The effect of 2-oxoindoline-3-glyoxylic acid derivatives on animals’ behavioral and autonomic reactions in the open field test

ABSTRACT Screening research on the effect of 2-oxoindoline-3-glyoxylic acid derivatives on the emotional and behavioral responses of rats in the open field test was performed. 2-oxoindoline derivatives were suspended in the injection water using the Tween-80 emulsifier (one drop with 25 mg of test substance) and intraperitoneally administered at a dose of 12 mg/kg 1 hour before the first test. Diazepam was administered in the dosage of 2 mg/kg and in the same way as the investigated compounds. Neuroethological indicators have been studied including the latent period of the first movement, ambulation (horizontal activity), number of upright postures (vertical activity), the number of times animals moved to the center, autonomic balance indicators such as the number of grooming acts and defecation acts calculated by the number of fecal balls (boluses). It was found that most 2-oxoindoline derivatives suppressed or did not affect animals’ orienting and investigatory responses in the open field test. Under these conditions, the compounds suppressed emotionality manifested as a reduced number of fecal balls and grooming acts indicating a decrease in anxiety and fear in a stressful situation caused by the novelty of the open field test. Based on the analysis, it can be argued that 2-oxoindoline derivatives can modify the psychophysiological features of intact rats, i.e. their neuropsychotropic properties.


Introduction
Mental health and well-being are important components of a high quality of life and its completeness [1].In the last decade, there has been a constant increase in mental pathology of the central nervous system (CNS) in almost all developed countries worldwide [2].Anxiety and fear states are leading components of neurotic, stress-related, and somatoform disorders.Pathogenetic links of these states, such as anxiety, fear, depression, and stress-related disturbances, have a close neurobiological basis [3].Clinical psychiatry and neurology require the development of new effective medications with a multicomponent spectrum of psychotropic action, as psychoemotional disorders and severe mental illnesses exhibit high heterogeneity and diverse clinical manifestations, although they are determined by various pathogenetic factors [4].
The open field is a classic test in which experimental animals demonstrate orienting, investigative, and protective autonomic behavioral responses.Animals' behavior changes when their body is exposed to extraordinary factors of different origin and biologically active substances.Based on these changes, it is possible to assess the specifics of the neuropsychotropic action of a particular factor or substance under study [5][6][7].
The chemistry of heterocyclic compounds has been one of the largest sources for developing new drugs in recent years.Developing drugs from this group enables researchers to synthesize a whole list of substances based on one specific compound, all of which have various physicochemical and biological properties capable of modifying the function of many different receptors [8].Oxoindole and its homologous structures are considered to be suitable substances for synthesizing new derivatives of the indole series.Studying oxoindoles is quite relevant as they show high physiological and pharmacological activity [9].New substances of this class include derivatives of 2-oxoindoline-3-glyoxylic acid with established antihypoxic, anxiolytic, antioxidant, and nootropic activity [1,10].Previous studies have shown that derivatives of 2-oxoindolin-3-glyoxylic acid are low-toxic [11,12].It has been demonstrated that derivatives of 2-oxoindolin-3-glyoxylic acid do not decrease the tone of skeletal muscles, and some compounds enhance animal endurance in the 'vertical screen' test, as well as exhibit hormone-like effects [13].This leads to studying the effect of 2-oxoindoline derivatives on animals' behavior as a part of a further search for neuropsychotropically active substances among those derivatives.
The open field test is a classic behavioral test that allows researchers to roughly assess hidden emotional tension in animals and the specific features of neurotropic action of newly studied compounds [14,15].
The study aims to conduct screening research on the effect of 2-oxoindoline-3-glyoxylic acid derivatives on rats' emotional and behavioral responses in the open field test.

Research materials and methods
One hundred and fifty adult male Wistar rats, weight 200-230 g, were used to perform the experiments.The rats have been kept in standard vivarium conditions with six animals per cage under 12-hour light for 2 weeks before the experiment.All experiments were performed in compliance with the Ukrainian legislation and the rules of the European Convention for the Protection of Vertebrate Animals applied in experimental research and for other scientific purposes.The study used 2-oxoindoline derivatives marked with the following symbols: 2 (2-Hydroxy-2-(2-oxo-1,2-dihydro-indol-3-ylidene)-N-phenethyl-acetamid), 18 (2-Hydroxy- The ex tempore compounds were suspended in the injection water using the Tween-80 emulsifier (one drop with 25 mg of test substance) and intraperitoneally administered at a dose of 12 mg/kg 1 hour before the first test.The control group of animals was injected with the same volume of solvent with the emulsifier.The reference drug was diazepam (Relanium injection solution in 2 ml (10 mg) ampoules branded as 'Polfa' by Tarchomin Pharmaceutical Works S.A., Poland) at a dose of 2 mg/kg, administered in the same way as the studied compounds.
The open field test was performed to assess the emotional and behavioral reactions of animals [6,16].A rectangular field of 140 × 70 cm divided into squares of 10 × 10 cm and illuminated by a lamp of 100 watts was applied.The front of the camera was transparent and was used to conduct observations.Neuroethological indicators have been studied including the latent period of the first movement, ambulation (horizontal activity), number of upright postures (vertical activity), the number of times animals moved to the center, autonomic balance indicators such as the number of grooming acts, and defecation acts calculated by the number of fecal balls (boluses).The results were processed with Microsoft Statistics 10.0 (StatSoft, Inc., USA) using ANOVA variance analysis [17].

Results
In the test, 'the open field of solvent and emulsifier (Twin-80)' (injection control) did not affect psychophysiological parameters such as the latency period of the first movement, the number of times animals moved to the center, and probably did not change the investigatory behavior of animals, i.e. the number of times animals crossed squares or rose upright on hind legs.This was also observed in autonomic reactions: the number of grooming acts and the number of boluses when compared to animals of the intact group (Table 1, Figure 1).
Introducing a 2-oxoindoline-3-glyoxylic acid derivative with laboratory code 2 did not significantly affect rats' behavioral and autonomic responses in the open field test.
Compound 18 modified the emotional and behavioral responses of animals.It reduced the number of upright postures by 4.8 times (p < 0.001) as well as the number of squares crossed by 2 times compared to the control animal group (p < 0.001).The number of animals entering the open field center probably decreased, but the latency period of the first movement probably did not change compared to the control under these conditions (Table 1).The effect of compound 18 on the autonomic function of rats was evaluated in addition to studying the motor activity of the experimental research reactions.It was found that in 3 hours the number of boluses decreased by 1.7 times compared with the introduction of solvent and emulsifier (p < 0,05) (Table 1).Compound 15 probably reduced the number of upright postures and the number of squares crossed compared to the injection control.The number of grooming acts decreased by 2.2 times compared to animals without correction (p < 0.001).
The introduction of 2-T suppressed rats' orienting and investigatory reactions which manifested as a decrease in the number of upright postures by 4.5 times (p < 0.001) and a decrease in the number of crossed squares by 2.2 times (p < 0.001) compared to a probable reduction in grooming in the injection control group.
Three hours after applying compound 1-F, the latency period of the first movement increased by 3.9 times (p < 0.001).At the same time, no animal from the group has moved to the center.Compound 1-F also significantly changed motor activity, as evidenced by a decrease in the number of crossed squares by 9.8 times (p < 0.001) and a probable decrease in the number of upright postures compared to that in the control (Table 1).It should be noted that the compound probably reduced the number of bolus balls and grooming acts compared to that in the group where saline was introduced.
Compound K probably suppressed all indicators of emotional and behavioral responses in the open field test except for the number of bolus balls.
It should be noted that the compound G increased only the number of times animals moved to the center of the open field.
The 2-oxoindoline derivative with laboratory code 3.85 increased the number of moves to the center by 2.7 times (p < 0.001) and probably reduced the emotional performance of test animals (Table 1).Compound 38 probably reduced the number of upright postures and grooming acts compared to the injection control (Table 1).
Compound M administration also changed animals' performance in the open field test, which was shown by a decrease in the number of upright postures by 1.9 times (p < 0.001), an increase in the number of bolus balls by 2.2 times (p < 0.001) and a probable decrease in grooming compared to animals who were not injected with compounds.Similar changes were observed when using the IK compound.
Three hours after administrating HIP-1, the number of crossed squares probably decreased, the number of bolus balls increased 1.5-fold (p < 0.05) and the number of grooming acts significantly decreased compared to injection control (Table 1).
Using diazepam as the reference drug prolonged the latency period of the first movement by 3.0 times (p < 0.001) and significantly reduced the number of upright postures compared to animals injected with saline.The effect of diazepam increased the number of times animals moved to the center of the open field by 2.2 times (p < 0.01) and decreased the number of grooming acts by 2.2 times (p < 0.001) compared with the injection control (Table 1).
The evaluation of the integrated indicator of total activity showed that compound 18 reduced this indicator by 1.8 times (p < 0.01) compared to the control injection.Introduction of compound 15 decreased the total activity by 1.5 times (p < 0.02), and compound 2-T decreased it by 2.2 times (p < 0.001) compared to the corresponding indicator in the control injection group.The most significant suppression of this indicator was observed with compounds 1-F and K, which reduced it by 6.4 times (p < 0.001) and 3.3 times (p < 0.001), respectively, compared to the values in the control group.The compound Gip-1 reduced the integrated indicator of all activities by 1.5 times (p < 0.02), and the reference drug diazepam reduced it by 1.4 times compared to the control, although these data were not statistically significant (Figure 2).

Discussion
Thus, in the open field test, 2-oxoindoline-3-glyoxylic acid derivatives marked as 1-F and K were highly likely to significantly extend the latency period of the first movement.Similarly to the reference drug, compounds 18, 15, 2-T, 1-F, K, 38, M, IK, HIP-1 reduced the number of times animals stood upright.Compounds 18, 1-F, and K reduced the number of moves to the center of the field, while compounds G, 3.85, M, IK, and diazepam had the opposite effect.2-oxoindoline derivatives reduced the number of crossed squares, such as compounds 18, 15, 2-T, 1-F, K, and HIP-1.Compared to this, compounds 18, 1-F, and 3.85 decreased, and compounds M and HIP-1 increased the number of bolus balls.Compounds 15, 2-T, 1-F, K, 3.85, 38, M, IK, HIP-1, and diazepam reduced the acts of grooming.The integrated indicator of all activities was reduced by compounds 18, 15, 2-T, 1-F, and K. Сompounds K and 1-F suppressed the activity the most, indicating their significant inhibitory effect on the central nervous system (CNS).
It was found that most 2-oxoindoline derivatives suppressed or did not affect animals' orienting and investigatory responses in the open field test.Under these conditions, the suppressed emotionality manifested as a reduced number of fecal balls and grooming acts indicates a decrease in anxiety and fear in a stressful situation caused by the novelty of the open field test [18].
Based on the analysis, it can be argued that 2-oxoindoline derivatives can modify the psychophysiological features of intact rats through their neuropsychotropic properties.Similar changes in emotional and behavioral responses in the open field test were found in a screening study of psychotropic properties of other new compounds that are actively studied and indicate the presence of moderate sedative and anxiolytic activity [1].It is known that anxiety is heterogeneous, and drugs can have different effects depending on the dose and the specifics of the behavioral test [18].Also, several tests need to be performed on the same animals to determine the individual level of anxiety or stressor resistance [10,19].
When comparing the features of emotional and behavioral reactions in the 'open field' test, the observed neurotropic activity may be associated with the peculiarities of the chemical structure of compounds.At the same time, the nature and pronounced character of such activity depend on the presence of an amide or ester bond and a substituted radical in the molecule.The oxindole ring is also important in the realization of neurotropic action, as amino acid-containing compounds showed higher activity compared to analogous amino acids [9,11,12].Therefore, for derivatives of 2-oxoindolin-3-glyoxylic acid, neurotropic activity may follow a pattern and be related to the nuclear structure of the compounds, while the nature and pronounced character of such activity depend on the presence of an ester or amide bond and substituted radicals.Considering that the compounds are derivatives of indole and the results of computer prediction, it can be hypothesized that the observed effects are due to the influence on specific subtypes of serotonin receptors in the central nervous system (CNS) and/or modulation of the activity of other receptors and/or agonists.
Considering the obtained results, it can be suggested that the presence of sedative and anxiolytic activity in the compounds allows positioning them for potential use in the treatment of anxiety disorders, anxiety-neurotic states (emotional lability, memory impairments, decreased concentration and attention), restlessness, fear, obsessive states, as well as for the prevention of stress-related conditions.
Therefore, it is advisable to use several various behavioral tests of different aversiveness for a more detailed study of the spectrum of neuropsychotropic action of 2-oxoindoline-3-glyoxylic acid derivatives.This is planned to be carried out in our upcoming research to further refine the direction of influence of the most active compounds.

Novelty
A number of promising compounds can be identified as a result of the conducted research, including compounds 3,85, 18, 1-F which significantly reduce the feeling of fear and increase novelty-seeking behavior, which may indicate potential anxiolytic properties that could be promising in terms of potential selectivity in the absence of impairments in motor and cognitive function.A similar interpretation of research findings is considered conventional and widely used in the study of new biologically active substances and toxicological research [16,20].

Conclusion
(1) 2-oxoindoline-3-glyoxylic acid derivatives administered at a dose of 12 mg/ kg change the behavioral and autonomic responses in intact rats.(2) Compounds 3, 85, 18, 1-F reduce feelings of anxiety and fear in a stressful situation associated with novelty.
The most active compounds may be promising for further study aimed at correcting anxiety disorders.

Table 1 .
The effect of 2-oxoinoline derivatives 3 hours after administration on the emotional and behavioral responses of rats in the open field test.