Ligand-Based Discovery of a New Scaffold for Allosteric Modulation of the μ‑Opioid Receptor

With the hope of discovering effective analgesics with fewer side effects, attention has recently shifted to allosteric modulators of the opioid receptors. In the past two years, the first chemotypes of positive or silent allosteric modulators (PAMs or SAMs, respectively) of μ- and δ-opioid receptor types have been reported in the literature. During a structure-guided lead optimization campaign with μ-PAMs BMS-986121 and BMS-986122 as starting compounds, we discovered a new chemotype that was confirmed to display μ-PAM or μ-SAM activity depending on the specific substitutions as assessed by endomorphin-1-stimulated β-arrestin2 recruitment assays in Chinese Hamster Ovary (CHO)-μ PathHunter cells. The most active μ-PAM of this series was analyzed further in competition binding and G-protein activation assays to understand its effects on ligand binding and to investigate the nature of its probe dependence

Solid Phase Synthesis of 1,5-Diarylpyrazole-4-carboxamides: Discovery of Antagonists of the CB-1 Receptor

We have developed a solid phase synthesis route to 1,5-substituted pyrazole-4-carboxamides with three diversity points aimed at the discovery of new compounds as potential G-Protein coupled receptor (GPCR) ligands. The new chemistry involves acylation of a resin bound secondary amine with a β-ketoester via transamidation, conversion of the resulting β-ketoamide to the corresponding vinylogous amide, pyrazole formation upon reaction with a aryl hydrzine, and cleavage of the product from the resin. Using the reported methodology, we describe the syntheses of multiple arrays of pyrazoles that were used collectively to construct a library of more than 1000 analogues. Several members of this library displayed submicromolar antagonist activities at the cannabinoid subtype 1 (CB-1) receptor

Solid Phase Synthesis of 1,5-Diarylpyrazole-4-carboxamides: Discovery of Antagonists of the CB-1 Receptor

We have developed a solid phase synthesis route to 1,5-substituted pyrazole-4-carboxamides with three diversity points aimed at the discovery of new compounds as potential G-Protein coupled receptor (GPCR) ligands. The new chemistry involves acylation of a resin bound secondary amine with a β-ketoester via transamidation, conversion of the resulting β-ketoamide to the corresponding vinylogous amide, pyrazole formation upon reaction with a aryl hydrzine, and cleavage of the product from the resin. Using the reported methodology, we describe the syntheses of multiple arrays of pyrazoles that were used collectively to construct a library of more than 1000 analogues. Several members of this library displayed submicromolar antagonist activities at the cannabinoid subtype 1 (CB-1) receptor

Solid Phase Synthesis of 1,5-Diarylpyrazole-4-carboxamides: Discovery of Antagonists of the CB-1 Receptor

We have developed a solid phase synthesis route to 1,5-substituted pyrazole-4-carboxamides with three diversity points aimed at the discovery of new compounds as potential G-Protein coupled receptor (GPCR) ligands. The new chemistry involves acylation of a resin bound secondary amine with a β-ketoester via transamidation, conversion of the resulting β-ketoamide to the corresponding vinylogous amide, pyrazole formation upon reaction with a aryl hydrzine, and cleavage of the product from the resin. Using the reported methodology, we describe the syntheses of multiple arrays of pyrazoles that were used collectively to construct a library of more than 1000 analogues. Several members of this library displayed submicromolar antagonist activities at the cannabinoid subtype 1 (CB-1) receptor

Solid Phase Synthesis of 1,5-Diarylpyrazole-4-carboxamides: Discovery of Antagonists of the CB-1 Receptor

We have developed a solid phase synthesis route to 1,5-substituted pyrazole-4-carboxamides with three diversity points aimed at the discovery of new compounds as potential G-Protein coupled receptor (GPCR) ligands. The new chemistry involves acylation of a resin bound secondary amine with a β-ketoester via transamidation, conversion of the resulting β-ketoamide to the corresponding vinylogous amide, pyrazole formation upon reaction with a aryl hydrzine, and cleavage of the product from the resin. Using the reported methodology, we describe the syntheses of multiple arrays of pyrazoles that were used collectively to construct a library of more than 1000 analogues. Several members of this library displayed submicromolar antagonist activities at the cannabinoid subtype 1 (CB-1) receptor

Activity of EI-1 in infectious virus assays.

a<p>Mean of ≥4 independent experiments.</p

Effect of EI-1 on HCV cell-to-cell spread.

<p>(A) Huh-7.5 cells were infected with 0.001 ffu/cell HCVcc-1a/2a at 37°C. At 12 hrs post infection, the inoculum was removed and replaced with medium +1% agarose overlay containing EI-1 (0.5 µM) or DMSO and the cultures were incubated at 37°C for 2, 3 or 4 days. Infected cells were labeled by indirect immunofluorescence using an anti-HCV core monoclonal antibody (green) and nuclei were stained with Hoechst 3325 (red). Images were captured using a Nikon Eclipse TE300 inverted epi-fluorescence microscope. (B) The mean number and standard deviation of infected cells/focus was determined from visual counting of infected cells in ≥100 foci for each time point. (C) The mean number and standard deviation of foci/well was determined at 2 and 4 days post infection.</p

Structure-activity-relationship of the EI-1 chemotype.

a<p>Mean of ≥3 independent experiments.</p>b<p>R, aniline ring substituent (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001086#ppat-1001086-g001" target="_blank">Fig 1A</a>).</p

Combination indices of EI-1 with HCV replication inhibitors.

a<p>NS5A, BMS-790052; NS3, BMS-605339.</p>b<p>Mean of 8 independent experiments.</p>c<p>Loewe combination index at the combined EC₅₀ level.</p

Schematic diagram of the HCV E2 protein and sequences of the region encompassing the EI-1 resistance residue.

<p>Previously defined regions of the protein are indicated by the shaded boxes. Numbers correspond to the HCV polyprotein amino acid positions in E2. HVR1, hypervariable region 1. HVR2, hypervariable region 2. pFP₁ and pFP₂, putative fusion peptide regions. igVR, intergenotypic variability region. HR, heptad repeat. TMD, transmembrane domain. The asterisk indicates the position of residue 719 that is involved in EI-1 resistance. The protein sequence alignment from amino acids 709–729 for each of the HCVpp genotype isolates from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001086#ppat-1001086-g002" target="_blank">Figure 2</a> is shown. The shaded residues are part of the TMD.</p