AU769578B2 - Pyrrolo(2,3-d)pyrimidine nucleoside analogs - Google Patents

Description

i -WO 01/27114 PCT/US00/22674 PYRROLO12,3-d]PYRIMIDINE NUCLEOSIDE ANALOGS Field of The Invention The field of the invention is nucleoside analogs.

Background of The Invention Nucleoside analogs have long been used as antimetabolites for treatment of cancers and viral infections. After entry into the cell, nucleoside analogs are frequently phosphorylated by nucleoside salvage pathways, in which the analogs are typically phosphorylated to the corresponding mono-, di-. and triphosphates. Among other intracellular destinations, triphosphorylated nucleoside analogs often are used as substrate for DNA or RNA polymerases and consequently incorporated into DNA or RNA. Where triphosphorylated nucleoside analogs are strong polymerase inhibitors, they may induce premature termination of a nascent nucleic acid molecule. Where triphosphorylated nucleoside analogs are incorporated into nucleic acid replicates or transcripts, gene expression or disruption of function may result.

On a more cellular level, the nucleoside analogs can also interfere with the cell cycle, and especially desirable effects of nucleoside analogs include induction of apoptosis of cancer cells.

Furthermore, nucleoside analogs are also known to modulate certain immune responses.

Various nucleoside analogs with relatively potent anti-cancer activity are known in the art. For example, known drugs include thymidylate synthase inhibitors such as adenosine deaminase inhibitors, including 2-chloroadenosine, and neplanocin A, which is an inhibitor of S-adenosylhomocysteine hydrolase. However, all or almost all of the known nucleoside analogs also imply a threat to normal mammalian cells, primarily because these nucleoside analog's lack adequate selectivity between normal cells and tumor cells.

Unfortunately, lack of adequate selectivity is frequently associated with severe side effects, and therefore often limits the potential of such analog therapeutics.

Although there are various nucleoside analogs known in the art, all or almost all of them, suffer from one or more disadvantage. Therefore, there is still a need to provide improved methods and compositions for nucleoside analogs.

U o/ LLD /4 Attorney Reference No.: 330.48-PCT PEAUS 6 MAR 2001 IPEANUS 6 MAR 2001 Summary of the InventionSummary of the Invention The present invention is directed to nucleoside analogs with modifications on the sugar moieties of the pyrrolo[2,3-d]pyrimidine nucleoside analogs, which can significantly reduce the toxicity of the nucleoside analogs to the mammalian cells while they also provide significant cytotoxicity to cancer cells. These modifications include but are not limited to substitutions at the C4' and C5' positions of ribofuranose moieties. The present invention also demonstrates that certain pyrrolo[2,3-d]pyrimidine nucleoside analogs have desired immunomodulation effects, including enhancement of Type 1 cytokines such as IL-2 and suppression of Type 2 cytokines such as IL-4. These immunomodulation properties can be useful in anticancer, antiviral and autoimmune diseases, treating inflammation and preventing graft rejection.

In one aspect of the inventive subject matter, the nucleoside analog is a pyrrolo[2,3]pyrimidine nucleoside having a structure according to the formula x z N N N (I)

R

R,

R

3

R

2 wherein A is O, S, or CH2; X is H, NH 2 or OH; Y is H, halogen or NH 2 Z is selected from the group consisting of H, halogen, R, OH, OR, SH, SR, NH 2 NHR, NR 2 CN, C(O)NH 2

COOH,

COOR, CH 2

NH

2

C(=NOH)NH

2 and C(=NH)NH 2 where R is alkyl, alkenyl, alkynyl, or aralkyl;

R

2 and R 3 are independently selected from the group consisting of H, F, and OH; R 4 is selected from the group consisting of a hydrogen, an alkyl, an alkenyl, an alkynyl, and an aralkyl, wherein

R

4 optionally has at least one of a heteroatom and a functional group; R 5 is H, OH, OP(O)(OH) 2

P(O)(OH)

2

OP(O)(OR')

2 or P(O)(OR') 2 wherein R' is a masking group; and R 5 is selected from the group consisting of an alkyl, an alkenyl, an alkynyl, and an aralkyl, wherein Rs. has at least two carbon atoms, and optionally has at least one of a heteroatom and a functional group.

In another aspect of the inventive subject matter, the nucleoside analog is a pyrrolo[2,3d]pyrimidine nucleoside having a structure according to the formula (II): 2 AMENDED

SHEET

P \OPER\Kbm2 00227.clims doc-24/11/03 -3- 0 z N N (II) R' O RS 0 3 2 wherein Z is CN, C(O)NH 2

C(=NH)NH

2 or C(=NOH)NH 2 and R 4 and R5. are independently selected from the group consisting of a hydrogen, an alkyl, an alkenyl, an alkynyl, and an aralkyl, wherein R 4 and Ry independently and optionally contain at least one of a heteroatom and a functional group; with the proviso that R4 and Rs 5 are not together hydrogen.

In a further aspect of the inventive subject matter, contemplated compounds are utilized to inhibit tumor growth or to modulate Type 1 and Type 2 cytokine, and chemokine production.

In a further aspect of the invention there is provided a method of changing secretion of a cytokine from a cell, comprising; providing a compound according to formula (Ia);

N

2 0 t N N (Ia)

R

R3 R2 25 wherein A is O, S, or CH 2 X is H, NH 2 or OH; Y is H; halogen or NH 2 Z is selected from the group consisting of H, halogen, R, OH, OR, SH, SR, NH 2 NHR, NR 2 CN, C(O)NH2, COOH, COOR, CH 2

NH

2 C(=NOH)NH2, and

C(=NH)NH

2 where R is alkyl, alkenyl, alkynyl, or aralkyl;

R

2 and R 3 are independently selected from the group consisting of H, F, and OH; P:\OPERUbmn2U00O227 cllims doc-24/11/03 3a

R

4 and R5. are independently selected from the group consisting of a hydrogen, an alkyl, an alkenyl, an alkynyl, and an aralkyl, and wherein R4 and Rs. independently and optionally contain at least one of a heteroatom and a functional group;

R

5 is H, OH, OP(O)(OH) 2

P(O)(OH)

2

OP(O)(OR')

2 or P(O)(OR') 2 wherein R' is a masking group; and presenting the cell with the compound at a concentration effective to change the secretion of the cytokine.

In yet a further aspect of the invention there is provided a method of changing secretion of a cytokine from a cell, comprising: providing a compound according to formula (III) 0 z

O

HN

N N (II)

RS

HO OH 20 wherein Z is CN, C(O)NH 2

C(=NH)NH

2

C(=NNH

2

)NH

2 or -C(=NOH)NH 2 wherein Rs is H, OH, OP(O)(OH) 2

P(O)(OH)

2

OP(O)(OR')

2 or P(O)(OR') 2 wherein R' is a masking group; and presenting the cell with the compound at a concentration effective to change the secretion of the cytokine.

In yet a further aspect of the invention there is provided a method of reducing growth of a hyperproliferative cell, comprising: providing a compound according to formula (IV); V S&S~j*5SVu a..Qi t C.f&V Aw P.\OPER\Kbm.\2S0227.claims doc.24/11103 -3bx z y

(V)

R

R

3

R

2 wherein A is O, S, or CH 2 X is H, NH 2 or OH; Y is H, halogen or NH 2 Z is selected from the group consisting of H, halogen, R, OH, OR, SH, SR, NH 2

NHR,

NR

2 CN, C(O)NH 2 COOH, COOR, CH 2

NH

2 C(=NOH)NH 2 and C(=NH)NH 2 where R is alkyl, alkenyl, alkynyl, or aralkyl;

R

2 and R 3 are independently selected from the group consisting of H, F, and OH;

R

4 is selected from the group consisting of a hydrogen, an alkyl, an alkenyl, an alkynyl, and an aralkyl, wherein R 4 optionally contains at least one of a heteroatom and a functional group; Rs 5 is selected from the group consisting of an alkyl, an alkenyl, an alkynyl, and an aralkyl, wherein R5. has at least two carbon atoms, and optionally contains at least one of a heteroatom and a functional group;

R

5 is H, OH, OP(O)(OH) 2

P(O)(OH)

2

OP(O)(OR')

2 or P(O)(OR') 2 wherein R' is a masking group, and presenting the hyperproliferative cell with the compound at a concentration effective to reduce the growth of the hyperproliferative cell.

25 In yet a further aspect of the invention there is provided a method of reducing a release of a growth factor from a cell, comprising; providing a compound of the invention; and presenting the cell with the compound at a concentration effective to reduce the release of the growth factor.

Various objects, features, aspects and advantages of the present invention will become IIIYI PN~J:~nlC~U I IUii~~Ylt~~~J~d~ P:\OPERVKX'2500227.claimns.doc-24/11/03 3c more apparent from the following detailed description of preferred embodiments of the invention, along with accompanying drawings.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a first exemplary synthetic scheme of reactions included in the production of compounds according to the inventive subject matter.

Fig. 2 is a second exemplary synthetic scheme of reactions included in the production of 2 compounds according to the inventive subject matter.

Fig. 3 is a third exemplary synthetic scheme of reactions included in the production of compounds according to the inventive subject matter.

Fig. 4 is a fourth exemplary synthetic scheme of reactions included in the production of compounds according to the inventive subject matter.

Fig. 5 is a fifth exemplary synthetic scheme of reactions included in the production of compounds according to the inventive subject matter.

o. 0* 0 o *ZVOCKAW 01M, v4WONANAWAIN% a N N V NI~ UbO 0/ 2 2 6 74 Attorney Reference No.: 330.48-PCT IPENUS O MAR 2001 Fig. 6 is a sixth exemplary synthetic scheme of reactions included in the production of compounds according to the inventive subject matter.

Fig. 7 depicts exemplary compounds according to the inventive subject matter.

Fig. 8A and 8B are graphs representing the effect of compounds according to the inventive subject matter on the expression of Type 1 and Type 2 cytokines, respectively.

Fig. 9 is a table indicating cytotoxicity of various compounds according to the inventive subject matter.

Fig. 10 is a table indicating rates of DNA synthesis in cells treated with of various compounds according to the inventive subject matter.

Fig. 11 is a graph depicting the inhibition of VEGF release from human prostate cancer cells upon treatment with compounds according to the inventive subject matter.

Fig. 12 is a graph depicting the inhibition of IL-8 release from human prostate cancer cells upon treatment with compounds according to the inventive subject matter.

Detailed DescriptionDetailed Description Pyrrolo[2,3-d]pyrimidine nucleoside analogs according to the general formulae and (II) were found to have various biological effects on normal and hyperproliferative cells.

4 AMENDED SHEEI A I: 2 A W W UIICM f 'rUIUO U U I t Attorney Reference No.: 330.48-PCT IPEA/US 2 NAR 2001 wherein A is O, S, or CH 2 X is H, NH 2 or OH; Y is H, halogen or NH 2 Z is selected from the group consisting of H, halogen, R, OH, OR, SH, SR, NH 2 NHR, NR 2 CN, C(O)NH 2

COOH,

COOR, CH 2

NH

2

C(=NOH)NH

2 and C(=NH)NH 2 where R is alkyl, alkenyl, alkynyl, or aralkyl;

R

2 and R 3 are independently selected from the group consisting of H, F, and OH; R 4 is selected from the group consisting of a hydrogen, an alkyl, an alkenyl, an alkynyl, and an aralkyl, wherein

R

4 optionally has at least one of a heteroatom and a functional group; Rs is H, OH, OP(O)(OH) 2

P(O)(OH)

2

OP(O)(OR')

2 or P(O)(OR') 2 wherein R' is a masking group; and R 5 is selected from the group consisting of an alkyl, an alkenyl, an alkynyl, and an aralkyl, wherein R 5 has at least two carbon atoms, and optionally has at least one of a heteroatom and a functional group.

It should be especially appreciated that the terms "alkyl", "alkenyl", "alkynyl", and "aralkyl" as used herein refer to both linear and branched species. With respect to the substituents R 2 and R 3 it should be appreciated that both R 2 and R 3 may be independently directed to the at- or p-face. Furthermore, where the substituents on C 5 y are non-identical, substitution on Cs may result in an R- or S-chiral center. The term "heteroatom", as used herein, refers to non-carbon atoms in an organic molecule, and particularly contemplated heteroatoms include halogens, nitrogen, oxygen, and sulfur. The term "functional group" as used herein refers to a reactive bond double or triple bond) or reactive group -OH, -SH, -NH 2

-N

3

-CN,

COOH, -CHO, -CONH 2 etc.).

Particularly contemplated pyrrolo[2,3-d]pyrimidine nucleoside analogs are those according to formula wherein Z is CN, C(O)NH 2 or C(=NH)NH 2 and wherein R 5 y has at least two carbon atoms and is selected from the group consisting of an alkyl, an alkenyl, an alkynyl, and an aralkyl.

The pyrrolo[2,3-d]pyrimidine nucleoside analog according to formula (II) has the following structure: 0 z R N (II) Rs'

O

R

3

R

AMENDED SHEET 711- 01R14f,11A~U~r U~~llil PAOPER\KtUh2500227.claimsdoc-2411/03 -6wherein Z is CN, C(O)NH 2

C(=NH)NH

2 or C(=NOH)NH2 and R4 and R 5 s are independently selected from the group consisting of a hydrogen, an alkyl, an alkenyl, an alkynyl, and an aralkyl, wherein R 4 and Rs5 independently and optionally contain at least one of a heteroatom and a functional group; with the proviso that R 4 and Rs 5 are not together hydrogen; and wherein the remaining substituents are as defined in formula It should be appreciated, however, that compounds according to the inventive subject matter may also be in forms and formulations other than previously described, and especially contemplated forms include prodrug forms or otherwise modified forms in which contemplated molecules are chemically and/or enzymatically modified to improve pharmacological and/or pharmacodynamical properties, including higher specificity towards target organs, cells or subcellular compartments and increased half-life time in the organism.

For example, cholesterol adducts may be formed to increase target specificity towards the liver, or apolipoprotein adducts may be formed to enhance penetration of the modified drug across the blood brain barrier to the brain. In another example, receptor ligand complexes may be synthesized to target the modified drug to a particular cell expressing a receptor specific for the ligand. Alternatively, antibody or antibody fragment 20 complexes may be formed to increase selective delivery of the modified drug to a subcellular location. There are many prodrug and modified forms known in the art.

Examples of prodrugs include charged or uncharged groups, lipophilic or polar groups may be added to contemplated molecules to increase the half-life time in serum or other target organs and/or cells. In further examples, it should be appreciated that the contemplated compounds, where phosphorylated at the C 5 atom, may also be di-, or tri-phosphorylated, or incorporate a thiophosphate.

While it is generally preferred that contemplated compounds have a sugar moiety in the D-configuration, it is also contemplated that the compounds may have a sugar 30 moiety in the L-configuration. Further stereochemical aspects especially include R and S configurations at the C 5 atom where appropriate, and it should be appreciated that the substituents in contemplated compounds may be directed to a or P phase.

Wr~?UT~Y-irrru ~,vrULPII~ ~IUIYIII ~iY WO 01/27114 WO 0127114PCTIUSOO/22674 It should also be appreciated that contemplated compounds can be formulated in various formulations, including liquid, syrup or gel forms for injection, ingestion, or topical administration) and solid forms for ingestion, injection, or deposition in a body cavity). For example. where it is contemplated that compounds according to the inventive subject matter are instable in the gastric environment, injection of a preferably isotonic solution is particularly contemplated. Alternatively, intranasal application or inhalation of a liquid form may be appropriate to circumvent acid degradation. On the other hand, where contemplated compounds are known to be resistant to digestive degradation. contemplated forms may be administered in form of a syrup or tablet. Depending on the particular use, contemplated compounds may also be formulated for topical or transdermal applications. There are many more formulations known in the art, all of which are also contemplated suitable in conjunction with the inventive subject matter presented herein, and particularly contemplated formulations are described in "Drug Products for Clinical Trials: An Intl. Guide to Formulation, Production, Quality Control" by Donald C. Monkhouse and Christopher T. Rhodes (Editors); ISBN:082479852X.

It should still further be appreciated that contemplated compounds and formulations may include functional and non-functional additives. For example, where transcutaneous drug delivery is desired, skin penetration enhancers may be added. Alternatively, pharmaceuticals including cytostatic. antiviral, or immunomodulatory agents, may be added to synergistically or additively improve the function of contemplated compounds. Examples for non-functional additives include fillers. antioxidants, flavor, or color agents to enhance a particular quality of contemplated compounds.

With respect to the concentration of contemplated compounds, it is preferred that the concentration is in a range of approximately I1pM to about I OOVM when measured at the site of action. However, and particularly where the affinity of contemplated compounds is below 1p M.

appropriate concentrations may also be in the range of 999nM to IlOaM, and less. On the other hand, where contemplated compounds exhibit relatively short half-life times, or have a high turnover, contemplated concentrations may be 0. 1 mM and I100mM. and more. Consequently, the dosage of contemplated compounds may vary significantly, but appropriate dosages can readily be determined in in vitro or animal experiments.

uiuOuu/ LC-Ul 4 Attorney Reference No.: 330.48-PCT IPEAUS 2 6 MAR 2001 Among the various biological effects of both and 4'-modified pyrrolopyrimidine nucleoside analogs, particularly significant biological effects include modulation of the Type 1 and Type 2 cytokine production, control of neoplastic conditions reduction of DNA synthesis or reduction in cell growth), and reduction of chemokine and growth factor release as described below.

Consequently, a contemplated method of changing secretion of a cytokine from a cell may comprise a step in which a compound according to formula is provided and has a further step in which the cell is presented with the compound according to formula at a concentration effective to change the secretion of the cytokine. While all possible combinations of substituents in formula are generally contemplated, particularly contemplated compounds are compounds according to formula wherein R 4 and R 5 S are independently selected from the group consisting of a hydrogen, an alkyl, an alkenyl, an alkynyl, and an aralkyl, and wherein R 4 and R 5 independently and optionally contain at least one of a heteroatom and a functional group, with the remaining substituents as defined above in formula In an alternative aspect, the compound employed to change the secretion of a cytokine from a cell may also be a compound according to the following structure:

R

5 0 Z Contemplated cytokines particularly include Type 1 IFNy) and Type 2 IL-4) cytokines. With respect to the cells, it is contemplated that all cells known to produce and/or secrete cytokines are appropriate, however, especially contemplated cells include lymphocytes and cancer cells prostate cancer cells, infra).

8 AMENDED SHEEI J4,"f I n n nrnrr l n~~nir n v l WO 01/27114 PCT/USO/22674 In a further aspect of the inventive subject matter, a method of reducing growth of a hyperproliferative cell may comprise a step in which a compound according to formula is provided, and another step in which the hyperproliferative cell is presented with the compound at a concentration effective to reduce the growth of the hyperproliferative cell. Particularly preferred compounds include compounds according to formula wherein R4 is selected from the group consisting of a hydrogen, an alkyl. an alkenyl, an alkynyl, and an aralkyl, wherein R 4 optionally contains at least one of a heteroatom and a functional group. and wherein R 5 is selected from the group consisting of a hydrogen, an alkyl. an alkenyl. an alkynyl. and an aralkyl.

wherein R 5 has at least two carbon atoms, and optionally contains at least one of a heteroatom and a functional group, with the proviso that R4 and R 5 are not together hydrogen, and with the remaining substituents as defined above in formula Particularly contemplated hyperproliferative cells include cancer cells, and an especially contemplated cancer cell is a prostate cancer cell. While not whishing to be bound to a particular theory. it is contemplated that the reduction of growth comprises reduction of DNA synthesis.

In a still further aspect of the inventive subject matter, it is contemplated that a method of reducing a release of a growth factor from a cell has a step in which a compound according to formula is provided, and another step in which the cell is presented with the compound at a concentration effective to reduce the release of the growth factor. It is contemplated that the release of various growth factors may be reduced by the method presented herein, however reduction of VEGF release is especially contemplated. Similarly. while all cells known to secrete growth factors are contemplated in conjunction with the method presented herein, particularly contemplated cells include cancer cells, and especially prostate cancer cells.

With respect to the synthesis of contemplated compounds. it should be appreciated that pyrrolo[2,3-dlpyrimidine nucleoside analogs according to the inventive subject matter can be synthesized via various synthetic routes, and the following procedures are provided by way of example only.

WO 01/27114 PCT/US00/22674 Synthesis ofC5'-modified pyrrolo[2.3-d]pyrimidine nucleoside analogs The 5'-substituted nucleoside analogs are prepared from the condensation of the pyrrolo[2,3-d]pyrimidine bases and the properly protected. 5'-substituted ribofuranoses. As shown in Figure 1, Compound 1. prepared according to a published procedure (Jones et al.

Methods in carbohydrate Chemistry (edited by Whistler and Moffat). vol. VI, pp315-322, Academic Press, New York, (1972)), was treated with a variety of nucleophiles such as Grignard reagents to give compound 2, which was benzoylated and subsequently treated with trifluoroacetic acid to give compound 4. Benzoylation and the following treatment with acetic anhydride/acetic acid in the presence of sulfuric acid to give compound 6, which was used for condensation with pyrrolo[2,3-d]pyrimidine bases.

Compound 7 (Jones et al. Methods in carbohydrate Chemistry (edited by Whistler and Moffat), vol. VI. pp 3 15-322, Academic Press. New York. (1972)), prepared according to a published procedure. was converted to a tosylate derivative, which was reduced with lithium aluminum hydride to give compound 8. By similar procedures shown in Figure 1. compound 8 was converted to compound 9. The condensation of 9 and the pyrrolo[2.3-d]pyrimidine 19 and the subsequent transformations as shown in scheme 2 gave compounds 10-15 as illustrated in Figure 2.

As shown in Figure 3, Compound 2 was converted to the sulfonate 16. which was subjected to nucleophilic replacement to give the configurationally inverted compound 17.

Deprotection of the isopropylidene and the subsequent acetylation gave the tetraacetate 18.

Condensation of the 5-C-substituted, protected ribofuranoses with nucleoside bases is depicted in Figure 4. 5-Cyanopyrrolo[2,3-d]pyrimidine 19, prepared according to a published procedure (Tolman et al. J. Org. Chem. 1969, 91, 2102-2108), was converted to the trimethylsilyl derivative and then condensed with compound 6 in the presence oftrimethylsilyl triflate by a similar procedure described for toyocamycin (Sharma et al. Nucleosides Nucleotides 1993, 12, 643-648). The resulting coupling product was subjected to debromination through hydrogenation to give compound 20. Treatment of 20 with ammonia in anhydrous methanol gave compounds 21 and 23. Compound 21 was oxidized to give compound 22. Compound 23 was converted to the carboxamide derivative 24. Compound 23 and 24 were oxidized to give compound Treatment of compound 25 with hydroxyamine yielded compound 26. which was hydrogenated r,~uuv ru nrx ,,,-r~irrilurr. ,uur*iur~ivii-l; WO 01/27114 PCT/US00/22674 over Raney Nickel to give 27. Alternatively, compound 27 was also prepared by heating compound 25 with ammonia in a pressured bomb.

Synthesis of C4 '-modifiedpyrrolo[2,3-d]pyrimidine nucleoside analogs In Figure 5, compound 1 was treated with formaldehyde in aqueous sodium hydroxide to give 4'-hydroxymethyl derivative 28, which was selectively protected to afford compound 29.

The subsequent protection with DMT and removal of TBS gave compound 31. which can be converted to a variety of substituents. The 4-C-substituted derivatives subjected to similar transformations as 5-C-substituted ribofuranoses (scheme 1) can be converted to compound which is used for condensation with nucleoside bases.

Similar to the C5'-substituted pyrrolopyrimidine nucleoiside analogs, the 4'-substituted analogs 36 can be obtained by condensation of compound 35 with compound 19 as depicted in Figure 6. The subsequent transformations can give the 4'-C-substituted pyrrolopyrimidine nucleoside 37-42.

Synthesis of2 '-modified. and other pyrrolo[2,3-d]pyrimidine nucleoside analogs The following pyrrolopyrimidine nucleoside analogs were prepared for biological testing, some of which were published (indicated as known compounds), and are shown in Figure 7.

The known compounds 43, 44, 52-55, and 57 were prepared according to a published procedure (Hinshaw et al. J. Org. Chem. 1970, 92, 236-241). Compound 56 was prepared by hydrogenation of compound 52. The known compound 49 (Krawczyk et al. Nucleosides Nucleotides 1989. 8.

97-115) was treated with sodium nitrite to give compound 50. The known compounds 45 and 48 were prepared according to a published procedure (Ramasamy et al. J. Heterocyclic Chem. 1988, 1043-1046). Compound 45 was treated with ammonia-methanol to give compound 46 and hydrogenated to give compound 47. Compounds 58-63 were prepared from compound 45 by similar procedures used for compounds 52-57. The known compound 64 (Krawczyk et al.

Nucleosides Nucleotides 1989, 8, 97-115) was converted to compounds 65-67. The known compound 68 (Ramasamy et al. Tetrahedron 1986, 42, 5869-5878) was converted to compounds 69 and 11 .im ~pun~.a rr *inl.i.~i~illsr WO 01/27114 PCT/USOO/22674 Preparation of human T-cells and activation in vitro Peripheral blood mononuclear cells were isolated from healthy donors by density gradient centrifugation followed by T cell enrichment using Lymphokwik (One Lambda. Canoga Park CA). Contaminating monocytes were removed by adherence to plastic. Purified T cells were 99% CD2+, HLA-DR+ and 5% CD25+ and were maintained in (RPMI1640 medium containing 5% autologous plasma. 1% L-glutamine, 1% penicillin/streptomycin and 0.05% 2-mercaptoethanol). For determination of cytokine protein levels, T-cells (0.2 x 10 6 cells in a volume of 0.2 ml) were activated by the addition of 2 ng phorbol myristate acetate plus 0.1 mg ionomycin (PMA-ION, both from Calbiochem, San Diego, CA) and incubated in 96 well plates in the presence of 0 or 10 1 M of various guanosine nucleosides for 48 h at 37 0 C. Following activation, supematants were analyzed for cell-derived cytokine production.

Extracellular cytokine analyses.

Human cytokine levels were determined in cell supernatants, following appropriate dilution, using ELISA kits specific for IFNy and IL-4 (Biosource. Camarillo. CA). All ELISA results were expressed as pg/mi.

Effect ofpyrrolo-[2.3.d]pyrimidine nucleoside analogs on extracellular cytokine levels in activated human T cells.

The effect of pyrrolo-[2,3-d]pyrimidine nucleoside analogs at 0 and 10 pM, on PMA/ionomycin stimulated T cell expression of the Type 1 cytokine. IFNy, and the Type 2 cytokine. IL-4, is shown in Figures 8A and 8B for 5 individual human donors. Cytokine levels were determined in cell free supematants by ELISA. The most potent effect was observed with 7-b-D-ribofuransyl-4-oxopyrrolo-[2,3-d] pyrimidine-5-carboxamidine. This compound enhanced activated IL-4 production by 498% 83 and suppressed IFNy by 43% 4 of the activated control levels of each cytokine. Data are shown as percentage of activated control calculated as the ratio of activated T cell cytokine level in the presence of test nucleosides over the cytokine level of untreated activated T cells x 100 Zero effect on cytokine levels by test nucleosides would give a percentage of activated control value of 100 The absolute level (pg/ml standard deviation) of PMA-ION-induced cytokine secretion was for IFNy, 22954 1 3391; and for IL-4, 162 40. Resting levels were 30 pg/ml for all cytokines tested.

WO 01/27114 PCT/US00/22674 Cytotoxicity of the pyrrolo[2.3-d]pyrimidine nucleoside analogs in vitro The pyrrolo[2,3-d]pyrimidine nucleoside analogs of the present invention are bioactive since they indicate some level of cytotoxicity in vitro. In these studies, the compounds tested were applied to cell culture of normal human fibroblasts, human Prostate cancer cells 81, human Melanoma cancer cells 140. Human Lung Cancer cells 177, and human Ovarian Cancer cells R and NR (all available from ATCC). In these experiments cells were plated at density of 2000 cells per 2004l of medium per well (96-well plate). The compounds tested were applied to the wells once, at concentration range 0.78-1 00pM, just after plating of cells. The colorimetric cytotoxicity assay MTS was performed after 72 hrs of treatment. EC50 was calculated based on readings collected and they are presented in Figure 9. Several compounds indicate lack of cytotoxicity in concentration below 100.M. In such cases EC50 is marked as >100. In other cases. EC50 indicates the concentration of the compound tested needed to damage 50% of cell population.

The pryrrolo[2,3-d]pyrimidine nucleoside analogs inhibit DNA synthesis in cells cultured in vitro in a dose-dependent manner Analogs of pyrrolo[2,3-d]pyrimidine nucleoside inhibit growth of human cells cultured in vitro as measured by the level of DNA. The experimental setup was the same as described above. The compounds were given once and DNA level was measured after 72 hrs. At that time, half of the medium was removed from culture wells and replaced by pure water. After that, the cells were transferred to -70 0 C for at least 12 hrs. In the next step, cells were transferred back from -70 0 C to room temperature and 1 uM of Hoechst 33342 was given to each well. After 2 hrs of incubation, the fluorescence signal (360-530nm) was measured. According to this method, intensity of florescence is proportional to amount of DNA due to presence of DNA-Hoechst 33342 complex formed. The results are presented in Figure 10. The numbers express fold of DNA amount increase compared to amount of DNA at the beginning of experiment (2 hrs after plating of cells). In the untreated prostate cancer cells and normal cells the DNA level increased 5.78 and 4.47 times, respectively, during 72 hrs of culture.

ia~ E iuj~i~ri~ui~va lwal h~ WO 01/27114 PCT/US00/22674 The compounds 23a(5 and 23a(5 inhibit secretion of VEGF from Human Prostate Cancer in vitro.

The compounds 23a(5'-R) and 23a(5'-S) are potent in inhibition of secretion of Vascular Endothelial Growth Factor (VEGF) from Human Prostate cancer cells, HTB81. VEGF is recognized as angiogenesis marker since this molecule is crucial for migration and growth of endothelial cells and microvessel formation in vivo. In order to prove this. 0.5x10 5 of the cells were plated in 5 ml of culture medium into a 10cm diameter petri dish. The compounds were applied just after plating for 72 hrs. After that, the medium was collected and the level of VEGF was measured using VEGF Elisa Assay (R&D Systems) and expressed as pg of VEGF per ml of the medium. The results are presented in Figure 11. According to these results, both compounds inhibit secretion of VEGF in a dose-dependent manner.

The compounds 23a(5'-R) and 23a(5'-S) inhibit release of'lL-8 from human prostate cancer cells cultured in vitro.

The compound 23a(5'-R) and 23a(5'-S) indicate an inhibitory effect on secretion of lnterleukin-8 (IL-8) from human prostate cancer cells, HTB81. IL-8 belongs to the class of chemo-attractant chemokines (type alpha), which are involved in inflammation processes due to attraction of neutrophils. Chemokines. in general, are known to be produced by various types of cancer. It is proven in several studies that inhibition of chemokine production by cancer cells is beneficial for the host. In order to prove the potency of these two compounds to inhibit IL-8 secretion from prostate cancer cells, prostate cancer cells HTB 81 were treated in vitro with compounds 23a in both the 5'-R and 5'-S configuration at concentrations indicated in the graph.

The medium collected from the culture was analyzed for IL-8 level using IL-8 Elisa Assay from R&D Systems. According to the results collected, both compounds are able to inhibit secretion of IL-8 in a dose-dependent manner, as depicted in Figure 12.

It should be appreciated, however, that the biological effects of contemplated compounds need not be limited to the particular effects as described above. In particular, it is contemplated that the compounds according to the inventive subject matter generally exhibit cytostatic effect in various hyperproliferative disorders, including localized and/or metastatic cancers lymphomas and carcinomas), benign prostate hyperplasia, and keratoses. While the inventors found substantial biological effects on IL-4 (a Type 2 cytokine) and IFNy (a Type 1 cytokine), it is generally contemplated that the compounds according to the inventive subject matter are biologically active in modulation of cytokines other than IL-4 and IFN-y. It is especially 14 I Mw -3 Vi1 11 WO 01/27114 PCT/US00/22674 contemplated that the compounds may increase or decrease the expression/secretion of a particular cytokine or set of cytokines. Therefore, it is contemplated that compounds according to the inventive subject matter may modulate the immune system of an organism such that a more pronounced Type 1 or Type 2 response may be achieved. Consequently, it is contemplated that the compounds according to the inventive subject matter may be effective to reduce the titer of a virus in a living system by either direct action as inhibitor of a viral polymerase and/or indirectly by activating the immune system to a particular humoral or cellular response. It is further contemplated that compounds according to the inventive subject matter may also be useful in reducing a response of an immune system towards an allo- or xenograft by reducing the severity of the cellular reponse towards the allo- or xenograft.

Examples The following protocols describe an exemplary synthesis of various compounds according to the inventive subject matter and are intended only to illustrate but not to limit the inventive concept presented herein.

Preparation of methyl 2.3-O-isopropylidene-5(R,S)-C-ethynyl-fl-ribofuranoside (2b) To a stirred solution of methyl 4-C,5-O-didehydro-2,3-O-isopropylidene-P-D-ribofuranoside (Jones et al. Methods in Carbohydrate Chemistry Vol 1, pp 3 15 3 2 2 (1972), 4.00 g, 19.78 mmol) in anhydrous THF (20 mL) at -42 °C under argon was added dropwise ethynylmagnesium bromide (0.5 M in THF. 80 mL, 40 mmol). Upon addition, the resulting mixture was slowly warmed up to 0 °C (-90 min.). The reaction was quenched by adding ice g)/water (50 mL) and the mixture was stirred for 30 min. After neutralization with 10 aq.

acetic acid, the mixture was extracted with ethyl acetate twice. The combined organic layer was dried (Na 2 SO4) and concentrated. Chromatography on silica (ethyl acetate-hexanes 1:4) gave 3.48 g of the titled compound (R/S ratio 1:1) as a white solid. The following compounds were prepared in a similar fashion: Methyl 2,3-O-isopropylidene-5(R)-C-methyl-p-D-ribofuranoside (2a) was prepared from methyl 4-C,5-O-dihehydro-2,3-O-isopropylidene-p-D-ribofuranoside and ethylmagnesium bromide. Methyl 2,3-O-isopropylidene-5(R)-C-vinyl-P-D-ribofuranoside (2c) was prepared from methyl 4-C,5-O-dihehydro-2,3-O-isopropylidene-p-D-ribofuranoside and vinylmagnesium bromide. Methyl 5(R)-C-allyl-2,3-O-isopropylidene-p-D-ribofuranoside (2d) was prepared from methyl 4-C,5-O-dihehydro-2,3-O-isopropylidene-3-D-ribofuranoside and allylmagnesium bromide.

kwiw"W-yyaw ifiauynh wg-Vom VNA PMA La SMOMMOW, WO 01/27114 PCT/US00/22674 Preparation of methyl 2,3-O-isoproplidene-5-O-methanesulfonyl-5(R)-C-methyl-f-Dribofuranoside (16) To a stirred solution of methyl 2,3-O-isopropylidene-5(R)-C-methyl-p-D-ribofuranoside (2a, 7.24 g, 33.17 mmol) in anhydrous pyridine (50 mL) at 0 *C was added methanesulfonyl chloride (3.1 mL, 39.92 mmol). The resulting mixture was stirred at room temperature for 1 h, cooled to 0 OC, quenched by adding water (1.0 mL), and stirred at room temperature for 30 min.

The solvent was evaporated and the residue was dissolved in ethyl acetate, washed with brine three times, dried (Na 2

SO

4 and concentrated. Chromatography on silica (30% EtOAc in hexanes) gave 8.62 g of the titled compound 16 as a colorless syrup.

Preparation of methyl 2,3-O-isopropylidene-5-O-acetyl-5(S)-C-methyl-f-D ribofuranoside (17) A stirred suspension of methyl 2,3-O-isopropylidene-5-O-methanesulfonyl-5(R)-Cmethyl-p-D-ribofuranoside (16, 8.62 g, 29.1 mmol) and NaOAc (anhydrous, 3.5 g, 42.5 mmol) in anhydrous DMF (350 mL) was heated at 125 'C under argon for 4 days. The solvent was evaporated and the residue chromatographed on silica (25% EtOAc in hexanes) to give 4.0 g of the titled compound 17 as a white solid.

Preparation of methyl 2.3-O-isopropylidene-4-C-hydroxymethyl-f3-D-ribofuranoside (28) To a stirred solution of methyl 4-C,5-O-didehydro-2,3-O-isopropylidene--Dribofuranoside 1 (20.22 g, 100 mmol) in dioxane (380 mL) at 0 *C was added dropwise formaldehyde (37 solution, 76 mL) and then 2 M NaOH (188 mL). The resulting reaction mixture was stirred at room temperature for 20 h, cooled to 0 neutralized (10% acetic acid), concentrated (-50 and extracted with methylene chloride twice. The combined organic layer was dried (Na 2

SO

4 and concentrated to dryness. Chromatography on silica (4 methanol in chloroform) gave 20.2 g of the titled compound 28 as a white solid.

Preparation of methyl 2,3-O-isopropylidene-5-deoxy-p-D-ribofuranoside (8) To a stirred solution of methyl 2,3-O-isopropylidene-p-D-ribofuranoside (14.2 g, 70.0 mmol) in anhydrous pyridine (250 mL) at 10 OC was added in portions (over 30 min) ptoluenesulfonyl chloride (19.1 g, 100 mmol). The resulting mixture was stirred at room temperature for 18 h, cooled to o0C, quenched by adding water (5.0 mL), and stirred at room temperature for 30 min. The solvent was evaporated. The residue was dissolved in ethyl acetate, 16 ~~mi~inilru~ ~iiil,~:~:~VIMrYrii.illl.~.r,;~,9~n h~u *y~.u.luiumuu~~..~ I*I .~arrrriinr..uWh~lViiWI~FIII~II~ WO 01127114 PTUO/27 PCTIUSOO/22674 washed with brine three times. dried (Na 2

SO

4 and concentrated to dryness. Chromatography on silica (ethyl acetate-hexanes 1:3) gave 24.1 g of the titled compound as a white solid.

To a stirred suspension of LiAIH 4 (4.58 g, 120.5 mmol) in anhydrous diethyl ether (120 mL) was added methyl 2,3 -O-isopropylidene-5--p-toluenesulfonyl-p-D-ribofuranaside (13.1 g, 36.55 mmol) in diethyl ether-toluene 140 mL). The resulting mixture was refluxed for 22 h. cooled to room temperature. diluted with ethyl acetate (25 mL) quenched by adding water mL). The solvent was evaporated. The residue was dissolved in ethyl acetate. washed with brine three times, dried (Na S0 4 and concentrated to dryness. Chromatography on silica (ethyl acetate hexanes 1:3) gave 3.58 g of the titled compound as a colorless liquid.

Preparalion of methyli S(R)-C-allvl-S-O-benzoyl-2. 3-O-isopropylidene-/3-D-ribofuranoside (3d) To a stirred solution of methyl 5(R)-C-allyl-2.3-0-isoproplidene-P3-D-ribofuranoside (4.49 g, 18.38 mmol) in anhydrous pyridine (40 mL) at 0 'C was added benzoyl chloride (2.7 mL, 23.0 mmcl). The resulting mixture was stirred at room temperature for 18 h, cooled with ice, quenched by adding water (I mL), and stirred at room temperature for 30 min. The solvent was evaporated and the residue was dissolved in ethyl acetate, washed with brine three times.

dried (NaSO 4 and concentrated. Chromatography on silica (12% ethyl acetate in hexanes) gave 6.26 g of the titled compound 3d as a colorless syrup. The following compounds were prepared in a similar fashion: Methyl 5-O-benzoyl-5(R,S)-C-ethynyl-2,3 -O-isoproplidene-o3-D-ribofuranoside (3b, RIS ratio: 1: 1) from methyl 5(RS)-C-ethynyl-2.3-O-isoproplidene-3-D-ribofuranoside Methyl 4-C-benzoyloxymcthyl-5-O-bcnzoyl-2 ,3-O-isoproplidene-o3-D-ribofuranoside from methyl 2.3-O-isoproplidene-4-C-hydroxymethyl-3-D-ribofuranoside.

Preparation of methyl 5(R)-C-allyl-S-O-benzoyl-fi.D-ribofuranoside (4d) A solution of methyl 5(R)-C-allyI-5-O-benzoyl-2,3-O-isopropylidene-3-D-ribofuranoside (3d, 6.2 g, 17.8 mmol) in TFA-H 2 0 mixture 1) was stirred at 0 'C for 90 min and concentrated to dryness at 0 0 C. The residue was dissolved in methanol-toluene mixture (20 mL.

1: 1) and concentrated to dryness. Chromatography on silica (ethyl acetate -hexanes 1: 1) gave 3.70 g of the titled compound 4d as a white solid. The following compounds were prepared in a similar fashion: Methyl 5-O-benzoyl-5(R,S)-C-ethynyl-p-D-ribofuranoside (4b, R/S ratio: 1: 1) from methyl 5-O-benzoyl-5(R,S)-C-ethynyl-2.3-O-isopropylidene-p-D-ribofuranoside (3b).

WO 01/27114 PCT/US00/22674 Methyl 5-O-benzoyl-4-C-benzoyloxymethyl--D-ribofuranoside from methyl 5-O-benzoyl-4-Cbenzoyloxymethyl-2.3-O-isopropylidene-3-D-ribofuranoside.

Preparation oj'methyl S(R)-C-allyl-2,3, S-tri-O-benzoyl-P-D-ribofuranoside (Sd) To a stirred solution of methyl 5(R)-C-allyl-5-O-benzoyl-P-D-ribofuranoside (4d, 3.60mg, 11.68 mmol) in anhydrous pyridine (80 mL) at 0 OC was added benzoyl chloride mL. 25.84 mmol). The resulting mixture was stirred at room temperature for 18 h, cooled with ice, quenched by adding water (I mL), then stirred at room temperature for 30 min. The mixture was concentrated, diluted with ethyl acetate, washed with brine three times, dried (Na 2

SO

4 and concentrated to dryness. Chromatography on silica (15% ethyl acetate in hexanes) gave 5.3 g of the titled compound 5d as a colorless syrup. The following compounds were prepared in a similar fashion: Methyl 5(R,S)-C-ethynyl-2.3,5-tri-O-benzoyl-1-D-ribofuranoside (5b, R/S ratio: 1:1) from methyl 5-O-benzolyl-5(R,S)-C-ethynyl-p-D-ribofuranoside Methyl 4-C-benzoyloxomethyl-2,3.5-tri-O-benzoyl-f-D-ribofuranoside from methyl benzoyl-p-D-ribofuranoside.

Preparation of 1-O-methyl-2,3, S-tri-O-benzoyl-S(R)-C-vinyl-f3-D-ribofuranose (Sc) A solution of methyl 2,3-O-isopropylidene-5(R)-C-vinyl--D-ribofuranosise (2c, 1.0 g, 4.3 mmol) in a mixture of trifluoroacetic acid and water v/v, 11 mL) was strirred at 0 OC for 30 min and concentrated to dryness. The residue was dissolved in methanol and concentrated to dryness (3 times), then dissolved in pyridine and evaporated, and finally was dissolved in anhydrous pyridine (11 mL). To this solution was added benzoyl chloride (1.9 mL, 16 mmol).

The reaction mixture was stirred at 25 OC for 16 h and poured into ice water (20 mL). The mixture was extracted with dichloromethane (20 mL) and the organic layer was dried over sodium sulfate, and concentrated to dryness. The residue was chromatographed on silica ethyl acetate in dichloromethane) to give 1.0 g of the titled compound Sc as a syrup.

Preparation of 1-O-acetyl-2,3,5-tri-O-benzoyl-S(R)-C-allyl-D-ribofuranose (6d) To a stirred solution of methyl 5(R)-C-allyl-2,3,5-tri-O-benzolyl-p-D-ribofuranoside g, 7.74 mmol) in acetic acid (14 mL) and acetic anhydride (1.75 mL, 18.36 mmol) at 0 'C ~ir*~rovvi~~~i;r~o~~n~uui *nil~~\;l~7~rr nr~,.uvnlr* U~U~~II~UB~W:~ W001/27114 PTU0127 PCTIUSOO/22674 was added concentrated sulfuric acid (200 pL, 3.79 mmol in 4.0 mL of acetic acid). The resulting mixture was stirred at room temperature for 20 h, cooled to 0 diluted with cold ethyl acetate, washed with water, 5% aq. NaHCO 3 and then brine. dried (Na 2

SO

4 and concentrated. Chromatography on silica (ethyl acetate-hexanes 1:4) gave 2.82 g of the titled compound 6d (ctlf3 ratio: 1:2) as a colorless foam. The following compounds were prepared in a similar fashion: I -O-Acetyl-5(R,S)-C-ethynyl-2,3 .5-tri-O-benzolyl-J3-D-riboftiranose (6b, RIS ratio: 1: 1 and Wj3P ratio: 1:2) from methyl 5(R,S)-C-ethynyl-2,3,5-tri-O-benzolyl43-Dribofuranoside I -O-Acethyl-4-C-benzoyloxymethyl-2,3 ,5-tri-O-bcnzoyl-D-ribofuranose P ratio: 1:3) from methyl 4-C-bcnzoyloxymethyl-2,35-tri-O-benzoyl-p3-D-ribofuranoside. C-Methyl-I ,2,3,5-tetra-O-acctyl-p3-D-ribofuranose from methyl 2,3-O-isopropylidene-5(R)-Cmethyl-I3-D-ribofuranoside.

I .2,3,5-Tetra-O-acetyl-5(S)-C-methyl-D-ribofuranose 6a from methyl 5-O-acetyl-2.3-Oisopropylidene-5(R)-C-methyl-j3-D-ribofuranoside. 5-deoxy- 1,2,3 -tri-O-acetyl-D-ribofuranose 9 from methyl 5-O-acetyl-2,3-O-isopropylidene-p3-D-ribofuranoside. I -O-Acetyl-2,3 benzoyl-5(R)-C-vinyl-p-D-ribofuranose 6c from methyl 2,3,5-tri-O-benzoyl-5(R)-C-viny-o3-Dribofuranoside.

Preparation of 4-amino-6-bromo-S-cyano- 3, S-Iri-O-benzoyl-S(R)-C-ally-f3-Dribofuranosyl)pyrrolo[2. 3-d]pyrimidine A suspension of 4-amino-6-bromo-5-cyanopyrrolo[2,3-d]pyrimidine (Tolman et al. J.

Org. Chem. 1969. 91, 2102-2108, 1.05 g, 4.41 mmol) and ammonium sulfate (50 mg) in HMDS mL) and anhydrous m-xylene (25 mL) was refluxed under argon for 18 h. Solvents were evaporated and the residue was dried under vacuum, The residue was dissolved in anhydrous 1,2-dichloroethane (80 mL) and mixed with Il-O-acetyl-2,3,5-tri-Q-benzoyl-5(R)-C-allyl-Dribofuranose (2.00 g, 3.67 mmol). Under cooling with ice, TMSOTf (1.3 mL, 7.30 mmol in mL of anhydrous 1 ,2-dicbloroethane) was added. The mixture under argon was stirred at room temperature for 30 min, then refluxed for 90 h, quenched by pouring it (cold) onto ice/NaHCO 3 mL), and filtered. The organic layer was separated, dried (Na 2

SO

4 and concentrated.

Chromatography on silica (EtOAc-hexanes 2:3) gave 1.81 g of the titled compound as a colorless solid. The following compounds were prepared in a similar fashion: 4-Amino-6bromo-5-cyano-7-(2,3,5-tri-O-benzoyl-5(R,S)-Cethynyl-p-D-ribofuranosyl)pyrrolo[2,3dlpyrimidine (R/S ratio: 1: 1) was prepared from I -O-acethyl-2,3,5-tri-O-benzolyl-5(R,S)-C- 1AY&MINI bit lljj Wlw t. WO 01/27114 PTUOI27 PCTIUSOO/22674 ethynyl-D-ribofuranose and 4 -amnino-6-bromo-5-cvanopyrrolo[2,3 -dlpyrimidine. 4-amino-6bromo-5-cyano.-7-(4-enzoyloxomethyl-2,3 ,5-tri-O-benzoyl-p-D-ribofuranosyl)pyrrolo[2,3d~pyrimidine was prepared from 1 -O-acetyl-4-benzoyloxymnethyl-2,3 ribofuranose and 4 -amino-6-bromo-5-cyanopyrrolo[2,3-djpyrimidine. 4-Amino-6-bromo-5cyano-7-(1I, 2 3 ,5-tetm-O-acethy1-5(R)-C-methy1-p3-D-ribofuaosyl)pyrrolo[2,3-d]pyrimidine was prepared from 1 ,2,3,5-tetra-O-acethvl-5(R)-C-methyl-D-ribofuranose and 4-amino-6bromo-5-cyanopyrrolo[2,3-d]pyrimidine. 4-Amino-6-bromo-5-cyano-7-(l ,2,3,5-tetra-Oacetyl-5(S)-C-methyl-13-D-ibofuranosyl)pyrrolo[2,3-dlpyrimidine was prepared from 1.2,3,5and 4-amino-6-bromo-5-cyanopyrrolo[2,3d4pyrimidine. 4-Amino-6-bromo-5-cyano-7-(2,3 -di-O-acetyI-5-deoxy-p3-Dribofuranosyl)pyrrolo[2,3-dlpyrimidine was prepared from 1 .2,3-tri-O-acetyl-5-deoxy-Dribofuranose and 4-amino-6-bromo-5 -cyanopyrrolo[2,3-d4pyr-imidine. 4-Amino-6-bromo-5cyano- 7 2 3 .5-tri-O-benzoyI-5(R)-C-vinyl-f-D.ribofuranosyl)pyrroo[2,3dlpyimidine was prepared from 1 -O-acetyl-2,3,5-tri-O-benzoyl.5(R)-C-vinyl.j3.D-rjbofuranose and 4-amino-6bromo-5-cyanopyrrolo[2,3-d]pyrimidine.

Preparation of 4-amino-5-cyano- 3, S-tri-O-benzoyl-S(R)-C-aly-3-Dribofuranosyl)pyrrolo[2 3-dipyrimidine To a solution of 4-amino-6-bromo-5-cyano-7-(2,3 ,5-tri-O-benzoyl-5(R)-C-allyl-o3-Dribofliranosyl)pyrrolo[2,3-djpyrimidine (738 mg 1.0 mmol) in acetic acid (25 mL) was added zinc dust (1.04 g, 16.0 mmol) in two portions (one hour apart). The reaction mixture was stirred at room temperature for 20 h and filtered. The filtrate was evaporated to dryness and the residue chromatographed on silica (ethyl acetate-hexanes 1: 1) to give 450 mg of titled compound 20e as a colorless foam. The following compounds were prepared in a similar fashion: cyano- 7 2 3 ,5-tri..O.benzoyl.5(R,S).CethynylpDribofuraosyl)pyrrolo[23djpyrimidine (R/S ratio: 1: 1) 20b from 4 -amino-6-bromo-5-cyano-7-(2,3,5-ti-O-benzoyl-5(R,S)-C-ethyny-p3- D-ribofuiranosyl)pyrrolo[2,3-d]pyrimidine. 4-Amino-5-cyano-7-(2,3,5-tri-O-benzoyl-5(R)-Cvinyl-P-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine 20c from 4-amino-6-bromo-5-cyano-7-(2,3,5tri-O-benzoyl-5(R)-C-vinyl-p-D-ribofuranosyl)pyrrolo[2,3-djpyrimidine.

Preparation of 4-amino-S-cyano- ribofuranosyl)pyrrolo[2, 3-dipyrimidine WO 01/27114 WO 0127114PCTIUSOO/22674 A suspension of 4-amino-6-bromo-5-cyano-7-(2,3 ,5-tri-O-benzoy1,-5(R)-C-aIlvl- P-Dibofuranosyl)pyrrolo[2,3-dJpyrimidine (400 mg, 0.54 mmol) and 10% Pd/C (100 mg, wvater) in dioxane (50 mL) and triethylamine (0.5 mL) was shaken in hydrogenation apparatus

(H

2 20 psi) for 4 h. The catalyst was filtered and washed (dioxane). The combined filtrate was concentrated and the residue chromatographed on silica (ethyl acetate-hexanes 1: 1) to give 340 mg of the titled compound 20f as a colorless foam. The following compounds were prepared in a similar fashion: 4-Amino- 5-cyano-7-(2,3 ,5-tri-O0-benzoyl -5 -ethyl P-Dribofuranosyl)pyrrolo[2,3-dlpyfimidine (R/S ratio: 1: 1) 20d from 4-amino-6-bromo-5-cyano-7- (2.3 ,5-tri-O-benzoyl-5(R,S)-C-ethynyl-p-D-ribofuranosyl)pyrrolo[2,3-d] pyrimidine. cyano-7-(4-benzoyloxomethyl-2,3.S-tri-O-benzoy-13-D-ribofuranosyl)pyrrolo[2,3-dlpyrimidine from 4-amino-6-bromo-5-cyano-7-(4-benzoyloxomethyl-2,3 ribofuranosyl)pyrrolo[2.3-d] pyrimidine. 4-Amlino-5-cyano-7- (1,2.3 ).5-tetra-0-acethyl-5(R)-Cmethyl-fl-D-ribofuranosyl)pyrrolo[2,3-djlpyrimidine 20a from 4-Amino-6-bromo-5-cvano-7- (1,2,3 ,5-tetra-O-acethyl-5(R)-C-methyl-p-D-ribofuranosyl)pyrrolo[2.3-dJpyrimidine. 4-Amino- 5-cyano- 1,2,3 ,5-tetra-O-acetyl-5(S)-C-methyl-p-D-ri bofuranosyl )pyrrolo[2.3 -dfpyri mi dine from 4-Amino-6-bromo-5-cyano-7-(1I,2.3.5-tetra-0-acethyl-5(S)-C-methyl-o-Dribofuranosyl)pyrrolo[2,3 -dfpyrimidine. 4-Amino-S-cyano-7-(1I.2,3-tri-O-acethyl-5-deoxy-o-Dribofuranosyl)pyrrolo[2,3-djpyrimidine from 4-amino-6-bromo-5-cyano-7-(1I,2.3-tri-O-acethyl- 5-deoxi-p-D-ribofuranosyl)pyrrolo[2,3)-dfpyrimidine. 4-Amino-5-cyano-7-(2,3 -dideoxy-3-Dglicero-pentofuranosyl)pyrrolo[2,3 pyrimidine was prepared from 4-amino-5-cyano-7-(2,3 dideoxy-1-D-pent-2-enofuranosvl)pyrrolo[2.3-dflpyrimidine.

Preparation of 4-amino-S -cyano- 7-('S'(R)-C-allyl-f-D-ribofrtranosyl~pyrrolo[2 3-dipyrimidine (23e) A solution of 4-amino-5-cyano-7-(2,3.5-tri-O-benzoyl-5(R)-C-allyl-p-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine (300 mg, 0.454 mmol) in methanol (40 mnL) at 0 *C was saturated with ammonia. The solution stood at room temperature for 2 days. Solvent was evaporated and the residue together with NaOAc (anhydrous, 20 mg) was suspended in DMF (20 mL). The mixture was stirred under argon at 120 *C for 5 h. Solvent was evaporated. The residue was adsorbed onto silica gel and eluted from silica gel column (methanol-ethyl acetate 1:25) to give 145 mg of the titled compound as a colorless solid.

WO 01/27114 PTU0127 PCT[tJSOO/22674 Before heating in DMF. the product contained two major compounds 21 and 23. which coulde be separated by chromatography on silica gel. Compounds 21 were prepared through this procedure. The following compounds were prepared in a similar fashion: 4-Amino-5-cyano-7- (5(R)-C-propy1-p-D-ribofuranosyl)pyrrolo[2.3-d~pyrimidine 23f from 4-amino-5-cyano-7-(2,3.5tri-O-benzoyl-5(R)-C-propyl-fp-D-ribofuranosyl)..pyrrolo[2,3-d~pyrimidine. 4-Amino-5-eyano-7- (5(RS)-C-ethynyl-f3-D-ribofuranosyl)pyrrolo[2.3 -d]pyrimidine (R-S ratio: 1: 1) 23b from 4-amino-S -cyano-7-(2,3 ,-tri-O-benzoyl-5(R.S)-C-ethynyl-p-D-ribofuranosvl)pyrolo[2.3.dlpy..

imidine. 4-mn--yno7( 3.C-ty---ibfrns.pyrl[,)d]pyrimidine (R-S ratio: 1: 1) 23d from 4 -amino-5-cyano-7-(23,5-triObenzoyI(R,S)-CethypDribofuaosyl)pyrrolo[2,3 -dlpyrimidine. 4 -Amino-5-cyano-7-(4-hydroxymethyl-p-D-ribofuranosyl)pyrrolo- [2,3-d~pyrimidine 33d from 4-mn--yn--4bnolxmehl235tiObnolpD ribofuranosyl)pyrrolo[2,3-dlpyrimidine. yl)pyrrolo[2,3 -dfpyrimidine 23a(5'-R) from 4-amino-5-cyano-7-( 1,2,3,5-tetra-O-acethyl-5(R)-Cmethyl-o-D-ribofuranosyI)pyrrolo[2,3-d]pyrimidine. 4-Amino-5-cyano-7-(5(S)-C-methyl-3-Dribofuranosyl)pyrrolo[2,3-dlpyrimidine 23a(5'-S) from 4-amino-5-cyano-7-( 1,2,3.5-tetra-Oacethyl-5(S)-C-methyl-13-D-ribofuranosyl)pyrrolo [2.3)-d~pyrimidine. 4-Amino-5-cyano-7-(5deoxy-p-D-ribofuranosyI)pyrrolo[2.3-dlpyrimidine 10 from 4-amino-5-cyano-7-( 1,2.3-tri-Oacethyl-5-deoxi-I3-D-ribofuranosyl)pyrrolo[2,3)d4pyrimidine. 4-Amino-5-cyano-7-(5(R)-Cvinyl-j3-D-ribofuranosyl)pyrrolo[2,3)-djpyrimidine 23c from 4-amino-5-cyano-7-(2,3 bezy-()CvnloDrbfrnslproo[.-lyiiie Preparation of 4-aniino-5-cyano- ribofuranosyl)pyrrolo[2. 3-dipyrimidine To a stirred solution of toyocamicin 43 (5.83 g, 20.0 mmol) in anhydrous pyridine (100 mL) at 0 0 C was added tert-butylchlorodiphenylsilane (6.2 mL, 24.0 mmol). The resulting mixture was stirred at room temperature for 18 h and then cooled to O 0 C, and methanesulfonyl chloride (3.4 mL, 44.0 mmol) was added. The resulting mixture was stirred at room temperature for 2 h, cooled with ice, quenched by adding water (2 mL), and stirred at room temperature for min. The solvent was evaporated. The residue was dissolved in ethyl acetate, washed with brine three times, dried (Na 2

SO

4 and concentrated. Chromatography on silica (ethyl acetatehexanes 3:2) gave 8.41 g of the titled compound as a colorless solid.

WO 01/27114 WOO1/7114PCT/USOO/22674 Preparation of 4-amino-5-cyano- 7-(5-O-tert-butvldiphenvlsilyl-2 .3-didehydro-2. 3-dideoxy-/3-Drib ofuranosyl)pyrrolo[2.3 -dipyrimidin e Tellurium powder (200 mesh. 640 mg 5.0 mmol) under argon was sealed, mixed with lithium triethylborohydrate (1.0 M in THF, 11.25 mL. 11.25 mmol). The mixture was stirred at room temperature for 6 h and then cooled to 5 and 4-amino-5-cyano-7-(2.3 -di-Olyl-o-D-ribofuranosy1)pyrrolo[2.3)-dApyrimidine (1.40 g, 2.09 mmol) in THF (12 mL) was added. The resulting mixture was stirred at room temperature for 18 h, cooled with ice. quenched by adding water (0 0 C. 5 mL). and stirred at room temperature for 30 min. Solvent was evaporated and the residue extracted with ethyl acetate. The extracts were concentrated and the residuc chromatographed on silica (15% ethyl acetate in hexanes) to give 640 mg of the titled compound as a colorless foam.

Preparation of 4-amino-S-cvano-l 712, 3-didehvdro-2. 3-dideoxy-/3-D- ribqfuranosyl)pyrrolof2, 3djpyrimidine (49) To a stirred solution of 4-amino-5-cyano-7-(5-O-ieri-butyldiphenylsilyl-2.3 -didehydro- 2,3-dideoxy-o-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine (2.55 g, 5.32 mmol) in anhydrous THIF (100 mL) at 5 0 C was added terabutylammonium fluoride (1.0 M in THF. 6.6 mL). The resulting mixture was stirred at room temperature for 3 h and concentrated. Chromatography on silica (6% methanol in ethyl acetate) gave 1.09 g of the titled compound 49 as a colorless solid.

Preparation of S-cvano- 7(5 (R)-C-melhvl-I3-D-ribofuranosyl)pyrrolo[2, 3-d]-4-pyrimidone To a stirred solution of 4-amino-5-cyano-7-(5(R)-C-methyl-p-D-ribofuranosyl )-pyrrolo- [2,3-dlpyrimidine (306 mg, 1.0 mmol) in water (30 mL) and acetic acid (2.0 mL) at 55 'C was added in portions sodium nitrite (590 mg, 8.55 mmol). The resulting mixture was stirred at C for 3 ht and more sodium nitrite (300 mg, 4.30 mmol) was added. The mixture was stirred at same temperature for additional 18 h. Solvent was evaporated and the residue chromatographed on silica (12 methanol in methylene chloride) to give 2 10 mg of the titled compound 25a(5'-R) as a colorless solid. Similarly, the following compounds were prepared: (2,3-d]-4-pyrimidone 25a(5'-S) from 4-amino-5-cyano-7-(5(S)-C-methyl-o-D-ribofuranosyl)-pyrrolo[2,3 -dlpyrimidine. cyano-7-(P-D-arabinofuranosyl)pyrrolo[2,3-dJ-4-pyrimidone 58 from 4-amino-5-cyano-7- (5-deoxi-p-D-arabinofuranosy)pyrrolo[2,3-d]pyrimidine. 4-Amino-5-cyano-7-(5 -deoxy-3-Dribofuranosyl)pyrrolo[2,3-d]-4-pyrimidone 11 from 4-amino-5-cyano-7-(5-deoxy-o3-Dt* WAAVY AMW "A I'll. I~M 11-A 1 11 h IM 1AA W III A 'l WO 01/27114 WO 0127114PCTIUSOOI22674 ribofuranosyl)pyrrolo[2,3-d]pyrimidine. 4-Amino-5-cyano-7-(2,3-dideoxy-2,3-didehydro-o3-Dglycero-pento-furanosyl) pyrrolo[2,3 -d-4-pyrimidone 50 from 4-amino-5-cyano-7-(2,3-dideoxy- 1-D-pent-2-enofuranosyl)pyrrolo-[2,3.dlpyrimidine. 4-Amino-5-cyano-7-(2,3-dideoxy-p3-Dglycero-pentofuranosyl)pyrrolo[2,3-d]-4-pyrimidone 65 from 4-amino-5-cyano-7-(2,3 -dideoxy-P -D-glycero-pentofuranosyl)pyrrolo[2,3-dlpyrimidine. 4-Amino-5-cyano-7-(2-deoxy-3-Dfuranosyl)pyrrolo[2,3-d]-4-pyrimidone 69 from 4-amino-5-cyano-7-(2-deoxy-p-Deritropentofuranosyl)pyrrolo[2,3-dlpyrimidine.

Preparation of 7-(S(R)-C-methyl-fl-D-ribofuranosyl)pyrrolo[2, 3-d]-4-pyrimidone-5carboxamidoxime (24a) A stirred suspension of 5-cyano-7-(5(R)-C-methyI-p-D-ribofuranosyl)pyrrolo[2,3-d]4pyrimidone (240 mg, 0.784 mmol), hydroxylamine hydrochloride (163 mg, 2.352 mmol), and potassim carbonate (162 mg, 1. 176 mmol) in ethanol (50 mL) was refluxed under argon for 18 h.

Preciptate was filtered and washed with warm ethanol. The filtrate was concentrated and the residue chromatographed on silica (20 methanol in methylene chloride) to give 170 mg of the titled compound 26a(5'-R) as a colorless solid. Similarly, the following compounds were prepared: 4 -Amino-5-cyano-7-(P-D-arabinofuranosyl)pyrrolo[2,3-d-4-pyrimidone-5carboxamidoxime 60 from 4-amino-5-cyano-7-(5-deoxi-p1-D-arabinofuranosy)-pyrrolo[2,3-d]-4pyrimidone.

4-Amino-5-cyano-7-(5-deoxy-3-D-ribofuranosyl)pyrrolo[2,3-dI-4-pyrimidone-5-carboxamidoxime 13 from 4-amino-5-cyano-7-( 5-deoxy-p-D-ribofuranosy1)pyrrolo[2,3-d]-4pyrimidone. 4-Amino-5-cyano-7-(2,3-didehydro-2.3-dideoxy-3-D-ribofuranosyl)pyrrolo[2,3-d- 51 from 4-amino-5-cyano-7-(2,3 -didehydro-2,3-dideoxy-p-Dribofuranosyl)pyrrolo[2,3-dJ-4-pyrimidone. 4-Amino-5-cyano-7-(2-deoxy-p3-Dribofuranosyl)pyrrolo[2,3-d]-4-pyrimidone-5-carboxamidoxime from 4-amino-5-cyano-7-(2deoxy-1-D-ribofuranosyl)pyrrolo[2,3-d]-4-pyrimidone.

Preparat ion of 7-(5(R)-C-methyl-f-D-ribofuranosyl)pyrrolo[2, 3-d]-4-pyrimidone-5carboxamidine hydrochloride (27a) A suspension of 7-(5(R)-C-methyl-Ip-D-ribofuranosyl)pyrrolo[2,3 carboxamidoxime (110 mg, 0.324 mmol), ammonium chloride (20 mg, 0.374 mmol), and Raney nickel (50% slurry in water, 200 mg) in water (75 mL) was shaken in a hydrogenation apparatus

(H

2 50 psi) at room temperature for 18 ht. The catalyst was filtered and washed (warm water).

%VVIA4 W a, JA,( I AM CK I 1 V .A oooo i~.AfJ~ 1, ,MOW W W O WO 01/27114 PCT/US00/22674 The combined filtrate was concentrated and the product was recrystallized from methanol to give 100 mg of the titled compound 27a(5'-R) as a colorless solid. The following compounds were prepared in similar fashion: 4 -Amino-5-cyano-7-(p-D-arabinofuranosyl)pyrrolo[2,3-d]-4hydrochloride 63 from 4-amino-5-cyano-7-(5-deoxi--Darabinofuranosy)pyrrolo[2,3-d-4-pyrimidone-5-carboxamidoxime. 4-Amino-5-cyano-7-(5deoxy-P-D-ribofuranosyl)pyrrolo[2,3-d]-4-pyrimidone-5-carboxamidine hydrochloride 15 from 4-amino-5-cyano-7-(5-deoxi-p-D-ribofuranosyl)pyrrolo[2,3-d]-4-pyrimidone-5carboxamidoxime. 4 -Amino-5-cyano-7-(2-deoxy-3-D-ribofuranosyl)pyrrolo[2,3-d]-4hydrochloride 70 from 4-amino-5-cyano-7-(2-deoxy-P-Dribofuranosyl)pyrrolo[2,3-dJ-4-pyrimidone-5-carboxamidoxime.

Thus, specific embodiments and applications of pyrrolo[2,3-d]pyrimidine nucleoside analogs have been disclosed. It should be apparent, however, to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. The inventive subject matter, therefore, is not to be restricted except in the spirit of the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms "comprises" and "comprising" should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements.

components, or steps that are not expressly referenced.

W 02 iiir.saa. .J.,Jir

Claims (5)

1. A nucleoside analog according to formula x z x Y N N R R 5 A R 3 R 2 wherein A is O, S, or CH 2 X is H, NH 2 or OH; Y is H, halogen or NH 2 Z is selected from the group consisting of H, halogen, R, OH, OR, SH, SR, NH 2 NHR, NR 2 CN, C(O)NH 2 COOH, COOR, CH 2 NH 2 C(=NOH)NH 2 and C(=NH)NH 2 where R is alkyl, alkenyl, alkynyl, or aralkyl; R 2 and R 3 are independently selected from the group consisting of H, F, and OH; R 4 is selected from the group consisting of a hydrogen, an alkyl, an alkenyl, an alkynyl, and an aralkyl, wherein R 4 optionally has at least one of a heteroatom and a functional group; R 5 is H, OH, OP(O)(OH) 2 P(O)(OH) 2 OP(O)(OR') 2 or P(O)(OR') 2 wherein R' is a masking group; and Rs' is selected from the group consisting of an alkyl, an alkenyl, an alkynyl, and an aralkyl, wherein R 5 s has at least two carbon atoms, and optionally has at least one of a heteroatom and a functional group.

2. The nucleoside analog of claim 1 wherein Z is CN, C(O)NH 2 or C(=NH)NH 2 and wherein R 5 s has at least two carbon atoms and is selected from the group consisting of an alkyl, an alkenyl, an alkynyl, and an aralkyl. 0 00 .Ak da1 -4 mg, p\OPER\Kbml2500227 clims doc-24/11/03 -27-

3. The nucleoside analog of claim 1 having the structure 9 z wherein Z is CN, C(O)NH 2 C(=NH)NH 2 or C(=NOH)NH 2 and R 4 is selected from the group consisting of a hydrogen, an alkyl, an alkenyl, an alkynyl, and aralkyl, and R 5 is selected from the group consisting of an alkyl, an alkenyl, and alkynyl and an aralkyl, wherein R 4 and Ry independently and optionally contain at least one of a heteroatom and a functional group.

4. A method of changing secretion of a cytokine from a cell, comprising; providing a compound according to formula (la); z (la) p 0 o p *00* 0 wherein A is O, S, or CH 2 X is H, NH 2 or OH; Y is H, halogen or NH 2 Z is selected from the group consisting of H, halogen, R, OH, OR, SH, SR, NH 2 NHR, NR 2 CN, C(O)NH 2 COOH, COOR, CH 2 NH 2 C(=NOH)NH 2 and C(=NH)NH2, where R 25 is alkyl, alkenyl, alkynyl, or aralkyl; R 2 and R 3 are independently selected from the group consisting of H, F, and OH; *P Pp

604.WA41, AW AdT.WMIZt- (uu UOf /LL674 Attorney Reference No.: 330.48-PCT s 2 6 4 1PEAIUS Z-6 MAR 2oo R 4 and Rs 5 are independently selected from the group consisting of a hydrogen, an alkyl, an alkenyl, an alkynyl, and an aralkyl, and wherein R 4 and Rs independently and optionally contain at least one of a heteroatom and a functional group; R 5 is H, OH, OP(O)(OH) 2 P(O)(OH) 2 OP(O)(OR') 2 or P(O)(OR') 2 wherein R' is a masking group; and presenting the cell with the compound at a concentration effective to change the secretion of the cytokine. The method of claim 4 wherein the cytokine is a Type 1 cytokine. 6. The method of claim 5 wherein the Type 1 cytokine is IFNy. 7. The method of claim 4 wherein the cytokine is a Type 2 cytokine. 8. The method of claim 7 wherein the Type 2 cytokine is IL-4. 9. The method of claim 4 wherein the cell is a lymphocyte. The method of claim 4 wherein the cell is a cancer cell. 11. The method of claim 10 wherein the cancer cell is a prostate cancer cell. 12. A method of changing secretion of a cytokine from a cell, comprising: providing a compound according to formula (III) N N (III) R O HO OH wherein Z is CN, C(O)NH 2 C(=NH)NH 2 C(=NNH 2 )NH 2 or -C(=NOH)NH 2 0 28 AMENDED SHEE1 NA AW5Wv VYUW.AZI M LI vlw lkat7 P \OPERU\b.\250022 7 do.24/I 1103 -29- wherein Rs is H, OH, OP(O)(OH) 2 P(O)(OH) 2 OP(O)(OR') 2 or P(O)(OR') 2 wherein R' is a masking group; and presenting the cell with the compound at a concentration effective to change the secretion of the cytokine. 13. A method of reducing growth of a hyperproliferative cell, comprising: providing a compound according to formula (IV); x z Y N (IV) R' A R 3 R, wherein A is O, S, or CH 2 X is H, NH 2 or OH; Y is H, halogen or NH 2 Z is selected from the group consisting of H, halogen, R, OH, OR, SH, SR, NH 2 NHR, NR 2 CN, C(O)NH 2 COOH, COOR, CH 2 NH 2 C(=NOH)NH 2 and C(=NH)NH 2 where R alkyl, alkenyl, alkynyl, or aralkyl; R 2 and R 3 are independently selected from the group consisting of H, F, and OH; R 4 is selected from the group consisting of a hydrogen, an alkyl, an alkenyl, an alkynyl, and an aralkyl, wherein R 4 optionally contains at least one of a heteroatom and a functional group; Rs is selected from the group consisting of an alkyl, an alkenyl, an alkynyl, and an aralkyl, wherein R 5 has at least two carbon atoms, and optionally contains at least one of a heteroatom and a functional group; R 5 is H, OH, OP(O)(OH) 2 P(O)(OH) 2 OP(O)(OR') 2 or P(O)(OR') 2 wherein R' is a masking group, and presenting the hyperproliferative cell with the compound at a fIIIi* ~t I~iVWU P:\OPER\Kbml2500227.claims.doc-24/ 103 concentration effective to reduce the growth of the hyperproliferative cell. 14. The method of claim 13 wherein the hyperproliferative cell is a cancer cell. 15. The method of claim 14 wherein the cancer cell is a prostate cancer cell. 16. The method of claim 13 wherein the reduction of growth comprises reduction of DNA synthesis. 17. A method of reducing a release of a growth factor from a cell, comprising; providing a compound according to claim 1; and presenting the cell with the compound at a concentration effective to reduce the release of the growth factor. 18. The method of claim 17 wherein the growth factor is VEGF. 19. The method of claim 17 wherein the cell is a cancer cell. 20. The method of claim 19 wherein the cancer cell is a prostate cancer cell. 21. A nucleoside analog according to claim 1, substantially as hereinbefore described with reference to the Examples. 22. A method of changing secretion of a cytokine from a cell according to claim 4 or claim 12, substantially as hereinbefore described with reference to the Examples. 23. A method of reducing growth of a hyperproliferative cell according to claim 13, substantially as hereinbefore described with reference to the Examples. a a* ~ir~~w~trrrLu P:\OPER\Kbm2500227.clims.doc-24/l 1/03 -31 24. A method of reducing release of a growth factor from a cell according to claim 17, substantially as hereinbefore described with reference to the Examples. DATED this 2 4 th day of November, 2003 ICN Pharmaceuticals, Inc. By DAVIES COLLISON CAVE Patent Attorneys for the Applicants ooo o°° o '~'*~"'lwcninr;u ni~zi V" "i~R""~".:uilYiLl?~