We have synthesized a series of benzazepinones (2) in order to determine the structure-activity relationships (SAR) for calcium channel blockers related to diltiazem. A prerequisite for calcium channel blocking activity in vitro and in vivo is the presence of two pharmacophores: a 4'-aryl methyl ether and a basic substituent appended to N1 with a pKa in the physiological range. When these constraints are satisfied, a wide variety of substitution is tolerated at C6, C7, and C3. The presence of an electron-withdrawing group at C6 appears to enhance potency in vitro and in vivo. For such benzazepinones, activity is primarily dependent upon lipophilicity, as measured by log P. We believe these compounds must partition into the cell membrane in order to access their receptor. The quaternary methiodide 15k was used to demonstrate that the binding site for benzazepinones is on the intracellular face of the membrane. This work represents the first comprehensive SAR of diltiazem-like calcium channel blockers.

We have shown that the pyrrolidinylmethyl substituent on the lactam nitrogen (N1) of benzazepinone and benzothiazepinone calcium channel blocking agents is resistant to metabolic deamination and generally increases the duration and potency of antihypertensive activity in spontaneously hypertensive rats (SHR) relative to (N,N-dimethylamino)ethyl analogs. Additionally, compounds possessing a substituent on the fused aromatic ring are more resistant to metabolic deacylation of the C3 hydroxy function, which may explain why aromatic substituents also frequently increase the potency and/or duration of antihypertensive activity. Our data also indicate the increased antihypertensive activity associated with these structural modifications is independent of any effects of potency in vitro. Overall, we interpret these results to indicate that these structural modifications improve antihypertensive activity as a result of increased metabolic stability and, consequently, oral bioavailability.

The peroxidase-anti-peroxidase immunoenzyme method was applied to red blood cell ghosts for the detection of ABO mixed-red blood cell populations. Red blood cell ghosts produced by acid-glycine lysis eliminated previous problems due to distorted red blood cell and hemoglobin-associated peroxidase activity. Anti-A,B was used to discriminate between various mixtures of test cells in group O cells. The positive-stained minor population of test cells stained dark brown following application of the peroxidase-anti-peroxidase method and the nonreactive group O cells were counterstained light blue with Coomassie brilliant blue which facilitated quantitation. The expected and observed numbers of positive-stained cells per 1000 cells were not significantly different as computed by X2. As low as a 0.5 percent minor population in an ABO red blood cell mixture could be accurately quantitated by this method.