Synthesis and evaluation of the antibacterial, antioxidant activities of novel functionalized thiazole and bis(thiazol-5-yl)methane derivatives

Novel functionalized thiazoles were prepared by the Hantzsch reaction from 3-[(4-hydroxyphenyl)carbamothioylamino]-2-methylpropanoic acid and the corresponding α -halocarbonyl compounds in good yields. A series of chemical transformations of the obtained products were carried out, and new functionalized thiazole derivatives with aliphatic, aromatic and heterocyclic substituents were synthesized. 4-Phenyl-substituted N -(4-hydroxyphenyl)- N -carboxyalkylaminothiazoles were used as precursors for the synthesis of bis(thiazol-5-yl)methane derivatives, which then were screened for their antibacterial, antioxidant activities.


Introduction
Thiazoles have found a wide spectrum of pharmacological and industrial applications.4][5] Aromatic ketone and enone compounds which form the central core for a variety of important biological compounds and which are known collectively as chalcones or chalconoids are typically found in plants.For example, naringenin chalcone found in the tomato peel shows the anti-allergic activity. 6Dihydrochalcone phlorizin found in cherry, apple, pears inhibits SGLT1 and SGLT2 proteins which are responsible for glucose transport. 7Because phlorizin can reduce glucose content in the blood, 8 it was studied as a potential pharmaceutical treatment for type II diabetes. 9Davidigenin extracted from Mascarenhasia arborescens shows antispasmodic and antioxidant activities. 10Chalcones have also good antibacterial, 11,12 anti-obesity, 13 immunosuppressant 14 properties.
β-Amino acid fragments are widely encountered in living nature and show a variety of biological activities.For example, a dipeptide containing histidine and β-alanine fragments, called anserine, which can be found in the muscles and brain of mammals, shows an antitumor activity. 15Anserine derivatives with β-cyclodextrin show good antioxidant properties. 16β-Amino acid derivatives show antiseizure, 17 antimalarial 18 activities.Endomorphin-2 analogs containing N-methylated amino acids exhibit a strong analgesic effect. 19,20As β-amino acid derivatives, compounds containing a thiazole ring also show strong bioactive properties, for example, vitamin B 1 , penicillins.In our previous papers, [21][22][23] we have reported the synthesis of N-aryl-N-carboxyethyl-2-aminothiazoles and their derivatives by the Hantzsch reaction, and some of the synthesized compounds showed antimicrobial activities.In continuation of our studies on the synthesis of functionalized thiazole derivatives, herein we report the synthesis of a new variety of functionalized thiazoles, bis(thiazol-5-yl)methanes and bis(thiazol-5yl)phenylmethanes and the evaluation of their antibacterial and antioxidant activities.

Results and Discussion
Chemistry.The synthetic sequence (Scheme 1) begins at the preparation of functionalized N,N-disubstituted 2-aminothiazoles 2a-h from the thioureido acid 1 and various α-haloketones.All reactions were carried out in refluxing 2-propanol, and the yield of the obtained products ranged within 59-87%.The increasing interest to the dihydroquinolone-type compounds as potential therapeutic agents encouraged to investigate the synthesis of compounds with this core in the structure.The effort to synthesize the above-mentioned compounds according to the described method, 24 i.e. to cyclize those by heating in the polyphosphoric acid, failed probably because of the interaction of the hydroxy group with the phosphoric acid.Therefore, another pathway was chosen.Primarily, the hydroxy group was protected by alkylation with dimethyl sulphate, then the synthesized products 3a,d were cyclized to 4a,d using PPA, and subsequent cleavage of the ether bond by heating 4a,d in the mixture of the acetic acid and hydrogen bromide afforded the desired products 5a,d in high yields.

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Treatment of thioureido acid 1 with 3-chloro-2,4-pentanedione yielded 57% of 3-((5-acetyl-4methylthiazol-2-yl)(4-hydroxyphenyl)amino)-2-methylpropanoic acid (6), which then was used for the synthesis of chalcone-type derivatives 7a-f.In order to perform a more detailed investigation of the structure of the synthesized compounds 7 containing one asymmetric carbon atom, the X-ray analysis was carried out.The ORTEP diagram of the structure of compound 7c is presented in Figure 1.It should be noted that in the crystalline state the compound 7c exists in two conformations; this leads to a structural disorder.For the prevailing conformation (its occupation g-factor is equal to 0.75) the torsion angle of N6−C7−C8−C10 is 62.3(5)°; for the second conformation (with g 0.25) this angle is −93.4 (9). Figure 1 shows the molecular structure in the dominant conformation.
The fused thiazole 8, containing thiazole and cyclohexane fragments, was synthesized by the interaction of thioureido acid 1 and 2-bromocyclohexane-1-one in 2-propanol, in the presence of sodium acetate.The naphthoquinone-fused thiazole 9 was prepared from compound 1 and 2,3-dichloro-1,4-naphthoquinone by stirring them in acetic acid at 80 °C for 24 h, in the presence of sodium acetate.Efforts to obtain NMR spectrum data, due to the very poor solubility of the target substance 9 in organic solvents, were unsuccessful.Therefore, the structure of compound 9 was established by the data of MS and IR spectroscopy.The mass spectrum data show a positive molecular ion [M+H] + with 409.0854 m/z, the calculated monoisotopic mass for compound 9 (C 21 H 16 N 2 O 5 S+H + ) being 409.0853.The IR spectrum data show three strong absorption bands at about 1712, 1634, 1616 cm −1 , corresponding to two C=O groups in the naphthoquinone fragment and one in the carboxyl group.
On the strength of the raised purposes of this study, we tried to synthesize polyfunctionalized derivatives with thiazole fragments in the structure.The attempts were successful: the interaction of compounds 2 with aromatic aldehydes in the molar ratio of 2:1 resulted in a high yield of bis(thiazol-5-yl)phenyl methanes 10-15 (Scheme 2).The reactions were carried out in acetone under reflux for 17 h in the presence of a catalytic amount of hydrochloric acid.The resulting products precipitate in the form of insoluble salts in acetone.The microanalysis data showed that in the first case the double hydrochloric salts were formed.In the reactions with 4-(dimethylamino)benzaldehyde, the triple salts were obtained.These salts were converted to their base by dissolving them in 5% aqueous sodium carbonate and acidifying the solution with acetic acid to pH 6. Scheme 2. Synthesis of polisubstituted bis(thiazolyl)phenylmethanes 10-15.
The 1 H NMR spectra of the synthesized compounds 10-15 showed a singlet at approx.5.66 ppm, which was assigned to the SCCHCS group proton.A singlet at approx.6.98 ppm characteristic of the CH group at the 5-position of the thiazole ring was not observed.The analysis of the aromatic region of the 1 H NMR spectra showed the additional spectral peaks characteristic of a p-substituted phenyl ring.These data approve the formation of bis(thiazol-5-yl)phenyl methanes 10-15.We have also found that thiazoles 2 easily react with formaldehyde to obtain bis(thiazol-5-yl)methanes 16a,d,e,g (Scheme 3).In this case, reactions were carried out in acetic acid, because in these conditions cleaner products are obtained.The structure of compounds 16 has been confirmed by methods of IR, 1 H, 13 C NMR spectroscopy and HRMS analysis.
Biology.All of the synthesized compounds (2-16) (50-1000 μg/mL) were evaluated for their antibacterial activity against the strains of Rhizobium radiobacter, Xanthomonas campestris, Escherichia coli by the diffusion technique. 25,26Only some of them appeared to be active against the investigated strains of bacteria.The activities of the tested compounds were compared with those of the known antibacterial agent ampicillin (50 μg/mL).The most active were derivatives 10a, 12e, 14e against Rhizobium radiobacter and 12e, 14e against Xanthomonas campestris at the concentration of 50 μg/mL.The evaluation of the antibacterial activity against Escherichia coli revealed that the most active appeared to be derivatives 10a, 12e, 14e at the concentration of 125 μg/mL.
The ferric reducing antioxidant power (FRAP) method is based on the reduction of a ferroin analog, the Fe 3+ complex of tripyridyltriazine Fe(TPTZ) 3+ , to the intensely blue coloured Fe 2+ complex Fe(TPTZ) 2+ by antioxidants in an acidic medium.The results are obtained as the absorbance increases at 593 nm and can be expressed as a Fe 2+ µmol/L concentration.The results revealed (Figure 3) that compounds 14e (88.72 Fe 2+ µmol/L), 15g (31.33 Fe 2+ µmol/L), 13d (30.67 Fe 2+ µmol/L) showed the highest antioxidative activity evaluated by FRAP method.In the reducing power assay, the presence of reductants (antioxidants) in a sample would result in the reducing of Fe 3+ to Fe 2+ by donating an electron.The amount of the Fe 2+ complex can then be monitored by measuring the formation of Perl's blue at 700 nm.The results of the reducing power assay (Figure 4) demonstrate that compounds 14e, 15g, 13d exhibit antioxidant effect.

Experimental Section
General.TLC was performed with Merck, Silica gel 60 F254 (Kieselgel 60 F254) silica gel plates.The 1 H and 13 C NMR spectra were recorded by the Bruker Ascend 400 ( 1 H 400 MHz, 13 C 101 MHz) and Bruker Ascend ( 1 H 700 MHz, 13 C 176 MHz) spectrometers.Chemical shifts are expressed as δ, ppm relative to TMS.The IR spectra (ν, cm -1 ) were recorded on a Perkin Elmer Spectrum Bx FT-IR spectrometer using KBr tablets.Elemental analyses were performed with a CE-440 elemental analyzer.Melting points were determined with a B-540 Melting Point Analyzer (Buchi Corporation, USA) and are uncorrected.Mass spectra were measured with the Xevo TQ-S and Bruker maXis 4G mass spectrometers.The X-ray crystallographic analysis was performed with a Bruker-Nonius KappaCCD diffractometer using the graphite monochromated Mo-Kα radiation (λ 0.71073 Å).

General procedure for the synthesis of compounds 2a-h.
A mixture of compound 1 (0.64 g, 2.5 mmol), the corresponding α-haloketone (2.75 mmol), sodium acetate (0.42 g, 5 mmol) and 2-propanol (10 mL) was refluxed for 4 h, cooled to room temperature and diluted with water (30 mL).The formed precipitate was filtered off, washed with water, dried.Purification was performed by dissolving crystals in 5% aqueous sodium hydroxide, filtering, and acidifying the filtrate with acetic acid to pH 6.

3,3'-((Methylenebis(4-phenylthiazole-5,2-diyl))bis((4-hydroxyphenyl)azanediyl))bis(2-methylpropanoic acid) (16a).
Blue crystals, yield 1.02 g (81%), mp 180-181 °C.IR (KBr, νmax, cm -1 ): 1511 (C=N), 1707 (C=O), 3392 (O-H). 1 H NMR (400 MHz, DMSO-d 6 ), δ, ppm, (J, Hz): 1.08 (6H, d, J 7.0, 2xCH 3 ), 2.61-2.85(2H X , m, 2xCH), 3.92 (2H B , dd, J BA 13.6, J BX 7.3, 2xCH 2 ), 4.00 (2H A , dd, J AB 13.6, J AX  Crystallography.X-ray crystallographic analysis of compound 7c.Diffraction data were collected at −90 °C on a Bruker-Nonius KappaCCD diffractometer using the graphite monochromated Mo-Kα radiation (λ 0.71073 Å).The crystal structure of 7c was solved by the direct method and refined by full-matrix least squares. 27Crystal data for 7c: monoclinic; a 11.2244(5), b 15.8918(8), c 12.6457(6) Å, β 102.351(3)°;V 2203.5(2)Å 3 , Z 4, μ 0.301 mm -1 ; space group is P2 1 /n.A total of 8565 reflection intensities were collected up to 2θ max 55°; for structure refinement, 2603 independent reflections with I > 2σ(I) were used.The final R-factor is 0.0839.For further details, see crystallographic data for 7c, deposited with the Cambridge Crystallographic Data Centre as the Supplementary Publication Number CCDC 1455153.Copies of the data can be obtained free of charge on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK.Antibacterial activity of the compounds.Antibacterial activity was tested using the disk diffusion technique.The microorganisms Rhizobium radiobacter, Escherichia coli, Xanthomonas campestris were commercially available from the German Collection of Microorganisms and Cell Cultures (DSMZ).The zone of the inhibition of bacterial growth was investigated.The main solution (1 mg/mL) of the synthesized compounds was prepared in DMSO and then diluted to various concentrations (50-1000 µg/mL) in DMSO.Cultures of Rhizobium radiobacter, Xanthomonas campestris, Escherichia coli were cultivated in Petri dishes for 24 h at 37 °C on the Luria-Bertani (LB) agar medium.A bacterial suspension was prepared from cultivated bacterial cultures, and 50 µL of the inoculum containing bacterial cells (10 8 CFU/mL) was spread over the LB agar medium.Filter paper disks were prepared by adding 25 µL of each compound solution, and then the disks were put on the LB agar medium.Ampicillin was used as the positive control, and DMSO was used as the negative control.The Petri dishes were incubated for 24 h at 37 °C, and the zones of inhibition were then ascertained for each sample.DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging assay.The free radical scavenging activity of compounds was measured by DPPH using the widely used method. 26Briefly, 1 mM solution of DPPH in ethanol was prepared, and this solution (1 mL) was added to the solutions of the tested compounds (1 mg/mL of dimethyl sulphoxide).The mixture was shaken vigorously and allowed to stand at room temperature for 20 min.Afterwards, the absorbance was measured at 517 nm with a UV-200-RS spectrophotometer (MRC Ltd., Israel).The lower absorbance of the reaction mixture indicated a higher free radical scavenging activity.The capability to scavenge the DPPH radical was calculated according to the following equation: DPPH scavenging effect (%) (A 0 -A 1 /A 0 ) × 100, where A 0 is the absorbance of the control reaction, and A 1 is the absorbance in the presence of the compounds.Reducing power assay.The tested compounds (1000 µg/mL) were mixed with the phosphate buffer (2.5 mL, 0.2 mol/L, pH 6.6) and potassium ferricyanide K 3 [Fe(CN) 6 ], (2.5 mL, 1%).The mixture was incubated at 50 °C for 20 min.10% TCA was added to the mixture, which was then centrifuged at 3000 rpm for 10 min.The upper layer of the solution was mixed with distilled water (2.5 mL), FeCl 3 (0.5 mL, 0.1%), and the absorbance was measured at 700 nm.The increased absorbance of the reaction mixture indicated an increased reducing power. 28erric reducing antioxidant power assay (FRAP).The principle of this method is based on the reduction of a ferric-tripyridyl triazine complex to its ferrous coloured form in the presence of antioxidants.Briefly, the FRAP reagent contained 2.5 mL of a 10 mmol/L TPTZ (2,4,6-tripyridyl-s-triazine) solution in 40 mmol/L HCl plus 2.5 mL of FeCl 3 (20 mmol/L) and 25 mL of acetate buffer (0.3 mol/L, pH 3.6).The aliquots of 100 μL tested compounds (1000 µg/mL) were mixed with 3 mL of the FRAP reagent, and the absorbance of the reaction mixture at 593 nm was measured spectrophotometrically after incubation at 37 °C for 10 min.For the construction of the calibration curve, five concentrations of FeSO 4 × 7 H 2 O (5, 10, 15, 20, 25 μmol/L) were used, and the absorbancies were measured as a sample solution. 29

Figure 1 .
Figure 1.The ORTEP diagram of the structure 7c.

procedure for the synthesis of compounds 4a,d
. A mixture of compound 3a or 3d (2.4 mmol) and polyphosphoric acid (15 mL) was stirred at 110 °C for 16 h.Then the reaction mixture was cooled to room temperature, and crushed ice was added.The formed yellow precipitate was filtered off, washed with water until the pH of the filtrate became neutral (pH 7), and recrystallized from the MeOH/H 2 O mixture.6