Synthesis, structure, and biological activity of 4-hetaryl-2-pyrrolidones containing a pyrazole ring

Single diastereomers of 4-hetaryl-2-pyrrolidone-3(5)-carbo- and 2-[4-hetaryl-2-pyrrolidon-1-yl]acetohydrazides were used in reactions with 2,4-pentanedione, providing (3R*,4S*)-3-, (4R*,5R*)-5-(3,5-dimethyl-1H-pyrazole-1-carbonyl)- and 1-[2-(3,5-dimethyl-1H-pyrazol-1-yl)-2-oxoethyl]-4-hetaryl-2-pyrrolidones. The structures of the synthesized compounds were confirmed by spectral methods and X-ray structural analysis. Some of the obtained compounds were shown to possess nootropic and anxiolytic activity. Supplementary Information The online version contains supplementary material available at 10.1007/s10593-022-03140-4.

The examples of currently approved synthetic drugs containing pyrazole rings include the anti-inflammatory drug celecoxib, the weight loss medication rimonabant, as well as fomepizole (an antidote against toxic alcohols) and others 4,6,7 (Fig. 1).
Thus, functionalized 2-pyrrolidones containing in their molecules hetaryl substituents along with two pharmacophoric moieties -lactam and pyrazole heterocycles can be considered as key structures for targeted synthesis of pharmacological agents with various types of activity.
Taking into account the fact that the chiral centers in the diastereomerically pure carbohydrazides 1a-e and 2a-g were not affected during their reactions with 2,4-pentanedione, the obtained compounds 5a-e had (3R*,4S*) configuration, while compounds 6a-g had (4R*,5R*) configuration. Indeed, the spin-spin coupling constants of 3,4-CH methine protons ( 3 J 3-4 = 10.7-11.2 Hz) in 1 Н NMR spectra of compounds 5a-e and 4,5-CH methine protons ( 3 J 4-5 = 8.2-8.7 Hz) in the spectra of compounds 6a-g indicated their trans and cis orientation relative to the lactam ring plane. This orientation corresponded to (3R*,4S*) and (4R*,5R*) configurations of the chiral centers and was in good agreement with the spin-spin coupling соnstants established by us for the starting carbohydrazides 1a-e, 2a-g, [20][21][22] as well as the literature data available for trans-and cis-isomers of similar molecules. [23][24][25] The formation of pyrazole ring was confirmed by the presence of characteristic =CH proton quartet signals in 1 Н NMR spectra of compounds 5a-e, 6a-g, and 7a-d in the region of 5.88-6.20 ppm. These quartets showed a long range spin-spin coupling ( 4 J = 0.5-0.7 Hz) to the pyrazole ring methyl group protons manifested as a doublet in the region of 1.98-2.44 ppm. The methylene and methine proton signals were assigned to the pyrrolidone and pyrazole rings of compounds 5a-e, 6a-g, and 7a-d according to the results of 1 H-13 C HMQC experiment.
The most downfield signals in 13 C NMR spectra of compounds 5a-e, 6a-g, and 7a-d were assigned to the carbonyl carbon atoms of lactam groups (172-177 ppm) and the side chain (169-171 ppm).
The spatial structure of the obtained compounds 5a-e, 6a-g, and 7a-d was examined by X-ray sructural analysis in the case of compounds 5-7 a (Figs. 3-7). According to the results of X-ray structural analysis, compounds 5-7 a crystallized in the form of racemates in centrosymmetric triclinic crystals.
The crystal structure of compound 5a contained a single molecule in the independent part of the unit cell, in which the pyrrolidone ring together with substituents at the С(3) and С(4) atoms were significantly disordered between two positions. The disordered atoms have been denoted with letters А and В. The relative configuration of the two chiral centers C(3) and С(4) in compound 5a was R* and S* (molecule А) or S* and R* (molecule В), respectively. Thus, the crystal of compound 5a can hold both enantiomers (RS/SR) or pair of diastereomers at the same position.
In the crystal structures of compounds 6a and 7a, the independent part of the unit cell contained two independent molecules of enantiomers А and В. For compound 6a, the chiral atoms С(4А) and С(5А) had S configuration, while C(4B) and C(5B) had R configuration. In the case of compound 7a, the chiral С(4А) atoms had R configuration and С(4В) had S configuration.
The molecules of compounds 5а, 6а in crystalline state formed centrosymmetric dimers linked by N-H···O hydrogen bonds. There were ordinary van der Waals contacts between the dimers (Fig. 6). In the crystal of compound 7а, С-Н···О type bonds were observed, mediated by dispersion interactions (Fig. 7).  Compounds 5a,c, 6a,b,d, 7a,c representing each group of the obtained products were tested as potentially biologically active compounds, anticipated to have psychotropic properties. Standard pharmacological testing methods were used, which were appropriate for the given type of activity: ''open field'' (OF), ''elevated plus maze'' (EPM), ''conditioned passive avoidance reflex'' (CPAr), ''extrapolation avoidance'' (EA), and Vogel conflict test (VCT). 26 The experiments were performed with male Wistar rats in accordance with the animal testing regulations set by the legislation of the Russian Federation and the EASC technical standards for Good Laboratory Practice (GOST R 53434-2009 and GOST R 51000. . Compounds 5a,c, 6a,b,d, 7a,c were injected into the animals intraperitoneally (IP, physiological saline) in equimolar amounts as a single dose equal to 1/10 of the molecular mass, 30 min prior to the test. The acute toxicity level (LD 50 , mg/kg) of compounds 5a,c, 6a,b,d, and 7a,c was evaluated from the survival rate of white mice and exceeded 2000 mg/kg, allowing to recognize them as relatively harmless materials, belonging to the IV class of toxicity. The selected reference drug was phenibut (20 mg/kg, IP), which possesses anxiolytic and nootropic activity and is prescribed for the treatment of anxiety, cognitive, and asthenic disorders. 7 Statistical processing of the test results was performed using the Prism 6 program according to Shapiro-Wilk, Kruskal-Wallis, and Dunn's criteria.
The OF test characterizes the locomotor activity (by the number of traversed squares) and orienting-exploratory activity of the animals (by the sum of rearing and headdipping acts). As shown in Table 1, compounds 5a,c, 6a,b,d, 7a,c did not produce a significant effect on the motor activity of animals in this test, pointing to the absence of psychostimulant or sedative activity associated with these compounds. It should be noted that, after the injection of compound 7c (32 mg/kg), the animals of experiment group showed a statistically significant increase in rearing and head-dipping behaviors, as well as the number of visits to the brightly lit central area (aversive for nocturnal rodents), pointing to the prevalence of orientingexploratory activity by the animals over the aversion toward their environment (Table 1). Thus, compound 7c in the OF test showed anxiolytic activity, comparable to that of phenibut.
The EPM test was used to evaluate the anxiety level in animals by recording the total time spent, time spent in the open arm, and the number of excursions into the open arm. After a single injection of compounds 5a,c, 6a,b,d, 7a,c, only compounds 6b and 7c statistically significantly increased the number and duration of excursions into the open arms of the apparatus, amount of rearing and headdipping behaviors in the open arms among the experiment group compared to the control group of animals. This indicated a reduction in the level of anxiety ( Table 1). The anxiolytic effect of compound 7c surpassed the activity of compound 6b and was comparable to that of phenibut.
The activity of the most promising compounds 6b, 7c was also studied according to the Vogel conflict test, which is a highly specific test for evaluating the anxiolytic action of new compounds. It was established from this test that, after the injection of compounds 6b, 7c, the number of animals in the experimental group punished by withdrawal of water was statistically significantly higher compared to the control group (Table 1). Remarkably, the anxiolytic activity of compound 6b exceeded that of compound 7c, the activity of which in the EPM test was higher than in the case of compound 6b and comparable to the activity of phenibut.
Within the framework of studying compounds 5a,c, 6a,b,d, and 7a,c with regard to their psychotropic activity, their effects on the learning and memory (formation and retrieval of memory trace) were also characterized using animals in CPAR and EA tests. During the CPAR test, the animals treated with compounds 5a,c, 7c prior to the training retained the memory trace and did not enter the * MA -motor activity (number of squares traversed in the experimental apparatus). ** EB -exploratory behavior (total number rearing and head-dipping acts). *** OA -the open arm of the EPM apparatus. * 4 The differences were statistically significant compared to the control group, at p < 0.05.   closed arm with electrode floor at 7 days after the training (Table 1). Along with that, the animals treated with compounds 5a,c solved the extrapolation task significantly faster during the EA test compared to the control group (Table 1). Thus, according to the results of both tests, from the series of compounds 5a,c, 6a,b,d, and 7a,c we selected compounds 5a,c that improved the formation and retrieval of memory trace in experimental animals and showed nootropic effects.

Compound
In conclusion, the results of this study allowed us to develop an effective method for the preparation of previously unknown diastereomerically pure 3(5)-(3,5-dimethyl-1H-pyrazole-1-carbonyl)-4-hetaryl-2-pyrrolidones and 4-aryl-1-[2-(3,5-dimethyl-1H-pyrazol-1-yl)-2-oxoethyl]-2-pyrrolidones -new members of the racetam family containing the pharmacophoric lactam and pyrazole rings in their molecules, along with hetaryl substituents. Some of these compounds were shown to possess nootropic and anxiolytic activity. The obtained data open further possibilities for more detailed study of these compounds as promising lead compounds in the drug development targeted toward the prevention and treatment of anxiety disorders.

Experimental
IR spectra were recorded on a Shimadzu IR Prestige-21 FT-IR spectrometer for samples in KBr pellets. Onedimensional 1 Н, 13 С, 15 N NMR spectra and twodimensional 1 H-13 C HMQC and 1 H-13 C HMBC spectra were acquired on a Jeol ECX400A spectrometer at the working frequencies of 400 MHz (for 1 H nuclei), 100 MHz (for 13 С nuclei), and 40 MHz (for 15 N nuclei) for samples in DMSO-d 6 solutions. The residual non-deuterated solvent signals (for 1 Н nuclei) or deuterated solvent signals (for 13 С nuclei) were used as standards, while 15 N NMR chemical shifts were recorded relative to MeNO 2 . Elemental analysis was performed on a EuroVector EA3000 (CHN Dual) analyzer. The reaction progress and purity of the obtained compounds were controlled by TLC on Silufol UV-254 plates using 9:1:2 i-PrOH-NH 4 OH-H 2 O mobile phase and visualization under UV light (λ = 254 nm).