Fragment-based lead generation has led to the discovery of a novel series of cyclic amidine-based inhibitors of beta-secretase (BACE-1). Initial fragment hits with an isocytosine core having millimolar potency were identified via NMR affinity screening. Structure-guided evolution of these fragments using X-ray crystallography together with potency determination using surface plasmon resonance and functional enzyme inhibition assays afforded micromolar inhibitors. Similarity searching around the isocytosine core led to the identification of a related series of inhibitors, the dihydroisocytosines. By leveraging the knowledge of the ligand-BACE-1 recognition features generated from the isocytosines, the dihydroisocytosines were efficiently optimized to submicromolar potency. Compound 29, with an IC50 of 80 nM, a ligand efficiency of 0.37, and cellular activity of 470 nM, emerged as the lead structure for future optimization.

Fragment-based lead discovery has been successfully applied to the aspartyl protease enzyme beta-secretase (BACE-1). Fragment hits that contained an aminopyridine motif binding to the two catalytic aspartic acid residues in the active site of the enzyme were the chemical starting points. Structure-based design approaches have led to identification of low micromolar lead compounds that retain these interactions and additionally occupy adjacent hydrophobic pockets of the active site. These leads form two subseries, for which compounds 4 (IC50 = 25 microM) and 6c (IC50 = 24 microM) are representative. In the latter series, further optimization has led to 8a (IC50 = 690 nM).

Alzheimer's disease is characterized by formation of neurofibrillary tangles and amyloid plaques in the regions of the central nervous system that are involved in learning and memory (1). It is believed that accumulation of A␤ 1 in plaques or as soluble aggregates initiates a pathological cascade leading to synaptic dysfunction and neuronal toxicity, with neurodegeneration and dementia as the final outcome (1, 2). Therefore, strategies to reduce the level of brain A␤ are being aggressively pursued as an approach likely to benefit Alzheimer's disease patients. A␤ is produced as the result of sequential proteolysis of a type I transmembrane protein APP by ␤-and ␥-secretases. ␤-Secretase cleaves APP in its extracellular domain at a site close to the membrane surface, a reaction that generates a membrane-bound APP C-terminal fragment of 99 amino acid residues (C99). A subsequent endoproteolysis within the transmembrane domain of C99 by ␥-secretase produces A␤. Whereas ␤-secretase, an aspartyl protease, has been well characterized (3-6), the identity and structure of ␥-secretase, also thought to be an aspartyl protease (7-12), remains elusive, and its kinetic and catalytic mechanisms are poorly understood. To a large extent, this is due to the highly complicated structural organization of this unusual protease. In contrast to other known proteases, ␥-secretase is composed of a high molecular weight multicomponent complex of transmembrane proteins (13,14). Primarily due to this structural complexity, the catalytic site and mechanism of action of ␥-secretase has not been unequivocally established. Early findings point to presenilin 1 or 2 as the catalytic subunit of ␥-secretase (15, 16). These multipass transmembrane proteins contain two essential aspartate residues in putatively adjacent transmembrane domains (15) and can be cross-linked by high affinity ␥-secretase inhibitors (16 -18). Recent advances have identified three additional proteins, nicastrin (19), aph-1 (20, 21), and pen-2 (20, 22), in the same multicomponent complex, whose co-expression with presenilin appears to be critical for ␥-secretase activity (19 -22). However, the precise roles of these additional protein subunits in the catalytic mechanism of ␥-secretase await further investigation.Associated with the structural complexity of ␥-secretase is the versatility of this protease in cleaving several type I transmembrane proteins. In addition to APP processing, ␥-secretase is required for proteolytic activation of Notch receptor (23-26), a signaling molecule essential for embryonic development of all metazoan species (27). Cleavage of Notch in the transmembrane domain by ␥-secretase generates Notch intracellular domain (NICD), which then translocates into the nucleus, where it regulates gene transcription (28,29). The list of other potential protein substrates for ␥-secretase has recently been expanded to include ErbB4 (30, 31), E-cadherin (32), and CD44 (33). However, the mechanisms by which ␥-secretase reacts with these different substrates remains un...

Pinane-thromboxane A2 (PTA2, [1a,2P(Z),-3a(1 E,3R*),5a]-7-1343-hydroxy-1-octenyl6,6-dimethylbicyclo[3.l.l]hept-2-ylJ-5-heptenoic acid) has been synthesized and tested for biological activity in systems responsive to thromboxane A2, stable prostaglandin endoperoxide (PGH2) analogs, and prostacyclin (PGI2). At low concentrations, PTA2 inhibited cat coronary artery constriction induced by stable prostaglandin endoperoxide analogs, and it stabilized liver lysosomes. At slightly higher concentrations, it inhibited platelet aggregation.At still higher concentrations, PTA2 inhibited thromboxane synthetase, but it had no effect on prostacyclin synthetase. The analog also had no effect on the inhibition of platelet aggregation by PGI2 or prostaglandin D2. It is suggested that PTA2 has a suitable biochemical profile for use as an antithrombotic agent.In 1975, Hamberg et al. (1) proposed the structure (Fig. 1) for an unstable (half-life of about 30 s at pH 7.4 and 370C) substance with potent thrombotic and smooth muscle contracting properties that they named thromboxane A2 (TA2). The isolation or chemical synthesis of this important member of the arachidonic acid cascade (2) has not yet been achieved. Furthermore, no analogs of this structurally unusual molecule have been reported to date. We now wish to report the synthesis of a thromboxane A2 analog (structure 8a) and some of its interesting biological properties. MATERIALS AND METHODSSynthesis of Pinane-Thromboxane A2, [la,2#(Z),3a-H(1 E,3R*),5aj -713 -(3-Hydroxy-l-octenyl-6,6 -dimethylbicyclo[3.1.1]hept-2-yll-5-heptenoic Acid. The thromboxane analog pinane-thromboxane A2 (PTA2, 8a) was synthesized from (-)-myrtenol (1) as outlined in Fig. 2 174.0, 139.6, 131.2, 130.2, 128.9, 73.0, 51.4, 48.8, 43.9, 41.7, 39.3, 38.6,37.4, 34.9, 34.3, 33.5, 32.4, 31.7, 28.2, 26.7, 25.2, 24.9, 23.0, 22.6, 14.0. Mass spectrum, parent ion massto-charge ratio 390. [aI122 +26.400 (methanol). Analysis, calculated for C25H4203: C 76.86% and H 10.87%; found: C 77.05% and H 11.08%. The 13C NMR spectra of 7a and 7b are of crucial value in assigning the stereochemistry of these compounds. In particular, the relatively low chemical shift for C-10 (PG numbering) in the 13C NMR spectra of 7a and 7b (533.3 ± 2 ppm) reveals the relative stereochemistry of the upper side chain by comparison to the corresponding model epimeric compounds from the myrtenol family, which exhibit this carbon at 3 33.2 ppm (C trans to the side chain) and 24.2 (C cis to the side chain) (4). PTA2 (8a)