Potent inhibition of norovirus 3CL protease by peptidyl α-ketoamides and α-ketoheterocycles

A series of structurally-diverse α-ketoamides and α-ketoheterocycles was synthesized and subsequently investigated for inhibitory activity against norovirus 3CL protease in vitro, as well as anti-norovirus activity in a cell-based replicon system. The synthesized compounds were found to inhibit norovirus 3CL protease in vitro and to also exhibit potent anti-norovirus activity in a cell-based replicon system.     

Synthesis and antiviral activity of novel HCV NS3 protease inhibitors with P4 capping groups

We have synthesized and evaluated a series of novel HCV NS3 protease inhibitors with various P4 capping groups, which include urea, carbamate, methoxy-carboxamide, cyclic carbamate and amide, pyruvic amide, oxamate, oxalamide and cyanoguanidine. Most of these compounds are remarkably potent, exhibiting single-digit to sub-nanomolar activity in the enzyme assay and cell-based replicon assay. Selected compounds were also evaluated in the protease-inhibitor-resistant mutant transient replicon assay, and they were found to show quite different potency profiles against a panel of HCV protease-inhibitor-resistant mutants.     

Synthesis of new acylsulfamoyl benzoxaboroles as potent inhibitors of HCV NS3 protease

HCV NS3/4A serine protease is essential for the replication of the HCV virus and has been a clinically validated target. A series of HCV NS3/4A protease inhibitors containing a novel acylsulfamoyl benzoxaborole moiety at the P1' region was synthesized and evaluated. The resulting P1-P3 and P2-P4 macrocyclic inhibitors exhibited sub-nanomolar potency in the enzymatic assay and low nanomolar activity in the cell-based replicon assay. The in vivo PK evaluations of selected compounds are also described.     

Potent inhibition of norovirus by dipeptidyl α-hydroxyphosphonate transition state mimics

The design, synthesis, and evaluation of a series of dipeptidyl α-hydroxyphosphonates is reported. The synthesized compounds displayed high anti-norovirus activity in a cell-based replicon system, as well as high enzyme selectivity.     

Design and synthesis of inhibitors of noroviruses by scaffold hopping

A scaffold hopping strategy was employed to identify new chemotypes that inhibit noroviruses. The replacement of the cyclosulfamide scaffold by an array of heterocyclic scaffolds lead to the identification of additional series of compounds that possessed anti-norovirus activity in a cell-based replicon system.     

Synthesis and antiviral activity of HCV NS3/4A peptidomimetic boronic acid inhibitors

A new series of NS3/4A protease boronic acid inhibitors is described. The compounds show good biochemical potency and cellular activity. The peptidomimetic inhibitors were evaluated against proteases from different HCV genotypes and clinically relevant NS3/4A mutants. Compound 28 displayed subnanomolar to single digit nanomolar potencies in the enzymatic assays and an EC₅₀ of 25 nM in the replicon cell-based assay.     

Inhibition of norovirus 3CL protease by bisulfite adducts of transition state inhibitors

Noroviruses are the most common cause of acute viral gastroenteritis, accounting for >21 million cases annually in the US alone. Norovirus infections constitute an important health problem for which there are no specific antiviral therapeutics or vaccines. In this study, a series of bisulfite adducts derived from representative transition state inhibitors (dipeptidyl aldehydes and α-ketoamides) was synthesized and shown to exhibit anti-norovirus activity in a cell-based replicon system. The ED₅₀ of the most effective inhibitor was 60 nM. This study demonstrates for the first time the utilization of bisulfite adducts of transition state inhibitors in the inhibition of norovirus 3C-like protease in vitro and in a cell-based replicon system. The approach described herein can be extended to the synthesis of the bisulfite adducts of other classes of transition state inhibitors of serine and cysteine proteases, such as α-ketoheterocycles and α-ketoesters.     

Potent and selective small molecule NS3 serine protease inhibitors of Hepatitis C virus with dichlorocyclopropylproline as P2 residue

Starting from a pentapeptide Hepatitis C virus NS3 protease inhibitor, a number of alpha-ketoamide inhibitors based on novel dichlorocyclopropylproline P2 core were synthesized and investigated for their HCV NS3 serine protease activity. The key intermediate 3,4-dichlorocyclopropylproline was obtained through a dichloro carbene insertion to 3,4-dehydroproline. The size of the molecules was reduced significantly through a series of truncations of the initial pentapeptide. By varying P1 side chain in length and size, potency and selectivity were improved. A variety of aliphatic carbamate and urea capping groups were examined. In general, compounds with urea cappings were more potent and selective than their carbamate counterparts. The most potent compound was a tert-butyl urea analog. Variations at P3 position were also investigated. Among the three residues incorporated, tert-leucine was clearly superior, leading to compounds that had excellent enzyme potency and selectivity. The most potent compound achieved cell-based replicon assay EC50 of 40 nM. The most promising compound of all had excellent potency in both enzyme (Ki* = 9 nM) and replicon assays (EC50 = 100 nM). Its bioavailabilities were above 10% in all three animal species (rats, monkeys, and dogs). It has provided a lead for future investigations.     

Novel macrocyclic HCV NS3 protease inhibitors derived from α-amino cyclic boronates

A novel series of P2–P4 macrocyclic HCV NS3/4A protease inhibitors with α-amino cyclic boronates as warheads at the P1 site was designed and synthesized. When compared to their linear analogs, these macrocyclic inhibitors exhibited a remarkable improvement in cell-based replicon activities, with compounds 9a and 9e reaching sub-micromolar potency in replicon assay. The SAR around α-amino cyclic boronates clearly established the influence of ring size, chirality and of the substitution pattern. Furthermore, X-ray structure of the co-crystal of inhibitor 9a and NS3 protease revealed that Ser-139 in the enzyme active site traps boron in the warhead region of 9a, thus establishing its mode of action.     

Potent norovirus inhibitors based on the acyclic sulfamide scaffold

The development of small molecule therapeutics to combat norovirus infection is of considerable interest from a public health perspective because of the highly contagious nature of noroviruses. A series of amino acid-derived acyclic sulfamide-based norovirus inhibitors has been synthesized and evaluated using a cell-based replicon system. Several compounds were found to display potent anti-norovirus activity, low toxicity, and good aqueous solubility. These compounds are suitable for further optimization of pharmacological and ADMET properties.     

3-heterocyclyl quinolone inhibitors of the HCV NS5B polymerase

The discovery and optimization of a novel class of quinolone small-molecules that inhibit NS5B polymerase, a key enzyme of the HCV viral life-cycle, is described. Our research led to the replacement of a hydrolytically labile ester functionality with bio-isosteric heterocycles. An X-ray crystal structure of a key analog bound to NS5B facilitated the optimization of this series of compounds to afford increased activity against the target enzyme and in the cell-based replicon assay system.     

Novel, potent, and orally bioavailable phosphinic acid inhibitors of the hepatitis C virus NS3 protease

A potent and novel class of product-like inhibitors of the HCV NS3 protease was discovered by employing a phosphinic acid as a carboxylate isostere. The replicon activity and pharmacokinetic profile of this series of compounds was optimized by exploring the substitution of the phosphinic acid, as well as conformationally constraining these compounds through macrocyclization. The syntheses and preliminary biological evaluation of these phosphinic acids is described.     

Synthesis and biological evaluations of P4-benzoxaborole-substituted macrocyclic inhibitors of HCV NS3 protease

We disclose here a series of P4-benzoxaborole-substituted macrocyclic HCV protease inhibitors. These inhibitors are potent against HCV NS3 protease, their anti-HCV replicon potencies are largely impacted by substitutions on benzoxaborole ring system and P21 groups. P21 2-thiazole-isoquinoline provides best replicon potency. The in vitro SAR studies and in vivo PK evaluations of selected compounds are described herein.     

Inhibition of noroviruses by piperazine derivatives

There is currently an unmet need for the development of small-molecule therapeutics for norovirus infection. The piperazine scaffold, a privileged structure embodied in many pharmacological agents, was used to synthesize an array of structurally-diverse derivatives which were screened for anti-norovius activity in a cell-based replicon system. The studies described herein demonstrate for the first time that functionalized piperazine derivatives possess anti-norovirus activity. Furthermore, these studies have led to the identification of two promising compounds (6a and 9l) that can be used as a launching pad for the optimization of potency, cytotoxicity, and drug-like characteristics.     

Synthesis and anti-HCV activity evaluation of anilinoquinoline derivatives

Hepatitis C virus (HCV) infection is a main cause of chronic liver disease, leading to liver cirrhosis and hepatocellular carcinoma (HCC). The objective of our research was to develop effective agents against viral replication. Here, we have synthesized a series of anilinoquinoline derivatives. Based on a cell-based HCV replicon system, we observed that 2-(3'-nitroanilino)quinoline (18) exhibited anti-HCV activity with a 50% effective concentration (EC(50)) value of 7μM and a selective index (SI) value of 10. In addition, compound 18 possessed the inhibitory effect on HCV NS3/4A protease activity. Therefore, we concluded that the compound 18 possessed a potent activity against HCV replication and could provide as a new lead compound as anti-HCV inhibitor.     

Synthesis and antiviral evaluation of novel peptidomimetics as norovirus protease inhibitors

A series of tripeptidyl transition state inhibitors with new P1 and warhead moieties were synthesized and evaluated in a GI-1 norovirus replicon system and against GII-4 and GI-1 norovirus proteases. Compound 19, containing a 6-membered ring at the P1 position and a reactive aldehyde warhead exhibited sub-micromolar replicon inhibition. Retaining the same peptidyl scaffold, several reactive warheads were tested for protease inhibition and norovirus replicon inhibition. Of the six that were synthesized and tested, compounds 42, 43, and 45 potently inhibited the protease in biochemical assay and GI-1 norovirus replicon in the nanomolar range.     

Synthesis and SAR of acyclic HCV NS3 protease inhibitors with novel P4-benzoxaborole moieties

We have synthesized and evaluated a new series of acyclic P4-benzoxaborole-based HCV NS3 protease inhibitors. Structure-activity relationships were investigated, leading to the identification of compounds 5g and 17 with low nanomolar potency in the enzymatic and cell-based replicon assay. The linker-truncated compound 5j was found to exhibit improved absorption and oral bioavailability in rats, suggesting that further reduction of molecular weight and polar surface area could result in improved drug-like properties of this novel series.     

Design and synthesis of spiro-cyclopentenyl and spiro-[1,3]-dithiolanyl substituted pyrrolidine-5,5-trans-lactams as inhibitors of hepatitis C virus NS3/4A protease

Using the pyrrolidine-5,5-trans-lactam template, we have designed small, neutral, mechanism-based inhibitors of hepatitis C NS3/4A protease. Compound 2b, with a spiro-cyclobutyl P1 substituent and an isopropyl carbonyl substituent at the lactam nitrogen, has an IC(50) value in the replicon cell-based assay of 3 microM.     

Discovery of pentacyclic compounds as potent inhibitors of hepatitis C virus NS5B RNA polymerase

We report a new series of inhibitors for hepatitis C virus NS5B RNA polymerase containing a constrained pentacyclic scaffold. Our SAR studies led to the identification of hexahydroindolo[2,1-a]pyrrolo[3,2-d][2]benzazepines exposing basic groups. The compounds displayed a high activity in the enzyme assay and displayed good activity in the cell-based (replicon) assay in the presence of serum proteins.