Development of HPLC Method for Quantitative Determination of Epimidin - New Perspective АPhI with Anticonvulsive Activity

The aim. Development of optimal, high-precision and reproducible methods for quantitative determination of the main substance in the substance Epimidin - 1-(4-methoxyphenyl)-5-[2-[4-(4-methoxyphenyl)piperazin-1-yl]-2-oxo-ethyl]pyrazolo[3,4-d]pyrimidin-4-one by high performance liquid chromatography.Materials and methods. High performance liquid chromatography (HPLC) was performed using a ShimadzuNexeraX2 LC-30AD system (Shimadzu, Japan) equipped with a SPD-M20A diode array detector (DAD). ACE C18 column, size 250 x 4.6 mm, YMC with pre-column, particle size 5 μm, filled with octylsilyl silica gel for chromatography P. During the work acetonitrile and trifluoroacetic acid of HPLC class (Sigma-AldrichGmbH, Switzerland) were used, other chemicals and solvents were of analytical grade. In the study an analytical ware class A were used that meet the requirements of SPhU.Results. The following optimal conditions of chromatographic distribution are established: column C18 (250*4.6 mm); the speed of the mobile phase 1 ml / min; column thermostat temperature 35 °С; injection volume 10 μl; mobile phase A - 0.1 % trifluoroacetic acid; mobile phase B - acetonitrile P; the detection wavelength is 270 nm, the retention time of the test compound is 7.22 minutes. The performance of the column was determined for its main indicators, such as the number of theoretical plates (more than 25410) and the coefficient of symmetry (about 1.00). The technique was tested for the influence of various factors, such as flow rate, mobile phase composition and column thermostat temperature. It was established that the influence of these factors is insignificant and does not affect the results obtained by this method. The method was validated in accordance with the recommendations of SPhU on the parameters of specificity, linearity, correctness, precision, robustness (stability).Conclusions. For the first time, a high-precision and reproducible method for quantitative determination of the main substance in the substance Epimidin with anticonvulsant activity by high-performance liquid chromatography was developed. Conditions for chromatographic analysis (HPLC) were standardized. The requirements for the test “System suitability test criteria for chromatographic methods” are set. Statistical processing of the experimental results shows that the relative uncertainty of the average result is within acceptable limits. The correctness of the method was confirmed by validation studies. The developed technique will be used for pharmaceutical development and standardization of dosage for

Design, Synthesis, Molecular Docking and Anticonvulsant Evaluation of 6-methyl-2-arylaminopyrimidin-4(3H)-one

The aim. Synthesis of 2-aminoaryl derivatives of 6-methyl-pyrimidin-4(3H)-one, target-based virtual screening followed by the study of anticonvulsant activity and the establishment of structure-activity patterns.Materials and methods. The standard methods of organic synthesis were used, synthesized compounds structure was proved with elemental analysis, 1H NMR spectroscopy, chromatography-mass spectrometry. Molecular docking was performed using AutoDockTools-1.5.6 and AutoDock Vina. Anticonvulsant activity was studied in а model of pentylenetetrazole seizures in rats.Results. Methylation of 6-methyl-2-thiopyrimidin-4 (3H)-one with dimethyl sulfate or methyl iodide gave a 2-thiomethyl derivative. By heating the latter with aromatic amines at 140 ºC, the target 2-aminoaryl derivatives of 6-methyl-pyrimidin-4 (3H)-one were obtained. The prospect of screening the synthesized compounds on the pentylenetetrazole model by seizure and the selection of the objects was performed by the results of binding energy and conformation evaluation at the active sites of GABA receptor and GABA-AT. The test substances did not show anticonvulsant activity: only 2 compounds tended to exhibit activity according to the criterion of integral protective index - a decrease in mortality compared to control, preventing mortality in 100 and 80 % of animals, respectively. Comparison with previous activity results of 2-thioacetanilide derivatives allowed to prove the positive role of thioacetamide and phenyl fragments, as well as 4-Br, 4-MeO radicals in the manifestation of anticonvulsant activity and increase of lethality in the presence of Cl atoms.Conclusions. The synthesis was performed and construction of the 2-aminoaryl derivatives of 6-methyl-pyrimidin-4(3H)-one was proved. PTZ seizures model in rats did not show anticonvulsant activity. However, the obtained results allowed us to identify a number of structural fragments that influence anticonvulsant activity. A positive correlation between in vivo studies on PTZ seizures model and docking results in active sites of GABAA and GABAAT enzyme was determine

Synthesis and Anticonvulsant Activity Evaluation of N-[(2,4-dichlorophenyl)methyl]-2-(2,4-dioxo-1h-quinazolin-3-yl)acetamide Novel 1-benzylsubstituted Derivatives

The aim. Synthesis of 1-benzylsubstituted derivatives of N-[(2,4-dichlorophenyl)methyl]-2-(2,4-dioxo-1h-quinazolin-3-yl)acetamide, and determination of affinity to GABAergic biotargets with the following anticonvulsant activity estimation using PTZ-induced seizures model in mice. Materials and methods. Standard organic synthesis methods were used; the structure of the synthesized compounds was proved by elemental analysis, 1H and 13C NMR spectroscopy, and LC/MS method; composition of the synthesized compounds – by elemental analysis, their individuality – by TLC and LC/MS methods. AutoDockTools-1.5.6, as well as AutoDock Vina software, was used to perform molecular docking. Anticonvulsant activity was studied using pentylenetetrazole-induced seizures in mice. Results. A targeted N-[(2,4-dichlorophenyl)methyl]-2-(1-(R-benzyl)-2,4-dioxo-quinazolin-3-yl)acetamides were obtained by alkylation of N-[(2,4-dichlorophenyl)methyl]-2-(2,4-dioxo-1H-quinazolin-3-yl)acetamide by corresponding 1-chloromethylbenzene in dimethylformamide environment with excess of potassium carbonate at a temperature 70-80 ˚С. Prediction of activity of 1-benzyl derivatives in the pentylenetetrazole-induced seizures in an in vivo experiment was carried out according to the obtained results of docking studies – affinity calculation for GABA receptor and GABA enzyme active sites, as well as analysis of conformational placement in them. In relation to the binding energy, the studied ligands were inferior to the reference drugs: GABA receptor positive allosteric modulators – benzamidine and diazepam, and GABA inhibitor – vigabatrin. The synthesized substances did not show anticonvulsant activity: only 2 compounds have shown a tendency to their activity manifestation according to the criterion of integral protective indicator – reduction of mortality by 17 % compared to control, as well as prolonging the time death of the animals. Comparison with the preliminary obtained results of the activity of the promising anticonvulsant N-[(2,4-dichlorophenyl)methyl] -2-(2,4-dioxo-1H-quinazolin-3-yl) acetamide N-[(2,4-dichlorophenyl)methyl]-2-(2,4-dioxo-1H-quinazolin-3-yl)acetamide made possible to prove the pharmacophore role of the cyclic amide fragment in anticonvulsant activity manifestation. Conclusion. The synthesis of N-[(2,4-dichlorophenyl)methyl]-2-(1-(R-benzyl)-2,4-dioxo-quinazolin-3-yl)acetamides, which have not still described in the literature, was carried out, as well as the structure of the mentioned compounds was proved. Unfortunately, the substances did not show anticonvulsant activity on the model of pentylenetetrazole-induced seizures. However, the obtained results allowed establishing the key role of the NHCO cyclic fragment on anticonvulsant activity. A positive correlation between the results of in vivo studies and in silico calculations was found – the model of pentylenetetrazole-induced seizures and docking into the active sites of PAMs GABAА receptor and enzyme inhibitor GABAАТ, which allows to recommend the given docking methodology as a tool to streamline and optimize the screening on the mentioned mode