In vitro activity: TH-302 is selectively potent under hypoxia and stable to liver microsomes. Substitution of the chlorine with bromine on the phosphorus mustard in 3b increases the potency by 10-fold and maintaines the high hypoxic selectivity [Hypoxia cytotoxicity ratio (HCR) = 270]. In both human lung cancer H460 cells and human colon cancer HT29 cells, potent cytotoxicity of TH-302 is observed under N2. TH-302 inhibits H460 cells and HT29 cells with IC90 of 0.1 μM and 0.2 μM, respectively. TH-302 shows much enhanced potency in H460 spheroids compared to H460 monolayer cells under normoxia. TH-302 exhibits potent cytotoxicity to MM cells with hypoxic selectivity and dose dependency. TH-302 can induce G0/G1 cell-cycle arrest under hypoxic conditions. The effect of TH-302 on cell-cycle machinery is mediated by down-regulating cyclin D1/2/3, CDK4/6, p21cip-1, p27kip-1, and pRb expression, whereas CDK2 expression remained undisturbed. TH-302 can induce dose-dependent apoptosis in both human and murine MM cells in hypoxic conditions. TH-302-activated apoptosis is mediated through down-regulating the antiapoptotic proteins BCL-2 and BCL-xL, as well as up-regulating the expression of cleaved proapoptotic protein caspase-3, -8, and -9 and poly ADP-ribose polymerase. In contrast to the hypoxia-specific toxicity, TH-302 shows very low toxicity in normoxic condition, even at high concentrations.

Cell Assay: Exponentially growing human H460 or HT29 cells are seeded into 60 mm notched glass plates at 3 × 105 cells per plate and grown in RPMI medium supplemented with 10% fetal bovine serum for 2 days prior to initiating treatment. On the day of the test, TH-302 stocks of known concentrations are prepared in complete medium and 2 mL of the desired stock is added to each plate. The plates are placed in either an anaerobic chamber or a standard tissue-culture incubator. The anaerobic chamber is evacuated and gassed with the anaerobic gas mixture (90% N2/5% CO2/5% H2) to create a hypoxic environment. Cells are then incubated with TH-302 for 2 hours at 37 °C. At the end of treatment, plates are removed from each vessel and washed with phosphate-buffered saline and a solution of trypsin-EDTA and then trypsinized for 5 min at 37 °C. Detached cells are neutralized with medium plus serum and spun for 5 min at 100g. Cells are resuspended at approximately 1 × 106 cells/mL and diluted 10-fold for plating. The exact concentration of each stock is determined. Known numbers of cells are plated and placed undisturbed in an incubator for between 9 and 13 days. Colonies are fixed and stained with a solution of 95% ethanol with 0.25% crystal violet stain. Colonies of greater than 50 cells are counted, and the surviving fraction is determined. Plating efficiencies (PEs) are determined by dividing the number of colonies by the actual number of cells plated. Surviving fractions are calculated by dividing the PEs of treated cells by the PEs of untreate.

J Med Chem. 2008 Apr 24;51(8):2412-20; Mol Cancer Ther. 2012 Mar;11(3):740-51; Clin Cancer Res. 2012 Feb 1;18(3):758-70.