SYNTHESIS OF 2-( ( 5-BENZYLIDENE-4-OXO-4 , 5-DIHYDROTHIAZOL-2-YL )-SUBSTITUTED AMINO ACIDS AS ANTICANCER AND ANTIMICROBIAL AGENTS

A series of Rhodanine derivatives were synthesized by Knoevenagel condensation. All the synthesized compounds were tested for their in vitro anticancer activity against MCF-7 and BT-474 human breast cancer cell lines. All the synthesized compounds were characterized and screened for their antimicrobial activity against the bacterial and fungal strain. Majority of the compounds showed good to moderate anticancer and antimicrobial activity. Among these compounds, one showed promising activity against gram-positive bacteria B. subtilis and S. aureus when compared with ampicillin. Some of the most potent compounds possessed selective antimicrobial activity.


MATERIALS AND METHODS
Rhodanine, benzaldehyde 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).IR spectra were recorded on an FT-IR (Bruker).Melting points were recorded on SRS Optimelt, melting point apparatus and are uncorrected.The 13  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.

Procedure for the synthesis of (Z)-5-benzylidene-2thioxothiazolidin-4-one (3)
A mixture of benzaldehyde 1 (1 mmol), 2thioxothiazolidin-4-one 2 (1 mmol), anhydrous sodium acetate (1 mmol) and glacial acetic acid (1 ml) was refluxed for 5 h.The progress of the reaction was monitored by TLC (20% ethyl acetate: n-hexane).After completion of the reaction, the reaction mixture was poured into the ice-cold water.The precipitate was filtered off and washed with water (3×10 ml), dried, and purified by recrystallization in ethanol as a solvent to give 82 % yield.

Antimicrobial activity
The antibacterial activity was evaluated against two Gram-positive bacteria namely, Bacillus subtilis (NCIM-2063)andStaphylococcus aureus (NCIM-2901), and one Gram-negative bacterium Escherichia coli (NCIM-2256).The antibacterial activity of compounds was monitored by observing their Minimum Inhibitory Concentration (MIC, μgmL -1 ) as previously mentionedby the broth dilution method using Ciprofloxacin and Ampicillin as standard drugs.The antifungal activity was evaluated against three fungal strains; Candida albicans (NCIM-3471), Aspergillus flavus (NCIM-539)and Aspergillus niger (NCIM-1196)using Fluconazole and Miconazole as standard drugs.Minimum inhibitory concentration (MIC, μgmL -1 ) values for antifungal were determined using standard agar dilution method. 43Methanol was used as solvent control for both antibacterial and antifungal testing.The MIC values of the tested compounds are presented in Table 3.

Anticancer activity
All the synthesized compounds were also tested for their anticancer activity on mammalian cell lines MCF-7 and BT-474 human breast cancer cell line.This test is performed as previously mentioned MTT colorimetric assay. 44The anticancer activity of the compounds was determined by calculating their IC50 values, the concentration of compound

RESULTS AND DISCUSSION
The synthetic protocols employed for the synthesis of Rhodanine derivatives 3, 4, and 6a-l is presented in Scheme 1.The compound (3) was prepared via a Knoevenagel condensation between and benzaldehyde (1) andRhodanine (2).The compound (4) (Scheme 1) was obtained via reaction of the compound (3) with iodomethane in dichloromethane using triethylamine as a catalyst.We synthesized and screening of model reaction (Z)-2-((5benzylidene-4-oxo-4,5-dihydrothiazol-2-yl)amino)propanoic acid (6a) (Scheme 2, Table 1).The reaction in which the compound 4 (1 mmol) and compound5a (1.2 mmol), catalyzed by various bases and various solvents were selected as a model reaction to optimize the reaction conditions.In terms of the effect of solvents and bases on the condensation reaction, potassium carbonate was found to be the better base and ethanol was found to be the best solvent for the reaction (Table 1, entry 11); other solvents, including methanol, acetic acid, N,N-dimethylformamide (DMF) and toluene were less efficient (Table 1, entries 2-5, 7-10 and 12-15).Nevertheless, all of these yields were best.Ethanol gave the corresponding product in 80-98% yield, which was the best among these solvents (Table 1, entries 1, 6 and 11).To increase the efficiency of the condensation reaction, the effects of different bases were investigated (Table 1, entries 1-15).Potassium carbonate exhibited the best performance with used solvents and gave a better yield, (Table 1, entries 11-15).Sodium acetate and triethylamine gave lower yields with other solvents but gave a better yield in combination with ethanol as a solvent (Table 1, entries 1 and 6).All the reactions were carried out in equimolar amounts of each compound in 1 mL of solvent.Among these reactions, same amounts of the solvent, namely 1 ml of ethanol turned out to be the best choice with yields of 82, 80 and 98% (Table 1, entries 1, 6 and 11).We would like to mention here that ethanol as a solvent with K2CO3 as base was the best choice with a yield of 98% and less time required for the completion of the reaction (Table 1, entry 11).Thus we decided to carry out the further reactions in ethanol with potassium carbonate.As a result, the reaction time was shortened; thermal decomposition was also minimized, at room temperature stirring, resulting in higher isolated yields.
Thus we decided the further series, substituted acid derivatives 6a-l(Scheme 3, Table 2) were synthesized reacting from 5-benzylidene-2-(methylthio)thiazol-4(5H)one (4) with various amino acids (5a-l) in ethanol by using K2CO3 as a catalyst.In this reaction, there was displacement of a methyl sulfinyl group by amino acids from the C2 position of the thiazolone ring.
The physical data of the synthesized compounds are presented in Table 2.All the reactions proceeded well in 20-60 min to give products in very good yields (82-98%).
The purity of the synthesized compounds was checked by TLC on silica gel precoated F254 Merck plates and melting points were recorded on SRS Optimelt, melting point apparatus and are uncorrected.The structure of the synthesized compounds was confirmed by IR, 1 H NMR, 13 C NMR and mass spectral analysis.

Antimicrobial activity
From the antibacterial activity data (Table 3), the synthesized compounds of present series showed moderate to good antibacterial activity.Amongst the synthesized series, the compounds 6g (MIC= 15 μgmL -1 and 12.5 μg mL -1 ), 6h (MIC= 10 μg mL -1 and 15 μg mL -1 )and 6j (MIC= 6μg mL -1 and 5.5 μg mL -1 )werefound to be most active molecules and they are found to specific towards the Grampositive bacteria, S. aureus and B. subtilis.The compound 6j (MIC= 6.0 and 5.5 μg mL -1 against B. subtilis and S. aureus, respectively) was more active than both standards; Ciprofloxacin (MIC= 6.25 μg mL -1 ) and Ampicillin (MIC= 12.5 μg mL -1 ).The compounds 6g (MIC=15 and 12.5 μg mL -1 against B. subtilis and S. aureus, respectively) and 6h (MIC= 10 and 15 μg mL -1 against B. subtilis and S. aureus, respectively) showed lower activity thanCiprofloxacin and comparative level of activity as Ampicillin.On the other hand, compound 6f (MIC= 15μg mL -1 )was found to be narrow spectrum molecule showing activity only against the bacterium S. aureus.Out of these, the remaining compounds of the series 3, 4, 6a, 6i, 6k,and 6l had very high MIC values and therefore they are inactive as antibacterial agents.It interesting to find out that bacterium E. coli is resistance to all compounds, suggest that molecules of the series may be inactive against Gram-negative bacteria.

Anticancer activity
The newly synthesized 14 compounds were screened for their in vitro growth inhibitory activities against two human barest cancer cells line MCF-7 and BT-474, 100 µM (micromol mL -1 ) by MTT assays method, (Table 4).The results are shown as percentage anticancer activity after 24 h.
The compounds found active in preliminary screening were further studied for their cytotoxic effect on human barest cancer cell line MCF-7 and BT-474 cell lines and the results are expressed as IC50.Among these 14 newly synthesized thiazole derivatives screened for their cytotoxic effect on MCF-7 and BT-474 cells, five compounds showed percentage cell death greater against cell lines used.Among the most active three compounds, two compounds exhibited cell death greater than 50% against both cell lines.The synthesized compounds 6e, 6f, 6g, 6h, 6k and 6l showed maximum percentage cytotoxicity 100 µM.The anticancer studies of compound (6g) and (6l), against MFC-7 and BT-474 cell lines exhibited IC50 values are 1.4,0.7, 1.2, and 1.3 µM,respectively.Table 3.In vitro antimicrobial evaluation of synthesizedRhodanine derivatives 3, 4, and 6a-l.
The data represents the mean values of three replicates; Standard errors were all within 10% of the mean; -denotes not tested.
While the compounds 6e, 6g, 6h, 6k and 6l against BT-474 cell lines exhibited IC50: 6.2, 0.7, 6.8, 12.1, and 1.3 µM mL -1 ,respectively.The IC50 of reference drug Adriamycin against MFC-7 and BT-474 cells was found to be 0.9 and 0.5 µM mL -1 ,respectively.The anticancer activity of all newly synthesized thiazole derivatives mainly depends on the type of substitution on thiazole moiety.The substitution pattern of amino acids showed variation in anticancer activity.The compounds with a substituted hydroxyl group attached to thiazole ring which contains amino acids showed the highest percentage of cell death.While the compounds having electron releasing alkyl chain, methyl-1H-imidazole ring group on thiazole rings resulted in the loss of activity.

CONCLUSIONS
We have reported at room temperature, less reaction time with good to excellent yields.All the synthesized compounds were also tested for their in vitro anticancer activity against MCF-7 and BT-474 human breast cancer cell lines.Among them most of the compounds show good to excellent anticancer activity, especially 6e, 6f, 6g, 6h, 6k and 6l are the most active compounds against tested cell line.The compound 6g activity value is very close to standard drug, which can be regarded as the promising drug candidate for development of anticancer drugs.The investigation has revealed that a number of Rhodanine derivatives have promising antimicrobial properties.

Figure 1 .
Figure 1.Previously reported antibacterial agents and synthesized compounds.

Table 4 .
In vitroanticancer activity of the studied compounds against the MCF-7 and BT-474cells, after 24 h a GI50(Growth inhibition of 50): Concentration of drug that decreases the growth of the cells by 50compared to a non-treated control cell.b Values are the average of three readings c MCF-7: Human breast cancer cell line d BT-474: Human breast cancer cell line e Adriamycin: Positive control compound C NMR spectra were recorded on a 400 MHz Varian NMR spectrometer.The 1 H NMR spectra were recorded on a 400 MHz Varian NMR spectrometer.The chemical shifts are reported as δ (ppm) units (tetramethylsilane).The following Eur. Chem.Bull.2019, 8(2), 63-70 DOI: 10.17628/ecb.2019.8.63-70 to inhibit 50% of cell growth compared to untreated control cells.The IC50 values were presented in micromol per milliliter (μM).The Adriamycin was used as a positive control for the comparison of the anticancer activity of synthesized compounds.

Table 1 .
Screening of catalyst, solvents, reaction time, and yield for the synthesis 6a a .
a All the reaction was carried out in equimolar amounts of each compound in 1 mL of solvent.b Isolated yield.