Tetrahydrofuro[3,2-b] pyrrol-3-one intermediates

General Procedures

[0418]Solvents were purchased from ROMIL Ltd, U.K. at SpS or Hi-Dry grade unless otherwise stated. 1H NMR and 13C NMR were obtained on a Bruker DPX400 (400 MHz 1H frequency and 100 MHz 13C frequency; QXI probe) or Bruker Avance 500 MHz (TXI probe with ATM) in the solvents indicated. Chemical shifts are expressed in parts per million (δ) and are referenced to residual signals of the solvent. Coupling constants (J) are expressed in Hz. All analytical HPLC were obtained on Phenomenex Jupiter C4, 5μ, 300 Å, 250×4.6 mm, using mixtures of solvent A (0.1% aq trifluoroacetic acid (TFA)) and solvent B (90% acetonitrile / 10% solvent A) on automated Agilent systems with 215 and / or 254 nm UV detection. Unless otherwise stated a gradient of 10 to 90% B in A over 25 min at 1.5 mL / min was performed for full analytical HPLC. HPLC-MS analysis was performed on an Agilent 1100 series LC / MSD, using automated Agilent HPLC systems, with a gradient of 10 to 90% B in A over 10 min on

Assays for Cysteine Protease Activity

[0635]The compounds of this invention may be tested in one of a number of literature based biochemical assays that are designed to elucidate the characteristics of compound inhibition. The data from these types of assays enables compound potency and the rates of reaction to be measured and quantified. This information, either alone or in combination with other information, would allow the amount of compound required to produce a given pharmacological effect to be determined.

In vitro Cathepsin KiInhibition Measurements

[0636]Stock solutions of substrate or inhibitor were made up to 10 mM in 100% dimethylsulfoxide (DMSO) (Rathburns, Glasgow, U.K.) and diluted as appropriately required. In all cases the DMSO concentration in the assays was maintained at less than 1% (vol. / vol.). The equilibrium inhibition constants (Kiss) for each compound were measured under steady-state conditions monitoring enzyme activity as a function of inhibitor concentration.