Structure-Guided Design of Potent Spirocyclic Inhibitors of Severe Acute Respiratory Syndrome Coronavirus-2 3C-like Protease

Abstract

The worldwide impact of the ongoing COVID-19 pandemic on public health has made imperative the discovery and development of direct-acting antivirals aimed at targeting viral and/or host targets. SARS-CoV-2 3C-like protease (3CL pro ) has emerged as a validated target for the discovery of SARS-CoV-2 therapeutics because of the pivotal role it plays in viral replication. We describe herein the structure-guided design of highly potent inhibitors of SARS-CoV-2 3CL pro that incorporate in their structure novel spirocyclic design elements aimed at optimizing potency by accessing new chemical space. Inhibitors of both SARS-CoV-2 3CL pro and MERS-CoV 3CL pro that exhibit nM potency and high safety indices have been identified. The mechanism of action of the inhibitors and the structural determinants associated with binding were established using high-resolution cocrystal structures.

Discovery and Crystallographic Studies of Trisubstituted Piperazine Derivatives as Non-Covalent SARS-CoV-2 Main Protease Inhibitors with High Target Specificity and Low Toxicity

“…The organic phase was washed by 0.5 M HCl, water, and saturated NaCl solution successively, dried over anhydrous Na 2 SO 4 , and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel to afford 13 N-(Thiophen-2-methyl)-1- (3,4-dichlorobenzoyl)piperazine-2-carboxyamide Trifluoroacetate (14). Compound 13 (4.0 g, 8.53 mmol) was dissolved in DCM (80 mL) under an ice bath and stirring.…”

“…The M pro inhibitors described so far are mainly based on peptidomimetics with electrophilic covalent warheads targeting Cys145. 12 − 14 This class of compounds includes Nirmatrelvir (PF-07321332), 15 PF-07304814, 16 GC-376, 17 and 11a, 18 which have been developed as clinical candidates or were already approved to treat COVID-19 ( Table 1 ). In addition, covalently binding small-molecule M pro inhibitors, such as chloropyridyl esters of indole derivatives, have been described.…”

“…In addition, covalently binding small-molecule M pro inhibitors, such as chloropyridyl esters of indole derivatives, have been described. 13 , 14 , 19 , 20 However, it was indicated that some of the developed covalently binding peptidomimetics additionally inhibit certain host proteases. 21 , 22 The reactive warhead-equipped peptidic scaffolds are responsible for those off-target activities, which have become a serious concern for most covalent peptidomimetic inhibitors (see Table 1 ).…”

“…The M pro inhibitors described so far are mainly based on peptidomimetics with electrophilic covalent warheads targeting Cys145. − This class of compounds includes Nirmatrelvir (PF-07321332), PF-07304814, GC-376, and 11a, which have been developed as clinical candidates or were already approved to treat COVID-19 (Table ). In addition, covalently binding small-molecule M pro inhibitors, such as chloropyridyl esters of indole derivatives, have been described. ,,, However, it was indicated that some of the developed covalently binding peptidomimetics additionally inhibit certain host proteases. , The reactive warhead-equipped peptidic scaffolds are responsible for those off-target activities, which have become a serious concern for most covalent peptidomimetic inhibitors (see Table ). , …”

Discovery of Chlorofluoroacetamide-Based Covalent Inhibitors for Severe Acute Respiratory Syndrome Coronavirus 2 3CL Protease

“…3CL pro recognizes glutamine and a hydrophobic amino acid residue at the S1 and S2 pockets in the active site, respectively, and cleaves amide bonds primarily within the Leu-Gln↓(Ser, Ala, Gly) sequence (↓: cleavage site) . This substrate specificity is conserved across other coronaviruses, including SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). , Therefore, initial efforts to develop SARS-CoV-2 3CL pro inhibitors have been extensively guided by previous molecular designs, especially those targeting SARS-CoV 3CL pro . , Most of these designs include peptidomimetics based on the Leu-Gln sequence combined with an electrophilic warhead, such as α-ketoamide, , aldehyde, − ketone, − vinyl sulfone, and nitrile, − for the covalent capture of the sulfhydryl group of the catalytic Cys145. In this category, nirmatrelvir (PF-07321332), developed by Pfizer, has been authorized for emergency use in combination with ritonavir as an oral medication (Figure ).…”