key: cord-0733497-lu31yn2o
authors: Catalano, Alessia; Rosato, Antonio; Salvagno, Lara; Iacopetta, Domenico; Ceramella, Jessica; Fracchiolla, Giuseppe; Sinicropi, Maria Stefania; Franchini, Carlo
title: Benzothiazole-Containing Analogues of Triclocarban with Potent Antibacterial Activity
date: 2021-07-01
journal: Antibiotics (Basel)
DOI: 10.3390/antibiotics10070803
sha: 2fc6010a4412a2a22eb98747c233da01d1efba19
doc_id: 733497
cord_uid: lu31yn2o

Triclocarban (TCC) is a polychlorinated, aromatic, antimicrobial agent commercially used since the 1950s in personal care products for the prevention of spoilage and infections. Humans are frequently exposed to TCC due to its widespread use, leading to its substantial release into the aquatic environment. With the recent ban of TCC from some personal care products, implemented in 2016, many replacement antimicrobial compounds have been studied by researchers. Herein, we report the synthesis and biological activity of a series of diarylureas, analogues of TCC that bear the benzothiazole nucleus as one of the two aryl moieties. Among the studied compounds, 2bF and 2eC showed the highest antimicrobial activity against Staphylococcus aureus, being also more active than TCC, with MIC values of 8 µg/mL versus 16 µg/mL of TCC. Moreover, compound 2bB was much more active than TCC against Enterococcus faecalis, a Gram-positive bacterium that is, unfortunately, strongly responsible for nosocomial infections. Finally, interesting results were found for compound 2bG that, even though less active than the others, exerts an interesting bactericidal action.

Triclocarban (TCC, Figure 1 ), N-(4-chlorophenyl)-N'- (3,4-dichlorophenyl) urea, is a polychlorinated aromatic antimicrobial that has been used in personal care products for over 60 years [1] for the prevention of spoilage and infections as it perturbs microbial fatty acid synthesis and cell membrane formation of microbes [2, 3] . It belongs to the class of diarylureas (or bis-phenylureas), commonly known for their antitumor properties [4] and numerous other biological activities, and it has been recently proposed for a repositioning to antimicrobial [5] and/or for the treatment of COVID-19 [6] . TCC is added, as a preservative, into consumer products including toothpastes, baby teethers, cosmetics, household sponges, plastic cutting boards, socks and undergarments [7, 8] . It is metabolized to hydroxylated species and glucuronides [2, 9] and detected in human tissue such as fingernails and body fluids, including blood, urine and seminal plasma [10] [11] [12] . Nevertheless, given its broad use in personal care products, some concerns about its endocrine disruptive properties were raised [13] . Its halogenated structure is responsible for the lack of biodegradability and its extensive utilization has led to substantial release into the aquatic environment, thus leading to a high environmental risk [14, 15] . Recently, it has been suggested that TCC can also affect embryonic development [16] . In September 2016, the U.S. Food and Drug Administration banned its use in over-the-counter wash products, driven by the concern that the use of these products contributes to antibiotic resistance and might negatively affect human health, either through endocrine disruption or modification of the by the concern that the use of these products contributes to antibiotic resistance and might negatively affect human health, either through endocrine disruption or modification of the human microbiota [17, 18] . Many antimicrobial replacement compounds for TCC have been then used, such as benzalkonium chloride, benzethonium chloride and chloroxylenol. However, it seems that these replacement compounds are not any safer than the banned antimicrobials [19] . Thus, the search for new antimicrobial agents that may substitute TCC and overcome antimicrobial resistance [20, 21] is a very important goal to pursue. Recently, we reported a series of small molecules, which are diarylureas, analogues of TCC, and evaluated their antibacterial activities against Gram-positive and Gram-negative bacteria. The most interesting compounds of the series were 1ab and 1bc (Figure 1) , which showed the same activity of TCC against Staphylococcus aureus (MIC = 16 µg/mL) [22] . They both bear the 2,6-xylyl moiety that is found in different compounds with various activities [23] [24] [25] . The two above-mentioned compounds also showed higher activity than TCC against Enterococcus faecalis (MIC = 32 µg/mL versus MIC = 64 µg/mL of TCC), a bacterium notoriously involved in nosocomial infections, which is the third most frequent cause of infective endocarditis (IE), a disease with high morbidity and mortality [26, 27] . In the search for new diarylurea analogues more potent as antibacterials than TCC, we sought to analyze new small molecules bearing an aryl moiety different from phenyl. Several nuclei for replacing the phenyl group were considered, finally choosing the benzothiazole (BTA) one that is known to have antimicrobial properties [28] [29] [30] . BTA is a fused benzoheterocyle present in many naturally occurring and synthetic products and responsible for the medicinal, pharmacological and pharmaceutical applications, including neuroprotective, antidiabetic, antitumor, antimalarial, antitubercular and antimicrobial [31] . Several diarylureas bearing a BTA group have been reported in the literature for diverse biological activities. For instance, frentizole (I, Figure 2 ) acts as an immunosuppressant and is used for the treatment of Alzheimer's disease (AD), behaving as a weak inhibitor of the Aβ-ABAD interaction (IC50 = 200 µM) [32] . Some diarylureas bearing a BTA moiety were studied for the treatment of Parkinson's disease (PD) as they showed high activity in alleviating haloperidol-induced catalepsy and oxidative stress in mice (II, Figure 2 ) [33] . The thiourea-containing benzothiazole derivative III, depicted in Figure 2 , showed high antiproliferative activity against the human cell line U-937 (human macrophage cell line) when compared to standard drug etoposide [34] . In the search for new diarylurea analogues more potent as antibacterials than TCC, we sought to analyze new small molecules bearing an aryl moiety different from phenyl. Several nuclei for replacing the phenyl group were considered, finally choosing the benzothiazole (BTA) one that is known to have antimicrobial properties [28] [29] [30] . BTA is a fused benzoheterocyle present in many naturally occurring and synthetic products and responsible for the medicinal, pharmacological and pharmaceutical applications, including neuroprotective, antidiabetic, antitumor, antimalarial, antitubercular and antimicrobial [31] . Several diarylureas bearing a BTA group have been reported in the literature for diverse biological activities. For instance, frentizole (I, Figure 2 ) acts as an immunosuppressant and is used for the treatment of Alzheimer's disease (AD), behaving as a weak inhibitor of the Aβ-ABAD interaction (IC 50 = 200 µM) [32] . Some diarylureas bearing a BTA moiety were studied for the treatment of Parkinson's disease (PD) as they showed high activity in alleviating haloperidol-induced catalepsy and oxidative stress in mice (II, Figure 2 ) [33] . The thiourea-containing benzothiazole derivative III, depicted in Figure 2 , showed high antiproliferative activity against the human cell line U-937 (human macrophage cell line) when compared to standard drug etoposide [34] . Based on the results obtained in our previous paper [22] , in this paper, we report the synthesis of several diarylureas (depicted in Figure 3 and detailed in Table 1 ) by a simple synthetic route, and the study of their antibacterial activity against Gram-positive and Gram-negative strains. 

In the search for new antimicrobials more active than TCC, we designed diphenylureas 2xY as benzothiazole analogues of TCC based on our previous results [22] . Several diarylureas, particularly diphenylureas, were tested. Interestingly, compounds 1ab and 1bc ( Figure 1 ) showed the same activity of TCC against S. aureus (MIC = 16 µg/mL) and higher activity than TCC against E. faecalis. Based on the results obtained in our previous paper [22] , in this paper, we report the synthesis of several diarylureas (depicted in Figure 3 and detailed in Table 1 ) by a simple synthetic route, and the study of their antibacterial activity against Gram-positive and Gram-negative strains. Based on the results obtained in our previous paper [22] , in this paper, we report the synthesis of several diarylureas (depicted in Figure 3 and detailed in Table 1 ) by a simple synthetic route, and the study of their antibacterial activity against Gram-positive and Gram-negative strains. 

In the search for new antimicrobials more active than TCC, we designed diphenylureas 2xY as benzothiazole analogues of TCC based on our previous results [22] . Several diarylureas, particularly diphenylureas, were tested. Interestingly, compounds 1ab and 

In the search for new antimicrobials more active than TCC, we designed diphenylureas 2xY as benzothiazole analogues of TCC based on our previous results [22] . Several diarylureas, particularly diphenylureas, were tested. Interestingly, compounds 1ab and 1bc ( Figure 1 ) showed the same activity of TCC against S. aureus (MIC = 16 µg/mL) and higher activity than TCC against E. faecalis.

Diphenylureas 2 were prepared as depicted in Scheme 1. Final products were obtained by reacting the commercial 2-aminobenzothiazoles (3) with the appropriate phenylisocyanate (4) in acetone, as reported in the literature [35] . Only for compounds 2eE and 2bF was the synthesis of the starting material (2-amino-6-phenoxybenzothiazole and 2-amino-5chloro-6-fluorobenzothiazole, respectively) needed, and it was accomplished as previously reported [36, 37] . 

Diphenylureas 2 were prepared as depicted in Scheme 1. Final products were obtained by reacting the commercial 2-aminobenzothiazoles (3) with the appropriate phenylisocyanate (4) in acetone, as reported in the literature [35] . Only for compounds 2eE and 2bF was the synthesis of the starting material (2-amino-6-phenoxybenzothiazole and 2-amino-5-chloro-6-fluorobenzothiazole, respectively) needed, and it was accomplished as previously reported [36, 37] . 

A first screening for the antimicrobial activity of our compounds was carried out by means of the Agar diffusion assay [38] . Afterwards, only compounds that showed activity in this test were studied with the microdilution test, using norfloxacin (NRF) as reference. The in vitro minimum inhibitory concentrations (MICs, µg/mL) were assessed by the broth microdilution method according to Clinical Laboratory Standards Institute (CLSI) guidelines [39] . MIC values were recorded as the lowest concentration of compounds at which there was no optically detectable microorganism growth and evaluated by comparing the growth in every well visually with that of the growth control well for bacteria. Compounds were dissolved in dimethyl sulfoxide (DMSO) and tested in a final concentration range from 512 µg/mL to 2 µg/mL. MICs for the reference antibiotic NRF against quality control strains were used to confirm the validity of the screening. According to CLSI guidelines, diphenylureas were tested in vitro against a panel of Gram-positive and Gram-negative bacteria belonging to the ATCC collection, using the standard microdilution test for the determination of MICs as previously reported [40] . Compounds 2eD, 2dA, 2aC, 2gC, 2fC, 2bA and 2bE demonstrated no activity in the Agar diffusion assay; thus, they were not analyzed via the microdilution assay. MICs of the other compounds, along with the parent compound TCC and the standard antibiotic NRF, are listed in Table 1 . Our results showed that the highest activity against S. aureus was obtained for compounds 2bF and 2eC, showing MIC values of 8 µg/mL, which were lower than the reference TCC (MIC = 16 µg/mL). A slightly lower activity, but on par with TCC, was observed for 2aA, 2eE and 2bB (MIC = 16 µg/mL). The other compounds were less active or inactive. Particularly, compound 2bB interestingly possessed the same activity of TCC against S. aureus and showed the highest activity against E. faecalis (MIC = 8 µg/mL versus 64 µg/mL of TCC), a commensal and nosocomial pathogen that is difficult to treat [41] . Finally, compound 2bG was interesting as it exerted bactericidal action. Based on these results, it can be easily deduced that the best substitutions for the aryl moiety are 3,4-dichloro and 2,6dimethyl. The former is the same as TCC, while the latter confirms our previously reported data [22] , corroborating the interesting activity of 2,6-xylyl also in terms of antibacterial activity. Then, the introduction of the BTA as the other aryl moiety leads to compounds with higher activity, especially when it bears halogens, such as chlorine and fluorine, as substituents (see 2bF, 2eC and 2bB). The absence of substituents on the BTA, or the introduction of a bulky group as the phenoxy, does not vary the antimicrobial activity (see 2aA and 2eE). Finally, in order to evaluate the cytotoxicity of the most active benzothiazole compounds (2eC, 2bG, 2bF and 2bB), we exposed the human non-malignant mammary epithelial MCF-10A cells to increasing concentrations (4, 8, 16, 32 and 100 Scheme 1. Reagents and conditions: (i) acetone, 100 • C, 6 h.

A first screening for the antimicrobial activity of our compounds was carried out by means of the Agar diffusion assay [38] . Afterwards, only compounds that showed activity in this test were studied with the microdilution test, using norfloxacin (NRF) as reference. The in vitro minimum inhibitory concentrations (MICs, µg/mL) were assessed by the broth microdilution method according to Clinical Laboratory Standards Institute (CLSI) guidelines [39] . MIC values were recorded as the lowest concentration of compounds at which there was no optically detectable microorganism growth and evaluated by comparing the growth in every well visually with that of the growth control well for bacteria. Compounds were dissolved in dimethyl sulfoxide (DMSO) and tested in a final concentration range from 512 µg/mL to 2 µg/mL. MICs for the reference antibiotic NRF against quality control strains were used to confirm the validity of the screening. According to CLSI guidelines, diphenylureas were tested in vitro against a panel of Gram-positive and Gram-negative bacteria belonging to the ATCC collection, using the standard microdilution test for the determination of MICs as previously reported [40] . Compounds 2eD, 2dA, 2aC, 2gC, 2fC, 2bA and 2bE demonstrated no activity in the Agar diffusion assay; thus, they were not analyzed via the microdilution assay. MICs of the other compounds, along with the parent compound TCC and the standard antibiotic NRF, are listed in Table 1 . Our results showed that the highest activity against S. aureus was obtained for compounds 2bF and 2eC, showing MIC values of 8 µg/mL, which were lower than the reference TCC (MIC = 16 µg/mL). A slightly lower activity, but on par with TCC, was observed for 2aA, 2eE and 2bB (MIC = 16 µg/mL). The other compounds were less active or inactive. Particularly, compound 2bB interestingly possessed the same activity of TCC against S. aureus and showed the highest activity against E. faecalis (MIC = 8 µg/mL versus 64 µg/mL of TCC), a commensal and nosocomial pathogen that is difficult to treat [41] . Finally, compound 2bG was interesting as it exerted bactericidal action. Based on these results, it can be easily deduced that the best substitutions for the aryl moiety are 3,4-dichloro and 2,6-dimethyl. The former is the same as TCC, while the latter confirms our previously reported data [22] , corroborating the interesting activity of 2,6-xylyl also in terms of antibacterial activity. Then, the introduction of the BTA as the other aryl moiety leads to compounds with higher activity, especially when it bears halogens, such as chlorine and fluorine, as substituents (see 2bF, 2eC and 2bB). The absence of substituents on the BTA, or the introduction of a bulky group as the phenoxy, does not vary the antimicrobial activity (see 2aA and 2eE). Finally, in order to evaluate the cytotoxicity of the most active benzothiazole compounds (2eC, 2bG, 2bF and 2bB), we exposed the human non-malignant mammary epithelial MCF-10A cells to increasing concentrations (4, 8, 16, 32 and 100 µg/mL) of the compounds for 24 h. The obtained results showed that 2eC did not interfere with normal cell growth, at least at the concentrations used in this assay (IC 50 > 100 µg/mL). Instead, 2bG, 2bF and 2bB exerted a slight cytotoxicity on the non-tumoral cells (IC 50 = 24.9 ± 1.2, 24.5 ± 0.9 and 27.8 ± 1.0 µg/mL, respectively), even though their IC 50 values were three-fold higher than their calculated antimicrobial activity (MIC = 8 µg/mL). Thus, at the determined MIC values (shown in Table 1 ), the most active compounds did not have significant cytotoxic effects against the MCF-10A cells. µg/mL) of the compounds for 24 h. The obtained results showed that 2eC did not interfere with normal cell growth, at least at the concentrations used in this assay (IC50 > 100 µg/mL). Instead, 2bG, 2bF and 2bB exerted a slight cytotoxicity on the non-tumoral cells (IC50 = 24.9 ± 1.2, 24.5 ± 0.9 and 27.8 ± 1.0 µg/mL, respectively), even though their IC50 values were three-fold higher than their calculated antimicrobial activity (MIC = 8 µg/mL). Thus, at the determined MIC values (shown in Table 1 ), the most active compounds did not have significant cytotoxic effects against the MCF-10A cells. µg/mL) of the compounds for 24 h. The obtained results showed that 2eC did not interfere with normal cell growth, at least at the concentrations used in this assay (IC50 > 100 µg/mL). Instead, 2bG, 2bF and 2bB exerted a slight cytotoxicity on the non-tumoral cells (IC50 = 24.9 ± 1.2, 24.5 ± 0.9 and 27.8 ± 1.0 µg/mL, respectively), even though their IC50 values were three-fold higher than their calculated antimicrobial activity (MIC = 8 µg/mL). Thus, at the determined MIC values (shown in Table 1 ), the most active compounds did not have significant cytotoxic effects against the MCF-10A cells. µg/mL) of the compounds for 24 h. The obtained results showed that 2eC did not interfere with normal cell growth, at least at the concentrations used in this assay (IC50 > 100 µg/mL). Instead, 2bG, 2bF and 2bB exerted a slight cytotoxicity on the non-tumoral cells (IC50 = 24.9 ± 1.2, 24.5 ± 0.9 and 27.8 ± 1.0 µg/mL, respectively), even though their IC50 values were three-fold higher than their calculated antimicrobial activity (MIC = 8 µg/mL). Thus, at the determined MIC values (shown in Table 1 ), the most active compounds did not have significant cytotoxic effects against the MCF-10A cells. µg/mL) of the compounds for 24 h. The obtained results showed that 2eC did not interfere with normal cell growth, at least at the concentrations used in this assay (IC50 > 100 µg/mL). Instead, 2bG, 2bF and 2bB exerted a slight cytotoxicity on the non-tumoral cells (IC50 = 24.9 ± 1.2, 24.5 ± 0.9 and 27.8 ± 1.0 µg/mL, respectively), even though their IC50 values were three-fold higher than their calculated antimicrobial activity (MIC = 8 µg/mL). Thus, at the determined MIC values (shown in Table 1 ), the most active compounds did not have significant cytotoxic effects against the MCF-10A cells. µg/mL) of the compounds for 24 h. The obtained results showed that 2eC did not interfere with normal cell growth, at least at the concentrations used in this assay (IC50 > 100 µg/mL). Instead, 2bG, 2bF and 2bB exerted a slight cytotoxicity on the non-tumoral cells (IC50 = 24.9 ± 1.2, 24.5 ± 0.9 and 27.8 ± 1.0 µg/mL, respectively), even though their IC50 values were three-fold higher than their calculated antimicrobial activity (MIC = 8 µg/mL). Thus, at the determined MIC values (shown in Table 1 ), the most active compounds did not have significant cytotoxic effects against the MCF-10A cells. µg/mL) of the compounds for 24 h. The obtained results showed that 2eC did not interfere with normal cell growth, at least at the concentrations used in this assay (IC50 > 100 µg/mL). Instead, 2bG, 2bF and 2bB exerted a slight cytotoxicity on the non-tumoral cells (IC50 = 24.9 ± 1.2, 24.5 ± 0.9 and 27.8 ± 1.0 µg/mL, respectively), even though their IC50 values were three-fold higher than their calculated antimicrobial activity (MIC = 8 µg/mL). Thus, at the determined MIC values (shown in Table 1 ), the most active compounds did not have significant cytotoxic effects against the MCF-10A cells. µg/mL) of the compounds for 24 h. The obtained results showed that 2eC did not interfere with normal cell growth, at least at the concentrations used in this assay (IC50 > 100 µg/mL). Instead, 2bG, 2bF and 2bB exerted a slight cytotoxicity on the non-tumoral cells (IC50 = 24.9 ± 1.2, 24.5 ± 0.9 and 27.8 ± 1.0 µg/mL, respectively), even though their IC50 values were three-fold higher than their calculated antimicrobial activity (MIC = 8 µg/mL). Thus, at the determined MIC values (shown in Table 1 ), the most active compounds did not have significant cytotoxic effects against the MCF-10A cells. µg/mL) of the compounds for 24 h. The obtained results showed that 2eC did not interfere with normal cell growth, at least at the concentrations used in this assay (IC50 > 100 µg/mL). Instead, 2bG, 2bF and 2bB exerted a slight cytotoxicity on the non-tumoral cells (IC50 = 24.9 ± 1.2, 24.5 ± 0.9 and 27.8 ± 1.0 µg/mL, respectively), even though their IC50 values were three-fold higher than their calculated antimicrobial activity (MIC = 8 µg/mL). Thus, at the determined MIC values (shown in Table 1 ), the most active compounds did not have significant cytotoxic effects against the MCF-10A cells. µg/mL) of the compounds for 24 h. The obtained results showed that 2eC did not interfere with normal cell growth, at least at the concentrations used in this assay (IC50 > 100 µg/mL). Instead, 2bG, 2bF and 2bB exerted a slight cytotoxicity on the non-tumoral cells (IC50 = 24.9 ± 1.2, 24.5 ± 0.9 and 27.8 ± 1.0 µg/mL, respectively), even though their IC50 values were three-fold higher than their calculated antimicrobial activity (MIC = 8 µg/mL). Thus, at the determined MIC values (shown in Table 1 ), the most active compounds did not have significant cytotoxic effects against the MCF-10A cells. µg/mL) of the compounds for 24 h. The obtained results showed that 2eC did not interfere with normal cell growth, at least at the concentrations used in this assay (IC50 > 100 µg/mL). Instead, 2bG, 2bF and 2bB exerted a slight cytotoxicity on the non-tumoral cells (IC50 = 24.9 ± 1.2, 24.5 ± 0.9 and 27.8 ± 1.0 µg/mL, respectively), even though their IC50 values were three-fold higher than their calculated antimicrobial activity (MIC = 8 µg/mL). Thus, at the determined MIC values (shown in Table 1 ), the most active compounds did not have significant cytotoxic effects against the MCF-10A cells. [39] .

Antibiotics 2021, 10, 803 6 of 12

All chemicals were purchased from Sigma-Aldrich or Lancaster at the highest quality commercially available. Solvents were reagent-grade unless otherwise indicated. Yields refer to purified products and were not optimized. The structures of the compounds were confirmed by routine spectrometric analyses ( 1 H NMR, 13 C NMR, LC-MS, IR). Only spectra for compounds not previously described are given. Melting points were determined on a Gallenkamp melting point apparatus in open glass capillary tubes and were uncorrected. 1 H and 13 C NMR spectra were recorded on a Varian VX Mercury spectrometer operating at 300 and 75 MHz for 1 H and 13 C, respectively, or an Agilent 500 MHz operating at 500 and 125 MHz for 1 H and 13 C, respectively, using DMSO-d 6 50 µL were taken, seeded on Mueller Hinton Agar (MHA) plates and incubated for 24 h at 37 • C. According to CLSI M26A guidelines [44] , the absence of growth for bacterial strains confirmed the bactericidal activity of this compound. Throughout the study, norfloxacin was used as reference antibiotic. Appropriate controls involved norfloxacin for bacteria as international reference standard. In addition, DMSO alone (used as diluent of tested compounds) was tested for its activity against bacteria and consequently quantities of this solvent under 5% concentration were used during the execution of the experiments. Each species was tested at least three times in duplicate [40] . The microtiter broth microdilution (MBM) assay for bacteria was based on the recommended procedures by CLSI [39] .

The MCF-10A human mammary epithelial cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). They were cultured in Dulbecco's modified Eagle's medium/nutrient mixture Ham F-12 (DMEM/F12), supplemented with 5% horse serum (HS, Thermo Fisher Scientific, Milan, Italy), 100 U mL −1 penicillin/streptomycin, 0.5 mg mL −1 hydrocortisone, 20 ng mL −1 human epidermal growth factor (hEGF), 10 mg mL −1 insulin and 0.1 mg mL −1 cholera enterotoxin (Sigma-Aldrich, Milan, Italy). Cells were maintained at 37 • C in a humidified atmosphere of 95% air and 5% CO 2 and periodically screened for contamination [45] .

Cell viability was determined using the 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetra zolium bromide (MTT, Sigma-Aldrich, Milan, Italy) assay [46] . Cells were seeded on 48-well plates and grown in complete medium. Before being treated, cells were starved in serum-free medium for 24 h to allow cell cycle synchronization. Then, cells were treated in phenol-red-free medium supplemented with 1% of serum with increasing concentrations of each compound for 24 h (4, 8, 16, 32 and 100 µg mL −1 ) and after fresh MTT was added to each well (final concentration 0.5 mg/mL). After 2 h incubation at 37 • C, cells were lysed with DMSO, and then optical density was measured at 570 nm using a microplate reader. For each sample, the mean absorbance was expressed as a percentage over the control and plotted versus drug concentrations to determine the IC 50 values by using GraphPad Prism 9 software (GraphPad Inc., San Diego, CA, USA). Data are representative of three independent experiments; standard deviations (SD) have been reported.

The increase in bacterial infections that are resistant to almost all known antibiotics is alarming and becoming a major problem worldwide. TCC is an antibacterial agent widely used years ago and then retired from the market in 2016. In the search for new compounds endowed with antibacterial activity that may substitute TCC, a series of diarylureas was synthesized and the obtained results strengthened our previous observations that the substitution of the 3,4-dichloro aryl moiety of TCC with a 2,6 xylyl one enhances the antibacterial activity. Particularly, we showed that the introduction of a benzothiazole group increased the antibacterial activity in compounds bearing a 2,6 xylyl or a 3,4-dichlorophenyl group. We found that the most active compounds were 2eC, which is the benzothiazole analogue of TCC (i.e., with the 3,4-dichlorophenyl and the 6-chloro-1,3-benzothiazole moieties), and 2bF (MIC = 8 µg/mL versus 16 µg/mL of TCC), which bears the 2,6-xylyl and the 5-chloro-6-fluoro-1,3-benzothiazol-2-yl moieties. Interestingly, the 2,6-xylylderivative 2bB was four-times more active than TCC against E. faecalis. The easy and cheap synthesis procedure adopted to obtain these small molecules and the improved antibacterial activity with respect to TCC, together with the absence of cytotoxicity on the normal cells, were the main goals of these studies. We are confident that the presented outcomes may pave the way for further studies of this interesting class of compounds.

Author Contributions: Conceptualization and writing-original draft preparation, A.C.; methodology, L.S. and J.C.; validation, A.R. and G.F.; writing-review and editing, D.I. and M.S.S.; supervision, C.F. All authors have read and agreed to the published version of the manuscript.

Funding: This study was supported by MIUR of Italy (ex 60%).

Informed Consent Statement: Not applicable.

The data presented in this study are available in manuscript.

(5-Chloro-6-fluoro-1,3-benzothiazol-2-yl)-3-(2,6-dimethylphenyl)urea (2bF). It was prepared as reported for 2eD starting from 2-amino-5-chloro-6-fluorobenzothiazole and 2,6-dimethylphenyl isocyanate

Benzothiazol-2-yl)-3-(4-methoxyphenyl)urea (2dA) [33]. It was prepared as reported for 2eD starting from 2-amino-benzothiazole and p-methoxyphenyl isocyanate

1601 (C=O) cm −1 ; 1 H NMR (500 MHz, DMSO-d 6 ): δ 3.71 (s, 3H, O-CH 3 ) 6.89 (d, J = 8.8 Hz, 2H, Ar), 7.21 (t

It was prepared as reported for 2eD starting from 2-amino-6-chlorobenzothiazole and p-tolylphenyl isocyanate. Yield: 98% (white solid): mp > 250 • C

-phenoxy-1,3-benzothiazol-2-yl)urea (2eE)

1696 (C=O) cm −1 ; 1 H NMR (300 MHz, DMSO-d 6 ): δ 6.98 (d

2H, Ar), 7.32-7.40 (m, 3H, Ar)

1-(6-Chloro-1,3-benzothiazol-2-yl)-3-(3,4-dichlorophenyl)urea (2eC) [42]. It was prepared as reported for 2eD starting from 2-amino-6-chlorobenzothiazole and 3,4-dichlorophenyl isocyanate

-methylphenyl)urea (2gC) [43]. It was prepared as reported for 2eD starting from 2-amino-6-chlorobenzothiazole and o-tolyl isocyanate

03 (s, 1H, Ar), 8.29 (d, J = 1.8 Hz, 1H, Ar), 8.56 (br s, exch. D 2 O, 1H, NH)

3209 (NH), 1566, 1666 (C=O) cm -1 ; 1 H NMR (300 MHz, DMSO-d 6 ): δ 7.05 (t, J = 7.3 Hz, 1H, Ar), 7.25-7.40 (m, 3H, Ar)

14 (s,1H, NH)

3-benzothiazol-2-yl)-3-(4-methoxyphenyl)urea (2dC) [43]. It was prepared as reported for 2eD starting from 2-amino-6-chlorobenzothiazole and 4-methoxyphenyl isocyanate. Yield: 54% (white solid): mp > 250 • C

72 (s, 3H, O-CH 3 ), 6.90 (d, J = 8.8 Hz, 2H, Ar), 7.36-7.41 (m, 3H, Ar), 7.61 (d, J = 8.8 Hz, 1H, Ar), 8.02 (d, J = 2.3 Hz, 1H, Ar), 8.97 (s, 1H, NH), 10.81 (s, 1H, NH)

-phenoxy-1,3-benzothiazol-2-yl)urea (2bE)

C=O) cm -1 ; 1 H NMR (300 MHz, DMSO-d 6 ): δ 2.21 (s, 6H

It was prepared as reported for 2eD starting from 6-chloro-7-fluoroaminobenzothiazole and 2,6-dimethylphenyl isocyanate

C=O) cm -1 ; 1 H NMR (300 MHz, DMSO-d 6 ): δ 2.19 (s, 6H

Afterwards, two-fold serial dilutions in the suitable test medium were carried out to obtain a set of concentrations from 512 µg/mL to 2 µg/mL in the wells. To obtain the stock solution, we employed as diluent DMSO 100%. The following bacterial strains, available as freeze-dried discs

McFarland Standard by spectrophotometric method (OD 625 nm 0.08-0.10), as indicated in CLSI protocol M7-A9 [39] and, further, the standardized suspension was diluted

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The Florence statement on triclosan and triclocarban

Environmental risk assessment of triclosan and triclocarban from personal care products in South Africa

Mass loading and removal of benzotriazoles, benzothiazoles, benzophenones, and bisphenols in Indian sewage treatment plants

tmbim4 protects against triclocarbaninduced embryonic toxicity in zebrafish by regulating autophagy and apoptosis

Safety and effectiveness of consumer antiseptics: Topical antimicrobial drug products for over-the-counter human use

Say goodbye to some antibacterials

Benzalkonium chloride, benzethonium chloride, and chloroxylenol-Three replacement antimicrobials are more toxic than triclosan and triclocarban in two model organisms

The global preclinical antibacterial pipeline

Biopolymeric self-assembled nanoparticles for enhanced antibacterial activity of Ag-based compounds

Searching for small molecules as antibacterials: Non-cytotoxic diarylureas analogues of triclocarban

Constrained analogues of tocainide as potent skeletal muscle sodium channel blockers towards the development of antimyotonic agents

N-aryl-2,6-dimethylbenzamides, a new generation of tocainide analogues as blockers of skeletal muscle voltage-gated sodium channels

Inhibition of skeletal muscle sodium currents by mexiletine analogues: Specific hydrophobic interactions rather than lipophilia per se account for drug therapeutic profile

The structure of Enterococcus faecalis thymidylate synthase provides clues about folate bacterial metabolism

Prevalence of infective endocarditis in Enterococcus faecalis bacteremia

A comprehensive review in current developments of benzothiazole-based molecules in medicinal chemistry

Effect of Methyl-beta-Cyclodextrin on the antimicrobial activity of a new series of poorly water-soluble benzothiazoles

1,3-Benzothiazoles as Antimicrobial Agents

Fernández, I.F. 2-Substituted benzothiazoles as antiproliferative agents: Novel insights on structure-activity relationships

Synthesis and evaluation of frentizole-based indolyl thiourea analogues as MAO/ABAD inhibitors for Alzheimer's disease treatment

Structure-based design, synthesis, and molecular modeling studies of 1-(benzo[d]thiazol-2-yl)-3-(substituted aryl) urea derivatives as novel anti-Parkinsonian agents

Synthesis and cytotoxic evaluation of thiourea and N-bisbenzothiazole derivatives: A novel class of cytotoxic agents

Synthesis and bological evaluation of new N-substituted-N -(3,5-di/1,3,5-trimethylpyrazole-4-yl)thiourea/urea derivatives

2-Aminobenzothiazole derivatives: Search for new antifungal agents

Synthesis and antimicrobial evaluation of a new series of N-1, 3-benzothiazol-2-ylbenzamides

Principles of assessing bacterial susceptibility to antibiotics using the agar diffusion method

Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically

Elucidation of the synergistic action of Mentha piperita essential oil with common antimicrobials

X-ray crystal structures of Enterococcus faecalis thymidylate synthase with folate binding site inhibitors

Benzothiazolyl ureas are low micromolar and uncompetitive inhibitors of 17β-HSD10 with implications to Alzheimer's disease treatment

Microwave Promoted Environmentally Benign Synthesis of 2-Aminobenzothiazoles and Their Urea Derivatives

Methods for Determining Bactericidal Activity of Antimicrobial Agents

Multifaceted properties of 1,4-dimethylcarbazoles: Focus on trimethoxybenzamide and trimethoxyphenylurea derivatives as novel human topoisomerase II inhibitors

Synthesis, inhibition of NO production and antiproliferative activities of some indole derivatives

The authors declare no conflict of interest.