STRUCTURE-ACTIVITY-RELATIONSHIPS OF MILRINONE ANALOGS DETERMINED IN-VITRO IN A RABBIT HEART MEMBRANE CA2-ATPASE MODEL()

The cardiac activity of a series of analogues of the positive inotropi c bipyridines amrinone (5-amino-[3,4'-bipyridin]-6(1H)-one) and milrin one (2-methyl-5-cyano-[3,4'-bipyridin]-6(1H) was evaluated in vitro in a rabbit myocardial membrane Mg2+-dependent, Ca2+-stimulable adenosin e triphosphatase (Ca2+-ATPase) model, and structure-activity relations hips were compared for nine closely related derivatives. In the presen t studies, a 5-bromo analogue of milrinone stimulated myocardial membr ane Ca2+-ATPase significantly (10(-7) M; P < 0.001 vs control, with 67 % of the activity of milrinone), whereas a 2'-methyl-2H-milrinone deri vative was inactive. Although amrinone was inactive in this assay, its 2-methyl analogue was stimulatory. However, analogues lacking a 2-sub stituent (with or without a 5-cyano group) or with the 3-N position bl ocked by a methyl group did not stimulate myocardial membrane Ca2+-ATP ase activity. Structural data for these bipyridines show that those wi th either a 2- or 2'-methyl substituent have a twist conformation, whe reas those without are nearly planar. Activity data reveal that those bipyridines with a nonplanar conformation are more active in the Ca2+- ATPase assay. Further study of milrinone analogues with a 2'-methyl su bstituent shows that even though the effect on the twist angle is equi valent to that of 2-methyl substitution, these analogues are less pote nt. Data for this series reveal that the prer equisites for Ca2+-ATPas e stimulation include not only a 2-methyl to maintain a twist conforma tion but also a free 3-N position and a g-substituent. This model for optimal activity in the m;yocardial membrane Ca2+-ATPase system differ s from those proposed for phosphodiesterase enzyme receptor recognitio n only in the requirement for a nonplanar molecule. We have previously shown that milrinone, but not amrinone, shares structural homology wi th thyroxine and was able to stimulate myocardial membrane Ca2+-ATPase activity in a manner similar to the thyroid hormone. Additionally, mi lrinone, but not amrinone, was an effective competitor for t;hyroxine binding to the s erum transp ort protein transthyretin. Analysis of th e milrinone - transthyretin crystal complex confirms the structural ho mology between milrinone and thyroid hormone which is not shared by am rinone. Modeling studies of the binding interactions of milrinone anal ogues indicate that the 2-desmethylmilrinone analogue, the most inhibi tory analogue, lacks the hydrophobic contacts present in milrinone in its transthyretin-bound complex. These? findings correlate with their structure-activity relationships in the Ca2+-ATPase assay. On the basi s of these data, it is predicted that increased inhibition of Ca2+-ATP ase activity could be achieved by the addition of a 2'-methyl to the 2 -desmethylmilrinone analogue, the most inhibitory in this series. Howe ver, such an analogue would be a weaker competitor for the binding of thyroxine to transthyretin.