Synthesis, Characterization, and Biological Evaluation of Novel N-{4-[(4-Bromophenyl)sulfonyl]benzoyl}-L-valine Derivatives

Abstract

In this article, we present the design and synthesis of novel compounds, containing in their molecules an L-valine residue and a 4-[(4-bromophenyl)sulfonyl]phenyl moiety, which belong to N-acyl-α-amino acids, 4H-1,3-oxazol-5-ones, 2-acylamino ketones, and 1,3-oxazoles chemotypes. The synthesized compounds were characterized through elemental analysis, MS, NMR, UV/VIS, and FTIR spectroscopic techniques, the data obtained being in accordance with the assigned structures. Their purities were verified by reversed-phase HPLC. The new compounds were tested for antimicrobial action against bacterial and fungal strains, for antioxidant activity by DPPH, ABTS, and ferric reducing power assays, and for toxicity on freshwater cladoceran Daphnia magna Straus. Furthermore, in silico studies were performed concerning the potential antimicrobial effect and toxicity. The results of antimicrobial activity, antioxidant effect, and toxicity assays, as well as of in silico analysis revealed a promising potential of N-{4-[(4-bromophenyl)sulfonyl]benzoyl}-L-valine and 2-{4-[(4-bromophenyl)sulfonyl]phenyl}-4-isopropyl-4H-1,3-oxazol-5-one for developing novel antimicrobial agents to fight Gram-positive pathogens, and particularly Enterococcus faecium biofilm-associated infections.

1. Introduction

Antimicrobial resistance represents a significant threat to global health and development today. Without effective antimicrobials (e.g., antibiotics, antifungals, and antiparasitics) and antiviral drugs, the success of modern medicine in preventing and treating infections would be at increased risk [1]. The discovery and development of new antimicrobial agents are therefore of considerable importance and one of the main focuses of today’s scientific community is the synthesis and identification of potent active substances.

An exhaustive survey of the literature on natural and synthetic oxazole-based molecules shows that they have numerous biological properties, which include antibacterial, antifungal, anti-inflammatory, antioxidant, and cytotoxic activities [2,3,4,5,6,7,8,9,10,11]. For example, a physiologically important active cyclic peptide with a 1,3-oxazole ring is dalfopristin, a semi-synthetic streptogramin A antibiotic analog marketed under the trade name Synercid in the combination with quinupristin, a streptogramin B derivative. The structures of some representative antimicrobial agents containing the 1,3-oxazole skeleton are shown in Figure 1.

The isolable 5-keto-tautomers of 1,3-oxazol-5-ols are 5-oxo-4,5-dihydro-1,3-oxazoles, known as 4H-1,3-oxazol-5-ones or 2-oxazolin-5-ones, which are also mentioned for their antimicrobial, cytotoxic, antiprotozoal, and antiviral properties [12,13,14,15]. Moreover, the N-acylated α-amino acids are reported to display many pharmaceutical activities, such as antimicrobial, antiviral, anticancer, mucolytic, antioxidant, and antihypertensive effects [16,17,18,19,20,21,22], and the 2-acylamino ketones present antiviral, anti-inflammatory, antithrombotic, and antihypertensive actions [23,24,25,26,27,28].

Furthermore, diaryl sulfones represent an important class of bioactive compounds with diverse biological properties, including antimicrobial and antioxidant actions [29,30,31,32,33,34,35,36,37]. The structural prototype of this class is dapsone (4,4′-sulfonyldianiline, 4,4′-diaminodiphenyl sulfone, DDS), which is used alone or as part of a multi-drug regimen in the treatment or prophylaxis of certain infectious diseases, such as leprosy (also known as Hansen’s disease). Currently, the therapeutic potential of organic compounds with a sulfonyl group in their molecules is being studied extensively [38,39]. In this regard, a new drug candidate from the organosulfones class is masupirdine (SUVN-502), a 1-[(2-bromophenyl)sulfonyl]-1H-indole derivative with a selective 5-HT₆ receptor antagonist effect, developed for the symptomatic treatment of Alzheimer’s disease [40].

Based on the promising therapeutic potential of these scaffolds and in the continuation of our research [41,42,43,44,45,46], we designed, obtained, and characterized novel L-valine-derived analogs with a 4-[(4-bromophenyl)sulfonyl]phenyl fragment and also evaluated their in silico and in vitro antimicrobial, antioxidant, and toxicity properties with the aim of identifying new biologically active compounds. The used synthesis methodology enables synthetic chemical diversification with the potential to prepare drug-like compounds.

The in silico approach to the biological activity of the new bromine compounds allowed the assessment of their toxicity and potential antimicrobial effect.

The in vitro antimicrobial testing of the new compounds included both qualitative screening (measuring the diameters of the zones of inhibition of microbial growth) and quantitative analysis (determining the values of minimal inhibitory concentration), as well as the assay of the antibiofilm action (determining the values of minimal biofilm inhibitory concentration). Particular emphasis was placed on evaluating the inhibition of microbial adhesion to surfaces as a possible means of reducing the burden of biofilm-associated infections.

The in vitro antioxidant activity of newly obtained compounds was investigated colorimetrically according to three electron transfer-based methods, namely 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation test, and ferric reducing power assay.

The compounds’ toxicity was assessed on Daphnia magna. This bioassay is a widely used method for the toxicity evaluation of natural and synthetic compounds, and it is in accordance with the “3 R’s” (reduction, refinement, and replacement) concept regarding experiments on vertebrates. It can be used as a prescreening method for rats and other mammals’ acute toxicity tests. The main advantage of this method is the crustacean reproduction mode by parthenogenesis, which leads to populations with lower variability than other invertebrate species [47,48].

2. Results

2.1. Drug Design Strategy

2.1.1. Structure-Based Similarity Analysis

Based on our previous research on N-{4-[(4-chlorophenyl)sulfonyl]benzoyl}-L-valine derivatives as antimicrobial and antibiofilm agents, we made modifications in the structures of the target molecules by replacing the chlorine atom with the more lipophilic bromine atom. The objective of this study was to increase the lipophilic character of the newly synthesized compounds while maintaining the substituent’s electronic effects. The proposed structures 5–8 were virtually explored on the ChEMBL database to demonstrate their original character and to assess their antimicrobial potential using similar compounds.

The search for similar compounds of N-acyl-L-valine 5 and α-acylamino ketones 7a and 7b returned 34 results and no similar structure for 4H-1,3-oxazol-5-one 6 and 1,3-oxazoles 8a and 8b, highlighting the originality of the proposed structures. No antimicrobial results were found for the 34 structurally similar compounds recorded in ChEMBL.

The substructure search based on the common 1-bromo-4-(phenylsulfonyl)benzene scaffold returned 32 compounds with 237 registered minimal inhibitory concentration (MIC) values, ranging from 2.5 up to 1024 μg/mL. All 32 compounds share an R-{4-[(4-bromophenyl)sulfonyl]phenyl} structure (Figure 2). The wide range of the MIC values indicates that the nature of the R group is very important for the potency of the antimicrobial effect. The lipophilic character of the compounds, expressed by the clogP value, seems to confirm the hypothesis of a better antimicrobial effect for most tested bacterial strains (Figure 2). The newly designed bromo derivatives have clogP values above the average of their similar compounds, indicating a potentially improved antimicrobial effect.

2.1.2. PASS Prediction

The 2D chemical structure of each target compound was input into the PASS application to obtain the probability of the corresponding compound to produce (Pa) or not (Pi) a certain biological effect. The application returns an array of Pa and Pi values for a very wide variety of pharmacological actions [49]. In the case of newly synthesized compounds 5, 6, 7a, 7b, 8a, and 8b, the 896 listed biological activities were filtered in relation to the antibacterial effect. The predicted probabilities Pa and Pi are presented in

Table 1. PASS predicted the probabilities that the new compounds 5, 6, 7a,b, and 8a,b to be active (Pa) as antimicrobial agents.

Target	5	6	7a	7b	8a	8b
Antibiotic glycopeptide-like	-	0.40	-	-	-	-
Antimycobacterial	0.49	0.58	0.51	0.51	0.36	0.37
Antituberculosis	0.48	0.41	0.49	0.49	0.30	0.30
Anti-infective	0.58	0.37	0.36	0.41	0.30	0.34

.

The two new α-acylamino ketones 7a and 7b were predicted to have very similar chances to produce antimycobacterial, antituberculosis, or anti-infective effects, and the corresponding values are significantly higher than those of the 1,3-oxazoles derivatives 8a and 8b. The Pa values reflect the likelihood of a certain effect to be produced, and not of its potency. Based on the Pa values, the addition of an aromatic fragment to N-acylated L-valine 5 has little impact on its predicted antimycobacterial and antituberculosis activities.

2.2. Chemistry

2.2.1. Synthesis of the New Compounds

The synthesis of new 4-[(4-bromophenyl)sulfonyl]benzoic acid derivatives 5–8 was performed according to the synthetic route, as outlined in Scheme 1.

The key starting material, 4-[(4-bromophenyl)sulfonyl]benzoic acid 3 was prepared by Friedel–Crafts sulfonylation of bromobenzene with commercially available tosyl chloride 1 in presence of aluminum trichloride, followed by oxidation of the sulfonation product (1-bromo-4-tosylbenzene) 2 with chromium trioxide in acetic acid [50]. The previously obtained compound 3 was then transformed by reaction with chlorinating reagent SOCl₂ to the corresponding acyl chloride 4 [46,51], which was used in the next step in the raw state. Subsequently, the 2-{4-[(4-bromophenyl)sulfonyl]benzamido}-3-methylbutanoic acid 5 was synthesized via Schotten–Baumann-type N-acylation of L-valine with 4-[(4-bromophenyl)sulfonyl]benzoyl chloride 4 in dichloromethane at room temperature with a reaction yield of 94%. The 2-{4-[(4-bromophenyl)sulfonyl]phenyl}-4-isopropyl-4H-1,3-oxazol-5-one 6 was produced by intramolecular cyclodehydration of compound 5 in presence of ethyl chloroformate and 4-methylmorpholine at room temperature in 90% yield. Then, the N-(1-aryl-3-methyl-1-oxobutan-2-yl)-4-[(4-bromophenyl)sulfonyl]benzamides 7a,b were obtained via Friedel–Crafts acylation of aromatic hydrocarbons (benzene, toluene) with 2-oxazolin-5-one 6 using anhydrous AlCl₃ as catalyst at room temperature in about 83% yield. Finally, the 5-aryl-2-{4-[(4-bromophenyl)sulfonyl]phenyl}-4-isopropyl-1,3-oxazoles 8a,b were prepared via Robinson–Gabriel-type intramolecular cyclization reaction of the 2-acylamino ketones 7a,b with phosphoryl trichloride at reflux in yields of 91% (8b) and 94% (8a). Structure elucidation of all newly synthesized L-valine derivatives was performed by means of spectral (UV/VIS, FTIR, NMR, MS) and elemental analyses.

2.2.2. Spectroscopic Characterization of the New Compounds

Various spectral analysis methods were applied to perform the detailed structural characterization of the newly obtained 4-[(4-bromophenyl)sulfonyl]benzoic acid derivatives 5–8, the resulting data being consistent with the depicted molecular structures.

Ultraviolet and Visible Absorption Spectroscopy Data

The UV/VIS absorption spectra of novel compounds 5–8 dissolved in methanol presented bands at 202.6 nm (5–8), at 227.3 nm (6), in the 249.3–255.5 nm region (5–8), and at 333.9 (8a) or 337.4 nm (8b). When acetonitrile was used as solvent, the first peak was shifted in the interval of 195.3–198.0 nm (5–8), while the rest of the bands were recorded at approximately the same wavelength values: at 228.2 nm (for 5, this peak appearing as a “shoulder” when methanol was used as solvent) or 229.1 nm (6), in the range of 248.6–254.8 nm (5–8), and at 335.6 (8a) or 340.1 nm (8b). The last absorption maximum was present only in the electronic spectra of compounds 8a and 8b due to the extension of the π-electron conjugation by the formation of the 1,3-oxazole chromophore.

Fourier-Transform Infrared Absorption Spectroscopy Data

The FTIR absorption spectra of acyclic precursors 5 and 7 showed a characteristic peak in the range of 3347–3281 cm⁻¹ due to the valence vibration of the N–H bond. In addition, the absorption maximum at 1746 cm⁻¹ due to carbonyl valence vibration, and another at 1635 cm⁻¹ due to stretching vibration of amidic carbonyl were remarked in the FTIR spectrum of 5. For α-acylamino ketones 7a and 7b, these two carbonyl absorption bands are overlapped, as suggested by the very strong single peak recorded in their FTIR spectra at 1655 cm⁻¹. A broad absorption band in the spectral region from 3300 to 2500 cm⁻¹, centered at ≈3000 cm⁻¹, due to stretching vibration of the O–H bond and two noticeably weak satellite peaks at 2676 and 2599 cm⁻¹ are also characteristic of the hydrogen-bonded N-acylated L-valine 5.

As evidence that the intramolecular cyclocondensations occurred, significant changes were observed in the FTIR absorption spectra of five-membered O,N-heterocyclic compounds 6, and 8a,b compared with the corresponding spectra of open-chain intermediates 5, and 7a,b, respectively. The FTIR spectrum of 4H-1,3-oxazol-5-one 6 presented a peak at 1825 cm⁻¹ due to carbonyl stretching vibration, which is shifted at a higher wavenumber compared with the C=O absorption maximum from the spectrum of 5. The FTIR absorption spectra of 5-membered heterocycles 6 and 8 showed an absorption band at 1650 (6), 1602 (8a), or 1601 cm⁻¹ (8b) due to the valence vibration of the C=N bond. The absorption maxima at 1099 (8a), 1097 (8b), or 1040 cm⁻¹ (6) due to the C–O–C symmetric stretching vibration and at 1280 (8a and 8b) or 1243 cm⁻¹ (6) due to the asymmetric stretching vibration of the C–O–C group were also observed.

Nuclear Magnetic Resonance Spectroscopy Data

The nuclear magnetic resonance spectroscopic data also confirmed the structures of the new L-valine analogs. Complete assignments of the signals from the ¹H and ¹³C NMR spectra of the new compounds 5–8 were performed using combinations of standard NMR spectroscopic techniques, namely 2D COSY and HETCOR experiments.

The molecular structures of the new bromine-containing derivatives 5–8 with the numbering of the atoms, used for the assignment of NMR signals, are presented in Figure 3.

• Proton NMR Spectroscopy Data

The proton NMR spectra of 5 and 7 showed a doublet signal in the region between 8.73 and 8.99 ppm, attributed to deshielded H-3 proton. The ¹H NMR spectrum of 5 revealed a signal assigned to the H-4 proton at 4.29 ppm as a doublet of doublets due to the vicinal coupling to H-3 and H-18 protons. The proton NMR spectra of α-acylamino ketones 7a and 7b showed the H-4 signal at 5.36 (7b) or 5.38 ppm (7a) as a triplet, due to the coupling to H-3 and H-18. The ¹H NMR spectra of 5, 7a, and 7b also highlighted characteristic signals for the isopropyl substituent, i.e., an octet (5) or a multiplet (7a,b) signal registered in the range of 2.17–2.28 ppm assigned to the methine proton (H-18) and two strongly shielded doublet signals, the first in the interval of 0.89–0.94 ppm and the second at 0.92 or 0.95 ppm, due to the non-equivalent protons (H-19, H-20) of the two methyl groups. The signal of proton of the carboxyl group of N-acyl-α-amino acid 5 was not recorded in the ¹H NMR spectrum probably due to hydrogen-deuterium exchange, but the presence of the COOH group was confirmed both by IR and ¹³C NMR spectral data.

The ¹H NMR spectrum of 6 displayed the H-4 doublet signal at 4.32 ppm due to the coupling only to the H-18 proton (with ³J = 4.7 Hz). The methine proton of the 1-methylethyl group was revealed by the presence of a septet of doublets signal at 2.40 ppm due to coupling to protons H-19, H-20 (with ³J = 6.9 Hz), and to H-4 proton (with ³J = 4.7 Hz). The ¹H NMR spectra of 8a and 8b showed for the isopropyl substituent a signal as septet at 3.26 (8b) or 3.29 ppm (8a) attributed to the H-18 proton, and a shielded doublet at 1.35 (8b) or 1.36 ppm (8a), due to the two CH₃ groups’ protons.

• Carbon-13 NMR Spectroscopy Data

The ¹³C NMR spectrum of N-acylated L-valine 5 presented a signal at 58.43 ppm attributed to the C-4, for the 1-methylethyl group revealed a signal at 29.48 ppm due to the methine carbon (C-18) and two signals at 18.61 and 19.25 ppm due to the C-19 and C-20 non-equivalent carbon atoms. The ¹³C NMR spectrum of 6 highlighted the C-4 signal at 71.04 ppm, which is deshielded by 12.61 ppm as a result of cyclization of the open-chain precursor 5. Further, the C-2 atom of 4H-1,3-oxazol-5-one 6 resonated at 160.36 ppm, being more shielded with 5.42 ppm compared with the C-2 of 5, and the C-5 at 176.98 ppm, being more deshielded with 4.19 ppm than the corresponding carbon atom of its precursor (5).

The carbon-13 NMR spectra of aromatic O,N-heterocycles 8a,b showed a signal assigned to the C-4 atom at 143.63 (8b) or 144.19 ppm (8a), which is more deshielded by ≈84.77 ppm than the corresponding signal of the compounds from which they were obtained by cyclization, from 59.05 (7b) or 59.23 ppm (7a). The cyclization of 7a,b to compounds 8a,b induced a shielding effect for the C-2 of the 1,3-oxazole ring, resulting in an about 7.87 ppm lower chemical shift. The C-5 signal of 1,3-oxazoles 8a,b was observed at 145.47 (8a) or 145.69 ppm (8b), whereas the corresponding signal of acyclic precursors 7a,b appeared at 198.57 (7b) or 199.19 ppm (7a), revealing a shift of this carbon signal at a smaller δ_C of approximately 53.30 ppm.

Gas Chromatography Coupled to Electron Ionization Mass Spectrometry Data

For saturated azlactone 6, supplementary evidence was obtained by its mass spectrum obtained by GC/EI–MS analysis. The two molecular ions of compound 6 corresponding to bromine isotopes (⁷⁹Br/⁸¹Br) were energetically unstable upon interaction with high-energy (70 eV) electrons and fragmented with the elimination of a molecule of propene. The base peak with m/z 381 and the corresponding radical cation with m/z 379 (with a relative abundance of 71.61%) were resulted according to the ⁷⁹Br/⁸¹Br isotopic ratio of ≈1:1. Other characteristic structural fragments of 6 are reported in the Materials and Methods section.

2.3. Evaluation of Antimicrobial Activity

2.3.1. Qualitative Assessment of Antimicrobial Activity

The agar diffusion assay showed that compounds 7a, 8a, and 8b did not interfere with microbial growth (

Table 2. Results of the qualitative analysis of the antimicrobial effect of compounds 5–8 tested at 5000 µg/mL, using an adapted disk diffusion method (diameters of growth inhibition zones were measured in mm).

Tested Compound	Gram-Positive Bacteria			Gram-Negative Bacteria		Fungus
	Bacillus subtilis ATCC 6683	Enterococcus faecium E5	Staphylococcus aureus ATCC 6538	Escherichia coli ATCC 8739	Pseudomonas aeruginosa ATCC 27857	Candida albicans 393
5	0	17	0	0	0	0
6	0	15	8	0	0	0
7a	0	0	0	0	0	0
7b	10	0	0	0	0	0
8a	0	0	0	0	0	0
8b	0	0	0	0	0	0
Ciprofloxacin	28	30	26	34	30	- *
Fluconazole	-	-	-	-	-	30

* -: not tested.

). Enterococcus faecium E5 was the most susceptible species to compounds 5 and 6 with a growth inhibition zone diameter of 17 and 15 mm, respectively. Compounds 6 and 7b also inhibited the growth of Staphylococcus aureus ATCC 6538 and Bacillus subtilis ATCC 6683, respectively with diameters of growth inhibition zones of 8 mm and of 10 mm.

2.3.2. Effects of Compounds on Antibiotic Susceptibility Profile

For compounds 5 and 6 only, their influence on the E. faecium E5 and S. aureus ATCC 6538 strains’ susceptibility to different antibiotics was evaluated. Although compound 7b was active against B. subtilis ATCC 6683, the influence on this strain’s susceptibility to antibiotics was not investigated, as there are no specific guidelines regarding the susceptibility breakpoints of this species.

No significant changes were noticed in the E. faecium E5 strain’s susceptibility to current antibiotics after culture in the presence of compound 5 tested at subinhibitory concentration (

Table 3. Antibiotic susceptibility testing of E. faecium E5 strain cultured in the presence of compounds 5 and 6 tested at a subinhibitory concentration of 250 µg/mL and of dimethyl sulfoxide (DMSO) (growth inhibition zone diameters were measured in mm).

Microbial Culture	Growth Inhibition Zone Diameter (In mm)
	Ampicillin	Linezolid	Penicillin	Vancomycin
Control *	24	27	14	19
5	22	25	13	17
6	29	39	15	23
DMSO	22	23	0	18

* liquid culture medium inoculated with standardized suspension of E. faecium E5.

), suggesting both a low selective pressure for resistance occurrence and a different mechanism of action. Regarding the E. faecium E5 strain cultured in the presence of compound 6, it was observed that it determined an increase in the antimicrobial effect of the tested antibiotics (ampicillin, penicillin, linezolid, and vancomycin). The diameters of growth inhibition zones (in mm) are shown in Table 3.

With respect to the S. aureus ATCC 6538 strain cultured in the presence of compound 6, a slight increase in the diameter of growth inhibition zone was observed in the case of cefoxitin (

Table 4. Antibiotic susceptibility testing of S. aureus ATCC 6538 strain cultured in the presence of compound 6 tested at 250 µg/mL and of DMSO control (growth inhibition zone diameters were measured in mm).

Microbial Culture	Growth Inhibition Zone Diameter (In mm)
	Azithromycin	Cefoxitin	Clindamycin	Linezolid	Penicillin	Rifampicin	Trimethoprim- Sulfamethoxazole	Vancomycin
Control *	15	20	26	32	0	21	0	23
6	15	21	23	29	0	19	0	21
DMSO	13	15	20	28	0	19	0	18

* liquid culture medium inoculated with standardized suspension of S. aureus ATCC 6538.

). Regarding other studied antibiotics, namely vancomycin, linezolid, clindamycin, and rifampicin, the susceptibility was reduced, suggesting a phenotypic change in the tested strain exposed to compound 6 at subinhibitory concentration. There were no changes in the growth inhibition zone diameters compared with those of the microbial growth control in the case of the studied antibiotics: azithromycin, penicillin, and trimethoprim-sulfamethoxazole.

2.3.3. Quantitative Testing of Antimicrobial Activity

The results of the quantitative antimicrobial testing by the standard broth microdilution method are presented in

Table 5. MIC (minimal inhibitory concentration) and MBIC (minimal biofilm inhibitory concentration) values in µg/mL determined for compounds 5–8 tested at concentrations between 500 and 0.97 µg/mL.

Tested Compound	Gram-Positive Bacteria						Gram-Negative Bacteria				Fungus
	Bacillus subtilis ATCC 6683		Enterococcus faecium E5		Staphylococcus aureus ATCC 6538		Escherichia coli ATCC 8739		Pseudomonas aeruginosa ATCC 27857		Candida albicans 393
	MIC	MBIC	MIC	MBIC	MIC	MBIC	MIC	MBIC	MIC	MBIC	MIC	MBIC
5	>500	>500	500	62.5	>500	>500	>500	>500	>500	>500	>500	>500
6	>500	>500	500	1.95	250	250	>500	>500	>500	>500	>500	>500
7a	>500	>500	>500	>500	>500	>500	>500	>500	>500	>500	>500	>500
7b	>500	>500	>500	>500	>500	>500	>500	>500	>500	>500	>500	>500
8a	>500	>500	>500	>500	>500	>500	>500	>500	>500	>500	>500	>500
8b	>500	>500	>500	>500	>500	>500	>500	>500	>500	>500	>500	>500
Ciprofloxacin	<0.03	<0.03	0.62	0.62	0.15	0.15	0.012	0.012	0.15	0.15	- *	-
Fluconazole	-	-	-	-	-	-	-	-	-	-	<0.12	<0.12

* -: not tested.

. The majority of the tested compounds exhibited MIC values equal to or higher than 500 µg/mL. The most active proved to be compound 6 which was found to have a moderate antimicrobial activity (MIC value of 250 µg/mL) against the S. aureus ATCC 6538 reference strain.

2.3.4. Evaluation of Antibiofilm Activity

The results obtained in this study showed that compounds 7a, 7b, 8a, and 8b did not interfere with microbial adherence on surfaces. Exceptional antibiofilm activity was observed for compounds 5 and 6 against E. faecium E5 strain, with MBIC values of 62.5 and 1.95 µg/mL, respectively (Table 5). Compound 6 also displayed a moderate antibiofilm effect in the case of the Gram-positive S. aureus ATCC 6538 strain, with an MBIC value of 250 µg/mL.

2.4. Evaluation of Antioxidant Activity

The antioxidant effect of newly synthesized derivatives 5–8 was studied using three methods based on electron transfer reactions, namely DPPH, ABTS, and ferric reducing power tests. The results were compared with those of the following standard antioxidant agents: ascorbic acid (AA), butylated hydroxyanisole (BHA, a mixture of 2-(tert-butyl)-4-methoxyphenol and 3-(tert-butyl)-4-methoxyphenol) and butylated hydroxytoluene (BHT, 2,6-di-tert-butyl-4-methylphenol), used as positive controls. The antioxidant capacity of the key starting materials L-valine and 4-[(4-bromophenyl)sulfonyl]benzoic acid 3 was also determined.

2.4.1. Antioxidant Activity Assay by DPPH Method

The results of the study concerning the antioxidant effect evaluation of the new 4-[(4-bromophenyl)sulfonyl]benzoic acid derivatives 5–8 by the DPPH method are presented in

Table 6. Results of the assessment of the antioxidant effect of the compounds tested by the DPPH method.

Compound	Concentration (μM)	Scavenging Effect (%)
5	250	4.70 ± 1.88
6	250	16.75 ± 1.18
7a	250	1.42 ± 0.06
7b	250	1.35 ± 1.72
8a	250	1.82 ± 0.36
8b	250	1.50 ± 0.11
L-Valine	250	7.87 ± 0.07
3	250	2.32 ± 0.22
AA	250	85.09 ± 1.67
BHA	250	77.99 ± 0.56
BHT	250	31.79 ± 1.52

. In terms of experimental results obtained by this test, of all the newly synthesized compounds, 4H-1,3-oxazol-5-one 6 had the best antioxidant activity with a DPPH inhibition rate of 16.75 ± 1.18%, its effect being better than that of key starting materials (L-valine and carboxylic acid 3), but lower than the standard antioxidants used. This compound was followed by N-acylated α-amino acid 5 with a DPPH inhibition percentage of 4.70 ± 1.88%. In contrast, compounds 7a,b, and 8a,b had the lowest scavenging effect values in the range of 1.35–1.82%.

2.4.2. Antioxidant Activity Assay by ABTS Method

The results of the assessment of the antioxidant potential of the compounds tested by the ABTS method are shown in

Table 7. Results of the evaluation of the scavenging effect of the compounds analyzed by the ABTS method.

Compound	Concentration (μM)	Scavenging Effect (%)
5	250	0.78 ± 0.91
6	250	1.49 ± 0.37
7a	250	7.14 ± 1.51
7b	250	0.40 ± 0.27
8a	250	1.77 ± 0.56
8b	250	7.66 ± 0.71
L-Valine	250	44.71 ± 0.66
3	250	0.17 ± 0.85
AA	250	99.93 ± 0.11
BHA	250	99.74 ± 0.08
BHT	250	98.46 ± 1.23

. The obtained values of the percentage scavenging effect of new compounds 5–8 are small in the interval of 0.40–7.66%. Among the new compounds, 1,3-oxazole 8b showed the best antioxidant activity (7.66 ± 0.71%), followed by α-acylamino ketone 7a (7.14 ± 1.51%), while compound 7b had the lowest (0.40 ± 0.27%). By this method, L-valine had a better effect (44.71 ± 0.66%) than the new products 5–8, but key raw material 3 showed a lower activity (0.17 ± 0.85%) than these.

2.4.3. Ferric Reducing Power Assay

The results of the investigation of the antioxidant capacity of the compounds tested by the ferric reducing power method are presented in

Table 8. Results of the screening of the antioxidant activity of the compounds tested by the ferric reducing power method.

Compound	Concentration (μM)	Reducing Power (Absorbance at 700 nm, A700)
5	500	0.0224 ± 0.0019
6	500	0.0461 ± 0.0088
7a	500	0.0722 ± 0.0013
7b	500	0.0437 ± 0.0105
8a	500	0.0369 ± 0.0060
8b	500	0.0439 ± 0.0057
L-Valine	500	0.0854 ± 0.0051
3	500	0.0568 ± 0.0078
AA	500	0.8727 ± 0.0315
BHA	500	1.1363 ± 0.0096
BHT	500	0.7282 ± 0.1686

. All the tested compounds possessed the ability to reduce iron(III) to iron(II). The α-acylamino ketone 7a was a better iron(III) reducer (absorbance at 700 nm, A₇₀₀ of 0.0722 ± 0.0013) than the other new derivatives, followed by three compounds, which showed similar values of the ferric reducing power, in descending order: 6 (A₇₀₀ = 0.0461 ± 0.0088), 8b (A₇₀₀ = 0.0439 ± 0.0057), and 7b (A₇₀₀ = 0.0437 ± 0.0105). Of the compounds tested, N-acyl-L-valine 5 was the least active tested compound (A₇₀₀ = 0.0224 ± 0.0019) and L-valine had the best antioxidant activity (A₇₀₀ = 0.0854 ± 0.0051). None of the activities of the analyzed compounds were comparable to those of the AA, BHA, and BHT positive controls.

2.5. Daphnia magna Toxicity Bioassay

The results of the Daphnia magna toxicity test are presented in

Table 9. Daphnia magna toxicity test results.

Tested Compound	Predicted LC50 1 (48 h) (µg/mL)	L % Max (48 h) 2	Determined LC50 (24 h) (µg/mL)	95% CI 3 of LC50 (24 h) (µg/mL)	Determined LC50 (48 h) (µg/mL)	95% CI of LC50 (48 h) (µg/mL)
5	0.51	60	ND 4 *	ND *	43.5	ND **
6	0.33	70	ND *	ND *	31.25	22.32 to 43.75
7a	0.11	100	42.93	38.14 to 48.31	ND ***	ND **
7b	0.11	90	58.83	46.28 to 74.78	21.07	12.92 to 34.35
8a	0.1	60	ND *	ND *	54.62	41.73 to 71.48
8b	0.04	30	ND *	ND *	ND *	ND *
L-Valine (Control 1)	1078.3	20	ND *	ND *	ND *	ND *
3 (Control 2)	11.8	100	31.11	ND **	1.144	0.13 to 9.93

¹ 50% lethal concentration; ² maximum lethality induced at 48 h; ³ 95% confidence interval; ⁴ not determined because of the results obtained; * lethality ranged between 0 and 40%; ** 95% CI could not be determined due to the results; *** lethality was higher than 50% at all concentrations.

, and the lethality curves are shown in Figure 4. At 24 h, the highest toxicity was induced by 7a and 7b, and the compounds 5, 6, 8a, and 8b were non-toxic on D. magna at the tested concentrations. At 48 h, the most toxic compound was 7a. At the lowest concentration, its lethality was 65%; therefore, the LC₅₀ being lower than 2 µg/mL, the estimated value by extrapolation is 0.21 µg/mL. The compounds 5, 6, 7b, and 8a induced medium to high toxicity, their LC₅₀ values varying from 21.07 to 54.62 µg/mL. Except for 7a, all compounds induced lower toxicity than the key starting material 3. The compound 8b at the highest concentration (50 µg/mL) induced a lethality of 25%, and at all other concentrations, the lethality varies from 0 to 10%. The predicted LC₅₀ values were significantly lower than those obtained experimentally, except for compound 3.

3. Discussions

The 1,3-oxazoles are five-membered heteromonocyclic scaffolds, containing two heteroatoms, O and N at positions 1 and 3, respectively, of particular importance in synthetic and medicinal chemistry, since representatives of this class have great structural diversity and are of significant biological interest.

Over time, researchers have paid particular attention to the preparation of new derivatives incorporating the 1,3-oxazole ring [52,53,54,55], as well as to the evaluation, among other pharmacological properties, of their antimicrobial and antioxidant activities, a large number of articles being published on this topic. Currently, various bioactive 1,3-oxazole-embedded heterocycles have been reported, such as 1,3-oxazole clubbed pyridyl-pyrazolines having good to excellent antimicrobial action [56], cytisine-containing 1,3-oxazoles as potential inhibitors of Candida spp. glutathione reductase [57], thioxo-1,3-oxazole analogs that prevent bacterial growth and with antioxidant ability [58], 1,3-oxazole-quinoxaline amine hybrids showing antibacterial activity [59], steroidal 1,3-oxazole derivatives displaying effective antimicrobial and antibiofilm properties [60], binaphthyl-based, functionalized 1,3-oxazole peptidomimetics with moderate to excellent antimicrobial activity [61], substituted 1,3-oxazole-benzamides as antibacterial inhibitors of the essential bacterial cell division protein FtsZ [62], N-(oxazolylmethyl)thiazolidinediones as selective inhibitors of Candida albicans biofilm formation [63], and some antimicrobial 2-amino-1,3-oxazoles [64]. Very recently, we investigated the in silico and in vitro antimicrobial and antibiofilm effects of a series of 1,3-oxazole-based compounds and their isosteric analogs derived from alanine and phenylalanine, some of them presenting a promising profile [65]. Quantitative studies also indicated that of all 2,5-diaryl-1,3-oxazoles tested, only some of those with a bromine atom in their molecules showed antimicrobial and/or antibiofilm action. In contrast, all other derivatives from the same class, unsubstituted or with a chlorine atom in their structures, proved to be inactive at the concentrations tested. Other researchers have also observed that the incorporation of the chlorine or bromine atom, usually grafted onto an aromatic nucleus, led to an improvement in the antimicrobial activity of compounds of different classes, as well as that brominated derivatives have shown greater efficacy than chlorinated analogs [66,67,68,69]. The antimicrobial effect due to the presence of halogen atoms appears to be related to the compounds’ relative hydrophobicity. It was found that hydrophobicity of the compounds enhanced with the addition of a halogen atom in their molecules and that chlorine derivatives displayed a less pronounced effect, while the incorporation of a bromine atom led to analogs with a higher hydrophobic profile [66]. In view of these findings, it is of interest to obtain new bromine-bearing 1,3-oxazoles-type molecules and to explore their application as biologically active agents.

In the present work, we synthesized novel organic compounds, carrying a bromine atom in their molecules, based on the 1,3-oxazole and diphenyl sulfone pharmacophores. The molecular structures of the newly obtained bromine-containing compounds (which are N-acyl-L-valine, 4-isopropyl-4H-1,3-oxazol-5-one, 2-acylamino ketone, and 4-isopropyl-1,3-oxazole derivatives) were elucidated based on spectroscopic investigations. Moreover, the synthesized analogs were assessed for antimicrobial properties, namely qualitative (zone of inhibition) and quantitative (MIC) activities, antibiofilm action (MBIC), and for antioxidant and toxic effects. Furthermore, in silico investigations on the toxicity and potential antimicrobial action were performed.

The structure-based similarity analysis of the new L-valine derivatives 5–8 highlighted the originality of the proposed structures and indicated that the presence of the bromine atom in the proposed structures of the new compounds can enhance the potential antimicrobial effect. The PASS prediction showed the probability that all new compounds to exhibit anti-infective, antimycobacterial, and antituberculosis activities and, in addition, that 4H-1,3-oxazol-5-one 6 to have a glycopeptide-like antibiotic effect.

The antimicrobial activity assays revealed a promising antimicrobial potential against Gram-positive cocci strains, particularly in the case of E. faecium biofilm, when the MBIC values were as from 8 to 256 times lower than the corresponding MIC values.

Concerning the results of quantitative tests for the evaluation of antimicrobial and antibiofilm activity, it was also found that the MIC and MBIC values of the standard broad-spectrum antibacterial drug ciprofloxacin and antifungal agent fluconazole, which served as positive controls, were lower than those determined for the compounds tested. This can be explained by the fact that the novel L-valine derivatives, which belong to four classes, may have distinct mechanisms of action from those of the used control drugs and, in addition, unlike them, are not standardized active ingredients in optimized drug formulations.

It was also found that all compounds analyzed show antioxidant activity through all spectroscopic tests used (DPPH, ABTS, and ferric reducing power methods), but lower than the bioactive standards (AA, BHA, and BHT). The tested compounds thus demonstrated weak electron-donating properties.

The in vitro evaluation regarding antimicrobial, antioxidant, and toxicity features highlighted that acyclic intermediate 5 is promising due to its very good antibiofilm activity, low antioxidant effect, and moderate toxicity. It is worth noting that by cyclodehydration of compound 5 to the corresponding 4H-1,3-oxazol-5-one 6, an increase in the antibiofilm effect and antioxidant activity was observed, but also an increase in its toxicity. Moreover, it appears that the conversion of 2-oxazolin-5-one 6 to α-acylamino ketone 7b and the cyclization of 7b to 1,3-oxazole 8b leads to a decrease in antimicrobial activity, and compound 7a and corresponding 1,3-oxazole 8a were found to be inactive at the tested concentrations. No regular variation in the antioxidant effect of compounds 7a, 7b, 8a, and 8b was noticed, but of all the new compounds tested, 7a and 8b stood out, which showed the best activity by all three methods used. It was also shown that by converting saturated azlactone 6 to α-acylamino ketones 7a,b, the toxicity increases, and by cyclization of open-chain intermediates 7a,b to 1,3-oxazoles 8a,b, the toxicity decreases significantly, 8b being non-toxic. However, the Daphnia magna assay showed medium to high toxicity for compounds 5, 6, and 7b, and significant-high toxicity for compounds 3 and 7a. Our findings indicate a high potential for biological targets, whereas the 1,3-oxazole-containing compound 8b showed no toxicity at concentrations lower than 50 µg/mL. The toxicity study also showed that, with the exception of the LC₅₀ value obtained for key intermediate 3, the experimental values of LC₅₀ for all other compounds were significantly higher than those predicted by the GUSAR analysis.

Recently, we reported the synthesis and results of the antimicrobial and antibiofilm assessment of a series of similar N-{4-[(4-X-phenyl)sulfonyl]benzoyl}-L-valine derivatives (X = H, Cl) [70,71]. The non-substituted analog of 7b, N-[3-methyl-1-oxo-1-(p-tolyl)butan-2-yl]-4-(phenylsulfonyl)benzamide presented a better antimicrobial effect compared with 7b or its chloro-derivative. The increase in the lipophilic character was perhaps detrimental because of the associated lower water solubility. The introduction into the molecule of the bromine atom proved slightly beneficial in the case of the 4H-1,3-oxazol-5-one 6.

Subsequent structural and biological investigations into these kinds of privileged scaffolds can lead to the discovery of new more potent derivatives that can act as optimized candidates for the development of new effective preventive and therapeutic agents. In this respect, we consider three critical positions on the molecular templates that may have an effect on biological action. A first possibility for the future improvement of the antimicrobial property of these analogs is the replacement of the bromine atom in the arylsulfonylphenyl fragment with different substituents, such as fluorine, iodine, trifluoromethyl, or nitro. For this purpose, other benzene analogs can be used as starting molecules in the Friedel–Crafts sulfonylation reaction. As a second optimization choice, in the N-acylation reaction instead of L-valine, we consider the use of other natural or unnatural α-amino acids (such as histidine and tryptophan) or the incorporation of several α-amino acid residues in the molecules as it is well-known that some natural polypeptides bearing the 1,3-oxazole ring (e.g., microcin B17) are inhibitors of DNA-gyrase [3]. The use of other aromatic compounds with different substituents (e.g., I, NO₂, CF₃, OCH₃) in the reaction with 6 is the third alternative route of synthesis to enhance the biological activity of these molecular scaffolds. The iodine atom proved more active than other halogens, CF₃-functionalized compounds showed higher antibacterial activity than those with a CH₃ group, hydrophobic substituents (e.g., benzyloxy, tert-butyl) [72], or electron-withdrawing groups (such as nitro) on aromatic cores increased the antibacterial effect [73], small substituents (e.g., hydroxy) at 4-position of the phenyl nucleus improved the antibacterial potency [62]. In addition, it was noticed that the electron-donating substituents (such as OCH₃) on the aromatic moiety enhanced the antioxidant activity compared with the electron-withdReversed-phase HPLC rawing groups and that, in general, this pharmacological action increased with the increasing electron-donating effect of the substituent [74,75,76,77].

4. Materials and Methods

4.1. Prediction of the Molecular Mechanism of Action and Toxicity

4.1.1. Structure-Based Similarity Analysis

Each target structure was inputted into the search engine of the ChEMBL database [78] and a similarity search was performed using a 50% threshold. The output structures were extracted together with any associated data of biological activity on bacteria. The DataWarrior v5.2.1 software (Actelion Pharmaceuticals Ltd., Allschwil, Switzerland, https://openmolecules.org/) [79] was used to filter the duplicate structures and to calculate the clogP values.

4.1.2. PASS Prediction

The web-based Prediction of Activity Spectra for Substances (PASS) application was used to evaluate the antimicrobial potential of the newly designed bromo derivatives based on their chemical structures. The array of results was analyzed for any effect with a predicted Pa value higher than the Pi value.

4.2. General Information

Melting points (mp) (in °C), were determined on a Boëtius hot plate microscope (VEB Wägetechnik Rapido, PHMK 81/3026, Radebeul, Germany) and are reported uncorrected. UV/VIS spectra were recorded for solutions of the new compounds in methanol at concentration of ≈0.025 mM and in acetonitrile at ≈0.015 mM on an Analytik Jena Specord 40 ultraviolet/visible spectrophotometer (Analytik Jena AG, Jena, Germany) using a quartz cuvette with a path length of 1.00 cm. Values of the wavelength (corresponding to the maximum absorbance), λ_max (in nm), and of the logarithm to the base 10 of the molar absorption coefficient, ε_max (M⁻¹ · cm⁻¹) are provided. FTIR spectra were collected with a Bruker Optics Vertex 70 Fourier-transform infrared spectrophotometer (Bruker Optics GmbH, Ettlingen, Germany) using the conventional KBr pellet technique. The position of the selected absorption bands in the FTIR spectra is reported using the wavenumber at the absorption maximum, $\tilde{\nu }$_max (in cm⁻¹). The intensity of the FTIR peaks is provided as very strong (vs); strong (s); medium (m); and weak (w). Nuclear magnetic resonance spectra were registered on a Varian Gemini 300 BB NMR spectrometer (Varian, Inc., Palo Alto, CA, USA) operating at 300 MHz for ¹H NMR and 75 MHz for ¹³C NMR, in deuterated solvents, i.e., dimethyl sulfoxide-d₆ (DMSO-d₆) or deuterochloroform (CDCl₃). Additional evidence was provided by the 2D HETCOR and COSY experiments. Chemical shifts, δ, were measured in ppm with respect to the ¹H or ¹³C resonance of (CH₃)₄Si (tetramethylsilane, TMS), and the coupling constants, J, are expressed in Hz. The multiplicity of ¹H NMR signals is provided as per the following convention: singlet (s); doublet (d); doublet of doublets (dd); triplet (t); triplet of triplets (tt); septet (sept); septet of doublets (septd); octet (oct); multiplet (m); and broad (br). Proton NMR spectroscopic data are quoted in the following order: ¹H chemical shift, multiplicity, proton number, signal/hydrogen atom assignment, coupling constants, and ¹³C NMR spectroscopic data are reported as follows: δ_C-value, signal/carbon atom attribution. GC/EI–MS data were acquired on a Fisons Instruments GC 8000 series gas chromatograph coupled to an MD 800 mass spectrometer detector equipped with an electron ionization source and with a quadrupole mass analyzer (Fisons Instruments SpA, Rodano, Milano, Italy), using an SLB-5ms capillary column (d_f 0.25 μm; L × I.D. 30 m × 0.32 mm), CH₂Cl₂ as solvent, and a flow rate of helium carrier gas of 2 mL/min. Reversed-phase high-performance liquid chromatography (HPLC) was performed on a Beckman Coulter System Gold 126 liquid chromatograph, equipped with a System Gold 166 UV/VIS spectrophotometer detector (Beckman Coulter, Inc., Fullerton, CA, USA), a Rheodyne injection system, and a LiChrosorb RP-18 column (d_p 5 μm; L × I.D. 25 cm × 4.6 mm). The flow rate of eluent, a mixture of CH₃OH–H₂O in different volume ratios, was 1 mL/min. The compounds’ purities (%) and retention times, t_R (in min), were reported. Elemental analysis was carried out on a Costech ECS 4010 instrument (Costech Analytical Technologies Inc., Valencia, CA, USA).

4.3. Chemistry

The chemicals and reagents were obtained from commercial sources of analytical grade and were used as received, except for CH₂Cl₂ which was dried over anhydrous CaCl₂.

4.3.1. Preparation and Characterization of 2-{4-[(4-Bromophenyl)sulfonyl]benzamido}-3-methylbutanoic Acid 5

The solution of 2.34 g (20 mmol) of L-valine in 20 mL (0.80 g, 20 mmol) of 1 N NaOH solution was cooled at 0–5 °C in an ice bath and then two other solutions were added simultaneously in droplets under magnetic stirring during half an hour, namely a solution of 7.19 g (20 mmol) of crude 4-[(4-bromophenyl)sulfonyl]benzoyl chloride 4 in 45 mL of anhydrous dichloromethane, and a 2 N NaOH solution (10 mL, 0.80 g, 20 mmol). The reaction mixture was stirred for another 1 h at room temperature and then the separated aqueous phase was acidified with 2 N HCl when a white precipitate appeared. The solid was filtered off at low-pressure, washed thoroughly with distilled water on the filter to remove traces of hydrochloric acid, air-dried, and recrystallized from water when white acicular crystals resulted.

Yield 94% (8.28 g, 18.80 mmol). mp 194–196 °C.

UV/VIS (CH₃OH) λ_max nm (${\log }_{10}\epsilon$_max): 202.6 (4.48); 252.0 (4.20); (CH₃CN) λ_max nm (${\log }_{10}\epsilon$_max): 196.2 (4.83); 228.2 (4.36); 252.2 (4.57).

FTIR (KBr disc) $\tilde{\nu }$_max cm⁻¹: 3347 s (νN–H); 3300–2500 vs (νO–H); 3092 s; 3069 s (νC–H, aromatic); 2966 vs (ν_asymCH₃); 2933 s (νC–H, aliphatic); 2876 s (ν_symCH₃); 2676 m; 2599 m (combination bands); 1746 vs (νO=C–O); 1635 vs (νO=C–N, amide I); 1599 s; 1573 vs; 1487 s; 1467 s (νC=C, aromatic); 1536 vs (δN–H, amide II); 1324 vs; 1296 vs; 1281 vs (ν_asymSO₂); 1161 vs (ν_symSO₂); 852 s (γC–H, aromatic); 613 vs; and 575 vs (νC–Br).

¹H NMR (300 MHz, DMSO-d₆) δ ppm: 8.73 (d, 1H, H-3, J = 8.0 Hz); 8.08 (d, 2H, H-7, H-11, J = 8.8 Hz); 8.04 (d, 2H, H-8, H-10, J = 8.8 Hz); 7.92 (d, 2H, H-13, H-17, J = 8.8 Hz); 7.85 (d, 2H, H-14, H-16, J = 8.8 Hz); 4.29 (dd, 1H, H-4, J = 8.1, 6.9 Hz); 2.17 (oct, 1H, H-18, J = 6.9 Hz); 0.95 (d, 3H, H-19, J = 6.9 Hz); and 0.94 (d, 3H, H-20, J = 6.9 Hz).

¹³C NMR (75 MHz, DMSO-d₆) δ ppm: 172.79 (C-5); 165.78 (C-2); 142.66 (C-9); 139.92 (C-12); 139.03 (C-6); 132.96 (C-14, C-16); 129.48 (C-7, C-11); 129.08 (C-8, C-10); 128.22 (C-15); 127.49 (C-13, C-17); 58.43 (C-4); 29.48 (C-18); 19.25 (C-19); and 18.61 (C-20).

Reversed-phase HPLC (CH₃OH–H₂O 30:70, V/V; flow rate 1 mL/min; λ 250 nm): purity 99.99%; t_R 4.47 min.

Elem. anal. (%) found: C, 49.06; H, 4.11; N, 3.19; and S, 7.28; calcd. for C₁₈H₁₈BrNO₅S (M_r 440.31): C, 49.10; H, 4.12; N, 3.18; and S, 7.28.

4.3.2. Preparation and Characterization of 2-{4-[(4-Bromophenyl)sulfonyl]phenyl}-4-isopropyl-4H-1,3-oxazol-5-one 6

An amount of 4.62 g (10.5 mmol) of 5 was suspended under magnetic stirring at room temperature in 50 mL of dry CH₂Cl₂ and then an equimolar quantity (1.15 mL, 1.06 g, 10.5 mmol) of 4-methylmorpholine was added. A volume of 1 mL (1.14 g, 10.5 mmol) of ethyl chloroformate was added dropwise with continuous stirring to the solution obtained above. The reaction mixture was stirred for an additional 30 min and further poured into 100 mL of ice water. The separated organic phase was washed with 5% NaHCO₃ solution, then with distilled water and dried (MgSO₄). After concentration under low-pressure, solid product 6 was recrystallized from cyclohexane when white crystals were obtained.

Yield 90% (3.99 g, 9.45 mmol). mp 144–145 °C.

UV/VIS (CH₃OH) λ_max nm (${\log }_{10}\epsilon$_max): 202.6 (4.49); 227.3 (4.14); 252.0 (4.35); (CH₃CN) λ_max nm (${\log }_{10}\epsilon$_max): 197.1 (4.93); 229.1 (4.48); 252.2 (4.64).

FTIR (KBr disc) $\tilde{\nu }$_max cm⁻¹: 3092 m; 3067 w (νC–H, aromatic); 2963 m (ν_asymCH₃); 2930 m (νC–H, aliphatic); 2875 m (ν_symCH₃); 1825 vs (νC=O); 1650 vs (νC=N); 1599 w; 1573 s; 1469 m (νC=C, aromatic); 1329 vs; 1293 s (ν_asymSO₂); 1243 m (ν_asymC–O–C); 1160 vs (ν_symSO₂); 1040 vs (ν_symC–O–C); 845 m (γC–H, aromatic); 614 vs; and 574 s (νC–Br).

¹H NMR (300 MHz, CDCl₃) δ ppm: 8.16 (d, 2H, H-7, H-11, J = 8.5 Hz); 8.05 (d, 2H, H-8, H-10, J = 8.5 Hz); 7.83 (d, 2H, H-13, H-17, J = 8.5 Hz); 7.68 (d, 2H, H-14, H-16, J = 8.5 Hz); 4.32 (d, 1H, H-4, J = 4.7 Hz); 2.40 (septd, 1H, H-18, J = 6.9, 4.7 Hz); 1.14 (d, 3H, H-19, J = 6.9 Hz); and 0.99 (d, 3H, H-20, J = 6.9 Hz).

¹³C NMR (75 MHz, CDCl₃) δ ppm: 176.98 (C-5); 160.36 (C-2); 144.89 (C-9); 139.92 (C-12); 132.97 (C-14, C-16); 130.58 (C-6); 129.49 (C-13, C-17); 129.23 (C-15); 128.99 (C-7, C-11); 128.19 (C-8, C-10); 71.04 (C-4); 31.42 (C-18); 18.90 (C-19); and 17.65 (C-20).

GC/EI–MS (70 eV) m/z (rel. abund. %): 379 (⁷⁹Br)/381 (⁸¹Br) (71.61/100, BP) [M-C₃H₆]·⁺; 323/325 (41.31/38.56) [⁷⁹BrC₆H₄SO₂C₆H₄CHNH]·⁺/[⁸¹BrC₆H₄SO₂C₆H₄CHNH]·⁺ or [⁷⁹BrC₆H₄SO₂C₆H₄CO]⁺/[⁸¹BrC₆H₄SO₂C₆H₄CO]⁺; 207 (29.87); 203 (40.89) [⁷⁹BrC₆H₄SO]⁺; 76 (24.15) [C₆H₄]·⁺; 43 (37.71) [C₃H₇]⁺; and t_R 36.53 min.

Reversed-phase HPLC (CH₃OH–H₂O 60:40, V/V; flow rate 1 mL/min; λ 250 nm): purity 94.16%; t_R 4.05 min.

Elem. anal. (%) found: C, 51.24; H, 3.81; N, 3.33; and S, 7.57; calcd. for C₁₈H₁₆BrNO₄S (M_r 422.29): C, 51.19; H, 3.82; N, 3.32; and S, 7.59.

4.3.3. General Procedure for the Preparation of 2-Acylamino Ketones 7a,b and Their Characterization

To a solution of raw 2-{4-[(4-bromophenyl)sulfonyl]phenyl}-4-isopropyl-4H-1,3-oxazol-5-one 6 (2.11 g, 5 mmol) in 25 mL of anhydrous arene (i.e., benzene and toluene, respectively), 2.00 g (15 mmol) of anhydrous aluminum trichloride were added portion-wise with magnetic stirring at room temperature. The reaction mixture was stirred for a further 20 h until hydrogen chloride emission ceased and then poured into 100 mL of ice water acidified with 5 mL of 37% HCl. A precipitate was obtained, which was isolated by filtration, washed on the filter with cold distilled water, and further with a cold ethanol/distilled water (1:1, V/V) mixture. The aqueous filtrate was extracted with CH₂Cl₂ (2 × 15 mL), then the organic phase was washed with distilled water, dried (Na₂SO₄), and concentrated to dryness by vacuum distillation, when the second fraction of 7 was isolated. Purification of the crude product by recrystallization from ethanol afforded colorless crystals.

4-[(4-Bromophenyl)sulfonyl]-N-(3-methyl-1-oxo-1-phenylbutan-2-yl)benzamide 7a

Compound 7a was synthesized by the reaction of 6 with benzene (25 mL, 21.85 g, 279.7 mmol).

Yield 80% (2.00 g, 4.00 mmol). mp 164–166 °C.

UV/VIS (CH₃OH) λ_max nm (${\log }_{10}\epsilon$_max): 202.6 (4.49); 252.0 (4.19); (CH₃CN) λ_max nm (${\log }_{10}\epsilon$_max): 198.0 (5.03); 250.2 (4.77).

FTIR (KBr disc) $\tilde{\nu }$_max cm⁻¹: 3301 s (νN–H); 3089 m; 3061 m; 3040 m (νC–H, aromatic); 2962 m (ν_asymCH₃); 2931 m (νC–H, aliphatic); 2872 m (ν_symCH₃); 1655 vs (νO=C–O and νO=C–N, amide I; overlapped); 1597 m; 1574 s; 1483 m; 1469 m; 1448 m (νC=C, aromatic); 1528 s (δN–H, amide II); 1323 vs; 1289 s (ν_asymSO₂); 1161 vs (ν_symSO₂); 854 m (γC–H, aromatic); 615 vs; and 580 s (νC–Br).

¹H NMR (300 MHz, DMSO-d₆) δ ppm: 8.99 (d, 1H, H-3, J = 8.0 Hz); 8.05 (m, 6H, H-7, H-8, H-10, H-11, H-22, H-26); 7.91 (d, 2H, H-13, H-17, J = 8.5 Hz); 7.84 (d, 2H, H-14, H-16, J = 8.5 Hz); 7.64 (br t, 1H, H-24, J = 7.6 Hz); 7.53 (br t, 2H, H-23, H-25, J = 7.6 Hz); 5.38 (t, 1H, H-4, J = 7.7 Hz); 2.28 (m, 1H, H-18); 0.92 (d, 3H, H-19, J = 7.1 Hz); and 0.90 (d, 3H, H-20, J = 7.1 Hz).

¹³C NMR (75 MHz, DMSO-d₆) δ ppm: 199.19 (C-5); 165.57 (C-2); 142.76 (C-9); 139.88 (C-12); 138.78 (C-6); 136.17 (C-21); 133.44 (C-24); 132.94 (C-14, C-16); 129.48 (C-13, C-17); 129.04 (C-7, C-11); 128.82 (C-23, C-25); 128.24 (C-22, C-26); 128.22 (C-15); 127.53 (C-8, C-10); 59.23 (C-4); 29.50 (C-18); 19.72 (C-19); and 18.36 (C-20).

Reversed-phase HPLC (CH₃OH–H₂O 60:40, V/V; flow rate 1 mL/min; λ 250 nm): purity 92.02%; t_R 4.35 min.

Elem. anal. (%) found: C, 57.63; H, 4.41; N, 2.81; and S, 6.40; calcd. for C₂₄H₂₂BrNO₄S (M_r 500.40): C, 57.60; H, 4.43; N, 2.80; and S, 6.41.

4-[(4-Bromophenyl)sulfonyl]-N-[3-methyl-1-oxo-1-(p-tolyl)butan-2-yl]benzamide 7b

Compound 7b was synthesized by the reaction of 6 with toluene (25 mL, 21.63 g, 234.8 mmol).

Yield 86% (2.21 g, 4.30 mmol). mp 154–155 °C.

UV/VIS (CH₃OH) λ_max nm (${\log }_{10}\epsilon$_max): 202.6 (4.47); 255.5 (4.18); (CH₃CN) λ_max nm (${\log }_{10}\epsilon$_max): 197.8 (5.01); 254.8 (4.78).

FTIR (KBr disc) $\tilde{\nu }$_max cm⁻¹: 3281 s (νN–H); 3086 m; 3058 m; 3037 m (νC–H, aromatic); 2962 m (ν_asymCH₃); 2929 m (νC–H, aliphatic); 2868 m (ν_symCH₃); 1655 vs (νO=C–O and νO=C–N, amide I; overlapped); 1606 m; 1572 s; 1483 m; 1467 m (νC=C, aromatic); 1530 s (δN–H, amide II); 1325 s; 1305 m; 1287 m (ν_asymSO₂); 1161 vs (ν_symSO₂); 858 m (γC–H, aromatic); 617 s; and 576 s (νC–Br).

¹H NMR (300 MHz, DMSO-d₆) δ ppm: 8.94 (d, 1H, H-3, J = 8.0 Hz); 8.06 (d, 2H, H-7, H-11, J = 8.8 Hz); 8.03 (d, 2H, H-8, H-10, J = 8.8 Hz); 7.95 (d, 2H, H-22, H-26, J = 8.2 Hz); 7.91 (d, 2H, H-13, H-17, J = 8.8 Hz); 7.84 (d, 2H, H-14, H-16, J = 8.8 Hz); 7.33 (d, 2H, H-23, H-25, J = 8.2 Hz); 5.36 (t, 1H, H-4, J = 7.7 Hz); 2.36 (s, 3H, CH₃); 2.27 (m, 1H, H-18); 0.92 (d, 3H, H-19, J = 7.4 Hz); and 0.89 (d, 3H, H-20, J = 7.4 Hz).

¹³C NMR (75 MHz, DMSO-d₆) δ ppm: 198.57 (C-5); 165.47 (C-2); 143.89 (C-24); 142.72 (C-9); 139.88 (C-12); 138.81 (C-6); 133.61 (C-21); 132.92 (C-14, C-16); 129.44 (C-13, C-17); 129.34 (C-23, C-25); 129.00 (C-7, C-11); 128.36 (C-22, C-26); 128.18 (C-15); 127.49 (C-8, C-10); 59.05 (C-4); 29.56 (C-18); 21.12 (CH₃); 19.71 (C-19); and 18.33 (C-20).

Reversed-phase HPLC (CH₃OH–H₂O 60:40, V/V; flow rate 1 mL/min; λ 250 nm): purity 99.99%; t_R 4.95 min.

Elem. anal. (%) found: C, 58.34; H, 4.69; N, 2.71; and S, 6.21; calcd. for C₂₅H₂₄BrNO₄S (M_r 514.43): C, 58.37; H, 4.70; N, 2.72; and S, 6.23.

4.3.4. General Procedure for the Preparation of 2,5-Disubstituted 4-Isopropyl-1,3-oxazoles 8a,b and Their Characterization

A mixture of 10 mmol of crude 7 and 20 mL (33.40 g, 217.8 mmol) of phosphoryl trichloride was heated under reflux during 4 h. The excess phosphoryl trichloride was evaporated under reduced pressure. The resulting oily residue was cooled, treated with an ice water mixture, and extracted with 2 × 20 mL CH₂Cl₂. The organic phase was separated and washed with 5% NaHCO₃ solution, then with distilled water and dried (Na₂SO₄). The solvent was evaporated off under vacuum. Purification of the resulting solid was performed by recrystallization from ethanol when colorless crystals of 8 were obtained.

2-{4-[(4-Bromophenyl)sulfonyl]phenyl}-4-isopropyl-5-phenyl-1,3-oxazole 8a

Compound 8a was synthesized from 5.00 g (10 mmol) of 2-acylamino ketone 7a.

Yield 94% (4.53 g, 9.39 mmol). mp 165–167 °C.

UV/VIS (CH₃OH) λ_max nm (${\log }_{10}\epsilon$_max): 202.6 (4.48); 249.3 (4.10); 333.9 (4.13); (CH₃CN) λ_max nm (${\log }_{10}\epsilon$_max): 196.2 (4.95); 248.6 (4.56); 335.6 (4.64).

FTIR (KBr disc) $\tilde{\nu }$_max cm⁻¹: 3092 m; 3063 m; 3041 w (νC–H, aromatic); 2963 s (ν_asymCH₃); 2930 m (νC–H, aliphatic); 2869 m (ν_symCH₃); 1602 m (νC=N); 1588 m; 1574 s; 1543 w; 1494 m; 1471 m; 1446 m (νC=C, aromatic); 1323 vs; 1293 m (ν_asymSO₂); 1280 m (ν_asymC–O–C); 1155 vs (ν_symSO₂); 1099 s (ν_symC–O–C); 844 m (γC–H, aromatic); 617 s; and 568 s (νC–Br).

¹H NMR (300 MHz, CDCl₃) δ ppm: 8.22 (d, 2H, H-7, H-11, J = 8.8 Hz); 8.00 (d, 2H, H-8, H-10, J = 8.8 Hz); 7.82 (d, 2H, H-13, H-17, J = 8.8 Hz); 7.65 (d, 2H, H-14, H-16, J = 8.8 Hz); 7.64 (dd, 2H, H-22, H-26, J = 7.4, 1.4 Hz); 7.48 (br t, 2H, H-23, H-25, J = 7.4 Hz); 7.37 (tt, 1H, H-24, J = 7.4, 1.4 Hz); 3.29 (sept, 1H, H-18, J = 6.9 Hz); and 1.36 (d, 6H, H-19, H-20, J = 6.9 Hz).

¹³C NMR (75 MHz, CDCl₃) δ ppm: 157.78 (C-2); 145.47 (C-5); 144.19 (C-4); 141.71 (C-12); 140.56 (C-9); 132.80 (C-14, C-16); 132.33 (C-6); 129.29 (C-13, C-17); 129.01 (C-23, C-25); 128.81 (C-21); 128.78 (C-15); 128.39 (C-24); 128.26 (C-7, C-11); 127.09 (C-8, C-10); 126.25 (C-22, C-26); 26.10 (C-18); and 22.07 (C-19, C-20).

Reversed-phase HPLC (CH₃OH–H₂O 70:30, V/V; flow rate 1 mL/min; λ 335 nm): purity 98.95%; t_R 5.97 min.

Elem. anal. (%) found: C, 59.71; H, 4.18; N, 2.91; and S, 6.67; calcd. for C₂₄H₂₀BrNO₃S (M_r 482.39): C, 59.76; H, 4.18; N, 2.90; and S, 6.65.

2-{4-[(4-Bromophenyl)sulfonyl]phenyl}-4-isopropyl-5-(p-tolyl)-1,3-oxazole 8b

Compound 8b was synthesized from 5.14 g (10 mmol) of 2-acylamino ketone 7b.

Yield 91% (4.52 g, 9.10 mmol). mp 215–217 °C.

UV/VIS (CH₃OH) λ_max nm (${\log }_{10}\epsilon$_max): 202.6 (4.48); 249.3 (4.11); 337.4 (4.09); (CH₃CN) λ_max nm (${\log }_{10}\epsilon$_max): 195.3 (4.96); 249.5 (4.59); 340.1 (4.64).

FTIR (KBr disc) $\tilde{\nu }$_max cm⁻¹: 3091 m; 3069 w; 3028 w (νC–H, aromatic); 2963 m (ν_asymCH₃); 2925 m (νC–H, aliphatic); 2869 m (ν_symCH₃); 1601 m (νC=N); 1592 m; 1574 m; 1545 w; 1508 m; 1471 m (νC=C, aromatic); 1322 s; 1292 m (ν_asymSO₂); 1280 m (ν_asymC–O–C); 1156 vs (ν_symSO₂); 1097 s (ν_symC–O–C); 846 m (γC–H, aromatic); 617 s; and 568 s (νC–Br).

¹H NMR (300 MHz, CDCl₃) δ ppm: 8.21 (d, 2H, H-7, H-11, J = 8.5 Hz); 8.00 (d, 2H, H-8, H-10, J = 8.5 Hz); 7.82 (d, 2H, H-13, H-17, J = 8.5 Hz); 7.65 (d, 2H, H-14, H-16, J = 8.5 Hz); 7.53 (d, 2H, H-22, H-26, J = 8.2 Hz); 7.28 (d, 2H, H-23, H-25, J = 8.2 Hz); 3.26 (sept, 1H, H-18, J = 6.9 Hz); 2.41 (s, 3H, CH₃); and 1.35 (d, 6H, H-19, H-20, J = 6.9 Hz).

¹³C NMR (75 MHz, CDCl₃) δ ppm: 157.52 (C-2); 145.69 (C-5); 143.63 (C-4); 141.57 (C-12); 140.61 (C-9); 138.48 (C-24); 132.80 (C-14, C-16); 132.43 (C-6); 129.70 (C-23, C-25); 129.29 (C-13, C-17); 128.76 (C-15); 128.25 (C-7, C-11); 127.03 (C-8, C-10); 126.22 (C-22, C-26); 126.01 (C-21); 26.08 (C-18); 22.07 (C-19, C-20); and 21.47 (CH₃).

Reversed-phase HPLC (CH₃OH–H₂O 70:30, V/V; flow rate 1 mL/min; λ 335 nm): purity 99.99%; t_R 7.32 min.

Elem. anal. (%) found: C, 60.52; H, 4.46; N, 2.81; and S, 6.48; calcd. for C₂₅H₂₂BrNO₃S (M_r 496.42): C, 60.49; H, 4.47; N, 2.82; and S, 6.46.

4.4. Assessment of Antimicrobial Activity

The evaluation of the influence of the compounds on microbial growth and biofilm formation was performed using an adapted disk diffusion assay, the standard broth microdilution method, and the microtiter plate test [80].

4.4.1. Pathogenic Microbial Strains

The pathogenic microorganisms tested included three Gram-positive bacteria, i.e., B. subtilis 6683, E. faecium E5, S. aureus ATCC 6538, and two Gram-negative bacteria, namely E. coli ATCC 8739, P. aeruginosa ATCC 27853, as well as a fungal C. albicans 393 strain.

4.4.2. Agar Diffusion Assay

The antimicrobial activity was screened using an adapted agar diffusion method [81]. Briefly, the bacterial and fungal suspensions were prepared in phosphate-buffered saline from fresh microbial cultures and their density was adjusted to 0.5 McFarland. The compounds were dissolved in DMSO at a concentration of 5 mg/mL and then a volume of 5 μL was spotted on the inoculated plates. The diameters of the growth inhibition zones were measured after incubation for 24 h at 37 °C. Standardized discs of ciprofloxacin, 5 µg (Oxoid), and fluconazole, 25 µg (Oxoid) were used as positive controls.

4.4.3. Evaluation of the Effects of the Tested Compounds on the Antibiotic Susceptibility Profile

Standardized microbial suspensions of E. faecium E5 and S. aureus ATCC 6538 were put in contact with subinhibitory concentrations (0.25 mg/mL) of tested compounds 5 and 6 prepared in broth. After incubation at 37 °C for 24 h, the bacterial cultures were used to evaluate their antibiotic susceptibility profile. The disk diffusion method was performed to assess the susceptibility to the following antibiotics: ampicillin, linezolid, penicillin, vancomycin for Gram-positive E. faecium E5 strain and azithromycin, cefoxitin, clindamycin, linezolid, penicillin, rifampicin, trimethoprim-sulfamethoxazole, and vancomycin (bioMérieux, Paris, France) for Gram-positive S. aureus ATCC 6538 strain. The reading of the results was performed following the Clinical and Laboratory Standards Institute (CLSI, Berwyn, PA, USA) guidelines.

4.4.4. Broth Microdilution Assay

The standard broth microdilution test was performed on 96-well microtiter plates. The compounds were solubilized in DMSO at a concentration of 10 mg/mL. Serial dilutions of the tested compounds were prepared in sterile broth and the obtained solutions of different concentrations were inoculated with each standardized microbial suspension. Ciprofloxacin and fluconazole (Oxoid) served as positive controls. After incubation for 24 h at 37 °C, the MIC values were determined as the lowest concentration of compound required to inhibit the microbial growth, as detected spectrophotometrically (Apollo LB 911 ELISA Reader, Berthold Technologies GmbH & Co. KG, Waltham, MA, USA) [82]. The assays were performed in triplicate.

4.4.5. Microplate Microtiter Assay

The influence of the compounds on the biofilm formation was determined using the microtiter plate assay. Briefly, after the determination of the MIC values, the microplates were emptied and washed three times with sterile distilled water for the removal of planktonic microbial cells. The biofilms adherent on the plastic wells were fixed with methanol for 5 min and stained with 1% crystal violet solution for a quarter of an hour. The excess dye was removed and thereafter the fixed dye was resuspended in 33% acetic acid. Ciprofloxacin (Oxoid) was used as a positive control for antibacterial tests and fluconazole (Oxoid) for antifungal testing. The absorbance of the colored solutions was recorded with an ELISA Reader (Apollo LB 911). The minimal biofilm inhibitory concentration (MBIC), defined as the lowest compound concentration required to inhibit biofilm formation, was determined. The results from three separate biological replicates were mediated [83].

4.5. Evaluation of Antioxidant Activity

The antioxidant activity of newly obtained derivatives 5–8 was studied spectrophotometrically by means of the DPPH, ABTS, and ferric reducing power methods [84,85,86].

4.5.1. Antioxidant Activity Assay by DPPH Method

The antioxidant effect of new compounds was determined according to the well-known DPPH assay based on the fact that the antioxidant samples react with stable 2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazin-1-yl (2,2-diphenyl-1-picrylhydrazyl, DPPH), an effective free radical trap with an absorption band at 517 nm, which converts to its reduced form, i.e., 1,1-diphenyl-2-(2,4,6-trinitrophenyl)hydrazine (2,2-diphenyl-1-picryl hydrazine), with a color change from purple to yellow accompanied by a decrease in absorption at 517 nm [84].

The antioxidant potential of the tested compounds was investigated according to the method previously described by Blois [87,88,89], with some modifications, and compared with the free radical scavenging activity of AA, BHA, and BHT standards.

Briefly, 2 mL of a solution of DPPH in ethanol at a concentration of 400 μM was added to 2 mL of each solution of the tested compound in DMSO at a concentration of 500 μM. After maintaining the samples in the dark at room temperature for 30 min, the absorbance of each sample at the wavelength of 517 nm was measured on a Specord 40 UV/VIS spectrophotometer (Analytik Jena AG, Jena, Germany). Then, the radical scavenging activity (RSA) in percent (%) was calculated using the following formula:

$$RSA (\%)=(1-\frac{A_{\mathrm{sample} }-A_{\mathrm{blank} \mathrm{sample}}}{A_{\mathrm{DPPH} \mathrm{control}}})\times 100$$

where A_sample is the absorbance of the tested compound solution with the DPPH solution, A_{blank sample} is the absorbance of the tested compound solution (without the DPPH solution), and A_{DPPH control} is the absorbance of the DPPH solution (without the tested compound solution) [90]. Each analysis was performed on three replicates and the results were averaged.

4.5.2. Antioxidant Activity Assay by ABTS Method

The ABTS discoloration test is based on the ability of the antioxidant agents to scavenge the long-life ABTS radical cation, ABTS^•+ [91]. The stable green-blue radical cationic chromophore, ABTS^•+, which has an absorption maximum at the wavelength of 734 nm, by reaction with most antioxidants turns into its colorless neutral form, i.e., 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) [85].

The antioxidant capacity of the tested compounds was assessed as described earlier by Re et al. [91], with some modifications. The ABTS^•+ is prepared by oxidation of ABTS with radical-initiator potassium persulfate. Initially, the stock solution of ABTS was obtained by dissolving it in water at a concentration of 7 μM. The ABTS radical cation (ABTS^•+) was then generated by reacting the 7 μM ABTS stock solution with a 2.45 μM K₂S₂O₈ solution (1:1, V/V) and kept in the dark at room temperature for 12–16 h. Before analysis, this solution was diluted with ethanol and equilibrated at 30 °C to have absorbance at 734 nm, A₇₃₄ of 0.7000 ± 0.02. Subsequently, 2 mL of ABTS^•+ solution was added to 2 mL of 500 μM tested compound solution in DMSO. After 6 min, the absorbance of each sample was read at 734 nm with a UV/VIS spectrophotometer (Specord 40, Analytik Jena AG, Jena, Germany) and converted into the percentage radical scavenging activity, %RSA, using the formula:

$$RSA (\%)=(1-\frac{A_{\mathrm{sample} }-A_{\mathrm{blank} \mathrm{sample}}}{A_{{\mathrm{ABTS}}^{•+}}{ }_{\mathrm{control}}})\times 100$$

where A_sample is the absorbance of the tested compound solution with the ABTS^•+ solution, A_{blank sample} is the absorbance of the tested compound solution (without the ABTS^•+ solution), and A_ABTS^•+ _control is the absorbance of the ABTS^•+ solution (without the tested compound solution) [89,90]. All determinations were undertaken in triplicate and the results were averaged. The same antioxidant agents (AA, BHA, and BHT) were used as positive controls.

4.5.3. Ferric Reducing Power Assay

In the ferric reducing power test, is measured the reduction in the bright red color potassium ferricyanide to the intense blue color ferric ferrocyanide (Prussian blue), which occurs by means of antioxidants in acidic environments, with an increase in absorbance at 700 nm [84].

The ability of all the newly synthesized compounds to reduce iron(III) to iron(II) was determined by the modified Oyaizu method [92,93,94]. The sample solutions were prepared in DMSO at a concentration of 500 μM. Two milliliters of each tested compound solution were mixed with 2 mL of 0.2 M phosphate buffer (pH 6.6) and 2 mL of 1% K₃[Fe(CN)₆] solution. After 20 min incubation at 50 °C, 2 mL of 10% trichloroacetic acid solution was added, and the mixture was centrifuged for 15 min at 4500 rpm. Finally, 2 mL of the upper layer was mixed with 2 mL of deionized water and 0.4 mL of 0.1% FeCl₃ solution, and after 5 min the absorbance was recorded at 700 nm with an Analytik Jena Specord 40 UV/VIS spectrophotometer. All analyses were performed on three replicates, and the results were averaged and compared with those of antioxidant standards used, i.e., AA, BHA, and BHT.

4.6. Daphnia magna Toxicity Bioassay

In ecotoxicology, the planktonic crustacean Daphnia magna Staus is specified for use in the “OECD Guidelines for the Testing of Chemicals”. Test No. 202: “Daphnia sp., Acute Immobilization Test” is an acute toxicity study, in which young daphnids, aged less than 24 h at the beginning of the test, are exposed to different concentrations of the chemical under test for 48 h and the half-maximal effective concentration (EC₅₀) determined [95].

In this study, the young D. magna Straus organisms were selected according to their size from a parthenogenetic culture. All compounds were tested at six concentrations ranging from 2 to 50 µg/mL, in duplicate, with each replicate having 10 individuals. The concentration range was selected based on the compounds’ solubilities and a pre-screening bioassay. L-Valine, compound 3, and a 1% DMSO solution were used as controls. All determinations were carried out using the same conditions (25 °C, a long day photoperiod of 16 h light/8 h dark cycle) in a Sanyo MLR-351H climate test chamber (Sanyo, San Diego, CA, USA) [48,96,97]. After 24 and 48 h of exposure, the lethality was evaluated. LC₅₀ was calculated for each compound based on interpolating on lethality curves which were obtained using the least square fit method. The 95% confidence intervals (95% CI) were calculated and the goodness of fit was also evaluated. The calculations were performed using GraphPad Prism v5.1 software (GraphPad Software, Inc., La Jolla, CA, USA). The prediction of LC₅₀ at 48 h was made using GUSAR online application (Institute of Biomedical Chemistry, Moscow, Russia, http://www.way2drug.com/gusar/) [98].

5. Conclusions

In order to identify new bioactive compounds, we designed and synthesized novel derivatives that incorporate in the same molecule the N-acyl-α-amino acid, 4H-1,3-oxazol-5-one, 2-acylamino ketone, or 1,3-oxazole template and, a biologically active fragment derived from diphenyl sulfone. The obtained compounds’ structures were confirmed on the basis of spectral studies and elemental analysis data. Taken together, the results of the qualitative and quantitative antimicrobial activity evaluation, antioxidant effect assessment, toxicity bioassay, as well as of in silico analysis revealed a promising potential of N-acyl-L-valine 5 and 2-substituted 4-isopropyl-4H-1,3-oxazol-5-one 6 for the development of novel antimicrobial agents to combat infections produced by Gram-positive bacterial strains, and in particular of E. faecium biofilm-associated infections.

6. Patents

Patent application no. a 2019 00668: Theodora-Venera Apostol, Ștefania-Felicia Bărbuceanu, Laura Ileana Socea, Ioana Șaramet, Constantin Drăghici, Valeria Rădulescu, Mariana-Carmen Chifiriuc, Luminița Gabriela Măruțescu, Octavian Tudorel Olaru, and George Mihai Nițulescu, 4-Isopropyl-1,3-oxazol-5(4H)-one Derivatives Containing a Diaryl sulfonyl Substituent in Position 2 with Antimicrobial Action, published in RO-BOPI No. 9/2021, from 30 september 2021.