Journal of Medicinal Chemistry Figure 5. Inhibitory profile of peptides and peptide analogues on the activity of streptococcal cysteine protease SpeB (A) and family class protease papain (B). Activity is expressed relative to enzyme activity in absence of putative inhibitors. (R) and (S) indicate the different stereoisomers of each analogue; “pip” represents the piperidine moiety replacing glycine residues. The enzyme activity was determined for papain at a final concentration of 0.9 μM and SpeB at final concentration of 0.28 μM in the presence of 4.8 × 103 molar excess of peptides/peptide analogues. However, both (S)-pipG ((S)-3) and (R)-LpipG ((R)-7) show promising selectivity profiles as they are both potent inhibitors of IdeS but are hardly affecting papain and do not inhibit SpeB. These compounds were investigated further to evaluate whether they might be a suitable starting point for the development of specific IdeS protease inhibitors. Various amounts of inhibitor and substrate were used to determine the inhibition constant (Ki) for these two compounds. Because a mechanism of competitive inhibition was postulated, initial velocities were determined in the presence or absence of inhibitor. The binding affinity of IdeS for IgG has been previously determined, and a high affinity was observed with a Kd of 2.5 μM and a Km for complete enzymatic cleavage ranging from 6.8 to 18.9 μM depending on the IgG subtype.12 Because of the high affinity substrate binding, a considerable excess of inhibitor was expected to be necessary to affect initial velocities and to successfully compete for IgG. The Ki values for (S)-pipG ((S)-3) and (R)-LpipG ((R)-7) were determined to approximately 6.7 and 5.7 mM, respectively. Although these concentrations are too high for in vivo evaluation, they were surprisingly low considering the high affinity of IdeS for IgG. Importantly, initial velocities of papain activity remained Article unchanged by the presence of the inhibitors and therefore Ki values could not be determined reliably, but the values exceed those observed for IdeS at least 16−267-fold, respectively. Thus, the aminopiperidine containing analogues (S)-pipG ((S)-3) and (R)-LpipG ((R)-7) exhibit effective and specific IdeS inhibitor properties and will serve as a starting point for further development of more potent inhibitors. Initially, an attempt to increase the rigidity of the compound will be undertaken which hopefully will increase the potency of the inhibitor. Synthesis of such compounds is ongoing. ■ CONCLUSION A series of 17 piperidine-containing peptide analogues have been efficiently synthesized. The peptide analogues were tested in inhibition assays addressing cysteine protease activities with focus on the streptococcal IgG degrading enzyme IdeS. Several of the synthesized piperidine-containing peptide analogues are active IdeS inhibitors, but the piperidine moiety does not necessarily improve the inhibitory capacity of the analogues toward other cysteine proteases. To the best of our knowledge, these are the first noncovalently bound inhibitors of IdeS, also showing selectivity toward SpeB and papain. ■ EXPERIMENTAL SECTION Synthesis. General. Starting materials and reagents were purchased from Sigma-Aldrich or Bachem and were used as such. The peptides were purchased from CASLO Laboratory ApS, Lyngby, Denmark. The purity of the peptides was >98%. Analytical TLC was performed on silica plated aluminum sheets (grade 60 F254, Merck). The spots were visualized by treatment with a dip solution [KMnO4 (1.5 g), K2CO3 (10 g), NaOH (10%) (1.25 mL) in water (200 mL)], followed by heating. Flash chromatography was performed on Merck silica gel 60 (0.040−0.063 mm). 1H and 13C NMR spectra were recorded on a JEOL Eclipse 400 spectrometer at 400 and 100 MHz, respectively, at ambient temperature and in CDCl3 if not otherwise stated. 13C NMR spectra were also recorded on a Varian 500 spectrometer at 125 MHz. Chemical shifts are reported in ppm, with the solvent residual peak as internal standard (CHCl3 δH 7.26, δC 77.0, CH3OH δH 3.30, δC 49.0). DEPT, COSY, TOCSY, and HSQC spectra were recorded to validate structural assignments of the NMR signals. Separation of the stereoisomers of 1 and 19 were performed on a preparative HPLC Shimadzu system equipped with a Chiralpak AD 250 mm × 20 mm column (Daicel Chemical CO., Tokyo, Japan) with a mobile phase consisting of hexane/ethanol 95:5 or hexane/2- propanol 90:10 at a flow rate of 15 or 10 mL/min, respectively. Detection was carried out at 240 nm. Chiral HPLC analysis was performed on a Varian system equipped with a Chiralpak AD 4.6 mm × 250 mm column (Daicel Chemical CO., Tokyo, Japan) at a flow rate of 1 mL/min. Detection was carried out at 240 nm. Analytical reversed phase HPLC was performed on a Waters 2690 system (photodiode array detector at 220 nm and flow rate 1.0 mL/min) equipped with a 250 mm × 4.6 mm Reprosil PUR C18aq column. Optical rotation was measured with a Perkin-Elmer 341 LC polarimeter at 20 °C. Infrared spectra were recorded on a Perkin-Elmer 16 PC FTIR spectrometer, and only the major peaks are listed. HRMS analyses were run at BioAnSer, Gothenburg, Sweden. The purity of all tested compounds was determined using analytical reversed phase HPLC on a Waters 2690 system (photodiode array detection at 220 nm and a flow rate of 1.0 mL/min) equipped with a 250 mm × 4.6 mm Reprosil PUR C18aq column. (S)- and (R)-pipG (3) were further analyzed using a reversed phase Waters Acquity UPLC system equipped with a BEC C18 column, connected to a MS instrument with a Waters SQD single quadrupole detector. All compounds showed a purity >95% except (S)-pipG ((S)-3) which was 90% pure. Measurement of the aqueous dissociation constant, pKa values, of (R)- and (S)-LpipGP (18) and (R)- and (S)-pipG (3) were performed at AstraZeneca, R&D Mölndal, Sweden.27 2555 dx.doi.org/10.1021/jm201517a | J. Med. Chem. 2012, 55, 2549−2560