Discovery of benzophosphadiazine drug candidate IDX375: A novel hepatitis C allosteric NS5B RdRp inhibitor.

Hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase (RdRp) plays a central role in virus replication. NS5B has no functional equivalent in mammalian cells, and as a consequence is an attractive target for selective inhibition. This paper describes the discovery of a novel family of HCV NS5B non-nucleoside inhibitors inspired by the bioisosterism between sulfonamide and phosphonamide. Systematic structural optimization in this new series led to the identification of IDX375, a potent non-nucleoside inhibitor that is selective for genotypes 1a and 1b. The structure and binding domain of IDX375 were confirmed by X-ray co-crystalisation study.

Synthesis of potent and broad genotypically active NS5B HCV non-nucleoside inhibitors binding to the thumb domain allosteric site 2 of the viral polymerase.

The hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase (RdRp) plays a central role in virus replication. NS5B has no functional equivalent in mammalian cells and, as a consequence, is an attractive target for selective inhibition. This Letter describes the discovery of a new family of HCV NS5B non-nucleoside inhibitors, based on the bioisosterism between amide and phosphonamidate functions. As part of this program, SAR in this new series led to the identification of IDX17119, a potent non-nucleoside inhibitor, active on the genotypes 1b, 2a, 3a and 4a. The structure and binding domain of IDX17119 were confirmed by X-ray co-crystallization study.

Synthesis and antiviral evaluation of a novel series of homoserine-based inhibitors of the hepatitis C virus NS3/4A serine protease.

We disclose here the synthesis of a series of macrocyclic HCV protease inhibitors, where the homoserine linked together the quinoline P2' motif and the macrocyclic moiety. These compounds exhibit potent inhibitory activity against HCV NS3/4A protease and replicon cell based assay. Their enzymatic and antiviral activities are modulated by substitutions on the quinoline P2' at position 8 by methyl and halogens and by small heterocycles at position 2. The in vitro structure activity relationship (SAR) studies and in vivo pharmacokinetic (PK) evaluations of selected compounds are described herein.

Discovery and structural diversity of the hepatitis C virus NS3/4A serine protease inhibitor series leading to clinical candidate IDX320.

Exploration of the P2 region by mimicking the proline motif found in BILN2061 resulted in the discovery of two series of potent HCV NS3/4A protease inhibitors. X-ray crystal structure of the ligand in contact with the NS3/4A protein and modulation of the quinoline heterocyclic region by structure based design and modeling allowed for the optimization of enzyme potency and cellular activity. This research led to the selection of clinical candidate IDX320 having good genotype coverage and pharmacokinetic properties in various species.

Serendipitous discovery of 2-((phenylsulfonyl)methyl)-thieno[3,2-d]pyrimidine derivatives as novel HIV-1 replication inhibitors.

We identified a novel class of 2-((phenylsulfonyl)methyl)-thieno[3,2-d]pyrimidine compounds as potent HIV-1 replication inhibitors serendipitously during the process of evaluation of triazolothienopyrimidine (TTPM) compounds. Herein, we report synthesis and biological evaluation of 2-((phenylsulfonyl)methyl)-thieno[3,2-d]pyrimidine compounds using a cell-based full replication assay to identify thienopyrimidines 6 and 30, which could be further utilized as viable lead compounds.

Structure-based design of a novel series of azetidine inhibitors of the hepatitis C virus NS3/4A serine protease.

Structural homology between thrombin inhibitors and the early tetrapeptide HCV protease inhibitor led to the bioisosteric replacement of the P2 proline by a 2,4-disubstituted azetidine within the macrocyclic ß-strand mimic. Molecular modeling guided the design of the series. This approach was validated by the excellent activity and selectivity in biochemical and cell based assays of this novel series and confirmed by the co-crystal structure of the inhibitor with the NS3/4A protein (PDB code: 4TYD).

Synthesis and biological evaluation of triazolothienopyrimidine derivatives as novel HIV-1 replication inhibitors.

We identified a novel class of triazolothienopyrimidine (TTPM) compounds as potent HIV-1 replication inhibitors during a high-throughput screening campaign that evaluated more than 200,000 compounds using a cell-based full replication assay. Herein, we report the optimization of the antiviral activity in a cell-based assay system leading to the discovery of aryl-substituted TTPM derivatives (38, 44, and 45), which exhibited significant inhibition of HIV-1 replication with acceptable safety margins. These novel and potent TTPMs could serve as leads for further development.

Discovery and characterization of a novel 7-aminopyrazolo[1,5-a]pyrimidine analog as a potent hepatitis C virus inhibitor.

We describe a novel 7-aminopyrazolo[1,5-a]pyrimidine (7-APP) derivative as a potent hepatitis C virus (HCV) inhibitor. A series of 7-APPs was synthesized and evaluated for inhibitory activity against HCV in different cell culture systems. The synthesis and preliminary structure-activity relationship study of 7-APP are reported.

A novel 3,4-dihydropyrimidin-2(1H)-one: HIV-1 replication inhibitors with improved metabolic stability.

Following the previous SAR of a novel dihydropyrimidinone scaffold as HIV-1 replication inhibitors a detailed study directed towards optimizing the metabolic stability of the ester functional group in the dihydropyrimidinone (DHPM) scaffold is described. Replacement of the ester moiety by thiazole ring significantly improved the metabolic stability while retaining antiviral activity against HIV-1 replication. These novel and potent DHPMs with bioisosteres could serve as advanced leads for further optimization.

Herpes simplex virus helicase-primase inhibitors are active in animal models of human disease.

Herpes simplex virus infections are the cause of significant morbidity, and currently used therapeutics are largely based on modified nucleoside analogs that inhibit viral DNA polymerase function. To target this disease in a new way, we have identified and optimized selective thiazolylphenyl-containing inhibitors of the herpes simplex virus (HSV) helicase-primase enzyme. The most potent compounds inhibited the helicase, the primase and the DNA-dependent ATPase activities of the enzyme with IC50 (50% inhibitory concentration) values less than 100 nM. Inhibition of the enzymatic activities was through stabilization of the interaction between the helicase-primase and DNA substrates, preventing the progression through helicase or primase catalytic cycles. Helicase-primase inhibitors also prevented viral replication as demonstrated in viral growth assays. One compound, BILS 179 BS, displayed an EC50 (effective concentration inhibiting viral growth by 50%) of 27 nM against viral growth with a selectivity index greater than 2,000. Antiviral activity was also demonstrated for multiple strains of HSV, including strains resistant to nucleoside-based therapies. Most importantly, BILS 179 BS was orally active against HSV infections in murine models of HSV-1 and HSV-2 disease and more effective than acyclovir when the treatment frequency per day was reduced or when initiation of treatment was delayed up to 65 hours after infection. These studies validate the use of helicase-primase inhibitors for the treatment of acute herpesvirus infections and provide new lead compounds for optimization and design of superior anti-HSV agents.

The discovery of thienopyridine analogues as potent IkappaB kinase beta inhibitors. Part II.

An SAR study that identified a series of thienopyridine-based potent IkappaB Kinase beta (IKKbeta) inhibitors is described. With focuses on the structural optimization at C4 and C6 of structure 1 (Fig. 1), the study reveals that small alkyl and certain aromatic groups are preferred at C4, whereas polar groups with proper orientation at C6 efficiently enhance compound potency. The most potent analogues inhibit IKKbeta with IC50s as low as 40 nM, suppress LPS-induced TNF-alpha production in vitro and in vivo, display good kinase selectivity profiles, and are active in a HeLa cell NF-kappaB reporter gene assay, demonstrating that they directly interfere with the NF-kappaB signaling pathway.

Synthesis and antiviral evaluation of thieno[3,4-d]pyrimidine C-nucleoside analogues of 2',3'-dideoxy- and 2',3'-dideoxy-2',3'-didehydro-adenosine and -inosine.

Several thieno[3,4-d]pyrimidine derivatives, including four hitherto unknown 2',3'-dideoxy- and 2',3'-dideoxy-2',3'-didehydro-C-nucleoside analogues of adenosine and inosine have been synthesized. When evaluated in cell culture experiments against human immunodeficiency virus, none of the tested compounds exhibited any significant antiviral effect, while two of them showed some cytotoxicity.

2'-C-Methyl branched pyrimidine ribonucleoside analogues: potent inhibitors of RNA virus replication.

RNA viruses are the agents of numerous widespread and often severe diseases. Their unique RNA-dependent RNA polymerase (RDRP) is essential for replication and, thus, constitutes a valid target for the development of selective chemotherapeutic agents. In this regard, we have investigated sugar-modified ribonucleoside analogues as potential inhibitors of the RDRP. Title compounds retain 'natural' pyrimidine bases, but possess a beta-methyl substituent at the 2'-position of the D- or L-ribose moiety. Evaluation against a broad range of RNA viruses, either single-stranded positive (ssRNA+), single-stranded negative (ssRNA-) or double-stranded (dsRNA), revealed potent activities for D-2'-C-methyl-cytidine and -uridine against ssRNA+, and dsRNA viruses. None of the L-enantiomers were active. Moreover, the 5'-triphosphates of the active D-enantiomers were found to inhibit the bovine virus diarrhoea virus polymerase. Thus, the 2'-methyl branching of natural pyrimidine ribonucleosides transforms physiological molecules into potent, broad-spectrum antiviral agents that merit further development.

Evolution of the thienopyridine class of inhibitors of IkappaB kinase-beta: part I: hit-to-lead strategies.

High-throughput screening is routinely employed as a method for the identification of novel hit structures. Large numbers of active compounds are typically procured in this way and must undergo a rigorous validation process. This process is described in detail for a collection of screening hits identified as inhibitors of IkappaB kinase-beta (IKKbeta), a key regulatory enzyme in the nuclear factor-kappaB (NF-kappaB) pathway. From these studies, a promising hit series was selected. Subsequent lead generation activities included the development of a pharmacophore hypothesis and structure-activity relationship (SAR) for the hit series. This led to the exploration of related scaffolds offering additional opportunities, and the various structural classes were comparatively evaluated for enzyme inhibition, selectivity, and drug-like properties. A novel lead series of thienopyridines was thereby established, and this series advanced into lead optimization for further development.

Polyadenylation-dependent screening assay for respiratory syncytial virus RNA transcriptase activity and identification of an inhibitor.

RNA-dependent RNA polymerase from respiratory syncytial virus (RSV) is a multi-subunit ribonucleoprotein (RNP) complex that, in addition to synthesizing the full 15 222 nt viral genomic RNA, is able to synthesize all 10 viral mRNAs. We have prepared crude RNP from RSV-infected HEp-2 cells, based on a method previously used for Newcastle disease virus, and established a novel polyadenylation-dependent capture [poly(A) capture] assay to screen for potential inhibitors of RSV transcriptase activity. In this homogeneous assay, radiolabeled full-length polyadenylated mRNAs produced by the viral RNP are detected through capture on immobilized biotinylated oligo(dT) in a 96-well streptavidin-coated FlashPlate. Possible inhibitors identified with this assay could interfere at any step required for the production of complete RSV mRNAs, including transcription, polyadenylation and, potentially, co-transcriptional guanylylation. A specific inhibitor of RSV transcriptase with antiviral activity was identified through screening of this assay.

Discovery of the Aryl-phospho-indole IDX899, a Highly Potent Anti-HIV Non-nucleoside Reverse Transcriptase Inhibitor.

Here, we describe the design, synthesis, biological evaluation, and identification of a clinical candidate non-nucleoside reverse transcriptase inhibitors (NNRTIs) with a novel aryl-phospho-indole (APhI) scaffold. NNRTIs are recommended components of highly active antiretroviral therapy (HAART) for the treatment of HIV-1. Since a major problem associated with NNRTI treatment is the emergence of drug resistant virus, this work focused on optimization of the APhI against clinically relevant HIV-1 Y181C and K103N mutants and the Y181C/K103N double mutant. Optimization of the phosphinate aryl substituent led to the discovery of the 3-Me,5-acrylonitrile-phenyl analogue RP-13s (IDX899) having an EC50 of 11 nM against the Y181C/K103N double mutant.

Design, synthesis and antiviral evaluation of 2'-C-methyl branched guanosine pronucleotides: the discovery of IDX184, a potent liver-targeted HCV polymerase inhibitor.

BACKGROUND: Ribonucleoside analogs possessing a ß-methyl substituent at the 2'-position of the d-ribose moiety have been previously discovered to be potent and selective inhibitors of hepatitis C virus (HCV) replication, their triphosphates acting as alternative substrate inhibitors of the HCV RdRp NS5B. Results/methodology: In this article, the authors detail the synthesis, anti-HCV evaluation in cell-based replicon assays and structure-activity relationships of several phosphoramidate diester derivatives of 2'-C-methylguanosine (2'-MeG). CONCLUSION: The most promising compound, namely the O-[S-(hydroxyl)pivaloyl-2-thioethyl]{abbreviated as O-[(HO)tBuSATE)]} N-benzylamine phosphoramidate diester derivative (IDX184), was selected for further in vivo studies, and was the first clinical pronucleotide evaluated for the treatment of chronic hepatitis C up to Phase II trials.

Isolation and characterization of herpes simplex virus type 1 resistant to aminothiazolylphenyl-based inhibitors of the viral helicase-primase.

The aminothiazolylphenyl-containing compounds BILS 179 BS and BILS 45 BS are novel inhibitors of the herpes simplex virus helicase-primase with antiviral activity in vitro and in animal models of HSV disease. To verify the mechanism of antiviral action, resistant viruses were selected by serial passage or by single-step plaque selection of HSV-1 KOS in the presence of inhibitors. Three resistant isolates K138r3, K22r5, and K22r1 were found to be 38-, 316-, and 2500-fold resistant to BILS 22 BS, a potent analog of BILS 45 BS. All three viruses had growth properties in vitro similar to wild-type HSV-1 KOS but they were sensitive to acyclovir. Cutaneous and intra-cerebral inoculation of mice with K22r1 or K22r5 resulted in pathogenicity equivalent to that of HSV-1 KOS. Both isolates were fully competent for reactivation from latency following corneal inoculation. Helicase-primase purified from cells infected with resistant viruses showed decreased inhibition in an in vitro DNA-dependent ATPase assay that correlated well with antiviral resistance. Marker transfer experiments and DNA sequence analysis identified single base pair mutations clustered in the N-terminus of the UL5 gene that resulted in single amino acid changes in the UL5 protein. Taken together, the results indicate that helicase-primase inhibitors prevent HSV growth by inhibiting HSV helicase-primase through specific interaction with the UL5 protein.

In vitro antiplasmodial activities and synergistic combinations of differential solvent extracts of the polyherbal product, Nefang.

Nefang, a polyherbal product composed of Mangifera indica (bark and leaf), Psidium guajava, Carica papaya, Cymbopogon citratus, Citrus sinensis, and Ocimum gratissimum (leaves), is a potential therapy against P. falciparum malaria. In vitro antiplasmodial activities of its constituent solvent extracts were analyzed on CQ-sensitive (3D7) and multidrug resistant (Dd2) P. falciparum strains. The interactions involving the differential solvent extracts were further analyzed using a variable potency ratio drug combination approach. Effective concentration 50 (EC50) values were determined by nonlinear regression curve-fitting of the dose-response data and used in calculating the fractional inhibitory concentration 50 (FIC50) and combination indices (CI) for each pair. The derived EC50 values (3D7/Dd2, µ g/mL) are Nefang-96.96/55.08, MiB-65.33/34.58, MiL-82.56/40.04, Pg-47.02/25.79, Cp-1188/317.5, Cc-723.3/141, Cs-184.4/105.1, and Og-778.5/118.9. Synergism was obtained with MiB/Pg (CI = 0.351), MiL/Pg (0.358), MiB/Cs (0.366), MiL/Cs (0.482), Pg/Cs (0.483), and Cs/Og (0.414) when analyzed at equipotency ratios. Cytotoxicity testing of Nefang and the solvent extracts on two human cell lines (Hep G2 and U2OS) revealed no significant toxicity relative to their antiplasmodial activities (SI > 20). Taken together, our data confirm the antimalarial activities of Nefang and its constituent plant extracts and identified extract pairs with promising synergistic interactions for exploitation towards a rational phytotherapeutic and evidence-based antimalarial drug discovery.

Discovery of carbohybrid-based 2-aminopyrimidine analogues as a new class of rapid-acting antimalarial agents using image-based cytological profiling assay.

New antimalarial agents that exhibit multistage activities against drug-resistant strains of malaria parasites represent good starting points for developing next-generation antimalarial therapies. To facilitate the progression of such agents into the development phase, we developed an image-based parasitological screening method for defining drug effects on different asexual life cycle stages of Plasmodium falciparum. High-throughput screening of a newly assembled diversity-oriented synthetic library using this approach led to the identification of carbohybrid-based 2-aminopyrimidine compounds with fast-acting growth inhibitory activities against three laboratory strains of multidrug-resistant P. falciparum. Our structure-activity relationship study led to the identification of two derivatives (8aA and 11aA) as the most promising antimalarial candidates (mean EC50 of 0.130 and 0.096 µM against all three P. falciparum strains, selectivity indices >600, microsomal stabilities >80%, and mouse malaria ED50 values of 0.32 and 0.12 mg/kg/day, respectively), targeting all major blood stages of multidrug-resistant P. falciparum parasites.