Synthesis, Characterization and Bioassay of Novel Substituted 1-(3-(1,3-Thiazol-2-yl)phenyl)-5-oxopyrrolidines

Thiazole derivatives attract the attention of scientists both in the field of organic synthesis and bioactivity research due to their high biological activity. In the present study, thiazole ring was obtained by the interaction of 1-(4-(bromoacetyl)phenyl)-5-oxopyrrolidine-3-carboxylic acid with thiocarbamide or benzenecarbothioamide, as well as tioureido acid. A series of substituted 1-(3-(1,3-thiazol-2-yl)phenyl)-5-oxopyrrolidines with pyrrolidinone, thiazole, pyrrole, 1,2,4-triazole, oxadiazole and benzimidazole heterocyclic fragments were synthesized and their antibacterial properties were evaluated against Gram-positive strains of Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes and Gram-negative Pseudomonas aeruginosa, Escherichia coli and Salmonella enterica enteritidis. The vast majority of compounds exhibited between twofold and 16-fold increased antibacterial effect against the test-cultures when compared with Oxytetracycline.


Chemistry
In this work, the initial compound 1 was prepared from 4-aminoacetophenone and itaconic acid by a known method [42]. In order to obtain 2,5-disubstituted thiazole derivatives 3a-c, compound 1 was brominated with Br 2 in acetic acid at room temperature (Scheme 1), and then used in the reactions with thiocarbonyl compounds. The bromination reaction resulted in the formation of α-bromocarbonyl compound 1-(4-(2-bromoacetyl)phenyl)-5-oxopyrrolidine-3-carboxylic acid (2). Cyclocondensation of α-bromoacyl derivative 2 with the corresponding thioamide, i.e., thiocarbamide, benzenecarbothioamide or thioureido acid, under different conditions was carried out to obtain the desired outcomes 3a-c. The a reaction was performed in acetic acid at 60 • C; in the cases of b and c, the reaction was carried out in refluxing acetone. A comparison of the 1 H and 13 C-NMR spectra of compounds 3a-c revealed the characteristic signals of the formed substituted thiazole moieties. In the spectra of compound 3a, the singlets at 6.96 (S-C=CH) and 7.07 (NH 2 , 1 H-NMR) ppm, and the resonance lines at 100.9 (C-S), 138.1 (S-C=CH), 168.2 (C=N, 13 C-NMR) ppm prove the presence of thiazole heterocycle in the molecule. The characteristic signals in the 1 H (8.20 ppm, S-C=CH) and 13 C (114.0, 154.7, 166.9 ppm, C-S, S-C=CH, C=N) NMR, as well as additional resonances in the aromatic field of the corresponding spectrum of 3b, provide the evidence of new 2-phenyl thiazole fragment. The formation of the 2-carboxyethyl-4-methylanilino moiety in compound 3c was confirmed by the presence of triplets at 2.40 (J = 7.9 Hz, NCH 2 CH 2 CO) and 4.10 (J = 7.9 Hz, NCH 2 CH 2 ) ppm in the 1 H-NMR spectrum. The signals of protons of the p-substituted benzene cycle resonated as two doublets at 7.26 and 7.32 ppm and the signal arising at 2.32 ppm indicated the presence of the methyl group. All NMR spectra of the synthesized compounds are given in Supplementary Materials. Compounds 5a,b were prepared following the standard synthesis route, i.e., 5-oxopyrrolidine-3-carboxylic acids 3a,b were esterified with methanol in the presence of a catalytic amount of sulfuric acid to obtain esters 4a,b. The prepared esters were easily converted to the corresponding acid hydrazides 5a,b, when treated with hydrazine hydrate in refluxing propan-2-ol.
The condensation of acid hydrazides 5a,b with aromatic aldehydes in dimethylformamide or 1,4-dioxane afforded hydrazone-type compounds (6-8)a,b, and (9-12)b in good to excellent yield (62-99%, Scheme 2; the yields of all synthesized compounds are given in Table 1). The synthesized hydrazones in DMSO-d 6 solution, due to the restricted rotation around the CO-NH bond, exist as a mixtures of E/Z-amide conformers with the prevailing Z conformational structure [43][44][45]. However, geometric isomers are also possible due to the presence of the N=C double bond. The academic literature indicates that hydrazones obtained from acid hydrazides and aromatic aldehydes favour the sterically less-hindered and more stable geometric E-geometrical isomer [46][47][48].
The reaction of acid hydrazides 5a,b with hexane-2,5-dione (2,5-HD) resulted in the formation of 2,5-dimethylpyrrole derivatives 13a,b, as expected. Using the method of the formation of pyrrolidinone ring from aromatic amines and itaconic acid, hydrazide 5b as an amino group-containing compound was reacted with this dicarboxylic acid. The reaction was performed in toluene at reflux, and the synthesized compound 14b containing two pyrrolidinone rings connected by the amide bond was proved by double sets of the protons ( 1 H-NMR) of the 2COCH 2 , 2CH and 2NCH 2 groups, which gave rise in the ranges of 2.54-2.88, 3.25-3.42 and 3.55-4.16 ppm, respectively. In the 13 C-NMR spectrum of 14b spectral lines at 31.3, 33.6, 34.2, 35.5, 49.7 and 50.3 ppm have been attributed to the COCH 2 , CH and NCH 2 groups of two pyrrolidinone rings, and resonances at 170.9, 171.4, 171.8 and 174.6 ppm approve the presence of four carbonyl groups in this structure. NMR spectral data of compound 14b showed the presence of only one isomer in the DMSO-d 6 solution. Unfortunately, attempts to grow single crystals suitable for stereochemical assignment by X-ray structural analysis were unsuccessful.
The reaction of hydrazide 5b with carbon disulfide in methanol under basic conditions (KOH/methanol) gave the intermediate, the potassium dithiocarbazate salt, which under the action of concentrated hydrochloric acid was cyclized to 5-thioxo-1,3,4-oxadiazole 17b. The treatment of potassium carbodithioate with hydrazine monohydrate afforded 4-aminotriazole 18b. The condensation of one equivalent of compound 18b containing free amino group with two equivalents of benzenecarbaldehyde in refluxing ethanol containing a catalytic amount of concentrated hydrochloric acid yielded the Schiff base 19b. The characteristic changes in the chemical shift of the resonances of the triazole moiety were observed because of the changed influence of the substituent at the nitrogen atom of the C-N-C=S fragment. In the case of 19b, the carbon atom of the C=S group resonated at 162.9 ppm, and the C=N-NH carbon atom peaked at 162.0 ppm in 13 C-NMR spectrum. Additional spectral lines were observed in the aromatic region and were assigned to the carbon atoms of the newly incorporated benzene ring and formed azomethine group. The 1 H-NMR spectrum of 1,2,4-triazole derivative 19b showed characteristic singlets at 10.14 ppm (N=CH) and 13.97 (NH), and the multiplet of the benzene rings integrated for 14 protons.
In the next stage of the work, the reactivity of acyl-1 and α-bromo acyl 2 derivatives was investigated by reacting them with benzene-1,2-diamine (Scheme 3). The reactions were performed in different solvents. The reaction of the derivative 1 with diamine in ethanol yielded the condensation product 1-(4-(1-((2-aminophenyl)iminoethyl)phenyl)-5-oxopyrrolidine-3-carboxylic acid (20), while the bromoacyl derivative 2 under the same conditions afforded the compound 21 containing the quinoxaline moiety. The condensation in the compound 1 occurred in the acyl moiety. This resulted in a conjugated system with double bonds in the chain of the benzene ring-iminoethyl fragment-benzene ring, which gave the brightly brown-orange crystalline compound 20. The obtained compound 20 was easily identified by the data of the NMR spectra. In the 1 H and 13 C-NMR spectra of compound 21, the increased number of spectral lines in the aromatic regions of the respective spectra, the HRMS (High-resolution mass spectrometry) data, and the elemental analysis approved the formation of quinoxaline moiety. The second step of the investigation was to find out the products of the same reaction in refluxing 4 M hydrochloric acid. The reaction of compound 1, having an acyl group on the benzene ring with benzene-1,2-diamine in refluxing 4 M hydrochloric acid, afforded the benzimidazole derivative 22 [49]. Resynthesis was carried out according to a well-known Philips method (the heating of reagents in a 4 M HCl); however, extending it to 16 h and neutralizing the mixture with aqueous ammonium hydroxide to pH 7. This resulted in the increased yield of the desired product 22 (from 40 to 92%). The condensation of brominated compound 2 with diamine under the same conditions led to the formation of the complex mixture, and product separation was unsuccessful.

Biological Activity
All synthesized compounds 2-19 and 21-23 were tested against Gram-positive bacteria strains of Staphylococcus aureus (ATCC 25923), Bacillus cereus (ATCC 10231), Listeria monocytogenes (ATCC 19111) as well as Gram-negative Pseudomonas aeruginosa (ATCC 10145), Escherichia coli (ATCC 8739) and Salmonella enterica enteritidis (ATCC 13076) bacteria for their in vitro antibacterial activity by broth dilution and spread-plate techniques [50][51][52]. Oxytetracycline was used as a control (C) for antibacterial activity screening. The determined values of the minimum inhibition (MIC, µg/mL) and the minimum bactericidal (MBC, µg/mL) concentrations are presented in Table 2. The investigations demonstrated that the synthesized compounds possessed higher antibacterial properties than those of the known antibiotic Oxytetracycline.
The in vitro evaluation of the above-mentioned compounds revealed the excellent antibacterial activity of compounds 3c, 5b, 15b and 16b. Their effect on Gram-positive bacteria strains (MIC 7.8 µg/mL and MBC 15.6 µg/mL) was 8 times higher, and against Gram-negative 16-fold higher, than those of the control Oxytetracycline.
It should also be noted that among the thiazoles 3a-c the amino thiazole 3c, containing the β-alanine moiety, displayed the best antibacterial results against all the tested bacteria strains.
Among all the tested compounds, methyl ester 4b, hydrazone 6b and oxadiazole derivative 17b should be distinguished. They demonstrated four times stronger inhibition effects against Gram-positive bacteria strains and showed 8-fold higher inhibition against Gram-negative bacteria in comparison with the control. Hydrazones 6a and (7-12)b in most cases displayed good to very good action against the tested bacteria species. However, the best activity showed hydrazone 9b with 4-fluorophenyl moiety in the molecule. The comparison of the antibacterial activity of both benzimidazoles showed that the alkylated derivative 22 was more potent only against the strain of S. aureus. In all other cases, the effect of benzimidazole 20 and its derivative 22 on test-bacteria strains was completely the same. It is also noteworthy that S. aureus and E. coli were the most sensitive to the action of benzimidazoles 20 and 22. The minimum inhibition and minimum bactericidal concentrations of 31.25 µg/mL (MIC 15.6 µg/mL for S. aureus in case of 22) were sufficient in comparison with 62.5 and 125 µg/mL of Oxytetracycline for the inhibition of the growth and bactericidal action of the indicated test-cultures.
The quinoxaline fragment containing derivative 21 exhibited a more marked effect than the control Oxytetracycline only against B. cereus, L. monocytogenes and S. enterica enteritidis. In other cases, resistance of the test-bacteria was found to be the same as that of the used control antibiotic (except for MIC in the case of L. monocytogenes).
Comparing the data of the biological activity of the compounds containing different substituents (amino (a) or phenyl (b)) at the 2-position of thiazole ring, it can be asserted that derivatives with 2-phenylthiazol-5-yl moiety (3-6 and 13)b, show stronger inhibition and bactericidal effects than the derivatives with 2-aminothiazol-5-yl fragment (3-6 and 13)a. The MIC of compounds with phenyl group changes in range of 7.8-62.5 µg/mL, with prevailing MIC of 15.6 µg/mL, and the MBC vary from 15.6 to 125 µg/mL, with the prevailing value of 31.25 µg/mL. Accordingly, the data of compounds with amino group are as follows: the MIC vary from 15.6 to 62.5 µg/mL, with the prevailing value of 31.25 µg/mL; and the MBC changes in range of 31.25-125 µg/mL, with the prevailing concentration of 62.5 µg/mL. It should be noted that, among the compounds of group a, the 2-aminothiazole derivative 3a exhibited stronger activity only against S. aureus and E. coli in comparison with its analogue 3b.

Synthesis
Reagents and solvents were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used without further purification. The reaction course and purity of the synthesized compounds were monitored by TLC (Thin layer chromatography) using aluminum plates pre-coated with Silica gel with F254 nm (Merck KGaA, Darmstadt, Germany). Melting points were determined with a Melt-Temp Melting Point Analyzer (Electrothermal, Bibby Scientific Company, Burlington, NJ, USA) and were uncorrected. NMR spectra were recorded on a Varian Unity Inova (300, 75 MHz) and Brucker BioSpin GmbH (400, 101 and 700, 175 MHz) spectrometers. Chemical shifts were reported in (δ) ppm relative to tetramethylsilane (TMS) with the residual solvent as internal reference ([D6]DMSO, δ = 2.50 ppm for 1 H and δ = 39.5 ppm for 13 C). The data are reported as follows: chemical shift, multiplicity, coupling constant [Hz], integration and assignment. IR spectra (ν, cm −1 ) were recorded on a Bruker TENSOR 27 spectrometer using KBr pellets. Mass spectra were measured on a Waters (Micromas) ZQ 2000 mass spectrometer (ESI 20 eV). Elemental analyses (C, H, N) were conducted using the Elemental Analyzer CE-440 (Exeter Analytical, Inc., North Chelmsford, MA, USA); their results were found to be in good agreement (± 0.3%) with the calculated values.

General Procedure for the Preparation of Hydrazones 6-12
A mixture of the corresponding hydrazide 5a,b (10 mmol), benzencarbaldehyde (11 mmol) or thiophene-2-carboxaldehyde (2.24 g, 20 mmol) and dimethylformamide (30 mL) was refluxed for 1-3 h. Afterwards, the reaction mixture was cooled down, diluted with water (50 mL), the obtained product was filtered off, washed with water and ether, and recrystallized from the indicated solvent.

Biological Activity
The determination of the antibacterial and antifungal activity by diffusion method in agar was carried out by diffusion in agar on a solid nutrient medium (beef-extract agar for bacteria, wort agar for fungi). Petri plates containing 20 mL of nutrient medium were used for all the tested microorganisms. The inoculum (the microbial loading 109 cells (spores)/1 mL) was spread on the surface of the solidified media and Whatman no. 1 filter paper discs (6 mm in diameter) impregnated with the test compound (0.1 and 0.5%) were placed on the plates. The duration of bacteria incubation was 24 h at 35 • C and that of fungi incubation was 48-72 h at 28-30 • C. The antimicrobial effect and degree of activity of the tested compounds were evaluated by measuring the inhibition zone diameters and the results were compared with the data for the well-known antibacterial/antifungal agent. Each experiment was repeated three times. The determination of minimal inhibitory (MIC) concentrations was achieved using the serial dilution method. The tested compounds were added to the nutrient medium (beef-extract broth for bacteria and wort for fungi) as solutions in dimethyl sulfoxide (DMSO) for ensuring the needed concentration (0.9-500.0 µg/mL). Bacteria and fungi inoculum were inoculated into nutrient medium (the microbial loading was 106 cells (spores)/1 mL). The duration of bacteria incubation was 24 h at 35 • C and that of fungi incubation was 48-72 h at 28-30 • C. The results were estimated by the microorganism growth measured by degree of microbial turbidity in nutrient medium. The MIC of any compound was defined as the lowest concentration, which completely inhibits visible growth (turbidity on liquid nutrient medium).

Conclusions
In this study, the condensation of 1-(4-(2-bromoacetyl)phenyl)-5-oxopyrrolidine-3-carboxylic acid with thiourea, benzenecarbothioamide and thioureido acid novel disubstituted thiazole derivatives were obtained. Some transformations of these compounds were carried out; compounds with pyrrolidine, pyrrole, triazole, oxadiazole fragments were synthesized; and their antibacterial properties were investigated. The antibacterial assay revealed that in most cases substituted phenylthiazole derivatives had stronger inhibition and bactericidal properties against the test-cultures Staphylococcus aureus (ATCC 25923), Bacillus cereus (ATCC 10231), Listeria monocytogenes (ATCC 19111), Pseudomonas aeruginosa (ATCC 10145), Escherichia coli (ATCC 8739) and Salmonella enterica enteritidis when compared with the corresponding aminothiazoles. Compounds 3c, 5b, 15b and 16 b were found to have an exceptional antibacterial potency against all tested bacteria strains. The MIC of these compounds was observed at 7.8 µg/mL, and MBC -at 15.6 µg/mL, while the minimum inhibitory and minimum bactericidal concentrations of Oxytetracycline for Gram-positive bacteria strains was determined to be 62.5 µg/mL, and 125 µg/mL for Gram-negative ones.