New pyrazolobenzothiazine derivatives as hepatitis C virus NS5B polymerase palm site I inhibitors

Abstract

We have previously identified the pyrazolobenzothiazine scaffold as a promising chemotype against hepatitis C virus (HCV) NS5B polymerase, a validated and promising anti-HCV target. Herein we describe the design, synthesis, enzymatic, and cellular characterization of new pyrazolobenzothiazines as anti-HCV inhibitors. The binding site for a representative derivative was mapped to NS5B palm site I employing a mutant counterscreen assay, thus validating our previous in silico predictions. Derivative 2b proved to be the best selective anti-HCV derivative within the new series, exhibiting a IC50 of 7.9 μM against NS5B polymerase and antiviral effect (EC50 = 8.1 μM; EC90 = 23.3 μM) coupled with the absence of any antimetabolic effect (CC50 > 224 μM; SI > 28) in a cell based HCV replicon system assay. Significantly, microscopic analysis showed that, unlike the parent compounds, derivative 2b did not show any significant cell morphological alterations. Furthermore, since most of the pyrazolobenzothiazines tested altered cell morphology, this undesired aspect was further investigated by exploring possible perturbation of lipid metabolism during compound treatment.

Figure 1

(Left) General formula of our first series of anti-NS5B pyrazolobenzothiazines. (Right) Hit compound 1.

Scheme 1

Scheme 2

Figure 2

Binding mode analysis of pyrazolobenzothiazine derivative 2a, depicted together with interacting NS5B residues and water molecules. Main interactions are represented schematically with their occupancies calculated in the time window 4–10 ns.

Figure 3

Huh-9-13 cells were stained with monodansylcadaverine (MDC) (a–c), Nile Red (d–f), or LipidTOX red (g–i) after mock treatment (DMSO) or treatment with either the HCV NS3 protease inhibitor VX-950 (at 15 μM) or compound 2a (at 90 μM). Monodansylcadaverine stains multilamellar bodies and autophagic vacuoles (blue). Nile Red stains both neutral lipids (yellow/gold color) and phospholipids (orange/red color). LipidTOX red stains neutral lipids (red).