5-(Indol-2-yl)pyrazolo[3,4-b]pyridines as a New Family of TASK-3 Channel Blockers: A Pharmacophore-Based Regioselective Synthesis

TASK channels belong to the two-pore-domain potassium (K_2P) channels subfamily. These channels modulate cellular excitability, input resistance, and response to synaptic stimulation. TASK-channel inhibition led to membrane depolarization. TASK-3 is expressed in different cancer cell types and neurons. Thus, the discovery of novel TASK-3 inhibitors makes these bioactive compounds very appealing to explore new cancer and neurological therapies. TASK-3 channel blockers are very limited to date, and only a few heterofused compounds have been reported in the literature. In this article, we combined a pharmacophore hypothesis with molecular docking to address for the first time the rational design, synthesis, and evaluation of 5-(indol-2-yl)pyrazolo[3,4-b]pyridines as a novel family of human TASK-3 channel blockers. Representative compounds of the synthesized library were assessed against TASK-3 using Fluorometric imaging plate reader—Membrane Potential assay (FMP). Inhibitory properties were validated using two-electrode voltage-clamp (TEVC) methods. We identified one active hit compound (MM-3b) with our systematic pipeline, exhibiting an IC₅₀ ≈ 30 μM. Molecular docking models suggest that compound MM-3b binds to TASK-3 at the bottom of the selectivity filter in the central cavity, similar to other described TASK-3 blockers such as A1899 and PK-THPP. Our in silico and experimental studies provide a new tool to predict and design novel TASK-3 channel blockers.     

Using magnetic core‐shell nanoparticles coated with an ionic liquid dispersion assisted by effervescence powder for the micro‐solid‐phase extraction of four beta blockers from human plasma by ultra high performance liquid chromatography with mass spectrometry detection

In this work, a novel, efficient, and green sorbent, SiO₂@Fe₃O₄ has been created and functionalized with 1‐butyl‐3‐methylimidazolium hexafluorophosphate as an ionic liquid. This sorbent was applied for microextraction of four beta blockers, propranolol, metoprolol, atenolol, and alprenolol with bupivacaine as internal standard from human plasma followed by liquid chromatography with mass spectrometric detection. A mixture of sodium bicarbonate and sodium dihydrogen phosphate was used as an extractant dispersive agent (effervescent power) to enhance the interaction between the magnetic sorbent and analytes. Main affecting parameters on microextraction and elution were optimized. Figures of merit for dispersive solid phase extraction with ionic liquid coated magnetic nanoparticles assisted by effervescent powder were calculated under the optimized conditions. The detection limits for propranolol, metoprolol, atenolol, and alprenolol were found at 0.33, 0.62, 0.03, and 0.44 ng/mL, respectively. For all analytes, good linearity was obtained. Intra‐ (n = 5) and interday (n = 10) precision were both under 6.3% while the preconcentration factors were obtained in the range between 15–18. The extraction efficiencies for each analyte ranged from 75 to 91%. The method was successfully applied for determination of trace amounts of the beta blockers in human plasma samples.     

Effect of chemically bonded stationary phases and mobile phase composition on beta-blockers retention in RP-HPLC

The effects of stationary and mobile phase on retention of 18 beta-adrenolytic drugs (beta-blockers) have been studied. Four 'deactivated surface' stationary phases (polar-embedded or end-capped) were examined. Special attention was drawn to the cholesterolic (SG-CHOL) and alkylamide (SG-AP) stationary phases, and their application for analysis of the compounds. The retention of analyzed substances was also examined in terms of mobile phase composition. Sixteen different configurations of mobile phases were prepared, all based on methanol and acetonitrile with ammonium acetate and ammonium formate. The difference in retention between ammonium formate and acetate water solutions, and peak shape changes related to the addition of triethylamine (TEA), were investigated. Principal component analysis was used to find the similarities between stationary phases. Polar-embedded phases synthesized on the same sorbent possess very similar properties. All phases based on silica gel compared with the monolithic column also showed similarities in retention of beta-blockers. The addition of TEA to the mobile phase did not influence strongly the retention, and analysis of asymmetry factors showed only a little peak broadening for a few compounds on the monolithic column.     

Simple, rapid, and sensitive determination of beta-blockers in environmental water using dispersive liquid-liquid microextraction followed by liquid chromatography with fluorescence detection

A novel method, dispersive liquid-liquid microextraction combined with liquid chromatography-fluorescence detection is proposed for the determination of three beta-blockers (metoprolol, bisoprolol, and betaxolol) in ground water, river water, and bottled mineral water. Some important parameters, such as the kind and volume of extraction and dispersive solvents, extraction time, pH, and salt effect were investigated and optimized. In the method, a suitable mixture of extraction solvent (60 μL carbon tetrachloride) and dispersive solvent (1 mL acetonitrile) were injected into the aqueous samples (5.00 mL) and the cloudy solution was observed. After centrifugation, the enriched analytes in the bottom CCl(4) phase were determined by liquid chromatography with fluorescence detection. Under the optimum conditions, the enrichment factors (EFs) for metoprolol, bisoprolol, and betaxolol were 180, 190, and 182, and the limits of detection (LODs) were 1.8, 1.4, and 1.0 ng L(-1) , respectively. A good linear relationship between the peak area and the concentration of analytes was obtained in the range of 3-150 ng L(-1) . The relative standard deviations (RSDs) for the extraction of 10 ng L(-1) of beta-blockers were in the range of 4.6-5.7% (n = 5). Compared with other methods, dispersive liquid-liquid microextraction is a very simple, rapid, sensitive (low limit of detection), and economical (only 1.06 mL volume of organic solvent) method, which is in compliance with the requirements of green analytical methodologies.     

Is the Antidepressant Activity of Selective Serotonin Reuptake Inhibitors Mediated by Nicotinic Acetylcholine Receptors?

It is generally assumed that selective serotonin reuptake inhibitors (SSRIs) induce antidepressant activity by inhibiting serotonin (5-HT) reuptake transporters, thus elevating synaptic 5-HT levels and, finally, ameliorates depression symptoms. New evidence indicates that SSRIs may also modulate other neurotransmitter systems by inhibiting neuronal nicotinic acetylcholine receptors (nAChRs), which are recognized as important in mood regulation. There is a clear and strong association between major depression and smoking, where depressed patients smoke twice as much as the normal population. However, SSRIs are not efficient for smoking cessation therapy. In patients with major depressive disorder, there is a lower availability of functional nAChRs, although their amount is not altered, which is possibly caused by higher endogenous ACh levels, which consequently induce nAChR desensitization. Other neurotransmitter systems have also emerged as possible targets for SSRIs. Studies on dorsal raphe nucleus serotoninergic neurons support the concept that SSRI-induced nAChR inhibition decreases the glutamatergic hyperstimulation observed in stress conditions, which compensates the excessive 5-HT overflow in these neurons and, consequently, ameliorates depression symptoms. At the molecular level, SSRIs inhibit different nAChR subtypes by noncompetitive mechanisms, including ion channel blockade and induction of receptor desensitization, whereas α9α10 nAChRs, which are peripherally expressed and not directly involved in depression, are inhibited by competitive mechanisms. According to the functional and structural results, SSRIs bind within the nAChR ion channel at high-affinity sites that are spread out between serine and valine rings. In conclusion, SSRI-induced inhibition of a variety of nAChRs expressed in different neurotransmitter systems widens the complexity by which these antidepressants may act clinically.     

Transversal hypergraphs to perfect matchings in bipartite graphs: Characterization and generation algorithms

A minimal blocker in a bipartite graph G is a minimal set of edges the removal of which leaves no perfect matching in G. We give an explicit characterization of the minimal blockers of a bipartite graph G. This result allows us to obtain a polynomial delay algorithm for finding all minimal blockers of a given bipartite graph. Equivalently, we obtain a polynomial delay algorithm for listing the anti‐vertices of the perfect matching polytope of G. We also provide generation algorithms for other related problems, including d‐factors in bipartite graphs, and perfect 2‐matchings in general graphs. © 2006 Wiley Periodicals, Inc. J Graph Theory 53: 209–232, 2006     

Monitoring of progestins: Development of immunochemical methods for purification and detection of levonorgestrel

A polyclonal antibody (Ab) for the progestin levonorgestrel (LNG) was generated, and immunochemical assays for its detection, clean-up and concentration were developed. A highly specific microplate diagnostic assay for the detection of LNG was developed that used the enzyme linked immunosorbent assay (ELISA) method. The LNG ELISA developed was sensitive and reproducible; it exhibited I₅₀ and I₂₀ values of 3.3 ± 1.8 ng mL⁻¹ and 0.6 ± 0.4 ng mL⁻¹, respectively, and the Abs did not cross react with any of the tested steroid hormones. The above Abs were used to develop a sol-gel-based immunoaffinity purification (IAP) method for concentration and clean-up of LNG that is compatible with subsequent immunochemical or instrumental chemical analytical procedures, such as liquid chromatography followed by mass spectrometry (LC-MS/MS). Development of the columns included successful entrapment of Abs within a tetramethoxysilane (TMOS)-based SiO₂ polymer network. The Abs could bind the free analyte from solution, and the bound analyte could be easily eluted from the sol-gel matrix at high recoveries. The Ab selectivity towards the antigen was high, in both ELISA and the sol-gel columns, but the entrapped Abs cross-reacted with two other steroid hormones - ethynylestradiol (EE2) and nortestosterone (NT) - which share similar epitopes with LNG, despite the lack of cross reactivity in the ELISA. The validity of the method was investigated by LC-MS/MS and a good analytical correlation was obtained.     

Singular value decomposition of torsional angles of analogs of the dopamine reuptake inhibitor GBR 12909

Analysis of large, flexible molecules, such as the dopamine reuptake inhibitor GBR 12909 (1), is complicated by the fact that they can take on a wide range of closely related conformations. The first step in the analysis is to classify the conformers into groups. Here, Singular Value Decomposition (SVD) was used to group conformations of GBR 12909 analogs by the similarity of their nonring torsional angles. The significance of the present work, the first application of SVD to the analysis of very flexible molecules, lies in the development of a novel scaling technique for circular data and in the grouping of molecular conformations using a technique that is independent of molecular alignment. Over 700 conformers each of a piperazine (2) and piperidine (3) analog of 1 were studied. Analysis of the score and loading plots showed that the conformers of 2 separate into three large groups due to torsional angles on the naphthalene side of the molecule, whereas those of 3 separate into nine groups due to torsional angles on the bisphenyl side of the molecule. These differences are due to nitrogen inversion at the unprotonated piperazinyl nitrogen of 2, which results in a different ensemble of conformers than those of 3, where no inversion is possible at the corresponding piperidinyl carbon.     

Hierarchical clustering analysis of flexible GBR 12909 dialkyl piperazine and piperidine analogs

Pharmacophore modeling of large, drug-like molecules, such as the dopamine reuptake inhibitor GBR 12909, is complicated by their flexibility. A comprehensive hierarchical clustering study of two GBR 12909 analogs was performed to identify representative conformers for input to three-dimensional quantitative structure–activity relationship studies of closely-related analogs. Two data sets of more than 700 conformers each produced by random search conformational analysis of a piperazine and a piperidine GBR 12909 analog were studied. Several clustering studies were carried out based on different feature sets that include the important pharmacophore elements. The distance maps, the plot of the effective number of clusters versus actual number of clusters, and the novel derived clustering statistic, percentage change in the effective number of clusters, were shown to be useful in determining the appropriate clustering level.Six clusters were chosen for each analog, each representing a different region of the torsional angle space that determines the relative orientation of the pharmacophore elements. Conformers of each cluster that are representative of these regions were identified and compared for each analog. This study illustrates the utility of using hierarchical clustering for the classification of conformers of highly flexible molecules in terms of the three-dimensional spatial orientation of key pharmacophore elements.     

Retention behaviour of beta-blockers in HPLC using a monolithic column

The chromatographic behaviour of a new HPLC-stationary phase (Chromolith RP-18e) is described for separation of beta-blockers. The effects of the different chromatographic conditions (buffer system, pH value, content of organic modifier, injection volume and flow rate) on the separation behaviour were studied. At higher flow rates the peaks seemed to be more symmetrical than at lower flow rates. The use of buffer or salt in the mobile phase for this separation is found to be very essential and the counter-anion type of this buffer or salt significantly affected the retention behaviour of beta-blockers while the cation type did not play the same important role.