Azepino-indazoles as calcitonin gene-related peptide (CGRP) receptor antagonists.

Calcitonin gene-related peptide (CGRP) receptor antagonists have been shown clinically to be effective treatments for migraine. Zavegepant (BHV-3500, BMS-742413) is a high affinity antagonist of the CGRP receptor (hCGRP Ki = 0.023 nM) that has demonstrated efficacy in the acute treatment of migraine with intranasal delivery in a Phase 2/3 trial, despite showing low oral bioavailability in rats (FPO = 1.7%). Using zavegepant as a template, we sought to improve oral bioavailability through a series of azepinones which were designed in an attempt to reduce the number of rotatable bonds. These efforts led to the discovery of compound 21 which was able to mostly maintain high affinity binding (hCGRP Ki = 0.100 nM) and in vivo efficacy in the marmoset facial blood flow assay, while greatly improving oral bioavailability (rat FPO = 17%).

Synthesis and SAR of calcitonin gene-related peptide (CGRP) antagonists containing substituted aryl-piperazines and piperidines.

Calcitonin gene-related peptide (CGRP) is a potent neuropeptide implicated in the pathophysiology of migraine. In the course of seeking CGRP antagonists with improved oral bioavailability, metabolic stability, and pharmacokinetic properties, lower molecular weight, structurally simpler piperidine and piperazine analogs of BMS-694153 were prepared. Several were found to have nM binding affinity in vitro. The synthesis and SAR of these substituted piperidine and piperazine CGRP antagonists are discussed.

Evidence for Classical Cholinergic Toxicity Associated with Selective Activation of M1 Muscarinic Receptors.

The muscarinic acetylcholine receptor subtype 1 (M1) receptors play an important role in cognition and memory, and are considered to be attractive targets for the development of novel medications to treat cognitive impairments seen in schizophrenia and Alzheimer's disease. Indeed, the M1 agonist xanomeline has been shown to produce beneficial cognitive effects in both Alzheimer's disease and schizophrenia patients. Unfortunately, the therapeutic utility of xanomeline was limited by cholinergic side effects (sweating, salivation, gastrointestinal distress), which are believed to result from nonselective activation of other muscarinic receptor subtypes such as M2 and M3. Therefore, drug discovery efforts targeting the M1 receptor have focused on the discovery of compounds with improved selectivity profiles. Recently, allosteric M1 receptor ligands have been described, which exhibit excellent selectivity for M1 over other muscarinic receptor subtypes. In the current study, the following three compounds with mixed agonist/positive allosteric modulator activities that are highly functionally selective for the M1 receptor were tested in rats, dogs, and cynomologous monkeys: (3-((1S,2S)-2-hydrocyclohexyl)-6-((6-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)methyl)benzo[h]quinazolin-4(3H)-one; 1-((4-cyano-4-(pyridin-2-yl)piperidin-1-yl)methyl)-4-oxo-4H-quinolizine-3-carboxylic acid; and (R)-ethyl 3-(2-methylbenzamido)-[1,4'-bipiperidine]-1'-carboxylate). Despite their selectivity for the M1 receptor, all three compounds elicited cholinergic side effects such as salivation, diarrhea, and emesis. These effects could not be explained by activity at other muscarinic receptor subtypes, or by activity at other receptors tested. Together, these results suggest that activation of M1 receptors alone is sufficient to produce unwanted cholinergic side effects such as those seen with xanomeline. This has important implications for the development of M1 receptor-targeted therapeutics since it suggests that dose-limiting cholinergic side effects still reside in M1 receptor selective activators.

Discovery, synthesis, and molecular pharmacology of selective positive allosteric modulators of the δ-opioid receptor.

Allosteric modulators of G protein-coupled receptors (GPCRs) have a number of potential advantages compared to agonists or antagonists that bind to the orthosteric site of the receptor. These include the potential for receptor selectivity, maintenance of the temporal and spatial fidelity of signaling in vivo, the ceiling effect of the allosteric cooperativity which may prevent overdose issues, and engendering bias by differentially modulating distinct signaling pathways. Here we describe the discovery, synthesis, and molecular pharmacology of δ-opioid receptor-selective positive allosteric modulators (δ PAMs). These δ PAMs increase the affinity and/or efficacy of the orthosteric agonists leu-enkephalin, SNC80 and TAN67, as measured by receptor binding, G protein activation, ß-arrestin recruitment, adenylyl cyclase inhibition, and extracellular signal-regulated kinases (ERK) activation. As such, these compounds are useful pharmacological tools to probe the molecular pharmacology of the δ receptor and to explore the therapeutic potential of δ PAMs in diseases such as chronic pain and depression.

Labware additives identified to be selective monoamine oxidase-B inhibitors.

Plastic labware is used in all processes of modern pharmaceutical research, including compound storage and biological assays. The use of these plastics has created vast increases in productivity and cost savings as experiments moved from glass test tubes and capillary pipettes to plastic microplates and multichannel liquid handlers. One consequence of the use of plastic labware, however, is the potential release of contaminants and their resultant effects on biological assays. We report herein the identification of biologically active substances released from a commonly used plastic microplate. The active contaminants were identified by gas chromatography-mass spectroscopy as dodecan-1-ol, dodecyl 3-(3-dodecoxy-3-oxopropyl)sulfanylpropanoate, and dodecanoic acid, and they were found to be selective monoamine oxidase-B inhibitors.

Characterization of efflux transporters involved in distribution and disposition of apixaban.

The studies reported here were conducted to investigate the transport characteristics of apixaban (1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxamide) and to understand the impact of transporters on apixaban distribution and disposition. In human permeability glycoprotein (P-gp)- and breast cancer resistance protein (BCRP)-cDNA-transfected cell monolayers as well as Caco-2 cell monolayers, the apparent efflux ratio of basolateral-to-apical (PcB-A) versus apical-to-basolateral permeability (PcA-B) of apixaban was >10. The P-gp- and BCRP-facilitated transport of apixaban was concentration- and time-dependent and did not show saturation over a wide range of concentrations (1-100 µM). The efflux transport of apixaban was also demonstrated by the lower mucosal-to-serosal permeability than that of the serosal-to-mucosal direction in isolated rat jejunum segments. Apixaban did not inhibit digoxin transport in Caco-2 cells. Ketoconazole decreased the P-gp-mediated apixaban efflux in Caco-2 and the P-gp-cDNA-transfected cell monolayers, but did not affect the apixaban efflux to a meaningful extent in the BCRP-cDNA-transfected cell monolayers. Coincubation of a P-gp inhibitor (ketoconazole or cyclosporin A) and a BCRP inhibitor (Ko134) provided more complete inhibition of apixaban efflux in Caco-2 cells than separate inhibition by individual inhibitors. Naproxen inhibited apixaban efflux in Caco-2 cells but showed only a minimal effect on apixaban transport in the BCRP-transfected cells. Naproxen was the first nonsteroidal antiinflammatory drug that was demonstrated as a weak P-gp inhibitor. These results demonstrate that apixaban is a substrate for efflux transporters P-gp and BCRP, which can help explain its low brain penetration, and low fetal exposures and high milk excretion in rats.

Ultrafast mass spectrometry based bioanalytical method for digoxin supporting an in vitro P-glycoprotein (P-gp) inhibition screen.

The evaluation of interactions between drug candidates and transporters such as P-glycoprotein (P-gp) has gained considerable interest in drug discovery and development. Inhibition of P-gp can be assessed by performing bi-directional permeability studies with in vitro P-gp-expressing cellular model systems such as Caco-2 (human colon carcinoma) cells, using digoxin as a substrate probe. Existing methodologies include either assaying (3)H-digoxin with liquid scintillation counting (LSC) detection or assaying non-labeled digoxin with liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis at a speed of several minutes per sample. However, it is not feasible to achieve a throughput high enough using these approaches to sustain an early liability screen that generates more than a thousand samples on a daily basis. To address this challenge, we developed an ultrafast (9 s per sample) bioanalytical method for digoxin analysis using RapidFire™, an on-line solid-phase extraction (SPE) system, with MS/MS detection. A stable isotope labeled analog, d3-digoxin, was used as internal standard to minimize potential ionization matrix effect during the RF-MS/MS analysis. The RF-MS/MS method was more than 16 times faster than the LC-MS/MS method but demonstrated similar sensitivity, selectivity, reproducibility, linearity and robustness. P-gp inhibition results of multiple validation compounds obtained with this RF-MS/MS method were in agreement with those generated by both the LC-MS/MS method and the (3)H-radiolabel assay. This method has been successfully deployed to assess P-gp inhibition potential as an important early liability screen for drug-transporter interaction.

Extraction, identification, and functional characterization of a bioactive substance from automated compound-handling plastic tips.

Disposable plastic labware is ubiquitous in contemporary pharmaceutical research laboratories. Plastic labware is routinely used for chemical compound storage and during automated liquid-handling processes that support assay development, high-throughput screening, structure-activity determinations, and liability profiling. However, there is little information available in the literature on the contaminants released from plastic labware upon DMSO exposure and their resultant effects on specific biological assays. The authors report here the extraction, by simple DMSO washing, of a biologically active substance from one particular size of disposable plastic tips used in automated compound handling. The active contaminant was identified as erucamide ((Z)-docos-13-enamide), a long-chain mono-unsaturated fatty acid amide commonly used in plastics manufacturing, by gas chromatography/mass spectroscopy analysis of the DMSO-extracted material. Tip extracts prepared in DMSO, as well as a commercially obtained sample of erucamide, were active in a functional bioassay of a known G-protein-coupled fatty acid receptor. A sample of a different disposable tip product from the same vendor did not release detectable erucamide following solvent extraction, and DMSO extracts prepared from this product were inactive in the receptor functional assay. These results demonstrate that solvent-extractable contaminants from some plastic labware used in the contemporary pharmaceutical research and development (R&D) environment can be introduced into physical and biological assays during routine compound management liquid-handling processes. These contaminants may further possess biological activity and are therefore a potential source of assay-specific confounding artifacts.

Solid-phase synthesis and anti-infective activity of a combinatorial library based on the natural product anisomycin.

The solid-phase synthesis of a library based on the natural product anisomycin is described. The resulting library was tested against a panel of bacterial and fungal targets, and active compounds were identified in a Staphylococcus aureus whole-cell assay and an efflux-deficient fungal whole-cell assay.