NSC-41589 is N-[2-(methylsulfanyl)phenyl]acetamide.

Direct PAI-I in vitro activity assays : The chromogenic assay is initiated by the addition of tiplaxtinin (10 – 100 μM final concentration, maximum DMSO concentration of 0.2%) to recombinant human PAI-1 (140 nM in pH 6.6 buffer). After a 15 minute incubation at 25°C, 70 nM of recombinant human t-PA is added, and the combination of tiplaxtinin, PAI-1 and tPA are incubated for an additional 30 minutes. After the second incubation, Spectrozyme tPA, is added and absorbance read at 405 nm at 0 and 60 minutes. Relative PAI-1 inhibitory activity is equal to the residual tPA activity in the tiplaxtinin / PAI-1 treatment. Control treatments include the complete inhibition of tPA by PAI-1 at the molar ratio employed (2:1), and the absence of any effect of the tiplaxtinin on t-PA alone. The immunofunctional assay is based upon the non-SDS dissociable interaction between tPA and active PAI-1. Assay plates are coated with 100 μl of a solution of t-PA (10 μg/ml in TBS), and kept at 4 °C overnight. Tiplaxtinin is dissolved in DMSO and diluted to a final concentration of 1-100 μM as described above. Tiplaxtinin is then incubated with human PAI-1 (50 ng/ml) for 15 minutes, and an aliquot of this solution added to the t-PA-coated plate for 1 h. The solution is aspirated from the plate, which is then washed with a buffer consisting of 0.05% Tween 20 and 0.1% BSA in TBS. This assay detects only active inhibitory PAI-1 (not latent or substrate) bound to the plate, and is quantitated using a monoclonal antibody against human PAI-1 (MA33B8). A 1000X dilution of MA33B8 is added to the plate and incubated at for one hour, aspirated and washed. A secondary antibody consisting of goat anti-mouse IgG (H+L)-AP alkaline phosphatase conjugate is added, incubated for one hour, aspirated and washed. A 100 μl aliquot of alkaline phosphatase solution is added, followed by determination of absorbance at 405 nm 60 minutes later.The quantitation of residual active PAI-1 bound to t-PA at varying concentrations of tiplaxtinin is used to determine the IC50 by fitting the results to a logistic dose-response program, with the IC50 defined as the concentration of compound required to achieve 50% inhibition of PAI-1 activity. The assay sensitivity is 5 ng/ml of human PAI-1 as determined from a standard curve ranging from 0-100 ng/ml of human PAI-1.