In vitro activity: Dimethylenastron, a quinazoline-thione analog, is a potent, cell-permeable, specific, and reversible mitotic kinesin 5 (Eg5) inhibitor, Eg5 is the the mitotic motor of microtubule-stimulated ATPase activity. As an Eg5 Inhibitor III, Dimethylenastron has potential anticancer activity and shows little effect on the ATPase activity of kinesin-1, -4, -7 and -10. Dimethylenastron also inhibits bipolar spindle formation in both HeLa cells and in Xenopus egg extracts and induces cell cycle arrest (~1 μM). Dimethylenastron (3 and 10 μM) concentration-dependently suppresses the migratory ability of the cancer cells in PANC1 pancreatic cancer cells after treatment for 24 h, but does not inhibit the proliferation of cancer cells at 24 h until 72 h. Dimethylenastron also reduces invasion ability of the cancer cells.

Kinase Assay: Dimethylenastron is a potent Eg5 inhibitor, with an IC50 of 200 nM. Dimethylenastron exhibits no inhibition of five other kinesin subfamilies (kinesin 1/4/7/10 and one ungrouped-originating from 4 different organisms). Dimethylenastron (0.5, 1 μM) causes accumulation of cells in G2/M in HeLa cells.

Cell Assay: Dimethylenastron also inhibits bipolar spindle formation in both HeLa cells and in Xenopus egg extracts and induces cell cycle arrest (~1 μM). Dimethylenastron (3 and 10 μM) concentration-dependently suppresses the migratory ability of the cancer cells in PANC1 pancreatic cancer cells after treatment for 24 h, but does not inhibit the proliferation of cancer cells at 24 h until 72 h. Dimethylenastron also reduces invasion ability of the cancer cells. Cell invasion in response to Dimethylenastron is carried out by transwell assays. The upper surface of the transwell filters is coated with matrigel or fibronectin. Cells suspended in 200 μL serum-free media are added to the chamber, and the chamber is placed in a 24-well plate containing complete medium. After 24 h of incubation at 37°C, the filters are gently taken out and matrigel on the upper surface of the filters is removed by cotton swabs. Cells on the underside of transwell filters are fixed with 4% paraformaldehyde for 30 min, stained with 0.1% crystal violet for 10 min, and then photographed. For quantitative assessment, the number of invading cells is counted in five random fields per filter. The extent of cell invasion is quantified as the number of invading cells in the drug-treatment group divided by the number of invading cells in the control group