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A series of new lawsone Mannich bases derived from salicylaldehydes or nitrofurfural were prepared and tested for their activities against Leishmania major, Toxoplasma gondii, and Trypanosoma brucei brucei parasites. The hydrochloride salts 5a and 6a of the Mannich bases 2a and 3a, derived from unsubstituted salicylaldehyde and longchained alkyl amines, were selectively and strongly active against T. gondii cells and appear to be new promising drug candidates against this parasite. Compound 6ashowed an even higher activity against T. gondii than the known lawsone Mannich base 1b. Compound 4a, derived from salicylaldehyde and 2-methylaminopyridine, was also distinctly active against T. gondii cells. The derivatives 3a (salicyl derivative), 3b(3,5-dichloro-2-hydroxyphenyl derivative), and 3d (5-nitrofuranyl derivative) as well as the hydrochlorides 6a and 6b were also efficacious against T. b. brucei cells with compounds 3a and 3b being more selective for T. b. brucei over Vero cells when compared with the known control compound 1b. The derivatives 5a, 5c, 6a, and 6c proved to be up to five times more active than 1b against L. major promastigotes and up to four times more efficacious against L. major amastigotes. K E Y W O R D Santiparasitic drugs, lawsone, Mannich base, neglected tropical diseases, salicyl derivatives

SUMMARY Cultured bovine endothelial cells were grown on microcarrier beads. Columns (0.2 ml) packed with microcarriers were perfused with oxygenated (20% OJ Tyrode's solution containing indomethacin (10 /xM), and the effluent was passed through precontracted, endothelium-denuded detector arteries. When the endothelial cells were stimulated with bradykinin (3-100 nM), adenosine 5'-triphosphate (0.3-30 /LAM), or calcium ionophore A23187 (10-300 nM), they released dose-dependently a nonprostanoid compound that dilated the detector vessel. The factor, probably identical to the endothelium-derived relaxing factor of native endothelium, evoked dilations of the same magnitude in different types of detector vessels (rabbit thoracic aorta, rabbit femoral artery, canine coronary artery). However, this relaxant factor was significantly more effective in arteries precontracted by norepinephrine or serotonin than in arteries precontracted by potassium depolarization. Thus, its dilator action resembles that of the nitrovasodilators. The factor is labile, with an apparent half-life in the range of 20 to 30 seconds. Its dilator potency was inhibited by dithiothreitol (0.2 mM), metyrapone (0.2 mM), nordihydroguaiaretic acid (20 /xM), and hemoglobin (1 /iM), all of which apparently inactivated the factor. Synthesis or release (or both) of the relaxant factor was abolished by methylene blue (1 /xM). High Po 2 levels (> 400 mm Hg) in the perfusate markedly reduced the release of the relaxant factor from the cultured cells. This study demonstrates that a vascular relaxant factor is released from endothelial cells in monoculture by adenosine 5'-triphosphate, bradykinin, and A23187 and establishes such a culture as a useful tool for analyzing the mechanisms of endotheliumdependent vasomotion. (Hypertension 9: 295-303, 1987) KEY WORDS triphosphate cultured endothelial cells bradykinin endothelium-derived relaxing factor • adenosine C ONSIDERABLE evidence suggests that the endothelium-dependent relaxation of vascular smooth muscle induced by acetylcholine, adenosine 5'-triphosphate (ATP), bradykinin, and various other vasoactive substances is mediated by a humoral signal.1 ' 2 However, the chemical nature of this endothelium-derived relaxing factor (EDRF) has not been identified. In transfer experiments, EDRF has been characterized as a labile factor that is inactivated by either antioxidants or substances interacting with carbonyl groups.3 Release of EDRF from native endothelium has been observed under basal conditions and under stimulation with acetylcholine.^ A vascular relaxing factor was shown to be released from cultured Received April 22, 1986; accepted August 28, 1986. endothelial cells 6 in response to bradykinin and the calcium ionophore A23187 and is probably identical to the EDRF produced by the endothelium of intact vascular segments. In several recent studies, however, it was emphasized that the phenotypic expression of agonist-induced EDRF synthesis and release from cultured endothelial cells requires mixed cultures...

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We investigated the effects of fendiline, calmidazolium and trifluoperazine, compounds described as calmodulin antagonists, on the release of the endothelial autacoids prostacyclin (PGI2) and endothelium-derived relaxant factor (EDRF). Cultured bovine aortic endothelial cells were grown on microcarrier beads and continuously superfused with Tyrode's solution. Samples collected from the superfusate were assayed for PGI2 concentration (6-keto PGF1 alpha radioimmunoassay) and for EDRF activity (stimulation of soluble guanylate cyclase in vitro). Stimulation of endothelial cells by ATP (3 microM) resulted in a 6.9 +/- 1.4-fold increase of PGI2 concentration in the superfusate (p less than 0.01) and an 8.6 +/- 3.4-fold enhanced guanylate cyclase activity (p less than 0.01). In the presence of calmidazolium (10 microM), the basal values of PGI2 concentration increased 28-fold (p less than 0.01) and the guanylate cyclase activity 10-fold (p less than 0.01). Further enhancement of both was observed after additional administration of ATP. Fendiline (30 microM) did not affect autacoid release by non-stimulated cells. However, the ATP-induced release of PGI2 and EDRF was more than doubled (p less than 0.01) in the presence of this drug compared to ATP-stimulation alone. Trifluoperazine (10 microM) had no enhancing effect on EDRF release, and the ATP-induced release of PGI2 was even significantly attenuated by 84 +/- 12% (p less than 0.01). Calmidazolium and fendiline were also applied to endothelial cells loaded with the fluorescent indicator of free calcium concentration (Ca2i+), indo-1. However, effects of calmidazolium on Ca2i+ could not be quantified since calmidazolium caused some leakage of indo-1 out of the cells. A smaller leakage was observed during the combined application of fendiline and ATP.(ABSTRACT TRUNCATED AT 250 WORDS)