Original article Discovery and kinetic evaluation of 6-substituted 4-benzylthio-1,3, 5-triazin-2(1H)-ones as inhibitors of cathepsin B Izidor Sosi  c a , Bojana Mirkovi  c a , Samo Turk a , Bogdan  Stefane b, c , Janko Kos a, d , Stanislav Gobec a, * a Faculty of Pharmacy, University of Ljubljana, A sker ceva 7, 1000 Ljubljana, Slovenia b Faculty of Chemistry and Chemical Technology, University of Ljubljana, A sker ceva 5, 1000 Ljubljana, Slovenia c EN-FIST Centre of Excellence, Dunajska 156, 1000 Ljubljana, Slovenia d Department of Biotechnology, Jo zef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia article info Article history: Received 3 June 2011 Received in revised form 30 July 2011 Accepted 2 August 2011 Available online 9 August 2011 Keywords: Cathepsin B Cysteine protease Noncovalent inhibitors 1,3,5-Triazine-2(1H)-ones Partial mixed-type inhibition abstract Cathepsin B is a lysosomal cysteine protease that has various physiological and pathophysiological functions. We present here the discovery of 6-substituted 4-benzylthio-1,3,5-triazin-2(1H)-ones as inhibitors of cathepsin B, starting from screening of a library of variously 2,4,6-trisubstituted 1,3,5- triazines and 1,3,5-triazin-2(1H)-ones on three different human cathepsins. The synthesis and enzy- matic evaluation of a focused library of new 1,3,5-triazin-2(1H)-ones is also described. The detailed kinetics analyses have shown that these compounds can act as reversible, partial mixed-type inhibitors of cathepsin B, with K i and K i 0 values in the low micromolar range. The inhibitory activities of selected compounds were also assessed against two related cysteine proteases, cathepsin H and cathepsin L, to estimate their selectivity; these compounds have a selective profile for catB and catL over catH. Ó 2011 Elsevier Masson SAS. All rights reserved. 1. Introduction Cathepsin B (catB) (EC 3.4.22.1) is a lysosomal cysteine protease that belongs to the papain family (C1) of clan CA of the cysteine proteases [1]. It is unique among cathepsins as it exists in two conformations, which are responsible for endopeptidase and dipeptidyl carboxypeptidase activities [2]. In the exopeptidase conformation access of the extended substrates into the active site of catB is limited by a 21 amino acid insertion termed the occluding loop (Ile105-Thr125) [3] that is held on to the body of the enzyme by two salt bridges (Asp22-His110 and Asp224-Arg116). Addition- ally, the loop provides two His residues (His110 and His111) that bind the substrate’s C-terminal carboxylate, enabling the exopep- tidase activity [4] that has a pH optimum around 5 [5], suitable for lysosomal compartments. However, catB can also act as an endo- peptidase since the occluding loop is flexible and can move away from the active-site cleft [6]. It appears that the conformation of the loop is pH dependent [3], with a prevalence of endopeptidase activity at neutral pH (typical of the environment of the membrane- bound or extracellular catB), suggesting an extralysosomal as well as an extracellular role for cathepsin B endopeptidase activity. The endopeptidase conformation of the occluding loop can also be stabilized with inhibitors such as cystatin C [7] and chagasin [8]. As well as participating in protein turnover in lysosomes [2], catB has some more specific physiological functions, such as bone resorption [9], liberation of thyroid hormones from thyroglobulin [10], antigen processing [11], and skin wound healing [12]. Alter- ations in catB expression, protein levels, activity and localization are associated with several disease states. Notably, catB has been shown to be critically involved in tumor metastasis, angiogenesis and progression [13]. These wide-ranging functions indicate that catB is a promising druggable target in cancers, rheumatoid arthritis and other important diseases. Inhibitors of catB include endogenous inhibitors, such as the cystatin superfamily of proteins, low molecular weight natural inhibitors, and synthetic inhibitors. The majority of synthetic inhibitors that have been described to date are peptidyl compounds containing an electrophilic functionality, which reacts reversibly or irreversibly with the catalytic cysteine in the active site of catB [14]. So far, none of these compounds have reached the clinical practice, mostly due to poor bioavailability, off-target side effects and high toxicity [15]. Therefore, due to the great number of possible Abbreviations: Z, benzyloxycarbonyl; AMC, 7-amido-4-methylcoumarin; catB, cathepsin B; EI, enzymeeinhibitor complex; ES, enzymeesubstrate complex; ESI, enzymeesubstrateeinhibitor complex. * Corresponding author. Tel.: þ386 1 476 9500; fax: þ386 1 425 8031. E-mail address: stanislav.gobec@ffa.uni-lj.si (S. Gobec). Contents lists available at ScienceDirect European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech 0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejmech.2011.08.005 European Journal of Medicinal Chemistry 46 (2011) 4648e4656