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Article

In Silico and In Vitro Assessment of Antimicrobial and Antibiofilm Activity of Some 1,3-Oxazole-Based Compounds and Their Isosteric Analogues

by
Theodora-Venera Apostol
1,
Mariana Carmen Chifiriuc
2,3,4,*,
George Mihai Nitulescu
1,*,
Octavian Tudorel Olaru
1,
Stefania-Felicia Barbuceanu
1,
Laura-Ileana Socea
1,
Elena Mihaela Pahontu
1,
Crina Maria Karmezan
2 and
Luminita Gabriela Marutescu
2
1
Faculty of Pharmacy, “Carol Davila” University of Medicine and Pharmacy, 6 Traian Vuia Street, District 2, 020956 Bucharest, Romania
2
Department of Botany and Microbiology, Faculty of Biology, University of Bucharest, 1–3 Aleea Portocalelor, District 6, 60101 Bucharest, Romania
3
Romanian Academy of Scientists, 54 Splaiul Independenței, District 5, 50085 Bucharest, Romania
4
The Romanian Academy, 125 Calea Victoriei, District 1, 010071 Bucharest, Romania
*
Authors to whom correspondence should be addressed.
Appl. Sci. 2022, 12(11), 5571; https://doi.org/10.3390/app12115571
Submission received: 16 May 2022 / Revised: 27 May 2022 / Accepted: 28 May 2022 / Published: 30 May 2022
(This article belongs to the Special Issue Synthesis of Novel Heterocyclic Compounds and Evaluation of Their Antimicrobial Activity)
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Versions Notes

Abstract

:
In this paper, we report on the antimicrobial activity assessment of 49 compounds previously synthesized as derivatives of alanine or phenylalanine that incorporate a 4-(4-X-phenylsulfonyl)phenyl fragment (X = H, Cl, or Br), namely 21 acyclic compounds (6 × N-acyl-α-amino acids, 1 × N-acyl-α-amino acid ester, and 14 × N-acyl-α-amino ketones) and 28 pentatomic heterocycles from the oxazole-based compound class (6 × 4H-1,3-oxazol-5-ones, 16 × 5-aryl-1,3-oxazoles, and 6 × ethyl 1,3-oxazol-5-yl carbonates). Both in silico and in vitro qualitative and quantitative assays were used to investigate the antimicrobial potential of these derivatives against planktonic and biofilm-embedded microbial strains. Some of the tested compounds showed promising antimicrobial and antibiofilm activity depending on their chemical scaffold and lipophilic character.

1. Introduction

The rise of multidrug-resistant pathogenic microorganisms is a major health concern. In response, there is an urgent need for the identification of novel antimicrobial agents.
The development of new sulfonyl-group-containing analogs is a hot research topic in medicinal chemistry [1,2,3,4,5,6,7]. Among these compounds, numerous diaryl sulfones have been found to exhibit a variety of biological activities, including antimicrobial, antioxidant, antimycobacterial, antimalarial, anticancer, anti-inflammatory, and anti-HIV effects [8,9,10,11,12,13,14,15,16,17]. Further, some representatives of this class selectively block the 5-HT6 receptors being developed as therapies for Alzheimer’s disease [18,19]. Recently, Alsaedi et al. synthesized a series of pyrazolo [1,5-a]pyrimidine derivatives containing the phenylsulfonyl moiety and evaluated their antimicrobial activities. The results revealed that several sulfone analogues showed effects exceeding the activity of the reference drug. Unexpectedly, it was observed that derivatives containing one sulfone group were more effective against different bacterial and fungal strains than those containing two sulfone groups [20]. Moreover, some unsaturated 4H-1,3-oxazol-5-ones bearing the arylsulfonylphenyl moiety in their molecules exhibited good antifungal and antibiofilm potential [21]. Very recently, the results reported by Rashdan et al. highlighted a synthetic 1,2,3-triazole-containing sulfone derivative structurally inspired by dapsone that exhibited outstanding antimicrobial properties against various bacterial strains [22].
In addition, the 1,3-oxazole is an important heterocyclic nucleus that is present in numerous active substances and displays a wide array of biological properties. Significant research has been conducted to synthesize 1,3-oxazole derivatives and to evaluate their pharmacological profile [23,24]. Therefore, over time, a large number of natural and synthetic 1,3-oxazole–based compounds, which have been associated with a wide spectrum of pharmacological activities, such as antimicrobial, antimalarial, antidiabetic, analgesic, anti-inflammatory, and antitumoral effects, have been reported [25,26,27,28,29,30,31,32]. It has been also shown that saturated 4H-1,3-oxazol-5-ones—the stable 5-oxo tautomers of 1,3-oxazol-5-ols—present antimicrobial, anticancer, antiviral, and trypanocidal actions [33,34,35,36].
N-acyl amino acids, in which the acyl moiety is derived from fatty acids, are similar to endogenous cannabinoids [37]. Among these N-fatty acyl-α-amino acids, N-arachidonoylserine has been reported to display antimicrobial and antibiofilm effects against methicillin-resistant Staphylococcus aureus (MRSA) strains. In addition, the staphylococcal-biofilm-associated virulence determinants are altered by this agent. N-arachidonoylserine is able to change the bacterial membrane potential and prevent biofilm formation without killing the bacterial cells [37,38]. It has been discovered that N-acyltyrosine derivatives act as bacterial metabolites that exhibit antibiotic effects against the Bacillus subtilis and an average inhibition of the Pseudomonas aeruginosa biofilm formation process [39]. N-acyl-α-amino acids exhibit other therapeutic effects, such as antihypertensive, mucolytic, anticancer, antianemic, antiulcer, and antioxidant actions [40,41,42,43,44,45,46].
Some representatives of the N-acyl-α-amino acid ester class have antibacterial, antileishmanial, antiproliferative, antidepressant, and monoamine oxidase inhibitory activities [47,48,49,50,51,52].
Several N-acyl-α-amino ketone derivatives show antiviral, antihypertensive, antithrombotic, and anti-inflammatory properties [53,54,55,56,57,58].
In an attempt to develop new antimicrobial agents, our group devoted considerable interest in the synthesis of compounds based on the 4-(4-X-phenylsulfonyl)phenyl fragment as the pharmacophore center [59,60,61,62,63,64,65]. In this study, the antimicrobial and antibiofilm effects of 49 compounds (1a–f, 2a–f, 3a, 4a–f, 5a–n, and 6a–p) from the classes mentioned above were evaluated against different bacterial and fungal strains. In silico prediction of the antimicrobial, pharmacokinetic, and toxicological features of the tested compounds was also performed.

2. Results

2.1. Chemistry

Following the described synthesis procedures, a series of new N-acyl-α-amino acids (1a–f), 4H-1,3-oxazol-5-ones (2a–f), N-acyl-α-amino ketones (5a–n), 1,3-oxazoles (4a–f, 6a–p), and one N-acyl-α-amino acid ester (3a) were synthesized [59,60,61,62,63,64,65]. The general structures of the compounds are presented in Scheme 1.
The chemical structures and purities (%) of the tested compounds 1a–f, 2a–f, 3a, 4a–f, 5a–n, and 6a–p are presented in Table 1.

2.2. Antimicrobial Activity Assessment

2.2.1. Qualitative Screening of Antimicrobial Activity

The qualitative screening tests showed a weak growth inhibitory effect of the tested compounds against the studied microorganisms with no clear inhibition zones, the limits of the growth inhibition not exceeding the compound solution deposition area on the agar layer. This could be explained by the poor diffusion into the agar media of the tested compounds. The growth inhibition zones were detected only for compounds 1a–f; 2a–f; 3a; 4a–f; 5a,b; 5d,e; 5g,h; 5j,k; 5m,n; 6g–k; 6m; and 6o,p, which were further evaluated by the quantitative method to determine the minimal inhibitory concentration (MIC) values.

2.2.2. Quantitative Assay of Antimicrobial Activity

The broth dilution method was used to quantitatively assess the in vitro antimicrobial profile of compounds 1a–f; 2a–f; 3a; 4a–f; 5a,b; 5d,e; 5g,h; 5j,k; 5m,n; 6g–k; 6m; and 6o,p against two Gram-positive bacteria (S. epidermidis 756 and B. subtilis ATCC 6683), two Gram-negative bacteria (E. coli ATCC 25922 and P. aeruginosa ATCC 27853), and one yeast strain (C. albicans 128).
Compounds 1a–c; 4b–f; 5a,b; 5d,e; 5g,h; 5j,k; 5m,n; 6g,h; 6k; 6m; and 6o,p did not inhibit the growth of any of the tested microbial strains up to a concentration of 225 μg/mL. In contrast, the MIC values of compounds 1d–f; 2a–f; 3a; 4a; and 6i,j were much lower in the case of some microbial strains with MICs in the range of 56.2 to 14 μg/mL being recorded (Table 2). Compound 1e exhibited a wide spectrum of antimicrobial activity, being active on both Gram-positive and -negative bacterial strains as well as against the fungal strain C. albicans 128, for which MICs ≤56.2 μg/mL were recorded. Compounds 1d,e; 3a; 4a; and 6i,j exhibited very good antifungal activity with MIC values of 14 μg/mL.

2.2.3. Effects of the Compounds on Biofilm Formation

To further evaluate the effects of the analyzed compounds against microbial biofilms, crystal violet microtiter assay was performed for compounds 1a–f; 2a–f; 3a; 4a–f; 5a,b; 5d,e; 5g,h; 5j,k; 5m,n; 6g–k; 6m; and 6o,p. The analysis showed that biofilm formation was not affected by compounds 1a–c; 4b–f; 5a,b; 5d,e; 5g,h; 5j,k; 5m,n; 6g,h; 6k; 6m; and 6o. However, decreased absorbance of the stained biomass was recorded for the biofilms grown in the presence of compounds 1d–f; 2a–f; 3a; 4a; 6i,j; and 6p, with the minimal biofilm inhibitory assay (MBIC) ranging from 14 to 225 µg/mL (Table 2). Compounds 1e, 2d–f, 3a, 4a, and 6j demonstrated an MBIC value of 14 µg/mL against the P. aeruginosa ATCC 27853 biofilm. Compound 1e inhibited the biofilm-forming capacity of the Gram-positive S. epidermidis 756 strain, the Gram-negative strains E. coli ATCC 25922 and P. aeruginosa ATCC 27853, and the fungal strain C. albicans 128.
Regarding the antimicrobial and antibiofilm activity of the standard antibiotic (ciprofloxacin) and antifungal (fluconazole) agents used as controls, the MIC values were much lower than those obtained for the tested compounds in all of the cases. This is somehow expected given the new compounds are not yet standardized in optimal formulations and their mechanisms of action could be different from those of the control drugs.

2.3. Prediction of the Biological Properties of the Compounds

2.3.1. In Silico Evaluation of the Molecular Mechanism of Action

Based on 2D structural descriptors, the PASS application was used to calculate the probability of the target compounds 1af, 2a–f, 3a, 4a–f, 5a–n, and 6a–p as being active (Pa) or inactive (Pi) on a large series of targets [66]. The analysis returned Pa values higher than the corresponding Pi values for 2097 pharmacological effects, of which 14 were directly related to antibacterial effects. The maximum and minimum predicted Pa values for the 49 compounds and the compounds with Pa values over 0.5 are displayed in Table 3.
A total of 33 compounds presented Pa values over 0.3 for anti-infective effect, and 37 compounds were predicted to have antimycobacterial effects with Pa value above 0.3.
Figure 1 presents the plotted Pa values for anti-infective, antimycobacterial, and antibacterial effects based on the main chemical scaffold.
The prediction results indicated a clear correlation between the chemical scaffolds of the studied compounds and the potential to have an anti-effect, with N-acyl-α-amino acids (scaffold 1) emerging as the most promising class (Figure 1b). In the case of antibacterial effects, the prediction indicated 4H-1,3-oxazol-5-one ring (scaffold 2) as the most favorable core structure (Figure 1d). The potential of the compounds to produce antimycobacterial effects was not correlated with the chemical scaffolds (Figure 1c).
The PASS application can be used to indicate a probable mechanism of action [67] of new compounds. Compounds 1a–d, 2a, and 2d had significant Pa values for the inhibition of peptidoglycan glycosyltransferase, a valuable target for new antimicrobial therapies [68]. UDP-N-acetylmuramate-L-alanine ligase inhibitor (MurC) is a member of the Mur enzymes family and, similar to peptidoglycan glycosyltransferase, is involved in synthesis of peptidoglycan [69]. Compounds 1a–c; 1e,f; 2d,e; and 3a presented small but significant Pa values towards this possible mechanism.

2.3.2. Structural Descriptors Analysis

DataWarrior v5.2.1 software [70] was used to calculate a series of structural descriptors, namely molecular weight (MW), logarithm of the octanol–water partition coefficient (cLogP), hydrogen bond donors count (HBD), hydrogen bond acceptor count (HBA), polar surface area (PSA), number of rotatable bonds (RB), and druglikeness (DLK). These descriptors are presented in Table 4 with the minimum and maximum values registered for the compounds.
In order to better understand the structure–activity relationships, each descriptor was plotted for both active and inactive compounds based on the MIC values presented in Table 2. The best difference of distribution of values was observed for cLogP (Figure 2). The results indicated that a lower lipophilic character was correlated with a higher antimicrobial effect. The chemical scaffold was also an important factor because some compounds were inactive despite a low cLogP value.
Four compounds with cLogP values in the range of 3.08–3.93 presented antimicrobial and antibiofilm effects towards S. epidermidis. Most of the inactive compounds had a cLogP value over 4, indicating that a high lipophilicity was detrimental. Except for the ethyl carbonate derivative 4a, the active compounds on S. epidermidis were all derivatives of phenylalanine. In the case of E. coli, the analysis of the structure–activity relationships indicated two major factors: the presence of the N-acyl phenylalanine scaffold or its cyclic 4H-1,3-oxazol-5-one analogue and a cLogP value under 4.

2.3.3. Predicted ADME-T Properties

A series of medicinal chemistry measures, ADME, and toxicity endpoints were estimated using the ADMETLab2.0 platform [71]. The data are presented in Table 5. Apart from compound 6p, all the compounds were predicted to have good medicinal chemistry-related scores (Lipinski’s rule, Pfizer rule, GSK rule, golden triangle). None of the compounds were estimated to be a pan-assay interference compound (PAINS). The new compounds were predicted to have a low therapeutic index because of their high plasma protein binding (%). For all the compounds, a significant hepatic toxicity was predicted. Except for compounds 1d–f and 3a, a carcinogen risk was estimated for all other analyzed compounds.
Based on the ADMET predictions, compound 3a had the best toxicological and pharmacokinetic profile of the new compounds. This compound was the ethyl ester of the derivative 1a, indicating that the transformation of compounds 1d–f could improve their toxicological profile and enhance their antibacterial properties.

3. Discussion

Over time, resistance of human pathogens to major antibiotics increases, and infectious agents that are resistant to most available antibiotics are rising globally [72]. An important strategy to help prevent and confront the resistance problem requires the discovery and development of new bioactive agents against both planktonic and adherent microorganisms. Medical devices and instruments are prone to microbial colonization and biofilm formation. Therefore, the discovery of agents that could prevent biofilm formation or adherence would be of great use. Very recently, we reported on the synthesis and antimicrobial and antibiofilm evaluation results of a series of compounds derived from valine [73,74].
In the present research, we examined the potential antimicrobial activity of some 1,3-oxazole derivatives and their isosteres sharing a 4-(4-X-phenylsulfonyl)phenyl moiety, which were synthesized using two other natural α-amino acids as raw materials, namely alanine and phenylalanine.
Preliminary qualitative antimicrobial screening revealed that compounds 1a–f; 2a–f; 3a; 4a–f; 5a,b; 5d,e; 5g,h; 5j,k; 5m,n; 6g–k; 6m; and 6o,p exhibited inhibitory growth effects, although the growth inhibition zones were detected only in contact with the agar layer. These compounds were further evaluated in vitro to determine their effects on planktonic and adherent microbial growth. Compounds 1a–c; 4b–f; 5a,b; 5d,e; 5g,h; 5j,k; 5m,n; 6g,h; 6k; 6m; and 6o,p did not exhibit growth inhibitory effects on any of the tested microbial strains up to a concentration of 225 μg/mL. Regarding the S. epidermidis 756 strain, among the different compounds, 1e,f; 2d; and 4a were the most effective with an MIC of 56.2 µg/mL. The most active compound against P. aeruginosa ATCC 27853 was 3a, the compounds 1d,e; 3a; 4a; and 6i,j were active against C. albicans 128 with a low MIC value of 14 µg/mL, and the compounds 1d,e; 2a–c; and 2e,f proved to be active against E. coli ATCC 25922 with an MIC of 28.1 µg/mL. Regarding B. subtilis ATCC 6683, compound 4a was found to be the best with an MIC of 56.2 µg/mL, while 3a had an MIC of 14 µg/mL against P. aeruginosa ATCC 27853.
The analyzed compounds affected the adherence and biofilm formation on inert surfaces at MBIC values in the range of 14–225 µg/mL. Our data demonstrated that compounds 1e, 2d–f, 4a, and 6j had a distinctly stronger effect on P. aeruginosa ATCC 27853 cells embedded in the biofilm (MBIC of 14 µg/mL) than on planktonic cells. We hypothesized that the tested compounds have a specific nonbactericidal mechanism that changes the bacterial cell surface rather than destroying the bacterial cell.
From the results obtained in the quantitative screening, it was observed that 2-[4-(4-X-phenylsulfonyl)benzamido]propanoic acids 1a–c were inactive at the concentrations used in the assay. However, by intramolecular cyclodehydration, biologically active 4H-1,3-oxazol-5-ones 2a–c were obtained, which displayed growth-inhibitory action with an MIC of 28.1 μg/mL and had an antibiofilm effect on E. coli ATCC 25922 with MBIC values of 56.2 (2b and 2c) and 225 (2a) μg/mL. The in silico prediction of the pharmacokinetic profile (ADME properties) indicated that compounds 2a–c had good pharmacokinetic profiles. Compounds 1a–d, 2a, and 2d were predicted to inhibit peptidoglycan glycosyltransferase.
By opening the 4H-1,3-oxazol-5-ones ring, the resulting N-acyl-α-amino ketones (5a–i) did not show antimicrobial properties up to a concentration of 225 μg/mL. Intramolecular cyclization of N-(1-aryl-1-oxopropan-2-yl)-4-(4-X-phenylsulfonyl)benzamides afforded the corresponding 1,3-oxazoles, which were inactive in the tested concentration range, with the exception of 2-{4-[(4-bromophenyl)sulfonyl]phenyl}-5-(2,4-dimethylphenyl)-4-methyl-1,3-oxazole 6i and 2-{4-[(4-bromophenyl)sulfonyl]phenyl}-5-mesityl-4-methyl-1,3-oxazole 6j, which showed antifungal action on C. albicans 128 (MIC = 14 μg/mL). Moreover, 6j inhibited the formation of biofilm by B. subtilis ATCC 6683 (MBIC = 225 μg/mL) and P. aeruginosa ATCC 27853 (MBIC = 14 μg/mL), and both 1,3-oxazoles presented antibiofilm effect against C. albicans 128 with an MBIC of 112.5 μg/mL. These properties were probably a consequence of the presence of a bromine atom in the para position of the arylsulfonylphenyl substituent linked to the C-2 and the m-xylyl or mesityl group grafted to the C-5 of the 1,3-oxazole ring. Esterification of 2-[4-(phenylsulfonyl)benzamido]propanoic acid 1a or ethanolysis of 4-methyl-2-[4-(phenylsulfonyl)phenyl]-1,3-oxazol-5(4H)-one 2a gave ethyl 2-[4-(phenylsulfonyl)benzamido]propanoate 3a, which was active on P. aeruginosa ATCC 27853 and C. albicans 128 (MIC = 14 μg/mL). It exhibited an antibiofilm effect on P. aeruginosa ATCC 27853 (MBIC = 14 μg/mL), S. epidermidis 756, and C. albicans 128 (MBIC = 112.5 μg/mL). The conversion of N-acylated α-amino acid 1a into its ester derivative 3a led to the appearance of antimicrobial properties. Based on in silico predictions, the obtained product also had a good pharmacokinetic and toxicological profile. Derivatization of the N-acyl-α-amino acid 1a also led to the ethyl {4-methyl-2-[4-(phenylsulfonyl)phenyl]-1,3-oxazol-5-yl} carbonate 4a, which showed antimicrobial activity on S. epidermidis 756 and B. subtilis ATCC 6683 (MIC = 56.2 μg/mL) and on C. albicans 128 (MIC = 14 μg/mL). Compound 4a was also capable of decreasing the biomass of S. epidermidis 756 (MBIC = 56.2 μg/mL), P. aeruginosa ATCC 27853 (MBIC = 14 μg/mL), B. subtilis ATCC 6683, and fungal strain C. albicans 128 (MBIC = 112.5 μg/mL).
All three N-acyl phenylalanines (1d–f) and all three corresponding 4H-1,3-oxazol-5-ones (2d–f) showed antimicrobial and antibiofilm activities. Thus, 3-phenyl-2-[4-(phenylsulfonyl)benzamido]propanoic acid 1d was active on E. coli ATCC 25922 (MIC = 28.1 μg/mL and MBIC = 225 μg/mL) and C. albicans 128 (MIC = 14 μg/mL and MBIC = 112.5 μg/mL). In contrast, 4-benzyl-2-[4-(phenylsulfonyl)phenyl]-1,3-oxazol-5(4H)-one 2d, which resulted from the cyclization of N-acyl-α-amino acid 1d, had an inhibitory effect on Gram-positive bacterium S. epidermidis 756 (MIC = 56.2 μg/mL). Moreover, 2d presented antibiofilm action against S. epidermidis 756 (MBIC = 56.2 μg/mL) and P. aeruginosa ATCC 27853 (MBIC = 14 μg/mL). The 2-{4-[(4-chlorophenyl)sulfonyl]benzamido}-3-phenylpropanoic acid 1e showed a broad antimicrobial spectrum on Gram-positive bacterium S. epidermidis 756 (MIC = 56.2 μg/mL), Gram-negative bacterium E. coli ATCC 25922 (MIC = 28.1 μg/mL), and fungus C. albicans 128 (MIC = 14 μg/mL). Compound 1e presented MBIC values of 56.2 µg/mL for S. epidermidis 756 and E. coli ATCC 25922, 14 µg/mL for P. aeruginosa ATCC 27853, and 112.5 μg/mL for C. albicans 128. These effects were probably a result of the presence of the phenylalanine fragment in the molecule and the chlorine atom in the para position of the arylsulfonylphenyl moiety. Intramolecular transformation of N-acyl-α-amino acid 1e led to 4-benzyl-2-{4-[(4-chlorophenyl)sulfonyl]phenyl}-1,3-oxazol-5(4H)-one 2e, which exhibited antimicrobial activity only on E. coli ATCC 25922 (MIC = 28.1 μg/mL) and antibiofilm effect on S. epidermidis 756 (MBIC = 112.5 μg/mL), B. subtilis ATCC 6683, E. coli ATCC 25922 (MBIC = 56.2 μg/mL), and P. aeruginosa ATCC 27853 (MBIC = 14 μg/mL). In addition, 2-{4-[(4-bromophenyl)sulfonyl]benzamido}-3-phenylpropanoic acid 1f had an inhibitory action on S. epidermidis 756 (MIC = 56.2 μg/mL) and inhibited biofilm formation of S. epidermidis 756 (MBIC = 56.2 μg/mL) and P. aeruginosa ATCC 27853 (MBIC = 28.1 μg/mL). By intramolecular cyclization of this N-acyl-α-amino acid (1f) to the isosteric 4H-1,3-oxazol-5-one analogue 2f, the antibacterial effect on S. epidermidis 756 disappeared, but the obtained compound inhibited the growth of E. coli ATCC 25922 (MIC = 28.1 μg/mL). Saturated azlactone 2f also had antibiofilm activity against S. epidermidis 756 (MBIC = 225 μg/mL), E. coli ATCC 25922 (MBIC = 56.2 μg/mL), and P. aeruginosa ATCC 27853 (MBIC = 14 μg/mL). All N-acyl-α-amino ketones 5j–n obtained by opening the ring of the 4-benzyl-2-[4-(4-X-phenylsulfonyl)phenyl]-1,3-oxazol-5(4H)-ones 2d–f were devoid of antimicrobial action up to a concentration of 225 μg/mL. By cyclization of N-(1-aryl-1-oxo-3-phenylpropan-2-yl)-4-(4-X-phenylsulfonyl)benzamides, the five-membered heterocycles of the 1,3-oxazoles class (6k–p) were synthesized, from which only 4-benzyl-2-{4-[(4-bromophenyl)sulfonyl]phenyl}-5-(2,4-dimethylphenyl)-1,3-oxazole 6p affected adherence and biofilm formation of S. epidermidis 756 on inert surfaces with an MBIC of 112.5 µg/mL. In the case of this compound, we hypothesized that the antibiofilm activity may be correlated to the substitution with bromine in the para position of the C-2-linked arylsulfonylphenyl fragment, and with the presence of the benzyl substituent bonded to the C-4, and the m-xylyl group grafted at position 5 of the 1,3-oxazole nucleus.
In the tested concentration range, {4-(benzyl/methyl)-2-[4-(4-X-phenylsulfonyl)phenyl]-1,3-oxazol-5-yl} ethyl carbonates 4b–f were proved to be inactive on the studied strains. The results were confirmed by PASS analysis with small probabilities to produce anti-infective, antimycobacterial, or antibacterial effects.
Taken together, the antimicrobial activity results indicate that compounds 1d,e,f; 2d,e,f; 3a; 4a; and 6i,j are the most promising candidates for further biological investigations and structural optimization as potential new anti-infective agents, as revealed by the lowest MIC and even MBIC values obtained.
From these compounds, the in silico assays predicted the anti-infective potential for 1e, also exhibiting the broadest antimicrobial spectrum, and 2f, which proved to successfully inhibit P. aeruginosa biofilm development. These compounds have also been predicted to have drug-like properties.

4. Materials and Methods

4.1. General Information

All solvents and reagents were purchased from commercial sources and used without further purification. The absorbance was measured on an Apollo LB 911 ELISA reader (Berthold Technologies GmbH & Co. KG, Waltham, MA, USA).

4.2. Chemistry

The tested compounds 1–6 were previously synthesized [59,60,61,62,63,64,65] according to the multiple-step strategy presented in Scheme 1. The N-acyl-α-amino acids 1a–f were obtained by Schotten–Baumann-type N-acylation of α-amino acids (alanine or phenylalanine) with 4-(4-X-phenylsulfonyl)benzoyl chlorides (X = H, Cl, or Br). The intramolecular cyclodehydration of compounds 1a–f using ethyl chloroformate in the presence of N-methylmorpholine (NMM) led to 4H-1,3-oxazol-5-ones 2a–f when the molar ratio of 1a–f/ClCO2C2H5/NMM was 1:1:1 and the reaction time was 30 min and to ethyl 1,3-oxazol-5-yl carbonates 4a–f when the molar ratio of the reactants was 1:1.5:1.5 and the reaction time was increased to 24 h. The carboxyl group of the N-acyl-α-amino acid 1a was highlighted by its transformation into the corresponding ethyl ester 3a, which was also obtained by O-acylation of the ethanol with 4H-1,3-oxazol-5-one 2a. The Friedel–Crafts acylation, catalyzed by AlCl3 of the aromatic hydrocarbons with the saturated azlactones 2a–f, yielded N-acyl-α-amino ketones 5a–n. These acyclic precursors underwent Robinson–Gabriel cyclization in the presence of phosphoryl trichloride with the formation of 5-aryl-1,3-oxazoles 6a–p. The structures of some of the compounds were confirmed by an additional method. The compounds were purified by recrystallization from water (1a–f), cyclohexane (2a–f and 5b,c), toluene (3a), ethanol (4a–f, 5a, 5d–g, 5i–n, and 6a–p), or ethanol–water (5h). Their purities were verified by RP-HPLC according to previously reported procedures [61,62,63,64,65], with the values ranging between 90.20 and 99.99% (Table 1). As shown in our previous works [59,60,61,62,63,64,65], all tested compounds were characterized using spectral methods (UV–vis, IR, MS, and 1H- and 13C-NMR) and elemental analyses, confirming the purity of the compounds.

4.3. Antimicrobial Activity Assessment

4.3.1. Microbial Strains

The antimicrobial activity of the synthesized compounds was tested against two Gram-positive bacteria (Staphylococcus epidermidis 756 and Bacillus subtilis ATCC 6683), two Gram-negative bacteria (Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853), and one yeast strain (Candida albicans 128).

4.3.2. Qualitative Screening of Antimicrobial Activity

The qualitative screening tests were performed using the agar diffusion method following the CLSI (Clinical and Laboratory Standards Institute) guidelines. The inoculums were prepared from 18–24 h microbial cultures obtained on tryptone soy broth (TSA) for bacteria and on yeast peptone glucose agar (YPGA) for yeast by direct colony suspension in sterile phosphate-buffered saline (PBS). The microbial suspensions turbidity was adjusted to 0.5 McFarland scale and used for the inoculation of the agar plates (Mueller–Hinton agar). Then, 5 μL of the solution of the tested compound at 450 μg/mL concentration, prepared in DMSO, was placed on the agar surface. The negative (DMSO) and positive (standardized antibiotic discs of ciprofloxacin 5 μg and fluconazole 25 μg) controls were prepared. The Petri dishes were incubated at 37 °C, and the diameters of inhibition growth zones were then measured.

4.3.3. Determination of the Minimal Inhibitory Concentration (MIC)

Quantitative analysis of the antimicrobial activity of the tested compounds was carried out using the broth microdilution method following the CLSI guidelines. Two-fold dilutions of the tested compounds were prepared in a liquid growth medium dispensed in a 96-well microplate. The range of final concentrations of the solutions in DMSO of all tested compounds was 1.7–225 µg/mL. Then, each well was inoculated with a microbial inoculum prepared in the same medium after dilution of the standardized microbial suspension adjusted to 0.5 McFarland scale. Binary serial dilutions for DMSO in the liquid growth medium were also prepared. The uninoculated media (MH broth or YPG) and inoculated media served as sterility controls and microbial growth controls. After mixing well, the inoculated 96-well microplates were incubated, without agitation, in aerobic conditions at 37 °C for 24 h. The MIC was measured as the lowest concentration of the tested compound showing no turbidity after 24 h, where turbidity was interpreted as visible bacterial growth. Ciprofloxacin, a broad-spectrum antibacterial agent, and antifungal fluconazole served as controls. The assays were performed in duplicate.

4.3.4. Determination of the Minimal Biofilm Inhibitory Concentration (MBIC)

The crystal violet assay was used to assess the biofilm’s susceptibility to the tested compounds. After determination of the MIC values, the 96-well microplates were emptied, washed gently three times with phosphate-buffered saline (PBS) to remove the planktonic microbial cells, and then fixed with cold methanol for 5 min. The adherent cells in the plastic wells were further stained with 1% violet crystal solution for 30 min. The excess dye was removed by washing with distilled deionized water. In each well, 200 μL of 30% acetic acid was added. After 10 min of incubation to release the dye, the biofilm was assessed by measuring the absorbance at 492 nm using a plate-reading spectrophotometer. The MBIC value was determined as the lowest concentration of the tested compounds showing biofilm inhibition compared to the untreated control. The experiment was performed in duplicate.

4.4. Prediction of the Biological Properties of the Compounds

4.4.1. In Silico Evaluation of the Molecular Mechanisms of Action

The study was executed using the PASS (Prediction of Activity Spectra for Substances) software, a product that predicts the pharmacological potential of new compounds. The structures were introduced as SMILES, and the results were considered only if the Pa values were higher than the corresponding Pi values.

4.4.2. Predicted ADME-T Properties

The ADMETlab 2.0 online platform was used to evaluate the in silico ADMET profile for the 49 compounds. Several physicochemical, medicinal chemistry, and ADME properties were computed, together with toxicity endpoints and toxicophore-based assessment.

5. Conclusions

A total of 49 derivatives that incorporate a 4-(4-X-phenylsulfonyl)phenyl fragment into their structure and are designed based on the 1,3-oxazole scaffold and its isosteric analogues were investigated for their antimicrobial and antibiofilm activity. The compounds belonged to the following chemotypes: N-acyl-α-amino acids, 4H-1,3-oxazol-5-ones, N-acyl-α-amino acid esters, N-acyl-α-amino ketones, and 1,3-oxazoles classes. The assays revealed that the tested compounds 1d,e, 3a, and 4a exhibited the best antimicrobial effects and could be considered as promising candidates for future biological investigations and structural optimization. Among the tested compounds, 1e exhibited the most intense and broad spectrum of antimicrobial activity, including for the Gram-positive, Gram-negative, and fungal strains, which is probably correlated with the presence of the phenylalanine moiety in its structure and the chlorine atom in the para position of the arylsulfonylphenyl fragment. The predictive studies indicate the inhibition of peptidoglycan glycosyltransferase and, to a less extent, the inhibition of the UDP-N-acetylmuramate-L-alanine ligase as possible mechanisms of action.

Author Contributions

Conceptualization, T.-V.A. and C.M.K.; methodology, T.-V.A., M.C.C., G.M.N., O.T.O., S.-F.B., L.-I.S., E.M.P., C.M.K. and L.G.M.; investigation, T.-V.A., M.C.C., and G.M.N., C.M.K. and L.G.M.; writing—original draft preparation, T.-V.A., M.C.C., G.M.N., C.M.K. and L.G.M.; writing—review and editing, T.-V.A., M.C.C., G.M.N., C.M.K. and L.G.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Publish Not Perish Grants from “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Conflicts of Interest

The authors declare no conflict of interest.

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Applsci 12 05571 sch001 550
Scheme 1. The general synthetic methodology as described in our previous papers [59,60,61,62,63,64,65]. Reagents and conditions: (a) (i) alanine or phenylalanine/NaOH, CH2Cl2, 0–5 °C, 30 min; (ii) room temperature (r.t.), 1 h; (iii) HCl; (b) ClCO2C2H5/N-methylmorpholine (NMM), CH2Cl2, r.t., 24 h (molar ratio of 1a–f/ClCO2C2H5/NMM = 1:1.5:1.5); (c) ClCO2C2H5/NMM, CH2Cl2, r.t., 30 min (molar ratio of 1a–f/ClCO2C2H5/NMM = 1:1:1); (d) benzene, toluene, m-xylene or mesitylene/anhyd AlCl3, r.t., 20 h; (e) POCl3, reflux, 4 h; (f) C2H5OH, reflux, 30 min; (g) C2H5OH/H2SO4, reflux, 12 h.
Scheme 1. The general synthetic methodology as described in our previous papers [59,60,61,62,63,64,65]. Reagents and conditions: (a) (i) alanine or phenylalanine/NaOH, CH2Cl2, 0–5 °C, 30 min; (ii) room temperature (r.t.), 1 h; (iii) HCl; (b) ClCO2C2H5/N-methylmorpholine (NMM), CH2Cl2, r.t., 24 h (molar ratio of 1a–f/ClCO2C2H5/NMM = 1:1.5:1.5); (c) ClCO2C2H5/NMM, CH2Cl2, r.t., 30 min (molar ratio of 1a–f/ClCO2C2H5/NMM = 1:1:1); (d) benzene, toluene, m-xylene or mesitylene/anhyd AlCl3, r.t., 20 h; (e) POCl3, reflux, 4 h; (f) C2H5OH, reflux, 30 min; (g) C2H5OH/H2SO4, reflux, 12 h.
Applsci 12 05571 sch001
Applsci 12 05571 g001 550
Figure 1. Pa values as predicted by the PASS application: (a) color codes for classification; (b) Pa values for anti-infective effect; (c) Pa values for antimycobacterial effect; (d) Pa values for antibacterial effect.
Figure 1. Pa values as predicted by the PASS application: (a) color codes for classification; (b) Pa values for anti-infective effect; (c) Pa values for antimycobacterial effect; (d) Pa values for antibacterial effect.
Applsci 12 05571 g001
Applsci 12 05571 g002 550
Figure 2. The cLogP values split by the active or inactive status against S. epidermidis, E. coli, and C. albicans. The horizontal lines represent the average values for each data column.
Figure 2. The cLogP values split by the active or inactive status against S. epidermidis, E. coli, and C. albicans. The horizontal lines represent the average values for each data column.
Applsci 12 05571 g002
Table
Table 1. The chemical structures and purities (%) of the tested compounds.
Table 1. The chemical structures and purities (%) of the tested compounds.
CompoundXRPurity (%)Ref.CompoundXRYPurity (%)Ref.
1aHCH399.99[59]5aHCH3H96.55[59]
1bClCH399.99[60]5bClCH3H98.02[60]
1cBrCH399.99[61]5cBrCH3H97.46[61]
1dHCH2C6H599.99[62]5dHCH34-CH395.10[59]
1eClCH2C6H599.05[63]5eClCH34-CH396.65[60]
1fBrCH2C6H599.63[64]5fBrCH34-CH392.28[61]
2aHCH391.75[59]5gHCH32,4-(CH3)297.16[59]
2bClCH392.92[60]5hClCH32,4-(CH3)291.10[60]
2cBrCH390.78[61]5iBrCH32,4-(CH3)297.55[61]
2dHCH2C6H592.03[62]5jHCH2C6H54-CH391.49[62]
2eClCH2C6H591.49[63]5kClCH2C6H54-CH398.30[63]
2fBrCH2C6H590.20[64]5lBrCH2C6H54-CH394.53[64]
3aHCH394.41[65]5mClCH2C6H52,4-(CH3)290.83[63]
4aHCH399.44[65]5nBrCH2C6H52,4-(CH3)292.40[64]
4bClCH393.29[65]6aHCH3H94.90[59]
4cBrCH397.80[65]6bClCH3H96.07[60]
4dHCH2C6H598.98[65]6cBrCH3H98.79[61]
4eClCH2C6H599.36[65]6dHCH34-CH399.50[59]
4fBrCH2C6H598.81[65]6eClCH34-CH397.96[60]
6fBrCH34-CH397.66[61]
6gHCH32,4-(CH3)298.68[59]
6hClCH32,4-(CH3)297.51[60]
6iBrCH32,4-(CH3)296.80[61]
6jBrCH32,4,6-(CH3)390.58[61]
6kClCH2C6H5H95.13[63]
6lHCH2C6H54-CH393.31[62]
6mClCH2C6H54-CH397.57[63]
6nBrCH2C6H54-CH397.70[64]
6oClCH2C6H52,4-(CH3)299.15[63]
6pBrCH2C6H52,4-(CH3)299.90[64]
Table
Table 2. The MIC and MBIC values (µg/mL) measured for compounds 1d–f; 2a–f; 3a; 4a; 6i,j; and 6p against the tested microbial strains.
Table 2. The MIC and MBIC values (µg/mL) measured for compounds 1d–f; 2a–f; 3a; 4a; 6i,j; and 6p against the tested microbial strains.
Tested
Compounds		S. epidermidis
756		B. subtilis
ATCC 6683		E. coli
ATCC 25922		P. aeruginosa ATCC 27853C. albicans
128
MICMBICMICMBICMICMBICMICMBICMICMBIC
1d>225>225>225>22528.1225>225>22514112.5
1e56.256.2>225>22528.156.2>2251414112.5
1f56.256.2>225>225>225>225>22528.1>225>225
2a>225>225>225>22528.1225>225>225>225>225
2b>225>225>225>22528.156.2>225>225>225>225
2c>225>225>225>22528.156.2>225>225>225>225
2d56.256.2>225>225>225>225>22514>225>225
2e>225112.5>22556.228.156.2>22514>225>225
2f>225225>225>22528.156.2>22514>225>225
3a>225112.5>225>225>225>225141414112.5
4a56.256.256.2112.5>225>225>2251414112.5
6i>225>225>225>225>225>225>225>22514112.5
6j>225>225>225225>225>225>2251414112.5
6p>225112.5>225>225>225>225>225>225>225>225
Ciprofloxacin0.150.15<0.03<0.030.0120.0120.150.15- *-
Fluconazole--------<0.12<0.12
* -, not tested.
Table
Table 3. The probability (Pa) for the compounds to produce biological effects related to antibacterial action as predicted by the PASS application.
Table 3. The probability (Pa) for the compounds to produce biological effects related to antibacterial action as predicted by the PASS application.
TargetPa MaxPa MinCompounds with
Pa > 0.5
Anti-infective0.7020.2181a, 1b, 1c, 1d, 1e, 1f, 3a
Antimycobacterial0.5740.1982c, 2f, 5c, 5f
Antituberculosis0.5260.1995c, 5f
Antibiotic glycopeptide-like0.4030.0830
Peptidoglycan glycosyltransferase inhibitor0.3230.2120
Antibacterial0.3120.1680
UDP-N-acetylmuramate-L-alanine ligase inhibitor (MurC)0.2250.1160
Antibacterial, ophthalmic0.1640.1220
Bacterial efflux pump inhibitor0.1190.1180
Antiseptic0.1180.1170
Antibiotic0.1060.1060
Peptidoglycan beta-N-acetylmuramidase inhibitor0.0930.0670
N-acetylmuramoyl-L-alanine amidase inhibitor0.0830.0540
UDP-N-acetylmuramoylalanine-D-glutamate ligase inhibitor (MurD)0.0790.0590
Bacterial leucyl aminopeptidase inhibitor0.0640.0510
Table
Table 4. Descriptive statistics for the molecular descriptors.
Table 4. Descriptive statistics for the molecular descriptors.
DescriptorMinMax
MW315.35576.51
cLogP1.297.61
HBD05
HBA02
RB39
PSA68.6108.9
DLK−19.74.9
Table
Table 5. ADMET predictions for the new compounds 1d–f; 2a–f; 3a; 4a; 6i,j; and 6p. yes for green, no for red.
Table 5. ADMET predictions for the new compounds 1d–f; 2a–f; 3a; 4a; 6i,j; and 6p. yes for green, no for red.
Property1d1e1f2a2b2c2d2e2f3a4a6i6j6p
Lipinski’s rule••••••••••••••
Pfizer rule••••••••••••••
GSK rule••••••••••••••
Golden triangle••••••••••••••
PAINSnononononononononononononono
Plasma protein binding (%)99.098.798.798.499.498.910010010094.398.2100100100
Volume distribution0.340.370.350.530.480.540.290.270.350.140.430.420.480.31
hERG blocker−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−
AMES toxicity−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−
Carcinogenicity−−−++++++++++++−−+++++
Hepatotoxicity++++++++++++++−−+++++
Drug-induced liver injury++++++++++++++++++++++++++++++++++++++++++
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Apostol, T.-V.; Chifiriuc, M.C.; Nitulescu, G.M.; Olaru, O.T.; Barbuceanu, S.-F.; Socea, L.-I.; Pahontu, E.M.; Karmezan, C.M.; Marutescu, L.G. In Silico and In Vitro Assessment of Antimicrobial and Antibiofilm Activity of Some 1,3-Oxazole-Based Compounds and Their Isosteric Analogues. Appl. Sci. 2022, 12, 5571. https://doi.org/10.3390/app12115571

AMA Style

Apostol T-V, Chifiriuc MC, Nitulescu GM, Olaru OT, Barbuceanu S-F, Socea L-I, Pahontu EM, Karmezan CM, Marutescu LG. In Silico and In Vitro Assessment of Antimicrobial and Antibiofilm Activity of Some 1,3-Oxazole-Based Compounds and Their Isosteric Analogues. Applied Sciences. 2022; 12(11):5571. https://doi.org/10.3390/app12115571

Chicago/Turabian Style

Apostol, Theodora-Venera, Mariana Carmen Chifiriuc, George Mihai Nitulescu, Octavian Tudorel Olaru, Stefania-Felicia Barbuceanu, Laura-Ileana Socea, Elena Mihaela Pahontu, Crina Maria Karmezan, and Luminita Gabriela Marutescu. 2022. "In Silico and In Vitro Assessment of Antimicrobial and Antibiofilm Activity of Some 1,3-Oxazole-Based Compounds and Their Isosteric Analogues" Applied Sciences 12, no. 11: 5571. https://doi.org/10.3390/app12115571

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