Synthesis, molecular docking studies, and antimicrobial evaluation of new structurally diverse ureas

Article abstract of DOI:10.1016/j.bioorg.2019.03.024

A series of new urea derivatives (3a-p) have been synthesized from readily available isocyanates and amines in good to high yields. All synthesized compounds were fully characterized using ¹H NMR, ¹³C NMR, IR, and mass spectrometry. Additionally, the structure of the compound (3n) was confirmed by single-crystal X-ray diffraction. Furthermore, all compounds were evaluated for antimicrobial activity against five bacteria and two fungi. Last but not the least, molecular docking studies with Candida albicans dihydropteroate synthetase were performed and the results are presented herein.

Full text of DOI:10.1016/j.bioorg.2019.03.024

Accepted Manuscript
Synthesis, Molecular Docking Studies, and Antimicrobial Evaluation of New
Structurally Diverse Ureas
Mahadev Patil, Anurag Noonikara Poyil, Shrinivas D. Joshi, Shivaputra A. Patil,
Siddappa A. Patil, Alejandro Bugarin
PII:
S0045-2068(19)30056-2
DOI:
https://doi.org/10.1016/j.bioorg.2019.03.024
YBIOO 2850
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
11 January 2019
9 February 2019
12 March 2019
Please cite this article as: M. Patil, A. Noonikara Poyil, S.D. Joshi, S.A. Patil, S.A. Patil, A. Bugarin, Synthesis,
Molecular Docking Studies, and Antimicrobial Evaluation of New Structurally Diverse Ureas, Bioorganic
Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.03.024
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Bioorganic Chemistry
Synthesis, Molecular Docking Studies, and Antimicrobial Evaluation of New
Structurally Diverse Ureas
a
b
c
d
a
Mahadev Patil , Anurag Noonikara Poyil , Shrinivas D. Joshi , Shivaputra A. Patil , Siddappa A. Patil *,
b
Alejandro Bugarin 
a
Centre for Nano & Material Sciences, Jain University, Jain Global Campus, Bangalore 562112, Karnataka, India.
Department of Chemistry & Biochemistry, University of Texas at Arlington, Arlington, TX 76019, USA.
b
c
Novel Drug Design and Discovery Laboratory, Department of Pharmaceutical Chemistry, S. E. T’s College of Pharmacy, Sangolly Rayanna Nagar, Dharwad
5
80 002, Karnataka, India.
d
Pharmaceutical Sciences Department, College of Pharmacy, Rosalind Franklin University of Medicine and Science, 3333 Green Bay Road, North Chicago, IL
6
0064, USA.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A series of new urea derivatives (3a-p) have been synthesized from readily available isocyanates
1
and amines in good to high yields. All synthesized compounds were fully characterized using H
13
NMR, C NMR, IR, and mass spectrometry. Additionally, the structure of the compound (3n)
was confirmed by single-crystal X-ray diffraction. Furthermore, all compounds were evaluated
for antimicrobial activity against five bacteria and two fungi. Last but not the least, molecular
docking studies with Candida albicans dihydropteroate synthetase were performed and the
results are presented herein.
Available online
Keywords:
Urea derivatives
Antimicrobial
2
019 Elsevier Ltd. All rights reserved.
Molecular docking
Synthesis and characterization
Single-crystal X-ray diffraction
—
——

Corresponding authors. Tel.: +1-817-272-9399: e-mail address: bugarin@uta.edu (A. B.) e-mail address: p.siddappa@Jainuniversity.ac.in

2
Bioorganic Chemistry
1. Introduction
Figure 1. Biologically active urea derivatives.
Wöhler synthesis of urea is one of the oldest synthetic
methods in which ammonium cyanate was converted into urea
Urea (I) is a simple and well-known naturally occurring
organic compound, which plays significant physiological and
biological roles in organisms [1]. The urea cycle in mammals
and other organisms helps to convert toxic ammonia into urea
[23]. Since then, several methods to prepare urea derivatives
have appeared in the literature. For example, it has been reported
that urea can be produced by the reaction of amines with carbon
monoxide and Pd/C [24], or amines with S,S-dimethyl
dithiocarbonate (DMDTC) as a phosgene substitute to yield N-
alkylureas [25], or amines with dialkyl carbonates using
zirconium(IV) catalyst and 2-hydroxypyridine [26], or amines
with phenyl 4,5-dichloro-6-oxopyridazine-1(6H)-carboxylate as
a carbonyl source [27], or amines in the presence of
stoichiometric quantities of trimethylaluminum [28], or amines
with ruthenium catalyst using methanol in closed vessel [29],
and also under solid phase urea synthesis, among others [30].
But most of these methods include the use of hazardous,
expensive organic reagents, solvents and catalysts. Some
methods using special metallic catalysts [31] have failed due to
the difficulties in recycling and recovery of the catalyst.
Currently, one the simplest and most efficient method for the
synthesis of urea is the reaction of primary or secondary amines
with isocyanates in organic solvents [32-34]. Therefore, our
synthetic approach to synthesize urea derivatives was to design
and develop a convenient method using commercially available
isocyanates and structurally diverse amines to produce our
desired ureas. This work also includes compounds’
characterization, molecular docking studies, and evaluation of
ureas’ antimicrobial activity.
[
2]. Urea derivatives have occupied key roles in medicinal
chemistry by regulating numerous pharmacological activities [3].
There are more than 6000 urea compounds registered in the Drug
Data Report (MDDR) database [4], which highlights the
importance of urea derivatives. In addition, the urea linkage is
often present in many pharmacologically active drugs [5,6].
Literature survey revealed that urea derivatives have
comprehensive spectrum of biological activities. For example, as
anti-tuberculosis, anti-viral, anti-HIV, HDL-elevating
antibacterial, analgesic, and even hold anticonvulsant properties
7-11]. Some of these urea derivatives have been used as enzyme
a
[
inhibitors, pseudo peptides, sedative-hypnotics, and anticancer
agents [12-14]. In addition, urea and its derivatives have been
employed as useful synthetic intermediates, agrochemicals, and
resin precursors [15]. Consequently, urea and its derivatives are
of a major interest for many organic and medicinal chemists that
seek to explore its various biological activities [16, 17].
In recent times, there is an increasing need for a new class of
antimicrobial agents due to the rise of new microbial strains that
have developed resistance to current chemotherapeutics and
antibiotics[18]. Urea derivatives bearing aryl groups belong to
the simplest chemicals used in medicine [3]. Urea derivatives
with important biological activity such as I: 1-(2-
hydroxyphenyl) (4-hydroxyphenyl) methyl) urea (antibacterial)
2. Results and discussion
[
19], II: 1-(1-arylimidazolidine-2-ylidene)-3-(4-chlorobenzyl)
urea derivative (antiviral) [20], III: aminoquinuride (antiseptic)
21], and IV: diflubenzuron (insecticide) [22] have been
2
.1. Chemistry
[
The synthetic route of urea derivatives containing alkyl/aryl
reported. Structures of these bioactive urea derivatives are
presented in Figure 1.
moieties (3a-p) is outlined in Scheme 1. The starting materials of
amine derivatives (1-amino-3,5-dimethyladmantane, 1-(bis (4-
fluorophenyl)
methyl)
piperazine,
4-(decyloxy)-3-
OH
O
ethoxybenzenamine
and
5,6-dimethoxy-2-(piperidin-4-
H2N
N
N
H
ylmethyl)-2,3-dihydroinden-1-one) were synthesized following
reported literature [35-38]. Urea derivatives containing alkyl/aryl
moieties (3a-p) were prepared in simple one-step method by the
reaction of isocyanates with amine derivatives in toluene as a
solvent, at 40-45 ºC. This method has the advantages of easier
work-up, mild reaction conditions and high yields (72 to 81%).
N
OH
O
Cl
III
N
NH2
H
II
1
-(2-Hydroxyphenyl)-(4-hydroxyphenyl)
1-(1-Arylimidazolidine-2-ylidene)-3-
(4-chlorobenzyl) urea derivative
methyl) urea
(
Antibacterial)
(Antiviral)
O
H2N
Me
NH2
I
Cl
Me
N
N
F
O
F
O
O
N
N
N
N
H
H
H
H
NH2
NH2
IV
V
Aminoquinuride (Antiseptic)
Diflubenzuron (Insecticide)

Scheme 1. Direct synthesis of urea derivatives.
2.2. Spectroscopic characterization
The newly synthesized urea derivatives containing alkyl/aryl
1
13
moieties (3a-p) were characterized using IR, H NMR, C NMR,
and mass spectroscopy. The spectral data of the newly
synthesized compounds (3a-p) are given in the experimental
section and are in accordance with the assigned structures of the
compounds. The FT-IR spectra for the urea derivatives were
-1
recorded in the region from 400 to 4000 cm . The bands at
-1
3
403-3272 cm in the infrared spectra of urea derivatives may
be assigned to the (NH) groups. The IR spectra of all urea
-1
derivatives show stretching frequencies around 1628-1707 cm ,
which correspond to the (C=O) groups. Weak to medium
-1
absorptions around 2900–2982 cm observed, corresponding to
the =C–H stretch of the aromatic ring. Formation of the
1
compounds was further established by using H NMR spectra.
1
3
The C NMR spectra provide additional support for the
1
3
structures of the compounds. In the C NMR spectra of urea
derivatives exhibit a signal characteristic of the (C=O) functional
groups between 155.6–152.2 ppm.

4
Bioorganic Chemistry
Table 1. Crystal data and structure refinement for 3n.
Table 2. Selected bond lengths [Å] and angles (°) for compound 3n.
Identification code
3n
Bond lengths [Å]
3n
Bond lengths [Å]
3n
Empirical formula
Molecular formula
Formula weight
Crystal system
Space group
Unit cell dimensions
C
C
24
H
H
27ClN
27ClN
2
2
O
O
4
4
, CH
, CH
2
Cl
Cl
2
24
2
2
O(1)-C(2)
O(1)-C(3)
O(2)-C(12)
O(3)-C(17)
1.439(2)
1.3581(18)
1.230(3)
1.2314(19)
1.371(2)
O(4)-C(21)
N(1)-C(11)
N(1)-C(12)
N(2)-C(12)
1.4287(19)
1.481(2)
1.388(2)
1.378(2)
1.415(2)
3n
527.9
Triclinic
P-1
a = 11.118(9)Å
b = 11.521(9)Å
c = 11.560(9)Å
α = 75.954(11)°
O(4)-C(20)
N(2)-C(13)
Bond angles [°]
C(2)-O(1)-C(3)
C(20)-O(4)-C(21)
C(11)-N(1)-C(12)
C(11)-N(1)-C(23)
C(12)-N(1)-C(23)
C(12)-N(2)-C(13)
O(1)-C(3)-C(4)
O(1)-C(3)-C(20)
O(2)-C(12)-N(1)
3n
Bond angles [°]
O(2)-C(12)-N(2)
N(1)-C(12)-N(2)
N(2)-C(13)-C(14) 116.29(17)
N(2)-C(13)-C(24) 124.30(14)
O(3)-C(17)-C(7)
O(3)-C(17)-C(18) 127.30(16)
O(4)-C(20)-C(3) 113.85(12)
O(4)-C(20)-C(19) 125.56(14)
C(3)-C(20)-C(19) 120.58(15)
117.97(13)
116.99(12)
115.02(17)
113.93(14)
123.22(13)
126.27(18)
124.56(14)
114.87(14)
122.46(15)
122.08(15)
115.43(17)

=79.076(10)
γ = 62.744(9)°
Volume
Z
3
124.49(14)
1
2
271.73(5) Å
Radiation type
Mo-K
Density (calculated)
Absorption coefficient
3
1
0
.3785 Mg/m
–1
.395 mm
F(000)
Crystal size
552
.24 x 0.23 x 0.09mm
2.02 to 30.21°.
–15<=h<=15, –16<=k<=16, –
6<=l<=16
3
0
Theta range for data collection
Index ranges
1
Diffractometer Bruker SMART
APEXII
Reflections collected
Independent reflections
Absorption correction Multi-scan
(SADABS; Bruker, 2014)
4505
4504 [R(int) = 0.0303]
Completeness to θmax
97 %
Max. and min. Transmission
Data / restraints / parameters
0.910and 0.965
7352 / 0 / 399
1.38
2
Goodness-of-fit on F
Final R indices [I>2sigma(I)]
R indices (all data)
R1 = 0.0385, wR2 = 0.0368
R1 = 0.0385, wR2 = 0.0368
Figure 2. ORTEP representation of the X-ray crystal structure of 3n
showing heteroatom labeling. 50% probability amplitude displacement
ellipsoids are shown. Additional data check CCDC–1878012.
-
3
-
3
-
3
ρmax,ρ min (e / Å )
0.38(e / Å ), -0.39(e / Å )
Computer programs: APEX2 and SAINT (Bruker, 2014) [39], SHELXS97
and SHELXL2013 (Sheldrick, 2008) [40], and JANA2006 [41].
2
.4. Antimicrobial activity
2
.3. X-ray crystallography
The antimicrobial activities of urea derivatives (3a-p) were
The crystal structure of the compound 3n has been
investigated against five bacterial [one gram-positive (S. aureus)
and four gram-negative (E. coli, P. aeruginosa, K. pneumonia,
and A. baumannii)] and two fungi (C. albicans and C.
neoformans) strains [42]. Colistin was used as positive inhibitor
standard for Gram-negative bacteria, Vancomycin was used for
Gram-positive bacteria, whereas Fluconazole was used as a
positive fungal inhibitor standard for both fungi. The results of
antimicrobial activity are tabulated in Table 3. In general, most
of the tested compounds displayed a certain degree of inhibition
of antimicrobial growth. From the results, compounds 3k and 3o
exhibited significant inhibition of antimicrobial growth towards
S. aureus. Compounds 3c and 3e showed moderate inhibition of
antimicrobial growth against E. coli. Compound 3h has shown a
moderate inhibition of antimicrobial growth against P.
aeruginosa. Compounds 3b-i and 3n exhibited good to high
inhibition of antimicrobial growth towards tested fungal C.
neoformans. Especially, compound 3b was observed as a lead
compound with promising inhibition of antimicrobial growth
against the tested pathogen C. neoformans.
determined. X-ray quality crystals of 3n were obtained by slow
evaporation of dichloromethane. The molecular structure of the
compound 3n is depicted in Figure 2. The crystal data and
refinement details of the compound 3n are found in Table 1,
whereas selected bond lengths and bond angles are depicted in
Table 2. Compound 3n crystallized in the triclinic space group
P-1 with two motifs in a unit cell. The X-ray structure of
compound 3n reveals that the molecule is nonplanar. The
crystals of compound 3n have organic solvent molecules
(
CH
bond distances in aromatic rings are in the normal range of 1.38–
.54 A˚, which is characteristic of delocalized aromatic rings.
2 2
Cl ) in the unit cell of the determined structure. The C–C
1
The C–C–C bond angles in aromatic rings are around 120 with
2
the variation being less than 3, which is characteristic of sp -
hybridized carbons. The compound 3n lies in three planes with
plane I [C(6) C(7) O(3) C(17) C(19) C(18) C(5) C(4) O(1) C(3)
C(20) and O(4)] making a dihedral angle of 84.46 and 19.82
with plane II [C(24) C(25) C(16) Cl(3) C(15) C(14) C(13) and
N(2)] and plane III [C(8) C(9) C(22) C(10) C(11) N(1) C(23)
and C(12)] whereas the plane II forms a dihedral angle of 77.14
with plane III. The molecular packing diagram shows two layers
of molecules, which are independently arranged in the unit cell.
Molecules forming each layer are not connected through
intermolecular hydrogen bonding.
Compound 3l showed moderate inhibition of antimicrobial
growth against fungal C. albicans. Moreover, all urea derivatives
(3a-p) demonstrated a poor inhibition of antimicrobial growth
against the germs K. pneumonia and A. baumannii.
Table 3. Antimicrobial activity of compounds (3a–p) with the concentration set at 32 g/mL in DMSO.

[a]
Percentage of inhibition of antibacterial and antifungal growth
Antibacterial activity
Antifungal activity
Compound
#)
Gram-positive
Staphylococcus
aureus
Gram-negative bacteria
(
Escherichia
coli
Pseudomonas
aeruginosa
Klebsiella
pneumoniae
Acinetobacter
baumannii
Candida
albicans
Cryptococcus
neoformans
3
a
-11.05.51
10.77.21
9.406.50
18.552.89
21.052.75
0.9011.45
1.350.77
-54.8518.31
-42.5519.30
1.803.25
2.852.89
2.895.79
3b
18.305.65
53.606.92
3
c
-5.5510.53
6.058.69
4.104.10
11.057.14
10.657.99
23.500.98
21.657.99
24.851.48
15.903.95
22.04.66
20.704.80
13.302.26
22.101.41
12.06.08
18.59.89
-2.0512.94
-10.21.13
-5.650.07
-0.6513.08
-5.600.00
-67.857.84
-56.7519.44
-33.9013.29
-70.1017.39
-54.7015.13
3.150.63
1.701.69
2.503.95
1.401.41
-0.402.40
31.202.54
34.305.09
36.307.21
30.552.05
33.603.39
3d
3e
3
f
3
3
g
h
11.356.43
20.700.70
21.300.98
17.954.03
17.405.09
13.201.13
17.654.31
21.854.87
11.452.33
14.153.88
23.501.97
5.552.47
12.754.59
7.052.19
1.750.63
16.950.49
19.600.98
17.01.97
15.752.61
4.2012.44
-5.9510.39
6.851.76
4.454.59
4.150.35
1.458.83
4.7512.51
7.052.75
8.002.40
-44.354.87
-67.1521.14
-4.450.07
0.452.05
-0.100.84
1.851.48
2.400.98
17.9526.09
2.700.98
1.352.61
3.103.11
1.255.16
29.554.87
25.451.76
0.650.07
-13.4014.84
3.754.03
14.1522.13
33.653.74
-11.454.17
-1.312.02
3
3
i
j
7.108.34
18.8511.95
25.2017.11
15.607.07
-1.604.10
12.109.05
3k
5.0031.11
3
l
-37.8515.06
-44.5532.73
-40.1517.46
10.7515.48
-26.252.47
3m
3
n
o
p
3
32.300.84
17.66.78
3
[a] Highest percentile of antibacterial/antifungal growth inhibition are highlighted
in bold. Data are expressed as the mean SD. SD = Standard Deviation
Table 4. Surflex docking score (kcal/mol) of the urea derivatives for Candida albicans (PDB ID: 1AI9)
a
b
c
d
e
f
g
Mol. #
C Score
Crash Score
Polar Score
D Score
PMF Score
G Score
Chem Score
Fluconazole
5.30
4.46
4.94
8.27
4.93
2.48
3.62
7.83
5.18
2.96
5.89
8.03
3.63
5.80
6.21
2.83
5.40
-2.19
-2.78
-2.51
-1.34
-1.77
-0.92
-0.34
-1.93
-1.34
-5.63
-0.88
-2.57
-1.89
-2.09
-3.77
-1.17
-0.48
1.85
1.78
3.05
1.71
0.85
1.16
1.07
1.87
2.11
1.18
1.03
1.63
0.04
2.13
2.52
1.61
1.45
-65.17
-9.24
-25.515
-24.118
-4.953
31.287
-8.431
9.698
-232.46
-176.254
-217.108
-214.756
-176.786
-83.324
-8.99
3a
3b
3c
3d
3e
-90.553
-106.697
-132.545
-102.067
-78.326
-77.092
-139.706
-93.096
-148.622
-109.082
-177.773
-98.471
-107.525
-146.593
-69.001
-97.416
-18.607
-28.428
-27.462
-22.703
-18.039
-17.351
-29.445
-22.665
-29.380
-20.835
-31.477
-23.738
-25.407
-30.057
-20.112
-19.500
3
f
-110.115
-242.152
-179.702
-230.697
-180.761
-297.270
-176.499
-240.611
-270.003
-137.682
-183.619
3
3
g
h
3.492
19.717
20.655
24.033
-0.555
14.101
-18.463
2.480
3
3
i
j
3
k
3
l
3
m
3
n
o
p
3
-24.673
7.259
3
a
CScore (Consensus Score) integrates a number of popular scoring functions for ranking the affinity of ligands bound to the active site of a receptor and reports
the output of total score.
b
Crash-score revealing the inappropriate penetration into the binding site. Crash scores close to 0 are favorable. Negative numbers indicate penetration.
Polar indicating the contribution of the polar interactions to the total score. The polar score may be useful for excluding docking results that make no hydrogen
c
bonds.
d
D-score for charge and van der Waals interactions between the protein and the ligand.
PMF-score indicating the Helmholtz free energies of interactions for protein-ligand atom pairs (Potential of Mean Force, PMF).
G-score showing hydrogen bonding, complex (ligand-protein), and internal (ligand-ligand) energies.
Chem-score points for H-bonding, lipophilic contact, and rotational entropy, along with an intercept term.
e
f
g
2
.5. Molecular docking studies
structural optimization. The docking study revealed that all the
compounds have shown very good docking score against
Candida albicans. Figure 3, represents the docked view of all
the synthesized compounds at the active site of the enzyme
Molecular docking was used to clarify the binding mode of
the compounds to elucidate new information for further

6
Bioorganic Chemistry
PDB ID 1AI9. As depicted in Figure 4, compound 3c makes
active site of the enzyme (PDB ID: 1AI9) and all the four-
hydrogen bonding interaction were raised from the nitrogen
atom of triazole ring with hydrogen atom of amino acid
residues SER78, LYS57& ARG56 (-N ---- H-SER78; H-
LYS57 & H-ARG56).
three hydrogen bonding interactions at the active site of the
enzyme (PDB ID: 1AI9), among those two interactions were
of hydrogen atoms of amino group oxygen of LYS22 (-NH----
--O-LYS22; 2.56 Å and 1.90 Å) and remaining another
hydrogen bonding interaction raised from the oxygen atom of
decyloxy group with hydrogen of GLU116 (O------H-
GLU116; 2.02 Å). As shown in Figure 5, compound 3k,
makes four hydrogen bonding interactions at the active site of
the enzyme (PDB ID: 1AI9), oxygen atom of ethoxy group
makes two hydrogen bonding interactions with hydrogens of
ARG79 (O-----H-ARG79; 2.71Å, 2.34Å), oxygen atom of
carbonyl group of urea makes hydrogen bonding interaction
with hydrogen of LYS57 (C=O------ H-LYS57, 2.11 Å) and
remaining another hydrogen bonding interaction raised from
the oxygen atom of decyloxy group with hydrogen of ARG79
Figure 7(A and B) represents the hydrophobic and
hydrophilic amino acids surrounded to the studied compounds
(3c and 3k) and also, we saw that the studied compounds have
shown the same type of interaction with amino acid residue as
that of Fluconazole standard drug. The comparative molecular
docking study of synthesized compounds and standard
Fluconazole drug highlighted that the synthesized compounds
exhibited high C-score value (Table 4). Fluconazole C-score
value 5.30 whereas the seven out of sixteen compounds
synthesized have higher C-score values than the Fluconazole.
Thus, from these studies, we can corroborate the experimental
findings, which suggest that urea derivatives may act by
inhibiting the dihydrofolate reductase enzyme.
(O------H-ARG79; 2.30 Å). As depicted in Figure 6,
fluconazole, makes four hydrogen bonding interaction at the
A
B
Figure
3.
Docked
view
of
all
the
compounds
at
the
active
site
of
the
enzyme
PDB
ID:
1AI9.
Figure 4. Docked view of compound 3c at the active site of the enzyme PDB ID: 1AI9.
A
C
B

A
C
B
Figure 5. Docked view of compound 3k at the binding site of the enzyme PDB ID: 1AI9.
A
B
C
B
Figure 6. Interaction of the Fluconazole at the binding site of the enzyme PDB ID: 1AI9.
Figure 7. A) Hydrophobic amino acids surrounded to compounds 3c (green color) and 3k (cyan color). B) Hydrophilic amino acids surrounded to compounds
3
c and 3k.
A B
A
B

8
Bioorganic Chemistry
3. Conclusion
To a solution of isocyanate (3.75 mmol) in toluene (5.0 mL) was
added a solution of amine (3.75 mmol) in toluene (2.0 mL). The
reaction mixture was heated at 40-45 °C for 30-60 min. Then,
cooled the reaction mixture to 25-30 °C and the resulting solids
were filtered, washed with toluene (2.0 mL). The wet solids were
then taken in toluene, stirred at 25-30 °C for about 30 min,
filtered and washed with toluene (2.0 mL) to get the crude urea
derivative. Finally, the crude urea derivative was purified by
silica gel column chromatography using DCM/MeOH (9:1).
In summary, we have designed and synthesized a new series
of urea derivatives containing alkyl/aryl moieties. The structures
1
of all the urea derivatives were confirmed through IR, H NMR,
1
3
C NMR, and mass spectroscopy. In addition, the molecular
structure of urea derivative 3n was unambiguously established
by single crystal X-ray diffraction analysis. The entire series of
urea derivatives were screened for antimicrobial properties.
Based on the biological evaluation results, the compounds 3b-i
and 3n showed promising inhibition of antimicrobial growth
towards the tested pathogenic strain C. neoformans. The present
synthetic protocol should, in principle, be applicable in
synthesizing various urea derivatives for use in the antimicrobial
activity. Finally, the molecular docking studies of the
synthesized compounds were carried out and the results of such
studies were reported. The docking study revealed that all the
urea derivatives showed very good docking score against
Candida albicans.
4.2.1.1 Synthesis of
4-(bis(4-fluorophenyl)methyl)-N-(p-
tolyl)piperazine-1-carboxamide (3a)
Compound (3a) was synthesized from 4-methyl phenyl
isocyanate (0.5 g, 3.75 mmol) and 1-(bis(4-fluorophenyl)
methyl) piperazine (1.08 g, 3.75 mmol) according to the general
1
procedure. White solid. Yield: 80% (1.26 g). H NMR (CDCl
3
,
5
=
00 MHz):  7.37-7.34 (m, 4H),7.20 (d, J = 8 Hz, 2H), 7.06 (d, J
8 Hz, 2H), 7.01-6.98 (m, 4H), 6.37 (s, 1H), 4.23 (s, 1H), 3.47-
13
3.45 (m, 4H), 2.39-2.36 (m, 4H), 2.28 (s, 3H). C NMR (CDCl ,
3
1
4
. Experimental section: Materials and methods
125 MHz):  161.9 (d, JC,F= 244.4 Hz), 155.2, 137.7, 136.2,
3
2
1
2
32.7, 129.3, 129.2 (d, JC,F= 8.4 Hz), 120.2, 115.5 (d, JC,F
1.5 Hz), 74.2, 51.4, 44.1, 20.7. IR (KBr): ῡ = 3277.2, 2965.4,
=
2
.1 General considerations
All chemicals including isocyanates were obtained from Sigma-
Aldrich chemical company and were used without further
purification. All solvents purchased were of analytical grade and
were used without further purification. All the reactions were
carried out under aerobic conditions in oven-dried glassware
with magnetic stirring. Heating was accomplished by either a
heating mantle or silicone oil bath. Reactions were monitored by
thin-layer chromatography (TLC) performed on 0.25 mm Merck
TLC silica gel plates, using UV light as a visualizing agent.
Purification of reaction products was carried out by flash column
chromatography using silica gel 60 (230-400 mesh). Yields refer
to the chromatographically pure adducts. Concentration in vacuo
refers to the removal of volatile solvent using a rotary evaporator
attached to a dry diaphragm pump (10-15 mm Hg) followed by
pumping to a constant weight with an oil pump (<300 mTorr).
2883.8, 2847.5, 1628.5, 1535.8, 1248.4, 1218.1 832.64, 807.76.
+
LC-MS for C25
25 2 3
H F N O: m/z = 422 [M+H] .
4.2.1.2 Synthesis of ethyl 2-(3-(p-tolyl)ureido)benzoate (3b)
Compound (3b) was synthesized from 4-methyl phenyl
isocyanate (0.5 g, 3.75 mmol) and ethyl 2-aminobenzoate (0.62
g, 3.75 mmol) according to the general procedure. White solid.
1
Yield: 76% (0.85 g). H NMR (DMSO-d , 500 MHz):  10.03 (s,
6
1H), 9.71 (s, 1H), 8.34 (d, J = 8.6 Hz, 1H), 7.94 (dd, J = 8.0, 1.7
Hz, 1H), 7.56 (td, J = 7.5, 1.7 Hz, 1H), 7.39 (d, J= 8.6 Hz, 2H),
7.10-7.04 (m, 3H), 4.35 (q, J = 7.5 Hz, 2H), 2.24 (s, 3H), 1.35 (t,
1
3
J = 7.5 Hz, 3H). C NMR (CDCl , 125 MHz):  168.5, 153.0,
3
142.6, 135.3, 134.3, 133.6, 130.6, 129.6, 121.2, 121.0, 119.9,
114.6, 61.2, 20.8, 14.1. IR (KBr): ῡ = 3272.2, 2982.6, 2938.9,
2918, 1704.5, 1665.9, 1549.4, 1513.2, 1253.9, 743.3. LC-MS for
1
+
H spectra were recorded on JEOL Eclipse Plus 500 (500 MHz),
C H N O : m/z = 299 [M+H] .
1
7
18
2
3
and are reported relative to CDCl
2
3
(δ 7.26) or DMSO-d6 (δ
.50). H NMR coupling constants (J) are reported in Hertz (Hz)
and multiplicities are indicated as follows: s (singlet), d
doublet), t (triplet), quint (quintet), m (multiplet). Proton-
1
4.2.1.3 Synthesis of 1-(4-(decyloxy)-3-ethoxyphenyl)-3-(p-
tolyl)urea (3c)
(
Compound (3c) was synthesized from 4-methyl phenyl
isocyanate (0.5 g, 3.75 mmol) and 4-(decyloxy)-3-
13
decoupled C NMR spectra were recorded on JEOL Eclipse
Plus 500 (125 MHz) and reported relative to CDCl (δ 77.00) or
3
ethoxybenzenamine (1.10 g, 3.75 mmol) according to the general
1
DMSO-d6 (δ 39.52). X-ray diffraction data for compound (3n)
was collected using Mo-Kα radiation and a Bruker SMART
APEXII diffractometer [40]. The structure was solved by the
direct method using SHELXS-97 and refined by full-matrix
least-squares on F2 for all data using SHELXL-97at 100 K [40].
An analytical absorption correction based on the shape of the
crystal was performed. All hydrogen atoms were added at
procedure. White solid. Yield: 80.5% (1.28 g). H NMR (CDCl
3
,
5
7
2
00 MHz):  7.5 (br, 1H), 7.09 (d, J= 8.0 Hz, 2H), 6.97 (d, J =
.5 Hz, 2H), 6.93 (s, 1H), 6.69-6.63 (m, 2H), 3.91-3.87 (m, 4H),
.23 (s, 3H), 1.78-1.73 (m, 2H), 1.41-1.26 (m, 18H), 0.90-0.87
13
(m, 3H). C NMR (CDCl
3
, 125 MHz):  154.8, 149.1, 145.6,
1
6
1
1
35.3, 133.3, 131.3, 129.5, 120.9, 114.2, 113.8, 108.1, 69.7,
4.5, 31.9, 29.6, 29.5, 29.4, 29.3, 29.3, 26.0, 22.6, 20.7, 14.7,
4.1. IR (KBr): ῡ = 3293.2, 2916.9, 2851.4, 1640.5, 1516.9,
calculated positions and refined using
a riding model.
Anisotropic thermal displacement parameters were used for all
non-hydrogen atoms. Further details about the data collection
and reliability factors are listed in Table 1. CCDC–1878012 (for
38 2 3
262.9, 1231.7, 805.1. LC-MS for C26H N O : m/z = 427
+
[M+H] .
3
n), contain the supplementary crystallographic data for this
paper. These data can be obtained free of charge from the
Cambridge Crystallographic DataCentre via
http://www.ccdc.cam.ac.uk/data_request/cif.
4.2.1.4 Synthesis of 1-((3R,5S,7r)-3,5-dimethyladamantan-1-yl)-
3-(p-tolyl)urea (3d)
Compound (3d) was synthesized from4-methyl phenyl
isocyanate (0.5 g, 3.75 mmol) and 1-amino-3,5-dimethyl
adamantane (0.67 g, 3.75 mmol) according to the general
4.2 Syntheses
1
procedure. White solid. Yield: 77% (0.92 g). H NMR (CDCl
3
,
5
2
00 MHz):  7.16 (d, J = 8.0 Hz, 2H), 7.05 (d, J = 8.0 Hz, 2H),
4
.2.1 General experimental procedure for the synthesis of urea
.27 (s, 3H), 2.10-2.08 (m, 1H), 1.79 (s, 2H), 1.61 (d, J = 12.0
derivatives
Hz, 2H), 1.57 (d, J = 11.5 Hz, 2H), 1.33-1.23 (m, 4H), 1.10 (s,

1
3
2
1
2
1
H), 0.80 (s, 6H). C NMR (CDCl
3
, 125 MHz):  155.6, 136.0,
2899.7, 2861.8, 1648.8, 1562.6, 1493.8, 1275.4, 1227.7, 833.9.
+
33.1, 129.7, 121.1, 52.9, 50.5, 48.1, 42.6, 40.6, 32.4, 30.1, 30.0,
0.8. IR (KBr): ῡ = 3330.5, 2945.5, 2898.9, 2861.4, 1647.2,
561.6, 1516.6, 1231, 816.8. LC-MS for C20
LC-MS for C19
H25ClN
2
O: m/z = 333 [M+H] .
4.2.1.9 Synthesis of
4-(bis(4-fluorophenyl)methyl)-N-(4-
28
H N
2
O: m/z = 313
+
fluorophenyl)piperazine-1-carboxamide (3i)
[M+H] .
Compound (3i) was synthesized from 4-fluoro phenyl isocyanate
4
.2.1.5 Synthesis of
4-(bis(4-fluorophenyl)methyl)-N-(4-
(0.5 g, 3.64 mmol) and 1-(bis (4-fluorophenyl) methyl)
chlorophenyl)piperazine-1-carboxamide (3e)
piperazine (0.30 g, 1.05 mmol) according to the general
1
Compound (3e) was synthesized from 4-chloro phenyl
isocyanate (0.5 g, 3.25 mmol) and 1-(bis (4-fluorophenyl)
methyl) piperazine (0.93 g, 3.25 mmol) according to the general
3
procedure. White solid. Yield: 76% (1.17 g). H NMR (CDCl ,
500 MHz):  7.36-7.33 (m, 4H), 7.30-7.24 (m, 2H), 7.01- 6.92
(m, 6H), 6.39 (s, 1H), 4.24 (s, 1H), 3.46 (t, J = 4.5 Hz, 4H), 2.38
1
13
procedure. White solid. Yield: 78% (1.43 g). H NMR (DMSO-
(t, J= 4.5 Hz, 4H). C NMR (CDCl
3
, 125 MHz):  161.9 (d,
1
1
d
2
4
1
8
5
1
6
, 500 MHz):  8.6 (s, 1H), 7.47-7.43 (m, 6H), 7.27-7.25 (m,
H), 7.14 (t, J = 9.0 Hz, 4H), 4.43 (s,1H), 3.45 (t, J = 4.5 Hz,
H), 2.29 (t, J = 4.5 Hz, 4H). C NMR (DMSO-d
JC,F= 245.9 Hz), 158.9 (d, JC,F= 242.3 Hz), 155.1, 137.6, 134.8,
3
3
2
129.2 (d, JC,F= 7.2 Hz), 122.0 (d, JC,F= 8.4 Hz), 115.6 (d, JC,F
=
1
3
2
6
, 125 MHz): 
21.6 Hz), 115.4 (d, JC,F= 21.6 Hz), 74.2, 51.3, 44.1. IR (KBr): ῡ
1
3
61.1 (d, JC,F= 242.0 Hz), 154.7, 139.5, 138.4, 129.5 (d, JC,F
=
= 3322.2, 2911.7, 2884.7, 2820.9, 1632.6, 1539.3, 1507.9,
2
.4 Hz), 128.1, 125.2, 121.0, 115.4 (d, JC,F= 21.5 Hz), 72.7,
1.2, 43.8. IR (KBr): ῡ = 3310.7, 2954.6, 2906.1, 2790, 1636.4,
591.5, 1505.3, 1236.8, 1213.7, 720.4. LC-MS for
1252.9, 1222.4, 830.9. LC-MS for C24
[M+H] .
22 3
H F N
3
O: m/z = 426
+
+
4.2.1.10 Synthesis of ethyl 2-(3-(4-fluorophenyl)ureido)benzoate
3j)
C
24 2
H22ClF N
3
O: m/z = 442 [M+H] .
(
4
.2.1.6 Synthesis of ethyl 2-(3-(4-chlorophenyl)ureido)benzoate
Compound (3j) was synthesized from 4-fluoro phenyl isocyanate
0.5 g, 3.64 mmol) and ethyl 2-aminobenzoate (0.60 g, 3.64
mmol) according to the general procedure. White solid. Yield:
(3f)
(
Compound (3f) was synthesized from 4-chloro phenyl
isocyanate (0.5 g, 3.25 mmol) and ethyl 2-aminobenzoate (0.53
g, 3.25 mmol) according to the general procedure. White solid.
Yield: 74% (0.76 g). H NMR (CDCl
1
3
77% (0.84 g). H NMR (CDCl , 500 MHz):  10.58 (s, 1H), 8.51
(dd, J = 8.5, 1.0 Hz, 1H), 8.00 (dd, J = 8.0, 2.0 Hz, 1H), 7.50 (td,
J = 7.0, 1.5 Hz, 1H), 7.40-7.37 (m, 2H), 7.03-6.97 (m, 4H), 4.31
1
3
, 500 MHz):  10.62 (s,
1
3
1H), 8.50 (d, J = 8.5 Hz, 1H), 8.01 (d, J = 8.0 Hz, 1H), 7.51 (t, J
(q, J = 6.5 Hz, 2H), 1.38 (t, J = 7.5 Hz, 3H). C NMR (CDCl ,
3
1
=
8.0 Hz, 1H), 7.39 (d, J = 8.5 Hz, 2H), 7.27 (d, J = 8.0 Hz, 2H),
125 MHz):  168.6, 159.5 (d, JC,F= 243.5 Hz), 152.9, 142.5,
134.5, 133.8, 130.7, 123.0 (d, JC,F= 7.2 Hz), 121.2, 119.8, 115.7
(d, JC,F= 22.8 Hz), 114.5, 61.3, 14.1. IR (KBr): ῡ = 3292.6,
3
7
1
1
1
2
.01 (t, J= 8.0 Hz, 1H), 6.98 (s, 1H), 4.33 (q, J = 7.5 Hz, 2H),
.39 (t, J = 7.0 Hz, 3H). C NMR (CDCl
52.4, 142.4, 136.8, 134.5, 130.8, 129.0, 128.7, 121.5, 121.3,
19.8, 114.5, 61.4, 14.1. IR (KBr): ῡ =3323.5, 2945.7, 2899.5,
1
3
2
3
, 125 MHz):  168.7,
2979.5, 2931.3, 1707.9, 1657.4, 1557.7, 1510.2, 1250.7, 1161.8,
+
745.3. LC-MS for C16
H15FN
2
O
3
: m/z = 303 [M+H] .
861.7, 1649, 1562.7, 1493.8, 1304.9, 1227.6, 1194.2, 830.1.
+
4.2.1.11 Synthesis of 1-(4-(decyloxy)-3-ethoxyphenyl)-3-(4-
fluorophenyl)urea (3k)
LC-MS for C16
H15ClN
2
O
3
: m/z = 319 [M+H] .
4
.2.1.7 Synthesis of 1-(4-chlorophenyl)-3-(4-(decyloxy)-3-
Compound (3k) was synthesized from4-fluoro phenyl isocyanate
ethoxyphenyl)urea (3g)
(0.5 g, 3.64 mmol) and 4-(decyloxy)-3-ethoxybenzenamine (1.0
Compound (3g) was synthesized from 4-chloro phenyl
isocyanate (0.5 g, 3.25 mmol) and 4-(decyloxy)-3-
ethoxybenzenamine (0.95 g, 3.25 mmol) according to the general
g, 3.64 mmol) according to the general procedure. White solid.
1
Yield: 72% (1.13 g). H NMR (DMSO-d , 500 MHz):  8.59 (s,
6
1H), 8.45 (s, 1H), 7.45-7.42 (m, 2H), 7.18 (d, J = 2.5 Hz, 1H),
7.12-7.08 (m, 2H), 6.86- 6.83 (m, 2H), 3.96 (q, J = 7.0 Hz, 2H),
3.88 (t, J = 6.0 Hz, 2H), 1.67-1.64 (m, 2H), 1.42-1.38 (m, 2H),
1
procedure. White solid. Yield: 78% (1.13 g). H NMR (DMSO-
d
6
, 500 MHz):  8.71 (s, 1H), 8.50 (s, 1H), 7.47 (dd, J = 6.8, 2.5
13
Hz, 2H), 7.30 (dd, J = 6.8, 2.5 Hz, 2H), 7.19 (d, J = 2.0 Hz, 1H),
1.33-1.25 (m, 15H), 0.85 (t, J= 7.0 Hz, 3H). C NMR (DMSO-
1
6
2
1
d
1
2
2
8
.86-6.15 (m, 2H), 3.98 (q, J = 7.0 Hz, 2H), 3.88 (t, J = 6.0 Hz,
H), 1.66 (quint, J = 7.0 Hz, 2H), 1.40 (quint, J = 7.5 Hz, 2H),
.33-1.25 (m, 15H), 0.85 (t, J = 7.0 Hz, 3H). C NMR (DMSO-
d , 125 MHz):  159.9 (d, JC,F= 246.7 Hz), 152.7, 148.5, 143.6,
6
3
2
136.2, 133.5, 119.9 (d, JC,F= 7.2 Hz), 115.2 (d, JC,F= 21.6 Hz),
114.9, 110.5, 106.6, 69.0, 63.8, 31.3, 29.1, 29.0, 28.9, 28.8, 28.7,
25.5, 22.1, 14.8, 14.0. IR (KBr): ῡ = 3296.4, 2946.5, 2921.3,
13
6
, 125 MHz):  152.5, 148.5, 143.7, 138.9, 133.3, 128.6, 125.1,
19.6, 114.8, 110.6, 105.6, 69.0, 63.8, 31.3, 29.1, 29.0, 28.9,
2849, 1628.8, 1562.5, 1517.1, 1265, 1233.1, 829.6.LC-MS for
+
8.8, 28.7, 25.5, 22.1, 14.8, 14.0. IR (KBr): ῡ = 3358.1, 3254.5,
C
25
H35FN
2
O
3
: m/z = 431 [M+H] .
951.5, 2920.3, 2848.1, 1647.5, 1554.8, 1517, 1262.3, 1231.7,
+
4.2.1.12 Synthesis of 1-(3,4-dichlorophenyl)-3-((3R,5S,7r)-3,5-
dimethyladamantan-1-yl)urea (3l)
65.7. LC-MS for C25
H35ClN
2
O
3
: m/z = 448 [M+H] .
4
.2.1.8 Synthesis of 1-(4-chlorophenyl)-3-((3R,5S,7r)-3,5-
Compound (3l) was synthesized from 3,4-dichloro phenyl
isocyanate (0.5 g, 2.65 mmol) and 1-amino-3, 5-dimethyl
dimethyladamantan-1-yl)urea (3h)
Compound (3h) was synthesized from 4-chloro phenyl
isocyanate (0.5 g, 3.75 mmol) and 1-amino-3,5-dimethyl
adamantane (0.58 g, 3.75 mmol) according to the general
adamantane (0.47 g, 2.65 mmol) according to the general
1
procedure. White solid. Yield:73% (0.71g). H NMR (DMSO-d
6
,
500 MHz):  8.54 (s, 1H), 7.85 (d, J = 3.0 Hz, 1H), 7.39 (d, J =
8.5 Hz, 1H), 7.11 (dd, J = 9.0, 2.5 Hz, 1H), 5.98 (s, 1H), 2.08-
2.06 (m, 1H), 1.74 (d, J = 2.0 Hz, 2H), 1.56 (s, 4H), 1.32-1.22
1
procedure. White solid. Yield: 72% (0.78 g). H NMR (DMSO-
d
=
6
, 500 MHz):  8.36 (s, 1H), 7.36 (d, J = 8.5 Hz, 2H), 7.22 (d, J
9.0 Hz, 2H), 5.90 (s, 1H), 2.08-2.06 (m, 1H), 1.75 (d, J = 2.0
1
3
6
(m, 4H), 1.09 (s, 2H), 0.80 (s, 6H). C NMR (DMSO-d , 125
Hz, 2H), 1.57 (s, 4H), 1.31 (d, J = 11.5 Hz, 2H), 1.24 (d, J = 12.0
MHz):  153.6, 140.8, 131.0, 130.3, 121.9, 118.4, 117.4, 51.7,
50.2, 47.5, 42.3, 40.0, 31.9, 30.0, 29.6. IR (KBr): ῡ = 3331,
13
Hz, 2H), 1.10 (s, 2H), 0.81 (s, 6H). C NMR (DMSO-d
MHz):  153.8, 139.6, 128.4, 124.1, 118.8, 51.5, 50.3, 47.6,
2.3, 40.1, 31.9, 30.1, 29.6. IR (KBr): ῡ = 3323.9, 2945.7,
6
, 125
2943.5, 2901.5, 2862.9, 1648, 1582, 1555.8, 1476.7, 1275.9,
+
4
1227.5, 859. LC-MS for C19
2
H24Cl N
2
O: m/z = 368 [M+H] .

1
0
Bioorganic Chemistry
4
.2.1.13 Synthesis of 4-((5,6-dimethoxy-1-oxo-2,3-dihydro-
H-inden-2-yl)methyl)-N-(p-tolyl)piperidine-1-carboxamide
3m)
The crystal structures used, Candida albicans dihydrofolate
reductase (PDB ID: 1AI9, X-Ray diffraction, 1.85 Å), for the
docking study was obtained from the Protein Data Bank. The
proteins were prepared for docking by adding polar hydrogen
atom with Gasteiger-Huckel charges and water molecules were
removed. The 3D structure of the ligands was generated by the
SKETCH module implemented in the SYBYL program (Tripos
Inc., St. Louis, USA) and its energy-minimized conformation
was obtained with the help of the Tripos force field using
Gasteiger-Huckel [43] charges and molecular docking was
performed with Surflex-Dock program that is interfaced with
Sybyl-X 2.0 [44] and other miscellaneous parameters were
assigned with the default values given by the software.
1
(
Compound (3m) was synthesized from4-methyl phenyl
isocyanate (0.5 g, 3.75 mmol) and 5, 6-dimethoxy-2-(piperidin-
4
-ylmethyl)-2,3-dihydroinden-1-one (1.08 g, 3.75 mmol)
according to the general procedure. White solid. Yield: 79%
1
(1.25 g). H NMR (CDCl
3
, 500 MHz):  7.23 (d, J = 8.0 Hz,
2
1
1
1
H), 7.14 (s, 1H),7.05 (d, J = 8.0 Hz, 2H), 6.85 (s, 1H), 6.78 (br,
H), 4.04-4.07 (m, 2H), 3.94 (s, 3H), 3.87 (s, 3H), 3.27-3.22 (m,
H), 2.89-2.84 (m, 2H), 2.71-2.67 (m, 2H), 2.26 (s, 3H), 1.92-
.87 (m, 1H), 1.80-1.75 (m, 3H), 1.38-1.32 (m, 1H), 1.29-1.24
13
(
1
5
m, 2H). C NMR (CDCl
3
, 125 MHz):  207.4, 155.5, 155.0,
49.4, 148.6, 136.4, 132.4, 129.2, 129.0, 120.1, 107.3, 104.2,
6.1, 56.0, 45.0, 44.7, 38.5, 34.3, 33.3, 32.4, 31.4, 20.6. IR
4.4 Antimicrobial studies
Samples were prepared in DMSO and water to a final testing
concentration of 32 μg/mL or 20 μM (unless otherwise indicated
in the data sheet), in 384-well, non-binding surface plate (NBS)
for each bacterial/fungal strain, and in duplicate (n=2), and
keeping the final DMSO concentration to a maximum of 1%
DMSO [45-49]. All the sample preparation for antimicrobial
studies where done using liquid handling robots.
(KBr): ῡ = 3403.7, 2932.7, 2841.8, 1684.5, 1659.4, 1532.5,
+
1259.2, 1215.8. LC-MS for C25
30 2 4
H N O : m/z = 423 [M+H] .
4.2.1.14 Synthesis of N-(4-chlorophenyl)-4-((5,6-dimethoxy-1-
oxo-2,3-dihydro-1H-inden-2-yl)methyl)piperidine-1-
carboxamide (3n)
Compound (3n) was synthesized from 4-chloro phenyl
isocyanate (0.5 g, 3.25 mmol) and 5, 6-dimethoxy-2-(piperidin-
Antimicrobial Assay
4
-ylmethyl)-2, 3-dihydroinden-1-one (0.94 g, 3.25 mmol)
Primary antimicrobial screening study was performed by whole
cell growth inhibition assays, using compounds (3a-p) at a single
concentration, in duplicate (n=2). The inhibition of growth is
measured against five bacteria: Escherichia coli (E. coli) ATCC
according to the general procedure. White solid. Yield: 74%
1
(1.06 g). H NMR (CDCl
3
, 500 MHz):  7.31 (d, J = 8.0 Hz,
2
1
1
1
H), 7.19 (d, J = 7.5 Hz, 2H), 7.13 (s, 1H), 6.85 (s, 1H), 6.79 (s,
H), 4.09-4.06 (m, 2H), 3.95 (s, 3H), 3.87 (s, 3H), 3.25 (dd, J =
8.0, 8.0 Hz, 1H), 2.86 (t, J = 12.5 Hz, 2H), 2.70-2.68 (m, 2H),
.90-1.86 (m, 1H), 1.80-1.74 (m, 3H), 1.38-1.32 (m, 1H), 1.24(t,
25922, Klebsiella pneumoniae (K. pneumoniae) ATCC 700603,
Acinetobacter baumannii (A. Baumannii) ATCC 19606,
Pseudomonas aeruginosa (P. Aeruginosa) ATCC 27853) and
Staphylococcus aureus (S. aureus) ATCC 43300, and two fungi:
Candida albicans (C. albicans) ATCC 90028 and Cryptococcus
neoformans (C. Neoformans) ATCC 208821.[50]
1
3
J = 12.5 Hz, 2H). C NMR (CDCl
3
, 125 MHz):  207.4, 155.6,
54.7, 149.5, 148.7, 137.9, 129.0, 128.6, 127.6, 121.1, 107.3,
04.3, 56.2, 56.0, 45.0, 44.5, 38.5, 34.3, 33.3, 32.5, 31.6. IR
1
1
(KBr): ῡ = 3310.8, 2922.3, 2846, 1689.7, 1635.1, 1591.6,
1
4
501.8, 1266.2, 1244.1, 827.8. LC-MS for C24
43 [M+H] .
H
27ClN
2
O
4
: m/z =
Procedure
+
All bacteria were cultured in Cation-adjusted Mueller Hinton
broth (CAMHB) at 37 ºC overnight. A sample of each culture
was then diluted 40-fold in fresh broth and incubated at 37 ºC for
4
.2.1.15
Synthesis
of
1-(4-bromophenyl)-3-(3,4-
dichlorophenyl)urea (3o)
1
.5-3 h. The resultant mid-log phase cultures were diluted
Compound (3o) was synthesized from 3,4-dichloro phenyl
isocyanate (0.5 g, 2.65 mmol) and 4-bromobenzenamine (0.45 g,
(CFU/mL measured by OD600), then added to each well of the
5
compound containing plates, giving a cell density of 5x10
CFU/mL and a total volume of 50 μL. All the plates were
covered and incubated at 37 ºC for 18 h without shaking.
2
.65 mmol) according to the general procedure. White solid.
1
Yield: 81% (0.77g). H NMR (DMSO-d
6
, 500 MHz):  9.02 (s,
1
1
H), 8.95 (s, 1H), 7.86 (d, J = 2.0 Hz, 1H), 7.50 (d, J = 9.0 Hz,
H), 7.46-7.42 (m, 4H), 7.33 (dd, J = 8.8, 2.5 Hz, 1H). C NMR
13
Analysis
(
1
2
DMSO-d6, 125 MHz):  152.2, 139.8, 138.7, 131.5, 131.1,
Inhibition of bacterial growth was determined measuring
absorbance at 600 nm (OD600), using a Tecan M1000 Pro
monochromator plate reader. The percentage of growth
inhibition was calculated for each well, using the negative
control (media only) and positive control (bacteria without
inhibitors) on the same plate as references. The significance of
the inhibition values was determined by modified Z-scores,
calculated using the median and MAD of the samples (no
controls) on the same plate. Samples with inhibition value above
80% and Z-Score above 2.5 for either replicate (n=2 on different
plates) were classed as actives. Samples with inhibition values
between 50 - 80% and Z-Score above 2.5 for either replicate
(n=2 on different plates) were classed as partial actives. Samples
with inhibition values between 50 - 80% and Z-Score above 2.5
for either replicate (n=2 on different plates) were classed as
partial actives.
30.6, 123.3, 120.4, 119.4, 118.4, 113.7. IR (KBr): ῡ = 3295.6
900, 2850, 1636.2, 1585.8, 1552.6, 1279.9, 1231.9, 816.8. LC-
+
MS for C13
H
9
BrCl
2
N
2
O: m/z = 361 [M+H] .
4.2.1.16
Synthesis
of
1-(3,4-dichlorophenyl)-3-(2,2,2-
trifluoroethyl)urea (3p)
Compound (3p) was synthesized from 3,4-dichloro phenyl
isocyanate (0.5 g, 2.65 mmol) and 2, 2, 2-trifluoroethanamine
(
0.26 g, 2.65 mmol) according to the general procedure. White
1
solid. Yield: 79% (0.60 g). H NMR (DMSO-d
9
7
6
, 500 MHz): 
.08 (s, 1H), 7.83 (d, J = 3.0 Hz, 1H), 7.47 (d, J = 9.0 Hz, 1H),
.29 (dd, J = 9.0, 2 Hz, 1H), 6.91 (t, J = 6.5 Hz, 1H), 3.95-3.88
1
3
(m, 2H). C NMR (DMSO-d
6
, 125 MHz):  154.6, 140.1, 131.0,
1
1
30.5, 125.0 (q, JC,F= 277.9 Hz), 123.0, 119.1, 118.1, 40.3 (q,
2
J
C,F= 33.4 Hz). IR (KBr): ῡ = 3330.6, 2900, 1651.4, 1589.9,
1
C
571.9, 1289, 1235.5, 1154.3, 832.95. LC-MS for
+
9
H
7
2 3
Cl F N
2
O: m/z = 288 [M+H] .
Antifungal Assay
Procedure
4.3 Docking simulations

Fungi strains were cultured for 3 days on Yeast Extract-Peptone
[6] P.C. Singh, S.K. Ballas, Emerging drugs for sickle cell anemia,
Expert Opin. Emerging Drugs, 20 (2015) 47-61.
6
Dextrose (YPD) agar at 30 ºC. A yeast suspension of 1 x 10 to 5
6
[7] J.D. Bloom, R.G. Dushin, K.J. Curran, F. Donahue, E.B. Norton,
x 10 CFU/mL (as determined by OD530) was prepared from
E. Terefenko, T.R. Jones, A.A. Ross, B. Feld, S.A. Lang, M.J.
DiGrandi, Thiourea inhibitors of herpes viruses. Part 2: N-Benzyl-
N'-arylthiourea inhibitors of CMV, Bioorg. Med. Chem. Lett., 14
five colonies. The suspension was subsequently diluted and
added to each well of the compound-containing plates giving a
3
final cell density of fungi suspension of 2.5 x 10 CFU/mL and a
(
2004) 3401-3406.
total volume of 50 μL. All plates were covered and incubated at
[8] J. Lee, J. Lee, M. Kang, M. Shin, J.M. Kim, S.U. Kang, J.O.
35 ºC for 24 h without shaking.
Lim, H.K. Choi, Y.G. Suh, H.G. Park, U. Oh, H.D. Kim, Y.H. Park,
H.J. Ha, Y.H. Kim, A. Toth, Y. Wang, R. Tran, L.V. Pearce, D.J.
Lundberg, P.M. Blumberg, N-(3-acyloxy-2-benzylpropyl)-N'-[4-
Analysis
Growth inhibition of C. albicans was determined measuring
absorbance at 530 nm (OD530), while the growth inhibition of
C. neoformans was determined measuring the difference in
absorbance between 600 and 570 nm (OD600-570), after the
addition of resazurin (0.001% final concentration) and
incubation at 35 ºC for additional 2 h. The absorbance was
measured using a Biotek Synergy HTX plate reader. The
percentage of growth inhibition was calculated for each well,
using the negative control (media only) and positive control
(
methylsulfonylamino)benzyl]thiourea analogues: novel potent and
high affinity antagonists and partial antagonists of the vanilloid
receptor, J. Med. Chem., 46 (2003) 3116-3126.
[9] M. Struga, J. Kossakowski, E. Kedzierska, S. Fidecka, J.
Stefanska, Synthesis and pharmacological activity of urea and
thiourea derivatives of 4-azatricyclo[5.2.2.0(2,6)]undec-8-ene-3,5-
dione, Chem. Pharm. Bull., 55 (2007) 796-799.
[10] R.S. Upadhayaya, G.M. Kulkarni, N.R. Vasireddy, J.K.
Vandavasi, S.S. Dixit, V. Sharma, J. Chattopadhyaya, Design,
synthesis and biological evaluation of novel triazole, urea and
thiourea derivatives of quinoline against Mycobacterium
tuberculosis, Bioorg. Med. Chem., 17 (2009) 4681-4692.
(fungi without inhibitors) on the same plate. The significance of
the inhibition values was determined by modified Z-scores,
calculated using the median and MAD of the samples (no
controls) on the same plate. Samples with inhibition value above
[11] T.K. Venkatachalam, C. Mao, F.M. Uckun, Effect of
stereochemistry on the anti-HIV activity of chiral thiourea
compounds, Bioorg. Med. Chem., 12 (2004) 4275-4284.
80% and Z-Score above 2.5 for either replicate (n=2 on different
plates) were classed as actives. Samples with inhibition values
between 50 - 80% and Z-Score above 2.5 for either replicate
[12] D.J. Kempf, K.C. Marsh, D.A. Paul, M.F. Knigge, D.W.
Norbeck, W.E. Kohlbrenner, L. Codacovi, S. Vasavanonda, P.
Bryant, X.C. Wang, et al., Antiviral and pharmacokinetic properties
of C2 symmetric inhibitors of the human immunodeficiency virus
type 1 protease, Antimicrob. Agents Chemother., 35 (1991) 2209-
(n=2 on different plates) were classed as partial actives.
Acknowledgments
2
[
214.
13] H.Q. Li, P.C. Lv, T. Yan, H.L. Zhu, Urea derivatives as
anticancer agents, Anticancer Agents Med. Chem., 9 (2009) 471-
80.
14] S. Mustafa, S. Perveen, A. Khan, Synthesis, enzyme inhibition
The University of Texas at Arlington and the American
Chemical Society Petroleum Research Fund partially supported
this research (grant
# 58261-ND1). The authors also
4
[
acknowledge Dr. Delphine Gout for X-ray data collection and
the NSF grants (CHE-0234811 and CHE-0840509) for
and anticancer investigation of unsymmetrical 1,3-disubstituted
ureas, J. Serb. Chem. Soc., 79 (2014) 1-10.
additional
instrumentation.
DST-Nanomission,
India
(
1
SR/NM/NS-20/2014), DST-SERB, India [SERB/F/1423/2017–
8 (File No. YSS/2015/000010)] and Jain University, India for
[15] G.M. Viana, L.C. de Sequeira Aguiar, J. de Araújo Ferrão,
A.B.C. Simas, M.G. Vasconcelos, The use of aqueous potassium
dichloroiodate for the synthesis of ureas, Tetrahedron Lett., 54
(2013) 936-940.
financial support. Antimicrobial screening was performed by
CO-ADD (The Community for Antimicrobial Drug Discovery),
funded by the Welcome Trust (UK) and The University of
Queensland (Australia).
[16] B. Sanjay, T. Zehra, M. Sudharshan, Medicinal Chemistry of
Ureido Derivatives as Anti-Infectives, Anti-Infect. Agents Med.
Chem., 5 (2006) 135-160.
[17] T.M. Wróbel, M. Kiełbus, A.A. Kaczor, V. Kryštof, Z.
Karczmarzyk, W. Wysocki, A. Fruziński, S.K. Król, A. Grabarska,
A. Stepulak, D. Matosiuk, Discovery of nitroaryl urea derivatives
with antiproliferative properties, J. Enzyme Inhib. Med. Chem., 31
Supplementary Material
1
13
Supplementary data [ H and C NMR data of all the
compounds (3a-p)] associated with this article can be found, in
the online version, at http://
(
2016) 608-618.
[
18] P. Padiyara, H. Inoue, M. Sprenger, Global Governance
References
Mechanisms to Address Antimicrobial Resistance, Infect. Dis.: Res.
Treat., 11 (2018) 1-4.
[1] I.D. Weiner, W.E. Mitch, J.M. Sands, Urea and Ammonia
[19] P. Umadevi, K. Deepti, I. Srinath, G. Vijayalakshmi, M.
Metabolism and the Control of Renal Nitrogen Excretion, Clin. J.
Am. Soc. Nephrol., 10 (2015) 1444-1458.
Tarakaramji, Synthesis and In-Vitro Antibatcterial Activity of some
new Urea, Thiourea and Thiosemicarbazide Derivatives, Int. J.
Pharm. Pharm. Sci., 4 (2012) 379-383.
[
2] J. Sidney M. Morris, Regulation of enzymes of the urea cycle
and arginine metabolism, Annu. Rev. Nutr., 22 (2002) 87-105.
3] P. Sikka, J.K. Sahu, A.K. Mishra, S.R. Hashim, Role of aryl urea
containing compounds in medicinal chemistry, Med. Chem., 5
2015) 479-483.
4] A.N. Acharya, A. Nefzi, J.M. Ostresh, R.A. Houghten, Tethered
[20] B. Rajtar, E. Szacoń, Ł. Świątek, M. Rządkowska, D. Matosiuk,
[
M. Polz-Dacewicz, Antiviral activity of 1-(1-arylimidazolidine-2-
ylidene)-3-(4-chlorobenzyl)urea derivatives, J. Pre-Clin. Clin. Res.,
(
[
7
[
(2013) 104-106.
21] S. Batra, Z. Tusi, S. Madapa, Medicinal chemistry of ureido
derivatives as anti-infectives, Anti-Infect. Agents Med. Chem., 5
2006) 135-160.
22] N. Soltani, J.P. Delbecque, J. Delachambre, Penetration and
Libraries:ꢀ Solid-Phase Synthesis of Substituted Urea-Linked
Bicyclic Guanidines, J. Comb. Chem., 3 (2001) 189-195.
(
[
[5] N. Kapuriya, K. Kapuriya, X. Zhang, T.-C. Chou, R. Kakadiya,
Y.-T. Wu, T.-H. Tsai, Y.-T. Chen, T.-C. Lee, A. Shah, Y. Naliapara,
T.-L. Su, Synthesis and biological activity of stable and potent
antitumor agents, aniline nitrogen mustards linked to 9-
anilinoacridines via a urea linkage, Bioorg. Med. Chem., 16 (2008)
insecticidal activity of diflubenzuron in Tenebrio molitor pupae,
Pestic. Sci., 14 (1983) 615-622.
[23] F. Wöhler, Ueber künstliche Bildung des Harnstoffs, Ann.
Phys. (Berlin, Ger.), 88 (1828) 253-256.
5
413-5423.

1
2
Bioorganic Chemistry
[
24] J. Zhao, Z. Li, S. Yan, S. Xu, M.-A. Wang, B. Fu, Z. Zhang,
[46] M. Balouiri, M. Sadiki, S.K. Ibnsouda, Methods for in vitro
evaluating antimicrobial activity: A review, J. Pharm. Anal., 6
(2016) 71-79.
Pd/C Catalyzed Carbonylation of Azides in the Presence of Amines,
Org. Lett., 18 (2016) 1736-1739.
[
25] E. Artuso, I. Degani, R. Fochi, C. Magistris, Preparation of
[47] L. Forbes, K. Ebsworth-Mojica, L. DiDone, S.-G. Li, J.S.
Freundlich, N. Connell, P.M. Dunman, D.J. Krysan, A High
Throughput Screening Assay for Anti-Mycobacterial Small
Molecules Based on Adenylate Kinase Release as a Reporter of Cell
Lysis, PLoS One, 10 (2015) 1-14.
[48] K.D. Greis, S. Zhou, R. Siehnel, C. Klanke, A. Curnow, J.
Howard, G. Layh-Schmitt, Development and Validation of a Whole-
Cell Inhibition Assay for Bacterial Methionine Aminopeptidase by
Surface-Enhanced Laser Desorption Ionization-Time of Flight Mass
Spectrometry, Antimicrob. Agents Chemother., 49 (2005) 3428-
3434.
Mono-, Di-, and Trisubstituted Ureas by Carbonylation of Aliphatic
Amines with S,S-Dimethyl Dithiocarbonate, Synthesis, (2007)
3
[
497-3506.
26] C. Han, Porco, Synthesis of Carbamates and Ureas Using
Zr(IV)-Catalyzed Exchange Processes, Org. Lett., 9 (2007) 1517-
520.
27] H.-G. Lee, M.-J. Kim, S.-E. Park, J.-J. Kim, B.R. Kim, S.-G.
1
[
Lee, Y.-J. Yoon, Phenyl 4,5-Dichloro-6-Oxopyridazine-1(6H)-
Carboxylate as Carbonyl Source: Facile and Selective Synthesis of
Carbamates and Ureas under Mild Conditions, Synlett, 2009 (2009)
2
809-2814.
[49] F. Nasrin, I. Bulbul, Y. Begum, In vitro antimicrobial and
cytotoxicity screening of n-hexane, chloroform and ethyl acetate
extracts of Lablab purpureus (L.) leaves, Agriculture and Biology
Journal of North America, 3 (2012) 43-48.
[28] S.-H. Lee, H. Matsushita, B. Clapham, K.D. Janda, The direct
conversion of carbamates to ureas using aluminum amides,
Tetrahedron, 60 (2004) 3439-3443.
[
29] S.H. Kim, S.H. Hong, Ruthenium-Catalyzed Urea Synthesis
Using Methanol as the C1 Source, Org. Lett., 18 (2016) 212-215.
30] A. Nefzi, N.A. Ong, R.A. Houghten, An efficient two-step
[50] I.A. Edwards, A.G. Elliott, A.M. Kavanagh, J. Zuegg, M.A.T.
Blaskovich, M.A. Cooper, Contribution of Amphipathicity and
Hydrophobicity to the Antimicrobial Activity and Cytotoxicity of β-
Hairpin Peptides, ACS Infect. Dis., 2 (2016) 442-450.
[
synthesis of mono-, di- and triureas from resin-bound amides,
Tetrahedron Lett., 41 (2000) 5441-5446.
[31] S.A.R. Mulla, C.V. Rode, A.A. Kelkar, S.P. Gupte, Activity of
homogeneous transition metal catalysts for oxidative carbonylation
of aniline to N,N′diphenyl urea, J. Mol. Catal. A: Chem., 122 (1997)
1
03-109.
[32] D. Carnaroglio, K. Martina, G. Palmisano, A. Penoni, C.
Domini, G. Cravotto, One-pot sequential synthesis of isocyanates
and urea derivatives via a microwave-assisted Staudinger-aza-Wittig
reaction, Beilstein J. Org. Chem., 9 (2013) 2378-2386.
[33] M. Mane, R. Balaskar, S. Gavade, P. Pabrekar, D. Mane, An
efficient and greener protocol towards synthesis of unsymmetrical
N,N′-biphenyl urea, Arabian J. Chem., 6 (2013) 423-427.
[34] E.V. Vinogradova, B.P. Fors, S.L. Buchwald, Palladium-
Catalyzed Cross-Coupling of Aryl Chlorides and Triflates with
Sodium Cyanate: A Practical Synthesis of Unsymmetrical Ureas, J.
Am. Chem. Soc., 134 (2012) 11132-11135.
[35] S. Shivprakash, G.C. Reddy, Stereoselective Synthesis of (Z)-1-
Benzhydryl-4-cinnamylpiperazines via the Wittig Reaction, Synth.
Commun., 44 (2014) 600-609.
[36] M. Subashini, K.K. Balasubramanian, S. Bhagavathy, Efficient
and Practical Synthesis of Dissymmetrical Ethers of 4-Nitrocatechol,
Synth. Commun., 38 (2008) 3088-3106.
[
37] S. Mills, E. Krumkalns, Adamantyel secondary amines, Eli
Lilly and Company, US3391142, 1968.
38] C.R. Elati, N. Kolla, S. Rao Chalamala, P.J. Vankawala, V.
[
Sundaram, H. Vurimidi, V.T. Mathad, New Synthesis of Donepezil
Through Palladium‐ Catalyzed Hydrogenation Approach, Synth.
Commun., 36 (2006) 169-174.
[
39] Bruker (2014). APEX2 and SAINT. Bruker AXS Inc.,
Madison, Wisconsin, USA.
40] G. Sheldrick, A short history of SHELX, Acta Crystallogr.,
Sect. A: Found. Adv., 64 (2008) 112-122.
41] V. Petříček, M. Dušek, L. Palatinus, Crystallographic
[
[
Computing System JANA2006: General features, Zeitschrift für
Kristallographie - Crystalline Materials, 229 (2014) 345-352.
[42] V. Patil, E. Barragan, S.A. Patil, S.A. Patil, A. Bugarin, Direct
Synthesis and Antimicrobial Evaluation of Structurally Complex
Chalcones, ChemistrySelect, 1 (2016) 3647-3650.
[
43] J. Gasteiger, M. Marsili, Iterative partial equalization of orbital
electronegativity—a rapid access to atomic charges, Tetrahedron, 36
1980) 3219-3228.
44] Sybyl-X Molecular Modeling Software Packages, Version 2.0,
TRIPOS Associates, Inc. St. Louis, MO, USA, 2012.
45] A.R. Ahameethunisa, W. Hopper, Antibacterial activity of
(
[
[
Artemisia nilagirica leaf extracts against clinical and
phytopathogenic bacteria, BMC Complementary Altern. Med., 10
(
2010) 1-6.

1
3
Synthesis, Molecular Docking Studies, and Antimicrobial Evaluation of New
Structurally Diverse Ureas
a
b
c
d
a
Mahadev Patil , Anurag Noonikara Poyil , Shrinivas D. Joshi , Shivaputra A. Patil , Siddappa A. Patil *,
b
Alejandro Bugarin 
HIGHLIGHTS:
1
2
3
4
5
. A practical method for the easy access to ureas is documented
. Good functional group tolerance
. Excellent docking scores were observed
. Extensive biological screening for both bacteria and fungi are reported
. Comprehensive adducts’ characterization was performed
—
——

Corresponding authors. Tel.: +1-817-272-9399: e-mail address: bugarin@uta.edu (A. B.) e-mail address: p.siddappa@Jainuniversity.ac.in

1
4
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Synthesis, Molecular Docking Studies, and
Antimicrobial Evaluation of New
Structurally Diverse Ureas
Leave this area blank for abstract info.
a
b
c
d
a
Mahadev Patil , Anurag Noonikara Poyil , Shrinivas D. Joshi , Shivaputra A. Patil , Siddapa A. Patil *,
b
and Alejandro Bugarin *
a
Centre for Nano & Material Sciences, Jain University, Jain Global Campus, Bangalore 562112, Karnataka,
India.
b
Department of Chemistry and Biochemistry, University of Texas at Arlington, Arlington, TX 76019, USA
c
Novel Drug Design and Discovery Laboratory, Department of Pharmaceutical Chemistry, S. E. T’s College of
Pharmacy, Sangolli Rayanna Nagar, Dharwad 580 002, Karnataka, India.
d
Pharmaceutical Sciences Department, College of Pharmacy, Rosalind Franklin University of Medicine and
Science, 3333 Green Bay Road, North Chicago, IL 60064, USA