Two series of derivatives of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), including a dechlorinated analog of W-7 (W-5) and various aminoalkyl chain analogs of W-7 (A-3, A-4, A-5, I-240, A-6) were synthesized and their structure-activity relationships with calmodulin antagonistic actions and their potencies in inhibiting human platelet aggregation in vitro were investigated. Their binding affinities to calmodulin in the presence of 100 microM Ca2+ were dependent both on the chlorination of the naphthalene ring and on the length of aminoalkyl chain. The ability of these derivatives to inhibit Ca2+-dependent phosphorylation of 20,000-dalton myosin light chain from platelets correlated well with the magnitude of their binding affinity to calmodulin. W-7(10-100 microM) inhibited in a dose-dependent manner platelet aggregation induced by collagen (2 micrograms/ml), ADP (5 microM), epinephrine (1 microgram/ml), sodium arachidonate (0.83 mM), thrombin (0.125 U/ml), and A-23187 (10 microM). The IC50 value (concentration producing 50% inhibition of aggregation) of W-7 was lower in arachidonate- and collagen-induced aggregation than in ADP- or epinephrine-induced aggregation. A good correlation between the potency in inhibition of collagen-induced aggregation by W-7 and its derivatives and their affinities to calmodulin was obtained (r = 0.94). Thus, the inhibitory mechanism of these compounds may be due to their effect on Ca2+-calmodulin-dependent processes, such as 20,000-dalton myosin light chain phosphorylation. These data also support the hypothesis that the calmodulin-mediated system has an important role in platelet function.