Study of Biological Activity and Toxicity of Thiosemicarbazides Carbohydrate Derivatives by in Silico, in Vitro and in Vivo Methods

Computer analysis of N-(β-D-galactopyranosyl)-thiosemicarbazide compounds by in silico method revealed high probability of antibacterial (antimycobacterial), anti-tuberculosis (antituberculosic), antiviral (Influenza), antitumor (antineoplastic) 9 > Pa > 0.5 and with a low probability of cytotoxic/cytostatic (cytostatic/cytotoxic) activities. An experimental study by in vitro and in vivo methods allowed us to conclude that studied new synthetic compound N-(β-D-galactopyranosyl)-thiosemicarbazide in the studied concentrations has a pronounced bactericidal and bacteriostatic effects.


Introduction
In recent years, computer programs have been widely used to predict biological activity and toxic effects of organic compounds. Among the programs that can be used to predict various types of biological activity is the PASS program developed by the V.N. Orekhovich RAMS [1].
The use of the PASS program makes it possible, among a wide group of analyzed compounds, to select those that with a high degree of probability have the required types of biological activity and, at the same time, with a low degree of probability, give undesirable toxic effects. When choosing promising compounds, not only main, but also side pharmacological effects were considered.
The fundamental problem of the relationship between the biological activity and the structure of chemical compounds and the search on this basis for new highly active medicinal substances is of fundamental importance for modern pharmacology. For this purpose, we have developed methods for the preparation of carbohydrate derivatives of thiosemicarbazides using the Lawesson reagent [2].
Molecule structure have an important role in the pharmacological activity of thiosemicarbazide derivatives [3], compounds having a pyridine ring and a thiosemicarbazide system as a well-known carrier antituberculosis agent with biological action were synthesized [4].

Research Methods and Materials
In this article, study of biological activity and toxicity of thiosemicarbazides carbohydrate derivatives was conducetd by in silico, in vitro and in vivo methods based on our previous works [5] [6]. Acute toxicity of N-(β-D-galactopyranosyl)-thiosemicarbazide was tested in the Biotechnology and Chemistry, Bacteriology Departments of Kyrgyz Republican Center for Diagnostics and Expertise.

Possibility Assessment in Using the PASS Сomputer System for Biological Activity
The results of assessing the possibility of using the PASS computer system to predict biological activity by structural formula for chemical compounds as a stage in pre-experimental screening of new substances are presented below. Table 1 shows the results of predicting 14 types of biological activity in the form of values of the probability of presence (Pa) and the probability of absence of this activity (Pi).
The results of computer predictions, we found that compounds 1.2 are predicted to have increased side effects, the ability to cause hyperuricemia an increased level of uric acid in the blood. Among the studied compounds, promising substances for experimental research identified with a high probability of antibacterial activity in N-(β-D-galactopyranosyl)-thiosemicarbazide compounds (Pa/Pi 0.799/0.004 antimycobacterial).

Studies on Acute Toxicity of N-(β-D-Galactopyranosyl)-Thiosemicarbazide
The acute toxicity of N-(β-D-galactopyranosyl)-thiosemicarbazide was tested in the Biotechnology and Chemistry, Bacteriology Departments of Kyrgyz Republican Center for Diagnostics and Expertise. Study aim of acute toxicity was to determine tolerable, toxic and lethal doses of a pharmacological substance and cause of animals' death [6]. When studying the toxicology of newly synthesized compounds, the first prerequisite is to determine the parameters of acute toxicity. These indicators are necessary to establish the degree of hazard of a chemical, as well as, for further research, where knowledge of the degree of acute toxicity is required.
Acute toxicity of pharmacological substances [7] [8] is determined by the following parameters: LD 0 is the maximum tolerated dose, LD 50 is an average lethal dose, LD 100 is minimum lethal dose [9]. LD 16 and LD 84 are also determined to establish the confidence limits of LD 50 the average lethal dose.
There are a number of classifications of chemical substances for the assessment of acute toxicity [10] [11] [12]. To assess the toxicity of antiparasitic drugs, the classification according to [12] [13] is more suitable. Based on the foregoing, Journal of Agricultural Chemistry and Environment The experiments were carried out on 36 clinically healthy white mice of both sexes with a live weight 18 to 22 grams. The substance was administered to animals orally in the form of a 10% solution using a syringe equipped with a special metal probe, in various doses. Control animals received an appropriate volume of sodium chloride saline solution.
During the experiment, the animals were not limited to feeding and watering. The experiments lasted 12 days, during which the general condition, the nature and degree of chemical toxicity, the time of death of the experimental and con-trol mice were observed. The corpses of the dead experimental animals were subjected to a visual pathoanatomical autopsy to establish the degree and nature of organ damage and the causes of death.
Statistical processing of digital materials was carried out by the method [14], modified [15], using ordinary graph paper [16].
The results of the experiments showed that the nature and degree of chemical toxicity in white mice were in direct proportion to the doses of the studied compound. Signs of poisoning when giving large doses appeared for the first time within minutes and were often fatal within 1 to 2 hours after giving. They were mainly expressed in the manifestation of an excited state (anxiety, increased reaction to external stimuli, tachycardia, rapid breathing), from feed tons of water.
Then this condition passed on to progressive oppression, leading to complete prostration. Breathing is shallow, frequent, and intermittent. Rapid palpitations, sometimes turning into arrhythmias. The death of animals that received large toxic doses of the compound occurred mainly on the first day of administration of the substance. The surviving animals showed mild diarrhea and poor appetite, which soon subsided.
The postmortem autopsy of dead white mice revealed: the back of corpses is soiled with liquid feces; the brain is swollen, hyperemic; the mucous membrane of the stomach and intestines is strongly hyperemic, in some places there are areas of extensive hemorrhage and necrosis; the liver is enlarged, dark red in color, its parenchyma is softened; kidneys of normal size, multiple punctate hemorrhages under the membrane; the heart is flabby, the myocardium is soft, the ventricles contain dark red blood clots, there are punctate hemorrhages on the epicardium and endocardium; the lungs are swollen of a dark red color, the blood vessels are dilated.

Results
Acute toxicity parameters, mg/kg Extinct Survived The results obtained indicate that, according to the current classification of the hazard of chemotherapeutic drugs according to the degree of impact on the body (GOST 12.1.007-76), N-(β-D-galactopyranosyl)-thiosemicarbazide belongs to substances of III class of moderate hazard [17] confirms the prospects for further study of this compound in this direction.

Study of Antibacterial Activity of N-(β-D-Galactopyranosyl)-Thiosemicarbazide
The experiments were carried out at the Department of Biotechnology and Chemistry and at the Department of Bacteriology of the Kyrgyz Republican Center for Diagnostics and Expertise by the in vitro method using generally accepted bacteriology techniques [18] [19] [20] [21] [22]. For this, by means of serial dilutions with distilled water (1:10; 1:20; 1:40 … 1:2650), various concentrations of the substance were prepared, the bactericidal effect of which was studied by plating Salmonella infection of lambs on agaragar in Petri dishes (Salmonellatyphimurium, 04), salmonellosis of calves (Salmonella Dublin, 09) and colipathogenic serotypes of Escherichia coli (Escherichiacoli 055, 026) and the addition of 0.1 ml of each dilution of the substance.
The results were taken into account after daily cultivation in a thermostat at t = 37˚C, by measuring the diameters of the zones (in mm), and by the absence of microorganism growth at the place where the compound was applied. Distilled water (i.e., solvent) served as a control.
The results of experiments carried out on a solid nutrient medium show ( Table 4) that the test compound showed bactericidal activity against the selected microbial cultures, although no pronounced species specificity was observed in its action.  The bacteriostatic activity of the substance was studied by diluting it in mesopatamia broth at the same concentrations as in the previous experiment, followed by sowing pure cultures in it. The results of this series of experiments showed ( Table 5) that the bacteriostatic substance acts on Esch. coli 055 from a dilution of 1:380, for other cultures from a dilution of 1:640.

Conclusion
Experiment results after analyses allow concluding, that studied new synthetic compound N-(β-D-galactopyranosyl)-thiosemicarbazide in studied concentrations has a pronounced bactericidal and bacteriostatic effects. Thus, it was proved that experimental study of biological activity coincided with the prediction data, which is an average accuracy of computer prediction with sliding control about 90%.