3.1.3  Inhibitors with Michael acceptor as a warhead group
The disclosure of the first crystal structure of the SARS‐CoV‐1 Mpro in complex with a peptidic inhibitor Cbz‐Val‐Asn‐Ser‐Thr‐Leu‐Gln‐chloromethyl ketone (also known as hexapeptide chloromethyl ketone; 28) 125  provided clues for the substrate‐based design. Although it is a substrate analog for the porcine transmissible gastroenteritis CoV (TGEV) Mpro, it offers a structural explanation for the P1‐Gln entering into the specific subsite S1 pocket and decreased P2‐leucine specificity in the hydrophobic S2 site of SARS‐CoV‐1 Mpro. Additionally, rupintrivir (29; AG7088), a peptidomimetic inhibitor of human rhinovirus 3C protease is oriented similar to inhibitor 28 in the binding pocket of TGEV Mpro. 129  These two molecules became prototype compounds for the development of SARS‐CoV‐1 Mpro inhibitors.
Compound 29 was only weakly active against SARS‐CoV‐1 Mpro (IC50, 800 µM) also in cellular antiviral assays. 130  However, systematic structural modifications led to a series of analogs that show moderate to good activity. 131  For example, compound 30 (Figure 11), in which the P1‐lactam was replaced by a phenyl ring, showed moderate activity. Compound 31, in which the larger P2 p‐fluorophenyl was replaced with a phenyl group, was even more effective. By taking 29 as a lead, Ghosh et al. designed new molecules mainly focusing on the replacement of the large P2 p‐fluorobenzyl group. Two of the resulting structures with P2‐benzyl (32) and prenyl (33) moieties showed decent inhibitory potencies at both enzymatic (K inact, 0.014 and 0.045 min−1, respectively) and cell‐based (IC50, 45 and 70 µM) assays. 132  Besides, no cytotoxicity was observed for these compounds up to 100 µM concentration. However, 32 and 33 were inactive at MERS‐CoV Mpro. 133  The same research group further modified the molecule with the introduction of P4 Boc‐serine, to establish additional hydrogen bond interactions as described in compound 34 (IC50, 75 µM). Unfortunately, the activity of the resulting compound was not improved. Further modification of the isobutyl group in compound 34 to isoprenyl group in compound 35 displayed potent activity with K i = 3.6 µM (Figure 11). 14
Figure 11  SARS‐CoV Mpro inhibitors containing Michael acceptor as a warhead group. Mpro, main protease; SARS‐CoV, severe acute respiratory syndrome coronavirus On the other hand, Yang et al. 134  reported a series of peptide inhibitors with a greater inhibitory potency. In general, they systematically changed the backbone of inhibitor 29. As a result, they were able to identify more specific residues for each subsite (compounds 36–38; Figure 12): At first, the P1‐lactam ring was identified as a more specific moiety for the S1‐site, forming multiple hydrogen‐bond interactions with the enzyme as can be seen in the crystal structure (36); P2‐leucine showed a fourfold increased inhibitory activity when compared to the P2‐phenylalanine or ‐4‐fluorophenylalanine (37). A lipophilic tert‐butyl residue was recognized to be a better P3‐moiety than the P3‐valine (38). Finally, the replacement of P4‐methylisoxazole with a benzyloxy group was the best option for activity enhancement (compare 29 vs 36). They all showed moderate to high antiviral activity against HCoV‐229E in cell‐based assays.
Figure 12  Broad‐spectral antiviral compounds containing a Michael acceptor Shie et al. 131  reported another series of peptide inhibitors with comparatively reduced molecular weight to increase drug‐like properties. These pseudo‐C2‐symmetric inhibitors consist of a Phe‐Phe‐dipeptidic α,β‐unsaturated ester. One of these inhibitors (39) had an outstanding inhibitory activity with an EC50 value of 0.52 µM (see Figure 12). Besides, it displayed remarkable antiviral activity with an EC50 value of 0.18 µM. Structurally, the presence of 4‐dimethylamine on the phenyl ring was found to be crucial for activity enhancement.
Another peptidic drug with a Michael acceptor was N3 (40), which was reported to inhibit SARS‐CoV‐1 3CLpro (K i, 9.0 µM) by Yang et al. It was observed to be a broad‐spectrum antiviral compound, also inhibiting other CoVs, such as MERS‐CoV Mpro (IC50, 0.28µM), 135  HCoV‐229E, HCoV‐NL63, and HCoV‐HKU1 Mpro. 135 ,  136 ,  137 ,  138  It has also exhibited high antiviral activity in an animal model of infectious bronchitis virus. 137  The CC50 of 40 is greater than 133 μM.
SARS‐CoV‐2 shares only 82% of its genome with its relative SARS‐CoV‐1. However, essential viral enzymes of both species show sequence similarities of greater than 90%. 137 ,  139 ,  140 ,  141 ,  142  SARS‐CoV‐2 3CLpro is highly similar to SARS‐CoV‐1 3CLpro, sharing 96% of its sequence. Therefore, one could expect that SARS‐CoV‐1 Mpro inhibitors are active against SARS‐CoV‐2 Mpro. Compound 40 was found to be active against SARS‐CoV‐2 Mpro and its value of kobs/[I] for the COVID‐19 virus Mpro was determined to be 11 300 ± 880 M−1·s−1. 143  Peptide N3 was co‐crystalized with SARS‐CoV‐1 Mpro at 2.1 Å resolution (see Figure 13). Its binding mode to SARS‐CoV‐2 Mpro is highly similar to that of other CoV main proteases. Some key features include the Cys‐His catalytic dyad and the substrate‐binding pocket situated in a gap between domain I and II.
Figure 13  The crystal structure of COVID‐19 virus Mpro in complex with N3. (A) Representation of the dimeric Mpro‐inhibitor complex. (B) Surface representation of the homodimer of Mpro. Protomer A (blue), protomer B (salmon), compound N3 is presented as green sticks. (C) Schematic view of compound N3 (40) in the substrate‐binding pocket. 143  Mpro, main protease [Color figure can be viewed at wileyonlinelibrary.com] In general, inhibitors possessing a Michael acceptor group as a warhead moiety could form an irreversible (covalent) bond with the catalytic cysteine residue in the following manner (Figure 14): First, the cysteine residue undergoes 1,4‐addition at the inhibitor's Michael acceptor group (warhead). Rapid protonation of the α‐carbanion from His‐H+ leads to the covalent bond formation between the warhead of the inhibitor and the cysteine residue.
Figure 14  Mechanism of inhibitors with Michael acceptor group [Color figure can be viewed at wileyonlinelibrary.com]