SK1772002A3 - Nucleoside analog, method of change of cytokine secreting from a cell and method for inhibiting the growth of hyperproliferative cells - Google Patents

Abstract

Compositions and methods for pyrrolo[2,3-d]pyrimidine nucleoside analogs having substituents at the C4' and C5' positions of the ribofuranose moiety are presented. Contemplated compositions exhibit, among other things, anti-cancer and immunomodulating effects at reduced cytotoxicity.

Description

Nucleoside analogue, method of altering cytokine secretion from a cell and method of reducing hyperproliferative cell growth

Technical field

The invention relates to nucleoside analogues.

BACKGROUND OF THE INVENTION

Nucleoside analogs have long been used as antimetabolites for the treatment of cancer and viral infections. Upon entry into a cell, nucleoside analogs are often phosphorylated by nucleoside pathways, in which the analogs are typically phosphorylated to the corresponding di- and triphosphates. In addition to other intracellular sites, triphosphorylated nucleoside analogs are often used as substrates for DNA and RNA polymerases and are subsequently incorporated into DNA or RNA. Where triphosphorylated nucleoside analogues are potent polymerase inhibitors, they may cause premature termination of the nascent nucleic acid molecule. Where triphosphorylated nucleoside analogs are inserted into nucleic acid replicators or transcripts, they may result in gene expression or impaired function.

At the multicellular level, nucleoside analogs may also affect the cell cycle, and in particular the desired effect of nucleoside analogs is the induction of apoptosis of tumor cells. In addition, nucleoside analogs are also known for their ability to alter certain immune responses.

Various nucleoside analogs with relatively potent anticancer activity are known in the art. For example, known drugs include thymidylate synthesis inhibitors such as 5-fluorouridine, adenosine deaminase inhibitors, including 2-chloroadenosine, and neplanocin A, which is a Sadenosylhomocysteine hydrolase inhibitor. However, all or nearly all known nucleoside analogues also pose a threat to normal mammalian cells, and in particular because the nucleoside analogue lacks sufficient selectivity between normal cells and tumor cells.

Unfortunately, the lack of this sufficient selectivity is often associated with various side effects and thus often limits the effect of such analog drugs.

Although many nucleoside analogs are known in the art, all or nearly all have one or more disadvantages. Therefore, there is still a need to provide improved methods and compositions of nucleoside analogs.

SUMMARY OF THE INVENTION

The present invention describes nucleoside analogs with changes in the sugar motifs of pyrrolo (2,3-d) pyrimidine nucleoside analogs that can greatly reduce the toxicity of nucleoside analogs to mammalian cells while also providing considerable cellular toxicity to tumor cells. These changes include, but are not limited to, the C4 'and C5' positions of ribofuranose motifs. The present invention also shows that certain pyrrolo (2,3-d) pyrimidine nucleoside analogs have the desired immunomodulatory effects, including enhancement of type 1 cytokines such as IL-2 and suppression of type 2 cytokines such as IL-4. These immunomodulatory properties can be used in anticancer and antiviral protection, in autoimmune diseases, in the treatment of inflammation and in preventing graft rejection.

In one aspect, the nucleoside analog is a pyrrolo (2,3-d) pyrimidine nucleoside having the structure of Formula I:

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 _? NHR, NR ^; CN, C (O) NH-, COOH, COOR, CH2NH ;, C (= NOH) NH-, and C (= NH) NH2, wherein R is alkyl, alkenyl, alkynyl or aralkyl; R 'and R' are independently selected from the group consisting of H, F and OH; R ⁴ is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, and aralkyl, wherein R ⁴ preferably has at least a heteroatom or 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 masking group; and R ⁵ 'is selected from the group consisting of alkyl, alkenyl, alkynyl and aralkyl, wherein R ⁵ ' has at least two carbon atoms and preferably has at least a heteroatom or functional group.

In another aspect, the nucleoside analog is a pyrrolo (2,3-d) pyrimidine nucleoside having the structure of Formula II:

wherein Z is CN, C (O) NH ₂ , C (= NH) NH ₂ or C (= NOH) NH ₂ and R ⁴ and R ⁵ 'are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl and aralkyl, wherein R ⁴ and R ⁵ 'independently and preferably contain at least a heteroatom or a functional group; with the proviso that R ⁴ and R ⁵ 'are not together hydrogen.

In another aspect of the invention, the contemplated ingredients are used to inhibit tumor growth or alter type 1 and type 2 cytokines and produce chemokines.

The various objects, features, aspects and advantages of the present invention will become more apparent from the following detailed description of preferred embodiments of the present invention, together with the accompanying drawings.

The pyrrolo (2,3-d) pyrimidine nucleoside analogs of formulas I and II have been found to have different biological effects on normal and hyperproliferative cells.

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 _Z 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, wherein 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 hydrogen, alkyl, alkenyl, alkynyl, and aralkyl, wherein R ⁴ preferably has at least a heteroatom or 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 masking group; and R ⁵ 'is selected from the group consisting of alkyl, alkenyl, alkynyl and aralkyl, wherein R ⁵ ' has at least two carbon atoms and preferably has at least a heteroatom or functional group.

In particular, it should be noted that the terms alkyl, alkenyl, alkynyl and aralkyl as used herein refer to both linear and branched species. In view of the substituents R ² and R ^3, it should be appreciated that both R ² and R ³ can be independently directed to the α- or β-side. In addition, where the substituents on C5 'are not the same, substitution at C5 may result in a R or S-asymmetric center. The term heteroatom, as used herein, refers to carbon atoms in an organic molecule, and in particular, heteroatoms include halogens, nitrogen, oxygen, and sulfur. As used herein, a functional group refers to a reactive bond (e.g., a double or triple bond) or a reactive group (e.g., -OH, -SH, -NH ₂ , -N ₃ , -CN, COOH, -CHO, -CONH _2, and the like) .

Particularly contemplated pyrrolo (2,3-d) pyrimidine nucleoside analogs are those of Formula I wherein Z is CN, C (O) NH _? or C (= NH) NH ₂ and wherein R ⁵ 'has at least two carbon atoms and is selected from the group consisting of alkyl, alkenyl, alkynyl and aralkyl.

The pyrrolo (2,3-d) pyrimidine nucleoside analogue of formula II has the following structure:

wherein Z is CN, C (O) NH ₂ , C (= NH) NH ₂ or C (= NOH) NH ₂ and R ⁴ and R ⁵ 'are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl and aralkyl, wherein R ⁴ and R ⁵ 'independently and preferably contain at least a heteroatom or a functional group; with the proviso that R ⁴ and R ⁵ 'are not together hydrogen; and wherein the increased substituents are as defined in Formula I.

However, it should be appreciated that the compounds of the present invention may also be in a different form and expression as described above, and in particular the contemplated forms include a prodrug form or otherwise altered form in which the molecules considered are chemically and / or enzymatically altered in order to improve the pharmacological and / or pharmacodynamic properties, which include a higher specificity for the target organs, cells or cellular parts and which increase the half-life in the body.

For example, cholesterol junctions may be established to increase the target specificity towards the liver, or apolipoprotein junctions that would increase the penetration of the altered drug through the blood brain barrier to the brain. In another example, ligand receptor complexes may be synthesized to target the altered drug to particular cells having a specific ligand receptor.

Alternatively, antibody complexes or antibody fragments may be formed that increase the selective targeting of the altered drug to the cell portions. Many drugs and altered forms are known in the art, and particularly the contemplated prodrug forms include those described in US 60/216 418, filed 04/17/00, and incorporated herein by reference. In other examples, charged or uncharged groups, lipophilic or polar groups may be added to the molecules of interest to achieve an increase in serum half-life or other target organs and / or cells. In other examples, it should be appreciated that contemplated compounds that are phosphorylated at the C ₅ atom may also be di- or tri-phosphorylated, or introduced as a thiophosphate.

Although it is generally preferred that the contemplated compounds have a sugar motif in the D-configuration, it is also contemplated that the compounds have a sugar motif in the L-configuration. Other stereochemical aspects in particular include the R and S configuration at the C ₅ atom, and it should be appreciated that the substituents in the contemplated compounds may be directed to the α or β phase.

Further, it should also be appreciated that the compounds contemplated may be in various forms, including liquid, syrup or gel forms (e.g., for injection, swallowing or topical administration) and solid forms (e.g., for swallowing, injection or storage in the body cavity) . For example, where the compounds of the invention are unstable in the gastric environment, the injection of the best isotonic solution is particularly contemplated. Alternatively, it may be administered by inhalation or by inhalation of a liquid form, which may be suitable to prevent acid degradation. On the other hand, where the contemplated compounds are known for their resistance to digestive disintegration, the contemplated forms may be administered in the form of a syrup or tablet. Depending on the particular use, contemplated compounds may be formulated for actual or dermal use. Many forms are known in the art, all of which are also contemplated are suitable in accordance with the spirit of the present invention described herein, and in particular the contemplated forms are described in Drug Products for Clinical Trials: An Intl. Guide to Formulation, Production

Quality Control, Donald C. Monkhouse, and Christopher T. Rhodes (Editors); ISBN: 082479852X.

It should further be appreciated that the contemplated compounds and forms may include functional and non-functional additives. For example, where it is desired to administer the drug through the skin, agents may be added to improve skin penetration. Or, for the purpose of synergistic or reverse enhancement of function of the contemplated compounds, medicaments including cytostatics, antiviral or immunomodulatory agents may be added. Examples of non-functional additives include fillers, antioxidants, flavorings or coloring agents that primarily enhance the quality of the compounds contemplated.

Based on the concentration of the compounds contemplated, a concentration range of about 1 μΜ to about 100 μΜ is preferred when measured at the site of action. However, particularly when the affinity of the compounds of interest is less than 1 μΜ, concentrations in the range of 999 nM to 10 nM or less may be appropriate. On the other hand, when the compounds of interest show a relatively short half-life or a high conversion, concentrations of 0.1 mM and 100 mM or more can be considered. Consequently, the dosage of the contemplated compounds may vary considerably, but appropriate dosages may be readily determined in vitro or in animal experiments.

Various biological effects of both 5'- and 4'-modified pyrrolopyrimidine nucleoside analogues, in particular significant biological effects include modulation of type 1 and type 2 cytokine production, control of neoplastic conditions (e.g. reduction of DNA synthesis or reduction of cell growth), and reduction of chemokines and release of growth factor as described below.

Consequently, the contemplated method for altering cytokine secretion from cells may comprise a phase in which a compound of formula I is provided and a phase in which a cell with a compound of formula I is present at a concentration effective to alter the cytokine secretion. Although in general all possible combinations of substituents of formula I are contemplated, especially the compounds contemplated are compounds of formula I, wherein R ⁴ and R ⁵ 'are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl and aralkyl, and wherein R ⁴ and R ⁵ 'independently and preferably contain at least a heteroatom and functional groups, with the remaining substituents as defined above in formula I. In another aspect, the compound used to alter the secretion of cytokines from the cell may also be a compound according to the following structure:

Z is CN, C (O) NH ₂ , C (= NH) NH ₂ , C (= NNH ₂ ) NH _2, or C (= NOH) NH ₂ , and wherein R ⁵ is H, OH, OP (0) ( 0H) _2, 0P (0) (0R ') ₂ or P (0) (0R) _2, wherein R' is a masking group. Specifically, contemplated cytokines include type 1 (e.g., IFNγ) and type 2 (e.g., IL-4) cytokines. With respect to the cells, it is contemplated that all cells known from the production and / or secretion of cytokines are suitable, but, in particular, the cells contemplated include lymphocytes and cancer cells (e.g., prostate cells).

In another aspect, the method of reducing hyperproliferative cell growth may comprise a phase in which a compound of Formula I is provided and another phase in which hyperproliferative cells are present with the compound at a concentration effective to reduce hyperproliferative cell growth. Particularly preferred compounds include compounds of formula I wherein R ⁴ is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, and aralkyl, wherein R ⁴ preferably comprises at least a heteroatom or a functional group, and wherein R ⁵ 'is selected from containing hydrogen, alkyl, alkenyl, alkynyl and aralkyl, and wherein R ⁵ 'has at least two carbon atoms and preferably contains at least a heteroatom or a functional group, provided that R ⁴ and R ⁵ ' are not together hydrogen, and with the remaining substituents as defined in formula I above.

Specifically, the hyperproliferative cells contemplated include cancer cells, and particularly the cancer cells contemplated are prostate cancer cells. Although not limited to a particular theory, growth reduction is believed to include reduction of DNA synthesis.

In yet another aspect of the present invention, it is contemplated that a method of reducing growth factor release from cells comprises a phase in which a compound of Formula I is provided and a further phase wherein the cell with the compound is present at a concentration effective to reduce growth factor release . It is contemplated that the release of various growth factors may be reduced in the manner described herein, but in particular consideration is given to reducing the release of VEGF. Similarly, although all cells known from secreting growth factors in conjunction with the method described herein are contemplated, the particular cells contemplated include cancer cells, and especially prostate cancer cells.

With respect to the synthesis of the contemplated compounds, it should be appreciated that pyrrolo (2,3-d) pyrimidine nucleoside analogs of the nature of the present invention can be synthesized by various synthetic routes. The following procedures are provided by way of example only.

Synthesis of C5'-modified pyrrolo (2,3-d) pyrimidine nucleoside analogs

The 5 'end-substituted nucleoside analogs are prepared by condensation of pyrrolo (2,3-d) pyrimidine bases and properly protected 5'-substituted ribofuranoses. As shown in FIG. 1, compound 1 prepared according to a published procedure (Jones et al., Methods in Carbohydrate Chemistry (edited by Whistler and Moffat, vol. VI, pp. 315-322, Academic Press, New York, (1972)), which has been treated with various nucleophiles such as the Grignard reagent afforded compound 2, which was benzoated and subsequently treated with trifluoroacetic acid to give compound 4. Benzoylation followed by treatment with acetic anhydride / acetic acid in the presence of sulfuric acid afforded compound 6, which was used to condense with pyrrole ( 2,3-d) pyrimidine bases.

Compound 7 (Jones et al., Methods in Carbohydrate Chemistry (edited by Whistler and Moffat, vol. VI, pp. 315-322, Academic Press, New York, (1972)), prepared according to a published procedure, was converted to a tosylate derivative which was reduced with lithium and aluminum hydride to give compound 8. By similar procedures shown in Figure 1, compound 8 was converted to compound 9. The condensation of compound 9 and pyrrolo (2,3-d) pyrimidine 19 and subsequent transformations are shown in the scheme. 2 which provides compounds 10 to 15 as shown in FIG. 2.

As shown in FIG. 3, compound 2 was converted to sulfonate 16, which was subjected to nucleophilic exchange to give the configuration inverse compound 17. Violation of isopropylidene protection and subsequent acetylation allowed formation of tetraacetate 18. The condensation of protected 5-terminal ribofuranoses with nucleoside bases is shown in FIG. 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 a trimethylsilyl derivative and then condensed with compound 6 in the presence of trimethylsilyl triflate by a similar procedure described for toyocamysin (Sharma et al., Nucleosides Nucleotides, 1993, 12, 643-648). The resulting combined product was subjected to debromination by hydrogenation to give compound 20. Treatment of compound 20 with ammonium in anhydrous methanol afforded compounds 21 and 23. Compound 21 was oxidized to give compound 22. Compound 23 was converted to the carboxamide derivative 24. Compounds 23 and 24 were oxidation to form compound 25. Treatment of compound 25 with hydroxyamine yields compound 26, which has been hydrogenated with Raney Nickel to produce compound 27. Or, compound 27 can be prepared by heating compound 25 with ammonia in a pressurized bomb.

Synthesis of C4'-modified pyrrolo (2,3-d) pyrimidine nucleoside analogs

In FIG. 5, compound 1 was treated with formaldehyde in liquid sodium hydroxide to give the 4'-hydroxymethyl derivative 28, which was selectively protected to give compound 29. Subsequent protection with DMT and removal of RBS allows the formation of compound 31, which was converted to various substituents. C4-substituted derivatives that have undergone a similar transformation to the C5-substituted ribofuranoses (Scheme 1) can be converted to compound 35, which is used for coupling with nucleoside bases.

Similar to the pyrrolopyrimidine nucleoside analogs substituted on C5 ', analogs substituted on C4' can be obtained by condensation of compound 35 with compound 19 as shown in FIG. 6. Subsequent transformations allow the formation of C4's-substituted pyrrolopyrimidine nucleosides 37-42.

Synthesis of C2´-modified and other pyrrolo (2,3-d) pyrimidine nucleoside analogs

The following pyrrolopyrimidine nucleoside analogs have been prepared for biological testing, some of which have been published (referred to as known compounds), shown in FIG. 7. Known compounds 43, 44, 52-55 and 57 were prepared according to published procedure 1970, 92, 236-241).

hydrogenating compound 52.

(Hinshaw et al., J. Compound 56 was known compound 49

Org. Chem., Prepared (Krawczyk et al., Nucleosides Nucleotides, 1989, 8, 97-115), was treated with sodium nitrite to give compound 50. Known compounds 45 and 48 were prepared according to a published procedure (Ramasamy et al., J. Heteroeyclic Chem., 1988, 25, 1043-1046).

Compound 45 was treated with ammonium with methanol to give compound 46 and hydrogenated to give compound 47. Compounds to 63 were prepared from compound 45 by similar procedures used for compound 52 to 57. 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 70.

Preparation of human T cells and activation in vitro

Peripheral blood mononuclear cells were isolated from human donors by density gradient centrifugation followed by T cell enrichment using Lymphokwik / One

Lambda, Canoga Park CA). The admixed monocytes were removed by adherence to the plastic. Purified T cells were> 99% CD2 +, <1% HLA-DR + and <5% CD25 + and left in RPMI-AP5 (RPMI1640 medium containing 5S self plasma, 1% L-glutamine, 1% penicillin / streptomycin and 0.05% 2-mercaptoethanol). To determine the protein level of cytokines, 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 and 0.1 mg ionomycin (PMA-ION, both from Calbiochem, San Diego, CA) and were incubated in a 96-well plate in the presence of 0 or 10 μΜ guanosine nucleosides for 48 hours at 37 ° C. After activation, the supernatants were analyzed for the production of cell-derived cytokines. Extracellular cytokine analyzes

After appropriate dilution using IFNγ and IL-4 specific ELISA (Biosource, Camarillo, CA), human cytokine levels were determined in the cell supernatants. All ELISA results were expressed as pg / ml.

Effect of pyrrolo (2,3-d) pyrimidine nucleoside analogues on extracellular cytokine levels in human T cells

The effect of pyrrolo (2,3-d) pyrimidine nucleoside analogs at concentrations of 0 and 10 μΜ on PMA / ionomycin-stimulated T cells and their expression of type 1 cytokines, IFNγ, and type 2 cytokines, IL4, is shown in FIG. 8A and 8B for 5 individual human donors. Cytokine levels were determined in cell-free supernatants by ELISA. The strongest effect was found in

7-b-D-ribofuranosyl-4-oxo-pyrrolo (2,3-d) pyrimidine-5-carboxamide. This compound increased activated IL-4 production to 498% ± 83 and suppressed IFNγ to 43% ± 4 activated control levels of each cytokine. Data is shown as the percentage of activated control calculated as the ratio of the activated T cell cytokine level x 100%. A zero effect on cytokine levels by the nucleoside assay would mean a percentage of activated control of 100%. Absolute level (pg / ml ± standard deviation)

PMA-ION-induced cytokine teaching was for IFNγ:

22954 ± 3391; and for IL-4: 162 ± 40. The immovable levels were for <30 pg / ml of all cytokines tested.

Cellular toxicity of pyrrolo (2,3-d) pyrimidine nucleoside analogs in vitro

The pyrrolo (2,3-d) pyrimidine nucleoside analogs of the present invention are biologically active as long as they indicate some level of in vitro toxicity. In these studies, test compounds were added to cell cultures of normal human fibroblasts, human prostate cancer cells 81, human melanoma cancer cells 140, human cancer lung cells 177, and human ovarian cancer cells R and NR (all available from AATCC). In these experiments, cells were coated at a density of 2000 cells per 20 μΐ of medium per well (96-well plate). Test compounds were added to the wells once at a concentration ranging from 0.78 to 100 μΜ, immediately after cell deposition. After 72 hours incubation, a colorimetric MTS cell toxicity assay was performed. The EC 50 was calculated based on the data obtained and the results are shown in FIG. 9. Many compounds indicate a lack of cellular toxicity at a concentration of less than 100 μΜ. In such cases, the EC50 is referred to as > 100. In other cases, the EC50 indicates the concentration of test compounds required to destroy 50% of the cell population.

Pyrolo (2,3-d) pyrimidine nucleoside analogs inhibit DNA synthesis in cells grown in vitro in a dose-dependent manner

Analogues of pyrrolo (2,3-d) pyrimidine nucleosides inhibit the growth of human cells grown in vitro, which is measured as DNA level. The experimental set-up was the same as described above. Compounds were added once and the DNA level was measured after 72 hours. After this time, half the medium was removed from the culture wells and this was replaced with pure water. Then the cells were transferred to -70 ° C for at least 12 hours. In the next step, the cells were moved back from -70 ° C to room temperature and 1 μΜ Hoechst 33342 was added to each well. After 2 hours of incubation, the fluorescence signal (360-530 nm) was measured. According to this method, the fluorescence intensity is proportional to the amount of DNA due to the presence of the DNA-Hoechst 33342 complex formed. The results are shown in FIG. 10. The numbers represent groups of DNA amount increased compared to DNA at the beginning of the experiment (2 hours after cell deposition). In untreated prostate cancer cells and normal cells, DNA levels increased 5.78 and 4.47 times, respectively, during 72 hours of culture.

Compounds 23a (5'-R) and 23a (5'-S) inhibit VEGF secretion from human prostate cancer in vitro

Compounds 23a (5'-R) and 23a (5'-S) are capable of inhibiting vascular endothelial growth factor (VEGF) secretion from human HTB81 prostate cancer cells. VEGF is recognized as an angionic trait as this molecule is essential for endothelial cell migration and growth and capillary formation in vivo. To prove this, 0.5 x 10 ⁵ cells were transferred to 5 ml culture medium in a 10 cm diameter Petri dish. 72 hours after coating, the compounds were applied. The medium was then harvested and the VEGF level was measured using VEGF Elisa Assay (R&D Systems) and expressed as pg VEGF per ml medium. The results are shown in FIG. According to these results, both compounds inhibit VEGF secretion in a dose-dependent manner.

Compounds 23a (5'-R) and 23a (5'-S) inhibit the release of IL-8 from human prostate cancer cells in vitro

Compounds 23a (5'-R) and 23a (5'-S) indicate an inhibitory effect on the secretion of interleukin 8 (IL-8) from HTB81 prostate cancer cells. IL-8 belongs to the class of chemoattractant chemokines (type a), which are involved in inflammatory processes due to the attraction of neutrophils. Chemokines are generally known to be produced by various types of cancers. It has been shown in various studies that inhibiting chemokine production by cancer cells is beneficial to the host. To demonstrate the ability of the two compounds to inhibit IL-8 secretion from prostate cancer cells, HTB81 prostate cancer cells were treated in vitro with compounds 23a in both the 5'-R and 5'-S configurations at the concentrations indicated in the graph. The harvested medium from the culture was analyzed for IL-8 level using IL-8 ELISA Assay from R&D Systems. According to the results obtained, both compounds are capable of inhibiting IL-8 secretion in a dose-dependent manner as shown in FIG. 12th

However, it should be appreciated that the biological effects of the contemplated compounds need not be limited to the specific effects as described above. In particular, it is contemplated that the compounds of the present invention generally exhibit cytostatic activity in a variety of hyperproliferative disorders including local and / or metastatic cancers (e.g., lymphomas and carcinomas), benign prostate hyperplasia, and keratosis. Although the inventors have found a significant biological effect on IL-4 (type 2 cytokine) and IFNγ (type 1 cytokine), it is generally considered that the compounds of the present invention are biologically active in modulating cytokines other than IL-4 and IFNγ. In particular, it is contemplated that the compounds may increase or decrease the expression / secretion of a particular cytokine or series of cytokines. Therefore, it is contemplated that the compounds of the present invention may affect the immune system of the organism so that a greater expression of the Type 1 or Type 2 response can be achieved. Finally, it is considered that the compounds of the present invention may be effective to reduce virus titer in a living system. as a direct action as a viral polymerase inhibitor and / or indirectly, by activating the immune system to a particular humoral or cellular response. It is further contemplated that the compounds of the present invention may also be useful in reducing the immune system response against allo- or xenografts by reducing the amount of cellular response against allo- or xenograft.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is the first Sample include production ingredients invention. Fig. 2 is the second Sample include production ingredients invention.

synthetic scheme of reactions which, according to the nature of the proposed synthetic scheme of reactions, which according to the nature of the proposed

Fig. 3 is the third Sample synthetic scheme reactions that include production ingredients by nature proposed invention. Fig. 4 is the fourth Sample synthetic scheme reactions that include production ingredients by nature proposed invention. Fig. 5 is the fifth Sample synthetic scheme reactions that include production ingredients by nature proposed invention. Fig. 6 is the sixth Sample synthetic scheme reactions that include production ingredients by nature proposed invention. Fig. 7 shows sample components by nature

of the present invention.

Fig. Figures 8A and 8B are graphs which represent the effect of the components of the present invention on the expression of type 1 and type 2 cytokines.

Fig. 9 is a table depicting the cellular toxicity of various compounds according to the present invention.

Fig. 10 is a table depicting DNA synthesis ratios in cells treated with various compounds according to the present invention.

Fig. 11 is a graph depicting the suppression of VEGF release by human prostate tumor cells after treatment with the compounds of the present invention.

Fig. 12 is a graph depicting the suppression of IL-8 release by human prostate tumor cells after treatment with the compounds of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The following protocols describe exemplary syntheses of various compounds according to the spirit of the present invention and are intended to illustrate but not limit the inventive concept presented herein.

Example 1

Preparation of methyl-2,3-O-isopropylidene-5 (R, S) -C-ethynyl-B-ribofuranoside 2b

To a stirred solution of methyl-4-C, 5-O-didehydro-2,3-isopropylidene-β-D-ribofuranoside (Jones et al., Methods in Carbohydrate Chemistry, Vol. 1, pp. 315-322, 1972, 4). , 0 g,

19.78 mmol) in aqueous THF (20 mL) at -42 ° C under argon, was added a drop of ethynyl magnesium bromide (0.5 M in THF, 50 mL, 40 mmol). After the addition, the resulting mixture was slowly warmed to 0 ° C (ca. 90 minutes). The reaction was quenched by the addition of ice (50 g) in 50 mL water and stirred for 30 minutes. After neutralization with 10% acetic acid, the mixture was extracted twice with ethyl acetate. The combined organic layer was dried (Na ₂ SO ₄ ) and concentrated. Chromatography on silica (ethyl acetate: hexanes 1: 4) gave 3.48 g of the title compound (R / S ratio 1: 1) as a white solid. The following compounds were prepared in a similar manner: methyl-2,3-O-isopropylidene-5 (R) C-methyl-BD-ribofuranoside 2a was prepared from methyl-4-C, 5-dihydro-2,3-O-isopropylidene- hD-ribofuranoside and ethyl magnesium bromide. Methyl-2,3-O-isopropylidene-5 (R) -C-vinyl-β-β-ribofuranoside 2c was prepared from methyl-4-C, 5-O-didehydro-2,3-isopropylidene-β-D-ribofuranoside and of magnesium magnesium bromide. Methyl-5 (R) -C-allyl-2,3-O-isopropylidene-5-D-ribofuranoside 2d was prepared from methyl-4-C, 5-O-didehydro-2,3-O-isopropylidene-fi- Dribofuranoside and allyl magnesium bromide.

Example 2

Preparation of methyl-2,3-O-isopropylidene-5-O-methanesulfonyl-5 (R) -C-methyl-β-D-ribofuranoside 16 methyl-2,3-O-isopropylidene-5 (R) -C (7.24) g, 33.17 mmol) in anhydrous ° C was added to the resulting reaction hour, followed by temperature

To a stirred solution of methyl-β-D-ribofuranoside 2a pyridine (50 mL) at methanesulfonyl chloride (3.1 mL, 39.92 mmol) the mixture was stirred at room temperature 1 cooled to 0 ° C, neutralized (10% acetic acid) , concentrated (ca. 50%) and extracted twice with methylene chloride. The combined organic layer was dried (Na ₂ SO ₄ ) and concentrated to dryness. Chromatography on silica (4% methanol in chloroform) afforded 20.2 g of compound 28 as a white solid.

Example 3

Preparation of methyl-2,3-O-isopropylidene-5-O-acetyl-5 (S) -C-methyl-β-Dribofuranoside 17

A stirred suspension of methyl-2,3-O-isopropylidene-5-O-methanesulfonyl-5 (R) -C-methyl-BD-ribofuranoside 16 (8.62 g, 29.1 mmol) and NaOAc (aq, 3.5 g, 42.5 mmol) in aqueous 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) afforded 4.0 g of the title compound 17 as a white solid.

Example 4

Preparation of methyl-2,3-O-isopropylidene-4-C-hydroxymethyl-B-D-ribofuranoside 28

To a stirred solution of methyl 4-C, 5-O-didehydro-2,3-O-isopropylidene-1H-ribofuranoside 1 (20.22 g, 100 mL) in dioxane (380 mL) at 0 ° C was added a drop formaldehyde (37% solution, 76 mL) followed by 2 M NaOH (188 mL). The resulting reaction mixture was stirred at room temperature for 20 hours, then cooled to 0 ° C, neutralized (10% acetic acid), concentrated (ca. 50%) and extracted twice with methylene chloride. The combined organic layer was dried (Na ₂ SO ₄ ) and concentrated to dryness. Chromatography on silica (4% methanol in chloroform) afforded 20.2 g of compound 28 as a white solid.

Example 5

Preparation of methyl-2,3-O-isopropylidene-5-deoxy-β-D-ribofuranoside 8

To a stirred solution of methyl-2,3-O-isopropylidene-5-D-ribofuranoside (14.2 g, 70.0 mol) in anhydrous pyridine (250 mL) at 10 ° C was added in portions (after 30 min). Ptoluenesulfonyl chloride (19.1 g, 100 mmol) was added. The resulting mixture was stirred at room temperature for 18 hours, cooled to 0 ° C, quenched by the addition of water (5.0 mL), and stirred at room temperature for 30 minutes. The solvent was evaporated.

The residues were dissolved in ethyl acetate, washed three times with brine, dried (Na ₂ SO ₄ ) and concentrated to dryness. Chromatography on silica (ethyl acetate: hexanes 1: 3) afforded 24.1 g of the title compound as a white solid.

To a stirred suspension of LiAlH ₄ (4.58 g, 120.5 mmol) in aqueous diethyl ether (120 mL) was added methyl 2,3-O-isopropylidene-5-O-p-toluenesulfonyl-β-ribofuranoside (13.1 g, 36.55 mmol) in diethyl ether: toluene (2.5: 1, 140 mL). The resulting mixture was poured over 22 hours, cooled to room temperature, diluted with ethyl acetate (25 mL), quenched by the addition of water (5.0 mL). The solvent was evaporated. The residues were dissolved in ethyl acetate, washed three times with brine, dried (Na ₂ SO ₄ ) and concentrated to dryness. Chromatography on silica (ethyl acetate: hexanes 1: 3) afforded 2.58 g of the title compound as a colorless liquid.

Example 6

Preparation of methyl-5 (R) -C-allyl-5-O-benzoyl-2,3-O-isopropylidene-b-D-furoside 3d

To a stirred solution of methyl 5 (R) -C-allyl-2,3-O-isopropylidene-beta-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 mmol). The resulting mixture was stirred at room temperature for 18 hours, cooled with ice, made up by addition of water (1 mL) and stirred at room temperature for 30 minutes. The solvent was evaporated. The residues were dissolved in ethyl acetate, washed three times with brine, dried (Na ₄ SO ₄ ), and concentrated. Chromatography on silica (12% ethyl acetate in hexanes) afforded 6.26 g of the title compound 3d as a colorless syrup. The following compounds were prepared in a similar manner: methyl 5-O-benzoyl-5- (R, S) -C-ethynyl-2,3-O-isopropylidene-BD-furanoside 3b (R / S ratio 1: 1) from methyl -5 (R, S) -C-ethynyl-2,3-isopropylidene-BD-ribofuranoside 2b. Methyl 4-C-benzoyloxymethyl-5-O-benzoyl-2,3-O-isopropylidene-6-D-furanoside from methyl-2,3-isopropylidene-4-C-hydroxymethyl-β-D-ribofuranoside.

Example Ί

Preparation of methyl-5 (R) -C-allyl-5-O-benzoyl-B-D-ribofuranoside 4d

A solution of methyl-5 (R) -C-allyl-5-O-benzoyl-2,3-O-isopropylidene-beta-D-furanoside 3d (6.2 g, 17.8 mmol) in TFA-H ₂ O mixture (9 1) was stirred at 0 ° C for 90 minutes and concentrated to dryness at 0 ° C. The residue was dissolved in a mixture of methanol and toluene (20 mL, 1: 1) and concentrated to dryness. Chromatography on silica (ethyl acetate: hexanes 1: 1) afforded 3.70 g of the title compound 4d as a white solid. The following compounds were prepared in a similar manner: Methyl 5-O-benzoyl (R, S) -C-ethynyl-BD-ribofuranoside 4b (R / S ratio 1: 1) from methyl 5-O-benzoyl- (R, S) - C-ethynyl-2,3-O-isopropylidene-ED-ribofuranoside 3b, methyl-5-O-benzoyl-4-C-benzoyloxymethyl-ED-ribofuranoside from methyl-5-O-benzoyl-4-C-benzoyloxymethyl-2 , 3-izopropylidénβ-D-ribofuranoside.

Example 8

Preparation of methyl-5 (R) -C-allyl-2,3,5-tri-O-benzoyl-B-O-ribofuranoside 5d

To a stirred solution of methyl 5 (R) -C-allyl-5-O-benzoyl-β-Dribofiranoside 4d (3.60 mg, 11.68 mmol) in anhydrous pyridine (80 mL) at 0 ° C was added benzoyl chloride (3.0 mL, 25.84 mmol). The resulting mixture was stirred at room temperature for 18 hours, cooled with ice, quenched by addition of water (1 mL), then stirred at room temperature for 30 minutes. 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) afforded 5.3 g of the title compound 5d as a colorless syrup. The following compounds were prepared in a similar manner: methyl 5 (R, S) -C-ethynyl-2,3,5-tri-O-benzoyl-BD-ribofuranoside 5b (1: 1 R / S ratio) from methyl 5- O-benzolyl-5 (R, S) -C-ethynyl-β-furanoside 4b. Methyl 4-C-benzoyl-oxomethyl-2,3,5-tri-O-benzoyl-D-ribofuranoside from methyl-4-C-benzoyloxymethyl-5-O-benzoyl-β-Dribofuranoside.

Example 9

Preparation of 1-O-methyl-2,3,5-tri-O-benzoyl-5 (R) -C-vinyl-E-D-ribofuranose 5c

A solution of methyl-2,3-O-isopropylidene-5 (R) -C-vinyl-ED-ribofuranose 2c (1.0 g, 4.3 mmol) in a mixture of trifluoroacetic acid and water (9: 1, v / v) (11 mL) was stirred at 0 ° C for 30 minutes and concentrated to dryness. The residue was dissolved in methanol and concentrated to dryness (three times), then dissolved in pyridine and evaporated, and finally 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 ° C for 16 h and cooled in 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 (0-5% ethyl acetate in dichloromethane) to give 1.0 g of the title compound 5c as a syrup.

Example 10

Preparation of 1-O-acetyl-2,3,5-tri-O-benzoyl-5 (R) -C-allyl-D-ribofuranose 6d

To a stirred solution of methyl 5 (R) -C-allyl-2,3,5-tri-O-benzolyl-ED-ribofuranoside 5d (4.0 g, 7.74 mmol) in acetic acid (14 mL) and anhydride acetic acid (1.75 mL, 18.36 mmol) was added concentrated sulfuric acid (200 μΐ) at 0 ° C,

3.79 mmol in 4.0 mL acetic acid). The resulting mixture was stirred at room temperature for 20 hours, cooled to 0 ° C, diluted with cold ethyl acetate, washed with water, 5% aqueous NaHCO ₃ and then brine, dried (Na ₂ SO ₄ ) and concentrated. Chromatography on silica (ethyl acetate in hexanes 1: 4) afforded 2.82 g of the title compound 6d (α / β ratio 1: 2) as a colorless foam. The following compounds were prepared in a similar manner: 10-acetyl-5 (R, S) -C-ethynyl-2,3,5-tri-O-benzolyl-β-D-ribufuranose 6b (R / S ratio 1: 1a α / β ratio 1: 2) from methyl-5 (R, S) -C-ethynyl-2,3,5-tri-OB-Dribofuranoside 5b. 1-O-acetyl-4-C-benzoyloxymethyl-2,3,5-tri-Obenzoyl-D-ribofuranose (α / β ratio 1: 3) from methyl-4-Cbenzoyloxymethyl-2,3,5-tri-0 benzoyl-.beta.-D-ribofuranoside. 5 (R) -C22 methyl-1,2,3,5-tetra-O-acetyl-1H-ribofuranose from methyl-2,3-isopropylidene-5 (R) -C-methyl-.beta.-D-furanoside. 1,2,3,5-tetra-O-acetyl-5 (S) -C-methyl-D-ribofuranose 6a from methyl-5-O-acetyl-2,3-isopropylidene-5 (R) -C-methyl -BD-ribofuranoside. 5-deoxy-2-1,2,3-tri-O-acetyl-D-ribofuranose 9 from methyl-5-O-acetyl-2,3-O-isopropylidene-BD-ribofuranoside. 10-acetyl-2,3,5-tri-O-benzoyl 5 (R) -C-vinyl-BD-ribofuranose 6c from methyl-2,3,5-tri-O-benzoyl 5 (R) -vinyl-RD-ribofuranoside .

Example 11

Preparation of 4-amino-6-bromo-5-cyano-7- (2,3,5-tri-O-benzoyl-5 (R) -Calyl-β-D-ribofuranosyl) pyrrolo (2,3-d) pyrimidine

A suspension of 4-amino-6-bromo-5-cyanopyrrolo (2,3-d) pyrimidine (Tol-man et al., J. Org. Chem., 1969, 91, 2102-2108) (1.05 g,

4.41 mmol) and ammonium sulfate (50 mg) in HMDS (75 mL) and anhydrous m-xylene (25 mL) were under argon for 18 hours. The solvents were evaporated and the residue was dried in vacuo. The residue was dissolved in anhydrous 1,2-dichloroethane (80 mL) and treated with 1-O-acetyl 2,3,5-tri-O-benzoyl-5 (R) -C-allyl-D-ribofuranose (2.0 g, 3.67 mmol). TMSOTf (1.3 mL, 7.30 mmol in 5 mL anhydrous 1,2-dichloroethane) was added under ice-cooling. The mixture was stirred at room temperature for 30 minutes under argon, then refluxed for 90 hours, cooled on ice / NaHCO ₃ (50 mL) and filtered. The organic layer was separated, dried (Na ₂ SO ₄ ) and concentrated. Chromatography on silica (EtOAc: hexanes 2: 3) afforded 1.81 g of the title compound as a colorless solid. The following compounds were prepared in a similar manner: 4-amino-6-bromo-5-cyano-7- (2,3,5-tri-O-benzoyl-5 (R, S) -C-ethynyl-BD-ribofuranosyl) pyrrole (2) 3-d) pyrimidine (R / S) ratio 1: 1) was prepared from 10-acetyl-2,3,5tri-O-benzoyl-5 (R, S) -C-ethynyl-D-ribofuranose and 4- amino-6-bromo-5-cyanopyrrolo [2,3-d] pyrimidine. 4-Amino-6-bromo-5-cyano-7- (4-benzoyloxomethyl-2,3,5-tri-O-benzoyl-BD-ribofuranosyl) pyrrolo (2,3-d) pyrimidine was prepared from 10-acetyl-4 -benzoyloxymethyl-2,3,5-tri-O-benzoyl-D-ribofuranose and 4-amino-6-bromo-5-cyanopyrrolo (2,3-d) pyrimidine. 4-amino-6-bromo-5-cyano-7- (1,2,3,5-tetra-O-acetyl-5 (R) -C-methyl-b-D-ribofuranosyl) pyrrolo (2,3-d) pyrimidine was prepared from 1,2,3,5-tetra-O-acetyl-5 (R) -C-methyl-D-ribofuranose and 4-amino-6-bromo-5-cyanopyrrolo [2,3-d] pyrimidine. 4-amino-6-bromo-5-cyano-7- (1,2,3,5-tetra-O-acetyl-5 (S) -C-methyl-β-D-ribofuranosyl) pyrrolo (2,3-d) pyrimidine prepared from 1,2,3,5-tetra-O-acetyl-5 (S) -C-methyl-D-ribofuranose and 4-amino-6-bromo-5-cyanopyrrolo (2,3-d) pyrimidine. 4-Amino-6-bromo-5-cyano-7- (2,3-di-acetyl-5-deoxy-β-ribofuranosyl) pyrrolo (2,3-d) pyrimidine was prepared from 1,2,3-tri -O-acetyl-5-deoxy-D-ribofuranose and 4 amino-6-bromo-5-cyanopyrrolo (2,3-d) pyrimidine. 4-Amino-6-bromo-5-cyano-7- (2,3,5-tri-O-benzoyl-5 (R) -C-vinyl-β-Dribofuranosyl) pyrrolo (2,3-d) pyrimidine was prepared from 1-O-acetyl-2,3,5-tri-O-benzoyl-5 (R) -β-D-ribofuranose and 4-amino-6-bromo-5-cyanopyrrolo (2,3-d) pyrimidine.

Example 12

Preparation of 4-amino-5-cyano-7- (2,3,5-tri-O-benzoyl-5 (R) -C-allyl-β-D-ribofuranosyl) pyrrolo (2,3-d) pyrimidine 20e

To a solution of 4-amino-6-bromo-5-cyano-7- (2,3,5-tri-O-benzoyl-5 (R) -C-allyl-1H-ribofuranosyl) pyrrolo (2,3-d) pyrimidine ( 738 mg, 1.0 mmol) in acetic acid (25 mL) was added zinc powder (1.04 g, 16.0 mmol) in two portions (after an hour). The reaction mixture was stirred at room temperature for 20 hours and filtered. The filtrate was evaporated to dryness and the residue allowed 450 mg of 20e to be obtained as a colorless foam by chromatography on silica (ethyl acetate: hexanes 1: 1). The following compounds were prepared in a similar manner: 4-amino-5-cyano-7- (2,3,5-tri-Obenzoyl-5 (R, S) -C-ethynyl-BD-ribofuranosyl) pyrrolo (2,3-d) ) Pyrimidine (R / S ratio 1: 1) 20b from 4-amino-6-bromo-5-cyano-7- (2,3,5-tri-O-benzoyl-5 (R, S) -C-ethynyl-BD -ribofuranozyl) pyrrolo (2,3-d) pyrimidine. 4-Amino-5-cyano-7- (2,3,5-tri-O-benzoyl-5 (R) -Cvinyl-BD-ribofuranosyl) pyrrolo (2,3-d) pyrimidine 20c from 4-amino6-bromo -5-cyano-7- (2,3,5-tri-O-benzoyl-5 (R) -C-vinyl-BD-ribofuranosyl) pyrrolo (2,3-d) pyrimidine.

Example 13

Preparation of 4-amino-5-cyano-7- (2,3,5-tri-O-benzoyl-5 (R) -C-propyl-beta-D-ribofuranosyl) pyrrolo (2,3-d) pyrimidine 20f

Suspension of 4-amino-6-bromo-5-cyano-7- (2,3,5-tri-O-benzoyl-5 (R) -C-allyl-BD-ribofuranosyl) pyrrolo (2,3-d) pyrimidine (400 mg, concentrated (ethyl acetate as a colorless foam. in a similar way:

x 10 Pa) for 4 hours. The catalyst was (dioxane). The combined filtrate was

0.54 mmol) and 10% Pd / C (100 mg, ca. 50% water) in dioxane (50 mL) and triethylamine (0.5 mL) were shaken in a hydrogenator (H ₂ , 1.78789 filtered and washed and the residue allowed to be chromatographed on silica hexanes 1: 1) to give 340 mg of the title compound 23f The following compounds were prepared

4-amino-5-cyano-7- (2,3,5-tri-O-benzoyl-5 (R, S) -C-ethyl-1H-ribofuranosyl) pyrrolo (2,3-d) pyrimidine (R / S ratio) 1: 1) 20d from 4-amino-6-bromo-5-cyano-7- (2,3,5-tri-Obenzoyl-5 (R, S) -C-ethynyl-ED-ribofuranosyl) pyrrole (2, 3-d) pyrimidine. 4-Amino-5-cyano-7- (4-benzoyloxomethyl-2,3,5-tri-O-benzoyl-beta-D-ribofuranosyl) pyrrolo (2,3-d) pyrimidine from 4-amino-6-bromo-5-cyano 7- (4-benzoyloxometyl-2,3,5-tri-0-benzoyl-HD-ribofuranosyl) pyrrolo (2,3-d) pyrimidine. 4-amino-5-cyano-7- (1,2,3,5-tetra-O-acetyl-5 (R) -C-methyl-.beta.-D-ribofuranosyl) pyrrolo (2,3-d) pyrimidine 20; 4-amino-6-bromo-5-cyano-7- (l, 2,3,5-tetra-O-acetyl-5 (R) C-methyl-bD-ribofuranosyl) pyrrolo (2,3-d) pyrimidine . 4-Amino-5-cyano-7- (1,2,3-tri-O-acetyl-5-deoxy-BD-ribofuranosyl) pyrrolo (2,3-d) pyrimidine from 4-amino-6-bromo-5-cyano 7- (1,2,3-tri-O-acetyl-5-deoxy-BD-ribofuranosyl) pyrrolo (2,3-d) pyrimidine.

4-Amino-5-cyano-7- (2,3-dideoxy-BD-glyceropentofuranosyl) pyrrolo (2,3-d) pyrimidine was prepared from 4-amino-5-cyano-7- (2,3dideoxy-ED- pent-2-énfuranozyl) pyrrolo (2,3-d) pyrimidine.

Example 14

Preparation of 4-amino-5-cyano-7- (5 (R) -C-allyl-E-D-ribofuranosyl) pyrrolo (2,3-d) pyrimidine 23e

A suspension of 4-amino-5-cyano-7- (2,3,5-tri-O-benzoyl-5 (R) -Calyl-BD-ribofuranosyl) pyrrolo (2,3-d) pyrimidine (300 mg, 0.454 mmol) ) in methanol (40 mL) at 0 ° C was saturated with ammonium. The solution was allowed to stand at room temperature for 2 days. The solvent was evaporated and the residue along with NaOAc (anhydrous, 20 mL) was suspended in DMF (20 mL). The mixture was stirred under argon at 120 ° C for 5 hours. The solvent was evaporated. The residue was adsorbed onto a silica gel and eluted with a silica goal column (methanol: ethyl acetate 1:25) to give 145 mg of the title compound as a colorless solid.

Prior to heating in DMF, the product contained two main compounds 21 and 23, which can be separated by chromatography on a silica gel. Compound 21 was prepared by this procedure. The following compounds were prepared in a similar manner: 4-amino-5-cyano-7 (5 (R) -C-propyl-5-D-ribofuranosyl) pyrrolo (2,3-d) pyrimidine 23f from

4-amino-5-cyano-7- (2,3,5-tri-0-benzoyl-5 (R) -C-propyl-p-D-ribofuranosyl) pyrrolo (2,3-d) pyrimidine. 4-amino-5-cyano-7- (5 (R, S) -Cetinyl-β-D-ribofuranosyl) pyrrolo (2,3-d) pyrimidine (R / S ratio 1:

1) 23b from 4-amino-5-cyano-7- (2,3,5-tri-O-benzoyl-5 (R, S) -Cetinyl-β-D-ribofuranosyl) pyrrolo (2,3-d) pyrimidine. 4-amino-5-cyano-7- (5 (R, S) -C-ethyl-β-D-ribofuranosyl) pyrrolo (2,3-d) pyrimidine (R / S ratio 1: 1) 23d from 4-amino- 5-cyano-7- (2,3,5-tri-Obenzoyl-5 (R, S) -C-ethyl-DE-ribofuranosyl) pyrrolo (2,3-d) pyrimidine. 4-Amino-5-cyano-7- (4-hydroxymethyl-b-D-ribofuranosyl) pyrrolo (2,3-d) pyrimidine 33d from 4-amino-5-cyano-7- (4-hydroxymethyl-2,3,5-tri) -0-benzoyl-.beta.-D-ribofuranosyl) pyrrolo (2,3-d) pyrimidine.

4-Amino-5-cyano-7- (5 (R) -C-methyl-5-D-ribofuranosyl) pyrrolo (2,3d) pyrimidine 23a (5'-R) from 4-amino-5-cyano-7 - (1,2,3,5-tetra-0acetyl 5 (R) -C-methyl-DE-ribofuranosyl) pyrrolo (2,3-d) pyrimidine.

4-Amino-5-cyano-7- (5 (S) -C-methyl-BD-ribofuranosyl) pyrrolo (2,3d) pyrimidine 23a (5'-S) from 4-amino-5-cyano-7- ( 1,2,3,5-tetra-acetyl-5 (S) -C-methyl-β-ribofuranosyl) pyrrolo (2,3-d) pyrimidine.

4-Amino-5-cyano-7- (5-deoxy-BD-ribofuranosyl) pyrrolo (2,3-d) pyrimidine 10 of 4-amino-5-cyano-7- (1,2,3-tri-O) acetyl-5-deoxy-fid-ribofuranosyl) pyrrolo (2,3-d) pyrimidine. 4-amino-5-cyano-7 (5 (R) -C-vinyl-β-D-ribofuranosyl) pyrrolo (2,3-d) pyrimidine 23c from

4-amino-5-cyano-7- (2,3,5-tri-0-benzoyl-5 (R) -C-vinyl-B-D-ribofuranosyl) pyrrolo (2,3-d) pyrimidine.

Example 15

Preparation of 4-amino-5-cyano-7- (2,3-di-O-methanesulfonyl-5-O-tert-butyldiphenylsilyl-β-D-ribofuranosyl) pyrrolo (2,3-d) pyrimidine

To a stirred solution of toyokamicin 43 (5.83 g, 20.0 mmol) in anhydrous pyridine (100 mL) at 0 ° C was added tert-butyl chlorodiphenylsilane (6.2 mL, 24.0 mmol). The resulting mixture was stirred at room temperature for 18 hours and then cooled to 0 ° C, then methanesulfonyl chloride (3.4 mL, 44 mmol) was added. The resulting mixture was stirred at room temperature for 30 minutes. The solvent was evaporated. The residue was ethyl acetate, washed three times with brine, concentrated.

: hexanes 3: 2) (Na ₂ SO ₄ ) and (ethyl acetate

Chromatography yielded 8.41 dissolved in silica dried g of the title compound as a colorless solid.

Example 16

Preparation of 4-amino-5-cyano-7- (5-O-tert-butyldiphenylsilyl-2,3didehydro-2,3-dideoxy-β-D-ribofuranosyl) pyrrolo (2,3-d) pyrimidine

Tellurium powder (640 mg, 5.0 mmol) was sealed under argon and treated with lithium triethyl borohydrate (1.0 M in THF, 11.25 mL, 11.25 mmol). The mixture was stirred at room temperature for 6 hours and then cooled to 5 ° C and 4-amino-5-cyano-7 (2,3-di-O-methanesulfonyl-5-O-tert-butyldiphenylsilyl-hD) was added. (Ribofuranosyl) pyrrolo (2,3-d) pyrimidine (1.40 g, 2.09 mmol) in THF (12 mL). The resulting mixture was stirred at room temperature for 18 hours and then cooled with ice, quenched by the addition of water (0 ° C, 5 mL) and then stirred at room temperature for 30 minutes. The solvent was evaporated and the residue was obtained with ethyl acetate. The yields were concentrated and the residue was chromatographed on silica (15% ethyl acetate in hexanes) to give 640 mg of the title compound as a colorless foam.

Example 17

Preparation of 4-amino-5-cyano-7- (2,3-didehydro-2,3-dideoxy-β-dribofuranosyl) pyrrolo (2,3-d) pyrimidine 49

To a stirred solution of 4-amino-5-cyano-7- (5-O-tert-butyldiphenylsilyl-2,3-didehydro-2,3-dideoxy-β-ribofuranosyl) pyrrolo (2,3-d) pyrimidine (2) , 55 g, 5.32 mmol) in anhydrous THF (100 mL) at 5 ° C was added tetrabutylammonium fluoride (1.0 M in THF, 6.6 mL). The resulting mixture was stirred at room temperature for 3 hours and concentrated. Chromatography on silica (6% methanol in ethyl acetate) afforded 1.09 g of the title compound 49 as a colorless solid.

Example 18

Preparation of 5-cyano-7- (5 (R) -C-methyl-β-D-ribofuranosyl) pyrrolo (2,3-

d) -4-pyrimidone 25a

To a stirred solution of 4-amino-5-cyano-7- (5 (R) -C-methyl-β-Dribofuranosyl) pyrrolo (2,3-d) pyrimidine (306 mg, 1.0 mmol) in water (30 mL) ) and acetic acid (2.0 mL) at 55 ° C were added in portions sodium nitrite (590 mg, 8.55 mmol). The resulting mixture was stirred at 70 ° C for 3 hours and more sodium nitrite (300 mg, 4.30 mmol) was added. The mixture was further stirred at the same temperature for an additional 18 hours. The solvent was evaporated and the residue allowed chromatography of silicon (12% methanol in methylene chloride) to give 210 mg of said compound 25a (5'-R) as a colorless solid. The following compounds were prepared in a similar manner: 4-Amino-5-cyano-7- (5 (S) -Cmethyl-1H-ribofuranosyl) pyrrolo (2,3-d) -4-pyrimidone 25a (5 * -S) from amino-5-cyano-7- (5 (S) -C-methyl-bD-ribofuranosyl) pyrrolo (2,3-d) pyrimidine-4. 4-Amino-5-cyano-7- (β-D-arabinofuranosyl) pyrrolo (2,3-d) -4-pyrimidone 58 from 4-amino-5-cyano-7- (5-deoxy-h-Darabinofuranosyl) pyrrolo (2,3-d) pyrimidine. 4-amino-5-cyano-7- (5-deoxy-BD-ribofuranosyl) pyrrolo (2,3-d) -4-pyrimidone 11 of 4 amino-5-cyano-7- (5-deoxy-BD-ribofuranosyl) pyrrole ( 2,3-d) pyrimidine-4-. 4-Amino-5-cyano-7- (2,3-dideoxy-2,3-didehydro-ED-glyceropentofuranosyl) pyrrolo (2,3-d) -4-pyrimidone 50 of 4-amino-5-cyano-7 - (2,3-dideoxy-DE-pent-2-enofuranosyl) pyrrolo (2,3-d) pyrimidine. 4-amino-5-cyano-7- (2,3-dideoxy-ED-glyceropentofuranosyl) pyrrolo (2,3-d) -4-pyrimidone 3dideoxy-BD-glyceropentofuranosyl) pyrrolo [2,3-d] pyrimidine. 4 amino-5-cyano-7- (2-deoxy-ED-furanosyl) pyrrolo (2,3-d) -4-pyrimidone 69 of 4-amino-5-cyano-7- (2-deoxy-ED-eritropentofuranosyl) pyrrolo (2,3-d) pyrimidine.

Example 19

Preparation of 7- (5 (R) -C-methyl-β-D-ribofuranosyl) pyrrolo (2,3-d) -4-pyrimidone-5-carboxamidoxime 24 a

A stirred suspension of 5-cyano-7- (5- (R) -C-methyl-BD-ribofuranosyl) pyrrolo (2,3-d) -4-pyrimidone (240 mg, 0.784 mmol), hydroxylamine hydrochloride (163 mg, 2.352 mmol) and potassium carbonate (162 mg, 1.176 mmol) in ethanol (50 mL) were refluxed under argon for 18 hours. The precipitate was filtered and washed with hot ethanol. The filtrate was concentrated and the residue allowed to chromatograph on silica (20% methanol in methylene chloride) to give 170 mg of the title compound 26a (5'-R) as a colorless solid. Similarly, the following compounds were prepared: 4-amino-5-cyano-7- (β-D-arabinofuranosyl) pyrrolo (2,3-d) -4-pyrimidone-5-carboxamidoxime 60 of 4-amino-5-cyano-7- (5-deoxy-DE-arabinofuranosyl) pyrrolo (2,3-d) -4-pyrimidone. 4-Amino-5-cyano-7- (5-deoxy-BD-ribofuranosyl) pyrrolo (2,3-d) 4-pyrimidone-5-carboxamidoxime 13 of 4-amino-5-cyano-7- (5-deoxyb) D-ribofuranosyl) pyrrolo (2,3-d) -4-pyrimidone. 4-Amino-5-cyano-7- (2,3-didehydro-2,3-dideoxy-β-ribofuranosyl) pyrrolo (2,3-d) -4-pyrimidone-5-carboxamidoxime 51 of 4-amino-5-cyano-7 - (2,3didehydro-2,3-dideoxy-.beta.-D-ribofuranosyl) pyrrolo (2,3-d) -4-pyrimidone. 4-Amino-5-cyano-7- (2-deoxy-ED-ribofuranosyl) pyrrolo (2,3d) -4-pyrimidone-5-carboxamidoxime from 4-amino-5-cyano-7- (2-deoxyβ-D) -ribofuranozyl) pyrrolo (2,3-d) -4-pyrimidone.

Example 20

Preparation of 7- (5 (R) -C-methyl-β-D-ribofuranosyl) pyrrolo (2,3-d) -4-pyrimidone-5-carboxamidine hydrochloride 27a

A suspension of 7- (5 (R) -C-methyl-BD-ribofuranosyl) pyrrolo (2,3-d) 4-pyrimidone-5-carboxamidoxime (110 mg, 0.324 mmol), ammonium chloride (20 mg, 0.374 mmol) and Nickel Raney (50% sludge in water, 200 mg) in water (75 mL) was shaken in a hydrogenation apparatus (H ₂ , 3.447 x 10 ⁵ Pa) at room temperature for 18 hours. The catalyst was filtered and washed (warm water). The total filtrate was concentrated and the product was recrystallized from methanol to give 100 mg of said compound 27a (5'-R) as a colorless solid. The following compounds were prepared in a similar manner: 4-amino-5-cyano-7- (β-D-arabinofuranosyl) pyrrolo (2,3d) -4-pyrimidone-5-carboxamidine hydrochloride 63 from 4-amino-5-cyano-7- ( 5-deoxy-.beta.-D-arabinofuranosyl) pyrrolo (2,3-d) -4-pyrimidone-5-carboxamidoxime. 4-Amino-5-cyano-7- (5-deoxy-β-D-ribofuranosyl) pyrrolo (2,3-d) -4-pyrimidone-5-carboxamidine hydrochloride 15 of 4-amino-5-cyano-7- (5-deoxy-BD-ribofuranosyl) pyrrolo (2,3-d) 4-pyrimidone-5-carboxamidoxime. 4-Amino-5-cyano-7- (2-deoxy-β-Dribofuranosyl) pyrrolo (2,3-d) -4-pyrimidone-5-carboxamidine hydrochloride 70 from 4-amino-5-cyano-7- (2 deoxy-.beta.-D-ribofuranosyl) pyrrolo (2,3-d) -4-pyrimidone-5-carboxamidoxime.

Thus, the particular processing and use of pyrrolo (2,3-d) pyrimidine nucleoside analogs have been included herein. However, it should be apparent to those skilled in the art that many of the not only described changes are also possible without departing from the inventive concept described herein. Accordingly, the spirit of the present invention is not limited to the scope of the appended claims. In addition, in the explanation of the specification and claims, all terms should be explained in a broader manner in accordance with the contents. In particular, the terms include and should be construed as referring to elements, components or phases in a non-exclusive manner to indicate that said elements, components or phases may be present or used, or combined with other elements, components or phases, which are not specified.

Claims (17)

PATENT CLAIMS 1. A nucleoside analogue having the formula (I): 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 , wherein 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 ¹¹ is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl and aralkyl, wherein R ⁴ preferably has at least a heteroatom or 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 masking group; and R ⁵ 'is selected from the group consisting of alkyl, alkenyl, alkynyl and aralkyl, wherein R ⁵ ' has at least two carbon atoms and preferably has at least a heteroatom or a functional group. The nucleoside analogue of claim 1, wherein Z is CN, C (O) NH ₂ , or C (= NH) NH _2, and wherein R ⁵ 'has at least two carbon atoms and is selected from the group consisting of alkyl, alkenyl, alkynyl and aralkyl. The nucleoside analogue of claim 1, having the structure: characterized in that wherein Z is CN, C (O) NH ₂ , C (= NH) NH ₂ or C (= NOH) NH _2, and R ⁴ and R ⁵ 'are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl and aralkyl, wherein R ⁴ and R ⁵ ' independently and preferably contain at least a heteroatom or a functional group; with the proviso that R ⁴ and R ⁵ 'are not together hydrogen. 4. A method for altering cytokine secretion from a cell comprising the provided compound of Formula II; and wherein A is O, S or CH ₂ ; X is H, NH ₂ or OH; Y is H, hydrogen or NH ₂ ; Z is selected from the group consisting of H, hydrogen, 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 wherein R is alkyl, alkenyl or aralkyl; R ² or R ³ are independently selected from the group consisting of H, F and OH; R ⁴ and R ⁵ 'are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl and aralkyl, wherein R ⁴ and R ⁵ ' independently and preferably contain at least a heteroatom or a functional group; R ⁵ is H, OH, OP (OHOH); P (O) (OH) _; , OP (O) (OR '), or P (O) (OR') ₂ , wherein R 'is a masking group; and represents a cell with the compound at a concentration effective to alter cytokine secretion. The method of claim 4, wherein the cytokine is a type 1 cytokine. The method of claim 5, wherein the type 1 cytokine is IFNγ. The method of claim 4, wherein the cytokine is a type 2 cytokine. The method of claim 7, wherein the type 2 cytokine is IL-4. 9. Method lymphocyte. by claim 4. characterized the because of cell is a 10. Method by claim 4. characterized the because of cell is a cancer cell. 11. Method by claim 10. characterized the because of cancer the cell is a prostate cancer cell. 12. A method of altering cytokine secretion from a cell comprising the compound of formula III provided: C (= NH) NH _2, C (= NNH ₂₎ NH _{2 R,} or wherein Z is CN, C (0) _NH2, C (= NOOH) NH ₂ , OP (O) (OR ') 2 or wherein R ⁵ is H, OH, OP (O) (OH) ₂ , P (O) (OH) ₂ , P (O) (OR ') 2, wherein R' is a masking group; and represents a cell with the compound at a concentration effective to alter cytokine secretion. 13. A method for reducing the growth of a hyperproliferative cell comprising: wherein A is O, S or CH ₂ ; X is H, NH ₂ or OH; Y is H, hydrogen or NH ₂ ; Z is selected from the group consisting of H, hydrogen, 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 , wherein R is alkyl, alkenyl or aralkyl; R ² or R ³ are independently selected from the group consisting of H, F and OH; R ⁴ is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl and aralkyl, wherein R ⁴ preferably comprises at least a heteroatom or a functional group; R ⁵ 'is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl and aralkyl, wherein R ⁵ ' has at least two carbon atoms and preferably contains at least a heteroatom or 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 masking group; with the proviso that R ⁴ and R ⁵ 'are not together hydrogen; and represents a hyperproliferative cell with a compound at a concentration effective to reduce hyperproliferative cell growth. The method of claim 13, wherein the hyperproliferative cell is a cancer cell. The method of claim 14, wherein the cancer cell is a prostate cancer cell. 16. The method of claim 13, wherein the reduction in growth comprises a reduction in DNA synthesis. 17. A method of reducing the release of growth factor from a cell comprising the provided compound of claim 1; and represents a cell with the compound at a concentration effective to reduce growth factor release. The method of claim 17, wherein the growth factor is VEGF. 19. The method of claim 17, wherein the cell is a cancer cell. The method of claim 18, wherein the cancer cell is a prostate cancer cell.