A series of Mannich bases and aminomethyl derivatives of ethyl [2,3-dichloro-4-(4-hydroxybenzoyl)phenoxy]acetate were synthesized and tested for saluretic and diuretic activities. The effects of nitrogen and aromatic nuclear substitution, reorientation of the aminomethyl group relative to that of the phenolic hydroxyl group, and replacement of either the phenolic hydroxyl or the aminomethyl group by other functional groups are described. Ethyl [2,3-dichloro-4-[3-(aminomethyl)-4-hydroxybenzoyl]phenoxy]acetate (27) was found to be a very potent, high-ceiling diuretic.

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Short communications killed 18 hr after administration of the labeled drug. Analysis of the various brain areas showed that with the exception of the cerebellum, residual E3H]reserpine concentrations were markedly iowered in all brain areas (Table 2), indicating that mutual ~eserpine-tetrabe~~ne sites are w~d~pread in the brain.The other way of establishing the specificity of reserpine binding was by taking advantage of the observation that specific reserpine binding sites are readily saturated and that pretreatment with unlabeled reserpine well in advance of the labeled drug greatly reduces the persistent binding of the latter.' Accordingly, rats were given unlabeled reserpine (0.5 mgjkg, i.m.) 6 hr before [jH]reserpine and were killed 18 hr after administration of the labeled compound. As was the case with tetrabenazine pretreatment, concentrations of [3H]reserpine were markedly lowered in all areas except the cerebellum (Table 2).These findings suggest that specific reserpine binding sites are widespread in the brain and that they are not well correlated with the ~atomic~ distribution of any single brain monoamine although the degree of reserpine binding in peripheral organs seems to be correlated with the degree of adrenergic innervation.' The present results suggest further that specific and persistent reserpine binding in cortex, which has a low content of any of the endogenous monoamines, may reveal the presence of m~noaminer~ic systems not associated with large amine storage pools. Other evidence that there may exist such systems has been presented by Snyder and Coyle* who showed that the cerebral cortex, a norepinephrine-poor area, takes up norepinephrine in vitro almost as well as the norepinep~rine* rich hypothalamus. If such hidden systems should exist, it would follow that the subcellular site of reserpine binding in brain may not necessarily be limited only to amine storage granules within monoamine&c neurones or, alternatively, that in some brain regions such granules have a low monoamine content.

The side-chain of cholesterol is oxidatively cleaved by a cytochrome P-450-dependent enzyme system present in mammalian adrenal mitochondria. The reaction was postulated to proceed via one or more hydroxylated intermediates. Inhibition of the reaction by the potent inhibitor 22-azacholesterol did not result in a build-up of the postulated intermediates: (ZOS)-20-hydroxycholesterol. (22R)-22.hydroxycholesterol, (20R,22R)-20,22-dihydroxycholesterol or (20S)-cholesterol hydroperoxide. Spectral studies indicate a modified type II spectrum for the reaction of 22-azacholesterol with adrenal mitochondrial cytochrome P-450. In addition, spectral and electron spin resonance studies indicate a common interaction of cholesterol and 22-azacholesterol with the cytochrome. Although this study does not resolve the question of the intermediacy of the oxygenated derivatives in the cleavage reaction. it indicates that the binding of 22-azacholesterol depends on its steroid structure rather than on its amine character. A discussion of the proximity of the heme to the substrate binding site is also presented.