Alpha-ketoamides as broad-spectrum inhibitors of coronavirus and enterovirus replication

Linlin Zhang; Daizong Lin; Yuri Kusov; Yong Nian; Qingjun Ma; Jiang Wang; Albrecht von Brunn; Pieter Leyssen; Kristina Lanko; Johan Neyts; Adriaan de Wilde; Eric J. Snijder; Hong Liu; Rolf Hilgenfeld

11u appeared so far the best compromise compound, yet for each of the individual viral enzymes, the following compounds proved superior: P2 = cyclopropylmethyl (compound 11s) for SARS-CoV M pro , P2 = isobutyl (compound 11n) and P2 = cyclopropylmethyl (11s) for HCoV-NL63 M pro , P2 = benzyl (11a) or cyclohexylmethyl (11r) for EV-A71 3C pro , and 11r for CVB3 3C pro . In other words, the nearly equipotent 11u is indeed a compromise. Therefore, in view of the surprisingly good antiviral activity of 11r against HCoV 229E in Huh7 cells, we relaxed the condition that the universal inhibitor should show good activity against the recombinant HCoV-NL63 M pro , and selected 11r (P2 = cyclohexylmethyl) as the lead compound for further development. This compound exhibited submicromolar IC50 values against CVB3 3C pro and SARS-CoV M pro , and IC50 = 1.7 µM against EV-A71 3C pro (Table 1) , as well as similarly low EC50 values in the replicons of these viruses (Table 2 ). In Huh7 cells infected with MERS-CoV, the performance of this compound was excellent, with EC50 = 0.0004 µM, and even against HCoV 229E in Huh7 cells and SARS-CoV in Vero E6 cells, EC50 values of 1.8 and 2.1 µM, respectively, were observed (Table 3) . Also in enterovirus-infected cell culture, the compound performed well, with EC50 values of 0.7 µM or below against HRV2, HRV14, and EV-D68 in HeLa (Rh) cells and selectivity values >15. The only concern is the activity of the compound against EV-A71 in RD cells, for which the EC50 value was 3.7 µM, resulting in too low a therapeutic index. On the other hand, only weak toxicity was detected for 11r in Vero or Huh-T7 cells. Preliminary pharmacokinetics tests with the compound in mice did not indicate a toxicity problem (to be published elsewhere).