The novel pyrazolin-5-one bearing thiazolidin-4-ones: synthesis, characterization and biological evaluation

© 2021 S. M. 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 UDC 616–093 + 547.789


Introduction
The pyrazolin-5-one scaffold is one of the oldest in medicinal chemistry and at the same time it does not lose its relevance and attractiveness for researchers. It could be aptly demonstrated by the examples of two worldknown drugs Antypyrine (Phenazone) [1] and Radicava (Edavaron) [2] (Fig. 1), which belong to the simplest pyrazolin-5-one derivatives but were introduced in pharmaceutical market with a difference of almost 120 years and represent different pharmacological groups. Nowadays pyrazolin-5-ones possess important place as a privilege heterocycles [3,4]. The pharmacological profile of these compounds is very wide, they are targeted to the treatment and improvement of many difficult pathologies and diseases. The novel molecules with pyrazolin-5-one/antipyryl-4 pharmacophore have been designed, synthesized and successfully studied as selective inhibitors to plausible anti-cancers targets such as vascular endothelial growth factor receptor 2 (VEGFR-2) [5,6]; c-mesenchymal epithelial transition factor (c-Met) [7,8]; Sirtuins (SIRTs) [9]; they display high and selective cytotoxic activity in the in vitro models [10]. Noteworthy, some pyrazolin-5-one/antipyr-4-yl bearing compounds are promising as lipid-lowering [11] and antihyperglycemic [12] agents.
The anti-inflammatory action/activity still remains an actual trend in the design of potential non-steroidal anti-inflammatory drugs (NSAIDs) using pyrazoline-5-one/antipyr-4-yl scaffold for more than 100 years of synthetic work. Nowadays the fragment/structure-based drug design (F/SBDD) [13] and hybrid-pharmocophore approach (HPhA) are useful tools for design of new drug-like molecules in this area of medicinal chemistry [14]. So, the pyrazolin-5-one bearing thiazolidin-4-one hybrids with promising anti-inflammatory (anti-exudative) activity in carrageenan/formaldehyde-induced inflammatory paw edema models of white rats with low-toxicity parameters have been synthesized and reported [15][16][17][18] (Fig. 1) using F/SBDD and/ or HPhA metho do logies.
The oxidative stress [19][20][21] and redox [22,23] processes play important role under inflammation condition. The ability of neutralization of the impacts of reactive element species (Oxygen, Nitrogen, free radicals) is a desirable feature for modern NSAIDs [24,25]. That is why an of the chemical groups with anti-scavenging/anti-oxidant or redox properties into the molecular structure of potential NSAIDs seems an attractive strategy for design. Taking into account the above-mentioned reasons and accordingly to our permanent interest to issues of medicinal chemistry of azoles [26][27][28], here in we report synthetic approaches to novel 5-substituted thiazolidin-4-ones with 4-aminoantipyrine fragments in the C-2 position of heterocycle, their characterization and biological evaluation.

Materials and Methods
Commercial reagents were purchased from Merck and used without purification. Melting points were measured in open capillary tubes on a BŰCHI B-545 melting point apparatus and are uncorrected. The elemental analyses (C, H, N) were performed using the Perkin-Elmer 2400 CHN analyzer; the results were within ±0.4 % of the theoretical values. The 1 H-NMR spectra were recorded on a Varian Gemini spectrometer at 400 MHz using a mixture of DMSO-d 6 +CCl 4 as a solvent and TMS as an internal standard. Chemical shift values are reported in ppm units with use of δ scale. Mass spectra were obtained using electrospray ionization (ESI) techniques on Agilent 1100 Series LCMS (column SUPELCO Ascentis Express C18 2.7 μm, 4.6 mm×15 cm, solvent -methanol). Infra-red spectra were recorded on OMNIC-510 spectrometer in KBr pellets. The purity of the compounds was checked by thin-layer chromatography performed with Merck Silica Gel 60 F 254 aluminum sheets. Spots were detected by their absorption under UV light.
Anti-inflammatory (antiexudative) assay. The male albino rats weighing 180-220 g were used for studying the anti-exudative activity. 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 experiment design and study protocol were approved by the Animal Ethics Committee of the Danylo Halytsky Lviv National Medical University, protocol No. 4, January 23, 2020. The carrageenaninduced hind paw oedema was produced by the method of Winter et al. [31].
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.
Estimation of ulcerogenic activity. The estimation of ulcerogenic activity was performed according to recommendations [32].
The 1 H NMR, IR spectroscopies and LC-MS spectrometry were used for structure determination of the synthesized compounds. The 2-R-imino/amino-thiazolidin-4-ones possess prototropic amine/imine tautomerism [33]. In the 1 H NMR spectra of compounds 4-15 the signals of both tautomeric forms were observed. The signals of amine-forms protons of compounds were observed at 9.00-10.70 ppm and the signals of imine-forms protons were observed at 11.0-14.06 ppm. The ratio of tautomers based on the integral intensity curve ranges from ~3:1 to ~1:3 (amine-imine forms respectively). The correlation of the prevalence amine-or imine-form with electron nature of substituent in C-5 was observed. The presence of electron withdrawing group (NO 2 -group) leads increasing of imine-form whereas the electron releasing groups (OMe, OH) result in [the] prevalence of amine-form. The 1 H NMR spectral patterns of derivatives 5, 8, 10-12, 15 are more complex and the appearance of 3 or 4 groups of signals which belong to possible rotameric forms was observed (Fig. 2). The studies of IR-spectra of compounds 7, 8, 11, 13, 14 confirmed the presence of amine/imine tautomerism in the solid state.

Anticancer activity
The last decades have witnessed a growing interest in the studies of anticancer properties of NSAIDs and compound with anti-inflammatory activity [34,35]. The prescreening of anti-cancer activity of compounds 7, 11, 13 was performed according to the US NCI protocol, as described elsewhere [29,30]. These substances were evaluated in the 3-cell line panel consisting of NCI-H460 (Lung), MCF7 (Breast), and SF-268 (CNS) cell lines. As a result all tested compounds were found inactive and in the pre-screening phase (Table 1). Only compound 11 showed moderate growth inhibition activity (69 %) against Lung cancer cell line NCI-H460. Despite, that presented anti-cancer activity results indicate that only derivative 11 possesses low activity, but the presence of one, as well as the prevailing impact on NCI-H460 line (Lung cancer), give opportunities for perspectives of rational design of anticancer agents among such type of pyrazolin-5-one/ thiazolidin-4-one hybrids.
Thus, the compounds 9, 15, 18 do not lead to the appearance of negative changes in the key liver enzymes function as well as do not induce ulcerogenic action. Summerizing our studies on the search for potential anti-inflammatory agents among antipyryl-4 based thiazolidin-4-ones we have observed the next SAR (Fig. 3): a) the hybrids with antipyryl-4 fragment linked in N-3 position of thiazolidin-4-one nucleus are optimal and possess anti-exudative activity higher than the relevant reference drugs; b) cinnamic aldehydes and p-chloro-benzaldehyde moieties in C-5 position of thiazolidin-4-one are pivotal and optimal for their activity; c) 2-thioxoderivatives of 3-(antipyryl-4)-thiazolidin-4-ones demonstrate a higher activity level compare[d] to 2-oxoanalogs; d) replacement of the aromatic aldehyde fragments at C-5 position by acetic acid or R-phenylacetamides residues provoke attenuation of the anti-exudative effect; e) introduction of the second pyrazolone fragment linked via imine/amine linker as well as it replacement from position 3 to 2 in the position 2 of thiazolidin-4-one does not enhance the anti-exudative effect; f) introduction of allyl group in N-3 position does not cause a positive impact on the effect.
The mechanism of anti-exudative action for 5-substituted antipyryl-4 based thiazolidin-4-ones (Fig. 3) seems very attractive for study due to the presence of set potential pharmacophore fragments in their molecules. It is known that pyrazolones act by the inhibition of both isoforms of cyclooxygenase (COX-1, COX-2, and COX-3) enzymes involved in the prostaglandin (PG) synthesis [4,36]. The inhibition of 5-lipoxygenase (5-LOX), COX-2 and phospholipases A2 (PLA2s) has been reported for 5-ene-thiazolid-4-one scaffold [37]. Nowadays the modulation of nuclear factor kappa-lightchain-enhancer of the activated B cells (NF-kB) signaling pathway is considered as the potential target for 5-ene-thiazolid-4-ones and as a promising molecular mechanism for antiinflammatory effect [38]. Also, it is also important and worth to note that the presence of cinnamic aldehydes residues resulting in the best activity level for 15 (as well as for previously reported analogues [15][16][17][18]) is a com-mon structure feature with known pharmacological agent Epalrestat (ONO-2235), which was originally developed as the aldose reductase (ALR) inhibitor and used for the treatment of diabetic neuropathy. According to modern insights the inhibition of aldose reductase is one of the prospective strategies for design of potential anti-inflammatory agents [39]. Thus, for the all above-mentioned reasons the multitarget anti-inflammatory action might be ex-  pected for this class of molecular hybrids and, undoubtedly, more comprehensive additional research is required.

Conclusions
In this paper the synthetic approaches to series of novel 2-imine/amine-(antipyryl-4)-thiazolidin-4-ones were proposed. The structure and tautomerism of synthesized compounds were confirmed and studied using LC-MS and 1 H NMR, IR spectra. The selected compounds were evaluated for their anticancer and antiinflammatory activities. The lead-compounds were identified, which were equal to the reference drug Diclofenac sodium by the anti-inflammatory activity but had neither negative impact on the key liver enzymes function nor ulcerogenic action. The SARs were formed and possible mechanisms of action were discussed. The obtained results are promising for the future design and synthesis of new pyrazolin-5-on/thiazolidin-4-one hybrids as the potential mo le cules with promising pharmacological pro perties.

Funding
This work was partially supported by COST Action NutRedOx-CA161112 "Personalized Nutrition in ageing society: redox control of major aged-related diseases" (for Holota S.) and Ministry of Healthcare of Ukraine (project 0121U100690).