Synthesis and Biological Activity of 3-[Phenyl(1,3-thiazol-2-yl)-amino]propanoic Acids and Their Derivatives

New N,N-disubstituted β-amino acids and their derivatives with thiazole, aromatic, and heterocyclic substituents were synthesized from N-phenyl-N-thiocarbamoyl-β-alanine by the Hantzsch method; derivatives with hydrazone fragments were also obtained. Some of the synthesized compounds exhibited discrete antimicrobial activity, and 3-[(4-oxo-4,5-dihydro-1,3-thiazol-2-yl)(phenyl)amino]propanoic acid was found to promote rapeseed growth and to increase seed yield and oil content.

The studies showed that the best yield was obtained when reactions were carried out in water in the presence of sodium carbonate. Then the reaction mixture was acidified with acetic acid to pH 6. In order to obtain more thiazole derivatives and to assess the influence of substituents on the biological activity, a series of 3-{[5-substituted 4-oxo-4,5-dihydro-1,3-thiazol-2-yl](phenyl)amino}propanoic acids 3 with aromatic and heterocyclic fragments was synthesized (Scheme 1) . The syntheses were performed by condensation of dihydrothiazolone 2 and aromatic or heterocyclic aldehydes. Since the starting compound 2 and the resulting products 3 are soluble in an alkaline medium, the reactions were carried out in aqueous sodium carbonate solution in the presence of glycine as a bifunctional catalyst. The target products from the reaction mixtures were separated by acidification of the aqueous solutions with acetic acid to pH 6. The preliminary laboratory tests showed that the compounds 2 and 3 had growth regulating effects for rapeseed (Brassica napus L.) and St. John's wort (Hypericum perforatum L.) [34]. In the same way, the compound 4 was synthesized from dihydrothiazolone 2 and terephthaldehyde.  The structure of the synthesized compounds 3a-i was confirmed by elemental analysis, 1 H-NMR, 13 C-NMR and IR spectral data. For example, in the 1 H-NMR spectrum of compound 3a, the CH 2 CO and NCH 2 group proton signals appear at 2.57 and 4.26 ppm, whereas aromatic and CH proton signals are observed in the range of 7.32 to 7.63 ppm. In the 13 C-NMR spectrum of compound 3a, the signals of the CH 2 CO and NCH 2 groups are observed at 34.57 and 51.44 ppm, respectively, whereas aromatic and CH= group carbon signals are observed in the range 111.96-140.42 ppm. The combination of CS, CN, CO, and COOH carbon signals are observed at 133.34, 171.94, 175.79, and 179.19 ppm respectively. In the IR spectrum, the C=N absorption band is observed at 1,540 cm −1 , two C=O bands at 1,686 and 1,725 cm −1 , and a band belonging to the OH group at 3,435 cm −1 are observed. The formation of isomeric structures in these reactions was not observed. According to data published in the literature, glycine used as a bifunctional catalyst gives a single isomer (Z) [35].
3-[Phenyl(1,3-thiazol-2-yl)amino]propanoic acid (5) was synthesized from compound 1 and chloroacetaldehyde in refluxing water solution after 2 hours. A water-soluble amino acid hydrochloride is formed, which is present in the base transferred by the addition of sodium acetate to the reaction mixture. The structure of compound 5 is confirmed by its NMR spectral data: in the 1 H-NMR spectrum two triplets at 2.62 and 4.11 ppm are assigned to the CH 2 CO and NCH 2 group protons, while two doublets at 6.71 and 7.18 ppm belong to the thiazole ring and the SCH NCH group protons. Aromatic ring proton signals are observed at 7.31-7.50 ppm, and broad proton singlets of the carboxyl group are detectable at 12.30 ppm.
Reactions of 1 with various haloketones were also performed, and a number of new thiazole derivatives 6-11 with aliphatic, aromatic, or heterocyclic substituents were thus synthesized. The compounds 6-10 were synthesized in acetone or 2-propanol at boiling temperature. Cyclic diketonesquinones-are interesting both for their chemical properties and practical uses. Besides, many compounds containing the quinone group are found in Nature. Various assays showed that these compounds are characterized by a broad spectrum of biological activities [36]. A quinone fragmentcontaining compound 11 in this work was synthesized from N-phenyl-N-thiocarbamoyl--alanine (1) and 2,3-dichloro-1,4-naphthoquinone. The reaction was carried out in acetic acid at 80 °C for 24 h in the presence of sodium acetate. Then the mixture was cooled and diluted with water. The synthesized 3-(N- (4,9-dihydro-4,9-dioxonaphtho[2,3-d]thiazol-2-yl)-N-phenylamino)propanoic acid (11) was purified by dissolving it in aqueous sodium hydroxide solution, filtering, and then acidifying the filtrate with acetic acid to pH 6.
The compound 8 was modified by introducing a hydrazone fragment into its molecule with the porpose of increasing its antimicrobial properties (Scheme 2). Firstly, acid 8 was esterified with methanol to obtain the corresponding ester 12, which afterwards was boiled in 2-propanol with hydrazine monohydrate. The resulting acid hydrazide 13 was condensed with aromatic and heterocyclic aldehydes. The NMR spectra of the synthesized hydrazones 14 showed that in DMSO-d 6 solution they exist as a mixture of E/Z isomers due the presence of a CO-NH fragment in the molecule and its hindered rotation around the CO-NH bond; however, the Z isomer was dominant.

Antimicrobial Evaluation of Synthesized Compounds
The antibacterial activity of the compounds was determined against Gram-positive spore-forming rods of Bacillus cereus (ATCC 11778), Gram-positive cocci of Staphylococcus (ATCC 9144), Gram-negative rods of E. coli (ATCC 8739) and Pseudomonas aeruginosa (NCTC 6750) by the broth and spread-plate methods. A range of concentrations for each compound was prepared according to the experimental procedure mentioned in experimental section. The minimal inhibition concentration (MIC, µg/mL) values are listed in Table 1. ni-no inhibition. The minimum inhibitory concentrations were determined by the micro broth dilution method.
The MIC values of the samples were obtained in spread-plate tests, considering compound concentrations demonstrating antibacterial activity. Antibacterial screening data revealed a prominent inhibitory activity of compounds 3a-g, and 3i against the tested strains of S. aureus, B. cereus, E. coli and P. aeruginosa (MIC = 250 µg/mL). The MIC value of 350 µg/mL was obtained against all bacteria for compounds 3a, 3b, 3c, 3d, 3f, 3e, 3i, 6, 7, 8, and 11, while for compounds 10 and 9, depending on bacterial species they were 500 or 1000 µg/mL. Compounds 2, 5 didn't show antibacterial activity against the test microorganisms. Based on these data, antibacterial activity of the investigated compounds may be arranged in the following order: Staphylococcus aureus: Increasing antibacterial activity Bacillus cereus: Increasing antibacterial activity Increasing antibacterial activity Pseudomonas aeruginosa: Increasing antibacterial activity The minimum bactericidal values (MBC) are presented in Table 2. These data also revealed pronounced inhibitory activity of some synthesized compounds against some bacteria species, particulalry S. aureus and B. cereus at 250 µg/mL. For instance, the lowest MBC values (250-500 µg/mL) were found for the compounds 3i, 3g, 11. However, the compounds 2, 3h, 5 didn't show any bactericidal activity against the tested microorganisms. Based on these experiments, bactericidal activity of the investigated compounds may be arranged in the following order: Decreasing MBC Values against Staphylococcus aureus:

Increasing bactericidal activity
Decreasing MBC Values against Bacillus cereus:

Increasing bactericidal activity
Decreasing MBC Values against Esherichia coli:

Increasing bactericidal activity
Decreasing MBC Values against Pseudomonas aeruginosa: Increasing bactericidal activity Among the all synthesized compounds, the highest antibacterial activity was exhibited by thiazole compound 11 containing a naphthoquinone ring. Among compounds 3a-i, the highest antibacterial activity was shown by the compounds with furan and bromothiophene substituents in their structure. Aromatic substituents with different groups (NO 2 , F, Cl) didn't show any inhibiting effect. Among thiazole compounds 5-9, the highest antibacterial activity was exhibited by compound 8 with a phenyl substituent in the 4-position of the thiazole ring.
Attempting to increase antimicrobial activity the structure of compound 8 was modified by replacing the carboxy group by hydrazone and hydrazide derivatives with aromatic and heterocyclic substituents, however, the results obtained suggest that modification of the carboxy group decreases the antimicrobial activity.

Evaluation of Synthesized Compounds on Rapeseed Yield and Biochemical Content
The effect of 3-[(4-oxo-4,5-dihydro-1,3-thiazol-2-yl)(phenyl)amino]propanoic acid (2) on the rapeseed yield and biochemical content is presented in Table 3. When rapeseed seedlings were sprayed with solutions of 2 of various concentrations, the yield of seeds ranged from 2.07 to 2.44 t/ha, i.e., 19%-40% higher in comparison with the control sample. The highest seed yield was obtained when rapeseed seedlings were sprayed with 150 mg/L of the test compound solution. Other quality characteristics were also affected by spraying rapeseed seedlings with the test compound; for instance, the highest mass of 1,000 seeds (4.17 g) was determined when 25 mg/L were applied, whereas at the same concentration the oil content increased by 13.3%-39.3% in comparison with the control sample. Table 3. The effect of 3-[(4-oxo-4,5-dihydro-1,3-thiazol-2-yl)(phenyl)amino]propanoic acid (2) on rapeseed yield and biochemical content. (2) Application of 3-[(4-oxo-4,5-dihydro-1,3-thiazol-2-yl)(phenyl)amino]propanoic acid (2) increased both the content of oil and protein. The highest content of oil (334 kg/t) and protein (31.2 mg/100g) was obtained upon using the 75 mg/L concentration of the study compound solution. The content of protein varied from 15.9 to 31.2 mg/100 g. When rapeseed seedlings were sprayed with 75 mg/L of the test compound solution, protein content was two times higher in comparison with the control sample.

The concentration of 3-[(4-oxo-4,5-dihydro-1,3-thiazol-2-yl)(phenyl)amino]propanoic acid
The effect of 3-[(4-oxo-4,5-dihydro-1,3-thiazol-2-yl)(phenyl)amino]propanoic acid (2) on the composition of fatty acids in rapeseed is presented in Table 4. The results showed that the variation of oleic acid content was insignificant (−0.09 to +0.77%) in comparison with the control sample. When rapeseed had been sprayed with 50 mg/L of the aforementioned solution, the highest content of oleic acid (61.51%) was obtained in the seeds. The content of saturated fatty acids (palmitic and stearic) varied insignificantly in comparison with the control sample. When rapeseed had been sprayed with the test compound solutions at various concentrations, some increase in the content of linolenic acid was observed; however, it was insignificant. Upon spraying rapeseed seedlings with 75 mg/L of the study compound solution, the highest content of linoleic acid (20.96%) was obtained in the seeds. Summarizing the data of the field experiments spraying rapeseed seedlings with different concentrations of 3-[(4-oxo-4,5-dihydro-1,3-thiazol-2-yl)(phenyl)amino]propanoic acid (2) solutions resulted in the following: (a) 100 mg/L: the highest number of pods and siliques was obtained; (b) 150 mg/L: the highest seed yield was obtained; (c) 25 mg/L: the highest seed mass was obtained.
During osmotic stress plants accumulate in the cytoplasm of their cells organic osmolytes such as proline, valine, isoleucine, ectoine, aspartic acid, betaine, glucose, fructose, sucrose, fructans, mannitol, pinitol, and myo-inositol (inositol) [37]. The regulating effect of compound 2 on rapeseed could be explained by the properties of typical structure osmolytes: they could exist in the zwitterionic form (Scheme 3).

General
The starting materials and solvents were obtained from Sigma-Aldrich Chemie GmbH (Munich, Germany) and Fluka (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) spectrometer (Varian, Inc.,Palo Alto, CA, USA) and Bruker Avance III/400 (400 MHz). Chemical shifts are expressed as , ppm relative to TMS. The J constants are given in Hz. The IR spectra (, cm −1 ) were recorded on a Perkin-Elmer BX FT-IR spectrometer (Perkin-Elmer Inc.,Waltham, MA, USA) using KBr tablets. Mass spectra were obtained on a Waters ZQ 2000 spectrometer (Waters, Eschborn, Germany) using the electrospray ionization (ESI) mode and operating at 25 V. Elemental analyses were performed with a CE-440 elemental analyzer (Exeter Analytical Inc., 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 Merck, Silica gel 60 F254 (Kieselgel 60 F254) plates. (2). A mixture of thioureido acid 1 (2.24 g, 0.01 mol), chloroacetic acid (1.04 g, 0.011 mol), sodium carbonate (1.16 g, 0.011 mol) and water (30 mL) was refluxed for 5 h. Then the mixture was acidified with acetic acid (6 mL) to pH 6. The precipitate was filtered off, washed with water, and crystallized.

Antimicrobial Activity
The following bacteria strains were used: gram-negative rods of Escherichia coli (ATCC 8739), Pseudomonas aeruginosa (NCTC 6750), gram-positive cocci of Staphylococcus aureus (ATCC 9144) and gram-positive spore forming rods of Bacillus cereus (ATCC 11778). 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 [39,40]. A range of concentrations, 1000, 500, 350, 250, and 125 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.
B. cereus, S. aureus, E. coli, and P. aeruginosa cultures containing 10 8 CFU/mL (colony-forming units corresponding to McFarland's 0.5) were diluted with TSB and used for the antibacterial test. The test organisms (100 µL) 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 [41]. The antibacterial compound 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 controls.

Field Trials and Rapeseed Biochemical Ontent
The influence of N-substituted β-alanine derivatives on the growth of rapeseed (Brassica napus L.) and St. John wort (Hypericum perforatum L.) in vitro was explored. According to the results, 3-[(4oxo-4,5-dihydro-1,3-thiazol-2-yl)(phenyl)amino]propanoic acid (2) was selected, because it had the most remarkable positive effect on the growth of rapeseed and St. John wort in vitro [34]. Following the data obtained during the in vitro experiments, compound 2 was selected for the field trials.
Experiments with 3-[(4-oxo-4,5-dihydro-1,3-thiazol-2-yl)(phenyl)amino]propanoic acid on spring rapeseed (Brassica napus L.) of the SW Landmark variety were carried out in field trials in 2012 at the Lithuanian Research Centre for Agriculture and Forestry branch Rumokai experimental station. Spring rapeseed was sprayed with the study compound solutions (concentrations 25-150 mg/L) before flowering. For each field, 1 L of the solution was used. The soil of the experimental field was carbonaceous shallow luvisols (Calc or epihypogleyic luvisol IDg8-k (LVg-w-cc) with the following charateristics: pH KCl 6.7, humus 1.82%, N total 0.12%, 308 mg kg −1 of mobile P 2 O 5 , 296 mg kg −1 of mobile K 2 O. Soil samples were collected on 25.04.2012. Barley was the preceding crop. The size of the initial and accounted fields was 2.7 × 11 m and 2.2 × 10 m, respectively. Experiments with rapeseed were performed in April-August. The average air temperature ranged from 7.9 to 19.3 °C, the rainfall ranged from 44.4 to 118.4 mm per month, and during the experiment it was 361.1 mm. The rainfall was by 11.1 mm higher than the average multi-annual precipitation. Biometric measurements were carried out after harvesting. The moisture of rape seeds was 8.5%. The oil content in rape seeds was measured by extraction with hexane for 3 hours in a Soxhlet apparatus (Behr Labor-Technik, Dusseldorf, Germany). Fatty acid composition was analysed with a HRGC 5300 Mega Series gas chromatograph Carlo Erba Strumentazione (Milano, Italy). The protein content in rape seeds was measured by the Bradford method.

Conclusions
The N-phenyl-N-thiocarbamoyl-β-alanine was used for the first time for the synthesis of biologically active 2-aminothiazole derivatives. By a new method of synthesizing the target structures we have established a convenient way to produce a number of compounds, which were used for biological testing. Some of the synthesized compounds exhibited promising antimicrobial activity, and 3-[(4-oxo-4,5-dihydro-1,3-thiazol-2-yl)(phenyl)amino]propanoic acid (2) was found to promote the growth, to increase seed yield and oil content in rapeseed.