Synthesis, neurotropic activity and SAR of new S-alkyl derivatives of 8- pyrazol-1-yl pyrano[3,4-c ]pyridines

8-Hydrazino-3,3-dimethyl-6-thioxo-3,4,6,7-tetrahydro-1 H -pyrano[3,4-c ]pyridine was synthesized by a newly developed method. Pyrazolo substituted 6-thioxo-and S-alkyl-derivatives of the pyrano[3,4-c ]pyridines have also been synthesized. In this work, 8-(3,5-dimethyl-1 H -pyrazol-1-yl)-3,3-dimethyl-6-oxo-3,4,6,7-tetrahydro-1 H - pyrano[3,4-c ]pyridine-5-carbonitrile was also synthesized via an intramolecular nucleophilic substitution (Smiles rearrangement). The study of the neurotropic activity of the synthesized compounds was carried out using convulsive models (pentylenetetrazole and maximal electroshock) and the rotating rod model was used to study central myorelaxation. Compounds were found to antagonize corazole, but did not exhibit muscle relaxation at the studied doses, while simultaneously, they showed anxiolytic and antidepressant psychotropic activity


Introduction
Synthesis of pharmaceuticals for the treatment of neuropsychiatric disorders, in particular epilepsy, is a serious challenge for medicinal synthetic organic chemistry.The antiepileptic agents which are used in medicine most frequently often cause toxic side responses affecting different organs and systems, which include emotional disturbances, impaired memory, etc.In this regard, the search for, and study of novel anticonvulsants possessing desirable psychotropic properties is of unquestionable interest.
However, during the reaction of thione 3 with alkyl halides the equilibrium is shifted toward the thiolactim form and S-alkyl derivatives 4a-e are formed in 82-91% yield (Scheme 2).The reaction was carried out at room temperature in 80% aqueous ethanolic KOH.The formation of S-alkyl derivatives 4a-e was confirmed by 1 H and 13 C NMR spectroscopy.For example, in the 1 H NMR spectra of compound 4a the signal of the SCH3 group is observed at 2.60 ppm, while the signal of the SCH2 group of the S-alkyl derivatives 4b-e is shifted to a lower field -3.32-4.06ppm.In the 13 C NMR spectra, the signals of SCH3 (for compound 4a) and SCH2 (for compounds 4b-e) are observed at 12.9 and 17.9-32.0ppm, accordingly.Finally, a Smiles rearrangement was performed on the 8- Rearrangement of the compound 4b occurred in the presence of a tenfold excess of sodium hydroxide in quite good yield (79%).The cleavage product -thiirane, is also formed in the reaction mixture and may polymerize under basic conditions.The structure of compound 5 was confirmed by IR and NMR spectroscopies.The IR spectra of compound 5 showed the amide carbonyl stretching vibration band at 1665 cm -1 , a nitrile group at 2222 cm -1 , and weak NH group vibration at 3474 cm -1 .In the 1 H NMR spectra of compound 5 the NH group signal appeared at 11.6-12.6ppm, while in the 13 C NMR spectra of compound 5 the CO group signal appeared at 161.9 ppm.A similar S-O Smiles rearrangement is observed in our previous work, 14 where we also described the proposed mechanism of reaction.In this case, we suggest that the reaction proceeds by the same mechanism, as depicted in Scheme The neurotropic activity of the new pyrazol-1-yl substituted pyrano [3,4-c]pyridine derivatives was next studied.6][17][18][19] The PTZ subcutaneous test is a petit mal (generalized clonic seizures) model of epilepsy, while MES is grand mal (generalized tonic seizures) model. 20Comparison with the prominent antiepileptic drugs ethosuximide and the tranquilizer diazepam was performed. 21The neurotoxicity of the compounds was studied on the ''rotating rod'' model 15 and the maximal tolerated dose (MTD) was also studied.To determine the ED50 (50% effective doses) and TD50 (50% neurotoxic doses), a statistical method of probity analysis by Litchfield and Wilcoxon was used. 22,23Finally, the protective (PI=TD50/ED50) index was identified for the active compounds.
The studied compounds, at a dose of 50 mg/kg, prevented subcutaneous PTZ clonic seizures in 40-80% of animals.However, compounds 3, 4d, 4e had more pronounced antagonism to PTZ seizures.The ED50 (with intraperitoneal injection) of these compounds ranged from 24 mg/kg to 34 mg/kg.It should be noted that the tested compounds are superior to ethosuximide (ED50=155 mg/kg), according to the anticorazole activity, but inferior to diazepam (ED50=0.5 mg/kg).The studied doses did not violate the coordination of movements on mice and the TD50 of the studied compounds ranged from 505 mg/kg to 590 mg/kg.Ethosuximide has the same neurotoxic effect, unlike diazepam, which causes muscle relaxation already at doses of 2-3 mg/kg.Maximal tolerated dose (MTD) of the studied compounds and ethosuximide were in the range of 1000-1350 mg/kg, and for diazepam − 200 mg/kg.The protective indexes of the selected compounds were determined as follows: 3 (PI = 21.7),4d (PI = 21.1),4e (PI = 22.3), and were found to be high and slightly exceed the indexes of ethosuximide (PI = 3.4) and diazepam (PI = 5.4).Finally, the compounds tested, as well as the comparison drug did not exhibit an anticonvulsant effect according to the MES test.
The structure-activity relationship (SAR) study on our small set of compounds revealed that replacement of the hydrazine fragment by the pyrazole ring (3) in the pyranopyridine system increased the anticonvulsant activity.The same activity is observed in the presence of ethylacetate (4d) and prop-2-yn-1-ylsubstituent on the sulfur atom of the 6 th position of the pyranopyridine ring, while replacement of these groups with methyl (4a), ethyl (4b) and methylacetate group decreased activity.
The most effective 3 compounds − 3, 4d, 4e − were studied for psychotropic activity using the following tests: ″open field″, ″elevated plus maze″ (EPM), ″forced swimming″.The efficacy of the compounds was studied at a dose of 50 mg/kg, since the ED50 of these compounds is in the range of 50 mg/kg.In the ″open field″ [24][25][26] exploratory behavioral model test in rats, the compounds 3, 4d and 4e exhibiting some sedative effect and antianxiety activity.
8][29][30] The administration of compound 4e resulted in a statistically reliable decrease in the time spent in closed arms and compound 3 also saw a decrease in the number of entries into the closed arms.All selected compounds had effects that were statistically significant, and when compared with the control increased the time spent by experienced animals in the center, which indicates sedative activity, and especially for compound 4e.After the administration of the selected compounds, experimental animals go to the open arms.The data obtained indicate the anxiolytic activity of all selected compounds, especially expressed in the compounds 3 and 4e.
Compounds 3, 4d and 4e also statistically significantly increased the time of latent period of the first immobilization and decreased the total time of immobilization in the forced swimming test (FST) 31 used to monitor depressive-like behavior.The data obtained thus indicates that compounds 3, 4d and 4e exhibit some antidepressant effects.

Conclusions
New pyrazolo substituted heterocyclic systems incorporating pyrano [3,4-c]pyridines 4a-e were synthesized by a new method and their neurotropic activity was studied.In addition, 8-(3,5-dimethyl-1H-pyrazol-1-yl)-3,3dimethyl-6-oxo-3,4,6,7-tetrahydro-1H-pyrano[3,4-c]pyridine-5-carbonitrile 5 was synthesized by a Smiles rearrangement.The structure of the synthesized compounds was confirmed by physicochemical methods.The evaluation of anticonvulsant activity of all the synthesized compounds revealed that compounds 3, 4d and 4e had a pronounced anticonvulsant action.It should be mentioned that replacement of the hydrazine fragment by the pyrazole ring in the pyranopyridine system appeared to increase the anticonvulsant activity, and that replacement of the sulfur atom by the oxygen in the pyranopyridine system decreased the anticonvulsant activity.Furthermore, substitution on the sulfur atom of the 6 th position of the pyranopyridine ring with larger alkyl and prop-2-yn-1-yl groups increased the anticonvulsant activity (4a4b4c4d=4e).The selected 3, 4d and 4e compounds exhibited pronounced anti-anxiety and some antidepressant activities, similar to diazepam, as well as sedative activity, the latter especially observed for compound 4e.

Experimental Section
General.All chemicals, reagents, and solvents were of commercially high purity grade purchased from Sigma-Aldrich.Melting points (M.p.) were determined on a Boetius microtable.They are expressed in degrees centigrade (°C). 1 H NMR and 13 C NMR spectra were recorded in DMSO/CCl4, 1/3 solution on a Varian mercury 300VX 300 ( 1 H) and 75.465MHz ( 13 C) spectrometer.Chemical shifts are reported as δ (parts per million) relative to TMS (tetramethylsilane) as the internal standard.IR spectra were recorded on Nicolet Avatar 330-FTIR spectrophotometer and the reported wave numbers are given in cm −1 .Elemental analyses were performed on a Euro EA 3000 Elemental Analyzer.

Biological evaluation
General.Compounds were studied for their possible neurotropic activities (anticonvulsant, sedative, antianxiety activity) as well as side effects on 450 white mice of both sexes weighing 18-24 g and 50 male rats of the Wistar line weighing 120-140 g.All groups of animals were maintained at 25 ± 2 ºC in the same room, on a common food ration.All  Evaluation of the anticonvulsant activity of the synthesized compounds.]19 The PTZ test is an experimental model for inducing myoclonic seizures, as well as for predicting the anxiolytic properties of compounds.Out bred mice (weight 18-22 g.) were used for the study The PTZ test was carried out in mice by subcutaneous administration of analeptic at a dose of 90 mg/kg and the effectiveness of the preparations was determined by the prevention of clonic seizures.The anticonvulsant activity of the compounds was also carried out to prevent the tonic-extensor phase of the convulsive seizure of maximal electroshock (MES).The parameters of the maximal electroshock were 50 mA, duration was 0.2 s, the oscillation frequency was 50 imp/s, the evaluation criterion was the warning of the tonic-extensor phase of a convulsive seizure.Substances were administered intraperitoneally in doses of 10, 25, 50, 75, 100 mg/kg in suspension with carboxymethylcellulose ("Viadi -Ingredients") with tween-80 ("FerakBerlin") 45 minutes before the injection of the convulsive agent PTZ causing electrical irritation.The control animals were administered an emulsifier.Each dose of compounds for each test was studied in 8 animals.Analogues of comparison were an anticonvulsant drug from the group of succinimide ethosuximide (neuraxpharmArzneimittel GmbH (Germany). 21Comparison drug, ethosuximide in doses from 100 to 300 mg/kg was administered intraperitoneally.Evaluation of the psychotropic properties of the synthesized compounds.Psychotropic properties of selected compounds (3, 4d, 4e) were studied by tests: ″open field″, ″elevated plus maze -EPM″, ″forced swimming″.Open field test.5][26] For this purpose, an installation was used, the bottom of which is divided into squares with holes (cells).Experiments were performed in the daytime with natural light.Within 5 minutes of the experiment, the indicators of sedative and activating behavior were determined -the number of horizontal movements, standing on the hind legs (vertical movements), sniffing of the cells.The number of animals on this model was 8 for each compound, control, and reference drug.The studied compounds were administered to rats in the most effective dose of 50 mg/kg intraperitoneally as a suspension with methylcarboxycellulose with Tween-80.Elevated plus maze -EPM test.Anti-anxiety and sedative effects were studied on a model of the ″elevated plus maze″ in mice. 27The labyrinth is a cruciform machine raised above the floor, having a pair of open and closed sleeves opposed to each other.Normal animals prefer to spend most of their time in the closed (dark) sleeves of the labyrinth.The anxiolytic effect of the compounds was estimated by the increase in the number of entries into open (light) sleeves and the time spent in, without increasing the total motor activity.This record the time spent in the closed sleeve, the number of attempts to enter the installation center.In the above model, the test compounds and the reference drug were injected intraperitoneally before the experiments.The control animals were administered an emulsifier.Results were processed statistically (P ≤ 0.05).Forced swimming test.To assess ″despair and depression″ the model ″compelling swimming″ 31 was used.Experimental animals were forced to swim in a glass container (height 22 cm, diameter 14 cm), filled 1/3 with water.Intact mice swim very actively, but soon they will be forced to immobilize.The latent period of immobilization, the total duration of active swimming, immobilization is fixed for 6 minutes.The experiments were conducted under natural light.Evaluation of incoordination of movements in the rotating rod test.Adverse neurotoxic (muscle relaxant) effect of compounds was studied in doses of 50 to 100 mg/kg when administered intraperitoneally, as well as reference drugs in effective anticonvulsant doses.Miorelaxation was investigated by the test of a ″rotating rod″ in mice. 15To this end, mice were planted on a metal rod with a corrugated rubber coating, which rotated at a speed of 5 revolutions per minute.The number of animals that cannot stay on it for 2 minutes was determined.To determine the ED50 and neurotoxic TD50, the statistical method penetration by Litchfield and Wilcoxon was used. 22,23 aximal tolerated doses (MTD) are also studied.The compounds by i.p. injection in doses from 500-1800 mg/kg were investigated.
the biological experiments were carried out in full compliance with the European Convention for the Protection of Vertebrate.All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of "(ETS No 123, Strasbourg, 03/18/1986): Strasbourg (France).European Treaty Series -No 123, March 18, 1986.11 P" University and Experiments were approved by the Animal Ethics Committee of Yerevan State Medical University after Mkhitar Heratsi (№7-5 from 18.03.2021).