Synthesis and in vivo evaluation of pyrazoline-thiazolidin-4-one hybrid Les-5581 as a potential non-steroidal anti-inflammatory agent

© 2019 S. М. Holota et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited


Introduction
Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most frequently prescribed drugs in modern medicine and represent diverse classes of chemical compounds [1]. Usually NSAIDs are taken systematically, for a long time and in the large doses. NSAIDs are characterized by different types of undesirable effects such as gastrointestinal (GI) and cardiovascular risks, hepatoand nephrotoxicity, negative impact on central nervous and blood systems etc [2]. The molecular mechanisms underlying this toxicity have not yet been fully elucidated but it is known that the oxidative stress (OS) and reactive oxygen species (ROS) are key factors which play an important role in the pathogenesis of "NSAIDs-gastropathy" [3], "NSAIDinduced hepatotoxicity" [4,5], etc. Thus, presen ce antioxidant properties are a desirable and useful characteristic for potential NSAIDs to overcome side effects [6].
New small molecules as modern anti-inflammatory agents with synergic action and minimizing toxicity were developed using molecular hybridization and multiple-ligand drugs techniques [7,8]. The combination of anti-oxidative and anti-inflammatory scaffolds in one molecule was shown to be a promising strategy for the search, further optimization and development of new hits/leads/candidates as potent and safe NSAIDs [8,9]. The study of hybridization of potential scaffolds with both antioxidant and anti-inflammatory properties is of particular interest.
Our experience in the search for potential pharmacological agents confirms the feasibility of synthesis and study of the properties of hybrid molecules with thiazlodin-4-one and pyrazoline moieties [18,19].
In the present work we describe synthesis and study of pyrazoline-thiazolidin-4-one hybrid Les-5581 -its acute toxicity, anti-inflammatory activity on the carragenin-and formaline-induced models, as well as influence on blood pattern, liver function and ulcerogenic impact under the treatment conditions. This work is a part of our research devoted to the search for anti-inflammatory agents and study on the inflammatory processes of different genesis [20][21][22][23].
The Animals. The experiment was performed on white male rats weighing 180-220 g and white mice of both sexes weighing 20-27 g. All the animals used for this study were kept in standard cages and maintained under controlled laboratory conditions of temperature (22±3°C), humidity and 12 hour day-12 hour night and had free access to food (standard pellet diet) and water ad libitum. The animals were treated humanely throughout the study period adhering to the guideline for use and care of animals in declaration of Helsinki (National Research Council, 2011). The ex-periment design and study protocol were approved by the Animal Ethics Committee of the Danylo Halytsky Lviv National Medical University, protocol No. 19, September 17, 2018.
Acute toxicity. The acute toxicity studying was performed to assess the prospects of the synthesized compounds as biologically active substances. The LD 50 value was determined by the method of Litchfield and Wilcoxon [25]. Test compounds were dissolved in tween-80 and purified water and administered intraperitoneally. The observation of the animals was performed for 14 days.
Anti-inflammatory (antiexudative) assay. The 54 male albino rats weighing 180-220 g were used for studying antiexudative activity. The selected animals were randomly divided into groups of six in each. The carrageenaninduced hind paw oedema was produced by the method of Winter et al. [26]. Carrageenan solution (1.0 % in sterile 0.9 % NaCl) was injected subcutaneosly into the subplanar region of the hind paw (0.1 mL to each paw) 1h after administration of the test compound. The synthesized compound was intraperitoneally injected in a dose 50 mg/kg (in saline solution with one drop of Tween-80™). Diclofenac (tablets "Diclofenac sodium", "Zdorovja narodu", Ukraine) in dose 8 mg/kg; Acetylsalicylic acid (tablets "Acetylsalicylic acid-Darnitsa", "Darnitsa", Ukraine) in dose 100 mg/kg, Ibuprofen (tablets "Ibuprofen-Darnitsa", "Darnitsa", Ukraine) in dose 50 mg/kg; Ketorolac (tablets "Ketanov", "Terapia SA", Romania) in dose 10 mg/kg were used as referen ce drug. Control rats received only saline solution with one drop of Tween-80™. The hind paw volume was measured with an elec-tronic onkograph immediately before and 4h after carrageenan injection. The formaldehydeinduced paw edema in rats was produced by the injecting of 0.1 ml 2 % formaldehyde solution into sub-plantar aponeurosis of the left hind limb one hour after the drug administration [27]. Paw volumes were measured 4h, 24 h, 72 h and 120 h after formaldehyde injection as described earlier. Diclofenac in dose of 8 mg/kg was used as a reference drug. The effect of test compounds on a decrease of paw oedema was compared with that in intact control. The antiexudative activity (АЕА) was expressed as a decrease of rats paw oedema, calculated using the equation and given in percentage: where ΔVcontrol and ΔVexperiment -the mean values of the volume difference for control and experimental animals hinds respectively. Hematological Assays. Sampling blood [from the lateral tail vein of the rats] into vacuum tubes with solution Na 2 EDTA was carried out for hematological studies in the appropriate experiment [28]. Level of hemoglobin and blood cells (erythrocytes, plates, leukocytes) was counted by automatic cell counter ABS-Micros 60-OT (Horiba Medical, Montpellier, France) in certified clinical laboratory of Danylo Halytsky Lviv National Medical University. White blood cell morphology was done (after staining by Romanowsky-Giemsa) by an experienced clinical laboratory specialist.
Assessment of liver function. The serum collected from the albino rats was used for estimation of biochemical parameters to determine the functional state of the liver. The levels of total alkaline phosphatase (ALP), gamma glutamyltransferase (γ-GGT), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were estimated photometrically according to the reported methods using CORMAY ACCENT-200 automatic analyzer (PZ Cormay, Poland).
Ulcerogenic activity estimation. The estimation of ulcerogenic activity was performed according to the recommendations [29]. All animals were sacrificed under deep anesthesia 6 hours after drug treatment, then their stomachs were removed, opened along the great curvature and rinsed with saline solution 0.9 %. The gastric mucosa was examined by means of a magnifying glass (2X) to assess the incidence of redness and spot ulcers. The mucosal damage was evaluated according to the following score: 0 -no visible damage; 1presence of edema or hemorrhages, 1-3 small ulcers; 2 -several (more than 3) small ulcers or 1 ulcer of considerable size; 3 -ulcer of considerable size (diameter up to 4 mm); 4several large ulcers; 5 -breakthrough ulcer. The gastric mucosal ulceration score was calculated by the difference between the mean score of each treated group and the mean score of control group.
Statistical Analysis. All data were processed using the statistical package Statistica 10.0 (Statsoft/Dell, Tulsa, OK, USA). The descriptive statistics of the data in tables include mean ± standard error of the mean (SEM) or mean ± standard deviation. Significance was assessed by using the one-way ANOVA followed by t-test. Values were considered statistically significant when P value is less than 0.05.

Results and discussion
The target hybrid Les-5581 has been synthesized in convenient and efficient aminolysis of 5-etoxymethylidenerhodanine (1) by 3,5-disubstituted-2-pyrazoline with satisfactory yield (74 %) and purity according to the scheme (Fig. 1): The acute toxicity (LD 50 value) of Les-5581 was studied by intraperitoneally administration in white mice. The approximate doses range from 300 mg/kg to 800 mg/kg was established based on series experiments. The LD 50 value for Les-5581 was determined to be 510 mg/kg. During the experiment with dose 510 mg/kg the animals were clean, active, had satisfactory appetite, responded to sound and light stimuli, the urinary and defecation processes and general behavioral parameters were normal, breathing disorder was noted. The animals deaths during the first day were observed in animal groups that received Les-5581 in a dose of more than 510 mg/kg.
Anti-inflammatory activity of Les-5581 was studied with dose 50 mg/kg, i.e. 0.1 LD 50 dose. The carrageenan-induced hind paw oedema as the acute process model was used for anti-inflammatory activity estimation at the first stage of experiment. The peak of pathology process was observed 4 hours after the inflammation induction. At this moment, the tested Les-5581 demonstrated a good level of the anti-inflammatory activity (40.2 % of inflammation inhibition), which was the same as with Ibuprofen, a little bit higher than with acetylsalicylic acid and ketanov, and only by 13.5 % less than the diclofenac sodium activity ( Table 1).
The formaldehyde test was used for the modeling of chronic inflammatory process. Les-5581 at dose of 50 mg/kg demonstrated good level of activity growing in dynamics over 5 days of experiment (Table 2). Noteworthy, a better anti-inflammatory acti vity (45.1 %) was observed on the 5 th day (120 h) compared to diclofenac sodium in these conditions. During the period from 4 h to 72 h the antiexudative activity of Les-5581 was a slightly lower than the diclofenac sodium effect.
So, Les-5581 displays promising anti-inflammatory activity in both acute and chronic inflammatory process models.
Hematological profile and impact on blood system of Les-5581 have been estimated in the end of experiment for both inflammation models in the blood test (Tables 3-5). No influence on blood pattern was observed.
An insignificant increase in the plate level, moderate an increase in the erythrocyte sedimentation rate and strong neutrophilia and  ESR -Erythrocyte sedimentation rate, * -p < 0.05; #p < 0.001 compared with intact control group lymphocytopenia were observed in experimental animals 4 hours after administration of phlogogenic agents. The application of Les-5581 and diclofenac sodium had a positive effect on the blood parameters normalization. The values of the parameters in the groups with treatment by Les-5581 and diclofenac sodium on the 5th day of the experiment reached the level of intact animals on the for maldehydeinduced paw oedema model. At the same time, in the "pathology model" group, the corresponding indicators on the 5 th day reached only the 4-hour level in the treated animals. A significant increase of liver enzymes levels was observed in the formaldehyde-induced paw oedema conditions for untreated animals ( Table 6). If compared with the intact control, in this group the enzymes levels were higher as follows: ALT 1.8-fold, AST 1.9-fold, ALP 1.7-fold, γ-GGT 1.5-fold.  At the same time, the administration of tested Les-5581 and reference drug does not show any negative impact and results in return from the biochemical changes to a normal level.
Les-5581 was evaluated for the ulcerogenic activity after repeated apllication in dose 50 mg/ kg in rats in the formaldehyde-induced paw oedema test. The results were compared with the intact control group and diclofenac sodium group ( Table 7). The second group showed significant ulcerogenic risk with a high ulceration score. The tested compound did not show any ulcerogenic activity.
So, the analysis of results suggests that Les-5581 is [a] good molecular platform for designing new potential anti-inflammatory agents with a low toxicity. The absence of ulcerogenic action and no influence on general blood parameters allow assumption that in the realization of anti-inflammatory effect of Les-5581 the inhibition of COX-2 or LOX-5 plays a key role rather than the COX-1 inhibition. Probably, the presence of half-saturated pyrazoline ring in the Les-5581 structure might have important impact on the compound redox properties. Therefore, the hybrid Les-5581 can reduce the "oxidative stress pathways", which occur under the inflammation process and are responsible for both anti-inflammatory effect realization and ulcerogenic action.

Conclusions
In the present study we report the anti-inflammatory activity evaluation and toxicity characterization of pyrazoline-thiazolidin-4-one hybrid (Les-5581) in vivo. Les-5581 demonstrated a good level of anti-inflammatory activity in the models of carrageenan-(acute pro-  cess) and formaldehyde-induced (chronic process) rat paw oedema; it was shown to be moderately toxic by intraperitoneally administration (LD 50 =510 mg/kg). The study of side effects indicates that use of Les-5581 does not provoke negative changes in blood test, liver enzymes function and does not manifest the ulcerogenic action. The studied pyrazolinethiazolidin-4-one hybrid is a good candidate for further investigations regarding its potential use in therapy of the inflammatory diseases.