The mitogen-activated protein kinase (MAPK) signal transduction pathway plays a central role in regulating tumor cell growth, survival, differentiation, and angiogenesis. The key components of the Ras/Raf/MEK/ERK signal module are frequently altered in human cancers. Targeting this pathway represents a promising anticancer strategy. Small molecule inhibitors targeting MEK1/2 have shown promise in the clinic; however, ultimate clinical proof-of-concept remains elusive. Here, we report a potent and highly selective non-ATPcompetitive MEK1/2 inhibitor, RO4927350, with a novel chemical structure and unique mechanism of action. It selectively blocks the MAPK pathway signaling both in vitro and in vivo, which results in significant antitumor efficacy in a broad spectrum of tumor models. Compared with previously reported MEK inhibitors, RO4927350 inhibits not only ERK1/2 but also MEK1/2 phosphorylation. In cancer cells, high basal levels of phospho-MEK1/2 rather than phospho-ERK1/2 seem to correlate with greater sensitivity to RO4927350. Furthermore, RO4927350 prevents a feedback increase in MEK phosphorylation, which has been observed with other MEK inhibitors. We show that B-Raf rather than C-Raf plays a critical role in the feedback regulation. The unique MAPK signaling blockade mediated by RO4927350 in cancer may reduce the risk of developing drug resistance. Thus, RO4927350 represents a novel therapeutic modality in cancers with aberrant MAPK pathway activation.

XIAP is a key regulator of apoptosis, and its overexpression in cancer cells may contribute to their survival. The antiapoptotic function of XIAP derives from its BIR domains, which bind to and inhibit pro-apoptotic caspases. Most known IAP inhibitors are selective for the BIR3 domain and bind to cIAP1 and cIAP2 as well as XIAP. Pathways activated upon cIAP binding contribute to the function of these compounds. Inhibitors selective for XIAP should exert pro-apoptotic effects through competition with the terminal caspases. This paper details our synthetic explorations of a novel XIAP BIR2-selective benzazepinone screening hit with a focus on increasing BIR2 potency and overcoming high in vivo clearance. These efforts led to the discovery of benzoxazepinone 40, a potent BIR2-selective inhibitor with good in vivo pharmacokinetic properties which potentiates apoptotic signaling in a manner mechanistically distinct from that of known pan-IAP inhibitors.

Ro 23-9424 is a dual-action cephalosporin with an aminothiazolylmethoxyimino-type side chain at the 7 position and fleroxacin esterified at the 3' position. The new compound has broad and potent antibacterial activity in vitro and in vivo, reflecting contributions from both the , (-lactam Ro 23-9424 is a cephalosporin with an aminothiazolylmethoxyimino-type side chain at the 7 position and fleroxacin esterified at the 3' position (1). Ro 23-9424 has broad and potent antibacterial activity in vitro (2, 9, 12) and in vivo (3). The antibacterial spectrum reflects contributions of both the cephalosporin moiety (notably, streptococci) and the quinolone moiety (notably, ,-lactamase-overproducing strains). Ro 23-9424 binds to penicillin-binding proteins and inhibits replicative DNA biosynthesis (8). These observations have led to the use of the term "dual-action cephalosporins" for Ro 23-9424 and analogous compounds (1,8).It was originally proposed by O'Callaghan et al. (15) that the second "warhead" of such a cephalosporin, in this case, the fleroxacin moiety of Ro 23-9424, is released when the ,-lactam ring is opened by a bacterial enzyme, such as a P-lactamase. In the case of Ro 23-9424, active quinolone may also be released by enzymatic or nonenzymatic hydrolysis of the ester linkage. It is well known that 3'-acetoxy cephalosporins, such as cephalothin, cephapirin, and cefotaxime, are deesterified in vivo to form 3-hydroxymethylcephalosporins (4, 5, 19). These metabolites have antibacterial activity, but they are less potent than the acetylated cephalosporins. Hydrolysis of the ester bond of Ro 23-9424 presumably results in the formation of fleroxacin and the 3-hydroxymethylcephalosporin desacetylcefotaxime, both of which have antibacterial activity. The extent to which Ro 23-9424 exerts the proposed mechanism of action in vivo (wherein the quinolone is released by a bacterial process), or acts as a combination of active metabolites, is therefore determined by its pharmacokinetic properties. This study *

<div>Abstract<p>The mitogen-activated protein kinase (MAPK) signal transduction pathway plays a central role in regulating tumor cell growth, survival, differentiation, and angiogenesis. The key components of the Ras/Raf/MEK/ERK signal module are frequently altered in human cancers. Targeting this pathway represents a promising anticancer strategy. Small molecule inhibitors targeting MEK1/2 have shown promise in the clinic; however, ultimate clinical proof-of-concept remains elusive. Here, we report a potent and highly selective non–ATP-competitive MEK1/2 inhibitor, RO4927350, with a novel chemical structure and unique mechanism of action. It selectively blocks the MAPK pathway signaling both <i>in vitro</i> and <i>in vivo</i>, which results in significant antitumor efficacy in a broad spectrum of tumor models. Compared with previously reported MEK inhibitors, RO4927350 inhibits not only ERK1/2 but also MEK1/2 phosphorylation. In cancer cells, high basal levels of phospho-MEK1/2 rather than phospho-ERK1/2 seem to correlate with greater sensitivity to RO4927350. Furthermore, RO4927350 prevents a feedback increase in MEK phosphorylation, which has been observed with other MEK inhibitors. We show that B-Raf rather than C-Raf plays a critical role in the feedback regulation. The unique MAPK signaling blockade mediated by RO4927350 in cancer may reduce the risk of developing drug resistance. Thus, RO4927350 represents a novel therapeutic modality in cancers with aberrant MAPK pathway activation. [Cancer Res 2009;69(5):1924–32</p></div>

Supplementary Table 1 from Characterization of a Novel Mitogen-Activated Protein Kinase Kinase 1/2 Inhibitor with a Unique Mechanism of Action for Cancer Therapy