| JDTic is a KOR selective antagonist belonging to the trans (3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine scaffold, which is known as an opioid antagonist pharmacophore for all receptors. A pharmacophore has been designed based on the crystalline structure of JDTic inside the KOR. In this study, by screening in silico over 1.9 million commercially available small molecules, we report a new class of MOR and KOR antagonist ligands which is representative of the successful outcome of virtual high throughput screening (HTS). To create these we applied a combination docking method that integrates ligand and receptor centric virtual screening search techniques in order to screen libraries of commercial compounds. Compound (1) produced a concentration-dependent inhibition of specific [3H]-U69,593 (KOR) binding with a Ki value of 445 nM.;Compound (1) also inhibited specific [3H]-DAMGO (MOR) with Ki values 44 nM, while no binding affinity toward DOR was observed. To characterize the relative efficacy and potency of compound (1), it was assayed in a GTPgamma[35S]S functional assays.;Compound 1 was tested for its ability to stimulate [35S]GTPgammaS binding mediated by the KOR and MOR. Compound (1) showed no activation of the G protein (i.e., was an antagonist) for both KOR and MOR in a GTPgammaS assay compared to full agonists U69,593 (KOR) and DAMGO (MOR). Antagonism was confirmed by running GTPgammaS assays with a fixed amount of agonist against a dilution series of compound (1) and known opioid antagonist naloxone hydrochloride. Both compound (1) and naloxone were able to antagonize 20 nM U69,593 (KOR) or 200 nM DAMGO (MOR) agonist activity, with IC50 values of 511.2+/-175.3 nM and 3.229+/- 0.942 nM, respectively, for KOR, and 774.9+/-107.3 nM and 11.78+/-1.70 nM, respectively, for MOR.;We developed an efficient six-step synthesis of a compound 1 starting from dimethyl itaconate utilizing a sequence of cyclization, reduction and coupling reactions. We also developed an easy derivatization approach utilizing convergent analog synthesis to study SAR around the lead structure.;Further SAR optimization led to the discovery of several low nanomolar binding affinity antagonists with preference for one of the opioid receptors. This new series of compounds can be further evaluated in in-vivo models to develop future drug candidates.;Radioligand binding assays for opioid and cannabinoid receptors have been a useful tool for the discovery of novel agents. Bioassay-guided fractionation of the Russian Sage Perovskia atriplicifolia resulted in the isolation of four known flavonoid derivatives, 5-hydroxy-3',4',6,7-tetramethoxy flavone (1), 5,7-dihydroxy-3',4',6,-trimethoxy flavone (2), 5-hydroxy-4',6,7-trimethoxy flavone (3) and 5,7-dihydroxy-4',6-dimethoxy flavone (4). The structures of these compounds were established on the basis of extensive 1D and 2D NMR spectroscopic analysis and mass spectrometric data. Compound 1, 3, 4 showed displacement of radioligand for the delta opioid receptor with IC50 values ranging from 6.1 to 49.5 muM. These studies have important implications in understanding non-nitrogenous delta opioid receptor ligands. |
