X-ray structural and biological evaluation of a series of potent and highly selective inhibitors of human coronavirus papain-like proteases

Abstract

Structure-guided design was used to generate a series of noncovalent inhibitors with nanomolar potency against the papain-like protease (PLpro) from the SARS coronavirus (CoV). A number of inhibitors exhibit antiviral activity against SARS-CoV infected Vero E6 cells and broadened specificity toward the homologous PLP2 enzyme from the human coronavirus NL63. Selectivity and cytotoxicity studies established a more than 100-fold preference for the coronaviral enzyme over homologous human deubiquitinating enzymes (DUBs), and no significant cytotoxicity in Vero E6 and HEK293 cell lines is observed. X-ray structural analyses of inhibitor-bound crystal structures revealed subtle differences between binding modes of the initial benzodioxolane lead (15g) and the most potent analogues 3k and 3j, featuring a monofluoro substitution at para and meta positions of the benzyl ring, respectively. Finally, the less lipophilic bis(amide) 3e and methoxypyridine 5c exhibit significantly improved metabolic stability and are viable candidates for advancing to in vivo studies.

Figure 1

Chemical structures of previously characterized SARS-CoV PLpro inhibitors: (A) hit (1) from a primary HTS from which lead 24 was developed; (B) hit (2) from a primary HTS from which 15g and 15h were developed. The chiral center for the nearly equipotent isomers derived from hit 2 is indicated with an asterisk.

Scheme 1. Synthesis of α-Substituted Naphthyl Analogues

Reagents and conditions: (a) 1-naphthyl-MgBr, ether, rt; (b) Aq HCl; (c) LAH, THF, rt; (d) Ms₂O, DIEA, DCM, 0 °C; (e) ethyl isonipecotate, rt; (f) aq NaOH, EtOH, THF; (g) piperonylamine, EDCI, HOBt, DIEA, rt.

Scheme 2. Synthesis of α-Hydroxymethyl Analogue 1b

Reagents and conditions: (a) LAH, THF, rt; (b) TBSCl, imidazole, ether, rt; (c) Ms₂O, DIEA, DCM, rt; (d) ethyl isonipecotate, rt; (e) aq NaOH, EtOH, 40 °C; (f) piperonylamine, EDCI, HOBt, DIEA, rt; (g) CsF, aq ACN, rt.

Scheme 3. Synthesis of α-Phenylmethyl Analogue 1d

Reagents and conditions: (a) BnMgCl, ether, rt; (b) 1 M HCl, ether, 90 °C; (c) CH₃ONH₂, pyridine, rt; then BH₃–THF, 80 °C; (d) dimethyl 2,2-bis((1,3-dioxolan-2-yl)methyl)malonate, HCl/THF; (e) 20, rt, 20 h; (f) H₂, Pd/C, 40 psi, rt; (g) NaCN, DMF, 145 °C; (h) 1 M aq NaOH, EtOH, 60 °C; (i) piperonylamine, EDCI, HOBt, DIEA, rt.

Scheme 4. General Synthesis of Amide Analogues

Reagents and conditions: (a) RNH₂, EDCI or HATU, DIEA.

Scheme 5. Synthesis of Quinolinyl- and Isoquinolinylamides

Reagents and conditions: (a) MeMgCl, THF, −78 °C; (b) Ms₂O, DIEA, DCM; (c) ethyl isonipecotate, DCM, rt; (d) 1 M NaOH, MeOH; (e) EDCI, HOBt, DIEA, 3-fluorobenzylamine, THF.

Scheme 6. Synthesis of Conformationally Restricted Analogue 7

Reagents and conditions: (a) NaBH₃CN, NH₄OAc, ⁱPrOH, reflux, 44 h; (b) dimethyl 2,2-bis((1,3-dioxolan-2-yl)methyl)malonate, HCl/THF; (c) 30, NaHCO₃, rt, 72 h ; (d) H₂, PtO₂, EA, 40 psi, 5 h; (e) NaCN, DMF, reflux, 16 h; (f) aq LiOH, THF/MeOH, rt; (g) 3-fluorobenzylamine, EDCI, HOBt, DIEA, rt, 16 h.

Figure 2

Chemical structure of compound 7. The conformationally restricted analogue 7 was designed to lock the conformation of compound 3k to the conformation observed in the X-ray crystal structure of SARS-CoV PLpro bound to 15g.

Figure 3

X-ray crystal structures of SARS-CoV PLpro in complex with 3k and 3j. Stereoviews of SARS-CoV PLpro (blue ribbon representation and gray surfaces) in complex with 3k (orange ball and sticks) are shown in (a) and (c), and SARS-CoV PLpro (cyan ribbon representation and gray surface) in complex with 3j (pink ball and sticks) are shown in (b) and (d). The corresponding F_o – F_c electron density omit maps (inhibitor atoms omitted) contoured at 3σ are shown as gray mesh. Important amino acids for inhibitor binding are shown, and the H-bonds between inhibitor atoms and amino acid residues are depicted as dotted lines. A superimposition of SARS-CoV PLpro–3k and PLpro–3j complexes is shown in (e) with conserved water molecules displayed as blue and cyan spheres for the 3k– and 3j– complexes, respectively. A superimposition of SARS-CoV PLpro–15g complex (SARS-CoV PLpro displayed as a gray ribbon and 15g displayed as gray balls and sticks, pdb:3MJ5) with SARS-CoV PLpro–3k and PLpro–3j complex is shown in (f).