NEW THIAZOLONE DERIVATIVES : DESIGN , SYNTHESIS , ANTICANCER AND ANTIMICROBIAL ACTIVITY

In order to explore the anticancer and antimicrobial activity associated with the thiazolone framework, several new (Z)-2-((5-(3fluorobenzylidene)-4-oxo-4,5-dihydrothiazol-2-yl)amino)carboxylic acid derivatives have been synthesized in water as a solvent. All synthesized compounds were evaluated for anticancer and antimicrobial activity in vitro. Amongst these, the 3-methylbutanoic and the 3or 4-methylpentanoic acid derivatives, the 3-hydroxy-, the 3-(1H-imidazol-4-yl) and the 3-(4-hydroxyphenyl)propanoic acid derivatives and the succinic acid derivative showed high antibacterial and antifungal activity. The unsubstituted propanoic acid derivative exhibited significant antibacterial activity against B. subtilis and significant antifungal activity against fungal strains, i.e., A. flavus. The in vitro anticancer studies revealed that the 3-(hydroxy)-, the 3-(1H-imidazol-4yl)and the 3-(4-hydroxyphenyl)propanoic acid, or the succinic acid derivatives are the most active compounds against MCF-7 and BT-474 human breast cancer cell lines.


INTRODUCTION
Since the last five decade very rapid progress has been made in the area of cancer cell biology, though most cancer treatments are still multimodal, involving the chemotherapy. 1Cancer is the second leading cause of death in the world after cardiovascular diseases and it is projected to begin the primary cause of death there within the coming year. 2,3The breast cancer may be one of the oldest known forms of cancerous tumors in humans.Worldwide, breast cancer is the most common cancer in women, after skin cancer, representing 16 % of all female cancers. 4e heterocyclic chemistry has great importance for the medicinal chemists due to the high therapeutic activity of heterocyclic compounds.The compounds containing the 2thioxothiazolidin-4-one (rhodanine) ring scaffold has been gaining prominence in recent years, because its derivatives are known to possess a broad spectrum of pharmacological activities, such as antimicrobial, [5][6][7][8][9] antidiabetic, 10 anticancer, [11][12][13][14] antiviral, 15,16 antifungal, 17 anticonvulsant, 18 anti-tuberculosis 19,20 and anti-HIV. 21,22The identification of new structures that can be potentially useful in designing new, potent selective and less toxic anticancer agent is still a significant challenge to medicinal chemistry researchers. 23,24he recent reports suggested that a chain containing free carboxyl group at the rhodanine nucleus had importance in the observed anticancer and antimicrobial activity. 25,26 initiated a program to synthesize thiazolone derivatives having amino acids chain as antimicrobial agents.

EXPERIMENTAL SECTION
The compounds 2-thioxothiazolidin-4-one, 3fluorobenzaldehyde, anhydrous sodium acetate, triethylamine, amino acids, dichloromethane, iodomethane and various solvents were commercially available (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).IR spectra were recorded on an FT-IR (Bruker).Melting points were recorded on SRS Optimelt, melting point apparatus and are uncorrected. 1H NMR spectra were recorded on a 400 MHz Bruker spectrometer and were recorded in DMSO-d6 solvent 13 C NMR spectra were recorded in DMSO-d6 solvent on a 100 MHz Bruker spectrometer.Chemical shifts are reported as δ ppm units (TMS).The following abbreviations are used; singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m) and broad (br).Mass spectra were taken with Micromass-QUATTRO-II of WATER mass spectrometer.

General procedure of (Z)-5-(3-fluorobenzylidene)-2-(methylthio)thiazol-4(5H)-one (4)
In a 100 ml round bottom flask, the compound (3) (1 mmol) and triethylamine (1.5 mmol) were added in dichloromethane (10 ml) at room temperature.Iodomethane (1.5 mmol) was added to the stirred reaction mixture and the mixture was stirred for 2 h at room temperature.The progress of the reaction was monitored by TLC (10 % chloroform: methanol).After completion of the reaction, the reaction mixture was concentrated in-vacuo.The residue was washed out with water (3×15 mL) to afford the crude product.The crude product was recrystallized using ethanol as solvent.
The antifungal study was carried by the standard agar dilution method and Fluconazole and Miconazole were used as control drugs.Ethanol was used as solvent control for both antibacterial and antifungal testing.
All the synthesized compounds were also tested for their general cytotoxicity on MCF-7 and BT-474 human breast cancer cell line.This test is performed as previously mentioned MTT colorimetric assay. 31Cytotoxicity of the compounds was determined by calculating their IC50 values (μM mL -1 ), the concentration of compound required to inhibit 50 % of cell growth compared to untreated control cells.The results are given as percentage cytotoxicity after 24 h.Adriamycin was used as positive control for the comparison of cytotoxicity of synthesized compounds.Assays were performed in triplicate on three independent experiments.

Isolated yields
The compound (3) was then subjected to a Knoevenagel condensation with the appropriate 2-thioxothiazolidin-4-one, to synthesize a new series of target compounds (6a-l).The structures of the desired compounds were confirmed by IR, 1 H NMR and 13 C NMR.The compound 3 was prepared in prominent good yields via a Knoevenagel condensation between the corresponding heterocyclic cores of Rhodanine and 3-fluorobenzaldehyde (Scheme 1).The 2-thioxothiazolidin-4-one based compounds were synthesized by conventional heating with sodium acetate, which acts as a base and glacial acetic acid as catalysts.In theory, of E and Z geometrical isomers may exist according to the configuration around the exocyclic double bond (CH=C) for (Z)-5-(3-fluorobenzylidene)-2-thioxothiazolidin-4-one (3).
The 1 H NMR spectrum of the compound 3 shows only one signal for the methine proton in the range δ 7.66, which show the presence of one isomer only, and at lower field values than those expected for the E-isomers, which was strongly indicated that the compounds have the Zconfiguration.The IR spectrum of compound 3, showed a strong absorption band at 1702 cm -1 that is due to a carbonyl group.The mass spectrum revealed a molecular ion peak at m/z=239.29 corresponding to a molecular formula C10H6FNOS2.The latter has been reported as thermodynamically more stable than the Econfiguration. 32,33mpound 4 was synthesized from compound 3 and the structures of the desired product were confirmed by IR, 1 H NMR, 13 C NMR and mass spectral analysis.The IR spectrum of (Z)-5-(3-fluorobenzylidene)-2-(methylthio)thiazol-4(5H)-one (4), showed a strong absorption band at 1704 cm -1 belongs to a carbonyl group.The mass spectrum revealed a molecular ion peak at m/z = 253.05corresponding to a molecular formula C11H8FNOS2.The 1 H NMR spectra of the compound 4 show only one signal for the methine proton in the range δ 7.82, sulfur attached methyl group proton shows a singlet at δ 2.85.
The IR spectrum of (Z)-2-((5-(3-fluorobenzylidene)-4oxo-4,5-dihydrothiazol-2-yl)-amino)propanoic acid (6a), showed a strong absorption band at 1742 cm -1 belongs to the carbonyl of a carboxylic group and 3397 cm -1 due to the presence hydroxyl group.The mass spectrum revealed a molecular ion peak at m/z=294.28 corresponding to a molecular formula C13H11FN2O3S.The 1 H NMR spectrum revealed that the signals of 6a (as a representative example), show the methyl group protons doublet in the range of δ 1.40-1.50, the methine proton (adjacent to carboxylic acid group) shows a quartet in the range of δ 4.50-4.70,phenyl ring protons show a multiplet in the range of δ 7.40-7.70,one signal appears for the alkene proton in the range δ 7.72, amine group proton shows singlet at δ 10.10, and the carboxylic acid proton shows singlet at δ 12.65.The antimicrobial activities of the synthesized compounds against selected Gram-positive and Gram-negative bacteria and multidrug-resistant bacteria are illustrated in Table 2 and Table 3.The majority of the synthesized compounds show a variety of antibacterial, antifungal and cytotoxic activity.The compounds 3 was found to be the most active against E. coli and the compounds 6c, 6g and 6k were found to be active against S. typhimurium.Some of the studied compounds are more potent against selected microorganisms than a standard antibacterial drug Ciprofloxacin.For example, against E. Coli the compound 3 while against B. subtilis the compounds 6b, 6e, 6f, and 6j have better MIC values than the values found for Ciprofloxacin.Against S. aureus, the compounds 6b, 6e, 6f,6i and 6j proved to be more active than Ciprofloxacin.
The compounds 6g and 6k showed antifungal activity against four fungus strains, namely A. oryzae, P. chrysogenum, C. albicans and A. flavus.The compound 6a showed activity against A. flavus, the compound 6c against P. Chrysogenum and F. oxysporum while the compound 6e was found to be active against P. chrysogenum.Remaining compounds of the series (3, 4, 6d, 6f, 6h, 6i, 6j and 6l were found to be inactive against fungi. All the synthesized compounds were tested for their cytotoxic activity against MCF-7 and BT-474 cell lines (Table 4).(Table 4).Three compounds, 6g, 6i and 6l showed good activity against the studied cancer cell lines, the IC50 values against MFC-7 and BT-474 cell lines were found to be 1.4 and 1.3, 1.5 and 0.6, or 1.1 and 1.4 µM mL -1 , respectively.The IC50 values for reference drug Adriamycin against MFC-7 and BT-474 cells were found to be 0.9 and 0.5 µM mL -1 , respectively.The cytotoxicity of the newly synthesized thiazolones depends on the type of substituents on thiazolone moiety.The compounds containing hydroxyl groups attached to the amino acid parts linked to the thiazolone ring have the highest cytotoxic activity.The presence of electron releasing alkyl chain, methyl-1Himidazole ring or thiol group on the amino acid attached to the thiazolone rings resulted in the loss of activity.

Structure-activity relationship (SAR)
The results of the antimicrobial screening demonstrated some facts about the structural-activity relationship (SAR) of the synthesized thiazolone derivatives.The notable highlights of structure-activity relationship are the followings: The biological activity profile of molecules is strongly affected by the branching pattern and chain length of alkyl moieties.Attachment of a methyl group at C2 position on the thiazolone moiety (6a) makes molecule active against bacterial and fungal strains, probably due to its small size and electron donating effects.When this methyl group is replaced by isopropyl (6b) and 2-methylbutyl (6c) groups, the molecules become active on a broader spectrum (against the majority of the studied strains).This shows that the presence of branching at the carbon located on the C2 position of the thiazolone ring has a positive effect on appearing or strengthening of antimicrobial activity.Attachment of 2-methylpropyl group (6f) at the same position makes the molecule to be specific towards the B. subtilis and S. aureus.Substitution of the alkane chain with a carboxylic group (6i), also resulted in specificity towards B. subtilis and S. aureus.Substitution by phenylmethyl group (6d) at the C2 position of the thiazolone moiety gave completely inactive molecule towards all tested strains, while the 4hydroxyphenyl (6k) substitution made the molecule to be active towards S. typhimurium, and P. chrysogenum, F. oxysporum, C. Albicans and A. flavus.The compound (6j) containing methyl-imidazole ring at the C2 position of the thiazolone moiety resulted in appearing of specific activity towards the Gram-positive bacteria B. subtilis and S. aureus.The compound (6e) with the terminal methylthio group is active toward.B. subtilis, S. aureus and P. chrysogenum, while compound (6h) with terminal mercapto group was proved to be inactive towards these bacterial and fungal strains.

CONCLUSION
The objective of our present study is to synthesize and investigation of the potent anticancer and antimicrobial activities of some new (Z)-2-((5-(3-fluorobenzylidene)-4oxo-4,5-dihydrothiazol-2-yl)amino) substituted organic acids.This is the first reported synthesis of the (Z)-2-((5-(3fluorobenzylidene)-4-oxo-4,5-dihydrothiazol-2-yl)amino) substituted organic acids in water as solvent with excellent yields in shorter reaction time making the process economically lucrative for industrial application.Some derivatives were found to be more active against several bacteria and fungi strains than the common antimicrobial agents.
In vitro anticancer studies revealed that the compounds 6g, 6i, 6k and 6l are most active against MCF-7 and BT-474 human breast cancer cell lines.
After completion of the reaction, the reaction mixture was poured into the ice-cold water.The precipitate was filtered, washed with water (3×10 mL), dried, and purified by recrystallization from ethanol as a solvent to give a 90 % yield.

Table 4 .
In vitro cytotoxicity of compounds towards the MCF-7 and BT-474 cells, after 24 h.Growth inhibition of 50 %): Concentration of drug that decreases the growth of the cells by 50 % compared to a nontreated control cell.bValuesare the average of three readings; Human Breast cancer cell line, d BT-474: Human Breast cancer cell line; e-Adriamycin: positive control compound a GI50(c MCF-7: