Synthesis and biological activity of 4'-C-hydroxymethyl-2'-fluro-D-arabinofuranosylpurine nucleosides.

A series of 4'-C-hydroxymethyl-2'-fluoro-D-arabinofuranosylpurine nucleosides was prepared and evaluated for cytotoxicity. The details of a convenient synthesis of the carbohydrate precursor 4-C-hydroxymethyl-3,5-di-O-benzoyl-2-fluoro-alpha-D-arabinofuranosyl bromide (13) are presented. Proof of the structure and configuration at all chiral centers of the sugars and the nucleosides were obtained by proton NMR. All five target nucleosides were evaluated for cytotoxicity in human tumor cell lines. The 4'-C-hydroxymethyl clofarabine analogue (16beta) showed slight cytotoxicity in CCRF-CEM leukemia cells.

Synthesis and antiproliferative activity of some 4'-C-hydroxymethyl-alpha- and -beta-D-arabino-pentofuranosyl pyrimidine nucleosides.

A suitably protected 4-C-hydroxymethyl-arabino-pentofuranose was prepared and condensed with the following nucleobases: uracil, 5-fluorouracil and thymine. The corresponding cytosine and 5-fluorocytosine derivatives have also been obtained respectively from the uracil and 5-fluorouracil nucleosides. Separation of the anomeric mixtures followed by deprotection afforded the target compounds that were found to be non-cytotoxic to CCRF-CEM leukemia cells.

Synthesis and biological activity of 4'-thio-L-xylofuranosyl nucleosides.

A series of 4'-thio-L-xylofuranosyl nucleosides were prepared and evaluated as potential anticancer and antiviral agents. The details of a convenient and high-yielding synthesis of the carbohydrate precursor 1-O-acetyl-2,3,5-tri-O-benzyl-4-thio-L-xylofuranose (6) are presented. Proof of structure and configuration at all chiral centers of the nucleosides was obtained by proton and carbon NMR. All target compounds were evaluated in a series of human cancer cell lines in culture and as antiviral agents.

Synthesis and biological activity of 4'-C-hydroxymethyl-2'-fluoro-D-arabinofuranosylpurine nucleosides.

A series of 4'-C-hydroxymethyl-2'-fluoro-D-arabinofuranosylpurine nucleosides was prepared and evaluated for cytotoxicity in human tumor cell lines. A convenient synthesis of the carbohydrate precursor 4-C-hydroxymethyl-3,5-di-O-benzoyl-2-fluoro-alpha-D-arabinofuranosyl bromide (13) was developed. Coupling of 13 with the sodium salt of 2,6-dichloropurine led to five target purine nucleosides.

Convenient syntheses of 6-methylpurine and related nucleosides.

Efficient methods for the synthesis of 6-methylpurine (3), 9-(2-deoxy-beta-D-erythro-pentofuranosyl)-6-methylpurine (8), and 6-methyl-9-beta-D-ribofuranosylpurine (5) are described. Methodology involving the (Ph3P)4Pd catalyzed cross-coupling reaction of CH3ZnBr with several different 6-chloropurine derivatives is described in high yield. This methodology now provides a facile and high-yielding synthesis of 8, which is needed in significant amounts for studies in cancer gene therapy.

A convenient synthesis of 2'-deoxy-2-fluoroadenosine; a potential prodrug for suicide gene therapy.

A convenient synthesis of 2'-deoxy-2-fluoro-adenosine (1) is described. Deaminative fluorination of 2-aminoadenosine (2) followed by silylation of the 3', 5'-hydroxyl groups gave the corresponding 2-fluoroadenosine derivative 4 in good yield. Thiocarbonylation of 4 to thiocarbonylimidazolyl derivative 5a followed by treatment with an excess of tris(trimethylsilyl)silane (TTMSS) and tert-butyl peroxide in toluene at 80 degrees C was found to affect an efficient deoxygenation to the corresponding 2'-deoxy derivative 6. Desilylation of 6 by Et4NF in CH3CN afforded 1 in high yield.

Synthesis of 4'-thio-beta-D-arabinofuranosylcytosine (4'-thio-ara-C) and comparison of its anticancer activity with that of ara-C.

4'-thio-beta-D-arabinofuranosylcytosine was synthesized by a facile route in high yields. It was evaluated for antitumor activity against a panel of human tumors, both in vitro and in vivo.

Preclinical antitumor activity of 2-chloro-9-(2-deoxy-2-fluoro-beta-D- arabinofuranosyl)adenine (Cl-F-ara-A).

Cl-F-ara-A, an analog of fludarabine, was evaluated against a spectrum of tumor systems in culture and in mice. The compound exhibited significant cytotoxicity against a variety of human tumor cell lines. More importantly, the compound showed selectivity in vivo, with excellent activity being demonstrated against human colon and renal tumors. Human nonsmall cell lung and prostate tumors were also sensitive in vivo to the compound, albeit at a reduced level.

Synthesis and structure activity relationships of 5-substituted-4'-thio-beta-D-arabinofuranosylcytosines.

Four 5-substituted (chloro, fluoro, bromo, methyl) 1-(4-thio-beta-D-arabinofuranosyl)cytosines and their alpha anomers were synthesized by a facile route in high yields. All of these nucleosides were evaluated for cytotoxicity against a panel of human tumor cell lines in vitro. Only 5-fluoro-1-(4-thio-beta-D-arabinofuranosyl)cytosine was found to be highly cytotoxic in all the cell lines and was further evaluated in vivo.

Synthesis and biological activity of 5-azacytosine nucleosides derived from 4-thio-2-deoxy-L-threo-pentofuranose and 4-thio-2-deoxy-D-erythro-pentofuranose.

1-O-Acetyl-2-deoxy-3,5-di-O-toluoyl-4-thio-D-erythro-pentofuranose and 2-deoxy-1,3,5-tri-O-acetyl-4-thio-L-threo-pentofuranose were coupled with 5-azacytosine to obtain alpha and beta anomers of nucleosides.

Synthesis and biological activity of 4'-thio-2'-deoxy purine nucleosides.

Coupling of 1-O-acetyl-2-deoxy-3,5-di-O-toluoyl-4-thio-D-ribofuranose with 6-chloropurine and 2,6-dichloropurine gave a mixture of 9 alpha and 9 beta anomers as major products. These anomers were separated and converted to 2'-deoxy-4'-thio analogues of adenosine, inosine, guanosine, 2-amino-adenosine, and 2-chloro adenosine as well as their alpha-anomers.

Gene therapy of cancer: activation of nucleoside prodrugs with E. coli purine nucleoside phosphorylase.

During the last few years, many gene therapy strategies have been developed for various disease targets. The development of anticancer gene therapy strategies to selectively generate cytotoxic nucleoside or nucleotide analogs is an attractive goal. One such approach involves the delivery of herpes simplex virus thymidine kinase followed by the acyclic nucleoside analog ganciclovir. We have developed another gene therapy methodology for the treatment of cancer that has several significant attributes. Specifically, our approach involves the delivery of E. coli purine nucleoside phosphorylase, followed by treatment with a relatively non-toxic nucleoside prodrug that is cleaved by the enzyme to a toxic compound. This presentation describes the concept, details our search for suitable prodrugs, and summarizes the current biological data.

Comparison of the mechanism of cytotoxicity of 2-chloro-9-(2-deoxy-2- fluoro-beta-D-arabinofuranosyl)adenine, 2-chloro-9-(2-deoxy-2-fluoro- beta-D-ribofuranosyl)adenine, and 2-chloro-9-(2-deoxy-2,2-difluoro- beta-D-ribofuranosyl)adenine in CEM cells.

In an effort to understand biochemical features that are important to the selective antitumor activity of 2-chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)adenine [Cl-F( upward arrow)-dAdo], we evaluated the biochemical pharmacology of three structurally similar compounds that have quite different antitumor activities. Cl-F( upward arrow)-dAdo was 50-fold more potent as an inhibitor of CEM cell growth than were either 2-chloro-9-(2-deoxy-2-fluoro-beta-D-ribofuranosyl)adenine [Cl-F( downward arrow)-dAdo] or 2-chloro-9-(2-deoxy-2, 2-difluoro-beta-D-ribofuranosyl)adenine [Cl-diF( upward arrow downward arrow)-dAdo]. The compounds were similar as substrates of deoxycytidine kinase. Similar amounts of their respective triphosphates accumulated in CEM cells, and the rate of disappearance of these metabolites was also similar. Cl-F( upward arrow)-dAdo was 10- to 30-fold more potent in its ability to inhibit the incorporation of cytidine into deoxycytidine nucleotides than either Cl-F( downward arrow)-dAdo or Cl-diF( upward arrow downward arrow)-dAdo, respectively, which indicated that ribonucleotide reductase was differentially inhibited by these three compounds. Thus, the differences in the cytotoxicity of these agents toward CEM cells were not related to quantitative differences in the phosphorylation of these agents to active forms but can mostly be accounted for by differences in the inhibition of ribonucleotide reductase activity. Furthermore, the inhibition of RNA and protein synthesis by Cl-F( downward arrow)-dAdo and Cl-diF( upward arrow downward arrow)-dAdo at concentrations similar to those required for the inhibition of DNA synthesis can help explain the poor antitumor selectivity of these two agents because all cells require RNA and protein synthesis.

Synthesis and biological activity of certain 4'-thio-D-arabinofuranosylpurine nucleosides.

A series of 4'-thio-D-arabinofuranosylpurine nucleosides was prepared and evaluated as potential anticancer agents. The details of a convenient and high-yielding synthesis of the carbohydrate precursor 1-O-acetyl-2,3,5-tri-O-benzyl-4-thio-D-arabinofuranose (6) are presented. Proof of structure and configuration at all chiral centers of the nucleosides was obtained through an X-ray crystal structure of 9alpha as well as through NOE experiments on 9beta and 9alpha. All six target compounds were evaluated in a series of human cancer cell lines in culture. Two target compounds, beta anomers with diaminopurine (12) and guanine (16) as the bases, had significant cytotoxicity. One of these compounds (12) was selected for animal studies but was found to have no selectivity at the maximum tolerated dose in the murine colon 36 tumor model.

Synthesis and antiviral evaluation of analogs of adenosine-N1-oxide and 1-(Benzyloxy)adenosine.

The activity of a series of compounds related to adenosine-N1-oxide (1) and 1-(benzyloxy)adenosine (42) against vaccinia virus has been determined both in vitro and in a vaccinia mouse tailpox model. Significant activities have been found both in vitro and in vivo for a number of the synthetic compounds.

In vivo gene therapy of cancer with E. coli purine nucleoside phosphorylase.

We have developed a new strategy for the gene therapy of cancer based on the activation of purine nucleoside analogs by transduced E. coli purine nucleoside phosphorylase (PNP, E.C. 2.4.2.1). The approach is designed to generate antimetabolites intracellularly that would be too toxic for systemic administration. To determine whether this strategy could be used to kill tumor cells without host toxicity, nude mice bearing human malignant D54MG glioma tumors expressing E. coli PNP (D54-PNP) were treated with either 6-methylpurine-2'-deoxyriboside (MeP-dR) or arabinofuranosyl-2-fluoroadenine monophosphate (F-araAMP, fludarabine, a precursor of F-araA). Both prodrugs exhibited significant antitumor activity against established D54-PNP tumors at doses that produced no discernible systemic toxicity. Significantly, MeP-dR was curative against this slow growing solid tumor after only 3 doses. The antitumor effects showed a dose dependence on both the amount of prodrug given and the level of E. coli PNP expression within tumor xenografts. These results indicated that a strategy using E. coli PNP to create highly toxic, membrane permeant compounds that kill both replicating and nonreplicating cells is feasible in vivo, further supporting development of this cancer gene therapy approach.

In vivo and in vitro pharmacologic activity of the purine nucleoside phosphorylase inhibitor BCX-34: the role of GTP and dGTP.

BCX-34 inhibits RBC PNP in vitro from humans, rats, and mice with IC50S ranging from 5 to 36 nM. BCX-34 also, in the presence but not in the absence of deoxyguanosine, inhibits human CCRF-CEM T-cell proliferation with an IC50 of 0.57 microM but not rat or mouse T-cell proliferation up to 30 microM. Inhibition of human T-cell proliferation is accompanied by an accumulation of intracellular dGTP with an associated reduction in GTP. These nucleotide changes do not occur in BC16A mouse T-cells and explain why proliferation is not inhibited by PNP inhibitors in this case. Reduction in intracellular GTP is not essential for the antiproliferative action of BCX-34. Oral bioavailability of BCX-34 in rats is 76%. BCX-34 is orally active in elevating plasma inosine in rats (2-fold at 30 mg/kg), in suppressing ex vivo RBC PNP activity in rats (98% at 3 h. 100 mg/kg), and in suppressing ex vivo skin PNP in mice (39% at 3 h, 100 mg/kg). The results demonstrate that BCX-34 inhibits human PNP and T-cell proliferation, is orally bioavailable in rodents, and pharmacologically active in vivo in rodents after oral dosing with no apparent side effects or toxicity. BCX-34 may, therefore, be useful in treating human T-cell proliferative inflammatory disorders.

Structure-based design of inhibitors of purine nucleoside phosphorylase.

Inhibitors of purine nucleoside phosphorylase may have therapeutic value in the treatment of T-cell proliferative diseases such as T-cell leukemia, in the suppression of host-versus-graft response in organ transplants, and in the treatment of T-cell-mediated autoimmune diseases. Competitive inhibitors of this enzyme have been designed using the three-dimensional structure of the enzyme determined by X-ray crystallography. This approach has resulted in the synthesis of the most potent and membrane-permeable inhibitors of purine nucleoside phosphorylase reported so far.

Effect of 9-benzyl-9-deazaguanine, a potent inhibitor of purine nucleoside phosphorylase, on the cytotoxicity and metabolism of 6-thio-2'-deoxyguanosine.

6-Thio-2'-deoxyguanosine (T-dGuo) has been reported to be both phosphorylated by deoxycytidine kinase and converted to 6-thioguanine by purine nucleoside phosphorylase (PNP). Combination of T-dGuo with an inhibitor of PNP would be expected to generate the 5'-triphosphate of T-dGuo and limit or prevent the formation of 6-thioguanosine triphosphate. Because the incorporation of 6-thioguanine into DNA is believed to be primarily responsible for the antitumor activity of the thiopurines, this treatment might result in enhanced activity against certain tumors, particularly those of T-cell origin. We have evaluated the metabolic basis of this strategy by examining the effects of 9-benzyl-9-deazaguanine (BDG), a potent inhibitor of PNP, on the metabolism of T-dGuo in CEM cells. The concentration of T-dGuo required to inhibit cell growth by 50% was approximately 50-fold greater in the presence of 8.0 microM BDG than in its absence. As expected, the addition of BDG to cells treated with T-dGuo prevented the metabolism of T-dGuo to 6-thio-guanine-containing ribo-nucleotides, but, unexpectedly, no 6-thio-2'-deoxyguanosine 5'-triphosphate was detected. In cells treated with T-dGuo plus BDG, the major phosphorylated metabolite was T-dGMP. These results indicated that even in the absence of PNP activity, T-dGuo cannot be phosphorylated directly to 6-thio-2'-deoxyguanosine 5'-triphosphate due to the inability of guanylate kinase to utilize T-dGMP as a substrate.

Phosphonate analogs of carbocyclic nucleotides.

Cyclopentadiene was converted in six steps to the key intermediate (+/-)-(1 alpha,2 beta,4 alpha)-4-amino-2-(benzyloxy)cyclopentanol (10), which in turn was converted to the carbocyclic nucleoside analogs 14 and 19 by standard procedures developed in these laboratories. Compounds 14 and 19 were then further converted to the target phosphonates 1b and 2b by modification of literature procedures. The phosphonate 1b was 40-fold more cytotoxic to HEp-2 cells than its parent, CDG, presumably after conversion to the diphosphoryl phosphonate.