A further pocket or conformational plasticity by mapping COX-1 catalytic site through modified-mofezolac structure-inhibitory activity relationships and their antiplatelet behavior

Cyclooxygenase enzymes have distinct roles in cardiovascular, neurological, and neurodegenerative disease. They are differently expressed in different type of cancers. Specific and selective COXs inhibitors are needed to be used alone or in combo-therapies. Fully understand the differences at the catalytic site of the two cyclooxygenase (COX) isoforms is still opened to investigation. Thus, two series of novel compounds were designed and synthesized in fair to good yields using the highly selective COX-1 Inhibitor mofezolac as the lead compound to explore a COX-1 zone formed by the polar residues Q192, S353, H90 and Y355, as well as hydrophobic Amino acids I523, F518 and L352. According to the structure of the COX-1:mofezolac complex, hydrophobic Amino acids appear to have free volume eventually accessible to the more sterically hindering groups than the methoxy linked to the phenyl groups of mofezolac, in particular the methoxyphenyl at C4-mofezolac isoxazole. Mofezolac bears two methoxyphenyl groups linked to C3 and C4 of the isoxazole core ring. Thus, in the novel compounds, one or both methoxy groups were replaced by the higher homologous ethoxy, normal and isopropyl, normal and tertiary butyl, and phenyl and benzyl. Furthermore, a major difference between the two sets of compounds is the presence of either a methyl or acetic moiety at the C5 of the isoxazole. Among the C5-methyl series, 12 (direct precursor of mofezolac) (COX-1 IC₅₀ = 0.076 μM and COX-2 IC₅₀ = 0.35 μM) and 15a (ethoxy replacing the two methoxy groups in 12; COX-1 IC₅₀ = 0.23 μM and COX-2 IC₅₀ > 50 μM) were still active and with a Selectivity Index (SI = COX-2 IC₅₀/COX-1 IC₅₀) = 5 and 217, respectively. The other symmetrically substituted alkoxyphenyl moietis were inactive at 50 μM final concentration. Among the asymmetrically substituted, only the 16a (methoxyphenyl on C3-isoxazole and ethoxyphenyl on C4-isoxazole) and 16b (methoxyphenyl on C3-isoxazole and n-propoxyphenyl on C4-isoxazole) were active with SI = 1087 and 38, respectively. Among the set of compounds with the acetic moiety, structurally more similar to mofezolac (SI = 6329), SI ranged between 1.4 and 943. It is noteworthy that 17b (n-propoxyphenyl on both C3- and C4-isoxazole) were found to be a COX-2 slightly selective inhibitor with SI = 0.072 (COX-1 IC₅₀ > 50 μM and COX-2 IC₅₀ = 3.6 μM). Platelet aggregation induced by arachidonic acid (AA) can be in vitro suppressed by the synthesized compounds, without affecting of the secondary hemostasia, confirming the biological effect provided by the selective inhibition of COX-1. A positive profile of hemocompatibility in relation to erythrocyte and platelet toxicity was observed. Additionally, these compounds exhibited a positive profile of hemocompatibility and reduced cytotoxicity. Quantitative structure activity relationship (QSAR) models and molecular modelling (Ligand and Structure based virtual screening procedures) provide key information on the physicochemical and pharmacokinetic properties of the COX-1 inhibitors as well as new insights into the mechanisms of inhibition that will be used to guide the development of more effective and selective compounds. X-ray analysis was used to confirm the chemical structure of 14 (MSA17).

Antiplatelet agent; Catalytic site mapping; Cyclooxygenase-1; Diarylisoxazoles; Drug design; Hemolysis; Inhibitors; QSAR.