Model of charge transfer collisions between C60 and slow ions.

A semiclassical model describing the charge transfer collisions of C60 fullerene with different slow ions has been developed to analyze available observations. These data reveal multiple Breit-Wigner-like peaks in the cross sections, with subsequent peaks of reactive cross sections decreasing in magnitude. Calculations of charge transfer probabilities, quasi-resonant cross sections, and cross sections for reactive collisions have been performed using semiempirical interaction potentials between fullerenes and ion projectiles. All computations have been carried out with realistic wave functions for C60's valence electrons derived from the simplified jellium model. The quality of these electron wave functions has been successfully verified by comparing theoretical calculations and experimental data on the small angle cross sections of resonant C60+C60 + collisions. Using the semiempirical potentials to describe resonant scattering phenomena in C60 collisions with ions and Landau-Zener charge transfer theory, we calculated theoretical cross sections for various C60 charge transfer and fragmentation reactions which agree with experiments.

Dodecafluoropentane Emulsion (DDFPe) Decreases Stroke Size and Improves Neurological Scores in a Permanent Occlusion Rat Stroke Model.

BACKGROUND: Dodecafluoropentane emulsion (DDFPe), given IV one hour after stroke, has been shown to greatly reduce the percent stroke volume (%SV) in rabbits. With repeated doses its effect continued for 24 hours. PURPOSE: Test DDFPe as neuroprotective agent in permanent occlusion rat stroke models in Sprague Dawley (SD) and Spontaneously Hypertensive Rats (SHR) measuring both %SV and neurological assessment scores (NAS). METHODS: The male rats received either saline (control), or one or four doses (1x or 4x) of DDFPe (0.6ml/kg IV) one hour post stroke. Treatment groups were SD (n=26) (control, 1x and 4x; n=12, 7 and 7) and SHR (n=14) (control, 1x and 4x; n=7, 3 and 4). The 4x doses were given at 1.5 hour intervals. At six hours post stroke, the rats received a NAS using standard tests for balance, reflexes, and motor performance. Then rats were euthanized and brains removed for TTC evaluation of %SV. RESULTS: For %SV analysis strain differences were not significant therefore strains were combined. DDFPe significantly decreased %SV in 1x and 4xDDFPe groups compared to control groups (2.59±1.81 and 0.98±0.88 vs. 9.24±6.06, p≤0.001 each; p≤0.0001 for the overall test for treatment effect). The 1x versus 4xDDFPe groups were not significantly different (p=0.40). In NAS analysis both strains showed significant improvement with 4xDDFPe therapy vs. controls, (SD: 5.00+2.45 vs. 9.36+3.56, p=0.01; SHR: 7.75+4.43 vs. 12.14+3.08, p=0.05). Differences between the 1x DDFPe group and controls were not significant (SD: 8.43+3.69; SHR: 9. 33+3.51). CONCLUSION: DDFPe treatment provides significant neuroprotection when assessed six hours post stroke.

Systematic structure-based design and stereoselective synthesis of novel multisubstituted cyclopentane derivatives with potent antiinfluenza activity.

The design and synthesis of novel, orally active, potent, and selective inhibitors of influenza neuraminidase differing structurally from existing neuraminidase inhibitors are described. X-ray crystal structures of complexes of neuraminidase with known five- and six-membered ring inhibitors revealed that potent inhibition of the enzyme is determined by the relative positions of the interacting inhibitor substituents (carboxylate, glycerol, acetamido, hydroxyl) rather than by the absolute position of the central ring. This led us to design potential neuraminidase inhibitors in which the cyclopentane ring served as a scaffold for substituents (carboxylate, guanidino, acetamido, alkyl) that would interact with the four binding pockets of the neuraminidase active site at least as effectively as those of the established six-membered ring inhibitors such as DANA (2), zanamivir (3), and oseltamivir (4). A mixture of the isomers was prepared initially. Protein crystallography of inhibitor-enzyme complexes was used to screen mixtures of isomers in order to identify the most active stereoisomer. A synthetic route to the identified candidate 50 was developed, which featured (3 + 2) cycloaddition of 2-ethylbutyronitrile oxide to methyl (1S,4R)-4[(tert-butoxycarbonyl)amino]cyclopent-2-ene-1-carboxylate (43). Structures of the synthetic compounds were verified by NMR spectroscopy using nuclear Overhauser effect methodology. Two new neuraminidase inhibitors discovered in this work, 50 and 54, have IC(50) values vs neuraminidase from influenza A and B of <1 and <10 nM, respectively. These IC(50) values are comparable or superior to those for zanamivir and oseltamivir, agents recently approved by the FDA for treatment of influenza. The synthetic route used to prepare 50 and 54 was refined so that synthesis of pure active isomer 54, which has five chiral centers, required only seven steps from readily available intermediates. Further manipulation was required to prepare deoxy derivative 50. Because the activities of the two compounds are comparable and 54 [RWJ-270201 (BCX-1812)] is the easier to synthesize, it was selected for further clinical evaluation.

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.

Purine nucleoside phosphorylase inhibitor BCX-1777 (Immucillin-H)--a novel potent and orally active immunosuppressive agent.

Patients with purine nucleoside phosphorylase (PNP) deficiency present a selective T-cell immunodeficiency. Inhibitors of PNP are, therefore, of interest as potential T-cell selective immunosuppressive agents. BCX-1777 is a potent inhibitor of PNP from various species including human, mouse, rat, monkey and dog, with IC50 values ranging from 0.48 to 1.57 nM. BCX-1777, in the presence of 2'-deoxyguanosine (dGuo, 3-10 microM), inhibits human lymphocyte proliferation activated by various agents such as interleukin-2 (IL-2), mixed lymphocyte reaction (MLR) and phytohemagglutinin (PHA) (IC50 values < 0.1-0.38 microM). BCX-1777 is a 10-100-fold more potent inhibitor of human lymphocyte proliferation than other known PNP inhibitors like PD141955 and BCX-34. Nucleotide analysis of human lymphocytes indicate that inhibition of proliferation by BCX-1777 correlates with dGTP levels in the cells. BCX-1777 has excellent oral bioavailability (63%) in mice. At a single dose of 10 mg/kg in mice, BCX-1777 elevates dGuo to approximately 5 microM. BCX-1777 was not effective in mouse T-cell models such as delayed type hypersensitivity (DTH) and splenomegaly because mouse T-cells do not accumulate dGTP as do human T-cells. However, in the human peripheral blood lymphocyte severe combined immunodeficiency (hu-PBL-SCID) mouse model, BCX-1777 was effective in prolonging the life span 2-fold or more. This is the first known example of a PNP inhibitor that elevates dGuo in mice similar to the levels observed in PNP-deficient patients. Furthermore, these dGuo levels are also required for in vitro T-cell inhibition by BCX-1777. Thus, BCX-1777 represents a novel class of selective immunosuppressive agents that could have therapeutic utility in various T-cell disorders.

Comparison of the anti-influenza virus activity of RWJ-270201 with those of oseltamivir and zanamivir.

We have recently reported an influenza virus neuraminidase inhibitor, RWJ-270201 (BCX-1812), a novel cyclopentane derivative discovered through structure-based drug design. In this paper, we compare the potency of three compounds, RWJ-270201, oseltamivir, and zanamivir, against neuraminidase enzymes from various subtypes of influenza. RWJ-270201 effectively inhibited all tested influenza A and influenza B neuraminidases in vitro, with 50% inhibitory concentrations of 0.09 to 1.4 nM for influenza A neuraminidases and 0.6 to 11 nM for influenza B neuraminidases. These values were comparable to or lower than those for oseltamivir carboxylate (GS4071) and zanamivir (GG167). RWJ-270201 demonstrated excellent selectivity (>10,000-fold) for influenza virus neuraminidase over mammalian, bacterial, or other viral neuraminidases. Oral administration of a dosage of 1 mg/kg of body weight/day of RWJ-270201 for 5 days (beginning 4 h preinfection) showed efficacy in the murine model of influenza virus infection as determined by lethality and weight loss protection. RWJ-270201 administered intranasally at 0.01 mg/kg/day in the murine influenza model demonstrated complete protection against lethality, whereas oseltamivir carboxylate and zanamivir at the same dose demonstrated only partial protection. In the delayed-treatment murine influenza model, oral administration of a 10-mg/kg/day dose of RWJ-270201 or oseltamivir (GS4104, a prodrug of GS4071) at 24 h postinfection showed significant protection against lethality (P < 0.001 versus control). However, when the treatment was delayed for 48 h, no significant protection was observed in either drug group. No drug-related toxicity was observed in mice receiving 100 mg/kg/day of RWJ-270201 for 5 days. These efficacy and safety profiles justify further consideration of RWJ-270201 for the treatment and prevention of human influenza.

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.

Striatal metabolism of hexanal, a lipid peroxidation product, in the rat.

Free radical induced lipid peroxidation may play a role in neurodegeneration and peroxidation leads to the formation of hexanal from omega-6 fatty acids. We have previously demonstrated in vitro that pyruvate dehydrogenase (PDH) catalyzes the condensation of saturated aldehydes with pyruvate to form acyloins. We have further shown in perfused rat heart that hexanal, presumably via PDH, is converted to 3-hydroxyoctan-2-one and that it in turn can be reduced to 2,3-octanediol. We now extend this work using intra-striatal microdialysis to show that this reaction also occurs in rat brain. The reduction of hexanal to hexanol was also evaluated. Microdialysis probes were implanted bilaterally in the striatum and were infused with hexanal with and in the absence of added pyruvate. Analysis of microdialysis samples showed a release of 3-hydroxyoctan-2-one (9.5-10.5 pmol/min), 2,3-octanediol (2.2-2.7 pmol/min) and hexanol (64-74 pmol/min). Pyruvate addition did not increase 3-hydroxyoctan-2-one or 2,3-octanediol production. In a second series of experiments where no exogenous hexanal was infused, endogenous production of 3-hydroxyoctan-2-one (1.0-1.3 pmol/min) and 2,3-octanediol (1.0-1.2 pmol/min) was still observed, although no hexanol was detected. We also investigated the possibility that oxidative stress induced by 1-methyl-4-phenylpyridinium (MPP+) would increase lipid peroxidation resulting in increased production of 3-hydroxyoctan-2-one. Analysis of samples collected following MPP+ infusion indicated no additional increase suggesting that brief exposure to MPP+ does not increase hexanal formation over baseline levels during the experimental period.

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.