The crystal form was determined to be monoclinic with a resolution of 2.0 ?. BACE 1) is definitely one of two proteases which cleaves -amyloid precursor protein (APP) and produces A and its aggregation product.3 There is considerable evidence that excess A leads to brain swelling, neuronal death, and AD.4 Consequently, -secretase has become a major therapeutic target for drug development.5,6 Since our design of initial transition-state inhibitor (1, Number 1) and subsequent determination of inhibitor-bound memapsin 2 X-ray structure, nearly a decade ago, steady progress Glutathione oxidized has been made for the evolution of small molecule potent and brain-penetrable inhibitor medicines.7,8 Recently, we have demonstrated that administration of -secretase inhibitor 2 rescued cognitive decrease in transgenic AD mice, validating -secretase as an important drug design target.9,10 However, the development of clinical -secretase inhibitor drug is faced with numerous formidable challenges, including lack of selectivity against additional physiologically important aspartic acid proteases and issues of poor pharmacological profiles including blood-brain penetration.7,8 In our continuing work towards the design of small molecule potent and selective inhibitors, we have been particularly interested in developing tools for selectivity H3FK against relevant physiologically important aspartic acid proteases especially cathepsin D and BACE 2. BACE 2 offers specificity similarity to BACE 1 and this is known to have important physiological functions.11 Cathepsin D takes on a key part in important biological functions like protein catabolism.12 The abundance of cathepsin D in various cells, especially in CNS cells cells, is very high. Furthermore, cathepsin D gene knock-out studies in mice showed designated phenotypic response including high mortality rate.13 Therefore, the selective inhibition of -secretase over cathepsin D and BACE 2 is very critical to reduce toxicity along with other side effects of -secretase inhibitor medicines. Open in a separate window Number 1 Constructions of -secretase inhibitors 1-3. As explained by us previously, the X-ray crystal structure of inhibitor 1-bound -secretase showed an interesting hydrogen bonding between P2-carbonyl and the hydroxyl of Tyr-198, forming a rare kink in the P2 site.8 We have exploited this interaction in the design and synthesis of very potent and highly selective -secretase inhibitors such as 3 by incorporating hydroxyethylene isosteres.14 However, cellular -secretase inhibitory activity of this class of inhibitors was only in micromolar range. In an attempt to design small molecule inhibitors with improved selectivity and cellular activity exploiting this unique interaction, we have further explored -secretase inhibitors with a reduced amide isostere and Glutathione oxidized integrated features to improve potency and selectivity. The basic amine features in the reduced amide isostere may also improve cell permeability.15 Herein, we report our structure-based design and synthesis of very potent and exceptionally selective inhibitors with excellent cellular inhibitory properties. A protein-ligand X-ray structure provided important molecular insight into the specific cooperative ligand-binding site relationships for selectivity. The inhibitors comprising reduced amide isostere have been reported however, they exhibited only marginal selectivity against memapsin 1 (BACE 2).6,16 A reduced amide Csecretase inhibitor 4 was synthesized by us and this compound offers exhibited a Glutathione oxidized BACE 1 Kof 27 nM and marginal selectivity against BACE 2 and cathepsin D in our in-house enzyme inhibitory assays. An energy-minimized model of 4 was created based upon the protein-ligand X-ray structure of 2-bound -secretase.9 Our preliminary model suggested that an introduction of a hydroxyl group with of 27 nM (entry 1). Inhibitor 5 with an allothreonine P1-part chain offers exhibited impressive BACE 1 inhibitory activity having a Kof 17 (nM)= 1.8 nM, IC50 = 2.5 nM with this assay. To gain further molecular insight, we have identified the Glutathione oxidized X-ray structure of 5-relationship to -secretase at a 2.2 ?.