Searching for Small Molecules as Antibacterials: Non-Cytotoxic Diarylureas Analogues of Triclocarban

Triclocarban (TCC), a broad-spectrum lipophilic antimicrobial agent, is a diarylurea derivative that has been used for more than 60 years as a major ingredient of toys, clothing, food packaging materials, food industry floors, medical supplies and especially of personal care products, such as soaps, toothpaste and shampoo. In September 2016, the U.S. FDA banned nineteen antimicrobial ingredients, including TCC, in over-the-counter consumer antiseptic wash products, due to their toxicity. Withdrawal of TCC has prompted efforts to search for new antimicrobial compounds. In this paper, we present the synthesis and biological evaluation, as antibiotic and non-cytotoxic agents, of a series of diarylureas, analogues of TCC. These compounds are characterized by an intriguingly simple chemistry and can be easily synthesized. Among the synthesized compounds, 1ab and 1bc emerge as the most interesting compounds as they show the same activity of TCC (MIC = 16 µg/mL) against Staphylococcus aureus, and a higher activity than TCC against Enterococcus faecalis (MIC = 32 µg/mL versus MIC = 64 µg/mL). Moreover, 1ab and 1bc show no cytotoxicity towards the human mammary epithelial cells MCF-10A and embryonic kidney epithelial cells Hek-293, in opposition to TCC, which exhibits a marked cytotoxicity on the same cell lines and shows a good antitumor activity on a panel of cell lines tested.


Introduction
The polychlorinated aromatic antimicrobials triclocarban (TCC) and triclosan (TCS, Figure 1) are organic compounds that have been widely used in a vast range of applications [1,2]. Triclocarban, N-(4-chlorophenyl)-N'-(3,4-dichlorophenyl)urea, is a highly effective and broad spectrum antimicrobial that has been successfully used in personal care products for over 60 years [3]. It belongs to the privileged class of diarylureas, which have been recently extensively reviewed for their multiple actions, including the anticancer [4], and have also been proposed for a repositioning as antimicrobial agents and/or for the treatment of new pandemics, including COVID-19 [5,6]. It is predominantly found in household necessities and personal care products, especially dermal cleaning products such as antibacterial bar/liquid soap, body lotion, deodorants, aftershave soaps, hand sanitizers, toothpaste, handwash and mouthwash [7]. Its concentration in the products can be as high as 1.5% [8]. It has been used for a long time to prevent food spoilage and infections because it perturbs microbial fatty acid synthesis and membrane formation [9]. TCC can be dechlorinated to 4,4 -dichlorocarbanilide (DCC), 1-(3-chlorophenyl)-3-phenylurea (MCC) and carbanilide (NCC), or either biologically or abiotically into 4-chloroaniline (4-CA) ( Figure 1) [10]. Although TCC has been widely used for over 50 years, only recently some concerns about its endocrine disruptive properties were raised [11,12]. As a halogenated hydrocarbon, TCC is hardly biodegradable, and many studies have reported the occurrence of TCC in wastewater effluent, surface water, biosolid, sediment and soil [13]. Risks of TCC in the environment, on plants and animals, humans and microorganisms have been recently extensively described [14]. TCC is ranked in the top 10 Contaminant of Emerging Concern (CEC) occurrence. In September 2016, the U.S. Food and Drug Administration banned its use in over-the-counter hand and body washes [15], but this compound still retained a large market demand [16]. More recent studies are devoted to the removal of TCC and its dechlorinated congeners from soil, given their toxicity [17]. Thus, the desirable properties of next-generation antimicrobials should include broad-spectrum action and high efficacy toward pathogens but low toxicity to humans and should pose no risk of bioaccumulation. Recently, several diarylureas analogues of TCC were reported as antibacterial [18] and antifungal agents [19] bearing pentafluorosulfanyl and trifluoromethylcoumarine groups, respectively. As small molecules, their design should take into account a good oral bioavailability, following the "Lipinski's rule of drug-likeness", also named "the Rule of 5" (Ro5) [20]. In this paper, we report the synthesis of a series of diarylureas, particularly diphenylureas or bis-phenylureas (Figure 2), inspired by TCC, bearing different aryl moieties that we have previously studied, such as 2,6-xylyl, p-methyl, 3-4-difluoro and others [21][22][23], and their antimicrobial activity evaluation against Gram-positive and Gram-negative bacteria. Moreover, the anticancer effect of TCC (1ce) and its active analogues (1ab and 1bc) was evaluated against several human cancer cell lines (breast cancer cells: MCF-7 and MDA-MB-231; uterine cancer cells: HeLa and Ishikawa; melanoma epithelial cells: A2058). Our outcomes clearly showed that TCC possesses a good antitumor activity against the panel of cell lines tested but, it is not selective because of its dramatic cytotoxic effects on normal human epithelial cells, MCF-10A, and embryonic kidney epithelial cells, Hek-293. Alternatively, both 1ab and 1bc do not possess any antitumor or cytotoxic activity, at least using the abovementioned cell lines and under our experimental conditions, but they exhibit an antibacterial activity comparable to or even higher than that of TCC. We are confident that these leads could represent promising tools for the development of new effective antibacterial agents and, whenever possible, also compounds with dual activity acting both as antibacterial and anticancer tools [24].

Chemistry
Diphenylureas were easily prepared as depicted in Scheme 1. Final products were obtained by reacting the commercial anilines with the appropriate phenylisocyanate in acetone as reported in the literature [25]. Scheme 1. Reagents and conditions: (i) acetone, 100 • C, 6 h.

Antibacterial Studies
The in vitro Minimum Inhibitory Concentrations (MICs, µg/mL) were determined by the broth microdilution method according to the Clinical Laboratory Standards Institute (CLSI) guidelines. 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 each well visually with that of the growth in the control well for bacteria. All compounds were dissolved in dimethyl sulfoxide (DMSO) and were tested in a final concentration range of from 512 to 2 µg/mL. MICs for the reference antibiotic norfloxacin against quality control strains were used to confirm the validity of the screen. According to the CLSI guidelines [26], diphenylureas were tested in vitro against a panel of Grampositive and Gram-negative bacteria belonging to the ATCC collection using the standard microdilution test for the determination of MICs. MICs of the synthesised compounds along with the parent compound TCC and with the standard antibiotic norfloxacin are listed in Table 1. The compounds were also analyzed in terms of molecular mass, logP (Table 1), number of hydrogen-bond donors and number of hydrogen-bond acceptors, and most of them obeyed Lipinski's rule [20]. Our results showed that the highest activity against S. aureus was obtained for compounds 1ab, 1bc, 1bd, 1fg, 1bf and 1ef, showing MIC values of 16 µg/mL, on a par with TCC. A slightly lower activity was observed for 1ad, 1be, 1gh and 1gl (MIC = 32 µg/mL). The other compounds were less active or inactive.
Based on our results, it seems that the 3,4-dichloro-disubstitution pattern, which is present in TCC, is not crucial for activity. Out of the six compounds bearing this substitution (TCC versus 1be, 1ef, 1eg, 1ae and 1de), only TCC showed high activity. Conversely, compounds 1ab, 1bc, 1bd and 1bf, bearing a 2,6-xylyl, showed the highest antimicrobial activity against Staphylococcus aureus. Moreover, the activity against Enterococcus faecalis was interesting. E. faecalis is a ubiquitous member of the healthy human gut microbiota and also a common opportunistic pathogen and leading cause of nosocomial infections [27,28]. It has a high incidence for most infections, and the case numbers have been increasing in recent decades, likely due to its greater ability to persist in healthcare facilities and its higher abundance in the human gut microbiome [29]. Kim et al. (2016) [30] compared the bactericidal effects of plain and antibacterial soap containing 0.3% TCC. The authors found no significative difference between the effects of plain and medicated soap at either temperature, with the only exception of E. faecalis ATCC 19433 at 40 • C. 1ab and 1bc showed antibacterial activity higher than TCC (MIC = 32 versus 64 µg/mL) against this Gram-positive bacterium.

Cytotoxicity Studies
In order to investigate the anticancer effects of compounds 1ab, 1bc and 1ce (TCC), we used different human cancer cell models, namely, two breast cancer cells (MCF-7 and MDA-MB-231), two uterine cancer cells (HeLa and Ishikawa) and the melanoma epithelial cells A2058. Performing the MTT assay, we determined that compounds 1ab and 1bc did not elicit any anticancer effect towards all the cancer cell lines used, at least until the concentration of 200 µg/mL (Table 2). On the contrary, TCC, used as the reference molecule, exerted a strong anticancer effect against the cancer cell lines, with IC 50 values between 0.64 and 1.68 µg/mL. Unfortunately, TCC also exhibited a marked cytotoxicity on the normal cells used in this assay, the human mammary epithelial cells MCF-10A and embryonic kidney epithelial cells Hek-293, with IC 50 values of 1.43 ± 0.7 and 1.60 ± 0.8 µg/mL, respectively. Instead, compounds 1ab and 1bc did not possess any cytotoxicity towards the same normal cancer cell lines at least below the concentration of 200 µg/mL. Table 2. IC 50 values (µg/mL) of compounds 1ab, 1bc and TCC on human breast, uterine and melanoma cancer cells as determined by using the MTT assay. IC 50 values are the mean ± SD of three independent experiments performed in triplicate.

Chemistry
Chemicals were purchased from Sigma-Aldrich or Lancaster. Yields refer to purified products and were not optimized. Compound stuctures were confirmed by ordinary spectrometric analyses ( 1 H NMR, 13 C NMR, LC-MS, IR, Elemental Analysis). Melting points were determined on a Gallenkamp melting point apparatus in open glass capillary tubes and are uncorrected. 1 H and 13 C NMR spectra were registered 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 DMSOd 6 as solvent. Chemical shifts are reported in parts per million (ppm) relative to solvent resonance: δ = 2.48 ppm ( 1 H NMR) and δ = 39.9 ppm ( 13 C NMR). J values are given in Hz. Abbreviations "s-singlet, d-doublet, t-triplet, m-multiplet" are used. Gas chromatography (GC)/mass spectroscopy (MS) was performed on a Hewlett-Packard 6890-5973 MSD at low resolution. Liquid chromatography (LC)/mass spectroscopy (MS) was performed on a spectrometer Agilent 1100 series LC-MSD Trap System VL. The molecular ion was designed as "M + ". Elemental analyses were performed on a Eurovector Euro EA 3000 analyzer. The data for C, H, N were within ± 0.4 of theoretical values.

Antibacterial In Vitro Evaluation
MICs (µg/mL) were determined by the broth microdilution method, using 96-well plates, according to CLSI [26]. Stock solutions of the investigated compounds were obtained by setting the concentration at the maximum possible value. Then, the stock solutions were diluted 1:10 with Mueller Hinton Broth (Oxoid, Italy). Afterwards, twofold serial dilutions in the suitable test medium were carried out to obtain a set of concentrations from 512 to 2 µg/mL in the wells. To obtain the stock solution, DMSO (100%) was employed as diluent. The following bacteria strains, available as freeze-dried discs, belonging to the ATCC collection, were used: Gram-positive: S. aureus ATCC 29213, 6538, and 6538P, E. faecalis ATCC 29212; Gram-negative: K. pneuomoniae ATCC 13883, P. aeruginosa ATCC 27853. To conserve the purity of cultures and to enable their reproducibility, cryovials of all microbial strains in the medium were set up and stored at −80 • C. Pre-cultures of each bacterial strain were prepared in Mueller Hinton Broth (MHB) and incubated at 37 • C for 3-5 h. The turbidity of bacterial cell suspension was calibrated to 0.5 McFarland Standard by the spectrophotometric method (OD 625 nm 0.08-0.10), as indicated in the CLSI protocol M7-A9 and, further, the standardized suspension was diluted (1:100) with MHB to reach 1-2 × 10 6 CFU/mL. All wells were seeded with 100 µl of the mentioned final inoculum, and some wells contained only inoculated broth as control growth. The plates were incubated at 37 • C for 24 h, and the 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. The MICs were determined by using the assay repeated twice in triplicate. Throughout the study, norfloxacin was used as reference antibiotic. Appropriate controls involved norfloxacin for bacteria as international reference standard. Additionally, 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. The MBM assay for bacteria was based on the recommended procedures by CLSI [26].

Cell Viability
Cell viability was determined using the 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, Milan, Italy) assay [48,49]. Cells were seeded on 48-well plates and grown in complete medium. Before treatment, 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 72 h; then, fresh MTT, re-suspended in phosphate-buffer saline (PBS), 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 (i.e., drug concentrations able to decrease cell viability by 50% with respect to control) for each cell line, using GraphPad Prism 8 software (GraphPad Inc., San Diego, CA, USA). Data are representative of three independent experiments; standard deviations (SD) are shown.

Conclusions
In the search for new compounds endowed with antibacterial activity but with more favorable toxicological properties than TCC, a series of diarylureas were synthesized. Structure-activity relationship studies showed that the 3,4-disubstitution pattern of TCC seems to be not essential for antibacterial activity. Indeed, the highest antimicrobial activity against S. aureus was found for compounds bearing a 2,6-xylyl moiety. Particularly, compounds 1ab and 1bc were the most interesting of the series showing the same activity as TCC against S. aureus and an even higher activity than TCC against E. faecalis. These compounds also displayed no cytotoxicity against MCF-10A and Hek-293 cell lines. Based on their simple preparation and interesting antibacterial activity profile, the newly prepared small molecules are promising candidates for antibacterial drug development to be introduced in personal care products in substitution of TCC. Funding: This study was supported by MIUR of Italy (ex 60%).

Data Availability Statement:
The data presented in this study are available in manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.