8-Cyclopentyl-1,3-dipropylxanthine (PD 116,948) is a very potent, very A1-selective adenosine antagonist, with a Ki of 0.46 nM in 3H-CHA binding to A1 receptors in rat whole brain membranes and 340 nM in 3H-NECA binding to A2 receptors in rat striatal membranes. Its 740-fold A1-selectivity is the highest reported for an adenosine antagonist. 3H-PD 116,948 (117 Ci/mmol) was prepared by reduction of the diallyl analog. 3H-PD 116,948 bound to a single site in rat whole brain membranes, with a Bmax of 46 pmol/g wet weight and Kd of 0.42 nM. Nonspecific binding was extremely low, amounting to about 3% of total binding under standard conditions and less than 1% when higher tissue concentrations were used. Affinities of compounds for inhibition of 3H-PD 116,948 binding were highly consistent with an A1 adenosine receptor. Antagonists were equally potent in 3H-PD 116,948 binding and in 3H-CHA binding, while agonists were consistently about 12-fold more potent in 3H-CHA binding. Hill coefficients were 1.0 for antagonists and about 0.65 for agonists. 3H-PD 116,948 should be a useful antagonist ligand for adenosine A1 receptors.

Overactivation of calcium-activated neutral protease (calpain) has been implicated in the pathophysiology of several degenerative conditions, including stroke, myocardial ischemia, neuromuscular degeneration, and cataract formation. Alpha-mercaptoacrylate derivatives (exemplified by PD150606), with potent and selective inhibitory actions against calpain, have been identified. PD150606 exhibits the following characteristics: (i) K; values for ,u-and m-calpains of 0.21 ,uM and 0.37 ,IM, respectively, (ii) high specificity for calpains relative to other proteases, (iii) uncompetitive inhibition with respect to substrate, and (iv) it does not shield calpain against inactivation by the active-site inhibitor trans-(epoxysuccinyl)-L-leucyl-amido-3-methylbutane, suggesting a nonactive site action for PD150606. The recombinant calcium-binding domain from each of the large or small subunits of ,u-calpain was found to interact with PD150606. In low micromolar range, PD150606 inhibited calpain activity in two intact cell systems. The neuroprotective effects of this class of compound were also demonstrated by the ability of PD150606 to attenuate hypoxic/hypoglycemic injury to cerebrocortical neurons in culture and excitotoxic injury to Purkinje cells in cerebellar slices.Calpain (EC 3.4.22.17) is a class of cytosolic cysteine protease that is activated by elevated intracellular calcium (1-3). Interest in this class of enzymes has grown substantially in recent years because it has been implicated in the pathophysiology of several degenerative conditions; including cerebral ischemia, myocardial ischemia, and cataract (4-8). The unifying features of these pathological conditions are that calcium serves as a trigger for cellular injury and that calpain may represent a crucial mediator of the degenerative response. Uncontrolled activation of calpain leads to cytoskeletal protein (e.g., spectrin) breakdown, degradation of many receptor proteins (e.g., epidermal growth factor receptor), and enzyme systems (e.g., protein kinase C and calmodulin-dependent kinases) and consequently cell death (1,8). Several studies have shown that peptidic inhibitors of calpain protect cells in models of ischemic, hypoxic and/or excitotoxic neuronal injury (6, 9-13). However, a clear interpretation of the role of calpain in these studies has been hampered by the lack of selectivity of these peptidic inhibitors (8).The predominant forms of calpain in mammalian tissues are ,u-calpain and m-calpain, requiring low and high micromolar calcium, respectively, for in vitro activation. Both of these isoforms are heterodimers in which the large subunit (80 kDa) contains a distinct cysteine protease domain and a calcium-binding domain with four helix-loop-helix (EF-hand) structures (14 MATERIALS AND METHODSProtease Assays and Kinetic Studies. The calpain microplate assay was carried out as described (15). Briefly, a mixture containing 0.5 mg/ml casein, 20 mM DTT, 50 mM Tris-HCl (pH 7.4), 0.01 unit purified ,u-calpain (human erythrocytes) or m-calpain (r...

A series of 2-amino-4H-3,1-benzoxazin-4-ones have been synthesized and evaluated as inhibitors of the complement enzyme C1r. C1r is a serine protease at the beginning of the complement cascade, and complement activation by beta-amyloid may represent a major contributing pathway to the neuropathology of Alzheimer's disease. Compounds such as 7-chloro-2-[(2-iodophenyl)-amino]benz[d][1,3]oxazin-4-one (32) and 7-methyl-2-[(2-iodophenyl)amino]benz[d][1,3]oxazin-4-one (37) show improved potency compared to the reference compound FUT-175. Many of these active compounds also possess increased selectivity for C1r compared to trypsin and enhanced hydrolytic stability relative to 2-(2-iodophenyl)-4H-3,1-benzoxazin-4-one (1).

PD 115,199, N-[2-(dimethylamino)ethyl]-N-methyl-4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3- dipropyl-1H-purin-8-yl)benzenesulfonamide, was found to have high affinity for the A2 adenosine receptor labeled by 3H-NECA in rat striatal membranes (Ki 15.5 nM). Unlike other potent adenosine antagonists, which always showed some degree of selectivity for the A1 receptor, PD 115,199 had equal affinity at A1 and A2 receptors (Ki in 3H-CHA binding to A1 receptors 13.9 nM). 3H-PD 115,199 (126 Ci/mmol) was prepared by reduction of the diallyl analog, and binding experiments were performed with 0.5 nM 3H-PD 115,199 at 25 degrees C in rat striatal membranes. By nonlinear least-squares analysis of the concentration-inhibition curve for the highly A1-selective adenosine antagonist PD 116,948 (8-cyclopentyl-1,3-dipropylxanthine), it could be demonstrated that about 11% of specific 3H-PD 115,199 binding was to A1 receptors, and the remainder to A2 receptors. A 20 nM concentration of PD 116,948 was included in subsequent experiments to eliminate the A1 component of binding. The remaining binding had a Kd of 2.6 nM and Bmax of 56 pmol/g wet weight. Specific binding was about 79% of total binding. Affinities of compounds in the 3H-PD 115,199 assay were consistent with binding to a high-affinity A2 receptor: antagonists were consistently about three times more potent in 3H-PD 115,199 binding than in 3H-NECA binding, whereas agonists were consistently about fivefold less potent.(ABSTRACT TRUNCATED AT 250 WORDS)

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