Chen Kevin X, Njoroge F George, Arasappan Ashok, Venkatraman Srikanth, Vibulbhan Bancha, Yang Weiying, Parekh Tejal N, Pichardo John, Prongay Andrew, Cheng Kuo-Chi, Butkiewicz Nancy, Yao Nanhua, Madison Vincent, Girijavallabhan Viyyoor
Schering-Plough Research Institute, 2015 Galloping Hill Road, K-15-3-3545, Kenilworth, NJ 07033, USA.
J Med Chem. 2006 Feb 9;49(3):995-1005. doi: 10.1021/jm050820s.
The hepatitis C virus (HCV) NS3 protease is essential for viral replication. It has been a target of choice for intensive drug discovery research. On the basis of an active pentapeptide inhibitor, 1, we envisioned that macrocyclization from the P2 proline to P3 capping could enhance binding to the backbone Ala156 residue and the S4 pocket. Thus, a number of P2 proline-based macrocyclic alpha-ketoamide inhibitors were prepared and investigated in an HCV NS3 serine protease continuous assay (K(i*)). The biological activity varied substantially depending on factors such as the ring size, number of amino acid residues, number of methyl substituents, type of heteroatom in the linker, P3 residue, and configuration at the proline C-4 center. The pentapeptide inhibitors were very potent, with the C-terminal acids and amides being the most active ones (24, K(i*) = 8 nM). The tetrapeptides and tripeptides were less potent. Sixteen- and seventeen-membered macrocyclic compounds were equally potent, while fifteen-membered analogues were slightly less active. gem-Dimethyl substituents at the linker improved the potency of all inhibitors (the best compound was 45, K(i*) = 6 nM). The combination of tert-leucine at P3 and dimethyl substituents at the linker in compound 47 realized a selectivity of 307 against human neutrophil elastase. Compound 45 had an IC(50) of 130 nM in a cellular replicon assay, while IC(50) for 24 was 400 nM. Several compounds had excellent subcutaneous AUC and bioavailability in rats. Although tripeptide compound 40 was 97% orally bioavailable, larger pentapeptides generally had low oral bioavailability. The X-ray crystal structure of compounds 24 and 45 bound to the protease demonstrated the close interaction of the macrocycle with the Ala156 methyl group and S4 pocket. The strategy of macrocyclization has been proved to be successful in improving potency (>20-fold greater than that of 1) and in structural depeptization.
丙型肝炎病毒（HCV）NS3蛋白酶对于病毒复制至关重要。它一直是密集药物研发研究的首选靶点。基于一种活性五肽抑制剂1，我们设想从P2脯氨酸到P3封端的大环化可以增强与主链Ala156残基和S4口袋的结合。因此，制备了许多基于P2脯氨酸的大环α-酮酰胺抑制剂，并在HCV NS3丝氨酸蛋白酶连续测定（K(i*)）中进行了研究。生物活性因环大小、氨基酸残基数、甲基取代基数、连接子中的杂原子类型、P3残基以及脯氨酸C-4中心的构型等因素而有很大差异。五肽抑制剂非常有效，其中C端酸和酰胺是活性最高的（24，K(i*) = 8 nM）。四肽和三肽的效力较低。十六元和十七元大环化合物效力相当，而十五元类似物的活性略低。连接子处的偕二甲基取代提高了所有抑制剂的效力（最佳化合物为45，K(i*) = 6 nM）。化合物47中P3位的叔亮氨酸与连接子处的二甲基取代相结合，实现了对人中性粒细胞弹性蛋白酶307的选择性。化合物45在细胞复制子测定中的IC(50)为130 nM，而24的IC(50)为400 nM。几种化合物在大鼠体内具有出色的皮下AUC和生物利用度。尽管三肽化合物40的口服生物利用度为97%，但较大的五肽通常口服生物利用度较低。与蛋白酶结合的化合物24和45的X射线晶体结构表明大环与Ala156甲基和S4口袋之间存在紧密相互作用。大环化策略已被证明在提高效力（比1高20倍以上）和结构去肽化方面是成功的。
