3.1.6  Inhibitors with electrophilic ketone
It was envisioned that a fluorinated ketone moiety could be utilized as a warhead for targeting proteases, because it forms a thermodynamically stable hemiketal or hemithioketal after nucleophilic attack by Ser‐OH or Cys‐SH residues, which are present in the active sites of serine or cysteine proteases, respectively (see Figure 22).
Figure 22  Peptide inhibitors containing electrophilic ketone warheads [Color figure can be viewed at wileyonlinelibrary.com] Initially, Hayashi et al. reported a series of natural‐substrate‐derived peptide inhibitors containing a trifluoromethyl ketone warhead targeting SARS‐CoV‐1 Mpro. Compound 69 (Figure 22) was the best of the series with a K i value of 116 µM against SARS‐CoV‐1 Mpro. 158  It was sequentially modified mainly focusing on the warhead moiety since the formation of a cyclic structure prevented the nucleophilic attack by cysteine at the active site. This study led to the discovery of 70 containing a P1‐lactam and P1'‐thiazole moiety with a >50‐fold increase in inhibitory activity compared to 69. 159  Docking studies of 70 to Mpro highlighted key H‐bond interactions with backbone amino acid residues Cys143, Ser144, and Cys145. The nitrogen atom of the thiazole warhead moiety also engaged in H‐bond interactions, and the P1‐lactam nicely fitted into the S1‐pocket.
Continued computer‐assisted structural design led to a tripeptide containing benzothiazole as a warhead group and an m‐N,N‐dimethylaminophenyl group as P4‐moiety (71). 160  This compound was extremely potent in inhibiting Mpro of SARS‐CoV‐1 with a K i value of 3.1 nM. Docking studies of 71 confirmed that the benzothiazole group was tightly bound to the active site. Consequently, the same research group disclosed a series of dipeptides with reduced molecular weight in an attempt to improve drug‐like properties. The P3‐valine in the tripeptide 71 was exchanged for a variety of functional groups. 161  The study determined N‐arylglycyl to be the optimal P3‐moiety. Compound 72 displayed the best inhibitory activity. Docking studies of 72 to the protease highlighted the amino hydrogen of the P3‐N‐phenyl glycyl forming a H‐bond with backbone Glu166 of Mpro, in addition to the best P2‐leucine and P1'‐benzthiazole moieties (see Figure 23A). Further structural optimization at the P3‐N‐arylglycyl moiety found the indole‐2 carbonyl group to be one of the best P3‐moeities, thus reaching inhibitors with low nanomolar potency, for example 73 (K i, 0.006 µM) against SARS‐CoV‐1 Mpro. 162  Docking studies of compound 73 to the protease revealed that the indole amino hydrogen and the carbonyl group attached to the 2‐position formed H‐bond interactions with the backbone Glu166 (see Figure 23B). These interactions are of great importance, seeing as shifting the position of the carbonyl group from position 2 to 3, or replacing the indole with benzofuran drastically reduced inhibitory potency.
Figure 23  (A) Docking poses of 72 and (B) 73 with SARS‐CoV‐1 Mpro. Mpro, main protease; SARS‐CoV, severe acute respiratory syndrome coronavirus [Color figure can be viewed at wileyonlinelibrary.com] Zhao et al. reported a series of trifluoromethyl ketones. Among them, 74, which has the same sequence as the peptide substrate from sites P1 to P4, exhibited moderate inhibitory activity with an IC50value of 10 µM. Inhibitor 74 also displayed time‐dependent inhibition (K i, 0.3 µM). 163
Zhang et al. described a series of dipeptides containing difluoromethyl ketone as SARS‐CoV‐1 Mpro inhibitors. Compound 75 displayed the best inhibitory activity in infected Vero and Caco‐2 cell cultures with an IC50 value of 2.5 µM. It also exhibited little toxicity. 164
A library of small peptide‐anilides was developed as anti‐SARS‐CoV‐1 Mpro agents (77–80; Figure 23). These inhibitors were basically designed from niclosamide (76) which was inactive at Mpro of SARS‐CoV‐1. Proper structural modifications led to the discovery of 77 (IC50, 0.06 µM). It behaved as a competitive, noncovalent inhibitor (K i, 0.03 µM). SAR investigations pointed out that the N,N‐dimethyl group on the phenyl ring, and electron‐withdrawing groups at the warhead phenyl are important. Structural modification of 77 resulted in compounds 78 – 80 displaying reduced potency. 165
A novel series of ketoglutamide tripeptides bearing a phthalhydrazido warhead group were identified as reversible SARS‐CoV‐1 Mpro inhibitors (81–84; Figure 24). 166  Among them, compound 83 showed the best inhibition (IC50, 0.6 µM). SAR studies revealed the presence of β and β'‐amino functionality adjacent to the keto and the intramolecular hydrogen bond to the carbonyl group made the keto center more electrophilic and inclined to build a hemithioacetal with Cys‐SH at the active site. Additionally, the hydrophobic P3‐benzyloxy moiety, the P1‐lactam, and the nitro group significantly contributed to the activity increment.
Figure 24  Small peptide anilides and ketoglutamide tripeptides as SARS‐CoV‐1 inhibitors. SARS‐CoV, severe acute respiratory syndrome coronavirus Wang et al. 167  described the development of selective and reversible SARS‐CoV‐1 Mpro inhibitors derived from HIV proteases inhibitors (Figure 25). The compound 85 as a SARS‐CoV‐1 Mpro lead inhibitor was continuously modified to obtain 86 and 87. These derivatives were highly selective toward SARS‐CoV‐1 Mpro versus HIV protease. Docking studies of 87 to Mpro demonstrated that both indole amino hydrogens establish H‐bond networks with side chain His142 and His41.
Figure 25  SARS‐CoV‐1 Mpro inhibitors derived from HIV proteases inhibitors. HIV, human immunodeficiency virus; Mpro, main protease; SARS‐CoV, severe acute respiratory syndrome coronavirus