1,3-Disubstituted urea derivatives: Synthesis, antimicrobial activity evaluation and in silico studies

Article abstract of DOI:10.1016/j.bioorg.2020.104104

The development of new antimicrobial compounds is in high demand to overcome the emerging drug resistance against infectious microbial pathogens. In the present study, we carried out the extensive antimicrobial screening of disubstituted urea derivatives. In addition to the classical synthesis of urea compounds by the reaction of amines and isocyanates, we also applied a new route including bromination, oxidation and azidination reactions, respectively, to convert 2-amino-3-methylpyridine to 1,3-disubstituted urea derivatives using various amines. The evaluation of antimicrobial activities against various bacterial strains, Candida albicans as well as Mycobacterium tuberculosis resulted in the discovery of new active molecules. Among them, two compounds, which have the lowest MIC values on Pseudomonas aeruginosa, were further evaluated for their inhibition capacities of biofilm formation. In order to evaluate their potential mechanism of biofilm inhibition, these two compounds were docked into the active site of LasR, which is the transcriptional regulator of bacterial signaling mechanism known as quorum sensing. Finally, the theoretical parameters of the bioactive molecules were calculated to establish their drug-likeness properties.

Full text of DOI:10.1016/j.bioorg.2020.104104

Journal Pre-proofs
1,3-Disubstituted Urea Derivatives: Synthesis, Antimicrobial Activity Evalua-
tion and In Silico Studies
Miyase Gözde Gündüz, Sümeyye Buran Uğur, Funda Güney, Ceren Özkul,
Vagolu Siva Krishna, Serdal Kaya, Dharmarajan Sriram, Şengül Dilem Doğan
PII:
S0045-2068(20)31401-2
DOI:
https://doi.org/10.1016/j.bioorg.2020.104104
YBIOO 104104
Reference:
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Bioorganic Chemistry
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29 June 2020
14 July 2020
Please cite this article as: M. Gözde Gündüz, S. Buran Uğur, F. Güney, C. Özkul, V. Siva Krishna, S. Kaya, D.
Sriram, S. Dilem Doğan, 1,3-Disubstituted Urea Derivatives: Synthesis, Antimicrobial Activity Evaluation and In
Silico Studies, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.104104
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1,3-Disubstituted Urea Derivatives: Synthesis, Antimicrobial Activity Evaluation and In
Silico Studies
Miyase Gözde Gündüza, Sümeyye Buran Uğurb, Funda Güneyb, Ceren Özkulc, Vagolu Siva
Krishnad, Serdal Kayae,f, Dharmarajan Sriramd, Şengül Dilem Doğanb*
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Hacettepe University, Sıhhiye,
06100 Ankara, Turkey
bDepartment of Basic Sciences, Faculty of Pharmacy, Erciyes University, Kayseri, Turkey
cDepartment of Pharmaceutical Microbiology, Faculty of Pharmacy, Hacettepe University,
Sıhhiye, 06100 Ankara, Turkey
dDepartment of Pharmacy, Birla Institute of Technology and Science-Pilani, Hyderabad, 500078,
India
eDepartment of Chemistry, Faculty of Arts and Sciences, Giresun University, 28200, Giresun,
Turkey
fDepartment of Aeronautical, Faculty of Aviation and Space Sciences, Necmettin Erbakan
University, 42140, Konya, Turkey
*Corresponding author
Dr. Şengül Dilem Doğan
Address: Erciyes University
Faculty of Pharmacy
Department of Basic Sciences
38039
Kayseri, TURKEY
E-mail address: dogandilem@gmail.com
Phone number: +90 352 2076666-28032

Abstract
The development of new antimicrobial compounds is in high demand to overcome the emerging
drug resistance against infectious microbial pathogens. In the present study, we carried out the
extensive antimicrobial screening of disubstituted urea derivatives. In addition to the classical
synthesis of urea compounds by the reaction of amines and isocyanates, we also applied a new
route including bromination, oxidation and azidination reactions, respectively, to convert 2-amino-
3-methylpyridine to 1,3-disubstituted urea derivatives using various amines. The evaluation of
antimicrobial activities against various bacterial strains, Candida albicans as well as
Mycobacterium tuberculosis resulted in new active agents. Among them, two compounds, which
have the lowest MIC values on Pseudomonas aeruginosa, were further evaluated for their
inhibition capacities of biofilm formation. In order to evaluate their potential mechanism of biofilm
inhibition, these two compounds were docked into the active site of LasR, which is the
transcriptional regulator of bacterial signaling mechanism known as quorum sensing. Finally, the
theoretical parameters of the bioactive molecules were calculated to establish their drug-likeness
properties.
Keywords: antibacterial, tuberculosis, antifungal, biofilm, molecular docking, quorum sensing

1. Introduction
Infectious diseases, caused by Gram-positive/Gram-negative, fungal and mycobacterial pathogens,
still seriously jeopardize human health despite existing for centuries [1]. Together with cancer and
cardiovascular diseases, bacterial infections stand among the leading causes of serious morbidity
and mortality all over the world [2]. Amongst different microbial infections, the incidence of fungal
infections has dramatically increased over the previous decades especially in immunocompromised
patients with AIDS diagnose, organ transplant history or cancer chemotherapy process [3].
Tuberculosis, caused by Mycobacterium tuberculosis, is an infectious pulmonary disease and is the
foremost reason for life lost owing to a single infectious agent. The World Health Organization
approximated ten million patients developed TB in 2017 [4].
The primary obstruction to the achievement of a successful chemotherapy regimen in infectious
diseases is the growing antimicrobial resistance to the present drugs [5]. Therefore, the discovery
of new and more effective therapeutic agents with favorable pharmacokinetic profiles is of utmost
importance.
Pseudomonas aeruginosa is a Gram-negative bacterium and is considered as one of the most
opportunistic pathogens causing life-threatening infections in diseases such as meningitis,
septicemia and cystic fibrosis [6]. It is also responsible for the considerable mortality in
immunocompromised patients. Additionally, P. aeruginosa is capable of producing biofilm which
reduces the effectiveness of therapeutic agents and increases the antibacterial resistance [7].
Biofilm formation is attributed to the communication of bacteria via the signaling mechanism
called quorum sensing (QS). LasR is the transcriptional regulator of QS and plays a key role in the
pathogenesis of P. aeruginosa [8]. N-Acyl homoserine lactones (AHLs) are a class of signaling
molecules employed in bacterial QS (Figure 1) [9].
O
O
O
O
R: Alkyl chain
R
N
H
Figure 1. General chemical structure of AHLs

The chemical structures of AHLs differ according to the bacteria species. 3-oxo-dodecanoyl
homoserine lactone (ODHL) is found in P. aeruginosa and is recognized by LasR with the defined
interactions (Figure 2) [9]. Therefore, inhibiting this system is a rational approach to decrease
bacterial virulence.
Figure 2. Molecular surface representation of LasR with the co-crystallized ligand ODHL (PDB
code: 3IX3) represented as pink stick (A). Identified interaction pattern of ODHL in the active site
of LasR (B). Pharmacophoric features are visualized as green arrow (hydrogen bond donor), red
arrows (hydrogen bond acceptor), and yellow sphere (hydrophobic interaction).
The urea functionality represents one of the most significant core structures incorporated into
organic and medicinal chemistry. This scaffold is inherent to diverse bioactive molecules such as
anticancer, antiviral, antibacterial, antihypertensive agents including clinically approved
therapeutics (Figure 3) [10].

O
NH2
O
Cl
O
O
O
O
N
H
O
S
O
N
NH2
F
F
N
N
N
HN
N
N
OH
H
H
N
H
H
F
Boceprevir
O
Sorafenib
Zileuton
O
O
N
O
N
H2N
N
Cl
O
NH
N
Cl
Cl
O
O
N
N
H
N
N
Lisuride
Cariprazine
Lenvatinib
N
H
N
H
H
F
F
F
F
O
F
OH
H
N
O
F
O
N
O
N
N
H
O
N
N
H
O
N
O
N
N
H
H
N
O
N
Carmofur
Celiprolol
Telcagepant
HN
O
Figure 3. Chemical structures of urea containing FDA approved drug molecules.
Hence, a remarkable amount of researchers focus on the synthesis of different derivatives using
urea functionality as an excellent building block. Among them, numerous substituted urea
derivatives were reported with their antibacterial, antifungal and antitubercular activities (Figure
4) [11–13].
F
O
O
F
F
N
O
O
O
N
CF3
CN
N
N
N
N
S
N
N
N
N
H
H
H
H
H
H
CF3
Antibacterial
Antifungal
Antitubercular
Figure 4. Some examples of disubstituted urea derivatives possessing antimicrobial activities.
Considering the great significance of urea-containing compounds with medicinal purposes as well
as different applications of organic reactions or material science, diverse methods have been
established for their syntheses [10]. The classical syntheses of urea derivatives include the reaction

of amines with isocyanate, carbamates or phosgene. However, especially due to the high toxicity
of phosgene, the researchers focus on finding alternative routes to obtain urea derivatives [14].
Based on the above-mentioned considerations, we aimed to synthesize various substituted urea
derivatives and test them against different Gram-positive and Gram-negative bacteria, Candida
albicans as well as Mycobacterium tuberculosis.
2. Results and Discussion
2.1. Chemistry
For the synthesis of the first subseries of the compounds (U1-U7), 2-bromo-3-methylpyridine (II)
was obtained by the bromination reaction of 2-amino-3-methylpyridine (I) in the presence of HBr
at low temperature. Oxidation of 2-bromo-3-methylpyridine (II) with KMnO4 in the presence of
water resulted in the formation of 2-bromonicotinic (III) acid. Afterward, 2-bromonicotinic acid
was converted into the corresponding azide (IV). For this reaction, 2-bromonicotinic was treated
with ethyl chloroformate in the presence of triethylamine followed by the addition of a solution of
NaN3 in water. The resulting azide moiety is an excellent starting compound to produce the
corresponding isocyanate. Therefore, acyl azide (IV) was refluxed in benzene with various amines.
Isocyanate was formed in the reaction medium and trapped with amine to give desired urea
1
derivatives U1-U7 (Figure 5). The structures of the compounds U1-U7 were elucidated by H
NMR, 13C NMR, HRMS and FTIR. 1H NMR and 13C NMR spectra of the synthesized compounds
are provided as supplementary materials.
O
CH3
CH3 b
Br
COOH
Br
CON3
Br
NH C NH R
a
c
d
N
I
NH2
N
II
N
N
N
Br
III
IV
U1-U7
Figure 5. Synthesis of urea derivatives U1-U7 a) Br2, NaNO2, 48% HBr, -20 °C b) KMnO4, H2O,
reflux c) i. Et3N, ethylchloroformate, THF ii. NaN3/H2O d) Substituted amines, benzene, reflux.
Second subseries of the target compounds (U8-U23) were synthesized in a simple one-step method.
The reaction of 2-aminothiophenol with equivalent amount of various isocyanate derivatives in
toluene at 80°C resulted in compounds U8-U23. The last derivative U24 was obtained in DMF at
0°C by the reaction of 2-aminothiophenol with two equivalent amounts of cyclohexyl isocyanate
(Figure 6).

O
NH2
SH
NH C NH R
Toluene
80 °C
+
R N C O
SH
U8-U23
O
N C O
NH2
SH
NH C NH
DMF
0 °C
+
S C NH
O
U24
Figure 6. Synthesis of urea derivatives U8-U24
The chemical structures of the studied compounds are provided in Table 1.
Table 1. The chemical structures of the tested compounds
Compound
X
N
N
N
N
N
N
N
C
R1
Br
Br
Br
Br
Br
Br
Br
SH
R2
U1
U2
U3
U4
U5
U6
U7
U8
phenyl
4-methoxyphenyl
2-pyridyl
2-aminophenyl
4-chlorophenyl
4-nitrophenyl
o-tollylphenyl
phenyl

U9
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
SH
SH
SH
SH
SH
SH
SH
SH
SH
SH
SH
SH
SH
SH
SH
4-fluorophenyl
4-cyanophenyl
U10
U11
U12
U13
U14
U15
U16
U17
U18
U19
U20
U21
U22
U23
4-bromophenyl
4-nitrophenyl
4-chlorophenyl
3-cyanophenyl
3,4,5-trimethoxyphenyl
4-methoxyphenyl
p-tollylphenyl
2-naphtyl
2,3,4-trimethoxyphenyl
3,4-dimethoxyphenyl
4-tert-butyl
2,4-dimethoxyphenyl
o-tollylphenyl
O
S C NH
U24
C
cyclohexyl
2.2. Antibacterial and Antifungal Activity Evaluation
The obtained urea derivatives were initially tested for their antibacterial and antifungal activities
by determining their minimum inhibitory concentration (MIC) values. Gentamicin, piperacillin-
tazobactam and fluconazole were employed as positive control. Table 2 represents the
antimicrobial activity of each compound against bacteria and C. albicans. The DMSO
concentration used to dissolve compounds had no antimicrobial effect.

Table 2. Minimum inhibitory concentrations (MIC) for bacteria and yeast (μg/mL)
Gram-positive bacteria Gram-negative bacteria
Yeast
P. aeruginosa C. albicans
S. aureus
MRSA
E.
E. coli
faecalis
Compound
ATCC
29213
ATCC
43300
ATCC
25922
ATCC
27853
ATCC
90028
ATCC
29212
512
U1
U2
256
512
512
512
256
256
256
256
256
8
256
256
256
512
512
512
256
128
256
16
256
256
256
256
256
512
256
256
256
128
64
256
256
512
512
256
512
256
64
16
32
64
64
32
64
32
16
32
32
32
64
64
32
32
64
64
64
64
64
256
256
256
256
256
128
256
64
U3
U4
U5
U6
U7
U8
U9
128
2
U10
U11
U12
U13
U14
U15
U16
U17
U18
U19
U20
16
64
64
4
32
512
128
256
64
512
128
256
64
512
32
512
128
256
128
512
512
512
256
256
512
128
32
256
64
0.5
128
128
32
256
256
128
256
256
256
256
128
256
512
64
64
128
128
256
128
128

U21
U22
256
256
512
512
256
256
16
256
128
128
64
256
256
256
256
0.25
0.25
256
128
64
64
64
U23
256
32
U24
128
64
8
Gentamicin
0.06
0.125
2
0.25
0.25
NA
NA
Piperacillin/
Tazobactam
NA
0.25
Fluconazole
NA
NA
NA
NA
NA
0.125
NA: Not applicable.
According to the obtained results, the synthesized derivatives possessed antimicrobial activity with
MIC values ranging between 0.5 and 512 µg/mL against all tested microorganisms. It is noteworthy
to mention that U15 was the most active compound on P. aeruginosa with a MIC value of 0.5
µg/mL. Furthermore, U10 was one of the most attractive compounds effective on P. aeruginosa
and S. aureus with MIC of 2 and 8 µg/mL, respectively. Additionally, U11 showed high
antimicrobial activity against E. faecalis possessing MIC of 4 µg/mL. U24 stands as the most active
compound as antifungal agent against C. albicans with 8 µg/mL MIC value.
When the obtained results are evaluated out in terms of the chemical structures of the compounds,
the substitution of the urea moiety with 2-bromopyridine ring (U1-U7) did not lead to an
improvement in the antimicrobial activity. It is interesting to note that all active compounds (U10,
U11 and U15) belong to the second subseries carrying 2-mercaptophenyl ring. Additionally, the
most effective antibacterial compound (U15) carries three methoxy groups on the 3’-phenyl ring.
U24, which has the lowest MIC value on C. albicans, is considered to be the most active antifungal
agent among all compounds. At the same time, it is the only compound carrying a bulky substituent
on the thiol group of the phenyl ring. Consequently, antifungal activity can be related to the
increased lipophilicity of U24.

2.3. Antitubercular Activity Evaluation
All synthesized urea derivatives were evaluated for their antitubercular activities against M.
tuberculosis H37Rv utilizing Microplate Alamar Blue Assay (MABA) method. Isoniazid,
rifampicin, and ethambutol were used as reference compounds. The antimycobacterial activity
screening results are reported as MIC values in Table 3.
Table 3. Antitubercular activities of the compounds
Compound MIC (μg/mL) Compound MIC (μg/mL)
U1
U2
>25
25
U13
U14
U15
U16
U17
U18
U19
U20
U21
U22
U23
U24
25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
6.25
U3
>25
>25
25
U4
U5
U6
>25
>25
>25
>25
12.5
>25
>25
U7
U8
U9
U10
U11
U12
İsoniazid: 0.05, Rifampicin: 0.1, Ethambutol: 1.56
The obtained MIC values revealed that U2, U5 and U13 were moderate inhibitors of M.
tuberculosis with MIC value of 25 µg/mL. Among the compounds, U10 and U24 were found to be
more active on M. tuberculosis showing 6.25 and 12.5 µg/mL MIC values, respectively. It is
remarkable that U24, the only active compound as antifungal agent, was found to be the best
inhibitor of M. tuberculosis growth inhibition at the same time.

2.4. Structure-Activity Relationship
According to the data obtained from biological activity determination experiments, we summarized
the structural requirements of the compounds possessing antimicrobial activities in Figure 7.
Phenyl ring at this locus optimizes
antimicrobial activity. The most
effective antibacterial derivative
possesses 3,4,5-trimethoxyphenyl
substituent as R2
O
The most active compounds
NH C NH R2
carry carbon at this position.
Changing carbon to nitrogen did
not make a positive contribution
to the antimicrobial activity
Bulky liphophilic substituents on
SH group increases antifungal and
antitubercular activity
X
R1
Thiol functionality rather than
bromine atom at this locus leads
to high antibacterial activity
Figure 7. Structure-activity relationships of the synthesized compounds
2.5. In-vitro Biofilm Inhibition
Among the tested compounds, U10 and U15 showed strong antibacterial activity against P.
aeruginosa. Moreover, the antimicrobial activity of U15 seems to be specific to P. aeruginosa
since its MIC values against other tested bacteria were relatively higher. Thus, we mainly focused
on these two compounds for further biofilm inhibition assay.
The MIC concentrations of U10 and U15 against PAO1 were 4 µg/mL and 1 µg/mL, respectively.
Thus we used MIC, MIC/2 and MIC/4 concentrations for each compound. Both inhibitory and sub-
inhibitory concentrations for U10 lead to a significant decrease in PAO1 biofilm formation when
compared to control. The percentage of biofilm inhibition was as follows: 56.83% for MIC, 48.92%
for MIC/2, 29.50% for MIC/4. U15 showed a diminished anti-biofilm effect when compared to
U10. However, both MIC and MIC/2 concentrations were significantly decreased PAO1 biofilm
formation (30.22% and 20.14% decrease, respectively) (Figure 8)

Figure 8. In-vitro biofilm inhibition assay. A) Representative pictures for crystal violet staining
microtiter biofilm formation assay (Control wells; PAO1 alone and NC:Negative control) B)
Inhibitory effect of MIC, MIC/2 and MIC/4 values of U10 and U15 against PAO1 biofilms, which
were quantified by crystal violet staining. P. aeruginosa PA01 alone was used as control. Statistical
comparisons were made between each group and control mean OD570 value by using two-way
ANOVA; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
2.6. Molecular Docking
Biofilm formation is directly quorum sensing-controlled mechanism in bacteria. Therefore,
inhibiting QS in P. aeruginosa decreases biofilm formation. Besides, it is a rational and frequently
used approach to mimic the signal molecules by using analog synthetic compounds for QS
inhibition. Therefore, we aimed to explain the activities of U10 and U15 by molecular docking
studies. To investigate the anti-biofilm activity mechanism of U10 and U15, we initially examined
the binding interactions of N-3-(oxododecanoyl)-L-homoserine lactone, 3-oxo-N-(2-
oxotetrahydrofuran-3-yl)octanamide, (ODHL) which is a co-crystallized ligand in LasR binding
site. This compound binds to LasR via four key hydrogen bonds and hydrophobic interactions. The
carbonyl group of lactone moiety and the amide functionality are responsible for forming these
hydrogen bonds. The long alkyl side chain occupies the hydrophobic pocket. The structural
requirements of ODHL to regulate QS in P. aeruginosa and the overlaid docking poses of U10 and
U15 within LasR active site are provided in Figure 9.

Figure 9. Identified pharmacophores of ODHL in LasR binding site (A). Superimposition of
ODHL (cyan stick), U10 (red stick) and U15 (orange stick) after docking to the active pocket of
LasR (B). Red arrow - hydrogen bond acceptor, green arrow - hydrogen bond donor, yellow sphere
- hydrophobic interaction
Figure 10 reports the detailed binding interactions of U10 and U15 in the active site of LasR, the
transcriptional regulator of QS. When the plausible binding modes of these compounds in the active
pocket of LasR were analyzed, it was observed that the urea functionality, the core scaffold, formed
three key hydrogen-bonding interactions to Tyr56, Asp73 and Ser129 in the same manner as the
amide functionality of ODHL. Additionally, thiol group in U10 and U15 served as a hydrogen bond
acceptor like the carbonyl of the lactone moiety in the original ligand. Also, phenyl rings of the test
compounds were responsible for the further hydrophobic contacts. The lack of a long alkyl side
chain in U10 and U15 leads to loss of the interaction in the hydrophobic pocket of LasR, where the
original ligand interacts to activate QS. In this manner, our compounds are stabilized in the binding
site by hydrogen bonds but cannot behave like an activator. The number of increased hydrophobic
contacts in U10 with LasR explains the higher biofilm inhibition capacitiy compared to U15.
Consequently, with these pharmacophore similarities between our compounds and ODHL, U10
and U15 are likely to inhibit LasR activation and act as QS and biofilm inhibitors in P. aeruginosa.
Although our in-silico studies indicate this potential mechanism of action in decreasing biofilm
formation, further mechanistic studies to confirm their direct interaction with QS signal receptors
will shed light on this issue.

Figure 10. Proposed docking poses of U10 (A) and U15 (B) in the binding site LasR and 2D
depictions of the formed interactions. Red arrow - hydrogen bond acceptor, green arrow - hydrogen
bond donor, yellow sphere - hydrophobic interaction
2.7. In Silico Prediction of Physicochemical Properties of Active Molecules
Some bioactive molecules may be unsuccessful to turn into good drug molecules because of their
unsuitable physicochemical properties. Therefore, it is a useful approach to predict the molecular
properties of novel drug candidates. Here, we carried out computational calculations to foresee the
parameters of the most active antimicrobial compounds and to determine their drug-likeness
properties (Table 4).
Lipinski’s rule of five [15] is applied to estimate drug-likeness and clarifiy the essential molecular
descriptors which are important to predict the oral bioavailability and cell permeability of new
bioactive molecules. Along with its descriptors, we also calculated the number of rotatable bonds

(nROTB) and topological polar surface area (TPSA), which are accepted to be important
parameters to determine the oral bioavailability of drug molecules [16].
Table 4. Calculated molecular properties of the most active molecules
Compound LogP[a] M.W.[b] HBA[c] HBD[d] TPSA[e] nROTB[f] %ABS Lipinski’s
Violation
U10
U11
U15
U24
3.08
4.13
2.96
5.29
269.33
323.21
334.40
375.54
4
3
6
5
2
2
2
3
64.92
41.12
68.83
70.22
2
2
5
5
86.60
94.81
85.25
84.77
0
0
0
1
aLogP: logarithm of n-octanol-water partition coefficient
bM.W: molecular weight
cHBA: number of hydrogen bond acceptors
dHBD: number of hydrogen bond donors
eTPSA: topological polar surface area
fNROTB: number of rotatable bonds
According to the calculated parameters, three of the compounds completely adhere to Lipinski’s
rule of five. Only U24 presented a slight violation with its LogP value. However, this value does
not exceed the accepted range excessively. Also, the increased lipophilicity can be a determining
factor for its high activity against M. tuberculosis and C. albicans compared to the other tested
compounds. TPSA values of the compounds were found to be ranging between 41.12 and 70.22,
which is an acceptable range considering that this value is below 140-150 Å2 for the most known
drug molecules. TPSA was also used to calculate the percentage of absorption using this equation:
%ABS=109-0.345xTPSA and the rates were found to be good for intestinal absorption. The
number of rotatable bonds is an important parameter that indicates molecular flexibility. To restrict
the conformational changes while interacting with the biological target, this value should be ≤ 10.
All of our active compounds have less than ten rotatable bonds.
Consequently, these compounds exhibit excellent physicochemical properties to serve as
antimicrobial drug candidates [17].
3. Conclusion
In this study, we synthesized and tested 1,3-disubstituted urea derivatives for their antimicrobial
activities. In addition to the classical urea synthesis, we reported a new route to convert methyl

group to substituted urea functionality. Extensive antimicrobial tests on bacterial strains, C.
albicans and M.tuberculosis resulted in new potential molecules against bacterial, fungal and
tubercular infections. The obtained results indicated that introducing 2-mercaptophenyl ring into
the core urea functionality led to an improvement in the antibacterial activities of the compounds.
Substitution of the thiol group with a bulky group increased the lipophilicity of the compounds and
provided better antifungal and antitubercular activities. Also, we carried out further studies on two
molecules, active on P. aeruginosa, via determining their biofilm inhibition capacities. According
to the obtained data from molecular docking studies, these compounds are likely to decrease
biofilm formation via quorum sensing inhibition. Calculated parameters of the most active
compounds showed that these compounds can be considered as drug candidates. Consequently, our
findings can be useful for the discovery of future molecules for the successful treatment of the
antimicrobial infections.
4. Experimental
4.1. Chemistry
4.1.2. Materials and Methods
Toluene and benzene were distilled from sodium-benzophenone just prior to use. All reagents were
commercially purchased and used without further purification. All reactions were monitored by
TLC performed on pre-coated 60 F254 plates. Visualization was effected with UV using Camag
Thin-Layer Chromatogram Lamp (254/366 nm). Melting points were determined using open glass
capillaries and are uncorrected. Infrared (IR) spectra were recorded in the range 4000–600 cm−1
1
13
via ATR diamond. H NMR and C NMR spectra were determined under ambient temperature
conditions in DMSO-d6 solution using a Bruker AM 400 MHz spectrophotometer. The chemical
shifts (δ) were reported in parts per million (ppm) and were relative to the central peak of the
solvent, which was DMSO-d6. MS spectra were carried out on an LC/MS High-Resolution Time
of Flight (TOF) Agilent 1200/6530 instrument at the Atatürk University-East Anatolian High
Technology Research and Application Center (DAYTAM).
4.1.3. General procedure for the synthesis of urea derivatives U1-U7

To a solution of 2-bromonicotinic acid [18–20] (0.5 g, 2.48 mmol) in 10 mL THF at -5 °C,
triethylamine (0.36 mL, 2.48 mmol) in THF (5 mL) was added dropwise and the resulting mixture
was stirred for 30 min. Cooled solution of ethyl chloroformate (0.31 mL, 2.98 mmol) in THF (5
mL) was added to the reaction mixture at the same temperature. After additional stirring for 30
min., a solution of sodium azide (0.32 g, 4.96 mmol) in water (5 mL) was added dropwise. The
reaction mixture was stirred at room temperature for 18h-24h. The mixture was extracted with ethyl
acetate (3×15 mL) and the organic phase was washed with saturated sodium bicarbonate (3×30
mL) and with water (2×25 mL) and dried over MgSO4. After the removal of ethyl acetate, azide
(0.41 g, 73%) was obtained as white solid. The obtained azide was used the next step without any
purification process. Acyl azide and one equimolar amine were heated at reflux in freshly distilled
benzene (10 mL) for 18-24h. The formed precipitate was filtered and washed with benzene to give
pure samples U1-U7.
1-(2-Bromopyridin-3-yl)-3-phenylurea (U1)
White solid, yield: 39%. mp 170-172 °C; Rf (EtOAc:Hexane=1:1):0.55; IR (ATR) 3270, 3065,
1636, 1549, 1391, 1203, 1043. 1H NMR (400 MHz, DMSO) δ 9.59 (s, 1H, -NH), 8.42 (dd, J = 8.2,
1.8 Hz, 1H), 8.27 (s, 1H, -NH), 8.03 (dd, J = 4.5, 1.8 Hz, 1H), 7.49 – 7.44 (m, 2H), 7.41 (dd, J =
13
8.1, 4.5 Hz, 1H), 7.34 – 7.26 (m, 2H), 7.01 (t, J = 7.4 Hz, 1H). C NMR (100 MHz, DMSO) δ
152.43, 143.77, 139.59, 135.35, 133.07, 129.60, 129.42, 124.12, 122.87, 118.75. HRMS (EI):
[M+H]+, found 292.0069. C12H11BrN3O requires 292.0085.
1-(2-Bromopyridin-3-yl)-3-(4-methoxyphenyl)urea (U2)
White solid, yield: 24%. mp 170-171 °C; Rf (EtOAc:Hexane=1:1):0.47; IR (ATR) 3663, 2987,
2906, 1708, 1623, 1517, 1384, 1231. 1H NMR (400 MHz, DMSO) δ 9.41 (s, 1H, -NH), 8.41 (dd,
J = 8.2, 1.9 Hz, 1H), 8.17 (s, 1H, -NH), 8.01 (dd, J = 4.6, 1.9 Hz, 1H), 7.54 – 7.25 (m, 3H), 7.01
13
– 6.81 (m, 2H), 3.71 (s, 3H). C NMR (100 MHz, DMSO) δ 155.26, 152.54, 143.56, 135.49,
132.90, 132.52, 129.39, 124.10, 120.58, 114.57, 55.62. HRMS (EI): [M+H]+, found 322.0193.
C13H13BrN3O2 requires 322.0191.
1-(2-Bromopyridin-3-yl)-3-(pyridin-2-yl)urea (U3)

White solid, yield: 53%. mp 227-228 °C; Rf (EtOAc:Hexane=1:1):0.46; IR (ATR) 2976, 1696,
1578, 1532, 1475, 1307, 1238. 1H NMR (400 MHz, DMSO) δ 12.16 (s, 1H), 10.17 (s, 1H), 8.64
(d, J = 8.2 Hz, 1H), 8.35 (d, J = 4.4 Hz, 1H), 8.06 (dd, J = 4.5, 1.7 Hz, 1H), 7.92 – 7.74 (m, 1H),
7.43 (dd, J = 8.2, 4.5 Hz, 1H), 7.18 (bd, J = 8.3 Hz, 1H), 7.06 (dd, J = 7.2, 5.1 Hz, 1H). 13C NMR
(100 MHz, DMSO) δ 152.92, 152.71, 146.72, 143.84, 139.63, 135.73, 132.88, 128.97, 124.14,
118.17, 112.47. HRMS (EI): [M+H]+, found 293.0014. C11H10BrN4O requires 293.1320.
1-(2-Aminophenyl)-3-(2-bromopyridin-3-yl)urea (U4)
White solid, yield: 42%. mp 170-172 °C; Rf (EtOAc:Hexane=1:1):0.55; IR (ATR) 3662, 2987,
2900, 1619, 1531, 1384, 1249, 1051. 1H NMR (400 MHz, DMSO) δ 8.67 (s, 1H, -NH), 8.45 (dd,
J = 8.3, 1.9 Hz, 1H), 8.31 (s, 1H, -NH), 8.00 (dd, J = 4.5, 1.8 Hz, 1H), 7.41 – 7.35 (m, 1H), 7.29
(dd, J = 7.9, 1.5 Hz, 1H), 6.87 (td, J = 7.7, 1.6 Hz, 1H), 6.77 – 6.72 (m, 1H), 6.56 (td, J = 7.6, 1.6
13
Hz, 1H), 4.87 (s, 2H). C NMR (100 MHz, DMSO) δ 153.18, 143.36, 142.03, 135.76, 132.62,
129.15, 125.70, 125.05, 124.09, 123.92, 117.02, 116.18. HRMS (EI): [M+H]+, found 307.0186.
C12H12BrN4O requires 307.0194.
1-(2-Bromopyridin-3-yl)-3-(4-chlorophenyl)urea (U5)
White solid, yield: 39%. mp 232-234 °C; Rf (EtOAc:Hexane=1:1):0.53; IR (ATR) 3280, 3200,
1
1637, 1592, 1572, 1544, 1390, 1243, 1203, 1049. H NMR (400 MHz, DMSO) δ 9.71 (s, 1H, -
NH), 8.39 (dd, J = 8.2, 1.9 Hz, 1H), 8.29 (s, 1H, -NH), 8.04 (dd, J = 4.6, 1.8 Hz, 1H), 7.53 – 7.45
13
(m, 2H), 7.41 (dd, J = 8.2, 4.6 Hz, 1H), 7.38 – 7.32 (m, 2H). C NMR (100 MHz, DMSO) δ
152.37, 144.00, 138.57, 135.16, 133.27, 129.80, 129.27, 126.39, 124.14, 120.29. HRMS (EI):
[M+H]+, found 325.9696. C12H10BrClN3O requires 325.9693.
1-(2-Bromopyridin-3-yl)-3-(4-nitrophenyl)urea (U6)
Yellow solid, yield: 58%. mp 254-256 °C; Rf (EtOAc:Hexane=1:1):0.47; IR (ATR) 3451, 3273,
1
1722, 1650, 1573, 1507, 1325, 1177, 1107. H NMR (400 MHz, DMSO) δ 10.30 (s, 1H, -NH),
8.54 (s, 1H, -NH), 8.37 (d, J = 8.3 Hz, 1H), 8.20 (d, J = 9.0 Hz, 2H), 8.08 (d, J = 4.9 Hz, 1H), 7.70
(d, J = 9.2 Hz, 2H), 7.52 – 7.29 (m, 1H). 13C NMR (100 MHz, DMSO) δ 152.20, 145.96, 144.79,
141.95, 134.56, 133.96, 130.78, 125.65, 124.23, 118.26. HRMS (EI): [M+H]+, found 336.9942.
C12H10BrN4O3 requires 336.9936.

1-(2-Bromopyridin-3-yl)-3-(o-tolyl)urea (U7)
White solid, yield: 62%. mp 234-235 °C; Rf (EtOAc:Hexane=1:1):0.61; IR (ATR) 2977, 1697,
1533, 1475, 1420, 1307, 1238. 1H NMR (400 MHz, DMSO) δ 8.78 (s, 1H), 8.62 (s, 1H), 8.37 (d,
J = 8.2 Hz, 1H), 8.21 – 7.94 (m, 1H), 7.72 (d, J = 8.3 Hz, 1H), 7.40 (dd, J = 8.1, 4.5 Hz, 1H), 7.28
13
– 7.11 (m, 2H), 6.99 (t, J = 7.5 Hz, 1H), 2.26 (s, 3H). C NMR (100 MHz, DMSO) δ 152.89,
143.81, 137.15, 135.55, 133.23, 130.78, 130.21, 129.12, 126.61, 124.04, 123.99, 122.70, 18.52.
HRMS (EI): [M+H]+, found 306.0263. C12H13BrN3O requires 306.0242.
4.1.4. General procedure for the synthesis of urea derivatives U8-U23
U8-U23 were synthesized according to the reported literature procedure [21]. Briefly, they are
obtained by the reaction of 2-aminothiophenole and corresponding phenyl isocyanate derivatives
in dry toluene. The resulting residue obtained upon the completion of the reaction was purified by
crystallization from ethanol to afford the target compounds U8-U23. These compounds were
included in this study as their biological activities were not studied before.
1-(2-Mercaptophenyl)-3-phenylurea (U8) 1H NMR (400 MHz, DMSO) δ 9.36 (s, 1H), 8.39 (s,
1H), 7.96 (d, J = 8.0 Hz, 1H), 7.46 (d, J = 7.5 Hz, 2H), 7.38 – 7.18 (m, 4H), 7.07 – 6.78 (m, 2H).
1-(4-Fluorophenyl)-3-(2-mercaptophenyl)urea (U9) 1H NMR (400 MHz, DMSO) δ 9.39 (s, 1H),
8.37 (s, 1H), 7.94 (d, J = 8.0 Hz, 1H), 7.49–7.40 (m, 2H), 7.31 (dd, J = 16.5, 7.5 Hz, 2H), 7.14 (dd,
J = 12.0, 5.5Hz, 2H), 6.95 (td, J = 7.5, 1.0Hz, 1H).
1
1-(4-Cyanophenyl)-3-(2-mercaptophenyl)urea (U10) H NMR (400 MHz, DMSO) δ 9.81 (s,
1H), 8.55 (s, 1H), 7.90 (d, J = 8.0 Hz, 1H), 7.75 (d, J = 8.5 Hz, 2H), 7.62 (d, J = 8.5 Hz, 2H), 7.38
(d, J = 7.5 Hz, 1H), 7.29 (t, J = 7.0 Hz, 1H), 7.00 (t, J = 7.5 Hz, 1H).
1
1-(4-Bromophenyl)-3-(2-mercaptophenyl)urea (U11) H NMR (400 MHz, DMSO) δ 9.48 (s,
1H), 8.41 (s, 1H), 7.92 (d, J = 8.2 Hz, 1H), 7.49 – 7.39 (m, 4H), 7.34 (dd, J = 7.5, 1.0 Hz, 1H), 7.29
(t, J = 7.5Hz, 1H), 6.96 (td, J = 7.5, 1.0Hz, 1H).

1
1-(2-Mercaptophenyl)-3-(4-nitrophenyl)urea (U12) H NMR (400 MHz, DMSO) δ 10.02 (s,
1H), 8.60 (s, 1H), 8.20 (d, J = 9.0 Hz, 2H), 7.91 (d, J = 8.0 Hz, 1H), 7.67 (d, J = 9.0 Hz, 2H), 7.39
(d, J = 7.5 Hz, 1H), 7.30 (t, J = 7.5 Hz, 1H), 7.01 (t, J = 7.0 Hz, 1H).
1
1-(4-Chlorophenyl)-3-(2-mercaptophenyl)urea (U13) H NMR (400 MHz, DMSO) δ 9.49 (s,
1H), 8.41 (s, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.47 (d, J = 9.0 Hz, 2H), 7.38 – 7.24 (m, 4H), 6.96 (t, J
= 7.5 Hz, 1H).
1
1-(3-Cyanophenyl)-3-(2-mercaptophenyl)urea (U14) H NMR (400MHz, DMSO) δ 9.67 (s,
1H), 8.51 (s, 1H), 7.98 (s, 1H), 7.90 (d, J = 7.5 Hz, 1H), 7.63 (d, J = 7.5 Hz, 1H), 7.50 (t, J = 8.0
Hz, 1H), 7.44 (d, J = 7.5 Hz, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.30 (t, J = 7.5 Hz, 1H), 6.99 (t, J = 7.0
Hz, 1H).
1
1-(2-Mercaptophenyl)-3-(3,4,5-trimethoxyphenyl)urea (U15) H NMR (400 MHz, DMSO) δ
9.37 (s, 1H), 8.36 (s, 1H), 7.98 (d, J = 8.0 Hz, 1H), 7.30 (d, J = 7.5Hz, 2H), 6.95 (t, J = 7.5 Hz,
1H), 6.80 (s, 2H), 3.75 (s, 6H), 3.61 (s, 3H).
1-(2-Mercaptophenyl)-3-(4-methoxyphenyl)urea (U16) 1H NMR (400 MHz, DMSO) δ 9.20 (s,
1H), 8.31 (s, 1H), 7.97 (d, J = 8.5 Hz, 1H), 7.36 (d, J = 8.5 Hz, 2H), 7.30 (d, J = 7.5 Hz, 2H), 6.93
(t, J = 7.5 Hz, 1H), 6.88 (d, J = 8.5Hz, 2H), 3.71 (s, 3H).
1
1-(2-Mercaptophenyl)-3-(p-tolyl)urea (U17) H NMR (400 MHz, DMSO) δ 9.27 (s, 1H), 8.35
(s, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.42 – 7.18 (m, 4H), 7.10 (d, J = 7.5 Hz, 2H), 6.94 (t, J = 7.0 Hz,
1H), 2.25 (s, 3H).
1
1-(2-Mercaptophenyl)-3-(naphthalen-2-yl)urea (U18) H NMR (400 MHz, DMSO) δ 9.39 (s,
1H), 8.86 (s, 1H), 8.18 (d, J = 8.0 Hz, 1H), 8.00 (d, J = 8.0 Hz, 1H), 7.95 (t, J = 7.0 Hz, 2H), 7.67
(d, J = 8.0 Hz, 1H), 7.63 – 7.51 (m, 2H), 7.48 (t, J = 7.5 Hz, 1H), 7.42 (d, J = 7.5Hz, 1H), 7.32 (t,
J = 7.5 Hz, 1H), 7.00 (t, J = 7.5 Hz, 1H).

1
1-(2-Mercaptophenyl)-3-(2,3,4-trimethoxyphenyl)urea (U19) H NMR (400 MHz, DMSO) δ
8.87 (s, 1H), 8.73 (s, 1H), 7.79 (d, J = 8.0 Hz, 1H), 7.71 (d, J = 9.0 Hz, 1H), 7.41 (dd, J = 7.5, 1.0
Hz, 1H), 7.29 (t, J = 7.5 Hz, 1H), 7.01 (t, J = 7.5Hz, 1H), 6.73 (d, J = 9.0 Hz, 1H), 3.80 (s, 3H),
3.77 (s, 3H), 3.75 (s, 3H).
1-(3,4-Dimethoxyphenyl)-3-(2-mercaptophenyl)urea (U20) 1H NMR (400 MHz, DMSO) δ 9.26
(s, 1H), 8.32 (s, 1H), 7.98 (d, J = 8.0 Hz, 1H), 7.37 – 7.24 (m, 2H), 7.17 (d, J = 1.5 Hz, 1H), 7.03
– 6.73 (m, 3H), 3.73 (s, 3H), 3.70 (s, 3H).
1-(Tert-butyl)-3-(2-mercaptophenyl)urea (U21) 1H NMR (400 MHz, DMSO) δ 7.93 (d, J = 8.0
Hz, 1H), 7.90 (s, 1H), 7.25 (dd, J = 14.0, 7.0 Hz, 2H), 6.91 – 6.78 (m, 3H), 1.29 (s, 9H).
1-(2,4-Dimethoxyphenyl)-3-(2-mercaptophenyl)urea (U22) 1H NMR (400 MHz, DMSO) δ 8.79
(s, 1H), 8.61 (s, 1H), 7.82 (t, J = 9.0 Hz, 2H), 7.39 (d, J = 7.5 Hz, 1H), 7.28 (t, J = 7.5 Hz, 1H),
6.99 (t, J = 7.0 Hz, 1H), 6.61 (d, J = 2.5Hz, 1H), 6.47 (dd, J = 8.5, 2.5 Hz, 1H), 3.85 (s, 3H), 3.73
(s, 3H).
1
1-(2-Mercaptophenyl)-3-(o-tolyl)urea (U23) H NMR (400 MHz, DMSO) δ 9.31 (s, 1H), 8.38
(s, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.35 – 7.27 (m, 3H), 7.24 (d, J = 8.0 Hz, 1H), 7.17 (t, J = 7.5 Hz,
1H), 6.95 (t, J = 7.5Hz, 1H), 6.80 (d, J = 7.0 Hz, 1H), 2.28 (s, 3H).
4.1.5. General procedure for the synthesis of urea derivative U24
S-(2-(3-cyclohexylureido)phenyl) cyclohexylcarbamothioate (U24) was synthesized according to
the modified literature procedure [22]. 2-Aminothiophenol was reacted with two equimolar
amounts of cyclohexyl isocyanate solution in DMF. The resulting residue was purified by
1
crystallization from Et2O to afford U24. H NMR (400 MHz, DMSO-d6) δ 8.17 (d, J = 7.5 Hz,
1H), 8.11 (d, J = 8.3 Hz, 1H), 7.81 (s, 1H), 7.39 – 7.25 (m, 2H), 7.12 (d, J = 7.6 Hz, 1H), 6.90 (t,
J = 7.5 Hz, 1H), 3.54 – 3.39 (m, 2H), 1.88 – 1.47 (m, 10H), 1.38 – 0.96 (m, 10H).
4.2. Determination of Antimicrobial Activity by Broth Microdilution

The minimum inhibitory concentrations (MIC) of the compounds were determined by broth
microdilution method according to the Clinical and Laboratory Standards Institute (CLSI)
recommendations [23,24]. Antibacterial activity testing was carried out with Staphylococcus
aureus ATCC 29213, Enterococcus faecalis ATCC 29212, methicillin resistant Staphylococcus
aureus ATCC 43300, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 and
antifungal activity was tested against C. albicans ATCC 90028. The stock solutions of the
compounds were prepared by dissolving in dimethyl sulphoxide (DMSO). MIC was defined as the
lowest concentration of the samples with no bacterial growth.
4.3. Microplate Alamar Blue Assay for Mycobacterium tuberculosis
The minimum inhibitory concentrations (MIC) of the synthesized compounds were tested using in-
vitro MABA (Microplate Alamar Blue Assay) assay applying the reported protocol [17]. Assay
was performed in duplicate for each compound and the interpretations were compared to standard
TB drugs isoniazid, rifampicin and ethambutol. Bacterial growth was observed by colour change
and the minimum concentration that didn’t change the colour was taken as its MIC value.
4.4. In-vitro Biofilm Inhibition Assay
We used biofilm positive laboratory strain of P. aeruginosa PAO1 for biofilm experiments. In-
vitro biofilm inhibition assay was performed as previously described with slight modifications [26].
MIC of compounds U10 and U15 against PAO1 was determined with broth microdilution method
as described above. MIC, MIC/2, MIC/4 concentrations for each compound were used for crystal
violet staining microtiter biofilm formation assay. Briefly, each well was filled with Brain Heart
Infusion (BHI) broth and inoculated with bacterial suspension in a final volume of 2X106 cfu/mL.
After adding compounds in triplicate, plates were subsequently incubated at 37 °C for 24 h under
static conditions. The same amount of sterile BHI was added to top and bottom rows to avoid edge
effects and to serve as negative controls. After the incubation period, the supernatant was removed
and nonadherent cells were discarded by washing 3 times. The plates were dried and biofilms were
stained with 0.1% crystal violet for 20 min and stained biofilms were solubilized with 33% acetic

acid. Absorbances were measured at 570 nm. The OD570 values of P. aeruginosa PA01 alone
were used as positive control.
4.5. Molecular Docking
The crystal structure of LasR with the co-crystallized ligand of N-3-(oxododecanoyl)-L-
homoserine lactone (ODHL), was retrieved from Protein Data Bank under the PDB code 3IX3,
with the resolution of 1.40 Å [27]. The chemical formulas of the molecules (U10 and U15)
decreasing PAO1 biofilm formation were drawn using ChemDraw Ultra 12.0. These urea
derivatives were docked into the active pocket of LasR using AutoDock Vina 1.1 [28], integrated
into LigandScout 4.2 [29], with default parameters. The obtained poses were visually analyzed
using LigandScout 4.2. The molecular docking figures in this study were generated by Maestro
[30] and LigandScout 4.2.
4.6. In silico Prediction of Physicochemical Properties
The chemical formulas of the most active compounds were drawn in ChemDraw Ultra 12.0 and
saved as Simplified Molecule Input Entry System (SMILES) file. Molinspiration
(https://www.molinspiration.com) [31] was used for the calculation of molecular and drug-likeness
properties of the compounds.
Declaration of Interest
The authors declared no potential conflicts of interest.
Acknowledgements
ŞDD is indebted to the Scientific and Technological Research Council of Turkey (Grant No:
115Z261) and the Faculty of Pharmacy at Erciyes University for their financial support of this
work. The authors would like to acknowledge the contribution of the COST (European Cooperation
in Science and Technology) Action CA 15106. MGG is grateful to Prof. Dr. Gerhard Wolber, Freie
Universitat Berlin, for providing the license for LigandScout 4.2.

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