Roflumilast analogues with improved metabolic stability, plasma protein binding and pharmacokinetic profile.

With the aim to study their in vitro and in vivo pharmacokinetics, new chromatographic methods were developed for the determination of three new roflumilast synthetic analogues (I-III) as PDE-4B inhibitors in rat liver S9 fraction, phosphate buffered saline, pH 7.4, human and rat plasma. The developed HPLC-UV methods were performed on a Zorbax Eclipse C8 column and UV detection was carried out at 215 nm. The three compounds were tested for their metabolic stability and they were found to be metabolically more stable than roflumilast especially the 2-mercaptobenzothiazol-6-yl analogue (III) which displayed an in vitro half-life time (247.55 min.) higher than that of roflumilast (12.29 min.) and a low in vitro clearance of 5.67 mL/min./kg. Possible phase I metabolites were investigated using UPLC-MS/MS showing hydroxylation of the unsubstituted benzothiazol-2-yl (I) and benzothiazole-6-yl (II) analogues and a cleaved benzothiazole metabolite of the 2-mercaptobenzothiazol-6-yl analogue (III). Plasma protein binding affinity was tested using equilibrium membrane dialysis method showing a very high percentage (more than 95%) of plasma protein binding of compounds I and II where compound III exhibited lower percentage (53.71%) demonstrating its accessibility for tissue distribution. Also, a UPLC-MS/MS method was developed using an Acquity UPLC BEH shield RP C18 column to be applied to an in vivo pharmacokinetic study in rats following a subcutaneous dose (1mg/kg). Compounds I-III showed improved in vivo pharmacokinetic parameters especially compound III which displayed a half-life greater than roflumilast by 3 folds (21 h) and a Cmax value of 113.958 ng/mL. Accordingly, this new chemical entity should be subjected for further investigation as it can be a good drug candidate for treating Chronic obstructive pulmonary disease.