Identification of SARS-CoV-2 Mpro inhibitors containing P1’ 4-fluorobenzothiazole moiety highly active against SARS-CoV-2

Abstract

COVID-19 caused by SARS-CoV-2 has continually been serious threat to public health worldwide. While a few anti-SARS-CoV-2 therapeutics are currently available, their antiviral potency is not sufficient. Here, we identify two orally available 4-fluoro-benzothiazole-containing small molecules, TKB245 and TKB248, which specifically inhibit the enzymatic activity of main protease (Mpro) of SARS-CoV-2 and significantly more potently block the infectivity and replication of various SARS-CoV-2 strains than nirmatrelvir, molnupiravir, and ensitrelvir in cell-based assays employing various target cells. Both compounds also block the replication of Delta and Omicron variants in human-ACE2-knocked-in mice. Native mass spectrometric analysis reveals that both compounds bind to dimer Mpro, apparently promoting Mpro dimerization. X-ray crystallographic analysis shows that both compounds bind to Mpro’s active-site cavity, forming a covalent bond with the catalytic amino acid Cys-145 with the 4-fluorine of the benzothiazole moiety pointed to solvent. The data suggest that TKB245 and TKB248 might serve as potential therapeutics for COVID-19 and shed light upon further optimization to develop more potent and safer anti-SARS-CoV-2 therapeutics.

Structure–Activity Relationship Studies of SARS-CoV-2 Main Protease Inhibitors Containing 4-Fluorobenzothiazole-2-carbonyl Moieties

“…The t 1/2 values of TKB245 ( 5 ) were 1.25 and 3.82 h when administered intravenously and orally, respectively. The oral bioavailability values of TKB245 ( 5 ) and TKB248 ( 6 ) were 48% and 72%, respectively …”

“…The original procedure for the syntheses of TKB245 ( 5 , Scheme ) and TKB248 ( 6 ) was described in a previous report . To develop a more efficient synthesis of derivatives of the most potent hybrid compound TKB245 ( 5 ), especially of derivatives at the P1′ moiety, we devised a novel synthetic scheme that allowed the insertion of benzothiazole derivatives of the P1′ moiety in the last step of the synthesis.…”

“…In order to meet this challenge, we previously reported highly potent M pro inhibitors ( 3 ) and ( 4 ) based on YH-53/5h ( 2 ), which was developed by Hayashi et al in 2013 as a SARS-CoV M pro inhibitor – (Figure ). More recently, our further studies led to the development of more potent inhibitors, TKB245 ( 5 ) and TKB248 ( 6 ) . In this study, we report our synthesis and evaluation of the anti-SARS-CoV-2 activity of derivatives of TKB245 ( 5 ) and TKB248 ( 6 ) and establishment of a diversity-oriented synthetic route for P1′ derivatization of TKB245 ( 5 ).…”

“…Structures of SARS-CoV-2 M pro inhibitors, nirmatrelvir ( 1 ), YH-53/5h ( 2 ), 3 , 4 , TKB245 ( 5 ), and TKB248 ( 6 ). , Fold change means a ratio of an EC 50 value to that of YH-53/5h ( 2 ).…”

Fluorine-a small magic bullet atom in the drug development: perspective to FDA approved and COVID-19 recommended drugs

“…Recently, fluorinated compounds like nirmatrelvir (PF-07321332) 247 etc. (Brewitz et al 2023;Higashi-Kuwata et al 2023;Abdallah et al 2022;Fang et al 2022).…”