Regorafenib is a novel diphenylurea oral multikinase inhibitor of angiogenic, stromal, and oncogenic kinases with potent preclinical antitumor activity and long-lasting antiangiogenic activity (as measured by dynamic contrast-enhanced magnetic resonance imaging). Regorafenib treatment has recently shown to result in a 31% partial response (PR) rate and 50% stabilization rate in patients with metastatic renal cell carcinoma in a Phase II study. Promising clinical activity was shown in a Phase I study in 27 evaluable patients with advanced refractory colorectal cancer (median 4 prior regimens) with a 74% overall disease control rate (PR + stable disease). The biotransformation of regorafenib was investigated in vitro with liver microsomes and hepatocytes, and in vivo in the plasma of several species. In man, the N-oxide (M-2) and the demethylated N-oxide (M-5) appear significant as both metabolites show systemic exposure (area under the curve [AUC], mg*h/L) at steady state similar to regorafenib (the parent compound) in patients at a dose of 160 mg in a 3 weeks on/1 week off Phase I study. Characterization of both M-2 and M-5 demonstrated potent pharmacologic activities. In biochemical kinase assays, M-2 and M-5 showed an inhibition profile similar to but distinct from regorafenib. In cellular assays, M-2 and M-5 inhibited key targets such as vascular endothelial growth factor (VEGF) receptor 2, TIE-2, and mutant and wild-type c-KIT and B-RAF, with IC50 values very similar to regorafenib for M-2 and somewhat higher for M-5. Regorafenib and both M-2 and M-5 dosed iv at 1 mg/kg showed significant activity in a rat VEGF hypotensive pharmacodynamic model. Both metabolites dosed orally exhibited potent dose-dependent tumor growth inhibition (TGI) in preclinical murine HT-29 colorectal and MDA-MB-231 breast cancer xenografts, achieving significant TGI of 62/58% and 54/50%, respectively, compared with vehicle controls at 10 mg/kg. Regorafenib revealed 66-77% TGI at 10 mg/kg in these models. On oral administration of regorafenib to mice, the N-oxide (M-2) plasma level accounted for ∼20% of the AUC of regorafenib; conversely, on administration of M-2, regorafenib exposure reached ∼20% of M-2 exposure, indicating that oxidation to and reduction of the N-oxide are metabolic pathways in vivo. In summary, these data demonstrate that the metabolites M-2 and M-5, exhibiting similar exposure as regorafenib, their parent compound, in man, are pharmacologically active and therefore most likely contribute to the clinical antitumor activity of regorafenib. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1666.