SI20819A - Pyrrolo/2,3-d/pyrimidine nucleoside analogs - 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

FIELD OF THE INVENTION

Field of the invention are nucleoside analogues.

BACKGROUND OF THE INVENTION

Nucleoside analogues have long been used as antimetabolites for the treatment of cancers and viral infections. Upon entry into the cell, nucleoside analogues are often phosphorylated by salvage pathways of nucleosides, in which analogs are typically phosphorylated into the corresponding mono-, di-. and triphosphates. Triphosphorylated nucleoside analogues, among other intracellular purposes, are often used as substrates for DNA or RNA polymerases and are therefore incorporated into DNA or RNA. Where triphosphorylated nucleoside analogues are potent polymerase inhibitors, they may cause premature completion of the emerging nucleic acid molecule. When triphosphorylated nucleoside analogues are incorporated into a nucleic acid, the consequence may be replicates or transcripts, gene expression, or disruption of function.

At a more cellular level, nucleoside analogues may also disrupt the cell cycle and the particularly desirable effects of nucleoside analogues include the induction of cancer cell apoptosis. Furthermore, nucleoside analogues are also known to modulate certain immune responses.

Various nucleoside analogues are known in the art with a relatively potent anti-cancer effect. For example, known drugs include thymidylate synthase inhibitors, such as 5-fluorouridine, adenosine deaminase inhibitors, including 2-chloroadenosine, and neplanocin A, which is an inhibitor of Sadenosylhomocysteine hydrolase. However, all or almost all of the known nucleoside analogues also present a risk to normal mammalian cells, primarily because these nucleoside analogues lack adequate selectivity between normal cells and tumor cells. Unfortunately, the lack of proper selectivity is often associated with severe side effects and therefore often limits the potential of such analogue drugs.

-2-2 Although various nucleoside analogues are known in the art, all or almost all of them have one or more drawbacks. Therefore, there is still a need to provide improved processes and compositions for nucleoside analogues.

Summary of the Invention

The present invention is directed to nucleoside analogues with modifications on the sugar moieties of the pyrrolo [2,3-d] pyrimidine nucleoside analogues, which can significantly reduce the toxicity of nucleoside analogues against mammalian cells while also providing 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 analogues have desirable immunomodulatory effects, including growth of type 1 cytokines such as IL-2 and suppression of type 2 cytokines, such as IL-4. These immunomodulatory properties can be useful against cancer, viruses and autoimmune diseases, treatment of inflammation and prevention of transplant rejection.

According to one aspect of the invention, the nucleoside analog is a pyrrolo [2,3] pyrimidine nucleoside having the structure of formula (I):

wherein AO, S or CH is 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 hydrogen, alkyl, alkenyl, alkynyl and aralkyl, wherein R ₄ optionally has at least one of the heteroatoms 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 , where R ' the masking group; and R _{5 is} selected from the group consisting of alkyl, alkenyl, alkynyl and aralkyl, wherein R _{5 is} . at least two carbon atoms and optionally having at least one of

-3-3 heteroatoms and a functional group.

According to another aspect of the present invention, the nucleoside analogue is a pyrrolo [2,3d] pyrimidine nucleoside having the structure of formula (II):

(H) 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 optionally containing at least one of the heteroatoms and a functional group; with the proviso that R ₄ and R ₅ are not simultaneously hydrogen.

According to a further aspect of the present invention, the present compounds are used to inhibit tumor growth or to modulate the production of type 1 and type 2 cytokines and 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 invention, together with the accompanying drawings.

Brief description of the drawings

Figure 1 is the first illustrative synthetic scheme of the reactions involved in the production of compounds of the present invention.

Figure 2 is another illustrative synthetic scheme of the reactions involved in the production of the compounds of the present invention.

Figure 3 is a third illustrative synthetic scheme of the reactions involved in the production of the compounds of the present invention.

Figure 4 is a fourth illustrative synthetic scheme of the reactions involved in the production of the compounds of the present invention.

-4-4Figure 5 is a fifth illustrative synthetic scheme of the reactions involved in the production of the compounds of the present invention.

Figure 6 is a sixth illustrative synthetic scheme of the reactions involved in the production of the compounds of the present invention.

Figure 7 shows typical compounds of the present invention.

8A and 8B are graphs representing the effect of the compounds of the present invention on the expression of type 1 and type 2 cytokines, respectively.

Figure 9 is a table showing the cytotoxicity of various compounds of the present invention.

Figure 10 is a table showing the rates of DNA synthesis in cells treated with various compounds of the present invention.

Figure 11 is a graph showing the inhibition of VEGF release from human prostate cancer cells after treatment with the compounds of the present invention.

Figure 12 is a graph showing the inhibition of IL-8 release from human prostate cancer cells after treatment with the compounds of the present invention.

Detailed description

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

wherein AO, S or CH is ₂ ; X is H, NH ₂ or OH; Y is H, halogen or NH ₂ ; Z is selected from the group

-5-5 which consists 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 ₄ is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl and aralkyl, wherein R ₄ optionally has at least one of the heteroatoms 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 , where R ' the masking group; and R ₅ . is selected from the group consisting of alkyl, alkenyl, alkynyl and aralkyl, wherein R _{5 is} . at least two carbon atoms and optionally having at least one of the heteroatoms and a functional group.

First of all, it should be borne in mind 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 can be independently directed at the α- or β-face. Furthermore, where the substituents on C ₅ are not identical, the substitution on C ₅ 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 the heteroatoms in particular include halogens, nitrogen, oxygen and sulfur. The term 'functional group, as used herein, refers to a reactive bond (e.g. double or triple bond) or a reactive group (e.g., -OH, -SH, -NH ₂ , -N ₃ , -CN, COOH, - CHO, -CONH ₂ , etc.).

The pyrrolo [2,3-d] pyrimidine nucleoside analogues discussed are particularly those of formula (I) wherein Z is CN, C (O) NH ₂ or C (= NH) NH ₂ and in which R _{5 is} . 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 analog 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 optionally containing at least one of the heteroatoms and a functional group; with the proviso that

-6-6R ₄ and R ₅ . are not hydrogen at the same time; and wherein the remaining substituents are as defined in formula (I).

However, it should be appreciated that the compounds of the present invention may also be present in other forms and preparations as described above, and the forms discussed above, in particular, include prodrug or other modified forms in which the molecules in question are chemically and / or enzymatically modified to pharmacological and / or pharmacodynamic properties are improved, including higher specificity against target organs or cells, or subcellular compartments, and increased half-life in the organism.

For example, cholesterol adducts may be formed to increase target specificity against the liver, or adducts of apolipoproteins may be formed to improve penetration of the modified drug across the blood-brain barrier. In another example, receptor-ligand complexes can be synthesized to target a modified drug to a single cell expressing a ligand-specific receptor. Alternatively, antibody complexes or antibody fragments may be formed to increase the selective delivery of the modified drug to the subcellular location. Many of the prodrugs and modified forms are known in the art, and the prodrugs discussed in particular include the prodrugs described in U.S. Provisional Application 60/216418, filed Apr. 17, 00 and incorporated herein by reference. In still further cases, charged or uncharged groups, lipophilic or polar groups may be added to the molecules in question to increase the half-life in serum or other target organs and / or cells. In further examples, it should be appreciated that the compounds considered, when phosphorylated at the C ₅ atom, may also be di-, or tri-phosphorylated or include thiophosphate.

While it is generally preferred that the compounds in question have a sugar moiety in the D-configuration, it is also contemplated that the compounds may have a sugar moiety in the L-configuration. Further stereochemical aspects include, in particular, the R and S configurations at the C ₅ atom, where appropriate, and it is to be appreciated that the substituents in the compounds under consideration may be oriented towards the β or β phases.

It should also be appreciated that the compounds in question may be formulated in various preparations, including in the form of liquids, syrups or gels (eg for injection, ingestion or topical administration) and in solid forms (eg for ingestion, injection or ingestion). cavity). For example, when the compounds of the present invention are expected to be unstable in the gastric environment, a particular injection of a preferably isotonic solution is contemplated. Alternatively, intranasal administration or inhalation of the liquid form may be suitable to prevent acid degradation. Second

-7-7page, if the compounds in question are known to be resistant to digestive digestion, the formulations in question may be in the form of a syrup or a tablet. Depending on the particular use, the compounds under consideration may also be formulated for topical or transdermal applications. Many other known art are known in the art, all of which are also expected to be suitable in connection with the subject matter disclosed herein, and particularly expected preparations 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: 082479852Χ.

It should further be appreciated that the compounds and preparations under consideration may include functional and non-functional additives. For example, where transcutaneous drug delivery is desirable, agents may be added to improve skin penetration. Alternatively, drugs may be added, including cytostatic, antiviral or immunomodulatory agents, that synergistically or further enhance the function of the compounds under consideration. Examples of non-functional additives include fillers, antioxidants, flavoring agents or colorants to enhance the individual quality of the compounds under consideration.

With respect to the concentration of the compounds under consideration, it is preferred that the concentration is in the range of about 1 μΜ to about 100 μΜ when measured at the site of action. However, suitable concentrations may also be in the range of 999 nM to 10 nM and less, and particularly where the affinity of the compounds considered is below 1 μΜ. On the other hand, where the compounds under consideration exhibit relatively short half-lives or have a strong conversion, concentrations of 0.1 mM and 100 mM and above may be predicted. Thus, the dosage of the compounds considered may vary greatly, but suitable dosages can be readily determined in vitro or in animal experiments.

Among the various biological effects of 5'- and 4'-modified pyrrolopyrimidine nucleoside analogues, important biological effects include, in particular, modulation of type 1 and type 2 cytokine production, regulation of neoplastic states (i.e., reduction of DNA synthesis or reduction of cell growth) and reduction of chemokine and growth factor release , as described below.

Thus, the present method of altering cytokine secretion from a cell may comprise the step at which a compound of formula (I) is provided and a further step at which a compound of formula (I) is administered to a cell at a concentration that acts to alter cytokine secretion. Although all possible combinations of substituents are generally expected in Formula (I), in particular compounds of formula (I) are expected, wherein R ₄ and R ₅ . independently selected from

-8-8 groups consisting of hydrogen, alkyl, alkenyl, alkynyl and aralkyl wherein R ₄ and R ₅ . independently and optionally contain at least one of the heteroatoms and a functional group, with the remaining substituents as defined above in formula (I). In an alternative aspect, a compound used to alter cytokine secretion from a cell may also be a compound of the following structure:

wherein 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 (O) (OH) ₂ , P (O) (OH) ₂ , OP (O) (OR ') ₂ or P (O) (OR') ₂ , with R 'being the masking group.

In particular, the cytokines considered include type 1 cytokines (eg IFNγ) and type 2 cytokines (eg IL-4). With respect to cells, all cells known to produce and / or secrete cytokines are expected to be suitable, but especially expected cells include lymphocytes and cancer cells (eg, prostate cancer cells, infra).

According to a further aspect of the present invention, the process of reducing the growth of a hyperproliferative cell may comprise the step of providing a compound of formula (I) and a second step of providing the compound with a hyperproliferative compound at a concentration that acts to reduce the growth of the hyperproliferative cell. 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 optionally contains at least one of the heteroatoms and a functional group, and wherein R ₅ . selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl and aralkyl, wherein R _{5 is} . at least two carbon atoms and optionally containing at least one of the heteroatoms and a functional group, with the proviso that R ₄ and R ₅ . are not simultaneously hydrogen and the remaining substituents as defined above.

Specially anticipated hyperproliferative cells include cancer cells, and a particularly expected cancer cell is a prostate cancer cell. Although we do not want to bind to the special theory, we assume that the decrease in growth involves the reduction of DNA synthesis.

According to a further aspect of the present invention, it is expected that the process of reducing growth factor release from a cell has the degree to which the compound of formula (I) is provided and

-9-9 the second step at which a compound is administered to a cell at a concentration that acts to reduce the release of growth factor. The release of various growth factors can be expected to be reduced by the process presented herein, but we particularly expect a decrease in VEGF release. Similarly, specifically expected cells include cancer cells, and especially prostate cancer cells, although we expect all cells known to secrete growth factors in connection with the process presented herein.

With respect to the synthesis of the compounds under consideration, it should be appreciated that the pyrrolo [2,3d] pyrimidine nucleosides analogues of the present invention can be synthesized by different synthesis pathways, and the following procedures are provided for example only.

Synthesis of C5'-modified analogs of pyrrolo [2 _r 3-d] pyrimidine nucleosides

5'-substituted nucleoside analogues were prepared by condensation of pyrrolo [2,3-d] pyrimidine bases and protected 5′-substituted ribofuranoses were added. As shown in Figure 1, compound 1 was prepared according to a published procedure (Jones et al., Methods in Carbohydrate Chemistry (edited by Vhistler and Moffat), vol. VI, pp. 315-322, Academic Press, New York, (1972 )) was treated with a large number of nucleophiles, such as Grignard reagents, to produce compound 2, which was benzoylated and subsequently treated with trifluoroacetic acid to produce compound 4. Benzoylation and subsequent treatment with acetic anhydride / acetic acid in the presence of sulfuric acids gave 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 Vhistler and Moffat), vol. VI, pp. 315-322, Academic Press, New York. (1972)), prepared according to a published process, was converted to a tosylate derivative , which was reduced with lithium aluminum hydride to produce compound 8. By the 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, as shown in Scheme 2 gave compounds 10-15 as illustrated in Figure 2.

As shown in Figure 3, compound 2 was converted to sulfonate 16, which was subjected to nucleophilic substitution to produce a configuration-reversed compound 17. Removal of isopropylidene protection and subsequent acetylation gave tetraacetate 18. Condensation of 5-C-substituted protected ribofuranoses with the nucleoside bases are shown in Figure 4.

5-cyanopyrrolo [2,3-d] pyrimidine 19 prepared by the published process (Tolman et al., J. Org.

-10-10Chem., 1969, 91, 2102-2108) was converted to trimethylsilyl derivative and then condensed with compound 6 in the presence of trimethylsilyl triflate by a similar procedure described for toyocamycin (Sharma et al., Nucleosides Nucleotides, 1993, 12, 643 -648). The resulting coupling product was debuted by hydrogenation to produce compound 20. Treatment of compound 20 with ammonia in anhydrous methanol gave compound 21 and 23. Compound 21 was oxidized to produce compound 22. Compound 23 was converted to carboxamide derivative 24. Compounds 23 and 24 were oxidized to produce compound 25. Treatment of compound 25 with hydroxyamine gave compound 26, which was hydrogenated over Raney nickel to produce 27. Alternatively, compound 27 was also prepared by heating compound 25 with ammonia in an autoclave. (orig.bomb), given the vacuum.

Synthesis of C4'-modified analogs of pyrrolol2,3-d} pyrimidine nucleosides

According to Figure 5, compound 1 was treated with formaldehyde in aqueous sodium hydroxide to produce 4'-hydroxymethyl derivative 28, which was selectively protected to prepare compound 29. Further protection with DMT and removal of TBS gave compound 31 which can convert to a large number of substituents. 4-C-substituted derivatives subjected to similar transformations to 5-C-substituted ribofuranoses (Scheme 1) can be converted to compound 35 used for condensation with nucleoside bases.

Similar to C5'-substituted analogs of pyrrolopyrimidine nucleosides, 4'-substituted analogs 36 can be obtained by condensation of compound 35 with compound 19, as shown in Figure 6. The following transformations can yield 4'-C-substituted pyrrolopyrimidine nucleoside 37-42.

Synthesis of 2′-modified and other analogs of pyrrolo [2,3-d] pyrimidine nucleosides

The following analogues of pyrrolopyrimidine nucleosides were prepared for bioassay, some of which were published (listed as known compounds) and shown in Figure 7. 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. Known compound 49 (Krawczyk et al., Nucleosides Nucleotides, 1989, 8, 97-115) was treated with sodium nitrite to produce compound 50. Known compounds 45 and 48 were prepared as reported procedure (Ramasamy et al., J. Heterocyclic Chem., 1988, 25, 1043-1046). Compound 45 was treated with ammonia-methanol to produce compound 46 and hydrogenated to produce compound 47. Compounds 58-63 were prepared from compound 45 s

-11-11similar 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 70.

Human T cell preparation and in vitro activation

Peripheral blood mononucleated cells from healthy donors were isolated 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 +, <1% HLA-DR + and <5% CD25 + and were maintained in RPMI-AP5 (RPMI1640 medium containing 5% autologous plasma, 1% L-glutamine, 1% penicillin / streptomycin and 0.05% 2-mercaptoethanol). To determine cytokine protein levels, T cells (0.2 χ 10 ⁶ cells in a volume of 0.2 ml) were activated by adding 2 ng forbol myristate acetate and 0.1 mg ionomycin (PMA-ION, both from Calbiochem, San Diego, respectively). CA) and incubated in 96-well plates in the presence of 0 or 10 μΜ of different guanosine nucleosides for 48 h at 37 ° C. After activation, the supernatants were analyzed for production from the cells of the originating cytokines.

Extracellular cytokine assays.

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

Effect of analogs of pyrrolo- [2,3, d] pyrimidine nucleosides on extracellular cytokine levels in activated human T cells.

The effect of the pyro- [2,3-d] pyrimidine nucleoside analogues at 0 and 10 μΜ on the expression of type 1, IFNγ and type 2, IL-4 cytokines on PMA / ionomycin-stimulated T cells is shown in Figures 8A and 8B for 5 individual human donors. Cytokine levels were determined by ELISA in cell-free supernatants. The strongest effect was observed with 7-b-D-ribofuranzyl-4-oxopyrro- [2,3-d] pyrimidine-5-carboxamidine. This compound increased the production of activated IL-4 by 498% ± 83 and suppressed IFNγ by 43% ± 4 relative to the levels of activated control of each cytokine. Data are shown as a percentage of the activated control calculated as the ratio of cytokine levels of activated T cells in the presence of test nucleosides with respect to cytokine levels

-12-12 untreated activated cells T x 100%. A null effect on cytokine levels due to test nucleosides would give a percentage value of 100% relative to the activated control. The absolute level (pg / ml ± standard deviation) of PMA-ION induced cytokine secretion was 22954 ± 3391 for lFNy; and for IL-4 162 ± 40. Resting levels were <30 pg / ml for all cytokines tested.

Cytotoxicity of anatogo to pyrrolo [2,3-d] pyrimidine nucleosides in vitro

The pyrrolo [2,3-d] pyrimidine nucleoside analogues of the present invention are bioactive because they exhibit some level of cytotoxicity! in vitro. In these studies, the tested compounds were applied to the cell culture of normal human tibroblasts, human prostate cancer cells 81, human melanoma 140 cancer cells, human lung cancer cells 177, and human ovarian cancer cells R and NR (all available from ATCC). In these experiments, cells were plated at a density of 2000 cells per 200 μΙ of medium per well (96-well plate). The test compounds were applied to the wells once, at a concentration range of 0.78-100 μΜ immediately after cell deposition. A colorimetric MTS cytotoxicity assay was performed after 72 hours of treatment. The EC50 was calculated on the basis of the readings collected and is presented in Figure 9. The absence of cytotoxicity is indicated at a concentration below 100 μΜ of several compounds. In such cases, the EC50 is designated> 100. In other cases, EC50 indicates the concentration of test compounds required to damage 50% of the cell population.

Pyrrolo [2,3-d] pyrimidine nucleoside analogues inhibit DNA synthesis in cells cultured in vitro in a dose-dependent manner

Pyrrolo [2,3-d] pyrimidine nucleoside analogues inhibit the growth of human cells cultured in vitro as measured by DNA level. The experimental mode was the same as described above. The compounds were administered once and DNA levels were measured after 72 hours. At this time, half of the medium was removed from the culture wells and replaced with clean water. After that, the cells were transferred to -70 ° C for at least 12 hours. In the next step, cells were transferred back from −70 ° C to room temperature and added to each well 1 μΜ Hoechst 33342. After 2 h of incubation, the fluorescence signal (360-530 nm) was measured. The fluorescence intensity according to this procedure is proportional to the amount of DNA based on the presence of the resulting DNA-Hoechst 33342. The results are presented in Figure 10. The numbers express many times the increase in the amount of DNA compared to the amount of DNA at the start of the experiment (2 hours after cell deposition). For untreated prostate cancer cells and normal cells, the DNA level increased 5.78 and 4.47 times, respectively, during 72 hours of culture.

-13-13 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 effective in inhibiting the secretion of vascular endothelial growth factor (VEGF) from human prostate cancer cells, HTB81. VEGF is recognized as a marker of angiogenesis because this molecule is crucial for migration and growth of endothelial cells and microbial formation in vivo. To prove this, 0.5x10 ⁵ cells were loaded into 5 ml of culture medium in a 10 cm diameter petri dish. The compounds were administered right after application for 72 hours. The medium was then collected and the VEGF level was measured using the VEGF Elisa assay (R&D Systems) and expressed as pg VEGF per ml of medium. The results are presented in Figure 11. 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 cultured in vitro.

Compounds 23a (5'-R) and 23a (5'-S) show an inhibitory effect on the secretion of interleukin-8 (IL-8) from human prostate cancer cells, HTB81. IL-8 belongs to the class of chemically attractive chemokines (alpha type), which are knitted into inflammatory processes due to the attraction of neutrophils. Generally, chemokines are known to be produced by various types of cancer. Various studies have shown that inhibition of chemokine production via cancer cells is beneficial for the host. To demonstrate the efficacy 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 the 5′-R and 5′-S configuration at the concentrations indicated in the graph. Culture media was analyzed for IL-8 levels using the 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 the figure.

12.

However, it should be borne in mind that the biological effects of the compounds in question need not be limited to the individual effects as described above. In particular, the compounds of the present invention are generally expected to exhibit a cytostatic effect in various hyperproliferative disorders, including localized and / or metastatic cancers (e.g., lymphomas and carcinomas), benign prostatic hyperplasia, and keratoses. Although the inventors have found significant biological effects on IL-4 (type 2 cytokine) and IFNγ (type 1 cytokine), it is generally expected that the compounds of the invention will be biologically active in modulating cytokines other than IL-4 and IFN-γ. In particular, the compounds are expected to increase or decrease the expression / secretion of a single cytokine or series of cytokines. Therefore, it is expected that the compounds of the present invention may be modulated immune

-14-14 system organism so that a more pronounced type 1 or type 2 response can be obtained. Therefore, it is expected that the compounds of the present invention may be successful in reducing the virus titer in the living system or by acting as a viral inhibitor directly polymerase and / or indirectly by activating the immune system to a single humoral or cellular response. It is further expected that the compounds of the present invention may also be useful in reducing the immune system response to an allo- or xenotransplant by reducing the severity of the cellular response against an allo- or xenotransplant.

Examples

The following protocols describe the illustrative synthesis of the various compounds of the present invention and are intended only to illustrate, not limit, the inventive concept presented herein.

Preparation of methyl 2,3-O-isopropylidene-5 (R, S) -C-ethynyl-p-ribofuranoside (2b) 1 H mixed solution of methyl 4-C, 5-O-didehydro-2,3- <9-Isopropylidene-pD-ribofuranoside (Jones et al., Methods in Carbohydrate Chemistiy, vol 1, pp. 315-322 (1972), 4.00 g, 19.78 mmol) in anhydrous THF (20 mL) at - 42 ° C under argon was added dropwise ethinylmagnesium bromide (0.5 M in THF, 80 mL, 40 mmol). After addition, the resulting mixture was slowly warmed to 0 ° C (~ 90 min.). The reaction was quenched by the addition of ice (50 g) / water (50 mL) and the mixture stirred for 30 min. After neutralization with 10% aqueous 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- <3-isopropylidene-5 (R) -Cmethyl-pD-ribofuranoside (2a) was prepared from methyl 4-C, 5-O-dihydro-2,3- <9-isopropylidene-pD-ribofuranoside and ethylmagnesium bromide. Methyl 2,3-Oisopropylidene-5 (R) -C-vinyl-p-Dribofuranoside (2c) was prepared from methyl 4-C, 5-O-dihydro-2,3- <9-isopropylidene-pD-ribofuranoside and vinylmagnesium of bromide. Methyl 5 (R) -C-allyl-2,3-h isopropylidene-pD-ribofuranoside (2d) was prepared from methyl 4-C, 5-O-dihydro-2,3-Oisopropylidene-pD-ribofuranoside and allylmagnesium bromide .

Preparation of 2,3-0-isopropridene-5-0-methanesulfonyl-5 (R) -C-methyl-p-Dribofuranoside criteria (16)

To a stirred solution of methyl 2,3- <9-isopropylidene-5 (R) 'C-methyl-β-D-ribofuranoside (2a, 7.24 g,

-15-1533.17 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 ° C, quenched by the addition of 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 three times with brine, dried (NaHSO4) and concentrated. Chromatography on silica (30% EtOAc in hexanes) gave 8.62 g of the title compound as a colorless syrup.

Preparation of methyl 2,3-0-isopropylidene-5-0-acetyl-5 (S) -C-methyl-β-D ribofuranoside (17)

A mixed suspension of methyl 2,3-isopropylidene-5- <3-methanesulfonyl-5 (R) -C-methyl-pD-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 was chromatographed on silica (25% EtOAc in hexanes) to give 4.0 g of the title compound as a white solid.

Preparing throws! 2,3-0-Isopropylidene-4-C-hydroxymethyl-fi-D-ribofuranoside (28)

To a stirred solution of methyl 4-C, 5-O-didehydro-2,3- <3-isopropylidene-pD-ribofuranoside 1 (20.22 g, 100 mmol) in dioxane (380 mL) at 0 ° C was added dropwise. formaldehyde (37% solution, 76 mL) followed by 2 M NaOH (188 mL). The resulting reaction mixture was stirred at room temperature for 20 h, cooled to 0 ° C, neutralized (10% acetic acid), concentrated (~ 50%) and extracted twice with methylene chloride. The combined organic layer was dried (Na2SO ₄₎ and concentrated to dryness. Chromatography on silica (4% methanol in chloroform) gave 20.2 g of the title 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-CMzopropylidene-pD-ribofuranoside (14.2 g, 70.0 mmol) in anhydrous pyridine (250 mL) at 10 ° C was added portionwise (over 30 min) p-toluenesulfonyl chloride ( 19.1 g, 100 mmol). The resulting mixture was stirred at room temperature for 18 h, cooled to 0 ° C, quenched by the addition of water (5.0 mL) and stirred at room temperature for 30 min. The solvent was evaporated. The residue was 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) gave 24.1 g of the title compound as a white solid.

-16-16V stirred suspension of LiAIH ₄ (4.58 g, 120.5 mmol) in anhydrous diethyl ether (120 mL) was added methyl 2,3-0-isopropylidene-5-0-p-toluenesulfonyl-pD-ribofuranoside ( 13.1 g, 36.55 mmol) in diethyl ether-toluene (2.5: 1, 140 mL). The resulting mixture was refluxed for 22 h, cooled to room temperature, diluted with ethyl acetate (25 mL), quenched by the addition of water (5.0 mL). The solvent was evaporated. The residue was 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) gave 3.58 g of the title compound as a colorless liquid.

Preparing throws! 5 (R) -C-Allyl-5-O-benzoyl-2,3-O-isopropylidene-β-D-ribofuranoside (3d)

Benzoyl chloride was added to a stirred solution of methyl 5 (R) -C-allyl-2,3-0-isoproplidene-pD-ribofuranoside (4.49 g, 18.38 mmol) in anhydrous pyridine (40 mL) at 0 ° C. (2.7 mL, 23.0 mmol). The resulting mixture was stirred at room temperature for 18 h, cooled with ice, quenched by the addition of water (1 mL) and stirred at room temperature for 30 min. The solvent was evaporated and the residue dissolved in ethyl acetate, washed three times with brine, dried (NagSO4 and concentrated. Silica chromatography (12% ethyl acetate in hexanes) gave 6.26 g of the title compound 3d as a colorless syrup. The following compounds were prepared: Methyl 5-0-benzoyl-5 (R, S) -C-ethynyl-2,3-0-isoproplidene-pD-ribofuranoside (3b, R / S ratio: 1: 1) from methyl 5 (R , S) -C-ethynyl-2,3-disopropylidene-pD-ribofuranoside (2b) Methyl 4-C-benzoyloxymethyl5-0-benzoyl-2,3-0-isoproplidene-pD-ribofuranoside from methyl 2,3 -O-isoproplidene-4-C-hydroxymethyl-β-D-ribofuranoside.

Preparing throws! 5 (R) -C-alkyl-5-O-benzoyl-β-D-ribofuranoside (4d)

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

-17-17Preparing targets! 5 (R) -C-N-2,3,5-tri-O-benzoyl- [1D-ribofuranoside (Sd) mixed solution of methyl 5 (R) -C-allyl-5-Obenzoyl-pD-ribofuranoside (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 h, cooled with ice, quenched by the addition of water (1 mL), then stirred at room temperature for 30 min. The mixture was concentrated, diluted with ethyl acetate, washed three times with brine, dried (Na ₂ SO ₄ ) and concentrated to dryness. Chromatography on silica (15% ethyl acetate in hexanes) gave 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-C> -benzoyl-pD-ribofuranoside (5b, R / S ratio: 1: 1) from methyl 5-6 &apos; -benzolyl-5 (R, S) -C-ethynyl-pD-ribofuranoside (4b). Methyl 4-C-benzoyl-oxomethyl-2,3,5-trifluorobenzoyl-pD-ribofuranoside from methyl 4-C-benzoyloxymethyl-5-Obenzoyl-pD-ribofuranoside.

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

A solution of methyl 2,3- <9-isopropylidene-5 (R) -C-vinyl-pD-ribofuranosise (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 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 dissolved in anhydrous pyridine (11 mL). Benzoyl chloride (1.9 mL, 16 mmol) was added to this solution. The reaction mixture was stirred at 25 ° C 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 (0-5% ethyl acetate in dichloromethane) to produce 1.0 g of the title compound 5c as syrup.

Preparation of 1-O-acetyl-2,3,5-tri-O-benzoyl-5 (R) -C-allyl-D-ribofuranose (6d) a mixed solution of methyl 5 (R) -C-allyl-2, 3,5-tri-tert -benzoyl-pD-ribofuranoside (5d, 4.0 g, 7.74 mmol) in acetic acid (14 mL) and acetic anhydride (1.75 mL, 18.36 mmol) at 0 Concentrated sulfuric acid (200 pL, 3.79 mmol in 4.0 mL acetic acid) was added at C. The resulting mixture was stirred at room temperature for 20 h, cooled to 0 ° C, diluted with cold ethyl acetate, washed with water, 5% aqueous NaHCO ₃ and then brine, dried (Na2SO4 and concentrated. Silica chromatography. (ethyl acetate-hexanes 1: 4) gave 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: 1-h> acetyl-5 (R. S) -C-ethynyl-2,3,5-tri-benzolyl-pD-ribofuranose (6b, R / S ratio: 1: 1

-18-18and α / β ratio: 1: 2) from methyl 5 (R, S) -C-ethynyl-2,3,5-tri-tert -benzolyl-p-D-ribofuranoside (5b). 1-Oacetyl-4-C-benzoyloxymethyl-2,3,5-tri-C> -benzoyl-D-ribofuranose (α / β ratio: 1: 3) from methyl 4-C-benzoyloxymethyl-2,3,5- tri-tb-benzoyl-pD-ribofuranoside. 5 (R) -C-methyl-1,2,3,5-tetra-3-acetylβ-D-ribofuranose from methyl 2,3- <9-isopropylidene-5 (R) -C-methyl-pD-ribofuranoside .

1.2.3.5-Tetra-acetyl-5 (S) -C-methyl-D-benzofuranose 6a from methyl 5- <3-acetyl-2,3-d-isopropylidene (R) -C-methyl-β-D-ribofuranoside . 5-deoxy-1,2,3-tri-3-acetyl-D-ribofuranoso 9 from methyl 5-Oacetyl-2,3- 3-isopropylidene-β-D-ribofuranoside. 1-acetyl-2,3,5-tri-benzoyl-5 (R) -C-vinyl-p-Dribofuranose 6c from methyl 2,3,5-tri-O-benzoyl-5 (R) - C-vinyl-pD-ribofuranoside.

Preparation of 4-amino-6-bromo-5-cyano-7- (2,3,5-tri-O-benzoyl-5 (R) -C-allyl-p-Dribofuranosyl) pyrrolo [2,3-d] pyrimidine

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 (75 mL) and anhydrous m-xylene (25 mL) were refluxed under argon for 18 h. The solvents were evaporated and the residue was dried in vacuo. The residue was dissolved in anhydrous 1,2-dichloroethane (80 mL) and mixed with 1-0-acetyl-2,3,5-tri-O-benzoyl-5 (R) -C-allyl-D-ribofuranose (2, 00 g, 3.67 mmol). TMSOTf (1.3 mL, 7.30 mmol in 5 mL of anhydrous 1,2-dichloroethane) was added while cooling with ice. The mixture under argon was stirred at room temperature for 30 min, then refluxed for 90 h, quenched by pouring (cold) into 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) gave 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 - (> benzoyl-5 (R, S) -C-ethynyl-pD-ribofuranosyl ) pyrrolo [2,3-d] pyrimidine (R / S ratio: 1: 1) was prepared from 1-acetyl-2,3,5-tri-3-benzolyl-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-enzoyloxomethyl-2,3,5-trifluoro-benzoyl-pD-ribofuranosyl) pyrrolo [2,3-d] pyrimidine was prepared from 1 -O-acetyl-4-benzoyloxymethyl-2,3,5-tri-3-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-acetyl-5 (R) -C-methyl-pD-ribofuranosyl) pyrrolo [2,3-oq-pyrimidine we prepared from

1.2.3.5-Tetra-O-ethyl-5 (R) -C-methyl-D-ribofuranose and 4-amino-6-bromo-5-cyanopyrrolo [2,3d] pyrimidine. 4-Amino-6-bromo-5-cyano-7- (1,2,3,5-tetra-acetyl-5 (S) -C-methyl-pD-ibofuranosyl) pyrrolo [2,3- </RTI> ] Pyrimidine was prepared from 1,2,3,5-tetra-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-O-acetyl5-deoxy-D-ribofuranosyl) pyrrolo [2,3-th] pyrimidine was prepared from 1,2,3- tri-C> -acetyl-5-deoxy-Dribofuranose and 4-amino-6-bromo-5-cyanopyrrolo [2,3-d] pyrimidine. 4-amino-6-bromo-5-cyano-7-19-19 (2,3,5-tri-3-benzoyl-5 (R) -C-vinyl-pD-ribofuranosyl) pyrrolo [2,3- and pyrimidine was prepared from 1-0acetyl-2,3,5-tri-O-benzoyl-5 (R) -C-vinyl-pD-ribofuranose and 4-amino-6-bromo-5-cyanopyrrolo- [2 , 3i /] pyrimidine.

Preparation of 4-amino-5-cyano-7- (2,3,5-tri-O-benzoyl-5 (Ft) -C-allyl-f-Dribofuranosyl) pyrrolo [2,3-d] pyrimidine (2Oe)

To a solution of 4-amino-6-bromo-5-cyano-7- (2,3,5-tri-3-benzoyl-5 (R) -C-allyl-pD-ribofuranosyl) pyrrolo [2,3-tf ] Pyrimidine (738 mg, 1.0 mmol) in acetic acid (25 mL) was added zinc powder (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 was chromatographed on silica (ethyl acetate-hexanes 1: 1) to give 450 mg of the title compound 20e as a colorless foam. The following compounds were prepared in a similar manner: 4-amino-5 cyano-7- (2,3,5-trifluoro-benzoyl-5 (R, S) -C-ethynyl-pD-ribofuranosyl) pyrrolo [2, 3-tf] pyrimidine (R / S ratio: 1: 1) 20b from 4-amino-6-bromo-5-cyano-7- (2,3,5-tri-C> -benzoyl-5 (R, S (-C-ethynyl-pD-ribofuranosyl) pyrrolo [2,3- d] pyrimidine. 4-amino-5-cyano-7- (2,3,5-tri-2-benzoyl-5 (R) -C-vinyl-pD-ribofuranosyl) pyrrolo [2,3-d] pyrimidine 20c from 4 -amino-6-bromo-5-cyano-7- (2,3,5-tri-3-benzoyl-5 (R) -C ^: vinyl-pD-ribofuranosyl) pyrrolo [2,3- </i>] pyrimidine .

Preparation of 4-amino-5-cyano-7- (2,3,5-tri-O-benzoyl-5 (R) -C-propyl-p-Dribofuranosyl) 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-pD-ribofuranosyl) pyrrolo [2,3-th] pyrimidine (400 mg, 0.54 mmol) and 10% Pd / C (100 mg, -50% water) in dioxane (50 mL) and triethylamine (0.5 mL) were shaken in a hydrogenation apparatus (H ₂ , 20 psi (= 137.9 kPa - transl. Note)) within 4 h. The catalyst was filtered and washed (dioxane). The combined filtrate was concentrated and the residue was chromatographed on silica (ethyl acetate-hexanes 1: 1) to produce 340 g of the title compound 20f as a colorless foam. The following compounds were prepared in a similar manner: 4-amino-5-cyano-7- (2,3,5-tri-9-benzoyl-5 (R, S) -C-ethyl-pD-ribofuranosyl) pyrrolo [2 , 3- <7] pyrimidine (R / S ratio: 1: 1) 20d from 4-amino-6-bromo-5-cyano-7- (2,3,5-trichloxy) benzoyl-5 (R, S) -C-ethynyl-pD-ribofuranosyl) pyrrolo [2,3-d] pyrimidine. 4-amino-5-cyano-7- (4-benzoyloxymethyl-2,3,5-tri-C3-benzoyl-p-ribofuranosyl) pyrrolo [2,3-d] pyrimidine from 4-amino-bromo-S- cyano-3H-benzoyloxomethyl ^ .SS-tri-CLbenzoyl-pD-ribofuranosylpyrrolo [.S-tf] pyrimidine. 4-amino-5-cyano-7- (1,2,3,5-tetra-Oacetyl-5 (R) -C-methyl-pD-ribofuranosyl) pyrrolo [2,3 </i>] pyrimidine 20a from 4-amino -6-bromo-5-cyano-7- (1,2,3,5-tetra-Oacetyl-5 (R) -C-methyl-pD-20-20ribofuranosyl) pyrrolo [2,3-h] pyrimidine. 4-amino-5-cyano-7- (1,2,3,5-tetra-0-acetyl-5 (S) -C-methyl-D-ribofuranosyl) pyrrolo [2,3-b] pyrimidine 20a from 4-amino-6-bromo-5-cyano-7- (1,2,3,5-tetra-acetyl-5 (S) -C-methyl-pD-ribofuranosyl) pyrrolo [2,3-t] ] pyrimidine. 4-amino-5-cyano-7- (1,2,3-tri-3-acetyl-5-deoxy-pD-ribofuranosyl) pyrrolo [2,3-d] pyrimidine from 4-amino-6-bromo-5 -cyano-7- (1,2,3tri-9-acetyl-5-deoxy-pD-ribofuranosyl) pyrrolo [2,3- b] pyrimidine. 4-amino-5-cyano-7- (2,3-dideoxy-p-glycero-pentofuranosyl) pyrrolo [2,3-c] pyrimidine was prepared from 4-amino-5-cyano-7- (2,3-dideoxy) -pD-pent-2-enofuranosyl) pyrrolo [2,3-h] pyrimidine.

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

A solution of 4-amino-5-cyano-7- (2,3,5-tri-3-benzoyl-5 (R) -C-allyl-pD-ribofuranosyl) pyrrolo [2,3d] pyrimidine (300 mg, 0.454 mmol) in methanol (40 ml_) at 0 ° C was saturated with ammonia. The solution was kept at room temperature for 2 days. The 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. The solvent was evaporated. The residue was adsorbed on silica gel and eluted from the silica gel column (methanol-ethyl acetate 1:25) to produce 145 mg of the title compound as a colorless solid.

Prior to heating in DMF, the product contained two major compounds 21 and 23 which could be separated by chromatography on silica gel. Compounds 21 were prepared by this procedure. The following compounds were prepared in a similar manner: 4-amino-5-cyano-7- (5 (R) -C-propyl-pD-ribofuranosyl) pyrrolo [2,3-c] pyrimidine 23f from 4-amino-5- cyano-7- (2,3,5-tri-O-benzoyl-5 (R) -C-propyl-p-Dribofuranosyl) -pyrrolo [2,3-d] pyrimidine. 4-amino-5-cyano-7- (5 (R, S) -C-ethynyl-p-ribofuranosyl) pyrrolo [2,3-d] pyrimidine (RS ratio: 1: 1) 23b from 4-amino- 5-cyano-7- (2,3,5-tri-O-benzoyl-5 (R, S) C-ethynyl-p-ribofuranosyl) pyrrolo [2,3-d] pyrimidine. 4-amino-5-cyano-7- (5 (R, S) -C-ethyl-pD-ribofuranosyl) pyrrolo [2,3-c] pyrimidine (RS ratio: 1: 1) 23d from 4-amino- 5-cyano-7- (2,3,5-tri-Obenzoyl-5 (R, S) -C-ethyl-pD-ribofuranosyl) pyrrolo [2,3-d] pyrimidine. 4-amino-5-cyano-7- (4-hydroxymethyl-p-ribofuranosyl) pyrrolo [2,3-c] pyrimidine 33d from 4-amino-5-cyano-7- (4-benzoyloxomethyl2,3,5-tri- O-benzoyl-pD-riboturanosyl) pyrrolo [2,3-d] pyrimidine. 4-amino-5-cyano-7- (5 (R) -C-methyl-D-ribofuranosyl) pyrrolo [2,3-b] pyrimidine 23a (5'-R) from 4-amino-5-cyano- 7- (1,2,3,5-tetra-O-acetyl5 (R) -C-methyl-pD-ribofuranosyl) pyrrolo [2,3-h] pyrimidine. 4-amino-5-cyano-7- (5 (S) -C-methyl-p-Dribofuranosyl) pyrrolo [2,3-d] pyrimidine 23a (5'-S) from 4-amino-5-cyano- 7- (1,2,3,5-Tetra-O-acetyl-5 (S) C-methyl-pD-ribofuranosyl) pyrrolo [2,3-b] pyrimidine. 4-amino-5-cyano-7- (5-deoxy-p-ribofuranosyl) pyrrolo [2,3-c] pyrimidine 10 from 4-amino-5-cyano-7- (1,2,3-tri-Oacetyl) -5-deoxy-pD-ribofuranosyl) pyrrolo [2,3-th] pyrimidine. 4-3ΐτίηο-5-α3ηο-7- (5 (Η) -6 ^: -νίηίΙ-ρ-Ο-Γ ^ οίυΓ3ηοζϊΙ) ρίΓθΙο [2,3-α ^, ] ρίΓίιτιί-21-21din 23c from 4-amino-5 -cyano-7- (2,3,5-tri-9-benzoyl-5 (R) -C-vinyl-pD-ribofuranosyl) pyrrolo [2,3-pyrimidine.

Preparation of 4-amino-5-cyano-7- (2,3-di-O-methanesulfonyl-5-0-tert-butyldiphenylsilyl-B-Dribofuranosyl) pyrrolo [2,3-d] pyrimidine

To a stirred solution of toyocamycin 43 (5.83 g, 20.0 mmol) in anhydrous pyridine (100 mL) at 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 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 the addition of water (2 mL) and stirred at room temperature for 30 min. The solvent was evaporated. The residue was dissolved in ethyl acetate, washed three times with brine, dried (Na ₂ SO ₄ ) and concentrated. Chromatography on silica (ethyl acetate-hexanes 3: 2) gave 8.41 g of the title compound as a colorless solid.

Preparation of 4-amino-5-cyano-7- (5-O-tert-butyldiphenylsilyl-2,3-didehydro-2,3-dideoxy-p-Dribofuranosyl) pyrrolo [2,3-d] pyrimidine

Tellurium powder (200 mesh screen, 640 mg, 5.0 mmol) was sealed under argon, 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 ° C, and 4-amino-5-cyano-7- (2,3-di- 3methanesulfonyl-5- <9-tert-butyldiphenylsilyl-pD was added -ribofuranosyl) pyrrolo [2,3-c] pyrimidine (1.40 g, 2.09 mmol) in THF (12 mL). The resulting mixture was stirred at room temperature for 18 h, cooled with ice, quenched by the addition of water (0 ° C, 5 mL) and stirred at room temperature for 30 min. The solvent was evaporated and the residue was extracted with ethyl acetate. The extracts were concentrated and the residue was chromatographed on silica (15% ethyl acetate in hexanes) to produce 640 mg of the title compound as a colorless foam.

Preparation of 4-amino-5-cyano-7- (2,3-didehydro-2,3-dideoxy-p-D-ribofuranosyl) pyrrolo [2,3-d] pyrimidine (49)

To a mixed solution of 4-amino-5-cyano-7- (5- <9-tert-butyldiphenylsilyl-2,3-didehydro-2,3-dideoxy-pD-ribofuranosyl) pyrrolo [2,3-d] pyrimidine ( 2.55 g, 5.32 mmol) in anhydrous THF (100 mL) at 5 ° 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)

-22-22 acetate) gave 1.09 g of the title compound 49 as a colorless solid.

Preparation of 5-cyano-7- (5 (R) -C-methyl-p-D-ribofuranosyl) pyrrolo [2,3-d] -4-pyrimidone (25a)

Into a mixed solution of 4-amino-5-cyano-7- (5 (R) -C-methyl-pD-ribofuranosyl) -pyrrolo [2,3-c] pyrimidine (306 mg, 1.0 mmol) in water (30 mL) and acetic acid (2.0 mL) at 55 ° C were added portionwise of sodium nitrite (590 mg, 8.55 mmol). The resulting mixture was stirred at 70 ° C for 3 h and sodium nitrite (300 mg, 4.30 mmol) was added. The mixture was stirred at the same temperature for an additional 18 h. The solvent was evaporated and the residue was chromatographed on silica (12% methanol in methylene chloride) to give 210 mg of the title compound 25a (5'-R) as a colorless solid. The following compounds were similarly prepared: 4-amino-5-cyano-7- (5 (S) -C-methyl-pD-ribofuranosyl) pyrrolo [2,3-th] -4-pyrimidone 25a (5'-S) from 4-amino-5-cyano-7- (5 (S) -C-methyl-pD-ribofuranosyl) -pyrrolo [2,3-b] pyrimidine. 4-amino-5-cyano-7- (p-arabinofuranosyl) pyrrolo [2,3-o] -4-pyrimidone 58 from 4-amino-5-cyano-7- (5-deoxy-pD-arabinofuranose) pyrrolo [2,3- £ /] pyrimidine. 4-amino-5-cyano7- (5-deoxy-pD-ribofuranosyl) pyrrolo [2,3-d] -4-pyrimidone 11 from 4-amino-5-cyano-7- (5-deoxy-p-Dribofuranosyl) pyrrolo [2,3- £ /] pyrimidine. 4-amino-5-cyano-7- (2,3-dideoxy-2,3-didehydro-p-Dglycero-pento-furanosyl) pyrrolo [2,3- d] -4-pyrimidone 50 from 4-amino- 5-cyano-7- (2,3-dideoxy-p-Dpent-2-enofuranosyl) pyrrolo [2,3-6] pyrimidine. 4-Amino-5-cyano-7- (2,3-dideoxy-p-D-glyceropentofuranosyl) pyrrolo [2,3-th] -4-pyrimidone 65 from 4-amino-5-cyano-7- (2,3- dideoxy-pD-glyceropentofuranosyl) pyrrolo [2,3-th] pyrimidine. 4-amino-5-cyano-7- (2-deoxy-pD-furanosyl) pyrrolo [2,3h] -4-pyrimidone 69 from 4-amino-5-cyano-7- (2-deoxy-pD-erythropentofuranosyl ) pyrrolo [2,3-cQyrimidine.

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

Mixed suspension of 5-cyano-7- (5 (R) -C-methyl-β-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 h. The precipitate 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 title compound 26a (5′-R) as a colorless solid. Similarly, the following compounds were prepared: 4-amino-5-cyano7- (pD-arabinofuranosyl) pyrrolo [2,3-th] -4-pyrimidone-5-carboxamidoxime 60 from 4-amino-5-cyano7- (5-deoxy- pD-arabinofuranose) -pyrrolo [2,3-d] -4-pyrimidone.

-23-234-amino-5-cyano-7- (5-deoxy-p-ribofuranosyl) pyrrolo [2,3-c] -4-pyrimidone-5-carboxamidoxime 13 from 4-amino-5-cyano-7 - (5-deoxy-pD-ribofuranosyl) pyrrolo [2,3-o]] - 4-pyrimidone. 4-amino-5-cyano-7- (2,3-didehydro-2,3-dideoxy-p-ribofuranosyl) pyrrolo [2,3-d] -4-pyrimidone-5-carboxamidoxime 51 from 4-amino-5- cyano-7- (2,3-didehydro-2,3-dideoxy-pD-ribofuranosyl) pyrrolo [2,3-d] 4-pyrimidone. 4-amino-5-cyano-7- (2-deoxy-p-ribofuranosyl) pyrrolo [2,3-c] -4-pyrimidone-5-carboxamidoxime from 4-amino-5-cyano-7- (2- deoxy-pD-ribofuranosyl) pyrrolo [2,3-b] -4-pyrimidone.

Preparation of 7- (5 (R) -C-methyl-t-D-ribofuranosyl) pyrrolo [2,3-d] -4-pyrimidone-5-carboxamidine hydroxy / oride (27a)

A suspension of 7- (5 (R) -C-methyl-pD-ribofuranosyl) pyrrolo [2,3-d] -4-pyrimidone-5-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 (= 344.7 kPa)) at room temperature for 18 hours. h. The catalyst was filtered and washed (warm water).

The combined filtrate was concentrated and the product was recrystallized from methanol to produce 100 mg of the title compound 27a (5'-R) as a colorless solid. In a similar manner, the following compounds were prepared: 4-amino-5-cyano-7- (p-arabinofuranosyl) pyrrolo [2,3-b] -4-pyrimidone-5carboxamidine hydrochloride 63 from 4-amino-5-cyano-7 - (5-deoxy-pD-arabinofuranose) pyrrolo [2,3r] -4-pyrimidone-5-carboxamidoxime. 4-amino-5-cyano-7- (5-deoxy-p-ribofuranosyl) -pyrrolo [2,3-th] -4-pyrimidone-5-carboxamidine hydrochloride 15 from 4-amino-5-cyano-7- ( 5-deoxy-pD-ribofuranosyl) pyrrolo [2,3-d] -4-pyrimidone-5-carboxamidoxime. 4-amino-5-cyano-7- (2-deoxy-p-Dribofuranosyl) pyrrolo [2,3-oQ-4-pyrimidone-5-carboxamidine hydrochloride 70 from 4-amino-5-cyano-7- (2deoxy- pD-ribofuranosyl) pyrrolo [2,3-d] -4-pyrimidone-5-carboxamidoxime.

Thus, we have described the specific embodiments and applications of the pyrrolo [2,3-d] pyrimidine nucleoside analogues. It will be apparent to those skilled in the art that, in addition to those already described, many modifications are possible without departing from inventive concepts here. Therefore, the object of the present invention is not to be limited, except in the spirit of the appended claims. In addition, 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 'scope' and 'scope' are to be construed as referring to elements, components, or stages in a non-exclusive manner indicating that those elements, components or stages may be present, or used,

-24-24or combined with other elements, components or grades not explicitly stated.

Claims (18)

PATENT APPLICATIONS 1. A nucleoside analog of formula (I): X Y R 1 - wherein AO, 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 hydrogen, alkyl, alkenyl, alkynyl and aralkyl, wherein R ₄ optionally has at least one of the heteroatoms and a functional group; R ₅ is OH, OP (O) (OH) ₂ , P (O) (OH) ₂ , OP (O) (OR ') ₂ or P (O) (OR') ₂ , wherein R ^{1 is a} 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 optionally has at least one of heteroatoms and 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 is} . 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 Rj R ₂ -26-26 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 optionally contain at least one of the heteroatoms and a functional group; with the proviso that R ₄ and R ₅ . are not hydrogen at the same time. Use of a compound of formula II in the manufacture of a medicament for altering cytokine secretion from a cell comprising: providing a compound of formula (II); and wherein AO, 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, afkenyl, alkynyl or aralkyl; R ₂ and 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 optionally containing at least one of the heteroatoms 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 , where R ' the masking group; and -27-2 Administration of a compound to a cell at a concentration that acts to alter cytokine secretion. The use of claim 4, wherein the cytokine is a type 1 cytokine. The use of claim 5, wherein the type 1 cytokine is IFNγ. The use of claim 4, wherein the cytokine is a type 2 cytokine. The use of claim 7, wherein the type 2 cytokine is IL-4. The use of claim 4, wherein the cell is a lymphocyte. The use of claim 4, wherein the cell is a cancer cell. The use of claim 10, wherein the cancer cell is a prostate cancer cell. Use of a compound of formula lil in the manufacture of a medicament for altering cytokine secretion from a cell comprising: providing a compound of formula (lil) (NI) HO OH wherein Z is CN, C (O) NH ₂ , C (= NH) NH ₂ , C (= NNH ₂ ) NH ₂ or -C (= NOH) NH ₂ ; wherein R _{5 is} H, OH, OP (O) (OH) ₂ , P (O) (OH) ₂ , OP (O) (OR ') _2, or P (O) (OR') ₂ , wherein R 'masking group; and administering the compound to a cell at a concentration that acts to alter cytokine secretion. -28-2813. Use of a compound of formula IV in the manufacture of a medicament for reducing the growth of a hyperproliferative cell, comprising: providing a compound of formula (IV); wherein AO, S or CH is 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 hydrogen, alkyl, alkenyl, alkynyl and aralkyl, wherein R ₄ optionally contains at least one of the heteroatoms and a functional group; R ₅ is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl and aralkyl, wherein R ₅ i has at least two carbon atoms and optionally contains at least one of the heteroatoms 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 , where R ' the masking group, with the proviso that R ₄ and R ₅ are not simultaneously hydrogen; and administering the compound to a hyperproliferative cell at a concentration that has the effect of reducing the growth of the hyperproliferative cell. The use of claim 13, wherein the hyperproliferative cell is a cancer cell. -29-2915. The use of claim 14, wherein the cancer cell is a prostate cancer cell. The use of claim 13, wherein the decrease in growth comprises a decrease in DNA synthesis. Use of a compound according to claim 1 in the manufacture of a medicament for reducing the release of growth factor from a cell comprising: providing a compound according to claim 1; and administering to the cell a compound at a concentration that acts to reduce the release of growth factor. The use of claim 17, wherein the growth factor is VEGF. The use of claim 17, wherein the cell is a cancer cell. The use of claim 19, wherein the cancer cell is a prostate cancer cell.