The enzymatic assays showed that ZINC3276278 displayed promising inhibitory potency against PL pro . Prior to more complete structure-activity relationship investigations, we thought to resynthesize this compound to confirm its activity as weak potencies can be induced by impurities rather than the main molecule. We developed a concise 3-step synthesis of the target molecule from commercially available building blocks (Scheme 3). The synthesis began with a carbodiimidecatalyzed peptide coupling between acetoxyacetic acid and 2-amino-5-chlorobenzophenone to give intermediate XZ-1, which was then deacetylated by treatment with potassium carbonate in methanol into intermediate XZ-2. The last step involved a Steglich esterification between 2-methoxycinnamic acid and intermediate XZ-2 to give XZ-3a, which is structurally identical to ZINC3276278. Optimization through synthesis. This resynthesis was followed by preliminary structure-activity relationship investigations to further explore the potential of these structures as PL pro inhibitors. We first thought to introduce diversity next to the covalent warhead (Scheme 3). Thus, the first set of analogues contained variation at the leftmost benzene ring of the original compound, where the methoxy group is at the ortho position. The first analogue (XZ-3b) contains a benzene ring where the ortho methoxy group was removed while the second analogue (XZ-3c) contains a trifluoromethyl moiety at the para position of the ring. These first two analogues will allow us to test the role of aromatic ring electronics in the activity. In particular, introducing an electron withdrawing trifluoromethyl group will lead to electronic effects on the Michael acceptor contrasting with those of the electron donating methoxy group. In the third analogue (XZ-3d) the benzene is removed further modulating the reactivity of the Michael acceptor but also significantly reducing the size of this part of the molecule. The fourth (XZ-3e) and fifth analogues (XZ-3f) with heterocycles, namely an imidazole ring and a furan ring, were designed to introduce hydrogen bond donors and acceptors at this position as water molecules have been observed in the cavity these are predicted to bind to. Scheme 3. a) DCC, DCM, rt, 4 h, 79%; b) K2CO3, MeOH, rt, 30 min, 74%; c) EDC•HCl, DMAP, DCM, rt, 24-48 h, 71% (XZ-3a), 74% (XZ-3b), 24 h, 80% (XZ-3c), 26 h, 35% (XZ-3d), 48 h, 36% (XZ-3e), 24 h, 54% (XZ-3f).