Shelby N. Waddell

Targeting the colchicine-binding site on tubulin represents a promising strategy for developing anticancer agents capable of overcoming drug resistance and alleviating limitations of current microtubule-targeting therapies. Building on the pharmacological potential of quinoline scaffolds, we designed pyrroloquinoline-based compounds aimed at targeting the colchicine-binding site on tubulin. A hypothesis-driven design strategy, guided by structure- and ligand-based molecular modeling, was employed to prioritize the designed molecules for strong predicted binding and pharmacophoric complementarity within the colchicine-binding site. A concise, efficient synthetic strategy was employed to access the target molecules, which were subsequently evaluated for antiproliferative activity across various cancer cell lines. The hit compounds reported in this paper, 4a and 5b, exhibited potent cytotoxicity, competitively inhibited the formation of the EBI−β-tubulin adduct, and induced pronounced disruption of the intracellular microtubule network. Compound 4a, in particular, consistently displayed significant biological activity, as mechanistic studies revealed that it triggers sustained G2/M phase arrest, suppresses proliferative signaling, and activates DNA damage–associated stress responses, followed by caspase-3 activation and PARP cleavage, indicative of caspase-dependent apoptosis. These results reinforce the utility of the quinoline scaffold for the development of colchicine-site microtubule inhibitors and highlight their potential in advancing new chemotherapeutic candidates.

Metastatic melanoma is the deadliest form of skin cancer, being responsible for 80% of skin cancer deaths. Small molecule colchicine-binding site inhibitors have previously demonstrated preclinical therapeutic efficacy in treating metastatic and paclitaxel-resistant melanoma. We report here on the design, synthesis, and biological evaluation of thirty-five novel triazoloquinoxaline-based compounds with promising therapeutic potential. The most potent analog, 9d, showed strong antiproliferative activity against a panel of melanoma cell lines (GI₅₀ = 15.4 nM against A375) and binds to the colchicine site. Treatment with 9d in vitro arrests the cell cycle in the G2/M phase and induces apoptosis. In vivo, compound 9d (10 mg/kg) demonstrated a good safety profile, significantly inhibited tumor growth and proliferation, and induced leukocyte infiltration along with apoptosis and necroptosis in an immunocompetent B16-F10 syngeneic mouse model. Further evaluation of 9d has characterized its unique mechanism of inducing both apoptosis and necroptosis in treated melanoma cells.

Background: Dietary supplements derived from botanicals are commonly consumed and investigated in biomedical studies for their potential health benefits. Accurate identification and quantification of key chemical constituents from botanical ingredients is necessary for consistent product preparations and reproducible research results. Manufacturers need quantitative reference materials of the chemical constituents of interest to verify the content of ingredients and products. The rigor and reproducibility of biomedical research is enhanced through thorough characterization of the interventions used in mechanistic, clinical, and safety investigations. Quantitative reference materials enable reliable product quality assessments and reproducible research results. Objective: Solution-based certified reference material (CRM) mixes were developed as calibrants for phytochemicals in ginger and kava. The kava CRM contained yangonin, desmethoxyyangonin, dihydrokavain, DL-kavain, methysticin, dihydromethysticin, flavokawain A, flavokawain B, and flavokawain C. The ginger CRM contained 6-gingerol, 8-gingerol, 10-gingerol, 6-shogaol, 8-shogaol, and 10-shogaol. Methods: Each phytochemical was sourced as an isolated compound and assigned a purity factor by a mass balance approach accounting for residual impurities. The solution standard mixes were formulated by gravimetric addition of each phytochemical incorporating the purity factor and diluting with acetonitrile to the target concentrations of 500 µg/mL for the gingerols and shogaols, 250 µg/mL for the kavalactones, and 25 µg/mL for the flavokawains. Results: The concentration accuracy of each component in the solution mixes was analytically verified by ultra high performance liquid chromatography with ultraviolet detection (UHPLC–UV) assay comparison to an independently prepared calibration solution. Each component in the ginger and kava CRMs were within 5 and 7% of the target concentrations, respectively. Conclusion: Homogeneous kava and ginger phytochemical solution mixes were produced with accurate constituent concentrations and demonstrated good stability over 2 years. These solution mixes were launched as commercially available CRMs. Highlights: These mixes can be used as accurate concentration stock solutions to prepare calibrators and controls for botanical dietary supplement product testing and standardization.