This article describes a set of 275 rules, developed over an 18-year period, used to identify compounds that may interfere with biological assays, allowing their removal from screening sets. Reasons for rejection include reactivity (e.g., acyl halides), interference with assay measurements (fluorescence, absorbance, quenching), activities that damage proteins (oxidizers, detergents), instability (e.g., latent aldehydes), and lack of druggability (e.g., compounds lacking both oxygen and nitrogen). The structural queries were profiled for frequency of occurrence in druglike and nondruglike compound sets and were extensively reviewed by a panel of experienced medicinal chemists. As a means of profiling the rules and as a filter in its own right, an index of biological promiscuity was developed. The 584 gene targets with screening data at Lilly were assigned to 17 subfamilies, and the number of subfamilies at which a compound was active was used as a promiscuity index. For certain compounds, promiscuous activity disappeared after sample repurification, indicating interference from occult contaminants. Because this type of interference is not amenable to substructure search, a "nuisance list" was developed to flag interfering compounds that passed the substructure rules.

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.

Central stimulant actions of 10 methylxanthines in mice correlate with affinities for adenosine receptors labeled with N6-[3H]cyclohexyladenosine. Affinities of methylxanthines for adenosine receptors are consonant with central levels attained at behaviorally effective doses. The much higher concentrations ofmethylxanthines required to influence benzodiazepine receptor binding do not correlate with behavioral potency. N6-(L-Phenylisopropyl)adenosine (L-PIA), a metabolically stable analog ofadenosine with high affinity for adenosine receptors, is an extremely potent behavioral depressant, reducing locomotor activity of mice at doses as little as 0.05 ,.mol/kg. The D isomer, which has much less affinity for adenosine receptors, is much less active as a central depressant. Theophylline stimulates locomotor activity and reverses depressant effects of L-PIA. Caffeine or 1,7-dimethylxanthine, when administered alone, elicits biphasic effects, with locomotor depression at lower doses and stimulation at higher doses. When administered with L-PIA, even low doses of caffeine produce marked stimulation. 3-Isobutyl-1-methylxanthine given alone elicits only behavioral depression. However, like theophylline and caffeine, isobutylmethylxanthine reverses the L-PIA-evoked depression, converting it into pronounced locomotor stimulation. The data strongly suggest that the behavioral stimulant effects of methylxanthines involve a blockade of central adenosine receptors. Although methylxanthines such as caffeine and theophylline are among the most widely used behavioral stimulant substances, molecular mechanisms for their stimulant effects are unclear. Methylxanthines can inhibit phosphodiesterase, and thus prevent inactivation of cyclic AMP (1), but the concentrations of caffeine and theophylline required to inhibit phosphodiesterases are substantially greater than those which occur in brain at behaviorally effective doses (2, 3). Moreover, several potent phosphodiesterase inhibitors lack behavioral stimulant actions and indeed are central depressants (4). Adenosine receptor activity is blocked by methylxanthines in concentrations similar to those that occur after stimulant doses (5, 6). Because the general neurophysiologic actions of adenosine are inhibitory (7), it is conceivable that methylxanthines exert stimulant actions by blocking adenosine effects.In several attempts to measure binding of adenosine-related ligands to membranes, binding sites largely lacked the specificity of physiologic adenosine receptors (8-12). Recently, we (13, 14) and others (15,16) have demonstrated binding of 3H-labeled ligands to adenosine receptors in brain and testes (15,17).In the present study we show a correlation between potencies of a series of methylxanthines in stimulating locomotor activity of mice and in competing at adenosine receptors labeled with [3H]CHA (14 Ci/mmol; 1 Ci = 3.7 x 101 becquerels) and[3H]flunitrazepam (79 Ci/mmol) were obtained from New England Nuclear. The sources ofxanthines were as described (13).Behavioral. ...

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