Substituted 2-oxo-azepane derivatives are potent, orally active gamma-secretase inhibitors.

A hydroxamic acid screening hit 1 was elaborated to 5,5-dimethyl-2-oxoazepane derivatives exhibiting low nanomolar inhibition of gamma-secretase, a key proteolytic enzyme involved in Alzheimer's disease. Early ADME data showed a high metabolic clearance for the geminal dimethyl analogs which could be overcome by replacement with the bioisosteric geminal difluoro group. Synthesis and structure-activity relationship are discussed and in vivo active compounds are presented.

Novel orally active, dibenzazepinone-based gamma-secretase inhibitors.

Structural modifications of the gamma-secretase inhibitor, LY411575, led to a malonamide analogue (S),(S)-1 with potent inhibitory activity in vitro, but disappointing activity in a mouse model of Alzheimer's disease. Identification and replacement of a metabolically labile position provided an improved compound (R/S),(S)-13 with high in vitro activity (IC(50)=1.7 nM), and in vivo activity after oral administration (MED=3 mg/kg). Further modifications gave an equipotent carbamate analogue 14 with improved molecular properties.

Endothelin-converting enzyme-1 inhibition and growth of human glioblastoma cells.

Endothelin-1 (ET-1) is mitogenic and/or antiapoptotic in human cancers, and antagonists to ET-1 receptors are under evaluation for cancer treatment. Inhibition of ET-1 activation by the endothelin-converting enzymes 1(a)(-)(d) (ECE-1(a)(-)(d); EC 3.4.24.71) represents another approach to block the ET-1 effect in cancer. To evaluate this potential, we synthesized and characterized a series of low nanomolar nonpeptidic thiol-containing ECE-1 inhibitors, and evaluated their effect, as well as the effect of inhibitors for the related metalloproteases neprilysin (NEP; EC 3.4.24.11) and angiotensin-converting enzyme (ACE; EC 3.4.15.1), on human glioblastoma cell growth. Only ECE-1 inhibitors inhibited DNA synthesis by human glioblastoma cells. Exogenous addition of ET-1 or bigET-1 to glioblastoma cells did not counterbalance the growth inhibition elicited by ECE-1 inhibitors, suggesting that ECE-1 inhibitors block the proliferation of human glioblastoma cells most likely via a mechanism not involving extracellular production of ET-1. This class of molecules may thus represent novel therapeutic agents for the potential treatment of human cancer.

SEAP expression in transiently transfected mammalian cells grown in serum-free suspension culture.

A transient transfection process was established using a novel 'in-house' developed transfection reagent, Ro-1539. It allows rapid production of large quantities of various recombinant proteins. Here we describe the transient expression of the secreted human placental alkaline phosphatase (SEAP) by HEK293EBNA and CHO cells in serum-free suspension culture. Unexpectedly, high expression levels of SEAP (150 mug/ml) were found 3-4 days post-transfection when placental alkaline phosphatase (AP) was used as the reference enzyme. To confirm these data, an SDS-PAGE analysis was performed and the visible SEAP protein band (MW of 65 kDa) was compared with co-migrated purified placental AP protein as reference. The scanning analysis of the gel showed that SEAP, a truncated form of AP, has a higher specific activity than the purified placental AP. A correction factor was introduced permitting a direct comparison of placental AP activity with the expression levels of SEAP. Scale-up of the transfection system from spinner flask to bioreactor was simple and straightforward, resulting in similar yields of SEAP. Finally, the effectiveness of Ro-1539 was compared to that of other transfection reagents.

Synthesis of triazole-Tethered pyrrolidine libraries: novel ECE inhibitors.

The solid-phase synthesis of substituted 1,2,4-triazoles tethered to a 4-mercaptopyrrolidine core 1 is described. This novel class of non-peptidic, Zn(2+) metallo-protease inhibitors was found to have inhibitory activity for the endothelin converting enzyme (ECE-1). The SAR of the substitution pattern in 1 is discussed.