2' AND/OR 5' AMINO-ACID ESTER PHOSPHORAMIDATE 3'-DEOXY ADENOSINE DERIVATIVES AS ANTI-CANCER COMPOUNDS

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 in homo sapiens. The compounds are derivatives of cordycepin (3′-deoxyadenosine).

Claims

1. A compound which is 3′-Deoxyadenosine-5′-O-[phenyl(benzyloxy-L-alaninyl)] phosphate: ##STR00033## or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1, in the form of a pharmaceutically acceptable salt thereof, wherein the salt is selected from the group consisting of acetate, benzenesulfonate, benzoate, bicarbonate, bitartarate, bromide, calcium edetate, camsylate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, glyceptate, gluconate, glutamate, glycollylarsanlate, hexylresourcinate, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, maleate, mandelate, mesylate, methylsulfate, mucate, napsylate, nitrate, pamoate, pantothenate, phosphate, diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, teoclate, tosylate, and triethiodide.

3. A pharmaceutical composition, comprising a compound of claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

4. The pharmaceutical composition of claim 3, in an oral dosage form.

5. The pharmaceutical composition of claim 3, in a liquid dosage form.

6. The pharmaceutical composition of claim 5, in an aqueous solution of suspension.

7. The pharmaceutical composition of claim 6, in an intravenous formulation

8. The pharmaceutical composition of claim 6, in a parenteral formulation.

9. The pharmaceutical composition of any one of claims 3, that contains from 50 mg to 700 mg of the compound.

10. A method of treating a cancer, comprising administering an effective amount of a compound of claim 1 or a pharmaceutically acceptable salt thereof, optionally in a pharmaceutically acceptable carrier, to a patient in need thereof

11. The method of claim 10, wherein the patient is a human.

12. The method of claim 11, wherein the cancer is a hematological cancer.

13. The method of claim 11, wherein the cancer is a solid cancer.

14. The method of claim 12, wherein the hematological cancer is selected from the group consisting of acute lymphoblastic leukemia, chronic myelogenous leukemia, acute myelogenous leukemia, acute monocytic leukemia, non-Hodgkin's lymphoma, Hodgkin lymphoma, human multiple myeloma, mantle cell lymphoma, erythroleukemia, acute T-cell leukemia, promyelocytic leukemia, and biphenotypic B myelomonocytic leukemia.

15. The method of claim 13, wherein the solid cancer is selected from the group consisting of plasmacytoma, colon adenocarcinoma, breast adenocarcinoma, liver cancer, head and neck cancer, thyroid carcinoma, skin cancer, oral squamous cell carcinoma, urinary bladder cancer, biliary cancer, colorectal cancer, and gynecological cancers.

16. The method of claim 11, wherein the cancer is comprised of cancer stem cells.

17. The method of claim 11, wherein the cancer is refractory.

18. The method of claim 11, wherein the cancer is relapsed.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0151] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

[0152] FIG. 1. Comparison of the LD.sub.50 values for Cordycepin, Compounds A, 2-F-Cordycepin, Compounds O, P, Q and R. All assays were carried out using KG1a cells and data are presented as mean(±SD) of five independent experiments.

[0153] FIG. 2. Analysis of the leukaemic stem cell (LSC) targeting capacity of Cordycepin and Compound A. The previously generated data (ii) is shown for comparison. All data are the mean(±SD) of three independent experiments.

[0154] FIG. 3. Analysis of the LSC targeting capacity of 2-F-Cordycepin and Compounds O, P, Q and. All data are the mean(±SD) of three independent experiments.

[0155] FIG. 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

[0156] As used herein, the term “alkyl” refers to a straight or branched saturated monovalent (except where the context requires otherwise) cyclic or acyclic hydrocarbon radical, having the number of carbon atoms as indicated (or where not indicated, an acyclic alkyl group 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 R.sub.1, R.sub.2, R.sub.3 and R.sub.4. By way of non-limiting examples, alkyl groups can include methyl, ethyl, propyl, butyl, pentyl, hexyl, octyl, nonyl and dodecyl.

[0157] As used herein, the term “alkenyl” refers to a straight or branched unsaturated monovalent (except where the context requires otherwise) acyclic or cyclic hydrocarbon radical having one or more C═C double bonds and having the number of carbon atoms as indicated (or where not indicated, an acyclic alkenyl group 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 substituents that may be present on R.sub.1, R.sub.2, R.sub.3 and R.sub.4. By way of non-limiting examples, alkenyl groups can include vinyl, propenyl, butenyl, pentenyl and hexenyl.

[0158] As used herein, the term “alkynyl” refers to a straight or branched unsaturated monovalent (except where the context requires otherwise) acyclic or cyclic hydrocarbon radical having one or more C↓C triple bonds and having the number of carbon atoms as indicated (or where not indicated, an acyclic 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 R.sub.1, R.sub.2, R.sub.3 and R4.

[0159] 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.

[0160] As used herein, the term “aryloxy” refers to the group aryl-O—, where aryl is as defined 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—.

[0161] As used herein, the term “alkoxyalkyl” refers to an alkyl group having an alkoxy substituent. Binding is through the alkyl group. The alkyl moiety and the alkoxy moiety are as defined herein with respect to the definitions of alkyl and alkoxy, respectively. The alkoxy and alkyl moieties may each be substituted by one, two or three substituents as set out above with regard to the definition of alkyl.

[0162] As used herein, the term “arylalkyl” refers to an alkyl group having an aryl substituent. Binding is through the alkyl group. The aryl moiety and the alkyl group are as defined herein with respect to the definitions of aryl and alkyl, respectively. The aryl and alkyl moieties may each be substituted by one, two or three substituents, the substituents being as defined herein with respect to the definitions of those substituents that may be present with respect to aryl and alkyl, respectively. In a preferred embodiment, arylalkyl is benzyl, which is Ph-CH.sub.2—.

[0163] As used herein, the term “alkoxyaryl” refers to an aryl group having an alkoxy substituent. Binding is through the aryl group. The alkoxy moiety and the aryl moiety are as defined herein with respect to the definitions of alkoxy and aryl, respectively. The alkoxy and aryl moieties may each be substituted by one, two 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.

[0164] As used herein, the term “cycloalkylaryl” refers to an aryl group having a cyclic alkyl substitutent. Binding is through the aryl group. The cycloalkyl moiety and the aryl moiety are as defined herein with respect to the definitions of cycloalkyl and aryl, respectively. The cycloalkyl moiety and the aryl moiety may each be optionally substituted by one, two or three substituents as set out herein with regard to the definitions of alkyl and aryl, respectively.

[0165] As used herein, the term “aryl” refers to a monovalent (except where the context requires otherwise) 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 containing 7, 8, 9 or 10 carbon atoms; or an aromatic fused tricyclic ring system containing 10, 11, 12, 13 or 14 carbon atoms. Non-limiting examples of aryl include phenyl and naphthyl. In a preferred embodiment, optional substituent groups on an aryl group can be independently selected from hydroxy, C.sub.1-6acyl, C.sub.1-6acyloxy, nitro, amino, carboxyl, cyano, C.sub.1-6alkylamino, diC.sub.1-6alkylamino, thiol, chloro, bromo, fluoro, iodo, SO.sub.3H, SH and SR′, wherein R′ is independently selected from the same groups as R.sub.1 with respect to formula Ia.

[0166] As used herein, the term “.sub.5-30heteroaryl” refers to a monovalent (except where the context requires otherwise) unsaturated aromatic 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 preferred embodiment has one, two or three fused rings. Available carbon atoms and/or heteroatoms in the ring system may be substituted on the ring with one, two, three, four or five substituents, as set out above with respect to the substituents that may be present on the group Ar. Heteroaryl groups can include an aromatic monocyclic ring system containing 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.

[0167] As used herein, the term “.sub.5-20heterocyclyl” refers to a monovalent (except where the context requires otherwise) saturated or partially unsaturated heterocyclic radical 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 embodiment, the radical has 5 to 10 ring members. Heterocyclyl radicals can include: a monocyclic ring system having five ring members of which at least one ring member is a N, O or S atom and which optionally contains one additional ring O atom or one, two or three additional ring N atoms; a monocyclic ring system having six ring members of which one, two or three ring members are a N atom and which optionally includes an O atom; a bicyclic fused ring system having nine ring members of which at least one ring member is a N, O or S atom and which optionally contains one, two or three additional ring N atoms; or a bicyclic fused ring system having ten ring members of which one, two or three ring members are a N atom. Examples include, and are not limited to, pyrrolinyl, pyrrolidinyl, 1,3-dioxolanyl, imidazolinyl, imidazolidinyl, pyrazolinyl, pyrazolidinyl, piperidinyl, morpholinyl or piperazinyl.

[0168] Available ring carbon atoms and/or ring heteroatoms of the “heterocyclyl” ring systems described above may be substituted with one, two, three, four or five substituents. Where the ring(s) is substituted with one or more heteroatoms, the heteroatom substituents are selected from halogen (F, Cl, Br and I) and from oxygen, nitrogen and sulphur, where the oxygen, nitrogen or sulphur form part of a sub stituent moiety. Where the ring(s) is substituted with one or more heteroatoms, preferably there are 1, 2, 3 or 4 heteroatom substituents selected from the group consisting of oxygen, nitrogen, sulphur and halogen. Examples of substituent groups that can be present on the heterocyclic ring system can be independently selected from hydroxy, C.sub.1-6acyl, C.sub.1-6acyloxy, nitro, amino, carboxyl, cyano, C.sub.1-6alkylamino, diC.sub.1-6alkylamino, thiol, chloro, bromo, fluoro, iodo, SO.sub.3H, SH and SR′, wherein R′ is independently selected from the same groups as R.sub.1 with respect to formula Ia.

[0169] As used herein, the term “acyl” refers to a straight or branched, saturated or unsaturated, substituted or unsubstituted, monovalent (except where the context requires otherwise) radical that includes the moiety —C(═O)—, where binding is through the —C— atom of —C(═O)— moiety, and has the number of carbon atoms indicated (or where not indicated, an acyl group has 1-6, or 1-4 or 1-2 carbon atoms, including the C atom of the —C(═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)—, CH.sub.3C(═O)—, C.sub.2H.sub.5C(═O)—, C.sub.3H.sub.7C(═O)—, C.sub.4H.sub.9C(═O)— and C.sub.5H.sub.11C(═O)—.

[0170] As used herein, the term “acyloxy” refers to a straight or branched, saturated or unsaturated, substituted or unsubstituted monovalent (except where the context requires otherwise) radical that includes the moiety —C(═O)—O—, where binding 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, including the carbon atom of the —C(═O)—O)— moiety), optionally substituted with one, two or three of the substituents that may be present on R.sub.1, R.sub.2, R.sub.3 and R.sub.4. By way of non-limiting examples, acyloxy groups include HC(═O)—O—, CH.sub.3C(═O)—O—, C.sub.2H.sub.5C(═O)—O—, C.sub.3H.sub.7C(═O)—O—, C.sub.4H.sub.9C(═O)—O— and C.sub.5H.sub.11C(—O)—O—.

[0171] As used herein, the term “C.sub.2-6ester” refers to a substituted or unsubstituted monovalent (except where the context requires otherwise) radical that comprises R.sub.18C(═O)—O—R.sub.19, where R.sub.18 is selected from the group consisting H and C.sub.1-4alkyl and R.sub.19 is selected from the group consisting of C.sub.1-5alkyl, subject to the maximum total number of C atoms, including the C atom of the —C(═O)—O— moiety, of R.sub.18C(═O)—O—R.sub.19 being six. Binding is through R.sub.18 or R.sub.18, with an H of the respective group absent such that the alkyl group through which binding occurs is divalent, or, when R.sub.18 is H, through the C of the —C(═O)—O— moiety. In a preferred embodiment, the C.sub.2-6ester, including the C atom of the —C(═O)—O moiety, has 2-5 carbon atoms. The C.sub.2-6ester can optionally be 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 R.sub.1, R.sub.2, R.sub.3 and R.sub.4. By way of a non-limiting example, C.sub.2-6ester can be —C.sub.2H.sub.4—C(═O)—O—C.sub.2H.sub.5, where the —C.sub.2H.sub.4— moiety is —CH.sub.2—CH.sub.2— and binding is through the —C.sub.2H.sub.4— moiety.

[0172] 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)—R.sub.20—, where binding is through —R.sub.20—, has the number of carbon atoms indicated, 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 R.sub.1, R.sub.2, R.sub.3 or R.sub.4. By way of non-limiting examples, aldehyde groups include HC(═O)—CH.sub.2—, HC(═O)—C.sub.2H.sub.4—, HC(═O)—C.sub.3H.sub.6—, HC(═O)—C.sub.4H.sub.8— and HC(═O)—C.sub.5H.sub.10—.

[0173] As used herein, the term “fluoroalkyl” refers to an alkyl group, where the alkyl group is a straight or branched saturated monovalent (except where the context requires otherwise) cyclic or acyclic hydrocarbon radical, having the number of carbon atoms as indicated (or where not indicated, an acyclic alkyl group has 1-6 or 1-4 carbon atoms and a cyclic alkyl group has 3-6 carbon atoms) substituted with 1 to 6 F atoms.

[0174] As used herein, the term “fluoroalkenyl” refers to an alkenyl group, where the alkenyl group is a straight or branched unsaturated monovalent (except where the context requires otherwise) acyclic or cyclic hydrocarbon radical having one or more C═C double bonds and having the number of carbon atoms as indicated (or where not indicated, an acyclic alkenyl group has 2-6 or 2-4 carbon atoms and a cyclic alkenyl group has 4-6 carbon atoms) substituted with 1 to 6 F atoms.

[0175] The process for preparing a compound of formula Ia or Ib is preferably carried out in the presence of a suitable solvent.

[0176] Suitable solvents include hydrocarbon solvents such as benzene and toluene; ether type solvents such as diethyl ether, tetrahydrofuran, diphenyl ether, anisole and dimethoxybenzene; halogenated hydrocarbon solvents such as methylene chloride, chloroform and chlorobenzene; ketone type solvents such as acetone, methyl ethyl ketone and methyl isobutyl ketone; alcohol type solvents such as methanol, ethanol, propanol, isopropanol, n-butyl alcohol and tert-butyl alcohol; nitrile type solvents such as acetonitrile, propionitrile and benzonitrile; ester type solvents such as ethyl acetate and butyl acetate; carbonate type solvents such as ethylene carbonate and propylene carbonate; and the like. These may be used singly or two or more of them may be used in admixture.

[0177] Preferably an inert solvent is used in the process of the present invention. The term “inert solvent” means a solvent inert under the conditions of the reaction being described in conjunction therewith including, for example, benzene, toluene, acetonitrile, tetrahydrofuran, dimethylformamide, chloroform, methylene chloride (or dichloromethane), diethyl ether, ethyl acetate, acetone, methylethyl ketone, methanol, ethanol, propanol, isopropanol, tert-butanol, dioxane, pyridine, and the like. Tetrahydrofuran is particularly preferred.

[0178] Preferably the process of the present invention is carried out under substantially dry conditions.

[0179] The phosphorochloridate may be prepared from an aryloxy phosphorodichloridate and a suitably protected amino acid derivative. Alternatively, phosphate chemistry may be used with suitable condensing agents.

[0180] 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 phosphoramidate is to be attached. For example, carrying out the reaction of the 3′-deoxynucleoside with the desired phosphorochloridate in the presence of .sub.t-BuMgCl allows the 2′-phosphoramidate to be prepared.

[0181] Reacting the 3′-deoxynucleoside with POC13 followed by a salt of N.sup.+R.sub.5R.sub.6H.sub.2 allows compounds to be prepared where each of U and V is —NR.sub.5R.sub.6. Suitable salts include chloride, tosylate, sulphonate and ester salts such as 4-methylbenzene sulphonate. Subsequent addition of a base such as diisopropylethyl amine can aid the process.

[0182] As used herein, the term “stereoisomer” defines all possible compounds made up of the same atoms bonded by the same sequence of bonds but having different three-dimensional structures which the compounds of the present invention may possess.

[0183] Where the compounds according to this invention have at least one chiral centre, they may accordingly exist as enantiomers. Where the compounds possess two or more chiral centres, they may additionally exist as diastereoisomers. Where the processes for the preparation of the compounds according to the invention give rise to mixture of stereoisomers, these isomers may be separated by conventional techniques such as preparative chromatography. The compounds may be prepared in stereochemically mixed form or individual enantiomers may be prepared by standard techniques known to those skilled in the art, for example, by enantiospecific synthesis or resolution, formation of diastereoisomeric pairs by salt formation with an optically active acid, followed by fractional crystallization and regeneration of the free base. The compounds may also be resolved by formation of diastereoisomeric esters or amides, followed by chromatographic separation and removal of the chiral auxiliary. Alternatively, the compounds may be resolved using a chiral HPLC column. It is to be understood that all such isomers and mixtures thereof are encompassed within the scope of the present invention.

[0184] Furthermore, it should be appreciated that the phosphate centre is chiral in the compounds of the present invention and the compounds may exist as R.sub.P and S.sub.P diastereoisomers. The composition of the compound may be mixed R.sub.P and S.sub.P or one pure diastereoisomer. In a preferred embodiment, the compound is a substantially pure single diastereoisomer of either R.sub.P or S.sub.P. By “substantially pure single diastereoisomer” is meant that the compound consists of 98% or more of either the R.sub.P or the S.sub.P diastereoisomer. In another embodiment, there may be a mixture of 1:1 R.sub.P to S.sub.P diastereoisomers. Alternatively, the compound may comprise a mixture of R.sub.P and S.sub.P diastereoisomers in a ratio of R.sub.P to S.sub.P diastereoisomers of 1:90 to 90:1, 1:50 to 50:1, 1:20 to 20:1, 1:15 to 15:1, 1:10 to 10:1, 1:9 to 9:1, 1:8 to 8:1, 1:7 to 7:1, 1:6 to 6:1, 1:5 to 5:1, 1:4 to 4:1, 1:3 to 3:1 or 1:2 to 2:1. In preferred embodiments, the compound of the invention may comprise a ratio of R.sub.P to S.sub.P diastereoisomers of greater than 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:50, 1:90, 1:95 or 1:99 or vice versa.

[0185] The term “solvate” means a compound of formula Ia or formula lb 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.

[0186] The compounds of the present invention may also be present in the form of pharmaceutical acceptable salts. For use in medicine, the salts of the compounds of this invention refer to “pharmaceutically acceptable salts.” FDA approved pharmaceutical acceptable salt forms (Ref. International J. Pharm. 1986, 33, 201-217; J. Pharm. Sci., 1977, January 66 (1)) include pharmaceutically acceptable acidic/anionic or basic/cationic salts.

[0187] Pharmaceutically acceptable acidic/anionic salts include, and are not limited to acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, glyceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, pamoate, pantothenate, phosphate, diphospate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, teoclate, tosylate and triethiodide.

[0188] Pharmaceutically acceptable basic/cationic salts include, and are not limited to aluminum, benzathine, calcium, chloroprocaine, choline, diethanolamine, ethylenediamine, lithium, magnesium, potassium, procaine, sodium and zinc.

[0189] The present invention includes within its scope prodrugs of the compounds of this invention. In general, such prodrugs will be functional derivatives of the compounds which are readily convertible in vivo into the required compound. Thus, in the methods of treatment of the present invention, the term “administering” shall encompass the treatment of the various disorders described with the compound specifically disclosed or with a compound which may not be specifically disclosed, but which converts to the specified compound in vivo after administration to the subject. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in “Design of Prodrugs”, ed. H. Bundgaard, Elsevier, 1985.

[0190] Pharmaceutically acceptable ester derivatives in which one or more free hydroxy groups are esterified in the form of a pharmaceutically acceptable ester are particular examples of prodrug esters that may be convertible by solvolysis under physiological conditions to the compounds of the present invention having free hydroxy groups.

[0191] Pharmaceutical compositions for use in accordance with the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. These pharmaceutical compositions may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Proper formulation is dependent upon the route of administration chosen.

[0192] The compound or pharmaceutical composition according to the present invention can be administered to a patient, which may be Homo sapiens or animal, by any suitable means.

[0193] The medicaments employed in the present invention can be administered by oral or parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal, vaginal and topical (including buccal and sublingual) administration.

[0194] For oral administration, the compounds of the invention will generally be provided in the form of tablets or capsules, as a powder or granules, or as an aqueous solution or suspension.

[0195] Tablets for oral use may include the active ingredient mixed with pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavouring agents, colouring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while cornstarch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.

[0196] Capsules for oral use include hard gelatin capsules in which the active ingredient is mixed with a solid diluent, and soft gelatin capsules wherein the active ingredient is mixed with water or an oil such as peanut oil, liquid paraffin or olive oil.

[0197] Formulations for rectal administration may be presented as a suppository with a suitable base comprising for example cocoa butter or a salicylate.

[0198] 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.

[0199] For intramuscular, intraperitoneal, subcutaneous and intravenous use, the compounds of the invention will generally be provided in sterile aqueous solutions or suspensions, buffered to an appropriate pH and isotonicity. Suitable aqueous vehicles include Ringer's solution and isotonic sodium chloride. Aqueous suspensions according to the invention may include suspending agents such as cellulose derivatives, sodium alginate, polyvinyl-pyrrolidone and gum tragacanth, and a wetting agent such as lecithin. Suitable preservatives for aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate.

[0200] The compounds of the invention may also be presented as liposome formulations.

[0201] 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 m.sup.2 of body surface area of recipient per day or 5 to 50 mg per m.sup.2 of body surface area of recipient per day. Suitable doses may be 6, 12, 24 or 48 mg per m.sup.2 of body surface area of recipient per day. The desired dose may be presented and administered as a single daily dose or as two, three, four, five or six or more sub-doses administered at appropriate intervals throughout the day. Doses may be administered in unit dosage forms, for example, containing 10 to 1500 mg, preferably 20 to 1000 mg, and most preferably 50 to 700 mg of active ingredient per unit dosage form. The total daily dose is suitably 1000 to 3000 mg, whether taken as a single dose or as sub-doses at intervals throughout the day.

[0202] “Cancer Stem Cells”

[0203] Cancer stem cells, which are sometimes otherwise referred to as “tumour initiating cells”, are well known to those skilled in the art. As used herein, the term “cancer stem cell” is to be interpreted in accordance with its widely accepted meaning, which is a cell that possesses the capacity to self-renew through asymmetric division, to initiate tumour formation, and to give rise to more mature non-stem cell cancer progeny by differentiation.

[0204] Cancer stem cells play a major role in the development, progression, recurrence and propagation of cancers. Accordingly, the finding that compounds of the invention are able to target cancer stem cells, and thereby reduce their numbers, offers therapeutic possibilities in preventing or treating these activities.

[0205] As discussed in more detail elsewhere in the specification, cancer stem cells are found in pre-cancerous conditions, where their presence is believed to contribute to the development of such conditions into cancers. Accordingly the methods of treatment and medical uses of the invention, in which a compound of the invention is used to target cancer stem cells, may be used to reduce cancer stem cell numbers in pre-cancerous conditions (such as myelodyplastic syndrome, or other conditions considered elsewhere in the specification), and thus to prevent progression of such pre-cancerous conditions into cancer.

[0206] 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.

[0207] The accumulation of such non-stem cancer cells plays a major role in the progression of cancers. Targeting of cancer stem cells by a compound of the invention is able to reduce cancer stem cell numbers, which in turn reduces the number of non-stem cancer cell progeny. Thus methods of treatment and medical uses of a compound of the invention in accordance with the present invention are of benefit in treating cancer by preventing cancer progression. Such embodiments are described in more details elsewhere in the present specification.

[0208] 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 removed (for example by surgery, radiotherapy, or chemotherapy, either alone or in combination), so that no observable signs of a cancer remain, the continued presence of cancer stem cells may nucleate the recurrence of the cancer over time. Targeting of cancer stem cells by a compound of the invention provides a new mode by which cancer stem cell numbers may be reduced and cancer stem cells killed. Accordingly, and as discussed in more detail elsewhere in the specification, in suitable embodiments the present invention provides methods and medical uses in which a compound of the invention prevents or delays recurrence of cancer.

[0209] 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.

[0210] In addition to their biological activities, cancer stem cells may be identified by their expression of certain characteristic cell surface markers. Cancer stem cells identified in haematological malignancies are typically CD34.sup.+, while in solid tumours, CD44.sup.+, CD133.sup.+ and CD90.sup.+ have been identified as cancer stem cell markers. The following table summarises examples of known cancer stem cell surface phenotypes. It is expected that each of these forms of cancer stem cell can be targeted using a compound of the invention in accordance with the invention, and so methods or uses employing a compound of the invention may be used in the prevention or treatment of cancers associated with cancer stem cells expressing any of these sets of markers.

TABLE-US-00001 Reported cell surface markers Tumour type for cancer stem cells Solid Tumours Breast CD44.sup.+/CD24.sup.−/.sup.low/Lineage.sup.−/ESA.sup.+ CNS CD133.sup.+ Colon CD133.sup.+ Colon ESA.sup.high/CD44.sup.+/Lineage.sup.− /(CD166.sup.+) Ewing’s CD133.sup.+ Head and Neck CD44.sup.+/Lineage.sup.− Melanoma ABCB5.sup.+ Liver CD90.sup.+/CD45.sup.−/(CD44.sup.+) Cholangiocarinoma CD44.sup.+/GLI1.sup.+ (Glioma-associated oncogene homolog-1) Ovarian CD44.sup.+/CD117.sup.+ Pancreas CD44.sup.+/CD24.sup.+/ESA.sup.+ Pancreas CD133.sup.+ Non-small-cell lung cancer CD44.sup.+/Ber-EP4.sup.+ Bladder cancer CD44.sup.+/ALDH1A1.sup.+ Haematological tumours Acute myeloid leukaemia Lin.sup.−/CD34.sup.+/CD38.sup.−/CD123.sup.+ B-Acute lymphoblastic leukaemia CD34.sup.+/CD10.sup.− or CD34.sup.+/CD19.sup.− B-Acute lymphoblastic leukaemia CD34.sup.+/CD38.sup.−/CD19.sup.+ Multiple myeloma CD34.sup.−/CD138.sup.− T-Acute lymphoblastic leukaemia CD34.sup.+/CD4.sup.− or CD34.sup.+/CD7.sup.−

[0211] The data presented in the Examples demonstrate that a compound of the invention is able to target cancer stem cells of leukaemic stem cell lines, specifically cancer stem cells present in the acute myeloid leukaemia cell line KG1a. This cell line manifests a minor stem cell-like compartment with a distinct immunophenotype (Lin.sup.−/CD34.sup.+/CD38.sup.−/CD123.sup.+) which is targeted by a compound of the invention. Accordingly, methods of treatment or medical uses of a compound of the invention in accordance with the present invention may be used to prevent or treat leukaemia or other cancers associated with cancer stem cells expressing these characteristic markers.

[0212] The present invention also provides methods and medical uses in which patients are selected for prevention or treatment of cancer, utilising a compound of the invention, on the basis of the identification of the presence of cancer stem cells in a biological sample representative of the patient's cancer or pre-cancerous condition. The markers set out above provide suitable examples that can be used to identify the presence of cancer stem cells in accordance with such embodiments of the invention. Suitable techniques by which expression of these markers may be investigated in a biological sample are considered further elsewhere in this specification.

[0213] “Targeting of Cancer Stem Cells”

[0214] The present invention provides the first indication that compounds of the invention can be used for targeting cancer stem cells. The ability of compounds of the invention to target cancer stem cells is illustrated in the Examples disclosed in this specification.

[0215] It can be seen that when a compound of the invention is provided to populations of cancer cells containing cancer stem cells it targets the cancer stem cells present, leading to a reduction in the total number of cancer cells. As discussed elsewhere in the present specification, certain compounds of the invention preferentially target cancer stem cells as opposed 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 proportion of total cancer cells that exhibit phenotypic markers of cancer stem cells.

[0216] 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 demonstrates that compounds of the present invention are phosphorylated to the triphosphate of cordycepin or a cordycepin derivate (e.g. 2-fluorocordycepin or 2-Cl-cordycepin) and it is believed that enzymatic cleavage within the cell converts a compound of the invention directly into 8-chloroadenosine monophosphate prior to phosphorylation to the triphosphate.

[0217] 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.

[0218] Without wishing to be bound by any hypothesis, the inventors believe that the reduction in cancer stem cell numbers arises as a result of targeted killing of the cancer stem cells among the cancer cell population. Thus compounds of the invention are able to cause the death of cancer stem cells. Furthermore, the results set out elsewhere in this specification illustrate that certain compounds 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.

[0219] While the inventors believe 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 targeting of these cells.

[0220] Merely by way of example, treatment with a compound of the invention may cause an increase in cancer stem cell differentiation, thereby reducing cancer stem cell numbers and also the proportion of total cancer cells represented by cancer stem cells. Alternatively, a compound of the invention may cause cancer stem cells to lose their stem cell phenotype, for example losing their ability to self-renew, thereby reducing cancer stem cell numbers.

[0221] References to targeting of cancer stem cells in the present disclosure should be interpreted accordingly. For the purposes of the present disclosure, “targeting” of cancer stem cells may be taken as encompassing any mechanism by which a compound of the invention reduces the number of cancer stem cells present in a population of cells, whether in vitro or in vivo. In particular targeting of cancer stem cells may be taken as encompassing preferential 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.

[0222] “Prevention or Treatment of Cancer”

[0223] The invention provides medical uses and methods of treatment in which a compound of the invention is used for the prevention or treatment of cancer. In the context of the present invention, “prevention” of cancer is to be considered as relating to prophylactic applications of a compound of the invention used before the development of cancer, and with an aim of stopping cancer from developing. On the other hand “treatment” of cancer is taken as concerning the use of a compound of the invention after cancer has occurred, with a view to ameliorating cancer by slowing or stopping cancer cell proliferation and tumour growth. Advantageously treatment of cancer may cause partial or total reduction in cancer cell numbers and tumour size. Effective treatment of cancer may bring about disease that either “stabilizes” or “responds” in accordance with the RECIST (Response Evaluation Criteria In Solid Tumours) guidelines.

[0224] As described in more detail below, prevention of cancer in accordance with the present invention may be of particular benefit in patients who have a pre-cancerous condition that increases their likelihood of developing cancer.

[0225] “Prevention of Cancer”

[0226] Prevention of cancer in accordance with the present invention may be effected by treatment of a pre-cancerous condition using a compound of the invention in accordance with the various aspects or embodiments of the invention described herein.

[0227] In particular, prevention of cancer, in the context of the present invention, may be achieved by the methods or medical uses of the invention in which a compound of the invention is provided to a patient with a pre-cancerous condition. Methods of treatment or medical uses in accordance with this embodiment may prevent development of the treated pre-cancerous condition into cancer, thereby providing effective prevention of cancer.

[0228] References to prevention of cancer in the context of the present invention may also encompass other prophylactic applications of a compound of the invention. For example, the ability of a compound of the invention to target cancer stem cells and thereby prevent the development of cancer, and/or prevent the progression of cancer, and/or prevent the recurrence of cancer, and/or prevent the propagation of cancer.

[0229] “Pre-Cancerous Conditions”

[0230] Cancer is frequently preceded by the development of a pre-cancerous condition, which is not itself cancerous, but is associated with an increased risk of cancer. Accumulation of genetic or epigenetic changes may cause previously normal cells to develop a cancer stem cell phenotype. Accordingly, cancer stem cells may also be present in such pre-cancerous conditions, as well as in cancerous conditions.

[0231] It is believed that the presence of cancer stem cells in pre-cancerous conditions contributes to the development of these conditions into cancer. The methods and medical uses of the invention may be employed to target cancer stem cells present in pre-cancerous conditions, and thereby treat such conditions. It will be appreciated that the new and unexpected finding that compounds of the invention target cancer stem cells means that treatment of 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.

[0232] Examples of pre-cancerous conditions that may be treated in accordance with the present invention include, but are not limited to, those selected from the group consisting of: actinic keratosis, Barrett's oesophagus, atrophic gastritis, dyskeratosis congenital, Sideropenic dysphagia, Lichen planus, oral submucous fibrosis, solar elastosis, cervical dysplasia, leukoplakia, erythroplakia, monoclonal gammopathy of unknown significance (MGUS), monoclonal B-cell lymphocytosis (MBL), myelodysplastic syndromes, as well as pre-cancerous conditions of the stomach such as atrophic gastritis, gastric ulcer, pernicious anaemia, gastric stumps, gastric polyps, and Menetrier's disease. Among the listed pre-cancerous conditions of the stomach, atrophic gastritis, pernicious anaemia, gastric stumps, and certain types of gastric polyp may have particularly heightened risk of developing into cancers.

[0233] Pre-cancerous conditions often take the form of lesions comprising dysplastic or hyperplastic cells. Accordingly, the presence of dysplasia or hyperplasia, as an alternative or addition to the presence of cells with expressed markers or phenotypes characteristic of cancer stem cells, may be used in the identification of pre-cancerous conditions.

[0234] The severity of dysplasia can vary between different pre-cancerous conditions, or with the development of a single pre-cancerous condition over time. Generally, the more advanced dysplasia associated with a pre-cancerous condition is, the more likely it is that the pre-cancerous 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.

[0235] In a suitable embodiment of the invention a compound of the invention is used to treat a patient with severe cervical dysplasia. Severe cervical dysplasia may be diagnosed by means of a smear test. In another embodiment of the invention a compound of the invention is used to treat severe oesophageal dysplasia (“Barrett's oesophagus”). Severe oesophageal dysplasia may be diagnosed following a tissue biopsy.

[0236] It has recently been reported that pre-malignancies can also be identified by detecting somatic mutations in cells in individuals not known to have cancer. In particular, it has been reported that age-related clonal haematopoiesis is a common pre-malignant condition that is associated with increased overall mortality and increased risk of cardiometabolic disease. The majority of mutations detected in blood cells occurred in three genes: DNMT3A, TET2, and ASXL1. Accordingly, patients that will benefit from the use of a compound of the invention to target cancer stem cells, and thereby treat a pre-cancerous condition, may be identified by assaying a sample comprising blood cells for the presence of genetic mutations indicative of a pre-cancerous condition in at least one of: DNMT3A and/or TET2 and/or ASXL1.

[0237] Pre-cancerous conditions that may benefit from treatment with a compound of the invention in accordance with the invention to target cancer stem cells may also be identified by determination of the presence of cancer stem cells with reference to any of the techniques based upon expression of markers characteristic of cancer stem cells, or cancer stem cell phenotypes, discussed elsewhere in the specification.

[0238] “Treatment of Cancer”

[0239] 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.

[0240] In certain embodiments, a compound of the invention may be used: to reduce the proportion of cancer stem cells in a population of cancer cells; and/or to inhibit tumour growth; and/or to reduce tumourigenicity; and/or to prevent or treat a primary cancer; and/or to prevent or treat a relapsed cancer; and/or to prevent or treat a metastatic or secondary cancer; and/or to treat, prevent or inhibit metastasis or recurrence; and/or to treat or prevent refractory cancer.

[0241] The ability of cancer treatment using a compound of the invention to bring about a reduction in tumour size, and also to maintain the reduction in tumour size during/after the period in which the treatment is administered represents a particularly relevant indication of effective cancer treatment.

[0242] As set out in the Examples, the treatments or medical uses of the invention have proven surprisingly effective in this respect, even in models using cells representative of relapsed or refractory cancers that have previously been resistant to treatment with other therapies.

[0243] The data presented in the Examples illustrate that treatment with a compound of the invention reduces the proportion of cancer stem cells in a population of cancer cells. Characteristic biological activities or cell surface markers by which cancer stem cells may be identified are described elsewhere in the specification. In a suitable embodiment, treatment of cancer in accordance with the present invention may give rise to a reduction in the proportion of cancer stem cells present in a patient's cancer of at least 10%, at least 20%, at least 30%, or at least 40%. In suitable embodiments treatment of cancer in accordance with the 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).

[0244] Asymmetric division of cancer stem cells contributes to the growth of tumours. Treatment of cancer with a compound of the invention in accordance with the present invention may bring about an inhibition of tumour growth of at least 10%, at least 20%, at least 30%, or at least 40%. Suitably treatment of cancer in accordance with the invention may give rise to an inhibition of tumour growth of at least 50%, at least 60%, at least 70%, or at least 80%. Treatment of cancer in accordance with the invention may give rise to an inhibition of tumour growth of at least 85%, at least 90%, or at least 95% in a patient so treated. Indeed, treatment of cancer in accordance with the invention may give rise to an inhibition of tumour growth of at least 96%, at least 97%, at least 98%, at least 99%, or even 100% in a treated cancer.

[0245] Tumour growth may be assessed by any suitable method in which the change in size of a tumour is assessed over time. Suitably the size of a tumour prior to cancer treatment may be compared with the size of the same tumour during or after cancer treatment. A number of ways in which the size of a tumour may be assessed are known. For example, the size of a tumour may be assessed by imaging of the tumour in situ within a patient. Suitable techniques, such as imaging techniques, may allow the volume of a tumour to be determined, and changes in tumour volume to be assessed.

[0246] As shown in the results set out in the Examples of this specification, the methods of treatment and medical uses of a compound of the invention 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 cancers. Suitably treatment of cancer in accordance with the present invention may give rise to a reduction in tumour volume of at least 10%, at least 20%, at least 30%, or at least 40%. In suitable embodiments, treatment of cancer 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%.

[0247] A reduction in tumour volume of the sort described above can be calculated with reference to a suitable control. For example in studies carried out in vitro, or in vivo in suitable animal models, the reduction in tumour 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 compound of the invention). It will be appreciated that such models requiring lack of treatment of a tumour may not be ethically acceptable in the context of clinical 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.

[0248] 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 cancers CEA may be a suitable biomarker.

[0249] Suitably treatment of cancer in accordance with the present invention may give rise to a reduction in cancer biomarkers 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 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%.

[0250] Beneficial effects, such as a reduction in the proportion of cancer stem cells present, reduction in tumour growth, or reduction in tumour volume or cancer biomarkers, observed on treatment of cancer in accordance with the present invention may be maintained for at least one month. Suitably such beneficial effects may be maintained for at least two months, at least three months, at least four months, at least five months, or at least six months. Indeed, such beneficial effects may be maintained for at least 12 months, at least 18 months, or at least 24 months. Suitably the beneficial effects may be maintained for at least three years, at least four years, at least five years, at least six years, at least seven years, at least eight years, at least nine years, or for ten years or more.

[0251] In a suitable embodiment of the invention a compound of the invention is used in a method of preventing or treating cancer or a pre-malignant condition, by targeting cancer stem cells. In a suitable embodiment the invention provides the use of a compound of the invention in a method of preventing or treating cancer or a pre-malignant condition, wherein the method reduces the tumourigenicity of one or more cancer stem cells. Suitably such methods may prevent the progression of cancer, or inhibit tumour growth.

[0252] When a compound of the invention is used in methods or medical uses of the present invention to prevent or treat the progression of a cancer, such prevention or treatment may cause the cancer progression to be slowed, delayed or stopped entirely.

[0253] 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 Ito IV to the cancer, with I being an isolated cancer and IV being a cancer that has spread to the limit of what the assessment measures. Specifics of staging vary between cancers, but the stage generally takes into account the size of a tumour, whether it has invaded adjacent organs, how many regional (nearby) lymph nodes it has spread to (if any), and whether it has appeared in more distant locations (metastasised).

[0254] Generally, Stage I is localised to one part of the body and may be treated by surgical resection (for solid tumours that are small enough). Stage II is locally advanced, and is treatable by chemotherapy, radiation therapy, surgery, or a combination thereof. Stage III is also locally advanced and the designation of Stage II or Stage III depends on the specific type of cancer, although Stage III is generally accepted to be “late” locally advanced. Stage IV cancers have often metastasised to a second organ. Treatment of cancer using a compound of the invention in the methods or medical uses of the present invention may be used to treat a stage I, II, III or IV cancer by targeting cancer stem cells. Treatment with a compound of the invention may be used to prevent the progression of a cancer from one stage to the next. In one embodiment, treatment with a compound of the invention is used to prevent progression from Stage I to Stage II. In another embodiment, treatment with a compound of the invention is used to prevent progression from Stage II to Stage III. In still another embodiment, treatment with a compound of the invention is used to prevent progression from Stage III to Stage IV.

[0255] Preventing or inhibiting progression of the cancer is particularly important for preventing the spread of the cancer, for example the progression from Stage Ito Stage II where the cancer spreads locally, or the progression from Stage III to Stage IV where the cancer metastasises to other organs. Cancer stem cells are tumourigenic and so are believed to play a critical role in the spread of cancer, both locally and metastatically. Methods of treatment or medical uses of the invention employing a compound of the invention can therefore be used to prevent the spread of cancer, by targeting tumourigenic cancer stem cells and thus reducing their numbers.

[0256] “Cancers”

[0257] 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.

[0258] Cancer stem cells play a role in the biological activity of a wide range of cancers. Accordingly, there are a wide range of cancers that may be prevented or treated in accordance with the present invention.

[0259] As discussed elsewhere herein, cancer stem cells are known to be present in many tumour types including liquid tumours (including haematological tumours such as leukaemias and lymphomas) and solid tumours (such as breast, lung, colon, prostate, ovarian, skin, bladder, biliary and pancreas tumours). Methods of treatment and medical uses of a compound of the invention to target cancer stem cells are therefore expected to be useful in the prevention or treatment of such cancers.

[0260] Suitably a compound of the invention may be used in the prevention or treatment of a cancer selected from the group consisting of: leukaemia, lymphoma, multiple myeloma, lung cancer, liver cancer, breast cancer, head and neck cancer, neuroblastoma, thyroid carcinoma, skin cancer (including melanoma), oral squamous cell carcinoma, urinary bladder cancer, Leydig cell tumour, biliary cancer, such as cholangiocarcinoma or bile duct cancer, pancreatic cancer, colon cancer, colorectal cancer and gynaecological cancers, including ovarian cancer, endometrial cancer, fallopian tube cancer, uterine cancer and cervical cancer, including epithelia cervix carcinoma. In suitable embodiments, the cancer is leukaemia and can be selected from the group consisting of acute lymphoblastic leukaemia, acute myelogenous leukaemia (also known as acute myeloid leukaemia or acute non-lymphocytic leukaemia), acute promyelocytic leukaemia, acute lymphocytic leukaemia, chronic myelogenous leukaemia (also known as chronic myeloid leukaemia, chronic myelocytic leukaemia or chronic granulocytic leukaemia), chronic lymphocytic leukaemia, monoblastic leukaemia and hairy cell leukaemia. In further preferred embodiments, the cancer is acute lymphoblastic leukaemia. In a particular embodiment, the leukaemia is refractory TdT-Positive Leukemia In a suitable embodiment the cancer is lymphoma, which may be selected from the group consisting of: Hodgkin's lymphoma; non-Hodgkin lymphoma; Burkitt's lymphoma; and small lymphocytic lymphoma.

[0261] Suitably targeting cancer stem cells in such cancers may achieve effective treatment of the cancer by preventing or treating the development of the cancer, by preventing or treating the progression of the cancer, by preventing or treating the recurrence of the cancer, or by preventing or treating the propagation of the cancer.

[0262] In a suitable embodiment the present invention provides a compound of the invention for use in targeting cancer stem cells in the prevention or treatment of metastatic cancer.

[0263] In a suitable embodiment the present invention provides a compound of the invention for use in targeting cancer stem cells in the treatment of relapsed or refractory cancer.

[0264] 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.

[0265] 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.

[0266] In a suitable embodiment the present invention provides a compound of the invention for use in targeting cancer stem cells, wherein the targeting of cancer stem cells prevents or inhibits: (i) recurrence of a cancer; (ii) occurrence of second primary cancer; or (iii) metastasis of a cancer.

[0267] Methods of treatment or medical uses in which a compound of the invention is employed on the basis of its ability to target cancer stem cells may be used in the treatment of relapsed or refractory cancer. The considerations regarding relapsed or refractory cancer in such embodiments are, except for where the context requires otherwise, the same as for the treatment of relapsed or refractory cancer in connection with the aspects of the invention.

[0268] “Relapsed or Refractory Cancer”

[0269] 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.

[0270] For the purposes of the present invention, refractory cancers may be taken as cancers that demonstrate resistance to treatment by anti-cancer therapies other than those utilising a compound of the invention. For example, a compound of the invention may be used in the treatment of refractory cancers that are resistant to treatment with radiotherapy. Alternatively, or additionally, a compound of the invention may be used in the treatment of refractory cancers that are resistant to biological agents used in the treatment of cancer. In a suitable embodiment a compound of the invention may be used in the treatment of refractory cancers that are resistant to treatment with chemotherapeutic agents other than a compound of the invention.

[0271] In particular, refractory cancers that may benefit from the methods of treatment of medical uses of the invention employing a compound of the invention include those cancers that are resistant to cordycepin or 2-fluorocordycepin.

[0272] Relapsed cancers (or recurrent cancers) are those that return after a period of remission during which the cancer cannot be detected. Cancer recurrence may occur at the site of the original cancer (local cancer recurrence), at a site close to that of the original cancer (regional cancer recurrence), or at a site distant from that of the original cancer (distal cancer recurrence). Cancer stem cells are believed to play a role in the recurrence of cancer, providing a source from which cells of the relapsed cancer are generated. Accordingly, the methods of treatment and medical uses of a compound of the invention in accordance with the invention, which enable targeting of cancer stem cells, may be of great benefit in the context of relapsed cancers. The ability of a compound of the invention to target cancer stem cells may be used to remove the populations of such cells that are able to give rise to recurrence, thus preventing incidences of relapsed cancer. The anti-cancer stem cell activity of a compound of the invention may also be used to target cancer stem cells in cancers that have recurred, as well as potentially exerting cytotoxic effects on non-stem cancer cells, thereby providing treatment of relapsed cancers.

[0273] In view of the above, it will be appreciated that a compound of the invention may be used in the methods or uses of the invention for the prevention or treatment of a relapsed cancer. A compound of the invention may be used in the methods or uses of the invention for the prevention or treatment of a local, regional or distant relapsed cancer.

[0274] A compound of the invention may be used in the methods or uses of the invention to prevent the recurrence of cancer by providing at least 2 months, at least 6 months, at least 12 months, at least 18 months, at least 24 months, or at least 30 months of remission. Indeed, a compound of the invention may be used to prevent recurrence of cancer by providing at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, or at least 10 years of remission.

[0275] 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.

[0276] The ability of the compounds of the invention to target cancer stem cells gives rise to the ability of these compounds to prevent or treat cancers in accordance with the medical uses or methods of treatment of the invention. However, it should be noted that compounds of the invention also exert a direct cytotoxic effect upon non-stem cancer cells that make up the bulk of tumours. While 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.

[0277] Compounds of the invention exert greater cytotoxic effects on non-stem cancer cells than does cordycepin or 2-fluorocordycepin, the chemotherapeutic molecule from which the compounds of the invention are derived. Accordingly, the mechanism by which a compound of the invention acts in the treatment of relapsed or refractory cancer may not be limited solely to the 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 non-stem 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.

[0278] Therapeutically Effective Doses of a Compound of the Invention

[0279] A therapeutically effective amount of a compound of the invention may be an amount sufficient to induce death of cancer cells. A therapeutically effective amount of a compound of the invention may be an amount sufficient to induce death of cancer stem cells. In some embodiments, particularly those relating to the treatment of relapsed or refractory cancer, a therapeutically effective amount of a compound of the invention may be an amount sufficient to induce death of cancer stem cells and also to induce death of non-stem cancer cells.

[0280] There are various different ways in which the amount of a therapeutically effective compound, such as a compound of the invention, to be administered to a patient may be calculated and expressed. One such way which is considered particularly relevant in doses of agents for the prevention or treatment of cancer, is in the amount of the agent to be administered per unit of body surface area of the patient. Such doses are typically expressed in terms of the amount of the agent (which may be determined by mass) per square meter (m.sup.2) of surface area.

[0281] Uses of a compound of the invention for the prevention or treatment of cancer may utilise a weekly dose of between 10 mg/m.sup.2 and 1000 mg/m.sup.2. Such treatments may, for example utilise a weekly dose of between 375 mg/m.sup.2 and 900 mg/m.sup.2. For example, effective treatment of relapsed or refractory cancers may be provided when patients are provided with weekly doses of a compound of the invention that range between approximately 500 mg/m.sup.2 and 825 mg/m.sup.2.

[0282] Without wishing to be bound by any hypothesis, the inventors believe that the ability of a compound of the invention to target cancer stem cells allows therapeutic effectiveness to be achieve using lower doses of this compound than would otherwise be expected. Merely by way of example, weekly doses of a compound of the invention that are as low as 825 mg/m.sup.2, 750 mg/m.sup.2, 600 mg/m.sup.2, or 500 mg/m.sup.2 may prove therapeutically effective in the uses and methods of the invention.

[0283] A chosen weekly dose of a compound of the invention may be provided in a single incidence of administration, or in multiple incidences of administration during a week. For example, a weekly dose of a compound of the invention may be provided in two incidences of administration, in three incidences of administration, or more. Thus, in the case of a weekly dose of 750 mg/m.sup.2, this may be achieved by three administrations of 250 mg/m.sup.2 over the course of a week, or two administrations of 375 mg/m.sup.2 during a week Similarly, in the case of a weekly dose of 600 mg/m.sup.2, this may be achieved by three administrations of 200 mg/m.sup.2 over the course of a week, or two administrations of 300 mg/m.sup.2 during a week.

[0284] A suitable amount of a compound of the invention to be administered in a single incidence of treatment in order to provide a required dose of this compound over the course of week may be between approximately 100 mg/m.sup.2 and 300 mg/m.sup.2.

[0285] The weekly dose of a compound of the invention provided may decrease over the course of treatment. For example, treatment may be started at a weekly dose of around 1000 mg/m.sup.2, 900 mg/m.sup.2, 825 mg/m.sup.2, 750 mg/m.sup.2, or 725 mg/m.sup.2, and over the course of treatment the dose needed may decrease to around 750 mg/m.sup.2 (in cases where the initial dose is above this amount), around 650 mg/m.sup.2, around 625 mg/m.sup.2, or even around 500 mg/m.sup.2 or around 375 mg/m.sup.2.

[0286] Doses of a compound of the invention can, of course, be presented in other manners. The most common of these is the amount of the active agent to be provided per unit body mass. It has been calculated that for an average human patient a dose of 1 mg/m.sup.2 is equivalent to approximately 0.025 mg/kg body mass. Accordingly, the data indicate that a compound of the invention is effective for the treatment of relapsed or refractory cancer at doses ranging from approximately 6.25 mg/kg to approximately 25 mg/kg. A suitable dose may, for example, be of between about 9.5 mg/kg and 22.5 mg/kg. In a suitable embodiment a compound of the invention achieves effective treatment of relapsed or refractory cancers when patients are provided with weekly doses ranging between approximately 12.5 mg/kg and 20.5 mg/kg.

[0287] Considerations regarding formulations of a compound of the invention suitable for use in the methods of prevention or treatment and medical uses of the present invention are described elsewhere in this disclosure. In the case of injectable formulations of a compound of the invention, these may be administered intravenously. Intravenous administration may be achieved over any suitable time frame, for example in a ten minute injection, or the like.

[0288] Types of Treatment

[0289] In a suitable embodiment a compound of the invention may be used for targeting cancer stem cells as a first line treatment of cancer.

[0290] However, the finding that compounds of the invention are able to target cancer stem cells and thereby treat relapsed or refractory cancer illustrates that a compound of the invention is able to provide effective treatment of cancer in contexts in which other treatments have proved ineffective. Accordingly, in a suitable embodiment the present invention provides a compound of the invention for targeting cancer stem cells as a second line treatment of cancer. Indeed, in a suitable embodiment the present invention provides a compound of the invention for targeting cancer stem cells as a third, or further, line treatment of cancer.

[0291] 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 surgical removal of cancer. A compound of the invention may be used as a neoadjuvant therapy for a patient who will subsequently undergo surgical treatment of cancer and/or radiotherapy for cancer.

[0292] Alternatively, or additionally, the invention provides a compound of the invention for use as an adjuvant in the treatment of cancer. An adjuvant is an agent provided to a patient after a “main” anti-cancer therapy, such as surgical removal of cancer, in order to prevent the return of cancer after the main therapy. A compound of the invention may be used as an adjuvant for a patient who has undergone surgical treatment of cancer and/or radiotherapy for cancer.

[0293] A compound of the invention may be employed in the methods or uses of the invention in a monotherapy, which is to say in preventions or treatments in which a compound of the invention provides substantially all of the therapeutic activity that is made use of in the prevention or treatment.

[0294] Alternatively, the methods or uses of the invention may employ a compound of the invention in a combination therapy. In such embodiments a compound of the invention is used in conjunction with at least one further cancer therapy. The further cancer therapy may comprise surgery and/or radiotherapy. Additionally, or alternatively, the further cancer therapy may comprise use of at least one further therapeutic agent that contributes to the prevention or treatment of cancer to be achieved. Suitably such an agent may be a chemotherapeutic agent or a biological agent used in the prevention or treatment of cancer.

[0295] In a suitable embodiment of a combination therapy a compound of the invention and a further therapeutic agent may be provided to a patient at the same time. In a suitable example, the compound of the invention and a further therapeutic agent may be formulated as part of the same pharmaceutical composition. Alternatively the compound of the invention and a further therapeutic agent may be formulated separately for provision to the patient at substantially the same time.

[0296] In another suitable embodiment of a combination therapy, a compound of the invention and a further therapeutic agent may be provided to a patient at different times. The compound of the invention and a further therapeutic agent may be provided to a patient sequentially. For example, the compound of the invention may be provided to the patient prior to provision of the further therapeutic agent. Alternatively a compound of the invention may be provided to the patient after provision of the further therapeutic agent.

[0297] “Further Therapeutic Agents”

[0298] A compound of the invention may be used in combination with a wide range of further therapeutic agents for the prevention or treatment of cancer. These include biological agents, immunotherapeutic agents, and chemotherapeutic agents that may be used for the prevention or treatment of cancer.

[0299] While specific examples of suitable further agents are considered in the following paragraphs, these should not be taken as limiting the range of further therapeutic agents suitable for use with a compound of the invention. Indeed, the ability of a compound of the invention to target cancer stem cells indicates that it may be beneficially used in combination with any further therapeutic agent used in the prevention or treatment of cancer, whether such further agent targets cancer stem cells, non-stem cancer cells, or other cells or constituents involved in the development, maintenance, recurrence, propagation or of cancer.

[0300] Examples of further therapeutic agents that may be used in combination with a compound of the invention include:

[0301] (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;

[0302] (b) an alkylating agent;

[0303] (c) an anti-metabolite;

[0304] (d) an anti-tumour antibiotic;

[0305] (e) a topoisomerase;

[0306] (f) a mitotic inhibitor;

[0307] (g) a monoclonal antibody;

[0308] (h) a metallic agent; or

[0309] (i) an active or passive immunotherapy.

[0310] 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.

[0311] Selection of Patients

[0312] The inventors' finding that a compound of the invention is able to target cancer stem cells makes possible a number of methods by which it is possible to determine whether a particular patient is likely to benefit from receiving a compound of the invention in the prevention or treatment of cancer, such as relapsed or refractory cancer.

[0313] Accordingly, the invention provides a method of determining whether a patient with cancer or a pre-cancerous condition will benefit from prevention or treatment of cancer with a compound of the invention, the method comprising: assaying a biological sample representative of cancer or a pre-cancerous condition in the patient for the presence of cancer stem cells; wherein the presence of cancer stem cells in the biological sample indicates that the patient will benefit from treatment with a compound of the invention.

[0314] The invention further provides a method of determining a suitable treatment regimen for a patient with cancer or a pre-cancerous condition, the method comprising: assaying a biological sample representative of cancer or a pre-cancerous condition in the patient for the presence of cancer stem cells; wherein the presence of cancer stem cells in the biological sample indicates that a suitable treatment regimen will comprise treatment of the patient with a compound of the invention.

[0315] The invention also provides a compound of the invention for use in the prevention or treatment of cancer in a patient selected for such treatment by a method comprising: assaying a biological sample representative of cancer or a pre-cancerous condition in the patient for the presence of cancer stem cells; wherein the presence of cancer stem cells in the biological sample indicates that the patient is suitable for treatment with a compound of the invention.

[0316] In suitable embodiments cancer stem cells in a biological sample may be identified by their expression of characteristic patterns of markers discussed previously in the application.

[0317] The skilled person will appreciate that there are many suitable examples of biological samples that may be used in embodiments of the invention such as those set out above. Suitably such a sample may include cells from the cancer or pre-cancerous condition. A suitable biological sample may be a tissue sample, such as a sample for use in histology. Cells in such samples may be directly assessed for their expression of cancer stem cell markers, such as those set out above.

[0318] Alternatively or additionally, a suitable biological sample may comprise target molecules representative of gene expression by cells of the cancer or pre-cancerous condition. Examples of such target molecules include proteins encoded by the genes expressed, or nucleic acids, such as mRNA, representative of gene expression.

[0319] Suitable examples of techniques by which expression of cancer stem cell markers may be assessed may be selected with reference to the sample type. Techniques for the investigation of expressed markers are frequently used in the context of clinical assessments (such as for diagnostic or prognostic purposes) and their use will be familiar to those required to practice them in the context of the present invention. Merely by way of example, in samples containing proteins the presence of cancer stem cell markers may be assessed by suitable techniques using antibodies that react with the cancer stem cell markers in question. Examples of such samples containing protein cancer stem cell markers include histology samples (where the presence of the markers may be visualised by suitable immunocytochemistry techniques), or samples derived from the circulation. Here the presence of circulating cancer stem cells (which are believed to contribute to the propagation of cancer through metastasis) may be assessed using techniques such as flow cytometry.

[0320] In samples containing nucleic acids representative of expression of cancer stem cell markers, such expression may be assessed by suitable molecule biology techniques, such as by polymerase chain reaction (PCR) amplification using suitable primers.

EXAMPLE 1

Synthetic Procedures

[0321] Compounds of the invention can be made according to or analogously to the following General Procedures and Exemplary Synthetic Procedures.

[0322] General Procedure 1 (For Compounds A-F and L-U)

[0323] N-methylimidazole (1.0 mmol) and a solution of the appropriate phosphorochloridate (0.6 mmol) in anhydrous THF (2 mL) were added dropwisely to a suspension 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. Purification 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.

[0324] General Procedure 2 (For Compound J)

[0325] 3′-Deoxyadenosine (0.80 mmol) was suspended in (CH.sub.3O).sub.3PO (5 mL), and POCl.sub.3 (0.80 mmol) was added dropwise at −5° C. 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 CH.sub.2Cl.sub.2 (5 mL) was added followed by diisopropyl ethyl amine (8.0 mmol) at −78° C. After stirring at room temperature for 20 hours, water was added and the layers were separated. The aqueous phase was extracted with dichloromethane and the organic phase washed with brine. The combined organic layers were dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by column chromatography (gradient elution of CH.sub.2Cl.sub.2/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.

[0326] General Procedure 3 (For Compounds G-I)

[0327] 3′-Deoxyadenosine (0.20 mmol) was suspended in anhydrous THF (5 mL) and .sup.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. Purification 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.

[0328] General Procedure 4 (For Compound V)

[0329] TertButyldimethylsilyl chloride (3.3 mol/eq.) and imidazole 6.6 (mol/eq) were added tp a solution of the appropriate 3′-deoxyadenosine derivate (1 mol/eq) in anhydrous DMF and the reaction mixture was stirred at room temperature overnight (16-20 h). Then NH.sub.4Cl was added to the mixture and washed twice with ethylacetate. Organic layers were combined, dried on Na.sub.2SO.sub.4 and solvent was removed under vacuum. Purification of the mixture by column chromatography afforded intermediate C1. Intermediate C1 was then dissolved at in an aqueous solution THF/H.sub.2O/TFA 4/1/1 (6 ml/eq) and was stirring at 0° C. for 4 h. The solution was then carefully neutralized with an aqueous saturated solution of NaHCO.sub.3 and the mixture was washed twice with ethylacetate. Organic layers were combined, dried on Na.sub.2SO.sub.4 and solvent was removed under vaccuo. Purification of mixture by column chromatography afforded intermediate C2. Then general procedure B was applied, and intermediate C3 was afforded. Intermediate C3 was dissolved in an aqueous solution of THF/H.sub.2O/TFA 1/1/1 (6 ml/eq) at 0° C. and was stirred at RT for 24 h. Purification by chromatography afforded the desired compounds as white solids.

[0330] General Procedure 5 (for Preparing 3′-deoxyadenosine and 3′-deoxy-2-chloroadenosine Employed in the Examples):

[0331] A solution of H.sub.2O/CH.sub.3CN 1:9 and then α-AIBBr (4.0 mol/eq) were added sequentially to a suspension of dried adenosine or 2-chloroadenosine in anhydrous CH.sub.3CN and stirring was continued at room temperature (20° C.). After 1 h, a saturated solution of NaHCO.sub.3 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 Na.sub.2SO.sub.4, filtered and evaporated to give a white gum. The crude mixture was dissolved in anhydrous MeOH and stirred for 1 h with Amberlite (2×OH.sup.−) 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′-dehydro-2-chloroadenosine as a white solid.

[0332] A solution of LiEt.sub.3BH (1M solution in THF 4-4.3 mol/eq) was added dropwise to a cold (4° C.) solution of 2′,3′-dehydroadenosine or 2′,3′-dehydro-2-chloroadenosine (1 mol/eq) in anhydrous 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/H.sub.2O), purged with N.sub.2 for 1 h (under the fume hood) to remove pyrophoric triethylborane, and evaporated. The residue was chromatographed to give 3′-deoxyadenosine or 3′-deoxy-2-chloroadenosine as a white powder.

[0333] Using General Procedure 5: 2′,3′-dehydroadenosine was prepared from 10.0 g (37.4 mmol) of adenosine, 7.5 mL of H.sub.2O/CH.sub.3CN (1/9), 22 mL (149.7 mmol) of α-AIBBr in 500 mL of anhydrous CH.sub.3CN, and 300 mL of Amberlite (2×OH.sup.−) 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 LiEt.sub.3BH/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%).

[0334] .sup.1H NMR (500 MHz, DMSO-d6) δ 8.37 (s, 1H, H8), 8.17 (s, 1H, H2), 7.29 (br s, 2H, NH.sub.2), 5.89 (d, J=2.5 Hz, 1H, H1′), 5.68 (d, J=4.5 Hz, 1H, OH-2′), 5.19 (t, J=6.0 Hz, 1H, OH-5′), 4.63-4.58 (m, 1H, H2′), 4.40-4.34 (m, 1H, H4′), 3.71 (ddd, J=12.0, 6.0, 3.0 Hz, 1H, H5′), 3.53-3.49 (ddd, J=12.0, 6.0, 4.0 Hz, 1H, H5′), 2.30-2.23 (m, 1H, H3′), 1.98-1.90 (m, 1H, H3′). .sup.13C NMR (125 MHz, DMSO-d6) δ 156.00 (C6), 152.41 (C2), 148.82 (C4), 139.09 (C8), 119.06 (C5), 90.79 (C1′), 80.66 (C4′), 74.56 (C2′), 62.61 (C5′), 34.02 (C3′).

[0335] Using General Procedure 5: 2′,3′-dehydro-2-chloroadenosine was prepared from 5.0 g (16.6 mmol) of 2-chloroadenosine, 3.0 mL of H.sub.2O/CH.sub.3CN (1/9), 9.7 mL (66.2 mmol) of α-AIBBr in 38 mL of anhydrous CH.sub.3CN, and 150 mL of Amberlite (2×OH.sup.−) resin in 200 mL of anhydrous methanol. 2′,3′-dehydro-2-chloroadenosine was obtained as a white solid (3.03 g, 60%). 3′-deoxy-2-chloroadenosine was prepared from 2.18 g (7.68 mmol) of 2′,3′-dehydro-2-chloroadenosine and 30.7 mL (30.7 mmol) of LiEt.sub.3BH/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′-deoxy-2-chloroadenosine as a white powder (1.20 g, 55%).

[0336] .sup.1H NMR (500 MHz, CD.sub.3OD): δH 8.41 (s, 1H, H8), 5.93 (d, J=2.5 Hz, 1H, H1′), 4.68-4.66 (m, 1H, H2′), 4.56-4.52 (m, 1H, H4′), 3.95 (dd, J=3, 12.5 Hz, 1H, H5′), 3.70 (dd, J=3, 12.5 Hz, 1H, H5′), 2.39-2.33 (m, 1H, H3′), 2.08-2.03 (m, 1H, H3′) .sup.13C NMR (125 MHz, CD.sub.3OD): 6C 158.14 (C6), 155.19 (C2), 151.15 (C4), 141.30 (C8), 119.56 (C5), 93.58 (C1′), 82.80 (C4′), 76.81 (C2′), 64.01 (C5′), 34.33 (C3′).

[0337] Preparation of 3′-deoxy-2-fluoroadenosine:

[0338] A solution of H.sub.2O/CH.sub.3CN (1:9; 1.4 mL) and then α-AIBBr (4.10 mL, 28.05 mmol) were added sequentially to a suspension of dried 2-fluoroadenosine (2.0 g, 7.01 mmol) in anhydrous CH.sub.3CN (50 mL) and stirring was continued at room temperature (20° C.). After 1 h, saturated solution of NaHCO.sub.3 was added cautiously and the solution was extracted with EtOAc (2×100 mL). The combined organic phase was washed with brine (1×50 mL). The aqueous phase was extracted with EtOAc (2×50 mL) and the combined organic phase was dried over Na.sub.2SO.sub.4, filtered and evaporated to give a white gum. The crude mixture was dissolved in a mixture of THF/H.sub.2O (4/1, 50 mL) and stirred for 1 h with 60 mL of Amberlite (2×OH.sup.−) resin (previously washed well with THF). The solution was then filtered and the resin carefully washed with THF. Evaporation of the combined filtrate and crystallisation of the residue from EtOH gave 2′,3′-dehydro-2-fluoroadenosine as a white solid (1.13 g, 60%).

[0339] A solution of LiEt.sub.3BH/THF (1M; 18.01 mL, 18.01 mmol) was added dropwise to a cold (4° C., ice bath) solution of 2′,3′-dehydro-2-fluoroadenosine (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 temperature overnight (16 h). The reaction mixture was carefully acidified (5% AcOH/H.sub.2O), purged with N.sub.2 for 1 h (under the fume hood) to remove pyrophoric triethylborane, and evaporated. The residue was chromatographed on silica gel (3-18% MeOH in DCM) to give 3′-deoxy-2-fluoroadenosine as a white powder (7.12 g, 77%).

[0340] .sup.19F NMR (470 MHz, DMSO-d6): δF -52.19. .sup.1H NMR (500 MHz, DMSO-d6) δH 8.34 (s, 1H, H8), 7.80 (br s, 2H, NH.sub.2), 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′). .sup.13C NMR (125 MHz, DMSO-d6) δC 158.51 (d, =202.7 Hz, C2), 157.55 (d, .sup.3J.sub.C-F=21.2 Hz, C6), 150.11 (d, .sup.3J.sub.C-F=20.3 Hz, C4), 139.22 (d, .sup.6J.sub.C-F=2.2 Hz, C8), 117.37 (d, .sup.4J.sub.C-F=4.1 Hz, C5), 90.67 (C1′), 80.90 (C4′), 74.73 (C2′), 62.35 (C5′), 33.89 (C3′).

[0341] Preparation of 3′-deoxy-2-methoxyadenosine:

[0342] A solution of H.sub.2O/CH.sub.3CN (1:9; 1.4 mL) and then α-AIBBr (4.10 mL, 28.05 mmol) were added sequentially to a suspension of dried 2-fluoroadenosine (2.0 g, 7.01 mmol) in anhydrous CH.sub.3CN (50 mL) and stirring was continued at room temperature (20° C.). After 1 h, saturated solution of NaHCO.sub.3 was added cautiously and the solution was extracted with EtOAc (2×100 mL). The combined organic phase was washed with brine (1×50 mL). The aqueous phase was extracted with EtOAc (2×50 mL) and the combined organic phase was dried over Na.sub.2SO.sub.4, 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 Amberlite (2×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 crystallisation of the residue from EtOH gave 2′,3′-dehydro-2-methoxyadenosine as a white solid (1.57 g, 84%).

[0343] A solution of LiEt.sub.3BH (1M solution in THF; 8.53 mL, 8.53 mmol) was added dropwise to a cold (4° C.) solution of 2′,3′-dehydro-2-methoxyadenosine (762 mg, 2.84 mmol) in 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/H.sub.2O), purged with N2 for 1 h (under the fume hood) to remove pyrophoric triethylborane, and evaporated. The residue was chromatographed on silica gel (3-17% MeOH in DCM) to give 3′-deoxy-2-methoxyadenosine as a white powder (650 mg, 81%).

[0344] .sup.1H NMR (500 MHz, CD.sub.3OD) δH 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, OCH.sub.3), 2.43-2.35 (m, 1H, H3′), 2.08-2.02 (m, 1H, H3′). .sup.13C NMR (125 MHz, CD.sub.3OD) δC 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 (OCH.sub.3), 34.81 (C3′).

[0345] Phosphorochloridates were prepared by published methods from aryl phosphorodichloridates and amino acid ester hydrochlorides.

3′-deoxyadenosine-5′-O-[phenyl(benzyloxy-L-alaninyl)]phosphate A

[0346] ##STR00010##

[0347] Compound A was prepared according to the General Procedure 1 using 3′-deoxyadenosine (50 mg, 0.20 mmol), N-methylimidazole (80 μL, 1.0 mmol) and phenyl(benzyloxy-L-alaninyl) phosphorochloridate (212 mg, 0.6 mmol). Purification by column chromatography (eluent system CH.sub.3OH/CH.sub.2Cl.sub.2 0/100 to 7/93) with gradient of CH.sub.2Cl.sub.2/MeOH (100% to 95:5%) and preparative TLC (1000 μm, eluent system CH.sub.3OH/CH.sub.2Cl.sub.2 5/95) afforded the title compound as a white solid (31 mg, 28%).

[0348] .sup.1H NMR (500 MHz, CD.sub.3OD): δH 8.26 (s, 0.5H, H8), 8.24 (s, 0.5H, H8), 8.22 (s, 0.5H, H2), 8.21 (s, 0.5H, H2), 7.34-7.25 (m, 7H, Ar), 7.21-7.13 (m, 3H, Ar), 6.01 (d, J=2.9 Hz, 1H, H1′), 6.00 (d, J=2.9 Hz, 1H, H1′), 5.15-5.04 (m, 2H, OCH.sub.2Ph), 4.73-4.63 (m, 2H, H2′, H4′), 4.43-4.35 (m, 1H, H5′), 4.27-4.20 (m, 1H, H5′), 4.03-3.91 (m, 1H, CHCH.sub.3), 2.35-2.28 (m, 1H, H3′), 2.09-2.02 (m, 1H, H3′), 1.32 (d, J=7.4 Hz, 1.5H, CHCH.sub.3), 1.28 (d, J=7.4 Hz, 1.5H, CHCH.sub.3).

[0349] .sup.13C NMR (125 MHz, CD.sub.3OD): δC 174.84 (d, .sup.3J.sub.C-P=4.5 Hz, C═O), 174.63 (d, .sup.3J.sub.C-P=4.5 Hz, C═O), 157.32 (C6), 157.31 (C6), 153.86 (C2), 153.84 (C2), 152.13 (C4), 152.07 (C4), 150.20 (C—Ar), 150.18 (C—Ar), 140.47 (C8), 137.26 (C—Ar), 137.19 (C—Ar), 130.76 (CH—Ar), 130.74 (CH—Ar), 129.57 (CH—Ar), 129.32 (CH—Ar), 129.31 (CH—Ar), 129.29 (CH—Ar), 129.26 (CH—Ar), 126.16 (CH—Ar), 126.14 (CH—Ar), 121.46 (d, .sup.3J.sub.C-P=4.7 Hz, CH—Ar), 121.38 (d, .sup.3J.sub.C-P=4.7 Hz, CH—Ar) 120.54 (C5), 120.53 (C5), 93.24 (C1′), 93.18 (C1′), 80.43 (d, .sup.3J.sub.C-P=3.6 Hz, C4′), 80.36 (d, .sup.3J.sub.C-P=3.6 Hz, C4′), 76.62 (C2′), 68.62 (d, .sup.2J.sub.C-P=5.3 Hz, C5′), 68.30 (d, .sup.2J.sub.C-P=5.3 Hz, C5′), 67.95 (OCH.sub.2Ph), 67.92 (OCH.sub.2Ph), 51.74 (CHCH.sub.3), 51.60 (CHCH.sub.3), 34.91 (C3′), 34.70 (C3′), 20.45 (d, .sup.3J.sub.C-P=7.0 Hz, CHCH.sub.3), 20.28 (d, .sup.3J.sub.C-P=7.0 Hz, CHCH.sub.3).

[0350] .sup.31P NMR (202 MHz, CD.sub.3OD): δP 3.9, 3.7.

[0351] MS (ES+) m/z: Found: 569.2 (M+H.sup.+), 591.2 (M+Na.sup.+), 1159.4 (2M+Na.sup.+), C.sub.26H.sub.29N.sub.6O.sub.7P required: (M) 568.2.

[0352] HPLC Reverse-phase HPLC eluting with H.sub.2O/CH.sub.3CN from 100/10 to 0/100 in 30 minutes, 1 ml/min, 1=254 nm, showed two peaks of the diastereoisomers with tR 14.02 min. and tR 14.26 min.

(2S)-benzyl 2-(((((2S,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)(naphthalen-1-yloxy)phosphoryl)amino)propanoate B

[0353] ##STR00011##

[0354] Using General Procedure 1 above, N-methylimidazole (240 μL, 3.0 mmol) and a solution of (2S)-benzyl 2-((chloro(naphthalen-1-yloxy)phosphoryl)amino)propanoate (727 mg, 1.8 mmol) in anhydrous THF (10 mL) were added dropwisely to a suspension of 3′-deoxyadenosine (150 mg, 0.6 mmol) in anhydrous THF and the reaction mixture was stirred at room temperature during a period of 16 hours. Purification by column chromatography (eluent system CH.sub.3OH/CH.sub.2Cl.sub.2 0/100 to 6/94) and preparative TLC (2000 μM, eluent system CH.sub.3OH/CH.sub.2Cl.sub.2 5/95) afforded the desired compound as white solid (45 mg, 12%).

[0355] MS (ES+) m/z: Found: 619.2 (M+H.sup.+), 641.2 (M+Na.sup.+), 1259.4 (2M+Na.sup.+) C.sub.30H.sub.31N.sub.6O.sub.7P required: (M) 618.58.

[0356] .sup.31P NMR (202 MHz, CH.sub.3OD): δP 4.3 (s), 4.1 (s).

[0357] .sup.1H NMR (500 MHz, CH.sub.3OD): δH 8.24 (s, 0.5H, H8), 8.22 (s, 0.5H, H8), 8.20 (s, 0.5H, H2), 8.19 (s, 0.5H, H2), 8.14-8.09 (m, 1H, Ar), 7.89-7.85 (m, 1H, Ar), 7.70-7.67 (m, 1H, Ar), 7.53-7.42 (m, 3H, Ar), 7.39-7.34 (m, 1H, Ar), 7.31-7.25 (m, 5H, Ar), 5.99 (d, J=2.0 Hz, 0.5H, H1′), 5.98 (d, J=2.0 Hz, 0.5H, H1′), 5.10-5.01 (m, 2H, CH.sub.2Ph), 4.72-4.61 (m, 2H, H2′, H4′), 4.47-4.40 (m, 1H, H5′), 4.33-4.24 (m, 1H, H5′), 4.09-3.98 (m, 1H, CH ala) 2.35-2.26 (m, 1H, H3′), 2.07-1.98 (m, 1H, H3′), 1.30-1.24 (m, 3H, CH.sub.3).

[0358] .sup.13C NMR (125 MHz, CH.sub.3OD): δC 174.85 (d, .sup.3J.sub.C-P=3.7 Hz, C═O), 174.56 (d, .sup.3J.sub.C-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, .sup.3J.sub.C-P=7.5 Hz, ‘ipso’ Nap), 147.95, (d, .sup.3J.sub.C-P=7.5 Hz, ‘ipso’ Nap), 140.56 (C8), 140.50 (C8), 137.22 (C—Ar), 137.17 (C—Ar), 136.28 (C—Ar), 129.55 (CH—Ar), 129.53 (CH—Ar), 129.30 (CH—Ar), 129.25 (CH—Ar), 128.88 (CH—Ar), 128.82 (CH—Ar), 127.91 (d, .sup.2J.sub.C-P=6.25 Hz, C—Ar), 127.83 (d, .sup.2J.sub.C-P=6.25 Hz, C—Ar), 127.77 (CH—Ar), 127.75 (CH—Ar), 127.49 (CH—Ar), 127.45 (CH—Ar), 126.48 (CH—Ar), 126.47 (CH—Ar), 126.02 (CH—Ar), 125.97 (CH—Ar), 122.77 (CH—Ar), 122.63 (CH—Ar), 120.58 (C5), 120.53 (C5), 116.35 (d, .sup.3J.sub.C-P=3.75 Hz, CH—Ar), 116.15 (d, .sup.3J.sub.C-P=3.75 Hz, CH—Ar), 93.22 (C1′), 93.20 (C1′), 80.30 (d, .sup.3J.sub.C-P=2.75 Hz, C4′), 80.24 (d, .sup.J.sub.C-P=2.75 Hz, C4′), 76.51 (C2′), 76.44 (C2′), 68.87 (d, .sup.2J.sub.C-P=5.2 Hz, C5′), 68.64 (d, .sup.2J.sub.C-P=5.2 Hz, C5′), 67.93 (OCH.sub.2Ph), 51.82 (CH ala), 51.73 (CH ala), 35.01 (C-3′), 34.76 (C3′), 20.41 (d, .sup.3J.sub.C-P=6.7 Hz, CH.sub.3 ala), 20.22 (d, .sup.3J.sub.C-P=6.7, CH.sub.3 ala).

[0359] HPLC Reverse-phase HPLC eluting with H.sub.2O/CH.sub.3CN from 100/10 to 0/100 in 30 minutes, 1 ml/min, 1=200 nm, showed two peaks of the diastereoisomers with tR 16.36 min. and tR 16.60 min.

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

[0360] ##STR00012##

[0361] Using General Procedure 1 above, N-methylimidazole (80 μL, 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 mixture was stirred at room temperature for 16 hours. Purification by column chromatography (eluent system CH.sub.3OH/CH.sub.2Cl.sub.2 0/100 to 6/94) and preparative TLC (500 μM, eluent system CH.sub.3OH/CH.sub.2Cl.sub.2 5/95) afforded the desired compound as a white solid (21 mg, 19%).

[0362] (ES+) m/z, found: 555.2 (M+H.sup.+), 577.2 (M+Na.sup.+), 1131.4 (2M+Na.sup.+). C.sub.25H.sub.27N.sub.6O.sub.7P required: (M) 554.2.

[0363] .sup.31P NMR (202 MHz, CH.sub.3OD) δ5.1, 4.9.

[0364] .sup.1H NMR (500 MHz, CH.sub.3OD) δ 8.27 (s, 0.5H, H8), 8.24 (s, 0.5H, H8), 8.22 (s, 0.5H, H2), 8.21 (s, 0.5H, H2), 7.37-7.26 (m, 7H, Ph), 7.22-7.13 (m, 3H, Ph), 6.02 (d, J=1.8 Hz, 0.5H, H1′), 6.00 (d, J=1.8 Hz, 0.5H, H1′), 5.14-5.11 (m, 2H, OCH.sub.2Ph), 4.73-4.64 (m, 2H, H2′, H4′), 4.50-4.39 (m, 1H, H5′), 4.36-4.24 (m, 1H, H5′), 3.53-3.71 (m, 2H, CH.sub.2 gly), 2.39-2.25 (m, 1H, H3′), 2.13-2.02 (m, 1H, H3′).

[0365] .sup.13C NMR (125 MHz, CH.sub.3OD) δ 172.30 (d, .sup.3J.sub.C-P=5.0 Hz, C═O), 172.27 (d, .sup.3J.sub.C-P=5.0 Hz, C═O), 157.34 (C6), 157.32 (C6), 153.88 (C2), 153.87 (C2), 152.08 (d, .sup.3J.sub.C-P=7.5 Hz, C—Ar), 152.05 (d, .sup.3J.sub.C-P=7.5 Hz, C—Ar), 150.20 (C4), 150.19 (C4), 140.52 (C8), 140.42 (C8), 137.15 (C—Ar), 130.79 (CH—Ar), 129.57 (CH—Ar), 129.55 (CH—Ar), 129.35 (CH—Ar), 129.34 (CH—Ar), 129.33 (CH—Ar), 126.22 (CH—Ar), 121.44 (d, J.sub.C-P=3.7 Hz, CH—Ar), 121.40 (d, J.sub.C-P=3.7 Hz, CH—Ar), 120.51 (C5), 120.49 (C5), 93.19, 93.14 (C1′), 80.46 (d, .sup.3J.sub.C-P=4.60 Hz, C4′), 80.39 (d, .sup.3J.sub.C-P=4.60, C4′), 76.66 (C2′), 68.68 (d, .sup.2J.sub.C-P=5.42 Hz, C5′), 68.24 (d, .sup.2J.sub.C-P=5.42 Hz, C5′), 67.95 (OCH.sub.2Ph), 67.93 (OCH.sub.2Ph), 43.90 (CH.sub.2 gly), 43.83 (CH.sub.2 gly), 34.83 (C3′), 34.54 (C3′).

[0366] HPLC Reverse-phase HPLC eluting with H.sub.2O/CH.sub.3CN from 100/10 to 0/100 in 30 minutes, 1 ml/min, 1=200 nm, showed two peaks of the diastereoisomers with tR 13.63 min. and tR 13.41 min.

(2S)-pentyl 2-(((((2S,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)(naphthalen-1-yloxy)phosphoryl)amino)-4-methylpentanoate D

[0367] ##STR00013##

[0368] Using General Procedure 1 above, N-methylimidazole (76 μL, 0.95 mmol) and a solution of (2S)-pentyl 2-((chloro(naphthalen-1-yloxy)phosphoryl)amino)-4-methylpentanoate (250 mg, 0.6 mmol) in anhydrous THF (1 mL) were added dropwisely to a suspension of 3′-deoxyadenosine (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 (eluent system CH.sub.3OH/CH.sub.2Cl.sub.2 0/100 to 5/95) and preparative TLC (1000 μM, eluent system CH.sub.3OH/CH.sub.2Cl.sub.2 4/96) afforded the desired compound as a white solid (27 mg, 22%).

[0369] MS (ES+) m/z: Found: 641.3 (M+H.sup.+), 663.3 (M+Na.sup.+), 1303.6 (2M+Na.sup.+) C.sub.31H.sub.41N.sub.6O.sub.7P required: (M) 640.3.

[0370] .sup.31P NMR (202 MHz, CH.sub.3OD) δ 4.64, 4.37.

[0371] .sup.1H NMR (500 MHz, CH.sub.3OD) δ 8.28 (s, 0.5H, H-8), 8.25 (s, 0.5H, H-8), 8.21 (s, 0.5H, H-2), 8.20 (s, 0.5H, H-2), 8.17-8.12 (m, 1H, Nap), 7.88-7.83 (m, 1H, Nap), 7.69-7.66 (m, 1H, Nap), 7.54-7.42 (m, 3H, Nap), 7.40-7.35 (m, 1H, Nap), 7.31-7.26 (m, 5H, Ar), 6.01 (d, J=2.1 Hz, 0.5H, H1′), 6.00 (d, J=2.1 Hz, 0.5H, H1′), 4.47-4.67 (m, 2H, H2′, H4′), 4.55-4.44 (m, 1H, H5′), 4.43-4.31 (m, 1H, H5′), 4.00-3.87 (m, 3H, CH leu, CH.sub.2 Pen), 2.44-2.30 (m, 1H, H3′), 2.14-2.04 (m, 1H, H3′), 1.66-1.39 (m, 5H, CH.sub.2CH leu, CH.sub.2Pen), 1.1.28-1.21 (m, 4H, CH.sub.2CH.sub.2 Pen), 0.86-0.81 (m, 3H, CH.sub.3 Pen), 0.81-0.68 (m, 6H, (CH.sub.3).sub.2 leu).

[0372] .sup.13C NMR (125 MHz, CH.sub.3OD) δ 175.42 (d, .sup.3J.sub.C-P=2.5 Hz, C═O), 175.04 (d, .sup.3J.sub.C-P=2.5 Hz, C═O), 157.32 (C6), 153.87 (C2), 153.86 (C2), 150.23 (C4), 147.97 (d, .sup.3J.sub.C-P=6.2 Hz, ‘ipso’ Nap), 140.55 (C8), 136.30 (C—Ar), 136.29 (C—Ar), 128.89 (CH—Ar), 128.84 (CH—Ar), 127.95 (C—Ar), 127.91 (C—Ar), 127.84 (C—Ar), 127.78 (CH—Ar), 127.76 (CH—Ar), 127.46 (CH—Ar), 126.50 (C—Ar), 126.48 (C—Ar), 126.46 (C—Ar), 126.01 (CH—Ar), 125.91 (CH—Ar), 122.80 (CH—Ar), 122.70 (CH—Ar), 120.58 (C5), 120.56 (C5), 116.40 (d, .sup.3J.sub.C-P=3.7 Hz, CH—Ar), 116.01 (d, .sup.3J.sub.C-P=3.7 Hz, CH—Ar), 93.31 (C1′), 93.27 (C1′), 80.35 (d, .sup.3J.sub.C-P=3.5 Hz, C4′), 80.29 (d, .sup.3J.sub.C-P=3.5 Hz, C4′), 76.54 (C2′), 76.50 (C2′), 69.07 (d, .sup.2J.sub.C-P=5.5 Hz, C5′), 68.85 (d, .sup.2J.sub.C-P=5.5 Hz, C5′), 66.33 (CH.sub.2Pent), 66.32 (CH.sub.2 Pent), 54.81 (CH leu), 54.71 (CH leu), 44.22 (d, .sup.3J.sub.C-P=7.6 Hz, CH2 leu), 43.93 (d, .sup.3J.sub.C-P=7.6 Hz, CH.sub.2 leu), 35.15 (C3′), 34.86 (C3′), 29.32 (CH.sub.2 pent), 29.30 (CH.sub.2 Pent), 29.11 (CH.sub.2 pent), 25.67 (CH leu), 25.45 (CH leu), 23.30 (CH.sub.2 pent), 23.12 (CH.sub.3 leu), 23.02 (CH.sub.3 leu), 22.04 (CH.sub.3 leu), 21.78 (CH.sub.3 leu), 14.28 (CH.sub.3 pent).

[0373] HPLC Reverse-phase HPLC eluting with H.sub.2O/CH.sub.3CN from 100/10 to 0/100 in 30 minutes, 1 ml/min, 1=200 nm, showed one peak of the two overlapping diastereoisomers with tR 20.84 min.

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

[0374] ##STR00014##

[0375] Using General Procedure 1 above, N-methylimidazole (24 μL, 3.0 mmol) and a solution of methyl 2-((chloro(naphthalen-1-yloxy)phosphoryl)amino)-2-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 CH.sub.3OH/CH.sub.2Cl.sub.2 0/100 to 7/93) and preparative TLC (1000 μM, eluent system CH.sub.3OH/CH.sub.2Cl.sub.2 4/96) afforded the desired compound as a white solid (20 mg, 6%).

[0376] MS (ES+) m/z: Found: 557.2 (M+H.sup.+), 579.2 (M+Na.sup.+), 1135.4 (2M+Na.sup.+) C.sub.25H.sub.29N.sub.6O.sub.7P required: (M) 556.51.

[0377] .sup.31P NMR (202 MHz, CH.sub.3OD) δ 2.73.

[0378] .sup.1H NMR (500 MHz, CH.sub.3OD) δ 8.28 (s, 0.5H, H8), 8.25 (s, 0.5H, H8), 8.21 (s, 0.5H, H2), 8.19 (s, 0.5H, H2), 8.18-8.14 (m, 1H, Nap), 7.90-7.84 (m, 1H, Nap), 7.71-7.66 (m, 1H, Nap), 7.53-7.47 (m, 3H, Nap), 7.41-7.35 (m, 1H, Nap), 6.03 (d, J=2.1 Hz, 0.5H, H1′), 5.99 (d, J=2.1 Hz, 0.5H, H1′), 4.76-4.67 (m, 2H, H2′, H4′), 4.52-4.44 (m, 1H, H5′), 4.42-4.33 (m, 1H, H5′), 3.65 (s, 1.5H, OCH.sub.3), 3.64 (s, 1.5H, OCH.sub.3), 2.48-2.41 (m, 0.5H, H3′), 2.37-2.30 (m, 0.5H, H3′), 2.15-2.09 (m, 0.5H, H3′), 2.08-2.02 (m, 0.5H, H3′), 1.47-1.44 (m, 6H, CH.sub.3).

[0379] .sup.13C NMR (125 MHz, CH.sub.3OD) δ 177.25 (d, .sup.3J.sub.C-P=3.7 Hz, C═O), 157.53 (C6), 157.51 (C6), 153.86 (C2), 150.28 (C4), 150.25 (C4), 148.06 (d, .sup.3J.sub.C-P=7.5 Hz, ‘ipso’ Nap), 148.04 (d, .sup.3J.sub.C-P=7.5, ‘ipso’ Nap), 140.67 (C8), 140.60 (C8), 136.28 (C—Ar), 136.27 (C—Ar), 128.82 (CH—Ar), 128.80 (CH—Ar), 127.93 (d, .sup.2J.sub.C-P=6.25 Hz, C—Ar), 127.92 (d, .sup.2J.sub.C-P=6.25 Hz, C—Ar), 127.71 (CH—Ar), 127.69 (CH—Ar), 127.32 (CH—Ar), 126.44 (CH—Ar), 125.84 (CH—Ar), 122.93 (CH—Ar), 120.56 (C5), 120.50 (C5), 116.38 (d, .sup.3J.sub.C-P=3.75 Hz, CH—Ar), 116.36 (d, .sup.3J.sub.C-P=3.75 Hz, CH—Ar), 93.25 (C1′), 80.40 (d, .sup.3J.sub.C-P=8.0 Hz, C4′), 80.33 (d, .sup.3J.sub.C-P=8.0 Hz, C4′), 76.57 (C2′), 76.43 (C2′), 68.99 (d, .sup.2J.sub.C-P=5.5 Hz, C5′), 68.84 (d, =5.5 Hz, C5′), 53.01 (OCH.sub.3), 35.22 (C-3′), 34.90 (C3′), 27.85 (d, .sup.3J.sub.C-P=6.0 Hz, CH.sub.3), 27.80 (d, .sup.3J.sub.C-P=6.0, CH.sub.3), 27.60 (d, .sup.3J.sub.C-P=6.0, CH.sub.3), 27.56 (d, .sup.3J.sub.C-P=6.0, CH.sub.3).

[0380] HPLC Reverse-phase HPLC eluting with H.sub.2O/CH.sub.3CN from 100/10 to 0/100 in 30 minutes, 1 ml/min, 1=254 nm, showed two peaks with tR 16.51 min, tR 16.75 min.

(2S)-benzyl 2-(((((2S,4R,5R)-5-(6-amino-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)(2-(3-ethoxy-3-oxopropyl)phenoxy)phosphoryl)amino)propanoate F

[0381] ##STR00015##

[0382] Using General Procedure 1 above, N-methylimidazole (32 μL, 4.2 mmol) and a solution of (2S)-benzyl 2-((chloro(2-(3-ethoxy-3-oxopropyl)phenoxy)phosphoryl)amino)propanoate (1.14 g, 2.5 mmol) in anhydrous THF (2 mL) were added dropwisely to a suspension of 3′-deoxyadenosine (210 mg, 0.84 mmol) in anhydrous THF (10 mL) and the reaction mixture was stirred at room temperature during a period of 16 hours. Purification by column chromatography (eluent system CH.sub.3OH/CHCl.sub.3 0/100 to 8/92) and preparative TLC (1000 μM, eluent system CH.sub.3OH/CH.sub.2Cl.sub.2 5/95) afforded the desired compound as a white solid (123 mg, yield=22%). MS (ES+) m/z: Found: 669.3 (M+H.sup.+), 691.3 (M+Na.sup.+) C.sub.31H.sub.37N.sub.6O.sub.9P required: (M) 668.63.

[0383] .sup.31P NMR (202 MHz, CH.sub.3OD): δP 3.95, 3.65.

[0384] .sup.1H NMR (500 MHz, CH.sub.3OD): δH 8.25 (s, 0.5H, H8), 8.21 (s, 1H, H8, H2), 8.20 (s, 0.5H, H2), 7.35-7.29 (m, 6H, Ph), 7.25-7.21 (m, 1H, Ph), 7.16-7.07 (m, 2H, Ar), 6.00 (d, J=1.9 Hz, 0.5H, H1′), 5.98 (d, J=1.9 Hz, 0.5H, H1′), 5.17-5.05 (m, 2H, OCH.sub.2Ph), 4.76-4.73 (m, 0.5H, H2′), 4.70-4.59 (m, 1.5H, H2′, H4′), 4.45-4.34 (m, 1H, H5′), 4.30-4.22 (m, 1H, H5′), 4.08-3.96 (m, 3H, CH.sub.2CH.sub.3, CH ala), 2.98-2.92 (m, 2H, CH.sub.2CH.sub.2), 2.62-2.56 (m, 2H, CH.sub.2CH.sub.2), 2.40-2.29 (m, 1H, H3′), 2.11-2.03 (m, 1H, H3′), 1.36 (d, J=6.9 Hz, 1.5H, CH.sub.3 ala), 1.33 (d, J=6.9 Hz, 1.5H, CH.sub.3 ala), 1.17 (t, J=7.0 Hz, 1.5H, CH.sub.2CH.sub.3), 1.16 (t, J=7.0 Hz, 1.5H, CH.sub.2CH.sub.3).

[0385] .sup.13C NMR (125 MHz, CH.sub.3OD): δC 174.82 (d, .sub.3J.sub.C-P=3.7 Hz, C═O), 174.62 (C═O), 174.58 (C═O), 174.55 (d, .sup.3J.sub.C-P=3.7 Hz, C═O), 157.34 (C6), 157.32 (C6), 153.86 (C2), 153.84 (C2), 150.48 (d, J.sub.C-P=2.5 Hz, C—Ar), 150.44 (C4), 150.22 (d, J.sub.C-P=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, .sup.3J.sub.C-P=8.7 Hz, C4′), 76.57 (C2′), 68.86 (d, .sup.2J.sub.C-P=5.0 Hz, C5′), 68.53 (d, .sup.2J.sub.C-P=5.0 Hz, C5′), 67.98 (OCH.sub.2Ph), 67.95 (OCH.sub.2Ph), 61.57 (CH.sub.2CH.sub.3), 51.76 (CH ala), 51.65 (CH ala), 35.37 (CH.sub.2CH.sub.2), 35.30 (CH.sub.2CH.sub.2), 35.08 (C3′), 34.85 (C3′), 26.77 (CH.sub.2CH.sub.2), 26.72 (CH.sub.2CH.sub.2), 20.55 (d, .sup.3J.sub.C-P=6.2 Hz, CH.sub.3 ala), 20.33 (d, .sup.3J.sub.C-P=6.2 Hz, CH.sub.3 ala), 14.53 (CH.sub.2CH.sub.3).

[0386] HPLC Reverse-phase HPLC eluting with H.sub.2O/CH.sub.3CN from 100/10 to 0/100 in 30 minutes, 1 ml/min, 1=245 nm, showed one peak with tR 15.99 min.

(2S)-benzyl 2-(((((2R,3R,5S)-2-(6-amino-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydro-furan-3-yl)oxy)(phenoxy)phosphoryl)amino)propanoate G

[0387] ##STR00016##

[0388] Using General Procedure 3 above, 3′-deoxyadenosine (50 mg, 0.20 mmol) was suspended in anhydrous THF (5 mL) and .sup.tBuMgCl (1.0 M solution in THF, 0.22 mL, 0.22 mmol) was added dropwisely at room temperature. A solution of (2S)-benzyl 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 (eluent system CH.sub.3OH/CH.sub.2Cl.sub.2 0/100 to 8/92) and preparative TLC (500 μM, eluent system CH.sub.3/CH.sub.2Cl.sub.2=5/95) afforded the desired compound as a white solid (6 mg, 5%).

[0389] MS (ES+) m/z: Found: 569.2 (M+H.sup.+), 591.2 (M+Na.sup.+), 1159.4 (2M+Na.sup.+) C.sub.26H.sub.29N.sub.6O.sub.7P required: (M) 568.2.

[0390] .sup.31P NMR (202 MHz, CH.sub.3OD): δP 2.44 (s), 2.92 (s).

[0391] .sup.1H NMR (500 MHz, CH.sub.3OD): δH 8.41 (s, 0.5H, H8), 8.28 (s, 0.5H, H8), 8.19 (s, 0.5H, H2), 8.18 (s, 0.5H, H2), 7.39-7.30 (m, 4H, Ar), 7.28-7.18 (m, 4H, Ar), 7.17-7.11 (m, 1H, Ar), 7.08-7.03 (m, 1H, Ar), 6.23 (d, J=2.0 Hz, 0.5H, H1′), 6.08 (d, J=3.4 Hz, 0.5H, H1′), 5.52-5.43 (m, 1H, C2′), 5.19-5.12 (m, 1H, CH.sub.2Ph), 5.07-4.95 (m, 1H, CH.sub.2Ph), 4.48-4.42 (m, 1H, H4′), 4.05-3.97 (m, 1H, CH ala), 3.95-3.87 (m, 1H, H5′), 3.69-3.61 (m, 1H, H5′), 2.59-2.45 (m, 1H, H3′), 2.31-2.23 (m, 1H, H3′), 1.36-1.27 (m, 3H, CH.sub.3 ala).

[0392] .sup.13C NMR (125 MHz, CH.sub.3OH): δC 174.76 (d, .sup.3J.sub.C-P=5.0 Hz, C═O), 174.52 (d, .sup.3J.sub.C-P=5.0 Hz, C═O), 157.44 (C6), 153.76 (C2), 151.93 (C4), 150.06 (C—Ar), 149.93 (C—Ar), 141.38 (C8), 141.18 (C8), 137.33 (C—Ar), 137.10 (C—Ar), 130.69 (CH—Ar), 130.79 (CH—Ar), 129.61 (CH—Ar), 129.51 (CH—Ar), 129.40 (CH—Ar), 129.30 (CH—Ar), 129.23 (CH—Ar), 126.33 (CH—Ar), 126.16 (CH—Ar), 121.53 (d, .sup.3J.sub.C-P=4.5 Hz, CH—Ar), 121.20 (d, .sup.3J.sub.C-P=4.5H, CH—Ar), 120.76 (C5), 91.56 (d, .sup.3J.sub.C-P=7.7 Hz, C1′), 91.45 (d, .sup.3J.sub.C-P=7.7 Hz, C1′), 82.78 (C4′), 82.28 (C4′), 81.83 (d, .sup.2J.sub.C-P=4.7 Hz, C2′), 80.96 (2×d, .sup.2J.sub.C-P=4.7 Hz, C2′), 67.95 (OCH.sub.2Ph), 67.92 (OCH.sub.2Ph), 64.13 (C5′), 63.59 (C5′), 51.88 (CH ala), 51.75 (CH ala), 33.75 (d, .sup.3J.sub.C-P=3.0 Hz, C3′), 33.59 (d, .sup.3J.sub.C-P=3.0 Hz, C3′), 20.33 (d, .sup.3J.sub.C-P=7.1 CH.sub.3 ala), 20.18 (d, .sup.3J.sub.C-P=7.1 CH.sub.3 ala).

[0393] HPLC Reverse-phase HPLC eluting with H.sub.2O/CH.sub.3OH from 90/10 to 0/100 in 30 minutes, 1 ml/min, 1=254 nm, showed two peaks of the diastereoisomers with tR 22.16 min. and tR 22.43 min.

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

[0394] ##STR00017##

[0395] Using General Procedure 3 above, 3′-deoxyadenosine (50 mg, 0.20 mmol) was suspended in anhydrous THF (5 mL) and .sup.tBuMgCl (1.0 M solution in THF, 0.22 mL, 0.22 mmol) was added dropwisely at room temperature. A solution of (2S)-benzyl 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 (eluent system CH.sub.3/CH.sub.2Cl.sub.2 0/100 to 8/92) and preparative TLC (500 μM, eluent system CH.sub.3/CH.sub.2Cl.sub.2 5/95) afforded the desired compound as a white solid (19 mg, yield =11%).

[0396] MS (ES+) m/z, found: 886.3 (M+H.sup.+), 1771.6 (2M+H.sup.+), 751.2 (molecule without nucleobase M). C.sub.42H.sub.45N.sub.7O.sub.11P.sub.2 required: (M+) 885.3.

[0397] .sup.31P NMR (202 MHz, CH.sub.3OD): δP 3.98, 3.88, 3.59, 3.12, 3.05, 2.45, 2.32.

[0398] .sup.1H NMR (500 MHz, CH.sub.3OD): δH 8.24-8.13 (m, 2H, H8, H2), 7.39-7.08 (m, 20H, Ph), 6.27-6.23 (m, 0.5H, Hi'), 6.16-6.13 (m, 0.5H, Hi'), 5.61-5.48 (m, 1H, H2′), 5.17-4.91 (m, 4H, CH.sub.2Ph), 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, CH.sub.3 ala).

[0399] .sup.13C NMR (125 MHz, CH.sub.3OD): δC 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 (C—Ar), 150.29 (C—Ar), 150.16 (C—Ar), 140.98 (C8), 140.91 (C8), 140.81 (C8), 137.31 (C—Ar), 137.28 (C—Ar), 137.22 (C—Ar), 137.09 (C—Ar), 130.86 (CH—Ar), 130.78 (CH—Ar), 130.77 (CH—Ar), 129.65 (CH—Ar), 129.61 (CH—Ar), 129.58 (CH—Ar), 129.55 (CH—Ar), 129.44 (CH—Ar), 129.42 (CH—Ar), 129.38 (CH—Ar), 129.34 (CH—Ar), 129.32 (CH—Ar), 129.30 (CH—Ar), 129.28 (CH—Ar), 129.23 (CH—Ar), 129.21 (CH—Ar), 12.42 (CH—Ar), 126.23 (CH—Ar), 126.20 (CH—Ar), 126.17 (CH—Ar), 121.65 (CH—Ar), 121.63 (CH—Ar), 121.61 (CH—Ar), 121.59 (CH—Ar), 121.52 (CH—Ar), 121.50 (CH—Ar), 121.47 (CH—Ar), 121.46 (CH—Ar), 121.40 (CH—Ar), 121.39 (CH—Ar), 121.36 (CH—Ar), 121.35 (CH—Ar), 121.30 (CH—Ar), 121.28 (CH—Ar), 121.26 (CH—Ar), 121.24 (CH—Ar), 120.61 (C5), 120.57 (C5), 120.56 (C5), 120.54 (C5), 91.56 (C1′), 91.51 (C1′), 91.45 (C1′), 91.25 (C1′), 91.20 (C1′), 81.84 (C2′), 81.82 (C2′), 81.79 (C2′), 81.27 (C2′), 81.22 (C2′), 81.18 (C2′), 80.49 (C4′), 80.43 (C4′), 80.06 (C4′), 79.99 (C4′), 68.29 (C5′, OCH.sub.2Ph), 68.25 (C5′, OCH.sub.2Ph), 68.00 (C5′, OCH.sub.2Ph), 67.96 (C5′, OCH.sub.2Ph), 67.94 (C5′, OCH.sub.2Ph), 67.90 (C5′, OCH.sub.2Ph), 67.71 (C5′, OCH.sub.2Ph), 67.67 (C5′, OCH.sub.2Ph), 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 (CH.sub.3 ala), 20.43 (CH.sub.3 ala), 20.39 (CH.sub.3 ala), 20.29 (CH.sub.3 ala), 20.27 (CH.sub.3 ala), 20.24 (CH.sub.3 ala), 20.21 (CH.sub.3 ala), 20.19 (CH.sub.3 ala).

[0400] HPLC Reverse-phase HPLC eluting with H.sub.2O/CH.sub.3CN from 100/10 to 0/100 in 30 minutes, 1 ml/min, 1=254 nm, showed one broad peak with tR 15.97 min.

(2S)-benzyl 2-(((((2R,3R,5S)-2-(6-amino-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydro-furan-3-yl)oxy)(naphthalen-1-yloxy)phosphoryl)amino)propanoate I

[0401] ##STR00018##

[0402] Using General Procedure 3 above, 3′-Deoxyadenosine (50 mg, 0.20 mmol) was suspended in anhydrous THF (5 mL) and .sup.tBuMgCl (1.0 M solution in THF, 0.3 mL, 0.3 mmol) was added dropwisely at room temperature. A solution of (2S)-benzyl 2-((chloro(naphthalen-1-yloxy)phosphoryl)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.

[0403] Purification by column chromatography (eluent system CH.sub.3/CH.sub.2Cl.sub.2 0/100 to 6/94) and preparative TLC (500 μM, eluent system CH.sub.3/CH.sub.2Cl.sub.2 5/95) afforded the desired compound as a white solid (14 mg, 11%).

[0404] (ES+) m/z, found: 619.2 (M+H.sup.+), 641.2 (M+Na.sup.+), 1259.4 (2M+Na.sup.+) C.sub.30H.sub.31N.sub.6O.sub.7P required: (M) 618.20.

[0405] .sup.31P NMR (202 MHz, CH.sub.3OD): δP 3.27 (s), 2.75 (s).

[0406] .sup.1H NMR (500 MHz, CH.sub.3OD): δH 8.37 (s, 1H, H8), 8.18 (s, 1H, H8), 8.14 (s, 1H, H2), 8.13-8.11 (m, 0.5H, Nap) 8.11 (s, 1H, H2), 7.94-7.90 (m, 0.5H, Ar), 7.90-7.87 (m, 0.5H, Ar), 7.86-7.82 (m, 0.5H, Ar), 7.74-7.70 (m, 0.5H, Ar), 7.66-7.61 (m, 0.5H, Ar), 7.57-7.47 (m, 1.5H, Ar), 7.46-7.37 (m, 2.5H, Ar), 7.34-7.27 (m, 4H, Ar), 7.25-7.17 (m, 1H, Ar), 6.19 (d, J=2.4 Hz, 0.5H, H1′), 6.04 (d, J=2.4 Hz, 0.5H, H1′), 5.60-5.54 (m, 0.5H, H2′), 5.50-5.42 (m, 0.5H, H2′), 5.16-4.99 (m, 2H, OCH.sub.2Ph), 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, CH.sub.3 ala).

[0407] .sup.13C NMR (125 MHz, CH.sub.3OH): δC 174.52 (C═O), 174.49 (C═O), 157.27 (C6), 153.58 (C2), 149.97 (C4), 149.93 (C-4), 147.70 (d, .sup.3J.sub.C-P=7.5, ‘ipso’ Nap), 147.48 (d, .sup.3J.sub.C-P=7.5, ‘ipso’ Nap), 141.36 (C8), 141.19 (C8), 137.25 (C—Ar), 137.05 (C—Ar), 136.31 (C—Ar), 136.20 (C—Ar), 129.58 (CH—Ar), 129.48 (CH—Ar), 129.37 (CH—Ar), 129.26 (CH—Ar), 129.22 (CH—Ar), 128.88 (CH—Ar), 127.84 (CH—Ar), 127.75 (CH—Ar), 127.49 (CH—Ar), 127.44 (CH—Ar), 126.48 (CH—Ar), 126.39 (CH—Ar), 126.26 (CH—Ar), 126.05 (CH—Ar), 122.76 (CH—Ar), 122.38 (CH—Ar), 120.68 (C5), 120.61 (C5), 116.64 (d, .sup.3J.sub.C-P=3.75 Hz, CH—Ar), 116.13 (d, .sup.3J.sub.C-P=3.75, CH—Ar), 91.60 (d, .sup.3J.sub.C-P=7.5 Hz, C1′), 91.43 (d, .sup.3J.sub.C-P=7.5 Hz, C1′), 82.74 (C4′), 82.27 (C4′), 81.99 (d, .sup.2J.sub.C-P=5.5 Hz, C2′), 81.12 (d, .sup.2J.sub.C-P=5.5 Hz, C2′), 67.97 (OCH.sub.2Ph), 67.94 (OCH.sub.2Ph), 64.16 (C5′), 63.51 (C5′), 51.96 (CH ala), 51.89 (CH ala), 33.89 (d, .sup.3J.sub.C-P=7.5 Hz, CH.sub.3 ala), 33.63 (d, .sup.3J.sub.C-P=7.5 Hz, CH.sub.3 ala).

[0408] HPLC Reverse-phase HPLC eluting with H.sub.2O/CH.sub.3OH from 100/10 to 0/100 in 30 minutes, 1 ml/min, 1=200 nm, showed two peaks of the diastereoisomers with tR 24.84 min. and tR 25.43 min.

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

[0409] ##STR00019##

[0410] Using General Procedure 2 above, 3′-deoxyadenosine (200 mg, 0.80 mmol) was suspended in (CH.sub.3).sub.3PO.sub.3 (5 mL), and POCl.sub.3 (75 μL, 0.80 mmol) was added dropwise at −5° C. The reaction mixture was allowed to reach room temperature and left stirring for 4 hours. A solution of (5)-1-(benzyloxy)-1-oxopropan-2-aminium 4-methylbenzenesulfonate (1.4 g, 4.0 mmol) dissolved in anhydrous CH.sub.2Cl.sub.2 (5 mL) was added followed by diisopropyl ethyl amine (1.4 mL, 8.0 mmol) at −78° C. After stirring at room temperature for 20 hours, water was added and the layers were separated. The aqueous phase was extracted with dichloromethane and the organic phase washed with brine. The combined organic layers were dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by column chromatography (gradient elution of CH.sub.2Cl.sub.2/MeOH=100/0 to 93/7) to give a white foam (256 mg, 49%).

[0411] MS (ES+) m/z: Found: 654.2 (M+H.sup.+), 676.2 (M+Na.sup.+), 1329.5 (2M+Na.sup.+) C.sub.30H.sub.36N.sub.7O.sub.8P required: (M) 653.62.

[0412] .sup.31P NMR (202 MHz, CH.sub.3OD) δ 13.9.

[0413] .sup.1H NMR (500 MHz, CH.sub.3OD) δ 8.28 (s, 1H, H8), 8.22 (s, 1H, H2), 7.37-7.26 (m, 10H, Ph), 6.00 (d, J=1.9 Hz, 1H, H1′), 5.15-5.05 (m, 4H, OCH.sub.2Ph), 4.74-4.70 (m, 1H, H2′), 4.63-4.56 (m, 1H, H4′), 4.24-4.18 (m, 1H, H5′), 4.11-4.05 (m, 1H, H5′), 3.97-3.87 (m, 1H, CH ala), 2.35-2.27 (m, 1H, H3′), 2.07-2.01 (m, 1H, H3′), 1.34-1.27 (m, 3H, CH.sub.3 ala).

[0414] .sup.13C NMR (125 MHz, CH.sub.3OD) δ 175.40 (d, .sup.3J.sub.C-P=5.0 Hz, C═O), 175.36 (d, .sup.3J.sub.C-P=5.0 Hz, C═O), 157.36 (C6), 153.91 (C2), 150.25 (C4), 140.64 (C8), 137.33 (C—Ar), 137.29 (C—Ar), 129.58 (CH—Ar), 129.57 (CH—Ar), 129.33 (CH—Ar), 129.31 (CH—Ar), 129.29 (CH—Ar), 120.55 (C5), 93.18 (C1′), 80.67 (d, .sup.3J.sub.C-P=8.4 Hz, C4′), 76.59 (C2′), 67.90 (OCH.sub.2Ph), 67.47 (d, .sup.2J.sub.C-P=5.2 Hz, C5′), 51.14 (d, .sup.2J.sub.C-P=1.7 Hz, CH ala), 51.11 (d, .sup.2J.sub.C-P=1.7 Hz, CH ala), 35.08 (C3′), 20.77 (d, .sup.3J.sub.C-P=6.5 Hz, CH.sub.3 ala), 20.59 (d, .sup.3J.sub.C-P=6.5 Hz, CH.sub.3 ala).

[0415] HPLC Reverse-phase HPLC eluting with H.sub.2O/CH.sub.3CN from 90/10 to 0/100 in 30 minutes, 1 ml/min, 1=254 nm, showed one peak with tR 13.87 min.

(2S)-benzyl 2-((((2S,4R,5R)-5-(6-amino-2-methoxy-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)(naphthalen-1-yloxy)phosphorylamino)propanoate K

[0416] ##STR00020##

[0417] Using General Procedure 1 above, N-methylimidazole (99 μL, 1.24 mmol) and a solution of (2S)-benzyl 2-((chloro(naphthalen-1-yloxy)phosphoryl)amino)propanoate (303 mg, 0.75 mmol) in anhydrous THF (5 mL) were added dropwisely 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 CH.sub.3/CH.sub.2Cl.sub.2 0/100 to 6/94) and preparative TLC (eluent system CH.sub.3/CH2Cl.sub.2 5/95) afforded the desired compound as white solid (96 mg, 60%).

[0418] MS (ES+) m/z: Found: 649.2 (M+H.sup.+) C.sub.31H.sub.33N.sub.6O.sub.8P required: 648.21(M). .sup.31P NMR (202 MHz, CD.sub.3OD): δP 4.38 (s), 4.08 (s). .sup.1H NMR (500 MHz, CD.sub.3OD): SH 8.14-8.11 (d, J=8.0 Hz, 0.5H, Ar), 8.07 (d, J=8.0 Hz, 0.5H, Ar), 8.05 (s, 0.5H, H8), 8.02 (s, 0.5H, H8), 7.82-7.80 (m, 1H, Ar), 7.61 (d, J=7.0 Hz, Ar), 7.47-7.44 (m, 4H, Ar), 7.35-7.29 (m, 2H, Ar), 7.24-7.22 (m, 3H, Ar), 5.88 (s, 1H, H1′), 4.71-4.68 (m, 1H, H4′), 4.65-6.60 (m, 1H, H2′), 4.42-4.40 (m, 1H, H5′), 4.30-4.27 (m, 1H, H5′), 4.08-3.98 (m, 1H, CH ala) 3.88 (s, 1.5H, OCH.sub.3), 3.86 (s, 1.5H, OCH.sub.3), 2.37-2.33 (m, 1H, H3′), 2.04-2.01 (m, 1H, H3′), 1.27 (d J=7.0 Hz, 1.5H, CH.sub.3), 1.24 (d J=7.0 Hz, 1.5H, CH.sub.3). .sup.13C NMR (125 MHz, CH.sub.3OD): δC 174.83 (d, .sup.3J.sub.C-P=3.7 Hz, C═O), 174.60 (d, .sup.3J.sub.C-P=3.7 Hz, C═O), 163.70 (C-2), 158.10 (C6), 151.95 (C4), 147.95 (d, .sup.3J.sub.C-P=7.5 Hz, ‘ipso’ Nap), 147.91, (d, .sup.3J.sub.C-P=7.5 Hz, ‘ipso’ Nap), 139.39 (C8), 139.37 (C8), 137.12, 137.17 (C-ipso CH.sub.2Ph), 136.22 (C—Ar), 129.57, 129.54, 129.48, 129.32, 129.27, 129.12, 129.24 128.89, 128.83, (CH—Ar), 127.85 (d,.sup.2J.sub.C-P=6.25 Hz, C—Ar), 127.86, 127.76, 127.51, 127.48, 126.49, 126.00, 125.97, 122.73, 122.63 (CH—Ar), 116.86 (C5), 116.72 (C5), 116.29 (d, .sup.3J.sub.C-P=3.75 Hz, CH—Ar), 116.22 (d, .sup.3J.sub.C-P=3.75 Hz, CH—Ar), 93.33 (C1′), 93.31(C1′), 80.24 (d, .sup.3J.sub.C-P=2.75 Hz, C4′), 76.29 (C2′), 76.26 (C2′), 69.09 (d, .sup.2J.sub.C-P=5.0 Hz, C5′), 68.16 (d, .sup.2J.sub.C-P=8.2 Hz, C5′), 67.95 (OCH.sub.2Ph), 55.28, 55.32 (OCH.sub.3), 51.79 (CH ala), 51.71 (CH ala), 35.40 (C-3′), 35.12 (C3′), 20.49 (d, .sup.3J.sub.C-P=6.7 Hz, CH.sub.3 ala), 20.35 (d, .sup.3J.sub.C-P=6.7, CH.sub.3 ala). HPLC Reverse-phase HPLC eluting with H.sub.2O/CH.sub.3CN from 100/10 to 0/100 in 30 minutes, F=1 ml/min, λ=280 nm, showed two peaks of the diastereoisomers with t.sub.R 16.22 min. and t.sub.R 16.48 min.

(2S)-benzyl 2-((((2S,4R,5R)-5-(6-amino-2-methoxy-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)(naphthalen-1-yloxy)phosphorylamino)propanoate L

[0419] ##STR00021##

[0420] Using General Procedure 1 above, N-methylimidazole (99 μL, 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 2-O-methyl-3′-deoxyadenosine (70 mg, 0.25 mmol) in anhydrous THF and the reaction mixture was stirred at room temperature for 16 hours. Purification by column chromatography (eluent system CH.sub.3OH/CH.sub.2Cl.sub.2 0/100 to 6/94) and preparative TLC (eluent system CH.sub.3OH/CH.sub.2Cl.sub.2 5/95) afforded the desired compound as a white solid (13 mg, 10%).

[0421] (ES+) m/z, found: 599.2 (M+H.sup.+), C.sub.27H.sub.31N.sub.6O.sub.8P required: 598.19 (M).

[0422] .sup.31P NMR (202 MHz, CD.sub.3OD) δ 3.97, 3.64. .sup.1H NMR (500 MHz, CD.sub.3OD) δ 8.06 (s, 0.5H, H8), 8.04 (s, 0.5H, H8), 7.33-7.28 (m, 7H, Ph), 7.20-7.14 (m, 3H, Ph), 5.92 (d, J=1.5 Hz, 0.5H, H1′), 5.90 (d, J=1.5 Hz, 0.5H, H1′), 5.14-5.04 (m, 2H, OCH.sub.2Ph), 4.78-4.76 (m, 0.5H, H4′), 4.74-4.72 (m, 0.5H, H4′), 4.63-4.59 (m, 1H, H2′), 4.10-4.34 (m, 1H, H5′a), 4.25-4.20 (m, 1H, H5′b), 3.94, 3.95 (OCH.sub.3), 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=7.0 Hz, CH.sub.3), 1.26 (d, J=7.0 Hz, CH.sub.3). .sup.13C NMR (125 MHz, CD.sub.3OD) S 174.82 (d, .sup.3J.sub.C-P=3.7 Hz, C═O), 174.62 (d, .sup.3J.sub.C-P=3.7 Hz, C═O), 163.80 (C-2), 158.16, 158.13 (C6), 152.15 (C4), 152.05 (d, .sup.3J.sub.C-P=4.8 Hz, C-ipso Ph), 152.00 (d, .sup.3J.sub.C-P=4.8 Hz, C-ipso Ph), 139.39 (C8), 137.30, 137.21 (C-ipso CH.sub.2Ph), 130.72, 129.57, 129.31,129.27, 126.122 (CH—Ar), 121.42 (d, .sup.2J.sub.C-P=4.5 Hz, CH—Ar), 121.37 (d, J.sub.C-P=4.5 Hz, CH—Ar), 116.72 (C5), 116.69 (C5), 93.33, 93.24 (C1′), 80.26 (d, .sup.3J.sub.C-P=8.87, C4′), 80.19 (d, .sup.3J.sub.C-P=8.87, C4′), 76.35 (C2′), 68.78 (d, .sup.2J.sub.C-P=5.0 Hz, C5′), 68.35 (d, .sup.2J.sub.C-P=5.0 Hz, C5′), 67.94 (OCH.sub.2Ph), 67.92 (OCH.sub.2Ph), 55.25, 55.28 (OCH.sub.3), 51.69, 51.57 (CH ala), 35.23 (C3′), 34.96 (C3′), 20.38 (d, .sup.3J.sub.C-P=6.7, CH.sub.3 ala), 20.26 (d, .sup.3J.sub.C-P=6.7, CH.sub.3 ala).

[0423] HPLC Reverse-phase HPLC eluting with H.sub.2O/CH.sub.3CN from 100/10 to 0/100 in 30 minutes, F=1 ml/min, λ=280 nm, showed two peaks of the diastereoisomers with t.sub.R 14.22 min. and t.sub.R 14.51 min.

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

[0424] ##STR00022##

[0425] Compound M was prepared according to the general procedure 1 using 2-O-methyl-3′-deoxyadenosine (70 mg, 0.25 mmol), N-methylimidazole (99 μL, 1.24 mmol) and naphthyl(pentyloxy-L-leucinyl) phosphorochloridate (330 mg, 0.75 mmol). Purification by column chromatography (eluent system gradient CH.sub.3/CH.sub.2Cl.sub.2 0/100 to 6/94) and preparative TLC (2000 μM, eluent system CH.sub.3/CH.sub.2Cl.sub.2 7/93) afforded the title compound as a white solid (50 mg, 30%).

[0426] .sup.31P NMR (202 MHz, CD.sub.3OD) δP 4.53, 4.28.

[0427] .sup.1H NMR (500 MHz, CD.sub.3OD) δH 8.04-7.96 (m, 1H, H8), 7.77-7.71 (m, 1H, Nap), 7.58-7.53 (m, 1H, Nap), 7.45-7.17 (m, 5H, Nap), 5.83-5.75 (m, 1H, H1′), 4.64-4.51 (m, 2H, H2′, H4′), 4.40-4.16 (m, 2H, H5′), 3.88-3.75 (m, 6H, OCH.sub.3, O(CH.sub.2).sub.4CH.sub.3, CHCH.sub.2CH(CH.sub.3).sub.2), 2.38-2.24 (m, 1H, H3′), 2.00-1.91 (m, 1H, H3′), 1.53-1.05 (m, 11H, O(CH.sub.2).sub.4CH.sub.3, CHCH.sub.2CH(CH.sub.3).sub.2), 0.77-0.55 (m, 9H, O(CH.sub.2).sub.4CH.sub.3, CHCH.sub.2CH(CH.sub.3).sub.2).

[0428] .sup.13C NMR (125 MHz, CD.sub.3OD) δC 175.02 (d, .sup.3J.sub.C-P=2.5 Hz, C═O), 174.78 (d, .sup.3J.sub.C-P=2.5 Hz, C═O), 163.76 (C2), 158.14 (C6), 151.03 (C4), 147.96 (d, .sup.3J.sub.C-P=7.2, ‘ipso’ Nap), 138.96 (C8), 136.30 (C—Ar), 136.28 (C—Ar), 136.22 (C—Ar), 128.93 (CH—Ar), 128.88 (CH—Ar), 128.81 (CH—Ar), 128.48 (CH—Ar), 127.77 (CH—Ar), 127.73 (CH—Ar), 127.44 (CH—Ar), 127.42 (CH—Ar), 127.06 (CH—Ar), 126.86 (CH—Ar), 126.45 (CH—Ar), 126.44 (CH—Ar), 126.31 (CH—Ar), 125.98 (CH—Ar), 125.88 (CH—Ar), 123.83 (CH—Ar), 123.43 (CH—Ar), 123.24 (CH—Ar), 122.81 (CH—Ar), 122.77 (CH—Ar), 122.69 (CH—Ar), 116.34 (d, .sup.3J.sub.C-P=3.7 Hz, CH—Ar), 116.02 (d, .sup.3J.sub.C-P=3.7 Hz, CH—Ar), 115.71 (C5), 93.42 (C1′), 93.32 (C1′), 80.22 (d, .sup.3J.sub.C-P=5.3 Hz, C4′), 80.15 (d, .sup.3J.sub.C-P=5.3 Hz, C4′), 76.29 (C2′), 76.27 (C2′), 69.22 (d, .sup.2J.sub.C-P=5.2 Hz, C5′), 69.028 (d, .sup.2J.sub.C-P=5.2 Hz, C5′), 66.31 (O(CH.sub.2).sub.4CH.sub.3), 66.30 (O(CH.sub.2).sub.4CH.sub.3), 55.29 (OCH.sub.3), 55.24 (OCH.sub.3), 54.79 (CHCH.sub.2CH(CH.sub.3).sub.2), 54.68 (CHCH.sub.2CH(CH.sub.3).sub.2), 44.20 (d, .sup.3J.sub.C-P=7.25 Hz, CHCH.sub.2CH(CH.sub.3).sub.2), 43.93 (d, .sup.3J.sub.C-P=7.25 Hz, CHCH.sub.2CH(CH.sub.3).sub.2), 35.49 (C3′), 35.17 (C3′), 29.31 (O(CH.sub.2).sub.4CH.sub.3), 29.11 (O(CH.sub.2).sub.4CH.sub.3), 25.67 (CHCH.sub.2CH(CH.sub.3).sub.2), 25.44 (CHCH.sub.2CH(CH.sub.3).sub.2), 23.30 (O(CH.sub.2).sub.4CH.sub.3), 23.10 (CHCH.sub.2CH(CH.sub.3).sub.2), 23.00 (CHCH.sub.2CH(CH.sub.3).sub.2), 22.94 (CHCH.sub.2CH(CH.sub.3).sub.2), 22.81 (CHCH.sub.2CH(CH.sub.3).sub.2), 14.27 (O(CH.sub.2).sub.4CH.sub.3).

[0429] (ES+) m/z, found: 671.3 (M+H.sup.+), C.sub.32H.sub.43N.sub.6O.sub.8P required: 670.69 (M).

[0430] HPLC Reverse-phase HPLC eluting with H.sub.2O/CH.sub.3CN from 100/10 to 0/100 in 30 minutes, 1 ml/min, 1=254 nm, showed two peaks of the diastereoisomers with tR 20.83 min. and tR 20.93 min.

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

[0431] ##STR00023##

[0432] Compound N was prepared according to the general procedure 1 using 2-o-methyl-3′-deoxyadenosine (70 mg, 0.25 mmol), N-methylimidazole (99 μL, 1.24 mmol) and phenyl(hexyloxy-L-alaninyl) phosphorochloridate (261 mg, 0.75 mmol). Purification by column chromatography (eluent system gradient CH.sub.3/CH.sub.2Cl.sub.2 0/100 to 6/94) and preparative TLC (1000 μM, eluent system CH.sub.3/CH.sub.2Cl.sub.2 7/93) afforded the title compound as a white solid (26 mg, 18%).

[0433] .sup.31P NMR (202 MHz, CD.sub.3OD) δP 3.87, 3.65.

[0434] .sup.1H NMR (500 MHz, CD.sub.3OD) δH 8.08 (s, 0.5H, H8), 8.07 (s, 0.5H, H8), 7.36-7.29 (m, 2H, Ph), 7.24-7.14 (m, 3H, Ph), 5.94 (d, J=2.0 Hz, 0.5H, H1′), 5.92 (d, J=2.0 Hz, 0.5H, H1′), 4.81-4.76 (m, 1H, H2′), 4.71-4.62 (m, 1H, H4′), 4.48-4.43 (m, 0.5H, H5′), 4.42-4.36 (m, 0.5H, H5′), 4.33-4.25 (m, 1H, H5′), 4.10-3.83 (m, 6H, OCH.sub.3, O(CH.sub.2).sub.5CH.sub.3, CHCH.sub.3), 2.48-2.40 (m, 1H, H3′), 2.13-2.07 (m, 1H, H3′), 1.61-1.51 (m, 2H, O(CH.sub.2).sub.5CH.sub.3), 1.33-1.24 (m, 9H, O(CH.sub.2).sub.5CH.sub.3, CHCH.sub.3), 0.89 (m, 3H, O(CH.sub.2).sub.5CH.sub.3).

[0435] .sup.13C NMR (125 MHz, CD.sub.3OD) δC 175.13 (d, .sup.3J.sub.C-P=4.3 Hz, C═O), 174.94 (d, .sup.3J.sub.C-P=4.3 Hz, C═O), 163.80 (C2), 163.78 (C2), 158.17 (C6), 158.15 (C6), 152.17 (d, .sup.2J.sub.C-P=6.3 Hz, C—Ar), 152.15 (d, .sup.2J.sub.C-P=6.3 Hz, C—Ar), 152.03 (C4), 151.99 (C4), 139.42 (C8), 139.39 (C8), 130.75 (CH—Ar), 130.74 (CH—Ar), 126.13 (CH—Ar), 121.43 (CH—Ar), 121.41 (CH—Ar), 121.39 (CH—Ar), 121.37 (CH—Ar), 116.74 (C5), 116.69 (C5), 93.40 (C1′), 93.27 (C1′), 80.30 (C4′), 80.23 (C4′), 76.40 (C2′), 68.85 (d, .sup.2J.sub.C-P=5.2 Hz, C5′), 68.42 (d, .sup.2J.sub.C-P=5.2 Hz, C5′), 66.43 (O(CH.sub.2).sub.5CH.sub.3), 55.30 (OCH.sub.3), 55.26 (OCH.sub.3), 51.64 (CHCH.sub.3), 51.54 (CHCH.sub.3), 35.30 (C3′), 35.04 (C3′), 32.58 (O(CH.sub.2).sub.5CH.sub.3), 29.67 (O(CH.sub.2)5CH.sub.3), 29.64 (O(CH.sub.2)5CH.sub.3), 26.61 (O(CH.sub.2).sub.5CH.sub.3), 23.59 (O(CH.sub.2).sub.5CH.sub.3), 20.56 (d, .sup.3J.sub.C-P=6.4 Hz, CHCH.sub.3), 20.41 (d, .sup.3J.sub.C-P=6.4 Hz, CHCH.sub.3), 14.36 (O(CH.sub.2).sub.5CH.sub.3).

[0436] (ES+) m/z, found: 593.3 (M+H.sup.+), C.sub.32H.sub.43N.sub.6O.sub.8P required: 592.58 (M).

[0437] HPLC Reverse-phase HPLC eluting with H.sub.2O/CH.sub.3CN from 100/10 to 0/100 in 30 minutes, 1 ml/min, 1=254 nm, showed two peaks of the diastereoisomers with tR 17.02 min. and tR 17.23 min.

2-Fluoro-3′-deoxyadenosine-5′-O-[1-naphthyhbenzyloxy-L-alaninyl)]phosphate O

[0438] ##STR00024##

[0439] Compound O was prepared according to the general procedure 1 using 2-Fluoro-3′-deoxyadenosine (50 mg, 0.18 mmol), N-methylimidazole (74 μL, 0.93 mmol) and phenyl(benzyloxy-L-alaninyl) phosphorochloridate (196 mg, 0.56 mmol). Purification by column chromatography (eluent system gradient CH.sub.3/CH.sub.2Cl.sub.2 0/100 to 6/94) and preparative TLC (500 μM, eluent system CH.sub.3/CH.sub.2Cl.sub.2 5/95) afforded the title compound as a white solid (5 mg, 4%).

[0440] .sup.31P NMR (202 MHz, CD.sub.3OD) δP 4.33, 4.08.

[0441] .sup.1H NMR (500 MHz, CD.sub.3OD) δH 8.17 (s, 0.5H, H8), 8.14 (s, 0.5H, H8), 8.14-8.09 (m, 1H, Ar), 7.89-7.85 (m, 1H, Ar), 7.70-7.66 (m, 1H, Ar), 7.54-7.42 (m, 4H, Ar), 7.40-7.24 (m, 5H, Ar), 5.89 (d, J=2.3 Hz, 0.5H, H1′), 5.88 (d, J=2.3 Hz, 0.5H, H1′), 5.08-5.01 (m, 2H, OCH.sub.2Ph), 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, CHCH.sub.3), 2.36-2.25 (m, 1H, H3′), 2.06-1.98 (m, 1H, H3′), 1.32-1.25 (m, 3H, CHCH.sub.3).

[0442] .sup.13C NMR (125 MHz, CD.sub.3OD) δC 175.54 (CO), 175.22 (CO), 161.02 (d, .sup.1J.sub.C-F=207.3 Hz, C2), 160.89 (d, .sup.1J.sub.C-F=207.3 Hz, C2), 158.45 (d, .sup.3J.sub.C-F=18.2 Hz, C6), 158.23 (d, .sup.3J.sub.C-F=18.2 Hz, C6), 150.63 (d, .sup.3J.sub.C-F=18.4 Hz, C4), 140.67 (C8), 136.26 (C—Ar), 131.62, 131.54, 129.56 (CH—Ar), 129.52 (CH—Ar), 129.37 (CH—Ar), 129.31 (CH—Ar), 129.26 (CH—Ar), 128.87 (CH—Ar), 128.81 (CH—Ar), 128.29 (CH—Ar), 128.02 (CH—Ar), 127.79 (CH—Ar), 127.76 (CH—Ar), 127.51 (CH—Ar), 127.49 (CH—Ar), 127.47 (CH—Ar), 126.47 (CH—Ar), 126.33 (C—Ar), 126.27 (C—Ar), 125.97 (CH—Ar), 122.78 (CH—Ar), 122.74 (CH—Ar), 122.64 (CH—Ar), 122.62 (CH—Ar), 116.35 (d, .sup.4J.sub.C-F=3.0 Hz, C5), 116.15 (d, .sup.4J.sub.C-F=3.0 Hz, C5), 93.25 (C1′), 93.20 (C1′), 80.41 (d, .sup.3J.sub.C-P=7.5 Hz, C4′), 80.33 (d, .sup.3J.sub.C-P=7.5 Hz, C4′), 76.43 (C2′), 76.35 (C2′), 68.84 (d, .sup.2J.sub.C-P=5.5 Hz, C5′), 68.45 (d, .sup.2J.sub.C-P=5.5 Hz, C5′), 67.92 (OCH.sub.2Ph), 67.92 (OCH.sub.2Ph), 51.75 (CHCH.sub.3), 51.52 (CHCH.sub.3), 34.97 (C3′), 34.74 (C3′), 20.42 (d, .sup.3J.sub.C-P=6.7 Hz, CHCH.sub.3), 20.20 (d, .sup.3J.sub.C-P=6.7 Hz, CHCH.sub.3).

[0443] .sup.19F NMR (470 MHz, CD.sub.3OD) δF −53.14, −53.22.

[0444] (ES+) m/z, found: 637.2 (M+H.sup.+), C.sub.30H.sub.30FN.sub.6O.sub.7P required: 636.57 (M).

[0445] HPLC Reverse-phase HPLC eluting with H.sub.2O/CH.sub.3CN from 100/10 to 0/100 in 30 minutes, 1 ml/min, 1=254 nm, showed two peaks of the diastereoisomers with tR 17.09 min. and tR 17.34 min.

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

[0446] ##STR00025##

[0447] Using General Procedure 1 above, N-methylimidazole (74 μL, 0.93 mmol) and a solution of (2S)-benzyl 2-((chloro(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 (eluent system CH.sub.3OH/CH.sub.2Cl.sub.2 0/100 to 6/94) and preparative TLC (eluent system CH.sub.3OH/CH.sub.2Cl.sub.2 5/95) afforded the desired compound as a white solid (5 mg, 7%).

[0448] (ES+) m/z, found: 587.1 (M+H.sup.+), C.sub.26H.sub.28FN.sub.6O.sub.7P required: 586.17 (M). .sup.19F NMR (470 MHz, CD.sub.3OD): δF −53.17, −53.23. .sup.31P NMR (202 MHz, CD.sub.3OD): δP 3.95 (s), 3.67 (s). .sup.1H NMR (500 MHz, CDCl.sub.3): δH 8.19 (s, 0.5H, H8), 8.16 (s, 0.5H, H8), 7.36-7.27 (m, 7H, Ar), 7.22-7.13 (m, 3H, Ar), 5.91 (d, J=1.5 Hz, 0.5H, H1′), 5.89 (d, J=1.7 Hz, 0.5H, H1′), 5.15-5.06 (m, 2H, OCH.sub.2Ph), 4.73-4.58 (m, 2H, H2′, H4′), 4.42-4.34 (m, 1H, H5′), 4.02-3.90 (m, 1H, H5′), 3.27-3.24 (m, 1H, H3′), 2.08-2.00 (m, 1H, H3′), 1.33 (d, J=7.1 Hz, 1.5H, CH.sub.3 ala), 1.29 (d, J=7.1 Hz, 1.5H, CH.sub.3 ala). .sup.13C NMR (125 MHz, CD.sub.3OD): δC 175.85 (d, .sup.3J.sub.C-P=3.7 Hz, C═O), 174.63 (d, .sup.3J.sub.C-P=5.0 Hz, C═O), 160.58 (d, .sup.1J.sub.C-F=207.5 Hz, C2), 160.53 (d, .sup.1J.sub.C-F=207.5 Hz, C2), 159.06 (d, .sup.3J.sub.C-F=18.7 Hz, C6), 159.05 (d, .sup.3J.sub.C-F=17.5 Hz, C6), 152.11 (d, .sup.2J.sub.C-P=8.75 Hz, C—Ar), 152.08 (d, .sup.2J.sub.C-P=8.7 Hz, C—Ar), 151.58 (d, .sup.3J.sub.C-F=19.7 Hz, C4), 151.56 (d, .sup.3J.sub.C-F=19.5 Hz, C4), 140.63 (C8), 137.28 (C—Ar), 137.21 (C—Ar), 130.78 (CH—Ar), 130.75 (CH—Ar), 129.58 (CH—Ar), 129.38 (CH—Ar), 129.34 (CH—Ar), 129.32 (CH—Ar), 129.28 (CH—Ar), 128.3 (CH—Ar), 128.02 (CH—Ar), 121.16 (CH—Ar), 121.18 (CH—Ar), 121.47 (CH—Ar), 121.51 (CH—Ar), 121.42 (CH—Ar), 121.39 (CH—Ar), 121.36 (CH—Ar), 118.75 (d, .sup.4J.sub.C-F=3.7 Hz, C5), 118.72 (d, .sup.4J.sub.C-F=3.7 Hz, C5), 93.25 (C1′), 93.18 (C1′), 80.48 (d, .sub.3J.sub.C-P=8.3 Hz, C4′), 80.46 (d, .sub.3Jc-p=8.1 Hz, C4′), 76.51 (C2′), 76.49 (C2′), 68.54 (d, .sup.2J.sub.C-P=5.2 Hz, C5′), 68.18 (d, .sup.2J.sub.C-P=5.6 Hz, C5′), 67.94 (CH.sub.2 Bn), 67.91 (CH.sub.2 Bn), 51.71 (CH ala), 51.56 (CH ala), 34.85 (C3′), 34.64 (C3′), 20.42 (d, .sup.3J.sub.C-P=7.1 Hz, CH.sub.3 ala), 20.25 (d, .sup.3J.sub.C-P=7.5 Hz, CH.sub.3 ala). HPLC Reverse-phase HPLC eluting with H.sub.2O/CH.sub.3CN from 100/10 to 0/100 in 30 minutes, 1 ml/min, 1=280 nm, showed two peaks of the diastereoisomers with t.sub.R 14.98 min. and t.sub.R 15.12 min.

2-Fluoro-3′-deoxyadenosine-5′-O-[1-naphthyhl(1-pentyloxy-L-leucinyl)]phosphate Q

[0449] ##STR00026##

[0450] Compound Q was prepared according to the general procedure 1 using 2-Fluoro-3′-deoxyadenosine (50 mg, 0.18 mmol), N-methylimidazole (74 μL, 0.93 mmol) and naphthyl(pentyloxy-L-leucinyl) phosphorochloridate (246 mg, 0.56 mmol). Purification by column chromatography (eluent system CH.sub.3OH/CHCl.sub.3 0/100 to 6/94) and preparative TLC (1000 μm, eluent system CH.sub.3/CH.sub.2Cl.sub.2 5/95) afforded the title compound as a white solid (65 mg, 53%).

[0451] .sup.31P NMR (202 MHz, CD.sub.3OD): 4.60, 4.35.

[0452] .sup.1H NMR (500 MHz, CD.sub.3OD): δH 8.23 (s, 0.5H, H8), 8.20 (s, 0.5H, H8), 8.18-8.12 (m, 1H, Ar), 7.92-7.86 (m, 1H, Ar), 7.73-7.68 (m, 1H, Ar), 7.57-7.46 (m, 3H, Ar), 7.42-7.36 (m, 1H, Ar), 5.93-5.91 (m, 1H, H1′), 4.74-4.62 (m, 2H, H2′, H4′), 4.55-4.50 (m, 0.5H, H5′), 4.49-4.44 (m, 0.5H, H5′), 4.43-4.37 (m, 0.5H, H5′), 4.36-4.31 (m, 0.5H, H5′), 4.02-3.86 (m, 3H, CHCH.sub.2CH(CH.sub.3).sub.2, O(CH.sub.2).sub.4CH.sub.3), 2.43-2.29 (m, 1H, H3′), 2.12-2.04 (m, 1H, H3′), 1.67-1.20 (m, 11H, O(CH.sub.2).sub.4CH.sub.3, CHCH.sub.2CH(CH.sub.3).sub.2), 0.89-0.67 (m, 9H, O(CH.sub.2).sub.4CH.sub.3, CHCH.sub.2CH(CH.sub.3).sub.2)

[0453] .sup.13C NMR (125 MHz, CD.sub.3OD): δC 175.03 (d, .sup.3J.sub.C-P=2.5 Hz, C═O), 174.93 (d, .sup.3J.sub.C-P=2.5 Hz, C═O), 161.45 (d, .sup.1J.sub.C-F=205.5 Hz, C2), 160.39 (d, .sup.1J.sub.C-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 (CH—Ar), 127.46 (CH—Ar), 126.48 (CH—Ar), 126.45 (CH—Ar), 126.02 (CH—Ar), 125.91 (CH—Ar), 123.03 (C—Ar), 122.81 (CH—Ar), 122.69 (CH—Ar), 116.39 (d, .sup.3J.sub.C-P=2.9 Hz, CH—Ar), 116.28 (C5), 116.26 (C5), 115.97 (d, .sup.3J.sub.C-P=2.9 Hz, CH—Ar), 93.29 (C1′), 93.23 (C1′), 80.45 (d, .sup.3J.sub.C-P=6.0 Hz, C4′), 80.38 (d, .sup.3J.sub.C-P=6.0 Hz, C4′), 76.45 (C2′), 76.41 (C2′), 68.99 (d, .sup.2J.sub.C-P=5.4 Hz, C5′), 68.78 (d, .sup.2J.sub.C-P=5.4 Hz, C5′), 66.31 (O(CH.sub.2).sub.4CH.sub.3), 66.29 (O(CH.sub.2).sub.4CH.sub.3), 54.78 (CHCH2CH(CH.sub.3).sub.2), 54.66 (CHCH2CH(CH.sub.3).sub.2), 44.16 (d, .sup.3J.sub.C-P=7.25 Hz, CHCH.sub.2CH(CH.sub.3).sub.2), 43.84 (d, .sup.3J.sub.C-P=7.3 Hz, CHCH.sub.2CH(CH.sub.3).sub.2), 35.09 (C3′), 34.79 (C3′), 29.31 (O(CH.sub.2).sub.4CH.sub.3), 29.12 (O(CH.sub.2).sub.4CH.sub.3), 25.65 (CHCH.sub.2CH(CH.sub.3).sub.2), 25.41 (CHCH.sub.2CH(CH.sub.3).sub.2), 23.33 (O(CH.sub.2).sub.4CH.sub.3), 23.11 (CHCH.sub.2CH(CH.sub.3).sub.2), 23.00 (CHCH.sub.2CH(CH.sub.3).sub.2), 21.95 (CHCH.sub.2CH(CH.sub.3).sub.2), 21.68 (CHCH.sub.2CH(CH.sub.3).sub.2), 14.29 (O(CH.sub.2).sub.4CH.sub.3).

[0454] .sup.19F NMR (470 MHz, CD.sub.3OD): δF −53.15, −53.20.

[0455] (ES+) m/z, found: 659.3 (M+H.sup.+), C.sub.31H.sub.40FN.sub.6O.sub.7P required: 658.66 (M).

[0456] HPLC Reverse-phase HPLC eluting with H.sub.2O/CH.sub.3CN 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.

(2S)-hexyl 2-(((((2S,4R,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate R

[0457] ##STR00027##

[0458] Using General Procedure 1 above, N-methylimidazole (74 μL, 0.93 mmol) and a solution of (2S)-hexyl 2-((chloro(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 (eluent system CH.sub.3/CH.sub.2Cl.sub.2 0/100 to 6/94) and preparative TLC (eluent system CH.sub.3/CH.sub.2Cl.sub.2 5/95) afforded the desired compound as a white solid (5 mg, 7%).

[0459] (ES+) m/z, found: 587.1 (M+H.sup.+), C.sub.26H.sub.28FN.sub.6O.sub.7P required: 586.17 (M). .sup.19F NMR (470 MHz, CD.sub.3OD): SF −53.15, −53.20. .sup.31P NMR (202 MHz, CD.sub.3OD): 3.91 (s), 3.73 (s). .sup.1H NMR (500 MHz, CDCl.sub.3): δH 8.21 (s, 0.5H, H8), 8.20 (s, 0.5H, H8), 7.37-7.29 (m, 7H, Ar), 7.26-7.13 (m, 3H, Ar), 5.94-5.91 (m, 1H, H1′), 4.76-4.64 (m, 2H, H2′, H4′), 4.49-4.44 (m, 0.5H, H5′), 4.43-4.37 (m, 0.5H, H5′), 4.33-4.26 (m, 1H, H5′), 4.11-3.99 (m, 2H, CH.sub.2 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, CH.sub.2 Hex), 1.37-1.23 (m, 9H, CH.sub.3 ala, CH.sub.2 Hex), 0.92-0.85 (m, 3H, CH.sub.3 Hex).

[0460] .sup.13C NMR (125 MHz, CD.sub.3OD): δC 175.15 (d, .sup.3J.sub.C-P=3.7 Hz, C═O), 174.96 (d, .sup.3J.sub.C-P=5.0 Hz, C═O), 160.59 (d, .sup.1J.sub.C-F=207.5 Hz, C2), 160.56 (d, .sup.1J.sub.C-F=207.5 Hz, C2), 159.09 (d, .sup.3J.sub.C-F=21.2 Hz, C6), 159.08 (d, .sup.3J.sub.C-F=20.0 Hz, C6), 152.16 (d, .sup.2J.sub.C-P=7.5 Hz, C—Ar), 152.14 (d, .sup.2J.sub.C-P=6.3 Hz, C—Ar), 151.71 (d, .sup.3J.sub.C-F=20.0 Hz, C4), 151.67 (d, .sup.3J.sub.C-F=20.0 Hz, C4), 140.70 (d, .sup.5J.sub.C-F=2.5 Hz, C8), 140.68 (d, .sup.5J.sub.C-F=2.5 Hz, C8), 130.77 (CH—Ar), 130.74 (CH—Ar), 126.16 (CH—Ar), 126.24 (CH—Ar), 121.48 (CH—Ar), 121.44 (CH—Ar), 121.41 (CH—Ar), 121.37 (CH—Ar), 118.80 (d, .sup.4J.sub.C-F=3.7 Hz, C5), 118.77 (d, .sup.4J.sub.C-F=3.7 Hz, C5), 93.37 (C1′), 93.25 (C1′), 80.52 (d,.sup.3J.sub.C-P=3.7 Hz, C4′), 80.45 (d, .sup.3J.sub.C-P=4.1 Hz, C4′), 76.52 (C2′), 76.49 (C2′), 68.69 (d, .sup.2J.sub.C-P=5.4 Hz, C5′), 68.30 (d, .sup.2J.sub.C-P=4.9 Hz, C5′), 66.46 (CH2 Hex), 51.68 (CH ala), 51.57 (CH ala), 35.02 (C3′), 34.80 (C3′), 32.58 (CH.sub.2 Hex), 29.65 (CH.sub.2 Hex), 26.61 (CH.sub.2 Hex), 23.59 (CH.sub.2 Hex), 20.60 (d, .sup.3J.sub.C-P=7.1 Hz, CH.sub.3 ala), 20.43 (d, .sup.3J.sub.C-P=7.5 Hz, CH.sub.3 ala), 14.35 (CH.sub.3 Hex). HPLC Reverse-phase HPLC eluting with H.sub.2O/CH.sub.3CN from 100/10 to 0/100 in 30 minutes, 1 ml/min, 1=280 nm, showed two peaks of the diastereoisomers with tR 17.83 min. and tR 18.02 min.

(2R)-benzyl 2-((((2S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-4-hydroxytetrahydrofuran-2-yl)methoxy)(naphthalen-1-yloxy)phosphorylamino)propanoate S

[0461] ##STR00028##

[0462] To a stirring solution of 2-chloro-3′-deoxyadenosine (100 mg, 1.0 mol/eq.) in 10 mL of anhydrous THF, 424 mg of (2S)-benzyl 2-(chloro(naphthalen-1-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 reduced pressure and the residue was purified by column chromatography with gradient of eluent (CH.sub.3/CH.sub.2Cl.sub.2 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.sup.+), 675 (M+Na.sup.+) C.sub.30H.sub.30ClN.sub.6O.sub.7P required: 652.16 (M); .sup.31P NMR (202 MHz, CD.sub.3OD): δP 4.39 (s), 4.12 (s); .sup.1H NMR (500 MHz, CD.sub.3OD): δH 8.10 (s, 0.5H, H8), 8.07 (s, 0.5H, H8), 8.02-7.97 (m, 3H, CH.sub.2Ph and Naph), 7.43-7.14 (m, 9H, CH.sub.2Ph and Naph), 5.80-5.81 m, 1H, H1′), 4.89-4.97 (m, 2H, CH.sub.2Ph) 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, CHCH.sub.3), 2.12-2.23 (m, 1H, H3′), 1.86-1.93 (m, 1H H3′), 1.14-1.17 (m, 3H, CHCH.sub.3); .sup.13C NMR (125 MHz, CD.sub.3OD): δC 174.85 (d J.sub.CP=4.0 Hz, C═O), 174.55 (d J.sub.CP=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 (d .sup.3J.sub.CP=7.25 Hz, C-ipso Naph), 147.90 (d .sup.3J.sub.CP=7.0 Hz, C-ipso Naph), 140.70 (C8), 137.21, 137.16 (C-ipso CH.sub.2Ph), 136.26 (C—Ar), 130.92, 130.80, 129.56, 129.53, 129.31, 129.27, 129.25, 128.88, 128.81 (CH—Ar), 127.78 (d J.sub.CP=4.7 Hz, CH—Ar), 127.50 (d J.sub.CP=6.2 Hz, CH—Ar), 126.48, 126.02, 125.97 (CH—Ar), 119.46, 119.42 (C5), 116.33 (d, J.sub.CP=3.0, CH—Ar), 116.16 (d, J.sub.CP=3.4, CH—Ar), 93.30, 93.27 (C1′), 80.56 (d J=8.3 Hz, C4′), 80.51 (d J=8.4 Hz, C4′), 76.61, 76.54 (C2′), 68.74 (d J.sub.CP=5.3 Hz, C5′), 68.54 (d J.sub.CP=5.1 Hz, C5′), 67.93, 67.90 (CH.sub.2Ph), 51.81, 51.70 (CHCH.sub.3), 34.79, 34.53 (C3′), 20.42 (d J.sub.CP=6.5 Hz, CHCH.sub.3), 20.23 (d J.sub.CP=7.7 Hz, CHCH.sub.3); HPLC Reverse-phase HPLC eluting with H.sub.2O/CH.sub.3CN from 90/10 to 0/100 in 30 minutes, F=1 ml/min, 1=254 nm, t.sub.R 18.03 min.

2-Chloro-3′deoxyadenosine 5′-O-[1-phenyl (2,2-dimethylpropoxy-L-alanine)]phosphate T

[0463] ##STR00029##

[0464] Compound T was prepared according to the general procedure 1 using 2-chloro-3′-deoxyadenosine (350 mg, 1.25 mmol), N-methylimidazole (490 μL, 6.15 mmol) and phenyl(2,2-dimethylpropoxy-L-alaninyl) phosphorochloridate (1231 mg, 3.69 mmol). Purification by column chromatography (eluent system CH.sub.3/CH.sub.2Cl.sub.2 0/100 to 5/95) and preparative TLC (1000 μm, eluent system CH.sub.3/CH.sub.2Cl.sub.2 4/96) afforded the title compound as a white solid (181 mg, 25%).

[0465] .sup.31P NMR (202 MHz, CD.sub.3OD): δP 3.93, 3.72.

[0466] .sup.1H NMR (500 MHz, CD.sub.3OD): δH 8.12 (s, 0.5H, H8), 8.10 (s, 0.5H, H8), 7.19-7.23 (m, 2H, Ph), 7.03-7.12 (m, 3H, Ph), 5.84 (d =2, 0.5H, H1′), 5.83 (d J=2, 0.5H, H1′), 4.54-4.60 (m, 2H, H4′and H2′), 4.34-4.38 (m, 0.5H, H5′), 4.27-4.31 (m, 0.5H, H5′), 4.16-4.23 (m, 1H, H5′), 3.80-3.90 (m, 1H, CHCH.sub.3), 3.57-3.73 (m, 2 H OCH.sub.2C(CH.sub.3).sub.3), 2.18-2.28 (m, 1H, H3′), 1.94-1.99 (m, 1H, H3′), 1.20-1.24 (m, 3H, CHCH.sub.3), 0.81 (s, 4.5 H OCH.sub.2(CH.sub.3).sub.3), 0.79 (s, 4.5 H OCH.sub.2C(CH.sub.3).sub.3).

[0467] .sup.13C NMR (125 MHz, CD.sub.3OD): δC 175.09 (d .sup.3J.sub.CP=4.75 Hz, C═O), 174.90 (d .sup.3J.sub.CP=5.37 Hz, C═O), 158.10, (C6), 155.31, 155.28 (C2), 152.14 (d .sup.2J.sub.CP=6.37 Hz, C-ipso Ph), 152.13 (d .sup.2J.sub.CP=6.25 Hz, C-Ipso 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 .sup.3J.sub.CP=11.75 Hz, CH—Ar), 121.41 (d .sup.3J.sub.CP=11.75 Hz, CH—Ar), 119.52, 119.48 (C5), 93.49, 93.35 (C1′), 80.67 (d .sup.3J=8.62 Hz, C4′), 80.65 (d .sup.3J=8.25 Hz, C4′), 76.70, 76.67 (C2′), 75.43, (OCH.sub.2C(CH.sub.3).sub.3), 68.68 (d .sup.2J.sub.CP=5.12 Hz, C5′), 68.42 (d .sup.2J.sub.CP=5.12 Hz, C5′), 51.77, 51.60 (CHCH.sub.3), 34.94, 34.67 (C3′), 32.36, 32.32 (OCH.sub.2C(CH.sub.3).sub.3), 26.78, 26.76 (OCH2C(CH.sub.3).sub.3), 20.83 (d J.sub.CP=6.25 Hz, CHCH.sub.3), 20.61 (d J.sub.CP=7.12 Hz, CHCH.sub.3).

[0468] MS (ES+) nth: Found: 583 (M+H.sup.+), 605 (M+Na.sup.+) C.sub.24H.sub.32ClN.sub.6O.sub.7P required: 582.18 (M).

[0469] HPLC Reverse-phase HPLC eluting with H.sub.2O/CH.sub.3CN from 90/10 to 0/100 in 30 minutes, F=1 ml/min, 1=254 nm, t.sub.R 16.37, 16.55 min.

2-Chloro-3′deoxyadenosine 5′-O-[1-naphtyl (2,2-dimethylpropoxy-L-alanine)]phosphate U

[0470] ##STR00030##

[0471] Compound U was prepared according to the general procedure 1 using 2-chloro-3′-deoxyadenosine (350 mg, 1.25 mmol), N-methylimidazole (490 μL, 6.15 mmol) and naphtyl(2,2-dimethylpropoxy-L-alaninyl) phosphorochloridate (1416 mg, 3.69 mmol). Purification by column chromatography (eluent system CH.sub.3/CH.sub.2Cl.sub.2 0/100 to 5/95) and preparative TLC (1000 μm, eluent system CH.sub.3/CH.sub.2Cl.sub.2 4/96) afforded the title compound as a white solid (264 mg, 34%).

[0472] .sup.31P NMR (202 MHz, CD.sub.3OD): δP 4.35, 4.20.

[0473] .sup.1H NMR (500 MHz, CD.sub.3OD): δH 8.23 (s, 0.5H, H8), 8.21 (s, 0.5H, H8), 8.11-8.16 (m, 1H, Naph), 7.86-7.89 (m, 1H, Naph), 7.69-7.70 (m, 1H, Naph), 7.54-7.46 (m, 3H, Naph), 7.37-7.41 (m, 1H, Naph), 5.95 (d J=2, 0.5H, H1′), 5.94 (d J=1.5, 0.5H, H1′), 4.67-4.73 (m, 2H, H4′ and H2′), 4.34-4.55 (m, 2H, H5′), 4.00-4.08 (m, 1H, CHCH.sub.3), 3.66-3.81 (m, 2H OCH.sub.2C(CH.sub.3).sub.3), 2.28-2.41 (m, 1H, H3′), 2.03-2.10 (m, 1H, H3′), 1.31-1.34 (m, 3H, CHCH.sub.3), 0.90 (s, 4.5 H OCH.sub.2C(CH.sub.3).sub.3), 0.89 (s, 4.5 H CH.sub.2(CH.sub.3).sub.3).

[0474] .sup.13C NMR (125 MHz, CD.sub.3OD): δC 175.11 (d J.sub.CP=4.1 Hz, C═O), 174.85 (d J.sub.CP=5.0 Hz, C═O), 158.10, 158.04 (C6), 155.32, 155.30 (C2), 151.33 (C4), 147.96 (d .sup.2J.sub.CP=7.25 Hz, C-ipso Naph), 147.93 (d.sup.2J.sub.CP=7.25 Hz, C-ipso Naph), 140.84, 140.76 (C8), 136.29 (C—Ar), 128.87, 128.82 (CH—Ar), 127.85 (C—Ar), 127.77, 127.74, 127.48, 127.45, 126.47, 125.99, 125.96, 122.74, 122.66 (CH—Ar), 119.47 (C5), 116.29 (d .sup.3J.sub.CP=3.4 Hz, CH—Ar), 116.17 (d .sup.3J.sub.CP=2.9 Hz, CH—Ar), 93.42, 93.34 (C1′), 80.57 (d .sup.3J.sub.CP=8.1 Hz, C4′), 80.53 (d .sup.3J.sub.CP=5.1 Hz, C4′), 76.61, 76.53 (C2′), 75.41, 75.38 (OCH.sub.2C(CH.sub.3).sub.3), 68.95 (d.sup.2J.sub.CP=5.3 Hz, C5′), 68.82 (d.sup.2J.sub.CP=5.2 Hz, C5′), 51.84, 51.73 (CHCH.sub.3), 35.04, 34.75 (C3′), 32.29 (OCH.sub.2C(CH.sub.3).sub.3), 26.70 (OCH.sub.2C(CH.sub.3).sub.3), 20.76 (d .sup.3J.sub.CP=6.4 Hz, CHCH.sub.3), 20.55 (d .sup.3J.sub.CP=7.2 Hz, CHCH.sub.3).

[0475] MS (ES+) m/z: Found: 633 (M+H.sup.+), 655 (M+Na.sup.+) C.sub.28H.sub.34ClN.sub.6O.sub.7P required: 652.16 (M).

[0476] HPLC Reverse-phase HPLC eluting with H.sub.2O/CH.sub.3CN from 90/10 to 0/100 in 30 minutes, F=1 ml/min, 1=254 nm, t.sub.R 19.16 min.

2-Chloro-3′deoxyadenosine 5′-O-[1-phenyl (ethoxy-L-alanine)]phosphate V

[0477] ##STR00031##

[0478] Compound V was prepared according to the general procedure 4 using 2-chloro-3′-deoxyadenosine (343 mg, 0.66 mmol), tertbutyldimethylsilyl chloride (328 mg 2.18 mmol) imidazole (297 mg, 4.36 mmol). Purification by column chromatography (eluent system CH.sub.3OH/CH.sub.2Cl.sub.2 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/H.sub.2O/TFA 4/1/1. Purification by column chromatography (eluent system CH.sub.3OH/CH.sub.2Cl.sub.2 0/100 to 12/88) afforded intermediate 2 (544 mg, 72%). Then, intermediate 2 (204 mg, 0.51 mmol) and was reacted with tertbutylmagnesium chloride and a solution of phenyl(ethyloxy-L-alaninyl) phosphorochloridate (348.56 mg, 1.02 mmol) in anhydrous THF (5 mL). Purification by column chromatography (eluent system CH.sub.3/CH2Cl.sub.2 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/H.sub.2O 1/1/1 (3 mL). Purification by preparative TLC (2000 μm, eluent system CH.sub.3/CH.sub.2Cl.sub.2 4/96) afforded the title compound as a white solid (50 mg, 66%). (Overall yield 13%)

[0479] .sup.31P NMR (202 MHz, CD.sub.3OD): δP 3.93, 3.72.

[0480] .sup.1H NMR (500 MHz, CD.sub.3OD): δH 8.12 (s, 0.5H, H8), 8.11 (s, 0.5H, H8), 7.18-7.23 (m, 2H, Ph), 7.03-7.12 (m, 3H, Ph), 5.85 (d 0.5H, H1′), 5.84 (d J=2, 0.5H, H1′), 4.55-4.62 (m, 2H, H4′and H2′), 4.34-4.38 (m, 0.5H, H5′), 4.28-4.32 (m, 0.5H, H5′), 4.16-4.22 (m, 1H, H5′), 3.93-4.03 (m, 2H, OCH.sub.2CH.sub.3), 3.70-3.84 (m, 1H, CHCH.sub.3), 2.20-2.28 (m, 1H, H3′), 1.95-1.99 (m, 1H, H3′), 1.15-1.21 (m, 3H, CHCH.sub.3), 1.06-1.11 (m, 3H, OCH.sub.2CH.sub.3).

[0481] .sup.13C NMR (125 MHz, CD.sub.3OD): δC 173.66 (d .sup.3J.sub.CP=4.5 Hz, C═O), 173.65 (d .sup.3J.sub.CP=5.3 Hz, C═O), 156.68, 156.70 (C6), 153.93, 153.88 (C2), 150.72 (d .sup.2J.sub.CP=6.7 Hz, C-ipso Ph), 150.71 (d.sup.2J.sub.CP=6.5 Hz, C-ipso Ph), 149.89, 149.94 (C4), 139.41, 139.35 (C8), 129.33 (CH—Ar), 124.74, 124.73 (CH—Ar), 120.03 (d .sup.3J.sub.CP=4.75 Hz, CH—Ar), 119.97 (d .sup.3J.sub.CP=4.87 Hz, CH—Ar), 118.07, 118.03 (C5), 92.02, 91.88 (C1′), 79.26, 79.19 (C4′), 75.26, 75.24 (C2′), 67.18 (d .sup.2J.sub.CP=5.25 Hz, C5′), 66.81 (d .sup.2J.sub.CP=5.12 Hz, C5′), 60.96 (OCH.sub.2CH.sub.3), 50.23, 50.12 (CHCH.sub.3), 33.46, 33.21 (C3′), 19.16 (d .sup.3J.sub.CP=6.3 Hz, CHCH.sub.3), 18.97 (d .sup.3J.sub.CP=7.2 Hz, CHCH.sub.3), 13.10, 13.07 (OCH2CH.sub.3).

[0482] MS (ES+) m/z: Found: 541 (M+H.sup.+), 563 (M+Na.sup.+) C.sub.21H.sub.26ClN.sub.6O.sub.7P required: 540 (M).

[0483] HPLC Reverse-phase HPLC eluting with H.sub.2O/CH.sub.3CN from 90/10 to 0/100 in 30 minutes, F=1 ml/min, 1=254 nm, t.sub.R 12.41, 12.83 min.

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

[0484] ##STR00032##

[0485] N-methylimidazole (240 μL, 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,5S)-2-(6-amino-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3-ol (150 mg, 0.6 mmol) in anhydrous THF (3 mL) and the reaction mixture was stirred at room temperature during a period of 16 hours. Purification by column chromatography (eluent system CH.sub.3OH/CH.sub.2Cl.sub.2 0/100 to 6/94) and preparative TLC (2000 μicron, eluent system CH.sub.3OH/CH.sub.2Cl.sub.2 5/95) afforded the desired compound as white solid (40 mg, 13%).

[0486] MS (ES+) m/z: Found: 521.2 (M+H.sup.+), 543.3 (M+Na.sup.+), 1063.4 (2M+Na.sup.+) C.sub.31H.sub.33N.sub.6O.sub.8P required: 520.18(M).

[0487] .sup.31P NMR (202 MHz, CD.sub.3OD): δP 3.99 (s), 3.82 (s).

[0488] .sup.1H NMR (500 MHz, CD.sub.3OD): δH 8.16 (s, 0.5H, H8), 8.15 (s, 0.5H, H8), 8.11 (s, 1H, H-2) 7.23-7.20 (m, 2H, Ph), 7.11-7.03 (m, 3H, Ph), 5.91 (d J=2.0 Hz, 0.5H, H1′), 5.90 (d J=2.0 Hz, 0.5H, H1′), 4.85-4.79 (m, 1H, CH(CH.sub.3).sub.2, 4.64-4.63 (m, 1H, H4′), 4.60-6.57 (m, 1H, H2′), 4.37-4.33 (m, 1H, H5′), 4.31-4.28 (m, 1H, H5′), 3.74-4.22-4.17 (m, 1H, H5′), 3.70 (m, 1H, CH ala), 2.02-1.97 (m, 1H, H3′), 2.04-2.01 (m, 1H, H3′), 1.18-1.14 (m, 3H, CH.sub.3), 1.24 (m,6H, CH(CH.sub.3).sub.2)

[0489] HPLC Reverse-phase HPLC eluting with H.sub.2O/CH.sub.3CN from 100/10 to 0/100 in 30 minutes, F=1 ml/min, □=200 nm, showed two peaks of the diastereoisomers with t.sub.R 11.58 min. and t.sub.R 11.92 min.

[0490] Solvents and Reagents. The following anhydrous solvents were purchased from Sigma-Aldrich: dichloromethane (CH.sub.2Cl.sub.2), trimethylphosphate ((CH.sub.3O).sub.3PO). Amino acid esters commercially available were purchased from Sigma-Aldrich. All reagents commercially available were used without further purification.

[0491] Thin Layer Chromatography (TLC).

[0492] 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.

[0493] Preparative TLC plates (20 cm×20 cm, 500-2000 μm) were purchased from Merck. Flash Column Chromatography. Flash column chromatography was carried out using silica gel supplied by Fisher (60 A, 35-70 μm). Glass columns were slurry packed using the appropriate eluent with the sample being loaded as a concentrated solution in the same eluent or preadsorbed onto silica gel. Fractions containing the product were identified by TLC, and pooled and the solvent was removed in vacuo.

[0494] High Performance Liquid Chromatography (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, 150×4.6 mm (as an analytic column) or II) Varian Prostar (LC Workstation-Varian Prostar 335 LC detector), Thermo SCIENTIFIC Hypersil Gold C18, 5 μ, 150×4.6 mm (as an analytic column). For the method of elution see the experimental part.

[0495] Nuclear Magnetic Resonance (NMR). .sup.1H NMR (500 MHz), .sup.13C NMR (125 MHz), .sup.31P NMR (202 MHz) and .sup.19F NMR (470 MHz) were recorded on a Bruker Avance 500 MHz spectrometer at 25° C. Chemical shifts (δ) are quoted in parts per million (ppm) relative to internal MeOH-d.sub.4 (δ 3.34 .sup.1H-NMR, δ 49.86 .sup.13C-NMR) and CHCl.sub.3-d.sub.4 (δ7.26 .sup.1H NMR, δ 77.36 .sup.13C NMR) or external 85% H.sub.3PO.sub.4 (δ 0.00 .sup.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 (apparent). The assignment of the signals in .sup.1H NMR and .sup.13C NMR was done based on the analysis of coupling constants and additional two-dimensional experiments (COSY, HSQC, HMBC, PENDANT).

[0496] Mass spectrometry (MS). Low resolution mass spectra were performed on Bruker Daltonics microTof-LC, (atmospheric pressure ionization, electron spray mass spectroscopy) in either positive or negative mode.

[0497] Purity of final compounds. The ≥95% purity of all the final compounds was confirmed using HPLC analysis.

EXAMPLE 2

Cytoxicity

[0498] Exemplified compounds embodying the present invention were assessed in the following procedures for their anti-cancer potency.

[0499] In vitro viability assays were performed to assess the effects of compounds on cell viability in 7 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 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 40 mM with thawed 100%. Compounds were serially diluted at 3.16 fold in thawed DMSO, and warmed to 37° C. before being dissolved in media (2 μl+200 μl). After compounds 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 (50 μl+50 μl), in duplicates. The compounds final concentrations were from 198M to 19.9 nM. All compound solubilities were checked and recorded again, then the plates were transferred to CO.sub.2 tissue culture incubator immediately and incubated for 3 days. DMSO final concentration is 0.5%.

[0500] The results of the initial screening are presented in Table II. A represents a relative IC.sub.50 of from 0.1 to 5 μM, B represents a relative IC.sub.50 greater than 5 μM and up to 15 μM, C represents an relative IC.sub.50 of greater than 15 μM and up to 100 μM; and D represents an relative IC.sub.50 of greater than 100 μM.

TABLE-US-00002 TABLE II MOLT-4.sup.a KG-1.sup.b HL-60.sup.c CCRF-CEM.sup.d K562.sup.e Cpnd IC50.sup.f M.I. %.sup.g IC50 M.I. % IC50 M.I. % IC50 M.I. % IC50 M.I. % cordycepin C 52 C 78 C 88 C 12 C 88 A A 98 B 92 B 97 A 100 A 92 B A 100 B 100 B 100 A 100 A 97 C A 93 D 65 C 82 B 94 B 92 D A 100 B 100 B 100 B 100 B 99 G C 75 C 63 C 57 C 65 C 78 H C 100 C 69 C 80 C 100 C 89 I C 100 C 93 C 99 C 99 C 87 E B 97 C 69 C 74 C 90 D 81 J A 100 C 29 C 98 C 100 C 97 F A 100 C 98 C 100 S 99 C 91 MCF-7.sup.h HepG2.sup.i 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 .sup.dMOLT-4: acute lymphoblastic leukaemia; .sup.bKG-1: acute myelogenous leukaemia; .sup.cHL-60: acute promyelocytic leukaemia; .sup.dCCRF-CEM: acute lymphoblastic leukaemia; .sup.eK562: chronic myelogenous leukaemia; .sup.fIC50 μM: relative IC.sub.50; .sup.gM.I. %: maximum percentage inhibition of cell viability. .sup.hMCF-7: breast adenocarcinoma; .sup.iHepG2: hepatocellular carcinoma

[0501] A subset of compounds of the invention were then assayed for their cytotoxic activity in a broader array of different solid tumours and haematological malignancies using the following assay.

[0502] Solid Tumour and Haematological Malignancy Assay

[0503] In 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 concentrations 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 40 mM with thawed 100%. Compounds were serially diluted at 3.16 fold in thawed DMSO, and warmed to 37° C. before being dissolved in media (2 μl+200 μl). 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 (50 μl+50 μl) in duplicates. The compounds' final concentrations were from 198M to 19.9 nM. All compound solubilities were checked and recorded again, then the plates were transferred to CO.sub.2 tissue culture incubator immediately and incubated for 3 days. DMSO final concentration is 0.5%.

[0504] The following cell lines were tested and are referred to in the Table IV below:

TABLE-US-00003 TABLE III Cell line Malignancy MOLT-4 Acute lymphoblastic leukaemia CCRFCEM Acute lymphoblastic leukaemia RL Non-Hodgkin’s lymphoma HS445 Hodgkin lymphoma RPMI8226 Human multiple myeloma K562 Chronic myelogenous leukaemia KG-1 Acute myelogenous leukaemia THP-1 Acute monocytic leukaemia Z-138 Mantle cell lymphoma NCI-H929 Plasmacytoma HEL92.1.7 Erythroleukaemia HL-60 Promyelocytic leukaemia MV4-11 Biphenotypic B myelomonocytic leukemia HepG2 Hepatocellular carcinoma HT29 Colon adenocarcinoma BxPC-3 Pancreatic cancer MCF-7 Breast adenocarcinoma MiaPaCa2 Breast adenocarcinoma SW620 Colon adenocarcinoma Jurkat acute T cell leukaemia

[0505] The results of the further screening are presented in Tables IV-VII. For Tables IV to VI: A represents an absolute IC.sub.50 of from 0.1 μM to 5 μM, B represents an absolute IC.sub.50 greater than 5 μM and up to 15 μM, C represents an absolute IC.sub.50 of greater than 15 μM and up to 100 μM; and D represents an absolute ICso of greater than 100 μM. For Table VII: A represents an absolute EC.sub.50 of from 0.1 μM to 5 μM, B represents an absolute EC.sub.50 greater than 5 μM and up to 15 μM, C represents an absolute EC.sub.50 of greater than 15 μM and up to 100 μM; and D represents an absolute EC.sub.50 of greater than 100 μM.

TABLE-US-00004 TABLE IV CCRFCEM MOLT-4 KG-1 Jurkat Cmpd IC.sub.50 MI % IC.sub.50 MI % IC.sub.50 MI % IC.sub.50 MI % Cordycepin D 41 D 46 D 69 D 20 A A 100 A 98 C 100 A 100 B A 100 A 98 B 97 A 100 C B 100 A 92 C 102 B 100 F A 101 A 98 C 95 A 100 E A 100 A 98 C 100 B 95 THP-1 RL HS445 NCI-H929 Cmpd IC.sub.50 MI % IC.sub.50 MI % IC.sub.50 MI % IC.sub.50 MI % Cordycepin D −3 D 17 D 2 D 24 A C 74 A 93 C 98 B 100 B C 99 A 96 B 96 A 99 C C 99 B 100 C 102 B 104 F C 100 B 90 C 92 B 100 E D 43 B 88 C 85 C 98 RPMI-8226 MV4-11 HEL92.1.7 K562 Cmpd IC.sub.50 MI % IC.sub.50 MI % IC.sub.50 MI % IC.sub.50 MI % Cordycepin D 1 D 1 C 88 C 78 A B 96 A 99 B 100 A 96 B B 102 A 99 A 98 A 99 C C 103 B 106 A 99 B 100 F C 106 A 100 B 99 A 93 E C 89 B 101 C 101 B 90 HL-60 Z138 BxPC-3 HepG2 Cmpd IC.sub.50 MI % IC.sub.50 MI % IC.sub.50 MI % IC.sub.50 MI % Cordycepin D 61 B 95 D 22 D 13 A B 99 C 95 C 81 C 75 B A 99 B 100 B 90 B 98 C B 100 B 100 C 99 C 99 F B 96 C 76 C 78 C 79 E B 95 C 93 C 71 C 67 HT29 MCF7 MiaPaCa-2 SW620 Cmpd IC.sub.50 MI % IC.sub.50 MI % IC.sub.50 MI % IC.sub.50 MI % Cordycepin D 44 D 77 D 35 D 10 A B 93 A 99 B 96 C 85 B B 98 A 96 A 98 B 93 C C 99 A 102 B 106 C 100 F C 89 B 103 B 101 C 90 E C 76 B 89 C 91 C 74

TABLE-US-00005 TABLE V CCRFCEM KG-1 K562 MOLT-4 Cmpd IC.sub.50 M.I. % IC.sub.50 M.I. % IC.sub.50 M.I. % IC.sub.50 M.I. % 2-OMe- D −4 D 1 D 40 D 1 Cordycepin L C 100 D 92 C 104 C 101 K B 102 C 99 C 100 C 104 M B 100 C 102 B 100 B 101 N C 93 C 96 C 78 C 97 HT29 MCF7 NCI-H929 RL Cmpd IC.sub.50 M.I. % IC.sub.50 M.I. % IC.sub.50 M.I. % IC.sub.50 M.I. % 2-OMe- D 19 D 8 D 51 D 0 Cordycepin L C 81 C 90 C 104 C 97 K C 98 B 99 B 102 C 104 M C 100 C 100 B 99 B 100 N C 69 C 73 C 99 C 88

TABLE-US-00006 TABLE VI HepG2 HL-60 HT29 Cmpd IC.sub.50 M.I. % IC.sub.50 M.I. % IC.sub.50 M.I. % 2-F- C 82 B 96 C 73 Cordycepin O B 83 A 101 C 84 P B 74 A 100 B 87 Q A 94 A 99 C 88 R B 82 A 100 C 92 CCRFCEM HEL92.1.7 KG-1 Cmpd IC.sub.50 M.I. % IC.sub.50 M.I. % IC.sub.50 M.I. % 2-F- C 101 B 99 B 99 Cordycepin O A 99 A 100 B 99 P A 99 A 99 A 97 Q A 101 A 99 B 103 R A 100 A 99 A 96 MiaPaCa-2 MCF7 K562 Cmpd IC.sub.50 M.I. % IC.sub.50 M.I. % IC.sub.50 M.I. % 2-F- C 100 C 97 C 97 Cordycepin O A 100 A 95 A 99 P A 98 A 95 A 97 Q A 100 A 99 A 100 R B 98 A 87 B 96

TABLE-US-00007 TABLE VII BxPC- CCRF- 3-Luc CEM HEL.92.1.7 HepG2 HL-60 HS445 HT29 K562 KG-1 2-Cl- EC.sub.50 D C B D C D D D D cordycepin MI % −1.3 98.1 94.3 31.1 84.4 −1.9 4.4 37.6 4.3 Cmpd. S EC.sub.50 C B B C B C C C C MI % 82.6 100 100.4 84.6 101.9 105.9 88.1 101.4 99 Mia-Pa- NCI- RPMI- MCF-7 Ca-2 MOLT-4 MV4-11 H929 RL 8226 SW620 THP-1 Z-138 2-Cl- EC.sub.50 D D D C B C D D D C cordycepin MI % −0.9 17.7 80.4 100.2 93.6 84.1 29 36.3 24.1 99.5 Compd. S EC.sub.50 C C B B B B C C C C MI % 100.9 98.2 100.1 100.9 102.3 100.2 99.6 92.1 97 90.2

[0506] All compounds tested showed cytotoxic activity against the cell lines tested. In most cases the compounds of the invention were more potent than the parent nucleoside against all cell lines.

EXAMPLE 3

Assessment of Cytotoxicity and Cancer Stem Cell Activity

[0507] A further comparative analysis of the toxicity of compounds in the acute myeloid leukaemia (AML) cell line KG1a 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 KG1a cell line, across the entire dose range.

[0508] Materials and Methods

[0509] KG1a Cell Culture Conditions

[0510] The KG1a cell line was maintained in RPMI medium (Invitrogen, Paisley, UK) supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin and 20% foetal calf serum. Cells were subsequently aliquoted (10.sup.5 cells/100 μl) into 96-well plates and were incubated at 37° C. in a humidified 5% carbon dioxide atmosphere for 72 h in the presence of nucleoside analogues and their respective proTides at concentrations that were experimentally determined for each series of compounds. In addition, control cultures were carried out to which no drug was added. Cells were subsequently harvested by centrifugation and were analyzed by flow cytometry using the Annexin V assay.

[0511] Measurement of In Vitro Apoptosis

[0512] Cultured cells were harvested by centrifugation and then resuspended in 195 μl of calcium-rich buffer. Subsequently, 5 μl of Annexin V (Caltag Medsystems, Botolph Claydon, UK) was added to the cell suspension and cells were incubated in the dark for 10 mins prior to washing. Cells were finally resuspended in 190 μl of calcium-rich buffer together with 10 μl of propidium iodide. Apoptosis was assessed by dual-colour immunofluorescent flow cytometry as described previously. Subsequently LD.sub.50 values (the dose required to kill 50% of the cells in a culture) were calculated for each nucleoside analogue and ProTide.

[0513] Immunophenotypic Identification of the Leukaemic Stem Cell Compartment

[0514] KG1a cells were cultured for 72 h in the presence of a wide range of concentrations of each compound assayed. Cells were then harvested and labelled with a cocktail of anti-lineage antibodies (PE-cy7), anti-CD34 (FITC), anti-CD38 (PE) and anti-CD123 (PERCP cy5). The sub-population expressing a LSC phenotype were subsequently identified and were expressed as a percentage of all viable cells left in the culture. The percentages of stem cells remaining were then plotted on a dose-response graph and the effects of the compounds were compared with each other, and with the parent nucleoside.

[0515] Statistical Analysis

[0516] 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. LD.sub.50 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, Calif.).

[0517] Results

[0518] The in vitro drug sensitivity was measured using the Annexin V/propidium iodide assay. Compound A showed increased in vitro potency when compared to Cordycepin (P<0.0001). 2-F-Cordycepin was significantly more potent than Cordycepin (P<0.0001) and all of the ProTides tested showed increased potency when compared to the parental nucleoside (FIG. 1).

[0519] These experiments confirmed that compound A showed evidence of increased potency in the stem cell compartment at concentrations above 1 mM. As can be seen from FIG. 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 compound A to preferentially target cancer stem cells. At the higher concentrations tested (1 mM and above), the ability of compound A to preferentially target LSCs was significantly greater than that of the parent compound.

[0520] 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 O was able to bring about a reduction in the proportion of LSCs present in treated cell populations (indicating an ability to target LSCs), its activity was not significantly different to 2-F-Cordycepin at any of the concentrations tested. FIG. 3 shows the comparison between 2-F-Cordycepin and all of the proTides tested, while individual comparisons are shown in the panels of FIG. 4.

EXAMPLE 4

Further Cytotoxicity Assessment and Inhibition Studies

[0521] Certain compounds of the invention were subjected to further studies to test the cytotoxic activity of certain compounds of the invention and also to measure their activity against 4 haematological cancer cell lines [0522] TdT positive CEM (Human ALL) [0523] TdT negative K562 (Human CML) [0524] TdT negative H1-60 (Human ANLL) [0525] RL (CRL-2261) non-HD lymphoma [0526] The concentrations of the active metabolite dATP (Cordycepin triphosphate) in these cell lines was also measured. [0527] The cytotoxic activity and the intracellular 3′-dATP concentrations were also studied in the presence of hENT1, Adenosine Kinase (AK) and Adenosine Deaminase pharmacological inhibitors in CEM and RL cancer cell lines. Said inhibitors that mimic known cancer resistance mechanisms.

[0528] Methods

[0529] Cell Culture

[0530] HL-60 (ATCC® CCL-240™), K562 (ATCC® CCL-243™), CCRF-CEM (ATCC® CRM-CCL-119™) and RL (ATCC® CRL-2261™) leukaemia cell lines, obtained from the American Type Culture Collection (ATCC), Middlesex. HL-60 and K562 cell lines are deoxynucleotidyl transferase-negative (TdT−ve), whereas CCRF-CEM cell line is TdT+ve.

[0531] 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.

[0532] Maintenance of Cell Lines

[0533] 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% CO.sub.2.

[0534] Adenosine 5′-triphosphate (ATP) Assay

[0535] The amount of ATP was used as a measurement of cell number and cell viability. ATP ViaLight™ plus assay kit (Lonza, USA: Product No. LT07-121) to detect ATP in cells treated in luminescence compatible 96 well plates (initial concentration of cells was 1×10.sup.4 cells/well) with cordycepin and ProTides at concentrations of: 0, 0.1, 0.5, 1, 5 and 10 μM, followed by incubation for 72 hours at 37° C. incubator with 5% CO.sub.2. For inhibitor studies, 10 μM of NBTI or 1 μM EHNA or A-134974 was added and left for 5 minutes before adding the drugs (see section 5 for inhibitor details).

[0536] After incubation, 50 μl of cell lysis reagent was added to the 96 well plates to release the intracellular ATP, followed by 100 μl 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.

[0537] Treating Cells and Extracting Samples for Intracellular triphosphate Analysis

[0538] Cell lines with 5×10.sup.6 cells/ml were used. Cells were treated with 1 μl of 50 μM of each of cordycepin and compounds A, B, D, E and F and incubated for 2 hours at 37° C. with 5% CO.sub.2. After incubation, cells were centrifuged (ambient, 1200 rpm, 5 minutes), the culture medium supernatants were removed, and the cell pellets were washed with 1 ml of PBS and centrifuged (ambient, 1200 rpm, 5 minutes). The supernatants were removed; the pellets were reconstituted in 100 μl of PBS and 100 μl of 0.8M perchloric acid and vortex mixed and kept on ice for 30 minutes. Then centrifuged (ambient, 1200 rpm, 5 minutes) and 180 μl of the supernatant was transferred to new tubes and stored at −80° C. until time of analysis.

[0539] During analysis, 90 μl of the extract was transferred to the fresh tubes. 25 μl of 1M ammonium acetate was added to the extract, and then neutralised by addition of 10 μl of 10% ammonia and 5 μl of deionised water, then transferred to LC-MS vials and 10 μl was injected into the UPLC-MS/MS system.

[0540] Inhibitor Studies

[0541] Cell lines were treated in the same way as described above but before treatment with drugs, a number of inhibitors were added: [0542] 1) Nitrobenzylthioinosine (NBTI) (Sigma-Aldrich, St. Louis, Mo., product # N2255) blocks nucleoside transporters [0543] 2) EHNA hydrochloride (Sigma-Aldrich, St. Louis, Mo., product # E114) blocks adenosine deaminase [0544] 3) Adenosine kinase inhibitor A-134974 dihydrochloride hydrate (Sigma-Aldrich, St. Louis, Mo., product # A2846): blocks adenosine kinase

[0545] Cells were treated with 10 μM of NBTI or 1 μM 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% CO.sub.2.

[0546] LC-MS/MS Analysis

[0547] The analytes were resolved using an ultra-performance liquid chromatography system (Accela UPLC, Thermo Scientific, UK) equipped with a Biobasic Ax5 μm, 50×2.1 mm column (Thermo Electron Corporation, Murrieta, Calif., USA) and mobile phase consisting of a mixture of 10 mM NH4Ac in ACN/H.sub.2O (30:70v/v), pH6.0 (A), and 1 mM NH4Ac in ACN/H.sub.2O (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 minutes, 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 500 μl/min.

[0548] Eluting compounds of interest were detected using a triple stage quadrupole Vantage mass spectrometry system (Thermo Scientific, UK) equipped with an electrospray ion source. Samples 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.fwdarw.392.1 (collision energy 19V) and the internal standard ChloroATP 539.9.fwdarw.442.2 (collision energy 24V).

[0549] Statistical Analysis

[0550] The dose-response curves of cytotoxicity of the drugs was determined using non-linear regression analysis of percentage cell viability versus concentration and EC.sub.50 values were obtained. The intracellular assay was conducted in five replicates for each condition. Intracellular assay was determined using paired t test (two-tailed) analysis of 3′ ATP/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.

[0551] Results

TABLE-US-00008 Summary IC.sub.50 Table (μM) A B D E F Cordycepin (FD) (FD) (FD) (FD) (FD) CEM 19.5 0.87 (22)   0.13 (150) 6.1 (3) 10.0 (2) 4.3 (5) (TdT.sup.+ve) K562 10.9 2.4 (5)  0.21 (52) 4.2 (3) 13.9 (1) 6.3 (2) HL-60 11.4 4.6 (3)  2.6 (4) 5.4 (2) 10.2 (1) 8.3 (1) CRL 24.5 2.1 (12)  0.4 (61) 4.8 (5) 11.0 (2) 3.4 (7) (FD) = fold difference compared to Cordycepin = Cordycepin IC.sub.50/ProTide IC.sub.50

Summary Mean Intracellular 3′-dATP levels (μg/ml)

[0552]

TABLE-US-00009 A B D E F Cordycepin (FD) (FD) (FD) (FD) (FD) CEM 0.2 3.7 (19) 11.5 (58) 2.9 (15)  0.2 (1)   1.1 (6)   K562 1.7 2.6 (13) 6.2 (4) 0.9 (0.5) 0.2 (0.1) 1.3 (0.8) HL-60 1.7 3.2 (16) 5.1 (3) 0.7 (0.4) 0.2 (0.1) 1.2 (0.7) CRL 0.8 6.6 (33) 10.8 (14) 1.7 (2)   1 (1) 3.4 (4)   (FD) = fold difference compared to Cordycepin = Protide Intracellular TP/Cordycepin Intracellular TP

[0553] Compounds A and B were the best performers with IC.sub.50 from 3 to 150-fold better than cordycepin Compounds A and B produced intracellular 3′-dATP concentrations 3 to 56-fold better than cordycepin.

TABLE-US-00010 Summary IC.sub.50 Table (All in μM) CEM CRL (FD) (FD) Cordycepin Control 11.5 7.3 NBTI 57.7 (5) 17.9 (2) EHNA 0.7 (−16) 13.2 (2) AK 29.2 (3) 28.6 (4) A Control 1.4 3.6 NBTI 2.0 (1) 2.6 (1) EHNA 3.1 (2.2) 2.9 (1) AK 3.6 (3) 10.2 (3) B Control 0.9 3.1 NBTI 1.4 (1) 2.6 (1) EHNA 1.3 (1) 5.2 (2) AK 1.3 (1) 2.8 (1) E Control 9.9 8.2 NBTI 17.1 (2) 8.2 (1) EHNA 13.4 (1) 5.9 (1) AK 10.3 (1) 7.1 (1) (FD) = fold difference compared to control

Summary Mean Intracellular 3′-dATP levels (μg/ml)

[0554]

TABLE-US-00011 CEM CRL (FD) (FD) Cordycepin Control 0.24 0.10 NBTI 0.14 (1) 0.06 (1) EHNA 9.01 (38) 1.85 (19) AK 0.31 (1) 0.16 (1) A Control 1.30 0.31 NBTI 0.99 (1) 0.32 (1) EHNA 1.35 (1) 0.27 (1) AK 1.20 (1) 0.30 (1) B Control 4.07 0.59 NBTI 3.14 (1) 0.68 (1) EHNA 3.62 (1) 0.67 (1) AK 2.99 (1) 0.77 (1) E Control 0.32 0.08 NBTI 0.17 (1) 0.12 (1) EHNA 0.21 (1) 0.07 (1) AK 0.19 (1) 0.06 (1) (FD) = fold difference compared to control

[0555] 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 compounds 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 invention will be less susceptible to cancer resistance mechanisms that cordycepin.