HETEROCYCLES

Paper | Regular issue | Vol. 91, No. 4, 2015, pp. 747-763
Received, 24th January, 2015, Accepted, 19th February, 2015, Published online, 25th February, 2015.
DOI: 10.3987/COM-15-13181

■ Synthesis and Antibacterial Activity of Novel N-Carboxyalkyl-N-phenyl-2-aminothia(oxa)zole Derivatives

Rita Vaickelioniene, Kristina Mickeviciene, Kazimieras Anusevicius, Jurate Siugzdaite, Kristina Kantminiene, and Vytautas Mickevicius*

Department of Organic Chemistry, Kaunas University of Technology, Radvilėnų pl. 19, Kaunas LT-50254, Lithuania

Abstract

N-Phenyl-N-thiocarbamoyl-α- and β-methyl-β-alanines were converted into a series of 1,3-thiazole derivatives by treatment with chloroacetaldehyde and haloketones. The reaction of N-phenyl-N-thiocarbamoyl-β-alanines and N-carbamoyl-N-phenyl-β-alanines with 2,3-dichloro-1,4-naphthoquinone and 2,3-dichloroquinoxaline provided naphthoquinone- and quinoxaline-fused thiazoles and oxazoles, respectively. A number of the synthesized compounds exhibited good antibacterial activity against Staphylococcus aureus and Salmonella enteritidis with MIC and MBC values (62.5 and 125 µg/mL, respectively) which are the same or even lower than those for the antibiotic oxytetracycline.

INTRODUCTION
Despite the rapid progress of science, the treatment of infectious diseases still remains a serious problem and concern to the scientific community, mainly because of the wide range of factors leading to the emergence of these diseases and also the increased number of pathogenic microorganisms with resistance towards multiple drugs.
The growing resistance of microorganisms requires the careful use of existing antimicrobial drugs. Besides, there is a need for the design of novel antimicrobial agents, particularly for the treatment of the infections.1-5 A potential approach to this problem is the design of innovative drugs with different mechanism of action in effort to avoid cross resistance to existing therapeuticals.6
N-Substituted β-amino acids are used for synthesis of heterocyclic compounds such as dihydrouracils and their 2-thioanalogues,7-10 quinolinones,11-12 and quinazolinones.13,14
Heterocycles containing nitrogen and sulphur or oxygen atom constitute an important class of compounds in the field of medicinal chemistry.15 The five-membered heterocyclic nucleus plays a vital role in many biological activities. For example, oxazoles are associated with antibacterial,16 antifungal, antitubercular,17 and antitumor18 activities. Thiazoles and their derivatives are found to exhibit various biological activities such as antibacterial,19-22 fungicidal,23 anti-inflammatory,24,25 antihypertensive,26,27 anti-HIV,28,29 antitumor,30 and antioxidant.31,32 Thiazole nucleus is also an integral part of all the available penicillins which have revolutionized the therapy of bacterial diseases.33
Recently, we have reported the application of N-aryl-N-thiocarbamoyl--alanines as excellent precursors for the synthesis of N-carboxyethylaminothiazoles.21,34 Not so long ago, N-substituted thioureido acids were employed just for synthesis of 1-substituted thiodihydrouracils7-9 or tetrahydropyridones.20,35 Herein, we report the synthesis of novel aminothiazole and aminooxazole derivatives and investigation of structure activity relationship revealing the influence of the substituents in thiazole ring and aliphatic moiety on antimicrobial activity of the synthesized compounds. The data obtained enables the purposeful further synthesis of aminothiazole and aminooxazole derivatives in the search of effective antibacterial agents.


RESULTS AND DISCUSSION
One of the most convenient methods for preparation of thiazoles is Hantzsch synthesis, i.e. condensation of α-halocarbonyl derivatives with thioamides or thiocarbamides. Thus, N,N-disubstituted aminothiazoles 2a,b were prepared by heating the corresponding N-phenyl-N-thiocarbamoyl-- or -methyl--alanines 1a,b with chloroethanal at 80 C for 2 h (Scheme 1). The reaction provided soluble in water amino acid hydrochlorides which were converted into insoluble bases by adding sodium acetate. When thioureido acids 1a,b were treated with chloropropanone at reflux temperature, the reaction was completed already in 1 h. Thiazole derivatives 3a,b were isolated by diluting the reaction mixture with water and treating the solution with sodium acetate.

The structures of the synthesized compounds were confirmed by the data of elemental analysis, 1H NMR, 13C NMR and IR spectra. Formation of the thiazole ring in compounds 2a,b has been proven by the doublets of SCH group proton at 7.15–7.17 ppm, whereas the same proton gave rise to singlets at 6.18–6.25 ppm in the 1H NMR spectra of 3a,b. In the 13C NMR spectra of these compounds, the resonances ascribed to SCH group carbon are observed in the range of 101–108 ppm. The signals of NCH group carbon in the 13C NMR spectra of 2a and 2b are observed at 139.1 ppm and 140.1 ppm, respectively, and the C=N group carbon gave rise to peaks at 169.5 ppm and 168.9 ppm in the spectra of 3a and 3b, respectively. In the IR spectrum of these compounds, the absorption band of the C=N group is observed in the range of 1506–1519 cm-1.
2,4-Disubstituted thiazole derivatives 4a,b–11a,b were synthesized from thioureido acids 1a,b and corresponding haloketones in 2-propanol at the reflux temperature of the reaction mixtures. Naphthoquinone and quinoxaline-fused thiazoles 12a,b and 13a,b were prepared in the reactions of 1a,b with 2,3-dichloroquinaxoline or 2,3-dichloro-1,4-naphthoquinone. These reactions were carried out in acetic acid in the presence of sodium acetate at 80 C for 24 h. The synthesized compounds were purified by dissolving them in the aqueous KOH solution, filtering the obtained solution and acidifying it with acetic acid to pH 6.

Reaction of N-carbamoyl--alanines 14a and 14c with 2,3-dichloro-1,4-naphthoquinone in water at reflux temperature provided a mixture of naphthoquinone-fused oxazoles 15a,c and 16 which were isolated by column chromatography (Scheme 2). The analogues reaction of 14b provided just 2-(phenylamino)naphtho[2,3-d]oxazole-4,9-dione (16) which was also prepared from N-phenylurea in order to prove the formation of such a compound and its structure (Scheme 3). In the 1H NMR spectra for 12a,b, 13a,b, 15a,c and 16, the number of resonances in the aromatic region has increased in comparison with the spectra of precursors. In the 1H NMR spectrum for 16, the resonances in the aliphatic region are absent in comparison with the spectra of the analogous compounds. In the IR spectra for 12a,b the absorption bands of three C=N groups are observed in the range of 1556–1683 cm-1 and three CO absorption bands are present in the 1623–1710 cm-1 region in the IR spectra for 13a,b.

ANTIBACTERIAL ACTIVITY
The antibacterial activity of the compounds 1a,b–13a,b, 15a,c, and 16 was screened by testing their different concentrations against Gram-positive cocci Staphylococcus aureus (ATCC 9144), Gram-negative

rods Escherichia coli (ATCC 8739), Salmonella enteritidis (ATCC 8739) and Pseudomonas aeruginosa (NCTC 6750) by the broth and spread-plate methods. A range of concentrations for each compound were prepared according to the experimental procedure described in Experimental. The minimum inhibition concentration (MIC, µg/mL) and minimum bactericidal concentration values (MBC, µg/mL) are listed in the Table. A broad-spectrum antibiotic oxytetracycline was used as a control in the antibacterial activity tests of the synthesized compounds.

As the screening data for antibacterial activity have shown, a number of the investigated compounds possess antibacterial properties. Compounds 3a, 5a, and 7a have shown significant bactericidal activity against S. aureus with MIC and MBC values of 62.5 µg/mL, which are the same as the ones for oxytetracycline. Compound 5a inhibits growth of this bacteria strain at 62.5 µg/mL and its MBC value is 125 µg/mL. Among tested compounds, just 4a did not suppress growth of S. aureus. Compounds 1b, 4b, 6b, 7b, 8a, 9a, 10a,b, 11a, and 12b have shown good activity against E. coli with MIC and MBC values of 125 µg/mL. Bacteristatic activity for 16 was also observed at 125 µg/mL, whereas its bactericidal action was noted at 250 µg/mL. Compounds 1b, 4b, 7a, 8a,b, 9a,b, and 10b have shown good bactericidal activity against S. enteritidis at 125 µg/mL, which is lower than MIC and MBC values for oxytetracycline. Compound 3b did not suppress growth of E. coli and S. enteritidis. P. aeruginosa was resistant towards action of more compounds, i.e. 1a, 2a,b, 3a,b, 4a,b, and 5a,b. On the other hand, this bacteria strain was sensitive to a number of compounds, i.e. 6b, 7b, 8a,b, 9a,b, 10a,b, 11a, 12b, and 13b, at 125 µg/mL.

CONCLUSIONS
A series of N-carboxyethylaminothiazoles and a few of their oxo analogues were synthesized. The analysis of the obtained data on antibacterial activity of the synthesized compounds has revealed that there is a relationship between structure of the synthesized compounds and their biological activity. Comparison of thus presented data with the ones published previously,21 indicates that introduction of the methyl group into - or -position in the -alanine fragment increases antibacterial efficacy of thiazole derivatives. In the case of the action against Gram-positive cocci S. aureus, a very clear trend can be noticed as an introduction of the methyl group into -position of the -alanine fragment enhances significantly antibacterial properties of thiazole and oxazole derivatives in comparison with the ones containing the methyl group in -position of the -alanine fragment. Regarding the group of Gram-negative bacterial strains, it can be stated that derivatives with the methyl group in the -position are more active and this tendency is strongly expressed for the efficacy against P. aeruginosa. As it could be expected, the antibacterial activity of thiazoles increases upon introduction of an aromatic substituent containing nitro group or halogen atom, and especially two of the latter as in the case of compounds 9a,b, into thiazole ring as well its fusion with naphthoquinone. The comparison of the data on the antibacterial activity of naphthoquinone-fused thiazole 13a and its oxo analogue 15a has revealed that the oxygen-containing naphthoquinone derivative is more active against S. aureus, whereas Gram-negative bacterial strains are more sensitive to sulfur-containing compound.
The obtained data is valuable for the further synthesis of the compounds of similar structure and search for the more effective substances possessing antibacterial properties.

EXPERIMENTAL
The starting materials and solvents were obtained from Sigma-Aldrich Chemie GmbH (Munich, Germany) and Fluka Analyticals (Buchs, Switzerland) and were used without further purification. The methods used to follow the reactions were TLC and NMR. The NMR spectra were recorded on a Varian Unity Inova (300 MHz) (Varian, Inc., Palo Alto, CA, USA) and Bruker Avance III/400 (400 MHz) spectrometers. Chemical shifts are expressed as δ, ppm relative to TMS. The J constants are given in Hz. The IR spectra (ν, cm-1) were recorded on a Perkin–Elmer BX FT–IR spectrometer (Perkin–Elmer Inc., Waltham, MA, USA) using KBr tablets. Elemental analyses were performed with a CE-440 elemental analyzer (Exeter Analytical Inc., North Chelmsford, MA, USA). Melting points were determined with a B-540 Melting Point Analyzer (Büchi Corporation, New Castle, DE, USA) and are uncorrected. TLC was performed using Silica gel 60 F254 (Kieselgel 60 F254) (Merck, Darmstadt, Germany) plates.

General procedure for preparation of 1,3-thiazoles 2a,b. A mixture of thioureido acid 1 (1.19 g, 5 mmol), 50% aqueous chloroethanal solution (0.79 g, 10 mmol), and water (20 mL) was heated at 80 C for 2 h. Afterwards, sodium acetate (0.82 g, 10 mmol) was added and the mixture was stirred for 5 min. The precipitate was filtered off and washed with water. Purification was performed by dissolving the crystals in 10% aqueous Na2CO3 (25 mL), filtering, and acidifying the filtrate with acetic acid to pH 6 (the procedure was repeated twice).
2-Methyl-3-[phenyl(1,3-thiazol-2-yl)amino]propanoic acid (2a). White solid, yield 0.86 g (66%), mp 117–118 °C; IR (KBr) νmax (cm-1): 3435 (OH), 1695 (C=O), 1519 (C=N); 1H NMR (400 MHz, DMSO-d6) δ 1.07 (d, 3H, J = 7.1 Hz, CH3), 2.762.84 (m, 1HX, CHCH3), 3.99 (dd, 1HA, JAM = 7.5 Hz, JAX = 13.8 Hz, NCH2), 4.09 (dd, 1HM, JMA = 7.2 Hz, JMX = 13.8 Hz, NCH2), 6.69 (d, 1H, J = 3.6 Hz, NCH), 7.17 (d, 1H, J = 3.6 Hz, SCH), 7.307.56 (m, 5H, HAr), 12.48 (s, 1H, OH); 13C NMR (100 MHz, DMSO-d6) δ 14.9 (CH3), 38.1 (CH), 55.2 (CH2), 108.0 (SCH), 126.7, 127.1, 130.0, 145.2 (CAr), 139.1 (NCH), 170.4 (C-N), 175.9 (CO). Anal. Calcd for C13H14N2O2S: C, 59.52; H, 5.38; N, 10.68%. Found: C, 59.71; H, 5.53; N, 10.84%.
3-[Phenyl(1,3-thiazol-2-yl)amino]butanoic acid (2b). White solid, yield 1.05 g (80%), mp 167–168 °C; IR (KBr) νmax (cm-1): 3388 (OH), 1702 (C=O), 1507 (C=N); 1H NMR (400 MHz, (CD3)2CO) δ 1.32 (t, 2H, J = 6.8 Hz, CH2CO), ), 2.49 (dd, 1HA, JAM = 7.9 Hz, JAX = 15.6 Hz, CH2CO), 2.93 (dd, 1HM, JMA = 6.6 Hz, JMX = 15.6 Hz, CH2CO), 5.05–5.13 (m, 1HX, CHCH3), 6.55 (d, 1H, J = 3.7 Hz, NCH), 7.15 (d, 1H, J = 3.7 Hz, SCH), 7.38–7.56 (m, 5H, HAr), 10.87 (br. s, 1H, OH); 13C NMR (100 MHz, (CD3)2CO) δ 19.0 (CH3), 40.0 (CH), 54.2 (CH2), 108.2 (SCH), 129.4, 131.0, 131.2, 143.8 (CAr), 140.1 (NCH), 171.9 (C-N), 172.7 (CO). Anal. Calcd for C13H14N2O2S: C, 59.52; H, 5.38; N 10.68%. Found: C, 59.73; H, 5.50; N 10.79%.
General procedure for preparation of 4-methyl-1,3-thiazoles 3a,b. A mixture of thioureido acid 1 (0.56 g, 2.5 mmol) and chloropropanone (0.29 g, 3 mmol) in acetone (10 mL) was refluxed for 1 h and diluted with water (50 mL). Afterwards, sodium acetate (0.98 g, 12 mmol) was added, and the reaction mixture was stirred for 5 min. The precipitate was filtered off and washed with water. Purification was performed by dissolving the crystals in 10% aqueous Na2CO3 (25 mL), filtering, and acidifying the filtrate with acetic acid to pH 6.
2-Methyl-3-[(4-methyl-1,3-thiazol-2-yl)(phenyl)amino]propanoic acid (3a). White solid, yield 0.56 g (81%), mp 95–96 °C; IR (KBr) νmax (cm-1): 3126 (OH), 1716 (CO), 1519 (C=N); 1H NMR (300 MHz, DMSO-d6) δ 1.08 (d, 3H, J = 7.0 Hz, CHCH3), 2.15 (s, 3H, CH3), 2.71–2.85 (m, 1HX, NCH), 3.97 (dd, 1HA, JAM = 7.4 Hz, JAX = 13.7 Hz, NCH2), 4.08 (dd, 1HM, JMA = 7.4 Hz, JMX = 13.7 Hz, NCH2), 6.25 (s, 1H, SCH), 7.30–7.49 (m, 5H, HAr), 12.29 (br. s, 1H, OH); 13C NMR (75 MHz, DMSO-d6) δ 14.9 (CHCH3), 17.5 (CH3), 37.9 (CHCO), 54.8 (NCH2), 102.1 (SCH), 126.9, 127.2, 130.0, 148.3 (CAr), 144.9 (CCH3), 169.5 (C=N), 175.8 (C=O). Anal. Calcd for C14H16N2O2S: C, 60.85; H, 5.84; N, 10.14%. Found: C, 60.72; H, 5.78; N, 10.26%.
3-[(4-Methyl-1,3-thiazol-2-yl)(phenyl)amino]butanoic acid (3b). White solid, yield 0.52 g (75%), mp 145–146 °C; IR (KBr) νmax (cm-1): 3123 (OH), 1717 (C=O), 1506 (C=N); 1H NMR (300 MHz, DMSO-d6) δ 1.20 (d, 3H, J = 6.8 Hz, CHCH3), 2.15 (s, 3H, CH3), 2.31 (dd, 1HA, JAM = 7.9 Hz, JAX = 15.6 Hz, CH2CO), 2.67 (dd, 1HM, JMA = 6.7 Hz, JMX = 15.6 Hz, CH2CO), 4.92–5.03 (m, 1HX, CHCH3), 6.18 (s, 1H, SCH), 7.30–7.54 (m, 5H, HAr), 12.31 (br. s, 1H, OH); 13C NMR (75 MHz, DMSO-d6) δ 16.8 (CHCH3), 17.8 (CH3), 37.9 (CH2CO), 51.6 (NCH), 101.0 (SCH), 127.8, 129.3, 129.5, 147.5 (CAr), 140.8 (CCH3), 168.9 (C=N), 171.6 (C=O). Anal. Calcd for C14H16N2O2S: C, 60.85; H, 5.84; N, 10.14%. Found: C, 60.63; H, 5.98; N, 10.32%.
General procedure for preparation of 4–11a,b. A mixture of thioureido acid 1 (1.12 g, 5 mmol), corresponding α-haloketone (5 mmol), sodium acetate (0.82 g, 10 mmol), and 2-propanol (10 mL) was refluxed for 5 h. Afterwards, it was diluted with water (30 mL). The precipitate was filtered off and washed with water. Purification was performed by dissolving the crystals in 10% aqueous Na2CO3 (30 mL), filtering, and acidifying the filtrate with acetic acid to pH 6.
2-Methyl-3-[phenyl(4-phenyl-1,3-thiazol-2-yl)amino]propanoic acid (4a) was prepared according to the general procedure from 1a and 2-bromo-1-phenylethan-1-one to afford light yellow solid, yield 1.52 g (90%), mp 123–124 °C; IR (KBr) νmax (cm-1): 3111 (OH), 1703 (CO), 1514 (CN); 1H NMR (300 MHz, DMSO-d6) δ 1.15 (d, 3H, J = 7.0 Hz, CH3), 2.86–2.98 (m, 1H, CHCO), 4.11 (dd, 1HA, JAM = 7.3 Hz, JAX = 13.8 Hz, NCH2), 4.21 (dd, 1HM, JMA = 7.3 Hz, JMX = 13.7 Hz NCH2), 7.12 (s, 1H, SCH), 7.26–7.89 (m, 10H, HAr), 12.22 (br. s, 1H, OH); 13C NMR (75 MHz, DMSO-d6) δ 15.0 (CH3), 38.1 (CHCO), 55.2 (NCH2), 102.5 (SCH), 125.6, 126.8, 127.3, 127.5, 128.5, 130.0, 134.6, 150.2 (CAr), 144.7 (NCH), 169.4 (CN), 175.9 (CO). Anal. Calcd for C19H18N2O2S: C, 67.43; H, 5.36; N, 8.28%. Found: C, 67.22; H, 5.15; N, 8.12%.

3-Phenyl(4-phenyl-1,3-thiazol-2-yl)amino]butanoic acid (4b) was prepared according to the general procedure from 1b and 2-bromo-1-phenylethan-1-one to afford light yellow solid, yield 1.32 g (78%), mp 144–145 °C; IR (KBr) νmax (cm-1): 3122 (OH), 1711 (C=O), 1510 (C=N); 1H NMR (300 MHz, DMSO-d6) δ 1.30 (d, 3H, J = 6.8 Hz, CH3), 2.45 (dd, 1HA, JAM = 6.9 Hz, JAX = 15.6 Hz, CH2CO), 2.84 (dd, 1HM, JMA = 6.9 Hz, JMX = 15.6 Hz CH2CO), 5.01–5.13 (m, 1H, CHCH3), 7.09 (s, 1H, SCH), 7.26–7.88 (m, 10H, HAr), 12.33 (br. s, 1H, OH); 13C NMR (75 MHz, DMSO-d6) δ 18.4 (CH3), 25.4 (CH2CO), 53.1 (CHCH3), 102.4 (SCH), 125.6, 127.4, 128.5, 128.6, 129.9, 130.1, 134.7, 150.2 (CAr), 141.8 (NCH), 169.4 (C=N), 172.4 (C=O). Anal. Calcd for C19H18N2O2S: C, 67.43; H, 5.36; N, 8.28%. Found: C, 67.20; H, 5.11; N, 8.10%.
3-{[4-(4-Fluorophenyl)-1,3-thiazol-2-yl](phenyl)amino}-2-methylpropanoic acid (5a) was prepared according to the general procedure from 1a and 2-bromo-1-(4-fluorophenyl)ethan-1-one to afford white solid, yield 1.59 g (89%), mp 138–139 °C; IR (KBr) νmax (cm-1): 3119 (OH), 1699 (C=O), 1519 (C=N); 1H NMR (400 MHz, (CD3)2CO) δ 1.25 (d, 3H, J = 7.1 Hz, CH3), 3.09–3.15 (m, 1H, CHCH3), 4.19 (dd, 1HA, JAM = 7.2 Hz, JAX = 13.8 Hz, NCH2), 4.32 (dd, 1HM, JMA = 7.2 Hz, JMX = 13.8 Hz NCH2), 6.94 (s, 1H, SCH), 7.13–7.98 (m, 9H, HAr), 10.65 (br. s, 1H, OH); 13C NMR (100 MHz, (CD3)2CO) δ 15.6 (CH3), 39.2 (NCH2), 56.6 (CHCH3), 102.6 (SCH), 116.0, 116.1, 128.2, 128.5, 128.7, 128.7, 131.0, 132.7, 151.0, 162.5, 163.9 (CAr), 146.4 (N-C), 171.1 (C=N), 176.3 (C=O). Anal. Calcd for C19H17FN2O2S: C, 64.03; H, 4.81; N, 7.86%. Found: C, 64.29; H, 4.87; N, 8.01%.
3-{[4-(4-Fluorophenyl)-1,3-thiazol-2-yl](phenyl)amino}butanoic acid (5b) was prepared according to the general procedure from 1b and 2-bromo-1-(4-fluorophenyl)ethan-1-one to afford white solid, yield 1.60 g (90%), mp 158–159 °C; IR (KBr) νmax (cm-1): 3122 (OH), 1710 (C=O), 1513 (C=N); 1H NMR (400 MHz, (CD3)2CO) δ 1.27 (d, 3H, J = 6.8 Hz, CH3), 2.43 (dd, 1HA, JAM = 7.6 Hz, JAX = 15.6 Hz, CH2CO), 2.88 (dd, 1HM, JMA = 6.8 Hz, JMX = 15.6 Hz CH2CO), 5.01–5.12 (m, 1HX, CHCH3), 6.76 (s, 1H, SCH), 6.97–7.87 (m, 9H, HAr), 10.67 (br. s, 1H, OH); 13C NMR (100 MHz, (CD3)2CO) δ 18.1 (CH3), 40.0 (CH2CO), 54.7 (CHCH3), 102.5 (SCH), 116.0, 116.2, 128.7, 128.8, 129.6, 131.1, 131.3, 133.0, 151.1, 162.0, 164.4 (CAr), 143.6 (N-C), 171.1 (C=N), 172.8 (C=O). Anal. Calcd for C19H17FN2O2S: C, 64.03; H, 4.81; N, 7.86%. Found: C, 64.25; H, 4.73; N, 7.98%.
3-{[4-(4-Chlorophenyl)-1,3-thiazol-2-yl](phenyl)amino}-2-methylpropanoic acid (6a) was prepared according to the general procedure from 1a and 2-bromo-1-(4-chlorophenyl)ethan-1-one to afford white solid, yield 1.70 g (91%), mp 128–129 °C; IR (KBr) νmax (cm-1): 3126 (OH), 1701 (C=O), 1508 (C=N); 1H NMR (400 MHz, DMSO-d6) δ 1.14 (d, 3H, J = 6.0 Hz, CH3), 2.763.08 (m, 1H, CHCH3), 3.924.43 (m, 2H, NCH2), 7.20 (s, 1H, SCH), 7.30–7.97 (m, 9H, HAr), 12.43 (s, 1H, OH); 13C NMR (100 MHz, DMSO-d6) δ 14.9 (CH3), 38.1 (CH), 55.2 (CH2), 103.4 (SCH), 126.9, 127.3, 127.5, 128.6, 130.1, 131.9, 133.5, 149.0 (CAr), 144.7 (S-CH=C), 169.7 (NCN), 175.8 (CO). Anal. Calcd for C19H17ClN2O2S: C, 61.20; H, 4.60; N, 7.51%. Found: C, 61.42; H 4.46; N, 7.68%.
3-{[4-(4-Chlorophenyl)-1,3-thiazol-2-yl](phenyl)amino}butanoic acid (6b) was prepared according to the general procedure from 1b and 2-bromo-1-(4-chlorophenyl)ethan-1-one to afford white solid, yield 1.51 g (81%), mp 182.5–183.5 °C; IR (KBr) νmax (cm-1): 3125 (OH), 1709 (C=O), 1533 (C=N); 1H NMR (400 MHz, (CD3)2CO) δ 1.42 (d, 3H, J = 6.8 Hz, CH3), 2.58 (dd, 1HA, JAM = 7.6 Hz, JAX = 15.6 Hz, CH2CO), 3.02 (dd, 1HM, JMA = 6.8 Hz, JMX = 15.6 Hz, CH2CO), 5.16–5.28 (m, 1H, CH), 7.00 (s, 1H, SCH), 7.30–8.06 (m, 9H, HAr), 10.08 (s, 1H, OH); 13C NMR (100 MHz, (CD3)2CO) δ 19.1 (CH3), 40.0 (CH), 54.7 (CH2), 103.5 (SCH), 128.4, 129.5, 129.7, 131.1, 131.3, 133.5, 135.2, 150.9 (CAr), 143.5 (S-CH=C), 171.2 (NCN), 172.8 (CO). Anal. Calcd for C19H17ClN2O2S: C, 61.20; H, 4.60; N, 7.51%. Found: C, 61.39; H, 4.75; N, 7.67%.
3-{[4-(4-Cyanophenyl)-1,3-thiazol-2-yl](phenyl)amino}-2-methylpropanoic acid (7a) was prepared according to the general procedure from 1a and 2-bromo-1-(4-isocyanophenyl)ethan-1-one to afford white solid, yield 1.47 g (81%), mp 173–174 °C; IR (KBr) νmax (cm-1): 3395 (OH), 2222 (CN), 1702 (C=O), 1513 (C=N); 1H NMR (400 MHz, DMSO-d6) δ 1.15 (d, 3H, J = 7.1 Hz, CH3), 2.832.93 (m, 1H, CHCH3), 4.09 (dd, 1HA, JAM = 7.2 Hz, JAX = 13.9 Hz, NCH2), 4.21 (dd, 1HM, JMA = 7.4 Hz, JMX = 13.8 Hz NCH2), 7.38 (t, 1H, J = 7.2 Hz, HAr), 7.44 (s, 1H, SCH), 7.458.07 (m, 8H, HAr), 12.32 (s, 1H, OH); 13C NMR (100 MHz, DMSO-d6) δ 15.0 (CH3), 38.0 (CH), 55.2 (CH2), 106.4 (SCH), 109.6, 126.2, 126.9, 127.6, 130.2, 132.7, 138.7, 148.5 (CAr), 119.0 (CN), 144.5 (S-CH=C), 169.9 (NC-N), 175.8 (CO). Anal. Calcd for C20H17N3O2S: C, 66.10; H, 4.72; N, 11.56%. Found: C, 65.87; H, 4.56; N, 11.45%.
3-{[4-(4-Cyanophenyl)-1,3-thiazol-2-yl](phenyl)amino}butanoic acid (7b) was prepared according to the general procedure from 1b and 2-bromo-1-(4-isocyanophenyl)ethan-1-one to afford white solid, yield 1.60 g (88%), mp 196–197 °C; IR (KBr) νmax (cm-1): 3396 (OH), 2222 (CN), 1707 (C=O), 1512 (C=N); 1H NMR (400 MHz, DMSO-d6) δ 1.30 (d, 3H, J = 6.8 Hz, CH3), 2.41 (dd, 1HA, JAM = 7.6 Hz, JAX = 15.5 Hz, CH2CO), 2.78 (dd, 1HM, JMA = 6.9 Hz, JMX = 15.5 Hz, CH2CO), 5.00–5.15 (m, 1H, CH), 7.37–8.08 (s, 10H, SCH and HAr), 12.51 (s, 1H, OH); 13C NMR (100 MHz, DMSO-d6) δ 18.5 (CH3), 40.0 (CH), 53.3 (CH2), 106.2 (SCH), 109.5, 126.2, 128.8, 129.9, 130.2, 132.6, 138.9, 148.5 (CAr), 119.1 (CN), 141.7 (S-CH=C), 169.8 (NCN), 172.6 (CO). Anal. Calcd for C20H17N3O2S: C, 66.10; H, 4.72; N, 11.56%. Found: C, 65.89; H, 4.59; N, 11.44%.
2-Methyl-3-{[4-(4-nitrophenyl)-1,3-thiazol-2-yl](phenyl)amino}propanoic acid (8a) was prepared according to the general procedure from 1a and 2-bromo-1-(4-nitrophenyl)ethan-1-one to afford yellow solid, yield 1.55 g (81%), mp 183–184 °C; IR (KBr) νmax (cm-1): 3128 (OH), 1710 (C=O), 1506 (C=N); 1H NMR (400 MHz, (CD3)2CO) δ 1.23 (d, 3H, J = 7.1 Hz, CH3), 3.05–3.11 (m, 1H, CHCH3), 4.18 (dd, 1HA, JAM = 7.4 Hz, JAX = 13.9 Hz, NCH2), 4.32 (dd, 1HM, JMA = 7.4 Hz, JMX = 13.9 Hz, NCH2), 7.30 (s, 1H, SCH), 7.47–8.25 (m, 9H, HAr), 10.81 (br. s, 1H, OH); 13C NMR (100 MHz, (CD3)2CO) δ 15.6 (CH3), 39.1 (CH), 56.6 (CH2), 107.4 (SCH), 124.8, 127.5, 128.3, 128.7, 131.1, 146.1, 147.8, 149.9 (CAr), 142.0 (S-CH=C), 171.4 (NCN), 176.1 (CO). Anal. Calcd for C19H17N3O4S: C, 59.52; H, 4.47; N, 10.96%. Found: C, 59.60; H, 4.34; N, 11.12%.
3-{[4-(4-Nitrophenyl)-1,3-thiazol-2-yl](phenyl)amino}butanoic acid (8b) was prepared according to the general procedure from 1b and 2-bromo-1-(4-nitrophenyl)ethan-1-one to afford yellow solid, yield 1.71 g (89%), mp 175–176 °C; IR (KBr), νmax (cm-1): 3392 (OH), 1701 (C=O), 1595 (NO2), 1504 (C=N); 1H NMR (400 MHz, (CD3)2CO) δ 1.44 (d, 3H, J = 6.8 Hz, CH3), 2.60 (dd, 1HA, JAM = 7.5 Hz, JAX = 15.6 Hz, CH2CO), 3.02 (dd, 1HM, JMA = 6.9 Hz, JMX = 15.6 Hz, CH2CO), 5.20–5.31 (m, 1H, CH), 7.32 (s, 1H SCH), 7.46–8.32 (s, 9H, HAr), 10.38 (s, 1H, OH); 13C NMR (100 MHz, DMSO-d6) δ 19.1 (CH3), 39.9 (CH), 54.7 (CH2), 107.3 (SCH), 124.8, 128.5, 129.9, 131.2, 131.3, 142.2, 147.8, 150.0 (CAr), 143.3 (S-CH=C), 171.5 (NCN), 172.8 (CO). Anal. Calcd for C19H17N3O4S: C, 59.52; H, 4.47; N, 10.96%. Found: C, 59.33; H, 4.55; N, 11.17%.
3-{[4-(3,4-Dichlorophenyl)-1,3-thiazol-2-yl](phenyl)amino}-2-methylpropanoic acid (9a) was prepared according to the general procedure from 1a and 2-bromo-1-(3,4-dichlorophenyl)ethan-1-one to afford white solid, yield 1.65 g (81%), mp 136–137 °C; IR (KBr) νmax (cm-1): 3117 (OH), 1703 (C=O), 1590 (NO2), 1506 (C=N); 1H NMR (400 MHz, DMSO-d6) δ 1.14 (d, 3H, J = 7.0 Hz, CH3), 2.802.94 (m, 1H, CHCH3), 4.07 (dd, 1HA, JAM = 7.1 Hz, JAX = 13.8 Hz, NCH2), 4.22 (dd, 1HM, JMA = 7.4 Hz, JMX = 13.8 Hz NCH2), 7.30–8.10 (m, 9H, HAr and SCH), 12.30 (s, 1H, OH); 13C NMR (100 MHz, DMSO-d6) δ 14.9 (CH3), 38.0 (CH), 55.17 (CH2), 104.8 (SCH), 125.7, 127.1, 126.9, 127.5, 129.7, 130.1, 130.8, 131.3, 135.2, 147.6 (CAr), 144.5 (S-CH=C), 169.8 (NCN), 175.7 (CO). Anal. Calcd for C19H16Cl2N2O2S: C, 56.03; H, 3.96; N, 6.88%. Found: C, 56.25; H, 4.09; N, 6.93%.

3-{[4-(3,4-Dichlorophenyl)-1,3-thiazol-2-yl](phenyl)amino}butanoic acid (9b) was prepared according to the general procedure from 1b and 2-bromo-1-(3,4-dichlorophenyl)ethan-1-one to afford white solid, yield 1.60 g (79%), mp 147–148 °C; IR (KBr) νmax (cm-1): 3387 (OH), 1703 (C=O), 1502 (C=N); 1H NMR (400 MHz, (CD3)2CO) δ 1.39 (d, 3H, J = 6.9 Hz, CH3), 2.55 (dd, 1HA, JAM = 7.5 Hz, JAX = 15.6 Hz, CH2CO), 2.79 (dd, 1HM, JMA = 6.9 Hz, JMX = 15.6 Hz, CH2CO), 5.19–5.27 (m, 1H, CH), 7.11 (s, 1H, SCH), 7.44–8.16 (m, 8H, HAr), 10.71 (s, 1H, OH); 13C NMR (100 MHz, (CD3)2CO) δ 19.1 (CH3), 39.9 (CH), 54.5 (CH2), 104.8 (SCH), 126.5, 128.5, 129.7, 131.1, 131.2, 131.5, 132.9, 136.8, 149.4 (CAr), 143.2 (S-CH=C), 171.3 (NCN), 172.8 (CO). Anal. Calcd for C19H16Cl2N2O2S: C, 56.03; H, 3.96; N, 6.88%. Found: C, 56.27; H, 4.11; N, 6.99%.
2-Methyl-3-{[4-(naphthalen-2-yl)-1,3-thiazol-2-yl](phenyl)amino}propanoic acid (10a) was prepared according to the general procedure from 1a and 2-bromo-1-(4-(naphthalen-2-yl)phenyl)ethan-1-one to afford yellow solid, 1.40 g (72%), mp 155–156 °C; IR (KBr), νmax (cm-1): 3355 (OH), 1740 (C=O), 1510 (C=N); 1H NMR (400 MHz, DMSO-d6) δ 1.10 (d, 3H, J = 7.0 Hz, CH3), 2.40–2.50 (m, 1H, CHCH3), 4.07–4.25 (m, 2H, NCH2), 7.24 (s, 1H, SCH), 7.30–8.40 (m, 8H, HAr), 10.75 (s, 1H, OH); 13C NMR (100 MHz, DMSO-d6) δ 16.3 (CH3), 25.5 (CH), 56.7, 62.0 (CH2), 102.9 (SCH), 124.0, 124.2, 125.9, 126.4, 126.8, 127.6, 128.0, 128.1, 129.8, 132.3, 132.4, 133.1, 150.2 (CAr), 144.9 (S-CH=C), 170.0 (NCN), 177.7 (CO). Anal. Calcd for C23H20N2O2S: C, 71.11; H, 5.19; N, 7.21%. Found: C, 69.86; H, 5.32; N, 7.06%.
3-{[4-(Naphthalen-2-yl)-1,3-thiazol-2-yl](phenyl)amino}butanoic acid (10b) was prepared according to the general procedure from 1b and 2-bromo-1-(4-(naphthalen-2-yl)phenyl)ethan-1-one to afford white solid, yield 1.63 g (84%), mp 147–148 °C; IR (KBr) νmax (cm-1): 3387 (OH), 1703 (C=O), 1502 (C=N); 1H NMR (400 MHz, (CD3)2CO) δ 1.39 (d, 3H, J = 6.9 Hz, CH3), 2.55 (dd, 1HA, JAM = 7.5 Hz, JAX = 15.6 Hz, CH2CO), 2.79 (dd, 1HM, JMA = 6.9 Hz, JMX = 15.6 Hz, CH2CO), 5.19–5.27 (m, 1H, CH), 7.11 (s, 1H, SCH), 7.44–8.16 (m, 12H, HAr), 10.71 (s, 1H, OH); 13C NMR (100 MHz, (CD3)2CO) δ 19.1 (CH3), 39.9 (CH), 54.5 (CH2), 104.8 (SCH), 126.5, 128.5, 129.7, 131.1, 131.2, 131.5, 132.9, 136.8, 149.4 (CAr), 143.2 (S-CH=C), 171.3 (NCN), 172.8 (CO). Anal. Calcd for C23H20N2O2S: C, 71.11; H, 5.19; N, 7.21%. Found: C, 69.89; H, 5.34; N, 7.03%.
2-Methyl-3-{[4-(2-oxo-2H-chromen-3-yl)-1,3-thiazol-2-yl](phenyl)amino}propanoic acid (11a) was prepared according to the general procedure from 1a and 3-(4-(2-bromoacetyl)phenyl)-2H-chromen-2-one to afford yellow solid, yield 1.81 g (89%), mp 198–199 °C; IR (KBr) νmax (cm-1): 3435 (OH), 1695 (C=O), 1492 (C=N); 1H NMR (400 MHz, DMSO-d6) δ 1.15 (d, 3H, J = 7.0 Hz, CH3), 2.742.86 (m, 1H, CHCH3), 4.104.24 (m, 2H, NCH2), 7.30–7.90 (m, 10H, HAr), 8.62 (s, 1H, SCH); 13C NMR (100 MHz, DMSO-d6) δ 15.2 (CH3), 38.30 (CH), 55.2 CH2), 109.5 (SCH), 115.8, 119.2, 120.3, 124.7, 127.5, 127.0, 128.8, 130.1, 131.5, 138.4, 143.7, 152.2 (CAr and chrom.), 144.5 (S-CH=C), 158.7 (O-C=O), 168.9 (NCN), 176.1 (CO). Anal. Calcd for C22H18N2O4S: C, 65.01; H, 4.46; N, 6.89%. Found: C, 64.77; H, 4.54; N, 6.80%.
3-{[4-(2-Oxo-2H-chromen-3-yl)-1,3-thiazol-2-yl](phenyl)amino}butanoic acid (11b) was prepared according to the general procedure from 1b and 3-(4-(2-bromoacetyl)phenyl)-2H-chromen-2-one to afford yellow solid, yield 1.79 g (88%), mp 178–179 °C; IR (KBr) νmax (cm-1): 3517 (OH), 1718, 1703 (2C=O), 1531 (C=N); 1H NMR (400 MHz, (CD3)2CO) δ 1.38 (d, J = 6.8 Hz, 3H, CH3), 2.53 (dd, 1HA, JAM = 7.3 Hz, JAX = 15.6 Hz, CH2CO), 2.95 (dd, 1HM, JMA = 6.9 Hz, JMX = 15.6 Hz, CH2CO), 5.195.26 (m, 1H, CH), 7.30–7.75 (m, 10H, HAr), 8.70 (s, 1H, SCH); 13C NMR (100 MHz, (CD3)2CO) δ 19.1 (CH3), 39.3 (CH), 54.4 CH2), 110.4 (SCH), 116.8, 120.7, 122.1, 125.5, 129.4, 129.7, 131.1, 131.3, 131.5, 132.2, 139.3, 143.1, 153.8 (CAr and chrom.), 145.4 (S-CH=C), 159.8 (O-C=O), 170.2 (NCN), 172.8 (CO). Anal. Calcd for C22H18N2O4S: C, 65.01; H, 4.46; N, 6.89%. Found: C, 64.80; H, 4.53; N, 6.73%.
General procedure for preparation of 12a,b and 13a,b. A mixture of corresponding thioureido acid 1a,b (1.19 g, 5 mmol), 2,3-dichloro-1,4-naphthoquinone (1.36 g, 6 mmol) or 2,3-dichloroquinoxaline (1.19 g, 6 mmol), sodium acetate (1,48 g, 18 mmol), and acetic acid (25 mL) was stirred at 80 °C for 24 h. Afterwards, it was cooled to room temperature and diluted with water (100 mL). The precipitate formed was filtered off and washed with water. Purification was performed by dissolving the crystals in 5% aqueous KOH (175 mL), filtering, and acidifying the filtrate with acetic acid to pH 6.
2-Methyl-3-[phenyl([1,3]thiazolo[4,5-b]quinoxalin-2-yl)amino]propanoic acid (12a). Yellow solid, yield 1.37 g (75%), mp 250–251 C (decomp.); IR (KBr) νmax (cm-1): 3048 (OH), 1766 (CO), 1683, 1614, 1595 (3 C=N); 1H NMR (400 MHz, DMSO-d6) δ 1.12 (d, 3H, J = 7.0 Hz, CH3), 2.933.00 (m, 1H, CHCH3), 3.72–3.85 (m, 2H, NCH2), 7.047.43 (m, 9H, HAr), 11.89 (br. s, 1H, OH). Anal. Calcd for C19H18N4O2S: C, 62.28; H, 4.95; N, 15.29%. Found: C, 62.17; H, 5.09; N, 15.42%.
3-[Phenyl([1,3]thiazolo[4,5-b]quinoxalin-2-yl)amino]butanoic acid (12b). Yellow solid, yield 1.57 g (86%), mp 280281 C (decomp.); IR (KBr) νmax (cm-1): 3559 (OH), 1708 (CO), 1608, 1590, 1556 (3C=N); 1H NMR (400 MHz, DMSO-d6) δ 1.22 (d, 3H, J = 7.0 Hz, CH3), 2.88–2.97 (m, 2H, CH2CO), 5.19–5.27 (m, 1H, CHCH3), 7.078.04 (m, 9H, HAr), 11.90 (br. s, 1H, OH). Anal. Calcd for C19H18N4O2S: C, 62.28; H, 4.95; N, 15.29%. Found: C, 62.13; H, 5.12; N, 15.46%.
3-[(4,9-Dioxo-4,9-dihydronaphtho[2,3-d][1,3]thiazol-2-yl)(phenyl)amino]-2-methylpropanoic acid (13a). Dark brown solid, yield 1.44 g (73%), mp 181–182 °C (decomp.); IR (KBr) νmax (cm-1): 3062 (OH), 1707, 1642, 1626 (3CO), 1522 (C=N); 1H NMR (400 MHz, DMSO-d6) δ 1.17 (d, 3H, J = 7.1 Hz, CH3), 2.732.86 (m, 1H, CHCH3), 4.234.30 (dd, 1HA, JAB = 7.2 Hz, JAX = 13.8 Hz, NCH2), 4.33–4.41 (dd, 1HB, JBA = 7.6 Hz, JBX = 13.8 Hz, NCH2), 7.327.99 (m, 9H, HAr), 12.19 (br. s, 1H, OH). Anal. Calcd for C21H16N2O4S: C, 64.27; H, 4.11; N, 7.14%. Found: C, 64.02; H, 4.29; N, 7.31%.
3-[(4,9-Dioxo-4,9-dihydronaphtho[2,3-d][1,3]thiazol-2-yl)(phenyl)amino]butanoic acid (13b). Dark brown solid, yield 1.57 g (80%), mp 195–196 °C (decomp.); IR (KBr) νmax (cm-1): 3375 (OH), 1710, 1637, 1623 (3CO), 1519 (C=N); 1H NMR (400 MHz, DMSO-d6) δ 1.34 (d, 3H, J = 6.8 Hz, CH3), 2.682.75 (dd, 1HA, JAB = 7.4 Hz, JAX = 15.0 Hz, CH2CO), 2.782.86 (dd, 1HB, JBA = 7.4 Hz, JBX = 15.0 Hz, CH2CO), 5.255.35 (m, 1H, CHCH3), 7.467.98 (m, 9H, HAr), 12.45 (br. s, 1H, OH). Anal. Calcd for C21H16N2O4S: C, 64.27; H, 4.11; N, 7.14%. Found: C, 64.05; H, 4.30; N, 7.33%.
General procedure for preparation of 15a,c and 16. A mixture of corresponding ureido acid 14a-c or N-phenylurea (4.5 mmol), 2,3-dichloro-1,4-naphthoquinone (1,04 g, 4.5 mmol, 98%) and water (30 mL) was stirred under reflux for 14 h. The precipitate was filtered off and washed with water. Products were isolated by column chromatography (acetone:hexane).
3-((4,9-Dioxo-4,9-dihydronaphtho[2,3-d]oxazol-2-yl)(phenyl)amino)-2-methylpropanoic acid (15a). Dark blue solid, yield 0.50 g (29%), mp 134–135 °C; Rf = 0.511 (acetone:hexane, 1:1); 1H NMR (400 MHz, DMSO-d6) δ 1.17 (d, 3H, J = 7.2 Hz, CH3), 2.842.88 (m, 1H, CHCH3), 3.75 (dd, 1HA, JAB = 4.6 Hz, JAX = 15.3 Hz, NCH2), 4.16 (dd, 1HB, JBA = 8.9 Hz, JBX = 15.3 Hz, NCH2), 6.917.40 (m, 5H, HAr), 7.928.09 (m, 4H, HAr), 12.31 (br.s., 1H, OH); 13C NMR (100 MHz, DMSO-d6) δ 15.89 (CH3), 35.43 (CH), 54.19 (CH2), 112.50, 117.03, 125.69, 126.24, 127.36, 129.10, 129.51, 131.88, 132.04, 134.72, 137.79, 146.62, 147.95 (CAr+oxazole), 176.89 (COOH), 178.33 (C=O), 180.84 (C=O); Anal. Calcd for C21H16N2O5: C, 67.02; H, 4.29; N, 7.44%. Found: C, 67.10; H, 4.15; N, 7.55%.
3-((4,9-Dioxo-4,9-dihydronaphtho[2,3-d]oxazol-2-yl)(phenyl)amino)propanoic acid (15c). Dark blue solid, yield 0.43 g (26%), mp 127-128 °C; Rf = 0.51 (acetone:hexane, 1:1); 1H NMR (400 MHz, DMSO-d6) δ 2.69 (t, 2H, J = 6.9 Hz, CH2CO), 4.02 (t, 2H, J = 6.9 Hz, NCH2), 6.877.25 (m, 5H, HAr), 7.888.10 (m, 4H, HAr), 12.27 (br.s., 1H, OH); 13C NMR (100 MHz, DMSO-d6) δ 33.32 (CH2), 46.80 (CH2), 117.10, 120.91, 126.79, 127.26, 129.57, 131.94, 132.09, 134.63, 134.70, 137.45, 145.80, 148.05 (CAr +oxazole), 173.48 (COOH), 178.43 (C=O), 181.05 (C=O); Anal. Calcd for C20H14N2O5: C, 66.30; H, 3.89; N, 7.73%. Found: C, 66.42; H, 3.95; N, 7.80%.
2-(Phenylamino)naphtho[2,3-d]oxazole-4,9-dione (16). Red solid, yield from 14a – 0.31 g (24 %), from 14b – 0,90 g (69%), from 14c – 0.47 g (36%), from N-phenylurea – 1.01 g (77%), mp 179–180 °C; Rf = 0.53 (acetone:hexane, 1:2); 1H-NMR (400 MHz, DMSO-d6) δ 7.137.31 (m, 5H, HAr), 7.748.02 (m, 4H, HAr), 9.28 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) δ 113.88, 123.59, 124.01, 125.71, 126.14, 127.54, 129.89, 131.59, 132.81, 134.41, 138.45, 142.76 (CAr +oxazole), 176.30 (C=O), 179.73 (C=O); Anal. Calcd for C17H10N2O3: C, 70.34; H, 3.47; N, 9.65%. Found: C, 70.41; H, 3.56; N, 9.74%.

Evaluation of antimicrobial activity. The following bacteria strains were used: Gram-positive cocci Staphylococcus aureus (ATCC 9144) Gram-negative rod Escherichia coli (ATCC 8739), Salmonella enteritidis (ATCC 8739), and Pseudomonas aeruginosa (NCTC 6750). Tryptic soy agar (TSA) and tryptic soy broth (TSB) were used for bacteria cultivation and antibacterial activity tests. Antibacterial activity of the compounds was determined by testing their different concentrations against B. cereus, S. aureus, P. aeruginosa and E. coli bacteria by the broth-dilution and spread plate methods.36,37 A range of concentrations, 1000, 500, 250, 125, and 62.5 µg/mL, were prepared for each sample. They were streaked out on TSA plates and incubated at 37 °C for 24 h. A representative colony was placed in 5 mL of TBS and incubated at 37 °C for 24 h. S. aureus, E. coli, S. enteritidis, and P. aeruginosa cultures containing 108 CFU/mL (colony-forming units corresponding to McFarland’s 0.5) were diluted with TSB and used for the antibacterial test. The test organisms (0.1 mL) were added to each tube and incubated at 37 °C for 24 h. At the end of this period, a small amount of the diluted mixture (different materials) from each tube was pulled out and spread on TSA. The plates were incubated at 37 °C for 48 h. The growth of bacterial cells was observed on agar plates. The lowest concentration of the bacterial material at which no growth was observed was considered as the minimum bactericidal concentration (MBC) value.38 Minimum inhibitory concentration (MIC) is the lowest concentration of an antimicrobial that inhibits the visible growth of a microorganism after overnight incubation. Oxytetracycline inoculated with each test bacterium in the tubes and plates was used as a control. The growth of the test bacteria was observed in all plates as positive control.

ACKNOWLEDGMENTS
Postdoctoral fellowship is being funded by European Union Structural Funds project ”Postdoctoral Fellowship Implementation in Lithuania” within the framework of the Measure for Enhancing Mobility of Scholars and Other Researchers and the Promotion of Student Research (VP1-3.1-ŠMM-01) of the Program of Human Resources Development Action Plan.

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