Synthesis and antimycobacterial screening of a novel series of α -amino acids containing thiazole linker

A small focused library of uncommon (S)-2-amino-3-(4-(((4-methyl-2-arylthiazol-5-yl)methyl)amino)phenyl) propanoic acid ( 5a-e ) and (S)-2-amino-3-(4-(((2-arylthiazol-4-yl)methyl)amino)phenyl)propanoic acid ( 9a-d ) derivatives have been efficiently synthesized by employing molecular simplification. The title compounds were screened for inhibitory activity against Mycobacterium tuberculosis H37Ra (MTB) and Mycobacterium bovis (BCG) strains. The cytotoxicity study was conducted against primary Human Umbilical Vein Endothelial Cells (HUVECs), on two different human tumor cells HeLa, and HCT 116 and was observed non-toxic to host cells.


Introduction
Tuberculosis (TB) is one of the top causes of death worldwide. 11][12][13][14][15] Due to its appreciable diversity in biological actions, thiazole is one of the influential scaffolds in drug discovery and development processes.

Synthesis of target amino acids
The synthetic strategies adopted for the synthesis of titled compounds 5a-e and 9a-d are depicted in Schemes 1 and 2, respectively.The 4-methyl-2-arylthiazole-5-carbaldehyde 1a-e on condensation with (S)-methyl 3-

Biological evaluation
The in vitro anti-tubercular activity against M. tuberculosis H37Ra (Dormant stage) and M. bovis (Dormant stage) revealed that most of the screened compounds showed moderate to good anti-tubercular activity against both strains.(Table 1) The compound 5c exhibited good anti-tubercular activity against M. bovis.The preliminary structure-activity relationship study revealed that the replacement of hydrogen atoms of phenyl ring by substituents like Cl, F, and CH3 significantly affects the anti-tubercular activity.The antibacterial activity of synthesized compounds was determined against the standard Gram-negative bacteria, E. coli (NCIM 2576), P. flurescence (NCIM 2059) and Gram-positive bacteria, S. aureus (NCIM 2602), B. subtilis (NCIM 2162).(Table 2) Unsubstituted phenyl at 2-position and methyl amino group at 5-position of thiazole ring showed good activity against both tubercular strains but found inactive against bacterial strains.The Br/Cl/F/CH3 substituted phenyl and methyl amino group at 5-position of thiazole ring at 2-position of thiazole showed good activity against M. tuberculosis H37Ra and all bacterial strains.The phenyl or substituted phenyl group at 2-position of thiazole and methyl amino group at 4-position found less active.From the antibacterial activity data, it is concluded that compounds 5c and 5e showed good activity against all tested strains.
To evaluate the cytotoxicity, the synthesized compounds 5a-e and 9a-d were further assayed for their cytotoxic activity in exponentially dividing primary (HUVECs) and human cancer cells (HeLa and HCT 116), the cells were treated with increasing concentrations of compounds, and cell viability was measured over time by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide MTT assay. 32,33Paclitaxel was used as a positive control.The cytotoxic effect of these compounds was checked on cancer cell lines using the concentration range between 30, 10, and 3 μg mL -1 to determine the growth inhibition.The results indicated that, in MTT cytotoxicity studies, most active compounds are leads as antimicrobials owing to no significant cell toxicity against HeLa, HCT 116 and HUVEC cell lines at the maximum concentration evaluated.It is noteworthy that compounds did not demonstrate cell toxicity towards HUVEC cells (>100 μg mL -1 ), implying its non-toxicity toward normal cells.

Experimental Section
General.All the reactions were monitored by thin layer chromatography (TLC).TLC was performed on Merck 60 F-254 silica gel plates.Melting points were determined in capillary tubes in a silicon oil bath using a Veego melting point apparatus and are uncorrected. 1H NMR and 13 C NMR spectra were recorded on Varian mercury XL-300 and Bruker at either 400/500 MHz ( 1 H NMR) or 100/126 MHz ( 13 C NMR), spectrometer instruments.Chemical shifts are reported from the internal tetramethylsilane standards and are given in δ units.Infrared spectra were recorded on Shimadzu FTIR (KBr) -408 in KBr.The chemicals and solvents used were laboratory grade and were purified as per literature methods.The starting compounds 4-methyl-2-arylthiazole-5carbaldehyde (1a-e) and 2-arylthiazole-4-carbaldehyde (6a-d) were synthesized by our previous methods. 34neral procedure for synthesis of methyl 3-( 4 ) propanoate (0.9 mmol) in dry methanol (5mL), acetic acid (0.5mL) was added and stirred for 10 min.The reaction mixture was cooled at 0-5 °C and sodium cyanoborohydride (1.84 mmol) was added portion-wise at 0-5 °C and the reaction mixture was further stirred for 12 hours.After completion of the reaction (TLC), solvent was removed under reduced pressure.The residue was dissolved in water (30mL) and the pH was adjusted to neutral using a saturated NaHCO3 solution.The aqueous layer was extracted with DCM (15mL x 2), and the organic layer was dried over anhydrous Na2SO4 and distilled under vacuum gave a crude product, which was purified by column chromatography, using ethyl acetate: hexane (3:7) as eluent.Compounds 8a-d were synthesized by applying similar experimental conditions.General procedure for synthesis of (S)-2-amino-3-(4-(((4-methyl-2-arylthiazol-5yl)methyl)amino)phenyl)propanoic acid (5a-e).The solution of methyl 3-(4-((4-methyl-2-arylthiazole-5yl)methylamino)phenyl)2-(1,3 dioxoisoindolin-2yl)propanoate (0.8 mmole), in conc.HCl (8N) was refluxed for about 8-12 h.The reaction mass was cooled and phthalic acid was removed by filtration.The filtrate was diluted with 25mL water and the reaction mixture was extracted with 10 mL diethyl ether.The pH of the aqueous layer was adjusted to 6-7 by using ammonia solution.The precipitated mass was stirred for 2 hours at room temperature.The product was filtered on a Buckner funnel, and washed with distilled water followed by acetone (10 mL).The product was dried in a vacuum drier.Compounds 9a-d were synthesized by using similar reaction conditions.

5a-e and 9a-d was
confirmed by spectral analysis.The synthesized compounds were screened for antimycobacterial activity.