Aza-nitrile Inhibitors With Trifluoroethylamine Group For Cathepsin K: Synthesis, Activity Assay, Cytotoxicity Test And Molecular Modeling

Osteoporosis is a disease characterized by an imbalance between bone resorptionand formation. Excessive bone resorption causes changes in the microstructure of thebone matrix which makes bonesprone to fracture. In1990, an estimated1.7millionosteoporosis-related hip fractures occurred worldwide from which approximately25%had a lethal outcome. Currently,44million people or55%of the US population aged50or older have osteoporosis or are at risk to develop the condition during theirlifespan. Direct and indirect costs of osteoporosis are staggering and estimated to be$18billion annually and increasing every year.Cathepsin K, a member of the papain family of cysteine proteases，is highlyexpressed by activated osteoclasts. It is specifically active against typeⅠcollagen, themajor component of the organic bone matrix. With such a major role in the initialprocess of bone resorption, cathepsin K has become a therapeutic target in thetreatment of osteoporosis.It have been reported that the inhibition of cathepsin K have the potential toprevent bone resorption without negatively impacting bone information, leading to anet anabolic effect and a more effective therapy for osteoporosis. Many derivatives ofkentone, aldehyde, ketoamide and nitrile have been reported as inhibitors of cathepsinK recently. Among them, peptidic nitriles, expecially azadipeptide nitriles, are knownto be excellently potent and reversible inhibitors and have attracted much interest.However, it is speculated that these peptidic nitriles, which have been reported in theprevious report, may have little biological utility for its deficiencies. One reason toinduce that is their potential toxicity resulting from barely satisfactory selectivity.Another major drawback of the reported aza-dipeptides, which contain scissile peptidebonds, is represented by their low bioavailability consequently. When thesecompounds are used as drugs in therapeutic classes, they have to be injected or administered in the form of expensive formulations. Therefore, great efforts have beentaken to improve their selectivity and stability toward cathepsin K.The modifications on the P2-P3linker are explored in the design of cathepsin Kinhibitors, and they have been proved to affect the profiles of inhibitors. It is reportedthat by replacing the P2-P3amide bond with a phenyl or aniline group, the selectivityor potency of the resulting inhibitors are improved more or less. Thus, thereplacement of amide bonds represents a viable and classic approach in the design ofnew inhibitors and appropriate replacement moiety becomes a crucial part to producebetter profiles of inhibitors.There are a few functional groups which are capable of preserving the hydrogenbond-donating properties of the amide. Among these replacements, thetrifluoroethylamine group, which C–CF3bond has similar polarity with the C=O,features low NH basicity. Moreover, the tetrahedral configuration oftrifluoroethylamine may allow the inhibitor to take a better posture in the active site ofcathepsin K than the planar amide bond, thus facilitating the interactions betweeninhibitor and cathepsin K.Herein, we use the stereogenic trifluoroethylamine to replace the carbonyl amideof the P2-P3linker in the azadipeptide inhibitors reported previously, generatingseveral new cathepsin K inhibitors with different P3groups.Besides, the MTT assay is used in the tested the cytotoxicity of differentcompounds on two distinct types of cells, human osteoblast-like line cell (MG63) andmouse vascular smooth muscle line cell (MOVAS). The MTT results show all thesecompounds were lowly toxic in the concentration range from1nM to1μM, at leaston the two types of test cell lines, illustrating their potential to be drugs foranti-osteoporosis in the future.Finally, the molecular docking program GOLD5.1, which has been successfullyapplied to the design and explanation of many bio-active molecules, is used to explorethe inhibition mechanism and structure activity relationship of inhibitors in ourresearch. The molecular docking shows that the stereoscopic trifluoroethylamine group and P3group affect the binding affinity and mode between inhibitors andenzymes.In conclution, replacing the amide bond with the trifluoroethylamino group isproven to be a promising method to explore other inhibitors, and the detailedinvestigation on the binding mechanism by using molecular docking lays thefoundation to further optimize the structure of more specific and potent inhibitors.