Synthesis of (Z)-5-(substituted benzylidene)-2-((substituted phenyl) amino)thiazol-4(5H)-one analogues with antitubercular activity

ABSTRACT We initiated a program to synthesize thiazolidinone derivatives as antitubercular agent by preparing hybrid molecules having the similar features of reported potent antitubercular agents. We desire to state the advancement and execution of a methodology allowing for the synthesis of some new (Z)-5-(substituted benzylidene)-2-((substituted phenyl) amino)thiazol-4(5H)-one analogues with antitubercular activity. A highly efficient protocol was developed for the synthesis with excellent yields as well as evaluated in vitro for their antimycobacterial activity against Mycobacterium tuberculosis MTB H37Ra and M. bovis BCG strains. Among these synthesized compounds 6a, 6c, 6e, 6f and 6i showed marginal antitubercular activity in the series along with no significant cytotoxicity against the MCF-7 and A549 human cancer cell lines. GRAPHICAL ABSTRACT


Introduction
The thiazole nucleus is a crucial constituent for medicinal agents having anticancer, antimicrobial and antiinflammatory activities [1,2]. The thiazole ring system is seen in a wide range of drugs used as cardiotonic, fungicidal, for the treatment of HIV infection, mental retardation in children, age-related neurodegenerative brain damage (Alzheimer's disease), Parkinson's disease. Thiazole is considered as the key nucleus in the class of heterocyclic compounds. Biologically potent as well as dynamic molecules such as Sulfathiazol (antimicrobial drug), Ritonavir (antiretroviral drug), Abafungin (antifungal drug), Bleomycine and Tiazofurin (antineoplastic drug) contain thiazoles. Thiazole pharmacophore along the combination with other rings possibly will provide biologically active compounds [3,4]. Thiazoles also show anti-tuberculosis activity. Tuberculosis (TB) is caused by the pathogen Mycobacterium tuberculosis (MTB), which is most infectious cause of humanity universally. Even though effective anti-TB drugs, such as isoniazid and rifampicin, are accessible, problems emerge as MTB develops resistance not only against the first line but also with the second line drugs. Hence there is an imperative need to develop novel anti-TB agents, which are synthetically possible, have less adverse effects and shorter duration of treatment [5]. The thiazolidinone and its derivatives are central part of biologically active compounds with various applications and uses [6][7][8] such as antibacterial [9][10][11], antifungal [12][13][14], anticancer [15], antitubercular [16,17] and antimalarial [18]. For years many medicinal chemists are in search of biologically active alkaloids containing substituted amine ring systems for their whole or partial synthesis. As by considering the medicinal and biological importance of thiazolidinone moiety, we initiated a program to synthesize thiazolidi- none derivatives as an antitubercular agent by preparing hybrid molecules having the similar features of reported potent antitubercular agents [19] (Figure 1).

General
Rhodanine, substituted benzaldehyde, substituted amines, anhydrous sodium acetate, triethylamine, dichloromethane, iodomethane and various solvents were commercially available. The major chemicals were purchased from Sigma Aldrich and Avra labs. Reaction courses were monitored by TLC on silica gel precoated F254 Merck plates. Developed plates were examined with UV lamps (254 nm). All compounds were purified by column chromatography. IR spectra were recorded on an FT-IR (Bruker). Melting points were recorded on SRS optimelt, melting point apparatus and are uncorrected. The 1 H NMR spectra were recorded on a 400 MHz varian NMR spectrometer. The 13 C NMR spectra were recorded on a 400 MHz varian NMR spectrometer. The chemical shifts are reported as δ ppm units. The following abbreviations are used: singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m) and broad (br). HRMS spectra were taken with micromass-QUATTRO-II of WATER mass spectrometer. Elemental analysis was performed on a Perkin-Elmer EAL-240 elemental analyser.

General procedure for the synthesis of (Z)-5-substituted benzylidene-2thioxothiazolidin-4-one (3a-e)
In a 50-mL round bottom flask, the compound substituted 2-thioxothiazolidin-4-one 1 (1 mmol), benzaldehyde 2a-e (1 mmol), glacial acetic acid (1 mL), anhydrous sodium acetate (1 mmol) were added to the reaction mixture. The mixture was stirred under reflux condition up to 2 h. The progress of the reaction was monitored by TLC (10% methanol: chloroform). After completion of the reaction, the reaction mixture was poured into the ice-cold water. The precipitate was filtered off and washed with water (3×15 mL), dried and purified by recrystallization in ethanol as solvent to give 90-94% yield.

Biological activity
All the chemicals such as sodium salt XTT, DMSO, sulfanilic acid, sodium nitrate, HCl, NEED and rifampicin were purchased from Sigma-Aldrich, USA. Dubos medium was purchased from DIFCO, USA. Compounds were dissolved in DMSO (10 µg/mL) and it was used as stock solution for further antimycobacterial testing. Microbial strains such as MTB H37Ra (ATCC 25177) and M. bovis BCG (ATCC 35734) were obtained from AstraZeneca, India. The stock culture was maintained at −80°C and subcultured once in a liquid medium before inoculation into an experimental culture. Cultures were grown in Dubos media (enrichment media). Mycobacterium pheli medium (minimal essential medium) was used for antimycobacterial assay. It contains 0.5 g KH 2 PO 4 , 0.25 g trisodium citrate, 60 mg MgSO 4 , 0.5 g aspargine and 2 mL glycerol in distilled water (100 mL) followed by pH adjustment to 6.6. All the newly synthesized compounds were screened in vitro against two Mycobacterium species such as MTB H37Ra and M. bovis BCG. Both species of Mycobacterium were grown in Mycobacterium pheli medium. Screening of MTB H37Ra was done by using XTT reduction menadione assay (XRMA) and M. bovis BCG screening was done by using NR (nitrate reductase) assay, both of them were developed [30]. Briefly 2.5 mL of these inhibitor solutions were added in a total volume of 250 mL of Mycobacterium pheli medium consisting of bacilli. The incubation was terminated on the 8th day for active MTB culture. The XRMA and NR was then carried out to estimate viable cells present in different wells of the assay plate. The optical density was read on a micro plate reader (Spectramax plus384 plate reader, Molecular Devices Inc.) at 470 nm filter for XTT and at 540 nm filter for NR against a blank prepared from cell-free wells. Absorbance given by cells treated with the vehicle alone was taken as 100% cell growth. Initially primary screening was done at 30, 10 and 3 µg/mL. Compounds showing 90% inhibition of bacilli at or lower than 30 µg/mL were selected for further dose response curve. All experiments were performed in triplicates and the quantitative value was expressed as the average ± standard deviation. MIC 50 values of selected compound were calculated from their dose response curves by using Origin 6 software. % Inhibition was calculated by using following formula: % Inhibition = [(absorbance of compound − absorbance of test)/(absorbance of control − absorbance of Blank)] × 100, where control is the medium with bacilli along with vehicle and blank is cell free medium.
In view of chemistry and easily available solvent, it was decided to prefer methanol as solvent in our initial study for optimization of the solvent. During this study, the model reaction was performed using methanol as a reaction medium at room temperature.
The desired product 6a was obtained in 90%, taking only 5 h in methanol at room temperature ( Table 2, entry 6). Other solvents like ethanol give good yield   65% in 6 h ( Table 2, entry 1). Among these, the solvents like THF, DMF, DCM, toluene gave a very low yield at 20%, 30%, 40%, 25% respectively. Methanol as a solvent was selected for further reaction. We have also screened different types of bases, reaction time and yield of the product. The model reaction (Z)-5-benzylidene-2-(phenylamino)thiazol-4(5H)-one (6a) (Scheme 2, Table 2). All of these yields were generally low before further optimizations. To increase the efficiency of the reaction, the effects of different bases were investigated (Table 2, entries 7-10). The yield obtained with potassium carbonate as a base and methanol as solvent was high (90%). Sodium carbonate, triethylamine and dimethylamine gave lower yields with methanol solvents (40%, 25% and 35% respectively). All the reactions were carried out in equimolar amounts of each compound in 1 mL of solvent. From the above conclusion, we decided to carry out the reaction in methanol as a solvent and potassium carbonate as a base. This condition was worked as a standard protocol for the further reactions ( Table 3).

Cytotoxicity assay
These findings inspired us to evaluate synthesized analogues cytotoxicity. The cytotoxicity results are presented in Table 5, where many compounds exhibit less cytotoxicity activity compared to Adriamycin as positive control. The compounds 6a, 6c, 6d and 6i were the less toxic, with MIC 50 values ranging from 34.2 to 45.6 µM. On the A-549 cell line the compounds which showed lower toxicity were 6b, 6f, 6 g (MIC 50 = 23.6, 46.3, 46.6 µM, respectively).
The synthesized analogues 6a-j were assayed for their cytotoxic effects in two different cell lines, MCF-7 and A549 using MTT assay [32,33]. The cell lines were maintained under standard cell culture conditions under 5% CO 2 at 37°C in 95% air humidified environment. Each concentration was tested in duplicates in a single experiment. MIC 50 values were calculated using OriginPro Software.

Conclusion
An effective method was developed which provided an easy access to a new series (Z)-5-(substituted benzylidene)-2-((substituted phenyl) amino)thiazol-4 (5H)-one analogues. The mild reaction conditions, good to excellent yields, ease of workup and easily available substrates make the reactions attractive for the preparation of compounds 6a-j. In vitro antitubercular studies revealed that the compounds 6a, 6c, 6e, 6f and 6i showed marginal antitubercular activity in the series along with no cytotoxicity against the MCF-7 and A-549 human cancer cell lines.