Antitumor agents. Part 226: synthesis and cytotoxicity of 2-phenyl-4-quinolone acetic acids and their esters

2-Phenyl-4-quinolone acetic acids and their esters were synthesized and evaluated for interaction with tubulin and for cytotoxicity against a panel of human tumor cell lines. 2-Phenyl- and 2-(2'-fluorophenyl)-4-quinolone-8-acetic acids (11 and 12) displayed potent cytotoxicity with ED(50) values at nanomolar concentrations, but had minimal activity against tubulin polymerization. 2-(2'-Fluorophenyl)-4-quinolone-6-acetic acid (3) and 2-(2'-fluorophenyl)-4-quinolone-8-acetic acid methyl ester (10) moderately inhibited tubulin polymerization.     

Mutation spectrum and splicing variants in the OPA1 gene

Optic atrophy type 1 (OPA1, MIM 165500) is a dominantly inherited optic neuropathy that features low visual acuity leading in many cases to legal blindness. We have recently shown, with others, that mutations in the OPA1 gene encoding a dynamin-related mitochondrial protein, underlie the dominant form of optic atrophy. Here we report that OPA1 has eight mRNA isoforms as a result of the alternative splicing of exon 4 and two novel exons named 4b and 5b. In addition, we screened a cohort of 19 unrelated patients with dominant optic atrophy by direct sequencing of the 30 OPA1 exons (including exons 4b and 5b) and found mutations in 17 (89%) of them of which 8 were novel. A majority of these mutations were truncative (65%) and located in exons 8 to 28, but a number of them were amino acid changes predominantly found in the GTPase domain (exons 8 to 15). We hypothesize that at least two modifications of OPA1 may lead to dominant optic atrophy, that is alteration in GTPase activity and loss of the last seven C-terminal amino acids that putatively interact with other proteins.     

Photoaffinity labeling of tubulin subunits with a photoactive analogue of vinblastine

A photoactive, radioactive analogue of vinblastine, N-(p-azido[3,5-3H]benzoyl)-N'-(beta-amino-ethyl)vindesine ([ 3H]NABV), was used to localize the Vinca alkaloid binding site(s) on calf brain tubulin after establishing that its in vitro interactions with tubulin were comparable to those of vinblastine. Microtubule assembly was inhibited by 50% with 2 microM NABV or vinblastine. At higher drug concentrations, NABV and vinblastine both induced tubulin aggregation, and both drugs inhibited tubulin-dependent GTP hydrolysis. Vinblastine and NABV inhibited each other's binding to tubulin, but the binding of neither drug was inhibited by colchicine. Two classes of binding sites for NABV and vinblastine were found on calf brain tubulin. High-affinity sites had apparent KD values of 4.2 and 0.54 microM for NABV and vinblastine, respectively, whereas the low-affinity binding sites showed apparent KD values of 26 and 14 microM for NABV and vinblastine, respectively. Mixtures of tubulin and [3H]NABV were irradiated at 302 nm and analyzed for incorporation of radioactivity into protein. Photolabeling of both the alpha- and beta-subunits of tubulin with increasing concentrations of [3H]NABV exhibited a biphasic pattern characteristic of specific and nonspecific reactions. Nonspecific labeling was determined in the presence of excess vinblastine. Saturable specific covalent incorporation into both subunits of tubulin was observed, with an alpha:beta ratio of 3:2 and maximum saturable incorporation of 0.086 and 0.056 mol of [3H]NABV/mol of alpha-tubulin and beta-tubulin, respectively. Such photolabeling of the tubulin subunits will permit precise localization of Vinca alkaloid binding sites, including identification of the amino acid residues involved, an essential requirement for understanding the interactions of these drugs with tubulin.     

Structure based drug design and in vitro metabolism study: Discovery of N-(4-methylthiophenyl)-N,2-dimethyl-cyclopenta[d]pyrimidine as a potent microtubule targeting agent

We report a series of tubulin targeting agents, some of which demonstrate potent antiproliferative activities. These analogs were designed to optimize the antiproliferative activity of 1 by varying the heteroatom substituent at the 4′-position, the basicity of the 4-position amino moiety, and conformational restriction. The potential metabolites of the active compounds were also synthesized. Some compounds demonstrated single digit nanomolar IC₅₀ values for antiproliferative effects in MDA-MB-435 melanoma cells. Particularly, the S-methyl analog 3 was more potent than 1 in MDA-MB-435 cells (IC₅₀?=?4.6?nM). Incubation of 3 with human liver microsomes showed that the primary metabolite of the S-methyl moiety of 3 was the methyl sulfinyl group, as in analog 5. This metabolite was equipotent with the lead compound 1 in MDA-MB-435 cells (IC₅₀?=?7.9?nM). Molecular modeling and electrostatic surface area were determined to explain the activities of the analogs. Most of the potent compounds overcome multiple mechanisms of drug resistance and compound 3 emerged as the lead compound for further SAR and preclinical development.