Dipartimento di Scienze Farmaceutiche, Università degli Studi di Perugia , Via A. Fabretti 48, 06123 Perugia, Italy.
J Med Chem. 2014 Apr 24;57(8):3247-62. doi: 10.1021/jm401688h. Epub 2014 Apr 2.
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.
我们之前已经确定了吡唑并苯并噻嗪骨架作为一种有前途的丙型肝炎病毒 (HCV) NS5B 聚合酶的化学型，聚合酶是一种经过验证且有前途的抗 HCV 靶点。在此，我们描述了新的吡唑并苯并噻嗪作为抗 HCV 抑制剂的设计、合成、酶和细胞特性。使用突变体反向筛选测定法将代表衍生物的结合位点映射到 NS5B 手掌 I 位置，从而验证了我们之前的计算机预测。衍生物 2b 被证明是新系列中最佳的选择性抗 HCV 衍生物，对 NS5B 聚合酶的 IC50 为 7.9 μM，抗病毒作用（EC50 = 8.1 μM；EC90 = 23.3 μM），同时在基于细胞的 HCV 复制子系统测定中没有任何代谢抑制作用（CC50 > 224 μM；SI > 28）。重要的是，显微镜分析表明，与母体化合物不同，衍生物 2b 不会引起任何明显的细胞形态改变。此外，由于大多数测试的吡唑并苯并噻嗪改变了细胞形态，因此通过探索化合物处理过程中脂质代谢可能发生的变化进一步研究了这一不理想的方面。
