Synthesis and study of chemically activated biradical precursors

The design, synthesis and study of molecules which produce 1,4-biradical intermediates upon thermal or chemical activation is described. The preparation and cyclization behavior of (Z)-1,2,4-heptatrien-6-yne and compounds that contain the (Z)-allene-ene-yne functional group or that form it in a serial reaction sequence are discussed. Evidence is presented that supports the thermal transformation of the (Z)-allene-ene-yne functional group to an α,3-dehydrotoluene intermediate that is best described as a singlet σ,π-biradical with substantial polar character. The partitioning between polar and free radical reaction pathways in these systems is shown to be influenced by biradical substitution and by the reaction medium in which the intermediate is generated. These results are discussed with reference to electrocyclization reactions occurring within the enediyne family of natural antitumor agents. The design, synthesis and reactivity of a system that produces a strained (Z)-enediyne moiety via the reductive activation of an anthraquinone-diacetylene conjugate in water with a flavin-based enzymatic system is described. The (Z)-enediyne thus produced is shown to undergo thermal rearrangement to form a naphthofuran product via a 1,4-dehydrobenzene biradical intermediate. The preparation of a molecule which forms a substituted 1,6-didehydro[10]annulene intermediate by nucleophilic addition of thiol is described. This intermediate is not observed, but rearranges to form two isomeric 1,5-dehydronaphthalene biradicals as evidenced by the isolation of the corresponding aromatized products.</p

Versatile Precursors for the Synthesis of Enynes and Enediynes

(Z)-Ethyl 2,3-dibromoproenoate, readily available in multigram quantities, undergoes selective replacement of the B-bromide in coupling reactions with trimethyl-silylacetylene to produce the (Z)-enyne 2. This product may be further elaborated to various functionalized enynes of defined geometry or may be coupled with a second acetylenic reactant to produce (Z)-enediynes.Chemistry and Chemical Biolog

Crystallographic Identification of a Noncompetitive Inhibitor Binding Site on the Hepatitis C Virus NS5B RNA Polymerase Enzyme

The virus-encoded nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) is an RNA-dependent RNA polymerase and is absolutely required for replication of the virus. NS5B exhibits significant differences from cellular polymerases and therefore has become an attractive target for anti-HCV therapy. Using a high-throughput screen, we discovered a novel NS5B inhibitor that binds to the enzyme noncompetitively with respect to nucleotide substrates. Here we report the crystal structure of NS5B complexed with this small molecule inhibitor. Unexpectedly, the inhibitor is bound within a narrow cleft on the protein's surface in the “thumb” domain, about 30 Å from the enzyme's catalytic center. The interaction between this inhibitor and NS5B occurs without dramatic changes to the structure of the protein, and sequence analysis suggests that the binding site is conserved across known HCV genotypes. Possible mechanisms of inhibition include perturbation of protein dynamics, interference with RNA binding, and disruption of enzyme oligomerization

Supplementation of Nicotinic Acid with NAMPT Inhibitors Results in Loss of In Vivo Efficacy in NAPRT1-Deficient Tumor Models

Nicotinamide adenine dinucleotide (NAD) is a metabolite essential for cell survival and generated de novo from tryptophan or recycled from nicotinamide (NAM) through the nicotinamide phosphoribosyltransferase (NAMPT)-dependent salvage pathway. Alternatively, nicotinic acid (NA) is metabolized to NAD through the nicotinic acid phosphoribosyltransferase domain containing 1 (NAPRT1)-dependent salvage pathway. Tumor cells are more reliant on the NAMPT salvage pathway making this enzyme an attractive therapeutic target. Moreover, the therapeutic index of NAMPT inhibitors may be increased by in NAPRT-deficient tumors by NA supplementation as normal tissues may regenerate NAD through NAPRT1. To confirm the latter, we tested novel NAMPT inhibitors, GNE-617 and GNE-618, in cell culture- and patient-derived tumor models. While NA did not protect NAPRT1-deficient tumor cell lines from NAMPT inhibition in vitro, it rescued efficacy of GNE-617 and GNE-618 in cell culture- and patient-derived tumor xenografts in vivo. NA co-treatment increased NAD and NAM levels in NAPRT1-deficient tumors to levels that sustained growth in vivo. Furthermore, NAM co-administration with GNE-617 led to increased tumor NAD levels and rescued in vivo efficacy as well. Importantly, tumor xenografts remained NAPRT1-deficient in the presence of NA, indicating that the NAPRT1-dependent pathway is not reactivated. Protection of NAPRT1-deficient tumors in vivo may be due to increased circulating levels of metabolites generated by mouse liver, in response to NA or through competitive reactivation of NAMPT by NAM. Our results have important implications for the development of NAMPT inhibitors when considering NA co-treatment as a rescue strategy