Synthesis, Biological Evaluation, and Molecular Modeling Studies of New Thiadiazole Derivatives as Potent P2X7 Receptor Inhibitors

Twenty new 2-(1H-pyrazol-1-yl)-1,3,4-thiadiazole analogs were synthetized to develop P2X7 receptor (P2X7R) inhibitors. P2X7R inhibition in vitro was evaluated in mouse peritoneal macrophages, HEK-293 cells transfected with hP2X7R (dye uptake assay), and THP-1 cells (IL-1β release assay). The 1-(5-phenyl-1,3,4-thiadiazol-2-yl)-1H-pyrazol-5-amine derivatives 9b, 9c, and 9f, and 2-(3,5-dimethyl-1H-pyrazol-1-yl)-5-(4-fluorophenyl)-1,3,4-thiadiazole (11c) showed inhibitory effects with IC50 values ranging from 16 to 122 nM for reduced P2X7R-mediated dye uptake and 20 to 300 nM for IL-1β release. In addition, the in vitro ADMET profile of the four most potent derivatives was determined to be in acceptable ranges concerning metabolic stability and cytotoxicity. Molecular docking and molecular dynamics simulation studies of the molecular complexes human P2X7R/9f and murine P2X7R/9f indicated the putative intermolecular interactions. Compound 9f showed affinity mainly for the Arg268, Lys377, and Asn266 residues. These results suggest that 2-(1H-pyrazol-1-yl)-1,3,4-thiadiazole analogs may be promising novel P2X7R inhibitors with therapeutic potential

Activity of C16:0-PAF and different <i>T. cruzi</i> LPC species on the aggregation of rabbit platelets.

<p>Platelet aggregation assays were performed as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003077#s2" target="_blank">Materials and Methods</a>, using synthetic 16:0-PAF and C16:0-, C18:0-, C18:1-LPC, and purified C18:2-LPC. Control platelets or platelets pre-treated for 30 min with 10 µM WEB 2086 were assayed in the absence or presence of 1 µM C16:0-PAF or the LPC species at 10 µM (C16:0-LPC, C18:0-LPC, C18:1-LPC, and C18:2-LPC) and 100 µM (C18:1-LPC). Each lipid was tested in duplicate as indicated by black and blue curves in each graph.</p

Proposed molecular structures of the three major LPC species of <i>T. cruzi</i>.

<p>Fragment ions obtained from the MSⁿ analysis of <i>T. cruzi</i> ion species at <i>m/z</i> 530, 528, and 526 are indicated. The tandem fragmentation of C16:0-PAF standard is included as a reference.</p

Distances between the nitrogen, phosphorous, and oxygen atoms in functional groups of C16:0-PAF and each LPC species.

<p>^<i>a</i>This refers to the nitrogen of the amino group, the phosphorous of the phosphate group, and the oxygen at <i>sn</i>-1, linking the glycerol backbone to the carbonyl group of the acyl chain.</p

The content of C18:2-, C18:1-, C18:0- and C16:0-LPC in different life-cycle stages of <i>T. cruzi</i>.

<p>^<i>a</i>The molar relative response factors (MRRF) of C10:0-LPC and LPC standards were used to calculate the amount of each LPC molecular species in Folch lower-phase fractions of <i>T. cruzi</i>.</p><p>^<i>b</i>The number of parasites was determined before lipid extraction by counting live parasites in a hemocytometer. Values are means of three determinations. The standard deviation in all cases was <15%.</p><p>^<i>c</i>Determined by multiplying the number of moles by the Avogadro's constant.</p><p>^<i>d</i>Estimated based on the parasite's length and diameter as determined by scanning electron microscopy, and assuming that each parasite is cylindrical, as previous described <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003077#pntd.0003077-PereiraChioccola1" target="_blank">[103]</a>.</p><p>^<i>e</i>Obtained by dividing the number of LPC molecules per cell by the surface area of each parasite form.</p><p>^<i>f</i>Obtained by dividing the amount of picomoles of LPC per 10⁶ cells of each parasite form (column 1) by the values obtained for Epi.</p><p>^<i>g</i>Not analyzed due to absence or trace amounts of the compound.</p