The antiplatelet activity of 6-piperazinyl-purine analogues bearing nitrate esters is determined by the length of the carbon side chain.

BACKGROUND

The nitrate ester-bearing 6-piperazinyl-purine analogue MK128 is a Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor with cardioprotective, anti-inflammatory, and antiplatelet properties. However, structure-activity relationships of this compound class have not been systematically explored. This study aimed to elucidate the impact of spacer length on the antiplatelet activity of closely related piperazinyl-purine analogues and to identify the most active candidate for further evaluation.

METHODS

Antiplatelet activity of piperazinyl-purine analogues bearing short (MK128), intermediate (MK176), and long (MK118) spacers was evaluated by lumi-aggregometry and flow cytometry in isolated platelets and whole blood. Platelets were activated by proteinase-activated receptor-1 agonist SFLLRN-OH or collagen. Binding interactions with ROCK1/ROCK2 were examined using SelectScreen™ Kinase Profiling, in silico docking, and molecular dynamics simulations.

RESULTS

MK128 exhibited the strongest inhibition of platelet aggregation and secretion in isolated platelets and whole blood, and rapidly promoted disaggregation of preformed aggregates. The rank order of potency in whole blood was MK128>MK176>MK118. All analogues inhibited ROCK1 and ROCK2 in cell-free assays, while broader kinase profiling demonstrated high selectivity. Docking studies suggested stable bidentate interactions between the purine core and hinge region, whereas molecular dynamics simulations identified spacer length-dependent differences in nitrate ester tail interactions. Reduced spacer flexibility in MK128 enabled productive interactions with catalytically relevant residues, providing a mechanistic explanation for its superior functional activity.

CONCLUSION

Spacer length critically determines the antiplatelet effect of piperazinyl-purine analogues. The one-carbon spacer analogue MK128 demonstrated superior platelet and ROCK inhibition, making it a promising lead candidate for further evaluation in ischemic disease models.