Hlavním cílem tohoto článku je prezentovat výsledky syntézy nových alkylových derivátů 5-(2-brom-4-fluorfenyl)-4-ethyl-4H-1,2,4-triazol-3-thiolu a molekulárního dokování studie proti COX-1 a COX-2. Předchozí studie prokázaly široký rozsah biologické aktivity 1,2,4-triazolových derivátů. Proto bylo důležité zjistit, jak nová řada 1,2,4-triazolových derivátů poskytne potenciální protizánětlivou aktivitu. K dosažení cíle byly připraveny alkylové deriváty 5-(2-brom-4-fluorfenyl)-4-ethyl-4H-1,2,4-triazol-3-thiolů (2a-2i) z 5-(2Byl získán brom-4-fluorfenyl)-4-ethyl-4H-l,2,4-triazol-3-thiol (1e). Struktura syntetizovaných sloučenin byla potvrzena 1H-NMR elementární analýzou. Totožnost a čistota sloučenin byla potvrzena metodou kapalinové chromatografie-hmotnostní spektrometrie. Tyto sloučeniny mají relativně jednoduché schéma syntézy, což jim dává výhodu v procesu tvorby potenciálního léčiva a výskyt alkylových radikálů v molekule by měl mít pozitivní vliv na farmakokinetické ukazatele, stabilitu, selektivitu a biologickou dostupnost. U syntetizovaných sloučenin byla provedena studie in silico, konkrétně molekulární dokování týkající se interakce s COX-1 a COX-2. Na základě indexů selektivity vazebných režimů pozorovaných pro vybrané sloučeniny (2e, 2g) s aktivními místy COX1 bylo zjištěno, že sloučeniny mohou pravděpodobně uplatňovat svůj protizánětlivý účinek cestou biosyntézy prostaglandinů, inhibicí COX-1 místo COX-2. Rovněž byl prokázán vliv hydrofobních interakcí alkylových skupin 1,2,4-triazolových derivátů na změnu afinity a selektivity k COX-1 nebo COX-2. Proto jsou deriváty 1,2,4 slibnými kandidáty na zlepšení, další studium a budoucí vývoj nových, účinnějších protizánětlivých léčiv pro terapeutické použití.
The main goal of this article is to present the results of the synthesis of new alkyl derivatives of 5-(2-bromo4-fluorophenyl)-4-ethyl-4H-1,2,4-triazole-3-thiol and molecular docking studies against COX-1 and COX-2. Previous studies have established a wide range of biological activity of 1,2,4-triazole derivatives. Therefore, it was essential to determine how a new series of 1,2,4-triazole derivatives would provide potential anti-inflammatory activity. To reach the goal, raw alkyl derivatives of 5-(2-bromo-4-fluorophenyl)-4-ethyl-4H-1,2,4-triazole-3-thiols (2a-2i) from 5-(2-bromo-4-fluorophenyl)-4-ethyl-4H-1,2,4-triazole3-thiol (1e) were obtained. The structure of the synthesized compounds was confirmed by 1H-NMR elemental analyses. The individuality and purity of compounds were confirmed by the method of liquid chromatography-mass spectrometry. These compounds have a relatively simple synthesis scheme, which gives them an advantage in creating a potential drug, and the appearance of alkyl radicals in the molecule should positively affect pharmacokinetic indicators, stability, selectivity, and bioavailability. An in silico study was conducted for the synthesized compounds, namely molecular docking, in relation to the interaction with COX-1 and COX-2. Based on the selectivity indexes of binding modes observed for the selected compounds (2e, 2g) with active COX-1 centers, it was found that compounds can reliably exhibit their anti-inflammatory effect through the prostaglandin biosynthesis pathway, inhibiting COX-1 instead of COX-2. The effect of hydrophobic interactions of alkyl groups of 1,2,4-triazole derivatives on changes in affinity and selectivity to COX-1 or COX-2 has also been proven. Therefore, derivatives of 1,2,4 are promising candidates for improvement, further study, and future development of new, more powerful antiinflammatory drugs for therapeutic use.
OBJECTIVES: The aim of the study was to use methods of pharmaceutical technology, and prepare carriers in the form of pellets suitable as a filling of detection tubes for enzymatic detection of cholinesterase inhibitors. The enzymatic detection was based on enzymatic hydrolysis of acetylthiocholine iodide and the subsequent colour reaction of its hydrolysis product with Ellman's reagent. The suitable carriers should be in the form of white, regular and sufficiently mechanically resistant particles of about 1 mm allowing it to capture the enzyme during the impregnation process and ensuring its high activity for enzymatic detection. METHODS: Carriers consisting of microcrystalline cellulose, lactose, povidone, and sodium carboxymethyl cellulose were prepared using extrusion-spheronization method under three different drying conditions in either a hot air oven or a microwave oven. Subsequently, the carriers were impregnated with acetylcholinesterase and their size, shape, mechanical resistance, bulk, tapped and pycnometric density, Hausner ratio, intraparticular and total tapped porosity, and activity were measured and recorded. RESULTS: In this procedure, carriers with different physical parameters and different acetylcholinesterase activity were evaluated. It was found that higher acetylcholinesterase activity was associated not only with a higher intraparticular porosity but also with more regular particles characterized by high sphericity and low total tapped porosity. CONCLUSION: This unique finding is important for the preparation of detection tubes based on enzymatic detection which is still irreplaceable especially in the field of detection and analysis of super-toxic cholinesterase inhibitors.
- MeSH
- Acetylthiocholine analogs & derivatives metabolism MeSH
- Cellulose analysis MeSH
- Cholinesterase Inhibitors metabolism MeSH
- Dithionitrobenzoic Acid MeSH
- Lactose analysis MeSH
- Porosity MeSH
- Povidone analysis MeSH
- Carboxymethylcellulose Sodium analysis MeSH
- Sulfhydryl Reagents MeSH
- Materials Testing MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
We have previously reported that the reducing agent dithiothreitol (DTT) strongly increases thermally induced activity of the transient receptor potential vanilloid receptor-1 (TRPV1) channel. Here, we show that exposure to oxidizing agents also enhances the heat-induced activation of TRPV1. The actions of sulfhydryl modifiers on heat-evoked whole-cell membrane currents were examined in TRPV1-transfected human embryonic kidney 293T cells. The sensitizing effects of the membrane-permeable oxidizing agents diamide (1 mM), chloramine-T (1 mM), and the copper-o-complex (100:400 microM) were not reversed by washout, consistent with the stable nature of covalently modified sulfhydryl groups. In contrast, the membrane-impermeable cysteine-specific oxidant 5,5'-dithio-bis-(2-nitrobenzoic acid) (0.5 mM) was ineffective. The alkylating agent N-ethylmaleimide (1 mM) strongly and irreversibly affected heat-evoked responses in a manner that depended on DTT pretreatment. Extracellular application of the membrane-impermeable reducing agent glutathione (10 mM) mimicked the effects of 10 mM DTT in potentiating the heat-induced and voltage-induced membrane currents. Using site-directed mutagenesis, we identified Cys621 as the residue responsible for the extracellular modulation of TRPV1 by reducing agents. These data suggest that the vanilloid receptor is targeted by redox-active substances that directly modulate channel activity at sites located extracellularly as well as within the cytoplasmic domains. The results obtained demonstrate that an optimal redox state is crucial for the proper functioning of the TRPV1 channel and both its reduced and oxidized states can result in an increase in responsiveness to thermal stimuli.
- MeSH
- Cell Line MeSH
- Diamide pharmacology MeSH
- Dithiothreitol pharmacology MeSH
- Ethylmaleimide pharmacology MeSH
- Financing, Organized MeSH
- Capsaicin pharmacology MeSH
- TRPV Cation Channels physiology genetics MeSH
- Rats MeSH
- Dithionitrobenzoic Acid pharmacology MeSH
- Humans MeSH
- Membrane Potentials drug effects MeSH
- Patch-Clamp Techniques MeSH
- Mutation, Missense genetics MeSH
- Mutation genetics MeSH
- Mutant Proteins physiology genetics MeSH
- Oxidants pharmacology MeSH
- Hydrogen Peroxide pharmacology MeSH
- Reducing Agents pharmacology MeSH
- Sulfhydryl Reagents pharmacology MeSH
- Transfection MeSH
- Hot Temperature MeSH
- Dose-Response Relationship, Drug MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Humans MeSH
- Animals MeSH
- MeSH
- Bacterial Proteins isolation & purification metabolism MeSH
- Chromatography, Ion Exchange MeSH
- Electrophoresis, Polyacrylamide Gel MeSH
- Lactate Dehydrogenases metabolism MeSH
- Membrane Proteins isolation & purification metabolism MeSH
- Paracoccus denitrificans enzymology MeSH
- Sulfhydryl Reagents MeSH
At a concentration of 0.5 to 3 mmol/l, ATP stimulates the activity of mitochondrial DNA polymerase of Neurospora crassa under the optimum reaction conditions; at higher concentrations, an inhibitory effect is observed. 4-Chloromercuribenzoate (1 mmol/L), a thiol inhibitor, decreases the enzyme activity two-fold, while N-ethylmalcimide (2 mmol/L) has no effect. Ethidium bromide (up to 10 mumol/L) and heparin (up to 0.4 micrograms/mL) reduce the activity by 60%. ddTTP does not affect the DNA polymerase reaction. The best in vitro template is the activated calf-thymus DNA.
- MeSH
- Adenosine Triphosphate pharmacology MeSH
- Enzyme Activation drug effects MeSH
- Dideoxynucleotides MeSH
- DNA-Directed DNA Polymerase metabolism MeSH
- Ethidium pharmacology MeSH
- Heparin pharmacology MeSH
- Nucleic Acid Synthesis Inhibitors * MeSH
- Mitochondria enzymology MeSH
- Neurospora crassa drug effects enzymology MeSH
- Sulfhydryl Reagents pharmacology MeSH
- Thymine Nucleotides pharmacology MeSH
- Publication type
- Journal Article MeSH
