Physicochemical and biological evaluation. To demonstrate the potential of 3,3-difluorooxetane for bioisosteric replacements in medicinal chemistry, we have prepared 3,3-diFOx-substituted analog 17c of Tenovin-6 (17g), a known selective inhibitor of histone deacetylases SirT1 and SirT2 (Table ). In this case, the 3,3-diFOx moiety was introduced instead of tert-butyl group. The synthesis of compounds 17c and 17g commenced from corresponding benzoic acids 12g and 9 and followed the procedure reported previously for compound 17g (see the Supporting Information for more details). Evaluation of the obtained products revealed that 3,3-diFOx-substituted analog 17c retained most biological activity of original Tenovin-6 (17g) against histone deacetylase SIRT1 (only 4-fold decrease of the IC50 value was observed). Furthermore, compound 17c was by 0.7-1.3 LogP / LogD7.4 units less lipophilic than parent Tenovin-6, while aqueous solubility of both compounds was similar (370 and 350 M, respectively). Caco-2 permeability test demonstrated moderate permeability through the cell membranes for both compounds. Nevertheless, the efflux of Tenovin-6 (17g) was strongly affected by P-glycoprotein (P-gp) inhibitor Verapamil, while 3,3-diFOx-Tenovin-6 (17c) was much less sensitive to the presence of this compound. In other words, introducing the 3,3-diFOx substituent decreased affinity of Tenovin-6 towards P-gp. Finally, metabolic stability (measured as intrinsic clearance, CLint) was somewhat improved for the 3,3-diFOx-substituted derivative (CLint = 2 µL/min/mg vs 9 µL/min/mg for Tenovin-6). These results show that 3,3-difluorooxetane can be indeed used as a bioisostere of tert-butyl (and likely other) groups to improve pharmacokinetic properties of optimized compounds.