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

Article abstract of DOI:10.3390/antibiotics10020204

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.

Full text of DOI:10.3390/antibiotics10020204

antibiotics
Article
Searching for Small Molecules as Antibacterials: Non-Cytotoxic
Diarylureas Analogues of Triclocarban
Alessia Catalano 1,
*
, Domenico Iacopetta 2 , Antonio Rosato 1 , Lara Salvagno 1 , Jessica Ceramella 2,
Francesca Longo 1, Maria Stefania Sinicropi 2,† and Carlo Franchini 1,†
1
Department of Pharmacy-Drug Sciences, University of Bari “Aldo Moro”, 70126 Bari, Italy;
antonio.rosato@uniba.it (A.R.); lara.salvagno89@gmail.com (L.S.); f.longo21@studenti.uniba.it (F.L.);
carlo.franchini@uniba.it (C.F.)
Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Arcavacata, Italy;
domenico.iacopetta@unical.it (D.I.); jessicaceramella@gmail.com (J.C.); s.sinicropi@unical.it (M.S.S.)
Correspondence: alessia.catalano@uniba.it; Tel.: +39-0805442731
2
*
†
These Authors equally contributed to this work.
Abstract: Triclocarban (TCC), a broad-spectrum lipophilic antimicrobial agent, is a diarylurea deriva-
tive 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
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Catalano, A.; Iacopetta, D.;
Rosato, A.; Salvagno, L.; Ceramella, J.;
Longo, F.; Sinicropi, M.S.; Franchini,
C. Searching for Small Molecules as
Antibacterials: Non-Cytotoxic
Diarylureas Analogues of
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.
Triclocarban. Antibiotics 2021, 10, 204.
https://doi.org/10.3390/
antibiotics10020204
Keywords: antibacterial activity; triclocarban; cytoxicity; diarylureas; small molecules; Staphylococcus
aureus; Enterococcus faecalis
Academic Editor: Anthony Clarke
Received: 3 February 2021
Accepted: 18 February 2021
Published: 19 February 2021
1. 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 appli-
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
cations [
effective and broad spectrum antimicrobial that has been successfully used in personal care
products for over 60 years [ ]. It belongs to the privileged class of diarylureas, which have
been recently extensively reviewed for their multiple actions, including the anticancer [ ],
and have also been proposed for a repositioning as antimicrobial agents and/or for the
treatment of new pandemics, including COVID-19 [ ]. It is predominantly found in
1,2]. Triclocarban, N-(4-chlorophenyl)-N’-(3,4-dichlorophenyl)urea, is a highly
3
4
5,6
Copyright:
© 2021 by the authors.
household necessities and personal care products, especially dermal cleaning products
such as antibacterial bar/liquid soap, body lotion, deodorants, aftershave soaps, hand sani-
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
tizers, toothpaste, handwash and mouthwash [
7]. Its concentration in the products can be
as high as 1.5% [ ]. It has been used for a long time to prevent food spoilage and infections
8
because it perturbs microbial fatty acid synthesis and membrane formation [9]. TCC can be
dechlorinated to 4,40-dichlorocarbanilide (DCC), 1-(3-chlorophenyl)-3-phenylurea (MCC)
and carbanilide (NCC), or either biologically or abiotically into 4-chloroaniline (4-CA)
Antibiotics 2021, 10, 204. https://doi.org/10.3390/antibiotics10020204
https://www.mdpi.com/journal/antibiotics

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(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 bioaccumula-
tion. 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].
Figure 1. Triclocarban (TCC), triclosan and TCC transformation products.
In this paper, we report the synthesis of a series of diarylureas, particularly dipheny-
lureas 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
and their antimicrobial activity evaluation against Gram-positive and Gram-negative bac-
teria. Moreover, the anticancer effect of TCC (1ce) and its active analogues (1ab and 1bc
–23],
)
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

Antibiotics 2021, 10, 204
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effective antibacterial agents and, whenever possible, also compounds with dual activity
acting both as antibacterial and anticancer tools [24].
Figure 2. Diarylureas (a–l) under study.
2. Results and Discussion
2.1. 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.
2.2. 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 compar-
ing 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 Gram-
positive 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.

Antibiotics 2021, 10, 204
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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.
Table 1. Physicochemical properties and antibacterial activity results of diarylureas.
Microorganisms (MIC, µg/mL)
Gram-Positive
Gram-Negative
E.c.
25922
P. a.
27853
K.p.
13883
milogP b
COMPD
clogP a
S.a. 29213 S.a. 6538 S.a. 6538P
E.f. 29212
1ab
1bc
1ad
1bd
1be
1af
1ag
4.24 ± 0.30
4.77 ± 0.33
3.27 ± 0.32
3.73 ± 0.32
5.68 ± 0.36
3.32 ± 0.29
3.78 ± 0.30
5.75 ± 0.38
2.86 ± 0.29
3.32 ± 0.29
3.78 ± 0.30
2.81 ± 0.31
3.83 ± 0.32
4.75 ± 0.35
3.85 ± 0.32
4.84 ± 0.35
5.22 ± 0.35
4.16 ± 0.52
4.91 ± 0.45
3.28 ± 0.40
3.70 ± 0.51
5.22 ± 0.35
4.70 ± 0.37
-
3.46
3.69
3.65
3.06
4.29
3.59
3.99
5.11
3.14
3.54
3.01
3.20
4.17
4.43
3.82
4.50
4.83
3.80
4.31
3.66
3.40
4.88
4.48
-
>256
256
128
>256
128
128
256
16
>256
256
>256
256
256
256
128
256
256
>256
256
128
256
128
128
0.5–2
16
16
32
32
32
16
16
32
32
32
128
128
64
64
128
64
>256
64
64
128
256
128
64
128
64
>256
>256
>256
>256
>256
>256
>256
256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
-
>256
>256
>256
>256
>256
>256
>256
256
>256
>256
>256
256
>256
256
>256
>256
>256
>256
256
>256
>256
>256
>256
>256
>256
>256
256
16
128
128
256
16
64
16
16
128
32
128
128
256
128
128
64
64
128
128
128
32
16
64
32
16
64
128
64
64
128
64
128
64
64
1ce (TCC)
1ff (NCC)
1fg
>256
256
1bf
1df
1gh
1ef
>256
>256
>256
>256
>256
>256
>256
>256
256
>256
256
>256
>256
0.5–1
1cf (MCC)
1cc (DCC)
1eg
1gj
1gk
1gl
1fj
1ae
1de
NRF
64
64
128
128
128
64
32
64
64
64
>256
-
>256
>256
>256
256
>256
>256
-
-
2–8
0.03–0.12
1–4
Antibacterial activity was assessed using the CLSI assay (2012) [26]. Abbreviations: S.a.: S. aureus; E.f.: E. faecalis; E.c.: E. coli; P.a.: P. aeruginosa;
K.p.: K. pneumoniae. NRF: norfloxacin; nt: not tested; a clogP values were computed with ACD Labs/Log P and ACD/ChemSketch 2012
from Advanced Chemistry Development (Toronto, Canada); b milogP values were obtained from Molinspiration property calculation
service FAQ, https://www.molinspiration.com/cgi-bin/properties (accessed on 2 February 2021); values according to CLSI (2012) [26].
2.3. 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,

Antibiotics 2021, 10, 204
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exerted a strong anticancer effect against the cancer cell lines, with IC50 values between 0.64
and 1.68
cells used in this assay, the human mammary epithelial cells MCF-10A and embryonic
kidney epithelial cells Hek-293, with IC50 values of 1.43 ,
µg/mL. Unfortunately, TCC also exhibited a marked cytotoxicity on the normal
±
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. IC50 values (µg/mL) of compounds 1ab, 1bc and TCC on human breast, uterine and
melanoma cancer cells as determined by using the MTT assay. IC50 values are the mean
three independent experiments performed in triplicate.
±
SD of
Cell Lines
IC50 (µg/mL)
1ab
1bc
1ce (TCC)
MDA-MB-231
MCF-7
HeLa
Ishikawa
A2058
MCF-10A
Hek-293
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
0.89 ± 0.8
0.64 ± 0.5
1.01 ± 1.1
1.68 ± 0.9
0.89 ± 0.7
1.43 ± 0.7
1.60 ± 0.8
3. Materials and Methods
3.1. 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 (1H NMR, 13C NMR, LC-MS, IR, Elemental Analysis). Melting
points were determined on a Gallenkamp melting point apparatus in open glass capillary
tubes and are uncorrected. 1H and 13C NMR spectra were registered on a Varian VX
Mercury spectrometer operating at 300 and 75 MHz for 1H and 13C, respectively, or an
Agilent 500 MHz operating at 500 and 125 MHz for 1H and 13C, respectively, using DMSO-
d6 as solvent. Chemical shifts are reported in parts per million (ppm) relative to solvent
resonance:
δ δ
= 2.48 ppm (1H NMR) and = 39.9 ppm (13C 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.
1-(2,6-Dimethylphenyl)-3-p-tolylurea (1ab) [31]. A solution of 2,6-dimethylphenyl iso-
cyanate (0.26 mL, 1.87 mmol) in acetone RP (4 mL) was added in three portions (in 1.5 h) to
a solution of p-toluidine (0.20 g, 1.87 mmol) in acetone RP (4 mL). The resulting mixture was
refluxed for 6 h. The solid was filtered to give 1ab as a white solid (0.41 g, 86%), which was
recrystallized from CHCl3 giving 0.35 g (73%) of white crystals: mp 242–243 ◦C; GC/MS
(70 eV) m/z (%): 254 (M+, 24), 107 (100); IR (KBr): 3288 (NH), 1642 (C=O) cm−1; 1H NMR
(300 MHz, DMSO-d6):
J = 8.2 Hz, 2H, Ar), 7.63 (br s, 1H, NH, exch. D2O), 8.57 (br s, 1H, NH, exch. D2O); 13C NMR
(75 MHz, DMSO-d6): 18.7 (2C), 20.8 (1C), 118.5 (2C), 126.3 (1C), 128.1 (2C), 129.5 (2C),
δ 2.18 (s, 6H, 2CH3), 2.21 (s, 3H, CH3), 6.98–7.10 (m, 5H, Ar), 7.31 (d,
δ
130.6 (1C), 135.9 (2C), 136.0 (1C), 138.2 (1C), 153.6 (1C). Anal. calcd for C16H18N2O
H2O (257.75)%: C 74.51; H 7.19; N 10.86. Found %: C 74.76; H 7.00; N 10.85.
·
0.20
1-(2,6-Dimethylphenyl)-3-(4-chlorophenyl)urea (1bc) [32]. Prepared from 4-CA and 2,6-
◦
dimethylphenyl isocyanate. Yield: 72% (needle-like white crystals): mp 244–245 C
(CHCl3); GC/MS (70 eV) m/z (%): 274 (M+, 13), 127 (100); IR (KBr): 3282 (NH), 1643
(C=O) cm−1; 1H NMR (300 MHz, DMSO-d6):
δ
2.18 (s, 6H, 2CH3), 7.02–7.08 (m, 3H, Ar),
7.27 (d, J = 8.8 Hz, 2H, Ar), 7.47 (d, J = 8.8 Hz, 2H, Ar) 7.73 (br s, 1H, NH, exch. D2O),
8.86 (br s, 1H, NH, exch. D2O); 13C NMR (125 MHz, DMSO-d6):
18.7 (2C), 119.8 (2C),
δ

Antibiotics 2021, 10, 204
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125.3 (1C), 126.5 (1C), 128.2 (2C), 129.0 (2C), 135.6 (1C), 136.0 (2C), 139.8 (1C), 153.5 (1C).
Anal. calcd for C15H15ClN2O (274.59)%: C 65.57; H 5.50; N 10.20. Found%: C 65.59; H 5.65;
N 10.59.
1-(4-Methoxyphenyl)-3-(p-tolyl)urea (1ad) [33]. Prepared from p-toluidine and p-methox-
yphenyl isocyanate. Yield: 65% (needle-like white crystals): mp 241–242 ◦C (EtOH 96%);
LC/MS (70 eV) m/z (%): 276 [M+, +23]; IR (KBr): 3283 (NH), 1644 (C=O) cm−1; 1H NMR
(300 MHz, DMSO-d6):
7.04 (d, J = 8.2 Hz, 2H, Ar), 7.26–7.36 (m, 4H, Ar), 8.38 (br s, 1H, NH, exch. D2O), 8.43 (br
s, 1H, NH, exch. D2O); 13C NMR (75 MHz, DMSO-d6):
20.8 (1C), 55.6 (1C), 114.4 (2C),
118.6 (2C), 120.4 (2C), 129.6 (2C), 130.8 (1C), 133.3 (1C), 137.7 (1C), 153.2 (1C), 154.9 (1C).
δ 2.21 (s, 3H, CH3), 3.68 (s, 3H, CH3), 6.84 (d, J = 8.8 Hz, 2H, Ar),
δ
Anal. calcd for C15H16N2O2
H 6.16; N 10.55.
·
0.33H2O (262.12)%: C 68.68; H 6.40; N 10.68. Found%: C 68.37;
1-(4-Methoxyphenyl)-3-(2,6-dimethylphenyl)urea (1bd) [32]. Prepared from p-methoxyphenyl
◦
isocyanate and 2,6-dimethylaniline. Yield: 66% (needle-like white crystals): mp 225–226 C
(CHCl3); LC/MS (70 eV) m/z (%): 293 [M+, +23]; IR (KBr): 3262 (NH), 1643 (C=O) cm−1
;
1H NMR (300 MHz, DMSO-d6):
2H, Ar), 7.03 (s, 2H, Ar), 7.33 (d, J = 8.8 Hz, 2H, Ar), 7.59 (s, 1H, Ar) 8.37 (br s, 1H, NH,
exch. D2O), 8.50 (br s, 1H, NH, exch. D2O); 13C NMR (75 MHz, DMSO-d6):
18.7 (2C),
δ 2.18 (s, 6H, 2CH3), 3.68 (s, 3H, CH3), 6.82 (d, J = 8.8 Hz,
δ
55.6 (1C), 114.4 (2C), 120.1 (2C), 120.3 (2C), 126.2 (1C), 128.1 (2C), 133.9 (1C), 135.9 (1C),
153.8 (1C), 154.6 (1C). Anal. calcd for C16H18N2O2 (270.33)%: C 71.09; H 6.71; N 10.36.
Found %: C 70.70; H 6.58; N 10.35.
1-(2,6-Dimethylphenyl)-3-(3,4-dichlorophenyl)urea (1be). Prepared from 2,6-dimethylaniline
and 3,4-dichlorophenyl isocyanate. Yield: 76% (white crystals): mp 249–250 ◦C (CHCl3/
MeOH/Acetone); LC/MS (70 eV) m/z (%): 331 [M+, +23]; IR (KBr): 3301 (NH), 1639
(C=O) cm−1; 1H NMR (300 MHz, DMSO-d6):
δ
2.17 (s, 6H, 2CH3), 7.05 (s, 3H, Ar), 7.31
(d, J = 8.8 Hz, 1H, Ar), 7.46 (d, J = 8.8 Hz, 1H, Ar), 7.84 (br s, 2H, Ar + NH, exch. D2O),
9.06 (br s, 1H, NH, exch. D2O); 13C NMR (75 MHz, DMSO-d6):
18.6 (2C), 118.5 (1C),
119.5 (1C), 123.0 (1C), 126.7 (1C), 128.2 (2C), 130.9 (1C), 131.4 (1C), 135.3 (1C), 136.1 (2C),
δ
141.0 (1C), 153.4 (1C). Anal. calcd for C15H14Cl2N2O
N 8.80. Found%: C 56.78; H 4.43; N 8.93.
·
0.50H2O (317.05)%: C 56.62; H 4.75;
1-(4-Methylphenyl)-3-phenylurea (1af) [34]. Prepared from p-toluidine and phenyl iso-
◦
cyanate. Yield: 80% (needle-like white crystals): mp 240–242 C (CHCl3/acetone); LC/MS
(70 eV) m/z (%): 249 [M+, +23]; IR (KBr): 3301 (NH), 1634 (C=O) cm−1; 1H NMR (500 MHz,
DMSO-d6):
(t, J = 7.5 Hz, 2H, Ar), 7.31 (d, J = 8.3 Hz, 2H, Ar), 7.42 (d, J = 8.3 Hz, 2H, Ar), 8.51 (s, 1H,
NH), 8.57 (s, 1H, NH); 13C NMR (125 MHz, DMSO-d6):
20.8 (1C), 118.6 (2C), 118.7 (2C),
δ 2.22 (s, 3H, CH3) 6.93 (t, J = 7.3 Hz, 1H, Ar), 7.06 (d, J = 8.3 Hz, 2H, Ar), 7.25
δ
122.1 (1C), 129.2 (2C), 129.6 (2C), 131.0 (1C), 137.6 (1C), 140.2 (1C), 153.0 (1C).
1-(2-Methylphenyl)-3-(4-methylphenyl)urea (1ag) [35]. Prepared from p-toluidine and
o-methylphenyl isocyanate. Yield: 74% (white solid): mp 241–242 ◦C; LC/MS (70 eV) m/z
(%): 263 [M+, +23]; IR (KBr): 3305 (NH), 1643 (C=O) cm−1; 1H NMR (300 MHz, DMSO-d6):
δ
2.21 (s, 6H, 2CH3), 6.91 (t, J = 7.6 Hz, 1H, Ar), 7.05–7.15 (m, 4H, Ar), 7.32 (d, J = 8.2 Hz,
2H, Ar), 7.82 (d overlapping s at 7.83 ppm, J = 8.8 Hz, 1H, Ar), 7.83 (s overlapping d at 7.82
ppm, 1H, NH, exch. D2O), 8.88 (s, 1H, NH, exch. D2O); 13C NMR (75 MHz, DMSO-d6):
δ
18.3 (1C), 20.8 (1C), 118.5 (2C), 121.4 (1C), 122.9 (1C), 126.6 (1C), 127.8 (1C), 129.6 (2C),
130.6 (1C), 130.9 (1C), 137.7 (1C), 137.9 (1C), 153.1 (1C). Anal. calcd for C15H16N2O
(242.87) %: C 74.18; H 6.76; N 11.53. Found%: C 74.52; H 6.56; N 11.61.
·
0.14H2O
1-(3,4-Dichlorophenyl)-3-(4-chlorophenyl)urea (1ce, TCC) [36]. Prepared from 3,4-dichloro-
aniline and p-chlorophenyl isocyanate. Yield: 29% (white solid): mp
≥
250 ◦C; LC/MS
1
(70 eV) m/z (%): 317 [M+]; IR (KBr): 3296 (NH), 1639 (C=O) cm−1; H NMR (500 MHz,
DMSO-d6):
8.92 (s, 1H, NH, exch. D2O), 8.99 (s, 1H, NH, exch. D2O); 13C NMR (125 MHz, DMSO-d6):
118.9 (1C), 119.8 (1C), 120.5 (1C), 123.7 (2C), 126.2 (1C), 129.1 (2C), 131.0 (1C), 131.5 (1C),
δ 7.31 (d, J = 8.8 Hz, 3H, Ar), 7.45–7.50 (m, 3H, Ar), 7.84 (d, J = 2.4 Hz, 1H, Ar),
δ
138.7 (1C), 140.3 (1C), 152.7 (1C). Anal. calcd for C13H9Cl3N2O (315.58) %: C 49.48; H 2.87;
N 8.88. Found%: C 49.48; H 3.02; N 8.79.

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1,3-Diphenylurea (1ff, NCC) [3◦7]. Prepared from aniline and phenyl isocyanate. Yield:
77% (white solid): mp 242–243 C; LC/MS (70 eV) m/z (%): 235 [M+, +23]; IR (KBr):
3328 (NH), 1647 (C=O) cm−1; 1H NMR (300 MHz, DMSO-d6):
δ
6.95 (t, J = 7.3 Hz, 2H, Ar),
7.26 (t, J = 7.9 Hz, 4H, Ar) 7.43 (d, J = 7.6 Hz, 4H, Ar), 8.65 (s, 2H, NH, exch. D2O); 13C NMR
(75 MHz, DMSO-d6): 118.6 (4C), 122.3 (2C), 129.2 (4C), 140.1 (2C), 152.9 (1C). Anal. calcd
δ
for C13H12N2O (212,24)%: C 73.56; H 5.70; N 13.20. Found%: C 73.63; H 5.61; N 13.19.
1-(2-Methylphenyl)-3-phenylurea (1fg) [33]. Prepared from aniline and o-tolyl isocyanate.
Yield: 77% (white solid): mp 241–242 ◦C; LC/MS (70 eV) m/z (%): 249 [M+, +23]; IR (KBr):
3269 (NH), 1631 (C=O) cm−1; 1H NMR (300 MHz, DMSO-d6):
δ 2.21 (s, 3H, CH3), 6.89–6.96
(m, 2H, Ar), 7.09–7.16 (m, 2H, Ar), 7.26 (t, J = 7.6 Hz, 2H, Ar), 7.44 (d, J = 7.6 Hz, 2H, Ar),
7.81 (d, J = 8.2 Hz, 1H, Ar), 7.90 (s, 1H, NH, exch. D2O), 9.01 (s, 1H, NH, exch. D2O);
13C NMR (75 MHz, DMSO-d6):
δ
18.3 (1C), 118.4 (2C), 121.5 (1C), 122.1 (1C), 123.1 (1C),
126.6 (1C), 128.0 (1C), 129.3 (2C), 130.6 (1C), 137.8 (1C), 140.3 (1C), 153.1 (1C). Anal. calcd
for C14H14N2O
N 12.27.
·
0.16H2O (229.27)%: C 73.34; H 6.30; N 12.22. Found%: C 73.65; H 6.08;
1-(2,6-Dimethylphenyl)-3-phenylurea (1bf) [32]. Prepared from 2,6-dimethylaniline and
phenyl isocyanate. Yield: quantitative (white solid): mp 240–241 ◦C; LC/MS (70 eV) m/z
(%): 263 [M+, +23]; IR (KBr): 3287 (NH), 1633 (C=O) cm−1; 1H NMR (300 MHz, DMSO-
d6):
2H, Ar), 7.43 (d, J = 7.6 Hz, 2H, Ar), 7.69 (s, 1H, NH, exch. D2O), 8.72 (s, 1H, NH, exch.
D2O); 13C NMR (75 MHz, DMSO-d6):
18.7 (2C), 118.3 (2C), 121.8 (2C), 126.4 (1C), 128.1
δ 2.18 (s, 6H, 2CH3), 6.91 (d, J = 7.0 Hz, 1H, Ar), 7.04 (s, 3H, Ar), 7.23 (t, J = 7.9 Hz,
δ
(2C), 129.1 (2C), 135.8 (1C), 136.0 (1C), 140.8 (1C), 153.6 (1C). Anal. calcd for C15H16N2O
(240.12)%: C 74.97; H 6.71; N 11.66. Found%: C 75.41; H 6.60; N 11.80.
1-(4-Methoxyphenyl)-3-phenylurea (1df) [38]. Prepared from phenyl isocyanate and
◦
p-anisidine. Yield: 64% (white needle-shaped fine crystals): mp 216–218 C; LC/MS (70 eV)
1
m/z (%): 265 [M+, +23]; IR (KBr): 3288 (NH), 1633 (C=O) cm−1; H NMR (300 MHz,
DMSO-d6):
7.24 (t, J = 7.6 Hz, 2H, Ar), 7.34 (d, J = 8.8 Hz, 2H, Ar), 7.42 (d, J = 8.2 Hz, 2H, Ar), 8.44 (s,
1H, NH, exch. D2O), 8.55 (s, 1H, NH, exch. D2O); 13C NMR (125 MHz, DMSO-d6):
55.6
(1C), 114.4 (2C), 118.5 (2C), 120.4 (2C), 122 (1C), 129.2 (2C), 133.2 (1C), 140.3 (1C), 153.2
δ 3.69 (s, 3H, O-CH3), 6.85 (d, J = 8.8 Hz, 2H, Ar), 6.93 (t, J = 7.0 Hz, 1H, Ar),
δ
(1C), 154.9 (1C). Anal. calcd for C14H14N2O2
Found %: C 67.43; H 5.76; N 11.14.
·
0.30 H2O (248.27)%: C 67.73; H 5.95; N 11.28.
1-(2-Chlorophenyl)-3-(2-methylphenyl)urea (1gh). Prepared from o-chloroaniline and o-
tolyl isocyanate. Yield: 87% (off-white crystals): mp 223–224 ◦C (acetone/CHCl3); LC/MS
(70 eV) m/z (%): 283 [M+, +23]; IR (KBr): 3284 (NH), 1643 (C=O) cm−1; 1H NMR (300 MHz,
DMSO-d6):
δ 3.33 (s, 3H, CH3), 6.96–7.03 (m, 2H, Ar), 7.11–7.18 (m, 2H, Ar), 7.24–7.30
(m, 1H, Ar), 7.44 (q, J = 3.2 Hz, 1H, Ar), 7.75 (d, J = 8.2 Hz, 1H, Ar), 8.11 (d, J = 1.7 Hz,
1H, Ar), 8.63 (d, J =12.4 Hz, 2H, NH exch. D2O); 13C NMR (125 MHz, DMSO-d6): 18.3
(1C), 121.1 (1C), 122.2 (1C), 123.4 (1C), 123.5 (1C), 126.3 (2C), 127.7 (1C), 128.5 (1C), 129.4
(1C), 130.5 (1C), 136.4 (1C), 137.2 (1C), 152.7 (1C). Anal. calcd for C14H13N2OCl (260.07)%:
C 64.49; H 5.03; N 10.74. Found%: C 64.20; H 4.97; N 10.68.
1-(3,4-Dichlorophenyl)-3-phenylurea (1ef) [39]. Prepared from 3,4-dichloroaniline and
◦
phenyl isocyanate. Yield: 76% (slightly greenish crystals): mp 215–216 C (acetone/CHCl3);
LC/MS (70 eV) m/z (%):280 [M+, –1]; IR (KBr): 3319 (NH), 1594 (C=O) cm−1; 1H NMR
(300 MHz, DMSO-d6):
3H, Ar), 7.86 (d, J = 2.3 Hz, 1H, Ar), 8.77 (s, 1H, NH, exch. D2O), 8.96 (s, 1H, NH, exch.
D2O); 13C NMR (125 MHz, DMSO-d6):
118.8 (1C), 118.9 (2C), 119.7 (1C), 122.7 (1C),
123.5 (1C), 129.2 (2C), 131.0 (1C), 131.5 (1C), 139.7 (1C), 140.4 (1C), 152.7 (1C). Anal. calcd
δ 6.97 (t, J = 7.3 Hz, 1H, Ar), 7.20–7.35 (m, 3H, Ar), 7.40–7.52 (m,
δ
for C13H10N2OCl2
N 9.86.
·
0.25 H2O (285.43)%:C 54.66; H 3.71; N 9.81. Found%: C 54.99; H 3.55;
1-(4-Chlorophenyl)-3-phenylurea (1cf, MCC) [40]. Prepared from 4-CA and phenyl
isocyanate. Yield: 90% (white crystals): mp 228–229 ◦C (acetone/CHCl3); LC/MS (70 eV)
1
m/z (%): 269 [M+, +23]; IR (KBr): 3302 (NH), 1636 (C=O) cm−1; H NMR (300 MHz,
DMSO-d6):
δ 6.95 (t, J = 7.3 Hz, 1H, Ar), 7.23–7.32 (m, 4H, Ar), 7.41–7.49 (m, 4H, Ar), 8.67 (s,

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1H, NH, exch. D2O), 8.86 (s, 1H, NH, exch. D2O); 13C NMR (125 MHz, DMSO-d6):
δ
118.8
(2C), 120.2 (2C), 122.4 (1C), 125.8 (2C), 129.0 (1C), 129.2 (2C), 139.2 (1C), 139.9 (1C), 152.9
(1C). Anal. calcd for C13H11N2OCl (246.58)%:C 63.29; H 4.49; N 11.36. Found%: C 63.67;
H 4.50; N 11.40.
1-(4-Chlorophenyl)-3-(4-chlorophenyl)urea (1cc, DCC) [41]. Prepared from 4-CA and
◦
4-chlorophenyl isocyanate. Yield: 86% (pinkish solid): mp 223–224 C; LC/MS (70 eV) m/z
(%): 281 [M+]; IR (KBr): 3296 (NH), 1651 (C=O) cm−1; 1H NMR spectrum was in agreement
with the literature [42]. Anal. calcd for C13H10ClN2O
N 9.81. Found%: C 54.85; H 3.51; N 9.80.
·
0.25 H2O (285.64)%: C 54.66; H 3.71;
1-(3,4-Dichlorophenyl)-3-(2-methylphenyl)urea (1eg). Prepared from 3,4-dichloroanili◦ne
and o-tolyl isocyanate. Yield: 81% (white needle-shaped fine crystals): mp 217–218 C
(acetone/CHCl3/MeOH); LC/MS (70 eV) m/z (%): 295 [M+]; IR (KBr): 3294 (NH), 1643
(C=O) cm−1; 1H NMR (500 MHz, DMSO-d6):
δ 3.32 (s, 3H, CH3), 6.96 (t, J = 7.8 Hz, 1H, Ar),
7.12–7.17 (m, 2H, Ar), 7.27–7.29 (m, 1H, Ar), 7.49 (d, J = 8.8 Hz, 1H, Ar), 7,74 (d, J = 7.8 Hz,
1H, Ar), 7.89 (d, J= 2.4Hz, 1H, Ar), 8.00 (s, 1H, NH exch. D2O), 9.27 (s, 1H, NH exch. D2O);
13C NMR (125 MHz, DMSO-d6):
δ 18.2 (2C), 118.6 (1C), 119.5 (1C), 122.1 (1C), 123.4 (1C),
126.6 (1C), 128.7 (1C), 130.7 (1C), 131.0 (1C), 131.5 (1C), 137.3 (1C), 140.5 (1C), 152.9 (1C).
Anal. calcd for C14H12Cl2N2O (295.14)%: C 56.97; H 4.10; N 9.49. Found%: C 57.03; H 4.19;
N 9.47.
1-(3,4-Difluorophenyl)-3-(2-methylphenyl)urea (1gj). Prepared from 3,4-difluoroaniline
and o-tolyl isocyanate. Yield: 79% (off-white crystals): mp 210–211 ◦C (acetone/CHCl3);
LC/MS (70 eV) m/z (%): 295 [M+, +23]; IR (KBr): 3306 (NH), 1632 (C=O) cm−1; 1H NMR
(300 MHz, DMSO-d6):
7.27–7.37 (m, 1H, Ar), 7.64–7.71 (m, 1H, Ar), 7.76 (d, J = 7.6 Hz, 1H, Ar), 7.96 (s, 1H, NH
exch. D2O), 9.19 (s, 1H, NH exch. D2O); 13C NMR (125 MHz, DMSO-d6):
18.3 (1C), 107.4
(1C), 114.5 (1C), 117.8 (1C), 121.9 (1C), 126.6 (1C), 123.5 (1C), 128.5 (1C), 130.7 (1C), 137.4
δ 2.21 (s, 3H, CH3), 6.95 (t, J = 7.3 Hz, 1H, Ar), 7.05–7.17 (m, 3H, Ar),
δ
(1C), 137.5 (1C), 145.8 (1C), 148.6 (1C), 153.0 (1C). Anal. calcd for C14H12N2OF2
(265.95)%: C 63.25; H 4.70; N 10.57. Found%: C 63.02; H 4.62; N 10.41.
·
0.20 H2O
1-(3-Chloro-4-fluorophenyl)-3-(2-methylphenyl)urea (1gk). Prepared from 3-chloro-4-
◦
fluoroaniline and o-tolyl isocyanate. Yield: 92% (off-white crystals): mp 210–211 C
(acetone/CHCl3); LC/MS (70 eV) m/z (%): 301 [M+, +23]; IR (KBr): 3323 (NH), 1642 (C=O)
1
cm−1; H NMR (300 MHz, DMSO-d6):
δ 3.33 (s, 3H, CH3), 6.95 (t, J = 7.3 Hz, 1H, Ar),
7.10–7.20 (m, 2H, Ar), 7.22–7.34 (m, 2H, Ar), 7.75 (d, J = 8.3 Hz, 1H, Ar), 7.82 (dd, J = 6.4,
2.3 Hz, 1H, Ar), 7.96 (s, exch. D2O, 1H, NH), 9.17 (s, exch. D2O, 1H); 13C NMR (125 MHz,
DMSO-d6):
δ 16.9 (1C), 113.6 (1C), 114.6 (1C), 120.9 (1C), 123.6 (1C), 124.2 (1C), 125.9 (1C),
129.3 (1C), 131.7 (1C), 131.9 (1C), 134.2 (1C), 135.2 (1C), 152.9 (1C), 154.8 (1C). Anal. calcd
for C14H12N2OFCl
N 9.84.
·
0.25 H2O (282.56)%: C 59.37; H 4.45; N 9.89. Found%: C 59.62; H 4.36;
1-(2-Metylphenyl)-3-(2-fluorophenyl)urea (1gl). Prepared from 2-fluoroaniline and o-
◦
tolyl isocyanate. Yield: quantitative yield (off-grey crystals); mp 224–225 C (acetone);
LC/MS (70 eV) m/z (%): 267 [M+, +23]; IR (KBr): 3290 (NH), 1644 (C=O) cm−1; 1H NMR
(300 MHz, DMSO-d6):
(d, J = 7.6 Hz, 1H, Ar), 8.18 (dt, J = 8.6, 1.5 Hz, 1H, Ar), 8.34 (s, 1H, NH exch. D2O), 8.96 (br
s, 1H, NH exch. D2O); 13C NMR (125 MHz, DMSO-d6):
18.4 (1C), 115.3 (d, JCF = 18.9 Hz
δ 2.24 (s, 3H, CH3), 6.91–7.09 (m, 2H, Ar), 7.11–7.25 (m, 4H, Ar), 7.84
δ
,
1C), 120.9 (1C), 121.6 (1C), 122.6 (d, JCF = 7.5 Hz, 1C), 123.3 (1C), 124.9 (1C), 126.6 (1C), 128.0
(1C), 128.2 (1C), 130.7 (1C), 137.6 (1C), 152.3 (d, JCF = 241.3 Hz, 1C), 152.9 (1C). Anal. calcd
for C14H12N2OF (244.26)%: C 68.84; H 5.36; N 11.47. Found%: C 68.74; H 5.34; N 11.37.
1-(3,4-Difluorophenyl)-3-phenylurea (1fj). Prepared from 3,4-difluoroaniline and phenyl
isocyanate. Yield: 58% (greyish crystals): mp 213–214 ◦C (acetone); LC/MS (70 eV) m/z
(%): 271 [M+, +23]; IR (KBr): 3298 (NH), 1630 (C=O) cm−1; 1H NMR (500 MHz, DMSO-d6):
δ
6.87 (t, J = 7.6 Hz, 1H, Ar), 7.05–7.15 (m, 1H, Ar), 7.24–7.32 (m, 3H, Ar), 7.42 (d, J = 7.3 Hz,
2H, Ar), 7.60–7.70 (m, 1H, Ar), 8.70 (s, exch. D2O, 1H, NH), 8.85 (s, 1H, exch. D2O, NH); 13
NMR (125 MHz, DMSO-d6): 107.6 (d, JCF = 21.9 Hz, 1C), 114.8 (dd, JCF = 5.6, 3.7 Hz, 1C),
117.8 (d, JCF = 1.7 Hz, 1C), 118.9 (1C), 122.5 (1C), 129.2 (1C), 137.3 (dd, JCF = 9.5, 2.8 Hz,
C
δ

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1C), 139.8 (1C), 143.8 (d, JCF = 12.4 Hz, 1C), 145.8 (d, JCF = 12.4 Hz,1C), 148.5 (d, JCF = 13.3
Hz, 1C), 150.5 (d, JCF = 13.4 Hz, 1C), 152.9 (1C). Anal. calcd for C13H10N2OF2 (248.23)%: C
62.90; H 4.06; N 11.29. Found%: C 62.61; H 4.08; N 11.19.
1-(3,4-Dichlorophenyl)-3-p-tolylurea (1ae) [43]. Prepared from p-toluidine and 3,4-
◦
dichlorophenyl isocyanate. Yield: 21% (white fine spicules crystals): mp 225–226 C
(acetone/CHCl3); LC/MS (70 eV) m/z (%): 317 [M+, +23]; IR (KBr): 3289 (NH), 1633 (C=O)
1
cm−1; H NMR (500 MHz, DMSO-d6):
δ 2.06 (s, 3H, CH3), 7.07 (d, J = 8.3Hz, 2H, Ar),
7.28–7.32 (m, 3H, Ar), 7.48 (d, J = 8.8 Hz, 1H, Ar), 7.86 (d, J = 2.4 Hz, 1H, Ar), 8.65 (s, exch.
D2O, 1H, NH), 8.91 (s, exch. D2O, 1H, NH); 13C NMR (125 MHz, DMSO-d6):
δ
20.8 (1C),
118.7 (1C), 119.1 (1C), 119.6 (1C), 123.4 (2C), 129.6 (2C), 130.9 (1C), 131.5 (1C), 131.6 (1C),
137.1 (1C), 140.5 (1C), 152.8 (1C). Anal. calcd for C14H12C12N2O (294.16)%: C 56.97; H 4.10;
N 9.49. Found%: C 56.56; H 4.09; N 9.36.
1-(3,4-Dichlorophenyl)-3-(4-methoxyl)phenylurea (1de) [44]. Prepared from 3,4-dichloroaniline
and p-methoxylphenyl isocyanate. Yield: 97% (greyish crystals): mp 234–235 ◦C (acetone);
LC/MS (70 eV) m/z (%): 333 [M+, +23]; IR (KBr): 3263 (NH), 1644 (C=O) cm–1; 1H NMR
(300 MHz, DMSO-d6):
(d, J = 8.8 Hz, 1H, Ar), 7.84 (d, J = 2.3 Hz, 1H, Ar), 8.58 (s, 1H, NH), 8.89 (s, 1H, NH); 13
NMR (125 MHz, DMSO-d6): 55.6 (1C), 114.4 (2C), 119.6 (1C), 120.4 (1C), 120.9 (2C), 123.4
δ 3.69 (s, 3H, CH3), 6.82–6.88 (m, 2H, Ar), 7.27–7.36 (m, 3H, Ar), 7.47
C
δ
(1C), 130.9 (1C), 131.5 (2C), 132.4 (1C), 140.4 (1C), 152.8 (1C), 155.2 (1C). Anal. calcd for
C14H12N2O2Cl2 (311.16)%: C 54.04; H 3.89; N 9.00. Found%: C 54.17; H 3.95; N 8.94.
3.2. 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 reproduci◦bility, 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
×
106 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].
3.3. Cell Cultures
Human MCF-7 and MDA-MB-231 breast cancer cells, HeLa and ISHIKAWA uterine
carcinoma cells, A2058 melanoma epithelial cells, MCF-10A mammary epithelial cells and

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Hek-293 embryonic kidney epithelial cells were obtained from American Type Culture
Collection (ATCC, Manassas, VA, USA). Estrogen receptor positive [(ER(+)] MCF-7 and
triple negative MDA-MB-231 breast cancer cells were maintained in Dulbecco’s modified
Eagle’s medium/nutrient mixture F-12 Ham (DMEM-F12) medium containing 2 mmol/L
L-glutamine, 1 mg/mL penicillin-streptomycin and supplemented with 5% fetal bovine
serum (FBS) [45]. HeLa epithelial cervix carcinoma cells, estrogen receptor negative [ER(-)],
and Ishikawa endometrial adenocarcinoma cell, estrogen receptor positive [ER(+)], were
kept in minimum essential medium (MEM), supplemented with 10% FBS, 100 U/mL peni-
cillin/streptomycin and 1% non-essential amino acid. A2058 highly metastatic melanoma
epithelial cells were kept in DMEM low glucose (1 g/L), supplemented with 20% FBS,
1% l-glutamine and 100 U/mL penicillin/streptomycin. MCF-10A mammary epithelial
cells, were cultured in DMEM/F12 medium, supplemented with 5% horse serum (HS),
100 U/mL penicillin/streptomycin, 0.5 mg/mL hydrocortisone, 20 ng/mL human epi-
dermal growth factor (hEGF), 10 mg/mL insulin and 0.1 mg/mL cholera enterotoxin
(Sigma-Aldrich, Milan, Italy). Hek-293 embryonic kidney epithelial cells were cultured in
DMEM high glucose (4.5 g/L), supplemented with 10% FBS, 1% l-glutamine and 100 U/mL
penicillin/streptomycin. Cells were conserved at 37 ◦C in a humid atmosphere of 95% air
and 5% CO2, and screened for contamination at regular intervals [46,47].
3.4. Cell Viability
Cell viability was determined using the 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyl-
tetrazolium 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 IC50 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.
4. 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 ac-
tivity 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 pre-
pared small molecules are promising candidates for antibacterial drug development to be
introduced in personal care products in substitution of TCC.
Author Contributions: Writing—Original Draft Preparation, A.C.; Data Curation, D.I.; Validation,
A.R.; Methodology, L.S., J.C. and F.L.; Writing—Review and Editing, C.F.; Supervision, M.S.S. All
authors have read and agreed to the published version of the manuscript.
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.

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Abbreviations
4-CA:4-Chloroaniline; CEC, Contaminant of Emerging Concern; CLSI, Clinical and Laboratory
Standards Institute; DCC, 4,40-Dichlorocarbanilide; DMSO, Dimethyl sulfoxide; hEGF, Human
epidermal growth factor; FBS, Fetal bovine serum; HS, Horse serum; IR, Infrared; LC-MS, Liq-
uid chromatography-mass spectrometry; MCC, 1-(3-Chlorophenyl)-3-phenylurea; MICs, Minimum
Inhibitory Concentrations; MEM, Minimum essential medium; MTT, 3-(4,5-Dimethylthiazol-2-y1)-2,5-
diphenyltetrazolium bromide; NCC, Carbanilide; NMR, Nuclear Magnetic Resonance; SD, Standard
deviations; TCC, Triclocarban.
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