All measurements were performed in duplicate with a single inhibitor concentration of 40 M

All measurements were performed in duplicate with a single inhibitor concentration of 40 M. accompanied by matriptase-2 inhibition. For example, 13 was inactive at matriptase-2, but highly active at thrombin [39]. Among the 3,4-dihydro-2 em H /em -1,4-benzoxazines with a methyl group at 4-position (13C21), several members inhibited matriptase-2 with IC50 values of less than 30 M. The presence of an oxamate moiety (in 13 and 14) appeared to be less favorable. This could be concluded from the results of the inactive compound 13 and of 16 (IC50 = 13.6 M). The higher flexibility of the glycine substructure (in 15C17) compared to the oxamate substructure (in 13 and 14) might account for Ergoloid Mesylates this effect. The position of the em N /em -substituted glycine moiety, as either 7- or 6-substituent, did not exert a remarkable influence on matriptase-2 inhibition (16 em versus /em 17). The common feature of the fluorine-free compounds 19 and 20 is the NHCO group at position 7. Both compounds were moderately active. The 3,4-dihydro-2 em H /em -1,4-benzoxazine derivatives 21C23 did not show an improved inhibitory activity, and ( em R /em )-24 and 25 were inactive. The finding that the latter two compounds did not affect matriptase-2 activity indicated that the presence of a benzamidine moiety does not necessarily lead to matriptase-2 inhibition. This was in accordance with the lack of inhibitory activity of benzamidine itself. On the TNR one hand, the absence of the benzo-fused heterocyclic core in ( em R /em )-24 and 25 was obviously unfavorable. On the other hand, since the majority of 2,3-dihydro-1,4-benzodioxines and 3,4-dihydro-2 em H /em -1,4-benzoxazines were active, these scaffolds are suitable for the positioning of various residues and for directing them to the targets binding pockets. In summary, representatives of three heterocyclic classes (4 em H /em -3,1-benzothiazin-4-ones, 2,3-dihydro-1,4-benzodioxines and 3,4-dihydro-2 em H /em -1,4-benzoxazines) were identified as inhibitors of matriptase-2. The three heterocyclic scaffolds are similar as they consist of a benzene ring fused to a six-membered heterocyclic ring. The results enabled us to assess the effect of certain residues on biological activity. Even though these compounds are not expected to be selective, this set of data can be used for the future design of new compounds in which such residues were placed at different positions at the bicyclic core in a combinatorial way. For example, the 4-benzamidino-oxymethylene group might be introduced into the 4 em H /em -3,1-benzothiazin-4-one scaffold. The first attempts to decorate the 4 Ergoloid Mesylates em H /em -3,1-benzothiazin-4-one heterocycle with a benzamidine moiety failed, because the scaffold was found to be unstable under the conditions used to convert a nitrile to an amidine group. Moreover, the substituents at positions 7 or 6 present in the active compounds ( em S /em )-12 and 17 might be introduced into the 4 em H /em -3,1-benzothiazin-4-one scaffold. The 6-substituent of 1 1 or the 2-substituent of 7 might also be considered for the design of new members of the 2 2,3-dihydro-1,4-benzodioxine and 3,4-dihydro-2 em H /em -1,4-benzoxazine series. Such investigations are planned for the future in our laboratories. 3. Experimental Section Ergoloid Mesylates 3.1. Assays for Human Matriptase-2 Inhibition The conditioned medium of HEK-MT2 cells was used as a source of matriptase-2 activity and assay conditions were as follows [11,19,25]. Assay buffer was 50 mM TrisCHCl, 150 mM NaCl, pH 8.0. The conditioned medium was collected and concentrated, and aliquots of the supernatant were stored at ?20 C. After thawing, it was diluted with assay buffer (1:10 or 1:20 depending on the enzyme activity) and kept at 0 C not longer than 8 h. The assays were performed at a FLUOstar OPTIMA PlateReader (BMG Labtech, Ortenberg, Germany). A 10 mM stock solution of the fluorogenic substrate Boc-Gln-Ala-Arg-AMC (Bachem, Bubendorf, Switzerland) in DMSO was diluted with assay buffer. The final concentration of the substrate was 40 M and of DMSO was 6%. The substrate concentration of 40 M refers to 1.24 em K /em m [19]. Into each well containing 163.8 L buffer, 11.2 L of an inhibitor solution in DMSO and 10 L of a substrate solution (800 M) were added and thoroughly mixed. At 37 C the reaction was initiated by adding 15 L of diluted conditioned medium and followed over 400 s. All measurements were performed in duplicate with a single inhibitor concentration of 40 M. Active inhibitors were investigated in duplicate with five different concentrations. Benzamidine hydrochloride.