From the commercially available analogues, modifications on the left-hand side of the molecules were investigated. The next step was to investigate the right-hand side of the molecule. We developed a simple 2-step strategy to synthesize CSSS28077995 without a nitro group (KH-2) to see the effect of the nitro group in binding affinity to the enzyme (Scheme 9). The synthesis started with ethyl 3-(4-nitrophenyl)-3-oxopropanoate, hydrolysis of the ethyl ester gave intermediate KH-1, which was then coupled with 5-amino-N-(3-chlorophenyl)-2methylbenzenesulfonamide in the presence of EDC.HCl and DMAP to give KH-2 as the product. However, it was not active in 50 μM with peptide as a substrate, which suggests the nitro group is necessary for binding, likely to activate the ketone for covalent bond formation. For in vitro assays of the commercially available analogues of CSSS28008898, the potency maintained the same (50 % inhibition at 50 μM with peptide as substrate) with the modifications on the left-hand side of the molecule (Table ). Since there is not much improvement with the changes on the molecule, we suggested that this may be the best potency that this series is able to achieve.