁltrate afforded 2’,3’- dehydroadenosine or 2’,3’-dehydrochloroadenosine as a white solid.

A on of LiEthH (1M solution in THF 4-4.3 mol/eq) was added dropwise to a cold (4 °C) on of 2’,3’-dehydroadenosine or 2’,3’-dehydrochloroadenosine (1 mol/eq) in ous DMSO/THF (1/ 10) under an argon atmosphere. Stirring was continued at 4 °C for 1 h and at room temperature overnight (16 h). The reaction mixture was carefully acidiﬁed (5% AcOH/HzO), purged with N2 for 1 h (under the fume hood) to remove pyrophoric triethylborane, and evaporated. The residue was chromatographed to give xyadenosine or xy chloroadenosine as a white powder.

Using General Procedure 5: 2’,3’-dehydroadenosine was prepared from 10.0 g (37.4 mmol) of adenosine, 7.5 mL of HzO/CH3CN (1/9), 22 mL (149.7 mmol) of d-AIBBr in 500 mL of anhydrous CH3CN, and 300 mL of Amberlite (2 x OH') resin in 400 mL of dry methanol. 2’,3’- dehydroadenosine was obtained as a white solid (9.12 g, 98%). 3’-Deoxyadenosine was prepared from the 9.12 g (36.6 mmol) of 2’,3’-dehydroadenosine and 159 mL (159 mmol) of LiEthH/THF 1M, in anhydrous DMSO/THF (1/10, 50 mL). Puriﬁcation by column chromatography on silica gel (eluent system 3-18% MeOH in DCM) gave 3’-deoxyadenosine as a white powder (7.12 g, 77%). 1H NMR (500 MHz, DMSO-d6) 5 8.37 (s, 1H, H8), 8.17 (s, 1H, H2), 7.29 (br s, 2H, NHz), 5.89 (d, J: 2.5 Hz, 1H, H1’), 5.68 (d, J: 4.5 Hz, 1H, OH-2’), 5.19 (t, J: 6.0 Hz, 1H, , 4.63 — 4.58 (m, 1H, H2’), 4.40 — 4.34 (m, 1H, H4’), 3.71 (ddd, J: 12.0, 6.0, 3.0 Hz, 1H, H5’), 3.53—3.49 (ddd, J: 12.0, 6.0, 4.0 Hz, 1H, H5’), 2.30—2.23 (m, 1H, H3’), 1.98-1.90 (m, 1H, H3’). 13C NMR (125 MHz, DMSO-d6) 5 156.00 (C6), 152.41 (C2), 148.82 (C4), 139.09 (C8), 119.06 (C5), 90.79 (Cl’), 80.66 (C4’), 74.56 (C2’), 62.61 (C5’), 34.02 (C3’).

Using General ure 5: 2’,3’-dehydrochloroadenosine was prepared from 5.0 g (16.6 mmol) of 2-chloroadenosine, 3.0 mL of HzO/CH3CN (1/9), 9.7 mL (66.2 mmol) of d-ATBBr in 38 mL of anhydrous CH3CN, and 150 mL of Amberlite (2 X OH') resin in 200 mL of anhydrous methanol. 2’,3’-dehydrochloroadenosine was obtained as a white solid (3.03 g, 60%). 3’- deoxychloroadenosine was prepared from 2.18 g (7.68 mmol) of 2’,3’-dehydro chloroadenosine and 30.7 mL (3 0.7 mmol) of LiEthH/THF 1M in anhydrous DMSO/THF (1/10 mL, 30 mL). Puriﬁcation by column chromatography on silica gel (eluent system 2-20% MeOH in DCM) gave 3’-deoxychloroadenosine as a white powder (1.20 g, 55%). 1H NMR (500 MHz, CD30D): 5H 8.41 (s, 1H, H8), 5.93 (d, J: 2.5 Hz, 1H, H1’), 4.68-4.66 (m, 1H, H2’), 4.56-4.52 (m, 1H, H4’), 3.95 (dd, J: 3, 12.5 Hz, 1H, H5’), 3.70 (dd, J: 3, 12.5 Hz, 1H, H5’), 2.39—2.33 (m, 1H, H3’), 2.08-2.03 (m, 1H, H3’) 13C NMR (125 MHz, CD30D): 6C 158.14 (C6), 155.19 (C2), 151.15 (C4), 141.30 (C8), 119.56 (C5), 93.58 (Cl’), 82.80 (C4’), 76.81 (C2’), 64.01 (C5’), 34.33 (C3’).

Preparation of 3 ’ -deoxyﬂuoroadenosine: A solution of HzO/CH3CN (1:9, 1.4 mL) and then d-ATBBr (4.10 mL, 28.05 mmol) were added sequentially to a suspension of dried 2-ﬂuoroadenosine (2.0 g, 7.01 mmol) in anhydrous CH3CN (50 mL) and stirring was ued at room temperature (20 °C). After 1 h, saturated solution of NaHCOg was added cautiously and the solution was extracted with EtOAc (2 X 100 mL). The ed organic phase was washed with brine (1 X 50 mL). The aqueous phase was extracted with EtOAc (2 X 50 mL) and the ed organic phase was dried over Nast4, ﬁltered and evaporated to give a white gum. The crude mixture was dissolved in a mixture of THF/HzO (4/ 1, 50 mL) and stirred for 1 h with 60 mL of ite (2 X OH') resin (previously washed well with THF). The on was then ﬁltered and the resin carefully washed with THF. ation of the combined ﬁltrate and crystallisation of the residue from EtOH gave 2’,3’-dehydro ﬁuoroadenosine as a white solid (1.13 g, 60%).

A solution of LiEthH/THF (1M, 18.01 mL, 18.01 mmol) was added dropwise to a cold (4 °C, ice bath) solution of 2’,3’-dehydroﬁuoroadenosine (1.13 g, 4.18 mmol) in anhydrous DMSO/THF (1/10, 15 mL) under an argon atmosphere. Stirring was continued at 4 °C for 1 h and at room ature overnight (16 h). The reaction mixture was carefully acidiﬁed (5% AcOH/HzO), purged with N2 for 1 h (under the fume hood) to remove oric triethylborane, and ated. The residue was chromatographed on silica gel (3-18% MeOH in DCM) to give 3’-deoxyﬁuoroadenosine as a white powder (7.12 g, 77%).

"P NMR (470 MHz, DMSO-d6): SF 6219. 1HNMR (500 MHz, DMSO-d6) 6H 8.34 (s, 1H, H8), 7.80 (br s, 2H, NHz), 5.78 (d, J: 2.25 Hz, 1H, H1’), 5.68 (br s, 1H, OH-2’), 5.01 (br s, 1H, OH-5’), 4.55—4.51 (m, 1H, H2’), 4.39—4.32 (m, 1H, H4’), 3.73-3.76 (m, 1H, H5’), 3.56-3.50 (m, 1H, H5’), 2.26-2.18 (m, 1H, H3’), 1.94-1.85 (m, 1H, H3’). 13C NMR (125 MHz, DMSO-d6) 6C 158.51 (d, 1JC.F = 202.7 Hz, C2), 157.55 (d, 3Jc.F = 21.2 Hz, C6), 150.11 (d, 3Jc.F = 20.3 Hz, C4), 139.22 (d, ﬁlm = 2.2 Hz, C8), 117.37 (d, 4Jc.F = 4.1 Hz, C5), 90.67 (C1’), 80.90 (C4’), 74.73 (C2’), 62.35 (C5’), 33.89 (C3’).

Preparation of 3 ’ -deoxymethoxyadenosine: A solution of HzO/CH3CN (1:9, 1.4 mL) and then d-AIBBr (4.10 mL, 28.05 mmol) were added sequentially to a suspension of dried 2-ﬁuoroadenosine (2.0 g, 7.01 mmol) in anhydrous CH3CN (50 mL) and stirring was continued at room temperature (20 °C). After 1 h, saturated solution of NaHCOg was added cautiously and the solution was extracted with EtOAc (2 x 100 mL). The combined organic phase was washed with brine (1 X 50 mL). The aqueous phase was extracted with EtOAc (2 x 50 mL) and the combined organic phase was dried over Na2SO4, ﬁltered and evaporated to give a white gum. The crude mixture was dissolved with anhydrous MeOH (50 mL) and stirred for 1 h with 60 mL of ite (2 x OH') resin (previously washed well with anhydrous MeOH). The solution was then ﬁltered and the resin carefully washed with THF.

Evaporation of the combined ﬁltrate and llisation of the residue from EtOH gave 2’,3’- dehydromethoxyadenosine as a white solid (1.57 g, 84%).

A solution of LiEthH (1M solution in THF, 8.53 mL, 8.53 mmol) was added dropwise to a cold (4 °C) solution of 2’,3’-dehydromethoxyadenosine (762 mg, 2.84 mmol) in anhydrous DMSO/THF (1/10, 15 mL) under an argon atmosphere. Stirring was continued at 4 °C for 1 h and at room temperature overnight (16 h). The reaction mixture was carefully acidiﬁed (5% AcOH/HzO), purged with N2 for 1 h (under the fume hood) to remove pyrophoric triethylborane, and evaporated. The e was chromatographed on silica gel (3-17% MeOH in DCM) to give 3’-deoxymethoxyadenosine as a white powder (650 mg, 81%). 1H NMR (500 MHz, CD30D) 6H 8.20 (s, 1H, H8), 5.90 (d, J: 2.4 Hz, 1H, H1’), 4.75—4.71 (m, 1H, H2’), 4.54-4.48 (m, 1H, H4’), 3.91 (dd, J: 12.3, 2.5 Hz, 1H, H5’), 3.69 (dd, J: 12.30, 4.0 Hz, 1H, H5’), 3.37 (s, 3H, OCH3), 2.43-2.35 (m, 1H, H3’), .02 (m, 1H, H3’). 13C NMR (125 MHz, CD30D) 6C 163.68 (C2), 158.12 (C6), 151.94 (C4), 139.71 (C8), 116.64 (C5), 93.36 (C1’), 82.53 (C4’), 76.59 (C2’), 64.24 (C5’), 55.29 (OCH3), 34.81 (C3’).

Phosphorochloridates were prepared by published s from aryl phosphorodichloridates and amino acid ester hydrochlorides. x ine-5’-O- hen lbenz 10x -L-alanin l hos hateA N \N o < I 4 II N N o—If—o NH 0 0% OH Compound A was prepared according to the General Procedure 1 using 3’-deoxyadenosine (50 mg, 0.20 mmol), N—methylimidazole (80 uL, 1.0 mmol) and phenyl(benzyloxy-L-alaninyl) phosphorochloridate (212 mg, 0.6 mmol). Puriﬁcation by column chromatography t system CH30H/CH2C12 0/100 to 7/93) with gradient of CHzClz/MeOH (100% to 95:5%) and preparative TLC (1000 um, eluent system CH30H/CH2C12 5/95) afforded the title compound as a white solid (31 mg, 28 %). 1H NMR (500 MHz, CD30D): 6H 8.26 (s, 0.5H, H8), 8.24 (s, 0.5H, H8), 8.22 (s, 0.5H, H2), 8.21 (s, 0.5H, H2), 7.34—7.25 (m, 7H, Ar), 7.21—7.13 (m, 3H, Ar), 6.01 (d, J: 2.9 Hz, 1H, H1’), 6.00 (d, J: 2.9 Hz, 1H, H1’), .04 (m, 2H, OCHgPh), .63 (m, 2H, H2’, H4’), 443— 4.35 (m, 1H, H5’), 4.27—4.20 (m, 1H, H5’), 4.03—3.91 (m, 1H, CHCH3), 2.35-2.28 (m, 1H, H3’), 2.09—2.02 (m, 1H, H3’), 1.32 (d, J = 7.4 Hz, 1.5 H, CHCHs), 1.28 (d, J = 7.4 Hz, 1.5 H, CHCHs). 13C NMR (125 MHz, CD30D): 8C 174.84 (d, 3Jc.p = 4.5 Hz, C=0), 174.63 (d, 3116.1) = 4.5 Hz, C=0), 157.32 (C6), 157.31 (C6), 153.86 (C2), 153.84 (C2), 152.13 (C4), 152.07 (C4), 150.20 (C-Ar), 150.18 (C-Ar), 140.47 (C8), 137.26 (C-Ar), 137.19 (C-Ar), 130.76 (CH-Ar), 130.74 (CH-Ar), 129.57 ), 129.32 (CH-Ar), 129.31 (CH-Ar), 129.29 (CH-Ar), 129.26 (CH-Ar), 126.16 ), 126.14 (CH-Ar), 121.46 (d, 3.16.1) = 4.7 Hz, CH-Ar), 121.38 (d, SJC-P = 4.7 Hz, CH-Ar) 120.54 (C5), 120.53 (C5), 93.24 (Cl’), 93.18 (Cl’), 80.43 (d, 3.16.1): 3.6 Hz, C4’), 80.36 (d, 3Jc.p= 3.6 Hz, C4’), 76.62 (C2’), 68.62 (d, 96.1): 5.3 Hz, C5’), 68.30 (d, 416.1): 5.3 Hz, C5’), 67.95 h), 67.92 (OCHzPh), 51.74 (CHCH3), 51.60 (CHCH3), 34.91 (C3’), 34.70 (C3’), .45 (d, 3Jc.p= 7.0 Hz, CHCH3), 20.28 (d, 3Jc.p= 7.0 Hz, CHCH3). 31P NMR (202 MHz, : 5P 3.9, 3.7.

MS (ES+) m/z.‘ Found: 569.2 (M + H), 591.2 (M + Na+), 1159.4 (2M + Na+) C26H29N607P required: (M) 568.2.

HPLC Reverse-phase HPLC eluting with H20/CH3CN from 100/10 to 0/100 in 30 minutes, 1ml/min, l = 254 nm, showed two peaks of the diastereoisomers with tR 14.02 min. and tR 14.26 min. 2- lmetho na hthalen-l- lox hos hor lamino r0 anoateB O N w MS (ES+) m/z: Found: 619.2 (M + H+), 641.2 (M + Na+), 1259.4 (2M + Na+) C30H31N607P required: (M) . 31P NMR (202 MHz, CH30D): 6P 4.3 (s), 4.1 (s). 1H NMR (500 MHz, CH30D): 6H 8.24 (s, 0.5H, H8), 8.22 (s, 0.5H, H8), 8.20 (s, 0.5H, H2), 8.19 (s, 0.5H, H2), 8.14-8.09 (m, 1H, Ar), 7.89-7.85 (m, 1H, Ar), 7.70-7.67 (m, 1H, Ar), 7.53- 7.42 (m, 3H, Ar), 7.39-7.34 (m, 1H, Ar), 7.31-7.25 (m, 5H, Ar), 5.99 (d, J: 2.0 Hz, 0.5H, H1’), .98 (d, J: 2.0 Hz, 0.5H, H1’), .01 (m, 2H, CHzPh), 4.72-4.61 (m, 2H, H2’, H4’), 4.47- 4.40 (m, 1H, H5’), 4.33-4.24 (m, 1H, H5’), 4.09-3.98 (m, 1H, CH a1a)2.35-2.26 (m, 1H, H3’), .98 (m, 1H, H3’), 1.30-1.24 (m, 3H, CH3). 13C NMR (125 MHz, CH30D): 6C 174.85 (d, 3JC-P = 3.7 Hz, C=O), 174.56 (d, 3Jc.p = 3.7 Hz, C=O), 157.33 (C6), 157.31 (C6), 153.87 (C2), 153.85 (C2), 150.24 (C4), 150.23 (C4), 147.91 (d, 3JC-P = 7.5 Hz, ‘ipso’ Nap), 147.95, (d, 3Jc-P = 7.5 Hz, ‘ipso’ Nap), 140.56 (C8), 140.50 (C8), 137.22 (C-Ar), 137.17 (C-Ar), 136.28 (C-Ar), 129.55 (CH-Ar), 129.53 (CH-Ar), 129.30 (CH- Ar), 129.25 (CH-Ar), 128.88 (CH-Ar), 128.82 (CH-Ar), 127.91 (d, ZJC-P = 6.25 Hz, C-Ar), 127.83 ((1, ch.P = 6.25 Hz, C-Ar), 127.77 (CH-Ar), 127.75 (CH-Ar), 127.49 (CH-Ar), 127.45 (CH-Ar), 126.48 (CH-Ar), 126.47 (CH-Ar), 126.02 (CH-Ar), 125.97 (CH-Ar), 122.77 (CH-Ar), 122.63 (CH-Ar), 120.58 (C5), 120.53 (C5), 116.35 (d, 3J01) = 3.75 Hz, CH-Ar), 116.15 (d, 3Jc-P = 3.75 Hz, CH-Ar), 93.22 (C1’), 93.20 (C1’), 80.30 (d, 3JC-P = 2.75 Hz, C4’), 80.24 (d, 3Jc-P = 2.75 Hz, C4’), 76.51 (C2’), 76.44 (C2’), 68.87 (d, 2Jc.P = 5.2 Hz, C5’), 68.64 (d, ZJC-P = 5.2 Hz, C5’), 67.93 (OCHzPh), 51.82 (CH ala), 51.73 (CH ala), 35.01 (C-3’), 34.76 (C3’), 20.41 (d, 3Jc-P = 6.7 Hz, CH3 ala), 20.22 (d, 3,161) = 6.7, CH3 ala).

HPLC Reverse-phase HPLC g with H20/CH3CN from 100/10 to 0/100 in 30 minutes, 1m1/min, 1 = 200 nm, showed two peaks of the diastereoisomers with tR 16.36 min. and tR 16.60 min.

Benz 12- 2S 4R 5R 6-amin0-9H- urin l h drox tetrah drofuran-Z- l methox henox hos hor 1 amino acetate C Using General Procedure 1 above, N—methylimidazole (80 uL, 1.0 mmol) and a solution of benzyl 2-((chloro(phenoxy)phosphoryl)amino)acetate (204 mg, 0.6 mmol) in anhydrous THF (2 mL) were added dropwisely to a suspension of 3’-deoxyadenosine (50 mg, 0.20 mmol) in anhydrous THF and the reaction e was stirred at room temperature for 16 hours. ation by column chromatography (eluent system CH30H/CH2C12 0/100 to 6/94) and preparative TLC (500 uM, eluent system CH3OH/CH2C12 5/95) afforded the d compound as a white solid (21 mg, 19 %).

(ES+) m/z, found: 555.2 (M + H+), 577.2 (M + Na+), 1131.4 (2M + Na+). C25H27N607P required: (M) 554.2. 31P NMR (202 MHz, CH30D) 5 5.1, 4.9. 1H NMR (500 MHz, CH30D) 6 8.27 (s, 0.5H, H8), 8.24 (s, 0.5H, H8), 8.22 (s, 0.5H, H2), 8.21 (s, 0.5H, H2), 7.37-7.26 (m, 7H, Ph), 7.22—7.13 (m, 3H, Ph), 6.02 (d, J = 1.8 Hz, 0.5H, H1’), 6.00 (d, J: 1.8 Hz, 0.5H, H1’), 5.14—5.11 (m, 2H, OCHzPh), 4.73-4.64 (m, 2H, H2’, H4’), 4.50—4.39 (m, 1H, H5’), .24 (m, 1H, H5’), 3.53—3.71 (m, 2H, CH2 gly), 2.39—2.25 (m, 1H, H3’), 213— 2.02 (m, 1H, H3’). 13C NMR (125 MHz, CH30D) 5 172.30 (d, 3,161: = 5.0 Hz, C=0), 172.27 (d, 3Jc.p = 5.0 Hz, C=0), 157.34 (C6), 157.32 (C6), 153.88 (C2), 153.87 (C2), 152.08 (d, {16.6 = 7.5 Hz, C-Ar), 152.05 (d, 3,161) = 7.5 Hz, C-Ar), 150.20 (C4), 150.19 (C4), 140.52 (C8), 140.42 (C8), 137.15 (C- Ar), 130.79 (CH-Ar), 129.57 (CH-Ar), 129.55 (CH-Ar), 129.35 (CH-Ar), 129.34 (CH-Ar), 129.33 ), 126.22 (CH-Ar), 121.44 (d, J61: = 3.7 Hz, CH-Ar), 121.40 (d, Jc-P = 3.7 Hz, CH-Ar), 120.51 (C5), 120.49 (C5), 93.19, 93.14 (Cl’), 80.46 (d, 3,161) = 4.60 Hz, C4’), 80.39 (d, 3,161) = 4.60, C4’), 76.66 (C2’), 68.68 (d, 2,161) = 5.42 Hz, C5’), 68.24 (d, 2Jc-P = 5.42 Hz, C5’), 67.95 (OCHzPh), 67.93 (OCHzPh), 43.90 (CH2 gly), 43.83 (CH2 gly), 34.83 (C3’), 34.54 (C3’).

HPLC Reverse-phase HPLC eluting with H20/CH3CN from 100/10 to 0/100 in 30 minutes, 1ml/min, l = 200 nm, showed two peaks of the diastereoisomers with tR 13.63 min. and tR 13.41 min. lmethox na hthalen-l- 10x hos hor lamino meth l entanoateD 06 N \ N <’ '2 9 N N O—FI’—O O Using General Procedure 1 above, N—methylimidazole (76 uL, 0.95 mmol) and a solution of (2S)—pentyl 2-((chloro(naphthalenyloxy)phosphory1)amino)methylpentanoate (250 mg, 0.6 mmol) in anhydrous THF (1 mL) were added dropwisely to a suspension of xyadenosine (48 mg, 19 mmol) in anhydrous THF (5 mL) and the reaction mixture was stirred at room temperature during a period of 16 hours. Puriﬁcation by column chromatography t system CH3OH/CH2C12 0/100 to 5/95) and preparative TLC (1000 M, eluent system CH30H/CH2C12 4/96) afforded the d compound as a white solid (27 mg, 22 %).

MS (ES+) m/z: Found: 641.3 (M + H+), 663.3 (M + Na+), 1303.6 (2M + Na+) C31H41N6O7P required: (M) 640.3. 31p NMR (202 MHz, CH30D) 8 4.64, 4.37. 1H NMR (500 MHz, CH30D) 5 8.28 (s, 0.5H, H-8), 8.25 (s, 0.5H, H-8), 8.21 (s, 0.5H, H-2), 8.20 (s, 0.5H, H-2), 8.17-8.12 (m, 1H, Nap), 7.88-7.83 (m, 1H, Nap), 7.69-7.66 (m, 1H, Nap), 7.54—7.42 (m, 3H, Nap), 7.40—7.35 (m, 1H, Nap), 7.31-7.26 (m, 5H, Ar), 6.01 (d, J: 2.1 Hz, 0.5H, H1’), 6.00 (d, J: 2.1 Hz, 0.5H, H1’), 4.47-4.67 (m, 2H, H2’, H4’), .44 (m, 1H, HS’), 4.43—4.31 (m, 1H, HS’), 4.00-3.87 (m, 3H, CH leu, CH2 Pen), .30 (m, 1H, H3’), 2.14—2.04 (m, 1H, H3’), 1.66-1.39 (m, 5H, CH2CH leu, CH2 Pen), 1.1.28-121 (m, 4H, CH2CH2 Pen), 0.86-0.81 (m, 3H, CH3 Pen), 0.81-0.68 (m, 6H, (CH3)2 leu). 13C NMR (125 MHz, CH30D) 6 175.42 (d, 3JC-P = 2.5 Hz, c=0), 175.04 (d, 3Jc.p = 2.5 Hz, c=0), 157.32 (C6), 153.87 (C2), 153.86 (C2), 150.23 (C4), 147.97 (d, 3.16.? = 6.2 Hz, ‘ipso’ Nap), 140.55 (C8), 136.30 (C-Ar), 136.29 (C-Ar), 128.89 (CH-Ar), 128.84 (CH-Ar), 127.95 (C- Ar), 127.91 (C-Ar), 127.84 (C-Ar), 127.78 (CH-Ar), 127.76 (CH-Ar), 127.46 (CH-Ar), 126.50 (C-Ar), 126.48 (C-Ar), 126.46 (C-Ar), 126.01 ), 125.91 (CH-Ar), 122.80 (CH-Ar), 122.70 (CH-Ar), 120.58 (C5), 120.56 (C5), 116.40 (d, 3.16.1) = 3.7 Hz, CH-Ar), 116.01 (d, 3Jc-P = 3.7 Hz, CH-Ar), 93.31 (C1’), 93.27 (C1’), 80.35 (d, 3.16.1) = 3.5 Hz, C4’), 80.29 (d, 3JC-P = 3.5 Hz, C4’), 76.54 (C2’), 76.50 (C2’), 69.07 (d, 2.16.1) = 5.5 Hz, C5’), 68.85 (d, ZJC-P = 5.5 Hz, C5’), 66.33 (CH2 Pent), 66.32 (CH2 Pent), 54.81 (CH leu), 54.71 (CH leu), 44.22 (d, ) = 7.6 Hz, CH2 leu), 43.93 (d, 3JC-P = 7.6 Hz, CH2 leu), 35.15 (C3’), 34.86 (C3’), 29.32 (CH2 pent), 29.30 (CH2 Pent), 29.11 (CH2 pent), 25.67 (CH leu), 25.45 (CH leu), 23.30 (CH2 pent), 23.12 (CH3 leu), 23.02 (CH3 leu), 22.04 (CH3 leu), 21.78 (CH3 leu), 14.28 (CH3 pent).

HPLC Reverse-phase HPLC eluting with H20/CH3CN from 100/10 to 0/100 in 30 s, n, l = 200 nm, showed one peak of the two overlapping diastereoisomers with tR 20.84 min.

Meth 12- 2S 4R 5R 6-amin0-9H— urin l h drox tetrah an-Z- l - methox na hthalen-l- 10x hos hor lamino meth 1 r0 anoateE 0,. <1,»’l Using General Procedure 1 above, N—methylimidazole (24 uL, 3.0 mmol) and a solution of methyl 2-((chloro(naphthalenyloxy)phosphoryl)amino)methylpropanoate (612 mg, 1.8 mmol) in anhydrous THF (1 mL) were added dropwisely to a suspension of 3’-deoxyadenosine (150 mg, 0.6 mmol) in anhydrous THF (15 mL) and the reaction mixture was stirred at room temperature during a period of 16 hours. Puriﬁcation by column chromatography (eluent system CH30H/CH2C12 0/100 to 7/93) and preparative TLC (1000 M, eluent system CH2C12 4/96) afforded the desired compound as a white solid (20 mg, 6%).

MS (ES+) m/z: Found: 557.2 (M + H), 579.2 (M + Na+), 1135.4 (2M + Na+) C25H29N6O7P required: (M) 556.51. 31P NMR (202 MHz, CH30D) 5 2.73. 1H NMR (500 MHz, CH30D) 6 8.28 (s, 0.5H, H8), 8.25 (s, 0.5H, H8), 8.21 (s, 0.5H, H2), 8.19 (s, 0.5H, H2), 8.18-8.14 (m, 1H, Nap), 7.90-7.84 (m, 1H, Nap), 7.71-7.66 (m, 1H, Nap), 7.53— WO 83830 2015/053628 7.47 (m, 3H, Nap), 7.41—7.35 (m, 1H, Nap), 6.03 (d, J: 2.1 Hz, 0.5H, H1’), 5.99 (d, J: 2.1 Hz, 0.5H, H1’), 4.76-4.67 (m, 2H, H2’, H4’), .44 (m, 1H, H5’), 4.42—4.33 (m, 1H, H5’), 3.65 (s, 1.5H, OCH3), 3.64 (s, 1.5H, OCH3), 2.48-2.41 (m, 0.5H, H3’), .30 (m, 0.5H, H3’), 2.15—2.09 (m, 0.5H, H3’), 2.08-2.02 (m, 0.5H, H3’), 1.47—1.44 (m, 6H, CH3). 13C NMR (125 MHz, CH30D) 5 177.25 (d, 3.16.1) = 3.7 Hz, C=O), 157.53 (C6), 157.51 (C6), 153.86 (C2), 150.28 (C4), 150.25 (C4), 148.06 (d, 3.16.? = 7.5 Hz, ‘ipso’ Nap), 148.04 (d, 3J61) = 7.5, ‘ipso’ Nap), 140.67 (C8), 140.60 (C8), 136.28 (C-Ar), 136.27 (C-Ar), 128.82 (CH-Ar), 128.80 (CH-Ar), 127.93 (d, 2.16.1) = 6.25 Hz, C-Ar), 127.92 (d, 2116.? = 6.25 Hz, C-Ar), 127.71 (CH-Ar), 127.69 (CH-Ar), 127.32 (CH-Ar), 126.44 (CH-Ar), 125.84 (CH-Ar), 122.93 ), 120.56 (C5), 120.50 (C5), 116.38 (d, 3.16.? = 3.75 Hz, CH-Ar), 116.36 (d, 3JC-P = 3.75 Hz, CH- Ar), 93.25 (C1’), 80.40 (d, 3.16.1) = 8.0 Hz, C4’), 80.33 (d, 3.16.1) = 8.0 Hz, C4’), 76.57 (C2’), 76.43 (C2’), 68.99 (d, 2.16.1) = 5.5 Hz, C5’), 68.84 (d, 2.16.1) = 5.5 Hz, C5’), 53.01 (OCH3), 35.22 (C-3’), 34.90 (C3’), 27.85 (d, 3Jc.p = 6.0 Hz, CH3), 27.80 (d, 3Jc.p = 6.0, CH3), 27.60 (d, 3Jc.p = 6.0, CH3), 27.56 (d, 3JC.P = 6.0, CH3).

HPLC Reverse-phase HPLC eluting with H20/CH3CN from 100/10 to 0/100 in 30 minutes, 1ml/min, l = 254 nm, showed two peaks with tR 16.51 min, tR 16.75 min. 2 -benz 12- 2S 4R 5R 6-amin0-9H- urin lh drox tetrah drofuran-Z- lmethox 2- 3-ethox oxo r0 1 henox hos hor lamino r0 anoateF OJ NHZ Qﬂo MS (ES+) m/z: Found: 669.3 (M + H+), 691.3 (M + Na+), N609P required: (M) 668.63. 31p NMR (202 MHz, CH30D): (SF 3.95, 3.65. 1H NMR (500 MHz, CH30D): 6H 8.25 (s, 0.5H, H8), 8.21 (s, 1H, H8, H2), 8.20 (s, 0.5H, H2), 7.35—7.29 (m, 6H, Ph), 7.25—7.21 (m, 1H, Ph), 7.16-7.07 (m, 2H, Ar), 6.00 (d, J: 1.9 Hz, 0.5H, H1’), 5.98 (d, J: 1.9 Hz, 0.5H, H1’), 5.17—5.05 (m, 2H, OCHzPh), 4.76-4.73 (m, 0.5H, H2’), 4.70—4.59 (m, 1.5H, H2’, H4’), 4.45—4.34 (m, 1H, HS’), 4.30—4.22 (m, 1H, HS’), 4.08-3.96 (m, 3H, CH2CH3, CH ala), .92 (m, 2H, CH2CH2), 2.62-2.56 (m, 2H, CH2CH2), 2.40—2.29 (m, 1H, H3’), 2.11—2.03 (m, 1H, H3’), 1.36 (d, J: 6.9 Hz, 1.5 H, CH3 ala), 1.33 (d, J: 6.9 Hz, 1.5 H, CH3 ala), 1.17 (t, J: 7.0 Hz, 1.5 H, CH2CH3), 1.16 (t, J: 7.0 Hz, 1.5 H, CH2CH3). 13C NMR (125 MHz, CH30D): 6C 174.82 (d, 3,161) = 3.7 Hz, C=0), 174.62 (C=0), 174.58 (C=0), 174.55 (d, 3,101): 3.7 Hz, C=0), 157.34 (C6), 157.32 (C6), 153.86 (C2), 153.84 (C2), 150.48 (d, J61) = 2.5 Hz, C-Ar), 150.44 (C4), 150.22 (d, J61) = 2.5 Hz, C-Ar), 140.49 (C8), 137.29 (C-Ar), 137.21 (C-Ar), 133.09 (d, J = 7.5 Hz, C-Ar), 132.94 (d, J = 7.5 Hz, C-Ar), 131.62 (CH-Ar), 131.59 (CH-Ar), 129.58 (CH-Ar), 129.34 (CH-Ar), 129.31 (CH-Ar), 129.28 (CH-Ar), 128.70 (d, J: 5.0 Hz, CH-Ar), 128.69 (d, J: 5.0 Hz, CH-Ar), 126.18 (CH-Ar), 121.02 (d, J: 2.5 Hz, CH-Ar), 120.49 (d, J: 2.5 Hz, CH-Ar), 120.58 (C5), 93.28 (C1’), 93.24 (C1’), 80.32 (d, : 8.7 Hz, C4’), 76.57 (C2’), 68.86 (d, 2,161): 5.0 Hz, C5’), 68.53 (d, 2Jc-P= 5.0 Hz, CS’), 67.98 (OCHzPh), 67.95 (OCHzPh), 61.57 (CH2CH3), 51.76 (CH ala), 51.65 (CH ala), 35.37 (CH2CH2), 35.30 (CH2CH2), 35.08 (C3’), 34.85 (C3’), 26.77 (CH2CH2), 26.72 2), 20.55 (d, 3Jc.1>= 6.2 Hz, CH3 ala), 20.33 (d, = 6.2 Hz, CH3 ala), 14.53 (CH2CH3).

HPLC Reverse-phase HPLC g with H20/CH3CN from 100/10 to 0/100 in 30 minutes, 1ml/min, l = 245 nm, showed one peak with tR 15.99 min. 3- lox henox hos hor lamino r0 anoateG Using General Procedure 3 above, 3’-deoxyadenosine (50 mg, 0.20 mmol) was suspended in anhydrous THF (5 mL) and lBuMgCl (1.0 M solution in THF, 0.22 mL, 0.22 mmol) was added dropwisely at room temperature. A solution of (25)-benzy1 2- ((chloro(phenoxy)phosphory1)amino)propanoate (212 mg, 0.6 mmol) in anhydrous THF (2 mL) was added dropwisely and the reaction e was stirred at room temperature during a period of 16 hours. Puriﬁcation by column chromatography (eluent system CH30H/CH2C12 0/100 to 8/92) and preparative TLC (500 11M, eluent system CH30H/CH2C12 = 5/95) afforded the desired compound as a white solid (6 mg, 5%).

MS (ES+) m/z: Found: 569.2 (M + H), 591.2 (M + Na+), 1159.4 (2M + Na+) C26H29N607P required: (M) 568.2. 311) NMR (202 MHz, CH30D): SF 2.44 (s), 2.92 (s). 1H NMR (500 MHz, CH30D): 6H 8.41 (s, 0.5 H, H8), 8.28 (s, 0.5 H, H8), 8.19 (s, 0.5H, H2), 8.18 (s, 0.5H, H2), 7.39—7.30 (m, 4H, Ar), 7.28-7.18 (m, 4H, Ar), 7.17—7.11 (m, 1H, Ar), 7.08- 7.03 (m, 1H, Ar), 6.23 (d, J: 2.0 Hz, 0.5H, H1’), 6.08 (d, J: 3.4 Hz, 0.5H,H1’), .43 (m, 1H, C2’), 5.19—5.12 (m, 1H, CHzPh), 5.07—4.95 (m, 1H, CHzPh), 4.48-4.42 (m, 1H, H4’), 405— 3.97 (m, 1H, CH ala), 3.95-3.87 (m, 1H, H5’), .61 (m, 1H, H5’), 2.59—2.45 (m, 1H, H3’), 2.31—2.23 (m, 1H, H3’), 1.36-1.27 (m, 3H, CH3 ala). 13C NMR (125 MHz, CH30H): 6C 174.76 (d, 3JC-P = 5.0 Hz, C=0), 174.52 (d, 3Jc.p = 5.0 Hz, C=0), 157.44 (C6), 153.76 (C2), 151.93 (C4), 150.06 (C-Ar), 149.93 (C-Ar), 141.38 (C8), 141.18 (C8), 137.33 (C-Ar), 137.10 (C-Ar), 130.69 ), 130.79 (CH-Ar), 129.61 (CH-Ar), 129.51 ), 129.40 (CH-Ar), 129.30 (CH-Ar), 129.23 (CH-Ar), 126.33 (CH-Ar), 126.16 (CH-Ar), 121.53 (d, 3Jc.p = 4.5 Hz, CH-Ar), 121.20 (d, 3Jc.p = 4.5 H, CH-Ar), 120.76 (C5), 91.56 (d, 3Jc.p = 7.7 Hz, Cl’), 91.45 (d, 3Jc.p = 7.7 Hz, Cl’), 82.78 (C4’), 82.28 (C4’), 81.83 (d, ZJC-P = 4.7 Hz, C2’), 80.96 (2 x d, 2J6.p = 4.7 Hz, C2’), 67.95 (OCHzPh), 67.92 (OCHzPh), 64.13 (C5’), 63.59 (C5’), 51.88 (CH ala), 51.75 (CH ala), 33.75 (d, 3,161) = 3.0 Hz, C3’), 33.59 (d, 3Jc.P = 3.0 Hz, C3’), 20.33 (d, 3,106 = 7.1 CH3 ala), 20.18 (d, 3,16? = 7.1 CH3 ala).

HPLC Reverse-phase HPLC g with H20/CH30H from 90/10 to 0/100 in 30 minutes, 1m1/min, 1 = 254 nm, showed two peaks of the diastereoisomers with tR 22.16 min. and tR 22.43 min.

Benz 12- 2S 4R 5R 6-amin0-9H- urin l 1- benz 10x 0x0 r0 an lamino henox hos hor lox tetrah drofuran-Z- lmethox henox hos hor l- aminomropanoate H Using General Procedure 3 above, xyadenosine (50 mg, 0.20 mmol) was ded in anhydrous THF (5 mL) and ’BuMgCl (1.0 M solution in THF, 0.22 mL, 0.22 mmol) was added dropwisely at room temperature. A solution of (2S)-benzy1 2- ((chloro(phenoxy)phosphoryl)amino)propanoate (212 mg, 0.6 mmol) in anhydrous THF (2 mL) was added dropwisely and the reaction mixture was stirred at room temperature during a period of 16 hours. Puriﬁcation by column chromatography t system CH30H/CH2C12 0/100 to 8/92) and ative TLC (500 M, eluent system CH30H/CH2C12 5/95) afforded the d compound as a white solid (19 mg, yield = 11%).

MS (ES+) m/z, found: 886.3 (M + H+), 1771.6 (2M + H+), 751.2 (molecule without nucleobase M). C42H45N7011P2 required: (M+) 885.3. 31P NMR (202 MHz, CH30D): SF 3.98, 3.88, 3.59, 3.12, 3.05, 2.45, 2.32. 1H NMR (500 MHz, CH30D): 6H 8.24-8.13 (m, 2H, H8, H2), 7.39-7.08 (m, 20H, Ph), 6.27- 6.23 (m, 0.5H, H1’), 6.16-6.13 (m, 0.5H, H1’), 5.61-5.48 (m, 1H, H2’), 5.17—4.91 (m, 4H, CHzPh), 4.57-4.49 (m, 1H, H4’), 4.41—4.29 (m, 1H, H5’), 4.25—4.15 (m, 1H, H5’), 4.10—4.01 (m, 1H, CH ala), 3.99-3.89 (m, 1H, CH ala),2.57-2.41 (m, 1H, H3’), 2.28-2.17 (m, 1H, H3’), 1.38- 1.23 (m, 6H, CH3 ala). 13C NMR (125 MHz, CH30D): 6C 174.88 (C=O), 174.83 (C=O), 174.79 (C=O), 174.73 (C=O), 174.61 (C=O), 174.57 (C=O), 174.53 (C=O), 157.36 (C6), 157.34 (C6), 157.32(C6), 157.29 (C6), 154.04 (C2), 154.01 (C2), 153.97 (C2), 153.94 (C2), 152.09 (C4), 152.04 (C4), 152.02 (C4), 151.97 (C4), 150.31 , 150.29 (C-Ar), 150.16 (C-Ar), 140.98 (C8), 140.91 (C8), 140.81 (C8), 137.31 (C-Ar), 137.28 (C-Ar), 137.22 (C-Ar), 137.09 , 130.86 (CH- Ar), 130.78 (CH-Ar), 130.77 (CH-Ar), 129.65 (CH-Ar), 129.61 (CH-Ar), 129.58 (CH-Ar), 129.55 (CH-Ar), 129.44 (CH-Ar), 129.42 (CH-Ar), 129.38 (CH-Ar), 129.34 (CH-Ar), 129.32 (CH-Ar), 129.30 (CH-Ar), 129.28 (CH-Ar), 129.23 (CH-Ar), 129.21 (CH-Ar), 12.42 (CH-Ar), 126.23 (CH-Ar), 126.20 (CH-Ar), 126.17 (CH-Ar), 121.65 (CH-Ar), 121.63 (CH-Ar), 121.61 (CH-Ar), 121.59 (CH-Ar), 121.52 (CH-Ar), 121.50 (CH-Ar), 121.47 (CH-Ar), 121.46 ), 121.40 ), 121.39 (CH-Ar), 121.36 (CH-Ar), 121.35 (CH-Ar), 121.30 (CH-Ar), 121.28 (CH-Ar), 121.26 (CH-Ar), 121.24 (CH-Ar), 120.61 (C5), 120.57 (C5), 120.56 (C5), 120.54 (C5), 91.56 (C1’), 91.51 (C1’), 91.45 (C1’), 91.25 (C1’), 91.20 (C1’), 81.84 (C2’), 81.82 (C2’), 81.79 (C2’), 81.27 (C2’), 81.22 (C2’), 81.18 (C2’), 80.49 (C4’), 80.43 (C4’), 80.06 (C4’), 79.99 (C4’), 68.29 (C5’, OCHzPh), 68.25 (C5’, ), 68.00 (C5’, OCH2Ph), 67.96 (C5’, OCHzPh), 67.94 (C5’, OCH2Ph), 67.90 (C5’, OCHzPh), 67.71 (C5’, OCH2Ph), 67.67 (C5’, OCHzPh), 51.91 (CH ala), 51.74 (CH ala), 51.70 (CH ala), 51.59 (CH ala), 34.22 (C3’), 34.20 (C3’), 34.16 (C3’), 33.97 (C3’), 33.94 (C3’), 33.91 (C3’), 20.44 (CH3 ala), 20.43 (CH3 ala), .39 (CH3 ala), 20.29 (CH3 ala), 20.27 (CH3 ala), 20.24 (CH3 ala), 20.21 (CH3 ala), 20.19 (CH3 ala).

HPLC e-phase HPLC eluting with H20/CH3CN from 100/10 to 0/100 in 30 minutes, 1ml/min, l = 254 nm, showed one broad peak with tR 15.97 min. furan 10x na hthalen-l- 10x hos hor lamino r0 anoateI Using General Procedure 3 above, 3’-Deoxyadenosine (50 mg, 0.20 mmol) was suspended in anhydrous THF (5 mL) and ’BuMgCl (1.0 M solution in THF, 0.3 mL, 0.3 mmol) was added dropwisely at room temperature. A solution of (2S)-benzy1 2-((chloro(naphtha1en y1oxy)phosphory1)amino)propanoate (323 mg, 0.8 mmol) in anhydrous THF (2 mL) was added dropwisely and the reaction mixture was stirred at room temperature during a period of 16 hours.

Puriﬁcation by column chromatography (eluent system CH2C12 0/100 to 6/94) and preparative TLC (500 uM, eluent system CH3OH/CH2C12 5/95) afforded the desired compound as a white solid (14 mg, 11 %).

(ES+) m/z, found: 619.2 (M + H+), 641.2 (M + Na+), 1259.4 (2M + Na+). C3oH31N6O7P required: (M) 618.20. 31P NMR (202 MHz, CH30D): SF 3.27 (s), 2.75 (s). 1H NMR (500 MHz, CH30D): 6H 8.37 (s, 1H, H8), 8.18 (s, 1H, H8), 8.14 (s, 1H, H2), 8.13- 8.11 (m, 0.5 H, Nap) 8.11 (s, 1H, H2), 7.94-7.90 (m, 0.5 H, Ar), 7.90-7.87 (m, 0.5 H, Ar), 7.86- 7.82 (m, 0.5 H, Ar), 7.74-7.70 (m, 0.5 H, Ar), 7.66-7.61 (m, 0.5 H, Ar), 7.57-7.47 (m, 1.5 H, Ar), 7.46-7.37 (m, 2.5 H, Ar), .27 (m, 4 H, Ar), 7.25-7.17 (m, 1 H, Ar), 6.19 (d, J: 2.4 Hz, 0.5H, H1’), 6.04 (d, J: 2.4 Hz, 0.5H, H1’), 5.60-5.54 (m, 0.5H, H2’), .42 (m, 0.5H, H2’), .16-4.99 (m, 2H, ), 4.46-4.40 (m, 0.5H, H4’), 4.36-4.30 (m, 0.5H, H4’), 4.13-4.04 (m, 1H, CH ala), 3.90-3.83 (m, 1H, H5’), 3.64-3.56 (m, 1H, H5’), 2.61-2.54 (m, 0.5H, H3’), 2.49- 2.41 (m, 0.5H, H3’), 2.35-2.27 (m, 0.5H, H3’), 2.22-2.16 (m, 0.5H, H3’), 1.35-1.24 (m, 3H, CH3 ala). 13C NMR (125 MHz, CH30H): 5C 174.52 (C=O), 174.49 (C=O), 157.27 (C6), 153.58 (C2), 149.97 (C4), 149.93 (C-4), 147.70 (d, 3Jc-p = 7.5, ‘ipso’ Nap), 147.48 (d, 3Jc-P = 7.5, ‘ipso’ Nap), 141.36 (C8), 141.19 (C8), 137.25 (C-Ar), 137.05 (C-Ar), 136.31 (C-Ar), 136.20 (C-Ar), 129.58 (CH-Ar), 129.48 (CH-Ar), 129.37 (CH-Ar), 129.26 (CH-Ar), 129.22 (CH-Ar), 128.88 (CH-Ar), 127.84 (CH-Ar), 127.75 (CH-Ar), 127.49 (CH-Ar), 127.44 ), 126.48 (CH-Ar), 126.39 (CH-Ar), 126.26 (CH-Ar), 126.05 (CH-Ar), 122.76 (CH-Ar), 122.38 (CH-Ar), 120.68 (C5), 120.61 (C5), 116.64 (d, 3Jc.p = 3.75 Hz, CH-Ar), 116.13 (d, 3JC-P = 3.75, CH-Ar), 91.60 (d, 3Jc-P = 7.5 Hz, C1’), 91.43 (d, 3JC.P = 7.5 Hz, C1’), 82.74 (C4’), 82.27 (C4’), 81.99 (d, ZJC-P = 5.5 Hz, C2’), 81.12 ((1, ch-p = 5.5 Hz, C2’), 67.97 (OCHzPh), 67.94 (OCHzPh), 64.16 (C5’), 63.51 (C5’), 51.96 (CH ala), 51.89 (CH ala), 33.89 (d, 3JC.P = 7.5 Hz, CH3 ala), 33.63 (d, 3JC.P = 7.5 Hz, CH3 ala).

HPLC Reverse-phase HPLC eluting with H20/CH3OH from 100/10 to 0/100 in 30 minutes, lml/min, 1 = 200 nm, showed two peaks of the diastereoisomers with tR 24.84 min. and tR 25.43 min.

Benz 12- 5- 6-amin0-9H- urin lh drox oxolan-Z- lmethox 1- benz 10x 0x0 r0 an lamino hos hor lamino r0 anoateJ Using General Procedure 2 above, 3’-deoxyadenosine (200 mg, 0.80 mmol) was suspended in (CH3)3PO3 (5 mL), and POC13 (75 uL, 0.80 mmol) was added dropwise at -5 oC. The reaction mixture was allowed to reach room temperature and left stirring for 4 hours. A solution of (S) (benzyloxy)—1-oxopropanaminium 4-methylbenzenesulfonate (1.4 g, 4.0 mmol) dissolved in ous CH2C12 (5 mL) was added ed by diisopropyl ethyl amine (1.4 mL, 8.0 mmol) at -78 0C. After stirring at room temperature for 20 hours, water was added and the layers were separated. The aqueous phase was extracted with romethane and the organic phase washed with brine. The combined organic layers were dried over NazSO4 and concentrated. The residue was puriﬁed by column chromatography (gradient elution of /MeOH=100/0 to 93/7) to give a white foam (256 mg, 49%).

MS (ES+) m/z: Found: 654.2 (M + H+), 676.2 (M + Na+), 1329.5 (2M + Na+) C30H36N708P required: (M) 653.62. 31P NMR (202 MHz, CH30D) 5 13.9. 1H NMR (500 MHz, CH30D) 5 8.28 (s, 1H, H8), 8.22 (s, 1H, H2), 7.37-7.26 (m, 10H, Ph), 6.00 (d, J: 1.9 Hz, 1H, Hl’), 5.15—5.05 (m, 4H, OCHzPh), 4.74—4.70 (m, 1H, H2’), 4.63-4.56 (m, 1H, H4’), 4.24-4.18 (m, 1H, H5’), 4.11—4.05 (m, 1H, H5’), .87 (m, 1H, CH a1a),2.35- 2.27 (m, 1H, H3’), 2.07—2.01 (m, 1H, H3’), .27 (m, 3H, CH3 ala). 13C NMR (125 MHz, CH30D) 5 175.40 (d, 3Jc.p = 5.0 Hz, c=0), 175.36 (d, 3Jop = 5.0 Hz, c=0), 157.36 (C6), 153.91 (C2), 150.25 (C4), 140.64 (C8), 137.33 (C-Ar), 137.29 (C-Ar), 129.58 ), 129.57 (CH-Ar), 129.33 (CH-Ar), 129.31 (CH-Ar), 129.29 (CH-Ar), 120.55 (C5), 93.18 (Cl’), 80.67 (d, 3JC-P= 8.4 Hz, C4’), 76.59 (C2’), 67.90 (OCHzPh), 67.47 (d, ZJC-P = .2 Hz, C5’), 51.14 (d, 2.16.1) = 1.7 Hz, CH ala), 51.11 (d, 2.16.1) = 1.7 Hz, CH ala), 35.08 (C3’), .77 (d, 3,161): 6.5 Hz, CH3 ala), 20.59 (d, 3,161): 6.5 Hz, CH3 ala).

HPLC Reverse-phase HPLC eluting with HzO/CH3CN from 90/10 to 0/100 in 30 minutes, 1m1/min, 1 = 254 nm, showed one peak with tR 13.87 min. h drox tetrah drofuran-Z- lmethox na hthalen-l- 10x hos hor lamino r0 anoate \ "H; 2¢,(\PMJ§5 {4N {"05" A G "~~~8~~-<;\ ..«.~r~>x $3,; {:1 ;\ CL g N."W]' ,2\ J, 3 \ 3g OH Using General ure 1 above, N—methylimidazole (99 uL, 1.24 mmol) and a solution of (25)—benzyl 2-((chloro(naphthalen-l-yloxy)phosphoryl)amino)propanoate (303 mg, 0.75 mmol) in anhydrous THF (5 mL) were added sely to a suspension of 2-O-methyl-3’- deoxyadenosine (70 mg, 0.25 mmol) in anhydrous THF (10 mL) and the reaction mixture was stirred at room temperature during a period of 16 hours. Puriﬁcation by column chromatography (eluent system CH3OH/CH2C12 0/100 to 6/94) and preparative TLC (eluent system CH30H/CH2C12 5/95) afforded the desired compound as white solid (96 mg, 60%).

MS (ES+) m/z.‘ Found: 649.2 (M + H) C31H33N608P required: 648.21(M). 31p NMR (202 MHz, : SF 4.38 (s), 4.08 (s). 1H NMR (500 MHz, CD30D): 6H 8.14-8.11 (d, J = 8.0Hz, 0.5H, Ar), 8.07 (d, J = 8.0Hz, 0.5H, Ar), 8.05 (s, 0.5H, H8), 8.02 (s, 0.5H, H8), 7.82-7.80 (m, 1H, Ar), 7.61 (d, J = 7.0Hz, Ar), 7.47—7.44 (m, 4H, Ar), .29 (m, 2H, Ar), 7.24—7.22 (m, 3H, Ar), 5.88 (s, 1H, Hl’), 4.71-4.68 (m, 1H, H4’), 4.65-6.60 (m, 1H, H2’), 4.42—4.40 (m, 1H, H5’), 4.30—4.27 (m, 1H, H5’), 4.08-3.98 (m, 1H, CH ala) 3.88 (s, 1.5H, OCH3), 3.86 (s, 1.5H, OCH3), 2.37-2.33 (m, 1H, H3’), 2.04—2.01 (m, 1H, H3’), 1.27 (d J: 7.0 Hz, 1.5H, CH3), 1.24 (d J = 7.0 Hz, 1.5H, CH3). 13C NMR (125 MHz, CH30D): 6C 174.83 (d, 3Jc-P = 3.7 Hz, C=O), 174.60 (d, 3J61) = 3.7 Hz, C=O), 163.70 (C-2), 158.10 (C6), 151.95 (C4), 147.95 (d, 3Jc.1> = 7.5 Hz, ‘ipso’ Nap), 147.91, (d, 3J61) = 7.5 Hz, ‘ipso’ Nap), 139.39 (C8), 139.37 (C8), 137.12, 137.17 (C—l'pso CHzPh), 136.22 (C-Ar), 129.57, 129.54, , 129.32, 129.27, 129.12, 129.24 128.89, 128.83, (CH-Ar), 127.85 (d, 2J61) = 6.25 Hz, C-Ar), , 127.76, 127.51, 127.48, 126.49, 126.00, 125.97, 122.73, 122.63 (CH-Ar), 116.86 (C5), 116.72 (C5), 116.29 (d, 3J61) = 3.75 Hz, CH-Ar), 116.22 (d, 3J61) = 3.75 Hz, CH-Ar), 93.33 (Cl’), C1’), 80.24 (d, 3Jc.1> = 2.75 Hz, C4’), 76.29 (C2’), 76.26 (C2’), 69.09 (d, 2J61) = 5.0 Hz, C5’), 68.16 (d, 2Jc-P = 8.2 Hz, CS’), 67.95 (OCHzPh), 55.28, 55.32 (OCH3), 51.79 (CH ala), 51.71 (CH ala), 35.40 (C-3’), .12 (C3’), 20.49 (d, 3Jc.P = 6.7 Hz, CH3 ala), 20.35 (d, 3Jc.P = 6.7, CH3 ala). HPLC Reverse- phase HPLC g with H20/CH3CN from 100/10 to 0/100 in 30 minutes, F in, 2» = 280 nm, showed two peaks of the diastereoisomers with tR 16.22 min. and tR 16.48 min. h drox tetrah drofuran-Z- lmethox na hthalen-l- 10x hos hor lamino r0 anoate v f\ xN ‘~ .r‘ﬂ’LQN «’ n \1’ E .3 \‘szx Q N"! \ Vii/NY)" OWFWCL §§7\\;, NH 0 \\ <§:,.J-‘\\ , 0,»fo Using General Procedure 1 above, N—methylimidazole (99 uL, 1.24 mmol) and a solution of (2S)—benzyl 2-((chloro(phenoxy)phosphoryl)amino)propanoate (264 mg, 0.75 mmol) in anhydrous THF (2 mL) were added dropwisely to a suspension of thyl-3’- deoxyadenosine (70 mg, 0.25 mmol) in anhydrous THF and the on mixture was stirred at room temperature for 16 hours. Puriﬁcation by column chromatography (eluent system CH3OH/CH2C12 0/100 to 6/94) and preparative TLC (eluent system CH30H/CH2C12 5/95) afforded the desired compound as a white solid (13 mg, 10%).

(ES+) m/z, found: 599.2 (M + H+), C27H31N608P required: 598.19 (M). 31p NMR (202 MHz, CD30D) 5 3.97, 3.64. 1H NMR (500 MHz, CD30D) 5 8.06 (s, 0.5H, H8), 8.04 (s, 0.5H, H8), 7.33-7.28 (m, 7H, Ph), 7.20—7.14 (m, 3H, Ph), 5.92 (d, J = 1.5 Hz, 0.5H, H1’), .90 (d, J = 1.5 Hz, 0.5H, H1’), .04 (m, 2H, OCHzPh), 4.78-4.76 (m, 0.5H, H4’), 474— 4.72 (m, 0.5H, H4’), 4.63-4.59 (m, 1H, H2’), .34 (m, 1H, H5’a), 4.25—4.20 (m, 1H, H5’b), 3.94, 3.95 (OCH3), 3.99—3.90 (m, 1H, CH ala), 2.40—2.37 (m, 1H, H3’), 2.07—2.04 (m, 1H, H3’), 1.31 (d J :70 Hz, CH3), 1.26 (d, J = 7.0 Hz, CH3). 13C NMR (125 MHz, CD30D) 5 174.82 (d, 3JC-P = 3.7 Hz, C=O), 174.62 (d, 3J61) = 3.7 Hz, C=O), 163.80 (C-2), 158.16, 158.13 (C6), 152.15 (C4), 152.05 (d, 3Jc.p = 4.8 Hz, C—l'pso Ph), 152.00 (d, 3Jc.p = 4.8 Hz, C-z'pso Ph), 139.39 (C8), 137.30, 137.21 (C—l'pso CHzPh), 130.72, 129.57, 129.31,129.27, 126.122 (CH-Ar), 121.42 (d, J61: = 4.5 Hz, CH-Ar), 121.37 (d, JC.1> = 4.5 Hz, CH-Ar), 116.72 (C5), 116.69 (C5), 93.33, 93.24 (Cl’), 80.26 (d, 3J61) = 8.87, C4’), 80.19 (d, 3J61) = 8.87, C4’), 76.35 (C2’), 68.78 (d, 2Jc-P = 5.0 Hz, C5’), 68.35 (d, 2116.? = 5.0 Hz, C5’), 67.94 h), 67.92 (OCHzPh), 55.25, 55.28 (OCH3), 51.69, 51.57 (CH ala), 35.23 (C3’), 34.96 (C3’), 20.38 (d, 3Jc-P = 6.7, CH3 ala), 20.26 (d, 3JC-P = 6.7, CH3 ala). HPLC Reverse-phase HPLC eluting with HzO/CHgCN from 100/10 to 0/100 in 30 minutes, F =1ml/min, 9» = 280 nm, showed two peaks of the diastereoisomers with tR 14.22 min. and tR 14.51 min. 2-0—meth l-3’-de0x adenosine—5’ 1-na hth 11- cut lox -L-leucin l hos hateM Compound M was prepared according to the general procedure 1 using 2-O-methyl-3’- deoxyadenosine (70 mg, 0.25 mmol), N-methylimidazole (99 uL, 1.24 mmol) and naphthyl(pentyloxy-L-leucinyl) phosphorochloridate (330 mg, 0.75 mmol). ation by column chromatography (eluent system gradient CH30H/CH2C12 0/100 to 6/94) and preparative TLC (2000 uM, eluent system CH30H/CH2C12 7/93) afforded the title compound as a white solid (50 mg, 30%). 31P NMR (202 MHz, CD30D) SF 4.53, 4.28. 1H NMR (500 MHz, CD30D) 5H 8.04-7.96 (m, 1H, H8), 7.77—7.71 (m, 1H, Nap), 7.58-7.53 (m, 1H, Nap), 7.45—7.17 (m, 5H, Nap), .75 (m, 1H, H1’), 4.64-4.51 (m, 2H, H2’, H4’), 4.40-4.16 (m, 2H, H5’), .75 (m, 6H, OCH3, O(CH2)4CH3, CHCHzCH(CH3)2), 2.38-2.24 (m, 1H, H3’), 2.00—1.91 (m, 1H, H3’), 1.53—1.05 (m, 11H, O(CH2)4CH3, CHCH2CH(CH3)2), 0.77-0.55 (m, 9H, O(CH2)4CH3, CHCHzCH(CH3)2). 13C NMR (125 MHz, CD30D) 6C 175.02 (d, 3JC-P = 2.5 Hz, C=0), 174.78 (d, 3Jc.p = 2.5 Hz, C=0), 163.76 (C2), 158.14 (C6), 151.03 (C4), 147.96 (d, 3,161) = 7.2, ‘ipso’ Nap), 138.96 (C8), 136.30 (C-Ar), 136.28 (C-Ar), 136.22 (C-Ar), 128.93 (CH-Ar), 128.88 (CH-Ar), 128.81 (CH- Ar), 128.48 (CH-Ar), 127.77 (CH-Ar), 127.73 (CH-Ar), 127.44 ), 127.42 (CH-Ar), 127.06 (CH-Ar), 126.86 (CH-Ar), 126.45 (CH-Ar), 126.44 ), 126.31 (CH-Ar), 125.98 ), 125.88 (CH-Ar), 123.83 (CH-Ar), 123.43 (CH-Ar), 123.24 (CH-Ar), 122.81 (CH-Ar), 122.77 (CH-Ar), 122.69 (CH-Ar), 116.34 (d, 3Jc.p = 3.7 Hz, CH-Ar), 116.02 (d, 3Jc-P = 3.7 Hz, CH-Ar), 115.71 (C5), 93.42 (C1’), 93.32 (C1’), 80.22 (d, 3Jc.p = 5.3 Hz, C4’), 80.15 (d, 3JC-P = .3 Hz, C4’), 76.29 (C2’), 76.27 (C2’), 69.22 (d, 2116.1) = 5.2 Hz, C5’), 69.028 (d, ZJC-P = 5.2 Hz, WO 83830 C5’), 66.31 (O(CH2)4CH3), 66.30 (O(CH2)4CH3), 55.29 (OCH3), 55.24 (OCH3), 54.79 (CHCHzCH(CH3)2), 54.68 (CHCHzCH(CH3)2), 44.20 (d, 3JC-P = 7.25 Hz, CHCHzCH(CH3)2), 43.93 (d, 3JC-P = 7.25 Hz, CHCHzCH(CH3)2), 35.49 (C3’), 35.17 (C3’), 29.31 (O(CH2)4CH3), 29.11 (O(CH2)4CH3), 25.67 (CHCHzCH(CH3)2), 25.44 (CHCHzCH(CH3)2), 23.30 (O(CH2)4CH3), 23.10 CH(CH3)2), 23.00 (CHCH2CH(CH3)2), 22.94 (CHCHzCH(CH3)2), 22.81 (CHCHzCH(CH3)2), 14.27 (O(CH2)4CH3).

(ES+) m/z, found: 671.3 (M + H+), C32H43N608P required: 670.69 (M).

HPLC Reverse-phase HPLC eluting with H20/CH3CN from 100/10 to 0/100 in 30 minutes, 1 ml/min, l = 254 nm, showed two peaks of the diastereoisomers with tR 20.83 min. and tR 20.93 min. 2-0—meth l-3’-de0x adenosine—5’ hen l 1-hex 10x -L-alanin l hos hateN Q N (ES+) m/z, found: 593.3 (M + H+), C32H43N608P required: 592.58 (M).

HPLC Reverse-phase HPLC eluting with H20/CH3CN from 100/10 to 0/100 in 30 minutes, 1 ml/min, l = 254 nm, showed two peaks of the diastereoisomers with tR 17.02 min. and tR 17.23 min. 2-Flu0r0-3’-de0x adenosine-5’ 1-na hth lbenz 10x -L-alanin l hos hateO Compound 0 was ed according to the general procedure 1 using 2-Fluoro-3’- deoxyadenosine (50 mg, 0.18 mmol), N—methylimidazole (74 uL, 0.93 mmol) and phenyl(benzyloxy-L-alaninyl) phosphorochloridate (196 mg, 0.56 mmol). Puriﬁcation by column tography t system gradient CH30H/CH2C12 0/100 to 6/94) and preparative TLC (500 uM, eluent system CH30H/CH2C12 5/95) afforded the title compound as a white solid (5 mg, 4%). 31P NMR (202 MHz, CD30D) SF 4.33, 4.08. 1H NMR (500 MHz, CD30D) 6H 8.17 (s, 0.5H, H8), 8.14 (s, 0.5H, H8), 8.14-8.09 (m, 1H, Ar), 7.89-7.85 (m, 1H, Ar), 7.70-7.66 (m, 1H, Ar), 7.54—7.42 (m, 4H, Ar), 7.40—7.24 (m, 5H, Ar), 5.89 (d, J = 2.3 Hz, 0.5H, H1’), 5.88 (d, J: 2.3 Hz, 0.5H, H1’), 5.08-5.01 (m, 2H, OCHzPh), 4.70— 4.60 (m, 2H, H2’, C4’), 4.46-4.39 (m, 1H, C5’), 4.32—4.24 (m, 1H, C5’), 4.09—3.97 (m, 1H, CHCH3), 2.36-2.25 (m, 1H, H3’), 2.06-1.98 (m, 1H, H3’), .25 (m, 3H, CHCHs). 13C NMR (125 MHz, CD30D) 6C 175.54 (CO), 175.22 (CO), 161.02 (d, 1JC.F = 207.3 Hz, C2), 160.89 (d, 1JC.F = 207.3 Hz, C2), 158.45 (d, 3Jc.F = 18.2 Hz, C6), 158.23 (d, 3Jc.F = 18.2 Hz, C6), 150.63 (d, 3Jc.F= 18.4 Hz, C4), 140.67 (C8), 136.26 (C-Ar), 131.62, 131.54, 129.56 (CH- Ar), 129.52 ), 129.37 (CH-Ar), 129.31 ), 129.26 (CH-Ar), 128.87 (CH-Ar), 128.81 (CH-Ar), 128.29 (CH-Ar), 128.02 (CH-Ar), 127.79 (CH-Ar), 127.76 (CH-Ar), 127.51 (CH-Ar), 127.49 (CH-Ar), 127.47 (CH-Ar), 126.47 (CH-Ar), 126.33 (C-Ar), 126.27 (C-Ar), 125.97 (CH-Ar), 122.78 (CH-Ar), 122.74 (CH-Ar), 122.64 (CH-Ar), 122.62 (CH-Ar), 116.35 (d, 4JC—F: 3.0 Hz, C5), 116.15 (d, 4JC—F: 3.0 Hz, C5), 93.25 (C1’), 93.20 (C1’), 80.41 (d, 3Jc-P: 7.5 Hz, C4’), 80.33 (d, 3,161): 7.5 Hz, C4’), 76.43 (c2’), 76.35 (C2’), 68.84 (d, 961:: 5.5 Hz, C5’), 68.45 (d, 2Jc.p = 5.5 Hz, C5’), 67.92 (OCHzPh), 67.92 (OCHzPh), 51.75 (CHCH3), 51.52 (CHCH3), 34.97 (C3’), 34.74 (C3’), 20.42 (d, 3JC.P = 6.7 Hz, CHCH3), 20.20 (d, 3Jc-P = 6.7 Hz, CHCH3). 19F NMR (470 MHz, CDsOD) 6F -53. 14, -53.22.

(ES+) m/z, found: 637.2 (M + H+), C30H30FN607P required: 636.57 (M).

HPLC Reverse-phase HPLC g with H20/CH3CN from 100/10 to 0/100 in 30 minutes, 1 ml/min, l = 254 nm, showed two peaks of the diastereoisomers with tR 17.09 min. and tR 17.34 min. h drox tetrah drofuran-Z- lmethox henox hos hor lamino r0 anoateP "i"? friﬁi:\ efﬂiﬁx‘E-f vi? 0"¥?70\, L NH EEO\ i W ‘1‘!" \ I‘VCNEE’J \, OH Using General Procedure 1 above, ylimidazole (74 uL, 0.93 mmol) and a on of (2S)-benzyl loro(phenoxy)phosphoryl)amino)propanoate (196 mg, 0.56 mmol) in anhydrous THF (2 mL) were added dropwisely to a suspension of 2-Fluoro-3’-deoxyadenosine (50 mg, 0.18 mmol) in anhydrous THF (5 mL) and the reaction mixture was stirred at room temperature for 16 hours. Puriﬁcation by column chromatography t system CH3OH/CH2C12 0/100 to 6/94) and preparative TLC (eluent system CH30H/CH2C12 5/95) afforded the desired compound as a white solid (5 mg, 7%).

(ES+) m/z, found: 587.1 (M + H), C26H28FN607P required: 586.17 (M). 19F NMR (470 MHz, CD30D): 6F , —53.23. 31p NMR (202 MHz, CD30D): SF 3.95 (s), 3.67 (s). 1H NMR (500 MHz, CDCls): 5H 8.19 (s, 0.5H, H8), 8.16 (s, 0.5H, H8), 7.36-7.27 (m, 7H, Ar), 7.22—7.13 (m, 3H, Ar), 5.91 (d, J = 1.5 Hz, 0.5H, H1’), 5.89 (d, J = 1.7 Hz, 0.5H, H1’), 5.15-5.06 (m, 2H, OCHzPh), 4.73-4.58 (m, 2H, H2’, H4’), 4.42—4.34 (m, 1H, H5’), .90 (m, 1H, H5’), 327— 3.24 (m, 1H, H3’), 2.08-2.00 (m, 1H, H3’), 1.33 (d, J = 7.1 Hz, 1.5H, CH3 ala), 1.29 (d, J = 7.1 Hz, 1.5H, CH3 ala). 13C NMR (125 MHz, CD30D): 6C 175.85 (d, 3Jc.P = 3.7 Hz, C=0), 174.63 (d, 3Jc.p = 5.0 Hz, C=0), 160.58 (d, {1m = 207.5 Hz, C2), 160.53 (d, {1m = 207.5 Hz, C2), 159.06 (d, 3JC.F = 18.7 Hz, C6), 159.05 (d, 3Jc.F = 17.5 Hz, C6), 152.11 (d, 2Jc.p = 8.75 Hz, C- Ar), 152.08 (d, 2Jc.P = 8.7 Hz, C-Ar), 151.58 (d, 3Jc.F = 19.7 Hz, C4), 151.56 (d, 3Jc.F = 19.5 Hz, C4), 140.63 (C8), 137.28 (C-Ar), 137.21 (C-Ar), 130.78 (CH-Ar), 130.75 ), 129.58 (CH- Ar), 129.38 (CH-Ar), 129.34 (CH-Ar), 129.32 (CH-Ar), 129.28 ), 128.3 (CH-Ar), 128.02 (CH-Ar), 121.16 (CH-Ar), 121.18 (CH-Ar), 121.47 (CH-Ar), 121.51 (CH-Ar), 121.42 ), 121.39 (CH-Ar), 121.36 (CH-Ar), 118.75 (d, 4JC.F = 3.7 Hz, C5), 118.72 (d, 4JC.F = 3.7 Hz, C5), 93.25 (Cl’), 93.18 (Cl’), 80.48 (d, 3Jc.p = 8.3 Hz, C4’), 80.46 (d, 3Jc-p = 8.1 Hz, C4’), 76.51 (C2’), 76.49 (C2’), 68.54 (d, 2116-1): 5.2 Hz, C5’), 68.18 (d, 96-1): 5.6 Hz, C5’), 67.94 (CH2 Bn), 67.91 (CH2 Bn), 51.71 (CH ala), 51.56 (CH ala), 34.85 (C3’), 34.64 (C3’), 20.42 (d, 3Jc-p = 7.1 Hz, CH3 ala), 20.25 (d, 3Jc.p = 7.5 Hz, CH3 ala). HPLC Reverse-phase HPLC eluting with HzO/CH3CN from 100/10 to 0/100 in 30 minutes, 1 ml/min, l = 280 nm, showed two peaks of the diastereoisomers with tR 14.98 min. and tR 15.12 min. 2-Fluoro-3’-deox adenosine-5’ 1-na hth l 1- cut 10x -L-leucin l hos hate Compound Q was prepared according to the general procedure 1 using 2-Fluoro-3’- deoxyadenosine (50 mg, 0.18 mmol), N—methylimidazole (74 uL, 0.93 mmol) and naphthyl(pentyloxy-L-leucinyl) phosphorochloridate (246 mg, 0.56 mmol). ation by column chromatography (eluent system CH30H/CHC13 0/100 to 6/94) and preparative TLC (1000 um, eluent system CH30H/CH2C12 5/95) afforded the title compound as a white solid (65 mg, 53%). 31P NMR (202 MHz, CD30D): 4.60, 4.35. 1H NMR (500 MHz, CD30D): 6H 8.23 (s, 0.5H, H8), 8.20 (s, 0.5H, H8), 8.18-8.12 (m, 1H, Ar), 7.92-7.86 (m, 1H, Ar), 7.73-7.68 (m, 1H, Ar), 7.57-7.46 (m, 3H, Ar), 7.42-7.36 (m, 1H, Ar), .93-5.91 (m, 1H, H1’), 4.74-4.62 (m, 2H, H2’, H4’), 4.55-4.50 (m, 0.5H, H5’), 4.49-4.44 (m, 0.5H, H5’), 4.43-4.37 (m, 0.5H, H5’), 4.36-4.31 (m, 0.5H, H5’), 4.02-3.86 (m, 3H, CHCH2CH(CH3)2, O(CH2)4CH3), 2.43-2.29 (m, 1H, H3’), 2.12-2.04 (m, 1H, H3’), 1.67-1.20 (m, 11H, 4CH3, CHCH2CH(CH3)2), 0.89-0.67 (m, 9H, O(CH2)4CH3, CHCH2CH(CH3)2) 13C NMR (125 MHz, : 6C 175.03 (d, 3JC-P = 2.5 Hz, C=O), 174.93 (d, 3Jc-P = 2.5 Hz, C=O), 161.45 ((1, 1JGF = 205.5 Hz, C2), 160.39 (d, IJC—F: 205.5 Hz, C2), 158.33 (C6), 151.60 (C4), 147.92 (C-Ar), 140.69 (C8), 136.30 (C-Ar), 128.88 (CH-Ar), 128.83 (CH-Ar), 127.80 (CH-Ar), 127.76 (CH-Ar), 127.49 ), 127.46 (CH-Ar), 126.48 (CH-Ar), 126.45 (CH-Ar), 126.02 ), 125.91 (CH-Ar), 123.03 (C-Ar), 122.81 (CH-Ar), 122.69 (CH-Ar), 116.39 (d, 3Jc-1> = 2.9 Hz, CH-Ar), 116.28 (C5), 116.26 (C5), 115.97 (d, 3Jc-1> = 2.9 Hz, CH-Ar), 93.29 (C1’), 93.23 (C1’), 80.45 (d, 3Jc-1> = 6.0 Hz, C4’), 80.38 (d, 3Jc-1> = 6.0 Hz, C4’), 76.45 (C2’), 76.41 (C2’), 68.99 (d, 2Jc-1> = 5.4 Hz, C5’), 68.78 (d, 2Jc.P = 5.4 Hz, C5’), 66.31 (O(CH2)4CH3), 66.29 )4CH3), 54.78 (CHCH2CH(CH3)2), 54.66 CH(CH3)2), 44.16 (d, 3Jc-P = 7.25 Hz, CHCH2CH(CH3)2), 43.84 (d, 3Jc-1> = 7.3 Hz, CHCH2CH(CH3)2), 35.09 (C3’), 34.79 (C3’), 29.31 (O(CH2)4CH3), 29.12 (O(CH2)4CH3), 25.65 (CHCH2CH(CH3)2), 25.41 (CHCHzCH(CH3)2), 23.33 (O(CH2)4CH3), 23.11 CH(CH3)2), 23.00 (CHCH2CH(CH3)2), 21.95 (CHCH2CH(CH3)2), 21.68 (CHCH2CH(CH3)2), 14.29 (O(CH2)4CH3). 19F NMR (470 MHz, CD30D): 6F -53.15, -53.20.

(ES+) m/z, found: 659.3 (M + H+), C31H40FN607P required: 658.66 (M).

HPLC Reverse-phase HPLC eluting with H20/CH3CN from 100/10 to 0/100 in 30 minutes, 1 ml/min, 1 = 254 nm, showed one peak of the overlapping diastereoisomers with tR 21.95 min. 2 -hex 12- 2S 4R 5R 6-amin0flu0r0-9H- urin lh drox tetrah drofuran- 2- lmethox henox hos hor lamino r0 anoateR r"' Nx‘V./’}‘\> 1‘ :’ e <3 \ " O "wax x.) \\ w" u "- ?!\ F U-~~P~O\ / 15m 40» \I A D (3 W \V \V.» \V/ \ {3H 2015/053628 Using General Procedure 1 above, N—methylimidazole (74 11L, 0.93 mmol) and a on of exyl 2-((chloro(phenoxy)phosphoryl)amino)propanoate (196 mg, 0.56 mmol) in anhydrous THF (2 mL) were added sely to a suspension of 2-Fluoro-3’-deoxyadenosine (50 mg, 0.18 mmol) in anhydrous THF (5 mL) and the reaction mixture was stirred at room temperature for 16 hours. Puriﬁcation by column chromatography (eluent system CH3OH/CH2C12 0/ 100 to 6/94) and preparative TLC (eluent system CH3OH/CH2C12 5/95) afforded the desired compound as a white solid (5 mg, 7%).

(ES+) m/z, found: 587.1 (M + H), C26H28FN6O7P required: 586.17 (M). 19F NMR (470 MHz, CD30D): SF 6315, —53.20. 311) NMR (202 MHz, CD30D): 3.91 (s), 3.73 (s). 1H NMR (500 MHz, CDCls): 6H 8.21 (s, 0.5H, H8), 8.20 (s, 0.5H, H8), 7.37—7.29 (m, 7H, Ar), 7.26-7.13 (m, 3H, Ar), 5.94—5.91 (m, 1H,H1’), 4.76-4.64 (m, 2H, H2’, H4’), 4.49—4.44 (m, 0.5H, H5’), 443— 4.37 (m, 0.5H, H5’), 4.33-4.26 (m, 1H, H5’), .99 (m, 2H, CH2 Hex), 3.97-3.83 (m, 1H, CH ala), 2.41—2.32 (m, 1H, H3’), 2.13-2.06 (m, 1H, H3’), 1.62-1.52 (m, 2H, CH2 Hex), 1.37—1.23 (m, 9H, CH3 ala, CH2 Hex), 0.92-0.85 (m, 3H, CH3 Hex). 13C NMR (125 MHz, CD30D): 6C 175.15 (d, 3,161) = 3.7 Hz, C=0), 174.96 (d, 3,161) = 5.0 Hz, C=0), 160.59 (d, 1JC.F = 207.5 Hz, C2), 160.56 (d, 1JC.F = 207.5 Hz, C2), 159.09 (d, SJC-F = 21.2 Hz, C6), 159.08 (d, {16.}: = 20.0 Hz, C6), 152.16 (d, 2,161: = 7.5 Hz, C-Ar), 152.14 (d, 2JC—P = 6.3 Hz, C-Ar), 151.71 (d, {16.}: = 20.0 Hz, C4), 151.67 (d, 3,167: = 20.0 Hz, C4), 140.70 (d, 5JC-F = 2.5 Hz, C8), 140.68 (d, 5JC.F = 2.5 Hz, C8), 130.77 (CH-Ar), 130.74 ), 126.16 (CH-Ar), 126.24 (CH-Ar), 121.48 (CH-Ar), 121.44 (CH-Ar), 121.41 (CH-Ar), 121.37 (CH-Ar), 118.80 (d, 4JC.F = 3.7 Hz, C5), 118.77 (d, 4JC.F = 3.7 Hz, C5), 93.37 (Cl’), 93.25 (Cl’), 80.52 (d, 3JC-P = 3.7 Hz, C4’), 80.45 (d, 3,161): 4.1 Hz, C4’), 76.52 (C2’), 76.49 (C2’), 68.69 (d, 2,161) = 5.4 Hz, C5’), 68.30 (d, 2,161) = 4.9 Hz, C5’), 66.46 (CH2 Hex), 51.68 (CH ala), 51.57 (CH ala), 35.02 (C3’), 34.80 (C3’), 32.58 (CH2 Hex), 29.65 (CH2 Hex), 26.61 (CH2 Hex), 23.59 (CH2 Hex), 20.60 (d, 3Jc.p = 7.1 Hz, CH3 ala), 20.43 (d, 3JC.P = 7.5 Hz, CH3 ala), 14.35 (CH3 Hex). HPLC Reversephase HPLC eluting with HzO/CH3CN from 100/10 to 0/100 in 30 minutes, 1 ml/min, l = 280 nm, showed two peaks of the diastereoisomers with tR 17.83 min. and tR 18.02 min. h drox tetrah drofuran-Z- lmethox na hthalen-l- 10x hos hor lamino r0 anoate To a stirring on of ro-3’-deoxyadenosine (100 mg, 1.0 mol/eq.) in 10 mL of anhydrous THF, 424 mg of (2S)-benzyl 2-(chloro(naphthalen yloxy)phosphorylamino)propanoate (3.0 eq/mol) dissolved in 10 mL of anhydrous THF were added dropwise. To that reaction mixture, 0.14 mL of NMI (5 mol/eq.) were added dropwise at room temperature under an argon atmosphere. The reaction mixture was stirred for 88 h. The solvent was removed under d pressure and the residue was d by column chromatography with gradient of eluent (CH30H/CH2C12 0/100 to 5/95) to give a desired product as a yellow solid. (7 mg, yield = 3%). MS (ES+) m/z: Found: 653 (M + H+), 675 (M + Na+) C30H30ClN607P required: 652.16 (M), 31P NMR (202 MHz, CD30D): SF 4.39 (s), 4.12 (s), 1H NMR (500 MHz, CD30D): 6H 8.10 (s, 0.5 H, H8), 8.07 (s, 0.5 H, H8), .97 (m, 3H, CHzPh and Naph), 7.43—7.14 (m, 9H, CHZPh and Naph), 5.80-5.81 (m, 1H, H1’), 4.89-4.97 (m, 2H, CHzPh) 4.49—4.53 (m, 2H, H4’and H2’), 4.30—4.35 (m, 1H, H5’), 4.15—4.21 (m, 1H, H5’), 3.87-3.95 (m, 1H, CHCH3), 2.12—2.23 (m, 1H, H3’), 1.86-1.93 (m, 1H H3’), 1.14—1.17 (m, 3H, CHCH3), 13C NMR (125 MHz, CD30D): 6C 174.85 (d JCP = 4.0 Hz, C=O), 174.55 (d JCP = 4.3 Hz, C=O), 158.07, 158.04 (C6), 155.31, 155.28 (C2), 151.34, 151.31 (C4), 149.69 (C-Ar), 147.96 ((1 3Jcp = 7.25 Hz, C-ipso Naph), 147.90 (d 3Jan: 7.0 Hz, C-ipso Naph), 140.70 (C8), 137.21, 137.16 (C—z'pso CHzPh), 136.26 (C-Ar), 130.92, 130.80, 129.56, 129.53, 129.31, 129.27, 129.25, 128.88, 128.81 (CH-Ar), 127.78 (d JCP = 4.7 Hz, CH-Ar), 127.50 (d JCP = 6.2 Hz, CH- Ar), , 126.02, 125.97 (CH-Ar), 119.46, 119.42 (C5), 116.33 (d, JCP = 3.0, CH-Ar), 116.16 (d, JCP = 3.4, CH-Ar), 93.30, 93.27 (C1’), 80.56 (d J: 8.3 Hz, C4’), 80.51 (d J: 8.4 Hz, C4’), 76.61, 76.54 (C2’), 68.74 (d Jan: 5.3 Hz, C5’), 68.54 (d J61): 5.1 Hz, C5’), 67.93, 67.90 (CH2Ph), 51.81, 51.70 (CHCH3), 34.79, 34.53 (C3’), 20.42 (d JCP = 6.5 Hz, CHCH3), 20.23 (d JCP = 7.7 Hz, , HPLC Reverse-phase HPLC g with HzO/CH3CN from 90/10 to 0/100 in 30 minutes, F = 1ml/min, l = 254 nm, tR 18.03 min. 2-Chlor0-3’de0x adenosine 5’-O- 1- hen l 22-dimeth 1 r0 0x -L-alanine hos hateT Compound T was prepared according to the general procedure 1 using 2-chloro-3’- deoxyadenosine (350 mg, 1.25 mmol), N—methylimidazole (490 uL, 6.15 mmol) and phenyl(2,2- dimethylpropoxy-L-alaninyl) phosphorochloridate (1231 mg, 3.69 mmol). Puriﬁcation by column chromatography (eluent system CH30H/CH2C12 0/100 to 5/95) and preparative TLC (1000 um, eluent system CH30H/CH2C12 4/96) afforded the title compound as a white solid (181 mg, 25 %). 31P NMR (202 MHz, : SF 3.93, 3.72. 1H NMR (500 MHz, CD30D): 6H 8.12 (s, 0.5 H, H8), 8.10 (s, 0.5 H, H8), 7.19—7.23 (m, 2 H, Ph), 7.03-7.12(m, 3 H, Ph), 5.84 (d J=2, 0.5 H, Hl’), 5.83 (d J=2, 0.5 H, Hl’), 4.54-4.60 (m, 2 H, H4’and H2’), 4.34-4.38 (m, 0.5 H, H5’), 4.27—4.31 (m, 0.5 H, H5’), 4.16-4.23 (m, 1 H, H5’), 3.80-3.90 (m, 1 H, CHCH3), 3.57—3.73 (m, 2 H OCH2C(CH3)3), 2.18-2.28 (m, 1 H, H3’), 1.94— 1.99 (m, 1 H, H3’), 1.20—1.24 (m, 3 H, CHCH3), 0.81 (s, 4.5 H OCH2(CH3)3), 0.79 (s, 4.5 H OCH2C(CH3)3). 13C NMR (125 MHz, : 6C 175.09 (d 3,161) = 4.75 Hz, C=O), 174.90 (d 3,161) = 5.37 Hz, C=O), 158.10, (C6), 155.31, 155.28 (C2), 152.14 (d 2,161): 6.37 Hz, C-l'pso Ph), 152.13 (d 2J6P= 6.25 Hz, C-l'pso Ph), 151.33, 151.30 (C4), 140.87, 140.76 (C8), 130.78, 130.77 (CH-Ar), 126.17, 126.42 (CH-Ar), 121.45 (d 3,161) = 11.75 Hz, CH-Ar), 121.41 (d 3,161) = 11.75 Hz, CH-Ar), 119.52, 119.48 (C5), 93.49, 93.35 (Cl’), 80.67 (d 3,1: 8.62 Hz, C4’), 80.65 (d 3,1: 8.25 Hz, C4’), 76.70, 76.67 (C2’), 75.43, (OCH2C(CH3)3), 68.68 (d 2J6p= 5.12 Hz, C5’), 68.42 (d 2J6p= 5.12 Hz, C5’), 51.77, 51.60 (CHCH3), 34.94, 34.67 (C3’), 32.36, 32.32 (OCH2C(CH3)3), 26.78, 26.76 (OCH2C(CH3)3), 20.83 (d JCP = 6.25 Hz, CHCH3), 20.61 (d JCP = 7.12 Hz, .

MS (ES+) m/z: Found: 583 (M + H+), 605 (M + Na+) C1N6O7P required: 582.18 (M).

HPLC e-phase HPLC g with H20/CH3CN from 90/10 to 0/100 in 30 minutes, F = 1ml/min, l = 254 nm, tR16.37, 16.55 min. 2-Chloro-3’deox adenosine 5’-O- 1-na ht l 22-dimeth 1 r0 0x -L-alanine hos hateU Compound U was prepared according to the general procedure 1 using ro-3’- deoxyadenosine (350 mg, 1.25 mmol), N—methylimidazole (490 uL, 6.15 mmol) and naphtyl(2,2-dimethylpropoxy-L-alaninyl) phosphorochloridate (1416 mg, 3.69 mmol).

Puriﬁcation by column chromatography (eluent system CH30H/CH2C12 0/100 to 5/95) and preparative TLC (1000 um, eluent system CH30H/CH2C12 4/96) afforded the title nd as a White solid (264 mg, 34 %). 31P NMR (202 MHz, CD30D): SF 4.35, 4.20. 1H NMR (500 MHz, CD30D): 6H 8.23 (s, 0.5 H, H8), 8.21 (s, 0.5 H, H8), 8.11-8.16 (m, 1 H, Naph), 7.86-7.89 (m, 1 H, Naph), 7.69-7.70 (m, 1 H, Naph), 7.54-7.46 (m, 3 H, Naph), 7.37—7.41 (m, 1 H, Naph), 5.95 (d J: 2, 0.5 H, H1’), 5.94 (d J: 1.5, 0.5 H, H1’), .73 (m, 2 H, H4’and H2’), 4.34—4.55 (m, 2 H, H5’), 4.00-4.08 (m, 1 H, CHCH3), 3.66-3.81 (m, 2 H OCH2C(CH3)3), 2.28-2.41 (m, 1 H, H3’), 2.03—2.10 (m, 1 H, H3’), 1.31—1.34 (m, 3 H, CHCH3), 0.90 (s, 4.5 H OCH2C(CH3)3), 0.89 (s, 4.5 H 3)3). 13C NMR (125 MHz, CD30D): 6C 175.11 (d JCP = 4.1 Hz, C=O), 174.85 (d JCP = 5.0 Hz, C=O), 158.10, 158.04 (C6), 155.32, 155.30 (C2), 151.33 (C4), 147.96 (d 2,161): 7.25 Hz, C-l'pso Naph), 147.93 (d 2,161): 7.25 Hz, C-l'pso Naph), 140.84, 140.76 (C8), 136.29 (C-Ar), , 128.82 ), 127.85 (C-Ar), 127.77, 127.74, 127.48, 127.45, , 125.99, 125.96, 122.74, 122.66 (CH-Ar), 119.47 (C5), 116.29 (d 3,161) = 3.4 Hz, CH-Ar), 116.17 (d 3,161) = 2.9 Hz, CH- Ar), 93.42, 93.34 (Cl’), 80.57 (d 3,161) = 8.1 Hz, C4’), 80.53 (d 3,161) = 5.1 Hz, C4’), 76.61, 76.53 (C2’), 75.41, 75.38 (OCH2C(CH3)3), 68.95 (d 2,161): 5.3 Hz, C5’), 68.82 (d 2,161): 5.2 Hz, C5’), 51.84, 51.73 (CHCH3), 35.04, 34.75 (C3’), 32.29 (OCH2C(CH3)3), 26.70 (CH3)3), 20.76 (d 3,161) = 6.4 Hz, CHCH3), 20.55 (d 3,161) = 7.2 Hz, CHCH3).

MS (ES+) m/z: Found: 633 (M + H+), 655 (M + Na+) C28H34C1N6O7P required: 652.16 (M).

HPLC Reverse-phase HPLC eluting with H20/CH3CN from 90/10 to 0/100 in 30 minutes, F = 1ml/min, l = 254 nm, tR 19.16 min.

Compound V was prepared ing to the general procedure 4 using ro-3’- deoxyadenosine (343 mg, 0.66 mmol), lertbutyldimethylsilyl chloride (328 mg 2.18 mmol) imidazole (297 mg, 4.36 mmol). Puriﬁcation by column chromatography (eluent system CH30H/CH2C12 0/ 100 to 12/88) afforded intermediate 1 in a quantitative yield. Next, intermediate 1 (970 mg, 1.89 mmol) was reacted with 12 mL of a solution THF/HzO/TFA 4/1/1.

Puriﬁcation by column chromatography (eluent system CH30H/CH2C12 0/100 to 12/88) afforded intermediate 2 (544 mg, 72 %). Then, intermediate 2 (204 mg, 0.51 mmol) and was reacted with lerlbutylmagnesium chloride and a solution of phenyl(ethyloxy-L-alaninyl) phosphorochloridate (348.56 mg, 1.02 mmol) in anhydrous THF (5 mL). cation by column chromatography (eluent system CH30H/CH2C12 0/100 to 8/92) afforded intermediate 3 (93 mg, 28 %). Finally intermediate 3 (93 mg, 0.14 mmol) was reacted with a solution of THF/TFA/HzO 1/1/1 (3 mL).

Purif1cation by preparative TLC (2000 um, eluent system CH30H/CH2C12 4/96) ed the title compound as a white solid (50 mg, 66 %). (Overall yield 13 %) 31P NMR (202 MHz, CD30D): SF 3.93, 3.72. 1H NMR (500 MHz, CD30D): 6H 8.12 (s, 0.5 H, H8), 8.11 (s, 0.5 H, H8), 7.18-7.23 (m, 2 H, Ph), 7.03—7.12 (m, 3 H, Ph), 5.85 (d J=1.5, 0.5 H, Hl’), 5.84 (d J=2, 0.5 H, Hl’), 4.55-4.62 (m, 2 H, H4’and H2’), 4.34-4.38 (m, 0.5 H, HS’), .32 (m, 0.5 H, HS’), 4.16-4.22 (m, 1 H, HS’), 3.93—4.03 (m, 2 H, OCH2CH3), 3.70-3.84 (m, 1 H, CHCH3), 2.20-2.28 (m, 1 H, H3’), 195 1.99 (m, 1 H, H3’), 1.15—1.21 (m, 3 H, CHCH3), 1.06-1.11 (m, 3 H, OCH2CH3). 13C NMR (125 MHz, CD30D): 6C 173.66 (d 3Jcp = 4.5 Hz, C=O), 173.65 (d 3JCP = 5.3 Hz, C=O), 156.68, 156.70 (C6), 153.93, 153.88 (C2), 150.72 (d 2,161): 6.7 Hz, C-l'pso Ph), 150.71 (d ZJCP: 6.5 Hz, C-l'pso Ph), , 149.94 (C4), 139.41, 139.35 (C8), 129.33 (CH-Ar), 124.74, 124.73 (CH-Ar), 120.03 (d 3,161) = 4.75 Hz, , 119.97 (d 3,161) = 4.87 Hz, CH-Ar), 118.07, 118.03 (C5), 92.02, 91.88 (Cl’), 79.26, 79.19 (C4’), 75.26, 75.24 (C2’), 67.18 (d 2,161): 5.25 Hz, CS’), 66.81 (d 2,161): 5.12 Hz, C5’), 60.96 (OCH2CH3), 50.23, 50.12 (CHCH3), 33.46, 33.21 (C3’), 19.16 (d 3J6? = 6.3 Hz, CHCH3), 18.97 (d 3JCP = 7.2 Hz, CHCH3), 13.10, 13.07 H3).

MS (ES+) m/z: Found: 541 (M + H+), 563 (M + Na+) C21H26C1N6O7P required: 540 (M).

HPLC Reverse-phase HPLC eluting with H20/CH3CN from 90/10 to 0/100 in 30 s, F = 1ml/min, l = 254 nm, tR 12.41, 12.83 min. 2 -iso r0 [ 2S 4R 5R 6-amin0-9H- urin lh drox tetrah drofuran-Z- lmethox henox hos hor lamino r0 anoateW Qno FN 0 NH OH V N—methylimidazole (240 uL, 5 mmol) and a solution of (2S)-isopropyl 2- ((chloro(phenoxy)phosphoryl)amino)propanoate (546 mg, 3 mmol) in anhydrous THF (5 mL) were added dropwisely to a suspension of (2R,3R,55)—2-(6-amino-9H-purinyl) (hydroxymethyl)tetrahydrofuranol (150 mg, 0.6 mmol) in anhydrous THF (3 mL) and the on mixture was stirred at room temperature during a period of 16 hours. Puriﬁcation by column chromatography (eluent system CH30H/CH2C12 0/100 to 6/94) and preparative TLC (2000 uicron, e1uent system CH30H/CH2C12 5/95) afforded the desired nd as white solid (40 mg, 13%).

MS (ES+) m/z.‘ Found: 521.2 (M + H+), 543.3 (M + Na+), 1063.4 (2M + Na+) C31H33N608P required: 520.18(M). 311) NMR (202 MHz, : SF 3.99 (s), 3.82 (s). 1H NMR (500 MHz, CDsOD): 5H 8.16 (s, 0.5H, H8), 8.15 (s, 0.5H, H8), 811 (s, 1H, H-2) 7.23—7.20 (m, 2H, Ph), 7.11—7.03 (m, 3H, Ph), 5.91 (d J = 2.0Hz, 0.5H, H1’), 5.90 (d J = 2.0Hz, 0.5H, H1’), 4.85-4.79 (m, 1H, CH(CH3)2, 4.64-4.63 (m, 1H, H4’), .57 (m, 1H, H2’), 437— 4.33 (m, 1H, H5’), 4.31-4.28 (m, 1H, H5’), 3.74—4.22—417 (m, 1H, H5’), 3.70 (m, 1H, CH ala), 2.02—1.97 (m, 1H, H3’), .01 (m, 1H, H3’), 1.18-1.14 (m, 3H, CH3), 1.24 (m,6H, CH(CH3)2) HPLC Reverse-phase HPLC eluting with H20/CH3CN from 100/10 to 0/100 in 30 minutes, F =1ml/min, 7» = 200 nm, showed two peaks of the diastereoisomers with tR 11.58 min. and 02 11.92 min.

Solvents and Reagents. The following anhydrous solvents were purchased from Sigma-Aldrich: dichloromethane (CH2C12), trimethylphosphate ((CH30)3PO). Amino acid esters commercially available were purchased from Sigma-Aldrich. All reagents cially available were used without further ation.

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 X 20 cm, 500-2000 um) were purchased from Merck.

Flash Column Chromatography. Flash column chromatography was carried out using silica gel supplied by Fisher (60A, 35-70 um). 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 t were identiﬁed by TLC, and pooled and the solvent was removed in vacuo.

High Performance Liquid tography (HPLC). 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 C18, 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, 5 u, 150 x 4.6 mm (as an analytic column).

For the method of elution see the experimental part.

Nuclear Magnetic Resonance (NMR). 1H NMR (500 MHz), 13C NMR (125 MHz), 31P NMR (202 MHz) and 19F NMR (470 MHz) were recorded on a Bruker Avance 500 MHz spectrometer at 25 °C. Chemical shifts (6) are quoted in parts per million (ppm) relative to internal MeOH-d4 (5 3.34 lH-NMR, 5 49.86 13C-NMR) and d4 (5 7.26 1H NMR, 5 77.36 13C NMR) or external 85 % H3PO4 (8 0.00 31P 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 ent). The assignment of the s in 1H NMR and 13C NMR was done based on the analysis of ng constants and additional mensional 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 oscopy) in either positive or negative mode.

Purity of ﬁnal compounds. The 295% purity of all the ﬁnal compounds was conﬁrmed using WO 83830 HPLC analysis.

Example 2 - Cytoxicity Exemplifled compounds embodying the present invention were assessed in the following procedures for their anti-cancer potency.

In vitro viability assays were performed to assess the effects of nds on cell viability in 7 selected cell lines over 72 hr using the CellTiterGlo (CTG, Promega-G7573) assay. The tests were performed in ates with treatment of compounds at 9 points, 3.16 folds ion in 96 well plates over ~72 hr. The compound starting concentrations were 198 mM. Cell viability assay using CellTiterGlo in 96-well plate were performed. The compound treatment was 72 hrs, under standard growth conditions, and in duplicate. 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 (2ul+200ul). After nds were dissolved in media (media was also warmed to 37°C). Media containing compounds were warmed to 37°C in incubator and then compounds in media were added to cell plates (50ul+50ul), in duplicates. The compounds ﬁnal concentrations were from 198M to 19.9nM. All compound solubilities were checked and ed again, then the plates were transferred to C02 tissue culture tor immediately and incubated for 3 days. DMSO final concentration is 0.5%.

The results of the initial screening are presented in Table II. A represents a relative ICso of from 0.1 to 5 uM, B represents a relative ICso r than 5 M and up to 15 uM, C represents an relative ICso of greater than 15 M and up to 100 M, and D represents an relative ICso of greater than 100 uM. 8.8.2 mm 8 8 8 88 8 a 8 5 8 8 350m "SE ﬂan—«9:8 32 o < < o m m o o o o o "Em—9:00 we Emo-§oo 8.8.2 EEDESB :oEn—EE S 02 02 8 02 8 02 88 8 02 88 3302085on omﬁnoaom 32 0 < < m m o o o o o m 55::me 8.8.2 "£3.28 "8-4m mm 8 02 g 02 8 ow 88 E 8 02 "SH—ﬂu memOH 32 MEEBNVEB oﬁﬁxe o m m o m 0 0 o 0 o o msonomoﬁob: H2180w 8.8.2 £70m 8 8 02 8 02 mo 8o 8 8o 8 8 350m 32 "TQM 0 m m o m 0 0 o 0 o o .ﬁﬁonéoﬁmsonomoﬁohﬁ : "8.8.2 MEEBNVEB 3:ch 28? ~85¢: mm 8 02 8 02 8 02 02 8 02 02 oumﬁn—oamﬁb "Nomvr 8 32 o < < < < o o o m < < 33m MEEBNVEB 28 888850 < m o o o m H m H m EN oumﬂn—oamﬁb Table II (cont) MCF—7h HepG2i Compound IC50 M.I.% IC50 M.I.% cordycepin C 78 C 66 A A 94 B 76 B A 99 B 95 C B 87 C 59 D A 100 B 99 G B 97 C 67 H A 94 B 55 I B 99 C 90 E C 78 C 59 J C 97 C 55 F B 99 C 84 hMCF-7: breast adenocarcinoma; iHepG2: cellular carcinoma A subset of compounds of the invention were then assayed for their xic activity in a r array of different solid tumours and haematological malignancies using the following assay.

Solid tumour and haematological malignancy assay [11 Vitro Viability assay were performed to assess the effects of compounds on cell Viability in selected cell lines over 72 hr 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 hr. The compound starting trations were 198 mM. 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 [2u1+200ul). 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 (50ul+50ul) in :luplicates. The compounds’ final trations were from 198M to . All nd solubilities were checked and recorded again, then the plates were transferred to C02 tissue culture incubator ately and incubated for 3 days. DMSO final concentration is 0.5%.

The following cell lines were tested and are referred to in the Table IV below: Table III Cell line Malignancy Cell line Malignancy VlOLT-4 Acute lymphoblastic leukaemia HEL92. 1.7 oleukaemia CCRFCEM Acute lymphoblastic leukaemia HL-60 Promyelocytic leukaemia RL Non-Hodgkin’s lymphoma MV4-ll Biphenotypic B myelomonocytic leukemia HS445 n lymphoma HepG2 cellular carcinoma RPMI8226 Human multiple myeloma HT29 Colon adenocarcinoma K562 Chronic myelogenous leukaemia BXPC-3 Pancreatic cancer KG-l Acute myelogenous leukaemia MCF-7 Breast adenocarcinoma THP-l Acute monocytic leukaemia MiaPaCa2 Breast adenocarcinoma Z-l38 Mantle cell lymphoma SW620 Colon adenocarcinoma 929 Plasmacytoma Jurkat acute T cell leukaemia The results of the further screening are presented in Tables . For Tables IV to VI: A represents an absolute ICso of from 0.1 uM to 5 uM, B represents an absolute ICso greater than 5 uM and up to 15 uM, C represents an absolute ICso of greater than 15 uM and up to 100 uM; and D represents an absolute ICso of greater than 100 uM. For Table VII: A represents an absolute EC50 of from 0.1 M to uM, B represents an absolute ECso greater than 5 uM and up to 15 uM, C ents an absolute EC50 of greater than 15 uM and up to 100 uM; and D represents an absolute EC50 of greater than 100 2015/053628 ,5: om 02 9: 02 02 mo ch: E 9: a 2: 02 ma REZ E3 omaioz Q 99 o m < m m 0 92 a2 o < < m < m ,5: go 02 8 NS 3 02 .52 N mm co S: mm mm IE 3m: .85: $2 o 0 m 0 o 0 $2 $2 o Q m 9 0 0 $2 E mo mo 02 8 mm AXLE $5 3 a mo Q 8 8 :52 38 o < < m m m :-§2 89 32 Q < < < < < xi m- E 3 8 02 9 :52 $5 Q 02 2: 2: 2: 02 zmoﬁoo 99 o o o o o a oﬁizé EB a2 o < < m < < 3.58 @858 3.58 >~ E 3:5 < m o m m E UQEU Bash 35 aaooéhoo < m o m m 28? 2%? WO 83830 8 8 8 2: 8 8 8% 2 8 8 8 8 3 8% S mm 8 9: 0 < < m < m N08: a2 o o m o o o 835 $2 o o m u Ma 2: 8 8 8 z: 8% 8 a 8 8 M: E $2 mm 8 8 we o m < < m 0 @8me 22 o u m o u o @83me a2 o m < m H 8 8 2: 02 § 8% 8 8 02 02 on 8 8% R 8 8 we 85 E02 a < < m < m a": m u m m u u a": o < < < 8 8 NS m: 2: 8 $2 G 8 8 03 8 8 83 E 8 8 8 a m m u o o 3-4: 8E a2 a m < m m m a2 a m m o 38828 388 :88 < m o m m E 35 88828 < m o m m E 3:5 m o 288 88828 < 288 8 E 3:2 H 2: E: z: 8 3% o 3 S: 02 mm o o #502 22 o o o m u a2 o u 0 m o o3: 3% E x: Q: 8 3 z: a .2 8 i: 2: 2: K Neg 39302 92 o u m m u m o 32 o 0 o m 0 £22 w 8 8 02 8-4: m: 3 .2 H mm a me cm E02 5M 92 a o m o o m m 22 o a 0 o 0 3:4 S a Ma 9: me a on .2 w- 02 Q: 2: mo 3E 8%: o 32 o o 0 o o o o 22 o o m m 0 :aooﬁoo-ozo-m :88 35 =58€50¢29~ A M 2 z A M 2 z m m > > 5 28? 28¢ 28¢ WO 83830 8a: 8 g a mm 8 8.8.2 8 8 8 m: 8 834 8 8 8 8va SQ 32 o o m o 0 32 m m < m < 38 o < < 8 z: 2: 8 92 8a: 8 9: 8 8 8 8.3 8a: 8 8 8 .8qu 562 a2 m < < < < a2 m < < < < o < < 8 2: 02 S 8 S 8.8.: z: 8 mm E 5.2 02 02 8 zmobso $8322 82 0 < 0 m m :2 < m < v < < < < 38828 388 £88300 3.38 3:5 o m o m o m o m o m E E 2:8 38828 H3 288 #3 28? .E 02 w 8 TOM m H u -N 208 Q u Nomm Tag g souso>ﬁ < m Q u H.N0 2: amok/m mo oNNwASEm Q u 0.00 mwssomﬁoo 3me 2: 8 a m NdOH 830 00-15 308 m m E 0N0 m.NOH < < EAUZ 68mg NOQQE u m 0 3:: 2-3/2 .03 :8 Q m 02 demqmm mm wFAOE HdOH 38me 3:: Emoimou :8 -moééz Q u N.w0 53:3 :m < m 2x885 3:506 0:4-mOmxm Q U biog 0.03 330% : @033 ":23 3.58 06m 30m— $3 2: o m :> 5 mwssomﬁoo 82: E39850 m 28a -_U-~ 250 28¢ ~ E39850 =< 3800 Example 3 - Assessment of cytotoxiciﬂ and cancer stem cell activity A further comparative analysis of the toxicity of compounds in the acute myeloid leukaemia (AML) cell line KGla over an extended dose range was carried out, and the relative effect assessed of the compounds on the leukaemic stem cell (LSC) compartment within the KGla cell line, across the entire dose range.

Materials and Methods KG]a cell culture conditions The KGla cell line was maintained in RPMI medium (Invitrogen, Paisley, UK) mented with 100 ml penicillin, lOOug/ml streptomycin and 20% foetal calf serum. Cells were subsequently aliquoted (105 cells/lOOul) into 96-well plates and were incubated at 37°C in a humidiﬁed 5% carbon dioxide here for 72h in the presence of side analogues and their respective proTides at concentrations that were experimentally determined for each series of compounds. In addition, l 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 ofin vitro apoptosis Cultured cells were harvested by centrifugation and then resuspended in 195 ul of calcium-rich buffer. Subsequently, 5 ul of Annexin V (Caltag Medsystems, h 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 190 ul of calcium-rich buffer together with lOul of propidium .

Apoptosis was assessed by dual-colour immunoﬂuorescent ﬂow try as described previously. Subsequently LDso values (the dose required to kill 50% of the cells in a e) were calculated for each nucleoside analogue and ProTide.

Immunophenotypic identification ofthe leukaemic stem cell compartment KGla cells were cultured for 72h in the presence of a wide range of concentrations of each compound d. Cells were then harvested and labelled with a cocktail of ineage antibodies (PE-cy7), anti-CD34 (FITC), anti-CD38 (PE) and anti-CD123 (PERCP cy5). The sub- population sing a 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 WO 83830 then plotted on a dose-response graph and the s of the compounds were compared with each other, and with the parent nucleoside.

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. LDso 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 The in vitro drug sensitivity was measured using the AnneXin idium iodide assay.

Compound A showed increased in vitro y when compared to Cordycepin (P<0.0001). 2- F-Cordycepin was significantly more potent than Cordycepin (P<0.0001) and all of the ProTides tested showed increased potency when compared to the parental nucleoside (Figure l).

These experiments conﬁrmed that compound A showed evidence of increased potency in the stem cell compartment at concentrations above lmM. As can be seen from Figure 2, compound A demonstrated an ability not only to reduce cancer stem cell numbers in total, but also to reduce numbers of such cells as a proportion of the total of cancer cells present in culture. This indicates the ability of nd A to preferentially target cancer stem cells. At the higher concentrations tested (lmM and above), the ability of compound A to entially target LSCs was signiﬁcantly greater than that of the parent compound.

The 2-F-Cordycepin proTides compounds P, Q and R also showed preferential targeting of LSCs that was significantly improved when compared with the parental nucleoside. In contrast, while compound 0 was able to bring about a reduction in the proportion of LSCs present in treated cell populations (indicating an y to target LSCs), its activity was not significantly different to 2- F-Cordycepin at any of the concentrations tested. Figure 3 shows the comparison between 2-FCordycepin and all of the proTides tested, while individual isons are shown in the panels ofFigure 4. e 4 — Further cytotoxicity assessment and Inhibition s Certain compounds of the invention were subjected to further studies to test the xic activity of certain compounds of the invention and also to e their activity against 4 haematological cancer cell lines ° TdT positive CEM (Human ALL) ° TdT negative K562 (Human CML) ° TdT negative Hl-6O (Human ANLL) ° RL (CRL-226l) non-HD ma The concentrations of the active metabolite dATP (Cordycepin triphosphate) in these cell lines was also measured.

The cytotoxic ty and the intracellular 3’-dATP concentrations were also studied in the presence of hENTl, Adenosine Kinase (AK) and Adenosine Deaminase pharmacological inhibitors in CEM and RL cancer cell lines. Said inhibitors that mimic known cancer resistance mechanisms.

W0 83830 Methods Cell Culture HL-60 (ATCC® 0TM), K562 (ATCC® CCL-243TM), CCRF-CEM (ATCC® CRM-CCL-119TM) and RL (ATCC® CRL-2261TM) leukaemia cell lines, obtained from the American Type Culture Collection (ATCC), MiddleseX. HL-60 and K562 cell lines are ucleotidyl transferase-negative (TdT-ve), whereas CCRF-CEM cell line is TdT+ve.

HL-60 cell line is of acute promyelocytic leukaemia; K562 is a CML cell line, CCRF-CEM cell line is of acute lymphoblastic leukaemia (ALL), and RL is non-Hodgkin’s lymphoma cell line.

Maintenance of Cell Lines HL-60, K562, CCRF-CEM and RL cell lines were cultured in RPMI-1640 medium (Sigma Aldrich, UK), which were 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% C02.

Aden0sine phosphate (ATP) Assay The amount of ATP was used as a measurement of cell number and cell viability. ATP ViaLightTM plus assay kit (Lonza, USA: t No. LT07-121) to detect ATP in cells d in luminescence compatible 96 well plates (initial tration of cells was 1X104 cells/well) with cordycepin and ProTides at concentrations of: 0, 0.1, 0.5, 1, 5 and 10 uM, followed by incubation for 72 hours at 37°C incubator with % C02. For inhibitor studies, 10 uM of NBTI or 1 uM EHNA or A-134974 was added and left for 5 minutes before adding the drugs (see n 5 for inhibitor details).

After incubation, 50ul of cell lysis reagent was added to the 96 well plates to release the intracellular ATP, followed by 100 pl of ATP monitoring reagent (AMR). The luminescent values of each well were determined using FLUOstar OPTIMA microplate reader (BMG Labtech) which convert ATP into light by using luciferase enzyme. Therefore, the amount of luminescence produced was directly proportional to the amount of ATP.

Treating Cells and Extracting Samplesfor Intracellular triphosphate analysis Cell lines with 5X106 cells/ml were used. Cells were treated with 1 ul of 50uM of each of cordycepin and compounds A, B, D, E and F and ted for 2 hours at 37°C with 5% C02. After incubation, cells were centriﬁJged (ambient, 1200 rpm, 5 minutes), the culture medium supematants were removed, and the cell pellets were washed with 1ml of PBS and centriﬁJged nt, 1200 rpm, 5 minutes). The supematants were removed, the pellets were reconstituted in 100ul of PBS and 100ul of 0.8M perchloric acid and vortex mixed and kept on ice for 30 minutes. Then centriﬁiged (ambient, 1200 rpm, 5 minutes) and 180ul of the supernatant was transferred to new tubes and stored at -80°C until time of analysis.

During is, 90ul of the extract was transferred to the fresh tubes. 25 ul of 1M ammonium acetate was added to the extract, and then neutralised by on of 10ul of 10% ammonia and 5 ul of deionised water, then transferred to LC-MS vials and 10ul was injected into the UPLC-MS/MS system.

Inhibitor Studies Cell lines were treated in the same way as described above but before treatment with drugs, a number of inhibitors were added: 1) Nitrobenzylthioinosine (NBTI) -Aldrich, St. Louis, MO, product # N2255) blocks side transporters 2) EHNA hydrochloride (Sigma-Aldrich, St. Louis, MO, product # E114) blocks adenosine 3) Adenosine kinase inhibitor A-134974 dihydrochloride hydrate (Sigma-Aldrich, St. Louis, MO, product # A2846): blocks adenosine kinase Cells were treated with 10 uM ofNBTI or 1 uM EHNA or A-134974 and left for 5 minutes before adding the drug. The cells were then incubated for 2 hours at 37°C with 5% C02.

LC—MSMSAnalysis The analytes were resolved using an ultra-performance liquid chromatography system (Accela UPLC, Thermo Scientific, UK) ed with a ic Ax5um, 50X2.1mm column (Thermo on Corporation, Murrieta, CA, USA) and mobile phase consisting of a mixture of 10mM NH4Ac in ACN/HzO (30:70v/v), pH6.0 (A), and 1mM NH4Ac in ACN/HzO (30:70v/v), pH10.5 (B). The mobile phase gradient will be employed, comprising: buffer A=95% at 0-0.5 min, from 95 to 0% over 1.25 s, held at 0% for 1.75 minutes, from 0-95% over 0.1 min, ending with 95% for 2.9 minutes, all at a ﬂow rate of 500ul/min.

Eluting compounds of interest were detected using a triple stage quadrupole Vantage mass spectrometry system (Thermo Scientific, UK) equipped with an electrospray ion source. s were analysed in the Multiple Reaction Monitoring, negative ion modes at a spray voltage of 3000V. Nitrogen was used as sheath and auxiliary gas at a ﬂow rate of 50 and 20 arbitrary units, respectively. Argon was used as collision gas with pressure of 1.5 mTorr. The optimum transitional daughter ions mass and collision energy of each analyst was as follows: 3’ATP 490.1—> 392.1 (collision energy 19V) and the internal standard ChloroATP 539.9 —> 442.2 (collision energy 24V).

Statistical Analysis The dose-response curves of cytotoxicity of the drugs was determined using non-linear regression is of percentage cell viability versus concentration and ECso values were obtained. The intracellular assay was ted in ﬁve replicates for each condition. Intracellular assay was determined using paired t test (two-tailed) analysis of ATP concentration and p-values were obtained. For all the analysis, Prism Software program (GraphPad Software) was used and Microsoft Powerpoint® 2013 was used to plot the results.

Results Summary ICso Table (11M) Cordycepin 19.5 CEM ) K562 (FD) :fold diﬂerence compared to Cordycepin : Cordycepin IC5o/Pr0Tide IC50 Summary Mean Intracellular 3'-dATP levels ("g/ml) A B D E F Cordycepin (FD) (FD) (FD) (FD) (FD) 0.2 3.7 11.5 2.9 0.2 1.1 CEM (19) (58) (15) (1) (6) CRL (33) (14) (2) (1) (4) (FD) : fold nce compared to Cordycepl'n : Protl'a’e ellular TP/Cora’ycepin Intracellular TP Compounds A and B were the best performers with ICso from 3 to ISO-fold better than cordycepin Compounds A and B produced intracellular 3’-dATP concentrations 3 to 56-fold better than cordycepin.

Summary ICso Table (A1] in 11M) CEM CRL (FD) (FD) 11.5 7.3 Cordycepin 57.7 (5) 17.9 (2) 0.7 (-16) 13.2 (2) 29.2 (3) 28.6 (4) 1.4 3.6 2.0 (1) 2.6 (1) 3.1 (2.2) 2.9 (1) 3.6 (3) 10.2 (3) 0.9 3.1 1.4 (1) 2.6 (1) 1.3 (1) 5.2 (2) 1.3 (1) 2.8 (1) 9.9 8.2 17.1 (2) 8.2 (1) 13.4 (1) .3 (1) (FD) :fold diﬂerence compared to control Summary Mean Intracellular 3'-dATP levels ("g/ml) CEM CRL (FD) (FD) Control 0.24 0.10 Cordycepin NBTI 0.14 (1) 0.06 (1) EHNA 9.01 (38) 1.85 (19) AK 0.31 (1) 0.16 (1) Control 1.30 0.31 A NBTI 0.99 (1) 0.32 (1) EHNA 1.35 (1) 0.27 (1) AK 1.20 (1) 0.30 (1) Control 4.07 0.59 B NBTI 3.14 (1) 0.68 (1) EHNA 3.62 (1) 0.67 (1) AK 2.99 (1) 0.77 (1) Control 0.32 0.08 E NBTI 0.17 (1) 0.12 (1) EHNA 0.21 (1) 0.07 (1) AK 0.19 (1) 0.06 (1) (FD) :fold diﬂerence compared to control NBTI, AK and EHNA did not affect the intracellular 3’-dATP generated by the three compounds of the invention tested indicating that these inhibitors do not interfere with the metabolism by which the nds of the invention generate the active agent 3’-dATP within the hematological cancer cell lines used in this study. As these inhibitors mimic known cancer resistance mechanisms, these results indicate that the compounds of the ion will be less susceptible to cancer ance mechanisms that cordycepin.

Claims

1. 3’-Deoxyadenosine-5’-O-[phenyl(benzyloxy-L-alaninyl)] phosphate or a pharmaceutically acceptable salt, ester, salt of an ester, or solvate of the compound thereof.

2. 3’-Deoxyadenosine-5’-O-[phenyl(benzyloxy-L-alaninyl)] phosphate according to claim 1

3. A compound according to claim 1 or 2 for use as a medicament.

4. Use of a compound according to claim 1 or 2 in the manufacture of a medicament for the prophylaxis or ent of .

5. The use according to claim 4 comprising targeting cancer stem cells.

6. The use according to claim 5, n the cancer is selected from leukaemia, lymphoma, breast cancer, lung cancer, colon cancer, prostate cancer, ovarian cancer, skin cancer, bladder cancer, y cancer and pancreatic cancer.

7. The use according to claim 5, wherein the cancer is selected from breast cancer, CNS cancer, colon cancer, Ewing’s sarcoma, melanoma, liver cancer, cholangiocarcinoma, ovarian cancer, pancreatic cancer, non small cell lung cancer, bladder cancer, acute myeloid leukaemia, B-acute blastic leukaemia, B-acute lymphoblastic leukaemia, multiple myeloma and T- acute lymphoblastic leukaemia.

8. The use according to claim 4 wherein the cancer is selected from the group ting of: leukaemia, lymphoma, multiple myeloma, lung cancer (including non small cell lung cancer and small cell lung cancer), liver cancer, breast cancer, bladder cancer, prostate cancer, head and neck cancer, neuroblastoma, sarcoma (including Ewing’s sarcoma), thyroid carcinoma, skin cancer (including melanoma), oral squamous cell carcinoma, urinary r cancer, Leydig cell tumour, biliary cancer, such as cholangiocarcinoma or bile duct cancer, pancreatic cancer, colon cancer, colorectal cancer and gynaecological s, including ovarian cancer, trial cancer.

9. The use ing to claim 8 wherein the cancer is leukaemia or lymphoma.

10. The use according to claim 9 wherein the leukaemia is ed from the group comprising acute lymphoblastic leukaemia, acute myelogenous leukaemia, acute promyelocytic leukaemia, acute lymphocytic leukaemia, chronic myelogenous leukaemia, chronic lymphocytic mia, monoblastic leukaemia, hairy cell mia, Hodgkin ma and non-Hodgkin lymphoma.

11. The use according to claim 10 wherein the leukaemia is acute lymphoblastic leukaemia.

12. The use according to any one of claims 4 to 11, wherein the cancer is relapsed or refractory cancer.

13. The use according to any one of claims 4 to 11, wherein the cancer is a atic cancer.

14. A pharmaceutical ition sing a compound according to claim 1 or 2 in combination with a pharmaceutically acceptable carrier, diluent or excipient.

15. The compound according to claim 1, substantially as herein described with reference to any one of the Examples and/or s thereof.