A series of tetrazole amide derivatives of (+/-)-2-dodecyl-alpha-phenyl-N-(2,4,6-trimethoxyphenyl)-2H-tetrazole-5- acetamide (1) was prepared and evaluated for their ability to inhibit acyl-CoA: cholesterol O-acyltransferase (ACAT) in vitro and to lower plasma total cholesterol in vivo. For this series of compounds, our objective was to systematically replace substituents appended to the amide and tetrazole moieties of 1 with structurally diverse functionalities and assess the effect that these changes have on biological activity. The ensuing structure-activity relationship (SAR) studies identified aryl (7b) and heteroaryl (7f,g) replacements for 2,4,6-trimethoxyphenyl that potently inhibit liver microsomal and macrophage ACAT in vitro and exhibit good cholesterol lowering activity (56-66% decreases in plasma total cholesterol at 30 mg/kg), relative to 1, when compared in the acute rat model of hypercholesterolemia. Replacement of the alpha-phenyl moiety with electron-withdrawing substituents (13e-h), however, significantly reduced liver microsomal ACAT inhibitory activity (IC50 > 1 microM). This is in contrast to electron-donating substituents (13ij,m-q), which produce IC50 values ranging from 5 to 75 nM in the hepatic microsomal assay. For selected tetrazole amides (1, 7b, 13n,o), reversing the order of substituents appended to the 2- and 5-positions in the tetrazole ring (36a-d), in general, improved macrophage ACAT inhibitory activity and provided excellent cholesterol-lowering activity (ranging from 65% to 77% decreases in plasma total cholesterol at 30 mg/kg) in the acute rat screen. The most potent isomeric pair in this set of unsubstituted methylene derivatives (13n and 36a) caused adrenocortical cell degeneration in guinea pigs treated with these inhibitors. In contrast, adrenal glands taken from guinea pigs treated with the corresponding alpha-phenyl-substituted analogs (7b and 36c) were essentially unchanged compared to untreated controls. Subsequent evaluation of 7b and 36c in a rabbit bioassay showed that both compounds and/or their metabolities were present in plasma after oral dosing. Unlike 7b and 36c, compound 1 and related 2,4,6-trimethoxyanilides (13j, 30c,d) showed poor oral activity in the rabbit bioassay. Nevertheless, in cholesterol-fed rabbits, both systemically available (7b, 36c) and poorly absorbed inhibitors (1, 36d) were more effective in lowering plasma total cholesterol than the fatty acid amide CI-976.